Drug-loaded oral biofilm and preparation method and application thereof

Abstract

The application adopts small intestinal submucosa SIS and animal membrane tissue AMT as raw materials, respectively carries out deantigen treatment on the small intestinal submucosa and the animal membrane tissue, and obtains a decellularized membrane. The SIS decellularized membrane is subjected to acidolysis and salt precipitation treatment to obtain a gelatin solution. The gelatin solution is subjected to micro-crosslinking under an EDC / NHS crosslinking system, and then the crosslinking agent is removed through dialysis. An antibiotic drug, PAMAM and glycerol are mixed with the micro-crosslinked gelatin solution, PAMAM is used as a drug loading medium to combine with the micro-crosslinked gelatin solution to form a drug loading solution. The SIS decellularized membrane is soaked in the drug loading solution, and then is combined with the AMT decellularized membrane to form a sandwich structure. The drug loading oral membrane has good barrier effect, is safe and effective, can release the antibiotic drug in the degradation process, has a stable slow-release process and length, thereby reducing the pain of patients, improving the success rate of operation and accelerating wound healing.

Description

A drug-loaded oral biofilm, its preparation method and application Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a drug-loaded oral biomembrane, its preparation method, and its application. Background Technology

[0002] This invention relates to an oral biomembrane, an implantable biodegradable medical device material widely used in GBR procedures. It is typically used in conjunction with inorganic bone powder to isolate the contact surfaces of soft and hard tissues, preventing soft tissue intrusion during bone defect repair and providing sufficient support space for bone regeneration in the defect area.

[0003] Oral biofilms are widely used in clinical practice due to their excellent biocompatibility. PTFE membranes are generally considered the first generation of oral membrane products. These products are easy to handle, have good biocompatibility, and relatively ideal mechanical strength, and are used as the gold standard for comparison with other guided tissue regeneration membrane materials. However, their disadvantages are also obvious. Due to their non-degradability, they require a second surgery for removal, increasing the cost and risk of treatment.

[0004] Absorbable biofilm materials, as second-generation oral membrane products, offer excellent biocompatibility and eliminate the need for secondary surgery, thus shortening treatment time and gaining popularity among doctors and patients. Considering the changing demands of the body's environment on oral membrane materials, researching intelligent oral membrane materials that adapt to environmental changes will be one of the research directions and development trends in this field. Third-generation oral membranes are gradually being applied clinically. They not only possess excellent barrier function but can also act as delivery devices to release special agents, such as antibiotics, growth factors, and adhesion factors, either at a specific time or continuously in the wound area, facilitating better coordination and guidance of wound healing.

[0005] 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) is a good carboxyl / amino crosslinking agent. EDC undergoes a nucleophilic addition reaction with the carboxylic acid of CMCS2 to form an active but unstable intermediate, which then reacts with its amino functional group to form an amide bond. However, the conversion rate of the reaction is very low, mainly because the intermediate is very unstable and easily decomposes. Furthermore, EDC is an unstable crosslinking agent and also decomposes in water. However, introducing N-hydroxysuccinimide (NHS) into the crosslinking system can form a metastable intermediate, which can improve the crosslinking conversion rate.

[0006] Polyamide-amine (PAMAM) dendritic polymers are a new type of polymer with a highly ordered, three-dimensional hyperbranched spatial geometric symmetry structure, consisting of a central core, internal cavities, abundant branching structures, and modifiable surface functional groups. PAMAMs are characterized by controllable molecular weight, multifunctionality, good solubility, and biocompatibility, and are widely used in fields such as biology, medicine, and industrial production.

[0007] Patent application CN102091057A discloses a method for preparing drug-loaded biofilms. The main process involves dissolving collagen in glacial acetic acid, then freeze-drying the resulting slurry to prepare a biofilm. The biofilm is then immersed in a drug aqueous solution and freeze-dried again to obtain the drug-loaded biofilm. However, the drug loading is achieved through biofilm adsorption, which is solely physical adsorption. This results in low drug loading and early drug release, failing to provide a slow and sustained drug release process and thus failing to achieve a prolonged drug effect. Summary of the Invention

[0008] The purpose of this invention is to provide a degradable drug-loaded oral biofilm with good barrier function, safety and effectiveness, and a slow and sustained drug release process, as well as its preparation method and application.

[0009] To achieve the above objectives, the present invention provides a drug-loaded oral biomembrane, which has a sandwich structure, specifically: it consists of a bottom layer of decellularized animal membrane tissue AMT sheet, a top layer of decellularized animal membrane tissue AMT sheet, and multiple layers of small intestinal submucosa SIS decellularized membrane sheets treated with a drug-loaded solution; wherein, both the bottom layer and the top layer of decellularized animal membrane tissue AMT sheet have a brushed surface formed by brushing on one side;

[0010] The first brushed surface of the bottom animal membrane tissue AMT decellularized membrane sheet is opposite to the second brushed surface of the top animal membrane tissue AMT decellularized membrane sheet;

[0011] The multilayer small intestinal submucosa SIS decellularized membrane sheet is composited between the first and second brushed surfaces.

[0012] A method for preparing the above-mentioned drug-loaded oral biofilm, characterized by comprising the following steps:

[0013] S1. Antigens were removed from the submucosal layer SIS of the small intestine and the AMT of the animal membrane tissue, respectively, to obtain decellularized membrane sheets of the submucosal layer SIS of the small intestine and decellularized membrane sheets of the AMT of the animal membrane tissue.

[0014] S2. The decellularized SIS submucosal membrane sheet of the small intestine is subjected to acid hydrolysis and salting out to obtain a gelatin solution;

[0015] S3. After micro-crosslinking the gelatin solution in the EDC / NHS crosslinking system, the crosslinking agent is removed by dialysis;

[0016] S4. Mix the antibiotic drug, PAMAM, glycerin and the micro-crosslinked gelatin solution prepared in S3, so that PAMAM can be used as a drug carrier to combine with the micro-crosslinked gelatin solution to form a drug-carrying solution.

[0017] S5. The submucosal layer of the small intestine (SIS) is immersed in a drug-loaded solution, and then combined with the AMT decellularized membrane sheet of animal membrane tissue according to the sandwich structure described in claim 1.

[0018] S1 includes the following steps:

[0019] S11. The animal small intestine is pre-treated using a scraper. The obtained submucosal layer SIS of the small intestine is soaked in 0.9% physiological saline and shaken to remove dirt.

