Letter pubs.acs.org/macroletters
Supramolecular Cyclodextrin Supplements to Improve the Tissue Adhesion Strength of Gelatin Bioglues Thai Thanh Hoang Thi,†,⊥ Jung Seok Lee,‡,⊥ Yunki Lee,† Hyun Bong Park,§ Kyung Min Park,∥ and Ki Dong Park*,† †
Department of Molecular Science and Technology, Ajou University, Suwon 443-749, Republic of Korea Department of Biomedical Engineering and §Department of Chemistry, Yale University, New Haven, Connecticut 06511, United States ∥ Division of Bioengineering, College of Life Sciences and Bioengineering, Incheon National University, Incheon 22012, Korea ‡
S Supporting Information *
ABSTRACT: A practical strategy to strengthen the inherent tissue adhesiveness of bioglues was investigated without compromising their chemical structure. A simple blending of α-cyclodextrins (α-CDs) in gelatin glues substantially improved the adhesiveness of the glues upon contact with porcine skin. Interestingly, the adhesiveness was even further enhanced when the glues were supplemented with γ-CDs through the formation of multivalent supramolecular networks inside of the glue. In contrast, the effect of β-CDs was rather limited because their water solubility is relatively low. A model study using modified gold substrates demonstrated that CDs also interacted with biomolecules naturally present in skin, thereby improving the adhesiveness. Altogether, we suggest that the supramolecular networking of CDs, both inside the glue and at the glue−skin interface, could reinforce tissue adhesiveness of bioglues, which allows them to be of great relevance to a primary treatment modality for larger skin incisions. field, CDs can form their host−guest complexes with hydrophobic therapeutics to improve the availability of the therapeutics in biological systems.6 Given their versatility, their assemblies or aggregates have been used for the fabrication of micelles, capsules, and hydrogels at the macro- and nanoscale levels.7 Bioglues are becoming emerging materials in diverse clinical applications, including hemostasis, wound closure, and healing,8 because they are fast and easy to use with relatively low technical skills as compared to suturing and stapling.9 The bioglues yield less traumatic closure and pain, excellent cosmetic results, and possibilities in delivery of therapeutic drugs and cells to the injury site.10 They are particularly important for friable tissues such as dura mater, lung, liver, spleen, and kidney, where suturing and stapling are impractical or ineffective to seal biological fluid leakage or reinforce anatomic integrity. 9 Despite the many aforementioned advantages, bioglues are continuously challenged with regard to the following clinical requirements: strong and rapid
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upramolecular forces (e.g., hydrogen bond (H-bond), host−guest insertion, metal−ligand coordination, van der Waals force, and aromatic stacking) are noncovalent interactions that are essential in nature as they participate in many biological processes and are critical in maintaining the three-dimensional structure of large organisms.1 Development of supramolecular chemistry helps to understand many biological events and has also brought new directions in designing biomaterials because of its several advantages. First, noncovalent interactions are flexible, dynamic, and reversible since they do not involve the sharing of electrons.2 They can simultaneously interact with multiple sites and rearrange their structures or morphologies by the internal/external stimuli.2 Second, supramolecular fabrications are less complicated as compared to covalent systems. The supramolecular structures could be built by simply blending building blocks in the solution to self-assemble into three-dimensional structures.2,3 Among supramolecules, cyclodextrins (CDs) have attracted tremendous attention for the past decades, and more than 10 000 papers have been published in the literature. CDs are structured with the hydrophilic outer surface, which favors the interaction with surrounding molecules through H-bonds, and the hydrophobic cavity, which can host a diverse range of hydrophobic guests to form host−guest inclusions in aqueous conditions and even in the solid state.4,5 In the pharmaceutical © XXXX American Chemical Society
Received: November 7, 2016 Accepted: January 9, 2017
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DOI: 10.1021/acsmacrolett.6b00841 ACS Macro Lett. 2017, 6, 83−88
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ACS Macro Letters
Scheme 1. Schematic Illustration of Gelatin Glue (Gelatin-(hydroxyphenyl)propionic Acid (Termed “GH”)) Formed by Oxidation Reaction Using HRP and Hydrogen Peroxide (H2O2) (a) and CD-Reinforced Cohesion and Adhesion Strength through H-Bond and Host−Guest Interactions with Functional Groups on Gelatin as Well as Skin (b)
Figure 1. Characterization of 5 wt % gelatin glues with CDs (GH5/CDs): the curing time of GH glues containing different amounts of α-CD (0, 2.5, 5, and 7.5 wt %) as a function of HRP concentration at H2O2 concentration of 0.03% (a), the residual H2O2 in GH5 and GH5/α-CD5 glues formed at HRP concentration of 0.003 and 0.009 mg/mL as a function of time (b), the α-CD effect on the curing time of gelatin glues in different media conditions (c), the G′ of GH5/α-CD glues and GH5 control (d), and in vitro proteolytic gelatin/CD glue degradation as a function of α-CD concentrations (0.005 mg/mL collagenase) (e) were measured (*P < 0.05; **P < 0.005; ***P < 0.001; NS P > 0.05).
