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ORIGINAL PAPER

Gemcitabine-loaded PLGA-PEG immunonanoparticlesfor targeted chemotherapy of pancreatic cancer

Sahil Aggarwal & Swati Gupta & Dilrose Pabla &R. S. R. Murthy

Received: 23 April 2013 /Revised: 8 July 2013 /Accepted: 5 September 2013 /Published online: 24 September 2013# Springer-Verlag Wien 2013

Abstract The aim of the present study was the directcovalent coupling of the epidermal growth factor receptor(EGFR)-specific monoclonal antibody (mAb) to the surfaceof poly(lactide)-co-glycolide (PLGA)-polyethylene glycol(PEG) nanoparticles in order to achieve a cell type-specificdrug carrier system against pancreatic cancer. The PLGA-PEG-NH2 diblock copolymer was synthesized by couplingreaction via amide linkage between PEG-diamine and activatedPLGA. PLGA and PLGA-PEG-NH2 nanoparticles loaded withgemcitabine were prepared using the double-emulsion solventevaporation method. PLGA-PEG immunonanoparticles wereprepared by glutaraldehyde mediated cross-linking method.The conjugated antibody was analysed by transmission elec-tron microscopy and sodium dodecyl sulfate-polyacrylamidegel electrophoresis (PAGE) analysis. Cell viability study wasperformed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bromide assay and cell uptake study was performedon fluorescein isothiocyanate-loaded formulations using con-focal microscopy. The PAGE results indicated that mAb integ-rity was remained intact in the formulations after conjugation.Biological activity was confirmed under cell culture conditions:antibody-conjugated nanoparticles showed specific targeting toEGFR-overexpressing MIA PaCa-2 cell lines as shown influorescence image using confocal microscopy. The obtaineddata provide the basis for the development of stable and bio-logically active carrier systems for direct targeting of tumourcells using antibody-conjugated PLGA-PEG nanoparticles.Direct covalent coupling of antibodies to nanoparticles usingglutaraldehyde as a cross-linker is an appropriate methodto achieve cell type-specific drug carrier systems based onPLGA-PEG nanoparticles and the anti-EGFR-decorated

PLGA-PEG nanoparticles have potentials to be applied fortargeted chemotherapy against EGFR positive cancers.

Keywords Adenocarcinoma . Epidermal growth factorreceptor . Targeting . Gemcitabine . Multifunctionalnanoparticles . MIA PaCa-2

1 Introduction

Adenocarcinoma of the exocrine pancreas is the fourth leadingcause of cancer deaths in the USA (Berger et al. 2004).Currently, surgery is the only treatment although, due to itslate presentation, only 9–15 % of the patients are suitable forsurgery. The median survival for all stages of pancreaticcancer is 3–5 months from diagnosis (Shore et al. 2004).The underlying problem in the use of anticancer drugs is theirtoxicity and poor bioavailability (Langer 1998). It is expectedthat if a delivery system-bearing anticancer drugs can bedelivered in a targeted fashion, inhibition of tumour growthwith reduced systemic toxicity will occur. Therefore, newtherapeutic strategies are necessary to combat this deadlydisease. Antibodies are well-established systems to targetdrugs or colloidal carriers to specific cell types. This principleis based on a defined receptor ligand interaction which enablesthe surface binding and subsequent cellular internalisation ofdrugs or drug carriers conjugated to the antibody. Chemicalmodification of the antibody is necessary to enable conjuga-tion with a drug carrier, but at the same time it is a prerequisitethat while performing conjugation reactions, biological activ-ity and full receptor binding of the antibody is maintained.Epidermal growth factor receptor (EGFR) monoclonal anti-body is directed against the extracellular domain of the recep-tor. EGFR belongs to a receptor tyrosine-specific proteinkinase family, the EGFR family, which consists of four EGFreceptors, EGFR (ErbB1), ErbB2 (Neu), ErbB3, and ErbB4.Members of the EGFR family contain a cytoplasmic tyrosinekinase domain, a single transmembrane domain, and anextracellular domain that is involved in ligand binding

S. Aggarwal : S. Gupta (*) :D. Pabla : R. S. R. MurthyNanomedicine Research Centre, Department of Pharmaceutics,I.S.F. College of Pharmacy, Moga 142-001, Punjab, Indiae-mail: [email protected]

S. GuptaSchool of Pharmaceutical Sciences, Apeejay Stya University,Sohna-Palwal Road, Sohna- 122103, Gurgaon (Haryana), India

Cancer Nano (2013) 4:145–157DOI 10.1007/s12645-013-0046-3

and receptor dimerisation (Ferguson et al. 2003; Olayioye et al.2000). Overexpression of EGFR at the cell surface may lead toa spontaneous formation of homodimers, but more likely theavailability of the receptor for ligand-driven heterodimerisationincreases (Jorissen et al. 2003). EGFR overexpression trans-lates into signals that potentiate dysregulated growth, oncogen-esis, metastasis, and possibly resistance against chemothera-peutic agents. EGFR is amplified at 50–70 % incidence inhuman pancreatic cancer (Dancer et al. 2007). The EGFRmonoclonal antibody (mAb) acts in a cytostatic mannerby blocking and downmodulation of the EGFR receptorand by antibody-dependent cellular cytotoxicity as majormechanism of antibody action (Ennis et al. 1991; Xiong andAbbruzzese 2002). It induces EGFR internalisation and deg-radation. EGFR mAb offers an excellent strategy for drugtargeting to cancer cells because EGF is an easily accessiblecell surface receptor overexpressed on the primary tumouras well as on metastatic sites (Bloomston et al. 2006).The internalisation ability of EGFR allows an efficient uptakeof the antibody alone as well as conjugated to drugs or drugcarrier systems.