[0020] S12. Use a gut dissection tool to cut open the tubular small intestine submucosa SIS, cut it into small segments, and disinfect it by soaking it in disinfectant at room temperature for 1-5 hours.

[0021] S13. After cleaning the disinfected submucosal SIS material of the small intestine with purified water by shaking, freeze it at -20℃~40℃ for 10~24h, and then thaw it at room temperature.

[0022] S14. Place the ablated submucosal SIS material of the small intestine in hypertonic saline and wash with shaking at room temperature for 5-20 minutes. Then place it in purified water and wash with shaking at room temperature for 5-20 minutes. Repeat this process 3-5 times for decellularization.

[0023] S15. Place the decellularized submucosal SIS material of the small intestine in 1-5% descaling agent and shake at room temperature for 4-16 hours. Then wash with purified water and store in 0.9% physiological saline.

[0024] S2 includes the following steps:

[0025] S21. Animal membrane tissue (AMT) is pretreated to remove surface stains, fascia and fat, processed by a splitting machine, and then washed with purified water or 0.9% physiological saline to remove blood stains.

[0026] S22. Place the pretreated animal membrane tissue AMT material in a defatting agent and shake it at room temperature for 4h to 30h. Then wash it with purified water to obtain the defatted sample.

[0027] S23. Place the defatted sample in a 1-5% enzyme solution and soak at 37°C for 1-5 hours, then wash with purified water.

[0028] S24. Next, place it in a 1% to 10% alkaline solution and let it stand at room temperature for 30 to 120 minutes. Then wash it with purified water or neutral phosphate buffer until the pH is close to neutral.

[0029] S25. Next, place it in a 3% to 30% oxidation solution and let it stand at room temperature for 30 to 120 minutes. Then wash it with purified water or neutral phosphate buffer until the pH is close to neutral to obtain a decellularized sample.

[0030] S26. Place the decellularized sample in a 1-5% detergent solution and shake at room temperature for 4-16 hours, then wash with purified water.

[0031] S27. The sample prepared in S26 was dehydrated using gradient ethanol solutions. The dehydration time for each gradient was 1 to 4 hours. After that, the sample was placed in a fume hood for ventilation and drying.

[0032] S28. The obtained animal membrane tissue AMT decellularized membrane sheet is subjected to single-sided texturing using a texturing machine and is ready for use.

[0033] S3 includes the following steps:

[0034] S31. The decellularized submucosal membrane sheet of small intestine obtained in S15 is air-dried and then pulverized using a pulverizer.

[0035] S32. Soak the SIS decellularized membrane sheet powder of the submucosal layer of small intestine in an acidic solution at room temperature for 10-24 hours, filter, and retain the filtrate.

[0036] S33. Add sodium chloride to the filtrate while stirring until no milky white flocculent gelatin is produced. Filter and dialyze the gelatin solution. The dialysate consists of alternating phosphate buffer and purified water.

[0037] S34. Place the gelatin solution in an oven at 35-45℃ and dry it until the viscosity of the gelatin solution is 100-500 mPa·s.

[0038] S35. Add 0.01% to 2% of an equimolar ratio EDC / NHS crosslinking system to the prepared gelatin solution. After stirring evenly, refrigerate and stand for 5 to 8 hours to obtain a micro-crosslinked gelatin solution. Dialyze the micro-crosslinked gelatin solution to remove excess crosslinking agent. The external dialysis solution is phosphate buffer and purified water alternately.

[0039] S36. Weigh out the following components according to the mass percentages: 10% micro-crosslinked gelatin solution, 1.0-5.0% glycerol, 0.001-0.2% PAMAM, and 0.5-2.0% antibiotic. Mix them thoroughly to obtain the drug-loaded solution, and refrigerate until ready for use.

[0040] S4 includes the following steps:

[0041] S41. After draining the small intestinal submucosal layer SIS decellularized membrane obtained in S15, place it in the drug-loaded solution obtained in S36 for soaking. Lay the soaked small intestinal submucosal layer SIS decellularized membrane flat on a horizontal mold, stacking 2 to 8 layers in total. The layers should be tightly attached without air bubbles and without wrinkles at the edges.

[0042] S42. Lay the fibrous AMT decellularized animal membrane tissue sheet flat on a horizontal mold, ensuring the unfibrous side is tightly fitted to the mold without air bubbles. Then place the stacked small intestinal submucosal layer SIS decellularized membrane sheet on the fibrous side of the animal membrane tissue AMT decellularized membrane sheet, ensuring a tight fit without air bubbles. Finally, cover the small intestinal submucosal layer SIS decellularized membrane sheet with the fibrous side of the animal membrane tissue AMT decellularized membrane sheet facing down, forming a sandwich structure.

[0043] S43. Place the composite drug-loaded biomembrane obtained in S42 into a freeze dryer for freeze drying;

[0044] S44. After drying, the drug-loaded biofilm is pressed by a film press, packaged, and sterilized with ethylene oxide or by irradiation to obtain the final product.

[0045] In S12, the disinfectant is one or two of 75% ethanol, sodium hypochlorite, 3% hydrogen peroxide, and peracetic acid.

[0046] In S14, the hypertonic brine is a mixed solution of sodium chloride with a concentration of 5-10% and sodium hydroxide with a concentration of 0.1-0.5%.

[0047] In S15, the descaling agent is one or a mixture of two or more of Triton X-100, sodium dodecyl sulfate, and sodium dodecyl sulfonate.

[0048] In S22, the degreasing agent is one or a mixture of two or more of acetone, methanol, ethanol, isopropanol, and chloroform;

[0049] In S23, the enzyme solution is one or more of the following: pancreatic enzyme, protease, lipase, and nuclease, in a phosphate buffer solution.

[0050] In S24, the alkaline solution is one or a mixture of two or more of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, and sodium bicarbonate.

[0051] In S25, the oxidizing solution is a hydrogen peroxide solution with a pH of 11 to 14;

[0052] In S26, the descaling agent is one or a mixture of two or more of Triton X-100, sodium dodecyl sulfate, and sodium dodecyl sulfonate;

[0053] In S27, the organic solvent is one or a mixture of two or more of acetone, isopropanol, n-hexane, chloroform, and petroleum ether.

