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Nanotechnologies in Cancer

Journal of Drug Delivery

Guest Editors Giuseppe De Rosa Michele Caraglia Stefano Salmaso and Tamer Elbayoumi




Guest Editors Giuseppe De Rosa Michele CaragliaStefano Salmaso and Tamer Elbayoumi

Copyright copy 2013 Hindawi Publishing Corporation All rights reserved

This is a special issue published in ldquoJournal of Drug Deliveryrdquo All articles are open access articles distributed under the Creative Com-mons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original workis properly cited

Editorial Board

Sophia Antimisiaris GreeceAbdul Basit UKE Batrakova USAShahrzad Bazargan-Hejazi USAHeather Benson AustraliaAndreas Bernkop-Schnrch AustriaGuru V Betageri USAMarıa J Blanco-Prieto SpainG Buckton UKYılmaz Capan TurkeyCarla Caramella ItalyRoberta Cavalli ItalyNevin Celeby TurkeyRita Cortesi ItalyAlekha K Dash USAZedong Dong USAMartin J DrsquoSouza USAJeanetta du Plessis South AfricaN D Eddington USAA Fadda ItalyJia You Fang TaiwanSven Froslashkjaeligr DenmarkSanjay Garg New Zealand

Andrea Gazzaniga ItalyRichard A Gemeinhart USALisbeth Illum UKJuan M Irache SpainBhaskara R Jasti USAHans E Junginger ThailandDae-Duk Kim Republic of KoreaVinod Labhasetwar USAClaus S Larsen DenmarkKang Choon Lee USALee-Yong Lim AustraliaRam I Mahato USAPhilippe Maincent FranceEdith Mathiowitz USAReza Mehvar USABozena Michniak-Kohn USATamara Minko USAAmbikanandan Misra IndiaAshim K Mitra USAS M Moghimi DenmarkA Mullertz DenmarkSteven H Neau USAAli Nokhodchi UK

Abdelwahab Omri CanadaRosario Pignatello ItalyViness Pillay South AfricaMorteza Rafiee-Tehrani IranMichael S Roberts AustraliaPatrick J Sinko USAJohn Smart UKQuentin R Smith USAHartwig Steckel GermanySnow Stolnik-Trenkic UKK Takayama JapanHirofumi Takeuchi JapanIstvan Toth AustraliaHasan Uludag CanadaClaudia Valenta AustriaJaleh Varshosaz IranSubbu S Venkatraman SingaporeS P Vyas IndiaChi H Wang SingaporeAdrian Williams UKTin Wui Wong MalaysiaSri Rama K Yellela USAP York United Kingdom

Contents

Nanotechnologies in Cancer Giuseppe De Rosa Michele Caraglia Stefano Salmaso and Tamer ElbayoumiVolume 2013 Article ID 604293 3 pages

Nanoparticle Albumin Bound Paclitaxel in the Treatment of Human Cancer Nanodelivery ReachesPrime-Time Iole Cucinotto Lucia Fiorillo Simona Gualtieri Mariamena Arbitrio Domenico CilibertoNicoletta Staropoli Anna Grimaldi Amalia Luce Pierfrancesco Tassone Michele Caragliaand Pierosandro TagliaferriVolume 2013 Article ID 905091 10 pages

Liposomal Doxorubicin in the Treatment of Breast Cancer Patients A Review Juan Lao Julia MadaniTeresa Puertolas Marıa Alvarez Alba Hernandez Roberto Pazo-Cid Angel Artal and Antonio Anton TorresVolume 2013 Article ID 456409 12 pages

Gene Therapy for Advanced Melanoma Selective Targeting and Therapeutic Nucleic AcidsJoana R Viola Diana F Rafael Ernst Wagner Robert Besch and Manfred OgrisVolume 2013 Article ID 897348 15 pages

Clinical Trials with Pegylated Liposomal Doxorubicin in the Treatment of Ovarian CancerCarmela Pisano Sabrina Chiara Cecere Marilena Di Napoli Carla Cavaliere Rosa Tambaro GaetanoFacchiniCono Scaffa Simona Losito Antonio Pizzolorusso and Sandro PignataVolume 2013 Article ID 898146 12 pages

Lipid-Based Nanovectors for Targeting of CD44-Overexpressing Tumor Cells Silvia ArpiccoGiuseppe De Rosa and Elias FattalVolume 2013 Article ID 860780 8 pages

Recent Trends in Multifunctional Liposomal Nanocarriers for Enhanced Tumor TargetingFederico Perche and Vladimir P TorchilinVolume 2013 Article ID 705265 32 pages

Stealth Properties to Improve Therapeutic Efficacy of Drug NanocarriersStefano Salmaso and Paolo CalicetiVolume 2013 Article ID 374252 19 pages

Bisphosphonates and Cancer What Opportunities from Nanotechnology Giuseppe De RosaGabriella Misso Giuseppina Salzano and Michele CaragliaVolume 2013 Article ID 637976 17 pages

Neoplastic Meningitis from Solid Tumors A Prospective Clinical Study in Lombardia and a LiteratureReview on Therapeutic Approaches A Silvani M Caroli P Gaviani V Fetoni R Merli M RivaM De Rossi F Imbesi and A SalmaggiVolume 2013 Article ID 147325 6 pages

Nanomaterials Toxicity and Cell Death Modalities Daniela De Stefano Rosa Carnuccioand Maria Chiara MaiuriVolume 2012 Article ID 167896 14 pages

Utilisation of Nanoparticle Technology in Cancer Chemoresistance Duncan Ayers and Alessandro NastiVolume 2012 Article ID 265691 12 pages

Hindawi Publishing CorporationJournal of Drug DeliveryVolume 2013 Article ID 604293 3 pageshttpdxdoiorg1011552013604293

EditorialNanotechnologies in Cancer

Giuseppe De Rosa1 Michele Caraglia2 Stefano Salmaso3 and Tamer Elbayoumi4

1 Department of Pharmacy University Federico II of Naples Via Montesano 49 80131 Naples Italy2 Department of Biochemistry Biophysics and General Pathology Second University of NaplesVia S M Costantinopoli 16 80138 Naples Italy

3 Department of Pharmaceutical and Pharmacological Sciences University of Padova Via F Marzolo 5 35131 Padova Italy4Department of Pharmaceutical Sciences Midwestern University 19555 North 59th Avenue Glendale AZ 85308 USA

Correspondence should be addressed to Giuseppe De Rosa gderosauninait

Received 9 April 2013 Accepted 9 April 2013

Copyright copy 2013 Giuseppe De Rosa et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Cancer is today themajor cause ofmorbidity andmortality inwestern and industrialized countriesThe use of drugs for thetherapeutic treatment of cancer raises important issues abouttheir toxicity on normal cells and more in general on theirsystemic side effects

Issues about systemic toxicities have been faced withboth first generation anticancer drugs and with more recentdrugs that operate through specific targets with the lattermaintaining the homeostasis of several normal tissues Theemergence of the nanomedicine has opened a novel scenarioin the use of all anticancer agents with the possibility toimprove their efficacy and to reduce their side effects dueto their distribution in normal tissues Products based onnanotechnological carriers have entered the clinical practiceand a huge number of studies have been performed inorder to optimize the application of nanomedicines in cancertreatment Although these nanotechnology-based systemsare still far to fully comply the idea of the ldquomagic bulletrdquo theadvantages offered by this approach are clearly promising

This special issue covers different aspects related to theexploitation of nanotechnology-based systems for cancertreatment including the design and features of multifunc-tional nanocarriers the drug targeting concept the gene ther-apy the toxicity of nanomaterials and themore recent clinicalstudies that have determined a glimmer of hope for cancerpatients

Liposomes are among the first nanotechnological-basedplatforms ever developed for cancer therapy One of the

major limitations in the clinical use of liposomes and othernanoparticles is their short plasma half-life due to therapid opsonization process that yields their removal frombloodstream and degradation by macrophages from reticularendothelial system On the basis of these considerationsldquostealthrdquo nanocarriers have been promptly developed throughconjugation of hydrophilic polymers such as polyethyleneglycol (PEG) on the particle surfaceThe review of S Salmasoand P Caliceti describes the basic concept underlining theldquostealthrdquo properties of drug nanocarriers the parametersinfluencing the polymer coating performance in terms ofopsoninsmacrophages interaction with the colloid surfacethe most commonly used materials for the coating processand the outcomes of this peculiar procedure

One of the first ldquostealthrdquo nanocarriers loaded with anti-cancer drug that has achieved the clinical practice was thepegylated liposomal doxorubicin (PLD) The paper by CPisano et al describes the role and clinical indications of PLDin ovarian cancer PLD was firstly approved for platinum-refractory ovarian cancer and then received full approvalfor platinum-sensitive recurrent disease Recently it wasdemonstrated that the combination of PLD with platinumhas similar activity but less toxicity than the combinationcontaining free doxorubicin triggering new interest on PLDalso in the first line of treatment of this tumour Anotherclinical indication of PLD is the treatment of metastatic orlocally advanced breast cancer when the maximal allowedcumulative doses of doxorubicin administered to patients is

2 Journal of Drug Delivery

reached The paper by J Lao et al summarizes the mainresults achieved with the use of PLD in this setting of patientsunderlining the loss of cardiotoxicity with the preservation ofclinical activity if compared to free doxorubicin Moreoverinteresting results have been recorded by combining anti-HER-2 antibodies (trastuzumab) with PLD in the treatmentof both locally advanced andmetastatic breast cancer enlight-ening the potential advantages of the combination of thesedrugs (both cardiotoxic) in these two clinical settings

An important concern that limits the therapeutic profileof doxorubicin and other anticancer agents is the devel-opment of innate or acquired tumour resistance that ismediated by several mechanisms The paper by D Ayers andANasti describes the differentmechanisms bywhich tumourcells generate the resistance to anticancer agents and thestrategies to overcome the refractoriness of cancer cells Indetails the authors discuss the limits and advantages ofdifferent nanotechnological devices used to deliver cytotoxicdrugs or nucleic acids (such as micro-RNAs or siRNAs) thattarget specific molecular resistance factors

Another important limitation to the effective therapeuticactivity of anticancer drugs is the inability of some moleculesto overcome anatomic barriers such as the blood-brain bar-rier and to accumulate in the subarachnoidal or leptomenin-geal spaces that can be sites of dissemination of brain or extra-brain tumoursThe paper byA Silvani et al describes the roleof liposomal arabinoside cytosine (AraC) in the treatment ofneoplastic meningosis including an unpublished prospectivetrial performed in the Italian region Lombardia and a shortreview of the data reported by other already published clinicalstudies

The paper from I Cucinotto et al reports and discussesthe most recent findings on the clinical use of nanoparticlealbumin-bound paclitaxel (nab-paclitaxel) also known withthe commercial name of Abraxane This drug is at the mo-ment approved for the treatment of metastatic breast cancerand nonsmall cell lung cancer However this nanotechnol-ogy-based drug is very promising also for the treatmentof other human neoplasms such as pancreatic cancer ormetastatic melanoma which generally are considered refrac-tory to treatment with conventional anticancer agents In thisview the paper of J R Viola et al provides a short intro-duction to the mechanisms of melanomagenesis discussingthe shortcomings of current therapeutic approaches ascribedto the existence of a wide range of mutations associatedwith this cancer Authors highlight alternative approaches fortreatment of melanomas based on the use of therapeutical-ly active nucleic acidsThe delivery of nucleic acid nanophar-maceutics is brought into perspective as a novel highlyselective antimelanoma therapeutic approachwhilst avoidingunwanted and toxic side effects The possibilities for mela-noma selective targeting are discussed together with latestreports of advanced clinical applications

Also target-based agents need to be specifically deliveredto tumour tissues and in this regard G De Rosa et al pro-vide a comprehensive article on the clinical applications ofbisphosphonates (BPs) starting from their use as inhibitors ofbone resorption up to their novel therapeutic indications asanticancer drugs In detail nitrogen-containing BPs (N-BPs)

induce apoptosis in a variety of cancer cells in vitro and inpreclinical settings and show a very intriguing antiangiogenicactivity Unfortunately clinical anticancer activity of N-BPs isfar to be demonstrated In this light the authors describe hownanotechnology can provide carriers to limit BP accumula-tion into the bone thus increasing drug level in extra-skeletalsites of the body to directly kill cancer cells On the otherhand BPs can also be used as targeting agents to specificallydeliver nanocarriers loadedwith anticancer drugs in the bonetissue for the treatment of bone tumours or metastases

The active targeting of nanoparticles is an effective strat-egy to increase the uptake of anti-cancer drug-loaded vehiclesby tumour cells It is based on the decoration of nanoparticleswith specific ligands such as peptides or antibodies raisedagainst tumour-associated antigens (molecules with higherexpression on tumour cells than in normal counterparts)

In this light S Arpicco et al review the use of hyaluronicacid (HA) as a unique targeting agent for the recognition ofcancer cells due to the high expression levels of its receptor(named CD44) on tumour cell surface The CD44 receptor isfound at low levels on the surface of epithelial haematopoi-etic and neuronal cells but it is overexpressed in manycancer cells and on cancer stem cells This review describesthe approaches used for the preparation and investigationof lipid-based nanovectors decorated with HA for the activedelivery of a variety of therapeuticmolecules in the treatmentof human cancer

Other strategies in the development of nanotechnologicaldevices include the multifunctional decoration with differentmoieties that allow both the detection and the treatmentof cancer cells (theranostic devices) In this view the paperby F Perche and V P Torchilin describes multifunctionalliposomal nanocarriers that combines long blood circulationand selective accumulation to the tumor lesions based uponremote-controlled or tumour stimuli-sensitive extravasationfrom blood to the tumour tissue and internalizationmotifs tomove from tumour bounds andor tumor intercellular spaceto the cytoplasm of cancer cells

Finally nanovectors are not completely inert materi-als and can be endowed with intrinsic cytotoxicity thatcauses sometimes potential deleterious effects in normaltissues In this light D De Stefano et al describe the mainmechanisms by which nanosized materials can induce celldeath such as apoptosis mitotic catastrophe authophagynecrosis and pyroptosis The understanding of these mech-anisms is mandatory for a safe use of nanocarriers Theauthors describe all the variables that can affect nanocarriercytotoxicity underlining the need for generally acceptedguidelines for the development and use of nanotechnologicaldevices

We believe that this special issue can be of great interestfor the readers in depicting the most recent advances gen-erated by basic translational and clinical research focusedon the development and use of nanocarriers for the deliveryof anticancer agents The special issue thoroughly reportsthe outcomes derived from basic and preclinical studies andthe main limitations emerged from both clinical trials andpractice The criticisms derived from the clinics need tobe regarded as crucial starting points for the optimization

Journal of Drug Delivery 3

of the nanotechnological drug delivery systems In otherwords bidirectional flow of information from the bench tothe bedside and back again to the bench is pivotal to offerimproved nanomedicine-based strategies of treatment ofcancer patients

Giuseppe De RosaMichele CaragliaStefano Salmaso

Tamer Elbayoumi


Review ArticleNanoparticle Albumin Bound Paclitaxel in the Treatment ofHuman Cancer Nanodelivery Reaches Prime-Time

Iole Cucinotto1 Lucia Fiorillo1 Simona Gualtieri1 Mariamena Arbitrio2 DomenicoCiliberto1 Nicoletta Staropoli1 Anna Grimaldi3 Amalia Luce3 Pierfrancesco Tassone1

Michele Caraglia3 and Pierosandro Tagliaferri1

1 Medical Oncology Unit Department of Experimental and Clinical Medicine University ldquoMagna Graeciardquo of Catanzaroand ldquoTommaso Campanellardquo Cancer Center Campus Salvatore Venuta Viale Europa 88100 Catanzaro Italy

2 Institute of Neurological Science (ISN-CNR) UOS of Pharmacology Roccelletta di Borgia 88021 Catanzaro Italy3 Department of Biochemistry Biophysics and General Pathology Second University of Naples 80138 Naples Italy

Correspondence should be addressed to Pierosandro Tagliaferri tagliaferriuniczit

Received 31 January 2013 Accepted 5 March 2013

Academic Editor Giuseppe De Rosa

Copyright copy 2013 Iole Cucinotto et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Nanoparticle albumin bound paclitaxel (nab-paclitaxel) represents the first nanotechnology-based drug in cancer treatment Wediscuss the development of this innovative compound and report the recent changing-practice results in breast and pancreaticcancer A ground-breaking finding is the demonstration that nab-paclitaxel can not only enhance the activity and reduce the toxicityof chromophore-diluted compound but also exert activity in diseases considered refractory to taxane-based treatment This is thefirst clinical demonstration of major activity of nanotechnologically modified drugs in the treatment of human neoplasms

1 Introduction

Current development of cancer treatment mainly relies onthree avenues

(a) the identification of molecular targets for selectiveblockade of driver pathways in cancer cells or intumour microenvironment

(b) immunemodulatory approaches which might en-hance the antitumor specific immune response

(c) new delivery approaches in order to achieve higherbioavailability of anticancer agents

The topic of the current review is the nanoparticle albu-min bound paclitaxel (nab-paclitaxel) development whichhas opened a novel scenario in cancer treatment by theenhancement of paclitaxel delivery by the use of nanotech-nology

2 Taxane (First) Revolution ofCancer Therapy

Taxanes are an important class of antitumor agents usingsolvent-based delivery vehicles Paclitaxel (Bristol-MyersSquibb (New York NY)) was identified in 1966 as an extractfrom Taxus brevifolia obtained in a pure form in 1969but its structure was published in 1971 Investigators facedseveral problems due to low concentration and structurecomplexities for low water solubility [1 2] (Figure 1)

In fact only in 1979 Susan Horwitz discovered thatpaclitaxel has a unique mechanism of action and interestwhich was additionally stimulated when impressive activitywas demonstrated in NCI tumor screening [3] Paclitaxelis a diterpenoid pseudoalkaloid with formula C

47H51NO14

(119872119882 = 853Da) whose activity was demonstrated in differ-ent preclinical models For antitumor activity the presenceof the entire taxane molecule is required (Figure 2) forthe inactivity of the ester and the tetraol formed by a lowtemperature cleavage of paclitaxel [4]

Although the development of paclitaxel was hampered bylimited availability of its primary source and the difficulties


O

O

OO

OO OO

O

OO

HN

HO HO

OH

Figure 1 Structure of paclitaxel (512057320-epoxy-121205724712057313120572-hex-ahydroxytan-11-en-9-one-410-diacetate2-benzoate-13-ester with(2R3S)-N-benzoyl-3-phenyllioserine)

Figure 2 Taxane nucleus

inherent to large-scale isolation extraction and its pooraqueous solubility interest was maintained after characteri-zation of its novel mechanism of cytotoxic action In order toafford new preclinical and clinical studies it was necessary tofind new andmore abundant and renewable resourcesThesestudies led to the development of docetaxel (Taxotere) asemisynthetic taxane analogue extracted from Taxus baccataa European yewDocetaxel differs frompaclitaxel in two posi-tions in its chemical structure and this small alterationmakesit more watersoluble Taxanes disrupt microtubule dynamicsby stabilizing the microtubule against depolymerizationenhancing their polymerization promoting the nucleationand elongation phases of the polymerization reaction andreducing the critical tubulin subunit concentration requiredfor microtubule assembly Moreover they alter the tubulindissociation rate at both ends of the microtubule This leadsto reduced dynamic instability whereas the association rateis not affected After the treatment with taxanes the micro-tubules are highly stable and resistant to depolymerizationby cold calcium ions dilution and other antimicrotubuleagents The final result is the impairment of dynamics ofmicrotubule depolymerization which is a critical event in themitotic process [5]

Paclitaxel is active against primary epithelial ovariancarcinoma breast cancer colon non-small-cell lung cancerand AIDS-related Kaposirsquos sarcoma in preclinical models[3 6 7] and is presently of common use in the treatmentof several important malignancies as lung cancer breast

cancer Kaposirsquos sarcoma squamous cell carcinoma of thehead and neck gastric cancer esophageal cancer bladdercancer and other carcinomas Despite being clinically veryactive paclitaxel and docetaxel are associated with manyserious sideeffects which often preclude the prolonged use inpatients A number of these side effects have been associatedwith the vehicles used for the formulation the cremophorEL (CrEL-polyethoxylated castor oil) [8] for paclitaxel andpolysorbate 80 (Tween 80) for docetaxel respectively thataltered also their pharmacokinetic profiles CrEL is consid-ered to be responsible for the hypersensitivity reactions seenin patients during paclitaxel therapy In vitro CrEL causedaxonal swelling demyelination and axonal degenerationand thus it may also contribute to the development ofneuropathy in patients receiving paclitaxel The use of CrELrequires premedication with antihistamines and corticos-teroids to prevent hypersensitivity reactions and despitethese premedications approximately 40 of all patients willhave minor reactions (eg flushing and rash) and 3 willhave life threatening reactions CrEL also causes leachingof the plasticizers from polyvinyl chloride (PVC) bags andinfusions sets thus paclitaxel must be infused via the useof special non-PVC infusion systems and in-line filtrationAnother effect induced by CrEL is the alteration of lipopro-tein pattern and the consequent hyperlipidemia MoreoverCrEL and polysorbate 80 interfere with efficacy by limitingtumor penetration through the formation of large polarmicelles which for CrEL-paclitaxel can lead to nonlinearpharmacokinetics and decreased unbound drug fraction [9]

To overcome the ideal dosage form and bypass allthe present limitations novel ldquocarrier delivery systemsrdquoincluding liposomes micelles and particulate drug deliverysystems were formulated as commonpractice for novel drugslike microRNAs [10ndash15]

Some of them have already reached the clinical practicelike liposomal doxorubicin or liposomal amphotericin BAnother example of nanotechnology applied to drug deliveryis the preclinical development of stealth liposomes encap-sulating zoledronic acid (LipoZOL) to reduce binding ofZOL to bone and increase its bioavailability in extraskeletaltumor sites [16] Natural human protein based carrier canalso be used to manufacture nanocarriers for drug deliverythis is the example of the paclitaxel albumin bound by whichit is possible to selectively deliver larger amounts of drugto tumors reducing the toxicities related to solvent-basedformulations Albumin is a natural carrier of hydrophobicendogenous molecules (such as vitamins hormones andother plasma constituents) in a noncovalent and reversiblebinding and allows for transport in the body and release atthe cell surface [17]

Abraxane (nab-paclitaxel ABI 007 or Abraxane CelgeneIncOdentonMDUSA)was the first to receive FDAapprovalin 2005 for the treatment of breast cancer in patientswho reported progressive disease after chemotherapy formetastatic cancer or relapse within 6 months of adjuvantchemotherapy

Nab-paclitaxel is a colloidal suspension of 130 nanome-ter particles solvent-free homogenized with human serumalbumin (3-4) by which it is possible to infuse higher


doses of drug than the standard dose used in paclitaxeltherapy with fewer side effects with less infusion time (30minutes) and without premedication The new formulationallows the delivery of paclitaxel to tumors with a 45-foldincrease in its transport coupled with albumin receptorsacross endothelial cells [18] with an enhanced intracellu-lar antitumor paclitaxel delivery and activity [19] In themechanism of drug delivery an albumin receptor (gp60) onendothelial cells seems to be involved which transports pacli-taxel into the extravascular space with subsequent invagi-nation of the cell membrane to form caveolae transcytoticvesicles and also tumor accumulation of nanoparticle boundto SPARC (secreted protein acidic and rich in cysteine)which is overexpressed in many solid tumors includingbladder prostate and pancreas cancers [20] Its intravenousinfusion is moremanageable and safe because it is performedby standard plastic intravenous infusion bags and can alsobe reconstituted in a much smaller volume of normal salinecompared to paclitaxel Preclinical studies have demonstratedthat nab-paclitaxel achieved higher intratumor concentra-tions compared to CrEL-paclitaxel with a better bioavailabil-ity and showed an improved efficacy and therapeutic index inmultiple animalmodels [21] Other new technologies recentlyused to deliver paclitaxel have led to the development ofinnovative formulations such as Nanoxel and liposomal andpolymeric paclitaxel

Nanoxel-PM is efficacious and less toxic than free doc-etaxel formulation and was evaluated in comparison withTaxotere in preclinical studies Nanoxel-PM can reducesideeffects of hypersensitivity reactions and fluid retentionwhile retaining antitumor efficacy in cancer patients [22]

Further studies led to the development of new formula-tions of liposomal paclitaxel The special composition of theliposomal membrane which contains high doses of paclitaxelcould reduce the aggregation giving the molecule higherstability and confers an increase of efficacy in animal modelsas in human tumors [23]

An hydrotropic polymer micelle system has also beendeveloped for delivery of poorly water-soluble drugs aspaclitaxel This polymer showed not only higher loadingcapacity but also enhanced physical stability in aqueousmedia and provides an alternative approach for formulationof poorly soluble drugs [24 25]

3 Nab-Paclitaxel in Breast Cancer Treatment

Breast cancer (BC) is the most common cancer in femalepatients and follows lung cancer as the most common causeof female cancer death While only 5ndash7 of BC patientspresent metastatic disease (mBC) at diagnosis and morethan 30 presenting localized disease will eventually recur5 year survival of advanced disease is less than 20 [33]Current treatment of advanced breast cancer is mainly aimedto ameliorate quality of life and prolong survival Treatmentchoice is not an easy task in terms of drug selection andcombination Chemotherapy plays an essential role for thetreatment of mBC Among anticancer drugs taxanes areconsidered the most effective while their use involves long

infusion time neurotoxicity and high risk of hypersensitivityreactions [8 34 35] These latter effects are due to allergicreactions induced by the use of solubilizing agents (as chro-mophores) and today are less common due to the use in theclinical practice of corticosteroids and antihistamines [36]In order to overcome these important limitations a majorinterest is devoted to novel drugs as nab-paclitaxel eribulinixabepilone PARP inhibitors and new HER 2 inhibitors aslapatinib pertuzumab TDM1 and neratinib [37ndash43]

Following phase I studies by Ibrahim et al in 2002[19] and by Teng et al in 2004 [44] which led to MTDidentification at 300mgm2 in the three weekly schedule withneurotoxicity as dose limiting toxicity Nyman et al in 2005[45] identify in the weekly schedule the MTD at 100mgsqmfor highly pretreated patients and 150mgm2 for nonhighlypretreated patients with grade 4 neutropenia and grade 3neuropathy as DLT with earlier onset at higher dosagesThe pivotal phase 3 study was published in 2005 whereGradishar et al [30] compared nab-paclitaxel (260mgm2)at three week schedule with CrEL-paclitaxel 175mgm2 alsoat three week schedule The study clearly demonstrateda survival advantage for nab-paclitaxel with an improvedtoxicity profile

In 2009 a phase II randomized study [26] compared threeweek docetaxel 100mgm2 with three week nab-paclitaxel300mgm2 weekly nab-paclitaxel 100mgsqm and weeklynab-paclitaxel 150mgsqm The 150 nab-paclitaxel weeklyschedule provided the best PFS (gt5months)which resulted tobe statistically significant An update of this study publishedby Gradishar et al in 2012 demonstrated a median overallsurvival (OS) of 338 months which statistically overcame theother treatment arms

All together these data demonstrated that nab-paclitaxelis superior to CrEL-paclitaxel in the three week scheduleand that nab-paclitaxel at weekly 150 schedule provides animpressive long term survival [27] Recently nab-paclitaxelwas administered in combination with biological agents inthe treatment of mBC In detail a safety analysis of thefirst ten enrolled patients treated for at least one cycle ofthe initial doses of nab-paclitaxel (125mgm2 iv on days1 8 and 15 every 28 days) in combination with lapatinib(1250mg orally once daily on a continuous basis) in a 4-weekcycle for a planned minimum of six cycles was performedHowever during the ongoing safety review of the first fivepatients Grade 3 toxicities were observed in all five patients(four with neutropenia and one with neutropenic fever anddiarrhea) and the decision was made to reduce the doseof both study drugs All subsequent patients (119899 = 55)received nab-paclitaxel (100mgm2 iv on days 1 8 and15 every 28 days) in combination with lapatinib (1000mgorally once daily on a continuous basis) in a 4-week cyclefor a minimum of six cycles RR was 53 with the majorityof patient responses demonstrating a partial response (PR)(47) Four (7) patient responses demonstrated a completeresponse (CR) and ten (17) demonstrated a stable diseaseThe progression-free survival (PFS) and time to progression(TTP) were 397 weeks (95 CI 341ndash639) and 41 weeks(95 CI 391ndash646) respectively Lapatinib 1000mg with


Table 1 Randomized phase II and III trials with nab-paclitaxel in mBC(a) Phase II

Arms Pts

RR ()INVRAD119875 = 047

RR ()INDRAD119875 = 047

PFS ()INVRAD119875 = 047

PFS ()IND RAD119875 = 047

OS(months)119875 = 47

Gradishar et al 2009[26]Gradishar et al 2012[27]Update OS(first line)

Nab-paclitaxel

300mgm2 q3w150mgm2 qw100mgm2 qw

767476

467463

374945

10914675

11129128

277338222

Docetaxel100mgm2 q3w 74 39 35 78 75 266

Arms Pts ORR () Median PFS (months) OS (months)119875 = 73 119875 = ND 119875 = 71

Blum et al 2007 [28](following lines)

Nab-paclitaxel125 mgm2 qw 75 16 35 91


Arm PtsRR I line

()119875 = ND

RR gt I line()119875 = ND

ORR()119875 = ND

Median TTP(weeks)119875 = ND

Median survival(weeks)119875 = ND

Ibrahim et al 2002[19](first andfollowing lines)

Nab-paclitaxel300mgm2 q3w 63 64 21 48 266 636

Arms Pts

MedianPFS

(months)119875 = ND

PFS at 6months()119875 = ND

MDR(months)119875 = ND

Median OS(months)119875 = ND

OS at 6 months()119875 = ND

Roy et al 2009 [29](first line)

Nab-paclitaxel125mgsqmGemcitabine1000mgsqmdays 1 and 8

50 79 60 69 Notreached 92

(b) Phase III

AEs () 119875 = 001

Arms Pts RR ()119875 = 001

TTPweeks119875 = 006

Grade IV neutropenia Grade III sensoryneuropathy

Gradisharet al 2005[30](first line)

Nab-paclitaxel260mgsqm 229 33 230 9 10

Paclitaxel175mgsqm 225 19 169 22 2

P P value nd not done AEs adverse events inv rad investigator radiologist ind rad independent radiologist ORR overall response rate RRresponse rate TTP time to progression PFS progression-free survival OS overall survival MDR median duration of response

nab-paclitaxel 100mgm2 iv is feasible with manageable andpredictable toxicity and an RR of 53 comparing favor-ably with other HER2-based combinations in this setting[50]

Two important points under investigation are the com-parison of weekly nab-paclitaxel with CrEL-paclitaxel bothat weekly schedules and the potential advantage of combi-nation with bevacizumab Finally nab-paclitaxel has shownsome activity also in CrEL-paclitaxel heavily pretreated andresistant patients [28] (Table 1)

4 Nab-Paclitaxel in PancreaticCancer Treatment

Pancreatic cancer (PC) is at present a big cancer killerwith an expected survival of 6 months in advanced stagePC (aPC) Till a recent report demonstrating good activ-ity of oxaliplatin irinotecan and fluorofolate (FOLFIRI-NOX combination) gemcitabine is still the mainstay treat-ment In a recent meta-analysis Ciliberto et al [51]described a statistically superiority in terms of survival


Table 2 Randomized phase III and III trials with nab-paclitaxel in aPC(a) Phase III

Arms Pts MTD RR ()119875 = ND

Median OS (months)119875 = ND

1 year survival ()119875 = ND

von Hoff et al 2011[31] (First line)

Gem

citabine

1000

mgsqm

Nab-paclitaxel

100mgm2 q3w125mgm2 q3w150mgm2 q3w

20443

X 48 122 48

(b) Phase III

Arms Pts ORR()

MedianTTP(MO)

PFS OS AEs () 119875 = 001

Median(MO)

1 yr()

Median(MO)

1 yr()

2 yr()

GradegeIIIn

eutro

penia

Fatig

ue

Neuropathy

119875 = lt001 119875 = lt001 119875 = lt001 119875 = 031 119875 = lt001 119875 = lt001 119875 = 02

Von Hoff etal 2011 [32](first line)

Nab-paclitaxel125mgm2 qw

followedGemcitabine1000mgsqm

qw

431 99 51 55 16 85 35 9 38 17 17

Gemcitabine1000mgsqm

qw430 31 36 37 9 67 22 4 27 7 1

P P value nd not done AEs adverse events MTD maximum tolerated dose ORR overall responce rate RR response rate TTP time to progressionPFS progression-free survival OS overall survival MDR median duration of response

and response rate for gemcitabine-based combination com-pared to gemcitabine alone Moreover this advantage wasmarginal and at the cost of an increased toxicity Theauthors concluded that in the era of targeted therapy newapproaches were possible only in presence of solid preclinicalfindings

A report by von Hoff et al [31] demonstrated in aphase III study an interesting activity of gemcitabinenab-paclitaxel combination at gemcitabine 1000mgm2 and nab-paclitaxel at 125mgm2 doses weekly for three doses ina 4 week schedule A 48 response rate was achieved atMTD The authors additionally demonstrated that SPARC-expressing tumors appeared more sensitive to the drugcombination

An interesting finding from a preclinical study reportedthat nab-paclitaxel demonstrated the capacity of increasingthe gemcitabine bioavailability inside the tumors Thesefindings led to the design of a phase III study wheregemcitabinenab-paclitaxel was compared to gemcitabinealone showing an advantage in OS PFS and RR This studypresented to ASCO GI 2013 (American Society of ClinicalOncology Gastrointestinal Cancer Symposium) by von Hoffis clearly a changing practice study and the gemcitabinenab-paclitaxel which led to an almost two month longer OSshould be now compared to FOLFIRINOX combination

(Table 2) The biological bases of the synergistic interac-tion between nab-paclitaxel and gemcitabine have recentlybeen elucidated by an in vivo study in animal modelsIn detail the combination treatment was administered toKPC mice that develop advanced and metastatic pancreasductal adenocarcinoma The authors have demonstrated anincrease of intratumoral gemcitabine levels attributable toa marked decrease in the primary gemcitabine metaboliz-ing enzyme cytidine deaminase Correspondingly paclitaxelreduced the levels of cytidine deaminase protein in culturedcells through reactive oxygen species-mediated degradationresulting in the increased stabilization of gemcitabine Thesefindings support the concept that suboptimal intratumoralconcentrations of gemcitabine represent a crucialmechanismof therapeutic resistance in PC [52] This study providesmechanistic insight into the clinical cooperation observedbetween gemcitabine and nab-paclitaxel in the treatment ofpancreatic cancer

5 Other Areas of Nab-Paclitaxel Development

Melanoma represents 5 and 4 of all cancers in malesin females respectively However the rates of incidence ofmelanoma are steadily increasing in the USA as in most partsof Europe [53]The survival rates ofmelanoma becomeworse


Table3Ra

ndom

ized

phaseIIand

IIItria

lswith

nab-paclitaxelinmelanom

a(a)Ph

aseII

Arm

sPts

RR(

)119875=05

PFS

OS

Median(M

O)

119875=ND

At6(

)119875=ND

Median

(MO)119875=ND

1year

()119875=ND

Hersh

etal2010

[46]

(firstlowastand

follo

winglowastlowastlin

e)

Nab-paclitaxel

lowast150m

gm

2q3w

lowastlowast100m

gm

2q3w

37 37216 27

45

35

34 2796 121

41 49

Arm

sPts

RR(

)119875=10

MedianPF

S(M

O)119875=ND

MedianOS(M

O)119875=ND

Kottschadee

tal2011[47]

(firstlowast

andfollo

winglowastlowastlin

e)

Nab-paclitaxel

lowast100m

gm

2q3w

Carbop

latin

AUC2

41256

43

111

lowastlowast100m

gm

2q3w

Carbop

latin

AUC2

3588

42

109

(b)Ph

aseIII

Arm

sPts

ORR

()

PFS

OS

AEs

gradege

III()119875=001

Median

(MO)

BRAFstatus

Median

(MO)

BRAFstatus

Neutropenia

Leukopenia

Fatigue

Neuropathy

WT

(MO)

V60

0Em

(MO)

Uk

(MO)

WT

(MO)

V60

0Em

(MO)

Uk

(MO)

119875=239119875=044119875=088119875=656119875=066119875=lt001119875=33119875=132119875=381

Hersh

etal

2010

[48]

(firstline)

Nab-paclitaxel

150m

gm

2qw

264

1548

54

53

37

128

127

169

111

2012

825

Dacarbazine

1000

mgsqm

q3w

265

1125

25

35

22

107

111

112

9910

72

0

PPvaluend

not

doneA

Esadverse

events

WT

wild

typeV

600E

mw

ithmutationof

V60

0EU

kun

know

nBR

AFmutation

ORR

overallrespon

cerateR

Rrespon

serateP

FSprogressio

n-fre

esurvivalOS

overallsurvival


Table4Ra

ndom

ized

phaseIIItrialswith

nab-paclitaxelinaN

SCLC

Arm

sPts

ORR

Median

PFS

(MO)

119875=lt214

Median

OS

(MO)

119875=lt271

AEs

gradeIIIlowast-IVlowastlowast(

)119875=lt001

Median

()

119875=005

SQ ()

119875=lt001

NSQ ()

119875=lt80

Neutro

peniaTh

rombo

cytopeniaFatig

ueAnemia

Socinskietal2012

[49]

(firstline)

Nab-paclitaxel100m

gm

2

521

3341

2663

121

33lowast

13lowast

4lowast22lowast

Carbop

latin

AUC6

q3w

14lowastlowast

5lowastlowast

lt1lowastlowast

5lowastlowast

Paclitaxel200

mgm

2

531

2524

2558

111

32lowast

7lowast6lowast

6lowast

Carbop

latin

AUC6

q3w

26lowastlowast

2lowastlowast

lt1lowastlowast

lt1lowastlowast

PPvaluend

not

donesqsquamou

shistolog

yof

NSC

LCnsqnon

squamou

shistolog

yof

NSC

LCA

Esadverse

events

ORR

overallrespon

cerateR

Rrespon

serateP

FSprogressio

n-fre

esurvivalOSoverall

survival


with advancing stage Therefore early diagnosis in additionto surgical treatment before its spread is the most effectivetreatment

Melanomas are a heterogeneous group of tumors char-acterized by specific genetic alterations including mutationsin kinase such as BRAF or c-kit Dacarbazine is commonlyused as a treatment for metastatic melanoma and has beenfor long time the standard of care for this disease Recentlynew approaches have completely changed the diagnosis andtreatment of melanoma New medications like vemurafenibhave been developed for the systemic therapy of advancedmelanomas in subpopulations identified by BRAF mutationtests Taxanes have been reported to have some limitedactivity in malignant melanoma [54ndash58] due to the hightoxicity attributed to their waterinsolubility In a phase IIclinical trial Hersh at al in 2010 [46] demonstrated thatnab-paclitaxel has activity not only in chemotherapy-naıvepatients with metastatic melanoma administered at a dose of150mgm2 but also in previously treated patients adminis-tered at a dose of 100mgm2 for 3 of 4 weeks In this studyPFS and OS were longer than the previous results reportedwith conventional standard of care In previously treated andchemotherapy-naıve patients PFS was 45 months and 35months respectively and similarly OS was 96 months and121 months (in respect to 16 months of PFS reported in theliterature for treatmentwith dacarbazine and temozolomide)In another phase II clinical trial Kottschade et al in 2011[59] demonstrated that in patients withmetastatic melanomathe combination of nab-paclitaxel 100mgm2 and carboplatinAUC2 administered in days 1 8 and 15 every 28 days ismoderately tolerated for the occurrence of adverse effects thatwere fatigue myelodepression and gastrointestinal toxicityThis study confirms that the efficacy and toxicity of nab-paclitaxel are similar to those of paclitaxel when combinedwith carboplatin for the treatment of patients with metastaticmelanoma Even if such regimens have not been formallycompared in a randomized study we can say that nab-paclitaxel is a good alternative for patients who cannottolerate conventional therapy with paclitaxel Last Novemberat the Society of Melanoma Research a preliminary analysisof a Phase III study by Hersh was presented which showsbenefit in terms of PFS in favor of nab-paclitaxel comparedto dacarbazine (48 versus 25 months) the same trendwas observed in the interim analysis that shows a trendfor better OS (128 versus 107 months) (Table 3) Recentlynab-paclitaxel was efficiently combined with temozolomideand oblimersen in the treatment of melanoma patients Indetail in a phase I trial chemotherapy-naıve patients withmetastatic melanoma and normal LDH levels were enrolledin 3 cohorts The treatment regimen consisted of 56-daycycles of oblimersen (7mgkgday continuous iv infusionon days 1ndash7 and 22ndash28 in cohort 1 and 2 900mg fixed dosetwice weekly in weeks 1-2 4-5 for cohort 3) temozolomide(75mgm2 days 1ndash42) and nab-paclitaxel (175mgm2 incohort 1 and 3 260mgm2 in cohort 2 on days 7 and 28)The RR in the 32 treated patients was 406 (2 CR and 11PR) and 11 patients had stable disease for a disease controlrate of 75 Haematological renal and neurologic toxicity

never exceeded grade 3 demonstrating a good tolerability ofthe schedule [60]

Lung cancer (LC) is the first cause of cancer death allover the world with a 5 year survival of 5 for metastaticdisease Treatment selection is based on different factorslike the performance status comorbidities histology andin the last years the molecular mutational profile whichis now mandatory to assess before deciding treatment Themost common chemotherapy approach is a platinum baseddoublet which is commonly combined with gemcitabinevinorelbine or pemetrexed [61] in Europe while in the USAthemost common combination is carboplatin paclitaxel dou-blet (RR 15ndash32) this combination is effective and relativelywell tolerated in the elderly [62ndash65] Bevacizumab addition tothis combination led to improved survival [66] Socinski et alreported in 2012 a phase III trial enrolling 1052 IIIb aNSCLC(advanced non-small-cell lung cancer) patients in the firstline of treatment which compared weekly nab-paclitaxel100mgm2 and carboplatinAUC6 every threeweekswith car-boplatin AUC6 and CrEL-paclitaxel 200mgm2 every threeweeks [49] The nab-paclitaxelcarboplatin combination wasmore active in terms of RR with a trend in PFS and OSimprovement and was also better tolerated (Table 4)

6 Conclusions and Future Developments

Nab-paclitaxel has produced a paradigm change in thetreatment of tumors like breast cancer pancreatic cancer andmelanoma and a large use in these important diseases can bepredicted Also in lung cancer nab-paclitaxel has produced agood safety profile and increase in RR

We think that nab-paclitaxel has opened a new way tohuman cancer treatment and indeed reached the prime-time

References

[1] M C Wani H L Taylor M E Wall P Coggon and A TMcPhail ldquoPlant antitumor agents VI The isolation and struc-ture of taxol a novel antileukemic and antitumor agent fromTaxus brevifoliardquo Journal of the American Chemical Society vol93 no 9 pp 2325ndash2327 1971

[2] A K Singla A Garg and D Aggarwal ldquoPaclitaxel and itsformulationsrdquo International Journal of Pharmaceutics vol 235no 1-2 pp 179ndash192 2002

[3] S B Horwitz ldquoMechanism of action of taxolrdquo Trends inPharmacological Sciences vol 13 no 4 pp 134ndash136 1992

[4] M E Wall and M C Wani ldquoCamptothecin and taxol fromdiscovery to clinicrdquo Journal of Ethnopharmacology vol 51 no1ndash3 pp 239ndash254 1996

[5] J J Correia and S Lobert ldquoPhysiochemical aspects of tubulin-interacting antimitotic drugsrdquo Current Pharmaceutical Designvol 7 no 13 pp 1213ndash1228 2001

[6] C M Spencer and D Faulds ldquoPaclitaxel A review of its phar-macodynamic and pharmacokinetic properties and therapeuticpotential in the treatment of cancerrdquo Drugs vol 48 no 5 pp794ndash847 1994

[7] E K Rowinsky and R C Donehower ldquoPaclitaxel (taxol)rdquo TheNew England Journal of Medicine vol 332 no 15 pp 1004ndash10141995


[8] H Gelderblom J Verweij K Nooter andA Sparreboom ldquoCre-mophor EL the drawbacks and advantages of vehicle selectionfor drug formulationrdquo European Journal of Cancer vol 37 no13 pp 1590ndash1598 2001

[9] A Sparreboom L van Zuylen E Brouwer et al ldquoCremophorEL-mediated alteration of paclitaxel distribution in humanblood clinical pharmacokinetic implicationsrdquoCancer Researchvol 59 no 7 pp 1454ndash1457 1999

[10] M Conti V Tazzari C Baccini G Pertici L P Serino andU De Giorgi ldquoAnticancer drug delivery with nanoparticlesrdquo InVivo vol 20 no 6 pp 697ndash702 2006

[11] M Rossi M R Pitari N Amodio et al ldquomiR-29b negativelyregulates human osteoclastic cell differentiation and functionimplications for the treatment of multiple myeloma-relatedbone diseaserdquo Journal of Cellular Physiology 2012

[12] N Amodio M T Di Martino U Foresta et al ldquomiR-29b sensi-tizes multiple myeloma cells to bortezomib-induced apoptosisthrough the activation of a feedback loop with the transcriptionfactor Sp1rdquo Cell Death and Disease vol 3 no 11 p e436 2012

[13] N Amodio M Leotta D Bellizzi et al ldquoDNA-demethylatingand anti-tumor activity of synthetic miR-29b mimics in multi-ple myelomardquo Oncotarget vol 3 no 10 pp 1246ndash1258 2012

[14] M T Di Martino E Leone N Amodio et al ldquoSynthetic miR-34a mimics as a novel therapeutic agent for multiple myelomain vitro and in vivo evidencerdquo Clinical Cancer Research vol 18pp 6260ndash6270 2012

[15] P Tagliaferri M Rossi M T Di Martino et al ldquoPromisesand challenges of MicroRNA-based treatment of multiplemyelomardquo Current Cancer Drug Targets vol 12 no 7 pp 838ndash846 2012

[16] M Marra G Salzano C Leonetti et al ldquoNanotechnologies touse bisphosphonates as potent anticancer agents the effects ofzoledronic acid encapsulated into liposomesrdquo Nanomedicinevol 7 no 6 pp 955ndash964 2011

[17] M Purcell J F Neault and H A Tajmir-Riahi ldquoInteractionof taxol with human serum albuminrdquo Biochimica et BiophysicaActa vol 1478 no 1 pp 61ndash68 2000

[18] N Authier J P Gillet J Fialip A Eschalier and F CoudoreldquoDescription of a short-term Taxol-induced nociceptive neu-ropathy in ratsrdquo Brain Research vol 887 no 2 pp 239ndash2492000

[19] N K Ibrahim N Desai S Legha et al ldquoPhase I and phar-macokinetic study of ABI-007 a Cremophor-free protein-stabilized nanoparticle formulation of paclitaxelrdquo Clinical Can-cer Research vol 8 no 5 pp 1038ndash1044 2002

[20] M S Surapaneni S K Das and N G Das ldquoDesigningPaclitaxel drug delivery systems aimed at improved patientoutcomes current status and challengesrdquo ISRN Pharmacologyvol 2012 Article ID 623139 15 pages 2012

[21] N Desai V Trieu Z Yao et al ldquoIncreased antitumor activityintratumor paclitaxel concentrations and endothelial cell trans-port of cremophor-free albumin-bound paclitaxel ABI-007compared with cremophor-based paclitaxelrdquo Clinical CancerResearch vol 12 no 4 pp 1317ndash1324 2006

[22] S W Lee M H Yun S W Jeong et al ldquoDevelopment ofdocetaxel-loaded intravenous formulation Nanoxel-PM usingpolymer-based delivery systemrdquo Journal of Controlled Releasevol 155 no 2 pp 262ndash271 2011

[23] P Kan C W Tsao A J Wang W C Su and H F LiangldquoA liposomal formulation able to incorporate a high contentof Paclitaxel and exert promising anticancer effectrdquo Journal ofDrug Delivery vol 2011 Article ID 629234 9 pages 2011

[24] Y W Cho J Lee S C Lee K M Huh and K Park ldquoHy-drotropic agents for study of in vitro paclitaxel release frompolymeric micellesrdquo Journal of Controlled Release vol 97 no2 pp 249ndash257 2004

[25] K M Huh S C Lee Y W Cho J Lee J H Jeong and K ParkldquoHydrotropic polymermicelle system for delivery of paclitaxelrdquoJournal of Controlled Release vol 101 no 1-3 pp 59ndash68 2005

[26] W J Gradishar D Krasnojon S Cheporov et al ldquoSignificantlylonger progression-free survival with nab-paclitaxel comparedwith docetaxel as first-line therapy for metastatic breast cancerrdquoJournal of Clinical Oncology vol 27 no 22 pp 3611ndash3619 2009

[27] W J Gradishar D Krasnojon S Cheporov et al ldquoPhase IItrial of nab-paclitaxel compared with docetaxel as first-linechemotherapy in patients with metastatic breast cancer finalanalysis of overall survivalrdquo Clinical Breast Cancer vol 12 no5 pp 313ndash321 2012

[28] J L Blum M A Savin G Edelman et al ldquoPhase II study ofweekly albumin-bound paclitaxel for patients with metastaticbreast cancer heavily pretreated with taxanesrdquo Clinical BreastCancer vol 7 no 11 pp 850ndash856 2007

[29] V Roy B R LaPlant G G Gross C L Bane and F MPalmieri ldquoNorth Central Cancer Treatment Group Phase IItrial of weekly nab (nanoparticle albumin-bound)-paclitaxel(nab-paclitaxel) (Abraxane) in combination with gemcitabinein patients with metastatic breast cancer (N0531)rdquo Annals ofOncology vol 20 no 3 pp 449ndash453 2009

[30] W J Gradishar S Tjulandin N Davidson et al ldquoPhase IIItrial of nanoparticle albumin-bound paclitaxel compared withpolyethylated castor oil-based paclitaxel in women with breastcancerrdquo Journal of Clinical Oncology vol 23 no 31 pp 7794ndash7803 2005

[31] D D von Hoff R K Ramanathan M J Borad et al ldquoGem-citabine plus nab-paclitaxel is an active regimen in patientswith advanced pancreatic cancer a phase III trialrdquo Journal ofClinical Oncology vol 29 no 34 pp 4548ndash4554 2011

[32] D D Von Hoff R K Ramanathan M J Borad et al ldquoGem-citabine plus nab-paclitaxel is an active regimen in patientswith advanced pancreatic cancer a phase III trialrdquo Journal ofClinical Oncology vol 29 no 34 pp 4548ndash4554 2011

[33] E L Mayer and H J Burstein ldquoChemotherapy for metastaticbreast cancerrdquo HematologyOncology Clinics of North Americavol 21 no 2 pp 257ndash272 2007

[34] G Capri E Tarenzi F Fulfaro and L Gianni ldquoThe role oftaxanes in the treatment of breast cancerrdquo Seminars inOncologyvol 23 no 1 pp 68ndash75 1996

[35] A J ten Tije J Verweij W J Loos and A SparreboomldquoPharmacological effects of formulation vehicles implicationsfor cancer chemotherapyrdquo Clinical Pharmacokinetics vol 42no 7 pp 665ndash685 2003

[36] R BWeiss R C Donehower P HWiernik et al ldquoHypersensi-tivity reactions from taxolrdquo Journal of Clinical Oncology vol 8no 7 pp 1263ndash1268 1990

[37] P G Morris ldquoAdvances in therapy eribulin improves survivalfor metastatic breast cancerrdquo Anti-Cancer Drugs vol 21 no 10pp 885ndash889 2010

[38] N Denduluri and S Swain ldquoIxabepilone clinical role inmetastatic breast cancerrdquoClinical Breast Cancer vol 11 pp 139ndash145 2011

[39] MKWeil andA PChen ldquoPARP inhibitor treatment in ovarianand breast cancerrdquoCurrent Problems in Cancer vol 35 no 1 pp7ndash50 2011


[40] J S Frenel E Bourbouloux D Berton-Rigaud S Sadot-Lebouvier A Zanetti and M Campone ldquoLapatinib in meta-static breast cancerrdquoWomenrsquos Health vol 5 no 6 pp 603ndash6122009

[41] M A Sendur S Aksoy and K Altundag ldquoPertuzumab inHER2-positive breast cancerrdquo Current Medical Research andOpinion vol 28 no 10 pp 1709ndash1716 2012

[42] M F Barginear V John and D R Budman ldquoTrastuzumab-DM1 a clinical update of the novel antibody-drug conjugate forHER2-overexpressing breast cancerrdquo Molecular Medicine vol18 no 1 pp 1473ndash1479 2012

[43] S Lopez-Tarruella Y Jerez I Marquez-Rodas and M MartinldquoNeratinib (HKI-272) in the treatment of breast cancerrdquo FutureOncology vol 8 no 6 pp 671ndash681 2012

[44] X Y Teng Z Z Guan Z W Yao et al ldquoA tolerability studyof A cremophor-free albumin bound nanoparticle paclitaxelintravenously administered in patients with advanced solidtumorrdquo Ai Zheng vol 23 no 11 pp 1431ndash1436 2004

[45] D W Nyman K J Campbell E Hersh et al ldquoPhase I andpharmacokinetics trial of ABI-007 a novel nanoparticle formu-lation of paclitaxel in patients with advanced nonhematologicmalignanciesrdquo Journal of Clinical Oncology vol 23 no 31 pp7785ndash7793 2005

[46] E M Hersh S J OrsquoDay A Ribas et al ldquoA phase 2 clinical trialof nab-paclitaxel in previously treated and chemotherapy-naivepatients with metastatic melanomardquo Cancer vol 116 no 1 pp155ndash163 2010

[47] L A Kottschade V J Suman T Amatruda III et al ldquoA phaseII trial of nab-paclitaxel (ABI-007) and carboplatin in patientswith unresectable stage IV melanoma a North Central CancerTreatment Group Study N057E(1)rdquo Cancer vol 117 no 8 pp1704ndash1710 2011


[49] M A Socinski I Bondarenko N A Karaseva et al ldquoWeeklynab-paclitaxel in combination with carboplatin versus solvent-based paclitaxel plus carboplatin as first-line therapy in patientswith advanced non-small-cell lung cancer final results of aphase III trialrdquo Journal of Clinical Oncology vol 30 no 17 pp2055ndash2062 2012

[50] D A Yardley L Hart L Bosserman et al ldquoPhase II study eval-uating lapatinib in combination with nab-paclitaxel in HER2-overexpressing metastatic breast cancer patients who havereceived no more than one prior chemotherapeutic regimenrdquoBreast Cancer Research and Treatment vol 137 no 2 pp 457ndash464 2013

[51] D Ciliberto C Botta P Correale et al ldquoRole of gemcitabine-based combination therapy in the management of advancedpancreatic cancer a meta-analysis of randomised trialsrdquo Euro-pean Journal of Cancer vol 49 no 3 pp 593ndash603 2013

[52] KK Frese ANeesseN Cook et al ldquonab-paclitaxel potentiatesgemcitabine activity by reducing cytidine deaminase levels in amousemodel of pancreatic cancerrdquoCancer Discovery vol 2 no3 pp 260ndash269 2012

[53] D CWhiteman C AWhiteman andA C Green ldquoChildhoodsun exposure as a risk factor for melanoma a systematic reviewof epidemiologic studiesrdquo Cancer Causes and Control vol 12no 1 pp 69ndash82 2001

[54] A Y Bedikian C Plager N Papadopoulos O Eton J Eller-horst and T Smith ldquoPhase II evaluation of paclitaxel by

short intravenous infusion inmetastatic melanomardquoMelanomaResearch vol 14 no 1 pp 63ndash66 2004

[55] S S Legha S Ring N Papadopoulos M Raber and R SBenjamin ldquoA phase II trial of taxol in metastatic melanomardquoCancer vol 65 no 11 pp 2478ndash2481 1990

[56] A I Einzig H Hochster P H Wiernik et al ldquoA phase II studyof taxol in patients with malignant melanomardquo InvestigationalNew Drugs vol 9 no 1 pp 59ndash64 1991

[57] S Aamdal I Wolff S Kaplan et al ldquoDocetaxel (Taxotere) inadvanced malignant melanoma a phase II study of the EORTCEarly Clinical Trials Grouprdquo European Journal of Cancer A vol30 no 8 pp 1061ndash1064 1994

[58] A Y Bedikian G R Weiss S S Legha et al ldquoPhase II trialof docetaxel in patients with advanced cutaneous malignantmelanoma previously untreated with chemotherapyrdquo Journal ofClinical Oncology vol 13 no 12 pp 2895ndash2899 1995

[59] L A Kottschade V J Suman T Amatruda et al ldquoA phase IItrial of nab-paclitaxel (ABI-007) and carboplatin in patientswith unresectable stage IV melanoma a North Central CancerTreatment Group Study N057E1rdquo Cancer vol 117 no 8 pp1704ndash1710 2011

[60] P A Ott J Chang K Madden et al ldquoOblimersen in com-bination with temozolomide and albumin-bound paclitaxel inpatients with advanced melanoma a phase I trialrdquo CancerChemotherapy and Pharmacology vol 71 no 1 pp 183ndash1912013

[61] G V Scagliotti P Parikh J von Pawel et al ldquoPhase IIIstudy comparing cisplatin plus gemcitabine with cisplatin pluspemetrexed in chemotherapy-naive patients with advanced-stage non-small-cell lung cancerrdquo Journal of Clinical Oncologyvol 26 no 21 pp 3543ndash3551 2008

[62] J R Jett S E Schild R L Keith and K A Kesler ldquoTreatmentof non-small cell lung cancer stage IIIB ACCP evidence-basedclinical practice guidelines (2nd edition)rdquo Chest vol 132 no 3pp 266Sndash276S 2007

[63] J H Schiller D Harrington C P Belani et al ldquoComparison offour chemotherapy regimens for advanced non-small-cell lungcancerrdquo The New England Journal of Medicine vol 346 no 2pp 92ndash98 2002

[64] K Kelly J Crowley P A Bunn Jr et al ldquoRandomized phaseIII trial of paclitaxel plus carboplatin versus vinorelbine pluscisplatin in the treatment of patients with advanced non-small-cell lung cancer a Southwest Oncology Group trialrdquo Journal ofClinical Oncology vol 19 no 13 pp 3210ndash3218 2001

[65] R C Lilenbaum J E Herndon M A List et al ldquoSingle-agentversus combination chemotherapy in advanced non-small-celllung cancer the cancer and leukemia group B (study 9730)rdquoJournal of Clinical Oncology vol 23 no 1 pp 190ndash196 2005

[66] A Sandler R Gray M C Perry et al ldquoPaclitaxel-carboplatinalone or with bevacizumab for non-small-cell lung cancerrdquoTheNew England Journal of Medicine vol 355 no 24 pp 2542ndash2550 2006


Review ArticleLiposomal Doxorubicin in the Treatment of Breast CancerPatients A Review

Juan Lao12 Julia Madani12 Teresa Pueacutertolas12 Mariacutea Aacutelvarez1 Alba Hernaacutendez1

Roberto Pazo-Cid12 Aacutengel Artal12 and Antonio Antoacuten Torres12

1 Medical Oncology Department Miguel Servet University Hospital Paseo Isabel la Catolica 1-3 50009 Zaragoza Spain2 Aragon Institute of Health Sciences Avda San Juan Bosco 13 planta 1 50009 Zaragoza Spain

Correspondence should be addressed to Antonio Anton Torres aantontsaludaragones

Received 1 December 2012 Accepted 10 February 2013

Academic Editor Michele Caraglia

Copyright copy 2013 Juan Lao et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Drug delivery systems can provide enhanced efficacy andor reduced toxicity for anticancer agents Liposome drug delivery systemsare able to modify the pharmacokinetics and biodistribution of cytostatic agents increasing the concentration of the drug releasedto neoplastic tissue and reducing the exposure of normal tissue Anthracyclines are a key drug in the treatment of both metastaticand early breast cancer but one of their major limitations is cardiotoxicity One of the strategies designed to minimize this sideeffect is liposome encapsulation Liposomal anthracyclines have achieved highly efficient drug encapsulation and they have provento be effective and with reduced cardiotoxicity as a single agent or in combination with other drugs for the treatment of eitheranthracyclines-treated or naıve metastatic breast cancer patients Of particular interest is the use of the combination of liposomalanthracyclines and trastuzumab in patients with HER2-overexpressing breast cancer In this paper we discuss the different studieson liposomal doxorubicin in metastatic and early breast cancer therapy

1 Background

In the past years we have seen significant advances in theunderstanding of neoplastic diseases and how they have beentranslated into improvements of therapy An increasing num-ber of more specific therapeutic options to manage differenttumour types are now available but classical chemotherapy(which is based on the administration of drugs that interferewith the cellrsquos cycle prevent its division and eventually des-troy them) remains in general a backbone option for manytumours Chemotherapy side effects must not however beunderestimated because its mechanism of action affects bothtumour and normal cells as well That is the reason whyefforts to improve chemotherapy treatments have focused ondesigning drugs that are more specific against cancer cells tominimize toxic side effects

Liposomes were conceived as drug delivery systems tomodify drug pharmacokinetics and distribution with the aimof reducing chemotherapyrsquos toxicity These liposomes im-prove the pharmacological properties of some cytostatic

agents allowing an increased proportion of the drug thatmaybe delivered within the tumour tissue whilst substantiallyreducing the exposure of normal tissues

Liposomes as a vehicle for delivering cytostatic agentswere first described in the 1960s They were initially used ascarriers for lipophilic cytostatic agents but their suitability forboth hydrophilic and hydrophobic drugs was soon assessedLiposomes can be either a membrane-based closed structureable to incorporate lipophilic drugs or may be built from thedirect encapsulation of hydrophilic compounds within theinternal aqueous compartment of vesicles [1ndash3]

Phospholipids are the major component of liposomeswhich make them to be less toxic biodegradable and bio-compatible The bilayer of phospholipids prevents also theactive form of the drug from breaking down before it reachesthe tumour tissue and in this way exposure of the normaltissue to the drug is minimized The therapeutic index of thedrug is then increased by two mechanisms on one hand agreater amount of the active drug reaches the tumour cellsand an increased cytotoxic effect is obtained and on the other


hand side effects are also reduced as a consequence of thedrug encapsulation Liposomal formulations have an addi-tional effect on drug metabolism by decreasing its enzymaticdegradation [4]

Liposomes can be produced by different methods Stabil-ity of both the bilayer and the incorporated drugs depends onlipid composition and cholesterol content Their size rangesfrom 25 to 100 nM and is determined by the maximum quan-tity of drug stored within the membrane and its flexibilityThe lower size limit avoiding liposomesmay enter the normalcapillary vessels whereas the upper limit is still within thetumour vasculature and enables the cytotoxic agent to reachthe tumour bed in order to produce its effect the activedrug needs to readily extravasate through the vascular defectspresent in the vessels surrounding cancer cells as a conse-quence of neoangiogenesis phenomena induced by neoplasticcells [5] In this way liposomes below this threshold have thepotential to accumulate in the tumour bed after passive drugentry and boosted by impaired lymphatic drainageThis phe-nomenon has been described as ldquoenhanced permeability plusretention effectrdquo [6] One more factor related to liposomersquossize is that the bigger they are the greater the uptake by thereticuloendothelial system and therefore more rapid thedrug is metabolized [7]

As the time liposomes are retained in the circulatorysystem is reduced the drug they are carryingmight not reachcytotoxic levels in the tumour tissue The size of the nan-otransporter could be reduced but then less drug quantityshould be transported One method that has proven to beeffective in overcoming this obstacle without compromisingthe quantity of chemotherapeutic agent delivered to thetumour consists in coating these delivery systems with poly-mers in particular with polyethylene glycol (PEG) whichallows liposomes to escape from the immune system andtherefore increase ldquoin vivordquo circulating time [8] Studies haveshown that when manufactured in this way pegylated lipo-somes have a longer half-life than nonpegylated (rangingfrom a few hours to 45 hours) [9] However the presence ofPEG may act as a barrier between the drug and the tumourcells hindering the delivery of the cytostaticTherefore futureimprovements should be directed to improve this aspectparticularly in the case of breast cancer

In this cancer new liposomal formulations have been de-veloped to facilitate the supply of the confined cytostatic agentusing thermosensitive molecules These formulations haveproven to be effective in this tumour and their design keepthem stable at normal body temperature of 37∘C but theybecome unstable at slightly higher temperatures as thoseexisting inside the tumours This system has also demon-strated a higher accumulation of the drug within the tumourand a facilitated release of the encapsulated drug [10]

An alternative strategy used to increase the therapeutic in-dex of liposome-based drugs is based on improving the colo-calization between the chemotherapeutic agent and the breastcancer cell In some cases this strategy can also include animprovement of the internalization of the drug into them aswhen cell surface receptors involved in endocytosis take part

In general these formulations involve modifications ofthe liposome surface to contain ligands that are specifically

recognized by receptors overexpressed in the breast cancercell surface Several of these strategies have been recentlypublished For example anti-HER2 immunoliposomes haveproven much more effective against HER2-overexpressingbreast cancer cells when compared with nontargeted lipo-somes In one study targeted liposomeswere formulatedwitha Fab of recombinant humanized anti-HER2 monoclonalantibody [11]

Estrogen receptor is a particularly attractive target as itis overexpressed in a large amount of breast cancer cell lines[12] Several studies incorporating either estradiol or estroneto liposomes to use them as a ligand against estrogen-ex-pressing breast cancer have been reported In one study theaccumulation of these estrogen-targeted liposomes wasapproximately six times higher than that observed with non-targeted liposomes [13]

2 Metastatic Breast Cancer Treatment andLiposomal Anthracyclines Pharmacology

Breast cancer is a heterogeneous disease that includes a vari-ety of biological types with different treatment options andclinical outcomes Metastatic breast cancer (MBC) is achronic and incurable disease with a median survival of ap-proximately 2-3 years Although advances have been made inthe management of MBC long-term survivors are rare with5-year survival rates varying from 5 to 10

At present prognosis and treatment selection are basedon tumor biology and molecular characterization In partic-ular multigene array and expression analyses have provided amolecular classification for breast tumorThemost importantsubtypes are luminal A and B Her2neu and basal like [1415]

Characterization of tumor biology (estrogen and proges-terone receptors Ki-67 and Her2) and clinical history (pasttreatment patient symptoms and functional status) is criticalfor selecting treatment inMBCQuality of life is an importantissue to consider when choosing a therapeutic option

The targeted therapies such as hormonal treatment ofpatients with hormone-sensitive tumors and trastuzumabin case of Her2 overexpression represent a treatment ofchoice for a subset of selected patients Nevertheless cyto-toxic chemotherapy remains the only therapeutic option inpatients with triple negative condition or in those who pro-gress after hormonotherapy Anthracyclines and taxanes arethe most active drugs for the treatment of MBC For manydecades conventional anthracyclines doxorubicin and epi-rubicin have been an important mainstay in the treatmentof breast cancer They have proven to be effective for bothmetastatic and early disease but their use has been limitedbecause of the intrinsic cardiotoxicity [16]

Many strategies have been designed to curtail this effectEncapsulating anthracyclines into liposomes which allowedpatients to receive much higher doses of an anthracyclinedelivered mainly into the tumour tissue with fewer sideeffects has been one of these Several formulations of lipo-some-encapsulated doxorubicin are available for its use in


the clinical practice [17] which differ in pharmacologicalcharacteristics

Pegylated liposomal doxorubicin (PLD) (Caelyx) is doxo-rubicin hydrochloride encapsulated in liposomes with sur-face-bound methoxypolyethyleneglycol (MPEG) Doxoru-bicin hydrochloride is a cytotoxic anthracycline antibioticderived from Streptomyces peucetius var caesius Pegylationavoiding liposomes may be detected by the mononuclearphagocyte system and thereby the blood circulating time isincreasedMean half-life of pegylated liposomes in humans is55 hours Its pharmacokinetic characteristics facilitate tissueaccumulation and this has been demonstrated in tumourbiopsies of Kaposirsquos sarcoma (KS) and bone metastases frombreast cancer [18 19]

Plasmatic pharmacokinetics of PLD in humans signif-icantly differ from the original doxorubicin Caelyx has alinear pharmacokinetic profile at lower doses (10ndash20mgm2)while in the dose interval of 20ndash60mgm2 PLD is nonlinearStandard doxorubicin hydrochloride displays extensive tissuedistribution (volume of distribution 700ndash1100 Lm2) andrapid clearance (24ndash73 Lhm2) On the contrary the distri-bution volume of PLD is limited mainly to the vascular fluidand the elimination of doxorubicin from the blood dependson the liposomal carrier doxorubicin becomes available forcatabolism once the liposomes are extravasated and enteredinto the tissular compartment

At equivalent doses plasma concentration and AUC val-ues of PLD are significantly higher than those achieved withdoxorubicin preparations The pharmacokinetic profile ofPLD determined in 18 patients with breast cancer (which wassimilar to a group of 120 patients with several tumour types)showed amean half-life of 715 hours (range 452ndash985 hours)

As already has been mentioned the pegylated liposomaldoxorubicin hydrochloride formulation allows the liposomesto circulate in the blood for extended periods of time Thesepegylated liposomes are small enough (mean diameter ofapproximately 100 nM) to pass intact through the defectiveblood vessels supplying tumoursThe entry of pegylated lipo-somes from blood vessels and their accumulation in tumourshave been tested in mice bearing C-26 colon carcinomatumours and in transgenic mice with KS-like lesions Thepegylated liposomes also combine a low permeability lipidmatrix with an internal aqueous buffer system that keepsdoxorubicin hydrochloride encapsulated as long as liposomesremain in the blood stream

Myocet (liposome-encapsulated doxorubicin citrate) isanother form of encapsulated doxorubicin hydrochlorideconsisting of a drug delivery system with a highly rigidbilayer [20]Myocet (LD) also provides amore prolonged cir-culating time than conventional doxorubicin and in addi-tion liposome-encapsulation significantly modifies the bio-distribution of doxorubicin resulting in reduced toxicityTheclearance of LD was 51 plusmn 48 Lh and steady-state volume ofdistribution (119881

119889)was 566plusmn615 Lwhereas after conventional

doxorubicin elimination and (119881119889) were 467 plusmn 96 Lh and

1451 plusmn 258 L respectively [21]In animals (Table 1) liposome-encapsulated doxorubicin

reduced the distribution to the heart and the gastrointestinal

mucosa compared to conventional doxorubicin while antitu-mor efficacy was maintained However when compared withconventional doxorubicin LDdid not prove to bemore activein doxorubicin-resistant cell lines

Doxorubicin plasma pharmacokinetics in patients receiv-ing LD showed a high degree of interpatient variabilityNonetheless as a rule total doxorubicin plasma levels weresignificantly higher with LD than with conventional doxoru-bicin while free doxorubicin peak plasma levels were lowerSimilarly the peak levels of the main circulating doxoru-bicin metabolite doxorubicinol (synthesized via aldo-keto-reductase) appeared in plasma later with LD than withconventional doxorubicin Available pharmacokinetic datapreclude settling strong conclusions regarding the relation-ship between plasma levels of totalfree doxorubicin and itsinfluence on the efficacysafety of LD

3 Anthracycline Toxicity

Anthracyclines have a well-known toxicity profile Theirmore frequent side effects includemyelosuppressionmucosi-tis alopecia and emesis Other less frequent although highlyrelevant side effects are cardiotoxicity and the occurrence ofsecondary leukemias

The emetogenic potential of anthracyclines is moderateeven though it is potentiated by other agents when admin-istered in combination The lowest blood cell count (nadir)is reached between 10 and 14 days after administrationDoxorubicin is a potent vesicant agent and its extravasationmay cause necrosis of the skin and soft tissue

Anthracycline-induced cardiotoxicity was described forthe first time in the 1970s [22] Cardiac side effects canbe divided into acute and late-onset events Acute toxicityencompasses phenomena that are usually reversible andnonfatal such as hypotension tachycardia and arrhythmiasThe occurrence of symptoms of myocarditis (with or withoutaccompanying pericarditis) in the immediate posttreatmentdays is less frequent but can lead to heart failure that is usuallyreversible

However late-onset cardiotoxicity is the most relevantproblem It results in dilated cardiomyopathy that causeslethal congestive heart failure (CHF) in 75 of cases in thefollowing 5 years and whose end-stage treatmentmay requirea heart transplant [23] This type of heart disease respondsto a dosing and regimen-dependent pattern [22] Toxicityis higher when anthracyclines are administered in boluscompared to regimens giving it as a continuous infusion andthis seems to be related to the higher dose peak reached whenadministered in a short period of time

A number of factors that predispose to this toxicity havebeen identified Specifically they are hypertension age below15 or over 70 years a history of radiotherapy to the medi-astinum and the concomitant use with other drugs such ascyclophosphamide paclitaxel or trastuzumab In particularwhen given with paclitaxel the risk of cardiotoxicity is higherwhen doxorubicin is administered just after paclitaxel insteadof the opposite sequence


Table 1 Comparison of AUC and 11990512

in various tissues in dogs following the administration of TLC D-99 and conventional doxorubicinSingle dose 15mgkg (30mg sdotmminus2) IV [18]

Tissues TLC D-99 Doxorubicin Ratio of AUC0rarr119879last

(TLC D-99Dox)AUC0rarr119879last (uM eq-h) 119879

12(h) AUC

0rarr119879last (uM eq-h) 11987912

(h)Liver 539 79 377 97 142Spleen 5087 92 559 52 907Bone marrow 1913 86 392 75 486Lymph nodes 896 211lowast 653 196lowast 138Myocardium (left ventricle) 208 59 313 50 066Myocardium (right ventricle) 189 62 282 54 067lowastDue to short sampling intervals relating to apparent 11990512 these values are estimated TLC D-99 nonpegylated liposomal doxorubicin

The earlier studies only recognized clinical-evident car-diac toxicity 3-4 of patients treated with cumulative dosesof 450mgm2 andup to 18of thosewho received 700mgm2presented with clinical heart failure [24] The incidence ofheart failure is lesser when epirubicin was used but occurredin a 07 of patients when cumulative doses of 660mgm2were reached [25]

Anthracyclines cause some pathological changes prior tothe occurrence of clinical cardiomyopathy that can be detect-ed by different techniques myocardial biopsy (Billinghamscale) isotope ventriculography (MUGA scan) and echocar-diography Billingham published in 1978 a histological classi-fication based on the findings observed in myocardial biop-sies Biopsy findings correlated fairly well with the cumulativedoses of anthracyclines and were able to detect early damageto the myocardial cells Early histological changes secondaryto anthracyclines include cytoplasmic vacuolization and lossof muscle fibres from myocytes due to dilated sarcoplasmicreticulum Inmore advanced stages changes occur in cellularremodelling leading to left ventricular failure [26] Such aninvasive method has had no widespread use in daily clinicalpractice

Isotope ventriculography (MUGA scan) has proven to bean easily reproducible and accurate technique in detectinganthracycline-induced cardiotoxicity [27] Echocardiogra-phy is another noninvasive test used in the study and followupof anthracycline-induced cardiotoxicity It is less accuratethan ventriculography in the early detection of systolic dys-function but allows assessing diastolic functionwhose declineseems to be a good predictor of early cardiac toxicity [28]Other techniques such as antimyosin antibody scintigraphyor biomarkers such as troponin have been unable to predictearly cardiotoxicity

Themajority of recent studies accept as cardiotoxicity cri-teria a gt20 reduction in the left ventricular ejection fraction(LVEF) as long as it remains above 50 a gt10 reduction ifthe resulting figure is below 50 or when symptoms ofCHF (congestive heart failure) occur [29] Using these cri-teria Swain calculated a 79 incidence of anthracycline-induced cardiotoxicity with a cumulative dose of 450mgm2157 with 500mgm2 26 with 550mgm2 and 48 with700mgm2 [30] Shapiro et al described cardiac toxicity inci-dence of 20 when the cumulative dose of doxorubicinin combination with cyclophosphamide reached 500mgm2

[31] Adjuvant chemotherapy studies in which cumulativedoses of doxorubicin did not exceed 300mgm2 showedan incidence of cardiomyopathy ranging from 02 to 09[32] Currently cumulative doses that do not exceed 450ndash500mgm2 of doxorubicin or 900ndash1000mgm2 of epirubicinare accepted to be safe [25]

The simultaneous administration of other drugs potenti-ates anthracycline toxicity The combined use of doxorubicinand paclitaxel was related to a rate of cardiotoxicity higherthan predicted despite relatively low cumulative doses of dox-orubicin [38] This increased toxicity appeared to be causedby a pharmacokinetic interference between paclitaxel anddoxorubicin resulting in higher doxorubicin and doxorubi-cinol plasma concentrations [39]

The combination of anthracyclines and trastuzumab hasalso been correlated with a higher rate of cardiotoxicity Inthe pivotal study that compared doxorubicin and cyclophos-phamide with or without trastuzumab in patients withoverexpression of HER-2 a 23 rate of cardiac toxicity wasobserved with the combination compared with 7 in thearm not receiving trastuzumab [40] Another study of thecombination of trastuzumab with epirubicin and cyclophos-phamide found that the combination with epirubicin90mgm2 translated into 5 cardiac toxicity compared withonly 17 when epirubicin was administered at 60mgm2[41]

4 Liposomal Anthracyclines andMetastatic Breast Cancer

In patients with MBC liposomal anthracyclines have shownsimilar efficacy and less toxicity when compared with con-ventional anthracyclines Currently three formulations withliposomal anthracyclines are available

(i) Myocet formulated with conventional liposomes(ii) DaunoXome liposomes with prolonged circulation

half-lives(iii) CaelyxDoxil with pegylated liposomes

According to their respective product labelling liposomaldoxorubicin (LD Myocet) was approved for the treatmentof metastatic breast cancer pegylated liposomal doxorubicin


(PLD Caelyx) for the treatment of advanced platinum-resistant ovarian cancer advanced breast carcinoma AIDS-related Kaposirsquos sarcoma and multiple myeloma

In June 2000 CaelyxDoxil received marketing authori-sation in the US and subsequently in Europe based on theresults of a pivotal randomised controlled and Phase IIItrial which compared the efficacy of PLD with topotecan inthe treatment of advanced ovarian cancer following failure ofa platinum-containing regimen [42]

In MBC both liposomal formulations have proven to beeffective as single agent or in combination with other drugsfor the treatment of either anthracycline-treated (progres-sion-free interval ofgt6ndash12months) or naıve patients [43ndash46]

Table 2 summarizes the trials that directly comparedliposomal anthracyclines with conventional anthracyclineseither as monotherapy or combinationWe shall review bothefficacy and toxicity emphasizing data related to cardiactoxicity Two Phase III studies have been published [33 34] inwhich efficacy and toxicity of liposomal anthracyclines havebeen directly compared to conventional doxorubicin Therewere no statistically significant differences between bothtreatments with respect to efficacy in terms of response rateprogression-free survival (PFS) or overall survival (OS)

OrsquoBrien et al [33] reported the results of a noninferiorityPhase III study in which 509 patients (p) with metastaticbreast cancer were randomized to receive PLD at a dose of50mgm2 every 4 weeks (254p) or conventional doxorubicin60mgm2 every 3 weeks (255p) The study met its objectiveof noninferiority with PFS being 69 versus 78 months res-pectively (HR 100 95 CI 082ndash122) OS was comparable21 and 22 months for PLD and doxorubicin respectively (HR094 95 CI 074ndash119) The objective response rate was alsosimilar for PLD (33) and doxorubicin (38) Remarkablythe risk of cardiotoxicity was significantly higher in the con-ventional doxorubicin group (HR 36 95 CI 158ndash631)forty-eight patients (196) treated with doxorubicin devel-oped cardiac toxicity compared with only 10p among thosereceiving PLD (119875 lt 0001) There were no patients withclinical heart failure in the PLD arm while 10 patients (4)in the conventional doxorubicin arm developed clinical heartfailure The number of patients to treat with PLD to avoid adoxorubicin-related cardiac event was 7 Also significant isthat 16 of patients in the PLD arm received treatment formore than 9 months compared with only 1 in the doxo-rubicin arm and this was not linked to an increase in cardiactoxicity with PLD In contrast hand-foot syndrome incidencewas higher in the PLD group (48 versus 2)

Harris et al [34] compared the efficacy and safety of LD(75mgm2 every 3 weeks) with conventional doxorubicin(75mgm2 every 3 weeks) in 224 patients with metastaticbreast cancer Of them 17 had received prior adjuvant orneoadjuvant treatment with anthracyclines Response ratewas 26 in both arms PFS was 38 months in the LD armcompared to 43 in the conventional doxorubicin arm (119875 =059) OS was 16 months in the LD arm versus 20 months inthe conventional doxorubicin arm (119875 = 009) Myocardialbiopsies were planned for patients with a LVEF reduction ofgt10 with absolute values above 50 or for those who hada LVEF reduction of gt6 if the resulting LVEF was lower

than 50 In addition to the standard criteria for identifyingcardiotoxicity the presence of a grade of 25 or greater onthe Billingham scale was included The rate of cardiac eventswas favourable to the liposomal anthracycline arm (13 versus29 119875 = 00001) with a clinical heart failure rate of 59versus 15 When the heart biopsies performed were ana-lyzed the proportion of patients with a value of 25 on theBillingham scale was 26 versus 71 (119875 = 002) favouring theliposomal formulation The mean cumulative dose until tox-icity occurred was calculated at 570mgm2 for doxorubicinand 785mgm2 for liposomal doxorubicin

Some other Phase III studies [35ndash37] compared efficacyand toxicity of liposomal anthracyclines in combination withother cytostatic agents (docetaxel or cyclophosphamide) withcombinations with conventional anthracyclines or otherdrugs Inclusion criteria for these studies were not identicalmainly regarding prior treatment allowed Studies by Chanet al and Batist et al included patients not previously treatedwith anthracyclines Sparano et al however randomizedpatients previously treated with anthracyclines during adju-vant or neoadjuvant therapy as long as progression-freeinterval was above 12 months As Table 2 shows we can seethat overall efficacy of liposomal anthracyclines is similar tothe efficacy of conventional formulations when combinedwith other cytostatic agents Of note in Chanrsquos study PFS waseven higher in the group treatedwithMyocet plusCyclophos-phamide

In Batistrsquos study [35] 30 of patients presented any car-diotoxicity risk factor and 10 had received prior anthracy-clines (adjuvant) with amean cumulative dose of 240mgm2Here 21 of patients treated with conventional doxorubicinhad some grade of cardiotoxicity compared to 6 in thegroup receiving liposomal doxorubicin (119875 = 00001) In thecontrol arm 32 of patients developed clinical heart failurecompared with 0 in the liposomal doxorubicin arm Theanalysis of patients with any cardiac risk factor showed aneven greater difference between both drugs with a HR of 161The mean cumulative dose calculated for 50 of patientspresenting with cardiotoxicity was much higher in thegroup receiving liposomal doxorubicin (2220mgm2 versus480mgm2)

Eventually the same author published in 2006 [47]retrospective data from the analysis of 68 patients that hadbeen included in the Phase III study and had been treatedwith adjuvant anthracyclines Cardiac toxicity was lower inpatients treated with liposomal doxorubicin (22 versus 39HR 54119875 = 0001) Four patients developed congestive heartfailure 3 of them in the doxorubicin arm The calculatedmean cumulative dose until cardiotoxicity occurrence was580mgm2 for doxorubicin and 780mgm2 for the liposomalformulation (HR 48 119875 = 0001)

A further Phase III study [36] randomized 160 patients toreceive cyclophosphamide 600mgm2 plus either epirubicin75mgm2 or liposomal doxorubicin 75mgm2 No significantdifferences were observed in the rate of asymptomatic reduc-tion in LVEF (11 versus 10) In this study no patient devel-oped clinical heart failure It must be noted that epirubicindosing was lower than the equipotent doxorubicin


Table 2 Trials that directly compared liposomal anthracyclines with conventional anthracyclines either in monotherapy or combination

Author Trial phase Treatment regimen Patientsrsquocharacteristics PFS OS RR Toxicity

OrsquoBrien et al[33]

IIIPLD (50mgm24w)

versusADR (60mgm23w)

Stage IV69mversus78m

21mversus22m

33versus38

Cardiac47 versus 196

CHF 0 versus 4

Harris et al[34]

IIILD (75mgm23w)


Stage IV(17 ADR previous)

38mversus43m

16mversus20m

26

Cardiac 13 versus 29CHF 59 versus 15Billinghamgt 2526 versus 71

Batist et al[35]

IIILD (60mgm2) + CTX (600mgm2)

versusADR (60mgm2) + CTX (600mgm2)


(30 CRF)

51mversus55m

19mversus16m

Cardiac 6 versus 21(119875 lt 005)

CRF 0 versus 32

Chan et al[36]


versusEPI (75mgm2) + CTX (600mgm2)

Stage IV(No ADR previous)

77mversus56m

183mversus16m

46 versus39

Cardiac 11 versus 10No CRF

Sparano et al[37]

IIIDocetaxel (75mgm2)

versusDocetaxel (60mgm2) + PLD (30mgm2)

Stage IV(100 ADRprevious)

7mversus98m

206mversus205m

Cardiac 4 versus 5PPS 0 versus 24

PLD pegylated liposomal doxorubicin LD liposomal doxorubicin ADR adriamycin EPI epirubicin CTX cyclophosphamide PFS progression-freesurvival OS overall survival RR response rate PPS plantar-palmar syndrome CHF clinical heart failure and CRF cardiac risk factor

In 2010 the Cochrane Library reported a systematic re-view of the different anthracycline compounds and their car-diotoxicity [48] Studies by Harris and Batist were analyzedtogether and authors concluded that nonpegylated liposomalanthracyclines reduced the overall risk of cardiotoxicity(RR = 038 119875 lt 00001) and the risk of clinical heart failure(RR = 020 119875 = 002)

Efficacy and safety of pegylated liposomal doxorubicin(PLD) combined with other cytostatic agents were studied intwo Phase III studies

Sparano et al [37] randomized 751 patients previouslytreated with anthracyclines (as adjuvant or neoadjuvant) witha PFI over 12 months to receive either docetaxel 75mgm2(373p) or the combination of PLD 30mgm2 plus docetaxel60mgm2 every 21 days (378p) until disease progression orunacceptable toxicity occurred Combined treatment im-provedPFS significantly from70 to 98months (HR065 95CI 055 minus077 119875 lt 000001) OS was similar 206 monthsin the docetaxel arm and 205 in the combined treatmentarm (HR 102 95CI 086ndash122)The incidence of hand-footsyndrome was higher in the combined treatment arm (24versus 0) and symptomatic cardiac toxicity was similar 4in the docetaxel group and 5 in the PLD-docetaxel group

Patients with metastatic breast cancer progressing aftertaxanes and anthracyclines had fewer treatment options andoften anthracyclines were not used again due to the cumula-tive risk of cardiotoxicity Based on the safety and efficacy datafor PLD a Phase III study was proposed [49] in which 301patients with metastatic breast cancer progressing to taxanes(lt6months) were randomized to receive one of the followingthree alternatives PLD 50mgm2 every 4 weeks (150p)vinorelbine 30mgm2 every week (129p) or mitomycin-C10mgm2 on days on 1 and 28 plus vinblastine 5mgm2 on

days 1 14 28 and 42 every 6ndash8 weeks (22p) 83 of patientshad received prior anthracyclines in 10 of them cumulativedoses above 450mgm2 had been reached No patient treatedwith PLD showed clinical symptoms of cardiotoxicity PFSwas similar (286 months in the PLD group versus 253months in the other two control groups) (HR 126 95CI 098ndash162) In the subgroup of patients not previouslytreated with anthracyclines (44p) PFS was higher in the PLDarm (58 months) compared with the control arms (21months) (119875 = 001) OS was slightly higher with PLD (11months) versus control arm (9months) albeit not statisticallysignificant (119875 = 093) The objective response rate wassimilar 10 for PLD versus 12 for the control arm

More recently an Austrian observational study was pub-lished [50] inwhich 129 patients withmetastatic breast cancertreated with PLD were analyzed 70 presented 2 or morecardiovascular risk factors Despite this only 4 of patientshad some degree of cardiotoxicity and only 2 cases of clinicalheart failure were reported

Alba et al [51] on behalf of GEICAM published a PhaseIII study exploring the role of PLD as maintenance therapyEligible patients had previously received a sequential schemebased on 3 cycles of doxorubicin 75mgm2 followed by 3more cycles of docetaxel 100mgm2 Patients who had notprogressed during this first part were randomized to receivepegylated liposomal doxorubicin 40mgm2 times 6 cycles ornothing TTP from randomization of the 155 p was 84 versus51 months favouring the maintenance treatment arm (119875 =00002) No differences in OS were found Six patients hadreduced LVEF ge 10 5 of them in the arm of PLD In 2 ofthe patients treated with PLD a LVEF reduction below 50during treatment was found although both recovered within6 months There was no clinical cardiac toxicity


5 Liposomal Anthracyclines and Trastuzumab

In HER2-postive breast cancer the addition of trastuzumabto chemotherapy significantly increases response rate time toprogression and overall survival compared with chemother-apy alone However when trastuzumab is combined withanthracyclines there is an increased risk of cardiac toxi-city Slamon et al [40] randomized 469p with metastaticbreast cancer and HER2 overexpression to receive standardtreatment (anthracyclinescyclophosphamide or paclitaxel)with or without trastuzumab The addition of trastuzumabincreased PFS (74 months versus 46 months 119875 lt 0001)and OS (251 versus 203 months 119875 = 0046) but with anincreased rate of cardiotoxicity in the group receiving theanthracycline and trastuzumab combination (27) Theseresults limited the use of anthracyclines in HER2-positivebreast cancer and in consequence non-anthracycline-basedregimens such as TCH [52 53] were designed As anthra-cyclines showed a high level of activity in this subgroupof patients other strategies were developed also to designregimens using less cardiotoxic anthracyclines such as epiru-bicin (a less cardiotoxic analog than doxorubicin) at lim-ited doses or liposomal anthracyclines in combination withtrastuzumab [54] which will be further analyzed

Several studies with a small number of patients exploredthe viability of combination regimens with liposomal anthra-cyclines and trastuzumab in metastatic breast cancer LD(Myocet) proved to be as effective as and less cardiotoxicthan conventional anthracyclines when combined withtrastuzumab in 4 Phase III studies

The first was a Phase III study by Theodoulou et al[55] that included 37 patients with HER2-positive metastaticbreast cancer 14 patients had been previously treated withadjuvant doxorubicin (lt240mgm2) and 17 patients with oneor two lines of prior chemotherapy for advanced disease(11 with trastuzumab) Myocet 60mgm2 was administeredevery 3 weeks plus trastuzumab 2mgKg weekly Responserate was 58 (95 CI 41ndash75) A LVEF reduction of gt10was observed in 10 patients (25) Five patients (12)presented with a LVEF lt 50 4 of them had been pretreatedwith anthracyclines 2 patients (5) withdrew from the trialdue to cardiac toxicity

Another Phase III trial [56] included 69 patients withlocally advanced or metastatic disease who had received noprior treatmentThe treatment regimen chosen for the PhaseII was trastuzumab combined with liposomal doxorubicin50mgm2 every 21 days and paclitaxel 80mgm2 weeklyResponse rate was 981 (95 CI 901ndash999) Median time toprogressionwas 221months (95CI 164ndash463) inmetastaticpatients and had not yet reached in locally advanced patientsby the time of publication No cases of treatment-relatedclinical heart failurewere observed Twelve patients presentedwith an asymptomatic reduced LVEF 8 of them recovering upto values of 50 or greater within a mean of 9 weeks

Venturini et al [57] conducted a Phase II study in 31patientswith first-linemetastatic disease to evaluate the safetyand efficacy of combining trastuzumab LD and docetaxelEight cycles of chemotherapy were administered followedby trastuzumab monotherapy to complete 52 weeks of

treatment The response rate was 655 with a TTP of 13months Five of the 31 patients experienced age 20 reductionfrom baseline or an absolute LVEF lt 45

Another Phase I-II trial with LD in combination withtrastuzumab and docetaxel was conducted by Amadori et al[58] Forty-five patients with metastatic breast cancer receiv-ed weekly trastuzumab associated with LD 50mgm2 every 3weeks and docetaxel 30mgm2 on days 2 and 9The responserate was 556 with a TTP of 109 months Only 2 patientshad a decrease in LVEF below 50

Similarly the use of PLD combined with trastuzumabmay reduce the incidence of cardiotoxicity while maintain-ing a similar efficacy We shall describe a series of smallPhase II studies that investigated this alternative Chia et al[59] included 30 patients with HER2-positive metastaticbreast cancer (MBC) 13 of thempreviously treatedwith adju-vant anthracyclines (lt300mgm2) PLD 50mgm2 was givenevery 4 weeks and trastuzumab 2mgKg weekly for 6 cyclesResponse rate was 52 and PFS 12 months The most freq-uent toxicities were grade 3 hand-foot syndrome (30) andgrade 34 neutropenia (27) Cardiac toxicity incidence was10 and in no case was symptomatic Andreopoulou et al[60] included 12 patients with MBC on first- and second-line therapy 7 treated with adjuvant anthracyclines and 7with prior trastuzumab for metastatic disease They receivedtreatment with PLD every three weeks and trastuzumabweekly achieving 66 disease stabilization 25 presentedwith grade 2 cardiac toxicity Stickeler et al [61] enrolled 16patients with HER2-positive metastatic breast cancer 5 hadreceived prior chemotherapy for advanced disease (2 of themreceived anthracyclines lt400mgm2) PLD 40mgm2 wasadministered every 4 weeks for 6ndash9 cycles plus trastuzumabweekly response rate was 50 PFS 967 months and OS1623 months Christodoulou et al [62] studied trastuzumabcombined with PLD administered at a dose of 30mgm2every three weeks All patients should have received first-linechemotherapy for advanced disease or have relapsed beforethe end of the year of taxane-based adjuvant treatment Theresponse rate was 22 PFS 65 months and OS 187 monthsThere were no episodes of LVEF reduction in any of thepatients

Wolff et al [63] published a Phase II study (ECOG E3198)in which 84 patients with HER2-positive or negativeMBC onfirst-line therapy were included and who had not been previ-ously treated with anthracyclines PLD was administered ata dose of 30mgm2 together with docetaxel 60mgm2 everythree weeks (maximum of 8 cycles) plus trastuzumab (46p)or without it (38p) according to HER2 expression Responserate was 474 in the armwithout trastuzumab (95 CI 310ndash642) and 457 in the armwith trastuzumab (95CI 309ndash61) PFS was 11 months (95 CI 86ndash128 months) and 106months (95 CI 156-157) respectively Median OS was 246months (95 CI 147ndash373) and 318 months (95 CI 237ndash449 months) There was only one case of heart failure whowas a HER2-negative patientThe addition of trastuzumab inpatients with HER2 overexpression was not associated withhigher cardiac toxicity but was related to a higher incidenceof hand-foot syndrome


Recently Martın et al [64] published a Phase II study(GEICAM 200405) which included 48 patients in first-linemetastatic disease PLD was administered at doses of 50mgm2 in combinationwith cyclophosphamide 600mgm2 every4 weeks along with weekly trastuzumab The response ratewas 688 the TTP was 12 months and OS of 342 monthsThere were no symptomatic cardiac events Eight patients(167) had decreased LVEF grade 2 six of them had beenpreviously treatedwith anthracyclines Seven of the 8 patientsrecovered cardiac function

6 Early Breast Cancer

A number of small studies of neoadjuvant treatment withliposomal anthracyclines for locally advanced breast cancerhave been publishedThePhase I study by Possinger et al [65]included 20 patients receiving a combination of LD60mgm2plus docetaxel 75mgm2 onday 1 and gemcitabine 350mgm2on day 4 every 3 weeksThe use of colony-stimulating factorswas mandatory Response rate was 88 No cardiotoxicitywas observed but there was significant haematological tox-icity (29) and stomatitis (28) Another Phase II studypublished by Gogas et al [66] included 35 patients receivingtreatment with PLD 35mgm2 in combinationwith paclitaxel175mgm2 every 3 weeks for 6 cycles Response rate was 71Grade 3 toxicity was cutaneous (11) hand-foot syndrome(9) and leukopenia (11)No cardiac toxicitywas observed

7 HER-2-Positive Early Breast Cancer

There has been a greater interest in the use of liposomalanthracyclines in early breast cancer overexpressing HER2oncogene as this subgroup of patients could obtain thegreatest benefit from treatment with anthracyclines [67] andcombining themwith trastuzumabmay be difficult due to thehigh cardiotoxicity that could be induced

Our group designed a Phase I-II study (GEICAM 2003-03) in patients with early breast cancer to be given as neoadju-vant therapy to deal with the dose variability of LD (Myocet)in combination with other drugs and the lack of evidence fora maximum tolerated dose when combined with docetaxeland trastuzumab [68 69] The results for Phase I after theinclusion of 19 patients with stages II and IIIA HER2-positive breast cancer determined the recommended dose forPhase II to be LD 50mgm2 plus docetaxel 60mgm2 everythree weeks with standard dose trastuzumab when prophy-lactic pegylated-filgrastim was administered Only one ofthe 19 patients presented with cardiac toxicity and it wasan asymptomatic grade 2 reduction in LVEF Pathologiccomplete response rate in the primary tumour and axillarylymph nodes was 33 With such stimulating data onactivity and safety Phase II of the study was com-pleted Fifty-nine patients with HER2-positive breast cancerwere included stages II 40p and IIIA 19p The recom-mended dose from prior Phase I was administered every 21days liposomal doxorubicin 50mgm2 docetaxel 60mgm2and trastuzumab 2mgkgweekly along with prophylactic

pegylated-filgrastim The clinical response rate was 86 andradiological response rate was 81 No patient progressedduring treatment All patients underwent surgery whichwas conservative in 42 cases Seventeen patients (29 95CI 172ndash404) obtained a pathologic complete response inthe breast tumour (G5 Miller and Payne) and 16 of them(27 95 CI 158ndash384) also obtained a pathologic completeresponse in the axillary lymph nodes An additional 15obtained a grade 4 Miller and Payne response in the primarytumour Neutropenia was the most significant grade 3-4haematological toxicity (17 patients 29) but only 3 devel-oped neutropenic fever Grade 3 nonhaematological toxicitywas infrequent asthenia in 5 patients nausea in 3 diarrhoeain 3 and stomatitis in one patient Grade 2 (gt20 reductionof the baseline value or reduction below the normal valueof 50) asymptomatic reduction of LEVF was observed in5 patients (9) and treatment was withheld in only one ofthem By the end of treatment 3 of the patients had recovereda LVEF greater than 50 There were no episodes of clinicalheart failure

Finally a Phase II randomized study published by Raysonet al [70] provided us with information regarding cardio-toxicity of the combination of PLD plus trastuzumab usedconcomitantly in adjuvant therapy for intermediate-riskbreast cancer with HER2 overexpression and either negativeor positive lymph nodes 181 patients with a baseline LVEFgt55 were included They were randomized (1 2) to arm Adoxorubicin 60mgm2 plus cyclophosphamide 600mgm2every 21days four cycles or arm B PLD 35mgm2 plus cyclo-phosphamide 600mgm2 every 21 days four cycles plustrastuzumab 2mgkgweekly for 12 weeks Both groups subse-quently received paclitaxel 80mgm2 plus trastuzumab for 12additional weeks followed by trastuzumab in monotherapyto complete one-year therapyThemain objective of the studywas cardiac toxicity comparing the rate of cardiac eventsandor the percentage of patients who were unable to com-plete one-year treatment with trastuzumab The incidence ofcardiac toxicity was 186 with doxorubicin (95 CI 97ndash309) versus 42 with PLD (95 CI 14ndash95) (119875 =00036) Among the 16 patients who had a cardiac event (11 inthe conventional doxorubicin arm and 5 in the PLD arm) 8were over 55 years old All the events occurred after the 4thcourse of therapy One of the events was a myocardial infarc-tion with subsequent clinical heart failure (this occurred inarm B) Of the remaining 15 cases 7 were recorded as gt10reduction from baseline LVEF with absolut values of lt50(3 of them developing clinical symptoms were classed asNHYA class II heart failure)The other 8 cases were classed asasymptomatic (NYHA class I) There were no cardiotoxicity-related deaths The LVEF mean value was similar in bothgroups (640 PLD+C+HT +H and 644 A +CT +H)Mean reduction of LVEF values after the 8th cycle (end ofchemotherapy) was significantly higher in patients receivingconventional doxorubicin (56 versus 21 119875 = 00014)Cardiac safety analysis for this study suggested that admin-istering trastuzumab concomitantly with PLD in the testedregimen was feasible caused less cardiotoxicity in the shortterm and avoided the premature interruption of treatment


with trastuzumab when compared with a standard regimensuch as A+CT+HThe authors concluded that this strategyof incorporating early and concomitantly a liposomal anthra-cycline plus trastuzumabwas safe but its possible clinical roleshould be properly investigated in a randomized Phase IIItrial versus a nonanthracycline regimen such as TCH

8 Conclusions

Liposome-based drug delivery systems are able to modifythe pharmacokinetics and pharmacodynamics of cytostaticagents enabling us to increase the concentration of the drugreleased into the neoplastic tissue and at the same timereducing the exposure of normal tissue to the drug

Anthracyclines are important agents in the treatment ofboth metastatic and early breast cancer but cardiotoxicityremains one of the major limitations for their use Liposomeencapsulation is one of the strategies designed to minimizethis side effect There are several liposome-encapsulateddoxorubicin formulations available which show differentpharmacological characteristics The most commonly usedare liposomal doxorubicin (Myocet) and pegylated liposomaldoxorubicin (Caelyx)

In patients with metastatic breast cancer liposomalanthracyclines have proven to be as effective and less toxicwhen compared face to face with conventional anthracy-clines allowing a longer period of treatment and a highercumulative dose of the anthracyclinesThe combined analysisof available data indicates an overall reduction in risk for bothcardiotoxicity (RR = 038 119875 lt 00001) and clinical heartfailure (RR = 020 119875 = 002) The safety of liposomal anthra-cyclines endorsed its use in patients with some cardiac riskfactors

In HER2-positive breast cancer the addition of trastu-zumab to chemotherapy significantly increased response rateprogression-free survival and overall survival Initial studiesdemonstrated synergywhen trastuzumabwas combinedwithanthracyclines but their excessive cardiac toxicity limitedtheir use and nonanthracycline therapeutic strategies weredesigned

Liposomal anthracyclines have proven to be effective andsafe when combined with trastuzumab both in advanced andearly breast cancer Of particular interest is the use of thecombination of liposomal anthracyclines plus trastuzumab inpatients with early and HER2-overexpressing breast canceras this is probably the subgroup that would benefit most froma treatment with anthracyclinesThe potential clinical benefitof anthracyclines in this setting should be investigated in aclinical trial comparing a regimen with liposomal anthra-cyclines versus a nonanthracyclines combination

Conflict of Interests

The authors declare no conflict of interests relating to thepublication of this paper

References

[1] D R Khan E M Rezler J Lauer-Fields and G B FieldsldquoEffects of drug hydrophobicity on liposomal stabilityrdquo Chemi-cal Biology and Drug Design vol 71 no 1 pp 3ndash7 2008

[2] New RRC Liposomes A Practical Approach Oxford UniversityPress Oxford UK 1st edition 1990

[3] E M Rezler D R Khan J Lauer-Fields M Cudic D Baronas-Lowell and G B Fields ldquoTargeted drug delivery utilizingprotein-like molecular architecturerdquo Journal of the AmericanChemical Society vol 129 no 16 pp 4961ndash4972 2007

[4] R Krishna and L D Mayer ldquoThe use of liposomal anticanceragents to determine the roles of drug pharmacodistribution andP-glycoprotein (PGP) blockade in overcoming multidrug resis-tance (MDR)rdquo Anticancer Research vol 19 no 4 B pp 2885ndash2891 1999

[5] H Maeda J Wu T Sawa Y Matsumura and K Hori ldquoTumorvascular permeability and the EPR effect in macromoleculartherapeutics a reviewrdquo Journal of Controlled Release vol 65 no1-2 pp 271ndash284 2000

[6] A A Gabizon ldquoStealth liposomes and tumor targeting onestep further in the quest for the magic bulletrdquo Clinical CancerResearch vol 7 no 2 pp 223ndash225 2001

[7] D C Drummond O Meyer K Hong D B Kirpotin andD Papahadjopoulos ldquoOptimizing liposomes for delivery ofchemotherapeutic agents to solid tumorsrdquo PharmacologicalReviews vol 51 no 4 pp 691ndash743 1999

[8] F K Bedu-Addo P Tang Y Xu and L Huang ldquoEffects ofpolyethyleneglycol chain length and phospholipid acyl chaincomposition on the interaction of polyethyleneglycol-phos-pholipid conjugates with phospholipid implications in liposo-mal drug deliveryrdquo Pharmaceutical Research vol 13 no 5 pp710ndash717 1996

[9] T M Allen ldquoLiposomes Opportunities in drug deliveryrdquoDrugs vol 54 no 4 pp 8ndash14 1997

[10] S Brown and R David Khan ldquoThe Treatment of Breast CancerUsing Liposome Technologyrdquo Journal of Drug Delivery vol2012 Article ID 212965 6 pages 2012

[11] J GaoWZhong JHe et al ldquoTumor-targetedPE38KDELdeliv-ery via PEGylated anti-HER2 immunoliposomesrdquo InternationalJournal of Pharmaceutics vol 374 no 1-2 pp 145ndash152 2009

[12] R S Tolhurst R S Thomas F J Kyle et al ldquoTransient over-ex-pression of estrogen receptor-120572 in breast cancer cells promotescell survival and estrogen-independent growthrdquo Breast CancerResearch and Treatment vol 128 no 2 pp 357ndash368 2011

[13] S R Paliwal R Paliwal N Mishra A Mehta and S P VyasldquoA novel cancer targeting approach based on estrone anchoredstealth liposome for site-specific breast cancer therapyrdquo CurrentCancer Drug Targets vol 10 no 3 pp 343ndash353 2010

[14] C M Perou T Soslashrile M B Eisen et al ldquoMolecular portraits ofhuman breast tumoursrdquoNature vol 406 no 6797 pp 747ndash7522000

[15] T Soslashrlie CM Perou R Tibshirani et al ldquoGene expression pat-terns of breast carcinomas distinguish tumor subclasses withclinical implicationsrdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 98 no 19 pp 10869ndash10874 2001

[16] H J Burstein J R Harris and M Morrow ldquoMalignant tumorsof the breastrdquo inDe Vita Hellman and Rosenbergrsquos Cancer Prin-ciplesampPractice ofOncology pp 1401ndash1446 LippincottWilliamsampWilkins 2011


[17] X Wang L Yang Z Chen and D M Shin ldquoApplication ofnanotechnology in cancer therapy and imagingrdquo CA CancerJournal for Clinicians vol 58 no 2 pp 97ndash110 2008

[18] D W Northfelt F J Martin P Working et al ldquoDoxorubicinencapsulated in liposomes containing surface-bound polyethy-lene glycol pharmacokinetics tumor localization and safetyin patients with AIDS-related Kaposirsquos sarcomardquo Journal ofClinical Pharmacology vol 36 no 1 pp 55ndash63 1996

[19] Z Symon A Peyser D Tzemach et al ldquoSelective delivery ofdoxorubicin to patients with breast carcinoma metastases bystealth liposomesrdquo Cancer vol 86 pp 72ndash78 1999

[20] T A Elbayoumi and V P Torchilin ldquoTumor-specific antibody-mediated targeted delivery of Doxil reduces the manifestationof auricular erythema side effect in micerdquo International Journalof Pharmaceutics vol 357 no 1-2 pp 272ndash279 2008

[21] ldquoPreclinical development tissue distribution of doxorubicin(DOX) and TLC D-99 and conventional doxorubicinrdquo Datafrom the Registration dossier

[22] D D Von Hoff M W Layard and P Basa ldquoRisk factors fordoxorubicin-induced congestive heart failurerdquo Annals of Inter-nal Medicine vol 91 no 5 pp 710ndash717 1979

[23] L J Steinherz P G Steinherz C T C Tan G Heller and M LMurphy ldquoCardiac toxicity 4 to 20 years after completing anthra-cycline therapyrdquo Journal of the American Medical Associationvol 266 no 12 pp 1672ndash1677 1991

[24] N G Fisher and A J Marshall ldquoAnthracycline-induced car-diomyopathyrdquo PostgraduateMedical Journal vol 75 no 883 pp265ndash268 1999

[25] A P Launchbury and N Habboubi ldquoEpirubicin and doxoru-bicin a comparison of their characteristics therapeutic activityand toxicityrdquo Cancer Treatment Reviews vol 19 no 3 pp 197ndash228 1993

