Polysaccharide-antibacterial peptide co-assembled hydrogel as well as preparation method and application thereof

Abstract

The invention discloses polysaccharide-antibacterial peptide co-assembled hydrogel as well as a preparation method and application thereof. The preparation method comprises the following steps: respectively dissolving an antibacterial peptide Temporin-SHF (SHF) and hyaluronic acid in water to obtain an antibacterial peptide solution with the concentration of 1mg / mL-50mg / mL and a hyaluronic acid solution with the concentration of 1%-10wt%; and mixing the antibacterial peptide solution and the hyaluronic acid solution in equal volume, fully stirring and uniformly mixing, and standing for 5-30 minutes to obtain the polysaccharide-antibacterial peptide co-assembled hydrogel. Hydrophobic interaction is used as a main driving force for formation of the hydrogel, hydrogen bonds and pi-pi stacking interaction provide additional structural stability, and a robust physical cross-linked network is jointly constructed; besides, the introduced SHF antibacterial peptide enables the material to have strong and broad-spectrum antibacterial ability and is beneficial to hemostasis, a multifunctional platform is finally formed, and infection can be inhibited and tissue healing can be supported while postoperative adhesion is effectively prevented.

Description

A polysaccharide-antimicrobial peptide co-assembled hydrogel, its preparation method and application Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically a polysaccharide-antimicrobial peptide co-assembled hydrogel, its preparation method, and its application. Background Technology

[0002] Postoperative tissue adhesions are a common and complex complication following surgery. Tissue damage, local inflammation, and foreign body stimulation can induce fibrin deposition and mesenchymal cell proliferation. If these fibrin components are not sufficiently degraded or abnormally remodeled during the healing process, they may form fibrous bands that connect previously separated tissues or organs, thereby interfering with normal anatomical structure and physiological function.

[0003] Clinically, the incidence of adhesions after abdominal surgery is extremely high, which can lead to serious consequences such as chronic pain, intestinal obstruction, and increased difficulty in subsequent surgeries. In gynecology, intrauterine adhesions can cause menstrual abnormalities, infertility, and recurrent miscarriages, severely impacting patients' quality of life and reproductive health. Although surgical procedures such as adhesiolysis can directly address existing adhesions, subsequent surgeries themselves can cause new tissue damage, increasing the risk of adhesion recurrence. While drug therapy (such as anti-inflammatory, anti-fibrotic, or hormonal drugs) can offer some benefits in suppressing inflammation and promoting regeneration, their efficacy is often insufficient and may be accompanied by systemic side effects. Therefore, developing effective, durable, and safe strategies for local prevention of tissue adhesions after surgery remains a significant clinical need.

[0004] Current physical barrier materials used for anti-adhesion (such as absorbable membranes and mechanical devices) aim to reduce adhesion formation by mechanically isolating the wound from surrounding tissues. However, existing products have significant limitations: commercially available membranes typically have poor conformability to irregular wounds, are prone to displacement, and are difficult to achieve complete coverage; their retention time in the body is limited, resulting in insufficient protective window. Mechanical devices (such as balloons and intrauterine devices) can provide immediate separation, but they are difficult to adapt to complex anatomical contours, and long-term mechanical compression may inhibit tissue regeneration and induce inflammatory responses. Therefore, an ideal anti-adhesion material should possess excellent tissue conformability and coverage, controllable degradation kinetics, good biocompatibility, and, when necessary, additional functions such as antibacterial or healing-promoting properties. Summary of the Invention

[0005] To overcome the shortcomings and deficiencies of the prior art, the purpose of this invention is to provide a polysaccharide-antimicrobial peptide co-assembled hydrogel (HA-SHF hydrogel), its preparation method, and its application.

[0006] The technical solution provided by the present invention is: a method for preparing a polysaccharide-antimicrobial peptide co-assembled hydrogel, the method comprising the following steps: (1) dissolving antimicrobial peptide SHF and hyaluronic acid in water respectively to obtain an antimicrobial peptide solution of 1 mg / mL to 50 mg / mL and a hyaluronic acid solution of 1% to 10% wt; (2) mixing the antimicrobial peptide solution and the hyaluronic acid solution in equal volumes, stirring thoroughly, and letting stand for 5 to 30 minutes to obtain a polysaccharide-antimicrobial peptide co-assembled hydrogel.

[0007] This invention further discloses the polysaccharide-antimicrobial peptide co-assembled hydrogel prepared by the above method.

[0008] The present invention further discloses the application of the above-mentioned polysaccharide-antimicrobial peptide co-assembled hydrogel in the preparation of medical devices or medical materials for wound hemostasis; wherein the wound includes, but is not limited to, surgical wounds and traumatic bleeding.

[0009] The present invention further discloses the application of the above-mentioned polysaccharide-antimicrobial peptide co-assembled hydrogel in the preparation of medical devices or medical materials for preventing tissue adhesion; wherein, the tissue adhesion includes, but is not limited to, peritoneal adhesion after surgery, tendon adhesion or organ surface adhesion.

[0010] The beneficial effects of this invention after adopting the above technical solution are as follows:

[0011] (1) No exogenous chemical crosslinking agent and simplified process: Hyaluronic acid and antimicrobial peptide Temporin-SHF (SHF) can be directly co-assembled to form a hydrogel system without the need for exogenous chemical crosslinking agents, reducing the risk of crosslinking agent residue and simplifying the hydrogel preparation process.

