Polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function and preparation method thereof

Abstract

The application provides a polyvinyl alcohol / chitosan composite compression hemostatic sponge with bacteriostatic function and a preparation method thereof, and belongs to the technical field of medical materials.The polyvinyl alcohol / chitosan composite compression hemostatic sponge with bacteriostatic function comprises a double-network hydrogel structure formed by carboxymethyl chitosan and aldehyde-based polyvinyl alcohol; the double-network hydrogel structure has high stability and fixity, so that the composite compression hemostatic sponge has excellent stability.The raw materials used in the application include carboxymethyl chitosan, aldehyde-based polyvinyl alcohol and antibacterial peptide, which are connected through covalent bonds and ionic bonds to form a double-network hydrogel structure.By introducing gallic acid and copper chloride, a metal-organic network structure can be formed, which can further improve the mechanical stability of the composite compression hemostatic sponge and further improve the antibacterial performance of the composite compression hemostatic sponge.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, and in particular to a polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function and its preparation method. Background Technology

[0002] During surgery, intraoperative bleeding is common. Hemostatic sponges can effectively reduce bleeding time, thereby shortening the operation time and improving patient prognosis. Blood contains a large amount of water, so the rapid water absorption and retention properties of the hemostatic sponge are crucial for its good hemostatic effect. In addition, while rapidly stopping bleeding, microbial infection can lead to inflammation; therefore, antibacterial properties are essential for hemostatic sponges.

[0003] Currently, researchers have developed several hemostatic sponges, such as chitosan sponges, gelatin sponges, and polyvinyl alcohol sponges. However, in practical applications, these hemostatic sponges still have certain shortcomings. For example, chitosan sponges alone can be used for minor bleeding wounds, but their hemostatic effect on severe bleeding wounds remains limited; gelatin sponges can be used for low to moderate oozing, however, their mechanical strength and support are poor, and their absorbency and expansion are limited; polyvinyl alcohol sponges have excellent mechanical stability, but they suffer from drawbacks such as rigidity, lack of antibacterial properties, and poor operability.

[0004] To address the problems with hemostatic sponges, studies have shown that introducing chitosan into polyvinyl alcohol (PVA) sponges via cross-linking can improve their antibacterial and hemostatic properties. However, due to the poor solubility of chitosan in neutral pH solutions, its reaction with PVA is limited, leading to the easy detachment of undissolved chitosan particles during use. This detachment not only renders the antibacterial properties ineffective but may also cause irritation to the body during use.

[0005] Therefore, how to provide a polyvinyl alcohol / chitosan composite compression hemostatic sponge with structural stability and excellent antibacterial function is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] The purpose of this invention is to provide a structurally stable polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function and its preparation method.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function.

[0009] The polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function comprises a double network hydrogel structure formed by carboxymethyl chitosan and aldehyde-modified polyvinyl alcohol.

[0010] The raw materials for preparing the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function, by mass percentage, include: 0.1-20% carboxymethyl chitosan, 0.4-50% aldehyde-modified polyvinyl alcohol, 0.1-10% antimicrobial peptide, 0.03-7% crosslinking agent, 0.1-5% gallic acid, 0.1-1% copper chloride, and the balance being water;

[0011] The degree of aldehyde alkylation of the aldehyde-modified polyvinyl alcohol is 10-30%.

[0012] Preferably, the method for preparing the aldehyde-modified polyvinyl alcohol includes: mixing an aqueous solution of polyvinyl alcohol with an oxidant and carrying out an oxidation reaction to obtain aldehyde-modified polyvinyl alcohol.

[0013] Preferably, the molar ratio of the oxidant to polyvinyl alcohol is 1:5~10.

[0014] Preferably, the oxidation reaction is carried out at a temperature of 40-60°C for 2-4 hours.

[0015] Preferably, the antimicrobial peptide includes one or more of polylysine, KR12, black soldier fly antimicrobial peptide, and nisin.

[0016] Preferably, the crosslinking agent includes one or more of formaldehyde, glutaraldehyde, glyoxal, and epichlorohydrin.

[0017] This invention also provides a method for preparing the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function as described in the above technical solution, comprising the following steps:

[0018] (1) Mix carboxymethyl chitosan, aldehyde-modified polyvinyl alcohol and water, and carry out Schiff base reaction to obtain graft copolymer solution;

[0019] The graft copolymer solution was mixed with a reducing agent, and then subjected to a reduction reaction and alcohol precipitation treatment in sequence to obtain the reduced graft copolymer.

[0020] (2) The reduced graft copolymer, water, antimicrobial peptide and crosslinking agent obtained in step (1) are mixed and subjected to ionic crosslinking reaction to obtain precursor gel solution;

[0021] (3) The precursor gel solution obtained in step (2) is mixed with gallic acid and copper chloride, and after reaction, a gel solution is obtained;

[0022] (4) After mixing the gel solution obtained in step (3) with gas, perform gradient freeze drying to obtain polyvinyl alcohol / chitosan composite compression hemostatic sponge;

[0023] The gradient freeze-drying process includes pre-freezing, initial drying, and desorption drying performed sequentially.

[0024] The pre-freezing temperature is -35~20℃, and the pre-freezing time is 5~20h;

[0025] The initial drying temperature is -10~10℃, and the initial drying time is 8~24h;

[0026] The temperature for the analytical drying process is 15~35℃, and the drying time is 8~24h.

[0027] Preferably, the temperature of the Schiff base reaction in step (1) is 30~50℃; the reaction time is 3~6h.

[0028] Preferably, the temperature of the ion crosslinking reaction in step (2) is 30~60℃; the time of the ion crosslinking reaction is 3~6h.

[0029] Preferably, the pre-freezing in step (3) is gradient freezing, which is a series of pre-freezing processes performed sequentially: cooling pre-freezing, first pre-freezing, second pre-freezing, third pre-freezing, and fourth pre-freezing.

[0030] The temperature for cooling and pre-freezing is 0~20℃; the time for cooling and pre-freezing is 0.5~4h;

[0031] The temperature for the first pre-freezing is -20~0℃; the temperature for the second pre-freezing is -35~-20℃; the temperature for the third pre-freezing is -20~0℃; and the temperature for the fourth pre-freezing is -35~-10℃.

[0032] The time for the first, second, third, and fourth pre-freezing cycles is independently 0.5 to 3 hours.

