European Journal of Pharmaceutical Sciences 47 (2012) 1006–1014

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European Journal of Pharmaceutical Sciences

journal homepage: www.elsevier .com/ locate /e jps

Site specific/targeted delivery of gemcitabine through anisamide anchoredchitosan/poly ethylene glycol nanoparticles: An improved understanding oflung cancer therapeutic intervention

Neeraj K. Garg a,b,⇑, Priya Dwivedi c, Christopher Campbell d, Rajeev K. Tyagi d,⇑a Drug Delivery Research Group, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh - 160 014, Indiab Department of Pharmaceutical Sciences, Dr. H.S. Gour University, Sagar 470 003, MP, Indiac Department of Biotechnology, TRS Colloege, Rewa 486 001, MP, Indiad Global Health Infectious Disease Research Program, Department of Global Health College of Public Health, University of South Florida, 3720 Spectrum BlvdTampa, FL33612-9415, USA

a r t i c l e i n f o

Article history:Received 28 May 2012Received in revised form 26 August 2012Accepted 12 September 2012Available online 4 October 2012

Keywords:AnisamideChitosanGemcitabineLung cancer targetingPoly(ethylene glycol)Nanoparticles

0928-0987/$ - see front matter Published by Elsevierhttp://dx.doi.org/10.1016/j.ejps.2012.09.012

⇑ Corresponding authors. Addresses: Drug DeliveryInstitute of Pharmaceutical Sciences, Panjab UniverIndia. Tel.: +91 9356268053 (N.K. Garg), Global HealtProgram, Department of Global Health College of PublFlorida, 3720 Spectrum Blvd, Tampa, FL 33612-9415(O); fax: +1 813 974 0992 (R.K. Tyagi).

E-mail addresses: neerajombiotech@gmail.com (Nedu (R.K. Tyagi).

a b s t r a c t

Gemcitabine (20, 20-difluorodeoxycytidine) is a deoxycytidine analog with significant antitumor activityagainst variety of cancers including non-small cell lung cancer. However, rapid metabolism and shorterhalf-life of drug mandate higher dose and frequent dosing schedule which subsequently results intohigher toxicity. Therefore, there is a need to design a vector which can reduce the burden of frequent dos-ing and higher toxicity associated with the use of gemcitabine. In this study, we investigated the possi-bility of improving the targeting potential by employing the surface modification on Chitosan/poly(ethylene glycol) (CTS/PEG) Nanoparticles. We demonstrate formulation and characterization ofchitosan/poly(ethylene glycol)-anisamide (CTS/PEG-AA) and compared its efficiency with CTS/PEG andfree gemcitabine. Our results reveal its sizeable compatibility, comparatively less organ toxicity andhigher antitumor activity in vitro as well as in vivo. This wealth of information surfaces the potential ofCTS/PEG-AA nanoparticles as a potent carrier for drug delivery. In brief, this novel carrier opens new ave-nues for drug delivery which better meets the needs of anticancer research.

Published by Elsevier B.V.

1. Introduction expressed in blood, liver and kidney (Immordino et al., 2004) and

Lung cancer is one of the most frequently occurring malignan-cies and is the leading cause of cancer-related death worldwide(Garbuzenko et al., 2010). Despite the recent advances in chemo-therapy, radiotherapy and surgery, survival rate from lung canceris still very low. Gemcitabine (GEM) is an anticancer agent thathas been demonstrated to be effective in the treatment of a widevariety of solid malignancies such as lung, colon, head and neck(Reddy and Couvreur, 2008). It primarily acts on cells undergoingDNA synthesis (S-phase of cell cycle) and also blocks the progres-sion through the G1/S phase boundary (Huang and Plunkett,1995). Despite of its efficient activity, the drug (GEM) has veryshort plasma circulation time (Burris 3rd et al., 1997). This drugis converted into its inactive and soluble metabolites, 20-difluoro-deoxyuridine (dFdU) by deoxycytidine deaminase (dCDA), which is

B.V.

Research Group, Universitysity, Chandigarh - 160 014,h Infectious Disease Researchic Health, University of South, USA. Tel.: +1 813 974 4243

.K. Garg), rtyagi@health.usf.

cleared rapidly out of body through renal excretion. Thus, a fre-quent administration scheduled at high doses is required, whichin turn leads to high levels of toxicity (hepatoxicity, nephrotoxicityand other toxicity) (Robinson et al., 2003). Therefore, novel thera-peutic strategies aiming at improved pharmacokinetics, reducedtoxicity and better bio-distribution are urgently needed.

Nanoparticles (NPs) have been emerging as key evolutionarycarrier system by providing a targeted approach for efficient deliv-ery of conventional chemotherapeutic drugs for cancer therapy.This targeted approach leads to an efficient delivery of drugs inlow dosages as well as in a sustained manner within the body(Sahoo and Labhasetwar, 2003). Chitosan (CTS) is considered tobe one of the most widely used bio-polymer for NPs preparationdue to its cationic, nontoxic, biodegradable and biocompatiblepolyelectrolyte properties (Agnihotri et al., 2004). Moreover, it pro-motes cross-linkage with multivalent anions, such as sodium tri-polyphosphate (TPP) to provide an efficient mesh-work to entrapthe water soluble drug molecules (Tiyaboonchai, 2003). NPs sur-face can be modified by target specific ligand anchoring to aug-ment the targetability of the carrier to specific cells or tissues.The surface modifications target the drug to the specific cell forthe desirable action and promote cellular uptake and prolong

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residence time in tumor within low dose (Das et al., 2009). Theencapsulation of GEM inside NPs prevents its untimely degrada-tion during intravenous administration, thereby increasing itstherapeutic competence.

