Fabrication of Self-Healing Hydrogel with On-demand Antimicrobial

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Biological and Medical Applications of Materials and Interfaces

Fabrication of Self-Healing Hydrogel with On-demand Antimicrobial Activity and Sustained Biomolecules release for Infected Skin Regeneration Ran Tian, Xinyu Qiu, Pingyun Yuan, Kai Lei, Lin Wang, Yongkang Bai, Shiyu Liu, and Xin Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01740 • Publication Date (Web): 25 Apr 2018 Downloaded from http://pubs.acs.org on April 25, 2018

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ACS Applied Materials & Interfaces

Fabrication of Self-Healing Hydrogel with On-demand Antimicrobial Activity and Sustained Biomolecules release for Infected Skin Regeneration

Ran Tiana#, Xinyu Qiub#, Pingyun Yuana, Kai Leic, Lin Wangc, Yongkang Baia, Shiyu Liub*, Xin Chena*

a. School of Chemical Engineering and Technology, Shanxi Key Laboratory of Energy Chemical Process Intensification, Institute of Polymer Science in Chemical Engineering, Xi’an Jiao Tong University, Xi’an, 710049, P. R. China E-mail: [email protected] (Corresponding-Author). b. State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi International Joint Research Center for Oral Diseases, Center for Tissue Engineering, School of Stomatology, Fourth Military Medical University, Xi’an, Shaanxi, 710032, China. E-mail: [email protected] (Corresponding-Author). c. College of Chemistry & Pharmacy, Northwest A&F University, Yangling, Shaanxi 712100, PR China [*] Corresponding-Author [#] These authors contributed equally to this work. Keywords: Self-healing hydrogel; Bacteria responsiveness; On-demand antimicrobial property; Sustained growth factor release; Infected tissue regeneration

Abstract: Microbial infection has been considered as one of the most critical challenges in bioengineering applications especially in tissue regeneration, which engender severe threat to public health. Herein, a hydrogel performing properties of rapid self-healing, on-demand antibiosis and controlled cargo release were fabricated by a simple assembly of Fe complex as crosslinker and hyaluronic acid as gel network. This hydrogel is able to locally degrade and release Fe3+ to kill bacteria as needed, due ACS Paragon Plus Environment

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to the hyaluronidase excreted by surrounding bacteria, resulting in efficient antibacterial activity to different types of bacteria. Sustained release property of certain type of growth factors was also observed from this hydrogel, owing to its dense network. Moreover, this hydrogel could repeatedly heal itself in minutes due to the coordination interaction between Fe3+ and COOH, exhibiting good potential in bioengineering applications on exposed tissue, where the materials are easy to be damaged during daily life. When topically applied onto damaged mouse skin with infection of staphylococcus aureus, the hydrogel is able to inhibit microbial infections meanwhile promote cutaneous regeneration, which formed the new skin with no inflammation within a 10-days treatment. These results demonstrate the potential application of this self-healing hydrogel for the integrated therapy of antibiosis and tissue regeneration. 1. Introduction Hydrogels are crosslinked polymer network containing a great deal of water which have been widely used for biomedical applications, because of their desirable biocompatibility, tunable architecture and negligible organism invasion1-2. The polymer network is not only able to encapsulate different cargos for targeted delivery, but also able to simulate the physical structure of extracellular matrix for promoting tissue regeneration3-4. However, the hydrogels are vulnerable to opportunistic microorganisms during implantation, leading to possible infection and inflammatory response in vivo, which is a serious threat for tissue engineering constructs5-7. Although inflammation could benefit wound healing during the early stage with the neutrophils and macrophages infiltration, the latter inhibition of inflammatory response is required to promote proliferation and remodeling for final tissue regeneration. Many studies showed that sustained and severe inflammatory reactions are detrimental to tissue regeneration in the later stages of the healing process

8-9

. Thus, effective control of the bacteria

induced inflammation was implied as an important issue for tissue engineering. To solve this problem, various antibacterial hydrogels have been fabricated by different ACS Paragon Plus Environment

