Synthesis and Properties of Poly [poly (ethylene glycol)-co-cyclic

Sep 12, 2007 - Maria Rikkou-Kalourkoti , Elena Loizou , Lionel Porcar , Krzysztof ... Shao Qiong Liu , Richie Tay , Majad Khan , Pui Lai Rachel Ee , J...
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Macromolecules 2007, 40, 7625-7632

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Synthesis and Properties of Poly[poly(ethylene glycol)-co-cyclic acetal] Based Hydrogels Sachiko Kaihara,† Shuichi Matsumura,† and John P. Fisher*,‡ Department of Applied Chemistry, Keio UniVersity, Yokohama, 223-8522 Japan, and Fischell Department of Bioengineering, UniVersity of Maryland, College Park, Maryland 20742 ReceiVed June 11, 2007; ReVised Manuscript ReceiVed July 23, 2007

ABSTRACT: The objective of this work is to synthesize and characterize a biomedical hydrogel based on the polyether-acetal, poly[poly(ethylene glycol)-co-cyclic acetal] (PECA). This hydrogel has been designed to possess two significant properties, a hydrolytically degradable cyclic acetal segment and a hydrophilic poly(ethylene glycol) (PEG) segment. The chemical structure of the synthesized PECA was analyzed by 1H NMR and matrixassisted laser desorption ionization time-of-flight mass spectrometry, while the weight average molecular weight (Mw) was measured by size exclusion chromatography. Results confirmed that PECA with Mw of 5000-44 000 g/mol was synthesized from a cyclic acetal and PEG, whose Mw ranged from 200 to 2000 g/mol. Differential scanning calorimetry confirmed that crystallization temperature and melting temperature increased as the Mw of PEG increased. After diacrylation of both hydroxyl terminal groups of the PECA chain, PECA hydrogels were prepared by cross-linking using redox initiators, ammonium persulfate, and N,N,N′,N′-tetramethylethylenediamine, and then characterized. The effects of polymer concentration and initiator concentration for cross-linking upon sol fraction and swelling degree were then investigated. Both sol fraction and swelling degree were found to be highly influenced by polymer concentration, while swelling degree and swelling rate were also influenced by Mw of PEG. Degradation kinetics of cyclic acetal segments were investigated in excessive acidic condition as well as simulated physiological conditions. Degradation rates of PECA in excessive acidic condition were significantly influenced by pH and temperature. Furthermore, the pH of buffer remained constant while the PECA hydrogel lost approximately 30% of its dry weight after 5 months of in vitro incubation. The ease of control over properties such as swelling degree and degradation rate indicates that the newly synthesized hydrogel is a promising material as a matrix for drug delivery and tissue engineering applications.

Introduction In recent years, a number of materials have been introduced for biomedical applications, including cell carriers in orthopedic tissue engineering and scaffolds for formation of cartilage.1-4 Synthetic biomaterials are advantageous due to their reproducibility as well as easily controlled properties and chemical structures. Many researchers are engaged in synthesis of new types of biomaterials by modifying conventional biomaterials for extended applications.5,6 Hydrogels are one of the most widely used biomaterials because of the similarity of their swelling property to soft tissues, such as cartilage. Several types of synthetic hydrogels are investigated for utilization as a scaffold of organs and drug delivery carriers.7-9 One of the most commonly used polymers for hydrogel fabrication is poly(ethylene glycol) (PEG), due to its biocompatibility and hydrophilicity. In fact, many copolymers based on PEG and other moieties have been synthesized, and their properties are widely investigated.10-12 These studies aimed to enhance the hydrophilicity of the material so as to produce a suitable matrix for cell encapsulation and formation of scaffolds. For example, copolymers of PEG and biodegradable polymers such as poly(lactic acid) have been fabricated for tissue engineering applications.13-15 Hydrogels obtained from PEG acrylate derivatives, including PEG methacrylates and PEG diacrylates, have also been developed and widely investigated.16-18 Degradable hydrogels are often based upon an ester or amide polymer backbone, such as poly(L-lactic acid) and poly* Corresponding author. Telephone: 301 405 7475. Fax: 301 405 0523. E-mail: [email protected]. † Keio University. ‡ University of Maryland.

