Preparation method and application of drug-loaded bee cocoon biological membrane

Abstract

The invention belongs to the field of medical nursing consumables, and particularly relates to a preparation method and application of a drug-loaded bee cocoon biological membrane. The drug-loaded bee cocoon biological membrane has good tensile strength and adhesive force, can be attached to the oral mucosa part, releases drugs in artificial saliva for not less than 4 hours, and does not degrade in the artificial saliva for 30 days; the drug-loaded bee cocoon biological membrane prepared by the method has good biocompatibility, can successfully load Kangfuxin drugs and slowly release the drugs, is convenient to carry, use and replace in daily life, does not influence the safety of the biological membrane to oral mucosa, and is suitable for home nursing and inter-diagnosis treatment of oral ulcers such as radioactive stomatitis, chemotherapeutic stomatitis and the like.

Description

Technical Field

[0001] The invention belongs to the field of medical care consumables, and specifically relates to a preparation method and application of a drug-loaded bee cocoon biofilm. Background Art

[0002] Oral mucositis (OM) refers to inflammation of the oral mucosa. It is an inflammatory reaction of the oral mucosal tissue caused by radiotherapy and chemotherapy in cancer patients, especially those undergoing head and neck radiotherapy. The incidence of OM is as high as 80%. Clinical manifestations include edema, erythema, ulcers, dysphagia, taste disorders and bleeding in the oral mucosa, accompanied by burning pain, which leads to malnutrition and communication disorders in patients, and reduces the quality of life. In severe cases, it can also cause systemic infection, and treatment has to be delayed or terminated. Therefore, it is necessary to focus on preventing and managing the occurrence of oral mucositis during tumor treatment.

[0003] The pathogenesis of OM is divided into direct and indirect mechanisms. The direct mechanism is the direct sensitivity of the oral mucosa to cytotoxic drugs, and the indirect mechanism is mainly the release of some pro-inflammatory factors that are indirectly toxic to the oral mucosa and the reduction of anti-inflammatory factors. At present, the recognized five-stage pathological model of Sonis is as follows: (1) Initiation stage: a large amount of reactive oxygen species is produced after the start of radiotherapy and chemotherapy, thereby inducing oxidative stress and destroying biological macromolecules, leading to cell apoptosis or triggering malignant transformation of normal cells. At this stage, the mucosal function is normal; (2) Signal transduction stage: nuclear transcription factor-KB and other genes are activated, leading to the production of pro-inflammatory cytokines; (3) Signal amplification stage: the inflammatory factors produced in the previous stage amplify or further activate nuclear transcription factors in other cells, leading to the transcription of genes encoding mitogen-activated protein kinase and cyclooxygenase, activating matrix metalloproteinases 1 and 3 in epithelial cells and lamina propria cells, and ultimately leading to tissue damage. At this stage, clinical symptoms of mucositis may occur; (4) Ulcer stage: mucosal ulcers, microbial invasion, activation of macrophages and neutrophils, etc. occur at this stage, further increasing the production of pro-inflammatory factors. (5) Healing stage: As mucosal cells are destroyed, epithelial cells migrate, proliferate, and differentiate. When the signal causing persistent mucosal damage disappears, the mucosa will gradually heal.

[0004] Traditional Chinese medicine refers to oral ulcers as "mouth sores". The pathogenesis is: first, insufficient vital energy, spleen deficiency and qi deficiency, second, excessive evil, Yin deficiency and fire in the body. Most cancer patients are weak in vital energy and strong in evil, unable to resist evil. The attacking force of radiotherapy and chemotherapy is too strong, which can easily damage the internal organs, causing imbalance of the five internal organs. The evil will turn into heat over time, and the fire will burn. In addition, anti-tumor drugs are drug toxic and hot in nature, which will damage the body's qi, blood and body fluids; radiation is a fiery and toxic evil, which will consume qi and damage yin, turn into fire and burn body fluids, thus inducing mouth sores.

[0005] In the treatment of oral mucositis, there is no effective drug, mainly symptomatic treatment, mostly local medication to promote wound healing and relieve pain, and there is a lack of specific drugs. The latest Western medicine treatment uses human granulocyte colony stimulating factor oral gargle, Jinyin peptide combined with semiconductor laser oral treatment, anti-infection rehydration combined with chitosan antibacterial film, selenium yeast and other treatments, and also adds new rehabilitation drugs with anti-ulcer effects.

[0006] Kangfuxin Liquid is a modern preparation of ethanol extract of American cockroach (Periplaneta americana Linn.). It is widely used in clinical practice. According to Shennong's Herbal Classic, American cockroach tastes salty and is cold in nature, and has the effects of breaking up accumulation and sore throat. Kangfuxin Liquid has the effects of nourishing yin, promoting muscle growth, and promoting blood circulation. It is used externally for wounds of various burns, ulcers, bedsores, etc. Kangfuxin Liquid, which is made from American cockroach, is a polypeptide preparation widely used in clinical practice to treat ulcers and promote damage repair. It is widely used in clinical practice. Kangfuxin Liquid is used to prevent and treat oral diseases and repair oral ulcers. It has a definite effect. In the treatment of oral mucositis caused by radiotherapy and chemotherapy, it has a significant effect without obvious adverse reactions and has good safety.

