Supramolecular Hydrogels Exhibiting Fast In Situ Gel Forming and

Biomacromolecules 2010, 11, 617–625

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Supramolecular Hydrogels Exhibiting Fast In Situ Gel Forming and Adjustable Degradation Properties Ngoc Quyen Tran, Yoon Ki Joung, Eugene Lih, Kyung Min Park, and Ki Dong Park* Department of Molecular Science and Technology, Ajou University, 5 Wonchon, Yeoungtong, Suwon 443-749, Republic of Korea Received October 16, 2009; Revised Manuscript Received February 5, 2010

Fast in situ forming supramolecular hydrogels consisted of the tyramine-conjugated supramolecular structures and chitosan derivative were prepared via an enzymatic reaction with horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). The gel formation was varied within a time period of 5 s to 10 min by controlling the concentrations of HRP, H2O2, and polymers. Tyramine conjugation at different sites of the supramolecular structure resulted in significant changes in physical properties and the degradation time of the hydrogels that were confirmed by water uptake, compressive strength and degradation tests. In addition, the hydrogels showed a good cytocompatibility in vitro. These hydrogels could be promising injectable biomaterials with adjustable degradation times to control both the cellular behaviors as a regenerative cell matrix and the drug release behavior as a drug delivery vehicle.

Introduction Hydrogels are three-dimensional networks capable of absorbing large amounts of water or biological fluids. These polymeric networks have attracted much attention as drug/cell delivery carriers and tissue regeneration scaffolds in biomedical applications because of their biocompatibility and biodegradation.1 For these applications, in situ formation of these hydrogels has been intensively investigated because this enables minimally invasive surgical implantation.2,3 In in situ, gel-forming systems, a polymer solution can be injected into the body and then forms a desired shape of hydrogel. The method has been developed by utilizing thermosensitive polymers or particular chemical reactions such as photochemical reaction, Michael-type reactions, and Schiff-base reactions.4-6 Moreover, the degradation of hydrogels is a crucial property that has also been mentioned. For drug delivery, hydrogels act as the matrix for the release of therapeutic agents, such as bioactive small molecules or proteins, in a sustained or controlled manner for a desired time period. Biodegradation of the hydrogels through hydrolysis, proteolysis, and physical erosion partially governs the release behavior of the drug from the hydrogel.7,8 For tissue regeneration, many hydrogels temporarily play the role of extracellular matrix (ECM) in the early stage of tissue formation from cells. As tissue formation progresses, the hydrogel matrix is replaced by naturally secreted ECMs from the cells.9 An interesting approach using an enzyme-catalyzed reaction to prepare hydrogels was recently suggested.10,11 This approach not only enables the hydrogel to form at a mild condition in a short period of time, but also strengths mechanical property of the hydrogel due to the chemical cross-link, which is a major issue in the development of in situ formation of hydrogels. In recent years, cyclodextrin (CD)-based supramolecular hydrogels have studied as advanced functional hydrogels in biomedical applications due to their potentially unique structures. Yui et al. cross-linked ester-terminated polyrotaxane with poly(ethylene glycol) (PEG) bis-amine using carbamate linkages * To whom correspondence should be addressed. Tel.: +82-31-219-1846. Fax: +82-31-219-1592. E-mail: [email protected].

to form hydrogel.12,13 In these studies, R-cyclodextrins (R-CDs) in polyrotaxane were used as cross-linked points that disappeared by supramolecular dissociation via hydrolysis of the ester terminal groups. Feng et al. produced a biodegradable thermoresponsive hydrogel by threading R-CD on methacrylateterminated poly(lactide)-poly(ethylene glycol)-poly(lactide) (PLAPEG-PLA) triblock copolymer and photopolymerized the inclusion complex in the presence of N-isopropylacrylamide (NiPAAm).14 They also produced a kind of biodegradable hydrogel formed in situ through the photocopolymerization of a methacrylamide-grafted gelatin with polypseudorotaxane made from R-CDs with PLA-PEG-PLA terminated with methacryloyl moieties.4 Although these poly(pseudo)rotaxane-based hydrogels were designed well, they completely degraded in several days because hydrolysis of the ester groups was easy at the endcapped points or ester bonds the in polymer chains and, subsequently, R-CD dethreaded resulting in network dissociation. In this study, two kinds of tyramine-terminated polypseudorotaxane-chitosan hydroxylphenyl acetamide (PRx-CHPA) and tyramine-unterminated polypseudorotaxane-chitosan hydroxylphenyl acetamide (PPRx-CHPA) hydrogels based on chitosan and polypseudorotaxane were examined. Chitosan is a well-known biodegradable and biocompatible polymer. The presence of chitosan in the supramolecular hydrogels was expected to enhance the biocompatibility as well as the crosslinking capability of the backbone to P(P)Rx. Chitosan was modified with 4-hydroxyphenylacetic acid to role-play to play the role of a soluble polymer for an enzymatic reaction with P(P)Rx in presence of HRP and H2O2. A chitosan-based hydrogel has recently developed by Sakai’s group, 4-hydroxyphenylpropionic acid had been conjugated to chitosan, and then the chitosan’s derivative were used to prepare chitosan-based hydrogel via an enzymatic reaction.15 In this enzymatic reaction, HRP readily combines with hydrogen peroxide (H2O2) and the resulting (HRP-H2O2) complex can oxidize substrates such as tyramine, homovanillic acid, 4-hydroxyphenyl acetic acid and tyrosine. Two hydroxyphenolic compounds can be linked together under the catalyst. This has been utilized to form crosslinking bonds between polymer chains in the preparation of