[0054] In S32, the acid solution is a hydrochloric acid or acetic acid solution with a concentration of 0.1% to 5%.

[0055] Application of drug-loaded oral biofilm in hard tissue defect repair during oral GBR surgery.

[0056] The beneficial effects of this invention are as follows: Using submucosal layer of the small intestine (SIS) and animal membrane tissue (AMT) as raw materials, the SIS and AMT are treated to remove antigens, respectively, to obtain decellularized membrane sheets. The SIS decellularized membrane sheets are then subjected to acid hydrolysis and salting-out treatment to obtain a gelatin solution. The gelatin solution is micro-crosslinked in an EDC / NHS crosslinking system, and the crosslinking agent is removed by dialysis. Antibiotic drugs, PAMAM, glycerol, and the micro-crosslinked gelatin solution are mixed, allowing PAMAM to bind with the micro-crosslinked gelatin solution as a drug-loaded medium, forming a drug-loaded solution. The SIS decellularized membrane sheet is immersed in the drug-loaded solution and then composited with the AMT decellularized membrane sheet to form a sandwich structure. This drug-loaded oral membrane has a good barrier function, is safe and effective, and releases antibiotic drugs during degradation, thereby reducing patient pain, improving surgical success rates, and accelerating wound healing. It has a rich porous structure, good hydrophilicity, and excellent adhesion and biocompatibility. The unique sandwich structure of drug-loaded oral biofilms can act as a physical barrier during the repair and treatment process. At the same time, the drug-loaded components in the middle of the sandwich structure can effectively and sustain the release of antibiotics for an ideal duration, which can effectively improve the inflammatory characteristics of the treatment process, increase the success rate of the operation, and reduce patient suffering. Attached Figure Description

[0057] Figure 1 is a schematic diagram of the structure of a drug-loaded oral biomembrane sandwich.

[0058] Figure 2. Electron micrograph of AMT decellularized membrane sheet after single-sided treatment by a brushing machine.

[0059] Figure 3 shows the antibiotic release rate data for each embodiment and comparative example under simulated conditions.

[0060] Figure 4 illustrates the peel strength test results for each embodiment.

[0061] Figure 5 shows the cytotoxicity detection results for each embodiment. In the figures, a–d represent Examples 1–4; e is the negative control; and f is the positive control. Detailed Implementation

[0062] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the specific implementation methods, structural features and effects of the present invention are described in detail below with reference to the accompanying drawings and embodiments.

[0063] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0064] Example 1:

[0065] 1. Preparation of SIS decellularized membrane sheets:

[0066] S11: The pig small intestine is pre-treated using a scraper. The obtained submucosal layer of small intestine (SIS) is then soaked in 0.9% saline solution and shaken to remove dirt.

[0067] S12: Use a guttural cutter to cut the tubular SIS open and cut it into small segments. Disinfect by soaking in 3% hydrogen peroxide at room temperature for 1 hour.

[0068] S13: After sterilizing the SIS material, rinse it with purified water by shaking, freeze it at -20℃ to 40℃ for 10 hours, and then thaw it at room temperature.

[0069] S14: Place the ablation-treated SIS material in a mixed solution of 5% sodium chloride and 0.1% sodium hydroxide, and wash with shaking at room temperature for 5 minutes. Then place it in purified water and wash with shaking at room temperature for 5 minutes. Repeat the above steps 3 times for decellularization.

[0070] S15: After decellularization, the SIS material was placed in 1% Triton X-100 solution and treated with shaking at room temperature for 4 hours. Then it was washed with purified water and stored in 0.9% physiological saline.

[0071] 2. Preparation of AMT decellularized membrane sheets:

[0072] S21. The porcine pericardium is pretreated to remove surface stains, fascia, fat, and other contaminants. After processing with a splitting machine, a membrane sheet with a thickness of 0.2-0.5 mm is obtained, and then washed with purified water by shaking to remove blood stains.

[0073] S22: Place the pretreated porcine pericardium in acetone solution and shake at room temperature for 4 hours. Discard the waste liquid and repeat the above operation twice. Then wash with purified water by shaking.

[0074] S23: Place the defatted sample in 1% trypsin phosphate buffer solution, let it stand at 37°C for 1 hour, and then wash it with purified water.

[0075] S24: Place the sample in a 1% sodium hydroxide solution and let it stand at room temperature for 30 minutes. Then wash with purified water until the pH is close to neutral.

[0076] S25: Place the sample in a 3% hydrogen peroxide solution with a pH of 12, let it stand at room temperature for 30 minutes, and then wash it with purified water until the pH is close to neutral.

[0077] S2:6: The decellularized sample was placed in a 1% Triton X-100 solution and treated with shaking at room temperature for 4 hours. This process was repeated twice. The sample was then washed with purified water and freeze-dried to obtain the oral biofilm.

[0078] S27: The sample was dehydrated using a gradient of ethanol solutions, with each gradient having a dehydration time of 1 hour. The ethanol gradient consisted of 50%, 75%, 90%, and anhydrous ethanol. After dehydration, the sample was immersed in an acetone / isopropanol (mass ratio 1:1) solution at room temperature for 10 minutes. The sample was then placed in a fume hood and ventilated to allow it to dry.

[0079] S28: The obtained AMT decellularized membrane sheets are subjected to single-sided napping treatment using a napping machine and set aside for later use.

[0080] 3. The preparation process of the drug-loaded solution is as follows:

[0081] S31: The decellularized SIS membrane obtained in S15 is air-dried and then pulverized using a pulverizer.

[0082] S32: Soak the SIS decellularized membrane powder in a 0.1% hydrochloric acid solution at room temperature for 10 hours, filter, and retain the filtrate.

[0083] S33: Add sodium chloride to the filtrate while stirring until no milky white flocculent gelatin is produced. Filter and dialyze the gelatin solution, alternating between phosphate buffer and purified water as the outer dialysate three times, with each treatment lasting 10 minutes.

[0084] S34: Dry the gelatin solution in a 35°C oven until the viscosity of the gelatin solution is 117 mPa·s.

[0085] S35: Add 0.01% of an equimolar ratio EDC / NHS crosslinking system to the prepared gelatin solution, stir until homogeneous, and then refrigerate for 5 hours. Dialyze the resulting micro-crosslinked gelatin solution to remove excess crosslinking agent. The peripheral dialysate is phosphate buffer and purified water, alternating three times, with each treatment lasting 10 minutes.

