A bio-cellulose anti-adhesion material and its preparation method

Abstract

This invention relates to the field of biomaterials technology, and in particular to a bio-cellulose anti-adhesion material and its preparation method. The preparation method involves in-situ grafting of N-vinylpyrrolidone onto bio-cellulose using oxidative coupling free radicals, followed by the introduction of cellulase and estrogen via nanoneedles to obtain the bio-cellulose anti-adhesion material. The bio-cellulose anti-adhesion material of this invention features controllable biodegradability, continuously providing the biochemical factors required for endometrial repair, and effectively reducing cell adhesion. This bio-cellulose anti-adhesion material solves the problems of other synthetic scaffold materials in the prior art, such as in vivo non-degradability leading to inflammation or foreign body reactions, resulting in endometrial adhesion and trauma during separation; and the lack of bioactivity in the materials themselves, preventing them from actively participating in and regulating the endometrial repair process.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a biocellulose anti-adhesion material and its preparation method. Background Technology

[0002] Intrauterine adhesions are a gynecological condition that seriously affects women's reproductive health. It is characterized by fibrous adhesions forming between the uterine walls due to damage to the endometrial tissue, causing partial or complete obstruction of the uterine cavity. This condition has a high incidence not only among women of reproductive age but also in high-risk groups such as women who have undergone curettage and those with recurrent miscarriages or infertility. Intrauterine adhesions can lead to increased rates of spontaneous abortion, decreased menstrual flow, or amenorrhea, and also increase the risk of pregnancy complications such as placenta previa and placenta accreta.

[0003] Currently, the main clinical approach is hysteroscopic adhesiolysis combined with estrogen therapy. This approach involves surgically removing the adhesions and using estrogen to promote endometrial regeneration. However, this treatment carries the risk of secondary adhesions during the surgical wound healing process. To prevent adhesion reformation, a strategy of implanting anti-adhesion biological scaffold materials post-surgery to form a physical barrier is employed. While these anti-adhesion materials have achieved some success in preventing recurrence of intrauterine adhesions, they still have several limitations.

[0004] In recent years, biocompatible cellulose (BC) has attracted much attention in the biomedical field due to its unique physicochemical properties and excellent biocompatibility. BC is characterized by high purity, high crystallinity, and high mechanical strength. Its biomembrane possesses extremely high water-holding capacity and flexibility approaching that of natural tissue, enabling it to adapt to different uterine cavity structures. Simultaneously, BC exhibits natural bioinertness, which can reduce inflammation and foreign body reactions. By regulating the structure and composition of BC, the controlled degradation of BC scaffolds can be achieved, and its degradation product, glucose, can be absorbed and utilized by the body without toxic side effects.

[0005] However, the following problems still urgently need to be solved in the existing technology:

[0006] (1) Biocompatibility issues: Some synthetic anti-adhesion materials may cause inflammatory reactions or foreign body reactions, affecting the treatment effect and patient recovery.

[0007] (2) The degradation performance of materials is not ideal: Synthetic anti-adhesion materials lack biodegradability and require a second surgery to remove them, which can easily cause trauma; the degradation time of some natural materials is uncontrollable, and too fast or too slow will affect the treatment effect; while natural BC cannot be degraded by the human body due to the lack of cellulose degradation enzyme system and needs to be removed after surgery.

[0008] (3) Single function: Most existing barrier materials only have physical isolation function and lack bioactivity to promote endometrial repair. They cannot actively participate in and regulate the endometrial repair process.

[0009] (4) Material interface problem: The highly hydrophilic nature of the surface of natural BC nanofibers leads to insufficient adhesion to the uterine wall, affecting its effect as an anti-adhesion barrier.

[0010] These technical limitations severely restrict the effectiveness of anti-adhesion treatment, thus necessitating the development of a novel anti-adhesion material. This material should possess good biocompatibility, controllable biodegradability, and the ability to actively promote endometrial repair, while simultaneously addressing the adhesion problem between the material and tissue. This is of significant clinical importance for reducing postoperative intrauterine adhesions and lowering the risk of pregnancy complications. Summary of the Invention

[0011] To address the aforementioned shortcomings of current biomaterials for preventing intrauterine adhesions in biomedical applications, this invention provides a bio-cellulose anti-adhesion material and its preparation method.

