Design, synthesis and evaluation of N-acetyl glucosamine (NAG)–PEG–doxorubicin targeted conjugates for anticancer delivery

International Journal of Pharmaceutics 436 (2012) 183–193

Contents lists available at SciVerse ScienceDirect

International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical Nanotechnology

Design, synthesis and evaluation of N-acetyl glucosamine (NAG)–PEG–doxorubicin targeted conjugates for anticancer delivery Smita K. Pawar a , Archana J. Badhwar b , Firuza Kharas b , Jayant J. Khandare b , Pradeep R. Vavia a,∗ a b

Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, N.P. Marg, Matunga (E), Mumbai 400019, India Piramal Life Sciences Ltd., 1 Nirlon Complex, Off Western Express Highway Goregaon (E), Mumbai, Maharashtra 400063, India

a r t i c l e

i n f o

Article history: Received 18 February 2012 Accepted 30 May 2012 Available online 18 June 2012 Keywords: Doxorubicin N-Acetyl glucosamine Prodrug Glutathione Cellular internalization

a b s t r a c t Efficacy of anticancer drug is limited by the severe adverse effects induced by drug; therefore the crux is in designing delivery systems targeted only to cancer cells. Toward this objectives, we propose, synthesis of poly(ethylene glycol) (PEG)–doxorubicin (DOX) prodrug conjugates consisting N-acetyl glucosamine (NAG) as a targeting moiety. Multicomponent system proposed here is characterized by 1 H NMR, UV spectroscopy, and HPLC. The multicomponent system is evaluated for in vitro cellular kinetics and anticancer activity using MCF-7 and MDA-MB-231 cells. Molecular modeling study demonstrated sterically stabilized conformations of polymeric conjugates. Interestingly, PEG–DOX conjugate with NAG ligand showed significantly higher cytotoxicity compared to drug conjugate with DOX. In addition, the polymer drug conjugate with NAG and DOX showed enhanced internalization and retention effect in cancer cells, compared to free DOX. Thus, with enhanced internalization and targeting ability of PEG conjugate of NAG–DOX has implication in targeted anticancer therapy. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Chemotherapeutic drugs like paclitaxel, doxorubicin (DOX), camptothecin, etc. are known to be highly potent; however, they are also known to induce severe systemic toxicity, intolerance, and resistance (Galletti et al., 2007). While the translation of such drugs into their polymeric prodrug conjugate offers reduction in systemic toxicity, improve therapeutic index, and enhanced targeting by a mechanism called enhance permeation and retention (EPR) effect (Matsumura and Maeda, 1986; Duncan, 2007). After following cellular uptake by the endocytic route they are potential to bypass mechanisms of drug resistance, leading to

Abbreviations: DOX, doxorubicin; NAG, N-acetyl glucosamine; PEG, polyethylene glycol; DMF, N,N-dimethyl formamide; DCM, dichloromethane; N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride; EDC.HCl, DMAP, N,N-diisopropyl-ethylamine,4-(methylamino) pyridine; EPR, enhance retention and permeation; LHRH, luteinizing hormone–releasing hormone; mAbs, monoclonal antibodies; TFA, trifluoroacetic acid; MAL, maleimide; GTS, glutathione; DIEA, N,N-diisopropyethyl amine; MS, material studio; PBS, physiological saline buffer solution; Fmoc, N-(9-fluorenylmethoxycarbonyl); HPMA, poly-2-hydroxypropylmethacrylamide; MTT, [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide]; DAPI, 4 ,6-diamidino-2-phenylindole. ∗ Corresponding author at: Center for Novel Drug Delivery Systems, Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, N.P. Marg, Matunga (E), Mumbai 400019, India. Tel.: +91 22 3361 2220; fax: +91 22 2414 5614. E-mail addresses: [email protected], [email protected] (P.R. Vavia). 0378-5173/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2012.05.078

enhance tumor targeting (>10–100-fold) compared to free drug (Duncan, 2006). Quite often the polymeric prodrug approach delivers anticancer drug with increased concentration of a drug at a tumor site due to overall passive targeting mechanism (Maeda et al., 1992). It is now very eminent fact that the tumors relatively possess altered physiology and molecular expression at cellular level compared to the normal tissues. Many studies have been focused in delivering anticancer drugs at the targeted sites using targeting peptides (e.g. LHRH (Dharap et al., 2005)), carbohydrates (e.g. fucose (Moriwaki and Miyosh, 2010), galactosamine (Pimm et al., 1993), sialic acid (Jayant et al., 2007), etc.), monoclonal antibodies (mAbs) (Singh et al., 1989) or ligands (e.g. folate) (Haizheng and Lin, 2008). The tumors are dense, with hampered heterogeneous vasculature and altered efflux mechanism thereby leading into lowering of drug disposition at the targeted site (Murray and Carmichael, 1995). Toward this direction, the criticalness in anticancer prodrug therapy is aimed with multifold of objectives: (a) enhance the aqueous solubility of the anticancer drugs, (b) design of suitable drug delivery system (DDS) to attain EPR effect (e.g. PEG, dendrimers, liposome, nanoparticle, etc.), and (c) utility of suitable targeting moiety with tumor cells internalization capacity. In addition, various synthetic strategies have been explored to improve the pharmacokinetic and pharmacodynamic profile for anticancer prodrug conjugates (Greco and Vicent, 2009). Recently, we reported multifunctional polymers in medicine to achieve cellular targetability and delivery (Khandare et al., 2012). Various architectures of polymers, assemblies, and prodrug conjugates having targeting components