(glycolic acid), where ester or amide hydrolysis provides a mechanism for degradation.19,20 The significant disadvantage of polyesters and polyamides is the formation of carboxylic acid as the degradation products. As a result, the acidity of the surrounding tissue is increased, and this rise in local acidity has been implicated in both the catalysis of further scaffold degradation and the elicitation of a pronounced inflammatory response.21-24 In this study, a novel class of hydrogels based on a polyether containing a cyclic acetal moiety was designed and synthesized. Hydrolysis of cyclic acetals produces hydroxyl and carbonyl terminals as degradation products, and therefore may not affect local acidity or promote inflammation. In particular, we recently synthesized a new material based solely on a cyclic acetal unit, with results indicating that their physical properties and cytocompatibility make them attractive candidates for biomedical applications.25 To fabricate a cyclic acetal based hydrogel with controlled molecular structure, we describe here the copolymerization of the cyclic acetal unit with PEG. Following synthesis, we then characterized the physicochemical properties of both the copolymer and the resulting cross-linked hydrogels. Here, we hypothesized that the molecular weight of the PEG unit in PECA will dominate the thermal properties of PECA. That is, both copolymer melting temperature and crystallization temperature of PECA will increase with the increasing of the molecular weight of PEG units. Similarly, we assumed that the increased molecular weight of PEG will lower the cross-link density of the resulting hydrogel and thus will lead to an increase in swelling properties. We evaluated the effect of polymer concentration upon swelling properties of the hydrogel, hypothesizing that increased polymer concentration of cross-linking

10.1021/ma071297p CCC: $37.00 © 2007 American Chemical Society Published on Web 09/12/2007

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Macromolecules, Vol. 40, No. 21, 2007 Scheme 1. Synthetic Route for PECA Hydrogel

reaction will be associated with a lower swelling degree due to the increased rigidity of the polymer chains of the hydrogel. Finally, we examined the dependence of the degradation rate of cyclic acetal segments on pH and temperature, hypothesizing that increased temperature and acidity will increase the degradation rate of cyclic acetal segments. Experimental Section Materials and Measurements. Acryloyl chloride, ammonium persulfate, and N,N,N′,N′-tetramethylethylenediamine (TEMED) were obtained from Sigma-Aldrich (Milwaukee, WI). Formaldehyde aqueous solution (37%), triethylamine, p-toluenesulfonyl chloride, and sodium hydride were purchased from Kanto Chemical Co. Inc. (Tokyo, Japan). Isobutyraldehyde, trimethylolpropane, PEGs (Mw ) 600, 1000, 2000), and tetraethylene glycol (TeEG) di-p-tosylate were purchased from TCI Co. Ltd. (Tokyo, Japan). Anhydrous methanol, dichloromethane, and tetrahydrofurane were used as purchased. All the other solvents and chemicals were used without further purification. The weight-average (Mw) and number-average (Mn) molecular weights as well as molecular weight distribution (Mw/Mn) of various products were determined by size exclusion chromatography (SEC) using SEC columns (Shodex K-804, Showa Denko Co., Ltd., Tokyo, Japan) with a refractive index detector. Chloroform was used as the eluent at 1.0 mL/min. The SEC system was calibrated with PEG standards (1050, 10 730, 30 000, and 49 000 g/mol). Molecular weights reported are relative to PEG calibration. The chemical structure of the polymer was analyzed by matrixassisted laser adsorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The MALDI-TOF MS was measured with a Bruker Ultraflex mass spectrometer (Bruker Daltonics, Billerica, MA) equipped with a nitrogen laser. Detection was in the reflectron mode. The matrix utilized was 2,5-dihydroxybenzoic acid, while the cation source was sodium bromide. Positive ionization was utilized in all procedures. The 1H NMR spectra were recorded on a JEOL Model Lambda 300 spectrometer (JEOL, Ltd., Tokyo, Japan) operating at 300 MHz. To determine the chemical structure of the cis and trans forms of 5-ethyl-5-(hydroxymethyl)-β,β-dimethyl-1,3-dioxane-2-ehtanol (EHD), nuclear Overhauser effect (NOE) NMR and 2D NOE spectroscopy (NOESY) were utilized. Thermal properties of the polymer were evaluated by differential scanning calorimetry (DSC). Samples placed in aluminum pans were analyzed with DSC-60 (Simadzu, Kyoto, Japan). All samples were heated at the rate of 10 °C/min from room temperature to 100 °C (first scan), quenched to -100 °C at the rate of -30 °C/ min, and then scanned at the same heating rate of 10 °C/min to 100 °C (second scan). The data were collected during the second heating scan. The glass transition temperature (Tg) was taken as the midpoint of the heat capacity change. The melting point (Tm) and the crystallization point (Tc) were taken as the top of the relevant peaks. Synthesis of 3-Hydroxy-2,2-dimethylpropionaldehyde. 3-Hydroxy-2,2-dimethylpropionaldehyde (HDP, Scheme 1) was synthe-