[0033] This invention provides a polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function. The sponge comprises a double-network hydrogel structure formed by carboxymethyl chitosan and aldehyde-modified polyvinyl alcohol. The raw materials for preparing the sponge, by mass percentage, include: 0.1-20% carboxymethyl chitosan, 0.4-50% aldehyde-modified polyvinyl alcohol, 0.1-10% antimicrobial peptide, 0.03-7% crosslinking agent, 0.1-5% gallic acid, 0.1-1% copper chloride, and the balance being water; the degree of aldehyde modification of the aldehyde-modified polyvinyl alcohol is 10-30%. The polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function provided by this invention comprises a double-network hydrogel structure formed by carboxymethyl chitosan and aldehyde-modified polyvinyl alcohol. The raw materials used in this invention include carboxymethyl chitosan, aldehyde-modified polyvinyl alcohol, and antimicrobial peptide, which are connected by covalent and ionic bonds to form a double-network hydrogel structure. The dual-network hydrogel structure exhibits high stability and fixation, enhancing the binding ability of chitosan and polyvinyl alcohol. The covalent bonds in the dual-network hydrogel structure result in a low cross-linking density, allowing water molecules to more easily penetrate the network, leading to hydrogel expansion. Furthermore, the non-uniformity of the covalently bonded cross-linked network results in lower cross-linking densities in certain areas, making them more susceptible to water molecule penetration. Simultaneously, the electrostatic interactions forming cross-linking points in the dual-network hydrogel possess a degree of dynamic reversibility. When the hydrogel is subjected to tensile or other mechanical stresses, these electrostatic cross-linking points can partially break and reform, thereby absorbing energy and dispersing stress, preventing damage to the network structure. This characteristic allows electrostatic interactions to enhance the network structure while maintaining a certain degree of flexibility and elasticity. Therefore, the composite compression hemostatic sponge provided by this invention possesses excellent stability. By introducing gallic acid and copper chloride, this invention can form a metal-organic network structure, which not only further enhances the mechanical stability of the composite compression hemostatic sponge but also achieves effective fixation of gallic acid within the sponge. The combined effect of gallic acid and copper ions further improves the antibacterial properties of the composite compression hemostatic sponge. The results of the examples show that the composite compression hemostatic sponge provided by the present invention has excellent stability and antibacterial properties. Attached Figure Description

[0034] Figure 1 The above is a bar chart showing the swelling ratio of the polyvinyl alcohol / chitosan composite compression hemostatic sponges prepared in Examples 1-4 and Comparative Examples 1 and 3 of this invention.

[0035] Figure 2 The results of in vitro blood coagulation index determination of the polyvinyl alcohol / chitosan composite compression hemostatic sponges prepared in Examples 1-4 of this invention;

[0036] Figure 3The bar chart shows the cytotoxicity of the polyvinyl alcohol / chitosan composite compression hemostatic sponges prepared in Example 1 and Comparative Example 2 of this invention. Detailed Implementation

[0037] This invention provides a polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function, wherein the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function comprises a double network hydrogel structure formed by carboxymethyl chitosan and aldehyde-modified polyvinyl alcohol;

[0038] The raw materials for preparing the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function, by mass percentage, include: 0.1-20% carboxymethyl chitosan, 0.4-50% aldehyde-modified polyvinyl alcohol, 0.1-10% antimicrobial peptide, 0.03-7% crosslinking agent, 0.1-5% gallic acid, 0.1-1% copper chloride, and the balance being water;

[0039] The degree of aldehyde alkylation of the aldehyde-modified polyvinyl alcohol is 10-30%.

[0040] The polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function provided by this invention comprises a dual-network hydrogel structure formed by carboxymethyl chitosan, aldehyde-modified polyvinyl alcohol, and antimicrobial peptides. This immobility allows covalent bonds to form fixed cross-linking points in the hydrogel network, limiting the network's dynamic adjustment ability and improving the binding ability of chitosan and polyvinyl alcohol. The cross-linking density formed by covalent bonds is generally low, which allows water molecules to penetrate the network more easily, leading to hydrogel expansion. In addition, the cross-linking network formed by covalent bonds is non-uniform, resulting in lower cross-linking density in some areas, making them easier for water molecules to penetrate. At the same time, the electrostatic interactions in the dual-network hydrogel form cross-linking points with a certain degree of dynamic reversibility. When the hydrogel is subjected to tensile or other mechanical stresses, these electrostatic cross-linking points can partially break and reform, thereby absorbing energy and dispersing stress, preventing damage to the network structure. This characteristic allows electrostatic interactions to enhance the network structure while maintaining a certain degree of flexibility and elasticity. Therefore, the composite compression hemostatic sponge provided by this invention has excellent stability.

[0041] In this invention, the raw material for preparing the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function includes 0.1-20% carboxymethyl chitosan by mass percentage. As one embodiment of this invention, the mass percentage of carboxymethyl chitosan can be 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, or 20%. The carboxymethyl chitosan used in this invention is rich in carboxyl groups, which can form a double-network hydrogel structure with aldehyde-modified polyvinyl alcohol.

[0042] This invention does not specifically limit the source of the carboxymethyl chitosan; any conventional commercially available product can be used. In an embodiment of this invention, the carboxymethyl chitosan can be sourced from Aladdin Chemical Reagent Network.

[0043] In this invention, the raw material for preparing the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function comprises 0.4-50% aldehyde-modified polyvinyl alcohol by mass percentage. As one embodiment of this invention, the mass percentage of the aldehyde-modified polyvinyl alcohol can be 0.4%, 0.5%, 1%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. This invention utilizes aldehyde-modified polyvinyl alcohol to form a double-network hydrogel structure with carboxymethyl chitosan.

[0044] In this invention, the degree of aldehyde alkylation of the aldehyde-modified polyvinyl alcohol is 10-30%. As one embodiment of this invention, the degree of aldehyde alkylation of the aldehyde-modified polyvinyl alcohol can be 10%, 15%, 20%, 25%, or 30%. By controlling the degree of aldehyde alkylation of the aldehyde-modified polyvinyl alcohol within the above range, this invention can prevent low aldehyde alkylation from resulting in low activity and an inability to form a stable double-network hydrogel structure with carboxymethyl chitosan, while also preventing excessively high aldehyde alkylation from causing the polyvinyl alcohol backbone to break.

[0045] In this invention, the preferred method for preparing aldehyde-modified polyvinyl alcohol includes: mixing an aqueous solution of polyvinyl alcohol with an oxidant to carry out an oxidation reaction to obtain aldehyde-modified polyvinyl alcohol.

[0046] In this invention, the molecular weight of the polyvinyl alcohol is preferably 50,000 to 100,000, more preferably 60,000 to 100,000.

[0047] In this invention, the oxidant preferably includes sodium periodate.

[0048] In this invention, the molar ratio of the oxidant to polyvinyl alcohol is preferably 1:5 to 10. As one embodiment of this invention, the molar ratio of the oxidant to polyvinyl alcohol can be 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. By adding an oxidant, this invention can selectively oxidize adjacent hydroxyl groups on the polyvinyl alcohol molecular chain to generate aldehyde groups.

[0049] In this invention, the mass concentration of the polyvinyl alcohol aqueous solution is preferably 5-8%. As one embodiment of this invention, the mass concentration of the polyvinyl alcohol aqueous solution can be 5%, 6%, 7%, or 8%. By controlling the mass concentration of the polyvinyl alcohol aqueous solution within the above range, this invention facilitates a more complete oxidation reaction and achieves a degree of aldehyde oxidation of 10-30% for the aldehyde-modified polyvinyl alcohol.

[0050] The present invention does not have any particular limitation on the method of mixing the polyvinyl alcohol aqueous solution with the oxidant, as long as the oxidant can be completely dissolved in the polyvinyl alcohol aqueous solution.

[0051] In this invention, the preferred temperature for the oxidation reaction is 40-60°C; the preferred reaction time is 2-4 hours. As one embodiment of this invention, the oxidation reaction temperature can be 40°C, 45°C, 50°C, 55°C, or 60°C; the reaction time can be 2 hours, 3 hours, or 4 hours. In this invention, the oxidation reaction selectively oxidizes adjacent hydroxyl groups on the polyvinyl alcohol molecular chain to generate aldehyde groups, with the reaction formula: -CH(OH)-CH(OH)-→-CHO-CHO- + H2O.

