Drug-loaded oral biofilm as well as preparation method and application thereof

Abstract

According to the preparation method, small intestinal submucosa SIS and animal membrane tissue AMT are adopted as raw materials, antigen removal treatment is performed on the small intestinal submucosa and the animal membrane tissue respectively, and the decellularized patch is obtained. Carrying out acidolysis and salting-out treatment on the SIS decellularized patch to obtain a gelatin solution; the gelatin solution is subjected to micro-crosslinking in an EDC / NHS crosslinking system, and a crosslinking agent is removed through dialysis. Mixing an antibiotic drug, PAMAM, glycerol and the micro-crosslinked gelatin solution, and combining PAMAM serving as a drug-loading medium with the micro-crosslinked gelatin solution to form a drug-loading solution. The SIS decellularized sheet is soaked in a drug-loading solution for treatment, and then is compounded with the AMT decellularized sheet to form a sandwich structure. The drug-loaded oral membrane has a good barrier effect, is safe and effective, can release antibiotic drugs in a degradation process, and has a stable slow release process and time length, so that the pain of a patient is relieved, the success rate of an operation is improved, and wound healing is accelerated.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to a drug-loaded oral biofilm and a preparation method and application thereof. Background Art

[0002] The oral biofilm of the present invention is an implantable degradable medical device material and is widely used in GBR surgery. It is usually used in combination with inorganic bone powder to isolate the contact surface between soft tissue and hard tissue, prevent soft tissue from invading during the bone defect repair period, and provide sufficient support space for bone regeneration in the defect area.

[0003] Oral biomembranes are widely used in clinical practice because of their good biocompatibility. PTFE membranes are generally considered to be the first generation of oral membrane products. This type of product is easy to operate and has good biocompatibility and relatively ideal mechanical strength. It is used as a gold standard for comparison with other guided tissue regeneration membrane materials. However, its disadvantages are also obvious. Due to its non-degradability, a second surgery is required to remove it, which increases the cost and risk of treatment.

[0004] As the second generation of oral membrane products, absorbable biofilm materials have good biosafety and do not require secondary surgery, shortening the treatment time, and are deeply loved by doctors and patients. Considering the requirements of oral membrane materials for changes in the body environment, research on intelligent oral membrane materials that can change with the environment will be one of the research directions and development trends in this field. The third generation of oral membranes are gradually being used in clinical practice. They not only have good barrier functions, but can also be used as delivery devices to release special preparations, such as antibiotics, growth factors, adhesion factors, etc., which are released at a certain time or continuously in the wound area, so as to coordinate and guide wound healing in a better way.

[0005] 1-Ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) is a good carboxyl / amino crosslinker. EDC undergoes nucleophilic addition reaction with the carboxylic acid of CMCS2 to form an active 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. At the same time, EDC is an unstable crosslinker and will also decompose in water. However, the introduction of N-hydroxysuccinimide (NHS) into the crosslinking system can form a metastable intermediate, which can improve the crosslinking conversion rate.

[0006] Polyamidoamine (PAMAM) dendrimers are a new type of polymer with a three-dimensional hyperbranched, highly ordered spatial geometric symmetry structure, consisting of a central core, an internal cavity, a rich branching structure, and modifiable surface functional groups. PAMAM has the characteristics of controllable molecular weight, multifunctionality, good solubility and biocompatibility, and is widely used in biology, medicine, health, industrial production and other fields.

[0007] The invention patent application with application publication number CN102091057A discloses a method for preparing a drug-loaded biofilm, the main process of which is to dissolve collagen in glacial acetic acid, then freeze-dry the resulting slurry to prepare a biofilm, then soak the biofilm in a drug aqueous solution, and then freeze-dry it to prepare a drug-loaded biofilm. The drug loading method is achieved through biofilm adsorption, that is, only physical adsorption, and there are problems of low drug loading and early drug release. It is impossible to provide a slow and lasting drug release process, and it is also impossible to achieve a lasting effect of the drug. Summary of the invention

[0008] The purpose of the present invention is to provide a degradable drug-loaded oral biofilm which has good barrier effect, is safe and effective, and can provide a slow and lasting drug release process, and a preparation method and application thereof.

[0009] To achieve the above-mentioned object, the present invention provides a drug-loaded oral biofilm, wherein the drug-loaded oral biofilm has a sandwich structure, specifically: it is composed of a bottom animal membrane tissue AMT decellularized membrane sheet, a top animal membrane tissue AMT decellularized membrane sheet, and a multi-layer small intestinal submucosal SIS decellularized membrane sheet treated by soaking in a drug-loaded solution; wherein the bottom animal membrane tissue AMT decellularized membrane sheet and the top animal membrane tissue AMT decellularized membrane sheet both have a single-sided velvet surface treated by velvet treatment;

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

[0011] The multi-layer small intestinal submucosal SIS decellularized membrane sheet is composited between the first velvet surface and the second velvet surface.