Sigma receptors are well known membrane-bound proteins,which are over expressed in a diverse set of human tumors, suchas melanoma, non-small cell lung carcinoma, breast tumors of neu-ral origin, and prostate cancer (Banerjee et al., 2004; Vilner et al.,1995). The high affinity of benzamide derivative such as anisamide(AA) to sigma receptor expressing cells drew attention for the pros-pect of using these sigma-receptor binding ligands for the diagno-sis and targeted therapy of a variety of tumors including lungcancer (Li and Huang 2006).

The present work is aimed to prepare and investigate the feasi-bility of using the chitosan/poly(ethylene glycol)-anisamide (CTS/PEG-AA) NPs as a drug carrier to target lung cancer. In this study,AA-modified NPs (CTS/PEG-AA) were prepared and characterizedfor particles size, zeta potential, and release property. The anti-neoplastic effect and value of AA modified CTS/PEG NPs wasassessed both in vitro and in vivo based on pharmacokinetics aswell as cytotoxicity studies.

2. Materials and methods

2.1. Materials

Chitosan (CTS, M.W = 50,0000, degree of deacetylation(DD) > 90%, Gemcitabine hydrochloride (GEM), COOH-PEG3500MW-NH2, N-(2-bromoethyl)-4-methoxy-benzamide, N,N-Diiso-propylethylamine (DIPEA), 3-[4,5-dimethylthiazol-2-yl]-3,5-di-phenyl tetrazolium bromide (MTT), fluorescein isothiocyanate(FITC), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),EDTA, Anisamide (AA) and other chemicals, were of analyticalgrade and procured from Sigma–Alderich (Germany). The lungepithelial cancer cell line (A549) was procured from the NationalCenter for Cell Sciences (NCCS), Pune, India. The deionized waterwas used for the experiments and filtered through membrane filter(Milli-Q Academic; Millipore, France).

2.2. Synthesis of PEG-AA

The AA was conjugated to PEG using the procedure previouslyreported by Banerjee et al. (2004) with minor modifications(Banerjee et al., 2004). Briefly, 100 mg of 0.4 mM AA was allowedto react with 100 mg of 23.2 lM COOH-PEG3500 MW-NH2 in ace-tonitrile (5 mL) in presence of DIPEA (30 lL, 0.2 mM) at 65–70 �Cfor 8 h. Five milliliter methanol was added to the above mixturefollowed by excess ether (50 mL) and it was then kept at �80 �Cfor 24 h. The precipitate was collected upon centrifugation andrecrystallization was repeated twice. The percentage of AA conju-gation was estimated by determining absorbance of PEG-AA and li-gand-free PEG at 255 nm (kmax for AA, e = 14,832). The amount ofAA attached to the PEG was then calculated based on the differencein the absorbance at 255 nm.

2.3. Preparation and characterization of NPs

NPs were prepared as previously reported by Calvo et al. (1997)with minor modifications based on the ionic gelation of CTS withTPP anions. (Calvo et al., 1997). The CTS/PEG-AA NPs were formedby addition of TPP solution (0.95 mg/mL in distilled water) to CTSsolution (18.95 mg dissolved in 10 mL of 1% acetic acid) containingvarious concentrations of PEG-AA (0, 5, 10, 15, 20 and 25 mg/mL)at a 2:4.5 (volume ratio), under constant magnetic stirring condi-tion. GEM-loaded NPs were formed by incorporation of the TPP

solution into the CTS and PEG-AA solution containing 6.5 mg/mlGEM at the same ratio. After incubation (under stirring), the solu-tion was centrifuged twice at 14300 rpm (Ultracentrifuge 3 (Opti-ma XL-100 K), Beckman, USA), for 20 min to remove excessamounts of TPP and unencapsulated GEM. The pellets were dis-persed in Milli-Q water and supernatant was pooled for the esti-mation of un-entrapped GEM. The resultant NPs were lyophilized(VirTis AdVantage) for further use. The CTS/PEG NPs were also pre-pared using similar procedure for the comparative studies. The NPswere formulated under a hygeinic condition (laminar flow) underaseptic conditions and attention was given to avoid the contamina-tion of formulation (samples) by external environment duringmanufacture and prior to injection into animals.

The drug (GEM) encapsulation efficiency of CTS/PEG-NPs andCTS/PEG-AA NPs was determined by using the method previousreported by Pasut et al. (2008) with slight modifications (Pasutet al., 2008). Briefly, the supernatant (as discussed in above section)was quantified for the presence of free GEM by RP-HPLC using C18column (Nova-Pak C18, 3.9 mm � 300 mm, Waters Associates, Mil-ford, Massachusetts) operated at 37 �C. Waters 2489 UV/VisibleDetector (Waters Associates) was used and setted at a wavelengthof 268 nm (kmax of GEM) for detection of free GEM. In concise, super-natant (1 mL) was taken out to estimate the amount of encapsulateddrug with a mobile phase consisting of phosphate buffer (2.5 mM),(pH 7.0) as eluant A and 50% of 2.5 mM phosphate buffer and 50% ofacetonitrile as eluant B in 95:5, v/v ratio with a constant flow rate(1 mL/min). The standard curve of drug (GEM) was prepared underidentical conditions and the amount of drug in CTS/PEG and CTS/PEG-AA NPs was calculated from the peak area correlated withthe standard curve. The total amount of drug incorporated intoCTS/PEG and CTS/PEG-AA NPs was expressed in terms of percentageentrapment efficiency and determined by following formula.

