Hemostatic antibacterial injectable multi-network hydrogel as well as preparation method and application thereof

Abstract

The invention provides hemostatic antibacterial injectable multi-network hydrogel as well as a preparation method and application thereof. The hydrogel is prepared from ColMA, HA-NB, CSMA and a photoinitiator LAP through photo-crosslinking. ColMA has a fibrous structure and is interwoven with an HA-NB branched chain structure to enhance the mechanical strength, and groups interact to regulate the microenvironment and resist bacteria; the HA-NB branched chain structure increases the space filling property and optimizes the property of the hydrogel; the CSMA modified group acts with other components to stabilize the structure, resist bacteria and regulate the microenvironment. The three components cooperate to form a multi-network system, and the effects of hemostasis, antibiosis, wound healing promotion, biocompatibility and the like are remarkable. The hydrogel disclosed by the invention can be used for preparing a reagent for improving hemostasis and antibiosis capabilities and promoting wound healing in medical application, has a wide application prospect and provides a new solution for skin wound treatment.

Description

[0001] This application claims priority to the prior application in China, application number: 202411940471.0, and filing date December 25, 2024; the specification, claims, abstract and set of drawings of that application are all cited as part of this application. Technical Field

[0002] The invention belongs to the technical field of biomaterials, and in particular relates to a hemostatic and antibacterial injectable multi-network hydrogel and a preparation method and application thereof. Background Art

[0003] At present, skin trauma is relatively common in daily life, which can be caused by a variety of reasons, such as accidental scratches, burns, surgical incisions, chronic ulcers, etc. Skin trauma not only causes symptoms such as pain and bleeding, but may also lead to serious complications due to problems such as wound infection and poor healing, affecting the patient's quality of life and even threatening life and health.

[0004] Wound infection is an important challenge in the healing process of skin wounds, which is mainly caused by the invasion of bacteria and other microorganisms into the wound. Common pathogens include Staphylococcus aureus, Escherichia coli, etc. These bacteria multiply in the wound, which will trigger an inflammatory response, causing wound redness and swelling, increased pain, and increased exudate, thereby delaying the wound healing process. Traditional treatment methods include wound cleaning, disinfection, and the use of antibiotics, but there are certain limitations. For example, the irrational use of antibiotics may lead to the emergence of drug-resistant bacteria.

[0005] In the field of skin wound treatment, injectable hydrogels have attracted widespread attention as a new type of wound dressing. They can fill irregular wounds, provide a moist healing environment, and promote cell proliferation and tissue regeneration. However, many existing injectable hydrogels still have deficiencies in antibacterial properties, hemostatic effects, tissue repair capabilities, and biocompatibility. For example, some hydrogels have a limited antibacterial spectrum and are difficult to effectively inhibit a variety of pathogens; some hydrogels have weak hemostatic abilities and cannot quickly control bleeding; some hydrogels have unsatisfactory mechanical properties and cannot adapt to the dynamic changes of the wound site; and some hydrogels may have biocompatibility issues and have adverse effects on surrounding tissues.

[0006] Therefore, there is an urgent need to find an injectable hydrogel with good antibacterial properties, excellent hemostatic effect, strong tissue repair ability, excellent biocompatibility, controllable photocrosslinking and good mechanical properties, which can effectively inhibit bacterial growth, quickly stop hemorrhage, promote cell proliferation and tissue regeneration, etc., thereby providing a more effective solution for the treatment of skin trauma, reducing the occurrence of complications and accelerating wound healing. Summary of the invention

[0007] In order to solve the problems existing in the prior art, the present invention provides a hemostatic and antibacterial injectable multi-network hydrogel and its preparation method and application, wherein the hydrogel is prepared by reacting a specific ratio of compounds ColMA, HA-NB, CSMA and a photoinitiator LAP through a photocrosslinking reaction. Among them, ColMA is prepared by reacting type I collagen and methacrylic anhydride in a specific ratio, and its unique fibrous structure is formed by orderly arrangement of type I collagen molecules, which not only provides attachment sites for cells to facilitate cell activity, but also interweaves with the HA-NB branched structure to enhance the mechanical strength of the hydrogel, and the interaction between its chemical groups can regulate the hydrogel microenvironment and exert an antibacterial effect; the branched molecular structure of HA-NB can increase the spatial filling of the hydrogel, interact with components such as ColMA to enhance the integrity of the network, optimize the physicochemical properties of the hydrogel, help maintain a moist environment on the wound surface, and promote cell metabolism and tissue regeneration; CSMA is based on a modified chitosan molecular chain, and interacts with other components through modified groups to stabilize the hydrogel structure, adjust the surface charge to achieve antibacterial function, and can also optimize the pore structure and hydration performance, providing a favorable microenvironment for cell growth. This multiple network structural design enables the hydrogel to have good biocompatibility, hemostatic and antibacterial properties, controllable photocrosslinking and degradation properties. It has broad application prospects in various tissue repair scenarios and can effectively promote wound healing and reduce complications.

[0008] In order to achieve the above object, the present invention adopts the following scheme:

[0009] In one aspect, the present invention provides an injectable multi-network hydrogel, wherein the hydrogel comprises compounds ColMA, HA-NB, CSMA and a photoinitiator LAP, and is prepared by photocrosslinking; the injectable multi-network hydrogel has the following chemical formula:

[0010] ;

[0011] Wherein, R1=(glycine-XY) n , R1 includes an amino group, the number of amino groups is an integer ≥3, X or Y is specifically a combination of any one or more of proline, hydroxyproline, lysine, hydroxylysine, methionine, serine, arginine, leucine, alanine, glutamine, phenylalanine or glutamic acid; n, a, b, c, d, e, f, g are integers including 0, 1, 2, 3, ...

[0012] In the field of skin trauma treatment, due to the many limitations of traditional wound treatment methods and existing injectable hydrogels, the present invention is committed to preparing a hemostatic and antibacterial injectable multi-network hydrogel. In daily life, skin trauma is extremely common, and its causes include accidental injuries, surgery, burns, etc., which not only cause pain and bleeding, but also easily cause complications such as infection and poor healing, seriously affecting the quality of life of patients. Although traditional treatment methods such as cleaning, disinfection and antibiotic use have certain effects, the abuse of antibiotics can easily lead to the production of drug-resistant bacteria. In terms of antibacterial properties, some existing injectable hydrogels have limited antibacterial spectra, making it difficult to resist the combined invasion of multiple pathogens and unable to create a safe and sterile environment for wounds; in terms of hemostatic effects, some hydrogels have slow hemostasis speed and low efficiency, making it difficult to quickly control bleeding in emergency situations, increasing the risk of hemorrhagic shock; in terms of tissue repair ability, some hydrogels cannot provide effective support for cell proliferation and tissue regeneration, resulting in a slow healing process; in terms of biocompatibility, some hydrogels have the potential to induce immune responses or stimulate surrounding tissues, which is not conducive to long-term implantation and repair.

[0013] The hemostatic and antibacterial injectable multi-network hydrogel prepared by the present invention is prepared by the coordinated synthesis of ColMA, HA-NB and CSMA.

[0014] ColMA is prepared by the reaction of type I collagen and methacrylic anhydride, and its unique fibrous structure originates from the orderly arrangement of type I collagen molecules. This structure provides excellent attachment sites for cells, which effectively promotes the adhesion, proliferation and differentiation of cells. In the hydrogel system, ColMA exhibits a variety of connection characteristics. It can not only connect with HA-NB through specific chemical reactions, such as under ultraviolet light irradiation, the groups formed by HA-NB can react with the amino groups on ColMA to produce Schiff bases to achieve cross-linking; it can also cross-link with CSMA, and the two can form a stable chemical bond connection under ultraviolet light conditions. In addition, ColMA itself also has the ability to cross-link. The intermolecular force enables the fiber structure formed by the orderly arrangement of type I collagen molecules to further cross-link with each other, building a more stable structural foundation, which plays a key role in enhancing the mechanical strength of the hydrogel, enabling the hydrogel to effectively maintain structural stability when subjected to force, and providing physical guidance similar to the extracellular matrix for cell migration and tissue regeneration. At the same time, the amino and other active groups in ColMA interact chemically with the functional groups of CSMA, such as by electrostatic attraction and hydrogen bonding, which can regulate the internal charge distribution and microenvironment of the hydrogel and enhance the structural stability. These active groups may also participate in the antibacterial process and interfere with the permeability and metabolism of bacterial cell membranes.

[0015] HA-NB presents a complex branched molecular structure with rich and diverse branched functional groups. Its branched structure can significantly improve the spatial filling capacity of the hydrogel, promote the formation of a dense network of the hydrogel, and effectively enhance the liquid absorption and retention capacity, thereby creating a suitable moist environment for the wound surface, which is beneficial to cell metabolism and tissue regeneration. In terms of cross-linking, the branched functional group NB of HA-NB can form a Schiff base with ColMA amino or skin amino to strengthen the integrity of the hydrogel network; it can also interact with the CSMA group to regulate the swelling performance and ion exchange capacity of the hydrogel, optimize the adaptability to the physiological environment, and bring better spatial structural characteristics and bioactivity regulation capabilities to the hydrogel.

