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Research review paper

Nanotechnology-based intelligent drug design for cancer

metastasis treatment

Yu Gao a,1, Jingjing Xie a,1, Haijun Chen a,b, Songen Gu a, Rongli Zhao a, Jingwei Shao a, Lee Jia a,⁎a Cancer Metastasis Alert and Prevention Institute, College of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou 350002, Chinab Department of Pharmaceutical Engineering, College of Chemistry and Chemical Engineering, Fuzhou University, Fujian 350108, China

a b s t r a c ta r t i c l e i n f o

Available online xxxx

Keywords:

Nanotechnology

Nanomedicine

Targeted drug delivery system

Cancer metastasis therapy

Nanoparticle platform

Traditional chemotherapy used today at clinics is mainly inherited from the thinking and designs made four de-

cades ago when the Cancer War was declared. The potency of those chemotherapy drugs on in-vitrocancer cells

is clearly demonstrated at even nanomolar levels. However, due to their non-specic effects in the body on nor-

mal tissues, these drugs cause toxicity, deteriorate patient's life quality, weaken the host immunosurveillance

system, and result in an irreversible damage to human's own recovery power. Owing to their unique physical

and biological properties, nanotechnology-based chemotherapies seem to have an ability to specically andsafe-

ly reach tumor foci with enhanced ef cacy and low toxicity. Herein, we comprehensively examine the current

nanotechnology-based pharmaceutical platforms and strategies for intelligent design of new nanomedicines

based on targeted drug delivery system (TDDS) for cancer metastasis treatment, analyze the pros and cons of

nanomedicines versus traditional chemotherapy, and evaluate the importance that nanomaterials can bring in

to signicantly improve cancer metastasis treatment.

© 2013 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2. Current established nanoparticle platforms as drug delivery systems for cancer therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2.1. Lipid-based nanoparticle platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2.2. Polymer-based nanoparticle platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2.3. Protein-based nanoparticle platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2.4. Inorganic nanoparticle platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

3. Strategies in designing intelligent nanomedicine for enhanced cancer treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

3.1. Active targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

3.2. Combination drug delivery approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

3.3. Environment-response controlled release strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

3.4. Multi-stage delivery nanovectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

3.5. Cancer nanotheranostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

4. Conclusions and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

Declaration of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

Reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

1. Introduction

Cancer remains a leading cause of death worldwide (Ferlay et al.,

2010). Although years of intense biomedical research and billions of

dollars in spending have increased our understanding of the underlying

mechanisms of tumorigenesis and biology of cancer, cancer mortality

surprisingly reached to the highest point as the top killer in the US pop-

ulation younger than 85 years old ( Jemal et al., 2010). Among them,

cancer metastasis attributes to approximately 90% of cancer-related

deaths (Veiseh et al., 2011). Although immunotherapy, thermal thera-

py, phototherapy ( Jia and Jia, 2012; Shao et al., 2013) and gene therapy

are available as cancer treatment modalities, surgery, radiation, and/or

Biotechnology Advances xxx (2013) xxx–xxx

JBA-06756; No of Pages 17

⁎ Corresponding author. Tel./fax: +86 591 8357 6921.

E-mail address: [email protected] (L. Jia).1 These authors contributed equally to this work.

0734-9750/$ – see front matter © 2013 Elsevier Inc. All rights reserved.

http://dx.doi.org/10.1016/j.biotechadv.2013.10.013

Contents lists available at ScienceDirect

Biotechnology Advances

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b i o t e c h a d v

Please cite this article as: Gao Y, et al, Nanotechnology-based intelligent drug design for cancer metastasis treatment, Biotechnol Adv (2013),http://dx.doi.org/10.1016/j.biotechadv.2013.10.013

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chemotherapy continue to be the therapeutic options for most cancers

over decades, each with its own limitations. Surgery and radiation ther-

apy could be effective for the primary tumor, however, they may not be

a good treatment choice for metastases. Chemotherapy with cytotoxic

agents is commonly used for the whole-body treatment of recurrent

disease. But the conventional anticancer drugs generally result in seri-

ous side effects in clinic (Sinha et al., 2006; Stortecky and Suter, 2010;

Tsuruo et al., 2003). The side effects are associated with the formulation

due to poor water solubility of the drug, non-speci

c distribution, se-vere toxicity to normal cells, inadequate drug concentrations at tumors

or cancerous cells, and the development of multidrug resistance. There-

fore, researchers are continuously seeking for improved anti-cancer

therapies that can selectively target tumor cells with minimal side

effects on normal tissues (Wang et al., 2008).

Nanotechnology is the understanding of materials in the nano(10−9)

size range, and involves imaging, measuring, modeling, and manipulat-

ing materials within that framework. Since its advent, nanotechnology

has revolutionized a wide range of medical products, generic tools and

biotechnology equipment. Nanomedicine focuses on application of

nanotechnology in medicine for diagnosis, prevention, detection, and

treatment of the disease. In particular, it has been used to design and de-

velopment of targeted drug delivery system (TDDS) which could safely

deliver therapeutic drugs to injury sites or specic cells. For formulations

intended for i.v. administration, effective TDDSs could retain therapeutic

drug in the vehicle, evade the reticuloendothelial system (RES) uptake,

target to intended sites of injury, and release drug at the intended sites

with required drug concentration (Mills and Needham, 1999). In

the eld of oncology, TDDS offers many potential benets such as

(1) avoiding the side effects of the clinical formulation for improving

solubility, (2) protecting the entrapped therapeutic drugfrom degrada-

tion, (3) modifying pharmacokinetic and tissue distribution prole to

increase drug distribution in tumor, (4) reducing toxicity to normal

cells, and (5) increasing cellular uptake and internalization in cancer

cells. In the past20 years, many nanomedicineshave been in preclinical

development and some of them have been approvedfor use in clinic in-

cluding for cancer therapy (Davis et al., 2008; Jain and Stylianopoulos,

2010; Peer et al., 2007). Besides used as drug delivery systems (DDSs)

for cancer therapy, nanoparticles loaded with imaging agents werealso found useful in imaging techniques applied for tumor diagnosis.

Here we will focus on TDDS designed for i.v. administration and for

delivering anticancer drugs including chemotherapeutic drugs and

therapeutic genes.

In this review, we rst outline the different types of nanoparticle

platforms currently being established for cancer treatment. We then

present various strategies that have been employed in designing new

effective TDDSs.

2. Current established nanoparticle platforms as drug delivery

systems for cancer therapy

There are diverse types of nanocarriers that have been synthesized

for drug delivery including dendrimers, liposomes, solid lipid nanopar-ticles, polymersomes, polymer-drug conjugates, polymeric nanoparti-

cles, peptide nanoparticles, micelles, nanoemulsions, nanospheres,

nanoshells, carbon nanotubes, and gold nanoparticles, etc. (Fig. 1). In

all these types, drugs can be entrapped inside, dissolved in the matrix,

covalently linked to the backbone, or absorbed on the surface. From

the aspect of the property, these nanocarriers could be divided into or-

ganic, inorganic, and organic/inorganic hybrid nanoparticles. From the

perspective of formulation type, they could be divided into liposomes,

micelles, emulsions, nanoparticles, etc ( Jia, 2005). Ljubimova and Holler

also proposed the term ‘nanopolymer’ meaning a single polymer mole-

cule in the nanoscale range, to distinguish with ‘nano-polymer compos-

ites’ such as micelles and other self-assembled or aggregated forms in

the point of whether they could dissociate in solutions (Ljubimova

and Holler, 2012). Here, we will categorize these current established

nanoparticle platforms basedon the differencein composition including

lipid-based nanomedicine, polymer-based nanomedicine, peptide-

based nanomedicine and inorganic nanomedicine for treating cancer.

Some examples of nanomedicines that are approved for commercial

use or still in clinical trials are listed in Table 1.

2.1. Lipid-based nanoparticle platforms

Lipid-based nanoparticles have attracted great attention as DDS dueto their attractive biological properties such as good biocompatibility,

biodegradability, low immunogenicity, and the ability to deliver hydro-

philic and hydrophobic drugs. Liposomes are the most widely used and

studied examples ( Jia et al., 2002), with bilayer membrane structures

composed of phospholipids for stabilizing drugs, directing their cargo

toward specic sites, and for overcoming barriers to cellular uptake.

Their aqueous reservoir and the hydrophobic membrane allow them

to encapsulate either hydrophilic or hydrophobic agents. The important

milestonethat ledto thedevelopmentof clinically suitable liposome for-

mulations could be the inclusion of PEGylated lipids in the liposomes to

protect liposomes from destruction by the RES, thus to increase circula-

tion time and increase drug accumulation in the tumors. It is worthy to

mention that Doxil®/caelyx, a PEGylated liposome formulation of the

anticancer drug doxorubicin (DOX), was the rst formulation approved

for application in the clinic (Barenholz, 2012). With the aim to site-

specic delivery of cancer drugs to the cancerous tissues, the surface of

liposomescan be modied with ligands or antibodies targetingthose re-

ceptors overexpressed on cancer cell membranes (Gabizon et al., 2006).

