http://informahealthcare.com/mncISSN: 0265-2048 (print), 1464-5246 (electronic)

J Microencapsul, Early Online: 1–9! 2014 Informa UK Ltd. DOI: 10.3109/02652048.2014.913725

Non-aggregated protamine-coated poly(lactide-co-glycolide)nanoparticles of cisplatin crossed blood–brain barrier, enhanceddrug delivery and improved therapeutic index in glioblastoma cells:in vitro studies

Neel Kamal Dhami1, Ravi Shankar Pandey2, Upendra Kumar Jain1, Ramesh Chandra3, and Jitender Madan1

1Department of Pharmaceutics, Chandigarh College of Pharmacy, Mohali, Punjab, India, 2SLT Institute of Pharmaceutical Sciences, Guru Ghasidas

University, Bilaspur, Chhattisgarh, India, and 3Dr. B.R Ambedkar Centre for Biomedical Research, University of Delhi, Delhi, India

Abstract

Background and objectives: Non-aggregated protamine impregnated poly(lactide-co-glycolide)nanoparticles of cisplatin (Pt-PLGA NPs) were synthesized to augment brain delivery. Methodsand results: The mean particle size of Pt-PLGA NPs and PLGA NPs were observed to be173.2 ± 7.9 nm and 140 ± 10.2 nm, respectively. The Pt-PLGA NPs significantly (p50.05, one-wayanalysis of variance; ANOVA) delivered higher amount (172.41 ± 15.04 mg) of cisplatin incomparison to 110.48 ± 4.71 mg by PLGA NPs and 20.83 ± 1.65mg by cisplatin solution acrossin vitro bovine brain microvessel endothelial cells. Cisplatin bearing Pt-PLGA NPs was found tobe highly cytotoxic to U87 glioblastoma cells with an IC50 of 2.1mM as compared (one-wayANOVA, p50.05) to PLGA NPs (3.9 mM) and cisplatin alone (13.33 mM). Impregnation with Ptenhanced the uptake of PLGA NPs in U87 glioblastoma cells as compared to PLGA NPs byfollowing endocytosis mechanism. Conclusion: Cisplatin-loaded Pt-PLGA NPs compel preclinicaltumour regression study to further improve its utility against glioblastoma.

Keywords

Blood–brain barrier, cisplatin, glioblastoma,poly(lactide-co-glycolide), protamine

History

Received 24 October 2013Revised 10 February 2014Accepted 31 March 2014Published online 20 June 2014

Introduction

Cisplatin, one of the components of platinum-based chemother-apy, induces apoptosis in glioblastoma cells by markeddecrease in BCL2L12 gene expression (Taghavi et al., 2013).Mechanistically, cisplatin cross-links DNA and produces intra-and inter-strand links that inhibit its synthesis. Therefore, rapidlydividing cells do not duplicate DNA for mitosis and consequently,apoptosis is activated (Kaminski et al., 2008). The human doseregimen of cisplatin (75 mg/m2 or 165 mg) in post intravenousadministration shows long plasma half-life of 32–100 h due to590% of protein binding. In addition, cisplatin does not crossblood–brain barrier (BBB) efficiently (Long and Repta, 1981).Moreover, deposition of cisplatin in renal parenchymal cells bymembrane transporter, Ctr1, causes nephrotoxicity (Zustovichet al., 2009). Neurotoxicity is associated with the formation ofcisplatin–dorsal root ganglia neurons adduct (Dzagnidze et al.,2007). Diffusion of cisplatin in cochlear hair cells stimulates thegeneration of reactive oxygen species and causes ototoxicity(Rybak et al., 2009). Coupled to this, cis isomer of cisplatin isonly cytotoxic to tumour cells. Hence, to reduce the toxicity andenhance the brain uptake, it is mandatory to encapsulate thecisplatin in a highly permeable brain targeted drug deliverysystem.

Recently, we have worked on a workable approach toregulate the multiple administration of chemotherapeutic drug

in glioblastoma by using brain-targeted drug delivery systems(Madan et al., 2013). Amongst prospective biomaterials for drugdelivery, poly(lactide-co-glycolide) (PLGA) has shown immensepotential in manufacturing of drug delivery nanoparticles(Kocbek et al., 2007). It is biocompatible, physically strong andapproved by US FDA for human consumption (Makadia andSiegel, 2011). PLGA, a co-polymer of poly-lactic acid and polyglycolic acid, is cleaved by hydrolysis into natural metabolitessuch as lactic acid and glycolic acid (Zhua et al., 2002). Thesemetabolites are eliminated from the body by citric acid cycle(Mainardes and Evangelista, 2005). Depending upon the com-position and molecular weight, PLGA shows a wide range ofdegradation rates, ranging from months to years (Lua et al., 2000).Moreover, PLGA nanoparticles (PLGA NPs) have the potentialto overcome P-glycoprotein-dependent drug resistance in glio-blastoma cells (Ke et al., 2013).