184

S.K. Pawar et al. / International Journal of Pharmaceutics 436 (2012) 183–193

are being reported to enhance the anticancer activity using folate, RGD peptide, VEGF, LHRH, etc. However, newer ligands are being explored and bioconjugation methodologies using established polymers (including, PEG and other newer dendritic polymers) are developed. Therefore in this work, NAG is being evaluated as a targeting moiety, considering NAG as a ligand for the NAG receptors (NAGRs) over expressed on cancer cells (Dhanikula et al., 2008; Vannucci et al., 2003). In addition, NAG being a carbohydrate is excessively aqueous soluble, thereby may increase the aqueous solubility to the resulting PEG conjugate and it is also likely to have greater cellular internalization capacity. Thus, NAG could be used as a targeting ligand for targeting anticancer drugs to tumors that over express NAG receptors. Tumor cells multiply rapidly and may over-express certain receptors for enhanced uptake of nutrients, including folic acid, vitamins, and sugars (Jayant et al., 2007; Haizheng and Lin, 2008). While active targeting can be achieved by conjugating a tumorspecific ligand or a polymer to the chemotherapeutic drug via a cleavable linker (Khandare and Minko, 2006). Furthermore, specific tumor uptake can occur through receptor-mediated endocytosis, where upon binding of the ligand-modified polymer with the cell-surface receptor leads to internalization of the entire polymer–receptor complex and vesicular transport through the endosomes (Garnett, 2001). In addition, the targeting moieties on to the delivery system are known to enhance the specificity toward tumor with increase in penetrating property of DDS (Minko et al., 2004). Overall, targeted prodrug conjugates can offer increase in efficacy and decrease in systemic toxicity in cancer therapy (Chun and Sidney, 2008). Therefore, the main objective of this work is to establish (a) design and synthesis polymeric prodrug conjugate containing PEG as a carrier, DOX as a anticancer drug, NAG as a targeting moiety and conjugation using a glutathione as a spacer, (b) to characterize the PEG conjugate, (c) evaluate cellular entry and dynamics for comparative internalization and localization, and (d) to evaluate and compare in vitro efficacy using human MCF-7 and MDA-MB-231 cell line. In the past, we have reported targeted prodrug conjugates consisting anticancer drugs; camptothecin, DOX, and paclitaxel using poly (ethylene glycol) linear polymer and hyperbranched dendrimers (Jayant et al., 2007; Saad et al., 2008). Furthermore, we reported varied targeting and internalization ligands (e.g. LHRH, and sialic acid) for increased cellular internalization ability and multicomponent DDS (Dharap et al., 2005; Jayant et al., 2007).

Fig. 1. Schematic depiction of targeted polymer prodrug conjugate. Glutathione (GTS), N-acetyl d-glucosamine (NAG), doxorubicin (DOX), and PEG-maleimide (30 kDa) (PEG).

Here we report design, synthesis, and in vitro evaluation of targeted PEG–DOX prodrug conjugates having NAG as a targeting moiety for enhanced cellular delivery ability and anticancer activity. We present PEGylated prodrug conjugate consisting PEGmaleimide 30,000 Da (MAL) as a carrier, DOX as an anticancer drug, and NAG as a targeting moiety and penetration enhancer (Fig. 1). In addition, we evaluated comparative cellular internalization dynamics of free DOX and PEG-drug conjugates using confocal microscopy. Drug release rate from the conjugates was analyzed by HPLC using esterase enzyme. Finally, in vitro cytotoxicity of the conjugates was evaluated and compared using human MCF-7 and MDA-MB-231 breast cancer cells.

2. Materials and methods 2.1. Materials Doxorubicin was provided as a gift sample by RPG Life sciences, India. Methoxy PEG–maleimide (30 kDa) (PEG–MAL) was procured from Sunbright, UK. N-Acetyl d-glucosamine (NAG), glutathione (GTS), succinic anhydride, N-(3-dimethylaminopropyl)N-ethylcarbodiimideHCl (EDC.HCl), N,N-diisopropyl-ethylamine, 4-(methylamino) pyridine (DMAP), and Sephadex G10 were purchased from Sigma–Aldrich, India. 9-Fluronyl methylcarbonyl formic acid (FmoC) was purchased from Merck, Germany. Dialysis membrane of molecular weight cut off 10–12 kDa was purchased from Hi-media, India. MCF-7 and MDA-MB-231 cells were procured from ATCC. 4 ,6-Diamidino-2-phenylindole (DAPI) and [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) were purchased from Invitrogen. All other chemicals and solvents were of analytical grade and used without purification.

2.2. Synthesis of PEG–DOX–NAG Synthesis of PEG–DOX–NAG conjugate was achieved by two steps. First, synthesis of succinate derivative of DOX and further conjugation of synthesized DOX-succinate with PEG–GTS–NAG.

2.2.1. Synthesis of N-Fmoc-DOX-14-0-Succinate Synthesis of N-Fmoc-DOX-14-0-succinate (compound 5) conjugate is schematically represented in Scheme 1. DOX (compound 1) was reacted with succinic anhydride (compound 4) by twostep method as previously reported (Chena et al., 2003). DOX (92 ␮M) and sodium bicarbonate (280 ␮M) were dissolved in distilled water (5 ml). The resulting clear solution having pH 8–9 was stirred continuously on magnetic stirrer at 5–10 ◦ C. 9-Fmoc chloride (compound 2) (110 ␮M) in ethyl acetate (3 ml) was added drop wise into the above solution. The reaction mixture was maintained in the pH range of 7–8 with an addition of saturated solution of sodium bicarbonate. After 5 h, the product was extracted in ethyl acetate and further recrystallized in 0.1% trifluroacetic acid (TFA). The crystals were further washed with cold ether to remove excess of Fmoc. After drying in a desiccator, N-Fmoc-DOX was recovered. This intermediate was reacted with succinic anhydride (100 ␮M) in 3 ml of anhydrous DMF in the presence of triethylamine (150 ␮M). The mixture was stirred at room temperature. After 12 h, the reaction was stopped, and the solvent was evaporated under vacuum. The residue was dissolved in ethyl acetate (15 ml) and washed with water (25 ml). After drying over anhydrous sodium sulfate, the solvent was removed under vacuum. The red colored crude product was purified by a silica gel chromatographic column using ethyl acetate (95):hexane (5) as an eluent. The both reactions were carried out in dark.

S.K. Pawar et al. / International Journal of Pharmaceutics 436 (2012) 183–193

185

Scheme 1. Synthetic scheme of anticancer polymer drug conjugate. The amine group of DOX (1) was protected with Fmoc (2) and further reacted with succinic anhydride (4) to obtain DOX-succinate (5). PEG (6) polymer was conjugated with compound GTS (7) to obtain PEG–GTS conjugate (8). PEG–GTS (8) was converted into its acid chloride form (9) by reaction with thionyl chloride and conjugated with NAG (10) to obtain PEG–GTS–NAG (11). Further, PEG–GTS–NAG conjugate (11) was coupled with DOX-succinate (5) using condensing agent EDC.HCl to get PEG–DOX–NAG (12).