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

[0013] S1. Respectively remove antigens from the small intestinal submucosal layer SIS and the animal membrane tissue AMT to obtain the small intestinal submucosal layer SIS decellularized membrane sheet and the animal membrane tissue AMT decellularized membrane sheet;

[0014] S2, subjecting the small intestinal submucosal SIS decellularized membrane sheet to acid hydrolysis and salting-out treatment to obtain a gelatin solution;

[0015] S3, micro-crosslinking the gelatin solution under the EDC / NHS crosslinking system, and then removing the crosslinking agent by dialysis;

[0016] S4, mixing the antibiotic drug, PAMAM, glycerol and the solution of micro-cross-linked gelatin prepared in S3, allowing PAMAM as a drug-carrying medium to combine with the solution of micro-cross-linked gelatin to form a drug-carrying solution;

[0017] S5. Soak the small intestinal submucosal layer SIS in a drug-loaded solution, and then compound the sandwich structure described in claim 1 with the animal membrane tissue AMT decellularized membrane sheet.

[0018] Wherein, S1 includes the following steps:

[0019] S11, pre-treating the small intestine of the animal with an intestine scraper, soaking the obtained small intestinal submucosal layer SIS in 0.9% physiological saline for shaking and washing to remove stains;

[0020] S12. Use an enterotomy device to cut open the tubular small intestinal submucosal layer SIS, cut it into small pieces, and soak it in disinfectant at room temperature for 1 to 5 hours for disinfection;

[0021] S13. After the disinfected small intestinal submucosal SIS material is cleaned by shaking with purified water, it is placed in a freezer at -20°C to 40°C for 10 to 24 hours, and then melted at room temperature;

[0022] S14. Place the ablated small intestinal submucosal SIS material in hypertonic saline, shake and wash at room temperature for 5 to 20 minutes, then place it in purified water, shake and wash at room temperature for 5 to 20 minutes; place the small intestinal submucosal SIS material in hypertonic saline again, shake and wash at room temperature for 5 to 20 minutes, then place it in purified water, shake and wash at room temperature for 5 to 20 minutes; repeat this step 3 to 5 times to perform decellularization treatment.

[0023] S15. Place the decellularized small intestinal submucosal SIS material in a 1-5% descaling agent, shake it at room temperature for 4h-16h, then wash it with purified water and store it in a 0.9% physiological saline.

[0024] S2 includes the following steps:

[0025] S21, pre-treating the animal membrane tissue AMT to remove surface stains, fascia and fat, processing it with a splitting machine, and then washing it with purified water or 0.9% saline to remove blood stains;

[0026] S22, placing the pre-treated animal membrane tissue AMT material in a degreasing agent, shaking it at room temperature for 4 hours to 30 hours, and then shaking and washing it with purified water to obtain a defatted sample;

[0027] S23, placing the defatted sample in a 1-5% enzyme solution, soaking it at 37°C for 1-5 hours, and then washing it with purified water;

[0028] S24, then place in 1% to 10% alkali solution, soak at room temperature for 30 to 120 minutes, and then wash with purified water or neutral phosphate buffer until the pH is close to neutral;

[0029] S25, then placing in a 3% to 30% oxidizing solution, soaking at room temperature for 30 to 120 minutes, and then washing with purified water or neutral phosphate buffer until the pH is close to neutral, to obtain a decellularized sample;

[0030] S26, placing the decellularized sample in a 1-5% descaling agent, shaking at room temperature for 4h-16h, and then washing with purified water;

[0031] S27, dehydrating the sample obtained in S26 with a gradient ethanol solution, the dehydration time of each gradient being 1 to 4 hours, and then placing in a fume hood for ventilation and drying;

[0032] S28, the obtained animal membrane tissue AMT decellularized membrane sheet is subjected to single-sided napping treatment by a napping machine and set aside.

[0033] S3 includes the following steps:

[0034] S31, air-drying the small intestinal submucosal SIS decellularized membrane sheet obtained in S15, and crushing it with a crusher;

[0035] S32, soaking the small intestinal submucosal SIS decellularized membrane powder in an acid solution at room temperature for 10 to 24 hours, filtering, and retaining the filtrate;

[0036] S33, adding sodium chloride to the filtrate while stirring until no milky white flocculent gelatin is produced, filtering, and dialyzing the gelatin solution, wherein the peripheral dialysate is alternating phosphate buffer and purified water;

[0037] S34, drying the gelatin solution in an oven at 35-45°C until the viscosity of the gelatin solution is 100-500 mPa·s;

[0038] S35, adding 0.01% to 2% of an equimolar ratio of EDC / NHS cross-linking system to the prepared gelatin solution, stirring evenly, refrigerating and standing for 5 to 8 hours to obtain a slightly cross-linked gelatin solution, and dialyzing the slightly cross-linked gelatin solution to remove excess cross-linking agent; the peripheral dialysate is alternating phosphate buffer and purified water;

[0039] S36. Weigh 10% of micro-cross-linked gelatin solution, 1.0-5.0% of glycerol, 0.001-0.2% of PAMAM and 0.5-2.0% of antibiotics according to their mass percentages, then mix them evenly to obtain a drug-loaded solution, and refrigerate for later use.