sized according to the method of Acerbis et al.26 Dipotassium carbonate (7.57 g, 55 mmol) was added to a mixture of isobutyraldehyde (10.0 mL, 110 mmol) and formaldehyde (37% aqueous solution, 8.2 mL, 110 mmol) and stirred overnight at room temperature under Ar. After the reaction, the product was extracted three times with chloroform and washed with water and brine. The chloroform layer was combined, dried with Na2SO4, and evaporated under reduced pressure to obtain HDP in 91% yield. The structure of the product was analyzed by 1H NMR. 1H NMR (CDCl3, ppm): δ 9.24 (1H, s, -CHO), 3.63 (2H, s, -CH2OH), 1.07 (6H, s, -C(CH2)3-). Synthesis of 5-Ethyl-5-(hydroxymethyl)-β,β-dimethyl-1,3-dioxane-2-ethanol. Trimethylolpropane (5.9 g, 44 mmol) was dissolved in 1 M hydrochloric acid (20.0 mL). HDP (3.0 g, 29 mmol) was added to the solution and stirred for 24 h at 90 °C under Ar. After the reaction, the product was extracted three times with chloroform and washed with water and brine. The chloroform layer was combined, dried with Na2SO4, and evaporated under reduced pressure. The crude product was further purified by silica gel column chromatography using a chloroform/methanol (50:1, v/v) as the eluent to obtain EHD as a white solid in 79% yield. The structure of the product was analyzed by 1H NMR. 1H NMR (CDCl3, ppm): δ (cis) 0.83 (3H, t, J ) 7.2 Hz, CH3CH2-), 0.94 (6H, s, -C(CH3)2CH2-), 1.20 (2H, q, J ) 7.5 Hz, CH3CH2-), 3.403.50 (4H, m, -C(CH3)2CH2OH, -CCH2O-), 3.86 (2H, d, J ) 6.0 Hz, -CCH2OH), 3.97 (2H, d, J ) 11.7 Hz, -CCH2O-), 4.26 (1H, s, -OCHO-); (trans) 0.88-0.94 (9H, m, CH3CH2-, -C(CH3)2CH2-), 1.76 (2H, q, J ) 8.3 Hz, CH3CH2-), 3.33 (2H, d, J ) 4.5 Hz, -CCH2OH), 3.46 (2H, d, J ) 6.0 Hz, -C(CH3)2CH2OH), 3.62 (2H, d, J ) 11.4 Hz, -CCH2O-), 3.86 (2H, d, J ) 11.7 Hz, -CCH2O-), 4.25 (1H, s, -OCHO-). Ditosylation of PEG. PEG(600) (Mw ) 600; 1.0 g, 1 equiv) was dissolved in anhydrous dichloromethane (20 mL), and triethylamine (3 equiv) was added. To the mixture p-toluenesulfonyl chloride (3 equiv) was added and stirred at room temperature under Ar overnight. After the reaction, the insoluble salt was removed by filtration, and the filtrate was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography using a chloroform/methanol (20:1, v/v) as the eluent to obtain PEG(600) ditosylate in 89% yield as a viscous liquid. In a similar procedure, PEG(1000) ditosylate and PEG(2000) ditosylate were prepared from PEG(1000) (Mw ) 1000) in 93% yield as a viscous liquid, and from PEG(2000) (Mw ) 2000) in 87% yield as a wax, respectively. The structure of the product was analyzed by 1H NMR. 1H NMR (CDCl , ppm): δ 2.48 (6H, s, Ar-CH ), 3.30-3.65 (xH, 3 3 m, -CH2O-), 7.34 (4H, d, J ) 8.4 Hz, Ar-H), 7.80 (4H, d, J ) 8.4 Hz, Ar-H). Polyetherification of EHD with PEG Ditosylate. EHD (1.0 g, 4.6 mmol, 1 equiv) was dissolved in anhydrous tetrahydrofuran (10 mL). Then, sodium hydride (600 mg, 3 equiv) was added to the solution at 0 °C and stirred at room temperature for 1 h. PEG ditosylate (1 equiv) was added into the solution, and the mixture was stirred at 50 °C under Ar for 24 h. EHD (100 mg, 0.1 equiv) was added at the end of the polyetherification to transform all the terminal groups to hydroxyl groups. Then, water was added and

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Figure 1. 1H NMR spectra of (a) trans-EHD and (b) PECA(TeEG). The labels e′ and f ′ correspond to peaks on e and f next to hydroxyl terminal groups, respectively. A peak at 7.26 ppm corresponds to chloroform in CDCl3.

