Core-shell microneedle patch loaded with tetracycline antibacterial drug and simvastatin as well as preparation method and application of core-shell microneedle patch

Abstract

The invention discloses a core-shell structure microneedle patch loaded with tetracycline antibacterial drugs and simvastatin as well as a preparation method and application of the core-shell structure microneedle patch, and belongs to the field of biomedical materials. An antibacterial drug doxycycline is loaded on a shell structure of a substrate layer and a needle body of the microneedle patch, and a core structure contains simvastatin-loaded polydopamine nanoparticles. The microneedle patch can release antibacterial drugs in stages, and the number of bacteria at local parts of a focus can be rapidly reduced in a short time through burst release of the antibacterial drugs in the first stage, so that inflammation spreading is controlled; in the second stage, the antibacterial medicine is slowly released, so that the formation of a new plaque biofilm is inhibited for a long time, meanwhile, simvastatin is slowly released, the effect of locally promoting osteogenesis is achieved, and the synergistic effect of antibiosis and bone regeneration is achieved. The compound has a good application prospect in the treatment of chronic periodontitis, especially intractable periodontitis combined with systemic diseases, such as chronic periodontitis combined with diabetes mellitus.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and in particular relates to a core-shell structure microneedle patch loaded with tetracycline antibiotics and simvastatin, and a preparation method and application thereof. Background Art

[0002] Periodontitis is the sixth largest chronic bacterial infectious disease in the world, affecting people of all ages. Its prevalence increases with age, causing inflammatory damage to periodontal supporting tissues and even tooth loss. Dental plaque biofilm is the initiating factor in periodontal disease, and both local and systemic risk factors can promote the occurrence and development of periodontitis. Local risk factors include: dental tartar, plaque, poor restorations, etc., while systemic risk factors include: smoking, obesity, diabetes, etc. Diabetes is an important contributing factor to periodontitis. High blood sugar can aggravate the destruction of alveolar bone and also increase the pathogenicity of oral microorganisms.

[0003] Currently, mechanical removal of dental plaque and biofilm is a conventional treatment for periodontitis, but it is less effective in treating periodontitis associated with diabetes, as diabetes can exacerbate the difficulty of treating periodontitis. Therefore, adjunctive antibiotic therapy is often necessary, but systemic antibiotic use carries the risk of developing drug resistance and gastrointestinal reactions. Topical antibiotic use, due to factors such as the complex anatomy of teeth, deep periodontal pockets, and saliva flushing, makes it difficult for drugs to achieve satisfactory therapeutic effects. Therefore, there is an urgent need to explore new treatment strategies for diabetic periodontitis.

[0004] Microneedles are a new type of transdermal drug delivery system that consists of multiple micron-sized tiny needle tips connected in an array on a base. The needle body pierces the skin's stratum corneum to allow drugs to enter the skin efficiently. Microneedle-based treatment strategies have broad application prospects in the biomedical field. They can not only reduce the discomfort caused by traditional drug delivery methods, but also deliver drugs more accurately and effectively. Compared with traditional drug delivery methods, microneedles have the advantages of being minimally invasive and painless, allowing for autonomous drug delivery, improving patient compliance, and improving treatment effects. However, the microneedle patches currently disclosed in the prior art have the following defects: for example, the designs of the microneedle patches disclosed in the prior art all use a structure of a backing and a microneedle body, so that they can only carry a single load of antibacterial drugs or drugs that promote osteogenesis, and cannot achieve the "two-pronged approach" of inhibiting periodontal pathogens while also promoting bone regeneration.

[0005] Based on the above reasons, this application is filed. Summary of the Invention

[0006] In view of the problems existing in the above-mentioned prior art, the present invention aims to design a drug delivery system that can simultaneously exert anti-inflammatory and osteogenesis-promoting effects, thereby playing a synergistic therapeutic role in diabetic periodontitis, and by improving the traditional drug administration method, improving patient compliance, and solving problems such as maintaining drug concentration and gastrointestinal side effects.

[0007] The purpose of the first aspect of the present invention is to provide a core-shell microneedle patch loaded with tetracycline antibiotics and simvastatin.

[0008] The purpose of the second aspect of the present invention is to provide a method for preparing the core-shell microneedle patch loaded with tetracycline antibiotics and simvastatin as described in the first aspect of the present invention.

[0009] The purpose of the third aspect of the present invention is to provide the use of the core-shell microneedle patch loaded with tetracycline antibiotics and simvastatin as described in the first aspect of the present invention in the preparation of drugs and / or drug delivery systems for treating diabetic periodontitis.

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

[0011] In order to achieve the above object, the technical solution of the present invention is:

[0012] The first aspect of the present invention provides a core-shell microneedle patch loaded with tetracycline antibiotics and simvastatin, wherein the microneedle patch comprises a substrate and a microneedle body disposed on the substrate, wherein the microneedle body has a core-shell structure; wherein the outer shell of the microneedle body and the substrate are both loaded with tetracycline antibiotics; and the inner core of the microneedle body comprises polydopamine nanoparticles (Sim@PDA) loaded with simvastatin.

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

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

[0015] Furthermore, in the above technical solution, the surface of the microneedle body has a threaded structure, which can effectively disperse stress concentration and reduce the risk of transverse fracture under axial pressure; at the same time, it significantly increases the surface area of ​​the needle body, thereby greatly increasing the effective contact area, which can effectively increase 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 vertical penetration force, thereby reducing penetration resistance, and can more effectively puncture the tissue without falling off, thereby improving the success of drug delivery.

[0016] Furthermore, in the above technical solution, the microneedle body, also known as a microneedle array, preferably adopts a 10×10 square array, and its three-dimensional size and the number of microneedles used can be flexibly adjusted according to the medication location and the required dosage.

[0017] Furthermore, in the above technical solution, the materials used to prepare the outer shell and inner core of the microneedle body also include a body-soluble polymer material; the body-soluble polymer material is at least one of hyaluronic acid or its salts, polyvinyl alcohol, chitosan, gelatin, sodium carboxymethyl cellulose, polyvinyl pyrrolidone, chondroitin sulfate, etc. The body-soluble polymer materials used to prepare the outer shell and inner core of the microneedle body can be the same or different.

[0018] Furthermore, in the above technical solution, in some embodiments of the present invention, the in vivo soluble polymer material for preparing the microneedle needle body shell is polyvinyl alcohol; in other embodiments of the present invention, the in vivo soluble polymer material for preparing the microneedle needle body core is polyvinyl pyrrolidone.

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

[0020] Furthermore, in the above technical solution, the molecular weight range of the in vivo soluble polymer material used to prepare the microneedle needle body shell is 10 to 200 kDa.