[0052] In this invention, ethylene glycol is preferably added to the system obtained after the oxidation reaction to terminate the oxidation reaction. The amount of ethylene glycol used is not particularly limited in this invention; adjustments can be made to achieve the desired termination of the oxidation reaction.

[0053] The present invention preferably removes residual reagents by dialysis to obtain aldehyde-modified polyvinyl alcohol. The present invention does not specifically limit the dialysis method; conventional dialysis methods can be used.

[0054] In this invention, the raw material for preparing the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function includes 0.1-10% antimicrobial peptides by mass percentage. As one embodiment of this invention, the mass percentage of the antimicrobial peptides can be 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In this invention, the antimicrobial peptides preferably include one or more of polylysine, antimicrobial peptide KR12, black soldier fly antimicrobial peptides, and nisin. In this invention, the nisin is a lactobacillus peptide. This invention does not have a specific limitation on the source of the antimicrobial peptides; conventional commercially available products can be used. The use of antimicrobial peptides in this invention enables aldehyde-modified polyvinyl alcohol to be covalently linked to carboxymethyl chitosan, forming a double-network hydrogel structure.

[0055] In this invention, the raw materials for preparing the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function include 0.03-7% crosslinking agent by mass percentage. As one embodiment of this invention, the mass percentage of the crosslinking agent can be 0.03%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7%. In this invention, the crosslinking agent preferably includes one or more of formaldehyde, glutaraldehyde, glyoxal, and epichlorohydrin. The use of a crosslinking agent in this invention enables further crosslinking of the dual-network hydrogel structure.

[0056] In this invention, the raw material for preparing the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function includes 0.1-5% gallic acid by mass percentage. As one embodiment of this invention, the mass percentage of gallic acid can be 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%. In this invention, the crosslinking agent preferably includes one or more of formaldehyde, glutaraldehyde, glyoxal, and epichlorohydrin.

[0057] In this invention, the raw materials for preparing the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function include 0.1-1% copper chloride by mass percentage. As one embodiment of this invention, the mass percentage of copper chloride can be 0.1%, 0.5%, or 1%. This invention uses gallic acid and copper chloride, which can further introduce a metal-organic network structure into the dual-network hydrogel structure. This not only further improves the mechanical stability of the composite compression hemostatic sponge but also achieves effective fixation of gallic acid in the composite compression hemostatic sponge, further enhancing the antibacterial properties of the composite compression hemostatic sponge through its interaction with copper ions.

[0058] In this invention, the raw materials for preparing the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function include the remainder water by weight percentage. Water is used as the reaction solvent in this invention.

[0059] This invention provides a method for preparing the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function as described in the above technical solution, comprising the following steps:

[0060] (1) Mix carboxymethyl chitosan, aldehyde-modified polyvinyl alcohol and water, and carry out Schiff base reaction to obtain graft copolymer solution;

[0061] The graft copolymer solution was mixed with a reducing agent, and then subjected to a reduction reaction and alcohol precipitation treatment in sequence to obtain the reduced graft copolymer.

[0062] (2) The reduced graft copolymer, water, antimicrobial peptide and crosslinking agent obtained in step (1) are mixed and subjected to ionic crosslinking reaction to obtain precursor gel solution;

[0063] (3) The precursor gel solution obtained in step (2) is mixed with gallic acid and copper chloride, and after reaction, a gel solution is obtained;

[0064] (4) After mixing the gel solution obtained in step (3) with gas, perform gradient freeze drying to obtain polyvinyl alcohol / chitosan composite compression hemostatic sponge;

[0065] The gradient freeze-drying process includes pre-freezing, initial drying, and desorption drying performed sequentially.

[0066] The pre-freezing temperature is -35~20℃, and the pre-freezing time is 5~20h;

[0067] The initial drying temperature is -10~10℃, and the initial drying time is 8~24h;

[0068] The temperature for the analytical drying process is 15~35℃, and the drying time is 8~24h.

[0069] In this invention, carboxymethyl chitosan, aldehyde-modified polyvinyl alcohol and water are mixed and subjected to a Schiff base reaction to obtain a graft copolymer solution.

[0070] In this invention, acetic acid is preferably added as a catalyst when carboxymethyl chitosan, aldehyde-modified polyvinyl alcohol, and water are mixed. This adjusts the pH of the mixed solution to be weakly acidic to neutral, which is beneficial for promoting the Schiff base reaction. In this invention, the pH of the mixed solution is preferably 6-7.

[0071] In this invention, the method of mixing carboxymethyl chitosan, aldehyde-modified polyvinyl alcohol and water preferably includes: dissolving carboxymethyl chitosan in an aqueous solution of acetic acid to obtain a carboxymethyl chitosan solution; and mixing the carboxymethyl chitosan solution with aldehyde-modified polyvinyl alcohol.

[0072] In this invention, the mass concentration of the aqueous acetic acid solution is preferably 1-3%, more preferably 2-3%. In this invention, the mass ratio of the carboxymethyl chitosan to the volume of the aqueous acetic acid solution is preferably 0.5-3 g:100 mL, more preferably 1-2 g:100 mL. In this invention, the mass concentration of the aldehyde-modified polyvinyl alcohol is preferably 1-5%, more preferably 2-4%. In this invention, the volume ratio of the carboxymethyl chitosan solution to the aldehyde-modified polyvinyl alcohol is preferably 1:5.

[0073] In this invention, the method of mixing the carboxymethyl chitosan solution with aldehyde-modified polyvinyl alcohol is preferably by stirring, and the stirring speed is preferably 30~800 rpm, more preferably 100~500 rpm.

[0074] In this invention, the preferred temperature for the Schiff base reaction is 30-50°C; the preferred reaction time is 3-6 hours. As one embodiment of this invention, the temperature for the Schiff base reaction can be 30°C, 35°C, 40°C, 45°C, or 50°C; the reaction time can be 3 hours, 4 hours, 5 hours, or 6 hours. This invention uses a Schiff base reaction to react the aldehyde group with the amino group of carboxymethyl chitosan to form an imine bond, creating a graft copolymer. The reaction principle is: -CHO + -NH2 → -C=N - +H2O.

[0075] After obtaining the graft copolymer solution, the present invention mixes the graft copolymer solution with a reducing agent and performs a reduction reaction and alcohol precipitation treatment in sequence to obtain a reduced graft copolymer.

[0076] In this invention, the reducing agent is preferably sodium borohydride. By adding a reducing agent, this invention can reduce the imine bond to a more stable CN single bond, avoiding hydrolysis and improving the stability of the graft copolymer.

[0077] In this invention, the preferred molar ratio of carboxymethyl chitosan to reducing agent is 1:0.5~1. Controlling the molar ratio of carboxymethyl chitosan to reducing agent within this range further facilitates the complete reduction of imine bonds.

[0078] In this invention, the temperature of the reduction reaction is preferably 30~50℃; the time of the reduction reaction is preferably 1~2h. As one embodiment of this invention, the temperature of the reduction reaction can be 30℃, 35℃, 40℃, 45℃ or 50℃; the time of the reduction reaction can be 1h, 1.5h or 2h.

