Amine-Functionalized Polylactide–PEG Copolymers - Macromolecules

Article Cite This: Macromolecules XXXX, XXX, XXX−XXX

Amine-Functionalized Polylactide−PEG Copolymers Mehmet Onur Arıcan,† Sezgi Erdoğan,† and Olcay Mert*,†,‡ †

Department of Polymer Science and Technology and ‡Department of Chemistry, Kocaeli University, 41380 Kocaeli, Turkey S Supporting Information *

ABSTRACT: The formation of halogenated carboxylic acid intermediate followed by a ring-closing reaction led to aminofunctionalized asymmetrical lactide monomer. PEG-based functional diblock and triblock polylactides were synthesized via a controlled ring-opening polymerization in a solvent-free medium with high conversions (up to 96%), low polydispersities as low as 1.06, monomodal GPC traces, and short reaction time (only 1 h). No polymerization of symmetrical monomer, synthesized via condensation of (S)-(+)-CBZ-4-amino-2hydroxybutyric acid, proved that the preferred site in the mechanism of ring-opening polymerization was found as a methyl site in asymmetrical lactide monomer. A highly efficient deprotection of copolymers was carried out in the presence of H2 gas and Pd/C catalyst without any degradation to obtain the corresponding free amine-functionalized aliphatic poly(α-hydroxy acid)s. These biodegradable thermosensitive polymers, suitable for any local therapy applications, were injectable around 42 °C (sol) and a gel just after cooling to body temperature. Faster hydrolytic degradation (up to 47% in 30 days) and more effective paclitaxel release from copolymer gels (up to 95% in 20 days) than well-known conventional PEG−PLA gels may make functional lactides a preferred candidate for developing controlled/ sustained release of drugs from delivery vehicles.

1. INTRODUCTION The technology of controlled drug delivery systems is one of the most rapidly developing fields of science in which medicinal, natural, and engineering departments make great contributions to human health care. Such delivery systems present many advantages including improved efficacy, reduced toxicity, and improved patient compliance and convenience when compared to conventional methods.1 Poly(α-hydroxy acid)s have drawn great attention in biological applications, especially in the field of drug delivery systems because of their biodegradable and biocompatible properties.2 Some formulations based on polylactide (PLA), polyglycolide (PGA), and poly(lactide-co-glycolide) (PLGA) approved by the Food and Drug Administration (FDA) are the most extensively studied class of poly(α-hydroxy acid)s (or poly(substituted glycolide)s) with the outstanding mechanical, physical, and thermal properties.2,3 But there is a significant need for the development of new biomaterials making an alternative to well-known polylactides. Contrary to the extensive literature for PLA, there were few reports on nonreactive poly(substituted glycolide)s, which are structural analogous of lactic acid.4−10 Various symmetric and/or asymmetric substituted glycolides (i.e., diethyl,5 diisobutyl,5,6 diisopropyl,4,6,7 dicyclohexyl,7 cyclohexyl methyl,7 dibenzyl,6,8,9 dihexyl,5,9,10 isopropylmethyl,9 butylmethyl,9 hexylmethyl,9−11 benzylmethyl,9,12 and trimethyl9 glycolide) were synthesized, and then their polymerization reactions were performed with various catalysts (i.e., Sn(Oct)2, DMAP) in the presence of monoalcohol (i.e., benzyl alcohol) by ring-opening polymerization. © XXXX American Chemical Society

A lack of reactive functional group along the polymer backbone is a major shortcoming for these conventional poly(α-hydroxy acid)s. Therefore, there remains a significant need for a diversity of functional groups like alkene,13,14 allyl,15−18 alkyne,19−21 carboxylic acid,22−28 hydroxy,23,24,29−31 and amine23,32,35−38 groups as a side chain in poly(α-hydroxy acid)s. This inevitable necessity greatly enhances the physical and chemical properties of conventional polymers and provides the opportunity to further modulate them. This is also better alternative to commonly studied aliphatic polyesters (i.e., PLA and PLGA) because functional side chains on the polymer backbone help to control the hydrophilicity, to adjust mechanical strength, to increase the degradation rate, and to bind biologically active compounds (i.e., RGD peptide) for active targeting.3,23,33,34 Among various presenting functionalities, especially aminefunctionalized poly(α-hydroxy acid)s have drawn increasing interests in scientific area due to its inherent properties.23,32,35−38 For example, cationic polylactides were specifically prepared from amine functionalization due to its unique basic character in different pathways for use in medicinal applications in recent years.35−38 Poly[α-(4-aminobutyl)-Lglycolic acid] (PAGA) homopolymer, prepared from polycondensation of Nε-cbz-L-oxylysine with the elimination of water under reduced pressure, binds DNA due to cationic character of amine group for use in gene delivery system. Received: December 29, 2017 Revised: March 24, 2018

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DOI: 10.1021/acs.macromol.7b02751 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules

methane (99%), triethylamine (99%), N,N-dimethylformamide (DMF) (99%), sodium bicarbonate (NaHCO3) (99.7%), diethyl ether (99.5%), toluene (99.7%), ethyl acetate (99.5%), hexane (95%), and benzene from Sigma-Aldrich (Germany) were used as received. (3S)-cis-3,6-Dimethyl-1,4-dioxane-2,5-dione (L-lactide (LLA)) (98%) was recrystallized three times from anhydrous ethyl acetate and dried under vacuum before use in polymerization. Poly(ethylene glycol) methyl ether (MePEG-2000) (Fluka), poly(ethylene glycol) (PEG2000) (Sigma), and tin(II) 2-ethylhexanoate (Sn(Oct)2) (Aldrich, 95%) were employed in the syntheses of copolymers. Palladium 10% on carbon (Pd/C (10%)) (TCI Europe) catalyst was employed for the removal of the protecting group from copolymers. The paclitaxel (ptx) anticancer drug (99.5%) was purchased from Alfa Aesar. The acetonitrile (Sigma-Aldrich, 99.9%), which was used as mobile phase, was filtrated with a filtration apparatus prior to use in high performance liquid chromatography (HPLC) for drug release experiments. Characterization. The attenuated total reflectance−Fourier transform infrared (ATR-FTIR) spectra of intermediate, monomers, and copolymers were obtained on an ATR Bruker-Tensor 27 spectrometer between 600 and 4000 cm−1. Nuclear magnetic resonance (NMR) spectra were acquired using Bruker Avance III 400 MHz and VarianUNITY INOVA 500 MHz spectrometer. Liquid chromatography/mass spectrometry-time-of-flight (LC/MS-TOF) data were recorded on a 1260 Infinity Agilent with TOF-MS unit (Model 6230). Gel permeation chromatography (GPC) measurements were performed on a Viscotek RI max system consisting of autosampler, pump, oven, Viscotek RI detector (VE3580), and two LT4000L Viscotek T-column columns (1500 A pore size, 7 μm particle size, 8 mm i.d., 300 mm length) in series. Tetrahydrofuran (THF) was used as an eluent at flow rate of 1.0 mL min−1 at 35 °C. Refractive index (RI) detector was calibrated with polystyrene (PS) standards having narrow-molecular-weight distribution between 1200 and 400 000 Da. Data were analyzed using Viscotek OmniSEC software. HPLC measurements were conducted on a 1260 Infinity Agilent system equipped with an ultraviolet (UV) detector and ZORBAX SB-C18 4.6 × 150 mm, 3.5 μm HPLC column. Thermal properties of the polymers were observed with differential scanning calorimetry (DSC) analysis using a Mettler Toledo DSC Star 1 instrument. The samples were heated from −60 to 180 °C with heating/cooling rate of 10 °C under a nitrogen atmosphere. Thermogravimetric analyses (TGA) were performed on a Mettler Toledo TGA 1 Star System under a nitrogen atmosphere between 25 and 600 °C at a heating rate of 40 °C min−1. Rigaku Miniflex II diffractometer with Cu Kα (λ = 1.5418 Å) radiation at 45 kV/40 mA was used for wide-angle X-ray diffraction (WAXD) analyses. The measurements were performed at a 2θ angle of 5°−50°, a scanning rate of 1°/min, and a scanning step of 0.02°. Synthesis of (S)-(+)-CBZ-4-Amino-2-hydroxybutyric Acid (2). (S)-(−)-4-Amino-2-hydroxybutyric acid (14.8 g, 0.124 mol) was dissolved in a solution of sodium hydroxide (10.4 g, 0.26 mol) in water (100 mL). Benzyl chloroformate (CBZ) (23.6 g, 0.136 mol) was added dropwise into the stirred solution over 2 h in an ice/salt bath, and the reaction was maintained for a further an hour at the same temperature. After the reaction mixture was washed with diethyl ether (100 mL), aqueous phase was treated with diluted HCl until pH 2 was achieved. Then, it was extracted with diethyl ether (4 × 100 mL). The combined organic extracts were washed with saturated NaCl and dried with Na2SO4. Solvent was removed by rotary evaporation under reduced pressure, and the residue was recrystallized from benzene to obtain (S)-(+)-CBZ-4-amino-2-hydroxybutyric acid (2)39 as a white solid (78%). 1H NMR (d6-DMSO, 500 MHz) δ: 1.54−1.7 (m, 1H), 1.74−1.92 (m, 1H), 3.05−3.17 (distorted q, 2H), 3.92−4.03 (m, 1H), 4.95−5.05 (s, 2H), 7.16−7.27 (t, J = 5.1 Hz, 1H), 7.28−7.43 (m, 5H), 11.2−14.0 (br, 1H). 13C NMR (d6-DMSO, 125 MHz) δ: 34.1, 37.1, 65.1, 67.6, 127.6, 127.7, 128.3, 137.2, 156.1, 175.6. ATR-FTIR (νmax/ cm−1): 3329 (NH), 3062, 3034, 2951 (CH), 1736, 1686 (CO). Synthesis of CBZ Protected 3-Aminoethyl-6-methyl-1,4dioxane-2,5-dione (ZNEtMG) (4). Synthesis of CBZ protected asymmetric glycolide monomer was performed in two steps. In the first step, 2-bromopropionyl bromide (0.84 mL, 8.06 mmol) was

However, poorly controlled molecular weights, broad molecular weight distributions, and unfavorable synthesis conditions (i.e., 5 days of reaction under very low pressure of 10−4 mmHg) reduce the usefulness of PAGA polymers.35 Aromatic polyester bearing pendant amine salt, capable of both cell penetration and gene delivery, was prepared from O-carboxyanhydrides via thiol−yne photochemistry. Multistep synthesis to the polymer (total of seven steps: O-carboxyanhydride monomer in five steps and polymer in two steps), overall 24% yield to final polymer from BOC-L-tyrosine (Boc = tert-butoxycarbonyl) starting material, some toxicity just after 100 μg/mL polymer concentration, and degradation problem due to consisting of aromatic group were major shortcomings of polymer.36 Tertiary amine-functionalized cationic polylactides (CPLAs) were also prepared from allyl-functionalized lactide, synthesized via multistep synthesis with 30% overall monomer yield, followed by thiol−ene click functionalization by using benzyl alcohol (BnOH) or α-methoxy-ω-hydroxyl PEG (mPEG-OH) as initiators for gene delivery.16,37,38 Although tertiary amine groups are suitable for electrostatic interaction, tertiary amine restricts the reactivity to any further functionalization with any ligand molecule for any other applications. Weck’s group prepared monomers of serine, lysine, and glutamic acid derivatives containing different lengths of side chains having amine, alcohol, and carboxylic acid groups.23 Then, their homopolymers and copolymers with lactide were obtained in bulk medium at 140 °C for 24 h (or 8 h for copolymers). However, serious deviations from the theoretical molecular weight and some oligomers due to low polymerization rate and/or possible transesterification reactions when compared to polylactide were observed. Chen et al. have reported the synthesis of O-carboxyanhydride monomer prepared from lysine and polymerize it to make polyesters bearing pendant amino groups.32 On the other hand, the side reaction products were observed due to misinsertion of the propagating chain end into the disfavored 2-position of the Ocarboxyanhydride ring with the formation of carboxylic acid end group having no ability further propagation. In this article, we describe the novel synthesis of free aminefunctionalized, ready to any further functionalization with ligands or any biological molecules, polylactide−PEG diblock and triblock copolymers by ring-opening polymerization (ROP) with very convenient and simple strategies (i.e., short step of monomer synthesis, solvent-free polymerization, high conversions, low polydispersities, and very short reaction times). As far as we know, our study is the first of a kind, establishing a new hydrogel-based platform composed of amine-functionalized polylactide for use in the local therapy. These tunable amine functional thermosensitive hydrogels showed liquid property (sol) around 42 °C, suitable for injection, and turned instantly into a gel form at body temperature to cover tumor surface. Fast hydrolytic degradation and effective paclitaxel release from copolymer gels may make these amine-functionalized lactides a preferred candidate for developing controlled/sustained release of drug from delivery vehicles.

2. EXPERIMENTAL SECTION Materials. (S)-(−)-4-Amino-2-hydroxybutyric acid (96%), benzyl chloroformate (CBZ) (98%), 2-bromopropionyl bromide (97%), ptoluenesulfonic acid monohydrate (PTSA.H2O) (98.5%), sodium hydroxide (NaOH) (98−100.5%), hydrochloric acid (HCl) (36.5− 38%), sodium chloride (NaCl), sodium sulfate (Na2SO4), dichloroB

