An Antifouling Hydrogel Contained Silver Nanoparticles for

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An Antifouling Hydrogel Contained Silver Nanoparticles for Modulating Therapeutic Immune Response in Chronic Wound Healing Guifang Shi, Wenting Chen, Yu Zhang, Xiaomei Dai, Xinge Zhang, and Zhongming Wu Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01834 • Publication Date (Web): 07 Aug 2018 Downloaded from http://pubs.acs.org on August 8, 2018

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An Antifouling Hydrogel Contained Silver Nanoparticles for Modulating Therapeutic Immune Response in Chronic Wound Healing

Guifang Shi,a Wenting Chen,a Yu Zhang,a Xiaomei Dai,b Xinge Zhang,b,* Zhongming Wua,*

a

2011 Collaborative Innovation Center of Tianjin for Medical Epigenetics (Ministry of

Health), Key Laboratory of Hormones and Development, Metabolic Diseases Hospital & Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin 300070, China. b

Key Laboratory of Functional Polymer Materials of Ministry Education, Institute of

Polymer Chemistry, College of Chemistry, Nankai University, Tianjin 300071, China

*

Corresponding author

E-mail: [email protected]; [email protected]

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Abstract

The patients with diabetic wounds have deficient local and systemic cellular immunity. Herein, a new silver nanoparticles-contained hydrogel with antifouling property was developed for enhancing immune response in diabetic wound healing. The antifouling property was obtained by adjusting the composition of cationic chitosan and anionic dextran to approach to zero charge. Furthermore, this hybrid hydrogel showed long-lasting and broad-spectrum antibacterial activity. The rapid wound contraction was observed after the treatment of hydrogel, which suggested its superior healing activity to promote fibroblast migration, granulation tissue formation and angiogenesis. The up-regulation of CD68+ and CD3+ expression levels demonstrated that the hydrogel could trigger immune responses in the treatment of wound healing. These results show that this antifouling hybrid hydrogel as a wound dressing provided a promising strategy for the treatment of diabetic ulcers.

Key words: antifouling hydrogel, silver nanoparticles, antibacterial activity, immune response, chronic wound healing


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1. Introduction

Diabetes mellitus is characterized by high blood glucose levels associating with the risk of developing severe complications and co-morbidities including blindness, heart disease, kidney disease, foot complications, stroke and nerve damage.1,2 Diabetic ulcer is a kind of severe and intractable complication of diabetes. In the most extreme cases, it can result in gangrene and amputation.3,4 Complete ulcers healing is a long-term and tardy process. There are several factors resisting wound healing, and certain problem can be attributed to the dysfunction of cells which participate in wound healing. These are resident cells such as langerhans cells, fibroblasts, keratinocytes and newly recruited immune cells, including dermal dendritic cells, macrophages and lymphocytes. These cells are important for the eradication of bacteria, the formation of epithelial tissue, and the regulation of different stages of the wound healing.5 The controlled aggregation of white blood cells is essential to ensure a regulated healing. Trafficking patterns of immune cells in the wound can be adjusted by nanomaterials.6,7

For diabetic ulcers, conventional wound dressings cannot provide a suitable environment for tissue regeneration and repair, tend to adhere to the wounds and cause secondary damage the new granulation tissue causing bleeding.8 Moreover, the wound infection is a tremendous challenge to the wound care, because such infection can result in forming exudate, facilitating false collagen deposition, delaying the wound healing, etc. Especially, because the blood perfusion around diabetic foot ulcer is blocked, leading the low drug concentration on the ulcer, it is difficult to achieve ACS Paragon Plus Environment

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the required antibacterial effect.9,10 Thus, wound dressings with antifouling and enhanced immune responses to infection are imperiously needed.

