An active wound dressing based on bacterial cellulose and chitosan microspheres encapsulating drugs and a method for its preparation

Abstract

The present application relates to a kind of active wound dressings based on bacterial cellulose and chitosan microspheres encapsulating drugs and its preparation method, the chitosan microspheres utilize bacterial cellulose nanometer network as dispersed phase, let chitosan be dispersed in bacterial cellulose matrix into ball by ionic crosslinking method, and drug is encapsulated in ball, microsphere is attached on the nanometer fiber inside bacterial cellulose film or network inside pore.The active wound dressings of the present application has broad-spectrum antibacterial property and biological safety;Method is simple and efficient and avoid the use of toxic crosslinking agent, can make chitosan microspheres evenly distributed in film.When the encapsulated drug is berberine, it has the potential to treat diabetic foot.

Description

Technical Field

[0001] This invention belongs to the field of functional materials, and specifically relates to an active wound dressing based on bacterial cellulose and chitosan microspheres encapsulating drugs and its preparation method. Background Technology

[0002] Skin wounds typically heal within three months through a highly ordered repair cycle. Factors such as disease, ischemia, and stress can delay healing and lead to chronic wounds, which, if not properly managed, can cause infection, amputation, or even death. Diabetic foot wounds are a typical example of chronic wounds. Besides creating a relatively sterile environment and protecting the wound, dressings, through close contact with the wound, provide appropriate healing-promoting measures tailored to the wound's current condition. Ideal wound dressings should be easy to use, inhibit bacterial growth, maintain a moist environment, facilitate gas exchange, promote tissue healing, and reduce scar formation. However, currently used clinical dressings do not meet these requirements and fall far short of the ideal.

[0003] Bacterial cellulose is a type of natural, high-purity cellulose produced by microorganisms. Freshly prepared bacterial cellulose membranes are smooth and colorless, becoming translucent after treatment with sodium hydroxide. They possess excellent water-holding capacity and a high swelling rate, providing a moist environment for wounds. Due to its simple production and purification process, high safety, good mechanical properties and vapor permeability, and its ability to effectively prevent infection caused by pathogenic microorganisms, bacterial cellulose is widely recognized by researchers as an ideal material for skin wound treatment.

[0004] Chitosan is a natural alkaline polysaccharide characterized by its non-toxicity, biodegradability, and non-antigenic properties, and has been recognized as a safe material by the FDA. It also exhibits numerous bioactivities, such as antibacterial, antifungal, antioxidant, hemostatic, and wound-healing properties, leading to its wide applications in pharmaceuticals and biomedicine. Chitosan microspheres possess the excellent properties of chitosan and can also be used for drug delivery.

[0005] Berberine is an alkaloid isolated from Coptis chinensis, also known as berberine extract. Currently, it is mainly used clinically as an antibacterial agent, and recent studies have shown promising applications in treating organ damage and metabolic diseases. Due to its antidiabetic, antibacterial, and anti-inflammatory pharmacological activities, berberine also demonstrates great potential in wound dressings.

[0006] There are many methods for preparing bacterial cellulose-based chitosan composites, mainly including impregnation and co-culture methods. Impregnation retains excellent mechanical properties, but due to the small pore size of bacterial cellulose, the composite efficiency is low, and uneven internal and external distribution is easily caused. Co-culture yields more ideal products, but the production efficiency is low, and the culture conditions are stringent. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide an active wound dressing based on bacterial cellulose and chitosan microspheres for encapsulating drugs, and a method for preparing the same. The in-situ spheroidization method of this invention allows chitosan to form microspheres on a fiber network, retaining the excellent properties of both materials. Simultaneously, the microspheres can serve as carriers for loading various drugs.

[0008] The present invention discloses a bacterial cellulose / chitosan microsphere material, wherein the bacterial cellulose / chitosan microsphere material comprises chitosan microspheres attached to the internal pores of a bacterial cellulose network and / or the internal fibers of bacterial cellulose.

[0009] The present invention relates to a bacterial cellulose / chitosan microsphere / drug material, wherein the bacterial cellulose / chitosan microsphere / drug material is a drug encapsulated within the chitosan microspheres of the bacterial cellulose / chitosan microsphere material.

[0010] Furthermore, the bacterial cellulose / chitosan microsphere / drug material is as follows: chitosan and divalent and polyvalent anions are electrostatically bonded under acidic conditions to form microspheres, the microspheres encapsulate the drug, and the microspheres are attached to the internal fibers of bacterial cellulose or the internal pores of the network.

[0011] A method for preparing bacterial cellulose / chitosan microspheres according to the present invention includes:

[0012] The bacterial cellulose membrane was impregnated with a chitosan solution, and then placed in an aqueous solution containing anionic substances to stand. After the ions had combined, the membrane was rinsed with deionized water to obtain a bacterial cellulose / chitosan microsphere active wound dressing.

[0013] The bacterial cellulose membrane is a commercially available membrane or a membrane obtained by culturing Acetobacter xylinum (ATCC23770) in liquid culture medium at a constant temperature of 25-30℃ for 2-6 days, followed by purification and washing until neutral. The purification process involves using a 1% (w / v) NaOH solution and culturing at 80-100℃. Washing until neutral is achieved by repeated washing with ultrapure water until neutral.

