A wet-adhesive injectable hydrogel and its preparation method and application

Abstract

This invention belongs to the field of biomedical materials, disclosing a wet-adhesive injectable hydrogel, its preparation method, and its applications. The hydrogel is obtained by mixing and separating the solid and liquid components, specifically an aqueous solution of component A and component B. The aqueous solution of component B is a bioadhesive molecular aqueous solution capable of cross-linking with component A and reaching the tissue interface. The aqueous solutions of component A and component B are, respectively, an aqueous solution of positively charged polyacrylamide hydrochloride and a negatively charged polymaleic acid. This hydrogel exhibits extremely high wet tissue adhesion strength, excellent sealing performance, rapid hemostasis, broad-spectrum antibacterial activity, and wound healing promotion function. As a wound dressing, it is suitable for the closure and repair of joint areas, high-pressure bleeding wounds, and infected wounds.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a wet-adhesive injectable hydrogel, its preparation method, and its application. Background Technology

[0002] Existing wound dressings (such as hydrogels, sponges, and films) exhibit significantly reduced adhesion in moist environments or active sites (such as joints and internal organs). This is primarily because moisture disrupts hydrogen bonds and dilutes electrostatic interactions, leading to dressing detachment, secondary bleeding, and infection. In recent years, inspired by the underwater adhesion mechanisms of marine organisms (such as mussels and sandcastle worms), the liquid-liquid phase separation (LLPS) strategy has been used to prepare polyelectrolyte composite hydrogels. This utilizes the electrostatic complexation of oppositely charged polymers to form an enriched condensed phase, resulting in strong adhesion at wet interfaces. However, reported hydrogel materials still have shortcomings in terms of adhesion strength, sealing pressure, and multifunctional integration: for example, most systems rely solely on electrostatic interactions, resulting in limited wet adhesion; they lack effective sealing capabilities for high-pressure bleeding wounds; their hemostatic effect is often lower than that of commercially available hemostatic powders (such as Celox™); and their single function makes it difficult to simultaneously meet clinical needs such as antibacterial and wound healing promotion. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a wet-adhesive injectable hydrogel, its preparation method, and its applications. This hydrogel possesses ultra-high wet tissue adhesion strength, excellent sealing performance, rapid hemostasis, broad-spectrum antibacterial activity, and wound healing promotion function. As a wound dressing, it is suitable for the closure and repair of joint areas, high-pressure bleeding wounds, and infected wounds.

[0004] The technical solution provided by this invention is as follows:

[0005] This invention provides a wet-adhesive injectable hydrogel, which is obtained by mixing and separating the solid and liquid components of an aqueous solution of component A and an aqueous solution of component B; wherein, the aqueous solution of component B is a bioadhesive molecular aqueous solution that can crosslink with component A and the tissue interface, and the aqueous solutions of component A and component B are respectively an aqueous solution of positively charged polyacrylamide hydrochloride and an aqueous solution of negatively charged polymaleic acid.

[0006] Furthermore, the volume ratio of the aqueous solution of component A to the aqueous solution of component B is 1:1.

[0007] Furthermore, the molar ratio of polyacrylamide hydrochloride to polymaleic acid is (1~4):(1~4).

[0008] Preferably, the molar ratio of polyacrylamide hydrochloride to polymaleic acid is 2:1.

[0009] Furthermore, the pH of both the aqueous solutions of component A and component B is adjusted to 6.0-8.0 before mixing.

[0010] This invention also provides a method for preparing a wet-adhesive injectable hydrogel, comprising the following steps: Polyacrylamine hydrochloride and polymaleic acid were dissolved in deionized water and adjusted to the same pH value to obtain aqueous solutions of polyacrylamine hydrochloride and polymaleic acid, respectively. Aqueous solutions of polymaleic acid are added to aqueous solutions of polyacrylamide hydrochloride at a preset rate, and the mixture is stirred to allow liquid-liquid phase separation, resulting in a mixed solution that forms a white emulsion-like condensed phase. The above mixed solution was centrifuged to remove the supernatant, and the lower precipitate was collected and washed with PBS buffer to obtain the hydrogel.

[0011] Furthermore, the pH values ​​of both the polyacrylamide hydrochloride aqueous solution and the polymaleic acid aqueous solution are adjusted to 6.0~8.0.

[0012] Furthermore, the pH values ​​of both the polyacrylamide hydrochloride aqueous solution and the polymaleic acid aqueous solution are adjusted to 7.

[0013] Furthermore, the concentration of the solute in the polyacrylamide hydrochloride aqueous solution is 50~300 g / L, and the concentration of the solute in the polymaleic acid aqueous solution is 30~200 g / L.

[0014] Furthermore, the concentration of the solute in the polyacrylamide hydrochloride aqueous solution is 168 g / L, and the concentration of the solute in the polymaleic acid aqueous solution is 100 g / L.