stirred for 1 h at room temperature. Water and all the organic solvents were evaporated under reduced pressure. The residue was dissolved in ethyl acetate, and insoluble salt was removed by filtration. The solvent was removed by evaporation under reduced pressure. The crude product was further purified by silica gel column chromatography using chloroform/methanol (10:1, v/v) as the eluent, yielding poly(PEG-co-cyclic acetal) (PECA). Thus, PECA(TeEG), PECA(600), PECA(1000), and PECA(2000) were obtained in 87 (viscous liquid), 75 (viscous liquid), 89 (wax), and 85% yield (crystal), respectively. The structure of PECA was analyzed by 1H NMR and MALDI-TOF MS. The molecular weight of PECA was measured by SEC. 1H NMR (CDCl3, ppm): δ (cis) 0.83 (3H, t, J ) 7.2 Hz, CH3CH2-), 0.94 (6H, s, -C(CH3)2CH2-),

Figure 2. MALDI-TOF MS spectrum of PECA-DA(TeEG).

Synthesis and Properties of PECA Hydrogels 7627 1.20 (2H, q, J ) 7.5 Hz, CH3CH2-), 3.27 (2H, s, -C(CH3)2CH2O-), 3.35 (2H, d, J ) 11.2 Hz, -CCH2O-), 3.41 (2H, d, J ) 6.0 Hz, -C(CH3)2CH2OH), 3.61-3.67 ((4x+2)H, m, -CCH2O-, -CH2O-), 3.84 (2H, d, J ) 4.8 Hz, -CCH2OH), 3.91 (2H, d, J ) 11.4 Hz, -CCH2O-), 4.27 (1H, s, -OCHO-); (trans) 0.86-0.93 (9H, m, CH3CH2-, -C(CH3)2CH2-), 1.71 (2H, q, J ) 8.3 Hz, CH3CH2-), 3.11 (2H, s, -CCH2O-), 3.26 (2H, s, -C(CH3)2CH2O-), 3.32 (2H, d, J ) 4.6 Hz, -CCH2OH), 3.46 (2H, d, J ) 6.0 Hz, -C(CH3)2CH2OH), 3.57-3.69 ((4x+2)H, m, -CCH2O-, -CH2O-), 3.82 (2H, d, J ) 11.4 Hz, -CCH2O-) 4.24 (1H, s, -OCHO-). Diacrylation of PECA. PECA (1.0 g, 1 equiv) and triethylamine (3 equiv) were dissolved in anhydrous dichloromethane (8.0 mL). Then, acryloyl chloride (3 equiv) was added dropwise at 0 °C, and the solution was stirred at room temperature for 2 h. The insoluble salt was removed by filtration and the solution was precipitated 3 times in n-hexane. The remaining solvent was removed by evaporation and further dried under reduced pressure to obtain diacrylated PECA (PECA-DA). Thus, PECA-DA(TeEG), PECADA(600), PECA-DA(1000), and PECA-DA(2000) were prepared in 94 (viscous liquid), 86 (viscous liquid), 94 (wax), and 90% yield (crystal), respectively. The structure of PECA-DA was analyzed by MALDI-TOF MS and 1H NMR. 1H NMR (CDCl3, ppm): δ (cis) 0.83 (3H, t, J ) 7.2 Hz, CH3CH2-), 0.94 (6H, s, -C(CH3)2CH2-), 1.20 (2H, q, J ) 7.5 Hz, CH3CH2-), 3.27 (2H, s, -C(CH3)2CH2O-), 3.35 (2H, d, J ) 11.2 Hz, -CCH2O-), 3.61-3.67 ((4x+2)H, m, -CCH2O-, -CH2O-), 3.91 (2H, d, J ) 11.4 Hz, -CCH2O-), 4.01 (2H, s, -CH2OCOCHCH2), 4.27 (1H, s, -OCHO-), 5.78 (1H, dd, J ) 10.0 Hz, 2 Hz, -OCOCHCH2), 6.20 (1H dd, J ) 16.5 Hz, 10 Hz, -OCOCH-), 6.43 (1H, dd, J ) 16.5 Hz, 2 Hz, -OCOCHCH2); (trans) 0.86-0.93 (9H, m, CH3CH2-, -C(CH3)2CH2O-), 1.71 (2H, q, J)8.3 Hz, CH3CH2-), 3.11 (2H, s, -CCH2O-), 3.26 (2H, s, -C(CH3)2CH2O-), 3.57-3.69 ((4x+2)H, m, -CCH2O-, -CH2O-), 3.82 (2H, d, J ) 11.4 Hz, -CCH2O-), 4.03 (2H, s, -CH2OCOCHCH2), 4.24 (1H, s, -OCHO-), 5.78 (1H, dd, J ) 10.0 Hz, 2 Hz, -OCOCHCH2), 6.20 (1H, dd, J)16.5 Hz, 10 Hz, -OCOCH-), 6.43 (1H, dd, J ) 16.5 Hz, 2 Hz, -OCOCHCH2). Cross-Linking of PECA-DA. PECA-DA was cross-linked by redox initiators, ammonium persulfate and TEMED. PECA-DA(600, 1000, or 2000) was first weighed in a mold (Wi), the ammonium persulfate aqueous solution was added, and the solution was stirred thoroughly. The TEMED aqueous solution was then added. The solution was stirred thoroughly, and placed at room temperature for 1 h, forming PECA hydrogel (600, 1000, or 2000). Fabricated hydrogels were soaked in phosphate-buffered saline (PBS, pH 7.4, 8 g/L of NaCl, 0.2 g/L of KCl, 1.44 g/L of Na2HPO4, 0.24 g/L of KH2PO4) at 25 °C for 20 h and then weighed (Ws). The swollen hydrogels were dried in the oven at 90 °C until the constant weight (Wd). The sol fraction of hydrogels was calculated with the following equation: sol fraction (s) ) (Wi -Wd)/ Wi × 100 (%). The swelling degree of hydrogels was calculated with the following equation: swelling degree (q) ) Ws/Wd (g/g). The swelling rate of hydrogels was calculated by the ratio of Ws at t ) t′ (min) and Ws at equilibrium state (t ) ∞), with the following equation: swelling ratio (r) ) Ws(t ) t′)/Ws(t )∞) × 100 (%). Two fabrication parameters were utilized to control the properties

