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Thiol-Mediated Synthesis of Hyaluronic Acid-Epigallocatechin-3-OGallate Conjugates for the Formation of Injectable Hydrogels with Free Radical Scavenging Property and Degradation Resistance Chixuan Liu, Ki Hyun Bae, Atsushi Yamashita, Joo Eun CHUNG, and Motoichi Kurisawa Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00788 • Publication Date (Web): 14 Aug 2017 Downloaded from http://pubs.acs.org on August 17, 2017
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Thiol-Mediated Synthesis of Hyaluronic AcidEpigallocatechin-3-O-Gallate Conjugates for the Formation of Injectable Hydrogels with Free Radical Scavenging Property and Degradation Resistance Chixuan Liu, Ki Hyun Bae, Atsushi Yamashita, Joo Eun Chung, and Motoichi Kurisawa* Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669, Singapore.
*Correspondence should be addressed to
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ABSTRACT
Hyaluronic acid (HA)-based biomaterials have demonstrated only limited in vivo stability as a result of rapid degradation by hyaluronidase and reactive oxidative species. The green tea catechin, (–)-epigallocatechin-3-O-gallate (EGCG), has received considerable attention because of its powerful antioxidant and enzyme-inhibitory activities. We describe here the synthesis of HA-EGCG conjugate using a thiol-mediated reaction and its use for the preparation of a longlasting injectable hydrogel. HA-EGCG conjugates with tunable degrees of substitution were synthesized by the nucleophilic addition reaction between EGCG quinone and thiolated HA under mild conditions. Contrary to unmodified HA, the conjugates exhibited free radical scavenging and hyaluronidase-inhibitory activities. Peroxidase-catalyzed coupling reaction between EGCG moieties was employed to produce in situ forming HA-EGCG hydrogel with surprisingly
high
resistance
to
hyaluronidase-mediated
degradation.
When
injected
subcutaneously in mice, HA-EGCG hydrogel was retained much longer than HA-tyramine hydrogel with minimal inflammation.
KEYWORDS: injectable; hydrogel; hyaluronic acid; antioxidant; green tea catechin
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INTRODUCTION Hyaluronic acid (HA) is a natural linear glycosaminoglycan serving as a major component of the extracellular matrix in mammalian connective tissues.1 Given the excellent hydrophilicity and biocompatibility, HA has been exploited for diverse biomedical applications including viscosupplementation, soft tissue augmentation, wound healing and drug delivery.2-5 However, the majority of HA-based biomaterials have demonstrated only limited in vivo stability due to rapid degradation of HA. For example, HA-based dermal fillers, accounting for more than 80% of currently used soft-tissue fillers, have a limited duration of effect ranging from 3 to 8 months.6 HA is known to have a short half-life in the body; 1-3 weeks in cartilage, 1-2 days in the epidermis of skin and 2-5 minutes in blood.2 The in vivo degradation of HA is attributed mostly to the action of hyaluronidase (HAase) and reactive oxidative species (ROS), such as hydroxyl radical (•OH) and superoxide anion radical (O2• ̶ ).7,8 To address the aforementioned limitations, extensive efforts have been devoted to improve the in vivo stability of HA through chemical modification.9-12 One representative example is benzylesterified HA produced via esterification of its carboxyl groups with benzyl alcohol.11 Although the introduction of benzyl groups to HA can delay its enzymatic degradation by hindering access of HAase to the HA backbone, excessive esterification is required to achieve desired in vivo stability. For instance, partially esterified HA with 75 mol% of esterification was degraded by over 90% for 15 days after subcutaneous implantation, while about 50% of a fully esterified HA implant remained in rats after 90 days.10 In addition, the excessive modification of HA may negatively influence its physical and biological properties. Indeed, the benzyl-esterified HA is hydrophobic due to the high degree of esterification (75-100 mol%) and thus requires dissolution in an organic solvent, which is unfavorable for clinical applications. More recent studies reveal