[0007] Although Kangfuxin Liquid has significant efficacy in the prevention and treatment of oral diseases when used alone or in combination, its natural defects in liquid dosage form are: it is generally administered by wet compress, gargle, smear, oral administration, spray, or oral and smear combination; with the physiological swallowing action in the oral cavity, a drug layer formed on the surface interface of the buccal mucosa, tongue periphery, periodontal gums, etc. will be gradually washed away by the saliva secreted by the salivary glands, and the effective ingredients in Kangfuxin Liquid are difficult to be stably retained on the patient's ulcer surface for a long time, resulting in a large amount of waste of drugs for treating ulcer surface. Therefore, patients need to smear, spray, or continuously apply Kangfuxin Liquid to the affected area multiple times to make up for the defects of the drug release technology. In summary, the market is in urgent need of a new formulation technology for the treatment of oral mucositis. It is hoped that academia and the industry can develop an innovative formulation of a new recovery liquid that can increase white blood cells and enhance immunity, can be applied to damaged mucosal areas for a long time, improve wound microcirculation, accelerate the shedding of necrotic cells, quickly repair ulcers and wounds, and simultaneously have the comprehensive effects of anti-inflammatory, detumescence, and antioxidant effects, and can reduce external stimuli.

[0008] In order to solve the market pain points mentioned above, the present invention aims to design and prepare a drug-containing film derived from a new rehabilitation drug, suitable for patients with oral mucosal inflammation caused by radiotherapy and chemotherapy, which is convenient for their daily carrying, use, and replacement without affecting the safety of the oral mucosa. The requirements are: a skeleton carrier that can be applied to the oral mucosa for a long time and can continuously release functional drugs such as increasing white blood cells, enhancing immunity, and promoting ulcer repair. The drug skeleton carrier needs to be a natural biological skeleton drug-carrying material that has good biocompatibility with the oral mucosa and does not have adverse reactions with tissues.

[0009] Bee cocoons are filaments covering the outside of bee pupae. Through testing, it is mainly composed of six major types of substances: alkanes, alkenes, acids, esters, ketones and other substances. Bee cocoons are similar to silkworm cocoons. After the queen bee lays eggs, the eggs hatch into larvae. The worker bees seal the mouth of their nest cells with wax caps. The larvae in the sealed nest cells spin silk to make bee cocoons. Li Shizhen, an ancient medical scientist in the Ming Dynasty of my country, recorded in the Compendium of Materia Medica: "The smell of bee roe is sweet and flat, slightly cold, and non-toxic. Indications: Wind-head, removing poison, replenishing deficiency and winning injury, long-term use makes people radiant, good complexion, anti-aging, light body and invigorating qi. Treat heart and stomach pain, yellow face, and five insects vomited from the mouth of children and adults. It can treat erysipelas, rubella, heat retention in the abdomen, facilitating urination and defecation, removing floating blood, promoting lactation, leucorrhea in women, and leprosy. The ancients used bee roe as food, and raising and using bee roe is a medicine for the Yangming and Taiyin meridians of the barefoot." Wasp adults, larvae, pupae and honeycombs can all be used as medicine, and can be taken internally or applied externally to treat various poisonous insect bites, various carbuncles, swellings and gynecological diseases.

[0010] At present, biocompatible sustained-release membrane-forming materials at home and abroad are roughly divided into natural and synthetic ones. However, natural bee cocoons are not common as membrane-forming materials for sustained-release membranes. The reason is that: although the biocompatibility of bee cocoons is second to none in the world, whether pure bee cocoons can curl up and assemble into a biological skeleton autonomously under specific conditions; whether the formed bee cocoon biocompatible membrane can carry drug molecules, and how to effectively mix Kangfuxin drugs with bee cocoons so that the self-assembled bee cocoon biomembrane can wrap the effective drug molecules in Kangfuxin and slowly release them in the oral environment, there is no research conclusion yet, which is a key common technology that needs to be urgently solved in the industry.

[0011] In order to solve the problem of biocompatible sustained-release drug-loaded film for oral mucositis and the industry gap, the inventors have conceived to explore a unique and effective new preparation method and technique, find out the combination of key technical parameters, so as to successfully load new rehabilitation drugs into natural bee cocoons with good biocompatibility, and innovatively form natural bee cocoon spider silk biofilms.

[0012] The present invention provides a pre-film drug loading method for blending Kangfuxin drug and natural bee cocoon in a solvent, and its application in the treatment of patients with oral mucositis after radiotherapy and chemotherapy. The research and development idea is to prepare the natural bee cocoon with the best biocompatibility in the biological world into a biocompatible film loaded with Kangfuxin drug, so that it can be applied to the ulcer surface. The Kangfuxin drug molecules embedded in the pores of the oral mucositis biocompatible drug loading film can be continuously released, solving the problem of insufficient action time of Kangfuxin. Summary of the invention

[0013] In view of the deficiencies in the prior art, the present invention provides a method for preparing a drug-loaded bee cocoon biofilm and its application.

[0014] The technical solution of the present invention is as follows: A drug-loaded bee cocoon biofilm is prepared by the following method:

[0015] 1) picking, removing impurities, washing and drying wasp cocoons to obtain pre-treated cocoon raw materials;

[0016] 2) weighing a raw material of bee cocoons and dissolving it in a hexafluoroisopropanol solution, stirring, heating and refluxing to dissolve it to obtain a bee cocoon solution;

[0017] 3) filtering the cocoon solution dissolved in step 2) while hot to obtain a pure drug-carrying material matrix cocoon filtrate;

[0018] 4) heating and concentrating the cocoon filtrate obtained in step 3) to a certain concentration, adding the drug thereto, and stirring evenly to obtain a drug-loaded cocoon extract solution;

[0019] 5) Spreading the drug-loaded bee cocoon extract solution obtained in step 4) in a flat-bottomed container, oscillating under heating to volatilize the solvent, and removing the film after annealing to obtain the drug-loaded bee cocoon biofilm.