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methods, including physical loading of antibacterial substances (antibiotics/silver nanoparticles) in the hydrogel and/or inclusion of antimicrobial ingredients onto the network of hydrogel through chemical reactions10-13. Although loading or grafting of antibacterial substances on hydrogel may be beneficial for inhibiting infection, it appears several negative impacts including bacterial resistance, limited effective time period, completed multi-step synthesis and especially wide toxicity to mammalian cells. Hence, hydrogels have intrinsic but hidden toxicity which could be triggered by local bacteria, is highly anticipated for tissue engineering13-17. Once applied for tissue engineering, the embedded hydrogels would always undergo the external force during daily life, which normally cause hydrogel break and the following function loss. Moreover, this procedure also opens a gate for bacteria invasion, resulting in an high chance of infection. Hydrogel formed through reversible supramolecular interactions may be a solution to the gel damage during use, due to its ability to completely heal itself. Thus, plenty of self-healing hydrogels were synthesized involving different reversible interactions, such as hydrogen bond

18-19

,

host−guest interaction20-22, charge interaction23-24 and coordinate bond 25-26. However, a multiple functionalized hydrogel synchronously performing on-demand antibiosis and self-healing ability still has not been achieved, which would have significant potential in bioengineering applications. Herein, to figure out this issue, we show an effective and simple physical strategy avoiding any chemical reactions to fabricate a hydrogel with germ-triggered antimicrobial property and rapid self-healing ability through EDTA−Fe3+ complexes crosslinked hyaluronic acid (Scheme 1). This dynamic coordinate bond formation between the EDTA−Fe3+ complexes and hyaluronic acid performed good self-healing properties of the hydrogel in minutes. Moreover, the hydrogel is able to locally release Fe3+ complexes where bacteria appear, due to the degradation of hyaluronic acid through the hyaluronidase excreted from these bacteria27. The Fe3+ complexes would be rapidly adsorbed by surrounding bacteria with subsequent reduction to Fe2+, which reacts with H2O2 to form a hydroxyl radical to damage proteins and nuclear

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acids of these bacteria28-29 (Scheme 2). The hydrogen peroxide (H2O2) was produced by both bacteria and inflammatory cells, which has been widely used as a spontaneous trigger for responsive drug release and selective Fenton reaction in the treatment of bacteria-related infections30-32. Furthermore, the nontoxic antibacterial agent (Fe3+) was designed to fill the whole hydrogel as part of physical cross-linker, which dramatically enhanced the loading amount of the antibacterial agent and prolonged its effective period, allowing the hydrogel to continuously release antibacterial agent until its fully decomposition by bacteria or the bacteria around hydrogel were all killed. This procedure exhibits high antibacterial activity with no bacteria resistance because hydroxyl radical cannot be detoxified by cellular enzyme33-34. In addition, the generated hydroxyl radicals are also prone to decompose biofilm components including proteins and polysaccharides35-36, which makes our hydrogel a promising candidate for inhibition of bacteria. In co-operation with the platelet derived growth factor (PDGF-BB) loading, this hydrogel was used for cutaneous regeneration under bacterial (staphylococcus aureus) infection in a rodent subcutaneous infection model, because the PDGF-BB is mitogenic for most mesenchymal derived cells including stem cells, fibroblasts and smooth muscle cells, which would effectively promote wound healing and vascular stabilization.36-41 In this case, our hydrogels were able to inhibit microbial infections meanwhile promote growth of blood vessels as well as cutaneous regeneration, which formed the new skin with no inflammation within a 10-days treatment. This study is expected to solve the problems for conventional hydrogel on low antibacterial efficiency, side effect to healthy tissue and gel function loss during use. 2. Experimental section 2.1 Materials Iron (III) Chloride (FeCl3), Ethylenediaminetetraacetic acid (EDTA) disodium salt, hyaluronidase (HAase, from bovine testes) and hyaluronic acid (HA, Mw 1000000) were purchased from Sigma-Aldrich, USA. Platelet derived growth factor BB (PDGF-BB) was purchased from Peprotech, USA. Triton-X100 and Bovine serum ACS Paragon Plus Environment

