Multifunctional Hydrogel Patch with Toughness, Tissue Adhesiveness

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Multifunctional Hydrogel Patch with Toughness, Tissue Adhesiveness, Antibacterial Activity for Sutureless Wound Closure Xinchen Du, Le Wu, Hongyu Yan, Lijie Qu, Lina Wang, Xin Wang, Shuo Ren, Deling Kong, and Lianyong Wang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.9b00130 • Publication Date (Web): 02 Apr 2019 Downloaded from http://pubs.acs.org on April 3, 2019

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ACS Biomaterials Science & Engineering

Multifunctional Hydrogel Patch with Toughness, Tissue Adhesiveness, Antibacterial Activity for Sutureless Wound Closure

Xinchen Du†, Le Wu†, Hongyu Yan†, Lijie Qu†, Lina Wang†, Xin Wang†, Shuo Ren†, Deling Kong†, Lianyong Wang*, †

†Key

Laboratory of Bioactive Materials, Ministry of Education, College of Life

Sciences, Nankai University, Tianjin 300071, P. R. China

*Corresponding author. Tel.: ＋86－137－0210－6876 (Lianyong Wang) E-mail: [email protected]

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Abstract A multifunctional hydrogel patch with a combination of high toughness, superior adhesion, and good antibacterial effect is a highly desired surgical material. In this study, we developed a novel hydrogel patch composed of poly(ethylene glycol) diacrylate/quaternized

chitosan/tannic

acid

(PEGDA/QCS/TA)

based

on

mussel-inspired chemistry. The physical and biological properties of the hydrogel patch were systematically evaluated in vitro and in vivo. The results indicated that this hydrogel patch possessed compact microstructure, low swelling ratio, tough mechanical properties, good antibacterial activities against S. aureus and E. coli, and excellent dry/wet adhesive ability to a wide range of substrates. The hydrogel patch could also be degraded and absorbed in vivo and used as a sutureless material for wound closure. All these findings demonstrate that the PEGDA/QCS/TA hydrogel patch with multifunctional properties has great potential for application in biomedical fields.

Keywords ： Quaternized chitosan (QCS), Poly(ethylene glycol) diacrylate (PEGDA), Tannic acid, Hydrogel patch, Anti-bacteria, Sutureless wound closure.

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1. Introduction The reconnection of wounded tissues is essential to restore their structures and functions.1 To date, several traditional fasteners, such as sutures, staples and wires, have been used in the clinic. However, these fasteners in some cases may suffer from some limitations, including the leakage of body fluid, suturing friable tissues and inability to be used in certain inaccessible organs.2-4 Their usage is time-consuming and liable to result in further tissue damage. The appearance of sutureless materials with rapidly reconnecting tissues ability can effectively resolve these limitations.4 Most importantly, sutureless reconnection of wounded tissues after surgery is beneficial to improve patient care quality.2 So far, various synthetic-based tissue adhesives, involving cyanoacrylate-based,5 polyurethane-based,6-7 poly(ethylene glycol)-based,8-10 polyester-based,11 and polyvinyl alcohol-based tissue adhesives,12-13 have been developed. But low wet-tissue adhesiveness, inferior mechanical properties, high swelling ratio, potential cytotoxicity and poor in vivo degradability have extremely restricted their applications. It was reported that mussels can produce a type of mucus molecule that contains abundant catechol groups that can confer strong ability to adhere to a variety of organic or inorganic materials in wet environments by oxidation reactions and chelation interaction.14-15 Inspired by the mussel adhesion chemistry, several water-resisted

tissue

adhesives

have

been

developed

by

introduction

of

L-b-3,4-dihydroxyphenyl-a-alanine (DOPA) containing a catechol group onto synthetic-based or natural-based polymers, such as catechol-modified poly(ethylene 3



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glycol),16 polyurethane,7 chitosan,17 sodium alginate,18 poly(gamma-glutamic acid),19 poly-L-lysine,20 gelatin,21-22 and albumin.23 Although the introduction of DOPA could give these polymers strong wet adhesive properties, the high cost and neurological effects of DOPA may pose concerns about its practical applications.24 On the other hand, when catechol-modified polymers are used as tissue adhesives, oxidants such as H2O2, Fe3+ and NaIO4 are necessary to transit the catechol to quinine groups.14, 19, 22, 25 The oxidants are liable to cause the too rapid oxidization of the catechol groups, leading to short-term adhesiveness.26 In addition, these adhesives cannot be used repeatedly. Fortunately, the low-cost and safe tannic acid (TA) derived from plants has been used as an alternative to DOPA due to the existence of a high content of dihydroxyphenyl (catechol) and trihydroxyphenyl (gallic acid, GA) groups in its molecular structure.24,