Hydrogels exhibiting swelling in aqueous solutions, characteristic three-dimensional network and splendid porous structure lay the foundation for their good biocompatible property and drug loading. A mass of hydrogels have been applied as hopeful materials for local drug delivery depots, wound healing, and surgical tissue adhesives as well as for hemostasis during surgery.11-13 However, it is pointed that hydrogels could not be used to improve the wound healing due to their weak antibacterial effect.

Silver nanoparticles (AgNPs) are the most effective antimicrobials because they have strong resistance to viruses, microbes, and other eukaryotic microorganisms14-16 It is known that the silver can damage the microbial cell membranes and inhibit the activity of enzyme, RNA and DNA by coordinating with electron-donating groups, such as amine, thiol, hydroxyl, etc, ultimately result in bacterial death.17,18 Moreover, silver possesses low cytotoxicity and rarely induces the bacterial resistance.19 However, the accumulation of AgNPs for minimizing their interface energy will decrease the surface area to volume and reduce antimicrobial activity because the smaller specific surface area will decrease the ability of AgNPs binding to the bacteria. The AgNPs can damage and kill bacteria by attaching to their cell membranes, and the binding of AgNPs to cell membranes depends on the superficial area available for mutual effect.20,21 Also, the aggregation of AgNPs will decrease the release of ionic silver, which is conducive to the antimicrobial activity of AgNPs.21 Hence, it is desired to incorporate silver nanoparticles into antifouling materials for increasing ACS Paragon Plus Environment

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bactericidal property.

Here, the anti-fouling hybrid hydrogels were synthesized with maleic acid-grafted dextran and thiolated chitoson, and utilized to accelerate wound healing. And the silver-contained hydrogels provided a slow and sustained Ag+ release. The porous structure could provide a humid microenvironment and absorb the exudation from the wound. The effect of the silver-contained hydrogels on wound occlusion and healing acceleration in diabetic SD rats was further evaluated. The hybrid hydrogel could directly kill bacteria, modulate immune responses, inhibit the development of inflammation and accelerate the healing of wound. In conclusion, this study focuses on developing a novel antifouling and infection-fighting hydrogel, and providing a promising strategy for the promotion of chronic wound healing.

2. Experimental Section

2.1 Materials Triethylamine (TEA) and dextran (Dex) were purchased from Sinopharm Chemical Reagent Co. Ltd.(Beijing, China). 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDAC·HCl) and 1-Hydroxybenzotriazole (HOBt) were acquired from Medpep Co. Ltd.(Shanghai, China). Chitosan (CS) was obtained from Golden-Shell Pharmaceutical Co. Ltd.(Jiangsu, China). Maleic anhydride (Ma) was gained from Chemical Reagent Co. Ltd.(Tianjin, China). N-Acetyl-L-cysteine (NAc), silver nitrate (AgNO3, 99.9%), acridine orange, and ethidium bromide were obtained from Alfa Aesar(USA). Heat-inactivated fetal bovine serum (FBS), trypsin, dulbecco's modified


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eagle's medium (DMEM), penicillin–streptomycin liquid and non-essential amino acid

were

purchased

from

Gibco(USA).

Butyl

bromide

and

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenylterazolium bromide (MTT) were bought from the J&K China Chemical Ltd. (Beijing, China). Hematoxylin and eosin (H&E) were obtained from Beyotime Institute of Biotechnology (Shanghai, China). Anti-CD68 antibody and anti-CD3 antibody were obtained from Abcam (USA). EDTA antigen retrieval solution and bovine serum albumin (BSA) were purchased from ZSGB-BIO Co. Ltd. (Beijing, China). Immunohistochemical kit DAB chromogenic agent was obtained from DAKO (Denmark). Other chemical reagents were of analytical grade and not purified. S.aureus and P. aeruginosa were donated by the Department of Microbiology of Nankai University (Tianjin, China). NIH 3T3 cells were obtained from Key Laboratory of Hormones and Development of Tianjin Medical University.

