Synthesis and Characterization of Clickable Cytocompatible Poly

Dec 18, 2012 - Water-soluble cytocompatible P(EAMO)-g-PEG brush polymers with controlled degrees of PEGylation were ... Yun Yu , Haotian Sun , Chong C...
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Synthesis and Characterization of Clickable Cytocompatible Poly(ethylene glycol)-Grafted Polyoxetane Brush Polymers Olga Yu. Zolotarskaya,† Quan Yuan,† Kenneth J. Wynne,‡ and Hu Yang*,†,§ †

Department of Biomedical Engineering, School of Engineering, Virginia Commonwealth University, Richmond, Virginia 23284, United States ‡ Department of Chemical and Life Science Engineering, School of Engineering, Virginia Commonwealth University, Richmond, Virginia 23284, United States § Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia 23298, United States S Supporting Information *

ABSTRACT: We report a new family of clickable poly(ethylene glycol) (PEG)grafted polyoxetane brush polymers as a potential modular platform for delivery of drugs and imaging agents. 3-Ethyl-3-hydroxymethyloxetane (EHMO) monomer reacted with propargyl benzenesulfonate in the presence of sodium hydride to yield alkyne-substituted monomer (EAMO). Subsequently, cationic ring-opening polymerization using boron trifluoride diethyl etherate catalyst and 1,4-butanediol initiator produced P(EAMO) homopolymer with a DP of ∼30 (30 alkynes per chain). Methoxypoly(ethylene glycol) azide (mPEG750-azide) prepared from mPEG750 (750 g mol−1) was grafted to P(EAMO) via copper(I)-catalyzed alkyne−azide cycloaddition (CuAAC) click chemistry. Water-soluble cytocompatible P(EAMO)-g-PEG brush polymers with controlled degrees of PEGylation were synthesized by varying the feed molar ratio of mPEG750-azide to alkyne (25:100, 50:100, 75:100, and 100:100). 1H NMR, GPC, end-group analysis, FTIR, and DSC were applied for polymer characterization. The utility of P(EAMO)-g-PEG for carrying imaging agents was demonstrated by preparing fluorescently labeled P(EAMO)-g-PEG. 5-(Aminoacetamido)fluorescein (AAF) was used as a model compound. Fluoresceincarrying P(EAMO)-g-PEG was synthesized by click coupling bifunctional spacer 6-azidohexanoic acid (AHA) to P(EAMO)-gPEG and subsequently coupling of AAF to AHA with EDC/NHS chemistry.

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INTRODUCTION Polymeric carriers have been adopted widely as an important class of vehicles for delivery of drugs and imaging agents. An ideal carrier is expected to possess not only biologically favorable properties such as nontoxicity and nonimmunogenicity but a high cargo capacity. In the covalent approach, efficient and controlled coupling reactions are crucial for success of drug coupling and functionalization of the delivery system. Click chemistry has had a profound impact on synthetic chemistry owing to modularity, orthogonality, and other appealing characteristics.1−3 In general, a click reaction is highly selective, gives high yields with few byproducts, and can be done with the use of organic solvents or aqueous solutions. The most well-known click reaction is Cu(I)-catalyzed azide−alkyne cycloaddition (CuAAC). Given that the reaction uses alkyne and azide functional groups that do not occur naturally, the application of CuAAC click chemistry in the rational design of drug delivery systems is well suited for coupling biomolecules such as peptides,4,5 proteins,6 and nucleic acids7 to the synthetic carrier. Linear,8 hyperbranched,9,10 and dendritic11 clickable polymers have been synthesized and used in drug delivery. Herein, we report a new family of clickable polyoxetanes as a modular delivery platform capable of carrying various functional entities such as drugs, © 2012 American Chemical Society