[0012] (2) Robust physical cross-linking network and structural stability: Hydrophobic interaction is the main driving force, and hydrogen bonds and π–π stacking provide additional stabilizing effects, forming a robust physical cross-linking network, which is beneficial to maintaining structural integrity and stability in humid environments.

[0013] (3) Adaptability and operability brought about by dynamic non-covalent assembly: The biomimetic dynamic non-covalent assembly mechanism endows the system with environmental adaptability, and can maintain network continuity under external disturbance conditions, thereby improving the adaptability to clinical operation.

[0014] (4) Multifunctional synergy of antibacterial, hemostatic, anti-adhesion and repair: The antimicrobial peptide SHF endows the hydrogel with strong and broad-spectrum antibacterial ability, and the membrane destruction mechanism reduces the risk of bacterial tolerance and helps to reduce the incidence of postoperative infection; the material has the potential to promote coagulation and rapid hemostasis, which can effectively control bleeding during or early postoperative period and reduce the conditions for adhesion initiation related to the continuous formation of fibrin network; the hyaluronic acid matrix combines antibacterial and hemostatic effects to build a local barrier platform, which provides a more favorable local microenvironment for tissue repair while preventing postoperative adhesion. Attached Figure Description

[0015] Figure 1 shows the formation, microstructure, and rheological properties of the polysaccharide-antimicrobial peptide co-assembled hydrogel; Figure 2 shows the cell compatibility of the polysaccharide-antimicrobial peptide co-assembled hydrogel; Figure 3 shows the in vivo toxicity of the polysaccharide-antimicrobial peptide co-assembled hydrogel; Figure 4 shows the swelling and degradation properties of the polysaccharide-antimicrobial peptide co-assembled hydrogel; Figure 5 shows the antifouling properties, antibacterial activity, and anti-inflammatory activity of the polysaccharide-antimicrobial peptide co-assembled hydrogel; Figure 6 shows the hemostatic properties of the polysaccharide-antimicrobial peptide co-assembled hydrogel; Figure 7 shows the anti-adhesion effect of the polysaccharide-antimicrobial peptide co-assembled hydrogel on a rat abdominal wall-cecal adhesion model; Figure 8 shows the therapeutic effect of the polysaccharide-antimicrobial peptide co-assembled hydrogel on a rat intrauterine adhesion model. Detailed Implementation

[0016] The technical solution of the present invention will be further described in detail below with reference to specific embodiments, but this does not constitute any limitation on the present invention.

[0017] I. Implementation Content

[0018] 1. Chemical reagents

[0019] The Fmoc-protected amino acids required for the synthesis of antimicrobial peptides and the hyaluronic acid used to construct the hydrogel in this invention were purchased from Maclean (Shanghai, China), Calcein-AM was purchased from Aladdin (Shanghai, China), and propidium iodide was purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China).

[0020] 2. Cell lines and bacterial strains used in experiments

[0021] The cell line used in this invention to test the cytotoxicity of the hydrogel was mouse fibroblast L929 cells, cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum. The bacterial strains used in this invention to test the antibacterial activity of the hydrogel were *Pseudomonas aeruginosa* (ATCC 27853), *Staphylococcus aureus* (ATCC 29213), and methicillin-resistant *Staphylococcus aureus* (MRSA).

[0022] 3. Laboratory animals

[0023] The male Kunming mice used in this invention weighed 18–22 g, and the SD rats weighed 180–220 g. All animals were housed in an animal room at 22 ± 1 °C, with ample food and water. All experiments in this invention were conducted in accordance with the European Community Council Directive of 24 November 1986 (86 / 609 / EEC).

[0024] 4. Synthesis of antimicrobial peptides and preparation of hydrogels

[0025] The antimicrobial peptide SHF was synthesized using the classic solid-phase antimicrobial peptide synthesis method. The specific process is as follows: (1) Add DCM to a certain amount of resin and stir for 15 min to allow it to swell fully. Wash the resin with DMF and add a deprotecting agent (20% piperidine) to remove the Fmoc protecting group. (2) Wash the resin with DMF, then add excess amino acids, HBTU, HOBT and DIEA and react fully for 1 hour. Wash with DMF and repeat the deprotection-amino acid condensation steps until the entire peptide chain is synthesized. (3) Add a cleavage reagent (TFA:Tris:H2O=95:2.5:2.5) and stir slowly for 3 h. Collect the cleavage solution, evaporate to dryness, and store at -20 ℃. (4) Add ice-cold diethyl ether to the cleavage solution to precipitate, extract with deionized water, collect the aqueous phase, and freeze-dry to obtain the crude peptide. (5) The antimicrobial peptide was purified by reversed-phase high performance liquid chromatography (RP-HPLC, Waters, USA) and its molecular weight was determined by ultra-high resolution time-of-flight mass spectrometry (TOF-MS, maXis 4G, Bruker, Germany).

[0026] The preparation method of hydrogel includes the following steps: (1) Dissolve the synthetic antimicrobial peptide SHF in deionized water to prepare antimicrobial peptide solutions of different concentrations; (2) Weigh an appropriate amount of hyaluronic acid powder and add it to deionized water at different mass percentage concentrations and stir evenly; (3) According to the peptide concentration and the percentage concentration of hyaluronic acid, take the corresponding concentration of antimicrobial peptide solution and hyaluronic acid solution in equal volumes, mix them, stir evenly, and let stand for a certain period of time to assemble a polysaccharide-antimicrobial peptide co-assembled hydrogel.