[0021] Furthermore, in the above technical solution, the molecular weight range of the in vivo soluble polymer material used to prepare the inner core of the microneedle body is 1 to 10 kDa.

[0022] Furthermore, in some embodiments of the present invention, the above technical solution comprises at least one of minocycline, doxycycline or other tetracyclines.

[0023] Furthermore, in some embodiments of the present invention, in the above technical solution, the raw materials for preparing the substrate include soluble biocompatible materials.

[0024] Furthermore, in the above technical solution, the soluble biocompatible material is hyaluronic acid or its salts (such as sodium salt), chitosan, agarose, alginate, maltose, galactose, fructose, polylactic acid, polyglycolic acid, polyvinyl alcohol, , polytrimethylene carbonate (PTMC), polydioxanone (PPDO), polyamino acid derived carbonate (PDTE), polyorthoester (POE), collagen, gelatin, silk fibroin, sodium carboxymethyl cellulose, chondroitin sulfate, polyvinyl pyrrolidone, etc.

[0025] Preferably, in the above technical solution, in some embodiments of the present invention, the soluble biocompatible material is silk fibroin.

[0026] Furthermore, in the above technical solution, the Sim@PDA is spherical and has an average particle size of 200 to 300 nm.

[0027] The second object of the present invention is to provide a method for preparing the core-shell microneedle patch loaded with tetracycline antibiotics and simvastatin, comprising the following steps:

[0028] Preparation of microneedle shells: tetracycline antibiotics and a body-soluble polymer material solution 1 are mixed according to a ratio to obtain a shell matrix solution; an appropriate amount of the shell matrix solution is then injected into a microneedle mold, centrifuged until fully filled, and dried to obtain a microneedle shell;

[0029] Preparation of microneedle cores: Sim@PDA nanoparticles are mixed with a solution of a body-soluble polymer material 2 according to a ratio to obtain a core matrix solution; an appropriate amount of the core matrix solution is then injected into the microneedle mold containing the microneedle shell, and centrifuged until fully filled; the injection and centrifugation process is repeated 0 to 3 times; and finally, the microneedle cores are dried.

[0030] Preparation of the substrate: tetracycline antibiotics and soluble biocompatible material solution are mixed in a ratio to obtain a substrate solution; then an appropriate amount of the substrate solution is laid on the above-mentioned microneedle mold containing the microneedle shell and the core, and the mold is demolded after vacuum drying to obtain the core-shell microneedle patch loaded with tetracycline antibiotics and simvastatin.

[0031] Furthermore, in the above technical solution, the concentration of the in vivo-soluble polymer material solution 1 is (5-15) g:100 mL. Its viscosity and concentration can be adjusted within a certain range to meet the mechanical strength requirements of the needle body. For example, when the molecular weight is small and the viscosity is low, the concentration can be higher; similarly, when 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, polyvinyl pyrrolidone, chondroitin sulfate, etc.; the solvent is deionized water.

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

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

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

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

[0036] Furthermore, in some embodiments of the present invention, the above technical solution comprises the following steps:

[0037] Polydopamine (PDA) nanoparticles are added to a simvastatin solution according to a ratio, and stirred in the dark for 5 to 12 hours. After the reaction, the obtained product is centrifuged, and the precipitate is collected, washed, and dried to obtain the Sim@PDA nanoparticles.

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

[0039] Furthermore, in the above technical solution, the PDA nanoparticles are spherical in shape, with uniform particle size and an average particle size of 150 to 250 nm. After the addition of simvastatin, the morphology of the Sim@PDA nanoparticles did not change significantly and remained spherical.

[0040] Furthermore, in the above technical solution, the preparation method of the PDA nanoparticles is as follows:

[0041] Dopamine hydrochloride and deionized water are mixed according to a ratio, stirred and dissolved evenly to obtain a dopamine hydrochloride solution; then, sodium hydroxide solution is slowly added dropwise to the dopamine hydrochloride solution at 40-60° C. according to the ratio, and after the addition is completed, the resulting reaction solution is stirred in the dark for 20-30 hours; after the reaction is completed, the resulting product is centrifuged, the precipitate is collected, and the resulting precipitate is further washed and freeze-dried to obtain the PDA nanoparticles.

[0042] Preferably, in the above technical solution, in one embodiment of the present invention, the usage ratio of dopamine hydrochloride to deionized water is 1 mg:1 mL.

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

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

[0045] Preferably, in the above technical solution, in one embodiment of the present invention, the time of the light-proof stirring reaction is 24 hours.

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

[0047] Furthermore, in the above technical solution, the ratio of the tetracycline antibiotic to the soluble biocompatible material solution in the base solution is (10-30) parts by mass: (1-5) parts by volume; in a preferred embodiment of the present invention, the ratio of the tetracycline antibiotic to the soluble biocompatible material solution in the base solution is 20 parts by mass: 2 parts by volume; wherein: the parts by mass and parts by volume are based on mg:mL.

[0048] The third aspect of the present invention provides the use of the core-shell microneedle patch loaded with tetracycline antibiotics and simvastatin according to the first aspect of the present invention in the preparation of drugs and / or drug delivery systems for treating diabetic periodontitis.

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

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

[0051] Simvastatin (Simvastatin) promotes the differentiation of mesenchymal stem cells into osteocytes, promoting osteogenesis while significantly inhibiting osteoclastogenesis and bone resorption, ultimately alleviating periodontitis and promoting periodontal regeneration. However, Sim is an anionic, lipophilic drug. Most oral Sim is likely eliminated through the hepatic and renal circulation, necessitating a higher dose to achieve effective concentrations at sites of alveolar bone resorption. While such high doses of Sim may be beneficial for bone formation, they may also produce systemic side effects, such as hepatic and renal adverse reactions. Furthermore, bone regeneration requires a long time, and daily local injections would significantly increase patient pain and rely heavily on patient compliance. Therefore, encapsulating Sim in polydimethylsiloxane (PDA) nanoparticles not only maintains the drug's robust bone regeneration activity but also enables controlled, sustained drug delivery within the local environment of the periodontal pocket. Furthermore, the surface phenolic groups of PDA nanoparticles can alleviate local oxidative stress by scavenging excess ROS at the site of inflammation. Therefore, using PDA nanoparticles loaded with simvastatin can not only serve as a drug delivery system, excellently achieving sustained and controlled drug release, but also has the advantage of antioxidant properties, which can help alleviate the inflammation of periodontitis. The main principles or mechanisms involved in the present invention are as follows:

