Improved cisplatin delivery in cervical cancer cells by utilizing folate-grafted non-aggregated gelatin nanoparticles

Biomedicine & Pharmacotherapy 69 (2015) 1–10

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Original Article

Improved cisplatin delivery in cervical cancer cells by utilizing folate-grafted non-aggregated gelatin nanoparticles Nishu Dixit a, Kumar Vaibhav b, Ravi Shankar Pandey c, Upendra Kumar Jain a, Om Prakash Katare d, Anju Katyal b, Jitender Madan a,* a

Department of Pharmaceutics, Chandigarh College of Pharmacy, Mohali, Panjab, India Dr. B.R. Ambedkar Centre for Biomedical Research, University of Delhi, Delhi, India SLT Institute of Pharmaceutical Sciences, Guru Ghasidas University, Bilaspur, India d University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 11 September 2014 Accepted 16 October 2014

Purpose: Cisplatin is highly effective in the treatment of cervical cancer. However, in therapeutic doses, cisplatin induces several adverse effects due to undesirable tissue distribution. Therefore, it is worth targeting cisplatin in cervical cancer cells by implicating non-aggregated ligand-modified nanotherapeutics. Methods and results: Here, we report the preparation of non-aggregated folic acid-conjugated gelatin nanoparticles of cisplatin (Cis-GNs-FA) by two-step desolvation method with mean particle size of 210.6  9.6 nm and 140.5  10.9 nm for Cis-GNs to improve the drug delivery in cervical cancer, HeLa cells. FTIR and DSC spectra confirmed the presence and stability of cisplatin in gelatin matrix. Furthermore, amorphization of cisplatin in nanoparticles was ascertained by PXRD. Drug release followed a first-order release kinetic at both pH  5.6 (cervical cancer pH) and pH  7.4. In addition, a significant (P < 0.05) decrease in IC50 value (8.3 mM) and enhanced apoptosis were observed in HeLa cells treated with Cis-GNs-FA as compared to Cis-GNs (15.1 mM) and cisplatin solution (40.2 mM). In contrast, A549 lung cancer cells did not discriminate between Cis-GNs-FA and Cis-GNs due to the absence of folate receptors-a (FR-a). Consistently, higher cellular uptake, 80.54  7.60% was promoted by Cis-GNs-FA significantly (two-way ANOVA, P < 0.05) greater than 51.68  9.78%, by Cis-GNs. This was also illustrated by CLSM images, which indicated that Cis-GNs-FA preferably accumulated in the cytoplasm of HeLa cells nearby nucleus by following receptor-mediated endocytosis pathway as compared to Cis-GNs. Conclusion: Therefore, Cis-GNs-FA warrants further in-depth in vitro and in vivo investigations to scale up the technology for clinical translation. ß 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Cisplatin Folate Gelatin nanoparticles Cervical cancer Cytotoxicity Apoptosis Cellular uptake

1. Introduction Cisplatin alone or in combination with paclitaxel has been recommended for long-term survival of cervical cancer patients [1]. It induces cross-linking in DNA by interfering with cell division followed by sensitization of repair mechanism that activates apoptosis [2]. In addition, cisplatin also suppresses Bcl2l2 protein in cervical cancer cells [3]. Both cis and trans adducts of cisplatin inhibit replication of cancer cells but only cis isomer is biologically

Abbreviations: TNBS, Trinitro benzene sulfonic acid; Cis-GNs, Cisplatin loaded gelatin nanoparticles; Cis-GNs-FA, Cisplatin loaded folate-grafted non-aggregated gelatin nanoparticles; FR-a, Folate receptors-a. * Corresponding author. Department of Pharmaceutics, Chandigarh College of Pharmacy, Mohali, Panjab, India Tel.: +91 172 3984209; fax: +91 172 3984209. E-mail address: [email protected] (J. Madan). http://dx.doi.org/10.1016/j.biopha.2014.10.016 0753-3322/ß 2014 Elsevier Masson SAS. All rights reserved.

active [4]. Cisplatin in therapeutic dose (75 mg/m2 or 165 mg by intravenous route in single administration) exhibited half-life of 43 min with approximately 1/4th being eliminated within the first 24 h (90% renal clearance) [5]. Continuation to this, accumulation of cisplatin in renal parenchymal cells by membrane transporter protein, Ctr1 causes nephrotoxicity [6]. Neurotoxicity is associated with the formation of cisplatin-DRG neuron adduct [7]. Moreover, diffusion of cisplatin in cochlear hair cells stimulates the generation of reactive oxygen species that grounds ototoxicity [8]. Hence, cisplatin displayed potent anticancer activity against cervical cancer cells but associated with serious side effects. Therefore, it is worth targeting cisplatin in cervical cancer cells by utilizing surface modified nanocomposites. Targeting drugs via functionalized drug delivery systems at molecular receptors, over-expressed on cancer cells through active transport could diminish the side effects [9]. Specific drug delivery to cancer cells

