Enhancing the Efficacy of Ara-C through Conjugation with PAMAM

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Enhancing efficacy of Ara-C by conjugation with PAMAM dendrimer and linear PEG: A Comparative Study Ugir Hossain Sk, Siva Pramodh Kambhampati, Manoj K Mishra, Wojciech G Lesniak, Fan Zhang, and Rangaramanujam M Kannan Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm3018615 • Publication Date (Web): 01 Feb 2013 Downloaded from http://pubs.acs.org on February 17, 2013

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Enhancing the efficacy of Ara-C through conjugation with PAMAM dendrimer and linear PEG: A Comparative Study

Ugir Hossain Sk,†,a Siva P. Kambhampati,†,a,b Manoj K. Mishra,†,a,b Wojciech G Lesniak,a,b, Fan Zhang,a,b and Rangaramanujam M. Kannana,b,*

Affiliations a

Department of Chemical Engineering and Materials Science, and Department of Biomedical Engineering, Wayne State University, Detroit, MI 48202 b

Center for Nanomedicine/Wilmer Eye Institute, Department of Ophthalmology, Johns Hopkins School of Medicine, Baltimore MD 21287. †

these authors contributed equally

*Corresponding author: Rangaramanujam M. Kannan (email: [email protected])

ACS Paragon Plus Environment

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ABSTRACT 1β-D-Arabinofuranosylcytosine (Cytarabine, Ara-C) is a key drug in the treatment of acute myeloid leukemia. Ara-C has a number of limitations such as a rapid deactivation by cytidine deaminase leading to the formation of a biologically inactive metabolite, Ara-U (1β-Darabinofuranosyluracil), a low lipophilicity, and fast clearance from the body. To address these problems, we developed a conjugate in which hydroxyl-terminated PAMAM dendrimer, G4-OH [“D”] and PEG were used as carriers for the drug (Ara-C). The conjugates were synthesized using an efficient multi-step protection/deprotection method resulting in the formation of a covalent bond between primary hydroxyl group of Ara-C and dendrimer/PEG. The structure, physicochemical properties, and drug release kinetics were characterized extensively. 1H NMR and MALDI-TOF mass spectrometry suggested covalent attachment of 10 Ara-C molecules to the dendrimer. The release profile of Ara-C in human plasma and in PBS buffer (pH 7.4) showed that the conjugates released the drug over 14 days in PBS, with the release speeded-up in plasma. In PBS, while most of the drug is released from PEG-Ara-C, the dendrimer continues to release the drug in a sustained fashion. The results also suggested that the formation of the inactive form of Ara-C (Ara-U) was delayed upon conjugation of Ara-C to the polymers. The inhibition of cancer growth by the dendrimer-Ara-C and PEG-Ara-C conjugates was evaluated in A549 human adenocarcinoma epithelial cells. Both dendrimer- and PEG-Ara-C conjugates were fourfold more effective in inhibition of A549 cells compared to free Ara-C after 72 h of treatment. Keywords: Cytarabine (Ara-C), PAMAM dendrimer, PEG, Drug delivery, A549 human lung epithelial cells.


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1. INTRODUCTION Cytarabine (Ara-C) is a widely used anticancer drug for the treatment of various forms of cancer such as acute myelogenous leukaemia, colon, breast and ovary carcinoma.1,2 As in other pyrimidine analogue nucleosides, the potency of Ara-C is limited by its low stability after intravenous administration and the narrow therapeutic index.3,4 Consequently, the minimum effective dose is high, and has to be increased regularly after the initial treatment. In this context, toxicity becomes the main limiting factor for treatment.4 Its rapid clearance from the body is due to the enzymatic conversion to the inactive and more soluble form of the drug (Ara-U) by cytidine deaminase, mainly in liver and kidney. Many efforts have been made to address the problem, including continuous intravenous infusion or frequent and high-dose schedules.5 To improve its in vivo stability, a combination therapy with tetrahydrouridine (a cytidine deaminase inhibitor) was used, which did not significantly improve the therapeutic efficacy of Ara-C.6 Moreover, the hydrophilic character of Ara-C strongly limits its intracellular uptake because of the low membrane permeability of the molecule.7 These issues raise the need for improved formulations, stability, and intracellular delivery, and provide new impetus for the present study. A variety of Ara-C conjugates have been synthesized to increase the stability and bioactivity. Macromolecular derivatives and prodrugs obtained by acylation at N-4 position of the nucleoside have been synthesized to prevent deamination and improve pharmacokinetics.8-11 In fact, conjugation of antitumor drugs to high molecular weight polymers, antibodies and polypeptides, are now representing a new and promising approaches in chemotherapy. These conjugates may act as classical prodrugs while taking advantage of longer circulation time and the enhanced permeability and retention (EPR) effect, resulting in improved accumulation in the tumor microvasculature and cellular internalization by endocytosis to reach their intracellular


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targets.12-14 It was also reported that Ara-C conjugated with polyethylene glycol (PEG) through a norleucine linker is less cytotoxic in human HeLa cells.15 This work builds on these efforts by using PAMAM dendrimer-based delivery of Ara-C. Poly(amidoamine) dendrimers (PAMAM) are promising drug delivery vehicles because of their multivalency, well-defined and globular structure, low polydispersity, and amenability to post-synthetic manipulation that are extensively investigated for various biomedical applications.16-19 PAMAM dendrimers are the most widely studied polymers used as vehicles for drug delivery,20-22 antiviral agents,23,24 MRI contrast agents,25 selective tumor targeting.26-30 Conjugation to a PAMAM dendrimer significantly improved the efficacy of methotrexate20 and paclitaxel.31 Khandare et al., have shown that PAMAM dendrimer-Paclitaxel conjugate was more efficacious than a PEG-Paclitaxel conjugate in A2780 human ovarian carcinoma cells.31 Folic acid-conjugated multifunctional PAMAM dendrimers were encapsulated into PEG-PLA nanoparticles to increase circulation time and decrease off-target or non-specific targeting.32 This formulation exhibited sustained release of dendrimers, which subsequently targeted overexpressed folate receptors on mouth epidermal carcinoma (KB) cells.32 In this study, we used the hydroxyl-functionalized generation-4 PAMAM dendrimer because of its improved cytotoxicity profile compared to other end functionalities, and its kidney-based clearance.18 We have previously reported extensively on the improved efficacies, based on this delivery platform. Dendrimer-antibiotic conjugates were prepared for the treatment of Staphylococcus aureus bacteria in periprosthetic inflammation and Chlamydia trachomatis infection in reactive arthritis.33,34 Conjugation of poorly soluble drugs to dendrimer can increase the bioavailability, and decrease the dose frequency.35-37 The most common covalent linkage of the drugs to the surface functional groups of dendrimers can be achieved through the ester or