[0012] To achieve the above objectives, the present invention provides the following technical solution:

[0013] A method for preparing a bio-cellulose anti-adhesion material involves in-situ grafting of N-vinylpyrrolidone onto bio-cellulose via oxidative coupling of free radicals, followed by the introduction of cellulase and estrogen into nanoneedles to obtain the bio-cellulose anti-adhesion material.

[0014] Specifically, it includes the following steps:

[0015] (1) TEMPO catalytic oxidation: Weigh out a biological cellulose membrane with a solid content of 5.0%, immerse it in a solution containing TEMPO and NaBr, adjust the pH with NaOH, add 10% sodium hypochlorite solution, place it on a plate shaker, and oxidize it at 25°C;

[0016] (2) In-situ oxidation of grafted N-vinylpyrrolidone initiated by two free radicals: Add ascorbic acid solution to the suspension of bio-cellulose membrane catalytically oxidized in step (1), then introduce nitrogen gas into the reaction vessel and seal it with a rubber stopper; under nitrogen protection, add 30% hydrogen peroxide solution, a mixed solution of TEMPO and NaBr, and 10% sodium hypochlorite solution, place it on a plate shaker, and initiate the generation of free radicals at 37°C;

[0017] Then, N-vinylpyrrolidone was added using a syringe, and the reaction vessel was placed in a shaker at 70°C for reaction. After the reaction was completed, the bio-cellulose membrane was removed and rinsed with deionized water until the pH of the rinsing solution was neutral and the conductivity was less than 5 μS / cm. The washed bio-cellulose membrane was then placed in autoclave, rinsed again, and stored at 4°C for later use.

[0018] (3) Nanoneedle delivery of cellulase and estrogen: The composite cellulase and β-estradiol were weighed and dissolved in pH 7.4 PBS solution at 4°C and filtered through a 0.2 μm microporous membrane for sterilization. The mixture of cellulase and estrogen was loaded into a syringe and uniformly delivered into the nanofiber membrane using a nanoneedle delivery device. Subsequently, it was freeze-dried to obtain the biocellulose anti-adhesion material.

[0019] In step (1), the mass ratio of the active ingredient in the biocellulose membrane to TEMPO is 1:0.02, the mass ratio of the active ingredient in the biocellulose membrane to NaBr is 1:0.1, and the mass ratio of the active ingredient in the biocellulose membrane to 10% sodium hypochlorite solution is 1:7.5. The solid content of the biocellulose membrane is 5.0%; the concentration of NaOH is 0.5M, and the pH is adjusted to 10.5.

[0020] Further, step (1) specifically involves weighing 100g of a bio-cellulose membrane with a solid content of 5.0%, immersing it in a 400mL solution containing 0.1g TEMPO and 0.5g NaBr, adjusting the pH to 10.5 with 0.5M NaOH, adding 37.5mL of 10% sodium hypochlorite solution, placing it on a flat shaker, and oxidizing it at 20rpm and 25℃ for 6-24 hours.

[0021] In step (2), the concentration of ascorbic acid solution is 0.5M, the concentration of TEMPO is 50mM, and the concentration of NaBr is 100mM.

[0022] Further, step (2) specifically involves adding 30 mL of 0.5 M ascorbic acid solution to the catalytically oxidized cellulose membrane suspension from step (1), then purging the reaction vessel with nitrogen gas and sealing it with a rubber stopper; under nitrogen protection, using a syringe, adding 5 mL of 30% hydrogen peroxide solution, 5 mL of a mixed solution of 50 mM TEMPO and 100 mM NaBr, and 5 mL of 10% sodium hypochlorite solution, placing it on a flat shaker, and initiating free radical generation for 10 minutes at 20 rpm and 37°C; subsequently, using a syringe, adding 5-10 mL of... For N-vinylpyrrolidone, place a three-necked flask in a shaker at 70°C and react at 20 rpm for 3-6 hours. After the reaction, remove the bio-cellulose membrane and rinse it with deionized water for 30 minutes each time until the pH of the rinsing solution is neutral and the conductivity is less than 5 μS / cm. After rinsing, autoclave the bio-cellulose membrane at 121°C for 30 minutes, then rinse it three times with sterile water for medical injection and store it at 4°C for later use.