[0079] This invention does not impose any particular limitation on the operation method of the alcohol precipitation treatment; conventional alcohol precipitation treatment can be used. In this invention, the preferred method for alcohol precipitation treatment is to add ethanol to the system after the reduction reaction. This invention does not impose any particular limitation on the amount of ethanol used, as long as it is sufficient to prevent further increase in the amount of precipitate in the system.

[0080] The present invention preferably involves washing the solid obtained from the alcohol precipitation treatment to obtain the reduced graft copolymer. The present invention does not specifically limit the washing method, as long as it can sufficiently remove unreacted carboxymethyl chitosan and aldehyde-modified polyvinyl alcohol.

[0081] After obtaining the reduced graft copolymer, the present invention mixes the reduced graft copolymer, water, antimicrobial peptide and crosslinking agent, and performs an ionic crosslinking reaction to obtain a precursor gel solution.

[0082] The present invention does not have any particular limitation on the method of mixing the reduced graft copolymer, water, antimicrobial peptide and crosslinking agent, as long as the reduced graft copolymer, antimicrobial peptide and crosslinking agent can be dissolved in water.

[0083] In this invention, the temperature of the ionic crosslinking reaction is 30~60℃; the time of the ionic crosslinking reaction is 3~6h. As one embodiment of this invention, the temperature of the ionic crosslinking reaction can be 30℃, 35℃, 40℃, 45℃, 50℃, 55℃ or 60℃; the time of the ionic crosslinking reaction can be 3h, 4h, 5h or 6h.

[0084] After obtaining the precursor gel solution, the present invention mixes the precursor gel solution with gallic acid and copper chloride, and reacts to obtain the gel solution.

[0085] The present invention does not specifically limit the method of mixing the precursor gel solution with gallic acid and copper chloride, as long as gallic acid and copper chloride are sufficiently dispersed in the precursor gel solution. In the present invention, the preferred method of mixing the precursor gel solution with gallic acid and copper chloride is stirring, and the stirring speed is preferably 100~300 rpm, more preferably 150~200 rpm.

[0086] In this invention, the reaction temperature is preferably 20-25°C, more preferably 22-25°C; the reaction time is preferably 10-30 min, more preferably 15-25 min. In this invention, the reaction is preferably carried out under stirring, and the stirring speed is preferably 300-500 rpm, more preferably 400-500 rpm.

[0087] After obtaining the gel solution, the present invention mixes the gel solution with gas and then performs gradient freeze-drying to obtain a polyvinyl alcohol / chitosan composite compression hemostatic sponge.

[0088] In this invention, the gas preferably includes one or more of an inert gas, carbon dioxide, and nitrogen. By mixing the gel solution with the gas, this invention enables the formation of bubbles in the gel solution, thereby reducing the anisotropy of the sponge.

[0089] In this invention, the volume ratio of the gel solution to the gas is preferably 10~20:1, more preferably 15~20:1.

[0090] In this invention, the method for mixing the gel solution with gas preferably includes: introducing gas into the gel solution, wherein the gas flow rate is preferably 10-50 mL / min, more preferably 15-30 mL / min. Introducing gas into the gel solution in this invention can form tiny bubbles, reducing the anisotropy of the sponge.

[0091] In this invention, the system obtained by mixing the gel solution with the gas is first poured into a mold, and then subjected to gradient freeze-drying. This invention does not impose any particular limitation on the size of the mold; it can be selected as needed. In an embodiment of this invention, the mold can be a cylindrical mold with an inner diameter of 1.2 cm and a height of 21 cm.

[0092] In this invention, the gradient freeze-drying includes pre-freezing, initial drying, and desorption drying performed sequentially.

[0093] In this invention, the pre-freezing temperature is -35~20℃, and the pre-freezing time is 5~20h.

[0094] In this invention, the pre-freezing is preferably gradient freezing, and the gradient freezing is preferably performed sequentially as cooling pre-freezing, first pre-freezing, second pre-freezing, third pre-freezing and fourth pre-freezing;

[0095] The preferred temperature for pre-freezing is 0~20℃; the preferred pre-freezing time is 0.5~4h. This invention lowers the temperature of the product after mixing the gel solution and gas to 0℃ through pre-freezing.

[0096] The preferred temperature for the first pre-freezing is -20 to 0°C, more preferably -20 to -10°C; the preferred temperature for the second pre-freezing is -35 to -20°C, more preferably -35 to -25°C; the preferred temperature for the third pre-freezing is -20 to 0°C, more preferably -20 to -10°C; the preferred temperature for the fourth pre-freezing is -35 to -10°C, more preferably -35 to -30°C; the preferred times for the first, second, third, and fourth pre-freezing stages are independently 0.5 to 3 hours, more preferably 1 to 2 hours. This invention enables the pre-freezing of the system containing the gel solution and gas to fully freeze and form a uniform porous structure.

[0097] In this invention, the initial drying temperature is -10~10℃, preferably -5~10℃; the initial drying time is preferably 8~24h, more preferably 12~20h. Through the initial drying process, this invention enables the pre-frozen sponge to be heated to a relatively low temperature, thereby improving the stability of the sponge's pores.

[0098] In this invention, the desorption drying temperature is 15~35℃, and the desorption drying time is 8~24h. As one embodiment of this invention, the desorption drying temperature can be 15℃, 20℃, 25℃, 30℃, or 35℃; the desorption drying time can be 8h, 10h, 12h, 15h, 18h, 20h, or 24h. In this invention, the desorption drying is preferably carried out under vacuum. This invention, through desorption drying, can effectively remove moisture.

[0099] The present invention preferably compresses the sponge obtained by gradient freeze-drying to obtain a polyvinyl alcohol / chitosan composite compressed hemostatic sponge. The present invention does not specifically limit the compression ratio; it can be adjusted as needed to form granules of the polyvinyl alcohol / chitosan composite compressed hemostatic sponge with dimensions meeting the requirements for use. In the present invention, the polyvinyl alcohol / chitosan composite compressed hemostatic sponge is preferably cylindrical, with a preferred height of 0.5~0.7cm and a preferred diameter of 1.2cm.

[0100] The polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function provided by this invention uses carboxymethyl chitosan, aldehyde-modified polyvinyl alcohol, and antimicrobial peptides as raw materials, which are covalently linked to form a double-network hydrogel structure. By introducing gallic acid and copper chloride, this invention can form a metal-organic network structure, which not only further improves the mechanical stability of the composite compression hemostatic sponge but also effectively immobilizes gallic acid within the sponge, further enhancing its antibacterial properties in conjunction with copper ions. In preparing the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function, this invention utilizes physical means to control the pore structure of the sponge by mixing the gel solution with gas and controlling the temperature and time of gradient freeze-drying. Therefore, the polyvinyl alcohol / chitosan composite compression hemostatic sponge provided by this invention exhibits excellent stability and antibacterial properties.