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amide bond, which can be cleaved hydrolytically or enzymatically.38 When a conjugate, appropriately designed to improve cell uptake and drug release profile is further combined with a targeting ligand, significant improvements in efficacy can be obtained.39 Interestingly, neutral PAMAM dendrimers with no targeting ligands have been shown to have an intrinsic ability to localize in cells associated with neuroinflammation in a rabbit model of cerebral palsy and a rat model of retinal degeneration.40,41 When an anti-inflammatory/anti-oxidant agent N-acetyl cysteine conjugated to the PAMAM dendrimer and administered intravenously to newborn rabbit kits with cerebral palsy (after birth), the dendrimer delivered the drug to activated microglia and astrocytes, resulting in significant improvements in motor function, myelination and neuronal injury by Day 5 of life.40,42 The aim of this study is to design and synthesize PAMAM dendrimer-Ara-C and polyethylene glycol-Ara-C (PEG-Ara-C) conjugates using appropriate protection/deprotection chemistry, to improve the drug stability, release profile and intracellular delivery. We report the synthesis, structural characterization, drug release, and the efficacy of the conjugates in an A549 human epithelial carcinoma cell line.

2. MATERIALS AND METHODS 2.1. Materials. Generation-four hydroxyl-terminated PAMAM dendrimer (G4-OH, “D”) in methanol was purchased from Dendritech, Inc. (Midland, Michigan). mPEG5000 (PEG) was purchased

from

Fluka

Chemicals.

Benzotriazol-1-yl-oxytripyrrolidinophosphonium

hexafluorophosphate (PyBOP), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 4(dimethylamino)pyridine

(DMAP),

1β-D-arabinofuranosylcytosine

(Ara-C),

tert-

butyldimethylsilyl chloride (TBDMS-Cl), di-tert-butyl dicarbonate, succinic anhydride,


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triethylamine (TEA), diisopropylethylamine (DIEA), acetonitrile (ACN), trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (St. Louis, Missouri). The anhydrous dimethylsulfoxide (DMSO), dimethylformamide (DMF), 1,4-dioxane, tetrahydrofuran (THF) and dichloromethane (DCM) solvents were purchased from Acros Organics (Morris Plains, New Jersey). Deuterated dimethylsulfoxide (DMSO-d6) and deuterated chloroform (CDCl3) were purchased from Cambridge Isotope Laboratories, Inc. (Andover, Massachusetts). All other solvents and chemicals were purchased from Fisher Scientific and were used without further purification. Regenerated cellulose (RC) dialysis membrane with molecular weight cut-off of 2000 Da was obtained from Spectrum Laboratories, Inc. (Rancho Dominguez, California). The reactions were carried out under nitrogen. Thin-layer chromatography (TLC) was performed on silica gel GF254 plates (Whatman, Piscataway, New Jersey), and the spots were visualized with UV light. NMR spectra were recorded on a Varian INOVA 400 spectrometer, using commercially available deuterated solvents. Proton chemical shifts are reported in ppm (δ) and tetramethylsilane (TMS) used for internal standard. Coupling constants (J) are reported in hertz (Hz). 2.2. Electrospray Ionization (ESI) and Matrix-Assisted Laser Desorption Ionizationtime-of-light (MALDI-TOF) Mass Spectrometry. Electrospray ionization (ESI) mass spectra were recorded on a Waters Micromass ZQ spectrometer (Waters Corporation, Milford, Massachusetts). Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectra were recorded on a Bruker Ultraflex system equipped with a pulsed nitrogen laser (337 nm) (Bruker Daltonics Inc., Billerica, Massachusetts), operating in positive ion reflector mode, using 19 kV acceleration voltage. 2,5-Dihydroxybenzoic acid was used as matrix and Cytochrome c (MW 12,361 g/mol) was used as external standard. The matrix solution was


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prepared by dissolving 20 mg of matrix in 1 mL of deionized water/ACN (0.1% TFA) (1:1). Analytical samples were prepared by mixing 10 µL of dendrimer solution (2 mg/mL in methanol) with 100 µL of matrix solution, followed by deposition of 1 µL of sample mixture onto the MALDI plate. This mixture was allowed to air dry at room temperature (22-25°C), and used for analysis. 2.3. High Performance Liquid Chromatography (HPLC) analysis. The conjugates were analyzed by Waters HPLC instrument (Waters Corporation, Milford, Massachusetts) equipped with binary pump, dual UV detector, and autosampler interfaced with breeze software. The HPLC chromatogram was monitored at 210 and 272 nm simultaneously using dual UV absorbance detector. The water/acetonitrile (0.14% w/w TFA) was freshly prepared, filtered, degassed and used as a mobile phase. Symmetry C-18 reverse phase column with 5µm particle size, 25cm length, 4.6 mm internal diameter was used. A gradient flow was used with initial condition 95:5 to 50:50 (H2O/ACN) in 30 minutes and returning to 95:5 (H2O/ACN) in 50 minutes with flow rate of 1 mL/min. 2.4. Size Exclusion Chromatography (SEC). SEC analysis was performed in a Waters GPC system using an isocratic pump (Waters 1515), a refractive index detector (Water 2414), a dual λ absorbance detector, an in-line degasser AF, and a column heater. Three Ultrahydrogel Waters columns (7.8 x 300 mm, 500, 250 and 150) along with ultrahydrogel guard column (6 x 40 mm) were used for analysis. Phosphate buffer (0.1 M, pH 7.4) with 0.025% sodium azide was used as mobile phase. Separations were run at 30oC and the flow rate was 0.8 mL/min. Elution was monitored at 210 nm (for dendrimer detection), 272 nm (for Ara-C detection) and refractive index (for the detection of all constituents of the conjugate).