Colloidal systems such as nanoparticles provide higherdrug-carrying capacities than antibodies and, therefore, areespecially suited for conjugation to antibodies. Poly(lactide)-co-glycolide (PLGA)-based nanoparticles represent a colloi-dal drug carrier systemwith high drug-loading capacity. Theseparticles are biodegradable, non-antigenic, non-irritative fortissues, and nontoxic (Mundargi et al. 2008). Drugs can beincorporated within the particle matrix, adsorbed on theparticle surface or bound by covalent linkage. Especiallyincorporation in the particle matrix protects the drug againstdegradation and enables controlled release.

The aim of the present study was the direct covalentcoupling of the EGFR mAb to the surface of PLGA-polyethylene glycol (PEG) nanoparticles in order to achievea cell type-specific drug carrier system against pancreaticcancer.

2 Materials and methods

Gemcitabine hydrochloride was generously gifted by SunPharma (SUPAC, Hyderabad). Poly (D,L -lactic-co-glycolicacid) 50:50, polyethylene glycol bis amine (PEG diamine),anti-EGFR mAb were purchased from Sigma ChemicalLaboratories Pvt. Ltd. Mumbai. All other chemicals werepurchased from HiMedia, Bombay and CDH LaboratoryReagents, New Delhi and were of analytical grade.

2.1 Cell line

The cell line, MIA PaCa-2 cell line (human pancreaticcarcinoma) was obtained from National Centre for Cell

Sciences, Pune, India. Cell line was cultured inDulbecco’s modified Eagle’s medium supplemented with10 % (v /v ) heat-inactivated foetal bovine serum and horseserum (2.5 %). Cells were maintained in 5 % CO2-humid-ified incubator at 37 °C. During subculture, cells weredetached by trypsinization when they reached 80 %confluency and split (1:4). Growth medium was changedevery 3 days.

2.2 Preparation of PLGA-PEG-NH2 diblock copolymer

The PLGA-PEG diblock copolymer was synthesized bycoupling reaction via amide linkage between PEG di-amine and activated PLGA (Esmaeili et al. 2008), withminor modifications. The carboxyl terminal end of thePLGA was first activated with N -hydroxysuccinimide(NHS) by using dicyclohexylcarbodiimide (DCC).Briefly, the PLGA (100 mg) was dissolved in DCM(5 ml); and then DCC (5 mg) and NHS (2.5 mg) wereadded under magnetic stirring. The activation reaction ofcarboxylic acid end group was accomplished for 12 h atroom temperature. Insoluble dicyclohexyl urea was fil-tered and the resultant solution was precipitated bydropping into ice-cold anhydrous diethyl ether. The acti-vated PLGA was completely dried under vacuum. TheNHS ester of the PLGA was conjugated with PEG di-amine via amide linkage. The activated PLGA (100 mg)was dissolved in DCM (5 ml) and excess of PEG di-amine (35 mg) was added in to this solution. The cou-pling reactionwas performed under magnetic stirring for 9 h atroom temperature. An excess amount of PEG diamine wasused to prevent the formation of PLGA-PEG-PLGA triblockcopolymers. The amine-terminated PEG-PLGA diblock co-polymer was precipitated with cold methanol and purifiedwith excess of ethanol (to eliminate unconjugated PEG di-amine) and finally dried under vacuum.

2.3 Preparation of nanoparticles

PLGA and PLGA-PEG-NH2 NPs loaded with gemcitabinewere prepared using the double emulsion solventevaporation method (Avgoustakis et al. 2002). PLGApolymer (40 mg) or PLGA-PEG-NH2 copolymer (40 mg)was dissolved in 4 ml DCM. An aqueous solution ofgemcitabine (V =1 ml, C =5 mg/ml) was emulsified in solu-tion while vortexing for 20 s and further sonicated using probesonicator (ultrasonic liquid processor, Misonix, USA) at anamplitude of 40W for 1 min to form primary water (w)/oil (o)emulsion. The emulsion was transferred dropwise to an aque-ous solution of sodium cholate (V =20 ml, 12 mM) and themixture was probe sonicated at an amplitude of 60 W for3 min to form w/o/w emulsion. The emulsion formed wasgently stirred at room temperature until the evaporation of the

146 S. Aggarwal et al.

organic phase was completed. The gemcitabine loaded PLGANPs (GEM-PLGA NPs) and GEM-PLGA-PEG-NH2 NPswere purified by applying two cycles of centrifugation(22,000 rpm for 20 min) using refrigerated centrifuge(Laborzentrifugen, Sigma 3K30) and reconstituted with de-ionized and distilled water. Blank NPs were also prepared bythe same method without adding gemcitabine at any stage ofthe preparation.

2.4 Characterization of nanoparticles

Characterization of NPs was done on the basis of size, zetapotential and entrapment efficiency. The method used forpreparation of NPs was optimized on the basis of change inamount of polymer, concentration of drug and surfactant(Govender et al. 1999; Acharya et al. 2009).