[0101] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0102] In an embodiment of the present invention, the preparation method of aldehyde-modified polyvinyl alcohol is as follows: Polyvinyl alcohol (molecular weight 50,000-100,000) aqueous solution is dissolved in water to obtain a 6% (w / w) polyvinyl alcohol solution. Then, an oxidant (sodium periodate) is added to the polyvinyl alcohol solution at a molar ratio of oxidant to polyvinyl alcohol of 1:5. An oxidation reaction is carried out at 50°C for 2-4 hours. The oxidation reaction is then terminated with ethylene glycol, and residual reagents are removed by dialysis to obtain aldehyde-modified polyvinyl alcohol. In this preparation method, an oxidation reaction time of 2 hours yields an aldehyde degree of approximately 15% for the obtained aldehyde-modified polyvinyl alcohol; an oxidation reaction time of 3 hours yields an aldehyde degree of approximately 18% for the obtained aldehyde-modified polyvinyl alcohol; and an oxidation reaction time of 4 hours yields an aldehyde degree of approximately 27% for the obtained aldehyde-modified polyvinyl alcohol.

[0103] Example 1

[0104] A polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function, wherein the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function comprises a double network hydrogel structure formed by carboxymethyl chitosan and aldehyde-modified polyvinyl alcohol.

[0105] The raw materials for preparing the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function by mass percentage are: 10% carboxymethyl chitosan, 15% aldehyde-modified polyvinyl alcohol, 6% antimicrobial peptide (polylysine), 5% crosslinking agent (glutaraldehyde), 3% gallic acid, 0.5% copper chloride, and the balance being water.

[0106] The degree of aldehyde alkylation of the aldehyde-modified polyvinyl alcohol is 15%.

[0107] The preparation method of the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function is as follows:

[0108] (1) Dissolve carboxymethyl chitosan in an aqueous solution of acetic acid with a mass concentration of 2% (the water in the aqueous solution of acetic acid is half of the total water) to obtain a carboxymethyl chitosan solution; mix the carboxymethyl chitosan solution with aldehyde-modified polyvinyl alcohol under stirring at 100 rpm, and then carry out a Schiff base reaction at 35°C for 5 h to obtain a graft copolymer solution.

[0109] The graft copolymer solution was mixed with a reducing agent (sodium borohydride) at a molar ratio of 1:0.5 for carboxymethyl chitosan and reducing agent, and the mixture was subjected to a reduction reaction at 35°C for 1.5 h. Then, ethanol was added for alcohol precipitation. After washing away the unreacted carboxymethyl chitosan and aldehyde-modified polyvinyl alcohol, the reduced graft copolymer was obtained.

[0110] (2) Dissolve the reduced graft copolymer, antimicrobial peptide (polylysine) and crosslinking agent (glutaraldehyde) obtained in step (1) in water (half of the total water volume), and carry out ionic crosslinking reaction at 35°C for 5 hours to obtain precursor gel solution;

[0111] (3) At a rotation speed of 200 rpm, the precursor gel solution obtained in step (2) is mixed with gallic acid and copper chloride, and the mixture is reacted at 23°C for 20 min to obtain a gel solution;

[0112] (4) Nitrogen gas is bubbled into the gel solution obtained in step (3). The volume ratio of gel solution to gas is 15:1 and the flow rate of nitrogen gas is 15 mL / min. Then the obtained system is placed in a mold for gradient freeze drying and then compressed to obtain polyvinyl alcohol / chitosan composite compression hemostatic sponge.

[0113] The gradient freeze-drying process consists of pre-freezing, initial drying, and desorption drying performed sequentially.

[0114] The pre-freezing is preferably gradient freezing, which is preferably performed sequentially as cooling pre-freezing, first pre-freezing, second pre-freezing, third pre-freezing and fourth pre-freezing.

[0115] The temperature for cooling and pre-freezing is 0°C; the cooling and pre-freezing time is 4 hours.

[0116] The temperature for the first pre-freezing is -20℃; the temperature for the second pre-freezing is -25℃; the temperature for the third pre-freezing is preferably -10℃; the temperature for the fourth pre-freezing is preferably -30℃; and the time for the first, second, third, and fourth pre-freezing is 1 hour.

[0117] The initial drying temperature is -5℃, and the initial drying time is 12 hours;

[0118] The temperature for the analytical drying process is 30°C, and the drying time is 24 hours.

[0119] Example 2

[0120] A polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function, wherein the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function comprises a double network hydrogel structure formed by carboxymethyl chitosan and aldehyde-modified polyvinyl alcohol.

[0121] The raw materials for preparing the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function by mass percentage are: 15% carboxymethyl chitosan, 18% aldehyde-modified polyvinyl alcohol, 6% antimicrobial peptide (polylysine), 5% crosslinking agent (glutaraldehyde), 3% gallic acid, 0.5% copper chloride, and the balance being water.

[0122] The degree of aldehyde alkylation of the aldehyde-based polyvinyl alcohol is 18%.

[0123] The preparation method of the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function is the same as that in Example 1.

[0124] Example 3

[0125] A polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function, wherein the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function comprises a double network hydrogel structure formed by carboxymethyl chitosan and aldehyde-modified polyvinyl alcohol.

[0126] The raw materials for preparing the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function by mass percentage are: 15% carboxymethyl chitosan, 20% aldehyde-modified polyvinyl alcohol, 6% antimicrobial peptide (polylysine), 5% crosslinking agent (glutaraldehyde), 4% gallic acid, 1% copper chloride, and the balance being water.

[0127] The degree of aldehyde alkylation of the aldehyde-based polyvinyl alcohol is 18%.

[0128] The preparation method of the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function is as follows:

[0129] (1) Dissolve carboxymethyl chitosan in an aqueous solution of acetic acid with a mass concentration of 2% (the water in the aqueous solution of acetic acid is half of the total water) to obtain a carboxymethyl chitosan solution; mix the carboxymethyl chitosan solution with aldehyde-modified polyvinyl alcohol under stirring at 100 rpm, and then carry out a Schiff base reaction at 35°C for 5 h to obtain a graft copolymer solution.

[0130] The graft copolymer solution was mixed with the reducing agent (sodium borohydride) at a molar ratio of 1:0.5, and the mixture was subjected to a reduction reaction at 40°C for 1 hour. Then, ethanol was added for alcohol precipitation. After washing away the unreacted carboxymethyl chitosan and aldehyde-modified polyvinyl alcohol, the reduced graft copolymer was obtained.

[0131] (2) Dissolve the reduced graft copolymer, antimicrobial peptide (polylysine) and crosslinking agent (glutaraldehyde) obtained in step (1) in water (half of the total water volume), and carry out ionic crosslinking reaction at 40°C for 6 hours to obtain precursor gel solution;

[0132] (3) At a rotation speed of 200 rpm, the precursor gel solution obtained in step (2) is mixed with gallic acid and copper chloride, and the mixture is reacted at 23°C for 20 min to obtain a gel solution;

[0133] (4) Nitrogen gas is bubbled into the gel solution obtained in step (3). The volume ratio of gel solution to gas is 15:1 and the flow rate of nitrogen gas is 15 mL / min. Then the obtained system is placed in a mold for gradient freeze drying and then compressed to obtain polyvinyl alcohol / chitosan composite compression hemostatic sponge.

[0134] The gradient freeze-drying process consists of pre-freezing, initial drying, and desorption drying performed sequentially.