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2.5. Potentiometry. Dendrimers were dissolved in NaCl (0.1 M) solution at a concentration of 0.6 mg/mL, the pH was adjusted to 3.0 with a small amount of 1M HCl. The titrations were performed at room temperature (3 mL volume), using a Mettler Toledo G20 automated titration system equipped with an InLab electrode, and NaOH (0.1 M) used as a titrant. Mettler Toledo LabX software was used to analyze the data. 2.6. Dynamic light scattering and Zeta potential. The particle size and ζ-potential of G4OH, PEG and their respective conjugates were determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instrument Ltd. Worchester, U.K) equipped with a 50 mW He-Ne laser (633 nm). The conjugates (D-Ara-C, PEG-Ara-C) and starting materials were dissolved in deionized water (18.2 Ω) to make the solution with the final concentration of 0.1 mg/mL. The solution was filtered through a cellulose acetate membrane (0.45 micron, PALL Life Science) and DLS measurements were performed at 25°C with a scattering detector angle of 173°. Zeta potentials were calculated using the Smolokowsky model and measurements were performed in triplicate. 2.7. Synthesis of compounds 1-3. The intermediates 1, 2a, 2b, 3a and 3b were synthesized using previously published procedures. 43 The detailed experimental procedure and 1H NMR and 13

C NMR spectra are described in the supplemental information section. 2.8. Synthesis of Boc-protected Ara-C succinic acid linker (4a/4b). Synthesis of mono

Boc-protected Ara-C succinic acid linker (4a). To a stirred solution of 3a (170 mg, 0.264 mmol) in DCM (10 mL), succinic anhydride (43 mg, 0.428 mmol) and DMAP (32 mg, 0.264 mmol) were added and the resulting mixture was stirred at room temperature for 6 h. The reaction mixture was concentrated on a rotary evaporator and the crude product was purified on a column packed with silica gel using chloroform:MeOH (10:1) as eluent to yield 4a as a white powder


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(150 mg, 71.4%). 1H-NMR (CDCl3) δ 7.85 (d, 1H, H-6, J = 6.4 Hz), 7.21 (d, 1H, H-5, J = 6.8 Hz), 6.28 (d, 1H, H-1′, J = 4.0 Hz ), 5.26 (m, 1H, H-2′), 4.87 (m, 1H, H-3′), 4.27-4.59 (m, 3H, H4′, H-5′ & H-5′′), 2.64-2.71 (m, 4H), 1.34-1.51 (m, 27H, -C(CH3)3). ESI-MS: calculated for C28H40N3O14, 642.2510; measured, 642.2509. Synthesis of (Boc)2-protected Ara-C succinic acid linker (4b). The compound 4b (80%) was synthesized by the same procedure as above for compound 4a. 1H-NMR (DMSO-d6) δ 7.99 (d, 1H, H-6, J = 7.6 Hz), 6.92 (d, 1H, H-5, J = 7.6 Hz), 6.21 (d, 1H, H-1′, J = 3.6 Hz), 5.22 (m, 1H, H-2′), 5.10 (m, 1H, H-3′), 4.43-4.28 (m, 3H, H-4′, H-5′ & H-5′′), 2.57-2.49 (m, 4H), 1.47 (s, 18H, -C(CH3)3), 1.44 (s, 9H, -C(CH3)3) and 1.28 (s, 9H, -C(CH3)3). ESI-MS: calculated for C33H50N3O16, 744.3191; measured, 744.3180. 2.9. Synthesis of Dendrimer-Ara-C Conjugate (6). To a stirred solution of PAMAM G4OH (50 mg, 0.0035 mmol) in anhydrous DMF (5 mL), PyBOP (72.8 mg, 0.14 mmol) was added under nitrogen. The mixture was stirred for 1 h at room temperature and 4a (45 mg, 0.07 mmol) dissolved in DMF was added and the reaction mixture was stirred for 48 h. The resulting solution was dialyzed with DMF (membrane MW cut-off 2000 Da) for 12 h. The dialyzed solution was concentrated on a rotary evaporator and dried under high-vacuum overnight. An oily product, 5a (51 mg, 68%) was isolated. 1H-NMR (DMSO-d6) δ 10.43 (s, -NH), 7.79-9.93 (m, 124H & 1H Ar-H-5), 7.02 (d, 1H, H-6, J = 8.0 Hz), 6.21 (m, 1H, H-1′), 5.19 (m, 1H, H-2′), 5.08 (m, 1H, H3′), 4.26-4.40 (m, 3H, H-4′, H-5′ & H-5′′), 4.07-4.11(m, 2H), 2.18-3.37 (dendrimer aliphatic proton), 1.42 (s, 18H, -C(CH3)3) and 1.26 (s, 9H, -C(CH3)3). The Boc-protected intermediate, 5a (50 mg, 0.0023mmol) was suspended in 10 mL of DCM and trifluoroacetic acid (5 mL) was added and stirred for 3 h at room temperature. The solvent of the reaction mixture was evaporated completely and dissolved in DMF (7 mL) and dialyzed against DMF for 12 h. On


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evaporation of solvent oily product was isolated. The crude product was further precipitated by anhydrous diethyl ether (50 mL) to afford a white solid dendrimer-Ara-C conjugate, (D-Ara-C, 6) (41 mg). 1H-NMR (DMSO-d6) δ 8.61-8.33 (m, 124H, amide protons of dendrimer), 7.99 (d, 1H, H-6, J = 7.2 Hz), 6.12 (d, 1H, H-5, J = 7.2 Hz), 5.97 (d, 1H, H-1′, J = 4.0 Hz), 4.36 (m, 1H, H-2′), 4.04 (m, 1H, H-3′), 3.9 (m, 1H, H-4′), 3.81(m, 1H, H-5′), 3.60-2.49 (m, dendrimer aliphatic proton). The dendrimer-Ara-C conjugate (D-Ara-C, 6) was prepared by reacting a bis-Boc protected Ara-C linker intermediate (4b) with G4-OH following by deprotection with TFA using similar procedure as described above. 2.10. Synthesis of PEG-Ara-C Conjugate (9). PEG-succinic acid linker. The mixture of mPEG5000 (0.85 g, 0.17 mmol), succinic anhydride (0.10 g, 1.02 mmol) and catalytic amount of DMAP in DCM (10 mL) were stirred at room temperature for 24 h. The solvent was evaporated and the residue was dissolved in DMF (10 mL) and dialyzed in DMF (membrane MW cut-off = 1000 Da) for 12 h. The resulting solution was evaporated to get an oily mass, which was then precipitated in anhydrous diethyl ether. The solid was collected by filtration and dried over high vacuum to get PEG-linker, 7 (0.75 g, 86.5%). 1H-NMR (CDCl3) δ 4.22 (t, 2H, -O-CH2-CH2-O-, J = 4.4 Hz), 3.42-3.80 (m, backbone CH2), 3.35 (s, 3H, -OCH3), 2.63-2.53 (m, 4H, linker CH2). Synthesis of Boc-protected Ara-C-PEG intermediate (8). To a solution of 7 (100 mg, 0.02 mmol) in DCM (10 mL), EDC (7.5 mg, 0.039 mmol) and DMAP (4.8 mg, 0.039 mmol) were added and stirred for 30 min. under nitrogen. The Boc-protected Ara-C (3b, 25.24 mg, 0.039 mmol) was added to the reaction mixture and stirred for 16 h at room temperature. The solvent was evaporated to get the oily crude product. The crude product was dissolved in DMF (7 mL) and purified by dialysis with DMF (membrane MW cut-off 1000 Da). The resulting dialyzed