[0052] The present invention uses microneedle patches as drug delivery carriers and innovatively designs a microneedle patch with a core-shell structure to achieve a "fast-acting + long-acting" two-phase release system: the outer shell of the microneedle patch is loaded with tetracycline antibiotics, which can reach the highest concentration of drug release within two hours. The burst release of antibacterial drugs in a short period of time can maximize the initial bactericidal intensity, quickly eliminate pathogenic microorganisms colonizing the infected root surface and block the formation of biofilms; the backing base structure of the microneedle patch is loaded with silk fibroin antibiotics, and through slow controlled release of drugs, it can maintain an effective concentration at the local site for 14 days, thereby inhibiting the regeneration of residual bacteria, preventing recurrence, and promoting tissue regeneration; at the same time, the inner core of the microneedle patch contains polydopamine nanoparticles loaded with simvastatin, which has a promoting effect on the regeneration of alveolar bone. By adopting a two-pronged strategy, the microneedle patch promotes periodontal tissue regeneration while exerting antibacterial and anti-inflammatory effects, thereby playing a synergistic therapeutic role in diabetic periodontitis.

[0053] Compared with the prior art, the advantages or beneficial effects of the present invention include at least:

[0054] The present invention innovatively designs a microneedle patch with a core-shell structure. The core-shell microneedle patch consists of a shell that can quickly release tetracycline antibiotics in a short period of time, and a core containing simvastatin polydopamine nanoparticles, which promotes the regeneration of alveolar bone. At the same time, the tetracycline antibiotics are slowly released through the backing substrate material, which has a long-term antibacterial effect and prevents recurrence. The core-shell microneedle patch provided by the present invention not only highlights the use of the design of the core-shell structure to achieve a treatment sequence of "elimination" first and then "regeneration", but also controls infection and reduces inflammatory response for diabetic periodontitis. More importantly, it promotes the regeneration of alveolar bone and reverses the course of periodontitis, thereby achieving the ultimate goal of improving the patient's occlusal function. On the other hand, the degradation and swelling rates of the shell material and the backing material are different, thereby achieving a staged two-phase release system, that is, the burst release layer quickly controls bacteria while the sustained-release layer has a synergistic mechanism of long-term antibacterial effect, which has the dual advantages of strong initial debridement and continuous prevention of recurrence.

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

[0056] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

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

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

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

[0060] Figure 4 FTIR spectra comparison of Sim@PDA nanoparticles, PDA nanoparticles, and simvastatin raw materials prepared in Example 1;

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

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

[0063] Figure 7 A is a scanning electron microscope image of the cross section of a single core-shell microneedle prepared in Example 1; B is an EDS point analysis result of the outer shell layer of the core-shell microneedle prepared in Example 1; C is an EDS point analysis result of the inner core layer of the core-shell microneedle prepared in Example 1;

[0064] Figure 8 The C and O element distribution diagram of the cross section of a single core-shell microneedle prepared in Example 1;

[0065] Figure 9 This is a fluorescence micrograph of the core-shell microneedle patch prepared in Example 1, wherein: from left to right are the core layer, the shell layer, and the core-shell bilayer;

[0066] Figure 10 This is an optical microscope image of pig gingival tissue in vitro after puncture with fluorescently labeled core-shell microneedles.

[0067] Figure 11is the drug concentration level of doxycycline released from the microneedle patch in vitro; A is the drug concentration level of doxycycline released from the outer shell layer, and B is the drug concentration level of doxycycline released from the basal layer;

[0068] Figure 12 Figure 2 shows the results of in vitro biosafety assessment of different microneedle patches;

[0069] Figure 13 Middle: A shows the results of the inhibition zone test after 7 days of co-culture of different microneedle patches with Staphylococcus aureus; B shows the results of the inhibition zone test after 7 days of co-culture of different microneedle patches with Porphyromonas gingivalis; C shows the live-dead staining and scanning electron microscopy observation of the microscopic changes of bacteria under the action of the microneedle patch after 7 days of co-culture of different microneedle patches with Staphylococcus aureus; D shows the live-dead staining and scanning electron microscopy observation of the microscopic changes of bacteria under the action of the microneedle patch after 7 days of co-culture of different microneedle patches with Porphyromonas gingivalis;

[0070] Figure 14 The results of ALP and ARS staining of C3H / 10 T1 / 2 cells after being treated with different microneedle patch extracts for 3 hours, and then induced into osteoblasts for 7 and 14 days.

[0071] Figure 15 This is a comparison of RUNX2 and OCN test results in C3H / 10 T1 / 2 cells treated with different microneedle patch extracts for 3 hours, 7 days after osteogenic induction.

[0072] Figure 16 C3H / 10 T1 / 2 cells were treated with different microneedle patch extracts for 3 hours, and then induced into osteoblasts for 7 days. q-PCR was used to detect the mRNA expression of osteoblast markers Ocn, Runx2, Alp, and Col1, followed by Alizarin red staining.

[0073] Figure 17 The results of OGTT experiments were performed on diabetic rats and healthy rats respectively.

[0074] Figure 18 After the diabetic model was established, random blood glucose measurements were performed on the rat tail vein on the 3rd, 5th, 7th and 10th days.

[0075] Figure 19 Middle A is a Micro-CT three-dimensional reconstruction and sagittal cross-sectional image of the rat maxillary bone; Middle B is the distance test result of the mesiodistal alveolar bone crest and the cementoenamel junction (ABC-CEJ) of the maxillary first molar of SD rats;

[0076] in: Figures 12-17The Control group involved is the negative control group, that is, no intervention was given in the C3H / 10-T1 / 2 cell culture; the Blank-MNs group is the blank microneedle patch prepared in Comparative Example 3 and incubated with C3H / 10-T1 / 2 cells for 24 hours. The blank microneedle patch only contains the matrix material of the synthetic microneedle patch and is not loaded with any drug; the SIM-MNs group is the microneedle patch prepared in Comparative Example 1. In addition to the matrix material, the needle body core contains Sim@PDA nanoparticles; the Doxy-MNs group is the microneedle patch prepared in Comparative Example 2. In addition to the synthetic matrix material, the needle body shell and the backing substrate contain doxycycline; the Doxy / SIM-MNs group is the microneedle patch prepared in Example 1. The outer shell and backing substrate of the needle body contain doxycycline, and the core contains Sim@PDA nanoparticles; the Gel group is minocycline hydrochloride ointment (trade name "Palio") which is currently widely used in periodontitis clinical practice; the Saline group is a normal saline group. DETAILED DESCRIPTION