10.1021/bm100047y  2010 American Chemical Society Published on Web 02/18/2010

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Figure 1. Schematics of the formation of supramolecular hydrogels via enzymatic cross-linking.

hydrogels.10,11 To prepare the P(P)Rx-CHPA hydrogels being adjustable degradation, P(P)Rx were modified with tyramine groups in two locations, at the terminal and external points on the tubular P(P)Rx as in Figure 1. In the case of the external TA-conjugated PPRx, The PPRx-formed hydrogel were rapidly degraded due to a gradual dethread of R-CD from the PPRx structure. For PRx, the TA molecules were conjugated to two hydroxyl end groups in the tubular PRx therefore the PRxformed hydrogels showed a lower degradation because of slow hydrolysis of the carbamate bonds at cross-linked sites and simultaneous dethreading of R-CD in another head of CHPAun-cross-linked PRx. Terminal tyramine in PRx was designed to play two roles, a partially bulky stopper that threaded R-CD from its saturated solution16,17 and a dethreading decrement for R-CD of PRx in the polymer solution prior to gelation because of a weak complex formation of the terminated tyramine with the inner cavity of R-CD.18-20 Rapid gelation between CHPA and modified P(P)Rx using an enzymatic reaction contributed to reduced dissociation of modified P(P)Rx in solution, con-

sidered to slowly dissociate in a large amount of water. Further, rapid gelation of these hydrogels has some advantages for in vivo injections.

Experimental Section Materials. Chitosan (low molecular weight, 75-85% deacetylation), 4-hydroxyphenylacetic acid, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), succinic anhydride, triethylamine (TEA), p-nitrophenyl chloroformate (NPC), tyramine (TA), hydrogen peroxide (H2O2), deuterium oxide (D2O), hexadeuterodimethyl sulfoxide (DMSO-d6), and horseradish peroxidase (HRP, type VI, 298 purpurogallin unit/mg solid) were purchased from Aldrich. R-CD was purchased from the Tokyo Chemical Industry. Poly(ethylene glycol) (PEG, Mw ) 3400 g/mol) was obtained from Polyscience Inc. Phosphate buffer (PBS, 150 mM, pH 7.4) was purchased from the B. Braun Co. All of the chemicals and solvents were used without further purification. Synthesis of Chitosan 4-Hydroxylphenylacetamide (CHPA). Chitosan (0.644 g, 4 mmol glucosamine) was dissolved in an aqueous solution of hydrochloride acid (100 mL, pH 3.5) into a 250 mL round

