ε-Poly- _L -lysine (EPL) (consisting of 25–30 _L-lysine residues) was purchased from Zhengzhou Bainafo Bioengineering Co. Ltd. (China). 2,3,4-trihydroxybenzaldehyde, sodium hydroxide, hydrogen peroxide (30%) and Tris-HCl buffer were all purchased from Shanghai Maclin Reagent Co., LTD. Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (P. aeruginosa) were derived from The Fifth Affiliated Hospital of Zunyi Medical University. Human skin fibroblast (BNCC337722) were derived from the cell sharing platform of Zhuhai Campus of Zunyi Medical University. DMEM culture medium and fetal bovine serum were derived from Kgi Reagent CD, LTD. NO kit is from Nanjing Jiancheng Bioengineering Institute. IL-6 and TNF-α kits were derived from Jiangsu Enzyme Free Industrial Co., LTD.

The hydrogels were created in a single step using a modified version of a previously reported method (Xu, et al., 2019). In short, TBA (247 mM, 371 mM and 494 mM) in Tris-HCl buffer (pH = 8.5, 1 M) in 25°C dissolves oscillations. Then add 1 M sodium hydroxide and EPL and shake to dissolve it. Finally, add 50 μL H₂O₂ (30 wt%) in glass bottles and incubate at room temperature for 5min to form a hydrogel. The hydrogels with different concentrations were named EPL-TBA-40, EPL-TBA-60 and EPL-TBA-80. The 40, 60 and 80 in EPL-TBA-40, EPL-TBA-60 and EPL-TBA-80 refer to the quality concentration of TBA (mg/mL), which equal to the molar concentration 247mM, 371mM and 494 mM of EPL-TBA-40, EPL-TBA-60 and EPL-TBA-80, respectively.

EPL-TBA hydrogels were recorded at 37°C on a rheometer (MCR302, Anton Paar, Austria). The rheometer is equipped with parallel plates with a diameter of 25 mm. The frequency scanning range was 0.1–15 Hz, the temperature was set at 37°C, and the constant strain was 1% to test the stability of EPL-TBA hydrogel.

The antibacterial activities of EPL and TBA against S. aureus and E. coli were determined by MIC method (Chen et al., 2019). Briefly, the chosen bacteria were able to grow in LB medium until the mid log phase. Dilute the bacterial working suspension to 10⁶ (CFU/mL). On a 96-well plate, 50 μL of EPL/TBA in LB (4,000 μg/mL) was 2-fold diluted and then mixed with 50 μL of a bacterial culture containing 10⁶ CFU/mL. The final bacterial concentration was 1 × 10⁵ CFU/mL. The OD value of bacteria was determined at OD600_nm. If no bacterial growth is observed, this concentration is the MIC value of the bacteria.

The Gram-positive bacteria S. aureus and MRSA and Gram-negative bacteria E. coli and P. aeruginosa were employed to assess the antibacterial efficacy of the EPL-TBA hydrogel (Zhou et al., 2022). Briefly, 1 mL of hydrogel was put into a clean glass bottle, and then 400 μL of bacteria in the mid-log phase in LB medium were diluted to 1 × 10⁵ CFU/mL and added to each bottle, which was then incubated at 37°C for 24 h. At OD_{600 nm}, the absorbance of bacteria at different time points was measured. Moreover, the bacterial suspension was daubed onto the agar plate, and the dish was cultured for an additional 24 h.

The morphology of bacteria treated with EPL-TBA hydrogel was observed using SEM. Briefly, The EPL-TBA hydrogel was incubated with bacteria for 4 h and fixed with 2.5% glutaraldehyde. The samples were freeze-dried by freeze-drying machine and observed by SEM after 24 h of freeze-drying.

The concentration of Gram-positive bacteria MRSA was diluted to 1 × 10⁸ CFU/mL, which was used as the bacterial working bacterial suspension (Zhi et al., 2017). Incubate the EPL-TBA hydrogel and quartz plate in a working bacterial suspension for 1 day to allow the biofilm to multiply. The surface of the EPL-TBA hydrogel and quartz plate was rinsed with PBS buffer to remove suspended bacteria. The hydrogel and quartz plates were then dyed with the LIVE/DEAD Backlight kit. The production of biofilm was observed under confocal microscope. Finally, the software was used for photography as well as data analysis.

