A core-shell microneedle, a microneedle patch and a preparation method and application thereof

Abstract

The application discloses a kind of core-shell microneedles, microneedle patch and preparation method and application thereof.The core-shell microneedle of the application includes the microneedle shell with perforated side wall, and the inner core filled in the cavity surrounded by the microneedle shell, the compound of the microneedle shell contains the group as shown in formula (I): wherein R1 is aliphatic hydrocarbon group;The inner core includes hydrogel and cells dispersed in the hydrogel.The core-shell microneedle and microneedle patch of the application can achieve better cell delivery function, make cell enrichment degree high at administration site, maintain cell function, deliver a large number of cells, and can continuously nourish cells to maintain cell activity, and further achieve better therapeutic effect, reduce cell resource waste, reduce the cost of cell therapy.

Description

Technical Field

[0001] This invention relates to a core-shell microneedle, a microneedle patch, its preparation method, and its application. Background Technology

[0002] Cell therapy is a method that utilizes cytokines produced by immune cells and related cells to regulate cellular function. These cytokines directly or indirectly kill cancer cells, leading to tumor regression and achieving therapeutic goals. As a novel form of immunotherapy, cell therapy has also been found to have good therapeutic effects on certain skin diseases and skin cancer in recent years. For example, psoriasis, recognized as an autoimmune disease caused by T-cell dysfunction, has traditional treatments using ultraviolet light or hormones that are ineffective, do not address the root cause, and have systemic toxicity. T-cell therapy has been shown to offer the potential to address the underlying issues of ineffective treatment, poor efficacy, and systemic toxicity in psoriasis, making it a current research hotspot.

[0003] However, current cell therapies all involve delivering cells into the human bloodstream via intravenous injection. As the blood flows, the cells are delivered to specific treatment sites. The main problem with this cell delivery method is that the cell concentration at the specific treatment site is low, resulting in most cells not being able to effectively exert their therapeutic effect, leading to resource waste and poor treatment results. Furthermore, changes in cell function during delivery may also lead to poor treatment outcomes.

[0004] In addition, cell therapy requires the delivery of nutrients along with the cells to nourish them, maintain their vitality and stable function, and the concentration of nutrients must be neither too low nor too high. Too low a concentration will not nourish the cells, while too high a concentration will kill them. If cells are delivered via subcutaneous injection, the amount that can be injected subcutaneously at one time is very limited, so the amount of nutrients in the injection solution is very small, resulting in a small number of oxygen-supplying cells and a short period of cell nourishment. The small number of cells and short cell viability time will lead to poor treatment results.

[0005] Transdermal drug delivery using microneedles has seen significant development and application in vaccine injection, cosmetic and hair care, and disease treatment. Microneedle drug delivery offers advantages such as stable transdermal absorption rates, the ability to control microneedle length to avoid contact with capillaries and nerve endings, reduced or eliminated pain, and convenient self-administration. However, the inherent incompatibility between the dry matrix of microneedles and the hydration environment required by cells has limited the potential for microneedles in the field of cell delivery.

[0006] Therefore, there is an urgent need for a better way to deliver cells so that they can accumulate in specific areas, effectively exert their therapeutic effects, not alter cell function, deliver a large number of cells, and continuously nourish the cells, thereby enabling cell therapy to achieve better therapeutic results. Summary of the Invention

[0007] The technical problem solved by this invention is to overcome the shortcomings of existing cell delivery technologies, such as low cell enrichment, waste of cell resources, altered cell function, small cell delivery volume, and short nourishment time, which lead to poor therapeutic effects and high treatment costs. This invention provides a core-shell microneedle, a microneedle patch, its preparation method, and its application. The core-shell microneedles and microneedle patches of this invention achieve better cell delivery, resulting in high cell enrichment at the drug delivery site, preservation of cell function, delivery of a large number of cells, and continuous nourishment of cells to maintain cell activity. This leads to better therapeutic effects, reduced cell resource waste, and lower costs for cell therapy.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] This invention provides a core-shell microneedle comprising a microneedle shell with perforated sidewalls and a core filling a cavity enclosed by the microneedle shell, wherein the compound of the microneedle shell contains groups as shown in formula (I):

[0010]

[0011] Wherein, R1 is an aliphatic hydrocarbon group; the core comprises a hydrogel and cells dispersed in the hydrogel.