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solution was evaporated and dissolved in 2 mL of DCM and added drop-wise to anhydrous diethyl ether to get white solid (90 mg, 80.3%) and dried over vacuum. 1H-NMR (CDCl3) δ 7.81 (d, 1H, Ar, J = 7.6 Hz), 7.07 (d, 1H, Ar, J = 7.6 Hz), 6.29 (d, 1H, H-1′, J = 3.2 Hz), 5.30 (d, 1H, H-2′, J = 3.2 Hz), 4.97 (d, 1H, H-3′, J = 2.4 Hz), 4.53-4.39 (m, 3H, H-4′ & H-5′), 4.26-4.22 (m, 2H,-O-CH2-CH2-O-), 3.80-3.43 (m, mPEG-backbone H), 3.35 (s, 3H, -OCH3), 2.69-2.63 (m, 4H), 1.53 (s, 18H, N-(Boc)2), 1.47 (s, 9H, O-Boc), 1.34 (s, 9H, O-Boc). PEG-Ara-C Conjugate (9). The Boc-protected PEG-Ara-C conjugate 8 (70 mg, 0.012 mmol) was dissolved in anhydrous DCM (3 mL), and trifluoroacetic acid (1 mL) was added drop-wise to it at 0°C and the solution was stirred for 3 h. The solvent was evaporated in vacuo and the residue was dissolved in DMF (10 mL) and purified through dialysis with DMF (membrane MW cut off 1000 Da). The resulting dialyzed solution was evaporated in vacuum and the obtained oily mass was precipitated in anhydrous diethyl ether to produce a white solid powder (55 mg, 84%). 1H-NMR (CDCl3). δ 7.79 (d, 1H, Ar, J = 7.6 Hz), 6.19 (d, 1H, H-1′, J = 3.2 Hz), 6.07 (d, 1H, Ar, J = 7.6 Hz), 4.55 (m, 1H, H-2′), 4.34 (s, 1H, H-3′), 4.24-4.14 (m, 4H, H-5′ & -OCH2CH2O-), 4.08 (s, 1H, H-4′) 3.80-3.42 (m, mPEG-backbone H), 3.35 (s, 3H, -OCH3), 2.70-2.60 (m, 4H). 2.11. Drug Release Study in PBS. Release of Ara-C from D-Ara-C and PEG-Ara-C conjugates was characterized in 0.1 M phosphate buffer (pH 7.4) at 37oC. A concentration of 1 mg/mL of the conjugate was maintained in a water bath with constant mixing, and at appropriate time points 300µL of the sample was withdrawn from the incubation mixture and directly analyzed by GPC, without further treatment. PBS solution (0.1M, pH 7.4) with 0.025% sodium azide was used as mobile phase and the release of Ara-C was monitored at 210 and 272 nm for


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both dendrimer and Ara-C respectively. Percentage of Ara-C and SA-Ara-C (Ara-C linker) released from the conjugates were quantified by respective calibration curves. 2.12. Drug Release Study in Plasma. D-Ara-C (2 mg/mL) and PEG-Ara-C (3 mg/mL) were incubated in human pooled plasma diluted to 80% with 0.1M PBS with constant mixing in water bath (Dual-action shaker; Polyscience, Niles, Illinois) at 37°C. At periodic intervals, plasma samples (200 µL) were withdrawn. The precipitation of protein was done by addition of 2M trichloroacetic acid, followed by addition of ice cold acetonitrile (200 µL) and centrifuged at 10000 rpm for 5 min at 4°C. The clear supernatant was then filtered through 0.2 µm filtered and stored at -80°C until GPC analysis. The samples were analyzed by GPC to evaluate the stability and release of Ara-C from the conjugates. 2.13. Cell Culture. Human lung adenocarcinoma epithelial cell line A549 was obtained from ATCC (Manassas, VA, USA) and was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% Fetal bovine serum (FBS) and 5% antibiotics (penicillin/streptomycin) in a humidified incubator at 37 °C with 5% CO2. 2.14. MTT Assay of Cell Inhibition. Human lung adenocarcinoma A549 epithelial cells (5 × 103) were plated and grown for 24 hrs in 200 µL of growth medium in 96-well microtiter plates (Costar, Cambridge, MA) and then the culture medium was replaced by fresh phenol red free DMEM medium containing different concentrations (0.005-2.0 µM) of Ara-C, D-Ara-C and PEG-Ara-C at increasing concentrations. The control cells were treated with medium containing fresh phenol-red free DMEM. The cells are incubated in standard conditions (explained in cell culture section) for 72 h. After a 72 hrs incubation period, the number of viable cells was determined by using MTT cell proliferation assay kit (Molecular probes, Invitrogen, Oregon, USA). Briefly, the medium was removed and replaced it with 100 µL of fresh culture medium


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and the cells were labeled with 10 µL of 12 mM MTT stock solution and incubated for 4 hrs at 37 °C in a humidified 5% CO2 atmosphere, the formazan crystals formed were dissolved using DMSO following the kit protocol. Absorbance was read at 540 nm using fluorescence microplate reader (BioTek Instruments, Winooski, Vermont, USA) and cell viability was determined as percent absorbance relative to untreated control cells. Background absorbance of the medium was measured in a triplicate set of control wells that contained medium and the MTT agent solution without added cells and was subtracted from the absorbance measured in each of the sample wells to provide a corrected absorbance for each of the wells. Triplicate wells with predetermined cell numbers were subjected to the above assay in parallel with the test samples; this also provided internal confirmation that the assay was linear over the range of absorbance and hence, cell numbers measured. IC50 values were calculated by GraphPad Prism version 5 for Windows (GraphPad Software, San Diego, CA; www.graphpad.com) using data from three independent experiments.

3. RESULTS AND DISCUSSIONS 3.1. Preparation of the Dendrimer-Ara-C Conjugate (D-Ara-C, 6). The synthetic strategies were aimed at synthesizing a Boc-protected Ara-C linker that would possess reactive functional groups suitable for efficient conjugation to hydroxy-terminated PAMAM dendrimer and PEG. The Ara-C contains heterofunctional reactive hydroxyl and amine groups. Our objective was to introduce a linker at C-5 position of Ara-C that could finally be used for subsequent reaction with dendrimer or PEG. Our previous studies suggested that linker plays a major role in releasing the drug from the dendrimer conjugates.38,44 Since the functional groups on the dendrimer used for conjugation on the surface are 'sterically crowded', enzymatic release