would improve the gelatin glue adhesiveness by forming additional supramolecular networks. Hydroxyl groups of CDs (18−24 groups per molecule) can form multiple H-bonds effectively with the gelatin backbone (cohesive bonding; Scheme 1bii) and even with the tissue surface (adhesive bonding; Scheme 1bv). Additionally, the hydrophobic cavity of CDs has strong complex affinity to phenol groups in the glues (cohesive complex; Scheme 1biii) and biomolecules on skin tissues (adhesive complex; Scheme 1bvi). To demonstrate our hypothesis, gelatin/CD glue adhesion strength on porcine skins or modified gold substrates with fatty acid and tyramine was measured using a universal testing machine (UTM). Additionally, gelatin/CD glues were characterized by their gelation time, elastic modulus (G′), and degradation kinetics. Human dermal fibroblasts (hDFBs) were cultured to test the glue toxicity using WST-1 and live/dead staining assay.
adhesion, biodegradability, high cell compatibility, and low immunogenicity. Herein, we described supramolecular interaction utility of CDs to strengthen the adhesion property of bioglues to skin tissues (Scheme 1), which has not yet been explored to the extent of our knowledge. The skin tissues are mainly composed of type 1 collagen, but gelatin was chosen as a base glue material because of its water solubility. The gelatin glues were rapidly formed by the horseradish peroxidase (HRP)-mediated oxidation reaction of 3-(4-hydroxyphenyl)propionic acid (HPA) that was conjugated onto the gelatin backbones (GH). This reaction took place through C−C bonds at the ortho-position of the phenol groups or C−O bonds between the ortho-carbon and phenolic oxygen (Scheme 1a, 1bi).11,12 The oxidation reaction could take place at the glue−skin interface as shown in Scheme 1biv. We hypothesized that CDs 84
DOI: 10.1021/acsmacrolett.6b00841 ACS Macro Lett. 2017, 6, 83−88
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ACS Macro Letters
Figure 2. Adhesive strength of gelatin glues containing different α-CD concentration (a), reduction in the adhesive strength of GH5 (white) or GH5/α-CD5 (black) glues in various preparation media (NaCl 1 M and PEG 2.5 wt %) as compared to the adhesive strength in distilled water (DIW) (b), the GH5 adhesiveness incorporated with α-CD, β-CD, or γ-CD (0 (white), 1.5 (gray), and 5 wt % (black)) (c), the GH10 adhesive strength with or w/o γ-CD compared to fibrin glue (d), and ESI-MS spectra of α-CD/tyramine (e) and γ-CD/tyramine mixtures (f) are represented (*P < 0.05; **P < 0.005; ***P < 0.001; NS P > 0.05).