[0016] CSMA is constructed based on modified chitosan molecular chains, and its molecular chain modification groups interact with other components in a variety of ways. When it interacts with ColMA, it can enhance the overall stability of the hydrogel, and at the same time adjust the surface charge properties of the hydrogel to make it positively charged so that it can interact with the negatively charged bacterial cell membrane, destroy the integrity of the cell membrane, and exert an antibacterial effect. When combined with HA-NB, it can regulate the pore structure and hydration properties of the hydrogel, affect the diffusion rate of nutrients and metabolic waste, create a favorable microenvironment for cell growth, and enrich the functional properties of the hydrogel in terms of antibacterial and microenvironment regulation. CSMA itself also has cross-linking ability, and certain groups on its molecular chain can interact under certain conditions to form a stable cross-linked structure, further enhancing the stability and functionality of the hydrogel.

[0017] When HA-NB, ColMA and CSMA work together, a highly ordered and interconnected complex multi-network system is formed. In terms of hemostasis, ColMA promotes platelet aggregation, HA-NB maintains the stability of the local environment, and CSMA activates coagulation factors, which work together to achieve rapid and effective hemostasis with lasting effects; in terms of antibacterial properties, the antibacterial ability of ColMA, the destruction of bacterial cell membranes by CSMA, and the unfavorable bacterial growth microenvironment created by HA-NB form a multi-faceted antibacterial system to enhance antibacterial properties; in terms of biocompatibility, ColMA provides cell attachment sites, HA-NB ensures spatial structure and moisture retention, and CSMA regulates biological activity, which together create an ideal growth microenvironment for cells and promote cell adhesion, proliferation and normal metabolism; in terms of mechanical properties, the ColMA fiber structure is interwoven with the HA-NB branched structure, combined with the chemical interaction with CSMA, to enhance the overall mechanical strength of the hydrogel, so that it can better adapt to the dynamic changes of the wound surface and provide long-term and effective support for wound healing.

[0018] At the same time, due to the presence of a variety of cross-linking combinations between the components, the hemostatic and antibacterial injectable multi-network hydrogel prepared by the present invention can present a variety of different multi-network structures. Figure 3One of the structural forms of the hydrogel of the present invention is shown, in which ColMA, HA-NB and CSMA interact with each other in a specific connection mode to form a relatively stable network architecture with specific functions. However, in actual situations, with the changes in the proportion of each component, reaction conditions and other factors, the cross-linking mode between the three components will be different, thereby producing different multiple network structure variants, and these variants may also have different performances in hemostasis, antibacterial, promotion of wound healing, etc. But in general, the multiple network structure formed by the present invention comprehensively improves the performance of the hydrogel in hemostasis, antibacterial, promotion of wound healing, etc., so that it shows great application potential in the field of skin wound repair.

[0019] Furthermore, the preparation of ColMA includes type I collagen and methacrylic anhydride, and the ratio of type I collagen to methacrylic anhydride is (1 g: 0.5 mL) to (1 g: 6 mL).

[0020] Furthermore, the ratio of type I collagen to methacrylic anhydride is 1 g:2.5 mL.

[0021] In some embodiments, in order to further improve the comprehensive performance of the hydrogel, the ratio of type I collagen and methacrylic anhydride in the preparation of ColMA was optimized and screened. Through a series of optimization experiments, other conditions were kept unchanged and the ratio of the two was gradually changed. The experimental results show that when the ratio of type I collagen to methacrylic anhydride is in the range of (1g: 0.5mL) to (1g: 6mL), the reaction activity and stability of the obtained ColMA product are relatively good. Within this range, the degree of chemical modification of ColMA is moderate, which can ensure the introduction of sufficient photo-crosslinkable groups without destroying the original structure and biological activity of collagen due to excessive modification. When the ratio is 1g: 2.5mL, the prepared ColMA has the best performance. At this time, ColMA exhibits good cross-linking ability in the subsequent hydrogel synthesis, and can effectively form a stable network structure with HA-NB and CSMA. The obtained hydrogel has high mechanical strength, and its Young's modulus is about 20%-30% higher than that of hydrogels prepared under other ratio conditions, while maintaining good biocompatibility. When cells are cultured on the surface of the hydrogel containing this ratio of ColMA, the cell survival rate can reach more than 95%, and the cell morphology is normal and the proliferation rate is fast. Therefore, it can be seen that in the preparation process of ColMA, it is crucial to accurately control the ratio of type I collagen to methacrylic anhydride to 1g:2.5mL for the construction of high-performance hemostatic and antibacterial injectable multi-network hydrogels. This ratio enables ColMA to give full play to its advantages in structural support, cell affinity and synergy with other components in the hydrogel system, thereby laying a solid foundation for improving the overall performance of the hydrogel.

[0022] Furthermore, in the hydrogel, the mass ratio of ColMA, HA-NB and CSMA is (1-5):(1-10):(1-10).

[0023] Preferably, in the hydrogel, the mass ratio of ColMA, HA-NB and CSMA is 1:1:1.

[0024] In some embodiments, in order to further enhance the comprehensive performance of the hydrogel, especially to achieve a better balance in terms of hemostasis, biocompatibility and mechanical properties, the ratio of ColMA, HA-NB and CSMA in the preparation of the present invention is optimized and screened. The experimental results show that when the mass ratio of ColMA, HA-NB and CSMA is in the range of (1-5): (1-10): (1-10), the hydrogel exhibits certain performance advantages, good injectability, is not easy to clog the needle during the injection process, and can be quickly formed in the body after injection; its rheological properties are stable, and the storage modulus and loss modulus are in an appropriate range, which can provide a certain support for the wound surface; the antibacterial properties are also guaranteed to a certain extent, and have a certain inhibitory effect on common bacteria. When the mass ratio is 1:1:1, the performance of the hydrogel reaches the best. From the perspective of hemostasis effect, the hydrogel prepared at this time can activate coagulation factors more quickly, form a more stable blood clot, significantly shorten the hemostasis time, and reduce the amount of bleeding. In terms of biocompatibility, the hydrogel at this ratio provides the most suitable microenvironment for cells, the cell proliferation rate is significantly improved, the cell survival rate is as high as 95%-100%, and the cell morphology is normal and the function is active. In terms of mechanical properties, its Young's modulus reaches 40-50kPa, and a tighter and more ordered network structure is formed inside the hydrogel. The synergy between the components is the strongest, which can better resist external force deformation and provide a stable mechanical environment for wound repair. Therefore, it can be seen that 1:1:1 is the optimal mass ratio of ColMA, HA-NB and CSMA in the preparation of the multi-network hydrogel of the present invention. At this ratio, the components cooperate with each other and give full play to their respective advantages, so that the hydrogel shows excellent performance in the hemostatic and antibacterial injectable multi-network hydrogel system, providing a solid material basis for its biomedical applications such as wound repair.

[0025] Furthermore, the mass fraction of the photoinitiator is 0.5-1%.

[0026] In another aspect, the present invention provides a method for preparing an injectable multi-network hydrogel, comprising the following steps:

[0027] (1) Preparation of ColMA: Type I collagen and methacrylic anhydride were mixed in a ratio of (1 g: 0.5 mL) to (1 g: 6 mL), stirred overnight, dialyzed for 1 week, and freeze-dried and stored at -20°C;

[0028] (2) Preparation of hydrogel precursor solution: HA-NB, ColMA and CSMA were mixed in a mass ratio of (1-5):(1-10):(1-10), and LAP was added to prepare a 1% hydrogel precursor solution;

[0029] (3) Preparation of hydrogel: A portion of the precursor solution was taken and slowly added dropwise into deionized water. The deionized water containing the precursor solution was placed under 365 nm ultraviolet light, and the hydrogel precursor solution was injected into the water at a slow and steady rate to prepare an injectable multi-network hydrogel.

[0030] Furthermore, in the step (2), the mass fraction of the photoinitiator is 0.5-1%.

[0031] Furthermore, in the preparation of the hydrogel precursor solution in step (2), the reaction time of HA-NB, ColMA and CSMA is 0.1-5 minutes.

[0032] In some embodiments, in order to accurately control the structure and performance of the hydrogel and ensure that it plays the best effect in hemostasis, antibacterial, and wound healing, the reaction time of HA-NB, ColMA, and CSMA in the preparation of the hydrogel precursor solution is also screened and optimized. The experimental results show that when the reaction time is in the range of 0.1-5 minutes, the various properties of the hydrogel are relatively stable and can meet the basic application requirements. Within this time range, the gelation process of the hydrogel is relatively complete, the network structure gradually tends to be perfect, and its physical properties such as elasticity and toughness gradually reach a good level. At the same time, the chemical stability can also be guaranteed, and it is not easy to have problems such as premature degradation or incomplete cross-linking. When the reaction time of HA-NB, ColMA, and CSMA is preferably 0.5-3 minutes, the performance of the hydrogel finally obtained is optimal. At this time, the hydrogel has the best controllable photo-crosslinking property, and it can quickly take shape under ultraviolet light, with a molding time of only 5-8 seconds, and the structure of the formed hydrogel is uniform and stable; its antibacterial property is the strongest, and the inhibitory effect on common pathogens such as Staphylococcus aureus is significantly enhanced; in the wound repair experiment, it can effectively promote wound healing, and the wound healing rate can reach 99%-100% on the 14th day, which is significantly better than hydrogels prepared under other reaction times.

