Synthesis, characterization and augmented anticancer potential of PEG-betulinic acid conjugate

Materials Science and Engineering C 73 (2017) 616–626

Contents lists available at ScienceDirect

Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Synthesis, characterization and augmented anticancer potential of PEG-betulinic acid conjugate Ankit Saneja a,b, Love Sharma a,c, Ravindra Dhar Dubey b, Mubashir Javed Mintoo d, Amrinder Singh a,c, Amit Kumar c, Payare Lal Sangwan a,e, Sheikh Abdullah Tasaduq a,c,⁎, Gurdarshan Singh a,b,c, Dilip M. Mondhe a,d, Prem N. Gupta a,b,c,⁎⁎ a

Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, 2 Rafi Marg, New Delhi 110001, India Formulation & Drug Delivery Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India c PK-PD-Toxicology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India d Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India e Natutal Product Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India b

a r t i c l e

i n f o

Article history: Received 18 October 2016 Received in revised form 30 November 2016 Accepted 20 December 2016 Available online 23 December 2016 Keywords: Betulinic acid Polyethylene conjugate Apoptosis Cytotoxicity Ehrlich ascites carcinoma

a b s t r a c t Betulinic acid (BA), a pentacyclic lupine-type triterpene, is reported to inhibit cell growth in a variety of cancers. However, its efficacy is limited by its poor aqueous solubility and relatively short half-life. In this study, BAmonomethoxy polyethylene glycol (mPEG) conjugate was synthesized by covalent coupling the C-28 carboxylic acid position of BA with amine groups of mPEG, in order to improve its solubility and anticancer efficacy. mPEGBA conjugate was characterized using various analytical techniques including NMR, FT-IR and MALDI-MS. The mPEG-BA conjugate was cytotoxic, demonstrated internalization and induced cell apoptosis in Hep3B and Huh7 hepatic cancer cells. The western-blot analysis revealed, marked decrease in Bcl-2/Bax ratio, and increase in cleaved-PARP and cleaved-caspase-3 expressions. In vivo studies in Ehrlich ascites tumor (EAT) model following intravenous administration demonstrated significant reduction in tumor volume in case of PEGylated BA as compare to native BA. Furthermore, PEGylated BA treated EAT mice showed no biochemical and histological toxicities. These findings demonstrate the potential of PEGylated BA in cancer therapy, with improved water solubility and efficacy. © 2016 Published by Elsevier B.V.

Pubchem classifications Betulinic acid (PubChem CID: 64971) 1-Ethyl-3 (3-dimethylaminopropyl) carbodiimide (PubChem CID: 15908) Hydroxybenzotriazole (PubChem CID: 75771) Methoxypolyethylene glycol amine (PubChem CID: 45157584) Diisopropylethylamine (PubChem CID: 81531)

1. Introduction Cancer, characterized by the uncontrolled growth of abnormal cells, is a leading cause of death worldwide [1]. It has been estimated by ⁎ Correspondence to: S.A. Tasaduq, PK-PD-Toxicology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-Tawi 180001, India. ⁎⁎ Correspondence to: P.N. Gupta, Formulation & Drug Delivery Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-Tawi 180001, India. E-mail addresses: [email protected] (S.A. Tasaduq), [email protected] (P.N. Gupta).

http://dx.doi.org/10.1016/j.msec.2016.12.109 0928-4931/© 2016 Published by Elsevier B.V.

International Agency for Research on Cancer that approximately 14 million new cases and 8.2 million cancer related deaths occurred in 2012 globally [2]. Chemotherapy i.e. the use of chemical agents to destroy cancer cells is a mainstay in the cancer treatment mostly for patients who do not respond to local excision or radiation treatment [3]. However, the effectiveness of cancer chemotherapy is limited due to poor aqueous solubility, poor bioavailability and toxicity of chemotherapeutic agents towards normal cells [4]. It is also worth emphasizing that more than one-third of the top-selling chemotherapeutic drugs in the world are natural products or their derivatives [5]. Betulinic acid (BA), a naturally occurring plant-derived pentacyclic triterpenoid has been reported to show a wide spectrum of pharmacological activities such as antimalarial, anti-inflammatory, anti-HIV, anticancer, hepatoprotective, anthelmintic, anti-depression and antioxidant [6,7]. BA has been shown to possess potent anticancer activity in various cancer cells, including hepatoma [6] breast cancer [8] prostate cancer [9] pancreatic cancer [10] colorectal cancer [11] and chronic myelogenous leukemia [12]. BA has been reported to induce cancer death via mitochondrial membrane permeabilization with the release of factors like cytochrome c, Smac or apoptosis inducing factor (AIF) in a permeability

A. Saneja et al. / Materials Science and Engineering C 73 (2017) 616–626

transition pore-dependent manner, activating caspases and nuclear fragmentation [13]. In spite of tremendous anticancer activity, it has some drawbacks, such as poor aqueous solubility and short half-life, which limits its efficacy. Therefore in recent years, much work has been focused on enhancing its solubility as well as efficacy by employing wide range of nanocarriers such as polymeric nanoparticles [14–16], polymeric conjugates [13,17,18], carbon nanotubes [19], cyclodextrins [20] and liposomes [21]. Medicinal chemists have also made efforts to enhance the efficacy of BA through various modifications on the C-3 and/or C-28 positions [22–25]. Our group has also been actively engaged in development of polymeric nanocarriers for delivery of natural products as well as approved chemotherapeutic drugs [26–29]. Recently, we have developed polymeric nanoparticles of pentacyclic triterpenediol (TPD) from Boswellia serrata and demonstrated augmented anticancer efficacy of nanoparticles both in vitro and in vivo [30,31]. Among the wide variety of nanocarriers employed by formulation scientists, PEG-drug conjugate (often referred as PEGylation) has emerged as one of the most propitious platform for the efficient delivery of chemotherapeutic agents. A wide variety of chemotherapeutic agents have been reinvented by PEGylation due to its capability to solubilise very insoluble drugs, prolong circulation time, and alter the bio-distribution of drugs through “enhanced permeability and retention (EPR)” effect [32–34]. Recently, efforts have been made to enhance the effectiveness of BA acid via conjugation with multi-arm PEGs via esterification at C-3 position for treatment of lung cancer [13]. Further, it has been demonstrated that biotransformation or modification at the C-28 carboxyl group of tri-terpenes leads to disappearance of hemolytic properties [20]. Therefore, taking these aspects into the consideration, in present study, we have conjugated the amine group of mPEG with C-28 carboxylic group of BA via amide bond. The characterization of PEGylated betulinic acid was done by different analytical techniques like 1H NMR, 13C NMR, FT-IR and UV-spectroscopy. Molecular weights of polymers have been determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The mPEG-BA conjugate was evaluated for solubility and in vitro hydrolysis. In vitro cytoxicity study was conducted using MTT assay in Hep3B and Huh7 hepatic cancer cells and in vivo efficacy of PEGylated BA was investigated in Ehrlich ascites tumor (EAT) model.

