Chitosan hydrogel loaded with copper-tin-sulfur nanosheet materials for skin wound healing

Chitosan hydrogel loaded with copper-tin-sulfur nanosheet materials for skin wound healing | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Chitosan hydrogel loaded with copper-tin-sulfur nanosheet materials for skin wound healing Mingfei Ren, Jingjing Yao, Dicheng Yang, Jingyao Zhu, Kun Dai, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5302334/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Apr, 2025 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Globally, burns are a serious health problem that disrupts the normal functioning of the skin and increases the risk of bacterial infections. Traditional burn dressings often have difficulties to achieve desired therapeutic results. Therefore, there is an urgent need to develop an ideal wound dressing with good antimicrobial properties, biocompatibility and rapid promotion of burn wound healing. Herein, we prepared a chitosan-based hydrogel loaded with copper-tin-sulfur nanosheet materials (CS/GP/Cu 3 SnS 4 ), and explored its biocompatibility and antibacterial properties in vitro. The results showed that the antibacterial rate of CS/GP/Cu 3 SnS 4 hydrogel can exceed 95% after contacting with Staphylococcus aureus and Escherichia coli for 4h. Meanwhile, the survival rate of L-929 cells was consistent with that of normal medium, revealing considerable antibacterial effect and biocompatibility which could be used for promoting wound repair. Furthermore, in vivo experiment was conducted to test its dressing properties, antibacterial properties, and the efficiency of promoting wound healing. Compared with control group and CS/GP hydrogel group, the wound healing rate was the highest since the 3rd days of treating with CS/GP/Cu 3 SnS 4 .While macrophage secretion factor 68 (CD68) decreased significantly and its expression level was lower than that of control group, and the expression level of VEGF increased significantly, with its expression being 1.18-fold, 3.61-fold, and 1.98-fold higher compared with the CS/GP hydrogel group. These results indicate that CS/GP/Cu 3 SnS 4 hydrogel has a potential application as a burn wound dressing. Biological sciences/Biochemistry Physical sciences/Materials science Physical sciences/Nanoscience and technology Burn wound dressing Chitosan Hydrogel CS/GP/Cu3SnS4 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Skin plays a variety of functions that are essential for human survival. It consists of epidermis and dermis. The epidermis is a barrier against bacterial invasion and water loss, which can rebuild shallow wounds. However, incomplete regeneration is inevitable for dermal lesions containing hair follicles, sweat glands and other skin appendages 1 – 3 . Burn is a disease that leads to the destruction or necrosis of skin cells, which poses a serious threat to human health, including psychological, physical disability and even death 4 – 6 , and is one of the most serious traumas in the world 7 , 8 . According to the symptoms, burns can be divided into first-degree burns, second-degree burns and third-degree burns. First-degree burns affect the epidermis, second-degree burns affect the dermis, and third-degree burns affect a larger area and reach the deep layer of the skin 9 , 10 . Burn wounds usually have more exudates and necrotic tissue than other types of wounds, providing conditions for bacterial reproduction which results in a higher probability of infection 11 – 13 . This not only causes long-term pain for the injured, but also affects aesthetics, and can even lead to psychological trauma. While the traditional wound dressing is not ideal in terms of antibacterial performance, especially when the wound area is large, the possibility of skin peeling and bacterial infection is also greatly increased due to water evaporation 14 , 15 . Therefore, the synthesis of burn dressings that can effectively combat bacteria and promote wound repair is of paramount importance 16 . Due to air permeability, low cost and easy operation, traditional burn dressings, such as gauze and skimmed cotton 17 , topical ointment 18 , are still widely applied to absorb wound fluid and prevent wound infection. However, wound adhesion, limited absorptive capacity and moisturizing properties may cause unbearable hurts and secondary injuries during frequently dressing replacing, otherwise, increasing possibilities of bacterial infection after absorption saturation of the dressings which is the ideal culture medium for bacterial growth. In order to meet the urgent demand of antibacterial effect and moisturization, as well as improving healing rate of wound, types of hydrogel dressings have been researched according to the abilities of biodegradability 20 , 21 , biocompatibility 22 , 23 , non-toxicity, antibacterial properties, bio-adhesion, bioactivity, and hemostasis 24 – 26 . Previous reports have proved the unique biological characteristics enable these hydrogels considerable materials used as wound dressings, and further indicating the potentiality as drug delivery system (DDS) to deliver antibacterial agents 27 , growth factors, stem cells, thereby, accelerating wound healing efficiency 28 , 29 at different stages of wound healing and alleviating factors hindering wound healing 30 . Moreover, hydrogel dressings containing different nanoparticles have been a research hotspot due to unique physical or biological nano effects. Chircov 31 et al., have developed a series of iron oxide nanoparticles uniformly dispersed chitosan dextran glycerol hydrogels by in situ formation of magnetite nanoparticles, which proves increased antimicrobial properties. However, in order to increase water absorption and permeability, glycerol is used and causing the reduction of antimicrobial potential. Besides, Puspita 32 et al., have developed a formulation of chloramphenicol microparticles (cpl-mp) and modified into chitosan hydrogel to improve the treatment efficiency against infection and create an optimal environment to support the healing process. Nevertheless, its antibacterial cycle was as long as 24 hours of direct contact. Further improving the antimicrobial properties and wound treatment efficacy of burn dressings still have been a research focus. 33 , 34 . Past studies have shown that copper ions have significant antibacterial effect on common bacteria such as Escherichia coli and Staphylococcus aureus, and can stimulate the production of collagen, which is the key protein required for wound healing 35 . Meanwhile, copper ion can promote the formation of new blood vessels and accelerate tissue regeneration 36 . Yang 37 et al., prepared a new type of BC based antibacterial wound dressing loaded with copper (Cu) ions through the co deposition of polydopamine (PDA) and copper ions, which showed considerable antibacterial properties and biocompatibility, and further proved that Cu 2+ @PBC-2 film could inhibit S. aureus infections and inflammatory effect, facilitating collagen deposition, capillary angiogenesis, hair follicle growth and wound healing. However, Excessive release of copper ions may hinder wound healing. Massoumi 38 et al., developed a wound dressing produced from gelatin sericin nanofibers containing HNT as well as copper or zinc ionophores. They discovered Cu 2+ -loaded dressing showing obviously faster bactericidal activities, however, reduced fibroblast viability which may hinder collagen secretion and blood vessels formation. Meanwhile, Zn 2+ -containing nanofibers showed ideal stimulus to induce fibroblast attachment, viability, and collagen biosynthesis, which may be attributed to iron release disparity between Cu 2+ and Zn 2+ , as iron release rate of Cu 2+ is 3 times higher than Zn 2+ . Therefore, scald dressings with ionic slow-release of Cu + still remains an ongoing goal of current research. In this work, we aimed to achieve the innovative integration of copper ions with chitosan-based hydrogels and develop a new type of scald wound dressing. To this end, we used a simple solvothermal method to prepare a polycrystalline Cu 3 SnS 4 nanosheet (Cu 3 SnS 4 NSs) and characterized its morphology and physical properties, then we loaded it on chitosan-based hydrogel (CS/GP). The potential use of copper ion in scald wound repair was evaluated by detecting its release rate, antibacterial properties and biocompatibility. Finally, it was applied to the repair of scalded wounds in mice to further prove the performance of the dressing. The results showed that this dressing (CS/GP/Cu 3 SnS 4 hydrogel) had excellent anti infection and accelerated wound repair ability, and had broad application prospects in the field of scald wound repair. Materials and methods Materials Chitosan, thioacetamide (TAA), N, N-dimethylformamide (DMF), copper acetate (CuAc 2 ), tin tetrachloride pentahydrate (SnCl 4 ⚫5H 2 O), and acetone were purchased from Shanghai Titan Technology Co. Sodium β-glycerophosphate(β-GP) was purchased from Shanghai McLean Biochemical Technology Co. CCK-8 kit, calcein-AM/PI were purchased from Shanghai Bi-Yun-tian Biotechnology Co. Preparation of the Cu 3 SnS 4 NSs Cu 3 SnS 4 NSs were synthesized through hydrothermal method. Briefly, 40ml DMF was poured into the flask, then 0.048g CuAc 2 , 0.056g SnCl 4 ⚫5H 2 O and 0.024g TAA were added successively. The reaction was stirred at 140℃ for 2h.The resulting solution was transferred into a reaction kettle and reacted at 220℃ for 12h. After the reaction, the solution was centrifugated at 6000 rpm, the resulting precipitate was thoroughly washed by acetone and deionized water to remove impurities, and finally resuspended in 1 mL deionized water for storage. Preparation of Chitosan based Hydrogel Loaded with Cu 3 SnS 4 NSs A typical chitosan solution was prepared by dissolving 100mg of chitosan powder (with a deacetylation degree of 95%) in 4.5ml of 0.1mol/L hydrochloric acid solution, and filtered and autoclaved at 121℃ for 15min. Then, 280mg of β-glycerophosphate sodium salt dissolved in 0.5ml deionized water was added drop by drop to the resulting solution, and the CS/GP hydrogel was prepared by incubated in water bath at 37℃. 