A chitosan composition with hemostatic and antibacterial efficacy and its preparation method and application

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to a chitosan composition with hemostatic and antibacterial effects, and a preparation method and application thereof. The chitosan is combined with one or two of water-soluble cellulose, sodium carboxymethyl starch, chondroitin sulfate, cationic hyaluronic acid, xanthan gum, porous starch, dextran, polyglycerol and cyclodextrin to prepare the chitosan composition. The chitosan has positive charges, can adsorb red blood cells to promote blood coagulation, and has natural antibacterial activity. The chondroitin sulfate and cationic hyaluronic acid are beneficial to improving the hydrophilicity of the system, enhancing the infiltration of the wound, reducing inflammation, and repairing the wound. Meanwhile, the interaction of the raw materials is beneficial to improving the water solubility and mechanical properties of the chitosan composition. Therefore, the chitosan composition provided by the present application not only has a simple preparation method, but also can be used to prepare hemostatic and / or antibacterial products, and has the effects of rapid hemostasis and persistent antibacterial effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a chitosan composition with hemostatic and antibacterial effects, its preparation method, and its application. Background Technology

[0002] In clinical trauma care, uncontrolled bleeding in the pre-traumatic period, secondary infections, and related complications are core risk factors leading to disability and death in trauma patients, and represent a critical clinical challenge that urgently needs to be addressed in the field of wound repair. Following severe trauma, ruptured blood vessels in the wound can cause persistent and uncontrolled bleeding, leading to hemorrhagic shock, insufficient tissue perfusion, and multiple organ dysfunction within a short period, significantly increasing early mortality. Simultaneously, trauma disrupts the natural barrier structure of the skin and mucous membranes, allowing external bacteria to easily invade the wound. Combined with the accumulation of wound exudate and decreased local immune function caused by bleeding, favorable conditions are created for bacterial proliferation, easily inducing secondary wound infections, increasing patient suffering and the difficulty of clinical treatment.

[0003] Developing bioproducts with hemostatic and antibacterial effects is a pressing challenge in this field. For example, Chinese patent CN105412975 A provides a biocompatible hemostatic product and tissue occlusive agent comprising polyoxyethylene particles. The viscosity-average molecular weight of the polyoxyethylene is between 100,000 and 7,000,000 Daltons, the particle size of the polyoxyethylene particles is between 0.5 μm and 2000 μm, and the water absorption ratio of the polyoxyethylene particles is 1 to 500. This hemostatic product forms a gel that seals bleeding wounds after absorbing water, promoting wound healing and stopping bleeding. However, the preparation method of the above-mentioned technical solution is relatively complex, and the product does not have antibacterial effects. That is, existing wound hemostasis, antibacterial, and healing-promoting treatment technologies are mostly single-function interventions, lacking a comprehensive treatment plan that can simultaneously achieve rapid hemostasis and broad-spectrum antibacterial effects. Therefore, providing a chitosan composition with a simple preparation method and both hemostatic and antibacterial effects has broad prospects for clinical application and industrialization. Summary of the Invention

[0004] The purpose of this invention is to provide a chitosan composition with hemostatic and antibacterial effects, its preparation method and application, which has the advantages of simple preparation method, rapid hemostasis and long-lasting antibacterial effect.

[0005] This invention provides a chitosan composition with hemostatic and antibacterial effects. The chitosan composition comprises the following raw materials in parts by weight: 5-8 parts chitosan, 3-5 parts substance A, 0.8-1.5 parts crosslinking agent, 3-5 parts auxiliary agent, and 100-120 parts water. The substance A includes one or two of the following: water-soluble cellulose, sodium carboxymethyl starch, chondroitin sulfate, cationic hyaluronic acid, xanthan gum, porous starch, dextran, polyglycerol, and cyclodextrin.

[0006] Preferably, when the substance A comprises chondroitin sulfate and porous starch, the mass ratio of chondroitin sulfate to porous starch is 0.8-1.3:1.2-2.8; When substance A includes cyclodextrin and cationic hyaluronic acid, the mass ratio of cyclodextrin to cationic hyaluronic acid is 1.2-2.2:0.5-1.1.

[0007] Preferably, the chitosan composition comprises the following raw materials in parts by weight: 8 parts chitosan, 3 parts substance A, 0.8 parts crosslinking agent, 3 parts auxiliary agent, and 100 parts water; wherein substance A is composed of chondroitin sulfate and porous starch, and the mass ratio of chondroitin sulfate to porous starch is 0.8:1.2. Alternatively, by mass, the chitosan composition comprises the following raw materials: 5 parts chitosan, 5 parts substance A, 0.8 parts crosslinking agent, 3 parts auxiliary agent, and 120 parts water; substance A is composed of cyclodextrin and cationic hyaluronic acid, with a mass ratio of cyclodextrin to cationic hyaluronic acid of 1.5:0.5.