Before being used for 3 days, the EPL-TBA hydrogel was made in a Petri dish, ground into a powder with a grinder, and disinfected with PBS. Around 5,000 human skin fibroblasts (BNCC337722) were plated per well in 96-well plates. In DMEM with 10% (v/v) fetal bovine serum, cells were grown (FBS). At 37°C, cells were grown in a humidified environment with 5% CO₂. The cytotoxicity of the EPL-TBA hydrogel toward human skin fibroblast (BNCC337722) cells was determined by CCK8. The percentage of cell survival was calculated using the Eq. After incubation with a hydrogel soak, BNCC337722 cells were stained with a LIVE/DEAD staining kit to evaluate the cytocompatibility of BNCC337722 cells. Incubate and stain at room temperature for 30 min, thoroughly clean the samples with PBS, and take photos of the staining results with an inverted microscope. C e l l   s u r v i v a l % = A s − A b A c − A b × 100 %

Where, A _s is the absorbance of experimental cells, A _c is the absorbance of control group cells. And A _b is the absorbance of blank group cells.

The cells were digested and incubated in 12-well plate. After the cells are filled with each septal hole, the 1 mL gun head was perpendicular to the hole plate to make cell scratches, and the width of each scratch was ensured as far as possible. The cell culture solution was removed, and the orifice plate was rinsed with PBS three times to wash away the cell debris generated by scratches. Add the hydrogel soak solution and culture medium. The culture plates were cultured in an incubator at 37°C, and photographs were taken with an inverted microscope after incubation for 0 h, 12 h, 24 h, and 36 h. M i g r a t i o n   R a t e % = S 1 S 0 × 100 %

Where, S ₀ is the scratch area on 0 h and S ₁ is the scratch area on different time.

Mouse mononuclear macrophages (RAW264.7) were inoculated in 96-well plates, LPS (5 μg/mL) was used to induce inflammation in the model for 24 h, and EPL-TBA-40 hydrogel of different concentration was added to the 96-well plates, and co-incubated for 24 h. The levels of NO, IL-6, and TNF-αin the supernatant were determined by the Griess reaction and ELISA kit.

The hemocompatibility test was based on a method published by (Zou et al., 2022). Briefly, the hemolytic activity of hydrogels was measured as follows. First, EPL-TBA hydrogel was added to a glass bottle and 4% of human red blood cells were added to the surface of the hydrogel. After incubating with the hydrogel for 1 hour, the suspension was centrifuged at 2,500 r/min for 3 min. After photographing the tubes, the supernatant was collected. Microplate reader was used to determine the optical density at 490 nm (OD_490nm) of the supernatant. The percentage of hemolysis was calculated using the Eq. H e m o l y s i s % = A 0 − A 1 A 2 − A 1 × 100 %

Where, A ₀ is the absorbance of experimental, A ₁ is the absorbance of negative control and A ₂ is the absorbance of positive control.

The wound healing assay was modified from (Xu, et al. 2019). Female Sprague-Dawley (SD) rats (weighing 200–250 g each) were used for constructing the model for a full-thickness skin wound that was performed on rats. The rats were divided into control group and EPL-TBA-40 group with four in each group. The Ethics Council at Zunyi Medical University authorized all animal testing (ZMU23-2,302-009). After anesthetizing healthy mice, full-layer skin wounds (1.0 cm × 1.0 cm) were made on the back of each rat. To keep the hydrogel samples in place, the lesion was treated with EPL-TBA-40 hydrogel before being bandaged with PU film. Using a smart phone, photograph wound healing on days 1, 3, 7, and 10. Image J software was used to measure the trauma area of each group. The percentage of wound area closure was calculated using the Eq. W o u n d   a r e a   c l o s u r e % = S 1 S 0

Where, S ₀ is the wound area on day 0, and S ₁ is the wound area on different days.

Wound site tissues were collected at days 3, 7, and 10, fixed with 10% paraformaldehyde solution and embedded in paraffin wax. Tissue sections were stained using hematoxylin and eosin (H&E) staining.

All data are expressed as mean ± standard deviation. Two-way analysis of variance (ANOVA) with the Geisser-Greenhouse correction and Tukey’s multiple comparisons test, one-way ANOVA and Holm-Sidak’s multiple comparisons test were used to calculate the significance of differences between groups under test conditions (GraphPad Prism 8.4.0 software, United States).