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that more than 45 mol% of HA modification greatly attenuates the ability of HA to recognize its cell surface receptors, such as cluster determinant 44 (CD44) and lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1).4,13 Since HA-cell surface receptor interactions are involved in many important biological processes, such as cell signaling, proliferation and differentiation,1 it would be highly desirable to develop an approach for improving the in vivo longevity of HAbased biomaterials while avoiding excessive chemical modification of HA. (–)-Epigallocatechin-3-O-gallate (EGCG), the main component of green tea catechin, has gained significant attention because of its potent antioxidant, free radical scavenging and antiinflammatory activities.14 In recent years, EGCG has been shown to inhibit the activity of nuclease and collagenase by binding to the enzymes via hydrogen bonding, π-π stacking and hydrophobic interactions.15,16 Several studies have reported that a pyrogallol moiety plays an important role in the ability of EGCG and other polyphenols (e.g., tannic acid and 5-pyrogallol 2-aminoethane) to form intermolecular hydrogen bonds and coordination bonds.17-19 Given the antioxidant and enzyme-inhibitory activities of EGCG, we hypothesized that HA could be endowed with ROS scavenging and HAase-inhibitory activities by conjugating with EGCG moieties. Based on this perspective, we previously synthesized HA-EGCG conjugates by conjugating ethylamine-bridged EGCG dimers to HA backbone through a carbodiimidemediated coupling reaction.20,21 HA-EGCG conjugates not only inhibited the enzymatic activity of HAase, but also scavenged ROS more effectively than native HA. However, the relatively high hydrophobicity of EGCG dimers allowed only limited modifications of HA (degree of substitution < 3). In the present study, we developed a new type of HA-EGCG conjugates consisting of EGCG monomers grafted to HA backbone. HA-EGCG conjugates with tunable degrees of substitution (3.7-9.4) were synthesized by the nucleophilic addition reaction between
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EGCG quinone and thiolated HA at pH 7.4. HA-EGCG conjugate exhibited superior ROS scavenging effect and HAase resistance over HA-tyramine conjugate with a comparable degree of substitution. Furthermore, long-lasting injectable HA-EGCG hydrogels were produced by a peroxidase-catalyzed oxidative coupling reaction between EGCG moieties. The cellular ROS scavenging property, in vitro degradation resistance and in vivo stability of HA-EGCG hydrogels were investigated to explore their potential for biomedical applications. Histological and hematological assessment was conducted to examine the biocompatibility of HA-EGCG hydrogels.
EXPERIMENTAL SECTION Materials. Sodium hyaluronate (Mw = 90 kDa) was kindly donated by JNC Corporation (Tokyo, Japan). EGCG was purchased from DSM Nutritional Products Ltd (Heerlen, the Netherlands). Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and 4-(4,6-dimethoxy-1,3,5triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) were purchased from Tokyo Chemical Industry (Tokyo , Japan). HAase from bovine testes, lipopolysaccharides from E. coli, xanthine, ascorbic acid, and 2-deoxy-D-ribose were purchased from Sigma-Aldrich (St. Louis, MO). EnzChek gelatinase/collagenase assay kit, Micro BCA protein assay kit and DyLight 800 maleimide were obtained from Thermo Fisher Scientific (Waltham, MA, USA). ROS Brite 570 dye was purchased from AAT Bioquest (Sunnyvale, CA, USA). Xanthine oxidase from buttermilk was purchased from Oriental Yeast Co. (Osaka, Japan). Boric acid was purchased from Bio-Rad Laboratories (Hercules, CA). Horseradish peroxidase (HRP) was purchased from Wako Pure Chemical Industries (Osaka, Japan). Hydrogen peroxide (H2O2) was obtained from Lancaster Synthesis (Lancashire, UK). AlamarBlue cell viability assay reagent (Life
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Technologies, USA) was used according to the manufacturer’s instruction. All other chemicals and reagents were of analytical grade. Synthesis of thiolated HA derivatives with tunable degrees of substitution. Thiolated HA derivatives were synthesized by modifying carboxyl groups in HA backbone with thiol groups. Typically, 1 g of sodium hyaluronate (2.5 mmol –COOH) was dissolved in 100 mL of 10 mM phosphate-buffered saline (PBS, pH 7.4). To this solution, equimolar amounts of DMTMM (1.04 g, 3.75 mmol) and cystamine dihydrochloride (845 mg, 3.75 mmol) were added to initiate the conjugation process. The reaction mixture was stirred for 24 h at 25°C. Next, 15 mL of 0.5 M TCEP solution (pH 7) was drop wisely added and the reaction mixture was stirred for