Cytocompatible Enzymatic Hydrogelation Mediated by Glucose and

Apr 12, 2017 - Division of Chemical Engineering, Department of Materials Engineering Science, Graduate School of Engineering Science, Osaka University...
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Cytocompatible Enzymatic Hydrogelation Mediated by Glucose and Cysteine Residues Enkhtuul Gantumur, Shinji Sakai,* Masaki Nakahata, and Masahito Taya* Division of Chemical Engineering, Department of Materials Engineering Science, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan S Supporting Information *

ABSTRACT: Hydrogels were obtained from aqueous solution containing polymer(s) possessing phenolic hydroxyl moieties through horseradish peroxidase (HRP)-catalyzed reaction without direct addition of H2O2. In this hydrogelation process, H2O2 was generated from HRP and glucose contained in the aqueous solution, that is, HRP functioned not only as a catalyst, but also as a source of H2O2. The gelation time and mechanical properties of the resultant hydrogel could be altered by changing the concentrations of HRP and glucose. Cytocompatibility of the hydrogelation process was confirmed from cell studies using mouse 10T1/2 fibroblast cells.

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In the present work, we report a method of preparing hydrogel through HRP-catalyzed reaction using an aqueous solution containing Polymer-Ph without direct addition of H2O2. Our method uses glucose and the reactive cysteine residue in HRP for generating H2O2. A notable point of this hydrogelation process is the function of HRP as both a catalyst and a supplier of H2O2. To the best of our knowledge, no attention has been paid to the use of HRP in such a way. The abundance in available Polymer-Phs derived from varieties of polysaccharides, proteins, and synthetic polymers4 is an attractive point of the hydrogelation method proposed here compared to the system using thiolated polymers.6 It enables to obtain hydrogels possessing characters suitable for a wide range of individual applications from existing Polymer-Phs. First, we confirmed the possibility of using glucose for hydrogelation of an aqueous solution containing Polymer-Ph. We used an alginate derivative possessing Ph moieties (Alg-Ph) as a model polymer. An aqueous solution of Alg-Ph (0.75 w/v %) was mixed with HRP (300 units/mL) and glucose (44 mg/ mL) in a phosphate-buffered saline solution (PBS, pH 7.4). After 20 min of standing at room temperature, only the mixture containing both HRP and glucose yielded a hydrogel (Figure 1a). It means that this hydrogelation was involved in HRPcatalyzed reaction. Since it is known that HRP oxidation occurs in the presence of H2O2, we predicted accelerated generation of H2O2 by adding glucose into the mixture. The detailed mechanism is not clear, but we hypothesized a cycle of redox reactions of thiol groups in HRP (Figure 1b). During the

ydrogels containing abundant water are useful materials for applications in biopharmaceutical and biomedical fields. Hydrogels have been prepared from a variety of materials through various processes.1−3 Recently, hydrogelation through enzymatic cross-linking of polymers has attracted an increasing interest due to the mildness of the reaction conditions such as at physiological temperatures, with negligible heat generation, and in aqueous milieu at neutral pH.4 Horseradish peroxidase (HRP) is one of the enzymes that has been applied to hydrogel preparation. HRP catalyzes the conjugation of phenol and aniline derivatives by consuming H2O2 as an electron donor. In this reaction, HRP promptly combines with H2O2, and the resultant complex can oxidize phenolic hydroxyl (Ph) moieties. The most common method for preparing hydrogels through this enzymatic reaction is addition of H2O2 aqueous solution directly to the mixture of HRP and polymer possessing Ph moieties (Polymer-Ph).5 HRP inactivation resulting from excess amount of H2O2 and inhomogeneity of the resultant hydrogel network resulting from temporal inhomogeneous H2O2 concentration just after H2O2 addition are considered as potential problems of this method.6 For overcoming these problems, several methods requiring no addition of H2O2 aqueous solution have been proposed, such as using glucose oxidase for producing H2O2 from glucose coexisting in an aqueous solution of HRP and Polymer-Ph.7,8 Moriyama et al. prepared hydrogel from an aqueous solution containing thiolated poly(ethylene glycol), HRP, and a phenolic compound.6 In this system, H2O2 was initially generated through the auto-oxidation of thiol groups, resulting in a disulfide-cross-linked network. It was reported that oxidation of the phenol compound through the consumption of H2O2 by HRP promoted the auto-oxidation of thiols.9 © XXXX American Chemical Society

Received: February 19, 2017 Accepted: April 11, 2017

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DOI: 10.1021/acsmacrolett.7b00122 ACS Macro Lett. 2017, 6, 485−488

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ACS Macro Letters

Figure 1. (a) Photographs of upturned glass vessels containing Alg-Ph solution (0.75 w/v %) with addition of HRP (300 units/mL), glucose (44 mg/mL) and both of them. The added components contained in each vessel are shown on the photographs. (b) Proposed scheme for the glucose-mediated hydrogelation of polymers possessing Ph moieties by HRP reaction.