[0023] In step (3), the enzyme activity of the composite cellulase is 50 U / mg, the needle of the nano-microneedle delivery instrument is 2.5 mm long and 125 μm in diameter, and the delivery speed is 1.0 mL / min; the freeze-drying conditions are -50℃, 0.1 mbar vacuum, and 72 hours.

[0024] Further, step (3) specifically involves weighing 400 mg of a composite cellulase with an enzyme activity of 50 U / mg and 100 mg of β-estradiol, dissolving them in 10 mL of pH 7.4 PBS solution at 4°C, and filtering them through a 0.2 μm microporous membrane for sterilization; loading the cellulase and estrogen mixture into a syringe, uniformly introducing it into a nanofiber membrane using a nanoneedle delivery device, and then freeze-drying it to obtain a biological cellulose anti-adhesion material.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] The bio-cellulose anti-adhesion material of this invention features controllable biodegradability, continuously providing the biochemical factors required for endometrial repair and effectively reducing cell adhesion. This bio-cellulose anti-adhesion material solves the problems of other existing synthetic scaffold materials that cannot degrade in vivo, causing inflammation or foreign body reactions, leading to endometrial adhesion and trauma during separation; and the lack of bioactivity in the materials themselves, preventing them from actively participating in and regulating the endometrial repair process. Attached Figure Description

[0027] Figure 1 In the figures, (a) to (e) show the infrared spectra of biocellulose before and after modification, the surface water contact angle, and the elemental analysis results of scanning electron microscopy-energy dispersive spectroscopy, respectively.

[0028] Figure 2 The in vitro degradation curve is the β-estradiol release curve.

[0029] Figure 3 Image of cell co-culture using laser confocal microscopy. Detailed Implementation

[0030] 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.

[0031] Example 1

[0032] A method for preparing a bio-cellulose anti-adhesion material, comprising the following steps:

[0033] (1) TEMPO catalytic oxidation: Weigh 100g of biological cellulose membrane with a solid content of 5.0% (provided by Hainan Guangyu Biotechnology Co., Ltd.), immerse it in 400mL of solution containing 0.1g TEMPO and 0.5g NaBr, adjust the pH to 10.5 with 0.5M NaOH, add 37.5mL of 10% sodium hypochlorite solution, place it on a plate shaker, and oxidize for 24 hours at 20rpm and 25℃.

[0034] (2) In-situ oxidation grafting of N-vinylpyrrolidone initiated by dual radicals: 30 mL of 0.5 M ascorbic acid solution was added to a three-necked flask containing the catalytically oxidized cellulose membrane suspension from step (1). Nitrogen gas was introduced for 5 minutes, and the flask was then sealed with a rubber stopper. Under nitrogen protection, 5 mL of 30% hydrogen peroxide solution, 5 mL of a mixed solution of 50 mM TEMPO and 100 mM NaBr, and 5 mL of 10% sodium hypochlorite solution were added using a syringe. The flask was placed on a flat shaker and the radicals were generated at 20 rpm and 37°C for 10 minutes. Subsequently, 10 mL of N-vinylpyrrolidone was added using a syringe, and the three-necked flask was placed on a shaker at 70°C and reacted at 20 rpm for 6 hours. After the reaction, the cellulose membrane was removed and rinsed with deionized water for 30 minutes each time until the pH of the rinsing solution was neutral and the conductivity was below 5 μS / cm. After being washed, the bio-cellulose membrane was autoclaved at 121°C for 30 minutes, then rinsed three times with sterile water for medical injection, and refrigerated at 4°C for later use.

[0035] (3) Nanoneedle delivery of cellulase and estrogen: 400 mg of a compound cellulase with an enzyme activity of 50 U / mg and 100 mg of β-estradiol were weighed and dissolved in 10 mL of pH 7.4 PBS solution at 4 °C. The solution was then filtered through a 0.2 μm microporous membrane for sterilization. The cellulase and estrogen mixture was loaded into a syringe and uniformly delivered into the nanofiber membrane using a nanoneedle delivery device (2.5 mm long, 125 μm diameter needle, delivery speed 1.0 mL / min). Subsequently, the material was freeze-dried (-50 °C, 0.1 mbar vacuum, 72 hours) to obtain the bio-cellulose anti-adhesion material.