[0012] This invention delivers cells via transdermal drug delivery using the aforementioned core-shell microneedles. The core-shell microneedles are applied to the skin surface, with the needle tip piercing the stratum corneum and epidermis, penetrating into the dermis. The hydrogel liquefies under 37°C heating in the body, connecting with the tissue fluid of the dermis to form a liquid system. Cells are released from the perforations into the tissue fluid, achieving cell delivery. Esterases are highly expressed in organisms with inflammatory diseases such as psoriasis. With the core-shell microneedles of this invention, esterases cleave fatty acid ester bonds to produce acidic substances, such as acetic acid, propionic acid, or butyric acid, fatty acids of any carbon chain length. These fatty acids nourish cells and maintain cell function. As the ester bonds in the shell continuously degrade, nutrient-rich fatty acids are continuously produced, thus continuously nourishing the cells without releasing nutrients all at once, preventing excessive nutrient concentrations that could kill cells. Furthermore, microneedle cell delivery concentrates cells at the drug delivery site, achieving higher enrichment and greater cell utilization.

[0013] In this invention, those skilled in the art will understand that the aliphatic hydrocarbons refer to compounds containing both carbon and hydrogen elements, with carbon atoms linked together in chains or rings, generally including straight-chain aliphatic hydrocarbons, branched-chain aliphatic hydrocarbons, and alicyclic hydrocarbons.

[0014] In this invention, the compound represented by formula (Ⅰ) is degraded by esterase to produce fatty acid R1COOH.

[0015] In this invention, R1 is preferably one or more of an aliphatic alkane group, an aliphatic olefin group, and an aliphatic alkyne group, more preferably an aliphatic alkane group, and even more preferably a C1 to C6 aliphatic alkane group, such as -CH3, -C2H5, -C3H7, or -C4H9, and even more preferably -CH3, -C2H5, or -C3H7.

[0016] In this invention, the mass percentage of the group represented by formula (I) in the compound of the microneedle shell is preferably 40-60%, more preferably 43.8-58.4%, for example 51%.

[0017] The inventors discovered that the content of the groups shown in Formula (I) above can ensure that the microneedle shell has good mechanical strength and release fatty acids at an appropriate rate, so as to produce an appropriate concentration of fatty acids in the tissue fluid to better and continuously nourish the cells.

[0018] In this invention, the compound of the microneedle shell can be a conventionally moldable compound in the art, preferably a polymer.

[0019] The polymer may be a homopolymer or a copolymer, preferably a copolymer of polyvinyl acetate, polyvinyl propionate, polyvinyl butyrate, a copolymer of ethylene glycol diacrylate and vinyl propionate, a copolymer of ethylene glycol diacrylate and vinyl butyrate, and a copolymer of methyl methacrylate (MMA) and vinyl propionate (VP), more preferably a copolymer of MMA and VP.

[0020] In a preferred embodiment of the present invention, the compound of the microneedle shell is a copolymer of methyl methacrylate (MMA) and vinyl propionate (VP), wherein the molar ratio of MMA to VP is (2:8) to (4:6), more preferably 3:7.

[0021] The copolymer of methyl methacrylate (MMA) and vinyl propionate (VP) is preferably obtained by polymerization of the methyl methacrylate (MMA), the vinyl propionate (VP), and a thermal initiator.

[0022] The thermal initiator may be conventional in the art, preferably one or more of 2,2'-azobis(isobutyronitrile), benzoyl peroxide, benzoyl peroxide, benzene sulfonate and alkyl sulfonate, and more preferably 2,2'-azobis(isobutyronitrile).

[0023] In this invention, the thickness of the microneedle shell can be conventional in the art, preferably 80-150 μm.

[0024] At the aforementioned microneedle shell thickness, the core-shell microneedles possess good mechanical strength, enabling them to successfully penetrate the dermis.

[0025] In this invention, the shape of the perforation can be conventional in the art, preferably circular or polygonal.

[0026] The polygon can generally be a triangle, a quadrilateral, or a pentagon.

[0027] In this invention, the aperture of the perforation can be any size that allows the cell to pass through, generally larger than the outer diameter of one cell, preferably larger than 10 μm, and more preferably 50-200 μm.

[0028] In this invention, the outer surface shape of the microneedle shell can be conventional in the art, preferably conical or pyramidal, and more preferably conical. The pyramidal shape is preferably a triangular or quadrangular pyramid.

