A tetracycline antibacterial drug and simvastatin-loaded core-shell microneedle patch, a preparation method and application thereof

Abstract

The application discloses a core-shell structure microneedle patch loaded with tetracycline antibacterial drugs and simvastatin, a preparation method and application thereof, and belongs to the field of biomedical materials. The base layer and the shell structure of the needle body of the microneedle patch are loaded with antibacterial drugs doxycycline, and the inner core structure contains polydopamine nanoparticles loaded with simvastatin. The microneedle patch can release the antibacterial drugs in stages, rapidly reduces the number of bacteria in the lesion locally in a short time through the first stage of explosive release of antibacterial drugs, and then controls the spread of inflammation; the second stage of slow release of antibacterial drugs inhibits the formation of new plaque biofilm for a long time, and slowly releases simvastatin, plays a role in promoting local bone formation, and realizes the synergistic effect of antibiosis and bone regeneration. In chronic periodontitis, especially in refractory periodontitis combined with systemic diseases, for example, chronic periodontitis combined with diabetes, the application has a good application prospect in treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical materials, and particularly relates to a core-shell structure microneedle patch loaded with tetracycline antibacterial drugs and simvastatin, a preparation method and application thereof. BACKGROUND

[0002] Periodontitis affects people of all ages, and its prevalence increases with age. It can cause inflammatory destruction of periodontal support tissue and even tooth loss. Dental plaque biofilm is the initiating factor of periodontal disease, and both local and systemic risk factors can promote the development of periodontitis. Local risk factors include dental calculus, plaque, and poor restorations, while systemic risk factors include smoking, obesity, and diabetes. Diabetes is an important factor that promotes periodontitis, and high blood sugar can exacerbate alveolar bone destruction and increase the pathogenicity of oral microorganisms.

[0003] Currently, mechanical methods for removing dental plaque biofilm are the conventional treatment for periodontitis, but the treatment effect is poor for periodontitis accompanied by diabetes, and diabetes can exacerbate the difficulty of treating periodontitis. Therefore, it is often necessary to assist in using antibiotics for treatment, but systemic use of antibiotics has the risk of developing drug resistance and gastrointestinal reactions. Local use of antibiotics is difficult to achieve satisfactory therapeutic effect due to the complex anatomy of teeth, deep periodontal pockets, and the flushing of saliva. Therefore, it is urgent to explore new strategies for treating periodontitis accompanied by diabetes.

[0004] Microneedles are a new type of transdermal drug delivery system composed of multiple micron-sized fine needle tips connected in an array on a base, which can efficiently deliver drugs into the skin by piercing the stratum corneum. The treatment strategy based on microneedles has broad application prospects in the field of biomedicine, which not only reduces the discomfort caused by traditional drug delivery methods, but also more accurately and effectively delivers drugs. Compared with traditional drug delivery methods, microneedles have the advantages of minimally invasive and painless, self-administration, improved patient compliance, and improved treatment effect. However, the microneedle patches disclosed in the prior art have the following defects: for example, the microneedle patches disclosed in the prior art are designed with a structure of backing and microneedle body, so they can only load single antibacterial drugs or drugs that promote bone formation, and cannot achieve the purpose of "two-pronged approach" of inhibiting periodontal pathogenic bacteria and promoting bone regeneration at the same time.

[0005] Based on the above reasons, the present application is proposed. SUMMARY

[0006] In view of the problems existing in the prior art, the present application aims to design a drug delivery system that can simultaneously exert anti-inflammatory and osteogenesis-promoting effects, thereby achieving a synergistic therapeutic effect on periodontitis accompanied by diabetes, and improving the traditional drug delivery method to improve patient compliance and solve problems such as maintenance of drug efficacy concentration and gastrointestinal side effects.

[0007] The first aspect of the present application aims to provide a core-shell microneedle patch loaded with tetracycline antibacterial drugs and simvastatin.

[0008] The second aspect of the present application aims to provide a preparation method of the core-shell microneedle patch loaded with tetracycline antibacterial drugs and simvastatin according to the first aspect of the present application.

[0009] The third aspect of the present application aims to provide the use of the core-shell microneedle patch loaded with tetracycline antibacterial drugs and simvastatin according to the first aspect of the present application in the preparation of a drug and / or drug delivery system for treating periodontitis with diabetes.

[0010] The fourth aspect of the present application aims to provide a drug and / or drug delivery system comprising the core-shell microneedle patch loaded with tetracycline antibacterial drugs and simvastatin described above.

[0011] In order to achieve the above-mentioned purposes, the technical solution of the present application is:

[0012] The first aspect of the present application provides a core-shell microneedle patch loaded with tetracycline antibacterial drugs and simvastatin, the microneedle patch comprising a substrate and a microneedle body arranged on the substrate, the microneedle body having a core-shell structure; wherein the shell of the microneedle body and the substrate are both loaded with tetracycline antibacterial drugs; the inner core of the microneedle body comprises simvastatin-loaded polydopamine nanoparticles (Sim@PDA).

[0013] Further, in the above technical solution, the microneedle body is conical or pyramidal in structure; the height of the microneedle body is 100-1000 μm, preferably 600-900 μm; the tip of the microneedle body is sharp, with a tip radius of less than 15 μm, preferably less than 10 μm; the center-to-center distance between adjacent microneedles is 50-1000 μm, preferably 300-800 μm; and the substrate is perpendicular to the microneedle body.

[0014] Further, in the above technical solution, when the microneedle body is conical, the diameter of the base circle is 100-600 μm, preferably 300-500 μm; and when the microneedle body is pyramidal in structure, the side length of the base polygon is 200-500 μm.

[0015] Further, the above technical solution, the micro-needle needle body surface is a threaded structure, which can effectively disperse stress concentration and reduce the risk of transverse fracture under axial pressure; at the same time, the surface area of the needle body is significantly increased, thereby greatly increasing the effective contact area and effectively improving the drug loading capacity; in addition, the threaded structure of the needle body is similar to the surface structure of a "screw", which can produce a "drilling effect", reduce the vertical penetration force, thereby reducing the penetration resistance, and more effectively penetrate the tissue without falling off after penetration, thereby improving the success rate of drug delivery.

[0016] Further, the above technical solution, the micro-needle needle body, also known as a micro-needle array, preferably adopts a 10x10 square array, and the three-dimensional size and the number of micro-needles used can be flexibly adjusted according to the medication location and the required dose.

[0017] Further, the above technical solution, the materials for preparing the shell and the core of the micro-needle needle body further include an in-vivo-dissolvable polymer material; the in-vivo-dissolvable polymer material is at least one of hyaluronic acid or its salt, polyvinyl alcohol, chitosan, gelatin, sodium carboxymethyl cellulose, polyvinyl pyrrolidone, chondroitin sulfate, etc. The in-vivo-dissolvable polymer materials for preparing the shell and the core of the micro-needle needle body can be the same or different.

[0018] Further, the above technical solution, in some embodiments of the present application, the in-vivo-dissolvable polymer material for preparing the shell of the micro-needle needle body is polyvinyl alcohol; in other embodiments of the present application, the in-vivo-dissolvable polymer material for preparing the core of the micro-needle needle body is polyvinyl pyrrolidone.

[0019] Preferably, the above technical solution, in some embodiments of the present application, the polyvinyl pyrrolidone is at least one of polyvinyl pyrrolidone-K30, polyvinyl pyrrolidone-K90, etc.

[0020] Further, the above technical solution, the in-vivo-dissolvable polymer material for preparing the shell of the micro-needle needle body has a molecular weight range of 10-200 kDa.