200 mg of copper tin sulfur nanosheet material was subsequently added to the mixture before gelation, and the resulting mixture was completely dispersed and then incubated in water bath at 37℃ for 10min to obtain CS/GP/Cu 3 SnS 4 . Material characterization Scanning electron microscopy (SEM) Morphologies of CS/GP hydrogel and CS/GP/Cu 3 SnS 4 hydrogel were analyzed by SEM(S-4800), and the lyophilized sample was sprayed with gold for 60s to ensure sufficient conductivity before image capture. Transmission electron microscope(TEM) The samples were fully dispersed in PBS solution and dropped onto the copper sheet for drying. Then morphology and size characterization of Cu 3 SnS 4 NSs was analyzed by TEM and HRTEM(JEM-2100F), and the elements were quantitatively analyzed with Energy Dispersive Spectrometer (EDS). Zeta potential of the Cu 3 SnS 4 NSs The surface potential of Cu 3 SnS 4 NSs was measured by Laser Particle Sizer (Zetasizer Nano ZS). Cu 3 SnS 4 NSs was dissolved in PBS solution and sonicated for half an hour for completely dispersion before measurement. Detection of copper ion release rate Inductively coupled plasma atomic emission spectrometer (ICP 710) was used to measure the release of copper ions in Cu 3 SnS 4 NSs and CS/GP/Cu 3 SnS 4 . Typically, 250mg of Cu 3 SnS 4 NSs and 5ml of chitosan-based hydrogel containing 250mg of Cu 3 SnS 4 NSs were added into two centrifuge tubes containing 5ml of PBS solution to prepare five tube suspensions each. Then, sequential sample were taken from the tube at the time interval of 0h, 5h, 10h, 20h and 40h for high-speed centrifugation (10000r, 10min), and the supernatants were used for copper ion concentration detection. Antibacterial experiment The antimicrobial activity of the materials was evaluated by selecting two bacteria that are prevalent in wound infections, Escherichia coli (E. coli, Gram-negative) and Staphylococcus aureus (S. aureus, Gram-positive). Simply, the two bacteria were resuscitated and incubated in broth on a shaker at 37°C and passaged at 8h time intervals. Cu 3 SnS 4 NSs, CS/GP hydrogel and CS/GP/Cu 3 SnS 4 hydrogel were added to the broth in contact with the bacterial fluids in the third generation, respectively. After contacting for 2h, 4h and 8h, bacterial fluids were inoculated on agar plates and then incubated for 12h to evaluate the anti-bacterial properties. Cell counting kit-8 assay The cytotoxicity of CS/GP/Cu 3 SnS 4 was evaluated using cell counting kit-8 (CCK-8) assay. Fibroblasts (L929 cells) cells were first seeded in a 96-well plate at the density of 1×104 cell/well, and the original culture medium was aspirated after incubation for 24h. The medium extracts of 250 mg Cu 3 SnS 4 NSs, 5 ml CS/GP hydrogel, and 5 ml CS/GP/Cu 3 SnS 4 hydrogel (obtained after incubation with normal medium for 24h) were used for further incubation. After cultured for 24, 48, and 72h, the culture medium was discarded, and each well was thoroughly washed with PBS. Then, 100 µl of culture medium containing 10% CCK-8 reagent was added to each well and incubated for another 1h, and the absorbance of 450 nm was recorded with a microplate reader. Finally, the cell survival rate was calculated according to the formula. Staining of live-dead cells Firstly, L-929 cells were seeded in a 24 well plate at the density of 2.5×104 cell/well. Then, the medium was aspirated after one day of incubation and the cells were continued to be cultured with the extraction solution of Cu 3 SnS 4 NSs, CS/GP hydrogel, and CS/GP/Cu 3 SnS 4 hydrogel. Then, 250ul of staining solution (1ulam + 1ulpi + 1ml buffer) was added to each well after another 24, 48 and 72h of incubation and take it out after incubation at 37℃ for 0.5h. Finally, fluorescence images were taken with a fluorescence microscope to calculate live and dead cells. Animal experiment Establishment and treatment of mouse burn model The experimental procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Shanghai Jiao Tong University and experiments were approved by the Animal Ethics Committee of IACUCs. All animal experimental procedures were conducted in accordance with applicable guidelines and regulations, as outlined in the ARRIVE guidelines. BALB/c mice of about 6 weeks were used as burn model to evaluate the wound healing efficiency. The mice were divided into blank group, CS/GP group CS/GP/Cu 3 SnS 4 group, and each group contained 5 rats. Each mouse was anesthetized with 7% chloral hydrate, and the back was depilated and disinfected. Then, 5g of weight was soaked in boiling hot water for 3min and taken out to cling to the depilation on the back of the mice for 15s to construct the second degree burn wound. Furthermore, the corresponding preparations were applied to the wounds of each group of mice, and the sterile gauze fixation agent was used for treatment. Afterwards, the body weight of mice was recorded every other day, and the wounds were cleaned with normal saline on the 1st, 4th, 7th and 14th days. The surgical procedures were performed under respiratory anesthesia throughout the animal experiments, and at the end of the experiments, the mice were euthanized by inhalation of carbon dioxide, after which the tissues were collected and analyzed. Observation wounds and calculation of closure rate On the 1st, 4th, 7th and 14th days after treatment with the experiment CS/GP/Cu 3 SnS 4 , the macroscopic pictures of the wounds were recorded with a camera, and the remaining wound length was measured with a standard graduated ruler. Then the wound area was calculated with Image J software. The wound closure rate of different groups at each time point was calculated according to the following formula: Wound healing rate (%) =(Ao-An)/Ao*100%, (1) where Ao is the starting wound area (d0) and An is the remaining wound area at postoperative nd. ELISA analysis At the 3rd, 7th and 14th day after treatment, one mouse in each group was sacrificed by cervical dislocation after anesthesia. The skin tissue at the wound edge was cut for immunohistochemical and immunofluorescence analysis, and the expression levels of CD68 and VEGF factors in the wound tissue were detected respectively. Results and discussion Basic characterization of samples Cu 3 SnS 4 NSs were prepared by solvothermal method and the morphologies were characterized. Firstly, from the electronic mapping image (Fig. 1 A), Cu, Sn and S atoms are co-existed and evenly distributed among lamellar structure, and the atomic ratio is calculated approximately 2.75:1:3.6, which is also basically in line with the stoichiometry of Cu 3 SnS 4 NSs. TEM and HRTEM images showing in Fig. 1 B, C indicate that the morphology of Cu 3 SnS 4 NSs is sheet-like and the crystal structure was polycrystalline. XRD characterization of Fig. 1 E verify the purity of Cu 3 SnS 4 NSs, the 2θ diffraction peaks at 28.5, 47.5 and 56.0 are consistent with the (112), (220) and (132) crystal planes of standard PDF card (JCPDS card, No.33–0501). No other impurity elements are found in the spectrum, indicating that the nanosheets are in pure phase. X-ray photoelectron spectroscopy (XPS) was used to further explore the valence states of the elements in the material. As depicted in Fig. 1 F- 1 , the XPS spectrum indicate Cu, Sn and S peaks are present in the nanosheets. The binding energies at Cu2p3/2 and Cu2p1/2 are concentrated at 932.1eV and 952.1eV, respectively (Fig. 1 F-2), which are typical binding energies of Cu + ions. In addition, the Cu2p3/2 satellite peak at 942.8eV can be identified as Cu 2+ ions, which indicates that Cu + and Cu 2+ ions exist simultaneously in Cu 3 SnS 4 . The binding energies of Sn3d5/2 and Sn3d3/2 are concentrated at 486.8eV and 495.2eV, respectively (Fig. 1 F-3), corresponding to the value of Sn 4+ . No Sn 2+ with a binding energy of 485.2 eV was detected in the sample. The binding energy at 161.9eV should be the core energy level spectrum of S2p (Fig. 1 F-4). In addition, an additional peak is detected at 168.9eV, which may be caused by the oxidation of the product. Zeta potential measurements showed that the surface of Cu 3 SnS 4 is positively charged in PBS solution (Fig. 1 D), while the surface of bacteria is usually negatively charged under the same condition, indicating that Cu 3 SnS 4 nanosheets have the potential of electrostatic adsorption with bacteria Then we characterized the microstructure of the synthesized CS/GP hydrogel and CS/GP/ Cu 3 SnS 4 hydrogel by scanning electron microscopy (SEM). From Fig. 2A-B, the addition of Cu 3 SnS 4 NSs did not affect the structure of CS/GP hydrogel, which is still a typical uniform porous structure. Meanwhile, Cu 3 SnS 4 NSs can uniformly dispersed in chitosan-based hydrogels. Figure 2. (A) SEM images of CS/GP hydrogel, (B) SEM images of CS/GP/ Cu 3 SnS 4 hydrogel. (C) Copper ion release from Cu 3 SnS 4 and CS/GP/ Cu 3 SnS 4 hydrogel. Copper ions have been recognized as an effective antimicrobial agent, while promoting wound healing through mechanisms such as stimulating cell migration and promoting collagen deposition and angiogenesis 39 , 40 . Here, the release of copper ions was studied by inductively coupled plasma optical emission spectroscopy (ICP-OES). As shown in Fig. 2C, 1.7µg/ml copper ion of Cu 3 SnS 4 NSs is released in 20h, and the release rate is slightly increased to 2.3µg/ml in 40h. Meanwhile, the CS/GP/Cu 3 SnS 4 hydrogel can release 1.4µg/ml and 2.1µg/ml of copper ions. It can be seen that due to the porous structure of chitosan-based hydrogels and the uniform dispersion of Cu 3 SnS 4 NSs in it, CS/GP/Cu 3 SnS 4 still has a considerable copper ion release rate in PBS solution. Compared to the previously mentioned study by Massoumi 38 et al. where the data provided showed that GSH5-Cu could reach a copper ion concentration of 5.71µg/ml after 3 days in PBS solution, in this study, the hydrogel acted as a retardant, resulting in a lower concentration of copper ions than the former in the same situation, further demonstrating the biocompatibility of the material. Therefore, CS/GP/Cu 3 SnS 4 hydrogel is expected to be an ideal wound dressing. Antibacterial experiment results Rapid bacterial growth increases the risk of infection in scald wounds, so the antimicrobial properties of scald dressings are of paramount importance41. In this work, E. coli and S. aureus were used to evaluate the anti-bacterial properties. As depicted in Fig. 3, obvious antibacterial performance of Cu3SnS4 NSs against E. coli and S. aureus is observed after treated for only 2 h, and the antibacterial rate could reach 99% as almost no colonies are visible. Meanwhile, the antibacterial efficiency is slightly decreased when Cu3SnS4 NSs loaded on chitosan-based hydrogel, which may be caused by the reduced release rate of copper ions, and the antibacterial rate reduces to 99% after 4 hours of contact. Furthermore, antibacterial performance against E. coli is comparatively less effective than that of S. aureus under same explosion conditions. The reason may be attributed to composition and structural