Fast In Situ Forming Supramolecular Hydrogels flask. The pH was adjusted to 5 and then 4-hydroxyphenylacetic acid (0.404 g, 2.6 mmol) was added to the flask. Then, EDC (0.768 g, 4 mmol) was added to the reaction mixture. The mixture was stirred at room temperature for 24 h. The solution was dialyzed (molecular weight cut off ) 8000) against 0.2 M NaCl aqueous solution at pH 4.5, followed by dialyzing against deionized water at pH 4.5. After dialysis, the modified chitosan was lyophilized. The degree of substitution (DS) was 19.8% (1H NMR). 1H NMR (D2O)/ppm: δ 1.86 (s, -COCH3 of chitosan), 2.68 (m, C2 (H) of chitosan and -CH2- of HPA), 3.20-4.00 (m, C3+4+5+6 (H) of chitosan), 6.68 and 7.00 (d, -CHCH- of HPA). The obtained CHPA was 0.68 g (yield: 65%). Synthesis of Tyramine-Terminated Poly(ethylene glycol) (TPEG). TPEG was synthesized using carbamate linkages. Dried PEG (3.06 g, 0.9 mmol) was completely dissolved in DMF (30 mL). The solution was placed into an ice bath holding the solution at a constant temperature of 0 °C. Then, TEA (0.36 g, 3.6 mmol) and NPC (0.72 g, 3.6 mmol) were added to the solution. The reaction mixture was stirred overnight under N2. Subsequently, TA (0.55 g, 4 mmol) was added to the mixture. The reaction mixture was stirred at room temperature for 12 h. The product was precipitated in excess diethyl ether to remove p-nitrophenol, a byproduct. The precipitate was dissolved in methylene choride and filtered. The TPEG solution was precipitated again in diethyl ether because the TEA salt and excess of TA did not dissolve in methylene chloride. The obtained white powder was dried under vacuum for 3 days. After drying, 2.86 g of TPEG was obtained (yield: 80.3%). The DS was 94.5% (1H NMR). 1H NMR CDCl3/ppm: δ 2.68 and 3.05 (m, -CH2CH2- of TA), 3.50 (s, -CH2CH2O- of PEG), 6.76 and 6.95 (d, -CHCH- of TA). Preparation of PRx-Sa. PRx was prepared according to the method described in previous reports.21,22 A total of 4 mL of an aqueous solution of TPEG (0.32 g, 0.085 mmol) was added to an aqueous solution saturated with R-CD (3.5 g, 3.6 mmol), which the stoichiometry of the ethylene oxide unit of PEG to R-CD is 2:1. The mixture was sonicated for 30 min and stirred for 24 h. Unthreaded R-CDs were removed by centrifugation and washing twice. The white powdery PRx was dried at room temperature under vacuum for 3 days. After dry, 2.34 g of PRx was obtained (yield: 61.5%). Approximately 26 units (1H NMR) of threaded R-CD were in PRx. PRx (2.3 g) was dissolved in the mixed solvent of DMF and dry pyridine (8 mL, v/v ) 3). Succinic anhydride (0.8 g, 8 mmol) was added to the solution under N2, and then the mixture was stirred at room temperature for 12 h. The reaction mixture was precipitated in a mixed solvent of diethyl ether and ethanol (v/v ) 2/1) and filtered. The product was dried at room temperature under vacuum for 3 days to obtain powdery PRx-Sa (yield: 1.67 g, 51.6%). The threading numbers of R-CD onto a PEG and DS was calculated to result in approximately 20 and 9% over the total hydroxyl groups of R-CD, respectively, by 1H NMR. 1H NMR DMSO-d6/ppm: δ 2.30-2.65 (m, -CH2CH2- of Sa), 3.10-3.50 (m, C2,4 (H) of R-CD), 3.50 (s, -CH2CH2O- of PEG), 3.55-4.40 (m, C3+5+6 (H) of R-CD), 4.50 (s, C6 (OH) of R-CD), 4.78 (s, C1 (H) of R-CD), 5.43 (m, C3 (OH) of R-CD), 5.57 (m, C2 (OH) of R-CD), 6.61 and 6.97 (m, -CHCH- of TA). Preparation of PPRx-Tsa. Polypseudorotaxane (PPRx) was also prepared in the similar manner to PRx. An aqueous solution saturated with R-CD (3.5 g, 3.6 mmol) was combined with 4 mL of an aqueous solution of PEG (0.31 g, 0.092 mmol). The weight, yield, and threading number of R-CD were determined as 2.85 g, 74.0%, and ∼31 by 1H NMR. TA was conjugated to the obtained PPRx via the carbanmate linkage. In the first step, PPRx (2.5 g) was dissolved in a mixed solvent of DMF and dry pyridine (8 mL, v/v ) 3). The solution was kept in an ice bath maintained at 0 °C. Then, NPC (0.4 g, 2 mmol) was added to the solution. The mixture was stirred under nitrogen for 4 h. Subsequently, succinic anhydride (1 g, 10 mmol) was added to the solution and followed by stirring at room temperature under N2 for 8 h. The reaction mixture was precipitated in excess diethyl ether to remove any unreacted succinic anhydride and a small amount of decomposed p-nitrophenol. After filtration, PPRx-Sa-NPC was dried


