Toleranogen vaccine microneedle patch for multiple sclerosis

Abstract

The invention belongs to the technical field of multiple sclerosis, and particularly relates to a tolerogen vaccine microneedle patch for multiple sclerosis, the tolerogen vaccine microneedle patch for multiple sclerosis comprises methacryloyl gelatin and zeolite imidazole skeleton-8 for encapsulating tolerogen polypeptides, the tolerogen polypeptides are encapsulated by using the large aperture and extremely high specific surface area of ZIF-8 nanoparticles, and the tolerogen vaccine microneedle patch for multiple sclerosis is prepared from the methacryloyl gelatin and the zeolite imidazole skeleton-8 for encapsulating the tolerogen polypeptides. According to the preparation method, efficient loading of a polypeptide drug is achieved, meanwhile, rapid crystallization of nanoparticles can prevent degradation of polypeptide and is beneficial to tolerating slow release of original polypeptide, the adopted GelMA hydrogel microneedle has enough mechanical strength, can penetrate through skin cuticle and form a diffusion channel beneficial to release of the polypeptide drug, and controllable slow release of the polypeptide drug in skin tissue can be achieved. The GelMA hydrogel microneedle drug release device can directly penetrate through the skin cuticle to deliver the polypeptide drug to the corium layer, so that the sustained and controlled release of the tolerance original polypeptide in the skin tissue is realized, the first-pass effect of the liver is avoided, and the treatment purpose is realized by inducing immune tolerance.

Description

Technical Field

[0001] The invention belongs to the technical field of multiple sclerosis, and in particular relates to a tolerogen vaccine microneedle patch for multiple sclerosis. Background Art

[0002] Multiple sclerosis (MS) is an autoimmune disease caused by the migration of myelin antigen-specific T cells into the central nervous system (CNS), leading to CNS demyelination and progressive neuropathy. Currently, the treatment of MS is mainly based on immunosuppression and immunomodulation, which may lead to adverse effects such as lifelong immunosuppression. Antigen-specific immunotherapy for autoimmune diseases aims to induce tolerance to self-antigens without suppressing systemic immune responses, and is an ideal immunotherapy strategy for MS.

[0003] Antigen-specific immunotherapy is the use of autoreactive T cell-specific peptides / proteins as therapeutic drugs to treat T cell-mediated autoimmune diseases by inducing T cell tolerance. Previous studies have shown that soluble autoantigen peptide drugs have certain therapeutic effects in animal models of autoimmune diseases, but the clinical trial results are not good. In addition, the systemic administration of large doses of soluble peptide tolerance agents may cause allergic reactions in mice and primates, which has aroused safety concerns about the soluble peptide strategy. In order to overcome the limitations of the above-mentioned soluble peptide drugs, new drug delivery technologies based on biomaterials have begun to be applied to the design of peptide drug delivery systems in recent years.

[0004] The induction of immune tolerance is affected by many factors, including antigen dose, route of antigen administration and delivery system. Studies have shown that inducing the tolerance phenotype of DCs is the key to reestablishing immune tolerance. Skin tissue has a variety of antigen presenting cells (APCs), including DCs. It is feasible and has great potential to use the skin immune system to regulate autoimmune diseases. At the same time, current clinical trials of tolerogenic vaccines mostly use intravenous administration, which may cause serious side effects such as anaphylactic shock. Summary of the invention

[0005] In order to solve the problems in the prior art, the present invention provides a tolerogen vaccine microneedle patch for multiple sclerosis, which achieves the purpose of loading the tolerogen vaccine with hydrogel microneedles, has high mechanical strength, can penetrate the stratum corneum of the skin, and forms a diffusion channel that is conducive to the release of polypeptide drugs, as well as the efficient loading and sustained release of polypeptide drugs.

[0006] Transdermal administration of tolerogen vaccines is an ideal way to improve bioavailability and reduce side effects. Microneedles (MN) are ideal delivery systems for transdermal administration of biomacromolecule drugs. Microneedles have significant advantages over traditional materials, such as painless and minimally invasive, easy to use, customizable and controllable drug release. Due to their large molecular weight, high hydrophilicity, skin barrier structure and other obstacles, transdermal administration of protein and peptide drugs has always been a difficult problem to be solved in the field. Improving permeability is one of the key issues in solving transdermal administration of biological drugs. Microneedles are a new type of transdermal drug delivery technology, generally composed of multiple micron-sized needle tips arranged in an array on a base, with a needle body height ranging from 10 to 2000μm. Microneedles can penetrate the stratum corneum without touching the pain nerves, forming a micron-sized drug delivery channel in the stratum corneum of the skin, delivering drugs to the epidermis or upper dermis, and promoting drug retention in specific parts.

