A microneedle submucosal controlled release drug delivery system and its preparation method and application

Abstract

The present application provides a microneedle submucosal controlled-release drug delivery system and a preparation method thereof. The microneedle submucosal controlled-release drug delivery system of the present application is in the form of a microneedle patch with gelatin as a matrix and chlorhexidine gluconate as a drug carrier. By controlling the drug loading amount and the amount of cross-linking agent used, the microneedle submucosal controlled-release drug delivery system of the present application achieves good microneedle physical properties and drug release performance, and can be used to treat diseases such as periodontitis.

Description

Field of the Invention

[0001] The present application belongs to the technical field of pharmaceutical preparations. Specifically, the present application provides a microneedle submucosal controlled-release drug delivery system and a preparation method thereof. Background Art

[0002] Drugs can help people fight diseases and improve their health. The routes of drug delivery are also rich and varied, and they are constantly evolving. Traditional routes of drug administration include oral, inhalation, injection, skin absorption, etc. However, these drug delivery routes all have corresponding limitations. Oral administration needs to consider gastrointestinal absorption issues, which will increase the burden on the liver and kidneys, and the first-pass effect will affect the therapeutic effect of the drug; inhalation administration mainly delivers drugs to the lungs through the respiratory tract, and this method can only deliver a limited number of drugs; drug injection, mainly including intramuscular injection, intravenous injection and subcutaneous injection, will cause pain and fear to patients; the skin absorption route absorbs the drugs applied to the skin, but the amount of drugs absorbed in this way is very limited, and only small molecules and fat-soluble drugs can be delivered.

[0003] The microneedle (MN) patch is a new type of drug delivery system that integrates an array of tiny needle-shaped structures on a chip. Microneedles can penetrate the skin or mucous membranes to achieve enhanced drug delivery. The skin is the barrier part of the human body that contacts the outside world and is the largest organ in the human body. Its structure includes the stratum corneum, epidermis and dermis. Microneedles can pass through the stratum corneum and epidermis to provide a channel for transdermal delivery of drugs without touching the blood vessels and nerves in the dermis, thus avoiding the patient's pain. The oral mucosa includes the epithelium and the lamina propria, of which the epithelium is only keratinized at the hard palate. Compared with the skin, it lacks the outermost stratum corneum, and has lower requirements on the mechanical properties of the inserted microneedles. The oral mucosa is rich in capillaries, nerve endings, and immune cells. Therefore, the oral mucosa has broad prospects in the field of microneedles. The application of microneedles in the oral cavity mainly includes drug delivery, vaccination, diagnosis and treatment of oral cancer, etc. This paper focuses on the application of microneedles in oral mucosal drug delivery, hoping to design a microneedle patch for the treatment of periodontitis, overcome the shortcomings of traditional periodontitis drug delivery methods, provide new ideas for periodontitis drug treatment, and contribute to maintaining periodontal health.

[0004] Periodontitis is a chronic infectious inflammatory disease, initially caused by infection with periodontal pathogens, which eventually leads to alveolar bone resorption and tooth loss, seriously affecting the patient's oral function and appearance, and causing a burden on the family and society. Studies have shown that periodontitis is not only directly related to oral health, but also to some systemic diseases, such as diabetes, cardiovascular and cerebrovascular diseases, chronic obstructive pulmonary disease, and Alzheimer's disease.

[0005] At present, the main treatment for periodontitis is to mechanically remove infected substances, scrape away tartar and plaque, and remove diseased cementum to form a smooth and flat root surface, which is conducive to the attachment and growth of periodontal tissues. However, pathogens deep in the periodontal pocket cannot usually be completely eliminated by physical measures, and the proliferation and metabolism of bacteria will seriously affect the treatment effect of periodontitis. In order to effectively inhibit bacteria, antibacterial drugs are usually required for auxiliary treatment. However, systemic application of antibiotics will produce side effects and drug resistance, and the actual drug concentration reaching the lesion site is very limited. Therefore, local administration in the periodontal pocket is a better choice, which can reduce the amount of drugs, reduce gastrointestinal side effects and reduce the frequency of administration, providing a safe and effective treatment mode. Therefore, many new local drug delivery systems have been developed, such as various local implants such as drug-loaded fibers, films, gels or particles. Researchers intend to directly administer drugs in the pocket to maintain the corresponding drug concentration at the lesion site and enhance the treatment effect. Although these methods achieve sustained release of drugs in the periodontal pocket, since the oral cavity is a dynamic environment often accompanied by functional movements such as pronunciation and chewing, stable adhesion of materials and tissues is a challenge. In addition, due to the destruction of periodontal tissues, invasion of pathogens and toxins can often be detected in the deep periodontal tissues. However, these current methods of drug administration make it difficult to achieve transepidermal delivery of drugs to deep tissues.