186

S.K. Pawar et al. / International Journal of Pharmaceutics 436 (2012) 183–193

2.2.2. Synthesis of PEG–MAL–GTS Synthesis of PEG–MAL–GTS conjugates (compound 8) is schematically shown in the Scheme 1. Here, glutathione is used as a spacer and is chemically (2S)-2-amino-4-{[(1R)-1[(carboxymethyl)carbamoyl]-2-sulfanylethyl]carbamoyl}butanoic acid containing reactive one thiol, two carboxyl and one amine functional groups for chemical conjugation. PEG–MAL (Mw 30,000 Da) (compound 6) (5 ␮M) was dissolved in 5 ml of distilled water and stirred for 30 min. GTS (Mw 307.32 Da) (compound 7) (5 ␮M) was added to the above solution. The pH of the reaction solution was adjusted in the range of 7.0–7.5 using tertiaryamine, N,N-diisopropyethyl amine (DIEA) and stirred for 12 h. PEG MAL–glutathione conjugate was purified using dialysis membrane (Mw cut off 12,000 Da) against distilled water for 24 h. In addition, the unreacted glutathione was removed using Sephadex G10 gel filtration column. 2.2.3. Synthesis of PEG–MAL–GTS–NAG Synthesis of PEG–MAL–GTS–NAG (compound 12) conjugates is schematically represented in Scheme 1. PEG–MAL–GTS was conjugated with NAG by two step reaction. PEG–MAL–GTS (compound 8) (5 ␮M) was dissolved in chloroform (5 ml), and thionyl chloride (compound 9) (15 ␮M) was added at cold condition. Further, the reaction mixture was subjected to reflux at 50–55 ◦ C. The reaction was stopped after 5 h, and the solvent was evaporated at a reduced pressure to get yellow colored viscous liquid. NAG (compound 10) (3 mg) and sodium bicarbonate (30 mg) were dissolved in water at temperature 5–10 ◦ C. PEG–MAL–GTS chloride (compound 11) in dichloromethane was added drop wise to NAG solution with pH maintained at 7–8. Reaction was stopped after 24 h and product

was extracted in ethyl acetate. Solvent was evaporated at a reduced pressure. The synthesized conjugate was purified by dialysis (Mw cut off 10–12 kDa). The unreacted NAG was removed by Sephadex G10. The obtained product was dried under a vacuum at room temperature. 2.2.4. Synthesis of PEG–MAL–GTS–NAG–DOX Synthesis of PEG–MAL–GTS–NAG–DOX (compound 13) conjugates is schematically presented in Scheme 1. Briefly, DOXsuccinate (compound 8) (8 ␮M) was dissolved in dimethylformamide (DMF):dichloromethane (DCM) (1:3) mixture and EDC.HCl (10 ␮M) was added to the above solution as a condensing agent, and DMAP (8 ␮M) as a catalyst. PEG–MAL–GTS–NAG (compound 12) (5 ␮M) was added to above the reaction mixture. The reaction was stirred continuously for 24 h at room temperature. The carbodiimide urea formed during the reaction was removed by filtration. The amine deprotection of PEG–MAL–GTS–NAG–DOXsuccinate was carried out by the reported method (Yoo et al., 2000) as treatment with 10% piperidine in DMF for 10 min followed by acidification with mixture of 0.3% (v/v) TFA, 0.7% (v/v) pyridine and DMF and further precipitated and washed with diethyl ether. On the other hand, unreacted DOX-succinate, piperidine and EDC.HCl was removed by dialysis using (Mw cutoff 10–12 kDa) in DMF as a solvent and further purified by Sephadex G-10. The conjugate was vacuum dried at room temperature. 2.3. Synthesis of PEG–DOX Synthesis of PEG–DOX conjugate was achieved by two steps (Scheme 2). First, synthesis of succinate derivative of DOX and

Scheme 2. Synthesis scheme of anticancer polymer drug conjugate without targeting ligand. The DOX-succinate (5) was conjugated with amine group of PEG–GTS (8) by EDC.HCl resulted in synthesis of PEG–GTS–DOX (13).

S.K. Pawar et al. / International Journal of Pharmaceutics 436 (2012) 183–193

further conjugation of DOX-succinate with PEG–GTS. Synthesis of succinate derivative of DOX and PEG–GTS is described earlier. The amino group of PEG–GTS was reacted with carboxyl group of DOX succinate using EDC.HCl as condensing agent. Briefly, DOX-succinate (compound 8) (8 ␮M) was dissolved in dimethylformamide (DMF):dichloromethane (DCM) (1:3) mixture and EDC.HCl (10 ␮M) was added to the above solution as a condensing agent, and DMAP (8 ␮M) as a catalyst. PEG–MAL–GTS (compound 8) (5 ␮M) was added to above the reaction mixture. The reaction was stirred continuously for 24 h at room temperature. The carbodiimide urea formed during the reaction was removed by filtration. The amine deprotection and purification of PEG–MAL–GTS–DOXsuccinate was carried out same as described above. 2.4. Characterization of prodrugs 1H

NMR spectra of synthesized conjugates were recorded on Varian 500-MHz spectrophotometer using DMSO-d6 and CDCl3 as a solvent. The synthesized conjugate was also characterized by FTIR. In addition, the conjugates were analyzed using HPLC, fluorescence spectroscopy, and UV spectrophotometry. The analytical HPLC system consisted of Waters HPLC device equipped with a UV detector. The analytical data was evaluated using Empower-2® software (Waters, USA). The conjugate and free DOX were monitored at 230 nm using HPLC column C18 4.6 mm × 250 mm (5 ␮m) at flow rate of 1 ml/min. Isocratic elution was employed using mobile phase of 50 mM potassium dihydrogen phosphate:acetonitrile (60:40) with injection volumes of 50 ␮l for free and DOX conjugate. 2.5. UV analysis of DOX and DOX conjugates The UV analysis was performed using Jasco V-530 UV/vis spectrophotometer. One milligram of standard DOX and PEG–DOX–NAG conjugates were dissolved in 1 ml of deionized water, and UV spectra were recorded from 200 to 600 nm. Further, the data was processed by Spectra Manager® software (Jasco, Japan).

