Preparation method of chitosan composite antibacterial material

Abstract

The invention discloses a preparation method of a chitosan composite antibacterial material, and belongs to the technical field of composite material preparation, chitosan and quaternary ammonium chitosan in a reasonable proportion are prepared to prepare chitosan composite gel, the chitosan composite gel is applied to the field of medical dressings of the composite antibacterial material, and the composite antibacterial material has good biocompatibility and a cell proliferation promoting function; the chitosan composite gel has the advantages that enough antibacterial performance is guaranteed while cytotoxicity of quaternary ammonium chitosan is reduced, a mixed solution and a 56w / v% sodium beta-glycerophosphate hydrate solution in a reasonable proportion are selected, effective substances of the chitosan composite gel are guaranteed to be released quickly, wound healing speed is increased, and wound healing time is shortened. The pore size of the porous structure in the prepared chitosan composite gel is similar to the size of granulation tissues of a human body, so that more fibroblasts, fibrocytes and collagen can be accommodated, loaded and adhered, the transportation of oxygen and nutrient substances at a wound can be enhanced, and the capability of adsorbing wound exudate is improved.

Description

Technical Field

[0001] The present invention discloses a composite material preparation technology, especially a preparation method of a chitosan composite antibacterial material. Background Art

[0002] Chitosan is a product obtained by removing part of the acetyl groups from natural polysaccharide chitin, and has various physiological functions such as biodegradability, biocompatibility, non-toxicity, bacteriostasis, anti-cancer, lipid-lowering, and immune enhancement. It is widely used in food additives, textiles, agriculture, environmental protection, beauty care, cosmetics, antibacterial agents, medical fibers, medical dressings, artificial tissue materials, drug sustained-release materials, gene transduction vectors, biomedical fields, medical absorbable materials, tissue engineering carrier materials, medical treatment, and drug development, etc., and other daily chemical industries.

[0003] Chitosan is widely used as an antibacterial material in medical dressings. However, the antibacterial performance of medical dressings prepared from conventional chitosan is insufficient and is easily affected by environmental conditions such as solubility, etc., resulting in the antibacterial property not being fully exerted. And the influence of chitosan on the wound cell healing activity needs to be considered. Therefore, a chitosan composite antibacterial material is needed. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method of a chitosan composite antibacterial material to solve the above problems.

[0005] To achieve the above purpose, the present invention provides the following technical solution: A preparation method of a chitosan composite antibacterial material, comprising the following steps:

[0006] S1, Mix chitosan and quaternary ammonium chitosan in proportion and dissolve them in an acetic acid solution. After adding nano-hydroxyapatite powder, stir and dissolve them fully with a magnetic stirrer to form a mixed solution. Place the 56 w / v% β-glycerophosphate hydrate solution and the mixed solution in a cooling box and cool them at 4°C for 30 min. Under an ice-water bath, add the β-glycerophosphate hydrate solution to the mixed solution, and then stir to obtain an emulsion solution;

[0007] S2, Dissolve polybutyl acrylate in a dimethyl sulfoxide solution and stir. Add EDC hydrochloride and hydroxy succinimide to react, then add a 10 w / v% gelatin solution and stir. Adjust the pH value to 5.0 with hydrochloric acid and continue to react for 24 h. After the reaction, dialyze with an industrial dialysis machine for 48 h. After dialysis, freeze-dry for 24 h and grind to obtain a white powder. Dissolve the white powder in deionized water, and then mix and stir it with a 10 w / v% polyvinyl alcohol solution until a solid gel is formed;

[0008] S3. Dissolve the milky solution and the blank liposomes modified with nano-hydroxyapatite in chloroform, and perform rotary evaporation at 38 °C using a rotary evaporator. After removing the organic solvent, a lipid film compound is obtained. Add a phosphate buffer solution with ascorbic acid, methacrylic acid, and 5-sulfosalicylic acid to the lipid film compound for hydration, transfer it to an ultrasonic machine for ultrasonic treatment, and then filter it through a filter membrane to obtain liposomes. The obtained liposomes are freeze-dried to obtain liposome raw materials;

[0009] S4. Add the liposome raw materials to the solid-state gel, mix them evenly, and then transfer them to a 10 w / v% polyvinyl alcohol solution and stir until the gel appears, thus preparing the chitosan composite gel.