[0015] Furthermore, the preset speed is 1~5 mL / min; the stirring speed is 300~500 rpm; the centrifugation conditions are 6000~10000 rpm for 5~15 min; and the PBS buffer soaking and washing time is 10~14 h.

[0016] The present invention also provides the application of hydrogels prepared using the hydrogels described above or the preparation methods described above in the preparation of wound dressings, the wound dressings being used to close tissue wounds, stop bleeding or fight infection, the tissue wounds including arterial injury wounds, irregular wounds or wounds in mobile areas.

[0017] Beneficial effects

[0018] The polymaleic acid of this invention, as a polycarboxylated polyelectrolyte, possesses high charge density and abundant carboxyl functional groups, enabling it to form a dense network structure with positively charged polymers through electrostatic interactions. Simultaneously, its carboxyl groups can form hydrogen bonds with amino and hydroxyl functional groups on the tissue surface, significantly enhancing tissue adhesion performance in wet environments. This invention, by introducing polymaleic acid into a liquid-liquid phase separation hydrogel system and utilizing its polycarboxyl structure to construct a highly cross-linked network, achieves for the first time high burst pressure performance (up to 143.7 mmHg) of this material under wet conditions, superior to existing wet adhesive dressings, and possesses the potential for application in high-pressure bleeding scenarios such as arterial hemorrhage.

[0019] In a rat model of incompressible hepatic hemorrhage, the hydrogel dressing described in this invention was significantly superior to the Celox™ hemostatic material reported in the literature (hemostasis time of about 60 seconds, with some experimental animals dying) in terms of hemostasis time (average 30.6 seconds) and blood loss (average 130.4 mg), indicating that it has excellent sealing effect and procoagulant ability on high-risk bleeding wounds.

[0020] The material of this invention possesses multiple functions, including strong adhesion to wet tissue, good shape adaptability, rapid hemostasis, broad-spectrum antibacterial activity, and promotion of wound healing. It simplifies wound care procedures, reduces the risk of infection, and is suitable for treating complex wounds. Furthermore, the preparation process described in this invention is simple, the raw materials are widely available and the cost is controllable, and the resulting material can be prepared as a dry powder for long-term storage. Its performance does not decrease after reconstitution, demonstrating good clinical operability and promising prospects for widespread application. Attached Figure Description

[0021] Figure 1 This is an analysis of the gelation phenomenon and yield of PAH / PMA hydrogels; where A is a diagram of the gelation state of hydrogels under different ratios; and B is a quantitative statistical diagram of the yield of hydrogels under different pH values.

[0022] Figure 2This section describes the adhesion properties of PAH / PMA hydrogels. A shows a schematic diagram of the tensile adhesion test; B shows the tensile adhesion test results (n=3); C shows a schematic diagram of the lap shear test; D shows the lap shear test results (n=3); E shows the adhesion strength test under dry, wet (pH = 7.0), and acidic (pH = 5.5) conditions (n=3); F shows the shear strength of the PP composite hydrogel on various substrates (steel, glass, and pigskin) at different pH values ​​measured by the lap shear test (n = 3); G shows photographs of the PAH / PMA composite hydrogel under repeated stretching: i) initial state, ii) stretched state, iii) recovered state, iv) re-stretched state; H shows the PAH / PMA hydrogel reformed by adding water to PAH / PMA powder on a polytetrafluoroethylene plate; I shows a schematic diagram of the reformation of PAH / PMA hydrogel from swollen powder; J shows the freshly prepared PAH / PMA hydrogel and the reformed PAH / PMA hydrogel on different substrates (steel, glass, and pigskin, n = 3). 3) Comparison of adhesion strength, data are mean ± standard error; K is a schematic diagram of burst pressure test; L is a quantitative statistical chart of burst pressure test; M is a photograph of liver, spleen, lung and kidney adhering to PP7 hydrogel.

[0023] Figure 3 A represents the hemostatic activity of PAH / PMA hydrogel; B represents the optical images of blood coagulation after adding different hemostatic materials; C represents the corresponding coagulation time (n = 3); D represents the schematic diagram of the hemostasis experiment in two rat models (tail amputation and liver hemorrhage), treated with PP6, PP7 and PP8 hydrogel powders respectively; E represents the hemostasis process of tail amputation (scale bar = 6 cm); F represents the blood loss of the rat tail amputation model (n = 5); G represents the hemostasis time of the rat tail amputation model (n = 5); H represents the blood loss of the rat liver hemorrhage model (n = 5); I represents the hemostasis time of the rat liver hemorrhage model (n = 5) (*** indicates p < 0.001).