[0021] Further, the above technical solution, the in-vivo-dissolvable polymer material for preparing the core of the micro-needle needle body has a molecular weight range of 1-10 kDa.

[0022] Further, the above technical solution, in some embodiments of the present application, the tetracycline antibacterial drug includes at least one of minocycline, doxycycline, or other tetracyclines, etc.

[0023] Further, the above technical solution, in some embodiments of the present application, the raw material for preparing the substrate includes a soluble biocompatible material.

[0024] Further, the soluble biocompatible material is at least one of hyaluronic acid or its salts (e.g. sodium salt), chitosan, agarose, alginate, maltose, galactose, fructose, polylactic acid, polyglycolic acid, polyvinyl alcohol, poly-epsilon-caprolactone (PCL), polytrimethylene carbonate (PTMC), poly-p-dioxanone (PPDO), polyamino acid derivative carbonate (PDTE), polyortho ester (POE), collagen, gelatin, silk fibroin, sodium carboxymethyl cellulose, chondroitin sulfate, polyvinylpyrrolidone, etc.

[0025] Preferably, in some embodiments of the present application, the soluble biocompatible material is silk fibroin.

[0026] Further, the Sim@PDA is spherical with an average particle size of 200-300 nm.

[0027] A second object of the present application is to provide a preparation method of the core-shell microneedle patch loaded with tetracycline antibacterial drugs and simvastatin as described above, comprising the following steps:

[0028] Preparation of the microneedle shell: mix tetracycline antibacterial drugs and solution 1 of in-vivo soluble polymer material according to the ratio to obtain a shell matrix solution; then inject an appropriate amount of the shell matrix solution into a microneedle mold, centrifuge until fully filled, and dry to obtain the microneedle shell;

[0029] Preparation of the microneedle core: mix Sim@PDA nanoparticles and solution 2 of in-vivo soluble polymer material according to the ratio to obtain a core matrix solution; then inject an appropriate amount of the core matrix solution into the microneedle mold containing the microneedle shell, centrifuge until fully filled; repeat the injection and centrifugation process 0-3 times; and finally dry to obtain the microneedle core;

[0030] Preparation of the substrate: mix tetracycline antibacterial drugs and a soluble biocompatible material solution according to the ratio to obtain a substrate solution; then lay an appropriate amount of the substrate solution on the microneedle mold containing the microneedle shell and core, vacuum dry, and demold to obtain the core-shell microneedle patch loaded with tetracycline antibacterial drugs and simvastatin.

[0031] Further, the concentration of the in-vivo soluble polymer material solution 1 is (5-15) g: 100 mL. The viscosity and concentration can be adjusted within a certain range to meet the mechanical strength required by the needle body. For example, if the molecular weight is small and the viscosity is low, the concentration can be higher; similarly, if the molecular weight is large and the viscosity is high, the concentration can be lower. The solute in the in-vivo soluble polymer material solution 1 is at least one of hyaluronic acid or its salts, polyvinyl alcohol, chitosan, gelatin, sodium carboxymethyl cellulose, polyvinylpyrrolidone, chondroitin sulfate, etc.; and the solvent is deionized water.

[0032] Further, in the above technical solution, the ratio of the tetracycline antibacterial drug to the soluble polymer material solution 1 in the shell matrix solution is (50-100) mass parts: (1-10) volume parts; in a preferred embodiment of the present application, the ratio of the tetracycline antibacterial drug to the soluble polymer material solution 1 in the shell matrix solution is 60 mass parts: 2 volume parts; wherein the mass parts and volume parts are based on mg:mL.

[0033] Further, in the above technical solution, the concentration of the in-vivo soluble polymer material solution 2 is (40-60) g:100 mL. The viscosity and concentration can be adjusted within a certain range to meet the mechanical strength required by the needle body. For example, when the molecular weight is small and the viscosity is low, the concentration can be high; similarly, when the molecular weight is large and the viscosity is high, the concentration can be low. The solute in the in-vivo soluble polymer material solution 2 is at least one of hyaluronic acid or its salts, polyvinyl alcohol, chitosan, gelatin, sodium carboxymethyl cellulose, polyvinylpyrrolidone, and chondroitin sulfate; and the solvent is deionized water.

[0034] Further, in the above technical solution, the ratio of the Sim@PDA nanoparticles to the in-vivo soluble polymer material solution 2 in the core matrix solution is (100-200) mass parts: (5-20) volume parts; in a preferred embodiment of the present application, the ratio of the Sim@PDA nanoparticles to the in-vivo soluble polymer material solution 2 in the core matrix solution is 150 mass parts: 10 volume parts; wherein the mass parts and volume parts are based on mg:mL.

[0035] Further, in the above technical solution, the concentration of the soluble biocompatible material solution is 5-15 g / mL.

[0036] Further, in some embodiments of the above technical solution, the Sim@PDA nanoparticles are prepared as follows:

[0037] The polydopamine (PDA) nanoparticles are added to the simvastatin solution according to the ratio, and the reaction is stirred in the dark for 5-12 h; after the reaction is completed, the product is centrifuged, the precipitate is collected, washed, and dried to obtain the Sim@PDA nanoparticles.

[0038] Further, in the above technical solution, the mass ratio of the PDA nanoparticles to simvastatin is 1:1-1:2, and the mass ratio of simvastatin does not exceed twice that of the PDA nanoparticles as the carrier. The free simvastatin not loaded on the nanoparticles will be removed in the centrifugal washing step.

[0039] Further, the PDA nanoparticles are spherical, have uniform particle size, and have an average particle size of 150-250 nm. After simvastatin is added, the shape of the Sim@PDA nanoparticles does not change significantly, and the nanoparticles remain spherical.

[0040] Further, the PDA nanoparticles are prepared according to the following method:

[0041] Dopamine hydrochloride and deionized water are mixed in a predetermined ratio, and stirred and dissolved uniformly to obtain a dopamine hydrochloride solution. Then, a sodium hydroxide solution is slowly added to the dopamine hydrochloride solution in a predetermined ratio at 40-60°C. After the addition is completed, the obtained reaction solution is stirred in the dark for 20-30 h. After the reaction is completed, the obtained product is centrifuged, and the precipitate is collected, washed, and freeze-dried to obtain the PDA nanoparticles.

[0042] Preferably, in one embodiment of the present application, the amount ratio of dopamine hydrochloride to deionized water is 1 mg:1 mL.

[0043] Preferably, in one embodiment of the present application, the concentration of the sodium hydroxide solution is 0.5-2 M.

[0044] Preferably, in one embodiment of the present application, the amount ratio of dopamine hydrochloride to the sodium hydroxide solution is 100 mg:1 mL.

[0045] Preferably, in one embodiment of the present application, the time for the reaction in the dark is 24 h.

[0046] Further, in the above technical solution, the concentration of the soluble biocompatible material solution is (5-15) g:100 mL.

[0047] Further, in the above technical solution, the amount ratio of the tetracycline antibacterial drug to the soluble biocompatible material solution in the base solution is (10-30) mass parts:(1-5) volume parts. In one preferred embodiment of the present application, the amount ratio of the tetracycline antibacterial drug to the soluble biocompatible material solution in the base solution is 20 mass parts:2 volume parts. The mass parts and volume parts are based on mg:mL.

[0048] In a third aspect, the present application provides a use of the Sim@tetracycline and simvastatin-loaded core-shell microneedle patch of the first aspect of the present application in the preparation of a drug and / or a drug delivery system for treating periodontitis with diabetes.

[0049] The fourth aspect of the present application aims to provide a medicine and / or a drug delivery system, comprising the core-shell microneedle patch loaded with tetracycline antibacterial drugs and simvastatin as described above.