2.2 Synthesis of thiolated chitosan

Thiolated chitosan (CS-NAc) was synthesized according to the method described by Wang et al.22 Briefly, chitosan (1 g) and HOBt (0.76 g) were dissolved in 100 mL of water under stirring. NAc (2.76 g) and EDAC·HCl (8.6 g) were then added to the above solution. The pH of the mixed solution was regulated to between 4 and 6, and the reaction was kept for 5 h in dark at room temperature. The resultant mixture was dialyzed against 5 mM HCl containing 0.2 % EDTA, 5 mM HCl containing 1 % NaCl and 1 mM HCl in sequence at 4 °C in dark. The dialyzed products were lyophilized


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and deposited at 4 °C. Thiol group content of CS-NAc was approximately 255 µmol/g, which was calculated through Ellman’s method.23

2.3 Synthesis of maleic acid modified dextran Maleic acid-grafted dextran (Dex-Ma) was prepared as described by Kim et al.24 Dextran (0.89 g) was added in a flask containing 18 mL of DMF and LiCl (2 g) at 90 °C under nitrogen. When the reaction temperature was cooled to 60 °C, 0.064 mL of TEA was added to the solution as a catalyst, and then 2.205 g of maleic acid was added. The reaction was kept for up to 10 h at 60 °C before the mixture was poured into cold isopropyl alcohol for precipitation. The precipitate was redissolved in deionized water, dialyzed (10 kDa cutoff) against water, lyophilized and stored at 4 °C before use. The substitution degree of maleic acid in Dex-Ma was about 30% calculated by the acid-base titration.

2.4 Synthesis of CS-NAc/Dex-Ma hydrogel (CNDM) and AgNPs@CNDM To determine the sol-gel transition, the phosphate buffer solution (PBS, pH 7.4, 0.02 M) of CS-NAc and Dex-Ma (various molar ratios of maleic group to thiol group, 3:1, 2:1, 1:1, 1:2 and 1:3) was incubated at 37 °C by vortexing, then the mixtures were evaluated by a test tube inverting method described by Jeong et al.25 Gel formation was considered when the sample did not flow for 3 min. AgNPs solution was synthesized as previously described.26 Briefly, 120 µL of 10 mg/mL AgNO3 was mixed with 5 mL of DI water, and 300 µL of 11.8 mmol/L fleshly NaBH4 was added, then the mixture was unceasingly stirred for 30 min. It was ACS Paragon Plus Environment

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suggested that the AgNPs were formed when the solution changed from yellow to brown. The synthesis of AgNPs@CNDM was similar to that of CNDM. The AgNPs solution was diluted to 1/3 of its concentration with PBS, then CS-NAc and Dex-Ma were added to the above solution for obtaining AgNPs@CNDM. All the AgNPs were encapsulated in the hydrogels and the concentration of AgNPs in AgNPs@CNDM hydrogels was 47μg/g.

2.5 Characterization of AgNPs@CNDM

FTIR spectra was made by FTS 6000 spectrometer (Bio-Rad, USA) to analyse the formation of AgNPs@CNDM. The dried hydrogels were mixed with KBr and pressed into tablets before use.

2.6 Release of ionic silver To determined the release of Ag+, AgNPs and AgNPs@CNDM with equal amounts of silver were dialyzed against 10 mL of deionized water at 25 °C. The solution was taken out every 2 h for the first 12 h, then taken out every 12 h, and finally taken out every 24 h. And an additional 10 mL of fresh deionized water was replenished into the dialysis tube every time. The solution was collected and measured by an inductively coupled plasma mass spectrometer (ICP-MS, Thermo Electron Corporation).

2.7 Morphology observation of AgNPs@CNDM The morphology of AgNPs@CNDM was filmed by scanning electron microscope (SEM, Shimadzu SS-550, Japan). To make samples, the synthetic AgNPs@CNDM


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were quickly frozen within liquid nitrogen and lyophilized until all the solvents were sublimed. The lyophilized hydrogels were sectioned carefully, fixed on conductive tape, coated with gold and observed under a SEM.