[0010] Preferably, in S1, the mass ratio of chitosan to quaternary ammonium chitosan is 0.85:0.15.

[0011] Preferably, in S1, the volume ratio of the mixed solution to the 56 w / v% β-glycerophosphate hydrate solution is 3:1.

[0012] Preferably, in S3, the addition amount of the phosphate buffer solution with ascorbic acid is 2 mg / ml.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] First, prepare a chitosan composite gel by configuring a reasonable ratio of chitosan and quaternary ammonium chitosan for use as a composite antibacterial material in the field of medical dressings. It has good biocompatibility and cell proliferation promotion function, reduces the cytotoxicity of quaternary ammonium chitosan while ensuring sufficient antibacterial performance, and selects a reasonable ratio of the mixed solution and the 56 w / v% β-glycerophosphate hydrate solution to ensure rapid release of the effective substances in the chitosan composite gel and improve the wound healing speed;

[0015] Second, the pore size of the porous structure in the prepared chitosan composite gel is similar to the size of human granulation tissue, which can accommodate and adhere more fibroblasts, fibrocytes, and collagen. This is beneficial to strengthening the transportation of oxygen and nutrients at the wound and improving the ability to adsorb wound exudate;

[0016] Third, form a sulfonic acid group spiropyran derivative through the reaction of methacrylic acid and 5-sulfosalicylic acid with the gel system, and realize the protection and release of ascorbic acid by means of the conversion charge change between the colorless closed-loop spiropyran SP and the colored open-loop merocyanine MC in the sulfonic acid group spiropyran derivative under light irradiation. Description of the Drawings

[0017] Figure 1 It is a fluorescence microscopy image of the gel prepared without using chitosan and quaternary ammonium chitosan corresponding to live / dead cell staining;

[0018] Figure 2 The figure shows the staining of live / dead cells of the chitosan composite gel prepared with pure chitosan under a fluorescence microscope;

[0019] Figure 3 The figure shows the staining of live / dead cells of the chitosan composite gel prepared with a mixture of chitosan and quaternary ammonium chitosan with a mass ratio of 0.95:0.05 under a fluorescence microscope;

[0020] Figure 4 The figure shows the staining of live / dead cells of the chitosan composite gel prepared with a mixture of chitosan and quaternary ammonium chitosan with a mass ratio of 0.90:0.10 under a fluorescence microscope;

[0021] Figure 5 The figure shows the staining of live / dead cells of the chitosan composite gel prepared with a mixture of chitosan and quaternary ammonium chitosan with a mass ratio of 0.85:0.15 under a fluorescence microscope;

[0022] Figure 6 The figure shows the staining of live / dead cells of the chitosan composite gel prepared with a mixture of chitosan and quaternary ammonium chitosan with a mass ratio of 0.80:0.20 under a fluorescence microscope;

[0023] Figure 7 The figure shows the swelling ratio chart of the chitosan composite gel prepared with different volume ratios of the mixed solution and 56 w / v% β-glycerophosphate hydrate solution;

[0024] Figure 8 The figure shows the chart of the proportion of residues after release of the chitosan composite gel prepared with different volume ratios of the mixed solution and 56 w / v% β-glycerophosphate hydrate solution;

[0025] Figure 9 The SEM image of the chitosan composite gel prepared by Scheme III at 5 μm;

[0026] Figure 10 The SEM image of the chitosan composite gel prepared by Scheme IV at 5 μm. Detailed implementation manners