[0050] The main raw materials used in the present application play the following roles in the present application:

[0051] Simvastatin (Sim) can promote mesenchymal stem cells to differentiate into bone cells, promote osteogenesis, and significantly inhibit osteoclastogenesis and bone resorption, ultimately reduce periodontitis and promote periodontal regeneration. However, Sim is an anionic lipophilic drug, and most of the drugs in oral treatment may be eliminated through liver and kidney circulation, which requires increasing the dosage of the drug to enable it to exert an effective concentration at the site of alveolar bone resorption. Such a high dose of Sim may be beneficial to bone formation, but it may have some adverse side effects on the whole body, such as liver and kidney adverse reactions. At the same time, the new bone needs a long time to form, and daily local injection will greatly increase the pain of the patient, and also greatly depends on the good compliance of the patient. Therefore, encapsulating Sim in PDA nanoparticles can not only maintain the good activity of the drug in promoting bone regeneration, but also achieve controlled and sustained drug delivery only in the local environment of the periodontal pocket. At the same time, the phenolic groups on the surface of the PDA nanoparticles can scavenge excess ROS at the inflammation site, thereby relieving local oxidative stress. Therefore, using PDA nanoparticles to load Sim not only serves as a drug delivery system, but also has the advantages of excellent sustained and controlled drug release and antioxidant properties, which can help to alleviate the inflammation of periodontitis. The main principles or mechanisms involved in the present application are as follows:

[0052] The present application selects a microneedle patch as a drug delivery carrier, and innovatively designs a microneedle patch with a core-shell structure to realize a biphasic release system of "fast-acting + long-acting": the needle body shell of the microneedle patch carries tetracycline antibacterial drugs, which can reach the highest concentration of drug release within two hours. The burst release of antibacterial drugs in a short time can maximize the initial bactericidal intensity, rapidly eliminate pathogenic microorganisms colonized on the root surface of infected teeth and block the formation of biofilms. The backing base structure of the microneedle patch carries antibiotics by loading silk fibroin, which can maintain an effective concentration at the local site for 14 days through slow and controlled drug release, thereby inhibiting the regeneration of residual bacteria, preventing recurrence, and promoting tissue regeneration. At the same time, the needle core of the microneedle patch contains polydopamine nanoparticles loaded with simvastatin, which has a promoting effect on the regeneration of alveolar bone. By using a double-barreled strategy, the microneedle patch can not only exert antibacterial and anti-inflammatory effects, but also promote periodontal tissue regeneration, thereby playing a synergistic therapeutic role in the treatment of periodontitis accompanied by diabetes.

[0053] Compared with the prior art, the present application has at least the following advantages or beneficial effects:

[0054] The application innovatively designs a microneedle patch with a core-shell structure, which comprises an outer shell capable of rapidly releasing tetracycline antibiotics in a short time, and an inner core containing simvastatin-loaded polydopamine nanoparticles, which plays a role in promoting alveolar bone regeneration, and slowly releases tetracycline antibiotics through the backing base material, thereby achieving long-acting antibacterial and preventing recurrence. The core-shell microneedle patch provided by the application not only highlights the use of the core-shell structure design to achieve the treatment sequence of "elimination first and regeneration second", but also controls infection and reduces inflammatory response for periodontitis with diabetes, and more importantly, promotes alveolar bone regeneration and reverses the course of periodontitis, thereby achieving the ultimate goal of improving the occlusal function of the patient. On the other hand, the degradation and swelling rates of the outer shell material and the backing material are different, so as to achieve a phased biphasic release system, that is, the synergistic mechanism of the rapid bacteria control of the burst layer and the long-acting bacteria inhibition of the slow release layer, which has the dual advantages of strong initial debridement and sustained prevention of recurrence.

[0055] The preparation method of the core-shell microneedle patch provided by the application has excellent biocompatibility of the raw materials, simple and easy-to-operate preparation method, is beneficial to clinical transformation, and has great clinical application value. BRIEF DESCRIPTION OF DRAWINGS

[0056] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.

[0057] Figure 1 Figure 1 is a graph of the measurement results of the hydration kinetic diameter of the Sim@PDA nanoparticles prepared in Example 1 by the nanoparticle size analyzer;

[0058] Figure 2 Figure 2 is a process flow diagram of the preparation of the PDA nanoparticles in Example 1; B is a transmission electron microscope graph of the PDA nanoparticles prepared in Example 1; C is a transmission electron microscope graph of the Sim@PDA nanoparticles prepared in Example 1; D is a scanning electron microscope graph of the PDA nanoparticles prepared in Example 1; E is a scanning electron microscope graph of the Sim@PDA nanoparticles prepared in Example 1;

[0059] Figure 3 Figure 3 is a comparison graph of the ultraviolet-visible absorption spectra of the Sim@PDA nanoparticles, the PDA nanoparticles and simvastatin prepared in Example 1;

[0060] Figure 4The FTIR spectrum comparison chart of Sim@PDA nanoparticles, PDA nanoparticles and Simvastatin raw material prepared in Example 1;

[0061] Figure 5 The basic morphology of the core-shell microneedle patch prepared in Example 1 was observed by using a body fluorescence microscope;

[0062] Figure 6 The scanning electron microscope image of the core-shell microneedle patch prepared in Example 1 is shown in A, and the scanning electron microscope image of a single needle body of the core-shell microneedle prepared in Example 1 is shown in B;

[0063] Figure 7 The cross-sectional scanning electron microscope image of a single needle body of the core-shell microneedle prepared in Example 1 is shown in A, the EDS point analysis result chart of the shell layer of the core-shell microneedle prepared in Example 1 is shown in B, and the EDS point analysis result chart of the core layer of the core-shell microneedle prepared in Example 1 is shown in C;

[0064] Figure 8 The cross-sectional C, O element distribution chart of a single needle body of the core-shell microneedle prepared in Example 1 is shown;

[0065] Figure 9 The fluorescence microscope image of the core-shell microneedle patch prepared in Example 1 is shown, wherein the core layer, the shell layer and the core-shell double layer are shown from left to right;

[0066] Figure 10 The optical microscope image of the porcine isolated gingival tissue after being punctured by the fluorescence-labeled core-shell microneedle is shown.

[0067] Figure 11 The drug concentration level of doxycycline released by the microneedle patch in vitro is shown, wherein A is the drug concentration level of doxycycline released by the shell layer, and B is the drug concentration level of doxycycline released by the base layer;

[0068] Figure 12 The in-vitro biological safety evaluation result chart of different microneedle patches is shown;

[0069] Figure 13 A is the inhibition zone test result chart of different microneedle patches after being co-cultured with Staphylococcus aureus for 7 days, B is the inhibition zone test result chart of different microneedle patches after being co-cultured with Porphyromonas gingivalis P. g for 7 days, C is the live and dead staining and the microscopic changes of bacteria under the action of microneedle patches observed by scanning electron microscope after different microneedle patches were co-cultured with Staphylococcus aureus for 7 days, and D is the live and dead staining and the microscopic changes of bacteria under the action of microneedle patches observed by scanning electron microscope after different microneedle patches were co-cultured with Porphyromonas gingivalis P. g for 7 days;

[0070] Figure 14ALP and ARS staining results of C3H / 10 T1 / 2 cells after stimulation with different microneedle patch extracts for 3 hours, osteogenic induction for 7 days and 14 days;

[0071] Figure 15 Comparison of RUNX2 and OCN test results in C3H / 10 T1 / 2 cells after 3 hours of stimulation with different microneedle patch extracts at 7 days of osteogenic induction.

