A composite hydrogel with hemostatic and antibacterial functions and a preparation method and application thereof

Abstract

This invention relates to a composite hydrogel with hemostatic and antibacterial functions, its preparation method, and its application. The nanocomposite hydrogel is prepared by copolymerizing graphene oxide loaded with silver nanoparticles and chitosan grafted with dihydroporphyrin E6 during the preparation of thermosensitive poly(N-isopropylacrylamide) hydrogel. This composite hydrogel integrates multiple functions such as antibacterial, hemostatic, and cell proliferation and migration promotion. It possesses excellent thermosensitivity, injectability, swelling properties, tissue adhesion, and biodegradability. It achieves highly efficient antibacterial activity through photothermal-photodynamic synergy and multiple antibacterial components. Furthermore, it effectively controls bleeding by utilizing the sealing effect of the hydrogel and the hemostatic properties of chitosan, while simultaneously promoting fibroblast proliferation and migration. It plays a positive role in all stages of wound healing and is suitable for use as a dressing for infected skin wounds.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and relates to composite hydrogel dressings for skin wound repair, particularly an injectable composite hydrogel that integrates rapid hemostasis and intelligent antibacterial functions and can dynamically adapt to the needs of different stages of wound healing, as well as its preparation method and application in accelerating the healing of infected skin wounds. Background Technology

[0002] As the largest organ in the human body, the skin is the first line of defense against external pathogens and physical and chemical damage. When the skin is damaged due to trauma, surgery, or disease, the wound healing process begins. Ideal wound healing is a complex and orderly physiological process, typically including four coordinated phases: hemostasis, inflammation, proliferation, and remodeling. Hemostasis is the first step in wound healing; a moderate amount of inflammation is essential for normal wound recovery, but during the inflammatory phase, wounds are susceptible to bacterial infection, leading to excessive inflammation and oxidative stress, which significantly delays wound healing. The proliferation and remodeling phases mainly involve the proliferation and migration of fibroblasts and epithelial cells, wound contraction, and collagen formation.

[0003] Abnormalities at any stage of wound healing can affect wound repair, leading to infection, suppuration, ulceration, and even life-threatening situations. Uncontrolled bleeding and subsequent bacterial infection following trauma or surgery are a major cause of trauma mortality worldwide. Therefore, finding wound dressings that can quickly stop bleeding and effectively inhibit bacterial infection is of great significance for promoting skin wound healing.

[0004] For wound bleeding, traditional hemostatic materials such as gauze, gelatin, and sponges mainly work by physical compression and blood absorption. However, these materials have limited functions and often lack the ability to actively promote coagulation. They are not very effective in stopping active bleeding or severe oozing wounds. Moreover, they cannot prevent the evaporation of moisture from the wound surface, which can easily lead to wound dehydration. After drying, they stick to the wound surface and can cause secondary damage when removed. At the same time, most of these materials do not have antibacterial properties and may cause bacterial infection.

[0005] In recent years, novel hemostatic materials, represented by chitosan-based materials and fibrin adhesives, have emerged. Chitosan (CS), as a natural cationic polysaccharide, possesses excellent biodegradability, antibacterial properties, antioxidant activity, and other biological activities. It can also be used as a wound hemostatic agent, stopping bleeding by adsorbing negatively charged red blood cells and causing them to aggregate into blood clots. It is a promising medical material. However, although they exhibit advantages in biocompatibility and procoagulant activity, they generally suffer from insufficient mechanical properties, a mismatch between degradation kinetics and wound healing processes, and limited functionality, which restricts their wider and more effective application.

[0006] In controlling wound infections, the local or systemic application of antibiotics is a routine strategy. While traditional antibiotics are effective against classic wound pathogens such as Staphylococcus aureus, antibiotic overuse has led to increasing bacterial resistance and the emergence of superbugs, making infection control increasingly difficult. Therefore, developing antibiotic-free antimicrobial strategies, such as those utilizing silver ions, antimicrobial peptides, quaternary ammonium compounds, or photothermal / photodynamic therapy, has become a hot research topic.

[0007] Silver nanoparticles (AgNPs), as a nano-antibacterial agent, have been widely used in biomedical fields such as water purification, dental restoration materials, and antibacterial dressings. AgNPs have a strong inhibitory effect on a large number of microorganisms and drug-resistant bacteria, and do not induce bacterial resistance. Their antibacterial mechanism is mainly to induce bacterial death by destroying the bacterial cell membrane, nucleic acid and other structures.

[0008] In addition, photothermal antibacterial therapy (PTT) and photodynamic antibacterial therapy (PDT) are emerging photonano antibacterial treatment strategies in recent years, with advantages such as strong efficacy, low side effects, and broad-spectrum antibacterial activity. PTT utilizes the photothermal effect of photothermal transducers such as graphene oxide (GO) to absorb near-infrared light energy and convert it into heat energy, thereby killing bacteria through local high temperature. PDT can transfer light energy to surrounding oxygen molecules through photosensitizers such as dihydroporphyrin e6 (Ce6), generating highly destructive singlet oxygen for sterilization.

[0009] However, simply loading or mixing these antibacterial ingredients into dressings often presents challenges such as burst release effects, local toxicity, short duration of action, or poor compatibility with the wound environment.