[0029] The length of the cone can be conventional in the art, preferably 1000-1500 μm, where the length refers to the vertical distance from the tip of the cone to the bottom.

[0030] The diameter of the base of the cone can be conventional in the art, preferably 300-800 μm, and the base refers to the side opposite to the tip of the cone.

[0031] In this invention, the volume of the cavity of each microneedle can be conventional in the art, preferably 0.05 to 0.5 μL, more preferably 0.1 to 0.2 μL, for example 0.148 μL.

[0032] In this invention, the core also includes a cell nutrient solution. The cell nutrient solution refers to nutrients used to maintain cell growth.

[0033] The cell nutrient solution can be conventional in the art, preferably a cell culture medium or a glucose solution.

[0034] The cell culture medium can be conventional in the art, but is preferably RPMI 1640 medium.

[0035] Those skilled in the art will understand that RPMI is an abbreviation for Roswell Park Memorial Institute, referring to the Roswell Park Memorial Institute; and that RPMI1640 culture medium is a type of cell culture medium developed by the institute.

[0036] The role of the cell culture medium is to supply nutrients to the cells before the microneedle shell degrades and produces fatty acids, thus maintaining the cell's vitality before release.

[0037] Those skilled in the art will understand that the cell nutrient solution is dispersed in the hydrogel after the hydrogel has solidified.

[0038] The concentration of the hydrogel in the cell nutrient solution is preferably 0.01–0.03 g / mL, and more preferably 0.02 g / mL.

[0039] The concentration of the hydrogel in the cell nutrient solution is 0.01 to 0.03 g / mL, which means that the amount of the hydrogel in each milliliter of the cell nutrient solution is 0.01 to 0.03 g.

[0040] In this invention, the cells may be conventional cells used in cell therapy in the art, preferably regulatory T cells.

[0041] Those skilled in the art will understand that the regulatory T cells are T cells that typically express CD4 and Foxp3 and have an inhibitory effect on conventional T cells.

[0042] In this invention, the cells can be extracted by conventional extraction methods in the art, preferably by magnetic bead adsorption extraction.

[0043] Preferably, the cells are not subjected to any biological treatment after extraction, wherein the biological treatment includes gene editing and / or in vitro amplification.

[0044] In this invention, those skilled in the art will understand that the hydrogel generally includes a dispersion medium of water and a cross-linked polymer. The cross-linked polymer is a water-soluble polymer with a network cross-linked structure. The water-soluble polymer contains some hydrophobic groups and hydrophilic residues. The hydrophilic residues combine with water molecules to connect the water molecules inside the network, while the hydrophobic residues swell when they come into contact with water.

[0045] Hydrogels are a type of highly hydrophilic structural gel that can swell rapidly in water, retain a large volume of water without dissolving, and can have a variety of shapes and strengths.

[0046] In this invention, the hydrogel is preferably one or more of gelatin, gelatin derivatives, cell matrix protein gel, sodium alginate gel and chitosan gel, and more preferably gelatin or gelatin derivatives.

[0047] Preferably, the cell matrix protein glue is glue and / or glue.

[0048] The gelatin derivative may be a product obtained by chemically modifying conventional gelatin in the art, preferably methacrylic anhydride gelatin (GelMA).

[0049] Gelatin has excellent biocompatibility. Its curing process is not chemical cross-linking but reversible curing under the influence of temperature, and its curing process does not introduce cell-killing factors.

[0050] In this invention, the core preferably fills the opening of the cavity.

[0051] In this invention, the concentration of the cells in the kernel is preferably 10. 6 ~10 8 / mL, more preferably 10 7 ~5x10 7 / mL, and even more preferably 2x10 7 / mL.

[0052] The concentration of the cells in the kernel is 10. 6 ~10 8 / mL refers to the presence of 10 per milliliter of the said kernel. 6 ~10 8 The cells mentioned.

[0053] In this invention, the number of cells loaded in each core-shell microneedle can be conventional in the art, preferably 1,000 to 5,000, more preferably 2,000 to 4,000, and even more preferably 2,500, 2,960 or 3,000.

[0054] The present invention also provides a method for preparing the microneedles as described above, which includes the following steps:

[0055] A1: Prepare a microneedle shell with perforated sidewalls as described above;

[0056] A2: Then apply the suspension of the cells and the hydrogel solution into the cavity of the microneedle shell to solidify the suspension.