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Figure 3. SEC profiles of (a)-1 TeEG ditosylate, (a)-2 PECA(TeEG) and (b)-1 PEG(600) ditosylate, and (b)-2 PECA(600).

Figure 5. Effect of (a) polymer concentration (b) and initiator concentration on sol fraction of PECA(600) hydrogels in PBS buffer at 25 °C. The mean and standard deviation (n ) 3) are reported.

Figure 4. Differential scanning calorimetry curves for PECA(2000), PECA(1000), and PECA(600).

of PECA hydrogels: polymer concentration and initiator concentration. The effects of Mw of PEG on swelling degree and swelling rate were also evaluated. Degradation Studies of Cyclic Acetal Segments. EHD and PECA(600) were degraded in buffers with pH values of 2 and 4 at 95, 80, and 65 °C. Each buffer was first prepared by dissolving boric acid (0.2 M), citric acid (0.05 M), and sodium phosphate dodecahydrate (0.1 M) in deionized water. EHD and PECA(600) (50 mg) were stirred in 10 mL of buffer at each temperature. At each time point, 1 mL of the solution was taken out and neutralized by NaOH aq (0.1 M). Water was evaporated under reduced pressure, and the product was dissolved in chloroform. The resulting insoluble salt was then removed by filtration, and the solvent was removed by reduced pressure. The ratio of degradation of EHD to nondegraded EHD at time t (h) was determined by comparing the integrated 1H NMR spectra of b of EHD and trimethylolpropane (see Figure 1) (δ 1.76 ppm (EHD), 1.31 ppm (trimethylolpropane)). The ratio of degradation of PECA(600) to non-degraded PECA at time t (h) was determined by comparing the area of GPC peaks of PECA(600) and PEG(600). The degradation rate coefficients k were calculated from the slope of the plot of the natural logarithm of the remaining monomer vs time for EHD and the variation of molecular weight of polymer vs time for PECA(600). To examine hydrogel degradation, PECA(600) hydrogel was incubated in PBS buffer (pH 7.4) at 37 °C. At each specified time point, the swollen hydrogels were weighed (W′s). The swollen hydrogels were dried in the oven at 90 °C until a constant weight (W′d) was achieved. The remaining mass of hydrogels was calculated with the following equation: remaining mass ) W′d/Wi

Figure 6. Effect of (a) polymer concentration (b) and initiator concentration on the swelling degree of PECA(600) hydrogels in PBS buffer at 25 °C. The mean and standard deviation (n ) 3) are reported.

× 100 (%). The swelling degree of degraded hydrogels was calculated with the following equation: swelling degree (q) ) W′s/ W′d (g/g). The pH of the buffer was measured using a pH meter at each time point, and the pH change was calculated with the following equation: pH change ) (pH of PBS buffer with samples) - (pH of PBS buffer without samples). Each measurement was carried out in triplicate.