[0072] Figure 16 The results of alizarin red staining analysis after q-PCR detection of the mRNA expression of osteogenic markers Ocn, Runx2, Alp, and Col1 in C3H / 10 T1 / 2 cells were obtained after stimulating them with different microneedle patch extracts for 3 hours and inducing osteogenic formation for 7 days.

[0073] Figure 17 Results of OGTT experiments performed on diabetic rats and healthy rats.

[0074] Figure 18 To establish a diabetes model, random blood glucose levels were measured in the tail vein of rats on days 3, 5, 7, and 10.

[0075] Figure 19 Image A shows the three-dimensional reconstruction and sagittal section image of the rat maxilla using Micro-CT; Image B shows the distance test results between the mesial and distal alveolar ridge crest and the cementoenamel junction (ABC-CEJ) of the first molar in the maxilla of SD rats.

[0076] in: Figures 12 to 17 The Control group involved was the negative control group, meaning that no intervention was given in the C3H / 10-T1 / 2 cell culture; the Blank-MNs group was the blank microneedle patch prepared in Comparative Example 3, which was co-incubated with C3H / 10-T1 / 2 cells for 24 hours. The blank microneedle patch contained only the matrix material of the synthetic microneedle patch and did not load any drugs; the SIM-MNs group was the microneedle patch prepared in Comparative Example 1, which contained Sim@PDA nanoparticles in its needle core in addition to the matrix material; the Doxy-MNs group was the microneedle patch prepared in Comparative Example 2, which contained doxycycline in the needle shell and backing base in addition to the synthetic matrix material; the Doxy / SIM-MNs group was the microneedle patch prepared in Example 1, which contained doxycycline in its needle shell and backing base and Sim@PDA nanoparticles in its core; the Gel group was minocycline hydrochloride ointment (trade name "Palio"), which is commonly used in clinical periodontitis treatment; and the Saline group was the physiological saline group. Detailed Implementation

[0077] The present application solves the pain point that current clinical treatment methods for periodontitis cannot effectively promote periodontal tissue regeneration and reverse the disease, whether through mechanical removal of dental plaque or through periodontal surgery. In addition, the outer layer of the core-shell microneedle structure of the present application is a water-soluble matrix, and the inner core is an organic solvent matrix. Compared with the prior art, the core-shell structure design can make the two drugs loaded in the inner core and the outer shell structure more stable, and the defect that part of the drug loaded in the inner core is dissolved into the outer shell structure does not occur. The microneedle patch of the present application can better achieve "programmed" release, and the core-shell structure microneedle of the present application has better application expansion: the outer shell structure of the core-shell structure of the present application is a water phase matrix, and the inner shell structure can load water-soluble drugs or fat-soluble drugs, and the release speed can be controlled by other means, such as responsive nanoparticles. In general, the microneedle patch of the present application focuses on periodontitis with diabetes disease model. Unlike simple periodontitis model, it has been clinically proven that periodontitis and diabetes can influence each other and aggravate the disease course. Periodontitis with diabetes has the characteristics of severe inflammation and difficult control. In addition to periodontal basic treatment, antibiotics and other drugs are often used clinically. Therefore, the microneedle patch proposed in the present application has better applicability for periodontitis with diabetes and provides more precise treatment strategies.

[0078] The present application realizes the release of antibacterial drugs and simvastatin with osteogenesis promotion effect in stages by designing a core-shell structure microneedle patch, thereby relieving the inflammation degree of periodontitis with diabetes and promoting the regeneration of periodontal tissue. The base layer and the outer shell structure of the needle body of the microneedle patch of the present application load the antibacterial drug doxycycline, and the inner core structure contains simvastatin-loaded polydopamine nanoparticles. The microneedle patch can release the antibacterial drug in stages. Through the first stage of explosive release of antibacterial drugs, the number of bacteria in the lesion is rapidly reduced in a short time, thereby controlling the spread of inflammation; the second stage slowly releases the antibacterial drug to inhibit the formation of new plaque biofilm for a long time, and slowly releases simvastatin to promote local osteogenesis, realizing the synergistic effect of antibiosis and bone regeneration. In chronic periodontitis, especially in refractory periodontitis combined with systemic diseases, such as chronic periodontitis combined with diabetes, it has good application prospect.

[0079] The present application will be further described in detail through the following implementation cases. The present implementation case is implemented on the premise of the present application technology, and the detailed implementation mode and specific operation process are given to illustrate the creativity of the present application, but the protection scope of the present application is not limited to the following implementation cases.

[0080] Various modifications in the precise arrangement of parts, as well as in the details thereof, can be made to the application within the spirit of the application, and with the scope of the application, as defined by the appended claims, in light of the above teachings. It is to be understood that the scope of the application is not limited to the processes, compositions or components specifically recited, but rather, the embodiments and examples are merely illustrative of specific ways to make and use the application.

[0081] For a better understanding of the present application, and not by way of limitation, all the numbers expressing quantities of components, percentages, and other numerical values used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the present application. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0082] The apparatus and materials used in the present application are commercially available or are commonly used in the art. The methods in the following examples are the conventional methods in the art, unless otherwise specified.

[0083] The molecular weight of the dopamine hydrochloride used in the following examples of the present application is 189.639; the CAS number is 62-31-7.

[0084] The CAS number of the simvastatin used in the following examples of the present application is 79902-63-9.

[0085] The CAS number of the doxycycline hydrochloride used in the following examples of the present application is 10592-13-9.

[0086] Example 1

[0087] A tetracycline antibiotic and simvastatin-loaded core-shell microneedle patch (Doxy / SIM-MNs, referred to as Doxy / SIM in the drawings) of the present embodiment has a length of 1 cm and a width of 1 cm, and comprises a substrate and a plurality of microneedle bodies arranged in an array on the substrate, the array being a 10x10 square array; each microneedle body has a core-shell structure and is conical in shape, has a clear thread structure on the surface of the needle body, a sharp needle tip less than 10 μm, a needle body height of 850 μm, a bottom diameter of 400 μm, and a center-to-center spacing of 700 μm between each microneedle; the substrate of the microneedle patch comprises silk fibroin and an antibacterial drug, the antibacterial drug being doxycycline; the needle body shell comprises polyvinyl alcohol and an antibacterial drug, the antibacterial drug being doxycycline; and the needle body core comprises polyvinylpyrrolidone and Sim@PDA.

[0088] The tetracycline antibiotic and simvastatin-loaded core-shell microneedle patch described above in the present embodiment is prepared by the following method, the steps being as follows:

[0089] (1) Preparation of silk fibroin

[0090] Weigh 20 mg of cocoon shell and cut into pieces, add to a 0.2 M Na2CO3 solution and heat to boiling for 20 min, adding deionized water as needed during the boiling process, and always maintaining the reaction system at 250 ml. After boiling, the cocoon is removed, rinsed with deionized water several times, and then placed again in a Na2CO3 solution, repeating the heating and boiling process. After rinsing, a crude silk fibroin extract is obtained. The crude silk fibroin extract obtained is dissolved in a 10 M LiBr solution, wherein the mass-volume ratio of the crude silk fibroin extract to the LiBr solution is 1 mg:20 mL; the silk fibroin fibers are fully dissolved by stirring for 4 h; the product obtained is filtered with a 300 mesh filter, then poured into a dialysis bag (12 kDa molecular weight cut-off) and dialyzed for 96 h, with the deionized water being changed every 4 h in the early stage and every 8 h in the later stage; after dialysis, the purified silk fibroin is obtained and stored by freeze-drying.