[0057] In A1, the method for preparing the microneedle shell can be conventional in the art, and preferably includes the following steps:

[0058] S1: Fill the cavity of the first female mold with the liquid of the compound of the microneedle shell;

[0059] S2: Press the first male mold into the cavity filled with the liquid compound, the end of the first male mold abutting against the side wall of the first female mold to form the perforation, and pull out the first male mold after the compound has solidified.

[0060] In S1, the liquid compound is preferably formed from the solid of the compound in a molten or glassy state after being heated in a vacuum environment.

[0061] The heating temperature can be conventional in the art, preferably higher than the glassy state temperature of the compound and lower than the carbonization temperature and ester bond breaking temperature of the compound, and more preferably 100-150°C higher than the glassy state temperature of the compound.

[0062] The vacuum environment is preferably a vacuum drying oven.

[0063] The vacuum level of the vacuum environment is 0 to 0.2 MPa, more preferably 0.1 MPa.

[0064] In S2, the preferred method of pressing is to first wait for the liquid of the compound in S1 to solidify, then invert the first male mold onto the solid of the compound, and then heat it to liquefy the solid of the compound, causing the first male mold to sink into the cavity by itself.

[0065] In A2, preferably, the hydrogel solution comprises the hydrogel and cell culture medium.

[0066] The hydrogel solution containing cell culture medium can supply the cells with the necessary nutrients during the microneedle preparation process and animal experiments, thus avoiding or reducing cell death.

[0067] In A2, the method of application can be conventional in the art, but loading is preferred.

[0068] The loading process typically involves filling the cavity of the microneedle shell with a solution of the mobile phase through vacuum or centrifugation.

[0069] In A2, preferably after application and before curing, the suspension remaining on the microneedle substrate is also removed.

[0070] The preferred method for removal is to first aspirate the fluid with a pipette and then scrape it off with a scraper.

[0071] In A2, the curing method can be conventional in the art, and preferably is cooling curing.

[0072] The temperature for cooling and curing can be conventional in the art, preferably 1-6°C, more preferably 2-4°C, and even more preferably 4°C.

[0073] The cooling and curing time can be conventional in the field, preferably 5 to 30 minutes, and more preferably 15 minutes.

[0074] The present invention also provides a microneedle patch comprising a plurality of core-shell microneedles as described above and a substrate connecting the core-shell microneedles.

[0075] In this invention, the material of the substrate can be conventional in the art, and preferably the same as the material of the microneedle shell.

[0076] In this invention, the arrangement of the plurality of microneedles can be conventional in the art, but preferably an array arrangement.

[0077] The present invention also provides a method for preparing a microneedle patch as described above, which includes the method for preparing core-shell microneedles as described above, wherein the compound of the microneedle shell is cured at the connection between the microneedles of the first negative mold to form the substrate.

[0078] In this invention, the preparation method of the substrate can be conventional in the art. Preferably, the substrate is prepared by the following method: placing the solid compound of the microneedle shell into the center of the microneedle array in the negative mold, heating and liquefying it, the liquid of the compound automatically filling the cavity, and the residual liquid of the compound between the microneedles solidifying to form the substrate.

[0079] The present invention also provides the application of the microneedles or microneedle patches described above in the preparation of medical devices for treating inflammatory skin diseases.

[0080] In this invention, the inflammatory skin disease can be conventional in the art, and preferably includes one or more of psoriasis, dermatitis, and systemic lupus erythematosus.

[0081] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0082] The reagents and raw materials used in this invention are all commercially available.

[0083] The positive and progressive effects of the present invention are as follows: (1) The core-shell microneedles or microneedle patches of the present invention achieve better cell delivery function, so that the cells are highly enriched at the drug delivery site, maintain cell function, deliver a large number of cells, and continuously nourish the cells to maintain cell activity, thereby achieving better skin disease treatment effect, reducing cell resource waste and reducing the cost of cell therapy.