[0014] The chitosan has a molecular weight of 5-8 million, a viscosity of 50-800 mPa·s, and a degree of deacetylation of 80-95%; the chitosan solution is a 5-20 g / L chitosan solution obtained by mixing chitosan and an aqueous acetic acid solution.

[0015] Furthermore, the chitosan solution is obtained by dissolving chitosan powder in a 0.1M acetic acid solution (natural pH 3-4) to prepare a 5-20 g / L chitosan solution, and then treating it at a high temperature of 105-121℃ for 10-30 min (preferably at 115℃ for 20 min).

[0016] The impregnation process is either vacuum negative pressure impregnation or atmospheric pressure impregnation; the vacuum negative pressure impregnation process specifically involves: using a bacterial cellulose membrane as a filter membrane under a pressure of 0-0.1 MPa, filtering the solution (chitosan solution) for 1-10 minutes, and then impregnating the membrane in the solution (chitosan solution) for 15-30 minutes.

[0017] The chitosan microspheres are obtained by dissolving divalent and polyvalent anionic substances such as sodium phosphate, sodium tripolyphosphate, sodium citrate, sodium sulfate, sodium tungstate, and sodium molybdate in deionized water to prepare a solution with a concentration of 5-30 g / L, and then adding the chitosan solution to the solution to allow the ions to combine.

[0018] A method for preparing bacterial cellulose / chitosan microspheres / drug material according to the present invention includes:

[0019] The bacterial cellulose membrane was impregnated with a chitosan / drug mixture, and then the membrane was placed in an aqueous solution containing anionic substances and allowed to stand until the ions bonded together. After rinsing with deionized water, the bacterial cellulose / chitosan microsphere active wound dressing was obtained.

[0020] The bacterial cellulose membrane is a commercially available membrane or a membrane obtained by culturing Acetobacter xylinum (ATCC23770) in liquid culture medium at a constant temperature of 25-30℃ for 2-6 days, followed by purification and washing until neutral. The purification process involves using a 1% (w / v) NaOH solution and culturing at 80-100℃. Washing until neutral is achieved by repeated washing with ultrapure water until neutral.

[0021] The chitosan / drug mixed solution is obtained by dissolving the drug in a chitosan solution and heating it at 30-50℃ to obtain a chitosan / drug mixed solution with a drug concentration of 0.125-0.5 g / L; the drug is berberine; the chitosan has a molecular weight of 5-8 million, a viscosity of 50-800 mPa·s, and a degree of deacetylation of 80-95%.

[0022] Further, the chitosan / drug mixed solution is obtained by dissolving the drug in a chitosan solution that has been treated at a high temperature of 105-121℃ for 10-30 minutes (preferably at 115℃ for 20 minutes), and then heating it at 30-50℃ to obtain a chitosan / drug mixed solution with a drug concentration of 0.125-0.5 g / L.

[0023] In the chitosan / drug mixed solution, the concentration of chitosan is 5-20 g / L.

[0024] The impregnation process is either vacuum negative pressure impregnation or atmospheric pressure impregnation; the vacuum negative pressure impregnation process specifically involves: using a bacterial cellulose membrane as a filter membrane under a pressure of 0-0.1 MPa, filtering the solution (chitosan / drug mixture) for 1-10 minutes, and then impregnating the membrane in the solution (chitosan / drug mixture) for 15-30 minutes.

[0025] The anionic substance is a divalent or polyvalent anionic substance; the anionic substance is one or more of sodium phosphate, sodium tripolyphosphate, sodium citrate, sodium sulfate, sodium tungstate, and sodium molybdate; the concentration of the aqueous solution of the anionic substance is 5-30 g / L, and the pH is 7.0-12.5; the pH of the aqueous solution of the anionic substance is adjusted to 5.0-6.5 with acid; the standing time is 10-20 min.

[0026] The anionic substance in the aqueous solution is sodium tripolyphosphate, sodium citrate, sodium sulfate, sodium tungstate, or sodium molybdate, and the pH is adjusted to 5.0-6.5 using hydrochloric acid or acetic acid; the anionic substance in the aqueous solution is sodium phosphate, and the pH is adjusted to 5.0-6.5 using phosphoric acid.

[0027] The present invention relates to the application of the bacterial cellulose / chitosan microspheres / drug material in the preparation of wound care and repair dressings, such as for the care and repair of diabetic foot wounds.

[0028] This invention provides a bacterial cellulose / chitosan microsphere active wound dressing and its preparation method. The method utilizes the fact that chitosan molecules contain numerous amino groups, existing in a polycationic state, while divalent and polyvalent anions such as sodium phosphate, sodium tripolyphosphate, sodium citrate, sodium sulfate, sodium tungstate, and sodium molybdate exist in a polyanionic state. The chitosan solution is uniformly distributed in bacterial cellulose through vacuum impregnation or atmospheric pressure impregnation, and then the anions and cations combine to form microspheres while simultaneously encapsulating the drug, thus preparing the wound dressing. This composite method is simple, mild, and efficient, and retains the excellent properties of all three materials.

[0029] This invention provides a bacterial cellulose / chitosan microsphere active wound dressing, characterized in that chitosan and divalent and polyvalent anionic substances such as sodium phosphate, sodium tripolyphosphate, sodium citrate, sodium sulfate, sodium tungstate, and sodium molybdate form microspheres through electrostatic bonding under acidic conditions. The drug is encapsulated within the microspheres, which are attached to the internal fibers of the bacterial cellulose or to the pores within the network.