27-30

TA also possesses excellent antibacterial, antioxidant,

anti-inflammatory, and biocompatible properties, which give it great potential for utilization in biomedical field.31-36 Ninan et al. developed a novel pH-sensitive TA-carboxylated

agarose

composite

hydrogel

with

antibacterial

and

anti-inflammatory activities for wound healing.35 Guo et al. developed an injectable hydrogel bioadhesive composed of TA and gelatin with silver nitrate used as a crosslinker.24 Wang et al. designed a promising TA/Pluronic F-68 drug-delivery nanovehicle delivering dexamethasone for inflammatory bowel disease therapy.37 Moreover, TA can improve the mechanical strength of polyvinyl alcohol-based, polyvinyl pyrrolidone-based and protein-based hydrogels through intermolecular interactions (hydrogen bonds and hydrophobic interactions) with different 4


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substrates.38-40 With increasing concern about bacterial infections in injured sites, there is a growing need for the development of tissue adhesives with antibacterial properties.1, 41-42

Antibiotic-modified and antibiotic-containing polymer-based adhesives have

been developed.43-46 Because the excessive usage of antibiotics is liable to result in the appearance of multiple-drug-resistant strains,47 the usage of inherently antibacterial polymers is considered the best choice. Quaternized chitosan (QCS) has been widely accepted and intensively studied to be used as an antibacterial polymer.48-49 The aim of this work is to develop a multifunctional hydrogel patch for sutureless wound closure. Toward this purpose, we synthesized QCS and poly(ethylene glycol) diacrylate (PEGDA). The mixture of QCS and PEGDA was gelled by ultraviolet light (UV) to form a hydrogel patch. The resultant hydrogel patch was immersed in TA solution to obtain a PEGDA/QCS/TA hydrogel patch. The properties of the hydrogel patch, including toughness, swelling ratio, wet adhesion abilities, antibacterial activity, degradation, and wound closure capability, were investigated in detail. Our results demonstrate that this hydrogel patch with multifunctional properties could act as an effective alternative to sutures to achieve sutureless closure of wounds.

2. Materials and Methods: 2.1 Materials 5



Chitosan (CS, molecular mass of ~50 kDa, deacetylation degree of 95%) was purchased from Jinan Haidebei Biotech Co., Ltd., China. Poly(ethylene glycol) (PEG, mean molecular mass of ~6 kDa) was from Shanghai 3A Chemicals Co., Ltd., China. Glycidyl trimethylammonium chloride (GTMAC, 99.5%), acetic acid, acryloyl chloride, tannic acid (TA, 99.8%), photoinitiator (Irgacure 2959), triethylamine (TEA), dichloromethane (CH2Cl2), ethyl alcohol and diethyl ether were from Shanghai Aladdin Co., Ltd., China. TEA and CH2Cl2 were distilled prior to use. All other reagents were of analytical grade and were used as received.

2.2 Synthesis and characterization of PEGDA The PEGDA was synthesized by the esterification reaction between PEG and acryloyl chloride. Briefly, PEG (4 g, 0.67 mmol) and TEA (1.4 mL, 10 mmol) were dissolved in 20 mL of dry CH2Cl2 followed by adding acryloyl chloride (1 mL, 12.3 mmol) predissolved in 5 mL of dry CH2Cl2 in an ice-water bath over 1 h. After that, the mixture reacted for 24 h at ambient temperature. At the end of the reaction, the undissolved substance was removed by centrifuging the mixture at 5000 rpm for 3 min at 4°C. PEGDA was obtained by repeatedly dissolving in CH2Cl2 and precipitating in precooled ethyl alcohol/diethyl ether (2/8, v/v) three times. The chemical structure of PEGDA was characterized by nuclear magnetic resonance (1H NMR, Mercury Vx-300) with D2O as solvent.