Figure 2. (a) Elapsed-time changes of fluorescence intensity after mixing H2O2 probe during hydrogelation. (b) Representative spectrum of solutions containing HRP + glucose and HRP alone. Absorbance at 412 nm attributed to the existence of thiol groups. The absorbance profile was normalized against a maximum value for each line. (c) Changes in absorbance at 412 nm occurred during 2 days. Absorbance recorded on day 2 was normalized against an initial value (day 0). Bars in graphs (a) and (c) show standard deviations (n = 3).

reaction, the self-oxidation of thiol was promoted by the Ph moieties in Polymer-Ph and generated H2O2. By adding glucose, disulfide could be reduced to thiol groups, leading to continuous H2O2 supply. It is known that glucose is a reducing sugar and HRP contains reactive cysteine residues.10 In order to clarify the proposed mechanism of this glucosemediated hydrogelation, we first checked the generation of H2O2 during the gelation of a Polymer-Ph solution. A fluorescent probe for H2O2 (BES-H2O2-Ac) was mixed with Alg-Ph (0.75 w/v %), HRP (100 units/mL) and glucose (88 mg/mL). The fluorescence intensity (excitation at 485 nm, emission at 515 nm) was measured for 50 min after mixing the components. As a result, the fluorescence intensity of an aqueous solution containing both of HRP and glucose increased with elapsed time (Figure 2a). This means that H2O2 was continuously generated during the hydrogelation. In the absence of glucose, the fluorescence intensity decreased during the first 10 min, and thereafter did not increase. A possible explanation for this result is that the initially generated H2O2 from the self-oxidation of thiol in HRP was consumed and no thiol regeneration could occur without glucose. The next step of our investigation was to determine the change in the content of thiol groups in HRP using the Ellman’s method.11 An Ellman’s reagent (5,5′-dithio-bis(2nitrobenzoic acid) mixed with HRP (600 units/mL) showed absorbance at 412 nm after its reaction with thiol groups (Figure 2b). In the presence of glucose, the absorbance at this wavelength decreased after 2 days of reaction, whereas there was no change in the absence of glucose (Figure 2c). This can be explained by the cycle of redox reactions of disulfide bond formation and its cleavage mediated by reduction with glucose. In fact, the hydrogelation did not occur when we used a sucrose, nonreducing sugar instead of glucose (see Figure S1 in the Supporting Information). These results indicate that our proposed mechanism is a possible route for glucose-mediated hydrogelation. We then studied the effect of concentrations of HRP and glucose on gelation time of Alg-Ph solution (0.75 w/v %) based

on a previously reported method.12 Figure 3a,b shows the effects of HRP and glucose concentrations on hydrogelation.

Figure 3. Effects of HRP and glucose concentrations (a, b) on gelation time and (c, d) on Young’s modulus of the resultant hydrogels obtained from Alg-Ph (0.75 w/v %) solution. The concentration of glucose in graphs (a) and (c) was fixed at 44 mg/mL and HRP in graphs (b) and (d) was fixed at 300 units/mL. Bars show standard deviations (n = 3).

The shortest gelation time of ∼6 min was observed at the highest concentration of HRP (600 units/mL). The gelation time decreased with increasing HRP concentration. We considered that the initial amount of thiol increased in proportion to the HRP concentration, and could be the reason for decreasing gelation time. For the effect of glucose concentration, the gelation time decreased with increasing glucose in the range of 11−44 mg/mL. Further increase in glucose concentration to 88 mg/mL did not induce drastic 486