It is reported that coating the nanovesicles with cationicpermeation enhancer increases the drug uptake across the BBB(Lu et al., 2006). Protamine sulphate (Pt), a 7-kD arginine rich,cationic protein facilitates adsorptive-mediated transcytosis(AMT)-dependent penetration of nanoparticles through non-specific binding with negatively charged membrane surface andsubsequent internalization through endocytosis pathway (Herveet al., 2008). However, aggregation of nanoparticles in theafferent vasculature critically hampers the tumour targeting(Chertok et al., 2011).

Therefore, in present investigation, we have innovativelysynthesized non-aggregated cisplatin-loaded protamine sulphate-coated poly(lactide-co-glycolide) nanoparticles (Pt-PLGA NPs),by emulsification solvent evaporation method (Pirooznia et al.,2012) with slight modification. Cisplatin-loaded PLGA NPswere also prepared for comparative study. The customized

Address for correspondence: Jitender Madan, PhD, Department ofPharmaceutics, Chandigarh College of Pharmacy, Mohali, Punjab,India. Tel: +91-172-3984209. Fax: +91-172-3984209. E-mail:jitenderpharmacy@gmail.com

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nanoformulations were tested for characterization of variousphysicochemical properties. The potential of tailored nanoformu-lations to cross BBB and enhanced cytotoxicity were testedin vitro in brain endothelial cells model and U87 glioblastomacells, respectively.

Materials and methods

Materials

Cisplatin (cis-diaminedichloroplatinum (II), molecular weight�300.05 Da and purity �99.5%) was a gift sample from Cipla,Mumbai, India. PLGA (lactide: glycolide¼ 75:25, molecularweight �78 000–85 000 Da), protamine sulphate (molecularweight �5000–10 000 Da) and magnesium hydroxide werepurchased from Sigma Aldrich, St Louis, MO. Methanol andacetonitrile (HPLC grade) were procured from Qualigens,Mumbai, India. Poly vinyl alcohol (POVAL 403) was purchasedfrom Kuraray, Tokyo, Japan. All other chemicals used wereof analytical grade and used without further purification.

Cell cultures

The brain microvessel endothelial cell line (ATCC, Manassas,VA) was maintained in culture medium consisting of 45%Dulbecco’s Modified Eagle’s Medium (DMEM) (Biologicals,Israel), 45% Ham’s F-12 nutrient mixture (Sigma Aldrich),10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid(pH� 7.4), 13 mM sodium bicarbonate, 10% plasma-derivedequine serum, 100 mg/mL heparin, 100 mg/mL streptomycin,100 mg/mL penicillin G, 50 mg/mL polymyxin B and 2.5 mg/mLamphotericin B (Landen et al., 2004). Human glioblastoma cellline, U87, was maintained in DMEM in 5% CO2 and 95% air,supplemented with 10% foetal calf serum (Ozolek et al., 2007).Both cultures were maintained at 37 �C.

Synthesis of cisplatin-loaded PLGA nanoparticles

Cisplatin-loaded PLGA NPs and Pt-PLGA NPs were synthesizedby double emulsion technique (Pirooznia et al., 2012) usingpartially hydrolyzed PVA instead of PVA (Sahoo et al., 2002).Briefly, solution A was prepared by dissolving 30 mg of POVAL-403 (a partially hydrolyzed PVA) and 15 mg of Mg (OH)2 in10 mL of 0.1% w/v (10 mg cisplatin) cisplatin aqueous solution.Solution B was prepared by dissolving 25 mg of PLGA in 3 mL ofdichloromethane. Then, solution B was poured into solution Aand sonicated (Trans-O-Sonic, D-250 Sonicator, Mumbai,Maharashtra, India) for 10 min. This is called solution C. Thesolution C was then kept overnight at room temperature (25 �C)for solvent (dichloromethane) evaporation to synthesize cisplatin-loaded PLGA NPs (Kashi et al., 2012). The Pt-PLGA NPs wereprepared by dissolving 2.5 mL of 1% w/v Pt in 2.5 mL of solutionC and mixture was stirred overnight at room temperature. Thenanoparticles were recovered by ultracentrifugation (ThermoScientific, Sorvall Ultra Centrifuge, Waltham, MA) at 40 000 rpmfor 30 min and washed twice with distilled water for purification.The purified nanoparticles were lyophilized (Lab India, Thane,India) using 2% w/v trehalose as cryoprotectant (Jeong et al.,2005). Blank PLGA NPs were prepared without incorporation ofcisplatin.

Fluorescent labelled PLGA NPs and Pt-PLGA NPs wereprepared by adding separately 5 mg/mL of rhodamine 6G insolutions B. To determine the labelling efficiencies, PLGA NPsand Pt-PLGA NPs were dissolved separately in phosphate buffersaline (pH� 7.4) until a final concentration of 0.10 mg/mL wasreached. The fluorescence intensity was measured at �exe� 553nm and �emi� 574 nm using a fluorometer (Perkin Elmer LS-5B,Cambridge, UK). A standard solution of 0.003–0.015mg/mL of

rhodamine 6G was prepared by diluting 100mg/mL of methanolicsolution of rhodamine 6G with PBS, pH� 7.4. Labellingefficiency was calculated as the percent weight of rhodamine6G to the weight of the PLGA NPs or Pt-PLGA NPs.

y ¼ 5:493xþ 0:398, R2 ¼ 0:990

Characterization of nanoparticles

Particle size and zeta potential

Each nanoparticle sample (100 mL) was dispersed in 5 mL of PBS,pH� 7.4, and the particle size was determined using MalvernNanoZS instrument (Malvern, Worcestershire, UK). A voltageof 150 mV was applied to measure the electrophoretic velocityof nanoparticles in PBS (pH� 7.4). All measurements werecarried out in triplicate (n¼ 3).