[0033] In another aspect, the present invention provides a use of an injectable multi-network hydrogel for preparing an agent for improving hemostatic and antibacterial capabilities, wherein the hydrogel comprises the injectable multi-network hydrogel as described in any one of the above technical solutions.

[0034] In some embodiments, a comparative study of in vitro coagulation experiment and antibacterial experiment was conducted to verify that the injectable multi-network hydrogel of the present invention has the excellent effect of significantly improving the hemostatic and antibacterial ability, and has important clinical application value. In the in vitro coagulation experiment, the results show that the hydrogel of the present invention can promote the formation of stable blood clots in a short time, and its coagulation time is significantly shortened compared with other different groups of hydrogels and control groups, and the amount of bleeding is also the least, indicating that the hydrogel of the present invention has excellent performance in promoting coagulation, which is due to its unique multi-network structure, wherein ColMA can promote platelet aggregation, HA-NB maintains local environmental stability, CSMA activates coagulation factors, and the synergistic effect of the three accelerates the hemostasis process. In terms of antibacterial experiments, the best hydrogel of the present invention and other different groups of hydrogels and control groups are co-cultured with common pathogens (such as Staphylococcus aureus, Escherichia coli, etc.), and the experimental results show that the best hydrogel prepared by the present invention has a strong inhibitory effect on the growth of various pathogens, and the bacterial growth rate is significantly slowed down, the number of colonies is greatly reduced, and the integrity of the bacterial cell membrane is destroyed, and deformation, death and other phenomena occur. This is because the CSMA in the hydrogel of the present invention can adjust the surface charge to destroy the bacterial cell membrane, and the three components synergistically create a microenvironment that is not conducive to bacterial growth, thereby effectively inhibiting the growth and reproduction of bacteria. These experiments fully demonstrate the excellent performance of the injectable multi-network hydrogel of the present invention in improving the hemostatic and antibacterial ability, and it is expected to become a key component of a new generation of highly effective hemostatic and antibacterial agents, providing strong support for controlling bleeding and preventing infection in clinical treatment.

[0035] In another aspect, the present invention provides a use of an injectable multi-network hydrogel for preparing an agent for promoting wound healing, wherein the hydrogel comprises the injectable multi-network hydrogel as described in any one of the above technical solutions.

[0036] In some embodiments, an animal wound model experiment was conducted, including making a full-thickness skin defect wound model on the back of rats, and randomly grouping the experimental animals, and treating them with the hydrogel of the present invention, the remaining different groups of hydrogels, and a blank control. At different time points after the wound treatment, the wound healing was observed. The results showed that the wound healing speed treated with the hydrogel of the present invention was significantly accelerated, the inflammatory reaction was mild in the early stage, the epithelialization process was accelerated in the middle stage, the collagen fiber deposition increased and the arrangement was more orderly in the late stage, and the new blood vessels were abundant. Compared with other groups, the wound healing time was significantly shortened, the histological structure was closer to normal skin tissue, the cytokine expression was more conducive to tissue repair, and the number of new blood vessels was more and the distribution was more reasonable. Therefore, it is verified that the injectable multi-network hydrogel of the present invention has an excellent effect of significantly promoting wound healing, can play an important role in the wound repair process, and provides an effective solution for clinical wound treatment.

[0037] The beneficial effects of the present invention are:

[0038] 1. The present invention provides a hemostatic and antibacterial injectable multi-network hydrogel and a preparation method and application thereof. The hydrogel (1) has excellent hemostatic properties. In the liver hemostasis experiment, the CHC group hydrogel can effectively control bleeding within 2-3 minutes, which is significantly better than other groups. It can quickly form a stable gel structure to seal the wound, promote the activation of coagulation factors and platelet aggregation, effectively reduce the amount of bleeding, and reduce the risk of complications such as hemorrhagic shock. It has obvious advantages in clinical trauma hemostasis; (2) It has excellent antibacterial ability, has a strong inhibitory effect on common pathogens such as Staphylococcus aureus, and the bacterial survival rate is as low as 20%. Its multi-network structure can create a microenvironment that is unfavorable for bacterial growth. CSMA can destroy the integrity of bacterial cell membranes, effectively reduce the risk of infection, and has a significant effect in preventing and treating wound infections; (3) It has good biocompatibility and is compatible with fibroblasts It is non-cytotoxic during co-culture and can promote cell proliferation. The cell proliferation rate is significantly improved, and the cell survival rate is as high as 98%-100%, providing an ideal growth environment for cells, facilitating cell adhesion, proliferation and differentiation, and promoting the wound healing process; (4) It has excellent mechanical properties, with a Young's modulus of 40-50 kPa, which can effectively resist deformation caused by external forces. It has a strong and stable structure and can adapt to complex mechanical environments in the body, such as joint motion sites or wound areas frequently subjected to external forces, providing long-term and stable mechanical support for wound repair; (5) It has controllable photocrosslinking and good injectability. It can be quickly formed within 8 seconds under ultraviolet light irradiation. The structure is uniform and stable. It can be precisely formed on demand at the wound site by precise injection of the precursor solution and local irradiation with ultraviolet light. It is easy to operate and can be injected into the wound with minimal invasion, alleviating the patient's pain and meeting the material requirements for wound repair applications.

[0039] 2. In the preparation method of a hemostatic and antibacterial injectable multi-network hydrogel provided by the present invention, by optimizing and screening the ratio of type I collagen and methacrylic anhydride in the preparation of ColMA, the mass ratio of each component in the hydrogel precursor solution and the reaction time, it is ensured that the components fully exert their synergistic effect, and a hydrogel with excellent comprehensive performance is obtained, thereby improving the product quality and performance stability, and providing a reliable technical guarantee for large-scale production.

[0040] 3. The hemostatic and antibacterial injectable multi-network hydrogel provided by the present invention can be used to prepare reagents for improving hemostatic and antibacterial capabilities in medical applications. It performs well in in vitro coagulation and antibacterial experiments and is expected to become a key component of a new generation of highly efficient hemostatic and antibacterial reagents, providing strong support for clinical treatment of bleeding and infection, improving treatment effects, and improving patient prognosis. At the same time, it can be used to prepare reagents for promoting wound healing. In animal wound model experiments, it significantly accelerates wound healing, reduces inflammatory reactions, promotes epithelialization, angiogenesis, and collagen fiber deposition, provides an effective solution for clinical wound treatment, shortens healing time, and promotes tissue repair to near normal skin tissue, with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 A is the verification and molding process of the controllable photocrosslinking and injectability of the hydrogel of the present invention.

[0042] Figure 1 B is a physical picture of the hydrogel of the present invention.

[0043] Figure 2 These are the structural formulas of ColMA, HA-NB, and CSMA in the present invention.

[0044] Figure 3 This is the structural formula of the hemostatic and antibacterial injectable multi-network hydrogel of the present invention.

[0045] Figure 4 This is a graph showing the rheological properties test results of the hydrogel of the present invention.

[0046] Figure 5 This is a graph showing the mechanical properties test results of different hydrogels of the present invention.

[0047] Figure 6 A shows the live-dead staining of fibroblasts after co-culture with different hydrogels.

[0048] Figure 6 B is the quantitative analysis of CCK-8 experiment after fibroblasts were co-cultured with different hydrogels.

[0049] Figure 7 A shows the live-dead staining of Staphylococcus aureus after co-culture with different hydrogels.

[0050] Figure 7 B is the quantitative analysis of bacterial absorbance after co-culture of Staphylococcus aureus with different hydrogels.

[0051] Figure 8 A is a real picture of liver hemostasis of hydrogels in different groups.

[0052] Figure 8 B is the quantitative analysis of the amount of bleeding after hemostasis by hydrogels in different groups.

[0053] Fig. 9 The figure compares the healing status of infected wounds in different groups of rats at 0, 3, 7, 11 and 14 days after wound treatment. DETAILED DESCRIPTION

[0054] The present invention will be further described in detail below in conjunction with the examples. It should be pointed out that the examples described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on the present invention.

[0055] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0056] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0057] Example 1 Preparation of hemostatic and antibacterial injectable multi-network hydrogel

[0058] Skin trauma is a common clinical problem, caused by a variety of factors such as accidental injuries, surgical incisions, burns, etc., often accompanied by bleeding and infection risks, which seriously affect the patient's quality of life and may cause complications. Traditional wound treatment methods have certain limitations, such as unsatisfactory hemostatic effects, limited antibacterial ability, slow tissue repair, etc. Therefore, the development of a new type of injectable hydrogel with hemostatic, antibacterial and tissue repair promoting functions has important clinical significance. This embodiment aims to provide an optimal preparation method for a hemostatic and antibacterial injectable multi-network hydrogel and to conduct an in-depth study of its performance. The specific preparation process is as follows:

[0059] 1. Preparation of ColMA

[0060] Type I collagen and methacrylic anhydride (MA) were mixed evenly in an optimal ratio of 1 g: 2.5 mL, stirred overnight at 4 ° C, and dialyzed with distilled water for 1 week at 4 ° C using a dialysis tube with a molecular weight cutoff of 8-14 kDa to remove excess MA, and then freeze-dried and stored at -20 ° C. For details of the preparation process, please refer to the literature "Qin Zhang, Qiaomei Tang, Yuwei Yang, Junzhi Yi, Wei Wei, Yi Hong, Xianzhu Zhang, Feifei Zhou, Xudong Yao, Hongwei Ouyang. Wound dressing gel with resisted bacterial penetration and enhanced re-epithelization for corneal epithelial-stromal regeneration. Applied Materials Today, 2021, 24: 101119." ColMA itself has the potential to form multiple network structures, and its unique molecular structure lays the foundation for the subsequent construction of more complex hydrogel networks.

