A method for preparing a bacterial cellulose antimicrobial wound dressing

Abstract

The application relates to the technical field of biomedical materials, and discloses a preparation method of a bacterial cellulose bacteriostatic wound dressing. Specifically, the method comprises the following steps: adding chitosan and glycerol into an ice acetic acid solution, adding bacterial cellulose after the chitosan is completely dissolved, adding genipin after the pH of the solution is adjusted, pouring into a mold for cross-linking reaction after ultrasonic treatment, and drying to obtain the bacterial cellulose wound dressing after the reaction is completed. The high mechanical support property of the bacterial cellulose is combined with the broad-spectrum antibacterial property of the chitosan, a natural cross-linking agent genipin is used to replace glutaraldehyde, and the biological safety and the functional synergy are considered.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically a method for preparing a bacterial cellulose antibacterial wound dressing. Background Technology

[0002] Wound dressings, as key medical materials for wound repair, have the core functions of protecting the wound, absorbing exudate, promoting healing, and preventing infection. With increasing clinical demands for treating complex wounds (such as diabetic foot ulcers and infected burn wounds), the limitations of traditional wound dressings such as gauze and sponges in terms of antibacterial properties and functionality are becoming increasingly apparent. While they can physically isolate the wound and absorb exudate, they cannot actively exert antibacterial effects, are prone to adhering to the wound, and ultimately lead to difficulties in wound healing.

[0003] In recent years, hydrogel dressings have become a research hotspot in the field of wound repair due to their high water content, good biocompatibility, and ability to load various functional components. Currently, domestic research mainly focuses on antibacterial hydrogels made of natural polymers.

[0004] Current research focuses on the innovation of intelligent responsive and multifunctional integrated hydrogels. For example, by designing lactic acid / enzyme-responsive self-activating hydrogels, targeted clearance of methicillin-resistant Staphylococcus aureus (MRSA) biofilms can be achieved; or peptide-based natural antibacterial agents can be used to replace traditional antibiotics, combined with nano-encapsulation technology to avoid the development of drug resistance; in addition, the bilayer Janus dressing achieves synergistic exudate directional flow and tissue repair through the structural design of a hydrophilic antibacterial outer layer and a hydrophobic tissue regeneration inner layer.

[0005] In summary, although progress has been made in the research and development of hydrogel dressings, they still face technical defects such as insufficient mechanical strength, poor biocompatibility caused by chemical cross-linking agents (such as glutaraldehyde), and unstable antibacterial effects, as well as challenges such as insufficient material stability, unclear in vivo mechanism of action, and difficulties in large-scale production. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for preparing a bacterial cellulose antibacterial wound dressing, which specifically includes the following steps: adding chitosan and glycerol to a glacial acetic acid solution, adding bacterial cellulose after the chitosan is completely dissolved, adjusting the pH of the solution to acidic, adding genipin, ultrasonicating, and then pouring the mixture into a mold to undergo a cross-linking reaction to obtain the bacterial cellulose wound dressing.

[0007] Preferably, the bacterial cellulose is derived from Novartis Hansenulatus (Hansenulatus spp.) Novacetimonas hansenii The Hansenula polymorpha (Hansenula polymorpha) was prepared by [preparation method]. Novacetimonas hanseniiIt was deposited on January 4, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37274. The suggested taxonomic name is... Novacetimonas hansenii .

[0008] Preferably, the mass ratio of bacterial cellulose to chitosan is (1:2) to (2:1).

[0009] Preferably, the mass ratio of chitosan to glycerol is 2:1.

[0010] Preferably, the concentration of chitosan in the glacial acetic acid solution is 20 g / L. Preferably, the volume concentration of glacial acetic acid in the glacial acetic acid solution is 2%.

[0011] Preferably, the amount of genipin used is 1.8% to 2.2% of the mass of chitosan.

[0012] Preferably, the pH of the solution is adjusted to 3.5~5.5.

[0013] Preferably, the crosslinking reaction is carried out at a temperature of 37℃±1℃ for 24~72 h.

[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention uses bacterial cellulose (BC) and chitosan (CS) produced by Hansenula polymorpha as the matrix, combined with the natural cross-linking agent genipin (GNP), to construct wound dressings through chemical cross-linking. It has successfully developed a wound dressing with excellent mechanical properties, functional characteristics and antibacterial activity. Compared with traditional dressings, the hydrogel prepared by this invention takes into account both biosafety and functional synergy. It not only provides a green solution for the repair of infected wounds, but also lays the foundation for the development of smart dressings.