[0036] Example 2

[0037] The difference between Example 2 and Example 1 is that in Example 2, the grafting of N-vinylpyrrolidone was initiated only by ascorbic acid-hydrogen peroxide-mediated single radical grafting, while in Example 1, N-vinylpyrrolidone was in-situ oxidative grafting initiated by both the TEMPO-NaBr-sodium hypochlorite system and the ascorbic acid-hydrogen peroxide system. The specific steps are as follows:

[0038] (1) TEMPO-catalyzed oxidation: Weigh 100g of a 5.0% solids content bio-cellulose membrane (provided by Hainan Guangyu Biotechnology Co., Ltd.), immerse it in 400mL of a solution containing 0.1g TEMPO and 0.5g NaBr, adjust the pH to 10.5 with 0.5M NaOH, add 37.5mL of 10% sodium hypochlorite solution, place on a plate shaker, and oxidize at 20rpm and 25℃ for 24 hours. After the reaction, remove the bio-cellulose membrane and rinse it with deionized water for 30 minutes each time until the pH of the rinsing solution is neutral and the conductivity is less than 5μS / cm.

[0039] (2) In-situ oxidative grafting of N-vinylpyrrolidone initiated by single free radicals: 30 mL of 0.5 M ascorbic acid solution was added to a three-necked flask containing the suspension of the bio-cellulose membrane washed in step (1). Nitrogen gas was introduced for 5 minutes, and the flask was then sealed with a rubber stopper. Under nitrogen protection, 5 mL of 30% hydrogen peroxide solution was added using a syringe, and the flask was placed on a plate shaker at 20 rpm and 37°C to initiate free radical generation for 10 minutes. Subsequently, 10 mL of N-vinylpyrrolidone was added using a syringe, and the three-necked flask was placed on a shaker at 70°C and reacted at 20 rpm for 6 hours. After the reaction, the bio-cellulose membrane was removed and rinsed with deionized water for 30 minutes each time until the pH of the rinsing solution was neutral and the conductivity was less than 5 μS / cm. The rinsed bio-cellulose membrane was then autoclaved at 121°C for 30 minutes, rinsed three times with sterile medical injection water, and stored at 4°C for later use.

[0040] (3) Nanoneedle delivery of cellulase and estrogen: 400 mg of a compound cellulase with an enzyme activity of 50 U / mg and 100 mg of β-estradiol were weighed and dissolved in 10 mL of pH 7.4 PBS solution at 4 °C. The solution was then filtered through a 0.2 μm microporous membrane for sterilization. The cellulase and estrogen mixture was loaded into a syringe and uniformly delivered into the nanofiber membrane using a nanoneedle delivery device (2.5 mm long, 125 μm diameter needle, delivery speed 1.0 mL / min). Subsequently, the material was freeze-dried (-50 °C, 0.1 mbar vacuum, 72 hours) to obtain the bio-cellulose anti-adhesion material.

[0041] Example 3

[0042] The difference between Example 3 and Example 1 is that, except that the amount of N-vinylpyrrolidone added is adjusted to 5 mL and the grafting reaction is carried out for 3 hours, the other parameters and steps are the same as in Example 1.

[0043] To highlight the beneficial effects of the present invention, the following comparative experiments are provided.

[0044] Comparative Example 1

[0045] Comparative Example 1 used bio-cellulose raw material, which was soaked only in deionized water for 30 minutes each time, until the pH of the soaking solution was neutral and the conductivity was below 5 μS / cm. It was then autoclaved at 121°C for 30 minutes and rinsed three times with sterile water for medical injection. It was then refrigerated at 4°C for later use. It was completely unmodified and unloaded with any drugs.

[0046] Comparative Example 2

[0047] Comparative Example 2 is identical to Example 1 except that the TEMPO, NaBr, and sodium hypochlorite solutions in steps (1) and (2) are replaced with an equal volume of deionized water. That is, Comparative Example 2 does not involve the TEMPO catalytic oxidation step.

[0048] Comparative Example 3

[0049] Comparative Example 3 is identical to Example 1 except that the N-vinylpyrrolidone solution in step (2) is replaced with an equal volume of deionized water. That is, Comparative Example 3 does not involve the N-vinylpyrrolidone grafting reaction step.