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Synthesis and Properties of PECA Hydrogels 7629

Table 1. Mw, Mw/Mn, and Thermal Properties of PECA Consisting of PEG Units with Molecular Weights of 600, 1000, and 2000 PECA Mw of PEG unit

Mw

Mw/Mn

Tc (°C)

Tm (°C)

Tg (°C)

176 600 1000 2000

5 000 12 000 29 000 44 000

11.6 5.8 10.0 8.5

-41.8 -18.1 8.1

12.6 32.6 44.5

-58.7

PECA(TeEG) PECA(600) PECA(1000) PECA(2000)

Table 2. Degradation Rate Coefficients of EHD and PECA at 95, 80, and 65 °C and pH 2 and pH 4 EHD

PECA(600)

T (°C)

pH 2

pH 4

pH 2

pH 4

95 80 65

0.84 0.80 0.10

0.10 0.05 0.01

2.30 0.75 0.20

0.15 0.03 0.01

Results and Discussion Synthesis of PECA Copolymer. A novel copolymer based on cyclic acetal and PEG has been designed and synthesized in a three-step reaction. To simplify the characterization of the chemical structure of the resulting polyether, molecularly pure TeEG (molecular weight, 194) was used for the synthesis of PECA instead of PEG with molecular weight distribution. The chemical structure of the polymer was analyzed by 1H NMR. The 1H NMR spectroscopy of trans-EHD as the monomer and PECA synthesized from trans-EHD and TeEG diacrylate are shown in Figure 1. These results confirmed that the protons at e, f, g and h in Figure 1 were all shifted, indicating that two hydroxyl groups of EHD were all reacted with TeEG ditosylate to produce ether linkages. It should be noted that protons at e and f in Figure 1b had two peaks, depending upon whether they are adjacent to terminal groups. The chemical structure of the copolymer backbone and terminal groups was also analyzed with MALDI-TOF MS. These results confirmed that alternating copolymers with hydroxyl terminal groups were indeed synthesized by the polyetherification reaction. After polyetherification, both hydroxyl terminal groups of the PECA chains were diacrylated to allow for the fabrication of cross-linked hydrogels. The completion of diacrylation was also confirmed by 1H NMR and MALDI-TOF MS (Figure 2). SEC was used to measure the molecular weight of the starting PEG ditosylate and the resulting copolymers. Typical examples are shown in Figure 3. The Mw of the obtained copolymers are shown in Table 1. These results showed that the Mw of PECA gradually increased with increasing initial Mw of PEG, where PEG(600) ditosylate and PEG(2000) ditosylate produced PECAs with Mws of 12 000 and 44 000, respectively. It was supposed that the PEG with a higher molecular weight would be less reactive due to the decrease in the relative number of terminal hydroxyl groups in the reaction media. However, as shown in Table 1, PEG(1000) ditosylate and PEG(2000) ditosylate effectively reacted with EHD to produce PECAs with relatively high molecular weights. There are few polyetherification reaction examples using two hydroxyls because it is difficult to synthesize high molecular weight polymers.27 In the work presented here, a high molecular weight polyether was actually obtained by the polyetherification of a highly reactive tosyl group and alkoxide group. We do note that there was no change in molecular weight after 24 h polymerization. This is due to the decrease in the relative number of the terminal hydroxyl groups of the polymer chain and the mobility of the polymer chain. Thermal Properties. Thermal properties of PECA were measured by DSC. Table 1 shows Tg, Tc, and Tm of each PECA. Figure 4 shows melting temperature curves for PECA with

