A sustained-release antibacterial composite film and a preparation method and application thereof

Abstract

The application discloses a slow-release antibacterial composite membrane and a preparation method and application thereof. First, hectorite and model drug kanamycin are loaded in an aqueous solvent, then mixed with a polylactic acid glycolic acid solution, and a porous membrane is prepared through a phase separation method; and then the prepared porous membrane is immersed in a chitosan solution with a certain concentration to form a three-dimensional porous composite membrane coated with chitosan; the prepared composite membrane has good drug slow-release effect, antibacterial property and mechanical property, can effectively promote angiogenesis and wound healing, and reduce inflammatory reaction.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, and particularly relates to a sustained-release antibacterial composite membrane, its preparation method, and its application. Background Technology

[0002] The skin plays a crucial role in preventing moisture loss and blocking the invasion of harmful substances and pathogenic microorganisms. As a vital interface between the body and its surrounding environment, it harbors a diverse microbial community, including bacteria, fungi, and viruses. In particular, different microbial communities significantly influence local and systemic immune responses through their interactions with host epithelial cells and immune cells. Chronic wounds caused by bacterial infections have become a major medical threat and challenge, as bacteria can compete with the immune system and subsequently invade living tissue. Staphylococcus aureus-associated skin wound infections, in particular, often lead to severe tissue damage. Wound dressings play a vital role in the management of skin wounds because they protect the wound and promote the regeneration of skin and epidermal tissue. A practical clinical problem is that dressings tend to adhere to the wound bed, requiring dressing changes every hour or more, accompanied by the destruction of new epithelial tissue, rebleeding, and delayed wound healing. Clinical statistics also show that pain during dressing changes has a significant negative impact on patient mood and wound healing. Therefore, the development of multifunctional and high-performance materials for treating wound bleeding and infection has long been urgently needed. Therefore, in order to ensure rapid and healthy wound healing, the use of biomaterial dressings for wound infection control and post-injury treatment is particularly important. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the present invention aims to provide a sustained-release antibacterial composite membrane, its preparation method, and its applications. This invention uses biocompatible polylactic-co-glycolic acid (PLGA) as the base material. The composite membrane is obtained by mixing lithium saponite-kanamycin with PLA and performing phase separation. The membrane is then impregnated in a chitosan solution, allowing it to coat the PLA framework. The resulting composite membrane possesses a uniformly sized pore structure, excellent sustained-release properties, antibacterial activity, and biocompatibility, and can promote angiogenesis, wound healing, and reduce inflammatory responses.

[0004] The objective of this invention is achieved through the following technical solution:

[0005] A sustained-release antibacterial composite membrane is formed by impregnating polylactic acid-glycolic acid copolymer and lithium soapstone-kanamycin in a chitosan solution after crosslinking reaction; the sustained-release antibacterial composite membrane has a three-dimensional porous framework structure.

[0006] The preparation method of the above-mentioned sustained-release antibacterial composite membrane includes the following steps:

[0007] S1. First, add lithium soapstone to water and stir to dissolve and swell. Then add kanamycin and stir to load the drug. After loading, centrifuge to obtain the centrifuged material. Then wash, separate and dry the centrifuged material to obtain lithium soapstone loaded with kanamycin.

[0008] S2. The polylactic acid-glycolic acid copolymer is dissolved in N,N-dimethylformamide, then lithium saponite loaded with kanamycin is added and stirred to obtain a solution; the mass ratio of the polylactic acid-glycolic acid copolymer to the lithium saponite loaded with kanamycin is 1:0.02-0.1.

[0009] The solution was placed in a container and spread out, then placed in water for phase separation to form a membrane. Finally, the prepared membrane was taken out and dried to obtain a polylactic acid-glycolic acid / lithium soapstone-kanamycin membrane.

[0010] S3. Chitosan, acetic acid and water are mixed to obtain a chitosan solution. The polylactic acid-glycolic acid / lithium soapstone-kanamycin membrane is immersed in the chitosan solution and dried after immersion to obtain the sustained-release antibacterial composite membrane.

[0011] Preferably, the mass ratio of lithium saponite to kanamycin in step S1 is 0.2 to 7:1.

[0012] Preferably, the mass ratio of lithium saponite to kanamycin in step S1 is 4:1.

[0013] Preferably, the amount of lithium saponite added to water in step S1 is 1 to 55 mg / mL.

[0014] Preferably, the stirring and swelling time in step S1 is 30 to 60 minutes.