[0077] The present invention solves the current clinical treatment methods for periodontitis, which are unable to effectively promote periodontal tissue regeneration and thus 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 invention is a water-soluble matrix, and the inner core is an organic solvent matrix. Compared with the prior art, the design of this core-shell structure can make the two drugs loaded by the inner core and the outer shell structure more stable, and there will be no defect that the drug loaded by the inner core is partially dissolved into the outer shell structure. The microneedle patch of the present invention can better achieve "programmed" release, and the core-shell structure microneedle of the present invention has better application expansion: the outer shell structure of the core-shell structure of the present invention is an aqueous matrix, and the inner shell structure can load water-soluble drugs or fat-soluble drugs, and the speed of its release can be regulated by other methods, such as responsive nanoparticles. In general, the microneedle patch of the present invention focuses on the periodontitis disease model associated with diabetes. Unlike the simple periodontitis model, it has been clinically proven that periodontitis and diabetes can affect each other and aggravate the course of the disease. Periodontitis associated with diabetes is characterized by severe inflammation and difficulty in control. In addition to basic periodontal treatment, clinically, auxiliary use of antibiotics and other drugs is often required. Therefore, the microneedle patch proposed in the present invention has better applicability for periodontitis associated with diabetes and provides a more precise treatment strategy.

[0078] The present invention designs a microneedle patch with a core-shell structure to achieve the phased release of antibacterial drugs and simvastatin, which has an osteogenesis-promoting effect, thereby alleviating the degree of inflammation associated with diabetic periodontitis while promoting the regeneration of periodontal tissue. The base layer and the outer shell structure of the microneedle patch of the present invention are loaded with the antibacterial drug doxycycline, and the inner core structure contains polydopamine nanoparticles loaded with simvastatin. The microneedle patch can release the antibacterial drug in stages. The first stage is a burst release of the antibacterial drug to quickly reduce the number of bacteria in the local lesion in a short period of time, thereby controlling the spread of inflammation; the second stage is a sustained release of the antibacterial drug, thereby inhibiting the formation of new plaque biofilms for a long time, while slowly releasing simvastatin to promote local osteogenesis, achieving a synergistic effect of antibacterial and bone regeneration. It has good application prospects in the treatment of chronic periodontitis, especially refractory periodontitis combined with systemic diseases, such as chronic periodontitis combined with diabetes.

[0079] The present invention is further described in detail below through an implementation case. This implementation case is implemented based on the technology of the present invention. Detailed implementation methods and specific operating procedures are now given to illustrate the creativity of the present invention, but the scope of protection of the present invention is not limited to the following implementation case.

[0080] Based on the information contained in this application, it will be readily apparent to those skilled in the art that various changes can be made to the precise description of the present invention. It should be understood that the scope of the present invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are provided merely to illustrate specific aspects of the present invention.

[0081] In order to better understand the present invention and not to limit the scope of the present invention, all numbers used in this application to express amounts, percentages, and other numerical values ​​should be understood to be modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification are approximate values, which may be changed according to the different ideal properties to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant figures and by conventional rounding methods.

[0082] The equipment and raw materials used in the present invention can be purchased from the market or are commonly used in the art. The methods in the following examples are all conventional methods in the art unless otherwise specified.

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

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

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

[0086] Example 1

[0087] This embodiment provides a core-shell microneedle patch (Doxy / SIM-MNs, referred to as Doxy / SIM in the accompanying drawings) loaded with tetracycline antibiotics and simvastatin. The microneedle patch is 1 cm long and 1 cm wide, and includes a substrate and a plurality of microneedle bodies arranged in an array on the substrate, wherein the array is a 10×10 square array; each microneedle body has a core-shell structure and is conical in shape, with a distinct 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 includes silk fibroin and an antibacterial drug, wherein the antibacterial drug is doxycycline; the needle body shell includes polyvinyl alcohol and an antibacterial drug, wherein the antibacterial drug is doxycycline; and the needle body core includes polyvinyl pyrrolidone and Sim@PDA.

[0088] The core-shell microneedle patch loaded with tetracycline antibiotics and simvastatin described in this embodiment was prepared by the following method, with the following steps:

[0089] (1) Preparation of silk fibroin

[0090] 20 mg of silkworm cocoon shells were weighed and cut into pieces. The mixture was added to a 0.2 M Na₂CO₃ solution and heated and boiled for 20 minutes. Deionized water was added as needed during the boiling process to maintain a 250 mL reaction volume. The cocoons were removed from the boiled cocoons, rinsed several times with deionized water, and then placed back into the Na₂CO₃ solution. The heating and boiling process was repeated, and the crude silk fibroin extract was obtained after rinsing. The crude silk fibroin extract was dissolved in a 10 M LiBr solution at a mass volume ratio of 1 mg to 20 mL. The reaction was stirred for 4 hours to fully dissolve the silk fibroin fibers. The resulting product was filtered through a 300-mesh filter and then dialyzed into a dialysis bag (molecular weight cutoff 12 kDa) for 96 hours. The deionized water was changed every 4 hours during the early stages of the dialysis process and every 8 hours during the later stages. The purified silk fibroin was then freeze-dried and stored.

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

[0092] Weigh 100 mg of dopamine hydrochloride and dissolve it in 100 ml of deionized water. Stir thoroughly until the solution turns from brown to light yellow. Place the mixed solution on a magnetic stirrer and heat it in an oil bath to 50°C. Slowly add 1 mL of 1 M sodium hydroxide solution dropwise. After the addition is complete, stir the resulting reaction mixture in the dark for 24 hours. After the reaction is complete, the solution turns black. Collect the solution and centrifuge it at 11,000 rpm for 15 minutes. Remove the supernatant. Repeat the washing and centrifugation process three times to collect the polydopamine (PDA) nanoparticles, freeze-dry them, weigh them, and store them.

[0093] Weigh 40 mg of simvastatin and dissolve it in 5 ml of anhydrous ethanol. Once the simvastatin is completely dissolved, a simvastatin solution is obtained. Then, weigh 30 mg of freeze-dried PDA nanoparticles and add them to the simvastatin solution. Stir and react in the dark for 12 hours. After the reaction, centrifuge the resulting product at 11,000 rpm for 15 minutes, collect the precipitate, and repeatedly wash it with anhydrous ethanol to remove unloaded simvastatin until the supernatant turns from yellow to clear and transparent. The precipitate is collected, freeze-dried, and weighed for later use.