2.3 Synthesis and characterization of QCS 6


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The QCS was synthesized as described.50 In short, 1 g CS was dispersed into 36 mL acetic acid aqueous solution (0.5%, v/v). After stirring for 5 h, 1.6067 g GTMAC predissolved in 10 mL acetic acid aqueous solution (0.5%, v/v) was dropwise added into the above mixture over 1 h. The reaction ran for 24 h at 55°C. At the end of the reaction, the mixture was centrifuged (12,000 rpm, 3 min) to remove undissolved substance. QCS was obtained after repeated dissolution in deionized water (DIW) and precipitation in ethyl alcohol three times. The chemical structure of QCS was characterized by 1H NMR (D2O as the solvent). The degree of substitution (DS) of GTMAC onto the CS backbone was determined by titrating the content of chlorine ion.51

2.4 Preparation of the hydrogel patch The PEGDA/QCS/TA hydrogel patch was prepared by the following procedures: (1) Preparation of PEGDA/QCS hydrogel patch: A 2 wt% solution of QCS dissolved in phosphate buffer solution (PBS, pH=7.4) and 5 wt%, 10 wt%, 20 wt% solutions of PEGDA predissolved in 0.1 wt% Irgacure 2959 aqueous solution were mixed intensively at the volume ratio of 1/2. The resultant mixtures were added into the Teflon model, followed by placing into the UV box installed on a UV curing lamp (425 nm, 1200 mW/cm2) for 1 min to obtain three kinds of hydrogel patches, which were called Gel1, Gel2 and Gel3, respectively. Gel0 was prepared using only 20 wt% solution of PEGDA under UV. For comparison, Gel1ʹ, Gel2ʹ and Gel3ʹ were prepared with the same compositions as Gel1, Gel2 and Gel3, but without UV treatment. (2) 7



Preparation of PEGDA/QCS/TA hydrogel patch: The Gel3 patch was immersed in 5 mL of 30 wt% TA solution for 24 h at ambient temperature to obtain the PEGDA/QCS/TA hydrogel (Gel3-TA) patch. The Gel3-TA patch was repeatedly washed by DIW prior to use.

2.5 Rheological properties The rheological properties were evaluated by a rotating rheometer (Bohlin Advanced Rheometer, Malvern Instruments) equipped with 25-mm parallel plate at 37°C. Briefly, the solutions or patches were first placed onto the plate’s surface, then contacted with another plate. Next, 200 μL of silicon oil was coated around the solutions or patches to prevent the evaporation of water. Finally, the dynamic frequency sweep model at angular velocities ranging from 1 to 100 rad/s at 1.0% strain amplitude was used to perform tests. These measurements were taken three times per sample.

2.6 Microstructure The microstructures of Gel3 and Gel3-TA patches were observed by using a scanning electron microscope (SEM, Quanta 200, Japan). The patches were lyophilized and cut. The surfaces and cross-sections of patches were sprayed with a gold layer, followed by visually observing by SEM.

2.7 Swelling ratio 8


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The swelling ratios of Gel3 and Gel3-TA patches were measured by the weighing method.52 Briefly, the patches were preweighed and then immersed in PBS. At various time points, the patches were picked out and contacted with filter paper to remove superfluous water, followed by weighing. The swelling ratio of patch was calculated using the following equation: Swelling ratio (%) = (Ws﹣Wo) / Wo × 100%

(equation 2.1)

Where Ws and Wo represent the weight of a patch after swelling and in the original state, respectively.

2.8 Mechanical properties The Gel3 and Gel3-TA patches with dumbbell shape were prepared (Schematic illustration in Fig. S3B). The mechanical properties of the patches were measured by tensile testing machine (Instron 5865, USA) equipped with a 500-N load cell. The tensile speed was 10 mm/min. The elongation ratio, fracture energy (defined as the area below the tensile strength (stress)-strain curve) and tensile strength of the patches were obtained from tensile curves. Each sample was tested three times.

2.9 Adhesive properties The adhesive properties of the Gel3-TA patch to various substrates were evaluated by qualitative and quantitative methods. Briefly, the adhesive behaviors of the Gel3-TA patch to native tissues (liver, kidney, lung and heart from mouse; intestine from pig), glass, plastic, metal and polycaprolactone (PCL) film were 9



qualitatively photographed with a digital camera. The adhesive force of the Gel3-TA patch (2 cm width, 2 cm length, 1 mm thickness) to pigskin (2 cm width, 5 cm length), glass (2 cm width, 4 cm length) and PCL film (2 cm width, 4 cm length) were quantitatively measured by a lap shear test, the bonding area was 2×2 cm2, the tensile speed was 10 mm/min. A 500 g metal block was used to bond the Gel3-TA to the different materials, the bonding time was set at 10, 20 and 30 minutes, respectively. The corresponding test specimens were called Gel3-TA-10, Gel3-TA-20 and Gel3-TA-30. Besides, the burst pressure strength of intestine with a hole sealed with the Gel3-TA patch (1.5 cm diameter, 1 mm thickness) was also examined as described.45 The Gel3 patch and 30 wt% TA solution were control groups. The mechanism of adhesion failure was investigated as described.53