[0030] This invention relates to an active wound dressing based on bacterial cellulose and chitosan microspheres encapsulating a drug, and its preparation method. The chitosan microspheres utilize a bacterial cellulose nanonetwork as the dispersed phase. Chitosan dispersed in the bacterial cellulose matrix is ​​spherically formed through ionic cross-linking, and the drug is encapsulated within the spheres. The microspheres adhere to the nanofibers inside the bacterial cellulose membrane or to the pores within the network. The active wound dressing of this invention exhibits broad-spectrum antibacterial activity and biocompatibility; the method is simple and efficient, avoids the use of toxic cross-linking agents, and allows for uniform distribution of the chitosan microspheres within the membrane. When the encapsulated drug is berberine, it has the potential to treat diabetic foot.

[0031] Beneficial effects

[0032] (1) The preparation method of the present invention is simple, mild and efficient. On the one hand, the uniform distribution of chitosan in the bacterial cellulose network can be achieved by vacuum negative pressure impregnation. On the other hand, while forming microspheres, the drug is encapsulated inside and is reversible under certain conditions. This process does not require the use of cross-linking agents, which can avoid the toxicity of chemical cross-linking agents and ensure the safety of the material.

[0033] (2) The main raw materials of this invention are bacterial cellulose, chitosan, and berberine, all of which are natural products. Bacterial cellulose is produced by bacterial fermentation, while chitosan and berberine can be extracted from insects and Coptis chinensis, respectively. The bacterial cellulose / chitosan microspheres / berberine wound dressing retains the excellent properties of the three materials. Uniform microspheres are visible inside the material, which has good mechanical properties, breathability, broad-spectrum antibacterial properties, and cell compatibility. It can promote blood coagulation in vivo and in vitro and promote wound healing, and has good application prospects. Attached Figure Description

[0034] Figure 1 The images show the morphology and structure of the bacterial cellulose / chitosan microsphere / berberine composite membrane of Example 1, the bacterial cellulose of Comparative Example 1, and the bacterial cellulose / chitosan microsphere composite membrane of Comparative Example 2; including an appearance photograph (A); a SEM micrograph (B); and a FI-IR spectrum (C). Wherein, BNC: Comparative Example 1; BNC / CSMS: Comparative Example 2; BNC / CSMS / BBR: Example 1;

[0035] Figure 2 The mechanical properties of the bacterial cellulose / chitosan microsphere / berberine composite membrane of Example 1, the bacterial cellulose of Comparative Example 1, and the bacterial cellulose / chitosan microsphere composite membrane of Comparative Example 2 are shown in the figure, including stress-strain curve (A), Young's modulus (B), tensile strength (C), and elongation at break (D); wherein, BNC: Comparative Example 1; BNC / CSMS: Comparative Example 2; BNC / CSMS / BBR: Example 1;

[0036] Figure 3The graph shows the liquid affinity results for the bacterial cellulose / chitosan microsphere / berberine composite membrane of Example 1, the bacterial cellulose of Comparative Example 1, and the bacterial cellulose / chitosan microsphere composite membrane of Comparative Example 2, including water content (A), water absorption (B), water loss curve (C), and water vapor transmission rate (D). Wherein, BNC: Comparative Example 1; BNC / CSMS: Comparative Example 2; BNC / CSMS / BBR: Example 1;

[0037] Figure 4 The release curves of berberine from the bacterial cellulose / chitosan microspheres / berberine composite membrane of Example 1 under different pH conditions are shown.

[0038] Figure 5 The images show the blood test results of the bacterial cellulose / chitosan microsphere / berberine composite membrane of Example 1, the bacterial cellulose of Comparative Example 1, and the bacterial cellulose / chitosan microsphere composite membrane of Comparative Example 2, including hemolysis rate (A), whole blood coagulation curve (B), erythrocyte adhesion plot (C), and platelet adhesion plot (D); wherein, BNC: Comparative Example 1; BNC / CSMS: Comparative Example 2; BNC / CSMS / BBR: Example 1;

[0039] Figure 6 The images show the antibacterial test results of the bacterial cellulose / chitosan microsphere / berberine composite membranes of Examples 1-3, the bacterial cellulose of Comparative Example 1, and the bacterial cellulose / chitosan microsphere composite membranes of Comparative Example 2. The results include inhibition zone (A), inhibition rate (B), and the growth of colonies eluted from different samples on the plate surface (C). Wherein I-V represent Comparative Examples 1-2 and Examples 1-3 respectively; BNC: Comparative Example 1; BNC / CSMS: Comparative Example 2; 0.125, 0.25, and 0.5-BNC / CSMS / BBR represent Examples 1-3 respectively.

[0040] Figure 7 The figures show the cell experiment results of the bacterial cellulose / chitosan microsphere / berberine composite membranes of Examples 1-3, the bacterial cellulose of Comparative Example 1, and the bacterial cellulose / chitosan microsphere composite membranes of Comparative Example 2, including cell proliferation and cytotoxicity of the 50% extract (A), cell proliferation and cytotoxicity of the 100% extract (B), and cell fluorescence staining results (C); where BNC: Comparative Example 1; BNC / CSMS: Comparative Example 2; 0.125, 0.25, and 0.5-BNC / CSMS / BBR are Examples 1-3 respectively.