Entrapment efficiency ð%Þ

¼ Total amount of Drug added� Amount of drug in supernatantTotal amount of Drug added

� 100 ð1Þ

2.4. SEM, TEM particle size and zeta potential

The shape and surface morphology was determined by Scan-ning Electron Microscopy (SEM) and Transmission ElectronMicroscopy (TEM). For SEM, a thin film of aqueous dispersion ofNPs was applied on double stick tape over an aluminum stuband air dried to get uniform layer of particles. These particles werecoated with gold using sputter gold coater, and then placed underscanning electron microscope (LEO 435 VP, Cambridge, UK) forexamination. For TEM, one drop of aqueous solution of samplewas placed on a membrane coated grid surface. A drop of 1% phos-photungstic acid was immediately added to the surface of the grid.After 1 min excess fluid was removed and the grid surface was airdried at room temperature and was examined with the microscope(FEI Morgagni 268 D).

The particle size, PDI (polydispersity index) and zeta potentialof CTS, CTS/PEG and CTS/PEG-AA NPs was measured by ZetasizerNano-ZS (Malvern Instruments, UK). The Particle size was deter-mined by using normal saline as dispersion medium where as1 mM HEPES buffer was used for the measurement of zetapotential.

2.5. In vitro release and ex vivo studies

2.5.1. Release studyThis study was carried out for checking in vitro release kinetics

of GEM from NPs. Gemcitabine release from NPs was performed

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in vitro by using PBS (10 mM, pH 5.8 and 7.4) as the release med-ium, and following the dialysis bag method. The bags were soakedin water for 12 h before use. The dialysis bag (cutoff 2000 Da; Spec-trum� Spectra/Por� 6 dialysis membrane tubing, USA) retained theNPs, but allowed the free drug to diffuse into the dissolution med-ium. An aliquot of 10 mg of each of CTS/PEG NPs and CTS/PEG-AANPs were placed separately into the dialysis bag with the two endsfixed by clamps. The bags were placed in a glass beaker containing50 mL of the dissolution medium and stirred at 200 rpm. The tem-perature was maintained at 37.0 ± 0.5 �C for all the drug releaseexperiments, which were performed in Penet. At prefixed timeintervals, 200 lL of the medium were withdrawn and analyzedfor the drug content using RP HPLC as mentioned in Section 2.3.An equal volume of PBS, maintained at the same temperature,was added after sample withdrawal to ensure the sink conditions.The same analytical procedure used for the estimation of drugloading was followed in this study.

2.5.2. Cytotoxicity assayThe anti-proliferative effect of the CTS/PEG-AA and CTS/PEG NPs

was assessed on lung epithelial cancer cell lines (A549 cells) fol-lowing MTT assay (Kolhe et al., 2003). The A549 cells were storedin DMEM medium (Himedia, India) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% streptomycin, 3 mM gluta-mine in a 37 �C humidified incubator and 5% CO2 atmosphere.Exponentially growing A549 was seeded at 3 � 104 cells/mL in 96well plates (Sigma, Germany). The cells were separately treatedwith GEM and GEM loaded CTS/PEG and CTS/PEG-AA and incu-bated under controlled environment (37 �C humidified incubatorand 5% CO2) for 72 h. Subsequently, MTT solution (5 mg/mL) wasadded to each well and incubated for 4 h at 37 �C, facilitatingMTT to be reduced by viable cells with the formation of purple for-mazan crystals. The formazan crystals were dissolved in DMSO andthe absorbance of individual wells was noted at 570 nm.

2.5.3. Cell uptake studyTo determine cellular binding cell uptake study was performed,

NPs containing a fluorescent dye (FITC) were prepared using theprocedure mentioned in Section 2.3 of this manuscript, except that300 lg FITC was added instead of GEM to the CTS solution beforeformation of NPs. The incorporated FITC acts as a probe (FITC doesnot change the cell uptake of the NPs) for NPs and offers a sensitivemethod to determine qualitative as well as quantitative cellularbinding (Arya et al., 2011).

The cells were cultured as reported in Section 2.6.2 of this man-uscript, detached by trypsinization, collected, counted, and thenseeded at a density of 2 � 106 cells/well in 96 well plates (Sigma,Germany). After 24 h incubation, the cell monolayer was washedwith RPMI 1640 medium, and FITC, FITC labeled CTS/PEG andCTS/PEG-AA NPs were added into the plates. With 2, 5 and 10 h fur-ther incubation, the cells were washed three times with PBS to re-move free FITC/FITC-labeled NPs. The cell-associated fluorescencewas measured by fluorescence activated cell sorters (BD Biosci-ences FACS Aria, Germany) (Agarwal et al., 2009). For inhibitionstudy, cells were pre-incubated for 2 h with 40 lM haloperidol,which is well known high affinity ligand for the sigma receptor,in complete media and NPs uptake study was similarly carriedout as described above.

