International Journal of Pharmaceutics 470 (2014) 51–62

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Synthesis, characterization and mechanistic-insight into the antiproliferative potential of PLGA-gemcitabine conjugate$ Vaibhav Khare a , Smit Kour b,c, Noor Alam a , Ravindra Dharr Dubey a , Ankit Saneja a,b , Mytre Koul b,c , Ajai Prakash Gupta d, Deepika Singh b,e, Shashank K. Singh b,c, ** , Ajit K. Saxena b,c, Prem N. Gupta a,b, * a

Formulation & Drug Delivery Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, 2 Rafi Marg, New Delhi 110001, India Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India d Quality Control & Quality Assurance Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India e Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, 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 18 March 2014 Received in revised form 2 May 2014 Accepted 3 May 2014 Available online 6 May 2014

Gemcitabine, a nucleoside analogue, is used in the treatment of various solid tumors, however, its efficacy is limited by rapid metabolism by cytidine deaminase and fast kidney excretion. In this study, a polymeric conjugate of gemcitabine was prepared by covalent coupling with poly(lactic-co-glycolic) acid (PLGA), in order to improve anticancer efficacy of the drug. The prepared conjugate was characterized by various analytical techniques including FTIR, NMR and mass spectroscopic analysis. The stability study indicated that the polymeric conjugate was more stable in plasma as compared to native gemcitabine. Further, in vitro cytotoxicity determined in a panel of cell lines including pancreatic cancer (MIAPaCa-2), breast cancer (MCF-7) and colon cancer (HCT-116), indicated that the cytotoxic activity of gemcitabine was retained following conjugation with polymeric carrier. In the nucleoside transportation inhibition assay, it was found that the prepared conjugate was not dependent on nucleoside transporter for entering into the cells and this, in turn, reflecting potential implication of this conjugate in the therapy of transporterdeficient resistance cancer. Further, the cell cycle analysis showed that the sub-G1 (G0) apoptotic population was 46.6% and 60.6% for gemcitabine and PLGA gemcitabine conjugate, respectively. The conjugate produced remarkable decrease in mitochondrial membrane potential, a marker of apoptosis. In addition, there was a marked increase in PARP cleavage and P-H2AX expression with PLGA gemcitabine conjugate as compared to native gemcitabine indicating improved apoptotic activity. The findings demonstrated the potential of PLGA gemcitabine conjugate to improve clinical outcome of gemcitabine based chemotherapy of cancer. ã 2014 Elsevier B.V. All rights reserved.

PubChem classifications: Gemcitabine hydrochloride (PubChem CID: 60749) poly(DL-lactic-co-glycolic acid) (PubChem CID: 23111554) Dicyclohexylcarbodiimide (PubChem CID: 10868) N-hydroxysuccinimide (PubChem CID: 80170) Dipyridamole (PubChem CID: 3108) Triethylamine (PubChem CID: 8471) Keywords: Gemcitabine PLGA Apoptosis Cytotoxicity Polymer drug conjugate

1. Introduction Gemcitabine (20 ,20 -difluoro-20 -deoxycytidine) is a nucleoside analogue approved by FDA for treatment of various solid tumors including pancreatic (Carmichael et al., 1996), breast (Yardley, 2005),

$

IIIM communication number: IIIM/1621/2013. * Corresponding author: Formulation & Drug Delivery Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-Tawi 180001, India. ** Corresponding author: Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-Tawi 180001, India. E-mail addresses: [email protected] (S.K. Singh), [email protected], [email protected] (P.N. Gupta). http://dx.doi.org/10.1016/j.ijpharm.2014.05.005 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.

ovarian (Ozols, 2005) and non-small lung cancers (Hoang et al., 2003). Unfortunately, rapid enzymatic metabolism and fast kidney excretion of gemcitabine compromised its efficient use. Gemcitabine is metabolized to its inactive uracil derivative (20 ,20 -difluorodeoxyuridine) by an enzyme, cytidine deaminase (CDA), present in plasma, kidney and liver, and over-expression of CDA is also associated with gemcitabine resistance (Heinemann et al.,1992; Neff and Blau,1996). The uptake of gemcitabine occurs mainly through nucleoside transporters (hENT1, hCNT1) (Mackey et al., 1998b). After entry into the cells, gemcitabine is phosphorylated to its monophosphate by deoxycytidine kinase and to its active triphosphate by pyrimidine nucleotide kinases (Abbruzzese et al., 1991; Plunkett et al., 1995). Due to short half-life (8–17 min) of gemcitabine, its frequent