187

or with 2.0 mg of esterase in separate vials. Both vials were stirred at 100 rpm continuously and 100 ␮l of the sample were withdrawn and replaced by an equivalent amount of PBS saline buffer at 0, 1, 3, 6, 48 h. The standard curve of DOX was obtained by a HPLC experiments and used for calculating the release of DOX from the PEG conjugate. The data was presented as a percentage of total DOX released from the conjugates over a 48 h period. 2.9. Cellular internalization Spatial distribution of DOX in breast cancer cell line, MCF-7, was visualized using fluorescence microscopy. MCF-7 cells were seeded on coverslips in six-well plates, and they were allowed to grow overnight. On the following day, cells were treated with DOX and prodrug conjugates and intracellular distribution, accumulation, and their modulation was evaluated in a time-dependent manner (2, 4, 18 and 24 h). After respective time-points, coverslips were removed and processed for confocal microscopy. Cells were washed with PBS and fixed with 3.7% paraformaldehyde for 15 min at room temperature. Cells were counterstained with DAPI for 5 min before being mounted in PBS/glycerol (1:1) containing antifading agent. Samples were then analyzed with a confocal laser microscope (LSN 510, version 2.01; Zeiss, Thornwood, NY). 2.10. In vitro cytotoxicity The cytotoxicity of DOX, PEG–GTS–DOX and PEG–DOX–NAG was determined in MCF-7 and MDA-MB-231 cell line using modified MTT assay. MCF-7 and MDA-MB-231 cells were seeded at a density of 3000 cells per well in a 96-well plate. Post 24 h, cells were treated with the respective compounds at varying concentrations (0.3–10 ␮M). After 48 h of compound treatment, cells were washed with PBS and propidium iodide (7 ␮g/ml) was added to each well. Plates were then frozen at −70 ◦ C for 24 h, and post freeze–thaw cycle, the fluorescence was measured at excitation 536 nm and emission 590 nm using a POLARstar optima plate reader. Background readings (blank) were obtained from cell-free wells containing media and propidium iodide.

2.6. Fluorescence measurements The concentration of DOX was estimated using fluorescence spectrophotometer with excitation at 478 nm and emission at 585 nm wavelength at the number of flashes 3. Varying concentrations of DOX were prepared with methanol as a solvent. The fluorescence intensity was measured, and the calibration curve was prepared. Amount of DOX in PEG–DOX–NAG conjugates was estimated using calibration curve. 2.7. Molecular modeling

3. Results 3.1. Characterization of synthesized prodrug conjugates DOX prodrug conjugates formed were characterized by FTIR, NMR. 1 H NMR spectra of PEG–DOX (data not shown) and PEG–DOX–NAG were recorded on 500 MHz spectrophotometer using DMSO-d6 as a solvent. A typical 1 H NMR spectrum of PEG–NAG–DOX was shown in Fig. 2. The peaks at ı1.23 (3H, d, CH3 C-5), 3.0–3.8 broad peaks consequences from PEG backbone, 3.25

1H

Chemical structures for free PEG, and its conjugates with NAG and DOX to represent ethylene repeat ( CH2 O CH2 )n units were drawn using Chemdraw (Cambridge, UK). The structure was stabilized for its minimum energy and terminated after 2000 optimization step using geometric force field. Distance between the first and last carbon atom was measured for the modeled structure of PEG–MAL and its conjugation with DOX and NAG. Molecular Modeling was explored using Material Studio (MS) (ver 4.1 Acclerys) Ms-1 Software to obtain the length of free drug, PEG and its conjugates. 2.8. In vitro release of DOX using HPLC The in vitro release of DOX from prodrug conjugate was assessed by HPLC using esterase at pH 7.4. 2.2 mg of PEG–DOX–NAG conjugate was dissolved in 2.0 ml of PBS saline buffer (pH 7.4) without

Fig. 2.

1

H NMR of PEG–DOX–NAG conjugate.

188

S.K. Pawar et al. / International Journal of Pharmaceutics 436 (2012) 183–193

Fig. 5. The UV spectra of free DOX (—) and PEG–DOX–NAG (—-) conjugates.

Fig. 3. FTIR spectra of PEG–DOX–NAG conjugate.

3.4. Release of DOX from PEG prodrug conjugate

(3H, s, O CH3 -H PEG), 3.92 (3H, s, CH3 -O-C1), 4.0–5.0 (2H, m, OH-C8 and OHC4 ), 7.21 (5H, m, aromatic), 7.95 (2H, d, CH-2 and CH-4), 8.30 (1H, s, NHCO) indicating formation of the PEG–DOX–NAG conjugate. A typical FTIR spectrum of PEG–DOX–NAG is represented in Fig. 3. The characteristic carbonyl C O stretch of DOX and PEG C H stretch were observed at 1720 and 2800–2900 cm−1 , respectively supporting the synthesis of polymer drug conjugate. DOX and their PEG-based prodrugs conjugates were analyzed by HPLC to verify the identity of compounds. Typical HPLC chromatograms were shown in Fig. 4. DOX.HCl had showed the retention at 9.4 min (Fig. 4A) and PEG–DOX–NAG had showed retention at about 7.8 min with major single peak (Fig. 4B) at detection wavelength of 230 nm.

The stability of conjugates in PBS saline buffer and rate of release of DOX from the PEG conjugates containing DOX and NAG after hydrolysis by esterase at pH 7.4 was assessed using HPLC (Fig. 7). The standard curve of DOX was obtained by using HPLC experiments and used for calculating drug release from the conjugates. Only 5–7% of DOX was released from the conjugate after incubation in buffer (7.4 pH) without using hydrolyzing enzyme. In contrast, approximately 40% of DOX was released from PEG–DOX–NAG conjugate in the presence of hydrolyzing enzymes at the end of 48 h (7.4 pH). The typical time-profile of DOX release from PEG–DOX–NAG conjugate in presence and absence of esterase is demonstrated in Fig. 7.