[0027] 5. Scanning electron microscopy observation

[0028] The prepared polysaccharide-antimicrobial peptide co-assembled hydrogel samples were frozen at -80°C, and then dried using a freeze dryer. The samples were fixed on the sample stage, sputtered with gold, and then observed using FE-SEM (SU8020, Hitachi, Japan).

[0029] 6. Rheological analysis of polysaccharide-antimicrobial peptide co-assembled hydrogels

[0030] Rheological tests were performed using a rotational rheometer (Thermo HAAKE RS6000, USA) at a temperature of 25°C. A 35mm plate and a 1mm height were used for the tests. (a) Strain scan data were obtained at a fixed frequency of 1Hz, ranging from 0.1% to 1000%. (b) Frequency scans were performed in the range of 0.1 to 100 rad / s. (c) The self-healing ability of the hydrogel was analyzed using continuous alternating high and low strain scans (1% / 1000%) at a fixed frequency of 1Hz. The shear thinning behavior of the hydrogel was determined in the range of shear rates from 0 to 100 1 / s.

[0031] 7. Biocompatibility of polysaccharide-antimicrobial peptide co-assembled hydrogels

[0032] The in vitro biocompatibility of the hydrogel was evaluated using hemolytic toxicity and cytotoxicity tests. For the hemolytic toxicity test, rat blood was used. Whole blood was centrifuged (800g, 10min) to obtain red blood cells, which were then diluted to a concentration of 8%. An equal volume of the 8% red blood cell suspension was incubated with the hydrogel at 37°C for 1 h. Physiological saline and 1% Triton X-100 were used as negative and positive controls, respectively. The supernatant red blood cell suspension was collected, centrifuged at 1200g for 10min, and the OD was measured using a multi-functional microplate reader (Synergy NEO2, Agilent, USA). 490 Calculate the hemolysis rate.

[0033] The cytotoxicity of the hydrogel against mouse fibroblast L929 cells was evaluated using the MTT assay and fluorescence staining. First, the hydrogel was soaked in DMEM for 24 h to obtain the hydrogel extract. L929 cells (8 × 10⁶ cells / year) were then... 4 L929 cells were seeded at 100 μL / well in 96-well plates and incubated overnight in a cell culture incubator to allow cell adhesion. Hydrogel extract was added to each well and co-incubated with the cells for 24, 48, and 72 h. Three parallel wells were set up. After incubation, 10 μL of MTT solution was added to each well. After 4 h of incubation, the supernatant was discarded, and 150 μL of DMSO was added to each well to fully dissolve the blue-purple formazan. The absorbance at 570 nm was measured using a multi-mode microplate reader, and cell viability was calculated by comparing the absorbance with the control group. For fluorescence staining, L929 cells were co-incubated with hydrogel extract for 24, 48, and 72 h, the supernatant was discarded, and double staining with Calcein-AM (5 μM, 30 min) and PI (50 μM, 15 min) was performed. Cell proliferation was observed under a fluorescence microscope, and photographs were taken.

[0034] The in vivo safety of the hydrogel was evaluated by injecting 2 mL of it into the peritoneal cavity of rats. Three days later, the rats were euthanized, and blood was collected. Complete blood count (CBC) parameters were measured using a Mindray BC-2800vet automated veterinary hematology analyzer. Plasma was collected after centrifugation, and six biochemical parameters (ALT, AST, ALB, CREA, BUN, and UA) were measured using a Rayto Chemray 800 automated biochemical analyzer. Furthermore, the levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α in the collected plasma were measured using an ELISA kit. Pathological examination of major organs (heart, liver, spleen, lungs, and kidneys) was performed after H&E staining.

[0035] 8. Determination of the antifouling properties of polysaccharide-antimicrobial peptide co-assembled hydrogels

[0036] The anti-adsorption capacity of hydrogel for bovine serum albumin (BSA) was studied using the BCA protein quantification method. First, 500 μL of hydrogel was prepared and placed in an EP tube. Then, a protein solution of 1 mg / mL was added on top and incubated at 37 °C for 2 h. After incubation, the protein solution layer was discarded, and the hydrogel was washed three times with physiological saline to remove unadsorbed protein from the surface. Subsequently, 500 μL of 1% sodium dodecyl sulfate was added to treat the hydrogel for 2 h, and the BSA adsorbed on the hydrogel was separated. The protein concentration in the supernatant was detected using a BCA protein assay kit (Solepro) and the amount of protein adsorbed was calculated.

[0037] The anti-cell adhesion properties of the hydrogel were investigated using L929 cells. Briefly, the hydrogel was prepared in a 48-well plate, and then 200 μL of L929 cells (8 × 10⁻⁶) were added. 4 The cells were seeded onto the surface of the hydrogel with a concentration of 1 / mL and cultured at 37°C for 12 hours. After washing three times with physiological saline, the adherent cells were stained with calcein. Finally, the cell morphology on the surface of the hydrogel was observed using a fluorescence microscope, and the cell number was counted to calculate the cell adhesion rate.

