Biodegradable and pH-Sensitive Hydrogels for Cell Encapsulation

Hydrogels for cell encapsulation and proliferation are of importance in tissue engineering(1-4) since hydrogels have excellent permeability, which all...
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Biomacromolecules 2008, 9, 1155–1162

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Biodegradable and pH-Sensitive Hydrogels for Cell Encapsulation and Controlled Drug Release De-Qun Wu, Yun-Xia Sun, Xiao-Ding Xu, Si-Xue Cheng, Xian-Zheng Zhang,* and Ren-Xi Zhuo* Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072, People’s Republic of China Received September 17, 2007; Revised Manuscript Received January 26, 2008

Hydrogels with pH-sensitive poly(acrylic acid) (PAAc) chains and biodegradable acryloyl-poly(-caprolactone)-2hydroxylethyl methacrylate (AC-PCL-HEMA) chains were designed and synthesized. The morphology of hydrogel was observed by scanning electron microscopy. The degradation of the hydrogel in the presence of Pseudomonas lipase was studied. The in vitro release of bovine serum albumin from the hydrogel was investigated. Cytotoxicity study shows that the AC-PCL-HEMA/AAc copolymer exhibits good biocompatibility. Cell adhesion and migration into the hydrogel networks were evaluated by using different cell lines. The hydrogel with a lower cross-linking density and a larger pore size exhibited a better performance for cells migration.

Introduction Hydrogels for cell encapsulation and proliferation are of importance in tissue engineering1–4 since hydrogels have excellent permeability, which allows diffusion and transport of essential materials, such as oxygen and nutrients, for cells. During the past decade, intelligent hydrogels that can alter their volumes and properties in response to ambient stimuli have drawn extensive research interest in biomedical and pharmaceutical applications, including controlled drug release,5–7 tissue culture substrates,8,9 and cell scaffolds.10,11 Among them, pHsensitive hydrogels have been extensively studied.6,12–14 Recently, numerous biodegradable hydrogels were developed to meet various applications including drug delivery and cell scaffolding.15,16 Furthermore, a wide range of biodegradable and biocompatible synthetic aliphatic polyesters such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and their copolymers are studied as biodegradable cross-linkers to achieve biodegradability of the hydrogels.17,18 However, PLA and PGA are stiff materials, which make them unsuitable as soft materials in tissue engineering. Among the biodegradable aliphatic polyesters, poly(-caprolactone) (PCL) and caprolactone (CL) copolymers have been widely developed, especially for drug delivery and cell proliferation owing to the high permeability of PCL to various molecules.19,20 To date, factors that affect the solute release from degradable cross-linked polymers, including cross-linker density, solute molecular weight, pH, hydrolytically labile spacers, and enzymes have been reported.21–24 Among them, the enzymatic degradation has been widely investigated. Pseudomonas lipase,25,26 rhizopus arrhizus lipase,27 and rhizopus delemer lipase28 have studied to accelerate the degradation of PCL. Owing to the three-dimensional network structure and viscoelasticity, which are similar to the extracellular matrix (ECM) in biological tissues, hydrogels are promising scaffolds not only for cell adhesion, spreading, and proliferating but also for repairing and regenerating varieties of tissues and organs.29,30 * Corresponding authors. Tel: 86-27-68754509. Fax: 86-27-68754509. E-mail addresses: [email protected] (X.-Z. Zhang), bmplab@ public.wh.hb.cn (R.-X. Zhuo).

Chen et al.31 reported that bovine fetal aorta endothelial cells could spread and proliferate on PAAc hydrogel without any cell adhesive protein modification. From the standpoint of applications, there is need to develop nontoxic, biodegradable hydrogels for biomedical applications without losing their smart properties, such as temperature sensitivity and/or pH sensitivity. In this study, we not only maintained the initial hydrogel’s biodegradability but also incorporated extra pH sensitivity to the biodegradable hydrogel. The resultant hydrogels exhibited better property in controlled release within varied pH. On the other hand, the hydrogels could act as a scaffold and wrap cells by the migration of the cells through the pore of hydrogels during the degradation of the hydrogels. The hydrogels can be a candidate as scaffolding for cell encapsulation and tissue regeneration applications.