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at room temperature under vacuum for 2 days. In the second step, TA was conjugated to PPRx-Sa-NPC to give PPRx-TSa. Briefly, PPRxSa-NPC was dissolved in DMF (8 mL) under N2. After 20 min, TA (0.27 g, 2 mmol) was added to the solution, followed by stirring at room temperature for 8 h. The product was precipitated in a mixed solvent of diethyl ether and ethanol (v/v ) 2:1). After drying at room temperature under vacuum for 3 days, 1.6 g (yield: 45.7%) of PPRxTSa with R-CD threading number of 22 was obtained. The degree of TA substitution was around 2.78% (1H NMR). 1H NMR DMSO-d6/ ppm: δ 2.40-2.60 (m, -CH2CH2-, Sa), 3.10-3.50 (m, C2+4 (H), R-CD), 3.52 (s, -CH2CH2O-, PEG), 3.53-3.84 (m, C3+5+6 (H), R-CD), 4.80 (m, C1 (H), R-CD), 5.20-5.80 (m, C2+3 (OH), R-CD), 6.62 and 6.94 (m, -CHCH-, TA). Polymer Characterizations. The structure and composition of CHPA, PRx-Sa, and PPRx-TSa were analyzed using 1H NMR (Varian, 400 MHz, U.S.A.) at 37 °C. D2O was used as a solvent for CHPA. The amount of substituted HPA on chitosan was determined with the integrals of signals attributed to aromatic protons HPA and glucosamine protons of chitosan. TPEG was measured in CDCl3. The degree of TA substitution was determined by the integrals of resonances from methylene protons adjacent to terminal carbamate bonds and the rest methylene protons of PEG. The average number of threaded R-CD of modified PRx and PPRx was calculated with the integral of signals attributed to the H1 protons of R-CD and the methylene protons of PEG. The degrees of succinylation and TA substitution relied on C1(H) of R-CD and succinated protons or aromatic TA protons. Wide-angle X-ray diffraction was carried out with a type of film as a specimen using a Rigaku Dmax-2200 PC type X-ray diffractometer (Ni-filtered, Cu KR radiation with a wavelength of 0.154 nm). The samples were scanned from 10 to 50° in 2 θ at a speed of 2° per min. Preparation and Characterization of Hydrogels. Hydrogels (1 mL) were prepared in vials at room temperature. In a typical example of a PRx-CHPA hydrogel, CHPA (4 mg) and PRx-Sa (36 mg) were dissolved in a PBS solution (pH 7.4, 850 µL). A PBS solution of HRP (42 µL of 0.6 mg/mL stock solution) and H2O2 (108 µL of 0.25 wt % stock solution) were added to the polymer solution with stirring gently at a vortex. The PPRx-CHPA hydrogel was prepared in a similar manner to PRx-CHPA. Briefly, PBS solutions of HRP (210 µL of 0.6 mg/mL of stock solution) and H2O2 (135 µL of 1 wt % stock solution) were added to the PBS solution (655 µL) of CHPA (20 mg) and PPRx-TSa (20 mg), respectively. The final concentration of the mixed solution was 4% (w/v). The time in which it took the gel to form (denoted by gelation time) was determined using the vial tilting method.11,23 The gelation time was determined by regarding the gel state when the solution did not flow for 1 min after inverting a vial. Equilibrium Water Content. Water uptake of P(P)Rx-CHPA hydrogels was determined by using agravimetric method. Samples of about 0.2 g of a hydrogel made from P(P)Rx/CHPA were lyophilized and weighted their dry state (Wd). These dried hydrogel samples were immersed in 1 mL buffer solutions at 37 °C for 1 day to reach equilibrium swelling. After removal of surface water, the samples were weighted (Ws). The water content of these dried hydrogels is expressed as (Ws - Wd)/Wd × 100. Mechanical Properties. Compressive tests of the hydrogels were performed on a Universal Testing Machine (Unitech TM, R&B, Korea). Hydrogels (n ) 4) were prepared in Teflon mold with uniform rectangular shapes and then placed on the metal plate, where they were pressed at a crosshead speed of 1 mm/min. Individual compressive strengths were obtained from the load-displacement curve at break. In Vitro Degradation Test. The PPRx-CHPA and PRx-CHPA hydrogels (1 mL) were prepared in vials according to the abovementioned procedure and accurately weighed (Wi). The samples were subsequently incubated in 2 mL of PBS solution (pH 7.4) at 37 °C. The hydrogels were weighed to determine the gel weight (Wt) at regular time intervals. After hydrogels were weighed, a fresh PBS solution was added to the samples. The weight ratio of hydrogels (Wt/Wi) was

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Scheme 1. Synthetic Scheme of CHPA

plotted as a function of incubation time. All experiments were carried out with each sample of hydrogels in triplicate. Cell Culture. Mouse embryonic fibroblasts (cell line NIH/3T3) were used for cytotoxicity evaluation. The NIH/3T3 cells were subcultured twice a week at 37 °C in an humidified atmosphere of 5% CO2 and maintained at low passage number (5-10). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) cell culture medium containing 10% (v/v) fetal bovine serum (FBS) and 1% antibiotics (100 U mL-1 penicillin and 0.1 mg · mL-1 streptomycin). The culture medium was replaced every 3 days. At confluence, the cells were detached using trypsin/EDTA in PBS, resuspended in DMEM, and used for the cytotoxicity. Cell Encapsulation within Hydrogels. To encapsulate the cells, solid polymers were first exposed to UV light for 30 min. HRP and H2O2 solutions in PBS were sterilized by filtration through 0.2 µm syringe filters. The cells were encapsulated in the hydrogels using the same procedure as for the gel formation in the absence of cells. Every hydrogel precursor solutions 2 wt %/v was prepared into two parts with the same volume ratio (one contained HRP and the other contained H2O2). The medium containing fibroblasts was added to the HRPcontained solution. DMEM was added to the other with the same ratio. Cellular hydrogels were prepared in 24 wells plate by mixing of the cellular HRP-contained solution and H2O2-contained solution. The cell seeding density in the gels was 1 × 106 cell/mL. The cellular hydrogels were immersed in cell culture medium and incubated at 37 °C and 5% CO2 for 24 h. Cytotoxicity Assay. A viability analysis of the encapsulated cells in the hydrogels was performed with a Live/Dead assay composed of fluorescein diacetate (FDA) and ethidium bromide (EB). FDA stains the cytoplasm of viable cells green, while EB stains the nuclei of nonviable cells red. After incubating 24 h, the cellular hydrogels were rinsed with PBS and stained with FDA/EB assay solution, according to the manufacturer’s instructions. The stained cells in hydrogel were visualized using Confocal laser scanning microscope (EZ-C1, Nikon, Japan). As a result, living cells fluoresce green and the nuclei of dead cells fluoresce red.