differences between E. coli and S. aureus. E. coli is a gram-negative bacterium, while S. aureus is a gram-positive bacterium, the presence of outer membrane barrier in gram-negative bacterium affects penetration of antibacterial agents, leading to relatively poor antibacterial effect. Finally, the inhibition rate exceeded 99% after 4 hours of contact, which was much better than the control group and CS/GP group, demonstrating its potential use as an antibacterial wound dressing. Figure 3. Antibacterial properties of materials in each group. (A) Antibacterial effect of different samples against E. coli, (B) Antibacterial effect of different samples against S. aureus. Toxicity test results According to “ISO 10993-5(1999) Evaluation Standards for Medical Instrument Biology 42 ”. In this paper, the biocompatibility and cytotoxicity of the materials were evaluated by the effect on the growth of mouse fibroblasts L-929. From Fig. 4A, the results showed that after cell culture with CS/GP/ Cu 3 SnS 4 hydrogel extract, the cell survival rate is basically the same as that of the control group. Even after 72h of incubation, the cell survival rate of the experimental group reached 104%, which was higher than that of the other groups, CCK-8 results revealed that no negative impact was caused by CS/GP/Cu 3 SnS 4 hydrogel on the growth of L-929 cells. Figure 4. Biocompatibility of each group of materials. (A) Activity of L929 cells after 24h, 48h and 72h incubation using normal medium and experimental medium of each group, (B) Live-dead staining of mouse fibroblasts cultured in various groups of media for 24, 48 and 72h. Double staining using calcineurin AM/PI is a routine method for diagnosing morphological changes in cells and allows further assessment of the cytocompatibility of the material. From Fig. 4B, after 24, 48 as well as 72 h of culture the green fluorescence of each group representing living cells occupied the majority of the fluorescence, with only a small amount of red fluorescence. The cells of each group were well defined with a spindle-like shape, suggesting that the cell growth was in a good condition 43 . These results further supported that CS/GP/ Cu 3 SnS 4 hydrogel had no effect on the growth of L-929 cells. Reflecting its good biocompatibility. This work also reflected the good biocompatibility of this hydrogel dressing by hemolysis experiments (Supplementary Fig. 1), H&E staining experiments (Supplementary Fig. 2), and blood analyses (Supplementary Tables 1,2,3) likewise Observation of mouse wounds and calculation of closure rate In vivo wound healing effect was further explored. As depicted in Fig. 5 A, after scalded for 4 days, the wound edges in each group are red and swollen, and the scab in the control group was invisible, while obvious scabs without obvious expansion are observed in the CS/GP and CS/GP/Cu 3 SnS 4 groups. We can see surroundings over the wound areas are dry, and barely tissue fluid exudation is present, indicating that both groups have effective antibacterial and anti-infection abilities. On the 7th day after scald, scab appeared in the control group, and the wound is slightly reduced. Meanwhile, the wound in the CS/GP group was significantly reduced with scabbing. This is consistent with existing research that CS may promote wound healing by promoting hemostasis, reducing inflammatory responses, and increasing cytokine expression 44 . While in the CS/GP/Cu 3 SnS 4 group, the scab has fallen off from wound, and the wound is significantly coalesced. On the 14th day, wound in CS/GP/Cu 3 SnS 4 group is basically healed without leaving any traces, and the repaired skin is relatively smooth, while, a small amount of scab is still present on the skin of the CS/GP group. Comparingly, the wounds size of the control group is still the largest and not completely healed, which verifying the better therapeutic effect for scald CS/GP/Cu 3 SnS 4 group than the former two groups. Furthermore, we used wound closure rate for quantitatively evaluating scald healing performances. Shown in Fig. 5 B, the healing rate of the blank control group is negative on the fourth day due to wound expanded, while the healing rate of CS/GP and CS/GP/Cu 3 SnS 4 groups increased to 1.4% and 6.5% respectively. On the 7th day after therapy, the wound healing rate of CS/GP/Cu 3 SnS 4 group is rapid increased and reached to 65%. On the 14th day, the healing rate of control group, CS/GP and CS/GP/Cu 3 SnS 4 are respectively 85% and 95%, revealing that the copper ions released by CS/GP/Cu 3 SnS 4 group can kill bacteria in time and reduce the inflammatory response. In addition, copper ions can stimulate the expression of matrix metalloproteinase-2 and collagen in fibroblasts, thus promoting wound healing 45 . The weight of mice in each group is relatively stable during the experimental cycle (Fig. 5 C), ‘which also show the biocompatibility of the material. Immunofluorescence chemistry We then use immunofluorescence analysis to clarify repair disparities of prepare hydrogel. VEGF plays an important role in wound healing, which is closely related to angiogenesis 46 . Depicted in Fig. 6 , at the 3rd day after scald, there is no significant difference in VEGF expression among the groups. On the 7th day, compared with the control group, the expression level of VEGF in CS/GP and CS/GP/Cu 3 SnS 4 groups increases significantly, reaching 2.06 and 3.62 times higher than that of the control group. On the 14th day, the expression level of VEGF in the CS/GP/Cu 3 SnS 4 group is still 1.99 times than that of the former two groups, and the immunofluorescence staining of VEGF also further reflect that more neovascularization formed in the wound area in the CS/GP/Cu 3 SnS 4 group. The new angiogenesis could provide more oxygen and nutrients for the local tissue, accelerate the migration of immune cells and humoral factors to the wound, thus promoting the formation of granulation tissue, collagen synthesis, and ultimately improve the healing of infected wounds. immunohistochemistry The wound repair process includes the inflammatory phase, but a long or excessive inflammatory reaction can lead to delayed healing and increased scar formation. Giant cell secreted factor 68 (CD68) is a macrophage biomarker of macrophage infiltration around the wound, which is essential for the endocytosis of tissue giant cells 47 . Through immunohistochemical analysis, CD68 in the skin tissue of burn wounds on the 3rd, 7th and 14th days were measured. As shown in the Fig. 7, the expression of CD68 in CS/GP and CS/GP/Cu 3 SnS 4 groups is obvious reduced. After incubation with wound healing materials for 14d, the expression of CD68 in CS/GP/Cu 3 SnS 4 group is significantly reduced to 34.28%, indicating that the wound inflammation in this group was suppressed. The reduction of inflammation can not only reduce the incidence of wound infection, but also advance the proliferation period of wound healing, thus accelerating the healing process. Figure 7. (A) Immunohistochemical staining of CD68 in wound tissues after different treatments, (B) Relative expression of CD68 at 3d, (C) Relative expression of CD68 at 7d, (D) Relative expression of CD68 at 14d. Conclusions In this work, we prepared chitosan-based hydrogels loaded with copper tin sulfur nanosheets, and found that CS/GP/Cu 3 SnS 4 has great antibacterial activity against E. coli and S. aureus, which has an obvious promoting effect on scald wound healing. We characterized the morphology and physicochemical properties of the synthesized Cu 3 SnS 4 and CS/GP/Cu 3 SnS 4 hydrogel, studied corresponding cell biocompatibility and evaluated the wound healing efficiency through burn model. The results showed that CS/GP/Cu 3 SnS 4 hydrogel as a scald wound dressing can reduce the inflammatory reaction of the early wound, promote the secretion of VEGF, and achieving rapid wound healing. Although drug resistance of CS/GP/Cu 3 SnS 4 hydrogel on bacteria are needed further estimated, the prepared NPs/hydrogel composites providing a possible solution to solve bacterial infection and meet urgent needs for wound healing. Declarations Conflicts of Interest The authors declare no conflicts of interest. Funding This project was financially supported by the Medical and Industrial cross research Fundation of "Star of Jiaotong University" Program of Shanghai Jiao Tong University, China (Grant No. YG2022ZD030), and the foundation of Shanghai Health and Family Planning Commission ((No. 20214Y008). Author Contribution X.Y. and R.M.F. wrote the main manuscript text. R.M.F. prepared all the pictures methodology. R.M.F., Z.J.Y. and D.K. used software to analyze data. Y.J.J., Z.Y.J. and X.Y. guided data analysis. T.L. and Y.J.M. completed supervision of various contents. All authors reviewed the manuscript. Acknowledgments Here, I would like to express my sincere gratitude to Xu Yan, Yang Dicheng, Yao Jingjing, Zhu Jingyao, Dai Kun, and Zhong Yujun for technical assistance. Data Availability All data supporting the findings of this study are available within the paper and its supplementary information or from the corresponding authors on request. References Dąbrowska, A. K. et al. 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Chitosan Hydrogel in combination with Nerolidol for healing wounds. Carbohydr. Polym. 152 , 409–418. 10.1016/j.carbpol.2016.07.037 (2016). Wen, H. P. et al. Facile formation of injectable quaternized chitosan/tannic acid hydrogels with antibacterial and ROS scavenging capabilities for diabetic wound healing. Int. J. Biol. Macromol. 195 , 190–197. 10.1016/J.IJBIOMAC.2021.12.007 (2022). Dong, X. Y. et al. Conformable hyaluronic acid hydrogel delivers adipose-derived stem cells and promotes regeneration of burn injury. Acta Biomater. 108 , 56–66. 10.1016/j.actbio.2020.03.040 (2020). Stoica, E. A., Chircov, C. & Grumezescu, M. A. Hydrogel Dressings for the Treatment of Burn Wounds: An Up-To-Date Overview. Materials . 13 , 2853. 10.3390/ma13122853 (2020). Abid, N. et al. Dressings for burn wound: a review. J. Mater. Sci. 57 , 6536–6572. 10.1007/S10853-022-07056-4 (2022). Elviri, L., Asadzadeh, M., Bianchera, C. 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Carbohyd Res . 488 , 107891. 10.1016/j.carres.2019.107891 (2020). Thanusha, A. V., Amit, K. D. & Veena, K. Evaluation of nano hydrogel composite based on gelatin/HA/CS suffused with Asiatic acid/ZnO and CuO nanoparticles for second degree burns. Mater. Sci. Eng. C Mater. 89 , 378–386. 10.1016/j.msec.2018.03.034 (2018). Bai, Q. et al. Chitosan and hyaluronic-based hydrogels could promote the infected wound healing. Int. J. Biol. Macromol. 