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. In the description of the present invention, it should be understood that the orientation or positional relationships indicated by the terms "upper", "lower", "front", "rear", "left", "right", "top", "bottom", "inner", "outer", etc. are based on the orientation or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0028] A preparation method of a chitosan composite antibacterial material includes the following steps:

[0029] S1, mixing chitosan and quaternary ammonium chitosan in a proportion and dissolving them in a 0.1 mol / L acetic acid solution. The quaternary ammonium chitosan has a substitution degree of 90%. After adding nano-hydroxyapatite powder, it is fully stirred and dissolved by a magnetic stirrer to form a mixed solution. A 56 w / v% β-glycerophosphate hydrate solution and the mixed solution are placed in a cooling box and cooled at 4°C for 30 min. Under an ice-water bath at a temperature of 4°C, the β-glycerophosphate hydrate solution is added to the mixed solution, and then stirred to obtain an emulsion solution;

[0030] The cell compatibility of the prepared chitosan composite gels was tested by mixing chitosan and quaternary ammonium chitosan in different proportions in each group (without using chitosan and quaternary ammonium chitosan, pure chitosan, 0.95:0.05, 0.90:0.10, 0.85:0.15, 0.80:0.20). Here, the volume ratio of the mixed solution and the 56 w / v% β-glycerophosphate hydrate solution used during preparation is 3:1. The test method is to select mouse embryo somatic cells in the logarithmic growth phase and grow them in a medium containing 10% fetal bovine serum, 1% penicillin, and streptomycin. The temperature of the incubator is set at 37°C, and the carbon dioxide concentration is 5%. The prepared chitosan composite gels in different groups are extracted and injected into a cell culture plate, and the cells grown in the medium are inoculated into the cell culture plate. After incubating in the incubator for 3 days, a live / dead cell staining experiment is carried out to detect the biocompatibility of each group;

[0031] The number of live cells and dead cells in each group was observed under a fluorescence microscope. As Figure 1-6 shown in the corresponding live / dead cell staining diagram, green represents live cells, and red represents dead cells. Except for not using chitosan and quaternary ammonium chitosan, from Figure 3-6It can be seen that as the proportion of quaternary ammonium chitosan increases, the number of dead cells shows an increasing trend. When the proportion reaches 0.80:0.20, the number of dead cells increases significantly. The reason is that a high proportion of quaternary ammonium chitosan has a certain toxicity to cells, and it is necessary to reduce the proportion of quaternary ammonium chitosan to reduce the toxicity to cells. From Figure 2 - Figure 5 It can be seen that both the pure chitosan or the chitosan composite gel prepared by mixing chitosan and quaternary ammonium chitosan can increase the number of live cells, have good biocompatibility, and can promote cell proliferation. Moreover, the quaternary ammonium chitosan has a better proliferation-promoting effect. Considering the cytotoxicity and cell compatibility, a 0.85:0.15 ratio of chitosan and quaternary ammonium chitosan is selected for preparation.

[0032] S2. Dissolve butyl acrylate in dimethyl sulfoxide solution and stir to prepare a 12.8 g / L butyl acrylate-dimethyl sulfoxide solution. Add 10 g / L EDC hydrochloride and 5 g / L hydroxy succinimide according to the solution volume and react for 30 min. Then add a 10 w / v% gelatin solution and stir. Adjust the pH value to 5.0 with hydrochloric acid and continue to react for 24 h. After the reaction, dialyze for 48 h with an industrial dialysis machine, changing the water every 2 h. After dialysis, freeze-dry for 24 h and grind to obtain a white powder. Dissolve the white powder in deionized water and then mix it with a 10 w / v% polyvinyl alcohol solution and stir until a solid gel is formed.