[0038] 9. In vitro and in vivo antibacterial activity assay of polysaccharide-antimicrobial peptide co-assembled hydrogels

[0039] Gram-positive Staphylococcus aureus, Gram-negative Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus were selected as representative strains to evaluate the antibacterial properties of the hydrogel. First, the bacterial suspension was diluted to 1×10⁻⁶ using MH liquid medium. 6CFU / mL, then the bacterial suspension and an equal volume of HA or hydrogel were co-incubated at 37℃ for 8 h. The supernatant was then serially diluted and evenly spread onto MH plates, and incubated at 37℃ for 18–24 h before colony counting and photographing. The in vivo antibacterial activity of the hydrogel was evaluated by establishing a wound infection model. Male Kunming mice aged 6–8 weeks (20±2g) were used, and the modeling began after one week of acclimatization. Before the experiment, the hair on the back of the mice was removed, and the mice were anesthetized with sodium pentobarbital. A full-thickness wound with a diameter of approximately 7 mm was created on the back of the mice, and then 50 μL of MRSA (1×10⁻⁶) was inoculated into the wound. 7 CFU / mL bacterial suspension was used to treat infected mice, which were randomly divided into a model group, an HA group, and an HA-SHF group. The model group was given physiological saline, and hydrogel was applied evenly to the wound every 12 hours. Three days after treatment, wound tissue was collected, weighed, and homogenized in physiological saline. The homogenate was then continuously diluted and evenly spread on MH plates. After incubation at 37°C for 18-24 hours, colony counts were performed and photographs were taken to calculate the bacterial load at the wound site.

[0040] 10. In vitro anti-inflammatory activity assay of polysaccharide-antimicrobial peptide co-assembled hydrogels

[0041] The anti-inflammatory activity of the hydrogel was evaluated using real-time quantitative PCR. First, a hydrogel with a density of 4 × 10⁻⁶ was prepared. 5RAW 264.7 cells were seeded at 2 mL / well in six-well plates. After cell adhesion, LPS was added to stimulate cell inflammation. The DMEM medium group without LPS served as the negative control group, the medium group containing LPS (100 ng / mL) served as the model group, and the group containing LPS (100 ng / mL) and hydrogel served as the experimental group. The cells were then incubated in a cell culture incubator for 24 h. After incubation, cells were collected and total RNA was extracted using the Trizol method. The RNA was reverse transcribed into cDNA, and the relative expression levels of IL-1β, IL-6, and TNF-α were determined using real-time quantitative PCR, with GADPH as an internal control. The primer sequences are as follows: GADPH-F: 5'-TGTGTCCGTCGTGGATCTGA-3', GADPH-R: 5'-TTGCTGTTGAAGTCGCAGGAG-3'; IL-1β-F: 5'-TGGTGTGTGACGTTCCCATT-3', IL-1β-R: 5'-TGTCGTTGCTTGGTTCTCCT-3'; IL-6-F: 5'-CGGCCTTCCCTACTTCACAA-3', IL-6-R: 5'-GCAAGTGCATCATCGTTGTTC-3'; TNF-α-F: 5'-ACTCCAGGCGGTGCCTATGT-3'; TNF-α-R: 5'-GTGAG GGTCTGGGCCATAGAA-3'.

[0042] 11. Evaluation of the in vitro and in vivo hemostatic properties of polysaccharide-antimicrobial peptide co-assembled hydrogels

[0043] The hemostatic properties of polysaccharide-antimicrobial peptide co-assembled hydrogels were evaluated using whole blood coagulation tests and in vivo bleeding models. For the whole blood coagulation test, whole blood from rats was first collected in sodium citrate collection tubes. GaCl2 (0.1 mM) was added to the whole blood at a ratio of 10:1 to activate coagulation. Then, the activated whole blood was added to the surface of the hydrogel pre-coated at the bottom of a 24-well plate (50 μL / well). At time points of 30 s, 60 s, 120 s, 180 s, 240 s, 300 s, 360 s, and 420 s, 2 mL of ultrapure water was added to each well to completely dissolve the uncoagulated red blood cells. Finally, 200 μL of the supernatant from each well was aspirated and added to a 96-well plate to measure A540. The OD540 measured by adding 50 μL of whole blood directly to deionized water was used as a reference value. BCI was calculated using the following formula: BCI = (At - A0) / (Ac - A0), where At represents the absorbance of the supernatant in the hydrogel, gauze-treated group, and TCP group, A0 represents the absorbance of deionized water, and Ac represents the absorbance of the reference value. A mouse model of liver hemorrhage was established to evaluate the in vivo hemostatic properties of a polysaccharide-antimicrobial peptide co-assembled hydrogel. Mice were first anesthetized with sodium pentobarbital, fixed, and their abdominal hair removed. The liver was surgically exposed, and a 21G needle was used to puncture the liver to induce bleeding. Hydrogel was immediately applied to stop the bleeding, and the hemostasis time was recorded. The amount of bleeding was measured using filter paper.

[0044] 12. Evaluation of the effect of polysaccharide-antimicrobial peptide co-assembled hydrogel on preventing postoperative abdominal wall adhesions in rats.

[0045] Male SD rats, weighing 200±20g, were selected and acclimatized for one week before modeling began. Rats were first anesthetized with sodium pentobarbital, and the abdominal hair was shaved. The abdomen was then incised along the midline to expose the cecum. The cecum was gently rubbed with sterile gauze until obvious bleeding points appeared. Subsequently, the abdominal wall surface corresponding to the cecum was scraped to create an abdominal wall defect of approximately 1×2cm. In the model group, physiological saline was sprayed onto the wound. A commercially available polylactic acid (PLA) anti-adhesion membrane was used as a positive control and adhered to the damaged cecum surface. In the HA and polysaccharide-antimicrobial peptide co-assembled hydrogel group, the cecal wound was covered with either HA or PLA co-assembled hydrogel. Finally, the abdominal incision was sutured with 4-0 sutures. On postoperative days 7 and 14, seven rats from each group were sacrificed, and postoperative adhesions were observed and photographed. The degree of adhesion was scored according to the previously reported adhesion scoring criteria: 0 points: no adhesion; 1 point: single thin membrane adhesion; 2 points: multiple thin membrane adhesions; 3 points: focal thick adhesions; 4 points: foot-like thick adhesions, or multiple focal thick adhesions; 5 points: vascularized thick adhesions, or multiple foot-like adhesions. Tissue from the abdominal wall-cecal adhesion sites was collected, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E and Masson's stain for histopathological analysis.