[0091] (2) Synthesis and preparation of Sim@PDA nanoparticles

[0092] Weigh 100 mg of dopamine hydrochloride, dissolve in 100 ml of deionized water, and stir until the solution changes from brown-yellow to light yellow. Place the mixed solution on a magnetic stirrer and heat to 50°C in an oil bath, and slowly add 1 mL of 1 M sodium hydroxide solution dropwise; after the addition is complete, the resulting reaction solution is stirred in the dark for 24 h, and the solution turns black after the reaction is complete. Centrifuge the solution at 11000 r / min for 15 min, and remove the supernatant. Repeat the washing and centrifugation process three times, collect the polydopamine (PDA) nanoparticles, freeze-dry, weigh, and store.

[0093] Weigh 40 mg of simvastatin and dissolve in 5 ml of anhydrous ethanol; after the simvastatin is completely dissolved, a simvastatin solution is obtained; weigh 30 mg of freeze-dried PDA nanoparticles and add to the simvastatin solution, and stir in the dark for 12 h; after the reaction is complete, centrifuge the resulting product at 11000 r / min for 15 min, and repeatedly wash the precipitate with anhydrous ethanol to remove the unloaded simvastatin until the supernatant changes from yellow to clear and transparent. Collect the precipitate, freeze-dry, and obtain Sim@PDA nanoparticles, which are weighed and stored for use.

[0094] (3) Preparation of core-shell microneedle patches

[0095] Firstly, 10 g of polyvinyl alcohol (PVA) was dissolved in 100 mL of deionized water to prepare a polyvinyl alcohol (PVA) solution with a concentration of 10 g / mL, and the weight average molecular weight (Mw) of the PVA was 14.5 KD; 60 mg of doxycycline hydrochloride was dissolved in 2 mL of the PVA solution to obtain a shell matrix solution; 50 μL of the shell matrix solution was injected into the microneedle mold to cover each micropore on the microneedle mold, and then the microneedle mold was placed in a high-speed centrifuge for centrifugation at 4200 rpm for 20 minutes; after centrifugation, the microneedle mold was dried at 28°C for 24 hours to obtain a microneedle shell.

[0096] 50 g of polyvinyl pyrrolidone K30 (PVP-K30) was dissolved in 100 mL of deionized water to prepare a polyvinyl pyrrolidone K30 (PVP-K30) solution with a concentration of 50 g / mL; 150 mg of Sim@PDA nanoparticles prepared in step (2) was resuspended in 10 mL of the PVP-K30 solution to obtain a core matrix solution; 100 μL of the prepared core matrix solution was added to the dried microneedle mold containing the microneedle shell, and centrifuged at 4200 rpm for 5 minutes, with the centrifugation temperature set to 4°C; the core matrix solution was repeatedly injected twice, and dried for 24 hours to obtain the needle body of the core-shell microneedle. Remove the excess PVP-K30 solution on the surface of the mold. 200 mg of silk fibroin lyophilized powder was dissolved in 2 mL of deionized water to prepare a 10% (w / v) silk fibroin solution, and 20 mg of doxycycline hydrochloride was dissolved in the above-mentioned 2 mL of silk fibroin solution with a concentration of 10 g / mL, and mixed uniformly as the base solution of the microneedle patch, and the dissolution process should not be shaken vigorously to avoid damaging the structure of the silk fibroin and accelerating gelation; 100 μL of the base solution was added to the surface of the dried microneedle mold, and vacuum was applied at a pressure of -0.1 MPa for 5 hours; finally, the mold was dried in a constant temperature and humidity dryer at 25°C and 5% humidity for 48 hours, and then demolded to obtain a complete core-shell microneedle.

[0097] Comparative Example 1

[0098] The core-shell microneedle patch loaded with simvastatin (SIM-MNs, referred to as SIM in the drawings) of this comparative example has the same structure as the core-shell microneedle patch loaded with tetracycline antibacterial drugs and simvastatin (Doxy / SIM-MNs) in Example 1, and the only difference is that the base and shell of the microneedle patch of this comparative example only contain matrix materials and do not contain antibacterial drugs, and the other parts of the structure and the preparation process are the same.

[0099] Comparative Example 2

[0100] A doxy simvastatin-loaded core-shell microneedle patch (Doxy-MNs, referred to as Doxy in the drawings) of the present comparative example has the same structure as the doxy / simvastatin-loaded core-shell microneedle patch Doxy / SIM-MNs of Example 1, except that the inner core of the microneedle patch of the present comparative example contains only the matrix material, and does not contain Sim@PDA. The other parts of the structure and the preparation process are the same.

[0101] Comparative Example 3

[0102] A blank microneedle patch (Blank-MNs, referred to as Blank in the drawings) of the present comparative example does not load any drug, and only contains the matrix material of the synthetic microneedle patch. The structure of the blank microneedle patch is the same as the doxy / simvastatin-loaded core-shell microneedle patch Doxy / SIM-MNs of Example 1, except that the inner core of the microneedle patch of the present comparative example does not contain Sim@PDA, and neither the base nor the shell contains the antibacterial drug.

[0103] Structure and performance characterization

[0104] (I) Structure characterization of Sim@PDA nanoparticles

[0105] The Sim@PDA nanoparticles prepared in step (2) of Example 1 were subjected to relevant analysis and detection. The Sim@PDA nanoparticles were dispersed in deionized water, and the sample concentration was controlled in a suitable range (scattered light intensity was 100 kcps-500 kcps). The sample was placed in a sample cell for particle size analysis. The Sim@PDA nanoparticles were dispersed with ethanol, 5 ml was added dropwise to a 100-mesh carbon film copper mesh, and after the sample was dried, transmission electron microscopy test was carried out under the condition of accelerating voltage of 80 Kv. The nanoparticles were uniformly dispersed in anhydrous ethanol solution, 20 μl was added dropwise on a silicon wafer, and after drying at room temperature, a 20 mA current was sprayed for 60 s with a gold spraying instrument. The microstructure of the nanoparticles was observed under a scanning electron microscope. The nanoparticle sample was analyzed by ultraviolet spectrophotometry, and the scanning wavelength was 200 nm-500 nm. The Sim@PDA nanoparticles were pressed into a thin sheet with a tablet press, and were placed in a sample chamber for Fourier infrared spectroscopy (FT-IR) test, and the scanning range was 4000 cm -1 ~500 cm -1 .

[0106] Figure 1 The Sim@PDA nanoparticles prepared in Example 1 have a diameter of about 244.4±3.4 nm.

[0107] From Figure 2 observation of the basic morphology and microstructure of Sim@PDA, the results show that the Sim@PDA prepared in Example 1 is spherical, and the particle size distribution is uniform.

[0108] From Figure 3 It can be seen that the Sim@PDA prepared in Example 1 has the same trend as PDA in the range of 200-400 nm, and the characteristic absorption peak at 290 nm has a trend of enhancement, indicating that due to the loading of simvastatin, the characteristic absorption peak of PDA has changed, and it is inferred that simvastatin is successfully modified on the PDA nanoparticles.

[0109] From Figure 4 It can be seen that the characteristic lactone carbonyl peak of simvastatin is 1698.59 cm -1 , the hydroxyl (O-H) stretching vibration peak of simvastatin is 3550.73 cm -1 , and multiple characteristic peaks in the range of 1300-1000 cm -1 prove that there is an ester group. In the infrared absorption spectrum of simvastatin-loaded polydopamine nanoparticles, the characteristic absorption peak of simvastatin disappears, and compared with the mesoporous polydopamine nanoparticles without drug loading, the absorption spectrum changes slightly, which may be due to the hydrogen bond formed between the carbonyl group of simvastatin and the carrier, indicating that simvastatin is successfully coated in the carrier material.

[0110] (II) Morphology characterization of microneedle patch

[0111] After preparing the microneedle patch according to Example 1, the morphology and structure of the microneedle patch were observed and analyzed.