differing lengths of PEG units. Results showed that the Tc increased from -41.8 to +8.1 °C as the molecular weight of PEG increased. Similarly, Tm increased from 12.6 to 44.5 °C as the molecular weight of PEG increased. It should be noted that the Tc was detected during the quenching process for PECA(1000) and PECA(2000), while the Tc was detected during the second heating scan for PECA(600). Tg could not be detected for PECA(1000) and PECA(2000) due to the high crystallinity of the high molecular weight PEG. Thermal properties of PECA were evaluated to determine the impact of the molecular weight of PEG. Results demonstrated that the molecular weight of PEG strongly influenced Tc and Tm. Tms of PEG were also measured to compare with those of PECA having the corresponding PEG chain length. Tms of PEG with the Mws of 600, 1000, and 2000 were 20.7, 37.1, and 55.8 °C, respectively. Interestingly, Tm of PECA was approximately 5-10 °C lower than that of PEG, even though the total molecular weight of PECA was much higher than that of PEG. That is due to the introduction of the cyclic acetal segment into PECA, which prevents PEG units from crystallizing. Swelling Degree and Sol Fraction of PECA Hydrogels. Hydrogels were successfully fabricated with ammonium persulfate and TEMED redox initiators by cross-linking the acryl terminal groups of PECA-DA. These initiators were utilized to prepare PECA hydrogels because of their solubility in water and relatively high cytocompatibility.28 PECA(TeEG) hydrogel was not formed with this initiator system by the cross-linking reaction of PECA-DA containing TeEG units due to the insolubility of the copolymer in water. Therefore, studies of further properties were carried out with PECA(600) hydrogel, PECA(1000) hydrogel, and PECA(2000) hydrogel. Sol fraction and mass swelling degree were measured to evaluate physicochemical properties of the fabricated hydrogels. The effects of polymer concentration and initiator concentration for cross-linking reaction upon sol fraction were first evaluated. The effect of polymer concentration upon sol fraction is shown in Figure 5a. These data indicate that the sol fraction increased from 19.3 ( 5.0 to 59.1 ( 8.5% as the polymer concentration increased from 0.09 to 1.00 g/mL. High polymer concentrations, associated with less mobile polymer chains, brought increased numbers of unreacted polymer chains. The effect of initiator concentration upon sol fraction was also evaluated (Figure 5b). It was found that initiator concentration did not significantly influence the sol fraction, where 55.4 ( 8.5 and 55.7 ( 10.4% were obtained at initiator concentration of 50 and 200 mM, respectively. The effects of polymer concentration and initiator concentration for cross-linking reaction upon swelling degree were also studied. The effect of polymer concentration upon swelling degree is shown in Figure 6a. Swelling degree increased from 6.2 ( 0.6 to 9.5 ( 0.1 g/g as the polymer concentration decreased from 1.00 to 0.09 g/mL. This increase in swelling was likely due to the mobility of the more dilute polymer chain during gelation. That is, intermolecular polymerization with cross-linking efficiently occurred by the radical reaction in a more dilute polymer solution, which resulted in the formation of a relatively large three-dimensional structure. On the other

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Figure 7. (a) Effect of molecular weight of PEG on the swelling degree (25 °C) of the synthesized PECA hydrogels. (b) Effect of molecular weight of PEG on the swelling rate (25 °C) of the synthesized PECA hydrogels: (2) PECA(2000) hydrogel; (9) PECA(1000) hydrogel; (b) PECA(600) hydrogel.

hand, due to the low mobility of polymer chain in a concentrated polymer solution, radicals might not be utilized efficiently in the cross-linking reaction. These results corresponded well to the effect of polymer concentration upon sol fraction. Overall, these results indicated that the mobility of the polymer chain during radical reaction most significantly affected the hydrogel network structure. The effect of initiator concentration upon swelling degree was also evaluated as shown in Figure 6b. The swelling degree was 6.7 ( 0.6 and 6.5 ( 0.1 g/g at initiator concentration of 50 and 200 mM, respectively. Results showed that the swelling degree was not significantly altered in this range of initiator concentration. Finally, the effects of molecular weight of the PEG unit in PECA-DA on swelling degree and swelling rate of PECA hydrogel were evaluated (Figure 7). Swelling degree was 3.4 g/g for PECA(600) hydrogel and 9.8 g/g for PECA(2000) hydrogel (Figure 7a). These results demonstrated that when PECA hydrogels were prepared under similar reaction conditions, the swelling degree increased significantly with increasing molecular weight of the PEG unit. These results indicated that the swelling properties were strongly influenced by the PEG polymer chain lengths. To compare the swelling properties between a PEG diacrylate type hydrogel and an PECA type hydrogel having the same PEG lengths, the PEG diacrylate type hydrogel was prepared by the cross-linking reaction of PEG diacrylate with the Mw ) 2000 using ammonium persulfate and TEMED. The swelling degree of a hydrogel prepared from PEG diacrylate was 3.5 g/g, which was relatively lower than that of PECA(2000) hydrogel. Assuming that the hydrophilicity of PEG diacrylate (Mw ) 2000) and PECA(2000) were similar, these results indicated that the molecular weight of the polymer backbone was the main factor of swelling rather than hydrophilicity. As the polymer chain lengths increased, the cross-

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Figure 8. Hydrolysis rate of (a) EHD and (b) PECA(600) at pH 2: (b) 95, (9) 80, and (2) 65 °C. Mx and Mo correspond to number average molecular weight of degradation product at time t (h) and minimum molecular weight of degradation product, respectively.