DOI: 10.1021/acs.macromol.7b02751 Macromolecules XXXX, XXX, XXX−XXX

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poly(LLA-co-ZNEtMG) (10), Sn(Oct)2 (0.05 mmol, 20 mg), MePEG-2000 (0.12 mmol, 240 mg), L-lactide (1.8 mmol, 260 mg), and ZNEtMG (0.2 mmol, 62 mg) were added into the polymerization tube. The polymerization reaction was kept at 120 °C for an hour under a nitrogen atmosphere. Copolymer 10 was purified by dissolving in methanol at 40 °C and then precipitated at −20 °C. 1H NMR (CDCl3, 400 MHz) δ: 1.4−1.7 (d, J = 6.9 Hz, 3H; m, 3H), 2.0−2.4 (m, 2H), 3.35−3.39 (br, 1H; s, 3H), 3.42−3.49 (m, 1H), 3.51−3.56 (m, 1H), 3.57−3.75 (s, 4H), 5.0−5.5 (q, J = 6.7 Hz, 1H; m, 1H; m, 1H; m, 2H), 7.28−7.46 (m, 5H). 13C NMR (CDCl3, 100 MHz) δ: 16.6, 20.5, 30.7, 36.7, 66.7, 68.8, 69.0, 69.2, 70.5, 128.1−134.9, 156.4 169.5, 169.6, 169.7. ATR-FTIR (νmax/cm−1): 2978, 2938, 2862 (CH), 1755 (CO). Similarly, the other copolymers (9, 11−18, 21, 22) were prepared with the same protocol as above except for the synthesis of triblock copolymers. The PEG homopolymer having two hydroxyl groups was used in place of MePEG homopolymer having a methoxy group on one end and a hydroxyl group on the other end for poly(LLA-coZNEtMG)−PEG−poly(LLA-co-ZNEtMG) triblock copolymer syntheses. Deprotection for Protected Copolymers. Deprotection of copolymers was accomplished with a modified protocol.29 50 mg of Pd/C (10%) was added into a solution of protected copolymers 10 or 15 (100 mg) in dichloromethane (8 mL). First, hydrogen gas (balloon) was passed from the system to remove the air. Catalytic hydrogenation was performed for 7 days with vigorously stirring at room temperature under hydrogen atmosphere. The mixture was filtrated over Celite to remove the catalyst from the reaction medium followed by evaporation of solvent under vacuum to obtain the deprotected copolymers 19 and 20. Gel−Sol Phase Transition. Gel−sol transition temperatures of copolymers were determined by following the procedures previously reported.4,40 Gels at given concentrations were prepared from copolymers and deionized water. The gel-to-sol transition temperature of the copolymers was observed visually by inverting the vials at different temperatures using a controlled water bath. Briefly, all copolymers with deionized water were vortexed to determine whether they form homogeneous mixture or not. Then, they were kept at 4 °C for 30 min in fridge for the equilibrium before immersing them in a temperature-controlled water bath. The gel-to-sol transition temperatures of the copolymers were examined from 4 to 80 °C with 2 °C increments. The vials were maintained in water bath for 3−4 min at each temperature before tilting. Degradation Study. Hydrolytic degradation of block copolymers was performed in phosphate buffered saline (PBS) at physiological conditions (pH: 7.4, 37 °C, 200 rpm). 15 mg of polymer was added into 5 mL of PBS in the test tubes followed by incubation. Samples were taken out at predetermined time intervals, and then, the supernatant was removed. They were washed thoroughly with deionized water to remove salt residues and then stored at −20 °C to lyophilize them. The resulting polymers were dissolved in THF for GPC analyses. The percentage of degradation products was determined by Lorentzian formulation using Origin 9 pro software according to the literature.49 In Vitro Paclitaxel Release Study. Release studies were conducted for poly(LLA-co-ZNEtMG)-PEG, poly(LLA-co-NEtMG)PEG, and PLLA−PEG block copolymers according to the same methodology that was described in a previously published work.4 Paclitaxel, which was selected as an anticancer drug, was loaded into copolymer gels effectively with loading ratio of 1.0%. Briefly, for the preparation of MePEG−poly(LLA-co-ZNEtMG) diblock copolymer 10 gel, 1.17 mg of paclitaxel, 117 mg of compound 10, and 233 μL of deionized water were added to the 1.5 mL vial, and the sample was vortexed at room temperature for 5 min to get homogeneous drug loaded gel. Then drug loaded gels were allowed to stand at 4 °C for 30 min in fridge for equilibrium. Other drug loaded copolymer gels were prepared in the same manner as shown in Table 1. Then, 650 μL of 2% Tween 80 in PBS buffer at pH 7.4 was added to the surface of the drug loaded gels at room temperature for drug release studies. These samples were kept in incubator at 37 °C with a constant speed of 200

added to a solution of (S)-(+)-CBZ-4-amino-2-hydroxybutyric acid (1.6 g, 6.32 mmol) in dichloromethane (80 mL) at 0 °C under a nitrogen atmosphere. Triethylamine (1.45 mL) in dichloromethane (5 mL) was added dropwise over a period of 1 h to a vigorously stirred solution in an ice/salt bath. The reaction was further stirred at 0 °C over 30 min after the addition was completed. The progress of the reaction was followed with thin layer chromatography (TLC, silica gel, 60 F254, CH2Cl2:CH3OH:CH3COOH (10:1:0.5)). Then, the TLC plate was treated with a ninhydrin solution (Rf: 0.69). The reaction mixture was diluted with more diethyl ether, washed with deionized water (3 × 10 mL), and dried over Na2SO4. Ether was evaporated in vacuo to give a pale yellow viscous liquid 4-(((benzyloxy)carbonyl)amino)-2-((2-bromopropanoyl)oxy)butanoic acid (3, intermediate) (70%). 1H NMR (CDCl3, 400 MHz) δ: 1.68−1.92 (m, 3H), 2.02− 2.32 (m, 2H), 3.18−3.48 (m, 2H), 4.28−4.54 (m, 1H), 4.98−5.12 (s, 2H), 5.12−5.2 (m, 1H), 5.2−5.3 (br, 1H), 7.22−7.44 (m, 5H), 8.5− 9.3 (br, 1H). 13C NMR (CDCl3, 100 MHz) δ: 21.4, 21.6, 21.7, 30.9, 37.1, 39.2, 39.6, 39.9, 67.1, 67.7, 70.9, 71.0, 128.1, 128.2, 128.3, 128.4, 128.6, 135.8, 136.2, 156.8, 158.2, 169.6, 169.8, 173.2, 174.6. ATRFTIR (νmax/cm−1): 3338 (NH), 3066, 3034, 2942 (CH), 1720 (CO). In the second step, to a vigorously stirred suspension of NaHCO3 (2.1 g, 25 mmol) in dimethylformamide (100 mL), a solution of intermediate 3 (5.82 g, 15 mmol) in dimethylformamide (40 mL) was added at 40 °C over 4 h. The reaction was monitored with thin layer chromatography (TLC, silica gel, 60 F254, hexane:ethyl acetate (2:1)) to avoid oligomeric species. After the mixture was stirred for further 3 h, the temperature was maintained, and solvent was evaporated under reduced pressure. The residue was dissolved with diethyl ether (50 mL). Obtained organic phase was washed with deionized water (3 × 10 mL) and dried over Na2SO4. After removing the ether, the resulting residue was recrystallized two times from diethyl ether to afford pure benzyl (2-(5-methyl-3,6-dioxo-1,4-dioxan2-yl)ethyl)carbamate (ZNEtMG) as a white solid 4 (finally recovered yield after double recrystallizations is 67%). 1H NMR (CDCl3, 500 MHz) δ: 1.5−1.7 (d, J = 6.5 Hz, 3H), 1.96−2.18 (m, 1H), 2.24−2.48 (m, 1H), 3.24−3.52 (distorted q, J = 6.1 Hz, 2H), 4.85−5.01 (q, J = 6.5 Hz, 1H), 5.01−5.06 (dd, J = 4.2, 7.5 Hz, 1H), 5.06−5.14 (s, 2H), 5.18−5.3 (br, 1H), 7.2−7.46 (m, 5H). 13C NMR (CDCl3, 125 MHz) δ: 15.7, 30.6, 36.7, 66.9, 72.4, 73.8, 128.2, 128.3, 128.6, 136.3, 156.7, 167.0, 167.3. ATR-FTIR (νmax/cm−1): 3346 (NH), 3032, 2947, 2895 (CH), 1767, 1693 (CO). LC/MS-TOF (C15H17NO6Na): theoretical: 330.10 g/mol; experimental: 330.08 g/mol. Synthesis of CBZ Protected 3,6-Diaminoethyl-1,4-dioxane2,5-dione (ZDNEtG) (5). Synthesis of dibenzyl ((3,6-dioxo-1,4dioxane-2,5-diyl)bis(ethane-2,1-diyl))dicarbamate (ZDNEtG) was performed by modifying the method in the literature.4,5 (S)(+)-CBZ-4-Amino-2-hydroxybutyric acid (1.5 g, 6 mmol) and ptoluenesulfonic acid monohydrate (30 mg, 0.15 mmol) mixture in toluene (90 mL) was refluxed with using Dean−Stark apparatus for 5 h in order to eliminate water. The proceeding of the reaction was monitored with thin layer chromatography (TLC, silica gel, 60 F254, hexane:ethyl acetate (3:1) eluent mixture). Then, the TLC plate was treated with a potassium permanganate (KMnO4) stain (Rf: 0.4). The flask was allowed to cool room temperature, and then toluene was evaporated under reduced pressure. The resulting residue was washed with cold toluene, and the solvent was separated by decantation. The obtained crystals were dissolved with ethyl acetate and recrystallized from hexane at −20 °C to eliminate impurities. After the solvent was removed, CBZ protected 3,6-diaminoethyl-1,4-dioxane-2,5-dione (ZDNEtG) was obtained (58%). 1H NMR (CDCl3, 400 MHz) δ: 1.91−2.1 (m, 2H), 2.4−2.52 (m, 2H), 2.9−3.48 (br, 2H), 3.53−3.64 (m, 2H), 3.86−3.96 (m, 2H), 4.35−4.45 (dd, J = 8.2, 10.5 Hz, 2H), 5.28−5.33 (s, 4H), 7.32−7.48 (m, 10H). 13C NMR (CDCl3, 100 MHz) δ: 27.1, 42.1, 68.4, 70.4, 128.3, 128.5, 128.6, 135.0, 151.2, 174.4. ATR-FTIR (νmax/cm−1): 3445 (NH), 3000, 2940, 2880 (CH), 1742 (CO). LC/MS-TOF (C24H26N2O8Na): theoretical: 493.16 g/mol; experimental: 493.18 g/mol. General Procedure for Copolymer Syntheses. Syntheses of diblock and triblock copolymers were carried out in bulk as previously described.4 Briefly, in order to perform the synthesis of MePEG− C