[0010] Hydrogels possess a three-dimensional network structure similar to the extracellular matrix and exhibit high hydrophilicity. Due to their high water content, good biocompatibility, adjustable physicochemical properties, and permeability to water and gas, they are considered near-ideal wound dressing matrices. They provide a moist healing environment for wounds, promote cell migration and epithelial regeneration, and reduce pain during dressing changes, significantly compensating for the shortcomings of traditional dry dressings. Among them, poly(N-isopropylacrylamide) (PNIPAM) hydrogel, in addition to possessing general hydrogel properties, is also a temperature-sensitive polymer. When the temperature is below its low critical phase transition temperature (LCST) of 32°C, PNIPAM absorbs water and swells; while when the temperature exceeds the LCST, PNIPAM gradually dehydrates and shrinks. Utilizing its temperature sensitivity, PNIPAM hydrogel can be used as a drug carrier to achieve controlled delivery of different drugs.

[0011] However, wound healing is a dynamic, continuous, and multi-stage process, with significantly different functional requirements for dressings at different stages. In the early stages of healing, rapid and effective hemostasis and infection control are paramount. During the mid-to-late stages of inflammation and the proliferative phase, dressings need to gently manage exudate, reduce inflammation, and promote angiogenesis and granulation tissue formation. In the remodeling phase, dressings are expected to degrade moderately without hindering tissue reconstruction. Simple PNIPAM hydrogels only possess basic adhesion and hemostasis functions, which cannot meet the needs of different stages of wound healing. Therefore, further modification and alteration are needed to endow them with multiple functions that accelerate the healing of infected skin wounds, such as antibacterial properties, hemostasis, and promotion of fibroblast proliferation and migration.

[0012] Although some studies have attempted to integrate hemostasis and antibacterial functions into the same hydrogel system, most of these systems are simply a superposition of functions, lacking the "intelligence" to dynamically respond to the healing process. For example, the degradation rate and drug release behavior of many hydrogels cannot be well synchronized with the healing stage, which may lead to insufficient release in the early stage when strong antibacterial effects are needed, while continuous release in the later stage when a mild environment is needed to promote regeneration may cause potential toxicity. In addition, how to balance the properties such as rapid adhesion and platelet aggregation required for strong hemostasis with good biocompatibility and appropriate mechanical support required for later cell migration and tissue regeneration in hydrogels remains a technical challenge.

[0013] Therefore, there is an urgent need to develop a new type of functionalized hydrogel that not only possesses immediate and efficient hemostatic capabilities and broad-spectrum and long-lasting antibacterial properties to address the core challenges in the early stages of wound healing, but also, through ingenious design of its composition or structure, enables its physical properties such as degradability and mechanical strength, as well as its biological functions such as drug release and bioactivity, to adapt to or respond to changes in the microenvironment at different stages of wound healing. This would provide dynamic and appropriate support throughout the entire wound healing process, thereby accelerating and promoting high-quality healing of infected skin wounds. Summary of the Invention

[0014] The purpose of this invention is to provide a composite hydrogel with hemostatic and antibacterial functions, its preparation method, and its application, in order to solve the shortcomings of existing single-function hydrogel materials in managing complex wounds, which cannot simultaneously meet the multiple needs of rapid physical sealing, efficient antibacterial and healing promotion. By integrating multiple mechanisms such as physical hemostasis, chemical antibacterial and photodynamic enhancement, the functionalized composite hydrogel can meet the needs of different stages of wound healing and accelerate the healing of infected skin wounds.

[0015] To achieve the aforementioned objectives, this invention first provides a composite hydrogel with hemostatic and antibacterial functions. Using the thermosensitive hydrogel PNIPAM as a carrier, the nanocomposite hydrogel GACCP is ultimately obtained through the synthesis of PNIPAM via free radical polymerization, combining the functional components graphene oxide-silver nanoparticle composite (GO@AgNPs) and chitosan-dihydroporphyrin e6 composite (CS-Ce6). This composite hydrogel is essentially a three-dimensional porous material synergistically constructed from a thermosensitive polymer network, inorganic-organic nanocomposite antibacterial units, and bioactive macromolecules through physical entanglement and chemical cross-linking.

[0016] Specifically, in the composite hydrogel of the present invention, the mass ratio of PNIPAM to the graphene oxide-silver nanoparticle composite and the chitosan-dihydroporphyrin E6 composite is 100-500:1:1.

[0017] Furthermore, the mass ratio of PNIPAM to the graphene oxide-silver nanoparticle composite and the chitosan-dihydroporphyrin E6 composite is more preferably 200:1:1.

[0018] GO@AgNPs, as the main functional component in the composite hydrogel of this invention, is a nanocomposite material with excellent antibacterial properties, which can endow the composite hydrogel with long-lasting and efficient antibacterial function.

[0019] The GO@AgNPs can be prepared using conventional methods well-known to those skilled in the art, such as, but not limited to, in-situ chemical reduction, where GO is uniformly dispersed in an aqueous solvent to form a dispersion, and after adding a silver source, a reaction is carried out under heating and stirring conditions with reducing agents such as sodium citrate and sodium borohydride, so that Ag... + The GO@AgNPs were reduced to AgNPs and loaded in situ onto GO sheets, and then post-processed to obtain the GO@AgNPs powder.

[0020] It should be noted that the present invention does not impose any special restrictions on the source of GO@AgNPs or the specific preparation process parameters. Any existing technical method that can achieve stable loading of nano-silver on graphene oxide and has good antibacterial activity is applicable.

[0021] CS-Ce6 is another key functional component in this invention that imparts photodynamic antibacterial and film-forming functions to the composite hydrogel. It is formed by the chemical covalent linkage between Ce6 and CS.