Results and Discussion Synthesis of CHPA. As shown in Scheme 1, the carboxyl group of HPA is covalently bound to amine groups of chitosan by activating with EDC, which the chemical structure could be confirmed. Figure 2 shows the 1H NMR spectrum of HPAconjugated chitosan (CHPA). Resonance signals at 6.64 and 7.00 ppm are attributed to the aromatic protons of HPA coupled to chitosan. The signal attributed to methylene group of HPA is

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overlapped with signals of chitosan near 2.7 ppm. Many broad, overlapped resonance signals at around 2-4 ppm are commonly observing characteristic signals of chitosan. From this NMR spectrum, the degree of HPA substitution to chitosan was estimated to be 19.8%, which could be calculated with the integrals of the HPA and glucosamine protons. HPA played an important role in the enzyme-catalyzed cross-linking reaction for hydrogel formation because HPA was easily dimerized in the presence of HRP and H2O2.10,11 The higher degree of HPA substitution facilitates to cross-link between chitosan chains. Furthermore, HPA conjugation improved the poor solubility of not modified chitosan because the conjugated HPA disturbs extended hydrogen bonds between chitosan chains. Preparation of PRx-Sa. A polyrotaxane conjugated with succinic anhydride (PRx-Sa) was prepared as shown in Scheme 2. Figure 3a demonstrates that TPEG was synthesized via carbamate bond between TA and PEG. Weak resonance signals at 2.68, 3.05, 6.76, and 6.95 ppm are assigned to methylene protons adjacent to carbamate group and phenyl protons of TA. A peak at δ 4.14 ppm is also assigned to methylene protons between ethylene glycol unit and carbamate group. TAterminated PEG (TPEG) could thread R-CD molecules to construct polyrotaxane-like structure (PRx). The TA groups of PRx will be cross-linked to HPA group of CHPA or other TA group of PRx. In the structure of PRx, TA also seems to play a role in reducing the dethreading of R-CD because of the weak complex formation between TA and R-CD. Figure 3b shows new signals for the R-CD protons beside the TPEG protons at many different resonances (3.20-3.50 C2,4 (H), 3.50-3.80 C3+5+6 (H), 4.51 C6 (OH), 4.79 C1 (H), 5.45 C3 (OH), and 5.57 C2 (OH) of R-CD). The result of 1H NMR ascertains the formation of the inclusion complex between TPEG and R-CD. The threading number of R-CD has used to assess the effectiveness of the threaded guest molecules onto polymers chain in the P(P)Rx preparations. In the preparation, 26 units of R-CD threaded onto TPEG, which was lower than the tyramineunterminated PEG backbone16 This can be explained that the weak complex formation between terminated tyramine groups of TPEG and R-CD partially disturbs the threading process.17 The obtained PPRx and PPX products were not dissolved in water due to the threaded R-CD. The threaded R-CD molecules interact with each other on a TPEG chain and construct a hydrophobic channel-like structure because of hydrogen bonds between hydroxyl groups. Poor solubility of their structures in water makes them not favorable for hydrogel formation. Thus, the succinylation of the PRx was carried out to increase the hydrophilicity of PRx. Figure 3c shows the NMR spectrum of succinylated PPRx. Broad signals from protons of the carboxyl ethylester group on R-CD were observed at 2-4 ppm, which have not been observed in the spectrum of PRx. This indicates that the succinylation on R-CD of PRx was really achieved, demonstrated by observing that poor soluble PRx became soluble in water. In addition to this, signals from R-CD protons in PRx-Sa became broader as compared to sharp those in PRx. This is ascribed to changes in interactions between R-CDs. Most of the hydroxyl groups of R-CD in PRx would interact with each other via a hydrogen bond. On the other hand, hydroxyl groups of R-CD in PRx-Sa might interact with water molecules as well as each other. This difference in interactions seems to make changes in resonance signals. It is well-known that modified R-CD molecules can rotate and slide on a linear polymer chain. Similarly, succinylated R-CD molecules could be hydrated and move around along the TPEG chain. Theoretically a maximum threading number of R-CD onto the PEG used

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Figure 2. 1H NMR spectrum of CHPA in D2O. Scheme 2. Synthetic Scheme of PRx-Sa

in this study is calculated to be ∼38 because the stoichiometry of R-CD inclusion to the ethylene glycol unit of PEG is known as 2:1. Therefore, the threading efficiency of R-CD is 68% of maximum inclusion with PEG. The threading number of R-CD in PRx-Sa (∼20) decreased as compared to that of PRx (∼26), which resulted from the dethreading of R-CD due to the succinylation. Preparation of PPRx-Tsa. Polypseudorotaxane conjugated with tyramine and succinic anhydride (PPRx-TSa) was prepared in the similar manner to PRx-Sa as shown in Scheme 3. Figure 4 shows the NMR spectra of PPRx and PPRx-TSa. As shown in Figure 4a, PPRx shows the same spectrum to that of PRx except the absence of signals from tyramine group. In Figure 4b, however, both signals from the carboxyl ethyl ester group at 2.4-2.6 ppm and signals from the phenyl group at 6.62 and 6.94 ppm are observed due to the conjugation of succinct anhydride and tyramine. In addition, signals derived from hydroxyl protons of R-CD are significantly changed. This evidently results from the conjugation of succinic anhydride and

Figure 3. 1H NMR spectra of TPEG in CDCl3 (a), PRx (b), and PRxSa (c) in DMSO-d6.