2.10 In vitro antibacterial and cytotoxic assays In vitro antibacterial assays: The antibacterial activities of patches against S.aureus (ATCC6538) and E.coli (ATCC25922) were investigated. Briefly, the pre-prepared patches were placed into wells in a 48-well microplate and rinsed with sterilized PBS for three times. Then, 10 μL bacterial suspension with a concentration of 108 colony-forming units per milliliter (108 CFU/mL) was uniformly spread onto each patch’s surface and incubated for 2 h at 37℃. After that, 1 mL of PBS was added into each well to re-suspend any survival bacteria. Then, 30 μL of the re-suspended bacterial suspension was picked out and diluted thrice by seven-fold dilution to obtain a final diluting bacterial suspension. 15 μL of the final diluting bacterial suspension 10


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was dropped onto surface of an LB agar gel with 30 degree of slope and incubated for 24 h at 37°C. The CFUs on the LB agar gel were counted. As control, 10 µL of bacterial suspension (108 CFU/mL) was spread onto a tissue culture plate (TCP) and put through the above experimental procedures. Tests were processed in triplicate, and the killing efficiency was calculated using the following formula50: Killing efficiency (%) = [(CFUs of control﹣survivor CFUs on patch) / CFUs of control] × 100%

(equation 2.2)

The antibacterial activities of patches also were evaluated by Live/Dead assay. The detailed methods were presented in Supporting Information. In vitro cytotoxicity assay: The cytotoxicity of the patches was evaluated by the cell counting kit-8 (CCK-8) and Live/Dead assays by 3T3 fibroblast cells culture. The detailed methods were presented in Supporting Information.

2.11 In vivo degradation The in vivo degradation of the Gel3-TA patch was evaluated by implanting each patch (5 mm diameter, 2 mm thickness) into subcutaneous tissue of a mouse. In short, the mouse (male, BALB/c, 6-8 weeks old) was paralyzed with 4 wt% chloral hydrate, then fixed on a surgical corkboard. After shaving and sterilizing with iodine, two full-thickness incisions (1 cm length) were made on the back of the mouse, followed by implanting preweighed patches into the subcutaneous tissue. Then, the incisions were sealed with sutures. At different time intervals, implanted patches were harvested for visual inspection and weight analysis. The weight loss ratio of each 11



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patch was calculated using the following formula: Weight loss (%) = (Wo﹣Wi) / Wo × 100%

(equation 2.3)

Where Wi and Wo represent the weight of the patch after implantation and the original weight, respectively. Three mice were used per time point.

2.12 In vivo wound closure The wound closure ability of the Gel3-TA patch was assessed by a full-thickness mouse skin incision model. Briefly, the mouse was paralyzed with 4 wt% chloral hydrate, then fixed on a surgical corkboard. After shaving and sterilizing with iodine, two full-thickness incisions (2 cm length) created on the back of the mouse were sealed with the Gel3-TA patches (2.5 cm length × 1 cm width). Incision without any treatment and sealing with suture acted as negative and positive control groups, respectively. Three mice were used per group. All tests were performed with the approval of the Animal Experimental Ethics Committee of Nankai University.

2.13 Histological analysis After day 7 post-surgery, all wound skins in different groups were harvested and immersed in 4 wt% paraformaldehyde for 4 h, followed by dehydrating in 30 wt% saccharose solution for 24 h. The dehydrated skins were cut into sections (6 μm thickness) and stained with hematoxylin and eosin (H&E), as well as Masson trichrome staining for evaluating the closure of wounds.

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2.14 Statistical analysis Statistical analysis was done using GraphPad Prism 5. Data are expressed as the mean ± SD. One-way ANOVA with the Newman-Keuls test was used to examine significant differences between groups. P

Multifunctional Hydrogel Patch with Toughness, Tissue Adhesiveness

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