imaging agents, and poly(ethylene glycol) (PEG) as a solubility and biocompatibility enhancer. Oxetanes have been recognized as an essential element in many drugs and play a pivotal role in facilitating desirable pharmacokinetic properties.12 Oxetanes can also be utilized as monomers to form linear13,14 or branched polymers15−18 via ring-opening polymerization (ROP). For the first time, we explored 3-ethyl-3-hydroxymethyloxetane (EHMO) (Scheme 1) as a monomer to make clickable polymeric drug carriers based on bifunctionality. EHMO hydroxyl side group is readily available for alkylation, while the oxetane ring readily undergoes Lewis acid-catalyzed ROP. Therefore, EHMO bifunctionality was used to synthesize alkyne-containing polyoxetane P(EAMO) homopolymer as a platform, which allows drug coupling and biofunctionalization via click chemistry. Previously, Emrick and co-workers reported azide modification of the anticancer drug camptothecin (CPT) and its coupling to alkyne-containing polymers via click chemistry.19 Herein, the focus is on the synthesis and characterization of a new alkynecontaining polyoxetane P(EAMO) platform. Because PEG is Received: October 10, 2012 Revised: November 25, 2012 Published: December 18, 2012 63

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Scheme 1. Synthesis of Clickable PEG-Grafted Polyoxetane Brush Polymers and Fluorescein-Carrying Brush Polymers

μm, 230−400 mesh) was purchased from Sigma-Aldrich (St. Louis, MO). Synthesis of EAMO. EAMO was synthesized following a method described in a patent24 with modifications. Briefly, sodium hydride NaH (17 g, 0.42 mol) was added portionwise to a solution of EHMO (37 g, 0.32 mol) in 800 mL of THF at 5 °C under N2. The suspension obtained was stirred for 1 h at 0 °C before a solution of propargyl benzenesulfonate (95 g, 0.48 mol) in 35 mL of THF was added dropwise. The reaction mixture was stirred at ambient temperature for 2 days followed by cooling to 5 °C, pouring into 200 mL of 5 wt % K2CO3, and stirring for 5 h. Upon removal of THF by rotary evaporation, the product was extracted with DCM, washed with brine, and dried over MgSO4. DCM was then removed under reduced pressure, and the product was distilled at 60−66 °C (1.8 mmHg) to obtain EAMO. EAMO was further purified by silica gel column chromatography using a hexane/ethyl acetate (7/1: v/v) mixture. Yield 20%. 1H NMR (CDCl3, 300 MHz): δ (ppm) 0.89 (t, J = 7.5 Hz, 3H), 1.76 (q, J = 7.5 Hz, 2H), 2.45 (t, J = 2.4 Hz, 1H), 3.67 (s, 2H), 4.20 (d, J = 2.4 Hz, 2H), 4.40 (d, J = 5.9 Hz, 2H), and 4.60 (d, 2H, J = 5.9 Hz, 2H). Synthesis of P(EAMO). BF3 (0.2 g, 1.4 mmol) was added to a solution of BD (72 mg, 0.8 mmol) in 3 mL of DCM; the mixture was stirred for 40 min at room temperature and then cooled to 0 °C. EAMO (2.5 g, 16 mmol) in 2 mL of DCM was added to the solution at a rate of 0.03 mL/min. After stirring for 12 h at 0 °C under N2, the reaction was quenched with 3 mL of water, and the DCM fraction was collected. The aqueous fraction was extracted again with DCM. The two DCM fractions were combined and dried with MgSO4. DCM was removed under reduced pressure, and the residue was precipitated from hexane to obtain P(EAMO) as a viscous liquid. 1H NMR (CDCl3, 300 MHz): δ (ppm) 0.84 (t, J = 7.4 Hz, 3H), 1.40 (q, J = 7.3 Hz, 2H), 2.40 (t, J = 2.3 Hz, 1H), 3.21 (s, 4H), 3.38 (s, 2H), and 4.10 (d, J = 2.3 Hz, 2H). Synthesis of P(EAMO)-g-PEG Brush Polymers. P(EAMO) was click grafted with mPEG750-azide (its synthesis is described in the Supporting Information) to make brush polymers. Briefly, a solution

commonly integrated into drug carriers to enhance water solubility and biocompatibility and to improve pharmacokinetics and efficacy drug delivery,4,20−22 PEG was click grafted to P(EAMO) to generate PEG-grafted brush polymers (P(EAMO)-g-PEGs) with controlled degrees of PEGylation. Characterization methods included 1H NMR, GPC, endgroup analysis, FTIR, and DSC. Cytocompatibility with human dermal fibroblasts was evaluated using the trypan blue assay. Finally, to demonstrate their ability to deliver imaging agents, 5-(aminoacetamido)fluorescein (AAF) was coupled to P(EAMO)-g-PEG via clickable bifunctional spacer 6-azidohexanoic acid. With the aid of AAF, cellular uptake of the fluorescein-labeled brush polymer by human dermal fibroblasts was examined.

