Drug-loaded guided periodontal tissue regeneration membrane and preparation and application thereof

Abstract

The application discloses a drug-loaded guided periodontal tissue regeneration membrane and a preparation and application thereof. The drug-loaded guided periodontal tissue regeneration membrane comprises a soluble composite matrix material and nano drugs loaded in the soluble composite matrix material. The application is based on the principle and manufacturing concept of periodontal GTR, conceives to design the interface implant into a structure with gradient change of components and structure, promotes bone growth, prevents bacterial infiltration and growth of gum tissue to the tissue defect area below, makes the characteristics of the membrane more and more tend to be "ideal", satisfies the local functional requirements, improves the multi-aspect performance of the membrane, and realizes more excellent tissue regeneration.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, in particular to a drug-loaded guided periodontal tissue regeneration membrane and preparation and application thereof. BACKGROUND

[0002] Periodontitis is a common chronic inflammatory disease characterized by destruction of periodontal tissue leading to tooth mobility and loss, which is the main cause of tooth loss in adults. The goal of periodontitis treatment is to achieve periodontal tissue regeneration, and the selection of appropriate carriers to carry active substances for slow release at the action site can effectively solve the problem of poor efficacy caused by direct injection of drug solution into the action site. The film or strip system belongs to the matrix type drug delivery system, which is prepared by mixing high molecular polymers and drugs to form a film or strip. The size can be determined according to the required shape and size, and it is easy to insert into the target site with less discomfort to the patient. Compared with other types of drug delivery systems, the high molecular film or strip also has the advantages of good stability, large surface area and slow degradation rate, and has been widely studied in the field of periodontal disease treatment.

[0003] The application of biological barrier membrane (BBM) is a key link of guided tissue regeneration (GTR) technology, which mainly separates the periodontal defect area from the gingival tissue to prevent the rapid growth of gingival epithelial tissue into the periodontal defect area, thereby creating a relatively closed and good environment for the regeneration of periodontal tissue. An ideal BBM should have sufficient mechanical strength, good osteogenesis and anti-inflammatory properties, etc., but the currently used BBMs cannot meet all the above requirements and have different degrees of shortcomings, resulting in unsatisfactory GTR effect. For example, periodontal pathogens such as Porphyromonas gingivalis can internalize into host cells to escape immune response and antibiotic action, and over time, periodontitis recurs, and bacteria release toxins to cause persistent inflammation, resulting in unsatisfactory GTR effect. Metronidazole (MTZ) is a commonly used nitroimidazole antibacterial drug for the treatment of periodontitis in clinical practice, and is widely used for the treatment of anaerobic bacterial infections. Although it can be taken orally as an adjunctive therapy for periodontitis treatment to eliminate and eradicate pre-existing infections, it can easily cause side effects and antibiotic resistance. Based on the above concept, during GTR, the BBM is not only a passive barrier, but also a biologically active space, and adjusting the composition and structure of the membrane can enhance the GTR effect. SUMMARY

[0004] The present application aims to at least solve one of the above technical problems in the prior art. To this end, the present application aims to provide a drug-loaded guided periodontal tissue regeneration membrane and a preparation method thereof, based on the principle and manufacturing concept of periodontal GTR, the interface implant is designed to promote bone growth, prevent bacterial infiltration and guide the growth of gingival tissue to the tissue defect area below, so that the characteristics of the membrane tend to be "ideal", to meet the local functional requirements, so that the performance of the membrane is improved, and better tissue regeneration is achieved.

[0005] To achieve the above-mentioned object, the technical solution adopted by the present application is:

[0006] The first aspect of the present application proposes a drug-loaded guided periodontal tissue regeneration membrane.

[0007] The second aspect of the present application proposes a preparation method of a drug-loaded guided periodontal tissue regeneration membrane.

[0008] The third aspect of the present application proposes an application of a drug-loaded guided periodontal tissue regeneration membrane.

[0009] According to the first aspect of the present application, a drug-loaded guided periodontal tissue regeneration membrane is proposed, comprising a soluble composite matrix material and a nano-drug loaded in the soluble composite matrix material, the soluble composite matrix material is composed of polyvinyl alcohol and polyvinylpyrrolidone; the nano-drug is composed of cyclodextrin modified polylactic acid-glycolic acid copolymer and a pharmaceutically active ingredient wrapped therein.

[0010] In some embodiments of the present application, the mass of the pharmaceutically active ingredient in the drug-loaded guided periodontal tissue regeneration membrane accounts for 0.5% to 7.0% of the total mass of the drug-loaded guided periodontal tissue regeneration membrane, preferably 0.5% to 5%.