[0041] Figure 8The graph shows the hypoglycemic effects of the bacterial cellulose / chitosan microsphere / berberine composite membranes of Examples 1-3, the bacterial cellulose of Comparative Example 1, and the bacterial cellulose / chitosan microsphere composite membranes of Comparative Example 2, including cell viability after 30 hours (A); and the glucose concentration decrease curve within 30 hours (B); where BNC represents Comparative Example 1; BNC / CSMS represents Comparative Example 2; and 0.125, 0.25, and 0.5-BNC / CSMS / BBR represent Examples 1-3 respectively.

[0042] Figure 9 Images show the wound healing results of rat skin using the bacterial cellulose / chitosan microsphere / berberine composite membrane of Example 1, the bacterial cellulose of Comparative Example 1, the bacterial cellulose / chitosan microsphere composite membrane of Comparative Example 2, and the Biataine silver ion alginate dressing of Comparative Example 3. The images include wound photographs at 0, 3, 7, and 14 days and the closure rate (A); HE staining results of wound tissue (B); MASSON staining results of wound tissue (C); where BNC: Comparative Example 1; BNC / CSMS: Comparative Example 2; BNC / CSMS / BBR: Example 1; Biataine® AlginateAg: Comparative Example 3.

[0043] Figure 10 This diagram illustrates the rat tail amputation and liver hemostasis of the bacterial cellulose / chitosan microsphere / berberine composite membrane of Example 1, the bacterial cellulose membrane of Comparative Example 1, and the bacterial cellulose / chitosan microsphere composite membrane of Comparative Example 2. It includes the tail amputation hemostasis process and material photographs (A); the liver hemostasis process and material photographs (B); the hemostasis time for each sample (C); and the wound blood loss (D). Wherein, BNC represents Comparative Example 1; BNC / CSMS represents Comparative Example 2; and BNC / CSMS / BBR represents Example 1.

[0044] Figure 11 The images show the spheroid formation effects of chitosan solutions and solutions containing polyvalent anions in Examples 4-6, including macroscopic images (top) and electron micrographs of microspheres (bottom);

[0045] Figure 12 Macroscopic images showing the spheroidizing effect of chitosan solution and sodium phosphate solution in Example 4 and Comparative Example 4;

[0046] Figure 13 Comparative graphs show the mechanical properties of the bacterial cellulose membrane in Comparative Example 1 and the bacterial cellulose / chitosan microsphere composite membranes in Examples 4-6, including tensile strength (A) and elongation at break (B). Detailed Implementation

[0047] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0048] Acetobacter xylinum ATCC23770, a commercially available strain, preserved in the American ATCC.

[0049] Example 1

[0050] (1) Using Acetobacter xylinum (ATCC23770) as the strain, after being cultured in liquid medium at 30℃ for 3 days, the membrane was placed in 1% (w / v) sodium hydroxide solution and repeatedly purified in an environment of 80℃. It was then rinsed with deionized water until neutral and finally sterilized by autoclaving to obtain bacterial cellulose membrane.

[0051] (2) Dissolve 1g of chitosan powder (viscosity 50-200mPa.s, molecular weight 50000, deacetylation 80%-90%, Rohn's reagent) in 100mL of 0.1M acetic acid solution. After dissolution, pH=4.2. After high temperature treatment at 115℃ for 20min, a chitosan solution of 10g / L is obtained.

[0052] (3) Dissolve 6.25 mg of berberine powder in 50 mL of the chitosan solution obtained in step (2), and heat at 50 °C to promote dissolution, to obtain a chitosan / berberine mixed solution containing 0.125 g / L of berberine.

[0053] (4) Dissolve 1g of sodium tripolyphosphate particles in 100mL of deionized water to obtain a 10g / L sodium tripolyphosphate solution. After dissolution, the pH is 9.2. Adjust the pH to 6.5 with hydrochloric acid.

[0054] (5) Place the bacterial cellulose membrane obtained in step (1) into a Buchner funnel, add the chitosan / berberine solution obtained in step (3) under a pressure of 0.1 MPa and perform vacuum filtration for 1 min. After the filtration is completed, place the membrane in the chitosan / berberine solution to recover for 30 min.

[0055] (6) After removing the membrane from step (5), place it in the sodium tripolyphosphate solution obtained in step (4) and let it stand for 15 minutes to complete the ion binding. Then rinse with deionized water to obtain bacterial cellulose / chitosan microspheres / berberine wound dressing.

[0056] Example 2

[0057] (1) Using Acetobacter xylinum (ATCC23770) as the strain, after being cultured in liquid medium at 30℃ for 3 days, the membrane was repeatedly purified in 1% (w / v) sodium hydroxide solution at 80℃, rinsed with deionized water, and finally sterilized by autoclaving to obtain bacterial cellulose membrane.

[0058] (2) Dissolve 1g of chitosan powder (viscosity 50-200mPa.s, molecular weight 50000, deacetylation 80%-90%, Rohn's reagent) in 100mL of 0.1M acetic acid solution. After dissolution, pH=4.2. After high temperature treatment at 115℃ for 20min, a chitosan solution of 10g / L is obtained.