The vial inversion experiment demonstrated that the EPL-TBA based visible hydrogel was formed with reaching steady state within 5 min (Figure 1A). EPL-TBA hydrogel is formed by cross-linking the amino group on EPL with the aldehyde group on TBA by the Schiff base reaction (Figure 1B). The schematic structure of the EPL-TBA hydrogel is shown in Figure 1C EPL’s concentration was set at 95 mM, and the levels of TBA (247 mM, 371 mM and 494 mM) were changed to control the amount of cross-linking. The chemical ingredients of EPL-TBA hydrogels, which were determined by FT-IR (Figure 2A). The amide I and II bands, which fell within the β-sheet conformation region (Maeda et al., 2003), were assigned the evident characteristic peaks of 1,654 and 1,532 cm⁻¹ in EPL, indicating that EPL backbones were already present in EPL-TBA hydrogels. EPL-TBA hydrogels show a peak at 2,452 cm⁻¹ due to the typical absorption of freshly synthesized Schiff bases by the amine groups of EPL and TBA, demonstrating that the hydrogel network was formed successfully (Khan et al., 2022). Moreover, compared to EPL, EPL-TBA hydrogels displayed a distinct absorption peak at 350 nm in the UV-vis spectroscopic analysis, demonstrating that the new structure was generated (Figure 2B). Rheological tests were performed to demonstrate the structural stability of EPL-TBA hydrogels (Figure 2C). The reserve modulus (G′) of the EPL-TBA hydrogel remained higher than its loss modulus (G″) over the entire strain range, indicating the gel-like behavior of the sample (Zhang F. et al., 2018). As shown in Supplementary Figure S1A–C, with the increase of shear frequency, the curves of G′ and G″ do not intersect in the strain range, indicating that the structure of hydrogel network will not be destroyed under high frequency shear conditions (Ren et al., 2019). Rheological tests demonstrated that the EPL-TBA gels have excellent structural stability even under high frequency shear conditions.

The liquid absorption ability of the hydrogels was tested by placing the EPL-TBA hydrogels into PBS solutions at 25°C, 37°C and 45°C. As shown in Figures 2D–F, within 1 h of soaking in PBS, all three EPL-TBA hydrogels exhibited rapid liquid absorption and swelling, with the EPL-TBA-40 hydrogel exhibiting more pronounced liquid absorption than the other two EPL-TBA hydrogels. At 25°C, the EPL-TBA-40 hydrogel reached a high swelling rate of 313.95%. The expansion rates of the other two hydrogels were 295.56% and 285.10%, respectively. The swelling rates of the three hydrogels at 25°C, 37°C and 45°C were not significantly different. EPL contains abundant hydrophilic amino groups, which is the main reason for the superior swelling properties of EPL-TBA hydrogels (Xu, et al., 2019). Since EPL-TBA-40 hydrogel has the highest content of unreacted EPL, it has the highest swelling rates. In addition to the swelling behavior of the hydrogel from the test, the moisturizing properties were also studied. The excellent moisturizing property of the hydrogel could maintain a moist microenvironment at the wound site for more time, which contributes to the formation of wound epithelialization (Santhini et al., 2022). As shown in Supplementary Figure S2, the three EPL-TBA hydrogels were still able to ensure more than 90% of the liquid, which was not lost after being placed at 37°C for 48 h. The EPL-TBA hydrogel can effectively prevent water evaporation and can always keep the EPL-TBA hydrogels in a moist state, therefore accelerating wound healing.

The antimicrobial activity is one of the most important properties of hydrogels used in wound dressings (Ghobril and Grinstaff, 2015). In addition to the ability of a wound dressing to resist external bacteria from entering its own wound tissue, an ideal wound healing dressing with effective antimicrobial properties would be more attractive (Zhao et al., 2017; Alves et al., 2020). Firstly, we tested the minimum inhibitory concentrations (MIC) of EPL and TBA, and the results showed that MIC of EPL for both S. aureus and E. coli was 4 μg/mL, and MIC of TBA for both S. aureus and E. coli was 30 μg/mL (Supplementary Table S1). In order to characterize the antibacterial ability of hydrogels, the Gram-positive bacteria S. aureus and MRSA, Gram-negative bacteria E. coli and P. aeruginosa were selected as experimental strains in our study. After 24 h incubation demonstrated that the EPL-TBA hydrogel experiment group had nearly no change in OD_600nm, while the control group S. aureus and E. coli gradually increase (Figure 3A). Meanwhile, S. aureus and E. coli treated with EPL-TBA hydrogel did not form bacterial colonies on the agar plates, while the control agar plates were filled with colonies, as shown in Figure 3B. Furthermore, the EPL-TBA hydrogel also inhibited MRSA and P. aeruginosa, and the absorbance of the bacteria in the experimental group remained basically unchanged, while that of the blank group gradually increased (Supplementary Figure S3A). After verification by agar plate coating, no colonies were formed on the experimental group, while the blank group was filled with colonies (Supplementary Figure S3B). These findings demonstrate that EPL-TBA hydrogel can inhibit various kinds of bacteria.