[0011] In some embodiments of the present application, the polyvinyl alcohol is type 17-88; the polyvinylpyrrolidone is type K30.

[0012] In some embodiments of the present application, the molecular weight of the polylactic acid-glycolic acid copolymer is 10000 to 30000; the molar ratio of lactic acid monomer (PLA) and glycolic acid monomer (PGA) in the polylactic acid-glycolic acid copolymer is 50 to 90: 50 to 10.

[0013] In some embodiments of the present application, the nano-drug is spheroid, with an average particle size of 50nm to 450nm, preferably 200nm to 300nm.

[0014] In some embodiments of the present application, the pharmaceutically active ingredient includes an anti-anerobic drug.

[0015] In some preferred embodiments of the present application, the anti-anerobic agent is a nitroimidazole, including at least one of metronidazole, tinidazole, ornidazole, and secnidazole.

[0016] In some preferred embodiments of the present application, the mass ratio of polyvinyl alcohol and polyvinylpyrrolidone in the soluble composite matrix material is 8-9: 1-2.

[0017] In some preferred embodiments of the present application, the thickness of the drug-loaded guided periodontal tissue regeneration membrane is 100-250 μm, preferably 150-200 μm.

[0018] According to a second aspect of the present application, a preparation method of the drug-loaded guided periodontal tissue regeneration membrane of the first aspect is provided, comprising the following steps:

[0019] The pharmaceutically active ingredient and the polylactic acid-glycolic acid copolymer are dissolved in an organic solvent to obtain an organic phase, the organic phase is injected into an aqueous phase containing cyclodextrin, the organic solvent is volatilized, and the obtained nanopharmaceutical solution is mixed with the soluble composite matrix material, thereby obtaining the drug-loaded guided periodontal tissue regeneration membrane.

[0020] In some embodiments of the present application, the organic solvent is one or more of acetone, ethanol, dichloromethane, and ethyl acetate, preferably acetone and ethanol, and more preferably a mixed solution of acetone and ethanol in a ratio of (2-4: 1, v / v).

[0021] In some embodiments of the present application, the mass ratio of the pharmaceutically active ingredient to the polylactic acid-glycolic acid copolymer is 0.1: 1-10.

[0022] In some embodiments of the present application, the mass fraction of the cyclodextrin in the aqueous phase is 1%-2% (w / v), and more preferably 1.5% (w / v).

[0023] In some embodiments of the present application, the aqueous phase further comprises a surfactant / emulsifier, and the mass fraction of the surfactant / emulsifier in the aqueous phase is 1%-3% (w / v), and more preferably 2% (w / v).

[0024] In some embodiments of the present application, the surfactant / emulsifier is selected from one of polyvinyl alcohol, gelatin, Tween-80, and poloxamer-188; and more preferably polyvinyl alcohol (type 05-88).

[0025] In some embodiments of the present application, the volume ratio of the organic phase to the aqueous phase is 1-2: 4.

[0026] In some embodiments of the present application, the mass volume ratio of the soluble composite matrix material to the nanopharmaceutical solution is 8-10: 100.

[0027] In some preferred embodiments of the present application, after the organic phase is injected into the water phase containing cyclodextrin, the process further comprises ultrasonic treatment for 30 minutes at intervals of 3 seconds; the ultrasonic treatment produces cavitation effect in the liquid, and the temperature of the liquid rises rapidly, so the ultrasonic treatment is performed for a short time and multiple times.

[0028] In some preferred embodiments of the present application, after the mixing of the nanodrug solution and the soluble composite matrix material, the process further comprises drying and forming, specifically comprising: casting the obtained mixed solution on a glass plate, and drying into a film at 25-30 DEG C, and peeling off from the glass plate.

[0029] According to a third aspect of the present application, there is provided a use of the drug-loaded guided periodontal tissue regeneration membrane of the first aspect or prepared by the preparation method of the second aspect in the preparation of a material for guided periodontal tissue regeneration.

[0030] In some preferred embodiments of the present application, the drug-loaded guided periodontal tissue regeneration membrane can be circular, square, etc., and when it is circular, the diameter is 4-8 mm, preferably 6 mm.

[0031] The present application has the following beneficial effects:

[0032] The raw materials of the present application are easy to obtain, the preparation method is simple, and the prepared drug-loaded guided periodontal tissue regeneration membrane has good biocompatibility; when applied to the periodontal region, the drug-loaded guided periodontal tissue regeneration membrane can enhance the drug permeability, thereby achieving the effects of high-efficiency antibiosis and inflammation relief. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 It is a preparation flowchart of the drug-loaded guided periodontal tissue regeneration membrane (MTZ-NPs BBM) of Example 1.