[0086] S36: Weigh the other components as follows:

[0087] Ingredient Name: Microcrosslinked Gelatin Solution, Glycerin, PAMAM, Antibiotic Component Percentage (m / m): 10, 10.00, 11.0 surface

[0088] Mix the weighed components thoroughly to obtain a drug-loaded solution, and store it under refrigeration until ready for use.

[0089] 4. The composite process of drug-loaded oral biofilm is as follows:

[0090] S41: After draining the decellularized SIS membrane obtained in S15, soak it in the drug-loaded solution obtained in S36. Lay the soaked decellularized SIS membrane flat on a horizontal mold, stacking two layers in total. The layers should fit tightly together without air bubbles and without wrinkles at the edges.

[0091] S42: Lay the brushed AMT decellularized membrane sheet flat on a horizontal mold, ensuring the unbrushed side is tightly fitted to the mold without air bubbles. Then place the stacked SIS decellularized membrane sheet on the brushed side of the AMT decellularized membrane sheet, ensuring a tight fit without air bubbles. Finally, cover the SIS decellularized membrane sheet with the brushed side of the AMT decellularized membrane sheet facing down, forming a sandwich structure.

[0092] S43: Place the compounded drug-loaded oral biofilm in a freeze dryer for freeze drying.

[0093] S44: The dried drug-loaded oral membrane is pressed by a film press and packaged for sterilization with ethylene oxide.

[0094] Example 2:

[0095] 1. Preparation of SIS decellularized membrane sheets:

[0096] S11: The pig small intestine is pre-treated using a scraper. The obtained submucosal layer of small intestine (SIS) is then soaked in 0.9% saline solution and shaken to remove dirt.

[0097] S12: The tubular SIS was cut open using a guillotine and cut into small segments. It was then immersed in 75% ethanol at room temperature for 2 hours, followed by immersion in 3% hydrogen peroxide for 5 hours for disinfection.

[0098] S13: After sterilizing the SIS material, rinse it thoroughly with purified water by shaking, then freeze it at -20℃ to 40℃ for 24 hours, and then thaw it at room temperature. Repeat the freeze-thaw cycle 3 times.

[0099] S14: Place the ablation-treated SIS material in a mixed solution of 10% sodium chloride and 0.5% sodium hydroxide, and wash with shaking at room temperature for 20 min. Then place it in purified water and wash with shaking at room temperature for 5 min. Repeat the above steps 5 times for decellularization.

[0100] S15: After decellularization, the SIS material was placed in a 5% sodium dodecyl sulfate solution and treated with shaking at room temperature for 8 hours. Then it was washed with purified water. The above operation was repeated twice. Then it was stored in 0.9% physiological saline.

[0101] 2. Preparation of AMT decellularized membrane sheets:

[0102] S21. The bovine pericardium is pretreated to remove surface stains, fascia, fat, and other contaminants. After being processed by a slicing machine, a membrane sheet with a thickness of 0.2-0.5 mm is obtained, and then it is rinsed with 0.9% physiological saline to remove blood stains.

[0103] S22: Place the pretreated bovine pericardium in a mixture of acetone / isopropanol (mass ratio 1:4), shake at room temperature for 10 hours, discard the waste liquid, repeat the above operation 3 times, and then wash with purified water by shaking.

[0104] S23: Place the defatted sample in 5% protease / lipase (mass ratio 1:1) phosphate buffer, let it stand at 37°C for 5 hours, and then wash it with purified water.

[0105] S24: Place the sample in a 10% sodium hydroxide solution and let it stand at room temperature for 120 minutes. Then wash with neutral phosphate buffer until the pH is close to neutral.

[0106] S25: Place the sample in a 30% hydrogen peroxide solution with a pH of 14 and let it stand at room temperature for 120 minutes. Wash with neutral phosphate buffer until the pH is close to neutral.

[0107] S26: The decellularized sample was placed in a 5% Triton X-100 solution and treated with shaking at room temperature for 8 hours. This process was repeated twice. The sample was then washed with purified water and freeze-dried to obtain the oral biofilm.

[0108] S27: The sample was dehydrated using a gradient of ethanol solutions, with each gradient lasting 4 hours. The ethanol gradient consisted of 50%, 75%, 90%, and anhydrous ethanol. After dehydration, the sample was immersed in petroleum ether at room temperature for 30 minutes. The sample was then placed in a fume hood and ventilated to allow it to dry.

[0109] S28: The obtained AMT decellularized membrane sheets are subjected to single-sided napping treatment using a napping machine and set aside for later use.

[0110] 3. The preparation process of the drug-loaded solution is as follows:

[0111] S31: The decellularized SIS membrane obtained in S15 is air-dried and then pulverized using a pulverizer.

[0112] S32: Soak the SIS decellularized membrane powder in a 5% acetic acid solution at room temperature for 24 hours, filter, and retain the filtrate.

[0113] S33: Add sodium chloride to the filtrate while stirring until no milky white flocculent gelatin is produced. Filter and dialyze the gelatin solution, alternating between phosphate buffer and purified water as the outer dialysate three times, with each treatment lasting 10 minutes.

[0114] S34: Place the gelatin solution in a 45°C oven and dry until the viscosity of the gelatin solution is 154 mPa·s.

[0115] S35: Add 2% of an equimolar ratio EDC / NHS crosslinking system to the prepared gelatin solution, stir until homogeneous, and then refrigerate for 8 hours. Dialyze the resulting micro-crosslinked gelatin solution to remove excess crosslinking agent. The peripheral dialysate is phosphate buffer and purified water, alternating three times, with each treatment lasting 10 minutes.

[0116] S36: Weigh the other components as follows:

[0117] Ingredient Name: Microcrosslinked Gelatin Solution, Glycerin, PAMAM, Antibiotic Component Percentage (m / m): 105.0, 0.2, 1.0 surface

[0118] Mix the weighed components thoroughly to obtain a drug-loaded solution, and store it under refrigeration until ready for use.

[0119] 4. The composite process of drug-loaded oral biofilm is as follows:

[0120] S41: After draining the decellularized SIS membrane obtained in S15, soak it in the drug-loaded solution obtained in S36. Lay the soaked decellularized SIS membrane flat on a horizontal mold, stacking two layers in total. The layers should fit tightly together without air bubbles and without wrinkles at the edges.