[26] M E Billingham J W Mason M R Bristow and J R DanielsldquoAnthracycline cardiomyopathy monitored by morphologicchangesrdquo Cancer Treatment Reports vol 62 no 6 pp 865ndash8721978

[27] R G Schwartz W B McKenzie J Alexander et al ldquoCongestiveheart failure and left ventricular dysfunction complicating dox-orubicin therapy Seven-year experience using serial radionu-clide angiocardiographyrdquo The American Journal of Medicinevol 82 no 6 pp 1109ndash1118 1987

[28] M F Stoddard J Seeger N E Liddell T J Hadley D MSullivan and J Kupersmith ldquoProlongation of isovolumetricrelaxation time as assessed by Doppler echocardiography pre-dicts doxorubicin-induced systolic dysfunction in humansrdquoJournal of the American College of Cardiology vol 20 no 1 pp62ndash69 1992

[29] W I Ganz K S Sridhar and T J Forness ldquoDetection of earlyanthracycline cardiotoxicity bymonitoring the peak filling raterdquoTheAmerican Journal of ClinicalOncology vol 16 no 2 pp 109ndash112 1993

[30] S M Swain F S Whaley and M S Ewer ldquoCongestive heartfailure in patients treated with doxorubicin a retrospectiveanalysis of three trialsrdquo Cancer vol 97 no 11 pp 2869ndash28792003

[31] C L Shapiro P H Hardenbergh R Gelman et al ldquoCardiaceffects of adjuvant doxorubicin and radiation therapy in breastcancer patientsrdquo Journal of Clinical Oncology vol 16 no 11 pp3493ndash3501 1998

[32] C L Shapiro and A Recht ldquoSide effects of adjuvant treatmentof breast cancerrdquoTheNew England Journal ofMedicine vol 344no 26 pp 1997ndash2008 2001

[33] M E OrsquoBrien N Wigler M Inbar et al ldquoReduced cardio-toxicity and comparable efficacy in a phase III trial of pegylatedliposomal doxorubicin HCI (CAELYXDoxil) versus conven-tional doxorubicin for first-line treatment of metastasic breastcancerrdquo Annals of Oncology vol 15 no 3 pp 440ndash449 2004

[34] LHarris G Batist R Belt et al ldquoLiposome-encapsulated doxo-rubicin compared with conventional doxorubicin in a random-ized multicenter trial as first-line therapy of metastatic breastcarcinomardquo Cancer vol 94 no 1 pp 25ndash36 2002

[35] G Batist G Ramakrishnan C S Rao et al ldquoReduced cardio-toxicity and preserved antitumor efficacy of liposome-encap-sulated doxorubicin and cyclophosphamide compared withconventional doxorubicin and cyclophosphamide in a random-ized multicenter trial of metastatic breast cancerrdquo Journal ofClinical Oncology vol 19 no 5 pp 1444ndash1454 2001

[36] S Chan N Davidson E Juozaityte et al ldquoPhase III trial ofliposomal doxorubicin and ciclophosphamide compared withepirrubicin and ciclophosphamide as first-line therapy formetastasic breast cancerrdquo Annals of Oncology vol 15 pp 1527ndash1534 2004

[37] J A Sparano A N Makhson V F Semiglazov et al ldquoPegylatedliposomal doxorubicin plus docetaxel significantly improvestime to progression without additive cardiotoxicity comparedwith docetaxel monotherapy in patients with advanced breastcancer previously treated with neoadjuvant-adjuvant anthra-cycline therapy results from a randomized phase III studyrdquoJournal of Clinical Oncology vol 27 no 27 pp 4522ndash4529 2009

[38] L Gianni E Munzone G Capri et al ldquoPaclitaxel by 3-hourinfusion in combinationwith bolus doxorubicin in womenwithuntreated metastatic breast cancer high antitumor efficacy andcardiac effects in a dose-finding and sequence-finding studyrdquoJournal of Clinical Oncology vol 13 no 11 pp 2688ndash2699 1995

[39] L Gianni L Vigano A Locatelli et al ldquoHuman pharmacoki-netic characterization and in vitro study of the interactionbetween doxorubicin and paclitaxel in patients with breastcancerrdquo Journal of Clinical Oncology vol 15 no 5 pp 1906ndash19151997

[40] D J Slamon B Leyland-Jones S Shak et al ldquoUse of chemother-apy plus a monoclonal antibody against her2 for metastaticbreast cancer that overexpresses HER2rdquo The New EnglandJournal of Medicine vol 344 no 11 pp 783ndash792 2001

[41] M Untch H Eidtmann A Du Bois et al ldquoCardiac safety oftrastuzumab in combination with epirubicin and cyclophos-phamide in women with metastatic breast cancer results of aphase I trialrdquo European Journal of Cancer vol 40 no 7 pp 988ndash997 2004

[42] A N Gordon J T Fleagle D Guthrie D E Parkin M E Goreand A J Lacave ldquoRecurrent epithelial ovarian carcinoma arandomized phase III study of pegylated liposomal doxorubicinversus topotecanrdquo Journal of ClinicalOncology vol 19 no 14 pp3312ndash3322 2001

[43] M S Rosati C Raimondi G Baciarello et al ldquoWeekly com-bination of non-pegylated liposomal doxorubicin and taxanein first-line breast cancer wALT trial (phase I-II)rdquo Annals ofOncology vol 22 no 2 pp 315ndash320 2011

[44] P Schmid J Krocker R Kreienberg et al ldquoNon-pegylatedliposomal doxorubicin and docetaxel in metastatic breast can-cer final results of a phase II trialrdquo Cancer Chemotherapy andPharmacology vol 64 no 2 pp 401ndash406 2009

[45] E Curtit P Nouyrigat N Dohollou E Levy et al ldquoMyotaxa phase II trial of docetaxel plus non-pegylated liposomaldoxorubicin as first-line therapy of metastatic breast cancer


previously treated with adjuvantrdquo European Journal of Cancervol 47 no 16 pp 2396ndash2402

[46] C Rochlitz T Ruhstaller S Lerch et al ldquoCombination ofbevacizumab and 2-weekly pegylated liposomal doxorubicinas first-line therapy for locally recurrent or metastatic breastcancer A multicenter single-arm phase II trial (SAKK 2406)rdquoAnnals of Oncology vol 22 no 1 pp 80ndash85 2011

[47] G Batist L Harris N Azarnia LW Lee and P Daza-RamirezldquoImproved anti-tumor response rate with decreased cardiotox-icity of non-pegylated liposomal doxorubicin compared withconventional doxorubicin in first-line treatment of metastaticbreast cancer in patients who had received prior adjuvantdoxorubicin results of a retrospective analysisrdquo Anti-CancerDrugs vol 17 no 5 pp 587ndash595 2006

[48] E C Van Dalen E M C Michiels H N Caron and L C MKremer ldquoDifferent anthracycline derivatives for reducing car-diotoxicity in cancer patientsrdquo Cochrane Database of SystematicReviews no 3 2010

[49] A M Keller R G Mennel V A Georgoulias et al ldquoRandom-ized phase III trial of pegylated liposomal doxorubicin versusvinorelbine or mitomycin C plus vinblastine in women withtaxane-refractory advanced breast cancerrdquo Journal of ClinicalOncology vol 22 no 19 pp 3893ndash3901 2004

[50] M Fiegl B Mlineritsch M Hubalek R Bartsch U Pluschnigand G G Steger ldquoSingle-agent pegylated liposomal doxoru-bicin (PLD) in the treatment of metastatic breast cancer resultsof an Austrian observational trialrdquo BMC Cancer vol 11 ArticleID 373 2011

[51] E AlbaM Ruiz-BorregoMMargelı et al ldquoMaintenance treat-ment with Pegylated liposomal doxorubicin versus observationfollowing induction chemotherapy for metastatic breast cancerGEICAM2001-01 studyrdquoBreast Cancer Research and Treatmentvol 122 no 1 pp 169ndash176 2010

[52] M D Pegram T Pienkowski D W Northfelt et al ldquoResultsof two open-label multicenter phase II studies of docetaxelplatinum salts and trastuzumab in HER2-positive advancedbreast cancerrdquo Journal of the National Cancer Institute vol 96no 10 pp 759ndash769 2004

[53] D Slamon W Eiermann N Robert et al ldquoAdjuvant trastuz-umab in her-2 positive breast cancerrdquoThe New England Journalof Medicine vol 365 no 14 pp 1273ndash1283 2011

[54] M Untch M Muscholl S Tjulandin et al ldquoFirst-line trastuz-umab plus epirubicin and cyclophosphamide therapy inpatients with human epidermal growth factor receptor 2-posi-tive metastatic breast cancer cardiac safety and efficacy datafrom the herceptin cyclophosphamide and epirubicin (HER-CULES) trialrdquo Journal of Clinical Oncology vol 28 no 9 pp1473ndash1480 2010

[55] M Theodoulou S M Campos L Welles et al ldquoTLC D99 (DMyocet) and Herceptin (H) is safe in advanced breast cancer(ABC) final cardiac safety and efficacy analysisrdquo Proceedings ofthe American Society of Clinical Oncology vol 21 Abstract 2162002

[56] J Cortes S DiCosimo M A Climent et al ldquoNonpegylatedliposomal doxorubicin (TLC-D99) Paclitaxel and Trastuz-umab in HER-2-overexpressing breast cancer a multicenterphase lll studyrdquo Clinical Cancer Research vol 15 no 1 pp 307ndash314 2009

[57] M Venturini C Bighin F Puglisi et al ldquoA multicentre phase IIstudy of non-pegylated liposomal doxorubicin in combinationwith trastuzumab and docetaxel as first-line therapy in meta-static breast cancerrdquo Breast vol 19 no 5 pp 333ndash338 2010

[58] D Amadori C Milandri G Comella et al ldquoA phase III trialof nonpegylated liposomal doxorubicin docetaxel and trastuz-umab as first-line treatment inHER-2-positive locally advancedor metastatic breast cancerrdquo European Journal of Cancer vol 47no 14 pp 2091ndash2098 2011

[59] S Chia M Clemons L A Martin et al ldquoPegylated liposomaldoxorubicin and trastuzumab in HER-2 overexpressing meta-static breast cancer a multicenter phase II trialrdquo Journal ofClinical Oncology vol 24 no 18 pp 2773ndash2778 2006

[60] E Andreopoulou D Gaiotti E Kim et al ldquoFeasibility andcardiac safety of pegylated liposomal doxorubicin plus trastuz-umab in heavily pretreated patients with recurrent HER2-over-expressing metastatic breast cancerrdquo Clinical Breast Cancer vol7 no 9 pp 690ndash696 2007

[61] E Stickeler M Klar D Watermann et al ldquoPegylated liposomaldoxorubicin and trastuzumab as 1st and 2nd line therapy inher2neu positive metastatic breast cancer a multicenter phaseII trialrdquo Breast Cancer Research and Treatment vol 117 no 3 pp591ndash598 2009

[62] C Christodoulou I Kostopoulos H P Kalofonos et al ldquoTra-stuzumab combined with pegylated liposomal doxorubicin inpatients with metastatic breast cancer phase II study of thehellenic cooperative oncology group (HeCOG) with biomarkerevaluationrdquo Oncology vol 76 no 4 pp 275ndash285 2009

[63] A C Wolff M Wang H Li et al ldquoPhase II trial of pegylatedliposomal doxorubicin plus docetaxel with and without trastuz-umab in metastatic breast cancer eastern cooperative oncologygroup trial E3198rdquo Breast Cancer Research and Treatment vol121 no 1 pp 111ndash120 2010

[64] M Martın M Munoz J M Baena-Canada et al ldquoPegylatedliposomal doxorubicin in combination with cyclophosphamideand trastuzumab in HER2-positive metastatic breast cancerpatients efficacy and cardiac safety from the GEICAM2004-05 studyrdquoAnnals of Oncology vol 22 no 12 Article IDmdr024pp 2591ndash2596 2011

[65] K Possinger J Krocker J Fritz et al ldquoPrimary chemotherapyfor locally advanced breast cancer (LABC) with gemcitabine(G) as prolonged infusion liposomal doxorubicin (M) andDocetaxel (T) results of a phase I trialrdquo Proceedings of theAmerican Society of Clinical Oncology vol 21 abstract 19712002

[66] H Gogas C Papadimitriou H P Kalofonos et al ldquoNeoadju-vant chemotherapy with a combination of pegylated liposomaldoxorubicin (Caelyx) and paclitaxel in locally advanced breastcancer a phase II study by the Hellenic cooperative oncologygrouprdquo Annals of Oncology vol 13 no 11 pp 1737ndash1742 2002

[67] A Gennari M P Sormani P Pronzato et al ldquoHER2 statusand efficacy of adjuvant anthracyclines in early breast cancera pooled analysis of randomized trialsrdquo Journal of the NationalCancer Institute vol 100 no 1 pp 14ndash20 2008

[68] A Anton A Ruiz M A Seguı et al ldquoPhase I clinical trialof liposomal-encapsulated doxorubicin citrate and docetaxelassociated with trastuzumab as neo-adjuvant treatment instages II and IIIA HER2-overexpressing breast cancer patientsGEICAM 2003-03 studyrdquo Annals of Oncology vol 20 no 3 pp454ndash459 2009

[69] A Anton A Ruiz A Plazaola et al ldquoPhase II clinical trial ofliposomal-encapsulated doxorubicin citrate and docetaxelassociated with trastuzumab as neoadjuvant treatment instages II and IIIA HER2-overexpressing breast cancer patients


GEICAM 2003-03 studyrdquo Annals of Oncology vol 22 no 1 pp74ndash79 2011

[70] D Rayson T M Suter C Jackisch et al ldquoCardiac safety ofadjuvant pegylated liposomal doxorubicin with concurrenttrastuzumab a randomized phase II trialrdquo Annals of Oncologyvol 23 no 7 Article ID mdr519 pp 1780ndash1788 2012


Review ArticleGene Therapy for Advanced Melanoma Selective Targeting andTherapeutic Nucleic Acids

Joana R Viola1 Diana F Rafael2 Ernst Wagner13 Robert Besch4 and Manfred Ogris13

1 Pharmaceutical Biotechnology Department of Pharmacy Ludwig-Maximilians-Universitat Butenandstraszlige 5-13 Munich Germany2Department of Nanomedicine and Drug Delivery Systems Faculty of Pharmacy iMEDUL Research Institute for Medicine andPharmaceutical Sciences University of Lisbon Avenida Professor Gama Pinto Lisbon Portugal

3 Center for NanoScience (CeNS) Ludwig-Maximilians-Universitat Munich Germany4Department of Dermatology and Allergology Ludwig-Maximilians-Universitat Munich Germany

Correspondence should be addressed to Joana R Viola joanaviolagmailcomand Manfred Ogris manfredogriscupuni-muenchende


Academic Editor Stefano Salmaso

Copyright copy 2013 Joana R Viola et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Despite recent advances the treatment of malignant melanoma still results in the relapse of the disease and second line treatmentmostly fails due to the occurrence of resistance A wide range of mutations are known to prevent effective treatment withchemotherapeutic drugs Hence approaches with biopharmaceuticals including proteins like antibodies or cytokines are appliedAs an alternative regimens with therapeutically active nucleic acids offer the possibility for highly selective cancer treatment whilstavoiding unwanted and toxic side effects This paper gives a brief introduction into the mechanism of this devastating diseasediscusses the shortcoming of current therapy approaches and pinpoints anchor points which could be harnessed for therapeuticintervention with nucleic acids We bring the delivery of nucleic acid nanopharmaceutics into perspective as a novel antimelanomatherapeutic approach and discuss the possibilities for melanoma specific targeting The latest reports on preclinical and alreadyclinical application of nucleic acids in melanoma are discussed

1 Introduction

Melanoma derivates frommelanocytesmdashpigment cells of theskin Melanoma most commonly arises from epidermal skinmelanocytes (cutaneous melanoma) but primary tumorscan also be found lining the choroidal layer of the eye(uveal melanoma) or the mucosal surfaces of the respi-ratory genitourinary and gastrointestinal surfaces Similarto other tumors the progression stage of melanoma ispredictive for therapeutic success Early stage melanomas(thin tumors) result in a 97 5-year survival rate of thepatients after surgical removal [1] Conversely advancedmelanoma patients comprising metastasis in regional lymphnodes or other organs face 5-year survival rates of lessthan 10 [1] Due to the intrinsic tendency of melanomato early metastasis even small primary tumors have alreadyled to metastasis and a substantial portion of diagnosedmelanoma cases are of late progression stages Treatment of

advanced or metastatic melanoma has proven a challenge asthe conventional therapeutic approaches failed to translateinto improved or significant survival rate in phase III clinicaltrials Newer treatments were established in the last years thatelicit unprecedented response rates in late stage melanomafor example up to 80 in the case of BRAF inhibitorsHowever almost all tumors become resistant within monthsand the treatment is available only for a subset of melanomasAltogether despite substantial improvements in therapeuticoptions during the last years there is still an urgent need foralternative approaches

Based on clinical and histopathological features mela-noma cancer cells undergo four sequential phases beforereaching metastasis [2] These phases ensue from severalgenetic epigenetic and microenvironmental modifications[3] In the last decade a number of reports have broughtsignificant insight into melanoma genetics and molecularmarkers which are essential for the development of therapies


and in particular targeted regimens This paper will focuson melanoma targeted gene delivery we aim at providinga general view on melanoma-targeting ligands and otherforms of specifically driving gene expression reported inthe literature as well as review the most recent andorrelevant nucleic acid therapeutics employed in this field Thecurrent paper will not dwell upon melanoma mutations orcancer transcriptional regulators (for reviews see [4 5])Instead the following melanoma section serves rather as acomprehensive overview on the key players of the neoplasiawhich is essential for the understanding of targeted therapies

2 From Melanocytes to Metastatic Melanoma

21 Four Steps Separate Melanocytes from MetastaticMelanoma Presently it is generally believed that melano-magenesis instigates from alterations in multiple moleculesor pathways rather than a single high-risk melanoma lociMoreover melanoma progression is a dynamic processinvolving several steps each requiring the activation ofdifferent genes First normal melanocytes undergo geneticalterations that lead to their transformation into benign neviBenign nevi differ from normal melanocytes in that theyhave initially proliferated in the basal layer of the epidermishowever they entered a long-term dormant status due to thelack of additional oncogenic alterations For example themost frequent activating mutation in the BRAF gene occursin the same frequency in nevi where it causes a dormantstatus called oncogene-induced senescence [6] Additionalalterations then allow bypassing senescence leading tocontinued tumor cell proliferation This progression stage ischaracterized by noninvasive horizontal growth and spreadthrough the epidermis and has been termed as radial growthphase (RGP) Further transformation is required for invasivetumor growth from the epidermis into the dermis Thisphase has been termed as vertical growth phase (VGP)For invasion alterations like loss of adhesive moleculestogether with an increase in extracellular matrix degradingenzymes are characteristic For metastasis cell populationshave to migrate to distant locations For this cells have toacquire more alterations that enable the complex processesunderlying metastasis These processes involve tissueinvasion entering and evasion of blood or lymphatic vesselsto reach distant location but also survival and proliferation atdistinct locations Hence melanocytic cells have to becomelargely independent from their normal microenvironment[7]

22 Melanoma Progression Risk Factors and BiologicalDrivers Themost important risk factor for melanoma is UVirradiation upon sun exposure Whole genome sequencingrevealed thatmelanoma is the tumor typewith themostDNAmutationsmdashmany being typical for UV-induced mutations[8] Despite the plethora of DNA alterations two genemutations were found to be rather common in melanoma Ageneral overview on thesemutations and their key players areschematically represented in Figure 1

With respect to mutation frequency the mitogen-activated protein kinase (MAPK) pathway plays a centralrole in melanoma Activation of growth factor receptorsleads to activation of RAS molecules which activate in adownstream phosphorylation cascade RAF MEK and ERKkinases ERK kinase phosphorylates a panel of substratesleading to increased cell proliferation and survival RASmolecules comprising HRAS KRAS and NRAS are smallGTPases or G proteins and activating mutations in NRASare found in 10ndash20 of melanomas RAS molecules acti-vate RAF family members consisting of ARAF BRAF andCRAF A single nucleotide mutation in BRAF at aminoacid 600mdashwhereupon a valine (V) aminoacid is replaced byglutamic acid (E)mdashrepresents the most common mutationin BRAF This mutant V600EBRAF leads to an alternativeprotein structure and to a constitutive active protein 50ndash60 of melanomas contain an activating mutation in BRAF[9] The outstanding importance of the RASRAF signalingpathway is documented by the observation that BRAF andNRAS mutationsmdashexclusively NRAS or BRAF is mutated ina tumormdashtogether are found in over 80 of melanomas andby inhibitors of mutated BRAF that are clearly effective inmelanoma therapy

Interestingly V600EBRAF has also been reported in mel-anocytic nevi [10ndash12] which rarely develop into melanomaNevi are described to be senescent and similarly expressionof V600EBRAF in melanocytes induces oncogene-inducedsenescence [6] These findings imply that BRAF mutationsare involved in the first transition state of melanoma pro-gression Hence this mutation per se is insufficient to drivetumorigenesis rather additional alterations are required toavoid dormancy

Several pathways have been shown to cooperate withRASRAF signaling and to reduce RASRAF-mediated senes-cence DNA damage due to oncogene-induced DNA repli-cation stress has been proposed as an important mecha-nism of senescence [13] Accordingly molecules involvedin DNA damage signaling have been shown to promoteoncogenesis together with BRAF for example the loss ofp53 [14] Most evidence for BRAF cooperation exists forphosphatase and tensin homolog (PTEN) PTEN is a tumorsuppressor gene that negatively modulates signal transduc-tion via phosphatidylinositol phosphatase (PIP

3 a cytosolic

second messenger) This gene encodes for a lipid proteinphosphatase that regulates cell growth and survival Allelicloss or altered expression of PTENcan be observed in tumorsIn melanoma this lostmodified expression is present in2040 of melanoma tumors respectively [15 16] In amouse model it was shown that expression of V600EBRAF inmelanocytes leads to benign lesions that do not progress tomelanoma However when PTEN was silenced these micedeveloped metastatic tumors with high penetrance [17]

Regarding the family history ofmelanoma a two-fold riskincrease has been reported [18] and it was associated to the9p12 chromosome [19] In 1994 the cyclin-dependent kinaseN2A (CDKN2A) gene was identified [20] and it is now holdas a high-risk melanoma locus The CDKN2A gene encodesfor two tumor suppressor proteins p16INK4a and p14ARF


RAS

qP12

Off

Off

BRAF On

(B) PTEN

ERK FAK

PKBAKTp130CAS

Off

OnOn

On

UV radiation

DNA mutations

V600EBRAF

Cell proliferation and survivaltumor metastasis

Cell survivalCell migrationP16INK4a

p14ARF

CDKN2A gene

(A) Family history

PIP3

Figure 1 Schematic summary of the most common mutations found in melanoma patients The most common risk for melanoma is UVand most DNA alterations are typically UV-induced Family history of melanoma accounts for a two-fold risk increase through mutations atthe level of CDKN2A gene These often affect the tumor suppressors p16INK4a or p14ARF which have roles in the cell cycle and apoptosisrespectively On the other hand there is the RASRAF signaling pathway which importance is underlined by the fact that exclusively NRASor BRAF is mutated in melanoma However the presence of BRAF mutations in benign nevi suggest that BRAF per se does not suffice forthe tumor progression Often mutations in PTEN pathways have been found to cooperate with RASRAF to reduce RASRAF-mediatedsenescence

involved in cell cycle and apoptosis respectively Explicitlyp14ARF directly promotes the degradation of human doubleminute 2 (MDM2) MDM2 promotes ubiquitinylation andproteasomal degradation of p53 Accordingly inactivation ofp14ARF leads to increased MDM2 levels leading to increaseddegradation of p53 [21] The other product of the CDKN2Alocus p16INK4a prevents cell cycle progression by bindingto CDK46 and through a series of events prevents therelease of E2F1 (a transcriptional inducer of S-phase genes)[22] Mutations of p16IK4a and similarly of CDK4 gene [2324] can therefore lead to increased cell cycle progressionHowever despite the contribution of CDKN2A mutationsfor oncogenesis the absolute risk of melanoma in mutationcarriers is still highly shaped by environmental and pedigreefactors [25] In close relation to pedigree structure is skin pig-mentation the positive connection between light skin colorand melanoma risks is well known Melanocortin-1 receptor(MC1-R) is responsible for the cutaneous pigmentation andinterestingly it has been reported as being overexpressed

in both melanotic and amelanotic melanomas [26] Thereare two forms of epidermal melanin eumelanin (with ablack-brown color) and pheomelanin (red-yellow color)Thesynthesis of eumelaninmdashin charge of UV attenuationmdashisstimulated by the activation of the MC1-R through thebinding of the tridecapeptide 120572-MSH or 120572-melanocortinstimulating hormone [27ndash29] The binding of 120572-MSH resultsin an increment of cAMP which in turn upregulatesthe microphthalmia-associated transcription factor (MITF)inducing the transcription of pigment synthetic genes and theproduction of eumelanin In addition some MC1-R variantshave been associated to melanoma risk [30] MITF on theother hand is also involved in the regulation of the cellcycle and proliferation and few variants of the gene havebeen found in melanoma patients [31 32] In particularMITF(E318 K) was reported to represent a gain-of-functionallele for the gene supporting MITFs role as an oncogeneHowever MITFs expression in melanoma metastasis isyet to be clarified as there are also studies showing that


downregulation and ablation of this gene create a moreinvasive phenotype in vitro [33] and increase tumor growthin vivo [34] respectively

The transcription factor activator protein-2120572 (AP2120572) hasbeen suggested as a major key player in the transition fromRGP to VGP [4] Similar to several other mediators AP2120572also modulates a variety of cellular processes including cellgrowth and apoptosis In tumors AP2120572 acts as a tumorsuppressor and high cytoplasmatic to nuclear expressionratiowas shown to correlatewith poor patientsrsquo prognosis [3536] In particular the promoters for the adhesion moleculeMCAMMUC18 [37] which is overexpressed in tumorsand tyrosinase kinase receptor c-KIT (silenced in 70 ofmetastatic tumors) [38] have AP2120572 binding sites AP2120572 hasbeen described to directly bind to MCAMMUC18 promoterand to inhibit its transcription whereas it promotes c-KITexpression Therefore the loss of this transcription factorduring melanoma results in high MCAMMUC18 levels andc-KIT downregulation In addition the loss of AP2120572 wasalso appointed as a probable cause for the upregulation ofthe G-protein-coupled receptor protease activated receptor-1 PAR-1 [10 39] In PAR-1 promoter region there are twobinding complexes forAP2120572 and SP1 In normalmelanocytesAP2120572 binds to PAR-1 inhibiting its transcription Howeverupon melanoma progression the levels of AP2120572 decreaseand SP1 binds to the PAR-1 promoter instead driving itsexpression RAS phosphoinositide-3 kinase (PI3 K) andMAPK pathways are all signaling events downstream PAR-1and hence closely related to tumor progression [40]

During the metastatic process following evasion into theblood circulation tumor cells adhere to the endotheliumat distant sites and herein adhesion molecules are neces-sary Together with selectins integrins have been found toplay crucial roles in these steps Integrins are a family oftransmembrane glycoproteins that mediate cell-cell and cell-matrix adhesion It is therefore expected that their expressionpattern changes during tumor growth metastasis and angio-genesis In particular 120572v1205733 and 12057241205731 (very late activationantigen-4 VLA-4) have been reported as overexpressed innumerous cancer types [41 42] and have served as therapeu-tic targets VLA-4 has been shown to be used by malignantmelanoma cells to adhere to the endothelium (binding tothe ligand VCAM-1) [43 44] and to promote transmigration[42 45] and metastasis [46 47]

3 Shortcomings of CurrentMelanoma Therapies

Overall melanoma incidence has been increasing over theyears reaching an annually increase of 31 during the pasttwo decades [48] Early prognosis permits 90 survival ratesby surgical removal Yet unresectable advanced melanomais characterized by an aggressive behaviour fast spread andmetastasis and a strong resistance to chemotherapy There-fore and in spite of the extensive research the current prog-nosis for patients with advanced melanoma is limited Theearlier conventional chemotherapeutic treatment approvedby US Food and Drug Administration (FDA) Dacarbazine

results in less than 10 response rate with median responsedurations of 4ndash8 months [49] Alternative chemotherapeuticagents include Fotemustine Temozolomide Paclitaxel (oftenin combination with carboplatin) and Docetaxel [50]mdashall not yielding larger progression-free survival (PFS) oroverall survival (OS) than Dacarbazine [50 51] Generallychemotherapeutics suffer from a lack of targeting specificitytheir low molecular mass results in easy and fast bodysecretion and thus the need of increased doses which leadsto inevitable toxicity Similarly immunotherapy based oninterleukine 2 (IL-2)mdashalso FDA approvedmdashhas comparableresponse rates and it is further restricted by the ensuingmul-tiorgan toxicity requiring management in specialized cancercenters Although combined therapies resulted in higherresponse rates they still failed to translate into improved sur-vival with no impact on PFS or OS compared to Dacarbazinealone [1 52] Another alternative is the combined treatmentwith the cytokine TNF120572 in combination with the alkylatingdrugmelphalan Although highly successful this treatment islimited to local treatment of melanoma in-transit metastasesin limbs by isolated limb perfusion due to live threaten-ing systemic toxicity of therapeutically active TNF120572 doses[53]

In the last decade much progress was achieved dueto the discovery of mutations in the BRAF gene This ledto the development of therapies interfering with RASRAFsignaling and to specific BRAF inhibitors In August 2011an alternative melanoma regimen for patients positive forBRAF mutations was brought into the market with the FDAapproval of Vemurafenib (Zelboraf PlexxikonRoche) InPhase II and III studies Vemurafenib showed a responserate up to 50 yet the response duration varied betweenthe phase studies [54ndash56] In addition Vemurafenib inducesacanthopapillomas keratoacanthomas and cutaneous squa-mous cell carcinomas in the early treatment [57 58] Unfortu-nately these unprecedented response rates are limited by thefact that almost all tumors become resistant to this therapyand the overall survival of patients was 67 months [59]In addition the treatment is only available for 50ndash60 ofpatients with mutated tumors because it is not effective intumors with wildtype BRAF Nevertheless this success hasled to the development of other RASRAFpathway inhibitorsfor example for mutated BRAF or downstream kinases likeMEK Alternative activation of RASRAF pathway has beenproposed as a resistance mechanism [60] In line with thisthe combination of BRAF inhibitionwithMEK inhibition ledto an improved survival of 94 months [61]

Other new therapies that add to the therapeutic optionsformelanoma patients are immunotherapies An anti-CTLA-4 antibody (Ipilimumab) improved survival of stage IIand IV melanoma patients (101 versus 64 months) [62]Cytotoxic T-lymphocyte Antigen 4 (CTLA-4) inhibits T-cell responses and respectively CTLA-4 blockade promotesimmune responses and antitumor activity In an early analysisof anti-PD-L1 antibody a 20 response rate in melanomawas observed Importantly these responses lasted for morethan 1 year [63] Similar to CTLA4 PD-1 reduces immuneactivation and its inhibition can lead to reactivation ofimmune responses


Altogether even with respect to the recent advances inmelanoma therapy the high resistance rates and the restric-tion to certain patient subgroups demonstrate that there isstill an urgent need to develop alternative therapies

4 Assets of Nucleic Acid Nanoparticles inAntitumoral Approaches

As also observed for other tumor entities melanoma treat-ment with low molecular weight chemotherapeutic drugsoften results in the rise of resistant cancers cells especially incase of relapsed disease A well-known mechanism of resis-tance is the elevated expression of multidrug transporter pro-teins like p-glycoprotein which actively pump chemothera-peutics out of the cell [64] Here macromolecular approachescan be a suitable approach to overcome such resistance As anexample the attachment of chemotherapeutics to polymersvia reversible covalent bonds helps to overcome this type ofresistance (for a recent review see [65]) Also biotherapeu-tics such as antibodies have been successfully applied inmelanoma therapy (see above) but also here resistance canoccur for example when blocking of one cellular pathwayresponsible for cancer cell proliferation can be replaced byanother [66] In this case the application of therapeuticallyactive nucleic acids comes into play Firstly they exhibit arelatively high molecular weight which prevents resistancemediated by p-glycoprotein upregulation Secondly nucleicacids can be designed to affect only malignant cells forexample by using promoter elements being only activatedin tumors or as RNA oligonucleotides (like siRNA) whichwill enable the knockdown of a specific protein overexpressedin tumor tissue Furthermore the delivery of more thanone siRNA targeting different pathways can prevent tumorresistance by blocking different resistance or escape strandsLast but not least nucleic acid delivery permits systemicdelivery of toxic agents such as diphtheria toxin A [67] ortumor necrosis factor (TNF) [68] as they only become toxicafter transcription in the target cell

Solid tumors exceeding a certain size rely on a func-tional blood supply for access to nutrients and oxygen Incontrast to nonmalignant tissues tumor vasculature oftenexhibits a leaky appearance which in principle also allowsnanosized particles to reach tumor cells [69] Being packedinto nanoparticles or polyplexes nucleic acids can be pro-tected from nucleases which are present in the blood-stream Nevertheless systemic delivery of nanopharmaceu-tics offers several pitfalls and obstacles such as aggrega-tion with blood cells undesired adherence to the vesselwall or opsonization with plasma proteins followed byclearance through tissue macrophages (a key componentof the reticulo-endothelial system) Blood proteins interactboth with negatively and positively charged nanosystemswhereas a neutral surface charge enables in principle bloodcirculation as it has been shown for small nanocrystalsso called quantum dots [70] Alternatively nanosystemscan be decorated with hydrophilic polymers which owingto their excessive hydration shield the particlesrsquo surfacecharge hereby preventing the aggregation with protein

Table 1 Common melanoma-targeting tools ligands for surfacecellular targeting and promoters for tissue-specific transcription

Targeting tool Target Reference

Ligand

[Nle4 dPhe7]-120572-MSH MC1-R [74ndash85]cRGD 120572v1205733 [86ndash90]LDV 12057211205734 [91]

Transferrin Transferrinreceptor [92]

Promoter Tyrosinase mdash [93ndash95]MIA mdash [96 97]

components From the group of hydrophilic polymers likeN-(2-hydroxypropyl)methacrylamide (HPMA) [71] hydrox-yethyl starch (HES) [72] or polyethyleneglycol (PEG) [73]PEG is the most commonly used one In addition targetingentities can be used to direct the nanocarrier to specific cellsCommonly these are ligands that bind to receptors or othercell surface molecules that are overexpressed in tumor cells

Macromolecular drugs which exceed the renal excretionlimit and are able to circulate in the blood stream canbenefit from the so-called enhanced permeability and reten-tion (EPR) effect nanopharmaceutics accumulate in tumortissue as they can penetrate the leaky vasculature but areretained within the tumor tissue due to incomplete lymphaticdrainage [98] This tumor deposition is a prerequisite forall steps that follow binding to and internalization of theparticles into target cells The latter can be promoted by theincorporation of the earlier mentioned cell-binding ligandsinto the carrier system Figure 2 summarizes the limitationsin nucleic acids delivery the solutions for such limitationsand the therapeutic advantages of nucleic acid nanosystems

5 On the Footsteps of Metastatic MelanomaCell Surface and Transcriptional Targeting

Directed approaches are of special interest as they havethe potential to specifically distress malignant cells caus-ing increased local concentrations of the active agent andavoiding undesired side effects Tracking down melanoma-associated molecular targets involves identifying signalingpathwaysrsquo key players earlier described as much as cancercell surface markers In particular for gene therapy cellsurface markers are important and these abide with theconception of a treatment addressing multiple melanomasubgroupsmdashas cells with different mutations can still exhibitcommon surface markers Ergo it is crucial to identifycritical and idiosyncratic targets for these cells Table 1summarizes the most common melanoma-targeting toolsherein described

Already reported in the early seventies [99] one of thelargely explored targets is the melanocortin-1 receptor (MC1-R) which is also overexpressed in numerousmelanoma casesMC1-R belongs to a class of G-coupled protein receptors(MC1-RndashMC5-R) where the different receptors allocate indifferent tissues reflecting their functions While MC1-R isfound in hair and skin [100] MC2-R is localized in adrenal


Aggregation with blood cells

Unwanted adherence to the vessel wall

Opsonization with plasma proteins

Clearance by macrophages

ChemotherapeuticsNucleic acids

Circumvent conventional resistance mechanisms

Reach tumor cells through EPR effect

Delivery of toxic elements

(eg TNF and DTA)

DNA transcriptional regulation

Specific targeting

Shielding of nanosystems (eg

HPMA PEG and HES)

AdvantagesProblems Solutions

Unspecific uptake

Specific targeting

Figure 2 Advantages and limitations in nucleic acid nanosystems delivery Particular advantages of nucleic acid therapies are (1) theability to include tissue specific targeting (or transcriptional targeting) and (2) the possibility to systemically deliver genes encoding forproteins with toxic properties Moreover as macromolecules nucleic acids can overcome resistance mechanisms such as that supported byp-glycoprotein However nucleic acids are vulnerable in blood circulation and hence they must be protected against enzyme degradationand condensed in the form of polyplexes Physiological barriers such as reticulo-endothelial system still present a threat for nanosystemsand these must be armed against possible interactions with blood cells that can result in opsonization or undesired blood vessel adhesionDecoration of nanocarriers with PEG or HPMA can provide shielding effect while decoration with ligands that can bind receptorsoverexpressed in tumors can assist in cellular targeting and internalization TNF tumor necrosis factor DTA Diphteria toxin A HPMAN-(2-hydroxypropyl)methacrylamide PEG polyethylene glycol HES hydroxyethyl starch

glands [101] whereas MC3-R and MC4-R are in hypothala-mus [102] and MC5-R in kidneys [103] However owing totheir similarity their binding domains may share commonaffinities and certain peptide motifs can bind to severalreceptors [74] For targeting purposes the most well-knownand used MCR-1 ligand is the synthetic [Nle4 D-Phe7]-120572-MSH or NDP-120572-MSH [75] The substitution of methioninein position four by norleucine (Nle4) and of phenylalaninefor its d-counterpart in position seven (d-Phe7) rendersthis peptide with higher affinity and resistance to enzymedegradation than its native form However NDP-120572-MSHwasshown to have a strong nanomolar binding affinity towardsMC3-R MC4-R and MC5-R [74] and for gene deliveryit is crucial to decrease off-target effects Aiming at thedesign of ligands suitable formicelle conjugation andwith anadequate selectivity to MC1-R Barkey et al have conducted acomparative study in which they screened several candidateligands [74] This paper allowed the following conclusions(1) free rotation of carbons that compose the peptidersquosbiding motif seems to be required for MC1-R avidity (2)alkyl modifications for the attachment of triblock polymermicelle at the N-terminal of the peptide did not affectbinding affinity in the short four amino acid peptide (3)for peptides twice as long C-terminal modifications formicellesrsquo attachment did not altered binding affinities In

addition the authors have synthesized micelles conjugatedto the short peptide version [4-phenylbutyril-Hist-dPhe-Arg-Trp-Gly-Lys(hex-5ynoyl)-NH

2] through a PEG linker

And importantly in vitro cell-uptake studies showed theability of conjugated micelles to selectively bind to MC1-R receptor and whether due to multivalent interactionsor other factors the micelles had higher avidity for thereceptor than the ligand alone Nevertheless further studies(ie by flow cytometry or confocal laser microscopy) toquantify the uptake of these conjugated micelles are neededto better evaluate the delivery efficiency of this platformMore recently 120572-MSH peptide has been conjugated to ananoplatform based on the heavy chain of the humanprotein ferritin (HFt) [76] Ferritin can be used to build ahollow nanocage that can transport materials such as Fe

3O4

Co3O4 Mn3O4 Pt and Au and hence be used for imaging

and therapeutic purposes The targeted ferritin nanocageshave been evaluated in vitro and in vivo Unfortunately theauthors have not analyzed the in vivo distribution of theirnanoparticles and the targeting efficiency was evaluated byimmunohistochemistry in the tumor tissue in relation tonormal skin In a similar approach to that of HFt nanocagesLu and collaborators have used hollow gold nanospheresconjugated to NDP-120572-MSH aiming at cancer photothermalablation [77] In this study nude mice were subcutaneously


inoculated with B16F10 murine melanoma cells and thenanoparticles were administered intravenously The authorshave collected different organs and were able to show thetargeting effect by the NDP-120572-MSH-gold nanospheres

Interestingly targeting of MC1-R by 120572-MSH peptidehas been mostly used in radionuclide therapy studies andfor diagnostic purposes Currently 2-[18F]fluoro-2-deoxy-d-glucose (18F-FDG) is the only radioactive probe used in theclinic to detect melanoma Be that as it may 18F-FDG isan unspecific positron emission tomography (PET) imagingagent with poor sensitivity towards micrometastatic sites[78 79] a fact that underlines the general insufficiency inmelanoma targeting

Regarding MC1-R targeting Yubin Miao and Thomas PQuinnrsquos extensive work is of particular interest reportingon two generations of an NDP-120572-MSH-based peptide usedfor melanoma imaging by single-photon emission-computedtomography (SPECT) and more recently by PET Whatdistinguishes the two 120572-MSH peptide generations is mostlythe peptidersquos length being twelve aminoacid-long in thefirst generation (CycMSH) [80ndash82] and six in the second(CycMSHhex) [83 84] In both generations the peptide iscyclized (Cyc) and the MC1-R binding motif (His-dPhe-Arg-Trp) is conserved The peptides have also undergonestructural modifications concerning the aminoacid linkerswhich are used to support the peptide cyclization and bridgethe targeting ligand and the radiometal chelator Interest-ingly the authors have observed that the exchange of singleaminoacids in these linkers [85] and the introduction ofmdashGlyGlymdashlinker between the chelator and the peptide [84]resulted in improved melanoma targeting with decreasedrenal excretion and liver uptake of the radiolabelled peptidein B16F1 melanoma-bearing C57 mice These studies under-score the structural role of the targeting moiety but also ofthe integral component being delivered In other words theaddition of a targeting entity to a carrier does not necessarilysuffice for efficient deliver the number of peptides conjugatedto the delivery platform the site of conjugation and the sizeand type of the linker play an important role

Integrin targeting has also been extensively exploredfor cancer gene delivery in general After the discovery ofadhesion molecules as mediators of tumor metastasis theidentification of their binding motifs opened the possibilitiesfor targeted therapies Several peptide fragments have beenemployed to target these mediators either as antagonists oras ligands for drug delivery purposes One of the utmosttargeted integrin is the 120572v1205733 120572v1205733 plays a central role inangiogenesismdashthe formation of new vesselsmdash and by servingas receptor for extracellular matrix proteins it mediatesmigration of endothelial cells into the basement membraneand regulates their growth survival and differentiation It istherefore no surprise that such integrin is found upregulatedin different tumor cells where it is involved in processes thatgovern metastasis The integrinrsquos binding peptide motif hasbeen identified in 1990 [121]mdashArginine-Glutamine-Aspartateor RGDmdashbut studies that followed have shown that thecyclic version of RGD (cRGD) has higher binding affinitiestowards the integrin [86 87] Either alone or in combination

with other ligands cRGD has been conjugated to severalnanocarriers for both diagnostic and therapeutic purposes[88ndash90]

Another integrin reported to have a dominant functionin the metastatic spread is 120572

41205731or VLA-4 Okumura and

more recently Schlesinger have shown in different settingsthat inhibition of VLA-4 by natalizumab (an antibody against1205724integrin) significantly decreased melanoma lung metas-

tases in murine models [42 44 122] In 1991 Makaremand Humphries have identified the Leucine-Aspartate-Valine(LDV) sequence as the integrinrsquos motif [123] and a fewyears later Vanderslice et al have reported on a series ofcyclized peptides based on LDV that were assayed for theinhibition of the integrin [124] However and despite thenumerous reports relating this agent to tumormetastasis andto melanoma in particular most of the literature relies onthe LDV sequence as an antagonist rather than for deliverpurposes where to our knowledge there is only one paperreporting on in vitro studies [91] Indeed VLA-4 is foundin multiple leukocyte populations VLA-4 is a vital receptorof leukocytes and it is involved in the immune responseHence a systemic application of VLA-4 inhibitors or bindingpeptides could induce undesired partially immunosuppres-sive effects In this context the application of transcriptional-targeting strategies could potentially prevent off-target effectsand prove this ligand a promising tool In fact tissue-specificelements as components of the DNA vector can providea tight control over gene expression and complement andstrengthen targeted-delivery Commonly tumor cellsrsquo surfacemarkers entail receptors that are also present in nontumorcells but are rather overexpressed in their malignant formThis is the case for both the integrins here described butalso the transferrin receptor [92]mdashall used as melanomatargets Therefore off-target effects can occur and for genedelivery purposes tissue-specific control elements are anelegant way to bypass undesired side effects These controlelements consist of nucleic acid sequences that are recognizedby proteins or other nucleic acids which hereby regulategene expression For the case of melanoma tissue specificpromoters have been described and these include tyrosinase[93ndash95] and melanoma inhibitory activity (MIA) [96 97]Gene expression is hence to be accomplished in tissues wheresuch promoters are activated

MicroRNA (miR) binding sites can also serve as tran-scriptional control elements MicroRNAs are a class ofshort (20ndash22 nucleotides long) regulatory RNAs which arebelieved to regulate as many as 30 of all genes SeveralmicroRNAs are tissue-specific and fine-tune genetic circuitssome of which are critical for normal development cellulardifferentiation and normal cellular homeostasis If the targetsequence and microRNA have perfect complementarity themRNA is eliminated by a RNA degradation pathway Inthe context of transcriptional control this means that aDNA vector that contains specific miR-binding sites is onlytranslated in cells where the miR in question is absent [125126] In tumor cells several microRNAs are deregulatedwhile miRs enrolled in cell homeostasis are downregulatedthose involved in cell proliferation and differentiation areupregulated [127] For the case of melanoma miR let-7b


miR-193b miR-34a miR-155 miR-205 miR148 miR-137and miR-152 have been found downregulated (for a reviewon melanoma microRNAs see [127]) and can therefore besuitable targets for transcriptional regulation when expressedin normal tissue

6 Therapeutic Nucleic Acids in Melanoma

As opposed to conventional therapy traditionally that is inthe case of loss of function gene therapy aims at permanentcorrection of a defected or missing gene by replacing with orproviding respectively the corrected versionmdashfor exampleby the introduction of plasmid DNA (pDNA) Ideally thisapproach translates into a single treatment or few initialtreatments rather than several (or life long) required toprovide the patients with the functional form of the proteinHowever this permanent correction treatment has provenvery challenging

In the last twenty years new nucleic acids with attractivetherapeutic properties were discovered notably siRNA andmicroRNAs Small interference RNA (siRNA) has the abilityto specifically silence protein expressionmdashan asset particu-larly valuable for antiviral and cancer regimens In generalalso miRNA negatively regulates gene expression althoughvia two different mechanism depending on the degree ofcomplementarity towards its mRNA target Nucleic acid-based approaches offer several advantages when comparedto treatment with small molecules or proteins They canbe seen as mostly inactive prodrugs which are activated atthe tumor site producing a therapeutically active protein orknocking down a specific target gene Importantly nucleicacid targeted delivery systems preferably also relying intranscriptional targeting decreasing off-target effects andtoxicity and permitting a systemic administration otherwisenot feasible with a therapeutic agent with toxic properties

In parallel with new therapeutic nucleic acid tools the lasttwo decades brought insight into tumorgenesis in general andunveiled a plethora of therapeutic concepts against cancer(Figure 3) The following paragraphs will deal with differentantimelanoma approaches based on nucleic acids

Despite the apparent tumor tolerance humoral andcellular immune responses are naturally generated againsttumor antigens Hence whether the tumor grows as a resultof stealth and nonrecognition or as the result of escapeand immunological shaping [128] its recognition by theimmune system can still be prompted Indeed at a laterstage during the progressive growth phase tumors maybecome more immune-activating for varies reasons damageor disruption of surrounding tissue generation of reactiveoxygen species upregulation of stress protective factors ordeath by necrosis or apoptosis However at this stage it isnot known whether the tumor still needs to escape immunerecognition as it is unclear that these immune responsescan cause tumor destruction [128] Therefore a number ofstudies have focused in eliciting earlier and suitable tumorrecognition by the immune system In a nucleic acid therapycontext this transliterates into genetic immunization orDNAvaccination the delivery and transcription of a gene encoding

antigens or immunestimulatory molecules that elicit animmune response As an example interleukine-12 (IL-12) hasbeen used and studied in different animal models [104 105]IL-12 is originally produced by mononuclear phagocytes anddendritic cells and is responsible for activating NK and CD4+T cells and inducing the production of high levels of inter-feron gamma (INF-120574) Interestingly IL-12 has been describedto increase antitumor immune responses [129 130] and laterstudies investigated its suitability for aDNAvaccine approachagainst melanoma [106] IL-12 effects appeared to be longlasting and efficient against tumor metastases although notmainly mediated by INF-120574 [106] The murine studies alsorevealed moderate toxicity caused by IL-12 and while lowerIL-12-encoding pDNA doses can be administered ideallythe gene expression should be controlled regarding thetissue and the durability of the expression Although DNAvaccination against a strongmelanoma tumor antigen shouldbe possible the authors have not seen an effect on lungmetastases when using melanoma-associated glycoprotein100 (gp 100)pmel17 pDNA alone Adjuvants appear to benecessary for a successful DNA vaccination the authors haveseen an effect when the gp 100-pDNA was administeredtogether with IL-12 similar to other murine study wheregranulocyte-macrophage colony-stimulating factor was used[107] Alternatively in a canine study the developed vaccinewas based on the human (rather than canine) gp 100 protein[108] where the human form of the antigen acted as adjuvantTogether with gp 100 and for the case of melanoma twomore tumor genes have been described for DNA vaccinationMART-1 and tyrosinase [108 109]

Also the expression of chemokines such as monocytechemoattractant protein-1 (MCP-1) and interferon-inducibleprotein-10 (IP-10) can mediate an immune response Inparticular IP-10 as been described by Sgadari et al as anantitumor agent and found to promote damage in establishedtumor vasculature as well as tissue necrosis in a murinemodel for the human Burkitt lymphomas [131] Based on thisand after their studies with IL-12 Keyser and collaboratorshave investigated the efficiency of IP-10-encoding pDNAtherapy in murine melanoma models [110] The authorshave used two murine tumor models whereupon cells havebeen injected subcutaneously (originating a solid tumor) orintravenously inducing lungmetastases When administeredalone and intramuscularly (resulting in systemic circula-tion) IP-10-encoding pDNA showed an antimetastatic effectreducing the number of lung metastases as compared to thecontrol-pDNA treated groupWhen administeredwith IL-12-encoding pDNA IP-10 pDNA enhanced the IL-12 effect anddecreased its earlier observed toxicity This anti-neoplasticeffect of IP-10 has been attributed to the engagement of NKcells and the inhibition of angiogenesis and cell proliferation

Alternative antitumor strategies aim at a direct destruc-tion of cancer cells through the delivery of pDNA encodingfor a toxic proteinmdashDNA-based strategies This is referred toas a suicide gene therapy or gene-directed enzyme prodrugtherapy (GDEPT) when the nucleic acid sequence encodesfor an enzyme which is not directly toxic but instead convertsa nontoxic prodrug into a cytotoxicmetaboliteThefirst proofof principle of GDEPT was presented in the mid-eighties and


DNA tumorantigen

expression (eggp100 and tyrosinase)

DNA based RNA based

Immune response

Suicide gene therapy

Cancer immunotherapy

DNA vaccination

Off

Gene silencing

Tumor progression

Tumor cell death

DNA vaccination based

On OnOnOffdsRNA mimic

pIC

On

Autophagy and apoptosis

Endolysosomal

machinery

Proliferationinvasion

factors (egPAR-1

N-cadherinand Bcl-2)

DNA cytokines(eg IL-12 and IP-10)

Toxic proteins(eg HSV-tk and DTA)

Figure 3 Different strategies used in antitumor nucleic acid approaches RNA-based strategies are commonly used to downregulate agentsthat are upregulated to favor cell proliferation or migration such as Bcl-2 Alternatively double stranded RNA (dsRNA) mimic polyinosinic-polycytidylic acid (pIC) can be used to engage the endosomal machinery resulting in autophagy and apoptosis Conversely pDNA deliveryaims at the expression of a protein that can (1) have toxic properties directly causing tumor cell apoptosis (pDNA-based approaches) (2)be a chemokine thus recruiting cell-mediated immunity or (3) be a tumor antigen recruiting humoral immunity (DNA vaccination-basedstrategies) Ultimately all strategies aim at putting an end to tumor progression and eventually tumor cell destruction

involved the herpes simplex thymidine kinase (HSV-tk) andthe prodrug ganciclovir (GCV) [132] Presently HSV-tk aswell as other approaches such as Diptheria toxin A chain(DTA) have been employed in the clinics themost successfulcases being reported in ovarian and prostate cancers [67133] As for melanoma treatments HSV-tk has been themost commonly used [111ndash113] although there is no humanclinical trial yet Suicide gene therapy has also been proveneffective when used in combined approaches such as withcytokine-enhanced vaccine in a clinical trial involving caninemelanoma patients [134] Despite promising this strategy iscurrently restrained by a poor delivery most nanocarriersare not as target-specific and efficient as required and thetoxic gene does not reach the tumor cells in efficaciousconcentrations

A number of studies have instead focused onmediators ofcell proliferation and differentiation which are upregulatedduring tumorgenesis aiming at their downregulation bymeans of siRNA delivery [114 135ndash137]mdashthese are RNA-based approaches As an example based on the fact that inepithelial cells N-cadherin induces changes in morphologyof a fibroblastic phenotype (rendering the cells more motileand invasive) the laboratory of Laidler has investigated theoutcome of N-cadherin silencing in human melanoma celllines [114] Although the results suggest that N-cadherinpositively affects the regulation of the cell cycle and pro-liferation through activation of the AKT kinase pathway

further investigations are needed to describe the mecha-nism Similarly Villares et al upon the observation thatthrombin receptor (or protease-activated receptor-1 PAR-1)is overexpressed in highlymetastaticmelanoma cell lines hasevaluated the therapeutic potential of siRNA against PAR-1[115] The authors have observed a significant reduction ofin vivo tumor growth as well as in the number of metastaticlung colonies This report showed that downregulation ofPAR-1 decreased the expression of matrix metallopeptidase-2 (MMP-2) interleukin 8 (IL-8) and vascular endothelialgrowth factor (VEGF) resulting in an overall decrease inangiogenesis and blood vessels In 2010 Davis et al reportedon the first human clinical trial (including three melanomapatients) on siRNA therapy against melanoma [92] ThesiRNA targeted the M2 subunit of ribonucleotide reductase(RRM2) and the protein knock down was confirmed at themRNA level but not corroborated to the same extend by theprotein analysis Nevertheless the fact that the authors useda delivery vector targeting the transferrin receptor withoutshowing analysis of such receptor expression in melanomacells was left to be explained [138]

Of special interests are combinatorial strategies involvingsiRNA delivery as these similar to other combinatorialtherapies cause the most significant outcomes Particu-larly Poeck and coauthors have used a simple and elegantsiRNA design [116] The authors targeted Bcl2 (an apoptosisregulator protein) which was reported to play a central


role in the resistance of melanoma cells to chemotherapy[7 116 139 140] By adding 51015840-triphosphate ends to theirsiRNA the authors also activated innate immune cellsinduced the expression of interferons and caused specificcell tumor apoptosis These actions are a consequence ofthe recognition of 51015840-triphosphate ends by the cytosolicretinoic acid-induced protein-1 (Rig-1) and synergized withthe silencing effects originated from siRNA resulting inmassive tumor destruction in the murine lung metastasesTwo years earlier aiming at RNA-based vaccination Tormoet al first reported on a promising double stranded RNA(dsRNA) mimic polyinisine-polycytidylic acid (pIC) [117]Importantly the therapeutic effect of the dsRNA was sig-nificantly increased when delivered in the form of a com-plex together with polyethyleneimine (PEI)-[pIC]PEI Ini-tially the dsRNA mimic was thought to engage toll-likereceptors (TLR) hereby mediating cellular tumor immunity[117] In turn further investigation studies showed that itmobilizes the endolysosomal machinery of melanoma cellsand through melanoma differentiation associated gene-5(MDA-5) induces self-degradation by (macro) autophagyand apoptosis following the MDA-5-mediated activationof proapoptotic factor NOXA [118] Interestingly at theexact same time MDA-5 and NOXA were also reported toplay a role in interferon-independent apoptosis in humanmelanoma cells by Besch and collaborators [141] Not onlywere these findings meaningful opening new windows forcancer therapy but also in particular in the Damıa Tormostudies was the murine model used very suited whereuponmice overexpressing hepatocyte growth factor (HGF) andcarrying an oncogenic mutation in the cyclin-dependentkinase-4 [(CDK4)R24C] developed invasive melanomas in theskin following neonatal exposure to carcinogenics

While a number of microRNA has been described toplay relevant roles in melanoma progression [127] only fewin vitro studies have reported on the miRNA potential forantimelanoma therapy [119 120] However pertinent ther-apeutic approaches targeting miRNAs described for othertumor types [142 143] foretell the potential and the thera-peutic window opportunities entailing these nucleic acids inmetastatic melanoma

As an overview of this section Table 2 presents thetherapeutic nucleic acids herein described and Figure 3schematically summarizes the different strategies in nucleicacid therapies

7 Conclusions and Future Perspectives

It is of general consensus that the last decade of cancerresearch significantly expanded our knowledge in tumordevelopment and progression Unfortunatelymdashsimilar to thetumor escape shaped by the immune surveillance in an earlygrowth phasemdashas new therapeutic strategies are appliedtumor cells undergo another round of selection giving riseto therapy-resistant cells It is therefore necessary to combineseveral approaches to attack different paths of tumor escapemdasha fact that is confirmed by the most significant resultsreported in studies where such strategies have been used

Table 2 Different therapeutic strategies againstmelanoma based onnucleic acids In the case of DNA-based approaches a therapeuticgene is delivered to induce a beneficial effect whereas with RNAbased generally the regimen is based on silencing of a tumor-active gene dsRNA mimetic pIC is as yet a recent and uniquefinding based on polyinosine-polycytidylic acid (pIC) complexedwith polyethyleneimine (PEI) that induces tumor cell autophagy andapoptosis As for the case of micro RNAs (miR) only few in vitrostudies have been conducted showing the therapeutic potential ofthe delivery of miRs that were found downregulated in tumor cells

Therapeuticsilencedupregulated gene Reference

DNA-based approaches

IL-12 [104ndash106]gp100 [107 108]

MART-1 [108]Tyrosinase [109]

IP-10 [110]HSV-tk [111ndash113]

N-Cadherin [114]PAR-1 [115]

RNA-based approaches RRM2 [92]Bcl2 [116]

dsRNA pIC [117 118]

miR Let-7b and miR 199a [119 120]

On this note nucleic acids deliveries are truly advantageoustools as they allow the systemic delivery of potentially toxicmolecules that can be combined with chemotherapy aimingat terminating possible resistant-tumor cells As an examplerecently Su and collaborators have reported on an antitumorstrategy combining TNF-encoding pDNA and chemotherapy[68] While systemically administered TNF is extremelytoxic in its genetic form and when reaching specific targetcells TNF revealed to be a powerful antitumor agent Specificand efficient are indeed key words in this type of targetedapproaches as in suicide gene delivery It is thus of extremeimportance to thoroughly evaluate the target options and toverify the levels of the target molecule in the cells of interestThe activation of possible target-receptors may be desiredsuch as in the case reported by Poeck et al [116] but onlywhen not hampering the therapeutic effect by activation ofpathways that can lead to cell proliferationdifferentiationenhanced cell migration or inhibition of apoptosis Asdescribed by Schafer et al this can be the case when targetingthe epidermal growth factor receptor (EGFR) and it isthen desirable to design a ligand that targets the receptorcircumventing its activation [144] On the other hand therelevance of analyzing the targeted receptor has been wellexposed in the short letter of Perris in response to thework published by Davis et al [138] To avoid other pitfallsin nanovector development also the in vivo distributionneeds to be assessed preferably by several approaches (egbioluminescence imaging positron emission tomography(PET) and magnetic resonance imaging (MRI)) To thisend immunohistochemistry studiesmay be suitable and very


convenient to corroborate and support data collected bydifferent means but also microscopy (mostly in vitro but alsohistochemistry analysis) has had its traps [145]

In summary already a number of promising nucleicacid strategies exist and these certainly present less hurdlesfor delivery than their protein counterpart as they aresmaller less antigenic and can bypass certain resistancemechanismsNevertheless further improvements in nonviraltargeted delivery appear required to increase the efficacy ofsuch therapies A small final note regarding the potentialof miRNA approaches microRNA therapies can aim at(1) miRNA upregulation when the target nucleic acid isenrolled in cell homeostasis and is found silenced in tumorcells (2) miRNA downregulation by antimiRs when it isupregulated in tumor cells due to its play in cell proliferation(3) alternatively miRNA can also have a role in cell-specifictranscription in pDNA vectors containing miRNA binding-sites allowing the expression of the gene of interest in cellswhere the miRNA is silenced All these assets make miRNAundoubtedly a very elegant and flexible tool


The authors state no conflict of interests

Acknowledgments

J R Viola was supported by a postdoctoral fellowship fromBayerischen Forschungsstiftung (PDOK-78-11) and there-after from Frauenbeauftragte at LMU D F Rafael wassupported by a doctoral fellowship of the Portuguese ScienceFoundation FCT (SFRHBD762702011)

References

[1] C M Balch J E Gershenwald S J Soong et al ldquoFinal versionof 2009 AJCC melanoma staging and classificationrdquo Journal ofClinical Oncology vol 27 no 36 pp 6199ndash6206 2009

[2] W H Clark Jr D E Elder D Guerry M N Epstein MH Greene and M van Horn ldquoA study of tumor progressionthe precursor lesions of superficial spreading and nodularmelanomardquo Human Pathology vol 15 no 12 pp 1147ndash11651984

[3] K Satyamoorthy and M Herlyn ldquoCellular and molecularbiology of human melanomardquo Cancer Biology andTherapy vol1 no 1 pp 14ndash17 2002

[4] A K Mobley R R Braeuer T Kamiya E Shoshan andM Bar-Eli ldquoDriving transcriptional regulators in melanomametastasisrdquo Cancer and Metastasis Reviews vol 31 no 3-4 pp621ndash632 2012

[5] H Tsao L Chin L A Garraway and D E Fisher ldquoMelanomafrom mutations to medicinerdquo Genes and Development vol 26pp 1131ndash1155 2012

[6] T Kuilman C Michaloglou W J Mooi and D S Peeper ldquoTheessence of senescencerdquo Genes and Development vol 24 no 22pp 2463ndash2479 2010

[7] A J Miller and M C Mihm Jr ldquoMelanomardquoThe New EnglandJournal of Medicine vol 355 no 1 pp 51ndash65 2006

[8] E Hodis I R Watson G V Kryukov et al ldquoA landscape ofdrivermutations inmelanomardquoCell vol 150 no 2 pp 251ndash2632012

[9] H Davies G R Bignell C Cox et al ldquoMutations of the BRAFgene in human cancerrdquo Nature vol 417 no 6892 pp 949ndash9542002

[10] M C Leslie and M Bar-Eli ldquoRegulation of gene expression inmelanoma new approaches for treatmentrdquo Journal of CellularBiochemistry vol 94 no 1 pp 25ndash38 2005

[11] P M Pollock K Cohen-Solal R Sood et al ldquoMelanomamouse model implicates metabotropic glutamate signaling inmelanocytic neoplasiardquo Nature Genetics vol 34 no 1 pp 108ndash112 2003

[12] R Kumar S Angelini E Snellman and K Hemminki ldquoBRAFmutations are common somatic events in melanocytic nevirdquoJournal of Investigative Dermatology vol 122 no 2 pp 342ndash3482004

[13] R Di Micco M Fumagalli A Cicalese et al ldquoOncogene-induced senescence is a DNA damage response triggered byDNAhyper-replicationrdquoNature vol 444 no 7119 pp 638ndash6422006

[14] E E Patton H R Widlund J L Kutok et al ldquoBRAFmutationsare sufficient to promote nevi formation and cooperate with p53in the genesis of melanomardquo Current Biology vol 15 no 3 pp249ndash254 2005

[15] P M Pollock G J Walker J M Glendening et al ldquoPTENinactivation is rare in melanoma tumours but occurs frequentlyin melanoma cell linesrdquo Melanoma Research vol 12 no 6 pp565ndash575 2002

[16] V K Goel A J F Lazar C L Warneke M S Redston and FG Haluska ldquoExamination of mutations in BRAF NRAS andPTEN in primary cutaneousmelanomardquo Journal of InvestigativeDermatology vol 126 no 1 pp 154ndash160 2006

[17] D Dankort D P Curley R A Cartlidge et al ldquoBrafV600Ecooperates with Pten loss to induce metastatic melanomardquoNature Genetics vol 41 no 5 pp 544ndash552 2009

[18] S Gandini F Sera M S Cattaruzza et al ldquoMeta-analysis ofrisk factors for cutaneousmelanoma III Family history actinicdamage and phenotypic factorsrdquo European Journal of Cancervol 41 no 14 pp 2040ndash2059 2005

[19] L A Cannon-Albright D E Goldgar L J Meyer et al ldquoAssign-ment of a locus for familial melanoma MLM to chromosome9p13-p22rdquo Science vol 258 no 5085 pp 1148ndash1152 1992

[20] M Serrano G J Hannon and D Beach ldquoA new regulatorymotif in cell-cycle control causing specific inhibition of cyclinDCDK4rdquo Nature vol 366 no 6456 pp 704ndash707 1993

[21] T Kamijo J D Weber G Zambetti F Zindy M F Rousseland C J Sherr ldquoFunctional and physical interactions of theARF tumor suppressor with p53 and Mdm2rdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 95 no 14 pp 8292ndash8297 1998

[22] J Koh G H Enders B D Dynlacht and E Harlow ldquoTumour-derived p16 alleles encoding proteins defective in cell-cycleinhibitionrdquo Nature vol 375 no 6531 pp 506ndash510 1995

[23] H Tsao E Benoit A J Sober C Thiele and F G HaluskaldquoNovel mutations in the p16CDKN2A binding region of thecyclin-dependent kinase-4 generdquo Cancer Research vol 58 no1 pp 109ndash113 1998

[24] A Molven M B Grimstvedt S J Steine et al ldquoA large Nor-wegian family with inherited malignant melanoma multipleatypical nevi and CDK4 mutationrdquo Genes Chromosomes andCancer vol 44 no 1 pp 10ndash18 2005


[25] D T Bishop F Demenais A M Goldstein et al ldquoGeograph-ical variation in the penetrance of CDKN2A mutations formelanomardquo Journal of the National Cancer Institute vol 94 no12 pp 894ndash903 2002

[26] J B Tatro Z Wen M L Entwistle et al ldquoInteraction ofan 120572-melanocyte-stimulating hormone-diphtheria toxin fusionprotein with melanotropin receptors in human melanomametastasesrdquoCancer Research vol 52 no 9 pp 2545ndash2548 1992

[27] C Dessinioti A J Stratigos D Rigopoulos and A D Kat-sambas ldquoA review of genetic disorders of hypopigmentationlessons learned from the biology of melanocytesrdquo ExperimentalDermatology vol 18 no 9 pp 741ndash749 2009

[28] V Chhajlani and J E S Wikberg ldquoMolecular cloning andexpression of the human melanocyte stimulating hormonereceptor cDNArdquo FEBS Letters vol 309 no 3 pp 417ndash420 1992

[29] K G Mountjoy L S Robbins M T Mortrud and R D ConeldquoThe cloning of a family of genes that encode the melanocortinreceptorsrdquo Science vol 257 no 5074 pp 1248ndash1251 1992

[30] C Kennedy J ter Huurne M Berkhout et al ldquoMelanocortin 1receptor (MC1R) gene variants are associated with an increasedrisk for cutaneous melanoma which is largely independent ofskin type and hair colorrdquo Journal of Investigative Dermatologyvol 117 no 2 pp 294ndash300 2001

[31] C Bertolotto F Lesueur S Giuliano et al ldquoA SUMOylation-defective MITF germline mutation predisposes to melanomaand renal carcinomardquoNature vol 480 no 7375 pp 94ndash98 2011

[32] S Yokoyama S L Woods G M Boyle et al ldquoA novel recur-rent mutation in MITF predisposes to familial and sporadicmelanomardquo Nature vol 480 no 7375 pp 99ndash103 2011

[33] A R Jeffs A C Glover L J Slobbe et al ldquoA gene expressionsignature of invasive potential in metastatic melanoma cellsrdquoPloS ONE vol 4 no 12 Article ID e8461 2009

[34] Y Cheli S Guiliano T Botton et al ldquoMitf is the key molecularswitch between mouse or human melanoma initiating cells andtheir differentiated progenyrdquoOncogene vol 30 no 20 pp 2307ndash2318 2011

[35] C S Tellez D W Davis V G Prieto et al ldquoQuantitativeanalysis of melanocytic tissue array reveals inverse correlationbetween activator protein-2120572 and protease-activated receptor-1expression during melanoma progressionrdquo Journal of Investiga-tive Dermatology vol 127 no 2 pp 387ndash393 2007

[36] A J Berger D W Davis C Tellez et al ldquoAutomated quanti-tative analysis of activator protein-2120572 subcellular expression inmelanoma tissue microarrays correlates with survival predic-tionrdquo Cancer Research vol 65 no 23 pp 11185ndash11192 2005

[37] D Jean J E Gershenwald S Huang et al ldquoLoss of AP-2 resultsin up-regulation of MCAMMUC18 and an increase in tumorgrowth and metastasis of human melanoma cellsrdquo The Journalof Biological Chemistry vol 273 no 26 pp 16501ndash16508 1998

[38] K Yamamoto A Tojo N Aoki andM Shibuya ldquoCharacteriza-tion of the promoter region of the human c-kit proto-oncogenerdquoJapanese Journal of Cancer Research vol 84 no 11 pp 1136ndash1144 1993

[39] C Tellez M McCarty M Ruiz and M Bar-Eli ldquoLoss of acti-vator protein-2120572 results in overexpression of protease-activatedreceptor-1 and correlates with the malignant phenotype ofhumanmelanomardquoThe Journal of Biological Chemistry vol 278no 47 pp 46632ndash46642 2003

[40] V O Melnikova G J Villares and M Bar-Eli ldquoEmergingroles of PAR-1 and PAFR in melanoma metastasisrdquo CancerMicroenvironment vol 1 no 1 pp 103ndash111 2008

[41] YMori N ShimizuM Dallas et al ldquoAnti-1205724 integrin antibodysuppresses the development of multiple myeloma and associ-ated osteoclastic osteolysisrdquo Blood vol 104 no 7 pp 2149ndash21542004

[42] H Okahara H Yagita K Miyake and K Okumura ldquoInvolve-ment of very late activation antigen 4 (VLA-4) and vascularcell adhesion molecule 1 (VCAM-1) in tumor necrosis factor 120572enhancement of experimental metastasisrdquoCancer Research vol54 no 12 pp 3233ndash3236 1994

[43] J Fritzsche D Simonis and G Bendas ldquoMelanoma cell adhe-sion can be blocked by heparin in vitro suggestion of VLA-4as a novel target for antimetastatic approachesrdquoThrombosis andHaemostasis vol 100 no 6 pp 1166ndash1175 2008

[44] M Schlesinger P Schmitz R Zeisig et al ldquoThe inhibition ofthe integrin VLA-4 in MV3 melanoma cell binding by non-anticoagulant heparin derivativesrdquo Thrombosis Research vol129 no 5 pp 603ndash610 2012

[45] S Liang and C Dong ldquoIntegrin VLA-4 enhances sialyl-Lewisxa-negative melanoma adhesion to and extravasationthrough the endothelium under low flow conditionsrdquo TheAmerican Journal of Physiology vol 295 no 3 pp C701ndashC7072008

[46] A Garofalo R G S Chirivi C Foglieni et al ldquoInvolvement ofthe very late antigen 4 integrin on melanoma in interleukin 1-augmented experimental metastasesrdquo Cancer Research vol 55no 2 pp 414ndash419 1995

[47] D Schadendorf J Heidel C Gawlik L Suter and B MCzarnetzki ldquoAssociation with clinical outcome of expression ofVLA-4 in primary cutaneous malignant melanoma as well as P-selectin and E-selectin on intratumoral vesselsrdquo Journal of theNational Cancer Institute vol 87 no 5 pp 366ndash371 1995

[48] F Spagnolo and P Queirolo ldquoUpcoming strategies for thetreatment of metastatic melanomardquo Archives of DermatologicalResearch vol 304 no 3 pp 177ndash184 2012

[49] E Atallah and L Flaherty ldquoTreatment of metastatic malignantmelanomardquo Current Treatment Options in Oncology vol 6 no3 pp 185ndash193 2005


[51] A P Algazi C W Soon and A I Daud ldquoTreatment of cuta-neous melanoma current approaches and future prospectsrdquoCancerManagement andResearch vol 2 no 1 pp 197ndash211 2010

[52] M B Atkins M T Lotze J P Dutcher et al ldquoHigh-doserecombinant interleukin 2 therapy for patients with metastaticmelanoma analysis of 270 patients treated between 1985 and1993rdquo Journal of Clinical Oncology vol 17 no 7 pp 2105ndash21161999

[53] J P Deroose A M Eggermont A N van Geel J H de Wilt JW Burger and C Verhoef ldquo20 years experience of TNF-basedisolated limb perfusion for in-transit melanoma metastasesTNF dose mattersrdquo Annals of Surgical Oncology vol 19 no 2pp 627ndash635 2012

[54] K T Flaherty I Puzanov K B Kim et al ldquoInhibition ofmutated activated BRAF in metastatic melanomardquo The NewEngland Journal of Medicine vol 363 no 9 pp 809ndash819 2010

[55] R A Kefford H Arkenau M P Brown et al ldquoPhase III studyof GSK2118436 a selective inhibitor of oncogenic mutant BRAFkinase in patients with metastatic melanoma and other solidtumorsrdquo Journal of Clinical Oncology vol 28 abstract no 85032010


[56] G V Long R F Kefford P Carr et al ldquoPhase 12 study ofGSK2118436 a selective inhibitor of V600 mutant (mut) BRAFkinase evidence of activity in melanoma brain metastases(mets)rdquo Annals of Oncology vol 21 Suppl 8 Article ID viii122010

[57] P A Oberholzer D Kee P Dziunycz et al ldquoRAS mutationsare associated with the development of cutaneous squamouscell tumors in patients treated with RAF inhibitorsrdquo Journal ofClinical Oncology vol 30 no 3 pp 316ndash321 2012

[58] F Su A Viros C Milagre et al et al ldquoRAS mutations incutaneous squamous-cell carcinomas in patients treated withBRAF inhibitorsrdquo The New England Journal of Medicine vol366 pp 207ndash215 2012

[59] J A Sosman K B Kim L Schuchter et al ldquoSurvival in BRAFV600-mutant advanced melanoma treated with vemurafenibrdquoThe New England Journal of Medicine vol 366 pp 707ndash7142012

[60] R Nazarian H Shi Q Wang et al ldquoMelanomas acquireresistance to B-RAF(V600E) inhibition by RTK or N-RASupregulationrdquo Nature vol 468 no 7326 pp 973ndash977 2010

[61] K T Flaherty J R Infante A Daud et al et al ldquoCombinedBRAF and MEK inhibition in melanoma with BRAF V600mutationsrdquo The New England Journal of Medicine vol 367 pp1694ndash1703 2012

[62] F S Hodi S J OrsquoDay D F McDermott et al ldquoImproved sur-vival with ipilimumab in patients with metastatic melanomardquoThe New England Journal of Medicine vol 363 no 8 pp 711ndash723 2010

[63] J R Brahmer S S Tykodi L Q Chow et al ldquoSafety and activityof anti-PD-L1 antibody in patients with advanced cancerrdquo TheNew England Journal of Medicine vol 366 pp 2455ndash2465 2012

[64] K G Chen J C Valencia J P Gillet V J Hearing and M MGottesman ldquoInvolvement of ABC transporters in melanogene-sis and the development of multidrug resistance of melanomardquoPigment Cell andMelanomaResearch vol 22 no 6 pp 740ndash7492009

[65] F Canal J Sanchis and M J Vicent ldquoPolymermdashdrug conju-gates as nano-sized medicinesrdquo Current Opinion in Biotechnol-ogy vol 22 no 6 pp 894ndash900 2011

[66] I Helfrich I Scheffrahn S Bartling et al ldquoResistance toantiangiogenic therapy is directed by vascular phenotype vesselstabilization and maturation in malignant melanomardquo Journalof Experimental Medicine vol 207 no 3 pp 491ndash503 2010

[67] S O Freytag H Stricker J Peabody et al ldquoFive-year follow-upof trial of replication-competent adenovirus-mediated suicidegene therapy for treatment of prostate cancerrdquo Molecular Ther-apy vol 15 no 3 pp 636ndash642 2007

[68] B Su A Cengizeroglu K Farkasova et al ldquoSystemic TNF120572gene therapy synergizes with liposomal doxorubicine in thetreatment of metastatic cancerrdquo Molecular Therapy vol 21 no2 pp 300ndash208 2013

[69] F Yuan M Dellian D Fukumura et al ldquoVascular permeabilityin a human tumor xenograft molecular size dependence andcutoff sizerdquo Cancer Research vol 55 no 17 pp 3752ndash3756 1995

[70] H SooChoiW Liu PMisra et al ldquoRenal clearance of quantumdotsrdquo Nature Biotechnology vol 25 no 10 pp 1165ndash1170 2007

[71] D OupickyM Ogris K A Howard P R Dash K Ulbrich andL W Seymour ldquoImportance of lateral and steric stabilizationof polyelectrolyte gene delivery vectors for extended systemiccirculationrdquoMolecularTherapy vol 5 no 4 pp 463ndash472 2002

[72] M Noga D Edinger W Rodl E Wagner G Winter and ABesheer ldquoControlled shielding and deshielding of gene deliverypolyplexes using hydroxyethyl starch (HES) and 120572-amylaserdquoJournal of Controlled Release vol 159 no 1 pp 92ndash103 2012

[73] Z Amoozgar and Y Yeo ldquoRecent advances in stealth coatingof nanoparticle drug delivery systemsrdquo Wiley InterdisciplinaryReviews Nanomedicine andNanobiotechnology vol 4 no 2 pp219ndash233 2012

[74] N M Barkey N K Tafreshi J S Josan et al ldquoDevelopmentof melanoma-targeted polymer micelles by conjugation of amelanocortin 1 receptor (MC1R) specific ligandrdquo Journal ofMedicinal Chemistry vol 54 no 23 pp 8078ndash8084 2011

[75] T K Sawyer P J Sanfilippo V J Hruby et al ldquo4-Norleucine 7-d-phenylalanine-120572-melanocyte-stimulating hormone a highlypotent 120572-melanotropin with ultralong biological activityrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 77 no 10 pp 5754ndash5758 1980

[76] L Vannucci E Falvo M Fornara et al ldquoSelective targetingof melanoma by PEG-masked protein-based multifunctionalnanoparticlesrdquo International Journal of Nanomedicine vol 7 pp1489ndash1509 2012

[77] W Lu C Xiong G Zhang et al ldquoTargeted photothermalablation of murine melanomas with melanocyte-stimulatinghormone analogmdashconjugated hollow gold nanospheresrdquo Clini-cal Cancer Research vol 15 no 3 pp 876ndash886 2009

[78] WDHolder Jr R LWhite Jr J H Zuger E J Easton Jr and FL Greene ldquoEffectiveness of positron emission tomography forthe detection of melanoma metastasesrdquo Annals of Surgery vol227 no 5 pp 764ndash771 1998

[79] B Krug A S Pirson R Crott and T V Borght ldquoThe diagnosticaccuracy of 18F-FDG PET in cutaneous malignant melanomardquoEuropean Journal of Nuclear Medicine and Molecular Imagingvol 37 no 7 pp 1434ndash1435 2010

[80] H Guo N Shenoy B M Gershman J Yang L A Sklarand Y Miao ldquoMetastatic melanoma imaging with an 111In-labeled lactam bridge-cyclized 120572-melanocyte-stimulating hor-mone peptiderdquo Nuclear Medicine and Biology vol 36 no 3 pp267ndash276 2009

[81] H Guo J Yang F Gallazzi E R Prossnitz L A Sklar andY Miao ldquoEffect of DOTA position on melanoma targetingand pharmacokinetic properties of 111In-labeled lactam bridge-cyclized 120572-melanocyte stimulating hormone peptiderdquo Biocon-jugate Chemistry vol 20 no 11 pp 2162ndash2168 2009

[82] Y Miao F Gallazzi H Guo and T P Quinn ldquo111In-labeled lac-tam bridge-cyclized 120572-melanocyte stimulating hormone pep-tide analogues formelanoma imagingrdquo Bioconjugate Chemistryvol 19 no 2 pp 539ndash547 2008

[83] H Guo J Yang F Gallazzi and Y Miao ldquoReduction of thering size of radiolabeled lactambridge-cyclized120572-MSHpeptideresulting in enhanced melanoma uptakerdquo Journal of NuclearMedicine vol 51 no 3 pp 418ndash426 2010

[84] H Guo J Yang F Gallazzi and Y Miao ldquoEffects of the aminoacid linkers on the melanoma-targeting and pharmacokineticproperties of 111In-labeled lactam bridge-cyclized 120572-MSH pep-tidesrdquo Journal of Nuclear Medicine vol 52 no 4 pp 608ndash6162011

[85] J Yang H Guo R S Padilla M Berwick and Y MiaoldquoReplacement of the Lys linker with an Arg linker resultingin improved melanoma uptake and reduced renal uptake ofTc-99m-labeled Arg-Gly-Asp-conjugated 120572-melanocyte stim-ulating hormone hybrid peptiderdquo Bioorganic and MedicinalChemistry vol 18 no 18 pp 6695ndash6700 2010


[86] M A Dechantsreiter E Planker B Matha et al ldquoN-methylatedcyclic RGD peptides as highly active and selective 120572(v)1205733integrin antagonistsrdquo Journal of Medicinal Chemistry vol 42no 16 pp 3033ndash3040 1999

[87] P L Barker S Bullens S Bunting et al ldquoCyclic RGD peptideanalogues as antiplatelet antithromboticsrdquo Journal of MedicinalChemistry vol 35 no 11 pp 2040ndash2048 1992

[88] J Yang H Guo F Gallazzi M Berwick R S Padilla andY Miao ldquoEvaluation of a novel Arg-Gly-Asp-conjugated 120572-melanocyte stimulating hormone hybrid peptide for potentialmelanoma therapyrdquo Bioconjugate Chemistry vol 20 no 8 pp1634ndash1642 2009

[89] MUchidaM L FlennikenMAllen et al ldquoTargeting of cancercells with ferrimagnetic ferritin cage nanoparticlesrdquo Journal ofthe American Chemical Society vol 128 no 51 pp 16626ndash166332006

[90] F Bianchini N Cini A Trabocchi et al ldquo(1)(2)(5)I-radi-olabeled morpholine-containing arginine-glycine-aspartate(RGD) ligand of 120572v120573(3) integrin as a molecular imaging probefor angiogenesisrdquo 2012Journal of Medicinal Chemistry vol 55pp 5024ndash5033

[91] S Zhong S Bhattacharya W Chan B Jasti and X LildquoLeucine-aspartic acid-valine sequence as targeting ligand anddrug carrier for doxorubicin delivery to melanoma cells invitro cellular uptake and cytotoxicity studiesrdquo PharmaceuticalResearch vol 26 no 12 pp 2578ndash2587 2009

[92] M E Davis J E Zuckerman C H J Choi et al ldquoEvidenceof RNAi in humans from systemically administered siRNA viatargeted nanoparticlesrdquo Nature vol 464 no 7291 pp 1067ndash1070 2010

[93] A C Fontecedro V Lutschg O Eichhoff R Dummer UF Greber and S Hemmi ldquoAnalysis of adenovirus trans-complementation-mediated gene expression controlled bymelanoma-specific TETP promoter in vitrordquo Virology Journalvol 7 article 175 2010

[94] D M Nettelbeck A A Rivera C Balague R Alemanyand D T Curiel ldquoNovel oncolytic adenoviruses targeted tomelanoma specific viral replication and cytolysis by expressionof E1A mutants from the tyrosinase enhancerpromoterrdquo Can-cer Research vol 62 no 16 pp 4663ndash4670 2002

[95] N S Banerjee A A Rivera M Wang et al ldquoAnalysesof melanoma-targeted oncolytic adenoviruses with tyrosinaseenhancerpromoter-driven E1A E4 or both in submerged cellsand organotypic culturesrdquo Molecular Cancer Therapeutics vol3 no 4 pp 437ndash449 2004

[96] M Golob R Buettner and A K Bosserhoff ldquoCharacterizationof a transcription factor binding site specifically activatingMIAtranscription in melanomardquo Journal of Investigative Dermatol-ogy vol 115 no 1 pp 42ndash47 2000

[97] A K Bosserhoff R Hein U Bogdahn and R Buettner ldquoStruc-ture and promoter analysis of the gene encoding the humanmelanoma-inhibiting protein MIArdquo The Journal of BiologicalChemistry vol 271 no 1 pp 490ndash495 1996

[98] H Maeda ldquoMacromolecular therapeutics in cancer treatmentthe EPR effect and beyondrdquo Journal of Controlled Release vol164 no 2 pp 138ndash144 2012

[99] L M Bershteın S V Patokin L M Khachaturian V N Gol-ubev andVMDilrsquoman ldquoAnahormone chimeras Conjugates ofmelanocyte-stimulating pituitary hormone (MSH) with humanmelanoma antigensrdquoDokladyAkademii Nauk SSSR vol 216 no6 pp 1402ndash1405 1974

[100] J C Garcıa-Borron B L Sanchez-Laorden and C Jimenez-Cervantes ldquoMelanocortin-1 receptor structure and functionalregulationrdquo Pigment Cell Research vol 18 no 6 pp 393ndash4102005

[101] T R Webb and A J L Clark ldquoMinireview the melanocortin 2receptor accessory proteinsrdquo Molecular Endocrinology vol 24no 3 pp 475ndash484 2010

[102] L H van der PloegW J Martin A D Howard et al ldquoA role forthe melanocortin 4 receptor in sexual functionrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 99 no 17 pp 11381ndash11386 2002

[103] A R Rodrigues D Pignatelli H Almeida and A M Gou-veia ldquoMelanocortin 5 receptor activates ERK12 through aPI3K-regulated signaling mechanismrdquo Molecular and CellularEndocrinology vol 303 no 1-2 pp 74ndash81 2009

[104] L M Heinzerling K Feige S Rieder et al ldquoTumor regressioninduced by intratumoral injection of DNA coding for humaninterleukin 12 into melanoma metastases in gray horsesrdquo Jour-nal of Molecular Medicine vol 78 no 12 pp 692ndash702 2000

[105] J Schultz L Heinzerling J Pavlovic and K Moelling ldquoInduc-tion of long-lasting cytokine effect by injection of IL-12 encod-ing plasmid DNArdquoCancer GeneTherapy vol 7 no 12 pp 1557ndash1565 2000

[106] J Schultz J Pavlovic B Strack M Nawrath and K MoellingldquoLong-lasting anti-metastatic efficiency of interleukin 12-encoding plasmid DNArdquo Human Gene Therapy vol 10 no 3pp 407ndash417 1999

[107] A L Rakhmilevich M Imboden Z Hao et al ldquoEffec-tive particle-mediated vaccination against mouse melanomaby coadministration of plasmid DNA encoding gp100 andgranulocyte-macrophage colony-stimulating factorrdquo ClinicalCancer Research vol 7 no 4 pp 952ndash961 2001

[108] A N Alexander M K Huelsmeyer A Mitzey et al ldquoDevel-opment of an allogeneic whole-cell tumor vaccine expressingxenogeneic gp100 and its implementation in a phase II clinicaltrial in canine patients with malignant melanomardquo CancerImmunology Immunotherapy vol 55 no 4 pp 433ndash442 2006

[109] P J Bergman J McKnight A Novosad et al ldquoLong-termsurvival of dogs with advancedmalignantmelanoma afterDNAvaccination with xenogeneic human tyrosinase a phase I trialrdquoClinical Cancer Research vol 9 no 4 pp 1284ndash1290 2003

[110] J Keyser J Schultz K Ladell et al ldquoIP-10-encoding plasmidDNA therapy exhibits anti-tumor and anti-metastatic effi-ciencyrdquo Experimental Dermatology vol 13 no 6 pp 380ndash3902004

[111] S David N Carmoy P Resnier et al ldquoIn vivo imaging of DNAlipid nanocapsules after systemic administration in amelanomamouse modelrdquo International Journal of Pharmaceutics vol 423no 1 pp 108ndash115 2012

[112] N Slade I Galetic S Kapitanovic and J Pavelic ldquoTheefficacy of retroviral herpes simplex virus thymidine kinasegene transfer and ganciclovir treatment on the inhibition ofmelanoma growth in vitro and in vivordquo Archives of Dermato-logical Research vol 293 no 10 pp 484ndash490 2001

[113] Y Liu and A Deisseroth ldquoOncolytic adenoviral vector carryingthe cytosine deaminase gene for melanoma gene therapyrdquoCancer Gene Therapy vol 13 no 9 pp 845ndash855 2006

[114] D Ciolczyk-Wierzbicka D Gil and P Laidler ldquoThe inhibitionof cell proliferation using silencing of N-cadherin gene bysiRNA process in human melanoma cell linesrdquo Current Medici-nal Chemistry vol 19 no 1 pp 145ndash151 2012


[115] G J Villares M Zigler H Wang et al ldquoTargeting melanomagrowth and metastasis with systemic delivery of liposome-incorporated protease-activated receptor-1 small interferingRNArdquo Cancer Research vol 68 no 21 pp 9078ndash9086 2008

[116] H Poeck R Besch CMaihoefer et al ldquo51015840-triphosphate-siRNAturning gene silencing and Rig-I activation against melanomardquoNature Medicine vol 14 no 11 pp 1256ndash1263 2008

[117] D Tormo A Ferrer P Bosch et al ldquoTherapeutic efficacy ofantigen-specific vaccination and toll-like receptor stimulationagainst established transplanted and autochthonous melanomain micerdquo Cancer Research vol 66 no 10 pp 5427ndash5435 2006

[118] D Tormo A Checinska D Alonso-Curbelo et al ldquoTargetedactivation of innate immunity for therapeutic induction ofautophagy and apoptosis in melanoma cellsrdquo Cancer Cell vol16 no 2 pp 103ndash114 2009

[119] DXu J TanMZhou et al ldquoLet-7b andmicroRNA-199a inhibitthe proliferation of B16F10 melanoma cellsrdquo Oncology Lettersvol 4 no 5 pp 941ndash946 2012

[120] T Y Fu C C Chang C T Lin et al ldquoLet-7b-mediatedsuppression of basigin expression and metastasis in mousemelanoma cellsrdquo Experimental Cell Research vol 317 no 4 pp445ndash451 2011

[121] J W Smith and D A Cheresh ldquoIntegrin (120572(v)1205733)-ligandinteraction Identification of a heterodimeric RGD binding siteon the vitronectin receptorrdquoThe Journal of Biological Chemistryvol 265 no 4 pp 2168ndash2172 1990

[122] A Higashiyama H Watanabe K Okumura and H YagitaldquoInvolvement of tumor necrosis factor 120572 and very late acti-vation antigen 4vascular cell adhesion molecule 1 interactionin surgical-stress-enhanced experimental metastasisrdquo CancerImmunology Immunotherapy vol 42 no 4 pp 231ndash236 1996

[123] R Makarem and M J Humphries ldquoLDV a novel cell adhesionmotif recognized by the integrin 12057241205731rdquo Biochemical SocietyTransactions vol 19 no 4 article 380S 1991

[124] P Vanderslice K Ren J K Revelle et al ldquoA cyclic hexapeptideis a potent antagonist of 1205724 integrinsrdquo Journal of Immunologyvol 158 no 4 pp 1710ndash1718 1997

[125] B D Brown B Gentner A Cantore et al ldquoEndogenousmicroRNA can be broadly exploited to regulate transgeneexpression according to tissue lineage and differentiation staterdquoNature Biotechnology vol 25 no 12 pp 1457ndash1467 2007

[126] B D Brown and L Naldini ldquoExploiting and antagonizingmicroRNA regulation for therapeutic and experimental appli-cationsrdquo Nature Reviews Genetics vol 10 no 8 pp 578ndash5852009

[127] V F Bonazzi M S Stark and N K Hayward ldquoMicroRNAregulation of melanoma progressionrdquoMelanoma Research vol22 no 2 pp 101ndash113 2012

[128] H T Khong and N P Restifo ldquoNatural selection of tumorvariants in the generation of ldquotumor escaperdquo phenotypesrdquoNature Immunology vol 3 no 11 pp 999ndash1005 2002

[129] G Dranoff E Jaffee A Lazenby et al ldquoVaccination with irra-diated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent spe-cific and long-lasting anti-tumor immunityrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 90 no 8 pp 3539ndash3543 1993

[130] M H Tao and R Levy ldquoIdiotypegranulocyte-macrophagecolony-stimulating factor fusion protein as a vaccine for B-celllymphomardquo Nature vol 362 no 6422 pp 755ndash758 1993

[131] C Sgadari A L Angiolillo B W Cherney et al ldquoInterferon-inducible protein-10 identified as a mediator of tumor necrosisin vivordquo Proceedings of the National Academy of Sciences of theUnited States of America vol 93 no 24 pp 13791ndash13796 1996

[132] F L Moolten ldquoTumor chemosensitivity conferred by insertedherpes thymidine kinase genes paradigm for a prospectivecancer control strategyrdquo Cancer Research vol 46 no 10 pp5276ndash5281 1986

[133] A Mizrahi A Czerniak P Ohana et al ldquoTreatment of ovariancancer ascites by intra-peritoneal injection of diphtheria toxinA chain-H19 vector a case reportrdquo Journal of Medical CaseReports vol 4 article 228 2010

[134] L M Finocchiaro and G C Glikin ldquoCytokine-enhancedvaccine and suicide gene therapy as surgery adjuvant treatmentsfor spontaneous caninemelanoma 9 years of follow-uprdquoCancerGene Therapy vol 19 pp 852ndash861 2012

[135] B Wang Z Liu M Zhang et al ldquoInterfering growth ofmalignant melanoma with Ang2-siRNArdquo Molecular BiologyReports vol 40 no 2 pp 1463ndash1471 2013

[136] Y Chen S R Bathula Q Yang and L Huang ldquoTargetednanoparticles deliver siRNA tomelanomardquo Journal of Investiga-tive Dermatology vol 130 no 12 pp 2790ndash2798 2010

[137] K P Hoeflich D C Gray M T Eby et al ldquoOncogenic BRAFis required for tumor growth and maintenance in melanomamodelsrdquo Cancer Research vol 66 no 2 pp 999ndash1006 2006

[138] R Perris C Borghese andGMagro ldquoPitfalling in nanomedicaltargeting of melanoma a ldquoclinicalrdquo case of misdelivered RNAirdquoPigment Cell and Melanoma Research vol 24 no 5 pp 980ndash982 2011

[139] N N Danial and S J Korsmeyer ldquoCell death critical controlpointsrdquo Cell vol 116 no 2 pp 205ndash219 2004

[140] G G McGill M Horstmann H R Widlund et al ldquoBcl2regulation by the melanocyte master regulator Mitf modulateslineage survival and melanoma cell viabilityrdquo Cell vol 109 no6 pp 707ndash718 2002

[141] R Besch H Poeck T Hohenauer et al ldquoProapoptotic signalinginduced by RIG-I and MDA-5 results in type I interferon-independent apoptosis in human melanoma cellsrdquo Journal ofClinical Investigation vol 119 no 8 pp 2399ndash2411 2009

[142] J Kota R R Chivukula K A OrsquoDonnell et al ldquoTherapeuticmicroRNA delivery suppresses tumorigenesis in a murine livercancer modelrdquo Cell vol 137 no 6 pp 1005ndash1017 2009

[143] A L Kasinski and F J Slack ldquoEpigenetics and geneticsMicroR-NAs en route to the clinic progress in validating and targetingmicroRNAs for cancer therapyrdquo Nature Reviews Cancer vol 11no 12 pp 849ndash864 2011

[144] A Schafer A Pahnke D Schaffert et al ldquoDisconnecting theyin and yang relation of epidermal growth factor receptor(EGFR)-mediated delivery a fully synthetic EGFR-targetedgene transfer system avoiding receptor activationrdquoHumanGeneTherapy vol 22 pp 1463ndash1473 2011

[145] A J North ldquoSeeing is believing A beginnersrsquo guide to practicalpitfalls in image acquisitionrdquo Journal of Cell Biology vol 172 no1 pp 9ndash18 2006


Review ArticleClinical Trials with Pegylated Liposomal Doxorubicin inthe Treatment of Ovarian Cancer

Carmela Pisano Sabrina Chiara Cecere Marilena Di NapoliCarla Cavaliere Rosa Tambaro Gaetano Facchini Cono Scaffa Simona LositoAntonio Pizzolorusso and Sandro Pignata

Department of Urology and Gynecology National Cancer Institute 80131 Naples Italy

Correspondence should be addressed to Sandro Pignata sandropignatagmailcom

Received 27 December 2012 Revised 29 January 2013 Accepted 29 January 2013


Copyright copy 2013 Carmela Pisano et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Among the pharmaceutical options available for treatment of ovarian cancer increasing attention has been progressively focusedon pegylated liposomal doxorubicin (PLD) whose unique formulation prolongs the persistence of the drug in the circulationand potentiates intratumor accumulation Pegylated liposomal doxorubicin (PLD) has become a major component in the routinemanagement of epithelial ovarian cancer In 1999 it was first approved for platinum-refractory ovarian cancer and then received fullapproval for platinum-sensitive recurrent disease in 2005 PLD remains an important therapeutic tool in the management ofrecurrent ovarian cancer in 2012 Recent interest in PLDcarboplatin combination therapy has been the object of phase III trials inplatinum-sensitive and chemonaıve ovarian cancer patients reporting response rates progressive-free survival and overall survivalsimilar to other platinum-based combinations but with amore favorable toxicity profile and convenient dosing scheduleThis papersummarizes data clarifying the role of pegylated liposomal doxorubicin (PLD) in ovarian cancer as well as researches focusing onadding novel targeted drugs to this cytotoxic agent

1 Introduction

Ovarian cancer (OvCa) is the leading cause of death fromgynaecological malignancies with an estimated 65697 newcases and 41448 deaths every year in Europe [1] Approxi-mately 15 of women present with disease localized in theovaries and in this group surgery allows a 5-year survival inmore than 90 of the cases However the majority of womenpresent at the diagnosis with advanced disease (InternationalFederation of Gynaecological Oncology (FIGO) stage III-IV)and their survival at 5 years is poor currently less than 30[2]

Themain reasons for the highmortality rate are the lack ofsymptoms accompanying this tumor in addition to the lackof an effective screening strategy for the overall populationand lastly the limited results obtained with standardmedicaltreatments

The standard of care for the management of OvCapatients includes surgery for staging and optimal cytoreduc-tion (no residual tumour) followed by a platinumtaxane

chemotherapy combination [3 4] Recently bevacizumab hasbeen approved in stage IIIb-IV cancer in combination andas a single-agent maintenance after carboplatin-paclitaxel [56] Although chemotherapy obtains high objective responserates even in patients with an advanced tumor stage the vastmajority of patients will experience tumor progression andrequire further therapy [7 8]

Many strategies have been implemented in order toimprove these unsatisfactory results and newdrugs have beeninvestigated

In this context among the pharmaceutical options cur-rently available for medical treatment of ovarian cancer(OvCa) greater emphasis has been placed progressively onpegylated liposomal doxorubicin (PLD) (Doxil in the USACaelyx in Canada and Europe) which was approved in 1999by the FDA and in 2000 by the European Medicines Evalua-tion Agency (EMA) as single agent for treatment of advancedOvCa patients failing first-line platinum-based treatmentMoreover phase III trials have been already conducted andresults suggest further role for PLD in salvage setting and in


front-line treatment in combination with other therapeuticdrugsThe aimof this paper is to summarize data showing therole of pegylated liposomal doxorubicin (PLD) in the mana-gement of epithelial ovarian cancer

2 Pegylated Liposomal Doxorubicin(PLD) Development Structure andPharmacokinetic Features

Anthracyclines have been for years among the drugs admin-istered for the majority of gynecologic cancers Before tax-anes were introduced into first-line therapy of ovarian can-cer anthracyclines demonstrated a comparable efficacy inmonochemotherapy with alkylating agents and superiorityof the combination of both when compared to single-agenttherapy Furthermore meta-analysis data suggest that theaddition of anthracyclines to cisplatinmight be advantageouscompared to using cisplatin alone [9 10]

Attempts have been made to introduce anthracyclines incombination with carboplatin-paclitaxel In the randomizedtrial conducted by the AGO group in collaboration with theFrench groupGINECO the addition of epirubicin (TECarm)to the platinumpaclitaxel (TC arm) combination in first-lineovarian cancer treatment patients showed a not statisticallysignificant advantage of about 5 months in median overallsurvival time (458 versus 410 months HR 093) [11] with noprogression-free survival benefit (184 versus 179months HR095) at the price of a greater toxicity of TEC versus TC arm(grade 34 hematologic nauseaemesis mucositis and infec-tions) Despite the antitumor activity in ovarian cancer theclinical use of conventional anthracyclines is limited by theirassociated side effects The haematological toxicity and thecumulative and irreversible cardiac damage (congestive heartfailure) are the more common side effects dose limiting ofanthracyclines As far as it is elucidated cardiotoxic eventstake place by increasing oxidative stress suppression of geneexpression and induction of apoptosis on cardiac tissue [12]with clinicalmanifestations reaching fromacute cardiac heartfailure to chronic cardiac insufficiency Several treatmentstrategies including the development of new formulations fordelivering the cytotoxic agents (as liposomes encapsulation)have been proposed to improve the therapeutic index ofanthracyclines [13] The inclusion of anthracyclines in a lipo-somal structure has been proposed to reduce side effectsand to enhance the antitumor activity In this paper we willfocus on the pharmacologic properties of pegylated lipo-somal doxorubicin (PLD) a new available formulation ofdoxorubicin that is encapsulated in a pegylated liposome [1415]The size of the liposomes approximately 100 nm preventsthem from entering tissues with tight capillary junctionssuch as the heart and gastrointestinal tract [16] In contrastto other nanoparticles the liposomal shell is surroundedby a polyethylene glycol (PEG) layer which represents ahydrophilic protective barrier between the liposome and themicroenvironment thus preventing the activation of the reti-culoendothelial system that leads to the destruction of theliposomal structure and release of the free drug Liposomaldrug delivery to cancer cells can occur in vivo by two different

pathways passive and active targeting In contrast to normalvessels the vessels of the tumor are tortuous dilated havemorphologically abnormal endothelial cells and are leakydue to large spaces between pericytes [17] These physicalcharacteristics allowmore extravasation of the liposomes intothe tumorwith higher cell concentration of the drugThe lackof functional lymphatic drainage in tumours prevents theoutflow of extravasated liposomes allowing doxorubicinaccumulation in the tumour extracellular fluid These lipo-someswill gradually release the entrapped drug in the vicinityof tumour cells thus increasing the tumour-drug exposure[18]Thismechanismof passive targeting is known as ldquoenhan-ced permeability and retention (EPR) effectrdquo [19]

The efficacy and safety of PLD has been evaluated in avariety of different tumor models including several humanxenograftmodels supporting its introduction in cancer treat-ment [15] In every model examined PLD was more effectivethan the same dose of free doxorubicin in inhibiting orhalting tumor growth in preventing metastasis andor inprolonging survival of the tumor-bearing animals [20 21]The pharmacokinetic and tissue distribution studies in thesemodels suggest that the greater persistence particularly intumor tissue achieved with PLD compared with conven-tional doxorubicin offers a therapeutic advantage PLD haswell-known pharmacokinetic features such as long circula-tion time minimal (lt5) drug leakage from circulating lipo-somes and half-lives of approximately 60ndash90 h for doses inthe range of 35ndash70mgm2 in patients with solid tumors [21]This translates into a PLDAUC approximately 250ndash1000-foldhigher than that of the free drug in humans [22] PLD phar-macokinetics is best modeled as a one-compartment modeldisplaying linear pharmacokinetics with C-max increasingproportionally with dose [23] It has also been described asa two-compartment model with an initial half-life of severalhours followed by a more prolonged terminal decline with ahalf-life of 2-3 days accounting for the majority of the AUC[22 24] After PLD administration nearly 100of the drug inthe plasma is in the encapsulated form Moreover comparedto free doxorubicin PLD plasma clearance is dramaticallyslower and its volume of distribution is very small androughly equivalent to the intravascular volume [22 24]

These properties which represent the rational basis forthe exploitation of nanoparticle technology represent themajor advantages of PLD compared to conventional doxoru-bicin in safety profile (lower cardiotoxicity and gastrointesti-nal toxicity compared to the free drug) [20ndash25]

Based on the previous evidences regarding the role ofanthracyclines and the modified toxicity profile of PLD thisagent has been a rational choice for further evaluation as asingle-agent and in combination with platinum agents in thetreatment of ovarian cancer

3 Pegylated Liposomal DoxorubicinActivity in Ovarian Cancer

31 Phase II Studies with PLDas a Single-Agent or in Combina-tion The initial studies evaluating PLD have been conductedin recurrent ovarian cancer as a single-agentmonotherapy or


in combinationwith platinum (carboplatin) and later onwithtrabectedin or other new drugs

A summary of phase II studies using PLD as a single agentor in combination regimens in ovarian cancer is presented inTable 1 [26ndash35]

Nonrandomized phase II trials of PLD in platinum-resistant ovarian cancer patients documented the biologicalactivity of this agent in this clinical setting with objectiveresponse rates of approximately 10ndash20 being reported inseveral trials [18 25 31] Data indicated that palmar-plantarerythrodysesthesia (PPE hand-foot syndrome toxic acralerythema) andmucositis were themost common toxicities ofPLD reported in up to 50 of treated patients PPE usuallyoccurs after two or more courses of treatment and the risk ofincidence increases with multiple repeated treatments PPEis related to dose intensity and dose interval rather thanto peak dose level Although not life threatening PPE cannegatively impact the quality of life and it is a major cause ofboth dose reduction and treatment discontinuation [61 62]As regards the cardiac toxicity in several trials PLD formu-lation has been related to a better safety profile comparedto conventional doxorubicin [63] Compared to the 75incidence of irreversible cardiotoxicity at cumulative dosesof 400ndash550mgm2 reported with doxorubicin [64] most ofthe studies of PLD showed a lower incidence of cardiacfailure even at doses higher than 500mgm2 [65 66] Ina prospective trial performed on patients with advancedgynecological malignancies treated with PLD the cardiacsafety was further assessed at histology (endomyocardial bio-psies) showing no myocardial damage in patients treatedwith PLD (median PLD dose of 708mgm2) [67] Thus theoptimal cardiac safety profile of PLD may allow a prolongedtreatment encouraging results from a phase II trial in AIDS-related Kaposirsquos sarcoma patients treated with PLD up to a2360mgm2 cumulative dose have been reported [68] Inmetastatic breast cancer patients also doses greater than450mgm2 were not associated with a significant decreasein LVEF from baseline compared to conventional doxoru-bicin [69] In relapsed ovarian cancer patient respondingto second-line chemotherapy a maintenance therapy withPLD for more than 1 year has been reported to be safe byAndreopoulou et al with no cardiac event reported [70]

Different schedules and doses have been investigated inan effort to improve tolerability while maintaining antitumorefficacy [28 35 36 71] Several studies have shown that amore acceptable toxicity profile in terms of decreased ratesof hand-foot syndrome and stomatitismucositis can beobtained with a PLD dose of 40mgm2 every 28 days com-pared to the traditional dose of 50mgm2 with comparableresponse rates and outcomes [26 32 33] According to thestudies published the optimal dose intensity appears to rangefrom 10mgm2 to 125mgm2 per week (given at doses of 40ndash50mgm2 every 4 weeks) when used as a single-agent ther-apy

The results obtained with a single-agent PLD in thesubgroup of platinum-resistant patients were the basis forthe development of PLDplatinum (cis- carbo- oxaliplatin)combinations

The trials that evaluated the combination regimen ofcisplatin or carboplatin with PLD showed an overall responserate ranging from 46 to 68 according to the platinum-free interval In the Rapoport trial the overall response rateswere about 65 in a population including platinum-sensitive(81) and partially sensitive patients (526) [38]