[0034] Figure 2 It is an appearance diagram of the metronidazole nanoparticle (MTZ-NPs) solution of Example 1.

[0035] Figure 3 It is a particle size distribution diagram of the metronidazole nanoparticle (MTZ-NPs) of Example 1.

[0036] Figure 4 It is a potential diagram of the metronidazole nanoparticle (MTZ-NPs) of Example 1.

[0037] Figure 5 It is a physical diagram of the MTZ-NPs BBM of Example 1.

[0038] Figure 6 It is a particle size distribution diagram of the particles in the MTZ-NPs BBM of Example 1.

[0039] Figure 7Zeta potential plot of the particles in the MTZ-NPs BBM of Example 1;

[0040] Figure 8 Stress-strain plot of the MTZ-NPs BBM of Example 1;

[0041] Figure 9 Infrared spectrum of the MTZ-NPs BBM of Example 1;

[0042] Figure 10 Differential scanning calorimetry plot of the MTZ-NPs BBM of Example 1;

[0043] Figure 11 X-ray diffraction plot of the MTZ-NPs BBM of Example 1;

[0044] Figure 12 Static water contact angle histogram of the MTZ-NPs BBM of Example 1;

[0045] Figure 13 Results of in vitro release of MTZ from the MTZ-NPs BBM of Example 1;

[0046] Figure 14 Results of in vitro cytotoxicity evaluation of the MTZ-NPs BBM of Example 1;

[0047] Figure 15 Results of periodontal probing depth evaluation of the MTZ-NPs BBM of Example 1 in a rat experimental periodontitis model;

[0048] Figure 16 Results of sulcus bleeding index evaluation of the MTZ-NPs BBM of Example 1 in a rat experimental periodontitis model;

[0049] Figure 17 Results of histological evaluation of the MTZ-NPs BBM of Example 1 in a rat experimental periodontitis model;

[0050] Figure 18 Results of inflammatory cell semi-quantitative scoring of the MTZ-NPs BBM of Example 1 in a rat experimental periodontitis model;

[0051] Figure 19 Actual photograph of the C6-NPs BBM;

[0052] Figure 20 Results of in vitro cytotoxicity evaluation of the C6-NPs BBM;

[0053] Figure 21 Results of fluorescence uptake of the C6-NPs BBM after 0.5 h co-incubation with L929 cells;

[0054] Figure 22 To determine the extent of C6-NPs uptake by cells at different time points. DETAILED DESCRIPTION

[0055] The present application will be further described in the following by specific examples. The raw materials, reagents or apparatus used in the examples and comparative examples are commercially available or can be obtained by prior art methods unless otherwise specified. The test or test method is a conventional method in the art unless otherwise specified.

[0056] The normal temperature referred to below is 25-30°C unless otherwise specified.

[0057] Example 1

[0058] This example prepared a drug-loaded guided periodontal tissue regeneration membrane, and the specific process is as follows:

[0059] (1) Preparation of nanoparticles by solvent evaporation method

[0060] Prepare a mixed aqueous solution of 1.5% (w / v) 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CD) and 2% (w / v) polyvinyl alcohol (PVA 05-88), filter through a 0.8 μm filter membrane, and take 20 mL as the water phase.

[0061] Precisely weigh 100 mg of metronidazole (MTZ) and 50 mg of poly(lactic-co-glycolic acid) (PLGA, PLA:PGA=75:25, Mw=23000) into a mixed solution of 10 mL of acetone:ethanol (4:1, v / v) as the organic phase.

[0062] Slowly inject the above organic phase into the water phase, and ultrasonicate in an ice water bath (set the amplitude rod to 6 mm, power to 180 W, interval of 3 s), 10 min / time, for a total of 30 min. At room temperature, magnetically stir at 450 rpm until the organic solvent is completely volatilized to obtain metronidazole nanoparticles (MTZ NPs). Dilute with deionized water to 20 mL to obtain a metronidazole nanoparticle (MTZ NPs) solution.

[0063] (2) Preparation of drug-loaded guided periodontal tissue regeneration membrane by solvent casting method

[0064] Accurately weigh a certain amount of polyvinyl alcohol (PVA 17-88) and polyvinylpyrrolidone (PVP-k30) and add them to 20 mL of the above MTZ NPs solution to make the concentration of the matrix material (PVA: PVP = 8:2, m / m) 9% (w / v), stir at 450 rpm until completely dissolved, and pour the entire solution on a glass plate (100x100 mm). Dry the glass plate on which the matrix solution is cast at room temperature, and peel off the dried and shaped film from the glass plate to obtain the drug-loaded guided periodontal tissue regeneration film MTZ-NPs BBM, The thickness of the film is 159 μm.