[0015] Preferably, in step S1, kanamycin is added and stirred for 12–24 hours to load the drug.

[0016] Preferably, after loading is completed in step S1, the solid is centrifuged at 6000-9000 r / min for 5-10 min to obtain the solid.

[0017] Preferably, the washing method in step S1 is to wash the material multiple times with water at a volume of 1 to 3 times that of the centrifuged material.

[0018] Preferably, the drying process in step S1 is freeze drying.

[0019] Preferably, the purity of the lithium saponite in step S1 is 90-99%, and the purity of the kanamycin is 90-99%.

[0020] Preferably, the amount of polylactic acid-glycolic acid copolymer added to N,N-dimethylformamide in step S2 is 0.5 to 1 g / mL.

[0021] Preferably, the stirring and mixing in step S2 lasts for 6 to 12 hours to obtain a solution.

[0022] Preferably, the drying method in step S2 is: drying at 37°C for 12 to 48 hours.

[0023] Preferably, the mass ratio of chitosan, acetic acid and water in step S3 is 1:0.05-2.5:20-200.

[0024] Preferably, the chitosan in step S3 has a purity of 90-99% and a molecular weight of 100,000-700,000.

[0025] Preferably, the soaking time in step S3 is 30 to 60 minutes.

[0026] Preferably, the drying method in step S3 is: drying at 37°C for 24–48 hours.

[0027] The above-mentioned slow-release antibacterial composite membrane is used in the preparation of medical wound repair materials.

[0028] The principles or mechanisms involved in this invention include:

[0029] Lithium saponite nanodisks possess a two-dimensional structure, with six octahedral magnesium ions sandwiched between two layers of four tetrahedral silicon atoms. Their high specific surface area, favorable nanoscale size, and suitable cell-cell interactions make them a promising drug carrier. This invention first loads kanamycin onto lithium saponite to form a chambered drug-carrying model, ensuring the feasibility of sustained-release kanamycin. Then, it is added to a polylactic acid-glycolic acid solution and mixed thoroughly. The mixture is then rapidly added to a beaker containing ultrapure water as a non-solvent to crosslink and form a film. After drying, it is immersed in a chitosan solution and dried again to obtain a three-dimensional porous composite membrane structure with sustained-release antibacterial properties. Its sustained-release antibacterial properties not only prevent wound infection, promote angiogenesis and cell proliferation, and accelerate wound healing, but also reduce the frequency of dressing changes to avoid secondary wound damage and reduce patient discomfort.

[0030] Compared with the prior art, the beneficial effects of the present invention include:

[0031] (1) The raw materials selected in this invention have good biocompatibility, are widely available and inexpensive; polylactic acid glycolic acid, lithium saponite and chitosan have been proven to be safe and non-toxic, and can be degraded or absorbed by the human body.

[0032] (2) The preparation process of the present invention is simple. By loading kanamycin with lithium saponite, a drug sustained-release carrier with high drug loading capacity is obtained. It is then added to polylactic acid glycolic acid to form a film in one step by phase separation, which is simple and fast. Finally, it is impregnated in chitosan solution to obtain a more uniform three-dimensional porous structure.

[0033] (3) The solvent used in the preparation process of this invention is green and environmentally friendly, and is widely available and easy to obtain. This invention provides a new method for preparing sustained-release wound dressings, promoting the development of advanced wound dressings.

[0034] (4) The composite membrane prepared by this invention can reduce the chance of chronic wound infection, reduce the number of dressing changes, and avoid secondary damage to the wound and patient discomfort. The composite membrane prepared by this invention has good sustained-release and antibacterial properties, can promote angiogenesis, and accelerate wound healing. Attached Figure Description

[0035] Figure 1 These are microscopic morphology images of the products prepared in Example 1, Comparative Example 1, and Comparative Example 3 of the present invention.

[0036] Figure 2 This is a comparison chart of the drug sustained-release test results of the products described in Example 1 and Comparative Example 3 of the present invention.

[0037] Figure 3 These are comparative photographs showing the effects of the products of Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 of the present invention in an antibacterial experiment (using Staphylococcus aureus as the test bacteria).

[0038] Figure 4 These are comparative photographs showing the effects of the products of Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 of the present invention in an antibacterial experiment (using Escherichia coli as the detection bacterium).

[0039] Figure 5 This is a comparison chart of the cytotoxicity test results of the products of Example 1, Comparative Example 1, and Comparative Example 2 of the present invention.