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

[0095] First, 10 g of polyvinyl alcohol (PVA) was weighed and dissolved in 100 mL of deionized water to prepare a polyvinyl alcohol (PVA) solution with a concentration of 10 g / mL. The mass average molecular weight (Mw) of PVA was 14.5 KD. 60 mg of doxycycline hydrochloride was weighed and dissolved in 2 mL of the PVA solution and mixed to obtain a shell matrix solution. 50 μL of the shell matrix solution was injected into the microneedle mold so that the shell matrix solution covered each micropore on the microneedle mold. The microneedle mold was then placed in a high-speed centrifuge and centrifuged 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] Weigh 50 g of polyvinylpyrrolidone K30 (PVP-K30) and dissolve it in 100 ml of deionized water to prepare a polyvinylpyrrolidone K30 (PVP-K30) solution with a concentration of 50 g / mL. Weigh 150 mg of the Sim@PDA nanoparticles prepared in step (2) and resuspend them in 10 mL of the PVP-K30 solution to obtain a core matrix solution. Take 100 μL of the prepared core matrix solution and add it to the dried microneedle mold containing the microneedle shell. Centrifuge at 4200 rpm for 5 minutes at 4°C. Repeat the injection of the core matrix solution twice and dry for 24 hours to obtain the core-shell microneedle body. Remove the excess PVP-K30 solution on the mold surface. Take 200 mg of lyophilized silk fibroin powder and dissolve it in 2 mL of deionized water to prepare a 10% (w / v) silk fibroin solution. Weigh 20 mg of doxycycline hydrochloride and dissolve it in the above 2 mL of 10 g / mL silk fibroin solution. Mix well and use it as the base solution for the microneedle patch. Do not shake violently during the dissolution process to avoid destroying the silk fibroin structure and accelerating gelation. Add 100 μL of base solution to the surface of the dried microneedle mold and evacuate at a pressure of -0.1 MPa for 5 hours. Finally, dry it in a desiccator at a constant temperature of 25°C and a constant humidity of 5% for 48 hours, and demold it to obtain a complete core-shell microneedle.

[0097] Comparative Example 1

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

[0099] Comparative Example 2

[0100] The structure of a core-shell microneedle patch loaded with simvastatin (Doxy-MNs, referred to as Doxy in the accompanying drawings) in this comparative example is the same as that of the core-shell microneedle patch Doxy / SIM-MNs loaded with tetracycline antibiotics and simvastatin in Example 1, with the only difference being that the inner core of the microneedle patch in this comparative example contains only matrix material and does not contain Sim@PDA. The other structures and preparation processes are the same.

[0101] Comparative Example 3

[0102] A blank microneedle patch (Blank-MNs, referred to as Blank in the accompanying drawings) in this comparative example does not load any drug and only contains the matrix material of the synthetic microneedle patch; its structure is the same as that of the core-shell microneedle patch Doxy / SIM-MNs loaded with tetracycline antibiotics and simvastatin in Example 1, with the only difference being that the needle core of the microneedle patch in this comparative example does not contain Sim@PDA, and neither the base nor the shell contains antibacterial drugs.

[0103] Structural and performance characterization

[0104] (1) Structural characterization of Sim@PDA nanoparticles

[0105] The Sim@PDA nanoparticles prepared in step (2) of Example 1 were subjected to correlation analysis and detection. The Sim@PDA nanoparticles were dispersed in deionized water, and the sample concentration was controlled within an appropriate range (scattered light intensity of 100 kcps-500 kcps). The sample was placed in a sample cell for particle size analysis. The Sim@PDA nanoparticles were dispersed in ethanol, and 5 ml was added dropwise to a 100-mesh carbon film copper grid. After the sample dried, transmission electron microscopy was performed at an accelerating voltage of 80 kV. The nanoparticles were evenly dispersed in anhydrous ethanol solution, and 20 μl was added dropwise to a silicon wafer. After drying at room temperature, the nanoparticles were sprayed with gold for 60 seconds using a 20 mA current gold spraying instrument. The microstructure of the nanoparticles was observed under a scanning electron microscope. The nanoparticle samples were analyzed using an ultraviolet spectrophotometer with a scanning wavelength of 200 nm-500 nm. The Sim@PDA nanoparticles were pressed into thin slices using a tablet press and placed in a sample chamber for Fourier transform infrared spectroscopy (FT-IR) testing with a scanning range of 4000 cm -1 ~500cm -1 .

[0106] Figure 1 It shows that the diameter of the Sim@PDA nanoparticles prepared in Example 1 is about 244.4±3.4 nm.

[0107] from Figure 2 The basic morphology and microstructure of Sim@PDA were observed. The results showed that the Sim@PDA prepared in Example 1 was spherical and had a uniform particle size distribution.

[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 an enhanced trend, indicating that the characteristic absorption peak of PDA has changed due to the loading of simvastatin. It is inferred that simvastatin has been successfully modified on the PDA nanoparticles.

[0109] from Figure 4 It can be seen that 1698.59 cm-1 It is the characteristic lactone carbonyl peak of simvastatin, 3550.73 cm -1 is the stretching vibration peak of simvastatin hydroxyl (OH), 1300-1000 cm -1 Multiple characteristic peaks within the range of 1:1 indicate the presence of ester groups. However, in the infrared absorption spectrum of simvastatin-loaded polydopamine nanoparticles, the characteristic absorption peak of simvastatin disappeared, and the absorption spectrum of the unloaded mesoporous polydopamine nanoparticles changed slightly. This may be due to the formation of hydrogen bonds between the carbonyl group of simvastatin and the carrier, indicating that simvastatin was successfully encapsulated in the carrier material.

[0110] (II) Morphological characterization of microneedle patches

[0111] After the microneedle patch was prepared 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 microneedles was observed using a stereofluorescence microscope. The microneedle shell was labeled with Nile Red (NR), and the microneedle core was labeled with Rhodamine 123 (Rh123). The fluorescently labeled microneedle patch was adhered to an acrylic plate, which was then mounted on a bevel mount. Under a stereofluorescence microscope, NR and Rh123 were excited at wavelengths of 510 nm to 560 nm and 455 nm to 490 nm, respectively, to image the fluorescence distribution of the shell and core of the core-shell microneedle patch. Subsequently, a single microneedle was cut horizontally and vertically with a blade, and the red and green fluorescence distributions of the cross-sections and longitudinal sections of the microneedles were imaged using a stereofluorescence microscope.

[0113] The microneedle microstructure was observed using a scanning disk electron microscope. The microneedles were bonded to a silicon wafer and gold-sprayed for 45 seconds at 20 mA to increase their conductivity. Finally, the gold-sprayed microneedles were placed in a scanning sample chamber, and the microstructure was observed at an accelerating voltage of 20 kV. Transmission scanning electron microscopy was used to scan and quantitatively analyze various elements, such as C, O, N, and Cl, in cross- and longitudinal sections of the microneedles to clearly visualize the outer shell and core structure of the core-shell microneedles.