232 , 123271–123271. 10.1016/J.IJBIOMAC.2023.123271 (2023). Shahzadi, L. et al. Triethyl orthoformate covalently cross-linked chitosan- (poly vinyl) alcohol based biodegradable scaffolds with heparin-binding ability for promoting neovascularization. J. Biomater. Appl. 31 , 582–593. 10.1177/0885328216650125 (2016). Antony, R. I. et al. Chitosan-Poly(hexamethylene) Biguanide Hydrogel for the Treatment of Infectious Wounds. J. Funct. Biomater. 14 , 528–528. 10.3390/JFB14100528 (2023). Cao, X. X. et al. A thermosensitive chitosan-based hydrogel for sealing and lubricating purposes in dental implant system. Clin. Implant Dent. Relat. Res. 21 , 324–335. 10.1111/cid.12738 (2019). Reza, K. E. et al. Chitosan hydrogel/silk fibroin/Mg(OH) 2 nanobiocomposite as a novel scaffold with antimicrobial activity and improved mechanical properties. Sci. Rep. 11 , 650–650. 10.1038/S41598-020-80133-3 (2021). Cristina, C. et al. Dextran-Glycerol Hydrogels Loaded with Iron Oxide Nanoparticles for Wound Dressing Applications. Pharmaceutics . 14 , 2620–2620. 10.3390/pharmaceutics14122620 (2022). Puspita, T. R. et al. Development of chloramphenicol wound dressing protein-based microparticles in chitosan hydrogel system for improved effectiveness of dermal wound therapy. Biomater. Adv. 143 , 213175–213175. 10.1016/j.bioadv.2022.213175 (2022). Tolstov, A. V., Kolsanov, A. V., Milyudin, E. S. & Kivaeva, O. L. Principles for the use of modern wound dressings for local treatment of limited borderline burns. Sci. Innovations Med. 8 , 307–312. 10.35693/2500-1388-2023-8-4-307-312 (2023). Amnah, A., Syafiqah, S., Helmi, M. S. & Hussien, R. A. A. Epidermal and fibroblast growth factors incorporated polyvinyl alcohol electrospun nanofibers as biological dressing scaffold. Sci. Rep. 11 , 5634–5634. 10.1038/S41598-021-85149-X (2021). Habibah, F. F. et al. Green synthesis of copper ions nanoparticles functionalized with rhamnolipid as potential antibacterial agent for pathogenic bacteria. Heliyon . 10 , e24242. 10.1016/j.heliyon.2024.e24242 (2024). Laura, M. E. & Valerio, V. Antimicrobial Nano-Agents: The Copper Age. ACS nano . 15 10.1021/ACSNANO.0C10756 (2021). Yang, F. Z. et al. Designment of polydopamine/bacterial cellulose incorporating copper (II) sulfate as an antibacterial wound dressing. Mat. Sci. Eng. C-mater . 134 , 112591–112591. 10.1016/J.MSEC.2021.112591 (2021). Massoumi, H. et al. Comparative study of the properties of sericin-gelatin nanofibrous wound dressing containing halloysite nanotubes loaded with zinc and copper ions. Int. J. Polym. Mater. 68 , 1142–1153. 10.1080/00914037.2018.1534115 (2019). Wang, X. et al. The synergistic antibacterial activity and mechanism of multicomponent metal ions-containing aqueous solutions against Staphylococcus aureus. J. Inorg. Biochem. 163 , 214–220. 10.1016/j.jinorgbio.2016.07.019 (2016). Wuliang, D. et al. Progress in copper-based materials for wound healing. Wound Repair. Regen . 32 , 314–322. 10.1111/WRR.13122 (2023). Ma, X. et al. Photo-crosslinking injectable Photothermal antibacterial hydrogel based on quaternary ammonium grafted chitosan and hyaluronic acid for infected wound healing. Mater. Today Bio . 29 , 101265–101265. 10.1016/J.MTBIO.2024.101265 (2024). Wang, Q. Q., Chen, S. Y. & Chen, D. J. Preparation and characterization of chitosan based injectable hydrogels enhanced by chitin nano-whiskers. J. Mech. Behav. Biomed. Mater. 65 , 466–477. 10.1016/J.CARBPOL.2021.119032 (2017). Wang, D. et al. The effect of form of carboxymethyl-chitosan dressings on biological properties in wound healing. Colloids Surf. B . 194 10.1016/j.colsurfb.2020.111191 (2020). Li, R. et al. Characterization and biological evaluation of a novel silver nanoparticle-loaded collagen-chitosan dressing. Regen Biomater. 7 , 371–380. 10.1093/rb/rbaa008 (2020). Xiao, J. S., Chen, S. Y., Yi, J., Zhang, H. & Ameer, G. A. A Cooperative Copper Metal-Organic Framework-Hydrogel System Improves Wound Healing in Diabetes. Adv Funct Mater . 27, n/a-n/a Doi: (2017). 10.1002/adfm.201604872 Zhang, D. Y. et al. Catechol functionalized chitosan/active peptide microsphere hydrogel for skin wound healing. Int. J. Biol. Macromol. 173 , 591–606. 10.1016/J.IJBIOMAC.2021.01.157 (2021). Zhao, X. et al. Antibacterial anti-oxidant electroactive injectable hydrogel as self-healing wound dressing with hemostasis and adhesiveness for cutaneous wound healing. Biomaterials . 122 , 34–47. 10.1016/j.biomaterials.2017.01.011 (2017). Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Published Journal Publication published 11 Apr, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 27 Nov, 2024 Reviews received at journal 27 Nov, 2024 Reviews received at journal 19 Nov, 2024 Reviewers agreed at journal 18 Nov, 2024 Reviews received at journal 13 Nov, 2024 Reviewers agreed at journal 13 Nov, 2024 Reviewers agreed at journal 13 Nov, 2024 Reviewers invited by journal 08 Nov, 2024 Editor assigned by journal 08 Nov, 2024 Editor invited by journal 08 Nov, 2024 Submission checks completed at journal 05 Nov, 2024 First submitted to journal 21 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5302334","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":379329172,"identity":"5b83158d-bd1a-4cd1-b66c-d8d1dfa1ecc5","order_by":0,"name":"Mingfei Ren","email":"","orcid":"","institution":"Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Mingfei","middleName":"","lastName":"Ren","suffix":""},{"id":379329173,"identity":"4fed94fb-7040-4b22-bbb7-d38dcfb5844a","order_by":1,"name":"Jingjing Yao","email":"","orcid":"","institution":"Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Jingjing","middleName":"","lastName":"Yao","suffix":""},{"id":379329174,"identity":"cb83633e-fd67-46df-9222-5d25454447db","order_by":2,"name":"Dicheng Yang","email":"","orcid":"","institution":"National Engineering Research Center for Nanotechnology","correspondingAuthor":false,"prefix":"","firstName":"Dicheng","middleName":"","lastName":"Yang","suffix":""},{"id":379329175,"identity":"59c861fc-da6e-4e3e-a6ec-db3af89d3cd0","order_by":3,"name":"Jingyao Zhu","email":"","orcid":"","institution":"Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Jingyao","middleName":"","lastName":"Zhu","suffix":""},{"id":379329176,"identity":"1535e9e9-5fc5-4aee-a556-8a91435e954c","order_by":4,"name":"Kun Dai","email":"","orcid":"","institution":"Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Kun","middleName":"","lastName":"Dai","suffix":""},{"id":379329177,"identity":"5a86a527-9fbc-420d-aa17-48db1b29cb33","order_by":5,"name":"Yujun Zhong","email":"","orcid":"","institution":"Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Yujun","middleName":"","lastName":"Zhong","suffix":""},{"id":379329178,"identity":"9a16148b-949e-4625-8c7a-2f1ddaa2930e","order_by":6,"name":"Jun Zhu","email":"","orcid":"","institution":"Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Zhu","suffix":""},{"id":379329179,"identity":"6c2facda-f668-41b4-9ad1-95e97734d659","order_by":7,"name":"Liang Tang","email":"","orcid":"","institution":"Tongren Hospital, Shanghai Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Tang","suffix":""},{"id":379329180,"identity":"f465a1c6-b1fa-42eb-8d19-de5fb8d66601","order_by":8,"name":"Yan Xu","email":"","orcid":"","institution":"National Engineering Research Center for Nanotechnology","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Xu","suffix":""},{"id":379329181,"identity":"58e626be-1fc9-4e29-ba3e-09336e3e7bf7","order_by":9,"name":"Jiangming Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApElEQVRIiWNgGAWjYFCCAyDChoefv4EkLQlpMpIzDpBkU8JhG4OGBCIVGxw8ncD488d5HgOGA4wfPuYQo+XA2Q3MPAm3ecyZG5glZ24jVgsDUItlwwE2Zl5itTD+SDjHY3AggQQtDDwJB0jQIgn2S1oyj+SMg83E+YXvBshhNnb2/PzNBz98JEaLwo0D7D8gTMYGItQDgXw/kQpHwSgYBaNgBAMA1po6vG054FoAAAAASUVORK5CYII=","orcid":"","institution":"Tongren Hospital, Shanghai Jiaotong University","correspondingAuthor":true,"prefix":"","firstName":"Jiangming","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2024-10-21 08:08:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5302334/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5302334/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-84416-x","type":"published","date":"2025-04-11T16:05:14+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69377272,"identity":"e82bd95e-7a8b-43de-b80c-a6e11b2dfd8e","added_by":"auto","created_at":"2024-11-19 17:26:59","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1282189,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs. (A) Electron mapping images of Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs, (B) TEM images of Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs, (C) HRTEM images of Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs, (D) Zeta potential of Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs, (E) XRD pattern of Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs, (F) XPS analyses of the Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5302334/v1/31a648319a74e1797359bf7b.jpeg"},{"id":69377461,"identity":"dbc8d71b-ac07-4df9-9da6-64d45f701451","added_by":"auto","created_at":"2024-11-19 17:34:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":128402,"visible":true,"origin":"","legend":"\u003cp\u003e(A) SEM images of CS/GP hydrogel, (B) SEM images of CS/GP/ Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e hydrogel. (C) Copper ion release from Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e and CS/GP/ Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e hydrogel.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5302334/v1/79589d71310d15a60d37623a.png"},{"id":69377462,"identity":"add65541-cd17-497b-b9e3-e1ee859d12fd","added_by":"auto","created_at":"2024-11-19 17:34:59","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1043993,"visible":true,"origin":"","legend":"\u003cp\u003eAntibacterial properties of materials in each group. (A) Antibacterial effect of different samples against E. coli, (B) Antibacterial effect of different samples against S. aureus.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5302334/v1/30eface4a7ea7b3dfb1a26c1.jpeg"},{"id":69377280,"identity":"91259b24-93c8-4e73-9a5d-836a6885ba1c","added_by":"auto","created_at":"2024-11-19 17:27:00","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":659523,"visible":true,"origin":"","legend":"\u003cp\u003eBiocompatibility of each group of materials. (A) Activity of L929 cells after 24h, 48h and 72h incubation using normal medium and experimental medium of each group, (B) Live-dead staining of mouse fibroblasts cultured in various groups of media for 24, 48 and 72h.