2.6. In vivo studies

2.6.1. Pharmacokinetic studyThe Institutional Animals Ethical Committee of Dr. Hari Singh

Gour University approved the protocols and animal experimentswere carried out in accordance to guidelines of Council for the Pur-pose of Control and Supervision of Experiments on Animals (CPC-

SEAs), Ministry of Social Justice and Empowerment, Governmentof India. Balb/c mice (4–5 weeks old, weight 25 ± 2 g) were pro-cured from CDRI, Lucknow, India.

This novel nanocarrier was deployed to study its effect on thebioavailability of drug, and therefore pharmacokinetic profiling ofdrug was inevitably determined (Paolino et al., 2010). Three groupsof three animals were employed in this study. This backgroundmouse strain was administered Plain GEM, GEM loaded-CTS/PEGand CTS/PEG-AA NPs a dose equivalent of 6 mg/kg body throughlateral tail vein. Blood samples (200 ll) were collected at differenttimes of the study from the retro-orbital plexus and collected plas-ma (5000 � g, 12 min) was frozen down. Glacial acetic acid (50 ll)was added to plasma samples to decrease hydrogen bonding be-tween nucleosides and proteins. Acetonitrile (1 ml) (HPLC grade)was added to plasma samples, which were then vortex-mixedand centrifuged at 900 � g for 15 min at 4 �C. The supernatantwas removed and collected in a glass tube and acetonitrile (1 ml)was added to the pellet. Three cycles of vortex mixing and centri-fugation procedure were performed. The supernatants werepooled, evaporated to dryness under nitrogen flux at 42 �C(thermostated water bath) and stored at �80 �C. Before RP-HPLCanalysis, the residue was re-suspended in water (1 ml, HPLCgrade), incubated for 5 min at 37 �C and then centrifuged at12,000 � g for 15 min at 20 �C. The supernatant was removed,filtered through a 0.22lmpore size Anotop 10 syringe filter(Whatman, Springfield Mill, UK) and placed in 4 ml HPLC glassvials for analytical determination. Detection of Gemcitabine inserum was carried out using a RP-HPLC method as mentioned inSection 2.3, and other Pharmacokinetic parameters from drugserum level were determined by Kinetica software version 5.

2.6.2. Biodistribution studyBiodistribution studies were carried out on tumor bearing

female Balb/c mice. Modified orthotopic model of lung tumorwas developed on mice as earlier reported (Garbuzenko et al.,2010). Briefly, A549 cells transfected with luciferase (5–8 � 106)were resuspended in 0.1 mL of RPMI medium containing 20%FBS, mixed with 5 lM EDTA and administered intratracheally tothe murine lung through a catheter. It was shown that a slight dis-ruption of the pulmonary epithelium or the surfactant layer by co-administration of EDTA allowed significantly better tumor engraft-ment. Four weeks after the instillation of tumor cells, mice wereused for biodistribution study. Biodistribution of the drug wasdetermined, with slight modifications as earlier reported (Yooand Park, 2004), to study the distribution of drug in different or-gans. Twenty-seven healthy Balb/c mice were selected and dividedinto three groups of nine mice each group for Free GEM, GEMloaded-CTS/PEG and CTS/PEG-AA NPs. Plain GEM, GEM loaded-CTS/PEG and CTS/PEG-AA NPs were administered through tail veinin a dose equivalent to 18 mg/kg body weight. Three animals fromeach group were sacrificed at 2, 12, and 24 h, and various organmasses such as hearts, livers, spleens and kidneys were collectedand washed with saline; and weighed prior to homogenization insaline. Tissue samples were cooled on ice after homogenizationprocedure; the homogenate was then centrifuged at 19,000 � gfor 12 min. Methanol and acetonitrile were added to the superna-tant (1:1) to precipitate unwanted proteins; samples were centri-fuged (19,000 � g, 10 min) as described above. The aliquots wereassayed for GEM levels using RP-HPLC to estimate the total amountof GEM was performed using RP-HPLC method according to themethod mentioned in Section 2.3.

2.6.3. In vivo anti-tumor activityBalb/c mice were provided with standard mouse food and water

ad libitum. A total of 2 � 106 exponentially growing A549 cellswere administered subcutaneously into each mouse at the upper

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portion of the right flank. After mice developed substantial solidtumors (average volume of �290 mm3), mice were divided intofour groups (3 mice for each group). Group I served as controland groups II, III and IV were injected with GEM, GEM loadedCTS/PEG, CTS/PEG-AA NPs respectively in a dose equivalent to18 mg/kg body weight on days 0, 2, 6, and 12 via the tail vein.The mice were monitored regularly for changes in tumor size.The sizes of tumor masses were measured with a caliper and tumorvolumes were calculated according to the formula: V = 0.5 � ab2,where a and b are the long and short diameter of the tumor,respectively.