Experimental Section Materials. -Caprolactone (CL) purchased from Sigma Chemical Co. (St. Louis, MO) was dried over CaH2 for 48 h and then distilled under reduced pressure. Lipase from pseudomonas cepacia was purchased from Fluka and used as received. 2-Hydroxylethyl methacrylate (HEMA), dimethyl sulfoxide (DMSO), acrylic acid (AAc), and stannous 2-ethyl hexanoate (SnOct2) were obtained from Shanghai Chemical Reagent Co. (China) and used after distillation under reduced pressure. Toluene and tetrahydrofuran (THF) were dried by refluxing over CaH2 and Na complex, and distilled just before use. Dubelcco’s Modified Eagle’s Medium (DMEM) was obtained from GIBCO Invitrogen Corp. Ammonium persulfate (APS), bovine serum albumin (BSA, MW ) 67000 g/mol), triethylamine, sodium azide, acryloyl chloride (AC), N,N,N′,N′-tetramethylethylenediamine (TEMED), and 2,6-di-tert-butyl-4-methylphenol were obtained from Shanghai Chemical Reagent Co. (China) and used directly. Synthesis of Acryloyl-Poly(E-caprolactone)-2-Hydroxylethyl Methacrylate (AC-PCL-HEMA) Macromonomer. PCL-HEMA was synthesized according to the literature32 with some modification. The synthesis of the AC-PCL-HEMA macromonomer as well as the ACPCL-HEMA/AAc hydrogel is illustrated in Scheme 1. Briefly, -caprolactone (17.10 g, 0.15 mol), HEMA (3.90 g, 0.03 mol), and catalyst SnOct2 (0.1221 g, 1 mol % with respect to HEMA) were added into a glass ampule with a magnetic bar. Then 2,6-di-tert-butyl-4-methylphe-

10.1021/bm7010328 CCC: $40.75  2008 American Chemical Society Published on Web 02/29/2008

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Scheme 1. Synthesis of the AC-PCL-HEMA Macromoner and AC-PCL-HEMA/AAc Hydrogel

Table 1. Feed Composition and Sample Code of the AC-PCL-HEMA/AAc Hydrogels sample codea component

gel 1

gel 2

gel 3

gel 4

gel 5

AC-PCL-HEMA/mg acrylic acid/mg APS/mg TEMED/µL DMSO/mL

20 380 20 10 3

40 360 20 10 3

60 340 20 10 3

80 320 20 10 3

120 280 20 10 3

a

Preparation of hydrogels was carried out at room temperature for 20 h.