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administration is required (1000–1200 mg/m2 as a 30 min intravenous infusion), which leads to serious haematological and gastrointestinal toxicities including myelosuppression, hepatotoxicity and nephrotoxicity (Bunn and Lilenbaum, 2003; Cullen, 2005; Immordino et al., 2004). Although, a number of efforts including preparation of nanoparticles (Li et al., 2009; Sandoval et al., 2012), liposomes (Dalla Pozza et al., 2013; Kim et al., 2009) and prodrugs (Kiew et al., 2010; Pasut et al., 2008; Vandana and Sahoo, 2010) have been made to improve the efficacy of gemcitabine, the synthesis of prodrug has provided greater therapeutic advantage (Chitkara et al., 2013). The polymeric conjugates of anticancer drugs are receiving growing recognition for the passive targeting to solid tumors (Duncan, 1992; Etrych et al., 2011; Mitra et al., 2001). The polymeric conjugate of the drug increases drug stability and reduces non-specific drug toxicity on normal tissue by preventing in vivo random dissemination of the drug (Duncan et al., 2005). Further, due to the enhanced permeation and retention effect, the polymeric drug conjugate can be preferentially localized in the tumor vasculature allowing selective release of drug in the desired site, while minimizing the glomerular excretion rate (Garnett, 2001). These attributes of polymeric conjugate, in turn, lead to reduction in dose administration frequency, which in turn, limit the possible adverse effects (Pasut and Veronese, 2007). Various prodrugs of gemcitabine have been reported so far, with a chemical modification on 4-(N)- and 50 -positions of gemcitabine and showed protection against deamination by CDA and prolonged drug release profile (Cavallaro et al., 2006; Chitkara et al., 2013; Kiew et al., 2010; Tao et al., 2012). Hydrophilic and lipophilic modifications have been demonstrated on gemcitabine in order to transform it in a more stable form. Various hydrophilic polymeric modifications, reported so far on gemcitabine, utilized polyethylene glycol (Pasut et al., 2008; Vandana and Sahoo, 2010), poly (N-2-hydroxypropyl) methacrylamide (Yang et al., 2011), poly (N-hydroxyethyl aspartamide) (Cavallaro et al., 2006) and polyglutamic acid (Kiew et al., 2010). These hydrophilic alterations showed improved plasma stability and cytotoxicity and exhibited long circulation effect. On the other hand, lipophilic modifications explored lipidic carriers such as squalene (Bildstein et al., 2010), cardiolipin (Ali et al., 2005) and linoleic acid (Tao et al., 2012). With the increased plasma stability and anticancer activity, most of the lipophilic gemcitabine modifications demonstrated nucleoside transporter independent pathways for entry into the cells, rather than transport through nucleoside transporter, as in case of native gemcitabine (Alexander et al., 2005; Tao et al., 2012). The change in the mechanism of absorption from the blood vessels to the cells is an important consideration, as reduction in number of nucleoside transporters is one of the resistance mechanisms of cancer cells for gemcitabine (Alexander et al., 2005). It is remarkable that blockage of amine group of gemcitabine could be a potential approach to protect deamination from CDA, which will also increase plasma half-life of the drug (Bender et al., 2009). In this study, we have designed a prodrug of gemcitabine by modifying its 4-(N) position with hydrophobic polymer (PLGA) through a hydrolysable amide linkage. It is also noteworthy that gemcitabine is a hydrophilic drug which relies on numerous transporters to pass through the cellular lipid bilayer to exert its cytotoxicity. The conjugation with hydrophobic polymer may enhance its lipophilicity, which in turn, could facilitates gemcitabine transport across the lipophilic bilayer and adding up values to its cytotoxic potential (Hung et al., 2012). The synthesized conjugate was characterized by FTIR, NMR and mass spectroscopic analysis and evaluated for its plasma stability profile and antiproliferative potential. Further, the mechanistic investigation of anti-proliferative activity of the conjugate was undertaken using cell cycle analysis, measurement of mitochondrial membrane potential and western blot analysis.

2. Materials and methods 2.1. Materials PLGA [Resomer1 RG503H] was purchased from Evonik Industries (Germany). N-Hydroxysuccinimide (NHS), N,N0 -dicyclohexylcarbodimide (DCC), dimethyl sulfoxide (DMSO), pyridine, diethyl ether, anhydrous dichloromethane (DCM), triethylamine and gemcitabine HCl was purchased from Sigma–Aldrich, India. All other chemicals used were of analytical grade. 2.2. Synthesis of PLGA gemcitabine conjugate Carbodiimide chemistry was utilized to mediate the formation of amide linkage between terminal carboxylic group of PLGA and 4-N amine group of gemcitabine. PLGA-gemcitabine conjugate was synthesized in two steps (Fig. 1), involving activation of terminal carboxylic group of PLGA by DCC/NHS, followed by conjugation of activated PLGA with gemcitabine. PLGA (1 equivalent) was activated by DCC/NHS (3 equivalents). Briefly, in 10 ml of anhydrous dichloromethane, DCC, NHS and PLGA was added and stirred for 12 h on magnetic stirrer under nitrogen atmosphere at room temperature. The insoluble by-product dicyclohexylurea was removed by filtration using 0.45 mm filter (Millipore), and the activated polymer was isolated by precipitation into cold anhydrous diethyl ether. The precipitate was washed 3–4 times to remove unreacted DCC and NHS and subsequently dried under reduced pressure. The degree of PLGA activation was determined by hydroxamate method, described separately. Gemcitabine hydrochloride (2 equivalents of PLGA), dissolved in 1 ml of anhydrous DMSO, was added drop wise in 5 ml of anhydrous DMSO solution containing activated PLGA and triethylamine (20 equivalents of gemcitabine) and kept on stirrer for 24 h at room temperature under nitrogen environment. Triethylamine was used as a catalyst to neutralize the hydrochloride salt and provided desalted gemcitabine, with freely available amine group for reaction with activated carboxylic group of PLGA (Yoo and Park, 2004). The reaction mixture was precipitated in deionized water to collect PLGA gemcitabine conjugate and washed 2–3 times to remove unreacted gemcitabine and the product was dried under reduced pressure. The percentage of gemcitabine conjugated to PLGA was determined by alkaline hydrolysis of conjugate followed by estimation of gemcitabine by LC–MS/MS. 2.3. Degree of PLGA activation The degree of PLGA activation was determined by hydroxamate method as reported earlier (Iwata et al., 1998). Briefly, 1 ml of sample solution with NHS moiety was mixed with 0.2 ml of 2 N NaOH and then the reaction mixture was incubated for 10 min at 40  C. For the preparation of the standard curve, 1 ml of solution containing known concentration of NHS was mixed with 0.2 ml of 2 N NaOH and incubated for 10 min at 40  C. All the solutions (sample and standard) were acidified by adding 1.5 ml of 0.85 N HCl and centrifuged to remove the precipitated PLGA. Subsequently, 2 ml of the supernatant was mixed with 0.5 ml of 5% FeCl3 in 0.1 N HCl. The NHS content in the sample solution was determined from the absorbance measured at 500 nm using UV–vis spectrophotometer (Shimadzu UV-2600 series). 2.4. Tandem mass spectroscopic analysis of gemcitabine The quantification of gemcitabine was carried by liquid chromatography with tandem mass spectrometry (QQQ LC–MS/ MS; Agilent 6410bB-QQQ LC–MS System). The mobile phase was 5 mM ammonium formate: acetonitrile (95:5) at flow rate of

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Fig. 1. Scheme for conjugation of PLGA and gemcitabine using DCC/NHS based coupling.