[0024] Figure 4 The images show the antibacterial properties of the PAH / PMA hydrogel; A represents the live / dead staining images of *Escherichia coli*, *Staphylococcus aureus*, and methicillin-resistant *Staphylococcus aureus* after 6 hours of co-incubation with the PAH / PMA hydrogel. Scale bar = 100 μm; B represents the survival rate of *E. coli*, *Staphylococcus aureus*, and MRSA after 6 hours of co-incubation with the PAH / PMA hydrogel (n=3); C represents the bacterial plate count results; D represents the SEM images of *E. coli*, *Staphylococcus aureus*, and MRSA after 6 hours of co-incubation with the PAH / PMA hydrogel, scale bar = 1 μm; E represents the biofilm inhibition test and corresponding quantitative analysis, scale bar = 4 mm.

[0025] Figure 5 A represents the in vitro cell compatibility of PAH / PMA hydrogels; B is a schematic diagram of the in vitro cell compatibility test; C represents the proliferation of L929 cells (days 1, 3, and 5) (n = 3); D represents the live / dead staining of L929 cells (days 1, 3, and 5); E represents the optical image of the PAH / PMA hydrogel hemolysis test; and E represents the hemolysis rate of each group (n = 3). *** indicates p < 0.001, and ns indicates no significant difference.

[0026] Figure 6 This is an assessment of the fit of PAH / PMA hydrogel to irregular wounds. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0028] The experimental materials used in the embodiments of this invention are as follows:

[0029] Polyallylamine hydrochloride (PAH): molecular weight 18,000-20,000, purchased from Shanghai Adamas Beta Chemical Reagent Co., Ltd.

[0030] Polymaleic acid (PMA): molecular weight 400-800, purchased from Aladdin Company (Shanghai, China).

[0031] Bacterial strains: Staphylococcus aureus, Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) were purchased from the North-South Collection Center (Beijing, China).

[0032] Cell line: Mouse fibroblasts (L929) were kindly provided by the Institute of Orthopedics, Soochow University.

[0033] Main kits and reagents: live / dead cell staining kit, live / dead bacteria staining kit containing DMAO and PI, CCK-8 assay kit, streptomycin-penicillin, 0.25% trypsin (all purchased from Beyotime Biotechnology Co., Ltd.); fetal bovine serum (FBS, purchased from Gibco); phosphate-buffered saline (PBS), paraformaldehyde (both purchased from Solarbio); 3M medical adhesive (vetbond, purchased from Beijing Jitian Biotechnology Co., Ltd.); fibrin glue (Fibrin Glue, purchased from Shanghai Caishi Blood Products Co., Ltd.).

[0034] This invention provides a wet-adhesive injectable hydrogel, which is obtained by mixing and separating an aqueous solution of component A and an aqueous solution of component B; wherein, the aqueous solution of component B is a bioadhesive molecular aqueous solution that can crosslink with component A and the tissue interface, and the aqueous solutions of component A and component B are respectively an aqueous solution of positively charged polyacrylamide hydrochloride and an aqueous solution of negatively charged polymaleic acid.

[0035] Specifically, the hydrogel is composed of positively charged polyacrylamide hydrochloride (PAH) aqueous solution and negatively charged polymaleic acid (PMA) aqueous solution, separated by liquid-liquid phase separation (LLPS). PMA, as the core functional component, has densely packed carboxyl groups (-COOH) on its molecular chain that partially ionize into -COO at physiological pH. - With the protonated amino group (-NH3) of PAH + Strong electrostatic complexation occurs, and unionized carboxyl groups form hydrogen bonds with the tissue surface, thereby giving the hydrogel excellent wet adhesion and cohesive force.

[0036] In this embodiment, the volume ratio of the aqueous solution of component A to the aqueous solution of component B is 1:1.

[0037] In this embodiment, the molar ratio of polyacrylamide hydrochloride to polymaleic acid is (1~4):(1~4).

[0038] Preferably, the molar ratio of polyacrylamide hydrochloride to polymaleic acid is 2:1.

[0039] In this embodiment, the pH of both the aqueous solutions of component A and component B is adjusted to 6.0~8.0 before mixing.

[0040] This invention also provides a method for preparing a wet-adhesive injectable hydrogel, comprising the following steps: Polyacrylamine hydrochloride and polymaleic acid were dissolved in deionized water and adjusted to the same pH value to obtain aqueous solutions of polyacrylamine hydrochloride and polymaleic acid, respectively. Aqueous solutions of polymaleic acid are added to aqueous solutions of polyacrylamide hydrochloride at a preset rate, and the mixture is stirred to allow liquid-liquid phase separation, resulting in a mixed solution that forms a white emulsion-like condensed phase. The above mixed solution was centrifuged to remove the supernatant, and the lower precipitate was collected and washed with PBS buffer to obtain the hydrogel.

[0041] In this embodiment, the pH values ​​of both the polyacrylamide hydrochloride aqueous solution and the polymaleic acid aqueous solution are adjusted to 6.0~8.0.