Cisplatin combination regimen (PLD at 50mgmq dos-age plus cisplatin at 60mgmq d1 q 28 days) was also deve-loped showing a moderate tolerability profile (10 grade 2neurotoxicity 18 grade 34 anemia 41 neutropenia and9 hand-foot syndrome) [34] Due to these results the PLDcarboplatin combination was considered more manageabledue to the lower neurotoxicity [37ndash39 72ndash74]

In two phase I-II trials PLD has been associated withcarboplatin AUC 5-6 in sensitive or partially sensitive (gt50)ovarian or other gynecological cancer patientsIn both stud-ies data of ORR (62 and 68 resp) PFS (92 and 116months) and median overall survival (OS 234 and 32months) substantially overlap [37 39]

Based on toxicity results the authors recommended aPLD dose of 40mgm2 when given in combination with car-boplatin AUC 5 both drugs administered on a 4-week sched-ule in epithelial ovarian or endometrial carcinoma

Gemcitabine is another drug studied in combinationwith PLD In several trials (PLD 30mgm2-gemcitabine1000mgm2 days 1ndash8 every 21 days) this combination hasbeen associated with overall response rates of about 30ndash35in the overall population (21ndash25 in platinum-resistant and50ndash53 in platinum-sensitive diseases) with an acceptabletoxicity profile Myelosuppression was the most commontoxicity and was found in 35 of patients [41 42]

Combinations of PLD with oxaliplatin (OXA) have beenalso reported with response rates that appear in the rangeof those reported with PLDcarboplatin In these trials avery acceptable rate of stomatitismucositis and hand-footsyndrome has been shown likely due to the use of the PLD atthe dosage of 30mgm2 every 21 or 28 days

Nicoletto et al [40] published a trial of pegylated lipo-somal doxorubicin dosed between 30 and 35mgm2 withoxaliplatin at 70mgm2 every 28 days The overall responserate was 54 with a median survival of 225 months Whenevaluated according to platinum sensitivity there was a res-ponse rate of 667 among the 29 platinum-sensitive patientsand of 286 in the 14 platinum-resistant patients Therewere 5 (12) grade 3 or 4 toxicities and only 3 patients(7) required dose reductionNeutropeniawas the treatmentlimiting toxicity

Some phase II studies explored the efficacy of PLD asso-ciated with topotecan (TPT) [43] as well as paclitaxel (PTX)[44] vinorelbine (VNR) [45] and ifosfamide (IFO) [46]Overall response rates of about 28 to 37 with a medianPFS of 55 to 75 months were found figures which are quitecomparable to those reported with other nonplatinum com-binations The association with weekly paclitaxel was welltolerated as was the PLDVNR combination [45] In contrastPLDTPT even if tested at different doses of the two drugswas characterized by an unacceptable rate of severe anemia(48) leukopenia (70) and thrombocytopenia (44) [43]


Table 1 Phase-II studies with pegylated liposomal doxorubicin (PLD) as a single agent or in combination regimens

Author Doseschedule Clinical setting PFI(mts) No pts RR () PFS (median) (mts)

Muggia et al [25] 50mgm2 q21 le6 35 257 57Gordon et al [18] 50mgm2 q21 ALL 89 168 48

Rose et al [26] 50mgm2 q28 le6 37 135 4040mgm2 q28 77 40

Katsumata et al [28] 50mgm2 q28 le6 63 209 56Markman et al [31] 40mgm2 q28 le6 44 91 mdash

ALL 135 72Lorusso et al [35] 35mgm2 q21 le6 17 189 mdash

ge6 20 100 mdash

Sehouli et al [36] 20mgm2 q15 ALL 64 109 43

Du Bois et al [37] PLD (40mgm2) d1CBDCA (AUC 6) d1 q28 ge6 67 68 116

Rapoport et al [38] PLD (50mgm2) d1CBDCA (AUC 5) d1 q28

ALL7ndash12

4019

675526

11997

Ferrero et al [39] PLD (30mgm2) d1CBDCA (AUC 5) d1 q28

ALL7ndash12ge12

964353

625mdashmdash

9479114

Nicoletto et al [40] PLD (30mgm2) d1OXA (70mgm2) d1 q28

le6ge6

1429

286667

5999

DrsquoAgostino et al [41] PLD (30mgm2) d1GEM (1000mgm2) d1 8 q21

le12ge12

3631

250452

mdashmdash

Ferrandina et al [42] PLD (30mgm2) d1GEM (1000mgm2) d1 8 q21

RESge12

6645

216537

587

Verhaar-Langereis et al [43]PLD (30mgm2) d1TPT (10mgm2)d1ndash5 q21 and PLD (40mgm2) d1 TPT(075mgm2) d1ndash5 q21

le12 27 280 75

Campos et al [44] PLD (30mgm2) d1 q21 PTX(70mgm2) weekly

ALLle12ge12

372413

290170540

mdash

Katsaros et al [45] PLD (30mgm2) d1vinorelbine (30mgm2) d1 q21 ALL 30 370 55

Joly et al [46] PLD (40mgm2) d1ifosfamide (1700mgm2) d1ndash3 q28

ALLRESSEN

985741

280190410

mdash

PFS progression-free survival RR response rate RES platinum-resistant recurrent disease (platinum sensitivity according to the cutoff of 12-monthplatinum-free interval) SEN platinum-sensitive recurrent disease q every d day CDDP cisplatin CBDCA carboplatin PFI platinum-free interval GEMgemcitabine PTX paclitaxel TPT topotecan OS overall survival

32 PLD Single-Agent Phase III Randomized Trials Table 2summarizes the results from randomized trials using PLDalone or in combination in phase III studies [47ndash52]

In the first trial [48] Gordon randomized 474 ovariancancer patients at first recurrence (stratified by PFI) to PLD(50mgm2 every 4 weeks) or topotecan (15mgm2day for 5consecutive days every 3 weeks) In platinum-resistant dis-ease (119899 = 255) no significant difference was seen in res-ponse rate PFS or OS between the two treatment armswhile in platinum-sensitive patients (119899 = 219) medianPFS and OS were significantly prolonged in PLD-treated

patients compared to TPT-treated patients (P value = 0037and P value = 0008 resp) More mature survival analysisconfirmed the long-term advantage for platinum-sensitivepatients receiving PLD versus TPT (median OS = 27 monthsversus 175months hazard ratio (HR) = 1432 P value = 0017)[49] Moreover for partially platinum-sensitive disease (119899 =122) the HR favored PLD versus TPT (HR = 158 P value= 0021) About the tolerability profile grade 34 haemato-logical toxicity occurred more frequently and more severelyin TPT compared to PLD in particular severe neutropeniawas documented in 77 of TPTndashtreated patients versus 12


Table 2 Phase-III studies with pegylated liposomal doxorubicin (PLD) as a single agent or in combination regimens

Author Doseschedule Clinical settingPFI (mts) No pts RR () PFS (median)

(mts) OS

OrsquoByrne et al [47] PLD (50mgm2) q28 versusPTX (175mgm2) q21

214REC 107 178 54 114

107 224 60 140

Gordon et al [48 49] PLD (50mgm2) d1 q28 versusTPT (15mgm2) d1ndash5 q21 RES

255130125

12365

2334

89103

Mutch et al [50] PLD (50mgm2) d1 q28 versusGEM (1000mgm2) d1 8 q21 RES

1959699

8361

3631

127135

Ferrandina et al [51] PLD (40mgm2) q28 versusGEM (1000mgm2) d1 8 15 q28 RES

1537677

1629

4050

14127lowast

Monk et al [52]OVA-301

TRAB (11mgm2) d1 q21 versusPLD (50mgm2) q28 ALL 672 280lowast

19073lowast59

205194

PLD (30mgm2) d1TRAB (11mgm2) d1 q21 versusPLD (50mgm2) q28

SEN 430

335337

35lowast23

92lowast75

mdashmdash

Markman et al [53]SWOG SO200

PLD (30mgm2) d1CBDCA(AUC 5) d1 q28 versusCBDCA (AUC 5) d1 q28

SEN6ndash24mts

3130

59lowast28

12lowast8

3118

Pujade-Lauraine et al[54]CALYPSO

PLD (30mgm2) d1JM8 (AUC 5) d1 q28 versusPTX (175mgm2) d1JM8 (AUC 5) d1 q21

SENgt6mts

467509

mdashmdash

113lowast94

mdashmdash

GEM gemcitabineOS overall survival PFS progression-free survival PTX paclitaxel REC not otherwise specified recurrent disease RES platinum-resistantrecurrent disease RR response rate SEN platinum-sensitive recurrent disease TRAB trabectedin q every d day lowastStatistically significant

of PLD-treated patients (119875 lt 0001) and thrombocytopeniawas found in 34 of TPT versus 1 of PLD cases (119875 lt 0001)No case of severe HFSwas documented in the TPT armwhileit was registered in 23 of PLD-treated patients (119875 lt 0001)with no difference in quality of life perceived by the patient

In a second randomized trial conducted by OrsquoByrne et al[47] 214 patients (not defined according to platinum sen-sitivity) were randomized to receive either PLD (50mgm2every 4 weeks) or paclitaxel (175mgm2 every 3 weeks) Apreliminary analysis of the data showed that there were nosignificant differences in response rates PFS OS or rate ofadverse eventsThe study was suspended due to poor accrualas paclitaxel became incorporated into first-line therapy sono definitive analysis was carried out

Several additional phase III trials have been reportedwhich directly compared single-agent PLD to other singleagents (paclitaxel gemcitabine) in platinum-resistant andpartially platinum-sensitive (platinum-free interval 6ndash12months) ovarian cancer patients [47 50 51]While side-effectprofiles of the agents often differed substantially these studiesessentially revealed the therapeutic equivalence for theseagents in this difficult clinical setting

Two phase III trials compared PLD with gemcitabinein recurrent platinum-resistant or partially sensitive ovariancancer patients [50 51]

In both trials there was no difference in the response ratesand median PFS between the two treatment arms Themedian OS in the MITO3 trial was greater in the PLD arm(14 versus 127 months respectively P value = 0048) Withthe limits inherent in the small sample series the survivaladvantage reported with PLD over GEM was maintained inthe subgroup of partially sensitive patients (P value = 0016)

Based on these results PLD at 40mgm2 seems to offerthe most favourable toxicity profile which is likely to sustainthe achievement of better quality of life (QoL) scores (atleast in comparison to GEM) and was adopted as a standardworldwide [50]

Other phase III trials have explored the combinationof PLD with other nonplatinum agents Among the mostintriguing novel drugs trabectedin (TRAB) (ET743 Yon-delis) has become relevant for treatment of sarcomas andother solid tumors for its unique mechanism of action inthat unlike most other agents it binds to the minor grooveof DNA thus affecting a variety of transcription factors cellproliferation and the nucleotide excision repair system andinhibits the MDR-1 gene coding for the protein responsiblefor chemoresistance [75ndash77]

Based on safety and efficacy results from phase-III stud-ies a phase-III trial (OVA-301 NCT00113607) has beenperformed to compare PLD (50mgm2 every 28 days) with


the combination PLD (30mgm2) and TRAB (11mgm2every 21 days) in second-line relapsed ovarian cancer patientsunsuitable for platinum therapy stratified according to thePFI (PFI lt 6 months versus PFI gt 6 months) After a medianfollowup of 474months in the whole series the response ratewas significantly higher in the combination compared to thePLD arm as was alsomedian PFS (HR= 079P value = 0019)[52]

However in platinum-resistant cases (119899 = 242) no sta-tistically significant difference was observed with the doubletin terms of response rate (134 versus 122 resp) and PFSwhile a clear advantage favouring the combination comparedto single-agent PLDwas evident in platinum-sensitive disease(RR 353 versus 226 119875 = 00042 median PFS 92 monthsversus 75 months HR = 073 119875 = 0017) and partially sensi-tive disease withmedian PFS of 74 months versus 55 monthsin PLDTRAB versus PLD arm (HR = 065 119875 = 00152)An unplanned hypothesis-generating analysis adjusting forthe PFI imbalance and other prognostic factors suggested animprovement inOS associated with the trabectedinPLD arm(HR = 082 95 CI 069ndash098 119875 = 00285) In anotherunplanned exploratory analysis the subset of patients witha PFI of 6ndash12 months had the largest difference in OS (HR =064 95 CI 047ndash086 119875 = 00027) Data showed a longertime to the following platinum therapy and this imbalance inplatinum-free interval was suggested as a possible cause of theincreasedOS [78]Thus these data suggest that the treatmentwith an effective nonplatinum combination may artificiallyprolong the platinum-free interval giving more chance ofactivity to further platinum therapy This hypothesis will beinvestigated in a phase III trial called INNOVATYION

As expected the combination regimen of TRABPLD hasbeen associated to a greater haematological toxicity (grade34 anaemia 14 neutropenia and thrombocytopenia 63)Among other toxicities short-lived grade 34 hypertransam-inasemia (38) and HFS were documented in 4 of thePLDTRAB arm compared to 20 in the PLD alone arm [79]In September 2009 based on these results which support thePLDTRAB combination as the most effective nonplatinum-based combination in platinum-sensitive disease the PLD(30mgm2) and TRAB (11mgm2) association every 3 weekshas been approved by the EMA for treatment of patients withrelapsed platinum-sensitive OvCa [80]

Based on the phase-II trials in platinum-sensitive OvCathe combination of PLDcarboplatin has been explored inphase-III trials [53] Markman et al compared single-agentcarboplatin to its combination with PLD in recurrent ovariancancer showing a statistically significant improvement of PFSwith carboplatinPLD without an overall survival benefitInterestingly for unknown reasons the association drasti-cally reduced the rate of hypersensitivity reactions comparedto carboplatin alone (9 versus 0 119875 = 00008) [53] Lateron the results of the CALYPSO trial have been reported [8182] This international open-label phase-III trial comparedcarboplatin PLD (CD) with carboplatin-paclitaxel (CP) inpatients with platinum-sensitive recurrent ovarian cancer(ROC) A total of 976 recurrent patients relapsing gt6 months

after first- or second-line therapy were randomized to receiveCD or CP for six cycles

Designed as a noninferiority trial CALYPSO demon-strated that the combination of CD was not only noninferiorto CP in terms of PFS but indeed it was more effective (HR =082119875 = 0005) in patients with platinum-sensitive recurrentovarian cancer Nevertheless with a median followup of49 months no statistically significant difference in OS wasobserved (hazard ratio = 099 (95 confidence interval 085116) log rank119875 = 094) with median survival times of307 (CD) and 330 months (CP) Treatment-related seriousadverse events weremore frequent in the CP arm (76 patients(30) versus 44 patients (18)) while the CD treatmentwas associated with more grade 34 thrombocytopenia andmore grade ge2 mucositis and PPE Interestingly even inthis trial as in other phase-II studies there was a lowerincidence of allergic reactions alopecia neuropathy andarthralgiamyalgia PLDcarboplatin represents a valid alter-native to other platinum-based regimens in recurrent plati-num-sensitive OvCa especially for patients whose QoL isrecognized to be heavily compromised by alopecia or whohad experienced or had not yet been rescued from taxane-induced neurotoxicity [81 82]

Attempts to include PLD in a front-line treatment havealso been made in particular with the aim of improvingstandard chemotherapy with carboplatin-paclitaxel doubletor triplet combinations including PLDhave been investigatedbased also on the very favourable and not overlapping tox-icity profile The potential efficacy of triplets and sequen-tial doublets (with TPT PLD and gemcitabine) has beeninvestigated in the GOG182ICON5 trial that enrolled 4312stage-IIIIVpatientswhowere randomized to 5-armfirst-linechemotherapy regimens and sequences with disappointingresults There was no PFS or OS advantage with sequentialdoublets or with triplets compared with the control arm Inthis trial PLD at a dosage of 30mgm2 was added to carbo-platin and paclitaxel at full dose every other cycle [83]

In the front-line setting MITO-2 was the first trial inves-tigating the PLDcarboplatin (30mgm2 AUC = 5 every21 days) combination compared to the standard treatmentthis trial was designed to show a superiority for the carbo-platinPLD combination Unfortunately there were no statis-tically significant differences in either PFS or overall survivalbetween the treatment arms with median PFS times of 190months versus 168 months (HR 095 95 CI 081 to 113119875 = 058) and median overall survival times of about 61 and53 months with carboplatinPLD and carboplatin-paclitaxelrespectively (HR 089 95 CI 072 to 112 119875 = 032) [84]CarboplatinPLD also produced a similar response rate butdifferent toxicities (less neurotoxicity and alopecia but morehematologic adverse effects)

Although the proposed combination has failed to under-mine the primacy of the standard carboplatin-paclitaxelgiven the observed confidence intervals and the differenttoxicity carboplatinPLD could be considered an alternativeto standard first-line therapy particularly in patients thatcannot receive paclitaxel


Table 3 Phase-I-II-III studies with pegylated liposomal doxorubicin (PLD) in combination with target agents


(mts)

Muggia et al [55]PLD 30mgm andBEV 15mgkg on cycles 2ndash7 (withoption to continue)

le6 48 Ongoing Ongoing

Pujade-Lauraine et al [56]

Arm1PTX (80mgm2) d1 8 15 and 22 q28orTPT (4mgm2) d1 8 15 q28orPLD (40mgm2) d1 q28

le6 166 126 34

Arm2BEV 10mkg d1 q15 or 15mgkg d1q21PTX (80mgm2) d1 8 15 and 22 q28orTPT (4mgm2) d1 8 15 q28orPLD (40mgm2) d1 q28

135 309 67

Del Carmen et al [57]PLD (30mgm2) d1 q28CBDCA (AUC5) d1 q28 Beva10mgkg d1 q14

ge6 54 722 139

Steffensen et al [58] PAN 6mgkg d1 15 q28PLD40mgm2 day 1 q28 le6 46 243 27ndash81

TRINOVA 2 [59]httpclinicaltrialsgovshowNCT01281254

Arm 1PLD 50mgm2 d1 q28 and blindedAMG 386 15mgkg qwArm 2PLD 50mgm2 d1 q28 and blindedAMG 386placebo qw

le12 Ongoing Ongoing Ongoing

Boers-Sonderen et al [60] T 15ndash20mgm2PLD 20ndash40mgmq ALL 20 3PR9SD

49

PFS progression-free survival PTX paclitaxel TPT topotecan T temsirolimus PAN panitumumab BEV bevacizumab RR response rate SEN platinum-sensitive recurrentdisease TRAB trabectedin q every d day lowastStatistically significant

4 PLD in Epithelial Ovarian CancerFuture Directions

Based on the excellent results obtained by the PLD alone orin combination with platinum as well as nonplatinum agentsin almost all clinical settings of ovarian cancer early phasetrials have begun to explore the potential of adding PLD toa variety of alternative drugs including bevacizumab (BEV)and other ldquotargeted agentsrdquo in the management of epithelialovarian cancer (Table 3)

Despite the encouraging results obtained in ovarian can-cer the combination of PLD with bevacizumab was intro-duced with caution because of the potential mechanism ofinterferenceWe know that the increased vascular permeabil-ity known as ldquoEPR effectrdquo greatly enhances liposome depo-sition in tumors enabling the increase of intratumoral deli-vering and concentration of PLD Normalization of the vas-culature induced by bevacizumab has been hypothesized tointerfere with liposomal tumour entry but a concomitantreduction in tumour interstitial pressure on the other handcould improve PLD delivery In a trial conducted by Muggiaet al the pharmacokinetic of PLD alone or in combination

with bevacizumab was investigated in order to evaluate thepostulated interferences Trial results show an increased PLDT 34 C7dCmax and PLD levels at day 21 after bevacizumabintroduction probably reflecting a greater delivery of PLD totumours [55] Preliminary results from a phase II study withthe PLDBEV combination in platinum-resistant patientshave been presented by the same authors The study wasconducted on 48 patients PLD (30mgm2 every 21 days)was administered alone at the first cycle and then with BEV(15mgkg every 21 days) for the following 6 cycles or untilprogression [85]

This proof-of-concept study was the first to report theefficacy and the tolerability of the combination of PLD andbevacizumab in the treatment of recurrent ovarian cancerTheORRobserved in this trial was 722 (95CI 584 835)The safety profile was consistent with the known toxicities ofthese agents with no sign of overlapping toxicities nor anyreports of cumulative-dose cardiotoxicity

Following these data a large phase III randomized study(AURELIA) in platinum-resistant setting assessed the effi-cacy of bevacizumab (10mgkg every 2 weeks or 15mgkgevery 3 weeks) combined to either dose-dense paclitaxel


(80mgm2 weekly) topotecan (4mgm2 on days 1 8 and15 of each 4-week cycle or 125mgm2 on days 1 through 5of each 3-week cycle) or pegylated liposomal doxorubicin(40mgm2 every 4 weeks) After a median followup (after301 PFS events) of 135 months the overall response rates(ORR) were 309 in the bevacizumab combination armcompared to 126 of chemotherapy alone (HR 048 CI95) In platinum-resistant OC bevacizumab combined tochemotherapy provided a statistically significant and clini-cally meaningful improvement in PFS and ORR comparedto chemotherapy alone with an acceptable safety profile alsodue to strict inclusion criteria that minimized the incidenceof BEV adverse events This is the first phase-III trial inplatinum-resistant ovarian cancer that shows a clear benefitwith a targeted agent combination regimen associated to animproved outcome compared to monotherapy [56] Takenoverall these data suggest that there is no pharmacologicdisadvantage of the combination of PLD with bevacizumab

In platinum-sensitive ovarian cancer relapse bevacizu-mab has been associated with carboplatinPLD regimen inanother phase-II trial with promising results Among the54 patients enrolled the ORR was 722 (95 CI 584835) the median duration of response was 119 monthsand median TTP was 139 months (95 CI 114 160) Thesafety profilewas consistent with the known toxicities of theseagents making this association a potential treatment optionfor platinum-sensitive ovarian cancer patients [57]

PLD is also under investigation with other antiangio-genetic drugs A phase-III ongoing trial (TRINOVA 2 study)compares PLD to PLD in association with AMG386 anangiopoietin inhibitor [59]

Panitumumab is a fully humanmonoclonal antibody spe-cific to the epidermal growth factor receptor (EGFR) Noprevious studies have evaluated the effect of panitumumabin ovarian cancer (OC) based on KRAS mutation statusThe main purpose of the PaLiDo study a phase-II non-randomized multicenter trial presented at ASCO 2012 [58]was to investigate the response rate in platinum-resistantKRAS wild-type OC patients treated with PLD and panitu-mumab Patients with relapsed and pretreated (no more thantwo lines) ovarian cancer were treated with panitumumab(6mgkg days 1 and 15) and with PLD (40mgm2 day 1)every 4 weeks Progression-free and overall survival in theintention-to-treat population (N 543) was 27 months (25ndash32 months 95 CI) and 81 months (56ndash117 months 95CI) respectively with a considerable skin toxicity grade 3 inabout 40 of patients

Other phase-I trials evaluated PLD in combination withthe mTOR inhibitor temsirolimus [60] and with the folatereceptor ligand farletuzumab [86] (humanized monoclonalantibody that binds to folate receptor-120572 a target which islargely absent in normal epithelium and overexpressed inEOC) showing feasibility and activity

Data regarding combinations are very preliminary butat least with antiangiogenetic drugs the combination seemstolerable and active

Another field of development is that of the patients withBRCAmutation BRCA1- or BRCA2-mutated ovarian cancer

patients are defective of the mechanisms of DNA repairingThis determines an improved chemosensitivity to someDNA-damaging agents [87] PLD that leads to DNA damageby inhibiting topoisomerase II may prove to bemore effectivein these patients [88] In a recent study from Kaye et al[89] the PARP inhibitor olaparib was compared with PLDin BRCA-mutated patients The study showed significantsingle-agent olaparib activity while PFS was not significantlyimproved compared to PLD Interestingly this negative resultwas hypothesis generating based on the unexpected high PFSfound in the control PLD arm In fact the 71-month PFSobserved in this study with PLDwas significantly higher thanthat expected for this drug in the general population Theseresults are in accordance with retrospective data publishedby Adams and colleagues on Gynecologic Oncology in 2011confirming the higher activity of PLD in BRCA-mutatedovarian cancer patients Although all these data are very pre-liminary it seems that PLDmay have a special role in patientswith BRCA mutation or BRCAness profile [90] In the samedirection are the results of a multicentre retrospective studyin relapsed ovarian patients BRCAmutation carriers treatedwith PLD where Safra et al showed an improved outcome interms ofmedian time to treatment failure (158months versus81 months in nonhereditary OC) and overall survival (568months versus 226 months) [91]

5 Conclusions

PLD plays an important role in the management of ovariancancer It represents the standard therapy in platinum-resis-tant recurrence and one of the standard options in platinum-sensitive patients Between the combination regimes due tothe results of efficacy achieved in phase-II and -III trialsand considering the favorable safety profile carboplatinPLDrepresents a valid alternative in both first-line (in patientsthat cannot receive paclitaxel) and recurrent ovarian cancercompared to actual standard options

Combinationwith nonplatinumagents (trabectedin) andantiangiogenetic drugs (bevacizumab) represents an alterna-tive treatment option in the recurrent setting associated incertain cases with remarkable toxicity New target therapy isunder evaluation in combination with PLD

Acknowledgments

The authors thank Dr Valeria Trocino for bibliographyassistance andMrs Balbina Apice and Antonietta Linardi forthe help in editing the paperThiswork has been partially sup-ported by the Associazione Italiana per la Ricerca sul Cancro(AIRC)

References

[1] B T Hennessy R L Coleman andMMarkman ldquoOvarian can-cerrdquoThe Lancet vol 374 no 9698 pp 1371ndash1382 2009

[2] F A Raja N Chopra and J A Ledermann ldquoOptimal first-linetreatment in ovarian cancerrdquo Annals of Oncology vol 23 sup-plement 10 pp x118ndashx127 2012


[3] S M Eisenkop N M Spirtos R L Friedman W C M Lin AL Pisani and S Perticucci ldquoRelative influences of tumor vol-ume before surgery and the cytoreductive outcome on survivalfor patients with advanced ovarian cancer a prospective studyrdquoGynecologic Oncology vol 90 no 2 pp 390ndash396 2003

[4] R F Ozols ldquoSystemic therapy for ovarian cancer current statusand new treatmentsrdquo Seminars in Oncology vol 33 no 2 sup-plement 6 pp S3ndashS11 2006

[5] R A Burger M F Brady M A Bookman et al ldquoIncorporationof bevacizumab in the primary treatment of ovarian cancerrdquoTheNew England Journal of Medicine vol 365 no 26 pp 2473ndash2483 2011

[6] T J Perren A M Swart J Pfisterer et al ldquoA phase 3 trial ofbevacizumab in ovarian cancerrdquo The New England Journal ofMedicine vol 365 no 26 pp 2484ndash2496 2011

[7] M Friedlander E Trimble A Tinker et al ldquoClinical trials inrecurrent ovarian cancerrdquo International Journal of GynecologicalCancer vol 21 no 4 pp 771ndash775 2011

[8] G C Stuart H Kitchener M Bacon et al ldquo2010 GynecologicCancer Inter Group (GCIG) consensus statement on clinicaltrials in ovarian cancer report from the FourthOvarian CancerConsensus Conference participants of 4th Ovarian CancerConsensus Conference (OCCC) Gynecologic Cancer Inter-grouprdquo International Journal of Gynecological Cancer vol 21 no4 pp 750ndash755 2011

[9] G A Omura M Buyse S Marsoni et al ldquoCyclophosphamideplus cisplatin versus cyclophosphomide doxorubicin and cis-platin chemotherapy of ovarian carcinoma a meta-analysisrdquoJournal of Clinical Oncology vol 9 no 9 pp 1668ndash1674 1991

[10] R ArsquoHern and M E Gore ldquoThe impact of doxorubicin on sur-vival in advanced ovarian cancerrdquo Journal of Clinical Oncologyvol 13 pp 726ndash732 1995

[11] H J Luck A Du Bois B Weber et al ldquoThe integration ofanthracyclines in the treatment of advanced ovarian cancerrdquoInternational Journal of Gynecological Cancer vol 11 supple-ment 1 pp 34ndash38 2001

[12] L Gianni E H Herman S E Lipshultz G Minotti N Sar-vazyan and D B Sawyer ldquoAnthracycline cardiotoxicity frombench to bedsiderdquo Journal of Clinical Oncology vol 26 no 22pp 3777ndash3784 2008

[13] AAGabzon ldquoPegylated liposomal doxorubicinmetamorpho-sis of an old drug into a new form of chemotherapyrdquo CancerInvestigation vol 19 no 4 pp 424ndash436 2001

[14] S T Duggan and G M Keating ldquoPegylated liposomal doxoru-bicin a review of its use in metastatic breast cancer ovariancancer multiple myeloma and AIDS-related Kaposirsquos sarcomardquoDrugs vol 71 no 18 pp 2531ndash2558 2011

[15] A Gabizon H Shmeeda and Y Barenholz ldquoPharmacokineticsof pegylated liposomal doxorubicin review of animal andhuman studiesrdquo Clinical Pharmacokinetics vol 42 no 5 pp419ndash436 2003

[16] D N Waterhouse P G Tardi L D Mayer and M B BallyldquoA comparison of liposomal formulations of doxorubicin withdrug administered in free form changing toxicity profilesrdquoDrug Safety vol 24 no 12 pp 903ndash920 2001

[17] R K Jain ldquoNormalization of tumor vasculature an emergingconcept in antiangiogenic therapyrdquo Science vol 307 no 5706pp 58ndash62 2005

[18] A N Gordon C O Granai P G Rose et al ldquoPhase II studyof liposomal doxorubicin in platinum- andpaclitaxel-refractoryepithelial ovarian cancerrdquo Journal of Clinical Oncology vol 18no 17 pp 3093ndash3100 2000

[19] H Maeda H Nakamura and J Fang ldquoThe EPR effect formacromolecular drug delivery to solid tumors improvement oftumor uptake lowering of systemic toxicity and distinct tumorimaging in vivordquo Advanced Drug Delivery Reviews vol 65 no1 pp 71ndash79 2012

[20] F J Martin ldquoPegylated liposomal doxorubicin scientific ratio-nale and preclinical pharmacologyrdquoOncology vol 11 no 10 pp11ndash20 1997

[21] A Gabizon ldquoApplications of liposomal drug delivery systems tocancer therapyrdquo in Nanotechnology for Cancer Therapy chapter29 pp 595ndash611 CRC Press New York NY USA 2006

[22] A Gabizon R Catane B Uziely et al ldquoProlonged circulationtime and enhanced accumulation in malignant exudates ofdoxorubicin encapsulated in polyethylene-glycol coated lipo-somesrdquo Cancer Research vol 54 no 4 pp 987ndash992 1994

[23] M Amantea M S Newman T M Sullivan A Forrest and PK Working ldquoRelationship of dose intensity to the induction ofpalmar-plantar erythrodysesthia by pegylated liposomal doxo-rubicin in dogsrdquo Human and Experimental Toxicology vol 18no 1 pp 17ndash26 1999

[24] M A Amantea A Forrest D W Northfelt and R MamelokldquoPopulation pharmacokinetics and pharmacodynamics ofpegylated-liposomal doxorubicin in patients with AIDS-relatedKaposirsquos sarcomardquo Clinical Pharmacology andTherapeutics vol61 no 3 pp 301ndash311 1997

[25] FMMuggia J DHainsworth S Jeffers et al ldquoPhase II study ofliposomal doxorubicin in refractory ovarian cancer antitumoractivity and toxicity modification by liposomal encapsulationrdquoJournal of Clinical Oncology vol 15 no 3 pp 987ndash993 1997

[26] P G Rose J HawthorneMaxson N Fusco and KMossbrugerldquoLiposomal doxorubicin in ovarian peritoneal and tubal car-cinoma a retrospective comparative study of single-agent dos-agesrdquo Gynecologic Oncology vol 82 no 2 pp 323ndash328 2001

[27] C Arcuri R Sorio G Tognon et al ldquoA phase II study of lipo-somal doxorubicin in recurrent epithelial ovarian carcinomardquoTumori vol 90 no 6 pp 556ndash561 2004

[28] N Katsumata Y Fujiwara T Kamura et al ldquoPhase II clin-ical trial of pegylated liposomal doxorubicin (JNS002) inJapanese patients with mullerian carcinoma (Epithelial ovariancarcinoma primary carcinoma of fallopian tube peritonealcarcinoma) having a therapeutic history of platinum-basedchemotherapy a phase II study of the Japanese gynecologiconcology grouprdquo Japanese Journal of Clinical Oncology vol 38no 11 pp 777ndash785 2008

[29] G Gorumlu Y Kucukzeybek M Kemal-Gul et al ldquoPegylatedliposomal doxorubicin in heavily pretreated epithelial ovariancancer patientsrdquo Journal of BUON vol 13 no 3 pp 349ndash3522008

[30] I Steppan D Reimer U Sevelda H Ulmer C Marth and AG Zeimet ldquoTreatment of recurrent platinum-resistant ovariancancer with pegylated liposomal doxorubicinmdashan evaluation ofthe therapeutic index with special emphasis on cardiac toxicityrdquoChemotherapy vol 55 no 6 pp 391ndash398 2009

[31] MMarkman A Kennedy KWebster G Peterson B Kulp andJ Belinson ldquoPhase 2 trial of liposomal doxorubicin (40mgm2)in platinumpaclitaxel-refractory ovarian and fallopian tubecancers and primary carcinoma of the peritoneumrdquoGynecologicOncology vol 78 no 3 pp 369ndash372 2000

[32] S M Campos R T Penson A R Mays et al ldquoThe clinicalutility of liposomal doxorubicin in recurrent ovarian cancerrdquoGynecologic Oncology vol 81 no 2 pp 206ndash212 2001


[33] S Wilailak and V Linasmita ldquoA study of pegylated liposomaldoxorubicin in platinum-refractory epithelial ovarian cancerrdquoOncology vol 67 no 3-4 pp 183ndash186 2004

[34] O Lyass A Hubert and A A Gabizon ldquoPhase I study of Doxil-cisplatin combination chemotherapy in patients with advancedmalignanciesrdquo Clinical Cancer Research vol 7 no 10 pp 3040ndash3046 2001

[35] D Lorusso A Naldini A Testa G DrsquoAgostino G Scambiaand G Ferrandina ldquoPhase II study of pegylated liposomaldoxorubicin in heavily pretreated epithelial ovarian cancerpatients may a new treatment schedule improve toxicity pro-filerdquo Oncology vol 67 no 3-4 pp 243ndash249 2004

[36] J Sehouli O Camara M Schmidt et al ldquoPegylated liposomaldoxorubicin (CAELYX) in patients with advanced ovariancancer results of a German multicenter observational studyrdquoCancer Chemotherapy and Pharmacology vol 64 no 3 pp 585ndash591 2009

[37] A du Bois J Pfisterer N Burchardi et al ldquoCombinationtherapy with pegylated liposomal doxorubicin and carboplatinin gynecologic malignancies a prospective phase II study ofthe Arbeitsgemeinschaft Gynaekologische Onkologie Studi-engruppe Ovarialkarzinom (AGO-OVAR) and KommissionUterus (AGO-K-Ut)rdquo Gynecologic Oncology vol 107 no 3 pp518ndash525 2007

[38] B L Rapoport D A Vorobiof C Slabber A S Alberts H SHlophe and C Mohammed ldquoPhase II study of pegylated lipo-somal doxorubicin and carboplatin in patients with platinum-sensitive and partially platinum-sensitive metastatic ovariancancerrdquo International Journal of Gynecological Cancer vol 19no 6 pp 1137ndash1141 2009

[39] JM Ferrero BWeber J F Geay et al ldquoSecond-line chemother-apy with pegylated liposomal doxorubicin and carboplatin ishighly effective in patients with advanced ovarian cancer in laterelapse a GINECO phase II trialrdquo Annals of Oncology vol 18no 2 pp 263ndash268 2007

[40] M O Nicoletto C Falci D Pianalto et al ldquoPhase II study ofpegylated liposomal doxorubicin and oxaliplatin in relapsedadvanced ovarian cancerrdquo Gynecologic Oncology vol 100 no2 pp 318ndash323 2006

[41] G DrsquoAgostino G Ferrandina M Ludovisi et al ldquoPhase IIstudy of liposomal doxorubicin and gemcitabine in the salvagetreatment of ovarian cancerrdquo British Journal of Cancer vol 89no 7 pp 1180ndash1184 2003

[42] G Ferrandina I Paris M Ludovisi et al ldquoGemcitabine andliposomal doxorubicin in the salvage treatment of ovarian can-cer updated results and long-term survivalrdquoGynecologic Oncol-ogy vol 98 no 2 pp 267ndash273 2005

[43] M Verhaar-Langereis A Karakus M Van Eijkeren E Voestand E Witteveen ldquoPhase II study of the combination of pegy-lated liposomal doxorubicin and topotecan in platinum-resis-tant ovarian cancerrdquo International Journal of Gynecological Can-cer vol 16 no 1 pp 65ndash70 2006

[44] S M Campos U A Matulonis R T Penson et al ldquoPhase IIstudy of liposomal doxorubicin and weekly paclitaxel for recur-rentMullerian tumorsrdquoGynecologic Oncology vol 90 no 3 pp610ndash618 2003

[45] D Katsaros M V Oletti I A Rigault de la Longrais et alldquoClinical and pharmacokinetic phase II study of pegylatedliposomal doxorubicin and vinorelbine in heavily pretreatedrecurrent ovarian carcinomardquo Annals of Oncology vol 16 no2 pp 300ndash306 2005

[46] F Joly E Sevin A Lortholary et al ldquoAssociation of pegylatedliposomal doxorubicin and ifosfamide in early recurrent ovar-ian cancer patients a multicenter phase II trialrdquo GynecologicOncology vol 116 no 3 pp 312ndash316 2010

[47] K J OrsquoByrne P Bliss J D Graham et al ldquoA Phase III study ofDoxilCaylex versus paclitaxel in platinum treated taxane naiverelapsed ovarian cancerrdquo Journal of Clinical Oncology vol 21abstract 808 2002 ASCO Annual Meeting


[49] A N Gordon M Tonda S Sun and W Rackoff ldquoLong-termsurvival advantage for women treated with pegylated liposomaldoxorubicin compared with topotecan in a phase 3 randomizedstudy of recurrent and refractory epithelial ovarian cancerrdquoGynecologic Oncology vol 95 no 1 pp 1ndash8 2004

[50] D G Mutch M Orlando T Goss et al ldquoRandomized phase IIItrial of gemcitabine compared with pegylated liposomal doxo-rubicin in patients with platinum-resistant ovarian cancerrdquoJournal of Clinical Oncology vol 25 no 19 pp 2811ndash2818 2007

[51] G Ferrandina M Ludovisi D Lorusso et al ldquoPhase III trial ofgemcitabine compared with pegylated liposomal doxorubicinin progressive or recurrent ovarian cancerrdquo Journal of ClinicalOncology vol 26 no 6 pp 890ndash896 2008

[52] B J Monk T Herzog S Kaye et al ldquoA randomized PhaseIII study of trabectedin with pegylated liposomal doxorubicin(PLD) versus PLD in relapsed ovarian cancer (OC)rdquo Annals ofOncology vol 22 no 1 pp 39ndash48 2011

[53] MMarkman J Moon SWilczynski et al ldquoSingle agent carbo-platin versus carboplatin plus pegylated liposomal doxorubicinin recurrent ovarian cancer final survival results of a SWOG(S0200) phase 3 randomized trialrdquo Gynecologic Oncology vol116 no 3 pp 323ndash325 2010

[54] E Pujade-Lauraine U Wagner E Aavall-Lundqvist et alldquoPegylated liposomal doxorubicin and carboplatin comparedwith paclitaxel and carboplatin for patients with platinum-sensitive ovarian cancer in late relapserdquo Journal of Clinical Onco-logy vol 28 no 20 pp 3323ndash3329 2010

[55] F M Muggia T Safra L Borgato et al ldquoPharmacokinetics(PK) of pegylated liposomal doxorubicin (PLD) given aloneand with bevacizumab (B) in patients with recurrent epithelialovarian cancer (rEOC)rdquo Journal of Clinical Oncology vol 28supplement abstract 5064 p 15s 2010 ASCOAnnual Meeting

[56] E Pujade-Lauraine F Hilpert and B Weber ldquoAURELIA arandomized phase III trial evaluating bevacizumab (BEV) pluschemotherapy (CT) for platinum (PT)-resistant recurrent ovar-ian cancer (OC)rdquo Journal of Clinical Oncology vol 30 supple-ment abstract LBA5002 2012 ASCO Annual Meeting

[57] M G del Carmen J Micha L Small et al ldquoA phase II clinicaltrial of pegylated liposomal doxorubicin and carboplatin plusbevacizumab in patients with platinum-sensitive recurrentovarian fallopian tube or primary peritoneal cancerrdquo Gyneco-logic Oncology vol 126 no 3 pp 369ndash374 2012

[58] K D Steffensen M Waldstroslashm N Pallisgard et al ldquoPani-tumumab and pegylated liposomal doxorubicin in platinum-resistant epithelial ovarian cancer with KRAS wild-type thePaLiDo study a phase II nonrandomized multicenter studyrdquoInternational Journal of Gynecological Cancer vol 23 no 1 pp73ndash80 2013

[59] httpclinicaltrialsgovshowNCT01281254


[60] M Boers-Sonderen I Desar W T A Van Der Graaf et alldquoA phase Ib study of the combination of temsirolimus (T)and pegylated liposomal doxorubicin (PLD) in advanced orrecurrent breast endometrial and ovarian cancerrdquo Journal ofClinical Oncology vol 30 supplement abstract 5061 2012ASCO Annual Meeting

[61] M Lotem A Hubert O Lyass et al ldquoSkin toxic effects ofpolyethylene glycol-coated liposomal doxorubicinrdquo Archives ofDermatology vol 136 no 12 pp 1475ndash1480 2000

[62] D S Alberts F M Muggia J Carmichael et al ldquoEfficacy andsafety of liposomal anthracyclines in Phase III clinical trialsrdquoSeminars in Oncology vol 31 supplement 13 pp 53ndash90 2004

[63] A A Gabizon ldquoLiposomal anthracyclinesrdquo HematologyOnco-logy Clinics of North America vol 8 no 2 pp 431ndash450 1994


[65] G Batist G Ramakrishnan C S Rao et al ldquoReduced car-diotoxicity and preserved antitumor efficacy of liposome-en-capsulated doxorubicin and cyclophosphamide compared withconventional doxorubicin and cyclophosphamide in a random-ized multicenter trial of metastatic breast cancerrdquo Journal ofClinical Oncology vol 19 no 5 pp 1444ndash1454 2001

[66] M E R OrsquoBrien N Wigler M Inbar et al ldquoReduced cardio-toxicity and comparable efficacy in a phase III trial of pegylatedliposomal doxorubicin HCl (CAELYXDoxil) versus conven-tional doxorubicin for first-line treatment of metastatic breastcancerrdquo Annals of Oncology vol 15 no 3 pp 440ndash449 2004

[67] A A Gabizon O Lyass G J Berry and M Wildgust ldquoCar-diac safety of pegylated liposomal doxorubicin (DoxilCaelyx)demonstrated by endomyocardial biopsy in patients withadvanced malignanciesrdquo Cancer Investigation vol 22 no 5 pp663ndash669 2004

[68] MHMustafa ldquoDecreased risk of cardiotoxicity with long-termuse of doxilcaelyx at high lifetime cumulative doses in patientswith AIDS-related KaposiEs sarcoma (KS)rdquo Journal of ClinicalOncology vol 20 abstract 2915 2001 ASCO Annual Meeting


[70] E Andreopoulou D Gaiotti E Kim et al ldquoPegylated liposomaldoxorubicin HCL (PLD CaelyxDoxil) experience withlong-term maintenance in responding patients with recurrentepithelial ovarian cancerrdquo Annals of Oncology vol 18 no 4 pp716ndash721 2007

[71] G Oskay-Oezcelik D Koensgen H J Hindenburg et alldquoBiweekly pegylated liposomal doxorubicin as second-linetreatment in patients with relapsed ovarian cancer after failureof platinum and paclitaxel results from a multi-center phase IIstudy of the NOGGOrdquo Anticancer Research vol 28 no 2 B pp1329ndash1334 2008

[72] B L Rapoport D A Vorobiof C Slabber G Cohen A SAlberts and H S Hlophe ldquoPhase 2 study of combination ther-apy with liposomal doxorubicin and carboplatin in patientswith relapsed platinum sensitive ovarian cancerrdquo Journal of Cli-nical Oncology vol 23 supplement abstract 5555 p 471s 2004ASCO Annual Meeting

[73] P Power G Stuart A Oza et al ldquoEfficacy of pegylated lipo-somal doxorubicin (PLD) plus carboplatin in ovarian cancer

patientswho recurwithin six to twelvemonths a phase II studyrdquoGynecologic Oncology vol 114 no 3 pp 410ndash414 2009

[74] B Weber A Lortholary F Mayer et al ldquoPegylated liposomaldoxorubicin and carboplatin in late-relapsing ovarian cancer aGINECO group phase II trialrdquo Anticancer Research vol 29 pp4195ndash4200 2009

[75] K N Ganjoo and S R Patel ldquoTrabectedin an anticancer drugfrom the seardquo Expert Opinion on Pharmacotherapy vol 10 no16 pp 2735ndash2743 2009

[76] M von Mehren R J Schilder J D Cheng et al ldquoA phaseI study of the safety and pharmacokinetics of trabectedin incombination with pegylated liposomal doxorubicin in patientswith advancedmalignanciesrdquoAnnals of Oncology vol 19 no 10pp 1802ndash1809 2008

[77] S McMeekin J M del Campo N Colombo et al ldquoTrabectedin(T) in relapsed advanced ovarian cancer (ROC) a pooled anal-ysis of three phase II studiesrdquo Journal of Clinical Oncology vol25 no 18 supplement p 5579 2007 ASCO Annual Meeting

[78] A Poveda I Vergote S Tjulandin et al ldquoTrabectedin pluspegylated liposomal doxorubicin in relapsed ovarian canceroutcomes in the partially platinum-sensitive (platinum-freeinterval 6ndash12 months) subpopulation of OVA-301 phase III ran-domized trialrdquoAnnals of Oncology vol 22 no 1 pp 39ndash48 2011

[79] C N Krasner A Poveda T Herzog et al ldquoHealth-related qual-ity of lifepatient-reported outcomes in relapsed ovarian cancerresults from a randomized phase III study of trabectedin withpegylated liposomal doxorubicin (PLD) versus PLD alonerdquoJournal of Clinical Oncology vol 27 no 15 supplement abstract5526 2009 ASCO Annual Meeting

[80] European Medicines Agency (EMA) ldquoAssessment reportfor Yondelisrdquo International non-proprietary nameCommonname trabectedin Procedure no EMEAHC000773II00082009

[81] U Wagner C Marth R Largillier et al ldquoFinal overall survivalresults of phase IIIGCIGCALYPSO trial of pegylated liposomaldoxorubicin and carboplatin vs paclitaxel and carboplatin inplatinum-sensitive ovarian cancer patientsrdquo British Journal ofCancer vol 107 no 4 pp 588ndash591 2012

[82] L Gladieff A Ferrero G De rauglaudre et al ldquoCarboplatin andpegylated liposomal doxorubicin versus carboplatin and pacli-taxel in partially platinum-sensitive ovarian cancer patientsresults from a subset analysis of the CALYPSO phase III trialrdquoAnnals of Oncology vol 23 no 5 pp 1185ndash1189 2012

[83] M A Bookman B E Greer and R F Ozols ldquoOptimal therapyof advanced ovarian cancer carboplatin and paclitaxel vscisplatin and paclitaxel (GOG 158) and an update onGOG0182-ICON5rdquo International Journal of Gynecological Cancer vol 13no 6 pp 735ndash740 2003

[84] S Pignata G Scambia G Ferrandina et al ldquoCarboplatin pluspaclitaxel versus carboplatin plus pegylated liposomal doxoru-bicin as first-line treatment for patients with ovarian cancerthe MITO-2 randomized phase III trialrdquo Journal of ClinicalOncology vol 29 no 27 pp 3628ndash3635 2011

[85] F M Muggia L Boyd L Liebes et al ldquoPegylated liposomaldoxorubicin (PLD) with bevacizumab (B) in second-line treat-ment of ovarian cancer (OC) pharmacokinetics (PK) safetyand preliminary outcome resultsrdquo Journal of Clinical Oncologyvol 27 supplement abstract 5548 p 15s 2009 ASCO AnnualMeeting

[86] K H Kim D Jelovac D Kay Armstrong et al ldquoPhase I safetystudy of farletuzumab carboplatin and pegylated liposomal


doxorubicin (PLD) in patients with platinum-sensitive epithe-lial ovarian cancer (EOC)rdquo vol 30 supplement abstract 50622012

[87] WD Foulkes ldquoBRCA1 andBRCA2 chemosensitivity treatmentoutcomes and prognosisrdquo Familial Cancer vol 5 pp 135ndash1422006

[88] S Lafarge V Sylvain M Ferrara and Y J Bignon ldquoInhibitionof BRCA1 leads to increased chemoresistance to microtubule-interfering agents an effect that involves the JNK pathwayrdquoOncogene vol 20 no 45 pp 6597ndash6606 2001

[89] S B Kaye J Lubinski U Matulonis et al ldquoPhase II open-labelrandomized multicenter study comparing the efficacy andsafety of olaparib a poly (ADP-ribose) polymerase inhibitorand pegylated liposomal doxorubicin in patients with BRCA1or BRCA2 mutations and recurrent ovarian cancerrdquo Journal ofClinical Oncology vol 30 no 4 pp 372ndash379 2012

[90] S F Adams E B MarshW Elmasri et al ldquoA high response rateto liposomal doxorubicin is seen among women with BRCAmutations treated for recurrent epithelial ovarian cancerrdquoGyne-cologic Oncology vol 123 no 3 pp 486ndash491 2011

[91] T Safra L Borgato M O Nicoletto et al ldquoBRCA mutationstatus and determinant of outcome in women with recurrentepithelial ovarian cancer treated with pegylated liposomaldoxorubicinrdquoMolecular CancerTherapeutics vol 10 no 10 pp2000ndash2007 2011


Review ArticleLipid-Based Nanovectors for Targeting of CD44-OverexpressingTumor Cells

Silvia Arpicco1 Giuseppe De Rosa2 and Elias Fattal3

1 Dipartimento di Scienza e Tecnologia del Farmaco University of Torino Via Giuria 9 10125 Torino Italy2 Dipartimento di Farmacia University Federico II of Naples Via Domenico Montesano 49 80131 Napoli Italy3 Institut Galien Paris Sud UMR CNRS 8612 University of Paris-Sud 5 Rue Jean-Baptiste Clement 92290 Chatenay-Malabry France

Correspondence should be addressed to Elias Fattal eliasfattalu-psudfr



Copyright copy 2013 Silvia Arpicco et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Hyaluronic acid (HA) is a naturally occurring glycosaminoglycan that exists in living systems and it is a major component ofthe extracellular matrix The hyaluronic acid receptor CD44 is found at low levels on the surface of epithelial haematopoietic andneuronal cells and is overexpressed inmany cancer cells particularly in tumour initiating cells HA has been therefore used as ligandattached to HA-lipid-based nanovectors for the active targeting of small or large active molecules for the treatment of cancer Thispaper describes the different approaches employed for the preparation characterization and evaluation of these potent deliverysystems

1 CD44 Receptor

CD44 (cluster of differentiation 44) is a widely expressed cellsurface hyaluronan receptor which consists in a single chaintransmembrane glycoprotein with a size that varies between80 and 200 kDa It is moreover an acidic molecule with anisoelectric point between 42 and 58 [1] CD44 receptorbelongs to the family of cell adhesion molecules (CAMs)together with selectins integrins and cadherins The CAMscontrol cell behavior by mediating contact between cells orbetween cells and the extracellular matrix and are essentialfor maintaining tissue integrity Because of these importantfunctions they are also involved in pathological conditionsincluding tumor progression and metastasis [2] It is wellknown that various tumors for example epithelial ovariancolon stomach and acute leukemia overexpress CD44 [3]

CD44 comprise a family of glycoproteins encoded bya single gene located on the short arm of chromosome11 and composed of 20 exons [4] Extensive alternativesplicing generatesmultiple variant isoforms of CD44 receptordenoted as CD44v The most abundant standard isoformof human CD44 protein is the smallest isoform that lacksany variant exons designated CD44s but some epithelial

cells also express a larger isoform called CD44E [5] Theexpression of CD44 isoforms containing combinations ofthe other variant exons is far more restricted in normaltissues In particular CD44s is abundantly expressed by bothnormal and cancer cells whereas the variant CD44 isoforms(CD44v) that contain a variable number of exon insertions(v1ndashv10) at the proximal plasma membrane external regionare expressed mostly by cancer cells

CD44 is endogenously expressed at low levels on variouscell types of normal tissues [6 7] but requires activationbefore binding to hyaluronan [8ndash11]

The CD44 structure of normal cells is distinct from thatof cancer cells because pathological conditions promote alter-nate splicing and posttranslational modifications to producediversified CD44 molecules with increased tumorigenicity[22 23]

The effect of native hyaluronan as well as of the catabolicenzymes and the degradation products of thismacromoleculeon tumor progression is complex Moreover the amountof intratumoral hyaluronan also varies depending on thecell type and on the degree of tumor cell differentiationThere are some good reviews that describe the associationof CD44 receptor with human cancer cells and underline the


D-glucuronic acid

119873-acetyl-D-glucosamine

O

O

OO

OO

O

O

OO

H

HH

H

HHNH

NHH

H

HH

H

HH

H

HH

HH

HH

OH

OH

OH

OHHO

HO HO

HO

CH3CH3

HOOCHOOC

119899

Figure 1 Chemical structure of HA

receptorrsquos role in the progression of the disease [10 24] thusthe overexpression of CD44 could be a good tool in drugdelivery approaches using the receptor as an anchor to attachthrough a ligand prodrugs or nanomedicine-based deliverysystems to increase the efficiency of anticancer drugs [25]

2 Hyaluronic Acid

Hyaluronic acid (hyaluronan HA) is a nonsulfated gly-cosaminoglycan polymer It is ubiquitous being the maincomponent of extracellular matrix [26] HA is composedof disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine linked together through alternating120573

13and 12057314

glycosidic bonds (Figure 1) HA is a biodegradable polymerwith a molecular weight of 106ndash107Da that is biocompatiblenontoxic hydrophilic and nonimmunogenic [27] MoreoverHA molecules have a number of sites suitable for chemi-cal modification such as hydroxyl carboxyl and 119873-acetylgroups

In adult tissues such as the vitreous synovial fluid anddermis hyaluronan plays an extracellular structural rolethat depends on its hydrodynamic properties as well as onits interactions with other extracellular matrix componentsHowever it is also concentrated in regions of high celldivision and invasion (during embryonic morphogenesisinflammation wound repair and cancer) Hyaluronic acid isthus also involved in tumorigenesis and its role is complexanddepends on various factors such as for example itsmolec-ular weight In fact lower molecular weight HA (10ndash100 kDa)stimulates angiogenesis but high molecular weight hyaluro-nan (gt1000 kDa) is inhibitory [28ndash30] High amount of HAproduction usually promotes tumor progression but it wasobserved that extremely high levels of hyaluronan productioncan be inhibitory [31] As also reported tumor progressionis often correlated with both hyaluronan and hyaluronidaselevels in human cancers [32] These considerations led to thehypothesis that the turnover of HA is strictly involved in thepromotion of tumor progression by HA [33ndash35]

In addition to its principal and previously describedreceptor CD44 HA also interacts with other cell sur-face receptors such as RHAMM (receptor for hyaluronan-mediated motility CD168) ICAM-1 (intracellular adhesion

molecule-1) TLR-4 (toll-like receptor-4) HARE (HA recep-tor for endocytosis) and LYVE-1 (lymphatic vessel endocyticreceptor)

The mechanism of HA-CD44 binding is still not fullyunderstood but it has been reported that the CD44 receptorcontains the specific binding domain for HA which consistsof 160 amino acid residues The binding affinity of CD44to HA was found to be dependent on the size of HAoligomers In fact hexamer and decamer are considered tobe the minimum size able to bind to CD44 while largeroligomers (20) have higher binding affinity because of theirmultiple interactions with more than one CD44 receptorsimultaneously [3 8 36 37]

It has also been reported that all the CD44 isoforms haveuniform affinity for HA [38] therefore HA can be used asvector for the active targeting of anticancer drugs Differentstrategies have been exploited with interesting results forexample in the preparation of bioconjugates obtained bycovalently linkingHA to a cytotoxic drug such as for examplepaclitaxel [39 40] or doxorubicin [41 42]These topics are outof the scope of this paper where only strategies consisting inthe design of HA decorated nanosystems will be discussed indepth

3 Chemical Conjugation of HA toLipid-Based Nanocarriers

Different approaches can be used to bind HA to the lipid-based nanocarriers depending on the molecular weight ofthe HA as well as on the need to start from preformednanocarriers or from pure lipids that will be then used toprepare particles

HA binding to preformed nanocarriers was the firstlyused method [43] and offers the advantage to conjugate theHA only on the external surface of the particle Of coursethis approach makes difficult the control of the density ofattachment of HA on the carrier surface Moreover thelower specificity of the linkage due to the possibility to binddifferent amino groups results in a consequent multipointattachment of the polymer on the nanocarrier that is thendifficult to characterize


Alternatively HA can be previously conjugated to apure lipid and then added in the lipid mixture during thepreparation of the nanoparticles This procedure permits theintroduction of a controlled amount of HA on nanocarriersbut could require a more elaborated synthetic method

31 HA Binding to Preformed Nanocarrier High molecularweight (HMW) HA was attached to the surface of preformedliposomes through amidation reaction between the aminore-active group of a lipid on the liposome surface generallya phosphatidylethanolamine (PE) and HA glucuronic car-boxylate (Figure 2) [13 14 43] The amidation reaction wasperformedpreactivatingHAby incubationwith the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) condensingagent in acidic medium and then adding the activated HA tothe nanocarrier suspension in a basic medium Eliminationof the excess of reagent and reaction byproducts was obtainedby centrifugation and repeated washing

32 Preparation of HA-PE Preformed Conjugates HA conju-gation to the lipid before nanocarrier preparation was carriedout with both high and low molecular weight (LMW) poly-mers [12 19] In all cases HA reacted with an aminoreactivegroup present on the lipid that was PE also in this case(Figure 2) Two different conjugation methods have beenproposed depending on the HA molecular weight Eliazand Szoka attached a mixture of oligosaccharide HA toPE by reductive amination using sodium cyanoborohydrideas reducing agent [12] Reductive amination is a chemicalreaction widely used in polysaccharide conjugation andconsists in two steps In the first step the aldehydic groupof the terminal residue of HA generated by opening thesugar ring reacts in acidic medium with the amino groupof PE forming the unstable imineThen the imine is reducedin the presence of a reducing agent to a secondary amineleading to the formation of the conjugate An improvementof this reaction was proposed by the same group in 2006 [44]The authors developed a methodology for the preparation ofaldehyde functionalized HA and reported that the reductiveamidination with this derivative is more efficient than thatperformed using the classical approach consisting in thereaction at the sugar reducing end

In these reactions involving LMW-HA only one PEmolecule was linked to the polymer Both kinds of conjugateswere purified by silica column chromatography and the latterwas characterized by MALDI and 1HNMR

HMW-HA-dioleoylphosphatidylethanolamine (DOPE)conjugate was prepared by EDC-mediated amidationreaction [19] In this conjugate the DOPE amino groupis randomly linked to the carboxylic residues of HA Theconjugate was purified by ultrafiltration and dialysis andits purity was assessed by capillary electrophoresis [20]This conjugate was introduced into cationic lipids duringliposome formation [19ndash21]

A similar synthetic approach was used by Toriyabeet al [45] for the preparation of a conjugate between HAand stearylamine (HA-SA conjugate) SA was linked via anamide linkage using EDC and NHS as coupling agents then

+

HA

(a)

+

PE HA-PE preformed conjugate

HA

(b)

Figure 2 Strategies to prepare HA-coated nanocarriers Aschematic representation (a) HA binding to preformed nanocarrierAmidation reaction betweenHA-carboxyl group and aminoreactivegroup of lipid on the liposome surface (b) Synthesis of HA-PE conjugates and following preparation of HA-coated lipidnanocarrier for postinsertion (i) Reductive amination at the HAreducing end (ii) Amidation reaction between HA-carboxyl groupand aminoreactive group of lipid (PE)

the solution of conjugate was added and incubated to theliposome suspension

Recently Cho et al described the preparation of anamphiphilic polymer obtained conjugating HA oligomersto a cellular component ceramide (CE) To obtain HA-CEconjugate HA was first activated by reaction with tetra-n-butylammoniumhydroxide (HA-TBA) and CE was previ-ously modified by esterification reaction with chloromethyl-benzoyl chloride used as linker Then linker CE was conju-gated to HA-TBA by ether bond formation [17]

4 Lipid-Based Nanocarriers for Targeting ofCD44-Rich Cells

First evidence of powerful delivery of chemotherapeuticsto cancer cells by HA-modified liposomes was providedby the group of Eliaz and Szoka [12] (Table 1) In thisstudy a low LMW-HA was bound onto the liposome sur-face The authors demonstrated B16F10 cells expressing highlevels of CD44 an avid cell-liposome binding followed byinternalization in a temperature-dependent manner Loweruptake was found in cells expressing low levels of CD44(CV-1) B16F10 cell association of the unilamellar vesicleswas found to depend critically on the density of HA onliposome surface These findings were observed after expos-ing cells to HA-modified liposomes in both transient (3 hand replacement with fresh cell medium) and continuousconditions for periods going up to 24 h [12] Moreover forgiven amounts of intracellular-delivered chemotherapeuticagent namely doxorubicin (DOX) the encapsulated formwas more efficient in killing B16F10 cells than the free form[12] Due to the enhanced potency of DOX encapsulatedinto HA-modified liposomes it was hypothesized that the


Table 1 Examples of HA-decorated lipid-based nanocarriers for targeting of CD44

Carrier Drug HA Main findings Reference

Liposomes DOX LMW-HA

Avid cell-liposome binding followed by internalization in cellsoverexpressing CD44Higher cytotoxicity compared with free drug onCD44-overexpressing cells

[12]

Liposomes MMCDOX HMW-HA

Higher affinity of HMW-HA to bind the CD44 receptorscompared to hyaluronan fragmentsLong-term circulation of HMW-HA liposomesHMW-HA can act as cryoprotectant thus allowing liposomelyophilizationLoading into the HA-modified liposomes generates a 100-foldincrease in drug potency in tumor cells overexpressing CD44receptorsHigher drug accumulation in tumor compared to free drug ordrug in unmodified liposomes

[13 14]

Self-assembled lipidnanoparticles PTX HMW-HA

Reduced PTX accumulation in liver and spleen and increaseddrug accumulation in the tumor compared to TaxolProlonged PTX half-lifeReduced PTX toxicity

[15]

HA-coatednanostructured lipidcarriers

PTX HMW-HAMore effective than Taxol with fewer side effectsProlonged PTX half-lifeIncreased PTX accumulation in tumors

[16]

Self-assemblednanoparticles DCT LMW-HA

Enhanced intracellular DCT uptake in theCD44-overexpressing cell linesMDR-overcoming effectsIn vivo specific CD44-mediated tumor targeting

[17]

PEGylated self-assemblednanoparticles DOX

Improved retention time in the bloodstream and nanoparticleaccumulation at the tumor sitePEGylation resulted in prolonged nanoparticle circulation andreduced DOX clearance rateHigher in vivo antitumor efficacy in the tumor xenograftmouse model in comparison to non-PEGylated nanoparticlesand DOX alone

[18]

Cationic liposomes DNA andsiRNA HMW-HA

The presence of HA-DOPE lipid conjugate in the liposomecomposition did not affect the lipoplex formationIncreased nucleic acid protection against enzymaticdegradationIncreased the level of transfection on CD44-highly expressingcells

[19ndash21]

Nanoparticles mdash Different molecularweights No induction of complement activation [18]

drug reaches a critical compartment more efficiently whencompared with the free form In particular the authorshypothesized that an uptake of the delivery system via a non-clathrin-coated endosome as already reported in the caseof hyaluronan catabolism could occur [46] This hypothesiswas recently confirmed by our group after incubating HA-modified cationic liposomes with CD44-expressing A549cells with different endocytosis inhibitors [20] It was foundthat the transfection efficiency of HA-modified cationic lipo-somes was not affected by a clathrin-mediated endocytosisinhibitor while it was significantly decreased by inhibitors of

caveolae-mediated endocytosis demonstrating that the latteris the main endocytosis pathway of HA-bearing lipoplexes Itis worthy of note that in the studies of Eliaz et al [47] andDufay Wojcicki et al [20] an LMW and an HMW-HA wereused respectively although a similar endocytotic pathwaycan be reasonably hypothesized

The targeting of cancer cells using HMW-HA bound toliposomes was firstly demonstrated by Peer and Margalit[13 14] HMW-HA should offer advantages such as to bindthe CD44 receptors with a higher affinity than hyaluronanfragments to provide long-term circulation through its many


hydroxyl residues and to allow liposome lyophilization dueto the properties of HA to act as a cryoprotectant [48]In particular in an in vivo study HA-modified liposomesresulted in long-circulating species over a time frame atleast equal to those reported for PEG-coated liposomes [13]Mitomycin C (MMC) a chemotherapeutic agent used indifferent form of tumors but also characterized by severeside effects was encapsulated into HA-modified liposomesand tested in vitro and in two experimental models of lungmetastases The in vitro studies showed that loading intothe HA-modified liposomes generates a 100-fold increase inMMC potency in tumor cells that overexpress hyaluronanreceptors but not in cells with poor expression of thesereceptors Moreover when using HA-modified liposomesMMC accumulated in the tumor 30-fold higher than whenthe drug was administered in free form and 4-fold higherthan when delivered via unmodified liposomes Interest-ingly liver uptake was significantly reduced when the drugwas delivered via the HA-modified liposomes that shouldcontribute to reducing the subacute toxicity associated withMMC administered as free drug [13] It is worthy of notethat in the case of MMC free or encapsulated in unmodifiedliposomes tumor size metastatic burden and survival timewere not much different than those observed in untreatedmice High positive responses were only reported in the caseof mice treated with MMC HA-modified liposomes Similarresults were obtained from different experimental modelof tumors with HA-modified liposomes but replacing theMMC with DOX thus demonstrating that the targeting iscarrier-specific rather than drug-specific [14] In this studythe HA-modified formulation was compared to free DOXDOX encapsulated in unmodified liposomes and pegylatedliposomes (Doxil) Drug accumulation in tumor-bearinglungs as well as key indicators of therapeutic responsessuch as tumor progression metastatic burden and survivalwas superior in animals receiving DOX-loaded HA-modifiedliposomes compared to the controls

HA-modified lipid-based nanoparticles encapsulatingpaclitaxel (PXT) were also proposed PXT is a chemothera-peutic agent largely used in the treatment of solid tumorsHowever its poor water solubility as well as the lack ofselective delivery approach represents important clinical lim-itations In vivo evidence of CD44 targeting by HA-modifiedlipid-based nanoparticles was also obtained by encapsulatingpaclitaxel (PXT) into self-assembled lipid nanoparticle-likeldquoclustersrdquo [15] Thus HA-coated PXT-encapsulating clusterswere administered in an experimental mice model of colonadenocarcinoma and their antitumor effect as well as thetoxicity was compared with that of FDA approved PXTformulations namely Taxol (PTX solubilized in the deter-gent Cremophor EL and in ethanol) and Abraxane (PXTencapsulated into albumin nanoparticles) Safety of the newHA-targeted formulation was demonstrated by any changein blood levels of enzymes released from the liver namelyalanine aminotransferase (ALT) and aspartate aminotrans-ferase (AST) respectively regarded as reliable indicatorsof liver tissue damage and more generally systemic tissuedamage This effect was not associated with any change inbody weight On the contrary multiple iv administrations

of Taxol resulted in changes of body weight and release ofhigh amounts of liver enzymes [15] Moreover when usingTaxol PXT was eliminated from the circulation within lessthan 1 h after iv injection while PTX administered withinHA-modified lipid clusters was still circulating even 24 hafter iv injection These findings still support the hypothesisthat HMW-HA when used as targetingmoieties also confersstealth properties on the nanoparticles Interestingly the HA-modified nanoparticles reduced PTX liver and spleen accu-mulation by almost 2-fold and increased PTX accumulationin the tumor by 10-fold compared to Taxol Finally tumorprogression was exponential in the case of 5mgKg bodyTaxol or Abraxane while it was arrested at the same dose ofPXT administered in HA-modified lipid clusters This effectwas also obtained with 20mgKg body of Taxol although itwas associated with a significant loss of body weight indi-cating global toxicity [15] Recently Yang et al proposed thepreparation of HA-coated nanostructured lipid carriers (HA-NLCs) for tumor targeting via electrostatic attraction [16] Inthis approach cationic NLCs loaded with PTXwere preparedby melt emulsion technology followed by coating with HA(300 kDa) the process of electrostatic attraction was simpleand controllable and no chemical reagents were neededThein vitro cytotoxicity and in vivo antitumoral activity studiesshowed that HA-PTX-NLCs were more effective than Taxolwith fewer side effects HA-NCL also prolonged the bloodcirculation time of PTX and increased its accumulation intumors

HA-modified nanoparticles have been proposed to over-come clinical limits of chemotherapeutics such as Docetaxel(DCT) DTC is a semisynthetic taxane derivative very effec-tive against different tumors but its clinical use causes severalside effects and other limitations regarding the appearanceof multidrug resistance (MDR) and its insolubility RecentlyCho et al described the preparation of HA-ceramide (CE)self-assembled nanoparticles for DCT and DOX active tar-geting [17 49] The in vitro cellular uptake studies showedthat nanoparticles enhanced intracellular DCT uptake in theCD44-overexpressing cell lines MCF-7 MDR-overcomingeffects of DCT nanoparticles were observed in cytotoxicitytest in CD44-positive MCF-7 breast cancer cells resistant todoxorubicin The in vivo tumor targetability was evaluatedusing a noninvasive fluorescence imaging system in the samecells xenografted in a mouse model To assess the uptakemechanismby a competitive inhibition assay CD44 receptorswere blocked with preinjection of high doses of HA Thefluorescence signal in the HA preinjected animal group waslower than that in no preinjection group for 24 h indicatinga probable reduction in nanoparticle uptake due to theblocking of CD44 The real-time imaging data showed thatthe fluorescent signal increased for the first 6 h and wasmaintained for 1 day Then the tumors were dissected 24 hfollowing injection and the observed fluorescence intensityof HA pre-injection group was only 439 of the no preinjec-tion group

The same research team described the preparation ofDOX-loaded self-assembled HA-CE-PEG-based nano-particles [18] In vitro tests were performed on two differentcell lines with different CD44 expression NIH3T3 (mouse


embryonic fibroblast cells CD44-negative) and SCC7(mouse squamous cell carcinoma cells CD44-positive) Thecytotoxicity studies showed that HA-CE-based nanoparticlescan be used as vehicle without important toxicity Thecellular uptake efficacy of DOX from nanoparticles viaHA and CD44 interaction was demonstrated by confocalmicroscopy analysis In vivo studies on SCC7 tumorxenograft mouse model showed improved retention timein the bloodstream and nanoparticle accumulation at thetumor site The pharmacokinetics evaluation confirmed thatPEGylation resulted in prolonged nanoparticle circulationand reduced DOX clearance rate Improved half-life of DOXwhen formulated as HA-CE-PEG nanoparticles led to higherin vivo antitumor efficacy in the tumor xenograft mousemodel in comparison to non-PEGylated nanoparticles andDOX alone

HA was also used to increase transfection efficiency ofcationic liposomes Plasmid DNA and siRNA were success-fully delivered to CD44-expressing cancer cells with thisapproach [19 21] The use of a lipid conjugate HA-DOPEinto the liposome composition did not affect the lipoplexformation upon liposome mixing with DNA [19] or siRNA[21] On the contrary the lipoplex zeta potential was stronglyaffected shifting from a positive to a negative value Thiswas consistent with the presence of HA at lipoplex surfaceMoreover the presence of HA in the liposome formulationled to increased nucleic acid protection from degradationagainst DNase I or RNAse V1 probably because the HMW-HA and cationic lipids prevent access of these enzymes tothe whole colloidal system [19 21] The presence of HA-DOPE did not modify the in vitro cytotoxicity on the MDA-MB-231 and MCF-7 breast cancer cell lines characterizedby high and low expressions of CD44 respectively Onthe contrary the use of HA strongly reduced the cytotoxicprofile of DOTAPDOPE liposomes in combination withsiRNA on A549 CD44-expressing cells [21] This effect wasattributed to the endogenous nature of HA that should bebiocompatible and when located on the lipoplex surfacemight avoid the direct contact of the cationic liposome withthe negatively charged cell surface and hence reduce itscytotoxic potential Finally HA-DOPE increased the level oftransfection on CD44-highly expressing cells (MDA-MB-231or A549) compared to the cells expressing low levels of CD44(MCF-7 or Calu-3) The involvement of the CD44 receptorswas confirmed by using anti-CD44 Hermes-1 antibody thathighly inhibited transfection efficiency this effect was notobserved by nonspecific anti-ErbB2 antibody [19 20]

HA-coated cationic liposomes were also prepared usinganHA-stearylamine (SA) conjugate and their ability to reachliver endothelial cells was evaluated [45] The pharmacoki-netics and biodistribution studies on HA-SA modified lipo-somes showed that liver accumulation was higher than thecorresponding value for nonmodified liposomes at every timepoint and increased depending on the extent of modificationof HA-SA On the contrary if free HA was introducedon liposomes surface via electrostatic interactions liveraccumulation decreased indicating that HA alone did notfully function as targeting ligand From confocal microscopyanalysis HA-SA modified liposomes accumulated along the

blood vessels to a greater extent than nonmodified liposomessuggesting that the HA-coated liposomes are distributedwithin endothelial cells in the liver

Recently the complement activation capacity of HAnanoparticles has been investigated [20 50] Complementactivation is an important aspect to consider since it mayinitiate adverse reactions among sensitive individuals andcould represent an obstacle for the clinical application of HA-decorated nanovectors Mizrahy et al evaluated the level ofthe terminal complement pathway activation markers C5aand SC5b-9 by ELISA on a panel of nanoparticles deco-rated with HA of different molecular weights (ranging from64 kDa to 1500 kDa) In these experiments no induction ofcomplement activation was observed and the marker levelsremained comparable with the baseline value [50] DufayWojcicki et al [20] evaluated the behavior of HA lipoplexesmade with increasing lipids DNA ratio (2 4 and 6) and theactivation of a protein of complement cascade the proteinC3 were determined by 2D immunoelectrophoresis Lowactivation of complement was observed in all the formula-tions although lipoplexes containing HA with lipids DNAratios of 4 and 6 give higher values than the respectivenonhyaluronate samples [20] These data suggest that HA-coated nanosystems could be an interesting alternative toPEG grafted particles since the latter were shown to activatecomplement after intravenous administration [51]

The impact of HA size and density of HA-graftednanoparticles on affinity toward CD44 was evaluated ina systematic manner [50 52] Qhattal and Liu preparedliposomes decorated with HA of both low and high molec-ular weights (5ndash8 10ndash12 175ndash350 and 1600 kDa) and withdifferent degree of grafting density They performed in vitrostudies (fluorescence microscopy analysis flow cytometricanalysis and competitive binding experiments) and statedthat cellular targeting efficiency of HA liposomes depends onHAmolecular weight grafting density and cell surface CD44receptor density In particular the HA liposomes binding andinternalization increased with increasing polymer molecularweight andor the grafting density [52] A small library ofHA-coated nanoparticles distinguished by the size of theirsurface HA was also described [50] The authors used HAof 5 different molecular weights (64 kDa 31 kDa 132 kDa700 kDa and 1500 kDa) and evaluated the nanoparticlesinteraction with CD44 receptor through surface plasmonresonance analysis Also in this case the affinity towardsCD44 was low for LMW-HA and increased with the polymermolecular weight [50]

5 Conclusions

HA represents a promising opportunity to develop new can-cer therapies A growing number of scientific works exploredthe possibility to target cancer cells overexpressing CD44receptor by usingHA-modified vectors HA is biocompatiblebiodegradable nontoxic and noninflammatory Moreover itcan easily undergo chemical modifications and conjugateswith drugs or other ligands HA targeting of cancer cells over-expressing CD44 receptor has been largely demonstrated In


addition HA coating has been recently proposed as a saferalternative to PEG grafting in order to increase the particlesrsquohalf-life The success of this strategy is demonstrated by anHA conjugate at the moment in clinical trials A phase IIIclinical trial based on a hyaluronic acid-Irinotecan conjugateis in the recruitment state and the final data collection isscheduled for January 2014 The possibility to conjugate HAto lipid-based nanocarriers such liposomes that are on longtime in the clinical practice should open new opportunitiesto target cancer cells also with drug that cannot be easilyconjugated to HA Further studies are certainly needed tounderstand the relations between the molecular weight andldquobiologicalrdquo properties of HA especially in the interaction ofHA-modified nanoparticles with the target

Moreover further information on the in vivo distributionof HA conjugated nanocarries as well as their tumor local-ization should be useful to design new anticancer therapiesbased on CD44 targeting

References

[1] S Goodison V Urquidi and D Tarin ldquoCD44 cell adhesionmoleculesrdquo Journal of Clinical Pathology vol 52 no 4 pp 189ndash196 1999

[2] V Orian-Rousseau ldquoCD44 a therapeutic target for metastasis-ing tumoursrdquo European Journal of Cancer vol 46 no 7 pp1271ndash1277 2010

[3] A J Day and G D Prestwich ldquoHyaluronan-binding proteinstying up the giantrdquo Journal of Biological Chemistry vol 277 no7 pp 4585ndash4588 2002

[4] P N Goodfellow G Banting M V Wiles et al ldquoThe geneMIC4 which controls expression of the antigen defined bymonoclonal antibody F10442 is on human chromosome 11rdquoEuropean Journal of Immunology vol 12 no 8 pp 659ndash6631982

[5] N Iida and L Y W Bourguignon ldquoNew CD44 splice variantsassociated with human breast cancersrdquo Journal of CellularPhysiology vol 162 no 1 pp 127ndash133 1995

[6] J Cichy and E Pure ldquoThe liberation of CD44rdquo Journal of CellBiology vol 161 no 5 pp 839ndash843 2003

[7] C R Mackay H J Terpe R Stauder W L Marston H Starkand U Gunthert ldquoExpression and modulation of CD44 variantisoforms in humansrdquo Journal of Cell Biology vol 124 no 1-2 pp71ndash82 1994

[8] J Lesley V C Hascall M Tammi and R Hyman ldquoHyaluronanbinding by cell surface CD44rdquo Journal of Biological Chemistryvol 275 no 35 pp 26967ndash26975 2000

[9] J Lesley and R Hyman ldquoCD44 can be activated to function asan hyaluronic acid receptor in normalmurine T cellsrdquoEuropeanJournal of Immunology vol 22 no 10 pp 2719ndash2723 1992

[10] R J S Sneath and D C Mangham ldquoThe normal structure andfunction of CD44 and its role in neoplasiardquo Journal of ClinicalPathology vol 51 no 4 pp 191ndash200 1998

[11] J Lesley Q He K Miyake A Hamann R Hyman and P WKincade ldquoRequirements for hyaluronic acid binding by CD44a role for the cytoplasmic domain and activation by antibodyrdquoJournal of Experimental Medicine vol 175 no 1 pp 257ndash2661992

[12] R E Eliaz and F C Szoka ldquoLiposome-encapsulated doxoru-bicin targeted to CD44 a strategy to kill CD44-overexpressingtumor cellsrdquoCancer Research vol 61 no 6 pp 2592ndash2601 2001

[13] D Peer and R Margalit ldquoLoading mitomycin C inside longcirculating hyaluronan targeted nano-liposomes increases itsantitumor activity in three mice tumor modelsrdquo InternationalJournal of Cancer vol 108 no 5 pp 780ndash789 2004

[14] D Peer andRMargalit ldquoTumor-targeted hyaluronan nanolipo-somes increase the antitumor activity of liposomal doxorubicinin syngeneic and human xenograft mouse tumor modelsrdquoNeoplasia vol 6 no 4 pp 343ndash353 2004

[15] I Rivkin K Cohen J Koffler D Melikhov D Peer andR Margalit ldquoPaclitaxel-clusters coated with hyaluronan asselective tumor-targeted nanovectorsrdquo Biomaterials vol 31 no27 pp 7106ndash7114 2010

[16] X-y Yang Y-x Li M Li L Zhang L-x Feng and NZhang ldquoHyaluronic acid-coated nanostructured lipid carriersfor targeting paclitaxel to cancerrdquo Cancer Letters 2012

[17] H J Cho H Y Yoon H Koo et al ldquoSelf-assembled nanoparti-cles based on hyaluronic acid-ceramide (HA-CE) and Pluronicfor tumor-targeted delivery of docetaxelrdquo Biomaterials vol 32no 29 pp 7181ndash7190 2011

[18] H-J Cho I-S Yoon H Y Yoon et al ldquoPolyethylene glycol-conjugated hyaluronic acid-ceramide self-assembled nanopar-ticles for targeted delivery of doxorubicinrdquo Biomaterials vol 33no 4 pp 1190ndash1200 2012

[19] C Surace S Arpicco A Dufay-Wojcicki et al ldquoLipoplexestargeting the CD44 hyaluronic acid receptor for efficient trans-fection of breast cancer cellsrdquo Molecular Pharmaceutics vol 6no 4 pp 1062ndash1073 2009

[20] A Dufay Wojcicki H Hillaireau T L Nascimento et alldquoHyaluronic acid-bearing lipoplexes physico-chemical charac-terization and in vitro targeting of the CD44 receptorrdquo Journalof Controlled Release vol 162 no 3 pp 545ndash552 2012

[21] S Taetz A Bochot C Surace et al ldquoHyaluronic acid-modifiedDOTAPDOPE liposomes for the targeted delivery of anti-telomerase siRNA to CD44-expressing lung cancer cellsrdquoOligonucleotides vol 19 no 2 pp 103ndash115 2009

[22] L Y W Bourguignon Z Hongbo L Shao and Y W ChenldquoCD44 interaction with Tiam1 promotes Rac1 signaling andhyaluronic acid- mediated breast tumor cell migrationrdquo Journalof Biological Chemistry vol 275 no 3 pp 1829ndash1838 2000

[23] D Naor S Nedvetzki I Golan L Melnik and Y FaitelsonldquoCD44 in cancerrdquo Critical Reviews in Clinical Laboratory Sci-ences vol 39 no 6 pp 527ndash579 2002

[24] R K Sironen M Tammi R Tammi P K Auvinen M Anttilaand V M Kosma ldquoHyaluronan in human malignanciesrdquoExperimental Cell Research vol 317 no 4 pp 383ndash391 2011

[25] S C Ghosh S Neslihan Alpay and J Klostergaard ldquoCD44 avalidated target for improved delivery of cancer therapeuticsrdquoExpert Opinion on Therapeutic Targets vol 16 no 7 pp 635ndash650 2012

[26] J W Kuo Practical Aspects of Hyaluronan Based MedicalProducts CRCTaylor amp Francis Boca Raton Fla USA 2006

[27] T C Laurent and J R E Fraser ldquoHyaluronanrdquo The FASEBJournal vol 6 no 7 pp 2397ndash2404 1992

[28] D C West and S Kumar ldquoHyaluronan and angiogenesisrdquo CibaFoundation Symposium vol 143 pp 187ndash201 1989

[29] R Montesano S Kumar L Orci andM S Pepper ldquoSynergisticeffect of hyaluronan oligosaccharides and vascular endothelialgrowth factor on angiogenesis in vitrordquo Laboratory Investiga-tion vol 75 no 2 pp 249ndash262 1996

[30] M Rahmanian H Pertoft S Kanda R Christofferson LClaesson-Welsh and P Heldin ldquoHyaluronan oligosaccharides


induce tube formation of a brain endothelial cell line in vitrordquoExperimental Cell Research vol 237 no 1 pp 223ndash230 1997

[31] N Itano T Sawai F Atsumi et al ldquoSelective expressionand functional characteristics of three mammalian hyaluronansynthases in oncogenic malignant transformationrdquo Journal ofBiological Chemistry vol 279 no 18 pp 18679ndash18687 2004

[32] V B Lokeshwar G L Schroeder M G Selzer et al ldquoBladdertumor markers for monitoring recurrence and screening com-parison of hyaluronic acid-hyaluronidase and BTA-stat testsrdquoCancer vol 95 no 1 pp 61ndash72 2002

[33] M A Simpson ldquoConcurrent expression of hyaluronan biosyn-thetic and processing enzymes promotes growth and vascu-larization of prostate tumors in micerdquo American Journal ofPathology vol 169 no 1 pp 247ndash257 2006

[34] D Liu E Pearlman E Diaconu et al ldquoExpression ofhyaluronidase by tumor cells induces angiogenesis in vivordquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 93 no 15 pp 7832ndash7837 1996

[35] B Delpech A Laquerriere C Maingonnat P Bertrand and PFreger ldquoHyaluronidase is more elevated in human brain metas-tases than in primary brain tumoursrdquo Anticancer Research vol22 no 4 pp 2423ndash2427 2002

[36] H Ponta L Sherman and P AHerrlich ldquoCD44 from adhesionmolecules to signalling regulatorsrdquo Nature Reviews MolecularCell Biology vol 4 no 1 pp 33ndash45 2003

[37] L Sherman J Sleeman P Herrlich andH Ponta ldquoHyaluronatereceptors key players in growth differentiation migration andtumor progressionrdquo Current Opinion in Cell Biology vol 6 no5 pp 726ndash733 1994

[38] L M Negi S Talegaonkar M Jaggi Z Iqbal and R KKhar ldquoRole of CD44 in tumour progression and strategies fortargetingrdquo Journal of Drug Targeting vol 20 no 7 pp 561ndash5732012

[39] Y Luo andG D Prestwich ldquoSynthesis and selective cytotoxicityof a hyaluronic acid-antitumor bioconjugaterdquo BioconjugateChemistry vol 10 no 5 pp 755ndash763 1999

[40] G Saravanakumar K Y Choi H Y Yoon et al ldquoHydrotropichyaluronic acid conjugates synthesis characterization andimplications as a carrier of paclitaxelrdquo International Journal ofPharmaceutics vol 394 no 1-2 pp 154ndash161 2010

[41] Y Luo N J Bernshaw Z R Lu J Kopecek and G D Prest-wich ldquoTargeted delivery of doxorubicin by HPMA copolymer-hyaluronan bioconjugatesrdquoPharmaceutical Research vol 19 no4 pp 396ndash402 2002

[42] L S Zhang W M Petroll H J Greyner and M E MummertldquoDevelopment of a hyaluronan bioconjugate for the topicaltreatment of melanomardquo Journal of Dermatological Science vol55 no 1 pp 56ndash59 2009

[43] N Yerushalmi A Arad and R Margalit ldquoMolecular andcellular studies of hyaluronic acid-modified liposomes as bioad-hesive carriers for topical drug delivery in wound healingrdquoArchives of Biochemistry and Biophysics vol 313 no 2 pp 267ndash273 1994

[44] D Ruhela K Riviere and F C Szoka ldquoEfficient synthesis ofan aldehyde functionalized hyaluronic acid and its applicationin the preparation of hyaluronan-lipid conjugatesrdquoBioconjugateChemistry vol 17 no 5 pp 1360ndash1363 2006

[45] N Toriyabe Y HayashiMHyodo andHHarashima ldquoSynthe-sis and evaluation of stearylated hyaluronic acid for the activedelivery of liposomes to liver endothelial cellsrdquo Biological andPharmaceutical Bulletin vol 34 no 7 pp 1084ndash1089 2011

[46] R Tammi K Rilla J P Pienimaki et al ldquoHyaluronan enterskeratinocytes by a novel endocytic route catabolismrdquo Journal ofBiological Chemistry vol 276 no 37 pp 35111ndash35122 2001

[47] R E Eliaz S Nir C Marty and F C Szoka ldquoDeterminationand modeling of kinetics of cancer cell killing by doxorubicinand doxorubicin encapsulated in targeted liposomesrdquo CancerResearch vol 64 no 2 pp 711ndash718 2004

[48] D Peer A Florentin and R Margalit ldquoHyaluronan is a keycomponent in cryoprotection and formulation of targetedunilamellar liposomesrdquo Biochimica et Biophysica Acta vol 1612no 1 pp 76ndash82 2003

[49] Y-J Jin U Termsarasab S-H Ko et al ldquoHyaluronic acidderivative-based self-assembled nanoparticles for the treatmentof melanomardquo Pharmaceutical Research vol 29 no 12 pp3443ndash3454 2012

[50] S Mizrahy S R Raz M Hasgaard et al ldquoHyaluronan-coatednanoparticles the influence of the molecular weight on CD44-hyaluronan interactions and on the immune responserdquo Journalof Controlled Release vol 156 no 2 pp 231ndash238 2011

[51] S M Moghimi I Hamad T L Andresen K Joslashrgensenand J Szebeni ldquoMethylation of the phosphate oxygen moi-ety of phospholipid- methoxy(polyethylene glycol) conjugateprevents PEGylated liposome-mediated complement activationand anaphylatoxin productionrdquoThe FASEB Journal vol 20 no14 pp 2591ndash2593 2006

[52] H S S Qhattal and X Liu ldquoCharacterization of CD44-mediated cancer cell uptake and intracellular distribution ofhyaluronan-grafted liposomesrdquo Molecular Pharmaceutics vol8 no 4 pp 1233ndash1246 2011


Review ArticleRecent Trends in Multifunctional Liposomal Nanocarriers forEnhanced Tumor Targeting

Federico Perche1 and Vladimir P Torchilin2

1 Center for Pharmaceutical Biotechnology and Nanomedicine Northeastern University 140 the Fenway Room 236360 Huntington Avenue Boston MA 02115 USA

2Center for Pharmaceutical Biotechnology and Nanomedicine Northeastern University 140 the Fenway Room 214360 Huntington Avenue Boston MA 02115 USA

Correspondence should be addressed to Vladimir P Torchilin vtorchilinneuedu


Academic Editor Tamer Elbayoumi

Copyright copy 2013 F Perche and V P Torchilin This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Liposomes are delivery systems that have been used to formulate a vast variety of therapeutic and imaging agents for the past severaldecades They have significant advantages over their free forms in terms of pharmacokinetics sensitivity for cancer diagnosis andtherapeutic efficacy The multifactorial nature of cancer and the complex physiology of the tumor microenvironment require thedevelopment of multifunctional nanocarriers Multifunctional liposomal nanocarriers should combine long blood circulation toimprove pharmacokinetics of the loaded agent and selective distribution to the tumor lesion relative to healthy tissues remote-controlled or tumor stimuli-sensitive extravasation from blood at the tumorrsquos vicinity internalization motifs to move from tumorbounds andor tumor intercellular space to the cytoplasm of cancer cells for effective tumor cell killing This review will focus oncurrent strategies used for cancer detection and therapy using liposomes with special attention to combination therapies

1 Introduction

Liposomes first described in 1965 [1 2] are establisheddrug and gene delivery carriers with clinical evidence ofefficacy [3ndash5] and several commercially available approvedclinical formulations [6] Liposomes are lipid vesicles eitherunilamellar or multilamellar with an aqueous compartmentThe structure of liposomes allows for delivery of a cargoloaded in the aqueous compartment or embedded in the lipidbilayer for cancer therapy noninvasive cancer imaging ortherapy [7 8] As recently reviewed [9] the most importantproperty of liposomal nanocarriers is protection from thedegradation and optimization of the pharmacokinetics ofthe encapsulated drug to improve tumor accumulation andtherapeutic efficacy while reducing the adverse effects asso-ciated with bolus administration [7 10 11] This paper willfocus on the use of liposomal nanocarriers in cancer therapyand diagnosis Cancer therapy targets the hallmark traitsof cancer deregulated cell growth evasion from apoptosissustained angiogenesis tissue invasion and metastasis [12]Liposomes remain one of the first drug delivery carrier tested

for improvement of pharmacokinetics of new anticancerdrugs withmore than 2000 papers and 200 reviews publishedin 2011 and many liposomal drugs approved for cancertherapy notably Doxil for doxorubicin (Johnson amp JohnsonNew Brunswick USA) Lipusu for paclitaxel (Luye PharmaGroup Yantai China) and Marqibo for vincristine (TalonTherapeutics South San Francisco USA) [7 13ndash15] Theliposomal platform has undergone continuous optimizationfor improved stability in vivo high drug andor imaging agentloading stimuli-targeted delivery of the cargo at the tumorsite for efficient uptake by cancer cells and intracellular pay-load release to engineer multifunctional liposomal nanocar-riers (Table 1 Figures 1ndash3) [16]Wewill describe themain axesof design of multifunctional liposomal nanocarriers

2 Stealth Targeted Liposomes

21 Stealth Liposomes Effective cancer treatment generallyimplies drug delivery to cancer cells after systemic adminis-tration by taking advantage of the leaky tumor vasculature


to deposit at the tumor site [17] Indeed liposome uptakeby tumors relies primarily on the enhanced permeabilityand retention (EPR) effect [13 17ndash19] EPR is dependenton large endothelial fenestrations in the tumor endothelialvasculature coupled with the incomplete pericyte coveragethat permits extravasation of large molecules and liposomesof size below 200 nm into tumors with an impaired lymphaticdrainage that is responsible for their retention [17 18 20]However after parenteral administration most liposomesare captured by the mononuclear phagocyte system (MPS)in the liver and spleen [21] This elimination is due to therecognition by serum proteins (opsonins) and complementcomponents which prime liposomes formacrophage removalfrom the circulation [21 22] The step required to increasethe probability of extravasation at the tumor site involvesextended stabilization decreased blood clearance and cap-ture by the MPS to favor their accumulation in tumors(Figure 2) [7 8 23]

To achieve this two approaches are currently used inpreclinical and clinical liposomal drug carriers [44] Decreaseof membrane fluidity through incorporation of cholesterol toimpede lipid extraction by high density lipoproteins in theblood associated with to liposome breakdown (approved for-mulations DaunoXome Myocet Depocyt Mariqibo Doxil)[44 45] The second approach is the incorporation of flexiblehydrophilicmoieties mainly polyethylene glycol(PEG) sincethis component is approved for use by the United StatesFood and Drug Administration and is currently used inseveral approved formulations (Doxil SPI-077 S-CDK602)[7 10 44 46] but also polyvinyl pyrrolidones [8] or Poly[N-(2-hydroxypropyl)methacrylamide] [47] The inclusion offlexible hydrophobic inert and biocompatible polyethyleneglycol (PEG) with a lipid anchor in liposome allows theformation of an hydrated steric barrier decreasing liposomeinteraction with blood-borne component increasing theirblood circulation time decreasing their spleen and livercapture [48 49] and their resistance to serum degradation[50] This lack of recognition by the MPS and decreasedelimination of PEGylated liposomes led to the term ldquostealthrdquoliposomes to qualify them [44]

Protection by PEG was shown to be dependent on boththe PEG molecular weight and density on the liposomesurfacewithsim5byweight allowing themaximal decrease inprotein adsorption and enhanced blood circulation time [51]Longer blood circulation time decreased spleen and livercapture and increased tumor accumulation after intravenousinjection have been reported for 111In-labeled liposomescontaining 6 PEG compared to 09 PEG [52] Lee etal compared the liver and spleen accumulation of 99mTc-labeled liposomes containing 0 5 96 or 137 PEG (molarratio) [53] While 5 or 96 PEG decreased spleen and liveraccumulation compared to unPEGylated liposomes spleenaccumulation increased again with 137 PEG indicating anupper limit to the effect of PEGylation When PEG chains ofdifferent lengths were appended to the surface of immunoli-posomes as short (750Da) intermediate (2000Da) or longPEG (5000Da) DSPE-PEG2000 was the best compromisefor extended blood circulation and target binding in vivo

PEG750 did not improve blood circulation and PEG5000decreased ligand binding [54]

Similarly superior interaction of cell penetrating peptide-modified PEGylated liposomes with cells was evidenced invitro after coupling of the peptide to PEG1000 over PEG750 orPEG3400 and was correlated with the architecture of ligandpresentation [55] The longer blood residency of PEGylatedliposomes associatedwith their lower elimination by theMPShas been correlated with increased tumor accumulation andefficacy [19 21 23 56] However liver spleen and bonemarrow remain the final destinations of empty or drug-loaded PEGylated liposomes [23 56] Improvement of drugpharmacokinetics and therapeutic efficacy after encapsula-tion in PEGylated liposomes was well illustrated by Yang etal [57] Indeed PEGylation of paclitaxel-loaded liposomesled to increased plasma and tumor levels of paclitaxel inparallel decreased liver and spleen paclitaxel levels over Taxolor conventional paclitaxel liposomes and resulted in the besttumor growth inhibition [57]

Interestingly albumin conjugation to drug-loaded PEGy-lated liposomes further enhanced their circulation time andresulting therapeutic activity [58 59] Indeed the blood clear-ance of doxorubicin after intravenous administration in ratsdecreased from 131mLh for free doxorubicin to 179mLh forPEGylated liposomal doxorubicin and decreased further to7mLh for PEGylated and albumin-conjugated doxorubicin-loaded liposomes Albumin also decreased opsonin bindingto PEGylated liposomes and improved the therapeutic activ-ity of doxorubicin-loaded liposomes against sarcoma

Inclusion of PEG in the liposome is achieved eitherby mixing a lipid-anchored PEG with the liposome form-ing lipids prior to liposome formation (preinsertion) orby insertion of PEG-lipid in already formed liposomes(postinsertion) These two approaches are currently usedin clinically approved formulations [44] Postinsertion ofDSPE-PEG2000 compared to its preinsertion in irinotecan-loaded liposomes revealed higher plasma concentration andslower drug release in rats [60] Of note this longer bloodcirculation time was correlated with better therapeutic effi-cacy of postinsertedDSPE-PEG2000 drug-loaded liposomesAlthough the lipid-PEG conjugates can be incorporated inliposomes before their formation (preinsertion) or insertedinto preformed liposomes the former strategy induces pre-sentation of the PEG groups both at the liposomal surfaceand in reverse orientation at the inner side of the lipidbilayer This results in decreased drug loading and stealthproperties of the liposomes Indeed when both strategiesof PEGylation were compared higher blood circulation andhigher therapeutic efficacy in vivo of postinsertion overpreinsertion modification were demonstrated [60 61]

A new alternative to increase the circulation time ofdrug-loaded liposomes is the use of superhydrophilic zwit-terionic polymers to create a hydrated shell around theliposome [62] Cao et al compared the therapeutic activity oftwo doxorubicin formulations Doxil where DSPE-PEG2000imparts blood stability and doxorubicin-loaded liposomescontaining the zwitterionic lipid DSPE-poly(carboxybetaine)for the same function Similar doxorubicin accumulationin tumors after intravenous administration was detected


Table 1 Examples of multifunctional liposomal nanocarriers

Encapsulated agent Targeting ligand Development stage ReferencesDoxorubicin None Approved (DoxilCaelyx) [13]Vincristine None Approved (Marqibo) [14]Paclitaxel None Approved (Lipusu) [15]Cytarabine and daunorubicin None Phase I (CPX-351) [24]Irinotecan and floxuridine None Phase I (CPX-1) [25]PKN3 siRNA None Phase I (Atu-027) [26]Irinotecan None Phase I (NL CPT-11) [27]Doxorubicin Stomach cancer-specific anti-GAH mAb Phase I (MCC-465) [28]Oxaliplatin Transferrin Phase II (MBP-426) [29]Liposomal p53 DNA and docetaxel Anti-Transferrin receptor scFv Phase I (SGT53-01) [30]Doxorubicin Thermoresponsive liposomes Phase III (ThermoDox) [31]Doxorubicin Cancer-specific 2C5 mAb preclinical [32]Doxorubicin Anti-CD22 mAb preclinical [33]Paclitaxel Anti-HER2 mAb preclinical [34]Vincristine mBAFF preclinical [35]Oxaliplatin Transferrin preclinical [36]Daunorubicin Transferrin and mannose preclinical [37]Vinorelbine NSCLC-specific peptide preclinical [38]Doxorubicin Metastasis-specific peptide preclinical [39]Doxorubicin MMP-29 detachable PEG preclinical [40]Irinotecan Folic acid preclinical [41]Doxorubicin Estrone preclinical [42]Etoposide Chondroitin sulfate preclinical [43]

for both formulations but poly(carboxybetaine) containingliposomes led to an earlier cure of tumor-bearing micevalidating this chemistry

211 Importance of Charge Neutralization for Passive Tar-geting Although neutral non-PEGylated radiolabeled lipo-somes were shown to accumulate in human tumors [63]PEGylation is required for effective tumor localizationPEGylation protected against aggregation of assembliesmadewith cationic lipids enhanced their tumor uptake anddecreased their accumulation in the liver [64] Campbell etal compared the biodistribution of negatively charged lipo-somes (minus20mV) and positively charged liposomes (+31mV)after intravenous injection to tumor-bearing mice [65]While liver was the major destination for both formula-tions with more than 50 of the injected dose positivelycharged liposomes showed lower spleen accumulation andhigher lung accumulation Interestingly in tumors positivelycharged liposomes showed higher association with tumorblood vessels than negatively charged ones Levchenko etal proposed the modulation of positively and negativelycharged liposomes biodistribution by different opsonins [66]Moreover neutral PEGylated liposomes encapsulating dox-orubicin showed superior therapeutic activity compared tocationic ones the decreased antitumor efficacy was correlatedwith reduced blood circulation and tumor accumulationof cationic liposomes [67] A critical correlation betweennegative liposome charge and uptake by liver and spleenhas been reported [66] charge shielding by PEG decreased

liver uptake and prolonged blood circulation Finally Huangand coworkers reported abolishment of liver uptake ofcationic liposomes after their neutralization by postinsertionof DSPE-PEG leading to an increased tumor accumulation[68]

212 Importance of Prior AdministrationAccelerated BloodClearance (ABC) Cancer treatments usually imply repeatedadministration of the same therapeutic agent to previouslytreated (predosed) patients Administration of radiolabeledPEGylated liposomes to animals pretreated with a first doseof PEGylated liposomes revealed a drastic decrease of theirblood concentration 4 h after injection from 50 of theinjected dose for naive animals to 06of the injected dose forpredosed animals [69] Noteworthy after the second admin-istration PEGylated liposomes were cleared from the circula-tion very rapidly (decrease in half-life from 24 h to 01 h) andthis decreased blood residency was mirrored by increasedaccumulation in liver and spleen supporting the acceleratedblood clearance of liposomes after their second administra-tionThis phenomenon is termed accelerated blood clearance(ABC) ABC is dependent on the time after initial injectionno ABC was reported for PEGylated liposomes injected dailyor with injection intervals less than 5 days in rats whereas aone week interval induced accelerated blood clearance in thesame study [69] This delay reflects the two phases of ABC[70 71] First anti-PEG IgM is secreted in the spleen duringthe effectuation phase [72 73] an organ where both drug-loaded PEGylated and non-PEGylated liposomes accumulate


[23 74] Second during the effectuation phase opsonisationof PEGylated liposomes by anti-PEG IgM primes them forelimination by liver macrophages [75] Tagami et al recentlydemonstrated that production of anti-PEG2000-DSPE IgMin mouse after administration of PEGylated lipoplexes washigher with PEGylated liposomes harboring siRNA on theirsurface over PEGylated liposome-wrapped siRNA lipoplexes[76] Moreover the same group reported higher anti-PEGIgM production after parenteral injection of PEGylated DNAlipoplexes prepared with adjuvant CpG motifs-containingpDNA over PEGylated lipoplexes prepared with pDNAdevoid of CpG motifs [77] This lower anti-PEG IgM pro-duction from CpG-free lipoplexes was correlated with loweraccelerated blood clearance Both of these studies suggestan important effect of the liposome cargo in anti-PEG IgMproduction and the ABC phenomenon

Anti-PEG IgM production is not limited to PEGylatedliposomes anti-PEG IgM was also detected in rats injectedwith PEGylated adenovirus bovine serum albumin or oval-bumin [78] Interestingly Laverman et al reported no ABCinduction of Doxil when rats were preinjected with Doxilone week before administration whereas preinjection withempty PEGylated liposomes induced ABC of Doxil [70]These data suggest prevention of ABCby doxorubicin entrap-ment in liposomes This has been attributed to a decreasedclearance capacity of Doxil-injected rats due to toxicity ofdoxorubicin for liver macrophages [79] By contrast VanEtten et al reported no decrease in bacterial clearance afterDoxil injection [80] suggesting a macrophage-independentmechanism Kiwada and coworkers reported the induction ofanti-PEG IgM production in the spleen after administrationof PEGylated liposomes priming them for elimination byliver macrophages and also demonstrated decreased ABC insplenectomized rats which was correlated with lower anti-PEG IgM titers [72]

Longer blood circulation of doxorubicin-loaded PEGy-lated liposomes after a second administration has beenobserved in mice dogs rats and patients [70 81ndash83] andwas proposed to be due to toxicity towards splenic B cells[70] The importance of toxicity in resistance to ABC byDoxil liposomes is supported by the suppression of IgMproduction after a second administration of oxaliplatin-loaded PEGylated liposomes compared to empty PEGylatedliposomes [84] and by the evidence of ABC induction withPEGylated topotecan-loaded liposomes that have a fast drugrelease rate [85] Additionally blood clearance of radiolabeledliposomes was inhibited by a preadministration of Doxilwhereas preinjection of free doxorubicin or empty liposomesdid not inhibit blood clearance [82] further supportinginhibition of the MPS as the mechanism of decreased bloodclearance of drug-loaded liposomes

However as pointed out recently by Suzuki et al thereis no report yet of ABC in patients [86] although PEGylatedliposomes such as Doxil have been in clinical use for morethan 20 years suggesting caution in interpretation of thepreclinical model data [86] Indeed Gabizon et al recentlyreported decreased blood clearance of Doxil after repeatedadministration in cancer patients [81] The high variabilityof pharmacokinetics of drug-loaded PEGylated liposomes

in cancer patients [87] should also be considered as it mayrender anABCphenomenon difficult to detect without a verylarge cohort Although complement activation by PEGylateddrug-loaded liposomes has been reported both in animalmodels and in patients (reviewed in [88]) its correlationwith accelerated blood clearance is still controversial [89]Finally ABC could be decreased after methylation of theanionic charge on the phosphate group of PEG [90] furtherimproving pharmacokinetics of PEGylated liposomes

22 Targeted Stealth Liposomes As recently reviewed PEGy-lation fails to lead to more than 5 of the administeredformulation accumulation in the tumor [23 91] Further-more although radiolabeled liposomes were shown to accu-mulate in solid tumors in patients they also distributed tonormal organs revealing the need for tumor targeting [63]Moreover most macromolecules free drugs and liposomeswithout an internalization moiety have an accumulationlimited to the periphery of a tumor due to the poor vasculardensity in tumors and the high tumor interstitial fluidpressure impeding transport of macromolecules [92ndash94] Ina direct comparison of doxorubicin-loaded PEGylated andnon-PEGylated liposomes PEGylation did not improve dox-orubicin accumulation in tumors with comparable therapeu-tic efficacy of PEGylated and non-PEGylated doxorubicin-loaded liposomes [95] On the contrary conjugation ofinternalizing antibodies with the surface of doxorubicin-loaded PEGylated liposomes dramatically improved theirtherapeutic efficacy [96 97] demonstrating the need forimproved internalization of antineoplastic agents for effectivetherapy [98] Similarly while Bartlett et al reported identicaltumor distribution of untargeted and transferrin-targetedsiRNA nanoparticles the latter achieved superior in vivosilencing [99]

To increase liposomal drug accumulation in the cancercells liposomes must combine small size and long circula-tion to reach the tumor (tumor site targeting) a targetingligand to discriminate between cancer cells and supportivecells (cancer cell targeting) and an internalizing moiety forintracellular delivery (Figure 3 Table 2) For a combinationof long blood circulation and targeting the ligand must beaccessible to the target for recognition while the liposomalsurface should be coated with PEG for long blood circulation[117] (Figure 1) Thus in addition to protection from sterichindrance of the liposome surface by the PEG chainspresentation of the ligand at the distal end of PEG allowsbetter ligand recognition [117 118] and multivalent bindingthanks to the flexibility of PEG [119] Such a combinationallowed ultimately superior therapeutic activity comparedto PEGylated drug-loaded liposomes without ligand [32ndash34 118 120 121] The rationale of targeting plus PEGylationfor antitumor efficacy has been well demonstrated by Yamadaet al using folate-linked PEGylated liposomal doxorubicin[122]They compared the in vitro cytotoxicity and in vivo anti-tumor efficacy of untargeted PEGylated doxorubicin-loadedliposomes non-PEGylated liposomes harboring folate andPEGylated liposomes with folate exposure at the liposomalsurfaceWhile the non-PEGylated folate-modified liposomes