[0065] The preparation process of the film of the present embodiment is shown in the following schematic diagram: Figure 1

[0066] The appearance of the metronidazole nanoparticles (MTZ-NPs) solution prepared in step (1) was observed, and the results are shown in the following figure: Figure 2 It can be seen from the figure that the appearance of the MTZ-NPs solution is light blue opalescent. Figure 2

[0067] The metronidazole nanoparticles (MTZ-NPs) prepared in step (1) were observed under a transmission electron microscope, and it was observed that the MTZ-NPs were spherical, with a particle size in the nanometer range, indicating that the nanoparticles were successfully prepared.

[0068] Figure 3 The particle size distribution of the metronidazole nanoparticles (MTZ-NPs) in the metronidazole nanoparticle (MTZ-NPs) solution prepared in step (1) is shown in the following figure. The particle size D 90 of the MTZ-NPs is 262.60 nm, and the PDI is 0.112, indicating that the MTZ-NPs have a small particle size and a narrow particle size distribution.

[0069] Figure 4 The potential of the metronidazole nanoparticles (MTZ-NPs) in the metronidazole nanoparticle (MTZ-NPs) solution prepared in step (1) is shown in the following figure. It can be observed from the figure that the Zeta potential of the MTZ-NPs is -4.40 mV, indicating that the nanoparticles are negatively charged.

[0070] The appearance, transparency, and smoothness of the MTZ-NPs BBM prepared in the present embodiment were observed by the naked eye, and the results are shown in the following figure: Figure 5 It can be observed from the figure that the appearance of the MTZ-NPs BBM is colorless and transparent, with a smooth and flat surface, indicating that the film matrix has good compatibility and can be successfully prepared by the method of the present embodiment.

[0071] ​​The particle size distribution range, polydispersity index (PDI) and Zeta potential of the MTZ-NPs BBM prepared in this embodiment before and after reconstitution were measured. The method was as follows: an appropriate amount of nanoparticle solution was diluted with deionized water by an appropriate multiple, and dynamic light scattering technology was used to measure the nanoparticle size, PDI and Zeta potential. The MTZ-NPs BBM was determined by the same method after the film was dissolved.

[0072] Figure 6 The particle size distribution graph of the MTZ-NPs BBM prepared in Example 1 was shown in the figure. It could be observed from the figure that the particle size D 90 was 402.40 nm, and the PDI was 0.131. Although the particle size distribution range and PDI of the system increased slightly, the reason might be that the viscosity of the system increased due to the addition of PVA and PVP during the preparation of the film, and the nanoparticles tended to aggregate. At the same time, it also indicated that the nanoparticles were embedded in the polymer matrix. However, the MTZ-NPs before and after the preparation of the BBM had a small particle size (<450 nm) and a narrow particle size distribution (PDI <0.2), and the nanoparticles were stably present in the film matrix.

[0073] Figure 7 The Zeta potential graph of the MTZ-NPs after the dissolution of the MTZ-NPs BBM prepared in Example 1 was shown in the figure. The Zeta potential of the MTZ-NPs loaded on the film was -4.22 mV, and the potential of the NPs before and after the preparation of the BBM had little difference, indicating that the nanoparticles were stably present in the film matrix.

[0074] The average weight and average thickness of the MTZ-NPs BBM prepared in this embodiment were measured to investigate the repeatability of the preparation process. The average weight was (5.15±0.21) mg, and the RSD was 3.98% (n=10). The average thickness was (159.3±3.1) μm, and the RSD was 1.92% (n=3). It could be known that the deviation range of the average weight and thickness of the film was small, indicating that the process was repeatable.

[0075] The Young's modulus, tensile strength, elongation at break and folding endurance were tested by tensile test to evaluate the mechanical properties of the MTZ-NPs BBM prepared in this embodiment.

[0076] Figure 8The stress-strain diagram of the MTZ-NPs BBM prepared in this embodiment is shown. The tensile strength of the MTZ-NPs BBM is 19.31 MPa and the elongation at break is 273.53%. The tensile strength of the unloaded blank BBM is 24.63 MPa and the elongation at break is 177.85%. Compared with the Blank BBM, the MTZ-NPs BBM has a lower tensile strength and a higher elongation at break. This may be because the addition of the drug affects the formation of intermolecular hydrogen bonds between PVA (-OH) and PVP (C=O) molecules, disrupting the polymer molecular chain entanglement and thus reducing the tensile strength. The prepared BBM has high mechanical strength, elasticity, and flexibility, and can be used for periodontal local drug delivery.