[0121] S42: Lay the brushed AMT decellularized membrane sheet flat on a horizontal mold, ensuring the unbrushed side is tightly fitted to the mold without air bubbles. Then place the stacked SIS decellularized membrane sheet on the brushed side of the AMT decellularized membrane sheet, ensuring a tight fit without air bubbles. Finally, cover the SIS decellularized membrane sheet with the brushed side of the AMT decellularized membrane sheet facing down, forming a sandwich structure.

[0122] S43: Place the compounded drug-loaded oral biofilm in a freeze dryer for freeze drying.

[0123] S44: The dried drug-loaded oral membrane is pressed by a film press, packaged, and then sterilized by irradiation.

[0124] Example 3:

[0125] 1. Preparation of SIS decellularized membrane sheets:

[0126] S11: The pig small intestine is pre-treated using a scraper. The obtained submucosal layer of small intestine (SIS) is then soaked in 0.9% saline solution and shaken to remove dirt.

[0127] S12: Use a guillotine cutter to cut the tubular SIS open and cut it into small sections. Disinfect by soaking in 5% sodium hypochlorite solution at room temperature for 1 hour.

[0128] S13: After sterilizing the SIS material, rinse it with purified water by shaking, then freeze it at -20℃ to 40℃ for 10 hours, and then thaw it at room temperature. Repeat the freeze-thaw cycle 3 times.

[0129] S14: Place the ablation-treated SIS material in a mixed solution of 7% sodium chloride and 0.1% sodium hydroxide, and wash with shaking at room temperature for 5 minutes. Then place it in purified water and wash with shaking at room temperature for 5 minutes. Repeat the above steps 3 times for decellularization.

[0130] S15: After decellularization, the SIS material was placed in a 1% sodium dodecyl sulfate solution and shaken at room temperature for 4 hours. Then it was washed with purified water and stored in 0.9% physiological saline.

[0131] 2. Preparation of AMT decellularized membrane sheets:

[0132] S21: The bovine pericardium is pretreated to remove surface stains, fascia, fat, and other contaminants. It is then processed using a slicing machine to obtain a membrane sheet with a thickness of 0.2-0.5 mm, followed by rinsing with purified water to remove blood stains.

[0133] S22: Place the pretreated bovine pericardium in a methanol / chloroform (mass ratio 1:1) mixture and shake at room temperature for 4 hours. Discard the waste liquid and repeat the above operation 3 times. Then wash with purified water by shaking.

[0134] S23: Place the defatted sample in 2% trypsin phosphate buffer, let it stand at 37°C for 3 hours, and then wash it with purified water.

[0135] S24: Place the sample in a 4% sodium hydroxide solution and let it stand at room temperature for 30 minutes. Then wash with neutral phosphate buffer until the pH is close to neutral.

[0136] S25: Place the sample in a 3% hydrogen peroxide solution with a pH of 12, let it stand at room temperature for 30 minutes, and wash it with neutral phosphate buffer until the pH is close to neutral.

[0137] S26: The decellularized sample was placed in a 5% sodium dodecyl sulfate solution and treated with shaking at room temperature for 4 hours. This process was repeated twice. The sample was then washed with purified water and freeze-dried to obtain the oral biofilm.

[0138] S27: The sample was dehydrated using a gradient of ethanol solutions, with each gradient having a dehydration time of 1 hour. The ethanol gradient consisted of 50%, 75%, 90%, and anhydrous ethanol. After dehydration, the sample was immersed in n-hexane at room temperature for 30 minutes. The sample was then placed in a fume hood and ventilated to allow it to dry.

[0139] S28: The obtained AMT decellularized membrane sheets are subjected to single-sided napping treatment using a napping machine and set aside for later use.

[0140] 3. The preparation process of the drug-loaded solution is as follows:

[0141] S31: The decellularized SIS membrane obtained in S15 is air-dried and then pulverized using a pulverizer.

[0142] S32: Soak the SIS decellularized membrane powder in a 3% acetic acid solution at room temperature for 24 hours, filter, and retain the filtrate.

[0143] S33: Add sodium chloride to the filtrate while stirring until no milky white flocculent gelatin is produced. Filter and dialyze the gelatin solution, alternating between phosphate buffer and purified water as the outer dialysate three times, with each treatment lasting 10 minutes.

[0144] S34: Dry the gelatin solution in an oven at 42°C until the viscosity of the gelatin solution is 210 mPa·s.

[0145] S35: Add 0.3% of an equimolar ratio EDC / NHS crosslinking system to the prepared gelatin solution, stir until homogeneous, and then refrigerate for 8 hours. Dialyze the resulting micro-crosslinked gelatin solution to remove excess crosslinking agent. The peripheral dialysate is phosphate buffer and purified water, alternating three times, with each treatment lasting 10 minutes.

[0146] S36: Weigh the other components according to the table below:

[0147] Ingredient Name: Microcrosslinked Gelatin Solution, Glycerin, PAMAM, Antibiotic Component Percentage (m / m): 102.0, 0.00, 51.0 surface

[0148] Mix the weighed components thoroughly to obtain a drug-loaded solution, and store it under refrigeration until ready for use.

[0149] 4. The composite process of drug-loaded oral biofilm is as follows:

[0150] S41: After draining the decellularized SIS membrane obtained in S15, soak it in the drug-loaded solution obtained in S36. Lay the soaked decellularized SIS membrane flat on a horizontal mold, stacking a total of 6 layers. The layers should fit tightly together without air bubbles and have no wrinkles at the edges.

[0151] S42: Lay the brushed AMT decellularized membrane sheet flat on a horizontal mold, ensuring the unbrushed side is tightly fitted to the mold without air bubbles. Then place the stacked SIS decellularized membrane sheet on the brushed side of the AMT decellularized membrane sheet, ensuring a tight fit without air bubbles. Finally, cover the SIS decellularized membrane sheet with the brushed side of the AMT decellularized membrane sheet facing down, forming a sandwich structure.

[0152] S43: Place the compounded drug-loaded oral biofilm in a freeze dryer for freeze drying.

[0153] S44: The dried drug-loaded oral membrane is pressed by a film press, packaged, and then sterilized by irradiation.

[0154] Example 4:

[0155] 1. Preparation of SIS decellularized membrane sheets:

[0156] S11: The pig small intestine is pre-treated using a scraper. The obtained submucosal layer of small intestine (SIS) is then soaked in 0.9% saline solution and shaken to remove dirt.