2.4.1 Optimization of process parameters

Optimization of concentration of drug NPs were optimizedfor different concentration of gemcitabine (5, 7, and 10mg/ml)with respect to particle size and entrapment efficiency.

Optimization of amount of polymer NPs were preparedvarying the amount of polymer using 20, 40, and 60 mgof PLGA while keeping the concentration of drug andsurfactant constant. The formulations were optimized onthe basis of size and entrapment efficiency.

Optimization of concentration of surfactant Different for-mulations were prepared using different concentrations ofsurfactants (8, 10, and 12 mM) while keeping concentrationof drug and polymer constant. The formulations were eval-uated for particle size and entrapment efficiency.

2.4.2 Size and zeta potential

The average particle size and zeta potential of optimizedformulations were determined by photon correlation spectros-copy using Zetasizer (Beckman Coulter, UK). The sampleswere kept into the cuvettes of zetasizer and the size, polydis-persity index (PDI), and zeta potential were measured.

2.4.3 Entrapment efficiency

The method adopted for estimating the entrapment efficiencywas a slight modification to the extraction method followed(Blanco and Alonso 1997). The amount of gemcitabineentrapped in NPs was estimated by adding 5 mg of NPs in1 ml of acetonitrile and shaking NPs vigorously in order tosolubilise the polymer so as to extract out the drug followed byaddition of 4 ml of PBS 7.4 to solubilise the drug. The amountof gemcitabine was measured by UV spectrophotometer

(Shimadzu, Japan) using a detection wavelength of 270 nm.The percentage drug entrapment was measured as:

% DEE ¼ amount of drug entrappedamount of drug added

� 100

2.4.4 Scanning electron microscopy

Scanning electron microscopy (SEM) was used to examinethe shape and morphology of the NPs (Govender et al. 1999;Acharya et al. 2009). The NPs were placed on sample holder,coated with gold-palladium alloy (150–250 Å) using a sputtercoater and then placed in scanning electron microscope.

2.4.5 In vitro drug release

The in vitro drug release profile of entrapped drug from NPswas studied using dialysis membrane. Each dialysis bag(pore size, 12 kDa; Sigma Chemical Co., USA) was loadedwith lyophilized preparation equivalent to 2.5 mg of drug. Thedialysis bag was then placed in 50 ml PBS (pH 7.4), contain-ing 1 % tween 80, at 37±1 °C and under 100 rpm stirring.Samples (5 ml) were withdrawn at predetermined time inter-val and replaced with the same volume of fresh dialyzingmedium and the withdrawn samples were assayed for drugcontent by measuring absorbance at 270 nm against the blankusing UV spectrophotometer (Li et al. 2001).

2.5 Preparation of PLGA-PEG immunonanoparticles

The PLGA-PEG immunonanoparticles (Ab-PLGA NPs)were prepared by two methods.

1. Glutaraldehyde mediated cross-linking.The Ab-PLGA NPs were prepared with slight mod-

ifications in reported method (Hun and Zhang 2009).PLGA-PEG-NH2 NPs were prepared using optimizedformulation of PLGA NPs. NPs (4 mg) were dispersedin PBS 7.4 containing glutaraldehyde (25 %) up to afinal concentration of 1.25 %, for about 2 h. The excessof glutaraldehyde was separated from NPs using dialy-sis membrane (12 kDa). The presence of free aldehydicgroup of glutaraldehyde was detected by addition ofSchiff reagent (25 μL of Schiff reagent was added to250 μL of NPs formulation). Glutaraldehyde-activatedNPs were further incubated with anti-EGFR antibody(1 mg/ml) for 12 h at 4 °C with continuous shaking.The anti-EGFR antibody conjugated NPs (glutaralde-hyde Ab-PLGA NPs) were centrifuged at 22,000 rpmat 4 °C for 20 min to remove excess antibody and keptat 4°C in PBS 7.4.

2. MBS cross-linkingThe antibody conjugation was also performed by

thiolation of mAb with slight modifications in reported

PLGA-PEG immunonanoparticles against pancreatic cancer 147

method (Steinhauser et al. 2006). The PLGA-PEG-NH2NPs were dispersed in 1 ml PBS (pH 7.2) and the cross-linker m -maleimidobenzoyl-N -hydroxysuccinimide(MBS; 3.14 mg) was dissolved in 1 ml of dimethylsulfoxide (DMSO). MBS solution (100 μl) wasadded in 1 ml NPs dispersion and incubated for 30 minat 25 °C in shaker incubator. The excess of MBS wasseparated from activated NPs using sephadex G25. Forthiolation of anti-EGFR antibody, 5.7 mg of 2-iminothiolane (Traut’s reagent) was dissolved in 5 mlPBS (pH 7.4) and 100 μl anti-HER-2 mAb was incubatedwith 4.2 μl of 2-iminothiolane for 2 h at 20 °Cunder constant shaking. Finally, 100 μl thiolated mAbwas added in 100 μl MBS-activated NPs for 30 min at25 °C under constant shaking for preparation of PLGA-PEG immunonanoparticles.