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Scheme 3. Synthetic Scheme of PPRx-TSa

Figure 5. X-ray diffraction patterns of PEG (a), TPEG (b), R-CD (c), PPRx (d), PPRx-TSa (e), PRx (f), and PRx-Sa (g).

tyramine to R-CD like the case of PRx-Sa. The signal for OH-6 protons is nearly gone. Broadened signals assigned to OH-2 and -3 protons are attributed to the hydrogen bonds between the hydroxyl groups and water molecules. The threading number of R-CD in PPRx was about 31. This number is larger than

Figure 4. 1H NMR spectra of PPRx (a) and PPRx-TSa (b) in DMSO-d6.

that in PRx, showing high threading efficiency of about 82%. Terminal tyramine groups of PEG in PRx can be included in the R-CD cavity to form inclusion complexes, in which subsequent inclusion by R-CD can be protected. Thus, the threading number in PRx could be lower than that in PPRx. In both PRx and PPRx, the conjugation processes induced decreases in the threading number of R-CD. The reduced percentages of threading number in PRx and PPRx were about 23 and 29, respectively. This indicates that structure of PRx terminated with tyramine is little more stable than that of PPRx without tyramine group because of the end-cappting effect of tyramine. This result also demonstrates that PPRx-TSa was prepared by suppressing the dethreading of R-CD from PEG, which will be confirmed in W-XRD data. The degree of TA substitution was 2.78%, indicating that 10 tyramine molecules were conjugated to a PPRx-TSa molecule. Crystalline Structures of Inclusion Complexes. Figure 5 shows the X-ray diffraction patterns of PEG, TPEG, R-CD, PPRx, PPRx-TSa, PRx, and PRx-Sa. The diffraction peaks of pure R-CD were observed at 12.02, 14.18, and 21.70°, as commonly reported. The crystalline structures of PEG and TPEG were confirmed by two diffraction peaks at 19.08 and 23.30°, derived from the crystalline PEG chains.7 The terminal tyramine group of TPEG probably affected the crystalline structure of PEG because slightly changed diffraction peaks were observed. The diffractograms of PPRxs and PRxs exhibited two main diffraction peaks at 19.82 and 22.62°, which are different from the crystalline structure of PEG (TPEG) and R-CD. These peaks were attributed to the presence of the tubular or channel-type crystalline structure in PRx and PPRx constructed by forming inclusion complexes of PEG (or TPEG) and R-CD.4,24 PPRxTSa and PRx-Sa show little different peaks, although those are similar peaks to PRx and PPRx at 19.82 and 22.62°. This indicates that the channel-like structures were partially destructed after the substitution of R-CD, though a channel-type crystalline structure still remained. It is known that the substitution of a molecule to hydroxyl groups of R-CD collapses the channeltyped structure of CDs because of disturbing the interaction between CDs. In addition, diffraction peaks of R-CD, PEG, and TPEG disappeared at the diffractograms of PPRxs and PRxs, demonstrating that not included, free molecules and polymers nearly did not exist together with inclusion complexes. These results suggest that most of the PEG or TPEG were included into R-CD molecules to form PPRx or PRx structures, and most



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Figure 6. Photographs showing in situ hydrogel formation from 4% (w/v) solutions of PRx-Sa (upper) and PPRx-TSa (lower) mixed with CHPA. Inversed vials demonstrate the formation of hydrogels within 10 and 20 s, respectively.

of their structures were still maintained in spite of the chemical modification of R-CD. In Situ Hydrogel Formation. Hydrogels were prepared via the HRP-mediated coupling reaction of phenol moieties in PRxSa (or PPRx-TSa) and CHPA, as shown in Figure 1. The coupling of phenols can take place either via a carbon-carbon bond at the ortho positions of phenol groups or via a carbon-oxygen bond between the carbon atom at the ortho position of phenol groups and the phenoxy oxygen.10,11,25-27 The enzymatic cross-linking between PRx-Sa and CHPA or PPRx-TSa and CHPA is carried out under mild reaction conditions containing room temperature, neutral pH, and aqueous solution. The mixed solutions formed an opaque solid state by adding HRP and hydrogen peroxide (see Figure 6). At the polymer concentration of 4% (w/v), the mixed solutions were opaque because the solutions contained crystalline structures of inclusion complexes, resulting in opaque hydrogel phases after cross-linking. The gelation time was very fast and changed at the wide ranges from tens to hundreds of seconds. In Figure 7a, the gelation times decreased from ∼170 to ∼10 s as the ratio of HRP/(TA+HPA) increased from 0.5 to 3 mg/mmol at a constant polymer concentration of 4% (w/v) and H2O2/ (TA+HPA) ratio ) 1:1 mol/mol. This is presumably ascribed to increases in the rate of the decomposition of hydrogen peroxide and the production of phenoxy radicals by HRP. In addition, the gelation times of PRx and CHPA are a little faster than those of PPRx and CHPA at the same HRP concentrations. These differences seem to be derived from structural differences between PRx-Sa and PPRx-TSa. The structural differences contain the amount of conjugated tyramine, conjugated positions of tyramine, and the threading number of R-CD. Figure 7b shows the effect of H2O2 concentration on gelation time. In Figure 7b, contrary to Figure 7a, the gelation times increased as the molar ratio of H2O2/(TA+HPA) increased from 0.5 to 3 under the same conditions containing a polymer concentration of 4% (w/v) and the HRP/(TA+HPA) ratio of 1.5 mg/mmol. At the highest concentration of H2O2, it took several minutes to complete the gelation process because an excess amount of H2O2 oxidized HRP to make an inactivated form, demonstrated by the fact that the gelation time significantly increased at that concentration.11 The fastest gelation time of 10-20 s was observed at the H2O2/(TA+HPA) ratio of 0.5 mol/mol in both hydrogels. At this ratio, the used concentration of H2O2 was 8 mM, which was nontoxic. A previous report described that the