Table 2 Examples of ligands used for targeting of liposomal nanocarriers

Type of ligand Ligand Target Reference(s)

AntibodyAnti-HER2 HER2 receptor overexpressed by cancer cells [34 98 100]Anti-CD19 CD19 overexpressed in B cell Lymphoma [101]

Nucleosome-specific 2C5 mAb Cancer cells surface-bound nucleosomes [32 102]

Protein Transferrin Transferrin receptor overexpressed by cancer cells [36 103]Interleukin 13 (IL-13) IL-13 receptor overexpressed in human gliomas [104]

PeptideOctreotide Somatostatin receptor type 2 overexpressed by cancer cells [105 106]

LHRH-derived peptide LHRH receptors overabundant on cancer cells [107]Arg-Gly-Asp (RGD) 120572V1205733overexpressed by endothelial tumor cells [108ndash110]

Small moleculeFolate Folate receptor on cancer cells [41 111]Estrone Estrogen receptors overexpressed in ovarian and breast cancers [42 112]

Anisamide Sigma receptors overexpressed by cancer cells [113]

Sugar Mannose Dendritic cells and macrophages to induce an immune response [114 115]Lactose Asialoglycoprotein receptors overexpressed by hepatocellular carcinomas [116]

HER2 human epidermal growth factor receptor 2 mAb monoclonal antibody LHRH luteinizing hormone releasing hormone

showed the highest toxicity in vitro the highest antitu-mor efficacy was reported with PEGylated folate-modifieddoxorubicin-loaded liposomes The need for targeted drugdelivery for the best antitumor efficacy is not limited toliposomes Indeed when Saad et al compared the therapeuticefficacy of targeted or untargeted paclitaxel delivery using alinear polymer dendrimer or PEGylated liposomes the besttumor accumulation and tumor suppression were obtainedwith targeted delivery systems over untargeted ones and freepaclitaxel for the three types of carriers [107] In agreementwith this study addition of a targeting moiety to PEGylatedliposomes containing the near infrared probe NIR-797 or111In improved tumor accumulation of the imaging agentsuggesting the benefit of targeting stealth liposomes for can-cer therapy and monitoring [123] Several ligands includingantibodies and peptides directed against molecular markersof tumor cells or their supportive endothelial cells presentin the tumor microenvironment have been employed fortargeted drug delivery [124] (Table 2)

221 Antibody-Targeted PEGylated Liposomes Targeted lipo-somes are obtained either by incorporation of ligand-lipidconjugates during liposome preparation incorporation oflipids with reactive groups during liposome preparationand subsequent ligand coupling and finally by insertion ofligand-lipid conjugates into preformed liposomes (postinser-tion) [125 126] For a comparison of techniques availablefor antibody conjugation to liposomes we refer the reader torecent reviews [97 127]

Coupling of the humanized anti-CD22 antibody targetingthe lymphocyte marker CD22 to PEGylated doxorubicin-loaded liposomes increased doxorubicin accumulation inNon-Hodgkinrsquos Lymphoma xenografts and increased sur-vival over untargeted doxorubicin-loaded liposomes [33]The p185HER2 (human epidermal growth factor receptor 2)receptor is upregulated in human cancers of several histology

(breast ovarian and prostate) with a low basal expres-sion in normal tissues allows cancer-specific delivery withHER2 monoclonal antibody conjugation [128 129] Conju-gation of a single-chain fragment antibody against HER2to doxorubicin-loaded liposomes led to higher doxorubicinaccumulation in breast cancer xenografts and better tumorcontrol than untargeted PEGylated doxorubicin-loaded lipo-somes [100] Conjugation of the recombinant humanizedanti-HER2 antibody Herceptin (Genentech San FranciscoCA USA) to paclitaxel-loaded PEGylated liposomes alsoincreased drug accumulation in tumors and therapeuticefficacy over untargeted paclitaxel-loaded liposomes [34]The potentiation of paclitaxel-loaded liposomes by HER2antibody was due to enhanced drug uptake by receptor-mediated endocytosis since a similar tissue distribution andantitumor activity were reported against breast xenograftsexpressing low levels of HER2 Indeed in a seminal studyKirpotin et al demonstrated that although HER2 antibody-targeted liposomes and untargeted liposomes had similaraccumulation profiles in tumors after intravenous injectionthey showed by flow cytometry and histological analysisof disaggregated tumors a 59-fold higher cancer cell accu-mulation of immunoliposomes versus untargeted liposomes[98] Antinuclear autoantibodies are present in both healthyelderly individuals and cancer patients [32]One of these anti-bodies 2C5 monoclonal antibody recognizing cell surface-bound nucleosomes specifically recognizes multiple tumorcell lines [32] Liposomes conjugated with 2C5 antibodyat the distal end of PEG3400-DSPE were preferentiallyaccumulated in tumors [32 130] and increased the therapeu-tic activity of doxorubicin-loaded (Doxil) liposomes [102]Tumor targeting of doxorubicin-loaded liposomes with theFabrsquo fragment of an anti-MT1-MMP (membrane type 1matrixmetalloproteinase expressed by cancer cells and endothelialcells) led to increased liposome uptake in vitro and highertherapeutic activity in vivo [120] It is noteworthy thatalthough the tumor accumulation of targeted and untargeted


liposomes was similar the MT1-MMP-targeted doxorubicin-loaded liposomes showed superior tumor protection thanksto enhanced uptake of the drug by tumor cells in agreementwith the results of Kirpotin et al with anti-HER2 targetedliposomes [98]

The conjugation of whole antibodies to the liposomesurface can induce complement activation and decrease theirblood circulation since the Fc fraction of immunoglobu-lins is recognized by macrophages [45 131] Thus conjuga-tion of Fabrsquo fragments instead of the whole antibody wasproposed While doxorubicin-loaded PEGylated immuno-liposomes harboring Fabrsquo fragments of an anti-CD19 anti-body had similar blood circulation and MPS accumula-tion than untargeted liposomes immunoliposomes har-boring the anti-CD19 IgG showed faster blood clearanceand a threefold accumulation in liver and spleen overuntargeted or Fabrsquo liposomes [101] Fabrsquo immunoliposomesalso resulted in superior therapeutic efficacy over untar-geted or anti-CD19 antibody-decorated immunoliposomes[101] Analogous with their results the blood circulationof pH-sensitive 1-D-arabinofuranosylcytosine-loaded lipo-somes harboring Fabrsquo fragments against CD33 was superiorto those decorated with the whole monoclonal antibody[121]

222 Protein-Targeted Liposomes Qi et al described a novelantineoplastic liposomal agent liposomal saposin C [132]Development of this agent is based on the observationthat patients suffering from lysosomal storage diseases fre-quently have saposin C deficiencies leading to accumula-tion of toxic glycosylceramide sphingolipids [133] and thatsaposin C inserts into negatively charged membranes atacidic pH [134] They prepared a saposin C-DOPS conjugatewhich assembled as 190 nm liposomes under sonicationat acidic pH Tumor targeting is based on activation ofmembrane fusion domains of saposin C at the acidic pH intumors leading to its internalization and glycosylceramide-induced apoptosis Intravenous injection into neuroblas-toma xenograft- bearing mice led to apoptosis inductionin tumors and tumor growth inhibition without systemictoxicity BAFF (B cell activating factor) is a cytokine whosereceptor is overexpressed in B-cell lymphomas conjugationof a BAFF mutant to vincristine-loaded PEGylated lipo-somes increased the survival of lymphoma-bearingmice overuntargeted vincristine-loaded liposomes or free drug [35]Cancer cells overexpress transferrin receptors [135] makingthe glycoprotein transferrin or antibodies to transferrinreceptor suitable ligands for tumor targeting [136] Addi-tion of transferrin to the surface of PEGylated oxaliplatin-loaded liposomes increased tumor accumulation over freeoxaliplatin or untargeted liposomes leading to the highesttumor growth inhibition against C26 colon carcinoma-bearing mice [36] In parallel to these studies conjugationof transferrin to doxorubicin-loaded liposomes resulted inhigher doxorubicin delivery to tumors and tumor growthinhibition over untargeted doxorubicin-loaded liposomes[103]

223 Peptide-Targeted Liposomes More and more tumor-specific ligands are being identified by combinatorial screen-ing of bacteriophage-borne peptide libraries phage displaybiopanning This is a strategy whereby the recombinantvirions able to bind cancer cells in vitro or tumors in vivoare purified before identification of the peptide and its usefor targeted drug delivery allowing identification of peptidesspecific for cancer cells tumor vasculature or both (reviewedin [137])

We previously described the selective exposure ofnucleohistones by cancer cells effective cancer therapy ofantinuclear-targeted doxorubicin-loaded liposomes [32] Ingood agreement with these studies Wang et al reportedtumor targeting of doxorubicin-loaded liposomes harboringthe histone H1-specific peptide ApoPep-1 [138] This peptideis selectively presented at the surface of tumor cells dueto spontaneous apoptosis in avascular tumors ApoPep-1conjugation to doxorubicin-loaded liposomes led to superiordoxorubicin distribution in lung xenografts and better tumorgrowth inhibition over untargeted liposomes Somatostatinreceptors particularly somatostatin receptor type 2 are over-expressed by cancer cells and endothelial cells of the tumorvasculature [139] Coupling of the somatostatin receptor type2 agonist to irinotecan-loaded liposomes improved their anti-tumor activity in amedullary thyroid carcinomamodel [105]Its coupling to PEGylated doxorubicin-loaded liposomesled to superior doxorubicin accumulation in tumors andenhanced anticancer efficacy against small cell lung cancertumors compared to untargeted liposomes [106]

Han and coworkers selected a peptide (HVGGSSV) byphage display which selectively bound to the tumor vas-culature of tumors that were regressing after radiotherapywhile no binding was detected before irradiation or inareas of tumor necrosis factor alpha-induced inflammationin mice [140] They proposed the peptide that recognizeda protein displayed only on tumor endothelial cells thatwere responding to therapy Interestingly they conjugatedthis peptide to the surface of doxorubicin-loaded liposomesfor ldquoradiation-guided tumor-targeted drug deliveryrdquo [141]Higher tumor accumulation of doxorubicin was achievedwith targeted liposomes after irradiation over untargeteddoxorubicin-loaded liposomes with or without irradiationand resulted in higher therapeutic efficacy in both Lewislung carcinoma and non-small cell lung carcinoma (HL460)tumors Identification of a non-small cell lung cancer-specificpeptide also identified by phage display to doxorubicinor vinorelbine-loaded PEGylated liposomes enhanced drugdistribution to tumors and resulted in increased therapeu-tic efficacy over untargeted drug-loaded liposomes [38]Another group reported higher therapeutic efficacy againstlung cancer xenografts of PEGylated doxorubicin-loadedliposomes conjugated with a large-cell cancer-specific pep-tide over untargeted doxorubicin-loaded liposomes [142]

Breast cancer-specific peptidephage fusion coat proteinpVIII chimeras have been used for tumor-targeted drugdelivery [143 144]Membranophilicmajor phage coat proteinpVIII fused with a targeting peptide identified by phage dis-play spontaneously inserts into liposomes The insertion of a


breast cancer-specific phage fusion protein into doxorubicin-loaded liposomes (Doxil) led to an increased binding tobreast tumor cells and enhanced cytotoxicity over untargetedDoxil liposomes in vitro [143 144] This is noteworthy sinceno chemical conjugation step is involved this method allowsfast and selective identification of tumor ligands

PEGylated paclitaxel-loaded liposomes harboring a syn-thetic luteinizing hormone-releasing hormone (LHRH) pep-tide designed to interact with the LHRH receptors that areoverabundant in the membrane of cancer cells [145] showedincreased tumor accumulation and therapeutic efficacy overuntargeted paclitaxel-loaded liposomes [107] Matrix met-alloproteinases (MMPs) are overabundant in tumor tissueswhere they act in angiogenesis matrix degradation andmetastasis [146]MoreoverMMP-2120572

1198811205733integrin complexes

and MMP-9 are present at the surface of angiogenic bloodvessels and cancer cells respectively and their targetingby inhibitory peptides showed antitumor effects [147 148]MMP-targeting of Caelyx doxorubicin-loaded liposomes byinsertion of a DSPE-PEG3400-CTT2 conjugate the CTT2peptide binding to MMP 2 and 9 led to increased doxoru-bicin accumulation in tumors and extended the survival ofovarian carcinoma xenograft-bearing mice over unmodifiedCaelyx liposomes [40]

224 Small Molecule-Mediated Tumor Targeting Aberranttumor growth is correlated with a greater demand for nutri-ents relative to healthy organs and has been exploited fortumor targeting To sustain their rapid growth tumor cellsoverexpress folate receptor to capture the folate required forDNA synthesis [149] The overexpression of folate receptorin cancers of several histology relative to normal tissues thelow cost of folic acid (FA) and the vast library of conjugationreactions available make it one of the most used ligands fortumor-targeted drug delivery and tumor imaging (reviewedin [150]) Inclusion of a FA-PEG-DSPE conjugate intoirinotecan-loaded liposomes enhanced drug concentration intumors after intravenous injection over untargeted liposomesor free irinotecan resulting in the highest anticancer activitywithout detected side toxicity [41] Similarly folate-targetingof doxorubicin-loaded liposomes increased the survival oftumor bearing mice by 50 over untargeted liposomes[111] Lee et al used tetraiodothyroacetic acid a competitiveinhibitor of thyroid hormone binding to the endothelialcell integrin 120572

1198811205733 as a new ligand for tumor-targeted drug

delivery This ligand increased liposomal accumulation intumors after intravenous injection and enhanced anticanceractivity of the encapsulated anticancer drug edelfosine [151]

Estrogen receptors are often overexpressed in breast andovarian cancers and conjugation of the ovarian estrogenichormone estrone to doxorubicin-loaded liposomes resultedin a dramatic increase in doxorubicin accumulation inbreast tumors after intravenous injection over free drug oruntargeted PEGylated doxorubicin-loaded liposomes (243and 60-fold resp) resulting in the highest therapeuticactivity [42 112] Similarly conjugation of a luteinizinghormone-releasing hormone (LHRH) analog to the surface

of docetaxel-loaded liposomes increased docetaxel accumu-lation in ovarian xenografts by 286-fold over untargeteddocetaxel-loaded liposomes with decreased liver and spleencapture though binding to the LHRH receptors highlyoverexpressed in ovarian cancer [152] The basic fibroblastgrowth factor (bFGF) receptor is also overexpressed in severalcancers [153] Electrostatic coating of cationic liposomesencapsulating doxorubicin or paclitaxel with a negativelycharged bFGF-derived peptide resulted in increased survivalofmelanoma or prostate tumor-bearingmice over untargetedliposomal formulations respectively [154] The use of chon-droitin sulfate which binds CD44 overexpressed by tumorcells has recently been introduced [43] Coupling of chon-droitin sulfate to the surface of etoposide-loaded liposomesincreased etoposide accumulation in breast cancer xenograftsafter intravenous injection 40-fold compared to free drug andby 8-fold compared to untargeted liposomes Presentationof lactose at the surface of doxorubicin-loaded PEGylatedliposomes using a lactose-DOPE conjugate to target theasialoglycoprotein receptors overexpressed in hepatocellularcarcinomas increased doxorubicin accumulation in tumorsand resulted in tumor growth inhibition over untargeteddoxorubicin-loaded liposomes [116]

Tan and coworkers introduced ternary nucleic acidcomplexes Liposome Polycation DNA (LPD) where nucleicacids are complexed by protamine before interaction withcationic liposomes to form a core nucleic acid complexsurrounded by two lipid bilayers [155] Sigma receptorsare ion channel regulators overexpressed in several can-cer types [156] Conjugation of the small molecular weightsigma receptor ligand anisamide [157] to the distal endof PEG2000-DSPE allowed 70ndash80 luciferase silencing inan experimental lung metastasis model [113] Moreoverparenteral injection of anisamide-armed LPD prepared witha combination of siRNA against the inhibitor of p53 MDM2(Murine Double Minute 2) against the Cmyc oncogene andthe other against the angiogenesis regulator VEGF (VascularEndothelial Growth Factor) were localized in tumors andallowed a 70ndash80 decrease in tumor load [68] Howeverwhile the common sigma receptor agonist haloperidol andanisamide recognize sigma receptor type 1 and 2 only sigmareceptor type 2 overexpression has been reported to be aprognostic indicator [158] The latter has low expressionin healthy tissues suggesting a higher therapeutic index ofsigma receptor 2 targeted therapies [158] Indeed binding ofthe sigma 2 receptor agonist SV119 to its receptor induced celldeath in vivo in a pancreatic cancer model and conjugationof SV119 to the surface of liposomes increased their uptake invitro in cell lines including lung breast and prostate cancercarcinoma whereas no increased uptake in normal cells wasreported [158]

3 Biological Targets

31 Brain Tumor Targeting Brain tumors are amajor concernfor both primary brain and brain metastases from primarylung melanoma breast and kidney cancers [159] Therapyagainst brain cancers is challenging since the brain is largely


isolated from the rest of the body by the blood brainbarrier (BBB) a dense barrier of endothelial cells pericytesastrocytes and extracellular matrix which limits moleculartransport into the brain [160] Several strategies to overcomethis barrier have been proposed for the treatment of braintumors either by targeted delivery of drug-loaded liposomesto the brain or by remote-controlled drug release within thebrain

Overexpression of IL-13 receptors has been reportedin human gliomas [161] and conjugation of IL-13 todoxorubicin-loaded liposomes allowed a 5-fold reduction intumor volume and extended survival of intracranial gliomatumor-bearing mice over untargeted doxorubicin-loadedliposomes [104] In the same vein the conjugation of IL-13 to PEGylated doxorubicin-loaded liposomes for astro-cytoma targeting dramatically improved brain delivery ofdoxorubicin compared to untargeted liposomes and resultedin increased survival of intracranial U87 glioma-bearingmice after intraperitoneal administration [104] To reinforcebrain drug delivery Du et al armed PEGylated topotecan-loaded liposomes with both wheat germ agglutinin for braincapillary targeting and tamoxifen to decrease drug efflux[162] These dual-targeted liposomes crossed a model BBBin vitro and increased the survival of brain tumor bearing-rats over free topotecan or untargeted topotecan-loadedliposomes [162]The need for dual-targeting for effective BBBcrossing in vivo is also exemplified in a study by Ying etal [163] They took advantage of the expression of glucosetransporter 1 and transferrin receptor by endothelial cells ofthe BBB for intracranial glioma therapy using mannose andtransferrin dual-targeted daunorubicin-loaded liposomesDual-targeting led to superior tumor growth inhibitionand increased life span over untargeted or single-targeteddaunorubicin-loaded liposomes

Gong et al used thermosensitive doxorubicin-loadedPEGylated liposomes capable of releasing 90 of drug after30min at 42∘C compared to less than 3 for unsensi-tive liposomes [164] They reported improved doxorubicindelivery to the brain after intravenous injection (34-foldover nonsensitive liposomes) and increased survival of C6glioma-bearing mice when heads of mice were heated in awater bath to 42∘C after injection [164] Another physicallycontrolled content release strategy has been described bythe group of Yang using focused ultrasounds for reversibledisruption of the BBB as evidenced by higher brain accu-mulation of Evanrsquos blue or gadolinium in ultrasound-treatedanimals over untreated ones [165] Administration of braintumor-targeted doxorubicin-loaded liposomes followed byultrasound-mediated BBB disruption allowed higher levelsof intracranial liposomes and doxorubicin accumulation overuntargeted liposomes in an intracranial glioblastoma model[166]

32 Vasculature Targeting The ldquoangiogenic switchrdquo whentumors establish their own blood supply by extensive neo-angiogenesis is critical for the progression of tumors froma dormant avascular nodule to an invasive carcinoma [167168]This dependence on blood supply for tumor growth and

the correlation between vascular permeability and accumu-lation of liposomal drug and therapeutic efficacy [169ndash171]supports research on liposomal tumor vasculature-targetingfor cancer therapy (reviewed in [172]) After intravenousinjection in mice PEGylated liposomes were shown toaccumulate in the perivascular space with limited tumorpenetration [94 173 174] Moreover when the tumor accu-mulation and therapeutic efficacy of PEGylated liposomaloxaliplatin were compared in animals bearing C26 coloncarcinoma Lewis lung carcinoma and B16BL6 melanoma acorrelation among tumor blood vessel permeability tumordrug accumulation and the resulting therapeutic efficacy havebeen reported [171] In vitro results were not predictive ofin vivo activity the least tumor accumulation and tumorgrowthwere detected in B16BL6 tumors whereas this cell linewas the most sensitive to liposomal oxaliplatin in vitro [171]Of note the lower tumor vessel permeability of melanomaxenografts compared to colon or lung carcinoma is clinicallyrelevant When the microvessel density of biopsies fromcancer patients was determined melanoma was also the leastvascularized (sim35 vesselsfield) compared to colon (sim70) orlung tumors (sim127) stressing the point that extravasationof agents from the tumor vasculature is a major barrier forliposomal drug delivery [175]

Targeting of selectin on endothelial cells with P-selectinglycoprotein ligand 1 allowed a 3-fold higher luciferin deliv-ery to B16F10 tumors after intravenous injection over untar-geted liposomes [176] The 120572

1198811205733integrin is overexpressed

by endothelial cells in the tumor vasculature [177] Thetripeptide Arg-Gly-Asp (RGD) and the cyclic RGD (Arg-Gly-Asp-D-Phe-Lys) are 120572

1198811205733

ligands used for tumor-targeted drug delivery [108] RGD-targeted paclitaxel ordoxorubicin-loaded PEGylated liposomes showed superiortherapeutic activity over free drug or untargeted liposomes[109 110] Antitumor activity of RGD-targeted liposomes isconsistent with tumor microvessel destruction after injec-tion of RGD-targeted paclitaxel-loaded liposomes reportedby another group [178] Functionalization of doxorubicin-loaded liposomes with a peptide targeted to bombesinreceptors overexpressed in cancers improved therapeuticefficacy over untargeted liposomes [179] 120572

51205731is another

integrin overexpressed in cancer in which the fibronectin-derived peptide antagonist ATN-161 showed antineoplas-tic and antimetastatic properties [180] Coupling of ATN-161 to doxorubicin-loaded PEGylated liposomes increasedtheir therapeutic activity in a melanoma model [181]Doxorubicin-loaded PEGylated liposomes were functional-ized with a NGR peptide at the distal end of PEG to targeta CD13 isoform overexpressed in the tumor neovasculature[182ndash184] In the study by Pastorino et al vasculature-targeted Caelyx showed superior apoptosis induction intumor xenografts and decreased blood vessel density leadingto increased survival of mice bearing lung ovarian orneuroblastoma xenografts compared to untargeted Caelyx[182]

To further improve the destruction of blood vessel sup-port of tumors Takara and coworkers recently developeda dual-ligand approach for antiangiogenic therapy using


liposomes targeted to CD13 (NGR-PEG2000-DSPE) func-tionalized with the stearylated cell penetrating peptide tetra-arginine at the liposome surface [183] They first comparedendothelial cell association in vivo in tumor-bearing miceafter intravenous injection of PEGylated doxorubicin-loadedliposomes measuring either 100 nm (small liposomes) or300 nm (large liposomes) Since a superior association withtumor blood vessels and lower extravasation was observedwith large liposomes over small ones they used the formerfor ligand conjugation Dual-ligand labeled liposomes accu-mulated sim3-fold more in tumors than unmodified or singleligand-modified liposomes revealing synergy of the twoligands Consistent with the tumor accumulation and bloodvessel association results only the dual-ligand doxorubicin-loaded liposomes allowed protection against tumor growthand induced tumor blood vessel destruction that revealed asynergy of endothelial cell targeting and enhanced uptake forantiangiogenic therapy

Cationic liposomes selectively bound to endothelial cellsin vivo with superior internalization over anionic or neu-tral liposomes due to the enrichment of tumor endothelialcell membranes with negatively charged lipids and heparansulfate proteoglycan [172 185 186] Superior accumulationof oxaliplatin in lung tumors was obtained after intravenousinjection of PEG-coated cationic drug-loaded liposomesover neutral liposomes [187] The same group used cationicliposomes for delivery of siRNA against the neoangiogen-esis regulator Argonaute 2 (Ago2) which resulted in Agosilencing in tumors together with apoptosis of tumor bloodvessels and decreased tumor growth while no therapeuticeffect was observed with cationic lipoplexes prepared with anirrelevant siRNA [188 189] In support of the effect of the neg-ative charge of angiogenic vessels paclitaxel-loaded cationicliposomes (EndoTAG-1) induced endothelial cell apoptosisin vivo retarded melanoma and pancreatic carcinoma tumorgrowth and decreased the number of melanoma lung metas-tases in vivo [190ndash192] Recently targeting of tumor vascu-lature by an aptamer directed against the tumor vasculaturemarker E-selectin has been reported [193] E-selectin aptamerconjugated liposomes accumulated in the tumor vascula-ture of breast cancer xenografts after intravenous injectionwhereas no untargeted liposomes were detected in tumorssupporting use of this selective approach for vasculature-targeted drug delivery The vasculature-targeting group usedmay be relevant only to a particular histology Indeedwhile the p15-RGR peptide which recognizes platelet-derivedgrowth factor receptor 120573 expressed by pericytes of the tumorvasculature identified by phage display against pancreaticcancer increased delivery of liposomes to pancreatic tumorsin vivo it did not direct liposomes to tumors in a melanomamodel [194 195] In the same study liposomes harboring p46-RGD 120572

119881-integrin-binding peptide targeting tumor endothe-

lial cells allowed a significant tumor accumulation over con-trols with higher therapeutic efficacy [195] Chang et al alsoused phage display to identify neovasculature peptides whichwhen conjugated to doxorubicin-loaded liposomes increaseddoxorubicin delivery to tumors and therapeutic efficacyover untargeted PEGylated doxorubicin-loaded liposomes[196]

Polyethylene glycol

Anticancer drugs

Targeting ligand

Cell penetrating peptide

Imaging agent

Inhibitor of metastasis or drug resistance

Stimuli-labile PEG-lipid linker

Stimuli-responsive lipids

Figure 1 Schematic picture of amultifunctional liposomal nanocar-rier

Pericytes are a critical conjunctive component of vas-culature aminopeptidase A (APA) has been identified as amarker of pericytes from orthotopic primary and metastatic(ovary) neuroblastoma in mice [197] Coupling of a peptideligand of APA to doxorubicin-loaded liposomes increaseddoxorubicin accumulation in neuroblastoma tumors overuntargeted doxorubicin with better therapeutic activitydemonstrating that pericytes are another critical target withinthe vasculature [198] Moreover coadministration of APA-targeted doxorubicin-loaded liposomes and aminopeptidaseN (APN a marker of tumor endothelial cells) targeteddoxorubicin-loaded liposomes led to superior doxorubicinaccumulation in tumors over either targeted formulationalone [198] The destruction of perivascular and endothelialcells in tumors resulted in a significant increase in survivalof neuroblastoma-bearing mice over either endothelial cell-targeted or pericyte-targeted liposomes alone [198]

Tumor lymphatics are also a therapeutic target since theysupport lymph node metastasis [199] Indeed lymph nodeinvasion is frequent in melanoma and is an indicator ofpoor prognosis [200] Laakkonen and coworkers identifieda tumor lymphatics-binding peptide (LyP-1) which afterintravenous injection in breast carcinoma-bearing mice wasshown to accumulate in hypoxic areas of primary tumorscofllocalize with lymphatic markers in primary tumors andlymph node metastases leading to tumor growth reductionand a decreased number of lymphatic vessels [201 202]Interestingly presentation of this peptide on doxorubicin-loaded liposomes increased tumor accumulation and ther-apeutic efficacy over untargeted liposomes and decreasedlymph node metastasis rate and growth [201 203ndash205]

A combination of targeting ligands may be needed foreffective antiangiogenic therapy Murase et al demonstratedsynergy in association with endothelial cells in vitro byliposomesmodified with two angiogenic vessel-targeted pep-tides (APRPG and GNGRG) identified by phage display andrevealed the more intense association with tumor blood ves-sels in vivo of dual-targeted liposomes over single-modifiedliposomes [206] Similarly Meng et al demonstrated synergyin tumor growth inhibition of non-small cell lung cancerof PEGylated paclitaxel-loaded liposomes targeted to tumorvasculature by both RGD and a neuropilin 1-specific peptideover untargeted or single-targeted liposomes [207] Theseresults are in accordance with the increased detection of


Drug effluxHeat light ultrasound stimuli

with or without MRI guidance

Activation of responsive lipids

(a) External physical stimuli

Low tumor pH tumor enzymesreductive environment

Selective internalization bycancer cells

Detachable protective polymer (PEG)

AntibodyCell penetrating peptide

Unmasking of ligand andor penetrating peptide

(b) Physiological tumor-environment stimuli induced PEG release

Figure 2 Schemes for tumor-specific liposome destabilization orendocytosis

Blood vessel

Step 1 tumor targetingEPR effect vasculature targeting

Tumor associated macrophages

Step 2 cancer cell targetingTargeting ligandInternalizing moiety

Cancer cells

PericyteEndothelial cell

Cancer-associated fibroblasts

Figure 3 Targeting mechanisms in liposomal cancer therapy

neoangiogenic blood vessels in surgical specimens from can-cer patients when using two neovasculature-specific peptidessimultaneously compared to individually used [196]

33 Targeting and Inhibition of Metastasis Metastasis isthe ultimate stage of clinical cancer and is the stage withthe least survival Treatment of metastasis is challengingbecause micrometastatic foci are hard to detect and moreaggressive than the primary tumors [208] Elimination ofmetastases is thus of utmost importance to prevent cancerrecurrence after chemotherapy or surgical removal of theprimary tumor Platelets have been proposed as shuttlesfor tumor cell metastasis by formation of platelets-tumorcell aggregates [209 210] This is consistent with the ele-vated platelet counts in patients with advanced cancer [210]Therefore Wenzel et al used PEGylated liposomes to code-liver the haemostatic inhibitor dipyridamole (DIP) and thecytotoxic drug perifosine (OPP) to inhibit platelet-tumorcell aggregate formation and kill tumor cells respectively[211] OPPDIP coloaded liposomes inhibited aggregation ofplatelets decreased formation of platelet-tumor cell aggre-gates in vitro and decreased the number of experimental lungmetastases when intravenously injected 6 h before parenteralinjection of tumor cells The metastasis-specific peptideTMPT1 [212] recognizes highly metastatic primary tumorsand metastases of prostate breast and lung cancers relativeto their nonmetastatic counterparts Conjugation of this

12

3

4

minus

+

+

++

minus

minus

minus

minusminus

Figure 4 Strategies for intracellular delivery Steps for intracellulardelivery (1) Stimuli-sensitive activationunmasking of internaliza-tion moiety (2) Cancer cell-specific endocytosis (3) Endosomalescape andor therapeutic agent release after activation of fusogenicpeptides or lipids (4) Binding to the highly negative mitochondrialouter membrane for mitochondria targeting Legends are the sameas in Figure 1

peptide to doxorubicin-loaded liposomes led to deeper tumorpenetration and greater induction of apoptosis with superiortumor growth inhibition against highly metastatic breastcancer xenografts [39] PAR-1 (Protease Activated Receptor1) a thrombin receptor is a major regulator of metastasisin melanoma through its roles in matrix degradation andangiogenesis [213] Villares et al reported for the first timea dramatic antimelanoma therapeutic activity after systemicdelivery of PAR-1 siRNA-loaded neutral DOPC liposomeswith tumor weight reduction and a decrease in experimentallung metastatic colonies [214] This was achieved via down-regulation of promoters of angiogenesis (VEGF and IL-8)and invasion (MMP-2) together with decreased tumor bloodvessel density (decreased CD31 staining)

34 Immune Cell Targeting For therapeutic vaccinationagainst cancer patientrsquos immune cells are stimulated bytumor cell antigens Since the development of effectiveadaptive immune responses by CD4+ T cells or CD8+T cells with cytotoxic activity (Cytotoxic T LymphocytesCTL) requires their activation by dendritic cells (DCs) thatpresent tumor antigen peptides [215] their targeting is oftherapeutic relevance [215ndash217] Altinrsquos group used a chela-tor lipid [Nickel3(nitrilotriacetic acid)-ditetradecylamine](Ni-NTA

3-DTDA) for functionalization of liposomes with

histidine-tagged peptides though polyhistidine binding tonitrilotriacetic acid in the presence of nickel [218 219]For antigen delivery Ni-NTA

3-DTDA functionalized lipo-

somes were prepared by preinsertion before conjugation withhistidine-tagged peptides derived from ICAM4 (IntercellularCell Adhesion Molecule 4) a ligand of the murine dendriticcell (DC) integrin CD11cCD18 [220] Ovalbumin-loadedPEGylated liposomes decorated with DC-targeting peptidesdistributed to splenic DC in vivo induced an adaptiveimmune response against ovalbumin and exhibited dramatictherapeutic activity against established B16-OVA melanoma


tumors with complete tumor regression in 80 of treatedmice [218]

In other studies Altinrsquos group reported on DC-targetedgene delivery in vivo and potent antitumor effects in theB16-OVA melanoma model after liposome functionalizationwith histidylated flagellin the major constituent of the bac-terial flagella recognized by the Toll Like Receptor 5 thatleads to their activation [221 222] LPR (Lipid-Polymer-RNA) mannosylated and histidylated lipopolyplexes loadedwith MART1 (Melanoma Antigen Recognized by T cells1) mRNA delayed the progression of B16F10 melanomamore effectively than untargeted LPR [223] This study alsoillustrated the importance of cytosolic delivery of nucleicacids for in vivo transfection of DC The authors used aternary formulation of mRNA or pDNA coding for thereporter gene EGFP (Enhanced Green Fluorescent Protein)complexed with PEGylated histidylated poly-L-Lysine andimidazole-rich liposomes both of which promote endosomalescape [224 225] While no in vivo transfection of splenicDC was observed with pDNA 12 were transfected withmRNA mannosylated LPR and 3 with untargeted LPRdemonstrating that nuclear delivery is a limiting step forDC transfection Liposomes targeted to dendritic cells bymannosylated ligands have recently been used as a platformfor effective cancer immunotherapy [114]The liposomes usedharbored mannosylated ligands at their surface for targetingof antigen presenting cells with a cytotoxic T lymphocytepeptide of the renal carcinoma antigen ErbB2 for induc-tion of an adaptive immune response Toll Like Receptors(TLRs) agonists as adjuvants and a T helper lymphocyteepitope peptide for improved immune activation Of notethe authors developed new functionalized lipid anchorsdevoid of adjuvant activity for their study dipalmitoylglyc-erol maleimide and dipalmitoylglycerol bromoacetate Theseliposomes induced an adaptive immune response againstthe ErbB2 antigen with high therapeutic activity Targetingof intraperitoneal macrophages by ovalbumin-loaded lipo-somes armed with dipalmitoylphosphatidylethanolamineconjugated mannotriose increased antigen-specific cell lysisinduction by splenocytes over untargeted liposomes resultingin therapeutic efficacy both as a preventive and therapeuticcancer vaccine [115] In addition to carrying tumor anti-gens liposomal vaccines are armed with immunostimulatorylipids usually derived from microorganisms recognized bypathogen recognition receptors leading to immunostim-ulation (reviewed in [226]) Zhong et al compared theantimetastatic efficacy of a basic Fibroblast Growth Factor(bFGF) vaccine in a mouse melanoma model when admin-istered as a Freundrsquos adjuvant mixture in cationic liposomesor cationic liposomes containing 025 of monophosphoryllipid A as adjuvant [227] They reported higher anti-bFGFIgG titers and higher pulmonary metastasis inhibition inmice treated with monophosphoryl lipid A bFGF-loadedliposomes over cationic liposomes or a bFGFFreundrsquos adju-vant mixture without the toxicity associated with administra-tion of free adjuvants

Selective depletion of tumor supporting cells repre-sents another approach to cell-specific cancer therapy

[228] The tumor environment is enriched in tumor sup-porting cells among the tumor-associated macrophagesthat constitute a predominant inflammatory populationinvolved both in resistance to therapy and metastasis[228]Dichloromethylenediphosphonate (DMDP) liposomesinduced macrophage depletion after intravenous injectionin mice [229] Intradermal injection of DMDP liposomesinto the tissues surrounding melanoma or squamous cellcarcinoma tumors led to a decrease in tumor-associatedmacrophages content and tumor rejection [230]

Ligand density was shown to influence both drug reten-tion and target recognition Zhang et al demonstratedincrease in liposome uptake in vitro as the ligand densitywas increased from 0 to 1 3 and 5 demonstratingenhanced ligand recognition [231] However increase of invitro drug release as a function of DSPE-PEG-RGD ligandmoiety has been reported by others [232] Moreover Saulet al evidenced increase of nonspecific uptake in vitro withligand density [233] Consistent with their results lowertumor accumulation of NGR (Asparagine-Glycine-Arginine)vasculature targeted liposomes has been evidenced in vivowith liposomes harboring 256mole NGR-PEG-DSPE than064 mole NGR-PEG-DSPE [234] Altogether these datasuggest the use of the lowest targeting ligand density allowingtarget binding for effective anticancer therapy

4 Liposomes for Combination Therapy

The prevalence of drug resistance in cancer patients bothprior to treatment and de novo [235 236] fueled the appli-cation of drug combinations to treat cancer as an alternativeto increased doses of chemotherapeutics associated with lifethreatening sideeffects [237ndash239]

Codelivery was well illustrated in a study by Chen etal [240] Using LPH-NP (liposome-polycation-hyaluronicacid) nanoparticles targeted by postinsertion of DSPE-PEG-GC4 (scFv selected by phage display against ovarian tumors[241]) they codelivered 3 different siRNA and one miRNAand obtained a 80 decrease in tumor load after treatmentThey simultaneously targeted proliferation pathways withCmyc siRNA and miR34a miRNA [242 243] apoptosiswith MDM2 siRNA [244] and angiogenesis using VEGFsiRNA [245] Liposomal codelivery of siRNA against theapoptosis regulator Mcl-1 (Myeloid cell leukemia sequence 1)and of theMEK (Mitogen-activatedExtracellularKinase) andapoptosis resistance inhibitor PD0325901 enhanced tumorgrowth inhibition compared to each treatment alone [246]The same group also developed trilysinoyl oleyamide (trily-sine peptide linked to oleyamine by a peptide bond) basedPEGylated liposomes for codelivery of Mcl-1 siRNA andthe histone deacytylase inhibitor suberoylanilide hydroxamicacid (SAHA) [247] Intravenous administration increasedthe tumor growth delay compared to liposomes with SAHAand an irrelevant siRNA Likewise Xiao and coworkersused targeted liposomes to codeliver doxorubicin and DNAencoding a dominant mutant of survivin [248] Liposomeswere targeted by a truncated basic fibroblast growth factor(tbFGF) peptide recognizing the bFGF receptor upregulated


in lung cancers and contained doxorubicin and pDNAencoding for a dominant negative mutant of survivin tocounter survivin-mediated apoptosis resistance [249] Theircodelivery produced a higher therapeutic efficacy againstLewis lung carcinoma tumors than liposomes with eitheragent alone

A further step in combination of an antineoplasticagent with modulation of drug resistance was achievedrecently by Minko and coworkers [250] by formulationof peptide-targeted liposomes containing doxorubicin orcisplatin together with oligonucleotides against the twomain drug resistance mechanisms Bcl-2 and MDR1 Theefficacy of this ldquocombined targeted chemo and gene ther-apyrdquo system was evaluated in xenografts established fromhuman ovarian malignant ascites While inclusion of eitherBcl-2 or MDR1 antisense oligonucleotides in cisplatin ordoxorubicin-loaded targeted liposomes decreased primarytumor volume and intraperitoneal metastases load furtherinhibition of tumor growth inhibition was obtained withtargeted liposomes containing doxorubicin or cisplatin Bcl-2 and MDR1 antisense oligonucleotides together with com-plete prevention of the development of detectable intraperi-toneal metastases or ascites Interestingly Minko et al pro-posed this system as a platform for personalized cancertherapy with liposomal formulations containing antisenseoligonucleotides targeting individually relevant resistancemechanism Sawant et al coloaded PEGylated liposomeswith a palmitoyl-ascorbate conjugate and paclitaxel [251]The therapeutic benefit of the coloading against 4T1 mam-mary carcinoma was evident at 10mgkg compared topalmitoyl-ascorbate or paclitaxel-loaded liposomes Atu027(Silence Therapeutics London UK) is a liposomal formu-lation of siRNA against protein kinase N3 a downstreameffector of the mitogenic PI3 KPTEN pathway involvedin prostate cancer metastasis [252 253] This formulationwas composed of 21015840-O-methyl-stabilized siRNA encapsu-lated in cationic liposomes (50mol cationic lipid -L-arginyl-23-L-diaminopropionic acid-N-palmitoyl-N-oleyl-amide trihydrochloride (AtuFECT01) 49mol co-lipid 12-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE)and 1molDSPE-PEG2000) [253]This formulation showedvery promising results in phase I clinical trial with tumorregressions in neuroendocrine and breast cancer patients[254]

Dai et al combined targeted delivery with antineoplasticand antiangiogenic agent delivery in PEGylated liposomes[255] Coloading of the antiangiogenic agent combretastinA-4 in the lipid bilayer and the anticancer drug doxoru-bicin in the aqueous core of PEGylated liposomes resultedin increased therapeutic activity Hu et al also combinedliposomal delivery of the antineoplastic and antiangiogenicagent honokiol with irradiation for maximal therapeuticefficacy [256] They hypothesized that this protocol wouldcombine the destruction of tumor cells by irradiation withinhibition of irradiation-induced neoangiogenesis by hon-okiol [257] The combination of PEGylated honokiol-loadedand radiotherapy showed increased survival of Lewis lungcarcinoma-bearing mice compared to radiotherapy or hon-okiol liposomes alone resulting in decreased angiogenesis

in vivo Maitani et al also combined an antineoplasticdrug (irinotecan) and an antiangiogenic agent (sunitinib)[258] The drug combination had more therapeutic efficacyagainst pheochromocytoma neuroendocrine tumors in vivowhen they were administered as sunitinib liposomes plusirinotecan liposomes or as coloaded liposomes than thecombination of the free drugs with higher drug accumu-lation as liposomes than as free drug In a similar fashionfolate-targeted doxorubicin-loaded liposomes coloaded witha bifunctional peptide capable of vascular disruption andantitumor activity were more effective against KB humannasopharyngeal carcinoma in vivo than untargeted coloadedliposomes than either monotherapy [259] RGD-targetedliposomes coloaded with doxorubicin and the vascular dis-rupting drug combrestatin A-4 increased tumor regressionof B16F10 melanoma compared to untargeted coloaded lipo-somes or targeted liposomes with either drug [260]

Zucker and coworkers have optimized the simultane-ous loading of vincristine and topotecan into PEGylatedliposomes (LipoViTo liposomes) and provided the readerwith the methods needed to characterize a liposomal drugcombination [261] Use of LipoViTo increased 100-fold thedrug distribution to tumors compared to free drug and ledto superior therapeutic efficacy over a free drug combinationor liposomes with a single drug PEGylated liposomes con-taining both vincristine and quercetin allowed reduced bloodclearance of both drugs in mice increased the therapeuticactivity over a combination of free drugs and decreased side-toxicity [262]

Celator Pharmaceuticals Inc (Princeton NJ) developeda liposomal formulation of cytarabine daunorubicin (CPX-351 5 1 molar ratio) [24 263 264] These PEGylated lipo-somes coloaded with the weak acid drug 5-fluoroorotic acid(FOA) and the amphiphatic drug irinotecan (CPT-11) at a5 1 ratio revealed a synergy between the two drugs withhigher therapeutic efficacy than the free drug cocktails inanimal models [264 265] To encapsulate both drugs theyfirst prepared liposomes before active loading of CPT-11 by apH gradient method with the protonated CPT-11 retained inliposomes after complex formation with FOA Mice treatedwith coloaded liposomes had increased survival comparedto the combination with separate liposomes However thetherapeutic efficacy was lower than with liposomes loadedwith FOA only probably because the FOA content had tobe lowered for CPT-11 coloading further demonstrating thedifficulty of reproducing a synergy with liposomes relative tofree drugs When tested in phase I trial with acute leukemiapatients the 5 1 ratio was maintained in plasma for 24 hand CPX-351 induced complete responses in 9 out of 43patients [24] The same group developed irinotecan floxuri-dine liposomes (CPX-1 1 1 molar ratio) In phase I clinicaltrial they demonstrated that the drug ratio was maintainedin plasma up to 12 h after infusion and showed positiveclinical responses in patients with colorectal cancer [25] Itis noteworthy that the high therapeutic efficacy of liposomesencapsulating two anticancer drugs was always correlatedwith the maintenance of their synergistic molar ratio inplasma in animal models [266] as well as in cancer patients[24 25 264] indicating optimization of drug loading and


liposomal stability as primary concerns for effective combina-tion therapy Ko et al codelivered the proapoptotic peptideD-(KLAKKLAK)

2and the Bcl-2 antisense oligodeoxynucleotide

G3139 [267] The authors took the advantage of the electro-static properties of these therapeutic molecules to codeliverthem by formation of a negatively charged complex betweenthe peptide and G3139 before mixing with positively chargedliposomes Intratumoral injection of coloaded liposomes ledto an enhanced tumor growth suppression

Finally the combined liposomal delivery of magneticfluid hyperthermia and photodynamic therapy using mag-netic fluid and zinc phthalocyanine as the photosensitizerdemonstrated superior toxicity in vitro of combined light andmagnetic stimuli over their separate applications suggestinga new treatment modality for enhanced tumor therapy [268]

5 Tumor Stimuli-Triggered PEG Release

The addition of PEG to the liposome surface was reported todecrease the interaction of the ligand-targeted liposomeswiththeir ligand either when small molecules were conjugatedto the liposome surface [269] or with antibody-targetedliposomes [48 118] by steric hindrance of the surface ligandMoreover PEGylation decreases targeted liposomal accumu-lation and drug release [270] Finally for gene delivery PEGy-lation has been shown to decrease intracellular traffickingof DNA [271] These drawbacks and the extensive researchin PEGylation chemistry (recently reviewed in [272 273])have led to the preparation of new multifunctional carrierswhere PEG release is promoted at the tumorrsquos vicinity aftera stimulus either by physiological stimuli (pH altered redoxpotential sensitivity to an enzyme overabundant in the tumormicroenvironment) or by physical external stimuli (lightheat and ultrasound) [8 274] (Figure 2)

51 pH-Sensitive PEG Release While normal tissues andblood have a physiological pH near 74 human tumors havelower pH values (sim6065) because of an elevated rate ofglycolysis [275 276] pH-sensitive bonds have been devel-oped for the coupling of PEG to liposomes [277] (Figure 1)pH-sensitive liposomes achieved a higher concentration ofcargo in the cytoplasm and nucleus than non-pH-sensitivePEGylated liposomes in vitro and allowed faster intratumoralcontent release in vivo [278 279] In addition to tumorsensitivity pH sensitive groups can potentiate the efficacy oftargeted drug-loaded liposomes

Folate-targeting of daunorubicin-loaded liposomes byincorporation of a pH-sensitive folate-PEG-cholesterolhemisuccinate (CHEMS) conjugate combined tumortargeting and increased drug release at the tumor sitewith improved chemotherapeutic activity over untargetedliposomes [280] Similarly untargeted cisplatin-loadedliposomes or EGFR-targeted gemcitabine-loaded liposomesincorporating CHEMS had superior antitumor activity overuntargeted drug-loaded liposomes or free drugs [281 282]Obata et al used a glutamic acid-based zwitterionic lipid (15-dihexadecyl NN-diglutamyl-lysyl-L-glutamate) as titratablelipid for doxorubicin delivery [283]These liposomes showed

a charge inversion from negative to positive at acidic pHwith endosomal escape leading to higher doxorubicindelivery in the cytoplasm and higher toxicity in vitro overconventional liposomes This resulted in superior antitumoractivity in vivo Biswas et al developed a new pH-sensitiveDSPE-PEG-hydrazone-PEG2000 conjugate for attachmentof ligands to the liposome surface [284] In their work thecell penetrating peptide (TATp) was unmasked after PEGrelease at acidic pH allowing efficient cellular uptake

Recently three new approaches for generation of pH sen-sitivity have been reported First by electrostatic adsorptionof negatively charged carboxyl-modified gold nanoparticlesto the surface of cationic liposomes (egg dipalmitoylphos-phatidylcholineDOTAP 9 1 weight ratio) at pH 7 (pKa of 5for the carboxylic group) [285] Authors reported detachmentof gold nanoparticles at acidic pH due to protonation ofthe carboxyl groups and speculated that a similar strat-egy could be applied with negative charged liposomes andamine-modified gold nanoparticles Second a platform forfinely tuned pH-induced PEG release was introduced usingphenyl-substituted-vinyl-ether-(PIVE)-PEG lipid conjugates[286] Liposomes containing PIVE showed pH-induced deP-EGylation and content release at acidic pH whereas theywere stable at physiological pH Third ligand unmaskingby acidic pH-induced membrane reorganization has beenintroduced as a reversible ligand-masking strategy Sofouand coworkers developed a new platform for pH-triggeredliposomal drug delivery [287 288] The rationale for theirdesign involves the increased permeability at the boundariesbetween lipid domains [289] Using lipid pairs of phospha-tidic acid as a titrable headgroup and phosphatidylcholineas the colipid headgroup with mismatched hydrophobicchain lengths (dipalmitoyl and distearoyl) they demonstratedthat formation of heterogeneous domains in PEGylatedliposomes containing 5 of cholesterol allowed faster pH-dependent content release than liposomes with matchedchains [288] They showed a pH-dependent membrane tran-sition due to the protonation of phosphatidylserine at lowerpH in cholesterol-richmembranes with protonation favoringtheir homologous interaction leading to the formation ofDSPS (12-distearoyl-sn-glycero-3[phosphor-L-serine]) lipiddomains PEG-lipid conjugates of matching hydrophobicanchor (DSPE-PEG) also segregated to these domains atacidic pH whereas no redistribution of unmatched chainDPPE-PEG was in evidence [290] The liposomes containeda ligand (biotin or an anti-HER2 peptide) harbored by anunmatched lipid (DPPE) which was masked by PEG atphysiological pH but freed from PEG shielding at acidicpH after formation of the lipid heterogeneities Applicationof this strategy to doxorubicin-loaded PEGylated (DSPE-PEG2000) liposomes harboring anHER2-specific peptide ledto pH-dependent doxorubicin release in vitro and superiortumor growth inhibition than did untargeted vesicles ortargeted vesicles devoid of pH-responsiveness [291]

52 MMP-Sensitive PEG Release Hatakeyama and cowork-ers introduced coupling of PEG to DOPE by an MMP-cleavable linker since MMPs are overexpressed in the tumor


environment [292 293] Transfection efficiency in vitrowas correlated with MMP levels and lipoplexes preparedwith a MMP-responsive PEG-lipid conjugate showed tumor-specific transgene expression when compared to PEGylatedlipoplexes with higher transgene expression for the samequantity of delivered lipoplexes To enhance tumor targetingZhu et al combined anMMP2-sensitive PEG-lipid conjugatewith antibody targeting and an intracellular penetratingmoiety (TaT peptide) [294] combining long circulation byPEGylation tumor targeting via antinuclear antibody 2C5and selective internalization by tumor cells through MMP-2triggered exposure of TaT peptide

53 Redox-Sensitive PEG Release Tumor cells have a higherconcentration of reductases than the extracellular environ-ment or normal cells and this feature has promoted the useof disulfide linkers both for the design of reduction-sensitivePEG-lipid conjugates and crosslinked nanoparticles sincethe linker is stable in the circulation and normal tissuesbut reduced in the tumor cells [295 296] Goldenbogen etal developed a versatile reduction-sensitive conjugate fortargeted delivery [297] Biotin was conjugated to a lipidanchor via a disulfide linker to prepare biotin-decoratedliposomes conjugation of streptavidin-HER2 monoclonalantibody allowed superior cellular uptake of doxorubicin invitro over untargeted liposomes Interestingly less intracel-lular doxorubicin was detected after incubation with unsen-sitive HER2 targeted doxorubicin-loaded liposomes thanreduction-sensitive targeted liposomes further demonstrat-ing the need for multifunctional liposomes A combinationof enhanced uptake and reduction-sensitivity was also doneusing reduction-detachable PEG and TAT [298] Cleav-age of DOPE-S-S-PEG5000 allowed unmasking of DOPE-PEG1600-TAT and superior uptake of calcein in vitro overuncleavable TAT-modified liposomes together with stabilityin the presence of serum Reduction-sensitive liposomes havealso been used for gene delivery and a linear correlationbetween intracellular glutathione content and transfectionefficiency has been recently demonstrated [299]

6 Intracellular Delivery

Internalization of anticancer drugs by cancer cells in tumorswas shown to be a barrier to be overcome for cancer therapy[98 101] The use of internalization modifications at theliposomal surface or exposed after release of a PEG coronain the tumor-environment for active transport into cells andeven subcellular delivery increased therapeutic activity [717 96 300] The influence of lipid composition on drugrelease and internalization endosomal escape strategies andmitochondria targeting is discussed below (Figure 4)

61 Importance of Lipid Composition Thepresence of choles-terol or rigid saturated lipids (DSPC HSPC) stabilizesthe liposomal membrane against liposomal dissociation byplasma proteins and limits drug leakage and thus mostdrug-loaded liposomes include cholesterol in the lipid bilayer

[45 288 301] These lipids have high gel-to-liquid crys-talline phase transition temperatures (55ndash58∘C) comparedto physiological temperature (37∘C) which prevents coex-istence of the two phases and contributes to improveddrug pharmacokinetics [13 45 302] In some studies thecouple sphingomyelincholesterol is used to further rigidifythe membrane through hydrogen bonding [303] Howevercholesterol inclusion can decrease drug loading Indeedpaclitaxel loading decreased form 993 at a 5 molarcontent of cholesterol to 665 at 17 cholesterol contentand 62 at a 37 molar content as a result of the hindereddrug penetration in the increasingly rigid lipid bilayer [304]The lipid composition is also important for the choice of thePEG-lipid conjugate used for PEGylation Indeed Kusumotoet al reported a 10-fold higher transfection using liposomesarmed with an endosomal-escape peptide (IFN7) harboringcholesteryl-PEG2000 over DSPE-PEG2000 [305] The supe-rior endosomal escape of liposomes preparedwith the formerwas attributed to the higher fluidity of cholesterol over DSPEa superior fluidity favoring interactionwith endosomalmem-branes and the resulting endosomal escape and transfectionefficiency Hydrophobicity was also shown to be a determi-nant for the design of smart multifunctional nanocarriersHansen et al compared UV-triggered TaT peptide-mediatedliposome internalization with a 16 or 12 carbons lipid anchor[306] In addition to better internalization liposomes with aC16 anchor were less prone to aggregation than those witha C12 anchor The authors suggested the more hydrophobicalkyl chain favored liposomal insertion and the burial of theTaT peptide in a PEG-loop for the best UV-responsivenessand stability in cell culturemediawith bovine serumalbumin

Insertion of negatively charged lipids such as cardi-olipin has been used to increase the retention of positivelycharged drugs in liposomes [45] This was recently wellillustrated for the preparation of mitoxantrone liposomes(mitoxantrone-complexed liposomes) by electrostatic com-plexation between anionic cardiolipin-based liposomes andcationic mitoxantrone [307] While loading efficiencies of95 were obtained with anionic liposomes using cardi-olipin (CA) cholesteryl hemisuccinate (CHEMS) egg L-120572-phosphatidylglycerol (PG) or L-120572-phosphatidylserine (PS)only 38 loading was achieved with neutral liposomesThe therapeutic activity of the different anionic liposomalmitoxantrone preparations was in good agreement withrelease ofmitoxantrone that is with themitoxantrone releasein vitro after heparin treatment CHEMS liposomes had thelowest retention capacity and had virtually no impact on thesurvival of peritoneal carcinoma-bearing mice and both PSand PG liposomes had intermediate mitoxantrone retentionand exhibited higher therapeutic activity than free drugalbeit still inferior to that of CA liposomes capable of thehighest mitoxantrone retention in vitro Inclusion of anioniclipids should be coupled with PEGylation since a negativecharge directs liposomes to liver and spleen [308]

Lipid composition is also determinant for stimuli-responsive drug release Goldenbogen et al reported nocalcein release from disulfide conjugated dipalmitoylphos-phatidylcholine liposomes after treatment with a reduc-ing agent whereas reduction-induced release was observed


from liposomes including 55 of unsaturated dioleoylphos-phatidylethanolamine [297] Note that Candiani et al alsoincorporated DOPE in the lipid composition for biore-ducible gene delivery stressing the importance of DOPE asa helper lipid for membrane destabilization [299] Increasedpermeability for thermosensitive drug release has beenaddressed by inclusion of 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (P-lyso-PC) due to its tendency to formmicelles and allow therapeutic efficacy in vivo of doxorubicin-loaded thermosensitive liposomes [309] Nevertheless thepharmacokinetics after administration in dogs was moresimilar to free doxorubicin than Doxil which demonstratesthe need to further optimize the lipid composition Althoughliposomal cisplatinwith 80hydrogenated soy phosphatidyl-choline (HSPC) showed increased cisplatin accumulation inpreclinical tumors over free drug [21] this did not translateinto therapeutic activity in patients [310 311] Absence ofclinical activity was correlated with a lack of detectablereleased drug in the serum of treated patients revealing theneed for a balance between modifying the free drug pharma-cokinetics for improved biodistribution to the diseased siteand bioavilability [96] PEGylation is required for enhancedblood residency and therapeutic efficacy but postinsertionof DSPE-PEG6000 into preformulated siRNA lipoplexes wasreported to induce siRNA release in vitro [312] and wasnicely overcome by the use of cholesterol grafted siRNA forincreased retention in liposomesThe combination of cellularuptake and targeting using a cholesterol-siRNA conjugateand cyclic RGD peptide allowed luciferase silencing in aB16F10-luc 2 experimental lung metastasis model validatingthis new system [313]

62 Cell Penetrating Peptides Cell penetrating peptides(CPPs) are amphiphatic peptides usually cationic eitherderived from viruses or synthetic that are able to improve thecellular internalization of the attached cargo [314] (Figure 4)The most frequently used CPPs are the TaT peptide derivedfrom the transcription-transactivating protein of humanimmunodeficiency virus type 1 and synthetic polyarginine[315 316] TaT peptide is a powerful internalization moi-ety However its endocytosis lacks cell-specificity and TaTpeptide exposure at the liposome surface can lead to MPSelimination after opsonin binding as well [317] For Tat-mediated internalization only in the tumor environmentmasking strategies have been proposed This concept wasproved by Kale and Torchilin using masked TaT peptidesurface-functionalized lipoplexes prepared with a plasmidcoding for GFP (DSPE-PEG1000-TAT) by a pH-sensitivePEG corona (DSPE-hydrazone-PEG2000) leading to highertransgene expression in tumor tissue after intratumoral injec-tion of pH-sensitive formulations [318] Kuai et al maskedTaT peptide at the liposome surface (TAT-PEG2000-DSPE)by a reduction-sensitive PEG corona (PEG5000-S-S-DSPE)to take advantage of the higher concentration of reductiveenzymes in tumors [319] This allowed higher tumor accu-mulation and less liver uptake than unmasked Tat peptide-modified liposomes after intravenous administration

More recently UV-triggered CPPs have been proposed[306] They added a CPP through incorporation of a TaTpeptide-lipid conjugatewith two lipid anchors a TaT peptide-PEG2000-DSPE conjugate linked to a less stable single chainhydrophobic group of 12 or 16 carbons via a UV-cleavablelinker They demonstrated a UV-dependent internalizationof liposomes (a 15-fold increase in cellular adhesion andinternalization only after irradiation) not observed withan uncleavable linker that reached levels comparable toDSPE-PEG2000-TaT peptide liposomes For the same pur-pose of cell-type selective CPP-mediated uptake Kibria etal functionalized liposomes with either RGD peptide orthe tumor endothelial cell-specific peptide KYND and theoctaarginine CPP and showed synergy of the combination oftargeting peptide and cell penetrating peptide for liposomeuptake in vitro with higher cell selectivity [320] The samegroup later demonstrated superior antitumor activity ofdoxorubicin-loaded liposomes harboring both the tumorendothelial cell-specific peptideNGRand the cell penetratingpeptide tetraarginine over untargeted liposomes or single-modified doxorubicin-loaded liposomes [183] Presentationof octaarginine at the surface of bleomycin-loaded liposomesincreased apoptosis induction in tumors and tumor growthinhibition over bleomycin-loaded liposomes devoid of theCPP [321] Superior tumor growth inhibition was evidencedover untargeted RTN (receptor-targeted nanocomplexesRTN) using lipopolyplexes decorated with an integrin-targeting peptide for delivery of pDNA encoding IL-2 andIL-12 to promote antitumor immunity [322 323] In theirstudy the complexes were optimized for disassembly in thetarget cell [323 324] The PEG-lipid conjugates used hadan esterase-cleavable bond for endosomal escape and theintegrin-targeting peptide was coupled to the polycationused for pDNA condensation by a linker cleavable by bothcathepsin B and along with furin for intracellular release ofthe nucleic acid and high transfection efficiency

In addition to enhancing cellular uptake TaT peptideconjugation allowed crossing of the blood brain barrier inin vitro models and increased drug delivery of doxorubicin-loaded liposomes resulting in prolonged survival of ortho-topic glioma-bearing animals after intravenous administra-tion [325]

63 Endosomal Escape After the endocytosis the cargo istransferred from endosomes (pH 65ndash6) to lysosomes (pH lt5) [326] in which enzymatic degradation occurs AlthoughPEGylation is required for extended blood circulation andtumor accumulation [7] this modification decreases cellularuptake and further increases endosomal degradation ofthe cargo thereby reducing its activity [327 328] Theseconflicting properties of PEG have been referred to as theldquoPEG dilemmardquo [292] The decreased endosomal pH hasbeen exploited as a means to escape degradation using eitherfusogenic lipids or peptides which destabilize membranesafter conformational activation at low pH amines protonableat acidic pH for endosome swelling and rupture by a buffereffect [329ndash338] (Figure 4) The peptides used are either


derived from viruses such as TATp from Human Immun-odeficiency Virus [339] IFN7 from the haemagglutinin ofinfluenza virus [340] or artificial peptides like GALA [341]Inclusion of these peptides leads to superior intracellu-lar drug accumulation and resulting in higher cytotoxicitythan liposomes devoid of endosomolysis properties As anew approach Kullberg et al attached the pore-formingprotein listeriolysin O to HER2-targeted bleomycin-loadedliposomes resulting in a higher toxicity in vitro over targetedbleomycin-loaded liposomes without listeriolysin O [342]

64 Mitochondrial Targeting Effective treatment of cancerfaces problems due to limited drug penetration and drugresistance [343ndash345] Since resistance to antineoplastic agentsinduced cell death is frequently associated with alteredmitochondrial function andor altered expression of mito-chondrial regulators of apoptosis [300 343] subcellularaccumulation of anticancer drugs in mitochondria can givea therapeutic advantage and has been exploited [300 346](Figure 4)

Mitochondria targeting of epirubicin-loaded liposomesby inclusion of the positively charged electrolytedequalinium increased their cytotoxicity in vitro andantitumor activity in vivo over untargeted liposomes[347] Hatakeyama and coworkers developed a Mito-Porter multifunctional envelope-type nanodevice (MEND)nanocarrier with sequential activation of essential functionsnecessary for mitochondria delivery [292 346 348]These formulations have a ldquoprogrammed packagingrdquotheir surface is functionalized with a targeting moiety(transferrin or antibody) a PEG-lipid conjugate for longblood circulation and a PEG-lipid bond that is cleavedin the tumor environment by matrix metalloproteinasesleading to exposure of a CPP (octaarginine tetraarginine)for tumor-selective endocytosis Once inside the cell afusogenic peptide (KALA or GALA) allows endosomalescape of positively charged liposomes by membrane fusionthe positive charge favoring their interaction with thelargely negative outer mitochondrial membrane and finallythe fusogenic lipid DOPE allows internalization of thecargo by the mitochondria [346] Although complex suchnanocarriers are produced in GMP conditions warrantingtheir clinical evaluation [348]

Instead of using one moiety for each step of intracel-lular targeting Zhang and coworkers designed a smartpH-responsive lipid (15-dioctadecyl-L-glutamyl-2-histidyl-hexahydroxybenzoic acid HHG2C

18) [349] The liposomes

generated are negatively charged at physiological pH andhave a sharp charge inversion at acidic pH (from minus229mVat pH 74 to +63mV at pH 65) for tumor-selective uptakeAfter uptake hexahydrobenzoic acid is released by cleavageof the 120573-carboxylic acid linker in the endosomes leadingto exposure of histidine and the endosomal escape of pos-itively charged liposomes electrostatically targeted to theouter mitochondrial membrane Liposomes containing theHHG2C

18lipid and encapsulating the anticancer drug Tem-

sirorimus showed higher renal cancer tumor growth inhibi-tion than free drug or nonresponsive liposomes Targeting

of topotecan-loaded PEGylated liposomes to mitochondriaby inclusion of dequalinium a lipophilic cation with a delo-calized charge center that is attracted by the mitochondrialtransmembrane potential [350] showed higher therapeuticefficacy than untargeted drug-loaded liposomes or free drugin two animal tumor models

In another study [351] postinsertion of the mitochon-driotropic dye Rh123-PEG2000-DSPE conjugate into PEGy-lated liposomes permitted their mitochondrial accumulationand increased the toxicity of paclitaxel-loaded liposomesover untargeted liposomes or free drug This result is inline with the activation of the intrinsic apoptosis pathwayby paclitaxel [352] Although these modifications lead tosuperior cytotoxicity the lack of cancer cell specificity candecrease their therapeutic index To address this challengethe same authors modified paclitaxel-loaded liposomes witha mitochondriotropic lipid (triphenylphosphonium TPP)TPP-PEG-PE conjugate [353] While the PEGylation of lipo-somes leads to their extravasation into the tumor by theEPR effect TPP modification allowed superior therapeu-tic activity of mitochondria-targeted liposomes since moredrug was intracellularly available Malhi et al developedldquomitocancerotropicrdquo doxorubicin-loaded liposomes combin-ing tumor targeting by folic acid and mitochondriotropismby TPP [354] Dual-targeted liposomes led to higher dox-orubicin accumulation in mitochondria and superior toxic-ity than single-targeted doxorubicin-loaded liposomes thuswarranting further evaluation of this strategy

7 Remote-Controlled Payload Release

To achieve release of the therapeutic agent at the tumor siteseveral strategies have been explored including ultrasound-triggered photo-triggered thermotriggered content releaseafter controlled destabilization of the lipid bilayer (Figure 2)

71 Ultrasonication Ultrasound-induced membrane perme-abilization has been used for external stimuli-triggered drugrelease form liposomes by thermal or nonthermal effects(reviewed in [355]) Using PEGylated cisplatin-loaded lipo-somes a 70 drug release after external ultrasound heatingand a 27-fold increase in drug content occured in vivowhereas only 3 cisplatin was released without ultrasoundexposure leading to the superior therapeutic activity of theformulation in ultrasound-treated mice [356] A correlationbetweenDSPE content in liposomemembranes and sonosen-sitivity has also been reported [357]

72 Photo-Sensitive Release and Photodynamic TherapyPhoto-sensitive liposomal drug delivery relies on photodesta-bilization of the liposomal bilayer to release the encapsulateddrug [358] The liposomes used should be able to routethe drug to the tumor and protect it from photodynamicdamage [359] Photodynamic therapy (PDT) consists of thedestruction of tumors by light-activation of a photosensitizerresulting in liberation of singlet oxygen that destroys thetumor by apoptosis necrosis or autophagy-induced celldeathmechanisms [360] Although the limited light diffusion


of this approach has been challenged by coupling of a lightsource to diffusing tips to treat deeper tumors [361] the areaof cell death induction is still restrained due to the shortlifetime of singlet oxygen (nanoseconds) [360] Moreover asthese agents are mainly hydrophobic their administration islimited by their aggregation and the technique is limited todetectable tumors due to the nonspecific photosensitization[360 362 363] Liposomal delivery of photosensitizers wouldallow treatment of both primary tumors and metastasesby enhanced uptake of the photosensitizer by tumor cellsYavlovich et al reported for the first time light-triggeredrelease of doxorubicin from PEGylated liposomes afterlaser irradiation including 10 of the photopolymerizablediacetylene phospholipid (12bis-(tricosa-10 12-diynoyl)-sn-glycero-3-phosphocholine DC

89PC) resulting in photo-

triggered cell killing in vitro [359] The encapsulation of zinctetraphenylporphyrin into PEGylated folate-targeted lipo-somes improved its uptake and cytotoxicity after irradiationcompared to untargeted liposomes in vitro [364] Bovis etal compared the pharmacokinetics of m-THPC [5101520-tetra-(m-hydroxyphenyl)chlorin] administered either in itsclinically approved ethanolpropylene glycol formulation(Foscan) or in PEGylated liposomes [363] Formulationof m-THPC in liposomes decreased its blood clearanceand decreased skin photosensitivity compared to FoscanFurthermore m-THPC showed superior tumor accumu-lation and higher tumor necrosis than Foscan support-ing its preclinical evaluation Using another m-THPC un-PEGylated liposomal formulation (dipalmitoylphosphatidyl-cholinedipalmitoylphosphatidylglycerol 9 1 molar ratio)Lasalle et al stressed the importance of optimization of thedelay between photosensitizer administration and irradiation[365] Indeed while no increase in survival of mammarycarcinoma-bearing mice was observed compared to controlfor 1 h and 3 h drug-light intervals 6 h and 15 h intervals cured79 and 63 of mice respectively

73 Thermoresponsive Preparations While lipids with hightransition temperatures (above 55∘C) are required for bloodstability and to decrease blood leakage inclusion of lipidswith transition temperatures closer to physiological bodytemperature (40ndash45∘C) allows induction of drug release afterexternal localized heating [45] Inclusion of low transitiontemperature lipids is a strategy used in tumor therapy formore than 30 years since the pioneering study of Wein-stein et al who used dipalmitoylphosphatidylcholine [366]Doxorubicin-loaded liposomes containing 2 of poly [2-(2-ethoxy)ethoxyethyl vinyl ether (EOEOVE)] (transitiontemperature 40∘C) exhibited a rapid doxorubicin release afterheating to 45∘C with limited release at 37∘C and allowedtumor growth suppression only after heating [367] Interest-ingly in their study thermoresponsiveness of poly (EOEOVE)liposomes was improved by coinclusion of DSPE-PEG5000in the liposome formulation and revealed an advantage ofmultifunctional liposome PEGylation Encapsulation of thedoxorubicin analog epirubicin into PEGylated thermore-sponsive liposomes increased blood residency and tumoraccumulation over unresponsive liposomes or free drug

resulting in a 20 higher tumor growth inhibition in animalstreated with thermoresponsive liposomes over unresponsiveepirubicin-loaded liposomes [368]

Paasonen et al used gold-nanoparticles as ldquoenergy col-lectorsrdquo to lower the threshold energy required to inducephoto-sensitive drug release [369] After heat transfer fromgold nanoparticles to lipids promoting liquid crystal-to-gel phase transition a UV-induced liberation of the modelcompound calcein was evidenced with virtually no releasewithout irradiation Magnetic fluid hyperthermia involvesheat transfer frommagnetic particles after exposure to amag-netic field that results in localized elevation of temperatureand induction of cell death [370] To improve the selectivitydoxorubicin thermo-responsive liposomes coloaded withdoxorubicin and magnetic nanoparticles were armed withfolic acid and resulted in improved cytotoxicity in vitro overnonresponsive liposomes or untargeted thermo-responsivedoxorubicin-loaded liposomes [371] Intra-tumoral injectionof anti-HER2 immunoliposomes containing magnetite fol-lowed by alternate magnetic field heating promoted ironretention in tumors in a HER2-specific manner 48 h afterinjection [372] A 3-fold higher iron content was detected inHER2-overexpressing BT474 breast cancer xenografts overlow HER2-expressing SKOV3 ovarian cancer xenografts andmagnetite retention in BT474 xenografts correlated withstable tumor regression [372] In line with these studies con-jugation of HER2 antibody to thermo-sensitive doxorubicin-loaded liposomes improved the doxorubicin-mediated toxic-ity over controls [373]

Boron capture neutron therapy relies on delivery of 10Bboron followed by 120574-irradiation and capture of neutrons by10B leading to the production of toxic 120572-particles 4H and7Li for cell death induction [374] Maruyama encapsulated10B into PEGylated transferrin-armed liposomes for targeteddelivery to colon carcinoma xenografts this led to higher 10Btumor accumulation compared to the free isotope or untar-geted liposomes and resulted in superior therapeutic efficacyafter irradiation over free isotope or untargeted 10B liposomes[36] Lastly the group led by Miyata reported a 36-foldhigher 10B tumor concentration in orthotopic gliomas afterintratumoral convection-enhanced delivery using PEGylatedtransferrin armed liposomes over untargeted liposomes witha lower retention in normal brains [375] Superior ther-apeutic activity was observed against intracranial gliomasafter intravenous injection of transferrin-targeted liposomesencapsulating sodium borocaptate over untargeted ones afterneutron irradiation [376]

8 Theranostic Liposomes

Simultaneous therapy and diagnosis following codelivery oftherapeutic and imaging agents theranostic are determinantfor the development of personalized medicine since it wouldallow clinicians to detect and characterize lesions and rapidlyevaluate tumor response and modify treatment accordingly(increase dose stop treatment or use an alternate drug)[377ndash379] Indeed liposomes are currently widely used fordiagnosis (see recent reviews) [380ndash382]


Kenny et al designed PEGylated liposome-entrappedsiRNA nanoparticles (LEsiRNA) loaded with gadolinium(III) for magnetic resonance imaging siRNA against theapoptosis inhibitor survivin for tumor therapy and labeledwith DOPE-rhodamine for fluorescence detection [383]Accumulation of LEsiRNA in ovarian cancer xenografts afterintravenous injection was demonstrated by MRI and con-firmed post mortem in tumor biopsies by fluorescence within vivo survivin silencing and tumor weight reduction Gd-labeled doxorubicin-loaded thermo-responsive liposomesallowed detection of both tumor imaging by MRI and tumorregression after localized heating [384] Note that to retainthermoresponsiveness after Gd-labeling a new Gd-chelate-dendron-based lipid was included in the lipid bilayer insteadof a standard Gd-lipid conjugate to decrease Gd-lipid contentto enhance thermosensitivity

The use of magnetic resonance imaging (MRI) to allowboth tumor visualization and temperature feedback forimaging-guided thermo-responsive drug delivery showedimproved therapy of the image-guided thermallyinduceddrug release [385 386] Labeling of prednisolone-labeledliposomes did not decrease its therapeutic activity allowedevaluation of in vivo drug biodistribution and responsemonitoring simultaneously with MRI signal detection 1week after injection [387] To combine the advantages ofthree imaging modalities (optical imaging CT imaging andMRI) Li et al and Mitchell et al developed liposomeslabeled with a fluorophore tracer with 99mTc 111In or 64Cuand Gd [388 389] Since most facilities do not possess allthe imaging equipment this system would allow a moreflexible followup of therapeutic activity by optical imagingwhile in depth studies would use CT or MRI without theneed of administration of another imaging agent Spatiallycontrolled thermallyinduced drug release was achieved withMRI-guided high intensity focused ultrasound heating of thetargeted tumor region resulting in deep tumor penetration ofdoxorubicin-loaded thermo-sensitive liposomes coloadingof liposomes with doxorubicin and gadolinium allowingtumor visualization and therapy [385 386 390]

The contrast agent used for the preparation of theranosticsiRNA liposomes must be chosen with care Mikhaylovaet al reported nonspecific protein downregulation in vitroafter incorporation of gadolinium of Magnevist into COX-2 (cyclooxygenase 2) siRNA-loaded liposomes while COX-2silencing without nonspecific downregulation was detectedwith liposomes coloaded with COX-2 siRNA and Feridex[391] Targeting drug-loaded liposomes in addition toenhancing their therapeutic activity enhances tumor detec-tion and response monitoring when they are coloaded withan imaging agent Addition of transferrin to 10B plus iodinecontrast agent coloaded liposomes allowed a 36-fold higher10B concentration in tumor tissues over untargeted coloadedliposomes [375] The selective retention of transferrin-targeted formulations led to better tumor detection 72 h afteradministration of liposomes a period duringwhich the signalfrom untargeted liposomes had washed out thus combiningmonitoring of drug delivery and tumor response with boronneutron capture therapy [375] Combined delivery of Gd and

doxorubicin in liposomes targeted with a neural cell adhe-sion molecule-specific peptide allowed higher concentrationof doxorubicin in tumor tissues correlated with increasedtumor growth inhibition over untargeted coloaded liposomestogether with better visualization of tumors by MRI [392]Targeting of iron oxide and doxorubicin coloaded liposomesto pancreatic tumors by conjugation of an antimesothelinantibody improved the antitumor activity and tumor signalenhancement over untargeted liposomes [393] Folate tar-geting of doxorubicin-loaded liposomes encapsulating ironoxide resulted in superior tumor growth inhibition of livercancer tumors than the standard formulation Doxil andsimultaneously allowed tumor imaging by MRI with highersensitivity than the commercial contrast agent Resovist[394]

9 Conclusions

In addition to the need for extended blood circulation andstimuli-controlled extravasation to the tumorrsquos niche mul-tifunctional liposomal nanocarriers must target at least onehallmark of cancer (aberrant cell growth drug resistance sus-tained angiogenesis and tissue invasion) for enhancement oftumor therapy andor diagnosis As described throughout thepaper this requires coordinated action of stealth targetingand internalizing moieties to achieve intracellular deliveryto cancer cells in tumors Moreover combined targeting oftumor cells and related neoangiogenesis is becoming a focusof research that allows destruction of both primary anddistant tumor nodules However targeted therapies rely onligands presented by a few types of tumors and must faceup to the fact of the heterogeneity of tumor cells and theirsurface markers [175 395 396] A possible direction may bethe coupling of ligands of different natures (antibody proteinpeptides and chimiokine hormone analogs) to target at leasttwo tumor cell populations for relapse-free cancer therapyand more sensitive malignant lesion detection


The authors declare that they have no conflict of interests

Acknowledgments

This work was supported by the NIH Grant U54CA151881 toV P Torchilin The authors are grateful to W C Hartner forcritical review of the paper

References

[1] A D Bangham M M Standish and J C Watkins ldquoDiffusionof univalent ions across the lamellae of swollen phospholipidsrdquoJournal of Molecular Biology vol 13 no 1 pp 238ndash252 1965

[2] G Gregoriadis ldquoLiposome research in drug delivery the earlydaysrdquo Journal of Drug Targeting vol 16 no 7-8 pp 520ndash5242008

[3] D J Porteous J R Dorin G McLachlan et al ldquoEvidencefor safety and efficacy of DOTAP cationic liposome mediated


CFTR gene transfer to the nasal epithelium of patients withcystic fibrosisrdquo Gene Therapy vol 4 no 3 pp 210ndash218 1997

[4] G J Nabel E G Nabel Z Y Yang et al ldquoDirect gene transferwith DNA-liposome complexes in melanoma expression bio-logic activity and lack of toxicity in humansrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 90 no 23 pp 11307ndash11311 1993

[5] N D James R J Coker D Tomlinson et al ldquoLiposomaldoxorubicin (Doxil) an effective new treatment for Kaposirsquossarcoma in AIDSrdquo Clinical Oncology vol 6 no 5 pp 294ndash2961994

[6] A Z Wang R Langer and O C Farokhzad ldquoNanoparticledelivery of cancer drugsrdquo Annual Review of Medicine vol 63pp 185ndash198 2012

[7] T M Allen and P R Cullis ldquoLiposomal drug deliverysystems from concept to clinical applicationsrdquo AdvancedDrug Delivery Reviews vol 65 no 1 pp 36ndash48 2012101016jaddr201209037

[8] V P Torchilin ldquoRecent advances with liposomes as pharmaceu-tical carriersrdquo Nature Reviews Drug Discovery vol 4 no 2 pp145ndash160 2005

[9] G SongHWuKYoshino andWC Zamboni ldquoFactors affect-ing the pharmacokinetics and pharmacodynamics of liposomaldrugsrdquo Journal of Liposome Research vol 22 pp 177ndash192 2012



[12] DHanahan andRAWeinberg ldquoHallmarks of cancerThenextgenerationrdquo Cell vol 144 no 5 pp 646ndash674 2011

[13] Y Barenholz ldquoDoxil(R)mdashthe first FDA-approved nano-druglessons learnedrdquo Journal of Controlled Release vol 160 pp 117ndash134 2012

[14] S M OrsquoBrien W Aulitzky D Ben Yehuda et al ldquoPhase IIstudy of marqibo in adult patients with refractory or relapsedphiladelphia chromosome negative (Ph-) acute lymphoblasticleukemia (ALL)rdquo Journal of Clinical Oncology Abstract 65072010 ASCO Annual Meeting 2010

[15] Q Zhang X E Huang and L L Gao ldquoA clinical study on thepremedication of paclitaxel liposome in the treatment of solidtumorsrdquo Biomedicine and Pharmacotherapy vol 63 no 8 pp603ndash607 2009

[16] V P Torchilin ldquoMultifunctional nanocarriersrdquo Advanced DrugDelivery Reviews vol 58 no 14 pp 1532ndash1555 2006

[17] D Peer J M Karp S Hong O C Farokhzad R Margalit andR Langer ldquoNanocarriers as an emerging platform for cancertherapyrdquo Nature Nanotechnology vol 2 no 12 pp 751ndash7602007

[18] Y Matsumura and H Maeda ldquoA new concept for macro-molecular therapeutics in cancer chemotherapy mechanism oftumoritropic accumulation of proteins and the antitumor agentsmancsrdquo Cancer Research vol 46 no 12 I pp 6387ndash6392 1986

[19] S Zalipsky M Saad R Kiwan E Ber N Yu and T MinkoldquoAntitumor activity of new liposomal prodrug of mitomycin Cinmultidrug resistant solid tumor insights of themechanism ofactionrdquo Journal of Drug Targeting vol 15 no 7-8 pp 518ndash5302007

[20] J Fang H Nakamura and H Maeda ldquoThe EPR effect uniquefeatures of tumor blood vessels for drug delivery factorsinvolved and limitations and augmentation of the effectrdquoAdvancedDrugDelivery Reviews vol 63 no 3 pp 136ndash151 2011

[21] M S Newman G T Colbern P K Working C Engbers andM A Amantea ldquoComparative pharmacokinetics tissue distri-bution and therapeutic effectiveness of cisplatin encapsulatedin long-circulating pegylated liposomes (SPI-077) in tumor-bearingmicerdquoCancer Chemotherapy and Pharmacology vol 43pp 1ndash7 1999

[22] H M Patel ldquoSerum opsonins and liposomes their interactionand opsonophagocytosisrdquo Critical Reviews in Therapeutic DrugCarrier Systems vol 9 no 1 pp 39ndash90 1992

[23] YH Bae andK Park ldquoTargeted drug delivery to tumorsmythsreality and possibilityrdquo Journal of Controlled Release vol 153 no3 pp 198ndash205 2011

[24] E J Feldman J E Lancet J E Kolitz et al ldquoFirst-in-manstudy of CPX-351 a liposomal carrier containing cytarabineand daunorubicin in a fixed 51 molar ratio for the treatmentof relapsed and refractory acute myeloid leukemiardquo Journal ofClinical Oncology vol 29 no 8 pp 979ndash985 2011

[25] G Batist K A Gelmon K N Chi et al ldquoSafety pharmacoki-netics and efficacy of CPX-1 liposome injection in patients withadvanced solid tumorsrdquo Clinical Cancer Research vol 15 no 2pp 692ndash700 2009

[26] A Santel M Aleku N Roder et al ldquoAtu027 prevents pul-monary metastasis in experimental and spontaneous mousemetastasis modelsrdquo Clinical Cancer Research vol 16 no 22 pp5469ndash5480 2010

[27] M Prados ldquoA Phase I trial of nanoliposomal CPT-11 (NLCPT-11) in patients with recurrent high-grade gliomasrdquo Clin-icalTrialsGov (NCT00734682) University of California SanFrancisco Calif USA

[28] T Hamaguchi Y Matsumura Y Nakanishi et al ldquoAntitumoreffect of MCC-465 pegylated liposomal doxorubicin taggedwith newly developedmonoclonal antibody GAH in colorectalcancer xenograftsrdquo Cancer Science vol 95 no 7 pp 608ndash6132004

[29] K K Sankhala A C Mita R Adinin et al ldquoA phase Ipharmacokinetic (PK) study of MBP-426 a novel liposomeencapsulated oxaliplatinrdquo Journal of Clinical Oncology vol 27Abstract 2535 no 15s 2009 ASCO Annual Meeting 2009

[30] I SynerGene Therapeutics ldquoSafety study of infusion of SGT-53to treat solid tumorsrdquo ClinicalTrialsGov (NCT00470613)

[31] Celsion ldquoPhase 3 study of thermoDox with RadioFrequencyAblation (RFA) in treatment of Hepatocellular Carcinoma(HCC)rdquo ClinicalTrialsGov (NCT00617981)

[32] V P Torchilin ldquoAntinuclear antibodies with nucleosome-restricted specificity for targeted delivery of chemotherapeuticagentsrdquoTherapeutic Delivery vol 1 no 2 pp 257ndash272 2010

[33] J M Tuscano S M Martin Y Ma W Zamboni and R TOrsquoDonnell ldquoEfficacy biodistribution and pharmacokinetics ofCD22-targeted pegylated liposomal doxorubicin in a B-cellnon-Hodgkinrsquos lymphoma xenograft mouse modelrdquo ClinicalCancer Research vol 16 no 10 pp 2760ndash2768 2010

[34] T Yang M K Choi F D Cui et al ldquoAntitumor effect ofpaclitaxel-loaded PEGylated immunoliposomes against humanbreast cancer cellsrdquo Pharmaceutical Research vol 24 no 12 pp2402ndash2411 2007

[35] L Zhang H Gao L Chen et al ldquotumor targeting of vincristineby mBAFF-modified PEG liposomes in B lymphoma cellsrdquoCancer Letters vol 269 no 1 pp 26ndash36 2008


[36] K Maruyama ldquoIntracellular targeting delivery of liposomaldrugs to solid tumors based on EPR effectsrdquo Advanced DrugDelivery Reviews vol 63 no 3 pp 161ndash169 2011

[37] X Ying H Wen W L Lu et al ldquoDual-targeting daunorubicinliposomes improve the therapeutic efficacy of brain glioma inanimalsrdquo Journal of Controlled Release vol 141 no 2 pp 183ndash192 2010

[38] DK Chang C T Lin CHWu andHCWu ldquoAnovel peptideenhances therapeutic efficacy of liposomal anti-cancer drugs inmice models of human lung cancerrdquo PLoS ONE vol 4 no 1article e4171 2009

[39] Z Wang Y Yu W Dai et al ldquoThe use of a tumor metastasistargeting peptide to deliver doxorubicin-containing liposomesto highly metastatic cancerrdquo Biomaterials vol 33 pp 8451ndash8460 2012

[40] O P Medina M Haikola M Tahtinen et al ldquoLiposomaltumor targeting in drug delivery utilizing MMP-2- and MMP-9-binding ligandsrdquo Journal of Drug Delivery vol 2011 ArticleID 160515 9 pages 2011

[41] Z Zhang and J Yao ldquoPreparation of irinotecan-loaded folate-targeted liposome for tumor targeting delivery and its antitu-mor activityrdquo AAPS PharmSciTech vol 13 pp 802ndash810 2012

[42] S R Paliwal R PaliwalHC Pal et al ldquoEstrogen-anchored pH-sensitive liposomes as nanomodule designed for site-specificdelivery of doxorubicin in breast cancer therapyrdquo MolecularPharmaceutics vol 9 pp 176ndash186 2012

[43] R Bagari D Bansal A Gulbake A Jain V Soni and S K JainldquoChondroitin sulfate functionalized liposomes for solid tumortargetingrdquo Journal of Drug Targeting vol 19 no 4 pp 251ndash2572011

[44] M L Immordino F Dosio and L Cattel ldquoStealth liposomesreview of the basic science rationale and clinical applicationsexisting and potentialrdquo International journal of nanomedicinevol 1 no 3 pp 297ndash315 2006

[45] D C Drummond C O Noble M E Hayes J W Park and DB Kirpotin ldquoPharmacokinetics and in vivo drug release rates inliposomal nanocarrier developmentrdquo Journal of PharmaceuticalSciences vol 97 no 11 pp 4696ndash4740 2008

[46] E H Kraut M N Fishman P M Lorusso et al ldquoFinalresults of a phase I study of liposome encapsulated SN-38(LE-SN38) safety pharmacogenomics pharmacokinetics andtumor responserdquo Journal of Clinical Oncology vol 23 no 16S2005 ASCO Annual Meeting Proceedings

[47] K R Whiteman V Subr K Ulbrich and V P TorchilinldquoPoly(HPMA)-coated liposomes demonstrate prolonged circu-lation in micerdquo Journal of Liposome Research vol 11 no 2-3 pp153ndash164 2001

[48] A L Klibanov K Maruyama A M Beckerleg V P Torchilinand L Huang ldquoActivity of amphipathic poly(ethylene glycol)5000 to prolong the circulation time of liposomes dependson the liposome size and is unfavorable for immunoliposomebinding to targetrdquo Biochimica et Biophysica Acta vol 1062 no2 pp 142ndash148 1991

[49] Y Maitani A Nakamura T Tanaka and Y Aso ldquoHydration ofsurfactant-modified and PEGylated cationic cholesterol-basedliposomes and corresponding lipoplexes by monitoring a fluo-rescent probe and the dielectric relaxation timerdquo InternationalJournal of Pharmaceutics vol 427 pp 372ndash378 2012

[50] V Reshetov V Zorin A Siupa M A DrsquoHallewin F Guilleminand L Bezdetnaya ldquoInteraction of liposomal formulations ofmeta-tetra(hydroxyphenyl)chlorin (Temoporfin) with serum

proteins protein binding and liposome destructionrdquo Photo-chemistry and Photobiology vol 88 pp 1256ndash1264 2012

[51] R Gref M Luck P Quellec et al ldquorsquoStealthrsquo corona-corenanoparticles surface modified by polyethylene glycol (PEG)influences of the corona (PEG chain length and surface density)and of the core composition on phagocytic uptake and plasmaprotein adsorptionrdquo Colloids and Surfaces B vol 18 no 3-4 pp301ndash313 2000

[52] T H Chow Y Y Lin J J Hwang et al ldquoImprovement of biodis-tribution and therapeutic index via increase of polyethyleneglycol on drug-carrying liposomes in anHT-29luc xenograftedmouse modelrdquoAnticancer Research vol 29 no 6 pp 2111ndash21202009

[53] C M Lee Y Choi E J Huh et al ldquoPolyethylene glycol (PEG)modified 99mTc-HMPAO-liposome for improving blood circu-lation and biodistribution the effect of the extent of PEGyla-tionrdquo Cancer Biotherapy and Radiopharmaceuticals vol 20 no6 pp 620ndash628 2005

[54] AMori A L KlibanovV P Torchilin andLHuang ldquoInfluenceof the steric barrier activity of amphipathic poly(ethyleneglycol)and ganglioside GM1 on the circulation time of liposomesand on the target binding of immunoliposomes in vivordquo FEBSLetters vol 284 no 2 pp 263ndash266 1991

[55] R R Sawant R M Sawant A A Kale and V P Torchilin ldquoThearchitecture of ligand attachment to nanocarriers controls theirspecific interaction with target cellsrdquo Journal of Drug Targetingvol 16 no 7-8 pp 596ndash600 2008

[56] W C Zamboni S Strychor E Joseph et al ldquoPlasma tumorand tissue disposition of STEALTH liposomal CKD-602 (S-CKD602) and nonliposomal CKD-602 in mice bearing A375humanmelanoma xenograftsrdquo Clinical Cancer Research vol 13no 23 pp 7217ndash7223 2007

[57] T Yang F D Cui M K Choi et al ldquoEnhanced solubility andstability of PEGylated liposomal paclitaxel in vitro and in vivoevaluationrdquo International Journal of Pharmaceutics vol 338 no1-2 pp 317ndash326 2007

[58] J I Yokoe S Sakuragi K Yamamoto et al ldquoAlbumin-conjugated PEG liposome enhances tumor distribution ofliposomal doxorubicin in ratsrdquo International Journal of Pharma-ceutics vol 353 no 1-2 pp 28ndash34 2008

[59] K Furumoto J I Yokoe K I Ogawara et al ldquoEffect ofcoupling of albumin onto surface of PEG liposome on its invivo dispositionrdquo International Journal of Pharmaceutics vol329 no 1-2 pp 110ndash116 2007

[60] K Yoshino K Nakamura Y Terajima et al ldquoComparativestudies of irinotecan-loaded polyethylene glycol-modified lipo-somes prepared using different PEG-modification methodsrdquoBiochimica et Biophysica Acta vol 1818 pp 2901ndash2907 2012

[61] K Nakamura K Yamashita Y Itoh K Yoshino S Nozawaand H Kasukawa ldquoComparative studies of polyethylene glycol-modified liposomes prepared using different PEG-modificationmethodsrdquo Biochimica et Biophysica Acta vol 1818 pp 2801ndash2807 2012

[62] Z Cao L Zhang and S Jiang ldquoSuperhydrophilic zwitterionicpolymers stabilize liposomesrdquo Langmuir vol 28 pp 11625ndash11632 2012

[63] K J Harrington SMohammadtaghi P S Uster et al ldquoEffectivetargeting of solid tumors in patients with locally advancedcancers by radiolabeled pegylated liposomesrdquo Clinical CancerResearch vol 7 no 2 pp 243ndash254 2001


[64] S D Li and LHuang ldquoPharmacokinetics and biodistribution ofnanoparticlesrdquoMolecular Pharmaceutics vol 5 no 4 pp 496ndash504 2008

[65] R B Campbell D Fukumura E B Brown et al ldquoCationiccharge determines the distribution of liposomes between thevascular and extravascular compartments of tumorsrdquo CancerResearch vol 62 no 23 pp 6831ndash6836 2002

[66] T S Levchenko R Rammohan A N Lukyanov K R White-man andV P Torchilin ldquoLiposome clearance inmice the effectof a separate and combined presence of surface charge andpolymer coatingrdquo International Journal of Pharmaceutics vol240 no 1-2 pp 95ndash102 2002

[67] W Zhao S Zhuang and X R Qi ldquoComparative study ofthe in vitro and in vivo characteristics of cationic and neutralliposomesrdquo International Journal of Nanomedicine vol 6 pp3087ndash3098 2011

[68] S D Li S Chono and L Huang ldquoEfficient oncogene silencingand metastasis inhibition via systemic delivery of siRNArdquoMolecular Therapy vol 16 no 5 pp 942ndash946 2008

[69] E T M Dams P Laverman W J G Oyen et al ldquoAcceleratedblood clearance and altered biodistribution of repeated injec-tions of sterically stabilized liposomesrdquo Journal of Pharmacologyand Experimental Therapeutics vol 292 no 3 pp 1071ndash10792000

[70] P Laverman M G Carstens O C Boerman et al ldquoFactorsaffecting the accelerated blood clearance of polyethylene glycol-liposomes upon repeated injectionrdquo Journal of Pharmacologyand Experimental Therapeutics vol 298 no 2 pp 607ndash6122001

[71] T Ishida and H Kiwada ldquoAccelerated blood clearance (ABC)phenomenon upon repeated injection of PEGylated liposomesrdquoInternational Journal of Pharmaceutics vol 354 no 1-2 pp 56ndash62 2008

[72] T IshidaM Ichihara XWang andHKiwada ldquoSpleen plays animportant role in the induction of accelerated blood clearanceof PEGylated liposomesrdquo Journal of Controlled Release vol 115no 3 pp 243ndash250 2006

[73] X Wang T Ishida and H Kiwada ldquoAnti-PEG IgM elicitedby injection of liposomes is involved in the enhanced bloodclearance of a subsequent dose of PEGylated liposomesrdquo Journalof Controlled Release vol 119 no 2 pp 236ndash244 2007

[74] A Gabizon R Chisin S Amselem et al ldquoPharmacokinetic andimaging studies in patients receiving a formulation of liposome-associated adriamycinrdquo British Journal of Cancer vol 64 no 6pp 1125ndash1132 1991

[75] T Ishida S Kashima and H Kiwada ldquoThe contribution ofphagocytic activity of liver macrophages to the acceleratedblood clearance (ABC) phenomenon of PEGylated liposomesin ratsrdquo Journal of Controlled Release vol 126 no 2 pp 162ndash165 2008

[76] T Tagami Y Uehara N Moriyoshi T Ishida and H KiwadaldquoAnti-PEG IgM production by siRNA encapsulated in a PEGy-lated lipid nanocarrier is dependent on the sequence of thesiRNArdquo Journal of Controlled Release vol 151 no 2 pp 149ndash1542011

[77] T Tagami K Nakamura T Shimizu N Yamazaki T Ishidaand H Kiwada ldquoCpG motifs in pDNA-sequences increaseanti-PEG IgM production induced by PEG-coated pDNA-lipoplexesrdquo Journal of Controlled Release vol 142 no 2 pp 160ndash166 2010

[78] T Shimizu M Ichihara Y Yoshioka T Ishida S Nakagawaand H Kiwada ldquoIntravenous administration of polyethylene

glycol-coated (PEGylated) proteins and PEGylated adenoviruselicits an anti-PEG immunoglobulin M responserdquo Biological ampPharmaceutical Bulletin vol 35 pp 1336ndash1342 2012

[79] T Daemen G Hofstede M T T Kate I A J M Bakker-Woudenberg and G L Scherphof ldquoLiposomal doxorubicin-induced toxicity depletion and impairment of phagocyticactivity of liver macrophagesrdquo International Journal of Cancervol 61 no 5 pp 716ndash721 1995

[80] E W M Van Etten M T T Kate S V Snijders and I A JM Bakker-Woudenberg ldquoAdministration of liposomal agentsand blood clearance capacity of the mononuclear phagocytesystemrdquo Antimicrobial Agents and Chemotherapy vol 42 no 7pp 1677ndash1681 1998

[81] A Gabizon R Isacson O Rosengarten D Tzemach HShmeeda and R Sapir ldquoAn open-label study to evaluate doseand cycle dependence of the pharmacokinetics of pegylatedliposomal doxorubicinrdquo Cancer Chemotherapy and Pharmacol-ogy vol 61 no 4 pp 695ndash702 2008

[82] A Gabizon D Tzemach L Mak M Bronstein and A THorowitz ldquoDose dependency of pharmacokinetics and thera-peutic efficacy of pegylated liposomal doxorubicin (DOXIL) inmurinemodelsrdquo Journal ofDrugTargeting vol 10 no 7 pp 539ndash548 2002

[83] M Amantea M S Newman T M Sullivan A Forrest and PK Working ldquoRelationship of dose intensity to the inductionof palmar-plantar erythrodysesthia by pegylated liposomaldoxorubicin in dogsrdquoHuman and Experimental Toxicology vol18 no 1 pp 17ndash26 1999

[84] A S Abu-Lila N E Eldin M Ichihara T Ishida and HKiwada ldquoMultiple administration of PEG-coated liposomaloxaliplatin enhances its therapeutic efficacy a possible mech-anism and the potential for clinical applicationrdquo InternationalJournal of Pharmaceutics vol 438 no 1-2 pp 176ndash183 2012

[85] C Li J Cao Y Wang et al ldquoAccelerated blood clearance ofpegylated liposomal topotecan influence of polyethylene glycolgrafting density and animal speciesrdquo Journal of PharmaceuticalSciences vol 101 pp 3864ndash3876 2012

[86] T Suzuki M Ichihara K Hyodo et al ldquoAccelerated bloodclearance of PEGylated liposomes containing doxorubicin uponrepeated administration to dogsrdquo International Journal of Phar-maceutics vol 436 pp 636ndash643 2012

[87] NM La-Beck B A Zamboni A Gabizon et al ldquoFactors affect-ing the pharmacokinetics of pegylated liposomal doxorubicinin patientsrdquo Cancer Chemother Pharmacol vol 69 pp 43ndash502012

[88] J Szebeni F Muggia A Gabizon and Y Barenholz ldquoActiva-tion of complement by therapeutic liposomes and other lipidexcipient-based therapeutic products prediction and preven-tionrdquo Advanced Drug Delivery Reviews vol 63 pp 1020ndash10302011

[89] J Szebeni and S M Moghimi ldquoLiposome triggering of innateimmune responses a perspective on benefits and adversereactionsrdquo Journal of LiposomeResearch vol 19 no 2 pp 85ndash902009

[90] S M Moghimi I Hamad T L Andresen K Joslashrgensenand J Szebeni ldquoMethylation of the phosphate oxygen moi-ety of phospholipid- methoxy(polyethylene glycol) conjugateprevents PEGylated liposome-mediated complement activationand anaphylatoxin productionrdquo FASEB Journal vol 20 no 14pp 2591ndash2593 2006


[91] I K Kwon S C Lee B Han and K Park ldquoAnalysis on thecurrent status of targeted drug delivery to tumorsrdquo Journal ofControlled Release vol 164 no 2 pp 108ndash114 2012

[92] C H Heldin K Rubin K Pietras and A Ostman ldquoHigh inter-stitial fluid pressuremdashan obstacle in cancer therapyrdquo NatureReviews Cancer vol 4 no 10 pp 806ndash813 2004

[93] A J Primeau A Rendon D Hedley L Lilge and I F TannockldquoThe distribution of the anticancer drug doxorubicin in relationto blood vessels in solid tumorsrdquo Clinical Cancer Research vol11 no 24 pp 8782ndash8788 2005

[94] F Yuan M Leunig S K Huang D A Berk D Papahadjopou-los and R K Jain ldquoMicrovascular permeability and interstitialpenetration of sterically stabilized (stealth) liposomes in ahuman tumor xenograftrdquo Cancer Research vol 54 no 13 pp3352ndash3356 1994

[95] M J Parr DMasin P R Cullis andM B Bally ldquoAccumulationof liposomal lipid and encapsulated doxorubicin in murineLewis Lung carcinoma the lack of beneficial effects by coatingliposomes with poly(ethylene glycol)rdquo Journal of Pharmacologyand Experimental Therapeutics vol 280 no 3 pp 1319ndash13271997

[96] T M Allen D R Mumbengegwi and G J R Charrois ldquoAnti-CD19-targeted liposomal doxorubicin improves the therapeuticefficacy inmurine B-cell lymphoma and ameliorates the toxicityof liposomes with varying drug release ratesrdquo Clinical CancerResearch vol 11 no 9 pp 3567ndash3573 2005

[97] R Wang R Xiao Z Zeng L Xu and J Wang ldquoApplicationof poly(ethylene glycol)-distearoylphosphatidylethanolamine(PEG-DSPE) block copolymers and their derivatives asnanomaterials in drug deliveryrdquo International Journal ofNanomedicine vol 7 pp 4185ndash4198 2012

[98] D B Kirpotin D C Drummond Y Shao et al ldquoAntibodytargeting of long-circulating lipidic nanoparticles does notincrease tumor localization but does increase internalization inanimal modelsrdquo Cancer Research vol 66 no 13 pp 6732ndash67402006

[99] D W Bartlett H Su I J Hildebrandt W A Weber and ME Davis ldquoImpact of tumor-specific targeting on the biodis-tribution and efficacy of siRNA nanoparticles measured bymultimodality in vivo imagingrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 104 no39 pp 15549ndash15554 2007

[100] K M Laginha E H Moase N Yu A Huang and T MAllen ldquoBioavailability and therapeutic efficacy of HER2 scFv-targeted liposomal doxorubicin in a murine model of HER2-overexpressing breast cancerrdquo Journal of Drug Targeting vol 16no 7-8 pp 605ndash610 2008

[101] P Sapra E H Moase J Ma and T M Allen ldquoImprovedtherapeutic responses in a xenograft model of human B lym-phoma (Namalwa) for liposomal vincristine versus liposomaldoxorubicin targeted via anti-CD19 IgG2a or Fab1015840 fragmentsrdquoClinical Cancer Research vol 10 no 3 pp 1100ndash1111 2004

[102] T A Elbayoumi and V P Torchilin ldquotumor-targetednanomedicines enhanced antitumor efficacy in vivo ofdoxorubicin-loaded long-circulating liposomes modifiedwith cancer-specific monoclonal antibodyrdquo Clinical CancerResearch vol 15 no 6 pp 1973ndash1980 2009

[103] X Li L Ding Y Xu YWang andQ Ping ldquoTargeted delivery ofdoxorubicin using stealth liposomesmodified with transferrinrdquoInternational Journal of Pharmaceutics vol 373 no 1-2 pp 116ndash123 2009

[104] A BMadhankumar B Slagle-Webb XWang et al ldquoEfficacy ofinterleukin-13 receptor-targeted liposomal doxorubicin in theintracranial brain tumor modelrdquo Molecular Cancer Therapeu-tics vol 8 no 3 pp 648ndash654 2009

[105] Y Iwase and Y Maitani ldquoOctreotide-targeted liposomes loadedwith CPT-11 enhanced cytotoxicity for the treatment ofmedullary thyroid carcinomardquoMolecular Pharmaceutics vol 8no 2 pp 330ndash337 2011

[106] J Zhang W Jin X Wang J Wang X Zhang and Q Zhang ldquoAnovel octreotide modified lipid vesicle improved the anticancerefficacy of doxorubicin in somatostatin receptor 2 positivetumor modelsrdquoMolecular Pharmaceutics vol 7 no 4 pp 1159ndash1168 2010

[107] M Saad O B Garbuzenko E Ber et al ldquoReceptor targetedpolymers dendrimers liposomes which nanocarrier is themost efficient for tumor-specific treatment and imagingrdquoJournal of Controlled Release vol 130 no 2 pp 107ndash114 2008

[108] F Danhier A L Breton and V Preat ldquoRGD-based strategiesto target alpha(v) beta(3) integrin in cancer therapy anddiagnosisrdquo Molecular Pharmaceutics vol 9 no 11 pp 2961ndash2973 2012

[109] H Zhao J C Wang Q S Sun C L Luo and Q ZhangldquoRGD-based strategies for improving antitumor activity ofpaclitaxel-loaded liposomes in nude mice xenografted withhuman ovarian cancerrdquo Journal of Drug Targeting vol 17 no1 pp 10ndash18 2009

[110] X B Xiong Y Huang W L Lu et al ldquoIntracellular delivery ofdoxorubicin with RGD-modified sterically stabilized liposomesfor an improved antitumor efficacy in vitro and in vivordquo Journalof Pharmaceutical Sciences vol 94 no 8 pp 1782ndash1793 2005

[111] K Riviere Z Huang K Jerger N MacAraeg and F C SzokaldquoAntitumor effect of folate-targeted liposomal doxorubicin inKB tumor-bearingmice after intravenous administrationrdquo Jour-nal of Drug Targeting vol 19 no 1 pp 14ndash24 2011


[113] S D Li S Chono and L Huang ldquoEfficient gene silencingin metastatic tumor by siRNA formulated in surface-modifiednanoparticlesrdquo Journal of Controlled Release vol 126 no 1 pp77ndash84 2008

[114] J SThomann B Heurtault S Weidner et al ldquoAntitumor activ-ity of liposomal ErbB2HER2 epitope peptide-based vaccineconstructs incorporating TLR agonists and mannose receptortargetingrdquo Biomaterials vol 32 no 20 pp 4574ndash4583 2011

[115] Y Ikehara N Shiuchi S Kabata-Ikehara et al ldquoEffective induc-tion of anti-tumor immune responses with oligomannose-coated liposome targeting to intraperitoneal phagocytic cellsrdquoCancer Letters vol 260 no 1-2 pp 137ndash145 2008

[116] X Zhou M Zhang B Yung et al ldquoLactosylated liposomes fortargeted delivery of doxorubicin to hepatocellular carcinomardquoInternational Journal of Nanomedicine vol 7 pp 5465ndash54742012

[117] G Blume G Cevc M D J A Crommelin I A J MBakker-Woudenberg C Kluft andG Storm ldquoSpecific targetingwith poly(ethylene glycol)-modified liposomes coupling ofhoming devices to the ends of the polymeric chains combineseffective target binding with long circulation timesrdquo Biochimicaet Biophysica Acta vol 1149 no 1 pp 180ndash184 1993