[0042] In this embodiment, the pH values ​​of both the polyacrylamide hydrochloride aqueous solution and the polymaleic acid aqueous solution are adjusted to 7.

[0043] In this embodiment, the concentration of solute in the polyacrylamide hydrochloride aqueous solution is 50~300 g / L, and the concentration of solute in the polymaleic acid aqueous solution is 30~200 g / L.

[0044] In this embodiment, the preset speed is 1~5 mL / min; the stirring speed is 300~500 rpm; the centrifugation conditions are 6000~10000 rpm for 5~15 min; and the PBS buffer soaking and washing time is 10~14 h.

[0045] Specifically, the preparation method in this embodiment is as follows: PAH and PMA aqueous solutions were adjusted to the same pH value (preferably 6.0-8.0, most preferably 7.0). At room temperature, the PMA solution was slowly added (1-5 mL / min) to the PAH solution with vigorous stirring, resulting in LLPS and a mixed solution forming a white emulsion-like condensed phase. The mixed solution was centrifuged (6000-10000 rpm, 5-15 min) to remove the supernatant. The precipitate was washed with PBS for 10-14 h to remove unreacted polymers, yielding the PAH / PMA hydrogel (named PPx hydrogel, where x represents the pH value). This hydrogel can be further freeze-dried into a powder, which rapidly re-gels upon contact with water or buffer, restoring its adhesive properties and facilitating storage and transportation.

[0046] The present invention also provides the above-described hydrogel or the above-described hydrogel in the preparation of wound dressings, the wound dressings being used to close tissue wounds, stop bleeding or fight infection, the tissue wounds including arterial injury wounds, irregular wounds or wounds in mobile areas.

[0047] The following detailed description, in conjunction with embodiments, illustrates the present invention providing a wet-adhesive injectable hydrogel, its preparation method, and its applications (e.g., ...). Figures 1-6 However, these should not be construed as limiting the scope of protection of this invention.

[0048] Example 1

[0049] Polyacrylamide hydrochloride and polymaleic acid were prepared at a molar ratio of 4:1 and added separately to water. The pH of each solution was adjusted to 7.0 with 1 M HCl / NaOH, resulting in a 214.4 g / L aqueous solution of polyacrylamide hydrochloride (solution A) and a 53.6 g / L aqueous solution of polymaleic acid (solution B). Solution B was added to solution A at 5 mL / min with stirring at 500 rpm at room temperature, resulting in a white emulsion-like condensate. The mixed solution was centrifuged (8000 rpm, 10 min), the supernatant was discarded, and the precipitate was washed with PBS for 12 h to obtain PAH / PMA hydrogel. Some samples were freeze-dried at -80℃ to obtain a pale yellow porous powder.

[0050] Example 2

[0051] The other steps are the same as in Example 1, except that polyacrylamide hydrochloride and polymaleic acid are prepared in a molar ratio of 3:1 and added to water respectively. The pH is adjusted to 7.0 with 1 M HCl / NaOH to form a polyacrylamide hydrochloride aqueous solution with a concentration of 201.0 g / L (solution A) and a polymaleic acid aqueous solution with a concentration of 67.0 g / L (solution B).

[0052] Example 3

[0053] The other steps are the same as in Example 1, except that polyacrylamide hydrochloride and polymaleic acid are prepared in a molar ratio of 2:1 and added to water respectively. The pH is adjusted to 7.0 with 1M HCl / NaOH to form a polyacrylamide hydrochloride aqueous solution with a concentration of 178.7 g / L (solution A) and a polymaleic acid aqueous solution with a concentration of 89.3 g / L (solution B).

[0054] Example 4

[0055] The other steps are the same as in Example 1, except that polyacrylamide hydrochloride and polymaleic acid are prepared in a 1:1 molar ratio and added to water respectively. The pH is adjusted to 7.0 with 1M HCl / NaOH to form a polyacrylamide hydrochloride aqueous solution (solution A) with a concentration of 134.0 g / L and a polymaleic acid aqueous solution (solution B) with a concentration of 134.0 g / L.

[0056] Example 5

[0057] The other steps are the same as in Example 1, except that polyacrylamide hydrochloride and polymaleic acid are prepared in a molar ratio of 1:2 and added to water respectively. The pH is adjusted to 7.0 with 1M HCl / NaOH to form an aqueous solution of polyacrylamide hydrochloride with a concentration of 89.3 g / L, i.e., solution A, and an aqueous solution of polymaleic acid with a concentration of 178.7 g / L, i.e., solution B.

[0058] Example 6

[0059] The other steps are the same as in Example 1, except that polyacrylamide hydrochloride and polymaleic acid are prepared in a molar ratio of 1:3 and added to water respectively. The pH is adjusted to 7.0 with 1M HCl / NaOH to form a polyacrylamide hydrochloride aqueous solution with a concentration of 67.0 g / L (solution A) and a polymaleic acid aqueous solution with a concentration of 201.0 g / L (solution B).