Transmission electron microscopy

The nanoparticle formulations viz cisplatin-loaded PLGA NPsand Pt-PLGA NPs were analysed for surface topography bytransmission electron microscopy (TEM, FTI Tecnai F20).The formulations were placed separately on to a carbon-coatedcopper grid, and the grid was air dried at 25 �C before loading intothe TEM, maintained at a voltage of 80 kV.

Fourier-transforms infrared spectroscopy

The infrared spectrum was recorded for cisplatin, PLGA NPs,physical mixture of cisplatin and PLGA NPs, cisplatin-loadedPLGA NPs and cisplatin-loaded Pt-PLGA NPs using SpectrumBX (Perkin Elmer, Waltham, MA) infrared spectrophotometer.Samples were prepared in a KBr disk (2 mg sample in 200 mgKBr) with a hydrostatic press at a force of 40 psi for 4 min.The scanning range used was 4400–400 cm�1 at a resolution of4 cm�1.

Differential scanning calorimetry

The endothermic peaks of pure cisplatin, PLGA NPs, physicalmixture of pure cisplatin and PLGA NPs, cisplatin-loaded PLGANPs and cisplatin-loaded Pt-PLGA NPs were recorded using thedifferential scanning calorimeter (Mettler-Toledo ThermalEquipment, Mumbai, India). Nitrogen was used as a carrier gasat the flow rate of 40 mL/min in the temperature range of 30–300 �C. A 10 mg quantity of each sample was employed at aheating rate of 20 �C/min. The PLGA NPs sample was heated andcooled for five cycles. The endothermic peaks were measuredbased on the last three heating thermograms.

Powder X-ray diffraction pattern

The crystalline geometry of cisplatin, PLGA NPs, physicalmixture of cisplatin and PLGA NPs, cisplatin-loaded PLGANPs and cisplatin-loaded Pt-PLGA NPs was compared by X-raydiffractometer (X’Pert PRO, Panalytical Company, Almelo, TheNetherlands) using Ni-filtered, CuKa-radiation, voltage of 60 Kvand a current of 50 mA. The scanning rate employed was 1 �/minover the 10� to 60� diffraction angle (2�) range.

Determination of encapsulation efficiency anddrug-loading capacity

The encapsulation efficiency (EE) and drug-loading capacity(DLC) were determined by dissolving 10 mg of cisplatin-loadedPLGA NPs and Pt-PLGA NPs formulations separately in 1 mLof dichloromethane and subsequently 5 mL of PBS (pH� 7.4)was added. After complete evaporation of dichloromethane,

2 N. K. Dhami et al. J Microencapsul, Early Online: 1–9

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ultracentrifugation (Thermo Scientific, Sorvall Ultra Centrifuge,Waltham, MA) at 40 000 rpm was done for 30 min. Furthermore,1 mL of supernatant was diluted with PBS (pH� 7.4) and passedthrough 0.22 mm membrane filter (MDI, Pune, India). Theconcentration of cisplatin was determined at 301 nm using UV/visible (UV-VIS) spectrophotometer (Shimadzu, Kyoto, Japan)(Carrielo et al., 1992). All measurements were carried out intriplicate (n¼ 3). The components of nanoparticles did notinterfere with the analytical estimation of cisplatin. EE andDLC were calculated as follows:

EE ð%Þ ¼ amount of drug entrapped

amount of drug added� 100

DLC ¼ amount of drug entrapped

amount of nanoparticles

In vitro drug release

The in vitro drug release of cisplatin-loaded PLGA NPs andPt-PLGA NPs was conducted using dialysis membrane method(Gupta et al., 1987) at pH� 7.4 and � 5.5 to harmonize thephysiological milieu. Briefly, cisplatin-loaded PLGA NPs andPt-PLGA NPs suspensions (� 75 mg/m2 or 165 mg of cisplatinbearing 264 mg of cisplatin-loaded PLGA NPs and 276.38 mg ofPt-PLGA NPs) were deposited separately in dialysis bag (12 KDa,Sigma Aldrich). The dialysis bags were then placed in 900 mLof PBS of pH� 7.4 (Paraskara et al., 2010) and pH� 5.5,previously maintained at 37 �C. Sodium lauryl sulphate (0.25%w/v) was added to enhance the solubility of cisplatin indissolution medium. The dissolution medium was stirred at50 rpm (as recommended for dissolution testing of parenteralproducts) (Anand et al., 2011). A 5 mL sample was collectedfrom the receptor chamber at specified time intervals andreplaced with fresh buffer of same pH to retain sink conditions.The drug concentration in the sample was estimated usingUV/VIS spectrophotometer (Shimadzu) at 301 nm (Carrieloet al., 1992). All measurements were carried out in triplicate(n¼ 3).