[0022] The CS-Ce6 can be prepared using a mature coupling chemistry method in the field. The core of this method is to utilize the condensation reaction between the carboxyl group at the end of the Ce6 molecule and the amino group on the CS chain. A typical preparation method is to couple Ce6 dissolved in a suitable solvent with a CS solution in the presence of carboxyl activators such as EDC or NHS, and then obtain the product through standard purification steps.

[0023] This invention does not impose any limitations on the specific process conditions for achieving the above coupling reaction. Any existing synthetic route or equivalent commercially available product that can stably graft Ce6 onto the CS molecular chain via covalent bonds and obtain the aforementioned function is applicable to this invention.

[0024] Secondly, this invention also provides a method for preparing the composite hydrogel with hemostatic and antibacterial functions. Through a multi-step, ordered physicochemical process, the thermosensitive polymer network PNIPAM, the functional nanocomposite material GO@AgNPs, and the functionalized biomacromolecule CS-Ce6 are integrated to construct a multifunctional three-dimensional composite hydrogel system that combines rapid hemostasis, long-lasting antibacterial properties, and photodynamic enhancement. The preparation method specifically includes: In an aqueous solution of ammonium persulfate (APS) initiator, NIPAM monomer and crosslinking agent N,N'-methylenebisacrylamide (BIS) were subjected to free radical polymerization to construct a basic thermo-responsive framework for hydrogel, forming a temperature-responsive PNIPAM prepolymer. According to the mass ratio of PNIPAM to GO@AgNPs and CS-Ce6, GO@AgNPs dispersion, CS-Ce6 dispersion and CS solution were added to the PNIPAM prepolymer solution under an inert atmosphere. After stirring evenly, alkali solution was added to induce deprotonation between CS molecular chains and enhance hydrogen bonding / electrostatic interaction in the system, which promoted the initial physical gelation of the system. Stirring and mixing were carried out to quickly and stably fix the functional components in their relative positions in the three-dimensional network, prevent sedimentation or phase separation, and form a uniform composite prepolymer. At a low temperature of 0–10°C, tetramethylethylenediamine (TEMED) catalyst is added to the composite prepolymer to accelerate the free radical crosslinking reaction of the PNIPAM network. After mixing evenly, the mixture is allowed to stand for crosslinking to form a structurally complete and uniform gel product. After purification to remove unreacted monomers, initiators, catalysts and other water-soluble impurities, the porous sponge-like composite hydrogel GACCP is prepared by freeze drying.

[0025] Specifically, in the above preparation method, the mass ratio of NIPAM monomer to crosslinking agent BIS is preferably 20-40:1, which can effectively balance the mechanical strength and swelling properties of the composite hydrogel. The amount of initiator APS is preferably 0.5-2% of the mass of NIPAM monomer, so as to initiate and fully complete the polymerization reaction under mild conditions.

[0026] More specifically, as a preferred embodiment, the concentrations of the GO@AgNPs dispersion and the CS-Ce6 dispersion are controlled at 1–2 mg / mL to ensure good dispersibility of the nanoparticles and effective loading of the photosensitizer; furthermore, the concentration of the CS solution is preferably 50–200 mg / mL to simultaneously exert its functions of bioadhesion, hemostasis, and participation in ionic cross-linking.

[0027] Furthermore, in the above preparation method of the present invention, the alkaline solution is preferably a sodium hydroxide solution.

[0028] Furthermore, the curing time for static crosslinking described in this invention is preferably 10 to 30 minutes.

[0029] Preferably, the purification method of the gel product of the present invention specifically involves soaking it in distilled water for 24 to 48 hours.

[0030] Finally, the present invention also provides the application of the composite hydrogel with hemostatic and antibacterial functions in the preparation of medical materials that promote the healing of skin infection wounds.

[0031] Furthermore, the present invention also provides a hemostatic and antibacterial medical material containing the GACCP composite hydrogel described in the present invention, and a pharmaceutically or materials-acceptable carrier.

[0032] The composite hydrogel with hemostatic and antibacterial functions provided by this invention exhibits a variety of combined advantages. As a high-performance moist wound dressing, it possesses excellent swelling properties and stable mechanical properties, effectively maintaining a moist wound environment and preventing tissue dehydration. Its soft texture facilitates clinical replacement. More importantly, through modification with various functional molecules, this nanocomposite hydrogel integrates multiple functions such as thermosensitive physical hemostasis, broad-spectrum and long-lasting antibacterial activity, photodynamic-enhanced antibacterial activity, and bioadhesion-promoting healing into a single gel system. It can rapidly and effectively repair wounds, outperforming traditional dry nonwoven wound dressings and ordinary hydrogel dressings.

[0033] In terms of functional mechanism, the composite hydrogel of this invention integrates multiple functions such as antibacterial, hemostatic, and promotion of cell proliferation and migration, achieving multiple synergistic effects. The broad-spectrum antibacterial effect of GO@AgNPs combined with the photodynamic effect of CS-Ce6 can produce a synergistic effect to exert a strong antibacterial effect and significantly enhance the killing ability against drug-resistant bacteria; the biological hemostatic mechanism of CS and the thermosensitive contractile physical hemostatic mechanism of PNIPAM complement each other, combined with the adhesive sealing effect of the hydrogel itself, which can quickly and effectively control wound bleeding; in addition, the bioactive components in the system also have the ability to promote fibroblast proliferation and migration to the damaged area; in summary, the composite hydrogel of this invention plays a significant positive role in all four stages of wound healing in skin infections.