[118] A Gabizon A T Horowitz D Goren et al ldquoTargeting folatereceptor with folate linked to extremities of poly(ethylene


glycol)-grafted liposomes in vitro studiesrdquo Bioconjugate Chem-istry vol 10 no 2 pp 289ndash298 1999

[119] K Loomis B Smith Y Feng et al ldquoSpecific targeting to B cellsby lipid-based nanoparticles conjugated with a novel CD22-ScFvrdquo Experimental and Molecular Pathology vol 88 no 2 pp238ndash249 2010

[120] H Hatakeyama H Akita E Ishida et al ldquotumor targetingof doxorubicin by anti-MT1-MMP antibody-modified PEGliposomesrdquo International Journal of Pharmaceutics vol 342 no1-2 pp 194ndash200 2007

[121] P Simard and J C Leroux ldquoIn vivo evaluation of pH-sensitivepolymer-based immunoliposomes targeting the CD33 antigenrdquoMolecular Pharmaceutics vol 7 no 4 pp 1098ndash1107 2010

[122] A Yamada Y Taniguchi K Kawano T Honda Y Hattori andY Maitani ldquoDesign of folate-linked liposomal doxorubicin toits antitumor effect in micerdquo Clinical Cancer Research vol 14no 24 pp 8161ndash8168 2008

[123] K H Chuang H E Wang F M Chen et al ldquoEndocytosisof PEGylated agents enhances cancer imaging and anticancerefficacyrdquo Molecular Cancer Therapeutics vol 9 pp 1903ndash19122010

[124] N Kamaly Z Xiao P M Valencia A F Radovic-Moreno andO C Farokhzad ldquoTargeted polymeric therapeutic nanoparti-cles design development and clinical translationrdquo ChemicalSociety Reviews vol 41 pp 2971ndash3010 2012

[125] B Frisch F S Hassane and F Schuber ldquoConjugation of ligandsto the surface of preformed liposomes by click chemistryrdquoMethods in Molecular Biology vol 605 pp 267ndash277 2010

[126] F Schuber F S Hassane and B Frisch ldquoCoupling of peptidesto the surface of liposomes-Application to liposome-basedsynthetic vaccinesrdquo in Liposome Technology G GregoriadisEd pp 111ndash130 Informa Healthcare New York NY USA 3rdedition 2007

[127] A S Manjappa K R Chaudhari M P Venkataraju et alldquoAntibody derivatization and conjugation strategies applicationin preparation of stealth immunoliposome to target chemother-apeutics to tumorrdquo Journal of Controlled Release vol 150 no 1pp 2ndash22 2011

[128] W Tai R Mahato and K Cheng ldquoThe role of HER2 incancer therapy and targeted drug deliveryrdquo Journal of ControlledRelease vol 146 no 3 pp 264ndash275 2010

[129] M F Press C Cordon-Cardo and D J Slamon ldquoExpressionof the HER-2neu proto-oncogene in normal human adult andfetal tissuesrdquo Oncogene vol 5 no 7 pp 953ndash962 1990

[130] S Erdogan Z O Medarova A Roby A Moore and V PTorchilin ldquoEnhanced tumor MR imaging with gadolinium-loaded polychelating polymer-containing tumor-targeted lipo-somesrdquo Journal of Magnetic Resonance Imaging vol 27 no 3pp 574ndash580 2008

[131] P Sapra and TM Allen ldquoLigand-targeted liposomal anticancerdrugsrdquo Progress in Lipid Research vol 42 no 5 pp 439ndash4622003

[132] X Qi Z Chu Y Y Mahller K F Stringer D P Witteand T P Cripe ldquoCancer-selective targeting and cytotoxicityby liposomal-coupled lysosomal saposin C proteinrdquo ClinicalCancer Research vol 15 no 18 pp 5840ndash5851 2009

[133] AMVaccaroMMottaM Tatti et al ldquoSaposinCmutations inGaucher disease patients resulting in lysosomal lipid accumu-lation saposin C deficiency but normal prosaposin processingand sortingrdquoHumanmolecular genetics vol 19 no 15 pp 2987ndash2997 2010

[134] X Qi and G A Grabowski ldquoDifferential membrane inter-actions of saposins A and C implications for the functionalspecificityrdquo Journal of Biological Chemistry vol 276 no 29 pp27010ndash27017 2001

[135] T R Daniels T Delgado J A Rodriguez G Helguera andM L Penichet ldquoThe transferrin receptor part I biology andtargeting with cytotoxic antibodies for the treatment of cancerrdquoClinical Immunology vol 121 no 2 pp 144ndash158 2006

[136] T R Daniels T Delgado G Helguera andM L Penichet ldquoThetransferrin receptor part II targeted delivery of therapeuticagents into cancer cellsrdquoClinical Immunology vol 121 no 2 pp159ndash176 2006

[137] T R Pearce K Shroff and E Kokkoli ldquoPeptide targeted lipidnanoparticles for anticancer drug deliveryrdquoAdvancedMaterialsvol 24 pp 3803ndash3822 2012

[138] K Wang M H Na A S Hoffman et al ldquoIn situ doseamplification by apoptosis-targeted drug deliveryrdquo Journal ofControlled Release vol 154 pp 214ndash217 2011

[139] L C Sun and D H Coy ldquoSomatostatin receptor-targeted anti-cancer therapyrdquo Current Drug Delivery vol 8 no 1 pp 2ndash102011

[140] ZHanA FuHWang et al ldquoNoninvasive assessment of cancerresponse to therapyrdquoNatureMedicine vol 14 no 3 pp 343ndash3492008

[141] A Lowery H Onishko D E Hallahan and Z Han ldquotumor-targeted delivery of liposome-encapsulated doxorubicin by useof a peptide that selectively binds to irradiated tumorsrdquo Journalof Controlled Release vol 150 no 1 pp 117ndash124 2011

[142] X He M H Na J S Kim et al ldquoA novel peptide probe forimaging and targeted delivery of liposomal doxorubicin to lungtumorrdquo Molecular Pharmaceutics vol 8 no 2 pp 430ndash4382011

[143] T Wang G G Drsquosouza D Bedi et al ldquoEnhanced binding andkilling of target tumor cells by drug-loaded liposomes modifiedwith tumor-specific phage fusion coat proteinrdquo Nanomedicinevol 5 no 4 pp 563ndash574 2010

[144] T Wang N Kulkarni D Bedi et al ldquoIn vitro optimization ofliposomal nanocarriers prepared from breast tumor cell specificphage fusion proteinrdquo Journal of Drug Targeting vol 19 pp 597ndash605 2011

[145] S S Dharap Y Wang P Chandna et al ldquotumor-specifictargeting of an anticancer drug delivery system by LHRHpeptiderdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 102 no 36 pp 12962ndash12967 2005

[146] K Kessenbrock V Plaks and Z Werb ldquoMatrix metallopro-teinases regulators of the tumor microenvironmentrdquo Cell vol141 no 1 pp 52ndash67 2010

[147] P C Brooks S Silletti T L Von Schalscha M Friedlanderand D A Cheresh ldquoDisruption of angiogenesis by PEX anoncatalytic metalloproteinase fragment with integrin bindingactivityrdquo Cell vol 92 no 3 pp 391ndash400 1998

[148] E Koivunen W Arap H Valtanen et al ldquotumor targeting witha selective gelatinase inhibitorrdquoNature Biotechnology vol 17 no8 pp 768ndash774 1999

[149] L E Kelemen ldquoThe role of folate receptor 120572 in cancer devel-opment progression and treatment cause consequence orinnocent bystanderrdquo International Journal of Cancer vol 119no 2 pp 243ndash250 2006

[150] P S LowWAHenne andDDDoorneweerd ldquoDiscovery anddevelopment of folic-acid-based receptor targeting for imagingand therapy of cancer and inflammatory diseasesrdquo Accounts ofChemical Research vol 41 no 1 pp 120ndash129 2008


[151] S Lee J Kim G Shim et al ldquoTetraiodothyroacetic acid-taggedliposomes for enhanced delivery of anticancer drug to tumortissue via integrin receptorrdquo Journal of Controlled Release vol164 no 2 pp 213ndash220 2012

[152] Y Qin Q G Song Z R Zhang et al ldquoOvarian tumor tar-geting of docetaxel-loaded liposomes mediated by luteinizinghormone-releasing hormone analogues in vivo distribution innude micerdquo Arzneimittel-ForschungDrug Research vol 58 no10 pp 529ndash534 2008

[153] T TeradaMMizobata S Kawakami Y Yabe F Yamashita andM Hashida ldquoBasic fibroblast growth factor-binding peptide asa novel targeting ligand of drug carrier to tumor cellsrdquo Journalof Drug Targeting vol 14 no 8 pp 536ndash545 2006

[154] X Chen X Wang Y Wang et al ldquoImproved tumor-targetingdrug delivery and therapeutic efficacy by cationic liposomemodified with truncated bFGF peptiderdquo Journal of ControlledRelease vol 145 no 1 pp 17ndash25 2010

[155] Y Tan M Whitmore S Li P Frederik and L Huang ldquoLPDnanoparticlesndashnovel nonviral vector for efficient gene deliveryrdquoMethods in molecular medicine vol 69 pp 73ndash81 2002

[156] B J Vilner C S John andW D Bowen ldquoSigma-1 and sigma-2receptors are expressed in a wide variety of human and rodenttumor cell linesrdquo Cancer Research vol 55 no 2 pp 408ndash4131995

[157] R Banerjee P Tyagi S Li and L Huang ldquoAnisamide-targetedstealth liposomes a potent carrier for targeting doxorubicin tohuman prostate cancer cellsrdquo International Journal of Cancervol 112 no 4 pp 693ndash700 2004

[158] D Spitzer P O Simon Jr H Kashiwagi et al ldquoUse ofmultifunctional sigma-2 receptor ligand conjugates to triggercancer-selective cell death signalingrdquo Cancer Research vol 72pp 201ndash209 2012

[159] P Boyle and B Levin EdsWorld Cancer Report InternationalAgency for Research on Cancer Lyon France 2008

[160] R Paolinelli M Corada F Orsenigo and E Dejana ldquoThemolecular basis of the blood brain barrier differentiation andmaintenance Is it still a mysteryrdquo Pharmacological Researchvol 63 no 3 pp 165ndash171 2011

[161] W Debinski B Slagle D M Gibo S K Powers and G YGillespie ldquoExpression of a restrictive receptor for interleukin13 is associated with glial transformationrdquo Journal of Neuro-Oncology vol 48 no 2 pp 103ndash111 2000

[162] J Du W L Lu X Ying et al ldquoDual-targeting topotecanliposomes modified with tamoxifen and wheat germ agglutininsignificantly improve drug transport across the blood-brainbarrier and survival of brain tumor-bearing animalsrdquoMolecularPharmaceutics vol 6 no 3 pp 905ndash917 2009

[163] X Ying H Wen H J Yao et al ldquoPharmacokinetics and tissuedistribution of dual-targeting daunorubicin liposomes inmicerdquoPharmacology vol 87 no 1-2 pp 105ndash114 2011

[164] W Gong Z Wang N Liu et al ldquoImproving efficiency ofadriamycin crossing blood brain barrier by combination ofthermosensitive liposomes and hyperthermiardquo Biological andPharmaceutical Bulletin vol 34 no 7 pp 1058ndash1064 2011

[165] F Y Yang and P Y Lee ldquoEfficiency of drug delivery enhancedby acoustic pressure during blood-brain barrier disruptioninduced by focused ultrasoundrdquo International Journal ofNanomedicine vol 7 pp 2573ndash2582 2012

[166] F Y Yang H E Wang R S Liu et al ldquoPharmacokineticanalysis of (111)in-labeled liposomal Doxorubicin in murineglioblastoma after blood-brain barrier disruption by focusedultrasoundrdquo PLoS One vol 7 article e45468 2012

[167] G Bergers and L E Benjamin ldquotumorigenesis and the angio-genic switchrdquo Nature Reviews Cancer vol 3 no 6 pp 401ndash4102003

[168] S M Weis and D A Cheresh ldquotumor angiogenesis molecularpathways and therapeutic targetsrdquo Nature Medicine vol 17 pp1359ndash1370 2011

[169] Q Chen A Krol A Wright D Needham M W Dewhirstand F Yuan ldquotumor microvascular permeability is a key deter-minant for antivascular effects of doxorubicin encapsulatedin a temperature sensitive liposomerdquo International Journal ofHyperthermia vol 24 no 6 pp 475ndash482 2008

[170] K I Ogawara K Un K Minato K I Tanaka K Higaki and TKimura ldquoDeterminants for in vivo anti-tumor effects of PEGliposomal doxorubicin importance of vascular permeabilitywithin tumorsrdquo International Journal of Pharmaceutics vol 359no 1-2 pp 234ndash240 2008

[171] A S Abu Lila H Matsumoto Y Doi H Nakamura T Ishidaand H Kiwada ldquotumor-type-dependent vascular permeabilityconstitutes a potential impediment to the therapeutic efficacy ofliposomal oxaliplatinrdquo European Journal of Pharmaceutics andBiopharmaceutics vol 81 pp 524ndash531 2012

[172] R B Campbell B Ying G M Kuesters and R HemphillldquoFighting cancer from the bench to bedside using secondgeneration cationic liposomal therapeuticsrdquo Journal of Pharma-ceutical Sciences vol 98 no 2 pp 411ndash429 2009

[173] D C Litzinger A M J Buiting N Van Rooijen and L HuangldquoEffect of liposome size on the circulation time and intraorgandistribution of amphipathic poly(ethylene glycol)-containingliposomesrdquo Biochimica et Biophysica Acta vol 1190 no 1 pp99ndash107 1994

[174] D C Drummond C O Noble Z Guo K Hong JW Park andD B Kirpotin ldquoDevelopment of a highly active nanoliposomalirinotecan using a novel intraliposomal stabilization strategyrdquoCancer Research vol 66 no 6 pp 3271ndash3277 2006

[175] S Taurin H Nehoff and K Greish ldquoAnticancer nanomedicineand tumor vascular permeability where is the missing linkrdquoJournal of Controlled Release vol 164 no 3 pp 265ndash275 2012

[176] R Carlisle L W Seymour and C C Coussios ldquoTargetingof liposomes via PSGL1 for enhanced tumor accumulationrdquoPharmaceutical Research vol 30 no 2 pp 352ndash361 2012

[177] L Vellon J AMenendez and R Lupu ldquo120572v1205733 integrin regulatesheregulin (HRG)-induced cell proliferation and survival inbreast cancerrdquo Oncogene vol 24 no 23 pp 3759ndash3773 2005

[178] S Meng B Su W Li et al ldquoIntegrin-targeted paclitaxelnanoliposomes for tumor therapyrdquo Medical Oncology vol 28pp 1180ndash1187 2011

[179] A Accardo G Salsano A Morisco et al ldquoPeptide-modifiedliposomes for selective targeting of bombesin receptors overex-pressed by cancer cells a potential theranostic agentrdquo Interna-tional Journal of Nanomedicine vol 7 pp 2007ndash2017 2012

[180] F Donate G C Parry Y Shaked et al ldquoPharmacology ofthe novel antiangiogenic peptide ATN-161 (Ac-PHSCN-NH2) observation of a U-shaped dose-response curve in severalpreclinical models of angiogenesis and tumor growthrdquo ClinicalCancer Research vol 14 no 7 pp 2137ndash2144 2008

[181] W Dai T Yang YWang et al ldquoPeptide PHSCNK as an integrinalpha(5)beta(1) antagonist targets stealth liposomes to integrin-overexpressing melanomardquo Nanomedicine vol 8 pp 1152ndash11612012


[182] F Pastorino D Di Paolo F Piccardi et al ldquoEnhanced antitumorefficacy of clinical-grade vasculature-targeted liposomal dox-orubicinrdquo Clinical Cancer Research vol 14 no 22 pp 7320ndash7329 2008

[183] K Takara H Hatakeyama G Kibria N Ohga K Hida and HHarashima ldquoSize-controlled dual-ligand modified liposomesthat target the tumor vasculature show promise for use in drug-resistant cancer therapyrdquo Journal of Controlled Release vol 162pp 225ndash232 2012

[184] G Colombo F Curnis G M S De Mori et al ldquoStructure-activity relationships of linear and cyclic peptides containingthe NGR tumor-homingmotifrdquo Journal of Biological Chemistryvol 277 no 49 pp 47891ndash47897 2002

[185] GThurston J W McLean M Rizen et al ldquoCationic liposomestarget angiogenic endothelial cells in tumors and chronicinflammation in micerdquo Journal of Clinical Investigation vol 101pp 1401ndash1413 1998

[186] S Ran and P E Thorpe ldquoPhosphatidylserine is a marker oftumor vasculature and a potential target for cancer imaging andtherapyrdquo International Journal of Radiation Oncology BiologyPhysics vol 54 no 5 pp 1479ndash1484 2002

[187] A S Abu Lila S Kizuki Y Doi T Suzuki T Ishida andH Kiwada ldquoOxaliplatin encapsulated in PEG-coated cationicliposomes induces significant tumor growth suppression viaa dual-targeting approach in a murine solid tumor modelrdquoJournal of Controlled Release vol 137 no 1 pp 8ndash14 2009

[188] T Tagami T Suzuki M Matsunaga et al ldquoAnti-angiogenictherapy via cationic liposome-mediated systemic siRNA deliv-eryrdquo International Journal of Pharmaceutics vol 422 pp 280ndash289 2012

[189] T Asai Y Suzuki S Matsushita et al ldquoDisappearance of theangiogenic potential of endothelial cells caused by Argonaute2knockdownrdquo Biochemical and Biophysical Research Communi-cations vol 368 no 2 pp 243ndash248 2008

[190] M E Eichhorn S Becker S Strieth et al ldquoPaclitaxel encap-sulated in cationic lipid complexes (MBT-0206) impairs func-tional tumor vascular properties as detected by dynamic con-trast enhanced magnetic resonance imagingrdquo Cancer BiologyandTherapy vol 5 no 1 pp 89ndash96 2006

[191] M Schmitt-Sody S Strieth S Krasnici et al ldquoNeovasculartargeting therapy paclitaxel encapsulated in cationic liposomesimproves antitumoral efficacyrdquo Clinical Cancer Research vol 9no 6 pp 2335ndash2341 2003

[192] C Bode L Trojan C Weiss et al ldquoPaclitaxel encapsulated incationic liposomes a new option for neovascular targeting forthe treatment of prostate cancerrdquo Oncology Reports vol 22 no2 pp 321ndash326 2009

[193] A P Mann R C Bhavane A Somasunderam et al ldquoThioap-tamer conjugated liposomes for tumor vasculature targetingrdquoOncotarget vol 2 pp 298ndash304 2011

[194] J Hamzah J G Altin T Herringson et al ldquoTargeted liposomaldelivery of TLR9 ligands activates spontaneous antitumorimmunity in an autochthonous cancer modelrdquo Journal ofImmunology vol 183 no 2 pp 1091ndash1098 2009

[195] T P Herringson and J G Altin ldquoIncreasing the antitumor effi-cacy of doxorubicin-loaded liposomes with peptides anchoredvia a chelator lipidrdquo Journal of Drug Targeting vol 19 pp 681ndash689 2011

[196] D K Chang C Y Chiu S Y Kuo et al ldquoAntiangiogenic tar-geting liposomes increase therapeutic efficacy for solid tumorsrdquoJournal of Biological Chemistry vol 284 no 19 pp 12905ndash129162009

[197] S Marchio J Lahdenranta R O Schlingemann et alldquoAminopeptidase A is a functional target in angiogenic bloodvesselsrdquo Cancer Cell vol 5 no 2 pp 151ndash162 2004

[198] M Loi S Marchio P Becherini et al ldquoCombined targeting ofperivascular and endothelial tumor cells enhances anti-tumorefficacy of liposomal chemotherapy in neuroblastomardquo Journalof Controlled Release vol 145 no 1 pp 66ndash73 2010

[199] J E Gershenwald and I J Fidler ldquoCancer targeting lymphaticmetastasisrdquo Science vol 296 no 5574 pp 1811ndash1812 2002

[200] A J Cochran R R Huang J Lee E Itakura S P L Leong andR Essner ldquoTumour-induced immune modulation of sentinellymph nodesrdquo Nature Reviews Immunology vol 6 no 11 pp659ndash670 2006

[201] P Laakkonen M E Akerman H Biliran et al ldquoAntitumoractivity of a homing peptide that targets tumor lymphatics andtumor cellsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 101 no 25 pp 9381ndash93862004

[202] P Laakkonen K Porkka J A Hoffman and E Ruoslahti ldquoAtumor-homing peptide with a targeting specificity related tolymphatic vesselsrdquo Nature Medicine vol 8 no 7 pp 751ndash7552002

[203] Z Yan C Zhan Z Wen et al ldquoLyP-1-conjugated doxorubicin-loaded liposomes suppress lymphatic metastasis by inhibitinglymph node metastases and destroying tumor lymphatics2011rdquoNanotechnology vol 22 no 41 article 415103

[204] Z Yan F Wang Z Wen et al ldquoLyP-1-conjugated PEGylatedliposomes a carrier system for targeted therapy of lymphaticmetastatic tumorrdquo Journal of Controlled Release vol 157 pp 118ndash125 2012

[205] T P Herringson and J G Altin ldquoEffective tumor targetingand enhanced anti-tumor effect of liposomes engrafted withpeptides specific for tumor lymphatics and vasculaturerdquo Inter-national Journal of Pharmaceutics vol 411 no 1-2 pp 206ndash2142011

[206] Y Murase T Asai Y Katanasaka et al ldquoA novel DDS strategyldquodual-targetingrdquo and its application for antineovascular ther-apyrdquo Cancer Letters vol 287 no 2 pp 165ndash171 2010

[207] S Meng B Su W Li et al ldquoEnhanced antitumor effect of noveldual-targeted paclitaxel liposomesrdquoNanotechnology vol 21 no41 Article ID 415103 2010

[208] S Valastyan and R A Weinberg ldquotumor metastasis molecularinsights and evolving paradigmsrdquo Cell vol 147 pp 275ndash2922011

[209] L Borsig R Wong J Feramisco D R Nadeau N M Varkiand A Varki ldquoHeparin and cancer revisited mechanisticconnections involving platelets P-selectin carcinoma mucinsand tumor metastasisrdquo Proceedings of the National Academy ofSciences of the United States of America vol 98 no 6 pp 3352ndash3357 2001

[210] D Buergy F Wenz C Groden andM A Brockmann ldquotumor-platelet interaction in solid tumorsrdquo International Journal ofCancer vol 130 pp 2747ndash2760 2012

[211] J Wenzel R Zeisig and I Fichtner ldquoInhibition of breast cancermetastasis by dual liposomes to disturb complex formationrdquoInternational Journal of Pharmaceutics vol 370 no 1-2 pp 121ndash128 2009

[212] W Yang D Luo S Wang et al ldquoTMTP1 a novel tumor-homing peptide specifically argeting metastasisrdquo Clinical Can-cer Research vol 14 no 17 pp 5494ndash5502 2008


[213] M Zigler T Kamiya E C Brantley G J Villares and M Bar-Eli ldquoPAR-1 and thrombin the ties that bind the microenviron-ment to melanoma metastasisrdquo Cancer Research vol 71 pp6561ndash6566 2011


[215] T R Petersen N Dickgreber and I F Hermans ldquotumor antigenpresentation by dendritic cellsrdquoCritical Reviews in Immunologyvol 30 no 4 pp 345ndash386 2010

[216] H Ueno E Klechevsky N Schmitt et al ldquoTargeting humandendritic cell subsets for improved vaccinesrdquo Seminars inImmunology vol 23 pp 21ndash27 2011

[217] L C Bonifaz D P Bonnyay A Charalambous et al ldquoInvivo targeting of antigens to maturing dendritic cells via theDEC-205 receptor improves T cell vaccinationrdquo Journal ofExperimental Medicine vol 199 no 6 pp 815ndash824 2004

[218] A Faham and J G Altin ldquoAg-bearing liposomes engraftedwith peptides that interact with CD11cCD18 induce potentAg-specific and antitumor immunityrdquo International Journal ofCancer vol 129 no 6 pp 1391ndash1403 2011

[219] A Faham D Bennett and J G Altin ldquoLiposomal Ag engraftedwith peptides of sequence derived from HMGB1 induce potentAg-specific and anti-tumour immunityrdquoVaccine vol 27 no 42pp 5846ndash5854 2009

[220] E Ihanus L M Uotila A Toivanen M Varis and C GGahmberg ldquoRed-cell ICAM-4 is a ligand for the mono-cytemacrophage integrin CD11cCD18 characterization of thebinding sites on ICAM-4rdquo Blood vol 109 no 2 pp 802ndash8102007

[221] A Faham T Herringson C Parish A Suhrbier A AKhromykh and J G Altin ldquopDNA-lipoplexes engrafted withflagellin-related peptide induce potent immunity and anti-tumour effectsrdquo Vaccine vol 29 pp 6911ndash6919 2011

[222] A Faham and J G Altin ldquoAntigen-containing liposomesengrafted with flagellin-related peptides are effective vaccinesthat can induce potent antitumor immunity and immunother-apeutic effectrdquo Journal of Immunology vol 185 no 3 pp 1744ndash1754 2010

[223] F Perche T Benvegnu M Berchel et al ldquoEnhancementof dendritic cells transfection in vivo and of vaccinationagainst B16F10 melanoma with mannosylated histidylatedlipopolyplexes loaded with tumor antigen messenger RNArdquoNanomedicine vol 7 no 4 pp 445ndash453 2011

[224] P Midoux andMMonsigny ldquoEfficient gene transfer by histidy-lated polylysinepDNA complexesrdquo Bioconjugate Chemistryvol 10 no 3 pp 406ndash411 1999

[225] M Mevel G Breuzard J J Yaouanc et al ldquoSynthesis andtransfection activity of new cationic phosphoramidate lipidshigh efficiency of an imidazolium derivativerdquo ChemBioChemvol 9 no 9 pp 1462ndash1471 2008

[226] D S Watson A N Endsley and L Huang ldquoDesign con-siderations for liposomal vaccines influence of formulationparameters on antibody and cell-mediated immune responsesto liposome associated antigensrdquo Vaccine vol 30 pp 2256ndash2272 2012

[227] Z Zhong X Wei B Qi et al ldquoA novel liposomal vaccineimproves humoral immunity and prevents tumor pulmonarymetastasis in micerdquo International Journal of Pharmaceutics vol399 no 1-2 pp 156ndash162 2010

[228] X Tang C Mo Y Wang D Wei and H Xiao ldquoAnti-tumour strategies aiming to target Tumour-associatedMacrophages2012rdquo Immunology vol 138 no 2 pp 93ndash104

[229] N Van Rooijen N Kors M V D Ende and C D DijkstraldquoDepletion and repopulation ofmacrophages in spleen and liverof rat after intravenous treatment with liposome-encapsulateddichloromethylene diphosphonaterdquo Cell and Tissue Researchvol 260 no 2 pp 215ndash222 1990

[230] T Takahashi M Ibata Z Yu et al ldquoRejection of intradermallyinjected syngeneic tumor cells frommice by specific eliminationof tumor-associated macrophages with liposome-encapsulateddichloromethylene diphosphonate followed by induction ofCD11b(+)CCR3(-)Gr-1(-) cells cytotoxic against the tumorcellsrdquo Cancer Immunology and Immunotherapy vol 58 no 12pp 2011ndash2023 2009

[231] Y Zhang Y Huang P Zhang X Gao R B Gibbs and S LildquoIncorporation of a selective sigma-2 receptor ligand enhancesuptake of liposomes by multiple cancer cellsrdquo InternationalJournal of Nanomedicine vol 7 pp 4473ndash4485 2012

[232] R Nallamothu G C Wood M F Kiani B M Moore F PHorton and L AThoma ldquoA targeted liposome delivery systemfor combretastatin A4 formulation optimization through drugloading and in vitro release studiesrdquoPDA Journal of Pharmaceu-tical Science and Technology vol 60 no 3 pp 144ndash155 2006

[233] J M Saul A Annapragada J V Natarajan and R V Bel-lamkonda ldquoControlled targeting of liposomal doxorubicin viathe folate receptor in vitrordquo Journal of Controlled Release vol92 no 1-2 pp 49ndash67 2003

[234] M Dunne J Zheng J Rosenblat D A Jaffray and C AllenldquoAPNCD13-targeting as a strategy to alter the tumor accumu-lation of liposomesrdquo Journal of Controlled Release vol 154 pp298ndash305 2011

[235] T Aas A L Boslashrresen S Geisler et al ldquoSpecific P53 mutationsare associated with de novo resistance to doxorubicin in breastcancer patientsrdquoNatureMedicine vol 2 no 7 pp 811ndash814 1996

[236] A Persidis ldquoCancer multidrug resistancerdquo Nature Biotechnol-ogy vol 17 no 1 pp 94ndash95 1999

[237] G Cavaletti G Bogliun L Marzorati et al ldquoPeripheral neu-rotoxicity of taxol in patients previously treated with cisplatinrdquoCancer vol 75 pp 1141ndash1150 1995

[238] P Parhi C Mohanty and S K Sahoo ldquoNanotechnology-basedcombinational drug delivery an emerging approach for cancertherapyrdquo Drug Discovery Today vol 17 pp 1044ndash1052 2012

[239] SWu and R K Singh ldquoResistance to chemotherapy andmolec-ularly targeted therapies rationale for combination therapy inmalignant melanomardquo Current Molecular Medicine vol 11 pp553ndash563 2011

[240] Y Chen X Zhu X Zhang B Liu and LHuang ldquoNanoparticlesmodified with tumor-targeting scFv deliver siRNA andmiRNAfor cancer therapyrdquo Molecular Therapy vol 18 no 9 pp 1650ndash1656 2010

[241] J XiaH BiQ Yao SQu andY Zong ldquoConstruction of humanScFv phage display library against ovarian tumorrdquo Journalof Huazhong University of Science and Technology [MedicalSciences] vol 26 pp 497ndash499 2006

[242] N Li H Fu Y Tie et al ldquomiR-34a inhibits migration andinvasion by down-regulation of c-Met expression in humanhepatocellular carcinoma cellsrdquo Cancer Letters vol 275 no 1pp 44ndash53 2009

[243] F De Nigris M L Balestrieri and C Napoli ldquoTargeting c-MycRas and IGF cascade to treat cancer and vascular disordersrdquoCellCycle vol 5 no 15 pp 1621ndash1628 2006


[244] M J Halaby and D Q Yang ldquop53 translational control a newfacet of p53 regulation and its implication for tumorigenesis andcancer therapeuticsrdquo Gene vol 395 no 1-2 pp 1ndash7 2007

[245] A Grothey ldquoFuture directions in vascular endothelial growthfactor-targeted therapy for metastatic colorectal cancerrdquo Semi-nars in Oncology vol 33 no 10 pp S41ndashS49 2006

[246] S H Kang H J Cho G Shim et al ldquoCationic liposomal co-delivery of small interfering RNA and a MEK inhibitor forenhanced anticancer efficacyrdquo Pharmaceutical Research vol 28pp 3069ndash3078 2011

[247] G Shim S E Han Y H Yu et al ldquoTrilysinoyl oleylamide-basedcationic liposomes for systemic co-delivery of siRNA and ananticancer drugrdquo Journal of Controlled Release vol 155 pp 60ndash66 2011

[248] W Xiao X Chen L Yang Y Mao Y Wei and L Chen ldquoCo-delivery of doxorubicin and plasmid by a novel FGFR-mediatedcationic liposomerdquo International Journal of Pharmaceutics vol393 no 1-2 pp 119ndash126 2010

[249] D Grossman P J Kim J S Schechner and D C AltierildquoInhibition of melanoma tumor growth in vivo by survivintargetingrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 2 pp 635ndash640 2001

[250] M Zhang O B Garbuzenko K R Reuhl L Rodriguez-Rodriguez and T Minko ldquoTwo-in-one combined targetedchemo and gene therapy for tumor suppression and preventionof metastasesrdquo Nanomedicine vol 7 pp 185ndash197 2012

[251] R R Sawant O S Vaze K Rockwell and V P TorchilinldquoPalmitoyl ascorbate-modified liposomes as nanoparticle plat-form for ascorbate-mediated cytotoxicity and paclitaxel co-deliveryrdquo European Journal of Pharmaceutics and Biopharma-ceutics vol 75 no 3 pp 321ndash326 2010

[252] K Unsal-Kacmaz S Ragunathan E Rosfjord et al ldquoTheinteraction of PKN3 with RhoC promotes malignant growthrdquoMolecular Oncology vol 6 pp 284ndash298 2012

[253] M Aleku P Schulz O Keil et al ldquoAtu027 a liposomalsmall interfering RNA formulation targeting protein kinase N3inhibits cancer progressionrdquoCancer Research vol 68 no 23 pp9788ndash9798 2008

[254] D Strumberg B Schultheis U Traugott et al ldquoFirst-in-humanphase I study of Atu027 a liposomal small interfering RNAformulation targeting protein kinase N3 (PKN3) in patientswith advanced solid tumorsrdquo Journal of Clinical Oncology vol29 Abstract 3057 2011 ASCO Annual Meeting 2011

[255] W Dai W Jin J Zhang et al ldquoSpatiotemporally con-trolled co-delivery of anti-vasculature agent and cytotoxicdrug by octreotide-modified stealth liposomesrdquoPharmaceuticalResearch vol 29 pp 2902ndash2911 2012

[256] JHu L J Chen L Liu et al ldquoLiposomal honokiol a potent anti-angiogenesis agent in combination with radiotherapy producesa synergistic antitumor efficacy without increasing toxicityrdquoExperimental and Molecular Medicine vol 40 no 6 pp 617ndash628 2008

[257] P E Huber M Bischof J Jenne et al ldquoTrimodal cancertreatment beneficial effects of combined antiangiogenesisradiation and chemotherapyrdquo Cancer Research vol 65 no 9pp 3643ndash3655 2005

[258] YMaitani H Saito Y Seishi et al ldquoA combination of liposomalsunitinib plus liposomal irinotecan and liposome co-loadedwith two drugs enhanced antitumor activity in PC12-bearingmouserdquo Journal of Drug Targeting vol 20 no 10 pp 873ndash8822012

[259] A Sochanik IMitrus R Smolarczyk et al ldquoExperimental anti-cancer therapy with vascular-disruptive peptide and liposome-entrapped chemotherapeutic agentrdquoArchivum Immunologiae etTherapiae Experimentalis vol 58 no 3 pp 235ndash245 2010

[260] Y F Zhang J C Wang D Y Bian X Zhang and Q ZhangldquoTargeted delivery of RGD-modified liposomes encapsulatingboth combretastatin A-4 and doxorubicin for tumor therapyin vitro and in vivo studiesrdquo European Journal of Pharmaceuticsand Biopharmaceutics vol 74 no 3 pp 467ndash473 2010

[261] D Zucker A V Andriyanov A Steiner U Raviv and YBarenholz ldquoCharacterization of PEGylated nanoliposomes co-remotely loaded with topotecan and vincristine relating struc-ture and pharmacokinetics to therapeutic efficacyrdquo Journal ofControlled Release vol 160 pp 281ndash289 2012

[262] M Y Wong and G N Chiu ldquoLiposome formulation of co-encapsulated vincristine and quercetin enhanced antitumoractivity in a trastuzumab-insensitive breast tumor xenograftmodelrdquo Nanomedicine vol 7 pp 834ndash840 2011

[263] E J Feldman J E Kolitz J M Trang et al ldquoPharmacokineticsof CPX-351 a nano-scale liposomal fixed molar ratio formu-lation of cytarabine daunorubicin in patients with advancedleukemiardquo Leukemia Research vol 36 pp 1283ndash1289 2012

[264] W S Lim P G Tardi N Dos Santos et al ldquoLeukemia-selective uptake and cytotoxicity of CPX-351 a synergistic fixed-ratio cytarabine daunorubicin formulation in bone marrowxenograftsrdquo Leukemia Research vol 34 no 9 pp 1214ndash12232010

[265] K Riviere H M Kieler-Ferguson K Jerger and F C SzokaldquoAnti-tumor activity of liposome encapsulated fluoroorotic acidas a single agent and in combination with liposome irinotecanrdquoJournal of Controlled Release vol 153 no 3 pp 288ndash296 2011

[266] P Tardi S Johnstone N Harasym et al ldquoIn vivo maintenanceof synergistic cytarabinedaunorubicin ratios greatly enhancestherapeutic efficacyrdquo Leukemia Research vol 33 no 1 pp 129ndash139 2009

[267] Y T Ko C Falcao and V P Torchilin ldquoCationic liposomesloaded with proapoptotic peptide D-(KLAKLAK)2 and Bcl-2antisense oligodeoxynucleotide G3139 for enhanced anticancertherapyrdquo Molecular Pharmaceutics vol 6 no 3 pp 971ndash9772009

[268] GC BolfariniM P Siqueira-MouraG J Demets P CMoraisand A C Tedesco ldquoIn vitro evaluation of combined hyperther-mia and photodynamic effects using magnetoliposomes loadedwith cucurbituril zinc phthalocyanine complex on melanomardquoJournal of Photochemistry and Photobiology B vol 115 pp 1ndash42012

[269] E P Botosoa M Maillasson M Mougin-Degraef et alldquoAntibody-hapten recognition at the surface of functionalizedliposomes studied by SPR steric hindrance of pegylated phos-pholipids in stealth liposomes prepared for targeted radionu-clide deliveryrdquo Journal of Drug Delivery vol 2011 Article ID368535 9 pages 2011

[270] V P Torchilin A L Klibanov L Huang S OrsquoDonnellN D Nossiff and B A Khaw ldquoTargeted accumulation ofpolyethylene glycol-coated immunoliposomes in infarcted rab-bit myocardiumrdquo FASEB Journal vol 6 no 9 pp 2716ndash27191992

[271] MKeller R PHarbottle E Perouzel et al ldquoNuclear localisationsequence templated nonviral gene delivery vectors Investiga-tion of intracellular trafficking events of LMD and LD vectorsystemsrdquo ChemBioChem vol 4 no 4 pp 286ndash298 2003


[272] G Pasut and F M Veronese ldquoState of the art in PEGylation thegreat versatility achieved after forty years of researchrdquo Journalof Controlled Release vol 161 pp 461ndash472 2012

[273] M J Roberts M D Bentley and J M Harris ldquoChemistryfor peptide and protein PEGylationrdquo Advanced Drug DeliveryReviews vol 54 no 4 pp 459ndash476 2002

[274] L Zhu and V P Torchilin ldquoStimulus-responsive nanoprepara-tions for tumor targetingrdquo Integrative Biology vol 5 pp 96ndash1072013

[275] R van Sluis Z M Bhujwalla N Raghunand et al ldquoInvivo imaging of extracellular pH using 1H MRSIrdquo MagneticResonance in Medicine vol 41 pp 743ndash750 1999

[276] I F Tannock and D Rotin ldquoAcid pH in tumors and its potentialfor therpeutic exploitationrdquo Cancer Research vol 49 no 16 pp4373ndash4384 1989

[277] D C DrummondM Zignani and J C Leroux ldquoCurrent statusof pH-sensitive liposomes in drug deliveryrdquo Progress in LipidResearch vol 39 no 5 pp 409ndash460 2000

[278] D D Castelli W Dastru E Terreno et al ldquoIn vivo MRImulticontrast kinetic analysis of the uptake and intracellulartrafficking of paramagnetically labeled liposomesrdquo Journal ofControlled Release vol 144 no 3 pp 271ndash279 2010

[279] E Ducat J Deprez A Gillet et al ldquoNuclear delivery of atherapeutic peptide by long circulating pH-sensitive liposomesbenefits over classical vesiclesrdquo International Journal of Pharma-ceutics vol 420 pp 319ndash332 2011

[280] S Xiong B Yu J Wu H Li and R J Lee ldquoPrepara-tion therapeutic efficacy and intratumoral localization oftargeted daunorubicin liposomes conjugating folate-PEG-CHEMSrdquo Biomedicine and Pharmacotherapy vol 65 no 1 pp2ndash8 2011

[281] I Y Kim Y S Kang D S Lee et al ldquoAntitumor activity ofEGFR targeted pH-sensitive immunoliposomes encapsulatinggemcitabine inA549 xenograftnudemicerdquo Journal of ControlledRelease vol 140 no 1 pp 55ndash60 2009

[282] E A Leite C M Souza A D Carvalho-Junior et al ldquoEncap-sulation of cisplatin in long-circulating and pH-sensitive lipo-somes improves its antitumor effect and reduces acute toxicityrdquoInternational Journal of Nanomedicine vol 7 pp 5259ndash52692012

[283] Y Obata S Tajima and S Takeoka ldquoEvaluation of pH-responsive liposomes containing amino acid-based zwitterioniclipids for improving intracellular drug delivery in vitro and invivordquo Journal of Controlled Release vol 142 no 2 pp 267ndash2762010

[284] S Biswas N S Dodwadkar R R Sawant and V P TorchilinldquoDevelopment of the novel PEG-PE-based polymer for thereversible attachment of specific ligands to liposomes synthesisand in vitro characterizationrdquo Bioconjugate Chemistry vol 22pp 2005ndash2013 2011

[285] D Pornpattananangkul S Olson S Aryal et al ldquoStimuli-responsive liposome fusion mediated by gold nanoparticlesrdquoACS Nano vol 4 no 4 pp 1935ndash1942 2010

[286] H K Kim J Van den Bossche S H Hyun and D H Thomp-son ldquoAcid-triggered release via dePEGylation of fusogenic lipo-somes mediated by heterobifunctional phenyl-substituted vinylethers with tunable pH-sensitivityrdquoBioconjugate Chemistry vol23 pp 2071ndash2077 2012

[287] A Bandekar S Karve M Y Chang Q Mu J Rotolo andS Sofou ldquoAntitumor efficacy following the intracellular andinterstitial release of liposomal doxorubicinrdquo Biomaterials vol33 pp 4345ndash4352 2012

[288] S Karve G B Kempegowda and S Sofou ldquoHeterogeneousdomains andmembrane permeability in phosphatidylcholinemdashphosphatidic acid rigid vesicles as a function of pH and lipidchainmismatchrdquo Langmuir vol 24 no 11 pp 5679ndash5688 2008

[289] A Carruthers and D L Melchior ldquoStudies of the relationshipbetween bilayer water permeability and bilayer physical staterdquoBiochemistry vol 22 no 25 pp 5797ndash5807 1983

[290] G B Kempegowda S Karve A Bandekar A Adhikari TKhaimchayev and S Sofou ldquopH-Dependent formation oflipid heterogeneities controls surface topography and bindingreactivity in functionalized bilayersrdquo Langmuir vol 25 no 14pp 8144ndash8151 2009

[291] A Bandekar C Zhu A Gomez M Z Menzenski M Semp-kowski and S Sofou ldquoMasking and triggered unmaskingof targeting ligands on liposomal chemotherapy selectivelysuppress tumor growth in vivordquo Molecular Pharmaceutics vol10 no 1 pp 152ndash160

[292] H Hatakeyama H Akita and H Harashima ldquoA multifunc-tional envelope type nano device (MEND) for gene delivery totumours based on the EPR effect a strategy for overcoming thePEG dilemmardquo Advanced Drug Delivery Reviews vol 63 no 3pp 152ndash160 2011

[293] H Hatakeyama H Akita K Kogure et al ldquoDevelopment of anovel systemic gene delivery system for cancer therapy with atumor-specific cleavable PEG-lipidrdquo Gene Therapy vol 14 no1 pp 68ndash77 2007

[294] L Zhu P Kate and V P Torchilin ldquoMatrix metalloprotease 2-responsivemultifunctional liposomal nanocarrier for enhancedtumor targetingrdquo ACS Nano vol 6 pp 3491ndash3498 2012

[295] N Ballatori S M Krance S Notenboom S Shi K Tieu and CL Hammond ldquoGlutathione dysregulation and the etiology andprogression of human diseasesrdquo Biological Chemistry vol 390no 3 pp 191ndash214 2009

[296] F Meng W E Hennink and Z Zhong ldquoReduction-sensitivepolymers and bioconjugates for biomedical applicationsrdquo Bio-materials vol 30 no 12 pp 2180ndash2198 2009

[297] B Goldenbogen N Brodersen A Gramatica et al ldquoReduction-sensitive liposomes from a multifunctional lipid conjugateand natural phospholipids reduction and release kinetics andcellular uptakerdquo Langmuir vol 27 pp 10820ndash10829 2011

[298] R Kuai W Yuan Y Qin et al ldquoEfficient delivery of payloadinto tumor cells in a controlled manner by TAT and thiolyticcleavable PEG Co-modified liposomesrdquoMolecular Pharmaceu-tics vol 7 no 5 pp 1816ndash1826 2010

[299] G Candiani D Pezzoli L Ciani R Chiesa and S RistorildquoBioreducible liposomes for gene delivery from the formula-tion to the mechanism of actionrdquo PLoS ONE vol 5 no 10article e13430 2010

[300] S Fulda L Galluzzi and G Kroemer ldquoTargeting mitochondriafor cancer therapyrdquo Nature Reviews Drug Discovery vol 9 no6 pp 447ndash464 2010

[301] J Damen J Regts and G Scherphof ldquoTransfer and exchangeof phospholipid between small unilamellar liposomes and ratplasma high density lipoproteins Dependence on cholesterolcontent and phospholipid compositionrdquo Biochimica et Biophys-ica Acta vol 665 no 3 pp 538ndash545 1981

[302] F Tokumasu A J Jin and J A Dvorak ldquoLipidmembrane phasebehaviour elucidated in real time by controlled environmentatomic force microscopyrdquo Journal of Electron Microscopy vol51 no 1 pp 1ndash9 2002

[303] M P Veiga J L R Arrondo F M Goni A Alonso and DMarsh ldquoInteraction of cholesterol with sphingomyelin inmixed


membranes containing phosphatidylcholine studied by spin-label ESR and IR spectroscopies A possible stabilization of gel-phase sphingolipid domains by cholesterolrdquo Biochemistry vol40 no 8 pp 2614ndash2622 2001

[304] J A Zhang G Anyarambhatla L Ma et al ldquoDevelopmentand characterization of a novel Cremophor EL free liposome-based paclitaxel (LEP-ETU) formulationrdquo European Journal ofPharmaceutics and Biopharmaceutics vol 59 no 1 pp 177ndash1872005

[305] K Kusumoto H Akita A El-Sayed and H Harashima ldquoEffectof the anchor in polyethylene glycol-lipids on the transfectionactivity of PEGylated cationic liposomes encapsulating DNArdquoBiological amp Pharmaceutical Bulletin vol 35 pp 445ndash448 2012

[306] M B Hansen E van Gaal I Minten G Storm J C vanHest and D W Lowik ldquoConstrained and UV-activatable cell-penetrating peptides for intracellular delivery of liposomesrdquoJournal of Controlled Release vol 164 no 1 pp 87ndash94 2012

[307] R S Chang J Kim H Y Lee et al ldquoReduced dose-limitingtoxicity of intraperitoneal mitoxantrone chemotherapy usingcardiolipin-based anionic liposomesrdquoNanomedicine vol 6 no6 pp 769ndash776 2010


[309] M L Hauck S M La Rue W P Petros et al ldquoPhase I trial ofdoxorubicin-containing low temperature sensitive liposomes inspontaneous canine tumorsrdquo Clinical Cancer Research vol 12no 13 pp 4004ndash4010 2006

[310] K JHarrington C R Lewanski ADNorthcote et al ldquoPhase I-II study of pegylated liposomal cisplatin (SPI-077Ů) in patientswith inoperable head and neck cancerrdquoAnnals of Oncology vol12 no 4 pp 493ndash496 2001

[311] W C Zamboni A C Gervais M J Egorin et al ldquoSystemic andtumor disposition of platinum after administration of cisplatinor STEALTH liposomal-cisplatin formulations (SPI-077 andSPI-077 B103) in a preclinical tumor model of melanomardquoCancer Chemotherapy and Pharmacology vol 53 no 4 pp 329ndash336 2004

[312] T Asai S Matsushita E Kenjo et al ldquoDicetyl phosphate-tetraethylenepentamine-based liposomes for systemic siRNAdeliveryrdquo Bioconjugate Chemistry vol 22 no 3 pp 429ndash4352011

[313] N Yonenaga E Kenjo T Asai et al ldquoRGD-based activetargeting of novel polycation liposomes bearing siRNA forcancer treatmentrdquo Journal of Controlled Release vol 160 pp177ndash181 2012

[314] I Nakase H Akita K Kogure et al ldquoEfficient intracellulardelivery of nucleic acid pharmaceuticals using cell-penetratingpeptidesrdquo Accounts of Chemical Research vol 45 pp 1132ndash11392012

[315] S Futaki W Ohashi T Suzuki et al ldquoStearylated arginine-rich peptides a new class of transfection systemsrdquo BioconjugateChemistry vol 12 no 6 pp 1005ndash1011 2001

[316] E Koren and V P Torchilin ldquoCell-penetrating peptides break-ing through to the other siderdquoTrends inMolecularMedicine vol18 pp 385ndash393 2012

[317] E Vives J Schmidt andA Pelegrin ldquoCell-penetrating and cell-targeting peptides in drug deliveryrdquo Biochimica et BiophysicaActa vol 1786 no 2 pp 126ndash138 2008

[318] A A Kale and V P Torchilin ldquoEnhanced transfection oftumor cells in vivo using ldquoSmartrdquo pH-sensitive TAT-modified

pegylated liposomesrdquo Journal of Drug Targeting vol 15 no 7-8pp 538ndash545 2007

[319] R KuaiWYuanW Li et al ldquoTargeted delivery of cargoes into amurine solid tumor by a cell-penetrating peptide and cleavablepoly(ethylene glycol) comodified liposomal delivery system viasystemic administrationrdquo Molecular Pharmacology vol 8 pp2151ndash2161 2011

[320] G Kibria H Hatakeyama and H Harashima ldquoA new peptidemotif present in the protective antigen of anthrax toxin exerts itsefficiency on the cellular uptake of liposomes and applicationsfor a dual-ligand systemrdquo International Journal of Pharmaceu-tics vol 412 no 1-2 pp 106ndash114 2011

[321] A Koshkaryev A Piroyan and V P Torchilin ldquoBleomycin inoctaarginine-modified fusogenic liposomes results in improvedtumor growth inhibitionrdquo Cancer Letters 2012

[322] S E Barker S M Grosse E K Siapati et al ldquoImmunotherapyfor neuroblastoma using syngeneic fibroblasts transfected withIL-2 and IL-12rdquo British Journal of Cancer vol 97 no 2 pp 210ndash217 2007

[323] A D Tagalakis S M Grosse Q H Meng et al ldquoIntegrin-targeted nanocomplexes for tumour specific delivery and ther-apy by systemic administrationrdquo Biomaterials vol 32 no 5 pp1370ndash1376 2011

[324] S M Grosse A D Tagalakis M F M Mustapa et al ldquotumor-specific gene transfer with receptor-mediated nanocomplexesmodified by polyethylene glycol shielding and endosomallycleavable lipid and peptide linkersrdquo FASEB Journal vol 24 no7 pp 2301ndash2313 2010

[325] Y Qin H Chen Q Zhang et al ldquoLiposome formulated withTAT-modified cholesterol for improving brain delivery andtherapeutic efficacy on brain glioma in animalsrdquo InternationalJournal of Pharmaceutics vol 420 pp 304ndash312 2011

[326] N Demaurex ldquopH homeostasis of cellular organellesrdquo News inPhysiological Sciences vol 17 no 1 pp 1ndash5 2002

[327] SMishra PWebster andM EDavis ldquoPEGylation significantlyaffects cellular uptake and intracellular trafficking of non-viralgene delivery particlesrdquoEuropean Journal of Cell Biology vol 83no 3 pp 97ndash111 2004

[328] K Remaut B Lucas K Braeckmans J Demeester and S CDe Smedt ldquoPegylation of liposomes favours the endosomaldegradation of the delivered phosphodiester oligonucleotidesrdquoJournal of Controlled Release vol 117 no 2 pp 256ndash266 2007

[329] A Makovitzki A Fink and Y Shai ldquoSuppression of humansolid tumor growth in mice by intratumor and systemic inoc-ulation of histidine-rich and pH-dependent host defense-likelytic peptidesrdquo Cancer Research vol 69 no 8 pp 3458ndash34632009

[330] P Midoux C Pichon J J Yaouanc and P A Jaffres ldquoChem-ical vectors for gene delivery a current review on polymerspeptides and lipids containing histidine or imidazole as nucleicacids carriersrdquo British Journal of Pharmacology vol 157 no 2pp 166ndash178 2009

[331] N D Sonawane F C Szoka and A S Verkman ldquoChlo-ride accumulation and swelling in endosomes enhances DNAtransfer by polyamine-DNA polyplexesrdquo Journal of BiologicalChemistry vol 278 no 45 pp 44826ndash44831 2003

[332] MThomas andAM Klibanov ldquoEnhancing polyethyleniminersquosdelivery of plasmid DNA into mammalian cellsrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 99 no 23 pp 14640ndash14645 2002


[333] Y Xu and F C Szoka Jr ldquoMechanism of DNA release fromcationic liposomeDNA complexes used in cell transfectionrdquoBiochemistry vol 35 no 18 pp 5616ndash5623 1996

[334] J P Behr ldquoSynthetic gene transfer vectors II back to the futurerdquoJournal of Drug Targeting vol 45 pp 980ndash984 2012

[335] W Zhang J Song B Zhang L Liu K Wang and R WangldquoDesign of acid-activated cell penetrating peptide for deliveryof active molecules into cancer cellsrdquo Bioconjugate Chemistryvol 22 no 7 pp 1410ndash1415 2011

[336] T Jiang Z Zhang Y Zhang et al ldquoDual-functional lipo-somes based on pH-responsive cell-penetrating peptide andhyaluronic acid for tumor-targeted anticancer drug deliveryrdquoBiomaterials vol 33 no 36 pp 9246ndash9258 2012

[337] VVKumar C PichonMRefregiers BGuerin PMidoux andA Chaudhuri ldquoSingle histidine residue in head-group regionis sufficient to impart remarkable gene transfection propertiesto cationic lipids evidence for histidine-mediated membranefusion at acidic pHrdquoGeneTherapy vol 10 no 15 pp 1206ndash12152003

[338] A K Varkouhi M Scholte G Storm and H J Haisma ldquoEndo-somal escape pathways for delivery of biologicalsrdquo Journal ofControlled Release vol 151 no 3 pp 220ndash228 2011

[339] V P Torchilin T S Levchenko R Rammohan N Volod-ina B Papahadjopoulos-Sternberg and G G M DrsquoSouzaldquoCell transfection in vitro and in vivo with nontoxic TATpeptide-liposome-DNA complexesrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 100 no4 pp 1972ndash1977 2003

[340] X Zhang L Collins and J W Fabre ldquoA powerful cooperativeinteraction between a fusogenic peptide and lipofectamine forthe enhancement of receptor-targeted non-viral gene deliveryvia integrin receptorsrdquo Journal of Gene Medicine vol 3 no 6pp 560ndash568 2001

[341] K Sasaki K Kogure S Chaki et al ldquoAn artificial virus-likenano carrier system enhanced endosomal escape of nanopar-ticles via synergistic action of pH-sensitive fusogenic peptidederivativesrdquoAnalytical and Bioanalytical Chemistry vol 391 no8 pp 2717ndash2727 2008

[342] M Kullberg K Mann and T J Anchordoquy ldquoTargeting Her-2+ breast cancer cells with bleomycin immunoliposomes linkedto LLOrdquoMolecular Pharmaceutics vol 9 no 7 pp 2000ndash20082012

[343] I R Indran G Tufo S Pervaiz and C Brenner ldquoRecentadvances in apoptosis mitochondria and drug resistance incancer cellsrdquo Biochimica et Biophysica Acta vol 1807 no 6 pp735ndash745 2011

[344] J Lankelma H Dekker R F Luque et al ldquoDoxorubicingradients in human breast cancerrdquoClinical Cancer Research vol5 no 7 pp 1703ndash1707 1999

[345] I F Tannock C M Lee J K Tunggal D S M Cowan andM J Egorin ldquoLimited penetration of anticancer drugs throughtumor tissue a potential cause of resistance of solid tumors tochemotherapyrdquo Clinical Cancer Research vol 8 no 3 pp 878ndash884 2002

[346] Y Yamada and H Harashima ldquoMitochondrial drug deliverysystems for macromolecule and their therapeutic application tomitochondrial diseasesrdquo Advanced Drug Delivery Reviews vol60 no 13-14 pp 1439ndash1462 2008

[347] YMen X XWang R J Li et al ldquoThe efficacy ofmitochondrialtargeting antiresistant epirubicin liposomes in treating resistantleukemia in animalsrdquo International Journal of Nanomedicinevol 6 pp 3125ndash3137 2011

[348] T Nakamura H Akita Y Yamada H Hatakeyama and HHarashima ldquoA multifunctional envelope-type nanodevice foruse in nanomedicine concept and applicationsrdquo Accounts ofChemical Research vol 45 pp 1113ndash1121 2012

[349] R Mo Q Sun J Xue et al ldquoMultistage pH-responsive lipo-somes for mitochondrial-targeted anticancer drug deliveryrdquoAdvanced Materials vol 24 pp 3659ndash3665 2012

[350] M J Weiss J R Wong and C S Ha ldquoDequalinium atopical antimicrobial agent displays anticarcinoma activitybased on selective mitochondrial accumulationrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 84 no 15 pp 5444ndash5448 1987

[351] S Biswas N S Dodwadkar R R Sawant A Koshkaryevand V P Torchilin ldquoSurface modification of liposomes withrhodamine-123-conjugated polymer results in enhanced mito-chondrial targetingrdquo Journal of Drug Targeting vol 19 no 7 pp552ndash561 2011

[352] C Ferlini L Cicchillitti G Raspaglio et al ldquoPaclitaxel directlybinds to Bcl-2 and functionally mimics activity of Nur77rdquoCancer Research vol 69 no 17 pp 6906ndash6914 2009

[353] S Biswas N S Dodwadkar P P Deshpande andV P TorchilinldquoLiposomes loaded with paclitaxel and modified with noveltriphenylphosphonium-PEG-PE conjugate possess low toxic-itytarget mitochondria and demonstrate enhanced antitumoreffects in vitro and in vivordquo Journal of Controlled Release vol159 pp 393ndash402 2012

[354] S S Malhi A Budhiraja S Arora et al ldquoIntracellular deliveryof redox cycler-doxorubicin to the mitochondria of cancercell by folate receptor targeted mitocancerotropic liposomesrdquoInternational Journal of Pharmaceutics vol 432 pp 63ndash74 2012

[355] A Schroeder J Kost andY Barenholz ldquoUltrasound liposomesand drug delivery principles for using ultrasound to controlthe release of drugs from liposomesrdquo Chemistry and Physics ofLipids vol 162 no 1-2 pp 1ndash16 2009

[356] A Schroeder R Honen K Turjeman A Gabizon J Kost andY Barenholz ldquoUltrasound triggered release of cisplatin fromliposomes in murine tumorsrdquo Journal of Controlled Release vol137 no 1 pp 63ndash68 2009

[357] T J Evjen E A Nilssen S Rognvaldsson M Brandl andS L Fossheim ldquoDistearoylphosphatidylethanolamine-basedliposomes for ultrasound-mediated drug deliveryrdquo EuropeanJournal of Pharmaceutics and Biopharmaceutics vol 75 pp 327ndash333 2010

[358] P Shum J M Kim and D H Thompson ldquoPhototriggeringof liposomal drug delivery systemsrdquo Advanced Drug DeliveryReviews vol 53 no 3 pp 273ndash284 2001

[359] A Yavlovich A Singh R Blumenthal and A Puri ldquoA novelclass of photo-triggerable liposomes containing DPPCDC89PC as vehicles for delivery of doxorubcin to cellsrdquoBiochimicaet Biophysica Acta vol 1808 no 1 pp 117ndash126 2011

[360] P Agostinis K Berg KA Cengel et al ldquoPhotodynamic therapyof cancer an updaterdquo CA A Cancer Journal for Clinicians vol61 pp 250ndash281 2011

[361] B C Wilson and M S Patterson ldquoThe physics biophysics andtechnology of photodynamic therapyrdquo Physics in Medicine andBiology vol 53 no 9 pp R61ndashR109 2008

[362] M Triesscheijn M Ruevekamp R Out et al ldquoThepharmacokinetic behavior of the photosensitizer meso-tetra-hydroxyphenyl-chlorin in mice and menrdquo CancerChemotherapy and Pharmacology vol 60 no 1 pp 113ndash1222007


[363] M J Bovis J H Woodhams M Loizidou D ScheglmannS G Bown and A J Macrobert ldquoImproved in vivo deliveryof m-THPC via pegylated liposomes for use in photodynamictherapyrdquo Journal of Controlled Release vol 157 pp 196ndash2052012

[364] M Garcıa-Dıaz S Nonell A Villanueva et al ldquoDo folate-receptor targeted liposomal photosensitizers enhance photody-namic therapy selectivityrdquo Biochimica et Biophysica Acta vol1808 no 4 pp 1063ndash1071 2011

[365] H P Lassalle D Dumas S Grafe M A DrsquoHallewin FGuillemin and L Bezdetnaya ldquoCorrelation between in vivopharmacokinetics intratumoral distribution and photody-namic efficiency of liposomal mTHPCrdquo Journal of ControlledRelease vol 134 no 2 pp 118ndash124 2009

[366] J N Weinstein R L Magin M B Yatrin and D S ZaharkoldquoLiposomes and local hyperthermia selective delivery ofmethotrexate to heated tumorsrdquo Science vol 204 no 4389 pp188ndash191 1979

[367] K Kono T Ozawa T Yoshida et al ldquoHighly temperature-sensitive liposomes based on a thermosensitive block copoly-mer for tumor-specific chemotherapyrdquo Biomaterials vol 31 no27 pp 7096ndash7105 2010

[368] Y Wu Y Yang F C Zhang C Wu W L Lu and X GMei ldquoEpirubicin-encapsulated long-circulating thermosensi-tive liposome improves pharmacokinetics and antitumor ther-apeutic efficacy in animalsrdquo Journal of Liposome Research vol21 pp 221ndash228 2011

[369] L Paasonen T Sipila A Subrizi et al ldquoGold-embedded pho-tosensitive liposomes for drug delivery triggering mechanismand intracellular releaserdquo Journal of Controlled Release vol 147pp 136ndash143 2010

[370] M Latorre and C Rinaldi ldquoApplications of magnetic nanopar-ticles in medicine magnetic fluid hyperthermiardquo Puerto RicoHealth Sciences Journal vol 28 no 3 pp 227ndash238 2009

[371] P Pradhan J Giri F Rieken et al ldquoTargeted temperature sensi-tive magnetic liposomes for thermo-chemotherapyrdquo Journal ofControlled Release vol 142 no 1 pp 108ndash121 2010

[372] T Kikumori T Kobayashi M Sawaki and T Imai ldquoAnti-cancer effect of hyperthermia on breast cancer by magnetitenanoparticle-loaded anti-HER2 immunoliposomesrdquo BreastCancer Research and Treatment vol 113 no 3 pp 435ndash4412009

[373] B Smith I Lyakhov K Loomis et al ldquoHyperthermia-triggeredintracellular delivery of anticancer agent to HER2+ cellsby HER2-specific affibody (ZHER2-GS-Cys)-conjugated ther-mosensitive liposomes (HER2+ affisomes)rdquo Journal of Con-trolled Release vol 153 no 2 pp 187ndash194 2011

[374] J W Hopewell G M Morris A Schwint and J A CoderreldquoThe radiobiological principles of boron neutron capture ther-apy a critical reviewrdquo Applied Radiation and Isotopes vol 69pp 1756ndash1759 2011

[375] S Miyata S Kawabata R Hiramatsu et al ldquoComputed tomog-raphy imaging of transferrin targeting liposomes encapsulatingboth boron and iodine contrast agents by convection-enhanceddelivery to F98 rat glioma for boron neutron capture therapyrdquoNeurosurgery vol 68 no 5 pp 1380ndash1387 2011

[376] A Doi S Kawabata K Iida et al ldquotumor-specific targeting ofsodium borocaptate (BSH) to malignant glioma by transferrin-PEG liposomes a modality for boron neutron capture therapyrdquoJournal of neuro-oncology vol 87 no 3 pp 287ndash294 2008

[377] J H Ryu H Koo I C Sun et al ldquotumor-targeting multi-functional nanoparticles for theragnosis new paradigm for

cancer therapyrdquo Advanced Drug Delivery Reviews vol 64 no13 pp 1447ndash1458 2012

[378] X Ma Y Zhao and X J Liang ldquoTheranostic nanoparticlesengineered for clinic and pharmaceuticsrdquo Accounts of ChemicalResearch vol 44 pp 1114ndash1122 2011

[379] R Weissleder and M J Pittet ldquoImaging in the era of molecularoncologyrdquo Nature vol 452 no 7187 pp 580ndash589 2008

[380] W T Al-Jamal and K Kostarelos ldquoLiposomes from a clinicallyestablished drug delivery system to a nanoparticle platform fortheranostic nanomedicinerdquo Accounts of Chemical Research vol44 pp 1094ndash1104 2011

[381] C Heneweer S E Gendy and O Penate-Medina ldquoLiposomesand inorganic nanoparticles for drug delivery and cancerimagingrdquoTherapeutic Delivery vol 3 pp 645ndash656 2012

[382] A L Petersen A E Hansen A Gabizon and T L AndresenldquoLiposome imaging agents in personalizedmedicinerdquoAdvancedDrug Delivery Reviews vol 64 pp 1417ndash1435 2012

[383] G D Kenny N Kamaly T L Kalber et al ldquoNovel mul-tifunctional nanoparticle mediates siRNA tumour deliveryvisualisation and therapeutic tumour reduction in vivordquo Journalof Controlled Release vol 149 no 2 pp 111ndash116 2011

[384] K Kono S Nakashima D Kokuryo et al ldquoMulti-functionalliposomes having temperature-triggered release and magneticresonance imaging for tumor-specific chemotherapyrdquo Biomate-rials vol 32 no 5 pp 1387ndash1395 2011

[385] A H Negussie P S Yarmolenko A Partanen et al ldquoFormu-lation and characterisation of magnetic resonance imageablethermally sensitive liposomes for use with magnetic resonance-guided high intensity focused ultrasoundrdquo International Journalof Hyperthermia vol 27 no 2 pp 140ndash155 2011

[386] A Ranjan G C Jacobs D L Woods et al ldquoImage-guided drugdelivery withmagnetic resonance guided high intensity focusedultrasound and temperature sensitive liposomes in a rabbit Vx2tumor modelrdquo Journal of Controlled Release vol 158 pp 487ndash494 2012

[387] E Cittadino M Ferraretto E Torres et al ldquoMRI evaluation ofthe antitumor activity of paramagnetic liposomes loaded withprednisolone phosphaterdquo European Journal of PharmaceuticalSciences vol 45 pp 436ndash441 2012

[388] S Li B Goins L Zhang and A Bao ldquoNovel multifunctionaltheranostic liposome drug delivery system construction char-acterization and multimodality MR near-infrared fluorescentand nuclear imagingrdquo Bioconjugate Chemistry vol 23 no 6 pp1322ndash1332 2012

[389] N Mitchell T L Kalber M S Cooper et al ldquoIncorporation ofparamagnetic fluorescent and PETSPECT contrast agents intoliposomes for multimodal imagingrdquo Biomaterials vol 34 no 4pp 1179ndash1192 2012

[390] M De Smet E Heijman S Langereis N M Hijnen and HGrull ldquoMagnetic resonance imaging of high intensity focusedultrasound mediated drug delivery from temperature-sensitiveliposomes an in vivo proof-of-concept studyrdquo Journal of Con-trolled Release vol 150 no 1 pp 102ndash110 2011

[391] M Mikhaylova I Stasinopoulos Y Kato D Artemov and ZM Bhujwalla ldquoImaging of cationic multifunctional liposome-mediated delivery of COX-2 siRNArdquo Cancer GeneTherapy vol16 no 3 pp 217ndash226 2009

[392] C Grange S Geninatti-Crich G Esposito et al ldquoCombineddelivery and magnetic resonance imaging of neural cell adhe-sion molecule-targeted doxorubicin-containing liposomes inexperimentally induced Kaposirsquos sarcomardquo Cancer Researchvol 70 no 6 pp 2180ndash2190 2010


[393] L Deng X Ke Z He et al ldquoA MSLN-targeted multifunctionalnanoimmunoliposome for MRI and targeting therapy in pan-creatic cancerrdquo International Journal of Nanomedicine vol 7 pp5053ndash5065 2012

[394] J H Maeng D H Lee K H Jung et al ldquoMultifunctionaldoxorubicin loaded superparamagnetic iron oxide nanoparti-cles for chemotherapy and magnetic resonance imaging in livercancerrdquo Biomaterials vol 31 no 18 pp 4995ndash5006 2010

[395] N A Saunders F Simpson E W Thompson et al ldquoRole ofintratumoural heterogeneity in cancer drug resistance molec-ular and clinical perspectivesrdquo EMBO Molecular Medicine vol4 pp 675ndash684 2012

[396] S Bhatia J V Frangioni R M Hoffman A J Iafrate and KPolyak ldquoThe challenges posed by cancer heterogeneityrdquo NatureBiotechnology vol 30 pp 604ndash610 2012


Review ArticleStealth Properties to Improve Therapeutic Efficacy ofDrug Nanocarriers

Stefano Salmaso and Paolo Caliceti

Department of Pharmaceutical and Pharmacological Sciences University of Padua Via F Marzolo 5 35131 Padova Italy

Correspondence should be addressed to Stefano Salmaso stefanosalmasounipdit



Copyright copy 2013 S Salmaso and P Caliceti This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Over the last few decades nanocarriers for drug delivery have emerged as powerful tools with unquestionable potential to improvethe therapeutic efficacy of anticancer drugs Many colloidal drug delivery systems are underdevelopment to ameliorate the sitespecificity of drug action and reduce the systemic side effects By virtue of their small size they can be injected intravenously anddisposed into the target tissues where they release the drug Nanocarriers interact massively with the surrounding environmentnamely endothelium vessels as well as cells and blood proteins Consequently they are rapidly removed from the circulationmostlyby the mononuclear phagocyte system In order to endow nanosystems with long circulation properties new technologies aimed atthe surface modification of their physicochemical features have been developed In particular stealth nanocarriers can be obtainedby polymeric coating In this paper the basic concept underlining the ldquostealthrdquo properties of drug nanocarriers the parametersinfluencing the polymer coating performance in terms of opsoninsmacrophages interaction with the colloid surface the mostcommonly used materials for the coating process and the outcomes of this peculiar procedure are thoroughly discussed

1 Introduction

Cancer is a leading cause of death worldwide as accounted for76 million deaths (around 13 of all deaths) in 2008 (sourceWHO Fact sheet N∘297 February 2012) About 70 of allcancer deaths occurred in low- andmiddle-income countriesDeaths caused by cancer are forecasted to rise to over 131millions in 2030 (Globocan 2008 IARC 2010)

Nevertheless over the past few decades significantadvances have been made in fundamental cancer biologyallowing for remarkable improvements in diagnosis andtherapy for cancer Beside the development of new drugs withpotent and selective activities nanotechnology offers novelopportunities to cancer fighting by providing adequate toolsfor early detection and personalized treatments

Over the last decades a number of different long circu-lating vehicles have been developed for theranostic purposesThese carriers are in the nanometer range size and most ofthem have been intended for the delivery of anticancer drugsto tissues affected by this pathology

The aimof this paper is to examine the features of ldquostealthrdquolong circulating nanocarriers and the pharmacokinetic

outcomes of stealthiness and it will showcase the mostinvestigated approaches yielding prolonged circulation ofsurface-engineered nanocarriers

2 The Opsonisation Process

The selective and controlled delivery of anticancer drugsto disease tissues is a requisite to prevent systemic toxic-ity enhance the pharmacological profiles and improve thepatient compliance which in turn provide for ameliorationof antitumour therapy

Due to the leaky vasculature and low lymph drainagesolid tumours present erratic fluid and molecular transportdynamics These features can yield specific accumulation ofcolloidal anticancer drug delivery systems into the tumourtissue by enhanced permeation and retention (EPR) effect[1] However in order to exploit the physiopathological andanatomical peculiarities of the tumour tissues the nanovehi-cles need prolonged circulation in the bloodstream ideallyover 6 hours [2]


Alternative

Pentraxin

Classical

Lectin

Ant

ibod

y (A

b) o

r not

Factor BC3

C3

convertase

Factor D

C3aC3bBb C3bBb3b

C1s

CRPSAP

MBLMASP1MASP2

C1r

C1q C1 complexC2

C4

minus

C4aC4b2a

C3bC3

C2a

C3b

C4b

C3a

C2b

C1 INH

iC3b

C4b2a3b

H

HI

C5 C5b

Proteolyticcascade

of C6 to C9proteins

Membrane lysis

C5ndash9Membrane

attackcomplex

minus

H2O

Mg2+

Mg2+

Ca2+

Ca2+

(minusAb)

Figure 1 Schematic representation of the different activation pathways of the complement system (Reprinted with permission fromBiomaterials 2006 27 4356ndash4373 Copyright copy2006 Elsevier Ltd)

The permanence in the bloodstream of nanovehiclesis strongly affected by physical interactions with specificblood circulating components opsonins These componentsprevalently include complement proteins such as C3 C4 andC5 laminin fibronectin C-reactive protein type I collagenand immunoglobulins [3]

Surface opsonisation promotes the removal of particlesfrom the circulation within seconds to minutes through themononuclear phagocytic system (MPS) also known as retic-uloendothelial system (RES) and by Kupffer cells phagocyticmacrophages permanently located in the liver [4]Thenaturalrole of opsonins is to promote the bacteria and virusesapproach by the phagocytic cells both systems having thesame negative charge that inhibits the interaction betweenbacteriaviruses and the phagocytes due to charge repulsion[5] After bacteria and virus coating opsonins undergo con-formational rearrangements that induce the biorecognitionby phagocytes through specific membrane receptors Thexenoparticle opsonisation by complement proteins over 30soluble and membrane-bound proteins induces the comple-ment activation through a cascade of physiological eventsThe opsonisation finally promotes the removal process byphagocytes [4]

The complement is a key component of innate immunitythat naturally monitors host invaders through three distinctactivation pathways described in Figure 1 [6]

The classical pathway is activated after the fixation ofC1q proteins to antibodies or to C1q receptors on the cellsurface The alternative pathway is spontaneously activatedby the binding of C3 fragments to the surface of thepathogen The lectin pathway is activated by the bindingof mannose-binding lectin on mannose contained on thesurface corona of bacteria and viruses Although a fewhypotheses have been proposed to explain the existence ofsupplementary activation pathways they have not been fullyelucidated

Regardless of the activation pathway the enzymatic cas-cade of the complement activation leads to the formation ofa common enzyme C3 convertase which cleaves the centralprotein of the complement system the third component C3[7] The fragment C3b of C3 is the crucial active componentthat triggers the cleavage of a variety of complement proteins(C5ndashC9) The assembly of these proteins contributes to theformation of the membrane attack complex (MAC) that isable to destabilize bacteria viruses and nanocarriers fordrug delivery C3b and its inactive fragment iC3b can berecognised by specific receptors on phagocytic cells leading tothe engulfing of opsonised particles and their removal fromthe bloodstream

Additionally the complement activation triggers a cas-cade of inflammatory and adverse complex reactions namedcomplement activation-related pseudoallergy (CARPA) that


reflect in symptoms of transient cardiopulmonary distressThese effects have been detailed by the literature [8ndash11]

The complement system is also finely regulated by thepresence of inhibitor proteins such as C1 INH Factor I andH [12]

Even though the natural role of opsonisation is directed tothe body protection from xenogeneic nanosystems this pro-cess promotes the removal of circulating drug nanocarriersThis represents a major obstacle to achieve adequate systemicand local therapeutic drug concentrations

21 Steric Shielding and Stealth Properties of Nanocarriers Inthe bloodstream opsonins interact with nanoparticles by vander Waals electrostatic ionic and hydrophobichydrophilicforces Therefore the surface features of the nanocarriershave a key role in the opsonisation process Hydrophobic andcharged particles undergo higher opsonisation as comparedto hydrophilic and neutrally charged particles [13ndash16]

In the last decades different theories have been attemptedto describe the pharmacokinetic profiles of nanosized drugdelivery systems namely liposomes and polymeric nanopar-ticles It is now recognised that long circulating nanocarriersldquostealthrdquo systems can be obtained by surface coating withhydrophilic polymers that prevent the opsonisation process[17ndash19] The consequence of avoiding opsonisation is theprolongation of the liposome and particle permanence in thebloodstream from few seconds to several hours [17 20 21]

Peppas described the effect of the hydrophilic polymershell on nanoparticle surface in terms of elastic forces Hefocused the attention on PEG that is the most representa-tive of the materials used to produce stealth nanocarriersAccording to their hydrophilic and flexible nature the PEGchains can acquire an extended conformation on particlesurface Opsonins attracted to the particle surface compressthe extended PEG chains that shift to a more condensed andhigher energy conformation As a consequence the repulsiveforces counterbalance the attractive forces between opsoninsand the particle surface [22]

At low polymer density on the particle surface when thepolymer chains cannot interact with the surrounding chainsand may freely collapse on the surface the polymer chainsprovide for steric repulsion at a distance h according to theequation

119865119898

st =(119896119879)

(1198632ℎ119888) (ℎ119888ℎ)83 (1)

In the equation 119865119898st is the steric repulsive force referred tothe ldquomushroomrdquo model (m) ℎ

119888is the extension of a polymer

above the surface = 119873119886(119886119863)23 D is the average distancebetween adjacent grafting points a is the size of the segmentand119873 is the degree of polymerization

At high polymer densities the polymer chains extend andinteract with each other exerting the steric repulsive force 119865brstreferred to the ldquobrushrdquo model (br)

119865brst =

(119896119879)

1198633 [(ℎ119888ℎ)94minus (ℎℎ

119888)34]

(2)

These equations describe repulsive phenomena occurringon flat surfaces However they can be properly elaborated togain information about repulsive steric barriers endowed byadsorbed polymers on curved surfaces of stealth nanoparti-cles [23]

22 Polymers Used to Coat Nanocarriers Long circulatingnanocarriers are usually obtained by polymer surface coatingthat endows systems with stealth properties [24] In drugdelivery the term ldquostealthrdquo translated from the ldquolow observ-able technologyrdquo applied to military tactics refers to nanove-hicles that are invisible to the biological system involved inclearance of particle from the bloodstream namely RES andKupffer cells

So far many efforts have been done to yield stealth prod-ucts by modification of the surface properties of nanocarrierswith polymers that prevent opsonin interactions [25] andsubsequent phagocyte clearance [26ndash28]

The polymers used to confer stealth properties tonanoparticles and nanovesicles have few basic common fea-tures high flexibility and high hydrophilicity Either naturaland semisynthetic polysaccharides or synthetic polymershave been used for these purposes Dextran (Dex) polysialicacid (PSA) hyaluronic acid (HA) chitosan (CH) and hep-arin are the most used natural polysaccharides Syntheticpolymers include polyvinyl pyrrolidone (PVP) polyvinylalcohol (PVA) polyacrylamide (Pam) poly(ethylene glycol)(PEG) and PEG-based copolymers such as poloxamerspoloxamines and polysorbates

221 PEG Poly(ethylene glycol) (PEG) is the polymer ofchoice to produce stealth nanocarriers This neutral flexibleand hydrophilic material can in fact properly produce surfacebarrier layers that reduce the adhesion of opsonins presentin the blood serum on the nanoparticles making themldquoinvisiblerdquo to phagocytic cellsThe protein repulsion operatedby PEG was also visualized by freeze-fracture transmissionelectron microscopy (TEM) [29]

A few physical protocols have been adopted to coatnanoparticle with PEG [22] even though these proceduresentail the risk of polymer desorption in the blood withconsequent loss of the beneficial contribution of the poly-mer [30] In order to overcome this problem covalentPEG conjugation protocols have been developed [31 32]Biodegradable nanoparticles with PEG covalently bound tothe surface have been produced using PEG derivatives ofpoly(lactic acid) poly(lactic acid-co-glycolic acid) [33] orpoly(alkylcyanoacrylates) [34] The nanoparticles are pre-pared by emulsion precipitation or dispersion protocolsin aqueous media These procedures allow for the PEGorientation toward the water phase while the biodegradablehydrophobic polymer fraction is physically entangled in theinner nanoparticle matrix [22] Alternatively PEG chainsmay be covalently conjugated to preformed nanoparticlesthrough surface functional groups [35 36]

222 Poloxamine and Poloxamer Poloxamines (Tetronics)and poloxamers (Pluronics) are amphiphilic block copoly-mers consisting of hydrophilic blocks of ethylene oxide (EO)


and hydrophobic blocks of propylene oxide (PO) monomerunits Poloxamers are a-b-a type triblock copolymers (PEO-PPO-PEO) while poloxamines are tetrablock copolymersof PEO-PPO connected through ethylenediamine bridges[(PEO-PPO)

2ndashNndashCH

2ndashCH2ndashNndash(PPO-PEO)

2] [37ndash39]

These polymers can be physically adsorbed on thenanocarrier surface through the hydrophobic PPO fraction[22]

Following intravenous injection to mice and ratspoloxamer- or poloxamine-coated sub-200 nm poly(phos-phazene) [40] PLGA nanoparticles [41] and liposomes[42 43] did not show prolonged circulation time as comparedto the uncoated counterparts This unexpected behaviourwas ascribed to the desorption of the polymers from thenanocarrier surface [30] as well as to the polymer capacity toadsorb opsonins [44] Indeed the polymer composition hasbeen found to affect the particle opsonisation as opsoninscan associate with the hydrophobic polymer fraction thatmay be partially exposed on the particle surface [45 46]This possible effect can further contribute to the clearance ofthe polymer-coated nanocarriers

For a given triblock polymer it was found that bothsurface polymer density and coating layer thickness areaffected by the particle size smaller particles (below 100 nm)adsorb fewer polymer molecules per unit area than largerparticles Therefore the polymer surface density decreases asthe particle size decreases Additionally Pluronic adsorptionon larger particles is relatively weaker than on smallerparticles which can affect the rate and extent of displacementof adsorbed polymers by blood components [47]

The surface adsorption efficiency and the stability ofthe polymer coating are strictly related to the polymercomposition namely POEO molar ratio and PPO and PEOchain length [44]

Pluronic F-108 NF (poloxamer 338) has a bulkier centralhydrophobic block and longer side hydrophilic arms (122monomers of PEO 56 monomers of PPO) as compared toPluronic F-68 NF (76 monomers of PEO 30 monomers ofPPO) Accordingly Pluronic F-108 NF forms more stablecoating layers than Pluronic F-68 NF In vivo Pluronic F-68NF-modified nanoparticles accumulate at 74 of the dose inthe liver in 1 h while the liver accumulation of Pluronic F-108NF-modified nanoparticles was 67 [48]

223 Dextran Dextran is a polysaccharide largely usedfor biomedical applications including for the decoration ofnanoparticulate drug delivery systems [49]

Dextran coating was found to bestow long circulatingproperties on liposomes [50] Similarly to PEG the stericbrush of the dextran on the vesicle surface reduces the proteinadsorption This effect results in enhanced liposome stabilityin the blood [50] which depends on the density of dextranmolecules

Interestingly 70 kDa dextran coating was also found toreduce the burst of drug release from liposomes [50]

Dextran was used to coat superparamagnetic iron oxidenanoparticles for magnetic resonance imaging [51 52] Par-ticles of 4 to 5 nm were coated with 20 to 30 dextranchains organized in ldquobrush-likerdquo structures which reduced

the removal from the bloodstream by Kupffer cells andsplenic macrophages The circulation half-life was prolongedto 3-4 hours [52] The slight macrophage recognition of thedextran-coated superparamagnetic iron oxide nanoparticleswas attributed to antidextran antibody opsonisation

224 Sialic Acid Derivatives to Mimic the Nature Sialicacid derivatives received considerable interest as potentialmaterials to confer stealth properties to nanoparticles fordrug delivery applications Sialic acid is a component ofeukaryotic cell surface and plays an important role in pre-venting the removal of self-tissue by low level of complementactivation through the alternative pathway Desialylationof erythrocyte membranes results in reduction of factorH binding on their membrane that switches them fromnonactivators to activators of the alternative complementpathway [53 54] Plasmatic circulating factor H adsorbed onbacteria or the surface of colloidal systems physiologicallyinhibits their complement-mediated destruction This resultis ascribable to factor H action as cofactor for the inactivationof the complement C3b factor and the alternative pathwayconvertase [55]Therefore factor H behaves as a dysopsonin

Surolia and Bachhawat demonstrated that liposomescoated with sialic acid derivatives are poorly recognised bythe macrophages as they mimic the mammalian cell surface[56]

Stealth nanocarriers have been obtained using a varietyof polysialic acid derivatives including gangliosides [57ndash61]ganglioside derivatives and glycophorin [62ndash64] On thecontrary the coating with orosomucoid protein a sialic acidrich protein did not yield stealth poly(isobutylcyanoacrylate)nanoparticles This effect was ascribed to the poor densityof the sialic acid on the particle surface that does not allowfor proper coating or to the inefficient conformation of theclustered glycans [65]

The liposome coating with the monosialogangliosideGM1 (Figure 2) a brain-tissue-derived monosialoganglio-side was found to inhibit the alternative complement path-way by promoting the association of factor H to C3b factoron the vesicle surface [66] In mice the liposome decorationwith 5ndash7mol of GM1 was found to increase the vesiclestability and inhibit the complement activation cascadewhich resulted in prolonged permanence in the circulation[67] As the molar ratio of GM1 in liposomes increasesthe macrophage uptake inhibition increases up to 90 with10mol GM1 [64]

Few studies postulated that the shielding of the negativecharges of GM1 by the bulky neutral hydrophilic sugarmoieties is paramount to its stealth activity [58] Never-theless other investigations showed that macromoleculesbearing unshielded negative charges namely the ganglio-side GM3 a sialic acid synthetic derivative and a GM1semisynthetic compound increase the blood circulation timeof sub-200 nm liposomes in mice [63] Therefore it can beconcluded that the sterical organization of the gangliosideresidues is primarily responsible for preventing the opsoni-sation of liposome containing glycolipids

Interestingly studies performed with mice and ratsshowed that the gangliosides have a specie-specific activity


O

OH

HOOH

O

OH

O

OH

NHO

OH

O O

OHO

OH

O

HOOH

O

OH

NHHO O

O

O

HO

HOOH

HN

O

GM1

GM2

GM3

1217

O

N-acetyl-a-neuraminidase(sialic acid)

galactose

Stearic acid

galactosamine

Sphingosine

CH3

H3C

120573-O-

120573-O-glucose

N-acetyl-120573-O-120573-O-galactose

Ominus

Figure 2 Chemical structure of the monosialoganglioside GM1

Indeed the GM1 decoration was effective in mice while itdid not have any beneficial effect on the circulation time ofliposomes in rats [63]

225 Zwitterionic Polymers Zwitterionic phospholipidderivatives have been demonstrated to reduce the comple-ment activation induced by liposomes [68]

Based on this evidence synthetic zwitterionic polymershave been used to produce stealth drug delivery systemsThese materials bind water molecules more strongly thanpolymers forming hydrogen bridges such as PEG Further-more they provide electrostatically induced hydration [69]that decreases the rate of adsorption of proteins cells andbacteria on surfaces [70 71] Conversely than amphiphilicpolymers namely PEG that can partially insert itself inthe lipid bilayer of liposomes [72 73] zwitterionic polymersenhance the hydration of lipid polar group regions on thesurface of liposomes and do not perturb the lipidic bilayerstability [74]

Liposomes coated with poly(zwitterionic) 2 and 5 kDapoly(carboxybetaine)-12-distearoyl-sn-glycero-3-phosphoe-thanolamine (poly(carboxybetaine)-DSPE) (Figure 3) pos-sess similar stability of PEGylated liposomes After 4 daysof incubation at 37∘C no aggregation was observed Theenhanced hydration and fluidity of the liposome membraneprovided by the poly(zwitterionic) component reduced itspermeability and accounted for prolonged drug releaseas compared to the PEGylated counterparts In vivo poly(zwitterionic) polymer and PEG-coated liposomes showed

similar pharmacokinetic profiles suggesting that the formermay be used as an alternative to PEG [75]

Poly(carboxybetaine) is more chemically stable thanPEG and has lower interactions with proteins over shortand long time [76] This material has been used to coata variety of nanoparticles including silica [77] gold [78]iron oxide [79] PLGA [80] and hydrogel nanoparticles [8182] In serum the coated nanoparticles showed excellentstability to aggregation indicating that negligible opsonisa-tion occurred as compared to other stealth particles [83]This behaviour translates in exceptionally low unspecificcellular uptake As an example internalization of cross-linkedpoly(carboxybetaine)iron oxide nanogels by HUVEC cellsand macrophages was barely detectable [79]

226 Polyglycerols Polyglycerols (PGs) are biocompatibleand flexible hydrophilic aliphatic polyether polyols with anantifouling effect comparable to PEG [84] By virtue of theirmultivalency that allows for the conjugation of targetingagents drugs labels and physical modifiers [85] thesepolymers have been extensively studied as drug carriers

Liposomes decorated with PGs exhibit extended bloodcirculation time and decreased uptake by liver and spleen[86]

Self-assembledmonolayers (SAMs) of dendritic PGsweredeposited on gold surface through a disulfide linker group(thioctic acid) Surface Plasmon resonance (SPR) measure-ments showed that PGs monolayers efficiently prevent theadsorption of proteins It was concluded that dendritic PGs


BrNH

OP

O

OO

O

O

HO

O

CH3

O

O

O15

15 CH3

N+

Ominus

Ominus119899

Figure 3 Chemical structure of poly(zwitterionic) poly(carboxybetaine)-DSPE derivative used to assemble poly-zwitterionic liposomes

behave as antiopsonic materials because they combine thecharacteristic structural features of several protein-resistantmaterials flexible aliphatic polyether structure hydrophilicsurface groups and a highly branched architecture [84] Theinhibition of protein adsorption of hyperbranched polyglyc-erol was more efficient than linear PEG of similar molecularweight [87] and dextran Furthermore PGs have enhancedresistance to heat and oxidative stress as compared to PEGwhichmakes them potential candidates for biomedical appli-cations [84]

227 Polyacrylic and Polyvinyl Polymers Synthetic poly-acrylic and polyvinyl polymers bearing hydrophobicmoietieshave been prepared to coat liposomes The hydrophobicfunction allows for the polymer anchoring on the particlesurface

Palmitoyl- or phosphatidylethanolamine- (PE-) termi-nated derivatives of poly(acryl amide) (PAA) poly(vinylpyrrolidone) (PVP) and poly(acryloyl morpholine) (PAcM)have been found to exert comparable stealth effects onliposomes in vivo This behaviour depends on the lengthof the hydrophobic alkyl function the polymer molecularweight and its surface density [88 89]

Comparative studies performed with palmitoyl-or PE-functionalized 6ndash8 kDa PAA PVP and PEG showed thatthe PEG derivative has slightly better performance ascompared to the other polymers Macromolecules con-taining shorter hydrophobic moieties than palmitoyl- orphosphatidylethanolamine- namely dodecyl alkyl chains orhigher polymermolecularweight (12ndash15 kDa) showed a lowereffect on circulation time of liposomes Short hydrophobicmoieties cannot efficiently anchor the polymer on the lipo-some surface as the energy of the polymeric chain motionis higher than the energy of the anchoring alkyl chaininteraction with the liposomal phospholipid bilayer [88 90]The higher the polymer molecular weight the higher thefree energy of the exposed polymer chains Therefore thepolymer can detach in vivo inducing liposome opsonisationand removal by the RES [91]

The layer thickness of poly(vinyl alcohol)s (6 9 and20 kDa PVA) derivatized with C

16H33ndashSndash as hydropho-

bic anchor (PVA-R) on the liposome surface was directlyproportional to the polymer molecular weight and to theconcentration of the polymer solution used for the coatingprocess Furthermore it was found that the PVA-R densityon the liposome surface increased as the molecular weightof the polymer decreased The PVA-R on liposomes wasnot detached by dilution or in presence of serum whilepreventing the adsorption of plasma proteins In vivo thePVA-R-coated liposomes showed prolonged permanence inthe circulation which increased as the PVAmolecular weightincreased The circulation time of liposomes coated with13 mol of 20 kDa PVA-R was comparable to that ofliposomes coated with 8 mol of 2 kDa PEG-12-distearoyl-sn-glycero-3-phosphoethanolamine (PEG-DSPE) Detailedinvestigations showed that the increased permanence in thebloodstream was strictly related to the PVA-R stability on theliposome surface that was higher compared to PEG-DSPE[92]

23 Surface Requirements to Set Up Long Circulating Nanocar-riers The capacity of hydrophilic polymers to repel proteinsis strictly related to the polymer composition polymermolecular weight density on the carrier surface thicknessof the coating conformation flexibility and architecture ofthe chains Furthermore this capacity depends also on thephysicochemical properties of the anchoring moieties thatallow for the attachment of the polymer on the particlesurface

231 Architecture and Molecular Weight of PEG DerivativesThe length of the polymer chains on stealth particle surfacemust exceed the range of the van der Waals attraction forceswith soluble proteins in the bulk and phagocytic cells [93]In the case of PEG 2 kDa molecular weight is considered thelower threshold to guarantee macrophage avoidance As thepolymer molecular weight increases the blood circulationhalf-life of the PEGylated particles increases [34 94] A study


carried out with nanoparticles assembled using PEG-PLAblock copolymer demonstrated that the 5 kDa PEG has themaximal capacity to reduce protein adsorption that yields tothe uptake by phagocytic cells [33 95]

High sensitivity differential scanning calorimetry wasused to evaluate the effect of PEG size and acyl chain lengthof the PEG-phospholipid conjugate on the physical stabilityof liposomes [96] The study was carried out with liposomesobtained using PEG-dipalmitoyl phosphatidylethanolamine(PEG-DPPE) and dipalmitoyl phosphatidylcholine (DPPC)A mixed lamellarmicellar phase was obtained with compo-sitions containing more than 7 mol of 1ndash3 kDa PEG-DPPEwhile the complete conversion tomicelles was achieved above17 mol of PEG-DPPE High molecular weight PEG-DPPEderivatives (12 kDa PEG-DPPE) could not be incorporatedin the DPPC bilayer at all concentrations The 5 kDa PEG-DPPE which has an intermediate molecular weight was par-tially miscible with DPPC at concentrations below 7 molPhase separation occurred above 7 mol 5 kDa PEG-DPPEwhile above 11 transition to micellar state was observedtogether with phase separation In conclusion stable stealthliposomes can be obtained with low ratio of 3ndash5 kDa PEG-DPPE

Concerning the hydrophobic anchoring moiety longeralkyl chains than DPPE yielded unstable liposomes PEG-DSPE embedded in a liposome distearoyl phosphatidyl-choline (DSPC) bilayer promoted the phase separation evenat low PEG-DSPE molar ratio (5) This is ascribable to thesteric restriction of the DSPE moiety within the bilayer dueto high van der Waals cohesive forces that limit its mobilityThis enhances dramatically the PEG chainchain interactionsthat result in high mixing energy and favour demixing ofthe PEG-DSPE accompanied by structural rearrangementsof the bilayer Lipid phase separation generates domainson the liposome surface with low PEG-DSPE density thatyields inhomogeneous PEG coating and poor sterical sta-bility with rapid opsonin-mediated clearance The phaseseparation would also lead to the leakage of encapsulateddrug On the other hand short phospholipid alkyl chainsnamely PEG-dimyristoyl phosphatidylethanolamine (PEG-DMPE) embedded in liposome dimyristoyl phosphatidyl-choline (DMPC) bilayer slightly delayed the formation ofmixed lamellaemicelles at higher PEG-DMPE molar ratio(above 10) than PEG-DPPE The extent of demixing ofPEG-phospholipid from bilayers decreases as the phospho-lipid alkyl chain decreases in the order of C180 gt C160 gtC140

232 PEG Density The polymer density on the nanocarriersurface is as much relevant as polymer molecular weightFew authors showed that the high polymer surface den-sity can compensate the low polymer molecular weightin obtaining stealth particles [25 95 97] Vittaz et alinvestigated complement consumption of PEGylated PLAnanoparticlesThe authors concluded that a distance betweentwo chains of 2 kDa PEG of 22 nm corresponding to 02PEG moleculesnm2 could achieve efficient 100 nm particlecoatingwithminimumcomplement consumption [98] Stud-ies carried out using human phagocytes demonstrated that

a distance of 14 nm between 5 kDa-PEG chains optimallyyielded stealth 190ndash270 nm PEG-PLA nanoparticles [33]However it is worth to note that the polymer densitythreshold depends on a number of parameters includingparticle size and surface curvature

Investigations carried out by decorating gold-coated silicaparticles with 750 and 2000Da methoxy-PEG suggested thata polymer density of 05 chainnm2 is a critical threshold toprevent the adsorption of plasma proteins [99]

Low complement consumption was observed in the caseof 15 kDa PEG-stearate-coated 26 nm nanocapsules Theprotein repulsion was found to depend on the polymerdensity rather than the polymer chain length [25 100] Thenanocapsule surface covered by one PEG 15 kDa-stearatemolecule was estimated to be about 28 nm2 correspondingto about 17 nm distance between two PEG chains whichis in fair agreement with the results described above As aresult of the low opsonisation and complement consumptionthese nanoparticles displayed prolonged residence time in theblood with 20 of the dose still present in the blood 24 h afterinjection [101]

The homogeneous surface polymer coating is togetherwith the polymer density a key parameter to obtain stealthparticles A study showed that 30 of PEGylated polystyrenenanoparticles underwent phagocytosis as a consequence ofthe inhomogeneous physical adsorption of the polymer onthe particle surface [102]

233 Liposome Rigidity and Cholesterol Effect Phospholipidmembrane rigidity is paramount to produce liposomes withstealth properties as well as to prevent rapid drug release

Decreased rigidity due to the use of phospholipids withlow melting temperature (Tm) for the preparation of lipo-somal formulation can lead to drug leakage and opsoninadsorption

The liposome membrane rigidity homogeneity and sta-bility can be optimised by selecting phospholipids withproper Tm and by introducing cholesterol in the phospho-lipid bilayer A minimum content of 30 mol cholesterolratio is required to prevent the formation of phase separatedlamellas and mixed micelles It also reduces the leakage ofencapsulated drug from liposomes [42 103] and decreasesthe interaction of liposome surface with plasma components[96 104]

234 Surface Polymer Conformation The polymer chainconformation on the particle surface plays a critical role inconferring improved stealth properties to nanocarriers

It was found that the optimal surface coverage to conferadequate stealth properties is the one that allows for apolymer chain conformation in between the ldquomushroomrdquoand ldquobrushrdquo configurations In this specific condition mostof the chains are in a slightly constricted configurationat a density to ensure no uncoated gaps on the particlesurface It is conceivable that predominant brush-like PEGconfigurations would sterically suppress the deposition oflarge proteins such as C3 convertase [25] However evenwhen PEG is in the brush-like conformation on the surface of


nanoparticles its capacity to prohibit the protein adsorptionon the surface is again affected by the obstruction capacityof the protecting layer Small molecules can in fact slide inbetween the polymeric chains For such a reason Papisovet al [105] highlighted the influence of (i) brush density(ii) brush rigidity (iii) brush molecular length (iv) substratesize and (v) cooperative character of interaction on stericrepulsion and obstruction

The polymer chains conformation is dictated by thedistance of the anchorage site of two polymer chains (D) andby the gyration radius of the polymer known as Flory radius(119877119892= 12057211989935 where 119899 is the number ofmonomers per polymer

chain and120572 is the length of onemonomer in angstromswhichcorresponds to 35 A for PEG) [106] The 119877

119892of 2 kDa PEG is

approximately 56 nm which can be compressed dependingon the surface grafting density At low surface density thePEG chains have higher mobility In the case of 119877

119892lt

119863 lt 2119877119892the polymer chain conformation corresponds to an

intermingled ldquomushroomrdquo configuration This conformationallows the polymer chain for closer interactions to the surfaceof the particle and formation of gaps in the PEG protectivelayer that yields nanoparticle opsonisation [107] High PEGdensity results in 119863 sim 119877

119892and limited polymer chain

motion that yields the transition from mushroom-like tomushroombrush conformationWhen119863 ≪ 119877

119892 the polymer

chains convert to a brush-like conformation The resultinglow PEG chain mobility and flexibility reduces the abilityof the polymer to repulse opsonins [23] The polymer chainmovement due to its high flexibility and mobility reducesboth of the accessible surface of the nanoparticles and theinteraction of the polymer with the cryptic pockets of theopsonins [108]

Studies performed with 100 nm liposomes coated with2 kDa PEG-DSPE showed that below 4 PEG-DSPE molarratio the PEG chains were arranged in a mushroom confor-mation while a brush conformation was obtained above 8PEG-DSPE molar ratio [109]

235 Polymeric Corona Thickness PEG layer thickness isparamount to obtain stealth nanoparticles The minimumcoating layer thickness required to guarantee efficient par-ticle coating depends on a number of parameters includingthe potential absorbable proteins and the nanocarrier size[110]

Studies have shown that a minimum effective hydrody-namic layer thickness is about 5 of the particle diameter[111] Moghimi et al demonstrated that efficient protectionof 60ndash200 nm polystyrene particles from complement activa-tion and protein adsorption can be obtained with 4 kDa PEGthat provides for a coating thickness of 5 nm [17]

The thickness of the polymer coating depends on thepolymer chemical composition In aqueous medium PEGcan provide for a maximum thickness corresponding to itsfull chain length For copolymer such as poloxamers andpoloxamines instead the thickness is linearly related to thenumber of EO monomers since only this function of thepolymer can extend outward from the nanocarrier surface[93]

A hydrophilic polymer can provide for a surface coatingthickness of ℎ

119888= 119886119873(119886119863)

1V where 119873 is the degree ofpolymerization a is the size of the monomer and 119863 is themean distance between grafting points [112] For a goodsolvent the exponent is 35

In general proper particle stabilization is achieved when119860(119887ℎ

119888) lt 119879 where T = temperature 119860 = Hamaker constant

and 119887 = particle radius As 119860119879 is typically in the order of110 a coating with a thickness corresponding to 10 of theparticle diameter is conventionally considered adequate toprovide for efficient steric stability [23]

236 Polymer Flexibility Studies have demonstrated thatpolymer chain mobility is required for repelling proteinsfrom polymer chains on particle surface yielding stealthnanocarrier [113] Accordingly the lower complement acti-vation of PEG as compared to dextran can be explainedon the basis of polymer chain flexibility In a CH50 assayan in vitro haemolytic complement consumption assay 10complement activation was obtained with 20 cm2of 5 kDadextran coated and 120 cm2 5 kDa PEG-coated polycaprolac-tone nanoparticles [114] The results normalized by the par-ticle surface area show that the PEG coated particle surfaceinduces a lower complement activation as compared to thedextran-coated surface This is due to continuous change ofthe well-hydrated PEG chain conformation that reduces theexposure of fixation sites for complement proteins The rapidmovement of the flexible chains allows for the polymer tooccupy a high number of possible conformations and leadsto a temporary squeezing out of water molecules making thesurface impermeable for other solutes such as plasmaproteins[108]Therefore the water cloud surrounding the PEG chainsconfers an interfacial free energy on the particle surface thatprotects the nanocarriers from opsonisation and recognitionby macrophages

237 Amphiphilic Polymer Architecture Thecoating polymerconformation on the nanocarrier surface is strongly affectedby the polymer architecture which influences the plasmaprotein adsorption and interactions with cells

Nanoparticles obtained with multiblock (PLA-PEG-PLA)119899copolymers were found to adsorb higher amounts of

proteins compared to nanoparticles obtained with polyeth-ylene-glycol-grafted poly-(DL) lactide (PEG-g-PLA) [115]The low protein adsorption on PEG-g-PLA nanoparticleswas ascribed to a higher surface PEG density Similarlynanoparticles obtained with copolymers with a PCL back-bone and PEO grafts (PCL-g-PEO) were more effective inpreventing protein adsorption as compared to PEO-b-PCLdiblock copolymer nanoparticles [116]

The PEG attached through both terminal groups to thenanoparticle surface formed a single-turned-coil arrange-ment which was found to provide compact conformationalstructures that endowed particles with high resistance againstblood protein adsorption [117]

The effect of linear and branched PEGs on stealth proper-ties of nanocarriers was also investigated by using liposomesdecorated with PEG-PE and PEG

2-PE PEG

2-PE was more


efficient in improving the blood circulation time than PEG-PE at a low content (3 mol) whereas at high molar ratio(7 mol) their effect on liposome blood clearance is almostidentical At higher ratio of protecting polymer (7 mol)even PEG-PE can provide complete coating of the liposomesurface that does not take place at low molar PEG-PE ratio[108]

24 Controversial Effect of Polymer Coating Many studieshave demonstrated that the particle opsonisation can bereduced by surface coating with hydrophilic flexible poly-mers and mathematical elaborations have been developedto describe this effect However it should be noted thatseveral controversial results have been reported in theliterature

In vitro studies showed that stealth vesicles obtained byPEG coating can associate with a pool of opsonic proteins ofserum and plasma such as components of the complementsystem and immunoglobulins Nevertheless it was not clear ifthe protein interaction occurred with the exposed or internalpart of the coating polymer [14 29 33 60 118ndash124] In vivo25ndash10 of the dose of PEG-coated vesicles and nanoparticleshas been found to dispose in the liver and spleen in the firsthour after intravenous administration [125ndash130] The limitedremoval of stealth particles from the bloodstream seems toindicate that a small amount of specific opsonic proteinscan target PEG-coated nanocarriers [124] This hypothesisis supported by the evidence that low doses (20 nmolkgbody weight) of PEGylated liposomes are rapidly cleared bymacrophages while the cleared dose fraction decreases asthe amount of the injected PEG-coated liposomes increased[125ndash127]

Stealth nanocarriers were found to display long circu-lation profiles even after extensive opsonisation A typicalexample is Doxil the PEGylated doxorubicin loaded lipo-some formulation which is efficiently opsonised by the C3bfactor and activates the complement Nonetheless Doxilpresents a biphasic circulation half-life with prolonged per-manence in the circulation [21]

Overall these data show that the stealth behaviour oflong circulating nanocarriers is a very complex mechanismand it cannot be reduced to the simple opsonin repulsionunderlining some additional and relevant effects operated bythe steric coating on the nanocarrier surface

241 PEG Induced Complement Activation PEG coating onone side reduces the opsonisation process while on the othercan induce the complement activation that is involved in thenanoparticle removal Liposomes are a typical example of thedouble effect of particle PEGylation

Liposomes with low surface charge obtained with sat-urated phospholipids and high cholesterol content whichendows rigid and uniform bilayer without surface defects arepoorly prone to opsonisation and structural destabilisation byC3 adsorption [121 128 131 132] On the contrary negativelycharged and flexible liposomes undergo rapid opsonisationand phagocytosis The incorporation of 5ndash75mol of PEG2 kDa-DSPE into the bilayer of anionic liposomes formed

by egg phosphatidyl-choline cholesterol and cardiolipin(35 45 20 mole ratio) was found to dramatically reducethe complement activation of these vesicles However thedegree of complement activation also depended on theliposomes concentration Indeed in vitro studies showedthat 15mMPEGylated liposomes concentration induced 40complement consumption [133]

Studies carried out with Doxil showed that 04mgmL ofPEGylated liposomes elicited the rapid complement activa-tion and generate the soluble terminal complement complex(SC5b-9) in 7 out of 10 human sera [134] These resultsunderline the individual effect of PEGylated liposomes on thecomplement activation

The complement activation by PEGylated liposomes wasfound to be responsible for several side effects In pigs Doxilwas demonstrated to activate the complement through boththe C1q-dependent classical and the alternative complementactivation pathways [135] which was responsible for thecardiopulmonary distress [136]

In few cases a transient in vivo response was observed inrabbits as a drop in the systemic arterial pressure at 10minafter liposome injection which is typical of the complementactivation [137] On the contrary no complement activationafter PEGylated liposome administration was evidenced bythe in vitro assay These evidences highlight that in vitrocomplement activation tests should be carefully evaluatedfor what concerns their sensitivity and response threshold inorder to obtain results that can be correlated with the in vivodata

Studies performed with PEGylated polymeric nanopar-ticles confirmed that PEG-coated systems can induce thecomplement activation regardless of the PEG chain lengthand surface densityThe complement activation was inverselycorrelated with the PEG molecular weight suggesting thatsteric hindrance on the particle surface due to the polymercoating reduces the approach and association of large pro-teins such as the C3 convertase [97 138]