[0059] (3) Dissolve 12.5 mg of berberine powder in 50 mL of the chitosan solution obtained in step (2), and heat at 50 °C to promote dissolution, so as to obtain a chitosan / berberine mixed solution containing 0.25 g / L of berberine.

[0060] (4) Dissolve 1g of sodium tripolyphosphate particles in 100mL of deionized water to obtain a 10g / L sodium tripolyphosphate solution. After dissolution, the pH is 9.2. Adjust the pH to 6.5 with hydrochloric acid.

[0061] (5) Place the bacterial cellulose membrane obtained in step (1) into a Buchner funnel, add the chitosan / berberine solution obtained in step (3) under a pressure of 0.1 MPa and perform vacuum filtration for 1 min. After the filtration is completed, place the membrane in the chitosan / berberine solution to recover for 30 min.

[0062] (6) After removing the membrane from step (5), place it in the sodium tripolyphosphate solution obtained in step (4) and let it stand for 15 minutes to complete the ion binding. Then rinse with deionized water to obtain bacterial cellulose / chitosan microspheres / berberine wound dressing.

[0063] Example 3

[0064] (1) Using Acetobacter xylinum (ATCC23770) as the strain, after being cultured in liquid medium at 30℃ for 3 days, the membrane was placed in 1% (w / v) sodium hydroxide solution and repeatedly purified in an environment of 80℃. It was then rinsed with deionized water until neutral and finally sterilized by autoclaving to obtain bacterial cellulose membrane.

[0065] (2) Dissolve 1g of chitosan powder (viscosity 50-200mPa.s, molecular weight 50000, deacetylation 80%-90%, Rohn's reagent) in 100mL of 0.1M acetic acid solution. After dissolution, pH=4.2. After high temperature treatment at 115℃ for 20min, a chitosan solution of 10g / L is obtained.

[0066] (3) Dissolve 25 mg of berberine powder in 50 mL of the chitosan solution obtained in step (2), and heat at 50 °C to promote dissolution, so as to obtain a chitosan / berberine mixed solution containing 0.5 g / L of berberine.

[0067] (4) Dissolve 1g of sodium tripolyphosphate particles in 100mL of deionized water to obtain a 10g / L sodium tripolyphosphate solution. After dissolution, the pH is 9.2. Adjust the pH to 6.5 with hydrochloric acid.

[0068] (5) Place the bacterial cellulose membrane obtained in step (1) into a Buchner funnel, add the chitosan / berberine solution obtained in step (3) under a pressure of 0.1 MPa and perform vacuum filtration for 1 min. After the filtration is completed, place the membrane in the chitosan / berberine solution to recover for 30 min.

[0069] (6) After removing the membrane from step (5), place it in the sodium tripolyphosphate solution obtained in step (4) and let it stand for 15 minutes to complete the ion binding. Then rinse with deionized water to obtain bacterial cellulose / chitosan microspheres / berberine wound dressing.

[0070] Example 4

[0071] (1) Using Acetobacter xylinum (ATCC23770) as the strain, after being cultured in liquid medium at 30℃ for 3 days, the membrane was placed in 1% (w / v) sodium hydroxide solution and repeatedly purified in an environment of 80℃. It was then rinsed with deionized water until neutral and finally sterilized by autoclaving to obtain bacterial cellulose membrane.

[0072] (2) Dissolve 1g of chitosan powder (viscosity 50-200mPa.s, molecular weight 50000, deacetylation 80%-90%, Rohn's reagent) in 100mL of 0.1M acetic acid solution. After dissolution, pH=4.2. After high temperature treatment at 115℃ for 20min, a chitosan solution of 10g / L is obtained.

[0073] (3) Dissolve 2.3g of sodium dodecahydrate phosphate particles in 100mL of deionized water to obtain a 10g / L sodium phosphate solution. After dissolution, the pH is 12.3. Adjust the pH to 6.5 with phosphoric acid.

[0074] (4) Place the bacterial cellulose membrane obtained in step (1) into a Buchner funnel, add the chitosan solution obtained in step (2) under a pressure of 0.1 MPa and perform vacuum filtration for 1 min. After the filtration is complete, place the membrane in the chitosan solution to recover for 30 min.

[0075] (5) After removing the membrane from step (4), place it in the sodium phosphate solution obtained in step (3) and let it stand for 15 minutes to complete the ion binding. Then rinse with deionized water to obtain bacterial cellulose / chitosan microsphere wound dressing.

[0076] Example 5

[0077] (1) Using Acetobacter xylinum (ATCC23770) as the strain, after being cultured in liquid medium at 30℃ for 3 days, the membrane was placed in 1% (w / v) sodium hydroxide solution and repeatedly purified in an environment of 80℃. It was then rinsed with deionized water until neutral and finally sterilized by autoclaving to obtain bacterial cellulose membrane.

[0078] (2) Dissolve 1g of chitosan powder (viscosity 50-200mPa.s, molecular weight 50000, deacetylation 80%-90%, Rohn's reagent) in 100mL of 0.1M acetic acid solution. After dissolution, pH=4.2. After high temperature treatment at 115℃ for 20min, a chitosan solution of 10g / L is obtained.

[0079] (3) Dissolve 1g of sodium tungstate particles in 100mL of deionized water to obtain a 10g / L sodium tungstate solution. After dissolution, the pH is 9.6. Adjust the pH to 6.5 with acetic acid.

