Technical Field

The invention relates to a new type of biodegradable and heat-sensitive copolymers prepared from amine substituted lactide and polyethylene glycol).

Prior Art

The controlled drug delivery systems are one of the most rapidly developing areas in medicine in order to make great contributions to human health care. Such delivery systems provide many benefits like enhanced efficacy, lower toxicity, and better patient compliance and convenience when compared to conventional ways. FDA approved poly(lactide-co-glycolide) (PLGA), polylactide (PLA), and polyglycolide (PGA) are the most popular of poly(a-hydroxy acid)s (or poly-(substituted glycolide)s due to their notable mechanical, physical and thermal characteristics. However, there is a still great need for the production of novel biomaterials which create an alternative to commonly studied polylactides because one of the major problems in conventional poly(a-hydroxy acid)s is the absence of reactive functional group along the polymer main backbone. On the other hand, when functional side groups like amines are introduced to the polymer backbone, it may help to control the hydrophilicity, degradation rate, and mechanical strength, and binding any biologically active compounds for further modulation. ¹ - ²

Amine-functionalized poly(a-hydroxy acid)s have increasingly gained much more attention in the scientific field due to its use in various applications. ^{2 6} Amine as an functional group helps to prepare cationic polylactides due to its basicity recently. ⁵ - ⁶ Polycondensation of Ne-cbz- _L -oxylysine with the elimination of water under low pressure yields poly[a-(4-aminobutyl)-L-glycolic acid] (PAGA) homopolymer that easily binds DNA via amine group for gene delivery applications. ⁵ PEG based cationic polylactides with tertiary amine-functionalization (CPLAs) were also obtained from allyl-functionalized lactide for gene delivery purposes. ⁶ The homopolymers of derivatives of serine, lysine, and glutamic acid containing amine, alcohol and carboxylic acid side chains together with their copolymers with lactide were synthesized in bulk conditions at 140 °C along one day for homo- and 8 hours for co-polymers. ³ Also, polyesters having pendant amino groups were obtained from the polymerization of O-carboxyanhydrides that had been synthesized from of lysine starting material. ⁴

The China patent document numbered CN101273965A of the prior art refers to a temperature-controlled sustained- release injection containing an anti-cancer drug and an amphiphilic block copolymer hydrogel. The injection form is flowable liquid in the environment that is lower than the body temperature and can be automatically converted to the water-insoluble gel that cannot flow. The drug is released through a tumor, therefore no systemic reaction of the drug to the rest of the body. The effective drug concentration is also maintained for a couple of weeks to months.

When the polymers of prior art were examined, requirement to develop a new type of biodegradable and heat- sensitive PEG based amine-functional polylactide copolymers have aroused.

Aims of the Invention

The aim of invention is to develop heat sensitive degradable copolymers, which has a new type of polylactide-PEG having amine functionality.

Detailed Description of the Invention

The results about the copolymers developed to achieve the aims of the invention are illustrated in the attached figures.

These Figures are; Figure 1 : A view of ¹ H NMR (a), ¹³ C NMR (b), HMQC 2-D NMR (c), COSY 2-D NMR (d) spectra of ZNEtMG II.

Figure 2: Proton NMR spectra of protected 10 (a) and deprotected diblock 19 (b), Carbon NMR spectrum of deprotected diblock 19 (c), and GPC chromatograms of MePEG 6, deprotected 19, and protected diblocks 9-11 . Figure 3: The gel to sol transition when temperature rise was applied (a, b, and c). Gel-sol curves of the copolymers 10, 15, 19, 20, and 21 (d). The graph of paclitaxel drug release from both diblock and triblock gels (e).

Figure 4: GPC chromatograms of copolymer 19 after (a), and before (b) degradation and copolymer 20 after (c) and before (d) degradation.