2.8 Swelling ratio of AgNPs@CNDM

Swelling measurement was performed to evaluate the hydrophilicity by immersing AgNPs@CNDM into PBS (pH 7.4) at 37 °C. At a certain time interval, the hydrogels were fetched and the surface moisture of them was wiped off, and then weighed on an electron microbalance (AE 240, Mettler, Switzerland) with the accuracy of ± 0.1 mg. The samples in each group were measured in triplicate. Swelling ratio was calculated as follows:

Swelling ratio (%) =

Wt − W0 × 100 % W0

where Wt is the weight of swollen hydrogels, and W0 is the primal weight of hydrogels. 2.9 Cytotoxic assay

NIH 3T3 cells were used to evaluate the cytotoxicity of AgNPs and hydrogels, and a standard MTT assay was carried out to analyze cell viability.27 Then the cells suspension (5 × 104 cells/mL) was seeded into 96-well culture plate and cultured in DMEM added with penicillin–streptomycin liquid and FBS at 37°C for 24 h. In order to get hydrogel leaching solution, the hydrogels were immersed in pH 7.4 PBS for 72 h, then the supernatant was taken out and filtrated. After 24 h of incubation, the culture medium was changed with new medium containing various hydrogel leaching


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solutions or AgNPs solutions, and an equal amount DMEM medium was added as control, then the cells were cultured for 48 h. Afterwards, MTT solution was added into the above media and cultured for another 4 h, and then the midia were sucked out and replenished with DMSO. Absorbance at 490 nm of each well was determined by a microplate reader (Molecular Devices, USA). The cell viability was calculated as follows:

The cell viability = (At–A0)/(Ac–A0) × 100% where At is the absorbance of treated cell, Ac is the absorbance of controlled cell and A0 is the absorbance of PBS.

2.10 Antibacterial assay The antibacterial property of AgNPs@CNDM was evaluated using LIVE/DEAD assay, and representative bacteria containing S. aureus and P. aeruginosa were chosen to evaluate the antimicrobial activity. Bacteria were diluted to 106 CFU/mL and incubated at 37 °C for up to 24 h with 10 mg of hydrogels (AgNPs@CNDM and CNDM) and AgNPs in experimental groups, and without hydrogels in control group, respectively. The content of AgNPs in the AgNPs@CNDM group was consistant with that of the AgNPs group, which was 762 μg. Then 0.1mg of each acridine orange and ethidium bromide was added to the bacteria for staining. Fifteen minutes later, the bacteria were washed with PBS and suspended with glycerol solution. The stained germs were viewed by a fluorescence microscope (Leica DMI4000B, Germany). Three repeats were performed for each material.


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The morphological change of bacteria was observed by scanning electron microscope (SEM, JEOL-JSM7500, Japan) before and after the treatment of nanoparticles and hydrogels.28 P. aeruginosa and S. aureus were incubated at 37 °C for up to 60 min with 10 mg of hydrogels (AgNPs@CNDM and CNDM) and AgNPs in experimental groups. The bacteria were performed without treatment as control. Afterwards, glutaraldehyde, PBS, and a stepwise of ethanol were used for bacteria to fixation, wash and dehydration, respectively. All the bacteira with extra procession were viewed under SEM.

2.11 The antibacterial study in vivo

The SD male rats used in research met the strict requirment of the Chinese Association of Accreditation of Laboratory Animal Care. All male SD rats were raised with a standard laboratory water and diet ad libitum in SPF environment under humidity-, lighting-, and temperature-controlled conditions. All rats were induced with STZ at a dosage of 65 mg/kg body weight and glucose monitoring was performed after 7 days. The SD rats models of diabetes were successfully establishedwhen the random blood glucose was greater than 16.7 mmol/L. These diabetic rats were randomly divided into three groups (n = 25), and then four full thickness-round wounds (1.5 cm in diameter) were prepared on the upper back of each rat with a surgical scalpel after anesthetization and sterilization.