[0040] S4 includes the following steps:

[0041] S41, drain the SIS decellularized membrane sheet of the small intestinal submucosal layer obtained in S15 and soak it in the drug-loaded solution obtained in S36, and lay the soaked SIS decellularized membrane sheet of the small intestinal submucosal layer flat on a horizontal mold, stacking 2 to 8 layers in total, and the layers should be tightly fitted without bubbles and without wrinkles on the edges;

[0042] S42, lay the brushed animal membrane tissue AMT decellularized membrane sheet flat on a horizontal mold, the non-brushed surface should fit tightly with the mold without bubbles, then place the superimposed small intestinal submucosa SIS decellularized membrane sheet on the brushed surface of the animal membrane tissue AMT decellularized membrane sheet, which should fit tightly without bubbles; then cover the small intestinal submucosa SIS decellularized membrane sheet with the brushed surface of the animal membrane tissue AMT decellularized membrane sheet facing downward to form a sandwich structure;

[0043] S43, placing the composite drug-loaded biofilm obtained in S42 in a freeze dryer for freeze drying;

[0044] S44, the dried drug-loaded biofilm is pressed by a laminator, packaged, and sterilized by ethylene oxide or radiation 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 saline is a mixed solution of 5-10% sodium chloride and 0.1-0.5% sodium hydroxide;

[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 pancreatic enzyme, protease, lipase, and nuclease in a phosphate buffer;

[0050] In S24, the alkali 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 the repair of hard tissue defects in oral GBR surgery.

[0056] The beneficial effects of the present invention are as follows: using small intestinal submucosa (SIS) and animal membrane tissue (AMT) as raw materials, respectively performing deantigen treatment on the small intestinal submucosa and animal membrane tissue to obtain a decellularized membrane sheet. Then the SIS decellularized membrane sheet is subjected to acid hydrolysis and salting-out treatment to obtain a gelatin solution. The gelatin solution is micro-crosslinked under an EDC / NHS crosslinking system, and then the crosslinking agent is removed by dialysis. Antibiotic drugs, PAMAM, and glycerol are mixed with the micro-crosslinked gelatin solution, and PAMAM is combined with the micro-crosslinked gelatin solution as a drug-carrying medium to form a drug-carrying solution. The SIS decellularized membrane sheet is immersed in the drug-carrying solution for treatment, and then compounded with the AMT decellularized membrane sheet to form a sandwich structure. The drug-carrying oral membrane has a good barrier effect, is safe and effective, and can release antibiotic drugs during the degradation process, thereby alleviating the patient's pain, improving the success rate of surgery, and accelerating wound healing. It has a rich pore structure, good hydrophilicity, good adhesion and biocompatibility. The unique sandwich structure of the drug-loaded oral biofilm can play a role of physical isolation during the repair and treatment process. At the same time, the drug-loaded component in the middle of the sandwich structure can effectively release antibiotics with an ideal sustained-release duration, which can effectively improve the inflammatory characteristics of the treatment process, increase the success rate of the operation, and reduce patient pain. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Schematic diagram of the drug-loaded oral biofilm sandwich structure.

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

[0059] Figure 3 Data graph of antibiotic release rate of each embodiment and comparative example under simulated environment.

[0060] Figure 4 The peel strength test results of each embodiment are shown graphically.

[0061] Figure 5 Cytotoxicity test results of various examples. Among them, a to d are examples 1 to 4; e is a negative control graph; and f is a positive control graph. DETAILED DESCRIPTION

[0062] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined purpose, the specific implementation methods, structural features and effects of the present invention are described in detail below in conjunction with the accompanying drawings and embodiments.

[0063] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be noted that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0064] Embodiment 1:

[0065] 1. Preparation of SIS decellularized membrane:

[0066] S11: Pre-treat the pig small intestine using an intestine scraper, and soak the obtained small intestinal submucosal layer (SIS) in 0.9% physiological saline for shaking cleaning to remove stains.

[0067] S12: Use an intestinal dissector to dissect the tubular SIS, cut it into small pieces, and immerse it in 3% hydrogen peroxide at room temperature for 1 hour for disinfection.

[0068] S13: After the sterilized SIS material is cleaned by shaking with purified water, it is placed in a freezer at -20℃--40℃ for 10 hours, and then thawed at room temperature.

[0069] S14: The ablated SIS material is placed in a mixed solution of 5% sodium chloride and 0.1% sodium hydroxide, and washed at room temperature for 5 minutes under shaking, and then placed in purified water, and washed at room temperature for 5 minutes under shaking. The above steps are repeated 3 times for decellularization.

[0070] S15: The decellularized SIS material was placed in a 1% Triton X-100 solution, shaken at room temperature for 4 hours, then washed with purified water and stored in a 0.9% physiological saline solution.

[0071] 2. Preparation of AMT decellularized membrane:

[0072] S21, pre-treating the porcine pericardium to remove surface stains, fascia, fat and other dirt, processing it with a layer splitter to obtain a membrane sheet with a thickness of 0.2-0.5 mm, and then washing it with purified water to remove blood stains.