(S)-(+)-CBZ-4-amino-2-hydroxybutyric acid. To a sodium hydroxide (10.4 g, 0.26 mol) solution of (S)-(-)-4-amino-2- hydroxybutyric acid (14.8 g, 0.124 mol) in 100 mL of distilled water, benzyl chloroformate (CBZ-CI) (23.6 g, 0.136 mol) was added dropwise over 2 h in an ice/salt bath. The reaction was stirred for a further an hour at the same temperature. After this time, the reaction mixture was washed with 100 mL of diethyl ether, pH of the resulting solution was adjusted to 2 with diluted HCI, and then it was extracted four times with 100 mL of diethyl ether. The combined organic fractions were washed with saturated NaCI and dried over Na ₂ S0 ₄ . After removing the diethyl ether, the residue was recrystallized from benzene to afford (S)-(+)-CBZ-4-amino-2-hydroxybutyric acid as a white solid (78%)

¹ H NMR (de-DMSO, 500 MHz) d: 1.54-1.7 (m, 1 H), 1 .74-1 .92 (m, 1 H), 3.05-3.17 (distorted q, 2H), 3.92-4.03 (m, 1 H), 4.95-5.05 (s, 2H), 7.16-7.27 (t, J = 5.1 Hz, 1 H), 7.28-7.43 (m, 5H), 1 1.2-14.0 (br, 1 H). ¹³ C NMR (de-DMSO, 125 MHz) d: 34.1 , 37.1 , 65.1 , 67.6, 127.6, 127.7, 128.3, 137.2, 156.1 , 175.6.

CBZ Protected 3-Aminoethyl-6-methyl-1 ,4-dioxane-2,5-dione (ZNEtMG). CBZ protected asymmetric amine substituted lactide monomer was synthesized via two-step reaction sequence. In the first step, to a solution of (S)-(+)- CBZ-4-amino-2-hydroxybutyric acid (1.6 g, 6.32 mmol) in 80 mL of dichloromethane, 2-bromopropionyl bromide (0.84 mL, 8.06 mmol) was added at 0 °C under a nitrogen atmosphere. Triethylamine (1.45 mL) dissolved in 5 mL of dichloromethane was added dropwise over a period of 1 h to a vigorously stirred mixture in an ice/salt bath. The mixture was further stirred at 0 °C over 30 min. The proceeding of the reaction was followed with thin layer chromatography (TLC, silica gel, 60 F254, dichloromethane/methanol/acetic acid (10/1/0.5)), and TLC plate was treated with a ninhydrin solution (Re 0.69). The mixture was diluted with more dichloromethane, washed with deionized water (3 x 10 mL), and dried with anhydrous Na2S04. Dichloromethane was removed by rotary evaporation under reduced pressure to obtain a pale yellow viscous liquid 4-(((benzyloxy)carbonyl)-amino)-2-((2- bromopropanoyl)oxy)butanoic acid (intermediate) (70%). ²

¹ H NMR (CDCI3, 400 MHz) d: 1.68-1 .92 (m, 3H), 2.02-2.32 (m, 2H), 3.18-3.48 (m, 2H), 4.28-4.54 (m, 1 H), 4.98-5.12 (s, 2H), 5.12-5.2 (m, 1 H), 5.2-5.3 (br, 1 H), 7.22-7.44 (m, 5H), 8.5-9.3 (br, 1 H). ¹³ C NMR (CDCI3, 100 MHz) d: 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.

In the second step, to a vigorously stirred suspension of NaHC03 (2.1 g, 25 mmol) in 100 mL of N,N- dimethylformamide, a solution of intermediate (5.82 g, 15 mmol) in 40 mL of N,N-dimethylformamide was added dropwise at 40 °C over 4 h. The progress of the reaction was followed with thin layer chromatography (TLC, silica gel, 60 F254, hexane/ethyl acetate (2/1 )). The reaction was maintained for a further 3 h at the same temperature, and solvent was removed under reduced pressure to give a residue that was extracted with diethyl ether (50 mL). Obtained organic fraction was washed three times with 10 mL of deionized water and dried over Na ₂ S0 ₄ . Diethyl ether was evaporated, and pure form of benzyl (2-(5-methyl-3,6-dioxo-1 ,4-dioxan-2-yl)ethyl)carbamate (ZNEtMG) was obtained after two times recrystallization with diethyl ether (finally recovered yield after double recrystallizations is 67%). ^{2 1} H NMR (CDCI3, 500 MHz) d: 1 .5-1 .7 (d, J = 6.5 Hz, 3H), 1 .96-2.18 (m, 1 H), 2.24-2.48 (m, 1 H), 3.24-3.52 (distorted q, J = 6.1 Hz, 2H), 4.85-5.01 (q, J = 6.5 Hz, 1 H), 5.01 -5.06 (dd, J = 4.2, 7.5 Hz, 1 H), 5.06-5.14 (s, 2H), 5.18-5.3 (br, 1 H), 7.2-7.46 (m, 5H). ¹³ C NMR (CDC , 125 MHz) d: 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. LC/MS-TOF (CisHiyNOeNa): theoretical: 330.10 g/mol; experimental: 330.08 g/mol.