2. Materials and methods

617

2.2. Synthesis of PEG-betulinic acid conjugate Betulinic acid (purity N97%) was isolated from 90% ethanolic extract of the stem bark of Platanus orientalis as described previously [35]. The PEGylated betulinic acid was synthesized using carbodiimide chemistry as reported previously [36]. Briefly, mPEG amine (1 equiv.), betulinic acid (2 equiv.), EDC (3 equiv.), HOBt (3 equiv.) and diisopropylethylamine (3 equiv.) were reacted in anhydrous DMF (2 mL) under N2 atmosphere for 48 h (Fig. 1). Subsequently, the reaction mixture was purified using dialysis (Mw cut off 2 kDa) performed against DMF for 24 h and deionized water (Millipore, India) for 48 h by replacing water at interval of every 2 h. Then the dialyzed solution was freeze-dried (Advantage freeze dryer, VirTis, Gardiner, NY, USA) using optimized protocol to obtain the powdered form of product. 2.3. Characterization of PEGylated betulinic acid The PEGylated betulinic acid was characterized using NMR, FT-IR, MALDI and UV–visible spectroscopy. 1H NMR (400 MHz) and 13C spectra (125 MHz) were recorded for betulinic acid, mPEG and PEGylated betulinic acid by NMR spectrometer (UXNMR, Bruker Analytische Messtechnik GmbH) using deuterated DMSO as the solvent. BA contents of PEGylated betulinic acid was measured by UV–Vis spectrophotometer (Shimadzu, UV-2600, Japan) at 210 nm as described previously [13]. The FTIR spectra (Perkin-Elmer, USA) were recorded using KBr pellet method over a range of 4000–400 cm−1. The matrix assisted laser desorption ionization (MALDI) mass spectroscopy (Applied Biosystems) was also performed in order to characterize PEGylated betulinic acid. 2.4. Solubility study The solubility of BA and PEGylated betulinic acid (mPEG-BA) was determined as described previously [13]. Briefly, BA and mPEG-BA were added in excess quantity in double distilled water and incubated at 37 °C for 24 h with gentle shaking at 80 rpm in thermo-mixture. Following incubation the samples were centrifuged (15,000 rpm for 10 min) and supernatant collected was diluted and BA content was determined by HPLC (Shimadzu Nexera, Japan). The HPLC analysis was conducted using C18 column (Lichrosphere RP-18, 250 mm × 4 mm × 5 μm) operated at 30 °C with UV detector set at 210 nm. The mobile phase was consisted of acetonitrile and water (75:25% v/v) at a flow rate of 1.0 mL/min.

2.1. Materials 2.5. In vitro stability of the conjugate Monomethoxy poly(ethylene glycol) (mPEG, MW 2 kDa), 1ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC), anhydrous N,N-dimethylformamide (DMF), hydroxybenzotriazole (HOBt), diisopropylethylamine (DIP) and thiazolylblue tetrazolium bromide (MTT) were obtained from Sigma–Aldrich, India. All other chemicals used were of analytical grade.

In vitro stability of mPEG-BA conjugate was examined in phosphate buffer saline (PBS) solution at physiological pH (7.4) and sodium acetate buffer (5.6). Briefly, the mPEG-BA conjugate was incubated in the buffers (1 mg/mL) at 37 °C and at scheduled time intervals aliquots were removed, centrifuged at 15,000 g for 15 min to get supernatant

Fig. 1. Synthetic scheme for conjugation of betulinic acid and mPEG.

618

A. Saneja et al. / Materials Science and Engineering C 73 (2017) 616–626

and analyzed by HPLC at 210 nm. The percentage of conjugate stability was generated by measuring the disappearance of the area of the conjugate peak vs initial area peak as reported previously [13]. 2.6. In vitro hemolysis The hemolysis assay was performed according to the previously reported protocol [29]. Fresh blood was collected from male Wistar rats and mixed added with EDTA-Na2 immediately to prevent coagulation. The collected blood was centrifuged at 3000 rpm for 10 min supernatant was discarded and settled red blood cells (RBC) were washed three times with phosphate buffer saline (PBS) solution (pH 7.4). The RBC collected following centrifugation was diluted 1:10 using 0.01 M PBS. The RBC suspension (0.1 mL) was added with 0.9 mL of mPEG-BA conjugate (0.1 mg/mL and 1 mg/mL in PBS) or PEI25K. The blank PBS and 1% Triton X-100 in PBS served as positive (100% lysis) and negative (0% lysis) control respectively. The samples were incubated at 37 ± 1 °C for 1 h in shaker incubator (New Brunswick Scientific, C76 water bath shaker, NJ-USA), centrifuged at 3000 rpm for 10 min and the supernatants were analyzed by using microplate spectrophotometer at 540 nm (Thermo Fisher Scientific, Multiscan, Finland) for hemoglobin release. The percent hemolysis was calculated using the following formula: Hem ð%Þ ¼

ABSSamples −ABS0 x 100 ABS100 −ABS0

where, ABSsamples, ABS100 and ABS0 are the absorbances of the samples, negative control (100% hemolysis) and positive control (0% hemolysis), respectively. Results are presented as mean of three measurements ± SD. 2.7. Cell culture, growth conditions and treatment conditions Human hepatoma cell lines Hep3b and Huh-7 were employed to study effects of BA and mPEG-BA on cell viability. Hep3b was procured from American Type Cell Culture (ATCC) while Huh-7 cells were obtained as kind gift from Prof. (Dr.) M. Charlton (Mayo Clinic USA). Both the cell lines were grown in 75 cm2 culture flask (Nunc Thermo Inc.) and maintained in Dulbecco's modified eagle medium (DMEM) supplemented with 200 U/mL penicillin, 270 μg/mL streptomycin and 10% (v/v) fetal bovine serum (FBS). Cells were incubated at 37 °C/5% CO2 and after attaining 80% confluency, cells were passaged three times before initiating cytotoxic assay.