[0084] (2) The core-shell microneedles of the present invention have good mechanical properties and can effectively penetrate the dermis to deliver cells; the overall structure of the degraded microneedles remains intact and will not remain on the skin. Attached Figure Description

[0085] Figure 1 This is a structural diagram of a perforated microneedle;

[0086] Figure 2 The left image is a view of the second female mold in Example 1; Figure 2 The right figure is a view of the second positive mold in Example 1;

[0087] Figure 3 Part A is an image of the microneedle shell obtained in Example 1; Figure 3 Part B consists of cross-sectional views of different cross-sections;

[0088] Figure 4 This is a graph showing the test results of the ability of the microneedle shell to degrade and produce fatty acids in Example 1;

[0089] Figure 5 This is a diagram showing the cell distribution in the core-shell microneedles of Example 1;

[0090] Figure 6 This is a graph showing the cell release performance test results of the core-shell microneedles in Example 1;

[0091] Figure 7 This is a schematic diagram of the animal experiment process;

[0092] Figure 8 Images showing changes in the skin on the backs of mice in each group at different time points;

[0093] Figure 9 The graph shows the changes in PASI in each group of experimental mice.

[0094] Figure 10 The changes in the skin on the lower back of three mice in each of groups G6 and G7 at different time points are shown in the graph.

[0095] Figure 11 The figure shows the results of the cell activity experiment with direct addition of fatty acids;

[0096] Figure 12 The effect of in vitro culture on the functional expression of regulatory T cells;

[0097] Figure 13 The left figure shows the experimental results of propionate enhancing Foxp3 expression; Figure 13 The right figure shows the results of propionate enhancing the inhibitory effect of Treg on Tcon;

[0098] Figure 14 The left figure shows the experimental results of the effect of microneedle shell degradation of fatty acids on enhancing Foxp3 expression in Example 1; Figure 14 The right figure shows the results of the degradation of fatty acids by the microneedle shell in Example 1, which enhances the inhibitory effect of Treg on Tcon.

[0099] Figure 15This is a scanning electron microscope image of the degraded core-shell microneedles in an animal experiment of Example 1.

[0100] Figure label:

[0101] A - Second female mold; B - Second male mold; A-1 is a three-dimensional view; A-2 is a longitudinal section view of JJ; A-3 is a top view; A-4 is a cross-sectional view of N; A-5 is a top view; A-6 is a longitudinal section view of BB; B-1 is a three-dimensional view; B-2 is a top view; B-3 is an enlarged view of part A of B-4; B-4 is a top view; A-2-1 is 0.05mm; A-2-2 is 0.50mm; A-2-3 is 0.40mm; A-2-4 is 0.92mm; A-4-1 is 0.17mm; A-4-2 is 0.15mm; A-4-3 is 0.2mm; A-6-1 The thickness of A-6-1 is 0.30mm; A-6-2 is 0.08mm; A-6-3 is 0.417mm; A-6-4 is 0.35mm; A-6-5 is 0.92mm; A-6-6 is 0.418mm; A-6-7 is 0.09mm; B-2-1 is 0.18mm; B-2-2 is 0.60mm; B-3-1 is 0.10mm; B-3-2 is 0.41mm; B-3-3 is 0.17mm; B-4-1 is 0.41mm; B-4-2 is 0.73mm; B-4-3 is 1.10mm; B-4-4 is 0.60mm. Detailed Implementation

[0102] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0103] The raw material information in the following examples and comparative examples is as follows:

[0104] Vinyl propionate (VP): Shanghai Aladdin Biochemical Technology Co., Ltd., containing stabilizer MEHQ, >98.0% (GC);

[0105] Methyl methacrylate (MMA): Shanghai Aladdin Biochemical Technology Co., Ltd., AR, 99%, containing 30ppm DMBP stabilizer.

[0106] Example 1

[0107] Preparation of core-shell microneedles that can be degraded by esterases:

[0108] S1: Preparation of VP-MMA copolymer

[0109] VP and MMA were separately distilled to remove the polymerization inhibitor, resulting in purified VP and MMA. The purified VP and MMA were mixed with 2,2'-azobis(isobutyronitrile) (AIBN) at a molar ratio of 7:3, with the AIBN added at a concentration of 0.01 g / mL (mL being the volume of the VP and MMA mixture). The mixture was reacted at 60 °C for 48 h to obtain a methyl methacrylate-vinyl propionate copolymer. Its structural formula is shown in formula (3) below, where a, b, c, d, and e represent H at different positions:

[0110]

[0111] The obtained product was subjected to NMR. 1 HNMR characterization yielded the hydrogen nuclear magnetic resonance spectrum, such as... Figure 1 As shown. Figure 1 Peak d in the figure is the characteristic peak of the ester bond, which proves that the copolymer contains ester bonds that can be degraded by esterases.