3.2. UV analysis of DOX and DOX prodrug conjugate

3.5. Cellular internalization of DOX conjugates by confocal microscopy

The UV analysis of DOX and DOX polymer conjugate were recorded, and it was observed that there was slight shift in the absorption maxima (Fig. 5). 3.3. Molecular modeling studies The structural stability of the linear PEG polymer along with DOX and NAG was analyzed by molecular modeling studies. The molecular dynamics conformational distance for free DOX (C1–C27) was observed to be 9.2 A˚ (data not shown). On the other hand, the molecular dynamics conformational distances for PEG–GTS–NAG (A), PEG–GTS–DOX (B) and PEG–DOX–NAG (C) con˚ 24.850 and 25.147 A, ˚ respectively jugates observed to be 19.822 A, (Fig. 6).

Intracellular drug accumulation is a complex process including drug uptake into the cell, retention and its distribution inside the cell, and efflux from the cell. At any given time, the net uptake (accumulation) of a drug in cells is a measure of difference between the amount of drug uptake and the efflux. Also, a limited number of techniques allow for direct visualization and assessment of these processes. Of the techniques, confocal microscopy is a widely used and valuable tool for such studies using varied cancer cells. Thus, a time kinetics study was performed to evaluate cellular internalization, intracellular distribution as well as accumulation of various conjugates of DOX on human breast cancer cell line, MCF-7. Cells were treated with the conjugates (DOX, PEG–GTS–DOX and PEG–DOX–NAG) and incubated over a period

Fig. 4. HPLC chromatograms of DOX (A) inset and PEG–DOX-NAG (B). Retention times were about 9.4 and 7.8 min for DOX. HCl and PEG–GTS–DOX conjugate, respectively.

S.K. Pawar et al. / International Journal of Pharmaceutics 436 (2012) 183–193

189

Fig. 6. Molecular modeling studies of PEG–DOX–NAG conjugates by Material studio (MS-1) software.

of 2, 4, 18 and 24 h. A significant DOX cellular internalization was seen at 4 h. Interestingly, free DOX is observed to be inside the cells, in cytoplasm as well in nucleus within short period of time, i.e. 2–4 h (Fig. 8A). While, at 18 h kinetic point the DOX intensity is lowered indicative of drugs efflux from the cell. Interestingly, DOX being a small molecule is entirely effluxed by cells within 24 h. However, it is to be noted that the free DOX is predominantly internalized and distributed inside cytoplasm as well as in nucleus. While, PEG–GTS–DOX conjugate was observed to internalize in cells by receptor mediated endocytosis (Chun and Sidney, 2008) and is primarily localized at the cell surface at 4 h. The intensity of PEG–GTS–DOX increases at later time points (18 h and 24 h), demonstrating EPR effect (Matsumura and Maeda, 1986) (Fig. 8B). However, conjugate was observed only in cytoplasm and around perinuclear regions. This observation remains consistent with past

reports of DOX prodrug conjugates. While, PEG–DOX–NAG conjugate showed presence of conjugate predominantly at cell surface and inside cytoplasm with lesser intensity at 2 and 4 h. However, NAG conjugate seems to have localized at cell surface with higher intensity at 18 and 24 h (Fig. 8C). 3.6. In vitro cytotoxicity The average cytotoxicity data of non-targeted and targeted PEG–GTS–DOX prodrug in MCF-7 and MDA-MB-231 is represented in Fig. 9. The results showed that after conjugation of DOX with PEG polymer showed significant decrease in cytotoxicity, resulting in increased IC50 dose (Table 1). IC50 value was not achieved in prodrug conjugates, so therefore the values are represented as a maximum toxicity indication. For example, PEG–DOX–NAG achieved maximum toxicity of 47% using MDA-MB-231 cell line. On incorporation of a targeting moiety NAG, led to a significant increase in the cytotoxicity of the whole polymer conjugate (Fig. 9A). Thus, the conjugate with NAG (IC50 = 9 ␮M) was observed to be more cytotoxic over a non-targeted PEG–DOX conjugate (IC50 > 10 ␮M) (p < 0.001). The study demonstrated that high cytotoxicity of NAG containing drug delivery system was associated

Table 1 Cytotoxicity of free DOX drug, PEG–DOX conjugates, and PEG–DOX–NAG conjugates against human cancer cells line (MCF-7 and MDA-MB-231) in vitro. IC50 (␮M) of DOX equivalent

Fig. 7. In vitro release of DOX from PEG–DOX–NAG by HPLC using esterase () and in absence of esterase () (pH 7.4).

DOX.HCl PEG–DOX PEG–DOX–NAG

MCF-7

MDA-MB-231

1.0 42% at 10 9.0

1.3 29% at 10 47% at 10

190

S.K. Pawar et al. / International Journal of Pharmaceutics 436 (2012) 183–193

Fig. 8. Kinetics of cellular uptake and internalization of free DOX (A), PEG–GTS–DOX (B), and PEG–DOX–NAG (C) at 2, 4, 18 and 24 h using MCF 7 human cancer cells, respectively. The intensity of the representative conjugate inside the cells is only qualitative, estimated by varied amount of drug DOX and NAG in each conjugates. The arrow indicates retention of DOX into the perinuclear region.

S.K. Pawar et al. / International Journal of Pharmaceutics 436 (2012) 183–193

Fig. 9. In vitro cytotoxicity of free DOX drug, PEG–GTS–DOX and PEG–DOX–NAG conjugates using MCF-7 (A) and MDA MB-231 (B) cancer cell lines (*p < 0.001 at 10 ␮M for PEG–DOX–NAG and PEG–GTS–DOX against MCF-7 cell line) (**p < 0.001 at 10 ␮M for PEG–DOX–NAG and PEG–GTS–DOX against MDA MB cell line).