[0034] At the structural and preparation level, this invention adopts a sequential molding strategy that combines "physical pregelation" and "low-temperature chemical crosslinking" to ensure the uniform distribution and stable immobilization of functional nanoparticles and biomacromolecules in the gel network, effectively avoiding phase separation and component leakage. The preparation method provided uses readily available raw materials, has mild reaction conditions, and a simple process flow, requiring no complex equipment, and has good prospects for industrialization in the field of medical wound dressings.

[0035] Therefore, the composite hydrogel of the present invention has good temperature sensitivity, injectability, swelling properties, tissue adhesion, biodegradability and biosafety, and integrates multiple functions such as antibacterial, hemostatic, and cell proliferation and migration promotion. It can play a positive role in the entire process of wound healing, meet the needs of different healing stages, and has good application prospects in the field of medical wound dressings. Attached Figure Description

[0036] Figure 1 This is the UV-Vis spectrum of the GO@AgNPs, CS-Ce6, PNIPAM and GACCP composite hydrogel.

[0037] Figure 2 The image shows the FTIR spectrum of the GO@AgNPs, CS-Ce6, PNIPAM and GACCP composite hydrogel.

[0038] Figure 3 This is a SEM image of the PNIPAM and GACCP composite hydrogel.

[0039] Figure 4 This is a swelling curve of the GACCP composite hydrogel.

[0040] Figure 5 The image shows the hemorrhage status (a) and hemorrhage volume (b) of mouse liver injury caused by the PNIPAM and GACCP composite hydrogel.

[0041] Figure 6It is a PNIPAM and GACCP composite hydrogel E. coli Photographs of plate coating (a) and bacterial survival results (b), as well as the results of plate coating (a) and bacterial survival results (b). S. aureus Photographs of plate coatings (c) and bacterial survival results (d).

[0042] Figure 7 The results show the cell proliferation rate of L929 cells induced by the PNIPAM and GACCP composite hydrogel.

[0043] Figure 8 The images (a) and (b) show the scratch images of L929 cells by the PNIPAM and GACCP composite hydrogel. Implementation

[0044] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and specific examples. It should be noted that the embodiments are merely illustrative and are intended to provide a thorough understanding of the technical solutions of the present invention and to provide guidance for those skilled in the art to implement and apply the present invention. It should be understood that these descriptions do not constitute any limitation on the scope of protection of the present invention.

[0045] Unless otherwise expressly stated, the production processes, experiments, tests or analysis methods involved in the embodiments of the present invention are all considered to be conventional methods known to those skilled in the art, and only need to be implemented in accordance with conventional conditions or relevant product instructions. The steps and names involved are also generally clear and unambiguous in the art.

[0046] The instruments, equipment, raw materials, reagents, or samples used in the embodiments are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels or prepared by known methods, and their source does not have a substantial impact on the implementation results of the present invention.

[0047] Unless otherwise expressly defined, the scientific and technical terms used in this invention have the meanings commonly understood by one of ordinary skill in the art. In case of any conflict, the definitions in this specification shall prevail.

[0048] The terms “comprising,” “including,” “having,” etc., used in this invention should be understood as open-ended, meaning “including but not limited to.” The term “and / or” includes any and all combinations of one or more of the associated listed items. Quantitative terms such as “a,” “one,” etc., do not exclude multiples; “multiple” or “a variety” refers to quantities greater than or equal to two.

[0049] The terms "preferred", "better", and "exemplary" used in this invention are only used to describe specific solutions or effects and are not intended to limit the necessary scope of the solution or the scope of protection.

[0050] This invention relates to the description of numerical parameters (such as quantity, concentration, temperature, time, etc.), and it should be understood that reasonable deviations naturally exist due to measuring instruments, operational errors, statistical fluctuations, etc. The range of such deviations should be within limits acceptable to those skilled in the art based on common sense.

[0051] The following embodiments of the present invention provide a composite hydrogel with hemostatic and antibacterial functions. The composition and mass ratio of the composite hydrogel GACCP are PNIPAM:GO@AgNPs:CS-Ce6=100~500:1:1.

[0052] The specific preparation method of the composite hydrogel GACCP includes the following steps: S1. PNIPAM prepolymer was prepared by vortex dissolution of NIPAM, BIS and APS in ultrapure water; S2. Add GO@AgNPs dispersion, CS-Ce6 dispersion, CS solution and appropriate amount of NaOH solution to the obtained PNIPAM prepolymer, and stir the reaction under N2 protection to obtain GACCP prepolymer; S3. Under ice-water bath conditions, TEMED was added to the prepared GACCP prepolymer, stirred and mixed, and allowed to stand for gelation. The obtained gel product was fully immersed in distilled water to remove unreacted reagents, and then freeze-dried to obtain the novel functionalized hydrogel GACCP.

[0053] Preferably, in step S2, the method for preparing GO@AgNPs includes the following steps: S2-1. Disperse GO in ultrapure water by ultrasonication to obtain GO dispersion; S2-2. Dissolve silver nitrate in ultrapure water and mix it with the prepared GO dispersion. Stir magnetically until homogeneous. Then dissolve sodium citrate in ultrapure water and add it to the above mixture. Stir the mixture at 100°C to obtain a reaction mixture. S2-3. After the reaction mixture cools, centrifuge to collect the precipitate, wash with ultrapure water, redisperse in ultrapure water, and freeze-dry to obtain the product GO@AgNPs.