[0006] Microneedle technology can achieve transdermal drug delivery while enhancing the stable adhesion between materials and tissues. Integrating a microneedle array that is tens to hundreds of microns high and can penetrate the surface of the skin or mucosa on a chip can provide a channel for drug transportation in a minimally invasive and painless manner, and the structure of the microneedle also enhances the adhesion strength between the material and the soft tissue. Since the concept of microneedles was proposed, people have invented various methods for making microneedles, including laser ablation, photolithography, additive manufacturing, micro-molding, etc.; the types of microneedles are also mainly divided into solid microneedles, hollow microneedles, coated microneedles and dissolving microneedles. Starting from the shortcomings of the existing periodontitis drug delivery system, analyzing the needs of periodontitis drug delivery, the materials need to meet the following conditions: good biocompatibility of the matrix material, good stability, sustained transdermal release, no tissue cytotoxicity, sufficient mechanical strength and effective sterilization ability. Summary of the invention

[0007] Therefore, this project intends to use the micro-molding method to construct microneedles. This method is relatively low-cost, easy to operate, and does not require specific instruments; gelatin with good biocompatibility and non-toxicity is used as the matrix of the dissolving microneedle membrane; and chlorhexidine gluconate, which has been widely used in clinical practice and has a broad-spectrum antibacterial effect on Gram-negative anaerobic bacteria, the main microorganisms of dental plaque, is used as an antibacterial agent. This study intends to design a submucosal microneedle drug delivery system to address the current problems of local application of oral drugs. After the basic treatment of periodontitis patients, the microneedle patch will be implanted in the periodontal pocket to improve the therapeutic effect of periodontitis through the sustained release effect of the microneedle and the enhanced transmucosal delivery effect. At the same time, this study also hopes to conduct follow-up research on the controlled release of microneedle patches and expand its application in pain relief after tooth extraction and promoting the healing of oral ulcers.

[0008] On the one hand, the present application provides a microneedle submucosal controlled-release drug delivery system, which uses gelatin as a matrix and contains an antibacterial agent.

[0009] Furthermore, the microneedle submucosal controlled-release drug delivery system is in the form of a microneedle patch.

[0010] Furthermore, the microneedle sub-membrane controlled-release drug delivery system is in the form of a microneedle patch suitable for periodontal pocket sites.

[0011] Furthermore, the microneedle submucosal controlled-release drug delivery system comprises a microneedle and a base portion.

[0012] Furthermore, the antibacterial agent is chlorhexidine gluconate.

[0013] Furthermore, the amount of the antibacterial agent contained in the microneedle and the base part is different.

[0014] Furthermore, the antibacterial agent content of the microneedle part is 10-25%, and the antibacterial agent content of the base is 30-50%.

[0015] Furthermore, the antibacterial agent content of the microneedle part is 13-16%, while the antibacterial agent content of the base is 35-45%.

[0016] Furthermore, the antibacterial agent content of the microneedle part is 14.3%, while the antibacterial agent content of the base is 40%.

[0017] Furthermore, the microneedle submucosal controlled-release drug delivery system is prepared using a micromolding method.

[0018] On the other hand, the present application provides a method for preparing a microneedle submucosal controlled-release drug delivery system, wherein the preparation method is a micro-molding method.

[0019] Furthermore, the micro-mold preparation process includes: preparing a mold; preparing a needle solution; and preparing a microneedle patch.

[0020] Furthermore, the mold is a PDMS mold.

[0021] Furthermore, the needle-forming solution comprises gelatin, glycerol, chlorhexidine gluconate and glutaraldehyde.

[0022] Furthermore, the preparation method of the needle-forming solution is as follows: (a) taking 1-4 g of gelatin, adding 20 mL of ultrapure water, stirring under heating conditions, and preparing a 5-20% gelatin solution; taking 0.5-2 g of glycerol, adding 0.5 g-2 g of a 98% mass volume fraction chlorhexidine gluconate solution, and pouring into the gelatin solution; (b) taking 0.3-0.5 mL of a 2.5% glutaraldehyde solution, adding 2 mL of ultrapure water, diluting it into a glutaraldehyde solution, pouring it into the solution obtained in (a), and stirring under heating conditions to obtain a needle-forming solution.

[0023] Furthermore, the steps for preparing the microneedle patch include: (A) loading the needle-forming solution into a mold; (B) evacuating the mold; (C) placing the mold in an oven for heating; (D) absorbing the needle-forming solution with bubbles and replenishing the needle-forming solution; (E) evacuating the mold; (F) repeating (C) (D); and (G) demolding after drying.

[0024] Furthermore, the vacuuming time in steps (B) and (E) is 5-20 seconds; and the step (C) is placed in an environment of 50-70° C. for 3-10 minutes.

[0025] Furthermore, the vacuuming time in steps (B) and (E) is 10 seconds; and the step (C) is placed in an environment of 60° C. for 5 minutes.

[0026] Furthermore, the microneedle patch preparation step includes a separate microneedle preparation step and a separate base preparation step.

[0027] Furthermore, the preparation method comprises:

[0028] (1) Take 1-4 g of gelatin and add 20 mL of ultrapure water to prepare a 5-20% gelatin solution;

[0029] (2) Take 0.5-2 g of glycerol and 0.5-2 g of 98% mass volume fraction chlorhexidine gluconate solution, pour into the gelatin solution to prepare a microneedle tip forming preparatory solution, recorded as solution A;

[0030] (3) Take 0.5-2 g of glycerol and 0.5-2 g of 98% mass volume fraction chlorhexidine gluconate solution, pour into the gelatin solution and prepare the microneedle base molding preliminary solution, which is recorded as solution B;

[0031] (4) Take 0.3-0.5 mL of 2.5% volume fraction glutaraldehyde solution, add 2 mL of ultrapure water, dilute to glutaraldehyde solution, prepare crosslinker solution, and record it as solution C;

[0032] (5) Solution C is added into solution A and solution B, respectively, to obtain microneedle tip forming solution AC and microneedle base forming solution BC, respectively.