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can be challenging compared to linear PEG. Here, we have used succinic acid as a spacer to conjugate Ara-C to the dendrimer surface to provide steric relief. The synthesis of Boc-protected Ara-C with succinic acid linker intermediates (4a, 4b) are depicted in Scheme 1. The intermediates 3a and 3b were synthesized by modification of the previously published procedure.43 In brief, Ara-C was reacted with TBDMS-Cl in pyridine in the presence of catalytic amount of imidazole to get a white solid product 1 in good yield. The intermediate 1 was then converted to Boc-protected Ara-C (2a) by using excess Boc anhydride in dioxane, and when we changed the solvent to dioxane-dichloromethane (DCM) mixture, it resulted in completely Bocprotected hydroxyl groups intermediate (2b). A free hydroxyl group was obtained by deprotecting the TBDMS group at C-5 position of 2a and 2b with triethylamine trihydrofluoride in THF to get 3a and 3b respectively.43 Finally, the carboxylic acid derivatives, 4a and 4b was obtained by reacting succinic anhydride with 3a and 3b in presence of catalytic amount of DMAP in DCM. The above intermediates were purified on silica gel by gradient elution and the structures of 1-4 were confirmed by 1H and 13C NMR, and high-resolution mass spectroscopy. The synthesis of the desired dendrimer-drug conjugate, D-Ara-C (6) is shown in Scheme 1. The Boc-protected dendrimer-drug conjugates (5a and 5b) were afforded by a coupling reaction between G4-OH and 4a and 4b in presence of PyBOP in DMF and the crude products were purified by dialysis (membrane MW cut off. 2000 Da). The intermediate 5a was characterized by 1

H-NMR, which suggested that 10 drug molecules were attached to the dendrimer based on

proton integration method.33,34, The final dendrimer-drug conjugate, D-Ara-C (6) was obtained by elimination of the Boc protecting groups of 5a and 5b with trifluoroacetic acid (TFA) in DCM, and subsequent purification by dialysis. The structure of D-Ara-C conjugate was established by 1H NMR; loading of the drug molecules was calculated by MALDI-TOF mass


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spectroscopy; purity was determined by reverse phase HPLC and size exclusion chromatography (SEC); and the number of amine groups originating from the Ara-C moieties and internal tertiary amines of the dendrimer were calculated by potentiometric analysis. In 1H NMR, the presence of two doublets correspond to aromatic protons of Ara-C at 7.76 and 6.04 ppm along with dendrimer peaks confirms the formation of the conjugate (Figure 1A). The loading of the drug molecules was calculated by comparing amidic protons peaks of the dendrimer (7.95-8.20 ppm) to the aromatic protons of the Ara-C, which indicated that an average of 10 molecules of Ara-C were conjugated to the dendrimer. A peak at 16,971 m/z in the MALDI-TOF mass spectrum indicated the presence of 10 drug molecules on the dendrimer, based on the molecular weight of the dendrimer (13,672 Da) and linker (118 Da) which is in agreement with the 1HNMR results (Figure 2A. The HPLC and SEC chromatograms of Ara-C, Ara-C linker (SA-AraC) and D-Ara-C indicate that there were no low molecular weights impurities present in the conjugate (Figure 3). Appearance of new peak of D-Ara-C at 18.78 min in HPLC chromatogram at 272 nm which is different from Ara-C (3.2 min) and SA-Ara-C (10.0 min) confirms the formation of the conjugate (Figure 3A). Additionally, in SEC chromatogram a peak at 29.4 min for D-Ara-C indicates the formation of the product, whereas Ara-C and SA-Ara-C elutes at 44.4 and 38.6 min respectively (Figure 3B). The difference in retention time of Boc-protected Ara-Cdendrimer and dendrimer-Ara-C conjugate in HPLC and SEC also confirm the formation of the product (data not shown). Finally, potentiometric titration measurements suggested that 10±2 Ara-C molecules (corresponding to 17% of the end OH groups being consumed in conjugation) were attached to the dendrimer, based on the titration curves of G4-OH dendrimer as the reference. This is in good agreement with 1H NMR and MALDI-TOF mass data (Figure 4). The number of tertiary amines present in the dendrimer does not change upon conjugation of Ara-C


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to the dendrimer, but at the same time there will be additional primary amine groups which are present in Ara-C to the conjugates. Using theoretical molecular weight of G4-OH (MW = 14277), the number of tertiary amines in the dendrimer was calculated to be 62 (Figure 4A), which is same in case of the D-Ara-C conjugate (Figure 4B). In addition, appearance of shoulder peak in the titration curve of the D-Ara-C conjugate indicates the presence primary amines, originating from Ara-C molecules and the number was calculated to be 10±2. 3.2. Preparation of the PEG-Ara-C Conjugate (PEG-Ara-C, 9). The synthesis of PEGAra-C conjugate is summarized in scheme 2. In this case, we attached a linker to the PEG molecules (PEG-linker, 7) instead of the Ara-C (as before for synthesis of D-Ara-Cconjugate). The desired PEG-Ara-C (9) was obtained by using EDC.HCl instead of PyBOP as the coupling agent followed by Boc deprotection. The purity of the final conjugate was confirmed by the chromatogram in HPLC and SEC (Figure 3), where no small amounts of starting material and other components were seen as impurities. The characterization of all intermediates and final compound were done by 1H-NMR (Figure 1B and supplement), and finally confirmed by the evident changes of molecular weight in MALDI-TOF MS spectroscopy (Figure 2B). 3.3. Particle size and Zeta potential. The surface properties and size of the dendrimer conjugates/nanoparticles play a crucial role in determining the potential of the drug carrier and its interaction with biological systems. We measured the particle size and zeta potential of G4OH (D), D-Ara-C, PEG and PEG-Ara-C (Table-1). There is no significant difference in diameter between G4-OH (4.3 nm) and D-Ara-C conjugate (3.4 nm). This indicates that the drug (with ketone and amine groups) may have folded back into the 'hydrophobic' interior of the dendrimer (amide and amine groups). It may also be due to the small size of the dendrimer and the conjugates, which are at the low end of the instrument sensitivity. It appears that Ara-C


