Thermosensitive Biodegradable Homopolymer of Trimethylene

Mar 19, 2012 - ... School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, ... the direct connection of OEG into trimethylene carbonate (TMC),...
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Thermosensitive Biodegradable Homopolymer of Trimethylene Carbonate Derivative at Body Temperature Hiroharu Ajiro,†,‡ Yoshikazu Takahashi,‡ and Mitsuru Akashi*,†,‡ †

The Center for Advanced Medical Engineering and Informatics, Osaka University, 2-2, Yamada-oka, Suita, Osaka 565-0871, Japan Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan

‡

S Supporting Information *

ABSTRACT: A thermosensitive biodegradable homopolymer with a lower critical solution temperature (LCST) at body temperature was developed, with a poly(trimethylene carbonate) (PTMC) backbone and oligoethylene glycol (OEG). The novel monomer was designed by the direct connection of OEG into trimethylene carbonate (TMC), and no other functional groups exist in the polymer structure. Such a welldefined monomer unit contributed to its homogeneous characteristics. Three units of ethylene glycol in the TMC derivative resulted in a water-soluble nature at room temperature, and the solution became cloudy at higher temperatures. The LCST ranged from 31 to 35 °C and was influenced by the molecular weight and polymer concentration. Four units of ethylene glycol, however, increased the LCST temperature to 72 °C. It is noteworthy that the present characteristics, thermosensitivity at body temperature, biodegradablility, and a well-defined homopolymer structure, are promising for biomedical applications as an essential material.

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dantly employed, combined with poly(organophosphazenes),22 polyacrylate derivatives,23 and poly(3-hydroxyalkanoate),24 as well as other original chemical structures like poly(amidoamine) dendrimers,25 poly[(vinylamine)-co-N-vinylisobutyramide],26 and poly[(N-vinylamide)-co-(vinyl acetate)].27 Some of these copolymers are fully biodegradable for the purposes of biomedical applications, such as poly(lactide)-copoly(ethylene glycol).28 On the other hand, aliphatic polycarbonate has received attention as a biodegradable polymer, and novel polymers have been developed. For example, the alternating copolymerization of limonene oxide and carbon dioxide has been achieved with a specific single site catalyst.29 PTMC is also known as a biodegradable synthetic polymer and has been actually used as a biomedical material in suture thread, taking advantage of its soft characteristics.30−32 Recently, research on novel PTMC derivatives and copolymers for biomaterial use has attracted attention, due to the organic catalyst system reported by Waymouth, Hedrick, and co-workers in 2007.33,34 Furthermore, micelle35 and polymersome36,37 preparations have predominantly been investigated as drug delivery systems by using copolymers with PTMC and hydrophilic polymers or by using trimethylene carbonate (TMC) derivatives with a protected carboxylic acid group at the side chain, such as 5-methyl-5benzyloxycarbonyl-1,3-dioxan-2-one38−40 and 3-chloropropyl 5methyl-2-oxo-1,3-dioxane-5-carboxylate.41 Moreover, temperature responsiveness was achieved with PTMC derivatives by

INTRODUCTION A large number of studies have been performed on thermosensitive synthetic polymers. Poly(N-isopropylacrylamide) (PNIPAm)1,2 is one of the most intensively studied polymers3−5 and possesses a LCST at 32 °C. PNIPAm was widely examined as a biomaterial6,7 because its LCST was near body temperature. Other than PNIPAm and its derivatives, poly(N-vinylisobutylamide) (PNVIBA),8,9 a poly(methyl methacrylate) (PMMA) derivative with oligoethylene glycol (OEG),10 and a poly(vinyl ether) derivative with OEG11−14 have been reported as thermosensitive synthetic homopolymers. These polymers exhibit two main features: a “welldefined structure” due to the homopolymer with clear monomer units and a “non-biodegradable characteristic” with the responsive temperature near body temperature. Referring to PNIPAm, many applications would be expected once any novel biodegradable homopolymer possessing a LCST at body temperature is discovered because the precise structure would usually have well-defined characteristics. However, to our knowledge, there is no such synthetic polymer thus far probably because it is necessary to have very a fine balance between hydrophilicity and hydrophobicity in the monomer structure. In general, copolymerization approaches with a combination of hydrophobic and hydrophilic moieties have been developed for thermosensitive polymers as a material, such as various copolymers of PNIPAM using poly(trimethylene carbonate) (PTMC),15 poly(methacrylic acid), 16 poly(propylacrylic acid),17 and poly(vinylpyrrolidone),18 and by introducing different moieties using radical polyaddition19 or macromonomer initiation20 approaches. Except for PNIPAM copolymers, poly(ethylene glycol) (PEG)21 has been abun© 2012 American Chemical Society

Received: January 24, 2012 Revised: March 6, 2012 Published: March 19, 2012 2668

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introducing a PEG domain into the block copolymer42,43 and into the side chain along the polymer main chain.44 Such thermosensitive PTMC derivatives are based on the copolymer structure, bearing both hydrophilic and hydrophobic regions in the polymer main chain separately (Figure 1a). Their polymer

macroinitiator,35,42,43,45 polymer reaction,38−41 and monomer synthesis.40,44,46−48 In this study, novel TMC derivatives, composed of different units of OEG groups directly connected into the TMC backbone were synthesized, and the turbidities of their polymers in water against temperature were examined (Scheme 1). In these screening tests, we discovered a novel thermosensitive biodegradable homopolymer that possessed a LCST at 33 °C. Herein we report this series of TMC derivatives and their polymerization (Figure 1c).