[0046] 13. Evaluation of the effect of polysaccharide-antimicrobial peptide co-assembled hydrogel on the prevention of postoperative intrauterine adhesions in rats.

[0047] Female SD rats (200±20g) were selected to establish a model of intrauterine adhesions. Rats were anesthetized with sodium pentobarbital, and a small incision was made in the lower abdomen to expose the uterus. The left uterus of all rats served as a control, while the right uterus was selected for modeling. A small incision was made at the uterine horn, and the endometrium was damaged using a combination of mechanical injury and ethanol perfusion. In short, the endometrium was first gently scraped with a uterine curette until visibly congested, followed by an injection of 200μL of 95% ethanol for 3 minutes to enhance endometrial damage. Finally, the uterus was rinsed with sterile saline. Rats were divided into five groups: the control group received no treatment; the model group received saline; the Film group received a polylactic acid anti-adhesion membrane; the HA group received HA injections; and the HA-SHF group received a polysaccharide-antimicrobial peptide co-assembled hydrogel. The uterine horn incision was then sutured with 8-0 sutures, and the abdomen was closed with 4-0 sutures. Fourteen days after the operation, all rats were euthanized, the intact uterus was separated for observation and photographic recording, the damaged side of the uterus was removed, fixed in 4% paraformaldehyde, embedded in paraffin, and transversely sectioned for histopathological analysis using H&E and Masson staining.

[0048] 14. Statistical Analysis

[0049] This invention uses GraphPad 8.0 software for statistical analysis. Data are expressed as mean ± standard error of the mean (SEM). Student's t-test was used to compare statistically significant differences between two groups, and one-way ANOVA was used to compare statistically significant differences among three or more groups. P < 0.05 was considered statistically significant.

[0050] II. Implementation Results

[0051] 1. Synthesis of antimicrobial peptides

[0052] This invention synthesizes the antimicrobial peptide SHF using a classic solid-phase peptide synthesis method based on the Fmoc protection strategy. Its sequence is FFFLSRIF-NH2, and its chemical structure is shown below:

[0053] 2. Construction of polysaccharide-antimicrobial peptide co-assembled hydrogels

[0054] Antimicrobial peptide SHF and hyaluronic acid were dissolved separately in water to obtain solutions of different concentrations (1 mg / mL to 50 mg / mL) for the antimicrobial peptide and hyaluronic acid (1% to 10%). Equal volumes of these solutions were then mixed thoroughly and allowed to stand for 5–30 minutes to obtain hydrogels with different concentrations of antimicrobial peptide and hyaluronic acid, as shown in Figure 1a (2% HA represents 2% hyaluronic acid; HA-SHF2, HA-SHF4, and HA-SHF8 represent hydrogels formed by mixing 2% HA with 2 mg / mL, 4 mg / mL, and 8 mg / mL antimicrobial peptide SHF, respectively). The formation of the hydrogels was observed in vials. Furthermore, nanofibers (Figure 1b) were observed using scanning electron microscopy (HA-SHF4 is representative). The structure of the hyaluronic acid is shown below:

[0055] Note: Unless otherwise specified, polysaccharide-antimicrobial peptide co-assembled hydrogel (HA-SHF hydrogel) in the following content refers to HA-SHF4.

[0056] 3. Rheological analysis of polysaccharide-antimicrobial peptide co-assembled hydrogels

[0057] In this invention, the rheological properties of the HA and polysaccharide-antibacterial peptide co-assembled hydrogel were studied by a rheometer. First, the HA and polysaccharide-antibacterial peptide co-assembled hydrogel was subjected to a frequency sweep within the range of 0.1 - 100 rad / s. As shown in Figure 1c, as the frequency increased, HA always remained in a state where the storage modulus (G') was less than the loss modulus (G''), indicating that HA was in a sol state. For the polysaccharide-antibacterial peptide co-assembled hydrogel, both G' and G'' gradually increased, and G' was always greater than G'', which represents the typical solid viscoelastic behavior of the hydrogel. Moreover, the non-covalent interactions within the hydrogel were continuously enhanced, the degree of co-assembly was continuously increased, the cross-linked network was tighter, and the mechanical properties were stronger (Figure 1e). Subsequently, the HA and polysaccharide-antibacterial peptide co-assembled hydrogel was subjected to a strain sweep. As shown in Figure 1d, HA always maintained G' < G'' within the strain range, indicating that HA was always in a sol state. For the polysaccharide-antibacterial peptide co-assembled hydrogel, when the strain was below 94%, G' > G'', and the hydrogel was in a gel state, maintaining its good elastic properties. However, when the strain exceeded 94%, G' < G'', and the hydrogel changed from the gel state to the sol state, indicating that its network structure collapsed, resulting in a gel-sol phase transition (Figure 1f). Continuous alternating high and low strain sweeps were used to further evaluate the self-healing performance of the hydrogel. As shown in Figure 1g, at low strain (1%), G' > G'', and the hydrogel was in a viscoelastic gel state. When switched to high strain (1000%), G' < G'', and the internal structure of the hydrogel was destroyed and changed to the sol state. After multiple high and low strain cycles, the hydrogel could still recover to the gel state with G' > G'', indicating its good self-healing performance, which was due to the fact that the hydrogel was formed by reversible non-covalent interactions. The viscosity sweep results showed that the viscosity of the hydrogel decreased rapidly with the increase of the shear rate (Figure 1h), indicating that the hydrogel had shear-thinning characteristics, confirming its injectability, which is a prerequisite for local drug delivery and helps it adapt to various irregular damaged areas.