[0112] First, the bright field morphology of the core-shell microneedle was observed under a stereoscopic fluorescence microscope. Nile red (NR) was used to label the outer shell of the microneedle, and rhodamine 123 (Rh123) was used to label the inner core of the microneedle. The fluorescence-labeled microneedle patch was adhered to an acrylic plate, and the acrylic plate was attached to an inclined plane holder. Under the stereoscopic fluorescence microscope, the wavebands of 510 nm ~ 560 nm and 455 nm ~ 490 nm were used to excite NR and Rh123 respectively, and the fluorescence distribution of the outer shell and the inner core of the entire core-shell microneedle patch was photographed respectively. Subsequently, a blade was used to cut a single microneedle along the horizontal and vertical angles, and the red and green fluorescence distribution of the cross-section and longitudinal section of the microneedle was photographed respectively under the stereoscopic fluorescence microscope.

[0113] The microstructure of the microneedle was observed under a scanning electron microscope. The microneedle was adhered to a silicon wafer, and a gold sprayer was used to spray gold for 45 seconds at a current of 20 mA to increase its conductivity. Finally, the gold-sprayed microneedle was placed in the scanning sample chamber, and the microstructure of the microneedle was observed under the condition of an acceleration voltage of 20 kV. The C, O, N, Cl and other different elements of the cross-section and longitudinal section of the microneedle were scanned and quantitatively analyzed to clearly observe the shell and core structure of the core-shell microneedle.

[0114] From Figure 5 It can be seen that the area of a single patch is about 0.5 cm2 , which consists of 10 x 10 microneedle array, Figure 9 The cross-section of single microneedle observed under fluorescence condition shows a core-shell structure. Figure 6 The microstructure of the core-shell microneedle was further observed by SEM. The whole microneedle patch consists of 100 microneedles. The single microneedle is sharp and conical. The diameter of the tip is less than 10 μm. The height of the conical needle is 850 μm. The diameter of the base is 400 μm. This design is very conducive to the penetration of the stratum corneum of the skin, achieving the purpose of minimally invasive drug delivery. Figure 7 and Figure 8 The cross-section of the microneedle was observed by SEM, and the elements of the shell and core of the microneedle were quantitatively analyzed by energy dispersive X-ray spectroscopy (EDS) and element mapping. As shown in the figure, the cross-section of the microneedle shows a clear layered structure. The EDS quantitatively analyzes the element composition of the cross-section of the microneedle. The shell is mainly composed of four elements: carbon (C), oxygen (O), nitrogen (N), and chlorine (Cl). The core is mainly composed of three elements: (C), oxygen (O), and nitrogen (N). The core does not contain chlorine, because only the shell is loaded with doxycycline hydrochloride, and the core does not contain it. This is exactly an indication that the core-shell structure has been successfully synthesized.

[0115] (III) Evaluation of the in vitro penetration ability of the microneedle patch

[0116] The microneedle patch prepared in Example 1 was vertically inserted into fresh porcine isolated gingiva. After pressing for 1 min, it was left for 20 min. The patch was removed, stained with crystal violet, and the penetration of the microneedle patch was observed under a microscope.

[0117] Figure 10 It is shown that the microneedle patch can effectively penetrate the skin, with a penetration success rate of 100 %.

[0118] (IV) In vitro drug release determination of microneedles

[0119] A gradient concentration of doxycycline hydrochloride solution was prepared. The absorbance value of doxycycline hydrochloride solution at a wavelength of 350 nm was determined by UV-vis, and the concentration was taken as the abscissa and the absorbance value was taken as the ordinate. The standard curve of doxycycline hydrochloride was drawn. The microneedle patch containing the doxycycline hydrochloride shell was prepared according to the method described in Example 1. The microneedle patch prepared in Example 1 was added to 6 ml of PBS buffer. Every 30 min, 600 μl of supernatant was taken and 600 μl of fresh PBS buffer was added to keep the total amount of release medium unchanged. After the collection was completed, the absorbance value of the supernatant at a wavelength of 350 nm was determined by UV-vis, and the cumulative release amount of doxycycline hydrochloride was calculated. The same method was used to determine the release curve of doxycycline hydrochloride in the microneedle base.

[0120] From Figure 11As can be seen from the middle A, the cumulative drug release amount of doxycycline in the microneedle body shell can reach 50% within 30 min, which can increase the concentration of antibiotics in the local environment in a short time, and play a role in quickly inhibiting bacteria. Figure 11 As can be seen from the middle B, the doxycycline in the backing silk fibroin can be slowly released within two weeks, thereby playing a long-acting antibacterial role and preventing the recurrence of diseases.

[0121] (Five) In vitro biological safety of microneedle patch

[0122] The biological compatibility of the microneedle patches prepared by example 1 and comparative examples 1-3 was verified by erythrocyte hemolysis experiment. First, the blood of healthy mice was taken, and physiological saline was centrifuged several times to wash the red blood cell precipitate until the supernatant was colorless. The obtained red blood cell precipitate was dispersed with physiological saline to obtain a red blood cell suspension. Then, the red blood cell suspension was added to ultrapure water (positive control group), physiological saline (negative control group) or different microneedle extraction solutions, respectively. The microneedle extraction solution was prepared by placing the microneedle patches (prepared by example 1 and comparative examples 1-3) in PBS after ultraviolet irradiation for 2 hours. After the microneedle patches gradually dissolved, the microneedle extraction solution was formed. Different samples were incubated at 37°C for 6 hours, and then centrifuged at 1500 rpm for 15 minutes. The supernatant of the sample was taken. Finally, the absorbance of the supernatant at 540 nm was measured by a microplate reader, and the erythrocyte hemolysis rate was calculated according to the formula

[0123]

[0124] Wherein, OD T represents the absorbance value of the experimental group, OD N represents the absorbance value of the negative control group, and OD P represents the absorbance value of the positive control group.

[0125] Figure 12 The results show that the red blood cells treated by the microneedle patches prepared by example 1 and comparative examples 1-3, including the Blank group, Doxy / SIM-MNs group, SIM-MNs group and Doxy-MNs group, are in good condition, and the hemolysis rate is less than 5%.

[0126] (Six) Study on the antibacterial performance of microneedle patch

[0127] Bacterial inhibition zone diameter assay was used to verify the antibacterial performance of microneedle patches. The microneedle patches sterilized by ultraviolet light were soaked in sterile PBS buffer for 5 days to obtain microneedle extract. Staphylococcus aureus (purchased from Beijing Microbial Culture Collection Center) and Porphyromonas gingivalis (purchased from Shanghai Microbial Culture Collection Center) were inoculated into liquid medium and cultured at 37°C for 12 hours to the logarithmic growth phase. The logarithmic growth phase bacteria were diluted to a concentration of 10 7 CFU / mL with PBS buffer for use. 25 g of LB powder (Hibio, Catalog No.: HBMU001) was dissolved in 1 L of deionized water, stirred and dissolved, and then divided into portions. The LB liquid medium was obtained by high-pressure sterilization at 121°C for 15-20 minutes. 40 g of premixed dry powder (Hibio, Catalog No.: HBMU002) was added to 1 L of deionized water, heated to boiling until completely dissolved, and then divided into portions. The LB solid medium was obtained by high-pressure sterilization at 121°C for 15-20 minutes, and then cooled to about 50°C. Pour the plate (15-20 mL per plate), and after solidification, the LB solid medium was obtained and stored at 4°C. 20 μL of liquid medium was added to the LB solid medium, and the bacteria were evenly coated with a coating rod. Different microneedle patches were gently attached to the center of the culture, and the solid medium was incubated at 37°C in a constant temperature incubator for 7 days. The diameters of the bacterial inhibition zones of each group were measured with a vernier caliper.