[0015] (2) By optimizing the ratio of bacterial cellulose (BC) to chitosan (CS) to 3:2, combined with 2% genipin crosslinking agent (GNP) and a crosslinking process of pH=4.5 and 48 hours, the nanofiber network of BC and the flexible molecular chain of CS form a dense interpenetrating structure through hydrogen bonding and covalent crosslinking, so that the tensile strength of the material reaches 0.750 MPa, and the elastic modulus and cohesiveness are synergistically improved. The wound dressing has outstanding water vapor transmission rate (3494 g / (m²·h)) and porosity (82.18%). The high porosity structure effectively promotes tissue fluid absorption and oxygen exchange, meeting the functional requirements of medical dressings. At the same time, the optimized porosity of BC in the wound dressing promotes the sustained release of chitosan, and the amino protonation at a lower pH enhances the antibacterial activity. It can inhibit Escherichia coli (inhibition zone 8.9±0.24 mm) and Staphylococcus aureus (7.5±0.55 mm), and the cell survival rate is 95.6%, showing good biocompatibility. Attached Figure Description

[0016] Figure 1 This is a schematic diagram showing the swelling rates of three types of wound dressings numbered 2, 3, and 9, where A represents wound dressing number 3; B represents wound dressing number 2; and C represents wound dressing number 9.

[0017] Figure 2 This is a schematic diagram showing the water vapor transmission rate of three types of wound dressings numbered 2, 3, and 9, where A represents wound dressing number 3; B represents wound dressing number 2; and C represents wound dressing number 9.

[0018] Figure 3 This is a schematic diagram showing the porosity of three types of wound dressings numbered 2, 3, and 9, where A represents wound dressing number 3; B represents wound dressing number 2; and C represents wound dressing number 9.

[0019] Figure 4 This is a schematic diagram showing the results of the inhibition zone experiment for three types of wound dressings numbered 2, 3, and 9, where A represents wound dressing number 3; B represents wound dressing number 2; and C represents wound dressing number 9. Figure 4 (a) is Escherichia coli; Figure 4 (b) is Staphylococcus aureus.

[0020] Figure 5 This is a schematic diagram of the Fourier transform infrared spectra of the purified bacterial cellulose gel in Example 1, Comparative Example 1, and three wound dressings numbered 9, where BC is the purified bacterial cellulose gel in Example 1; CS / JNP is the wound dressing in Comparative Example 1; and BC / CS / JNP are the three wound dressings numbered 9. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Example 1 The specific steps for the preparation and purification of bacterial cellulose are as follows: (1) Prepare the culture medium according to the following ratio: 4 g / L glucose, 0.5 g / L yeast extract, 0.1 g / L potassium dihydrogen phosphate, and 1.5 g / L magnesium sulfate heptahydrate in an Erlenmeyer flask, add distilled water to dissolve, adjust the pH of the solution to 7.0, autoclave at 121℃ for 20 min, and set aside.

[0023] (2) Take Hansenula polymorpha isolated and screened from sugarcane juice and stored at 4℃. Novacetimonas hansenii ) Plate, pick 1 loop of the strain and inoculate it into 200 mL of the culture medium prepared in step (1). Incubate at 28°C until bacterial cellulose with a thickness of 4 mm and a radius of 50 mm is obtained.

[0024] (3) Take out the bacterial cellulose and rinse it three times with distilled water. Immerse it in a 0.2 mol / L NaOH solution and keep it at 80°C until the solution is colorless and transparent. Replace the NaOH solution every 30 minutes during this period. Wash the bacterial cellulose with distilled water until it is neutral to obtain white transparent gelatinous bacterial cellulose.

[0025] Example 2 In this embodiment, the mass ratio of bacterial cellulose to chitosan was set to 2:1, 3:2, 1:1, and 1:2, respectively. The amount of genipin used was 2% of the mass of chitosan. The pH of the reaction solution was 4.5, and the crosslinking time was 48 hours. The specific preparation method is as follows: (1) Add water to the bacterial cellulose membrane prepared in Example 1, and homogenize it for 5 minutes at 12000 rpm using a homogenizer (cooling is required for every 1 minute of homogenization) to form a bacterial cellulose suspension. Dry the bacterial cellulose at 80°C for 24 hours.