Therapeutic efficacy testing of cisplatin-loaded Pt-PLGANPs and PLGA NPs

In vitro bovine brain microvessel endothelial cell assay

Bovine brain microvessel endothelial cells (6� 104 cells) wereseeded on to the tissue culture dishes (35� 12 mm) and placed onthe polycarbonate membrane (Landen et al., 2004). The polycar-bonate membrane was then placed in a side-by-side diffusion cellcontaining 5 mL of continuously stirred PBS (pH� 7.4) on eachside at 37 �C. Cisplatin, cisplatin-loaded PLGA NPs and Pt-PLGANPs (equivalent to 270mg of cisplatin, a single intravenous doseof mice, encapsulated in PLGA NPs and Pt-PLGA NPs) wasadded to the donor chamber separately. A 200mL of sample atvarious time points (5, 10, 15, 30, 60 and 120 min) was removedfrom the receptor chamber, replaced with equal quantity of freshbuffer to mimic sink condition and stored at �20 �C (Schuldeset al., 1997) for high-performance liquid chromatography (HPLC)assay.

Reverse phase-HPLC (RP-HPLC) was used to determinethe concentration of cisplatin (Flores et al., 2005). The chroma-tographic system (ELICO�) consisted of a column (VARIAN,Microsorb-MV 100-5 C18, 250� 4.6 mm), an UV-VISdetector (HD 462) and an isocratic pump (HP 466). Columnelution was carried at 25 �C. Mobile phase was consistedof methanol:acetonitrile:water (40:29:31). A fixed flow rate of1.6 mL/min was maintained. Detection was performed at 254 nm

(Flores et al., 2005). A calibration curve ranging from 0.2 to11 mg/mL was prepared in aqueous phase.

y ¼ 12:44xþ 13:06, R2 ¼ 0:999

In vitro cytotoxicity assay

3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide(MTT) assay was used to determine the cytotoxicity ofnanoformulations of cisplatin using 96-well microtitre plate(Mosmann, 1983). Briefly, 4� 103 U87 glioblastoma cells wereplaced in 200mL of the serum DMEM. The medium was replacedwith serum free-DMEM after 24 h of incubation period.Subsequently seeded U87 glioblastoma cells were incubatedwith a gradual concentration of cisplatin, cisplatin-loaded PLGANPs, Pt-PLGA NPs and respective blank nanoparticles equiva-lent to cisplatin concentration of 0.1 to 30 mM for 72 h. Afterpredetermined incubation period, MTT (0.5 mg/mL) was added,and the plate was incubated for 4 h at 37 �C. The formazoncrystals, formed after cell lysis, were dissolved using 100mL ofdimethylsulfoxide. The absorbance was read at 570 nm using630 nm as reference wavelength by ELISA reader (Tecan,Mannedorf, Switzerland).

In vitro cellular uptake assay: quantitative andqualitative analysis

U87 glioblastoma cells were plated in Lab-Tek II ChamberSlideTM System (Nalge Nune, Penfield, NY) at a densityof 4� 103 cells per chamber. Dosing solutions of rhodamine6G-labeled PLGA NPs and Pt-PLGA NPs formulations (� 0.1–30 mM) were prepared using PBS (pH� 7.4) and diluted withDMEM. The cell monolayers were rinsed thrice and pre-incubated with 1 mL of DMEM at 37 �C for 1 h. Uptake wasinitiated when 1 mL of specified dosing solution was exchangedwith DMEM, followed by incubation of the cells at 37 �C for 24 h.The experiment was terminated by washing the cell monolayerthree times with ice-cold PBS (pH� 7.4) and lysing the cellswith 1 mL of 0.5% Triton X-100. Cell-associated rhodamine6G-labeled PLGA NPs and Pt-PLGA NPs were quantifiedby analyzing the cell lysate in a fluorimeter (Spectra Fluor,Tecan, �exe� 553 nm, �emi� 574 nm) (Huang et al., 2002). Theprotein content of the cell lysate was measured using the BCAprotein assay kit (Brown et al., 1989).

After 24 h incubation period, the medium was removedand plates were washed thrice with sterile PBS (pH� 7.4).After the final wash, the cells were fixed with 4% paraformalde-hyde, and individual cover slips were mounted on cleanglass slides with Fluoromount-G mounting medium (SouthernBiotechnology, Birmingham, AL). The slides were viewed undera fluorescent microscope (�exe� 553 nm, �emi� 574 nm, Leica,Heidelberg, Germany) (Wang et al., 2005).

Statistical analysis

One-way and two-way analysis of variance (ANOVA) wereperformed for statistical significance analysis by GraphPad Prism04 Software (Sao Paulo, Brazil). The results are presented as themean ± SD for n¼ 3. The significance level of difference wastaken as p50.05.