[0033] Furthermore, the preparation method comprises:

[0034] (1) Take 2 g of gelatin and add 20 mL of ultrapure water to prepare a 10% gelatin solution;

[0035] (2) Take 1 g of glycerol and 0.5 g of 98% mass volume fraction chlorhexidine gluconate solution, pour into the gelatin solution to prepare the microneedle tip forming preparatory solution, recorded as solution A;

[0036] (3) Take 1 g of glycerol and 2 g of 98% mass volume fraction chlorhexidine gluconate solution, pour into the gelatin solution and prepare the microneedle base molding preparatory solution, which is recorded as solution B;

[0037] (4) Take 0.5 mL of 2.5% volume fraction glutaraldehyde solution, add 2 mL of ultrapure water, dilute to 0.5% glutaraldehyde solution, prepare cross-linking agent solution, recorded as solution C;

[0038] (5) 2.5 mL of solution C was added to solution A and solution B, respectively, to obtain microneedle tip forming solution AC and microneedle base forming solution BC, respectively. The concentration of glutaraldehyde in microneedle tip forming solution AC and microneedle base forming solution BC was 0.05%.

[0039] Furthermore, the preparation method also includes

[0040] (6) Transfer solution AC into the microneedle PDMS mold and evacuate the mold;

[0041] (7) placing the microneedle PDMS mold containing solution AC after vacuum suction in an oven until the gel solution is completely liquefied;

[0042] (8) Aspirate the solution containing bubbles at the base of the microneedle PDMS mold;

[0043] (9) Pour solution BC into the microneedle PDMS mold using a dropper and evacuate;

[0044] (10) Remove the microneedle mold and repeat steps (7)-(8);

[0045] (11) Drying and demoulding.

[0046] Furthermore, the vacuuming time in steps (6) and (9) is 5-20 seconds; the oven temperature in step (7) is 50-70° C., and the mixture is placed in the oven for 3-10 minutes.

[0047] Furthermore, the vacuuming time in steps (6) and (9) is 10 seconds; the oven temperature in step (7) is 60° C., and the mixture is placed in the oven for 5 minutes.

[0048] Furthermore, the microneedle tip is in the shape of a quadrangular pyramid, the microneedle height is 500-1000 μm, and the length of the bottom side of the quadrangular pyramid is 100-300 μm. The size of the substrate of the microneedle base is 0.2-1×0.2-1 cm 2 , base thickness 200μm-500μm.

[0049] Furthermore, the microneedle tip is in the shape of a quadrangular pyramid, the microneedle height is 800 μm, and the length of the bottom side of the quadrangular pyramid is 200 μm. The size of the substrate of the microneedle base is 0.5×0.5 cm 2 , the base thickness is between 200μm-500μm.

[0050] On the other hand, the present application provides the use of the above-mentioned microneedle submucosal controlled release drug delivery system in the preparation of drugs or devices for treating oral inflammatory diseases.

[0051] Furthermore, the oral infectious disease is periodontitis.

[0052] Furthermore, the oral infectious disease is gingivitis.

[0053] Furthermore, the medicine or device also includes packaging BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Schematic diagram of a microneedle submucosal controlled release drug delivery system;

[0055] Figure 2 The degradation rates of microneedles with different cross-linking degrees in an ideal environment (left) and a simulated oral environment (right);

[0056] Figure 3 The drug release rates of microneedles with different cross-linking degrees in an ideal environment (left) and an oral simulated environment (right).

[0057] Figure 4 The results of CCK8 proliferation experiment (the bar graphs from left to right are A. control group, B. non-drug-loaded microneedle group (no drug, other parts are the same as drug-loaded microneedles), C. drug-loaded microneedle group, D. drug-loaded ordinary membrane group (the ingredients are the same as drug-loaded microneedles, but without microneedle parts), E. Periochip Group);

[0058] Figure 5 The results of antibacterial experiment.

[0059] Figure 6 is the Young's modulus of the microneedle patch at different drug loadings;

[0060] Figure 7 The simulation results of deformation and load of the tip of the microneedle patch at different drug loadings;

[0061] Figure 8 These are the results of the microneedle patch transdermal test at different drug loading levels. DETAILED DESCRIPTION

[0062] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments are for demonstration purposes only, and the protection scope of the present invention is defined by the claims and is not limited to the following embodiments.

[0063] Example 1 Preparation of mold

[0064] Common preparation processes for microneedles include laser ablation, photolithography, micro-molding, injection molding, additive manufacturing, etc. Among them, the micro-molding method has become a common preparation method for dissolving microneedles due to its simple operation, low cost, good effect, and large-scale production. The micro-molding method uses polydimethylsiloxane (PDMS) as a mold because of its low cost, easy use, low surface energy, and good thermal stability.