[0026] (2) Chitosan was added to a 2% glacial acetic acid solution, and then glycerol was added. The mass ratio of chitosan to glycerol was 2:1. The concentration of chitosan in the glacial acetic acid solution was 20 g / L. The mixture was magnetically stirred at 150 r / min for 8 hours until the chitosan was dissolved. The bacterial cellulose prepared in step (1) was added. The pH of the solution was adjusted to 4.5. Genipin was added. The mixture was sonicated at 80 Hz for 10 minutes. The mixture was poured into a 90 mm membrane and crosslinked in a 37 ℃ incubator for 48 hours. Finally, four wound dressings with different mass ratios of chitosan to bacterial cellulose were prepared in this embodiment. The dressings were numbered as 1 (mass ratio of bacterial cellulose to chitosan was 2:1), 2 (mass ratio of bacterial cellulose to chitosan was 3:2), 3 (mass ratio of bacterial cellulose to chitosan was 1:1), and 4 (mass ratio of bacterial cellulose to chitosan was 1:2).

[0027] Example 3 In this embodiment, the mass ratio of bacterial cellulose to chitosan was set to 1:1, the amount of genipin was 1.8%, 1.9%, and 2.2% of the chitosan mass, the pH of the reaction solution was 4.5, the crosslinking time was 48 h, and the specific preparation method was the same as in Example 1. Finally, the three wound dressings with different genipin contents prepared in this embodiment were numbered 5 (genipin content was 1.8% of the chitosan mass), 6 (genipin content was 1.9% of the chitosan mass), and 7 (genipin content was 2.2% of the chitosan mass).

[0028] Example 4 In this embodiment, the mass ratio of bacterial cellulose to chitosan was set to 1:1, the amount of genipin was 2% of the mass of chitosan, the pH of the reaction solution was 3.5, 5.0, and 5.5, the crosslinking time was 48 h, and the specific preparation method was the same as in Example 1. Finally, the three wound dressings prepared in this embodiment under different pH conditions were numbered 8 (pH of reaction solution was 3.5), 9 (pH of reaction solution was 5.0), and 10 (pH of reaction solution was 5.5).

[0029] Example 5 In this embodiment, the mass ratio of bacterial cellulose to chitosan was set to 1:1, the amount of genipin was 2% of the mass of chitosan, the pH of the reaction solution was 4.5, and the cross-linking time was 24h, 60h, and 72h. The specific preparation method was the same as in Example 1. Finally, the three wound dressings prepared in this embodiment with different cross-linking times were numbered 11 (cross-linking time of 24h), 12 (cross-linking time of 60h), and 13 (cross-linking time of 72h).

[0030] Comparative Example 1 The only difference between this comparative example and the wound dressing in Example 2, No. 1, is that it does not use bacterial cellulose. The specific preparation method is as follows: Chitosan was added to a 2% glacial acetic acid solution, followed by 2.5g of glycerol. The mixture was magnetically stirred at 150r / min for 8 hours until the chitosan dissolved. The pH of the solution was adjusted to 4.5, genipin was added, and the mixture was sonicated at 80Hz for 10 minutes. The mixture was then poured into a 90mm membrane and crosslinked in a 37℃ incubator for 48 hours to obtain chitosan wound dressing.

[0031] Comparative Example 2 The only difference between this comparative example and the wound dressing in Example 2, No. 1, is that it does not use chitosan. The specific preparation method is as follows: (1) Add water to the bacterial cellulose membrane prepared in Example 1, homogenize it for 5 minutes at 12000 rpm (cooling is required for every 1 minute of homogenization) to form a bacterial cellulose suspension, and dry it at 80°C for 24 hours to obtain bacterial cellulose.

[0032] (2) Add the bacterial cellulose prepared in step (1) to a 2% glacial acetic acid solution, then add 2.5g of glycerol, stir magnetically at 150r / min for 8 hours, adjust the pH of the solution to 4.5, add genipin, sonicate at 80Hz for 10 minutes, pour into a 90mm membrane, and crosslink in a 37℃ incubator for 48 hours.

[0033] Comparative Example 3 The only difference between this comparative example and the wound dressing in Example 2, No. 1, is that genipin is replaced with glutaraldehyde. The specific preparation method is as follows: (1) Add water to the bacterial cellulose membrane prepared in Example 1, homogenize it for 5 minutes at 12000 rpm (cooling is required for every 1 minute of homogenization) to form a bacterial cellulose suspension, and dry it at 80°C for 24 hours to obtain bacterial cellulose.

[0034] (2) Add chitosan to a 2% glacial acetic acid solution, then add 2.5g glycerol, stir magnetically at 150r / min for 8 hours until the chitosan dissolves, add the bacterial cellulose prepared in step (1), adjust the pH of the solution to 4.5, add glutaraldehyde, sonicate at 80Hz for 10 minutes, pour into a 90mm membrane, and crosslink in a 37℃ incubator for 48 hours.