For tumor site-specic triggeringdrug release, liposomes were designed

with responsive to changes in light (Leung and Romanowski, 2012),

temperature (Park et al., 2013), acid (Mamasheva et al., 2011) or en-

zymes (Andresen et al., 2005). Thoughthe work on modication of lipo-

somes has achieved great progress, the application of liposomes in the

clinic still poses several challenges including rapid clearance from the

bloodstream, instability of the carrier, high production cost, and fast

oxidation of some phospholipids.

Solid lipid nanoparticles (SLN) is an alternative to liposomes, the

matrix of which comprisesof solid lipids. They exhibit major advantages

such as less cytotoxicity than polymeric counterparts; stable formula-tions, excellent reproducibility, avoidance degradation of incorporated,

controlled drug release, and potential application in intravenous, oral,

dermalor topical routes (Uner and Yener, 2007). However, some limita-

tions still exist such as undesired particle growth by agglomeration or

coagulation,ineffective drug loading capacity, rapid drug expulsion dur-

ing storage due to lipid crystallization and high water contents of the

dispersions. Thus, modied SLN, so-called nanostructured lipid carriers

(NLC) were developed to overcome these limitations and combine the

advantages associated with SLN. In contrast to SLN which are made

from solid lipids core containing triglycerides, glyceride mixtures, or

waxes, NLC were composed of liquid lipid and solid lipid (preferably

in a ratio of 30:70 up to 0.1:99.9) to form a nanosized solid particle ma-

trix. The imperfect crystalor amorphous structure assures them to have

enhanced drug loading and less drug expulsion during storage (Iqbalet al., 2012). Till now, SLN and NLC as colloidal drug carriers have

been successfully multi-functionalized to transport drugs to the

targeted cancer cells and achieve ef cient drug release in a controlled

manner, which conrm their promising application in cancer therapy.

2.2. Polymer-based nanoparticle platforms

Polymer-based nanoparticle platforms show enormous potential for

treating disease or repairing damaged tissues especiallyfor cancer treat-

ment, which relies on their remarkable properties including small size,

excellent biocompatibility and biodegradability, prolonged circulation

time in the bloodstream, enhanced drug loading capacity, and easy

chemical modication or surface functionalization. The last two charac-

ters are the utmost important criteria for their clinical use. Generally

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speaking, polymer-based nanomedicine can be categorized into three

groups based on drug-incorporation mechanisms including polymer-

drug conjugates by covalent conjugation, polymeric micelles by hydro-

phobic interactions, and polyplexes or polymersomes by encapsulation.

Most of the polymers are approved by FDA as the commonly explored

carriers for targeted drug delivery.Polymer-drug conjugates using water-soluble polymers as carriers

have produced expected results, including a water-soluble polymeric

carrier (natural or synthetic), a biodegradable linkage and an anticancer

agent. Because polymer-drug conjugates can passively target to tumor

cells by enhanced permeability and retention (EPR) effect (Matsumura

and Maeda, 1986), many polymer-drug conjugates were under clinical

evaluation. The most attractive representative of synthetic polymer-

drug conjugates is poly (L -glutamic acid)-paclitaxel (CT-2103, Xyotax®),

which has advanced to Phase III clinical trials (Bonomi, 2007). Polymer-

drug conjugates have exhibited several superiorities such as enhanced

therapeutic ef ciency, fewer side effects, exible drug administration

and even increased patient compliance. However, many challenges still

exist in the development of new generation of polymer-drug conjugates,

includingthe design of novel polymersthat havemodulated degradationcharacteristics, polymerization methods allowing for controlling the

weight distribution, and conjugation techniques available for site-

specic attachment.

Amphiphilic block copolymers can self-assemble into different kinds

of mesoscopic structures (micelles and vesicles), which is just up to the

control about the volume ratio of hydrophilic to hydrophobic blocks

(Antonietti and Förster, 2003; Zupancich et al., 2006). Polymersomes

are self-assembled polymer vesicles formed by amphiphilic copolymers

containing hydrophilic and hydrophobic segments, which are different

from liposomes formed by amphiphilic phospholipids. The hydrophilic

interior structure is suitable for encapsulating with water-soluble

agents such as DNAs or proteins while the hydrophobic exterior bilayer

membrane can be simultaneously entrapped with poorly water-soluble

drugs. Compared with liposomes, polymersome exhibited more

prominent features such as higher loading capabilities, greater stabili-

ties, and longer circulation time. The improvement of storage abilities

is attributed to their own large hydrophic core and surface functionality

through chemical synthesis and modication (Ghoroghchian et al.,

2005, 2006).

Polymeric micelles are self-assembling monolayers formed sponta-neously under certain conditions including the concentrations of am-

phiphilic surfactants, pH, temperatures and ionic strength with a

hydrophobic core and hydrophilic shell in the nanometer range. The

properties of polymeric micelles such as small size, hydrophilic shell

avoiding the uptake by the mononuclear phagocyte system (MPS),

and the high molecular weight evading renal excretion made them

effective passive targeting systems. Ligands such as small organic mole-

cules, DNA/RNA aptamers, peptides, carbohydrates and monoclonal an-

tibodies could be attached to the surface of micelles, not only increasing

the accumulation at tumor sites but also increasing the cellular uptake

in cancer cells via receptor-mediated endocytosis (Farokhzad et al.,

2006; Sethuraman and Bae, 2007; Torchilin et al., 2003; Yoo and Park,

2004).

Dendrimers are kinds of nanomaterials with super biological char-acteristics: small size (1–15 nm), high water solubility, regularly

and highly branched three-dimensional architecture, nearly perfect

monodispersibility in nature, and high payload. All these facilitate

their applications in cancer or disease prevention and therapy.

Polyamidoamine (PAMAM) dendrimer was one of the most studied

dendrimers. It possesses multiple amine surface groups, and the num-

ber of the groups could be precisely controlled. Therefore, the multiva-

lent conjugation could be achieved by attachment of targeting ligands,

therapeutics agents, drugs, imaging contrast agents, genes or even

chemical sensors to their terminal functional groups. M.H. Li et al.

(2012) prepared the G5 PAMAM dendrimer-based multivalent metho-

trexates as dual acting nanoconjugates for cancer cell targeting. The

study demonstrated that re-engineering dendrimer conjugates not

only target KB cancer cells, but also inhibited dihydrofolate reductase.

Fig. 1. Schematic illustration of representative nanoparticle platforms that have been synthesized for drug delivery for cancer therapy.

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Thomas and his co-workers used antibody-conjugated dendrimers to

bind antigen-expressing cells. The conjugates specically bound to the

antigen-expressing cells in a dose-and time-dependent manner with af-

nity similar to that of the free antibody ( Thomas et al., 2008).

2.3. Protein-based nanoparticle platforms

Protein-based nanomedicine platforms as one of the representatives

have been paid serious attention owing to their biocompatibility, biode-

gradability as well as low toxicity. Protein-based nanomedicine plat-

forms are usually consisted of naturally protein subunits of the same

protein or the combination of natural or synthetic protein, and different

types of drugmolecules. There are a variety of proteins used and charac-

terized for DDSs such as the plant-derived viral capsids (Liepold et al.,

2007; Suci et al., 2007), the small Heat shock protein (sHsp) cage

(Flenniken et al., 2005, 2006), albumin (Kratz, 2008; W. Lu et al.,

2007), soy and whey protein (Chen et al., 2008; Gunasekaran et al.,

2006), casein (Latha et al., 2000), collagen (Metzmacher et al., 2007)

and the ferritin/apoferritin protein cage (Wu et al., 2008a, 2008b). The

protein cage with hierarchical architectures derived from viruses hasits various advantages on the cage's uniform nanometer size for drug

loading and for avoidance of macromolecular aggregation, multifunc-

tional groups on the surface available for conjugation with drugs, and

superior biological characteristics benecial for pharmacokinetics

study. Albumin as a versatile protein carrier for improving drug targeting

and pharmacokinetic properties is playinga vital role in the development

of protein-based nanoparticles. It demonstrates prominent features of

stability in a broad range of pH (4–9) and temperature (4 °C–60 °C),

preferential uptake by tumor, and non-toxicity. Methotrexate-albumin

conjugate, albumin-binding prodrug of DOX and albumin PTX nanoparti-

cle (Abraxane) have been designed and now are under clinical trials

(Miele et al., 2009).

2.4. Inorganic nanoparticle platforms

Organic nanoparticles such as liposomes, dendrimers, polymeric mi-

celles have made great advances in cancer diagnosis and therapy

(Khemtong et al., 2009; Ljubimova et al., 2008). In contrast, inorganic

nanoparticles such as gold nanoparticles (AuNPs), carbon nanotubes

(CNTs), silica nanotubes, quantum dots (QDs), and super-paramagnetic

iron oxide nanoparticles (SPIOs) have also been extensively developed

and studied for biomedical applications due to their intrinsic unparallel

physical and biological properties such as optical, electrochemical, mag-

netic characteristics.