[0077] The presence forms of metronidazole and the carrier material in the MTZ-NPsBBM prepared in Example 1 were determined by infrared spectroscopy, differential scanning calorimetry and X-ray diffraction; the hydrophilicity and hydrophobicity of the membrane were characterized by contact angle measurement.

[0078] Figure 9 The infrared spectrum of the MTZ-NPs BBM prepared in Example 1 is shown; the infrared spectrum of metronidazole shows its characteristic absorption peak at 3220 cm⁻¹. -1 (OH vibration), 3100cm -1 (=CH vibration), 1535cm -1 (-NO2 asymmetric stretching vibration) and 1074 cm -1 (CO vibration); from Figure 9 Blank BBM and MTZ-NPs BBM can be observed to be between 3385 and 3394 cm. -1 The red shift of the characteristic absorption peak of metronidazole indicates that metronidazole may form intermolecular hydrogen bonds with PVA and PVP, suggesting good compatibility between metronidazole and the polymers. The characteristic peak of metronidazole in MTZ-NPs BBM ranges from 1535 to 1074 cm⁻¹. -1 The peak intensity weakens, and some characteristic peaks even almost disappear in the spectrum, which may be due to the low amount of metronidazole in the membrane or the metronidazole being encapsulated in the nanoparticles and membrane matrix.

[0079] Figure 10 This is a differential scanning calorimetry (DSC) curve of the MTZ-NPs BBM prepared in Example 1. It can be observed from the figure that the metronidazole endothermic peak (160℃) in the MTZ-NPs BBM curve disappears, indicating that metronidazole may be dispersed in the film in an amorphous state.

[0080] Figure 11X-ray diffraction pattern of MTZ-NPs BBM prepared in Example 1; from the figure, it can be observed that there is no metronidazole diffraction peak (12.4° and 14.0°) in MTZ-NPs BBM, indicating that metronidazole can be dispersed in the film in an amorphous state.

[0081] Figure 12 Static water contact angle histogram of MTZ-NPs BBM prepared in Example 1; from the figure, it can be observed that the static water contact angle of Blank BBM and MTZ-NPs BBM is less than 90°, indicating that the prepared BBM has good hydrophilicity and wettability; compared with Blank BBM, the static water contact angle of MTZ-NPs BBM is significantly reduced (p<0.05), indicating that the addition of metronidazole increases the hydrophilicity of BBM. Figure 12

[0082] The in vitro release behavior of MTZ-NPs BBM prepared in this example was studied by dialysis bag method. MTZ-NPs BBM (equivalent to about 2.5 mg of metronidazole) and 0.5 mL of artificial saliva (8.00 g of NaCl, 0.19 g of KH2PO4 and 2.38 g of Na2HPO4 were weighed and dissolved in 1000 mL of deionized water, and the pH was adjusted to 6.8) were placed in a pre-treated dialysis bag (MW 7000), the two ends of the dialysis bag were tightly tied, and then placed in a 50 mL centrifuge tube containing 10 mL of artificial saliva, and then placed in a shaking incubator at a temperature of (37±1) °C and a shaking frequency of 100 rpm. At the predetermined time point, 2 mL of release medium was taken, and immediately 2 mL of artificial saliva at the same temperature was supplemented. The concentration of metronidazole was determined by high performance liquid chromatography (mobile phase: methanol-water (20:80, v / v); detection wavelength: 320 nm; flow rate: 1.0 mL·min -1 ; column temperature: 30 °C; injection volume: 10 μL), the cumulative release percentage Q was calculated, the release curve was drawn, and the release kinetics was fitted.

[0083]

[0084] (In the formula: Q—cumulative release percentage, %; V s —sample volume, mL; V0—release medium volume, mL; C i —drug concentration in the release medium at the i th sampling time, μg·mL -1 ; M—total drug amount in the dialysis bag at time 0, μg; n—number of sampling times)

[0085] Figure 13 ​Results of in vitro release of MTZ from MTZ-NPs BBM prepared in this example (n = 6); the control group MTZ released rapidly, 47% in 5 min, 70% in 15 min, and almost completely in 1 h. The release of MTZ fitted a first-order kinetic model (Q = 89.3955(1-e -0.1198t ), R 2 = 0.9426). In contrast, the MTZ-NPs BBM had a lower release rate, 17% in 5 min, the initial release was due to the diffusion of free metronidazole to the surface of the film during the film drying preparation process, when the film was in contact with artificial saliva, the free metronidazole on the surface of the film was released rapidly. The release of MTZ-NPs BBM fitted a first-order kinetic model (Q = 91.2228(1-e -0.0335t ), R 2 = 0.9966), indicating that the release was a combination of diffusion and dissolution, and the amount of drug released was proportional to the concentration of the remaining drug in the preparation. The dissolution of the soluble matrix material (PVA and PVP) affected the drug release, and the release of metronidazole may be delayed due to drug-polymer interaction. Water penetrates and swells, the MTZ-NPs BBM dissolves, the MTZ-NPs are released from the film, the nanoparticles are rapidly taken up by cells, and the drug is released intracellularly.