2.7. Toxicity profile

2.7.1. Nephrotoxicity and hepatotoxicityThe serum samples collected for pharmacokinetic study were

also used to determine liver and kidney functioning. Simulta-neously, three mice were injected normal saline and consideredas control. Serum urea concentration was estimated by urease glu-tamate dehydrogenase method and creatinine by the modified Jaf-fe’s method using the diagnostic kits of Agappe Diagnostic, IndiaPvt. Ltd. (Ajith et al., 2008). Serum activities of alanine aminotrans-ferase (ALT), aspartate aminotransferase (AST) and alkaline phos-phatase (ALP) were evaluated using commercially available testkits from Randox Laboratories Ltd. (UK) (Injac et al., 2008).

2.8. Statistical analysis

The results were expressed as mean ± SD and the statisticalanalysis was done by one-way analysis of variance (ANOVA) withTukey–Kramer multiple comparison post-test using GraphPad In-Stat™ software (GraphPad Software Inc., San Diego, California).Statistical differences are denoted as ⁄p < 0.05, ⁄⁄p < 0.01 and ⁄⁄⁄-p < 0.001, respectively. NS = not significant (p > 0.05).

3. Results and discussion

3.1. Formulation and characterization of the nanoparticles

The CTS/PEG-AA NPs were prepared by two-step method(Fig. 1). In first step, anisamide was conjugated with PEG (overall reaction yield of PEG-AA was 65%) and secondly the CTS/PEG-AA NPs were formulated using ionic gelation method. In this meth-od, the electrostatic interaction between TPP (negatively charged)and CTS (positively-charged) is used as driving force for NPs for-mulation. The average diameter of CTS NPs was found to be160.2 ± 8.5 nm (Table 1). Results showed that the addition ofPEG-AA resulted in the increase in the average diameter of the par-ticles indicating the association of PEG-AA with the particles. It wasobserved that the addition of higher concentration of the PEG-AAresulted in higher particles diameter (data not shown). Accordingto the earlier studies the NPs with diameters larger than 200 nmare known to induce non-specific scavenging by monocytes andthe reticuloendothelial system (Gabizon et al., 1990; Na et al.,2003). Since, the average particles size with the formulation con-taining 10 mg/mL of PEG-AA was found to be 182.3 ± 14.6 nm; itwas therefore selected for further studies to ensure reduced toxic-ity and better tumor targetability. It is noteworthy that we couldobtain a homogeneous NP dispersion (Table 1) with smooth sur-face (Fig. 2).

In this study, zeta potential was determined as a function ofsurface characteristic of the developed formulations (Table 1).The zeta potential of CTS particles was found to be 38.3 ± 2.9 mV,whereas in the case of CTS/PEG and CTS/PEG-AA, it was found tobe 21.1 ± 1.8 mV and 24.1 ± 1.8 mV, respectively. The lower zeta

potential of CTS/PEG particle may be attributed to the stearic hin-drance created by PEG chains present on the surface of particlesobtained. It has been reported that PEG interacts with CTS to forma CTS/PEG semi-interpenetrating network through intermolecularhydrogen bonding between the electropositive amino hydrogenof CTS and electronegative oxygen atom of polyethers (Calvoet al., 1997). Polydispersity index (PDI) of NPs before and afterlyophilization was found to be <0.2. The lyophilized formulationdemonstrated more or less similar particle size upon reconstitution(Table 1).

The drug encapsulation efficiency of CTS/PEG-AA and CST/PEGwas found to be 30.2 ± 2.1% and 32.6 ± 2.3%, respectively. Thelow encapsulation efficiency observed with CTS/PEG and CTS/PEG-AA due to cationic nature of the drug (GEM) and CTS, whicheventually results into the electrostatic repulsive interaction be-tween GEM and CTS and thereby low association efficiency (Aryaet al., 2011).

3.2. In vitro release and ex vivo studies

3.2.1. Release studyThe total amount of the GEM released from the nanoparticles

over 10 days was nearly 79.11% (CTS/PEG) and 75.29% (CTS/PEG-AA) at pH 5.8, 52.56% (CTS/PEG) and 50.65% (CTS/PEG-AA) at pH7.4. After 15 days, the cumulated release amounts of GEM (CTS/PEG-AA NPs) release were 95.3 ± 3.9% and 75.4 ± 4.5% at pH 5.8and pH 7.4 respectively (Data not shown). This in vitro releasestudy indicates that CTS/PEG and CTS/PEG-AA particles exhibit abiphasic drug release profile (Fig. 3) typical of this polymer (Koet al., 2002; Wilson et al., 2010). This release behavior consistedof an initial fast (burst) drug release, the remaining being releasedin a sustained manner in PBS (pH 7.4 and pH 5.8). It was apparentthat GEM release rate from NPs at pH 5.8 was rapid compared tothat at pH 7.4 (Fig. 3). This faster release at lower pH (PBS, pH5.8) was related to the higher solubility of CTS at lower pH values,thus, allowing GEM to leak out at a faster rate. Hence, CTS/PEG-AAand CTS/PEG NPs could be expected to provide a pH-responsive re-lease profile for entrapped GEM in vivo and, therefore, might helpdelivering higher drug concentrations intracellularly as well as intotumor interstitium.