DOI: 10.1021/acs.macromol.7b02751 Macromolecules XXXX, XXX, XXX−XXX

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there were no oligomeric products in the purified monomers. In addition, the purified monomer was analyzed with GPC to check oligomeric species, and no oligomeric peaks were obtained. Chemical analysis of intermediate 3 was performed by ATRFTIR, 1H, and 13C NMR techniques. A single broad band at 1720 cm−1 as a combination of three carbonyl groups was observed in ATR-FTIR spectrum of intermediate 3, whereas two carbonyl stretching vibrations in the starting material 2 separately appeared at 1686 and 1736 cm−1 (Supporting Information Figures S1 and S2). Also, two new resonances at 1.68−1.92 ppm (CH3) and 4.28−4.54 ppm (CH) in the 1H NMR spectrum and three new resonances at 21.4−21.6−21.7 ppm (CH3), 39.2−39.6−39.9 ppm (CH), and a new carbonyl peak at 169.6−169.8 ppm were observed in the 13C NMR spectrum of intermediate 3 when compared to starting material 2. Moreover, the shifting of the CH proton and carbon in starting material 2 from 3.92−4.03 and 67.6 ppm to 5.12−5.2 and 70.9−71.0 ppm in 1H and 13C NMR proved the formation of intermediate 3, respectively (Figures S13, S14, S22, and S23). ZNEtMG 4 monomer was analyzed by ATR-FTIR, 1H, 13 C, COSY 2-D, HMQC 2-D NMR, and LC/MS-TOF techniques. Its chemical structure was confirmed by the shifting of the CH proton and carbon in intermediate 3 from 4.28−4.54 to 4.85−5.01 ppm and from 39.2−39.6−39.9 to 72.4 ppm in the ZNEtMG 4 in 1H and 13C NMR, respectively (Figure 1a,b and Figures S14, S23). The formation of ZNEtMG 4 was also observed in 13C NMR by the shifting from 173.2 to 174.6 ppm (acid carbonyl group of intermediate 3) to 167.3 ppm (ester carbonyl group of ZNEtMG 4) (Figure 1b and Figure S23). The HMQC 2-D NMR spectrum was recorded to indicate the direct proton−carbon shift correlation in monomer 4. It has been proven from HMQC 2-D NMR spectrum that a-coded peaks are bound to CH3 carbon and protons; b1 and b2 coded protons are bound to b carbon; c and f-coded peaks refer to peaks of CH2 carbon and protons; d and e coded peaks refer to peaks of CH carbons and protons; and the h, i, j, and k coded peaks show the peaks of the carbon and protons of the aromatic ring. The l, m, and n coded peaks are also the peaks of carbonyl groups (Figure 1c). In the COSY 2-D NMR spectrum of the compound 4 shown in Figure 1d, the interactions of the neighboring protons which indicate spin−spin coupling

Table 1. Gel Preparation with Paclitaxel Drug at 1% Drug Loading Ratio ID MePEG−poly(LLA-co-ZNEtMG), 10 MePEG−poly(LLA-co-NEtMG), 19 poly(LLA-co-ZNEtMG)−PEG− poly(LLA-co-ZNEtMG), 15 poly(LLA-co-NEtMG)−PEG− poly(LLA-co-NEtMG), 20 MePEG−PLLA, 21 PLLA−PEG−PLLA, 22

copolymer (mg)

drug (mg)

deionized water (μL)

117 117 115

1.17 1.17 1.15

233 233 235

115

1.15

235

112 140

1.12 1.4

238 210

rpm. At different time periods, 650 μL of supernatant was taken out from the vial, and the same amount of fresh supernatant was added. Before the measurements, collected supernatants in Eppendorf tubes were kept in a fridge at −20 °C. The amounts of paclitaxel in the supernatants were analyzed via HPLC at 227 nm using a UV detector. HPLC assays were repeated three times for each release group, and drug-free gels were also analyzed to eliminate the influence of low characteristic signals of copolymers.

3. RESULTS AND DISCUSSION Syntheses of Protected Functional Monomer. CBZ protected 3-aminoethyl-6-methyl-1,4-dioxane-2,5-dione (ZNEtMG) (4) was synthesized via two-step reaction sequence: the formation of halogenated carboxylic acid intermediate 3 from (S)-(+)-CBZ-4-amino-2-hydroxybutyric acid39 with 2-bromopropionyl bromide in dichloromethane (DCM) at 0 °C and cyclocondensation reaction of the intermediate 3 with NaHCO3 in dimethylformamide at 40 °C (Scheme 1). According to NMR analyses, intermediate 3 yielded a 1:1 mixture of diastereomers (R,S and S,S) while a stereoselectivity was observed for final monomer 4 (R,S or S,S). Overall synthetic yield of functional lactide monomer 4 and 5 starting from commercially available compound 2 was 47% and 58%, respectively. Some oligomerizations have been observed at longer reaction times, whereas at shorter reaction times, the starting material has not been converted to the monomers at high rates, as reported previously.4 Therefore, the conversion of the monomers to the oligomeric species was minimized by monitoring the reactions by TLC, and thus higher yields of the monomers were obtained. NMR measurements proved that

Scheme 1. Synthesis of CBZ Protected Amine-Functionalized Monomer 4

D

DOI: 10.1021/acs.macromol.7b02751 Macromolecules XXXX, XXX, XXX−XXX

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Figure 1. 1H NMR (a), 13C NMR (b), HMQC 2-D NMR (c), and COSY 2-D NMR (d) spectra of ZNEtMG 4.