[0050] Verification Experiment 1: Performance Analysis and Characterization of Bio-cellulose and its Modified Materials

[0051] To verify the surface-modified biocellulose prepared in Examples 1-3 and Comparative Examples 1-3, the nitrogen content and contact angle of the modified materials were detected by scanning electron microscopy-energy dispersive spectroscopy and surface contact angle measurement, respectively. The degradation rate of the materials was analyzed by quantitatively analyzing the residual solids after 14 days of degradation. The release rate was calculated by the ratio of β-estradiol release after 14 days to the initial loading. The adhesion efficiency of fibroblasts (NIH 3T3) was evaluated by in vivo microscopy counting method after 5 days of co-culturing of cellulose materials with cells (with blank control as reference). The results are shown in Table 1:

[0052] Table 1 Performance analysis and characterization of bio-cellulose and its modified materials

[0053]

[0054] As can be seen from the table, the functionally modified biocellulose prepared in Example 1 has a high nitrogen content on its surface and a high water contact angle. Furthermore, after loading with cellulase and β-estradiol, it can be controlled to degrade and continuously release the drug within two weeks of a normal intrauterine adhesion treatment cycle. In addition, fibroblasts have a low adhesion rate to its surface, exhibiting anti-adhesion effects.

[0055] Therefore, the materials prepared in Example 1 were further analyzed and characterized in depth, and the results are as follows.

[0056] Verification Experiment 2: TEMPO-catalyzed oxidation - dual radical initiation grafting of N-vinylpyrrolidone characterization and verification

[0057] To verify the surface-modified biocellulose prepared in Example 1, the material was analyzed using Fourier transform infrared spectroscopy, a surface contact angle meter, and scanning electron microscopy-energy dispersive spectroscopy. The results are as follows: Figure 1 As shown.

[0058] Compared to biological cellulose raw materials, the modified biological cellulose exhibits a newly appearing absorption peak at 1290 cm⁻¹ in its infrared spectrum. -1 The peak at 1650 cm⁻¹ represents the C-N stretching vibration absorption peak of the secondary amine N-vinylpyrrolidone. -1 The enhanced absorption peak is attributed to the -C==O stretching vibration absorption peak of polyvinylpyrrolidone and the stretching vibration absorption peak of -COO' introduced into the carboxyl group by TEMPO catalytic oxidation. (1736 cm⁻¹) -1 The stretching vibration absorption peak of the carboxyl group (-COOH) was introduced to the TEMPO-catalyzed oxidation. Following this, N-vinylpyrrolidone was successfully introduced onto the surface of bio-cellulose through TEMPO-catalyzed oxidation and free radical-initiated polymerization.

[0059] Analysis of the water contact angle on the material surface shows that the water contact angle significantly increases after introducing N-vinylpyrrolidone onto the cellulose surface. Compared to the hydrophilic surface of biological cellulose, the modified biological cellulose surface exhibits increased hydrophobicity, which can reduce cell adhesion on the surface and lay the foundation for the material's anti-adhesion properties.

[0060] SEM analysis of the surface morphology of the material revealed that the surface of the unmodified bio-cellulose was an ultrafine fibrous network structure with a rough and porous surface. Although the modified material exhibited a wrinkled structure, its surface remained smooth, reducing cell adhesion and preventing tissue aggregation during application. Energy dispersive spectroscopy (EDS) analysis of the C, N, and O content of the material surface showed that the bio-cellulose surface contained 1.26% nitrogen, which originated from residual bacterial proteins or nucleic acids within the membrane. The significant increase in nitrogen content on the modified cellulose surface further validated the successful introduction of N-vinylpyrrolidone into the cellulose surface.

[0061] Verification Experiment 3: Introduction of Cellulase and Estrogen via Nanoneedles

[0062] To verify the delivery of cellulase and estrogen via nanoneedles in Example 1, 100 mg of the prepared material was weighed and placed in a 6-well cell culture plate. 5 mL of PBS solution was added, and the plate was incubated on a shaker at 20 rpm and 37°C. Samples were taken and photographed every 12 hours during incubation. The samples were then centrifuged at 5000 rpm for 5 minutes, and the absorbance of the supernatant was measured at 280 nm. Based on the β-estradiol absorbance standard curve and the amount of β-estradiol delivered via the nanoneedles, the β-estradiol release rate in the material was calculated. After centrifugation, the supernatant was aspirated, and the solid sample was transferred to a 105°C oven and dried to constant weight. The degradation rate of the material was calculated based on the initial sample weight. An equal mass of unmodified biocellulose was used as a control under the same conditions. The results are as follows: Figure 2 As shown.