with enhanced intracellular entry of NAG conjugate in cells compared to non-targeted. Higher toxicity of NAG containing polymer drug conjugate may be associated with binding of NAG to NAGspecific receptors expressed in the plasma membrane of the tumor cells (Fig. 9A). The free DOX, targeted and non-targeted polymer conjugate of DOX were also evaluated against human MDA-MB-231 resistant breast carcinoma cell line. The results are shown in Fig. 9B. It was observed that free DOX showed higher cytotoxicity (IC50 1.3 ␮M) compared to polymer conjugate and is in good agreement with the past reports on prodrug conjugation efficacies. In particular, targeted polymer drug conjugate of NAG ligand was observed to be significantly higher cytotoxic (47% at 10 ␮M) compared to nontargeted polymer conjugate (29% at 10 ␮M) (p < 0.001). However, it should be noted that the lowered % cell toxicity for these conjugates may be due to delayed release of drug DOX from its polymeric conjugate. The presence of enzymes or pH gradient would enhance the cell toxicity, i.e. in vivo milieu. More studies in this direction are being carried out and will be reported separately. However, it is noted that NAG shows EPR effect and greater residential time inside the cells compared to free form of drug. In addition, the targeting efficiency of NAG in resistant carcinoma cell line is observed. These results demonstrate carbohydrate–lectin interactions or transport of NAG through glucose transport channel at cellular level which resulted in increased endocytosis/permeability and higher cytotoxicity (Haberkorn et al., 1994; Duncan et al., 2001). 4. Discussion Here, we report design, synthesis and evaluation of PEG–DOX–NAG prodrug conjugate for cellular targeting in cancer. The synthesized polymer drug conjugate consists of DOX, PEG–MAL, GTS and NAG. There are several strategies reported to

191

conjugate DOX with PEG (Veronese et al., 2005; Bronchud et al., 1989). However, the chemical stability of adding two components onto a PEG simultaneously necessitates additional incorporation of multi-reactive spacer. Therefore, we incorporated GTS due to availability of reactive amine, thiol, and carboxyl groups. PEG–MAL is conjugated with GTS utilizing SH group at pH 7–8 thereby leaving amine and carboxyl groups for further reactions. While, DOX is covalently conjugated by Fmoc translation reaction and NAG is conjugated by its acid chloride form (Scheme 1). In characterization by 1 H NMR, UV, HPLC demonstrated conjugation of drug on PEG. The anthracycline antibiotic, DOX is one of the most effective anticancer drug and extensively used for many neoplastic diseases. The DOX is known to generate reactive oxygen species involved in a quinone group in the anthracycline ring, to induce intracellular oxidative stress; DNA intercalation and inhibition of topoisomerase II (Pereverzeva et al., 2007). This cytotoxic effect of DOX lacks specificity to the cancer cells and hence leads to severe adverse side effects such as acute toxicity to bone marrow cells and intestinal epithelial cells and chronic toxicity affects cardiac and hepatic tissues (Davis and Robinson, 2002). Many attempts to reduce cardiotoxicity have been reported and practiced (e.g. Doxil liposomes). Other strategies limit cell specific targetability. Such non-selective cytotoxicity of DOX demands for targeted drug delivery. Carbohydrates (for e.g. sialic acid) have been used as a signaling molecule/targeting ligand for the cells over expressing sialic acid receptors (Jayant et al., 2007). Carbohydrates (e.g. fucose, sialic acid) have major role as signaling molecule, recognition molecule and adhesion molecule (Ghazarian et al., 2011; Couldrey and Green, 2002). Experimental studies on different cancer cell systems have revealed that malignant transformation is associated with a variety of altered cell glycosylation patterns (Hatanaka, 1974). The NAG has a high affinity toward the C-type of lectin receptors and it is reported that cancer cells are upregulating this type of receptors or there may be increase in glucose transport channel which ultimately results in increase in demand of carbohydrates (Luciani et al., 2004; Dhanikula et al., 2008). Earlier studies have revealed the possible targetability of NAG against cancer cell lines once incorporated into the niosomes and dendrimers (Luciani et al., 2004; Vannucci et al., 2003). However, NAG has not been used as targeting cell intending units with PEG. Thus, NAG was considered as targeting moiety/penetration enhancer to limit the acute and chronic toxicity of drug on a normal organ and enhances the endocytosis of polymer drug conjugate, specifically by cancerous cells. PEG–MAL with molecular weight of 30 kDa was used as synthetic polymer for the present polymer drug conjugate. High molecular weight PEG polymers are known to result in passive targeting across the cancerous cell, which signifies the enhanced EPR effect. The targeting was further expected to be improved by incorporation of tumor targeting moiety (NAG) into the polymer drug conjugate. The present synthesis approach is based on the use of GTS as a branched spacer. The advantage of GTS as a branched linker comprises chemical flexibility and versatility due to the presence of one thiol, one amine and two carboxyl functional groups for conjugation with PEG–MAL, DOX, and NAG, respectively. Earlier, many spacers have been used in polymer conjugation strategies for linking of drug or bioactives (Khandare et al., 2005). To our knowledge GTS has not been used as linker in PEG drug conjugate for conjugating DOX simultaneously. In addition, GTS as a spacer may reduce the steric hindrance between bulkier drugs and high molecular weight linear polymers in chemical conjugation. MAL group is highly reactive toward thiol group of peptide and this chemistry was used in conjugation of PEG–maleimide with thiol group of GTS (Scheme 2) (Chilkoti et al., 1994). The resulting PEG–MAL–GTS is used for conjugating DOX and two copies of NAG by varying the molar ratios of reactant and coupling agent. To carry