[0020] Preferably, in step 2), the ratio of the bee cocoon raw material to hexafluoroisopropanol is 1 mg: (2-6) ml.

[0021] Preferably, in step 2), the stirring, heating and reflux time of the bee cocoon raw material and hexafluoroisopropanol is 6 to 8 hours.

[0022] Preferably, in step 3), filtration is performed using a stainless steel metal filter of 800 mesh.

[0023] Preferably, in step 4), the cocoon filtrate is concentrated to 12-18 mg / ml.

[0024] Preferably, the drug in step 4) is selected from Kangfuxin solution or American cockroach extract.

[0025] Preferably, in step 4), the mass ratio of the drug to the raw material of the bee cocoon is (0.12-0.16):1.

[0026] Preferably, in step 5), the drug-loaded bee cocoon extract is evaporated to form a film at 45° C. and 50 rpm on a shaker.

[0027] Preferably, the thickness of the drug-loaded bee cocoon biofilm is 0.2 mm to 0.68 mm.

[0028] The present invention also provides application of the drug-loaded bee cocoon biofilm in preparing a drug for treating oral mucositis.

[0029] Beneficial effects of the present invention: (1) The inventors applied the new Kangfu medicine liquid that can repair ulcer surface, repair damaged skin and resist infection to the long-acting product for ulcer surface required by patients with oral mucositis during radiotherapy and chemotherapy, forming an innovative device that can have both home care and in-clinic treatment application functions.

[0030] (2) The natural honey bee cocoon membrane prepared by the preparation method of the present invention has good biocompatibility and will not cause adverse reactions such as rejection and inflammation when in contact with mucous membranes and tissues for a long time;

[0031] (3) The natural drug-loaded bee cocoon biofilm prepared according to the preparation method of the present invention can be used as a semi-embedded fixed biological skeleton material drug storage capsule, and the Kangfuxin drug solution can be infused into the bee cocoon membrane at regular intervals; thereby, the repair drug solution can continuously penetrate into the oral mucosa, play a role in repairing damage, and reduce the pain and trouble of surgical implantation;

[0032] (4) The natural drug-loaded bee cocoon biofilm prepared by the present invention can effectively improve the speed, convenience and effectiveness of drug administration, dressing change and nursing for patients with oral mucositis. It is suitable for both in-clinic treatment of patients with oral mucositis and self-care of patients at home.

[0033] (5) The present invention adopts a pre-film-forming drug-loading method to prepare a drug-loaded material matrix bee cocoon biofilm and a drug-loaded bee cocoon membrane, and tests the 30-day degradation properties of the two in artificial saliva. The biocompatibility in cells is good; it has good water permeability and swelling properties, certain tensile strength and adhesion, which ensures the applicability of the membrane as a medical care consumable for oral medicine requirements.

[0034] (6) The drug-loaded bee cocoon biofilm of the present invention has good oral biocompatibility, no adverse reactions, and can release drugs for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a scanning electron microscope (SEM) image of the drug-carrying material matrix spider silk protein film prepared in Example 1 (2000x).

[0036] Figure 2 This is a graph of cell viability when the 100% final concentration extract of the drug-loaded spider silk protein Kangfu new drug extract film prepared in Example 2 was co-incubated with mouse fibroblast L-929 cells for 24 hours.

[0037] Figure 3 The drug release curve of the drug-loaded spider silk protein American cockroach extract film prepared in Example 3 in artificial saliva within 180 minutes. DETAILED DESCRIPTION

[0038] The following embodiments can enable those skilled in the art to more fully understand the present invention, but the present invention is not limited to the scope of the embodiments.

[0039] Example 1: Preparation of drug-carrying material matrix cocoon membrane

[0040] This embodiment provides a method for preparing a drug-carrying material matrix bee cocoon protein membrane, that is, a blank membrane without any drug. The wasp cocoons used in the experiment were from a wasp breeding base in Yunnan Province.

[0041] 1.1 Preparation of drug-carrying material matrix cocoon solution: Pick, remove impurities, wash and dry wasp cocoons, weigh 100 mg of cocoon raw material and dissolve it in 600 mL of hexafluoroisopropanol (HFIP) solution, heat to 58°C with magnetic stirring and reflux for 8 hours to dissolve it; filter the dissolved cocoon solution with a 400-mesh stainless steel metal filter while it is hot to obtain a pure drug-carrying material matrix cocoon filtrate.

[0042] 1.2 Preparation of drug-carrying material matrix cocoon membrane: Spread the cocoon filtrate prepared in 1.1 in a flat-bottomed glass dish with a diameter of 6 cm, shake in a 40°C water bath to evaporate the solvent, and peel off the membrane after annealing to obtain a drug-carrying material matrix cocoon biofilm with uniform texture and an average thickness of 0.2 mm (see Figure 1 ).

[0043] Example 2: Preparation of drug-loaded honey bee cocoon protein recovery new drug extract film

[0044] This embodiment provides a method for preparing a drug-loaded bee cocoon protein Kangfuxin drug extract film. The wasp cocoons used in the experiment come from the wasp breeding base in Yunnan Province, and Kangfuxin drug in this embodiment refers to Kangfuxin drug extract (i.e., Kangfuxin liquid raw material extract) provided by Sichuan Good Doctor Panxi Pharmaceutical Co., Ltd.