0.4 ml/min in a Chromolith RP-18e column (E-Merck 4.6 mm  50 mm). The experiment on mass spectrometer was set to perform in multiple reactions monitoring mode. The detection of gemcitabine was performed using electrospray ionization in positive ion mode by monitoring the ion transitions m/z 264.0 ! 112.0. The tandem mass chromatogram showed a precursor mass of peak of 264.0 corresponds to gemcitabine [M + 1] and a dominant mass peak of 112.0 mass which corresponds to the 4-aminopyrimidin-2 (1H)-one [M + H] + 1 fragment (Fig. 2).

2.5.3. MALDI-TOF MS analysis PLGA and PLGA gemcitabine conjugate were characterized by MALDI-TOF MS analysis. A mass spectrum was acquired using MALDI/TOF/TOF (Applied Biosystems/AB Sciex) equipped with a ND-YAG diode pump laser (355 nm/200 Hz). The instrument was operated in a positive ion reflection mode extracting ions at 10 different regions of sample spot and each was the result of the accumulation of at least 1000 laser shots having 8–12 sub-spectra with 100 shots per sub-spectra, optimized to give good signal-tonoise ratio.

2.5. Characterization of PLGA gemcitabine conjugate 2.5.1. Fourier transform infrared spectroscopy (FTIR) The FTIR spectra for native gemcitabine, PLGA and PLGA gemcitabine conjugate were obtained by using IR spectrometer (PerkinElemer Spectrum Version 10.03.06). Briefly, the samples were pressed into a potassium bromide pellet, and the spectra were detected over a range of 4000–500 cm1. 2.5.2. 1H NMR spectroscopy 1 H NMR spectra were recorded for native gemcitabine, PLGA, and PLGA gemcitabine conjugate by NMR spectrometer (UXNMR, Bruker Analytische Messtechnik GmbH, 500 MHz) using deuterated DMSO as the solvent and trimethylsilane as an internal standard.

2.5.4. Degree of conjugation The degree of gemcitabine conjugated to PLGA was determined by alkaline hydrolysis method as reported previously (Schiavon et al., 2004). Briefly, PLGA gemcitabine conjugate was mixed with 1 ml of 2 N NaOH and incubated at 40  C in water bath. The solution was kept at room temperature for 3–4 h and sonicated for 10 min on bath sonicator (Elmasonic PH500EL). The solution was centrifuged, and the supernatant was estimated for gemcitabine content using LC–MS/MS. 2.5.5. In vitro release and stability of gemcitabine in plasma The in vitro release and stability of gemcitabine in plasma was investigated by method as reported previously (Tao et al., 2012) with slight modifications. PLGA gemcitabine conjugate (1.2 mg in

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Fig. 2. The tandem mass spectra of gemcitabine at multiple reactions monitoring mode in 5 mM ammonium formate: acetonitrile (95:5). A clear response is observed at m/z of 264 corresponding to protonated molecules (M + H)+ of gemcitabine and product ion at m/z of 112.

20 ml DMSO) was added to 480 ml of rat plasma and samples were kept at 37 C for 24 h. At scheduled time intervals 50 ml of plasma was removed and replaced with equal amount of fresh plasma. For deproteinization, 450 ml of acetonitrile was added to 50 ml of each plasma sample, the resulting mixture was centrifuged at 1500  g for 5 min at 4  C, and supernatant was filtered through Amicon filter unit (Millipore) and quantified by LC–MS/MS. Further, to investigate gemcitabine stability in plasma, gemcitabine was added to 500 ml of rat plasma. At scheduled time intervals 50 ml of plasma was removed and replaced with equal amount of fresh plasma and concentration of gemcitabine was determined by using LC–MS/MS. 2.5.6. Cell culture, growth conditions and treatment conditions The human cell lines for pancreatic cancer (MIAPaCa-2), breast cancer (MCF-7) and colon cancer (HCT-116) were purchased from European Collection of Cell Cultures (ECACC). The human cancer cell lines were grown in tissue culture flasks in complete growth medium (DMEM medium supplemented with 10% fetal calf serum, 100 mg/ml streptomycin and 100 units/ml penicillin) in carbon dioxide incubator (New Brunswick, USA) at 37  C, 5% CO2 and 98% RH. 2.5.7. In vitro anti-proliferative assay MTT assay was performed to determine the in vitro antiproliferative activity of gemcitabine and PLGA gemcitabine conjugate. A panel of human cancer cell lines of various tissue origins were used to evaluate the anti-proliferative activity. The cell suspension of optimum cell density (3000–4000 cells/ 100 ml) was seeded in 96 well flat bottom plates and incubated for 24 h at 37  C, 5% CO2 and 98% RH. The test compound at different concentrations in complete growth medium (100 ml) were added after 24 h of incubation. For IC50 calculation different concentrations (1, 0.1, 0.01, 0.001 and 0.0001 mg/ml) of test compound were used and kept for 72 h. Twenty microlitres of MTT (2.5 mg/ml) was added 4 h before completion of incubation time. Excess media was blotted off, and 150 ml of DMSO was added to dissolve the purple formazan crystals. The amount of formazan formed is indicative of the number of viable cells. The plates were kept on the shaker for 5 min to solubilize the dye completely, and finally the reading was taken at 570 nm on