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albumin (BSA) were purchased from MP, USA. All the products were analytical grade and used without further treatment. 2.2 Preparation of the HA-Fe-EDTA hydrogel Hyaluronic acid (HA) was dissolved in DI water at the concentration of 3% (wt/vol) at room temperature. Separately, the FeCl3 and EDTA with the mole ratio of 2:1 were dissolved in DI water to form the crosslinker (Fe3+-EDTA complex). As to tune the crosslinking degree, the Fe3+-EDTA complex solution was prepared with different concentrations. Then 400 uL HA solution and 100ul Fe3+-EDTA complex solutions with different concentrations were vortex mixed for 10 s to make homogenous mixture. After that the mixture was kept at room temperature without shaking for complete gel formation. 2.3 PDGF-BB loading in HA-Fe-EDTA hydrogel The resulted HA-Fe-EDTA hydrogel was lyophilized at first. Meanwhile 0.33 ug of PDGF-BB was dissolved in 500 ul PBS to get PDGF-BB solution with concentration of 0.66 ug/ml. Then the dry HA-Fe-EDTA hydrogel was incubated in the PDGF-BB solution for 12 h at room temperature to load PDGF-BB. 2.4 Rheology test of the HA-Fe-EDTA hydrogel Rheological properties of the hydrogels were tested on a strain-controlled AR-G2 rheometer (TA Instruments Inc., New Castle, DE) with parallel-plate geometry of 22 mm in diameter. The homogeneous mixture, which contained 400 uL of HA solution and 100 uL of Fe3+-EDTA solution, was placed between the parallel plates of 22 mm diameter and with a gap of1000 um. Oscillatory rheological measurements were performed in the linear viscoelastic regime after the complete hydrogel formation. A frequency of 1 rad/s with a dynamic strain sweep from 0.1% to 10%and a strain of 1% with a dynamic frequency sweep from 1 to 100 rad/s were respectively conducted to measure the storage modulus G' and loss modulus G''. 2.5 Self-healing performance of the HA-Fe-EDTA hydrogel



Two methods were used to testify the self-healing properties of the HA-Fe-EDTA hydrogel through macroscopic self-healing experiments. (1) Several pieces of the hydrogel disks (10 mm diameter, 2 mm thickness) stained with red or blue dyes were cut into half respectively. 2 pieces of alternate colors were combined into blended integral hydrogel disks, and then placed at 25 oC for several minutes. After that, the healed hydrogel disk was hold up to check their healing ability. (2) Several pieces of the hydrogel disks (10 mm diameter, 2 mm thickness) stained with red or blue dyes were brought into contact with each other, and then placed at 25 oC for several minutes. After that, the healed hydrogel disk was hold up to check their healing ability. 2.6 In vitro antimicrobial activity of the HA-Fe-EDTA hydrogel The antimicrobial activity of the HA-Fe-EDTA hydrogel was evaluated based on the colony count method. Two types of bacteria were used (S. aureus and E. coli) according to an adjusted standard test method (ISO 14729:2001(E)). Prior to in vitro antimicrobial tests, the bacteria were grown aerobically overnight in 5 mL of MHB at 37 oC under shaking at 200 rpm. The bacteria were re-cultured by taking 10 uL of the overnight bacterial suspensions from the tube and adding 5 mL fresh MHB. The samples were sequentially incubated at 37 oC and shaken at 200 rpm until mid-log phase (OD600~0.5). 1 mL of the bacteria suspension was collected in sterile Eppendorf tubes, centrifuged at 1000g and washed with PBS to remove the nutrient culture medium. This process was repeated three times. Finally, the testing bacteria were resuspended in PBS at a concentration of 109 CFU mL-1, and 100 uL each was spread onto the surfaces of the LB broth (control group) and hydrogel covered LB broth, respectively. After a certain contact time, the samples were placed in 1 mL PBS. The mixture was vigorously shaken for 10 min to remove all the bacteria, which was verified by microscope. Then the suspensions were plated in LB agar with appropriate dilutions. The LB agar plates were incubated at 37 oC for 24 h and the number of colonies was recorded. The antimicrobial activity was represented by log reduction, which was calculated by the below equation. The experiments were carried out in


