Chitosan-g-hematin: Enzyme-mimicking polymeric catalyst for adhesive hydrogels
Graphical abstract
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.
Section snippets
Materials
Chitooligosaccharide (MW 3–5 kDa, 95.2% deacetylated) was purchased from Kitto Life Co., Ltd. (Gyeonggi-do, Republic of Korea). Chitosan 100 (Mv = 1.31 MDa [40], 80% deacetylated) was purchased from Wako Pure Chemical Co. (Osaka, Japan). Hyaluronic acid was obtained from Amore Pacific (Seoul, Republic of Korea). Poly(vinyl alcohol) (MW 85–123 kDa), Hematin, hydrocaffeic acid (3,4-dihydroxy hydrocinnamic acid), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), 3,3-dithiodipropionic
Synthesis of chitosan-g-hem
We hypothesized that a water-soluble polymer might increase the poor water solubility of hematin. We chose water-soluble, low-molecular weight chitosan (MW 3–5 kDa) as the polymeric backbone. The chitosan was reacted with 3,3-dithiodipropionic acid, resulting in the formation of chitosan-S-S-chitosan conjugates. To reduce the disulfide bonds, DTT was added to the solution, generating thiolated chitosan (chitosan-SH). The chitosan-SH was further reacted with hematin via radical coupling using
Conclusions
In this study, a novel enzyme-mimicking chitosan-g-hem biocatalyst was developed to fabricate adhesive catechol-containing hydrogels. Using chitosan-g-hem, the gelation effectively occurred at physiological conditions even without the use of the enzyme HRP. Chitosan-g-hem biocatalysts showed enhanced solubility and activity compared with the unmodified hematin. The catechol-containing hydrogels prepared by chitosan-g-hem exhibited enhanced tissue adhesion compared with the hydrogels formed by
Acknowledgements
This study was supported by the grants from the Molecular-level Interface Research Center (2011-0001319), and Future Fundamental Technology Development Program (2010-0028765) funded by the Ministry of Education, Science and Technology, Republic of Korea. This study was also supported by the grant of the Korea Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (Grant Number: A120170).
References (52)
Hydrogels for biomedical applications
Adv Drug Deliv Rev
(2012)- et al.
Selfassembled and nanostructured hydrogels for drug delivery and tissue engineering
Nanotoday
(2009) Hydrogels for tissue engineering and delivery of tissueinducing substances
J Pharm Sci
(2007)- et al.
Novel crosslinking methods to design hydrogels
Adv Drug Deliv Rev
(2002) - et al.
Enzyme-mediated fast in situ formation of hydrogels from dextran–tyramine conjugates
Biomaterials
(2007) - et al.
Photopolymerizable hydrogels for tissue engineering applications
Biomaterials
(2002) - et al.
Enzyme-mediated cross-linking of pluronic copolymer micelles for injectable and in situ forming hydrogels
Acta Biomater
(2011) - et al.
Enzyme-mediated fast in situ formation of hydrogels from dextran–tyramine conjugates
Biomaterials
(2007) - et al.
Rapidly curable chitosan-PEG hydrogels as tissue adhesives for hemostasis and wound healing
Acta Biomater
(2012) - et al.
Enzymatically crosslinked carboxymethylcellulose–tyramine conjugate hydrogel: cellular adhesiveness and feasibility for cell sheet technology
Acta Biomater
(2009)
Horseradish peroxidase: a modern view of a classic enzyme
Phytochemistry
Chitosan – a versatile semi-synthetic polymer in biomedical applications
Prog Polym Sci
Structure and interactions in covalently and ionically crosslinked chitosan hydrogels for biomedical applications
Eur J Pharm Biopharm
Prevention of postsurgical adhesions with an autocrosslinked hyaluronan derivative gel
J Surg Res
Hydrophilic gels for biological use
Nature
Mechanism of forming organic/inorganic network structures during in-situ free-radical polymerization in PNIPA–clay nanocomposite hydrogels
Macromolecules
In situ crosslinkable hyaluronan hydrogels for tissue engineering
Biomaterials
Synthesis and physicochemical characterization of end-linked poly(ethylene glycol)-co-peptide hydrogels formed by Michael-type addition
Biomacromolecules
Network formation and degradation behavior of hydrogels formed by Michael-type addition reactions
Biomacromolecules
Selected properties of pH-sensitive, biodegradable chitosan–poly(vinyl alcohol) hydrogel
Polym Int
Rheological characterization of in situ crosslinkable hydrogels formulated from oxidized dextran and N-carboxyethyl chitosan
Biomacromolecules
Rational design of transglutaminase substrate peptides for rapid enzymatic formation of hydrogels
J Am Chem Soc
Enzyme-catalyzed gel formation of gelatin and chitosan: potential for in situ applications
Biomaterials
Injectable biodegradable hydrogels composed of hyaluronic acid–tyramine conjugates for drug delivery and tissue engineering
Chem Commun
An injectable enzymatically crosslinked hyaluronic acid–tyramine hydrogel system with independent tuning of mechanical strength and gelation rate
Soft Matter
In situ hydrogelation and RGD conjugation of tyramine-conjugated 4-arm PPO–PEO block copolymer for injectable bio-mimetic scaffolds
Soft Matter
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