nol (0.067 g) was added, and the ampule was sealed under vacuum ( gel 4 > gel 5. Figure 5B shows that the order of weight loss for different hydrogels was similar to that in the 0.2 mg/mL enzyme solution in Figure 4A. However, the degradation rates in the 0.1 mg/mL enzyme solution were lower than these in the 0.2 mg/mL enzyme solution, which demonstrated the degradation rate highly depended on the concentration of enzyme solution. In general, the degradation of hydrogel is linked to several network parameters such as number of crosslinks per backbone chain, number of vinyl groups on the crosslinking molecule, molecular weight of backbone, and proportion of degradable groups in the main and side chain.38–41 With the hydrogel degrading, polymer chains freed by enzyme cleavage migrated out of the hydrogel and dissolved in solution, and the weight of the remaining cross-linked network decreased. The degradation rate decreased with increasing cross-linking density. In other words, the hydrogel with a higher swelling ratio had a faster degradation rate in this study. The morphologies of the swollen hydrogel and the hydrogel after enzymtic degradation were observed by SEM. As shown in Figure 5A, the SEM images of freeze-dried AC-PCL-HEMA/ AAc hydrogels before degradation exhibited a highly macroporous spongelike structure. With the increasing of cross-linking density, the pore size decreased attributed to the decreasing distance between polymer chains with increasing cross-linker concentration. After enzymatic degradation, the PCL chains in the network degraded and the pores of the hydrogel increased (panels B and C of Figure 5). The porous structures of these hydrogels suggest their potential as scaffolds for cell infiltration and growth. In Vitro Release of BSA. Figure 6A shows the release of BSA from AC-PCL-HEMA/AAc hydrogels in PBS solution (pH ) 7.4, I ) 0.2 M). It was found that there was a burst release at the initial stage. The BSA located near the hydrogel surface could be released immediately from the hydrogel to the medium as soon as the hydrogel was placed into the buffer solution. The initial burst release of BSA from other drug carriers was also reported elsewhere.42–44 After the initial burst, the consequent release seemed to be a diffusion process. Due to the fact that the cross-link density was arranged in a sequence of gel 5 > gel 4 > gel 3 > gel 2 > gel 1, the drug release rate was also arranged in the same order. Among the different hydrogels, gel 1 has the highest release rate because of its lowest cross-linking density. It could be seen that after 11 h, about 85% of BSA was released from gel 1. With a further increase in the release

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time, the release slowed down since the concentration gradient was the driving force for BSA diffusion and the higher BSA concentration gradient between the hydrogel surface and the release medium during the early stage led to a higher initial fast release rate. In addition, the in vitro drug release in the presence of enzyme was further investigated in PBS solution (pH 7.4) as exhibited in Figure 6B. In contrast to the release profile in PBS solution (pH 7.4) without enzyme (Figure 6A), it was found that the initial burst release and the release rate were almost the same within the initial 9 h. For example, in the presence of Pseudomonas lipase (0.1 mg/mL), after the initial 5 h, the cumulative release of BSA from gel 1, gel 2, gel 3, gel 4, and gel 5 was 77.4%, 31.4%, 28.5%, 27.1%, and 24.3%, respectively. While without the enzyme, the corresponding cumulative release was 76.3%, 30.9%, 27.3%, 26.0%, and 19.9%, respectively. But after 9 h, the release rate of the hydrogels was improved in the presence of enzyme. In detail, at 48 h, the cumulative release in PBS (pH 7.4) without enzyme was 92.8%, 66.5%, 64.6%, 59.7%, and 49.5% for gel 1, gel 2, gel 3, gel 4, and gel 5, respectively, but such value increased to 94.6%, 76.3%, 73.3%, 70.5%, and 63.2% for the corresponding hydrogel in the presence of Pseudomonas lipase. It was obvious that the cumulative drug release in the presence of enzyme increased around 10% from gel 2 to gel 5. The improved drug release rate was ascribed to the accelerated degradation of the cross-linkers in the hydrogels in the presence of the Pseudomonas lipase. Besides, the pH-triggered drug release behavior of the hydrogels was also investigated at different pH values (pH 7.4 and pH 5.0) as revealed in parts A and C of Figure 6. It was found that the release rate of the hydrogels at pH 5.0 was slightly decreased in comparison with that in pH 7.4 buffer solution. The decreased drug release in an acidic medium is easily understood due to the hydrogen bonding interactions as well as the constrained network at pH 5.0. However, the pH-dependent drug release was not as obvious as the pH-dependent swelling ratios (Figure 2), which was ascribed to the open porous hydrogel matrix allowing fast release of loaded drug even at pH 5.0. Cytotoxicity Study. MTT assay was performed to investigate the cytotoxicity of the hydrogel. The effect of the hydrogel concentration on the proliferation of 3T3 mouse fibroblasts was studied (Figure 7). The results demonstrated there was a nonsignificant decrease in cell viability when the concentration of the hydrogel was between 20 and 160 g/L. It was clear that the hydrogel had no apparent cytotoxicity. Cells Cultured on and Embedded in the Hydrogels. Because AC-PCL-HEMA/AAc hydrogels are translucent, the morphology of cells adhering to the hydrogels was evaluated by phase-contrast inverted light microscopy (Figure 8). Different cell lines, Hela, A549, and ECV304 cells, were seeded in gel 4 and gel 5. After 72 and 120 h of incubation, the cells formed colonies on the hydrogel surfaces, which demonstrated the hydrogels were suitable for cell adhesion. Bovine serum albumin is amphoteric polyelectrolyte with both acidic and basic peptide which could behave as a bridge between positive or negative groups on a cell or gel surface. This result was in accordance with previous studies.31 Since the hydrogels we synthesized were porous and biodegradable materials, if the cells seeded on the hydrogels for a suitable time, the cells might migrate into the hydrogel networks. In this study, the hydrogels with cells cultured on them were paraffin embedded,45 sectioned, and stained with hematoxylin, erythrosin, and safran (HES) in PBS. In this study, we used the