Studies carried out using PEGylated erythrocytes showedthat the complement activationmay bemediated by anti-PEGIgG and IgM [139]

Anti-PEG IgM elicited by a first administration of PEGy-lated liposome forms immunocomplexes with the seconddose of liposomes [140] These complexes activate the com-plement and convert the C3 component into C3b Thecomplex formed by C3b with other complement componentsis involved in the antibody-mediated complement activationpathway [134 141] that yields C3b fragmentation to iC3boperated by factors H and I iC3b is a proteolytically inactiveproduct of the complement fragment C3b that can stillopsonise However it cannot participate in the complementcascade since it does not associate with factor B a componentof the alternative activation pathway in the early stage of theactivation The generation of iC3b prevents the amplificationof the complement cascadeOverall the PEGmolecules on theliposome surface do not interfere with production of opsoniccomponents from the C3 component

Complement activation has been suggested to accountfor the clearance of PEGylated liposomes by the macrophageuptake of the RES [142]


Furthermore the extent of the accelerated blood clear-ance (ABC) of PEGylated liposomes is inversely proportionalto the dose probably because of the saturation of themononu-clear phagocytic system [143]

242 Poloxamine Induced Complement Activation Similarlyto PEG Poloxamines and Poloxamers have been extensivelyused to endownanocarriers with stealth properties Nonethe-less even these materials have been found to activate thecomplement to some extent thus reducing the beneficial effecton particle opsonisation

Poloxamine-908-coated polystyrene nanoparticles werefound to activate the complement through a complicatedpathway The adsorbed poloxamine-908 on the polystyrenenanoparticles rearranges from flat mushroom-like to brush-like conformation as the density of the polymer on theparticle surface increases As the polymer packs on par-ticle surface the surface area occupied by poloxaminedecreases from 45 to 15 nm2poloxamine chainThe interme-diate mushroom-brush poloxamine conformation inducedremarkable complement activation that decreased when thepolymer rearranged to a brush-like structure Uncoatednanoparticles and particles coated with poloxamine in themushroom-like conformation promote surface association ofthe C1q fragment of the complement protein C1 and acti-vate the complement through the classical pathway Nakedand poloxamine-coated nanoparticles in the mushroom andmushroom-brush conformation also activate the comple-ment through the alternative pathway by covalent conju-gation of properdin to poloxamine and the C3 componentadsorption Conversely particles coated with poloxamine inthe mushroom-brush and fully brush conformation activatethe complement via the lectin pathway which involvesthe opsonisation of mannose-binding lectin protein (MBL)andor ficolins This complement activation pathway wasattributed to the structural similarities between the EOmonomers of poloxamine and a region of D-mannose [144]The brush-like conformation minimizes the MBL and ficolinbinding to PEG backbone and consequently reduces thecomplement activation via the lectin pathway [145]

Thus the conformation and the mobility of surfaceprojected PEO chains of poloxamine on nanoparticles areparamount to modulate the complement activation pathway[146]

25 ldquoLong Circulationrdquo Revealed PEG-and poloxamine-coated nanocarriers have been demonstrated to undergoimmunoglobulin fibronectin and apolipoprotein associa-tion [14 29 33 118 122ndash124 147] as well as C3 opsonisationthat mediates the biorecognition by macrophages throughspecific complement receptors (CR1 and CR3 CD11bCD18)[18] However these systems possess long-lasting profiles inblood [148] The prolonged circulation in the bloodstreamis due to the steric hindrance of the surface polymers [134]that prevents the macrophage approach [124] Furthermorethe C3b adsorbed on the polymer corona of the particlesurface can be proteolytically degraded to fragments thatby assembling with other cofactors inhibit the recognition

by the macrophage receptors [149] The factor C3bn ofthe complement adsorbed on PEG-coated liposomes mayalso bind CR1 receptor associated with the erythrocytesmembrane which can also explain the prolonged circulationtime of PEGylated liposomes [150]

The steric shielding effect conveyed by polymer coatingon long circulation properties of stealth nanocarriers wasdemonstrated by Moghimi using poloxamine-908-coatedparticles These particles incubated with serum obtainedfrom a poloxamine-908 preinjected animal showed a higherprotein adsorption as compared to particles incubated withserum obtained from animals that were not preexposed topoloxamine The protein-coated nanoparticles showed sim-ilar pharmacokinetic profiles when administered to animalsnever exposed to poloxamine This evidence reinforces theexplanation that the improved circulation time of stealthnanoparticles is not solely ascribable to reduced proteinadsorption on particle surface [151] which surely takes placefor sterically stabilized nanocarriers Improved circulationtime can be mainly attributable to the prohibited biorecog-nition of the adsorbed opsonic proteins by the macrophages

26 Nanocarrier Coating with Hydrophilic Polymers Physicaland Chemical Strategies Sterically protective polymer canbe physically or chemically conjugated to the nanocarriersurface Physically conjugation involves the hydrophobicadsorption of polymer fragments on the particle surfacewhilethe chemical conjugation is obtained by chemical reaction ofpolymers with surface functions to yield covalent bonds

So far a variety of protocols have been set up to con-jugate PEG to small molecules and biologically active pro-teins These methods have been translated to obtain stealthnanoparticles with other materials [152 153]

261 Physical Coating of Polymeric Nanoparticles and Lipo-somes Surface PEG coating of PLGA nanoparticles was car-ried out using 2 kDa PEG-DSPE as emulsifier during oil-in-water microemulsion nanoparticle preparation The processallows for the embedding of the PEG-DSPE phospholipidfraction in the PLGA matrix by hydrophobic interactionswhereas the hydrophilic PEG chain extends outward thenanoparticle surface forming a polymeric brush that sta-bilizes the system Drug loaded 120 nm PEGylated PLGAnanoparticles were successfully used for the treatment of acystic fibrosis murine model by intranasal administration[154]

An original multistep technique for physical PEGyla-tion of doxorubicin loaded PLGA nanoparticles involvesthe surface adsorption of palmitate-avidin on the particlesthrough the avidin alkyl chain anchor during the parti-cle preparation by emulsion The avidinated particles aresubsequently PEGylated by exposure to PEG-biotin Theparticle coating with 5 and 10 kDa PEG reduced proteinadsorption by 50 and 75 respectively compared to thenon-PEGylated PLGA nanoparticles Approximately 3 ofthe initial dose of the doxorubicin loaded nanoparticlesintravenously administered was detected in the serum after48 hours from administration This corresponds to a twofold


OON

H

OP

O

OO

O

O

HO

O

4415

15

CH3Ominus

Figure 4 Structures of PEG-lipid conjugates used in preparing stealth liposomes The derivative is obtained with a PEG chain of 45monomers corresponding to a molecular weight of approximately 2000Da PEG units are capped at the distal end with a methoxy groupand conjugated to a DSPE lipid

residual doxorubicin plasma concentration as compared tothat obtained with non-PEGylated particles [155]

Protective PEG layer on liposomes can be achievedthrough two very conventional strategies

In the first approach PEG is conjugated with a hydropho-bic moiety (usually the residue of PE or a long chainfatty acid is reacted with methoxy-PEG-hydroxysuccinimideester) [156 157] (Figure 4) Subsequently a dry mixture filmof phospholipids and the mPEG-PE is rehydrated to yieldliposomes that spontaneously expose the PEG chains on theirsurface [158]

A second approach to coat liposomes with PEG is calledthe ldquopostinsertionmethodrdquo and consists in the conjugation ofactivated PEG to preformed liposomes

262 Polymer Coating of Magnetic Iron Oxide Nanoparticles Specific coating protocols have been set up to produce stealthinorganic nanoparticles

The incorporation of a polymer coating on the nanopar-ticle surface can be achieved either via ldquoone-potrdquo methodswhere the nanoparticles are coated by a polymer dissolved inthe particle productionmixture or by ldquotwo-steprdquo or ldquopostpro-ductionrdquomethod where nanoparticles are first generated andthen coated with a polymer

Magnetic nanoparticles coated with PEG-based copoly-mers have been prepared in one pot by Fe

3O4nucleation

and growth Poly(ethylene glycol) monomethyl ether-b-poly(glycerol monoacrylate) (PEG-b-PGA) was added toFe2+Fe3+ solutions and the coprecipitation of the iron ionswas inducedThe iron atoms on the nanoparticle surface werecoordinated via the 12-diols of the PGAblock which resultedin particle stabilization [159]

Iron oxide nanoparticles stabilized by carboxyl coordina-tion of the surface oxide molecules were prepared by high-temperature decomposition of tris(acetylacetonate) iron(III)[Fe(acac)

3] in the presence of monocarboxyl-terminated

PEG [160]Postproduction iron oxide nanoparticle decoration was

performed using silane-terminating PEG The silane groupstrongly interact with the oxide on the nanoparticle surface[161] PEGs derivatised with amino propyl trimethoxy silane(APTMS) or amino propyl triethoxy silane (APTES) wereused

Phosphonic acid-terminated poly(oligoethylene glycolacrylate) [poly(OEGA)] was grafted to iron oxide nanopar-ticles through the phosphonic acid end group that pro-vide strong interaction with iron oxide nanoparticles The

poly(OEGA-) stabilized iron oxide nanoparticles showed sig-nificant stealth properties and exhibited low BSA adsorption(lt30mg gminus1 nanoparticles) over a wide range of proteinconcentration (005 to 10 g Lminus1) [162]

Iron oxide nanoparticles synthesized by Fe(acac)3dec-

omposition in high-boiling organic solvents were postpro-duction PEGylated by the ligand exchange method Thenanoparticles produced with oleic acid hexane or trioctylphosphine oxide (TOPO) coating were combined with PEG-silanes PEG-PEI PEG-PAMAM PEG-fatty acid to allow forthe coating exchange in aqueous medium [163ndash168]

Dopamine has been proposed as an alternative anchoringgroup to silane to coat magnetic nanoparticles Dopaminehas high affinity for the iron oxide and can be conjugatedto PEG through the amino group PEG-dopamine was usedto displace the oleateoleylamine coating on the particlesproduced by high-temperature decomposition of Fe(acac)

3

thereby converting the particle surface from hydrophobic tohydrophilic according to a postproduction protocol [169]

ldquoGrowing fromrdquo approaches based on living radicalpolymerization techniques such as Atom-Transfer Radi-cal-Polymerization (ATRP) and Reversible Addition-Frag-mentation chain-Transfer (RAFT) polymerization have beenlargely investigated to coat preformed iron oxide nanopar-ticles with PEG copolymers ATRP polymerization of PEG-methacrylate (PEG-MA) was performed in aqueous solventafter a silane initiator (4-(chloromethyl) phenyl trichlorosi-lane) immobilization on iron oxide nanoparticle surfaceAfter poly(PEG-MA) grafting the uptake of the nanoparti-cles by macrophages was reduced from 158 to less than 2 pgper cell confirming the excellent shielding capacity of thisnovel material [170]

Alternatively the ATRP polymerization of the PEG-MA was performed according to a solvent-free protocolThe macroinitiator on the surface of the magnetic ironoxide nanoparticles was introduced by exchanging the sur-factant (oleic acid) on the nanoparticle surface with 3-chloropropionic acid The exchange made the nanoparticlessoluble in PEG-MA that was then polymerized by ATRPNo difference in terms of capacity to evade macrophageuptake was detected when poly(PEG-MA-) coated iron oxidenanoparticles were prepared in water or by the solvent-freemethod [171]

Hyperbranchedpolyglycerol (HPG)has recently emergedas a biocompatible and resistant material to protein adsorp-tion which was ascribed to its hyperbranched nature [84]HPG-grafted magnetic iron oxide nanoparticles have been


prepared by surface-initiated anionic polymerization of gly-cidol Iron oxide nanoparticles were first functionalizedwith 3-mercaptopropyltrimethoxysilane that in the anionicform promotes the ring opening polymerization of glyci-dol in toluene A 13wt HPG coating was obtained bythis procedure The protein adsorption was very low andcomparable to that of nanoparticles grafted with silanatedmethyloxy-PEG (MW = 750Da) at a similar grafting den-sity [172] Glycidol polymerization can be also initiated byaluminium isopropoxide grafted to 6-hydroxycaproic acidcoated iron oxide nanoparticles The resulting 24 nm HPG-grafted nanoparticles are very stable in PBS and culturemediaand their uptake bymacrophageswas very low (lt3 pg Fecell)over a 3-day contact time [173]

263 Polymer Coating of Gold Nanoparticles Gold nanopar-ticles have been PEGylated according to ldquoone-potrdquo methodsAuCl3

minus in solution can in fact be reduced by the amino groupsof the PEI block of poly(ethylenimine)-poly(ethylene glycol)block copolymer (PEI-b-PEG) [174]

Postproduction PEGylation strategies have relied mostlyon the use of thiol (-SH) terminated PEGs because of the veryhigh specific binding affinity of thiol groups to metal gold(S-Au bond energy = 47 kcal molminus1) Thiol-PEG can react insolution with gold nanoparticles providing colloidally stableand biocompatible gold nanoparticles [175]

Bidentate PEGs (PEG-thioctic acid and PEG-dihydroli-poic acid) conjugated on gold nanoparticle surface substan-tially improved the stability in biological media [176] Goldnanoparticles PEGylated with thioctic-modified 5 kDa PEGwere shown to perform better in vivo than gold nanoparticlescoated with thiol-PEG since the latter can release the PEG byexchange with thiolated compounds in the body [177]

The in vivo performance of gold nanorods stabilizedwith thiol-PEG depends on the polymer molecular weightAccordingly stable nanorods for blood circulation wereobtained with 5 and 10 kDa PEGs while smaller or largerPEGs were poorly flexible or bend into a mushroom-likeconfiguration respectively [34 178]

The maximum achievable density of PEG chains on goldnanoparticles was 22 nm2 per chain which is comparableto the hydrodynamic size of the mPEG-thiol molecule [179]At saturation the PEG molecules are so tightly packed thatopsonins will be prevented from adsorbing on the coatinglayer thus prohibiting the binding to macrophage receptors

Layer-by-layer (LBL) coating approaches relying on elec-trostatic interactions between polymer chains and goldnanoparticle surface have been investigated to build upa hydrophilic polymer corona on gold nanoparticles Thecolloidal core of gold nanoparticles was coated with lay-ers of poly(allylamine) (PAH) and poly-(styrenesulfonate)(PSS) F-HPMA a hydrophilic terpolymer composed by90 mol of N-(2-hydroxypropyl) methacrylamide was thenconjugated to the amino groups of PAH to yield coreshellmultifunctional nanoparticles The terpolymer provides ahighly water-solvated corona layer that minimizes the opson-isation process and bestows remarkable stealth propertieson nanoparticles The multifunctional nanoparticles did not

show a significant degree of adsorption on the macrophagemembrane or internalization by the cells [180]

PEG was grafted on gold nanoparticle surface accord-ing to a process named physisorption PEG-NH

2and 12-

distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) wereconjugated to the backbone of polyglutamic acid (PGA) at60 and 10 mol ratio with respect to the PGA monomersrespectively Gold nanoparticle coating was achieved byexchanging the citrate adsorbed on gold particles obtainedby tetrachloroauric acid reduction with the multifunctionalpolymer PGA-DSPE-mPEG These functionalized colloidalsystems showed high stability to aggregation over 48 hoursof incubation in 50 fetal calf serum [181]

Polyethylene glycol-block-poly(2NN-dimethylamino)ethyl methacrylate (PEG-b-PAMA) was shown to improvethe long-term stability of gold nanoparticles The tertiaryamino group of PAMA can strongly adsorb to the surfaceof gold nanoparticles even though the mechanism ofimmobilization is not clear yet The alkylation of pendantamino groups along the polymer backbone seems to favourthe interaction of the nitrogen atom with gold The colloidalsystem was physically stable over 4 days of storage in 95human serum [182]

Gold nanoshell can also be coated with a variety ofpolymers according to the same postproduction strategiesreported for gold nanoparticles and nanorods

264 Polymer Coating of Silica Nanoparticles Silica nano-particles possessing an organosilica core and a PEG shellwere prepared according to a one-pot procedureThe processincludes the co-hydrolysis and copolycondensation reactionsof120596-methoxy-(polyethyleneoxy)propyltrimethoxysilane andhydroxymethyltriethoxysilane mixtures in the presence ofsodium hydroxide and a surfactant [183]

Alternatively silica nanoparticles were also PEGylated bya postproduction procedure bymesoporus silica nanoparticlereaction with PEG-silanes It was reported that the PEGcoating inhibits the nonspecific binding of human serumproteins to PEGylated silica nanoparticlesThis is a guaranteeif the molecular weight of the polymer is higher than 10 kDaand the polymer density (defined as wt of the coating on themesoporous silica nanoparticles) is 075 wt and 0075wtfor PEG 10 kDa and PEG 20 kDa respectively The humanserumalbumin adsorptionwas only 25wtwhenPEGylatedsilica nanoparticles were tested compared to 187 for non-PEGylated nanoparticles [184]

PEG coating on silica nanoparticles can also beachieved via electrostatic adsorption of polyethyleneimine-polyethylene glycol (PEI-PEG) copolymer The polymericcoating was stable and tightly associated with the particlesurface by virtue of the strong electrostatic interactionsbetween the polyamino backbone of the copolymer and thenegatively charged silica surface The PEI-PEG copolymerinvestigated had 34 PEG chains (5 kDa) per PEI chain Theefficiency of the PEG coating in preventing the adsorption ofserum proteins on the nanoparticle surface was remarkablyhigh Protein adsorption was at the limit of sensitivity forX-ray photoelectron spectroscopy (XPS) detection and noaggregation was observed for the coated nanoparticles [185]


The synthesis of PEOon silica nanoparticles has also beenperformed resulting in a 40wt of grafted PEOThemethodhas been carried out first by a two-step conjugation process ofprehydrolyzed 3-glycidoxypropyl trimethoxysilane and alu-minium isopropoxide to the particle surface The subsequentpolymerization of ethylene oxide was carried out at 55∘CThe density of the polymer chains was found to be strictlydependent on the conjugation efficiency of themetal alkoxideon the particle surface [186 187]

3 Conclusions

The therapeutic advantages of nanotechnology-based drugdelivery systems include improved drug bioavailabilityextended duration of action reduced frequency of admin-istration and lower systemic toxicity with beneficial effectson the patient acceptance The medical management ofmalignancies has already benefited from the outcomes of fewnanotechnology-based delivery systems However followingintravenous administration drug-loaded nanocarriers arerapidly opsonised by a variety of proteins most of thembelonging to the complement system and undergo very rapidclearance via the MPS cells

In this paper the main aspects of polymer coatingtechnology applied to colloidal drug delivery systems havebeen reviewed A number of studies and examples reportedin the literature showing that stealthiness can be conferred tonanocarriers by a proper formulation design and predicatedby precise physicochemical determinants have been detailedand critically discussed

The evidence reported in the literature shows that theresidence time in the blood of nanocarriers can be prolongedby surface coatingwith neutral or zwitterionic polymers char-acterized by high hydrophilicity and high flexibility Further-more the stealth character of the nanocarriers depends on thepolymer organization on the particle surface namely densitythickness and association stability The beneficial effect ofnanocarrier polymer coating in promoting stealth propertiesgenerates predominantly from the polymer ability to confer aphysical barrier to the biorecognition of adsorbed opsoninsby macrophages On the other hand the paper underlinesthat the components of the hydrated polymeric corona arenot completely inert to the biological environment and thesematerials do not totally prohibit the protein opsonisation[124]

In conclusion while many discoveries in the field ofnanotechnology have allowed to clearly improve the perfor-mances of stealth nanocarriers a significant amount of workneeds to be done before these systems achieve the requiredlevel of safety for use in humans Studies are required tofully profile at the molecular level the interactions of thenanocarriers with the biological environment and the MPScell response that is triggered upon contact with a specificnanocarrier

References

[1] Y Matsumura and H Maeda ldquoA new concept for macro-molecular therapeutics in cancer chemotherapy mechanism of

tumoritropic accumulation of proteins and the antitumor agentsmancsrdquo Cancer Research vol 46 no 12 part 1 pp 6387ndash63921986

[2] K Greish J Fang T Inutsuka A Nagamitsu and H MaedaldquoMacromolecular therapeutics advantages and prospects withspecial emphasis on solid tumour targetingrdquo Clinical Pharma-cokinetics vol 42 no 13 pp 1089ndash1105 2003

[3] B D Ratner A S Hoffman F J Schoen and J E LemonsBiomaterials Science An Introduction to Materials in MedicineElsevier Academic Press Amsterdam The Netherlands 2ndedition 2004

[4] M M Frank and L F Fries ldquoThe role of complement ininflammation and phagocytosisrdquo Immunology Today vol 12 no9 pp 322ndash326 1991

[5] L E van Vlerken T K Vyas and M M Amiji ldquoPoly(ethyleneglycol)-modified nanocarriers for tumor-targeted and intracel-lular deliveryrdquo Pharmaceutical Research vol 24 no 8 pp 1405ndash1414 2007

[6] T Kinoshita ldquoBiology of complement the overturerdquo Immunol-ogy Today vol 12 no 9 pp 291ndash295 1991

[7] A Sahu and J D Lambris ldquoStructure and biology of comple-ment proteinC3 a connecting link between innate and acquiredimmunityrdquo Immunological Reviews vol 180 pp 35ndash48 2001

[8] MMMarkiewski B Nilsson K Nilsson Ekdahl T EMollnesand J D Lambris ldquoComplement and coagulation strangers orpartners in crimerdquo Trends in Immunology vol 28 no 4 pp184ndash192 2007

[9] B Nilsson K N Ekdahl T E Mollnes and J D Lambris ldquoTherole of complement in biomaterial-induced inflammationrdquoMolecular Immunology vol 44 no 1ndash3 pp 82ndash94 2007

[10] D Ricklin and J D Lambris ldquoComplement-targeted therapeu-ticsrdquo Nature Biotechnology vol 25 no 11 pp 1265ndash1275 2007

[11] P Gros F J Milder and B J C Janssen ldquoComplement drivenby conformational changesrdquo Nature Reviews Immunology vol8 no 1 pp 48ndash58 2008

[12] A Vonarbourg C Passirani P Saulnier and J P BenoitldquoParameters influencing the stealthiness of colloidal drug deliv-ery systemsrdquo Biomaterials vol 27 no 24 pp 4356ndash4373 2006

[13] H Carstensen R H Muller and B W Muller ldquoParticlesize surface hydrophobicity and interaction with serum ofparenteral fat emulsions and model drug carriers as parametersrelated to RES uptakerdquo Clinical Nutrition vol 11 no 5 pp 289ndash297 1992

[14] M E Norman P Williams and L Illum ldquoHuman serumalbumin as a probe for surface conditioning (opsonization) ofblock copolymer-coated microspheresrdquo Biomaterials vol 13no 12 pp 841ndash849 1992

[15] R H Muller K HWallis S D Troster and J Kreuter ldquoIn vitrocharacterization of poly(methyl-methaerylate) nanoparticlesand correlation to their in vivo faterdquo Journal of ControlledRelease vol 20 no 3 pp 237ndash246 1992

[16] M Roser D Fischer and T Kissel ldquoSurface-modifiedbiodegradable albumin nano- and microspheres II effect ofsurface charges on in vitro phagocytosis and biodistribution inratsrdquo European Journal of Pharmaceutics and Biopharmaceuticsvol 46 no 3 pp 255ndash263 1998

[17] S M Moghimi I S Muir L Illum S S Davis and VKolb-Bachofen ldquoCoating particles with a block co-polymer(poloxamine-908) suppresses opsonization but permits theactivity of dysopsonins in the serumrdquo Biochimica et BiophysicaActa vol 1179 no 2 pp 157ndash165 1993


[18] S M Moghimi A C Hunter and J C Murray ldquoLong-circulating and target-specific nanoparticles theory to prac-ticerdquo Pharmacological Reviews vol 53 no 2 pp 283ndash318 2001

[19] X Yan G L Scherphof and J A A M Kamps ldquoLiposomeopsonizationrdquo Journal of Liposome Research vol 15 no 1-2 pp109ndash139 2005

[20] T M Allen ldquoLong-circulating (sterically stabilized) liposomesfor targeted drug deliveryrdquo Trends in Pharmacological Sciencesvol 15 no 7 pp 215ndash220 1994

[21] M C Woodle and G Storm Long Circulating Liposomes OldDrugs NewTherapeutics Springer New York NY USA 1998

[22] D E Owens III and N A Peppas ldquoOpsonization biodis-tribution and pharmacokinetics of polymeric nanoparticlesrdquoInternational Journal of Pharmaceutics vol 307 no 1 pp 93ndash102 2006

[23] G Storm S O Belliot T Daemen and D D Lasic ldquoSurfacemodification of nanoparticles to oppose uptake by themononu-clear phagocyte systemrdquo Advanced Drug Delivery Reviews vol17 no 1 pp 31ndash48 1995

[24] A E Stuart ldquoPhagocytic engulfment and cell adhesiveness ascellular surface phenomenardquo Journal of Clinical Pathology vol30 no 6 article 592 1977

[25] S I Jeon and J D Andrade ldquoProtein-surface interactions in thepresence of polyethylene oxide II Effect of protein sizerdquo Journalof Colloid and Interface Science vol 142 no 1 pp 159ndash166 1991

[26] L IllumNWThomas and S S Davis ldquoEffect of a selected sup-pression of the reticuloendothelial system on the distribution ofmodel carrier particlesrdquo Journal of Pharmaceutical Sciences vol75 no 1 pp 16ndash22 1986

[27] Y Tabata and Y Ikada ldquoPhagocytosis of polymer microspheresby macrophagesrdquo Advances in Polymer Science vol 94 pp 106ndash141 1990

[28] A Gabizon and D Papahadjopoulos ldquoThe role of surfacecharge and hydrophilic groups on liposome clearance in vivordquoBiochimica et Biophysica Acta vol 1103 no 1 pp 94ndash100 1992

[29] M T Peracchia S Harnisch H Pinto-Alphandary et al ldquoVisu-alization of in vitro protein-rejecting properties of PEGylatedstealth polycyanoacrylate nanoparticlesrdquo Biomaterials vol 20no 14 pp 1269ndash1275 1999

[30] J C Neal S Stolnik E Schacht et al ldquoIn vitro displacement byrat serum of adsorbed radiolabeled poloxamer and poloxam-ine copolymers from model and biodegradable nanospheresrdquoJournal of Pharmaceutical Sciences vol 87 no 10 pp 1242ndash12481998

[31] G R HarperM C Davies S S Davis T F Tadros D C Taylorand M P J A I Waters ldquoSteric stabilization of microsphereswith grafted polyethylene oxide reduces phagocytosis by ratKupffer cells in vitrordquo Biomaterials vol 12 no 7 pp 695ndash7001991

[32] D Bazile C PrudrsquoHomme M T Bassoullet M Marlard GSpenlehauer and M Veillard ldquoStealth MePEG-PLA nanopar-ticles avoid uptake by the mononuclear phagocytes systemrdquoJournal of Pharmaceutical Sciences vol 84 no 4 pp 493ndash4981995

[33] R Gref M Luck P Quellec et al ldquolsquoStealthrsquo corona-corenanoparticles surface modified by polyethylene glycol (PEG)influences of the corona (PEG chain length and surface density)and of the core composition on phagocytic uptake and plasmaprotein adsorptionrdquo Colloids and Surfaces B vol 18 no 3-4 pp301ndash313 2000

[34] M T Peracchia E Fattal D Desmaele et al ldquoStealth PEGylatedpolycyanoacrylate nanoparticles for intravenous administra-tion and splenic targetingrdquo Journal of Controlled Release vol 60no 1 pp 121ndash128 1999

[35] K Bergstrom E Osterberg K Holmberg et al ldquoEffects ofbranching and molecular weight of surface-bound poly(ethy-lene oxide) on protein rejectionrdquo Journal of Biomaterials Science(Polymer Edition) vol 6 no 2 pp 123ndash132 1994

[36] S E Dunn A Brindley S S Davis M C Davies and L IllumldquoPolystyrene-poly (ethylene glycol) (PS-PEG2000) particles asmodel systems for site specific drug delivery 2 The effect ofPEG surface density on the in vitro cell interaction and invivo biodistributionrdquo Pharmaceutical Research vol 11 no 7 pp1016ndash1022 1994

[37] M Yokoyama ldquoBlock copolymers as drug carriersrdquo CriticalReviews inTherapeutic Drug Carrier Systems vol 9 no 3-4 pp213ndash248 1992

[38] N KumarM N V Ravikumar and A J Domb ldquoBiodegradableblock copolymersrdquoAdvancedDrugDelivery Reviews vol 53 no1 pp 23ndash44 2001

[39] M L Adams A Lavasanifar and G S Kwon ldquoAmphiphilicblock copolymers for drug deliveryrdquo Journal of PharmaceuticalSciences vol 92 no 7 pp 1343ndash1355 2003

[40] J Vandorpe E Schacht S Dunn et al ldquoLong circulatingbiodegradable poly(phosphazene) nanoparticles surface mod-ified with poly(phosphazene)-poly(ethylene oxide) copolymerrdquoBiomaterials vol 18 no 17 pp 1147ndash1152 1997

[41] S Stolnik S EDunnMCGarnett et al ldquoSurfacemodificationof poly(lactide-co-glycolide) nanospheres by biodegradablepoly(lactide)-poly(ethylene glycol) copolymersrdquo Pharmaceuti-cal Research vol 11 no 12 pp 1800ndash1808 1994

[42] M C Woodle and D D Lasic ldquoSterically stabilized liposomesrdquoBiochimica et Biophysica Acta vol 1113 no 2 pp 171ndash199 1992

[43] K Kostarelos T F Tadros and P F Luckham ldquoPhysicalconjugation of (Tri-) block copolymers to liposomes toward theconstruction of sterically stabilized vesicle systemsrdquo Langmuirvol 15 no 2 pp 369ndash376 1999

[44] S M Moghimi and A C Hunter ldquoPoloxamers and poloxam-ines in nanoparticle engineering and experimental medicinerdquoTrends in Biotechnology vol 18 no 10 pp 412ndash420 2000

[45] M E Norman P Williams and L Illum ldquoInfluence of blockcopolymers on the adsorption of plasma proteins to micro-spheresrdquo Biomaterials vol 14 no 3 pp 193ndash202 1993

[46] Y Chang W L Chu W Y Chen et al ldquoA systematic SPR studyof human plasma protein adsorption behavior on the controlledsurface packing of self-assembled poly(ethylene oxide) triblockcopolymer surfacesrdquo Journal of Biomedical Materials ResearchA vol 93 no 1 pp 400ndash408 2010

[47] J Lee P A Martic and J S Tan ldquoProtein adsorption onpluronic copolymer-coated polystyrene particlesrdquo Journal ofColloid and Interface Science vol 131 no 1 pp 252ndash266 1989

[48] D B Shenoy and M M Amiji ldquoPoly(ethylene oxide)-modifiedpoly(120576-caprolactone) nanoparticles for targeted delivery oftamoxifen in breast cancerrdquo International Journal of Pharmaceu-tics vol 293 no 1-2 pp 261ndash270 2005

[49] R Weissleder A Bogdanov E A Neuwelt and M PapisovldquoLong-circulating iron oxides for MR imagingrdquoAdvanced DrugDelivery Reviews vol 16 no 2-3 pp 321ndash334 1995

[50] D Pain P K Das P Ghosh and B K Bachhawat ldquoIncreasedcirculatory half-life of liposomes after conjunction with dex-tranrdquo Journal of Biosciences vol 6 no 6 pp 811ndash816 1984


[51] H H Bengele S Palmacci J Rogers C W Jung J Crenshawand L Josphson ldquoBiodistribution of an ultrasmall superparam-agnetic iron oxide colloid BMS 180549 by different routes ofadministrationrdquoMagnetic Resonance Imaging vol 12 no 3 pp433ndash442 1994

[52] S M Moghimi and B Bonnemain ldquoSubcutaneous and intra-venous delivery of diagnostic agents to the lymphatic systemapplications in lymphoscintigraphy and indirect lymphogra-phyrdquo Advanced Drug Delivery Reviews vol 37 no 1ndash3 pp 295ndash312 1999

[53] M K Pangburn and H J Muller-Eberhard ldquoComplement C3convertase cell surface restriction of 1205731H control and genera-tion of restriction on neuraminidase-treated cellsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 75 no 5 pp 2416ndash2420 1978

[54] M D Kazatchkine D T Fearon and K F Austen ldquoHumanalternative complement pathway membrane-associated sialicacid regulates the competition between B and 1205731H for cell-bound C3brdquo Journal of Immunology vol 122 no 1 pp 75ndash811979

[55] D T Fearon and K F Austen ldquoActivation of the alternativecomplement pathway due to resistance of zymosan boundamplification convertase to endogenous regulatory mecha-nismsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 74 no 4 pp 1683ndash1687 1977

[56] A Surolia and B K Bachhawat ldquoMonosialoganglioside lipo-some entrapped enzyme uptake by hepatic cellsrdquo Biochimica etBiophysica Acta vol 497 no 3 pp 760ndash765 1977

[57] T M Allen and A Chonn ldquoLarge unilamellar liposomes withlow uptake into the reticuloendothelial systemrdquo FEBS Lettersvol 223 no 1 pp 42ndash46 1987

[58] A Gabizon and D Papahadjopoulos ldquoLiposome formulationswith prolonged circulation time in blood and enhanced uptakeby tumorsrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 85 no 18 pp 6949ndash6953 1988

[59] T M Allen C Hansen and J Rutledge ldquoLiposomes withprolonged circulation times factors affecting uptake by reticu-loendothelial and other tissuesrdquo Biochimica et Biophysica Actavol 981 no 1 pp 27ndash35 1989

[60] A Chonn S C Semple and P R Cullis ldquoAssociation of bloodproteins with large unilamellar liposomes in vivo Relation tocirculation lifetimesrdquo The Journal of Biological Chemistry vol267 no 26 pp 18759ndash18765 1992

[61] D Liu Y K Song and F Liu ldquoAntibody dependent com-plement mediated liver uptake of liposomes containing GM1rdquoPharmaceutical Research vol 12 no 11 pp 1775ndash1780 1995

[62] Y S Park and L Huang ldquoEffect of chemically modified G(M1)and neoglycolipid analogs of G(M1) on liposome circula-tion time evidence supporting the dysopsonin hypothesisrdquoBiochimica et Biophysica Acta vol 1166 no 1 pp 105ndash114 1993

[63] H Yamauchi H Kikuchi K Yachi M Sawada M Tomikawaand S Hirota ldquoEffects of glycophorin and ganglioside GM3 onthe blood circulation and tissue distribution of liposomes inratsrdquo International Journal of Pharmaceutics vol 90 no 1 pp73ndash79 1993

[64] H Yamauchi T Yano T Kato et al ldquoEffects of sialic acidderivative on long circulation time and tumor concentration ofliposomesrdquo International Journal of Pharmaceutics vol 113 no2 pp 141ndash148 1995

[65] J C Olivier C VauthierM Taverna F Puisieux D Ferrier andP Couvreur ldquoStability of orosomucoid-coated polyisobutyl-cyanoacrylate nanoparticles in the presence of serumrdquo Journalof Controlled Release vol 40 no 3 pp 157ndash168 1996

[66] M T Michalek E G Bremer and C Mold ldquoEffect of gan-gliosides on activation of the alternative pathway of humancomplementrdquo Journal of Immunology vol 140 no 5 pp 1581ndash1587 1988

[67] T M Allen ldquoThe use of glycolipids and hydrophilic polymersin avoiding rapid uptake of liposomes by the mononuclearphagocyte systemrdquoAdvanced Drug Delivery Reviews vol 13 no3 pp 285ndash309 1994

[68] P Vermette and L Meagher ldquoInteractions of phospholipid-and poly(ethylene glycol)-modified surfaceswith biological sys-tems relation to physico-chemical properties andmechanismsrdquoColloids and Surfaces B vol 28 no 2-3 pp 153ndash198 2003

[69] S Chen S Chen S Jiang et al ldquoStudy of zwitterionic sulfo-propylbetaine containing reactive siloxanes for application inantibacterial materialsrdquo Colloids and Surfaces B vol 85 no 2pp 323ndash329 2011

[70] S Jiang and Z Cao ldquoUltralow-fouling functionalizable andhydrolyzable zwitterionic materials and their derivatives forbiological applicationsrdquo Advanced Materials vol 22 no 9 pp920ndash932 2010

[71] Z Cao N Brault H Xue A Keefe and S Jiang ldquoManipulatingsticky and non-sticky properties in a single materialrdquo Ange-wandte ChemiemdashInternational Edition vol 50 no 27 pp 6102ndash6104 2011

[72] D Massenburg and B R Lentz ldquoPoly(ethylene glycol)-inducedfusion and rupture of dipalmitoylphosphatidylcholine largeunilamellar extruded vesiclesrdquo Biochemistry vol 32 no 35 pp9172ndash9180 1993

[73] R Saez A Alonso A Villena and F M Goni ldquoDetergent-like properties of polyethyleneglycols in relation to modelmembranesrdquo FEBS Letters vol 137 no 2 pp 323ndash326 1982

[74] Y He J Hower S Chen M T Bernards Y Chang and S JiangldquoMolecular simulation studies of protein interactions withzwitterionic phosphorylcholine self-assembled monolayers inthe presence of waterrdquo Langmuir vol 24 no 18 pp 10358ndash10364 2008

[75] Z Cao L Zhang and S Jiang ldquoSuperhydrophilic zwitterionicpolymers stabilize liposomesrdquo Langmuir vol 28 no 31 pp11625ndash11632 2012

[76] Z G Estephan J A Jaber and J B Schlenoff ldquoZwitterion-stabilized silica nanoparticles toward nonstick nanordquo Lang-muir vol 26 no 22 pp 16884ndash16889 2010

[77] G Jia Z Cao H Xue Y Xu and S Jiang ldquoNovel zwitterionic-polymer-coated silica nanoparticlesrdquo Langmuir vol 25 no 5pp 3196ndash3199 2009

[78] W Yang L Zhang S Wang A D White and S JiangldquoFunctionalizable and ultra stable nanoparticles coated withzwitterionic poly(carboxybetaine) in undiluted blood serumrdquoBiomaterials vol 30 no 29 pp 5617ndash5621 2009

[79] L Zhang H Xue C Gao et al ldquoImaging and cell tar-geting characteristics of magnetic nanoparticles modified bya functionalizable zwitterionic polymer with adhesive 34-dihydroxyphenyl-l-alanine linkagesrdquo Biomaterials vol 31 no25 pp 6582ndash6588 2010

[80] Z Cao Q Yu H Xue G Cheng and S Jiang ldquoNanoparticlesfor drug delivery prepared fromamphiphilic PLGAzwitterionicblock copolymers with sharp contrast in polarity between two


blocksrdquoAngewandte ChemiemdashInternational Edition vol 49 no22 pp 3771ndash3776 2010

[81] G Cheng L Mi Z Cao et al ldquoFunctionalizable and ultrastablezwitterionic nanogelsrdquo Langmuir vol 26 no 10 pp 6883ndash68862010

[82] L Zhang H Xue Z Cao A Keefe J Wang and S JiangldquoMultifunctional and degradable zwitterionic nanogels for tar-geted delivery enhancedMR imaging reduction-sensitive drugrelease and renal clearancerdquo Biomaterials vol 32 no 20 pp4604ndash4608 2011

[83] J Ladd Z Zhang S Chen J C Hower and S Jiang ldquoZwit-terionic polymers exhibiting high resistance to nonspecificprotein adsorption from human serum and plasmardquo Biomacro-molecules vol 9 no 5 pp 1357ndash1361 2008

[84] C SiegersM Biesalski and R Haag ldquoSelf-assembledmonolay-ers of dendritic polyglycerol derivatives on gold that resist theadsorption of proteinsrdquoChemistry vol 10 no 11 pp 2831ndash28382004

[85] M Calderon M A Quadir S K Sharma and R HaagldquoDendritic polyglycerols for biomedical applicationsrdquoAdvancedMaterials vol 22 no 2 pp 190ndash218 2010

[86] KMaruyama SOkuizumiO IshidaHYamauchiHKikuchiandM Iwatsuru ldquoPhosphatidyl polyglycerols prolong liposomecirculation in vivordquo International Journal of Pharmaceutics vol111 no 1 pp 103ndash107 1994

[87] P Y J Yeh R K Kainthan Y ZouMChiao and J N Kizhakke-dathu ldquoSelf-assembled monothiol-terminated hyperbranchedpolyglycerols on a gold surface a comparative study on thestructure morphology and protein adsorption characteristicswith linear poly(ethylene glycol)srdquo Langmuir vol 24 no 9 pp4907ndash4916 2008

[88] V P Torchilin M I Shtilman V S Trubetskoy K Whitemanand A M Milstein ldquoAmphiphilic vinyl polymers effectivelyprolong liposome circulation time in vivordquo Biochimica et Bio-physica Acta vol 1195 no 1 pp 181ndash184 1994

[89] V P Torchilin and V S Trubetskoy ldquoWhich polymers canmakenanoparticulate drug carriers long-circulatingrdquo AdvancedDrug Delivery Reviews vol 16 no 2-3 pp 141ndash155 1995

[90] V P Torchilin V S Trubetskoy K R Whiteman P CalicetiP Ferruti and F M Veronese ldquoNew synthetic amphiphilicpolymers for steric protection of liposomes in vivordquo Journal ofPharmaceutical Sciences vol 84 no 9 pp 1049ndash1053 1995

[91] D Feldman ldquoPolymers in solution Their modelling and struc-ture by J des Cloizeaux and G Jannink Oxford universitypress New York 1991 944 pp $19500rdquo Journal of PolymerScience A vol 30 no 2 pp 343ndash343

[92] H Takeuchi H Kojima H Yamamoto and Y KawashimaldquoEvaluation of circulation profiles of liposomes coated withhydrophilic polymers having different molecular weights inratsrdquo Journal of Controlled Release vol 75 no 1-2 pp 83ndash912001

[93] L Illum L O Jacobsen and R H Muller ldquoSurface charac-teristics and the interaction of colloidal particles with mouseperitoneal macrophagesrdquo Biomaterials vol 8 no 2 pp 113ndash1171987

[94] J C Leroux F de Jaeghere B Anner E Doelker and R GurnyldquoAn investigation on the role of plasma and serum opsoninson the internalization of biodegradable poly(DL-lactic acid)nanoparticles by human monocytesrdquo Life Sciences vol 57 no7 pp 695ndash703 1995

[95] W R GombotzWGuanghui T AHorbett andA S HoffmanldquoProtein adsorption to poly(ethylene oxide) surfacesrdquo Journal

of Biomedical Materials Research vol 25 no 12 pp 1547ndash15621991

[96] F K Bedu-Addo and L Huang ldquoInteraction of PEG-phospholipid conjugates with phospholipid implicationsin liposomal drug deliveryrdquo Advanced Drug Delivery Reviewsvol 16 no 2-3 pp 235ndash247 1995

[97] V C F Mosqueira P Legrand A Gulik et al ldquoRelationshipbetween complement activation cellular uptake and surfacephysicochemical aspects of novel PEG-modified nanocapsulesrdquoBiomaterials vol 22 no 22 pp 2967ndash2979 2001

[98] MVittaz D Bazile G Spenlehauer et al ldquoEffect of PEO surfacedensity on long-circulating PLA-PEO nanoparticles which arevery low complement activatorsrdquoBiomaterials vol 17 no 16 pp1575ndash1581 1996

[99] L D Unsworth H Sheardown and J L Brash ldquoProtein-resistant polyethylene oxide-grafted surfaces chain density-dependent multiple mechanisms of actionrdquo Langmuir vol 24no 5 pp 1924ndash1929 2008

[100] C Passirani and J P Benoit ldquoComplement activation byinjectable colloidal drug carriersrdquo in Biomaterials for Deliveryand Targeting of Proteins and Nucleic Acids CRC Press NewYork NY USA 2004

[101] A Beduneau P Saulnier N Anton et al ldquoPegylated nanocap-sules produced by an organic solvent-freemethod evaluation oftheir stealth propertiesrdquo Pharmaceutical Research vol 23 no 9pp 2190ndash2199 2006

[102] S M Moghimi ldquoChemical camouflage of nanospheres witha poorly reactive surface towards development of stealth andtarget-specific nanocarriersrdquo Biochimica et Biophysica Acta vol1590 no 1ndash3 pp 131ndash139 2002

[103] P S Uster ldquoLiposomes as drug carriers recent trends andprogress Edited by Gregory Gregoriadis John Wiley Chich-ester UK 1988 xxvi + 885 pp 22 times 16 cm ISBN 0-471-91654-4Price not givenrdquo Journal of Pharmaceutical Sciences vol 78 no8 pp 693ndash693 1989


[105] M I Papisov ldquoTheoretical considerations of RES-avoidingliposomes molecular mechanics and chemistry of liposomeinteractionsrdquo Advanced Drug Delivery Reviews vol 32 no 1-2pp 119ndash138 1998

[106] P M Claesson E Blomberg J C Froberg T Nylander and TArnebrant ldquoProtein interactions at solid surfacesrdquo Advances inColloid and Interface Science vol 57 no C pp 161ndash227 1995

[107] AKKenworthy S A Simon andT JMcIntosh ldquoStructure andphase behavior of lipid suspensions containing phospholipidswith covalently attached poly(ethylene glycol)rdquo BiophysicalJournal vol 68 no 5 pp 1903ndash1920 1995

[108] V P Torchilin ldquoPolymer-coated long-circulating microparticu-late pharmaceuticalsrdquo Journal of Microencapsulation vol 15 no1 pp 1ndash19 1998

[109] S D Li and L Huang ldquoStealth nanoparticles high densitybut sheddable PEG is a key for tumor targetingrdquo Journal ofControlled Release vol 145 no 3 pp 178ndash181 2010

[110] S Rudt and R H Muller ldquoIn vitro phagocytosis assay of nano-and microparticles by chemiluminescence III Uptake of dif-ferently sized surface-modified particles and its correlation toparticle properties and in vivo distributionrdquo European Journalof Pharmaceutical Sciences vol 1 no 1 pp 31ndash39 1993


[111] S Stolnik L Illum and S S Davis ldquoLong circulating micropar-ticulate drug carriersrdquo Advanced Drug Delivery Reviews vol 16no 2-3 pp 195ndash214 1995

[112] P G de Gennes ldquoPolymer solutions near an interface 1Adsorption and depletion layersrdquo Macromolecules vol 14 no6 pp 1637ndash1644 1981

[113] S W Shalaby and A C S Meeting Polymers As BiomaterialsPlenum Press New York NY USA 1984

[114] C Lemarchand R Gref C Passirani et al ldquoInfluence ofpolysaccharide coating on the interactions of nanoparticleswithbiological systemsrdquoBiomaterials vol 27 no 1 pp 108ndash118 2006

[115] S Sant S Poulin andPHildgen ldquoEffect of polymer architectureon surface properties plasma protein adsorption and cellularinteractions of pegylated nanoparticlesrdquo Journal of BiomedicalMaterials Research A vol 87 no 4 pp 885ndash895 2008

[116] J Rieger C Passirani J P Benoit K van Butsele R Jeromeand C Jerome ldquoSynthesis of amphiphilic copolymers ofpoly(ethylene oxide) and poly(120576-caprolactone) with differentarchitectures and their role in the preparation of stealthynanoparticlesrdquoAdvanced FunctionalMaterials vol 16 no 11 pp1506ndash1514 2006

[117] M T Peracchia C Vauthier C Passirani P Couvreur and DLabarre ldquoComplement consumption by poly(ethylene glycol)in different conformations chemically coupled to poly(isobutyl2-cyanoacrylate) nanoparticlesrdquo Life Sciences vol 61 no 7 pp749ndash761 1997

[118] T Blunk D F Hochstrasser J C Sanchez B W Muller andR H Muller ldquoColloidal carriers for intravenous drug targetingplasma protein adsorption patterns on surface-modified latexparticles evaluated by two-dimensional polyacrylamide gelelectrophoresisrdquo Electrophoresis vol 14 no 12 pp 1382ndash13871993

[119] R Gref A Domb P Quellec et al ldquoThe controlled intra-venous delivery of drugs using PEG-coated sterically stabilizednanospheresrdquo Advanced Drug Delivery Reviews vol 16 no 2-3pp 215ndash233 1995

[120] S C Semple A Chonn and P R Cullis ldquoInfluence of choles-terol on the association of plasma proteins with liposomesrdquoBiochemistry vol 35 no 8 pp 2521ndash2525 1996

[121] S C Semple A Chonn and P R Cullis ldquoInteractions ofliposomes and lipid-based carrier systems with blood proteinsrelation to clearance behaviour in vivordquoAdvancedDrugDeliveryReviews vol 32 no 1-2 pp 3ndash17 1998

[122] M E Price RMCornelius and J L Brash ldquoProtein adsorptionto polyethylene glycol modified liposomes from fibrinogensolution and from plasmardquo Biochimica et Biophysica Acta vol1512 no 2 pp 191ndash205 2001

[123] S Stolnik B Daudali A Arien et al ldquoThe effect of surfacecoverage and conformation of poly(ethylene oxide) (PEO)chains of poloxamer 407 on the biological fate of modelcolloidal drug carriersrdquo Biochimica et Biophysica Acta vol 1514no 2 pp 261ndash279 2001

[124] S M Moghimi and J Szebeni ldquoStealth liposomes and longcirculating nanoparticles critical issues in pharmacokineticsopsonization and protein-binding propertiesrdquo Progress in LipidResearch vol 42 no 6 pp 463ndash478 2003

[125] P Laverman A H Brouwers E T M Dams et al ldquoPreclinicaland clinical evidence for disappearance of long-circulatingcharacteristics of polyethylene glycol liposomes at low lipiddoserdquo Journal of Pharmacology and Experimental Therapeuticsvol 293 no 3 pp 996ndash1001 2000

[126] P Laverman O C Boerman W J G Oyen F H M Corstensand G Storm ldquoIn vivo applications of PEG liposomes unex-pected observationsrdquo Critical Reviews in Therapeutic DrugCarrier Systems vol 18 no 6 pp 551ndash566 2001


[128] T M Allen C Hansen F Martin C Redemann and A FYau-Young ldquoLiposomes containing synthetic lipid derivativesof poly(ethylene glycol) show prolonged circulation half-livesin vivordquo Biochimica et Biophysica Acta vol 1066 no 1 pp 29ndash36 1991

[129] D R Utkhede and C P Tilcock ldquoEffect of lipid dose onthe biodistribution and blood pool clearance kinetics of PEG-modified technetium-labeled lipid vesiclesrdquo Journal of LiposomeResearch vol 8 no 3 pp 381ndash390 1998

[130] M C Woodle K K Matthay M S Newman et al ldquoVersatilityin lipid compositions showing prolonged circulation withsterically stabilized liposomesrdquo Biochimica et Biophysica Actavol 1105 no 2 pp 193ndash200 1992

[131] J T PDerksenHWMMorselt DKalicharan C EHulstaertand G L Scherphof ldquoInteraction of immunoglobulin-coupledliposomes with rat liver macrophages in vitrordquo ExperimentalCell Research vol 168 no 1 pp 105ndash115 1987

[132] U R Nilsson K E Storm H Elwing and B Nilsson ldquoCon-formation epitopes of C3 reflecting its mode of binding to anartificial polymer surfacerdquo Molecular Immunology vol 30 no3 pp 211ndash219 1993

[133] A J Bradley D V Devine S M Ansell J Janzen and D EBrooks ldquoInhibition of liposome-induced complement activa-tion by incorporated poly(ethylene glycol)-lipidsrdquo Archives ofBiochemistry and Biophysics vol 357 no 2 pp 185ndash194 1998

[134] J Szebeni L Baranyi S Savay et al ldquoThe role of complementactivation in hypersensitivity to pegylated liposomal doxoru-bicin (doxil)rdquo Journal of Liposome Research vol 10 no 4 pp467ndash481 2000

[135] S M Moghimi I Hamad T L Andresen K Joslashrgensen andJ Szebeni ldquoMethylation of the phosphate oxygen moiety ofphospholipid-methoxy(polyethylene glycol) conjugate preventsPEGylated liposome-mediated complement activation and ana-phylatoxin productionrdquo FASEB Journal vol 20 no 14 pp 2591ndash2593 2006

[136] J Szebeni L Baranyi S Savay et al ldquoComplement activation-related cardiac anaphylaxis in pigs role of C5a anaphylatoxinand adenosine in liposome-induced abnormalities in ECG andheart functionrdquo The American Journal of Physiology vol 290no 3 pp H1050ndashH1058 2006

[137] D R Utkhede and C P Tilcock ldquoStudies upon the toxicity ofpolyethylene glycol coated lipid vesicles acute hemodynamiceffects pyrogenicity and complement activationrdquo Journal ofLiposome Research vol 8 no 4 pp 537ndash550 1998

[138] J K Gbadamosi A C Hunter and S M Moghimi ldquoPEGyla-tion of microspheres generates a heterogeneous population ofparticles with differential surface characteristics and biologicalperformancerdquo FEBS Letters vol 532 no 3 pp 338ndash344 2002

[139] A J Bradley S T Test K L Murad J Mitsuyoshi and M DScott ldquoInteractions of IgM ABO antibodies and complementwith methoxy-PEG-modified human RBCsrdquo Transfusion vol41 no 10 pp 1225ndash1233 2001


[140] K Taguchi Y Urata M Anraku et al ldquoHemoglobin vesiclespolyethylene glycol (PEG)ylated liposomes developed as a redblood cell substitute do not induce the accelerated blood clear-ance phenomenon in micerdquo Drug Metabolism and Dispositionvol 37 no 11 pp 2197ndash2203 2009

[141] H U Lutz P Stammler E Jelezarova M Nater and P JSpath ldquoHigh doses of immunoglobulin G attenuate immuneaggregate-mediated complement activation by enhancing phys-iologic cleavage of C3b in C3b(n)-IgG complexesrdquo Blood vol88 no 1 pp 184ndash193 1996

[142] E T M Dams W J G Oyen O C Boerman et al ldquo99mTc-PEG liposomes for the scintigraphic detection of infection andinflammation clinical evaluationrdquo Journal of Nuclear Medicinevol 41 no 4 pp 622ndash630 2000


[144] S M Moghimi A J Andersen D Ahmadvand P P WibroeT L Andresen and A C Hunter ldquoMaterial properties incomplement activationrdquo Advanced Drug Delivery Reviews vol63 no 12 pp 1000ndash1007 2011

[145] T Blunk M Luck A Calvor et al ldquoKinetics of plasma proteinadsorption on model particles for controlled drug deliveryand drug targetingrdquo European Journal of Pharmaceutics andBiopharmaceutics vol 42 no 4 pp 262ndash268 1996

[146] I Hamad O Al-Hanbali A C Hunter K J Rutt T LAndresen and S M Moghimi ldquoDistinct polymer architecturemediates switching of complement activation pathways at thenanosphere-serum interface implications for stealth nanopar-ticle engineeringrdquoACSNano vol 4 no 11 pp 6629ndash6638 2010

[147] M Luck W Schroder S Harnisch et al ldquoIdentificationof plasma proteins facilitated by enrichment on particulatesurfaces analysis by two-dimensional electrophoresis and N-terminal microsequencingrdquo Electrophoresis vol 18 no 15 pp2961ndash2967 1997


[149] D L GordonGM Johnson andMKHostetter ldquoCharacteris-tics of iC3b binding to human polymorphonuclear leucocytesrdquoImmunology vol 60 no 4 pp 553ndash558 1987

[150] J B Cornacoff L A HebertW L SmeadM E VanAman D JBirmingham and F J Waxman ldquoPrimate erythrocyte-immunecomplex-clearing mechanismrdquo Journal of Clinical Investigationvol 71 no 2 pp 236ndash247 1983

[151] S M Moghimi ldquoHumoral-mediated recognition of ldquophagocyteresistantrdquo beads by lymph node macrophages of poloxamine-treated ratsrdquo Clinical Science vol 95 no 3 pp 389ndash391 1998

[152] S Zalipsky ldquoFunctionalized poly(ethylene glycol) for prepara-tion of biologically relevant conjugatesrdquo Bioconjugate Chem-istry vol 6 no 2 pp 150ndash165 1995

[153] C Monfardini and F M Veronese ldquoStabilization of substancesin circulationrdquo Bioconjugate Chemistry vol 9 no 4 pp 418ndash450 1998

[154] N Vij T Min R Marasigan et al ldquoDevelopment of PEGylatedPLGA nanoparticle for controlled and sustained drug deliveryin cystic fibrosisrdquo Journal of Nanobiotechnology vol 8 article22 2010

[155] J Park P M Fong J Lu et al ldquoPEGylated PLGA nanoparticlesfor the improved delivery of doxorubicinrdquo Nanomedicine vol5 no 4 pp 410ndash418 2009

[156] A L Klibanov K Maruyama V P Torchilin and L HuangldquoAmphipathic polyethyleneglycols effectively prolong the circu-lation time of liposomesrdquo FEBS Letters vol 268 no 1 pp 235ndash237 1990


[158] K Kostarelos and A D Miller ldquoSynthetic self-assembly ABCDnanoparticles a structural paradigm for viable synthetic non-viral vectorsrdquo Chemical Society Reviews vol 34 no 11 pp 970ndash994 2005

[159] S R Wan Y Zheng Y Q Liu H S Yan and K L LiuldquoFe3O4nanoparticles coated with homopolymers of glycerol

mono(meth)acrylate and their block copolymersrdquo Journal ofMaterials Chemistry vol 15 no 33 pp 3424ndash3430 2005

[160] Z Li L Wei M Gao and H Lei ldquoOne-pot reaction tosynthesize biocompatible magnetite nanoparticlesrdquo AdvancedMaterials vol 17 no 8 pp 1001ndash1005 2005

[161] Y Zhang N Kohler and M Zhang ldquoSurface modification ofsuperparamagnetic magnetite nanoparticles and their intracel-lular uptakerdquo Biomaterials vol 23 no 7 pp 1553ndash1561 2002

[162] C Boyer V Bulmus P Priyanto W Y Teoh R Amal and TP Davis ldquoThe stabilization and bio-functionalization of ironoxide nanoparticles using heterotelechelic polymersrdquo Journal ofMaterials Chemistry vol 19 no 1 pp 111ndash123 2009

[163] U I Tromsdorf N C Bigall M G Kaul et al ldquoSize and surfaceeffects on the MRI relaxivity of manganese ferrite nanoparticlecontrast agentsrdquo Nano Letters vol 7 no 8 pp 2422ndash2427 2007

[164] M Ji W Yang Q Ren and D Lu ldquoFacile phase transfer ofhydrophobic nanoparticles with poly(ethylene glycol) graftedhyperbranched poly(amido amine)rdquo Nanotechnology vol 20no 7 Article ID 075101 2009

[165] E KU Larsen T Nielsen TWittenborn et al ldquoSize-dependentaccumulation of pegylated silane-coated magnetic iron oxidenanoparticles in murine tumorsrdquo ACS Nano vol 3 no 7 pp1947ndash1951 2009

[166] C Barrera A P Herrera and C Rinaldi ldquoColloidal disper-sions of monodisperse magnetite nanoparticles modified withpoly(ethylene glycol)rdquo Journal of Colloid and Interface Sciencevol 329 no 1 pp 107ndash113 2009

[167] E K Lim J Yang M Y Park et al ldquoSynthesis of watersoluble PEGylated magnetic complexes using mPEG-fatty acidfor biomedical applicationsrdquoColloids and Surfaces B vol 64 no1 pp 111ndash117 2008

[168] H B Na I S Lee H Seo et al ldquoVersatile PEG-derivatizedphosphine oxide ligands for water-dispersible metal oxidenanocrystalsrdquoChemical Communications no 48 pp 5167ndash51692007

[169] J Xie C Xu N Kohler Y Hou and S Sun ldquoControlledPEGylation of monodisperse Fe

3O4nanoparticles for reduced

non-specific uptake by macrophage cellsrdquo Advanced Materialsvol 19 no 20 pp 3163ndash3166 2007

[170] F Hu K G Neoh L Cen and E T Kang ldquoCellular response tomagnetic nanoparticles ldquoPEGylatedrdquo via surface-initiated atomtransfer radical polymerizationrdquo Biomacromolecules vol 7 no3 pp 809ndash816 2006


[171] Q L Fan K G Neoh E T Kang B Shuter and S C WangldquoSolvent-free atom transfer radical polymerization for thepreparation of poly(poly(ethyleneglycol) monomethacrylate)-grafted Fe

3O4nanoparticles synthesis characterization and

cellular uptakerdquo Biomaterials vol 28 no 36 pp 5426ndash54362007

[172] S Wang Y Zhou S Yang and B Ding ldquoGrowing hyper-branched polyglycerols on magnetic nanoparticles to resistnonspecific adsorption of proteinsrdquoColloids and Surfaces B vol67 no 1 pp 122ndash126 2008

[173] L Wang K G Neoh E T Kang B Shuter and S C WangldquoSuperparamagnetic hyperbranched polyglycerolgrafted Fe

3O4

nanoparticles as a novel magnetic resonance imaging contrastagent an in vitro assessmentrdquo Advanced Functional Materialsvol 19 no 16 pp 2615ndash2622 2009

[174] L M Bronstein S N Sidorov A Y Gourkova et al ldquoInter-action of metal compounds with ldquodouble-hydrophilicrdquo blockcopolymers in aqueous medium and metal colloid formationrdquoInorganica Chimica Acta vol 280 no 1-2 pp 348ndash354 1998

[175] D Shenoy W Fu J Li et al ldquoSurface functionalization of goldnanoparticles using hetero-bifunctional poly(ethylene glycol)spacer for intracellular tracking and deliveryrdquo InternationalJournal of Nanomedicine vol 1 no 1 pp 51ndash57 2006

[176] B C Mei K Susumu I L Medintz and H MattoussildquoPolyethylene glycol-based bidentate ligands to enhance quan-tum dot and gold nanoparticle stability in biological mediardquoNature Protocols vol 4 no 3 pp 412ndash423 2009

[177] A S Karakoti S Das S Thevuthasan and S Seal ldquoPEGylatedinorganic nanoparticlesrdquo Angewandte ChemiemdashInternationalEdition vol 50 no 9 pp 1980ndash1994 2011

[178] M T Peracchia ldquoStealth nanoparticles for intravenous admin-istrationrdquo STP Pharma Sciences vol 13 no 3 pp 155ndash161 2003

[179] J C Y Kah K Y Wong K G Neoh et al ldquoCritical parametersin the pegylation of gold nanoshells for biomedical applicationsan in vitro macrophage studyrdquo Journal of Drug Targeting vol 17no 3 pp 181ndash193 2009

[180] G F Schneider V Subr K Ulbrich and G Decher ldquoMultifunc-tional cytotoxic stealth nanoparticles A model approach withpotential for cancer therapyrdquoNano Letters vol 9 no 2 pp 636ndash642 2009

[181] G Prencipe S M Tabakman K Welsher et al ldquoPEG branchedpolymer for functionalization of nanomaterials with ultralongblood circulationrdquo Journal of the American Chemical Societyvol 131 no 13 pp 4783ndash4787 2009

[182] D Miyamoto M Oishi K Kojima K Yoshimoto and YNagasaki ldquoCompletely dispersible PEGylated gold nanopar-ticles under physiological conditions modification of goldnanoparticles with precisely controlled PEG-b-polyaminerdquoLangmuir vol 24 no 9 pp 5010ndash5017 2008

[183] H Du P D Hamilton M A Reilly A drsquoAvignon P Biswasand N Ravi ldquoA facile synthesis of highly water-soluble core-shell organo-silica nanoparticles with controllable size via sol-gel processrdquo Journal of Colloid and Interface Science vol 340no 2 pp 202ndash208 2009

[184] Q He J Zhang J Shi et al ldquoThe effect of PEGylation ofmesoporous silica nanoparticles on nonspecific binding ofserum proteins and cellular responsesrdquo Biomaterials vol 31 no6 pp 1085ndash1092 2010

[185] B Thierry L Zimmer S McNiven K Finnie C Barbe and HJ Griesser ldquoElectrostatic self-assembly of PEG copolymers ontoporous silica nanoparticlesrdquo Langmuir vol 24 no 15 pp 8143ndash8150 2008

[186] M Joubert C Delaite E Bourgeat-Lami and P Dumas ldquoHairyPEO-silica nanoparticles through surface-initiated polymeriza-tion of ethylene oxiderdquoMacromolecular RapidCommunicationsvol 26 no 8 pp 602ndash607 2005

[187] K G Neoh and E T Kang ldquoFunctionalization of inorganicnanoparticles with polymers for stealth biomedical applica-tionsrdquo Polymer Chemistry vol 2 no 4 pp 747ndash759 2011


Review ArticleBisphosphonates and CancerWhat Opportunities from Nanotechnology

Giuseppe De Rosa1 Gabriella Misso2 Giuseppina Salzano1 and Michele Caraglia2

1 Department of Pharmacy Universita degli Studi di Napoli Federico II Via Domenico Montesano 49 8013 Naples Italy2 Department of Biochemistry Biophysics and General Pathology Seconda Universita degli Studi di NapoliVia Costantinopoli 16 80138 Naples Italy


Received 4 December 2012 Accepted 22 January 2013



Bisphosphonates (BPs) are synthetic analogues of naturally occurring pyrophosphate compoundsThey are used in clinical practiceto inhibit bone resorption in bonemetastases osteoporosis and Pagetrsquos disease BPs induce apoptosis because they can bemetabol-ically incorporated into nonhydrolyzable analogues of adenosine triphosphate In addition the nitrogen-containing BPs (N-BPs)second-generation BPs act by inhibiting farnesyl diphosphate (FPP) synthase a key enzyme of the mevalonate pathway Thesemolecules are able to induce apoptosis of a number of cancer cells in vitro Moreover antiangiogenic effect of BPs has also beenreported However despite these promising properties BPs rapidly accumulate into the bone thus hampering their use to treatextraskeletal tumors Nanotechnologies can represent an opportunity to limit BP accumulation into the bone thus increasing druglevel in extraskeletal sites of the body Thus nanocarriers encapsulating BPs can be used to target macrophages to reduce angio-genesis and to directly kill cancer cell Moreover nanocarriers can be conjugated with BPs to specifically deliver anticancer agent tobone tumorsThis paper describes in the first part the state-of-art on the BPs and in the following part the main studies in whichnanotechnologies have been proposed to investigate new indications for BPs in cancer therapy

1 The Bisphosphonates

Bisphosphonates (BPs) synthetic analogues of naturallyoccurring pyrophosphate compounds represent the treat-ment of choice for different diseases such as metabolic bonedisease osteoporosis Pagetrsquos disease and bonemetastases [1]In the 1960s Fleisch et al proposed that inorganic pyrophos-phate a naturally occurring polyphosphate and a knownproduct of many biosynthetic reactions in the body mightbe the bodyrsquos own natural ldquowater softenerrdquo that normallyprevents calcification of soft tissues and regulates bone min-eralization by binding to newly forming crystals of hydrox-yapatite [2 3] It subsequently became clear that calcifica-tion disorders might be linked to disturbances in inorganicpyrophosphate (PPi)metabolism [2 3] Alkaline phosphatasepresent in bone destroys pyrophosphate locally therebyallowing amorphous phase calcium phosphate to crystallizeand inducingmineralization of bone [2]Themajor limitation

of pyrophosphate is that when orally administered it isinactive because of its hydrolysis in the gastrointestinal tractDuring the search for more stable analogues of pyrophos-phate that might also have the antimineralization propertiesof pyrophosphate but would be resistant to hydrolysis severaldifferent chemical classes were studied The bisphosphonates(at that time called diphosphonates) characterized by PndashCndashP motifs were among these classes [1ndash4] The fundamentalproperty of BPs which has been exploited by industry andmedicine is their ability to form bonds with crystal surfacesand to form complexes with cations in solution or at asolid-liquid interface Since BPs are synthetic analogues ofpyrophosphates they have the same chemical activity butgreater stability [1ndash4] Like pyrophosphates BPs had highaffinity for bone mineral and they were found to preventcalcification both in vitro and in vivo but unlike pyrophos-phate they were also able to prevent experimentally inducedpathologic calcification when given orally to rats in vivo This


property of being active orally was key to their subsequent usein humans [4] Perhaps the most important step toward thesuccessful use of BPs occurred when their ability to inhibithydroxyapatite crystals dissolution was demonstrated Thisfinding led to following studies designed to determine if theymight also inhibit bone resorption [5] The clarification ofthis property made BPs the most widely used and effectiveantiresorptive agents for the treatment of diseases in whichthere was an increase in the number or activity of osteoclastsincluding tumor-associated osteolysis and hypercalcemia [6]After more than three decades of research first- second-and third-generation bisphosphonates have been developedChanges in chemical structure have resulted in increasedpotency without demineralization of bone [1] There is nowa growing body of evidence regarding the efficacy of thesedrugs in clinical settings All BPs that act significantly on theskeleton are characterized as stated above by PndashCndashP bond(Figure 1(a)) in contrast to pyrophosphate which has a PndashOndashP bond (Figure 1(b))

This peculiarity confers stability both to heat and to mostchemical reagents and is one of the most important prop-erties of these compounds [4] Extensive chemical researchprograms have produced a wide range of molecules withvarious substituents attached to the carbon atom Variationsin potency and in the ability of the compounds to bind tocrystals in bone one determined by the chemical and three-dimensional structure of the two side chains R

1and R

2

attached to the central geminal carbon atom [1ndash4]Thebioac-tive moiety comprising the R

2chain of the molecule is con-

sidered primarily responsible for BPsrsquo effect on resorptionand small changes in this part of the structure can resultin large differences in their antiresorptive potencies [4] Theuptake and binding to bone mineral is determined by thebi- or tridentate ligand (hydroxybisphosphonate) of themolecule which is also thought to be responsible for thephysicochemical effects the most important being the inhi-bition of growth of calcium crystalsThemost effective struc-tures for binding to bone mineral consist of the two phos-phonate groups attached to the central carbon and the sub-stitution at R

1with a hydroxyl or amino group that provides

tridentate binding [4] In fact the addition of a hydroxyl(OH) or primary amino (NH

2) group increases the affinity

for calcium ions resulting in preferential localization of thesedrugs to sites of bone remodelling Increasing the number ofcarbon atoms in the side chain initially increases and thendecreases the magnitude of the effect on bone resorption [1ndash4] The early compounds clodronate (CLO) and etidronate(ETI) contained simple substituents (H OH Cl CH

3) and

lacked a nitrogen atom (Figure 2)Subsequently more complex and potent compounds

were produced by the insertion of a primary secondary ortertiary nitrogen function in the R

2side chain for example

pamidronate (PAM) alendronate (ALN) ibandronate (IBA)and incadronate (INC) which have an alkyl R

2side chain

or risedronate (RIS) zoledronate (ZOL) and minodronate(MIN) which have heterocyclic rings in the R

2side chain

(Figure 2) Variation of the substituents modulates the phar-macologic properties and gives each molecule its uniqueprofile [7]

OOH

OH

OH

OH

P

O

O

P

Inorganic pyrophosphate

(a)

OH

OH

OH

OH

P

C

PR1

R2

O

O

Geminal bisphosphonate

(b)

Figure 1 Structures (a) and (b) show the basic structures of inor-ganic pyrophosphate and geminal bisphosphonate respectivelywhere R

1and R

2represent different side chains for each bisphos-

phonate

2 Intracellular Effect and Pharmacodynamicsof Bisphosphonates

Extensive structureactivity studies have resulted in severalvery useful drugs that combine potent inhibition of osteo-clastic bone resorption with good clinical tolerability [5ndash8] The pronounced selectivity of BPs for bone rather thanother tissues is the basis for their value in clinical practiceThe antiresorptive effect cannot be accounted simply byadsorption of BPs to bone mineral and prevention of hydrox-yapatite dissolution It became clear that BPs must inhibitbone resorption by cellular effects on osteoclasts rather thansimply by physicochemical mechanisms [5] Bisphosphonatemoiety and R

1group are both essential for hydroxyapatite

affinity [8] The BPs bind to hydroxyapatite crystals in thearea of osteoclast-mediated bone erosion during resorptionthe dissolution of hydroxyapatite crystals by osteoclast deter-mines the consequent release of the bisphosphonate that mayindeed come into contact with osteoclasts and inhibit theirabsorption capacity [8] Incorporation of an aminoalkyl sidechain at R

2increases antiresorptive potency by 10-fold also

the length of carbon chain is important (alendronate is about1000-fold more potent than etidronate while pamidronate isonly 100-fold more active than etidronate) [4 8] In additionincorporation of a nitrogen heterocycle (third-generationagents) further enhances antiresorptive potency the mostactive compound in this class is ZOL a BP containing an imi-dazole ring which is up to 10000-fold more potent than bothCLO and ETI in some experimental systems During boneresorption BPs are probably internalized by endocytosisalong with other products of resorption [4 8] Many studieshave shown that BPs can affect osteoclast-mediated boneresorption in a variety of ways including effects on osteoclastrecruitment differentiation and resorptive activity and mayinduce apoptosis [7] Because mature multinucleated osteo-clasts are formed by the fusion of mononuclear precursors ofhematopoietic origin BPs could also inhibit bone resorptionby preventing osteoclast formation in addition to affectingmature osteoclasts In vitro BPs can inhibit dose-dependentlythe formation of osteoclast-like cells in long-term cultures of


Cl

Cl

OOH

OHOH

OH

P

O

O

Clodronate(CLO)

HO

P

P

O

OH

OH

OH

OH

Etidronate(ETI)

1st generation

P

H3C

OOH

OHOH

OH

P

O

HO P

OH

OHOH

OH

P

O

O

HO P

N

OH

OHOH

OH

H2N

Alendronate(ALN)

Ibandronate(IBA)

Pamidronate(PAM)

CH3P

PHO

H2N

O

O

2nd generation

OOH

OHOH

OH

P

O

HO P

OH

OHOH

OH

N

P

PHO

N

N

N

NHO

O

O

OH

OHOH

OH

P

P

O

O

Zoledronate(ZOL)

Minodronate(MIN)

Risedronate(RIS)

3rd generation

Figure 2 Structures of simple bisphosphonates (1st generation) N-BPs with primary secondary or tertiary nitrogen function in the R2alkyl

side chain (2nd generation) and N-BPs with heterocyclic rings in the R2side chain (3rd generation)

human bone marrow [7] In organ culture also some BPscan inhibit the generation of mature osteoclasts possibly bypreventing the fusion of osteoclast precursors [5] In contrastto their ability to induce apoptosis in osteoclasts which con-tributes to the inhibition of resorptive activity some exper-imental studies suggest that BPs may protect osteocytes andosteoblasts from apoptosis induced by glucocorticoids [9]

Since the early 1990s there has been a systematic effortto elucidate the molecular mechanisms of action of BPs andnot surprisingly it has been found that they could be dividedinto 2 structural subgroups [10 11] The first group comprisesthe nonnitrogen-containing bisphosphonates such as CLOand ETI that perhaps most closely resemble pyrophosphateThese can be metabolically incorporated into nonhydrolyz-able analogues of adenosine triphosphate (ATP) methylene-containing (AppCp) nucleotides by reversing the reactions

of aminoacyl-transfer RNA synthetases [12] The resultingmetabolites contain the PndashCndashP moiety in place of the 120573120574-phosphate groups of ATP [13] Intracellular accumulation ofthese metabolites within osteoclasts inhibits their functionand may cause osteoclast cell death most likely by inhibitingATP-dependent enzymes such as the adenine nucleotidetranslocase a component of the mitochondrial permeabilitytransition pore [14] Induction of osteoclast apoptosis seemsto be the primary mechanism by which the simple BPsinhibit bone resorption since the ability of CLO and ETI toinhibit resorption in vitro can be overcome when osteoclastapoptosis is prevented using a caspase inhibitor [15]

In contrast the second group comprising the nitro-gen-containing bisphosphonates (N-BPs) which are sev-eral orders of magnitude more potent at inhibiting boneresorption in vivo than the simple bisphosphonates is not


Prenylation

Mevalonic acid

FPPS

Cholesterol

Geranyl-Geranyl-PP

GGTase

Ras

Ras

Ras

FTase

HMG-CoA

Farnesyl-PP C-CH3

-A-A-X

C-A-A-X

C-A-A-X

CH3

Zoledronic acid

Figure 3 Isoprenoids are synthesized from the mevalonate pathway that starts from reaction catalyzed by the 3-hydroxy-3-methylglutarylCoA (HMG-CoA) reductase (the rate-limiting reaction in cholesterol biosynthesis) which catalyzes the conversion of HMG-CoA tomevalonic acid The pathway triggered by this reaction can lead to the synthesis of a key isoprenoid molecule the farnesyl-pyrophosphate(Farnesyl-PP) whose formation is catalyzed by the farnesylpyrophosphate synthase (FPPS) Farnesyl-PP can be either converted by a series ofreactions in cholesterol or can be transferred on target cellular proteins as Farnesyl-PP itself (reaction catalyzed by farnesyltransferase FTase)or firstly converted in geranyl-geranyl-pyrophosphate (Geranyl-Geranyl-PP) and then transferred on cellular proteins by type I or typeII geranylgeranyl-transferase (GGTase) FTase and GGTase-I catalyze the prenylation of substrates with a carboxy-terminal tetrapeptidesequence called a CAAX box where C refers to cysteine A refers to an aliphatic residue and X typically refers to methionine serine alanineor glutamine for FTase or to leucine for GGTase-I Following prenylation of physiological substrates the terminal three residues (AAX) aresubsequently removed by aCAAXendoprotease and the carboxyl group of the terminal cysteine ismethyl esterified by amethyltransferase Atthismoment prenyl substrates such as Ras are ready to be located on the inner side of the biologicalmembranes to receive signalsmediated byexternal factors ZOL specifically inhibits the FPPS activity required for the synthesis of farnesyl and geranylgeranyl lipidic residues blockingprenylation of Ras that regulates the proliferation invasive properties and proangiogenic activity of human tumour cells

metabolized to toxic analogues of ATP [16] N-BPs act byinhibiting farnesyl diphosphate (FPP) synthase a key enzymeof the mevalonate pathway (Figure 3)

This enzyme is inhibited by nanomolar concentrations ofN-BPs ZOL and the structurally similar MIN are extremelypotent inhibitors of FPP synthase [6] and inhibit the enzymeeven at picomolar concentrations Importantly studies withrecombinant human FPP synthase revealed that minor mod-ifications to the structure and conformation of the R

2side

chain that are known to affect antiresorptive potency alsoaffect the ability to inhibit FPP synthase These studiesstrongly suggest that FPP synthase is the major pharmaco-logic target of N-BPs in osteoclasts in vivo and help to explainthe relationship between bisphosphonate structure andantiresorptive potency [6] Clinical and experimental evi-dence indicates that N-BPs suppress the progression of bonemetastases and recent observations suggest that this effectmay be independent of the inhibition of bone resorption [17]

Tumour progression and metastasis formation are criticallydependent on tumour angiogenesis [18] Antiangiogenictreatments suppress tumour progression in animal modelsand many antiangiogenic substances are currently beingtested in clinical trials for their therapeutic efficacy againsthuman cancer [19] Recent research indicates that ZOL pos-sesses antiangiogenic activities [20]

The exact mechanism by which N-BPs inhibit FPP syn-thase is only just becoming clear The recent generation ofX-ray crystal structures of the human FPP synthase enzymecocrystallized with RIS or ZOL [51] revealed that N-BPsbind the geranyl diphosphate (GPP) binding site of theenzyme with stabilizing interactions occurring between thenitrogen moiety of the N-BP and a conserved threonineand lysine residue in the enzyme Enzyme kinetic analysiswith human FPP synthase indicates that the interaction withN-BPs is highly complex and characteristic of ldquoslow tightbindingrdquo inhibition [51] By inhibiting FPP synthase N-BPs


prevent the synthesis of FPP and its downstream metabo-lite geranylgeranyl diphosphate [11] These isoprenoid lipidsare the building blocks for the production of a variety ofmetabolites such as dolichol and ubiquinone but are alsorequired for posttranslational modification (prenylation) ofproteins including small GTPases [11] The loss of synthesisof FPP and geranylgeranyl diphosphate therefore prevents theprenylation at a cysteine residue in characteristic C-terminalmotifs of small GTPases such as Ras Rab Rho and Rac(Figure 3) Small GTPases are important signaling proteinsthat regulate a variety of cell processes important forosteoclast function including cell morphology cytoskeletalarrangement membrane ruffling trafficking of vesicles andapoptosis Prenylation is required for the correct function ofthese proteins because the lipid prenyl group serves to anchorthe proteins in cell membranes and may also participate inprotein-protein interactions [3 20]

3 Pharmacokinetics of Bisphosphonates

Recent studies with a fluorescently labelled bisphosphonatehave shown that macrophages and osteoclasts internalizebisphosphonates into membrane-bound vesicles by fluid-phase endocytosis endosomal acidification then seems to beabsolutely required for exit of bisphosphonate from vesiclesand entry into the cytosol [52] This mechanism of uptakesuggests that large amounts of N-BP is in intracellular vesiclesbut probably only very small amounts of bisphosphonate thenenter in the cytosol or in other organelles for inhibition of FPPsynthase Even though the relatively poor uptake of bispho-sphonates into the cytosol is overcome by their extremelypotent inhibition of FPP synthase [6 11] Bisphosphonates arepoorly absorbed in the intestine due to their negative chargehindering their transport across the lipophilic cellmembranethey are therefore givenmainly intravenously A pharmacoki-netic evaluation of ZOL for treatment of multiple myelomaand bonemetastases carried out by Ibrahim et al exhibited athree-compartment model [53] The distribution half-life (120572-11990512

) was 14min followed by a 120573-phase of 19 h A prolongedterminal phase with a half-life of at least 146 hmight indicatea slow release of ZOL from the bone back into the plasmaZOL pharmacokinetics were dose proportional from 2 to16mg based on peak plasma concentration (119862max) and areaunder the curve (AUC

24 h) ZOL dosed every 21 days didnot demonstrate significant plasma accumulation In vitrostudies indicated that 22 of ZOL is protein bound Theexcretion of ZOL was primarily renal Approximately 40of the radiolabeled ZOL dose was recovered in urine within24 h Only traces of ZOL were observed in the urine after twodays suggesting a prolonged period of ZOL binding to bonePopulation modeling described the ZOL clearance as a func-tion of creatinine clearance On the basis of a comparison ofAUC24 h patients with mild or moderate renal impairment

had 15 and 43 higher exposure respectively than patientswith normal renal function However no significant relation-ship between ZOL exposure (AUC) and adverse events mightbe established The use of ZOL in patients with severe renalfailure was not recommended In vitro studies showed no

inhibition of or metabolism by cytochrome P-450 enzymes[53]

One of the most important limits of N-BPs which makesthe direct anticancer activity difficult to demonstrate in vivois just their pharmacokinetic profile This issue is demon-strated by also other pharmacological studies performed ondifferent N-BPs In fact after intravenous administration(4mg over 15min) of ZOL an immediate increase of itsconcentration in peripheral blood was recorded as shownby estimations of the early distribution and elimination ofthe drug which resulted in plasma half-lives of the drug ofabout 15min (119905

12120572) and of 105min (119905

12120573) respectively The

maximum plasma concentration (119862max) of ZOL was about1 120583M that was from 10- to 100-fold less than that requiredin in vitro studies to induce apoptosis and growth inhibitionin tumour cell lines while the concentrations required foranti-invasive effects were in the range of those achieved afterin vivo administration Moreover approximately 55 of theinitially administered dose of the drug was retained in theskeleton and was slowly released back into circulation result-ing in a terminal elimination half-life (119905

12120574) of about 7 days

[54 55] Other studies performed on ALN demonstrate thatN-BP concentration in noncalcified tissues declined rapidlyat 1 h (5 of the initial concentration) On the other hand itsconcentration in the bone continuously increased reachingits peak at 1 h demonstrating that a significant redistributionof the drug from noncalcified tissues to bone occurred Thedrug was retained in bone tissue for a long time and wasslowly released into plasma with a terminal half-life of about200 days [56] Similar data were obtained with IBA and ZOL[54ndash57] demonstrating that long-lasting accumulation inbone is a common feature of N-BPs The rapid redistributionof N-BPs results not only in a short exposure of noncalcifiedtissues to the drug but also in a prolonged accumulation inbone where N-BPs can also reach higher and tumoricidalconcentrations These considerations explain the relativeefficacy of N-BPs on tumours placed in bone tissues [20] Inbiodistribution studies by Weiss et al performed in rats anddogs administered with single or multiple intravenous dosesof 14C-labeled ZOL its levels rapidly decreased in plasmaand noncalcified tissue but higher levels persisted in boneand slowly diminished with a half-life of approximately 240days In contrast the terminal half-lives (50 to 200 days)were similar in bone and noncalcified tissues consistent withZOL rapidly but reversibly binding to bone being rapidlycleared from the plasma and then slowly released frombone surfaces back into circulation over a longer time Theresults suggested that a fraction of ZOL is reversibly takenup by the skeleton the elimination of drug is mainly byrenal excretion and the disposition in blood and noncalcifiedtissue is governed by extensive uptake into and slow releasefrom bone [58] It is important to consider that ZOL is nottaken up by tumor cells but prevalently by cells with increasedendocytosis processes such as osteoclasts and macrophagesHowever owing to the intrinsic pharmacokinetics limitationsof ZOL more efforts were required to increase the anticanceractivity of both this drug and the other members of N-BPsfamily


4 Bisphosphonate and CancerIn Vitro Studies

FPP synthase is a highly conserved ubiquitous enzymetherefore N-BPs have the potential to affect any cell type invitro Among BPs recent advances suggest that ZOL beyondthe strongest activity of antibone resorption has directanticancer effects In fact extensive in vitro preclinical studiessupport that ZOL can inhibit tumor cell adhesion to extra-cellular matrix proteins thereby impairing the process oftumour-cell invasion and metastasis [59] moreover it wasdemonstrated that ZOL has a direct effect on angiogenesis invitro [60 61] and an in vitro stimulation of 120574120575T lymphocyteswhich play important roles in innate immunity against cancer[62] One of the crucial mechanisms responsible for theantitumor activity of ZOL is the induction of tumor cellapoptosis [63]

Inhibition of protein prenylation by N-BPs can be shownby measuring the incorporation of 14C mevalonate intofarnesylated and geranylgeranylated proteins [64] The mostpotent FPP synthase inhibitor ZOL almost completelyinhibits protein prenylation in J774 cells at a concentration of10 120583molL which is similar to the concentration that affectsosteoclast viability in vitro [65] Alternatively the inhibitoryeffect of N-BPs on the mevalonate pathway can be shownby detecting accumulation of the unprenylated form of thesmall GTPase Rap1A which acts as a surrogate marker forinhibition of FPP synthase and which accumulates in cellsexposed to N-BPs Roelofs et al have shown the abilityof N-BPs to inhibit the prenylation of Rap1A in a widerange of cultures of different types of primary cells and celllines such as osteoclasts osteoblasts macrophages epithelialand endothelial cells and breast myeloma and prostatetumor cells [16] Macrophages and osteoclasts were the mostsensitive to low concentrations of N-BPs (1ndash10120583M) in vitroMoreover treatment with 100 120583M N-BP caused a detectableaccumulation of unprenylated Rap1A already after few hoursConcerning myeloma cells in order to detect the unpreny-lated form of Rap1A longer times of in vitro treatments andhigher concentrations were required [16]

BPs have also been shown to inhibit adhesion of tumorcells to extracellular matrix (ECM) proteins and to pro-mote invasion and metastasis Inhibition of the mevalonatepathway and induction of caspase activity are importantmechanisms in explaining the inhibitory effects of N-BPs ontumor cells adhesion to the ECM and on invasiveness [66]In vitro findings have demonstrated that N-BPs particularlyZOL can affect endothelial cells exerting a suppressive effecton angiogenesis [67 68] In fact N-BPs inhibit the expressionof vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) that induce the proliferationof endothelial cells and enhance the formation of capillary-like tubes

Recent evidence suggests that ZOL is a potent inducer ofapoptosis in several cancer cell types [69] It has recently beendemonstrated in vitro that N-BPs PAM and ZOL induceapoptosis and growth inhibition in human epidermoid cancercells together with depression of Ras-dependent Erk andAkt survival pathways These effects occurred together with

poly(ADP-ribose) polymerase (PARP) fragmentation and theactivation of caspase 3 [70] Moreover the latter seems to beessential for apoptosis induced byN-BPs in this experimentalmodel Furthermore it was reported that ZOL inducedgrowth inhibition on both androgen-dependent LnCaP andandrogen-independent PC3 prostate cancer cell lines withG1 accumulation Recent studies showed that the effects ofZOL were caspase dependent In human breast cancer celllines ZOL induced a modulating expression of Bcl-2 andsubsequent caspase 3 activation These events might beprecipitated by inhibition of Ras activation which requiresprotein farnesylation [71]

In human colon carcinoma HCT-116 cells ZOL stronglyinhibited the proliferation paralleled by a G1 cell cycleaccumulation and induction of apoptosis via a caspase-dependent mechanism [72] Recent studies by Fujita et aldemonstrated the involvement of the mevalonate pathwayin the antiproliferative and proapoptotic effects of ZOL onACHN renal cell carcinoma cells [73]

The sensitivity of different cell types to N-BPs mostlikely depends largely on their ability to internalize sufficientamounts of N-BPs to inhibit FPP synthase In view of thepharmacokinetic concerns that limit the anticancer activity ofZOL in the last decade the scientists have defined a series ofpharmacological and molecular strategies Some approachwas represented by the design of rationale-based drug combi-nations and the improvement of the pharmacokinetic profileEvidence from both in vitro and in vivo models indicated asynergistic antitumor activity of N-BPs when used in com-bination with either cytotoxic drugs or targeted moleculartherapies [69] Based on the relevance of the farnesylationinhibitory effects on antitumour activity of N-BPs the farne-syl transferase inhibitor (FTI) R115777was used together withPAM or ZOL and the effects of the combination treatmenton growth inhibition and apoptosis were evaluated N-BPsand FTI given in combination were strongly synergistic [70]Notably low concentrations of FTI induced a strong increaseof Ras expression with only a moderate reduction of Rasactivity that was on the other hand significantly reducedby the combined treatment [70] These data suggested thatescapemechanisms for the inhibition of isoprenylation of Rasmight be based on the geranylgeranylation or other prenylat-ing processes [74] The addition of farnesol to cells treatedwith the combination abolished the effects of the N-BPsFTIcombination on apoptosis and on the activity of the signalingmolecules suggesting that the synergistic growth-inhibitoryand proapoptotic effects produced by the N-BPsFTI combi-nation involved the inhibition of both Erk and Akt survivalpathways acting in these cells in a Ras-dependent fashion[70]

A synergistic interaction between R115777 and ZOL wasalso found on both androgen-independent PC3 and andro-gen-dependent LNCaP prostate cancer cell lines [70] and theeffectswere attributed to enhanced apoptosis and inactivationof Erk and Akt Several papers reported the significant cyto-static and cytotoxic effects of docetaxel (DTX) and ZOL onthe hormone- sensitive prostate cancer cell line LNCaP [1775 76] In details the highest inhibition of cell proliferationwas observed after DTX exposure and was already evident


at concentrations 200-fold lower than the plasma peak levelFabbri et al hypothesized the use of low DTX doses inconcomitance with and followed by a prolonged ZOL expo-sure to reduce the prostatic tumour cell population and torapidly induce eradication of hormone-resistant cells presentin hormone-responsive tumours without compromising theuse of conventional-dose DTX for the first-line treatmentfor hormone-sensitive prostate cancer The principal molec-ular mechanisms involved were found to be apoptosis anddecreased pMEK and Mcl-1 expression [77] FurthermoreKarabulut et al found that the combination treatment ofDTXand ZOL in hormone and drug refractory PC-3 and DU-145prostate cancer cells synergistically inhibited cell growth byinducing the apoptotic pathways through the downregulationof the antiapoptotic protein Bcl-2 [78]

A further strategy for the implementation of ZOL activ-ity is the interference of its molecular targets The recentanalysismdashperformed by cDNAmicroarray platformmdashof genemodulation induced by ZOL in androgen-resistant prostatePC3 cell line showed a significant dose- and time-dependentreduction of transcriptional activity of CYR61 after exposureto ZOL as demonstrated by the reduction of the transcrip-tional activity of Cyr61 promoter [79] This result is consid-ered of interest in designing new therapeutical approaches inandrogen-independent prostate cancer

5 Bisphosphonate and CancerIn Vivo Studies

In addition to the established in vitro induction of tumorcell apoptosis also emerging in vivo evidence supports N-BPs anticancer activity Preclinical studies support that ZOLdisplays an antitumor activity including direct antitumorin vivo effects such as inhibition of tumor cell adhesionto mineralized bone invasion and effects on angiogenesis(animal models) probably due to the modification of variousangiogenic properties of endothelial cells [59ndash61] effects onthe metastatic process (animal models) [60] stimulationof 120574120575 T lymphocytes in humans [62] N-BPs may targetseveral steps involved in the metastatic process extracellularmatrix extravasation into distant tissues angiogenesis andavoidance of immune surveillance [80]

Roelofs et al detected the unprenylated form of Rap1Ain osteoclasts purified from ALN-treated rabbits usingimmunomagnetic beads thereby showing that N-BPs inhibitprotein prenylation in vivo [16]

Many animal studies have focused on models of multiplemyeloma breast cancer and prostate cancer showing that thenewer N-BPs can significantly reduce the number and sizeof osteolytic lesions in tumor-bearing mice reduce skeletaltumor burden induce tumor cell apoptosis in bone lesionsreduce serum levels of tumormarkers and prevent formationof bone metastases [81ndash83]

A recent study utilizing a plasmacytoma xenograftmodelwithout complicating skeletal lesions demonstrated thattreatment with ZOL led to significant prolongation of sur-vival in severe combined immunodeficiency mice inocu-lated with human INA-6 plasma cells Following treatment

with ZOL histological analysis of tumors revealed extensiveareas of apoptosis associated with poly(ADP-ribose) poly-merase cleavage Furthermore western blot analysis of tumorhomogenates demonstrated the accumulation of unpreny-lated Rap1A indicative of the uptake of ZOL by nonskeletaltumors and inhibition of farnesyl pyrophosphate synthase[84]This is one of the few evidence of direct antitumor effectsof N-BPs in plasma cell tumors in vivo In fact it is generallybelieved that the reduction in tumor burden observed insome animal models may be due to inhibition of osteoclastactivity [85] For example bisphosphonates including IBAand ZOL acid were shown to inhibit the development ofosteolytic bone lesions in the 5T2MM model and alternativemodels of myeloma bone disease [86] Moreover the effectof bisphosphonates on the osteoclast stimulatory activity(OSA) was evaluated in the marrow of patients with multiplemyeloma For this purpose the effects of IBA treatmentprior to the development of bone disease were examinedin a murine model of human myeloma Sublethally irradi-ated severe combined immunodeficient (SCID) mice weretransplanted with ARH-77 cells on day 0 These ARH-77mice were treated daily with subcutaneous injections of N-BP started before or at different times after tumor injectionARH-77micewere sacrificed after they developed paraplegiaand the data demonstrated that early treatment of ARH-77micewith IBAprior to development ofmyeloma bone diseasedecreases OSA and possibly retards the development of lyticlesions but not eventual tumor burden [87] Numerousstudies in breast cancer models have also been reportedA study using MDA-MB-231 human breast tumour cellsinjected directly into the femoral artery of male athymic ratsalso showed that IBA (10 120583gkgday days 18 to 30) reducedthe extent of the osteolytic lesions [88] This study alsoprovided evidence that once tumours have reached a certainsize (gt6mm in this model) they become less dependenton the bone microenvironment for their further expansionand hence less sensitive to BP therapy A study by van derPluijm and colleagues showed that BPs modify tumourgrowth primarily through effects on bone rather thanthrough targeting tumour cells directly [89] MDA-231-Bluc+ breast cancer cells were implanted by intracardiacinjection and olpadronate given as a preventive (subcuta-neous 16 120583molkgday from 2 days before implantation) ora treatment (days 3 to 43) schedule Effects on the formationof new bone metastases and osteolysis were assessed as wellas tumour burden both inside and outside the bone mar-row cavity However the reduction in tumour growth wasonly transient and did not affect progression of establishedtumours Studies in a prostate cancermodel have also recentlybeen reported In those studies PC-3 and LuCaP cells wereinjected directly into the tibia of mice [81] PC-3 cells formosteolytic lesions and LuCaP cells form osteoblastic lesionsThe treatment group receiving ZOL (5 120583g sc twice weekly)either at the time of tumor cell injection or after tibial tumorswas established (7 days for PC-3 tumors and 33 days forLuCaP tumors) Treatment with ZOL significantly inhibitedgrowth of both osteolytic and osteoblastic metastases byradiographic analysis and also reduced skeletal tumor bur-den as evidenced by a significant decrease in serum levels of


prostate-specific antigen in animals bearing LuCaP tumorsThe observed reduction in serum prostate-specific antigenlevels provides compelling direct evidence of the antitumoractivity of ZOL in this animal modelThe potential of ZOL toprevent bone metastasis was also demonstrated in an animalmodel of prostate cancer [90]

In order to separate the direct antitumour effects of BPsfrom those mediated via bone the sequential or combinedtreatment with other antitumor agents were investigated

The synergistic interaction between R115777 and ZOL onboth androgen-independent PC3 and androgen-dependentLNCaP prostate cancer cell lines was also found to inducecooperative effects in vivo on tumour growth inhibition ofprostate cancer xenografts in nude mice with a significantsurvival increase [70]These in vivo and in vitro effectswere inboth cases attributed to enhanced apoptosis and inactivationof Erk and Akt

On the basis of preliminary results about sequence-dependent synergistic effects of ZOL and DTX combinationon growth inhibition and apoptosis of human prostate cancercells the closely related taxane paclitaxel (PTX) has shownsynergistic inhibitory activity with ZOL in animal modelsfor lung cancer Compared with vehicle and ZOL alonecancerous cells in the bone of mice treated with PTX + ZOLexpressed higher levels of Bax and lower levels of Bcl-2and Bcl-xl Moreover this drug combination produced asignificant reduction in serum n-telopeptide of type I colla-gen which levels correlate with the rate of bone resorptionThe results of this study indicated that ZOL enhanced theefficacy of PTX synergistically by reducing the incidence ofbone metastasis from lung cancer and prolonging survivalin a mouse model of nonsmall cell lung cancer with a highpotential for metastasis to bone [91]

Ottewell et al also showed that the treatment with ZOLafter exposure to doxorubicin (DOX) elicited substantial anti-tumor effects in amousemodel of breast cancer Interestinglythe treatment induced an increase in the number of caspase-3-positive cells paralleled by a decrease in the number oftumour cells positive for the proliferation marker Ki-67Moreover the sequential treatment with clinically relevantdoses of DOX followed by ZOL reduced intraosseous butnot extraosseous growth of breast tumours in mice injectedwith a clone of MDA-MB-231 [92]

The findings of synergy of interaction between ZOL andother agents could reduce the ZOL concentrations requiredfor antitumour activity and then could allow the achievementof its effective in vivo levels overcoming the limits associatedwith the pharmacokinetics of ZOL

Another strategy to potentiate the antitumor effects ofchemotherapeutic agents and ZOL could be also the admin-istration of the drugs at repeated low doses (ldquometronomicrdquoway) Santini et al recently demonstrated that weekly admin-istration of ZOL has higher antitumor effects as comparedwith conventional 3 weekly administration in nude micexenografted with breast cancer cells even if the total admin-istered dose is the same [93] Moreover a single dose of 1mgZOL is able to induce a significant reduction of circulatingVEGF in patients with bone metastases suggesting an in vivobiological activity of low ZOL concentrations in humans [93]

6 Nanotechnology and BPsMacrophage Targeting

Macrophages are the major differentiating cell of the mono-nuclear phagocyte system (MPS) They derive from mono-cytes that migrate from the peripheral blood to extravas-cular tissue where they differentiate into macrophages [94]Macrophages play a critical role in host defense becausethey migrated to an infected focus following attraction bya variety of substances such as components from bacteriacomplement components immune complexes and collagenfragments Once at the infected focus macrophages mayphagocytose and kill infectious agents by a variety of mech-anisms [95] Moreover following uptake of protein anti-gens macrophages generated immunogenic fragments acti-vating and regulating the immune response [96] Finallymacrophages infiltrate tumors where they represent animportant mechanism of host defense against tumor cellseither inhibiting tumor cell division or killing the cellsfollowing secretion of soluble mediators or by other means[97 98] However most tumors can be infiltrated by a differ-ent macrophage phenotype which provides an immunosup-pressive microenvironment for tumor growth Furthermorethese tumor-associated macrophages (TAM) secrete manycytokines chemokines and proteases which promote tumorangiogenesis growth metastasis and immunosuppression[99]

Thus due to their pivotal role in a number of physio-logical and pathological processes including tumors macro-phages represent an attractive target for therapy While in thecase of small soluble drug only a small fraction can reachthe macrophages these latter can be the preferential accu-mulation site for intravenously injected colloidal carriersIndeed once into the bloodstream plasma proteins adsorb onparticle surface and this process also named opsonizationfacilitates particle recognition and clearance from the bloodby circulating phagocytes as well as tissue macrophagesthat are in direct contact with the blood [100] Thus thelocalization of intravenously injected nanocarriers in cells ofthe mononuclear phagocytes system (MPS) offers a potentialand powerful method to target therapeutic agents to thesecells Nowadays various lipid and polymeric carriers such asliposomes and nanoparticle are under investigation to deliverdrugs to macrophages However nanocarrier characteristicsin terms of size shape and particle surface affect the phar-macokinetics of the nanocarrier and need to be carefully eval-uated when designing nanocarriers for macrophage target-ing Formore details the readers are directed tomore specificreviews on this theme for example an excellent review byMoghimi [100]

The powerful effect of BPs against osteoclasts suggestsa possible activity on cells with a common lineage such asthe macrophages However pharmacokinetics of BPs requiredelivery method to escape bone and to target macrophagesLiposomes encapsulating CLO were successfully used toachieve temporary macrophage depletion in the spleen [21]The authors demonstrated that once phagocytosed the lipo-somal membranes were disrupted by the phospholipases ofthe lysosomes and the drug is released into the cell Other


studies confirmed macrophage elimination from the spleenfollowing intravenous (iv) injection of CLO entrapped intoliposome by the absence of lysosomal acid phosphataseactivity [21 22] and surface markers of macrophages [23] aswell as by the absence of cells with the capacity to ingest andaccumulate carbon particles from the circulation [22] Ultra-structural studies also confirmed that macrophages not onlylose some of their functional characteristics but are also phys-ically removed from the circulation [26] Growth inhibitionof macrophages-like cells by using liposomes encapsulatingBP was also confirmed with other BPs namely PAM andETI on RAW 264 and CV1 cells [24] In this study free BPswere found to be even 1000 times less active compared withthe corresponding liposome-based formulations Interest-ingly the use of high calcium extracellular concentrationresulted in a stronger macrophage depletion suggesting therole of calcium to mediate BP cell uptake [24 27] The lipo-some type affected macrophage depletion which was higherwhen using negatively charged unilamellar liposomes [27]however this effect was found only in the case of CLOand ETI but not in the case of PAM Finally the use ofcalciumbisphosphonate complex was found to lead to anenhanced uptake into cells but not to an inhibitory effecton the cytokine production by macrophages [27] BP-encapsulating liposomes when intravenously administeredled to elimination of macrophages from spleen and liver [25]but not those in other organs [23] reflecting the pharma-cokinetics of the carrier Accordingly subcutaneous footpadadministration of the BP-encapsulating liposomes resultedin macrophage elimination in draining lymph nodes [28]while intratracheal administration exclusively eliminatesmacrophages from lung tissues [29]

Liposome encapsulating BPs were used to enhance tumorgrowth in an experimental model of liver metastasis [30] Ratinoculation with colon carcinoma cells resulted in a strongenhanced tumor growth in the liver only when the animalswere pretreated with an iv injection of CLO-encapsulatingliposomes This effect was attributed to the effective elimina-tion of all Kupffer cells that are preferential accumulation sitefor colloidal carriers Accordingly in the same experimentnonphagocytic cells into the liver were not affected [30] Incontrast liposome encapsulating CLO have been successfullyused to inhibit the tumor growth In different experimentalanimal models of cancer this effect was accompanied bydrastic reduction of the blood vessel density in the tumortissue [31ndash33 101] CLO-encapsulating liposomes were alsoused in combination therapy with VEGF-neutralizing anti-body The treatment led to significant reduction of angio-genesis as demonstrated by blood vessel staining and vesselquantification that was associated to a significant reduc-tion of the TAM and tumor-associated dendritic cells [31]Liposomes encapsulating CLO were also investigated incombinationwith sorafenib in two human hepatocellular car-cinoma xenograft nudemousemodels [34]Mice treatedwithsorafenib showed a significant inhibition of tumor growthand lung metastasis but associated to significant increaseof macrophage recruitment in peripheral blood as well asincreased intratumoral infiltration A combination therapywith sorafenib and liposome containing CLO or sorafenib

and free ZOL also led to reduced tumor angiogenesis withthe highest effects found with ZOL This effect could besurprising when considering that zoledronic acid was usedas free however the strong activity of ZOL at very low con-centrations compared with CLO could explain the highesteffect found in this study In the same study the authorsfound toxic effects in animals treated with liposomes encap-sulating CLO while ZOL appeared as more promising espe-cially because already in the clinical practice Macrophagedepletion by using BP-containing liposomes has also beenproposed as adjuvant agent in the cancer radiotherapyIndeed radiotherapy although directly inducing tumor celldeath may upregulate proangiogenic and prosurvival factorswithin the tumor microenvironment In particular uponradiation upregulation of tumor cells and cells of themyeloidlineage can occur with consequent TNF120572 production [35]followed by the induction of macrophage-secreted vascularendothelial growth factor (VEGF) with consequent radiopro-tective effect Radiotherapy association with the treatmentwith CLO-containing liposomes resulted in the improve-ments in the therapeutic index as determined by a delay oftumor regrowth [36] The use of CLO-containing liposomeswas also useful to reducemetastasis of human prostate cancerin bone thus confirming the role of TAM in regulation oftumor tissue homeostasis [37] The effect was potentiatedwhen mice were inoculated with cancer cells previouslyknocked down of IL-6 thus confirming the role of IL-6 asa strong chemotactic factor that recruits TAM to the tumorlesion

7 Nanotechnology and BPsTargeting of Cancer Cells

Although many research papers are focused on the use ofnanocarriers targeting macrophages the delivery of bispho-sphonates directly to cancer cells has been recently investi-gated

Tumors characterized by cells derived from myeloidlineage cells can be targeted with BP This has been recentlydemonstrated in a model of malignant histiocytosis [38]CLO-containing liposomes were firstly assayed in vitro oncanine malignant histiocytosis cells demonstrating a signifi-cant inhibition of cell growthThis effect was also found evenin nonphagocytic cells although for these cells free CLOwas more efficient In vivo dogs with spontaneous malignanthistiocytosis and treated with CLO-containing liposomeselicited significant tumor regression in two of five treated ani-mals The authors also reported an antitumor activity follow-ing iv administration of CLO-containing liposomes inseveral different nonhistiocytic mouse tumor models thussuggesting the antitumor activity may have beenmediated bya combination of both direct and indirect tumor effects [38]

Liposomes have been used to deliver BPs directly tocancer cells (Table 1) Neridronate (NER) encapsulated intoliposomes increased the inhibition activity on cell growthon human breast cancer cells (MDA-MB-231) by 50 timescompared to the free drug [39]


Table 1 Summary of the most meaningful studies published on nanotechnology to deliver BPs in cancer

Delivery system Strategy Bisphosphonate Main findings References

Liposomes Macrophage depletion Clodronate Macrophage elimination in the spleenand liver following iv administration [21ndash25]

Liposomes Macrophage depletion Clodronatepamidronate etidronate

Macrophage elimination in thebloodstream following iv

administration[26]


BPs were found to be even 1000 times lessactive compared with the corresponding

liposome-based formulations highcalcium extracellular concentrationresulted in a stronger macrophage

depletion negatively charged unilamellarliposomes favour macrophage depletion

[23 24 27]

Liposomes Macrophage depletion ClodronateMacrophage elimination in draininglymph nodes following subcutaneous

footpad administration[28]

Liposomes Macrophage depletion ClodronateIntratracheal administration exclusively

eliminates macrophages from lungtissues

[29]

Liposomes Macrophage depletion Clodronate Enhanced tumor growth in anexperimental model of liver metastasis [30]