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EXPERIMENTAL SECTION

Materials. Methoxyethanol, triethylene glycol monomethyl ether, tetraethylene glycol monomethyl ether, p-toluenesulfonyl chloride, benzaldehyde, p-toluenesulfonic acid, 2-methyoxyethyl p-toluenesulfonate, sodium hydride in oil (20 wt %), 1,1′-carbonyldiimidazole (CDI), benzyl alcohol, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and trimethylolethane were purchased from Tokyo Chemical Industry Co., Ltd., Japan. Anhydrous dimethylformamide (DMF) was purchased from Aldrich. Tetrahydrofuran (THF) and dichloromethane (CH2Cl2) for synthesis and polymerization solvents were distilled with calcium hydride before use. Tosylation of Oligoethylene Glycol Monomethyl Ether. The tosylation of oligoethylene glycol monomethyl ether was performed according to previous reports.49 The typical procedure was described for 2-{2-(2-methoxyethoxy)ethoxy}ethyl tosylate as follows: 21 mL of triethylene glycol monomethyl ether (131 mmol) was dissolved in an aqueous NaOH solution (5 mol/L, 40 mL) and kept at 0 °C. A ptoluenesulfonyl chloride (25 g, 131 mmol) solution in THF (120 mL) was slowly added into the mixture and stirred for 22 h. The reaction mixture was extracted with CH2Cl2 and NaCl(aq) to recover the 2-{2(2-methoxyethoxy)ethoxy}ethyl tosyleate (40.2 g, yield 96%). The same procedure for the synthesis of 2-[2-{2-(2-methoxyethoxy)ethoxy}ethoxy]ethyl tosylate as per 2-{2-(2-methoxyethoxy)ethoxy}ethyl tosyleate was carried out, except that tetraethylene glycol monomethyl ether was used instead of triethylene glycol monomethyl ether (40.1 g, yield 92%) Synthesis of 5-Hydroxymethyl-5-methyl-2-phenyl-1,3-dioxane (HMPD). Protection of the diol moiety of trimethylolethane was achieved according to previous reports.48 Trimethylolethane (24.7 g, 205 mmol) was added into anhydrous THF (480 mL) in the flask and stirred to dissolve. Benzaldehyde (25 mL, 247 mmol) was slowly introduced by a dropping funnel, and the mixture was stirred for 16 h at room temperature. The reaction was stopped by adding 0.38 mL of aqueous ammonium solution (28%) for neutralization. The reaction

Figure 1. Schematic illustration and chemical structure of polymers. The model of block copolymer with hydrophilic and hydrophobic domains (a), the model of homopolymer composed of hydrophilic and hydrophobic parts (b), and the chemical structures of novel trimethylene carbonate derivatives and their polymerization (c).

designs are excellent for the creation of nanomaterials, such as micelle and lamellar structures, which need domain separation. However, we were interested in the synthesis of a novel thermosensitive biodegradable homopolymer as an essential material (Figure 1b). Thus, we chose the monomer design from three possible strategies to modify PTMC, which are a