[0033] S3. Dissolve the emulsion solution and the blank liposomes modified with nano-hydroxyapatite in chloroform at a mass ratio of 1:5. The concentration of the solution after dissolution is 0.1 g / ml. Rotate and evaporate the solution at 38 °C with a rotary evaporator to remove the organic solvent and obtain a lipid film compound. Add 2 mg / ml phosphate buffer solution with ascorbic acid, methacrylic acid, and 5-sulfosalicylic acid to the lipid film compound for hydration. Transfer it to an ultrasonic machine and ultrasonically treat it at a power of 200 w for 10 min, and then filter it through a 0.22 μm filter membrane to obtain liposomes. Freeze-dry the obtained liposomes to obtain liposome raw materials.

[0034] S4. Add the liposome raw materials to the solid gel. The concentration after dissolution is 5 mg / ml. Mix well and transfer it to a 10 w / v% polyvinyl alcohol solution and stir until a gel appears, thus obtaining the chitosan composite gel.

[0035] The swelling ratio and release performance of the prepared chitosan composite gel were tested using mixed solutions with different volume ratios in each group and a 56 w / v% β-glycerophosphate hydrate solution (① 4:1, ② 3.5:1, ③ 3:1, ④ 2.5:1). The test method for the swelling ratio was to freeze-dry the chitosan composite gel prepared in each group until its mass remained unchanged, weigh its initial mass with an electronic balance, soak it in PBS solution at 37 °C, take it out at regular intervals, dry the surface moisture with filter paper and weigh its mass. Each group was repeated 3 times, and the swelling ratio was calculated using the initial mass and the mass after soaking. As Figure 7 shown in the swelling ratio chart measured, as the volume ratio of the 56 w / v% β-glycerophosphate hydrate solution increased, the swelling ratio of the corresponding prepared chitosan composite gel increased, reaching the maximum when using ④;

[0036] The test method for the release performance was to load rhodamine basic with blank liposomes when preparing the chitosan composite gel, so that the liposome raw materials had red fluorescence. The chitosan composite gel prepared by this method was soaked in glucose and PBS solution at 37 °C, and fluorescence images were taken after centrifuging to remove the supernatant at regular intervals. The above steps were repeated three times. As Figure 8 shown in the release curve plotted based on the fluorescence intensity in the taken fluorescence images (Ⅰ - 4:1, Ⅱ - 3.5:1, Ⅲ - 3:1, Ⅳ - 2.5:1), as time increased, the proportion of the residue after release decreased accordingly. Among them, as the volume ratio of the mixed solution decreased, the proportion of the residue after release increased;

[0037] For Schemes Ⅲ and Ⅳ, the proportion of the residue after release of Scheme Ⅳ was greater than that of Scheme Ⅲ in the first 4 hours of release, but as time increased, the proportion of the residue after release was close and finally exceeded that of Scheme Ⅳ, and the residual rate after the final release of Scheme Ⅲ was less than that of Scheme Ⅳ. This was due to the manifestation of the release performance under the micro-structure. As Figure 9 and 10 shown are the SEM images of the chitosan composite gels prepared by Schemes Ⅲ and Ⅳ at 5 μm respectively. Scheme Ⅳ presented a dense porous structure, and Scheme Ⅲ presented a loose porous structure, and the porous structure was continuous, indicating that as the mixed solution decreased and the proportion of the β-glycerophosphate hydrate solution increased, the cross-linking density could be reduced, thereby accelerating the release performance, and the phosphate ester groups and three-dimensional porous structure formed by the reaction of polyvinyl alcohol and β-glycerophosphate were not damaged. Moreover, the pore size of the porous structure was similar to the size of human granulation tissue, which could accommodate and adhere more fibroblasts, fibrous cells and collagen, which was beneficial to strengthening the transportation of oxygen and nutrients at the wound site, so as to maintain the best healing environment at the wound site. In addition, excessive wound exudate would increase the risk of wound infection and delay wound healing, while the loose porous structure would improve the ability to adsorb wound exudate.

[0038] While ensuring a low proportion of residues after release, it is also necessary to ensure a high swelling performance so that wound exudate can be fully absorbed, improving the wound healing speed. Therefore, the volume ratio of the mixed solution to the 56 w / v% sodium β-glycerophosphate hydrate solution is selected as 3:1 as the preparation ratio.