3.2.2. Cell uptake studyThe cell uptake of GEM is a critical step in ensuring the cyto-

toxic efficacy of the drug as it acts by incorporation of its active tri-phosphate form (50-triphosphate GEM) into DNA strand, halting itselongation and causing cell death (Huang and Plunkett, 1995.). Thecellular uptake of NPs was evaluated on A549 cancer cell lines(Figs. 4A and 4B). The uptake of FITC labeled CTS/PEG-AA wasfound substantially higher compared to FITC labeled CTS/PEG andplain FITC. Thus, higher cellular binding with eventual uptake ob-served with CTS/PEG-AA NPs is presumably due to their greaterintracellular delivery by receptor-mediated endocytosis (Barefordand Swaan, 2007). The cellular uptake of CTS/PEG-AA was signifi-cantly inhibited by pretreatment with haloperidol, a known sigmareceptor antagonist (Li and Huang 2006; Banerjee et al., 2004)(Fig. 4A). The cellular uptake of CTS/PEG-AA is comparable withthe cellular uptake of CTS/PEG. This suggests that the enhancedcellular uptake of CTS/PEG-AA NPs was specifically mediated bythe sigma receptors.

3.2.3. Cytotoxicity studyThe MTT assay was performed to investigate the comparative

cytotoxic response of plain GEM, GEM loaded CTS/PEG and CTS/PEG-AA NPs (Fig. 4C). The result indicates that CTS/PEG-AA NPswere most cytotoxic when compared with CTS/PEG NPs and plainGEM. This is possibly due to receptor–ligand interaction between

Fig. 1. Schematic representation of the synthesis of PEG-AA and formulation of CTS/PEG-AA NPs.

Table 1Table showing particle size, zeta potential and encapsulation efficiency of the NPs. Data represented as mean ± SD (n = 5).

Formulation Before lyophilization After lyophilization % Encapsulation efficiency

Diameter (nm) PDI Zeta potential (mV) Diameter (nm) PDI Zeta potential (mV)

CTS 160.2 ± 8.5 0.18 ± 0.01 38.3 ± 2.9 161.1 ± 9.3 0.17 ± 0.01 37.4 ± 3.9 –CTS/PEG 168.3 ± 16.6 0.16 ± 0.01 21.1 ± 1.8 169.1 ± 15.5 0.19 ± 0.02 21.2 ± 1.9 32.6 ± 2.3CTS/PEG-AA 182.3 ± 14.6 0.16 ± 0.01 24.1 ± 1.7 181.3 ± 10.6 0.16 ± 0.01 24.3 ± 1.7 30.2 ± 1.1

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sigma receptors (at the cell surface) and AA ligand (at the surface ofthe NPs), which resulted into better interaction between cell andCTS/PEG-AA NP and thereby efficient internalization ultimatelyresulting into higher cytotoxic response. The in vitro cell uptakestudies also support the toxicity data as presence of AA on CTS/PEG NPs significantly increase the cell uptake when compare toNPs (CTS/PEG) without AA. Therefore, it was concluded that AAplays a major role in the uptake of NPs, and the AA modifiedCTS/PEG NPs have a higher affinity to the A549 cells compared toCTS/PEG, leading to the increased uptake of the CTS/PEG-AA nano-particles and subsequently the higher cytotoxic responses.

3.3. In vivo study

3.3.1. Pharmacokinetic studyThe in vivo study further surfaces the importance of NPs be-

cause of alteration in pharmacokinetic and bio-distribution GEMwhen delivered through this nano-carrier. The naked/free GEMsolution was rapidly cleared from blood. However, GEM deliveredthrough NPs was retained for significant period of time than that offree GEM solution and was augmented to 10–12 h for CTS/PEG andCTS/PEG-AA (Fig. 5A). This sustained release indicates markedretainability and obviates the prolonged circulation and stealth

Fig. 2. Surface morphology of CTS/PEG-AA NPs. (A) TEM; (B) SEM.

Fig. 3. In vitro release study. Graph showing % cumulative drug release with respect to time in PBS (pH 7.4) and PBS (pH 5.8). The data represent the mean ± SD (n = 5).

Fig. 4A. Ex vivo studies. Graph showing cell % Fluorescence in cells followingincubation with CTS/PEG and CTS/PEG-AA NPs at various time intervals. The datarepresent the mean ± SD (n = 6).

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characteristics of CTS/PEG and CTS/PEG-AA NPs. In particular,in vivo experiments evidenced that the half-life (t1/2) of free GEMwas about 0.5 ± 0.06 h, while that t1/2 of GEM shielded from CTS/PEG and CTS/PEG-AA was higher 2.09 ± 0.16 h and 2.59 ± 0.19 hrespectively (Table 2). Free GEM was not detectable 3 h postadministration, while GEM shielded with CTS/PEG and CTS/PEG-AA NPs was found for up to 12 h. These findings attest the abilityof CTS/PEG and CTS/PEG-AA to prolong the half-life of this drug.

In addition other pharmacokinetic parameters, i.e. MRT (h), rateof elimination (Kel), AUMC0�t and the area under the curve(AUC0�t) (Table 2) singles out the usefulness of this carrier. Phar-macokinetic parameters confirmed encapsulation of gemcitabinein CTS/PEG and CTS/PEG-AA NPs. This drug confined and retainedin the systemic circulation eventually decreasing the amount ofthis antitumor agent that was removed from blood stream. Thedata obtained from pharmacokinetic studies reveals higher serumconcentration of CTS/PEG-AA time as a function as compared toCTS/PEG. The difference in serum drug concentration profile fol-lowing CTS/PEG and CTS/PEG-AA treatment may be associatedwith the difference in surface characteristics, selectivity andslightly different shielding effect. Therefore, in vivo biodistributionstudy was performed to establish NPs fate in the body.