According to the treatment, the four wounds of each rat were divided into four groups: (I) treatment group, (II) model group, (III) treatment control group and (IV) blank


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control group. In the treatment group and the model group, 100 µL of bacterial suspension of S. aureus and P. aeruginosa (2.0 × 107 CFU/mL) was mixed to be directed at the wound surface. Three days later, we observed the wounds with yellow purulent fluid, indicating that the diabetic wound infection model was established. The treatment groups were treated by AgNPs@CNDM, AgNPs or CNDM. The model group was treated by 0.9% NaCl solution, and the blank control group was treated without any intervention.

After the wound model was established, three treatment groups were AgNPs@CNDM group, AgNPs group and CNDM group (n = 25 wounds per group), respectively. The wounds of AgNPs@CNDM group were treated with AgNPs@CNDM hydrogel, and the wounds of AgNPs group were smeared with AgNPs solution, as well as the wounds of CNDM group were covered with CNDM hydrogel.

All the animals were observed every day during the experiment period. After days 1, 4, 7 and 10, the wound areas were measured individually. And the measurement results in the next days were presented as percent change in wound area relative to day 1. Then all rats were sacrificed, and wounds with surrounding tissue were excised and fixed for histological and immunohistochemical analysis.

2.12 Histology analysis For histological preparation,

the excised

skins were immersed in 4

%

paraformaldehyde overnight for fixation, dehydrated in a stepwise of ethanol (70%~100%) and embedded in paraffin. Each wound tissue was sectioned into 5 µm


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thickness slices for H&E staining. The photos of sections were taken by a light microscope (Nikon Eclipse TE2000-U) for histological analysis.

2.13 Immunohistochemistry staining

Wound tissues fixed with paraffin were sectioned into slices of 5 µm thickness, then attached to slides, deparaffinized and rehydrated. In order to block endogenous peroxidase stainning, the sections were immersed in H2O2 for 15 min and washed with PBS at room temperature. The tissue sections were placed in antigen retrieval buffer for antigen repairing, and blocked with 5 % BSA for 30 min at room temperature. The sections were incubated with anti-CD3 antibody and anti-CD68 antibody at approximately 4 °C all night. Then HRP secondary antibodies were used for 30 min in moist chamber to visualize the primary antibody. Afterwards, the sections were treated with a DAB chromogen kit and hematoxylin in sequence, and then dehydrated, cleared and sealed with cover slip for subsequent microscopic observations.

2.14 Statistical analysis

The obtained data were calculated and analyzed by mean and standard deviation, and Student’s t-test was used to determine the statistical difference with SPSS for Windows (version 13.0; SPSS Inc., Chicago, IL, USA). In statistical analysis, p < 0.05 was regarded as significant difference.


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3. Results and discussion

3.1 Synthesis and characterization of anti-fouling AgNPs@CNDM

Hydrogels are

promising materials in biomedical applications for their three

dimension crosslinked network structure. Here, the CNDM hydrogels were synthesized through Michael addition of an aqueous dispersion of Dex-Ma and CS-NAc in PBS at 37 °C. The hydrogels with different components were obtained by changing the molar ratio of thiol group to maleic group. The molar ratios of CNDM for CN1DM3, CN1DM2, CN1DM1, CN2DM1 and CN3DM1 were 1:3, 1:2, 1:1, 2:1 and 3:1, respectively. The antifouling hydrogel was obtained when the molar ratio of CS-NAC to Dex-Ma was 1:3, and the total zeta potential of the mixture was close to zero. The AgNPs@CNDM hydrogels were synthesized by mixing CS-NAc and Dex-Ma with AgNPs solution. To confirm the synthesis of these materials, the obtained samples were characterized by 1H NMR (400 MHz, D2O, TMS). As displayed in Figure 1A, the peak at 2.87 ppm indicated that -SH of NAc was successfully coupled to CS chain, and the peaks at 6.35 and 7.91 ppm were attributed to proton of the double bond of maleic acid (Figure 1B), indicating the successful synthesis of Dex-Ma.