Figure 7. Gelation time of the hydrogels under various conditions: (a) Gelation time of a 4% (w/v) polymer solution with H2O2/(HPA+TA) ) 1 mol/mol; HPA/TA ) 1.5 mol/mol as a function of HRP/HPA+TA. (b) Gelation time of a 4% (w/v) polymer solution with HRP/(HPA+TA) ) 1.5 mg/mmol; HPA/TA ) 1.5 mol/mol as a function of H2O2/ HPA+TA. (c) Gelation time of polymer solutions with HRP/(HPA+TA) ) 1.5 mg/mmol; HPA/TA ) 1.5 mol/mol; H2O2/(HPA+TA) ) 0.5 mol/ mol as a function of polymer concentration.

hyaluronic acid-TA hydrogel formed in the presence of 70 mM H2O2 was nontoxic and biocompatible.10 In this study, the HRP/ (TA+HPA) ratio of 1.5 mg/mmol and the H2O2/(TA+HPA) ratio of 0.5 mol/mol were used to investigate the effect of the polymer concentration on gelation time. Figure 7c shows the effect of polymer concentration on gelation time, the time decreased as the polymer concentration increased. The behavior was caused by increasing cross-linking density in polymer solutions This was also found in Feijen’s study when they investigated the gelation of dextran-tyramine via the HRPmediated condition. At lower polymer concentrations, the gelation times significantly increased, whereas the gelation time

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Figure 9. Compressive strength of P(P)Rx-CHPA hydrogels at different polymer concentrations. Figure 8. Water uptake of hydrogels at different polymer concentrations.

slightly decreased at the higher concentrations. On the contrary, at higher concentrations, the concentration effect on gelation time is not so significant. These may explain that polymer solutions increased in viscosity, resulting in the inhibition of the catalytic dispersion. The high viscosity of CHPA also made it impossible to prepare the PPRx-CHPA hydrogel at higher precursor polymer concentration. Under the same conditions for preparation of P(P)Rx-CHPA hydrogels, we tried to prepare the CHPA hydrogel with the low and high concentration, but we could not control the gelation. The CHPA hydrogel immediately formed but it was unstable and heterogeneous. There has been more water released from the obtained gels after some minutes (images are not shown here). These may be contributed by both a high density of the crosslinking groups and a low solubility of CHPA. The gelation times at all conditions were comparatively fast and controllable at a wide range of times, suggesting that hydrogels may be applied to in situ forming injectable biomaterials. Water Uptake. Water absorbance were assessed for the dried hydrogels at different precursor polymer solutions. Figure 8 shows a significantly difference in equibrilium water content of hydrogels. As the polymer concentrations increased from 2 to 4%, the amount of water uptake increased. This indicates that network formation is more efficient at higher polymer concentrations This might also be contributed by increment in content of the succinilated P(P)Rx in hydrogel structures. The water uptake of the PRx-formed hydrogels were higher than that of PPRx-formed hydrogels. This indicates that the PRx-CHPA hydrogels network is more stable than that in PPRx-CHPA hydrogels due to the dethread of R-CD in PPRx-CHPA. This results in a decrement in the ability to hold water inside the PPRx-CHPA network structures as well as a fast degradation of PPRx-CHPA hydrogels. Compressive Strength. The obtained compressive strength of the hydrogels on the cubic shapes demonstrated that the precursor polymers concentration in the P(P)Rx-CHPA hydrogel increased, resulting in increasing mechanical strength of the materials, as shown in Figure 9. In PPRx-CHPA, the values were insignificantly different as the polymer concentration was increased. As prepared, the external cross-linking sites on PPRx were connected based on physical bonds of the host-guest interaction. The interaction was considered less stable, which resulted in a low mechanical property of the PPRx-CHPA hydrogels. On the contrary, the values were significantly different in PRx-CHPA as the polymer concentration increased. This may be contributed by increasing the cross-linking density in the stabilized PRx and CHPA. Degradation Behaviors of Hydrogels. Hydrogels revealed quite different degradation profiles as varying the polymer con-