Results

Synthesis and characterization of cisplatin-loadedprotamine coated PLGA nanoparticles

Cisplatin-loaded Pt-PLGA NPs were prepared using doubleemulsion technique (Pirooznia et al., 2012) with slight

DOI: 10.3109/02652048.2014.913725 Pt-PLGA NPs were synthesized to augment brain delivery 3

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modification. This technique is reported for encapsulation ofhydrophilic drugs. Cisplatin and PLGA were made soluble in PVA(partially hydrolyzed) aqueous phase and dichloromethane solv-ent, respectively. The nanocoacervate phase was induced by slowevaporation of dichloromethane, kept for overnight stirring atroom temperature. Furthermore, Pt formed thin layer over thecisplatin-loaded PLGA NPs due to interaction between positiveand negative charge of Pt and PLGA NPs, respectively (Figure 1).

Characterization of nanoparticles

Particles size and zeta potential analysis

Particle size analysis plays a crucial role in determination of theeffectiveness of a nanoparticle formulation for targeting tumourcells. The anatomical location of the BBB is the endothelial cellsof arterioles, capillaries, veins and the epithelial cell surface of thechoroid plexus that form impermeable tight junctions. Endothelialcells bear negative charge due to presence of glycocalyx,constituted by sialo-glycoconjugates and heparan sulphate pro-teoglycans. In this manner, endothelial cells pave the way for thedelivery of cationic moieties to the brain via AMT mechanism(Bickel, 1995). Hydrophobic nanoparticles in the range of 150–600 nm can efficiently transport the drug to brain by diffusionmechanism (Georgieva et al., 2011). The prepared nanoformula-tions, cisplatin-loaded Pt-PLGA NPs and PLGA NPs have shownthe mean nanoparticle size of 173.2 ± 7.9 nm and 140 ± 10.2 nm,respectively, and thereby hold the great potential to cross BBB.A significant (two way ANOVA test, p50.05) fall in zetapotential was also observed for cisplatin-loaded Pt-PLGA NPs(+ 10.33 ± 0.03 mV) as compared to PLGA NPs (–4.69 mV) ofcisplatin (Figure 2A and B and Table 1).

TEM

TEM photographs were found to be consistent with the resultsof particle size analysis. Both types of nanoparticles were smooth,spherical in shape and the brushing of Pt over the surfaceof PLGA NPs was clearly visible (Figure 2A and B).

Fourier-transforms infrared

Fourier-transforms infrared (FTIR) spectrum peaks were recordedto examine the formation of any new chemical linkage during theencapsulation of cisplatin in nanoparticle matrix. FTIR spectrumpeaks of cisplatin, PLGA NPs, physical mixture of cisplatin andPLGA NPs and cisplatin-loaded PLGA NPs and Pt-PLGA NPsare listed in Table 2. The pure cisplatin sample showed thecharacteristics peaks such as –N–H stretching of –NH3

(3450 cm�1), –O–H-stretching of –H2O (3282.54 cm�1) and –C–Cl stretching of –Cl2 (743 cm�1). The peaks obtained in PLGANPs were due to OH stretching (3200–3500 cm�1), –CHstretching (2850–3000 cm�1), –C¼O stretching (1700–1850 cm�1) and C–O stretching (1050–1250 cm�1). Physicalmixture showed the peaks of individual components. The peaksobtained for cisplatin-loaded PLGA NPs were –N–H stretching of–NH3 (3450 cm�1), –C–Cl-stretching of –Cl2 (743 cm�1), OHstretching (3200–3500 cm�1), –CH stretching (2850–3000 cm�1),–C¼O stretching (1700–1850 cm�1) and C–O stretching (1050–1250 cm�1), which indicated the presence of a stable andintact cisplatin in PLGA NPs. Continuation to this, Pt-PLGANPs indicated the peak between 1500 and 1600 cm�1 becauseof the presence of polypeptide (–CONH– bond) chain.Moreover, peak for arginine residue was obtained at 1110 cm�1

in addition to the peaks for cisplatin and PLGA NPs (Awotwe-Otoo et al., 2012). Hence, FTIR data specified that cisplatinmolecules did not interact chemically with PLGA NPs orPt-PLGA NPs.

Differential scanning calorimetry

Differential scanning calorimetry (DSC) was performed todetermine the physical state of cisplatin in nanoparticle matrix.We observed a sharp endothermic peak at 270.83 �C in purecisplatin (Figure 3). The thermogram of PLGA NPs showeda broad peak at 52.66 �C. Physical mixture of cisplatin andPLGA NPs indicated that endothermic peaks were slightlyshifted to 268 �C and 50.83 �C, respectively, from its originalpeaks. However, cisplatin-loaded PLGA NPs and Pt-PLGA NPs

Figure 1. Schematic representation of thesynthesis of cisplatin-loaded non-aggregatedpoly(lactide-co-glycolide) nanoparticles,PLGA NPs and protamine sulphate-coatedpoly(lactide-co-glycolide) nanoparticles,Pt-PLGA NPs.

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showed broad peaks at 58.33 �C and 75.34 �C respectively,indicated that cisplatin was molecularly dispersed in tailorednanoformulations (Dong and Feng, 2007; Awotwe-Otooet al., 2012).