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can be achieved by adding a ligand on to the surface of drug delivery systems [10]. These targeting moieties then penetrate the cancer cell compartments through over-expressed receptors and release the payload [11]. During the last decade, biodegradable polymeric nanoparticles ranging between 10–200 nm have gained lot of attention in anticancer drug delivery due to self-stability, high drug loading capacity and ability to deliver both hydrophobic and hydrophilic drugs [12,13]. Recently, we gained success in delivery and targeting cytotoxic drugs in cancer cells via functionalized nanovesicles [14–16]. Both low molecular weight (folate, biotin, galactose, glucose and mannose) and high molecular weight (transferrin and antibodies) ligands can be decorated over the surface of nanocomposites [17]. Targeting folate receptors (FRs), over-express in human cervical cancer cells, is a highly lucrative technology to deliver chemotherapeutic drugs. FRs may serve as a front door for the entry of drug delivery cargo in cervical cancer cells. Functional FRs (FRs-a) highly over-express in cervical cancer cells [18]. The FR-a is a glycosyl phosphatidyl inositol (GPI)linked protein that belongs to single-chain GPI-anchored membrane. Expression of FR-a in normal tissue is restricted to the apical surfaces of polarized epithelial cells that do not exposed to blood stream [19]. FR-a allows internalization of folate-conjugated nanovesicles due to high affinity (Kd1010M) and penetration via receptor-mediated endocytosis pathway [20]. In several investigations, folate-conjugated nanopharmaceuticals gained nondestructive intracellular delivery via FR-a receptors [21,22]. The cellular protein synthesis was inhibited in a time and dose dependent manner by folate-toxin conjugate into the cells, providing compelling evidence that conjugate reaches the cytoplasm in a functionally active form [23]. The noteworthy advantage of FR-a mediated endocytosis is the recognition of folate by the cells as essential and retained by the endocytic compartments rather than lysosomes [22,23]. Gelatin is a non-toxic, natural, hydrophilic polymer that is approved by FDA for human consumption. Its unique properties like biodegradability and non-immunogenicity offered fabrication of nanoscale based drug delivery systems for the delivery of cytotoxic drugs in cancer cells [15,24,25]. However, major obstructions, which limit the use of nanocomposites, are physical (aggregation/ particle fusion) and chemical instability (drug leakage and chemical reactivity) noticed on long-term storage of nanoparticles aqueous suspension [26]. Moreover, aggregation of nanoparticles also diminishes both therapeutic and diagnostic potentials of drug delivery systems. Aggregation reduces solubility, stability, and cellular uptake in cancer cells, since aggregated nanoparticles exhibit different traits as compared to non-aggregated vesicles. Therefore, in present investigation, gelatin was used to synthesize and optimize cisplatin loaded folate-grafted gelatin nanoparticles (Cis-GNs-FA) for targeting FR-a positive, human cervical cancer, HeLa cells. Furthermore, in vitro characterization was carried out using spectral and biological techniques to define the utility of customized and optimized nanoformulation against cervical cancer. 2. Materials and methods 2.1. Materials Cisplatin (purity  99.5%) and 2,4,6-Trinitrobenzene sulfonic acid (TNBS) were the gift samples from Cipla, Mumbai, and Panjab University, Chandigarh, India, respectively. Gelatin (Type B, 33.0 mol amine content/gelatin molecule) was purchased from Sigma-Aldrich, USA. Glutaraldehyde (25%v/v aqueous solution), folic acid (Mw  441.4), EDAC 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride and N-hydroxy succinimide (NHS) were purchased from Loba Chemie, Mumbai, India. All other

chemicals used were of highest analytical grade and used without further purification. 2.2. Cell culture Human cervical cancer cell line (FR-a positive, HeLa) and nonsmall cell lung cancer (FR-a-negative, A549) were grown separately in folic acid free Dulbecco’s Modified Eagles Medium (FA-free-DMEM) supplemented with 10% fetal bovine serum at 37oC in a humidified incubator with 5% CO2 [27]. 2.3. Synthesis of cisplatin loaded non-aggregated gelatin nanoparticles Cisplatin loaded gelatin nanoparticles (Cis-GNs) were prepared by previously optimized two-step desolvation method [24]. Briefly, 25 mL of gelatin B solution (5% w/v) was prepared at room temperature and desolvated using 25 mL of acetone to precipitate high molecular weight gelatin. The solution was then kept for sedimentation. Subsequently, supernatant liquid was discarded and the sediment was re-dissolved in 25 mL of distilled water containing 1 mg/mL of cisplatin at pH  2.5. Acetone was again added drop-wise to form cisplatin loaded gelatin nanoparticles (Cis-GNs). Nanoparticles were further cross-linked with 500 mL of glutaraldehyde. The excess of glutaraldehyde was neutralized by adding 500 mg of glycine. Purification was done by centrifugation at 40,000 rpm in an ultracentrifuge (Thermo Scientific, Sorvall Ultra Centrifuge). The resultant pellet of nanoparticles was lyophilized (Lab India, Thane, India) in the presence of 5% w/v trehalose (lyoprotectant) to produce non-aggregated Cis-GNs. 2.4. Synthesis and optimization of non-aggregated folate-grafted cisplatin loaded gelatin nanoparticles Non-aggregated folate-grafted cisplatin loaded gelatin nanoparticles (Cis-GNs-FA) were synthesized using covalent coupling technique [28]. Briefly 10 mg of folic acid (FA) was added to a mixture of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDAC.HCl) and N-hydroxy succinimide (NHS) (6 mg:11 mg) prepared in 10 mL of DMSO. The activation was carried out under inert atmosphere for 30 min. The activated FA solution was then added drop-wise to different aliquots of nanoparticle solution, prepared by suspending 40 mg of Cis-GNs in 10 mL of sodium carbonate buffer (pH  10) and stirred for 3, 6, 12 and 24 h. Reaction was stopped by adding 500 mg/mL of hydroxylamine to each aliquot. In last, all Cis-GNs-FA nanoformulations were ultracentrifuged (Thermo Scientific, Sorvall Ultra Centrifuge) at 40,000 rpm for 2 h. The resultant pellet of Cis-GNsFA in each aliquot was lyophilized in presence of 5% w/v trehalose that consequently produced non-aggregated nanoparticles. The supernatant liquid in each aliquot was analyzed for un-reacted FA by using an UV/Visible spectrophotometer (1800, Shimadzu, Kyoto, Japan) at 363 nm [29]. The FA content was calculated from the standard curve and expressed in terms of n.mol/mg of nanoformulation. The conjugation of FA over the surface of optimized Cis-GNs-FA was also confirmed by differential scanning calorimeter (Mettler-Toledo Thermal Equipment, USA). Nitrogen was used as a carrier gas at the flow rate of 40 mL/min. A 10 mg quantity of sample was scanned at a heating rate, 20 8C/min in the temperature range of 30 8C to 300 8C. 2.5. Determination of free amine groups The free amine groups in Cis-GNs and optimized Cis-GNs-FA were determined by a 2, 4, 6-trinitro benzene sulfonic acid (TNBS) assays [30]. TNBS, being a sensitive reagent, rapidly