The biomedical applications of CNTs have been gradually proposed

and recognized through preliminary studies in vitro and in vivo and

even clinical tests, which is ascribed to their prominent physical and

chemical properties. In general, CNTs canbe classied to twocategories:

single-walled carbon nanotubes (SWNTs, 0.4–2.0 nm in diameters,

20–1000 nm in lengths) and multi-walled carbon nanotubes (MWNTs,

1.4–100 nm in diameters, ≥1 μ m in lengths). MWNTs provide potential

platforms for large biomolecules delivery such as plasmids into cells,

which is mainly due to the multiple layers of grapheme and larger di-

ameters (Gao et al., 2006; Liu et al., 2005). SWNTs exhibit more attrac-

tive optical properties suitable for biological imaging (Cherukuri et al.,

2004; O'Connell et al., 2002; Welsher et al., 2008). Functionalized

SWNTs by covalent binding, adsorption, and electrostatic interaction

can serve as novel drug delivery carriers in cancer therapy owing to

their biocompatibility, little toxicity and enhanced water solubility

(Feazell et al., 2007; Liu et al., 2007). Liu et al. (2008) prepared the

SWNT-PTX conjugate by coupling PTX to the branched PEG chains on

SWNTs and studied its antitumor effects in a xenograft murine 4T1

breast cancer model. They showed that SWNT delivery of PTX into

xenograft tumors could have 10-fold higher tumor suppression ef cacy

than the clinical drug formulation Taxol.

Distinction from other nanomaterials, mesoporous silica nanoparti-

cles (MSNs) showed unique properties such as tunable particle size

from 50 to 300 nm convenient for cell endocytosis; stable and rigidframework resistant to degradations induced by pH, heat, and mechan-

ical stress; uniform and tunable pore size adjusted between 2 and 6 nm

for the loading of different drug molecules; high surface area and large

porevolume allowing for high drug loading; internal and external func-

tional surfaces available for selective modication. MSNs could be func-

tionalized through co-condensation, grafting, and imprint coating

methods (Burleigh et al., 2001; Chen et al., 2006). The different surface

functionalization of MSNs has great effects on the cellular uptake mech-

anism and the internalization ef ciency of MSNsas well as the ability to

escape the endolysosomes (Slowing et al., 2006). MSNs were reported

having better biocompatibility compared with other silica-based mate-

rials. Theviability of mammaliancellswasn't affected by theinternaliza-

tion of MSNs at concentrations below 100 μ g/ml (Slowing et al., 2008).

Similar results were found that injecting MSNs to the animals didn'tpose any toxic side effects for 42 days (Kortesuo et al., 2000). Therefore,

MSNs were widely employed as promising intracellular controlled re-

lease drug delivery carriers in cancer treatment (Slowing et al., 2007;

Trewyn et al., 2007; Vallet-Regi et al., 2007). J. Lu et al. (2007) incorpo-

rated the hydrophobic anticancer drug camptothecin (CPT) into the

pores of the prepared uorescent mesoporous silica nanoparticles

(FMSNs) and successfully achieved the controlled drug release to

human cancer cells and induced tumor cell death.

Magnetic nanoparticles (MNPs) have their own unique physical and

biological features including controllable size distribution ranging from

nanometers to micrometers, high magnetic ux densitywith the intrin-

sic penetrability for drug targeting, the ability to convert magnetic to

heat, non-toxicity, biocompatibility, and injectability (Ito et al., 2005;

Pankhurst et al., 2003). The magnetic nanoparticle-based DDSs could

Table 1

Examples of current established nanoparticle platforms approved for commercial use or undergoing clinical investigation for cancer therapy.

Product Nanoparticle platform Drug Current stage of development Type of cancer

Doxil/caelyx PEGylated liposomes Doxorubicin Approved by FDA Refractory Kaposi's sarcoma, recurrent breast cancer,

ovarian cancer

Myocet Non-PEGylated liposomes Doxorubicin Approved in Europe and Canada Combinational therapy of metastatic breast cancer,

ovarian cancer, Kaposi's sarcoma

DaunoXome Liposomes Daunorubicin Approved in the USA Advanced HIV-associated Kaposi's sarcoma

Abraxane Albumin-bound nanoparticles Paclitaxel Approved by FDA Various cancers

ALN-VSP Lipid nanoparticles siRNA Phase I Liver cancerCRLX101 Cyclodextrin nanoparticles Camptothecin Phase II Various cancers

CALAA-01 Cyclodextrin-containing linear polymer,

decorated with PEG and transferrin

siRNA Phase I Solid melanoma tumors

NK-911 Polymer micelle Doxorubicin Phase I Various cancers

BIND-014 PSMA-targeted polymeric nanoparticles Docetaxel Phase I Advanced solid tumor cancers

Aurimune Pegylated colloidal gold nanoparticles TNFi Phase II Solid tumors

ThermoDox Heat-activated liposome Doxorubicin Phase III Hepatocellular carcinoma

CPX-1 Liposomes Irinotecan and oxuridine Phase II Advanced solid tumors




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be constructed by loadingdrug onto theparticle coat via physical means

such as electrostatic interaction instead of covalent conjugation (Kievit

et al., 2011; Medarova et al., 2007). The physical, hydrodynamic, and

physiological parameters have great effects on the drug delivery ef -

ciencies of magnetic nanoparticles. Among the MNPs, SPIOs with the di-

ameter of 5–100 nm, which show high magnetization in an external

magnetic eld, have demonstrated attractive possibilities in biomedical

application. They could serve as good “nanotheranostics” for both

targeted drug delivery and magnetic resonance imaging of tumor cells(Lee et al., 2013; Mouli et al., 2013; Yang et al., 2008; Zou et al., 2010 ).

Usually, SPIOs are loaded with small-molecule-based therapeutics into

polymer-based matrices (Quan et al., 2011).

Great interest has been paid to gold nanoparticles (AuNPs) in recent

years for their attractive properties including the strong and attractive

optical properties in the near-infrared (NIR) region from 700 to

900 nm ( Jain, 2009; Xia et al., 2011), easy modication with functional

groups through formation of stable gold-thiolate bonds (Au\S) by

reacting with disulde (S\S) or thiol (\SH) groups (Huang et al.,

2013), controllable particle size, shape and geometry (Kim et al.,

2009), and diversely multi-functionalization with desired targeting li-

gands, specic antibodies or drugs. The routine applications of AuNPs

in cancer therapy were photothermal therapyand radiation therapy, re-

spectively, for theirstrong absorption cross-sections and X-rayemission

characteristics (Sperling et al., 2008). AuNPs were also used as

nanocarriers for drug delivery. Several strategies have been used to im-

prove AuNPs accumulation in tumor cells specically and ef cientintra-

cellular drug release, including the conjugation of AuNPs with

appropriate surface ligands (membrane-translocating peptides) or spe-

cic antibodies (Huang et al., 2008), the coupling drugs of AuNPs

through non-covalent (available for drug release) or covalent binding

(requiring for second release), the external triggering methods such as

glutathione (Hong et al., 2006), light or photothermal-mediated release

(Agasti et al., 2009; Bikram et al., 2007), and the surface modication

with amphiphilic reagents (PEG). Though advances have been made

in the research eld of AuNPs as TDDS for cancer therapy, more chal-

lenges are still confronted. The suitable types of AuNPs used as drug de-

livery (Caiet al., 2008; Chithrani et al., 2006), thedelivery ef ciency, the

accuracy of targetingas well as the toxicity (Panet al., 2009) were underre-evaluation and optimization prior to clinical application.

3. Strategies in designing intelligent nanomedicine for enhanced

cancer treatment

3.1. Active targeting

Active targeting utilizes targeting moieties to peripherally conjugate

to nanoparticlesystems for specically targeting to tumor tissue, specif-

ic cancer cells, or even cellular organelles (Fig. 2). The most common

used active targeting strategy involves the attachment of the targeting

ligands such as folic acid, antibodies, aptamers, or proteins to the nano-

particles which recognizes receptors over-expressed on cancer cells

(Ruoslahti et al., 2010). For example, in FR positive KB cells, uptake of folate-targeted liposomal arsenic PEGylated liposomes inserted with a

small amount of DSPE-PEG3350-folate (0.3 mol%) was three to six

times higher than that of nontargeted liposomal arsenic, leading to a

28-fold increase in cytotoxicity (H. Chen et al., 2009).