[0135] The pre-freezing is preferably gradient freezing, which is preferably performed sequentially as cooling pre-freezing, first pre-freezing, second pre-freezing, third pre-freezing and fourth pre-freezing.

[0136] The temperature for cooling and pre-freezing is 0°C; the cooling and pre-freezing time is 4 hours.

[0137] The temperature for the first pre-freezing is -20℃; the temperature for the second pre-freezing is -35℃; the temperature for the third pre-freezing is preferably -20℃; the temperature for the fourth pre-freezing is preferably -35℃; and the time for the first, second, third, and fourth pre-freezing is 1 hour.

[0138] The initial drying temperature is -10℃, and the initial drying time is 12 hours;

[0139] The temperature for the analytical drying process was 35°C, and the drying time was 24 hours.

[0140] Comparative Example 1

[0141] A polyvinyl alcohol / chitosan composite compression hemostatic sponge, the raw materials for preparing the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function by weight percentage are: 10% chitosan, 15% polyvinyl alcohol, 5% crosslinking agent (glutaraldehyde) and the balance water.

[0142] The preparation method of the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function is as follows:

[0143] (1) Chitosan is dissolved in an aqueous solution of acetic acid with a mass concentration of 2% (the water in the aqueous solution of acetic acid is half of the total water) to obtain a chitosan solution; the chitosan solution is mixed with polyvinyl alcohol (50,000 to 100,000) under stirring at 100 rpm, and then Schiff base reaction is carried out at 35°C for 5 h to obtain a graft copolymer solution. Then ethanol is added for alcohol precipitation treatment. After washing the unreacted chitosan and polyvinyl alcohol, the graft copolymer is obtained.

[0144] (2) Dissolve the graft copolymer and crosslinking agent (glutaraldehyde) obtained in step (1) in water (half of the total water volume), and carry out an ionic crosslinking reaction at 35°C for 5 hours to obtain a gel solution;

[0145] (3) Nitrogen gas is bubbled into the gel solution obtained in step (2), the volume ratio of gel solution to gas is 15:1, the flow rate of nitrogen gas is 15 mL / min, and then the obtained system is placed in a mold for gradient freeze drying, and then compressed to obtain polyvinyl alcohol / chitosan composite compression hemostatic sponge.

[0146] The gradient freeze-drying method and parameters are the same as in Example 1.

[0147] Comparative Example 2

[0148] A polyvinyl alcohol / chitosan composite compression hemostatic sponge, wherein the raw materials for preparing the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function are, by mass percentage: 10% carboxymethyl chitosan, 15% aldehyde-modified polyvinyl alcohol, 6% antimicrobial peptide (polylysine), 5% crosslinking agent (glutaraldehyde), 3% gallic acid, 0.5% copper chloride, and the balance being water.

[0149] The degree of aldehyde alkylation of the aldehyde-modified polyvinyl alcohol is 15%.

[0150] The preparation method of the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function is as follows:

[0151] (1) Dissolve carboxymethyl chitosan in an aqueous solution of acetic acid with a mass concentration of 2% (the water in the aqueous solution of acetic acid is half of the total water) to obtain a carboxymethyl chitosan solution; mix the carboxymethyl chitosan solution with aldehyde-modified polyvinyl alcohol under stirring at 100 rpm, and then carry out a Schiff base reaction at 35°C for 5 h to obtain a graft copolymer solution.

[0152] The graft copolymer solution was mixed with a reducing agent (sodium borohydride) at a molar ratio of 1:0.5 for carboxymethyl chitosan and reducing agent, and the mixture was subjected to a reduction reaction at 35°C for 1.5 h. Then, ethanol was added for alcohol precipitation. After washing away the unreacted carboxymethyl chitosan and aldehyde-modified polyvinyl alcohol, the reduced graft copolymer was obtained.

[0153] (2) Dissolve the reduced graft copolymer, antimicrobial peptide (polylysine) and crosslinking agent (glutaraldehyde) obtained in step (1) in water (half of the total water volume), and carry out ionic crosslinking reaction at 35°C for 5 hours to obtain precursor gel solution;

[0154] (3) At a rotation speed of 200 rpm, the precursor gel solution obtained in step (2) is mixed with gallic acid and copper chloride, and the mixture is reacted at 23°C for 20 min to obtain a gel solution;

[0155] (4) Nitrogen gas is bubbled into the gel solution obtained in step (3). The volume ratio of the gel solution to the gas is 15:1 and the flow rate of nitrogen gas is 15 mL / min. Then the obtained system is placed in a mold and freeze-dried at -35°C for 48 h. After natural thawing, it is compressed to obtain polyvinyl alcohol / chitosan composite compression hemostatic sponge.

[0156] Comparative Example 3

[0157] A polyvinyl alcohol / chitosan composite compression hemostatic sponge, the raw materials for preparing the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function by weight percentage are: 10% carboxymethyl chitosan, 15% aldehyde-modified polyvinyl alcohol, 5% crosslinking agent (glutaraldehyde) and the balance water.

[0158] The degree of aldehyde alkylation of the aldehyde-modified polyvinyl alcohol is 15%.

[0159] The preparation method of the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function is as follows:

[0160] (1) Dissolve carboxymethyl chitosan in an aqueous solution of acetic acid with a mass concentration of 2% (the water in the aqueous solution of acetic acid is half of the total water) to obtain a carboxymethyl chitosan solution; mix the carboxymethyl chitosan solution with aldehyde-modified polyvinyl alcohol under stirring at 100 rpm, and then carry out a Schiff base reaction at 35°C for 5 h to obtain a graft copolymer solution.

[0161] The graft copolymer solution was mixed with a reducing agent (sodium borohydride) at a molar ratio of 1:0.5 for carboxymethyl chitosan and reducing agent, and the mixture was subjected to a reduction reaction at 35°C for 1.5 h. Then, ethanol was added for alcohol precipitation. After washing away the unreacted carboxymethyl chitosan and aldehyde-modified polyvinyl alcohol, the reduced graft copolymer was obtained.

[0162] (2) Dissolve the reduced graft copolymer and crosslinking agent (glutaraldehyde) obtained in step (1) in water (half of the total water volume), and carry out an ionic crosslinking reaction at 35°C for 5 hours to obtain a gel solution;

[0163] (3) Nitrogen gas is bubbled into the gel solution obtained in step (2), the volume ratio of gel solution to gas is 15:1, the flow rate of nitrogen gas is 15 mL / min, and then the obtained system is placed in a mold for gradient freeze drying, and then compressed to obtain polyvinyl alcohol / chitosan composite compression hemostatic sponge.

[0164] The gradient freeze-drying process is the same as in Example 1.

[0165] Comparative Example 4

[0166] A polyvinyl alcohol / chitosan composite compression hemostatic sponge, wherein the raw materials for preparing the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function are, by mass percentage: 10% carboxymethyl chitosan, 15% aldehyde-modified polyvinyl alcohol, 6% antimicrobial peptide (polylysine), 5% crosslinking agent (glutaraldehyde), and the balance being water.

[0167] The degree of aldehyde alkylation of the aldehyde-modified polyvinyl alcohol is 15%.