concentrations of 1 μM of BA and mPEG-BA for 24 and 72 h. For microscopy, cells were washed with DPBS twice and incubated with Lyso Tracker Red DND-99 solution prepared in incomplete DMEM, (2 μl of 1 mM DMSO solution in 20 mL of DMEM) for 30 min in the dark, washed with DPBS, fixed with 10% buffered formaldehyde. The images were acquired on Evios fluorescent microscope (Life Technology, USA). 2.10. Western blot analysis Protein isolation and analysis was performed on Hep3b cell line, for which it was grown in 90 mm of culture dishes (Nunc, Thermo-Fischer Inc. USA). After attaining required confluency, treatment was given with mPEG-BA at concentrations of 15 μM and 30 μM respectively for 72 h. Cells were trypsinised using 1× Trypsin-EDTA solution (Sigma-Aldrich St. Louis USA) and harvested in DPBS (pH 7.4), centrifuged at 1000 rpm for 5 min, resuspended in RIPA buffer (Sigma-Aldrich St. Louis USA) along with fresh addition of protease inhibitor cocktail, phosphatase inhibitor cocktail, and phenyl methyl sulfonyl fluoride (PMSF), (Sigma-Aldrich St. Louis USA) all at 1% of total buffer volume. After 40 min of incubation at 4 °C, lysates were centrifuged at 14000 rpm for 40 min at 4 °C, followed by collection of supernatant and leaving behind cellular debris. Protein quantification was performed by Bradford assay. Equal amount (ranging 40–70 μg) of protein from each sample was denatured at 100 °C, for 4 min in Laemmeli buffer. Protein samples were resolved on 7–12% SDS-PAGE gels at 70–100 V, following trans-blotting on poly-vinylene difluoride (PVDF) membrane (EMD-Millipore, MA, USA), at 100–150 V for 2 h. Blocking of membrane was done by 5% defatted high protein milk in Tris-buffer saline with Tween 20 (TBST), pH 8.0 for 2 h with overnight incubation with primary antibodies (anti-PARP, anti-Bcl-2, anti-Bax, anti-caspase-3, and antibeta actin, Sanatacruz Biotechnology, USA) followed by incubation with secondary HRP-conjugated antibodies of either anti-mouse or anti-rabbit origin. Membrane visualization was done by ChemiDoc ™ XRS + (Bio-Rad, USA) and densitometry analysis was performed by Image Lab software version 3.0. 2.11. In vivo anticancer activity of BA and mPEG-BA in Ehrlich tumor (solid)

The cytotoxic effect of native BA and mPEG-BA was assayed using colorimetry by the MTT assay as described previously with slight modifications [37]. Briefly cells were seeded in 96 well plates and after attaining desired confluency, treatments with different concentrations of BA and mPEG-BA were given. After 72 h of corresponding treatments, cells were incubated with MTT solution (0.25 mg/mL in Dulbecco's Phosphate Buffer Saline (DPBS), pH 7.4) for 3 h at 37 °C. Formazan crystals formed were dissolved in dimethyl sulfoxide (DMSO), and optical density was measured at 570 nm using Mutiskan spectrum (ThermoElectron Corporation USA). The IC50 was calculated by plotting concentration versus percent inhibition values in Graphpad Prism software (v5.0).

In vivo anticancer activity of BA and mPEG-BA was performed according to the protocols approved by Institutional Animal Ethics Committee (IAEC). The Swiss albino male mice (20–25 g) were grouped and housed in poly acrylic cages. The Ehrlich ascites carcinoma (EAC) cells were maintained in swiss albino mice in peritoneal cavity. The EAC cells obtained from peritoneal cavity were injected (1 × 107 EAC tumor cells) in vivo by intramuscularly inoculation in the right thigh of the Swiss male mice at the day 0. The mice were randomly categorized into four groups, respectively (n = 7); (i) control group treated with normal saline, (ii) positive control group treated with 5-fuorouracil (5-FU) (22 mg kg− 1 i.p.), (iii) BA solution (10 mg kg− 1) and (iv) PEGylated BA (10 mg kg−1 equivalent to BA). Normal saline and 5-FU were regularly injected from day 1 to 9 via the intraperitoneal (IP) route. BA and mPEG-BA were intravenously injected as multiple doses of 10 mg kg 1 (every 2 days, q2d × 5). In the observation phase, the body weight of the mice was noted on days 1, 5, 9 and 13. At the end of the experiment (on day 13), the tumor bearing thigh of mice was shaved and longest (a) and shortest diameter (b) of the tumor were measured by using vernier caliper and the tumor volume (V) was calculated by using the formula: V = 1/2(a × b2) [39].

2.9. Sub-cellular fate of mPEG-BA

2.12. Evaluation of biochemical parameters

The intracellular localization of native BA and mPEG-BA was analyzed by using Lysotracker-Red DND-99 (Molecular Probes, Oregon, USA) [38]. Briefly, Hep3b cells were seeded in 6-well cell culture plate in 2 mL of DMEM and incubated overnight for attachment. After reaching suitable confluency, cells were treated with respective

At the end of the experiment various biochemical parameters were evaluated. For examination of the biochemical parameter, blood samples were allowed to clot and plasma was separated by centrifugation of blood samples at 3000g for 10 min at 4 °C. Plasma samples were analyzed for various biochemical parameters such as Alanine

2.8. In vitro cytotoxicity

A. Saneja et al. / Materials Science and Engineering C 73 (2017) 616–626

transaminase (ALT), Aspartate aminotransferase (AST), urea and creatinine by using automated biochem analyzer (Erba EM360, Japan). 2.13. Histopathological examination of organs of EAT bearing mice after the therapy For histopathological examination, mice were sacrificed by cervical dislocation and organs viz. liver and kidney were excised, fixed in 10% neutral buffer formalin, paraffinised and sectioned (5 μM), using microtome. Slides were stained with hematoxylin-eosin (H&E) staining for histopathological examination. Images of histopathological examination were observed under Nikon Eclipse E200 microscope (Nikon, Korea). 2.14. Statistical analysis All the results were expressed as mean ± standard deviation. The statistical analysis was carried out by using Student's t-test (for two groups) and p-value b 0.05 were considered as statistically significant. 3. Results 3.1. Synthesis and characterization of PEGylated betulinic acid PEGylated betulinic acid was synthesized by direct coupling of carboxylic group of betulinic acid with –NH2 group of mPEG as illustrated in Fig. 1. Betulinic acid was coupled to mPEG using EDC and HOBT coupling chemistry and purified conjugate was characterized by 1H NMR and 13C NMR using DMSO-d6 as the solvent. The 1H NMR of mPEG (Fig. 2b) give signals at the range of δ 3.4–3.6 ppm due to protons of \\O-CH2-CH2\\ (PEG chain) and a signal at δ 3.24 ppm due to protons of CH3 group in mPEG. 1H NMR of PEGylated betulinic acid (Fig. 2c) give the signals of both BA and mPEG at δ 4.65–4.53 ppm (\\CH2\\ at C-29 of BA), 1.62 ppm (\\CH3 in 30 of BA), 3.43–3.63 ppm (\\O-CH2CH2\\ of PEG chain) and 3.24 ppm (s, CH3 of mPEG). Further, the appearances of an amide signal δ 7.59 ppm confirms the covalent bond formation in betulinic acid and mPEG. The 13C NMR of mPEG (Fig. 3b) give signal at δ 70 ppm due to \\O-CH2-CH2\\ of PEG chain and at δ 59 (CH3 of mPEG). 13C NMR of PEGylated betulinic acid (Fig. 3c) gave