another 2 h. The resulting solution was transferred to dialysis tubes with a molecular weight cut-off of 3,500 Da. The tubes were dialyzed against 0.1 M NaCl solution for 2 days, 25% ethanol for 1 day and deionized water for 2 days under nitrogen atmosphere. The purified solution was lyophilized to obtain thiolated HA. The degree of substitution (DS), defined as the number of substituents per 100 repeating disaccharide units in HA, was determined by Ellman’s assay. The thiolated HA derivatives had about 85% yield. Synthesis and characterization of HA-EGCG conjugates. HA-EGCG conjugates were synthesized by conjugating EGCG to thiolated HA derivatives. In all cases, 0.5 g of thiolated HA was dissolved in 70 mL of PBS (pH 7.4) under nitrogen atmosphere. This solution was added dropwise to 30 mL of PBS (pH 7.4) containing excess of EGCG with continuous stirring. The pH of the mixture was adjusted to 7.4 by dropwise addition of 1 M NaOH. After reaction for 3 h at 25ºC, the pH of the mixture was adjusted to 6. The resultant solution was transferred to dialysis tubes with a molecular weight cutoff of 3,500 Da. The tubes were dialyzed against 25% ethanol for 1 day and deionized water for 2 days under nitrogen atmosphere. The purified
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solution was lyophilized to obtain HA-EGCG conjugates (about 95% yield). UV-visible spectra were monitored by using a Hitachi U-2810 spectrophotometer. The structure of HA-EGCG conjugate was confirmed by 1H NMR spectroscopy. DS was determined by comparing the relative peak area of two protons from D ring of EGCG (6.9–7.0 ppm) with that of three methyl protons of HA (2.0 ppm). 1H NMR (D2O, 400 MHz): δ 2.0 (s, 3H, –C=OCH3 from HA), 3.3–4.0 (m, protons of HA backbone), 4.3–4.7 (d, 2H, anomeric protons of HA), 5.6–5.9 (s, 2H, H-2 and H-3 of C ring), 6.2–6.3 (s, 2H, H-6 and H-8 of A ring), 6.6–6.7 (s, 1H, H-2’ and H-6’ of B ring), 6.9–7.0 (s, 2H, H-2’’ and H-6’’ of D ring). Evaluation of hydroxyl radical scavenging activity. HA-EGCG conjugate with DS of 6.6 was synthesized as described above. For comparison, HA-tyramine (HA-Tyr) conjugate with comparable DS (DS = 6) was synthesized according to the previous report.22 Hydroxyl radical (•OH) generated through the Fenton reaction was treated with HA-EGCG conjugate and the amount of remaining •OH was determined by the deoxyribose method.20 Briefly, 80 µM FeSO4 solution and 20 mM 2-deoxy-D-ribose solution were separately prepared by using a reaction buffer (pH 7.4) containing 0.2 M potassium phosphate and 0.3 M NaCl. Then, 50 µL of aqueous solution containing HA-EGCG, HA-Tyr, HA or EGCG at various concentrations were mixed with 50 µL of 882 µM H2O2, 50 µL of FeSO4 solution and 50 µL of 2-deoxy-D-ribose solution. After incubation at 37°C for 1 h, 50 µL of the solution was mixed with 50 µL of thiobarbituric acid (10 mg mL-1) and 50 µL of trichloroacetic acid (28 mg mL-1). The mixture was heated at 99°C for 15 min and then cooled down to 25°C. Subsequently, 145 µL of the mixture was transferred to a 96-well plate and the absorbance at 532 nm was measured using a Tecan Infinite 200 microplate reader (Tecan Group, Switzerland). The •OH scavenging activity was calculated by the following equation:
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(%) =
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−
! "
× 100
where Acontrol and Asample represent the absorbance of control and sample, respectively. For a control experiment, the reaction buffer (pH 7.4) containing 0.2 M potassium phosphate and 0.3 M NaCl was used instead of 2-deoxy-D-ribose solution. Evaluation of superoxide anion radical scavenging activity. Superoxide anion radical (O2• ̶ ) was generated by incubating xanthine with xanthine oxidase and then measured by using p-nitro blue tetrazolium chloride (NBT) according to the previous report.23 Briefly, 60 µL of 1 mM xanthine solution and 60 µL of 0.1 mM NBT solution were mixed with various concentrations of HA-EGCG, HA-Tyr, HA or EGCG (60 µL) in a UV-transparent 96-well plate. Then, 20 µL of xanthine oxidase (0.4 units mL-1) in 0.1 M phosphate buffer (pH 7) was added to each well to initiate the reaction. After incubation at 37°C for 15 min in a dark place, the absorbance at 560 nm was measured using a Tecan Infinite 200 microplate reader. The O2• ̶ scavenging activity was calculated by the following equation: &'(
(%) =
−
! "
× 100