CBZ Protected 3,6-Diaminoethyl-1 ,4-dioxane-2,5-dione (ZDNEtG). Dibenzyl ((3,6-dioxo-1 ,4-dioxane-2,5- diyl)bis(ethane-2, 1 -diyl))dicarbamate (ZDNEtG) was synthesized with a modified protocol. ¹ (S)-(+)-CBZ-4-amino-2- hydroxybutyric acid (1 .5 g, 6 mmol) and p-toluenesulfonic acid monohydrate (30 mg, 0.15 mmol) were dissolved in 90 ml. of toluene. The solution was refluxed for 5 h in order to eliminate water with Dean-Stark apparatus. The progress of the reaction was followed with thin layer chromatography (TLC, silica gel, 60 F , hexane/ethyl acetate (3/1 )), and TLC plate was treated with a potassium permanganate (KMn04) solution (/=¾: 0.4). The toluene was removed, and the resulting residue was washed with cold toluene to eliminate impurities. Then, the obtained crystals were dissolved with ethyl acetate and recrystallized from hexane at -20 °C to afford pure CBZ protected 3,6-diaminoethyl-1 ,4- dioxane-2,5-dione (ZDNEtG) (58%). ²

¹ H NMR (CDCb, 400 MHz) d: 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). ¹³ C NMR (CDCb, 100 MHz) d: 27.1 , 42.1 , 68.4, 70.4, 128.3, 128.5, 128.6, 135.0, 151 .2, 174.4. LC/MS-TOF (C ₂₄ H ₂₆ N ₂ 0 ₈ Na): theoretical: 493.16 g/mol; experimental: 493.18 g/mol.

Amine-Functional Polylactide-PEG Copolymers. Syntheses of diblock and triblock copolymers were performed in bulk under a nitrogen atmosphere as described in the previously published work. ¹ Briefly, for the synthesis of MePEG- poly(LLA-co-ZNEtMG) (IV b), tin(ll) 2-ethylhexanoate (0.05 mmol, 20 mg), polyethylene glycol) methyl ether-2000 (0.12 mmol, 240 mg), L-lactide (1.8 mmol, 260 mg), and ZNEtMG (0.2 mmol, 62 mg) were placed into the polymerization tube, respectively. The reaction was performed at 120 °C for an hour. The copolymer product (IV b) was purificated by dissolving in methanol at 40 °C and then precipitated at -20 °C.

¹ H NMR (CDCb, 400 MHz) d: 1 .4-1 .7 (d, J = 6.9 Hz, 3H; m, 3H), 2.0-2.4 (m, 2H), 3.35-3.39 (br, 1 H; s, 3H), 3.42-3.49 (m, 1 H), 3.51 -3.56 (m, 1 H), 3.57-3.75 (s, 4H), 5.0-5.5 (q, J = 6.7 Hz, 1 H; m, 1 H; m, 1 H; m, 2H), 7.28-7.46 (m, 5H). ¹³ C NMR (CDCb, 100 MHz) d: 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.

Syntheses of the other copolymers were performed with the similar conditions as MePEG-poly(LLA-co-ZNEtMG) (IV b) but the PEG homopolymer having two hydroxyl groups was used instead of MePEG homopolymer having a methoxy group on one end and a hydroxyl group on the other end for triblock copolymer syntheses.

Deprotection of copolymers was performed by catalytic hydrogenolysis. To a solution of protected copolymers IV b and VI b (100 mg) in 8 mL of dichloromethane, 50 mg of palladium 10% on carbon (Pd/C (10%)) was added. First, hydrogen gas (balloon) was passed from the system to remove the air. Catalytic hydrogenation was carried out for seven days with vigorously stirring at room temperature under a hydrogen atmosphere. The solution was filtrated over celite to remove the Pd/C (10%) from the reaction medium followed by removing of dichloromethane to obtain the deprotected copolymers V and VII. ²

Gel-Sol Phase Transition. Gel-sol transition temperatures of copolymers were examined according to the procedures previously reported. ¹ Gels at given concentrations were obtained from copolymers and deionized water in 1 .5 mL vials. The gel-to-sol transition temperature of the copolymers was determined by inverting the vials at different temperatures using a controlled water bath. Firstly, 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 determined from 4 to 80 °C with 2 °C increments. The vials were kept in water bath for 3-4 min. at each temperature before tilting.