619

the signals of both BA and mPEG. The signal in 13C NMR at δ 178 ppm (due to carboxylic group) was shifted slightly to δ 175 ppm in PEGylated betulinic acid due to amide bond formation. The peaks of 13C NMR was also correlated by DEPT (distortionless enhancement of polarisation transfer)-135 spectra (see Supplementary information, Fig. S1). DEPT spectra demonstrated all the 11 \\CH2 groups of BA in negative side. PEGylated betulinic acid demonstrated 11 \\CH2 groups of BA along with the CH2 groups of PEG in the negative side whereas the signal of \\CH3 group of mPEG was observed at δ 59 at positive side. The FT-IR spectra of mPEG (Fig. 4a) exhibits its characteristic peaks at 1115 cm−1 due to C\\O\\C units of PEG backbone along with signals at 2884 cm− 1 for C\\H stretching of repeating ethylene groups. Betulinic acid exhibit the characteristic peaks at 3467 and 1686 cm−1 due to \\OH and \\COOH functional groups stretch of carboxylic groups. The peaks at 2942 and 1450 cm−1 imply both the asymmetric and symmetric C\\H stretching vibrations which arise from the methyl and methylene group of BA. PEGylated betulinic acid displayed the characteristic peaks at 2884 and 1105 cm−1 similar to mPEG. Also, the spectral peak at 1711 and 1645 cm− 1 exhibit the amide bond formation between betulinic acid and mPEG. The conjugation between PEG and BA was also characterized by MALDI-TOF MS (Fig. 5). The MALDI spectra of both mPEG and PEGylated BA exhibited a difference of 44 units between each peak due to the repeating monomer unit of CH2CH2O of mPEG. Belulinic acid exhibits mass of 456 Da (Supplementary Fig. S2) and its conjugate with mPEG demonstrated increase in each mass peak to approximately 438 Da compared with that of free mPEG, which indicated BA has been conjugated to the mPEG. For example, the peaks of mPEG at 2168.5, 2080.4, 1992.4 has been shifted to 2606.8, 2518.7, 2430.7 in PEGylated BA, which confirms that one mPEG molecule has been successfully conjugated to one molecule of BA. The content of BA bound to mPEG was 15.68 ± 1.04% as determined using UV spectroscopy. 3.2. Solubility study The BA had a very poor aqueous solubility (~21 μg/mL) [13]. In our study, after conjugation with mPEG, the solubility of BA has been increased significantly, from 16.53 ± 0.85 μg/mL to the 749.15 ± 6.03 μg/mL. This enhancement in solubility could be attributed to the

Fig. 2. 1H NMR (400 MHz) spectra of (a) betulinic acid, (b) mPEG and (c) mPEG-betulinic acid conjugate in deuterated dimethyl sulphoxide.

620

A. Saneja et al. / Materials Science and Engineering C 73 (2017) 616–626

Fig. 3. 13C NMR (125 MHz) spectra of (a) betulinic acid, (b) mPEG and (c) mPEG-betulinic acid conjugate in deuterated dimethyl sulphoxide.

presence of PEG spacer, which constitutes almost 84% of the synthesized conjugate.

indicated by presence of N 98%, 96% and 90% drug following 24, 48 and 72 h respectively, indicating low potential for off-target effect. 3.4. In vitro hemolysis

3.3. In vitro stability The in vitro stability study of PEGylated BA conjugate was measured in buffers at different pH value (5.6 and 7.4) using HPLC (Table 1). The PEGylated BA was highly stable under physiological pH conditions as

For an intravenously administered drug delivery system, the formulation should be biocompatible with blood components. Betulinic acid has been reported to be cytotoxic to RBC [40]. Therefore, hemolytic activity of the mPEG-BA was examined and the RBC lysis profiles were

Fig. 4. Fourier transform infrared spectra of (a) betulinic acid, (b) mPEG and (c) mPEG-betulinic acid in the region 4000 to 500 cm−1.

A. Saneja et al. / Materials Science and Engineering C 73 (2017) 616–626

621

Fig. 5. MALDI-TOF mass spectrum of (a) mPEG and (b) mPEG-betulinic acid conjugate (mPEG-BA). The single mass peaks of mPEG-BA are shifted by a factor of 438 Da, equivalent to one BA moleculea. aMW (mPEG-BA) = MW (mPEG-NH2) + MW (BA) − MW (H2O) = MW (mPEG-NH2) + 438.

expressed as the percentage of hemoglobin released relative to the positive and negative controls. At the tested concentrations PEGylated BA demonstrated b 2% hemolysis (Fig. 6). 3.5. In vitro cytotoxicity Cytotoxicity assay was performed to evaluate anti-proliferative effect of mPEG-BA and native BA after 72 h of exposure with Hep3b and Huh-7 cells. Cell viability analysis revealed that mPEG-BA and BA were cytotoxic to both the cell lines in concentration dependent manner (Fig. 7). BA showed cytotoxicity with IC50 values for Hep3bas 5.67 μM and 7.45 μM for Huh-7 while IC50 values for mPEG-BA were19.93 μM for Hep3b and 17.25 μM for Huh-7.

endosomes and lysosomes, mainly by localizing in acidic organelle of the cells. The experiment was done on Hep3b cells with concentration 30 μM for BA and mPEG-BA. Fig. 8 showed the comparative punctate fluorescence co-localized with LysoTracker Red dye indicating the formation of lysosomes. After 72 h of treatment, mPEG-BA showed much enhanced punctate fluorescence as compared to native BA. The control untreated cells with very few red labels show basal levels of lysosomes inside the cells.