[0040] Figure 6 These are photographs showing the repair results of Example 1 and Comparative Example 1 of the present invention in a rat skin infection wound model.

[0041] Figure 7 This is the infrared spectrum of the lithium saponite loaded with kanamycin prepared in step S1 of Example 1.

[0042] Figure 8 This is the infrared spectrum of lithium saponite. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0044] Example 1

[0045] A method for preparing a sustained-release antibacterial composite membrane, comprising the following steps:

[0046] S1. First, weigh 20 mg of lithium saponite and add it to 5 ml of ultrapure water. Stir and swell for 30 min. Then add 3 mg of kanamycin and continue stirring for 24 h. Centrifuge at 8000 r / min for 10 min. Wash the precipitate with ultrapure water 3 times and centrifuge. Finally freeze-dry for 48 h to obtain kanamycin-loaded lithium saponite.

[0047] S2. Weigh 0.5g of polylactic acid-glycolic acid copolymer and add it to 5ml of N,N-dimethylformamide. Dissolve for 12h, then add 10mg of lithium saponite loaded with kanamycin and stir for 2h to obtain a mixture. Take 1ml of the mixture and place it on a 9mm culture plate. Spread it evenly and quickly put it into a 1000ml ultrapure water beaker. Separate the phases to form a membrane. Finally, dry it at 37℃ for 48h to obtain the polylactic acid-glycolic acid / lithium saponite-kanamycin membrane.

[0048] S3. Weigh 0.2g of chitosan and add it to 9.3g of ultrapure water, then add 0.5g of acetic acid and stir to dissolve for 2h to obtain a 2wt% chitosan solution. Then, immerse the polylactic acid-glycolic acid / lithium soapstone-kanamycin membrane prepared in S2 in the chitosan solution for 60min, take it out, and then dry it at 37℃ for 48h to obtain the sustained-release antibacterial composite membrane.

[0049] Example 2

[0050] Except for step S1, where the mass ratio of lithium saponite to kanamycin is 4:1 (i.e., 20 mg of lithium saponite and 5 mg of kanamycin), the other steps are the same as in Example 1.

[0051] Example 3

[0052] Except for the stirring and swelling time of 60 min as described in step S1, the other steps are the same as in Example 1.

[0053] Comparative Example 1

[0054] Weigh 0.5g of polylactic acid-glycolic acid copolymer and add it to 5ml of N,N-dimethylformamide. Dissolve for 12h to obtain a mixture. Take 1ml of the mixture and place it on a 9mm culture plate. Quickly pour it into a 1000ml ultrapure water beaker to crosslink and form a membrane. Finally, dry it at 37℃ for 48h to obtain a porous membrane.

[0055] Comparative Example 2

[0056] Weigh 0.5g of polylactic acid-glycolic acid copolymer and add it to 5ml of N,N-dimethylformamide. Dissolve for 12h, then add 10mg of lithium soapstone and stir for 2h to obtain a mixture. Take 1ml of the mixture and place it on a 9mm culture plate. Quickly pour it into a 1000ml ultrapure water beaker to crosslink and form a membrane. Finally, dry it at 37℃ for 48h to obtain a porous membrane.

[0057] Comparative Example 3

[0058] S1. Weigh 20 mg of lithium saponite and add it to 5 ml of ultrapure water. Stir and swell for 30 min. Then add 3 mg of kanamycin and stir for 24 h. Centrifuge at 8000 r / min for 10 min. Wash the precipitate with ultrapure water 3 times and centrifuge. Finally freeze-dry for 48 h to obtain kanamycin-loaded lithium saponite.

[0059] S2. Weigh 0.5g of polylactic acid-glycolic acid copolymer and add it to 5ml of N,N-dimethylformamide. Dissolve for 12h, then add 10mg of lithium soapstone loaded with kanamycin and stir for 2h to obtain a mixture. Take 1ml of the mixture and place it on a 9mm culture plate. Quickly pour it into a 1000ml ultrapure water beaker to crosslink and form a membrane. Finally, dry it at 37℃ for 48h to obtain a porous membrane.

[0060] Characterization tests were performed on the examples and comparative examples.

[0061] 1. Microscopic morphological observation

[0062] The products prepared in Example 1, Comparative Example 1 and Comparative Example 3 were cut into pieces of 0.5cm × 0.5cm and examined with an S-4800 microscope at an accelerating voltage of 5kV, with a magnification range of 5000 to 100000X.