[0008] Preferably, the water-soluble cellulose includes one or more of sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, methylcellulose, hydroxyethyl cellulose, and hydroxypropyl cellulose.

[0009] Preferably, the crosslinking agent includes: epichlorohydrin, tetra-arm polyethylene glycol glycidyl ether, pentaerythritol tetraglycidyl ether, octa-arm polyethylene glycol glycidyl ether, 1,4-butanediol diglycidyl ether, or glycerol triglycidyl ether.

[0010] Preferably, the additives include acetic acid, formic acid, or glacial acetic acid.

[0011] This invention provides a method for preparing the chitosan composition described above, comprising the following steps: Chitosan, substance A, additives and water were mixed evenly and sterilized by irradiation to obtain an aqueous system; a cross-linking agent was added to the aqueous system for cross-linking and homogenization to obtain a homogeneous system; the homogeneous system was freeze-dried to obtain a chitosan composition.

[0012] Preferably, the freeze-drying temperature is -30°C to -50°C, and the time is 6-12 hours; The irradiation dose for the irradiation sterilization is 3-8 kGy.

[0013] The present invention provides the application of the chitosan composition described above in the preparation of hemostatic and / or antibacterial products.

[0014] This invention provides a product with hemostatic and antibacterial effects, the product comprising: the chitosan composition described in the above technical solution.

[0015] Beneficial effects: This invention provides a chitosan composition with hemostatic and antibacterial effects, achieved by selecting chitosan in combination with one or two of the following: water-soluble cellulose, sodium carboxymethyl starch, chondroitin sulfate, cationic hyaluronic acid, xanthan gum, porous starch, dextran, polyglycerol, and cyclodextrin. Chitosan, carrying a positive charge, can adsorb red blood cells and promote coagulation, while also possessing natural antibacterial activity. Chondroitin sulfate, cationic hyaluronic acid, and other substances improve the system's hydrophilicity, enhance wound infiltration, reduce inflammation, and repair wounds. Furthermore, the interaction of these raw materials enhances the water solubility and mechanical properties of the chitosan composition. Therefore, the chitosan composition provided by this invention is not only simple to prepare but can also be used to prepare hemostatic and / or antibacterial products, exhibiting rapid hemostasis and long-lasting antibacterial effects. Detailed Implementation

[0016] In this invention, unless otherwise specified, the equipment, reagents and methods used are all conventional selections.

[0017] The porous starch described in this invention is corn porous starch, with a porosity of 50%-60% and an average pore size of 0.8-1.2 μm.

[0018] The molecular weight of the chitosan used in this invention is preferably 150-700 kDa. In specific embodiments and comparative examples, the molecular weight of the chitosan used is 300 kDa.

[0019] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0020] Example 1 The steps for preparing a chitosan composition with hemostatic and antibacterial effects are as follows: (1) Raw material composition The chitosan composition comprises, by weight, the following raw materials: 8 parts chitosan, 3 parts substance A, 0.8 parts crosslinking agent, 3 parts additives, and 100 parts water; The substance A is composed of chondroitin sulfate and porous starch, with a mass ratio of chondroitin sulfate to porous starch of 0.8:1.2. The crosslinking agent is four-arm polyethylene glycol glycidyl ether; The auxiliary agent is acetic acid.

[0021] (2) Preparation method Chitosan, substance A, additives and water are mixed evenly to obtain a mixture; The mixture was sterilized by irradiation at 3 kGy to obtain an aqueous system; A crosslinking agent was added to an aqueous system to perform crosslinking and homogenization, resulting in a homogeneous system. The homogenized system was freeze-dried at -30℃ for 12 hours to obtain a chitosan composition.

[0022] Example 2 The steps for preparing a chitosan composition with hemostatic and antibacterial effects are as follows: (1) Raw material composition The chitosan composition comprises, by weight, the following raw materials: 5 parts chitosan, 5 parts substance A, 0.8 parts crosslinking agent, 3 parts additives, and 120 parts water; The substance A is composed of cyclodextrin and cationic hyaluronic acid, with a mass ratio of cyclodextrin to cationic hyaluronic acid of 1.5:0.5; The crosslinking agent is four-arm polyethylene glycol glycidyl ether; The auxiliary agent is acetic acid.