[0063] As can be seen from the degradation curves, the unmodified biocellulose, lacking cellulase, showed no significant mass loss even after 14 days of incubation. The approximately 1% mass loss observed between 7 and 14 days likely originated from the collapse of the material's edge structure. In contrast, the material prepared in the example showed a gradual increase in mass loss rate with prolonged incubation time, gradually reaching equilibrium after 10 days. Simultaneously, the overall structure of the material began to thin and gradually disintegrate from day 7, becoming almost completely broken after 12 days. This demonstrates that the material prepared by this method can have its structure destroyed by the degradation action of its own embedded cellulase, thus solving the problem of biocellulose materials being unable to degrade autonomously in vivo.

[0064] As can be seen from the β-estradiol release curve, the β-estradiol released from the material prepared in the examples exhibits a rapid release in the first 7 days, followed by a slow and sustained release over 7-12 days until reaching equilibrium. The rapid release of β-estradiol in the first 7 days is attributed to partial surface adsorption and shallower β-estradiol release via microneedle delivery. Simultaneously, with the degradation of the material and the disintegration of its structure, the β-estradiol loaded in deeper layers is continuously released. After 14 days of incubation, the β-estradiol release rate reaches 97%, demonstrating good loading capacity. The released estrogen is expected to provide a stable and continuous biomolecular signal for endometrial repair, thereby achieving the goal of actively participating in and regulating endometrial repair.

[0065] Experiment 4: Verification of the anti-adhesion potential and immune safety of bio-cellulose

[0066] To verify the anti-adhesion potential and immunological safety of the bio-cellulose prepared in Example 1, the prepared material was co-cultured with NIH 3T3 fibroblasts and Raw 264.7 macrophages, respectively. After 5 days of culture, immunofluorescence staining and laser confocal microscopy were used to analyze cell adhesion and growth status. The results are as follows: Figure 3 As shown.

[0067] As shown in the laser confocal microscope images of macrophage growth, after co-culturing macrophages with the biocellulose and the biocellulose anti-adhesive material prepared in the examples for 5 days, the macrophages showed good growth and exhibited the same cell morphology as the blank control. No pro-inflammatory or anti-inflammatory polarization was observed in the cells, indicating that the material does not cause an inflammatory response or have anti-inflammatory function, and is an immune-inert material. At the same time, the normal growth of macrophages indicates that the material has no cytotoxicity and good biocompatibility.

[0068] As seen in the laser confocal microscopy images of fibroblast growth, fibroblasts almost completely adhered to and covered the surface of the unmodified biocellulose, exhibiting good cell growth. The unmodified biocellulose surface did not possess anti-adhesion properties. In contrast, the biocellulose anti-adhesion material prepared in the examples showed only a small amount of cell adhesion and growth on its surface, and the cytoskeleton structure was not fully unfolded, indicating that the material exhibited better anti-adhesion properties.

[0069] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a bio-cellulose anti-adhesion material, characterized in that: In situ grafting of N-vinylpyrrolidone to modify biocellulose by oxidative coupling free radicals, followed by the introduction of cellulase and estrogen into nanoneedles, yields a biocellulose anti-adhesion material. Includes the following steps: (1) TEMPO catalytic oxidation: Weigh out a biological cellulose membrane with a solid content of 5.0%, immerse it in a solution containing TEMPO and NaBr, adjust the pH with NaOH, add 10% sodium hypochlorite solution, place it on a plate shaker, and oxidize it at 25°C; (2) In-situ oxidation of N-vinylpyrrolidone initiated by dual radicals: Add ascorbic acid solution to the suspension of bio-cellulose membrane catalytically oxidized in step (1), then introduce nitrogen into the reaction vessel and seal it with a rubber stopper; under nitrogen protection, add 30% hydrogen peroxide solution, a mixed solution of TEMPO and NaBr, and 10% sodium hypochlorite solution, place it on a plate shaker, and initiate the generation of free radicals at 37°C; Then, N-vinylpyrrolidone was added using a syringe, and the reaction vessel was placed in a shaker at 70°C for reaction. After the reaction was completed, the bio-cellulose membrane was removed and rinsed with deionized water until the pH of the rinsing solution was neutral and the conductivity was less than 5 μS / cm. The washed bio-cellulose membrane was then placed in autoclave, rinsed again, and stored at 4°C for later use. (3) Nanoneedle delivery of cellulase and estrogen: weigh the compound cellulase and β-estradiol separately, dissolve them in pH 7.4 PBS solution at 4°C, and filter them through a 0.2μm microporous membrane for sterilization; load the cellulase and estrogen mixture into a syringe, and use a nanoneedle delivery device to deliver it evenly into the nanofiber membrane; then freeze dry to obtain the biocellulose anti-adhesion material.