192

S.K. Pawar et al. / International Journal of Pharmaceutics 436 (2012) 183–193

out the coupling of PEG–GTS and DOX, it was necessary to form DOX-succinate which was reacted with amine group of PEG–GTS. The conjugates formed were characterized by 1 H NMR. In addition, DOX and their PEG conjugates were studied by HPLC, for their difference in eluting time points. The synthesized polymer drug conjugate has shown single major chromatographic peak. The synthesized polymer conjugate has lesser retention time over DOX indicating the more hydrophilic nature of PEG–DOX–NAG. Evaluation of the synthesized polymer conjugate confirms the formation of prodrug with NAG, DOX and PEG–MAL. NAG is a 8-carbon monosaccharide. The primary hydroxyl group at C-6 position or hydroxyl group at C-2 position in the NAG was used for chemical conjugation with carboxyl terminal of the PEG–GTS conjugate. Such type of reactions, which form a conjugate with NAG and sialic acid have been reported earlier (Jayant et al., 2007; Kulkarni and Khandare, 2006). The two moles of NAG were conjugated with PEG-GTS by converting carboxyl group of GTS into acid chloride and then NAG was reacted with it at basic condition. DOX is chemically an anthraquinone derivatives used in various delivery forms. There were various approaches used to formulate DOX prodrug such as conjugation of HPMA–DOX–hyluaronic acid (Luo et al., 2002), HPMA–DOX–galactosamine (Pimm et al., 1993), etc. In most of the cases, the DOX was conjugated through its amino group of glycosidic ring. However, it should be noted that the amine group of DOX is essential for intercalation with DNA, thus it may result in decreased cytotoxicity toward the cancer cell and increased in adverse toxicity (Zeman et al., 1998). The hydroxyl group at C-8 position of DOX can be used for conjugation. On the other hand, the glycosidic amine group of DOX is in HCl salt form and cannot be used for coupling unless protected. The protection of amine in DOX is performed by Fmoc which is treated with succinic anhydride to form C-8 succinate DOX. Thus, synthesized DOX-succinate is coupled with amine group of GTS with equimolar concentration of condensing agent. Later, the synthesized amine protected polymer drug conjugate was deprotected with 10% piperidine in DMF. The polymer drug conjugate was purified by dialysis and Sephadex G10. A further light is brought into accounting PEG-based polymer conjugate reactions by molecular modeling. The modeling results designate the stable structural conformations for PEG–MAL, DOX and its conjugates. Conformational distances for PEG–DOX–NAG ˚ and 24.850 A˚ which provides and PEG–GTS–DOX were 25.147 A, the steric stabilization. These findings may support in designing of reactions using larger polymers to avoid steric hindrance and to increase reactivity ratios of the reactions. In vitro cytotoxicity study revealed an enhanced anticancer activity of PEG–DOX–NAG than PEG–GTS–DOX prodrug conjugates. The targeting moiety enhances internalization of the prodrugs by cancer cells and substantially increases their cytotoxicity. Such results were most likely related to the change in the mechanism of internalization of the prodrugs. The free drug are internalized by simple diffusion but high molecular weight polymer conjugate are internalized by endocytosis, which was relatively very slow process and required high concentration of drug across plasma membrane to enter into the cell as compare to diffusion process (Garnett, 2001). The internalization process could be accelerated by using targeting moiety as ligand to over expressed receptors on cancer cells. The addition of targeting moiety can change the internalization mechanism from simple endocytosis to receptor mediated endocytosis or increased into cellular permeability, results into enhancement of cytotoxicity of the whole prodrug (Minko et al., 2000). However, more studies in this direction are suggested. Overall presence of hydrophilic component in polymer prodrug (e.g. carbohydrates) may improve cellular internalization. This is of interest, in particular when the drug of choice is not aqueous soluble, thereby enhancing aqueous solubility of the final PEGylated

conjugate. The synthesized conjugate retained into cancerous cell compare to free DOX which was mainly signifies the EPR effect. Thus, the synthesized PEG–DOX–NAG has proven the hypothesis related to targeting cancerous cells with NAG. 5. Conclusion In this study, we have designed and synthesized PEG–DOX conjugate for targeting cells. In vitro cytotoxicity study shows that NAG targeted polymer conjugates is more potent than nontargeted polymer conjugate against MCF-7 cells and MDA-MB-231 resistant cancer cells. Hence, glucosylated PEG conjugate can serve as potential delivery system for the treatment of cancer. Acknowledgments The authors thank UGC for providing financial assistance during the research work. The authors thank AICTE/NAFETIC for providing the facility to conduct research. The authors thank Prof. P.M. Bhate for conducting fluoroscence spectroscopy study. The authors thank to Dr. Anil Patil for molecular modeling studies. References Bronchud, M.H., Howell, A., Crowther, D., Hopwood, P., Souza, L., Dexter, T.M., 1989. The use of granulocyte colony-stimulating factor to increase the intensity of treatment with doxorubicin in patients with advanced breast and ovarian cancer. Br. J. Cancer 60, 121–125. Chena, Q., Sowaa, D.A., Gabathuler, R., 2003. Synthesis of doxorubicin conjugates through 14-hydroxy group to melanotransferrin P97. Synth. Commun. 33, 2391–2400. Chilkoti, A., Ghen, G., Stayton, P.S., Hoffman, A.S., 1994. Site-specific conjugation of a temperature-sensitive polymer to a genetically engineered protein. Bioconjugate Chem. 5, 504–507. Chun, L., Sidney, W., 2008. Polymer–drug conjugates: recent development in clinical oncology. Adv. Drug Deliv. Rev. 60, 886–898. Couldrey, C., Green, J.E., 2002. Metastases: the glycan connection. Breast Cancer Res. 2, 321–323. Davis, B.G., Robinson, M.A., 2002. Drug delivery systems based on sugarmacromolecule conjugates. Curr. Opin. Drug Discov. Devel. 5, 279–288. Dhanikula, R.S., Argaw, A., Bouchard, J.F., Hildgen, P., 2008. Methotrexate loaded polyether-copolyester dendrimers for the treatment of gliomas: enhanced efficacy and intratumoral transport capability. Mol. Pharm. 5, 105–116. Dharap, S.S., Wang, Y., Chandna, P., Khandare, J.J., Qiu, B., Gunaseelan, S., Sinko, P.J., Stein, S., Farmanfarmaian, A., Minko, T., 2005. Tumor-specific targeting of an anticancer drug delivery system by LHRH peptide. Proc. Natl. Acad. Sci. U.S.A. 102, 12962–12967. Duncan, R., 2006. Polymer conjugates as anticancer nanomedicines. Nat. Rev. Cancer 6, 688–701. Duncan, R., 2007. Designing polymer conjugates as lysosomotropic nanomedicines. Biochem. Soc. Trans. 35, 56–60. Duncan, R., Breton, S.G., Keane, R., Musila, R., Sat, Y.N., Satchi, R., Searle, F., 2001. Polymer-drug conjugates, PDEPT and PELT: basic principles for design and transfer from the laboratory to clinic. J. Control. Release 74, 135–146. Galletti, E., Magnani, M., Renzulli, M., Botta, M., 2007. Paclitaxel and docetaxel resistance: molecular mechanisms and development of new generation taxanes. ChemMedChem 2, 920–942. Garnett, M.C., 2001. Targeted drug conjugates: principles and progress. Adv. Drug Deliv. Rev. 53, 171–216. Ghazarian, H., Idoni, B., Oppenheimer, S.B., 2011. A glycobiology review: carbohydrates, lectins and implications in cancer therapeutics. Acta Histochem. 113, 236–247. Greco, F., Vicent, M.J., 2009. Combination therapy: opportunities and challenges for polymer–drug conjugate as anticancer nanomedicines. Adv. Drug Deliv. Rev. 61, 1203–1213. Haberkorn, U., Ziegler, S., Oberdorfer, F., Trojan, H., Haag, D., Peschke, P., Berger, M., Altmann, A., Kaick, G., 1994. FDG uptake, tumor proliferation and expression of glycolysis associated genes in animal tumor models. Nucl. Med. Biol. 21, 827–834. Haizheng, Z., Lin, Y.L., 2008. Selectivity of folate conjugated polymer micelles against different tumor cells. Int. J. Pharm. 349, 256–268. Hatanaka, M., 1974. Transport of sugars in tumor cell membranes. Biochim. Biophys. Acta 355, 77–104. Jayant, S., Khandare, J.J., Wang, Y., Singh, A.P., Vorsa, N., Minko, T., 2007. Targeted sialic acid–doxorubicin prodrugs for intracellular delivery and cancer treatment. Pharm. Res. 24, 2120–2130. Khandare, J.J., Minko, T., 2006. Polymer–drug conjugates: progress in polymeric prodrugs. Prog. Polym. Sci. 31, 359–397.