[0063] Figure 1 These are microscopic morphology images of the products prepared in Example 1, Comparative Example 1, and Comparative Example 3 of the present invention; Figure 1 It can be seen that the pore size of the pure PLGA membrane is relatively large. After adding LAP (lithium saponite) / KAKA (kanamycin), the pore size becomes smaller and LAP / KANA particles are distributed, indicating that the composite carrier is successfully loaded onto PLGA. After being coated with chitosan solution, the pore size of the composite membrane becomes smaller and the edges of the pores become smoother.

[0064] 2. Drug sustained-release test

[0065] The in vitro release kinetics of kanamycin from the membrane samples prepared in Example 1 and Comparative Example 3 were investigated using ultraviolet-visible spectroscopy. Specifically, 25 mg of the membranes prepared in Example 1 and Comparative Example 3 were immersed in sample vials containing 5 mL of phosphate-buffered saline (PBS) solution (pH = 7.4), with all samples in triplicate and incubated for different times in a steam bath shaker at 37°C. At each time interval, 3 mL of PBS solution was removed from each vial and replenished with an equal volume of fresh PBS solution. The optical density (OD) value was measured using a UV-Vis spectrophotometer, and the cumulative release was calculated.

[0066] Figure 2 This is a comparison chart of the drug sustained-release test results of the products described in Example 1 and Comparative Example 3 of the present invention; Figure 2 It can be seen that both Comparative Example 3 and Example 1 have good sustained-release effects, while Example 1, being a composite membrane coated with chitosan, has a more obvious sustained-release effect, with a slower burst release within 24 hours, achieving a better sustained-release effect.

[0067] 3. Antibacterial test:

[0068] The antibacterial properties of the material were characterized by observing the size of the inhibition zone after the bacterial solution was coated using the agar diffusion method.

[0069] The bacteria used for testing were Staphylococcus aureus (ATCC6538) and Escherichia coli (ATCC25922).

[0070] The membrane samples prepared in Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were cut into small pieces of equal weight, each approximately 0.6 cm in diameter. Then, 150 μL of Staphylococcus aureus suspension and Escherichia coli suspension in LB liquid medium + bacteria (bacterial OD value 0.25-0.35) were inoculated onto solid agar plates, covering the membrane samples of Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3, respectively, and their antibacterial activity was measured. All these agar plates were incubated at 37°C for 24 h. The antibacterial activity of the samples was assessed by visually observing the bacterial inhibition zone.

[0071] Figure 3 These are comparative photographs showing the effects of the products of Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 of the present invention in an antibacterial experiment (using Staphylococcus aureus as the test bacteria). Figure 3 It can be seen that Comparative Examples 1 and 2 have no antibacterial effect, while Comparative Example 3 and Example 1 show better antibacterial effects, indicating that the drug composite carrier can be released from the composite membrane and achieve a certain antibacterial effect.

[0072] Figure 4These are comparative photographs showing the effects of the products of Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 of the present invention in an antibacterial experiment (using Escherichia coli as the detection bacteria). Figure 4 It can be seen that Comparative Examples 1 and 2 have no antibacterial effect, while Comparative Example 3 and Example 1 show better antibacterial effects, indicating that the drug composite carrier can be released from the composite membrane and achieve a certain antibacterial effect.

[0073] 4. In vitro cytotoxicity test: The composite membranes prepared in Example 1, Comparative Example 1, and Comparative Example 2 were sterilized and then subjected to a 6mm... 2 The cells were soaked in complete culture medium (10% FBS (fetal bovine serum) + 1% PS (penicillin-streptomycin) + 89% DMEM) for 24 h at a concentration of / mL. Then, fibroblasts (NIH3T3 cells) were seeded in 96-well plates at a density of 5000 cells / well. After incubating the plates at 37°C for 24 h, the culture medium was aspirated, and the medium soaked in the composite membrane was transferred to the 96-well plates for further incubation. After 24 h, the culture medium was discarded, and the cells were washed twice with PBS buffer. A mixture containing MTT (thiazolyl blue) and complete culture medium (1:9 ratio) was added to each well in the dark. After incubation at 37°C for 4 h, the supernatant was carefully aspirated, and 200 μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the blue-purple crystals at the bottom of the plate. The absorbance was measured at 490 nm using a microplate reader. The complete culture medium served as the negative control, and the mixture containing 10% phenol and complete culture medium served as the positive control.