Microplate Reader (BioTek Synergy HT). The IC50 values were determined by using Prism, version 5.04, from GraphPad Software (La Jolla, CA). 2.5.8. Nucleoside transporter inhibition To examine the variation in nucleoside transporter-dependent membrane permeation, between native gemcitabine and prepared conjugate, dipyridamole was used as nucleoside transporter inhibitor, during in vitro cytotoxicity evaluation (Alexander et al., 2005). MTT assay as described above was performed with slight modifications. MIAPaCa-2 (3000 cells/100 ml) cell lines were seeded in 96 well flat bottom plates and incubated for 24 h. Dipyridamole (10 mM) was added 30 min before sample addition. Different concentrations (1, 0.1, 0.01, 0.001 and 0.0001 mg/ml) of test compound were used, and IC50 was calculated by using Prism, version 5.04, from GraphPad Software (La Jolla, CA). 2.5.9. Cell cycle analysis by flow cytometry The effect on cell cycle following treatment with native gemcitabine and PLGA gemcitabine conjugate was studied using flow cytometry. MIAPaCa-2 cells (1.5  105 cells/ml/six-well plate) were seeded for 24 h and subsequently, exposed to native gemcitabine (0.25 mg/ml) and equivalent amount of conjugate. After 72 h incubation, the cells were washed with PBS and fixed in 70% ethanol at 20  C, overnight. The cells were thereafter washed, digested with DNase free RNase (100 mg/ml) at 37  C for 45 min and stained with propidium iodide to determine the cell cycle phase distribution. The DNA fluorescence was measured on a flow cytometer FACS Aria (Becton Dickinson, USA) (Zhu and Gooderham, 2006). 2.5.10. Mitochondrial membrane potential (MMP) The changes in mitochondrial transmembrane potential (C mt) as a result of apoptosis-inducing conditions were measured by flow cytometry after staining with Rhodamine-123. MIAPaCa-2 cells (1.5  105 cells/ml/six-well plate) were seeded for 24 h and subsequently, incubated with gemcitabine (0.25 mg/ml) and equivalent amount of conjugate for 72 h. Rhodamine-123 (1 mM) was added 1 h before the termination of experiment. The cells were collected at 3000 rpm for 5 min, washed once with PBS, resuspended in 500 ml of PBS, and mitochondrial membrane

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potential was measured by BD-FACS Aria flow cytometer with an excitation wavelength of 488 nm and an emission wavelength of 525 nm in FITC channel (Bhushan et al., 2007).

3. Results

2.5.11. Western blot analysis The effect of native gemcitabine and PLGA gemcitabine conjugate on cell cycle arrest and apoptosis was further examined at molecular level by western blot analysis. MIAPaCa-2 cells (2.5  105 cells/ml) were seeded in 25 cm2 culture flasks (Nunc) containing 5 ml media. After 24 h, gemcitabine (0.25 mg/ml) and equivalent amount of conjugate were added and incubated for 72 h. The untreated cells were taken as control for the experiment. After 72 h, the cells were trypsinized and washed with PBS. The cell lysates were prepared by using RIPA buffer and protease and phosphatase inhibitor cocktail. The protein concentration was determined with the QuantiPro BCA assay kit (Sigma). The cell lysates were diluted in 5 loading buffer and boiled for 5 min. An equal amount of protein (70 mg) was subjected to 10% SDS-PAGE analysis and transferred to PVDF membrane (Millipore). The membrane was blocked with 5% non-fat milk made in TBST buffer for 1 h at RT. The membrane was then incubated with primary PARP (1:1000), PH2AX (1:1000) and b-actin (1:1000) and HRP linked respective secondary antibodies (Santa Cruz). The signals were detected by using ECL and exposed to film.

PLGA gemcitabine conjugate was synthesized using zero length cross-linkers, DCC and NHS. It is the most common activation method of carboxylic group, and the formed active esters are suitable for coupling with primary amines (Veronese and Morpurgo, 1999). The method involves activation of carboxylic acid of PLGA by DCC and NHS, and the formed O-acyl derivative of carboxylic acid is more prone for reaction with a nucleophile (Valeur and Bradley, 2009), i.e. amine group of gemcitabine, which leads to amide bond formation between gemcitabine and PLGA. Various analytical techniques were utilized for characterization of polymeric conjugate. The FTIR spectra (Fig. 3) of gemcitabine showed characteristics bending vibrations of amines at 1702 cm1 and 1658 cm1 and stretching vibration of amine at 3261 cm1 which is in accordance to previous reports (Cavallaro et al., 2006; Tao et al., 2012). In the FTIR spectra of pure PLGA, the peak at 1753 cm1 is exhibited due to the absorbance of carbonyl group in PLGA matrix, and peaks at 2998 cm1, 2953 cm1and 2853 cm1 correspond to (C H) bending vibrations (Deniz, 1999; Stevanovic et al., 2008). On the other hand, the FTIR spectrum of PLGA gemcitabine conjugate showed the characteristic peak of carbonyl group of PLGA at 1750 cm1 and C H bands at 2954 cm1, 2924 cm1 and 2854 cm1. The peaks at 1665 cm1 and 1536 cm1 confirm the formation of amide bond between PLGA and gemcitabine. PLGA gemcitabine conjugate showed a peak at 3417 cm1 attributed to stretching ( N H) vibration band of secondary amide (Cavallaro et al., 2006; Chitkara et al., 2013; Tao et al., 2012).