dimension of 4.3 Å can fit into the cavity of α-CD with diameter of 4.7 Å,13 and the calculated binding constant was KTyrosine/α‑CD = 27 M−1. When the complexation was inhibited by PEG (poly(ethylene glycol)) (KPEG/α‑CD = 102 M−1 > KTyrosine/α‑CD),14 the original curing time was obtained, confirming the formation of HPA/CD complexes (Figure 1c). In contrast, H-bonds did not influence the curing process. GH/α-CD glues were formed in NaCl solution (1 M) to discreate the H-bond formation, but the glues exhibited the comparable curing time to those formed in DIW. A faster glue formation was obtained when more HRPs were involved in the cross-linking reaction, as the rate of the phenoxy radical generation was accelerated (Figure 1a). The average G′ increased as more α-CDs were incorporated (Figure 1d), due to the additional supramolecular cross-linking in the glue matrix associated with CDs. This result was aligned with the degradation of glues that was slower with higher CD contents (Figure 1e). The CDs tightened the glue matrix and likely limited the accessibility of enzymes to the cleavage sites. The porcine skin was used to evaluate tissue adhesion properties of GH/CD glues as it has similarities to human skin in terms of general structure, thickness, pigmentation, and composition.15 The adhesion strength was measured by UTM using the method modified from ASTM F2255-03 (“Test Method for Strength Properties of Tissue Adhesives in LapShear by Tension Loading”) 9 (Figure S5, Supporting Information). Compared to gelatin alone, gelatin/α-CD glues exhibited significantly high adhesion strength (Figure 2a). As more CDs were incorporated in the glues, the stronger adhesiveness was obtained. GH5 glues containing 5 wt % of αCD showed the adhesiveness of 27.2 ± 0.8 kPa that was nearly 2.4-fold higher than that of GH5 (11.1 ± 0.2 kPa). Considering the fact that G′ of gelatin glues was also increased by incorporation of CDs (Figure 1d), it could be seen that higher
In situ gelatin glue formation takes less than 30 s at HRP concentration of 0.010 mg/mL without phase separation (Figure 1a). Interestingly, the curing time of GH5 (5 wt % of GH) glue was prolonged as α-CD concentration increased. One mechanism behind this could be that HPAs required for the oxidation reaction by HRP were occupied by α-CDs. This hypothesis was proved by measuring the concentration of H2O2, an oxidizing agent, during the glue formation to monitor the kinetics of oxidation reaction. At the same concentration of HRP, GH5/α-CD5 glues consumed H2O2 slower than GH5, demonstrating the delayed oxidation reaction by formation of inclusion complexes of α-CDs and HPAs (Figure 1b). However, the influence of α-CDs on the oxidation reaction shown in Figure 1a and 1b became minor when the glue was rapidly catalyzed with higher concentration of HRPs. Notably, a similar curing time was obtained when 5 wt % of α-CD (αCD5) and 7.5 wt % of α-CD (α-CD7.5) were used, meaning that CDs were already excessive to phenol groups (GH5 phenol: 7.3 mM; α-CD5: 51.5 mM). A 2D-ROESY (rotating frame nuclear Overhauser effect spectroscopy) NMR measurement was performed to provide evidence of the proposed inclusion complexes. For the measurement, HPA was dispersed in D2O, yielding a turbid dispersion because HPA is poorly water soluble. The dispersion became a clear solution when α-CD was added, indicating the formation of a complex. In Figure S3, strong ROESY NMR correlations from two HPA aromatic protons H-5′ and H-9′ to a proton H-5 which is located in α-CD, along with two from HPA methylene protons H-2′ and H-3′ to a proton H-3, unambiguously indicated both HPA and α-CD are spatially placed on the closed face, supporting the formation of the guest HPA and host α-CD complexes. The complexation between αCD and phenol compounds is also well-known in the literature. Evan et al. reported that the phenolic ring of tyrosine with 85
DOI: 10.1021/acsmacrolett.6b00841 ACS Macro Lett. 2017, 6, 83−88
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ACS Macro Letters
Figure 3. UTM measurement on gold substrates is illustrated (a). GH5 glue was compared with GH5/α-CD5 glue in the stickiness on porcine skins (b). Pieces of gold substrates (bare gold substrate (Au) (i), 11-mercaptoundecanoic acid-functionalized gold surface (Au-fatty acid) (ii), and tyramine-conjugated to the Au-fatty acid substrate (Au-phenol) (iii)) were used to compare GH5/α-CD5 with GH5/γ-CD5 (c). The photo images show contact angle measurement of a water drop on gelatin (GH5) or gelatin/α-CD glue (GH5/α-CD5) (d) (*P < 0.05; NS P > 0.05).