[0035] Performance tests of the wound dressings prepared in Examples 2-5 and Comparative Examples 1-3.

[0036] (1) Tensile property test: The prepared hydrogel sample was cut into regular specimens with dimensions of 50mm×10mm×1mm (length×width×thickness). Before the test, the two ends of the specimen were fixed in the upper and lower clamping ranges of the h / TG type tensile clamp of the texture analyzer, with a clamping distance of 2.5cm. The instrument operating parameters were set as follows: the speed before the test was 1.0mm / s; the test speed was 1.0mm / s; the speed after the test was 2.0mm / s; the trigger contact force threshold was 20.0gf. Each sample was measured three times in parallel. The mean and standard deviation of tensile strength and elongation at break were calculated. The tensile strength (σ) was calculated according to formula (1), and the elongation at break (ε) was calculated according to formula (2).

[0037] In the formula: σ represents tensile strength (MPa); ε represents elongation at break (%); F represents fracture strength (gf); A represents sample cross-sectional area (mm2); ΔL represents elongation at break (mm); L0 represents initial gauge length (mm).

[0038] (2) Texture analysis: The prepared hydrogel samples were cut into regular specimens with dimensions of 50mm×50mm×2.5mm (length×width×thickness), and fixed horizontally on the test platform. A cylindrical probe was selected as the test fixture to test the hardness, elastic modulus and cohesiveness of the hydrogel. The instrument operating parameters were set as follows: speed before test was 1.0mm / s; test speed was 1.0mm / s; speed after test was 2.0mm / s; contact force was 20.0gf. Each sample was measured three times in parallel. The mean and standard deviation of hardness, elastic modulus and cohesiveness were calculated. The multivariate influence diagram of the hydrogel samples was plotted to characterize the material properties change law.

[0039] (3) Swelling ratio test: After freeze-drying for 48 hours, the hydrogel sample was cut into regular samples with dimensions of 30mm×30mm×2.5mm (length×width×thickness). Each sample was placed in a beaker containing 50mL of distilled water and soaked. The samples were removed every 12 hours, and the surface moisture was absorbed with filter paper. The mass was recorded using an electronic balance until the mass of the sample reached a stable swelling equilibrium state. The swelling rate was calculated according to formula (3).

[0040] In the formula: SR represents the swelling rate (%); M0 represents the mass of the hydrogel sample after freeze-drying (g); M represents the mass of the hydrogel sample after reaching swelling equilibrium (g).

[0041] (4) Water vapor transmission rate test: The water vapor transmission rate test refers to the People's Republic of China Pharmaceutical Industry Standard YY / T0471.2-2004. After modification, the hydrogel sample was cut into regular samples with dimensions of 50mm×50mm×2.5mm (length×width×thickness). 2mL of distilled water was added to a centrifuge tube with an inner diameter of 10mm, and the sample was covered to the tube opening. The sample was sealed to the tube opening wall with sealing glue and weighed. The mass was recorded as W0. The entire sample was placed in a constant temperature incubator at 37℃ and the sample was weighed every 24 hours. The water vapor transmission rate was calculated according to formula (4).

[0042] In the formula, WVTR represents water vapor transmission rate (%); W t W0 represents the total mass (g) of the centrifuge tube, distilled water, and hydrogel sample at different time points; A represents the area of ​​the centrifuge tube opening (m2); and t represents the weighing time interval (h).

[0043] (5) Porosity determination: Cut the product into regular samples with dimensions of 50mm×10mm×2.5mm (length×width×thickness), and immerse them in 96% ethanol for 10 minutes; the porosity is calculated according to formula (5).

[0044] In the formula, P represents porosity (%); V e0 V represents the initial volume (mL) of ethanol. e1 V represents the volume (mL) after immersion in the sample. e2 This indicates the volume (mL) of ethanol after the sample was removed.

[0045] (6) Antibacterial ability test: The antibacterial performance test selected Escherichia coli (E. coli) Escherichia coli Gram-negative bacteria) and Staphylococcus aureus ( taphylococcus aureus Gram-positive bacteria were used as the model strain and cultured in LB broth medium. The size of the inhibition zone was used for comparison.