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conjugation to the dendrimer did not change the size appreciably. G4-OH has small positive zeta potential (+4.5 mV) which is due to the presence of tertiary amines in its core. The zeta potential of D-Ara-C increased to +15.0 mV due to the presence of amine functionality in the Ara-C molecule. There are 10 Ara-C molecules attached to the dendrimer. Since PEG is a linear molecule, the hydrodynamic diameter of PEG and its conjugates are in between 4.5 to 7.0 nm. Linear PEG and PEG-SA have negative zeta potential (Table-1). After conjugation with Ara-C, the zeta potential of PEG-Ara-C increases to +6.9 mV due to the presence of amine group in the Ara-C. 3.4. Release Study. In vitro release characteristics of the prepared conjugates were studied to investigate their stability in physiologically relevant solutions such as PBS (pH 7.4) and 80% human plasma. Human plasma is used to simulate the biological condition of intravenous injection where degradation may occur by hydroxyl ions and enzymes.15 Free Ara-C released from the conjugates binds to plasma proteins and gets deaminated converting to Ara-U, an inactive form of Ara-C. Hence the peak corresponding to Ara-U was also taken into account for total drug release. We found that both the conjugates release the drug in two forms (i) free AraC, and (ii) SA-Ara-C both in plasma and PBS, but not Ara-U. Release in PBS: In PBS, both the conjugates released the drug 'relatively slowly', and had different release kinetics. The released Ara-C (elution time of 44.4 min SEC in Figure 5C) and conjugated Ara-C (elution time of ~30 min for both PEG-Ara-C and D-Ara-C in SEC Figure 5C) could be identified, based on their different elution times. For PEG-Ara-C in PBS, the release is nearly zero order (20% in 100 hours), with no initial burst (Figure 5A). The release from D-AraC shows an initial burst (~10% in 4 hours), followed by a sustained release (nearly zero order20% over the next 350 hours). This estimation based on free Ara-C is consistent with the


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estimation based on the conjugated Ara-C (Figure 5B). Figure 5C illustrates this through the increase in the SEC peak associated with free Ara-C and the decrease of signal for conjugated Ara-C. Faster release was observed from PEG conjugate. Over 14 days approximately 79% (56% as free Ara-C and 23% as SA-Ara-C); whereas D-Ara-C released only 42% (35% and 6.6% of free Ara-C and SA-Ara-C) respectively, suggesting that ester bonds in linear PEG conjugate were more susceptible to hydrolysis due to their increased size in water, and the fact that the drug is attached to one end of the linear PEG. In the dendrimer conjugate, there are 10 Ara-C molecules attached to the dendrimer, and the steric crowding on the dendrimer surface may hinder enzymatic cleavage of the ester bond (Figure 5A and 5B). In the dendrimer conjugate, the ester linkage is surrounded by electron rich hydroxyl groups, which makes the ester bond less electron deficient and less accessible to nucleophile like hydroxyl ions or water molecules. So the rate of hydrolysis slows down in case of the dendrimer conjugate whereas PEG being a linear molecule has no such effect. Since the formation of the inactive form of AraU requires the deaminase, there is no Ara-U formation in PBS. Release in Plasma: In human plasma, the extent of release was relatively higher for both PEG and dendrimer conjugates, compared to PBS at the same time point. This is expected because of the presence of enzymes (e.g. esterases), which can specifically cleave the ester bond between the linker and the drug. One of the limitations of Ara-C as a drug is its low stability in plasma and rapid conversion into a more inactive and soluble form, Ara-U. Such conversion is mediated by deaminase enzyme in plasma. PEG conjugate showed higher release rates (approximately 73% of the drug in 5 hrs in the form of Ara-U, Ara-C and SA-Ara-C), and almost all the drug molecules were released within 4 days (Figure 5D). The Ara-C and SA-Ara-C released from the PEG conjugate was converted to Ara-U in plasma, which was confirmed by the decrease of Ara-


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C peak and appearance of Ara-U peak in the GPC chromatogram. From 5 hours to 5 days, there appears to be a conversation of ~35% of the released Ara-C and SA-Ara-C into the inactive AraU form, suggestive of instability of Ara-C in plasma. This suggests that the expected increased circulation time of PEG-Ara-C may not be able to directly address the conversion of Ara-C to Ara-U. The release from D-Ara-C conjugate was comparatively slower, releasing approximately 70% of its payload in 5 days in the form of free Ara-C (30%), Ara-U (35%) and SA-Ara-C (5%) (Figure 5E). Interestingly, at the end of 5-days, 30% of the released Ara-C from the dendrimer is still in the active form, whereas only 10% of that released from the PEG conjugate was in the active form. This could be attributed to the initial rapid release of Ara-C from PEG compared to the dendrimer. From 5 days to the end of 10 days, there is a decrease in the free Ara-C associated with an increase in Ara-U. At the end of 10 days, ~ 20% of the Ara-C is still conjugated to the dendrimer (Figure 5F). In both plasma and PBS, the release of Ara-C from dendrimer conjugate was comparatively lower than that of PEG conjugate suggesting a steric hindrance at the dendrimer surface, affecting the enzymatic action on the linker between the dendrimer and the drug. On the other hand, PEG, being a linear polymer, is more susceptible to the enzymatic cleavage, since the drug is attached to only one end of the monofunctional-PEG. It was also noticed that, in plasma, SA-Ara-C was released at a much slower rate from dendrimer conjugates compared to that of PEG conjugates. The half-life (t1/2) of Ara-C in plasma is reported to be 2 mins and hence the conversion of Ara-C to Ara-U is unavoidable.15 We have also seen that the free Ara-C was completely converted to Ara-U after an incubation of 15 min in human plasma. In the release study of Ara-C from dendrimer conjugate, no Ara-U was detected for the first 2 hrs and negligible amount was observed after 5 hrs of incubation. In case of the


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PEG conjugate, a considerable amount of Ara-U was detected within first 2 hrs. These studies suggest that the advantage of using dendrimers as drug carriers since resulting conjugates are stable for a longer period of time and can reach the target site before considerable amount of drug is released from them. 3.5. In vitro Drug efficacy. The efficacy of prepared conjugates in inhibiting the growth of cancer cells was evaluated in A549 human lung adenocarcinoma epithelial cells after 72 hours of incubation. The cells were treated with dendrimer/PEG conjugates based on equivalent Ara-C concentrations. Our results suggested that both D-Ara-C and PEG-Ara-C conjugates were four fold more efficacious in inhibiting cell growth, based on IC50 values (Table 2), suggesting superior intracellular transport and release from the conjugates, compared to cell uptake of free drug. The free dendrimer and PEG at concentrations equivalent to those used in the conjugates did not have any effect on the A549 cells. After 24h and 48h incubation, the dendrimer and PEG conjugates showed lower inhibition activity compared to that of free Ara-C. This lower efficacy is probably due to the low amount of free Ara-C hydrolytically released from the conjugates inside cells at short times (up to 48h), rather than due to slower uptake of the dendrimer conjugates.15 We have previously shown that neutral PAMAM dendrimers are taken up by A549 cells by non-specific interactions, relatively rapidly.45 Moreover these dendrimers are mostly localized in the lysosomes of the cytosol21 where the ester bonds are susceptible to cleavage by the action of lysosomal enzymes in acidic pH. The delayed efficacy of the conjugates can be correlated with the stability of dendrimer conjugates in plasma where ~50% of the incubated conjugates (Figure 5F) were stable up to 50h, releasing ~60% of its payload in the form of Ara-C and SA-Ara-C, whereas PEG conjugates were comparatively less stable in plasma, releasing more that 70% of the drug within 5h.