3.3.2. Biodistribution studyThe sustainability and increased residence time of drug in dif-

ferent organs was determined by employing CTS/PEG and CTS/PEG-AA as carriers. The drug (GEM) distribution was investigatedby efficacious anisamylated CTS/PED NPs to target tumor tissuesas well as bypass non-tumor tissues (spleen, kidney, heart and li-ver). The concentration of free GEM was found to be greatest inkidney where up to 25.68 ± 2.32% drug was localized after 2 h(Fig. 5B). Almost negligible free GEM was detectable in kidneyand tumors at the end of 24 h. Further, in heart and liver,1.03 ± 0.06% and 2.76 ± 0.29% of free GEM was detected respec-tively, 24 h post administration. Conversely, after 24 h of adminis-tration, the concentrations of GEM loaded in CTS/PEG were

Fig. 4B. Ex vivo studies. Fluorescent microscopy images showing the uptake of dye (FITC) loaded NPs in A549 cell line. 1, 2, 3 for free FITC; 4, 5, 6 for CTS/PEG NPs; 7, 8, 9 forCTS/PEG-AA NPs.

Fig. 4C. Ex vivo studies. Graph showing toxicity of different concentration of plainGEM, CTS/PEG and CTS/PEG-AA NPs on A549 cell line. The data represent themean ± SD (n = 6). Asterisk over bars indicated degree of significance.

Fig. 5A. In vivo studies. Pharmacokinetic study: Graph showing serum concentra-tion of GEM attained at various intervals. The data represent the mean ± SD (n = 3).

Table 2Data represented as mean ± SD (n = 3).

Parameters Free GEM CTS/PEG CTS/PEG-AA

Pharmacokinetic parameters in serum of Balb/c mice timeCmax (lg/ml) 30.03 ± 2.63 17.55 ± 1.13 18.55 ± 2.63Kel 1.31 ± 0.15 0.33 ± 0.04 0.27 ± 0.02AUC0�t (lg h/ml) 16.79 ± 1.45 62.24 ± 0.19 93.44 ± 2.63AUMC0�t (lg h2/ml) 18.37 ± 1.73 229.878 ± 8.19 379.99 ± 7.63T1/2 (h) 0.5 ± 0.06 2.09 ± 0.163 2.59 ± 0.19MRT (h) 1.09 ± 0.13 3.69 ± 0.23 4.07 ± 0.39

Fig. 5B. In vivo studies. Biodistribution study: Graph showing biodistribution ofFree GEM, CTS/PEG and CTS/PEG-AA in different tissues at various time intervals.The data represent the mean ± SD (n = 3).

1012 N.K. Garg et al. / European Journal of Pharmaceutical Sciences 47 (2012) 1006–1014

monitored to be 2.67 ± 0.26%, 3.37 ± 0.23%, 0.32 ± 0.0.03% and4.67 ± 0.44% in liver, kidney, heart and tumor respectively. In caseof CTS/PEG-AA, 15.96 ± 1.11% of GEM was found in tumor tissues,5.57 ± 0.51% in liver and no GEM in kidney and heart after 24 h.

The GEM content was found higher in kidney and, being theprincipal organ of drug clearance, may be accredited to kidney.However, the free drug concentration was observed to decline sig-nificantly as compared to GEM that loaded in CTS/PEG and CTS/PEG-AA. In addition, sizeable decrease in the delivery of GEM to

Fig. 5C. In vivo studies. In vivo antitumor activity: Graph showing antitumoractivity of GEM, CTS/PEG and CTS/PEG-AA NPs on 0, 3,7,11 Day. The data representthe mean ± SD (n = 3). Where [⁄⁄⁄p < 0.001].

N.K. Garg et al. / European Journal of Pharmaceutical Sciences 47 (2012) 1006–1014 1013

heart indicating efficiency of CTS/PEG-AA and, in turn, reduced car-diotoxicity due to gemcitabine therapy. Further, preferential local-ization of CTS/PEG-AA in tumors led reduced entry of drug in non-targeted organs. As a consequence of which augmented level ofGEM when delivered through anisamylated CTS/PEG in contrastto plain drug was observed in tumor tissues. Results indicated thatCTS/PEG-AA NPs resulted into the accumulation of significantlyhigher amount of drug in tumor as compared to other non-targetedorgans (Fig. 5B). On the contrary, unanchored CTS/PEG resultedinto normal distribution of drug amongst various organs. As previ-ously reported, several factors, such as particle size, polymer com-position, molecular weight and surface characteristic of NPsdetermine the particle distribution in the body (Bareford andSwaan, 2007; Gref et al., 1994; Storm et al., 1995; Gao et al.,2004; Cho et al., 2007). However, we report the similar particlesizes and zeta potentials of CTS/PEG NPs and CTS/PEG-AA NPs(Table 1). The over expression of sigma receptors for AA on cellularmembrane of cancer cells (Walker et al., 1990) has been reported.We also believe AA plays a key role in selective localization of theCTS/PEG-AA NPs in the lung cancer cells. The CTS/PEG NPs modi-fied with AA could be recognized by the sigma receptors and even-tually transferred into lung cancerous cells via receptor-mediatedendocytosis. The later enhances their ability to target lung cancercells. On the contrary, unmodified CTS/PEG NPs accumulatedsparely in tumor when compared to CTS/PEG-AA NPs (Tian et al.,2010). The residual CTS/PEG NPs in various organs was due tonon-specific binding and uptake by cells of reticulo-endothelialsystem (RES) (Banerjee et al., 2002).