Hydrolytic degradation of block copolymers was carried out in phosphate buffered saline (PBS, pH 7.4) at physiological conditions (37 °C, 200 rpm). About 15 mg of polymer was immersed into 5 mL of PBS in the test tubes followed by incubation. Samples were taken out at different time periods, 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 tetrahydrofuran for GPC analyses. The percentage of degradation products was determined by Lorentzian formulation. ²

In vitro drug release studies were performed for poly(LLA-co-ZNEtMG)-PEG, poly(LLA-co-NEtMG)-PEG and PLLA-PEG block copolymers according to the same methodology that we described previously. ¹ The anticancer drug paclitaxel was loaded into copolymer gels effectively with loading ratio of 1.0%. Briefly, for the preparation of MePEG-poly(LLA-co-ZNEtMG) diblock copolymer IV b hydrogel, paclitaxel (1.17 mg), compound IV b (1 17 mg), and deionized water (233 mI_) were added into the 1 .5 mL vial, and the sample was vortexed at room temperature for 5 min to get homogeneous drug-loaded hydrogel. Then drug-loaded hydrogels were kept at 4 °C for 30 min for equilibrium. Other drug-loaded copolymer hydrogels were prepared in the same manner as shown in Table 1. Then, 650 pL of 2% Tween 80 in PBS buffer (pH 7.4) was added to the surface of the drug-loaded hydrogels at room temperature for release experiments. These samples were kept in incubator at 37 °C with a constant speed of 200 rpm. At different time intervals, 650 pL 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. ¹ - ²

Table 1. Gel Preparation with Paclitaxel Drug at 1% Drug Loading Ratio

CBZ protected 3-aminoethyl-6-methyl-1 ,4-dioxane-2,5-dione (ZNEtMG) (Formula II) was synthesized via two-step reaction sequence: the formation of halogenated carboxylic acid intermediate from (S)-(+)-CBZ-4-amino-2- hydroxybutyric acid with 2-bromopropionyl bromide in dichloromethane (DCM) at 0 °C and cyclocondensation reaction of the intermediate with NaHCC>3 in dimethylformamide at 40 °C (Scheme 1). According to NMR analyses, intermediate yielded a 1 :1 mixture of diastereomers (R,S and S,S) while a stereoselectivity was observed for final monomer (Formula II) (R,S or S,S). Overall synthetic yield of functional lactide monomer II and III starting from commercially available (S)-(+)-CBZ-4-amino-2-hydroxybutyric acid 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. ¹

Scheme 1 :

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 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 was performed by ¹ H and ¹³ C NMR techniques. Two new resonances at 1.68-1 .92 ppm (CH3) and 4.28-4.54 ppm (CH) in the ¹ H 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 ¹³ C NMR spectrum of intermediate when compared to starting material. Moreover, the shifting of the CH proton and carbon in starting material from 3.92-4.03 and 67.6 ppm to 5.12-5.2 and 70.9-71.0 ppm in ¹ H and ¹³ C NMR proved the formation of intermediate, respectively. ZNEtMG II monomer was analyzed by ¹ H, 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 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 II in ¹ H and ¹³ C NMR, respectively (Figure 1 a, 1 b). The formation of ZNEtMG II was also observed in ¹³ C NMR by the shifting from 173.2 to 174.6 ppm (acid carbonyl group of intermediate) to 167.3 ppm (ester carbonyl group of ZNEtMG II) (Figure l b).