[0046] Escherichia coli and Staphylococcus aureus slant strains were picked and streaked onto LB agar medium and activated by incubation at 37°C for 19 hours. The activated Escherichia coli and Staphylococcus aureus were then inoculated into 100 mL of LB liquid medium and incubated at 37°C with shaking at 180 r / min for 15 hours. The bacterial suspension was diluted until the absorbance at 620 nm on a spectrophotometer reached 0.8 (±0.01).

[0047] Sample preparation: Cut the prepared wound dressing into circular slices with a diameter of 6 mm and a thickness of 2.5 mm, and place them in a clean bench for ultraviolet irradiation for 20 min for later use.

[0048] Antibacterial activity test: Under aseptic conditions, 100 μL of bacterial suspension was aspirated onto LB agar medium. The bacterial suspension was evenly distributed on the plate using a spreader. Three hydrogel samples were placed in each petri dish and incubated at 37°C for 24 hours. After incubation, the presence of inhibition zones around the samples was checked and measured and photographed to determine whether the prepared hydrogel samples had antibacterial activity.

[0049] (7) Biocompatibility test: The biocompatibility of the prepared hydrogel with L929 mouse fibroblasts was tested according to the CCK-8 experiment. All materials were sterilized by UV for 30 min. When preparing the hydrogel extract, the hydrogel sample cut to an appropriate size was placed in a sterile centrifuge tube, and 5 mL of DMEM complete medium containing 10% fetal bovine serum was added. The mixture was incubated at 37℃ and 5% CO2 for 24 hours. The supernatant was used as the extract. The cells were co-cultured with 100 μL of the extract diluted 1:4 for 24 hours. Then, 10 μL of CCK-8 reagent was added and the cells were incubated for another 2 hours. The absorbance was measured at 450 nm using an ELISA reader to analyze the cell proliferation rate. The control group was replaced with an equal volume of fresh medium.

[0050] Table 1. Performance test results of the wound dressings prepared in Examples 2-5 and Comparative Examples 1-3 As can be seen from the data in Table 1, the bacterial cellulose antibacterial wound dressing prepared by the method of the examples has better performance than the wound dressing prepared by the method of the comparative examples. The wound dressing prepared without bacterial cellulose in Comparative Example 1 has significantly lower antibacterial performance, tensile strength, hardness, water vapor transmission rate and porosity than other products. The wound dressing prepared by glutaraldehyde in Comparative Example 3 has the lowest cell survival rate and elasticity. Although the wound dressing prepared by Comparative Example 2 does not have the lowest values, it is still lower than the examples. This shows that the wound dressing prepared by the method of this invention has good product performance, antibacterial properties and biocompatibility. Moreover, this invention reduces cell death rate through the synergistic effect between the three substances. Therefore, the dressing prepared by this invention has better safety.

[0051] Figure 5 The image shows the Fourier transform infrared spectra of the purified bacterial cellulose gel in Example 1, the three wound dressings in Comparative Example 1 and number 9. The image shows that the composite gel was successfully prepared.

Claims

1. A method for preparing a bacterial cellulose antibacterial wound dressing, characterized in that: The specific steps include: adding chitosan and glycerol to a glacial acetic acid solution; after the chitosan is completely dissolved, adding bacterial cellulose; adjusting the pH of the solution to acidic; adding genipin; ultrasonic treatment; and then pouring the solution into a mold for cross-linking reaction to obtain bacterial cellulose wound dressing.

2. The method for preparing the bacterial cellulose antibacterial wound dressing according to claim 1, characterized in that: The bacterial cellulose is produced by *Novartis hansenii* (… Novacetimonas hansenii The Hansenula polymorpha (Hansenula polymorpha) was prepared by [preparation method]. Novacetimonas hansenii The accession number for this object is CGMCC No. 37274, and its taxonomic name is [missing information]. Novacetimonas hansenii .

3. The method for preparing the bacterial cellulose antibacterial wound dressing according to claim 1, characterized in that: The mass ratio of bacterial cellulose to chitosan is (1:2) to (2:1).

4. The method for preparing the bacterial cellulose antibacterial wound dressing according to claim 1, characterized in that: The mass ratio of chitosan to glycerol is 2:

1.

5. The method for preparing the bacterial cellulose antibacterial wound dressing according to claim 1, characterized in that: The dosage of genipin is 1.8% to 2.2% of the chitosan mass.

6. The method for preparing the bacterial cellulose antibacterial wound dressing according to claim 1, characterized in that: Adjust the pH of the solution to 3.5~5.

5.

7. The method for preparing the bacterial cellulose antibacterial wound dressing according to claim 1, characterized in that: The cross-linking reaction was carried out at a temperature of 37℃±1℃ for 24~72 h.

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