[0073] S22: Place the pre-treated porcine pericardium in an acetone solution, shake it at room temperature for 4 hours, discard the waste liquid, repeat the above operation twice, and then shake and wash it with purified water.

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

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

[0076] S25: The sample is then placed in a 3% hydrogen peroxide solution with a pH of 12, left to soak at room temperature for 30 minutes, and then washed 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 shaken at room temperature for 4 hours, and the above operation was repeated twice. Then, the sample was washed with purified water and freeze-dried to obtain the oral biofilm.

[0078] S27: Dehydrate the sample using a gradient ethanol solution, the dehydration time for each gradient is 1 hour, and the ethanol gradient is 50%, 75%, 90% and anhydrous ethanol. Soak the dehydrated sample in an acetone / isopropanol (mass ratio 1:1) solution at room temperature for 10 minutes. Place the sample in a fume hood and ventilate to dry the sample.

[0079] S28: The obtained AMT decellularized membrane sheet is subjected to single-sided napping treatment by a napping machine for standby use.

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

[0081] S31: The SIS decellularized membrane sheet obtained in S15 is naturally air-dried and crushed by a crusher.

[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, with the peripheral dialysate being phosphate buffer and purified water alternately treated 3 times, each treatment time being 10 minutes.

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

[0085] S35: Add 0.01% of an equimolar ratio of EDC / NHS cross-linking system to the prepared gelatin solution, stir evenly, and refrigerate for 5 hours. The obtained slightly cross-linked gelatin solution is dialyzed to remove excess cross-linking agent. The peripheral dialysate is phosphate buffer and purified water alternately treated 3 times, each treatment time is 10 minutes.

[0086] S36: Weigh other ingredients as follows:

[0087] Ingredient name Micro-cross-linked gelatin solution glycerin PAMAM antibiotic Component ratio (m / m) 10 1 0.001 1.0

[0088] The weighed components are mixed evenly to obtain a drug-loaded solution, which is then placed under refrigeration for later use.

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

[0090] S41: Drain the SIS decellularized membrane obtained in S15 and soak it in the drug-loaded solution obtained in S36. Spread the soaked SIS decellularized membrane flat on a horizontal mold, stacking 2 layers in total. The layers should fit tightly without bubbles and without wrinkles on the edges.

[0091] S42: Lay the AMT decellularized membrane sheet after velvet on a horizontal mold. The non-velvet surface should fit tightly with the mold without bubbles. Then place the superimposed SIS decellularized membrane sheet on the velvet surface of the AMT decellularized membrane sheet. It should fit tightly without bubbles. Then cover the SIS decellularized membrane sheet with the velvet surface of the AMT decellularized membrane sheet facing down to form a sandwich structure.

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

[0093] S44: The dried drug-loaded oral film is pressed by a film pressing machine, and packaged for ethylene oxide sterilization.

[0094] Embodiment 2:

[0095] 1. Preparation of SIS decellularized membrane:

[0096] S11: Pre-treat the pig small intestine using an intestine scraper, and soak the obtained small intestinal submucosal layer (SIS) in 0.9% physiological saline for shaking cleaning to remove stains.

[0097] S12: Use an intestinal dissector to dissect the tubular SIS and cut it into small pieces. Soak it in 75% ethanol at room temperature for 2 hours, and then transfer it to 3% hydrogen peroxide and soak it for 5 hours for disinfection.

[0098] S13: After the disinfected SIS material is cleaned by shaking with purified water, it is placed in a freezer at -20℃--40℃ for 24 hours, and then thawed at room temperature. Repeat the freeze-thaw process 3 times.

[0099] S14: The ablated SIS material is placed in a mixed solution of 10% sodium chloride and 0.5% sodium hydroxide, and washed at room temperature for 20 minutes under shaking, and then placed in purified water, and washed at room temperature for 5 minutes under shaking. The above steps are repeated 5 times for decellularization.

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

[0101] 2. Preparation of AMT decellularized membrane:

[0102] S21, pre-treating the bovine pericardium to remove surface stains, fascia, fat and other dirt, and processing it with a layer splitter to obtain a membrane sheet with a thickness of 0.2-0.5 mm, and then washing it with 0.9% physiological saline to remove blood stains.

[0103] S22: The pre-treated bovine pericardium is placed in a mixture of acetone / isopropanol (mass ratio 1:4), shaken at room temperature for 10 hours, the waste liquid is discarded, the above operation is repeated 3 times, and then washed with purified water by shaking.

[0104] S23: The defatted sample was placed in a 5% protease / lipase (mass ratio 1:1) phosphate buffer, soaked at 37°C for 5 hours, and then washed with purified water.

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

[0106] S25: The sample is then placed in a 30% hydrogen peroxide solution with a pH of 14, left to soak at room temperature for 120 minutes, and then washed with a 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 shaken at room temperature for 8 hours, and the above operation was repeated twice. The sample was then washed with purified water and freeze-dried to obtain an oral biofilm.