3.6. Intracellular fate The cellular uptake and subsequent internalization of conjugate is important consideration influencing pharmacological effect of drug. We studied the sub-cellular fate of mPEG-BA in comparison to native BA. Lysotracker Red DND-99 dye was employed which labels the

Table 1 Stability of mPEG-BA conjugate in different media at 37 °C. Time (days)

Sodium acetate buffer (pH 5.6)

PBS buffer (pH 7.4)

0 1 2 3

100 ± 0.35% 97.84 ± 0.33% 95.11 ± 0.39% 84.62 ± 1.84%

100 ± 0.67% 98.70 ± 0.72% 96.85 ± 0.27% 90.72 ± 0.47%

Fig. 6. Graphical representation of hemolysis percentage (error bars represent the standard deviation). Hemolytic potential of mPEG-BA conjugate was compared to PEI25K and Triton X-100 by measuring absorbance at 540 nm.*p b 0.05, and **p b 0.01.

622

A. Saneja et al. / Materials Science and Engineering C 73 (2017) 616–626

Fig. 7. IC50 and dose dependent cytotoxicity of native BA and mPEG-BA. IC50 value was calculated on Human hepatoma cell lines: (a) Hep3b, and (b) Huh-7. Cells were incubated with different concentrations of BA and mPEG-BA for 72 h. MTT (0.25 mg/mL in DPBS) was added for 3 h, and OD was taken at 570 nm. The inhibition was calculated with respect to their respective controls, and IC50 was calculated using Graphpad Prism software (v5.0).

3.7. Western blot analysis Hep3b cells were treated with 15 μM (dose below IC50) and 30 μM (dose above IC50) concentrations of mPEG-BA for 72 h and effect of mPEG-BA was evaluated on protein expressions of few apoptotic markers by western blotting (Fig. 9). mPEG-BA decreased the bcl2/bax ratio as compared to untreated, control cells in dose dependent manner. At 15 μM, the ratio was decreased to 2.5 fold as compared to control cells, while at 30 μM it was reduced to 3.3 fold. Further, we analyzed two important markers of cell death i.e. cleaved PARP and cleaved caspase-3. At both the concentrations of mPEG-BA, the increase in protein expression of cleaved PARP was observed in dose dependent manner. It was increased significantly by 1.7 fold at 15 μM and 2.4 fold at 30 μM mPEG-BA, hence indicating apoptosis in treated cells. mPEG-BA induced apoptosis was also evident by increased expression of cleaved caspase-3 at both the concentrations. The cleaved caspase-3 (p-17) which is mature form of caspase-3 was increased significantly to 1.4 fold and 1.7 fold respectively at 15 μM and 30 μM concentrations of mPEG-BA, as compared to untreated control cells.

3.8. In vivo anticancer activity of BA and mPEG-BA in EAT bearing mice model The in vitro cytotoxicity and western blot analysis demonstrated anticancer potential of mPEG-BA. Thus inspired by the in vitro results, in vivo antitumor efficacy of mPEG-BA was evaluated in EAT bearing Swiss mice. The tumor growth inhibition and tumor volume were used as indicators for measuring in vivo effectiveness. The tumor growth

inhibition was compared after multiple injections of respective treatments i.e. normal saline, 5-FU (22 mg kg−1), BA (10 mg kg− 1) and mPEG-BA (10 mg kg−1 equivalent to BA), post EAT tumor inoculation in the mice which is schematically represented in Fig. 10a. At day 13, average tumor growth inhibition of the group treated with 5-FU (standard), BA and mPEG-BA was 50.40%, 44.04% and 55.29% (Fig. 10b). The average tumor volume of normal saline, 5-FU (standard), BA and mPEG-BA was approximately 1143, 567, 639 and 511 mm3 (Fig. 10c) thus demonstrating improved in-vivo efficacy of the mPEG-BA conjugate as compare to BA. This may be attributed to selective localization by EPR effect and prolonged release of drug at desired site. Further, there was no significant change in the body weight and no mortality in mice. 3.9. Biochemical and histopathological examination of EAT bearing mice after therapy Various biochemical parameters such as ALT, AST, urea and creatinine were also determined as per standard procedures to examine organ toxicity (Fig. 11A). All the biochemical parameters were found to be in the normal range, demonstrating safety profile of BA and mPEG-BA. In order to investigate the organ toxicity in mice after the therapy most essential visceral organs i.e. liver and kidney were excised for histopathological examination. Despite the promising anticancer efficacy of mPEG-BA, no obvious histopathological changes were found in these organs as compared to normal control groups (Fig. 11B). The tissue sections of the mice treated with the native BA were also normal. Thus, these results demonstrated the biocompatibility of the mPEG-BA in the in vivo biological system under the experimental conditions.

Fig. 8. Sub-cellular localization of BA and mPEG-BA by Lysotracker Red DND dye staining. Images of Hep3b cells stained with Lysotracker Red DND dye after 72 h of individual treatment with 30 μM of BA and mPEG-BA (equivalent to BA). The control cells, with very few red dots of fluorescence, indicating basal levels of lysosomes. The higher fluorescence was observed with mPEG-BA treatment as compared to native BA. The concentration of LysoTracker Red DND-99 was used as 0.1 mM. The images (20×) were taken on EVOS fluorescent imaging system, Life Technology USA.

A. Saneja et al. / Materials Science and Engineering C 73 (2017) 616–626

623

Fig. 9. Effect of mPEG-BA on protein expression of apoptotic markers. Images show western blots and densitometric graphs of indicated proteins of Hep3b cells which were treated with 15 and 30 μM concentrations of mPEG-BA for 72 h. mPEG-BA induced apoptosis in Hep3b cells, shown by dose dependent reduction in bcl2/bax ratio, [2.5 fold (15 μM) & 3.3 fold (30 μM)] as compared to control un-treated cells, (*** indicate p b 0.001) and increased in expression of cleaved caspase-3 [1.4 fold (15 μM) & 1.7 fold (30 μM)], (* indicates p b 0.05), and cleaved PARP [1.7 fold (15 μM), * indicates p b 0.05, & 2.4 fold (30 μM), ** indicate p b 0.001]. All the values were normalized with β-actin, and fold change was calculated with respect to control untreated cells, considering their value as 1.

4. Discussion The anticancer efficacy of betulinic acid is compromised due to its poor aqueous solubility and short half-life. There are several advantages reported for PEGylation of betulinic acid at C-3 position such as

enhancing the solubility, stability and in-vivo efficacy [13]. BA showed hemolytic potential and it has been reported that modification of C-28 position of BA could limit this drawback. Therefore we synthesized mPEG conjugate of betulinic acid by coupling with C-28 carboxyl groups via EDC/HOBT based chemistry. The characterization of the conjugate

Fig. 10. Evaluation of in vivo anticancer activity of BA and mPEG-BA in Ehrlich ascites tumor (EAT) bearing Swiss mice. (a) Schematic representation of the experimental design, (b) graph demonstrating tumor growth inhibition (%) and (c) average tumor volume after therapy. 1 × 107 Ehrlich ascites carcinoma (EAC) cells were intramuscularly inoculated in the right thigh of the Swiss male mice at day 0 and then divided randomly into four groups: normal control, 5-FU (22 mg kg−1), BA (10 mg kg−1) and mPEG-BA (10 mg kg−1 equivalent to BA). Data are represented as mean value ± SD (n = 7). The statics were applied using student t-test. *** indicates p b 0.0001, mPEG-BA versus BA.