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4. CONCLUSIONS We have developed conjugates of Ara-C with a hydroxyl-terminated PAMAM dendrimer, and a linear polymer (PEG), using an ester linkage, and evaluated them in human adenocarcinoma epithelial cells. Our results suggest a significant increase in the inhibition of growth of A549 cells in both the conjugates compared to free drug. Release studies of Ara-C from the conjugates in PBS and in human plasma indicated their stability against hydrolytic cleavage and susceptibility in plasma. Release of the drug from PEG-Ara-C conjugate in plasma is much faster compared to D-Ara-C conjugate. Results also indicate the formation of the inactive form of Ara-C (Ara-U) is delayed upon polymeric conjugation compared to its free form. In MTT assay, both D-Ara-C and PEG-Ara-C conjugates were found to be four-fold more efficacious than Ara-C in inhibiting the growth of A549 cells (based on IC50), suggesting a superior intracellular transport and release compared to free drug. Hence, it could be expected that both of the Ara-C conjugates would have much better stability and longer circulation time than free Ara-C in vivo. It is evident from the study that dendrimer plays a crucial role in increasing the stability as well as the efficacy of the drug, addressing a key need for treatment with Ara-C related to poor blood stability. Conjugation with dendrimer, in addition to further ligand targeting to the target site, may improve the effectiveness of Ara-C in vivo, an important drug in the fight against cancer.

ACKNOWLEDGMENTS


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The authors wish to thank the Ralph Wilson Foundation for Biomedical Engineering, and the Wayne State University Nanotechnology Initiative (Nano Incubator) for financial support; Admira Bosnjakovic for MALDI-TOF mass spectroscopy; and Fan Zhang for Potentiometric analysis and Size Exclusion Chromatography.

SUPPORTING INFORMATION Preparation of compounds 3a and 3b are described in method section of supplement materials. The 1H and 13C-NMR spectra of the intermediates were presented in Figures S1-S13. This material is available free of charge via the Internet at http://pubs.acs.org.


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(17) Lee, C. C.; Mackay, J. A.; Frechet, J. M.; Szoka, F. C. Nat Biotechnol. 2005, 23, 1517– 1526. (18) Menjoge, A. R.; Kannan, R. M.; Tomalia, D. A. Drug Discov Today. 2010, 15, 171–185. (19) Patri, A. K.; Kukowska-Latallo, J. F.; Baker, Jr. J. R. Adv Drug Deliv Rev. 2005, 57, 2203–2214. (20) Gurdag, S.; Khandare, J.; Stapels, S.; Matherly, L. H.; Kannan, R. M. Bioconjug Chem. 2006, 17, 275–283. (21) Khandare, J.; Kolhe, P.; Pillai, O.; Kannan, S.; Lieh-Lai, M.; Kannan, R. M. Bioconjug Chem. 2005, 16, 330–337. (22) Kolhe, P.; Khandare, J.; Pillai, O.; Kannan, S.; Lieh-Lai, M.; Kannan, R. M. Biomaterials. 2006, 27, 660–669. (23) McCarthy, T. D.; Karellas, P.; Henderson, S. A.; Giannis, M.; O'Keefe, D. F.; Heery, G.; Paull, J. R.; Matthews, B. R.; Holan, G. Mol Pharm. 2005, 2, 312–318. (24) Clayton, R.; Hardman, J.; LaBranche, C. C.; McReynolds, K. D. Bioconjug Chem. 2011, 22, 2186–97. (25) Talanov, V. S.; Regino, C. A. S.; Kobayashi, H.; Bernardo, M.; Choyke, P. L.; Brechbiel, M. W. Nano Lett. 2006, 6, 1459–1463. (26) Kirkpatrick, G. J.; Plumb, J. A.; Sutcliffe, O. B.; Flint, D. J.; Wheate, N. J. J Inorg Biochem. 2011, 105, 1115–1122. (27) Zhang, Y.; Thomas, T. P.; Lee, K. H.; Li, M.; Zong, H.; Desai, A. M.; Kotlyar , A.; Huang, B.; Holl, M. M.; Baker, J. R. Jr. Bioorg Med Chem. 2011, 19, 2557–64. (28) Vijayalakshmi, N.; Ray, A.; Malugin, A.; Ghandehari, H. Bioconjug Chem. 2010, 21, 1804–1810. (29) Lesniak, W. G.; Kariapper, M. S. T.; Nair, B. M.; Balogh, L. P.; Khan, M. K. Bioconjug Chem. 2007, 18, 1148–1154. (30) Lesniak, W.; Shi, X.; Bielinska, A. U.; Janczak, K. W.; Sun, K.; Baker J. R. Jr.; Balogh, L. P. Nano Lett. 2005, 5, 2123–2130.


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(31) Khandare, J. J.; Jayant, S.; Singh, A.; Chandna, P.; Wang, Y.; Vorsa, N.; Minko T. Bioconjug Chem. 2006, 17, 1464–72. (32) Sunoqrot, S.; Bae, J. W.; Pearson, R. M.; Shyu, K.; Liu, Y.; Kim, D. H.; Hong, S. Biomacromolecules. 2012, 13, 1223-1230. (33) Bosnjakovic, A.; Mishra, M. K.; Ren, W.; Kurtoglu, Y. E.; Shi, T.; Fan, D.; Kannan, R. M. Nanomedicine. 2011, 7, 284–94. (34) Mishra, M. K.; Kotta, K.; Hali, M.; Wykes, S.; Gerard, H. C.; Hudson, A. P.; WhittumHudson, J. A.; Kannan, R. M. Nanomedicine. 2011, 7, 935–44. (35) Gillies, E. R.; Frechet, J. M. J. Drug Discov Today. 2005, 10, 35–43. (36) Chauhan, A. S.; Sridevi, S.; Chalasani, K. B.; Jain, A. K.; Jain, S. K.; Jain, N. K.; Diwan, P. V. J Control Release. 2003, 90, 335–343. (37) Jain, N. K.; Gupta, U. Expert Opin Drug Metab Toxicol. 2008, 4, 1035–1052. (38) Kurtoglu, Y. E.; Mishra, M. K.; Kannan, S.; Kannan, R. M. Int J Pharm. 2010, 384,189– 194. (39) Lee, C. C.; Gillies, E. R.; Fox, M. E.; Guillaudeu, S. J.; Fréchet, J. M.; Dy, E. E.; Szoka, F. C. Proc Natl Acad Sci U S A. 2006, 103, 16649–54. (40) Kannan, S.; Dai, H.; Navath, R. S.; Balakrishnan, B.; Jyoti, A.; Janisse, J.; Romero, R.; Kannan, R. M. Sci Transl Med. 2012, 4(130), 130ra46. (41) Iezzi, R.; Guru, B. R.; Glybina, I. V.; Mishra, M. K.; Kennedy, A.; Kannan, R. M. Biomaterials. 2012, 33, 979–88. (42) Dai, H.; Navath, R. S.; Balakrishnan, B.; Guru, B. R.; Mishra, M. K.; Romero, R.; Kannan, R. M.; Kannan, S. Nanomedicine. 2010, 5, 1317–1329. (43) Bazzanini, R.; Gouy, M.; Peyrottes, S.; Gosselin, G.; Périgaud, C.; Manfredini, S. Nucleosides Nucleotides Nucleic Acids. 2005, 24, 1635–1649. (44) Navath, R. S.; Kurtoglu, Y. E.; Wang, B.; Kannan, S.; Romero, R.; Kannan, R. M. Bioconjug Chem. 2008, 19, 2446–55.