Scheme 1. Syntheses of Novel Trimethylene Carbonate Derivatives

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Synthesis of 5-(2-Methoxyethyoxymethyl)-5-methyl-1,3dioxa-2-one (TMCM-MOE1OM). Under a nitrogen atmosphere, 6.46 g (36.3 mmol) of Diol-OEG1 was placed in a three-necked flask and dissolved in anhydrous CH2Cl2 (25 mL) with molecular sieve 4A (MS4A) powder and stirring at room temperature for 3 h. A 160 mL aliquot of 0.4 M CDI solution in CH2Cl2 (40 mmol) was introduced into the mixture, and stirring was maintained at room temperature for 20 h. After filtration and evaporation, the crude mixture was purified by silicagel chromatography to obtain 3.9 g of the novel monomer TMCM-MOE1OM (19.1 mmol, yield 53%). 1H NMR (CDCl3, 400 MHz) δ: 1.10 (s, 3H, CH3C), 3.37 (s, 3H, CH3O), 3.45 (s, 2H, OCH2CCH3), 3.52−3.54 (m, 2H, OCH2CH2O), 3.60−3.62 (m, 2H, OCH2CH2O), 4.08 (d, 2H, J = 10.9 Hz, OCOCH2), 4.36 (d, 2H, J = 10.9 Hz, OCOCH2). FT-IR (cm−1): 2875, 1745, 1473, 1401, 1175, 1107, 1090. Anal. Calcd for C9H16O5: C, 52.93; H, 7.90; O, 39.17. Found: C, 52.78; H, 7.72, O, 39.50. CI-MS: m/z = 205 ([M + 1]+). Synthesis of 5-[2-{2-(2-Methoxyethoxy)ethyoxy}ethoxymethyl]-5-methyl-1,3-dioxa-2-one (TMCM-MOE3OM). The same procedure as per the TMCM-MOE1OM synthesis was repeated, except that Diol-OEG3 (10.5 g, 37.4 mmol) was used instead of Diol-OEG1. A total of 3.84 g of the novel monomer TMCMMOE3OM was obtained (12.5 mmol, yield 34%). 1H NMR (CDCl3, 400 MHz) δ: 0.94 (s, 3H, CH3C), 3.38 (s, 3H, CH3O), 3.45 (s, 2H, OCH2CCH3), 3.54−3.66 (m, 12H, OCH2CH2O), 4.07 (d, 2H, J = 10.8 Hz, OCOCH2), 4.36 (d, 2H, J = 10.8 Hz, OCOCH2). FT-IR (cm−1): 2871, 1748, 1472, 1401, 1174, 1105. Anal. Calcd for C13H24O7: C, 53.42; H, 8.22; O, 38.36. Found: C, 52.94; H, 9.05, O, 38.01. CI-MS: m/z = 293 ([M + 1]+). Synthesis of 5-(2-[2-{2-(2-Methoxyethoxy)ethyoxy}ethoxy]ethyoxymethyl)-5-methyl-1,3-dioxa-2-one (TMCM-MOE4OM). The same procedure as per the TMCM-MOE1OM synthesis was repeated, except that Diol-OEG4 (7.30 g, 23.5 mmol) was used instead of Diol-OEG1. A total of 1.29 g of the novel monomer TMCMMOE4OM was obtained (3.76 mmol, yield 16%). 1H NMR (CDCl3, 400 MHz) δ: 1.09 (s, 3H, CH3C), 3.38 (s, 3H, CH3O), 3.45 (s, 2H, OCH2CCH3), 3.54−3.66 (m, 16H, OCH2CH2O), 4.07 (d, 2H, J = 10.8 Hz, OCOCH2), 4.36 (d, 2H, J = 10.8 Hz, OCOCH2). FT-IR (cm−1): 2870,1747, 1472, 1402, 1176, 1104. Anal. Calcd for C15H28O8: C, 53.57; H, 8.33; O, 38.10. Found: C, 52.66; H, 8.48, O, 38.86. CIMS: m/z = 337 ([M + 1]+). Polymerization. Polymerization of TMC derivatives were achieved by the literature.33 A typical polymerization procedure was described for TMCM-MOE1OM. In the three-necked flask, 0.5 g of TMCM-MOE1OM (2.45 mmol) was dissolved in about 5 mL of anhydrous CH2Cl2 with CaH2 to stir for overnight. Using a cannula with glass filter to remove CaH2, the monomer solution was transferred to the other flask with a three-way cock, and the solvent CH2Cl2 was evaporated under reduced pressure. Then, the required amount of anhydrous CH2Cl2 under nitrogen atmosphere was introduced. Into the monomer solution, 0.6 mL of benzyl alcohol (0.049 mmol) solution in CH2Cl2 as initiator and 0.6 mL of DBU (0.049 mmol) solution in CH2Cl2 as catalyst were added to start the polymerization at room temperature for 8 h. The reaction was stop by adding small portion of acetic acid, and then the reaction mixture was poured into a large amount of hexane/2-propanol (9/1, v/v). The product was recovered by decantation and centrifugation and dried under vacuum (73% yield). Measurements. 1H NMR and 13C NMR spectra were measured by a JEOL JNM-GSX400 system. Attenuated total reflection (ATR) IR spectra were obtained with a Spectrum 100 FT-IR spectrometer (Perkin-Elmer). The interferograms were coadded 64 times and Fourier-transformed at a resolution of 4 cm−1. The number-average molecular weights and their distribution were measured by gel permeation chromatography. A Tosoh System HLC-8120GPC was used with PMMA standards at 40 °C. Two commercial columns (TSKgel SuperH4000 and TSKgel GMHXL) were connected in series and tetrahydrofuran was used as an eluent. The mass spectra were measured on a JEOL JSM-700 mass spectrometer for novel monomers. MALDI-TOF/MS were measured with a Bruker Autoflex