[0060] Example 7

[0061] The other steps are the same as in Example 1, except that polyacrylamide hydrochloride and polymaleic acid are prepared in a molar ratio of 1:4 and added to water respectively. The pH is adjusted to 7.0 with 1M HCl / NaOH to form a polyacrylamide hydrochloride aqueous solution with a concentration of 53.6 g / L (solution A) and a polymaleic acid aqueous solution with a concentration of 214.4 g / L (solution B).

[0062] The PAH / PMA hydrogels with different molar ratios prepared in Examples 1-7 were compared and observed.

[0063] result( Figure 1 The results show that when the molar ratio of PAH to PMA is 4:1 and 3:1, the amount of precipitate formed in the system is relatively small. However, when the molar ratio is 1:1, 2:3, and 1:4, the high proportion of PMA leads to turbidity of the solution, a loose and non-dense gel structure, and poor adhesion. In contrast, the molar ratio of 2:1 yields the best results, as the electrostatic complexation is most efficient, resulting in the most dense gel structure.

[0064] Example 8

[0065] The other steps are the same as in Example 3, except that the pH of both PAH aqueous solution and PMA aqueous solution is adjusted to 6.0 to prepare PP6 hydrogel.

[0066] Example 9

[0067] The other steps are the same as in Example 3, except that the pH of both PAH aqueous solution and PMA aqueous solution is adjusted to 8.0 to prepare PP8 hydrogel.

[0068] Performance tests were conducted on Examples 3, 8, and 9:

[0069] 1. Effect of different pH values ​​on hydrogel yield

[0070] Yield (%) = (Actual output / Theoretical output) × 100%

[0071] The results showed that, Figure 2 As shown in Figure B, the yields of PP6 and PP7 (~69%) were significantly higher than those of PP8 (41.0%).

[0072] 2. Adhesion strength test

[0073] 2.1 Tensile test

[0074] Referring to ASTM F2258-05 and F2256-05 standards, commercially available pigskin was used as the biological tissue material, and tensile adhesion tests were conducted on PP6, PP7, and PP8 hydrogels using a universal testing machine. Figure 2 (A) Before testing, the tissue surface was coated with water or blood, and then the PAH / PAA hydrogel was placed between the two tissues and fixed for 1 minute. The hydrogel was placed between two pieces of pigskin and stretched at a rate of 1 mm / min, and the maximum force value at separation was recorded.

[0075] The results showed that, Figure 2 As shown in Figure B, the tensile adhesion strength of PP7 hydrogel is greater than that of PP6 and PP8 hydrogels, reaching 95.33 ± 4.33 kPa.

[0076] 2.2 Overlap Cutting

[0077] The lap shear test was performed according to ASTM F2255-05 standard. Figure 2 (C)

[0078] The results showed that, Figure 2 As shown in Figure D, the lap shear strength of PP7 hydrogel is greater than that of PP6 and PP8 hydrogels, reaching 65.67 ± 4.06 kPa.

[0079] 2.3 Adhesion performance under different environmental conditions

[0080] The adhesion stability of PP6, PP7, and PP8 hydrogels under different environments was investigated. Figure 2 As shown in Figure E, the hydrogel maintained good adhesion strength under dry, humid (pH 7.4), and acidic (pH 5.5, simulating the wound microenvironment) conditions. Further testing of its adhesion properties to different substrates was conducted. Figure 2 (F) The results showed that PP7 hydrogel exhibited excellent adhesion to steel, glass and pigskin.

[0081] 2.4 Shape adaptability and reversible deformation

[0082] PP7 hydrogel was coated onto pigskin and subjected to bending and twisting; it maintained stable adhesion throughout the mechanical deformation process. The entire process was monitored using a 4x magnification camera to evaluate its shape-adaptive performance. Figure 2As shown in G, the hydrogel can fully recover from the initial state (i), through the stretched state (ii), to the recovered state (iii), and then be stretched again to the stretched state (iv). Throughout the process, the hydrogel structure remains intact without any damage or debonding, demonstrating excellent flexibility and shape adaptability.

[0083] 2.5 Injectability

[0084] PP7 hydrogel was loaded into a 1.5 mL syringe and injected into a glass vial at a rate of 0.1 mL / s. Then, "NMU" was written on a PTFE plate. The entire injection process was observed using a 4x magnification camera to assess its injectability. PP7 hydrogel was successfully injected into the glass vial, and the letters "NMU" were clearly formed on the PTFE plate. No instability was observed during the injection process under 4x magnification. Furthermore, PP7 hydrogel can be precisely applied to irregular wounds (such as joint flexion areas) using a syringe and closely conforms to the wound contour. Figure 6 ).