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(45) Perumal, O. P.; Inapagolla, R.; Kannan, S.; Kannan, R. M. Biomaterials. 2008, 29, 3469– 3476.


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SCHEMES, FIGURES AND TABLE

Scheme 1. Schematic representation of the synthesis of the Boc-protected Ara-C with succinic acid linker (4a and 4b); Boc-protected Ara-C-dendrimer conjugate (5a); and dendrimer-Ara-C conjugate (D-Ara-C, 6). “D” stands for generation four, hydroxyl terminated PAMAM dendrimer.

Scheme 2. Synthesis of polyethylene glycol-succinic acid linker (PEG-linker, 7); Boc-protected Ara-C-PEG conjugate (8); and (PEG-Ara-C, 9) conjugate using Boc-protection/deprotection chemistry.


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Figure 1. Proton NMR spectra of (A) dendrimer-Ara-C conjugate (D-Ara-C, 6) in DMSO-d6 and (B) polyethylene glycol-Ara-C conjugate (PEG-Ara-C, 9) in CDCl3.


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Figure 2. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectra of (A) D-Ara-C (6) shows the relative increase of molecular weight upon Ara-C conjugation. The difference in the molecular weights of main fraction of G4-OH (13672 Da) and D-Ara-C (16971 Da) indicates 10 Ara-C molecules are attached. (B) MALDI-TOF mass spectra of PEGlinker (PEG-SA; MW 5117 Da) and PEG-Ara-C (MW 5338 Da) conjugates shows one molecule is attached to the PEG.


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Figure 3. (A) Elution profile in HPLC chromatograms of Ara-C (3.29 min), SA-Ara-C (10.03 min), D-Ara-C conjugate (18.78 min) and PEG-Ara-C conjugate (35.81 min) are different from each other monitored at 272 nm. Unmodified G4-OH (13.8 min) and PEG (30.49 min) monitored at 210 nm are different from their respective conjugates (monitored at 272 nm). There is no measureable amount of free Ara-C, dendrimer or PEG was observed indicating the purity of the desired products; (B) SEC chromatograms of conjugates with different retention time; D-Ara-C (29.49 min), PEG-Ara-C conjugate (29.55 min), unmodified Ara-C (44.44 min) and SA-Ara-


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C(38.68 min). Both G4-OH and PEG exhibited very low intensity at 28.76 min and 29.89 min respectively monitored at 272 nm (for clarity data not shown).

Figure 4. Potentiometric titration curves of dendrimer (G4-OH) and dendrimer-Ara-C (D-AraC) conjugate. D-Ara-C conjugate shows increase of the molecular weight upon conjugation with Ara-C. Number of tertiary amine (3′-N) in the dendrimer was found to be 62, based on the theoretical molecular weight (MW=14277). The number of 3′-N and primary amine (1′-N) in the D-Ara-C conjugate was calculated considering the relative increase of the molecular weight (MW=17527) and number of moles of titrant used for titration. The number of primary amine is found out to be 10 and the number of tertiary amines did not change upon Ara-C conjugation with dendrimer.


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Figure 5. Controlled release profile of the Ara-C from D-Ara-C and PEG-Ara-C conjugates, monitored by size exclusion chromatography (SEC) at 272 nm. (A) Release profiles of Ara-C and SA-Ara-C from D-Ara-C and PEG-Ara-C conjugates in PBS buffer (pH 7.4), and (B) showing the decrease of the concentration of both conjugates with time; (C) The representative chromatograms of the D-Ara-C conjugate incubated in phosphate buffer (pH 7.4) at concentration of 1 mg/mL for 0h, 1h, 4h and 48h, showing increase of the signals related to AraC and SA-Ara-C, and decrease of the peak associated with D-Ara-C conjugate; (D) Graph showing the relative percentage of Ara-C, SA-Ara-C and inactive form of Ara-C (Ara-U) from PEG conjugate in 80% human plasma; (E) Graph showing the relative percentage of Ara-C, SAAra-C and inactive form of Ara-C (Ara-U) release from D-Ara-C in 80% human plasma; (F) Decrease of D-Ara-C concentration in percentage with time in 80% human plasma.


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Figure 6. Evaluation of cell-cytotoxicity of the conjugates against human lung adenocarcinoma epithelial (A549) cell line using MTT assay at 72 h; (A) D-Ara-C conjugates, physical mixture of dendrimer (D) and Ara-C (D + Ara-C); and Ara-C alone; and (B) PEG-Ara-C conjugates, physical mixture of PEG and Ara-C (PEG + Ara-C), and Ara-C alone.


Biomacromolecules

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Table 1. Particle Size and Zeta Potential of Dendrimer and PEG Conjugates Compound

Diameter (nm)

Zeta potential (mV)

G4-OH (D)

4.28±0.24

+4.54±0.10

D-Ara-C (6)

3.39±0.57

+15.03±1.62

PEG

4.68±0.69

-6.97±0.76

PEG-SA

6.89±1.01

-13.50±2.14

PEG-Ara-C (9)

5.89±1.78

+6.89±1.10

Table 2. IC50 Values of Ara-C and Ara-C Conjugates Compounds

IC50 (µM)

Ara-C

0.66 ± 0.11

D-Ara-C (6)

0.17 ± 0.05*

Dendrimer + Ara-C

0.46 ± 0.13

PEG-Ara-C (9)

0.14 ± 0.04*

PEG + Ara-C

0.42 ± 0.08

*IC50 values of Ara-C conjugates in human lung adenocarcinoma epithelial cells after 72 hrs treatment (MTT assay). Values represent the average from three independent experiments; Values are mean ± S.E.


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TABLE OF CONTENT


Enhancing the Efficacy of Ara-C through Conjugation with PAMAM

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