[0045] 2.1 Preparation of drug-carrying material matrix bee cocoon protein solution: Pick, remove impurities, wash and dry wasp cocoons, weigh 500 mg of cocoon raw materials and place them in a 2000 mL round-bottom flask, measure 1000 mL of hexafluoroisopropanol solution and pour it into the round-bottom flask, heat to 60°C with magnetic stirring and reflux for 8 hours to dissolve it; filter the dissolved cocoon solution with an 800-mesh stainless steel metal filter while it is hot to obtain a pure drug-carrying material matrix cocoon filtrate.

[0046] 2.2 Preparation of drug-loaded bee cocoon Kangfuxin drug extract solution: Place the bee cocoon filtrate obtained under 2.1 in a round-bottom flask, control the recovery temperature at 70°C, heat and condense the reflux to recover the solvent, and concentrate the bee cocoon filtrate to 1mL of bee cocoon concentrate equivalent to 18.0mg of bee cocoon raw material. Weigh 80.0mg of Kangfuxin drug extract, pour the concentrated drug-loaded material matrix bee cocoon concentrate into Kangfuxin drug extract, and stir magnetically for 80 seconds to make the solution uniform to obtain the drug-loaded bee cocoon Kangfuxin drug extract solution.

[0047] 2.3 Preparation of drug-loaded Bee Cocoon Kangfu New Drug Extract Film: Spread the drug-loaded Bee Cocoon Kangfu New Drug Extract solution prepared under Item 2.2 in a flat-bottomed glass dish with a diameter of 12 mm, heat and oscillate at 40°C to evaporate the solvent, and peel off the film after annealing to obtain a drug-loaded Bee Cocoon Kangfu New Drug Extract Film with uniform texture and an average thickness of 0.68 mm.

[0048] Example 3: Preparation of drug-loaded cocoon American cockroach extract film

[0049] This embodiment provides a method for preparing a drug-loaded American cockroach extract film of wasp cocoon. The wasp cocoons used in the experiment come from the wasp breeding base in Yunnan Province, and the American cockroach extract of the raw material drug for preparing Kangfuxin liquid in this embodiment is prepared and provided by Sichuan Good Doctor Panxi Pharmaceutical Co., Ltd.

[0050] 3.1 Preparation of drug-carrying material matrix cocoon solution: Pick, remove impurities, wash and dry wasp cocoons, weigh 1000 mg of cocoon raw material, dissolve it in 2000 mL of hexafluoroisopropanol solution, heat to 65°C with magnetic stirring and reflux for 6 hours to dissolve it; filter the dissolved cocoon solution while hot with an 800-mesh stainless steel metal filter to obtain a pure drug-carrying material matrix cocoon filtrate.

[0051] 3.2 Preparation of drug-loaded cocoon American cockroach extract solution: Place the cocoon filtrate obtained under 3.1 in a round-bottom flask, control the recovery temperature at 70°C, heat and condense the reflux to recover the solvent, and concentrate the cocoon filtrate to 1mL of cocoon protein concentrate equivalent to 12.0mg of cocoon raw material. Weigh 120.0mg of American cockroach extract, pour it into the prepared drug-loaded material matrix cocoon concentrate, and stir magnetically for 60s to evenly disperse the solution to obtain a drug-loaded cocoon American cockroach extract solution.

[0052] 3.3 Preparation of drug-loaded bee cocoon and American cockroach extract film: Spread the drug-loaded bee cocoon and American cockroach extract solution prepared under 3.2 in a flat-bottomed glass dish with a radius of 10 cm, place it on a shaker at 50 rpm in a fume hood under 45°C water bath heating to evaporate the solvent to form a film, and obtain a uniform drug-loaded bee cocoon and American cockroach extract film with a thickness of 0.35 mm.

[0053] Example 4: Degradability test of membrane in artificial saliva

[0054] In this embodiment, the drug-loaded material matrix bee cocoon membrane (hereinafter referred to as blank membrane) prepared in Example 1 and the drug-loaded bee cocoon Kangfuxin drug extract membrane (hereinafter referred to as drug-loaded membrane) prepared in Example 2 were respectively subjected to degradation tests in artificial saliva.

[0055] 4.1 Degradation of blank film and drug-loaded film in artificial saliva (30 days): Artificial saliva was prepared according to the method in the standard "GB / T18886-2019", and the blank film prepared in Example 1 and the drug-loaded film prepared in Example 2 were cut into squares of 1 cm × 1 cm in size, and each was placed in a 10 mL test tube. 2 mL of freshly prepared artificial saliva at 37°C ± 0.5°C was added to the test tubes containing the blank film and the drug-loaded film, respectively, to ensure that the artificial saliva covered the test film surface and the above test tubes were placed in a constant temperature and humidity incubator, and a 30-day degradation test was carried out at a constant temperature of 37°C. Before the test, the morphological characteristics and pore size of the membrane before the degradation test were observed and recorded under a scanning electron microscope at a magnification of 1000x; thereafter, during the 30-day test, the artificial saliva in the test tube was completely removed every 24 hours, and freshly prepared artificial saliva of equal volume at isothermal temperature was added to continue the degradation test until the end of the test; during the test, the membrane was taken out at the same time every day, the artificial saliva on the surface was blotted dry, and the mass change of the membrane was weighed and recorded using the same electronic balance, and the degradation test was observed and recorded whether the morphology of the membrane was degraded during the corresponding number of days (results are shown in Table 1). If the membrane is observed to have gaps, the pores of the membrane are enlarged, the membrane becomes thinner, and the volume is reduced, it means that the membrane has degraded; it should be pointed out that the release of drugs in the drug-loaded membrane under artificial saliva conditions is a normal phenomenon, and the change in the morphology of the membrane caused by drug release does not belong to the category of membrane degradation.