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Figure 5. SEM images of the AC-PCL-HEMA/AAc hydrogels before degradation (A), after enzymatic degradation for 7 days (with the enzyme concentration of 0.2 mg/mL) (B), and after enzymatic degradation for 14 days (with the enzyme concentration of 0.1 mg/mL) (C).

Figure 6. Cumulative release of BSA from the AC-PCL-HEMA/AAc hydrogels at 37 °C in PBS solution (pH 7.4) (A), in PBS solution (pH 7.4) in the presence of Pseudomonas lipase with the concentration of 0.1 mg/mL (B), and in SAABS solution (pH 5.0) (C).

method of paraffin embedding described by Underhill et al.46 to obtain intact sections and observe the cell migration into

hydrogels. As shown in Figure 9, all the cells could migrate inside the hydrogels, and the number of cells in gel 4 was more

Biodegradable and pH-Sensitive Hydrogels

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Figure 7. Cytotoxicity study of the AC-PCL-HEMA/AAc hydrogel with different concentrations.

Figure 9. Micrographs of cell lines cultured inside the hydrogel networks for 5 days: (A) Hela cells in gel 4; (B) Hela cells in gel 5; (C) A549 cells in gel 4; (D) A549 cells in gel 5; (E) ECV304 cells in gel 4; (F) ECV304 cells in gel 5 (magnification: 400×).

hydrogels showed that the cells can adhere and spread on the hydrogel surfaces as well as migrate inside the hydrogel networks. In comparison with the conventional biodegradable hydrogels, the novel pH-sensitive and biodegradable AC-PCLHEMA/AAc hydrogels would have great potential for biological and biomedical applications. Figure 8. Micrographs of different cell lines cultured on the hydrogel surfaces for 3 days: (A) Hela cells on Gel4, (B) Hela cells on gel 5; (C) A549 cells on gel 4; (D) A549 cells on gel 5; (E) ECV304 cells on gel 4; (F) ECV304 cells on gel 5 (magnification: 100×).

Acknowledgment. Financial support from the National Natural Science Foundation of China (50633020, 20504024) and Ministry of Science and Technology of China (2005CB623903) is gratefully acknowledged.

References and Notes than that in gel 5, which was ascribed to the fact that the pore size of gel 4 was larger than that of the latter. Due to the biodegradability and biocompatibility, the hydrogel is suitable for scaffolds for cell encapsulation and tissue regeneration applications in vivo.

Conclusion A series of biodegradable AC-PCL-HEMA/AAc hydrogels were synthesized. The degradation of hydrogel was obviously accelerated in the presence of Pseudomonas lipase due to the catalytic effect of the lipase on the degradation of PCL chains in the hydrogel network. The hydrogel exhibited pH sensitivity, and a higher swelling ratio was observed in the solution with a higher pH value. The in vitro release of BSA from the hydrogel demonstrated that the hydrogel with a lower cross-linking density had a higher release rate. The cell culture on the

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