[0065] The PDMS mold is made by molding or micromachining, and the prepared microneedle material solution is poured into the PDMS mold, and then vacuumed or centrifuged to allow the solution to completely fill the needle body of the mold. After the solution is dry, the microneedle can be demoulded.

[0066] Example 2 Preparation of injection solution

[0067] After obtaining the PDMS mold, the first step in preparing the microneedle is to prepare the solution for needle formation. The solution is composed of gelatin, glutaraldehyde, chlorhexidine gluconate, and glycerol. The specific preparation method is as follows: 1. Take 2g of gelatin, add 20mL of ultrapure water, and stir continuously under heating conditions at 60°C to make a 10% gelatin solution. 2. Take 1g of glycerol and 2g of chlorhexidine gluconate solution (98% mass volume fraction), pour into the gelatin solution and stir evenly. 3. Take 0.5mL of glutaraldehyde solution (2.5% volume fraction), add 2mL of ultrapure water, dilute to 0.5% glutaraldehyde solution, pour in the solution, stir for 30s under heating conditions, and you can get the needle-forming solution.

[0068] During the preparation of the solution, the amounts of chlorhexidine gluconate and glutaraldehyde added can be adjusted. Changing the amount of chlorhexidine gluconate added can adjust the drug loading of the microneedle patch; changing the amount of glutaraldehyde added can adjust the cross-linking degree of the microneedle patch, thereby changing its mechanical properties, sustained-release properties, etc.

[0069] In this embodiment, the solution prepared by the above data has a drug loading of 40% and a cross-linking agent concentration of 0.05%, which can be freely adjusted.

[0070] Example 3 Preparation of microneedle patch

[0071] The prepared solution should be used for the production of microneedle patches as soon as possible. The specific production process is as follows: 1. Use a dropper to transfer the prepared solution into the PDMS mold. 2. Place the mold in a vacuum container, turn on the vacuum pump, and evacuate for 10 seconds. It should be noted that the temperature in the container will drop during the vacuum process, and the solution will solidify rapidly due to the drop in temperature during the vacuum process. The results of microneedle preparation show that the sol solution can enter the tiny needle body part of the PDMS mold, but the part of the microneedle base will be irregular in shape due to the rapid solidification of the solution into a gel state, and many holes and bubbles will remain.

[0072] To solve this problem. 3. Put the solidified gel solution after vacuuming together with the PDMS mold into an oven and place it at 60°C for 5 minutes. After the gelatin of the microneedle base is completely liquefied, take it out, use a dropper to suck out the solution with bubbles, and then add the solution to fill the mold. 4. Vacuum again for 10 seconds and repeat step 3. 5. Place the PDMS mold filled with microneedle solution for 24 hours, and demold it after it is completely dry to obtain a microneedle patch. Since chlorhexidine gluconate has good stability at room temperature, the obtained microneedle patch can be stored at room temperature for a considerable period of time.

[0073] The content of chlorhexidine gluconate or glutaraldehyde can be changed in the process of needle solution configuration to adjust the mechanical properties and sustained-release characteristics of the patch. The rise of drug loading can significantly reduce the elastic modulus of the material, and the rise of glutaraldehyde content can increase the elastic modulus of the material and delay the time of drug release. The applicant has obtained through experiments that the maximum concentration of glutaraldehyde cross-linking is 0.05%. If it exceeds this concentration, the solution configured will solidify, which is very viscous and difficult to pour into the PDMS mold. Therefore, in order to extend the sustained-release time of the microneedle patch as much as possible, the concentration of glutaraldehyde is set to 0.05% when cross-linking.

[0074] In the exploration of the mechanical properties of microneedles, only the axial force test results are often not enough to fully demonstrate that they have excellent mechanical strength. In order to ensure that the microneedles can penetrate the skin or mucous membranes, the actual microneedle insertion situation needs to be tested. When the microneedle patches without drug loading and with a drug loading of 14.3% were removed, the microneedle array left obvious array marks on the skin, accompanied by skin redness, which verified the penetration of the microneedles on the skin surface; after removal, the skin redness gradually weakened over time until it disappeared, which proved the minimally invasive nature of the microneedles. When the microneedle patches with drug loadings of 25% and 40% were pressed, the marks left on the skin were not obvious, and it can be considered that it is difficult to penetrate the stratum corneum and epidermis. As the drug loading gradually increases, the mechanical properties of the microneedles decrease.

[0075] In order to ensure the mechanical strength of the microneedle body and increase the drug loading of the microneedle, the following microneedle submucosal controlled release drug delivery system was prepared, wherein the microneedle patch needle tip carries 14.3% chlorhexidine gluconate to ensure sufficient skin penetration strength of the microneedle; wherein the drug loading concentration of the microneedle patch base is 40% to increase the overall drug loading of the microneedle patch. The specific preparation of the microneedle submucosal controlled release drug delivery system is as follows:

[0076] 1. Take 2g of gelatin, add 20mL of ultrapure water, heat at 60℃ and stir continuously to make a 10% gelatin solution;

[0077] 2. Take 1g glycerol and 0.5g chlorhexidine gluconate solution (98% mass volume fraction), pour into the gelatin solution and stir evenly to prepare the microneedle tip forming preparatory solution, which is recorded as solution A;