[0058] 4. Biocompatibility of Polysaccharide-Antibacterial Peptide Co-Assembled Hydrogel

[0059] Biomaterials used in the biomedical field should possess good biocompatibility. Therefore, this invention tested their in vitro and in vivo biocompatibility. First, the hemolytic toxicity of the hydrogel was evaluated by co-incubating it with rat erythrocytes, using physiological saline and 0.1% Triton X-100 as negative and positive controls, respectively. As shown in Figure 2a, the supernatant after treatment with the hydrogel group was colorless, and the hemolysis rate of all hydrogel groups was less than 5%, meeting international standards for biomaterials. Furthermore, the cytotoxicity of the hydrogel was determined using the MTT assay and live-death staining method. As shown in Figure 2b, after co-incubation with the hydrogel for 24h, 48h, and 72h, the survival rate of L929 cells was above 80%. The results of live-death staining with calcein and PI showed abundant green fluorescence and almost no red fluorescence under a fluorescence microscope (Figure 2c), indicating that the hydrogel has good in vitro biocompatibility. The biocompatibility of the hydrogel was evaluated by direct injection into the peritoneal cavity of rats. As shown in Figure 3a, within 14 days after hydrogel injection, the rats showed a slow weight gain trend, consistent with the weight gain trend of the control group. Furthermore, three days after hydrogel injection, the concentrations of inflammatory factors IL-1β, IL-6, and TNF-α in the serum of rats in the HA and HA-SHF groups, measured by ELISA, showed no significant difference compared to the control group (Figure 3b). As shown in Figures 3c and 3d, there were no significant differences in blood routine indicators (RBC, MCH, HGB, PLT, MCV, WBC) and biochemical indicators (ALT, AST, BUN, CREA, ALB, UA) in the blood of rats in the HA and HA-SHF groups compared to the control group. No significant pathological changes were observed in the major organs (heart, liver, spleen, lung, kidney) of rats treated with HA and HA-SHF (Figure 3e). This indicates that the polysaccharide-antimicrobial peptide co-assembled hydrogel can be safely applied in vivo without causing major adverse reactions. All of the above results demonstrate that the hydrogel has good safety and biocompatibility both in vitro and in vivo.

[0060] Furthermore, in vitro swelling experiments showed that the hydrogel stopped swelling after 36 hours, with a swelling rate of approximately 30% (Figure 4a). In vitro degradation experiments showed that HA could be completely degraded within 16 days, while the HA-SHF4 hydrogel was completely degraded in approximately 45 days (Figure 4b). This indicates that the polysaccharide-antimicrobial peptide co-assembled hydrogel has better stability and can remain in vivo for a longer period, thus effectively exerting its biological activity.

[0061] 5. Antifouling properties, antibacterial efficacy, and anti-inflammatory activity of polysaccharide-antimicrobial peptide co-assembled hydrogels

[0062] The formation of adhesions is a complex process influenced by multiple factors. Upon trauma, the body immediately initiates an inflammatory response, with a large number of inflammatory cells and fibrinogen seeping to the damaged surface. Fibrinogen is converted into fibrin and deposited on the tissue surface, leading to adhesion formation. Subsequently, the large number of cytokines released by inflammatory cells causes abnormal activation of fibroblasts, attracting their migration and adhesion, thereby producing large amounts of collagen and forming fibrous, permanent adhesions. Furthermore, bacterial infections often exacerbate the inflammatory response and prolong the inflammatory cycle, further aggravating adhesion formation. Therefore, preventing protein and fibroblast adhesion, fighting infection, and reducing inflammation are key to preventing adhesions and fibrosis. Hyaluronic acid molecules contain numerous hydrophilic groups, which can bind a large number of water molecules to form a hydration layer, thereby isolating proteins and cells from adhesion. As shown in Figure 5c, the amount of protein adhering to HA was significantly lower than that to TCPS, while the amount of protein adhering to HA-SHF was further reduced, decreasing with increasing antimicrobial peptide concentration. The antimicrobial peptide concentrations of 4 mg / mL and 8 mg / mL exhibited the best anti-protein adhesion performance, which may be due to the increased density and stability of the hydration layer after HA and SHF binding. Similarly, the hydrogel also showed good anti-cell adhesion ability. As shown in Figures 5a and 5b, the adhesion of L929 cells was significantly reduced on the surface of the HA and polysaccharide-antimicrobial peptide co-assembled hydrogel, especially on the polysaccharide-antimicrobial peptide co-assembled hydrogel surface, where almost no L929 cell adhesion was observed, while a large number of L929 cells adhered to TCPS. These results indicate that the polysaccharide-antimicrobial peptide co-assembled hydrogel has good antifouling properties and can effectively prevent the adhesion of proteins and fibroblasts.