The bacteriostatic effect of cationic peptide hydrogels is achieved by disrupting the cell membranes of anionic bacteria through electrostatic action (Li et al., 2011; Yan, et al., 2021). Scanning Electron Microscope (SEM) was used to observe the morphology characteristics of bacteria in contact with the EPL-TBA hydrogel in order to further investigate the mechanism of killing bacteria. The morphology characteristics of bacteria changed significantly after contact with the EPL-TBA-40 hydrogel, when compared to control bacteria with intact cells (Figure 4A). The cell membranes of bacteria that had been incubated with hydrogels differed from the smooth cell membranes of intact bacteria (Figure 4A), and the cell membrane surfaces of E. coli and MRSA were collapsed or even broken (Figure 4A). These results demonstrated that the cell membrane of EPL-TBA incubated bacteria underwent collapse and breakage, which in turn led to the death of the bacteria.

The bacterial biofilms encapsulate the bacterial body in layers, delaying or inhibiting the penetration of antimicrobial drugs and compromising the antimicrobial effect, leading to further difficulties in wound healing (Sharma et al., 2016; Johani et al., 2017). Inhibiting the biofilm formation remains a key strategy to prevent wound infection. In our study, MRSA was cultured using EPL-TBA-40 hydrogel and control quartz slides to form biofilms. Bacterial biofilm formation was observed using confocal microscopy and LIVE/DEAD staining. Biofilm formation diagram using quartz slide incubation (Figure 4B). A biofilm formed when a lot of living bacteria and several dead bacteria adhered to the quartz plate. Nevertheless, on the surface of the EPL-TBA-40 hydrogel, no bacterial biofilms were observed. (Figure 4B). This occurrence is explained by the fact that when bacteria come into contact with EPL-TBA hydrogel, they are already killed by the EPL-TBA hydrogel and cannot form a biofilm, thus preventing the formation of a biofilm. EPL-TBA hydrogel can remove the biofilm and prevent the production of drug-resistant bacteria.

The hemolytic activity of EPL-TBA hydrogels on human erythrocytes and their cytotoxicity to BNCC337722 cells were determined. Hemolysis may occur when erythrocytes rupture after direct contact with different hydrogels in blood. The hemolysis assay evaluates the degree of in vitro hemolysis of the material by measuring the amount of hemoglobin. The average hemolysis rates of EPL-TBA-40, EPL-TBA-60, and EPL-TBA-80 for red blood cells are 2.61%, 3.73%, and 12.93%. Respectively, as shown in Figure 5A, indicating that EPL-TBA-40 and EPL-TBA-60 have excellent biocompatibility for red blood cells. The toxicity of EPL-TBA hydrogel to cells is one of the key factors in determining whether the hydrogel can be used as a wound dressing. Furthermore, we carefully investigated the toxicity of different concentrations of EPL-TBA hydrogels on BNCC337722 cells by CCK-8 assay (Figures 5B–D). These findings indicated that different concentrations of EPL-TBA hydrogels did not exhibit toxicity to BNCC337722 cells below the concentration of 25 μg/mL. Different concentrations of EPL-TBA hydrogels at 50–100 μg/mL showed slight cytotoxicity, but the cell survival rate was still higher than 80%. BNCC337722 cells were co-cultured with EPL-TBA hydrogel for 0 h, 12 h, and 24 h and stained with LIVE/DEAD Backlight Cell Viability Kit. BNCC337722 cells incubated with EPL-TBA hydrogel had complete morphology and a clear structure. After 36 h of incubation, the number of BNCC337722 cells increased, indicating that EPL-TBA hydrogel has excellent biocompatibility and can promote cell proliferation (Figure 5E). These findings indicated that EPL-TBA hydrogel has excellent biocompatibility and is likely to be a wound dressing.