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EXPERIMENTAL SECTION

Materials. 3-Ethyl 3-hydroxymethyloxetane (EHMO) was generously provided by Perstorp Polyols (Toledo, OH). NaH (60% dispersion in oil), propargyl benzenesulfonate, boron trifluoride diethyl etherate (BF3), 1,4-butanediol (BD), trifluoroacetic anhydride (TFAA), p-toluenesulfonyl chloride, methoxypoly(ethylene glycol) (750 g mol−1) (mPEG750), sodium azide (NaN3), copper(I) iodide, N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDTA), copper(II) sulfate pentahydrate, (+)-sodium L-ascorbate, methyl 6-bromohexanoate, lithium hydroxide, N-hydroxysuccinimide (NHS), N-(3dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), magnesium sulfate (MgSO4), trifluoroacetic anhydride (TFAA), deuterated solvents (CDCl3 and D2O), and other solvents were purchased from Acros (Morris Plains, NJ). 5-(Aminoacetamido)fluorescein (AAF) was purchased from Invitrogen (Grand Island, NY). SnakeSkin dialysis tubing (cellulose membrane, 3.5K MWCO) was obtained from Thermo Fisher Scientific (Pittsburgh, PA). Prior to use, tetrahydrofuran (THF) was distilled over Na in the presence of benzophenone. Dichloromethane (DCM) was washed according to the standard procedure23 and distilled over CaH2. Silica gel 60 (40−63 64

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Figure 1. 1H NMR spectra of EAMO and P(EAMO) in CDCl3. al.25 with modifications (see Supporting Information) and click coupled to the polymer to serve as a spacer for AAF. For instance, AAF-labeled brush polymer on the basis of B-72% was synthesized as follows. Briefly, CuSO4 (8.0 mg, 0.05 mmol) and sodium ascorbate (19.8 mg, 0.1 mmol) were added to a solution of B-72% (100 mg, 4.7 μmol) and excess AHA (30 mg, 0.2 mmol) in THF/H2O (2:1, v/v). The mixture was stirred for 24 h at room temperature under N2. Upon removal of THF by rotary evaporation, the remaining residue was dialyzed against water and then freeze-dried to obtain AHA-modified B-72%. 1H NMR spectrum of AHA-modified B-72% (CDCl3, 600 MHz): δ (ppm) 0.75 (s, 3H, CH3), 1.32 (br, 4H; CH2), 1.64 (s, 2H, CH2), 1.90 (s, 2H, CH2), 2.3 (s, 2H, CH2), 3.16 (s, 4H, CH2), 3.35 (m, 2H, CH2OCH2 + 3H, OCH3), 3.64 (br m, CH2CH2O of PEG), 3.86 (s, 2H, CH2), 4.33 (s, 2H, CH2), 4.51 (s, 4H, CH2), 7.58 (s, 1H), 7.66 (s, 1H). To a solution of AHA-modified B-72% (0.6 μmol) in a mixture of DMF/H2O (1 mL/0.4 mL), NHS (460 μg, 4 μmol) was added followed by addition of EDC (764 μg, 44 μmol). Following overnight reaction at room temperature, the solvents were removed by rotary evaporation. The obtained NHS ester was dissolved in 4 mL of 0.1 M NaHCO3, to which AAF (3.6 μmol) was added. The reaction mixture was stirred overnight in dark at room temperature, followed by dialysis in water and freeze-drying. To further remove unreacted AAF, the product was dissolved in chloroform and filtered through 5 μm filter.