The 1H NMR spectrum of MePEG−poly(LLA-co-ZNEtMG) diblock copolymer 10 with the peak assignments is shown in Figure 2a. Resonances appearing at 1.4−1.7 ppm, 2.0−2.4 ppm, 3.35−3.39 ppm, 3.42−3.49 and 3.51−3.56 ppm, 5.0−5.5 ppm, and 7.28−7.46 ppm were assigned to methyl protons (a, b) of poly(LLA-ZNEtMG) block, methylene protons (c) of ZNEtMG, both amine protons (d) of ZNEtMG and methoxy protons (e) at the end of the MePEG block, methylene protons (f) of ZNEtMG, methine protons (h, i, j) of poly(LLAZNEtMG) and methylene protons (k) of protecting group (−O−CH2−C6H5), and aromatic protons (l, m, n), respectively. The singlet signal appearing at 3.57−3.75 ppm belongs to methylene protons (g) of MePEG block in the copolymer. The multiplet peak at 4.18−4.42 ppm is attributed to methylene protons of LLA-ZNEtMG units connected to the MePEG block (poly(LLA-co-ZNEtMG)−COO−CH2−CH2−) and the methine proton neighboring the hydroxyl end-group (−COO−CH(CH2CH2NHCBZ)OH), which prove copolymer formation (Figure 2a). 1H NMR spectroscopy also verifies the polymerization of the ZNEtMG monomer by the appearance of a methine protons peak at 5.0−5.5 ppm, too. In addition, very high conversions (>90%) were obtained from the 1H NMR spectra of copolymers by integration of the polymer peaks relative to monomer peaks (Table 2). The 13C NMR spectrum of compound 10 also showed resonances of main peaks at 16.6 (CH3), 69.0 (CH), 169.6 (CO) for lactide units, 70.5 (CH2) for MePEG, and 20.5 (CH3), 30.7 (CH2−CH2−NH), 36.7 (CH2−CH2−NH), 66.7 (−CH2C6H5), 68.8 (CH−CH3), 69.2 (CH−CH 2 −), 128.1−134.9 (aromatic ring), 156.4 ( −N H C O O −) , 1 69 . 5 (− C O C H C H 3 ) , a n d 16 9 . 7 (−COCHCH2−) for the ZNEtMG unit, and the end units, the neighboring units to the end, and some low quantity of

interactions between the correlated nuclei in the structure 4 were observed in the cross-peaks of horizontal and vertical axes. The presence or absence of these cross-peaks has been employed effectively in the assignment of skeleton connectivities in the monomer structure. The diagonal peaks here serve only as reference points. The off-diagonal peaks at points 1, 2, 3, 4, and 5 represent coupling of protons of “a” with “d”, “b1, b2” with “c and e”, “c” with “b1, b2, and g”, “e” with “b1, b2”, and “b1” with “b2” in ZNEtMG 4. There is no cross-peak for “k, l, m, and n” because it does not possess any hydrogens. Polymerization and Deprotection. MePEG−poly(LLAco-ZNEtMG) diblock and poly(LLA-co-ZNEtMG)−PEG− poly(LLA-co-ZNEtMG) triblock copolymers were obtained via the ring-opening polymerization using monomers of ZNEtMG 4 and L-lactide and the terminal MePEG or PEG as an initiator in the presence of Sn(Oct)2. All polymerization reactions done under solvent-free medium at 120 °C are presented in Scheme 2. The benzyl protective groups were removed and free amine side chains were obtained via catalytic hydrogenolysis by using H2 gas to give functional copolymers 19 and 20. Molecular weights of copolymers were specifically kept in a specific range (generally less than 10K) in order to be suitable for sol−gel preparation by changing the mole ratio of ester monomers while keeping a constant mole ratio of initiator and catalyst, as seen in Table 2. The repeating unit of the ZNEtMG and LLA in the block copolymers were determined from 1H NMR spectra by comparing the intensity of the methine proton signal of polymers at δ = 5.0−5.5 ppm, the phenyl proton signal of ZNEtMG unit at δ = 7.28−7.46 ppm and methylene proton signal of MePEG or PEG at δ = 3.57− 3.75 ppm. E

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Macromolecules Scheme 2. Syntheses of Protected and Deprotected Block Copolymers

heterotacticity (Figure S26). The reaction of compound 10 with 33% HBr/AcOH led to partial deprotection (∼60%) with significant degradation. Complete removal of the protecting group without backbone degradation was accomplished with H2 gas using Pd/C catalyst. GPC analysis of the deprotected polymer 19 confirmed that there was no chain scission under these reaction conditions (Figure 2d). Deprotection of compound 10 was easily confirmed by the disappearance of phenyl protons and carbons at 7.28−7.46 and 128.1−134.9 ppm in 1H and 13C NMR, respectively (Figure 2b,c and Figure S26). The molecular weight distribution of copolymers was determined with GPC. These copolymers exhibited a monomodal peaks with polydispersity indices varying from 1.06 to 1.26, indicating the monodisperse properties of the synthesized copolymer chains (Table 2). On the other hand, LLA-ZNEtMG feed ratio was optimized at 90/10% because beginning of homopolymerization occurred with increasing feeding ratio of ZNEtMG monomer for copolymers 12 and 13 (Table 2). A lower reactivity of ZNEtMG than L-lactide in the copolymerization was due to more bulky side groups in ZNEtMG. GPC analysis also showed that longer reaction times lead to decreasing the molecular weight and increasing the polydispersity value of copolymer (data not shown). Therefore, the polymerization time was optimized as 1 h. As can be seen from the Figure 2d, MePEG homopolymer was not detected on

the GPC traces of copolymers, proving that syntheses of copolymers were effectively accomplished with absence of unreacted MePEG homopolymer. Also, MePEG−poly(LLA-coZNEtMG) (10) was analyzed with ATR-FTIR spectroscopy. The peak at 1755 cm−1 corresponds to the stretching vibration of carbonyl groups, while peaks at 2978, 2938, and 2862 cm−1 are related to C−H stretching vibrations (Figure S6). Gel−Sol Transition Properties. The gel−sol transition temperatures were determined for PEG based polyester diblock and triblock copolymers 10, 15, 19, 20, 21, and 22 by the changing of the concentration of copolymers.4,40 Our object was specifically to prepare appropriate copolymers that display a sol behavior at around 42 °C, which is proper for injection, and then a gel with quick cooling to body temperature. Thus, each component’s length in the diblock and triblock PEG based polyesters was adjusted with a great attention during the chemical syntheses. The gel-to-sol transition upon heating can be seen in Figure 3a−c. Both 44 and 42 °C at 33.5% for MePEG−poly(LLA-co-ZNEtMG) (10) and MePEG−poly(LLA-co-NEtMG) (19) and 46 and 42 °C at 33% for poly(LLA-co-ZNEtMG)−PEG−poly(LLA-co-ZNEtMG) (15) and poly(LLA-co-NEtMG)−PEG−poly(LLA-co-NEtMG) (20) were found as an appropriate critical gel−sol transition temperature, respectively. These results indicated that the gel− sol transition temperatures were slightly lower for copolymers 19 and 20 due to removal of the hydrophobic CBZ protecting F