mixture was evaporated to remove the THF and extracted by CH2Cl2 and NaCl(aq) to collect the organic layer (32.2 g, yield 75%). Synthesis of 5-(2-Methoxyethoxymethyl)-5-methyl-2-phenyl-1,3-dioxane (MPD-OEG1). Sixty percent sodium hydride in oil (5.03 g, 126 mmol) was placed in a three-necked flask under nitrogen and washed by anhydrous THF (10 mL) three times. Anhydrous DMF (14 mL) and anhydrous THF (70 mL) were combined in the flask, and HMPD (20.5 g, 98.5 mmol) was introduced with stirring at 60 °C for 5 h. Next, 2-methyoxyethyl p-toluenesulfonate (25 g, 109 mmol) was introduced under a nitrogen atmosphere with stirring at 60 °C for 14 h. The reaction mixture was then extracted by CH2Cl2 and NaCl(aq) to collect the organic layer. The crude mixture was purified by silica gel column chromatography to obtain 18.7 g of MPD-OEG1 (70.3 mmol, yield 71%). 1H NMR (CDCl3, 400 MHz) δ:0.82 (s, 3H, CH3C), 3.39 (s,3H, CH3O), 3.55−3.58 (m, 2H, OCH2CH2O), 3.61 (d, 2H, J = 11.7 Hz, CCH2OCPh), 3.66−3.68 (m, 2H, OCH2CH2O), 3.72 (s, 2H, OCH2C), 4.06 (d, 2H, J = 11.7 Hz, CCH2OCPh), 5.42 (s, 1H, CHPh), 7.36 −7.48 (m, 5H, Ph). FT-IR (cm−1): 2850, 1454, 1387, 1358, 1311, 1208, 1095. CI-MS: m/z = 267 ([M + 1]+). S y n t h es is of 5 - [ 2 -{ 2 - ( 2 -M e t h o x y e t h o x y ) e t h o x y } ethoxymethyl]-5-methyl-2-phenyl-1,3-dioxane (MPD-OEG3). The same procedure as per the MPD-OEG1 synthesis was repeated, except that 2-{2-(2-methoxyethoxy)ethoxy}ethyl tosyleate (20.0 g, 60.2 mmol) was used instead of 2-methyoxyethyl p-toluenesulfonate. The crude mixture was purified by silica gel column chromatography using hexane/ethyl acetate (1/2, v/v). A total of 19.3 g of MPD-OEG3 was obtained at Rf = 0.62 (52.4 mmol, yield 87%). 1H NMR (CDCl3, 400 MHz) δ: 0.81 (s, 3H, CH3C), 3.37 (s, 3H, CH3O), 3.53−3.71 (m, 16H, OCH2H2O, OCH2CCH3), 4.05 (d, 2H, J = 11.7 Hz, OCH2CCH3), 5.41 (s, 1H, CHPh), 7.33−7.49 (m, 5H, Ph). FT-IR (cm−1): 2860, 1454, 1387, 1208, 1097, 1025. FAB-MS: m/z = 355 ([M + 1]+). Synthesis of 5-(2-[2-{2-(2-Methoxyethoxy)ethoxy}ethoxy]ethoxymethyl)-5-methyl-2-phenyl-1,3-dioxane (MPD-OEG4). The same procedure as per the MPD-OEG1 synthesis was repeated, except that 2-[2-{2-(2-methoxyethoxy)ethoxy}ethoxy]ethyl tosylate (11.5 g, 55.2 mmol) was used instead of 2-methyoxyethyl ptoluenesulfonate. A total of 15.2 g of MPD-OEG4 was obtained (38.1 mmol, yield 69%). 1H NMR (CDCl3, 400 MHz) δ: 0.81 (s, 3H, CH3C), 3.37 (s, 3H, CH3O), 3.53−3.74 (m, 20H, OCH2H2O, OCH2CCH3), 4.06 (d, 2H, J = 11.7 Hz, OCH2CCH3), 5.41 (s, 1H, CHPh), 7.33−7.49 (m, 5H, Ph). FT-IR (cm−1): 2861, 1454, 1387, 1208, 1097, 1025, 970. CI-MS: m/z = 399 ([M + 1]+). Synthesis of 2-(2-Methoxyethoxymethyl)-2-methylpropandiol (Diol-OEG1). According to the previous literature,46 the diol deprotection of MPD-OEG1 (18.7 g, 70.3 mmol) was achieved by refluxing in MeOH (70 mL) and 5 M HCl(aq) (70 mL) at 110 °C for 8 h. The reaction mixture was extracted by CH2Cl2 and ultrapure water to collect the aqueous layer. The crude mixture was then purified by distillation to obtain 6.46 g of Diol-OEG1 (36.3 mmol, yield 52%). 1 H NMR (D2O, 400 MHz) δ:0.89 (s, 3H, CH3C), 3.41 (s, 3H, CH3O), 3.44 (s, 2H, OCH2CCH3), 3.51 (s, 4H, CH2OH), 3.66−3.68 (m, 4H, OCH2CH2O). FT-IR (cm−1): 3332, 2877, 1455, 1358, 1088, 1033. CI-MS: m/z = 179 ([M + 1]+). S y n t h es is of 2 - [ 2 -{ 2 - ( 2 -M e t h o x y e t h o x y ) e t h o x y } ethoxymethyl]-2-methylpropan-diol (Diol-OEG3). The same procedure as per the Diol-OEG1 synthesis was repeated except that MPD-OEG3 (19.3 g, 52.4 mmol) was used instead of MPD-OEG1. A total of 10.5 g of Diol-OEG3 was obtained (37.4 mmol, yield 71%). 1H NMR (CDCl3, 400 MHz) δ: 0.79 (s, 3H, CH3C), 3.39 (s, 3H, CH3O), 3.52−3.74 (m, 18H, OCH2CCH3, OCH2CH2O). FT-IR (cm−1): 3323, 2880, 1456, 1350, 1079, 1024. CI-MS: m/z = 267 ([M + 1]+). Synthesis of 2-(2-[2-{2-(2-Methoxyethoxy)ethoxy}ethoxy]ethoxymethyl)-2-methylpropan-diol (Diol-OEG4). The same procedure as per the Diol-OEG1 synthesis was repeated, except that MPD-OEG4 (15.2 g, 38.2 mmol) was used instead of MPD-OEG1. A total of 7.30 g of Diol-OEG4 was obtained (23.5 mmol, yield 62%). 1H NMR (CDCl3, 400 MHz) δ: 0.75 (s, 3H, CH3C), 3.38 (s, 3H, CH3O), 3.53−3.74 (m, 22H, OCH2CCH3, OCH2CH2O). FT-IR (cm−1): 2873, 1455, 1350, 1297, 1092, 1037, FAB-MS: [M + 1]+ = 311. 2670