[0074] Figure 5 This is a comparison chart of the cytotoxicity test results of the products of Example 1, Comparative Example 1, and Comparative Example 2 of the present invention; Figure 5 It can be seen that the cell survival rates of Comparative Example 1, Comparative Example 2 and Example 1 are 97.32%, 96.41% and 98.21%, respectively, and the material toxicity level is 1.

[0075] 5. Experimental procedure for a rat skin infection wound model: After anesthetizing the rats, the backs were shaved and disinfected with 75% ethanol. Three 6mm diameter wounds were then created on the backs, and each wound was inoculated with 10μL of 10⁸ CFU / mL Staphylococcus aureus for one hour. The first wound was left untreated as a blank control group. The second and third wounds were treated with membrane samples prepared in Comparative Example 1 and Example 1, respectively (8mm diameter discs were prepared, placed over the wounds, and then uniformly bandaged with ordinary gauze). The results are as follows: Figure 6 As shown, the wound treated with the composite membrane of Example 1 showed the best healing effect. The composite membrane of Example 1 can promote angiogenesis and accelerate the wound healing process.

[0076] Figure 7This is the infrared spectrum of the lithium saponite loaded with kanamycin prepared in step S1 of Example 1. Figure 8 This is the infrared spectrum of lithium saponite. (Comparison) Figures 7-8 It can be seen that: [the following is a list of measurements] were found at 2945 and 1515 cm. -1 The appearance of new infrared characteristic peaks is attributed to the vibration of the CH bond and the bending vibration of the amino group in the kanamycin molecule, thus proving that kanamycin has been successfully loaded onto lithium saponite.

[0077] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a sustained-release antibacterial composite membrane, characterized in that, Includes the following steps: S1. First, add lithium soapstone to water and stir to dissolve and swell. Then add kanamycin and stir to load the drug. After loading, centrifuge. Wash, separate and dry the centrifuged material to obtain lithium soapstone loaded with kanamycin. The mass ratio of lithium saponite to kanamycin in step S1 is 0.2~7:1; The amount of lithium saponite added to water in step S1 is 1~55 mg / mL; The stirring and swelling time in step S1 is 30~60 min; In step S1, kanamycin is added and stirred for 12-24 hours to load the drug. S2. The polylactic acid-glycolic acid copolymer is dissolved in N,N-dimethylformamide, then lithium saponite loaded with kanamycin is added and stirred to obtain a solution; the mass ratio of the polylactic acid-glycolic acid copolymer to the lithium saponite loaded with kanamycin is 1:0.02~0.

1. The solution was placed in a container and spread out, then placed in water for phase separation to form a membrane. Finally, the prepared membrane was taken out and dried to obtain a polylactic acid-glycolic acid / lithium soapstone-kanamycin membrane. S3. Chitosan, acetic acid and water are mixed to obtain a chitosan solution. The polylactic acid-glycolic acid / lithium soapstone-kanamycin membrane is immersed in the chitosan solution and dried after immersion to obtain the sustained-release antibacterial composite membrane. The amount of polylactic acid-glycolic acid copolymer added to N,N-dimethylformamide in step S2 is 0.5~1g / mL; The mass ratio of chitosan, acetic acid and water in step S3 is 1:0.05~2.5:20~200.

2. The method for preparing the sustained-release antibacterial composite membrane according to claim 1, characterized in that, After loading is completed as described in step S1, centrifuge at 6000~9000 r / min for 5~10 min to obtain a solid; The washing method described in step S1 is as follows: wash the material multiple times with water at a volume of 1 to 3 times that of the centrifuged material; The drying process described in step S1 is freeze drying.

3. The method for preparing the sustained-release antibacterial composite membrane according to claim 2, characterized in that, Step S2 involves stirring and mixing for 6-12 hours to obtain a solution; The drying method described in step S2 is: drying at 37°C for 12~48 hours.

4. The method for preparing the sustained-release antibacterial composite membrane according to claim 1, characterized in that, The chitosan in step S3 has a purity of 90-99% and a molecular weight of 100,000-700,000.

5. The method for preparing a sustained-release antibacterial composite membrane according to claim 4, characterized in that, The soaking time in step S3 is 30-60 minutes; The drying method described in step S3 is: drying at 37°C for 24~48 hours.

6. A sustained-release antibacterial composite membrane, characterized in that, It is prepared by the method for preparing the sustained-release antibacterial composite membrane according to any one of claims 1 to 5.

7. The application of the sustained-release antibacterial composite membrane according to claim 6 in the preparation of medical wound repair materials.

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