The HMQC 2-D NMR spectrum was recorded to indicate the direct proton-carbon shift correlation in monomer II. It has been proven from HMQC 2-D NMR spectrum that a-coded peaks are bound to CH3 carbon and protons; bi 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 I, m and n coded peaks are also the peaks of carbonyl groups (Figure l c). In the COSY 2-D NMR spectrum of the compound II shown in Figure 1 d, the interactions of the neighboring protons which indicate spin-spin coupling interactions between the correlated nuclei in the structure II 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 connectivity’s 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”,“bi , b” with“c and e”,“c” with“bi, b ₂ , and g”,“e” with“bi, b ₂ ”, and“bT’ with“b ₂ ” in ZNEtMG II. There is no cross-peak for“k, I, m, and n” because it does not possess any hydrogens. Table 2. Conditions and characterization of the diblock and triblock copolymers

Conditions for synthesis of copolymers Characterization of diblock and triblock copolymers

LLA-ZNEtMG

RU ^b RU ^b of

ID Copolymer |_LA ZNEtMG Feed Mw ^a Mn ^a Mn ^b Conv.

Mw/Mn ^a Of

Ratio (g/mol) (g/mol) (g/mol) % ^b ZNEtMG (mmol) (mmol) LLA

(%)

IV a 0.9 0.1 90/10 4290 3880 3270 3340 1.10 96 0.9 7 IV b 1 .8 0.2 90/10 5180 4240 4460 4670 1.22 92 1.6 13 IV c Diblock 2.7 0.3 90/10 5850 4850 5780 6000 1.21 91 2.4 19 IV d 2.55 0.45 85/15 4870 3840 6210 1.27

IV e 2.25 0.75 75/25 4710 3780 6000 1.25

VI a 0.9 0.1 90/10 4780 4510 2990 3340 1.06 90 0.7 5 VI b Triblock 1.8 0.2 90/10 5830 4640 4290 4670 1.26 92 1.6 13 VI c 2.7 0.3 90/10 6910 5750 5310 6000 1.20 91 2.2 18 ^a Determined by GPC, ^b Determined by ¹ H NMR spectrum (The calculation of conversion was performed using the signal from the CH (d 4.85-5.01 , 5.01-5.06 ppm) of the unreacted monomer, and the CH (d 5.0-5.5 ppm) of the polymer), RU: Repeating unit.

Moles of MePEG/PEG and Sn(Oct are 0.12 mmol and 0.05 mmol, respectively.

All polymerization reactions were performed at 120 °C for an hour under a nitrogen atmosphere.

MePEG-poly(LLA-co-ZNEtMG) diblock and poly(LLA-co-ZNEtMG)-PEG-poly(LLA-co-ZNEtMG) triblock copolymers were synthesized via bulk polymerization using monomers of ZNEtMG II and L-lactide and the initiators of terminal MePEG or PEG in the presence of stannous octoate at 120 °C, as shown in Scheme 2. Catalytic hydrogenolysis was performed to remove benzyl protective groups in order to obtain free amine functional groups in copolymers V and

VII. The specific moles of hydrophobic monomers were used to obtain desired molecular weights of copolymers, especially for less than 10K for sol-gel experiments, while maintaining a constant mole ratio of PEG or MePEG, as indicated in Table 2. The intensities of the methine proton signal of polymers at d = 5.0-5.5 ppm, the phenyl proton signal of ZNEtMG unit at d = 7.28-7.46 ppm, and methylene proton signal of MePEG or PEG at d = 3.57- 3.75 ppm in proton NMR of copolymer IV b were compared to determine the repeating unit of the ZNEtMG and LLA in both di- and tri-block copolymers in Figure 2a.

Scheme 2:

-CH3 protons (a, b) of poly(LLA-ZNEtMG) block, -CH2- protons (c) of ZNEtMG, both amine protons (d) of ZNEtMG and -OCH3 protons (e) at the end of the MePEG block, methylene protons (f) of ZNEtMG, -CH- protons (h, i, j) of poly(LLA-ZNEtMG) and methylene protons (k) of protected group (-O-CH2-C6H5), and aromatic protons (I, m, n) gave resonances 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, respectively.