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triplicate for each formulation. log reduction = log (cell count of control) - log (survivor count on hydrogel surface). 2.7 Stimulated Fe3+ release and sustained PDGF delivery In the HAase triggered Fe3+ release experiment, a certain amount of HA-Fe-EDTA hydrogel was immersed in 10 mL of 2 different types of PBS buffer (i: in the presence of 4 mg/L HAase and ii: in the absence of 4 mg/L HAase) at 37°C. Subsequently, 50 uL of supernatant were taken at predetermined time intervals from the release medium and replaced with the same volume of the fresh release medium (PBS with and without HAase). The collections were measured by atomic absorption spectroscopy to quantitatively analyze the released Fe3+. In vitro release profiles of PDGF from the HA-Fe-EDTA hydrogel were determined as follows. 10 mg of hydrogel were incubated in 5 ml PBS (pH=7.4) at 37oC with shaking. At designated times, 100 uL release medium was collected by centrifugation and replaced with equal amount of fresh PBS. The released amount was measured by Elisa kit. 2.8 Construction of rodent cutaneous wound model with infection A total of twenty-four 8-week-old female C57BL/6 mice were randomized to four groups. Each mouse was anesthetized intraperitoneally with 1% sodium pentobarbital, and the skin was prepared. Then the full-thickness wound including the panniculus carnosus muscle was made on the mid back. After that 1-cm diameter punch biopsy instrument was placed with moderate force onto the dorsum of the mouse to create an impression of the circumference. Next, the middle of the outline region of skin was sharply excised along the outline with a pair of scissors. The excised tissue was full-thickness skin in depth, leaving subcutaneous dorsal muscle exposed after excision. After that, 100 µL of inoculums containing 109 CFU/mL of E. coli was uniformly smeared on the wound. The gels were then placed into the dorsal wound. The wound was then covered by two layers of Vaseline gauze with discontinuous suture onto the marginal recipient mice skin of the defect area by 4-0 silk suture. ACS Paragon Plus Environment


2.9 In vivo assay of the antimicrobial activity and wound healing property To determine the rate of wound healing in wound area, the wounds were imaged by digital camera after surgery at 0–10 days. Photographs were uploaded to an appropriate computer platform and were analyzed using the Image Pro Plus image analysis software (Media Cybernetics, USA) from three randomly selected views of each specimen, respectively. All photographs were taken with the experimental mouse placed adjacent to a metric ruler that was used for distance calibration and standardization, allowing subsequent quantitative analysis. The percentage of wound closure was calculated as follows: (area of original wound–area of actual wound)/area of original wound×100%. “100% wound healing rate” means the defective tissues were completely healed. The harvested skins were fixed in 4% phosphate-buffered paraformaldehyde for 12h, embedded in paraffin. 10 um thick serial sections were cut from the paraffin embedded blocks and underwent H&E staining. The pathology index from 0 to 5 was determined based on the area infiltrated by inflammatory cell. Photographs were uploaded to an appropriate computer platform and were analyzed using the Image Pro Plus image analysis software (Media Cybernetics, USA) from three randomly selected views of each specimen, respectively. 2.10 Immunofluorescence staining. The sections from tissue were firstly fixed and rinsed. After rinsing, they were permeabilized with 0.03% Triton-X100 for 10 min at room temperature and blocked in 3% BSA at 37 ℃ for 30 min. The sections were incubated overnight (at least 8 hours) at 4℃ with the primary antibody for CD31 (1:200, Abcam, USA), respectively. After rinsing, the sections were incubated with fluorescence secondary antibody (Abcam, USA) at room temperature for 1 hour. The nuclei were counterstained by Hoechst 33342 (Sigma-Aldrich, USA) for 10 min at room temperature. The results were examined under a confocal microscope (Olympus, Japan). The photographs were evaluated by Image-Pro Plus 6.0 (Media Cybernetics, USA) for micro vessels


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density from three randomly selected views of each specimen, respectively. 2.11 RNA extraction and real-time RT-PCR of mRNA. The total RNA was extracted by Trizol reagent (Invitrogen, USA) according to the manufacture’s protocol from skin. 1000ng total RNA wan reverse transcribed to cDNA using a PrimeScript RT reagent kit (TaKaRa, Japan). Real-time RT-PCR analysis was performed using the SYBR Premix Ex Taq II kit (TaKaRa, Japan) and tested by CFX96TM Real-time RT-PCR System (Bio-Rad, USA). β-actin was used as the internal control for quantitation of the target mRNA. The primer sequences for real-time RT-PCR were given in Supplementary table 1. 2.12 Statistical analysis All the results are representative of data generated in three independent experiments. All numerical values were expressed as the mean ± SD. Comparisons of two groups were done with two-tailed Student’s t tests and comparisons of multiple groups were done with ANOVA using the Statistical Program for Social Science. P-values less than 0.05 were considered statistically significant. 2.13 Characterization The overall morphology of the microspheres was examined using scanning electron microscopy (SEM) (Hitachi S-4800 with energy dispersive spectrometer) after gold coating of the microsphere samples on a stub. The infrared (IR) spectra were measured by AVATAR 320 FT-IR using KBr pellets. All pH value measurements were carried out on a Sartorius BECKMAN F 34 pH meter. 3. Results and Discussion 3.1 Preparation of the HA-Fe-EDTA Hydrogel The hydrogel was fabricated through self-assembly of precoordinated Fe3+-EDTA complexes and hyaluronic acid via metal−ligand interactions. Briefly, the EDTA and FeCl3 solution with molar ratios of 1:2 were first mixed to form complexes, followed by pouring into a hyaluronic acid solution to generate hydrogel, where these ACS Paragon Plus Environment