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determines the free amine groups in gelatin polymer. Briefly, 10 mg quantity of Cis-GNs and optimized Cis-GNs-FA were mixed separately with 1 mL of 4% w/v sodium bicarbonate (pH  8.5) solution and 1 mL of 0.5% TNBS reagent. Both solutions were heated at 40 8C for 4 h with mild shaking. This was followed by addition of HCl (6N, 3 mL) to both mixtures and heating at 70 8C for 1 h to hydrolyze and dissolve any insoluble material. The hydrolysate of both samples was then diluted with 5 mL of distilled water followed by extraction with diethyl ether. A 5 mL quantity of the aqueous phase was removed from each sample and heated for 15 min in a hot water bath. Subsequently samples were cooled to room temperature and diluted with 15 mL of distilled water. The absorbance in each sample was measured at 346 nm by using an UV/visible spectrophotometer (1800, Shimadzu, Kyoto, Japan). 2.6. Conjugation of fluorescein isothiocynate to nanoparticles Both Cis-GNs and Cis-GNs-FA were conjugated to fluorescein isothiocynate (FITC) by utilizing primary amines group [31]. In brief, 100 mg of Cis-GNs and optimized Cis-GNs-FA were dispersed separately in 3 mL of borate buffer (pH  8.5) and labeled as solution A and B, respectively. Separately FITC solution in borate buffer (pH  8.5) was prepared (1 mg/3 mL) and labeled as solution C. Both solutions A and B were then mixed separately with solution C and incubated for 3 h at room temperature. The mixture was then dialyzed against distilled water until the residual FITC was not completely removed. To determine the labeling efficiencies, the fluorescence intensity of solutions of nanoparticles dissolved in PBS, pH  7.4 was measured. The fluorescence intensity (Spectra Fluor, Tecan, Switzerland, lexe  485 nm, lemi  535 nm) was calibrated with standard solutions of 0.005 to 0.013 mg/mL of FITC prepared by diluting 100 mg/mL of methanolic solution of FITC with PBS, pH  7.4. Labeling efficiency (%) was calculated as the percent weight of FITC to weight of the FITC-labeled nanoparticles.Y = 1605x – 8.699, R2 = 0.994 Furthermore, the release of FITC from FITC conjugated Cis-GNs and Cis-GNs-FA was performed in acetone, PBS (pH  7.4) and FAfree-DMEM [32] for 24 h to exhibit the stability of fluorescent nanoformulations in cell culture medium. The fluorescence intensity was measured using a fluorometer (Spectra Fluor, Tecan, Switzerland, lexe  485 nm, lemi  535 nm). 2.7. Characterization of nanoparticles 2.7.1. Particle size distribution and zeta-potential Particle size distribution and surface charge analysis of nanoparticles were performed by a laser doppler anemometry using a zeta-sizer (Malvern, Instruments, Worcestershire, UK). Briefly, 200 mL quantity of each nanoparticle suspension was dispersed in 5 mL of PBS (10 mM, pH  7.4) and the mean particle size was determined. An electric field of 150 mV was applied to observe the zeta-potential of nanoparticles samples. All measurements were made in triplicate (n  3) at 25 8C. 2.7.2. Transmission electron microscopy (TEM) Particle shape and surface topography were examined using transmission electron microscopy (TEM, FTI Tecnai F20). An aqueous dispersion of each nanoparticles suspension was drop cast onto a carbon-coated grid which was then air dried at room temperature before loading into microscope, maintained at a voltage of 80 kV. 2.7.3. Fourier-transforms infrared (FTIR) spectroscopy Fourier-transforms infrared (FTIR) spectroscopy was employed to record the spectrum of cisplatin, GNs, physical mixture of

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cisplatin and GNs and Cis-GNs using infrared spectrophotometer (Perkin Elmer, Massachusetts, USA). Samples were prepared in a KBr disk (2 mg sample in 200 mg KBr) with a hydrostatic press at a force of 40 psi for 4 min. The scanning range employed was 4400– 400 cm1 at a resolution of 4 cm1. 2.7.4. Differential scanning calorimetry (DSC) The endothermic peaks of cisplatin, GNs, physical mixture of cisplatin and GNs and Cis-GNs were recorded using differential scanning calorimeter (Mettler-Toledo Thermal Equipment, USA) according to the conditions specified earlier. 2.7.5. Powder X-ray diffraction (PXRD) The crystalline structure of cisplatin, GNs, physical mixture of cisplatin and GNs, and Cis-GNs was compared by x-ray diffractometer (X’Pert PRO, Panalytical Company, Netherlands) using Nifiltered, CuKa-radiation, voltage of 60 Kv and current of 50 mA. The scanning rate employed was 18/min over the 108 to 608 diffraction angle (2u) range. 2.7.6. Nanoencapsulation efficiency and drug loading capacity The nanoencapsulation efficiency of Cis-GNs and optimized Cis-GNs-FA was determined by dissolving 50 mg quantity of each nanoparticles sample in 10 mL distilled water. Both samples were warmed for few minute and kept for 48 h. After this treatment, samples were ultracentrifuged (Thermo Scientific, Sorvall Ultra Centrifuge) at 40,000 rpm for 2 h. The supernatant of each sample was filtered with 0.22 mm membrane filters (MDI, India). The absorbance of each filtered aliquot was measured at 301.2 nm by using an UV/Visible Spectrophotometer (1800, Shimadzu, Kyoto, Japan) [33]. All measurements were made in triplicate (n  3). The nanoencapsulation efficiency and drug loading capacity were calculated using the following formulas: Nanoencapsulation efficiency (%) = amount of drug extracted/ amount of drug added  100 Drug loading capacity = amount of drug entrapped/amount of nanoparticles 2.7.7. In vitro drug release Release kinetics of drug from suspension of nanoparticles was examined using dialysis technique, a method for the quantization of drug transport across a dialysis membrane [34]. In brief, 2 mL sample of Cis-GNs (165 mg of cisplatin  208 mg of CisGNs) and optimized Cis-GNs-FA (165 mg of cisplatin  224 mg of optimized Cis-GNs-FA) was filled separately in dialysis bags (12 kDa, Sigma) and dialyzed against 250 mL of PBS (10 mM/L at pH  5.6 and pH  7.4) [35] maintained at 37 8C. Both dissolution mediums were stirred at 100 rpm as recommended for the dissolution testing of parenteral products [36]. A 5 mL sample was withdrawn at different time intervals and replaced with fresh buffer of same pH to mimic sink conditions. The cisplatin concentration in the each sample was measured at 301.2 nm using an UV/Visible Spectrophotometer (1800, Shimadzu, Kyoto, Japan) [33]. Quantification of drug release was done using a mathematical model based on first-order release kinetic. This calculates the diffusion of drug from a dialysis membrane into the external dissolution medium as a function of time [24,25]. ln [C1–Qmo/VT] = –kMt + ln [kcQMo/kM–kCV]V1V2], where, C1 is the concentration of the drug in the dialysate (outer solution), QMo is the total amount of drug associated with the nanoparticles at time zero, kM is the first-order release rate constant, kC is the apparent permeability constant of dialysis tubing. V1 and V2 are volumes of dissolution medium outside and inside the dialysis bag, VT = V1 + V2, and KCV = kCVT = V1V2.