that γ-CD enormously promoted the adhesiveness (36.5 kPa) of the glues even more than α-CD did. This betterment could be explained by the difference in the size of their cavities. γ-CD is more than 2-fold larger than α-CD in the size of cavity, which allows it to accommodate more than one guest molecule. This assertion was evidenced by the mass spectroscopy of CD/ tyramine mixtures. The parent ions detected in the mass spectrum of α-CD/tyramine (Figure 2e) at m/z 1112 correspond to protonated 1:1 complexes of α-CD/tyramine, while the peaks of both 1:1 and 1:2 complexes of γ-CD/ tyramine were represented at m/z 1435 and 1572, respectively (Figure 2f). This agreed with the findings in the literature that linoleic acid (fatty acid) and salicylic acid (phenolic compound) have been reported to form a complex with one γ-CD.20,21 Unlike α-CDs and γ-CDs, however, β-CD showed minor effects on the adhesion strength probably due to its relatively low water solubility. By using GH10 (10 wt % of GH) and 10 wt % of γ-CDs, the highest adhesiveness of GH/CD glues was achieved (Figure 2d), which was almost 10-fold higher than the adhesiveness of fibrin glues (Tissucol Duo 500). Interestingly, GH glue was completely detached from one of the skin patches, while GH/α-CD glue was pulled apart on two skin patches, adhering to both skin surfaces (Figure 3b). This phenomenon is indicative that the GH/α-CD glue may possess stronger interaction with the skin. To understand the glue−skin interactions, fatty acids (Figure 3aii) and/or phenol groups (Figure 3aiii) were introduced on gold substrates as they are known to be present in proteins and structural proteoglycans of skin22,23 and have a complex affinity to CDs.24 The adhesion strength of the GH/CD glues was measured using the modified gold substrates and compared to bare gold (Figure 3ai). For both GH/α-CD and GH/γ-CD glues, the adhesiveness was increased by the following order: bare Au < Au-fatty acid < Auphenol (Figure 3c). The presence of CDs in gelatin glue enabled the complexations with fatty acids or phenols on gold,
mechanical strength of the glues may contribute to the greater adhesiveness. When two skin tissues were sliding in different directions, separation of the skins can occur due to the glue cohesion failure. It is conceivable that high cohesion strength of glues is crucial to sustain a given amount of strain and achieve strong adhesion strength. We hypothesized that the adhesiveness increment is due to H-bond formation. In Figure 2b, a significant reduction in tissue adhesiveness of GH5/α-CD glues was found when the glues were prepared in saline. NaCl salts in the saline solution broke H-bonds, reducing the adhesive strength of 9.5 kPa. In the literature, it is well accepted that the H-bond is one of the most important and powerful interactions for the tissue adhesiveness.16,17 The H-bond forces between OH and COOH are known to be as strong as the single bond forces (0.151−0.171 nN).18 Another predominant factor to explain the adhesive enhancement was the CD−phenol complexation in the glues. The complexation effect was examined by the adhesion test, where PEG was added in the GH5/α-CD glues. The PEG was applied to countervail the complexation, and the GH5/α-CD5 adhesion strength dramatically decreased. GH5 glues alone also showed that the reduction in the adhesiveness may be due to the lubricative nature of PEG,19 but the difference between GH5 and GH5/α-CD glues was clear enough. Investigating how structural variation of CDs influences the gelatin/CD glue adhesion properties was another important subject of the study. Among innumerable CDs that are different in their sizes and functionalities, nonfunctionalized α-CD, βCD, and γ-CD were selected as representatives. The adhesion property of gelatin glues that were supplemented with α-CD, βCD, and γ-CD was evaluated as a function of the CD concentrations (0, 1.5, and 5 wt %). As expected, the gelatin/αCD adhesiveness was in the manner proportional to the α-CD concentrations (Figure 2c). However, what was not expected is 86
DOI: 10.1021/acsmacrolett.6b00841 ACS Macro Lett. 2017, 6, 83−88
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ACS Macro Letters yielding tremendous enhancement in adhesion strength (Figure 3aiv). The higher adhesion strength on the Au-phenol surface than Au-fatty acid is likely because the phenol groups present on the gold surface were also possibly involved in the HRP reaction when the glue was formed. The glues containing γCDs were stickier than α-CD glues due to more efficient complexation. H-bond formation on the glue surface was estimated by the wettability of the glues with or without CD incorporation. It was found in the literature that changes in Hbond extent are closely correlated to the changes in the wettability.25 Although both glues were hydrophilic (their contact angle