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5302334/v1/8197b3c67abf561cf2b7370e.jpeg"},{"id":69377277,"identity":"91fc8ad5-729f-40fa-b223-47e81a123048","added_by":"auto","created_at":"2024-11-19 17:26:59","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":654633,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of wound surface and body weight in mice. (A) Wound changes in mice, (B) Wound closure rate in mice, (C) Changes in body weight of mice.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5302334/v1/ff87fc5d7c0e1cac717c8cd9.jpeg"},{"id":69377274,"identity":"989552cc-11d9-419c-8998-520d7a65e1e3","added_by":"auto","created_at":"2024-11-19 17:26:59","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":792606,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Immunofluorescence analysis of VEGF in wound regeneration after different treatments; (B) Relative expression of VEGF at 3d, (C) Relative expression of VEGF at 7d, (D) Relative expression of VEGF at 14d.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5302334/v1/3ee54b345f8f07045b3f82d3.jpeg"},{"id":69377275,"identity":"0e3afeff-8e90-4486-a91d-6ec24f7e8ad8","added_by":"auto","created_at":"2024-11-19 17:26:59","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":507636,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Immunohistochemical staining of CD68 in wound tissues after different treatments, (B) Relative expression of CD68 at 3d, (C) Relative expression of CD68 at 7d, (D) Relative expression of CD68 at 14d.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5302334/v1/d51c4949cd1b079c34973669.jpeg"},{"id":80558882,"identity":"e07fc22e-f62a-4e3f-832b-c86c67cb2a66","added_by":"auto","created_at":"2025-04-14 16:16:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5748856,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5302334/v1/28f4c034-3ad1-40c4-81f1-c76d31968529.pdf"},{"id":69377463,"identity":"1c4b8114-3f49-4acf-a43e-f66a9644253b","added_by":"auto","created_at":"2024-11-19 17:34:59","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1381781,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-5302334/v1/67e66558bfea7ef5071dfb83.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chitosan hydrogel loaded with copper-tin-sulfur nanosheet materials for skin wound healing","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSkin plays a variety of functions that are essential for human survival. It consists of epidermis and dermis. The epidermis is a barrier against bacterial invasion and water loss, which can rebuild shallow wounds. However, incomplete regeneration is inevitable for dermal lesions containing hair follicles, sweat glands and other skin appendages\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. Burn is a disease that leads to the destruction or necrosis of skin cells, which poses a serious threat to human health, including psychological, physical disability and even death\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e, and is one of the most serious traumas in the world\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. According to the symptoms, burns can be divided into first-degree burns, second-degree burns and third-degree burns. First-degree burns affect the epidermis, second-degree burns affect the dermis, and third-degree burns affect a larger area and reach the deep layer of the skin\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. Burn wounds usually have more exudates and necrotic tissue than other types of wounds, providing conditions for bacterial reproduction which results in a higher probability of infection\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. This not only causes long-term pain for the injured, but also affects aesthetics, and can even lead to psychological trauma. While the traditional wound dressing is not ideal in terms of antibacterial performance, especially when the wound area is large, the possibility of skin peeling and bacterial infection is also greatly increased due to water evaporation\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. Therefore, the synthesis of burn dressings that can effectively combat bacteria and promote wound repair is of paramount importance\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDue to air permeability, low cost and easy operation, traditional burn dressings, such as gauze and skimmed cotton\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e, topical ointment\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e, are still widely applied to absorb wound fluid and prevent wound infection. However, wound adhesion, limited absorptive capacity and moisturizing properties may cause unbearable hurts and secondary injuries during frequently dressing replacing, otherwise, increasing possibilities of bacterial infection after absorption saturation of the dressings which is the ideal culture medium for bacterial growth. In order to meet the urgent demand of antibacterial effect and moisturization, as well as improving healing rate of wound, types of hydrogel dressings have been researched according to the abilities of biodegradability\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e, biocompatibility\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e, non-toxicity, antibacterial properties, bio-adhesion, bioactivity, and hemostasis\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. Previous reports have proved the unique biological characteristics enable these hydrogels considerable materials used as wound dressings, and further indicating the potentiality as drug delivery system (DDS) to deliver antibacterial agents\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e, growth factors, stem cells, thereby, accelerating wound healing efficiency \u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e at different stages of wound healing and alleviating factors hindering wound healing\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. Moreover, hydrogel dressings containing different nanoparticles have been a research hotspot due to unique physical or biological nano effects. Chircov\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e et al., have developed a series of iron oxide nanoparticles uniformly dispersed chitosan dextran glycerol hydrogels by in situ formation of magnetite nanoparticles, which proves increased antimicrobial properties. However, in order to increase water absorption and permeability, glycerol is used and causing the reduction of antimicrobial potential. Besides, Puspita\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e et al., have developed a formulation of chloramphenicol microparticles (cpl-mp) and modified into chitosan hydrogel to improve the treatment efficiency against infection and create an optimal environment to support the healing process. Nevertheless, its antibacterial cycle was as long as 24 hours of direct contact. Further improving the antimicrobial properties and wound treatment efficacy of burn dressings still have been a research focus. \u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePast studies have shown that copper ions have significant antibacterial effect on common bacteria such as Escherichia coli and Staphylococcus aureus, and can stimulate the production of collagen, which is the key protein required for wound healing\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. Meanwhile, copper ion can promote the formation of new blood vessels and accelerate tissue regeneration\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. Yang\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e et al., prepared a new type of BC based antibacterial wound dressing loaded with copper (Cu) ions through the co deposition of polydopamine (PDA) and copper ions, which showed considerable antibacterial properties and biocompatibility, and further proved that Cu\u003csup\u003e2+\u003c/sup\u003e@PBC-2 film could inhibit S. aureus infections and inflammatory effect, facilitating collagen deposition, capillary angiogenesis, hair follicle growth and wound healing. However, Excessive release of copper ions may hinder wound healing. Massoumi\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e et al., developed a wound dressing produced from gelatin sericin nanofibers containing HNT as well as copper or zinc ionophores. They discovered Cu\u003csup\u003e2+\u003c/sup\u003e-loaded dressing showing obviously faster bactericidal activities, however, reduced fibroblast viability which may hinder collagen secretion and blood vessels formation. Meanwhile, Zn\u003csup\u003e2+\u003c/sup\u003e-containing nanofibers showed ideal stimulus to induce fibroblast attachment, viability, and collagen biosynthesis, which may be attributed to iron release disparity between Cu\u003csup\u003e2+\u003c/sup\u003e and Zn\u003csup\u003e2+\u003c/sup\u003e, as iron release rate of Cu\u003csup\u003e2+\u003c/sup\u003e is 3 times higher than Zn\u003csup\u003e2+\u003c/sup\u003e. Therefore, scald dressings with ionic slow-release of Cu\u003csup\u003e+\u003c/sup\u003e still remains an ongoing goal of current research.\u003c/p\u003e \u003cp\u003eIn this work, we aimed to achieve the innovative integration of copper ions with chitosan-based hydrogels and develop a new type of scald wound dressing. To this end, we used a simple solvothermal method to prepare a polycrystalline Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e nanosheet (Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs) and characterized its morphology and physical properties, then we loaded it on chitosan-based hydrogel (CS/GP). The potential use of copper ion in scald wound repair was evaluated by detecting its release rate, antibacterial properties and biocompatibility. Finally, it was applied to the repair of scalded wounds in mice to further prove the performance of the dressing. The results showed that this dressing (CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e hydrogel) had excellent anti infection and accelerated wound repair ability, and had broad application prospects in the field of scald wound repair.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eMaterials\u003c/p\u003e \u003cp\u003eChitosan, thioacetamide (TAA), N, N-dimethylformamide (DMF), copper acetate (CuAc\u003csub\u003e2\u003c/sub\u003e), tin tetrachloride pentahydrate (SnCl\u003csub\u003e4\u003c/sub\u003e⚫5H\u003csub\u003e2\u003c/sub\u003eO), and acetone were purchased from Shanghai Titan Technology Co. Sodium β-glycerophosphate(β-GP) was purchased from Shanghai McLean Biochemical Technology Co. CCK-8 kit, calcein-AM/PI were purchased from Shanghai Bi-Yun-tian Biotechnology Co.