[0168] The preparation method of the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function is as follows:

[0169] (1) Dissolve carboxymethyl chitosan in an aqueous solution of acetic acid with a mass concentration of 2% (the water in the aqueous solution of acetic acid is half of the total water) to obtain a carboxymethyl chitosan solution; mix the carboxymethyl chitosan solution with aldehyde-modified polyvinyl alcohol under stirring at 100 rpm, and then carry out a Schiff base reaction at 35°C for 5 h to obtain a graft copolymer solution.

[0170] The graft copolymer solution was mixed with a reducing agent (sodium borohydride) at a molar ratio of 1:0.5 for carboxymethyl chitosan and reducing agent, and the mixture was subjected to a reduction reaction at 35°C for 1.5 h. Then, ethanol was added for alcohol precipitation. After washing away the unreacted carboxymethyl chitosan and aldehyde-modified polyvinyl alcohol, the reduced graft copolymer was obtained.

[0171] (2) Dissolve the reduced graft copolymer, antimicrobial peptide (polylysine) and crosslinking agent (glutaraldehyde) obtained in step (1) in water (half of the total water volume), and carry out ionic crosslinking reaction at 35°C for 5 hours to obtain a gel solution;

[0172] (3) Nitrogen gas is bubbled into the gel solution obtained in step (2), the volume ratio of gel solution to gas is 15:1, the flow rate of nitrogen gas is 15 mL / min, and then the obtained system is placed in a mold for gradient freeze drying, and then compressed to obtain polyvinyl alcohol / chitosan composite compression hemostatic sponge.

[0173] The gradient freeze-drying process is the same as in Example 1.

[0174] Test case

[0175] (1) The polyvinyl alcohol / chitosan composite compression hemostatic sponges prepared in Examples 1-4 and Comparative Examples 1 and 3 were subjected to swelling tests in physiological saline, and the results are as follows: Figure 1 As shown. From Figure 1 It can be seen that the swelling ratio of the polyvinyl alcohol / chitosan composite compression hemostatic sponges prepared in Examples 1-4 of this invention is significantly higher than that of Comparative Examples 1 and 3. In Comparative Example 1, since polyvinyl alcohol and chitosan are directly used as raw materials, they cannot form a double gel network, resulting in a lower swelling ratio of the prepared sponge. The swelling ratio of the polyvinyl alcohol / chitosan composite compression hemostatic sponge prepared in Comparative Example 3 is higher than that of Comparative Examples 1 and 2, but lower than that of the examples. This is because the absence of antimicrobial peptides is not conducive to the formation of a double-network gel, thus affecting the swelling ratio of the polyvinyl alcohol / chitosan composite compression hemostatic sponge.

[0176] (2) The BET test results of the polyvinyl alcohol / chitosan composite compression hemostatic sponges prepared in Examples 1-3 and Comparative Examples 1 and 4 are shown in Table 1:

[0177] Table 1. BET test results of polyvinyl alcohol / chitosan composite compression hemostatic agents prepared in Examples 1-3 and Comparative Examples 1 and 4

[0178]

[0179] As can be seen from the results in Table 1, the average pore size of the polyvinyl alcohol / chitosan composite compression hemostatic sponges prepared in Examples 1-3 is lower than that in Comparative Examples 1 and 4, and the pore size distribution is uniform.

[0180] (3) Determination of the in vitro blood coagulation index of the polyvinyl alcohol / chitosan composite compression hemostatic sponges prepared in Examples 1-4: First, accurately weigh 0.2g of each polyvinyl alcohol / chitosan composite compression hemostatic sponge prepared in each example and place it in a plastic dish, preheating it at 37℃ for 5min. Add 100μL of citric acid-anticoagulated whole blood to the hemostatic agent, then add 10μL of 0.2mol / L CaCl2 solution, and incubate the hemostatic agent at 37℃ for 5min. Then, add 25mL of deionized water to the plastic dish, shake and incubate at 37℃ for 10min to rinse the uncoagulated blood on the hemostatic agent. Finally, measure the absorbance of the uncoagulated blood solution at 540nm (Abs 1) using an ELISA reader (Varioskan LUX, ThermoFisher). Use the absorbance of 100μL of citric acid-anticoagulated whole blood in 25mL of deionized water as a blank control (Abs 0). The blood coagulation index (BCI) is calculated using the following formula I.

[0181] Formula I

[0182] Experimental results are as follows Figure 2 As shown, compared with the negative control group (blank group), the polyvinyl alcohol / chitosan composite compression hemostatic sponges prepared in each example all had in vitro procoagulant effects, and no significant effect was observed on the in vitro blood coagulation index of the samples by the liquid absorption ratio, antimicrobial peptide content and microporous structure.

[0183] (4) Antibacterial test: Take 10 mL of polyvinyl alcohol / chitosan composite compressed hemostatic sponge particles from Example 1 and add them to 10 mL of ... 7 Centrifuge tubes from the experimental and control groups were incubated at 37°C and 180 rpm for 21 hours in a constant temperature shaking incubator with cfu / mL Staphylococcus aureus and Escherichia coli bacterial suspensions. Since the concentrations of Staphylococcus aureus and Escherichia coli bacterial suspensions are positively correlated with the absorbance values ​​of the bacterial suspensions at 610 nm, the absorbance values ​​of each group were measured at a wavelength of 610 nm.

[0184] The results showed that in Example 1, the polyvinyl alcohol / chitosan composite compressed hemostatic sponge was placed at the bottom of a centrifuge tube and completely immersed in the mixture of bacterial solution and culture medium. After the material absorbed the liquid completely, it expanded to its maximum volume, and the bacterial solution became turbid. The absorbance values ​​of the bacterial solution after incubation are shown in Table 2. The results indicate that the hemostatic sponge has a certain antibacterial effect compared with the negative control group, and the comparison results are significantly different (P < 0.01).

[0185] Table 2. Antibacterial test results of the polyvinyl alcohol / chitosan composite compression hemostatic sponges prepared in Example 1 and Comparative Examples 1-4

[0186]

[0187] Note: * indicates p < 0.05

[0188] (5) Cytotoxicity experiment of polyvinyl alcohol / chitosan composite compression hemostatic sponges prepared in Example 1 and Comparative Example 2 of the present invention: The polyvinyl alcohol / chitosan composite compression hemostatic sponges prepared in Example 1 and Comparative Example 2 were sterilized by cobalt source irradiation and then immersed in RPMI 1640 culture medium (without serum, purchased from Thermo Fisher Scientific, USA) at 37°C for 24 h. The supernatant was filtered to obtain the extract. L929 cells (mouse fibroblasts, obtained from the National Experimental Cell Resource Sharing Platform) were seeded in 96-well plates (the wells were pre-filled with complete culture medium, including RPMI 1640 culture medium and fetal bovine serum), 5 × 10⁶ cells per well. 4 The 96-well plate was then incubated at 37°C for 24 hours. The plate was removed, the old solution was discarded, and 100 μL of the collected 100% and 50% concentration extracts (the 50% extract was obtained by mixing the 100% extract with an equal volume of RPMI 1640 medium) were added to each well. The plate was then incubated at 37°C for another 48 hours. After 48 hours, the plate was removed, and 20 μL of 5 mg / ml MTT (thiazolyl blue, purchased from Beijing Jingke Hongda) aqueous solution was added to each well. The plate was then incubated at 37°C for another 4 hours. Finally, the 96-well plate was removed, the old solution was carefully discarded, and 150 μL of DMSO (dimethyl sulfoxide) was added to each well. After shaking for 10 minutes, the OD value was read using a microplate reader. The test wavelength was 570 nm, and the reference wavelength was 630 nm. Six replicates were set for each sample. Blank culture medium (procedure was the same as the experimental group except hemostatic material was not added) and 0.5 wt% phenol aqueous solution served as the negative control and positive control, respectively. The relative growth rate (RGR%) was calculated as follows: RGR% = OD sample / OD negative control × 100%.