624

A. Saneja et al. / Materials Science and Engineering C 73 (2017) 616–626

Fig. 11. (A) Investigation of various biochemical parameters measured in serum after treatment in EAT tumor in Swiss mice. Data are represented as mean value ± SD (n = 5). (B) Histopathological examination of liver and kidney of Ehrlich ascites tumor (EAT) bearing mice after the therapy experiment of BA and mPEG-BA ((Olympus, 20× magnification). At the experimental end point, the organs from each group were excised and the tissue sections were preceded for H & E staining. No significant difference was observed by applying one way ANOVA followed by Student's t-test.

performed using NMR, FT-IR and MALDI demonstrated conjugation of betulinic acid with mPEG. The conjugate demonstrated enhanced solubility, stability and diminished hemolytic activity. The reduced hemolytic potential of the developed conjugate may be attributed to the little hydrolysis during the incubation period (1 h) resulting in meager availability of free drug in systemic circulation leading to good biocompatibility. The hemolysis of betulinic acid at equivalent concentration of mPEG-BA at 0.1 mg/mL was slightly greater than that of mPEG-BA and it has been shown that 60 μM of the BA exhibited approximately 38% hemolysis [40]. Therefore, developed pegylated version of BA is advantageous in having non-hemolytic nature. The pegylation of BA was performed at C-3 position in an earlier report [13] and the drug loading was around 11.81%, while in our study, higher drug loading was observed (15.68%), which could be attributed to the lower molecular weight of mPEG used. In another investigation, betulinic acid nanocomposite was prepared with chitosan which exhibited drug loading as 6.7% [16]. Therefore, mPEG-BA is advantageous in terms of higher drug loading. Further, PEG is a biocompatible polymer approved by FDA, which offer additional advantage in comparison to other drug delivery polymer (i.e. chitosan). The mPEG-BA was compared with the native BA for its anti-proliferative activity in the hepato-carcinoma cell lines Hep3B and Huh-7. The BA is known to induce cytotoxicity in hepatoblastoma cells [6], and in our study also, BA showed its cytotoxic effects on both cells lines with IC50 as 5.67 μM for Hep3b and IC50 as 7.45 μM on Huh-7 cells. The synthesized conjugate also showed good cytotoxicity in both the cells

lines, when compared with the un-treated control cells. Although, mPEG-BA had higher IC50 than native BA, but its cytotoxicity was significant enough. Further, it is anticipated that the developed conjugate could offer more advantages in in-vivo condition owing to prolong residence time and sustained drug release in cancer cell compared to native BA. To further confirm the cytotoxic action and mechanistic events related to mPEG-BA, we performed the apoptotic protein expression analysis on Hep3b cells, treated with mPEG-BA for 72 h at two relevant doses of 15 μM (lower than IC50) and 30 μM (higher than IC50). Role of caspases is significant in apoptosis [41], therefore we investigated cleavage of caspase-3 which was significantly induced by mPEG-BA, at both the given doses. The bcl-2/bax balance is also critical to decide the fate of cell, from apoptotic stimuli [41]. Lower bcl-2/bax ratio signifies apoptosis, while a balanced ratio suggests cell resistant to apoptosis or a healthy cell. mPEG-BA, induced apoptotic Hep3b cells revealed significantly lower ratio of bcl-2/bax, in western blotting as compared to untreated cells. Along with this, mPEG-BA at both the given doses, induced the cleavage of PARP in Hep3b cells, which also represent significant apoptosis. Despite of higher IC50 values, the apoptotic activity of mPEG-BA was not compromised, as shown by in-vitro apoptotic protein expression analysis. Cells capture polymeric drug conjugates, which is further entrapped by lysosomes and endosomes, leading to degradation of conjugate and ensuing release of active moiety for desired therapeutic outcome [38]. In our study, Hep3b cells, when incubated for 72 h with mPEG-BA and native BA showed increased fluorescence with mPEG-

A. Saneja et al. / Materials Science and Engineering C 73 (2017) 616–626

BA as compared to native BA. It showed enhanced formation of lysosomes and endosomes thus indicating fair internalization of BA via polymeric conjugate compared to native BA. After, in-vitro confirmation of cytotoxic activity of developed conjugate, we further investigated its safety and efficacy in in-vivo mouse model of EAC as described in our previous study [31]. EAC is a model mimicking human tumor and it highly sensitive to the chemotherapeutic agents because of their undifferentiated nature coupled with rapid growth rate. EAC is further characterized by high transplantable capability, and short life span [42]. In our study mPEG-BA induced significant tumor growth suppression (p = 0.0001) as compared to native BA. At day 13, mPEG-BA treated mice showed average tumor volume of 511 mm3, which was highly significant (p = 0.0008) as compared to native BA treated group having average tumor volume of 639 mm3, indicating enhanced anti-tumor activity of mPEG-BA. The 5-FU, and normal saline group were kept as positive and negative controls respectively (Fig. 10). In our in-vivo study, we did not observe any sort of drug induced toxicity. There were no significant body weights changes among the four groups, and no mortality in case of mPEG-BA drug conjugate. This was further confirmed by the end point analysis of blood biochemistry data, and histopathological examination of major organs involved in drug metabolism viz. liver and kidney. The serum enzymes ALT, AST, and nephrotoxic markers viz. urea and creatinine were also in the recommended range in all four groups. The histopathological examination of liver and kidney sections of all groups showed no signs of inflammation, or any other drug related toxicological manifestations. Hence our findings suggest that the polymeric conjugate of BA i.e. mPEG-BA, proved to be highly efficacious with enhanced solubility, augmented efficacy and no in vivo toxic manifestations. Conclusively, in the present study, conjugate of betulinic acid with PEG was prepared which exhibited improved solubility, stability and enhanced cellular internalization. The synthesized conjugate was devoid of hemolytic toxicity and also demonstrated its potent anti-cancer potential in in-vitro hepatocarcinoma cell lines. Further, this conjugate showed enhanced anticancer potential as compared to native drug in in-vivo EAT tumor bearing mice, with no signs of toxicity. The improved efficacy of PEG-BA conjugate could be attributed to the prolonged tumor residence time and slow drug release facilitating tumor drug accumulation through the EPR effect. Conflict of interest Authors declare that they have no conflicts of interest. Acknowledgements Authors are grateful to Director, CSIR-IIIM, Jammu for providing necessary support for carrying out this work. The study was supported by funding from Council of Scientific and Industrial Research (CSIR), New Delhi, India (MLP6006 and BSC0106). The institutional publication number for this manuscript is IIIM/1963/2016. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2016.12.109. References [1] D. Arora, S. Jaglan, Nanocarriers based delivery of nutraceuticals for cancer prevention and treatment: a review of recent research developments, Trends Food Sci. Technol. 54 (2016) 114–126. [2] J. Ferlay, I. Soerjomataram, M. Ervik, S. Eser, C. Mathers, M. Rebelo, GLOBOCAN 2012 v1. 0, Cancer Incidence and Mortality Worldwide: IARC Cancer Base No. 11 2013, International Agency for Research on Cancer, Lyon, France, 2015.