2.5.12. Statistical analysis The results were expressed as mean  standard deviation. The statistical analysis was carried out by using Student’s t-test, and the statistical significance was designated as P < 0.05.

3.1. Synthesis and characterization of PLGA gemcitabine conjugate

Fig. 3. Fourier transform infrared spectra of PLGA, gemcitabine and PLGA-gemcitabine conjugate (PLGA-GEM) in the region 4000 to 500 cm1.

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Fig. 4. 1H NMR (500 MHz) spectra of PLGA, gemcitabine and PLGA-gemcitabine conjugate (PLGA-GEM) in deuterated dimethyl sulphoxide.

The polymeric conjugate of gemcitabine was also characterized by 1H NMR (Fig. 4). The recorded 1H NMR spectra of pure PLGA showed the typical signal approximately at 1.48 ppm, attributed to the methyl groups of the D- and L-lactic acid monomers, and the multiplets around 5.23 and 4.92 ppm, due to the CH groups of lactic acid and CH2 groups of glycolic acid, respectively. The findings are in accordance to previous investigations (Cenni et al., 2012; Pignatello et al., 2009). The 1H NMR spectra of gemcitabine gives signal for the protons of 50 - and 30 OH groups at 6.05 ppm and 6.16 ppm, respectively (Cavallaro et al., 2006; Pasut et al., 2008). The signal of 40  NH2 group of gemcitabine was registered at 8.07 ppm, which is in accordance to a previous report (Vandana and Sahoo, 2010). On the other hand, 1H NMR spectra of PLGA gemcitabine conjugate showed signal of methyl groups of PLGA at 1.48 ppm and CH of lactic acid and CH2 of glycolic acid at 5.18 ppm and 4.8 ppm, respectively. Further, 1H NMR spectra also showed the chemical shifts at 6.17 ppm and 6.34 ppm corresponding to 5’and 3’–OH groups. The appearance of signal at 8.29 ppm confirms the formation of amide bond which is shifted from 8.07 of amine group of gemcitabine (Bondioli et al., 2010; Vandana and Sahoo, 2010). The conjugation between PLGA and gemcitabine was also characterized by MALDI-TOF MS (Fig. 5). In particular, MS spectrum PLGA gemcitabine conjugate showed increase abundance of the ion at m/z 1276 with respect to pure PLGA m/z 1032. The conjugation between PLGA and gemcitabine was further confirmed by the appearance of other ions at m/z 1233, 1219 and 1161 with respect to pure PLGA at m/z 988, 974 and 916, respectively. The degree of PLGA activation as determined by hydroxamate method was about 73%, and the extent of conjugation was 0.124 mol of gemcitabine/mol of PLGA.

3.2. In vitro stability and release profile of gemcitabine in plasma The rate of gemcitabine release from polymeric conjugate and its stability in plasma are important considerations as drug is metabolized to its inactive form when administered intravenously. The rate of release of the gemcitabine in plasma from the PLGAgemcitabine conjugate is shown in Fig. 6. Initially, during the 1 h incubation period, approximately 16% of the gemcitabine was available in plasma. Further, after 72 h the release from polymeric conjugate increases and about 40% of gemcitabine was available in plasma, whereas in the case of native gemcitabine only 7% of the drug was detected at this time point. The availability of gemcitabine in the plasma suggested that after incubation in plasma the drug in the polymeric conjugate was more stable as compared to native gemcitabine. It was observed that more than 50% of the native gemcitabine was degraded in just 1 h and negligible amount was detected after 72 h. The results showed that the stability of gemcitabine in the plasma was improved following conjugated with PLGA. 3.3. In vitro cytotoxicity The gemcitabine and PLGA gemcitabine conjugate were tested for their in vitro cytotoxicity in a panel of cell lines including MIAPaCa-2, MCF-7 and HCT-116 at different concentrations for 72 h (Fig. 7). In HCT-116 cell line, PLGA gemcitabine conjugate showed similar activity to native gemcitabine, whereas in MCF-7 and MIAPaCa-2 cell lines, PLGA gemcitabine conjugate showed an enhanced efficacy than native gemcitabine. An IC50 of 2.4 ng/ml and 2.8 ng/ml were observed in HCT-116 cell line for native gemcitabine and PLGA gemcitabine conjugate, respectively. In

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Fig. 5. MALDI-TOF mass spectrum of PLGA and PLGA-gemcitabine conjugate (PLGA-GEM) in positive ion mode.

varying concentrations of native gemcitabine and PLGA gemcitabine conjugate. The IC50 values were obtained for the human pancreatic cancer cell line, MIAPaCa-2. In MIAPaCa-2 IC50 of 464 ng/ml was obtained for native gemcitabine in the presence of dipyridamole which is approximately 19 folds greater than the value when cells were not pre-incubated with the nucleoside inhibitor. However, for PLGA gemcitabine conjugate an IC50 of 7.0 ng/ml was observed which is similar to the values when cells were not pre-incubated with the nucleoside inhibitor (Table 1). Alternatively, it can be stated that in MIAPaCa-2 cell line, dipyridamole markedly decreased sensitivity to native gemcitabine, while sensitivity to PLGA gemcitabine conjugate was not altered. The results suggest that gemcitabine activity was many folds lower when pre-incubated with dipyridamole, however similar cytotoxic effect was observed in the case of PLGA

MCF-7 cell line, IC50 in the order of 132 ng/ml and 15 ng/ml were observed for native gemcitabine and PLGA gemcitabine conjugate, respectively. Similarly, MIAPaCa-2 cell line displayed higher cytotoxicity for PLGA gemcitabine conjugate with IC50 value in the order of 7.5 ng/ml as compared to 24 ng/ml for native gemcitabine. 3.4. Nucleoside transporter inhibition The gemcitabine enters the cells through human nucleoside transporters (Mackey et al., 1998b); therefore in order to investigate whether the PLGA gemcitabine conjugate also rely on one or more of these transporters, the nucleoside transporter inhibition study was performed. Dipyridamole (a nucleoside transporter inhibitor) was used 30 min before the addition of

Drug present in plasma (%)

100

Gemcitabine

90

PLGA-Gem

80 70 60 50 40 30 20 10 0

0

10

20

30

40 Time (h)

50

60

70

80

Fig. 6. Time dependent availability of gemcitabine in plasma following release from PLGA-gemcitabine conjugate (2.4 mg/ml) and stability of native gemcitabine (0.2 mg/ml) in plasma.