[0157] S12: Use a guillotine cutter to cut the tubular SIS open and cut it into small sections. Disinfect by soaking in 5% sodium hypochlorite solution at room temperature for 3 hours.

[0158] S13: After sterilizing the SIS material, rinse it with purified water by shaking, then freeze it at -20℃ to 40℃ for 12 hours, and then thaw it at room temperature. Repeat the freeze-thaw cycle 5 times.

[0159] S14: Place the ablation-treated SIS material in a mixed solution of 7% sodium chloride and 0.3% sodium hydroxide, and wash with shaking at room temperature for 10 min. Then place it in purified water and wash with shaking at room temperature for 5 min. Repeat the above steps 3 times for decellularization.

[0160] S15: After decellularization, the SIS material was placed in a 2% sodium dodecyl sulfate solution and treated with shaking at room temperature for 6 hours. Then it was washed with purified water and stored in 0.9% physiological saline.

[0161] 2. Preparation of AMT decellularized membrane sheets:

[0162] S21: The bovine pericardium is pretreated to remove surface stains, fascia, fat, and other contaminants. It is then processed using a slicing machine to obtain a membrane sheet with a thickness of 0.2-0.5 mm, followed by rinsing with purified water to remove blood stains.

[0163] S22: Place the pretreated bovine pericardium in a methanol / chloroform (mass ratio 2:1) mixture and shake at room temperature for 4 hours. Discard the waste liquid and repeat the above operation 3 times. Then wash with purified water by shaking.

[0164] S23: Place the defatted sample in 1% trypsin phosphate buffer, let it stand at 37°C for 4 hours, and then wash it with purified water.

[0165] S24: Place the sample in a 5% sodium carbonate solution and let it stand at room temperature for 30 minutes. Then wash with neutral phosphate buffer until the pH is close to neutral.

[0166] S25: Place the sample in a 3% hydrogen peroxide solution with a pH of 14, let it stand at room temperature for 30 minutes, and wash it with neutral phosphate buffer until the pH is close to neutral.

[0167] S26: The decellularized sample was placed in a 5% sodium dodecyl sulfate solution and treated with shaking at room temperature for 6 hours. This process was repeated twice. The sample was then washed with purified water and freeze-dried to obtain the oral biofilm.

[0168] S27: The sample was dehydrated using a gradient of ethanol solutions, with each gradient lasting 2 hours. The ethanol gradient consisted of 50%, 75%, 90%, and anhydrous ethanol. After dehydration, the sample was immersed in hexane / petroleum ether (mass ratio 1:1) at room temperature for 15 minutes. The sample was then placed in a fume hood and ventilated to allow it to dry.

[0169] S28: The obtained AMT decellularized membrane sheets are subjected to single-sided napping treatment using a napping machine and set aside for later use.

[0170] 3. The preparation process of the drug-loaded solution is as follows:

[0171] S31: The decellularized SIS membrane obtained in S15 is air-dried and then pulverized using a pulverizer.

[0172] S32: Soak the SIS decellularized membrane powder in 1% hydrochloric acid solution at room temperature for 24 hours, filter, and retain the filtrate.

[0173] S33: Add sodium chloride to the filtrate while stirring until no milky white flocculent gelatin is produced. Filter and dialyze the gelatin solution, alternating between phosphate buffer and purified water as the outer dialysate three times, with each treatment lasting 10 minutes.

[0174] S34: Dry the gelatin solution in a 42°C oven until the viscosity of the gelatin solution is 380 mPa·s.

[0175] S35: Add 0.3% of an equimolar ratio EDC / NHS crosslinking system to the prepared gelatin solution, stir until homogeneous, and then refrigerate for 8 hours. Dialyze the resulting micro-crosslinked gelatin solution to remove excess crosslinking agent. The peripheral dialysate is phosphate buffer and purified water, alternating three times, with each treatment lasting 10 minutes.

[0176] S36: Weigh the other components according to the table below:

[0177] Ingredient Name: Microcrosslinked Gelatin Solution, Glycerin, PAMAM, Antibiotic Component Percentage (m / m): 10, 1.5, 0.00, 31.0 surface

[0178] Mix the weighed components thoroughly to obtain a drug-loaded solution, and store it under refrigeration until ready for use.

[0179] 4. The composite process of drug-loaded oral biofilm is as follows:

[0180] S41: After draining the decellularized SIS membrane obtained in S15, soak it in the drug-loaded solution obtained in S36. Lay the soaked decellularized SIS membrane flat on a horizontal mold, stacking a total of 4 layers. The layers should fit tightly together without air bubbles and have no wrinkles at the edges.

[0181] S42: Lay the brushed AMT decellularized membrane sheet flat on a horizontal mold, ensuring the unbrushed side is tightly fitted to the mold without air bubbles. Then place the stacked SIS decellularized membrane sheet on the brushed side of the AMT decellularized membrane sheet, ensuring a tight fit without air bubbles. Finally, cover the SIS decellularized membrane sheet with the brushed side of the AMT decellularized membrane sheet facing down, forming a sandwich structure.

[0182] S43: Place the compounded drug-loaded oral biofilm in a freeze dryer for freeze drying.

[0183] S44: The dried drug-loaded oral membrane is pressed by a film press, packaged, and then sterilized by irradiation.

[0184] Comparative Example 1:

[0185] The SIS decellularized membrane, AMT decellularized membrane, and drug loading solution are the same as in Example 2. The composite process of the drug-loaded oral biomembrane is as follows:

[0186] S41: After draining the decellularized SIS membrane obtained in S15, soak it in the drug-loaded solution obtained in S36. Lay the soaked decellularized SIS membrane flat on a horizontal mold, stacking a total of 4 layers. The layers should fit tightly together without air bubbles and have no wrinkles at the edges.

[0187] S42: Lay the brushed AMT decellularized membrane sheet flat on a horizontal mold, ensuring the unbrushed side is tightly fitted to the mold without air bubbles. Then place the stacked SIS decellularized membrane sheet on the brushed side of the AMT decellularized membrane sheet, ensuring a tight fit without air bubbles. No sandwich structure.

[0188] S43: Place the compounded drug-loaded oral biofilm in a freeze dryer for freeze drying.

[0189] S44: The dried drug-loaded oral membrane is pressed by a film press, packaged, and then sterilized by irradiation.