[0054] Preferably, in step S2, the method for preparing CS-Ce6 includes the following steps: S2-a. Dissolve Ce6 in DMSO, add EDC solution and stir until homogeneous, then add NHS solution and continue stirring to activate, thus obtaining Ce6 activated solution. S2-b: Dissolve CS in acetic acid solution and add it to the prepared Ce6 activation solution. Stir the mixture at room temperature in the dark to obtain a reaction mixture. The S2-c reaction mixture was placed in a dialysis bag, dialyzed in ultrapure water, and then freeze-dried to obtain CS-Ce6. Example

[0055] Example 1

[0056] Weigh 5 mg of graphene oxide (GO) and ultrasonically disperse it in 10 mL of ultrapure water to obtain a GO dispersion.

[0057] Weigh 50 mg of silver nitrate and dissolve it in 16 mL of ultrapure water. Mix it with the above GO dispersion and stir magnetically for 10 min. Then weigh 120 mg of sodium citrate and dissolve it in 40 mL of ultrapure water. Continue to add it to the above solution and heat it to 100 °C. Stir and react for 2 h to prepare the reaction mixture.

[0058] After the reaction mixture cooled to room temperature, it was centrifuged at 12,000 rpm for 20 min, the precipitate was collected, washed three times with ultrapure water, redispersed in ultrapure water, and freeze-dried to prepare the product GO@AgNPs.

[0059] Example 2

[0060] Weigh 28 mg of dihydroporphyrin e6 (Ce6) and dissolve it in 5 mL of anhydrous dimethyl sulfoxide (DMSO) to prepare Ce6 stock solution.

[0061] Weigh 70 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 80 mg of N-hydroxysuccinimide (NHS), and dissolve them in 5 mL of 0.1 M MES buffer (pH 5.5) to prepare aqueous solutions of EDC and NHS.

[0062] Add all of the EDC aqueous solution to the Ce6 stock solution, stir for 20 min, then add all of the NHS aqueous solution, and continue stirring to activate for 4 h to obtain the Ce6 activated solution.

[0063] Weigh 120 mg of chitosan (CS) and dissolve it in 100 mL of 1 wt% acetic acid solution. Add the solution to Ce6 activation solution and stir at 450 rpm for 24 h at room temperature in the dark to obtain a reaction mixture.

[0064] The reaction mixture was placed in a dialysis bag with a molecular weight cutoff of 8–14 kDa, dialyzed in ultrapure water for 3 days, and then freeze-dried to prepare CS-Ce6.

[0065] Example 3

[0066] GO@AgNPs prepared in Example 1 were dispersed in ultrapure water to prepare a GO@AgNPs dispersion with a concentration of 2 mg / mL; CS-Ce6 prepared in Example 2 was dispersed in ultrapure water to prepare a CS-Ce6 dispersion with a concentration of 2 mg / mL.

[0067] Weigh 1g of N-isopropylacrylamide (NIPAM), 2mg of N,N'-methylenebisacrylamide (BIS) and 30mg of ammonium persulfate (APS), and vortex dissolve them in 10mL of ultrapure water to prepare poly(N-isopropylacrylamide) (PNIPAM) prepolymer.

[0068] GACCP prepolymer was prepared by adding 2.5 mL of GO@AgNPs dispersion, 2.5 mL of CS-Ce6 dispersion, 10 mL of CS solution with a concentration of 100 mg / mL, and 200 μL of 1 mol / L NaOH solution to the PNIPAM prepolymer and stirring for 20 min under N2 protection.

[0069] Under ice-water bath conditions, N,N,N',N'-tetramethylethylenediamine (TEMED) was added to the GACCP prepolymer, stirred until homogeneous, and allowed to stand for 20 min. The solution then gelled. The obtained gel product was immersed in distilled water for 24 h to remove unreacted reagents, and then freeze-dried to prepare the composite hydrogel GACCP with hemostatic and antibacterial functions.

[0070] Example 4

[0071] The preparation method is the same as in Example 3, except that the volume of GO@AgNPs dispersion and CS-Ce6 dispersion used in the GACCP prepolymer preparation step is 1.0 mL. All other raw material types, amounts and preparation conditions are the same as in Example 3.

[0072] Example 5

[0073] The preparation method is the same as in Example 3, except that the volume of GO@AgNPs dispersion and CS-Ce6 dispersion used in the GACCP prepolymer preparation step is 5.0 mL. All other raw material types, amounts and preparation conditions are the same as in Example 3.

[0074] Application Example 1

[0075] The UV-Vis absorption spectra of the GO@AgNPs prepared in Example 1, the CS-Ce6 prepared in Example 2, and the PNIPAM and GACCP composite hydrogel prepared in Example 3 were analyzed, and the results are as follows: Figure 1The UV absorption curve of GO@AgNPs shows both the absorption peak of GO at 290 nm and the absorption peak of AgNPs at 400 nm, indicating that AgNPs were successfully loaded onto the GO surface. The UV absorption curve of CS-Ce6 clearly shows the characteristic peaks of Ce6, located at 400 nm, 510 nm and 670 nm, respectively. Further copolymerization of GO@AgNPs, CS-Ce6 and PNIPAM resulted in the GACCP composite hydrogel exhibiting all the characteristic absorption peaks of GO@AgNPs (290 nm, 405 nm) and CS-Ce6 (405 nm, 525 nm, 640 nm) on its absorption curve, proving the successful crosslinking of GO@AgNPs, CS-Ce6 and PNIPAM.