In the cell migration experiment (Figures 6A, B), BNCC337722 cells gradually migrated to the blank area after 36 h of incubation. Compared with the control group, EPL-TBA-40 hydrogel can significantly promote cell migration. The cell mobility of EPL-TBA-40 (98.9%) was higher than that of cells completely culture-induced (80.02%). Furthermore, the anti-inflammatory effect of EPL-TBA-40 hydrogel was investigated by inducing inflammation in RAW264.7 cells by LPS. Compared with the blank group, different concentrations of EPL-TBA-40 hydrogel could significantly reduce the concentrations of NO, IL-6, and TNF-α in a concentration dependent manner (Figures 6C–E). These findings demonstrate that EPL-TBA hydrogels promote cell migration while also inhibiting the production of inflammatory cytokines, which would be benefit for wound healing.

To assess the therapeutic effect of EPL-TBA hydrogel on the entire skin normal wound, rats were divided into two groups, a blank control group and EPL-TBA-40 hydrogel as an experimental group, and the hydrogel was changed at 1st, 3rd, 7th, and 10th days after surgery. We tracked and statistically analyzed wound closure markers using smart photographs of the wound and quantitative displays of closure rate (Figures 7A, B). As shown in Figure 7A, depicts the physical map of wound healing at various time periods, and it is obvious that the wound healing rate of the EPL-TBA hydrogel group was much faster than the other groups throughout the treatment period. The initial wound size for both groups was about the same at day 0. On day 3, the blank control wounds did not reduce appreciably, whereas the wounds in the EPL-TBA-40 hydrogel group all crusted and shrank. On day 7, the blank group began to show significant wound contraction, and the hydrogel group basically healed, with better wound healing in the EPL-TBA-40 hydrogel group and wound closure rates 58.64% and 86.21%, respectively. On day 10, the wound closure area of the blank control group increased, a small amount of hair appeared around the wound, and the wound closure rate was only 83.47%, respectively (Figure 7C). In contrast, in the EPL-TBA-40 hydrogel group, wound healing was nearly complete, and new hairs began to cover the wound surface.

Wound healing was assessed by histological study of wound tissue on days 3, 7, and 10. (Figure 7D). H&E staining of the surrounding wound tissue helps better understand the effects of EPL-TBA hydrogel treatment. The wound tissue was infiltrated by abundant of inflammatory cells such as neutrophils, which can be seen in blue in H&E staining. On the third day, both the blank group and the EPL-TBA-40 hydrogel group had some areas of scab, but the EPL-TBA-40 hydrogel group had significantly more scab than the blank group. Additionally, a small amount of capillary regeneration could be seen microscopically in the EPL-TBA-40 hydrogel group. The fact that there were relatively fewer inflammatory cells in the wounds that were coated by EPL-TBA-40 hydrogel, in comparison to the wounds in the blank group, indicating EPL-TBA-40 hydrogel may be able to reduce the inflammation that is present around the wounds. After 7 days, the skin of the blank group did not entirely return and showed signs of perhaps still being necrotic tissue with a small amount of fluid collection, more inflammatory cells and fibroblasts were evident, and hair follicles, glands, and capillaries were apparent. Compared with the blank group, the epidermal layer of the wound covered by EPL-TBA-40 hydrogel had recovered, the epidermal layer was thickened, and inflammatory cells and fibroblasts were visible microscopically, while hair follicles, glands, and capillaries could be observed. After 10 days, the local epidermal layer of the blank group did not recover; crusting and bleeding were visible; the epidermal layer was slightly thickened; and more inflammatory cell infiltration was visible in the dermis. Compared with the blank group, the wound recovered to nearly normal skin; the epidermis was slightly thicker; granules were discharged from the epidermis layer; a small amount of inflammatory cells and fibroblasts were visible in the dermis layer; and hair follicles and glands were visible. On days 7 and 10, the epidermal layer of the EPL-TBA-40 hydrogel group was seen to have recovered or recovered to near-normal skin, with granular discharge visible in the spiny cell layer (consolidation of the epidermal layer) and new hair follicles, glands, and capillaries visible. The recovery effect was significant compared to the blank group. Those findings demonstrate that EPL-TBA hydrogel can promote wound healing.