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Table 1. Polymerization of TMC Derivativesa entry

monomer

[monomer]/[initiator]

conc (mol/L)

time (h)

yieldb (%)

Mnc × 103

PDIc

1 2 3d 4 5

TMCM-MOE1OM TMCM-MOE3OM TMCM-MOE3OM TMCM-MOE3OM TMCM-MOE4OM

50 50 100 300 200

1.44 0.80 0.73 1.98 0.74

8 8 36 36 36

73 72 60 97 16

2.5 0.96 7.4 11 2.3

1.06 1.42 1.28 1.36 1.41

a

Initiator = benzyl alcohol. Catalyst = DBU. [Initiator]/[Cat.] = 1.0. Solvent = CH2Cl2. Monomer = 0.5 g. bHexane/isopropanol (9/1, v/v) insoluble-part. cDetermined by SEC by PMMA standard in THF. dMonomer = 0.3 g.

III. LCST was measured by the JASCO V-550 ETC-505S and JSSCO V-550 ETC-505T system.

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RESULTS AND DISCUSSION

During the design of the monomer structure for the biodegradable thermosensitive homopolymer, we selected OEG as the thermosensitive parts according to the literature.7,10,14,28−30 As the polymer main chain, a PTMC backbone was believed to be suitable because it contains no ester groups which might cause inflammation due to increased acidity upon degradation. For the same reason, we avoided ester groups24−27 to join the OEG to the TMC in the monomer structure (Figure 1c), although it is a convenient functional group for converting polymer characteristics. In this manner, the novel TMC derivatives, TMCM-MOE1OM, TMCM-MOE3OM, and TMCM-MOE4OM were designed and synthesized, in order to control their thermosensitive characteristics by the length of the OEG units. Trimethylolethane was employed as the starting material, and five steps were required for the synthesis. Carbonylation reaction with diol50 was confirmed by Fourier transform infrared (FT-IR) spectra (Supporting Information, Figure S1). The detailed monomer synthesis procedures and analyses are described in the Supporting Information. The results of the polymerization are summarized in Table 1. The polymerization in CH2Cl2 at 25 °C was achieved with benzyl alcohol as the initiator and DBU as the catalyst.33 When compared to the polymerization of TMCM-MOE1OM, the number-average molecular weight (Mn) of poly(TMCMMOE3OM) tended to decrease under dilute concentrations (Table 1, entries 1 and 2) as determined by size exclusion chromatography (SEC). By changing the ratio of the [monomer]/[initiator] and the concentration over a long reaction time, a larger Mn for poly(TMCM-MOE3OM)s was obtained, although they were still smaller than the theoretical values (Table 1, entries 3 and 4). One of the possible explanations for this discrepancy is a substituent effect in the monomer structure by a reverse reaction during the equilibrium polymerization.51 Alternatively, the OEG units near the growing chain end might affect the polymerizability at the closed position because a much lower Mn value was observed in the polymerization of TMCM-MOE4OM bearing the longest OEG units. Figure 2 shows the SEC traces of poly(TMCM-MOE3OM)s with different Mns (Table 1, entries 2−4). When the ratio of the [monomer]/[initiator] was 50, the amount of DBU required increased work due to the same amount of DBU used as the initiator. This amount of DBU could attenuate a living polymerization system or cause polymer degradation, thus resulting in a broad polydispersity index (PDI) and the possible aggregation of species at high Mw values (Figure 2a). On the other hand, the PDI became narrow with a smaller amount of

Figure 2. SEC charts of poly(TMCM-MOE3OM)s obtained with [monomer]/[initiator] = 50 (Table 1, entry 2) (a), 100 (Table 1, entry 3) (b), and 300 (Table 1, entry 4) (c).