Fe3+-EDTA complexes would act as robust linkers. As to explore the optimal condition for gel formation, we maintained the amount of hyaluronic acid at 30 mg/mL and mixed with four different ratios of Fe3+-EDTA complexes. The instant formation of coordination bond was evidenced by the infrared (IR) spectra (Figure 1a). It is obvious in figure 1a, the increasing new signal at 1740 cm-1 against the concentrations of Fe3+-EDTA complexes indicates coordination bonding formation between the Fe3+ and carboxyl groups from EDTA and HA, which is considered as main force to drive gelation. To get the mechanical property of these hydrogels, we did the rheological analysis at varied frequencies and strains (Figure 1b and 1c). As can be seen from these figures, the value of G′′ was less than G′ during the whole measurement, which means the generation of a self-standing hydrogel. Moreover, the frequency of the measurement remarkably affects the dynamic modulus of all these hydrogels, owing to the repeatable interaction between carboxyl groups and Fe3+ (Figure 1b). While there is no visible change of the elastic modulus against the strain increase, which exhibits the HA-Fe3+-EDTA coordination bond is stable and all these hydrogels are homogeneous (Figure 1c). The highest G' and G" appeared at sample with mole of Fe3+-EDTA complexes and HA units equal to 1:2, indicating the best mechanical properties, which were chosen for further investigations. The structure of the optimal hydrogel was further measured by scanning electron microscope (SEM, Figure 2), exhibiting that the hydrogel is fully filled by porous structure with 10-20 µm in diameter. Its chemical component was obtained by energy-dispersive X-ray spectroscopy (EDS), which shows obvious signal of iron and nitrogen (EDTA), indicating the existence of Fe3+-EDTA complexes as crosslinker in the hydrogel (Figure 2d).

3.2 Macroscopic Self-healing and Responsive Degradation of HA-Fe-EDTA Hydrogel


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Based on this reversible metal−ligand interaction, we investigated the self-healing ability of the HA-Fe-EDTA hydrogel (Figure 3). Several column-shaped hydrogels with different color were first cut into two pieces equally. After that, two pieces were randomly connected to form one gel. After standing for only 1 min, the crack completely disappeared, resulting in a stable Janus hydrogel (Figure 3IV and 3V). When several pieces of these hydrogels were brought into contact with each other, the hydrogel stuck together to form an aggregate (Figure 3VI). The metal−ligand interactions between Fe-EDTA and HA was strong enough to hold the shape of the gel assembly. Moreover, the boundaries between three different color hydrogels became blurred and the red color originally at edges appeared in the middle of the gel-assembly with the increase of incubation time (Figure 3VII), indicating the free mass transport among adjacent hydrogel pieces, which not only provided a further evidence of the complete self-healing, but demonstrate the possible unhindered circulation of metabolites and nutrient substance in our hydrogel for effective tissue regeneration. To mimic the environment of bacterial infection, we evaluated the degradability and Fe3+ release property of the HA-Fe-EDTA hydrogel in PBS with and without HAase to respectively simulate the bacterial infected tissue and healthy tissue, due to the behavior of bacteria for sustained secretion of HAase. Figure S1 shows similar morphology of the HA-Fe-EDTA hydrogel before and after 3 days incubation in PBS only. While the adding of HAase would immediately trigger the hydrogel degradation, which nearly completed in 3 days with only few residues. This HAase responsive degradation of the hydrogel would cause on-demand Fe3+ delivery to local bacteria, resulting in a prolonged antibacterial activity. The Fe3+ release curve was obtained by measuring the concentration of the Fe3+ under different simulated physiological conditions (PBS at pH 6.8, 7.4 and 8.5 with and without HAase) against incubation time by atomic absorption spectroscopy (Figure S2). Figure S2 shows that the Fe3+ release from HA-Fe-EDTA hydrogel is mainly relied on the addition of HAase rather than the pH value. In PBS without HAase at pH 6.8, pH 8.5 and pH 7.4, the Fe3+