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2.8. Therapeutic efficacy testing of cisplatin loaded gelatin and folategrafted gelatin nanoparticles against cervical cancer cells

(Beckman Coulter, Inc., Fullerton, CA). The extent of apoptosis (in percent) was assessed by the formula:

2.8.1. In vitro cytotoxicity assay The cytotoxicity of tailored nanoformulations against cervical cancer cells (HeLa, FR-a positive) and non-small cell lung cancer (A549, FR-a- negative, as a negative control) cells was analyzed by using standard cell viability assay [37]. Briefly, 5  103 HeLa cells or A549 cells were separately placed in 200 mL of the serum DMEM (FA-free) filled in 96 wells microtitre plates. Subsequently, seeded HeLa cells or A549 cells were incubated with a gradient concentration of cisplatin, Cis-GNs and Cis-GNs-FA and respective blank nanoparticles equivalent to cisplatin concentration of 10 to 70 mM for 72 h. The cytotoxicity against HeLa cells was also determined in the presence of FA. FA (2 mM) was added in the microtitre plate containing HeLa cells 24 h before the addition of cisplatin solution, Cis-GNs, Cis-GNs-FA and respective blank nanoparticles. In last, 0.5 mg/mL quantity of MTT (3-[4,5dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) dye was added to each well of the microtitre plate and incubated for 4 h at 37 8C. The formazon crystals, formed after cell lysis, were dissolved using 100 mL of dimethylsulfoxide. The absorbance was read at 570 nm using 630 nm as reference wavelength by ELISA reader (Tecan, Switzerland).

Cell viability in control group
 Cell viability in drug treated group  100 % Apoptosis ¼ ðCell viability in control groupÞ

2.8.2. Determination of platinum concentration in HeLa cells The platinum (Pt) concentration in HeLa cells treated with tailored nanoformulations was determined by atomic absorption spectroscopy (AAS) [38]. Briefly 50  103 HeLa cells were grown in FA-free-DMEM in tissue culture petridishes. The cells were then treated with 10 mM quantity of cisplatin in cisplatin solution, CisGNs and optimized Cis-GNs-FA and subsequently incubated for 72 h at 37 8C. After incubation, cells were washed thrice with sterile PBS (pH  7.4) and trypsinized. Cell suspension was centrifuged at 6000 rpm (Thermo Scientific Corporation, USA) for 20 min and the pellet obtained was redispersed in 100 mL of icecold lysis buffer (1.0 mM DTT, 1.0 mM PMSF, 10 mM KCl, 10 mM MgCl2, pH  7.5) for 15 min. The process was repeated and finally the pellets were redispersed in 50 mL of ice-cold lysis buffer. The resulting suspension was centrifuged at 15000 rpm (Thermoscientific Corporation, USA) for 30 min and the cytosolic fraction of the cells was collected as supernatant. The pellet was resuspended in 40 mL of extraction buffer (1.0 mM DTT, 1.0 mM PMSF, 1.5 mM MgCl2, 0.2 M EDTA, 0.42 M NaCl, 25% glycerol, pH  7.9) and lysed. The lysate was shaken at 1000 rpm for 1 h at 4 8C and then centrifuged at 35000 rpm (Thermo Scientific, Sorvall ultracentrifuge) for 25 min at 4 8C. The supernatant containing nuclear fractions was used for determination of Pt concentration by AAS. The protein concentration in each sample was determined using bicinchoninic acid (BCA) dye [39]. The Pt concentration was expressed in ng of Pt/mg of protein. 2.8.3. Analysis of apoptosis The apoptosis analysis was carried out using standard fluorescence-activated cell sorting (FACS) assay [40]. In brief, 50  104 HeLa cells were treated with cisplatin solution, Cis-GNs, and optimized Cis-GNs-FA samples for 48 h at a concentration equivalent to  10 mM of cisplatin. The cells were then harvested with trypsin-EDTA, centrifuged and washed twice with ice-cold PBS. Subsequently, binding buffer (1 ) was added to each tube to make the final concentration of 5  104 cells/mL. Then 100 mL of cell suspension from each tube was taken in to 5 mL capacity FACS tube. Finally, 5 mL PE Annexin V + 5 mL 7-AAD was added to all FACS tubes. Cells were vortexed and incubated for 15 min at room temperature. After mixing, 400 mL of 1  binding buffer was added to each tube before analyses on a FACSCalibur flow cytometer

At least 10,000 cells were characterized by flow cytometry for apoptosis analysis. All tests were performed in doublets. 2.8.4. Cellular uptake assay: quantitative and qualitative analysis Both quantitative and qualitative cellular uptake assays were performed by fluorometry. In brief, HeLa were plated in Lab-Tek II Chamber SlideTM System (Nalge Nune, USA) at a density of 5  103 cells per chamber. Dosing solutions of FITC-labeled Cis-GNs and optimized Cis-GNs-FA (equivalent to 10 to 70 mM concentration of cisplatin) were prepared using PBS (pH  7.4) and diluted with FAfree-DMEM. Cell monolayers were rinsed thrice and pre-incubated with 1 mL of FA-free-DMEM at 37 8C for 1 h. Uptake was commenced when 1 mL of specified dosing solution was exchanged with culture medium, followed by incubation of the cells at 37 8C for 24 h. The experiment was terminated by washing the cell monolayer thrice with ice-cold PBS (pH  7.4) and cells were lysed with 1 mL of 0.5% Triton X-100. Cell associated FITC was quantified by analyzing the cell lysate in a fluorometer (Spectra Fluor, Tecan, Switzerland, lexe  485 nm, lemi  535 nm) [41]. The protein content of the cell lysate was measured using the BCA protein assay kit [39]. After 24 h incubation period, the medium was removed and plates were washed thrice with sterile PBS (pH  7.4). After the final wash, the cells were fixed with 4% paraformaldehyde and individual cover slips were mounted on clean glass slides with fluoromount-G mounting medium (Southern Biotechnology, Birmingham, AL). The slides were viewed under a confocal laser-scanning microscope (lexe  485 nm, lemi  535 nm). DAPI (4,6-diamidino-2-phenylindole) was used for nucleus staining [42]. Statistics Statistical analysis was performed using unpaired ‘‘t’’ test, oneway analysis of variance (ANOVA) and two-way analysis of variance tests using Graph Pad Prism04 Software. The results are presented as the mean  SD for n  3. The significance level of difference was taken as P < 0.05. 3. Results 3.1. Preparation and optimization of non-aggregated cisplatin loaded folate-grafted gelatin nanoparticles Cisplatin loaded gelatin nanoparticles (Cis-GNs) were prepared by optimized two-step desolvation method [24]. Trehalose was used as a lyoprotectant, which prevented aggregation of nanoparticles and reduced mechanical stress during freezing and drying processes [43]. Subsequently, folic acid (FA-COOH) was anchored onto the surface of drug loaded nanoparticles (–NH2) using conjugation chemistry [28]. This conjugation reaction yielded a stable amide bond with FA. The FA content was measured to be 6.3, 15.1, 25.4 and 25.9 n.mol/mg of Cis-GNs at the incubation period of 3, 6, 12 and 24 h, respectively. The Cis-GNs-FA nanoformulation with 25.9 n.mol FA and modification of 32% amine groups (preferably lysine) was designated as optimized nanoformulation. 3.2. Size and surface charge characterization of nanoparticles The mean particle size and zeta-potential of nanoparticles were determined to predict the in vitro and in vivo behavior of