[0080] (4) Place the bacterial cellulose membrane obtained in step (1) into a Buchner funnel, add the chitosan solution obtained in step (2) under a pressure of 0.1 MPa and perform vacuum filtration for 1 min. After the filtration is complete, place the membrane in the chitosan solution to recover for 30 min.

[0081] (5) After removing the membrane from step (4), place it in the sodium tungstate solution obtained in step (3) and let it stand for 15 minutes to complete the ion binding. Then rinse with deionized water to obtain bacterial cellulose / chitosan microsphere wound dressing.

[0082] Example 6

[0083] (1) Using Acetobacter xylinum (ATCC23770) as the strain, after being cultured in liquid medium at 30℃ for 3 days, the membrane was placed in 1% (w / v) sodium hydroxide solution and repeatedly purified in an environment of 80℃. It was then rinsed with deionized water until neutral and finally sterilized by autoclaving to obtain bacterial cellulose membrane.

[0084] (2) Dissolve 1g of chitosan powder (viscosity 50-200mPa.s, molecular weight 50000, deacetylation 80%-90%, Rohn's reagent) in 100mL of 0.1M acetic acid solution. After dissolution, pH=4.2. After high temperature treatment at 115℃ for 20min, a chitosan solution of 10g / L is obtained.

[0085] (3) Dissolve 1g of sodium citrate granules in 100mL of deionized water to obtain a 10g / L sodium citrate solution. After dissolution, the pH is 8.7. Adjust the pH to 6.5 with hydrochloric acid.

[0086] (4) Place the bacterial cellulose membrane obtained in step (1) into a Buchner funnel, add the chitosan solution obtained in step (3) under a pressure of 0.1 MPa and perform vacuum filtration for 1 min. After the filtration is complete, place the membrane in the chitosan solution to recover for 30 min.

[0087] (5) After removing the membrane from step (4), place it in the sodium citrate solution obtained in step (3) and let it stand for 15 minutes to complete the ion binding. Then rinse with deionized water to obtain bacterial cellulose / chitosan microsphere wound dressing.

[0088] Comparative Example 1

[0089] Using Acetobacter xylinum (ATCC23770) as the strain, the membrane was cultured in liquid medium at a constant temperature of 30°C for 3 days. Then, the membrane was repeatedly purified in 1% (w / v) sodium hydroxide solution at 80°C. After rinsing with deionized water until neutral, the membrane was finally sterilized by autoclaving to obtain the bacterial cellulose membrane.

[0090] Comparative Example 2

[0091] (1) Using Acetobacter xylinum (ATCC23770) as the strain, after being cultured in liquid medium at 30℃ for 3 days, the membrane was placed in 1% (w / v) sodium hydroxide solution and repeatedly purified in an environment of 80℃. It was then rinsed with deionized water until neutral and finally sterilized by autoclaving to obtain bacterial cellulose membrane.

[0092] (2) Dissolve 1g of chitosan powder (viscosity 50-200mPa.s, molecular weight 50000, deacetylation 80%-90%, Rohn's reagent) in 100mL of 0.1M acetic acid solution. After dissolution, pH=4.2. After high temperature treatment at 115℃ for 20min, a chitosan solution of 10g / L is obtained.

[0093] (3) Dissolve 1g of sodium tripolyphosphate particles in 100mL of deionized water to obtain a 10g / L sodium tripolyphosphate solution. After dissolution, the pH is 9.2. Adjust the pH to 6.5 with hydrochloric acid.

[0094] (4) Place the bacterial cellulose membrane obtained in step (1) into a Buchner funnel, add the chitosan solution obtained in step (2) under a pressure of 0.1 MPa and perform vacuum filtration for 1 min. After the filtration is complete, place the membrane in the chitosan solution to recover for 30 min.

[0095] (5) After removing the membrane from step (4), place it in the sodium tripolyphosphate solution obtained in step (3) and let it stand for 15 minutes to complete the ion binding. Then rinse with deionized water to obtain the bacterial cellulose / chitosan microsphere composite membrane.

[0096] Comparative Example 3

[0097] Bairtan silver ion alginate dressing ( Alginate Ag)

[0098] Comparative Example 4

[0099] (1) Using Acetobacter xylinum (ATCC23770) as the strain, after being cultured in liquid medium at 30℃ for 3 days, the membrane was placed in 1% (w / v) sodium hydroxide solution and repeatedly purified in an environment of 80℃. It was then rinsed with deionized water until neutral and finally sterilized by autoclaving to obtain bacterial cellulose membrane.

[0100] (2) Dissolve 1g of chitosan powder (viscosity 50-200mPa.s, molecular weight 50000, deacetylation 80%-90%, Rohn's reagent) in 100mL of 0.1M acetic acid solution. After dissolution, pH=4.2. After high temperature treatment at 115℃ for 20min, a chitosan solution of 10g / L is obtained.

[0101] (3) Dissolve 2.3g of sodium dodecahydrate granules in 100mL of deionized water to obtain a 10g / L sodium phosphate solution. After dissolution, the pH is 12.3. Adjust the pH to 6.5 with hydrochloric acid.

[0102] (4) Place the bacterial cellulose membrane obtained in step (1) into a Buchner funnel, add the chitosan solution obtained in step (2) under a pressure of 0.1 MPa and perform vacuum filtration for 1 min. After the filtration is complete, place the membrane in the chitosan solution to recover for 30 min.