[0056] Table 1: Blank membrane and drug-loaded membrane in artificial saliva (7 days)

[0057]

[0058] *Note: Blank membrane refers to the drug-loaded material matrix bee cocoon membrane, and drug-loaded membrane refers to the drug-loaded bee cocoon Kangfuxin drug extract membrane.

[0059] 4.2 Result explanation: Neither the drug-carrying material matrix honey cocoon membrane nor the drug-carrying honey cocoon Kangfu new drug extract membrane degraded in artificial saliva at 37°C within 7 days. The degradation test results show that the drug-carrying material matrix honey cocoon membrane and the drug-carrying honey cocoon Kangfu new drug extract membrane prepared by this method can exist stably in the oral saliva environment for a long time.

[0060] Example 5: In vitro cytotoxicity test of membranes

[0061] In this embodiment, the drug-carrying material matrix bee cocoon membrane prepared in Example 1 and the drug-carrying bee cocoon Kangfuxin drug extract membrane prepared in Example 2 were respectively subjected to in vitro cytotoxicity evaluation tests. This test method is in accordance with the requirements of GB / T16886.5-2017 "Biological Evaluation of Medical Devices", using mouse fibroblast L-929 cell line, and using CCK-8 method to determine the cytotoxicity of the test sample.

[0062] 5.1 Preparation of test samples: Sample 1 (drug-carrying material matrix bee cocoon membrane), sample 2 (drug-carrying bee cocoon Kangfuxin liquid extract membrane) and negative control group (high-density polyethylene membrane) were extracted under the conditions of 6 cm 2 :1mL sampling ratio, cut out 20 pieces with a surface area of ​​6cm 2 The 20 pieces of test sample 1, 20 pieces of test sample 2 and 20 pieces of negative control were cut into corresponding culture bottles, and 20 mL of MEM culture medium containing 10% fetal bovine serum was poured into each bottle. The cells were incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours to obtain sample extracts. The extracts of each sample in the test sample group and the negative control were clear before and after leaching, among which the extract of test sample 2 was light brown, and the extracts of the other samples had no obvious visible color changes. The extracts were used for the test immediately. The blank control group was MEM culture medium containing only 10% fetal bovine serum, and the cell group was normal cultured cells in MEM culture medium containing 10% fetal bovine serum.

[0063] 5.2 Experimental method: In the experiment, cells grown to the logarithmic phase were digested with trypsin and resuspended, and the cell concentration was counted and diluted to about 1×10 5 The cell suspension was inoculated into a 96-well plate at 100 μL per well and cultured in a cell culture incubator (37° C., 5% CO 2 , >90% humidity). The cell morphology was observed under an inverted microscope. When the cells grew to about 30% by adhering to the wall, the original culture medium was discarded, and 100 μL of the test sample group (the extract of the test sample 1 (final concentration of 100%, 75%, 50%, 25%) and the extract of the test sample 2 (final concentration of 100%), blank control group and negative control group were added to the corresponding wells of the 96-well plate. The 96-well plate was placed in a cell culture incubator and taken out after culturing for 24 hours, and the growth morphology of the cells was observed under an inverted microscope. After observing the cell morphology of the 96-well plate after culturing for 24 hours, the culture medium was removed, 100 μL of CCK8 solution was added to each well, and the plate was placed in a cell culture incubator and cultured for 2 hours under the above-mentioned culture conditions. The absorbance value at a wavelength of 450 nm was measured on an ELISA instrument, and the cell viability was calculated. The test sample was determined to have potential cytotoxicity based on the degree of difference between the cell viability of the test sample and the cell viability of the blank control group.

[0064] Test sample group cell viability % = (test sample group OD450 - blank sample group OD450) / (cell group OD450 - blank sample group OD450 × 100%);

[0065] Negative sample group cell viability % = (negative sample group OD450-blank sample group OD450) / (cell group OD450-blank sample group OD450×100%);

[0066] The cell viability of the test sample group (test sample 1 and test sample 2) under the action of the extract is taken as 100% extraction result as the final result; if the cell viability of the test sample group is less than 70% of the cell viability of the cell group, the test sample is considered to have potential cytotoxicity.

[0067] 5.3 Test results and explanation: Under the test conditions, the cell viability of the extract of test sample 1 (final concentration of 100%, 75%, 50%, 25%) in the test sample group was 100.31%, 103.85%, 101.06%, 104.89%, 106.5% respectively; the cell viability of the extract of test sample 2 in the test sample group was 101.10%; the cell viability of the negative control group was 98.6%. From the results, it can be seen that the cell viability of the extract of 100% concentration of the two test samples in the test sample group meets the requirements (i.e., greater than 70% of the cell viability of the blank group), so it can be considered that the blank matrix honey cocoon membrane and the honey cocoon Kangfuxin extract membrane of the drug-loaded material prepared by the patent method have no significant potential cytotoxicity to L-929 cells.

[0068] Example 6: Oral mucosal irritation test of film

[0069] In this embodiment, the skin irritation test was conducted using the drug-carrying material matrix bee cocoon protein film (hereinafter referred to as blank film) prepared in Example 1 and the drug-carrying bee cocoon protein Kangfuxin drug extract film (hereinafter referred to as drug-carrying film) prepared in Example 2. This test was conducted using Kunming mice as test animals using the direct contact method.