[0085] 2.6 Powder rehydration properties

[0086] After freeze-drying the PP7 hydrogel, a pale yellow porous powder was obtained. Figure 2 (H). For example Figure 2 As shown in Figure I, this powder can rapidly rehydrate upon contact with water, reforming into an adhesive hydrogel. Importantly, as... Figure 2 As shown in Figure J, the adhesion strength of the reformed PP7 hydrogel on steel, glass, and pigskin was not significantly different from that of the freshly prepared hydrogel, demonstrating its good reversible gelation properties, which facilitates storage and transportation.

[0087] 3. Burst Pressure Test

[0088] Burst pressure testing was performed according to the standard burst pressure testing procedure (ASTM F2392-04). A 2 mm diameter hole was created in the center of the pigskin using a punch, and the area was covered with a 12 mm diameter sample. Pressure was applied by pumping PBS solution at a rate of 10 mL / min, and the maximum pressure (burst pressure) was recorded using a digital pressure gauge (XINTEST HT-1895). Figure 2 (Middle K).

[0089] The results showed that the burst pressure of PP7 was 143.7 ± 5.2 mmHg (n=5). Figure 2 In the L group, PP6 and PP8 were worse than PP7, and PP7 was significantly higher than the 3M Vetbond group (85.2±4.1 mmHg) and the fibrin glue group (61.3±3.8 mmHg).

[0090] 4. Multi-organ tissue adhesion

[0091] Qualitative assessment of tissue adhesion was performed using rat organs (including heart, liver, spleen, lung, and kidney). PP7 hydrogel (0.1 g) was applied to the fingertips of rubber gloves, and the adhesion was observed and photographed. As shown in Figure 2M, the hydrogel formed strong adhesions with all tested organs, demonstrating its good universal adhesion ability to a variety of solid tissues.

[0092] 5. Hemostatic performance test

[0093] 5.1 In vitro coagulation test

[0094] In the control group (untreated rat whole blood), 1g of PMA, PAH, PP6, PP7, and PP8 powders were incubated with rat whole blood, and coagulation was observed. Figure 3 As shown in Figure A, the blood group co-incubated with the hydrogel all developed obvious blood clots, while the blood in the control group remained flowing. The corresponding clotting time statistics ( Figure 3 Figure B shows that PP series hydrogels can significantly shorten clotting time.

[0095] 5.2 In vivo hemostasis experiment

[0096] The experimental procedure is as follows Figure 3 As shown in Figure C, the hemostatic effect of PAH / PMA hydrogel powder was evaluated using tail amputation and non-compressible liver wound models in male SD rats (200–250 g). This study was approved by the Animal Care and Use Committee of Nanjing Medical University. All animal husbandry and experimental procedures strictly followed the National Research Council's guidelines on laboratory animal care and use. The hemostatic properties of PAH / PMA hydrogel powder were investigated, with an untreated rat group serving as a control. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital. For the tail amputation model, the tail was amputated 2 cm from the tail tip using a scalpel. The amputated tail was exposed to air for 15 seconds to allow blood to drain naturally, and then the wound was covered with the test sample. For the non-compressible liver hemorrhage model, after anesthesia, an open abdominal surgery was performed to expose the liver. A deep and narrow wound (3 mm deep, 5 mm in diameter) was created on the liver using a biopsy punch, and then the sample was covered at the wound site. Blood loss (mg) and bleeding time (seconds) were recorded until complete hemostasis was achieved (n = 5).

[0097] Experimental results:

[0098] (1) Tail truncation model: such as Figure 3 As shown in Figure D, PP6, PP7, and PP8 powders can all rapidly cover the wound and effectively stop bleeding. Quantitative analysis shows ( Figure 3Among the F and G groups, the PP7 group had the least blood loss (52.8 mg) and the shortest hemostasis time (89.2 s), showing better efficacy than PP6 and PP8.

[0099] (2) Liver hemorrhage model: such as Figure 3 As shown in Figure E, PP series powders also exhibited rapid hemostatic ability in the non-compressible liver hemorrhage model. Quantitative results showed that ( Figure 3 (Among H and I), the hemostasis time of PP7 group was only 30.6s, and the blood loss was 130.4 mg, which was better than PP6 and PP8.

[0100] Furthermore, compared with the hemostatic effect of Celox™ reported in the literature (Tithy et al., Carbohydr. Polym. 2024 and Zhang et al., Int. J. Biol. Macromol. 2025) (approximately 90 s in the tail amputation model and approximately 60 s in the liver model with some animal deaths), PP7 showed significantly better hemostasis time and blood loss in the liver model than Celox™. This is attributed to the strong physical barrier formed by its high burst pressure and the adsorption and aggregation effect of PAH positive charge on blood cells / platelets, and also indicates that the multi-carboxyl structure of PMA is crucial for high adhesion and high sealing performance.