[0112] S2: Fabrication of a microneedle shell with perforated sidewalls

[0113] The microneedle structure was designed using 3D software, and then utilized... S140 (Chongqing Mofang Precision Technology Co., Ltd.) 3D printing technology was used to print an 8*8 array of second male molds and an 8*8 array of second female molds made of HTL (Chongqing Mofang Precision Technology Co., Ltd.) material. The shape and size of the individual microneedles in the second female mold and the second male mold are as follows: Figure 2 As shown. The second positive mold and the second negative mold were first soaked in an excess of isopropanol for 12 hours. Afterward, the molds were removed and baked in a 90-degree oven for 6 hours to ensure complete drying. Then, the prepared... 184 (Dow) polydimethylsiloxane (PDMS) is introduced into the grooves of two second molds and subjected to a vacuum of -0.1 MPa for 5-10 minutes to completely remove air bubbles and ensure that the PDMS adheres perfectly to the molds. The second molds containing PDMS are then placed in a 60-degree oven for 1 hour. Demolding yields the first female mold corresponding to the second male mold and the first male mold corresponding to the second female mold. 158 mg of the polymethyl methacrylate-vinyl propionate copolymer obtained from S1 was placed in the center of the first female mold microneedle array. The array was then placed in a vacuum drying oven at 160°C for 20 minutes under vacuum. After the liquefied polymethyl methacrylate-vinyl propionate copolymer filled the cavity of the first female mold, the first female mold was allowed to return to room temperature. At this point, the polymethyl methacrylate-vinyl propionate copolymer solidified. The first male mold was then inverted onto the solid copolymer, forming a "sandwich" structure. Both were placed in the 160°C drying oven, where the solid copolymer reliquefied. Due to gravity, the first male mold sank into the female mold, and the protruding first end of the first male mold contacted the inner surface of the first female mold, forming a perforation. The portion not in contact with the inner surface, along with the inner surface, clamped out the wall of the microneedles. After this structure was returned to room temperature, the first male mold was removed, resulting in the following structure. Figure 3 The microneedle housing shown has perforated sidewalls, and a connecting substrate is formed between the individual microneedle housings. Figure 3 As can be seen in part A (i.e. the upper part), the microneedle array is clearly visible, and the perforated hole structure can be observed from various angles.

[0114] Figure 3 Part B (the lower half) consists of four images from left to right, which are cross-sectional views of the microneedle shell at different heights from the tip to the bottom. It can be seen that the microneedle shell has a distinct cavity structure, and the cavity structure is symmetrical and has a moderate wall thickness. Figure 3 The fifth image from left to right in part B (i.e., the lower half) is a schematic diagram of different cross-sections.

[0115] S3: Preparation of the core of core-shell microneedles

[0116] The cultured regulatory T cells were centrifuged, the supernatant was removed, and the pellet was added to a 0.02 g / mL gelatin solution (mL being the volume of RPMI medium) at 37°C. The gelatin solution was prepared by mixing solid gelatin and RPMI medium. The cell pellet was repeatedly pipetted to resuspend the cells in the solution, resulting in a cell concentration of 2 x 10⁻⁶ cells / mL. 7 / mL (mL is the volume of the gelatin solution) of gelatin cell suspension. At 37°C, the gelatin suspension is liquid. 100 μL of the suspension is taken with a pipette and spread evenly in the center of the microneedle array after the first positive mold has been removed (at this time, the microneedles have not detached from the first negative mold). The microneedle shell with cell suspension is then placed in a vacuum chamber at 37°C with saturated water vapor. Vacuum is applied at 0.1 MPa for 2 minutes to allow the suspension to sink into the hollow of the perforated microneedle shell, with each microneedle filled with 0.148 μL. After restoring atmospheric pressure, the suspension on the microneedle substrate that has not entered the needle tip is first aspirated with a pipette and then scraped off with a cell scraper. The microneedles are placed in a 4°C refrigerator for 15 minutes to solidify the gelatin cell suspension into a hydrogel, thus obtaining the core-shell microneedles and microneedle patch of the present invention (the microneedle patch includes an array of microneedles and a substrate connecting each microneedle). It is then stored at 4°C.