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release amount only ranges from 17% to 22% even after 7 days, which comes from the unbonded Fe3+. However, the release percentage all increased to over 95% after addition of HAase, indicating a HAase triggered Fe3+ release in thrin acidic, neutral or alkalinous

conditions.

The

HAase

induced

Fe3+

release

could

form

a

microenvironment rich of Fe3+ around the bacteria adhered on/in the hydrogel, following with the high bacteria toxicity performed by Fe3+ uptake, reduction (Fe3+ to Fe2+) and Fenton oxidation (Fe2+ and H2O2).

3.3 In Vitro Antimicrobial Properties and sustained cargo delivery of the HA-Fe-EDTA hydrogel The antimicrobial property of the HA-Fe-EDTA hydrogel was assessed using two common germs of wound infection in our life, Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Figure 4 shows that the hydrogel performed remarkable antimicrobial property to both E. coli (log reduction = 2.193) and S. aureus (log reduction = 2.564), demonstrating a efficient antibacterial material for different germs. After verified the antimicrobial activity of the HA-Fe-EDTA hydrogel, platelet derived growth factor (PDGF-BB) was loaded in this hydrogel for the further application of infected skin regeneration, due to its property for promoting tissue regeneration and wound healing42-43. The release profile of PDGF-BB from the HA-Fe-EDTA hydrogel was measured in PBS at pH 6.8, 7.4 and 8.5 with and without HAase, which were used to simulate physiological environments, through monitoring the PDGF-BB concentration in the supernatant fluid against incubation time (Figure 5). Figure 5 shows that the HA-Fe-EDTA hydrogel could always perform sustained PDGF-BB release up to over 95 % in 10 days even under different pH values, while the release speed of PDGF-BB changes with the pH value, where the rate at pH 6.8 > the rate at 8.5 > the rate at 7.4, indicating the alkalinous wound environment would further enhance the PDGF-BB release. This release procedure of PDGF-BB highly


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matched with the skin regeneration period44-45, making our HA-Fe-EDTA hydrogel a good candidate for cutaneous regeneration.

3.4 Biocompatibility of the HA-Fe-EDTA hydrogel As biomaterials for tissue regeneration, the biocompatibility is also important. Thus, we assessed the biocompatibility of the HA-Fe-EDTA hydrogel through cell cytotoxicity investigation, which was measured by DNA quantification kit (Sigma-Aldrich, USA) according to the manufacture's protocol after 3 days, 7 days and 10 days co-culture with L-929 mouse fibroblast. The fluorescence of DNA content was read by using Varioskan Flash multimode reader (Thermo Fisher Scientific, USA), where 360 nm was chosen as excitation wavelength and 460 nm was chosen as emission wavelength

46

(Figure S3). Figure S3 shows no obvious

cytotoxicity of the hydrogel with over 90% cell viability even after 10 days incubation, indicating that the hydrogel has high biocompatibility for biomedical applications.

3.5 In Vivo Evaluation of Inflammation Inhibition and Cutaneous Regeneration The in vivo experiment was performed on mice after being removed the back skin and daubed with 100ul bacteria (109 CFU) to evaluate the ability of the PDGF loaded HA-Fe-EDTA hydrogel patch, which was expected to present high antimicrobial activity to inhibit infection and inflammation, meanwhile provide sufficient growth factors (PDGF) in long term to promote cutaneous regeneration. Beside the mouse treated by PDGF loaded hydrogel, the mouse treated by hydrogel only and untreated mouse were chosen as negative controls. After that the photographs of each group at different time points were taken to evaluate the effect of transplantation (Figure 6). Figure 6 shows that the hydrogel with the PDGF-BB loaded had the best regenerative effect among all the groups. During the wound healing procedure, the release of PDGF-BB after antimicrobial performance has exerted the good conditions for granulation formation and angiogenesis. Meanwhile, ACS Paragon Plus Environment