link density of the network structure decreased resulting in an increase of water retention. The effect of the molecular weight of PEG unit on swelling rate was also evaluated, and it was confirmed that the swelling rate increased as the molecular weight of PEG decreased (Figure 7b). This is likely due to the increased mobility of the shorter polymer main chains as well as the reduced cross-linking density of the resulting hydrogels that lead to faster diffusion of water in the cross-linked structure. Overall, hydrogel swelling appears to be well-controlled by changing either the PEG length or PECA-DA concentration. Degradation Studies of Cyclic Acetal Segments. Degradation kinetics of cyclic acetal was investigated to evaluate the dependence of the degradation behavior of novel degradation segments upon pH as well as temperature. The decrease of nondegraded EHD and PECA were monitored by 1H NMR and GPC, respectively. The fraction of EHD, which hydrolytically degrades into trimethylolpropane and HDP, was expressed as the remaining monomer. Parts a and b of Figure 8 show the time variation of the remaining EHD monomer and the number average molecular weight for the reactions in acidic condition (pH ) 2), respectively. The degradation rate coefficients (k) for EHD and PECA(600) were determined from the slope of the plot of the natural logarithm of the remaining monomer vs time for EHD and the variation of molecular weight of polymer vs time for PECA (600). The linear relationship between logarithmic remaining product and time indicated pseudo-firstorder degradation kinetics for cyclic acetal hydrolysis. It was confirmed that the degradation rate coefficients increased as the decrease of temperature and acidity of the buffering environments (see Table 2). This is likely due to the dependence of the cyclic acetal hydrolysis rate upon hydronium ion concentration.

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(Figure 9). The remaining mass of the hydrogel after 5 months incubation was 65.8 ( 3.3%, while the swelling degree gradually increased from 3.0 ( 0.2 to 5.4 ( 0.4 g/g. The pH change of PBS buffer was measured to be -0.3 ( 0.1 after 5 months incubation. Results confirmed that the pH of the surrounding environment remained constant as the hydrogels gradually degraded. The increase of swelling degree was associated with the degradation of polymer main chains due to the increase of cross-linking density. This constancy of pH during degradation of cyclic acetal segments indicated that it can be a new candidate for degradable segments that can be utilized in biomaterials.30,31 Conclusions In this study, we have successfully synthesized a novel type of hydrogels based on polyethers consisting of cyclic acetal segments and PEG units. The thermal properties of synthesized polyethers were strongly influenced by the molecular weight of PEG units. In particular, Tm of PECA was approximately 10 °C lower than that of PEG, although the total molecular weight was significantly different. These results were attributed to the prevention of PEG crystallization by cyclic acetal segments. On the other hand, the swelling properties of the prepared hydrogels were influenced by polymer concentration, initiator concentration, and molecular weight of PECA-DA. These factors influenced the cross-link density of the hydrogels and mobility of the polymers in the solution. The degradation of cyclic acetal segments were significantly influenced by the acidity and temperature of the buffering environment. In addition, the buffer pH remained constant after incubation of PECA hydrogel for 5 months. Overall, results demonstrated that PECA was readily synthesized and fabricated into hydrogels, whose properties were mostly controlled by PECA-DA concentration and constituent PEG length.

Figure 9. Degradation of PECA(600) hydrogels in PBS buffer at 37 °C: (a) remaining mass, (b) swelling degree, and (c) pH change of PBS buffer.

The temperature dependency of the reaction rate was also evaluated. The activation energy of degradation of EHD and PECA(600) at pH 4 were calculated by the Arrhenius relationship, where ln(k) ) ln(A) - Ea/RT, where A is the frequency factor, Ea is the activation energy, and R is the universal gas constant. The activation energies of degradation of EHD and PECA(600) were determined to be 77 and 89 kJ/mol, respectively. Assuming the activation energy for degradation of cyclic acetal is constant at the investigated temperatures, the degradation rate coefficient of cyclic acetal at 37 °C and pH 4 for EHD was estimated to be 1.0 × 10-3 h-1 with a half-life of 29 days. For PECA(600) the degradation rate coefficient was 5.5 × 10-4 h-1 with a half-life of 53 (day). The longer half-life of PECA than EHD could be due to the higher stability and lower mobility of cyclic acetal in high molecular weight polymer chains. The results indicate that the cyclic acetal EHD degrades more slowly than linear acetal such as benzylidene acetal cross-linker and amine-functionalized acetal monomers.29 This is likely due to the higher stability of cyclic acetal compared to linear acetal. Finally, hydrogels were degraded in vitro to demonstrate the degradation behavior under simulated physiological conditions

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