[0076] Further FTIR analysis was performed on the GO@AgNPs, CS-Ce6, PNIPAM, and GACCP composite hydrogels, and the results are as follows: Figure 2 As shown, in the infrared spectrum of GO@AgNPs, the hydroxyl stretching vibration peak of GO is at 3444 cm⁻¹. -1 At this location, the epoxy absorption peak is at 1630 cm⁻¹. -1 Location, 1091cm -1 The peak at [location] corresponds to the stretching vibration of COC, while the absorption peak of the stretching vibration of C=O in the carboxyl group (1728 cm⁻¹) is [missing information]. -1 The almost complete disappearance of AgNPs indicates that the incorporation of AgNPs affected the oxygen-containing groups on the GO surface; in the infrared spectrum of CS-Ce6, 3442 cm⁻¹ -1 The peak at 1390 cm⁻¹ is attributed to the stretching vibrations of OH and NH. -1 The peak at 1166 cm⁻¹ is attributed to the CH stretching vibration of CS. -1 The peak at 1634 cm⁻¹ is attributed to the asymmetric stretching vibration of the ether oxygen bond and the CN stretching vibration of the amide III band. -1 and 1551cm -1 The absorption peak at 3439 cm⁻¹ is caused by the stretching vibration of C=O in amide I and the bending vibration of NH in amide II, indicating that CS and Ce₆ are successfully coupled through amide bonds; in the PNIPAM infrared spectrum, 3439 cm⁻¹ -1 The absorption peak corresponding to the stretching vibration of NH in the macromolecular chain structure is 1640 cm⁻¹. -1 1543cm -1 and 1172cm -1 The vibrational absorption peaks at 1366 cm⁻¹ belong to the C=O, NH, and CN groups in the amide group, respectively. -1The absorption peak at 3436 cm⁻¹ belongs to the -CH bending vibration of the isopropyl group; while the infrared absorption curve of the GACCP composite hydrogel shows a similar trend and characteristic peaks to PNIPAM, suggesting that the introduction of GO@AgNPs and CS-Ce6 did not affect the overall structure of the gel network. Therefore, compared with PNIPAM, the GACCP composite hydrogel exhibits a higher absorption peak at 3436 cm⁻¹. -1 The broadening of the absorption peak at this point may be due to the crosslinking of PNIPAM affecting the stretching vibrations of OH and NH in GO@AgNPs and CS-Ce6, further proving the successful preparation of the GACCP composite hydrogel.

[0077] Figure 3 The microstructure of the PNIPAM and GACCP composite hydrogels observed by scanning electron microscopy (SEM) is presented. It can be seen that both hydrogels exhibit a three-dimensional porous network structure. However, because PNIPAM only exhibits chemical crosslinking, its crosslinking mechanism is singular, resulting in a higher porosity (47.991%). In contrast, the GACCP composite hydrogel, in addition to the chemical crosslinking effect of BIS, utilizes GO as a physical crosslinking agent. Its surface oxygen-containing functional groups can form hydrogen bonds with the amide groups of NIPAM, and CS, as a linear macromolecule, can intertwine with NIPAM to form a semi-interpenetrating polymer network structure. These various crosslinking mechanisms increase the number of crosslinking points and the density of the crosslinked network within the GACCP composite hydrogel, thus reducing its porosity to 42.743%.

[0078] The swelling properties of the GACCP composite hydrogel were further tested, and the results are as follows: Figure 4 The hydrogel exhibits a rapid liquid absorption rate within 3.5 hours, which gradually slows down. The swelling rate reaches 300% at 24 hours, demonstrating that the GACCP composite hydrogel prepared in this invention has excellent water retention properties and helps maintain a moist environment in the wound.

[0079] Application Example 2

[0080] The in vivo hemostatic properties of the PNIPAM / GACCP composite hydrogel were tested using a mouse liver injury model, and the rapid hemostatic effect of the GACCP composite hydrogel prepared in this invention in a live animal model was evaluated.

[0081] A mouse model of liver injury was established. A pre-weighed filter paper was placed at the bottom of the liver, and 50 mg of PNIPAM or GACCP composite hydrogel was immediately injected into the wound surface. Bleeding was observed, and the hemostasis time was recorded. Finally, the filter paper that absorbed blood was weighed, and the amount of bleeding was calculated. Mice that did not undergo any material treatment served as a negative control group.

[0082] Bleeding status and amount in mice under different treatment conditions are as follows: Figure 5As shown, the Control group experienced significant bleeding for 120 seconds, with a blood loss of 393 mg. The PNIPAM hydrogel group experienced substantial bleeding at 30 seconds, but by 120 seconds, there was no more flowing blood on the filter paper, indicating a slight relief of bleeding, with the bleeding amount decreasing to 327 mg. This may be attributed to the hydrogel's ability to achieve hemostasis by adhering to and sealing the wound. The GACCP composite hydrogel group consistently exhibited minimal liver bleeding throughout 120 seconds, with a total bleeding amount of only 188 mg, significantly lower than the other two groups. This phenomenon is mainly attributed to the fact that, in addition to its adhesive hemostatic effect, the positively charged CS in the GACCP composite hydrogel can also bind to the negatively charged erythrocyte membrane, causing erythrocytes to aggregate and form blood clots, thus achieving coagulation. This demonstrates that the GACCP composite hydrogel prepared in this invention has excellent hemostatic performance. The introduction of CS and AgNPs promotes erythrocyte aggregation and blood clot formation, which, combined with the physical sealing effect of PNIPAM, produces a synergistic hemostatic effect, possessing the potential to promote wound healing through rapid hemostasis.