[0070] 6.1 Experimental preparation: Six healthy Kunming mice weighing 20.0±5g were selected for this experiment. Before the experiment, the blank film and the drug-loaded film were cut into squares of 2.5cm×2.5cm and immersed in saline for 24h. Cotton balls were soaked in the extract, and cotton balls of the same size were soaked in saline as a control.

[0071] 6.2 Test method: The test sample and control sample are contacted with the oral mucosa of mice respectively, and then the mice are fixed for contact for 15 to 30 minutes. After the contact period, the test sample and control sample are removed. Stimulate three times a day for a total of 7 days. Observe the reaction of the stimulated area and surrounding skin tissue of the test animals, including erythema, edema and necrosis, and record them (see Table 3 and Table 4 for evaluation criteria).

[0072] Table 3 Scoring criteria for oral mucosal irritation response in tested mice

[0073] erythema Scoring Edema Scoring No erythema 0 No edema 0 Mild erythema (barely visible) 1 Mild edema (barely noticeable skin thickening) 1 Obvious erythema (light red) 2 Obvious edema (prominent and well-defined) 2 Moderate erythema (bright red) 3 Severe edema (uplift of approximately 1 mm) 3 Severe erythema (purple with slight eschar formation) 4 Severe edema (bulge greater than 1 mm) 4

[0074] Table 4 Evaluation of stimulation response types of tested mice

[0075] Reaction Type Scoring No irritation 0-0.4 Mild irritation 0.5-1.9 Moderate stimulation 2.0-4.9 Severe irritation 5-8

[0076] 6.3 Test results and explanation: During the test that ended on day 7, the test animals did not show abnormal symptoms or die. The oral mucosa of the animals treated with the two experimental factors of test sample 1 blank film and test sample 2 drug-loaded film remained intact after day 7, without any reactions such as erythema, edema and necrosis; on day 7, the mucosal reactions of both groups did not exceed those of the control group, and the primary irritation index was 0. Result explanation: The drug-loaded material matrix honey cocoon film and the drug-loaded honey cocoon Kangfuxin drug extract film should both be classified as non-irritation reaction types in the evaluation type of oral mucosal irritation reaction of mice.

[0077] Example 7: Film tensile properties and adhesion test

[0078] In this embodiment, the drug-carrying material matrix bee cocoon membrane (sample 1) prepared in Example 1 and the drug-carrying bee cocoon Kangfuxin drug extract membrane (sample 2) prepared in Example 2 were used to perform membrane tensile tests and adhesion tests respectively.

[0079] 7.1 Tensile test of membrane: According to the tensile performance test standard of GB / T1040-2022, the membranes prepared in Example 1 and Example 2 were cut into three 1mm×4mm rectangular specimens, and the drug-loaded material matrix bee cocoon membrane and the drug-loaded bee cocoon Kangfuxin drug-loaded membrane were subjected to tensile tests respectively using the HT tensile strength testing machine of Dongguan Huitai Machinery Co., Ltd. The test conditions were temperature of 25°C, relative humidity of 65%, and tensile speed of 50mm / min.

[0080] Table 5 Tensile performance material mechanical data of each tested sample film (x±s; n=3)

[0081] sample Tensile strength / Mpa Elongation at break / % Sample 1 27.488±0.098 1.378±0.062 Sample 2 41.562±0.107 2.270±0.073

[0082] 7.2 Adhesion test of membrane: Select mouse buccal mucosa membrane as biofilm material, soak it in artificial saliva for 45 minutes, cut out a small square of 2cm×2cm, and stick it to the test board with double-sided tape. Cut three 1cm×1cm films of sample 1 prepared in Example 1 and sample 2 prepared in Example 2 as the test sample membrane; moisten the test sample membrane with artificial saliva, and quickly stick it to the mouse buccal mucosa, hang an S-shaped iron hook under the test sample membrane, and hang a ziplock bag on the other end of the iron hook; then hang the test board vertically, so that the test board, the composite of the buccal mucosa membrane and the test sample membrane, the S-shaped iron hook, and the ziplock bag are on the same horizontal plane and vertical to the ground. Use a 10mL syringe to slowly drip ultrapure water into the open ziplock bag until the test sample membrane falls off from the mucosa. The weight of the water in the ziplock bag when the adhesive sheet falls off is weighed. The adhesion of the 1 cm × 1 cm area of ​​the test sample film is expressed as the total mass of the collected ultrapure water, S-shaped iron hook and ziplock bag (g / cm 2 ).

[0083] Table 6 Adhesion force of each test sample film (x±s; n=3)

[0084] sample Adhesion (g / ㎝2) Sample 1 35.09±0.13 Sample 2 26.02±0.09

[0085] 8.3 Result explanation: The test results in Table 5 and Table 6 show that the film prepared by the method of the present invention has certain tensile properties and adhesion, and can meet the relevant requirements for oral film administration.

[0086] Example 8: Drug release test of drug-loaded bee cocoon American cockroach extract film in artificial saliva

[0087] In this embodiment, the drug-loading bee cocoon American cockroach extract film prepared in Example 3 was used to conduct a drug release test in artificial saliva.