Powder X-ray diffraction pattern

Powder X-ray diffraction pattern (PXRD) was used to study thecrystal structure of drug in nanoparticles. PXRD pattern ofcisplatin, PLGA NPs, physical mixture of cisplatin and PLGANPs and cisplatin-loaded PLGA NPs was recorded and comparedfor any change in crystalline geometry. The PXRD pattern ofcisplatin showed sharp and intense peaks indicating its crystallineconfiguration (Figure 4). PLGA NPs showed diffused peaks withlow intensities. Physical mixture showed undefined diffusedpeaks with low intensities. However, cisplatin-loaded PLGA NPsand Pt-PLGA NPs showed peaks of diminished intensity

indicating the deformation of crystalline geometry of drug toamorphous state in customized nanoformulations.

Determination of EE and DLC

The impregnation of Pt over PLGA NPs did not influence theEE and DLC. We did not observe any significant difference(two way ANOVA test, p40.05) between EE (62.5 ± 3.4% and59.7 ± 4.6%) and DLC (6.25 mg/10 mg and 5.97 mg/10 mg)of cisplatin-loaded PLGA NPs and Pt-PLGA NPs, respectively(Table 1).

In vitro drug release

In vitro drug release test was conducted by dynamic dialysismethod (Gupta et al., 1987) to determine the pH-dependent releaseof cisplatin from tailored nanoformulations. Cisplatin-loaded

Figure 2. Particle size distribution, transmis-sion electron microscopy and zeta potentialanalysis of (A) cisplatin-loaded PLGA NPsand (B) cisplatin-loaded Pt-PLGA NPs. Scalebar� 100 nm. All measurements were carriedout in triplicate (n¼ 3).

Table 1. Parameters for characterization of cisplatin bearing poly(lactide-co-glycolide) nanoparticles.

Sample code Particle sizea (nm)Entrapmenta

Efficiency (%)Drug-loading

capacitya (mg/10 mg) Zeta potentiala,b (mV)

PLGA NPs 135.4 ± 8.4 – – �18.45 ± 0.07Cisplatin-loaded PLGA NPs 140.0 ± 10.2 62.5 ± 3.4 6.25 ± 0.27 �4.69 ± 0.06Cisplatin-loaded Pt-PLGA NPs 173.2 ± 7.9 59.7 ± 4.6 5.97 ± 0.08 10.33 ± 0.03

Notes: aAll observations are carried out in triplicate (mean ± SD, n� 3).bp50.05 (one-way ANOVA test).

DOI: 10.3109/02652048.2014.913725 Pt-PLGA NPs were synthesized to augment brain delivery 5

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PLGA NPs and Pt-PLGA NPs released 95.41 ± 3.63% and85.32 ± 3.27% of cisplatin at pH� 5.5 significantly (one-wayANOVA test, p50.05) higher than the cisplatin released fromcisplatin-loaded PLGA NPs (75.34 ± 2.92%) and Pt-PLGA NPs(70.53 ± 2.53%), respectively, at pH� 7.4 (Figure 5).

In vitro bovine brain microvessel endothelial cell assay

In vitro bovine brain microvessel endothelial cell assay was usedto examine the ability of cisplatin, cisplatin-loaded PLGA NPsand Pt-PLGA NPs to cross BBB. The concentration of cisplatinwas determined by RP-HPLC (Flores et al., 2005). A negligibleconcentration of 5.25 ± 0.45 mg of cisplatin was observed at 5 mintime interval from cisplatin solution sample that was further

enhanced up to 20.83 ± 1.65 mg at 120 min (Figure 6). Coupledwith this, cisplatin-loaded PLGA NPs and Pt-PLGA NPs signifi-cantly (one-way ANOVA, p50.05) crossed cell monolayersof bovine brain microvessel endothelial cells and documentedabout 110.48 ± 4.71 mg and 172.41 ± 15.04 mg of the drug at120 min, respectively.

In vitro cytotoxicity assay

Standard colorimetry-based MTT assay was used to determine theanti-proliferation capacity of cisplatin, cisplatin-loaded PLGANPs, Pt-PLGA NPs and respective blank formulations againstU87 glioblastoma cells (Figure 7A). The IC50 (concentrationof drug required to kill 50% of cells) value of cisplatin-loaded

Table 2. FTIR assignments of cisplatin, PLGA NPs, physical mixture of cisplatin and PLGA NPs, cisplatin-loaded PLGA NPs and cisplatin-loaded Pt-PLGA NPs scanned between 400 and 4400 cm�1.