2. The method for preparing the bio-cellulose anti-adhesion material according to claim 1, characterized in that: In step (1), the mass ratio of the active ingredient in the biocellulose membrane to TEMPO is 1:0.02, the mass ratio of the active ingredient in the biocellulose membrane to NaBr is 1:0.1, and the mass ratio of the active ingredient in the biocellulose membrane to 10% sodium hypochlorite solution is 1:7.

5.

3. The method for preparing the bio-cellulose anti-adhesion material according to claim 2, characterized in that: In step (1), the solid content of the biocellulose membrane is 5.0%; the concentration of NaOH is 0.5 M, and the pH is adjusted to 10.

5.

4. The method for preparing the bio-cellulose anti-adhesion material according to claim 3, characterized in that: The specific steps (1) are as follows: weigh 100g of biological cellulose membrane with a solid content of 5.0%, immerse it in 400 mL of solution containing 0.1g TEMPO and 0.5g NaBr, adjust the pH to 10.5 with 0.5 M NaOH, add 37.5 mL of 10% sodium hypochlorite solution, place it on a flat shaker, and oxidize it at 20 rpm and 25°C for 6-24 hours.

5. The method for preparing the bio-cellulose anti-adhesion material according to claim 1, characterized in that: In step (2), the concentration of ascorbic acid solution is 0.5M, the concentration of TEMPO is 50 mM, and the concentration of NaBr is 100 mM.

6. The method for preparing the bio-cellulose anti-adhesion material according to claim 5, characterized in that: Step (2) specifically involves adding 30 mL of 0.5 M ascorbic acid solution to the catalytically oxidized cellulose membrane suspension from step (1), then purging the reaction vessel with nitrogen gas and sealing it with a rubber stopper; under nitrogen protection, using a syringe, add 5 mL of 30% hydrogen peroxide solution, 5 mL of a mixed solution of 50 mM TEMPO and 100 mM NaBr, and 5 mL of 10% sodium hypochlorite solution, place it on a flat shaker, and initiate free radical generation for 10 minutes at 20 rpm and 37°C; subsequently, using a syringe, add 5-10 mL of N-vinylpyrrolidone, place the three-necked flask in a shaker at 70°C, and react for 3-6 hours at 20 rpm; after the reaction, remove the cellulose membrane and rinse it with deionized water for 30 minutes each time until the pH of the rinsing solution is neutral and the conductivity is below 5. μS / cm; After washing, the bio-cellulose membrane was autoclaved at 121℃ for 30 minutes, then rinsed three times with sterile water for medical injection, and stored at 4℃ for later use.

7. The method for preparing the bio-cellulose anti-adhesion material according to claim 1, characterized in that: In step (3), the enzyme activity of the composite cellulase is 50 U / mg, the nanoneedle delivery device has a needle length of 2.5 mm, a diameter of 125 μm, and a delivery speed of 1.0 mL / min; the freeze-drying conditions are -50℃. 0.1 mbar vacuum, 72 hours.

8. The method for preparing the bio-cellulose anti-adhesion material according to claim 7, characterized in that: The specific steps (3) are as follows: 400 mg of a compound cellulase with an enzyme activity of 50 U / mg and 100 mg of β-estradiol are weighed and dissolved in 10 mL of pH 7.4 PBS solution at 4°C. The solution is then filtered through a 0.2 μm microporous membrane for sterilization. The mixture of cellulase and estrogen is loaded into a syringe and uniformly introduced into a nanofiber membrane using a nanoneedle delivery device. The membrane is then freeze-dried to obtain a biological cellulose anti-adhesion material.

9. The biocellulose anti-adhesion material prepared by the preparation method according to any one of claims 1-8.

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