[0117] The core-shell microneedles obtained in this invention can be applied to the skin surface. The tips of the core-shell microneedles pierce the stratum corneum and epidermis, penetrating into the dermis. The perforations of the core-shell microneedles are located in the dermis, allowing the cell-loaded hydrogel to connect with the tissue fluid of the dermis through the pores. Since the body temperature is approximately 37 degrees Celsius, the gelatin hydrogel is heated and liquefied by the body, thus forming a liquid system with the tissue fluid of the dermis and the hydrogel inside the perforated microneedles. Due to the high cell concentration inside the microneedles, cells enter the dermal tissue fluid through active migration and passive diffusion, achieving cell delivery. Simultaneously, esterases highly expressed in the dermis and epidermis of inflamed areas such as psoriasis directly contact the microneedle shell, catalyzing the breakage of ester bonds to continuously produce free propionate. Propionate can be taken up by regulatory T cells, continuously nourishing them and enhancing their performance in suppressing inflammation.

[0118] Examples 2-9

[0119] The volume ratio of VP and MMA and the addition ratio of AIBN in Example 1 were changed as shown in Table 1. All other parameters were the same as S1 and S2 in Example 1, resulting in a microneedle shell with perforated sidewalls.

[0120] Comparative Example 1

[0121] Only S1 and S2 of Example 1 were performed to obtain blank microneedles.

[0122] Comparative Example 2

[0123] S1: Preparation of PMMA polymer

[0124] Commercially available MMA (Shanghai Aladdin Biochemical Technology Co., Ltd., AR, 99%, containing 30 ppm DMBP stabilizer) was distilled to remove the polymerization inhibitor, yielding purified MMA. Each milliliter of purified MMA was added to 0.01 g of AIBN and mixed, then reacted at 60°C for 48 h to obtain polymethyl methacrylate (PMMA).

[0125] S2: Fabrication of PMMA microneedle shell

[0126] The temperature of the vacuum drying oven in S2 is 190°C, the polymer is PMMA obtained in S1, and everything else is the same as S2 in Example 1, to obtain a microneedle shell of PMMA.

[0127] S3: Preparation of the core of core-shell microneedles

[0128] S3 is the same as S3 in Example 1, and MMA microneedles are obtained.

[0129] Effect Example

[0130] 1. Mechanical performance testing

[0131] The mechanical properties of the microneedles were determined using a pressure testing instrument. Specifically, 1 to 4 microneedles obtained in Examples 1 to 9, along with their substrates, were placed on a testing platform. The descent speed of the pressure platform was set to 0.1 mm per minute, and the stress recording threshold was 0.025 Newtons. After the machine was started, the pressure platform moved towards the testing platform, pressing the microneedles until they were in close contact with the platform. When the stress exceeded the recording threshold, the displacement of the pressure platform was recorded, and the stress-displacement curve was plotted to determine the maximum force that each needle could withstand.

[0132] The results are shown in Table 1. Table 1 shows that when the volume ratio of VP to MMA is 3:1, changing the AIBN addition ratio results in a consistently soft microneedle shell with almost no load-bearing capacity. As the volume ratio of VP to MMA decreases, the maximum load-bearing capacity of the microneedle shell increases accordingly. When the volume ratio of VP to MMA is 7:3 or 13:7, the maximum load-bearing capacity of the microneedle shell decreases with increasing AIBN addition ratio.

[0133] Table 1

[0134]

[0135]

[0136] 2. Test on the ability to degrade and produce fatty acids

[0137] The microneedle shells obtained in S2 of Example 1 were placed in 1 mL of 100 IU (International Units of Enzyme Activity) esterase / PBS solution and PBS solution without esterase, respectively, and reacted at 37°C. At the corresponding sampling time points, all PBS solutions were taken out and replaced with the same volume of PBS solution to continue the reaction.

[0138] Since propionate is difficult to detect with conventional liquid chromatography and other instruments, it is necessary to derivatize it to obtain derivatives, which can then be analyzed using liquid chromatography coupled with tandem mass spectrometry. For PBS solutions taken at different time points, 20 μL of 200 mM 3-nitrophenylhydrazine hydrochloride solution (dissolved in 50% v / v acetonitrile aqueous solution) and 20 μL of 120 mM 1-(3-dimethylaminopropyl)-3-ethylcarboimide solution (dissolved in 6% v / v pyridine aqueous solution) were added to every 40 μL of solution, and the mixture was reacted at 40 °C for 30 minutes to obtain propionic acid derivatives.