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nanoformulation in physiological milieu. The mean particle size of optimized Cis-GNs-FA was significantly (Unpaired ‘‘t’’ test, P < 0.05) higher than Cis-GNs and measured to be 210.6  9.6 nm and 140.5  10.9 nm, respectively (Table 1 and Fig. 1A–B). Continuation to this, zeta-potential of about – 11.79  0.49 mV was measured for optimized Cis-GNs-FA, significantly (unpaired ‘‘t’’ test, P < 0.05) lesser than –85.82  0.28 mV of Cis-GNs. 3.3. Surface topography analysis The TEM images of optimized Cis-GNs-FA and Cis-GNs corroborated the non-aggregation of nanoparticles. In addition, nanoparticles were smooth and spherical in shape (Fig. 1A–B). The coating of ligand, FA is clearly visible over the surface of nanoparticles. Photomicrographs also indicated that ultracentrifugation and freeze-drying steps did not influence the nanoparticle texture. 3.4. Fourier-transform infrared (FTIR) spectroscopy FTIR spectra were recorded to analyze any chemical incompatibility between cisplatin and gelatin polymer during encapsulation process. The assignment of FTIR scans indicated that cisplatin exhibits its characteristic peak at 3450 cm1 (v,–N–H stretching vibrations of NH2), 3282 cm1(v, –O–H stretching of –H2O) and 743 cm1 (v,–C–Cl–stretching of –Cl2) (Table 2). The spectrum of GNs showed the characteristics protein backbone between 1540 cm1 and 1650 cm1 (multiple peak absorption patterns, typical of protein backbone). Another peak was observed at 1462 cm1 (vibrational, aldimine stretching, cross-linking of gelatin). These peaks were almost unchanged in physical mixture as well as in Cis-GNs. Hence, the FTIR analysis indicated the presence and stability of cisplatin in tailored Cis-GNs. 3.5. Differential scanning calorimetry DSC was performed to confirm the physical state of cisplatin in nanoparticle matrix. We observed a sharp endothermic peak at 270.83 8C for cisplatin (Fig. 2A–E) close to its melting point (270 8C). The thermogram of GNs showed a broad peak at 65.3 8C. The physical mixture of cisplatin and GNs indicated that the endothermic peak was slightly shifted to 260.32 8C and 65.41 8C, respectively from its original peaks. However, Cis-GNs exhibited a very broad peak at 69.98 8C indicating the loading of cisplatin in molecularly dispersed state in GNs. The modification of Cis-GNs with FA also displayed a broad endothermic peak without any significant shift [44].

Fig. 1. Particle size distribution, zeta-potential analysis and transmission electron microscopy of (A) cisplatin loaded gelatin nanoparticles (Cis-GNs), and (B) optimized cisplatin loaded folate-grafted gelatin nanoparticles (Cis-GNs-FA). All measurements were carried out in triplicate (n  3).

displayed few sharp peaks beside some undefined broad, diffused peaks of low intensities, characteristics of both components. In last, PXRD of Cis-GNs demonstrated deformed and diffused peaks of very low intensities. This confirmed the amorphous configuration of cisplatin in GNs. 3.7. Cisplatin-loading onto gelatin nanoparticles There was no significant difference (Unpaired ‘‘t’’ test P > 0.05) noticed between nanoencapsulation efficiency (63.5  3.6% and 61.2  5.9%) and drug loading capacity (7.9 mg/10 mg and 7.37 mg/ 10 mg) of Cis-GNs and optimized Cis-GNs-FA, respectively (Table 1).

3.6. Powder X-ray diffraction (PXRD) pattern 3.8. Drug release kinetics from gelatin nanoparticles The crystalline architecture of cisplatin in tailored nanoformulation was elucidated using PXRD technique. The PXRD pattern of cisplatin showed peaks that were intense and sharp, indicating its crystalline state (Fig. 3). Subsequently GNs exhibited few sharp peaks of low intensities. The physical mixture of cisplatin and GNs

In vitro release of cisplatin from nanoparticles was analyzed by dynamic dialysis method [34] in PBS of pH  5.6 and 7.4 to simulate the cancer cell compartment [35] and normal body physiology milieu. Release kinetic of a drug from the dialysis

Table 1 Characterization parameters for cisplatin loaded nanoparticles. Sample

Particle sizea (nm)

Zeta-potentiala (mV)

Nanoencapsulationb Efficiency(%)

Drug loading capacityb (cisplatin/nanoparticles)

Cis-GNs Cis-GNs-FA

140.5  10.9 nm 210.6  9.6 nm

–85.82  0.28 –11.79  0.14

63.5  3.6 61.2  5.9

7.93 mg/10 mg 7.37 mg/10 mg

Note: Cis-GNs: cisplatin loaded gelatin nanoparticles; Cis-GNs-FA: cisplatin loaded folate-grafted gelatin nanoparticles. a P < 0.05 (unpaired ‘‘t’’ test). b P > 0.05 (unpaired ‘‘t’’ test).