[0007] The present invention solves the technical problem by adopting the following technical solutions:

[0008] The present invention aims to provide a tolerogen vaccine microneedle patch for multiple sclerosis, comprising methacryloyl gelatin and zeolite imidazole framework-8 encapsulating tolerogen polypeptide.

[0009] Furthermore, the preparation method of the microneedle patch comprises the following steps:

[0010] Step 1: Preparation of PZNPs: weigh MOG polypeptide and add it to 2-methylimidazole solution to prepare a 2-methylimidazole solution containing polypeptide; prepare zinc acetate solution and slowly add it to the 2-methylimidazole solution containing polypeptide; mix well and obtain PZNPs by coprecipitation method;

[0011] Step 2: Preparation of MN@PZNPs: Weigh the GelMA solid, and use the photoinitiator solution to dissolve it by stirring under water bath heating, and prepare a gel solution after ultrasonic defoaming. Weigh the PZNPs solid containing the polypeptide and add it to the above gel solution and mix well to obtain a gel solution containing PZNPs. Inject the gel containing PZNPs into the microneedle mold for molding and demolding to obtain MN@PZNPs.

[0012] Furthermore, the concentration of the 2-methylimidazole solution is 1200 mM, and the prepared 2-methylimidazole solution contains 1 mg / mL of the polypeptide.

[0013] Furthermore, the PZNPs obtained by the co-precipitation method were washed with a washing solution prepared by anhydrous ethanol and pure water in a ratio of 1:1, and the precipitate after washing was freeze-dried to obtain the final PZNPS.

[0014] Furthermore, the concentration of the photoinitiator solution used is 0.25% (w / v), the water bath heating temperature is 50° C.-60° C., and the concentration of the gel solution prepared after ultrasonic debubbling is 15%-20% (w / v).

[0015] Furthermore, the mold forming method in step 2 is: injecting the gel containing PZNPs into the PDMS microneedle mold, and placing it in a vacuum to allow it to deposit at the bottom of the mold, scraping off excess gel after 10 minutes, and after filling, using a light curing device for light curing for 2-3 minutes. After curing, adding a 15%-20% (w / v) polyvinyl alcohol solution to the mold as a microneedle base layer, and then adding color-changing silica gel to the container where the microneedle mold is placed and placing it in a 4°C dryer for drying for 10-15 days.

[0016] The microneedle patch is arranged in a 10x10 array, and the patch size is 8.9x8.9mm. The microneedle body is a cone, with a needle body height of 800μm, a cone bottom diameter of 300μm, and a needle tip distance of 650μm. After the microneedle patch is applied to the skin, it is fixed with medical tape and maintained on the skin for 3-5 days.

[0017] Compared with the prior art, the beneficial technical effects of the present invention are:

[0018] 1. The present invention designs hydrogel microneedles loaded with tolerogenic vaccines, and its main raw material is methacryloyl gelatin (GelMA), which has the advantages of good biocompatibility, formability, degradability and adjustable mechanical properties, and is widely used in the fields of tissue engineering and drug delivery. The GelMA hydrogel microneedles designed by us have sufficient mechanical strength, can penetrate the stratum corneum of the skin, and form a diffusion channel that is conducive to the release of polypeptide drugs. In order to achieve efficient loading and sustained release of polypeptide drugs, we use metal-organic framework structures (MOFs) to encapsulate tolerogenic polypeptides. MOFs materials are assembled from organic ligands and their coordinated metal ions, and have a controllable porous network structure and a large specific surface area. Among them, the zeolite imidazole framework-8 (ZIF-8) used in the present invention has the characteristics of high porosity structure, adjustable pore size, room temperature preparation, high biocompatibility, effective encapsulation of biomacromolecules, pH-responsive degradation, etc., and shows good application prospects in the delivery of biomacromolecule drugs.