625

[3] A. Saneja, V. Khare, N. Alam, R.D. Dubey, P.N. Gupta, Advances in P-glycoproteinbased approaches for delivering anticancer drugs: pharmacokinetic perspective and clinical relevance, Expert Opin. Drug Deliv. 11 (2014) 121–138. [4] A. Saneja, R. Dhar Dubey, N. Alam, V. Khare, P.N. Gupta, Co-formulation of P-glycoprotein substrate and inhibitor in nanocarriers: an emerging strategy for cancer chemotherapy, Curr. Cancer Drug Targets 14 (2014) 419–433. [5] R. Watkins, L. Wu, C. Zhang, R.M. Davis, B. Xu, Natural product-based nanomedicine: recent advances and issues, Int. J. Nanomedicine 10 (2015) 6055. [6] M. Eichenmuller, D. Von Schweinitz, R. Kappler, Betulinic acid treatment promotes apoptosis in hepatoblastoma cells, Int. J. Oncol. 35 (2009) 873. [7] H.Y. Quan, S.J. Kim, H.K. Jo, G.W. Kim, S.H. Chung, Betulinic acid alleviates non-alcoholic fatty liver by inhibiting SREBP1 activity via the AMPK–mTOR–SREBP signaling pathway, Biochem. Pharmacol. 85 (2013) 1330–1340. [8] R. Tiwari, A. Puthli, S. Balakrishnan, B. Sapra, K. Mishra, Betulinic acid-induced cytotoxicity in human breast tumor cell lines MCF-7 and T47D and its modification by tocopherol, Cancer Investig. 32 (2014) 402–408. [9] J. Shin, H.-J. Lee, D.-B. Jung, J.H. Jung, H.-J. Lee, E.-O. Lee, S.G. Lee, B.S. Shim, S.H. Choi, S.G. Ko, Suppression of STAT3 and HIF-1 alpha mediates anti-angiogenic activity of betulinic acid in hypoxic PC-3 prostate cancer cells, PLoS One 6 (2011), e21492. . [10] L. Li, Y. Du, X. Kong, Z. Li, Z. Jia, J. Cui, J. Gao, G. Wang, K. Xie, Lamin B1 is a novel therapeutic target of betulinic acid in pancreatic cancer, Clin. Cancer Res. 19 (2013) 4651–4661. [11] W. Ren, L. Qin, Y. Xu, N. Cheng, Inhibition of betulinic acid to growth and angiogenesis of human colorectal cancer cell in nude mice, Chin.-Ger. J. Clin. Oncol. 9 (2010) 153–157. [12] D.R. Gopal, A.A. Narkar, Y. Badrinath, K. Mishra, D. Joshi, Betulinic acid induces apoptosis in human chronic myelogenous leukemia (CML) cell line K-562 without altering the levels of Bcr-Abl, Toxicol. Lett. 155 (2005) 343–351. [13] L. Dai, D. Li, J. Cheng, J. Liu, L.-H. Deng, L.-Y. Wang, J.-D. Lei, J. He, Water soluble multiarm-polyethylene glycol–betulinic acid prodrugs: design, synthesis, and in vivo effectiveness, Polym. Chem. 5 (2014) 5775–5783. [14] L. Dai, C.-X. Li, K.-F. Liu, H.-J. Su, B.-Q. Chen, G.-F. Zhang, J. He, J.-D. Lei, Self-assembled serum albumin–poly (L-lactic acid) nanoparticles: a novel nanoparticle platform for drug delivery in cancer, RSC Adv. 5 (2015) 15612–15620. [15] L. Dai, T. Yang, J. He, L. Deng, J. Liu, L. Wang, J. Lei, L. Wang, Cellulose-graft-poly (Llactic acid) nanoparticles for efficient delivery of anti-cancer drugs, J. Mater. Chem. B 2 (2014) 6749–6757. [16] S.H. Hussein-Al-Ali, P. Arulselvan, S. Fakurazi, M.Z. Hussein, The in vitro therapeutic activity of betulinic acid nanocomposite on breast cancer cells (MCF-7) and normal fibroblast cell (3T3), J. Mater. Sci. 49 (2014) 8171–8182. [17] L. Dai, X. Cao, K.-F. Liu, C.-X. Li, G.-F. Zhang, L.-H. Deng, C.-L. Si, J. He, J.-D. Lei, Self-assembled targeted folate-conjugated eight-arm-polyethylene glycol–betulinic acid nanoparticles for co-delivery of anticancer drugs, J. Mater. Chem. B 3 (2015) 3754–3766. [18] L. Dai, K.-F. Liu, C.-L. Si, J. He, J.-D. Lei, L.-Q. Guo, A novel self-assembled targeted nanoparticle platform based on carboxymethylcellulose co-delivery of anticancer drugs, J. Mater. Chem. B 3 (2015) 6605–6617. [19] J.M. Tan, G. Karthivashan, P. Arulselvan, S. Fakurazi, M.Z. Hussein, Sustained release and cytotoxicity evaluation of carbon nanotube-mediated drug delivery system for betulinic acid, J. Nanomater. 2014 (2014) 1. [20] S. Xiao, Q. Wang, L. Si, Y. Shi, H. Wang, F. Yu, Y. Zhang, Y. Li, Y. Zheng, C. Zhang, Synthesis and anti-HCV entry activity studies of β-cyclodextrin–pentacyclic triterpene conjugates, ChemMedChem 9 (2014) 1060–1070. [21] F.B. Mullauer, L. van Bloois, J.B. Daalhuisen, M.S. Ten Brink, G. Storm, J.P. Medema, R.M. Schiffelers, J.H. Kessler, Betulinic acid delivered in liposomes reduces growth of human lung and colon cancers in mice without causing systemic toxicity, AntiCancer Drugs 22 (2011) 223–233. [22] B. Chakraborty, D. Dutta, S. Mukherjee, S. Das, N.C. Maiti, P. Das, C. Chowdhury, Synthesis and biological evaluation of a novel betulinic acid derivative as an inducer of apoptosis in human colon carcinoma cells (HT-29), Eur. J. Med. Chem. 102 (2015) 93–105. [23] Z. Dang, K. Qian, P. Ho, L. Zhu, K.-H. Lee, L. Huang, C.-H. Chen, Synthesis of betulinic acid derivatives as entry inhibitors against HIV-1 and bevirimat-resistant HIV-1 variants, Bioorg. Med. Chem. Lett. 22 (2012) 5190–5194. [24] K. Qian, I.D. Bori, C.-H. Chen, L. Huang, K.-H. Lee, Anti-AIDS agents 90. Novel C-28 modified bevirimat analogues as potent HIV maturation inhibitors, J. Med. Chem. 55 (2012) 8128–8136. [25] T.A.D. Thi, N.T.K. Tuyet, C.P. The, H.T. Nguyen, C.B. Thi, H.T. Phuong, L. Van Boi, T. Van Nguyen, M. D'hooghe, Synthesis and cytotoxic evaluation of novel amide–triazolelinked triterpenoid–AZT conjugates, Tetrahedron Lett. 56 (2015) 218–224. [26] N. Alam, V. Khare, R. Dubey, A. Saneja, M. Kushwaha, G. Singh, N. Sharma, B. Chandan, P.N. Gupta, Biodegradable polymeric system for cisplatin delivery: development, in vitro characterization and investigation of toxicity profile, Mater. Sci. Eng., C, Mater. Biol. Appl. 38 (2014) 85–93. [27] R.D. Dubey, N. Alam, A. Saneja, V. Khare, A. Kumar, S. Vaidh, G. Mahajan, P.R. Sharma, S.K. Singh, D.M. Mondhe, P.N. Gupta, Development and evaluation of folate functionalized albumin nanoparticles for targeted delivery of gemcitabine, Int. J. Pharm. 492 (2015) 80–91. [28] V. Khare, S. Kour, N. Alam, R.D. Dubey, A. Saneja, M. Koul, A.P. Gupta, D. Singh, S.K. Singh, A.K. Saxena, P.N. Gupta, Synthesis, characterization and mechanistic-insight into the anti-proliferative potential of PLGA-gemcitabine conjugate, Int. J. Pharm. 470 (2014) 51–62. [29] V. Khare, A. Singh, G. Mahajan, N. Alam, S. Kour, M. Gupta, A. Kumar, G. Singh, S.K. Singh, A.K. Saxena, D.M. Mondhe, P.N. Gupta, Long-circulatory nanoparticles for gemcitabine delivery: development and investigation of pharmacokinetics and invivo anticancer efficacy, Eur. J. Pharm. Sci. 92 (2016) 183–193.