2.6 Characterization of Ab-PLGA NPs

2.6.1 Transmission electron microscopy

Transmission electron microscopy (TEM) was done forboth the prepared Ab-PLGA NP conjugates (Acharyaet al. 2009). Dispersions were visualized under TEM withan accelerating voltage of 100 kV. A drop of the samplewas placed on to a carbon-coated copper grid to leave athin film on the grid. The film was negatively stained with1 % phosphotungastic acid solution and dried. A drop ofthe staining solution was added on to the film and theexcess of the solution was drained off. The grid wasallowed to dry at ambient temperature and samples wereviewed under transmission electron microscope (Hitachi,Japan) and photographs were taken at suitable magnification.


The size, PDI, and zeta potential of optimized formula-tions were determined by photon correlation spectroscopymethod using zetasizer (Beckman Coulter). The sampleswere loaded into the cuvettes of zetasizer and the size,PDI, and zeta potential were measured (Acharya et al.2009).


The amount of gemcitabine entrapped in NPs was esti-mated by adding 5 mg of NPs in 1 ml of acetonitrile andshaking NPs vigorously in order to solubilise the polymerso as to extract out the drug followed by addition of 4 mlof PBS 7.4 to solubilise the drug. The amount ofgemcitabine was measured by UV spectrophotometerusing a detection wavelength of 270 nm (Blanco andAlonso 1997).

2.6.4 Sodium dodecyl sulfate-polyacrylamide gelelectrophoresis

Sodium dodecyl sulphate (SDS) gel electrophoresis iswidely used for the analysis and characterization of proteinsamples. It is useful in molecular weight determination,relative estimation of purity, and amino acid sequencingof proteins. In the present study, conjugation efficiencieswere evaluated by SDS-polyacrylamide gel electrophoresis(PAGE), allowing comparison of free mAb fragment vs.conjugated NPs (Flatman et al. 2007). SDS-PAGE was donefor both the prepared conjugated NPs.

2.6.5 Optimized Ab-PLGA NPs

The method for the preparation of immunonanoparticleswas optimized on the basis of morphology (TEM), size,zeta potential, and entrapment efficiency. The optimizedformulation was used for cell line studies.

2.7 In vitro evaluation

In vitro evaluation of prepared formulations was carried outon cell lines using MIA PaCa-2 cell line (human pancreaticcarcinoma). The studies included cell viability assay (using3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide (MTT) dye) and cellular uptake studies (using confocalmicroscopy).

2.7.1 Cell viability assay

Cell viability was tested using MTT assay (Wang et al.1996; Acharya et al. 2009) based on the cleavage ofyellow tetrazolium salt MTT by metabolically active cellsto form an orange formazan dye which was quantifiedusing ELISA reader (Biorad, USA, model 680). Cellswere seeded in 96-well microtitre plates (2×104 cells/200 μL growth medium/well) followed by overnightincubation. Supernatants from the wells were aspiratedout and fresh aliquots of growth medium (containingNPs, i.e., GEM-PLGA NPs, blank PLGA NPs, GEM-Ab-PLGA NPs, and blank Ab-PLGA NPs) in desiredconcentrations in the range of 2–32 μg/mL were added.Blank formulations were used as control. After 24 h,supernatants were aspirated out and the cell monolayersin the wells were washed with 200 μL PBS (0.1 M, pH 7.4).Subsequently, MTT reagent (150 μL, 0.8 mg/mL) wasadded in each well, incubated for 5 h. DMSO (100 μL) wasadded in each well after aspirating out the supernatantand incubated at 37 °C for 1 h. Absorbance at twowavelengths (570 nm for soluble dye and 630 nm forcells) were recorded using ELISA reader. Concentrationsof samples showing 50 % reduction in cell viability (i.e.,


IC50 values) were then calculated. An optical density value ofcontrol cells (unexposed cells) was taken as 100 % viability(0 % cytotoxicity).

2.7.2 Cell uptake studies

Preparation of fluorescence marker loaded nanoparticles TheNPs encapsulating fluorescence marker (fluorescein iso-thiocyanate, FITC) was prepared by the double emulsi-fication solvent evaporation technique as described ear-lier. Polymer or copolymer (40 mg) was dissolved in 4 mldichloromethane. An aqueous solution of FITC (V =1 ml,C =1 mg/ml) was emulsified in solution while vortexing for20 s and further sonicated using probe sonication (ultrasonicliquid processor, Misonix, USA) at 10 W for 1 min toform primary w/o emulsion. The emulsion was trans-ferred dropwise to an aqueous solution of sodium cho-late (V =20 ml, 12 mM) and the mixture was probesonicated at 18 W for 3 min to form w/o/w emulsion.The emulsion formed was gently stirred at room tem-perature until the evaporation of the organic phase wascomplete. The NPs were purified by applying two cy-cles of centrifugation (22,000 rpm for 20 min) usingrefrigerated centrifuge (Laborzentrifugen, Sigma 3 K30)and reconstituted with deionized-distilled water. Theprepared NPs were further conjugated to anti-EGFRantibody using glutaraldehyde-mediated cross-linkingdescribed previously.

Procedure for cell uptake studies EGFR positive MIAPaCa-2 cells were seeded on glass cover slips (18×18 mm) placed into 35 mm tissue culture plates at a densityof 2×105 cells (in 2 mL growth medium). After overnightgrowth, supernatants from the culture plates were aspiratedout and fresh aliquots of growth medium containing suspen-sion of FITC-loaded PLGA NPs and FITC-loaded Ab-PLGANPs were added at the concentration of 0.25 mg/ml andincubated at 37 °C for 6 h. Upon incubation for 6 h, cellswere washed with PBS, fixed in 4 % formaldehyde for20 min, and then washed twice with PBS and observed underconfocal laser scanning microscope (CLSM) (Fluoview 1000,Olympus, USA) after excitation at 490 nm (Acharya et al.2009).