[0086] The L929 cell line (mouse fibroblasts) was selected as the experimental object of cytotoxicity, and the CCK-8 method was used to evaluate the in vitro cytotoxicity of the MTZ-NPs BBM prepared in this example (MTZ concentration 114.2-570.9 μg·mL -1 ).

[0087] Figure 14 Results of in vitro cytotoxicity evaluation of MTZ-NPs BBM prepared in this example (n = 3); it can be observed from the figure that the cell survival rate of each group is > 70%, there is no cytotoxicity, and there is no significant difference between groups of different concentrations, indicating that the MTZ-NPs BBM has good biocompatibility.

[0088] A rat experimental periodontitis model was established by silk ligation combined with high-sugar viscous diet, and the effectiveness of the film in regulating periodontal inflammation and promoting periodontal tissue healing was evaluated by periodontal indexes such as periodontal probing depth and sulcus bleeding index, and histological analysis, wherein the rats were specifically 30 SPF level SD rats aged 5-6 weeks, half male and half female, weighing 120-150 g, purchased from Guangdong Medical Laboratory Animal Center (Experimental Animal Qualification Certificate No. SCXK (Guangdong) 2022-0002).

[0089] The MTZ group was intragastrically administered metronidazole solution ( Before use, the tablets were configured into 0.348 mg·mL -1 solution, and 0.87 mg·kg-1 MTZ BBM group was given MTZ BBM at the gum margin of the first maxillary molar of rats (membrane content measured by high performance liquid chromatography, 0.87 mg / kg dose of MTZ was administered); MTZ-NPs BBM group was given MTZ-NPs BBM at the gum margin of the first maxillary molar of rats (membrane content measured by high performance liquid chromatography, 0.87 mg·kg -1 MTZ-NPs BBM group was given MTZ-NPs BBM at the gum margin of the first maxillary molar of rats (membrane content measured by high performance liquid chromatography, 0.87 mg / kg dose of MTZ was administered); MTZ-NPs BBM group was given MTZ-NPs BBM at the gum margin of the first maxillary molar of rats (membrane content measured by high performance liquid chromatography, 0.87 mg·kg

[0090] Figure 15 The results of evaluation of probing depth in the experimental periodontitis model of rats by the MTZ-NPs BBM prepared in this example (n = 6, ****p < 0.0001, *p < 0.05). Probing depth (PD) is used to assess the depth of the periodontal pocket and check the level of periodontal attachment, etc. From Figure 16 It can be seen that after treatment, the probing depth of the MTZ-NPs BBM group is reduced and significantly lower than that of the model group (p < 0.05); the probing depth of the MTZ group and the MTZ BBM group has no statistical difference compared with the model group, indicating that local administration of MTZ-NPs BBM in the periodontal area can relieve inflammation and promote the recovery of periodontal tissue, and its treatment effect is better than that of oral administration of metronidazole tablets and local application of metronidazole-loaded membrane (MTZ BBM) in the periodontal area.

[0091] Figure 16 The results of evaluation of gingival sulcus bleeding index in the experimental periodontitis model of rats by the MTZ-NPs BBM prepared in this example (n = 6, ****p < 0.0001, *p < 0.05). Gingival sulcus bleeding index (BI) is an index for evaluating inflammatory conditions. Normal healthy gums do not bleed after probing. If bleeding occurs after probing, it indicates inflammation and loss of periodontal attachment. The scoring criteria for gingival sulcus bleeding index are as follows: 0 = healthy gums, no bleeding after probing; 1 = mild inflammation of gums, no bleeding after probing; 2 = mild inflammation of gums; color change, no edema, pinpoint bleeding after probing; 3 = moderate inflammation of gums; color change, mild edema, bleeding after probing but still in the gingival sulcus; 4 = severe inflammation of gums; color change, severe edema, bleeding and overflow of gingival sulcus; 5 = severe inflammation of gums; color change, severe edema, ulcer, spontaneous bleeding and overflow of gingival sulcus after probing. From Figure 16It can be seen that after administration of the treatment, the sulcus bleeding index of the MTZ-NPs BBM group is reduced, and is significantly lower than that of the model group (p<0.05); the sulcus bleeding indexes of the MTZ group and the MTZ BBM group have no statistical difference compared with the model group, indicating that the MTZ-NPs BBM periodontal local administration can relieve inflammation and promote the recovery of periodontal tissue, and its treatment effect is better than that of oral metronidazole tablets and periodontal local metronidazole film.