DOI: 10.1021/acsmacrolett.7b00122 ACS Macro Lett. 2017, 6, 485−488

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ACS Macro Letters

Furthermore, cells seeded on a fresh cell culture dish after being collected from the Alg-Ph hydrogel by degradation through alginate lyase showed a similar morphology and growth behavior to those of nontreated control cells (Figure 4c). These results indicate that the process of hydrogelation by glucose and HRP-catalyzed reaction has no specific harmful effects on the tested cells, that is, a cytocompatible process. In this study, we rinsed the resultant hydrogel several times for removing the remaining glucose because the concentration required for hydrogelation is higher than the normal amount in animal blood and cell culture medium. The studies for the effect of exposure time of cells to a high glucose concentration are out of the scope of this paper. However, it is theorized that exposure time should be as short as possible. In the applications through in situ hydrogelation in vivo, the volume of hydrogels should be decided considering the effect of glucose released from the hydrogels on blood glucose level. As long as we know, there are no literatures describing the obvious adverse effect of HRP in hydrogels in the applications in vivo. However, the effect should also be investigated before applying in vivo because of the higher concentration of HRP in the current system than those in previous systems. In summary, we developed a method for preparing hydrogel through HRP-catalyzed reaction without direct addition of H2O2. Glucose and reactive cysteine residues in HRP were used to promote H2 O2 generation. The gelation time and mechanical property of the resultant hydrogel could be controlled easily by changing the concentrations of HRP and glucose. Additionally, the hydrogel could be prepared from different polymers possessing Ph moieties. Cytocompatibility of the hydrogelation process was demonstrated by cell studies. The method reported in this study has potential for a wide range of biomedical applications.

decrease in gelation time (Figure 3b). A possible reason for this result is that the self-oxidation of thiol group was a rate-limiting step at that glucose concentration. Inactivation of HRP through the reaction of thiols with H2O2 might also have contributed to the result. We also evaluated the effects of concentrations of HRP and glucose on the mechanical property of the resultant hydrogel by measuring Young’s modulus. As shown in Figure 3c,d, the Young’s modulus increased significantly with increasing HRP and glucose concentrations. This is understandable as the amount of generated H2O2 is supposed to increase with increasing HRP and glucose concentrations. Therefore, crosslinking between Ph moieties was promoted, resulting in the formation of a stiff hydrogel. These results demonstrate the possibility of controlling the mechanical properties and gelation time by changing the concentrations of HRP and glucose. Finally, we evaluated the cytocompatibility of the hydrogelation process by measuring the viability and behavior of enclosed cells in hydrogel, and the morphology and growth of recovered cells from the hydrogel using mouse 10T1/2 fibroblast cells. In these cell studies, we also prepared hydrogel through this method of hydrogelation from an aqueous solution containing hyaluronic acid (HA-Ph) and gelatin (gelatin-Ph) derivatives possessing Ph moieties. The viability of cells after 24 h of being enclosed in hydrogel derived from a mixture of AlgPh (0.75 w/v%), HRP (300 units/mL), and glucose (22 mg/ mL) was 94.6 ± 2.2% (n = 4, Figure 4a). In addition, the cells enclosed in the hydrogel obtained from a mixture of HA-Ph (0.75 w/v%), gelatin-Ph (0.3 w/v%), HRP (300 units/mL), and glucose (22 mg/mL) grew and migrated in the hydrogel during 5 days of incubation (Figure 4b, Movie S1).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.7b00122. Supporting video (AVI). Experimental details and additional data of synthesis of Alg-Ph, HA-Ph, and gelatin-Ph, determination of H2O2 generation, determination of change in the content of thiol group in HRP, measurements of gelation time and mechanical property, cell studies, and gelation with nonreducing sugar (PDF).

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID Figure 4. (a) Photograph of 10T1/2 cells enclosed in Alg-Ph hydrogel after 24 h of cultivation. (b) Confocal fluorescence image of 10T1/2 cells grown inside of HA-Ph + gelatin-Ph hydrogel after 5 days of incubation. (c) Cells seeded on a fresh cell culture dish after degradation of Alg-Ph hydrogel by using alginate lyase (enclosed cells) and seeded without exposure to treatment (control). Live and dead cells in photograph (a) exhibit green and red fluorescence, respectively. Bars in photographs (a) and (c) show 200 μm and (b) shows 300 μm.

Shinji Sakai: 0000-0002-1041-4798 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Japan Society for the Promotion of Science KAKENHI Grant Numbers 15H04194 and 16H02423. 487

DOI: 10.1021/acsmacrolett.7b00122 ACS Macro Lett. 2017, 6, 485−488

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DOI: 10.1021/acsmacrolett.7b00122 ACS Macro Lett. 2017, 6, 485−488