2.8 Statistical analysis

All experiments were carried out three times, independent-ly. The data obtained were expressed in terms of “mean±standard deviation” values. Wherever appropriate, the datawere also subjected to unpaired two-tailed Student’s t test.A value of p

the various parameters for optimized formulations of PLGANPs and PLGA-PEG NPs.

3.3 In vitro release studies

The in vitro release profile of entrapped drug from NPs wasdetermined in PBS (pH 7.4) using dialysis bag. The releasestudy of both PLGA and PLGA-PEG-NH2NPswas performedthree times to check the reproducibility. The release behavioursof both the formulations were comparatively different fromeach other. The release behaviour involved two different mech-anisms, one was the diffusion of drug and the other was thedegradation of polymer matrix. The burst release of drug wasdue to the drug which had been adsorbed at the surface ofpolymer, which diffused out easily during initial incubationtime (Asadishad et al. 2010). It was observed that 6.93±1.00%of drug was released within initial 0.5 h and 36.9±1.10 %was released in 48 h from PLGA NPs. In contrast to PLGANPs, PLGA-PEG-NH2 NPs showed release of 9.3±1.54 and47±1.80 % in 0.5 and 48 h, respectively. Figure 2 shows therelease profile of both GEM-PLGA and GEM-PLGA-PEG-NH2 NPs.

3.4 Characterization of Ab-PLGA NPs

3.4.1 Morphological study (TEM)

The conjugation of antibody to the surface of nanoparticlewas confirmed by TEM. TEM was performed for PLGANPs as well as Ab-PLGA NPs prepared by MBS-mediatedcross-linking and glutaraldehyde cross-linking. The imagesrevealed that conjugation was successfully done in both theconjugates but the conjugation with glutaraldehyde wasmore efficient than MBS crosslinked Ab-PLGA NPs, asmore antibodies were conjugated to the PLGA-PEG NPsfollowing glutaraldehyde conjugation. Figures 3 and 4a,b

shows the TEM images of PLGA nanoparticle, glutaralde-hyde, and MBS cross-linked Ab-PLGA NPs, respectively.


The size of prepared Ab-PLGA NPs should be in the suitablerange for intravenous administration, after conjugation of theantibody on the NPs. Particle charge determines the stabilityof NPs. The particle size, size distribution, and zeta potentialof the glutaraldehyde mediated Ab-PLGA NPs are shown inFig. 5a,b, respectively. Figure 6a,b shows the size, size distri-bution, and zeta potential of MBS cross-linked Ab-PLGANPs.


The entrapment efficiency of gemcitabine-loaded Ab-PLGA NPs was estimated and repeated three times. Theformulation showed good reproducible results. Table 5

Table 3 Optimization of concentration of surfactant (n =3)

Code Concentration(mM)

Size (nm) Entrapmentefficiency (%)

S1 8 243±7.5 22±1

S2 12 178±10 33.6±1.2

S3 16 204±9.5 30.6±1.4

Fig. 1 a SEM photograph of PLGA NPs. b SEM photograph ofPLGA-PEG NPs

Table 2 Optimization of amount of polymer (n=3)

Code Amount (mg) Size (nm) Entrapment efficiency (%)

P1 20 206.6±9.5 20.5±1.4

P2 40 178±8.5 34.9±0.89

P3 60 392±7 34±0.94

Table 4 Various parameters of optimized formulation of PLGA andPLGA-PEG-NH2 NPs

S. No. Parameters PLGAnanoparticles

PLGA-PEG-NH2nanoparticles

1 Size (nm) 178±8.5 132.5±10

2 Polydispersity index (PDI) 0.095±0.04 0.072±0.03

3 Zeta potential (mV) −59.88±3.5 −38.43±2.34 % Drug entrapment

efficiency35.16±1.04 34.56±1.35


shows the entrapment efficiency of Ab-PLGA NPs alongwith other characterization parameters.

3.4.4 SDS-PAGE

The integrity of anti-EGFR antibody after conjugation on theNPs surface was analyzed by SDS-PAGE in comparison withthe native anti-EGFR antibody (mAb). SDS-PAGE was runfor both the conjugated NPs that is MBS crosslinked Ab-PLGA NPs and glutaraldehyde crosslinked Ab-PLGA NPs.Figure 7 shows SDS-PAGE of glutaraldehyde cross-linkedand MBS cross-linked Ab-PLGA NPs. Lane 1 shows theSDS-PAGE for MBS crosslinked NPs, lane 2 representsPAGE for glutaraldehyde crosslinked NPs, and lane 3 showsPAGE run of standard anti-EGFR mAb. The PAGE resultsindicate that mAb integrity remained intact in both the sam-ples after conjugation.