[0114] from Figure 5 It can be seen that the area of ​​a single patch is about 0.5 cm 2 , consisting of a 10×10 microneedle array, Figure 9 The cross-section of a single microneedle clearly observed under fluorescence conditions shows a core-shell structure. Figure 6 The core-shell microneedle microstructure was further observed using SEM. The entire microneedle patch consists of 100 microneedles, each of which is sharp and conical, with a tip diameter of less than 10 μm, a height of 850 μm, and a base diameter of 400 μm. This design facilitates microneedles to penetrate the stratum corneum of the skin, achieving minimally invasive drug delivery. Figure 7 and Figure 8 The microneedle cross-section was observed using SEM, and the elements of the microneedle shell and core were quantitatively analyzed using energy dispersive X-ray spectroscopy (EDS) and elemental mapping. As shown in the figure, the cross-section of the microneedle shows a distinct layered structure. EDS quantified the elemental composition of the microneedle cross-section. The shell is primarily composed of four elements: carbon (C), oxygen (O), nitrogen (N), and chlorine (Cl), while the core is primarily composed of (C), oxygen (O), and nitrogen (N). The core does not contain chlorine. This is because only the shell is loaded with doxycycline hydrochloride, while the core does not, which confirms the successful synthesis of the core-shell structure.

[0115] (III) In vitro puncture ability evaluation of microneedle patches

[0116] The microneedle patch prepared in Example 1 was vertically inserted into fresh porcine ex vivo gums, pressed for 1 minute, and then allowed to stand for 20 minutes. The patch was removed, stained with crystal violet, and the puncture condition of the microneedle patch was observed under a microscope.

[0117] Figure 10 It shows that the microneedle patch can effectively puncture the skin, with a puncture success rate of 100%.

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

[0119] Doxycycline hydrochloride solutions with gradient concentrations were prepared. The absorbance values ​​of the different concentrations of doxycycline hydrochloride solutions at a wavelength of 350 nm were measured using UV-visible light. A standard curve for doxycycline hydrochloride was plotted with concentration as the horizontal axis and absorbance as the vertical axis. A microneedle patch containing a 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 minutes, 600 μl of supernatant was aspirated and supplemented with 600 μl of fresh PBS buffer to maintain the total amount of release medium. After collection, the absorbance value of the supernatant at a wavelength of 350 nm was measured using UV-visible light, and the cumulative release of doxycycline hydrochloride was calculated. The release curve of doxycycline hydrochloride from the microneedle substrate was determined using the same method.

[0120] from Figure 11 As can be seen from Figure A, doxycycline can reach 50% of the cumulative drug release in the microneedle shell within 30 minutes, which can increase the antibiotic concentration in the local environment in a short period of time and play a rapid antibacterial role. Figure 11 As can be seen from Figure B, doxycycline in the backing silk protein can be slowly released within two weeks, thereby playing a long-lasting antibacterial role and preventing the recurrence of the disease.

[0121] (V) In vitro biosafety of microneedle patches

[0122] The biocompatibility of the microneedle patches prepared in Example 1 and Comparative Examples 1-3 was verified by an erythrocyte hemolysis assay. First, blood was collected from healthy mice and washed repeatedly with normal saline by centrifugation until the supernatant was colorless, yielding an erythrocyte precipitate. The resulting erythrocyte precipitate was then dispersed with normal saline to obtain an erythrocyte suspension. The erythrocyte suspension was then added to ultrapure water (positive control), normal saline (negative control), or different microneedle extracts. The microneedle extracts were prepared from different microneedle patches (prepared in Example 1 and Comparative Examples 1-3) after 2 hours of UV irradiation. The extracts were then placed in PBS and allowed to dissolve gradually. The samples were incubated at 37°C for 6 hours and centrifuged at 1500 rpm for 15 minutes. The supernatants were then collected. Finally, the absorbance of the supernatants at 540 nm was measured using a microplate reader, and the erythrocyte hemolysis rate (HCR) was calculated according to the formula.

[0123]

[0124] Among them, OD T Indicates the absorbance value of the experimental group, OD N Indicates the absorbance value of the negative control group, OD P represents the absorbance value of the positive control group.

[0125] Figure 12 The results showed that after treatment with the microneedle patches prepared in Example 1 and Comparative Examples 1-3, including the Blank group, Doxy / SIM-MNs group, SIM-MNs group, and Doxy-MNs group, the red blood cell status was good and the hemolysis rate was less than 5%.

[0126] (VI) Study on the antibacterial properties of microneedle patches

[0127] The inhibition zone diameter measurement was used to verify the antibacterial properties of the microneedle patch. The microneedle patch, which had been disinfected by ultraviolet light, was immersed in sterile PBS buffer for 5 days to obtain a microneedle extract. Staphylococcus aureus (purchased from Beijing Microbiological Collection Center) and Porphyromonas gingivalis (P. g) (purchased from Shanghai Microbiological Collection Center) were inoculated into the liquid culture medium and cultured at 37°C for 12 hours until the logarithmic growth phase. The bacteria in the logarithmic growth phase were diluted to a concentration of 10 using PBS buffer. 7CFU / mL, set aside. Weigh 25 g of LB powder (Haibo Biotechnology, Catalog No. HBMU001) and dissolve it in 1 L of deionized water. Stir to dissolve, then aliquot and sterilize by autoclaving at 121°C for 15-20 minutes to obtain LB liquid medium for later use. Weigh 40 g of premixed dry powder (Haibo Biotechnology, Catalog No. HBMU002) and dissolve it in 1 L of deionized water. Heat and boil until completely dissolved, then aliquot and sterilize by autoclaving at 121°C for 15-20 minutes. Cool to approximately 50°C, then pour onto plates (15-20 mL per plate). Once solidified, obtain LB solid medium and store at 4°C until needed. Add 20 μL of liquid medium dropwise to the LB solid medium and spread the bacteria evenly with a spreading rod. Gently place different microneedle patches on the center of the culture medium. Place the solid medium in a 37°C incubator for 7 days. Measure the diameter of the inhibition zone for each group using a vernier caliper.

[0128] from Figure 13 Doxy-MNs can be seen in both A and B. The Doxy / SIM-MNs group has obvious antibacterial rings on the S. aureus and P. gingivalis plates, with diameters of 31.43±1.56mm and 79±9.73mm, respectively, which are not significantly different from the Palio ointment group. In contrast, the diameters of the antibacterial rings in the control group and the Blank-MNs group are almost 0mm. This shows that Doxy-MNs and Doxy / SIM-MNs have very good antibacterial functions, especially the inhibitory effect on the anaerobic bacteria P. gingivalis. Figure 13 As can be seen in Figures C and D, compared with the bacteria in other groups, a large number of bacteria died after treatment with the microneedles containing doxycycline (Doxy-MNs, Doxy / SIM-MNs group) and the Palio ointment group, and the bacterial survival rate was less than 10%.