DBU, and the order of the Mw conformed to the theoretical values (Figure 2b,c). In order to examine the polymer model, various SEC standards and solvents were examined, whereas the values were listed with the PMMA standards. For example, the Mn was estimated as 5500, 4900, and 4500 in the case of poly(TMCM-MOE3OM) in chloroform with PMMA, PEG, and polystyrene standards (Table 1, entry 3). An estimation of the Mn by 1H NMR on the basis of the phenyl groups of the initiator resulted in larger values than the theoretical values, whereas matrix-assisted laser desorption ionization/time-offlight mass spectroscopy (MALDI-TOF/MS) did not detect higher molecular weight species (Supporting Information, Figure S2). The Mn of some PTMC copolymers, which were obtained with the DBU system, were analyzed by 1H NMR using protons from the macroinitiator52 and the aromatic group in the initiator.53 The estimation of the Mn by 1H NMR with the initiator group was not in good agreement with the results from SEC in this study, and hence the possibility of a secondary initiation cannot be excluded. Further systematic studies under various polymerization conditions would make the mechanism clearer. In any case, the polymers were successfully recovered as a hexane/isopropanol (9/1, v/v) insoluble part as a highly sticky liquid. Figure 3 shows the 1H NMR spectra of the monomers and their polymers. The splitting spectral pattern of the monomers around 4.2 ppm (Figure 3a,c,e), which was based on their ring structures, became a single peak after polymerization (Figure 3b,d,f), thus confirming that the ring-opening polymerization had occurred. The peaks between 3 and 4 ppm corresponded to the OEG groups, and the methyl protons in the polymer main chain appeared at around 1 ppm, together with a split peak around 0.9 ppm, in all polymers. Considering the amount of methyl groups, the split peak at around 0.9 ppm should be assigned as part of the methyl group. Because of the low Mn of the polymers, the split peak at around 0.9 ppm could be a 2671

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Figure 3. 1H NMR spectra of monomer TMCM-MOE1OM (a), poly(TMCM-MOE1OM) (b), TMCM-MOE3OM (c), poly(TMCM-MOE3OM) (d), TMCM-MOE4OM (e), and poly(TMCM-MOE4OM) (f).

LCST of poly(TMCM-MOE4OM) with the increased hydrophilicity due to the longer OEG units; the LCST was 72 °C at a 2300 Mn (Figure 4a, open squares), showing improved solubility in water. Therefore, it was determined that the true LCST of poly(TMCM-MOE3OM) originated from the polymer main chain and was 33 °C near body temperature. Poly(TMCM-MOE3OM) is a promising novel thermosensitive biodegradable homopolymer with a similar essential polymer structure as PNIPAm. Finally, the concentration effect was examined with poly(TMCM-MOE3OM) in comparison to Figure 4a, which was set at 2.0 mg/mL. The lower concentrations of 1.0 and 0.5 mg/ mL broadened the transition behaviors and increased the LCST temperatures to 34 and 35 °C, respectively. In contrast, the higher concentrations of 4.0 and 8.0 mg/mL resulted in values of 32 and 31 °C. No more concentrated solution could be prepared. Obviously, the LCST was influenced by the polymer concentration, suggesting a coacervate structure rather than a coil−globule transition. At the same time, differential scanning calorimetry (DSC) analysis indicated a very small endothermic peak at 33 °C (Supporting Information, Figure S3). The

hydroxyl group terminated species, which was recognized in the precursor. It is known that PTMC is insoluble in methanol, and thus the hydrophilicity of poly(TMCM-MOE1OM) was confirmed by being soluble in methanol as well as acetone and ethanol (Supporting Information, Table S1). Poly(TMCM-MOE3OM) and poly(TMCM-MOE4OM) were even soluble in water at 25 °C, and therefore the thermosensitivities of the polymers were measured by their turbidity. The results of the light transmittance of the poly(TMCMMOE3OM) and poly(TMCM-MOE4OM) solutions in ultrapure water against temperature are depicted in Figure 4. The LCST of poly(TMCM-MOE3OM) with a 960 Mn was 43 °C during the heating process, and this value decreased slightly during the cooling process (Figure 4a, closed squares). On the other hand, poly(TMCM-MOE3OM) with Mn values of 7400 and 11 000 showed LCST values at 33 °C (Figure 4a, closed triangles and closed circles). Given that a 960 Mn is low and that there may possibly be a hydroxyl group at the polymer chain end, the result of 43 °C should be ascribed to the slightly increased hydrophilicity effect. This is also supported by the 2672

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Figure 4. Transmittance vs temperature plots of poly(TMCM-MOE3OM) and poly(TMCM-MOE4OM) solutions in water at 2.0 mg/mL in heating and cooling processes (a). Closed square: poly(TMCM-MOE3OM) with 960 of Mn. Closed triangle: poly(TMCM-MOE3OM) with 7400 of Mn. Closed circle: poly(TMCM-MOE3OM) with 11 000 of Mn. Open square: poly(TMCM-MOE4OM) with 2300 of Mn. Transmittance vs temperature plots of poly(TMCM-MOE3OM) solution with 7400 of Mn with 0.5, 1.0, 2.0, 4.0, and 8.0 mg/mL (b).

enthalpy was estimated as 2.20 J/g in the region from 30 to 45 °C, using a 2 °C/min heating process with 4.7 mg of poly(TMCM-MOE3OM). Direct observation of the cloudy suspension was also performed by microscopy, revealing a phase separation (Supporting Information, Figure S4). At present, poly(TMCM-MOE3OM) itself is a highly viscous liquid, and the synthesis of higher Mn of poly(TMCMMOE3OM)s is currently underway.