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Figure 1. 1H NMR spectra of (A) CS-NAc and (B) Dex-Ma.

FTIR spectra of CNDM and AgNPs@CNDM were shown in Figure 2. The results depicted the absorption peak of AgNPs@CNDM at 1630 cm-1 became weaker than that of CNDM, which can be assigned to the bind of AgNPs with CNDM by amino bonds.

Figure 2. FT-IR spectra of (A) CNDM and (B) AgNPs@CNDM.

3.2 Morphology of AgNPs@CNDM

AgNPs@CNDM with three-dimensional cross-linked network had unique antifouling property. Internal morphology of AgNPs@CNDM hydrogels was viewed by using a


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SEM. The morphology images of AgNPs@CNDM exhibited a highly porous three-dimensional meshy structure, and the pore diameter was distributed around 10 µm (Figure 3). These capillary channels allowed hydrogel stronger permeation and adsorption to keep moist wounds and prevent hydrogels adhesion to wounds, better gas exchange to improve the permeability of wound skin, and preferable load ability to bond with AgNPs.

Figure 3. SEM images of AgNPs@CNDM and CNDM.

3.3 Swelling assay

Hydrophilic property is very important for the hydrogels, and it can help hydrogels to facilitate exudation absorption and drug leakage. The hydrophilicity was determined by exploring the swelling of hydrogels. As shown in Figure 4, the swelling speed of these hydrogels increased prominently when the molar ratio of maleic group to thiol group changed from 1:3 to 3:1, illustrating that Dex-Ma was vital for the swelling property of AgNPs@CNDM. The swelling ratio of these hydrogels was increased rapidly in the first 5 min of immersion, then the swelling speed gradually slowed down, and reached swelling equilibrium within 60 min of incubation, the average


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maximum swelling rate was about 850%, which showed that hydrogel had excellent performance of water absorption. A properly moistened environment can promote drug release and absorbsion of excessive drainage around the wound tissue, which helps to reduce the adhesion to the wound tissue, facilitate the formation of granulation tissue, improve the wound healing and prevent bleeding and pain when dressings are exchanged.29

Figure 4. Swelling kinetic curves of AgNPs@CNDM in pH 7.4 PBS at 37 °C.

3.4 Release of ionic silver The release of Ag+ from AgNPs@CNDM and AgNPs was measured by ICP-MS. As shown in Figure 5, the concentration of silver ion release in both AgNPs@CNDM and AgNPs was the highest at initial 2 h and then declined in the following time. After 6 hours, the release of ionic silver in AgNPs@CNDM group was higher than that in AgNPs group. It was possible that AgNPs@CNDM could slow down the speed of Ag+ release. Eight hours later, the release of ionic silver in AgNPs@CNDM group almost reached a constant level, indicating that AgNPs@CNDM displayed a sustained


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and slow Ag+ release, which may make hydrogel continuous antibacterial activity.

Figure 5. Release of silver ions from AgNPs and AgNPs@CNDM.

3.5 Biocompatibility assay

The MTT method was used to evaluate the biocompatibility of AgNPs and AgNPs@CNDM against NIH3T3 cells. The cells were cultured without the hydrogel in control group, and with AgNPs@CNDM and AgNPs with various concentrations for 48 h in experiment groups. The cell viability of AgNPs@CNDM was higher than that of AgNPs (Figure 6A), indicating that AgNPs@CNDM could decrease the cytotoxicity of AgNPs. We speculated that as the carrier of AgNPs, CNDM hydrogels could release Ag+ slowly to avoid the sudden increase of Ag+ level, thus weakening the cytotoxicity of AgNPs. As shown in Figure 6B, the viability of NIH3T3 cells was more than 80% after treatment with AgNPs@CNDM. It can be seen that there was no statistical difference in cell viability among various concentrations of hydrogels, which displayed good biocompatibility of AgNPs@CNDM hydrogels, so the hybrid


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hydrogel could be used as a wound dressing.