\u003c/p\u003e \u003cp\u003ePreparation of the Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs\u003c/p\u003e \u003cp\u003eCu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs were synthesized through hydrothermal method. Briefly, 40ml DMF was poured into the flask, then 0.048g CuAc\u003csub\u003e2\u003c/sub\u003e, 0.056g SnCl\u003csub\u003e4\u003c/sub\u003e⚫5H\u003csub\u003e2\u003c/sub\u003eO and 0.024g TAA were added successively. The reaction was stirred at 140℃ for 2h.The resulting solution was transferred into a reaction kettle and reacted at 220℃ for 12h. After the reaction, the solution was centrifugated at 6000 rpm, the resulting precipitate was thoroughly washed by acetone and deionized water to remove impurities, and finally resuspended in 1 mL deionized water for storage.\u003c/p\u003e \u003cp\u003ePreparation of Chitosan based Hydrogel Loaded with Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs\u003c/p\u003e \u003cp\u003eA typical chitosan solution was prepared by dissolving 100mg of chitosan powder (with a deacetylation degree of 95%) in 4.5ml of 0.1mol/L hydrochloric acid solution, and filtered and autoclaved at 121℃ for 15min. Then, 280mg of β-glycerophosphate sodium salt dissolved in 0.5ml deionized water was added drop by drop to the resulting solution, and the CS/GP hydrogel was prepared by incubated in water bath at 37℃. 200 mg of copper tin sulfur nanosheet material was subsequently added to the mixture before gelation, and the resulting mixture was completely dispersed and then incubated in water bath at 37℃ for 10min to obtain CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eMaterial characterization\u003c/p\u003e \u003cp\u003eScanning electron microscopy (SEM)\u003c/p\u003e \u003cp\u003eMorphologies of CS/GP hydrogel and CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e hydrogel were analyzed by SEM(S-4800), and the lyophilized sample was sprayed with gold for 60s to ensure sufficient conductivity before image capture.\u003c/p\u003e \u003cp\u003eTransmission electron microscope(TEM)\u003c/p\u003e \u003cp\u003eThe samples were fully dispersed in PBS solution and dropped onto the copper sheet for drying. Then morphology and size characterization of Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs was analyzed by TEM and HRTEM(JEM-2100F), and the elements were quantitatively analyzed with Energy Dispersive Spectrometer (EDS).\u003c/p\u003e \u003cp\u003eZeta potential of the Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs\u003c/p\u003e \u003cp\u003eThe surface potential of Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs was measured by Laser Particle Sizer (Zetasizer Nano ZS). Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs was dissolved in PBS solution and sonicated for half an hour for completely dispersion before measurement.\u003c/p\u003e \u003cp\u003eDetection of copper ion release rate\u003c/p\u003e \u003cp\u003eInductively coupled plasma atomic emission spectrometer (ICP 710) was used to measure the release of copper ions in Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs and CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e. Typically, 250mg of Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs and 5ml of chitosan-based hydrogel containing 250mg of Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs were added into two centrifuge tubes containing 5ml of PBS solution to prepare five tube suspensions each. Then, sequential sample were taken from the tube at the time interval of 0h, 5h, 10h, 20h and 40h for high-speed centrifugation (10000r, 10min), and the supernatants were used for copper ion concentration detection.\u003c/p\u003e \u003cp\u003eAntibacterial experiment\u003c/p\u003e \u003cp\u003eThe antimicrobial activity of the materials was evaluated by selecting two bacteria that are prevalent in wound infections, Escherichia coli (E. coli, Gram-negative) and Staphylococcus aureus (S. aureus, Gram-positive). Simply, the two bacteria were resuscitated and incubated in broth on a shaker at 37\u0026deg;C and passaged at 8h time intervals. Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs, CS/GP hydrogel and CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e hydrogel were added to the broth in contact with the bacterial fluids in the third generation, respectively. After contacting for 2h, 4h and 8h, bacterial fluids were inoculated on agar plates and then incubated for 12h to evaluate the anti-bacterial properties.\u003c/p\u003e \u003cp\u003eCell counting kit-8 assay\u003c/p\u003e \u003cp\u003eThe cytotoxicity of CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e was evaluated using cell counting kit-8 (CCK-8) assay. Fibroblasts (L929 cells) cells were first seeded in a 96-well plate at the density of 1\u0026times;104 cell/well, and the original culture medium was aspirated after incubation for 24h. The medium extracts of 250 mg Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs, 5 ml CS/GP hydrogel, and 5 ml CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e hydrogel (obtained after incubation with normal medium for 24h) were used for further incubation. After cultured for 24, 48, and 72h, the culture medium was discarded, and each well was thoroughly washed with PBS. Then, 100 \u0026micro;l of culture medium containing 10% CCK-8 reagent was added to each well and incubated for another 1h, and the absorbance of 450 nm was recorded with a microplate reader. Finally, the cell survival rate was calculated according to the formula.\u003c/p\u003e \u003cp\u003eStaining of live-dead cells\u003c/p\u003e \u003cp\u003eFirstly, L-929 cells were seeded in a 24 well plate at the density of 2.5\u0026times;104 cell/well. Then, the medium was aspirated after one day of incubation and the cells were continued to be cultured with the extraction solution of Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs, CS/GP hydrogel, and CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e hydrogel. Then, 250ul of staining solution (1ulam\u0026thinsp;+\u0026thinsp;1ulpi\u0026thinsp;+\u0026thinsp;1ml buffer) was added to each well after another 24, 48 and 72h of incubation and take it out after incubation at 37℃ for 0.5h. Finally, fluorescence images were taken with a fluorescence microscope to calculate live and dead cells.\u003c/p\u003e \u003cp\u003eAnimal experiment\u003c/p\u003e \u003cp\u003eEstablishment and treatment of mouse burn model\u003c/p\u003e \u003cp\u003e The experimental procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Shanghai Jiao Tong University and experiments were approved by the Animal Ethics Committee of IACUCs. All animal experimental procedures were conducted in accordance with applicable guidelines and regulations, as outlined in the ARRIVE guidelines. BALB/c mice of about 6 weeks were used as burn model to evaluate the wound healing efficiency. The mice were divided into blank group, CS/GP group CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e group, and each group contained 5 rats. Each mouse was anesthetized with 7% chloral hydrate, and the back was depilated and disinfected. Then, 5g of weight was soaked in boiling hot water for 3min and taken out to cling to the depilation on the back of the mice for 15s to construct the second degree burn wound. Furthermore, the corresponding preparations were applied to the wounds of each group of mice, and the sterile gauze fixation agent was used for treatment. Afterwards, the body weight of mice was recorded every other day, and the wounds were cleaned with normal saline on the 1st, 4th, 7th and 14th days. The surgical procedures were performed under respiratory anesthesia throughout the animal experiments, and at the end of the experiments, the mice were euthanized by inhalation of carbon dioxide, after which the tissues were collected and analyzed.\u003c/p\u003e \u003cp\u003eObservation wounds and calculation of closure rate\u003c/p\u003e \u003cp\u003eOn the 1st, 4th, 7th and 14th days after treatment with the experiment CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e, the macroscopic pictures of the wounds were recorded with a camera, and the remaining wound length was measured with a standard graduated ruler. Then the wound area was calculated with Image J software. The wound closure rate of different groups at each time point was calculated according to the following formula:\u003c/p\u003e \u003cp\u003eWound healing rate (%) =(Ao-An)/Ao*100%, (1)\u003c/p\u003e \u003cp\u003ewhere Ao is the starting wound area (d0) and An is the remaining wound area at postoperative nd.\u003c/p\u003e \u003cp\u003eELISA analysis\u003c/p\u003e \u003cp\u003eAt the 3rd, 7th and 14th day after treatment, one mouse in each group was sacrificed by cervical dislocation after anesthesia. The skin tissue at the wound edge was cut for immunohistochemical and immunofluorescence analysis, and the expression levels of CD68 and VEGF factors in the wound tissue were detected respectively.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003eBasic characterization of samples\u003c/p\u003e \u003cp\u003eCu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs were prepared by solvothermal method and the morphologies were characterized. Firstly, from the electronic mapping image (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), Cu, Sn and S atoms are co-existed and evenly distributed among lamellar structure, and the atomic ratio is calculated approximately 2.75:1:3.6, which is also basically in line with the stoichiometry of Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs. TEM and HRTEM images showing in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C indicate that the morphology of Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs is sheet-like and the crystal structure was polycrystalline. XRD characterization of Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE verify the purity of Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs, the 2θ diffraction peaks at 28.5, 47.5 and 56.0 are consistent with the (112), (220) and (132) crystal planes of standard PDF card (JCPDS card, No.33\u0026ndash;0501). No other impurity elements are found in the spectrum, indicating that the nanosheets are in pure phase. X-ray photoelectron spectroscopy (XPS) was used to further explore the valence states of the elements in the material. As depicted in Fig.