626

A. Saneja et al. / Materials Science and Engineering C 73 (2017) 616–626

[30] N. Alam, A. Qayum, A. Kumar, V. Khare, P.R. Sharma, S.S. Andotra, S.K. Singh, S. Koul, P.N. Gupta, Improved efficacy of cisplatin in combination with a nano-formulation of pentacyclic triterpenediol, Mater. Sci. Eng., C, Mater. Biol. Appl. 68 (2016) 109–116. [31] R.D. Dubey, A. Saneja, A. Qayum, A. Singh, G. Mahajan, G. Chashoo, A. Kumar, S.S. Andotra, S.K. Singh, G. Singh, PLGA nanoparticles augmented the anticancer potential of pentacyclic triterpenediol in vivo in mice, RSC Adv. 6 (2016) 74586–74597. [32] A.K. Choubey, C.P. Dora, T.D. Bhatt, M.S. Gill, S. Suresh, Development and evaluation of PEGylated enoxaparin: a novel approach for enhanced anti-Xa activity, Bioorg. Chem. 54 (2014) 1–6. [33] M. Vandana, S.K. Sahoo, Long circulation and cytotoxicity of PEGylated gemcitabine and its potential for the treatment of pancreatic cancer, Biomaterials 31 (2010) 9340–9356. [34] M. Vandana, S.K. Sahoo, Reduced folate carrier independent internalization of PEGylated pemetrexed: a potential nanomedicinal approach for breast cancer therapy, Mol. Pharm. 9 (2012) 2828–2843. [35] R. Majeed, P.L. Sangwan, P.K. Chinthakindi, I. Khan, N.A. Dangroo, N. Thota, A. Hamid, P.R. Sharma, A.K. Saxena, S. Koul, Synthesis of 3-O-propargylated betulinic acid and its 1, 2, 3-triazoles as potential apoptotic agents, Eur. J. Med. Chem. 63 (2013) 782–792.

[36] C. Genet, A. Strehle, C. Schmidt, G. Boudjelal, A. Lobstein, K. Schoonjans, M. Souchet, J. Auwerx, R. Saladin, A. Wagner, Structure–activity relationship study of betulinic acid, a novel and selective TGR5 agonist, and its synthetic derivatives: potential impact in diabetes, J. Med. Chem. 53 (2009) 178–190. [37] M.V. Berridge, P.M. Herst, A.S. Tan, Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction, Biotechnol. Annu. Rev. 11 (2005) 127–152. [38] D. Chitkara, A. Mittal, S.W. Behrman, N. Kumar, R.I. Mahato, Self-assembling, amphiphilic polymer–gemcitabine conjugate shows enhanced antitumor efficacy against human pancreatic adenocarcinoma, Bioconjug. Chem. 24 (2013) 1161–1173. [39] N.F. Khedr, R.M. Khalil, Effect of hesperidin on mice bearing Ehrlich solid carcinoma maintained on doxorubicin, Tumor Biol. 36 (2015) 9267–9275. [40] M. Gao, P. Lau, S. Kong, Mitochondrial toxin betulinic acid induces in vitro eryptosis in human red blood cells through membrane permeabilization, Arch. Toxicol. 88 (2014) 755–768. [41] J.C. Reed, Mechanisms of apoptosis, Am. J. Pathol. 157 (2000) 1415–1430. [42] M. Ozaslan, I.D. Karagoz, I.H. Kilic, M.E. Guldur, Ehrlich ascites carcinoma, Afr. J. Biotechnol. 10 (13) (2011) 2375–2378.