Liposomes Macrophage depletion Clodronate

Inhibition of the tumor growth indifferent experimental animal models ofcancer reduction of the blood vessel

density in the tumor tissue reduction ofthe tumor-associated macrophages and

tumor-associated dendritic cells

[31ndash33]

Liposomes Macrophage depletionClodronate in

combination withsorafenib

Significant inhibition of tumor growthand lung metastasis reduced tumor

angiogenesis[34]

Liposomes Macrophage depletion Clodronate as adjuvantagent in radiotherapy

Adjuvant agent in the cancer radiotherapywith delayed tumor regrowth [35 36]

Liposomes Macrophage depletion Clodronate Reduced metastasis of human prostatecancer in bone [37]

Liposomes Inhibitory effect oncancer cells Clodronate Significant tumor regression [38]

Liposomes Inhibitory effect oncancer cells Neridronate Inhibition of cell growth [39]

PEGylated liposomes Targeting ofextraskeletal tumors Zoledronate

Enhanced cytotoxic effect in vitroenhanced inhibition of tumor growth

(prostate cancer and multiple myeloma)[40 41]

Folate-coupled PEGylatedliposomes

Targeting ofextraskeletal tumors Zoledronate Enhanced cytotoxic effect in vitro [42]

Self-assembling NPs Targeting ofextraskeletal tumors Zoledronate


(prostate cancer)[41 43]

Superparamagnetic ironoxide nanocrystals Theranostic purposes Alendronate

zoledronate

Decrease cell proliferation in vivo andinhibition of tumour growth in vivo onlyin combination with a magnetic field

[44ndash46]

LiposomesTargeting of

doxorubicin to bonetumors

Bisphosphonate headgroup in a novel

amphipathic molecule

Increased cytotoxicity in vitro on humanosteosarcoma cell line associated to

hydroxyapatite[47]

Poly(lactide-co-glycolide)NPs

Targeting ofdoxorubicin to bone

tumors

Alendronate conjugatedon the nanocarrier

surface

Reduced incidence of metastasesassociated to a significant reduction ofthe osteoclast number at the tumor site

[48]


Table 1 Continued


Poly(lactide-co-glycolide) NPs Targeting of docetaxelto bone tumors

Zoledronate conjugatedon the nanocarrier surface Enhanced cytotoxic effect in vitro [49]

Poly(ethylene glycol)-dendrimer Targeting of paclitaxelto bone tumors

Alendronate conjugatedto the nanocarrier

Significant improvement of paclitaxelin vivo half-life [50]

Moreover even at a lower concentration liposomal NERshowed a suppressive effect on tumor cell mobility in vitrowhereas free NER showed almost no effect Reasonably lipo-somes should mediate the enhanced bisphosphonate uptakeinto the cells although this hypothesis was demonstratedonly by indirect evidence by co-encapsulation of fluorescentdye together with the drug

In order to directly deliver BP in tumor cells accumu-lation in MPS should be avoided Thus nanocarriers withstealth properties able to avoid opsonization should bepreferred In the light of this consideration stealth liposomesencapsulating ZOL (lipoZOL) designed for tumor targetingwere developed [40 42] ZOL was encapsulated into lipo-somes by different strategies and the reverse-phase evapo-ration technique was selected to achieve the highest encap-sulation efficiency (unpublished data) With this techniquethe use of an alkaline buffer improved the ZOL solubilityinto the aqueous phase of liposomes thus increased the drugencapsulation efficiency up to about 5 [40] Liposomeswereable to significantly prolong ZOL circulation time Free ZOLwas quickly cleared from blood with 01-02 of the injecteddose still present 1 h after injection ZOL encapsulationinto liposomes especially PEGylated liposomes significantlyincreased ZOL circulation time with more than 10 of theinjected dose still present into the blood 24 h following theinjection [42] Concerning the in vitro activity of lipoZOLcontrasting results have been found In particular our groupdemonstrated that the use of lipoZOL compared with freeZOL increased the cytotoxic effect until a potentiation factorof about 20 [40] The effect was confirmed in cell lines ofdifferent cancer namely prostate breast headneck lung andpancreas and multiple myeloma with an IC50 ranging from4 to about 200120583M These data are in contrast with thosereported by other authors who found that stealth liposomescontaining ZOL did not elicited any significant inhibitoryeffect on cell from 001 to 200120583M [42] Significant cytotox-icity was found only by using folate-conjugated lipoZOLespecially in cell overexpressing the folate receptor Thediscrepancy among the two studies could be ascribed to thedifferent formulations used aswell as to the different cell lines

The in vivo antitumor activity of lipoZOL was demon-strated in two different model of tumors namely prostatecancer and multiple myeloma [40 41] In these experimentsmice treated with lipoZOL compared to animal with freeZOL showed a higher tumor weight inhibition and tumorgrowth delay together with increased mice survival As inthe case of non-stealth nanocarriers also stealth liposomesallowed to obtain reduced number of TAM as well as inhi-bition of the neoangiogenesis [40 41] Moreover no signif-icant changes were found in serum creatinine urea and

calcium in animals treated with lipoZOL suggesting theabsence of potential adverse effects [40] In order to overcometechnological limits of the lipoZOL such as low encapsu-lation efficiency and stability issue of the liposomal formu-lation our group recently developed a new nanovector todeliver ZOL in extraskeletal tumor The new system consistsof self-assembling NPs encapsulating ZOL and designed tobe prepared before use thus avoiding storage issues [43 102]In particular the formulation can be prepared by mixingtwo components namely an aqueous solution of ZOLCa2+PO

4

3minus NPs and cationic PEGylated liposomes Ca2+PO4

3minus have already been used to deliver other negativelycharged molecules such as nucleic acids [103] In the caseof BPs an encapsulation process driven by ionic interactionsallowed to overcome the loading issues observed with lipo-somes Indeed in the case of self-assembling NPs a ZOLencapsulation efficiency 12-fold greater compared with thatobtained with ZOL-containing liposomes was achieved Theself-assembling NPs increased the growth inhibition of ZOLondifferent cancer cell lines compared to freeZOLThehigh-est cell growth inhibition was observed on breast cancer cellsThe anticancer activity of this formulation was also demon-strated in vivo in an animal model of prostate cancer ZOLencapsulated into self-assembling NPs elicited a markedantitumor activity while free ZOL did not show a significantreduction of tumor growth [43]The in vivo anticancer activ-ities of two different ZOL-containing nanocarriers namelylipoZOL and self-assembling NPs were compared [41] Inthis study self-assembling NPs encapsulating ZOL inducedthe complete remission of tumour xenografts and an increaseof survival time higher than that observedwith lipoZOLThiseffect was paralleled by a significant increase of both necroticand apoptotic indexes NPs more than lipoZOL also causeda statistically significant reduction of TAM and displayed ahigher neoangiogenesis inhibition With both nanovectorstoxic effects affecting the mice weight or inducing deathswere not found Finally the histological examination of somevital organs such as liver kidney and spleen did not find anychanges in terms of necrotic effects or modifications in theinflammatory infiltrate [41]

The ability of BPs to bind metal ions was used to prepareBP-complexing superparamagnetic iron oxide nanocrystalswith theranostic purposes [44ndash46] In a first study a 5-hydroxy-5 5-bis(phosphono) pentanoic acid was used whilein the following works more powerful BPs such as ALE andZOL were used Amino fluorescein or rhodamine were cova-lently coupledwith the nanocrystal thus allowing to visualizean efficient uptake of the nanovector into two different celllines [44 104] However cell viability assays demonstratedthat ZOL alone had an IC50 at 48 h that was 1 order of


magnitude lower than with 120574Fe2O3-ZOL nanocrystals

According to the authors cell proliferation decreases to 75under an applied magnetic field compared to 40 withoutmagnetic field [45] 120574Fe

2O3-ALE NPs were investigated on

different cell lines however a clear advantage of the NPswas found only on breast cancer cell [104] These NPs werealso investigated in vivo in an experimental model of breastcancer [104] In this study tumour growth in animals treatedwith free ALE and 120574Fe

2O3-ALE NPs was not significantly

different than in control group NPs used in combinationwith a magnetic field significantly inhibited tumour growthby about 60 after 5 weeks with all mice treated that werealive 5 weeks after treatment and did not present significantloss of body weight However the lack of control experimentswith 120574Fe

2O3NPs (NPs without ALE) hampers to affirm that

ALE could be responsible for the antitumor affect while thephysical effect of NPs under the magnetic field could bethe main responsible of anticancer effect described by theauthors

8 Nanotechnology and BPsTargeting of Bone Tumors

Bonemetastasis especially originating by breast and prostatecancer are the most frequent form of skeletal neoplasia Inthe majority of patients treatments of bone metastasis arepalliative being aimed to relieve pain improve function andprevent complications such as spinal cord compression andpathological fracture The development of anticancer thera-pies with high affinity for bone and reduced distribution toother sites is certainly attractive To this aim nanovectors tar-geting hydroxyapatite have been proposed Hydroxyapatite(Ca10(PO4)6(OH)2) is the major inorganic mineral phase

present in bone and teeth and not found in other tissuesunder normal circumstances Thus the use of nanocarriersconjugated to BPs that are characterized by high affinity forhydroxyapatite have been proposed

A novel amphipathic molecule bearing a bisphospho-nate head group 4-N-(35-ditetradecyloxybenzoyl)-aminob-utane-1-hydroxy-11-bisphosphonic acid disodium salt (BPA)was synthesized and used at different concentrations toprepare liposomes [47] The presence of the bisphosphonateson the liposome surface was suggested by a zeta poten-tial that was as negative as high the amount of the BPAused in the preparation BPA-containing liposomes boundhydroxyapatite in vitro depending on the BPA concentrationinto the carrier while no binding was found in the case ofliposomes prepared without BPA In vitro studies on humanosteosarcoma cell line associated to hydroxyapatite demon-strated an increased cytotoxicity of BPA-containing lipo-somes encapsulating doxorubicin compared to liposome notcontaining BPA this effect being dependant on the amountof BPA used in the preparation [47] Liposomes containingdoxorubicin (DOX) were also conjugated to CLO to tar-get osteosarcoma [105] DOX-encapsulating BP-conjugatedliposomes showed similar antitumor effect on two differentosteosarcoma cell lines compared to DOX in free formor encapsulated into PEGylated liposomes Moreover in

an experimental model of osteosarcoma a higher inhi-bition rate of tumor growth together with a prolongedsurvival was observed when comparing mice treated withDOX-encapsulating BP-conjugated liposomes with the othergroups

ALE has also been coupled to poly(lactide-co-glycolide)(PLGA)NPs encapsulating doxorubicin [48]TheseNPswereinvestigated in a panel of human cell lines representative ofprimary and metastatic bone tumors on which doxorubicinas free or encapsulated in ALE-conjugated NPs induceda concentration-dependent inhibition of cell proliferationIn vivo studies on an orthotopic mouse model of breastcancer bone metastases demonstrated a reduced incidence ofmetastases in the case of mice treated with doxorubicin asfree or encapsulated in ALE-conjugated NPs However in thecase of ALE-conjugated NPs independently on the presenceof doxorubicin a significant reduction of the osteoclastnumber was found at the tumor site reasonably attributedto the ALE activity [48] PLGA NPs conjugated with ZOLhave been recently developed to deliver docetaxel (DCX) tobone [49] ZOL was conjugated to PLGA-PEG-NH2 and theresulting PLGA-PEG-ZOL was used to prepare the NPs Invitro bone binding affinity showed that PLGA-PEG-ZOLNPshave affinity with human bone powder comparable to thatobserved for ZOL in solution On two different breast cancercell lines PLGA-PEG-ZOLNPs exhibited significantly highercytotoxicity compared to DCX DCX associated to ZOL andunconjugated NPs at all drug concentrations and differenttime points Interestingly the authors demonstrated that thepresence of ZOL on the NP surface affected the pathway forthe intracellular uptake In particular PEGylated PLGA NPspredominantly followed lysosome through early endosomeswhich displayed significant colocalization of NPs and lyso-somes On the other hand ZOL-modified NPs were endo-cytosed by both clathrin-mediated and caveolae-mediatedendocytosis mechanism where caveolae pathway followeda non-lysosomal route The different intracellular traffickingof ZOL-coupled and ZOL-free NPs was also confirmed by theprolonged time needed for the exocytosis [49] Finally ZOL-coupled NPs showed an enhanced cytotoxic effect that hasbeen attributed to the higher uptake via ZOL-mediated endo-cytosis Finally ALE was also conjugated to a poly(ethyleneglycol) (PEG) dendrimer in combination with paclitaxel totarget bone tumors [50] The pharmacological activity ofpaclitaxel in terms of inhibition of cell growth and cellmigration was not altered by conjugation with PEG den-drimer Moreover in vivo half-life of paclitaxel was signif-icantly improved when administering the conjugate ALE-dendrimer-paclitaxel compared with free paclitaxel

9 Concluding Remarks

In vitro results have clearly demonstrated that BPs in additionto inhibiting osteoclast-mediated bone resorption can exertmarked proapoptotic and antiproliferative effects on tumorcells especially when combined with other standard antineo-plastic therapy In vivo this antitumor effect appears to bebetter experienced in tumor cells of bone metastases at least


in the majority of experiments performed to date This maybe explained by the high local concentration of BPs in bonerelative to the much lower one in other organs and plasmathis feature makes bisphosphonates the drugs of choice inthe treatment of bone problems associated with malignancyHowever large-scale clinical trials have investigated thebenefit of bisphosphonate therapy in reducing the incidenceof SRE inmyeloma in breast cancer metastases in metastaticprostate cancer in lung cancer in renal cell carcinoma andin other solid tumors Many in vivo tumor models havedemonstrated ZOL PAM CLO and IBA antitumor efficacycompared with control

The use of nanotechnology can open new therapeutic sce-nario for BPs Nanocarriers such as conventional liposomesallow to use the BP as potent agent formacrophage depletionPreferential accumulation of BP in extraskeletal tissue can beachieved by using long circulating nanocarriers such as lipo-ZOL and self-assembling NPs The functionalization of theseNPs with ligand that is folate or transferrin able to targetcancer cells can be used to enhance the antitumor activityand to increase the selectivity of the treatment BP can beconjugated on the surface of nanocarriers that is PEGylatedPLGANPs or PEG dendrimer conjugated with the anticanceragent to be used as targeting moieties for the treatment ofbone cancers

Taking together all the scientific papers cited in thispaper the role of BPs in therapy appears underestimatedThisclass of molecules especially the third-generation N-BPs asZOL can certainly represent a new weapon against canceralthough today they are approved only as antiresorptionagent Of course new therapeutic indications cannot leaveaside the design of a specific delivery system able to changebiopharmaceutical characteristics of BPs In line with thisnanotechnology can certainly represent an attractive oppor-tunity

10 Future Perspectives

Several strategies could be developed in the next future therational use of N-BPs in combination with other target-basedagents to overcome escape mechanism occurring in cancercells the sequential combination of N-BPs with conventionalcytotoxic agents to strengthen their apoptotic and antiangio-genic potential the administration of N-BPs in metronomic-like modality (low doses for protracted time) the discoveryand the targeting of new intracellular molecules foundthrough the use of new advanced molecular technologiessuch as DNA microarray In all these possible perspectivesnanotechnologywill represent a valid support also contribut-ing tomake thesemoleculesmore specific thus reducing con-traindications for example osteonecrosis of the jaw due tothe excessive N-BP accumulation in sites where their actionis not required Studies in progress in our labs suggest futureapplications of BPs also in form of cancer hard to kill likeglioma and for other applications in the central nervous sys-tem like the treatment of neuropathic pain (data submittedfor publication)

Authorsrsquo Contribution

G D Rosa and G Misso equally contributed to the paper

References

[1] J R Ross Y Saunders P M Edmonds et al ldquoA systematicreview of the role of bisphosphonates in metastatic diseaserdquoHealth Technology Assessment vol 8 no 4 pp 1ndash176 2004

[2] H Fleisch R G G Russell S Bisaz P A Casey and RC Muhlbauer ldquoThe influence of pyrophosphate analogues(diphosphonates) on the precipitation and dissolution of cal-cium phosphate in vitro and in vivordquo Calcified Tissue Researchvol 2 no 1 p 10 1968

[3] R G Russell ldquoBisphosphonates the first 40 yearsrdquo Bone vol49 no 1 pp 2ndash19 2011

[4] L Widler W Jahnke and J R Green ldquoThe chemistry of bis-phosphonates from antiscaling agents to clinical therapeuticsrdquoAnticancer Agents inMedicinals Chemistry vol 12 no 2 pp 95ndash101 2012

[5] R G Russell ldquoBisphosphonates mode of action and pharma-cologyrdquo Pediatrics vol 119 supplement 2 pp S150ndashS162 2007

[6] J E Dunford K Thompson F P Coxon et al ldquoStructure-acti-vity relationships for inhibition of farnesyl diphosphate syn-thase in vitro and inhibition of bone resorption in vivo bynitrogen-containing bisphosphonatesrdquo Journal of Pharmacol-ogy and ExperimentalTherapeutics vol 296 no 2 pp 235ndash2422001

[7] J R Green ldquoAntitumor effects of bisphosphonatesrdquoCancer vol97 no 3 pp 840ndash847 2003

[8] F H Ebetino A M Hogan S Sun et al ldquoThe relationshipbetween the chemistry and biological activity of the bisphos-phonaterdquo Bone vol 49 no 1 pp 20ndash33 2011

[9] L I Plotkin R S Weinstein A M Parfitt P K Roberson S CManolagas and T Bellido ldquoPrevention of osteocyte and osteo-blast apoptosis by bisphosphonates and calcitoninrdquoThe Journalof Clinical Investigation vol 104 no 10 pp 1363ndash1374 1999

[10] M J Rogers D JWatts RGG Russell et al ldquoInhibitory effectsof bisphosphonates on growth of amoebae of the cellular slimemold Dictyostelium discoideumrdquo Journal of Bone and MineralResearch vol 9 no 7 pp 1029ndash1039 1994

[11] M J Rogers ldquoFrom molds and macrophages to mevalonatea decade of progress in understanding the molecular mode ofaction of bisphosphonatesrdquo Calcified Tissue International vol75 no 6 pp 451ndash461 2004

[12] M J Rogers R J Brown VHodkin R GG Russell D JWattsand G M Blackburn ldquoBisphosphonates are incorporated intoadenine nucleotides by human aminoacyl-tRNA synthetaseenzymesrdquo Biochemical and Biophysical Research Communica-tions vol 224 no 3 pp 863ndash869 1996

[13] J C Frith J Monkkonen S Auriola H Monkkonen and M JRogers ldquoThemolecular mechanism of action of the antiresorp-tive and anti-inflammatory drug clodronate evidence for theformation in vivo of a metabolite that inhibits bone resorptionandcauses osteoclast and macrophage apoptosisrdquo Arthritis ampRheumatism vol 44 no 9 pp 2201ndash2210 2001

[14] P P LehenkariM Kellinsalmi J P Napankangas et al ldquoFurtherinsight into mechanism of action of clodronate inhibition ofmitochondrial ADPATP translocase by a nonhydrolyzableadenine-containing metaboliterdquo Molecular Pharmacology vol61 no 5 pp 1255ndash1262 2002


[15] J M Halasy-Nagy G A Rodan and A A Reszka ldquoInhibitionof bone resorption by alendronate and risedronate does notrequire osteoclast apoptosisrdquo Bone vol 29 no 6 pp 553ndash5592001

[16] A J Roelofs K Thompson S Gordon and M J RogersldquoMolecular mechanisms of action of bisphosphonates currentstatusrdquo Clinical Cancer Research vol 12 no 20 part 2 pp6222sndash6230s 2006

[17] J R Berenson ldquoAntitumor effects of bisphosphonates fromthe laboratory to the clinicrdquo Current Opinion in Supportive ampPalliative Care vol 5 no 3 pp 233ndash240 2011

[18] P Carmeliet and R K Jain ldquoAngiogenesis in cancer and otherdiseasesrdquo Nature vol 407 no 6801 pp 249ndash257 2000

[19] P Carmeliet ldquoAngiogenesis in health and diseaserdquo NatureMedicine vol 9 pp 653ndash660 2003

[20] M Caraglia D Santini M Marra B Vincenzi G Tonini andA Budillon ldquoEmerging anti-cancer molecular mechanisms ofaminobisphosphonatesrdquo Endocrine-Related Cancer vol 13 no1 pp 7ndash26 2006

[21] E Claassen and N van Rooijen ldquoThe effect of elimination ofmacrophages on the tissue distribution of liposomes containing[3H]methotrexaterdquo Biochimica et Biophysica Acta vol 802 no3 pp 428ndash434 1984

[22] N vanRooijen andR vanNieuwmegen ldquoElimination of phago-cytic cells in the spleen after intravenous injection of liposomeencapsulated dichloromethylene diphosphonate An enzyme-histochemical studyrdquo Cell and Tissue Research vol 238 no 2pp 355ndash358 1984

[23] N van Rooijen ldquoThe liposome-mediated macrophage lsquosuicidersquotechniquerdquo Journal of ImmunologicalMethods vol 124 no 1 pp1ndash6 1989

[24] J Monkkonen N Pennanen S Lapinjoki and A UrttildquoClodronate (dichloromethylene bisphosphonate) inhibits LPS-stimulated IL-6 and TNF production by RAW 264 cellsrdquo LifeSciences vol 54 no 14 pp PL229ndashPL234 1994

[25] N van Rooijen and E Claassen ldquoIn vivo elimination of macro-phages in spleen and liver using liposome encapsulated drugsmethods and applicationsrdquo in Liposomes as Drug CarriersTrends and Progress G Gregoriadis Ed chapter 9 pp 131ndash143John Wiley amp Sons Chichester UK 1988

[26] N van Rooijen R van Nieuwmegen and E W A KamperdijkldquoElimination of phagocytic cells in the spleen after intravenousinjection of liposome-encapsulated dichloromethylene diphos-phonate Ultrastructural aspects of elimination of marginalzone macrophagesrdquo Virchows Archiv B vol 49 no 1 pp 375ndash383 1985

[27] N Pennanen S Lapinjoki A Urtti and J Monkkonen ldquoEffectof liposomal and free bisphosphonates on the IL-1120573 IL-6 andTNF120572 secretion from RAW 264 cells in vitrordquo PharmaceuticalResearch vol 12 no 6 pp 916ndash922 1995

[28] F G A Delemarre N Kors G Kraal and N van RooijenldquoRepopulation of macrophages in popliteal lymph nodes ofmice after liposome-mediated depletionrdquo Journal of LeukocyteBiology vol 47 no 3 pp 251ndash257 1990

[29] TThepen N van Rooijen and G Kraal ldquoAlveolar macrophageelimination in vivo is associated with an increase in pul-monary immune response inmicerdquoThe Journal of ExperimentalMedicine vol 170 no 2 pp 499ndash509 1989

[30] G Heuff H S A Oldenburg H Boutkan et al ldquoEnhancedtumour growth in the rat liver after selective elimination ofKupffer cellsrdquo Cancer Immunology and Immunotherapy vol 37no 2 pp 125ndash130 1993

[31] S M Zeisberger B Odermatt C Marty A H Zehnder-Fjallman K Ballmer-Hofer and R A Schwendener ldquoClo-dronate-liposome-mediated depletion of tumour-associatedmacrophages a new and highly effective antiangiogenic therapyapproachrdquo British Journal of Cancer vol 95 no 3 pp 272ndash2812006

[32] Y N Kimura K Watari A Fotovati et al ldquoInflammatory stim-uli from macrophages and cancer cells synergistically promotetumor growth and angiogenesisrdquo Cancer Science vol 98 no 12pp 2009ndash2018 2007

[33] S Gazzaniga A I Bravo A Guglielmotti et al ldquoTarget-ing tumor-associated macrophages and inhibition of MCP-1reduce angiogenesis and tumor growth in a human melanomaxenograftrdquo Journal of Investigative Dermatology vol 127 no 8pp 2031ndash2041 2007

[34] W Zhang X D Zhu H C Sun et al ldquoDepletion of tumor-associated macrophages enhances the effect of sorafenib inmetastatic liver cancer models by antimetastatic and antiangio-genic effectsrdquo Clinical Cancer Research vol 16 no 13 pp 3420ndash3430 2010

[35] M L Sherman R Datta D E Hallahan R R Weichselbaumand D W Kufe ldquoRegulation of tumor necrosis factor geneexpression by ionizing radiation in human myeloid leukemiacells and peripheral blood monocytesrdquo The Journal of ClinicalInvestigation vol 87 no 5 pp 1794ndash1797 1991

[36] Y Meng M A Beckett H Liang et al ldquoBlockade of tumornecrosis factor 120572 signaling in tumor-associated macrophages asa radiosensitizing strategyrdquo Cancer Research vol 70 no 4 pp1534ndash1543 2010

[37] S W Kim J S Kim J Papadopoulos et al ldquoConsistentinteractions between tumor cell IL-6 and macrophage TNF-120572enhance the growth of human prostate cancer cells in the boneof nudemouserdquo International Immunopharmacology vol 11 no7 pp 862ndash872 2011

[38] S Hafeman C London R Elmslie and S Dow ldquoEvaluation ofliposomal clodronate for treatment of malignant histiocytosisin dogsrdquo Cancer Immunology and Immunotherapy vol 59 no3 pp 441ndash452 2010

[39] I Chebbi E Migianu-Griffoni O Sainte-Catherine M Lecou-vey and O Seksek ldquoIn vitro assessment of liposomal ner-idronate on MDA-MB-231 human breast cancer cellsrdquo Interna-tional Journal of Pharmaceutics vol 383 no 1-2 pp 116ndash1222010


[41] M Marra G Salzano C Leonetti et al ldquoNew self-assemblynanoparticles and stealth liposomes for the delivery of zole-dronic acid a comparative studyrdquo Biotechnology Advances vol30 no 1 pp 302ndash309 2012

[42] H Shmeeda Y Amitay J Gorin et al ldquoDelivery of zoledronicacid encapsulated in folate-targeted liposome results in potentin vitro cytotoxic activity on tumor cellsrdquo Journal of ControlledRelease vol 146 no 1 pp 76ndash83 2010

[43] G Salzano M Marra M Porru et al ldquoSelf-assembly nanopar-ticles for the delivery of bisphosphonates into tumorsrdquo Interna-tional Journal of Pharmaceutics vol 403 no 1-2 pp 292ndash2972011

[44] Y Lalatonne C Paris J M Serfaty P Weinmann M Lecouveyand L Motte ldquoBis-phosphonates-ultra small superparamag-netic iron oxide nanoparticles a platform towards diagnosis


and therapyrdquo Chemical Communications no 22 pp 2553ndash25552008

[45] F Benyettou Y Lalatonne O Sainte-Catherine M Mon-teil and L Motte ldquoSuperparamagnetic nanovector with anti-cancer properties 120574Fe

2O3Zoledronaterdquo International Journal

of Pharmaceutics vol 379 no 2 pp 324ndash327 2009[46] F Benyettou EGuenin Y Lalatonne and LMotte ldquoMicrowave

assisted nanoparticle surface functionalizationrdquo Nanotechnol-ogy vol 22 no 5 Article ID 055102 2011

[47] T Anada Y Takeda Y Honda K Sakurai and O SuzukildquoSynthesis of calcium phosphate-binding liposome for drugdeliveryrdquo Bioorganic amp Medicinal Chemistry Letters vol 19 no15 pp 4148ndash4150 2009

[48] M Salerno E Cenni C Fotia et al ldquoBone-targeted doxorubi-cin-loaded nanoparticles as a tool for the treatment of skeletalmetastasesrdquoCurrent Cancer Drug Targets vol 10 no 7 pp 649ndash659 2010

[49] K Ramanlal Chaudhari A Kumar V K Megraj Khandelwalet al ldquoBone metastasis targeting a novel approach to reachbone using Zoledronate anchored PLGAnanoparticle as carriersystem loaded with Docetaxelrdquo Journal of Controlled Releasevol 158 no 3 pp 470ndash478 2012

[50] C Clementi KMiller AMero R Satchi-Fainaro andG PasutldquoDendritic poly(ethylene glycol) bearing paclitaxel and alen-dronate for targeting bone neoplasmsrdquo Molecular Pharmaceu-tics vol 8 no 4 pp 1063ndash1072 2011

[51] K L Kavanagh K Guo J E Dunford et al ldquoThe molecularmechanism of nitrogen-containing bisphosphonates as anti-osteoporosis drugs crystal structure and inhibition of farnesylpyrophosphate synthaserdquo Proceedings of the National Academyof Sciences of the United States of America vol 103 no 20 pp7829ndash7834 2006

[52] K Thompson M J Rogers F P Coxon and J C CrockettldquoCytosolic entry of bisphosphonate drugs requires acidificationof vesicles after fluid-phase endocytosisrdquoMolecular Pharmacol-ogy vol 69 no 5 pp 1624ndash1632 2006

[53] A Ibrahim N Scher GWilliams et al ldquoApproval summary forzoledronic acid for treatment of multiple myeloma and cancerbone metastasesrdquo Clinical Cancer Research vol 9 no 7 pp2394ndash2399 2003

[54] T Chen J Berenson R Vescio et al ldquoPharmacokinetics andpharmacodynamics of zoledronic acid in cancer patients withbone metastasesrdquo Journal of Clinical Pharmacology vol 42 no11 pp 1228ndash1236 2002

[55] A Skerjanec J Berenson C HHsu et al ldquoThe pharmacokinet-ics and pharmacodynamics of zoledronic acid in cancer patientswith varying degrees of renal functionrdquo Journal of ClinicalPharmacology vol 43 no 2 pp 154ndash162 2003

[56] J H Lin ldquoBisphosphonates a review of their pharmacokineticpropertiesrdquo Bone vol 18 no 2 pp 75ndash85 1996

[57] J Barrett E Worth F Bauss and S Epstein ldquoIbandronate aclinical pharmacological and pharmacokinetic updaterdquo Journalof Clinical Pharmacology vol 44 no 9 pp 951ndash965 2004

[58] H MWeiss U Pfaar A Schweitzer H Wiegand A Skerjanecand H Schran ldquoBiodistribution and plasma protein binding ofzoledronic acidrdquo Drug Metabolism and Disposition vol 36 no10 pp 2043ndash2049 2008

[59] L M Pickering and J L Mansi ldquoAdhesion of breast cancercells to extracellular matrices is inhibited by zoledronic acidand enhanced by aberrant Ras signalingrdquo American Society ofClinical Oncology vol 22 p 863 2003

[60] J Wood K Bonjean S Ruetz et al ldquoNovel antiangiogeniceffects of the bisphosphonate compound zoledronic acidrdquoJournal of Pharmacology and Experimental Therapeutics vol302 no 3 pp 1055ndash1061 2002

[61] P I Croucher H de Raeve M J Perry et al ldquoZoledronic acidtreatment of 5T2MM-bearing mice inhibits the development ofmyeloma bone disease evidence for decreased osteolysis tumorburden and angiogenesis and increased survivalrdquo Journal ofBone and Mineral Research vol 18 no 3 pp 482ndash492 2003

[62] F Dieli N Gebbia F Poccia et al ldquoInduction of 120574120575 T-lymphocyte effector functions by bisphosphonate zoledronicacid in cancer patients in vivordquo Blood vol 102 no 6 pp 2310ndash2311 2003

[63] D Santini S Galluzzo B Vincenzi et al ldquoNew developmentsof aminobisphosphonates the double face of Janusrdquo Annals ofOncology vol 18 supplement 6 pp vi164ndashvi167 2007

[64] H L Benford J C Frith S Auriola J Monkkonen and MJ Rogers ldquoFarnesol and geranylgeraniol prevent activation ofcaspases by aminobisphosphonates biochemical evidence fortwo distinct pharmacological classes of bisphosphonate drugsrdquoMolecular Pharmacology vol 56 no 1 pp 131ndash140 1999

[65] F P Coxon M H Helfrich R vanrsquot Hof et al ldquoProtein geranyl-geranylation is required for osteoclast formation function andsurvival inhibition by bisphosphonates andGGTI-298rdquo Journalof Bone andMineral Research vol 15 no 8 pp 1467ndash1476 2000

[66] S Boissier M Ferreras O Peyruchaud et al ldquoBisphosphonatesinhibit breast and prostate carcinoma cell invasion an earlyevent in the formation of bone metastasesrdquo Cancer Researchvol 60 no 11 pp 2949ndash2954 2000

[67] G Misso M Porru A Stoppacciaro et al ldquoEvaluation of thein vitro and in vivo antiangiogenic effects of denosumab andzoledronic acidrdquo Cancer Biology andTherapy vol 13 no 14 pp1491ndash1500 2012

[68] M Bezzi M Hasmim G Bieler O Dormond and C RueggldquoZoledronate sensitizes endothelial cells to tumor necrosisfactor-induced programmed cell death evidence for the sup-pression of sustained activation of focal adhesion kinase andprotein kinase BAktrdquo The Journal of Biological Chemistry vol278 no 44 pp 43603ndash43614 2003

[69] M Marra A Abbruzzese R Addeo et al ldquoCutting the limitsof aminobisphosphonates new strategies for the potentiation oftheir anti-tumour effectsrdquo Current Cancer Drug Targets vol 9no 7 pp 791ndash800 2009

[70] M Caraglia A M DrsquoAlessandro M Marra et al ldquoThefarnesyl transferase inhibitor R115777 (Zarnestra) synergisti-cally enhances growth inhibition and apoptosis induced onepidermoid cancer cells by Zoledronic acid (Zometa) andPamidronaterdquo Oncogene vol 23 no 41 pp 6900ndash6913 2004

[71] S G Senaratne J L Mansi and K W Colston ldquoThe bispho-sphonate zoledronic acid impairs Ras membrane [correctionof impairs membrane] localisation and induces cytochrome crelease in breast cancer cellsrdquo British Journal of Cancer vol 86no 9 pp 1479ndash1486 2002

[72] L Sewing F Steinberg H Schmidt and R Goke ldquoThe bispho-sphonate zoledronic acid inhibits the growth of HCT-116 coloncarcinoma cells and induces tumor cell apoptosisrdquo Apoptosisvol 13 no 6 pp 782ndash789 2008

[73] M Fujita M Tohi K Sawada et al ldquoInvolvement of the meval-onate pathway in the antiproliferative effect of zoledronate onACHN renal cell carcinoma cellsrdquoOncology Reports vol 27 no5 pp 1371ndash1376 2012


[74] G Ferretti A Fabi P Carlini et al ldquoZoledronic-acid-inducedcirculating level modifications of angiogenic factors metallo-proteinases and proinflammatory cytokines inmetastatic breastcancer patientsrdquo Oncology vol 69 no 1 pp 35ndash43 2005

[75] R S Herbst and F R Khuri ldquoMode of action of docetaxelmdasha basis for combination with novel anticancer agentsrdquo CancerTreatment Reviews vol 29 no 5 pp 407ndash415 2003

[76] A Ullen L Lennartsson U Harmenberg et al ldquoAdditivesynergistic antitumoral effects on prostate cancer cells in vitrofollowing treatment with a combination of docetaxel andzoledronic acidrdquo Acta Oncologica vol 44 no 6 pp 644ndash6502005

[77] F Fabbri G Brigliadori S Carloni et al ldquoZoledronic acidincreases docetaxel cytotoxicity through pMEK and Mcl-1inhibition in a hormone-sensitive prostate carcinoma cell linerdquoJournal of Translational Medicine vol 6 article 43 2008

[78] B Karabulut C Erten M K Gul et al ldquoDocetaxelzoledronicacid combination triggers apoptosis synergistically throughdownregulating antiapoptotic Bcl-2 protein level in hormone-refractory prostate cancer cellsrdquo Cell Biology International vol33 no 2 pp 239ndash246 2009

[79] M Marra D Santini G Meo et al ldquoCYR61 downmodulationpotentiates the anticancer effects of zoledronic acid in andro-gen-independent prostate cancer cellsrdquo International Journal ofCancer vol 125 no 9 pp 2004ndash2013 2009

[80] H K Koul S Koul and R B Meacham ldquoNew role foran established drug Bisphosphonates as potential anticanceragentsrdquo Prostate Cancer and Prostatic Diseases vol 15 no 2 pp111ndash119 2012

[81] E Corey L G Brown J E Quinn et al ldquoZoledronic acidexhibits inhibitory effects on osteoblastic and osteolytic metas-tases of prostate cancerrdquo Clinical Cancer Research vol 9 no 1pp 295ndash306 2003


[83] E Alvarez M Westmore R J S Galvin et al ldquoProperties ofbisphosphonates in the 13762 rat mammary carcinoma modelof tumor-induced bone resorptionrdquo Clinical Cancer Researchvol 9 no 15 pp 5705ndash5713 2003

[84] A Guenther S Gordon M Tiemann et al ldquoThe bispho-sphonate zoledronic acid has antimyeloma activity in vivoby inhibition of protein prenylationrdquo International Journal ofCancer vol 126 no 1 pp 239ndash246 2010

[85] Y Zheng H Zhou K Brennan et al ldquoInhibition of boneresorption rather than direct cytotoxicity mediates the anti-tumour actions of ibandronate and osteoprotegerin in a murinemodel of breast cancer bonemetastasisrdquo Bone vol 40 no 2 pp471ndash478 2007

[86] P I Croucher C M Shipman B van Camp and K Vanderk-erken ldquoBisphosphonates and osteoprotegerin as inhibitors ofmyeloma bone diseaserdquo Cancer vol 97 no supplement 3 pp818ndash824 2003

[87] J C CruzMAlsina F Craig et al ldquoIbandronate decreases bonedisease development and osteoclast stimulatory activity in an invivomodel of humanmyelomardquo Experimental Hematology vol29 no 4 pp 441ndash447 2001

[88] M Neudert C Fischer B Krempien F Bauss and M J SeibelldquoSite-specific human breast cancer (MDA-MB-231) metastases

in nude rats model characterisation and in vivo effects of iban-dronate on tumour growthrdquo International Journal of Cancer vol107 no 3 pp 468ndash477 2003

[89] G van der Pluijm I Que B Sijmons et al ldquoInterference withthemicroenvironmental support impairs the de novo formationof bone metastases in vivordquo Cancer Research vol 65 no 17 pp7682ndash7690 2005

[90] S S Padalecki M Carreon B Grubbs Y Cui and T AGuise ldquoAndrogen deprivation therapy enhances bone loss andprostate cancer metastases to bone prevention by zoledronicacidrdquo Oncology vol 17 no supplement 3 p 32 2003

[91] S Lu J Zhang Z Zhou et al ldquoSynergistic inhibitory activityof zoledronate and paclitaxel on bone metastasis in nude micerdquoOncology Reports vol 20 no 3 pp 581ndash587 2008

[92] P D Ottewell B Deux H Monkkonen et al ldquoDifferentialeffect of doxorubicin and zoledronic acid on intraosseous versusextraosseous breast tumor growth in vivordquo Clinical CancerResearch vol 14 no 14 pp 4658ndash4666 2008

[93] D Santini B Vincenzi S Galluzzo et al ldquoRepeated intermit-tent low-dose therapy with zoledronic acid induces an earlysustained and long-lasting decrease of peripheral vascularendothelial growth factor levels in cancer patientsrdquo ClinicalCancer Research vol 13 no 15 part 1 pp 4482ndash4486 2007

[94] M J Auger and J A Ross ldquoThe biology of the macrophagerdquo inTheMacrophageThe Natural Immune System C E Lewis and JOrsquoDonnellMcGee Eds pp 3ndash74OxfordUniversity Press NewYork NY USA 1992

[95] D P Speert ldquoMacrophages in bacterial infectionrdquo in TheMacrophage The Natural Immune System C E Lewis and JOrsquoDonnell McGee Eds pp 215ndash263 Oxford University PressNew York NY USA 1992

[96] E R Unanue and P M Allen ldquoThe basis for the immuno-regulatory role of macrophages and other accessory cellsrdquoScience vol 236 no 4801 pp 551ndash557 1987

[97] I J Fidler ldquoTargeting of immunomodulators to mononuclearphagocytes for therapy of cancerrdquo Advanced Drug DeliveryReviews vol 2 no 1 pp 69ndash106 1988

[98] RC Rees andH Parry ldquoMacrophages in tumour immunologyrdquoinTheMacrophageTheNatural Immune System C E Lewis andJ OrsquoDonnellMcGee Eds pp 314ndash335 OxfordUniversity PressNew York NY USA 1992

[99] N B Hao M H Lu Y H Fan et al ldquoMacrophages in tumormicroenvironments and the progression of tumorsrdquo Clinicaland Developmental Immunology vol 2012 Article ID 94809811 pages 2012

[100] S MMoghimi A C Hunter and T L Andresen ldquoFactors con-trolling nanoparticle pharmacokinetics an integrated analysisand perspectiverdquoAnnual Review of Pharmacological Toxicologyvol 52 pp 481ndash503 2012

[101] S Halin S H Rudolfsson N van Rooijen and A BerghldquoExtratumoral macrophages promote tumor and vasculargrowth in an orthotopic rat prostate tumor modelrdquo Neoplasiavol 11 no 2 pp 177ndash186 2009

[102] G Salzano M Marra C Leonetti et al ldquoNanotechnologies touse zoledronic acid as a potent antitumoral agentrdquo Journal ofDrug Delivery Science and Technology vol 21 no 3 pp 283ndash284 2011

[103] E V Giger J Puigmartı-Luis R Schlatter B Castagner P SDittrich and J C Leroux ldquoGene delivery with bisphosphonate-stabilized calcium phosphate nanoparticlesrdquo Journal of Con-trolled Release vol 150 no 1 pp 87ndash93 2011


[104] F Benyettou Y Lalatonne I Chebbi et al ldquoA multimodal mag-netic resonance imaging nanoplatform for cancer theranosticsrdquoPhysical Chemistry Chemical Physics vol 13 no 21 pp 10020ndash10027 2011

[105] D Wu and M Wan ldquoMethylene diphosphonate-conjugatedadriamycin liposomes preparation characteristics and tar-geted therapy for osteosarcomas in vitro and in vivordquo Biomedi-cal Microdevices vol 14 no 3 pp 497ndash510 2012


Review ArticleNeoplastic Meningitis from Solid TumorsA Prospective Clinical Study in Lombardia and a LiteratureReview on Therapeutic Approaches

A Silvani1 M Caroli2 P Gaviani1 V Fetoni3 R Merli4 M Riva5

M De Rossi5 F Imbesi6 and A Salmaggi7

1 Fondazione IRCCS Istituto Neurologico C Besta Milano Italy2 Clinica Neurochirurgica Ospedale Policlinico Milano Italy3Ospedale Melegnano Milano Italy4Ospedali Riuniti Bergamo Milano Italy5Ospedale di Lodi Lodi Italy6Ospedale Niguarda Milano Italy7Ospedale Lecco Lombardy Italy

Correspondence should be addressed to A Salmaggi asalmaggiospedaleleccoit

Received 7 December 2012 Accepted 20 December 2012


Copyright copy 2013 A Silvani et alis is an open access article distributed under theCreativeCommonsAttributionLicense whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Neoplastic dissemination to the leptomeninges is an increasingly common occurrence in patients with both haematologicaland solid tumors arising outside the central nervous system oth renement of diagnostic techniques (Magnetic resonanceimaging) and increased survival in patients treated with targeted therapies for systemic tumors account for this increasedfrequency Cerebrospinal uid cytological analysis and MRI conrm clinical diagnosis based on multifocal central nervous systemsignssymptoms in a patient with known malignancy Overall survival in patients with leptomeningeal neoplastic disseminationfrom solid tumors is short rarely exceeding 3-4 months However selected patients may benet from aggressive therapies Apartfrom symptomatic treatment intrathecal chemotherapy is used with both free (methotrexate iotepa AraC) and liposomalantitumor agents (liposomal AraC) Palliative radiotherapy is indicated only in cases of symptomatic bulky disease surgery islimited to positioning of Ommaya recervoirs or C5F shunting We report clinical data on a cohort of 26 prospectively followedpatients with neoplastic leptomeningitis followed in Lombardia Italy in 2011 Prognostic factors and pattern of care are reported

1 Introduction

Neoplastic meningitis is due to dissemination of malignantcells to the leptomeninges and the subarachnoid space Itoccurs in 10ndash15 of haemolymphoproliferative malignan-cies and in 5ndash10 of solid cancers [1]

It more frequently represents late complication of long-standing neoplastic disease but in 10ndash15 of patients maybe the rst-ever manifestation of otherwise occult cancer [1]

e pathways for tumor dissemination to the lep-tomeninges and subarachnoid space include haematogenous

route perineural bloodlymphatic vessels and direct inltra-tion from contiguous sites (for instance dural andor bonemetastases close to the brain and spinal cordroot surface)

Not only extra-CNS tumors but also tumors arisingwithin the CNS (among which gliomas ependymomasmedulloblastomas and germinomas) display relapses andormultifocal presentations with distant foci and a supposedlyintra-CSF pathway of dissemination of neoplastic cells

Guidelines for effective treatment of neoplastic menin-gitis are lacking due to the low levels of evidence which ismostly present for haemolymphoproliferative disease


In meningeal dissemination from solid extra-CNStumors and more so in distant spread of primitive CNStumors there is a lack of uniform approach due to a numberof factors among these the belief of oncologists thatneoplastic meningitis invariably implies a dismal prognosisin the short-term has limited patient recruitment in clinicaltrials

Although this assumption holds true in a high number ofcases it does not apply to the totality of patients however

is consideration together with the more widespreadavailability of MRI facilities in neurooncological diagnosisand with the progress in survival in extra-CNS cancersachieved by chemotherapy and molecularly targeted thera-pies [2] increases the need for accurate diagnosis of neo-plastic meningitis as a prerequisite for accurate validation ofprognostic factors and for enrollment of patients in clinicaltrials

2 Diagnosis of Neoplastic Meningitis

e clinical signs and symptoms of neoplastic meningitis areclassically subdivided in those pointing to cerebral cranialnerve or spinal cordroots involvement Typically in a highproportion of patients symptoms are present suggestingsimultaneous involvement of both cerebral and spinal levelsbut some patients present with isolated decits (for instancean isolated cranial nerve defect)

Cerebral signs and symptoms may either be localized (asin the case of focal seizures) or suggestive of a widespreadbrain dysfunction (for instance drowsiness in hydrocephalusor encephalopathic features in diffuse sulcal enhancement)or be even more unspecic such as headache

e literature reports that the presence of signs at theneurological examination is more frequent as compared tothe reporting of symptoms by the patients during historycollection

Neoplastic meningitis not infrequently coexists withintraparenchymal or dural metastases especially in the caseof breast cancer and leukemialymphoma

e diagnosis of neoplastic meningitis is straightforwardin the majority of cases but a number of cases may posediagnostic challenges

is happens more frequently when the gold standard fordiagnosis (ie CSF cytology) does not yield unequivocallypositive results is may be the casemdashaccording to theliteraturemdashin a proportion of patients ranging from 20 to50ndash60 reasons for this include too little volume of CSFanalyzed distance of the CSF sampling site from the bulkof leptomeningeal disease and delay in CSF processing andanalysis [3 4] e diagnostic yield of CSF cytology increasesignicantly from the rst to the second lumbar puncture torise only negligibly thereaer [5]

In such cases CSF analysis may yield negative results formalignant cells yet display other abnormal features (howeverless specic) such as increase in total proteins and reducedglucose levels as well as moderate reactive pleocytosis

Such CSF pattern may pose serious difficulties in differ-ential diagnosis with CNS infections which may mimic the

neuroradiological picture of NM and are not unexpected inheavily treated cancer patients (for instance chronic fungalandor mycobacterial meningitis)

Some reports have stressed that the closer the CSFsampling to the site of disease the higher the percentage ofpositivity for CSf malignant cells ventricular CSF or lumbarCSFmay thus provide different information as far as cytologyis concerned

In exceptional cases leptomeningeal biopsy is deemednecessary

In neoplastic meningitis from heamatological malignan-cies CSF cytouorimeter analysis has been reported to bemore oen diagnostic as compared to standard cytomorpho-logical analysis [6 7]

As far as the role of MRI is concerned the features of lep-tomeningeal dissemination include both indirect and directevidence of neoplastic cell CSF seeding Among the formerhydrocephalus is not rare duemostly to alterations in theCSFow and particularly in CSF reabsorption at the skull vaultDirect evidence of neoplastic dissemination includes linearor nodular enhancement at leptomeningealependymal level

More subtle signs of alterations in the CSF dynamicsinclude exclusion of part of cerebral sulci with limitedvolumes with increased protein content

3 Management of Neoplastic Meningitis

e role of surgery is limited to resection of symptomaticbulky disease andor biopsy in order to achieve diagnosis inselected cases in some patients positioning of an Ommayarecervoir may allow intraventricular chemotherapy withoutthe need for repeated lumbar punctures but the dynamics ofCSF ow need to be carefully assessed in order to possiblyachieve tumoricidal drug concentrations in the sites ofdisease Ventriculoperitoneal shunting procedures to relievesymptomatic hydrocephalus carry a risk for the developmentof neoplastic dissemination to the peritoneum and are oencomplicated by shunt dysfunctionocclusion

Intrathecal chemotherapy should preferably be deliveredin patients with good PS (see below) with limited extra-CNSdisease and with linear contrast enhancement at MRI (thepenetration of drugs within bulky disease areas is limited to2-3mm)

e NCCN 2012 Guidelines for diagnosis and manage-ment of CNS tumors include brain and spine MRI as well asCSF examination in the workup of patients with suspectedleptomeningeal tumor dissemination According to theseguidelines either positivity of CSF cytology alone or positiveradiologic ndings with supportive clinical ndings or elsesigns and symptoms with suggestive CSF in a patient knownto have a malignancy may be sufficient for diagnosis

Aer diagnosis patients are stratied in either poorrisk (low PS multiple serious major neurologic decitsextensive systemic disease with few treatment options bulkyCNS disease and encephalopathy) or else good risk (highPS no major neurologic decits minimal systemic diseaseand reasonable systemic treatment options)


In the former group only fractionated external beam RTis considered to symptomatic sites and palliative care is thestandard An exception is possible in patients with highlychemosensitive tumors such as lymphoma and SCLC

On the other hand in good risk patients both radiother-apy to bulky disease or symptomatic sites may be deliveredand intrathecal chemotherapy is a worthwhile option

Of note assessment of CSF ow is strongly recommendedbefore initiating intrathecal chemotherapyis assessment ismore frequently performed in northern America while it isless a frequent practice in Europe

With normal CSF ow either craniospinal irradia-tionmdashin the case of breast cancer or lymphomamdashorhigh dosemethotrexate iv in the case of breast cancer or lymphomaor intrathecal chemotherapy with methotrexate or AraC orliposomal AraC are the treatment of choice

Unless an Ommaya recervoir is positioned by the neuro-surgeon repeated intrathecal administration of antineoplas-tic drugs is usually performed via lumbar punctures Withmethotrexate twice weekly administrations are performedduring the induction phase due to the short half life of thedrug in the CSF

Analogous schedules are needed with nonliposomalcytarabine whereas a pegylated formulation of cytarabineallows sustained tumoricidal concentrations in the CSFwhich make once every 2 weeks treatment possible edevelopment of cytarabine encapsulated in multivesicularliposomes has led to detection of CSF concentrations of morethan 01 120583120583GmL persisting at 14 days

In this technology microscopic particles made of aque-ous chambers separated from each other by bilayer lipidmembranes (with synthetic analogs of natural lipids) delivergradually the incorporated drug with subsequent metabo-lization of the membrane remnants via normal pathwaysCytarabine a highly hydrophyilic compound is an idealmolecule for this approach [8]

e achievement of tumoricidal concentrations of cytara-bine in the CSF is of crucial importance since cytarabine is aphase-specic drug affecting only cells in the S phase In theCSF very little activity of the inactivating enzyme cytidinedeaminase enables cytarabine to persist in its biologicallyactive form for longer time as compared to systemic delivery[9]

Only few randomized trials have been conducted onthe effectiveness and toxicity of intrathecal chemotherapy inneoplastic meningitis (reviewed in [10])

In the 1999 published trial by Glantz et al on neoplasticmeningitis from solid tumors [11] intrathecal methotrexatewas compared to liposomal cytarabine in 61 patients Aerthe induction phase a slight increase in the frequency ofpatients attaining a response in the liposomal AraC group(26 versus 20) was seen Overall median survival reached73 days in the latter group and 105 in the former with anonsignicant advantage e only parameter displaying adenite benet in the liposomal AraC group was the timeto neurological progression which was of 58 versus 30 dayswith a statistically signicant difference It remains to be seenwhether this statistically signicant improvement translatesinto a clinically meaningful effect but in this respect the

OS of the whole group of pts

100

80

60

40

20

0

Surv

ival

pro

bab

ilit

y (

)

OS 22 weeks

0 20 40 60 80

Weeks

F 1

studies conducted so far lack detailed quality of life data andthis makes conclusions difficult

Also the 2006 trial by Shapiro and colleagues providesdata pointing to a nonsignicantly different effect of liposo-mal AraC versusmethotrexate in 103 patients with neoplasticmeningitis froms solid tumors [12]

In the other 1999 paper by Glantz et al [13] liposomalAraC was compared to AraC in the treatment of neoplasticmeningitis in a low number (28) of patients with lymphoma-tous meningitis is trial showed an increase in time totumor progression in survival time and in response rate inthe liposomal AraC treated subgroup

Other nonrandomized studies have been performed [1415] investigating the effectiveness and side effects of lipo-somal cytarabine in neoplastic meningitis Overall a fairtolerability prole has emerged e frequent occurrenceof chemical meningitis may be prevented by concomitantsteroid treatment

e main reason for continuing use of liposomal AraCin these patientsmdashapart from the lack of a consolidatedand effective standard of caremdashis the need for less frequentlumbar punctures in oen severely ill patients However thelevels of evidence in favour of this approach are weak Arecent determination of EMA has temporarily suggested toconsider alternative therapies to liposomal AraC aer aninspection to the production site of the drug in Californiatreating physicians are waiting for a solution of this possibilytemporary problem

Other widely adopted intrathecal treatments apart fromliposomal AraC include methotrexate and thiotepa

Preliminary experiences show the feasibility of associ-ating rituximab with liposomal cytarabine in patients withrecurrent neoplastic meningitis [16] Also systemic beva-cizumabmay be effective in some cases on neoplastic menin-gitis [17] in combination with other systemic chemothera-peutic agents

Some effect has been reported for systemic treatmentwith systemic getinib or erlotinib in SCLCwith neoplastic


F 2 Postcontrast T1-weighted MRI images of diffuse enhancement in cerebral sulci and linear enhancement surrounding thedorsolumbar spinal cord and the lumbosacral roots in a 28-yr-old female with breast cancer

F 3 CSF cytology with stain with peroxidase-conjugated anti-cytokeratin antibody and counterstain with haematoxylin (courtesyof Dr E Corsini Fondazione IRCCS Istituto Neurologico BestaMilano)

meningitis and with sorafenib in renal cancer whereasthe role of trastuzumab in breast cancer with neoplasticmeningitis is still debatable (reviewed in [18])

4 Prospective Collection of Newly DiagnosedNeoplastic Meningitis Cases from SolidTumors in Lombardia

In 2011 a prospective collection of patients diagnosed withneoplastic meningitis from solid tumors was started in anumber of Centers in Lombardia e aim of this study is toassess the pattern of care in this oen underdiagnosed andundertreated condition Previous work from an analogousinitiative in Piedmont [19] supports the concept that a higherindex of suspect for diagnosis may lead to earlier diagnosis of

this condition Increase in frequency of neoplastic meningitismay indeed be a consequence of survival increase in a numberof systemic malignancies thanks to advances in targetedtherapies as well as of more widespread use of MRI in thefollowup of these patients

In 12months 26 patients with neoplasticmeningitis fromsolid extra-CNS tumors have been diagnosed eir clinicalfeatures are reported in Tables 1 and 2

Cerebrospinal uid analysis was performed in 22 out of26 patients yielding the following results in 1822 patientsCSF analysis revealed malignant cells Mean values of CSFtotal protein were 152mg (normal values 10ndash45mg)whereas mean CSF glucose was 515mgdL (normal values40ndash80mgdL for normal glycemic levels) Lower than normalglucose levels were only seen in 3 patients out of 22

As reported in Table 3 11 out of the 26 patients weretreated by intrathecal liposomal AraC and 2 by systemicchemotherapy

In this cohort no patientwas treated by radiotherapy aerdiagnosis of neoplastic meningitis

Figure 1 reports overall survival in the entire cohortisattained a median value of 22 weeks in line with data fromthe literature

Assessment of possible prognostic factors showed thatat univariate analysis higher performance status primaryhistology (breast versus others) less elevated CSF proteinand linear contrast enhancement at MRI versus nodular dis-ease as well as intrathecl chemotherapy versus no intrathecalchemotherapy were associated withmore prolonged survival

However probably due to the low number of patients nostatistically signicant differences were detected in subgroupsat multivariate analysis

In Figure 2 the MRI images of a young female affectedby neoplastic meningitis from breast cancer are reportedthis 28-yr-old woman had a 2-year history of ductal carci-noma Her2- hormone receptor-negative with positive lym-phnodes at diagnosis She had been treated with systemicchemotherapy surgery second-line chemotherapy associ-ated with antiangiogenic therapy for relapse and with RT


T 1 Demographic features site of primary tumor and PS

Extra CNS tumor 26Breast 13Lung 7lowast (lowast1 pt lung and colon tumor)Digestive system 3lowast

Melanoma 2Unknown 1Median age (range) 53 yrs (30ndash82)Median KPS (range) 60 (20ndash100)

T 2 Clinical signs and symptoms at onset of neoplasticmeningitis

Signs and symptoms and PS in extra CNS tumorsSpinal cord and root symptoms and signs 926Headache Mental status change 626Meningeal signs and headache 626Cranial nerve symptoms and signs 426Seizures 226

T 3 erapeutic management in the 26 patients of the cohort

Control at primary site of disease 16 yes10 no

Steroids 2226Radiotherapy 026Systemic Chemotherapy 226

Intrathecal Depocyte 1126( median 3 injections)

on lymhnodes 18 months aer diagnosis she developedfever and headache with subsequent rapid development ofconfusion cognitive deterioration behavior abnormalitiesand progression to stupor On neurological examination atadmission the patients was responsive but not oriented inspace and time with signs of meningeal irritation She couldnot walk the sitting position was maintained with difficultyCerebrospinal uid analysis disclosed 90 cells (of which 85malignant cells cytokeratin-positive) with negative culturesextremely low glucose levels (4mg) and slightly increasedtotal proteins (64mg) Due to the very poor conditionsonly palliative care was chosen for this patient who died 4weeks aer diagnosis

Figure 3 shows her CSF cytology with a representativecytokeratin-positive tumor cell

is case underscores the heterogeneity of clinical coursein neoplastic meningitis since it conicts with 2 other cases(both from a primary breast cancer) who are still alive at thepresent followup Differences in themolecular biology proleof tumors within the same histotype are well known and mayindeed play a role also in the more aggressive or indolentcourse of neoplastic meningitis Note that in this case seriesthe majority of patients did not present meningeal irritationsignssymptoms at disease onset

When considering the toxicity prole only one grade4 toxicity occurred In a melanoma patient an inamma-tory encephalopathy picture with seizures stupor signs ofmeningeal irritation nausea moderate increase in temper-ature took place starting 24 hours aer intraventricularadministration of 50mg of liposomal AraC concomitantly aslight intraventricular CSF lymphocytosis was detected eencephalopathy improved progressively leading to recoveryof the premorbid status within 72 hours CSF culture wasnegative for infectious complications

4 more patients displayed moderate postinjectionheadache and slight fever usually starting within 24 hoursfrom intrathecal delivery of liposomal AraC and receding in1 to 2 days

2 patientsmdashboth affected bymetastatic breast cancermdasharealive at a followup ranging from 11 to 23 months

5 Future Developments

Intrathecal chemotherapy for neoplastic meningitis may bea worthwhile option for a number of patients with thisvery serious disease Technological developments allowingslow-release delivery of potentially active drugs may in thefuture be combined with targeted treatments (monoclonalantibodies small molecule inhibitors) focused on multistepinhibition of neoplastic cell survival growth and spreadingwithin the neuraxis

However a better basic knowledge of the biologicalmechanisms underlying selective homing of neoplastic cellsto the leptomeninges together with strict monitoring of theriskbenet ratio [20 21] will be needed before routineadoption of these approaches becomes a standard of care

is is very important since increased survival times are(also) the consequence of more aggressive systemic treat-ments which may signicantly enhance the neurotoxicity ofintrathecal therapies [22ndash24]

References

[1] B Gleissner and M C Chamberlain ldquoNeoplastic meningitisrdquoe Lancet Neurology vol 5 no 5 pp 443ndash452 2006

[2] S Kesari and T T Batchelor ldquoLeptomeningeal metastasesrdquoNeurologic Clinics vol 21 no 1 pp 25ndash66 2003

[3] L M DeAngelis ldquoCurrent diagnosis and treatment of lep-tomeningeal metastasisrdquo Journal of Neuro-Oncology vol 38 no2-3 pp 245ndash252 1998

[4] M C Chamberlain P A Kormanik and M J Glantz ldquoA com-parison between ventricular and lumbar cerebrospinal uidcytology in adult patients with leptomeningeal metastasesrdquoNeuro-Oncology vol 3 no 1 pp 42ndash45 2001

[5] W R Wasserstrom J P Glass and J B Posner ldquoDiagnosisand treatment of leptomeningeal metastases from solid tumorsexperience with 90 patientsrdquoCancer vol 49 no 4 pp 759ndash7721982

[6] U Hegde A Filie R F Little et al ldquoHigh incidence ofoccult leptomeningeal disease detected by ow cytometry innewly diagnosed aggressive B-cell lymphomas at risk for centralnervous system involvement the role of ow cytometry versuscytologyrdquo Blood vol 105 no 2 pp 496ndash502 2005


[7] A Orfao S uijano A Lpez et al ldquoIdentication ofleptomeningeal disease in aggressive B-Cell non-Hodgkinrsquoslymphoma improved sensitivity of ow cytometryrdquo Journal ofClinical Oncology vol 27 no 9 pp 1462ndash1469 2009

[8] D J Murry and S M Blaney ldquoClinical pharmacology ofencapsulated sustained-release cytarabinerdquo Annals of Pharma-cotherapy vol 34 no 10 pp 1173ndash1178 2000

[9] S Zimm J M Collins and J Miser ldquoCytosine arabinosidecerebrospinal uid kineticsrdquo Clinical Pharmacology and er-apeutics vol 35 no 6 pp 826ndash830 1984

[10] M C Chamberlain ldquoLeptomeningeal metastasisrdquo CurrentOpinion in Oncology vol 22 no 6 pp 627ndash635 2010

[11] M J Glantz K A Jaeckle M C Chamberlain et al ldquoArandomized controlled trial comparing intrathecal sustained-release cytarabine (DepoCyt) to intrathecal methotrexate inpatients with neoplastic meningitis from solid tumorsrdquo ClinicalCancer Research vol 5 no 11 pp 3394ndash3402 1999

[12] W R Shapiro M Schmid M Glantz et al ldquoA randomizedphase IIIIV study to determine benet and safety of cytarabineliposome injection for treatment of neoplastic meningitisrdquoJournal of Clinical Oncology vol 24 p 1528 2006

[13] M J Glantz S LaFollette K A Jaeckle et al ldquoRandomized trialof a slow-release versus a standard formulation of cytarabine forthe intrathecal treatment of lymphomatousmeningitisrdquo Journalof Clinical Oncology vol 17 no 10 pp 3110ndash3116 1999

[14] W Boogerd M J Van Den Bent P J Koehler et al ldquoerelevance of intraventricular chemotherapy for leptomeningealmetastasis in breast cancer a randomised studyrdquo EuropeanJournal of Cancer vol 40 no 18 pp 2726ndash2733 2004

[15] I Gil-Bazo J Rodriguez J Espinos et al ldquoe safety andefficacy of intrathecal liposomal cytarabine in patients with car-cinomatous meningitis from solid tumorsrdquo European Journal ofCancer Supplements vol 7 abstract 501 2009

[16] M C Chamberlain S K Johnston A Horn and M J GlantzldquoRecurrent lymphomatous meningitis treated with intra-CSFrituximab and liposomal ara-Crdquo Journal of Neuro-Oncology vol91 no 3 pp 271ndash277 2009

[17] G Y Ku G Krol and D H Ilson ldquoSuccessful treatment ofleptomeningeal disease in colorectal cancer with a regimenof bevacizumab temozolomide and irinotecanrdquo Journal ofClinical Oncology vol 25 no 13 pp e14ndash16 2007

[18] G Lombardi F Zustovich P Farina et al ldquoNeoplasticmeningitis from solid tumors new diagnostic and therapeuticapproachesrdquoe Oncologist vol 16 pp 1175ndash1188 2011

[19] L Bertero E Picco E Trevisan et al ldquoFrequenza opzioniterapeutiche e sopravvivenza della meningite neoplastica (mn)da tumori solidi nella regione Piemonte studio prospet-tico di una rete oncologicardquo in 15th Congressi Nazionali-nazionalemdashAssociazione Italiana di Neuro-Oncologia (AINOrsquo10) pp 3ndash6 Fiuggi Italy ottobre 2010

[20] A G Mammoser and M D Groves ldquoBiology and therapy ofneoplastic meningitisrdquo Current Oncology Reports vol 12 no 1pp 41ndash49 2010

[21] J Grewal M Garzo Saria and S Kesari ldquoNovel approachesto treating leptomeningeal metastasesrdquo Journal of Neuro-Oncology vol 106 pp 225ndash234 2012

[22] E Jabbour S OrsquoBrien H Kantarjian et al ldquoNeurologic compli-cations associated with intrathecal liposomal cytarabine givenprophylactically in combination with high-dose methotrexateand cytarabine to patients with acute lymphocytic leukemiardquoBlood vol 109 no 8 pp 3214ndash3218 2007

[23] B McClune F K Buadi N Aslam and D PrzepiorkaldquoIntrathecal liposomal cytarabine for prevention of meningealdisease in patients with acute lymphocytic leukemia and high-grade lymphomardquo Leukemia and Lymphoma vol 48 no 9 pp1849ndash1851 2007

[24] J Watterson I Toogood M Nieder et al ldquoExcessive spinalcord toxicity from intensive central nervous system-directedtherapiesrdquo Cancer vol 74 pp 3034ndash3041 1994

Hindawi Publishing CorporationJournal of Drug DeliveryVolume 2012 Article ID 167896 14 pagesdoi1011552012167896

Review Article

Nanomaterials Toxicity and Cell Death Modalities

Daniela De Stefano1 Rosa Carnuccio1 and Maria Chiara Maiuri1 2

1 Dipartimento di Farmacologia Sperimentale Facolta di Scienze Biotecnologiche Universita degli Studi di Napoli Federico IIVia D Montesano 49 80139 Napoli Italy

2 INSERM U848 IGR 39 Rue C Desmoulins 94805 Villejuif France

Correspondence should be addressed to Maria Chiara Maiuri mcmaiuriuninait

Received 10 September 2012 Accepted 7 November 2012


Copyright copy 2012 Daniela De Stefano et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

In the last decade the nanotechnology advancement has developed a plethora of novel and intriguing nanomaterial applicationin many sectors including research and medicine However many risks have been highlighted in their use particularly relatedto their unexpected toxicity in vitro and in vivo experimental models This paper proposes an overview concerning the cell deathmodalities induced by the major nanomaterials

1 Introduction

Nanotechnologies are emerging for important new appli-cations of nanomaterials in various fields Nanomaterialsare defined as substances which have one or more externaldimension in the nanoscale (1ndash100 nm) Nanomaterialsespecially nanoparticles and nanofibres show higher physicaland chemical activities per unit weight These propertiesexplain their large application not only in industry but alsoin the scientific and medical researches In fact in theseareas the use of many kinds of manufactured nanoparticlesproducts is in development such as metal oxide nanopar-ticles (cerium dioxide cupric oxide titanium dioxide zincoxide etc) metal nanoparticles (gold silver platinum pal-ladium etc) C60 fullerenes nanocrystals carbon nanotubes(CNTs) and quantum dots Initially the nanomaterials werebelieved to be biologically inert but a growing literaturehas highlighted the toxicity and potential risks of their useExtrapolations from the field of toxicology of particulatematter (less than 10 nm) confirm that nanoparticles present arange of harmful effects [1 2] In most cases enhanced gen-eration of reactive oxygen species (ROS) leading to oxidativestress which in turn may trigger proinflammatory responsesis assumed to be responsible for nanomaterials toxicityalthough nonoxidative stress-related mechanisms have alsobeen recently reported (see the extensive and interesting

reviews [3ndash10]) However despite intensive investigationsthe understanding of nanomaterials-induced cellular damageremains to be clarified The literature in the field suggestscorrelations between different physicochemical propertiesand the biological and toxicological effects of cells and tissuesexposure to nanomaterials First of all nanomaterials arecharacterized by high specific surface area that correlateswith high interfacial chemical and physical reactivity thatin turn translates to biological reactivity [11] The additionof different types of nanoparticles to various primary cellcultures or transformed cell lines may result in cell deathor other toxicological outcomes depending on the size ofthe nanomaterial Quantum dots were reported to localize todifferent cellular compartment in a size-dependent manner[12] Silica nanoparticles (40ndash80 nm) can enter into thenucleus and localize to distinct subnuclear domains inthe nucleoplasm whereas thin and coarse ones locatedexclusively in the cytoplasm [13] Gold nanocluster (14 nm)intercalates within the major groove of DNA and is a potentinducer of cell death in human cancer cells [14] Growingevidence suggests that the state of nanoparticles aggregationcannot be ignored in fact the toxicity may depend on thesize of the agglomerate and not on the original nanoparticlesize itself [15 16] For example in rats exposed by inhalationto 20 nm or 250 nm titanium dioxide (TiO2) particles thehalf-times for alveolar clearance of polystyrene test particles


accumulation

Surfacechanges

Biodegradability

Cell-site specific

(nucleuscytoplasm)

SizeshapeNanomaterials features leading

to toxicity

Adsorption of

proteins ions etc

Chemical natureAggregation

Figure 1

were proportional to the TiO2 particle surface area permillion of macrophages [17 18] Clearly a surface impurityresulting from air or water contaminants such as bacterialendotoxin could contribute to the cellular responses inducedby nanomaterials in particular immunological responses[16] The same consideration is true for residual materials(surfactants or transition metals) arising from the syntheticprocess [6 19 20] Nevertheless the adsorption ability andsurface activity are also involved in cellular influences ofnanomaterials When dispersed in culture medium somemetal oxide nanoparticles and CNTs could adsorb proteinsoften called ldquoprotein coronardquo such as serum albumin orcalcium which could change the biological activity of nano-materials This adsorption could be particle size and timedependent In these conditions many nanoparticles formsecondary particles which are a complex of nanoparticlesand medium components [21ndash26] For example adsorbedalbumin on the CNT was involved in phagocytosis ofthe macrophage via scavenger receptor [27] A surface-engineered functionalization also may be linked with thebiological nanomaterials activity although in this item that isa wanted effect Moreover examples of dose-dependent tox-icity also are evaluated [6 28 29] As pointed out in a recentreview [6] the degree of recognition and internalization ofnanomaterials likely influences their distribution and maydetermine also their toxic potential It has been reported thatthe number of internalized quantum dots (the intracellulardose) correlates with the toxicity in human breast cancercell line [30] Furthermore the toxicity and cell death fateappear to correlate with the type of crystal structures [1631] Finally the nanomaterials degradability should also betaken into account (Figure 1) Nondegradable nanomaterialscan accumulate into the cells andor organs and exertdamage effect as well as their degradation products [32ndash34]However it is not yet clear which of these parameters mainlyinfluences the nanomaterials toxicity or if all of these featuresact together [35] It is important to note that in the literatureconflicting results are present These are likely caused byvariations in type composition size shape surface chargeand modifications of nanoparticles employed use of variousin vivo and in vitro models (the cell death mode may be

also cell type dependent) experimental procedures (differentmethods to evaluate cell death nanomaterials dose concen-trations and efficiency of cellular uptake and time of expo-sure) This paper aims to give a critical overview concerningthe different cell death modalities induced by nanomaterials

Deregulated cell death is a common element of severalhuman diseases including cancer stroke and neurodegen-eration and the modulation of this cellular response can bean optimal target for an effective therapeutic strategy Manycytotoxic agents are potent anticancer therapeutics whereascytoprotective compounds may be used to elude unwantedcell death in the context of stroke myocardial infarction orneurodegenerative disorders [36 37] The complex molec-ular mechanisms and signalling pathways that control celldeath are increasingly becoming understood and it is nowclear that different cell death subroutines play a critical rolein multiple diseases In many instances the modality bywhich cells die is crucial to the cell death achievement atthe organism level The Nomenclature Committee on CellDeath (NCCD) has recently formulated a novel systematicclassification of cell death based on morphological char-acteristics measurable biochemical features and functionalconsiderations [38] We will consider these definitions of celldeath in order to summarize and organize the molecularmechanisms underlying the nanomaterials toxicity We couldnot report all the studies and we apologize for this we willdescribe the most recently accurate and representative onesin term of the described molecular mechanisms

2 Nanomaterials and Apoptosis

Apoptosis is a form of cellular suicide that can be classifiedinto extrinsic and intrinsic apoptosis Extrinsic apoptosisindicates the cell death caspase dependent stimulated byextracellular stress signals that are sensed and propagatedby specific transmembrane receptors Three major lethalsignalling cascades have been reported (i) death receptorsignalling and activation of the caspase-8 (or -10) andthen caspase-3 cascade (ii) death receptor signalling andactivation of the caspase-8 then BH3-interacting domain


death agonist (BID) mitochondrial outer membrane perme-abilization (MOMP) caspase-9 and caspase-3 pathways and(iii) ligand deprivation-induced dependence receptor sig-nalling followed by (direct or MOMP-dependent) activationof the caspase-9 and after caspase-3 cascade [38] Intrinsicapoptosis can be triggered by a plethora of intracellular stressconditions such as DNA damage oxidative stress and manyothers It results from a bioenergetic and metabolic catastro-phe coupled to multiple active executioner mechanisms Thisprocess could be caspase-dependent or- independent andis mediated by MOMP associated with the generalized andirreversible dissipation of the mitochondrial transmembranepotential release of mitochondrial intermembrane spaceproteins into the cytosol (and their possible relocalizationto other subcellular compartments) and the respiratorychain inhibition [38] Apoptosis plays a fundamental rolein development and for maintenance of tissue homeostasisin the adult organism In addition impairment of apoptosismay contribute to tumour progression

Nanomaterials are described as triggers of extrinsic andintrinsic apoptotic pathways however the oxidative stressparadigm of nanomaterials-induced cell death linked tointrinsic apoptotic network is by far the most accepted infact many in vitro studies have identified increased ROSgeneration as an initiating factor of toxicity in nanomaterialsexposed cells [3 6 7 10 39] Although it is well establishedthat the mode of cell death depends on the severity of the cel-lular insult (which may in turn be linked to mitochondrialfunction and intracellular energy) it has been difficult to setup a comprehensive mechanism of nanomaterials cell deathbased on conflicting observations present in the literatureFurthermore in most of the studies the molecular mech-anisms underlying cell death are not investigated Finallyanother problem is the nonhomogeneity of the studies interms of materials and experimental methods used whichmakes it difficult to compare

Sarkar and colleagues showed that the nano-copperinduces intrinsic apoptotic cell death in mice kidney tissue(via the increase of ROS and reactive nitrogen speciesproduction regulation of Bcl-2 family protein expressionrelease of cytochrome c from mitochondria to cytosol andactivation of caspase-3) but in addition they observed theactivation of FAS caspase-8 and tBID suggesting also theinvolvement of extrinsic pathways [40] The exposure tonano-copper dose-dependently caused oxidative stress andled to hepatic dysfunction in vivo Nano-copper caused thereciprocal regulation of Bcl-2 family proteins disruption ofmitochondrial membrane potential release of cytochrome cformation of apoptosome and activation of caspase-3These results indicate that nano-copper induces hepatic dys-function and cell death via the oxidative stress-dependentsignalling cascades and mitochondrial event [41]

Metallic nickel nanoparticles induced apoptotic celldeath through an FAScaspase-8BID mediated cytochromec-independent pathway in mouse epidermal cells [42] Nickeloxide nanoparticles excited in dose-dependent mannerthe increase of ROS production lipid peroxidation andcaspase-3 activation in human airway epithelial and breastcancer cells [43] Moreover nickel ferrite nanoparticles

provoked apoptosis in human lung epithelial cells throughROS generation via upregulation of p53 and Bax as well asthe activation of caspases cascade [44]

In vitro silicon dioxide (SiO2) nanoparticles increasedROS and RNS (reactive nitrogen species) production thatin turn can induce the intrinsic apoptotic machinery [45]Furthermore Wang and collaborators showed that p53plays a key role in silica-induced apoptosis in vitro (mousepreneoplastic epidermal cells and fibroblasts) and in vivo(p53 wild-type and deficient mice) [46]

TiO2 nanoparticles sized less than 100 nm triggeredapoptotic cell death through ROS-dependent upregulation ofFAS and activation of Bax in normal human lung fibroblastand breast epithelial cell lines [47] Moreover it was alsodemonstrated that TiO2 nanoparticles induced apoptosisthrough the caspase-8BID pathway in human bronchialepithelial cells and lymphocytes as well as in mouse preneo-plastic epidermal cells [48 49] Some reports indicated thatTiO2 induced also lipid peroxidation p53-mediated damageresponse and caspase activation [50 51] In contrast thereare also reports demonstrating that TiO2 nanoparticles didnot induce oxidative stress on mouse macrophages [52] aswell as did not shown cytotoxicity in human dermal fibrob-lasts and lung epithelial cells [31]

A number of studies have been published concerning theeffects of CNTs on apoptosis Multiwall carbon nanotubes(MWCNTs) induced an increase of ROS cell cycle arrestdecrease in mitochondrial membrane potential determiningapoptosis in different in vitro models [53ndash56] In contrastanother study reported that these nanotubes were nontoxic[57] Accordingly it has been observed that MWCNTs didnot stimulate cell death in vitro after acute exposure andneither after the continuous presence of their low amountsfor 6 months [58] Instead apoptotic macrophages have beenobserved in the airways of mice after inhalation of SWCNTs(single-walled carbon nanotubes) [6] Accordingly severalstudies in vivo suggest that the exposure to SWCNTs leadsto the activation of specific apoptosis signalling pathways[59 60] For more details recent interesting reviews focuson the nanomaterials toxicity in vivo studies [6 34]

Nanoparticles are frequently detected in lysosomes uponinternalization and a variety of nanomaterials have beenassociated with lysosomal dysfunction [61] It has beenestablished that lysosomal destabilization triggers the mito-chondrial pathway of apoptosis [62 63] Carbon nanotubeswere shown to induce lysosomal membrane permeabiliza-tion and apoptotic cell death in murine macrophages andhuman fibroblasts [64 65] Carbon black nanoparticleselicited intrinsic apoptosis in human bronchial epithelialcells with activation of Bax and release of cytochrome c frommitochondria whereas TiO2 nanoparticles induced apopto-sis through lysosomal membrane destabilization and cathep-sin B release suggesting that the pathway of apoptosisdiffers depending on the nanomaterials chemical nature [66]The lysosomal destabilization induced by TiO2 is also con-firmed in mouse fibroblasts [67] SiO2 and several cationicnanoparticles such as cationic polystyrene nanospheresand cationic polyamidoamine (PAMAM) dendrimers havealso shown the same mode of action [68ndash70] However


also the micromaterials are able to destabilize lysosomesin fact silica microparticles have been demonstrated toinduce apoptosis in mouse alveolar macrophages by thismolecular mechanism [70] A comparative study of nano-versus microscale gold particles demonstrated that nanopar-ticles present a higher potency in the induction of lysosomalmembrane destabilization [71]

Chronic or unresolved endoplasmic reticulum (ER)stress can also cause apoptosis [72 73] Zhang and colleaguesreported that the ER stress signalling is involved in silvernanoparticles-induced apoptosis in human Chang liver cellsand Chinese hamster lung fibroblasts [74] Using omictechniques and systems biology analysis Tsai and collabo-rators demonstrated that upon ER stress cellular responsesincluding ROS increase mitochondrial cytochrome c releaseand mitochondria damage chronologically occurred inthe gold nanoparticles-treated human leukemia cells Thistreatment did not induce apoptosis in the normal humanperipheral blood mononuclear cells [75] It has been shownthat poly(ethylene glycol)-phosphoethanolamine (PEG-PE)an FDA-approved nonionic diblock copolymer widely usedin drug delivery systems accumulated in the ER andinduced ER stress and apoptosis only in cancer cells (humanadenocarcinomia alveolar basal epithelial) whereas it did nothave effect in normal cells (secondary human lung fibroblastsand embryonic kidney cells) [76]

The predisposition of some nanoparticles to target mito-chondria ER or lysosomes and initiate cell death could beused as a new cancer chemotherapy principle

Interestingly nanoparticles (polystyrene nanoparticles of20ndash40 nm with two different surface chemistries carboxylicacid and amines) may also induce apoptosis in individualcells (differentiated human colorectal adenocarcinoma) thatthen propagates to other neighbouring cells through aldquobystander killing effectrdquo The authors of this study suggestthat ingested nanoparticles represent a potential health riskdue to their detrimental impact on the intestinal membraneby destroying their barrier protection capability over time[77]

Surely in this context a common incentive to synchroni-ze the studies and research efforts is needed The understandwhy cancer cells and distinctive normal cells have differentcell fates as a result of nanomaterials exposure focusing onthe underlying mechanisms will allow a better prediction ofthe consequences of exposure to nanomaterials and a saferassessment of the risks (Figure 2)

3 Nanomaterials and Mitotic Catastrophe

Recently Vitale and colleagues suggested a novel definition ofmitotic catastrophe based on functional consideration [78]They proposed to consider mitotic catastrophe not a ldquopurerdquocell death executioner pathway but as an oncosuppressivemechanism that is triggered by perturbations of the mitoticapparatus is initiated during the M phase of the cell cycle isparalleled by some degree of mitotic arrest and induces celldeath (apoptosis or necrosis) and senescence [78]

It has been reported that several nanomaterials suchas SiO2 TiO2 cobalt-chrome (CoCr) metal particles and

carbon nanotubes interact with structural elements of thecell with an apparent binding to the cytoskeleton andin particular the tubulins [79 80] In this setting someevidence in vitro demonstrated that carbon nanotubes mimicor interfere with the cellular microtubule system therebydisrupting the mitotic spindle apparatus and leading toaberrant cell division [81ndash83] In particular the perturbationof centrosomes and mitotic spindles dynamics caused bythese nanoparticles results in monopolar tripolar andquadripolar divisions that in turn could determinateaneuploidy [78] an event closely linked to the carcinogen-esis Tsaousi and collaborators found that alumina ceramicparticles increase significantly in micronucleated binucleatecells [84] which is considered a morphological markerof mitotic catastrophe [78] Interestingly this increase wasmuch greater after exposure of primary human fibroblaststo CoCr metal particles suggesting that these nanoparticlesare particularly efficient in affecting the mitotic machinery[84] Apparently the genotoxic effect of CoCr nanoparticlesis size dependent Indeed CoCr nanoparticles induced moreDNA damage than microsized ones in human fibroblasts(Figure 3) In fact the mechanism of cell damage appearsto be different after nano- or microparticles exposure Theenhanced oxidative DNA damage by the microparticles mayresult from a stronger ability of large particles to activateendogenous pathways of reactive oxygen species formationfor example involving NADPH oxidases or mitochondrialactivation It also suggests that the observed genotoxic effectof the nanoparticles in the comet assay and the micronucleusassay (ie stronger aneugenic effect) is due to mechanismsother than oxidative DNA attack A different mechanism ofDNA damage by nanoparticles and microparticles is furthersuggested by measures of DNA damage from the cometand micronucleus assays The comet assay revealed moredamage in nanoparticle-exposed than in microparticle cellsIn contrast the micronucleus assay revealed slightly lesscentromere-negative micronuclei in nanoparticle exposedthan in microparticle-exposed cells This assay measuresclastogenic that is double strand breakage events Althoughsome micronuclei in nanoparticle-exposed cells might nothave been seen as a result of inhibition of cell division fromgreater cytotoxicity these results point to a greater com-plexity of DNA damage caused by exposure to nanoparticlescompared to microparticles [85] A genotoxic effect has alsodescribed for silver nanoparticles that induced chromosomalaberrations damage of metaphases and aneuploidy in med-aka (Oryzias latipes) cell line [86]

Further studies are needed to validate this dangerouspotential effect of the nanomaterials Obviously close atten-tion to safety issues will be required also in the light ofthe potential interference between engineered nanomaterialsand the environment

4 Nanomaterials and Autophagy orldquoAutophagic Cell Deathrdquo

Autophagy is a highly conserved homeostatic processinvolved in the recognition and turnover of damagedaged


Lysosomaldysfunction

Mithocondrial

apoptosis

Lysosomalmembrane

permeabilization

nanoparticles

ROS

ER stress

nanoparticlesNanoparticles Carbon black PEG-PEAu

Cancer cells

Apoptosis

Cathepsin B

pathway of

CNTsTiO2SiO2

Figure 2

Alumina ceramic

Binding to

cytoskeletal tubulin

Mitotic catastrophe

Disruption

division

carcinogenesisCoCr

CoCrTiO2SiO2CNTsof mitoticapparatus

perturbationof centromers

Aberrant cell

Aneuplody

Figure 3

proteins and organelles During autophagy parts of thecytoplasm are sequestered within characteristic double- ormulti-membraned autophagic vacuoles (named autophago-somes) and are finally delivered to lysosomes for bulkdegradation This process is dynamically regulated by ATG(Autophagy-related gene) gene family and is finely controlledby several signalling pathways [87] Autophagy constitutes acytoprotective response activated by cells in the challenge tocope with stress In this setting pharmacological or geneticinhibition of autophagy accelerates cell death On the basis ofmorphological features the term ldquoautophagic cell deathrdquo haswidely been used to indicate instances of cell death that areaccompanied by a massive cytoplasmic vacuolization [38]The expression ldquoautophagic cell deathrdquo is highly prone tomisinterpretation and hence must be used with caution butdiscussion this problem is beyond the scope of this paperand an excellent paper concerning this subject has beenpublished [88] In any case ldquoautophagic cell deathrdquo is usedto imply that autophagy would execute the cell demise Inthe literature it has been reported that several classes ofnanomaterials induce elevated levels of autophagic vacuoles

in different animals and human cell culture as well as invivo models (masterfully summarized in two recent reviews[10 61]) Such nanomaterials include alumina europiumoxide gadolinium oxide gold iron oxide manganeseneodymium oxide palladium samarium oxide silica ter-bium oxide titanium dioxide ytterbium oxide and yttriumoxide nanoparticles nanoscale carbon black fullerene andfullerene derivate and protein-coated quantum dots Theinduction of autophagy was evaluated using panoply ofestablished methods including the electron microscopydetection of autophagic vacuoles the immunoblot detectionof ATG expression level andor LC3-I to LC3-II conversion(an established marker of autophagy activity) andor cellularimmunolabeling of punctate LC3-II in cytoplasmic vacuolesThese studies were performed in vivo but mainly in primarycells andor cell lines from rat (alveolar macrophages kidneydopaminergic neuron and glioma) mouse (macrophagesand neuroblasts) porcine (kidney) and human (lung oralcolon breast cervical and epithelial cancer cells as well asfibroblasts peripheral blood mononuclear and endothelialand mesenchymal stem cells) Nanomaterials may induce


autophagy via an oxidative stress mechanism such as accu-mulation of damaged proteins and subsequent endoplasmicreticulum or mitochondrial stress [39 89ndash92] and alteringgeneprotein expression andor regulation and interferingwith the kinase-mediated regulatory cascades [93ndash103] Theincrease in autophagic vacuoles in response to nanomaterialsmay be an adaptive cellular response There is evidence thatautophagy can selectively compartmentalize nanomaterialsIn fact nanoparticles are commonly observed within theautophagosome compartment suggesting that activation ofautophagy is a targeted exertion to sequester and degradethese materials following entrance into the cytoplasm [104]It is possible that the cells might perceive nanomaterials as anendosomal pathogen or an aggregation-prone protein (bothcommonly degraded by the autophagy machinery) Recentevidence supports ubiquitination of nanomaterials directlyor indirectly via colocalization with ubiquitinated proteinaggregates suggesting that cells may indeed select nanoma-terials for autophagy through a pathway similar to invadingpathogens [13 98 105] Additionally ubiquitinated pro-teins accumulate concomitantly with nanomaterial-inducedautophagic vacuoles [106]

It is important to underlie that nanoscale was a signifi-cant factor in eliciting the autophagic response Autophagywas not induced by quantum dots that had a tendencyto aggregate to microscale particles into the cells [107]Nanoscale size dependence was also reported for neodymiumoxide nanoparticle with larger particles inducing less auto-phagy [108] Apparently modifications of the surface prop-erties might be able to alter the autophagy-inducing activityof the nanomaterials Cationic PAMAM dendrimers elicitedautophagy more than anionic ones in vitro [94] Carbon nan-otubes with carboxylic acid group could induce autophagywhile those functionalized with poly-aminobenzene sulfonicacid and polyethylene glycol groups were not [100] Recentlyit has been published that a short synthetic peptide RE-1 binds to lanthanide-based nanocrystals forms a stablecoating layer on the nanoparticles surface and significantlyabolishes their autophagy-inducing activity Furthermorethe addition of an arginine-glycine-aspartic acid motif toRE-1 enhances autophagy induced by lanthanide-basednanocrystals [109]

It is also possible that nanomaterials cause a state ofautophagic dysfunction correlated with a blockade of auto-phagy flux and this may be involved in their mechanismof toxicity [110 111] Nanoparticles could give rise toautophagy dysfunction by overloading or directly inhibitinglysosomal enzymes or disrupting cytoskeleton-mediatedvesicle trafficking resulting in diminished autophagosome-lysosome fusion [112] Nanoparticles could also directlyaffect lysosomal stability by inducing lysosomal oxidativestress alkalization osmotic swelling or causing detergent-like disruption of the lysosomal membrane (see the completereview of Stern and colleagues [61] about this subject)Disruption in autophagosome trafficking to the lysosomehas been implicated in several human pathologies includingcancer development and progression as well as neurodegen-erative diseases As exposure to airborne pollution has beenassociated with Alzheimer and Parkinson-like pathologies

and nanoparticles are the primary particle number andsurface area component of pollution-derived particulatesStern and Johnson have recently postulated a relationshipbetween nanoparticle-induced autophagy dysfunction andpollution-associated neurodegeneration [113]

Several studies have been suggested also that thenanomaterial-induced autophagy dysfunction is correlatedwith mitochondrial damage [102 114ndash118]

In the majority of the studies autophagosome accu-mulation induced by nanomaterials treatment was asso-ciated with cell death unfortunately the possibility ofautophagy inhibition was not often investigated (the blockof autophagy flux and autophagy induction both can deter-minate autophagosome accumulation) [119] and the mech-anism of nanomaterial-induced autophagy accumulation inmany cases is unclear

Interestingly nanomaterials have been proposed also astools to monitor autophagy [120 121] In conclusion agrowing body of the literature indicates that nanomaterialsimpact the autophagy pathways then the possible autophagicresponse should be always taken into consideration in thedevelopment of novel nanomaterials systems (Figure 4)Moreover further studies should be performed to clarify themolecular mechanisms underlying the interaction betweennanomaterials and the autophagy machinery as well as toexpand the knowledge of the implications and biologicalsignificance of this modulation

5 Nanomaterials and Necrosis

Necrosis was for a long time considered as an accidentalform of cell death but in recent years several studies clarifiedthat this process is regulated and may play a role in multiplephysiological and pathological settings [122] Several triggerscan induce regulated necrosis including alkylating DNAdamage excitotoxins and the ligation of death receptors [38122] Indeed when caspases are genetically or pharmacolog-ically inhibited RIP1 (receptor-interacting protein kinase 1)and its homolog RIP3 are not degraded and engage inphysical and functional interactions that ultimately activatethe execution of necrotic cell death [38 122] It shouldbe noted that RIP3-dependent and RIP1-independent casesof necrosis have been described suggesting that there areseveral subprograms of regulated necrosis [38 122ndash124]In a genome-wide siRNA screen Hitomi and colleagueselucidated the relationship between appotosis and necrosispointing out that some components of the apoptotic pathway(eg the BH3-only protein Bmf) are also crucial in thenecrotic machinery [125] Moreover recent studies provideevidence that apoptosis and necrosis are closely linked [126ndash128] The term ldquonecroptosisrdquo has been used as a synonymof regulated necrosis but it was originally introduced toindicate a specific case of necrosis which is induced by deathreceptor ligation and can be inhibited by the RIP-1 targetingchemical necrostatin-1 [38 122 129]

In the literature there are confused and inconsistentexamples of necrosis induced by nanomaterials because onone hand only the loss of cell viability is often evaluatedwithout focalising into the cell death modalities and on


Aluminametal oxidesCNTsfullerene

Alteration of geneprotein

expressionregulation

Damaged proteins

Interfering with kinase

cascades

Oxidative stress

stress

lysosomalenzymes

Reduced

function

Autophagy

dysfunction

ERmithocondria

Inhibitionoverloading of

autophagosome-lysosome

cytoskeletal-mediatedvesicle trafficking

Disruption of

Figure 4

the other hand there are no single discriminative bio-chemical markers available yet Moreover it should not beunderestimated that the induction of apoptosis in cell cultureis inevitably followed by secondary necrosis and this couldlead to a misinterpretation of results However a recent studydemonstrated that water-soluble germanium nanoparticleswith allylamine-conjugated surfaces (4 nm) induce necroticcell death that is not inhibited by necrostatin-1 in Chinesehamster ovary cells [130] Although the mechanisms of lig-and and surface chemistry surface charge and crystallinity-based toxicity are complex studies are beginning to elucidatecertain surface functional groups and properties that caneffectively alter biological responses In fact the crystal struc-ture with the different forms of nanomaterials can dictate itscytotoxic potential Braydich-Stolle and coworkers identifythat both size and crystal structure (rutile anatase andamorphous) of TiO2 nanoparticles affect the mechanism ofcell death in mouse keratinocyte cell line [131] They foundthat 100 anatase TiO2 nanoparticles induced necrosis insize-independent manner whereas the rutile TiO2 nanopar-ticles elicited apoptosis Pan and collaborators investigatedthe size-dependent cytotoxicity exhibited by gold nanopar-ticles (stabilized with triphenylphosphine derivatives) inseveral human cell lines All cell types internalised goldnanoparticles and showed signs of stress Smaller particles(lt14 nm) were more toxic than their larger equivalentsHowever 14 nm nanoparticles cause predominantly rapidcell death by necrosis while closely related particles 12 nm indiameter affect predominantly apoptosis [132 133] Besidesit has been reported that small (10 nm) silver nanoparticleshad a greater ability to induce apoptosis than other-sizedones (50 and 100 nm) in mouse osteoblastic cell line andinduce necrosis in rat phaeochromocytoma cells [134] Theshape-dependent toxicity of polyaniline (PANI) nanomateri-als with four different aspect ratios on human lung fibroblastcells was evaluated The toxicity increased with decreasingaspect ratio of PANI nanomaterials low aspect ratio PANI

nanomaterials induced more necrosis than others [135]Furthermore the surface charge seems to be a major factorof how nanoparticles impact cellular processes It has beendemonstrated that charged gold nanoparticles induced celldeath via apoptosis whereas neutral nanoparticles causednecrosis [136] Clearly other parameters may influence thecell death modalities induced by nanomaterials such as thedose or the time of exposure Depending on the concen-tration nano-C60 fullerene caused ROS-mediated necrosis(high dose) or ROS-independent autophagy (low dose) inrat and human glioma cell cultures [137] The type of celldeath induced by silver ions (Ag+) and silver nanoparticlecoated with polyvinylpyrrolidone were also dependent on thedose and the exposure time with Ag+ being the most toxic ina human monocytic cell line [138] The silver nanoparticlesconcentrations required to elicit apoptosis were found to bemuch lower than the concentrations required for necrosis inhuman fibrosarcoma skin and testicular embryonal carci-noma cells [139 140] In conclusion although the reportsare often contradictory the cell death appears roughly celltype material composition and concentration dependentFor instance it has been reported that TiO2 (5ndash10 nm)SiO2 (30 nm) and MWCNTs (with different size lt8 nm 20ndash30 nm and gt50 nm but same length 05ndash2 μm) induce cell-specific responses resulting in variable toxicity and subse-quent cell fate in mouse fibroblasts and macrophages as wellas telomerase-immortalized human bronchiolar epithelialcells Precisely the macrophages were very susceptible tonanomaterial toxicity while fibroblasts are more resistant atall the treatments whereas only the exposure of SiO2 andMWCNT (lt8 nm) induce apoptosis in human bronchiolarepithelial cells In the experimental conditions of this studythe investigated nanomaterials did not trigger necrosis [65]In the same mouse macrophage cell line it has beendemonstrated that MWCNT (10ndash25 nm) and SWCNTs (12ndash15 nm) induced necrosis in a concentration-dependentmanner [141] CNTs have been demonstrated to induce


both necrosis and apoptosis in human fibroblasts [142] Incontrast Cui and co-workers found that SWNTs upregulateapoptosis-associated genes in human embryo kidney cells[143] and Zhu and colleagues showed that MWCNTsinduce apoptosis in mouse embryonic stem cells [144] whilePulskamp and collaborators assert that commercial CNTsdo not induce necrosis or apoptosis in rat macrophages[145] Recently a multilevel approach including differenttoxicity tests and gene-expression determinations was usedto evaluate the toxicity of two lanthanide-based luminescentnanoparticles complexes with the chelating agent EDTAThe study revealed that these nanomaterials induced necrosisin human lymphoblasts and erythromyeloblastoid leukemiacell lines while no toxicity was observed in human breastcancer cell line Moreover no in vivo effects have beenobserved The comparative analysis of the nanomaterials andtheir separated components showed that the toxicity wasmainly due to the presence of EDTA [146]

The knowledge advances concerning the molecular char-acterization of necrosis will make necessary more precise andaccurate studies to confirm the ways in which nanomaterialsmight cause necrotic death

6 Nanomaterials and Pyroptosis

Pyroptosis described the peculiar death of macrophages inf-ected by Salmonella typhimurium [147] Several other bac-teria triggering this atypical cell death modality have beenidentified Pyroptosis neither constitutes a macrophage-spe-cific process nor a cell death subroutine that only results frombacterial infection Pyroptotic cells can exhibit apoptoticandor necrotic morphological features The most distinctivebiochemical feature of pyroptosis is the early caspase-1 activation associated with the generation of pyrogenicmediators such as Interleukin-1β (IL-1β) [38]

Recently it has been shown that the exposure of macrop-hages (both a mouse macrophage cell line and primaryhuman alveolar macrophages) to carbon black nanoparticlesresulted in inflammasome activation as defined by cleavageof caspase-1 to its active form and downstream IL-1β releaseThe carbon black nanoparticles-induced cell death wasidentified as pyroptosis through the inhibition of caspase-1 and pyroptosis by specific pharmacological inhibitorsThe authors showed that in this setting TiO2 particles didnot induce pyroptosis or significantly activate the inflam-masome [148] In contrast it has been shown that nano-TiO2 and nano-SiO2 but not nano-ZnO (zinc oxide) andcarbon nanotubes induced inflammasome activation butnot cell death in murine bone marrow-derived macrophagesand human macrophages cell line Although the caspase-1cleavage and IL-1β release was induced the inflammationcaused by nanoparticles was largely caused by the biologicaleffect of IL-1α [149] This apparent discrepancy could beexplained considering the different concentration and kindof nanomaterials used in these studies moreover it ispossible that different macrophages perform differently inresponse to nanomaterials Future studies should address thisissue However the identification of pyroptosis as a cellular

response to carbon nanoparticles exposure is novel andrelates to health impacts of carbon-based particulates

7 Conclusions and Perspectives

The continued expansion of the nanotechnology field req-uires a thorough understanding of the potential mecha-nisms of nanomaterial toxicity for proper safety assessmentand identification of exposure biomarkers With increasingresearch into nanomaterial safety details on the biologi-cal effects of nanomaterials have begun to emerge Thenanomaterials intrinsic toxicity has been attributed to theirphysicochemical characteristics that is their smallness andthe remarkably large surface area per unit mass and highsurface reactivity In fact their type composition andmodifications size shape and surface charge should beconsidered However the complex death paradigms may alsobe explained by activation of different death pathways in acontext-dependent manner In vitro experiments could beinfluenced by a cell type-specific response and ones in vivocould be affected by the animal species and the model usedor by pharmacokinetic parameters (administration distribu-tion metabolism etc) Moreover the dose concentrationsand the time of exposure of a nanomaterial employed areessential In effect the efficiency of cellular uptake of nano-materials and the resultant intracellular concentration maydetermine the cytotoxic potential Elucidating the molecularmechanisms by which nanosized particles induce activationof cell death signalling pathways will be critical for thedevelopment of prevention strategies to minimize the cyto-toxicity of nanomaterials Unfortunately in the literaturethere are many conflicting data the most plausible reason iscertainly the discrepancy of nanomaterials and experimentalmodels engaged Although some authors have recentlyalerted colleagues on these issues [3 5 8 9 150ndash152] ithas not yet been put in place a guideline generally acceptedby the scientific community in the field to address thesematters In fact harmonization of protocols for materialcharacterization and for cytotoxicity testing of nanomaterialsis needed In addition parallel profiling of several classesof nanomaterials combined with detailed characterizationof their physicochemical properties could provide a modelfor safety assessment of novel nanomaterials [153] Duringthe past decade owing to major technological advancesin the field of combinatorial chemistry in addition to thesequencing of an ever increasing number of genomes high-content chemical and genetic libraries have become availableraising the need for high-throughput screening (HTS) andhigh-content screening (HCS) approaches In response tothis demand multiple conventional cell death detectionmethods have been adapted to HTSHCS and many novelHTSHCS-amenable techniques have been developed [37154] In the last years several authors started to study thenanotoxicity with this tools and highlighted the potentialof these approaches [9 60 75 155ndash161] An overall aimshould identify HTSHCS assays that can be used routinelyto screen nanomaterials for interaction with the cell deathmodalities system HTSHCS may accelerated the analysison a scale that commensurates with the rate of expansion


of new nanomaterials but in any case is a first validationstep then it remains to confirm whether the same identifiedmechanisms in vitro are responsible for their in vivo toxicityIn conclusion a multilevel-integrated uniform and consis-tent approach should contemplate for nanomaterial toxicitycharacterization

In spite of the recent advances in our understanding ofcell death mechanisms and associated signalling networksmuch work remains to be done before we can fully elucidatethe toxicological behaviour of the nanomaterials as well asunderstand their participation in the determination of cellfate More and accurate results are needed for apoptosisautophagy and necrosis induction by nanomaterials furtherstudies are necessary to test if the novel strategic targetsidentified could be affected either directly or indirectly bynanomaterials Moreover no data are present in the literatureconcerning the nanomaterials exposure and other forms ofcell death including anoikis entosis parthanatos netosisand cornification For example although numerous studieshave been performed on keratinocytes none of these hasrated cornification a cell death subroutine restricted tokeratinocytes and functionally linked to the generation ofthe stratum corneum of the epidermis [38] It will be ofconsiderable interest to establish whether these various celldeath modalities are associated with the intent of identi-fying a structure-activity relationship and delineating themechanisms by which these interactions occur In additionto the established paradigms of nanomaterials toxicity thestudy of their interactions with the death signalling pathwayscould potentially have many important human pathologicaloutcomes including cancer metabolic disorders and neu-rodegenerative disorders

Abbreviations

Ag+ Silver ionsATG Autophagy-related geneBcl-2 B-cell lymphoma 2BH3 Bcl-2 homology domain 3BID BH3-interacting domain death agonistBmf Bcl-2-modifying factorCNTs Carbon nanotubesCoCr Cobalt-chromeDNA Deoxyribonucleic acidEDTA Ethylenediaminetetraacetic acidER Endoplasmic reticulumFDA Food and Drug AdministrationHCS High-content screeningHTS High-throughput screeningIL InterleukinMOMP Mitochondrial outer membrane

permeabilizationMWCNTs Multiwall carbon nanotubesNADPH Nicotinamide adenine dinucleotide phosphateNCCD Nomenclature Committee on Cell DeathPAMAM Cationic polyamidoaminePANI PolyanilinePEG-PE Poly(ethylene glycol)-phosphoethanolamineRIP Receptor-interacting protein kinase

RNA Ribonucleic acidRNS Reactive nitrogen speciesROS Reactive oxygen speciesSiO2 Silicon dioxidesiRNA Small interfering RNASWCNTs Single-walled carbon nanotubestBID Truncated BIDTiO2 Titanium dioxideZnO Zinc oxide



Acknowledgment

This work is supported by the Italian Ministry of the Univer-sity and Scientific Research

References

[1] G Oberdorster ldquoNanotoxicology in vitro-in vivo dosime-tryrdquo Health Perspect vol 120 no 1 p 13 2012

[2] S Gangwal J Brown A Wang KA Houck and DJ DixldquoInforming selection of nanomaterial concentrations forToxCast in vitro testing based on occupational exposurepotentialrdquo Health Perspect vol 119 no 11 pp 1539ndash15462011

[3] A Nel T Xia L Madler and N Li ldquoToxic potential of mat-erials at the nanolevelrdquo Science vol 311 no 5761 pp 622ndash627 2006

[4] T Xia N Li and A E Nel ldquoPotential health impact of nano-particlesrdquo Annual Review of Public Health vol 30 pp 137ndash150 2009

[5] E J Petersen and B C Nelson ldquoMechanisms and measure-ments of nanomaterial-induced oxidative damage to DNArdquoAnalytical and Bioanalytical Chemistry vol 398 no 2 pp613ndash650 2010

[6] A A Shvedova V E Kagan and B Fadeel ldquoClose encountersof the small kind adverse effects of man-made materialsinterfacing with the nano-cosmos of biological systemsrdquoAnnual Review of Pharmacology and Toxicology vol 50 pp63ndash88 2010

[7] S Orrenius P Nicotera and B Zhivotovsky ldquoCell deathmechanisms and their implications in toxicologyrdquo Toxicolog-ical Sciences vol 119 no 1 pp 3ndash19 2011

[8] M Horie H Kato K Fujita S Endoh and H Iwahashi ldquoInvitro evaluation of cellular response induced by manufac-tured nanoparticlesrdquo Chemical Research in Toxicology vol 25no 3 pp 605ndash619 2012

[9] A A Shvedova A Pietroiusti B Fadeel and V E KaganldquoMechanisms of carbon nanotube-induced toxicity focus onoxidative stressrdquo Toxicology and Applied Pharmacology vol261 no 2 pp 121ndash133 2012

[10] F T Andon and B Fadeel ldquoProgrammed cell death molec-ular mechanisms and implications for safety assessment ofnanomaterialsrdquo Accounts of Chemical Research In press

[11] C F Jones and D W Grainger ldquoIn vitro assessments ofnanomaterial toxicityrdquo Advanced Drug Delivery Reviews vol61 no 6 pp 438ndash456 2009


[12] I Nabiev S Mitchell A Davies et al ldquoNonfunctionalizednanocrystals can exploit a cellrsquos active transport machinerydelivering them to specific nuclear and cytoplasmic compart-mentsrdquo Nano Letters vol 7 no 11 pp 3452ndash3461 2007

[13] M Chen and A Von Mikecz ldquoFormation of nucleoplasmicprotein aggregates impairs nuclear function in response toSiO2 nanoparticlesrdquo Experimental Cell Research vol 305 no1 pp 51ndash62 2005

[14] M Tsoli H Kuhn W Brandau H Esche and G SchmidldquoCellular uptake and toxicity of Au55 clustersrdquo Small vol 1no 8-9 pp 841ndash844 2005

[15] A M Schrand L K Braydich-Stolle J J Schlager L Dai andS M Hussain ldquoCan silver nanoparticles be useful as potentialbiological labelsrdquo Nanotechnology vol 19 no 23 Article ID235104 2008

[16] S M Hussain L K Braydich-Stolle A M Schrand et alldquoToxicity evaluation for safe use of nanomaterials recentachievements and technical challengesrdquo Advanced Materialsvol 21 no 16 pp 1549ndash1559 2009

[17] G Oberdorster J Ferin and B E Lehnert ldquoCorrelationbetween particle size in vivo particle persistence and lunginjuryrdquo Environmental Health Perspectives vol 102 no 5 pp173ndash179 1994

[18] C Monteiller L Tran W MacNee et al ldquoThe pro-inflammatory effects of low-toxicity low-solubility particlesnanoparticles and fine particles on epithelial cells in vitrothe role of surface areardquo Occupational and EnvironmentalMedicine vol 64 no 9 pp 609ndash615 2007