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5 13 18 0.7 1.6 2.2 90 92 91

7 13 19 0.9 1.6 2.4 96 92 91

1.10 1.22 1.21 1.27 1.25 1.06 1.26 1.20 2990 4290 5310

3340 4670 6000 6210 6000 3340 4670 6000 3270 4460 5780

3880 4240 4850 3840 3780 4510 4640 5750 0.05

1

120

4290 5180 5850 4870 4710 4780 5830 6910 120 1 0.05

90/10 90/10 90/10 85/15 75/25 90/10 90/10 90/10 0.1 0.2 0.3 0.45 0.75 0.1 0.2 0.3 0.12

0.12

diblock

triblock

9 10 11 12 13 14 15 16

group. Triblock copolymers 15 and 20 showed higher gel−sol temperature than diblock copolymers 10 and 19 at a constant 33% concentration due to more hydrophobic nature in both sides of triblock copolymer. Also, amine-functionalized diblock copolymers 10 and 19 were compared with MePEG−PLLA 21 in terms of gel−sol temperature and concentration. Copolymer 21, as expected, reached the desired gel−sol temperature (40 °C) at lower concentration and showed higher gel−sol temperature at a constant 33% concentration due to hydrophobic nature of MePEG−PLLA 21 when compared to copolymer 10 and 19. (Figure 3) On the other hand, PLLA− PEG−PLLA triblock 22 copolymer indicated transitions from gel to sedimentation41 except for very high concentrations. A higher mole of hydrophobic ester relative to the PEG moiety in the copolymers expectedly resulted in nonhomogenous suspensions even at 10% concentration (i.e., copolymer 11). Therefore, the molecular weight of the copolymers (2 mol of ester content) was purposely kept within a specific range for the gel−sol experiments. As a result, the aqueous polymer solutions form a gel at high concentration levels and at a lower temperature and became a sol at lower concentration and a higher temperature (Figure 3d). In addition, deionized water has a weakly acidic medium, possibly leading to the protonation of amine groups in copolymer in the preparations of gels;50 therefore, the gel−sol transitions were also determined in a buffer of pH: 7.4. It was observed that the change in pH led to a decrease in gel−sol transitions of around 4.0 °C for protected diblock copolymer 10, 2.0 °C for deprotected diblock copolymer 19, 6.0 °C for protected triblock copolymer 15, and 4.0 °C for deprotected triblock copolymer 20. Release Studies. The release behavior of paclitaxel anticancer drug from the MePEG−poly(LLA-co-ZNEtMG) diblock 10, poly(LLA-co-ZNEtMG)−PEG−poly(LLA-coZNEtMG) triblock 15, MePEG−poly(LLA-co-NEtMG) diblock 19, and poly(LLA-co-NEtMG)−PEG−poly(LLA-coNEtMG) triblock 20 gels were examined with HPLC over 20 days at 37 °C. They were compared with MePEG−PLLA diblock 21 and PLLA−PEG−PLLA triblock 22 copolymers. There was an initial burst release of 15% to 23% for copolymers 10, 15, 19, and 20 after 48 h. It was about 6.5% and 13.5% for PLLA−PEG copolymers 21 and 22, respectively. Briefly, side chain length (1) and hydrophilicity (2) independently affect the paclitaxel release rate. Longer length of the side chain (1) increases free volume in the polymeric matrix which is a prerequisite for a primarily diffusion controlled drug delivery. An increase in free volume also leads to a decrease in the glass transition temperature (i.e., copolymer 19: Tg: 17 °C). Lower glass transition temperature than release temperature causes more flexible and mobile chains, creating much more free volume in the polymeric matrix,42−44 resulting in faster release of the paclitaxel drug. These effects appear in drug release of copolymers 19 and 21 as follows: A burst release of about 23% paclitaxel from copolymer 19 was observed while only 6.5% drug was released from copolymer 21. After 20 days, similar behavior for copolymers 19 and 21 was observed as a drug release of 72% and 28%, respectively. A similar behavior about a close relation between the glass transition temperature and the temperature at which the release takes place was reported for flurbiprofen and 5,10,15,20-tetra(m-hydroxyphenyl)porphyrin (mTHPP) loaded PLA and PLGA nanoparticles in the literature.42 Copolymers with CBZ group 10 and 15 had very close Tg values to copolymers 19 and 20, and all Tgs were below the release temperature (data not shown). All

a Determined by GPC. bDetermined by 1H NMR spectrum (the calculation of conversion was performed using the signal from the CH (δ 4.85−5.01, 5.01−5.06 ppm) of the unreacted monomer and the CH (δ 5.0−5.5 ppm) of the polymer); RU: repeating unit.

(mmol) copolymer ID

0.9 1.8 2.7 2.55 2.25 0.9 1.8 2.7

RUb of ZNEtMG convb (%) Mw/Mna theor Mnb (g/mol) time (h) Sn(Oct)2 (mmol) feed ratio (%)

LLA-ZNEtMG

ZNEtMG (mmol) L-lactide

PEG (mmol)

conditions for synthesis of copolymers

Table 2. Conditions and Characterization of the Diblock and Triblock Copolymers

temp (°C)

Mwa (g/mol)

Mna (g/mol)

characterization of diblock and triblock copolymers

RUb of LLA

Macromolecules

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Figure 2. 1H NMR spectrum of protected diblock 10 (a), deprotected diblock 19 (b), 13C NMR spectrum of deprotected 19 (c), and GPC curves of MePEG 6, deprotected diblock 19, and protected diblock 9−11 (d).

rate for copolymers with CBZ group 10 and 15 is that they are not close-packed since they have longer side chains. The drug was released very rapidly due to the wide spacing of these copolymer chains in the irregular structure. The CBZ groupremoved copolymers 19 and 20 have shorter side chains and hydrogen bonding via amino groups so they have fewer spaces between the chains. Therefore, the drug release from the CBZ group-removed copolymers is slower. The hydrophilicity (2) of the copolymer 19 and 20 also leads to faster drug release because the higher hydrophobic character in MePEG−PLLA 21 and PLLA−PEG−PLLA 22 causes a lower release due to the hydrophobic−hydrophobic interactions between the polyester block and the paclitaxel. The limiting factor that hinders release in the first stage is the limited water absorption by hydrophobic unfunctionalized PLA copolymers.45 Therefore, a hydrophilic side chain, ethylamine, was added to enhance water absorption. On the other hand, the release results showed that the factor of chain length was more effective than hydrophilicity when copolymers 10 and 15 were compared with copolymers 19 and 20. Functional copolymers (Figure 4) show remarkable improvement in drug release during the period of 3 weeks. As a result, paclitaxel release was increased considerably; after 20 days, a cumulative 72%−95% drug in the copolymers 10, 15, 19, and 20 was released as opposed to only 28%−29% in pure PLA−PEG−PTX matrix. Thermal Properties and X-ray Diffraction. The characteristics of the copolymers featuring different polyester/ PEG contents were further assessed by TGA analyses, as illustrated in Figure 5. Two significant weight loss steps belonging to each block were observed when thermal stabilities of MePEG−poly(LLA-co-ZNEtMG) diblock 9, 10, and 11

Figure 3. Image of the gel-to-sol transition upon heating (a−c); gel− sol curves of the diblock and triblock copolymers 10, 15, 19, 20, and 21 (d). The upper left region of each curve represented the sol phase and the opposite region (lower right) the gel phase.

copolymers 10, 15, 19, and 20 gave faster drug release, as expected, than copolymers 21 and 22. When the release rate of copolymers with CBZ group 10 and 15 was compared with the copolymers without CBZ group 19 and 20 according to chain lengths and the effect of groups, the reason for highest release H

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Table 3. Characteristics of the Copolymers by TGA Analyses at Different Polyester/PEG Contents

a

Figure 4. Paclitaxel release curves from the diblock and triblock gels. Each point of the plot is the result of an average of at least three independent reproductions.