[0061] 2. Preparation of hydrogel precursor solution

[0062] HA-NB (Haining Jurassic Biotechnology Co., Ltd.) and CSMA (Yongchiquan Intelligent Equipment Co., Ltd.) were purchased respectively. HA-NB, ColMA prepared in step 1 and CSMA were mixed according to a specific optimal ratio. In this embodiment, the mass ratio of HA-NB:ColMA:CSMA was 1:1:1. During the mixing process, 0.5-1% (mass fraction) of the photoinitiator LAP was gradually added to prepare a hydrogel precursor solution with a concentration of 1% (CHC group). At the same time, in order to conduct comparative studies in subsequent experiments, the ColMA group (1% ColMA solution) and the CH group (1% ColMA and HA-NB solution) were prepared as control groups.

[0063] 3. Preparation of hemostatic and antibacterial injectable multi-network hydrogel

[0064] A portion of the precursor solution was taken up with a pipette, specifically 200 μL in this embodiment, and slowly added to the pre-prepared deionized water. The deionized water containing the precursor solution was placed under 365 nm ultraviolet light, and the hydrogel precursor solution was injected into the water at a slow and steady rate using a microsyringe. At this time, it can be observed that under the irradiation of ultraviolet light, the hydrogel gradually takes shape (e.g. Figure 1In addition, using the same method, a portion of the precursor solution was sucked into the mold with a pipette, and after irradiation with 365nm ultraviolet light for 30s, the mold was quickly removed, and it can also be seen that the hydrogel has been successfully formed ( Figure 1 B).

[0065] The structural formulas of ColMA, HA-NB and CSMA are as follows: Figure 2 As shown. For ColMA, it is mainly composed of glycine and two other amino acids, R1 = (glycine-XY) n , X or Y includes any one or more combinations of proline, hydroxyproline, lysine, hydroxylysine, methionine, serine, arginine, leucine, alanine, glutamine, phenylalanine or glutamic acid. Figure 2 In the above, R includes any one or more of H or 2-methylpropylene-carbonyl. m and n are integers including 0, 1, 2, 3, ..., respectively.

[0066] The structure of the injectable hemostatic and antibacterial multi-network hydrogel is as follows: Figure 3 As shown. It is composed of three components: ColMA, HA-NB, and CSMA. In the present invention, a specific structural formula is shown, but in fact, based on the chemical structural characteristics of these three components, there are many possible connection methods. Figure 3 In addition to the connection form shown in , there are other feasible connection modes: for example, CSMA may not be connected to the NB group, but may be connected to ColMA near f to form a new network architecture; or, ColMA may not be connected to the NB group, but may be connected to CSMA near g to form a new network architecture; or, based on the existing connection position (the existing structure is that CSMA and ColMA are connected to the NB group respectively, CSMA is grafted in the upper left corner through the NB group, and ColMA is grafted in the upper right corner through the NB group), the connection positions of CSMA and ColMA can be exchanged with each other, thereby producing different structural variants. These different connection methods may affect the performance and function of the hydrogel, and the structural formula shown in the present invention is one of the representative structural forms, which provides an important basis for the subsequent research and application of the hydrogel.

[0067] In forming the present invention Figure 3 When the hydrogel is shown, the structure of ColMA needs to further satisfy the following specific conditions: R1 = (glycine-XY) n , R1 includes an amino group, and the number of amino groups is an integer ≥3. In this embodiment, X or Y is specifically a combination of any one or more of lysine, arginine or glutamine, and n, a, b, c, d, e, f, and g are integers including 0, 1, 2, 3, ... respectively.

[0068] The hydrogel prepared by the present invention has many significant advantages. First, its unique multiple network structure is the key to integrating multiple excellent properties. By cleverly combining the three components of hyaluronic acid modified NB group (HA-NB), type I collagen modified MA group (ColMA) and chitosan modified MA group (CSMA), the synergistic enhancement of multiple functions such as antibacterial, hemostasis and promotion of wound healing is achieved. Compared with traditional hydrogels, in terms of biocompatibility, since all natural polymer components are used, the immunogenicity risk is greatly reduced, the biosafety is improved, and it is conducive to long-term implantation in the body for wound repair. In terms of antibacterial performance, the presence of CSMA ensures a good antibacterial effect, and will not produce drug resistance problems like traditional antibiotic antibacterial materials, providing a reliable guarantee for long-term control of wound infection. In terms of hemostasis and promotion of wound healing, the synergistic effect of HA-NB and ColMA provides a more comprehensive and efficient solution. The combination of the adhesion performance of HA-NB and the tissue regeneration promotion ability of ColMA can not only stop bleeding quickly, but also provide continuous support for cell activity and tissue reconstruction throughout the healing process.

[0069] In summary, this embodiment successfully prepared an injectable multi-network hydrogel with excellent hemostatic and antibacterial performance through a specific raw material ratio and processing method, laying a solid foundation for further developing efficient and multifunctional wound repair materials and promoting their wide application in the clinical medical field.

[0070] Example 2 Material Characterization of the Hemostatic and Antibacterial Injectable Multi-Network Hydrogel of the Present Invention

[0071] In this example, the material properties of the hemostatic and antibacterial injectable multi-network hydrogel prepared in Example 1 were verified, including controllable photocrosslinking, injectability, rheology and mechanical properties, to determine whether the hydrogel prepared by the present invention meets the material requirements in the biomedical field, especially in wound repair applications.

[0072] 1. Controllable photocrosslinking and injectability testing

[0073] A portion of the precursor solution was taken up with a pipette and placed in deionized water. Under 365 nm ultraviolet light, the hydrogel precursor solution was slowly introduced into the water ( Figure 1 A), the hydrogel was formed after irradiation with UV light, and not formed without UV light. In addition, a portion of the precursor solution was taken up with a pipette, irradiated with 365nm UV light for 30s, and then the mold was removed. It can be seen that the hydrogel has been formed, with a complete morphology and a stable structure. It can be seen that the hydrogel has good controllable photocrosslinking and injectability ( Figure 1 B).

[0074] 2. Rheological properties test

[0075] The hydrogel precursor solution was evenly placed on a rheometer (MCR302, Anton Paar, Austria), and the rheometer was started to perform a scanning test on the hydrogel precursor solution. The change curves of the hydrogel storage modulus (G') and loss modulus (G") with time before and after the UV light was turned on were recorded. Figure 4 As shown, from Figure 4 It can be seen from the rheological behavior curve that after the ultraviolet light is turned on, the storage modulus (G') of the hydrogel rises rapidly and exceeds the loss modulus (G") in a short time, which indicates that the hydrogel begins to form a stable network structure and the solution gradually changes from liquid to solid. After gelation, both G' and G" can be maintained at a relatively stable level, indicating that the hydrogel has solid-like mechanical properties after molding and can withstand certain external forces without obvious deformation.

[0076] 3. Mechanical properties test

[0077] By using a nanoindenter (MCT 3 , Anton Paar, Austria) tested the mechanical properties of the hydrogel and recorded its Young's modulus. The specific results are as follows Figure 5 As shown, from Figure 5 It can be seen that with the addition of HA-NB and CSMA, the mechanical strength of the hydrogel shows a trend of gradual enhancement, indicating that the ability of the hydrogel to resist deformation by external force is improved, and its structure is more solid and stable.

[0078] Therefore, it can be known that the hemostatic and antibacterial injectable multi-network hydrogel prepared by the present invention has excellent material properties. Its good controllable photocrosslinking property enables the hydrogel to accurately control the occurrence of crosslinking reaction under specific ultraviolet light conditions, and realize on-demand molding, which is of great significance in the repair application of complex-shaped wounds. For example, when repairing irregular skin trauma or deep tissue damage, the hydrogel can be accurately molded at the wound site by accurately injecting the precursor solution and performing local ultraviolet light irradiation to provide fitting repair support. Injectability ensures the convenience of operation of the hydrogel in clinical applications, and it can be injected into the wound site in a minimally invasive manner to reduce the pain of patients. In terms of rheological properties, the solid-like properties after gelation enable it to provide stable support for the wound, prevent the wound from being deformed due to external pressure or its own activities during the healing process, and is conducive to the growth, proliferation of tissue cells and reconstruction of the extracellular matrix. The mechanical properties can be effectively enhanced by adding HA-NB and CSMA. This property enables the hydrogel to better adapt to the complex mechanical environment in the body, such as in the joint movement area or the wound area frequently subjected to external forces, and still maintain its structural integrity and continue to play its hemostatic, antibacterial and tissue repair functions. Therefore, the hydrogel fully meets the various requirements of materials science as an injectable multi-network hydrogel, and provides a solid material foundation for its further application in the biomedical field, especially in the field of wound repair.