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Table 2 FTIR assignments of cisplatin, gelatin nanoparticles, physical mixture and cisplatin loaded gelatin nanoparticles. Formulation

FTIR peaks

Assignments

Cisplatin

3450 cm1 3282 cm1 743 cm1 1425, 1388 cm1 3549 cm1 2932 cm1 1646 cm1 1540–1650 cm1

v, –N–H stretching of –NH3 v, –O–H– stretching of –H2O v, –C–Cl– stretching of –Cl2 v, N–CH3 v, N–H stretching of CONH– v, C–H stretching v, C = O– Multipeak absorption pattern, (Typical of protein backbone) Aldimine stretching, cross-linking of gelatin v, –N–H stretching of –NH3

Blank GNs

1462 cm1 Physical mixture

3451 cm1 1

1646 cm 1540–1650 cm1

Cis-GNs

741 cm1 3500–3100 cm1 1650 cm1 1540–1650 cm1 742 cm1

V, > C = O Multiple peak absorption pattern, (Typical of protein backbone) v, –C–Cl– stretching of –Cl2 v, N–H amide bond with carboxylic acid v, C = O– Multiple peak absorption pattern, (Typical of protein backbone) v, –C–Cl– stretching of –Cl2

Note: GNs: gelatin nanoparticles; Cis-GNs: cisplatin loaded gelatin nanoparticles.

membrane depends on the permeability constant, which was measured by inserting 100 mg/mL of cisplatin inside the dialysis bag followed by measuring the drug concentration in the receptor chamber (C1) as a function of time. Using the equation for

Fig. 2. Differential scanning calorimetry of (A) cisplatin, (B) gelatin nanoparticles (GNs), (C) physical mixture of cisplatin and gelatin nanoparticles (D) cisplatin loaded gelatin nanoparticles (Cis-GNs) and, (E) optimized cisplatin loaded folategrafted gelatin nanoparticles (Cis-GNs-FA).

Fig. 3. Powder X-ray diffraction pattern of cisplatin, gelatin nanoparticles (GNs), physical mixture of cisplatin and gelatin nanoparticles (Cisplatin and GNs), and cisplatin loaded gelatin nanoparticles (Cis-GNs).

permeability constant, KCV was calculated (0.052/h/mL). The intercepts on the axis gave the value of the original amount of cisplatin present inside the dialysis membrane, which was approximately 99.8 mg, almost identical to the original amount showing that adsorption of the drug by the dialysis membrane was negligible. A representative graph of the release kinetics of cisplatin from Cis-GNs and optimized Cis-GNs-FA is shown in Fig. 4A. Drug release was found to follow a first-order release

Fig. 4. In vitro (A) release kinetic, and (B) release profile of cisplatin loaded gelatin nanoparticles (Cis-GNs) and optimized cisplatin loaded folate-grafted gelatin nanoparticles (Cis-GNs-FA) in phosphate buffer saline (pH  5.6 and pH  7.4). Optimized Cis-GNs-FA and Cis-GNs at pH  5.6 released 85.32% and 95.41% of cisplatin, significantly (one-way ANOVA, P < 0.05) greater than 70.53% and 75.34% of the drug released at pH  7.4. The dissolution testing was carried out in triplicate (n  3).

N. Dixit et al. / Biomedicine & Pharmacotherapy 69 (2015) 1–10

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Table 3 Release rate constants as determined by dynamic dialysis method for cisplatin loaded nanoparticles. Samples

pH  5.6

pH  7.4

km1 (mg/h)

km2 (mg/h)

km = km1–km2

km1

km2

km = km1–km2

Cisplatin Cis-GNs Cis-GNs-FA

5.14 2.7  101 4.7  101

5.3  102 4.3  102 1.04  101

5.09 2.3  101 3.7  101

7.68 4.5  101 8.0  101

4.1  101 1.2  101 3.0  101

7.26 3.3  101 4.9  101

Note: Cis-GNs: cisplatin loaded gelatin nanoparticles; Cis-GNs-FA: cisplatin loaded folate-grafted gelatin nanoparticles. km1: initial release rate constant. km2: terminal release rate constant.

kinetic at both, pH  5.6 and pH  7.4 with characteristics of biphasic release. Release rate constants for two phases were given by the slopes and are presented in Table 3. The Cis-GNs and optimized Cis-GNs-FA released 95.41  3.63% and 85.32  3.27% of cisplatin at pH  5.6, that were significantly (one-way ANOVA, P < 0.05) higher than the 75.34  2.92% and 70.53  2.53% of cisplatin released by Cis-GNs and Cis-GNs-FA, respectively at pH  7.4 (Fig. 4B). 3.9. Optimized Cis-GNs-FA exhibited enhanced efficacy against HeLa cells

did not observe any significant difference (Unpaired ‘‘t’’ test, P < 0.05) between IC50 of Cis-GNs-FA (21.8 mM) and Cis-GNs (20.2 mM), observed against A549 cells. Subsequently, platinum (Pt) concentration was measured by atomic absorption spectroscopy (AAS) [37] in HeLa cells (Fig. 5B). The optimized Cis-GNs-FA nanoformulation reported 76.8  8.3 ng of Pt/mg of protein significantly (one-way ANOVA test, P < 0.0001) higher than CisGNs (45.1  5.9 ng of Pt/mg of protein) and cisplatin alone (9.6  2.3 ng of Pt/mg of protein) incubated at the concentration equivalent to  10 mM of cisplatin for 72 h. 3.10. Optimized Cis-GNs-FA induced apoptosis in HeLa cells

MTT assay [37] was used to determine the cytotoxicity induced by cisplatin, Cis-GNs, optimized Cis-GNs-FA and respective blank nanoformulations in human cervical cancer, HeLa (FR-a positive) cell line cultured in FA-free-DMEM. A549 (FR-a negative) was taken as negative control. The IC50 (concentration of the drug required to kill 50% of the cells) value of cisplatin solution (40.2 mM) and Cis-GNs (15.1 mM) was significantly (one-way ANOVA, P < 0.05) higher than the optimized Cis-GNs-FA (8.3 mM), measured against HeLa cells (Fig. 5A). Furthermore, the IC50 of cisplatin solution, Cis-GNs, and Cis-GNs-FA was measured in the presence of FA. Cisplatin solution, Cis-GNs and Cis-GNs-FA exhibited the IC50 of 38.4 mM, 16.8 mM and 14.9 mM respectively, against HeLa cells when cultured in presence of FA. In contrast, we

The extent of apoptosis was determined by measuring the cell viability using flow cytometry assay [40]. The optimized nanoformulation, Cis-GNs-FA induced 60% apoptosis, significantly (oneway ANOVA, P < 0.05) greater than Cis-GNs (42%) and cisplatin solution (24%) in HeLa cells. 3.11. Optimized Cis-GNs-FA enhanced cellular accumulation in HeLa cells: quantitative and qualitative analysis We determined quantitatively and qualitatively the accumulation of nanoparticles in HeLa cells by trafficking FITC-labeled CisGNs and optimized Cis-GNs-FA using a fluorometer and CLSM