[0088] 8.1 Prepare artificial saliva according to the method in the standard "GB / T18886-2019". Cut 6 1cm×1cm squares of the drug-loaded bee cocoon American cockroach extract film prepared under 3.3 in Example 3 for parallel testing. Put the 6 films into 6 2mL centrifuge tubes of the same shape, add 1mL of artificial saliva at 37℃±0.5℃, so that the test film is completely submerged by artificial saliva. Place the floating plate with the above centrifuge tubes on a shaker, control the shaker speed to 50rpm, and keep the temperature of the drug release system constant at 37℃±0.5℃ for drug release test. Set the cumulative drug release time to 0, 5, 15, 30, 60, 90, 120, and 180min. Take samples of the drug release solution at each drug release time point: after the drug release solution in the centrifuge tube is completely taken out, add 1 mL of freshly prepared artificial saliva at 37°C ± 0.5°C to the centrifuge tube, and continue the drug release test until the preset cumulative drug release time is reached. The sampling operation at each cumulative drug release time point is the same as the sampling operation at the first time point.

[0089] 8.2 Drug-loaded Bee Cocoon American Cockroach Extract Film Using inosine as an index component to establish its content detection method: accurately weigh 25.0 mg of American Cockroach extract, dissolve it with artificial saliva, and place it in a 25 mL volumetric flask to make up to a concentration of 1.0 mg / mL American Cockroach extract mother solution. Take 8, 6, 4, 2, and 1 mL of the American Cockroach extract mother solution in a 10 mL volumetric flask, make up to 10 mL with artificial saliva, and prepare a series of American Cockroach extract solutions with concentration gradients of 0.8, 0.6, 0.4, 0.2, and 0.1 mg / mL; using inosine as an index component, use inosine as a standard substance to do the external standard method to determine the peak corresponding to inosine in the high-performance liquid chromatogram of American Cockroach extract. According to the high-performance chromatographic separation conditions of American Cockroach extract (see Table 7), a series of concentration gradient American Cockroach extract solutions were tested. Finally, under the condition of 254nm wavelength, the concentration of American cockroach extract was taken as the horizontal coordinate (x), and the peak area corresponding to inosine in the spectrum was taken as the vertical coordinate (y) to obtain the detection curve of the content of American cockroach extract with inosine as the indicator component: y=181.99x-13.153; R 2 =0.9996.

[0090] 8.3 Detection of drug release in the drug release solution of drug-loaded bee cocoon American cockroach extract film: Detect the drug release solution obtained in Item 9.1 under the conditions of high-performance chromatography of American cockroach extract. Calculate the content of American cockroach extract in the drug release solution based on the content detection curve of drug-loaded bee cocoon American cockroach extract film with inosine as the indicator component obtained under Item 9.2. Draw the drug release curve of drug-loaded bee cocoon American cockroach extract film in artificial saliva (see Figure 3 ).

[0091] Instrument: Agilent 1260Ⅱ Chromatographic column: Sepax HP-C18 column;

[0092] Detector: DAD detector, double-side column temperature: 30°C;

[0093] Sample volume: 10 μL Detection wavelength: 254 nm Flow rate: 0.5 mL / min;

[0094] Mobile phase A: 0.1% trifluoroacetic acid (TFA)-water; mobile phase B: 0.1% trifluoroacetic acid (TFA)-methanol.

[0095] Table 7: Gradient elution conditions of American cockroach extract

[0096] Time / min A% (0.1% TFA-water) B% (0.1% TFA-methanol) 0 98 2 15 80 20 30 98 2

[0097] 8.4 Results: The drug-loaded Periplaneta americana extract film has a certain sustained release effect in artificial saliva. Figure 3 It can be seen that the drug is continuously released from the membrane during the 180-min drug release period, and the drug release of the membrane has not yet entered the drug release plateau period. Therefore, it is speculated that the drug-loaded protein American cockroach extract membrane prepared by the patented method can continuously release the drug in human saliva for more than 4 hours.

[0098] Example 9: Oral mucositis pharmacodynamics test of the film

[0099] In this embodiment, the drug-loaded honey bee cocoon protein Kangfu new drug extract film (hereinafter referred to as drug-loaded film) prepared in Example 2 was used for pharmacodynamics test. In this test, Kunming mice were used as test animals and the direct contact method was used for the test.

[0100] 9.1 Experimental preparation: Six healthy Kunming mice weighing 20.0±5g were selected for this experiment. Before the experiment, the drug-loaded films were cut into squares of 2.5cm×2.5cm and immersed in saline for 24h. Cotton balls were soaked in the extract, and cotton balls of the same size were soaked in saline as a control.

[0101] 9.2 Experimental method: The mice were adaptively fed for 1 week and fed a normal diet. Except for the blank group, mice in each experimental group received an intraperitoneal injection of 5-fluorouracil (60 mg / kg) on ​​the first and third days of the experiment. Then the mice were anesthetized by inhalation of sevoflurane, and a cotton ball was placed at the front end of a 1 mL syringe with an inner diameter of about 5 mm and the bottom flush with the tube mouth. 50% glacial acetic acid was dripped to just soak the cotton ball. The cotton ball of the syringe was placed flat on the right buccal membrane of the mouse and burned for 60 seconds to establish an oral ulcer animal model, and the wound size and depth of each rat were kept consistent as much as possible. Ulcers appeared 48 hours after mucosal injury, and medication was started. The blank group was fed conventionally; the control group was given the blank film of Example 1 to be applied on the ulcer surface; the positive drug group was given an equal amount of Kangfuxin liquid to be evenly applied on the ulcer surface; the drug group was given a drug-loaded film once a day, which was attached to the ulcer surface to ensure that the drug stayed in the mouth for 3 minutes; and the drug was administered continuously for 7 days. The main performance indicators and grading table of the oral ulcer animal model were used to evaluate the oral ulcer situation of mice.