Various antibodies that target receptors over-expressed on the sur-

face of cancer cells such as vascular endothelial growth factor (VEGF),

human epidermal receptor-2 (HER-2), tumor necrosis factor-α (TNF-

α), and epidermal growth factor receptor (EGFR) have been attached

to nanoparticulate materials to achieve selective cancer cell targeting

(Fayand Scott, 2011). Whendeveloping the antibody-conjugated nano-

particles, the af nity and the conguration of the antibodies, as well

as the method to attach to the nanoparticles, are all key design factors

that should be taken into consideration (Cheng and Allen, 2008; Firer

and Gellerman, 2012; Rizk et al., 2009; Rudnick et al., 2011). The

characteristics of the antibodies will have great effects on the circulation

time, cellular uptake, tolerability, and ef cacyof the nanoparticulate sys-

tems. In a recent study,two gold nanoparticles with thesurfacepartially-

covered and surface fully-covered by EGFR antibody cetuximab were

designed to determine the cellular uptake mechanism of cetuximab-

conjugated nanoparticles. The endocytosis mechanism could be

switched from a Cdc42-dependent pinocytosis/phagocytosis to original

Dyn-2-dependent caveolar pathway when the nanoparticles were fully

coated with cetuximab (Bhattacharyya et al., 2012). Compared with an-tibodies, antibodyfragments suchas Fab' andscFv aremore widelyused

for active targeting because they have a smaller size easy to conjugate

into nanoparticles. More importantly, they could keep their targeting

specicity while reducing nonspecic antigen binding from Fc (Ansell

et al., 2000; Chapman, 2002; Rothdiener et al., 2010; Sapra et al.,

2004). Conjugation of the antibody on nanoparticles can be carried

out by coupling the carboxylic acid groups or primary amine groups of

the amino acid in the antibody to the primary amine groups or the car-

boxylic acid groups on the surface of the nanoparticles ( Chapman,

2002). The interaction between biotin and avidin or streptavidin has

also been exploited for designing antibody-conjugated nanoparticles.

Wartlick et al. covalently modied the nanoparticles based on gelatin

and HSA with the biotin-binding protein NeutrAvidin followed by

the biotinylated herceptin conjugation to the surface of the nanoparti-

cles. These nanoparticles could effectively internalize into HER-2-

overexpressing cells via receptor-mediated endocytosis observed by

confocal laser scanning microscopy (Wartlick et al., 2004).

Aptamers are kinds of oligonucleotidesthat arecapable of bindingto

a large numberof targets with high af nityand specicity. Since the ad-

vent of aptamer technology (Ellington and Szostak, 1990; Tuerk and

Gold, 1990), aptamers have represented an interesting class of modern

pharmaceuticals for therapy and diagnostics. Known as “chemical

antibodies”, aptamers show many similar properties to traditional anti-

bodies, however, they demonstrated a number of advantages over anti-

bodies such as low immunogenic potential, easier to synthesize and

modify, structural exibility, higher af nity and specicity, and good

stability (Majumder et al., 2009). Recently, aptamers have been conju-

gated to many types of molecules such as siRNAs, miRNA, proteins,

and nanoparticles to improve their targeting ef ciency, stability, andbiodistribution prole (Kanwar et al., 2011). The chimeric aptamer-

nanoparticle conjugates can bind to the target cells by the interaction

of aptamer–receptor interaction, and nally enter into the target cells,

resulting in release of the entrapped drugs (Estevez et al., 2010). In a

study, branched PEI-PEG polyplexes modied with an anti-prostate-

specic membrane antigens (PSMA) aptamer were used to co-deliver

DOX and Bcl-xL-specic shRNA.The polyplexes could inducea synergis-

tic and selective cancer cell death in PSMA-overexpressingprostate can-

cer cells (E. Kim et al., 2010).

With the development of different kinds of biomolecular ligands,

more and more new molecular-targeted nanoparticles are designed to

target different receptors. Considering that one kind of ligand is com-

monly specic to only one or a limited few target receptors, and conju-

gation of large targeting molecules to nanoparticles can change theirbehaviors in vivo, pre-targeting strategy has been utilized to avoid

development of multiple nanoparticle formulations with different

targeting ligands and accommodate different targeting ligands without

alternating pharmacokinetic prole of the nanoparticles. Pre-targeting

is a multi-step process that rst has a targeting ligand localized within

a tumor by virtue of its anti-tumor binding site, followed by treatment

with nanoparticles that recognize the targeting ligand conjugate on

the cell surface. Using a cell targeting recombinant fusion protein (FP)

composed of a single-chain antibody (scFv) and streptavidin (SA) to

specically pre-label the targeted cells, followed by application of a bio-

tinylated nanoparticle that binds to the SA of the FP on the target cells,

the nanoparticle system with two FPs, anti-CD20 and anti-TAG-72

CC49, could specically target two model cancer cell lines, i.e., Ramos

and Jurkat, respectively (Gunn et al., 2011). Besides using the




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interaction between biotin and avidin or streptavidin, bioorthogonal

chemistry such as tetrazine/trans-cyclooctene cycloaddition reaction

has also been employed to nanoparticles recognition of pre-labeled

cells (Devaraj et al., 2009; Haun et al., 2010; Rossin et al., 2010 ).

Dual-targeting strategyhas beenapplied to develop nanoparticles for

treatment of brain tumor. The treatment of brain tumor entails ef cient

delivery of therapeutic agents to specic regions of the brain after pene-

trating the blood–brain barrier (BBB). The BBB is a physiological barrier

that selectively allows the entry of certain molecules. Many strategies

have been employed to across the BBB such as the disruption BBB integ-

rity by osmotic means or by ultrasound means, the use of endogenous

carrier-mediated transporters, receptor-mediated transcytosis, and

blocking of active ef ux transporters. Nanotechnology is considered to

be one of the most promising methods to deliver drugs across the BBBby attaching BBB-penetrating ligands to the surface of nanoparticles

(Yang, 2010). To targeting inltrated glioma cells or the glioblastoma

multiform after crossing the BBB, nanoparticles were modied with

dual ligands such as angiopep, transferrin, wheat germ agglutinin,

which recognize the receptors over-expressed on both BBB and glioma

cells for transporting the drug across the BBB and then targeting brain

glioma (Du et al., 2009; He et al., 2011; Xin et al., 2011; Y. Li et al.,

2012; Ying et al., 2010). Recently, a sequential-targeting strategy

has been applied to develop nanoparticles that can come across the

BBB and recognize the glioma cells subsequently. The exterior of the

micelle was conjugated with transferrin to enhance the cellular uptake

and BBB-penetrating through receptor mediated endocytosis. Then

the loaded drug cyclo-[Arg–Gly–Asp–d–Phe–Lys] (c[RGDfK])-PTX con-

jugate (RP) was released from micelle subsequently to target integrin

over-expressed glioma cells (Zhang et al., 2012). This sequential-

targeting nanoparticulate system could not only protect the ligands

from degradation during transportation across the BBB (Knisely et al.,

2008), but also overcome the non-specic recognition of the receptors

that are highly expressed throughout the brain (Bu et al., 1994).

Many research groups have studied the mechanism of active

targeting in solid tumors with ligand-modied nanoparticles. The im-

provement of cellular uptake of ligand-modied nanoparticles could

be achieved through receptor-mediated endocytosis by tumor cells

over-expressing corresponding receptors on the surface. However,

whether ligand-modied nanoparticles could increase drug accumula-

tion at the tumor site is largely dependent on the ligands. In a subcuta-

neous KB-3-1 xenograft model, the administration of the nanoparticles

formed by ternary conjugate heparin-folic acid-PTX and additional PTX(HFT-T) enhanced the specic delivery of PTX into tumor tissues (Wang

et al., 2009). Using transferrin-containing gold nanoparticles as study

model, Choi et al. found that the content of targeting ligands signicant-

ly inuences the number of nanoparticles localized within the cancer

cells (Choi et al., 2010). Some contrary results were also found in

some nanoparticulate systems such as antibody targeting of long-

circulating lipidic nanoparticles and chlorotoxin labeled magnetic

nanovectors that the targeting ligand only enhanced cellular uptake of

nanoparticles, but did not affect the accumulation of nanoparticles at

the tumor site (Kievit et al., 2010; Kirpotin et al., 2006).

Besides the cancer cells, tumor vasculature is also a potential target

for drug delivery ( Jain and Stylianopoulos, 2010). In vivo phase display

is a very useful tool to identify numerous peptides targeting the tumor

vasculature (Li and Cho, 2012; Trepel et al., 2008). Several peptides

Fig. 2. Schematic illustration of active targeting strategies that have been used for design intelligent drug delivery systems for cancer therapy. The active targeting strategy involves the

attachment of the targeting ligands such as folic acid, antibodies, aptamers, or proteins to the nanoparticles for speci cally targeting to tumor tissue, tumor vasculature, specic cancer

cells, or even cellular organelles (nucleus, cytoplasm, mitochondria). Pre-targeting is a multi-step process that rst has a targeting ligand localize within a tumor by virtue of its anti-

tumor binding site, followed by treatment with nanoparticles that recognize the targeting ligand conjugate on the cell surface. Dual-targeting strategy and sequential-targeting strategy

have been applied to develop nanoparticles that can both penetrate the BBB and target the glioma cells.




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such as arginine–glycine–aspartic acid (RGD), asparagine–glycine–

arginine (NGR) (Arap et al., 1998), TCP-1 (Li et al., 2010), iRGD

(Sugahara et al., 2009; Sugahara et al., 2010), F3 (Porkka et al., 2002),

and LyP-1 (Laakkonen et al., 2002) have been identied to specically

target tumor blood vessels or simultaneously recognize the tumor vas-

culature and cancer cells. Nanoparticles modied with peptides that

targeting the tumor vasculature could enhance ef cacy of the chemo-

therapeutic agents against human cancer xenografts (Chang et al.,

2009), suppress tumor growth (Hood et al., 2002), and metastasis inmice (Murphy et al., 2008).