[0101] 6. Antibacterial performance test

[0102] 6.1 Staining of live / dead bacteria

[0103] After incubating Escherichia coli, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus (MRSA) with PP6, PP7, and PP8 hydrogels for 6 hours, live / dead bacteria staining was performed. Figure 4 As shown in Figure A, the control group bacteria (untreated bacteria) mainly appeared green (live bacteria), while the vast majority of bacteria co-incubated with PP6, PP7, and PP8 were stained red (dead bacteria), indicating that the hydrogel has broad-spectrum and highly efficient bactericidal ability. Quantitative analysis was performed on the live / dead staining results. Figure 4 As shown in Figure B, after 6 hours of co-incubation with PP6, PP7, and PP8 hydrogels, the survival rates of Escherichia coli, Staphylococcus aureus, and MRSA were all less than 1%, with a kill rate exceeding 99%.

[0104] 6.2 Plate Count Results

[0105] The steps for preparing LB (Luria Bertani) medium are as follows: Dissolve 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl separately in 950 mL of deionized water. Shake the mixture until all components are completely dissolved. Then adjust the pH to the range of 7.35-7.45, and add deionized water to bring the total volume to 1 L. The LB medium is then sterilized by autoclaving. The co-incubated bacterial suspension is spread onto LB solid medium and incubated for 24 hours to observe colony formation. Figure 4 As shown in Figure C, almost no colonies grew on the plates of the PP7 treatment group, while the control group plates had dense colonies, further confirming the strong bactericidal ability of PP7 hydrogel.

[0106] 6.3 Bacterial Morphology Observation

[0107] Bacterial morphological changes were observed using scanning electron microscopy (SEM). For example... Figure 4 As shown in Figure D, the control group bacteria had smooth surfaces and intact morphology; however, after co-incubation with PP6, PP7, and PP8 hydrogels, the cell membranes of Escherichia coli, Staphylococcus aureus, and MRSA all showed severe shrinkage, rupture, and leakage of contents, indicating that the hydrogels exert their bactericidal effect by disrupting the integrity of bacterial cell membranes.

[0108] 6.4 Biofilm Inhibition Test

[0109] The inhibitory effects of PP6, PP7, and PP8 hydrogels on bacterial biofilm formation were evaluated. 1 mL of a 10% concentration was added to each 24-well plate. 6 Staphylococcus aureus at CFU / mL was co-cultured with a hydrogel at 37°C and 5% CO2 for 24 hours. The bacterial suspension from each well was then transferred to a new 24-well plate and incubated for another 48 hours. The biofilm was fixed with 4% paraformaldehyde (PFA) for 20 minutes and stained with 1% crystal violet solution for 10 minutes. Afterward, 300 μL of 95% ethanol was added to each well, and the sample was shaken for destaining for 10 minutes before being transferred to a 96-well plate. The absorbance at 595 nm was measured using a microplate reader. Figure 4 As shown in Figure E, after crystal violet staining, a distinct purple biofilm was visible at the bottom of the control group wells, while the PP7-treated group showed very light staining. Corresponding quantitative analysis revealed that the OD of the PP7-treated group... 595 The value was significantly lower than that of the control group, proving that hydrogels can effectively inhibit the formation of biofilms.

[0110] 7. In vitro cell compatibility test

[0111] 7.1 Cell proliferation and activity

[0112] The cell compatibility of PP6, PP7, and PP8 hydrogels was evaluated using the Transwell co-culture system. Figure 5 (A) NCTC clone L929 cells were cultured in α-MEM medium supplemented with 1% streptomycin-penicillin and 10% fetal bovine serum. These cells were then cultured at 1 × 10⁶ cells per well. 5 A density of 1000 cells / well was seeded in the lower chamber of a 6-well Transwell plate. After 24 hours, the hydrogel was placed in the upper chamber. A control group was set up, whose upper chamber was not filled with hydrogel.

[0113] L929 cells were co-cultured with hydrogel for 1, 3, and 5 days, and cell proliferation was detected by the CCK-8 assay. Figure 5 As shown in Figure B, there were no significant differences in cell viability between the PP6 and PP7 groups and the control group at any time point, while PP8 showed a significant difference. L929 was toxic, indicating that the PP6 and PP7 hydrogels were safe, while PP8 was toxic to L929. Live / dead cell staining results ( Figure 5 Further investigation by C) confirmed that after co-culturing for 1, 3, and 5 days, the cells in the PP7 group showed good morphology, dense green fluorescence (live cells), and almost no red fluorescence (dead cells), consistent with the control group.