[0108] S27: Dehydrate the sample using a gradient ethanol solution, the dehydration time of each gradient is 4 hours, and the ethanol gradient is 50%, 75%, 90% and anhydrous ethanol. Soak the dehydrated sample in petroleum ether at room temperature for 30 minutes. Place the sample in a fume hood for ventilation to dry the sample.

[0109] S28: The obtained AMT decellularized membrane sheet is subjected to single-sided napping treatment by a napping machine for standby use.

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

[0111] S31: The SIS decellularized membrane sheet obtained in S15 is naturally air-dried and crushed by a crusher.

[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, with the peripheral dialysate being phosphate buffer and purified water alternately treated 3 times, each treatment time being 10 minutes.

[0114] S34: drying the gelatin solution in an oven at 45°C until the viscosity of the gelatin solution reaches 154 mPa·s.

[0115] S35: Add 2% of the equimolar ratio of EDC / NHS cross-linking system to the prepared gelatin solution, stir evenly, and refrigerate for 8 hours. The obtained slightly cross-linked gelatin solution is dialyzed to remove excess cross-linking agent. The peripheral dialysate is phosphate buffer and purified water alternately treated 3 times, each treatment time is 10 minutes.

[0116] S36: Weigh other ingredients as follows:

[0117] Ingredient name Micro-cross-linked gelatin solution glycerin PAMAM antibiotic Component ratio (m / m) 10 5.0 0.2 1.0

[0118] The weighed components are mixed evenly to obtain a drug-loaded solution, which is then placed under refrigeration for later use.

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

[0120] S41: Drain the SIS decellularized membrane obtained in S15 and soak it in the drug-loaded solution obtained in S36. Spread the soaked SIS decellularized membrane flat on a horizontal mold, stacking 2 layers in total. The layers should fit tightly without bubbles and without wrinkles on the edges.

[0121] S42: Lay the AMT decellularized membrane sheet after velvet on a horizontal mold. The non-velvet surface should fit tightly with the mold without bubbles. Then place the superimposed SIS decellularized membrane sheet on the velvet surface of the AMT decellularized membrane sheet. It should fit tightly without bubbles. Then cover the SIS decellularized membrane sheet with the velvet surface of the AMT decellularized membrane sheet facing down to form a sandwich structure.

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

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

[0124] Embodiment 3:

[0125] 1. Preparation of SIS decellularized membrane:

[0126] S11: Pre-treat the pig small intestine using an intestine scraper, and soak the obtained small intestinal submucosal layer (SIS) in 0.9% physiological saline for shaking cleaning to remove stains.

[0127] S12: Use an intestinal dissector to dissect the tubular SIS, cut it into small pieces, and immerse it in 5% sodium hypochlorite at room temperature for 1 hour for disinfection.

[0128] S13: After the disinfected SIS material is cleaned by shaking with purified water, it is placed in a freezer at -20℃--40℃ for 10 hours, and then thawed at room temperature. Repeat the freeze-thaw process for 3 times.

[0129] S14: The ablated SIS material is placed in a mixed solution of 7% sodium chloride and 0.1% sodium hydroxide, and washed at room temperature for 5 minutes under shaking, and then placed in purified water, and washed at room temperature for 5 minutes under shaking. The above steps are repeated 3 times for decellularization.

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

[0131] 2. Preparation of AMT decellularized membrane:

[0132] S21: The bovine pericardium is pre-treated to remove surface stains, fascia, fat and other dirt. It is processed by a layer splitter to obtain a membrane sheet with a thickness of 0.2-0.5 mm, and then washed with purified water to remove blood stains.

[0133] S22: Place the pre-treated bovine pericardium in a methanol / chloroform mixture (mass ratio 1:1), shake it at room temperature for 4 hours, discard the waste liquid, repeat the above operation 3 times, and then shake and wash it with purified water.

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

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

[0136] S25: The sample is then placed in a 3% hydrogen peroxide solution with a pH of 12, left to soak at room temperature for 30 minutes, and then washed with a 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 shaken at room temperature for 4 hours, and the above operation was repeated twice. Then, the sample was washed with purified water and freeze-dried to obtain an oral biofilm.

[0138] S27: Dehydrate the sample using a gradient ethanol solution, the dehydration time for each gradient is 1 hour, and the ethanol gradient is 50%, 75%, 90% and anhydrous ethanol. Soak the dehydrated sample in n-hexane at room temperature for 30 minutes. Place the sample in a fume hood for ventilation to dry the sample.

[0139] S28: The obtained AMT decellularized membrane sheet is subjected to single-sided napping treatment by a napping machine for standby use.

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

[0141] S31: The SIS decellularized membrane sheet obtained in S15 is naturally air-dried and crushed by a crusher.

[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, with the peripheral dialysate being phosphate buffer and purified water alternately treated 3 times, each treatment time being 10 minutes.

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

[0145] S35: Add 0.3% of the equimolar ratio EDC / NHS cross-linking system to the prepared gelatin solution, stir evenly, and refrigerate for 8 hours. The obtained slightly cross-linked gelatin solution is dialyzed to remove excess cross-linking agent. The peripheral dialysate is phosphate buffer and purified water alternately treated 3 times, each treatment time is 10 minutes.