Fig. 2 In vitro release of PLGAand PLGA-PEG-NH2nanoparticles

Fig. 3 TEM image of PLGA NPsFig. 4 a TEM image of Glu-Ab-PLGA NPs. b TEM image of MBS-Ab-PLGA NPs


Fig. 5 a The size and size distribution. b Zeta potential of Glu-GEM-Ab-PLGA NPs


Fig. 6 a The size and size distribution. b Zeta potential of MBS cross-linked Ab-PLGA NPs


3.4.5 Optimization of conjugation method

On the basis of morphology, size, and zeta potential, theconjugation method of antibody to the NPs was selected andused for further experimentation. Out of both the prepared Ab-PLGA NPs, glutaraldehyde cross-linked immunonanoparticlewere found to be optimum and used further for cell line studies.

3.5 In vitro evaluation

3.5.1 Cell viability assay

The cell viability assay was performed on MIA PaCa-2 celllines. IC50 values were found to be 16 and 31 μg/ml for drug-loaded PLGA NPs and drug-loaded Ab-PLGA NPs, respec-tively. Plain drug showed an IC50 of 15 μg/ml (Fig. 8).

3.5.2 Cell uptake studies

NPs encapsulated with fluorescent dye were used to studycellular uptake. FITC, fluorescent dye was incorporated inNPs as a marker for fluorescence microscopy to study the

NPs uptake in cells. The fluorescence image shows the uptakeof NPs in the cells. CLSM images of the MIA PaCa-2 cellsafter incubation of FITC loaded NPs and Ab-PLGA NPssuspension for 6 h at 37 °C are shown in Fig. 9.

4 Discussion

The prime objective of the study was to prepare and eval-uate Ab-PLGA NPs derived from PLGA-PEG diblock co-polymer. Both the polymer and copolymer were biocom-patible and biodegradable biomaterial. The PLGA-PEGdiblock copolymer was synthesized by coupling reactionbetween primary amine group of PEG diamine and terminalcarboxylic end group of PLGAvia amide linkage. The w/o/wdouble emulsification solvent evaporation method was usedfor encapsulating hydrophilic drug, gemcitabine. The methodis reported to be the most suitable for encapsulating water-soluble drugs in polymeric carriers. An important step in thepreparation of NPs was the formation of a stable homogenousprimary emulsion, as the droplet size of dispersed phase inprimary emulsion is directly related to the final NPs size. Byapplying sonication, which induced ultrasonic waves, emul-sion droplets smaller than 0.5 μm were obtained leading toNPs of the nanosize. Several process parameters were variedto identify their influence on the particle size and entrapment

Table 5 Characteriza-tion parameters of Ab-PLGA NPs

Nanoparticle Size (nm) PDI Zeta potential(mV)

%Drug entrapmentefficiency

Glutaraldehyde-mediated Ab-PLGA NPs 146.5 0.167 −32.65 31.43±1.58MBS mediated-Ab-PLGA NPs 446.5 0.216 −10.13 29.58±2.14

Fig. 7 SDS-PAGE of antibody-conjugated PLGA nanoparticles. 1The SDS-PAGE for MBS cross-linked NPs, 2 PAGE for glutaralde-hyde cross-linked NPs, and 3 PAGE run of standard anti-EGFR mAb

Fig. 8 Percent viability measured by MTT assay on human pancreaticcells. An optical density value of unexposed cells, PLGA NPs, and AbPLGA-PEG NPs was taken as 100 % viability (0 % cytotoxicity) fordrug, drug-loaded PLGA, and drug-loaded Ab PLGA-PEG NPs, respec-tively. *p

efficiency keeping the other parameters constant (primarysonication amplitude (40 W) for 2 min and secondary sonica-tion amplitude (60 W) for 4 min; volume of DCM (4 ml) andvolume of sodium desoxycholate solution (20 ml)). Theamount of drug, amount of polymer, and concentration ofsurfactant used in formulation were optimized.

The concentration of drug that is added in the formulationduring emulsification process also affects the morphology andphysicochemical properties of the resultant NPs. The drug thatis added in the primary emulsion shows the tendency torapidly partition out into the outer aqueous phase (beinghydrophilic in nature), thus higher drug amount would resultin low entrapment efficiency. The amounts of drug used foroptimization were 5, 7.5, and 10 mg. Out of the three formu-lations prepared, i.e., D1, D2, and D3; formulation D2 wasfound to be most suitable on the basis of size as well asentrapment efficiency. The amount of polymer used for thepreparation of NPs also caused variation in size and entrap-ment. Increasing the amount of polymer, beyond an optimumrange would result in increase in size of the formulation and anadditional amount of energy would be required to reduce thesize to desired range. Thus the amount of polymer used forformulating NPs was also optimized and three formulationsP1, P2, and P3 were prepared using 20, 40, and 60 mg ofpolymer. Of the prepared formulations, P2 responded well tothe chosen parameters. The size of the formulation preparedusing 40 mg of polymer was lesser and entrapment efficiencywas higher of all the three formulations. Increasing the amountof polymer, i.e., 60 mg resulted in increase in size beyondacceptable range. Furthermore, the concentration of surfactantwould result in variation in selected parameters. Surfactantin less concentration would not be able to stabilize the NPs

whereas, in high concentration, it results in aggregation ofNPs. Thus, for optimizing concentration of surfactant, againthree formulations S1, S2, and S3 were prepared using 8, 12,and 16 mM of sodium desoxycholate. Of the three formula-tions, S2 was optimized on the basis of desired size andmaximum entrapment of all the three formulations.