[0190] Result detection

[0191] (1) Drug release rate: The antibiotic content in the samples of each example and comparative example was detected. The samples prepared in Example 1, Example 2, Example 3, Example 4 and Comparative Example 1 were cut into 15×15mm samples, and then placed in 20ml of phosphate buffer solution with pH 7.2. They were observed in a water bath at 37℃ for 120 days, and the antibiotic content was detected and the release rate was calculated.

[0192] (2) Peel strength: The samples of Example 1, Example 2, Example 3 and Example 4 were cut into 30×10mm specimens. The specimens were peeled from the middle with a scalpel, and the peeling length was 15mm. The peeled specimens were then clamped on a mechanical testing machine and stretched and peeled at a speed of 50mm / min. The maximum force was recorded.

[0193] (3) Cytotoxicity: according to 6cm 2 / ml of the samples from Examples 1, 2, and 3 were extracted, and then cytotoxicity was tested according to GB / T16886.5.

[0194] The test results show that the antibiotic release in the comparative examples was significantly faster than in the examples, indicating that each example does indeed have a sustained-release effect. The peel strength of each example is around 4N, which meets the requirements for clinical application. The cytotoxicity of each example is satisfactory.

Claims

1. A drug-loaded oral biofilm, characterized in that, The drug-loaded oral biofilm has a sandwich structure, specifically: it consists of a bottom layer of decellularized pericardial membrane, a top layer of decellularized pericardial membrane, and multiple layers of small intestinal submucosal SIS decellularized membrane treated with a drug-loaded solution; wherein, both the bottom and top layers of decellularized pericardial membrane have a single-sided brushed surface; the first brushed surface of the bottom layer of decellularized pericardial membrane faces the second brushed surface of the top layer of decellularized pericardial membrane; the multiple layers of small intestinal submucosal SIS decellularized membrane are composited between the first and second brushed surfaces; the multiple layers of small intestinal submucosal SIS decellularized membrane are multiple layers of drug-loaded small intestinal submucosal SIS decellularized membrane after being soaked in a drug-loaded solution; the drug-loaded solution consists of, by weight percentage: 10% micro-crosslinked gelatin solution, 1.0-5.0% glycerol, 0.001-0.2% PAMAM, and 0... The mixture consists of 0.5%–2.0% antibiotics; the micro-crosslinked gelatin solution is prepared by adding 0.01%–2% of an equimolar ratio EDC / NHS crosslinking system to a gelatin solution, stirring until homogeneous, and then refrigerating and standing for 5–8 hours. The micro-crosslinked gelatin solution is then dialyzed to remove excess crosslinking agent; the peripheral dialysis solution consists of alternating phosphate buffer and purified water. The gelatin solution is prepared by soaking small intestinal submucosal layer SIS decellularized membrane powder in an acidic solution at room temperature for 10–24 hours, filtering, and retaining the filtrate; sodium chloride is added to the filtrate while stirring until no milky white flocculent gelatin is produced, followed by filtration and dialysis; the peripheral dialysis solution consists of alternating phosphate buffer and purified water; and the solution is dried in an oven at 35–45°C until the viscosity of the gelatin solution is 100–500 mPa·s.

2. A method for preparing a drug-loaded oral biofilm, characterized in that, Includes the following steps: S1. Antigens were removed from the submucosal layer (SIS) of the small intestine and the pericardium, respectively, to obtain decellularized membrane sheets of the submucosal layer (SIS) of the small intestine and decellularized membrane sheets of the pericardium. S2. The decellularized SIS membrane sheet of the small intestinal submucosa is subjected to acid hydrolysis and salting out to obtain a gelatin solution; S3. The gelatin solution is micro-crosslinked in an EDC / NHS crosslinking system, and then the crosslinking agent is removed by dialysis; S4. Mix the antibiotic drug, PAMAM, glycerin, and the micro-crosslinked gelatin solution prepared in S3, allowing PAMAM to bind with the micro-crosslinked gelatin solution as a drug-carrying medium to form a drug-carrying solution; S5. Immerse the small intestinal submucosal layer SIS decellularized membrane sheet in the drug-carrying solution, and then prepare the drug-carrying oral biomembrane according to the following structure, which has a sandwich structure, specifically: it consists of a bottom pericardial decellularized membrane sheet, a top pericardial decellularized membrane sheet, and multiple layers of small intestinal submucosal layer SIS decellularized membrane sheets treated with the drug-carrying solution; wherein, the bottom... Both the layered pericardial decellularized membrane and the top layer pericardial decellularized membrane have a brushed surface on one side; the first brushed surface of the bottom layer pericardial decellularized membrane faces the second brushed surface of the top layer pericardial decellularized membrane; the multilayer submucosal SIS decellularized membrane is composited between the first and second brushed surfaces; the multilayer submucosal SIS decellularized membrane is a multilayer drug-loaded submucosal SIS decellularized membrane soaked in a drug-loaded solution; the drug-loaded solution consists of, by weight percentage: 10% micro-crosslinked gelatin solution, 1... The mixture consists of 0.0-5.0% glycerol, 0.001-0.2% PAMAM, and 0.5-2.0% antibiotics. The micro-crosslinked gelatin solution is prepared by adding 0.01%-2% of an equimolar ratio EDC / NHS crosslinking system to a gelatin solution, stirring until homogeneous, and then refrigerating and allowing it to stand for 5-8 hours. The micro-crosslinked gelatin solution is then dialyzed to remove excess crosslinking agent; the peripheral dialysis solution consists of alternating phosphate buffer and purified water. The gelatin solution is prepared by soaking small intestinal submucosal layer SIS decellularized membrane powder in an acidic solution at room temperature for 10-24 hours, filtering, and retaining the filtrate. Sodium chloride is added to the filtrate while stirring until no milky white flocculent gelatin is produced. The solution is then filtered, dialyzed, and the peripheral dialysis solution consists of alternating phosphate buffer and purified water. Finally, the solution is dried in an oven at 35-45°C until the viscosity of the gelatin solution is 100-500 mPa·s.