[0128] From Figure 13 A and B in FIG. 6 can see Doxy-MNs, Doxy / SIM-MNs group has obvious inhibition zone on S. aureus and P. gingivalis plate, the diameter is 31.43 ± 1.56 mm, 79 ± 9.73 mm, respectively, and there is no significant difference with the Periocline ointment group, while the diameter of the inhibition zone in the control group and the Blank-MNs group is almost 0 mm. This shows that Doxy-MNs and Doxy / SIM-MNs have very good antibacterial function, especially the inhibition effect on anaerobic bacteria P. gingivalis is significant. From Figure 13 C and D in FIG. 6 can be seen that compared with other groups of bacteria, the bacteria treated with microneedles containing doxycycline (Doxy-MNs and Doxy / SIM-MNs groups) and Periocline ointment group died in large numbers, and the survival rate of bacteria was less than 10%.

[0129] (VII) In vitro evaluation of the osteogenic effect of microneedle patches

[0130] (1) Alkaline phosphatase staining experiment

[0131] C3H / 10-T1 / 2 cells (purchased from Wuhan Punsai Biological Company) were collected and the cell concentration was adjusted The sterile crawling pieces in the 12-hole plate were inoculated, and the culture system in each hole was 1 ml. After overnight culture, the culture medium was replaced in different groups, Control group (cell culture medium containing only osteogenic induction liquid), Blank-MN group (cell culture medium of blank microneedle extract and osteogenic induction liquid), Doxy-MN group (cell culture medium of doxycycline-loaded microneedle patch extract and osteogenic induction liquid), SIM-MN group (cell culture medium of simvastatin-loaded microneedle patch extract and osteogenic induction liquid), Doxy / SIM-MN group (cell culture medium of doxycycline and simvastatin-loaded microneedle patch extract and osteogenic induction liquid), and Gel group (cell culture medium of Palio cream extract and osteogenic induction liquid) were induced for 14 days. The culture supernatant was removed at 7 days and 14 days, respectively, the cells were washed with PBS for 2 times, 1 mL of 4% paraformaldehyde was added to each hole to fix the cells for 30 min, the paraformaldehyde was absorbed, the cells were washed with PBS for 2-3 times, and the staining solution was prepared according to the staining kit instructions of alkaline phosphatase (ALP quantitative kit, purchased from Biyun Tian Biological Company) to perform staining; a scanner was used to scan the 6-hole plate, an inverted white light microscope was used to take pictures of the cells, and the ALP staining level was analyzed by Image J software.

[0132] (2) Alizarin red staining experiment

[0133] C3H / 10-T1 / 2 cells (purchased from Wuhan Punuo Sai Biological Company) were collected and the cell concentration was adjusted The sterile crawling pieces in the 12-hole plate were inoculated, and the culture system in each hole was 1 ml. After overnight culture, the culture medium was replaced in different groups, Control group (cell culture medium containing only osteogenic induction liquid), Blank-MNs group (cell culture medium of blank microneedle extract and osteogenic induction liquid), Doxy-MNs group (cell culture medium of doxycycline-loaded microneedle patch extract and osteogenic induction liquid), SIM-MNs group (cell culture medium of simvastatin-loaded microneedle patch extract and osteogenic induction liquid), Gel group (cell culture medium of Palio cream extract and osteogenic induction liquid) were induced for 14 days. The culture supernatant was removed at 7 days and 14 days, respectively, the cells were washed with PBS for 2 times, 1 mL of 4% paraformaldehyde was added to each hole to fix the cells for 30 min, the paraformaldehyde was absorbed, the cells were washed with PBS for 2-3 times, and the staining solution was prepared according to the staining kit instructions of alkaline phosphatase (ALP quantitative kit, purchased from Biyun Tian Biological Company) to perform staining; a scanner was used to scan the 6-hole plate, an inverted white light microscope was used to take pictures of the cells, and the ALP staining level was analyzed by Image J software.

[0134] (3) Immunofluorescence labeling of the expression of osteogenic factors OCN and RUNX2

[0135] On the 7th day of induction of C3H / 10-T1 / 2 cells by the above method, the C3H / 10-T1 / 2 cells were digested and collected, and the cells were washed twice with PBS. 1 mL of 4% paraformaldehyde was added to each well for cell fixation for 10 minutes. The paraformaldehyde was removed, and the cells were washed with PBS for 3 times. The paraformaldehyde was removed, and the cells were washed with PBS for 2 times. 1 ml of 0.25% Triton X-100 was added to each well for nuclear breakage treatment. The cell slides were gently removed from the 6-well plate with cell forceps, with the cell surface facing upwards, and placed on a glass slide. The corresponding primary antibody diluent (rabbit anti-OCN monoclonal antibody at a volume ratio of 1:500, purchased from Wuhan Aibote Biological Technology Co., Ltd., item number A2851; rabbit anti-RUNX2 monoclonal antibody at a volume ratio of 1:500, purchased from Wuhan Aibote Biological Technology Co., Ltd., item number A20800) was prepared with 5% BSA and incubated at 4°C for 12 hours. The primary antibody diluent was removed, and the cell slides were gently washed with PBS for 2 times, each time for 10 minutes. Then, the fluorescent secondary antibody diluent (at a volume ratio of 1:200) corresponding to the attribute of the primary antibody was incubated in the dark for 2 hours. The glass slide was taken out in the dark, and the cells were washed with PBS for 2 times. 50 μL of DAPI staining solution was added to stain the cell nucleus, and incubated in the dark for 10 minutes. In the dark environment, the cells were washed with PBS for 2 times, and then 20 μL of anti-fluorescence quencher was added for mounting treatment. Laser confocal photography was used, and ImageJ software was used for quantitative analysis

[0136] Figure 14 As shown in the staining results, on the 7th day, all groups except the control group and the Blank-MNs group generated a certain amount of ALP. On the 14th day, the ALP of all groups increased significantly, and the ALP content of the Doxy / SIM-MNs group was higher than that of the other groups on the 7th day and the 14th day. Alizarin red (ARS) staining was performed on the 7th day and the 14th day after osteogenic differentiation induction of C3H10T1 / 2 cells in each group. In both time periods, the Doxy / SIM-MNs group had the most calcium nodules, followed by the SIM-MNs group. The calcium nodule content of the Doxy-MNs and Gel groups was also significantly improved compared with the control group. The Blank-MNs group had the least red calcium nodules. This indicates that simvastatin has more advantages in promoting osteogenic differentiation.

[0137] Figure 15 Through statistical analysis of the intensity of positive expression, it can be concluded that the osteogenic differentiation marker factors RUNX2 and OCN also have Figure 14The results of ALP staining and alizarin red staining were similar. The Doxy / SIM-MNs group had the strongest fluorescence intensity, which was significantly higher than the SIM-MNs group, the Doxy-MNs group, and the Gel group.

[0138] From Figure 16 As can be seen, compared with the control group, the expression of four osteogenic differentiation marker mRNAs in the Doxy / SIM-MNs group and the SIM-MNs group was higher.