[0092] Figure 17 The histological evaluation results of the MTZ-NPs BBM prepared in this example in a rat experimental periodontitis model (yellow arrow indicates inflammatory cells, red arrow indicates hyperemia, and blue arrow indicates cementum resorption). It can be observed from the figure that the periodontal tissue structure of the normal group (Control) is complete, there is no inflammatory cell infiltration, the periodontal membrane fibers are arranged in order, and there is no destruction (Fig. A, A-1, A-2). The periodontal tissue of the model group (Model) is hemorrhagic and edematous, the capillaries are increased, and there is inflammatory cell infiltration (Fig. B-1), the periodontal fibers are loose and arranged in disorder, the connective tissue attachment is lost, and cementum resorption can be observed (Fig. B-2). The MTZ group and the MTZ BBM group have inflammatory cell infiltration and fiber arrangement disorder, but the fiber arrangement of the MTZ BBM group is more orderly than that of the MTZ group (Fig. C and D). The MTZ-NPs BBM group has partial dispersed inflammatory cells, the inflammatory infiltration is the lightest, the inflammation is relieved, and the fiber arrangement is normal (Fig. E), and its treatment effect is better than that of oral metronidazole tablets (MTZ group) and periodontal local metronidazole film (MTZ BBM group). The reason may be that the MTZ-NPs BBM is directly absorbed through the mucosa, the MTZ-NPs effectively penetrate into the cells, kill intracellular bacteria, effectively relieve inflammation, and reduce periodontal tissue damage.

[0093] Figure 18 The semi-quantitative score results of inflammatory cells of the MTZ-NPs BBM prepared in this example in a rat experimental periodontitis model (n=6, ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05). It can be observed from the figure that the inflammatory cell score of the MTZ-NPs BBM group is significantly lower than that of the model group (p<0.001), the MTZ group (p<0.01), and the MTZ BBM group (p<0.05), indicating that the MTZ-NPs BBM can effectively relieve inflammation, and its treatment effect is better than that of oral metronidazole tablets and periodontal local metronidazole film.

[0094] Comparative Example 1

[0095] A metronidazole-loaded film was prepared in this comparative example, and the specific process was as follows:

[0096] Precisely weigh 100 mg metronidazole (MTZ) into 20 mL deionized water, accurately weigh a certain amount of polyvinyl alcohol (PVA 17-88) and polyvinylpyrrolidone (PVP-K30) into 20 mL of the above MTZ solution, so that the concentration of the matrix material (PVA: PVP = 8:2, m / m) is 9% (w / v), and stir at 450 rpm until completely dissolved; 20 mL of the above solution is cast on a glass plate (100x100 mm), and the glass plate with the cast matrix solution is dried at room temperature, and the dried film is peeled off from the glass plate to obtain a metronidazole-loaded film MTZ BBM,

[0097] Test Example

[0098] The fluorescence probe technology, combined with fluorescence microscopy observation and flow cytometry are used to evaluate the efficiency of the drug-loaded guide periodontal tissue regeneration film in crossing the cell membrane and entering the cell.

[0099] Coumarin-6 (0.06 mg) is used to replace metronidazole in Example 1, and a coumarin-6 nanoparticle-loaded regeneration film (C6-NPs BBM) is prepared by the method of Example 1, and the results are shown in Figure 19 The C6-NPs BBM is observed to have a green transparent appearance, a smooth and flat surface, and softness, indicating that the C6-NPs BBM is successfully prepared.

[0100] The CCK-8 method is used to evaluate the in vitro cytotoxicity of the C6-NPs BBM (C6 concentration is 47.0-141.1 ng·mL -1 ) in vitro: 1x10 5 cells / mL L929 cell suspension is inoculated into a 24-well plate, 1 mL per well, and incubated at 37°C, 5% CO2 for 24 h.

[0101] Figure 20 The in vitro cytotoxicity evaluation results of the C6-NPs BBM (n=3) are shown in the figure, and it can be observed that the cell survival rate of each group is >70%, without cytotoxicity, and there is no significant difference between different concentrations, indicating that the C6-NPs BBM has good biocompatibility.

[0102] The C6-NPs BBM is incubated with L929 cells for 0.5 h, and after incubation, 4% paraformaldehyde solution is used for fixation for 15 min, and DAPI staining is performed for 15 min. The sample is observed under a fluorescence microscope to observe the uptake of C6 by cells.