ID

Mna

polyester/ PEGa (%)

polyester/ PEGb (%)

char (%)

T1p (°C)

T2p (°C)

9 10 11 14 15 16 19 20 MePEG PEG

1270−2000 2460−2000 3780−2000 990−2000 2290−2000 3310−2000 2260−2000 2090−2000 0−2000 0−2000

39:61 55:45 65:35 33:67 53:47 62:38 53:47 51:49 0:100 0:100

40:60 57:43 65:35 35:65 55:45 64:36 55:45 52:48 0:100 0:100

5.2 4.2 5.3 7.2 3.8 4.1 0.5 0.9 0.7 0.6

286 295 302 283 285 283 320 335

421 424 427 415 416 415 413 420 415 421

Determined by the 1H NMR spectrum. bDetermined by TGA.

char yield of copolymer 10 was found to be 4.2%. The molar ratios of ester and ether blocks in the copolymers 9, 10, 11, 14, 15, and 16 have been calculated from the weight loss percentage. The values thus acquired from TGA analyses were in good agreement with those based on 1H NMR spectra (Table 3). Also, the deprotection of amine groups in polyester segments caused less distinctive transition between both polyester and PEG segments (10 vs 19 and 15 vs 20 in Figure 5c) at ca. 55% mass loss, lower char yields because of the absence of aromatic groups, and higher inflection points (Table 3).

(Figure 5a and Table 3), and poly(LLA-co-ZNEtMG)−PEG− poly(LLA-co-ZNEtMG) triblock 14, 15, and 16 (Figure 5b and Table 3) copolymers were investigated. For example, the first one in diblock 10 was due to the decomposition of poly(LLAco-ZNEtMG) segment with 57% weight loss (T1p = 295 °C, inflection point, temperature at which greatest rate of change on the weight loss in the peak of first derivative has occurred). The second stage, appearing at higher temperatures, was due to MePEG decomposition with 43% weight loss with T2p = 424 °C (Tp = 415 °C for pure MePEG).4 After heating to 600 °C, the

Figure 5. Thermal degradation profiles of (a) MePEG−poly(LLA-co-ZNEtMG) 9, 10, and 11 diblock copolymers; (b) poly(LLA-co-ZNEtMG)− PEG−poly(LLA-co-ZNEtMG) 14, 15, and 16 triblock copolymers; and (c) comparison of protected 10, 15 vs deprotected 19, 20 amine di- and triblock copolymers. I

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Figure 6. DSC thermograms of (a) MePEG 6, (b) MePEG−PLLA 21, and (c) copolymer 19 in the first run and (d) MePEG 6, (e) MePEG−PLLA 21, and (f) copolymer 19 in the second run.

Thermal characteristics of MePEG−poly(LLA-co-NEtMG) diblock 19 (MePEG 6 and MePEG−PLLA 21 for the comparison) were also examined by DSC analysis (Figure 6). The broadness between 30 and 48 °C in MePEG−PLLA 21 is probably due to overlapping melting point (Tm) of PEG units and glass transition temperature (Tg) of polyester units. Similar results have been reported in the literature about the values of Tg of PLA and Tm of PEG being close to each other.46 Tg for copolymer 19 was lower and more distinctive at 17 °C due to increased flexibility of polyester chains. On the other hand, the Tm value of MePEG was found to be 40.2 °C for copolymer 19 while the melting temperature of pure MePEG 6 was around 56 °C (Figure 6a vs 6c). Therefore, the presence of the poly(LLAco-NEtMG) blocks attached to the PEG blocks diminished the melting temperature of the corresponding PEG. This situation proved that the crystallization of each block was remarkably affected by the presence of the other block. Two different melting endotherms due to crystallization behaviors of poly(LLA-co-NEtMG) blocks were observed for copolymer 19 at 60 and 100−105 °C in the second run of DSC. The former was probably for crystals of poly(LLA-co-NEtMG) segments, and the latter was for only lactide units due to high mole of lactide in the copolymer 19. Poly(LLA-co-NEtMG)− PEG−poly(LLA-co-NEtMG) triblock 20 copolymer showed similar behavior with diblock copolymer 19. The crystalline structures of MePEG 6, MePEG-PLLA 21, MePEG−poly(LLA-co-ZNEtMG) 10, and MePEG−poly(LLAco-NEtMG) 19 were analyzed by wide-angle X-ray diffraction as indicated in Figure 7. MePEG 6 showed two sharp characteristic peaks at 2θ angles of 19.3° and 23.4° while MePEG−PLLA 21 gives an additional signal at 2θ angle of 16.9° for PLLA segment in the copolymer. PLLA and MePEG crystallinities are present in the copolymers 10 and 19. However, the peaks belonging to PLLA crystallites appeared much less intense due to the existence of ZNEtMG or NEtMG in copolymers 10 and 19 as compared with MePEG−PLLA 21. The intensity of crystallites decreased in the order MePEG 6 > MePEG−PLLA

Figure 7. Wide-angle X-ray diffraction (WAXD) patterns of compounds of 6, 21, 10, and 19.

21 > MePEG−poly(LLA-co-NEtMG) 19 ≥ MePEG−poly(LLA-co-ZNEtMG) 10. Degradation. Hydrolytic degradation study of copolymers 19 and 20 was performed according to molecular weight change during one month at pH: 7.4 in PBS at 37 °C. The formation of oligomeric species was observed for diblock 19 and triblock 20 copolymers due to random chain scissions (Figure 8). Also, their degradation performances were compared with well-known MePEG−PLLA 21, PLLA−PEG− PLLA 22 block copolymers, and PAGA homopolymer.35 The GPC curves in the degradation study in Figure 8 were analyzed by Origin 9 pro software, and the three identifiable peaks were J