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REFERENCES

(1) Pelton, R. Adv. Colloid Interface Sci. 2000, 85, 1−33. (2) Kikuchi, A.; Okano, T. Prog. Polym. Sci. 2002, 27, 1165−1193. (3) Winnik, F. M. Macromolecules 1990, 23, 233−242. (4) Ringsdorf, H.; Simon, J.; Winnik, F. M. Macromolecules 1992, 25, 7306. (5) Bokias, G.; Staikos, G.; Iliopoulos, I. Polymer 2000, 41, 7399− 7405. (6) Dube, D.; Francis, M.; Leroux, J.-C.; Winnik, F. M. Bioconjugate Chem. 2002, 13, 685−692. (7) Garbern, J. C.; Hoffman, A. S.; Stayton, P. S. Biomacromolecules 2010, 11, 1833−1839. (8) Suwa, K.; Wada, Y.; Kikunaga, Y.; Morishita, K.; Kishida, A.; Akashi, M. J. Polym. Sci., Part A: Polym. Chem. 1997, 35, 1763−1768. (9) Yamamoto, K.; Serizawa, T.; Muraoka, Y.; Akashi, M. Macromolecules 2001, 34, 8014−8020. (10) Han, S.; Hagiwara, M.; Ishizone, T. Macromolecules 2003, 36, 8312−8319. (11) Kono, K.; Murakami, T.; Yoshida, T.; Haba, Y.; Kanaoka, S.; Takagishi, T.; Aoshima, S. Bioconjugate Chem. 2005, 16, 1367−1374. (12) Aoshima, S.; Kanaoka, S. Adv. Polym. Sci. 2008, 210, 169−208. (13) Sugihara, S.; Kanaoka, S.; Aoshima, S. Macromolecules 2005, 38, 1919−1927. (14) Sugihara, S.; Kanaoka, S.; Aoshima, S. Macromolecules 2004, 37, 1711−1719. (15) Lee, R.; Chen, W.-H. React. Funct. Polym. 2011, 71, 455−462. (16) Brazel, C. S.; Peppas, N. A. Macromolecules 1995, 28, 8016− 8020. (17) Yin, X.; Hoffman, A. S.; Stayton, P. S. Biomacromolecules 2006, 7, 1381−1385. (18) Siu, M.; He, Ch.; Wu, C. Macromolecules 2003, 36, 6588−6592. (19) Mizutani, M.; Satoh, K.; Kamigaito, M. Macromolecules 2011, 44, 2382−2386. (20) Alli, S.; Alli, A.; Hazer, H. J. Appl. Polym. Sci. 2012, 124, 536− 548.

ASSOCIATED CONTENT

S Supporting Information *

FT-IR spectra of monomers. MALDI/TOF-MS of polymers; solubility test; DSC analysis of poly(TMCM-MOE3OM); microscope observation of poly(TMCM-MOE3OM) in water; photos of poly(TMCM-MOE3OM). This material is available free of charge via the Internet at http://pubs.acs.org.

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ACKNOWLEDGMENTS

The authors are grateful to the fruitful discussion with Drs. T. Kida, M. Matsusaki, and T. Akagi. The authors are also grateful to MDs. K. Yamamoto and S. Nanto. This work was partly supported by a Grant-in-Aid for Young Scientists (B) No. 21750220.

CONCLUSIONS In conclusion, a novel thermosensitive biodegradable homopolymer with a LCST at body temperature was prepared with a PTMC backbone and OEG units. The polymer was constructed from a repeating well-defined monomer structure without the inclusion of any ester groups, which generate acidic compounds during degradation. Although poly(TMCMMOE1OM) was insoluble, poly(TMCM-MOE3OM) and poly(TMCM-MOE4OM) were soluble in water at 25 °C, showing a LCST. Poly(TMCM-MOE3OM) was a highly viscous liquid below a Mn of 11 000, and its LCST in water ranged from 31 and 35 °C, depending on the concentration. The LCST of poly(TMCM-MOE4OM) appeared at 72 °C and differed greatly from poly(TMCM-MOE3OM). We believe that poly(TMCM-MOE3OM) is promising as an essential polymer material for various biomedical applications.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; Ph +81-6-6879-7356; Fax +81-6-6879-7359. Notes