S.K. Pawar et al. / International Journal of Pharmaceutics 436 (2012) 183–193 Khandare, J., Kolhe, P., Pillai, O., Kannan, S., Lieh-Lai, M., Kannan, R.M., 2005. Synthesis, cellular transport, and activity of polyamidoamine dendrimermethylprednisolone conjugates. Bioconjugate Chem. 16, 330–337. Khandare, J.J., Calderon, M., Dagia, N.M., Haag, R., 2012. Multifunctional dendritic polymers in nanomedicine: opportunities and challenges. Chem. Soc. Rev., http://dx.doi.org/10.1039/C1CS15242D. Kulkarni, M.G., Khandare, J.J., 2006. Polymerizable macromers and preparation thereof. United States Patent 6,822,064. Luciani, A., Olivier, J., Clement, O., Siauve, N., Brillet, P.Y., Bessoud, B., Gazeau, F., Uchegbu, L.F., Kahn, E., Frija, G., Cuenod, C.A., 2004. Glucose-receptor MR imaging of tumors: Study in mice with PEGylated paramagnetic niosomes. Radiology 231, 135–142. Luo, Y., Bernshaw, N.J., Lu, Z.R., Kopecek, J., Prestwich, G.D., 2002. Targeted delivery of doxorubicin by HPMA copolymer-hyaluronan bioconjugates. Pharm. Res. 19, 396–402. Maeda, H., Seymour, L.W., Miyamoto, Y., 1992. Conjugates of anticancer agents and polymers: advantages of macromolecular therapeutics in vivo. Bioconjug. Chem. 3, 351–362. Matsumura, Y., Maeda, H., 1986. A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent SMANCS. Cancer Res. 46, 6387–6392. Minko, T., Kopeckova, P., Kopecek, J., 2000. Efficacy of the chemotherapeutic action of HPMA copolymer-bound doxorubicin in a solid tumor model of ovarian carcinoma. Int. J. Cancer 86, 108–117. Minko, T., Dharap, S.S., Pakunlu, R.I., Wang, Y., 2004. Molecular targeting of drug delivery systems to cancer. Curr. Drug Targets 5, 389–406. Moriwaki, K., Miyosh, E., 2010. Fucosylation and gastrointestinal cancer. World J. Hepatol. 27, 151–161. Murray, J.C., Carmichael, J., 1995. Targeting solid tumor: challenges, disappointments and opportunities. Adv. Drug Deliv. Rev. 17, 117–127.

193

Pereverzeva, E., Treschalin, I., Bodyagin, D., Maksimenko, O., Langer, K., Dreis, S., Asmussen, B., Kreuter, J., Gelperina, S., 2007. Influence of the formulation on the tolerance profile of nanoparticle-bound doxorubicin in healthy rats: focus on cardio- and testicular toxicity. Int. J. Pharm. 337, 346–356. Pimm, M.V., Perkins, A.C., Duncan, R., Ulbrich, K., 1993. Targeting of N-(2hydroxypropyl) methacrylamide copolymer-doxorubicin conjugate to the hepatocyte galactose-receptor in mice: visualisation and quantification by gamma scintigraphy as a basis for clinical targeting studies. J. Drug Target. 1, 125–131. Saad, M., Garbuzenko, O.B., Ber, E., Chandna, P., Khandare, J.J., Pozharov, V.P., Minko, T., 2008. Receptor targeted polymers, dendrimers, liposomes: which nanocarrier is the most efficient for tumor-specific treatment and imaging? J. Control. Release 130, 107–114. Singh, M., Ghose, T., Faulkner, G., Kralovec, J., Meze, M., 1989. Targeting of methotrexate-containing liposomes with a monoclonal antibody against human renal cancer. Cancer Res. 49, 3976–3984. Vannucci, L., Fiserova, A., Sadalapure, K., Lindhorst, T., Kuldova, M., Rossmann, P., Horvath, O., Kren, V., Krist, P., Bezouska, K., Luptovcova, M., Mosca, F., Pospisil, M., 2003. Effects of N-acetyl-glucosamine-coated glycodendrimers as biological modulators in the B16F10 melanoma model in vivo. Int. J. Oncol. 23, 285– 296. Veronese, F.M., Schiavon, O., Pasut, G., Mendichi, R., Andersson, L., Tsirk, A., Ford, J., Wu, G., Kneller, S., Davies, J., Ducan, R., 2005. PEG–doxorubicin conjugates: influence of polymer structure on drug release, in vitro cytotoxicity, biodistribution, and antitumor activity. Bioconjugate Chem. 16, 775–784. Yoo, H.S., Lee, K.H., Oh, J.E., Park, T.G., 2000. In vitro and in vivo anti-tumor activities of nanoparticles based on doxorubicin–PLGA conjugates. J. Control. Release 68, 419–431. Zeman, S.M., Phillips, D.R., Crothers, D.M., 1998. Characterization of covalent adriamycin–DNA adducts. Proc. Natl. Acad. Sci. U.S.A. 95, 11561–11565.