compared to the untreated group, with only HA-Fe-EDTA hydrogel patch application also can improve the regenerative effect on wound healing, which indicate bacteriostasis play an important role in cutaneous regeneration. Considering the number of neovascularization is directly related to the rate and quality of wound healing, we also investigated the angiogenesis of the wound area after treatment by different samples. As can be seen from figure 7, a large number of microvessels appeared in the group of PDGF loaded HA-Fe-EDTA hydrogel, which is way above the level of pure hydrogel group and untreated group. The pure HA-Fe-EDTA hydrogel group also promoted a bit of the neovascularization, indicating that the effective suppression of the bacterial level is also significant for establishment of a good blood supply. More importantly, with the release of PDGF-BB, there is also a synergistic effect on the migration and recombination of the endothelial cells as well as the promotion of their proliferation and differentiation, which further enhanced the formation of new tissue, resulting in a rapid skin regeneration. As to demonstrate the designed performance of the PDGF loaded HA-Fe-EDTA hydrogel for infected skin regeneration, we focus our study on inflammation control. It can be seen from figure 8 that the inflammatory cells were significantly reduced after 10 days in both the pure hydrogel group and the PDGF-BB loaded hydrogel group. At high magnification, we found some of skin appendages were nascent and epithelization was completed partly. Through the detection and analysis of tissue RNA, we found that the pro-inflammation related genes were significantly down-regulated by application of HA-Fe-EDTA, which indicate that the inflammation has been effectively and consistently suppressed. Thus, the well-conditioned immune microenvironment improves the success rate of the cutaneous regeneration. All these results revealed that our HA-Fe-EDTA could achieve the on-demand release of Fe3+ and sustained release of growth factors to synergistically promote wound healing meanwhile inhibit surrounding bacteria, which is a promising candidate for applications of infected tissue regeneration.


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The skin samples from the infected wounds with and without HA-Fe-EDTA hydrogel treatment were collected, and the number of bacteria were counted after 24 h culture to investigate the antimicrobial activity of the HA-Fe-EDTA hydrogel in vivo (Figure S4). Figure S4 shows that the samples without hydrogel treatment have high content of bacteria with a number of 6.1 log CFU. However, a much lower number of for 1.5 log CFU was collected for the hydrogel treated group, which indicates a remarkable in vivo antimicrobial activity of the HA-Fe-EDTA hydrogel with a log reduction of 4.6. The microbial/skin metabolites of the infected wound before and after HA-Fe-EDTA treatment (Figure S5) and the chemical analyses of hydrogel residues generated during this therapy (Figure S6 and Figure S7) were all measured by mass spectrometry and atomic absorption spectroscopy to provide in vivo evidence for the antibacterial mechanism of our hydrogel. As can be seen from figure S5, the signals of infection relative microbial/skin metabolites decreased dramatically against the time period of HA-Fe-EDTA treating, which is finally equal to the uninfected sample (healthy control) after 7 days treatment, indicating the ability of our hydrogel to effectively inhibit the bacteria infection. Figure S6 showed the mass spectrometry of all hydrogel residues after different time of use. It is observed in figure S6, the HA-Fe-EDTA hydrogel contains no molecules with molecular weight between 1000 and 2000, because the molecular weight of HA100K, Fe3+ and EDTA are all not in this range. However, few molecules with molecular weight between 1000 and 2000 appeared after 1-day treatment, and the quantity gradually increased against the treating time. At day 7, obvious signals could be observed on the mass spectrometry, indicating the degradation of our hydrogel by the bacteria secreted HAase, which is the prerequisite to release Fe3+ for bacterial inhibition. The in vivo change of Fe3+ content in the HA-Fe-EDTA hydrogel residues was also investigated by the atomic absorption spectroscopy (Figure S7). It is observed in figure S7, the content of Fe3+ in the HA-Fe-EDTA hydrogel gradually drop with the ACS Paragon Plus Environment


increase of treating time, indicating the effective release of Fe3+ from hydrogel in infection environment, which is in accordance with the degradation process of the hydrogel demonstrated by MS results. The continuously released Fe3+ is able to kill bacteria as need, which would result in inhibition of the bacteria infection and decrease of the infection relative microbial/skin metabolites (Figure S5). All these results indicate the potential of our hydrogel as a new class of bioactive materials for bacteria killing and infection inhibition.