-CH - protons (g) in methoxy polyethylene glycol) moiety of the copolymer gave a singlet signal at 3.57-3.75 ppm. Copolymer formation was proved by new signals at 4.18-4.42 ppm of -CH - protons of LLA-ZNEtMG units connected to the MePEG block (poly(LLA-co-ZNEtMG)-COO-CH -CH -) and the -CH- proton neighboring the -OH end-group (-COO-CH(CH CH NHCBZ)OH), as shown in Figure 2a. The polymerization of ZNEtMG monomer was verified by the appearance of a methine protons peak at 5.0-5.5 ppm in ¹ H NMR spectrum, too. Moreover, high conversions in copolymers (>90%) were confirmed from the ¹ H NMR via comparing the integrations of polymer and monomer peaks (Table 2). Some low quantity of heterotacticity was observed in ¹³ C-NMR spectrum of compound IV b, and there are resonance peaks at 16.6 (CH ), 69.0 (CH), 169.6 (C=0) for lactide units, 70.5 (CH ) for MePEG, and 20.5 (CH ₃ ), 30.7 (CH -CH -NH), 36.7 (CH -CH -NH), 66.7 (-CH C H ), 68.8 (CH-CH ), 69.2 (CH-CH -), 128.1 -134.9 (aromatic ring), 156.4 (-NHCOO-), 169.5 (-COCHCH ) and 169.7 (-COCHCH -) for the ZNEtMG unit, and the end units, the neighboring units to the end. The partial deprotection (—60%) and significant degradation were observed when the compound IV b was reacted with 33% HBr/AcOH. No backbone degradation and fully removal of the protected group by the disappearance of phenyl protons and carbons at 7.28-7.46 and 128.1 -134.9 ppm were succeeded in presence of hydrogen gas and Pd/C catalyst according to ¹ H and ¹³ C NMR, respectively (Figure 2b, 2c). No chain scission of the deprotected copolymer V was also confirmed from GPC chromatograms under the latter conditions (Figure 2d). Monomodal peaks, narrow PDI values (1.06 to 1.26), and expected molecular weights distribution for copolymers were observed according to GPC analyses (Table 2). On the other hand, feed ratio of LLA-ZNEtMG was selected as 90/10% for the syntheses of copolymers IV d and IV e because some homopolymerization was observed at higher feed ratios of ZNEtMG (Table 2). More bulky substituents in ZNEtMG than the one in L-lactide caused a lower reactivity of copolymerization. The fact that MePEG peak was not detected on the GPC traces of copolymers, as indicated in Figure 2d, proved effective syntheses of desired copolymers.

The determination of gel-sol transition temperatures were carried out by the changing of the concentration of all of PEG based copolymers IV b, VI b, V, VII, MePEG-PLLA, and PLLA-PEG-PLLA. ² Our aim was especially to prepare suitable gels that exhibit a fluidic character at around 42 °C for injection, and then a gel with rapid cooling to 37 °C. Suitable critical gel-sol transition temperature were 44 °C and 42 °C at 33.5% for both MePEG-poly(LLA-co- ZNEtMG) (IV b) and MePEG-poly-(LLA-co-NEtMG) (V), and 46 °C and 42 °C at 33% for poly(LLA-co- ZNEtMG)-PEG-poly(LLA-co-ZNEtMG) (VI b) and poly(LLA-co-NEtMG)-PEG-poly(LLA-co-NEtMG) (VII), respectively. Obtained results showed that the gel-sol transition temperatures were relatively lower for copolymers V and VII because of removal of the hydrophobic CBZ protected group. Higher gel-sol temperatures for triblock copolymers VI b and VII than diblock ones IV b and V were observed at 33% concentration. MePEG-PLLA was also synthesized for the comparison of gel-sol temperatures of copolymers IV b and V. The desired gel-sol temperature (40 °C) was obtained at lower concentrations for MePEG-PLLA. More hydrophobic nature of MePEG-PLLA than copolymer IV b and V caused higher gel-sol temperature at 33% concentration (Figure 3 d).

Non-homogenous suspensions even at concentrations as low as 10% were observed if copolymers were prepared with a higher mole of hydrophobic monomer at a constant PEG ratio (i.e., copolymer IV c). Therefore, the gel-sol experiments were performed with the polymers prepared from 2 mole of ester content. Finally, a gel was obtained from the aqueous polymer solutions at levels of higher concentrations while a fluid form was observed at lower concentrations and higher values of temperature (Figure 3 d).