[0078] 3. Take 1g glycerol and 2g chlorhexidine gluconate solution (98% mass volume fraction), pour into the gelatin solution and stir evenly to prepare the microneedle base molding preparatory solution, which is recorded as solution B;

[0079] 4. Take 0.5 mL of glutaraldehyde solution (2.5% volume fraction), add 2 mL of ultrapure water, dilute to 0.5% glutaraldehyde solution, prepare crosslinker solution, record as solution C;

[0080] 5. Add 2.5 mL of solution C to solution A and solution B respectively, and stir for 30 seconds under heating conditions to obtain microneedle tip forming solutions, which are referred to as solution AC and microneedle base forming solutions, which are referred to as solution BC;

[0081] 6. Use a dropper to transfer solution AC into the microneedle PDMS mold and place it in a vacuum pump to evacuate for 10 seconds;

[0082] 7. Place the vacuum-sucked microneedle PDMS mold containing solution AC in an oven for 5 min until the gel solution is completely liquefied;

[0083] 8. Use a dropper to suck out the solution with bubbles at the base of the microneedle PDMS mold;

[0084] 9. Use a dropper to transfer solution BC into the microneedle PDMS mold and place it in a vacuum pump to evacuate for 10 seconds.

[0085] 10. Remove the microneedle mold and repeat steps 7-8.

[0086] 11. Dry overnight at room temperature and demold to obtain a microneedle submucosal controlled release drug delivery system with different drug loading amounts on the needle body and base.

[0087] Therefore, in order to take into account both the drug loading capacity and mechanical strength of the microneedle, the drug loading capacity of the microneedle body is set to 14.3%, and the drug loading capacity of the base can be freely selected according to actual usage requirements.

[0088] Example 4 Microneedle drug loading performance test

[0089] After simulating and testing the mechanical properties of the microneedles and ensuring their transdermal performance, other properties of the microneedles need to be tested. After the microneedles are implanted in the periodontal pocket, they will slowly degrade in the periodontal pocket and continuously release the drug. In order to determine how long the microneedles can maintain sustained release in the mouth and the concentration of the released drug, it is necessary to test the sustained release performance of the microneedles, which includes the degradation rate of the patch and the drug release rate. At the same time, in order to verify that the drugs carried by the microneedles can be released smoothly and effectively inhibit bacteria, the antibacterial properties of the microneedle patch also need to be tested.

[0090] A major feature of dissolving microneedles made of polymers is that the microneedles can gradually dissolve in the body to achieve drug release and diffusion. In this study, the applicant selected gelatin as the microneedle matrix material, which is a water-soluble material that can slowly degrade in the body and release drugs slowly. The use of glutaraldehyde cross-linking can effectively improve the stability of the gelatin material and slow down its degradation rate. It is very meaningful to explore the sustained-release properties of microneedle patches. It is necessary to confirm the time the patch can stay in the periodontal pocket and the time it can maintain an effective antibacterial concentration, and use this as a reference to determine the time to replace the patch.

[0091] Changing the concentration of the cross-linking agent will change the degree of cross-linking of gelatin and change its sustained-release properties. Therefore, the applicant conducted sustained-release tests on a series of microneedle patches with different degrees of cross-linking, including patch degradation rate and drug release rate. In terms of test conditions, two environments were considered: an ideal environment consisting of excess water and an oral simulation environment consisting of an appropriate amount of artificial saliva.

[0092] The gelatin-based microneedle patch will slowly degrade in an aqueous environment. In order to test its specific degradation rate, the mass of the patch before and after degradation is measured, and the mass is compared to obtain the degradation rate of the microneedle patch at different times. The specific experimental process is as follows: 1. Microneedle patch production. The microneedle patch is made according to the microneedle patch production method. The microneedle patch needs to change the content of glutaraldehyde added to prepare patches with different cross-linking degrees. The cross-linking concentrations selected by the applicant are 0, 0.03%, and 0.05%, respectively, so the amount of glutaraldehyde added is 0, 0.3, and 0.5mL (before dilution). 2. Construction of two test environments. Ideal environment with excess water: take 10mL of ultrapure water and place it on a 37°C hot stage; simulate the oral environment: take 3mL of artificial saliva (CMC) and place it on a 37°C hot stage. 3. Rate test. Weigh the microneedle patch and put it in the test environment. After a period of time, take out the microneedle patch, remove the liquid in the test environment, and dry the remaining undegraded part to remove moisture. Weigh it afterwards. 4. The degradation rate of the patch can be obtained by comparing the mass of the degraded patch with the mass of the original patch.

[0093] Depend on Figure 2 It can be observed that in both environments, as the concentration of the cross-linking agent increases, the degree of cross-linking increases, and the degradation rate of the patch decreases significantly. Under the simulated oral environment test, the microneedle patch cross-linked by 0.05% glutaraldehyde degraded by about 60% within a week, indicating that the degradation performance of the patch is better than the commonly used commercial drug Palio. It is hoped that the obtained patch can stay in the oral periodontal pocket for as long as possible to achieve continuous degradation and drug release, so a microneedle patch made with a cross-linking agent concentration of 0.05% was selected.

