Injectable citrate-based mussel-inspired tissue bioadhesives with high wet strength for sutureless wound closure
Introduction
In the past two decades, clinical surgical practices have significantly benefited from using bioadhesives, tissue sealants, and hemostatic agents to control blood loss and promote tissue healing [1]. For example, biologically-derived fibrin glues, which mimic the last stage of physiological coagulation cascade, and synthetic cyanoacrylate adhesives are two well-known tissue adhesives that have been widely utilized in many applications [2], [3], [4]. Despite having advantages, such as fast curing and biodegradability, fibrin glue has relatively poor adhesion and tensile strength and its utilization involves risks of blood-borne disease transmission and potential allergic reactions to patients [4], [5], [6], [7], [8], [9], [10]. Cyanoacrylate adhesives offer advantageous properties, such as strong adhesion, rapid setting time, instantaneous adhesion to tissue, and ease of use [11]. However, concerns about complications that might occur due to the slow degradation of cyanoacrylates, the exothermic polymerization reaction, and the toxicity of degradation products, have limited the applications of cyanoacrylates mainly to topical uses [4], [12]. Furthermore, both fibrin glue and cyanoacrylates work best when applied to a dry surgical field, which greatly restricts their applications for wet tissue adhesion and hemostasis required in many internal organ surgeries [13]. Currently no commercially available tissue adhesives and sealants can be broadly applied for both external and internal tissue adhesion and hemostatic applications.
Inspired by the adhesion strategy employed by some maritime creatures, such as blue mussel Mytilus edulis [14], [15], researchers developed a new family of adhesives, which can adhere to non-specific surfaces in aqueous condition. The strong adhesion ability of the mussels has been ascribed to the presence of a catechol-containing amino acid called l-3,4-dihydroxyphenylalanine (l-DOPA), a post-translational hydroxylation of tyrosine, found in the structure of secreted mussels adhesive foot proteins [14], [16], [17]. Under oxidizing or alkaline condition, DOPA is believed to promote the crosslinking reactions of these adhesive proteins through the oxidation of catechol hydroxyl groups to ortho-quinone, which subsequently triggers intermolecular crosslinking, rendering cohesion and bulk elastic properties to the network of proteins. Recent studies revealed that oxidized DOPA also contributes to strong adhesion to biological surfaces, through the formation of covalent bonds with available nucleophile groups on these surfaces such as –NH2, –SH, –OH and –COOH groups [16], [18], [19], [20], [21], [22]. By incorporating catechol-containing species into the structure of polymers, wet tissue adhesive hydrogel materials have been synthesized [23], [24], [25], [26], [27], [28]. However, the syntheses of these catecholic polymers require costly multi-step preparation/purification techniques and the use of toxic reagents. In spite of the appealing wet tissue adhesion properties, existing mussel-inspired adhesive polymers [29], [30] are essentially non-degradable thus substantially limiting their potential uses in a variety of medical applications, including tissue engineering and drug delivery. Inspired by another natural adhesion mechanism – gecko adhesion which is mainly effective on dry surfaces, biodegradable elastomer, poly(glycerol sebacate acrylate) (PGSA) was fabricated into adhesive tape with nano-pillar structures to simulate the nano-scale setae on gecko food pads [31]. To improve wet tissue adhesion, a strategy has been developed recently to coat wet tissue adhesives such as mussel-inspired adhesives [32] and aldehyde-functionalized starch [31] on gecko-adhesive structures to achieve mechanical interlocking and covalent chemistry simultaneously. Despite these exciting progresses, none of the existing bioinspired adhesives alone possess sufficient wet tissue adhesion strength and controlled degradability for sutureless wound closure application.
In the present work, we synthesized a new family of injectable citrate-based mussel-inspired bioadhesives, iCMBAs (Fig. 1). The rationale behind the iCMBA strategy was to react citric acid, poly(ethylene glycol) (PEG), and catechol-containing monomers such as dopamine or l-DOPA via a one-step polycondensation reaction. Such an approach allows us to fabricate new adhesive materials with great wet adhesion strength, controllable degradability, improved biocompatibility, and substantially reduced manufacturing costs. Citric acid, a non-toxic metabolic product of the body (Krebs cycle), was a key in our established methodology in the development of citrate-based biodegradable polymers (CBBPs) including poly(diol citrates) [33], [34], [35], [36], crosslinked urethane-doped polyesters (CUPE) [37], [38], poly(alkylene maleate citrates) (PAMC) [39], [40], [41], and biodegradable photoluminescent polymers (BPLP) [42], [43] for applications in tissue engineering, drug delivery, medical devices, and bioimaging. Citric acid was mainly used to facilitate degradable ester-bond formation in biomaterials, while enhancing hemocompatibility and hydrophilicity of the polymers and providing pendant binding sites for bioconjugation to confer additional functionality such as optical properties. For iCMBA synthesis, citric acid was used to not only form degradable polyesters with PEG, but also provide valuable pendant reactive carboxyl groups to conjugate dopamine or l-DOPA. Thus, using highly reactive multifunctional citric acid enables a one-step synthesis to prepare biodegradable polyesters with pendant catechol functionalities via a facile condensation reaction. All the monomers used for iCMBA syntheses are inexpensive, readily available, and safe for in vivo uses and have been documented in many FDA (US Food and Drug administration)-approved devices and applications.
Section snippets
Materials
All chemicals, cell culture medium, and supplements were purchased from Sigma Aldrich (St. Louis, MO), except where mentioned otherwise. All chemicals were used as received.
Synthesis and characterization of iCMBAs
iCMBAs are oligomers based on citric acid (CA) and PEG that have been functionalized by catechol-containing compounds, such as dopamine and l-DOPA (l-3,4-dihydroxyphenylalanine) as illustrated in Fig. 1. CA and PEG were placed in a 250 mL three-necked round-bottom flask and heated to 160 °C until a molten clear mixture was
Synthesis and characterization of iCMBA pre-polymers
iCMBA pre-polymers were synthesized in a convenient one-step polycondensation reaction between citric acid, PEG, and dopamine without requiring any organic solvent or toxic reagent. The FTIR spectra of some iCMBA pre-polymers as well as the spectrum of poly (citrate-PEG) are shown in Fig. 2A. The peak at 1527 cm−1 were assigned to amide group (C(O)–NH) which is not observed in CA-PEG. Peaks between 1700 and 1750 cm−1 were assigned to carbonyl group (CO) in amide and ester bonds. The relatively
Discussion
iCMBAs were synthesized by a single-step polycondensation reaction between citric acid, polyethylene glycol and dopamine (Fig. 1). The FTIR and 1H NMR characterizations confirmed the esterification reaction between CA and PEG, and formation of amid linkage between the unreacted –COOH groups of CA and dopamine's –NH2 groups (Fig. 2A, B). 1H NMR also revealed that the compositional ratio of iCMBA pre-polymers was consistent with the feeding ratio, a sign of high yield of this synthesis method (
Conclusion
The experimental results presented above demonstrate the syntheses of a family of injectable citrate-based mussel-inspired biodegradable adhesives, iCMBAs, from safe and inexpensive constituents and via a one-step synthesis technique without involvement of any toxic reagents, and their applications in hemostasis and sutureless wound closure. The syntheses of iCMBA enrich the methodology of citrate-based biomaterial development and represent an innovation for tissue adhesive biomaterial design.
Acknowledgments
This work was supported in part by R01 awards (EB012575 (to J.Y.) and EB007271 (to L.T.)) from the National Institute of Biomedical Imaging and Bioengineering (NIBIB), and a National Science Foundation (NSF) CAREER award 0954109 (to J.Y.).
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