[0146] S36: Weigh other ingredients according to the following table:

[0147] Ingredient name Micro-cross-linked gelatin solution glycerin PAMAM antibiotic Component ratio (m / m) 10 2.0 0.005 1.0

[0148] The weighed components are mixed evenly to obtain a drug-loaded solution, which is then placed under refrigeration for later use.

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

[0150] S41: Drain the SIS decellularized membrane obtained in S15 and soak it in the drug-loaded solution obtained in S36. Spread the soaked SIS decellularized membrane flat on a horizontal mold, stacking 6 layers in total. The layers should fit tightly without bubbles and the edges should be free of wrinkles.

[0151] S42: Lay the AMT decellularized membrane sheet after velvet on a horizontal mold. The non-velvet surface should fit tightly with the mold without bubbles. Then place the superimposed SIS decellularized membrane sheet on the velvet surface of the AMT decellularized membrane sheet. It should fit tightly without bubbles. Then cover the SIS decellularized membrane sheet with the velvet surface of the AMT decellularized membrane sheet facing down to form a sandwich structure.

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

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

[0154] Embodiment 4:

[0155] 1. Preparation of SIS decellularized membrane:

[0156] S11: Pre-treat the pig small intestine using an intestine scraper, and soak the obtained small intestinal submucosal layer (SIS) in 0.9% physiological saline for shaking cleaning to remove stains.

[0157] S12: Use an intestinal dissector to dissect the tubular SIS, cut it into small pieces, and immerse it in 5% sodium hypochlorite at room temperature for 3 hours for disinfection.

[0158] S13: After the disinfected SIS material is cleaned by shaking with purified water, it is placed in a freezer at -20℃--40℃ for 12 hours, and then thawed at room temperature. Repeat the freeze-thaw process 5 times.

[0159] S14: The ablated SIS material is placed in a mixed solution of 7% sodium chloride and 0.3% sodium hydroxide, and washed at room temperature for 10 minutes under shaking, and then placed in purified water, and washed at room temperature for 5 minutes under shaking. The above steps are repeated 3 times for decellularization.

[0160] S15: The decellularized SIS material was placed in a 2% sodium dodecyl sulfate solution, shaken at room temperature for 6 hours, then washed with purified water and stored in a 0.9% physiological saline solution.

[0161] 2. Preparation of AMT decellularized membrane:

[0162] S21: The bovine pericardium is pre-treated to remove surface stains, fascia, fat and other dirt. It is processed by a layer splitter to obtain a membrane sheet with a thickness of 0.2-0.5 mm, and then washed with purified water to remove blood stains.

[0163] S22: Place the pre-treated bovine pericardium in a methanol / chloroform mixture (mass ratio 2:1), shake it at room temperature for 4 hours, discard the waste liquid, repeat the above operation 3 times, and then shake and wash it with purified water.

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

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

[0166] S25: The sample is then placed in a 3% hydrogen peroxide solution with a pH of 14, left to soak at room temperature for 30 minutes, and then washed with a 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 shaken at room temperature for 6 hours, and the above operation was repeated twice, and then washed with purified water and freeze-dried to obtain an oral biofilm.

[0168] S27: Dehydrate the sample using a gradient ethanol solution, the dehydration time for each gradient is 2 hours, and the ethanol gradient is 50%, 75%, 90% and anhydrous ethanol. Soak the dehydrated sample in n-hexane / petroleum ether (mass ratio 1:1) at room temperature for 15 minutes. Place the sample in a fume hood for ventilation to dry the sample.

[0169] S28: The obtained AMT decellularized membrane sheet is subjected to single-sided napping treatment by a napping machine for standby use.

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

[0171] S31: The SIS decellularized membrane sheet obtained in S15 is naturally air-dried and crushed by a crusher.

[0172] S32: Soak the SIS decellularized membrane powder in a 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, with the peripheral dialysate being phosphate buffer and purified water alternately treated 3 times, each treatment time being 10 minutes.

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

[0175] S35: Add 0.3% of the equimolar ratio EDC / NHS cross-linking system to the prepared gelatin solution, stir evenly, and refrigerate for 8 hours. The obtained slightly cross-linked gelatin solution is dialyzed to remove excess cross-linking agent. The peripheral dialysate is phosphate buffer and purified water alternately treated 3 times, each treatment time is 10 minutes.

[0176] S36: Weigh other ingredients according to the following table:

[0177] Ingredient name Micro-cross-linked gelatin solution glycerin PAMAM antibiotic Composition ratio (m / m) 10 1.5 0.003 1.0

[0178] The weighed components are mixed evenly to obtain a drug-loaded solution, which is then placed under refrigeration for later use.

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

[0180] S41: Drain the SIS decellularized membrane obtained in S15 and soak it in the drug-loaded solution obtained in S36. Spread the soaked SIS decellularized membrane flat on a horizontal mold, stacking 4 layers in total. The layers should fit tightly without bubbles and the edges should be free of wrinkles.