The authors declare no competing financial interest. 2673

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Article

(21) Lutz, J.-F.; Akdemir, O.; Hoth, A. J. Am. Chem. Soc. 2006, 128, 13046−13047. (22) Song, S.-C.; Lee, S. B.; Jin, J.-I.; Sohn, Y. S. Macromolecules 1999, 32, 2188−2193. (23) Qiao, Z.-Y.; Du, F.-S.; Zhang, R.; Liang, D.-H.; Li, Z.-C. Macromolecules 2010, 43, 6485−6494. (24) Hazer, B.; Lenz, R. W.; Cakmakli, B.; Borcakli, M.; Kocer, H. Macromol. Chem. Phys. 1999, 200, 1903−1907. (25) Haba, Y.; Kojima, C.; Harada, A.; Kono, K. Macromolecules 2006, 39, 7451−7453. (26) Yamamoto, K.; Serizawa, T.; Muraoka, Y.; Akashi, M. J. Polym. Sci., Part A: Polym. Chem. 2000, 38, 3674−3681. (27) Yamamoto, K.; Serizawa, T.; Akashi, M. Macromol. Chem. Phys. 2003, 204, 1027−1033. (28) Jeong, B.; Bae, Y. H.; Lee, D. S.; Kim, S. W. Nature 1997, 388, 860−862. (29) Byrne, C. M.; Allen, S. D.; Lobkovsky, E. B.; Coates, G. W. J. Am. Chem. Soc. 2004, 126, 11404−11405. (30) Cheng, S.-X.; Miao, Z.-M.; Wang, L.-S.; Zhuo, R.-X. Macromol. Rapid Commun. 2003, 24, 1066−1069. (31) Kashio, M.; Sugizaki, T.; Tanaka, Y.; Yamamoto, S.; Moriya, O.; Nishikubo, T. Polym. J. 2011, 43, 525−530. (32) Danquah, M.; Fujiwara, T.; Hahato, R. I. Biomaterials 2010, 31, 2358−2370. (33) Nederberg, F.; Lohmeijer, B. G. G.; Leibfarth, F.; Pratt, R. C.; Choi, J.; Dove, A. T.; Waymouth, R. M.; Hedrick, J. L. Biomacromolecules 2007, 8, 153−160. (34) Coulembier, O.; Sanders, D. P.; Nelson, A.; Hollenbeck, A. N.; Horn, H. W.; Rice, J. E.; Fujiwara, M.; Dubois, P.; Hedrick, J. L. Angew. Chem., Int. Ed. 2009, 48, 5170−5173. (35) Zhang, H.-H.; Huang, Z.-Q.; Sun, B.-W.; Guo, J.-X.; Wang, J.-L.; Chen, Y.-Q. J. Polym. Sci., Part A: Polym. Chem. 2008, 46, 8131−8140. (36) Sanson, C.; Schatz, C.; Meins, J.-F. L.; Brulet, A.; Soum, A.; Lecommandoux, S. Langmuir 2010, 26, 2751−2760. (37) Sanson, C.; Schatz, C.; Meins, J.-F. L.; Soum, A.; Thevenot, J.; Garanger, E.; Lexommandoux, S. J. Controlled Release 2010, 147, 428− 435. (38) Seow, W. Y.; Yang, Y. Y. J. Controlled Release 2009, 139, 40−47. (39) Lu, J.; Shoichet, M. S. Macromolecules 2010, 43, 4943−4953. (40) Li, F.; Danquah, M.; Mahato, R. I. Biomacromolecules 2010, 11, 2610−2620. (41) Nederberg, F.; Zhang, Y.; Tan, J. P. K; Xu, K.; Wang, H.; Yang, C.; Gao, S.; Guo, X. D.; Fukushima, K.; Li, L.; Hedrik, J. L.; Yang, Y.-Y. Nat. Chem. 2011, 3, 409−414. (42) Zhang, Z.; Gripma, D. W.; Feijen, J. J. Controlled Release 2006, 112, 57−63. (43) Kim, S. Y.; Kim, H. J.; Lee, K. E.; Han, S. S.; Sohn, Y. S.; Jeong, B. Macromolecules 2007, 40, 5519−5525. (44) Kim, S. H.; Tan, J. P. K; Fukushima, K.; Nederberg, F.; Yang, Y. Y.; Waymoutgh, R. M.; Hedrick, J. L. Biomaterials 2011, 32, 5505− 5514. (45) Watanabe, J.; Kotera, H.; Akashi, M. Macromolecules 2007, 40, 8731−8736. (46) Wang, X.-L.; Zhuo, R.-X.; Liu, L.-J.; He, F.; Liu, G. J. Polym. Sci., Part A: Polym. Chem. 2002, 40, 70−75. (47) Sanders, D. P.; Fukushima, K.; Coady, D. J.; Nelson, A.; Fujiwara, M.; Yasumoto, M.; Hedrick, J. L. J. Am. Chem. Soc. 2010, 132, 14724−14726. (48) Chen, W.; Yang, H.; Wang, R.; Cheng, R.; Meng, F.; Wei, W.; Zhong, Z. Macromolecules 2010, 43, 201−207. (49) Lin, X.; Li, W.; Zhang, J.; Sun, H.; Yan, Y.; Wu, L. Langmuir 2010, 26, 13201−13209. (50) Davies, S. G.; Roberts, P. M.; Stephenson, P. T.; Storr, H. R.; Thomson, J. E. Tetrahedron 2009, 65, 8283−8296. (51) Matsuo, J.; Aoki, K.; Sanda, F.; Endo, T. Macromolecules 1998, 31, 4432−4438. (52) Lee, A. L. Z.; Venkataraman, S.; Sirat, S. B. M.; Gao, S.; Hedrick, J. L. Biomaterials 2012, 33, 1921−1928.

(53) Qiao, Y.; Yang, C.; Coady, D. J.; Ong, Z. Y.; Hedrick, J. L.; Yang, Y. Y. Biomaterials 2012, 33, 1146−1153.

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