[0063] Previous studies have shown that the antimicrobial peptide SHF possesses broad-spectrum antimicrobial activity. Therefore, this invention determined the antimicrobial properties of the polysaccharide-antimicrobial peptide co-assembled hydrogel. As shown in Figures 5d-g, the hydrogel exhibited good antimicrobial effects against both Gram-positive and Gram-negative bacteria. After co-incubation of the bacterial solution with the hydrogel for 12 hours, the bacterial count was lower than that in the control group, with only a few colonies observed on the plates. This invention established a mouse wound infection model to evaluate the in vivo antimicrobial activity of the polysaccharide-antimicrobial peptide co-assembled hydrogel. As shown in Figures 5h and i, after 3 days of hydrogel treatment, the bacterial load at the mouse skin wound was significantly lower than that in the model group and the HA group, indicating that the polysaccharide-antimicrobial peptide co-assembled hydrogel has good in vivo antimicrobial activity.

[0064] Studies have shown that hyaluronic acid (HA) possesses anti-inflammatory properties, playing a crucial role in tissues such as synovial fluid and skin by inhibiting the expression of inflammatory mediators like prostaglandins and regulating the function of immune cells such as macrophages. Therefore, this invention evaluates the anti-inflammatory activity of a polysaccharide-antimicrobial peptide co-assembled hydrogel by establishing an LPS-induced RAW264.7 inflammation model, as shown in Figures 5j, 5k, and 5l. The hydrogel significantly reduces the expression levels of LPS-induced IL-1β, IL-6, and TNF-α, which is attributed to the anti-inflammatory activity of HA. In summary, the polysaccharide-antimicrobial peptide co-assembled hydrogel exhibits excellent antifouling properties, antimicrobial activity, and anti-inflammatory activity. It can effectively prevent protein and fibroblast adhesion, resist infection, and shorten the inflammatory cycle, thereby inhibiting collagen production, which is crucial for its application in preventing tissue adhesion.

[0065] 6. Hemostatic properties of polysaccharide-antimicrobial peptide co-assembled hydrogels

[0066] After tissue injury and bleeding, fibrinogen in the blood is converted into fibrin cross-links to form a network that binds the tissue together. This is the initial form of adhesion. Therefore, effective hemostasis is a key step in preventing postoperative adhesions. This invention investigates the hemostatic properties of a polysaccharide-antimicrobial peptide co-assembled hydrogel. The coagulation ability of the hydrogel was tested using a whole blood coagulation test. As shown in Figure 6a, after whole blood was incubated with the polysaccharide-antimicrobial peptide co-assembled hydrogel for 60 seconds, the blood began to coagulate, and its supernatant began to become clear. In contrast, blood clots appeared in the gauze group after 240 seconds, while the coagulation time of normal blood was 300 seconds. The BCI results calculated by measuring the absorbance of the supernatant further verified these results (Figure 6b). These results indicate that the hydrogel has good blood coagulation-promoting properties. Studies have shown that many anionic polysaccharides, such as sodium alginate and hyaluronic acid, can activate factor XII in the intrinsic coagulation pathway when in contact with blood, thereby activating the coagulation cascade and promoting blood coagulation. Subsequently, a mouse liver hemorrhage model was established using gauze as a positive control. As shown in Figures 6c-e, the hydrogel exhibited the best hemostatic effect, achieving hemostasis within 50 seconds with a bleeding volume of only 24±6 mg, significantly less than the 197±71 mg in the model group. The hemostatic properties of the polysaccharide-antimicrobial peptide co-assembled hydrogel are due to the synergistic effect of multiple hemostatic mechanisms. Firstly, the anionic polysaccharides in the hydrogel can activate the intrinsic coagulation pathway and promote the coagulation process. In addition, the hydrogel can achieve hemostasis through the physical barrier effect. These results fully demonstrate that the hydrogel can effectively control wound bleeding, showing great potential as a rapid and effective hemostatic material.

[0067] 7. Postoperative peritoneal anti-adhesion ability of polysaccharide-antimicrobial peptide co-assembled hydrogels

[0068] A rat abdominal wall defect-cecal abrasion model was constructed to evaluate the effectiveness of HS-SHF hydrogel in preventing postoperative abdominal adhesions. As shown in Figure 7a, on postoperative days 7 and 14, almost no obvious adhesions were found in the hydrogel group, and the injured abdominal wall and cecum recovered normally. However, the model group, anti-adhesion membrane group, and HA group all showed varying degrees of adhesions. The severity of tissue adhesions was scored across all groups, as shown in Figures 7b-e. The model group exhibited the most severe abdominal adhesions, with average scores of 4.6±0.5 and 4±0.6 on days 7 and 14, respectively. The anti-adhesion membrane group scored 2.1±1.3 and 2.9±1.6, respectively, lower than the model group, but still showed significant adhesions, indicating an overall unsatisfactory effect. This may be because the anti-adhesion membrane, being a solid sheet, could not completely cover the wound surface, and the degradation products of polylactic acid (PLA) caused a mild inflammatory response. The HA group scored 2.4±1.5 and 2.3±1.9, similar to the anti-adhesion membrane, primarily because pure hyaluronic acid degrades rapidly in vivo and has poor mechanical properties. Conversely, the hydrogel-treated rats showed almost no adhesions, with average scores as low as 0.4±0.5 and 0.3±0.5, indicating effective adhesion prevention. Previous degradation performance tests showed that HA significantly improved the stability and mechanical properties of SHF after cross-linking with it to form a hydrogel. This is the main reason why the polysaccharide-antimicrobial peptide co-assembled hydrogel can exert a good anti-adhesion effect.