Active targeting not only candirect the nanoparticles to specic cells

of the tumor, but also been applied to deliver cargos to specic cellular

organelles. As oncogenesand tumor suppressor genes playa crucial role

in the processes of cancer development, gene therapy provides a tool to

cure the disease at its source. TheDNA-based medicines require carriers

ef ciently and safely carry the plasmid DNA into the nucleus of the de-

sired cells. The cationic non-viral vectors which could interact with

negatively charged DNA through electrostatic interactions to form

polyplexes or lipoplexes, have been considered to be the most promis-

ing gene delivery systems compared to naked DNA or viral vectors

(Al-Dosari and Gao, 2009). To successfully deliver genes into the nucle-

us, the cationic nanoparticles should circumvent a series of barriers in-

cluding survival in the bloodstream, extravasation into the tissue,

binding and internalization into the target cells, escaping from endo-

some, subcellular traf cking, and nally entry into nucleus (Khalil

et al., 2006; Wiethoff and Middaugh, 2003). Numerous efforts have

been made to develop effective and safe gene delivery systems and a

large amount of strategies has been employed to improve systemic de-

livery and intracellular traf ckingof cationic nanoparticles including at-

tachment of ligand for cell-specic targeting and receptor-mediated

endocytosis, use of protein transduction domain (PTD) such as TAT

peptides to mediate cellular transduction, incorporation of pH-sensitive

endosomolytic peptides, fusogenic peptides or membrane-destabilizing

compounds to facilitate endosomal escape, employment of nuclear lo-

calization sequence to improve the uptake of plasmid DNA into the nu-

cleus (Morille et al., 2008). Some multifunctional nanoparticles that

could circumvent several biological barriers have been designed.For ex-

ample, a multifunctional nano device consisting of poly(folate-poly(-ethylene glycol)cyanoacrylate-co-hexadecylcyanoacrylate), dioleoyl

phosphatidylethanolamine (DOPE), and DNA condensed by protamine

sulfate (PS) was developed for nucleus delivery of DNA. The folic acid

on the surface of the nano devicecould increase its active targeting abil-

ity to cancer cells. The PEG chain within the polymer could decrease its

macrophages recognition and extend its half-life in blood circulation.

DOPE could facilitate endosomal escape and PS could be served for nu-

clear transfer (Gao et al., 2007). Unlike the pDNA delivery to the cell nu-

cleus, siRNA delivery involves fewer barriers because the target of

delivery of siRNA is in the cytoplasm (Nguyen and Szoka, 2012). It is

worthy to mention that a complex nanoparticle formulation CALAA-

01 (Calando Pharmaceuticals, Inc.), which consists of cyclodextrin-

based polymer, transferrin targeting ligand, a hydrophilic PEG chain,

and siRNA targeting ribonucleotide reductase M2, a critical biomoleculein DNA synthesis, has been recently shown to effectively deliver siRNA

to humans (Davis et al., 2010).

Mitochondria are playing an important role in regulating cell metab-

olism and cell death, and are involved in diverse physiological activities

in the course of cancer development and progression. As an alternative

subcellular target, some novel mitochondrial TDDSs have been devel-

oped (Yamada and Harashima, 2008). Torchilin's group developed

mitochondrial-targeted liposomal drug-delivery system by incorpora-

tion a mitochondriotropic dye rhodamine-123 (Rh123)-PEG-DOPE

into the liposomal lipid bilayer (Biswas et al., 2011) or by modication

of a mitochondria-targeting triphenylphosphonium cation to liposome

surfaces (Boddapati et al., 2008). The mitochondria-targetingliposomes

could ef ciently deliver the model drug to mitochondria to enhance its

activity.

3.2. Combination drug delivery approaches

To achieve better treatment ef cacy, multimodality treatment or

combination treatment is commonly used to treat cancer. Compared

with single-modality treatment, multimodality treatment can do an ex-

cellent job with additive or even synergistic ef cacy. On the one hand,

using drugs acting through different molecular targets could delay or

block the cancer adaptation processes from different aspects. On the

other hand, the drugs with the same molecular target could functionsynergistically for higher therapeutic ef cacy. The combination of che-

motherapy, biologic therapy, endocrine therapy and/or thermotherapy

has been investigated for their synergistic effects recently (Goodwin

et al., 2012; Mehta et al., 2012).

Although combination therapy has synergistic ef cacy, it could also

lead to increased toxicity in some cases. Some anticancer agents are

mixed together for administration but they are eliminated indepen-

dently, which could cause the additive adverse effects. The combination

of taxanes withanthracyclines inrst-line chemotherapy for metastatic

breast carcinoma produces a signicant benet in activity, but with a

signicant cost in hematologic toxicity (Bria et al., 2005). In phase III

trial, combination of PTX and bevacizumab signicantly prolonged

progression-free survival as compared with PTX alone, however, grade

3 or 4 hypertension, proteinuria, headache, and cerebrovascular ische-

mia were more frequent in patients receiving PTX plus bevacizumab

(Miller et al., 2007). In addition, combination of anticancer agents

which have different routes of administration would decrease patient

satisfaction and compliance. For example, when employing the combi-

nation therapy of lapatinib and trastuzumab to treat ErbB2-positive

breast cancer, patients needed to receive doses of lapatinib adminis-

tered once daily (continuous) in combination with trastuzumab weekly

(Storniolo et al., 2008).

Nanomedicine can provide a fantastic platform for multimodality

treatment. Nanoparticulate DDSs demonstrate many advantages over

conventional formulations by physically blending multiple drugs to-

gether including: (1) improved solubility and bioavailability, (2) escape

of elimination by macrophages and prolonged drug circulation half-life,

(3)increased tumor site accumulation by passive or activetargeting, (4)

ef cient internalization with the mechanism of endocytosis, (5) con-trolled pharmacokinetics of each drug, resulting in enhanced drug ef -

cacy and reduced side effects. Many nanoparticulate platforms have

been developed as co-delivery systems that can deliver a combination

of small molecule drugs or a combination of small molecule drugs and

macromolecular therapeutics.

Liposomes are commonly used as co-delivery carriers with the abil-

ity to load both hydrophilic and hydrophobic drugs. In a study by Wong

et al., co-encapsulation of vincristine and quercetin into a liposome for-

mulation exhibited signicant antitumor activity than free vincristine/

quercetin combinations (Wong and Chiu, 2011). Stealth liposomes

(Ong et al., 2011) and targeted liposomes (Wu et al., 2007) were also

developed as co-delivery carriers for cancer therapy. Some combination

DDSs based on liposomes are currently in clinical trial. Liposomes en-

capsulated with irinotecan and oxuridine (CPX-1) (Batist et al.,2009) and liposomes containing cytarabine and daunorubicin (CPX-

351) (Feldman et al., 2011) have been entered into phase I trials. To en-

sure the product quality of complex liposome formulations, Zuckeret al.

showed the methods to characterize the critical features of LipoViTo

where vincristine (VCR) and topotecan (TPT) were encapsulated in

the same nanoliposome. The characterization methods are useful for a

rational clinical development of liposomal formulations alike (Zucker

et al., 2012).

Cationic lipoplexes were initially proposed to co-delivery of antican-

cer drug and gene. The lipoplexes developed by L. Huang's team could

co-deliver DNA and inammatory suppressors into one immune cell

(Liu et al., 2004). Co-delivery of antitumor agent docetaxel and DNA

which suppress surviving protein, was achieved by a folate-modied

multifunctional lipoplexes as a therapeutic approach for human




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hepatocellular carcinoma (Xuet al., 2010). Cationic liposomes were also

applied to co-deliver siRNA and an anticancer drug (Saad et al., 2008;

Shim et al.,2011). In thepreparation of lipoplexes,a condenseris always

needed to condense pDNA to form the core of the cationic liposomes.

But the cationic liposome:siRNA complexes are always formed by

mixing siRNA and cationic liposomes together.

Polymer-based nanoparticles have been extensively studied as co-

delivery carriers as the drug can be entrapped inside or covalently linked

to the polymermatrix. Polyalkylcyanoacrylate nanoparticles were used toentrapped DOX and chemo-sensitizing compound cyclosporin A to

achieve the synergistic effect in multidrug resistance (MDR) cancer cells

(Soma et al., 2000) Amphiphilic block copolymers such as PEG-PLGA

and PEG-PLA could self-assemble into micelles to co-deliver two

chemotherapeutic agents(H. Wanget al., 2011; Shinet al., 2009). Micelles

formed by cationic copolymers such as amphiphilic copolymer poly{(N-

methyldietheneamine sebacate)-co-[(cholesteryl oxocarbonylamido

ethyl) methyl bis(ethylene) ammonium bromide] sebacate} (P(MDS-

co-CES)) (Wang et al., 2006) and triblock copolymers poly(N,N-

dimethylamino-2-ethylmethacrylate)-polycaprolactone-poly(N,N-

dimethylamino-2-ethyl methacrylate) (PDMAEMA-PCL-PDMAEMA)

(Zhu et al., 2010) have been used to co-deliver genes and drugs to the

same cells, in which hydrophobic anticancer drugPTX wasentrapped in

the core of the micelles, and genes were complexed onto their surface.