[0129] (VII) In vitro evaluation of the osteogenesis-promoting effect of the microneedle patch

[0130] (1) Alkaline phosphatase staining experiment

[0131] Digest and collect C3H / 10-T1 / 2 cells (purchased from Wuhan Punosai Biotechnology Co., Ltd.) and adjust the cell concentration. Cells were seeded onto sterile slides in 12-well plates, with 1 ml of culture medium per well. After overnight culture, the culture medium was replaced for the following groups: control group (cell culture medium containing osteogenic induction solution alone), Blank-MN group (cell culture medium containing blank microneedle extract and osteogenic induction solution), Doxy-MN group (cell culture medium containing doxycycline-loaded microneedle patch extract and osteogenic induction solution), SIM-MN group (cell culture medium containing simvastatin-loaded microneedle patch extract and osteogenic induction solution), Doxy / SIM-MN group (cell culture medium containing doxycycline- and simvastatin-loaded microneedle patch extract and osteogenic induction solution), and Gel group (cell culture medium containing Palio ointment extract and osteogenic induction solution). Induction was continued for 14 days. On the 7th and 14th days, the culture supernatant was removed, the cells were washed twice with PBS, and 1 mL of 4% paraformaldehyde was added to each well to fix the cells for 30 min. The paraformaldehyde was aspirated, and the cells were washed 2-3 times with PBS. The cells were stained according to the instructions of the alkaline phosphatase (ALP) quantification kit (purchased from Beyotime Biotechnology Co., Ltd.). The 6-well plate was scanned with a scanner, and the cells were photographed with an inverted white light microscope. The ALP staining level was analyzed using Image J software.

[0132] (2) Alizarin red staining experiment

[0133] Digest and collect C3H / 10-T1 / 2 cells (purchased from Wuhan Punosai Biotechnology Co., Ltd.) and adjust the cell concentration. Cells were seeded onto sterile slides in 12-well plates, with 1 ml of culture medium per well. After overnight culture, the culture medium was replaced for the following groups: Control group (cell culture medium containing osteogenic induction solution alone), Blank-MNs group (cell culture medium containing blank microneedle extract and osteogenic induction solution), Doxy-MNs group (cell culture medium containing doxycycline-loaded microneedle patch extract and osteogenic induction solution), SIM-MNs group (cell culture medium containing simvastatin-loaded microneedle patch extract and osteogenic induction solution), Doxy / SIM-MNs group (cell culture medium containing doxycycline- and simvastatin-loaded microneedle patch extract and osteogenic induction solution), and Gel group (cell culture medium containing Palio ointment extract and osteogenic induction solution). Induction was continued for 14 days. On the 7th and 14th days respectively; the culture supernatant was removed, the cells were washed twice with PBS, and 1 mL of 4% paraformaldehyde was added to each well to fix the cells for 30 minutes; the paraformaldehyde was aspirated, the cells were washed 2-3 times with PBS, and stained according to the operating instructions of the Alizarin Red Staining Kit (purchased from Beyotime Biotechnology Co., Ltd.); the 6-well plate was scanned with a scanner, the cells were photographed with an inverted white light microscope, and the Alizarin Red staining level was analyzed using Image J software.

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

[0135] On the 7th day after induction, C3H / 10-T1 / 2 cells were digested and collected, washed twice with PBS, and 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 three times with PBS. The paraformaldehyde was aspirated and the cells were washed twice with PBS. 1 mL of 0.25% TritonX-100 was added to each well for nuclear disruption. The cell slides were gently removed from the 6-well plate using cell forceps and placed on a glass slide with the cell surface facing up. 5% BSA was used to stain the cells. The prepared corresponding primary antibody dilution (rabbit anti-OCN monoclonal antibody volume ratio 1:500, purchased from Wuhan Abotek Biotechnology Co., Ltd., catalog number A2851; rabbit anti-RUNX2 monoclonal antibody volume ratio 1:500; purchased from Wuhan Abotek Biotechnology Co., Ltd., catalog number A20800) was incubated at 4°C for 12 hours; the primary antibody dilution was aspirated, and the cell slides were gently washed twice with PBS for 10 minutes each time, and then incubated in the dark for 2 hours with the fluorescent secondary antibody dilution corresponding to the primary antibody (volume ratio 1:200); the slides were removed in a dark environment, the cells were washed twice with PBS, 50 μL of LDAPI stain was added to stain the cell nuclei, and incubated in the dark for 10 minutes; in a dark environment, the cells were washed twice with PBS, and then 20 μL of anti-fluorescence quencher was added for sealing; laser confocal microscopy was used to take pictures and quantitative analysis was performed with ImageJ software.

[0136] Figure 14 Staining results showed that all groups except the control and Blank-MNs groups produced a certain amount of ALP on day 7. ALP levels increased significantly in all groups by day 14. The Doxy / SIM-MNs group showed significantly higher ALP levels than the other groups at both days 7 and 14. Alizarin red (ARS) staining of C3H10T1 / 2 cells in each group after osteogenic differentiation induction revealed the highest number of calcium nodules in the Doxy / SIM-MNs group, followed by the SIM-MNs group, at both time points. The Doxy-MNs and Gel groups also showed significantly higher levels of calcium nodules compared to the control group. The Blank-MNs group had the fewest red calcium nodules. This suggests that simvastatin is more effective in promoting osteogenic differentiation.

[0137] Figure 15 Through the statistical analysis of the intensity of positive expression, it can be concluded that the osteogenic differentiation markers RUNX2 and OCN are also Figure 14ALP staining and Alizarin red staining showed similar results. That is, the fluorescence intensity of the Doxy / SIM-MNs group was the strongest, significantly higher than that of the SIM-MNs group, Doxy-MNs group, and Gel group.

[0138] from Figure 16 It can be seen that compared with the control group, the mRNA expression levels of the four osteogenic differentiation markers in the Doxy / SIM-MNs group and the SIM-MNs group were higher.