[0103] (5) After removing the membrane from step (4), place it in the sodium phosphate solution obtained in step (3) and let it stand for 15 minutes to complete the ion binding. Then rinse with deionized water to obtain bacterial cellulose / chitosan microsphere wound dressing.

[0104] To compare the beneficial effects of the above embodiments and comparative examples, the physicochemical, morphological, and surface properties of the materials were characterized according to ISO 10993-19:2006, including SEM and FTIR. Static tensile tests were performed on the samples using a universal material testing instrument, based on general methods for testing the mechanical properties of materials and considering the characteristics of hydrogels. At room temperature, a 50N sensor was used, the tensile speed was set to 10 mm / min, and the distance between the upper and lower clamps was 1 cm. The sample was stretched until complete fracture, then the stretching was stopped and the sample automatically returned to its initial position. Parameters involved included stress-strain curves, tensile strength, Young's modulus, and elongation at break. The antibacterial properties of the materials were tested using the agar diffusion method and co-contact method, according to the People's Republic of China National Standard GB / T 20944.2-2007. In vitro cytotoxicity experiments were performed on the materials according to ISO 10993-5:2009, including CCK-8 cytotoxicity assays and cell fluorescence staining.

[0105] like Figure 1 As shown, bacterial cellulose has a loose structure, with fibers interwoven into an ultra-fine network structure and large gaps between the fibers. In contrast, the bacterial cellulose / chitosan microspheres and bacterial cellulose / chitosan microspheres / berberine composite membranes, due to vacuum filtration, have smaller gaps between the fibers and are more dense, with visible nano-sized microspheres around 60 nm in diameter between the fibers. The characteristic absorption peaks of bacterial cellulose, chitosan, and berberine in the bacterial cellulose / chitosan microspheres / berberine composite membranes prove that the three materials have been successfully combined.

[0106] like Figure 2 As shown, the introduction of chitosan microspheres increases the Young's modulus of the material, thus enhancing its resistance to deformation and improving its tensile strength. The material can withstand greater forces before fracture, while the elongation at break remains unchanged. This is mainly due to the high rigidity of chitosan, which adheres to the fibers and forms spheres, thickening the fibers and thus improving both the Young's modulus and tensile strength. The introduction of berberine has virtually no effect on the material's mechanics.

[0107] like Figure 3 As shown, the moisture content of all three materials is higher than 95%, the water absorption rate is higher than 80%, and the water vapor transmission rate of bacterial cellulose / chitosan microspheres / berberine is 2177 g / m³. 2 / d, which is close to the ideal dressing range. This is mainly due to the large number of hydrophilic groups inside bacterial cellulose, and the many "channels" formed by the interlacing of internal fibers, which gives it high water content, high water absorption and good vapor permeability.

[0108] like Figure 4 As shown, berberine is released rapidly within 1 hour, and then the release rate gradually slows down, with the release rate remaining above 65% until 24 hours. Under acidic conditions, berberine is more easily released from the material. This is because the presence of amino groups in the sample causes the microspheres to expand under acidic conditions, and acidic conditions are more conducive to the erosion and dissolution of the microspheres.

[0109] like Figure 5 As shown, all three materials exhibit good blood compatibility and do not cause hemolysis. Within 10 minutes, the clotting rates of bacterial cellulose / chitosan microspheres and bacterial cellulose / chitosan microspheres / berberine were significantly higher than those of pure bacterial cellulose, reaching the same endpoint at 20 minutes, indicating that the blood had essentially coagulated. When the materials came into contact with blood, platelet counts showed no significant change, while the red blood cells in Comparative Example 2 and Example 1 exhibited deformation and clumping, suggesting that the chitosan microspheres may accelerate blood clotting by promoting red blood cell aggregation.

[0110] like Figure 6As shown, no inhibition zones appeared around Comparative Examples 1-2 and Example 1. The inhibition zones gradually expanded with increasing berberine concentration. The bacterial cellulose / chitosan microspheres / berberine composite membrane further improved the antibacterial activity compared to Comparative Example 2, exhibiting broad-spectrum antibacterial effects and achieving inhibition rates of over 85% against both Staphylococcus aureus and Escherichia coli. The antibacterial effect was positively correlated with the berberine concentration.

[0111] like Figure 7 As shown, at appropriate berberine concentrations, the materials do not exhibit cytotoxicity, and fluorescence staining results indicate that all materials promote cell proliferation. This is mainly because bacterial cellulose and chitosan have good cytocompatibility, and berberine does not produce cytotoxicity at low concentrations, thus promoting fibroblast growth.

[0112] like Figure 8 As shown, there was no significant difference in cell viability among the groups after 30 hours of culture. Under these conditions, however, there were significant differences in the uptake of glucose from the culture medium by the cells. Within 6 hours, the glucose concentrations of Examples 1-3 were all lower than those of Controls 1-2, and this trend remained consistent at the endpoint. This indicates that the addition of berberine reduced the glucose concentration in the culture medium to some extent, and its application in dressings can effectively promote the reduction of glucose concentration on the wound surface.