Formulation ATR peaks Assignments

Cisplatin 3450 cm�1 v, –N–H stretching of –NH3

3282 cm�1 v, –O–H-stretching of –H2O743 cm�1 v, –C–Cl-stretching of –Cl2

PLGA NPs 3200–3500 cm�1 v, OH stretching2850–3000 cm�1 v, –CH stretching1700–1850 cm�1 v, –C¼O stretching1050–1250 cm�1 v, C–O stretching

Physical mixture 3200–3500 cm�1 v, OH stretching2850–3000 cm�1 v, –CH stretching1700–1850 cm�1 v, –C¼O stretching1050–1250 cm�1 v, C–O stretching3450 cm�1 v, –N–H stretching of –NH3

3282 cm�1 v, –O–H stretching of –H2O743 cm�1 v, –C–Cl stretching of –Cl2

Cisplatin-loaded PLGA NPs 3200–3500 cm�1 v, OH stretching2850–3000 cm�1 v, –CH stretching1700–1850 cm�1 v, –C¼O stretching1050–1250 cm�1 v, C–O stretching3450 cm�1 v, –N–H stretching of –NH3

743 cm�1 v, –C–Cl stretching of –Cl2

Cisplatin-loaded Pt-PLGA NPs 3200–3500 cm�1 v, OH stretching2850–3000 cm�1 v, –CH stretching1700–1850 cm�1 v, –C¼O stretching1500–1600 cm�1 v, –CO–NH bond in protamine1050–1250 cm�1 v, C–O stretching1110 cm�1 v, Arginine content of protamine3450 cm�1 v, –N–H stretching of –NH3

743 cm�1 v, –C–Cl stretching of –Cl2

Figure 3. Differential scanning calorimetry of cisplatin, PLGA NPs,physical mixture of cisplatin and PLGA NPs, cisplatin-loaded PLGA NPsand cisplatin-loaded Pt-PLGA NPs.

Figure 4. Powder X-ray diffraction patterns of cisplatin, PLGA NPs,physical mixture of cisplatin and PLGA NPs, cisplatin-loaded PLGA NPsand cisplatin-loaded Pt-PLGA NPs.

6 N. K. Dhami et al. J Microencapsul, Early Online: 1–9

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Pt-PLGA NPs (2.1 mM) was significantly (one-way ANOVA test,p50.05) lower than PLGA NPs (3.9 mM) and cisplatin solution(13.33 mM). Blank PLGA NPs and Pt-PLGA NPs did not induceany cytotoxicity in U87 glioblastoma cells.

In vitro cellular uptake assay

We determined quantitatively the accumulation of nanoparticlesby tracking rhodamine 6G labelled PLGA NPs (44.2 ± 6.03%) andPt-PLGA NPs (51.9 ± 6.76%) using a fluorimeter and observed asignificant (two way ANOVA; p50.05) difference in bindingcapacity (Figure 7B). We further used fluorescent microscopeto visualize the particle internalization and observed redcoloured fluorescence of rhodamine 6G-labeled PLGA NPs(1440� 10�5 g) and Pt-PLGA NPs (1500� 10�5 g) in the cyto-plasm of U87 glioblastoma cells (Figure 7C).

Discussion

In this investigation, PLGA NPs and Pt-PLGA NPs of cisplatinwere designed and prepared to enhance the passage across BBBfor the better management of glioblastoma. Double emulsiontechnique (Pirooznia et al., 2012) with slight modification wasused to encapsulate the cisplatin in PLGA NPs as it is a simple,fast and employs non-toxic solvents. In addition, inclusion of bothhydrophilic and hydrophobic drugs is possible with this method.Next, pore formations at the surface of nanoparticles play acentral role in drug release mechanism. PLGA is composed oflactic acid that is known for its pH lowering effect in systemiccirculation. To prevent this fall in pH, magnesium hydroxide wasincorporated to neutralize the effect of lactic acid (Jaganathanet al., 2004). Adhesive property of PVA promotes aggregationof PLGA nanoparticles. This causes difficulty in scaling up theformulation for tumour targeting. Hence, we included a partiallyhydrolyzed PVA (PVA 403) to synthesize non-aggregated PLGANPs (Figure 1). The particle size distribution and zeta potential