Fig. 5. (A) In vitro cytotoxicity analysis of cisplatin, cisplatin loaded gelatin nanoparticles (Cis-GNs) and optimized cisplatin loaded folate-grafted gelatin nanoparticles (CisGNs-FA) carried out against human cervical cancer, HeLa cells and A549 cells, in presence and absence of folic acid in culture medium. The IC50 of optimized Cis-GNs-FA (8.3 mM) was significantly (one-way ANOVA, P < 0.05) lower than the Cis-GNs (15.1 mM) and cisplatin solution (40.2 mM), analysis carried out in absence of folic acid in culture medium. Continuation to this, cisplatin solution, Cis-GNs and Cis-GNs-FA exhibited the IC50 of 38.4 mM, 16.8 mM and 14.9 mM respectively, against HeLa cells when cultured in the presence of folic acid in culture medium. In contrast, no significant difference (unpaired ‘‘t’’ test) between IC50 of Cis-GNs-FA (21.8 mM) and Cis-GNs (20.2 mM) was observed against A549 cells, (B) Platinum (Pt) concentration in HeLa cells incubated with Cis-GNs-FA, Cis-GNs and cisplatin solution for 72 h at the concentration  10 mM of cisplatin, measured by atomic absorption spectroscopy (AAS), (C) Apoptosis assay carried out using fluorescent-activated cell sorting (FACS) protocol for control HeLa cells, cisplatin solution ( 10 mM cisplatin), Cis-GNs ( 10 mM cisplatin), and Cis-GNs-FA ( 10 mM cisplatin) treated HeLa cells, at 48 h incubation period. Percentage of viable cells is indicated. Dot plots show flow cytometric analysis of cells stained with PE Annexin and 7-AAD. Y-axis denotes 7-AAD and X-axis FITC-Annexin V.

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Fig. 6. Cellular uptake analysis in human cervical cancer, HeLa cells (A) quantitative analysis indicated significantly (P < 0.05, two-way ANOVA) higher cellular accumulation (80.54  7.60%) by FITC-labeled optimized cisplatin loaded folate-grafted gelatin nanoparticles (Cis-GNs-FA) as compared to 51.68  9.78% by cisplatin loaded gelatin nanoparticles (Cis-GNs) expressed in terms of mean fluorescence intensity and percent cellular accumulation, Fluorescence value of nanoformulations at time ‘0 h’ was designated as 100%. (B) Confocal laser-scanning microscope (CLSM) images of cellular uptake of FITC-labeled optimized Cis-GNs-FA that showed higher accumulation preferentially in the cytoplasm of HeLa cells as compared to Cis-GNs. DAPI was used for nucleus staining. Nanoparticles are showing green color fluorescence. Scale bar  20 mm.

(Spectra Fluor, Tecan, Switzerland) [41,42]. Both fluorescent nanoformulations were stable in cell culture medium, as determined by in vitro release of FITC from FITC-labeled Cis-GNs and optimized Cis-GNs-FA. We observed negligible, 2.5% and 3.1% release of FITC in FA-free-DMEM and PBS (pH  7.4) respectively, as compared to 34.5% in acetone in 24 h. Furthermore, optimized, Cis-GNs FA showed maximally (P < 0.05, two-way ANOVA) 80.54  7.60% cellular accumulation as compared to 51.68  9.78% by Cis-GNs in HeLa cells, treated with a gradient concentration of nanoparticles at the concentration equivalent to 10–70 mM of cisplatin (Fig. 6A). Consistent with the quantitative results, photomicrographs of cellular uptake of optimized Cis-GNs-FA and Cis-GNs in HeLa cells confirmed the qualitative analysis (Fig. 6B). Optimized Cis-GNs-FA exhibited higher accumulation preferentially in the cytoplasm of HeLa cells, therefore, depicted comparatively elevated fluorescence, as compared to Cis-GNs. 4. Discussion Use of antineoplastic agents through intravenous route of administration presents the possibility of severe side effects and

toxicity due to undesirable biodistribution. In addition, poor vascularization and impaired blood supply pose impediments for the delivery of antitumor drugs in tumor tissue. Eventually, an abnormal extracellular matrix offered increased frictional resistance to antitumor drug penetration [45,46]. Active targeting promotes the delivery of cytotoxic drugs at site of action and minimizes its exposure to normal tissues. Therefore, we have synthesized, optimized and constructed non-aggregated folateanchored gelatin nanoparticles of cisplatin (Cis-GNs-FA) for active targeting in human cervical cancer cells by using two-step desolvation method [24,28]. This method is associated with high practical yield, greater entrapment efficiency and high stability. Furthermore, trehalose was incorporated as a lyoprotectant during lyophilization step to prevent the aggregation of nanoparticles. Density of the ligand, FA over the surface of nanoparticles remarkably influenced the therapeutic efficacy of encased cytotoxic drug [29]. Hence, optimized Cis-GNs-FA was tested by using various spectral and biological techniques for establishing its targeting potential against cervical cancer cells. Particle size and zeta-potential play important roles in determining the nanoparticles fate following intravenous administration [47]. Previous