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V. Khare et al. / International Journal of Pharmaceutics 470 (2014) 51–62 Table 1 Effect of nucleoside transporter inhibitor on cytotoxic activity of gemcitabine and PLGA- gemcitabine conjugate in MIAPaCa-2 cell lines following incubation for 72 h. Compound(s)

MIAPaCa-2 IC50 (mg/ml)

Gemcitabine Gemcitabine + dipyridamole* PLGA-gemcitabine PLGAgemcitabine + dipyridamole *

0.024 0.464 0.0075 0.007

   

0.005 0.100 0.004 0.003

Relative gemcitabine resistance – 19.33 – 0.933

Statistical significant (P < 0.05 vs. gemcitabine).

MIAPaCa-2 cells were analysed. The percentage of apoptotic cells was greater in PLGA gemcitabine conjugate than native gemcitabine. The sub-G1 (G0) apoptotic population was observed to be 46.6% and 60.6% for native gemcitabine and PLGA gemcitabine conjugate, respectively, as compared to 3.55% in control cells (Fig. 8). 3.6. Mitochondrial membrane potential At the induction of apoptotic condition, inner mitochondrial transmembrane potential is disrupted, eventually leading to outer mitochondrial membrane rupture and release of proapoptotic proteins into the cytoplasm. Rh-123, a cationic fluorophore, is actively accumulated by cells and is useful in monitoring the membrane potential of mitochondria. The energization of mitochondria induces quenching of Rh-123 fluorescence and is directly proportional to mitochondrial membrane potential. The effect of gemcitabine and conjugated gemcitabine was observed on MIAPaCa-2 cells at a concentration of 0.25 mg/ml following incubation for 72 h. The untreated cells showed 8.4% loss of MMP after 72 h incubation. The gemcitabine showed 25.9% loss of MMP whereas 42.1% MMP loss was observed for PLGA gemcitabine conjugate (Fig. 9). These results indicated that PLGA gemcitabine conjugate induced greater cell death implicated through greater loss of MMP in comparison to native gemcitabine. 3.7. Western blot analysis

Fig. 7. Percent of cell viability on different concentrations of gemcitabine and PLGA-gemcitabine conjugate (PLGA-GEM) against different cell lines. The cells were incubated with gemcitabine and PLGA-GEM conjugate for 72 h. The results represent mean  SD of three experiments. *P < 0.05 vs. GEM.

gemcitabine conjugate, suggesting that they do not enter the cells via the nucleoside transporters. 3.5. Cell cycle analysis In order to investigate the cell death caused by native gemcitabine and PLGA gemcitabine conjugate in MIAPaCa-2 cells, the cell cycle analysis using propidium iodide staining was performed. The DNA specific fluorochrome propidium iodide discriminates apoptotic cells (sub-G1 cell population) wherein apoptosis-associated endonucleases degrade the DNA. By measuring the DNA content using flow cytometry, sub-G1 cell population of 72 h gemcitabine and PLGA-gemcitabine conjugate treated

To maintain homeostasis in normal cells apoptosis plays a major role. Apoptosis is characterized by various morphological changes including chromatin condensation, cell shrinkage, membrane blebbing and changes at the molecular level including internucleosomal DNA fragmentation and cleavage of poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 plays an important role in repairing damaged DNA. It is activated, and its level increases when DNA damage is induced in cells. It contains an active site and a DNA binding domain that are separated on cleavage into 89 kDa and 24 kDa fragments by caspase-3 following induction of apoptosis (Kaufmann et al., 1993). The western blot of PARP cleavage in MIAPaCa-2 cancer cell line was performed following 72 h of native gemcitabine and PLGA gemcitabine conjugate treatment. The PARP cleavage was observed in gemcitabine and PLGA gemcitabine conjugate, with latter showing greater cleavage in comparison to the native gemcitabine. The western blot analysis of P-H2AX further corroborated these results. The histone variant H2AX is phosphorylated at Ser 139 (g-H2AX) in response to DNA damage during apoptosis. P-H2AX senses the DNA double strand breaks and serves as docking site for DNA damage checkpoint protein 1 (facilitates recruitment of various proteins for DNA repair) at the damage foci, thus is important for initiating early DNA damage response (Sharma et al., 2012). The gemcitabine

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-ve control

*

% Cell population

60

Gemcitabine

59

PLGA-GEM

50 40

*

30 20 10 0

* sub G1

G1 S Different phases of cell cycle

G2/M

Fig. 8. Effect of gemcitabine and PLGA-gemcitabine conjugate (PLGA-GEM) on cell cycle distribution of human pancreatic cancer cell (MIAPaCa-2). Flow cytometric analysis of MIAPaCa-2 cells was performed after propidium iodide staining. The cells were incubated for 72 h in the presence of gemcitabine (0.25 mg/ml) and PLGA-gemcitabine conjugate (0.25 mg/ml). Figure shows the representative mean  SD of percent population of cells in each phase of cell cycle of three experiments. *P < 0.05 vs. GEM.

inhibits DNA synthesis, causing DNA damage thus elevating the levels of P-H2AX. The western blot analysis of P-H2AX in MIAPaCa2 following 72 h of gemcitabine and PLGA gemcitabine treatment revealed greater expression of P-H2AX in response to PLGA gemcitabine conjugate in comparison to native gemcitabine. The results suggested enhanced efficacy and biological action of PLGA gemcitabine conjugate than the native gemcitabine (Fig. 10). 4. Discussion

Fig. 9. Effect of gemcitabine and PLGA-gemcitabine conjugate on mitochondrial membrane potential loss in human pancreatic cancer cell line (MIAPaCa-2). The cells were incubated at equivalent gemcitabine concentration of 0.25 mg/ml in 6 well plates for 72 h treatment. Figures show the representative staining profile of one of two similar experiments.