[0023] (2) Preparation method Chitosan, substance A, additives and water are mixed evenly to obtain a mixture; The mixture was sterilized by irradiation at 8 kGy to obtain an aqueous system; A crosslinking agent was added to an aqueous system to perform crosslinking and homogenization, resulting in a homogeneous system. The homogenized system was freeze-dried at -50℃ for 6 hours to obtain a chitosan composition.

[0024] Example 3 The only difference from Example 1 is that in Example 3, the tetra-arm polyethylene glycol glycidyl ether in Example 1 is replaced with 1,4-butanediol diglycidyl ether.

[0025] Example 4 The only difference from Example 2 is that in Example 4, the tetra-arm polyethylene glycol glycidyl ether in Example 2 is replaced with 1,4-butanediol diglycidyl ether.

[0026] Comparative Example 1 The only difference from Example 1 is step (1). Step (1) of Comparative Example 1 is as follows: (1) Raw material composition The chitosan composition comprises, by weight, the following raw materials: 8 parts chitosan, 1 part substance A, 0.8 parts crosslinking agent, 3 parts additives, and 100 parts water; The substance A is composed of chondroitin sulfate and porous starch, with a mass ratio of chondroitin sulfate to porous starch of 0.8:1.2. The crosslinking agent is four-arm polyethylene glycol glycidyl ether; The auxiliary agent is acetic acid.

[0027] Comparative Example 2 The only difference from Example 1 is step (2). Step (2) of Comparative Example 2 is as follows: (2) Preparation method Chitosan, substance A, additives and water are mixed evenly to obtain an aqueous system; A crosslinking agent was added to an aqueous system to perform crosslinking and homogenization, resulting in a homogeneous system. The homogenized system was freeze-dried at -30℃ for 12 hours to obtain a chitosan composition.

[0028] Comparative Example 3 The only difference from Example 1 is step (1). Step (1) of Comparative Example 3 is as follows: (1) Raw material composition The chitosan composition comprises, by weight, the following raw materials: 8 parts chitosan, 3 parts substance A, 0.8 parts crosslinking agent, 3 parts additives, and 100 parts water; The substance A is composed of chondroitin sulfate and porous starch, with a mass ratio of chondroitin sulfate to porous starch of 4:1. The crosslinking agent is four-arm polyethylene glycol glycidyl ether; The auxiliary agent is acetic acid.

[0029] Comparative Example 4 The only difference from Example 1 is step (1). Step (1) of Comparative Example 4 is as follows: (1) Raw material composition The composition comprises, by weight parts, the following raw materials: 8 parts dextran, 3 parts substance A, 0.8 parts crosslinking agent, 3 parts additives, and 100 parts water; The substance A is composed of chondroitin sulfate and porous starch, with a mass ratio of chondroitin sulfate to porous starch of 0.8:1.2. The crosslinking agent is four-arm polyethylene glycol glycidyl ether; The auxiliary agent is acetic acid.

[0030] Comparative Example 5 The only difference from Example 2 is step (1). Step (1) of Comparative Example 5 is as follows: (1) Raw material composition The chitosan composition comprises, by weight, the following raw materials: 5 parts chitosan, 10 parts substance A, 0.8 parts crosslinking agent, 3 parts additives, and 120 parts water; The substance A is composed of cyclodextrin and cationic hyaluronic acid, with a mass ratio of cyclodextrin to cationic hyaluronic acid of 1.5:0.5; The crosslinking agent is four-arm polyethylene glycol glycidyl ether; The auxiliary agent is acetic acid.

[0031] Comparative Example 6 The only difference from Example 2 is step (2). Step (2) of Comparative Example 6 is as follows: (2) Preparation method Chitosan, substance A, additives and water are mixed evenly to obtain an aqueous system; A crosslinking agent was added to an aqueous system to perform crosslinking and homogenization, resulting in a homogeneous system. The homogenized system was freeze-dried at -30℃ for 12 hours to obtain a chitosan composition.

[0032] Comparative Example 7 The only difference from Example 2 is step (1). Step (1) of Comparative Example 7 is as follows: (1) Raw material composition The chitosan composition comprises, by weight, the following raw materials: 5 parts chitosan, 5 parts substance A, 0.8 parts crosslinking agent, 3 parts additives, and 120 parts water; The substance A is composed of cyclodextrin and cationic hyaluronic acid, with a mass ratio of cyclodextrin to cationic hyaluronic acid of 1:5; The crosslinking agent is four-arm polyethylene glycol glycidyl ether; The auxiliary agent is acetic acid.