[0094] Example 5 Drug Release Rate

[0095] Experimental Materials:

[0096] In the drug-loaded microneedle prepared in Example 3, the drug loading amount of the needle tip is 14.3%, while the drug loading amount of the microneedle base is 40%.

[0097] The gelatin microneedle patch carries chlorhexidine gluconate as an antibacterial drug, which can inhibit microorganisms in dental plaque in the periodontal pocket. In addition to patch degradation, the more important thing about the sustained-release performance of the patch is to test the release rate of the drug in the patch. For this reason, the drug release rate of chlorhexidine gluconate was tested under two different environments.

[0098] Similar to the test of patch degradation rate, microneedle patches with cross-linking concentrations of 0 and 0.05% were made and placed in two constructed test environments. Sampling and spectrophotometric testing were performed after a certain period of time. The drug concentration at different times was obtained according to the standard concentration curve, and then the drug release rate was obtained.

[0099] The experimental results are as follows Figure 3As shown: It can be observed that after cross-linking with glutaraldehyde, the drug release rate of the microneedle is significantly reduced. In the drug release test simulating the oral environment, about 80% of chlorhexidine gluconate was released within a week of release. Therefore, the effective antibacterial concentration can be maintained in the oral cavity for up to one week. This reflects the excellent sustained-release performance of the microneedle patch. After using the microneedle patch, patients can avoid continuous medication within a week.

[0100] Example 6 CCK8 proliferation assay and antibacterial assay

[0101] Experimental Materials:

[0102] In the drug-loaded microneedle prepared in Example 3, the drug loading amount of the needle tip is 14.3%, while the drug loading amount of the microneedle base is 40%.

[0103] CCK8 proliferation assay procedures

[0104] Periodontal ligament stem cells (PDLSCs) were plated at 4*10 per well. 4 / well were inoculated in a 24-well plate and divided into: A. control group, B. non-drug-loaded microneedle group (no drug, other parts are the same as drug-loaded microneedles), C. drug-loaded microneedle group, D. drug-loaded ordinary membrane group (the composition is the same as drug-loaded microneedles, but without microneedle parts), E. Periochip Groups; 3 replicates per group. After 12 hours, cells were observed under a microscope to be attached to the wall and to have normal morphology. Different membrane materials were added to each well according to the grouping situation. CCK8 reagent (Beijing Langbolid Trading Co., Ltd., CK001) was added at baseline, 12 hours, 24 hours, and 48 hours, respectively. After 2 hours, the absorbance at 450nm was measured with an enzyme-labeled instrument and recorded. The results are shown in Figure 4 shown.

[0105] Antibacterial experimental steps

[0106] Porphyromonas gingivalis suspension was added into sterile centrifuge tubes at a rate of 10 ml per tube and divided into different groups: A. control group, B. drug-loaded microneedle membrane group, C. periochip Different membranes were added to the bacterial solution in each group, with 3 replicates per group. The absorbance of the bacterial solution at 660 nm was detected and recorded at baseline, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and 7 days. Figure 5 shown.

[0107] Example 7 Young's modulus test of microneedle patch molding material

[0108] Experimental Materials:

[0109] The drug-loaded microneedle preparation process was used as in Example 3, but the drug loading amount of the molding solution used for the needle tip part and the base part was the same, and 0 / 0.5 / 1 / 2g chlorhexidine gluconate solution (98% mass volume fraction) was used to mold microneedle patches with drug loading amounts of 0, 14.3%, 25%, and 40%, respectively.

[0110] Use a tensile tester to perform a tensile test on the material. The patch is stretched at a speed of 0.67 mm / s, and the force changes over time are recorded to obtain the force (F)-deformation (l) curve of the material. At the same time, the thickness (h), length (L), and width (D) of the patch are measured, and the Young's modulus of the material can be obtained from the calculation formula of the Young's modulus.

[0111]

[0112] Where E is Young's modulus, σ is stress, and ε is strain. The elastic modulus of the microneedle patch at different drug loadings was obtained using a tensile tester. Figure 6 shown.

[0113] Example 8 Simulation of axial mechanical properties of microneedle patch

[0114] Use SolidWorks to create Figure 1 The microneedle model shown, where the substrate size is 0.5×0.5 cm 2 , the array contains 100 (10×10) microneedles, and the horizontal and vertical spacing of the array is 500μm. Each microneedle is a quadrangular pyramid with a sharp tip. The microneedle height is 800μm and the length of the bottom side of the quadrangular pyramid is 200μm. The material parameters are set according to the Young's modulus of the microneedle material tested in Example 7. Among them, the material with a drug loading of 0 has a Young's modulus of 25.74MPa; the material with a drug loading of 14.3% has a Young's modulus of 21.29MPa; the material with a drug loading of 25% has a Young's modulus of 12.90MPa; the material with a drug loading of 40% has a Young's modulus of 10.37MPa. The model is meshed using the tetrahedral meshing method. A fixed constraint is applied to the lower surface of the microneedle, which means that the lower surface of the microneedle is fixed and cannot be displaced. A force is applied to the upper surface of the cuboid above the microneedle, and the direction is toward the microneedle. Observe the deformation degree of the microneedle after solving, and the deformation-load relationship of the microneedle tip is obtained as follows: Figure 7 Shown

[0115] pass Figure 7 The mechanical properties of microneedles with different drug loadings can be analyzed, and it can be observed that the mechanical properties of microneedles with a drug loading of 14.3% are similar to those of microneedles without drug loading; and when the drug loading is greater than 25%, the mechanical strength of the microneedles decreases significantly.