PEI-graft-poly(ε-caprolactone) copolymer was also reported to co-

deliver DOX and a reporter gene(Qiu and Bae, 2007). A core-shell nano-

particle formed from folate coated PEGlated lipid shell and PLGA core

was also developed for targeted co-delivery of drug and gene (Wang

et al., 2010). Recently, a nanoparticle system formed by blend of

poly(lactide)-D-α-tocopheryl polyethylene glycol succinate and car-

boxyl group-terminated TPGS (TPGS-COOH) copolymer was developed

for triple modality treatment of cancer. The nanoparticles were formu-

lated with docetaxel, herceptin and iron oxides for the chemo, bio, and

thermo therapies (Mi et al., 2012). Besides entrapped inside the

polymer-based nanoparticle systems, two or more drugs could be con-

jugated to one kind of polymer carrier for combination delivery. The

aromatase inhibitor aminoglutethimide and DOX were simultaneously

conjugated to HPMA copolymer. The results showed that the conjugate

carrying two drugs wasmorepotent than thecombination of two poly-mer conjugates carrying only one drug (Vicent et al., 2005). The HPMA-

based polymer was also used to simultaneously deliver DOX and anti-

inammatory drug dexamethasone (DEX) (Krakovicova et al., 2009).

In another study, DOX and gemcitabine were co-conjugated to the

same HPMA polymer, which increased the ef cacy of the combination

of gemcitabine and DOX without increasing its toxicity ( Lammers

et al., 2009). Dendrimers have also been used for successfully synchro-

nous delivery of two therapeutic agents by either complexation of two

drugs or by conjugation of two drugs in the same polymer, as well as

by complexation of one drug and simultaneous conjugation of another

drug (Clementi et al., 2011; Kaneshiro and Lu, 2009; Kim et al., 2011;

Lee et al., 2011). Also, nanoparticles formed by blending of self-

assembled amphilic copolymer in the presence of a chemotherapeutic

agent and polymer-prodrug conjugate could be used to achieve combi-nation drug delivery (Kolishetti et al., 2010).

Some inorganic nanoparticulate systems have been successfully

employed for co-delivery. Successfully synchronous delivery of chemo-

therapeutic drug and siRNA was achieved by mesoporous silica nano-

particles coating with the cationic polymer. MSNs modied with

PAMAM dendrimers were utilized to simultaneously deliver DOX

and a Bcl-2 siRNA into MDR cancer cells (A. M. Chen et al., 2009). PEI

functionalized MSNs were also used to synchronously deliver DOX

and P-gp siRNA to reverse drug resistance in MDR cancer cells ( Meng

et al., 2010). The in vivo ef cacy of the use of this dual drug/siRNA

nanocarrier was also tested in a xenograft to overcome DOX resistance

(Meng et al., 2013). Positively charged ammonium-functionalized

MSNs could also immobilize negatively charged single strand DNA

(ssDNA) for drug/ssDNA co-delivery (X. Ma et al., 2012). In a recent

work,hydrophilic-hydrophobic anticancer drug pairs, suchas doxorubi-

cin–paclitaxel and doxorubicin–rapamycin, could be loaded into mag-

netic mesoporous silica nanoparticles for simultaneous delivery of

hydrophilic and hydrophobic drugs for combination treatment (Q. Liu

et al., 2012). Magnetic nanoparticles embedded in polylactide-co-

glycolide matrixes were also designed as drug delivery and imaging

vector for loading both hydrophilic and hydrophobic drugs (A. Singh

et al., 2011). Layer-by-layer assembled charge-reversal functional gold

nanoparticles were employed to co-deliver siRNA and plasmid DNAinto cancer cells (Guo et al., 2010).

Some hybrid systems such as lipid-polymer hybrid systems, lipid-

inorganic silica hybrid systems were developed for combination drug

delivery. A polymer-caged nanobin (PCN) formed by liposomal core en-

capsulated with DOX and a polymer shell conjugated with cisplatin

prodrug was designed to co-deliver of DOX and cisplatin. The PCN

could exert synergistic cytotoxic effects of each drug against cancer

cells at reduced doses (Lee et al., 2010). In another study, the protocells

were designed with nanoporous silicacoresenveloped by a lipid bilayer,

which was further functionalized with poly(ethylene glycol),targeting

peptides, and pH-responsive peptides, to deliver combinations of di-

verse drug cargos such as quantum dots, small molecules and oligonu-

cleotides (Ashley et al., 2011).

3.3. Environment-response controlled release strategies

Development of stimuli-responsive nanoparticles is a particularly

appealing approach for the goal of increasing the specicity of drug de-

livery in vivo. Environmentally-responsive nanoparticles have the abil-

ity to produce physicochemicalchanges that regulate drug release at the

target site when exposed to external stimuli. The differences between

tumor environment and normal tissue such as pH value, protease ex-

pression, and the change of external conditions such as the local appli-

cation of heat, ultrasound, light, magnetic eld, or electric eld could

be served as stimuli (Fig. 3). The environmentally-responsive nanopar-

ticles could improve tumor accumulation, tumor penetration of cancer

therapeutics, and increase the intracellular localization of anticancer

therapeutics, and thus further enhance the ef cacy of antitumor thera-

peutics (MacEwan et al., 2010).Among these environmentally-responsive nanoparticles, pH-

responsive nanoparticulate DDSs have been widely studied in the eld

of cancer therapy. A change in pH can be used in two ways to trigger

drug release. First, the extracellular pH values of most human tumors

(pHe) are found to be at an average value of ~7.0, which is a

distinguishing phenotype of solid tumor to normal tissue ( Vaupel,

2004). A second is used after cellular uptake when nanoparticles

reached the endosomal and lysosomal compartments with low pH of

about 5–6 (Murphy et al., 1984).

Nanoparticles could be formulated with pH-responsive polymers that

could change their physical and chemical properties in response to differ-

ent pH values for pH-dependent drug release. One strategy is to take

advantage of the changes in polymer protonation states to make pH-

dependent hydrophobic-to-hydrophilic transitions to affect polymerswelling or solubility, which willthen acquire pH-responsive drug release.

Expansile nanoparticles formulated by acrylate-based hydrophobic poly-

mers modied with pH-labile protecting groups were stable at neutral

pH, but in a mildly acidic pH, the protecting group was cleaved to reveal

hydroxyls, which corresponded to a hydrophobic-to-hydrophilic trans-

formation, resulting in swelling of the polymeric structure to create a hy-

drogel and subsequent drug release. The expanded nanoparticles can act

as an intracellular or intratumoral depot for the chemotherapeutic agent

PTX, affording higher intracellular drug concentrations (Zubris et al.,

2012). PTX loaded expanded nanoparticles showed superior in vivo

ef cacy in murine tumor models including non-small cell lung cancer

(Griset et al., 2009) and mesothelioma (Colson et al., 2011). PEG-

poly(β-amino ester) polymers have also been used to design for

pH-responsive nanoparticles targeting the low pH present in the




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extratumoral and cellular microenvironment. The polymers are de-

signed to incorporate some amines that are unprotonated at pH 7.4

to make the polymer insoluble in water, but are protonated at

pH 6.4–6.8 to increase polymer solubility and induce a sharp micelliza-

tion–demicellization transition for drug release (Shenoy et al., 2005;

X.L. Wu et al., 2010). The micellization–demicellization transition was

also found in DDSs formulated by combination of poly(L -histidine)-b-

PEG and PLLA-b-PEG (Lee et al., 2003). The low pH tumor microenvi-

ronment could also serve as stimulus to trigger a change in surface

charge of nanoparticle which could facilitate uptake of nanoparticles

by tumor cells. Poon et al. designed the trilayer nanoparticles which

consist of iminobiotin modied PLL, the linker protein neutravidin,

and biotin end-functionalized PEG layer. The PEG layer in the nanopar-ticles could selectively deshield when localized in low pH tumor micro-

environment to expose positive charged nanoparticles for improving

cellular uptake by tumor cells and decreasing non-specic cellular up-

take by normal cells (Poon et al., 2011). Another work based on a

charge-reversal nanogel triggered by the pHe was reported by Du

et al. At physiological pH values, the nanogel was negatively charged

with high positively charged drug loading capacity and was relatively

inert to tumor cells. When exposed to the tumor microenvironment,

the nanogel was turned to be positively charged to strengthen

nanogel-cell interaction and enhance cellular uptake by cancer cells

(Du et al., 2010).