[0139] (Eight) Evaluation of the effect of microneedle patches in a rat diabetic periodontitis model

[0140] (1) Establishment of a rat diabetic periodontitis model and intervention

[0141] Six-week-old Sprague Dawley rats were fed a high-sugar high-fat diet for two weeks. After fasting for 12 hours, the rats were intraperitoneally injected with streptozotocin (STZ) solution at a dose of 35 mg / kg. On the 3rd, 5th, 7th, and 10th days after STZ injection, the rats' tail vein random blood glucose was measured, and an oral glucose tolerance test (OGTT) was performed. The standard for successful modeling of diabetic rats was a random blood glucose level of ≥16.7 mmol / L. If the random blood glucose level did not meet the standard, the rat would not be used in subsequent experiments. On the second day of STZ intraperitoneal injection, the periodontitis model was also established. The rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (40 mg / kg), the initial gingival separation was performed using an oral probe, and the interproximal space between the first and second maxillary molars was appropriately increased using an insulin needle. A 3-0 surgical suture was wrapped around a 0.2 mm orthodontic ligature wire, and a needle holder was used to gently thread the 0.2 mm ligature wire between the first and second molars, around the first molar, and tie it off. Subsequently, 1x10 8Gingival pocket and suture on the ligature wire were injected with P. gingivalis suspension at a concentration of 108 CFU / mL every 3 days, and the loosening of the ligature wire was checked. After 2 weeks of placing the ligature wire in the gingival sulcus of the first molar of the rats, the ligature wire was removed, and different experimental interventions were given according to different experimental groups. 36 SD rats with established diabetic periodontitis model were randomly divided into 6 groups, named Control group, Blank group, Doxy group, SIM group, Doxy / SIM group, and Gel group. Among them, the control group was not given any treatment, the blank group was placed with blank microneedle patches without any drug, the Doxy group was placed with microneedle patches with doxycycline in the shell, the SIM group was placed with microneedle patches with simvastatin and dopamine nanoparticles in the core, the Doxy / SIM group was placed with microneedle patches with doxycycline and simvastatin, and the Gel group was placed with Plurio ointment (minocycline hydrochloride ointment). After 2 weeks of treatment, the rats were sacrificed, and the bilateral maxillary bones of the rats were collected.

[0142] From Figure 17 It can be seen that the glucose levels of both diabetic rats and healthy rats increased within 30 minutes of gavage, and then gradually decreased. However, 2 hours after the experiment, the glucose level of healthy rats returned to normal, while the glucose level of diabetic rats remained at 16.7 mmol / L, indicating that the diabetic rat model was successfully established.

[0143] Figure 18 To detect the random blood glucose level of rats after establishing a diabetic model through tail vein, rats with blood glucose level < 16.7 mmol / L will not be subjected to subsequent experiments.

[0144] (2) In vivo evaluation of the therapeutic effect of microneedle patches on diabetic periodontitis

[0145] The specimens were scanned, three-dimensional image reconstructed and analyzed by micro-computed tomography (Micro-CT). The scanning parameters were set as voltage 70 kVp, current 114 μA, and pixel 17.5 μm. The distance between the enamel cementum junction (CEJ) and the alveolar bone crest (ABC) was measured to determine the amount of alveolar bone loss (CEJ-ABC). The distance between the alveolar bone crest (ABC) and the cement-enamel junction (CEJ) was measured to determine the amount of alveolar bone loss (CEJ-ABC). The specimens to be embedded were trimmed, washed in physiological saline, decalcified in ethylenediaminetetraacetic acid (EDTA) for 4 weeks, dehydrated in 70%-100% gradient alcohol, transparentized in xylene, immersed in wax, embedded with a paraffin embedding machine, sliced at a thickness of 5 μm, and the slices were subjected to H&E staining.

[0146] From Figure 19It can be seen that after the Doxy / SIM-MNs microneedle patch is used to treat the diabetic periodontitis rat model, the distance from the cement-enamel junction (CEJ) to the alveolar bone crest (ABC) is significantly reduced from 4.2 mm to 1.7 mm, which indicates that after the Doxy / SIM-MNs microneedle patch is used for treatment, the absorption degree of the alveolar bone in the diabetic periodontitis rat can be greatly improved, and the periodontal tissue regeneration is promoted.

Claims

1. A core-shell microneedle patch loaded with tetracycline antibacterial drugs and simvastatin, characterized in that: The microneedle patch comprises a substrate and microneedle bodies arranged on the substrate, the microneedle bodies have a core-shell structure; wherein the shell of the microneedle body and the substrate are both loaded with tetracycline antibacterial drugs; the inner core of the microneedle body comprises polydopamine nanoparticles loaded with simvastatin Sim@PDA; the tetracycline antibacterial drug is doxycycline; the preparation raw material of the substrate comprises soluble biocompatible material silk fibroin; the preparation raw material of the shell of the microneedle body comprises in-vivo soluble polymer material polyvinyl alcohol.

2. The core-shell microneedle patch of claim 1, wherein: The microneedle body is in a conical or pyramidal structure; the height of the microneedle body is 100-1000 μm; the needle tip of the microneedle body is sharp, the tip radius of the needle body is less than 15 μm, the center distance between adjacent microneedles is 50-1000 μm, and the substrate is perpendicular to the microneedle body.

3. The core-shell microneedle patch of claim 1, wherein: The Sim@PDA is in a spherical shape, and the average particle size is 200-300 nm.

4. The method of making the core-shell microneedle patch of claim 1, characterized by: The method comprises the following steps: Preparation of the shell of the microneedle: mix tetracycline antibacterial drugs and in-vivo soluble polymer material solution 1 according to a proportion to obtain a shell matrix solution; then inject a proper amount of the shell matrix solution into a microneedle mold, centrifuge until fully filled, and dry to obtain the shell of the microneedle; Preparation of the inner core of the microneedle: mix Sim@PDA nanoparticles and in-vivo soluble polymer material solution 2 according to a proportion to obtain an inner core matrix solution; Then inject a proper amount of the inner core matrix solution into the microneedle mold containing the shell of the microneedle, centrifuge until fully filled; repeat the injection and centrifugation process 0-3 times; finally dry to obtain the inner core of the microneedle; Preparation of the substrate: mix tetracycline antibacterial drugs and soluble biocompatible material solution according to a proportion to obtain a substrate solution; then lay a proper amount of the substrate solution on the microneedle mold containing the shell and the inner core of the microneedle, vacuum dry, and demold to obtain the core-shell microneedle patch loaded with tetracycline antibacterial drugs and simvastatin.

5. The method of claim 4, wherein: The preparation method of the Sim@PDA nanoparticles is as follows: Mix polydopamine (PDA) nanoparticles and simvastatin solution according to a proportion, stir and react in the dark for 5-12 h; after the reaction is completed, centrifuge the obtained product, collect the precipitate, wash, and dry to obtain the Sim@PDA nanoparticles.

6. The method of claim 5, wherein: The mass ratio of the PDA nanoparticles to simvastatin is 1:1-1:

2.

7. The method of claim 5, wherein: The preparation method of the PDA nanoparticles is as follows: Mix dopamine hydrochloride and deionized water according to a proportion, stir and dissolve uniformly to obtain a dopamine hydrochloride solution; then slowly drop in sodium hydroxide solution into the dopamine hydrochloride solution according to a proportion at 40-60°C, after the dropping is completed, stir the obtained reaction solution in the dark for 20-30 h; after the reaction is completed, centrifuge the obtained product, collect the precipitate, continue to wash the obtained precipitate, and freeze-dry to obtain the PDA nanoparticles.

8. The method of claim 7, wherein: The dosage ratio of dopamine hydrochloride to sodium hydroxide solution is 100 mg:1 mL.

9. Use of the core-shell microneedle patch loaded with tetracycline antibacterial drugs and simvastatin according to any one of claims 1-3 in the preparation of a drug for treating periodontitis accompanied by diabetes.

10. A pharmaceutical composition, characterized by: The core-shell microneedle patch comprising the tetracycline antibacterial drug and simvastatin according to any one of claims 1 to 3. The core-shell microneedle patch comprising the tetracycline antibacterial drug and simvastatin according to any one of claims 1 to 3.

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