[0039] In the field of wound healing, there is a physical therapy method that uses light therapy to treat diseases by means of the radiant energy of light to achieve effects such as anti-inflammatory, promoting blood circulation, and promoting tissue regeneration. Ascorbic acid can promote collagen synthesis and improve the wound healing speed, but it is easily decomposed when exposed to light. Therefore, a sulfonic acid group spiropyran derivative is formed by the reaction of methacrylic acid and 5-sulfosalicylic acid with the gel system. Among them, the colorless closed-loop spiropyran SP and the colored open-loop merocyanine MC in the sulfonic acid group spiropyran derivative will cause clusters to form between the hydrophobic groups of phospholipids to protect ascorbic acid. When light therapy is performed, the spiropyran SP molecules absorb light and are converted into merocyanine MC molecules. The proportion of merocyanine MC molecules in the sulfonic acid group spiropyran derivative increases after light irradiation, and its corresponding charge increases. Due to the electrostatic repulsion, clusters will not form. At the same time, ascorbic acid is released, and the chromophore is used to protect ascorbic acid from photodegradation. At the same time, the increase in charge density helps the volume expansion and diffusion of the chitosan composite gel, improving the diffusion rate of the chitosan composite gel.

[0040] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be encompassed within the present invention. Any reference signs in the claims should not be regarded as limiting the claimed rights.

[0041] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A preparation method of a chitosan composite antibacterial material, characterized in that, It includes the following steps: S1. Chitosan and quaternary ammonium chitosan are mixed in a certain proportion and dissolved in acetic acid solution. After adding nano-hydroxyapatite powder, it is fully stirred and dissolved by a magnetic stirrer to make a mixed solution. A 56 w / v% β-glycerophosphate hydrate solution and the mixed solution are placed in a cooling box and cooled at 4 °C for 30 min. Under an ice-water bath, the β-glycerophosphate hydrate solution is added to the mixed solution, and then stirred to obtain an emulsion solution; S2. Polybutyl acrylate is dissolved in dimethyl sulfoxide solution and stirred. EDC hydrochloride and hydroxy succinimide are added for reaction. Then, a 10 w / v% gelatin solution is added and stirred. The pH value is adjusted to 5.0 with hydrochloric acid and the reaction continues for 24 h. After the reaction, it is dialyzed by an industrial dialysis machine for 48 h. After dialysis, it is freeze-dried for 24 h and ground to obtain a white powder. The white powder is dissolved in deionized water and then mixed and stirred with a 10 w / v% polyvinyl alcohol solution until a solid gel is formed; S3. The emulsion solution and the blank liposome modified with nano-hydroxyapatite are dissolved in chloroform and rotary evaporated at 38 °C to remove the organic solvent to obtain a lipid film compound. In the lipid film compound, a phosphate buffer solution containing ascorbic acid, methacrylic acid and 5-sulfosalicylic acid are added for hydration with the lipid film compound. After transferring to an ultrasonic machine for ultrasonic treatment and filtering through a filter membrane, liposomes are obtained. The obtained liposomes are freeze-dried to obtain liposome raw materials; S4. The liposome raw materials are added to the solid gel, mixed evenly and then transferred to a 10 w / v% polyvinyl alcohol solution and stirred until a gel appears, thus obtaining the chitosan composite gel.

2. The preparation method of a chitosan composite antibacterial material according to claim 1, characterized in that, In S1, the mass ratio of chitosan to quaternary ammonium chitosan is 0.85:0.

15.

3. The preparation method of a chitosan composite antibacterial material according to claim 1, wherein In S1, the volume ratio of the mixed solution to the 56 w / v% β-glycerophosphate hydrate solution is 3:

1.

4. The preparation method of a chitosan composite antibacterial material according to claim 1, characterized in that In S3, the addition amount of the phosphate buffer solution containing ascorbic acid is 2 mg / ml.

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