data indicated that non-aggregated nanoparticles of PLGAhold great potential in drug delivery across BBB and tumourtargeting. An increase in zeta potential substantiated the brushingof arginine rich cationic protamine over the surface of PLGA NPsshowing negative charge due to –COOH group (Figure 2 andTable 1). The EE and DLC was not affected in Pt-PLGANPs (Table 1). Next, we performed FTIR, DSC and PXRD ofindividual components, physical mixtures, drug-loaded nanopar-ticles and protamine modified nanoparticles. FTIR assured thepresence of unmodified functional groups of cisplatin in bothPLGA NPs and Pt-PLGA NPs and indicated the absence ofany chemical linkage between drug and polymer (Table 2). Thephysical state and structural geometry of cisplatin in PLGA NPsand Pt-PLGA NPs were confirmed by DSC and PXRD, whichsubstantiated the amorphization of drug in nanoparticle matrix,a highly bioavailable state (Figures 3 and 4). The therapeuticpotential of tailored nanoformulations of cisplatin to releasethe drug in physiological milieu (pH� 5.5) of cancer cellcompartments was tested in vitro in terms of pH dependentrelease. Cisplatin-loaded PLGA NPs and Pt-PLGA NPs showedinitial burst followed by a slow release over three weeks at bothpH� 5.5 and pH� 7.4 (Figure 5). Protamine sulphate suppressedthe initial burst release and prolonged the releasing period withoutaltering the principal dissolution pattern of PLGA NPs(Jiang et al., 2011). Dissolution testing performed at pH� 5.5demonstrated enhanced release as compared to pH� 7.4, whichmight be attributed to faster degradation of PLGA in acidiccascade (Carrielo et al., 1992). Next, we performed in vitro bovinebrain microvessel endothelial cell assay that demonstratedsignificantly greater uptake of cisplatin-loaded Pt-PLGA NPs incomparison of PLGA NPs and cisplatin due to AMT mechanism(Figure 6). The AMT mechanism allows electrostatic complex-ation of cationic protamine-impregnated PLGA NPs at thenegatively charged endothelial tight junctions of BBB and therebyfacilitates enhanced uptake in comparison of unmodifiednanoformulations (Kumagai et al., 1987). In continuation tothis, the higher cytotoxicity and lower IC50 of cisplatin-loadedPt-PLGA NPs against U87 glioblastoma cells as compared toPLGA NPs and cisplatin solution might be attributed to greateruptake of nanoparticles through endocytosis mechanism(Figure 7A). Further cellular uptake of Pt-PLGA NPs andPLGA NPs was determined by the use of fluorescent microscopy.Consistent with the results of cytotoxicity assay, the quantitativeand qualitative cellular accumulation of Pt-PLGA NPs in U87glioblastoma cells was higher than the PLGA NPs (Figure 7Band C). This affirms that tethering the nanoparticle surface withcationic peptide allows greater uptake through endocytosispathway due to the presence of net negative charge on surfaceof cancer cells. It is reported that cationic cell-penetratingpeptides have the ability to deliver a wide range of nanoparticlesinto tumour cells (Regberg et al., 2012). Protamine anchored/surface coated nanoparticles promote electrostatic interactionbetween the positively charged protamine and the negatively

Figure 6. Permeation of cisplatin, cisplatin-loaded PLGA NPs and cisplatin-loaded Pt-PLGA NPs from in vitro bovine brainmicrovessel endothelial cells barrier withrepresentative HPLC chromatograms.Cisplatin solution documented20.83 ± 1.65mg of cisplatin, while cisplatin-loaded PLGA NPs and Pt-PLGA NPs docu-mented significantly (one-way ANOVA,p50.05) higher amount of about110.48 ± 4.71mg and 172.41 ± 15.04 mg,respectively, at 120 min. The study wascarried out in triplicate (n¼ 3).

Figure 5. In vitro release profile of cisplatin-loaded PLGA NPs andcisplatin-loaded Pt-PLGA NPs in phosphate buffer saline at pH� 7.4 and�5.5. Cisplatin-loaded Pt-PLGA NPs and PLGA NPs at pH� 5.5released 85.32% and 95.41% of the drug, respectively, which wassignificantly (one-way ANOVA test, p50.05) higher than 70.53% and75.34% of cisplatin released at pH� 7.4. The study was carried out intriplicate (n¼ 3).

DOI: 10.3109/02652048.2014.913725 Pt-PLGA NPs were synthesized to augment brain delivery 7

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charged cell surface proteoglycans. Cellular uptake then pro-ceeds by mechanisms of lipid raft-mediated endocytosis anddirect translocation (Xia et al., 2011). Thus, cisplatin-loadedPt-PLGA NPs have shown promising in vitro results in drugdelivery across BBB.

Conclusion

We have innovatively constructed non-aggregated Pt-PLGA NPsto augment the brain uptake by AMT mechanism. The tailoredcisplatin nanoformulation exhibited a sustained release effect overthree weeks time period. Potentially, Pt-PLGA NPs crossedin vitro BBB model and improved the therapeutic index inglioblastoma cells. Hence, this investigation demonstrated alucrative technology for the synthesis of peptide modifiednon-aggregated nanoparticles of cisplatin for brain targeting.In conclusion, research outcome of this investigation has pavedthe way for a systematic and rationale approach towards the

development of potential non-aggregated nanoparticles of cis-platin. Thus, cisplatin-loaded Pt-PLGA NPs could be used asa roadmap for preclinical tumour regression study to furtherimprove its utility against glioblastoma.

Acknowledgements

We acknowledge, National Institute of Pharmaceutical Education andResearch, Mohali, Panjab, India, for the providing the facility of TEM.

Declaration of interest

The authors report no conflict of interest. The authors alone areresponsible for the content and writing of this article.

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Figure 7. (A) Cell viability assay performed in U87 human glioblastoma cell line. The IC50 value of cisplatin, cisplatin-loaded PLGA NPs andcisplatin-loaded Pt-PLGA NPs was found to be 13.33 mM43.9mM and42.1 mM (one-way ANOVA, p50.05). Blank nanoparticles did not induce anycytotoxicity to U87 glioblastoma cells; (B) cellular uptake expressed in terms of mean fluorescence intensity and percent cellular accumulation.The accumulation of PLGA NPs and Pt-PLGA NPs were accounted to be 44.2 ± 6.03% and 51.9 ± 6.76% with a significant (two-way ANOVA;p50.05) difference in the binding capacity; (C) representative differential interference contrast and fluorescent image of cellular uptake of PLGA NPsand Pt-PLGA NPs at 24-h incubation period. Scale bar �20 mm. The study was carried out in triplicate (n¼ 3).

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