containing P(EAMO) (0.65 mmol of alkyne equivalent) in 15 mL of THF was prepared, and mPEG750-azide addition was adjusted to achieve the following molar feed ratios of mPEG750-azide to alkyne: 25/100, 50/100, 75/100, and 100/100. CuI (one tenth of molar amount of mPEG750-azide) was added to the solution followed by addition of PMDTA (the same molar amount as mPEG750-azide). The reaction mixture was stirred under N2 overnight at room temperature. Upon removal of THF by rotary evaporation, the remaining residue was dialyzed against water and then freeze-dried to obtain brush polymers B-#, where # indicates percentage of alkynes per polymer coupled to PEG. According to 1H NMR analysis, brush polymers B-25, 49, 72 and 100% were obtained. 1H NMR spectrum of B-100% (CDCl3, 300 MHz): δ (ppm) 0.75 (s, 3H), 1.32 (br, 2H), 3.16 (br, 4H), 3.38 (s, 2H, CH2OCH2 + 3H, OCH3), 3.64 (br m, CH2CH2O of PEG), 3.86 (s, 2H), 4.51 (s, 4H), 7.65 (s, 1H). 1H NMR spectrum of B-25%, B-49%, and B-72% (CDCl3, 300 MHz): δ (ppm) 0.75 (s, 3H), 1.34 (br, 2H), 2.44 (s,1H), 3.17 (br, 4H), 3.38 (s, 2H, CH2OCH2 + 3H, OCH3), 3.64 (br. m, CH2CH2O of PEG), 3.88 (t, 2H), 4.10 (s, 2H), 4.51 (s, 4H), and 7.65 (s, 1H). Synthesis of Fluorescein-Carrying Brush Polymers. To use AAF to label the polymer, carboxylate groups were introduced to P(EAMO) using click chemistry. To this end, 6-azidohexanoic acid (AHA), a bifunctional molecule containing a terminal azide and a carboxyl group, was synthesized following a method reported by Lee et 65

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The filtrate was evaporated under reduced pressure to obtain semisolid product (i.e., AAF-labeled B-72%). The product was kept in dark prior to use. AAF density in the labeled polymer was determined by fluorometry. Instrumentation. 1H NMR spectra were recorded on either Varian Mercury-300 MHz or Bruker AVANCEIII 600 MHz Instruments. FTIR spectra were obtained on a Megna-IR 760 spectrometer using KBr pellets. Gel permeation chromatography (GPC) was performed using a Viscotek GPC system equipped with a TriSEC triple detector. THF was the mobile phase, and flow rate was 1 mL/min. Universal calibration by polystyrene standards was used for determination of molecular weight and polydispersity. Differential scanning calorimetry was performed on a TA-Q 1000 Series instrument (TA Instruments). The measurements were done at a heating rate of 10 °C/min and a cooling rate of 5 °C/min over a temperature range from −90 to 60 °C. Fluorescence spectra were recorded on a Varian Cary Eclipse fluorescence spectrophotometer. End-Group Analysis. End-group analysis was conducted as described previously14 to determine number-average molecular weight (Mn) of P(EAMO). Briefly, excess TFAA was added to a solution of P(EAMO) in CDCl3 and stirred for 30 min at 40 °C. Mn was then calculated from the ratio of proton integrals for the shifted methylene protons next to the trifluoroacetyl end groups (α for P(EAMO) and β for butanediol) against methyl proton integral (see Supporting Information). Cell Cytotoxicity Assay. Cytotoxicity of P(EAMO)-g-PEG brush polymers was examined using human dermal fibroblasts. The cells were seeded in 24-well plates (5 × 103 cells/well) in 1 mL of growth medium (DMEM medium containing 10% fetal bovine serum (FBS), 100 UI/mL penicillin−streptomycin) at 37 °C in an atmosphere of 10% CO2. After 24 h incubation, the growth medium was replaced with 1 mL of fresh culture medium containing different concentrations of polymer (i.e., 2, 1, 0.5, or 0.25 mg/mL). At 48 h, cell viability was determined using the Trypan blue dye exclusion assay (n = 3).26 Intracellular Uptake Studies. Fibroblasts were seeded on borosilicate glass coverslips in 12-well plates at a density of 5 × 103 cells/well and allowed to grow for 24 h in 1 mL of growth medium supplemented with 10% FBS. The cells were incubated for various lengths of time (3, 6, and 24 h) with 100 μg of AAF-labeled B-72% in 1 mL of fresh growth medium supplemented with 10% FBS, fixed with ice-cold methanol for 10 min, counterstained with 4′,6-diamidino-2phenylindole (DAPI), and then rinsed with PBS buffer. True-color fluorescent images of the transfected cells were taken under a Zeiss Axiovert 200 inverted fluorescence microscope (Carl Zeiss Microimaging, Inc., Thornwood, NY). Statistical Analysis. Data were analyzed by analysis of variance (ANOVA), followed by Tukey’s test for pairwise comparison of subgroups. P values