An alternate pH-triggered strategy involves the use of acid-labile

linkers upon cleavage at a specic pH. Commonly used acid-labile

crosslinkers include ester,hydrazone, carboxy dimethylmaleic anhydride,

orthoester, imine, vinylether, phosphoramidate, and so on (Gao et al.,

2010). The prodrug strategy often conjugates drug molecules to macro-

molecular chains via pH-labile linkers. In response to the acidic extracel-

lular or intracellular environment, these macromolecular carriers are

able to release the drug to exert its ef cacy (Ulbrich and Subr, 2004). In

an example of this approach, cisplatin was conjugated to poly(ethylene

glycol)-b-poly(L -lactide) using hydrazone cross-linkers to allow release

of the drug after the nanoparticles were endocytosed by the target cells

(Aryal et al., 2010). Likewise, a hydrazone linker was used to link the

doxorubicinyl group andthePEG chain to the surface of the goldnanopar-

ticles, which released DOX in acidic organelles after endocytosis (F. Wang

et al., 2011). Acid-labile linkers were also used to synthesize acid-labile

polymers for pH triggered rapid degradation to release the entrappeddrugs. One example is the use of nanoparticles composed of a copolymer

(poly-β-aminoester ketal-2) for ef cient gene delivery. The nanoparticles

can respond to endosomal pH and undergo a hydrophobic-hydrophilic

switchanda rapid degradation“in series”, resulting in increased cytoplas-

mic delivery (Morachis et al., 2012). As many chemotherapeutics and

some macromolecules such as DNA, exert their action in the nucleus,

the pH-labile linkers havebeen used to develop charge-reversal polymers

for the nuclear-targeted drug delivery. Modication of the positive

amines in the cationic polymers such as PLL (Zhou et al., 2009) and

PAMAM dendrimers (Shen et al., 2010) with negatively charged groups

with acid-labile amides so that the polymer was negatively charged at

physiological condition, but was hydrolyzed at low pH to restore its pos-

itive charge, canobtain ef cientnuclear-targeted gene delivery. For favor-

ing gene expression, pH labile linker was also employed to construct a

Fig. 3. Schematicillustrationof environmentalresponsedrug delivery systems for cancer therapy. The low extracellular pH or up-regulated protease expression at the tumorenvironment,

and the local application of heat, electric eld, magnetic eld, ultrasound or light could be utilized to trigger drug release from nanoparticulate drug delivery systems.




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smart nanoassembly which has the ability for self-dePEGylation. The

nanoassembly containing lipid envelope based on PEG-vinyl ether-

DOPE can self-remove PEG and recover the fusogenic ability of DOPE at

low pH, resulting a higher transfection ef ciency and much lower cyto-

toxicity than that of commercial Lipofectamine 2000 in several cancer

cell lines (Xu et al., 2008).

Over-expressed cancer-associated enzymes are also utilized to trig-

ger drug release. Many kinds of nanomaterials such as liposomes,

polymer-based nanoparticles, mesoporous silica nanoparticles andgold nanoparticles have been designed with high specicity for the en-

zyme stimulus (Andresen et al., 2010; Ghadiali and Stevens, 2008; Ulijn,

2006). The general strategy to design enzyme-responsive nanoparticle

systems is to employ biological motifs that can be degraded by en-

zymes. By this approach, the self-assembled polymers could be synthe-

sized with enzyme-responsive motifs to the encapsulated drug or

the conjugated drug using enzyme-responsive linkers. When these

nanomaterials encounter the enzyme, they could degrade to release

the encapsulated drug or conjugated drug. In other cases, the nanopar-

ticles can be designed to generate a change in the physical properties

uponenzymatic stimulation, which could facilitate cellular uptake or in-

tracellular delivery of nanoparticles. Matrix metalloproteinases (MMPs)

especially MMP-2 and MMP-9, have been regarded to be up-regulated

in the tumor microenvironment (Roy et al., 2009). Banerjee et al. had

integrated an MMP-cleavable lipopeptide into the liposome formula-

tion to prepare MMP-sensitive liposomes which can rapidly release

their contents by MMP-9 (Banerjee et al., 2009). In another work,

PEG-mesoporous silica nanoparticles (MSNPs) to respond to MMP for

controlled drug delivery were engineered. The proteases present at

the tumor site could trigger DOX release from the MSNPs, resulting in

signicant cellular apoptosis (N. Singh et al., 2011). Basel et al. incorpo-

rated peptides with a sequence that can be recognized by cancer-

associated proteases into a polymer-stabilized liposome formulation.

The liposomes were much stable and resistant to osmotic swelling,

but released their payloads quickly in response to the protease present-

ed at the tumor site (Basel et al., 2011). As abnormally high concentra-

tions of phospholipase A2 (PLA2) have been found in the evading zone

of tumors (Yamashita et al., 1993), several liposome systems consisting

prodrugs of antitumor ether lipids (proAELs) were investigated forPLA2-triggered degradation, resulting in the release of antitumor

ether lipids (AELs). As the AELs possess the ability to enhance trans-

membrane drug diffusion, encapsulation of the conventional chemo-

therapeutic drugs in liposomes containing proAELs can enhance the

intracellular distribution of drug in cancer cells (Andresen et al.,

2004). Sugar-based nanocarrier has also been designed to release

anti-cancer drugs selectively to tumors. Bernardos et al. used saccharide

derivatives which could respond to the lyzosomal amylase to modify

the pore of the MSNPs for specic release of drugs by enzymatic stimu-

lus. After the internalization of the nanoparticle, the saccharide molec-

ular gate opened in the presence of the lyzosomal amylase, and the

cytotoxicagent consequently released to attain a decreasedcell viability

(Bernardos et al., 2010).

Differences in the reducing potential between the extra- and intra-cellular environment provide another stimulus for drug delivery. The di-

sulde linkage has been extensively employed to designredox sensitive

nanocarriers for drug delivery due to their stability in the typically oxi-

dizing extracellular environment and their lability in the elevated re-

ducing intracellular environment (high glutathione concentration) to

ef ciently release entrapped cargos (Saito et al., 2003). Gao et al. syn-

thesized a linear cationic click polymer containing disulde bonds via

the “click chemistry”. The polymer could facilitate ef cient gene deliv-

ery through releasing DNA ef ciently by the cleavage of disulde

bonds under the reduction condition (Gao et al., 2011). The disulde

bond was also used to link poly(epsilon–caprolactone) and poly(ethyl

ethylene phosphate) to synthesize diblock copolymer. The micelles

formed by the diblock copolymer could load drug in its inner core

in aqueous solution, while release drug rapidly under glutathione

stimulus, leading to enhanced drug toxicity to tumor cells (Tang et al.,

2009). The disulde linkage was also applied to design micelles with

the capability to self-remove PEG chain under the intracellular reducing

environment. The successful detachment of PEG in endosome is bene-

cial for endosomal escape of micelles and enhanced gene transfection

ef ciency (Takae et al., 2008).

An extrinsicstimuli canalso be utilized to enhance drugdistribution at

the tumor site or improve intracellular drug accumulation by selectively

release of its payload at the tumor tissue or cancer cells. Thermosensitivepolymers that exhibit a volume phase transition in response to tempera-

ture have been developed for triggering drug release and local accumula-

tion by application of heat. Ultrasound and electromagnetic (EM) elds

have been employed as external stimuli to produce heat (O'Neill and

Rapoport, 2011). Polymers based on N-isopropylacrylamide (NIPAm), N,

N-diethylacrylamide and N-vinylcaprolactam monomers have a lower

critical solution temperature (LCST) and polymers based on a combina-

tion of acrylamide and acrylic acid monomers showed a upper critical so-

lution temperature (UCST) (Schmaljohann, 2006). Thermoresponsive

chitosan-g-poly (N-vinylcaprolactam) polymers loaded with curcumin

can specically kill cancer cells at above their LCST (Rejinold et al.,

2011). Hoare et al. produced thermosensitive nanoparticles by wrap-

ping superparamagnetic iron oxide nanoparticles with a lm formed

by PNIPAm-based nanogels and ethyl cellulose. The entrapped drug

molecules could transport across the lm through heating the

superparamagnetic nanoparticles to dissolve of the PNIPAm (Hoare

et al., 2011). Inducing a local hyperthermia effect by the magnetic

eld at the level of the polymersome membrane could achieve trig-

gered drug release from polymersomes encapsulate DOX together

with superparamagnetic iron oxide nanoparticles (USPIO; γ-Fe2O3)

(Oliveira et al., 2013). Similarly, ultrasound can be employed to trigger

the release of DOX and other hydrophobic drugs from polymeric mi-

celles at denite time and space (Husseini and Pitt, 2008, 2009).