[0116] Example 9 Microneedle patch skin penetration performance test

[0117] Experimental Materials:

[0118] The drug-loaded microneedle preparation process was used as in Example 3, but the drug loading amount of the molding solution used for the needle tip part and the base part was the same, and 0 / 0.5 / 1 / 2g chlorhexidine gluconate solution (98% mass volume fraction) was used to mold microneedle patches with drug loading amounts of 0, 14.3%, 25%, and 40%, respectively.

[0119] The microneedle patches with different drug loadings were pressed firmly on the skin surface for 30 seconds. After the microneedles were removed for 5 minutes, the effects of microneedle pressing on the skin surface were observed and the skin marks were recorded. Figure 8 shown.

[0120] When the microneedle patches without drug loading and with drug loading of 14.3% were removed, the microneedle array left obvious array marks on the skin, accompanied by skin redness, which verified the penetration of the microneedles on the skin surface; after removal, the skin redness gradually weakened over time until it disappeared, which proved the minimally invasive nature of the microneedles. When the microneedle patches with drug loading of 25% and 40% were pressed, the marks left on the skin were not obvious, which can be considered that it is difficult to penetrate the stratum corneum and epidermis.

[0121] Example 10 Microneedle patch thinning process

[0122] The microneedle submucosal controlled-release drug delivery system is thinned as a whole according to the space requirement of the periodontal pocket patch, and the thinning process is as follows:

[0123] 1. Perform operations 1-5 as described in Example 3 to prepare a microneedle tip forming solution AC and a microneedle base forming solution BC;

[0124] 2. Use a dropper to transfer solution AC into the microneedle PDMS mold and place it in a vacuum pump to evacuate for 10 seconds;

[0125] 3. Place the vacuum-sucked microneedle PDMS mold containing solution AC in an oven for 5 min until the gel solution is completely liquefied;

[0126] 4. Use a dropper to suck out the solution with bubbles at the base of the microneedle PDMS mold, and further suck out the solution AC remaining on the microneedle PDMS mold until it is almost completely removed from the non-needle body mold of the microneedle mold, leaving only the solution AC at the needle body of the microneedle mold;

[0127] 5. Use a dropper to transfer solution BC into the microneedle PDMS mold and place it in a vacuum pump to evacuate for 10 seconds.

[0128] 6. Remove the microneedle mold and repeat step 3.

[0129] 7. Use a dropper to suck out the solution with bubbles at the base of the microneedle PDMS mold, and further absorb the solution BC remaining on the microneedle PDMS mold until the liquid level of solution BC in the microneedle mold drops to 1 mm from the bottom of the mold.

[0130] 8. Dry overnight at room temperature and demold to obtain a microneedle submucosal controlled-release drug delivery system with different drug loading amounts on the needle body and base.

[0131] According to the different drying degrees of the microneedle molding solution in the final mold, a thinned microneedle submucosal controlled release drug delivery system is obtained, in which the microneedle tip is a quadrangular pyramid with a sharp tip, the microneedle height is 800μm, and the length of the bottom side of the quadrangular pyramid is 200μm. The size of the substrate of the microneedle base is 0.5×0.5cm 2 , the base thickness is between 200μm-500μm.

Claims

1. A microneedle submucosal controlled release drug delivery system, characterized in that: The microneedle submucosal controlled-release drug delivery system is in the form of a microneedle patch suitable for periodontal pockets, which uses gelatin as a matrix and contains an antibacterial agent; the microneedle submucosal controlled-release drug delivery system includes a needle tip and a base part; the antibacterial agent content in the needle tip part is 13-16%, while the antibacterial agent content in the base is 35-45%; the antibacterial agent is chlorhexidine gluconate; the preparation process of the microneedle submucosal controlled-release drug delivery system includes preparing a needle-forming solution, the needle-forming solution contains gelatin, glycerol, chlorhexidine gluconate and glutaraldehyde, and the concentration of the cross-linking agent glutaraldehyde in the needle-forming solution is 0.05%.

2. The microneedle submucosal controlled release drug delivery system according to claim 1, wherein the content of the antibacterial agent in the needle tip is 14.3%, and the content of the antibacterial agent in the base is 40%.

3. The microneedle submucosal controlled-release drug delivery system according to claim 1 or 2, wherein the microneedle submucosal controlled-release drug delivery system is prepared using a micromolding method.

4. The method for preparing the microneedle submucosal controlled release drug delivery system according to claim 1 or 2, characterized in that: The preparation method is a micro-molding method.

5. The preparation method according to claim 4, wherein the micro-molding preparation process comprises: preparing a mold; Prepare a solution for injection; Microneedle patch preparation. The preparation method according to claim 5 , wherein the mold is a PDMS mold.

