Chitosan-g-hematin: Enzyme-mimicking polymeric catalyst for adhesive hydrogels

Abstract

Phenol derivative-containing adhesive hydrogels has been widely recognized as having potential for biomedical applications, but their conventional production methods, utilizing a moderate/strong base, alkaline buffers, the addition of oxidizing agents or the use of enzymes, require alternative approaches to improve their biocompatibility. In this study, we report a polymeric, enzyme-mimetic biocatalyst, hematin-grafted chitosan (chitosan-g-hem), which results in effective gelation without the use of alkaline buffers or enzymes. Furthermore, gelation occurs under mild physiological conditions. Chitosan-g-hem biocatalyst (0.01%, w/v) has excellent catalytic properties, forming chitosan–catechol hydrogels rapidly (within 5 min). In vivo adhesive force measurement demonstrated that the hydrogel formed by the chitosan-g-hem activity showed an increase in adhesion force (33.6 ± 5.9 kPa) compared with the same hydrogel formed by pH-induced catechol oxidation (20.6 ± 5.5 kPa) in mouse subcutaneous tissue. Using the chitosan-g-hem biocatalyst, other catechol-functionalized polymers (hyaluronic acid–catechol and poly(vinyl alcohol)–catechol) also formed hydrogels, indicating that chitosan-g-hem can be used as a general polymeric catalyst for preparing catechol-containing hydrogels.

Introduction

Hydrogels have received increased attention as implantable materials since the 1960s, demonstrating an ability to be used for the sustained release of therapeutic drugs in the body (i.e. drug depot) and as biocompatible tissue engineered scaffolds [1], [2], [3], [4], [5], [6]. Methods for preparing hydrogels are primarily divided into two categories. The first category uses physical crosslinking between the polymer chains. Physically crosslinked hydrogels are formed under mild conditions, with the in situ gelation properties driven by hydrogen bonds, hydrophobic interactions and ionic associations. These types of bonds have low mechanical strength with a rapid dissociation of the polymer chains under physiological conditions. Another class of hydrogels is formed by chemical crosslinking. These types of hydrogels typically exhibit excellent mechanical strength and chain-dissociation-resistant properties. The gelation time for chemically crosslinked hydrogels can be varied by controlling the molecular factors involved in the crosslinking chemistry. Methods for the preparation of chemically crosslinked hydrogels include free radical polymerization [7], [8], Michael-type addition [9], [10], [11], Schiff base formation [12], [13] and enzymatic crosslinking [14], [15], [16].

Among the aforementioned chemical crosslinking methods, crosslinkings between phenolic derivatives have recently become popular. Tyramine (tyr), 4-(2-aminoethyl)phenol, has been conjugated to a variety of polymers, generating hyaluronic acid–Tyr [17], [18], dextran–tyr [19], pluronic F127–tyr [16], tetronic–tyr [20] and other types of derivative [21], [22], [23]. To prepare these types of hydrogel, horseradish peroxidase (HRP) is added to the polymer solutions. The catalytic action of HRP, a heme-containing enzyme with oxidoreductase properties, results in the formation of di-tyramine (di-tyr) with the consumption of hydrogen peroxide (H₂O₂). Tyramine is a chemically stable compound that is easy to conjugate to the carboxylic acids present in many biopolymers. For the hydrogel preparation process, HRP must be used to crosslink the tyramine groups. In general, the use of enzymes for hydrogel preparations has the intrinsic challenge of the unavoidable entrapment of the enzymes within the hydrogels, which thus requires the use of significant amounts of the enzymes. Therefore, time and labor for large-scale bacterial culture are required.

Another class of phenolic derivative used for crosslinking functional groups includes catechol-containing small molecules, such as dopamine and 3,4-dihydroxyhydrocinnamic acid. The pH-induced oxidation of catechol results in catecholquinone, which subsequently forms catechol–catechol adducts in the absence of enzymes [24], [25]. Other routes for catecholquinone-involved chemical crosslinkings involve the formation of catechol–amine and catechol–thiol adducts [26], [27]. Catechol-containing hydrogels have recently been used for the preparation of adhesive hydrogels, generating interesting underwater adhesive properties from the conjugated catechol [28]. A variety of hydrogels formed by a catechol-mediated chemical crosslinking have been reported, including end-functionalized poly(ethylene glycol) (PEG)–catechol [29], catechol-grafted PEG [30], catechol-conjugated hyaluronic acid (HA-C) [31], HA-C/pluronic-thiol [32] and catechol-conjugated chitosan (CHI-C)/pluronic-thiol [33] hydrogels. Catechol-mediated hydrogels are formed by a non-enzymatic process, which is an obvious advantage compared with tyramine-containing hydrogels. However, the disadvantages of the catechol-mediated hydrogels include a gelation that is triggered by relatively high pH values (>8) or by the presence of highly concentrated oxidizing agents, such as periodate ions, often resulting in the cytotoxicity of the encapsulated cells (Fig. 1a). The development of a new approach that can effectively trigger gelation in the physiological pH range without using enzymes, such as HRP, is necessary for further improvement of the biocompatibility of catechol-containing hydrogels (Fig. 1b).

We hypothesize that the use of a water-soluble biocatalyst to initiate catechol oxidation and subsequent crosslinking cold be a possible solution to overcome the aforementioned challenges in catechol hydrogel formation. In this study, a new polymer-based biocatalyst, hematin-grafted chitosan (chitosan-g-hem) (Fig. 2), was investigated. Hematin, an iron-containing heme group, has been studied as an oxidative catalyst for phenol compounds [34], [35]. HRP and hematin commonly catalyze covalent crosslinks between phenolic compounds, consuming hydrogen peroxide via radical transfer reactions occurring in the heme group [34], [35], [36], [37], [38]. One major problem encountered with hematin is its poor solubility in water, because the porphyrin structure in hematin is a water-insoluble functional group [35], [39]. Because of this poor solubility, the effective use of hematin is significantly hindered. Previously, hematin has been dissolved in alkaline buffer, with the pH of the solution being subsequently reduced to 7.4 for further use [35]. This method is useful for the simplicity of the hematin dissolution, but its solubility is nevertheless limited, resulting in crude aggregates, and the time needed to dissolve the hematin is significantly long. As a potential solution to the poor solubility, we have investigated conjugating hematin to chitosan. Chitosan-g-hem is highly water soluble, but retains the HRP-mimicking catalytic activity of hematin to facilitate the rapid gelation of catechol-containing polysaccharides. With this conjugate, the resulting hydrogels exhibited tissue adhesion properties, demonstrating increased adhesiveness compared with the same hydrogels formed by conventional pH-induction processes.