[0103] Figure 21The results of the fluorescence uptake of C6-NPs BBM and L929 cells co-incubated for 0.5 h are shown in the graph. It can be observed from the graph that both free coumarin-6 (Free C6) and C6-NPs can be taken up by the cells, and the uptake is mainly distributed in the cytoplasm. The fluorescence intensity of C6-NPs BBM is significantly higher than that of the Free C6 group, indicating that the drug in the form of nanoparticles is more easily taken up into the cells than in the free state, and the drug permeability is increased.

[0104] The fluorescence intensity of the cell uptake was quantitatively analyzed by flow cytometry. C6-NPs BBM and L929 cells were incubated for 1, 2 and 4 h, the culture medium was discarded, and the cells were washed twice with PBS. The cells were trypsinized, resuspended in 0.2 mL PBS, and the average fluorescence intensity of 10,000 cells was analyzed by flow cytometry to compare the uptake at different time points. The results are shown in Figure 22 It can be seen from the graph that both free C6 and C6-NPs can be taken up by the cells, and the uptake of L929 cells increases with increasing uptake time. The cell uptake of C6-NPs BBM is significantly higher than that of the Free C6 group, indicating that the drug in the form of nanoparticles is more easily taken up into the cells than in the free state, and the drug permeability is increased, and the cells are quickly taken up to saturation.

[0105] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods, and are included in the protection scope of the present application.

Claims

1. A drug-loaded guided periodontal tissue regeneration membrane, characterized in that: The drug-loaded membrane for guided periodontal tissue regeneration comprises a soluble composite matrix material and a nanodrug loaded in the soluble composite matrix material, the soluble composite matrix material is composed of polyvinyl alcohol and polyvinylpyrrolidone; the nanodrug is composed of a polylactic acid-glycolic acid copolymer modified by cyclodextrin and a pharmaceutically active ingredient wrapped by the polylactic acid-glycolic acid copolymer modified by cyclodextrin; the nanodrug is spheroid, and the average particle size is 50 nm to 450 nm; the pharmaceutically active ingredient comprises an anti-anerobic bacterium drug, and the anti-anerobic bacterium drug is a nitroimidazole, and at least one of metronidazole, tinidazole, ornidazole and secnidazole. The preparation method of the drug-loaded membrane for guided periodontal tissue regeneration comprises the following steps: dissolving the pharmaceutically active ingredient and the polylactic acid-glycolic acid copolymer in an organic solvent to obtain an organic phase, injecting the organic phase into an aqueous phase containing cyclodextrin, volatilizing the organic solvent, mixing the obtained nanodrug solution with a soluble composite matrix material, casting the obtained mixed solution on a glass plate, drying the mixed solution into a film at 25 DEG C to 30 DEG C, and peeling off the film from the glass plate.

2. The drug-loaded guided periodontal tissue regeneration membrane according to claim 1, characterized in that: The mass of the pharmaceutically active ingredient in the drug-loaded membrane for guided periodontal tissue regeneration accounts for 0.5% to 7.0% of the total mass of the drug-loaded membrane for guided periodontal tissue regeneration.

3. The drug-loaded guided periodontal tissue regeneration membrane according to claim 1, characterized in that: The thickness of the drug-loaded membrane for guided periodontal tissue regeneration is 100 µm to 250 µm.

4. The method for preparing the drug-loaded guided periodontal tissue regeneration membrane according to any one of claims 1 to 3, characterized in that, The preparation method comprises the following steps: dissolving the pharmaceutically active ingredient and the polylactic acid-glycolic acid copolymer in an organic solvent to obtain an organic phase, injecting the organic phase into an aqueous phase containing cyclodextrin, volatilizing the organic solvent, mixing the obtained nanodrug solution with a soluble composite matrix material, casting the obtained mixed solution on a glass plate, drying the mixed solution into a film at 25 DEG C to 30 DEG C, and peeling off the film from the glass plate.

5. The production method according to claim 4, characterized by, The mass ratio of the pharmaceutically active ingredient to the polylactic acid-glycolic acid copolymer is 0.1 to 1:

10.

6. The preparation method according to claim 4, characterized in that, The mass-volume ratio of the soluble composite matrix material to the nanodrug solution is 8 to 10:

100.

7. The production method according to claim 4, characterized by, The mass-volume fraction of the cyclodextrin in the aqueous phase is 1% to 2% (w / v), and the volume ratio of the organic phase to the aqueous phase is 1 to 2:

4.

8. The drug-loaded membrane for guided periodontal tissue regeneration according to any one of claims 1 to 3 is used for preparing a material for guided periodontal tissue regeneration.

9. The drug-loaded membrane for guided periodontal tissue regeneration according to any one of claims 1 to 3 is used for preparing a drug for treating periodontitis.

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