[19] E E Connor J Mwamuka A Gole C J Murphy and MD Wyatt ldquoGold nanoparticles are taken up by human cellsbut do not cause acute cytotoxicityrdquo Small vol 1 no 3 pp325ndash327 2005

[20] V E Kagan Y Y Tyurina V A Tyurin et al ldquoDirect andindirect effects of single walled carbon nanotubes on RAW2647 macrophages role of ironrdquo Toxicology Letters vol 165no 1 pp 88ndash100 2006

[21] T Cedervall I Lynch S Lindman et al ldquoUnderstanding thenanoparticle-protein corona using methods to quntify excha-nge rates and affinities of proteins for nanoparticlesrdquo Pro-ceedings of the National Academy of Sciences of the UnitedStates of America vol 104 no 7 pp 2050ndash2055 2007

[22] M Horie K Nishio K Fujita et al ldquoProtein adsorption ofultrafine metal oxide and its influence on cytotoxicity towardcultured cellsrdquo Chemical Research in Toxicology vol 22 no3 pp 543ndash553 2009

[23] M S Ehrenberg A E Friedman J N Finkelstein G Obe-rdorster and J L McGrath ldquoThe influence of protein adso-rption on nanoparticle association with cultured endothelialcellsrdquo Biomaterials vol 30 no 4 pp 603ndash610 2009

[24] M Horie K Nishio H Kato et al ldquoCellular responsesinduced by cerium oxide nanoparticles induction of intra-cellular calcium level and oxidative stress on culture cellsrdquoThe Journal of Biochemistry vol 150 no 4 pp 461ndash4712011

[25] M Lundqvist J Stigler T Cedervall et al ldquoThe evolution ofthe protein corona around nanoparticles a test studyrdquo ACSNano vol 5 no 9 pp 7503ndash7509 2011

[26] M P Monopoli D Walczyk A Campbell et al ldquoPhysical-Chemical aspects of protein corona relevance to in vitro andin vivo biological impacts of nanoparticlesrdquo Journal of theAmerican Chemical Society vol 133 no 8 pp 2525ndash25342011

[27] D Dutta S K Sundaram J G Teeguarden et al ldquoAdsorbedproteins influence the biological activity and molecular tar-geting of nanomaterialsrdquo Toxicological Sciences vol 100 no1 pp 303ndash315 2007

[28] N Lewinski V Colvin and R Drezek ldquoCytotoxicity of nano-partidesrdquo Small vol 4 no 1 pp 26ndash49 2008

[29] S T Stern and S E McNeil ldquoNanotechnology safety con-cerns revisitedrdquo Toxicological Sciences vol 101 no 1 pp 4ndash21 2008

[30] E Chang N Thekkek W W Yu V L Colvin and RDrezek ldquoEvaluation of quantum dot cytotoxicity based onintracellular uptakerdquo Small vol 2 no 12 pp 1412ndash14172006

[31] C M Sayes R Wahi P A Kurian et al ldquoCorrelating nano-scale titania structure with toxicity a cytotoxicity and inf-lammatory response study with human dermal fibroblastsand human lung epithelial cellsrdquo Toxicological Sciences vol92 no 1 pp 174ndash185 2006

[32] G Jones and P M Brooks ldquoInjectable gold compounds anoverviewrdquo British Journal of Rheumatology vol 35 no 11 pp1154ndash1158 1996

[33] M L Schipper N Nakayama-Ratchford C R Davis et alldquoA pilot toxicology study of single-walled carbon nanotubesin a small sample of micerdquo Nature Nanotechnology vol 3 no4 pp 216ndash221 2008

[34] K L Aillon Y Xie N El-Gendy C J Berkland and M LForrest ldquoEffects of nanomaterial physicochemical propertieson in vivo toxicityrdquo Advanced Drug Delivery Reviews vol 61no 6 pp 457ndash466 2009

[35] C Buzea I I Pacheco and K Robbie ldquoNanomaterials andnanoparticles sources and toxicityrdquo Biointerphases vol 2 no4 pp MR17ndashMR71 2007

[36] J M Brown and L D Attardi ldquoThe role of apoptosis in can-cer development and treatment responserdquo Nature ReviewsCancer vol 5 no 3 pp 231ndash237 2005

[37] O Kepp L Galluzzi M Lipinski J Yuan and G KroemerldquoCell death assays for drug discoveryrdquo Nature Reviews DrugDiscovery vol 10 no 3 pp 221ndash237 2011

[38] L Galluzzi I Vitale J M Abrams et al ldquoMolecular def-initions of cell death subroutines recommendations of theNomenclature Committee on cell deathrdquo Cell Death ampDifferentiation vol 19 no 1 pp 107ndash120 2012

[39] N Li T Xia and A E Nel ldquoThe role of oxidative stressin ambient particulate matter-induced lung diseases and itsimplications in the toxicity of engineered nanoparticlesrdquo FreeRadical Biology and Medicine vol 44 no 9 pp 1689ndash16992008

[40] A Sarkar J Das P Manna and P C Sil ldquoNano-copperinduces oxidative stress and apoptosis in kidney via both ext-rinsic and intrinsic pathwaysrdquo Toxicology vol 290 no 2-3pp 208ndash217 2011

[41] P Manna M Ghosh J Ghosh J Das and P C Sil ldquoContri-bution of nano-copper particles to in vivo liver dysfunctionand cellular damage role of IeBaNF-eB MAPKs andmitochondrial signalrdquo Nanotoxicology vol 6 no 1 pp 1ndash212012

[42] J Zhao L Bowman X Zhang et al ldquoMetallic nickel nano-and fine particles induce JB6 cell apoptosis through a cas-pase-8AIF mediated cytochrome c-independent pathwayrdquoJournal of Nanobiotechnology vol 7 article 2 2009

[43] M A Siddiqui M Ahamed J Ahmad et al ldquoNickel oxidenanoparticles induce cytotoxicity oxidative stress and apop-tosis in cultured human cells that is abrogated by the dietary


antioxidant curcuminrdquo Food and Chemical Toxicology vol50 no 3-4 pp 641ndash647 2012

[44] M Ahamed M J Akhtar M A Siddiqui et al ldquoOxidativestress mediated apoptosis induced by nickel ferrite nanopar-ticles in cultured A549 cellsrdquo Toxicology vol 283 no 2-3 pp101ndash108 2011

[45] B Fubini and A Hubbard ldquoReactive oxygen species (ROS)and reactive nitrogen species (RNS) generation by silicain inflammation and fibrosisrdquo Free Radical Biology andMedicine vol 34 no 12 pp 1507ndash1516 2003

[46] L Wang L Bowman Y Lu et al ldquoEssential role of p53 insilica-induced apoptosisrdquo American Journal of Physiology vol288 no 3 pp L488ndashL496 2005

[47] K C Yoo C H Yoon D Kwon et al ldquoTitanium dioxideinduces apoptotic cell death through reactive oxygen species-mediated Fas upregulation and Bax activationrdquo InternationalJournal of Nanomedicine vol 7 pp 1203ndash1214 2012

[48] S J Kang B M Kim S H Hong and H W ChungldquoTitanium dioxide nanoparticles induce apoptosis throughthe JNKp38-caspase-8-Bid pathway in phytohemagglutinin-stimulated human lymphocytesrdquo Biochemical and Biophysi-cal Research Communications vol 386 no 4 pp 682ndash6872009

[49] Y Shi F Wang J He S Yadav and H Wang ldquoTitanium diox-ide nanoparticles cause apoptosis in BEAS-2B cells throughthe caspase 8t-Bid-independent mitochondrial pathwayrdquoToxicology Letters vol 196 no 1 pp 21ndash27 2010

[50] S J Kang B M Kim Y J Lee and H W Chung ldquoTitaniumdioxide nanoparticles trigger p53-mediated damage responsein peripheral blood lymphocytesrdquo Environmental and Molec-ular Mutagenesis vol 49 no 5 pp 399ndash405 2008

[51] E J Park J Yi K H Chung D Y Ryu J Choi and K ParkldquoOxidative stress and apoptosis induced by titanium dioxidenanoparticles in cultured BEAS-2B cellsrdquo Toxicology Lettersvol 180 no 3 pp 222ndash229 2008

[52] T Xia M Kovochich J Brant et al ldquoComparison of the abil-ities of ambient and manufactured nanoparticles to inducecellular toxicity according to an oxidative stress paradigmrdquoNano Letters vol 6 no 8 pp 1794ndash1807 2006

[53] L Ding J Stilwell T Zhang et al ldquoMolecular characteriza-tion of the cytotoxic mechanism of multiwall carbon nan-otubes and nano-onions on human skin fibroblastrdquo NanoLetters vol 5 no 12 pp 2448ndash2464 2005

[54] M Bottini S Bruckner K Nika et al ldquoMulti-walled carbonnanotubes induce T lymphocyte apoptosisrdquo Toxicology Let-ters vol 160 no 2 pp 121ndash126 2006

[55] C Grabinski S Hussain K Lafdi L Braydich-Stolle and JSchlager ldquoEffect of particle dimension on biocompatibilityof carbon nanomaterialsrdquo Carbon vol 45 no 14 pp 2828ndash2835 2007

[56] R K Srivastava A B Pant M P Kashyap et al ldquoMulti-walled carbon nanotubes induce oxidative stress and apop-tosis in human lung cancer cell line-A549rdquo Nanotoxicologyvol 5 no 2 pp 195ndash207 2011

[57] J Chłopek B Czajkowska B Szaraniec E Frackowiak KSzostak and F Beguin ldquoIn vitro studies of carbon nanotubesbiocompatibilityrdquo Carbon vol 44 no 6 pp 1106ndash11112006

[58] T Thurnherr C Brandenberger K Fischer et al ldquoA compar-ison of acute and long-term effects of industrial multiwalledcarbon nanotubes on human lung and immune cells invitrordquo Toxicology Letters vol 200 no 3 pp 176ndash186 2011

[59] L Wang S Luanpitpong V Castranova et al ldquoCarbon nan-otubes induce malignant transformation and tumorigenesis

of human lung epithelial cellsrdquo Nano Letters vol 11 no 7pp 2796ndash2803 2011

[60] Y Y Tyurina E R Kisin A Murray et al ldquoGlobal phospho-lipidomics analysis reveals selective pulmonary peroxidationprofiles upon inhalation of single-walled carbon nanotubesrdquoACS Nano vol 5 no 9 pp 7342ndash7353 2011

[61] S T Stern P P Adiseshaiah and R M Crist ldquoAutophagy andlysosomal dysfunction as emerging mechanisms of nanoma-terial toxicityrdquo Particle and Fibre Toxicology vol 9 no 1 p20 2012

[62] M H Eesen K Pegan A Spes and B Turk ldquoLysosomalpathways to cell death and their therapeutic applicationsrdquoExperimental Cell Research vol 318 no 11 pp 1245ndash12512012

[63] U Repnik V Stoka V Turk and B Turk ldquoLysosomes andlysosomal cathepsins in cell deathrdquo Biochimica et BiophysicaActa vol 1824 no 1 pp 22ndash33 2012

[64] Y Tahara M Nakamura M Yang M Zhang S Iijima andM Yudasaka ldquoLysosomal membrane destabilization inducedby high accumulation of single-walled carbon nanohorns inmurine macrophage RAW 2647rdquo Biomaterials vol 33 no 9pp 2762ndash2769 2012

[65] S K Sohaebuddin P T Thevenot D Baker J W Eaton andL Tang ldquoNanomaterial cytotoxicity is composition size andcell type dependentrdquo Part Fibre Toxicol vol 21 no 7 p 222010

[66] S Hussain L C J Thomassen I Ferecatu et al ldquoCarbonblack and titanium dioxide nanoparticles elicit distinct apop-totic pathways in bronchial epithelial cellsrdquo Particle and FibreToxicology vol 7 article 10 2010

[67] C Y Jin B S Zhu X F Wang and Q H Lu ldquoCytotoxicityof titanium dioxide nanoparticles in mouse fibroblast cellsrdquoChemical Research in Toxicology vol 21 no 9 pp 1871ndash1877 2008

[68] T Xia M Kovochich M Liong J I Zink and A E NelldquoCationic polystyrene nanosphere toxicity depends on cell-specific endocytic and mitochondrial injury pathwaysrdquo ACSNano vol 2 no 1 pp 85ndash96 2008

[69] T P Thomas I Majoros A Kotlyar D Mullen M MBanaszak Holl and J R Baker ldquoCationic poly(amidoamine)dendrimer induces lysosomal apoptotic pathway at therapeu-tically relevant concentrationsrdquo Biomacromolecules vol 10no 12 pp 3207ndash3214 2009

[70] M S Thibodeau C Giardina D A Knecht J Helble andA K Hubbard ldquoSilica-induced apoptosis in mouse alveolarmacrophages is initiated by lysosomal enzyme activityrdquoToxicological Sciences vol 80 no 1 pp 34ndash48 2004

[71] S Tedesco H Doyle J Blasco G Redmond and D SheehanldquoOxidative stress and toxicity of gold nanoparticles in Mytilusedulisrdquo Aquatic Toxicology vol 100 no 2 pp 178ndash186 2010

[72] I Tabas and D Ron ldquoIntegrating the mechanisms of apop-tosis induced by endoplasmic reticulum stressrdquo Nature CellBiology vol 13 no 3 pp 184ndash190 2011

[73] A M Gorman S J Healy R Jager and A Samali ldquoStressmanagement at the ER regulators of ER stress-inducedapoptosisrdquo Pharmacology amp Therapeutics vol 134 no 3 pp306ndash316 2012

[74] R Zhang M J Piao K C Kim et al ldquoEndoplasmic reticu-lum stress signaling is involved in silver nanoparticles-ind-uced apoptosisrdquo The International Journal of Biochemistry ampCell Biology vol 44 no 1 pp 224ndash232 2012

[75] Y Y Tsai Y H Huang Y L Chao et al ldquoIdentification ofthe nanogold particle-induced endoplasmic reticulum stress


by omic techniques and systems biology analysisrdquo ACS Nanovol 5 no 12 pp 9354ndash9369 2011

[76] J Wang X Fang and W Liang ldquoPegylated phospholipidmicelles induce endoplasmic reticulum-dependent apoptosisof cancer cells but not normal cellsrdquo ACS Nano vol 6 no 6pp 5018ndash5030 2012

[77] A Thubagere and B M Reinhard ldquoNanoparticle-inducedapoptosis ropagates through hydrogen-peroxide-mediatedbystander killing insights from a human intestinal epithe-lium in vitro modelrdquo ACS Nano vol 4 no 7 pp 3611ndash36222010

[78] I Vitale L Galluzzi M Castedo and G Kroemer ldquoMitoticcatastrophe a mechanism for avoiding genomic instabilityrdquoNature Reviews Molecular Cell Biology vol 12 no 6 pp385ndash392 2011

[79] A E Porter M Gass K Muller J N Skepper P A Midgleyand M Welland ldquoDirect imaging of single-walled carbonnanotubes in cellsrdquo Nature Nanotechnology vol 2 no 11 pp713ndash717 2007

[80] L Gonzalez I Decordier and M Kirsch-Volders ldquoInductionof chromosome malsegregation by nanomaterialsrdquo Biochem-ical Society Transactions vol 38 no 6 pp 1691ndash1697 2010

[81] L M Sargent A A Shvedova A F Hubbs et al ldquoInductionof aneuploidy by single-walled carbon nanotubesrdquo Environ-mental and Molecular Mutagenesis vol 50 no 8 pp 708ndash717 2009

[82] L M Sargent S H Reynolds and V Castranova ldquoPotentialpulmonary effects of engineered carbon nanotubes in vitrogenotoxic effectsrdquo Nanotoxicology vol 4 no 4 pp 396ndash4082010

[83] L M Sargent A F Hubbs S H Young et al ldquoSingle-walledcarbon nanotube-induced mitotic disruptionrdquo MutationResearch vol 745 no 1-2 pp 28ndash37 2012

[84] A Tsaousi E Jones and C P Case ldquoThe in vitro genotoxicityof orthopaedic ceramic (Al2O3) and metal (CoCr alloy)particlesrdquo Mutation Research vol 697 no 1-2 pp 1ndash9 2010

[85] I Papageorgiou C Brown R Schins et al ldquoThe effect ofnano- and micron-sized particles of cobalt-chromium alloyon human fibroblasts in vitrordquo Biomaterials vol 28 no 19pp 2946ndash2958 2007

[86] J P Wise B C Goodale S S Wise et al ldquoSilver nanospheresare cytotoxic and genotoxic to fish cellsrdquo Aquatic Toxicologyvol 97 no 1 pp 34ndash41 2010

[87] D J Klionsky ldquoThe molecular machinery of autophagy andits role in physiology and diseaserdquo Seminars in Cell andDevelopmental Biology vol 21 no 7 p 663 2010

[88] G Kroemer and B Levine ldquoAutophagic cell death the storyof a misnomerrdquo Nature Reviews Molecular Cell Biology vol 9no 12 pp 1004ndash1010 2008

[89] Q Zhang W Yang N Man et al ldquoAutophagy-mediatedchemosensitization in cancer cells by fullerene C60 nanocrys-talrdquo Autophagy vol 5 no 8 pp 1107ndash1117 2009

[90] J J Li D Hartono C N Ong B H Bay and L Y LYung ldquoAutophagy and oxidative stress associated with goldnanoparticlesrdquo Biomaterials vol 31 no 23 pp 5996ndash60032010

[91] B Halamoda Kenzaoui C Chapuis Bernasconi S Guney-Ayra and L Juillerat-Jeanneret ldquoInduction of oxidativestress lysosome activation and autophagy by nanoparticles inhuman brain-derived endothelial cellsrdquo Biochemical Journalvol 441 no 3 pp 813ndash821 2012

[92] Z M Markovic B Z Ristic K M Arsikin et al ldquoGraphenequantum dots as autophagy-inducing photodynamic agentsrdquoBiomaterials vol 33 no 29 pp 7084ndash7092 2012

[93] S T Stern B S Zolnik C B McLeland J Clogston JZheng and S E McNeil ldquoInduction of autophagy in porcinekidney cells by quantum dots a common cellular responseto nanomaterialsrdquo Toxicological Sciences vol 106 no 1 pp140ndash152 2008

[94] C Li H Liu Y Sun et al ldquoPAMAM nanoparticles promoteacute lung injury by inducing autophagic cell death throughthe Akt-TSC2-mTOR signaling pathwayrdquo Journal of Molecu-lar Cell Biology vol 1 no 1 pp 37ndash45 2009

[95] L Yu Y Lu N Man S H Yu and L P Wen ldquoRare earthoxide nanocrystals induce autophagy in hela cellsrdquo Small vol5 no 24 pp 2784ndash2787 2009

[96] N Man L Yu S H Yu and L P Wen ldquoRare earth oxidenanocrystals as a new class of autophagy inducersrdquo Autopha-gy vol 6 no 2 pp 310ndash311 2010

[97] C M Lee S T Huang S H Huang et al ldquoC60 fullerene-pentoxifylline dyad nanoparticles enhance autophagy toavoid cytotoxic effects caused by the β-amyloid peptiderdquoNanomedicine vol 7 no 1 pp 107ndash114 2011

[98] H Li Y Li J Jiao and H M Hu ldquoAlpha-aluminananoparticles induce efficient autophagy-dependent cross-presentation and potent antitumour responserdquo Nature Nan-otechnology vol 6 no 10 pp 645ndash650 2011

[99] H L Liu Y L Zhang N Yang et al ldquoA functionalized single-walled carbon nanotube-induced autophagic cell death inhuman lung cells through Akt-TSC2-mTOR signalingrdquo CellDeath and Disease vol 19 no 2 article e159 2011

[100] J X Yu and T H Li ldquoDistinct biological effects of differentnanoparticles commonly used in cosmetics and medicinecoatingsrdquo Cell amp Bioscience vol 19 no 1 p 1 2011

[101] M Reale G Vianale L V Lotti et al ldquoEffects of palladiumnanoparticles on the cytokine release from peripheral bloodmononuclear cells of palladium-sensitized womenrdquo Journalof Occupational and Environmental Medicine vol 53 no 9pp 1054ndash1060 2011

[102] M I Khan A Mohammad G Patil SA Naqvi L K Cha-uhan and I Ahmad ldquoInduction of ROS mitochondrialdamage and autophagy in lung epithelial cancer cells by ironoxide nanoparticlesrdquo Biomaterials vol 33 no 5 pp 1477ndash1488 2012

[103] T Sun Y Yan Y Zhao F Guo and C Jiang ldquoCopper oxidenanoparticles induce autophagic cell death in a549 cellsrdquoPLoS ONE vol 7 no 8 Article ID e43442 2012

[104] T Yokoyama J Tam S Kuroda et al ldquoEGFR-targeted hybridplasmonic magnetic nanoparticles synergistically induceautophagy and apoptosis in non-small cell lung cancer cellsrdquoPLoS ONE vol 6 no 11 Article ID e25507 2011

[105] L Calzolai F Franchini D Gilliland and F Rossi ldquoProtein-nanoparticle interaction identification of the ubiquitin-goldnanoparticle interaction siterdquo Nano Letters vol 10 no 8 pp3101ndash3105 2010

[106] H Yamawaki and N Iwai ldquoCytotoxicity of water-solublefullerene in vascular endothelial cellsrdquo American Journal ofPhysiology vol 290 no 6 pp C1495ndashC1502 2006

[107] O Seleverstov O Zabirnyk M Zscharnack et al ldquoQuantumdots for human mesenchymal stem cells labeling a size-dependent autophagy activationrdquo Nano Letters vol 6 no 12pp 2826ndash2832 2006

[108] Y Chen L Yang C Feng and L P Wen ldquoNano neodymiumoxide induces massive vacuolization and autophagic celldeath in non-small cell lung cancer NCI-H460 cellsrdquo Bio-chemical and Biophysical Research Communications vol 337no 1 pp 52ndash60 2005


[109] Y Zhang F Zheng T Yang et al ldquoTuning the autophagy-inducing activity of lanthanide-based nanocrystals throughspecific surface-coating peptidesrdquo Nature Materials vol 11no 9 pp 817ndash826 2012

[110] P Wei L Zhang Y Lu N Man and L Wen ldquoC60(Nd)nanoparticles enhance chemotherapeutic susceptibility ofcancer cells by modulation of autophagyrdquo Nanotechnologyvol 21 no 49 Article ID 495101 2010

[111] X Ma Y Wu S Jin et al ldquoGold nanoparticles induce auto-phagosome accumulation through size-dependent nanopar-ticle uptake and lysosome impairmentrdquo ACS Nano vol 5 no11 pp 8629ndash8639 2011

[112] D N Johnson-Lyles K Peifley S Lockett et al ldquoFullerenolcytotoxicity in kidney cells is associated with cytoskeletondisruption autophagic vacuole accumulation and mito-chondrial dysfunctionrdquo Toxicology and Applied Pharmacol-ogy vol 248 no 3 pp 249ndash258 2010

[113] S T Stern and D N Johnson ldquoRole for nanomaterial-autophagy interaction in neurodegenerative diseaserdquo Auto-phagy vol 4 no 8 pp 1097ndash1100 2008

[114] M M Monick L S Powers K Walters et al ldquoIdentificationof an autophagy defect in smokersrsquo alveolar macrophagesrdquoJournal of Immunology vol 185 no 9 pp 5425ndash5435 2010

[115] H Afeseh Ngwa A Kanthasamy Y Gu N Fang V Anan-tharam and A G Kanthasamy ldquoManganese nanoparticleactivates mitochondrial dependent apoptotic signaling andautophagy in dopaminergic neuronal cellsrdquo Toxicology andApplied Pharmacology vol 256 no 3 pp 227ndash240 2011

[116] H L Herd A Malugin and H Ghandehari ldquoSilica nanocon-struct cellular toleration threshold in vitrordquo Journal ofControlled Release vol 153 no 1 pp 40ndash48 2011

[117] Y N Wu L X Yang X Y Shi et al ldquoThe selective growthinhibition of oral cancer by iron core-gold shell nanoparticlesthrough mitochondria-mediated autophagyrdquo Biomaterialsvol 32 no 20 pp 4565ndash4573 2011

[118] H Eidi O Joubert C Nemos et al ldquoDrug delivery by poly-meric nanoparticles induces autophagy in macrophagesrdquoInternational Journal of Pharmaceutics vol 422 no 1-2 pp495ndash503 2012

[119] S Barth D Glick and K F Macleod ldquoAutophagy assays andartifactsrdquo Journal of Pathology vol 221 no 2 pp 117ndash1242010

[120] O Seleverstov J M Phang and O Zabirnyk ldquoChap-ter 18 semiconductor nanocrystals in autophagy researchMethodology improvement at nanosized scalerdquo Methods inEnzymology vol 451 pp 277ndash296 2009

[121] K M Choi H Y Nam J H Na et al ldquoA monitoring methodfor Atg4 activation in living cells using peptide-conjugatedpolymeric nanoparticlesrdquo Autophagy vol 7 no 9 pp 1052ndash1062 2011

[122] P Vandenabeele L Galluzzi T Vanden Berghe and G Kroe-mer ldquoMolecular mechanisms of necroptosis an orderedcellular explosionrdquo Nature Reviews Molecular Cell Biologyvol 11 no 10 pp 700ndash714 2010

[123] D W Zhang J Shao J Lin et al ldquoRIP3 an energy metabo-lism regulator that switches TNF-induced cell death fromapoptosis to necrosisrdquo Science vol 325 no 5938 pp 332ndash336 2009

[124] J W Upton W J Kaiser and E S Mocarski ldquoVirus inhibi-tion of RIP3-dependent necrosisrdquo Cell Host and Microbe vol7 no 4 pp 302ndash313 2010

[125] J Hitomi D E Christofferson A Ng et al ldquoIdentification ofa molecular signaling network that regulates a cellular necro-tic cell death pathwayrdquo Cell vol 135 no 7 pp 1311ndash13232008

[126] W J Kaiser J W Upton A B Long et al ldquoRIP3 mediatesthe embryonic lethality of caspase-8-deficient micerdquo Naturevol 471 no 7338 pp 368ndash372 2011

[127] A Oberst C P Dillon R Weinlich et al ldquoCatalytic activityof the caspase-8-FLIP L complex inhibits RIPK3-dependentnecrosisrdquo Nature vol 471 no 7338 pp 363ndash367 2011

[128] H Zhang X Zhou T McQuade J Li F K M Chan andJ Zhang ldquoFunctional complementation between FADD andRIP1 in embryos and lymphocytesrdquo Nature vol 471 no7338 pp 373ndash376 2011

[129] A Degterev Z Huang M Boyce et al ldquoChemical inhibitorof nonapoptotic cell death with therapeutic potential forischemic brain injuryrdquo Nature Chemical Biology vol 1 no2 pp 112ndash119 2005

[130] Y H Ma C P Huang J S Tsai M Y Shen Y K Li and L YLin ldquoWater-soluble germanium nanoparticles cause necroticcell death and the damage can be attenuated by blockingthe transduction of necrotic signaling pathwayrdquo ToxicologyLetters vol 207 no 3 pp 258ndash269 2011

[131] L K Braydich-Stolle N M Schaeublin R C Murdock etal ldquoCrystal structure mediates mode of cell death in TiO2

nanotoxicityrdquo Journal of Nanoparticle Research vol 11 no 6pp 1361ndash1374 2009

[132] Y Pan S Neuss A Leifert et al ldquoSize-dependent cytotoxicityof gold nanoparticlesrdquo Small vol 3 no 11 pp 1941ndash19492007

[133] Y Pan A Leifert D Ruau et al ldquoGold nanoparticles ofdiameter 14 nm trigger necrosis by oxidative stress andmitochondrial damagerdquo Small vol 5 no 18 pp 2067ndash20762009

[134] T H Kim M Kim H S Park U S Shin M S Gong and HW Kim ldquoSize-dependent cellular toxicity of silver nanoparti-clesrdquo Journal of Biomedical Materials Research A vol 100 no4 pp 1033ndash1043 2012

[135] W K Oh S Kim O Kwon and J Jang ldquoShape-dependentcytotoxicity of polyaniline nanomaterials in human fibrob-last cellsrdquo Journal of Nanoscience and Nanotechnology vol 11no 5 pp 4254ndash4260 2011

[136] N M Schaeublin L K Braydich-Stolle A M Schrand et alldquoSurface charge of gold nanoparticles mediates mechanismof toxicityrdquo Nanoscale vol 3 no 2 pp 410ndash420 2011

[137] L Harhaji A Isakovic N Raicevic et al ldquoMultiple mech-anisms underlying the anticancer action of nanocrystallinefullerenerdquo European Journal of Pharmacology vol 568 no 1ndash3 pp 89ndash98 2007

[138] R Foldbjerg P Olesen M Hougaard D A Dang H JHoffmann and H Autrup ldquoPVP-coated silver nanoparticlesand silver ions induce reactive oxygen species apoptosis andnecrosis in THP-1 monocytesrdquo Toxicology Letters vol 190no 2 pp 156ndash162 2009

[139] S Arora J Jain J M Rajwade and K M Paknikar ldquoCellularresponses induced by silver nanoparticles in vitro studiesrdquoToxicology Letters vol 179 no 2 pp 93ndash100 2008

[140] N Asare C Instanes W J Sandberg et al ldquoCytotoxic andgenotoxic effects of silver nanoparticles in testicular cellsrdquoToxicology vol 291 no 1ndash3 pp 65ndash72 2012

[141] M L Di Giorgio S D Bucchianico A M Ragnelli PAimola S Santucci and A Poma ldquoEffects of single andmulti walled carbon nanotubes on macrophages cyto and


genotoxicity and electron microscopyrdquo Mutation Researchvol 722 no 1 pp 20ndash31 2011

[142] F Tian D Cui H Schwarz G G Estrada and H KobayashildquoCytotoxicity of single-wall carbon nanotubes on humanfibroblastsrdquo Toxicology in Vitro vol 20 no 7 pp 1202ndash12122006

[143] D Cui F Tian Y Kong I Titushikin and H Gao ldquoEffectsof single-walled carbon nanotubes on the polymerase chainreactionrdquo Nanotechnology vol 15 no 1 pp 154ndash157 2004

[144] L Zhu D W Chang L Dai and Y Hong ldquoDNA damageinduced by multiwalled carbon nanotubes in mouse embry-onic stem cellsrdquo Nano Letters vol 7 no 12 pp 3592ndash35972007

[145] K Pulskamp S Diabate and H F Krug ldquoCarbon nanotubesshow no sign of acute toxicity but induce intracellularreactive oxygen species in dependence on contaminantsrdquoToxicology Letters vol 168 no 1 pp 58ndash74 2007

[146] L Galluzzi L Chiarantini E Pantucci et al ldquoDevelopmentof a multilevel approach for the evaluation of nanomaterialsrsquotoxicityrdquo Nanomedicine vol 7 no 3 pp 393ndash409 2012

[147] M A Brennan and B T Cookson ldquoSalmonella inducesmacrophage death by caspase-1-dependent necrosisrdquo Molec-ular Microbiology vol 38 no 1 pp 31ndash40 2000

[148] A C Reisetter L V Stebounova J Baltrusaitis et al ldquoInduc-tion of inflammasome-dependent pyroptosis by carbon blacknanoparticlesrdquo Journal of Biological Chemistry vol 286 no24 pp 21844ndash21852 2011

[149] A S Yazdi G Guarda N Riteau et al ldquoNanoparticlesactivate the NLR pyrin domain containing 3 (Nlrp3) inflam-masome and cause pulmonary inflammation through releaseof IL-1α and IL-1βrdquo Proceedings of the National Academy ofSciences of the United States of America vol 107 no 45 pp19449ndash19454 2010

[150] J M Hillegass A Shukla S A Lathrop M B MacPhersonN K Fukagawa and B T Mossman ldquoAssessing nanotoxicityin cells in vitrordquo Wiley Interdisciplinary Reviews Nanomedici-ne and Nanobiotechnology vol 2 no 3 pp 219ndash231 2010

[151] A M Schrand M F Rahman S M Hussain J J SchlagerD A Smith and A F Syed ldquoMetal-based nanoparticles andtheir toxicity assessmentrdquo Wiley Interdisciplinary ReviewsNanomedicine and Nanobiotechnology vol 2 no 5 pp 544ndash568 2010

[152] R Damoiseaux S George M Li et al ldquoNo time to losemdashhigh throughput screening to assess nanomaterial safetyrdquoNanoscale vol 3 no 4 pp 1345ndash1360 2011

[153] S Y Shaw E C Westly M J Pittet A Subramanian SL Schreiber and R Weissleder ldquoPerturbational profiling ofnanomaterial biologic activityrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 105no 21 pp 7387ndash7392 2008

[154] L Galluzzi S A Aaronson J Abrams et al ldquoGuidelines forthe use and interpretation of assays for monitoring cell deathin higher eukaryotesrdquo Cell Death and Differentiation vol 16no 8 pp 1093ndash1107 2009

[155] B T Mossman J Bignon M Corn A Seaton and J B LGee ldquoAsbestos scientific developments and implications forpublic policyrdquo Science vol 247 no 4940 pp 294ndash301 1990

[156] B W S Robinson and R A Lake ldquoAdvances in malignantmesotheliomardquo The New England Journal of Medicine vol353 no 15 pp 1591ndash1603 2005

[157] T Zhang J L Stilwell D Gerion et al ldquoCellular effectof high doses of silica-coated quantum dot profiled withhigh throughput gene expression analysis and high content

cellomics measurementsrdquo Nano Letters vol 6 no 4 pp 800ndash808 2006

[158] A Zollanvari M J Cunningham U Braga-Neto and ER Dougherty ldquoAnalysis and modeling of time-course gene-expression profiles from nanomaterial-exposed primaryhuman epidermal keratinocytesrdquo BMC Bioinformatics vol10 supplement 11 p S10 2009

[159] Y Y Tyurina V A Tyurin V I Kapralova et al ldquoOxidativelipidomics of γ-radiation-induced lung injury mass spectro-metric characterization of cardiolipin and phosphatidylser-ine peroxidationrdquo Radiation Research vol 175 no 5 pp610ndash621 2011

[160] J G Teeguarden B J Webb-Robertson K M Waters et alldquoComparative proteomics and pulmonary toxicity of instilledsingle-walled carbon nanotubes crocidolite asbestos andultrafine carbon black in micerdquo Toxicological Sciences vol120 no 1 pp 123ndash135 2011

[161] Y Zhang Y Xu Z Li et al ldquoMechanistic toxicity evaluationof uncoated and PEGylated single-walled carbon nanotubesin neuronal PC12 cellsrdquo ACS Nano vol 5 no 9 pp 7020ndash7033 2011


Review Article

Utilisation of Nanoparticle Technology inCancer Chemoresistance

Duncan Ayers1 and Alessandro Nasti2

1 Department of Pathology Faculty of Medicine amp Surgery University of Malta Msida MSD 2060 Malta2 School of Medicine Kanazawa University Hospital University of Kanazawa Kanazawa 920-1192 Japan

Correspondence should be addressed to Duncan Ayers duncanayersgooglemailcom

Received 6 August 2012 Revised 11 October 2012 Accepted 11 October 2012


Copyright copy 2012 D Ayers and A Nasti This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The implementation of cytotoxic chemotherapeutic drugs in the fight against cancer has played an invariably essential role forminimizing the extent of tumour progression andor metastases in the patient and thus allowing for longer event free survivalperiods following chemotherapy However such therapeutics are nonspecific and bring with them dose-dependent cumulativeadverse effects which can severely exacerbate patient suffering In addition the emergence of innate andor acquired chemo-resistance to the exposed cytotoxic agents undoubtedly serves to thwart effective clinical efficacy of chemotherapy in the cancerpatient The advent of nanotechnology has led to the development of a myriad of nanoparticle-based strategies with the specificgoal to overcome such therapeutic hurdles in multiple cancer conditions This paper aims to provide a brief overview and recollec-tion of all the latest advances in the last few years concerning the application of nanoparticle technology to enhance the safe andeffective delivery of chemotherapeutic agents to the tumour site together with providing possible solutions to circumvent cancerchemoresistance in the clinical setting

1 Introduction

It is definitely not a matter of dispute that chemotherapy andits constituent cytotoxic agents play a vital role in the clinicalmanagement of the vast majority of cancer conditionsChemotherapy measures focus on eradication of tumourpresence or (at least) control the degree of tumour progres-sion and metastasis However this therapy has its own criticalflaws due to two major issues namely dose-dependentadverse conditions and the emergence of chemoresistanceproperties within the tumour

2 Dose-Dependent Cumulative Adverse Effects

The issue of dose-dependent cumulative adverse effectsderives from the pharmacological properties of cytotoxicchemotherapeutic agents which are not tissue-specific andthus affect all tissues in a widespread manner In addi-tion tissues having increased turnover rates such as thegastro-intestinal system and skin are more vulnerable to

cytotoxic drug activity and are the most prevalent dose-limiting cumulative adverse effects in patients undergoingchemotherapy Table 1 describes in brief the pharmacologyand adverse effects of a few of the most commonly prescribedchemotherapeutic agents that are implemented in manycancer chemotherapy strategies

3 Tumour Chemoresistance Properties

The emergence of chemoresistance within tumour cells ofsolid tissues is sadly one of the main reasons for treatmentfailure and relapse in patients suffering from metastatic can-cer conditions [1] Resistance of the tumour cell to chemo-therapeutic agent exposure may be innate whereby thegenetic characteristics of the tumour cells are naturally resis-tant to chemotherapeutic drug exposure [2] Alternativelychemoresistance can be acquired through development of adrug resistant phenotype over a defined time period of expo-sure of the tumour cell to individualmultiple chemotherapycombinations [1 2] (see Figure 1)


Table 1 Overview of a selection of cytotoxic drugs commonly used in chemotherapy

Cytotoxic drug Mechanism of action Major adverse effects References

CisplatinInterintrastrand cross-link formation on nucleophilicN7 sites of adjacent adenine and guanine bases leading

to apoptosis

Dose-dependent ototoxicitynephrotoxicity neurotoxicity and

myelosuppression[3ndash9]

CarboplatinInterintrastrand cross-link formation on nucleophilicN7 sites of adjacent adenine and guanine bases leading

to apoptosisDose-dependent myelosuppression [3 4]

Cyclophosphamide

Oxazaphosphorine DNA-alkylating pro-drug activatedby liver P450 cytochrome-induced 4-hydroxylation

thus forming DNA cross-linking phosphoramidemustard

Neurotoxicity and nephrotoxicity due tochloroacetaldehyde formation by P450

cytochrome-induced oxidation[10]

Doxorubicin

Anthracycline-glucuronide conjugate prodrug activatedby tumour β-glucuronidase whereby the drugDNA

adduct possibly induces apoptosis by topoisomerase 2inhibition or by a caspase cascade

Dose-dependent cardiotoxicityhepatotoxicity and myelosuppression

[11ndash15]

EtoposideTopoisomerase II inhibitor by raising the stability of

the enzymeDNA cleavage complex ultimately leadingto DNA strand breaks and apoptosis

Possible secondary leukaemia due tochromosomal translocations induced by

etoposide strand break activitymyelosuppression

[16ndash22]

Ifosfamide (insevere NB cases)

Oxazaphosphorine DNA-alkylating prodrug activatedby liver P450 cytochrome-induced 4-hydroxylation


Marked neurotoxicity and nephrotoxicitydue to increased chloroacetaldehyde

formation by P450 cytochrome-inducedoxidation

[10]

Cisplatin Recovery

Parent cell line Surviving cells Resistant subline

Cisplatin-sensitive cellCisplatin-resistant cell intrinsic resistanceCisplatin-resistant cell new mutation

(a)

Recovery



Cisplatin

(b)

Figure 1 Overview of chemoresistance emergence using cisplatin as an example for a conventional chemotherapeutic drug Intrinsicchemoresistance (a) demonstrates the presence of tumour cell colonies that possess the optimal genetic and phenotypic characteristicsto withstand exposure to cytotoxic agent activity These characteristics were present in such cells prior to initial chemotherapy exposure andhence the term intrinsic chemoresistance In acquired chemoresistance (b) the tumour cell line develops chemoresistance due to mutationaldriving forces following prolonged exposure to chemotherapeutic agents

The biological routes by which the tumour cell is able toescape death by chemotherapy are numerous and complexHowever the major pathways enabling chemoresistance incancer have been studied in detail and are summarised inTable 2

4 Nanoparticle Technology

The introduction of nanotechnology in the last few decadeshas led to an undisputed boom in the conception anddevelopment of innovative methods for effective and safedelivery of small-molecule drugs and gene-based therapiesto their intended target tissues

The advantages of exploiting nanoparticle delivery sys-tems are many such as the possibility to protect nuclease-labile drug therapies such as short interfering RNAs (siR-NAs) and microRNAs (miRNAs) during transit withinthe bloodstream [87 88] In addition implementation ofnanoparticle-based delivery systems has led to improvedpharmacokinetic profiles for the specific drug being carriedwithin such a system together with enhanced targetingof the site of action of the drug [89ndash91] The excellentreview by Hu and Zhang [92] highlighted that nanoparticlesalso have the capacity to carry combination therapies oftwo drugssmall molecules and have demonstrated to beparticularly effective in circumventing multidrug resistance(MDR) issues in multiple cancer models


Table 2 Overview of methods adopted by tumour cells for acquiring chemoresistance properties

Chemoresistancemethod

DescriptionKey player genes proteins andor signalling

pathwaysReferences

Drug effluxmechanisms

Utilisation of drug efflux active pump proteins forexpulsion of multiple cytotoxics from tumour cell

cytoplasm thus inducing multidrug resistance(MDR)

ATP-dependent binding cassette (ABC)transporter proteins multidrug resistance 1

(MDR1) gene P-glycoprotein (P-gp) multidrugresistance 1 protein (MRP1) ABCG2

[23ndash26]

Drug modulation

Tumour cell ability to inactivate or at leastattenuate drug activation through the

modulation of expression of key enzymesinvolved in the target cytotoxic drugrsquos

pharmacological and pharmacokinetic pathways

Decreased expression or impairment offolylpoly-gamma glutamate-synthetase activityresulting in antifolate drug resistance Effect ofglutathione on cisplatin inactivation-mediated

chemoresistance

[27ndash29]

Modification of drugtargets

Upregulated expression or amplification of atarget proteinenzyme which may prove crucial

for drug potency and effectivenessβ-catenin thymidylate synthase [30 31]

Repair mechanismsfollowing DNAdamage

Exacerbated activity of components of thenucleotide excision repair pathway following

tumour cell DNA damage

Excision repair cross complementing 1 proteinmicrosatellite instability phenotype due tomutations in DNA mismatch repair genes

[32ndash37]

DNA methylationmechanisms

Inhibition of key tumour suppressor genesleading to DNA methylations

Caspase-8 promoter hypermethylation inneuroblastoma

[38 39]

p53 statusDysfunction or loss of DNA damageother stress

induced p53 pathway-mediated apoptotic activityMouse double minute 2 (Mdm2) p53 encoding

gene (TP53)[40ndash46]

Apoptotic pathwaydefects

Dysfunction or inactivation of the cytotoxic drugtargeted intrinsicextrinsic proapoptotic pathways

in tumour cells

Bcl-2 protein family cellular FADD-likeinterleukin 1 beta converting enzyme-inhibitoryprotein (c-FLIP) cellular inhibitors of apoptosis

proteins (cIAPs)

[47ndash59]

Proliferative pathwayactivation

Stimulation of cell proliferation throughmodulation of the PI3K and extracellular

signal-regulated kinase (ERK) survival signallingpathways

Protein tyrosine kinases (PTKs) familiesepidermal growth factor receptor (EGFR) family

transcription factor kappa B (NFκB) Sirtuins(SIRTs)

[60ndash68]

Electrostatic network setupamongst polymers stabilizingagents and siRNA

Figure 2 Representative example of a chitosan-based nanoparticle designed for the loading of individual siRNAs within the electrostaticnetwork created by the nanoparticle internal infrastructure

The chemical composition of nanoparticles both fromnatural occurring compounds (see Figure 2) and syntheticones (see Table 3) is varied and the selection of which nano-particle to utilize for any individual drug delivery system isvery much dependent on a multitude of factors such as thechemical nature of the drug to be transported the loadingcapacity of the nanoparticle and resultant pharmacokineticand pharmacodynamics properties of the nanoparticle fol-lowing drug loading [93]

It is beyond the scope of this review to delve into thespecific technical details regarding each individual type ofnanoparticle utilized at present as this has been alreadydiscussed extensively in other technical reviews and researcharticles within the literature [83 84 94 95] Howevera brief summary encompassing the spectrum of vary-ing nanoparticle compositions key advantages togetherwith toxicity profiles can be viewed in Table 3 and Figure3


Table 3 Overview of the major classes of nanoparticles utilised for chemotherapeutic drug delivery

Nanoparticle(NP)composition

Unique characteristics and advantagesAdverse effectstoxicity of nanoparticle

componentsReferences

Solid lipidAcidic pH of MDR tumour cells favours drug

release from NPNo haemolytic activity in human erythrocytes [69]

Polymer-based Versatile acid-responsive drug release kineticsMinimal cytotoxicity observed on ovarian cancer

cell lines[70]

HydrogelsEasy synthesis peptide-attachment facility for

targeted deliveryNontoxic [71]

Magnetic (ironoxide)

Allows for physical (magnetic) enhancement ofthe passive mechanisms implemented for theextravastation and accumulation within the

tumour microenvironment

L-glutamic acid coated iron oxide nanoparticlesdemonstrated in vitro biocompatibility

[72ndash74]

Micelle-basedCapable of solubilizing a wide range of

water-insoluble drugs

Relatively safe though elevated doses can inducedose-dependent adverse effects such as

hyperlipidaemia hepatosplenomegaly andgastrointentinal disorders

[75ndash77]

Gold

Lack of complexity in their synthesischaracterization and surface functionality Gold

nanoparticles also have shapesize-dependentoptoelectronic characteristics

Can induce cellular DNA damage [78ndash80]

Quantum dotsCapacity to be tracked in real time within specific

areas of the target cells due to their intrinsicfluorescence properties

Potential long-term toxicity due to release of toxiccomponents (eg Cadmium) and generation of

reactive oxygen species[81 82]

ChitosanNaturally occurring compound derived from

crustacean shellsHigh biocompatibility properties [83 84]

Mesoporoussilica

Physical characteristics (eg size shape) can beeasily modified to induce bespoke

pharmacokineticpharmacodynamics profiles

Possible membrane peroxidation glutathionedepletion mitochondrial dysfunction andor

DNA damage[85 86]

5 Recent Advances inNanoparticle-Based Cancer ChemoresistanceCircumvention Methodologies

The study carried out by Kang et al [69] demonstrated thatadministration of solid lipid nanoparticles containing dox-orubicin (SLN-Dox) to the adriamycin-resistant breast can-cer cell line MCF-7ADR which also overexpressed P-glyco-protein (P-gp) allowed for chemosensitisation of the cellline This was induced due to enhanced accumulation ofdoxorubicin within the cell line contributed by the nano-particle-based delivery method and thus the degree of apop-tosis was enhanced [69]

The same principle of exploiting nanoparticle deliveryto substantiate chemotherapeutic drug accumulation withinthe target cancer cell with the ultimate goal of enhancingtumour chemosensitivity was adopted in the study by Aryalet al [70] Polymer-cisplatin conjugate nanoparticles weredeveloped and consequently delivered to A2780 human ovar-ian carcinoma cell line [70] The added potential of thisdelivery system relied on the cisplatin analogue prodrugcovalently linked to a poly(ethylene glycol)-based polymerwhich only released its therapeutic payload in a low pHenvironment [70] Consequently clinical administration ofsuch a delivery system would ensure that the drug will remain

complexed whilst in transit within the bloodstream due to itsneutral pH environment [70]

Additionally RNAi therapeutics have come to rely muchfurther on the utilization of nanoparticle delivery systems toexert their biological effects The study by Dickerson et al[71] elucidated the efficiency to knock-down genes such asepidermal growth factor receptor (EGFR) by the delivery ofEGFR-specific siRNAs contained within coreshell hydrogelnanoparticles (nanogels) The nanogels were also coated withpeptides targeting the EphA2 receptor to enhance deliveryof anti-EGFR siRNAs within the targeted Hey tumour cells[71] Consequently the knock-down effect on EGFR led toenhanced chemosensitivity of cancer cells to taxane chemo-therapy [71]

The implementation of nanoparticle technology has alsodemonstrated to aid the clinical effect of other therapiesthat were previously unsuccessful due to poor drug deliveryissues Jin et al [98] developed transferrin conjugated pH-sensitive lipopolyplex nanoparticles with the capacity to bindspecific oligodeoxynucleotides (GTI-2040 in this case) Thisdelivery system allowed GTI-2040 to exert its effect on theR2 subunit of the chemoresistance factor ribonucleotidereductase in acute myeloid leukaemia cell line models [98]The influence of ultilising such a delivery system was evidentin that the 50 inhibitory concentration (IC(50)) for 1 μMGTI-2040 decreased from 4769 nM to 905 nM [98]


Target cell cytoplasm

NP

Rx NP

NPs

EGFR

Enhanced drug

accumulation

Ribonucleotide

reductase

MDR1

Jagged1

MDR1

quantum dotPolymer-

SLN-Rx

P-gp

GTI-2040

Lipopolyplex-

Danuorubicin-

iron

Anti-

siRNAs-

Anti-

-chitosan NP

Jagged1 siRNA

Anti-VLA-4 peptide

-micelle NP

VLA-4

oxide NP

siRNA-

Figure 3 Visual representation of a selection of varying nanoparticle-based drug (Rx) delivery systems adopted for averting cancer chemo-resistance properties Polymer-based [70] and solid lipid nanoparticle-based [69] delivery systems (blue) allow for bypass of the drug effluxpump acquired chemoresistance pathways and allow for enhanced drug accumulation within the target cell cytoplasm together with P-gpdownregulation [96] RNA interference methods utilising short interfering RNAs (purple) have been incorporated in hydrogel nanoparticlesfor targeting of epidermal growth factor receptor a key player in mediating cell adhesion methods of chemoresistance [71] Another majorMDR gene targeted by short interfering RNAs includes P-gp [97] Lipopolycomplex nanoparticles were successful in enhancing the pharma-codynamic properties of the GTI-2040 oligonucleotide targeting ribonucleotide reductase [98] Ferromagnetic nanoparticles (black) havealso been deployed for downregulation of the major chemoresistance gene MDR1 [72] Micelle-based nanoparticles (orange) were found tobe effective in delivering doxorubicin and VLA-4-specific peptides in multiple myeloma cells [76] Quantum dots (green) containing siRNAswere also successfully deployed for downregulating MDR1 and P-gp expression in HeLa cell lines [81] Chitosan nanoparticles (grey)incorporating Jagged1 siRNAs were also highly effective in circumventing MDR properties in taxane-resistant ovarian cell lines [99]

An additional nanoparticle delivery system adoptedagainst MDR in leukaemic conditions was investigated byCheng et al [72] This system combined magnetic iron oxidenanoparticles together with daunorubicin and 5-bromo-tetrandrin which proved to possess a sustained release phar-macokinetic drug profile when administered to K562A02multidrug resistant leukaemic cell lines [72] The principlebehind the utilization of magnetic nanoparticles is due tothe effects of magnetic field gradients positioned in a non-parallel manner with respect to flow direction within thetumour vasculature [73] This allows for physical (mag-netic) enhancement of the passive mechanisms implementedfor the extravastation and accumulation of such magnet-ically responsive nanoparticles within the tumour micro-environment followed by cellular uptake of the nanoparti-cles within the target tumour cell cytoplasm [73] The mag-netically responsive nanoparticle itself is composed of one or

a combination of the three ferromagnetically active elementsat physiological temperature namely iron nickel and cobalt[73] The delivery system described by Cheng et al [72]also aided in providing a dose-dependent antiproliferativeeffect on such cell lines together with enhanced intracellularaccumulation of daunorubicin and downregulated transcriptexpression of MDR1 gene the main factor for induction ofMDR in most cancer models [72] These factors all contri-buted to a reduction in MDR and were directed by the levelof endosomal-mediated cellular uptake properties of suchnanoparticles [100]

In chronic myelogenous leukaemia (CML) a Bcr-Ablpositive status induces MDR properties through multiplepathways including resistance to p53 and Fas ligand-inducedapoptotic pathways [101] The delivery system devised bySingh et al [101] consisted of magnetic nanoparticles com-bined with paclitaxel and was consequently administered


to Bcr-Abl positive K562 leukaemic cell lines [101] Theaddition of lectin functional groups to the nanoparticlecomplex served to aid cellular uptake by the target K562 cellline and also demonstrated a reduction in the IC(50) forpaclitaxel within this cell line model [101]

Multiple myeloma is an additional tumour model thathas seen benefit from the exploitation of nanoparticle tech-nology in its therapeutic avenues [76] The study by Kiziltepeet al [76] succeeded in developing a micelle-based nanopar-ticle delivery system containing doxorubicin and very lateantigen-4 (VLA-4) antagonist peptides [76] This deliverymethod not only accomplished enhanced cytotoxic activitywhen compared to doxorubicin alone but also the additionof VLA-4 antagonist peptides served well in circumventingthe phenomenon of cell-adhesion-mediated drug resistancedue to the resultant impaired VLA-4 mediated adhesion ofmultiple myeloma cells to the stroma of bone marrow withinCB17 SCID murine multiple myeloma xenograft models[76] Additionally drug accumulation within the stroma ofthe multiple myeloma murine xenograft models was alsotenfold higher than the control murine model [76]

Yet another tumour model that has been investigated forthe application of nanoparticle-based chemotherapy for thepurpose of avoidance of chemoresistance is prostate cancer[102] Gold nanoparticles are an attractive avenue for drugdelivery researchers primarily due to their lack of complexityin their synthesis characterization and surface functionality[78] Gold nanoparticles also have shapesize-dependentoptoelectronic characteristics [78] The endosomal-basedroute for gold nanoparticle cellular uptake can be viewed asthe primary advantage for circumventing MDR within thetumour cell since the drug efflux pump is bypassed and thenanoparticle-held chemotherapeutic agent is released withinthe acidic environment of the endosome and allowed topenetrate the tumour cell cytoplasm [79] Consequentlytumour progression phenotypes such as cell proliferationand level of apoptosis are affected to direct an ameliorationof patient prognosis

Gold nanoparticleantiandrogen conjugates were devel-oped by Dreaden et al [102] with the capacity to selectivelybind to two surface receptors which are upregulated inprostate tumour cell surface Thus allowing accumulationof the nanoparticle conjugate specifically within treatment-resistant prostate tumour cells [102] Gold nanoparticleswere also exploited in the study conducted by Tomuleasaet al [103] for the purpose of reducing MDR hepatocellu-lar carcinoma-derived cancer cells The gold nanoparticleswere loaded with doxorubicin capecitabine and cisplatinfollowed by nanoparticle stabilization by L-aspartate [103]The resultant cellular proliferation rates of the hepatocellularcarcinoma cells treated with this nanoparticle-based therapywere found to be lowered drastically [103]

In the study carried out by Punfa et al [104] the cyto-toxic properties of curcumin on multidrug resistant cervicaltumours were maximized through the development of ananoparticle-curcumin drug delivery system Curcumin wassuccessfully entrapped within poly (DL-lactide-co-glycolide)(PLGA) nanoparticles followed by the incorporation ofthe amino-terminal of anti-P-gp [104] Consequently the

curcumin-nanoparticle conjugates were deployed onto theKB-V1 cervical cancer cell line having upregulated P-gpexpression together with the KB-3-1 cell line that has areduced P-gp expression level [104] The results of this studydemonstrated that nanoparticle conjugates bearing anti-P-gp surface markers were highly efficient in binding tothe MDR-inducing surface protein allowing enhanced cel-lular uptake and ultimately aid in the cytotoxic efficacyof curcumin due to increased accumulation of the drugparticularly within the KB-V1 cell line due to its exacerbatedP-gp expression status [104]

Curcumindoxorubicin-laden composite polymer nano-particles were also developed in other studies [105] as ameans of enhancing the pharmacokinetic and pharmacody-namics properties of curcumin thus enhancing its MDR-modulating effect in the target tumour cells The resultantnanoparticle complex was deployed onto several MDRtumour models such as acute leukaemia multiple myelomaand ovarian cancers both in vitro and in vivo [105] Theresults of this study highlighted the possibility of adminis-tration of lower doses of doxorubicin due to the circum-vention of tumour MDR by efficient curcumin activity thusenhancing the toxicity profile for doxorubicin in clinical usestemming from the reduction in cardiotoxicity and haema-tological toxicity dose-dependent adverse effects [105]

Retinoblastoma therapeutic avenues have also beenincreased due to the introduction of nanoparticle drug deliv-ery technology The study by Das and Sahoo demonstratedthe effectiveness of utilising a nanoparticle delivery systemwhich was dual loaded with curcumin together with nutlin-3a (which has been proven to stimulate the activity of thetumour suppressor protein p53) [106] The results of thisparticular investigation highlighted an enhanced level oftherapeutic efficacy on utilizing the nanoparticle-curcumin-nutlin-3a conjugates on the target retinoblastoma Y79 celllines [106] In addition a downregulation of bcl2 and NFκBwas also observed following cell line exposure to the nano-particle conjugates [106]

The nanoparticle-based drug delivery system designed bySaxena and Hussain [96] for its application against multidrugresistant breast tumours was novel in that the actual compo-nents of the nanoparticle biomaterials namely poloxamer407 and D-α-tocopheryl polyethylene glycol 1000 succinate(TPGS) are both known to exert pharmacological activityagainst P-gp [96] The drug utilized for nanoparticle loadingin this case was gambogic acid a naturally occurring cyto-toxic agent though laden with issues of poor bioavailabilityand severe dose-limiting adverse effects [96] Similarly toother studies mentioned above the incorporation of a nano-particle-based drug delivery system allowed for enhancedcellular uptake by the target breast cancer cell line MCF-7thus leading to elevated drug accumulation on the intracel-lular level and ultimately inducing enhanced cytotoxic effectsin the target breast cancer cell line [96]

A separate nanoparticle-based drug delivery system foruse in circumventing MDR effects in breast cancer is the onedeveloped by Li et al [107] In this study the nanoparticledrug delivery system consisted of a dimethyldidodecylam-monium bromide (DMAB)-modified poly(lactic-co-glycolic


acid) (PLGA) nanoparticle core that was conjugated to dox-orubicin then consequently coated with a 12-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) shell [107] This sys-tem has been described to be specifically effective againstMCF-7 breast cancer cell lines overexpressing P-gp [107]The results obtained from this particular study indicatedan elevated accumulation of doxorubicin released from thenanoparticle complex within the nuclei of the drug resistantMCF-7 cell line [107] In comparison the level of accumula-tion of freely administered (ie not utilising a nanoparticle-based drug delivery system) doxorubicin attained lowerdrug concentration levels within the same cell line [107]Finally the IC(50) levels for doxorubin on adriamycin-resistant MCF-7 have been observed to be lowered by 30-fold following the incorporation of this nanoparticle deliverysystem [107]

Apart from delivery of conventional chemotherapeu-tic drugs in drug resistant breast cancer cell line mod-els researchers also delved into the possibility of adoptingsiRNA therapeutic approaches using the aid of nanoparticledrug delivery systems [97] The study conducted by Navarroet al [97] developed a nanoparticle-based delivery systemfor siRNAs targeting P-gp expression with the nanoparticleconstituent biomaterials being dioleoylphosphatidylethanol-amine and polyethylenimine (PEI) [97] Again the reductionin P-gp expression led the path to enhanced cytoxic effectsbrought about by the exposure of the MCF-7 cell line todoxorubicin thus this nanoparticle-siRNA therapy was suc-cessful in drastically reducing MDR in this cancer model[97]

Quantum dots have also been implemented as novel andeffective drug delivery systems for circumventing multidrugresistance in cancer chemotherapy [81] Researchers in thisstudy developed a quantum dot-based drug delivery systemthat allowed anti-MDR1 siRNA and doxorubicin incorpora-tion to two cadmium-seleniumzinc-selenium quantum dotsthat were eventually functionalized by β-cyclodextrin coupl-ing to L-arginine or L-histamine [81] Following deploymentof these dual loaded quantum dots in the HeLa cervical can-cer cell line model elevated accumulation of doxorubicinwithin the tumour cells was denoted together with a markedreduction in MDR1 and P-gp expression on analysis byreverse transcription real time quantitative polymerase chainreaction and western blotting [81] In line with magneticand gold nanoparticle platforms quantum dots rely mainlyon the endosomal method of tumour cellular uptake andtherefore the drug efflux pump system is bypassed withconsequent reduction in MDR properties by the tumour cells[82] Finally the additional benefit of utilizing quantum dotsas a drug delivery system is their capacity to be tracked inreal time within specific areas of the target cells due to theirintrinsic fluorescence properties [81]

Apart from cell line studies researchers have also lookedinto the feasibility of implementing nanoparticle-based drugdelivery systems within in vivo models [108] The study byMilane et al [108] investigated the efficacy of utilising aEGFR-targeting polymer blend nanoparticles loaded withpaclitaxel and the mitochondrial hexokinase 2 inhibitor loni-damine The nanoparticle polymer blend consisted of 70

polycaprolactone (PCL) incorporating a PLGA-polyethyleneglycol-EGFR specific peptide that helped enable nanoparticleactive targeting efficiency [108]

Following nanoparticle development four groups oforthotopic MDR breast cancer murine models (MDA-MB-231 in nude mice) were treated with free paclitaxelfree lonidamine free paclitaxellonidamine combination ornanoparticle complexes containing paclitaxellonidaminecombination [108] The degree of toxicity of such treatmentswas also monitored through body weight change measu-rements liver enzyme plasma levels and white blood cellplatelet counts together with H amp E staining of tumour sec-tions was carried out [108]

Tumour weight and other clinical parameters such asMDR protein marker (P-gp Hypoxia Inducible factor αHexokinase 2 EGFR Stem Cell factor) were observed overthe course of 28 days after-treatment [108] Following this28-day period the results demonstrated that only the murinemodel sample group exposed to the nanoparticle-basedpaclitaxellonidamine combination treatment was the onlygroup to experience statistically significant tumour volumeand density reduction together with overall alteration of theMDR phenotype [108] Toxicity effects due to paclitaxel andlonidamine were also drastically reduced when administeredwithin the nanoparticle-based delivery system which canultimately provide enhanced tolerance by the cancer patient[108]

Other in vivo studies in this field include the investiga-tions carried out by Shen et al [109] which focused onthe codelivery of paclitaxel and survivin short hairpin RNA(shRNA) for circumventing chemoresistance in lung cancerThe investigators utilized the pluronic block co-polymer P85combined with D-α-Tocopheryl polyethylene glycol 1000succinate (P85-PEITPGS) for developing the nanoparti-cles to be implemented in this study [109] These nano-particles were based upon triblock structural formation ofhydrophilic poly(ethylene oxide) (PEO) blocks and hydro-phobic poly(propylene oxide) (PPO) blocks which alsogives enhanced capacity to revert chemoresistance due todrug efflux pump inhibition properties downregulation ofATPase activity and P85-induced inhibition of the glutha-thione S-transferase compound detoxification enzyme at thesubcellular level [109] Paclitaxel and surviving shRNA wereselected as the ideal drugs for nanoparticle delivery due to theformer having poor efficacy due to chemoresistance withinthe tumour and survivin was identified as highly expressedwithin chemoresistant tumours [109] The in vivo activityof such nanoparticle systems (withwithout paclitaxel andsurvivin shRNA) was evaluated on BALBc nude miceinjected with viable paclitaxel-resistant A549T lung ade-nocarcinoma epithelial cells [109] The results of this studydemonstrated that deployment of the nanoparticle-basedchemotherapeutic drug proved to have distinct enhancementof antitumour efficacy when compared to deployment of thedrugs alone [109]

Chemoresistance to the aromatase inhibitor letrozole inpostmenopausal breast cancer is another major therapeutichurdle which was investigated in vivo [110] BiodegradablePLGA-polyethylene glycol copolymer nanoparticles were


developed by nanoprecipitation and designed to incorporatehyaluronic acid-bound letrozole (HA-Letr-NPs) [110] Theaddition of hyaluronic acid served to enhance letrozole bind-ing specificity to CD44 on the target tumour cell surface withthe expected consequences of enhanced drug accumulationwithin the target tumour cell cytoplasm and resultant re-sensitization of the target tumour cells to letrozole activity[110] Such HA-Letr-NPs once produced at a size of lessthan 100 nm diameter were deployed within a letrozole-resistant murine xenograft tumour model [110] The resultsof this study demonstrated a highly efficient nanoparticle-based drug delivery system with the IC(50) for HA-Letr-NPs within the murine xenograft model being only 5 μMwhen compared to the control groups thus enhancing thein vivo aromatase enzyme activity within the xenograft andultimately inducing a prolonged resensitising of the breastcancer tumour to letrozole activity [110]

The naturally occurring compound chitosan was alsoutilized for the development of in vivo nanoparticle-basedtherapies to circumvent ovarian cancer chemoresistanceproperties induced by overexpression of the Jagged1 notchligand [99] Murine orthotopic models utilising femaleathymic nude mice were injected with SKOV3Trip2 taxane-resistant ovarian cancer cell line and consequently followingone week subjected to anti-Jagged1 siRNAchitosan nano-particle complexes (5 μg dose of siRNA) withwithout taxaneapplied via intraperitoneal route twice weekly for a totalperiod of five weeks [99] The results of this study indicatedthat such nanoparticle-based complexes had the capacity toreduce tumour weight by over 70 within such murinemodels and also induced taxane sensitization within thetumour [99]

In a similar study cationic liposome-polycation-DNA(LPD) and anionic liposome-polycation-DNA (LPD II)nanoparticle systems were developed to incorporate dox-orubicin and VEGF siRNA within a murine ovarian canceranimal model [111] Female athymic nude mice were treatedwith 5 times 106 cells of the MDR ovarian cancer cell line NCIADR-RES [111] Once the murine tumours reached a sizeof approximately 16ndash25 mm2 the mice were consequentlyinjected with individual nanoparticle complexes bearingeither siRNA or doxorubicin at a dose of 12 mgKg in bothcases once daily for three consecutive days [111] The resultsof this study demonstrated the effectiveness of such nano-particle complexes for inhibiting tumour progression withinthe treated murine model groups mainly due to impairedVEGF expression-related MDR [111]

Other human cancer conditions which were investigatedfor circumvention of tumour MDR properties throughnanoparticle delivery include uterine sarcomas [112] In thestudy carried out by Huang et al [112] pH-sensitive meso-porous silica nanoparticles incorporating hydrazine anddoxorubicin were developed for in vivo testing on murinemodels of doxorubicin-resistant uterine sarcoma Since thecomposition of such nanoparticles specifically allow for cel-lular uptake through endocytosis bypassing of the P-gpefflux pump induced a marked reduction in P-gp dependentMDR properties [112] Consequently the murine MDRtumour model treated with such nanoparticles demonstrated

enhanced tumour apoptotic effects which were clearly con-firmed by active caspase-3 immunohistochemical validationanalysis [112]

6 Conclusion

The latest studies described above undoubtedly serve asa testament to the immense clinical value represented bynanoparticle technology The ability of such nanoparticlesirrelevant of biomaterial composition to efficiently load indi-vidual or combinations of chemotherapeutic drugs andorchemosensitising agents (such as curcumin) and novel RNAinterference-based therapies has been clearly demonstratedabove This property provides an excellent escape mecha-nism for circumventing target tumour cell multidrug resis-tance properties based on drug efflux pump activity on thetumour cell surface such as that exerted by P-gp The overalladvantage of deploying nanoparticles includes the drasticreduction in the IC(50) parameter for most of the carriedchemotherapy agents due to marked intracellular accumu-lation pharmacodynamics This in turn would lead to areduction in the clinical doses of the conventional cytotoxicagents required for chemotherapy ultimately demonstratinga striking reduction in dose-dependent adverse effects in theoncology patient

Presently this does not mean that nanotechnology-basedtranslational therapies are not fraught with challenges suchas biocompatibility issues of the nanoparticle componentsand the level of complexity required for cost-effectively trans-lating these novel therapies to the patient bedside Howeverit is the firm belief of the authors that through constantaccumulation of marginal gains in knowledge derived frompersistent and motivated researchers on a global scale willultimately overcome such scientific hurdles thus nanopar-ticle-based drug delivery aided therapies will eventuallybecome commonplace in the oncology clinic in the nearfuture

Acknowledgment

The authors would like to thank Dr Jennifer Logan (Uni-versity of Manchester UK) for the initial design of Figure 1utilised in this paper

References

[1] D B Longley and P G Johnston ldquoMolecular mechanismsof drug resistancerdquo Journal of Pathology vol 205 no 2 pp275ndash292 2005

[2] R S Kerbel H Kobayashi and C H Graham ldquoIntrinsicor acquired drug resistance and metastasis are they linkedphenotypesrdquo Journal of Cellular Biochemistry vol 56 no 1pp 37ndash47 1994

[3] D S Goodsell ldquoThe molecular perspective cisplatinrdquo Oncol-ogist vol 11 no 3 pp 316ndash317 2006

[4] G N Kaludjerovic D Miljkovic M Momcilovic et alldquoNovel platinum(IV) complexes induce rapid tumor celldeath in vitrordquo International Journal of Cancer vol 116 no3 pp 479ndash486 2005


[5] A L Berg J B Spitzer and J H Garvin ldquoOtotoxic impact ofcisplatin in pediatric oncology patientsrdquo Laryngoscope vol109 no 11 pp 1806ndash1814 1999

[6] Y Li R B Womer and J H Silber ldquoPredicting cisplatin oto-toxicity in children the influence of age and the cumulativedoserdquo European Journal of Cancer vol 40 no 16 pp 2445ndash2451 2004

[7] J Sastry and S J Kellie ldquoSevere neurotoxicity ototoxicityand nephrotoxicity following high-dose cisplatin and amifos-tinerdquo Pediatric Hematology and Oncology vol 22 no 5 pp441ndash445 2005

[8] I Arany and R L Safirstein ldquoCisplatin nephrotoxicityrdquoSeminars in Nephrology vol 23 no 5 pp 460ndash464 2003

[9] M Jiang X Yi S Hsu C Y Wang and Z Dong ldquoRole of p53in cisplatin-induced tubular cell apoptosis dependence onp53 transcriptional activityrdquo American Journal of Physiologyvol 287 no 6 pp F1140ndashF1147 2004

[10] C-S Chen J T Lin K A Goss Y A He J R Halpertand D J Waxman ldquoActivation of the anticancer prodrugscyclophosphamide and ifosfamide identification of cyto-chrome P450 2B enzymes and site-specific mutants withimproved enzyme kineticsrdquo Molecular Pharmacology vol 65no 5 pp 1278ndash1285 2004

[11] A Atessahin G Turk I Karahan S Yilmaz A O Ceribasiand O Bulmus ldquoLycopene prevents adriamycin-induced tes-ticular toxicity in ratsrdquo Fertility and Sterility vol 85 no 1pp 1216ndash1222 2006

[12] M J Ferguson F Y Ahmed and J Cassidy ldquoThe role ofpro-drug therapy in the treatment of cancerrdquo Drug ResistanceUpdates vol 4 no 4 pp 225ndash232 2001

[13] L P Swift A Rephaeli A Nudelman D R Phillips and SM Cutts ldquoDoxorubicin-DNA adducts induce a non-topo-isomerase II-mediated form of cell deathrdquo Cancer Researchvol 66 no 9 pp 4863ndash4871 2006

[14] Chemocare Doxorubicin Adriamycin RubexmdashChemothe-rapy Drugs Chemo Drug Side Effects [Internet] 2011httpwwwchemocarecombiodoxorubicinasp

[15] P K Singal and N Iliskovic ldquoDoxorubicin-induced cardio-myopathyrdquo The New England Journal of Medicine vol 339no 13 pp 900ndash905 1998

[16] K R Hande ldquoEtoposide four decades of development of atopoisomerase II inhibitorrdquo European Journal of Cancer vol34 no 10 pp 1514ndash1521 1998

[17] M Duca D Guianvarcrsquoh K Oussedik et al ldquoMolecular basisof the targeting of topoisomerase II-mediated DNA cleavageby VP16 derivatives conjugated to triplex-forming oligonu-cleotidesrdquo Nucleic Acids Research vol 34 no 6 pp 1900ndash1911 2006

[18] The Chemical Heritage Foundation Magic Bullets Chem-istry vs Cancer Cancer Chemotherapy a chemical needlein a haystack [Internet] 2001 httpwwwchemheritageorgEducationalServicespharmchemoreadingsageshtm

[19] D A Burden P S Kingma S J Froelich-Ammon et alldquoTopoisomerase II-etoposide interactions direct the forma-tion of drug- induced enzyme-DNA cleavage complexesrdquoJournal of Biological Chemistry vol 271 no 46 pp 29238ndash29244 1996

[20] R Bagatell P Rumcheva W B London et al ldquoOutcomesof children with intermediate-risk neuroblastoma after treat-ment stratified by MYCN status and tumor cell ploidyrdquoJournal of Clinical Oncology vol 23 no 34 pp 8819ndash88272005

[21] Electronic Medicines Compendium Whatrsquos Newmdashelectro-nic Medicines Compendium (eMC) [Internet] 2010 httpwwwmedicinesorgukEMC

[22] A R Mistry C A Felix R J Whitmarsh et al ldquoDNAtopoisomerase II in therapy-related acute promyelocyticleukemiardquo The New England Journal of Medicine vol 352 no15 pp 1529ndash1538 2005

[23] R W Robey P R Massey L Amiri-Kordestani and S EBates ldquoABC transporters unvalidated therapeutic targetsin cancer and the CNSrdquo Anti-Cancer Agents in MedicinalChemistry vol 10 no 8 pp 625ndash633 2010

[24] R Krishna and L D Mayer ldquoMultidrug resistance (MDR)in cancerMechanisms reversal using modulators of MDRand the role of MDR modulators in influencing the pharma-cokinetics of anticancer drugsrdquo European Journal of Pharma-ceutical Sciences vol 11 no 4 pp 265ndash283 2000

[25] M Colone A Calcabrini L Toccacieli et al ldquoThe multidrugtransporter P-glycoprotein a mediator of melanoma inva-sionrdquo Journal of Investigative Dermatology vol 128 no 4pp 957ndash971 2008

[26] M D Norris S B Bordow G M Marshall P S Haber S LCohn and M Haber ldquoExpression of the gene for multidrug-resistance-associated protein and outcome in patients withneuroblastomardquo The New England Journal of Medicine vol334 no 4 pp 231ndash238 1996

[27] Y G Assaraf ldquoMolecular basis of antifolate resistancerdquoCancer and Metastasis Reviews vol 26 no 1 pp 153ndash1812007

[28] T R Wilson D B Longley and P G Johnston ldquoChemore-sistance in solid tumoursrdquo Annals of Oncology vol 10 sup-plement 10 pp x315ndashx324 2006

[29] C Meijer N H Mulder H Timmer-Bosscha W J SluiterG J Meersma and E G E De Vries ldquoRelationship of cel-lular glutathione to the cytotoxicity and resistance of sevenplatinum compoundsrdquo Cancer Research vol 52 no 24 pp6885ndash6889 1992

[30] J Yeung M T Esposito A Gandillet et al ldquoβ-catenin medi-ates the establishment and drug resistance of MLL leukemicstem cellsrdquo Cancer Cell vol 18 no 6 pp 606ndash618 2010

[31] S Copur K Aiba J C Drake C J Allegra and E ChuldquoThymidylate synthase gene amplification in human coloncancer cell lines resistant to 5-fluorouracilrdquo BiochemicalPharmacology vol 49 no 10 pp 1419ndash1426 1995

[32] P A Bradbury M H Kulke R S Heist et al ldquoCisplatin phar-macogenetics DNA repair polymorphisms and esophagealcancer outcomesrdquo Pharmacogenetics and Genomics vol 19no 8 pp 613ndash625 2009

[33] S Arora A Kothandapani K Tillison V Kalman-Malteseand S M Patrick ldquoDownregulation of XPF-ERCC1 enhancescisplatin efficacy in cancer cellsrdquo DNA Repair vol 9 no 7pp 745ndash753 2010

[34] L Shen and J-P J Issa ldquoEpigenetics in colorectal cancerrdquoCurrent Opinion in Gastroenterology vol 18 no 1 pp 68ndash73 2002

[35] H Kim J Y An S H Noh S K Shin Y C Lee and HKim ldquoHigh microsatellite instability predicts good prognosisin intestinal-type gastric cancersrdquo Journal of Gastroenterologyand Hepatology vol 26 no 3 pp 585ndash592 2011

[36] M Takahashi M Koi F Balaguer C R Boland and AGoel ldquoMSH3 mediates sensitization of colorectal cancer cellsto cisplatin oxaliplatin and a poly(ADP-ribose) polymeraseinhibitorrdquo The Journal of Biological Chemistry vol 286 no14 pp 12157ndash12165 2011


[37] L P Martin T C Hamilton and R J Schilder ldquoPlatinumresistance the role of DNA repair pathwaysrdquo Clinical CancerResearch vol 14 no 5 pp 1291ndash1295 2008

[38] J Ren B N Singh Q Huang et al ldquoDNA hypermethylationas a chemotherapy targetrdquo Cellular Signalling vol 23 no 7Article ID 213453 pp 1082ndash193 2011

[39] T Teitz T Wei M B Valentine et al ldquoCaspase 8 is deletedor silenced preferentially in childhood neuroblastomas withamplification of MYCNrdquo Nature Medicine vol 6 no 5 pp529ndash535 2000

[40] N J Maclaine and T R Hupp ldquoHow phosphorylationcontrols p53rdquo Cell Cycle vol 10 no 6 pp 916ndash9121 2011

[41] A Macchiarulo N Giacche F Mancini E Puxeddu FMoretti and R Pellicciari ldquoAlternative strategies for target-ing mouse double minute 2 activity with small moleculesnovel patents on the horizonrdquo Expert Opinion on TherapeuticPatents vol 21 no 3 pp 287ndash294 2011

[42] M R Buchakjian and S Kornbluth ldquoThe engine drivingthe ship metabolic steering of cell proliferation and deathrdquoNature Reviews Molecular Cell Biology vol 11 no 10 pp715ndash727 2010

[43] R Garcıa-Escudero A B Martınez-Cruz M Santos et alldquoGene expression profiling of mouse p53-deficient epidermalcarcinoma defines molecular determinants of human cancermalignancyrdquo Molecular Cancer vol 9 article 193 2010

[44] A Mogi and H Kuwano ldquoTP53 mutations in nonsmall celllung cancerrdquo Journal of Biomedicine and Biotechnology vol2011 Article ID 583929 9 pages 2011

[45] S Stilgenbauer and T Zenz ldquoUnderstanding and managingultra high-risk chronic lymphocytic leukemiardquo Hematologyvol 2010 pp 481ndash488 2010

[46] F Al-Ejeh R Kumar A Wiegmans S R Lakhani M PBrown and K K Khanna ldquoHarnessing the complexity ofDNA-damage response pathways to improve cancer treat-ment outcomesrdquo Oncogene vol 29 no 46 pp 6085ndash60982010

[47] J Plati O Bucur and R Khosravi-Far ldquoApoptotic cellsignaling in cancer progression and therapyrdquo Integrative Bio-logy vol 3 no 4 Article ID 213400 pp 279ndash296 2011

[48] Y Kushnareva and D D Newmeyer ldquoBioenergetics and celldeathrdquo Annals of the New York Academy of Sciences vol 1201pp 50ndash57 2010

[49] L A Allan and P R Clarke ldquoApoptosis and autophagyregulation of caspase-9 by phosphorylationrdquo FEBS Journalvol 276 no 21 pp 6063ndash6073 2009

[50] S G Rolland and B Conradt ldquoNew role of the BCL2 familyof proteins in the regulation of mitochondrial dynamicsrdquoCurrent Opinion in Cell Biology vol 22 no 6 pp 852ndash8582010

[51] L Gandhi D R Camidge M R de Oliveira et al ldquoPhase Istudy of navitoclax (ABT-263) a novel bcl-2 family inhibitorin patients with small-cell lung cancer and other solidtumorsrdquo Journal of Clinical Oncology vol 29 no 7 pp 909ndash916 2011

[52] W J Placzek J Wei S Kitada D Zhai J C Reed and MPellecchia ldquoA survey of the anti-apoptotic Bcl-2 subfamilyexpression in cancer types provides a platform to predict theefficacy of Bcl-2 antagonists in cancer therapyrdquo Cell Deathand Disease vol 1 no 5 article e40 2010

[53] U Testa ldquoTRAILTRAIL-R in hematologic malignanciesrdquoJournal of Cellular Biochemistry vol 110 no 1 pp 21ndash342010

[54] J Liu X Q Fu W Zhou H G Yu J P Yu and H S LuoldquoLY294002 potentiates the anti-cancer effect of oxaliplatin forgastric cancer via death receptor pathwayrdquo World Journal ofGastroenterology vol 17 no 2 pp 181ndash190 2011

[55] Z Yu R Wang L Xu S Xie J Dong and Y Jing ldquoβ-elemenepiperazine derivatives induce apoptosis in human leukemiacells through downregulation of c-FLIP and Generation ofROSrdquo PLoS ONE vol 6 no 1 Article ID e15843 2011

[56] W C Earnshaw L M Martins and S H Kaufmann ldquoMam-malian caspases structure activation substrates and func-tions during apoptosisrdquo Annual Review of Biochemistry vol68 pp 383ndash424 1999

[57] S L Petersen M Peyton J D Minna and X Wang ldquoOver-coming cancer cell resistance to Smac mimetic induced apop-tosis by modulating cIAP-2 expressionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 107 no 26 pp 11936ndash11941 2010

[58] P Lanuti V Bertagnolo L Pierdomenico et al ldquoEnhance-ment of TRAIL cytotoxicity by AG-490 in human ALL cellsis characterized by downregulation of cIAP-1 and cIAP-2through inhibition of Jak2Stat3rdquo Cell Research vol 19 no9 pp 1079ndash1089 2009

[59] C Gill C Dowling A J OrsquoNeill and R W G WatsonldquoEffects of cIAP-1 cIAP-2 and XIAP triple knockdown onprostate cancer cell susceptibility to apoptosis cell survivaland proliferationrdquo Molecular Cancer vol 8 article 39 2009

[60] R Avraham and Y Yarden ldquoFeedback regulation of EGFRsignalling decision making by early and delayed loopsrdquoNature Reviews Molecular Cell Biology vol 12 no 2 pp 104ndash117 2011

[61] F Vidal W M de Araujo A L S Cruz M N TanakaJ P B Viola and J A Morgado-Dıaz ldquoLithium reducestumorigenic potential in response to EGF signaling in humancolorectal cancer cellsrdquo International Journal of Oncology vol38 no 5 pp 1365ndash1373 2011

[62] Q Sheng and J Liu ldquoThe therapeutic potential of targetingthe EGFR family in epithelial ovarian cancerrdquo British Journalof Cancer vol 1041 no 8 pp 1241ndash1245 2011

[63] G Metro G Finocchiaro L Toschi et al ldquoEpidermal growthfactor receptor (EGFR) targeted therapies in non-small celllung cancer (NSCLC)rdquo Reviews on Recent Clinical Trials vol1 no 1 pp 1ndash13 2006

[64] S E Al-Batran M Ruppert and E Jager ldquoTrastuzumab pluschemotherapy in gastric cancer overexpressing HER-2 andEGFR a case reportrdquo Onkologie vol 34 no 1-2 pp 42ndash452011

[65] S E Chuang P Y Yeh Y S Lu et al ldquoBasal levels andpatterns of anticancer drug-induced activation of nuclearfactor-κB (NF-κB) and its attenuation by tamoxifen dex-amethasone and curcumin in carcinoma cellsrdquo BiochemicalPharmacology vol 63 no 9 pp 1709ndash1716 2002

[66] Y Olmos J J Brosens and E W F Lam ldquoInterplay betweenSIRT proteins and tumour suppressor transcription factorsin chemotherapeutic resistance of cancerrdquo Drug ResistanceUpdates vol 14 no 1 pp 35ndash44 2011

[67] B Peck C Y Chen K K Ho et al ldquoSIRT inhibitors inducecell death and p53 acetylation through targeting both SIRT1and SIRT2rdquo Molecular Cancer Therapeutics vol 9 no 4 pp844ndash855 2010

[68] E Lara A Mai V Calvanese et al ldquoSalermide a Sirtuininhibitor with a strong cancer-specific proapoptotic effectrdquoOncogene vol 28 no 6 pp 781ndash791 2009


[69] K W Kang M K Chun O Kim et al ldquoDoxorubicin-loadedsolid lipid nanoparticles to overcome multidrug resistance incancer therapyrdquo Nanomedicine vol 6 no 2 pp 210ndash2132010

[70] S Aryal C M J Hu and L Zhang ldquoPolymer-cisplatinconjugate nanoparticles for acid-responsive drug deliveryrdquoACS Nano vol 4 no 1 pp 251ndash258 2010

[71] E B Dickerson W H Blackburn M H Smith L B Kapa LA Lyon and J F McDonald ldquoChemosensitization of cancercells by siRNA using targeted nanogel deliveryrdquo BMC Cancervol 10 article 10 2010

[72] J Cheng J Wang B Chen et al ldquoA promising strategy forovercoming MDR in tumor by magnetic iron oxide nanopar-ticles co-loaded with daunorubicin and 5-bromotetrandrinrdquoInternational Journal of Nanomedicine vol 6 pp 2123ndash21312011

[73] J Klostergaard and C E Seeney ldquoMagnetic nanovectors fordrug deliveryrdquo Nanomedicine vol 73 supplement 1 pp S37ndashS50 2012

[74] T Zhang L Qian M Tang et al ldquoEvaluation on cytotoxicityand genotoxicity of the L-glutamic acid coated iron oxidenanoparticlesrdquo Journal of Nanoscience and Nanotechnologyvol 12 no 3 pp 2866ndash2873 2012

[75] V P Torchilin ldquoMicellar nanocarriers pharmaceutical per-spectivesrdquo Pharmaceutical Research vol 24 no 1 pp 1ndash162007

[76] T Kiziltepe J D Ashley J F Stefanick et al ldquoRationallyengineered nanoparticles target multiple myeloma cellsovercome cell-adhesion-mediated drug resistance and showenhanced efficacy in vivordquo Blood Cancer Journal vol 2 no 4article e64 2012

[77] S B Lim A Banerjee and H Onyuksel ldquoImprovement ofdrug safety by the use of lipid-based nanocarriersrdquo Journal ofControlled Release vol 163 no 1 pp 34ndash45 2012

[78] R R Arvizo S Bhattacharyya R A Kudgus K Giri RBhattacharya and P Mukherjee ldquoIntrinsic therapeutic appli-cations of noble metal nanoparticles past present andfuturerdquo Chemical Society Reviews vol 41 no 7 pp 2943ndash2970 2012

[79] L Vigderman and E R Zubarev ldquoTherapeutic platformsbased on gold nanoparticles and their covalent conjugateswith drug moleculesrdquo Advanced Drug Delivery Reviews Inpress

[80] C Di Guglielmo J De Lapuente C Porredon D Ramos-Lopez J Sendra and M Borras ldquoIn vitro safety toxicologydata for evaluation of gold nanoparticles-chronic cytotox-icity genotoxicity and uptakerdquo Journal of Nanoscience andNanotechnology vol 12 no 8 pp 6185ndash6191 2012

[81] J-M Li Y-Y Wang M-X Zhao et al ldquoMultifunctional QD-based co-delivery of siRNA and doxorubicin to HeLa cellsfor reversal of multidrug resistance and real-time trackingrdquoBiomaterials vol 33 no 9 pp 2780ndash2790 2012

[82] C E Probst P Zrazhevskiy V Bagalkot and X Gao ldquoQuan-tum dots as a platform for nanoparticle drug delivery vehicledesignrdquo Advanced Drug Delivery Reviews In press

[83] N M Zaki A Nasti and N Tirelli ldquoNanocarriers for cyto-plasmic delivery cellular uptake and intracellular fate ofchitosan and hyaluronic acid-coated chitosan nanoparticlesin a phagocytic cell modelrdquo Macromolecular Bioscience vol11 no 12 pp 1747ndash1760 2011

[84] A Nasti N M Zaki P De Leonardis et al ldquoChitosanTPPand chitosanTPP-hyaluronic acid nanoparticles systematicoptimisation of the preparative process and preliminary

biological evaluationrdquo Pharmaceutical Research vol 26 no8 pp 1918ndash1930 2009

[85] V Mamaeva C Sahlgren and M Linden ldquoMesoporous silicananoparticles in medicine-Recent advancesrdquo Advanced DrugDelivery Reviews In press

[86] T Asefa and Z Tao ldquoBiocompatibility of mesoporous silicananoparticlesrdquo Chemical Research in Toxicology In press

[87] C Alabi A Vegas and D Anderson ldquoAttacking the genomeemerging siRNA nanocarriers from concept to clinicrdquo Cur-rent Opinion in Pharmacology vol 12 no 4 pp 427ndash4332012

[88] K A Howard ldquoDelivery of RNA interference therapeuticsusing polycation-based nanoparticlesrdquo Advanced Drug Deliv-ery Reviews vol 61 no 9 pp 710ndash720 2009

[89] L Zhang F X Gu J M Chan A Z Wang R S Langer andO C Farokhzad ldquoNanoparticles in medicine therapeuticapplications and developmentsrdquo Clinical Pharmacology ampTherapeutics vol 83 no 5 pp 761ndash769 2008

[90] A Z Wang F Gu L Zhang et al ldquoBiofunctionalized targetednanoparticles for therapeutic applicationsrdquo Expert Opinionon Biological Therapy vol 8 no 8 pp 1063ndash1070 2008

[91] C-M J Hu S Kaushal H S T Cao et al ldquoHalf-antibodyfunctionalized lipid-polymer hybrid nanoparticles for tar-geted drug delivery to carcinoembryonic antigen presentingpancreatic cancer cellsrdquo Molecular Pharmaceutics vol 7 no3 pp 914ndash920 2010

[92] C-M J Hu and L Zhang ldquoNanoparticle-based combina-tion therapy toward overcoming drug resistance in cancerrdquoBiochemical Pharmacology vol 83 no 8 pp 1104ndash11112012

[93] A Shapira Y D Livney H J Broxterman and Y G AssarafldquoNanomedicine for targeted cancer therapy towards theovercoming of drug resistancerdquo Drug Resistance Updates vol14 no 3 pp 150ndash163 2011

[94] S Dufort L Sancey and J-L Coll ldquoPhysico-chemical param-eters that govern nanoparticles fate also dictate rules for theirmolecular evolutionrdquo Advanced Drug Delivery Reviews vol64 no 2 pp 179ndash189 2012

[95] A Bitar N M Ahmad H Fessi and A Elaissari ldquoSilica-based nanoparticles for biomedical applicationsrdquo Drug Dis-covery Today vol 17 no 19-20 pp 1147ndash1154 2012

[96] V Saxena and M D Hussain ldquoPoloxamer 407TPGS mixedmicelles for delivery of gambogic acid to breast and multi-drug-resistant cancerrdquo International Journal of Nanomedi-cine vol 7 pp 713ndash721 2012

[97] G Navarro R R Sawant S Biswas et al ldquoP-glycoproteinsilencing with siRNA delivered by DOPE-modified PEIovercomes doxorubicin resistance in breast cancer cellsrdquoNanomedicine vol 7 no 1 pp 65ndash78 2012

[98] Y Jin S Liu B Yu et al ldquoTargeted delivery of antisenseoligodeoxynucleotide by transferrin conjugated pH-sensitivelipopolyplex nanoparticles a novel oligonucleotidemdashbasedtherapeutic strategy in acute myeloid leukemiardquo MolecularPharmaceutics vol 7 no 1 pp 196ndash206 2010

[99] A D Steg A A Katre B Goodman et al ldquoTargeting thenotch ligand JAGGED1 in both tumor cells and stroma inovarian cancerrdquo Clinical Cancer Research vol 17 no 17 pp5674ndash5685 2011

[100] O Osman L F Zanini M Frenea-Robin et al ldquoMonitoringthe endocytosis of magnetic nanoparticles by cells usingpermanent micro-flux sourcesrdquo Biomed Microdevices vol 14no 5 pp 947ndash954 2012


[101] A Singh F Dilnawaz and S K Sahoo ldquoLong circulatinglectin conjugated paclitaxel loaded magnetic nanoparticlesa new theranostic avenue for leukemia therapyrdquo PLoS ONEvol 6 no 11 Article ID e26803 2011

[102] E C Dreaden B E Gryder L A Austin et al ldquoAntiandrogengold nanoparticles dual-target and overcome treatment resis-tance in hormone-insensitive prostate cancer cellsrdquo Bioconju-gate chemistry vol 23 no 8 pp 1507ndash1512 2012

[103] C Tomuleasa O Soritau A Orza et al ldquoGold nanoparticlesconjugated with cisplatindoxorubicincapecitabine lowerthe chemoresistance of hepatocellular carcinoma-derivedcancer cellsrdquo Journal of Gastrointestinal and Liver Diseasesvol 21 no 2 pp 187ndash196 2012

[104] W Punfa S Yodkeeree P Pitchakarn C Ampasavate and PLimtrakul ldquoEnhancement of cellular uptake and cytotoxicityof curcumin-loaded PLGA nanoparticles by conjugationwith anti-P-glycoprotein in drug resistance cancer cellsrdquo ActaPharmacologica Sinica vol 33 no 6 pp 823ndash831 2012

[105] D Pramanik N R Campbell S Das et al ldquoA compositepolymer nanoparticle overcomes multidrug resistance andameliorates doxorubicin-associated cardiomyopathyrdquo Onco-target vol 3 no 6 pp 640ndash650 2012

[106] M Das and S K Sahoo ldquoFolate decorated dual drug loadednanoparticle role of curcumin in enhancing therapeuticpotential of nutlin-3a by reversing multidrug resistancerdquoPLoS ONE vol 7 no 3 Article ID e32920 2012

[107] B Li H Xu Z Li et al ldquoBypassing multidrug resistance inhuman breast cancer cells with lipidpolymer particle assem-bliesrdquo International Journal of Nanomedicine vol 7 pp 187ndash197 2012

[108] L Milane Z Duan and M Amiji ldquoTherapeutic efficacyand safety of paclitaxellonidamine loaded EGFR-targetednanoparticles for the treatment of multi-drug resistant can-cerrdquo PLoS ONE vol 6 no 9 Article ID e24075 2011

[109] J Shen Q Yin L Chen Z Zhang and Y Li ldquoCo-deliveryof paclitaxel and survivin shRNA by pluronic P85-PEITPGScomplex nanoparticles to overcome drug resistance in lungcancerrdquo Biomaterials vol 33 no 33 pp 8613ndash8624 2012

[110] H B Nair S Huffman P Veerapaneni et al ldquoHyaluronicacid-bound letrozole nanoparticles restore sensitivity toletrozole-resistant xenograft tumors in micerdquo Journal ofNanoscience and Nanotechnology vol 11 no 5 pp 3789ndash3799 2011

[111] Y Chen S R Bathula J Li and L Huang ldquoMultifunc-tional nanoparticles delivering small interfering RNA anddoxorubicin overcome drug resistance in cancerrdquo Journal ofBiological Chemistry vol 285 no 29 pp 22639ndash22650 2010

[112] I-P Huang S-P Sun S H Cheng et al ldquoEnhanced chemo-therapy of cancer using pH-sensitive mesoporous silicananoparticles to antagonize P-glycoprotein-mediated drugresistancerdquo Molecular Cancer Therapeutics vol 10 no 5 pp761ndash769 2011







Editorial Board




Contents










































Tamer Elbayoumi












1 Introduction









47H51NO14




O

O

OO

OO OO

O

OO

HN

HO HO

OH























Arms Pts

RR ()INVRAD119875 = 047

RR ()INDRAD119875 = 047

PFS ()INVRAD119875 = 047

PFS ()IND RAD119875 = 047

OS(months)119875 = 47


Nab-paclitaxel


767476

467463

374945

10914675

11129128

277338222

Docetaxel100mgm2 q3w 74 39 35 78 75 266





Arm PtsRR I line

()119875 = ND


ORR()119875 = ND





Arms Pts

MedianPFS

(months)119875 = ND








(b) Phase III

AEs () 119875 = 001

Arms Pts RR ()119875 = 001

















Gem

citabine

1000

mgsqm

Nab-paclitaxel


20443

X 48 122 48

(b) Phase III

Arms Pts ORR()

MedianTTP(MO)

PFS OS AEs () 119875 = 001

Median(MO)

1 yr()

Median(MO)

1 yr()

2 yr()

GradegeIIIn

eutro

penia

Fatig

ue

Neuropathy

119875 = lt001 119875 = lt001 119875 = lt001 119875 = 031 119875 = lt001 119875 = lt001 119875 = 02




qw

431 99 51 55 16 85 35 9 38 17 17


qw430 31 36 37 9 67 22 4 27 7 1









Table3Ra

ndom

ized

phaseIIand

IIItria

lswith


a(a)Ph

aseII

Arm

sPts

RR(

)119875=05

PFS

OS

Median(M

O)

119875=ND

At6(

)119875=ND

Median

(MO)119875=ND

1year

()119875=ND

Hersh

etal2010

[46]

(firstlowastand

follo

winglowastlowastlin

e)

Nab-paclitaxel

lowast150m

gm

2q3w

lowastlowast100m

gm

2q3w

37 37216 27

45

35

34 2796 121

41 49

Arm

sPts

RR(

)119875=10

MedianPF

S(M

O)119875=ND

MedianOS(M

O)119875=ND

Kottschadee

tal2011[47]

(firstlowast

andfollo

winglowastlowastlin

e)

Nab-paclitaxel

lowast100m

gm

2q3w

Carbop

latin

AUC2

41256

43

111

lowastlowast100m

gm

2q3w

Carbop

latin

AUC2

3588

42

109

(b)Ph

aseIII

Arm

sPts

ORR

()

PFS

OS

AEs

gradege

III()119875=001

Median

(MO)

BRAFstatus

Median

(MO)

BRAFstatus

Neutropenia

Leukopenia

Fatigue

Neuropathy

WT

(MO)

V60

0Em

(MO)

Uk

(MO)

WT

(MO)

V60

0Em

(MO)

Uk

(MO)

119875=239119875=044119875=088119875=656119875=066119875=lt001119875=33119875=132119875=381

Hersh

etal

2010

[48]

(firstline)

Nab-paclitaxel

150m

gm

2qw

264

1548

54

53

37

128

127

169

111

2012

825

Dacarbazine

1000

mgsqm

q3w

265

1125

25

35

22

107

111

112

9910

72

0

PPvaluend

not

doneA

Esadverse

events

WT

wild

typeV

600E

mw

ithmutationof

V60

0EU

kun

know

nBR

AFmutation

ORR

overallrespon

cerateR

Rrespon

serateP

FSprogressio

n-fre

esurvivalOS

overallsurvival


Table4Ra

ndom

ized

phaseIIItrialswith

nab-paclitaxelinaN

SCLC

Arm

sPts

ORR

Median

PFS

(MO)

119875=lt214

Median

OS

(MO)

119875=lt271

AEs


)119875=lt001

Median

()

119875=005

SQ ()

119875=lt001

NSQ ()

119875=lt80

Neutro

peniaTh

rombo

cytopeniaFatig

ueAnemia

Socinskietal2012

[49]

(firstline)

Nab-paclitaxel100m

gm

2

521

3341

2663

121

33lowast

13lowast

4lowast22lowast

Carbop

latin

AUC6

q3w

14lowastlowast

5lowastlowast

lt1lowastlowast

5lowastlowast

Paclitaxel200

mgm

2

531

2524

2558

111

32lowast

7lowast6lowast

6lowast

Carbop

latin

AUC6

q3w

26lowastlowast

2lowastlowast

lt1lowastlowast

lt1lowastlowast

PPvaluend

not

donesqsquamou

shistolog

yof

NSC

LCnsqnon

squamou

shistolog

yof

NSC

LCA

Esadverse

events

ORR

overallrespon

cerateR

Rrespon

serateP

FSprogressio

n-fre

esurvivalOSoverall

survival









References
















































































1 Background













































12(h) AUC

0rarr119879last (uM eq-h) 11987912






























IIIPLD (50mgm24w)



21mversus22m

33versus38


CHF 0 versus 4

Harris et al[34]

IIILD (75mgm23w)



38mversus43m

16mversus20m

26


Batist et al[35]




(30 CRF)

51mversus55m

19mversus16m


CRF 0 versus 32

Chan et al[36]




77mversus56m

183mversus16m

46 versus39


Sparano et al[37]




7mversus98m

206mversus205m
































8 Conclusions








References






















































































1 Introduction












3 a cytosolic




RAS

qP12

Off

Off

BRAF On

(B) PTEN

ERK FAK

PKBAKTp130CAS

Off

OnOn

On

UV radiation

DNA mutations

V600EBRAF



p14ARF

CDKN2A gene

(A) Family history

PIP3




















Ligand


















(eg TNF and DTA)


Specific targeting


HPMA PEG and HES)


Unspecific uptake

Specific targeting






3O4

























DNA tumorantigen


DNA based RNA based

Immune response



DNA vaccination

Off

Gene silencing

Tumor progression

Tumor cell death



pIC

On


Endolysosomal

machinery


factors (egPAR-1


















IL-12 [104ndash106]gp100 [107 108]


IP-10 [110]HSV-tk [111ndash113]



dsRNA pIC [117 118]








Acknowledgments


References































































































































































1 Introduction







































ge6 20 100 mdash




ALL7ndash12

4019

675526

11997


ALL7ndash12ge12

964353

625mdashmdash

9479114


le6ge6

1429

286667

5999


le12ge12

3631

250452

mdashmdash


RESge12

6645

216537

587


le12 27 280 75


ALLle12ge12

372413

290170540

mdash



ALLRESSEN

985741

280190410

mdash








(mts) OS


214REC 107 178 54 114

107 224 60 140


255130125

12365

2334

89103


1959699

8361

3631

127135


1537677

1629

4050

14127lowast



19073lowast59

205194


SEN 430

335337

35lowast23

92lowast75

mdashmdash



SEN6ndash24mts

3130

59lowast28

12lowast8

3118



SENgt6mts

467509

mdashmdash

113lowast94

mdashmdash























(mts)





le6 166 126 34


135 309 67


ge6 54 722 139






49

















5 Conclusions



Acknowledgments


References











































































































1 CD44 Receptor








D-glucuronic acid


O

O

OO

OO

O

O

OO

H

HH

H

HHNH

NHH

H

HH

H

HH

H

HH

HH

HH

OH

OH

OH

OHHO

HO HO

HO

CH3CH3

HOOCHOOC

119899



2 Hyaluronic Acid


13and 12057314

















+

HA

(a)

+


HA

(b)











[12]



[13 14]



[15]



[16]



[17]



[18]



[19ndash21]


















5 Conclusions





References

































































1 Introduction











































































1198811205733


51205731is another








Polyethylene glycol

Anticancer drugs

Targeting ligand


Imaging agent




















Blood vessel




Cancer cells






12

3

4

minus

+

+

++

minus

minus

minus

minusminus

















































































9 Conclusions




Acknowledgments


References











































































































































































































































































































































































































































1 Introduction










Alternative

Pentraxin

Classical

Lectin

Ant

ibod

y (A

b) o

r not

Factor BC3

C3

convertase

Factor D

C3aC3bBb C3bBb3b

C1s

CRPSAP

MBLMASP1MASP2

C1r

C1q C1 complexC2

C4

minus

C4aC4b2a

C3bC3

C2a

C3b

C4b

C3a

C2b

C1 INH

iC3b

C4b2a3b

H

HI

C5 C5b

Proteolyticcascade

of C6 to C9proteins

Membrane lysis

C5ndash9Membrane

attackcomplex

minus

H2O

Mg2+

Mg2+

Ca2+

Ca2+

(minusAb)
















119865119898

st =(119896119879)

(1198632ℎ119888) (ℎ119888ℎ)83 (1)





119865brst =

(119896119879)

1198633 [(ℎ119888ℎ)94minus (ℎℎ

119888)34]

(2)










2ndashNndashCH


2] [37ndash39]


















O

OH

HOOH

O

OH

O

OH

NHO

OH

O O

OHO

OH

O

HOOH

O

OH

NHHO O

O

O

HO

HOOH

HN

O

GM1

GM2

GM3

1217

O


galactose

Stearic acid

galactosamine

Sphingosine

CH3

H3C

120573-O-

120573-O-glucose


Ominus












BrNH

OP

O

OO

O

O

HO

O

CH3

O

O

O15

15 CH3

N+

Ominus

Ominus119899































119892lt





119892 the polymer







119888= 119886119873(119886119863)











2-PE PEG

2-PE was more






























OON

H

OP

O

OO

O

O

HO

O

4415

15

CH3Ominus









3O4nucleation











3
















2and 12-









3 Conclusions





References




























































































































































































3O4































1and R

2








3) and





2side chain


2side chain


OOH

OH

OH

OH

P

O

O

P


(a)

OH

OH

OH

OH

P

C

PR1

R2

O

O


(b)


1and R


phonate








Cl

Cl

OOH

OHOH

OH

P

O

O

Clodronate(CLO)

HO

P

P

O

OH

OH

OH

OH

Etidronate(ETI)

1st generation

P

H3C

OOH

OHOH

OH

P

O

HO P

OH

OHOH

OH

P

O

O

HO P

N

OH

OHOH

OH

H2N

Alendronate(ALN)

Ibandronate(IBA)

Pamidronate(PAM)

CH3P

PHO

H2N

O

O

2nd generation

OOH

OHOH

OH

P

O

HO P

OH

OHOH

OH

N

P

PHO

N

N

N

NHO

O

O

OH

OHOH

OH

P

P

O

O

Zoledronate(ZOL)

Minodronate(MIN)

Risedronate(RIS)

3rd generation








Prenylation

Mevalonic acid

FPPS

Cholesterol

Geranyl-Geranyl-PP

GGTase

Ras

Ras

Ras

FTase

HMG-CoA

Farnesyl-PP C-CH3

-A-A-X

C-A-A-X

C-A-A-X

CH3

Zoledronic acid




2side













12120572) and of 105min (119905


















































administration[26]





[23 24 27]





[29]






[31ndash33]




angiogenesis[34]















zoledronate


[44ndash46]






hydroxyapatite[47]



tumors


surface


[48]


Table 1 Continued











4





























References




























































































































1 Introduction











































100

80

60

40

20

0

Surv

ival

pro

bab

ilit

y (

)

OS 22 weeks

0 20 40 60 80

Weeks

F 1












































References



























Review Article










1 Introduction




accumulation

Surfacechanges

Biodegradability

Cell-site specific

(nucleuscytoplasm)


to toxicity

Adsorption of

proteins ions etc


Figure 1































Mithocondrial

apoptosis

Lysosomalmembrane

permeabilization

nanoparticles

ROS

ER stress


Cancer cells

Apoptosis

Cathepsin B

pathway of

CNTsTiO2SiO2

Figure 2

Alumina ceramic

Binding to


Mitotic catastrophe

Disruption

division

carcinogenesisCoCr



Aberrant cell

Aneuplody

Figure 3


















Damaged proteins


cascades

Oxidative stress

stress

lysosomalenzymes

Reduced

function

Autophagy

dysfunction

ERmithocondria




Disruption of

Figure 4















Abbreviations






Acknowledgment


References














































































































































































Review Article









1 Introduction











to apoptosis





Cyclophosphamide





Doxorubicin




[11ndash15]





[16ndash22]






[10]

Cisplatin Recovery



(a)

Recovery



Cisplatin

(b)










pathwaysReferences






[23ndash26]

Drug modulation





chemoresistance

[27ndash29]








[32ndash37]




[38 39]






in tumour cells


proteins (cIAPs)

[47ndash59]






[60ndash68]













cell lines[70]







[72ndash74]





[75ndash77]

Gold










Mesoporoussilica




DNA damage[85 86]









NP

Rx NP

NPs

EGFR

Enhanced drug

accumulation

Ribonucleotide

reductase

MDR1

Jagged1

MDR1

quantum dotPolymer-

SLN-Rx

P-gp

GTI-2040

Lipopolyplex-

Danuorubicin-

iron

Anti-

siRNAs-

Anti-

-chitosan NP

Jagged1 siRNA

Anti-VLA-4 peptide

-micelle NP

VLA-4

oxide NP

siRNA-
































6 Conclusion



Acknowledgment


References






















































































































nanotechnologies in cancer · 2019. 8. 7. · juan m. irache, spain bhaskara r. jasti, usa hans e....

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