Figure 6. Cell viability of NIH3T3 cells incubated with AgNPs and AgNPs@CNDM.

3.6 Antimicrobial activity

The LIVE/DEAD baclight bacterial viability kit in combination with the SEM was used to evaluate antimicrobial activity of the hybrid hydrogels. In this assay, bacteria which showed red or green were dead or live with damaged or intact membranes when observed under fluorescence microscopy. Figure 7 depicted that after treated with AgNPs@CNDM for 60 min, almost all bacteria presented red, while some of bacteria still presented green in the CNDM and AgNPs groups, and all bacteria were green in control group, indicating that CNDM and AgNPs presented weak antibacterial activity compared with AgNPs@CNDM.

The SEM images were performed before and after treated with CNDM, AgNPs and AgNPs@CNDM, and the results were shown in Figure 8. For the bacteria without treatment, the bacterial surface presented smooth. On the contrary, the bacteria were shriveled and even died after treated with CNDM, AgNPs and AgNPs@CNDM. It was observed that the bacteria were severely damaged after treated with


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AgNPs@CNDM, compared with those treated with AgNPs and CNDM. Therefore, we concluded that AgNPs@CNDM exhibited strong impact on cell walls and membranes of bacteria, which eventually brought about bacterial death. Here, the antibacterial activity of AgNPs was decreased, which could be caused by its easy aggregation in aqueous solution.

Chitosan is a cationic linear copolymer polysaccharide, and its binding to the bacterial cell membranes results in cell leakage on account of the stronger interaction of O-chains and the outer membrane structure.30 AgNPs was proved to kill bacteria by associating with bacterial membranes and releasing Ag+.31,32 The results could be used to prove that AgNPs@CNDM possessed potent antimicrobial activity.

Figure 7. Fluorescence micrograph of P. aeruginosa and S. aureus before and after the treatment of CNDM and AgNPs@CNDM.


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Figure 8. SEM images of P. aeruginosa and S. aureus before and after the treatment of CNDM and AgNPs@CNDM.

3.7 Effect of silver nanoparticles-loaded hydrogel on the wound healing in vivo Traditional dressingis easy to adhere to the tissue, and damage newborn granulation tissue to cause bleeding when dressing is changed. In order to solve the problem, we adjusted material proportion to make it antifouling activity. When the molar ratio of CS-NAc to Dex-Ma was 1:3, the total zeta potential was close to zero, which was proved its antifouling property. Moreover, the antifouling property of the CN1DM3 hydrogel had been confirmed by anti-bacterial and anti-cellular adhesion experiment and the CN1DM3 hydrogel could resistant infection from pathogenic bacteria and prevent further damage during the replacement of wound dressings.33 The new antifouling hydrogel with 1:3 mass ratio of CS-NAc to Dex-Ma and AgNPs was developed, which was equivalent to zwitterionic materials. The antibacterial activities of AgNPs, CNDM and AgNPs@CNDM in vivo were evaluated with diabetic wounds on rats’ back. On day 1, 4, 7 and 10 post-treatments, the changes in wound area of rats were shown in Figure 9. On day 1 post-treatment, the condition of the wounds was


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consistent in four groups. After 7-day treatment, it was observed that the wounds of AgNPs@CNDM, CNDM and AgNPs groups contracted compared with those of blank control and model groups. After 10-day treatment, the wounds in the model group were closed slower than those in other groups. Compared with the wounds in AgNPs and CNDM goups, the wounds in AgNPs@CNDM group were almost completely healed.