3.3.3. In vivo anti-tumor activityThe antitumor activity of plain GEM, GEM loaded CTS/PEG-AA

and CTS/PEG NPs was assessed against A549 subcutaneous tumorin mice (Fig 5C). It was observed that plain GEM exhibited limitedantitumor activity. This may be attributed to rapid deamination bydeoxycytidine deaminases into inactive uracil derivative (Abbruzz-ese et al., 1991), thus, leading to a dramatic decrease in the thera-peutic activity. Also, being hydrophilic in nature, GEM is actively

Table 3Table showing the toxicity profile of GEM, CTS/PEG and CTS/PEG-AA. Data represented as

S.no. Formulation Kidneya

Urea (mM/L) Creatinine (mM/

1 Control 7.02 ± 0.56 40.87 ± 3.172 GEM 13.54 ± 0.98 75.213 ± 5.843 CTS-PEG 8.52 ± 0.65 48.53 ± 3.344 CTS-PEG-AA 9.01 ± 0.73 50.25 ± 5.32

a Serum from mice treated with NS was considered as control.

transported into the cells through membrane nucleoside transport-ers, which is a saturable process (Mackey et al., 1991). Comparableto plain GEM, the improved anticancer activity of CTS/PEG-AA andCTS/PEG NPs was significantly higher (p < 0.001) at all time pointfrom second day. It is expected that incorporation of GEM intothe NPs protects the drug from being metabolized in the systemiccirculation, while sustained release from NPs matrix may have im-proved the anticancer activity as well. It was also observed thatCTS/PEG-AA exhibited significantly higher antitumor activity whencompared to CTS/PEG from day 12 to all time point. This may be acharacteristic feature of selective accumulation (Fig. 5C) of CTS/PEG-AA NPs in tumor followed by receptor-mediated endocytosisconsequently leading enhanced/improved antitumor activity ascompared to CTS/PEG.

3.4. Toxicity profile

3.4.1. Nephrotoxicity and hepatotoxicitySerum levels of urea/creatinine are used as indicators of renal

function and ALT/AST/ALP are markers of liver function (Table 3).This study indicates that GEM loaded CTS/PEG-AA and CTS/PEGresulted into minimal alteration in serum kidney (creatinine andurea) and liver (AST, ALT and ALP) function parameters, when com-pared to plain GEM. This indicates that the free GEM was presentand exposed to liver and kidney cells all at once without any shieldto non-target cells thereby leading to cytotoxic actions. Whereas,CTS/PEG-AA and CTS/PEG NPs due to its sustained release behaviorand shielding effect could significantly reduce hepato and nephro-toxicity. It is noteworthy, that the kidney and liver function param-eters were slightly higher (p > 0.05) in case of CTS/PEG-AA, whencompared to CTS-PEG. This may be attributed to the presence ofsigma receptor present in liver and kidney (Banerjee et al., 2004).

4. Conclusions and perspectives

A lung-targeted drug delivery NPs (CTS/PEG-AA) composed ofPEG-AA and CTS could be prepared conveniently by the ionic gela-tion process. The results of the study indicate targeting prospec-tive, spatial delivery, amplified bioavailab1ility and elevatedretention potential of the formulation in tumor tissues. The pres-ence of sigma receptors presents an exclusive platform and accord-ingly manifold replica of ligand can be used to facilitate targeting.These NPs accumulated particularly in the tumor, and maintainedat a high level. This is remarkably higher than that of the NPs with-out the AA. Moreover, the in vitro cell uptake results showed thatthe introduction of AA to the CTS/PEGNPs could significantly in-crease the affinity of particles and the content therein to humanlung carcinoma cells. In addition, the GEM-loaded CTS/PEG-AANPs showed remarkable cytotoxicity towards A549 cells(in vitro), and could effectively inhibit tumor growth in A549cell-bearing mice. Finally the present study reveals the prospectiveof anisamylated CTS/PEG NPs as efficient vectors to ferry largedoses of anti-cancer drug. In vitro studies depict the sustained re-lease nature of formulation. The developed CTS/PEG-AA nanopar-

mean ± SD (n = 3).

Livera

L) ALP (Units/L) ALT (Units/L) AST (Units/L)

53.31 ± 4.21 13.08 ± 0.96 11.93 ± 0.9271.65 ± 5.93 35.62 ± 2.68 31.51 ± 2.5356.52 ± 3.64 16.76 ± 1.13 13.75 ± 1.1058.53 ± 5.43 18.63 ± 1.57 15.75 ± 1.79

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ticulate system demonstrated minimal toxicity in the tested area.This indicates that AA facilitates targeted delivery of anti-cancerdrug to tumor sites, with reduced access to non-tumor tissues.Thus optimal therapeutic response, improved therapeutic efficacymay be attained with the interception of minimal side effects.

Acknowledgment

Authors are thankful to All India Council of Technical Education(AICTE, New Delhi) for providing Research Fellowship to carry outthe research work.

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