[0181] S42: Lay the AMT decellularized membrane sheet after velvet on a horizontal mold. The non-velvet surface should fit tightly with the mold without bubbles. Then place the superimposed SIS decellularized membrane sheet on the velvet surface of the AMT decellularized membrane sheet. It should fit tightly without bubbles. Then cover the SIS decellularized membrane sheet with the velvet surface of the AMT decellularized membrane sheet facing down to form a sandwich structure.

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

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

[0184] Comparative Example 1:

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

[0186] S41: Drain the SIS decellularized membrane obtained in S15 and soak it in the drug-loaded solution obtained in S36. Spread the soaked SIS decellularized membrane flat on a horizontal mold, stacking 4 layers in total. The layers should fit tightly without bubbles and the edges should be free of wrinkles.

[0187] S42: Lay the AMT decellularized membrane after velvet on a horizontal mold. The non-velvet surface should fit tightly with the mold without bubbles. Then place the superimposed SIS decellularized membrane on the velvet surface of the AMT decellularized membrane. It should fit tightly without bubbles. No sandwich structure.

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

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

[0190] Results

[0191] (1) Drug release rate: The antibiotic content in each example and comparative example sample was detected. The samples prepared in Example 1, Example 2, Example 3, Example 4 and Comparative Example 1 were cut into 15×15 mm size specimens, and then placed in 20 ml of phosphate buffer solution with a pH of 7.2, and observed in a 37°C water bath 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 specimens of 30×10 mm in size. The specimens were peeled off from the middle with a scalpel to a peeling length of 15 mm. The peeled specimens were then clamped on a mechanical testing machine and stretched and peeled off at a speed of 50 mm / min. The maximum force was recorded.

[0193] (3) Cytotoxicity: According to 6cm 2 The samples of Example 1, Example 2 and Example 3 were extracted with 1% ethanol / ml and then the cytotoxicity test was performed according to GB / T16886.5.

[0194] From the test results, it can be seen that the antibiotic release of the comparative example is significantly faster than that of the embodiment, indicating that each embodiment has the effect of sustained release of antibiotics. The peel strength of each embodiment is about 4N, which can meet the requirements of clinical application. The cytotoxicity of each embodiment is qualified.

Claims

1. A drug-loaded oral biofilm, characterized in that: The drug-loaded oral biofilm has a sandwich structure, specifically: it is composed of a bottom animal membrane tissue AMT decellularized membrane sheet, a top animal membrane tissue AMT decellularized membrane sheet, and a multi-layer small intestinal submucosal SIS decellularized membrane sheet treated by soaking in a drug-loaded solution; wherein the bottom animal membrane tissue AMT decellularized membrane sheet and the top animal membrane tissue AMT decellularized membrane sheet both have a single-sided velvet surface treated by velvet treatment; The first velvet surface of the bottom animal membrane tissue AMT decellularized membrane sheet is opposite to the second velvet surface of the top animal membrane tissue AMT decellularized membrane sheet; The multi-layer small intestinal submucosal SIS decellularized membrane sheet is composited between the first velvet surface and the second velvet surface.

2. A method for preparing the drug-loaded oral biofilm according to claim 1, characterized in that: The following steps are involved: S1. Respectively remove antigens from the small intestinal submucosal layer SIS and the animal membrane tissue AMT to obtain the small intestinal submucosal layer SIS decellularized membrane sheet and the animal membrane tissue AMT decellularized membrane sheet; S2, subjecting the small intestinal submucosal SIS decellularized membrane sheet to acid hydrolysis and salting-out treatment to obtain a gelatin solution; S3, micro-crosslinking the gelatin solution under the EDC / NHS crosslinking system, and then removing the crosslinking agent by dialysis; S4, mixing the antibiotic drug, PAMAM, glycerol and the solution of micro-cross-linked gelatin prepared in S3, allowing PAMAM as a drug-carrying medium to combine with the solution of micro-cross-linked gelatin to form a drug-carrying solution; S5. Soak the small intestinal submucosal layer SIS in a drug-loaded solution, and then compound the sandwich structure described in claim 1 with the animal membrane tissue AMT decellularized membrane sheet.

3. The preparation method according to claim 2, characterized in that: The S1 comprises the following steps: S11, pre-treating the small intestine of the animal with an intestine scraper, soaking the obtained small intestinal submucosal layer SIS in 0.9% physiological saline for shaking and washing to remove stains; S12. Use an enterotomy device to cut open the tubular small intestinal submucosal layer SIS, cut it into small pieces, and soak it in disinfectant at room temperature for 1 to 5 hours for disinfection; S13. After the disinfected small intestinal submucosal SIS material is cleaned by shaking with purified water, it is placed in a freezer at -20°C to 40°C for 10 to 24 hours, and then melted at room temperature; S14. Place the ablated small intestinal submucosal SIS material in hypertonic saline, shake and wash at room temperature for 5 to 20 minutes, then place it in purified water, shake and wash at room temperature for 5 to 20 minutes; place the small intestinal submucosal SIS material in hypertonic saline again, shake and wash at room temperature for 5 to 20 minutes, then place it in purified water, shake and wash at room temperature for 5 to 20 minutes; repeat this step 3 to 5 times to perform decellularization treatment. S15. Place the decellularized small intestinal submucosal SIS material in a 1-5% descaling agent, shake it at room temperature for 4h-16h, then wash it with purified water and store it in a 0.9% physiological saline.