[0069] On days 7 and 14 of treatment, tissue samples from the adhesion sites were collected and histologically analyzed using hematoxylin and eosin (H&E) staining and Masson's trichrome (MT) staining. As shown in Figure 7f, HE staining results revealed severe tissue adhesions between the cecum and abdominal wall in the model group on postoperative days 7 and 14, accompanied by extensive inflammatory cell infiltration and connective tissue accumulation. Moderate adhesions were observed in the anti-adhesion membrane and HA groups, consistent with the scoring results. Conversely, after hydrogel treatment, almost no tissue adhesions or inflammatory cell infiltration were found between the abdominal wall and cecum tissues. Furthermore, MT staining results showed minimal collagen deposition in the hydrogel group (Figures 7g and 7h), while extensive collagen deposition was observed in the model group, although partial tissue adhesions and collagen were still observed despite the isolation and protection provided by the anti-adhesion membrane and HA to the abdominal wall and cecum.

[0070] 8. Anti-adhesion effect of polysaccharide-antimicrobial peptide co-assembled hydrogel on postoperative intrauterine adhesions in rats.

[0071] Given the excellent therapeutic effect of hydrogel in preventing abdominal wall-cecal adhesions, this invention established a rat model of intrauterine adhesions to further evaluate the anti-adhesion and therapeutic effects of hydrogel. The intrauterine adhesion model was established through a combination of mechanical injury (endometrial curettage) and ethanol induction. Only one side of the uterus was injured in each rat, with the other side serving as a control, to better observe the therapeutic effect. As shown in Figure 8a, 14 days post-surgery, the uterus of rats in the model group, anti-adhesion membrane group, and HA group all showed swelling and significant intrauterine fluid accumulation, a common complication of intrauterine adhesions. Counting the number of rats with intrauterine fluid accumulation revealed that 4 rats in the model group had intrauterine fluid accumulation, while two rats each in the anti-adhesion membrane and HA groups had it. All rats in the hydrogel group had normal uterine appearance, similar to the control group, and no intrauterine fluid accumulation was observed. As shown in Figures 8b-8d, H&E staining revealed that in the model group, the endometrium was significantly thinner (467±57 μm), with denser endometrial stroma and a significantly reduced number of glands. These are typical characteristics of endometrial fibrosis. In contrast, the endometrial thickness in the hydrogel group was 729±55 μm, comparable to the control group (754±64 μm), and the number of glands showed no significant difference compared to the control group. The anti-adhesion membrane group and the HA group showed some improvement in endometrial thickness and gland number, but these were still lower than those in the polysaccharide-antimicrobial peptide co-assembled hydrogel group. As shown in Figures 8e and 8f, Masson staining revealed significant collagen deposition in the endometrium of the rats in the model group, a marker of fibrosis. Statistical analysis of collagen area showed that the model group had the largest collagen area. In contrast, the hydrogel group showed a significant reduction in collagen area, indicating that hydrogel can significantly alleviate endometrial fibrosis, thereby preventing intrauterine adhesions.

[0072] III. Conclusion

[0073] This invention, based on a polysaccharide-antimicrobial peptide co-assembly strategy, successfully constructed a multifunctional polysaccharide-antimicrobial peptide co-assembled hydrogel, which can be used to prevent postoperative adhesions in the peritoneal and uterine cavities. The hydrogel's initial assembly is driven by hydrophobic interactions, and its conformation is stabilized via hydrogen bonding and π–π stacking interactions, achieving cross-linking and molding without the introduction of any harmful chemical cross-linking agents. This material possesses excellent injectability and self-healing properties, allowing for convenient injection into target sites and thorough adherence to irregular tissue areas. The polysaccharide-antimicrobial peptide co-assembled hydrogel exhibits significant antibacterial activity, effectively inhibiting bacterial infection; it also possesses excellent antifouling properties, preventing protein adsorption and fibroblast migration. Furthermore, the hydrogel demonstrates good anti-inflammatory effects, significantly inhibiting LPS-induced expression of inflammatory factors such as IL-1β, IL-6, and TNF-α in RAW264.7 macrophages. Notably, it shows excellent biocompatibility in both in vitro and in vivo experiments, without inducing any organ pathological changes. In rat abdominal wall-cecal adhesion models and intrauterine adhesion models, its anti-adhesion effect was superior to that of commonly used polylactic acid anti-adhesion films and hyaluronic acid control groups. In conclusion, the polysaccharide-antimicrobial peptide co-assembled hydrogel integrates antifouling, antibacterial, and anti-inflammatory functions, while also possessing good biocompatibility and injectability, demonstrating broad clinical application prospects as a novel anti-adhesion material.

[0074] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a polysaccharide-antimicrobial peptide co-assembled hydrogel, characterized in that, The method includes the following steps: (1) dissolving antimicrobial peptide SHF and hyaluronic acid in water to obtain an antimicrobial peptide solution of 1 mg / mL to 50 mg / mL and a hyaluronic acid solution of 1% to 10% wt; (2) mixing the antimicrobial peptide solution and the hyaluronic acid solution in equal volumes, stirring thoroughly, and letting stand for 5 to 30 minutes to obtain a polysaccharide-antimicrobial peptide co-assembled hydrogel.

2. The polysaccharide-antimicrobial peptide co-assembled hydrogel prepared by the method of claim 1.

3. The application of the polysaccharide-antimicrobial peptide co-assembled hydrogel according to claim 2 in the preparation of medical devices or medical materials for wound hemostasis; wherein, The trauma includes, but is not limited to, surgical wounds and traumatic bleeding.

4. The use of the polysaccharide-antimicrobial peptide co-assembled hydrogel of claim 2 in the preparation of medical devices or medical materials for preventing tissue adhesion; wherein, The tissue adhesions include, but are not limited to, peritoneal adhesions after surgery, tendon adhesions, or organ surface adhesions.

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