[0019] 2. The present invention utilizes the larger pore size and extremely high specific surface area of ​​ZIF-8 nanoparticles to encapsulate the tolerogen polypeptide, thereby achieving efficient loading of polypeptide drugs. Meanwhile, the rapid crystallization of the nanoparticles can prevent polypeptide degradation, thereby facilitating the sustained release of the tolerogen polypeptide.

[0020] 3. The GelMA hydrogel microneedle of the present invention has sufficient mechanical strength, can penetrate the stratum corneum of the skin, form a diffusion channel that is conducive to the release of polypeptide drugs, and can achieve controlled and sustained release of polypeptide drugs in skin tissue. The GelMA hydrogel microneedle drug release device can directly penetrate the stratum corneum of the skin to deliver polypeptide drugs to the dermis, achieve sustained and controlled release of tolerogenic polypeptides in skin tissue, avoid the first-pass effect of the liver, and achieve the therapeutic purpose by inducing immune tolerance.

[0021] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented according to the contents of the specification. In order to make the above contents of the present invention and its objectives, features and advantages more obvious and easy to understand, the specific implementation methods of the present invention are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a scanning electron microscope image of PZNPs of the present invention.

[0023] Figure 2 This is the particle size distribution diagram of PZNPs of the present invention.

[0024] Figure 3 This is a picture of MN@PZNPs taken by the optical microscope of the present invention.

[0025] Figure 4 This is a scanning electron microscope image of MN@PZNPs of the present invention.

[0026] Figure 5 This is the mechanical strength diagram of MN@PZNPs of the present invention.

[0027] Figure 6 This is a diagram of MN@PZNPs of the present invention inducing BMDC cell tolerance.

[0028] Figure 7 This is a diagram of the tolerance of antigen presenting cells in the skin tissue of EAE mice induced by MN@PZNPs of the present invention. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0030] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0031] A tolerogen vaccine microneedle patch for multiple sclerosis, the preparation method comprising the following steps:

[0032] Step 1: Preparation of PZNPs

[0033] 1. Weigh 1 mg of MOG polypeptide and add it to 1 ml of 1200 mM 2-methylimidazole solution to prepare a 2-methylimidazole solution containing 1 mg / mL of polypeptide.

[0034] 2. Prepare 60 mM zinc acetate solution and slowly add it to the solution prepared in step 1, mix well, and obtain PZNPs by co-precipitation method.

[0035] 3. Prepare a cleaning solution with anhydrous ethanol and pure water in a ratio of 1:1 and use the cleaning solution to clean the precipitate obtained in step 2 (3-4 times), and then freeze-dry the cleaned precipitate to obtain PZNPS.

[0036] Take a small amount of PZNPs, spray gold, and use a scanning electron microscope to photograph and perform elemental analysis on the PZNPs. Figure 1 The particle size distribution of the samples in the SEM images was statistically analyzed using the particle size statistics software (Nano Measurer 1.2). Figure 2 Particle size distribution of PZNPs.

[0037] Step 2: Preparation of MN@PZNPs

[0038] 1. Weigh a certain amount of GelMA solid, and use 0.25% (w / v) photoinitiator solution (LAP) to stir and dissolve it in a 50℃-60℃ water bath, and prepare a gel solution with a concentration of 15%-20% (w / v) after ultrasonic defoaming.

[0039] 2. Weigh 1 mg of PZNPs solid containing polypeptide and add it to the above gel solution and mix well to obtain a gel solution containing PZNPs.

[0040] 3. Inject the gel containing PZNPs into the PDMS microneedle mold and place it in a vacuum to allow it to deposit at the bottom of the mold. After 10 minutes, scrape off the excess gel (repeat the above filling operation 1-2 times).

[0041] 4. After filling, use a light curing device for light curing, and the curing time is 2-3 minutes.

[0042] 5. After curing, add a 15%-20% (w / v) polyvinyl alcohol solution to the mold as the microneedle base layer, then add some color-changing silica gel to the container where the microneedle mold is placed and place it in a 4°C dryer for drying for 10-15 days.

[0043] 6. Demolding to obtain MN@PZNPs.