Figure 9. General wound observations after treated with AgNPs, CNDM, AgNPs@CNDM and the normal saline control groups at different time (A). The wounds were divided into four groups: (I) treatment group, (II) model group, (III) treatment control group and (IV) blank control group. Wound open areas at different time after the treatment of CNDM (B), AgNPs (C) and AgNPs@CNDM (D), respectively. ACS Paragon Plus Environment

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3.8 Histological analysis The wound tissue was obtained for H&E-stained histological evaluation on 10th day to determine the host immune responses. As shown in Figure 10, after 10 days treatment, both blank and model control groups had more inflammatory cells compared with AgNPs@CNDM treatment and the control groups, showing the shortening of the inflammatory process characterized with less inflammatory cells in the latter two groups, indicating that the wound healing process was advanced. In contrast, the wounds with mass inflammatory cells in model group were not healed. The results demonstrated that AgNPs@CNDM could promote the host immune response during wound healing in vivo.

Figure 10. H&E staining of the rat dermal wound for (I) AgNPs@CNDM treatment group, (II) model group, (III) AgNPs@CNDM treatment control group and (IV) blank control group

3.9 Immunohistochemistry staining

In the experiment, CD68+ and CD3+ was chosen as the most reliably specific marker


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of macrophages and T lymphocytes, respectively, which played a vital function in the healing process.34,35 To detect the expression of CD68+ and CD3+, tissue sections were stained with antibodies to CD68+ and CD3+ by immunohistochemistry. The CD68+ positive cells were brown in the cytoplasm (Figure 11A), and the CD3+ positive cells were brown in the nuclei (Figure 11B). As exhibited in Figure 11C, the CD68+ expression levels in both treatment control and treatment groups were obviously higher than those in both model and blank control groups. Such plentiful macrophages could promote pro-inflammatory activities, help to eliminate invading microbia and accelerate healing.

Figure 11. Micrographs of immunocytal marker staining in the wound: positive cells of CD68+ (A) and CD3+ (B). The higher CD68+ expression levels (C) in both the


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treatment control and treatment groups were statistical significant in contrast with model and blank control groups, respectively (** p < 0.05). The higher CD3+ expression levels (D) in both the treatment and treatment control groups were statistical significant in contrast with model and blank control groups, respectively (** p < 0.05).

Similarly, as shown in Figure 11D, the CD3+ T lymphocytes in both the treatment control and treatment groups were obviously higher in contrast with other groups. The fact of rapid wound healing in the treatment group and the results of up-regulation of CD68+ and CD3+ expression levels suggested that AgNPs@CNDM could promote specific immunocytes activation. However, few studies are performed about the mechanism of immune response modulated by this material. Moreira Neto et al. reported that metallic nanoparticles could promote immunocytes activation, cytokines secretion, and induce a host response.6 Moreover, AgNPs displayed a vital function in recruiting and activating immunocytes especially macrophagus.36 Previous studies also showed that AgNPs could directly activate the innate immune responce.37,38 All results considered, we could draw a conclusion that the novel AgNPs@CNDM hydrogel could promote immune cells to gather in wound regions, fuel the immune response and accelerate wound healing.

4. Conclusions A new AgNPs@CNDM hydrogel for modulating therapeutic immune response was developed by mixing chitosan and dextran with AgNPs. The hybrid hydrogel with antifouling property lays the foundation for improving wound healing. This hybrid


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hydrogel basically fulfills the requirement of ideal wound dressings, not only preventing adhesion to the wounds, but also providing a slow and sustained Ag+ release for antibacteria. The hydrogel could stimulate immune system against infection and accelerate diabetic wound repair. Hence, the antifouling hydrogel contained silver nanoparticles provided a new approach for the treatment of chronic wound infection.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant No. 81671835, 21474055, 21774062 and 51673102) and the Natural Science Foundation of Tianjin City, China (Grant No. 18JCYBJC29300).

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The graphic abstract


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An Antifouling Hydrogel Contained Silver Nanoparticles for

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