The potential of gemcitabine as an effective therapeutic agent in solid tumors has been hampered because of its deamination by CDA, fast kidney excretion and resistance among tumor cell lines. Therefore, novel drug delivery strategies are required to address these issues. In this study, we have reported a novel polymeric conjugate of gemcitabine, which is more stable against CDA present in plasma and does not depend on nucleoside transporters for entry into the cells, one amongst the resistance parameter for gemcitabine. The prepared PLGA gemcitabine conjugate was successfully characterized by IR, NMR and MALDI-TOF. The first step in the conjugation involves reaction of carboxylic acid with DCC to form an intermediate O-acylisourea, which yields amide bond on reaction with amine, along with a by product, dicyclohexylurea. Further, the intermediate O-acylisourea can also yield carboxylic acid anhydride which subsequently forms amide when reacted with amine (Valeur and Bradley, 2009). The intermediate O-acylisourea may undergo spontaneous rearrangement to more stable inactive N-acylisourea, and the rate of this rearrangement is dramatically increased in aprotic solvent such as DMSO. Therefore, NHS was used which reacts with O-acylisourea to form O-succinimidyl ester, which is more reactive towards amine group of gemcitabine (Stewart and Young, 1984). In addition, the formation of N-acylisourea is also hindered as succinimidyl ester cannot undergo O ! N displacement. The mechanism involves a nucleophilic (dissociated hydroxyl group of NHS) attack on O-acylisourea to yield a urea derivative and succinimidyl ester, which can then react with a non-dissociated primary amine, resulting in the formation of amide and regeneration of NHS (Grabarek and Gergely, 1990). The efficiency of gemcitabine is compromised with short halflife and metabolism through an enzyme, cytidine deaminase present in plasma, liver and kidney. The blockade of deamination site in gemcitabine exhibited increase in half-life (Bender et al., 2009). In this study, the conjugation of gemcitabine with PLGA was

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Fig. 10. (a) Protein expression of PARP and P-H2AX detected by western blot of the MIAPaca-2 cells treated with gemcitabine and PLGA-gemcitabine conjugate (PLGA-GEM). The cells were treated with equivalent gemcitabine concentration of 0.25 mg/ml for 72 h. Equal amount of the proteins were fractionated on SDS-polyacrylamide gels and transferred to PVDF membrane, followed by immunobloting with anti-PARP and anti-P-H2AX antibodies. b-actin is shown as a loading control. (b) Densitometric analysis of cleaved PARP and P-H2AX expression on untreated cells, gemcitabine treated cells and PLGA-GEM treated cells. *P < 0.05 vs. GEM.

investigated in order to limit the enzymatic deactivation of this drug. The stability studies indicated that PLGA gemcitabine conjugate was more stable in plasma as compared to native gemcitabine. It was observed that PLGA gemcitabine conjugate could resist deamination in plasma while the native drug could be liberated in the controlled pattern. In the case of PLGA gemcitabine conjugate, after 72 h, the amount of free gemcitabine available in plasma was approximately 40%, on the other hand, native gemcitabine degraded significantly, and the amount estimated was only 7% at the same time point. The study indicated that the polymeric conjugate of gemcitabine retarded its deamination from cytidine deaminase and as a result elevated level of the drug was detected in the plasma when compared to native gemcitabine. The chemotherapy with the gemcitabine requires high doses to maintain optimum concentration of the drug in the body because gemcitabine undergo enzymatic degradation in the blood and rapid renal excretion (Immordino et al., 2004). The current gemcitabine therapy is associated with adverse effects, high cost and discomfort to the patients. The application of biodegradable PLGA gemcitabine conjugate may mitigate these limitations of gemcitabine therapy. The PLGA gemcitabine conjugate showed controlled drug release via gradual drug liberation from the conjugate, thereby, allowing a constant level of the drug in the blood. Further, the polymeric conjugate possesses tendency for selective localization in the tumor vasculature due to the enhanced permeation and retention effect, before releasing bulk of the drug at the required site. This type of delivery platform may reduce premature release of drug in the systemic circulation and thereby limit drug degradation and non-specific tissue toxicity. The in vitro cytotoxicity study on various cell lines (MCF-7, HCT116, and MIAPaCa-2) was performed to investigate the effect of conjugation, if any, on the cytotoxicity profile of gemcitabine. It was found that in the case of human colon carcinoma cell line (HCT-116), the activity of gemcitabine was not altered after the conjugation with PLGA. The polymeric conjugate of gemcitabine and native gemcitabine showed similar activity in HCT116 cells in respect to IC50 value in MTT assay. However, in the human pancreatic cancer cell line (MiaPaCa-2), the PLGA gemcitabine conjugate showed lower IC50 value in comparison to native gemcitabine, displaying a greater effect on cellular cytotoxicity. Further, in the human breast cancer cell line (MCF-7) also PLGA gemcitabine conjugate showed promising cytotoxic activity in