where Acontrol and Asample represent the absorbance obtained with deionized water and sample, respectively. To examine the potential inhibitory effect of EGCG moieties on xanthine oxidase, the activity of xanthine oxidase was measured by monitoring the formation of uric acid from xanthine at 295 nm, as reported previously.23 Examination of the resistance of HA-EGCG conjugate to HAase-mediated degradation. HAase (50 units mL-1) was treated to various concentrations of HA-EGCG or HA-Tyr conjugate in 0.1 M phosphate buffer (pH 6) at 37°C for 24 h. A physical mixture containing equivalent amounts of HA and EGCG was also tested for comparison with HA-EGCG conjugate. As a
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control, HA was subjected to HAase treatment in the same manner. The extent of HA degradation was determined by measuring the concentration of HA fragments with N-acetyl-Dglucosamine reducing ends using the Morgan-Elson assay.24 Briefly, 32 µL of each sample was heated at 100°C for 5 min and then cooled down to 4°C. To this solution, 8 µL of borate solution (0.741 g of boric acid and 0.297 g of potassium hydroxide dissolved in 15 mL of deionized water) was added. The mixture was heated at 100°C for another 4 min and then cooled down to 4°C. Subsequently, 160 µL of p-dimethylaminobenzaldehyde (DMAB) solution (0.1 g of DMAB dissolved in a mixture of 125 µL of 10 N HCl and 9.775 mL of glacial acetic acid) was added to the mixture and incubated at 37°C for 20 min. After centrifugation at 1000 × g for 15 min at 4°C, 150 µL of the supernatant was taken and the absorbance at 585 nm was measured using a Tecan Infinite 200 microplate reader. The resistance of HA-EGCG or HA-Tyr conjugate to HAasemediated degradation was calculated by the following equation: ) ℎ ' (%) =
+, − +,
! "
× 100
where AHA and Asample represent the absorbance obtained with HA and sample, respectively. Evaluation of collagenase-inhibitory activity. Collagenase-inhibitory activity of HA-EGCG conjugate was characterized by using the EnzChek gelatinase/collagenase assay kit according to the manufacturer’s instruction. Briefly, DQ gelatin (a fluorogenic substrate for collagenase) was mixed with various concentrations of HA-EGCG, HA-Tyr or HA at a volume ratio of 4:1. Then, 100 µL of the mixtures was treated with 100 µL of collagenase solution (0.2 units mL-1) for 2 h at 37°C in a dark place. The fluorescence intensity was measured using a Tecan Infinite 200 microplate reader with an excitation wavelength at 420 nm and an emission wavelength at 515 nm. The extent of collagenase inhibition was calculated by the following equation:
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- ℎ . (%) =
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(/012 − /032 ) − (/0 ! " − /042 ) × 100 /012 − /032
where FIPC, FINC, FISample and FISC represent the fluorescence intensity (FI) of positive control (DQ gelatin + collagenase), negative control (DQ gelatin), sample solution (DQ gelatin + sample + collagenase) and sample control (DQ gelatin + sample), respectively. Formation and characterization of HA-EGCG hydrogels. A stock solution of HA-EGCG conjugate was prepared by dissolving the conjugate in deionized water at a concentration of 11.5 mg mL-1 at 25ºC. The resultant solution was sterilized by filtration through a 0.22 µm syringe filter. To form HA-EGCG hydrogels, 780 µL of the filtered stock solution was mixed with 15 µL of HRP solution, 15 µL of H2O2 solution and 90 µL of 100 mM PBS (final ionic strength: 0.16 M, pH 7.4) in LoBind tubes (Eppendorf, Germany). The final concentrations of HA-EGCG, HRP and H2O2 were 10 mg mL-1, 0.24 units mL-1 and 0.76 mM, respectively. The mixture was immediately vortexed and 850 µL of which were injected into a cylindrical plastic mold (14 mm inner diameter; 6 mm height). Gelation was allowed to proceed for 24 h at 37°C in a humid chamber. For comparison, HA-Tyr hydrogels were prepared using HA-Tyr solution (11.5 mg mL-1) instead of HA-EGCG solution. The final concentrations of HRP and H2O2 used to form HA-Tyr hydrogels were 0.15 units mL-1 and 0.32 mM, respectively. The resulting cylindershaped hydrogel was taken from the mold and its dimension was measured with a digital caliper. Compression test of the hydrogel was performed with an Instron 5848 microtester (Instron Corporation, Norwood, MA). The measurement was carried out in a compression mode, with a compressive strain rate of 0.5 mm/min and no preload, using a 50 N load cell. Young’s modulus was determined using the slope of the stress versus strain curve at low strains (