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reports have provided compelling evidences that intracellular uptake of nanoparticles in tumor tissue was influenced by the size of nanoparticles [48,49]. Nanoparticles of diameter between 100– 300 nm preferentially accumulated in tumor tissue rather than normal tissues. This may be attributed to increased permeability of tumor vasculature and enhanced permeation and retention effect (EPR) [48]. The particle size of Cis-GNs and optimized Cis-GNs-FA was measured to below > 200 nm with remarkable decrease in zeta-potential in ligand anchored nanoparticles (Table 1 and Fig. 1A–B). The isoelectric point of gelatin B is equivalent to 4.5. A reduction in pH (pH  2.5) during desolvation promoted protonation of the free amine groups (+NH3 groups) and facilitated cross-linking of gelatin with glutaraldehyde. It is reported that increasing or decreasing the pH from isoelectric point of gelatin causes a shift either in net negative or positive charge owing to ionization of either –COOH or –NH2 group, respectively [24]. Addition of a buffer of pH  7.4 exhibited the base effect only on the carboxyl groups, as the amine groups are already cross-linked with glutaraldehyde. It ionized the –COOH in to COO and H+ ions. Hence, ionization at pH  7.4 indicated the higher negative zetapotential of Cis-GNs. Compared to this, Cis-GNs-FA reduced the higher negative zeta-potential to lower value and confirmed the coating of ligand over the surface of nanoparticles (Table 1 and Fig. 1A–B). Modification with ligand also enhanced the particle size but retained the original texture of nanoparticles (Fig. 1A–B). In addition, ligand coating did not alter the nanoencapsulation efficiency and drug loading capacity significantly (Table 1). Next, we attempted to describe the chemical and physical stability of drug in gelatin matrix using FTIR, DSC and PXRD spectral techniques. FTIR spectra confirmed that cisplatin even after encapsulation in nanoparticles did not form any chemical linkage with the gelatin macromolecule (Table 2). DSC thermogram of drug loaded nanoparticles as compared to individual components and physical mixture assured that cisplatin was molecularly dispersed in gelatin matrix without any significant alteration in physical and chemical profile (Fig. 2A–E). The crystalline configuration of cisplatin in nanoparticle matrix was ascertained by PXRD (Fig. 3). The crystal structure of cisplatin was deformed into amorphous phase after encasing in the nanoformulation. Amorphous state due to the absence of an ordered crystal structure requires minimal energy and thus offers maximal solubility advantage to further enhance the bioavailability of drugs in vivo. However, drawbacks of amorphous system such as physical instability and higher chemical reactivity act as barriers in their extensive commercialization. Therefore, polymeric nanoparticles are usually employed to tender an amorphous matrix in which the drug can dissolve, whereby the viscosity of the matrix is usually high enough to prevent recrystallization on storage [24,25]. Next, the therapeutic efficacy of tailored cisplatin nanoformulations was analyzed in vitro following dissolution testing, cytotoxicity assay, flow cytometry and cellular uptake assay. Dissolution studies displayed variation in the release rate at pH  5.6 and  7.4 (Fig. 4A–B). We observed first-order release kinetic, characterized by initial burst followed by a slow release, for both optimized Cis-GNs-FA and CisGNs, respectively. The drug release from hydrophilic polymeric network depend on the rate of water uptake, drug dissolution and polymer glass-rubbery transition including matrix erosion or degradation rate [24,25]. The optimized, Cis-GNs-FA owing to coating of ligand rendered the water uptake and thereby suppressed the release rate. The initial (or first phase) release rate constant was greater than the terminal (or second phase) release rate constant showing that both the surface adsorbed (free drug) and the entrapped drug were released in the first phase, whereas only the entrapped drug was released in the second phase (Table 3). Nanoparticles meant for targeting solid tumors must be able to pass through the systemic circulation and reach the tumor

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mass in therapeutic concentration, get transported across the microvessels and diffuse into the cancer cells [35]. However, irregular blood supply, high interstitial pressure, acidified microenvironment of tumor cells, hypoxia, and lack of lymphatic system lead to sub-therapeutic uptake of nanoparticles Moreover, significant hydrostatic pressure gradient across the cancer cell also restricts the diffusion of cytotoxic drugs [50]. Thus, Cis-GNsFA formulation has illustrated maximum release of cisplatin at acidic milieu rather than physiological pH. Next, in vitro cytotoxicity assay was performed against human cervical cancer, HeLa cells cultured in FA-free-DMEM. The optimized Cis-GNs-FA exhibited enhanced cytotoxicity against HeLa cells in terms of low IC50 and higher Pt concentration as compared to Cis-GNs and cisplatin solution (Fig. 5A–B). However, in vitro cytotoxicity assay carried out in presence of FA did not reduce the IC50 of Cis-GNs-FA remarkably as compared to Cis-GNs due to blockage of FR-a on HeLa cells (Fig. 5A). Hence, selective uptake of Cis-GNs-FA due to over-expressed FR-a receptors on HeLa promoted active targeting while Cis-GNs and cisplatin did not [29]. On the other hand; A549 cells prohibited the active uptake of Cis-GNs-FA due to absence of FR-a. Thus, Cis-GNs-FA and Cis-GNs were found to be almost equally potent against A549 cells (Fig. 5A). Apoptosis analysis determined by flow cytometry [40] in HeLa cells treated with three nanoformulations illustrated that optimized Cis-GNs-FA induced greater extent of apoptosis due to enhanced drug delivery exposure, as compared to Cis-GNs and cisplatin solution (Fig. 5C). In continuation to determination of therapeutic efficacy, our next step was to perform quantitative and qualitative cellular uptake assay of optimized Cis-GNs-FA and Cis-GNs nanoformulations in HeLa cells. HeLa cells are known to recognize FA via surface bound FR-a [22,23]. Mechanistically ligand-receptor complex (FAFR-a complex) collects in specialized area of the plasma membrane, known as coated pits. These coated pits penetrate the cytoplasm and form coated vesicles. After acidification of the endosome, ligand dissociates from the receptor (FR-a) and transports to the other side of membrane. In this way, it recycles back to the cell surface. The quantification of Cis-GNs and optimized Cis-GNs-FA in HeLa cells was done by fluorometry after incubation for 24 h (Fig. 6A–B). The percent accumulation of Cis-GNs-FA in HeLa cells was remarkably greater than Cis-GNs. The percentage of incorporated Cis-GNs-FA in HeLa cells was increased as a function of the concentration of nanoparticles. These results indicated that Cis-GNs-FA had a specific affinity for the cancerous, HeLa cells owing to ligand-receptor (FA-FR-a) recognition [51]. Qualitatively, it is also depicted in photomicrographs that optimized Cis-GNs-FA preferentially accumulated in the cytoplasm of HeLa cells nearby nucleus via receptor-mediated endocytosis pathway after 24 h of incubation period, whilst CisGNs were not incorporated sufficiently. These results confirmed that optimized Cis-GNs-FA might potentially be used for targeting cisplatin in cervical cancer cells. 5. Conclusions In summary, we have synthesized ligand-modified nonaggregated nanotherapeutics of cisplatin using folic acid and biocompatible polymer, gelatin for targeting human cervical cancer cells. Gelatin was successfully employed to prepare cisplatin loaded nanoparticles < 200 nm using optimized twostep desolvation method. Quantitative and qualitative studies revealed that ligand-modified nanotheraputics of cisplatin exhibited higher therapeutic efficacy against HeLa cells in terms of low IC50, higher degree of apoptosis and cellular uptake via receptormediated endocytosis pathway. Thus, we propose that Cis-GNs-FA warrants in-depth in vivo study for their safety, efficacy, and potency in clinical settings.

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