The optimized method was utilized to formulate the NPsusing both PLGA and PLGA-PEG diblock copolymer fordrug delivery. The use of PLGA-PEG diblock copolymerenables the preparation of non-aggregating particles with-out the need of an additional stabilizer such as poly (vinylalcohol). Furthermore, PEG is an uncharged, highly flexiblepolymer that is known to decrease drug adsorption and cellinteractions when present at the surface. PEG has outstandingphysiochemical and biological properties including solubilityin water and in organic solvents. Furthermore, it is non-toxic,non-antigenic, and non-immunogenic. Morphological studieswere performed utilizing SEM. The particles were observed tobe spherical, smooth and morphologically similar. The size ofthe optimized formulations was also found to be in nanorange,i.e., 178±8.5 and 132.5±10 nm for PLGA and PLGA-PEGNPs, respectively.

The drug entrapment in case of optimized formulations wasalso found to bemore than other formulations and was 35.16±1.04 % for PLGA and 34.56±1.35 % for PLGA-PEG-NH2NPs. The polydispersity index was also found to be very less,i.e., 0.095 and 0.072 for PLGA and PLGA-PEG-NH2 NPs,respectively, depicting a uniform size distribution for both theformulations. PLGA NPs exhibited highest zeta potential(−59.88±3.5 mV) due to the presence of free carboxyl endgroup on particle surface whereas in case of PLGA-PEG-NH2NPs the zeta potential (−38.43±2.3 mV) was found to get

Fig. 9 Confocal laser scanningmicrographs of MIA PaCa-2pancreatic cancer cells after 6 hincubation with the FITC-loaded(a) PLGA NPs (b ) Ab-PLGANPs at 0.25 mg/ml nanoparticleconcentration at 37 °C


shifted to lesser negative value due to the presence of amineterminated PEG chains because the coating layer shields andmove the shear plane outwards from the particle surface. Amarked difference in the surface charge between PLGA andPLGA-PEG-NH2 NPs was observed. The result confirms thatthe amine terminated PEG chains were situated on the surfaceof the NPs. The hydrophilic NH2-PEG moiety was orientedoutside towards aqueous medium forming corona type, whichwas anchored to hydrophobic PLGA polymer backbone as acore material.

The in vitro release profile of PLGA and PLGA-PEG-NH2NPs was determined in PBS (pH 7.4) at 37 °C using a constantshaking incubator. The drug release profile was studied forvarious time intervals, i.e., 0.5, 1, 2, 4, 6, 8, 16, 24, 36, and48 h. Both formulations exhibited a biphasic release patternthat was characterized by an initial burst up to 10 h, followedby a slower sustained release and remained constant up to48 h. At the end of 48 h, the PLGA-PEG-NH2 NPs showedhigher release rate of drug than PLGA NPs. The PLGA-PEG-NH2 NPs degraded quickly and released the drug subsequent-ly. This could be explained by the degradation mechanisms;the PEG chains (being hydrophilic) enhanced the water pen-etration into the matrix and the NPs were degraded homoge-neously over time. In contrast, PLGA formulation releasedonly 36.9±1.1 % drug due to its hydrophobicity and hencepoor degradation in aqueous environment. It is inferred thatthe gradual degradation of the polymer resulted in sustainedrelease of the drug. However, the release of gemcitabinedepends on several factors including the drug to polymer ratio,drug solubility inside the matrix, and the interaction betweenthe core and drug.

The aim of this studywas to prepareAb-PLGANPs derivedfrom PLGA-PEG-NH2 block copolymers containing function-al groups at the PEG chain end allowing NPs surface modifi-cation for targeting purposes. Amine groups were chosen tocouple targeting units by using two different conjugation tech-niques, one involving glutaraldehyde as a cross-linker andother method involved MBS modification of NPs. The formerinvolved applying aldehyde chemistry in aqueous media. NPswere prepared from PLGA-PEG-NH2 copolymers and thecoupling of anti-EGFR mAb (as an antibody for pancreaticcell for targeting units) to glutaraldehyde-activated NPs at thesurface was studied. The excess of glutaraldehyde was re-moved after the activation of NPs using dialysis membrane.The free aldehyde group present on the activated NPs wasmade to react with amine moiety of antibody. The removal ofexcess of glutaraldehyde was confirmed using Schiff reaction.At different time intervals, 250 μl of glutaraldehyde mixedformulation was taken and 25 μl of Schiff reagent was added,which turns deep purple if glutaraldehyde is present. Thedecrease in intensity of colour showed almost removal ofexcess glutaraldehyde. The second technique for conjugationinvolved activation of NH2 moiety of NPs using MBS and

thiolation of mAb using Traut’s reagent, respectively. Later,both the active moieties were incubated for 30 min to allowconjugation of both the units.

Both the conjugated Ab-PLGA NPs were characterized onthe basis of size, PDI, zeta potential, and entrapment efficien-cy. The size of MBS cross-linked NPs was much higher andwas beyond acceptable range. Also, zeta potential depictedcomparatively lower stability of MBS-conjugated NPs. Theentrapment efficiency of former was also lower which mightbe due to dissolution of NPs because of use of DMSO ascosolvent during activation of PLGA-PEG-NH2. TEM imagesalso revealed that favourably more antibody was coupledusing glutaraldehyde as a cross-linker as compared to MBS-mediated conjugation.