[0033] Comparative Example 8 The only difference from Example 2 is step (1). Step (1) of Comparative Example 8 is as follows: (1) Raw material composition The composition comprises, by weight parts, the following raw materials: 5 parts dextran, 5 parts substance A, 0.8 parts crosslinking agent, 3 parts additives, and 120 parts water; The substance A is composed of cyclodextrin and cationic hyaluronic acid, with a mass ratio of cyclodextrin to cationic hyaluronic acid of 1.5:0.5; The crosslinking agent is four-arm polyethylene glycol glycidyl ether; The auxiliary agent is acetic acid.

[0034] Hemostasis function test Products prepared in Examples 1-4 and Comparative Examples 1-8 were tested. Each product was labeled sequentially as Example 1-4 and Comparative Examples 1-8. Cell viability was assessed according to GB / T 16886.5-2017 to evaluate the cytotoxicity of the corresponding products. Hemostasis time was tested according to YY / T 1477.5-2020. The results are shown in Table 1.

[0035] Table 1 Hemostatic function of different products

[0036] Based on the results in Table 1, both the products prepared in the examples and the yields prepared in the comparative examples have good hemostatic effects and high cell survival rates.

[0037] Antibacterial efficacy test (1) Antibacterial effect of Staphylococcus aureus The products prepared in Examples 1-4 and Comparative Examples 1-8 were tested respectively. Each product was labeled as Test Sample 1-Test Sample 12. LB liquid culture medium was used as a negative control.

[0038] Staphylococcus aureus (ATCC 25923) was used as the test strain. 1 mL of the test sample was added to each well of a 24-well plate (each test sample constituted one treatment, and each treatment was performed in triplicate). The negative control group underwent the same procedure. 10 μL (10 μL of the test sample) was added to the surface of each test sample (or negative control). 6 A suspension of Staphylococcus aureus (ATCC 25923) was prepared and incubated for 24 hours. The bactericidal rate of each treatment was determined by viable cell counting. The formula for calculating the bactericidal rate is as follows: 24-hour sterilization rate = (live bacteria concentration in negative control group) (24h live bacteria concentration in the test group) / (live bacteria concentration in the negative control group) × 100%.

[0039] Staphylococcus aureus (ATCC 25923) was used as the test strain. 1 mL of the test sample was added to each well of a 24-well plate (each test sample constituted one treatment, and each treatment was performed in triplicate). The negative control group underwent the same procedure. 10 μL (10 μL of the test sample) was added to the surface of each test sample (or negative control). 6 A suspension of Staphylococcus aureus (ATCC 25923) was prepared and incubated for 72 hours. The bactericidal rate of each treatment was determined by viable cell counting. The formula for calculating the bactericidal rate is as follows: 72h sterilization rate = (live bacteria concentration in negative control group) (72h viable bacterial concentration in the test group) / (viable bacterial concentration in the negative control group) × 100%.

[0040] The sterilization rates of the above treatments are shown in Table 2 (Table 2 shows the mean values).

[0041] Table 2. Bactericidal rates of different treatments against Staphylococcus aureus

[0042] Based on the data in Table 2, both the samples prepared in the examples and the comparative examples showed good bactericidal effects against Staphylococcus aureus within 24 hours. However, as time went on, the antibacterial effect of the comparative examples decreased to varying degrees, while the samples in the examples still maintained an antibacterial effect of over 95%.

[0043] (2) Antibacterial effect of Escherichia coli The products prepared in Examples 1-4 and Comparative Examples 1-8 were tested respectively. Each product was labeled as Test Sample 1-Test Sample 12. LB liquid culture medium was used as a negative control.

[0044] Using *Escherichia coli* (ATCC 25922) as the test strain, 1 mL of the test sample was added to each well of a 24-well plate (each test sample constituted one treatment, and each treatment was performed in triplicate); the negative control group underwent the same procedure. 10 μL (10 μL of the test sample) was added to the surface of each test sample (or negative control). 6 A bacterial suspension of *Escherichia coli* (ATCC 25922) was prepared and incubated for 24 hours. The bactericidal rate of each treatment was determined using a viable count method. The formula for calculating the bactericidal rate is as follows: 24-hour sterilization rate = (live bacteria concentration in negative control group) (24h live bacteria concentration in the test group) / (live bacteria concentration in the negative control group) × 100%.