[0139] (8) Evaluation of the efficacy of microneedle patches in a rat model of diabetic periodontitis

[0140] (1) Establishment of diabetic periodontitis model in rats and intervention treatment

[0141] Six-week-old Sprague Dawley rats were fed a high-glucose, high-fat diet for two weeks. After a 12-hour fast, streptozotocin (STZ) solution was intraperitoneally injected at a dose of 35 mg / kg body weight. Random blood glucose levels were measured via tail vein on days 3, 5, 7, and 10 after STZ injection, and an oral glucose tolerance test (OGTT) was performed. Successful diabetic modeling was established when random blood glucose levels ≥16.7 mmol / L. Rats that did not meet this standard were excluded from further experiments. On the second day after STZ intraperitoneal injection, the periodontitis model was established. Anesthesia was performed by intraperitoneal injection of 1% sodium pentobarbital (40 mg / kg). The gingiva was initially separated using an oral probe, and the gap between the maxillary first molar and the second molar was appropriately increased with insulin. 3-0 surgical sutures were wrapped around the 0.2 mm orthodontic ligature. The 0.2 mm ligature was gently passed between the first and second molars using a needle holder, and then the first molar was encircled and fixed with a ligature. Subsequently, a solution containing 1×10 8A suspension of Porphyromonas gingivalis (CFU / mL) was injected into the gingival sulcus and the sutures of the ligature. Injections were performed every three days, and the ligature was checked for loosening. If the ligature became loose or fell off, it was promptly re-ligated. After two weeks of placement of the ligature in the gingival sulcus of the rats' first molars, the ligature was removed, and the rats were given different experimental interventions based on their respective experimental groups. Thirty-six SD rats with established diabetic periodontitis models were randomly divided into six groups: Control, Blank, Doxy, SIM, Doxy / SIM, and Gel. The control group received no treatment; the Blank group received a blank microneedle patch without any drug; the Doxy group received a microneedle patch with doxycycline as the outer shell; the SIM group received a microneedle patch with simvastatin-dopamine nanoparticles as the inner core; the Doxy / SIM group received a microneedle patch loaded with both doxycycline and simvastatin; and the Gel group received Palio Ointment (minocycline hydrochloride ointment). Rats were sacrificed after 2 weeks of treatment, and bilateral maxillary bones were harvested.

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

[0143] Figure 18 In order to detect the random blood glucose level in the tail vein after the diabetic model is established in rats, rats with blood glucose levels <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 in animals

[0145] Specimens were scanned, reconstructed, and analyzed using microcomputed tomography (Micro-CT) with scanning parameters set at 70 kVp, 114 μA, and a pixel size of 17.5 μm. Alveolar bone loss (CEJ-ABC) was determined by measuring the distance between the cemento-enamel junction (CEJ) and the alveolar bone crest (ABC) of the maxillary first molar. Specimens for embedding were trimmed and rinsed in saline. Samples were decalcified in ethylenediaminetetraacetic acid (EDTA) for four weeks, dehydrated through a gradient of 70% to 100% alcohol, cleared in xylene, and immersed in paraffin. Blocks were embedded in paraffin using a paraffin embedding machine and sectioned at a thickness of 5 μm. Hematoxylin and Erythrocyte Embedding (H&E) staining was performed.

[0146] from Figure 19 It can be seen that after the diabetic periodontitis rat model was treated with Doxy / SIM-MNs microneedle patches, the distance from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) was significantly reduced from 4.2 mm to 1.7 mm. This shows that the use of Doxy / SIM-MNs microneedle patches can greatly improve the degree of alveolar bone absorption in diabetic periodontitis rats and promote periodontal tissue regeneration.

Claims

1. A core-shell microneedle patch loaded with tetracycline antibiotics and simvastatin, characterized by: The microneedle patch includes a substrate and a microneedle body arranged on the substrate, wherein the microneedle body has a core-shell structure; wherein the outer shell of the microneedle body and the substrate are both loaded with tetracycline antibiotics; and the inner core of the microneedle body contains polydopamine nanoparticles Sim@PDA loaded with simvastatin.

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

3. The core-shell microneedle patch according to claim 1, characterized in that: The Sim@PDA is spherical and has an average particle size of 200 to 300 nm.

4. The method for preparing the core-shell microneedle patch according to claim 1, wherein: The steps include: Preparation of microneedle shells: tetracycline antibiotics and a body-soluble polymer material solution 1 are mixed according to a ratio to obtain a shell matrix solution; an appropriate amount of the shell matrix solution is then injected into a microneedle mold, centrifuged until fully filled, and dried to obtain a microneedle shell; Preparation of microneedle core: Sim@PDA nanoparticles and body-soluble polymer material solution 2 are mixed according to the ratio to obtain a core matrix solution; Then, an appropriate amount of the core matrix solution is injected into the microneedle mold containing the microneedle shell, and centrifuged until fully filled; the injection and centrifugation process is repeated 0 to 3 times; and finally, drying is performed to obtain the microneedle core; Preparation of the substrate: tetracycline antibiotics and soluble biocompatible material solution are mixed in a ratio to obtain a substrate solution; then an appropriate amount of the substrate solution is laid on the above-mentioned microneedle mold containing the microneedle shell and the core, and the mold is demolded after vacuum drying to obtain the core-shell microneedle patch loaded with tetracycline antibiotics and simvastatin.

5. The preparation method according to claim 4, characterized in that: The preparation method of the Sim@PDA nanoparticles is as follows: Polydopamine (PDA) nanoparticles are added to a simvastatin solution according to a ratio, and stirred in the dark for 5 to 12 hours. After the reaction, the obtained product is centrifuged, and the precipitate is collected, washed, and dried to obtain the Sim@PDA nanoparticles.

6. The preparation method according to claim 5, characterized in that: The mass ratio of the PDA nanoparticles to simvastatin is 1:1 to 1:

2.

7. The preparation method according to claim 5, characterized in that: The preparation method of the PDA nanoparticles is specifically as follows: Dopamine hydrochloride and deionized water are mixed according to a ratio, stirred and dissolved evenly to obtain a dopamine hydrochloride solution; then, sodium hydroxide solution is slowly added dropwise to the dopamine hydrochloride solution at 40-60° C. according to the ratio, and after the addition is complete, the resulting reaction solution is stirred in the dark for 20-30 hours; after the reaction is completed, the resulting product is centrifuged, the precipitate is collected, and the resulting precipitate is further washed and freeze-dried to obtain the PDA nanoparticles.

8. The preparation method according to claim 7, characterized in that: The dosage ratio of the dopamine hydrochloride to the sodium hydroxide solution is 100 mg:1 mL.

9. Use of the core-shell microneedle patch loaded with tetracycline antibiotics and simvastatin according to any one of claims 1 to 3 in the preparation of a drug and / or a drug delivery system for treating diabetic periodontitis.

10. A drug and / or drug delivery system, characterized in that: A core-shell microneedle patch loaded with tetracycline antibiotics and simvastatin according to any one of claims 1 to 3.

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