[0114] 6.2 Hemolytic performance test

[0115] The blood compatibility of PP7 hydrogel was assessed using a hemolysis test. Whole blood from rats was collected and stored in test tubes containing sodium heparin. The samples were diluted with PBS, centrifuged at 3000 rpm for 8 minutes, and then resuspended in deionized water and PBS, respectively, to prepare red blood cell suspensions. 1.5 mL of red blood cell suspension diluted with deionized water, 1.5 mL of red blood cell suspension diluted with PBS, and 1.5 mL of red blood cell suspension diluted with PBS and co-cultured with PT hydrogel were incubated at 37°C. After incubation at 37°C for 4 hours, the mixture was centrifuged at 3000 rpm for 5 minutes. Subsequently, the absorbance of the supernatant was measured at 540 nm using a spectrophotometer. The control group treated with deionized water served as a positive control, indicating significant hemolysis; the red blood cells treated with PBS served as a negative control, indicating no hemolysis. The hemolysis rate was calculated using the formula: [(sample group OD...]]. 540 - Negative control group OD 540 ) / (Positive control group OD 540 - Negative control group OD 540 )] × 100%.

[0116] The results showed that, Figure 5 As shown in Figure D, the supernatant of the positive control group (DI water, deionized water) was bright red due to red blood cell rupture; the supernatants of the negative control group (PBS) and the PP7 group were both clear and colorless. Quantitative statistics of hemolysis rate ( Figure 5E) showed that the hemolysis rate in the PP7 group was far below the international standard threshold of 5% and there was no statistical difference compared with the negative control group (ns indicates no significant difference), while the hemolysis rate in the positive control group was close to 100% (p<0.001), indicating that PP7 hydrogel has excellent blood compatibility.

[0117] This invention successfully constructed a PAH / PMA composite hydrogel with ultra-high burst pressure (143.7 mmHg), strong wet adhesion, rapid hemostasis (superior to Celox™), and multifunctional integration by introducing polymaleic acid (PMA) as a key anionic component. This material demonstrates outstanding clinical application potential in high-pressure bleeding wounds, active sites, and infected wounds, providing a novel high-performance solution for the field of wound dressings.

[0118] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A wet-adhesive injectable hydrogel, characterized in that, The hydrogel is obtained by solid-liquid separation after mixing aqueous solutions of component A and component B; wherein, the aqueous solution of component B is a bioadhesive molecular aqueous solution that can crosslink with component A and tissue interface, and the aqueous solutions of component A and component B are respectively an aqueous solution of positively charged polyacrylamide hydrochloride and an aqueous solution of negatively charged polymaleic acid.

2. The wet-adhesive injectable hydrogel according to claim 1, characterized in that, The volume ratio of the aqueous solution of component A to the aqueous solution of component B is 1:

1.

3. The wet-adhesive injectable hydrogel according to claim 1, characterized in that, The molar ratio of polyacrylamide hydrochloride to polymaleic acid is (1~4):(1~4).

4. The wet-adhesive injectable hydrogel according to claim 1, characterized in that, The pH of both the aqueous solutions of component A and component B is adjusted to 6.0-8.0 before mixing.

5. A method for preparing a wet-adhesive injectable hydrogel, characterized in that, Includes the following steps: Polyacrylamine hydrochloride and polymaleic acid were dissolved in deionized water and adjusted to the same pH value to obtain aqueous solutions of polyacrylamine hydrochloride and polymaleic acid, respectively. Aqueous solutions of polymaleic acid are added to aqueous solutions of polyacrylamide hydrochloride at a preset rate, and the mixture is stirred to allow liquid-liquid phase separation, resulting in a mixed solution that forms a white emulsion-like condensed phase. The above mixed solution was centrifuged to remove the supernatant, and the lower precipitate was collected and washed with PBS buffer to obtain the hydrogel.

6. The method for preparing a wet-adhesive injectable hydrogel according to claim 5, characterized in that, The pH values ​​of both the polyacrylamide hydrochloride aqueous solution and the polymaleic acid aqueous solution were adjusted to 6.0-8.

0.

7. The method for preparing a wet-adhesive injectable hydrogel according to claim 6, characterized in that, The pH values ​​of both the polyacrylamide hydrochloride aqueous solution and the polymaleic acid aqueous solution were adjusted to 7.

8. The method for preparing a wet-adhesive injectable hydrogel according to claim 5, characterized in that, The concentration of solute in the polyacrylamide hydrochloride aqueous solution is 50~300 g / L, and the concentration of solute in the polymaleic acid aqueous solution is 30~200 g / L.

9. The method for preparing a wet-adhesive injectable hydrogel according to claim 5, characterized in that, The preset speed is 1~5 mL / min; the stirring speed is 300~500 rpm; the centrifugation conditions are 6000~10000 rpm for 5~15 min; and the PBS buffer soaking and washing time is 10~14 h.

10. The application of the hydrogel according to any one of claims 1-4 or the hydrogel prepared by the preparation method according to any one of claims 5-9 in the preparation of wound dressings, characterized in that, The wound dressing is used to close tissue wounds, stop bleeding, or prevent infection. The tissue wounds include arterial injury wounds, irregular wounds, or wounds in mobile areas.

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