\u0026nbsp;\u0026lt;link rid=\"fig1\"\u0026gt;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u0026lt;/link\u0026gt;\u003c/span\u003eF-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the XPS spectrum indicate Cu, Sn and S peaks are present in the nanosheets. The binding energies at Cu2p3/2 and Cu2p1/2 are concentrated at 932.1eV and 952.1eV, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-2), which are typical binding energies of Cu\u003csup\u003e+\u003c/sup\u003e ions. In addition, the Cu2p3/2 satellite peak at 942.8eV can be identified as Cu\u003csup\u003e2+\u003c/sup\u003e ions, which indicates that Cu\u003csup\u003e+\u003c/sup\u003e and Cu\u003csup\u003e2+\u003c/sup\u003e ions exist simultaneously in Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e. The binding energies of Sn3d5/2 and Sn3d3/2 are concentrated at 486.8eV and 495.2eV, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-3), corresponding to the value of Sn\u003csup\u003e4+\u003c/sup\u003e. No Sn\u003csup\u003e2+\u003c/sup\u003e with a binding energy of 485.2 eV was detected in the sample. The binding energy at 161.9eV should be the core energy level spectrum of S2p (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-4). In addition, an additional peak is detected at 168.9eV, which may be caused by the oxidation of the product. Zeta potential measurements showed that the surface of Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e is positively charged in PBS solution (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), while the surface of bacteria is usually negatively charged under the same condition, indicating that Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e nanosheets have the potential of electrostatic adsorption with bacteria\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e Then we characterized the microstructure of the synthesized CS/GP hydrogel and CS/GP/ Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e hydrogel by scanning electron microscopy (SEM). From Fig.\u0026nbsp;2A-B, the addition of Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs did not affect the structure of CS/GP hydrogel, which is still a typical uniform porous structure. Meanwhile, Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs can uniformly dispersed in chitosan-based hydrogels.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 2.\u003c/b\u003e (A) SEM images of CS/GP hydrogel, (B) SEM images of CS/GP/ Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e hydrogel. (C) Copper ion release from Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e and CS/GP/ Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e hydrogel.\u003c/p\u003e \u003cp\u003eCopper ions have been recognized as an effective antimicrobial agent, while promoting wound healing through mechanisms such as stimulating cell migration and promoting collagen deposition and angiogenesis\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. Here, the release of copper ions was studied by inductively coupled plasma optical emission spectroscopy (ICP-OES). As shown in Fig.\u0026nbsp;2C, 1.7\u0026micro;g/ml copper ion of Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs is released in 20h, and the release rate is slightly increased to 2.3\u0026micro;g/ml in 40h. Meanwhile, the CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e hydrogel can release 1.4\u0026micro;g/ml and 2.1\u0026micro;g/ml of copper ions. It can be seen that due to the porous structure of chitosan-based hydrogels and the uniform dispersion of Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e NSs in it, CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e still has a considerable copper ion release rate in PBS solution. Compared to the previously mentioned study by Massoumi\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e et al. where the data provided showed that GSH5-Cu could reach a copper ion concentration of 5.71\u0026micro;g/ml after 3 days in PBS solution, in this study, the hydrogel acted as a retardant, resulting in a lower concentration of copper ions than the former in the same situation, further demonstrating the biocompatibility of the material. Therefore, CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e hydrogel is expected to be an ideal wound dressing.\u003c/p\u003e \u003cp\u003eAntibacterial experiment results\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRapid bacterial growth increases the risk of infection in scald wounds, so the antimicrobial properties of scald dressings are of paramount importance41. In this work, E. coli and S. aureus were used to evaluate the anti-bacterial properties. As depicted in Fig.\u0026nbsp;3, obvious antibacterial performance of Cu3SnS4 NSs against E. coli and S. aureus is observed after treated for only 2 h, and the antibacterial rate could reach 99% as almost no colonies are visible. Meanwhile, the antibacterial efficiency is slightly decreased when Cu3SnS4 NSs loaded on chitosan-based hydrogel, which may be caused by the reduced release rate of copper ions, and the antibacterial rate reduces to 99% after 4 hours of contact. Furthermore, antibacterial performance against E. coli is comparatively less effective than that of S. aureus under same explosion conditions. The reason may be attributed to composition and structural differences between E. coli and S. aureus. E. coli is a gram-negative bacterium, while S. aureus is a gram-positive bacterium, the presence of outer membrane barrier in gram-negative bacterium affects penetration of antibacterial agents, leading to relatively poor antibacterial effect. Finally, the inhibition rate exceeded 99% after 4 hours of contact, which was much better than the control group and CS/GP group, demonstrating its potential use as an antibacterial wound dressing.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e \u003cb\u003eFigure 3.\u003c/b\u003e Antibacterial properties of materials in each group. (A) Antibacterial effect of different samples against E. coli, (B) Antibacterial effect of different samples against S. aureus.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eToxicity test results\u003c/p\u003e \u003cp\u003e According to \u0026ldquo;ISO 10993-5(1999) Evaluation Standards for Medical Instrument Biology\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e\u0026rdquo;. In this paper, the biocompatibility and cytotoxicity of the materials were evaluated by the effect on the growth of mouse fibroblasts L-929. From Fig.\u0026nbsp;4A, the results showed that after cell culture with CS/GP/ Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e hydrogel extract, the cell survival rate is basically the same as that of the control group. Even after 72h of incubation, the cell survival rate of the experimental group reached 104%, which was higher than that of the other groups, CCK-8 results revealed that no negative impact was caused by CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e hydrogel on the growth of L-929 cells.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e \u003cb\u003eFigure 4.\u003c/b\u003e Biocompatibility of each group of materials. (A) Activity of L929 cells after 24h, 48h and 72h incubation using normal medium and experimental medium of each group, (B) Live-dead staining of mouse fibroblasts cultured in various groups of media for 24, 48 and 72h.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eDouble staining using calcineurin AM/PI is a routine method for diagnosing morphological changes in cells and allows further assessment of the cytocompatibility of the material. From Fig.\u0026nbsp;4B, after 24, 48 as well as 72 h of culture the green fluorescence of each group representing living cells occupied the majority of the fluorescence, with only a small amount of red fluorescence. The cells of each group were well defined with a spindle-like shape, suggesting that the cell growth was in a good condition\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. These results further supported that CS/GP/ Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e hydrogel had no effect on the growth of L-929 cells. Reflecting its good biocompatibility. This work also reflected the good biocompatibility of this hydrogel dressing by hemolysis experiments (Supplementary Fig.\u0026nbsp;1), H\u0026amp;E staining experiments (Supplementary Fig.\u0026nbsp;2), and blood analyses (Supplementary Tables\u0026nbsp;1,2,3) likewise\u003c/p\u003e \u003cp\u003eObservation of mouse wounds and calculation of closure rate\u003c/p\u003e \u003cp\u003eIn vivo wound healing effect was further explored. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, after scalded for 4 days, the wound edges in each group are red and swollen, and the scab in the control group was invisible, while obvious scabs without obvious expansion are observed in the CS/GP and CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e groups. We can see surroundings over the wound areas are dry, and barely tissue fluid exudation is present, indicating that both groups have effective antibacterial and anti-infection abilities. On the 7th day after scald, scab appeared in the control group, and the wound is slightly reduced. Meanwhile, the wound in the CS/GP group was significantly reduced with scabbing. This is consistent with existing research that CS may promote wound healing by promoting hemostasis, reducing inflammatory responses, and increasing cytokine expression\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. While in the CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e group, the scab has fallen off from wound, and the wound is significantly coalesced. On the 14th day, wound in CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e group is basically healed without leaving any traces, and the repaired skin is relatively smooth, while, a small amount of scab is still present on the skin of the CS/GP group. Comparingly, the wounds size of the control group is still the largest and not completely healed, which verifying the better therapeutic effect for scald CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e group than the former two groups. Furthermore, we used wound closure rate for quantitatively evaluating scald healing performances. Shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, the healing rate of the blank control group is negative on the fourth day due to wound expanded, while the healing rate of CS/GP and CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e groups increased to 1.4% and 6.5% respectively. On the 7th day after therapy, the wound healing rate of CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e group is rapid increased and reached to 65%. On the 14th day, the healing rate of control group, CS/GP and CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e are respectively 85% and 95%, revealing that the copper ions released by CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e group can kill bacteria in time and reduce the inflammatory response. In addition, copper ions can stimulate the expression of matrix metalloproteinase-2 and collagen in fibroblasts, thus promoting wound healing\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. The weight of mice in each group is relatively stable during the experimental cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), \u0026lsquo;which also show the biocompatibility of the material.