[0045] Using *Escherichia coli* (ATCC 25922) as the test strain, 1 mL of the test sample was added to each well of a 24-well plate (each test sample constituted one treatment, and each treatment was performed in triplicate); the negative control group underwent the same procedure. 10 μL (10 μL of the test sample) was added to the surface of each test sample (or negative control). 6 A bacterial suspension of *Escherichia coli* (ATCC 25922) was prepared and incubated for 72 hours. The bactericidal rate of each treatment was determined using a viable count method. The formula for calculating the bactericidal rate is as follows: 72h sterilization rate = (live bacteria concentration in negative control group) (72h viable bacterial concentration in the test group) / (viable bacterial concentration in the negative control group) × 100%.

[0046] The sterilization rates of the above treatments are shown in Table 3 (Table 3 shows the mean values).

[0047] Table 3. Sterilization rate of different treatments against Escherichia coli

[0048] Based on the data in Table 3, all tested samples showed good bactericidal effects against Escherichia coli at 24h, but the antibacterial rate of the comparative sample decreased rapidly at 72h, and it could not exert its antibacterial effect for a long time.

[0049] In summary, the present invention combines chitosan, chondroitin sulfate, and porous starch in a specific ratio; or combines chitosan, cyclodextrin, and cationic hyaluronic acid in a specific ratio, which helps to enhance the hemostatic effect of chitosan, reduce inflammation, and provide sustained antibacterial activity. Therefore, the product prepared by the present invention has the effects of rapid hemostasis and long-lasting antibacterial activity.

[0050] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A chitosan composition with hemostatic and antibacterial effects, characterized in that, The chitosan composition comprises, by weight, 5-8 parts chitosan, 3-5 parts substance A, 0.8-1.5 parts crosslinking agent, 3-5 parts additives, and 100-120 parts water; The substance A includes one or two of the following: water-soluble cellulose, sodium carboxymethyl starch, chondroitin sulfate, cationic hyaluronic acid, xanthan gum, porous starch, dextran, polyglycerol, and cyclodextrin.

2. The chitosan composition according to claim 1, characterized in that, When substance A comprises chondroitin sulfate and porous starch, the mass ratio of chondroitin sulfate to porous starch is 0.8-1.3:1.2-2.8; When substance A includes cyclodextrin and cationic hyaluronic acid, the mass ratio of cyclodextrin to cationic hyaluronic acid is 1.2-2.2:0.5-1.

1.

3. The chitosan composition according to claim 1 or 2, characterized in that, The chitosan composition comprises the following raw materials in parts by weight: 8 parts chitosan, 3 parts substance A, 0.8 parts crosslinking agent, 3 parts auxiliary agent, and 100 parts water; substance A is composed of chondroitin sulfate and porous starch, with a mass ratio of chondroitin sulfate to porous starch of 0.8:1.

2. Alternatively, by mass, the chitosan composition comprises the following raw materials: 5 parts chitosan, 5 parts substance A, 0.8 parts crosslinking agent, 3 parts auxiliary agent, and 120 parts water; substance A is composed of cyclodextrin and cationic hyaluronic acid, with a mass ratio of cyclodextrin to cationic hyaluronic acid of 1.5:0.

5.

4. The chitosan composition according to claim 1, characterized in that, The water-soluble cellulose includes one or more of sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, methylcellulose, hydroxyethyl cellulose, and hydroxypropyl cellulose.

5. The chitosan composition according to claim 1, characterized in that, The crosslinking agent includes: epichlorohydrin, tetra-arm polyethylene glycol glycidyl ether, pentaerythritol tetraglycidyl ether, octa-arm polyethylene glycol glycidyl ether, 1,4-butanediol diglycidyl ether, or glycerol triglycidyl ether.

6. The chitosan composition according to claim 1, characterized in that, The additives include acetic acid, formic acid, or glacial acetic acid.

7. A method for preparing the chitosan composition according to any one of claims 1-6, characterized in that, Includes the following steps: Chitosan, substance A, additives and water were mixed evenly and sterilized by irradiation to obtain an aqueous system; a cross-linking agent was added to the aqueous system for cross-linking and homogenization to obtain a homogeneous system; the homogeneous system was freeze-dried to obtain a chitosan composition.

8. The preparation method according to claim 7, characterized in that, The freeze-drying temperature is -30℃ to -50℃, and the time is 6-12 hours; The irradiation dose for the irradiation sterilization is 3-8 kGy.

9. The use of the chitosan composition according to any one of claims 1-6 in the preparation of hemostatic and / or antibacterial products.

10. A product with hemostatic and antibacterial effects, characterized in that, The product comprises: the chitosan composition according to any one of claims 1-6.

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