Figure 10. Weight ratios of hydrogels having different concentrations for a 30 day incubation in aqueous media: Hydrogels prepared from 10, 4, and 2% solutions of PRx and CHPA, and 4 and 2% solutions of PPRx and CHPA at the same molar ratios of HPA to TA (1.5), of HRP to (HPA+TA) (1.5), and of H2O2 to (HPA+TA) (0.5).

centration and supramolecular structure at the same conditions containing molar ratios of HPA to TA (1.5), of HRP to (HPA+TA) (1.5), and of H2O2 to (HPA+TA) (0.5). Figure 10 shows the degradation profiles of hydrogels with different polymer concentrations and supramolecular structure. After only a few days, all hydrogels show increases in the weight ratio, although the degree of increases is varied depending on preparation conditions. Distinct difference in degradation behaviors between PPRx and PRx hydrogels was observed in their degradation profiles. At the same polymer concentrations, the degradation rate of PPRx hydrogels is remarkably higher than that of PRx hydrogels. In 4% hydrogels (w/v), PRx hydrogel is much more slowly degraded than PPRx hydrogel. This difference in their degradation rates result from distinct different supramolecular structure between PPRx and PRx. In the structure of PPRx, threaded R-CD molecules onto PEG are linked to the modified chitosan chain (CHPA) without end-capping of the terminal groups of PEG. On the contrary, in the structure of PRx, the two ends of PEG threaded with R-CD are linked to the CHPA. Thus, it is suggested that PPRx hydrogels show an extremely fast degradation rate in particular at the initial period of incubation due to dethreading of R-CD molecules, resulting in the disruption of a supramolecular network between PPRx and CHPA. In PRx hydrogels, the degradation rate also susceptibly depends on the polymer concentration. PRx hydrogel (10%) exhibits



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PRx-based hydrogel and PPRx-based hydrogel. These hydrogels had several advantages compared to many in situ forming hydrogels of stimuli-sensitive polymers, including a comparatively lower polymer concentration for in situ gelation, a fast and controllable gelation time, and precise degradation control. These features were evidently attributed to peculiar supramolecular structures and efficient enzymatic cross-linking. In particular, the controlled degradation and cytocompatibility of these hydrogels may play crucial roles in governing the release mechanism of encapsulated drugs by diffusion for drug delivery and the regeneration process of encapsulated cells for regenerative cell therapy. Figure 11. Fluorescent microscopy images of fibroblasts in the P(P)Rx-CHPA hydrogels after a 24 h incubation using Live/Dead assay (scale bar: 100 µm).

increased gel weight up to 25 days because of high swelling capacity due to relatively higher polymer concentration, whereas 2% PRx hydrogel shows significant weight loss, in which the hydrogel is completely degraded at 20 days. However, this degradation behavior did not occur in PPRx hydrogels, showing that the difference in degradation rate between 2 and 4% hydrogels was almost not observed. In PRx hydrogels, the higher concentration of polymers results in more robust and dense hydrogel network. The degradation of hydrogels is mainly governed by hydrolysis at their cross-linked linkages, which depends on their concentration. In contrast, in PPRx hydrogels, the degradation of hydrogels could be dominated by dethreading of R-CD molecules, giving rise to dissociation of the cross-linked structure between PPRxs and CHPA, which is likely to not be dependent on polymer concentration.28,29 The interaction between chitosan chains could also affect the degradation time of hydrogels. It is well-known that chitosan binds each other into an extensive range of chain via hydrogen bond, resulting in insolubility in water. Interestingly, in 2% hydrogels, PRx hydrogel shows more weight loss than PPRx hydrogels after 15 days, while 4% hydrogels result in much lower weight loss of PRx hydrogel than PPRx hydrogel. This is probably attributed to the lack of cross-linking degree, which is dependent on polymer concentration rather than structural difference. PPRx hydrogels remained at about 20% of the initial hydrogel weight up to 30 days after significant weight loss at the initial stage of degradation, resulting from the presence of remaining threaded R-CD molecules cross-linked with CHPA. These results on degradation behavior of hydrogels may provide a clue to employ their combinations in controlling a more diverse degradation profile of hydrogels. Biocompatibility of Hydrogels. To study a biocompatibility of P(P)Rx-CHPA hydrogels, fibroblasts were encapsulated in the gels and stained using a Live/Dead assay kit following one day of encapsulation. The viable and dead cells (stained green and red) were visualized by a Confocal laser scanning microscope. Figure 11 shows that over 95% of living cells were observed in both hydrogels. These obtained results show that the polypseudorotaxane and chitosan-based hydrogels have a good cytocompatibility.

Conclusions Novel in situ forming supramolecular hydrogels were prepared with CD/PEG inclusion complexes and a chitosan derivative via a fast enzymatic reaction. 1H NMR and W-XRD were used to confirm the threaded CDs and chemical structures in the supramolecular structures and chemically modified chitosan. Fast hydrogel formation was achieved using a unique enzymatic reaction. Physical and mechanical properties showed the significantly different behaviors in water absorbance and compressive strength between

Acknowledgment. This study was supported by grants of the Korean Health Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A091120) and the Pioneer Research Center Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2009-0082804).

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Supramolecular Hydrogels Exhibiting Fast In Situ Gel Forming and

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