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presence of p-toluenesulfonic acid monohydrate catalyst in toluene via removing water from the reaction medium with a Dean−Stark apparatus (Scheme 3). The synthesis of ZDNEtG 5 was completed in a short time like 5 h with the 58% yield. The proceeding of the reaction was monitored with thin layer chromatography (Rf: 0.4). Cyclization of α-hydroxyacid was a time-dependent reaction described previously by us.4 It should be carefully optimized because longer reaction times lead to form oligomeric species while shorter ones cause unreacted starting material in the reaction medium. For this reason, formation of monomer 5 was also followed with GPC (data were not shown). The full characterization of monomer 5 was performed with 1H NMR, 13C NMR, HMQC 2-D NMR, COSY 2-D NMR, ATR-FTIR, and LC/MS-TOF, as shown in Figures S4, S15, S24, S32, S33, and S39 of the Supporting Information. Then, the polymerization of monomer 5 with MePEG and lactide was attempted under the same polymerization conditions with monomer 4 (Scheme 3). Although the polymerization of disubstituted lactides catalyzed by Sn(Oct)2 has been previously reported by us and others,4−10,24 no polymerization of disubstituted monomer 5 with MePEG (Supporting Information, Figures S45 and S46, the molecular weight of MePEG was only observed) is probably due to the functional long side chain of monomer 5 having interactions with the catalyst preventing the polymerization of itself23 or high steric hindrance of bulky substituents in both sides, which is known to lessen the ΔG of the polymerization.23 These findings were consistent with the results of works performed by Cohen-Arazi et al.6 and Hall et al.,47 who reported that the polymerization of dilactones strongly depends on substitution in the ring. For example, the polymerization rate of lactide (−CH3 substituent) is slower than glycolide (−H substituent) probably due to the steric hindrance of the −CH3 groups that hinders nucleophilic attack on the carbonyl groups of the ring.6 More methyl substituent in the dilactone ring makes it hard to polymerize. Trimethyl glycolide was polymerized in very high temperature (180 °C) and long reaction time (24 h).48 Even more ring substitution on monomer, i.e., tetramethyl glycolide, may stop polymerization in the presence of Sn(Oct)2 catalysis.47 Therefore, an increase in the substituent’s bulkiness causes to decrease the polymerization rate. Thus, steric bulky groups in both sides in monomer 5 hindering nucleophilic attack at the carbonyl carbon in the ring led to no polymerization. That is why the

Figure 8. GPC chromatograms of copolymer 19 after (a) and before (b) degradation and copolymer 20 after (c) and before (d) degradation.

fitted by a Lorentzian formulation.49 The fractional areas of the three peaks at different degradation times were summarized. Both diblock 19 (47%, M n, P (L L A‑NEt MG) : 1180 and Mn,P(LLA‑NEtMG): 310) and triblock 20 (28%, Mn,P(LLA‑NEtMG): 1215 and Mn,P(LLA‑NEtMG): 320) copolymers (Figure 8) showed faster degradation than conventional MePEG−PLLA diblock 21 (0.6% degradation, Mn,PLLA: 920, data shown in Figure S50) and PLLA−PEG−PLLA triblock 22 (13.8% degradation, Mn,PLLA: 2190, data shown in Figure S50) copolymers by the end of a month, respectively. The poor hydrolytic degradability of MePEG−PLLA and PLLA−PEG−PLLA copolymers is likely due to being highly crystalline and hydrophobic nature of the PLLA chains. Thus, the faster degradation rate of synthesized novel functional copolymers that make them very useful in variety of biological applications like drug delivery or tissue scaffolding was due mainly to disruption of crystallinity by the NEtMG residues and hydrophilic amine groups in the copolymers.34 On the other hand, the copolymers 19 and 20 (17% and 19% of degradation in 30 min according to GPC analyses) showed slower degradation than PAGA homopolymer35 (interestingly, only 30 min for the intact polymer to halve according to MALDI analyses). The difference in degradation rate between PAGA and copolymers 19 and 20 was probably diversity in polymer structure (due to the existence of highly lactide units in copolymer); water penetration (i.e., the solution degradation) was studied in Park’s work,35 and the gel was studied in the current work. Mechanism. In order to elucidate the polymerization mechanism of ZNEtMG 4, we also synthesized symmetrical version of the monomer, CBZ protected 3,6-diaminoethyl-1,4dioxane-2,5-dione (ZDNEtG) 5, from compound 2 in the

Scheme 3. Synthesis of MePEG−Poly(LLA-co-ZDNEtG) Diblock Copolymers

K

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Macromolecules Scheme 4. Polymerization Mechanism of Asymmetrical Monomer 4 with MePEG and Lactide

functional group was found to be faster than those with free amine groups, and the copolymers with only L-lactide showed the slowest release. In this study, novel thermosensitive copolymers containing free amine groups can be injected at 42 °C and can be used to treat tumors and cancerous tissues in the local regions by forming gels at body temperature.

polymerization of ZNEtMG 4 probably continues from only methyl side as seen in Scheme 4. On the other hand, in monomer 4, the temporary coordination of oxygenes in carbonyl group of ZNEtMG 4 and in the end of MePEG homopolymer into metal alkoxide catalyst (Sn(Oct)2) increases the nucleophilicity of oxygen of the hydroxyl group which helps the cleavage of the bond between the carbonyl carbon and the endocyclic oxygen atom. Electrophilicity of the carbonyl group in ZNEtMG 4 also plays an important role in the cleavage during this stage. In following steps, ZNEtMG 4 continues to open and place in the metal−oxygen bond for the growth of copolymer chain.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.7b02751. Additional data of ATR-FTIR spectra, NMR spectra, LC/MS-TOF spectra, and GPC chromatograms (PDF)

4. CONCLUSION PEG-based polylactide and poly(lactide-co-glycolide) copolymers are among the most widely used thermosensitive biodegradable polymers. Synthesis, characterization, and various properties of these polymers have been extensively studied in the literature. On the other hand, there are very few studies on glycolide family polymers prepared by using glycolic acid derivatives as monomers, and new biomaterials that can undergo various modifications easily by means of free functional groups are highly needed. For this purpose, a new type of asymmetric substituted CBZ protected amine groups (ZNEtMG) was synthesized for the first time. PEG-based polymerization studies with L-lactide were carried out by the ring-opening reaction method in the presence of Sn(Oct)2 catalyst. New biodegradable diblock and triblock copolymers were obtained with different molar ratios of the monomers at various molecular weights, with the keeping amounts of initiator and catalyst constant. Removal of the CBZ protecting group after polymer synthesis was successfully carried out under H2 atmosphere in the presence of Pd/C (10%) catalyst. Suspensions of various concentrations of copolymers 10, 15, 19, and 20 were prepared, and optimal concentrations for each critical gel−sol temperature were determined. The copolymers 19 and 20 without protective groups were observed to have gel−sol transitions at lower temperatures. Twenty days of drug release studies were performed with using 1.0% of the anticancer drug loaded the copolymer gels after gel-to-sol transition temperatures and concentrations were determined. The drug release from the copolymers with protective amine

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AUTHOR INFORMATION

Corresponding Author

*(O.M.) E-mail [email protected]; Tel +902623032054; Fax +902623032003. ORCID

Olcay Mert: 0000-0002-5769-8994 Author Contributions

M.O.A. and S.E. contributed equally to this work. Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS This study was granted by TUBITAK, Turkey, with project number 112T865. REFERENCES

(1) Uhrich, K. E.; Cannizzaro, S. M.; Langer, R. S.; Shakesheff, K. M. Polymeric Systems for Controlled Drug Release. Chem. Rev. 1999, 99, 3181−3198. (2) Yu, Y.; Zou, J.; Cheng, C. Synthesis and biomedical applications of functional poly(α-hydroxyl acid)s. Polym. Chem. 2014, 5, 5854− 5872. (3) Lavik, E. B.; Hrkach, J. S.; Lotan, N.; Nazarov, R.; Langer, R. A Simple Synthetic Route to the Formation of a Block Copolymer of Poly(lactic-co-glycolic acid) and Polylysine for the Fabrication of Functionalized, Degradable Structures for Biomedical Applications. J. Biomed. Mater. Res. 2001, 58, 291−294.

L

DOI: 10.1021/acs.macromol.7b02751 Macromolecules XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.macromol.7b02751 Macromolecules XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.macromol.7b02751 Macromolecules XXXX, XXX, XXX−XXX