[0189] Depend on Figure 3 As can be seen from the negative control group, the relative cell proliferation rate of the 50% extract group of the polyvinyl alcohol / chitosan composite compressed hemostatic sponge prepared in Example 1 was greater than 100%, indicating that the polyvinyl alcohol / chitosan composite compressed hemostatic sponge of the present invention has no cytotoxicity. However, the polyvinyl alcohol / chitosan composite compressed hemostatic sponge prepared in Comparative Example 2 exhibited slight cytotoxicity. This was due to the addition of copper ions in Comparative Example 2, which produced slight toxicity to cells. However, according to the national standard for medical devices GB / T 16886.5 "Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Tests", it belongs to Class I cytotoxicity and is acceptable.

[0190] The results above demonstrate that the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function provided by this invention exhibits excellent antibacterial properties and stability. This is because, by controlling the raw materials and preparation method, the composite compression hemostatic sponge obtained by this invention comprises a double-network hydrogel structure formed by carboxymethyl chitosan and aldehyde-modified polyvinyl alcohol. The double-network hydrogel structure possesses high stability and fixation, enhancing the binding ability between chitosan and polyvinyl alcohol. The double-network hydrogel structure is covalently bonded, resulting in a low cross-linking density, allowing water molecules to more easily penetrate the network, leading to hydrogel expansion. Furthermore, the non-uniformity of the covalently bonded cross-linked network results in lower cross-linking densities in certain areas, making them more susceptible to water molecule penetration. Simultaneously, the electrostatic interactions in the double-network hydrogel form cross-linking points with a certain degree of dynamic reversibility. When the hydrogel is subjected to tensile or other mechanical stresses, these electrostatic cross-linking points can partially break and reform, thereby absorbing energy and dispersing stress, preventing damage to the network structure. This characteristic allows the electrostatic interactions to enhance the network structure while maintaining a certain degree of flexibility and elasticity. Therefore, the composite compression hemostatic sponge provided by this invention possesses excellent stability. The raw materials used in this invention include carboxymethyl chitosan, aldehyde-modified polyvinyl alcohol, and antimicrobial peptides, which are covalently linked to form a double-network hydrogel structure. By introducing gallic acid and copper chloride, this invention can form a metal-organic network structure, further improving the antibacterial properties of the composite compression hemostatic sponge.

[0191] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A polyvinyl alcohol / chitosan composite compression hemostatic sponge with bacteriostatic function, characterized in that, The polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function comprises a double network hydrogel structure formed by carboxymethyl chitosan and aldehyde-modified polyvinyl alcohol. The raw materials for preparing the polyvinyl alcohol / chitosan composite compression hemostatic sponge with antibacterial function, by mass percentage, include: 0.1-20% carboxymethyl chitosan, 0.4-50% aldehyde-modified polyvinyl alcohol, 0.1-10% antimicrobial peptide, 0.03-7% crosslinking agent, 0.1-5% gallic acid, 0.1-1% copper chloride, and the balance being water; The degree of aldehyde alkylation of the aldehyde-modified polyvinyl alcohol is 10-30%.

2. The polyvinyl alcohol / chitosan composite compression hemostatic sponge with bacteriostatic function according to claim 1, characterized in that, The method for preparing aldehyde-modified polyvinyl alcohol includes: mixing an aqueous solution of polyvinyl alcohol with an oxidant and carrying out an oxidation reaction to obtain aldehyde-modified polyvinyl alcohol.

3. The polyvinyl alcohol / chitosan composite compression hemostatic sponge with bacteriostatic function according to claim 2, characterized in that, The molar ratio of the oxidant to polyvinyl alcohol is 1:5~10.

4. The polyvinyl alcohol / chitosan composite compression hemostatic sponge with bacteriostatic function according to claim 2, characterized in that, The oxidation reaction is carried out at a temperature of 40-60℃ for 2-4 hours.

5. The polyvinyl alcohol / chitosan composite compression hemostatic sponge with bacteriostatic function according to claim 1, characterized in that, The antimicrobial peptides include one or more of polylysine, KR12, black soldier fly antimicrobial peptides, and nisin.

6. The polyvinyl alcohol / chitosan composite compression hemostatic sponge with bacteriostatic function according to claim 1, characterized in that, The crosslinking agent includes one or more of formaldehyde, glutaraldehyde, glyoxal, and epichlorohydrin.

7. The method for preparing the polyvinyl alcohol / chitosan composite compression hemostatic sponge with bacteriostatic function according to any one of claims 1-6, characterized in that, Includes the following steps: (1) Mix carboxymethyl chitosan, aldehyde-modified polyvinyl alcohol and water, and carry out Schiff base reaction to obtain graft copolymer solution; The graft copolymer solution was mixed with a reducing agent, and then subjected to a reduction reaction and alcohol precipitation treatment in sequence to obtain the reduced graft copolymer. (2) The reduced graft copolymer, water, antimicrobial peptide and crosslinking agent obtained in step (1) are mixed and subjected to ionic crosslinking reaction to obtain precursor gel solution; (3) The precursor gel solution obtained in step (2) is mixed with gallic acid and copper chloride, and after reaction, a gel solution is obtained; (4) After mixing the gel solution obtained in step (3) with gas, perform gradient freeze drying to obtain polyvinyl alcohol / chitosan composite compression hemostatic sponge; The gradient freeze-drying process includes pre-freezing, initial drying, and desorption drying performed sequentially. The pre-freezing temperature is -35~20℃, and the pre-freezing time is 5~20h; The initial drying temperature is -10~10℃, and the initial drying time is 8~24h; The temperature for the analytical drying process is 15~35℃, and the drying time is 8~24h.

8. The preparation method according to claim 7, characterized in that, The Schiff base reaction temperature in step (1) is 30~50℃; the Schiff base reaction time is 3~6h.

9. The preparation method according to claim 7, characterized in that, The temperature of the ion crosslinking reaction in step (2) is 30~60℃; the time of the ion crosslinking reaction is 3~6h.

10. The method of claim 7, wherein, In step (4), the pre-freezing is gradient freezing, which is a series of pre-freezing processes performed in sequence: cooling pre-freezing, first pre-freezing, second pre-freezing, third pre-freezing, and fourth pre-freezing. The temperature for cooling and pre-freezing is 0~20℃; the time for cooling and pre-freezing is 0.5~4h; The temperature for the first pre-freezing is -20~0℃; the temperature for the second pre-freezing is -35~-20℃; the temperature for the third pre-freezing is -20~0℃; and the temperature for the fourth pre-freezing is -35~-10℃. The time for the first, second, third, and fourth pre-freezing cycles is independently 0.5 to 3 hours.

Images