terms of lower IC50 value as compared to native gemcitabine. The results are in accordance to previous investigations (Tao et al., 2012; Vandana and Sahoo, 2010), where gemcitabine exhibited improved cytotoxic potential following conjugation with polymer. The cytotoxic activity of the conjugate could be related to the improved lipophilicity of the gemcitabine following conjugation with PLGA, which could enhance the intracellular uptake and inturn, attributed to improved anti-proliferative effect. The observation was further supported by the previous investigation, where a squalenoyl prodrug of gemcitabine (SQdFdC) exhibited passive diffusion into cancer cells and predominantly accumulated within cellular membrane including those of organelles such as the endoplasmic reticulum. Subsequently, SQdFdC released from this transient reservoir into cell cytoplasm and liberate gemcitabine, which either led to the build-up of its biologically active triphosphate metabolite or to the efflux of gemcitabine through equilibrative membrane transporters (Bildstein et al., 2010). The uptake of another conjugate, gemcitabine-cardiolipin was also proven transport-independent and attributed to passive diffusion through the plasma membrane (Chen et al., 2006). Various other investigations have shown that polymeric conjugate of small molecules provides a sustained release of therapeutic agent in the cells due to prolonged retention, thereby contributing to higher cytotoxicity (Duncan and Kopecek, 1984). On the other hand, lower cytotoxic effect of native gemcitabine may be attributed to its poor internalization and rapid degradation in the cancer cells (Cavallaro et al., 2006; Giovannetti et al., 2010). The gemcitabine is dependent on nucleoside transporters to enter into the cells. It is a major drawback as an anticancer agent to rely on transporters to reach their target sites as lack of nucleoside transporters can lead to resistance (Mackey et al., 1998b). The gemcitabine, being a hydrophilic compound, does not readily cross the cell membrane by diffusion (Mackey et al., 1998a). This necessitates the presence of specialized transport protein such as human equilibrative nucleoside transporters (hENT1 or hENT2) for efficient transport of gemcitabine (Damaraju et al., 2003). It has been demonstrated that deficiency in hENT1 confers resistance to gemcitabine toxicity in vitro (Possinger, 1995). We have shown that in the presence of dipyridamole, a nucleoside transporter inhibitor, the activity of gemcitabine conjugate was not much affected indicating their transport is independent of the nucleoside transporters. On the contrary, the gemcitabine is heavily dependent on the transporters as indicated by 19 folds increase in IC50 in

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MIAPaCa-2 cells confirming that gemcitabine enters into the cells through the nucleoside transporter. From the cell cycle analysis, it could be inferred that gemcitabine has a cytotoxic effect on cells as seen by increase in sub-G1 population. A greater percent of sub-G1 population was observed in PLGA gemcitabine conjugate as compared to native gemcitabine, displaying a significantly greater cytotoxic effect in the pancreatic cancer cell line. The mitochondrion plays an important role in inducing apoptosis. The permeability transition is an early marker in apoptosis which leads to decrease in mitochondrial membrane potential (MMP). Cytochrome-c is released when mitochondrial permeability transition pore (MPTP) located on inner mitochondrial membrane opens resulting in mitochondrial membrane potential loss (Vander Heiden et al., 1997). It has already been reported that gemcitabine induced apoptosis through MMP loss (Bortner and Cidlowski, 1999). PLGA gemcitabine conjugate was able to induce MMP loss in MIAPaCa-2 cells and showed greater effect as compared to native gemcitabine. Thus, it can be inferred that PLGA gemcitabine conjugate causes cell death by apoptosis through mitochondrial damage at lower concentrations in comparison to native gemcitabine. Furthermore, we studied the action of gemcitabine and its conjugate at the molecular level on proteins responsible for DNA damage and apoptosis. The western blot result (Fig. 10) for 72 h indicated that the expression of P-H2AX was greater in response to DNA damage by PLGA gemcitabine conjugate as compared to native gemcitabine. Further, the proteolytic cleavage of nuclear protein (PARP), owing to DNA degradation is characteristics of apoptosis. Gemcitabine is a known inducer of apoptosis and PARP-1 cleavage and P-H2AX over expression are known markers of apoptosis. The results indicated that gemcitabine conjugate induces apoptosis at levels greater than native gemcitabine. 5. Conclusion The gemcitabine is useful in the treatment of a variety of solid tumors, however, its efficacy is limited due to the enzymatic metabolism by cytidine deaminase and fast kidney excretion. In this study, PLGA gemcitabine conjugate was synthesized and characterized by various techniques including FTIR, NMR and mass spectroscopy. The conjugate retained anticancer activity of the gemcitabine and it exhibited increased drug stability in the plasma. The mechanistic studies including cell cycle analysis, mitochondrial membrane potential and western blotting analysis showed potential anticancer activity of developed polymeric conjugate. PLGA gemcitabine conjugate possesses potential for reduced kidney clearance and prevention of drug degradation by cytidine deaminase enzyme, therefore, higher bioavailability of the drug can be expected. On the other hand, it is also noteworthy that in vitro assays are not offering an evaluation of prolonged in vivo circulation that could be associated with polymeric drug conjugates. Another, important feature of the PLGA gemcitabine conjugate is that it is not rely on nucleoside transporters for entering into the cells and therefore may be useful in the cases of gemcitabine resistance, which may arise from an impaired gemcitabine membrane transport. Acknowledgments We are grateful to Director, CSIR-IIIM, Jammu for providing necessary support for carrying out this work. The research funding provided by Science & Engineering Research Board, Department of Science and Technology (DST), New Delhi, India, is gratefully acknowledged. The authors (VK, RDD) also acknowledge DST for the financial assistance in the form of INSPIRE Fellowship.

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