4. Conclusion We have fabricated a self-healing hydrogel which is able to perform on-demand antibiosis and prolonged growth factors release for the integrative procedure of infection inhibition and tissue regeneration. The hydrogel could rapidly heal itself in minutes, through the dynamic coordination interaction between Fe3+ and COOH. In addition, this hydrogel can effectively kill E. coli and S. aureus as needed due to the bacteria triggered Fe3+ release and succedent fenton oxidation in bacteria. After preloading the platelet derived growth factor (PDGF-BB), the hydrogel performed ability on inhibiting microbial infections meanwhile promoting angiogenesis, resulting in the rapid formation of new skin with no inflammation within a 10-days treatment. The integration of self-healing, on-demand antibiosis and sustained growth factor delivery into one single hydrogel developed a new approach for effective therapeutics of the bacterial infected tissue defects.

Acknowledgements This work was supported by the National Natural Science Foundation of China (81601606 to XC and 81400498 to ZL), the "Young Talent Support Plan" of Xi'an Jiaotong University (XC), the Technology Foundation for Selected Overseas Chinese Scholar of Shaanxi Province (XC), the Fundamental Research Funds for the Central Universities (2016qngz02 to XC), and the One Hundred Talents Program of Shaanxi ACS Paragon Plus Environment

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Province (XC). Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: ****** (1) Photographs of HA-Fe-EDTA hydrogels under different conditions (Figures S1); (2) Stimuli-responsive Fe3+ release (Figure S2); (3) Cytotoxicity of HA-Fe-EDTA hydrogel (Figure S3); (4) In vivo antibacterial property of HA-Fe-EDTA hydrogel (Figure S4); (5) Mass spectrometry of HA-Fe-EDTA hydrogel residues (Figure S5 and Figure S6); (6) In vivo content change of Fe3+ in HA-Fe-EDTA hydrogel (Figure S7); (7) Gene Primer sequence used in experiments (Table S1) Reference 1.

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Scheme 1. Schematic illustration of formation and self-healing mechanism of the HA-Fe-EDTA hydrogel


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Scheme 2. Schematic illustration of the bacterial triggered on-demand antimicrobial behavior of the HA-Fe-EDTA hydrogel

Figure 1. Formulation and rheological characterizations of HA-Fe-EDTA hydrogels. FTIR spectra of hydrogel samples made with four different cross-linker (Fe3+-EDTA) concentrations (a). The storage modulus G' and loss modulus G" were plotted logarithmically against frequency (b) and strains (c) of the corresponding hydrogel samples.



Figure 2. (a-c) Scanning electron microscope (SEM) images of the HA-Fe-EDTA hydrogel with mole of cross-linker (Fe3+-EDTA) and HA units equal to 1:2. (d) The energy dispersive X-Ray (EDX) spectrum of corresponding hydrogel sample.

Figure 3. Photographs of self-healing performance of the HA-Fe-EDTA hydrogels: 1) hydrogel samples were cut in half (II and III), and then the two fragments after being


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brought together (III) to contact for several seconds could heal into one integral piece (IV and V). 2) several hydrogel samples were brought into contact with each other, the hydrogel stuck together to form an aggregate (VI), which has free mass transport among adjacent hydrogel pieces (VII)

Figure 4. In vivo antibacterial activity test of the HA-Fe-EDTA hydrogels. Photographs of actual CFUs of E. coli (a) and S. aureus (b) on agar plates from diluted bacterial suspension without (i) and with (ii) HA-Fe-EDTA hydrogel treatment. (c) Log reduction of the HA-Fe-EDTA hydrogels against E. coli and S. aureus calculated from the photographs (pristine Luria−Bertani broth was used as control group) (mean ± SD, n = 3).



Figure 5. Release profiles of PDGF-BB from the HA-Fe-EDTA hydrogels in vitro in PBS buffer with pH 6.8, pH 7.4 and pH 8.5. Each data plot was measured from 3 samples.


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Figure 6. Wound healing rate. (a) Photographs of wound regions were taken on days 0, 3, 7 and 10 after the creation of excisional wounds and application of the different hydrogel. (b) Analysis of wound healing rates. The data was collected with n=6 per group and data are shown as mean ± SD. * means P

Fabrication of Self-Healing Hydrogel with On-demand Antimicrobial

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