4. The preparation method according to claim 3, characterized in that: The S2 comprises the following steps: S21, pre-treating the animal membrane tissue AMT to remove surface stains, fascia and fat, processing it with a splitting machine, and then washing it with purified water or 0.9% saline to remove blood stains; S22, placing the pre-treated animal membrane tissue AMT material in a degreasing agent, shaking it at room temperature for 4 hours to 30 hours, and then shaking and washing it with purified water to obtain a defatted sample; S23, placing the defatted sample in a 1-5% enzyme solution, soaking it at 37°C for 1-5 hours, and then washing it with purified water; S24, then place in 1% to 10% alkali solution, soak at room temperature for 30 to 120 minutes, and then wash with purified water or neutral phosphate buffer until the pH is close to neutral; S25, then placing in a 3% to 30% oxidizing solution, soaking at room temperature for 30 to 120 minutes, and then washing with purified water or neutral phosphate buffer until the pH is close to neutral, to obtain a decellularized sample; S26, placing the decellularized sample in a 1-5% descaling agent, shaking at room temperature for 4h-16h, and then washing with purified water; S27, dehydrating the sample obtained in S26 with a gradient ethanol solution, the dehydration time of each gradient being 1 to 4 hours, and then placing in a fume hood for ventilation and drying; S28, the obtained animal membrane tissue AMT decellularized membrane sheet is subjected to single-sided napping treatment by a napping machine and set aside.

5. The preparation method according to claim 4, characterized in that: The S3 comprises the following steps: S31, air-drying the small intestinal submucosal SIS decellularized membrane sheet obtained in S15, and crushing it with a crusher; S32, soaking the small intestinal submucosal SIS decellularized membrane powder in an acid solution at room temperature for 10 to 24 hours, filtering, and retaining the filtrate; S33, adding sodium chloride to the filtrate while stirring until no milky white flocculent gelatin is produced, filtering, and dialyzing the gelatin solution, wherein the peripheral dialysate is alternating phosphate buffer and purified water; S34, drying the gelatin solution in an oven at 35-45°C until the viscosity of the gelatin solution is 100-500 mPa·s; S35, adding 0.01% to 2% of an equimolar ratio of EDC / NHS cross-linking system to the prepared gelatin solution, stirring evenly, refrigerating and standing for 5 to 8 hours to obtain a slightly cross-linked gelatin solution, and dialyzing the slightly cross-linked gelatin solution to remove excess cross-linking agent; the peripheral dialysate is alternating phosphate buffer and purified water; S36. Weigh 10% of micro-cross-linked gelatin solution, 1.0-5.0% of glycerol, 0.001-0.2% of PAMAM and 0.5-2.0% of antibiotics according to their mass percentages, then mix them evenly to obtain a drug-loaded solution, and refrigerate for later use.

6. The preparation method according to claim 5, characterized in that: The S4 comprises the following steps: S41, drain the SIS decellularized membrane sheet of the small intestinal submucosal layer obtained in S15 and soak it in the drug-loaded solution obtained in S36, and lay the soaked SIS decellularized membrane sheet of the small intestinal submucosal layer flat on a horizontal mold, stacking 2 to 8 layers in total, and the layers should be tightly fitted without bubbles and without wrinkles on the edges; S42, lay the brushed animal membrane tissue AMT decellularized membrane sheet flat on a horizontal mold, the non-brushed surface should fit tightly with the mold without bubbles, then place the superimposed small intestinal submucosa SIS decellularized membrane sheet on the brushed surface of the animal membrane tissue AMT decellularized membrane sheet, which should fit tightly without bubbles; then cover the small intestinal submucosa SIS decellularized membrane sheet with the brushed surface of the animal membrane tissue AMT decellularized membrane sheet facing downward to form a sandwich structure; S43, placing the composite drug-loaded biofilm obtained in S42 in a freeze dryer for freeze drying; S44, the dried drug-loaded biofilm is pressed by a laminator, packaged, and sterilized by ethylene oxide or radiation to obtain the final product.

7. The preparation method according to claim 3, 4, 5 or 6, characterized in that: In S12, the disinfectant is one or two of 75% ethanol, sodium hypochlorite, 3% hydrogen peroxide, and peracetic acid; In S14, the hypertonic saline is a mixed solution of 5-10% sodium chloride and 0.1-0.5% sodium hydroxide; 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.

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 more of pancreatic enzyme, protease, lipase, and nuclease in a phosphate buffer; In S24, the alkali 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 to 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%.

10. Use of the drug-loaded oral biofilm according to any one of claims 1 to 6 in repairing hard tissue defects in oral GBR surgery.

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