7. The preparation method according to claim 5, wherein the preparation method of the needle-forming solution is: (a) taking 1-4 g of gelatin, adding 20 mL of ultrapure water, stirring under heating conditions, and preparing a 5-20% gelatin solution; taking 0.5-2 g of glycerol, adding 0.5 g-2 g of 98% mass volume fraction chlorhexidine gluconate solution, and pouring into the gelatin solution; (b) taking 0.3-0.5 mL of 2.5% glutaraldehyde solution, adding 2 mL of ultrapure water, diluting it into a glutaraldehyde solution, pouring it into the solution obtained in (a), stirring under heating conditions, and obtaining a needle-forming solution.

8. The preparation method according to claim 5, wherein The steps for preparing the microneedle patch include: (A) loading a needle-forming solution into a mold; (B) evacuating the mold; (C) placing the mold in an oven for heating; (D) sucking the needle-forming solution with bubbles and replenishing the needle-forming solution; (E) evacuating the mold; (F) repeating (C) and (D); and (G) demolding after drying.

9. The preparation method according to claim 8, wherein the vacuuming time in steps (B) and (E) is 5-20 seconds; and the step (C) is placed at 50-70°C for 3-10 minutes.

10. The preparation method according to claim 9, wherein the vacuuming time in steps (B) and (E) is 10 seconds; and the step (C) is placed at 60°C for 5 minutes.

11. The preparation method according to claim 5, wherein the microneedle patch preparation step comprises a separate needle tip preparation step and a separate base preparation step.

12. The preparation method according to claim 11, wherein the microneedle patch preparation step comprises: (1) Take 1-4 g of gelatin and add 20 mL of ultrapure water to prepare a 5-20% gelatin solution; (2) Take 0.5-2 g of glycerol and 0.5-2 g of 98% mass volume fraction chlorhexidine gluconate solution, pour into the gelatin solution to prepare a microneedle tip forming preparatory solution, recorded as solution A; (3) Take 0.5-2 g of glycerol and 0.5-2 g of 98% mass volume fraction chlorhexidine gluconate solution, pour into the gelatin solution to prepare a microneedle base molding preparatory solution, recorded as solution B; (4) Take 0.3-0.5 mL of 2.5% volume fraction glutaraldehyde solution, add 2 mL of ultrapure water, dilute to glutaraldehyde solution, prepare crosslinker solution, and record it as solution C; (5) Solution C is added into solution A and solution B, respectively, to obtain microneedle tip forming solution AC and microneedle base forming solution BC, respectively.

13. The preparation method according to claim 12, comprising: (1) Take 2 g of gelatin and add 20 mL of ultrapure water to prepare a 10% gelatin solution; (2) Take 1 g of glycerol and 0.5 g of 98% mass volume fraction chlorhexidine gluconate solution, pour into the gelatin solution to prepare the microneedle tip forming preparatory solution, recorded as solution A; (3) Take 1 g of glycerol and 2 g of 98% mass volume fraction chlorhexidine gluconate solution, pour into the gelatin solution to prepare the microneedle base molding preparatory solution, which is recorded as solution B; (4) Take 0.5 mL of 2.5% volume fraction glutaraldehyde solution, add 2 mL of ultrapure water, dilute to 0.5% glutaraldehyde solution, prepare crosslinker solution, record as solution C; (5) 2.5 mL of solution C was added to solution A and solution B, respectively, to obtain microneedle tip forming solution AC and microneedle base forming solution BC, respectively. The concentration of glutaraldehyde in microneedle tip forming solution AC and microneedle base forming solution BC was 0.05%.

14. The preparation method according to claim 13, further comprising (6) Transfer solution AC into the microneedle PDMS mold and evacuate the mold; (7) placing the microneedle PDMS mold containing solution AC after vacuum suction in an oven until the gel solution is completely liquefied; (8) Aspirate the solution containing bubbles at the base of the microneedle PDMS mold; (9) Pour solution BC into the microneedle PDMS mold using a dropper and evacuate; (10) Remove the microneedle mold and repeat steps (7)-(8); (11) Drying and demoulding.

15. The preparation method according to claim 14, wherein the vacuuming time in steps (6) and (9) is 5-20 seconds; the oven temperature in step (7) is 50-70°C, and the mixture is placed in the oven for 3-10 minutes.

16. The preparation method according to claim 15, wherein the vacuuming time in steps (6) and (9) is 10 seconds; and the oven temperature in step (7) is 60°C and the mixture is placed in the oven for 5 minutes.

17. The microneedle submucosal controlled release drug delivery system according to claim 1, wherein the microneedle tip is in the shape of a quadrangular pyramid, the microneedle height is 500-1000 μm, the length of the bottom side of the quadrangular pyramid is 100-300 μm; the substrate size of the microneedle base is 0.2-1×0.2-1 cm 2 , base thickness 200μm-500μm.

18. The microneedle submucosal controlled release drug delivery system according to claim 17, wherein the microneedle tip is in the shape of a quadrangular pyramid, the microneedle height is 800 μm, and the length of the bottom side of the quadrangular pyramid is 200 μm; the substrate size of the microneedle base is 0.5×0.5 cm 2 , the base thickness is between 200μm-500μm.

19. Use of the microneedle submucosal controlled release drug delivery system according to any one of claims 1-3, 17, and 18 in the preparation of drugs or devices for treating periodontitis or gingivitis.

20. The use according to claim 19, wherein the medicine or device further comprises packaging.

Images