[0079] Example 3 Cytological characterization of the hemostatic and antibacterial injectable multi-network hydrogel of the present invention

[0080] In this example, the cytological properties of the hemostatic and antibacterial injectable multi-network hydrogel prepared in Example 1 were deeply evaluated, specifically including precise verification of its biocompatibility and antibacterial properties, so as to clarify the performance of the hydrogel in the process of interaction with cells and bacteria, and provide a cytological basis for its application in the biomedical field.

[0081] 1. Biocompatibility testing

[0082] Select a fibroblast cell line, inoculate it in a culture dish containing complete culture medium, and culture it in a cell culture incubator at 37°C and 5% CO2 to ensure that the cells are in the logarithmic growth phase to ensure the reliability of the experimental results. After the cells grow to an appropriate density, use trypsin to digest and collect the cells, resuspend the cells in culture medium, and set aside.

[0083] The collected fibroblasts were inoculated into culture plates containing three different hydrogels (CHC group, ColMA group, CH group), and a control group without hydrogel was set up. Multiple replicate wells were set up in each group to reduce experimental errors. The culture plates were placed in a cell culture incubator for continued culture. During the culture process, the cell growth status was regularly observed.

[0084] After the co-culture, CCK-8 kit and AM / PI stain were used to detect the proliferation ability and activity of cells. Figure 6 A and Figure 6 As shown in B, Figure 6 A and Figure 6 As shown in Figure B, compared with the control group, the cell activity of fibroblasts remained good and the morphology was normal after co-culture with the three hydrogels for days, indicating that the best hydrogel prepared by the present invention has no cytotoxicity. At the same time, the CKK-8 experiment showed that compared with the control group, the cell proliferation curves of the three hydrogel co-culture groups all showed an upward trend, and the cell proliferation rate of the CHC group was relatively faster, indicating that the hydrogel can not only maintain the normal activity of fibroblasts, but also has the ability to promote cell proliferation, which is of great significance for the regeneration and repair of tissues in the wound healing process; and as time goes by, the cell proliferation performance is significantly improved.

[0085] 2. Antibacterial test

[0086] The suspension of Staphylococcus aureus (Beijing Sanyao Technology Co., Ltd.) was co-cultured and mixed with the three hydrogels, placed in a sterile culture container, and incubated for 24 hours at 37°C and 220 rpm in a constant temperature shaker to allow the bacteria to fully contact the hydrogel. At the same time, a bacterial control group without hydrogel was set up to ensure the consistency of experimental conditions.

[0087] The bacterial activity was detected by AM / PI staining. The bacterial staining was observed under a fluorescence microscope. Live bacteria showed green fluorescence, and dead bacteria showed red fluorescence. The bacterial activity was qualitatively evaluated by observing the distribution and number of bacteria of different colors.

[0088] A portion of the bacterial solution was taken for quantitative absorbance analysis. The absorbance value of the bacterial solution was measured at a specific wavelength of 600 nm, and the number of remaining live bacteria after co-culture was calculated based on the pre-drawn bacterial concentration and absorbance standard curve, thereby quantitatively evaluating the antibacterial properties of the hydrogel.

[0089] Specific results such as Figure 7 A and Figure 7 As shown in B, Figure 7 As shown in Figure A, compared with the control group, in the bacterial samples co-cultured with the hydrogel, the number of dead bacteria with red fluorescence increased significantly, and the number of live bacteria with green fluorescence decreased significantly, indicating that the hydrogel has a significant inhibitory effect on the growth of bacteria and can effectively destroy the integrity of the bacterial cell membrane, leading to bacterial death. Figure 7 As shown in B, compared with the control group, the absorbance value of the bacterial solution after co-culture was significantly reduced, and the number of remaining live bacteria calculated according to the standard curve was significantly reduced, further indicating that the hydrogel can effectively reduce the survival rate of bacteria and has a good antibacterial effect.

[0090] Therefore, it can be seen that the hemostatic antibacterial injectable multi-network hydrogel of the present invention shows excellent performance in cytological characterization. In terms of biocompatibility, the hydrogel has no cytotoxicity to fibroblasts and can promote cell proliferation, which provides a strong guarantee for its good interaction with surrounding tissue cells in the wound repair process. As a key cell type in the wound healing process, the maintenance of its activity and the promotion of its proliferation help to accelerate the synthesis of collagen and the reconstruction of the extracellular matrix, thereby promoting the wound healing process. In terms of antibacterial properties, both qualitative AM / PI staining observation and quantitative absorbance analysis show that the hydrogel has a significant antibacterial effect and can effectively inhibit the growth and reproduction of bacteria. This antibacterial property is crucial for preventing and treating wound infection, because wound infection is one of the main factors affecting wound healing, and reducing the number and activity of bacteria can reduce the risk of infection and create a favorable microenvironment for wound healing. Therefore, based on the cytological characterization results of the present embodiment, the hydrogel prepared by the present invention has broad application prospects in the biomedical field, especially in skin wound repair, and is expected to become a kind of efficient and safe wound repair material.

[0091] Example 4 Biological Characterization of the Hemostatic and Antibacterial Injectable Multi-Network Hydrogel of the Present Invention

[0092] In this example, the biological properties of the hemostatic and antibacterial injectable multi-network hydrogel prepared in Example 1 in vivo were deeply evaluated, including precise verification of its hemostatic performance and wound repair effect, thereby providing a solid experimental basis for the application of the hydrogel in clinical trauma treatment and evaluating its potential to promote wound healing and restore tissue function under actual physiological conditions.

[0093] 1. Hemostasis performance test

[0094] Twelve healthy adult SD rats were selected and randomly divided into four groups, namely CHC group, ColMA group, CH group and control group. The rats were anesthetized with anesthetics to ensure that the rats were in a painless state and their vital signs were stable during the experiment. A razor blade was used to make a wound of about 1 cm in the rat liver, and three types of hydrogels were used to stop bleeding at the incision. The control group did not receive any treatment. After hydrogel treatment, the bleeding of the wound was carefully observed and the time when the bleeding stopped was recorded. The specific results are as follows Figure 8 A and Figure 8B. Combined with actual observations, it was found that the bleeding stop time of the wounds treated with hydrogels in the CHC group, ColMA group and CH group was significantly shorter than that in the control group. Among them, the CHC group had the fastest hemostasis speed, and the bleeding could be effectively controlled within 2-3 minutes. The CH group and ColMA group also showed good hemostasis effects. In contrast, the bleeding duration of the control group was longer, indicating that the hydrogel has a significant hemostatic effect (such as Figure 8 As shown in A, it can be seen that the blood exudation from the wound was reduced after the hydrogel treatment). Through precise measurement and statistical analysis of the amount of bleeding, the results showed that the CHC group and the CH group had the least bleeding, which was significantly different from the control group (P<0.01). The bleeding amount of the ColMA group was also significantly lower than that of the control group (P<0.05). This further confirms that the hydrogel can effectively reduce the amount of bleeding from liver wounds and has excellent hemostatic properties ( Figure 8 B).

[0095] 2. Wound repair effect test

[0096] A wound with a diameter of about 1 cm was made on the back of SD rats, and Staphylococcus aureus (200 μL, 10 8 CFU / mL) were used to make the infected wound model. The next day, the rats were randomly divided into the control group, ColMA group, CH group and CHC group according to the different treatments, with the same number of rats in each group.

[0097] According to the grouping, different hydrogels (ColMA group, CH group, CHC group) were injected into the wound surface of the corresponding group of rats and irradiated with ultraviolet light to form gel. The control group did not use hydrogel (no treatment was performed).

[0098] At specific time intervals after wound treatment (e.g., 0, 3, 7, 11, and 14 days), the wounds of rats were observed and evaluated. The observations included changes in wound size, wound healing (e.g., whether there was scab, degree of epithelialization, etc.), degree of inflammatory response of tissues around the wound (e.g., redness, swelling, exudation, etc.), and whether there were signs of recurrence of infection.

[0099] Specific results such as Fig. 9As shown, during the wound repair process, regular observations found that the wound healing speed of the CHC group, ColMA group and CH group was significantly faster than that of the control group. The wound of the CHC group began to show obvious scab and epithelialization in the early stage (such as 7-11 days), and the wound area gradually shrank; over time (11-14 days), the wound of the CHC group was basically healed, the epithelial tissue was intact and smooth, and the inflammatory reaction of the surrounding tissue was mild. Although the wound healing of the ColMA group and the CH group was inferior to that of the CHC group, it was also significantly better than that of the control group. The wound healing of the control group was relatively slow, and some of them were still not completely healed at 14 days, and the inflammatory reaction around the wound was more obvious, which further proved that the optimal hydrogel prepared by the present invention can effectively promote the repair of infected wounds.

[0100] Therefore, it can be seen that the hemostatic and antibacterial injectable multi-network hydrogel of the present invention exhibits excellent performance in biological characterization. In terms of hemostatic performance, the hydrogel can significantly shorten the bleeding time of liver wounds and reduce the amount of bleeding. Its hemostatic effect is better than that of traditional treatment methods or control groups, which is of great significance for clinical trauma hemostasis, especially in emergency situations or during surgery. It can effectively control bleeding and reduce the risk of complications such as hemorrhagic shock. In terms of wound repair effect, the healing of the wound appearance shows that the hydrogel can effectively promote the repair of infected wounds. It can accelerate the epithelialization process of the wound, promote angiogenesis and collagen fiber deposition, and at the same time reduce inflammatory reactions, creating a good microenvironment for wound healing. On the whole, the advantages of the hydrogel in biological properties give it broad prospects in clinical applications, and it is expected to become an ideal wound repair material, providing strong support for improving the effect of wound treatment and improving patient prognosis.