[0083] Application Example 3

[0084] Using Escherichia coli ( E. coli ) and Staphylococcus aureus ( S. aureus The in vitro antibacterial properties of the PNIPAM and GACCP composite hydrogel were tested to verify the photothermal-photodynamic synergistic antibacterial effect of the GACCP composite hydrogel under near-infrared light irradiation.

[0085] Take 12 vials containing 5 mL of bacterial culture (1 × 10⁻⁶) 8 EP tubes (CFU / mL) were divided into three groups: PNIPAM hydrogel group, GACCP composite hydrogel group, and Control group. The corresponding hydrogel materials were added to each group for appropriate treatment. The Control group was used as a negative control without hydrogel.

[0086] Three groups of bacterial cultures were incubated on a shaker at 37°C for 4 hours. During this period, the EP tubes in each group were irradiated with laser every hour. The specific treatment method for the four EP tubes in each group was as follows: 1) The first tube was not irradiated at all; 2) The second tube was irradiated with an 808nm near-infrared laser for 4 minutes (2.0W / cm²). 2 ); 3) The third tube was irradiated with a 660nm red laser for 4 minutes (13mW / cm). 2 ); 4) The fourth tube uses 808nm (2.0W / cm) sequentially. 2 ) and 660nm (13mW / cm 2 Laser irradiation for 4 minutes.

[0087] After incubation, 100 μL of bacterial suspension was taken from each tube, plated, and incubated overnight at 37°C. The plate colony count was then recorded, and the bacterial viability was calculated.

[0088] E. coli The antibacterial results are as follows Figure 6 As shown in (a) and (b), the Control group and the PNIPAM hydrogel group had a relatively high number of colonies under both light and dark conditions; while the bacterial survival rate of the GACCP composite hydrogel group dropped to 23.66% under dark conditions, mainly due to the shrinkage of the thermosensitive GACCP composite hydrogel at 37°C, which released the potent antibacterial substances AgNPs and CS; when the GACCP composite hydrogel was irradiated with both 808nm and 660nm lasers, the bacterial survival rate further dropped to 1.32%, which was significantly lower than the 10.55% bacterial survival rate of the photothermal effect of the 808nm laser alone or the 14.81% bacterial survival rate of the photodynamic effect of the 660nm laser.

[0089] S. aureus The antibacterial results are as follows Figure 6 As shown in (c) and (d), the bacterial survival rate of the GACCP composite hydrogel group decreased to the lowest level, only 1.93%, under combined irradiation with 808nm and 660nm lasers. E. coli The results were consistent. The above experimental results demonstrate that the GACCP composite hydrogel prepared in this invention can exert the strongest antibacterial effect through the synergistic effect of multiple antibacterial materials and photothermal combined with photodynamic antibacterial methods, which helps to promote the healing of skin infection wounds by inhibiting the growth of bacteria on the wound surface. Its synergistic mechanism lies in the fact that the thermal effect generated by PTT not only directly kills bacteria, but also enhances the permeability of the bacterial membrane, making it easier for ROS generated by Ce6 to enter the bacterial cell, triggering more lethal oxidative stress. Through the synergistic effect of photothermal-photodynamic, it exhibits highly efficient and broad-spectrum antibacterial properties against both Gram-negative and Gram-positive bacteria.

[0090] Application Example 4

[0091] The proliferative properties of PNIPAM and GACCP composite hydrogel were tested using mouse fibroblasts (L929). The effect of GACCP composite hydrogel on the proliferation activity of L929 cells was evaluated using the CCK-8 assay.

[0092] L929 cells were used at a rate of 5 × 10⁻⁶ 3The cells were seeded at a density of 1 / 2 well in a 96-well plate and cultured at 37°C until adherent. 20 μL of PNIPAM or GACCP composite hydrogel suspension (3 mg / mL) was added and the cells were cultured for another 24 h. Wells without any treatment were used as the negative control group.

[0093] Cells were counted using a hemocytometer at 0 h and 24 h of co-culture with the material, and cell proliferation rate was calculated. The results are as follows: Figure 7 As shown.

[0094] Compared to the 122.26% proliferation rate in the Control group and the 120.65% proliferation rate in the PNIPAM hydrogel group, the GACCP composite hydrogel group achieved a cell proliferation rate of 151.46%, demonstrating significantly superior cell proliferation activity compared to the Control and PNIPAM groups. This indicates that the GACCP composite hydrogel prepared in this invention possesses excellent ability to promote fibroblast proliferation, which is beneficial for accelerating wound tissue repair. This proliferative effect is attributed to the cell growth-promoting properties of CS degradation products and the gentle release of low-concentration Ag. + Upregulation of the expression of growth factors associated with proliferation and migration in fibroblasts indicates that GACCP composite hydrogel can provide a favorable microenvironment for tissue repair while effectively fighting bacteria.

[0095] Application Example 5

[0096] The cell scratch assay was used to test the fibroblast migration-promoting properties of the PNIPAM and GACCP composite hydrogel and to evaluate the effect of the GACCP composite hydrogel on the migration ability of L929 cells.