3. The preparation method according to claim 2, characterized in that, S1 includes the following steps: S11, pre-treating the animal's small intestine using a gut scraper, immersing the obtained submucosal SIS in 0.9% physiological saline solution and shaking to remove impurities; S12, using a gut-dissecting instrument to cut the tubular submucosal SIS into small segments, and immersing it in a disinfectant at room temperature for 1-5 hours for disinfection; S13, after disinfection, rinsing the submucosal SIS material with purified water, freezing it at -20℃ to -40℃ for 10-24 hours, and then thawing it at room temperature; S14, rinsing the thawn submucosal SIS material in hypertonic saline solution at room temperature for 5-20 minutes. Then place it in purified water and wash with shaking at room temperature for 5-20 minutes; place the small intestinal submucosal SIS material in hypertonic saline again and wash with shaking at room temperature for 5-20 minutes, then place it in purified water and wash with shaking at room temperature for 5-20 minutes; repeat this step 3-5 times for decellularization; S15, place the decellularized small intestinal submucosal SIS material in 1-5% descaling agent and treat with shaking at room temperature for 4-16 hours, then wash with purified water and store in 0.9% physiological saline.

4. The preparation method according to claim 3, characterized in that, S2 includes the following steps: S21. Pre-treating the pericardium to remove surface stains, fascia, and fat, processing it with a lamina extractor, and then washing it with purified water or 0.9% saline solution to remove blood stains; S22. Placing the pre-treated pericardium material in a defatting agent and shaking it at room temperature for 4-30 hours, then washing it with purified water to obtain a defatted sample; S23. Placing the defatted sample in a 1-5% enzyme solution and soaking it at 37°C for 1-5 hours, then washing it with purified water; S24. Next, placing it in a 1-10% alkaline solution and soaking it at room temperature for 30-120 minutes, then washing it with purified water or neutral phosphate buffer until the pH is close to neutral; S25. Next, place the sample in a 3%–30% oxidizing solution and let it stand at room temperature for 30–120 minutes. Then wash it with purified water or neutral phosphate buffer until the pH is close to neutral to obtain a decellularized sample. S26. Place the decellularized sample in a 1–5% detergent solution and shake it at room temperature for 4–16 hours. Then wash it with purified water. S27. Dehydrate the sample obtained in S26 using a gradient ethanol solution. The dehydration time for each gradient is 1–4 hours. After that, place the dehydrated sample in an organic solvent and soak it at room temperature for 10, 15, or 30 minutes. Then place it in a fume hood for ventilation and drying. S28. Perform single-sided flocking treatment on the obtained decellularized pericardial membrane using a flocking machine for later use.

5. The preparation method according to claim 4, characterized in that, S3 includes the following steps: S31, air-dry the small intestinal submucosal layer SIS decellularized membrane sheet obtained in S15, and then pulverize it using a pulverizer; S32, soak the small intestinal submucosal layer SIS decellularized membrane sheet powder in an acidic solution at room temperature for 10-24 hours, filter, and retain the filtrate; S33, add sodium chloride to the filtrate while stirring until no milky white flocculent gelatin is produced, filter, and dialyze the gelatin solution, using alternating phosphate buffer and purified water as the external dialysate; S34, dry the gelatin solution in an oven at 35-45℃ until the viscosity of the gelatin solution is 100-500 mPa·s; S35, add 0... A micro-crosslinked gelatin solution was prepared by mixing an equimolar ratio of EDC / NHS in a 0.01%–2% EDC / NHS crosslinking system and then refrigerating it for 5–8 hours. The micro-crosslinked gelatin solution was then dialyzed to remove excess crosslinking agent. The external dialysate consisted of alternating phosphate buffer and purified water. S36. The following ingredients were weighed according to the mass percentages of 10% micro-crosslinked gelatin solution, 1.0–5.0% glycerol, 0.001–0.2% PAMAM, and 0.5–2.0% antibiotic, and then mixed thoroughly to obtain the drug-loaded solution, which was then refrigerated for later use.

6. The preparation method according to claim 5, characterized in that, S4 includes the following steps: S41, after draining the small intestinal submucosal layer SIS decellularized membrane obtained in S15, soak it in the drug-loaded solution obtained in S36. Then, lay the soaked small intestinal submucosal layer SIS decellularized membrane flat on a horizontal mold, stacking 2-8 layers in total. The layers should be tightly fitted without air bubbles and the edges should be wrinkle-free. S42, lay the flocculent pericardial decellularized membrane flat on the horizontal mold, ensuring the unflocculent side is tightly fitted to the mold without air bubbles. Then, stack the layers... The small intestinal submucosal layer SIS decellularized membrane sheet is placed on the fleece surface of the pericardial decellularized membrane sheet, ensuring a tight fit without air bubbles; then, the pericardial decellularized membrane sheet is placed on top of the small intestinal submucosal layer SIS decellularized membrane sheet with the fleece surface facing down, forming a sandwich structure; S43, the composite drug-loaded biomembrane obtained in S42 is placed in a freeze dryer for freeze drying; S44, the dried drug-loaded biomembrane is pressed using a film press, packaged, and sterilized with ethylene oxide or by irradiation to obtain the final product.

7. The preparation method according to claim 3, 4, 5, or 6, characterized in that, In step S12, the disinfectant is one or two of 75% ethanol, sodium hypochlorite, 3% hydrogen peroxide, and peracetic acid; in step S14, the hypertonic brine is a mixed solution of sodium chloride with a concentration of 5-10% and sodium hydroxide with a concentration of 0.1-0.5%; in step S15, the descaling agent is one or a mixture of two or more of Triton X-100, sodium dodecyl sulfate, and sodium dodecyl sulfonate.

8. The preparation method according to claim 4, 5, or 6, characterized in that, In S22, the degreasing agent is one or a mixture of two or more of acetone, methanol, ethanol, isopropanol, and chloroform; in S23, the enzyme solution is one or a mixture of two or more of trypsin, protease, lipase, and nuclease in a phosphate buffer solution; in S24, the alkaline solution is one or a mixture of two or more of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, and sodium bicarbonate; in S25, the oxidizing solution is a hydrogen peroxide solution with a pH of 11-14; in S26, the descaling agent is one or a mixture of two or more of Triton X-100, sodium dodecyl sulfate, and sodium dodecyl sulfonate; in S27, the organic solvent is one or a mixture of two or more of acetone, isopropanol, n-hexane, chloroform, and petroleum ether.

9. The preparation method according to claim 5 or 6, characterized in that, In S32, the acid solution is a hydrochloric acid or acetic acid solution with a concentration of 0.1%-5%.

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