The particle size of Ab-PLGA NPs was slightly in-creased than plain PLGA-PEG NPs. Moreover, polydisper-sity index of the Ab-PLGA NPs was higher. The reasoncould be that, during immobilization, a small fraction of theglutaraldehyde-activated NPs might have reacted with otherNPs, leading to bridging between two particles. The anti-EGFR antibody attachment showed comparatively littleeffect on drug entrapment efficiency.

Anti-EGFR antibody might become inactive due to irre-versible denaturation and aggregation during the immuno-nanoparticle preparation process. The integrity of anti-EGFR antibody after conjugation on the NPs surface wasanalyzed by SDS-PAGE in comparison with the native anti-EGFR antibody. The SDS-PAGE showed that the molecularweight of both the antibody conjugated NPs was shiftedtowards the higher side as compared to native anti-EGFRantibody. From the comparison of the native antibody andantibody conjugated on surface of PLGA-PEG NPs, it canbe concluded that antibody conjugated on both the NPssurface was almost same in structure as the native EGFRantibody, which also validates the feasibility of Ab-PLGANPs for EGFR overexpressed cancer targeting. On the basisof results obtained, the glutaraldehyde-mediated conjuga-tion was selected for further in vitro cell line studies.

In cytotoxicity study, gemcitabine concentrations in all theformulations were adjusted to be the same. Percentage cellviability of both the formulations at different concentrations inMIA PaCa-2 cell line was determined. Ab-PLGANPs showedsignificantly higher cytotoxic effect (IC50 16 μg/ml) thanPLGA NPs (IC50, 31 μg/ml) after 24-h incubation period inMIA PaCa-2 cell line, which might be due to lower cellularuptake of PLGA NPs as compared to Ab-PLGA NPs. Theanti-EGFR monoclonal antibody attached on the surface ofNPs for targeting EGFR positive MIA PaCa-2 pancreaticcancer cells produced a synergistic effect with anticancer drugdepicting possible role of antibody in blocking cellular signal-ing, thus leading to inhibition of cellular proliferation. In vitrocytotoxicity results of Ab-PLGA NPs (without drug) alsosupport this finding. The blank PLGA-PEGNPs also exhibited


low cytotoxicity in MIA PaCa-2 cell lines. It implies that theseNPs could be useful as drug carriers without any significantcytotoxic effects. The significant cytotoxicity was shown byantibody alone and synergistic effect was shown along withgemcitabine.

NPs encapsulated with fluorescent dyes are frequentlyused to study cellular uptake. FITC, fluorescent dye, wasincorporated in NPs as a marker for fluorescence micros-copy to study the NPs uptake in cells. Aggregates of thegreen colour of FITC-loaded NPs in cell cytoplasm indicatethat NPs were internalised by the cells. It can thus beconcluded that the fluorescent NPs were located inside thecells. In the present study, the cellular uptake of Ab-PLGANPs was higher than that of PLGA NPs as shown by highintensity fluorescence developed by antibody-conjugatedNPs as compared to PLGA NPs, in EGFR-positive MIAPaCA-2 cells. The results reveal that anti-EGFR antibody-conjugated NPs were successfully developed for specifical-ly targeting EGFR receptors in pancreatic cancer cells.

5 Conclusion

The study provide the basis for the development of stableand biologically active carrier systems for direct targetingof tumour cells using antibody-conjugated PLGA-PEGNPs. Direct covalent coupling of antibodies to NPs usingglutaraldehyde as a cross-linker is an appropriate methodto achieve cell type-specific drug carrier systems basedon PLGA-PEG NPs and the anti-EGFR decorated PLGA-PEG NPs have potentials to be applied for targeted chemo-therapy against EGFR-positive cancer. Such a nanoparticlesystem for drug delivery is multifunctional, which provides aplatform to formulate anticancer drugs which reduces the sideeffects of the formulated anticancer drug, promotes synergistictherapeutic effects, and achieves targeted delivery for thetherapy.

Acknowledgments The authors acknowledge the chairman of theISF College of Pharmacy, Moga for providing the necessary facilitiesrequired to carry out this research work.

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http://dx.doi.org/10.1016/S0939-6411(97)00056-8http://dx.doi.org/10.1016/S0939-6411(97)00056-8

Gemcitabine-loaded PLGA-PEG immunonanoparticles for targeted chemotherapy of pancreatic cancerAbstractIntroductionMaterials and methodsCell linePreparation of PLGA-PEG-NH2 diblock copolymerPreparation of nanoparticlesCharacterization of nanoparticlesOptimization of process parametersSize and zeta potentialEntrapment efficiencyScanning electron microscopyIn vitro drug release

Preparation of PLGA-PEG immunonanoparticlesCharacterization of Ab-PLGA NPsTransmission electron microscopySize and zeta potentialEntrapment efficiencySodium dodecyl sulfate-polyacrylamide gel electrophoresisOptimized Ab-PLGA NPs

In vitro evaluationCell viability assayCell uptake studies

Statistical analysis

ResultsOptimization parameters of PLGA NPsOptimization of concentration of drugOptimization of amount of polymerOptimization of concentration of surfactant

Morphological study (SEM)In vitro release studiesCharacterization of Ab-PLGA NPsMorphological study (TEM)Size and zeta potentialEntrapment efficiencySDS-PAGEOptimization of conjugation method

In vitro evaluationCell viability assayCell uptake studies

DiscussionConclusionReferences

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