[0097] L929 cells were used at a rate of 5 × 10⁻⁶ 5 The cells were seeded at a density of 90% in 6-well plates. When the cell density reached 90%, a sterile white pipette tip was used to draw a vertical line along a ruler. After drawing the line, the old solution was discarded and the cells were washed twice with PBS. Serum-free culture medium and 0.4 mL of PNIPAM or GACCP composite hydrogel (3 mg / mL) were added and the cells were cultured for another 24 h. The wells without any material treatment were used as the negative control group.

[0098] Scratch changes were recorded by photographing the cells at 0h and 24h of co-culture, and the scratch area ratio was calculated using ImageJ software. The scratch images and scratch area ratios for each group of cells under different treatment conditions are shown below. Figure 8 As shown in (a) and (b).

[0099] After 24 hours of culture, L929 cells in the Control group and PNIPAM hydrogel group showed no significant migration, with remaining scratch areas of 87.99% and 83.57%, respectively. In contrast, the GACCP composite hydrogel group exhibited stronger cell migration ability, with a remaining scratch area of ​​only 39.36%. This indicates that the GACCP composite hydrogel of this invention can effectively promote fibroblast migration and further aid wound healing by guiding cells from both sides of the wound towards the center. The significant promotion of cell migration by the GACCP composite hydrogel may be achieved through the following mechanisms: CS and its oligosaccharide fragments can act as chemotactic signals, attracting cells to move directionally towards the damaged area; the material degradation process mimics the dynamic characteristics of the extracellular matrix, providing more suitable physical support and biochemical cues for cell migration; Ag... + The antibacterial properties of CS alleviated the inhibition of cell migration by bacterial toxins in a simulated infection environment. This result strongly demonstrates that the GACCP composite hydrogel can not only inhibit infection but also actively guide repair cells to accumulate in the wound, thereby accelerating the healing process.

[0100] In summary, the GACCP composite hydrogel prepared in this invention achieves a synergistic effect of hemostasis, antibacterial properties, and wound repair through its unique composition and structure. In vivo hemostasis experiments demonstrated its synergistic ability to rapidly block wounds physically and promote biochemical coagulation, creating a stable initial environment for wound healing. In vitro antibacterial experiments revealed its multi-mode synergistic mechanism of photothermal-photodynamic-chemical bactericidal action, which can effectively eliminate the risk of infection. Cell experiments further confirmed that the material is not only harmless to fibroblasts at antibacterial doses but also significantly promotes their proliferation and migration. Therefore, the GACCP composite hydrogel is a highly promising biomaterial suitable for integrated treatment of acute traumatic hemostasis and infected wounds.

[0101] The above embodiments of the present invention do not describe all details exhaustively, nor do they limit the present invention to the embodiments described above. Various changes, modifications, substitutions, and variations made by those skilled in the art to these embodiments without departing from the principles and spirit of the present invention should be included within the scope of protection of the present invention.

Claims

1. A composite hydrogel with hemostatic and antibacterial functions is a nanocomposite hydrogel obtained by combining functional components graphene oxide-silver nanoparticle composite and chitosan-dihydroporphyrin e6 composite during the preparation of PNIPAM by free radical polymerization using thermosensitive hydrogel PNIPAM as a carrier. The mass ratio of PNIPAM to graphene oxide-silver nanoparticle composite and chitosan-dihydroporphyrin e6 composite is 100-500:1:

1.

2. The composite hydrogel according to claim 1, characterized in that... The mass ratio of PNIPAM to the graphene oxide-silver nanoparticle composite and the chitosan-dihydroporphyrin E6 composite is 200:1:

1.

3. A method for preparing the composite hydrogel according to claim 1 or 2, comprising: A thermosensitive PNIPAM prepolymer was prepared by free radical polymerization of NIPAM monomer and crosslinking agent N,N'-methylenebisacrylamide in an aqueous solution of ammonium persulfate initiator. According to the mass ratio of PNIPAM to graphene oxide-silver nanoparticle composite and chitosan-dihydroporphyrin e6 composite, under an inert atmosphere, graphene oxide-silver nanoparticle composite dispersion, chitosan-dihydroporphyrin e6 composite dispersion and chitosan solution were added to PNIPAM prepolymer solution. After stirring evenly, chitosan gelled in an alkali-induced system was added and stirred to prepare composite prepolymer. At 0–10°C, tetramethylethylenediamine catalyst was added to the composite prepolymer. After mixing evenly, the mixture was allowed to stand for crosslinking to obtain a gel product. The product was then purified and freeze-dried to prepare a composite hydrogel.

4. The preparation method according to claim 3, characterized in that: The mass ratio of NIPAM monomer to crosslinking agent is 20-40:1, and the amount of initiator ammonium persulfate is 0.5-2% of the mass of NIPAM monomer.

5. The preparation method according to claim 3, characterized in that: The concentration of the graphene oxide-silver nanoparticle composite dispersion is 1-2 mg / mL, the concentration of the chitosan-dihydroporphyrin E6 composite dispersion is 1-2 mg / mL, and the concentration of the chitosan solution is 50-200 mg / mL.

6. The preparation method according to claim 3, characterized in that: The alkaline solution is a sodium hydroxide solution.

7. The preparation method according to claim 3, characterized in that: The static crosslinking time is 10 to 30 minutes.

8. The preparation method according to claim 3, characterized in that: The gel product was purified by soaking it in distilled water for 24–48 hours.

9. The application of the composite hydrogel with hemostatic and antibacterial functions as described in claim 1 in the preparation of medical materials that promote the healing of skin infection wounds.

10. A hemostatic and antibacterial medical material comprising the composite hydrogel of claim 1 and a pharmaceutically or materials-acceptable carrier.

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