Some smart nanoparticles are designed to respond to a mixture of

two or more stimuli. A nanoparticle system composed of a conducting

polymer (polypyrrole) and a temperature-sensitive hydrogel matrix

(PLGA-PEG-PLGA) were developed with response to temperature and

electric eld dual-stimulus for programmed drug delivery (Ge et al.,

2012). Superparamagnetic maghemite (γ-Fe2O3) nanoparticles modi-ed with poly (2-(dimethylamino)ethyl methacrylate) (pDMAEMA)

which exhibit a pH- and temperature-dependent reversible agglomera-

tion showed remarkable gene delivery ef ciency in CHO-K1 cells

(Majewski et al., 2012). A novel PEG and cRGD peptide modied

poly(2-(pyridin-2-yldisulfanyl)ethyl acrylate) nanoparticle loaded with

DOX (RPDSG/DOX) was designed to be both pH-responsive and redox

sensitive. The RPDSG/DOX nanoparticle is stable in physiological condi-

tion while releasing DOXfast with the trigger of acidic pH andredox po-

tential (Bahadur et al., 2012). Micelles formed by block copolymer

which consisted of an acid-sensitive tetrahydropyran-protected 2-

hydroxyethyl methacrylate (HEMA) hydrophobic chain and a

temperature-sensitive poly(N-isopropylacrylamide) (PNIPAM) hydro-

philic chain can respond to triple stimuli including temperature, pH

and redox potential (Klaikherd et al., 2009).

3.4. Multi-stage delivery nanovectors

Between the point of intravenous administration and the tumor

tissue, systemically administered nanoparticles should go through a

three-step process: blood delivery to the blood vessels of the tumor,

transported across the vessel wall into the interstitium, and migrated

through the interstitium to reach cancer cells in tumor ( Jain, 1999). To

circumvent the multiplicity of biological barriers that the nanoparticles

encounter after administration, and maximize drug localization and

release in cancer cells, multistage nanovectors have been developed

recently. The rationale for the multistage approach is based on the

arrangement of different tasks to a single nanoparticle which could

complete the tasks at different stages.




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Thisconcept wasrstapplied to design a “nanocell”which overcomes

the barriers unique to solid tumors. The nanocell was composed of a nu-

clear polymer-based nanoparticle containing a chemotherapy drug and a

pegylated-lipid envelope, which entrapped an anti-angiogenesis agent.

The anti-angiogenesis agent was rst released from the outer envelope

causing a vascular shutdown, and the chemotherapy drug was then re-

leased from the inner nanoparticle, which is trapped inside the tumor.

The multistage release prole within a tumor results in improved thera-

peutic effects with reduced toxicity (Sengupta et al., 2005).Based on mathematical modeling, mesoporous silicon nanoparticles

could be designed to carry the payload traf cking ef ciently and con-

trolling the release rate of the burden. Recently, Ferrari's group devel-

oped a multistage silicon nanocarrier system which was composed of

mesoporous silican particles (also known as the rst stage) and the

entrapped nanoparticles (the second stage) loaded with anticancer

therapeutics (the third stage). The rst stage mesoporous silican parti-

cles could protect and ferry the inner nanoparticles until they recognize

and dock at the tumor vasculature. Then the second stage nanoparticles

released from the MSP with the biodegradation of porous multistage

particles under physiological conditions. The released nanoparticles

were able to extravasate through fenestrations of vessels and enter

the tumor parenchyma, thus concentrating diagnostic and therapeutic

agents within the target microenvironment (Tasciotti et al., 2008).

This kind of multistage nanovectors was also applied to gene delivery.

Liposomes consisted of dioleoyl phosphatidylcholine containing siRNA

targeted against the EphA2 oncoprotein (the second-stage carriers)

were loaded into mesoporous biodegradable silicon particles (the

rst-stage carriers). The mesoporous silicon particles allowed for the

loading and release of second-stage nanocarriers in a sustained manner.

Compared withthe one-stage neutral nanoliposomes that require twice

weekly injections to achieve continuous gene silencing, the multistage

delivery methods could achieve sustained EphA2 gene silencing which

could last for at least 3 weeks after a single i.v. administration (Tanaka

et al., 2010). Similarly, superparamagnetic CaCO3 mesocrystals were

used to encapsulate DOX, Au–DNA, and Fe3O4@silica nanoparticles for

the co-delivery of drug and gene via a multistage method for treatment

of cancer. The stage-one nanoparticles-CaCO3 system protected the en-

capsulated payloads from degradation and phagocytosis during naviga-tion in the blood. After they docked to the vascular walls, the stage-one

nanoparticles degraded to gradually release the stage-two nanoparti-

cles and drugs (Zhao et al., 2010). A multistage system with size-

shrinking property was designed for deep tumor tissue penetration.

The 100-nm multistage nanoparticles are composed of a gelatin core

with surface covered with 10-nm QDs. After they exposed to the

tumor microenvironment, the 100-nm nanoparticles “shrink” to 10-

nm nanoparticles due to the hydrolysis of gelatin by the MMPs. This

shrinkable multistage system can combine the advantage of large 100-

nm nanoparticles that are suitable for the EPR effect, and small 10-nm

nanoparticles that are suitable for diffusion in the collagen matrix of

the interstitial space and penetration into the tumor parenchyma

(Stylianopoulos et al., 2012; Wong et al., 2011).

3.5. Cancer nanotheranostics

Cancer nanotheranostics is the use of nanotechnology for the com-

bined therapeutics and diagnostics for cancer (Sumer andGao, 2008). Be-

sides for therapeutic purposes, potential applications of nanotheranostics

range from the visualizing the blood circulation, biodistribution of drugs

in real time, noninvasively assessing drug accumulation and drug release

at the target site, facilitating triggered drug release and monitoring drug

distribution, to predicting drug responses and evaluating drug ef cacy

longitudinally (Lammers et al., 2010). Integration of imaging capability

into the design of nanoparticles made it possible to evaluate the fate of

nanoparticles in real time, which will allow physicians to adjust the

type and dosing of drugs for more personalized treatment regimens

(Diou et al., 2012). The currently accessible imaging techniques include

MRI, single photon emission computed tomography (SPECT), positron

emission tomography (PET),computer tomography (CT),and ultrasonog-

raphy (US) (Mura and Couvreur, 2012). Nanotheranostics can be obtain-

ed by either attaching different imaging moieties (i.e. NIR probes,

radionuclides) to the available nanocarriers or taking advantage of the in-

trinsic properties of some nanoparticle materials such as SPIOs for MRI

and QDs for uorescence imaging(Brigger et al., 2002). Till now, a variety

of nanotheranostics that combine anti-cancer therapeutics with afore-

mentioned imaging modalities has been developed using liposomes, mi-celles, polymers, gold-based nanomaterials, magnetic nanomaterials,

carbon nanomaterials, and silica-based nanomaterials. From a recent

summary of selected papers published between 2009 and 2012 on

nanotheranostics, optical and MRI are the preferable modalities per-

formed for imaging functionality, through use of NIR emission and mag-

netic agents, respectively (Wang et al., 2012).

Magnetic nanoparticles have been used as “nanotheranostics” for

both targeted drug delivery and tumor imaging due to their magnetic

property as nanostructured contrast probes for MRI. Among the mag-

netic nanoparticles, SPIOs are the most commonly used nanomaterials.

A number of polymers, including dextran, dendrimer, polyaniline, and

polyvinylpyrrolidone, have been utilized to coat magnetic nanoparti-

cles. Pluronic polymer F127 and β-cyclodextrin (β-CD) were coated

onto the iron oxide core nanoparticles by a multi-layer approach for en-

capsulation of the anti-cancer drugs and for sustained drug release, re-

spectively. The optimized water-dispersible SPIOs formulation showed

improved MRI characteristics and improved therapeutic effects

(Yallapu et al., 2011). By tuning the properties of the coating polymers,

the triggered drug release could be realized in magnetic theranostic

nanoparticles. A poly (beta-amino ester) (PBAE) copolymer was used

to entrap SPIO and DOX for sensitive detection and effective treatment

of cancer by pH sensitive controlled drug release (Fang et al., 2012).

The magnetic nanoparticle formulation loaded with other imaging moi-

eties could allow formultimodal imaging. Foyet al.loaded near-infrared

dyes into a magnetic nanoparticle (MNP) formulation stabilized by an

amphiphilic block copolymer to provide for both tumor MRI and optical

imaging (Foy et al., 2010). To achieve targeted delivery, a variety of li-

gands has been attached to the outside layer of magnetic theranostic

nanoparticles such as folate (Santra et al., 2009), cRGD, (Nasongklaet al., 2006) and antibody (Zou et al., 2010). Yang et al. designed an

FR-targeted multifunctional polymer vesicle nanocarrier system loaded

with SPIO and DOX to increase specic cellular uptake by FR positive

cancer cells (Yang et al., 2010). Similarly, a multifunctional antibody-

and uorescence-labeled HuCC49ΔCH2-SPIO “nanotheranostics” was

developed for combined targeted anticancer drug delivery and

multimodel imaging of cancer cells (Zou et al., 2010). Cyclo(Arg–Gly–

Asp–d–Phe–Cys) (c(RGDfC)) peptides were also employed to modify

SPIO nanocarriers for targeted drug delivery and dual PET/MRI imaging

(Yang et al., 2011). Magnetic theranostic nanoparticles can also be used

for gene delivery. An MRI visible gene delivery system developed with a

core of SPIO nanocrystals and a shell of biodegradable ste

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