[0044] The morphology was observed using an optical microscope and a scanning electron microscope. Figure 3 and 4 The mechanical strength is tested by using a microcomputer-controlled universal tensile machine in tension-compression mode. The results are shown in Figure 5 shown.

[0045] The tolerogen microneedle patch of the present invention was used to detect the maturation and activation of BMDC cells: 2 μL LPS (500X) was added to each well of a 24-well plate to pre-stimulate BMDC for 3 hours, and then PBS / PBS+LPS / P / MNP / MNPZ / PZ were added to the cells and incubated for 20 hours. The cell suspension was obtained by trypsin digestion and flow cytometry antibody staining (CD80 / CDB6) was performed, and the maturation and activation of BMDC cells was detected by flow cytometry. The results are shown in FIG. Figure 6 shown.

[0046] Establishment of multiple sclerosis mouse model (EAE) and administration of tolerogen microneedle patch:

[0047] 1. Female C57BL / 6 mice (8 weeks old) were immunized with 200 μl of emulsion containing complete Freund's adjuvant (CFA, Sigma-Aldrich), MOG35-55 peptide (100 μg) and Mycobacterium tuberculosis extract H37Ra (Difco) (400 μg). 200 ng pertussis toxin (PTX) (List Biological Laboratories) was injected into the mice 4 hours and 24 hours after immunization.

[0048] 2. The microneedle patch of the present invention was inserted into the dorsal skin area of ​​EAE mice and fixed with medical tape. The skin tissue of the microneedle implantation site of the mice was taken on the next day, the tissue-infiltrating immune cells were separated, and the phenotype of antigen-presenting cells was analyzed by flow cytometry. The results were as follows: Figure 7 shown.

[0049] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0050] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A tolerogen vaccine microneedle patch for multiple sclerosis, characterized in that: Includes methacryloyl gelatin and a zeolitic imidazole framework-8 encapsulating tolerogenic polypeptides.

2. A tolerogen vaccine microneedle patch for multiple sclerosis according to claim 1, characterized in that: The preparation method of the microneedle patch comprises the following steps: Step 1: Preparation of PZNPs: weigh MOG polypeptide and add it to 2-methylimidazole solution to prepare a 2-methylimidazole solution containing polypeptide; prepare zinc acetate solution and slowly add it to the 2-methylimidazole solution containing polypeptide; mix well and obtain PZNPs by coprecipitation method; Step 2: Preparation of MN@PZNPs: Weigh the GelMA solid, and use the photoinitiator solution to dissolve it by stirring under water bath heating, and prepare a gel solution after ultrasonic defoaming. Weigh the PZNPs solid containing the polypeptide and add it to the above gel solution and mix well to obtain a gel solution containing PZNPs. Inject the gel containing PZNPs into the microneedle mold for molding and demolding to obtain MN@PZNPs.

3. A tolerogen vaccine microneedle patch for multiple sclerosis according to claim 2, characterized in that: The concentration of the 2-methylimidazole solution is 1200 mM, and the prepared 2-methylimidazole solution contains 1 mg / mL of the polypeptide.

4. A tolerogen vaccine microneedle patch for multiple sclerosis according to claim 2, characterized in that: The PZNPs obtained by the co-precipitation method were washed with a washing solution prepared by mixing anhydrous ethanol and pure water in a ratio of 1:

1. After washing, the precipitate was freeze-dried to obtain the final PZNPS.

5. A tolerogen vaccine microneedle patch for multiple sclerosis as described in claim 2, characterized in that: The concentration of the photoinitiator solution used was 0.25% (w / v), the water bath heating temperature was 50° C.-60° C., and the concentration of the gel solution prepared after ultrasonic debubbling was 15%-20% (w / v).

6. A tolerogen vaccine microneedle patch for multiple sclerosis according to claim 2, characterized in that: The mold forming method in step 2 is: inject the gel containing PZNPs into the PDMS microneedle mold, and place it in a vacuum to allow it to deposit at the bottom of the mold. After 10 minutes, scrape off the excess gel. After filling, use a light curing device for light curing for 2-3 minutes. After curing, add a 15%-20% (w / v) polyvinyl alcohol solution to the mold as the microneedle base layer, then add color-changing silica gel to the container where the microneedle mold is placed and place it in a 4°C dryer for drying for 10-15 days.

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