[0101] Example 5 Screening of injectable multi-network hydrogels constructed with other different groups

[0102] In order to verify that the hemostatic and antibacterial injectable multi-network hydrogel constructed by the present invention has the best effect, in this embodiment, different hydrogels are constructed respectively using a single component, a two-component and other similar combinations, and their various properties are comprehensively tested and compared and analyzed. The specific operation is as follows (in this embodiment, except for the different components for preparing the hydrogel, the other preparation conditions are the same as the optimal conditions in Example 1):

[0103] 1. Preparation of different hydrogels

[0104] 1. Single-component hydrogels: Hydrogels containing only ColMA, HA-NB, or CSMA were prepared and labeled as ColMA group, HA-NB group, and CSMA group, respectively.

[0105] 2. Two-component hydrogel: ColMA and HA-NB mixed hydrogel (CH group), ColMA and CSMA mixed hydrogel (CC group), and HA-NB and CSMA mixed hydrogel (HC group) were accurately prepared.

[0106] 3. The best multi-network hydrogel of the present invention: a hydrogel containing three components of HA-NB, ColMA and CSMA prepared according to the method of Example 1 (CHC group).

[0107] 4. Comparative combination hydrogels: Sodium alginate (SAMA), a natural polymer commonly used in the biomedical field with good biocompatibility, was selected to prepare a hydrogel (HCS group) composed of HA-NB, CSMA and SAMA.

[0108] 2. Performance Testing and Result Analysis

[0109] Using the different hydrogels prepared above, the controllable photocrosslinking, injectability, rheological properties, mechanical properties, antibacterial properties, hemostatic properties, and wound repair effects of the different hydrogels were tested according to the steps in Examples 2 to 4, and the specific results are as follows:

[0110] 1. Controllable photocrosslinking and injectability testing

[0111] The experimental results show that the ColMA group can be formed under ultraviolet light, but the forming speed is relatively slow, and it takes an average of about 15-20 seconds to be completely formed. The structural stability of the formed hydrogel is general and it is easy to deform under slight external force. The fluidity of its precursor solution during injection is acceptable, but occasionally a small amount of flocs will clog the needle. When the HA-NB group is used alone, the ultraviolet cross-linking effect is poor and the forming is difficult. Even if the ultraviolet light exposure time is extended to 60 seconds, it is still difficult to form a complete and stable shape. The injectability is poor, and the solution easily clogs the needle during injection. The forming effect of the CSMA group alone is also not ideal. The texture of the formed hydrogel is brittle and easy to break. Its precursor solution is relatively smooth during injection, but the structural integrity of the formed hydrogel is poor.

[0112] Among the two-component hydrogels, the moldability and injectability of the CH and CC groups were improved compared with the single component. The molding time of the CH group was shortened to about 10-15 seconds, and the structural stability of the hydrogel after molding was improved, but it was still not as good as the CHC group. The CH group had better structural stability after molding, but the molding speed was slightly slower than the CC group, which took about 12-18 seconds. The molding speed and stability of the HC group were between the HA-NB group and the CHC group, with a molding time of about 15-20 seconds, and the hydrogel structure after molding was relatively brittle.

[0113] The CHC group hydrogel showed the best controllable photo-crosslinking and injectability. It was quickly formed under ultraviolet light, taking only 5-8 seconds on average. The formed hydrogel structure was uniform and stable, with good elasticity. It could be easily injected into molds simulating complex wound shapes and maintaining shape integrity.

[0114] The molding speed of the HCS group was similar to that of the CHC group, with an average molding time of about 8 seconds. The structural strength of the hydrogel after molding was worse than that of the CHC group. This may be because SAMA lacks amino groups and cannot undergo Schiff base reaction with HA-NB, resulting in the inability to effectively construct a stable multiple network structure, thereby affecting the photocrosslinking and injection performance.

[0115] 2. Rheological properties testing

[0116] The experimental results show that after the UV light is turned on, the G' rise rate of the ColMA group is relatively slow, and the equilibrium G' value is finally reached after about 15-20 seconds, and the G' value is low, indicating that the hydrogel network structure formed is not tight enough and the mechanical strength is limited. When the HA-NB group is alone, the changes in G' and G" are extremely small, and it is almost impossible to form an effective solid-like state during the entire test process, indicating that it is difficult to construct a stable hydrogel network alone. Although the G' rise of the CSMA group is slightly better than that of the HA-NB group, it is still weaker than that of the CHC group, and the stability after gelation is poor, and there are certain fluctuations in G' and G".

[0117] In the two-component hydrogels, the rheological properties of the CH and CC groups were improved, and the G' rise rate and equilibrium value were better than the corresponding single components, but still inferior to the CHC group. The CH group reached the final equilibrium G' value about 15 seconds after the UV light was turned on, which was improved compared with the ColMA group, but still inferior to the CHC group. The CC group had relatively good stability after gelation, but the G' rise rate and final value were not as good as the CHC group. The G' rise and stability performance of the HC group were between the HA-NB group and the CHC group.

[0118] After the CHC group hydrogel was turned on by ultraviolet light, G' could rise rapidly and exceed G" within 5-8 seconds. After gelation, both were extremely stable with almost no fluctuation during long-term testing, showing ideal solid properties, indicating that it has excellent mechanical support capabilities.

[0119] The rheological curve of the HCS group showed that its G' rose slightly slower than that of the CHC group, indicating that the gelation performance was not as good as that of the CHC group. It could form a hydrogel structure, but its strength was slightly weaker and could not provide reliable mechanical support for the wound. This is because SAMA lacks amino groups and cannot interact with HA-NB and CSMA, resulting in the inability to effectively build a stable multiple network structure.

[0120] 3. Mechanical properties testing

[0121] The experimental results show that the ColMA group has a relatively low Young's modulus of about 10-16 kPa, limited mechanical strength, and can deform by 30%-40% when subjected to a pressure of 5 mN, and cannot effectively maintain its shape and structure. The HA-NB group and the CSMA group have weaker mechanical properties, with Young's moduli of about 5-10 kPa and 8-15 kPa, respectively, and larger deformations of about 40%-50% and 35%-45%, respectively.

[0122] Although the mechanical strength of the two-component hydrogel was improved, the Young's modulus of the CH and CC groups were about 16-25 kPa and 18-30 kPa, respectively, and the deformation was 25%-35% and 20%-30% under 5 mN pressure, respectively, but it was still significantly lower than that of the CHC group. The Young's modulus of the HC group was about 10-20 kPa, and the deformation was about 30%-40%.

[0123] The mechanical strength of the CHC group hydrogel was significantly enhanced after adding HA-NB and CSMA, and its Young's modulus could reach 40-50kPa. The deformation under the same pressure was only 10%-20%, which was able to resist deformation due to external forces and had a stronger structure. This is because the three components work together to form a more complex and stable multi-network structure, which enhances the interaction force inside the hydrogel.

[0124] The Young's modulus of the HCS group was about 20-30 kPa, which was lower than that of the CHC group. In the mechanical property test, it showed poor compressive resistance and deformation as high as 20%-30%. This may be because there is no amino group in SAMA, which cannot form a close and effective multi-network structure with HA-NB and CSMA like ColMA, thus affecting the mechanical properties.

[0125] 4. Antibacterial performance testing

[0126] The experimental results showed that the ColMA group had a certain antibacterial ability, with a bacterial survival rate of about 50%-60%, the HA-NB group had a weaker antibacterial effect, with a bacterial survival rate of about 30%-40%, and the CSMA group had relatively good antibacterial performance, with a bacterial survival rate of about 15%-20%.

[0127] In the two-component hydrogel, the antibacterial properties of CC and HC groups were better than those of the single component, with bacterial survival rates of approximately 25%-35% and 25%-35%, respectively, but still lower than those of CHC group. The bacterial survival rate of CH group was approximately 45%-55%.

[0128] The CHC hydrogel had the strongest inhibitory effect on Staphylococcus aureus, and the bacterial survival rate was significantly reduced to 15%-20%, indicating that the chitosan and multiple network structures in the CHC hydrogel can effectively inhibit bacterial growth and reproduction. This may be because the three components work together to form a microenvironment that is unfavorable to bacterial growth, such as changing the permeability of the cell membrane, destroying the integrity of the cell wall, changing the normal internal structure of the cell or the physical barrier function, etc., which limits the growth and spread of bacteria.

[0129] The antibacterial performance of the HCS group was slightly weaker than that of the CHC group, with a bacterial survival rate of approximately 20%-30%. The addition of SAMA may have a certain impact on the strength of the hydrogel and the overall structural stability, thereby reducing the overall antibacterial effect.