[0102] 9.3 Evaluation indicators: Evaluation indicators: 1) Changes in food intake of rats in each group: Before medication and 1 to 7 days after medication, the weight of each rat was weighed every morning with a small high-precision electronic scale. 2) Observation of rat ulcer area: After successful modeling and 1 to 7 days after medication, take photos and record with a camera to observe the changes in the area of ​​oral ulcers in rats. 3) Comparison of ulcer area: After successful modeling and 1 to 7 days after medication, use a vernier caliper to measure the maximum transverse diameter (d1) and maximum longitudinal diameter (d2) of the ulcer to calculate the ulcer area. 4) Serum inflammatory factor level: 7 days after medication, anesthetize the mice, take heart blood samples, and measure the serum inflammatory factor level. 5) Tissue inflammatory factor level: 7 days after medication, anesthetize the rats, cut the oral mucosal tissue, process and measure the tissue inflammatory factor level. 6) Comparison of oral ulcer scores: After successful modeling and 1 to 7 days after medication, observe the changes in rat mucosa every morning, and use the main performance indicators and grading table of oral ulcer animal model to evaluate the ulcer score.

[0103] Ulcer area = π × d1 × d2 × 1 / 4.

[0104] 9.4 Test results and explanation: From the 1st to the 3rd day, there was no statistically significant difference in the food intake of rats in each group (P>0.05). Starting from the 4th day, the changes in the food intake of mice in each group showed differences, and the oral healing effect of each drug-treated group was more significant than that of the control group. The ulcer surface of the mice after successful modeling was recorded by taking pictures, and the surface of the oral ulcer of the mice was continuously observed. The drug was administered for 7 consecutive days. The results showed that the oral mucosa of the blank group was intact and smooth, without obvious damage. The ulcer area of ​​each drug-treated group was reduced with the extension of time. On the 1st and 3rd days, there was no significant difference in the ulcer area of ​​each group of mice. Starting from the 5th day, the ulcer area of ​​the drug-treated group was significantly smaller than that of the control group, and the ulcer area of ​​the drug-treated group was also significantly smaller than that of the positive group, which was related to the slow release of the drug-loaded film and the long-term maintenance of a certain blood drug concentration. Observation under the light microscope showed that the oral mucosa of the blank group mice was intact, the contours of each layer of cells were clear, and the morphology was normal. The subepithelial lamina propria was loose connective tissue, rich in capillaries, and there was no obvious inflammatory cell infiltration. The cells of the oral mucosal epithelium of the mice in the control group dissolved, necrotized and fell off, and there was dense inflammatory cell infiltration under the mucosal lamina propria. The mucosal epithelium of the drug-loaded film group tended to be intact, with a small amount of necrotic tissue, reduced inflammatory cell infiltration, and a large number of fibroblasts proliferated under the epithelium.

Claims

1. A drug-loaded bee cocoon biofilm, which is prepared by the following method: 1) picking, removing impurities, washing and drying wasp cocoons to obtain pre-treated cocoon raw materials; 2) weighing a raw material of bee cocoons and dissolving it in a hexafluoroisopropanol solution, stirring, heating and refluxing to dissolve it to obtain a bee cocoon solution; 3) filtering the cocoon solution dissolved in step 2) while hot to obtain a pure drug-carrying material matrix cocoon filtrate; 4) heating and concentrating the cocoon filtrate obtained in step 3) to a certain concentration, adding the drug thereto, and stirring evenly to obtain a drug-loaded cocoon extract solution; 5) Spreading the drug-loaded bee cocoon extract solution obtained in step 4) in a flat-bottomed container, oscillating under heating to volatilize the solvent, and removing the film after annealing to obtain the drug-loaded bee cocoon biofilm.

2. The drug-loaded bee cocoon biofilm according to claim 1, characterized in that: In step 2), the ratio of the raw material of Chinese honey bee cocoon to hexafluoroisopropanol is 1 mg: (2-6) ml.

3. The drug-loaded bee cocoon biofilm according to claim 1, characterized in that: Step 2) The stirring, heating and reflux time of the Chinese honey bee cocoon raw material and hexafluoroisopropanol is 6 to 8 hours.

4. The drug-loaded bee cocoon biofilm according to claim 1, characterized in that: In step 3), filter with a stainless steel metal filter of 800 mesh.

5. The drug-loaded bee cocoon biofilm according to claim 1, characterized in that: In step 4), the cocoon filtrate is concentrated to 12-18 mg / ml.

6. The drug-loaded bee cocoon biofilm according to claim 1, characterized in that: The drug in step 4) is selected from Kangfuxin solution or American cockroach extract.

7. The drug-loaded bee cocoon biofilm according to claim 1, characterized in that: The mass ratio of the drug to the raw material of the honey cocoon in step 4) is (0.12-0.16):

1.

8. The drug-loaded bee cocoon biofilm according to claim 1, characterized in that: In step 5), the drug-loaded bee cocoon extract is evaporated to form a film at 45° C. and 50 rpm on a shaker.

9. The drug-loaded bee cocoon biofilm according to claim 1, characterized in that: The thickness of the drug-loaded bee cocoon biofilm is 0.2mm to 0.68mm.

10. Use of the drug-loaded bee cocoon biofilm according to claim 1 in preparing a drug for treating oral mucositis.

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