[0113] like Figure 9 As shown, on day 3, the wound in the bacterial cellulose / chitosan microspheres / berberine group showed significant shrinkage, with a closure rate of 54%, 18% higher than the worst group; at day 7, the closure rate was 85%, 17% higher than the worst bacterial cellulose group; by day 14, wounds in all groups had basically healed, while the bacterial cellulose group showed poor healing with only 89%. At all stages, the wound healing in the bacterial cellulose / chitosan microspheres / berberine group was superior to other control groups, and it exhibited low inflammatory response, demonstrating the material's excellent healing-promoting properties and its ability to accelerate the recovery of damaged skin wounds.

[0114] like Figure 10 As shown, Comparative Example 1 exhibits a longer hemostasis time, exceeding 3 minutes in both models, and involves a larger amount of bleeding. Comparative Example 2, similar to Example 1, shows less bleeding from the severed tail and a hemostasis time within 2-3 minutes, indicating that the introduction of chitosan contributes to the hemostatic effect of the material. In summary, the bacterial cellulose / chitosan microspheres / berberine composite material can achieve effective hemostasis within 3 minutes, demonstrating its ability to control wound bleeding in emergency situations.

[0115] like Figure 11As shown, this method is also applicable to the formation of spheres from chitosan and sodium phosphate, sodium tungstate, and sodium citrate. The mixture becomes turbid after mixing, which proves that a reaction has occurred. Sodium phosphate and sodium citrate show spherical structures under SEM, while sodium tungstate appears as flakes.

[0116] like Figure 12 As shown, the solution adjusted to pH with phosphoric acid formed spheres, while the solution adjusted to hydrochloric acid did not. This indicates that the sphere-forming effect is related to the pH adjustment medium and the ions in the solution.

[0117] like Figure 13 As shown, the composite membrane containing chitosan microspheres has stronger tensile properties than the pure bacterial cellulose membrane. However, the introduction of chitosan leads to a decrease in elongation, indicating that while chitosan microspheres enhance mechanical properties, they also reduce the elasticity of the material.

[0118] This invention utilizes an ion crosslinking method to prepare chitosan microspheres. The process is simple and mild, allowing bacterial cellulose, chitosan, and drugs to be stably combined, preserving their excellent properties and forming a wound dressing with antibacterial, hemostatic, and highly biosafety properties, which has the potential to be applied to the treatment of chronic wounds.

Claims

1. A method for preparing a bacterial cellulose / chitosan microsphere material, comprising: immersing a bacterial cellulose membrane in a chitosan solution, and then placing the membrane in an aqueous solution containing an anion to stand, rinse, and obtain a bacterial cellulose / chitosan microsphere material; the aqueous solution containing the anion is an aqueous solution of sodium dodecahydrate phosphate, and the anion in the aqueous solution is sodium dodecahydrate phosphate with a pH adjusted to 5.0-6.5 by phosphoric acid.

2. The preparation method according to claim 1, characterized in that, the chitosan has a molecular weight of 5-80 million, a viscosity of 50-800 mPa·s, and a degree of deacetylation of 80-95%; and the chitosan solution is obtained by mixing chitosan and an aqueous acetic acid solution to obtain a 5-20 g / L chitosan solution. 3.A method for preparing a bacterial cellulose / chitosan microsphere / drug material, comprising: immersing a bacterial cellulose membrane in a chitosan / drug mixed solution, and then placing the membrane in an aqueous solution containing an anion to stand, rinse, and obtain a bacterial cellulose / chitosan microsphere material; the aqueous solution containing the anion is an aqueous solution of sodium dodecahydrate phosphate, and the anion in the aqueous solution is sodium dodecahydrate phosphate with a pH adjusted to 5.0-6.5 by phosphoric acid; and the drug is berberine.

4. The preparation method according to claim 3, characterized in that, the chitosan / drug mixed solution is obtained by dissolving a drug in a chitosan solution, heating at 30-50℃, and obtaining a chitosan / drug mixed solution with a drug concentration of 0.125-0.5 g / L; and the chitosan has a molecular weight of 5-80 million, a viscosity of 50-800 mPa·s, and a degree of deacetylation of 80-95%.

5. The method of claim 1 or 3, wherein the step of preparing the mixture is performed at a temperature of 20 to 30°C. the immersion treatment is vacuum negative pressure immersion or atmospheric pressure immersion; and the vacuum negative pressure immersion treatment is specifically as follows: under a pressure of 0-0.1 MPa, the bacterial cellulose membrane is used as a filter membrane, a solution is filtered for 1-10 min, and then the membrane is immersed in the solution for 15-30 min.

6. The method of claim 1 or 3, wherein the compound is prepared by the method of claim 5. the concentration of the aqueous solution containing the anion is 5-30 g / L; and the standing time is 10-20 min.

7. The bacterial cellulose / chitosan microsphere material prepared by the method of claim 1, characterized in that, the bacterial cellulose / chitosan microsphere material is chitosan microspheres attached to the internal pores of the bacterial cellulose network and / or the internal fibers of the bacterial cellulose.

8. The bacterial cellulose / chitosan microsphere / drug material prepared by the method of claim 3. the bacterial cellulose / chitosan microsphere / drug material is the drug encapsulated in the chitosan microspheres of the bacterial cellulose / chitosan microsphere material of claim 7. 9.Use of the bacterial cellulose / chitosan microsphere / drug material of claim 8 in the preparation of a wound care and repair dressing for diabetic foot.

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