[0130] 5. Hemostatic performance test

[0131] The experimental results showed that the hemostatic effect of ColMA, HA-NB and CSMA groups was limited when used alone. The bleeding stopped in ColMA group for about 5-7 minutes, and the amount of bleeding was about 500-700 mg; the hemostatic effect of HA-NB group was not obvious, the bleeding stopped in about 9-11 minutes, and the amount of bleeding was about 1000-1300 mg; although the CSMA group had some hemostatic ability, it was not as good as the CHC group, the bleeding stopped in about 4-6 minutes, and the amount of bleeding was about 450-600 mg.

[0132] The hemostatic effect of the two-component hydrogel was improved, with the bleeding stopping time of CH group and HC group being about 3-5 minutes and 3-4 minutes, and the bleeding volume being about 300-500 mg and 250-400 mg, respectively, but still inferior to that of CHC group. The bleeding stopping time of CC group was about 4-6 minutes, and the bleeding volume was about 400-550 mg.

[0133] The CHC group hydrogel can effectively control bleeding in the shortest time, the bleeding stops in about 2-3 minutes, and the amount of bleeding is about 150-300 mg. Its hemostatic effect is significantly better than that of other groups. This may be due to the synergistic effect of the three components. On the one hand, it quickly forms a stable gel structure to seal the wound, and on the other hand, HA-NB has an adhesive effect that can react with the amino group on the wound surface to further enhance the adhesion, thereby accelerating the hemostatic process.

[0134] The hemostatic performance of the HCS group was slightly worse, with a bleeding stop time of about 3-4 minutes and a bleeding volume of about 200-350 mg, which was higher than that of the CHC group. This may be because the presence of SAMA affected the strength of the hydrogel and the effectiveness of the hemostatic mechanism.

[0135] 6. Wound repair effect detection

[0136] The experimental results showed that the wound healing speed of the ColMA group, HA-NB group and CSMA group was slow. On the third day, the wound healing rate of the ColMA group was about 20%-30%, the HA-NB group was about 10%-25%, and the CSMA group was about 20%-35%; on the 11th day, the healing rate of the ColMA group was about 70%-80%, the HA-NB group was about 60%-70%, and the CSMA group was about 75%-85%.

[0137] The wound healing of the two-component hydrogel was better than that of the single-component hydrogel. The wound healing rates of the CH and CC groups were approximately 30%-40% and 33%-38% on the third day, and approximately 75%-85% and 70%-80% on the 11th day, respectively, but still inferior to the CHC group. The healing rates of the HC group were approximately 35%-43% on the third day and approximately 75%-85% on the 11th day.

[0138] The wounds treated with hydrogel in the CHC group showed a faster epithelialization process in the early stage, and the inflammatory response was milder. The wound healing rate was about 50%-60% on the 3rd day, about 90%-95% on the 11th day, and up to 99%-100% on the 14th day. Histological observation of the wound showed abundant neovascularization, dense and orderly deposition of collagen fibers, and good epithelial tissue regeneration. This may be because the three components work together, with good adhesion to reduce wound bleeding, and the unique three-dimensional structure provides effective mechanical support, providing a favorable microenvironment for cell migration, proliferation and tissue regeneration. At the same time, its antibacterial properties help control infection and reduce the interference of inflammation on wound healing.

[0139] The wound healing effect of the HCS group was significantly worse than that of the CHC group. The wound healing rate was about 40%-50% on the 3rd day and about 80%-90% on the 14th day. The wound inflammatory reaction was more severe, and the formation of new blood vessels and collagen fibers was less. This may be because the addition of SAMA may have changed the biological properties of the hydrogel, resulting in a lack of effective support for cell migration and adhesion, and unable to effectively promote wound repair.

[0140] Through the above comprehensive performance testing and comparative analysis of the hydrogels constructed with different components, it can be seen that the CHC group hydrogel containing the three components of HA-NB, ColMA and CSMA of the present invention shows significant advantages in controllable photocrosslinking, injectability, rheological properties, mechanical properties, biocompatibility, antibacterial properties, hemostatic properties and wound repair effects. Although the single-component and two-component hydrogels have certain performances in various properties, the overall effect is obviously not as good as the CHC group. When ColMA is replaced with SAMA to form the HCS group hydrogel, its various properties are also poor. This shows that only when the three components of HA-NB, ColMA and CSMA are synergistically combined can an injectable multi-network hydrogel with optimal performance be constructed. The reason may be that there is a unique interaction between the three components. ColMA, as the skeleton of the hydrogel, provides support and foundation for the overall structure; HA-NB not only provides good adhesion, but its chemical changes during the photocrosslinking process can also interact with other components to further enhance the network structure; CSMA plays an antibacterial role, and its synergistic effect with other components helps to optimize the physical and chemical properties of the hydrogel. However, the combination of SAMA with HA-NB and CSMA failed to form a similar effective synergistic effect, thus affecting the overall performance of the hydrogel.

[0141] At the same time, when the three components of HA-NB, ColMA and CSMA are used synergistically to prepare the hydrogel of the present invention, the prepared hydrogel has a multiple network structure, in which each component plays a unique and synergistic role.

[0142] Among them, ColMA has a unique fibrous structure, which is formed by the orderly arrangement of type I collagen molecules through intermolecular forces. Type I collagen itself has good biocompatibility and biodegradability, providing cells with suitable attachment sites, which is conducive to cell adhesion, proliferation and differentiation. In the hydrogel system, on the one hand, the carbon-carbon double bonds of ColMA and CSMA can form cross-links under ultraviolet light; on the other hand, HA-NB forms aldehyde groups under ultraviolet light, which can react with the amino groups on ColMA and CSMA to produce Schiff bases to form cross-links. The three components interact to form a physical support framework with multiple networks, which enhances the overall mechanical strength of the hydrogel. This combination not only enables the hydrogel to maintain a stable structure when subjected to external forces and avoid excessive deformation or rupture, but also provides physical guidance similar to the extracellular matrix for cell migration and tissue regeneration, promoting cell activity during wound healing. At the same time, there are chemical interactions between the active groups such as amino groups in ColMA and the functional groups in CSMA, such as electrostatic attraction and hydrogen bonding. This interaction helps to regulate the charge distribution and microenvironment inside the hydrogel, further enhancing the stability of the hydrogel structure, and may be involved in the inhibition of bacterial growth, exerting antibacterial effects by affecting the permeability of bacterial cell membranes or interfering with their metabolic processes. ColMA itself has a multi-network structure based on a collagen fiber network, which, when working in synergy with other components, becomes an important basis for building a stable and functional network for the entire hydrogel.

[0143] HA-NB has a complex branched molecular structure, and the rich functional groups on its branches give it a variety of properties. On the one hand, the branched structure of HA-NB can increase the spatial filling capacity of the hydrogel system, forming a denser network structure, thereby enhancing the hydrogel's ability to absorb and retain liquids, which helps maintain a moist environment on the wound surface during wound repair and is beneficial to cell metabolism and tissue regeneration. On the other hand, specific functional groups on the HA-NB branches (such as aldehyde groups, etc.) can form hydrogen bonds and other interactions with amino groups in ColMA or amino groups on the skin, strengthening the connection between the two and improving the integrity of the hydrogel network. At the same time, these functional groups may also interact with amino groups in CSMA, jointly affecting the physicochemical properties of the hydrogel, such as regulating the swelling properties and ion exchange capacity of the hydrogel, thereby optimizing its adaptability in the physiological environment. HA-NB's own unique branched multi-network structure, when synergistically acting with ColMA and CSMA, brings better spatial structural characteristics and bioactivity regulation capabilities to the hydrogel.

[0144] CSMA has a modified chitosan molecular structure, and the polysaccharide structure of chitosan itself provides it with certain biological activity and biodegradability. In the hydrogel, CSMA interacts with other components through the modified groups on its molecular chain. For example, the interaction between CSMA and ColMA enhances the overall stability of the hydrogel. At the same time, the presence of CSMA can adjust the surface charge properties of the hydrogel, making it have a certain positive charge, which is conducive to interacting with the negatively charged bacterial cell membrane and destroying the integrity of the bacterial cell membrane, thereby exerting an antibacterial effect. In addition, the combination of CSMA and HA-NB can regulate the pore structure and hydration properties of the hydrogel, affect the diffusion rate of nutrients and metabolic waste, and provide a more favorable microenvironment for cell growth. CSMA itself is based on the multiple network structure of the modified chitosan molecular chain. When it is used to construct a hydrogel with HA-NB and ColMA, it further enriches the functional properties of the hydrogel, especially in terms of antibacterial and microenvironment regulation.

[0145] When the three components are used to prepare the hydrogel of the present invention in a coordinated manner, a highly ordered and interrelated complex multiple network system is formed. This system further improves the various functions and effects of the hydrogel. In terms of hemostasis, the synergistic effect of each component can quickly aggregate platelets and activate coagulation factors, while forming a stable gel structure to block blood vessel damage and accelerate the hemostasis process; in terms of antibacterial, through the synergistic effect of multiple antibacterial mechanisms, such as physical barriers, charge effects, and chemical inhibition, the growth and reproduction of bacteria are inhibited in all directions, effectively reducing the risk of infection; in terms of wound repair, it provides cells with a suitable microenvironment for growth, migration, and differentiation, promotes angiogenesis, collagen fiber deposition, and epithelial tissue regeneration, and significantly improves the speed and quality of wound healing. At the same time, the multiple network structure also enhances the mechanical properties and stability of the hydrogel, enabling it to better adapt to the dynamic changes of the wound site and provide long-term and effective support for wound healing.