The drug delivery from MePEG-poly(LLA-co-ZNEtMG) diblock IV b, poly(LLA-co-ZNEtMG)-PEG-poly(LLA-co- ZNEtMG) triblock VI b, MePEG-poly(LLA-co-NEtMG) diblock V, and poly(LLA-co-NEtMG)-PEG-poly(LLA-co- NEtMG) triblock VII gels were monitored by HPLC over 20 days at 37 °C. Their performances were evaluated with MePEG-PLLA diblock and PLLA-PEG-PLLA triblock copolymers. Initial burst releases of 15% to 23% were observed for copolymers IV b, VI b, V, and VII in 48 h. On the other hand, there were about 6.5% and 13.5% burst releases for PLLA-PEG copolymers diblock and triblock, respectively.

When the release profiles of copolymers having CBZ group IV b and VI b was compared with the copolymers without CBZ group V and VII according to chain lengths and the effect of groups, the reason for highest release rate for copolymers with CBZ group IV b and VI b is that they are not close-packed since they get longer side chains.

The hydrophilicity (2) of the copolymer V and VII also leads to faster drug release because the higher hydrophobic character in MePEG-PLLA and PLLA-PEG-PLLA causes a lower release due to the hydrophobic-hydrophobic interactions between the hydrophobic block and the drug. The limiting factor that prevents release in the first stage is the restricted water absorption by hydrophobic unfunctionalized PLA copolymers. Functional copolymers (Figure 3 e) displayed remarkable improvement in drug release during the period of 3 weeks. As a result, paclitaxel anticancer drug release was increased notably; after 20 days, a cumulative 72%-95% drug in the copolymers IV b, VI b, V, and VII was released as opposed to only 28%-29% in pure PLA-PEG-PTX matrix.

Hydrolytic degradation work of copolymers V and VII was carried out according to molecular weight change during a month at pH: 7.4 in PBS at 37 °C. The formation of oligomeric species was observed for diblock V and triblock VII copolymers because of random chain scissions. Also, their degradation performances were compared with well- known MePEG-PLLA, PLLA-PEG-PLLA block copolymers, and PAGA homopolymer. ⁵ The gel permeation chromatograms in the degradation work in Figure 4 were evaluated.

The poor hydrolytic degradability of MePEG-PLLA and PLLA-PEG-PLLA copolymers is probably due to being highly crystalline and hydrophobic nature of the polylactide chains. Thus, the faster degradation rate of synthesized novel functional copolymers that make them very useful in many biological applications like drug delivery systems or tissue scaffolding was due mainly to disruption of crystallinity by the NEtMG residues and hydrophilic amine groups in the copolymers.

In order to explain the polymerization mechanism of ZNEtMG II, we also performed the synthesis of symmetrical version of the monomer, CBZ protected 3,6-diaminoethyl-1 ,4-dioxane-2,5-dione (ZDNEtG) III, from (S)-(+)-CBZ-4- amino-2-hydroxybutyric acid in the presence of PTSA catalyst in toluene via removal of water from the reaction medium with a Dean-Stark apparatus (Scheme 3). The synthesis of ZDNEtG III was completed in a short time like 5 hours with the 58% yield. The proceeding of the reaction was followed with TLC (/¾: 0.4). Cyclization of a-hydroxy acid was a time-dependent reaction studied previously by us. ¹ It should be carefully optimized because longer reaction times lead to form oligomeric species when shorter ones cause unreacted starting material in the reaction medium. For this reason, formation of monomer III was also followed with gel permeation chromatography (data not shown). The full characterization of monomer III was carried out ¹ H NMR, ¹³ C NMR, HMQC 2-D NMR, COSY 2-D NMR, and LC/MS-TOF.

Scheme 3:

MePEG-Poly(LLA-co-ZDNEtG) (VIII) CBZ protected 3,6-diaminoethyl-1 ,4-dioxane-2,5-dione (ZDNEtG) (III)

Although the polymerization of disubstituted lactides catalyzed by stannous octoate has been previously reported by us and others, ¹ · ³ no polymerization of disubstituted monomer III with MePEG is probably due to the functional long side chain of monomer III having interactions with the catalyst preventing the polymerization of itself ³ or high steric hindrance of bulky substituents in both sides, which is known to lessen the AG of the polymerization. ³ These findings were consistent with the data obtained by Cohen-Arazi et al. and Hall et al., who reported that the polymerization of dilactones strongly depends on substitution in the ring. ²

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