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImmunofluorescence chemistry\u003c/p\u003e \u003cp\u003eWe then use immunofluorescence analysis to clarify repair disparities of prepare hydrogel. VEGF plays an important role in wound healing, which is closely related to angiogenesis\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. Depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e6\u003c/span\u003e, at the 3rd day after scald, there is no significant difference in VEGF expression among the groups. On the 7th day, compared with the control group, the expression level of VEGF in CS/GP and CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e groups increases significantly, reaching 2.06 and 3.62 times higher than that of the control group. On the 14th day, the expression level of VEGF in the CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e group is still 1.99 times than that of the former two groups, and the immunofluorescence staining of VEGF also further reflect that more neovascularization formed in the wound area in the CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e group. The new angiogenesis could provide more oxygen and nutrients for the local tissue, accelerate the migration of immune cells and humoral factors to the wound, thus promoting the formation of granulation tissue, collagen synthesis, and ultimately improve the healing of infected wounds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eimmunohistochemistry\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe wound repair process includes the inflammatory phase, but a long or excessive inflammatory reaction can lead to delayed healing and increased scar formation. Giant cell secreted factor 68 (CD68) is a macrophage biomarker of macrophage infiltration around the wound, which is essential for the endocytosis of tissue giant cells\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. Through immunohistochemical analysis, CD68 in the skin tissue of burn wounds on the 3rd, 7th and 14th days were measured. As shown in the Fig.\u0026nbsp;7, the expression of CD68 in CS/GP and CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e groups is obvious reduced. After incubation with wound healing materials for 14d, the expression of CD68 in CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e group is significantly reduced to 34.28%, indicating that the wound inflammation in this group was suppressed. The reduction of inflammation can not only reduce the incidence of wound infection, but also advance the proliferation period of wound healing, thus accelerating the healing process.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e \u003cb\u003eFigure 7.\u003c/b\u003e (A) Immunohistochemical staining of CD68 in wound tissues after different treatments, (B) Relative expression of CD68 at 3d, (C) Relative expression of CD68 at 7d, (D) Relative expression of CD68 at 14d.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this work, we prepared chitosan-based hydrogels loaded with copper tin sulfur nanosheets, and found that CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e has great antibacterial activity against E. coli and S. aureus, which has an obvious promoting effect on scald wound healing. We characterized the morphology and physicochemical properties of the synthesized Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e and CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e hydrogel, studied corresponding cell biocompatibility and evaluated the wound healing efficiency through burn model. The results showed that CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e hydrogel as a scald wound dressing can reduce the inflammatory reaction of the early wound, promote the secretion of VEGF, and achieving rapid wound healing. Although drug resistance of CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e hydrogel on bacteria are needed further estimated, the prepared NPs/hydrogel composites providing a possible solution to solve bacterial infection and meet urgent needs for wound healing.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis project was financially supported by the Medical and Industrial cross research Fundation of \"Star of Jiaotong University\" Program of Shanghai Jiao Tong University, China (Grant No. YG2022ZD030), and the foundation of Shanghai Health and Family Planning Commission ((No. 20214Y008).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eX.Y. and R.M.F. wrote the main manuscript text. R.M.F. prepared all the pictures methodology. R.M.F., Z.J.Y. and D.K. used software to analyze data. Y.J.J., Z.Y.J. and X.Y. guided data analysis. T.L. and Y.J.M. completed supervision of various contents. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eHere, I would like to express my sincere gratitude to Xu Yan, Yang Dicheng, Yao Jingjing, Zhu Jingyao, Dai Kun, and Zhong Yujun for technical assistance.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data supporting the findings of this study are available within the paper and its supplementary information or from the corresponding authors on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDąbrowska, A. K. et al. The relationship between skin function, barrier properties, and body-dependent factors. \u003cem\u003eSkin. Res. 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Antibacterial anti-oxidant electroactive injectable hydrogel as self-healing wound dressing with hemostasis and adhesiveness for cutaneous wound healing. \u003cem\u003eBiomaterials\u003c/em\u003e. \u003cb\u003e122\u003c/b\u003e, 34\u0026ndash;47. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.biomaterials.2017.01.011\u003c/span\u003e\u003cspan address=\"10.1016/j.biomaterials.2017.01.011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Burn wound dressing, Chitosan, Hydrogel, CS/GP/Cu3SnS4","lastPublishedDoi":"10.21203/rs.3.rs-5302334/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5302334/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGlobally, burns are a serious health problem that disrupts the normal functioning of the skin and increases the risk of bacterial infections. Traditional burn dressings often have difficulties to achieve desired therapeutic results. Therefore, there is an urgent need to develop an ideal wound dressing with good antimicrobial properties, biocompatibility and rapid promotion of burn wound healing. Herein, we prepared a chitosan-based hydrogel loaded with copper-tin-sulfur nanosheet materials (CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e), and explored its biocompatibility and antibacterial properties in vitro. The results showed that the antibacterial rate of CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e hydrogel can exceed 95% after contacting with Staphylococcus aureus and Escherichia coli for 4h. Meanwhile, the survival rate of L-929 cells was consistent with that of normal medium, revealing considerable antibacterial effect and biocompatibility which could be used for promoting wound repair. Furthermore, in vivo experiment was conducted to test its dressing properties, antibacterial properties, and the efficiency of promoting wound healing. Compared with control group and CS/GP hydrogel group, the wound healing rate was the highest since the 3rd days of treating with CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e.While macrophage secretion factor 68 (CD68) decreased significantly and its expression level was lower than that of control group, and the expression level of VEGF increased significantly, with its expression being 1.18-fold, 3.61-fold, and 1.98-fold higher compared with the CS/GP hydrogel group. These results indicate that CS/GP/Cu\u003csub\u003e3\u003c/sub\u003eSnS\u003csub\u003e4\u003c/sub\u003e hydrogel has a potential application as a burn wound dressing.\u003c/p\u003e","manuscriptTitle":"Chitosan hydrogel loaded with copper-tin-sulfur nanosheet materials for skin wound healing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-19 17:26:54","doi":"10.21203/rs.3.rs-5302334/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-27T15:48:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-27T10:12:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-19T10:15:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"29814330480472133980144476065515771805","date":"2024-11-18T08:32:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-13T19:42:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"83991453629753240803577102358246019692","date":"2024-11-13T14:04:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"77041080817055561956110410231585285595","date":"2024-11-13T13:37:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-08T09:51:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-08T09:48:17+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-11-08T09:28:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-06T04:58:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-10-21T08:04:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6600ff74-bac3-4cd0-8798-536988d9d25c","owner":[],"postedDate":"November 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":40389185,"name":"Biological sciences/Biochemistry"},{"id":40389186,"name":"Physical sciences/Materials science"},{"id":40389187,"name":"Physical sciences/Nanoscience and technology"}],"tags":[],"updatedAt":"2025-04-14T16:12:31+00:00","versionOfRecord":{"articleIdentity":"rs-5302334","link":"https://doi.org/10.1038/s41598-024-84416-x","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-04-11 16:05:14","publishedOnDateReadable":"April 11th, 2025"},"versionCreatedAt":"2024-11-19 17:26:54","video":"","vorDoi":"10.1038/s41598-024-84416-x","vorDoiUrl":"https://doi.org/10.1038/s41598-024-84416-x","workflowStages":[]},"version":"v1","identity":"rs-5302334","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5302334","identity":"rs-5302334","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}