[0146] Therefore, it can be known that the best synergistic effect can only be achieved when the three components of HA-NB, ColMA and CSMA of the present invention are synergistically prepared into the hemostatic and antibacterial injectable multi-network hydrogel of the present invention. In terms of hemostasis, ColMA contributes to platelet aggregation, HA-NB provides wound adhesion hemostasis, and CSMA promotes the activation of coagulation factors. The three work together to quickly and effectively stop bleeding with lasting effects; in terms of antibacterial properties, the certain antibacterial ability of ColMA, the destruction of bacterial cell membranes by CSMA, and the unfavorable bacterial growth microenvironment synergistically created by HA-NB form a multi-faceted antibacterial system to enhance antibacterial properties; in terms of biocompatibility, ColMA provides cell attachment sites, HA-NB guarantees spatial structure and moisture retention, and CSMA regulates biological activity, which together create an ideal growth microenvironment for cells and promote cell adhesion, proliferation and normal metabolism; in terms of mechanical properties, the fiber structure of ColMA and the branched chain structure of HA-NB are intertwined, and the chemical interaction with CSMA enhances the overall mechanical strength of the hydrogel, enabling it to better adapt to the dynamic changes of the wound surface and provide long-term and effective support for wound healing.

[0147] Example 6 Screening of reaction conditions for synthesizing the hemostatic and antibacterial injectable multi-network hydrogel of the present invention

[0148] In order to obtain the best preparation method in Example 1, a large number of screening experiments were conducted in this example to screen and optimize the reaction conditions and reagents in the synthesis process of ColMA and the final hydrogel, which is crucial for obtaining a hemostatic and antibacterial injectable multi-network hydrogel with excellent performance. Different reaction conditions and reagent ratios will significantly affect the structure and properties of the hydrogel. Only through careful screening can we ensure that each component fully exerts its synergistic effect and achieve the ideal comprehensive effects of hemostasis, antibacterial, biocompatibility and physical properties. The specific screening process is as follows (in this example, the best preparation method is the same as in Example 1):

[0149] 1. Optimization of the ratio of type I collagen and methacrylic anhydride (MA) in the preparation of ColMA:

[0150] In the preparation process of ColMA, the ratio of type I collagen and MA is one of the key factors. Through a series of preliminary experiments, other conditions were kept unchanged and the ratio of the two was changed. It was found that when the ratio of type I collagen to MA was in the range of (1g: 0.5mL) to (1g: 6mL), the reaction activity and stability of the obtained ColMA product were relatively good. Within this range, the degree of chemical modification of ColMA was moderate, which could not only ensure the introduction of sufficient photo-crosslinkable groups, but also would not destroy the original structure and biological activity of collagen due to excessive modification. And when the ratio of type I collagen to MA was 1g: 2.5mL, the prepared ColMA had the best performance. At this time, ColMA showed good cross-linking ability in the subsequent hydrogel synthesis and could effectively form a stable network structure with HA-NB and CSMA. The obtained hydrogel has high mechanical strength, and its Young's modulus is about 20%-30% higher than that of hydrogels prepared under other proportions, while maintaining good biocompatibility. When cells are cultured on the surface of the hydrogel containing this proportion of ColMA, the cell survival rate can reach more than 95%, and the cell morphology is normal and the proliferation rate is faster.

[0151] 2. Screening and optimization of the mass ratio of HA-NB, ColMA and CSMA in the preparation of hydrogel precursor solution:

[0152] In this embodiment, extensive screening experiments were conducted on the mass ratios of the three main components HA-NB, ColMA and CSMA in the hydrogel precursor solution. By changing the mass ratios of the three, multiple groups of hydrogel samples were prepared and their performance was tested. The results show that when the mass ratio of ColMA:HA-NB:CSMA is in the range of (1-5):(1-10):(1-10), the hydrogel can exhibit good comprehensive performance. Within the mass ratio range, the hydrogel has good injectability, is not easy to clog the needle during injection, and can be quickly formed in the body after injection; at the same time, its rheological properties are stable, and the storage modulus and loss modulus are in an appropriate range, which can provide a certain support for the wound surface; the antibacterial properties are also guaranteed to a certain extent, and it has a certain inhibitory effect on common bacteria. When the mass ratio of ColMA:HA-NB:CSMA is 1:1:1, the hydrogel prepared at this time has the best effect, its hemostatic effect is significantly improved, and its biocompatibility is further improved. The cell proliferation rate can reach 200%-250% after 5 days of culture, and the cell survival rate is as high as 95%-100%; the mechanical properties are also optimal, with a Young's modulus of 40-50kPa, which can better resist external deformation and provide a stable mechanical environment for wound repair.

[0153] 3. Optimization of reaction time of HA-NB, ColMA and CSMA in preparation of hydrogel precursor solution:

[0154] The reaction time has an important influence on the structure formation and performance of the hydrogel of the present invention. While keeping other reaction conditions consistent, different reaction times are set to prepare the hydrogel. The experiment found that when the reaction time is within the range of 0.1-5 minutes, the various properties of the hydrogel are relatively stable and can meet the basic application requirements. Within the reaction time range, the gelation process of the hydrogel is relatively complete, the network structure gradually tends to be perfect, and its physical properties such as elasticity and toughness gradually reach a good level. At the same time, the chemical stability can also be guaranteed, and it is not easy to have problems such as premature degradation or incomplete crosslinking. At the same time, when the reaction time of HA-NB, ColMA, and CSMA is preferably 0.5-3 minutes, the performance of the hydrogel finally obtained is the best. At this time, the controllable light crosslinking of the hydrogel is the best, and it is quickly formed under ultraviolet light irradiation. The molding time only takes 5-8 seconds, and the structure of the formed hydrogel is uniform and stable; its antibacterial property is the strongest, and in the wound repair experiment, it can effectively promote wound healing. The wound healing rate can reach 99%-100% on the 14th day, which is significantly better than the hydrogel prepared under other reaction times.

[0155] Therefore, it can be seen that the optimization method in this embodiment is the best method for preparing the hemostatic and antibacterial injectable multi-network hydrogel of the present invention, which significantly improves the performance of the hydrogel.

[0156] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An injectable multi-network hydrogel, characterized in that: The hydrogel comprises compounds ColMA, HA-NB, CSMA and photoinitiator LAP, and is prepared by photocrosslinking; the injectable multi-network hydrogel has the following chemical structure: ； Wherein, R1=(glycine-XY) n , R1 includes an amino group, the number of amino groups is an integer ≥3, X or Y is specifically a combination of any one or more of proline, hydroxyproline, lysine, hydroxylysine, methionine, serine, arginine, leucine, alanine, glutamine, phenylalanine or glutamic acid; n, a, b, c, d, e, f, g are integers including 0, 1, 2, 3, ...

2. The injectable multi-network hydrogel according to claim 1, characterized in that: The preparation of ColMA includes type I collagen and methacrylic anhydride, and the ratio of type I collagen to methacrylic anhydride is (1 g: 0.5 mL) to (1 g: 6 mL).

3. The injectable multi-network hydrogel according to claim 2, characterized in that: In the hydrogel, the mass ratio of ColMA, HA-NB and CSMA is (1-5):(1-10):(1-10).

4. The injectable multi-network hydrogel according to claim 3, characterized in that: In the hydrogel, the mass ratio of ColMA, HA-NB and CSMA is 1:1:

1.

5. The injectable multi-network hydrogel according to claim 4, characterized in that: The mass fraction of the photoinitiator is 0.5-1%.

6. A method for preparing an injectable multi-network hydrogel, characterized in that: The following steps are involved: (1) Preparation of ColMA: Type I collagen and methacrylic anhydride were mixed in a ratio of (1 g: 0.5 mL) to (1 g: 6 mL), stirred overnight, dialyzed for 1 week, and freeze-dried and stored at -20°C; (2) Preparation of hydrogel precursor solution: HA-NB, ColMA and CSMA were mixed according to (1-5): (1-10): The mass ratio of (1-10) was mixed, and LAP was added to prepare a 1% hydrogel precursor solution; (3) Preparation of hydrogel: A portion of the precursor solution was taken and slowly added dropwise into deionized water. The deionized water containing the precursor solution was placed under 365 nm ultraviolet light, and the hydrogel precursor solution was injected into the water at a slow and steady rate to prepare an injectable multi-network hydrogel.

7. The preparation method according to claim 6, characterized in that: In the step (2), the mass fraction of the photoinitiator is 0.5-1%.

8. The preparation method according to claim 7, characterized in that: In the step (2) of preparing the hydrogel precursor solution, the reaction time of HA-NB, ColMA and CSMA is 0.1-5 minutes.

9. Use of an injectable multi-network hydrogel for preparing an agent for improving hemostatic and antibacterial capabilities, characterized in that: The hydrogel comprises the injectable multi-network hydrogel according to any one of claims 1 to 5.

10. Use of an injectable multi-network hydrogel for preparing an agent for promoting wound healing, characterized in that: The hydrogel comprises the injectable multi-network hydrogel according to any one of claims 1 to 5.

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