[0139] The release of propionic acid derivatives was analyzed using liquid chromatography coupled with tandem mass spectrometry (LC-MS / MS), and the release of propionate was calculated from the release of propionic acid derivatives, resulting in a plot of the cumulative release of propionate. The results are as follows: Figure 3 As shown in the figure, the copolymer shell of Example 1 of this application exhibits a relatively uniform fatty acid release rate under 100 IU esterase degradation, producing an average of approximately 100 μg of fatty acids per hour, which can continuously nourish cells. It shows virtually no degradation in PBS solution, producing very little fatty acid.

[0140] 3. Cell distribution test in core-shell microneedles

[0141] The core-shell microneedles are basically the same as S1 to S3 in Example 1, except that: the freshly extracted regulatory T cells are first incubated with 5 μM (molar concentration of CFSE) 5-carboxyfluorescein succinimide (CFSE) / DPBS solution at 37 degrees Celsius for 30 minutes, and then centrifuged at 300 centrifugation to discard the supernatant and remove the precipitate.

[0142] The nucleoshell microneedles loaded with cells were placed tip-down in the center of a confocal dish, which was then placed under the lens of a confocal microscope. The results are as follows: Figure 5 As shown.

[0143] Depend on Figure 4 As can be seen, under a confocal microscope, regulatory T cells stained with CFSE fluorescence can be seen on different cross sections of the nucleoshell microneedles (bright areas). It can be seen that the cells fill the lumen of the nucleoshell microneedles at a high concentration. The uneven cross-section in the fusion image of the bright field channel and the fluorescence channel shows the hydrogel state after the gelatin has solidified, indicating that it can fix the cells in the lumen without them flowing out.

[0144] 4. Cell release performance test in core-shell microneedles

[0145] The experimental method is basically the same as that for the "cell distribution assay in nucleoshell microneedles", the difference being that: the perforated microneedles loaded with cells are inserted tip-down into the hydrogel in the center of the confocal dish, and the hydrogel in the dish is pre-filled with 300 μL of water. The hydrogel was cured by incubating at 37°C for 2 hours. The confocal dish was then placed under a confocal microscope lens. The results are as follows: Figure 6 As shown.

[0146] from Figure 6 As can be seen, cell release at different time points was observed under a confocal microscope on the same cross-section, perpendicular to the perforated microneedles. Bright circles mark the microneedle shells; it can be seen that as time progresses, cells gradually spread beyond the bright circles and migrate out of the perforations. When release ceases, the total number of released cells can be displayed through 3D reconstruction; in the side view and bird's-eye view, it can be seen that the released cells permeate the entire environment.

[0147] 5. Animal experiments

[0148] A schematic diagram of the animal experiment treatment process is shown below. Figure 7 As shown. 8-9 week old mice (strain name BALB / c) were induced with psoriasis-like inflammation by continuous application of 5% imiquimod ointment (Mingxinlidi) (Day 0–Day 6). Mice were first treated with hair removal on their backs, rested for two days, and mice whose backs had grown hair were excluded from the experiment. Mice whose backs had not grown hair were divided into 7 groups (G1-G7), with 4–6 mice in each group:

[0149] The normal group (G1) applied 40 mg of petroleum jelly ointment daily; the untreated group (G2) applied 40 mg of 5% imiquimod ointment daily, and on the second day (Day 2), 4 hours after applying the ointment, they wrapped their backs with bio-adhesive tape.

[0150] The blank microneedle group (G3) was treated with 40 mg of 5% imiquimod ointment every day. On the second day (Day 2), 4 hours after the ointment was applied, the blank microneedle patch obtained from Comparative Example 1 was applied to the back of the mice and wrapped with bio-adhesive tape.

[0151] The systemic injection group (G4) received daily application of 40 mg 5% imiquimod ointment, followed by a tail vein injection of 4 × 10 mg ointment 4 hours after application on Day 2. 7 Cells / kg mouse;

[0152] The intradermal injection group (G5) received daily application of 40 mg 5% imiquimod ointment. On Day 2, four hours after applying the ointment, two intradermal injection sites were administered on the back, with each site receiving 20 μL of 8 × 10⁻⁶ solution.6 A mixture of cells / mL complete T-cell culture medium (without interleukin-2);

[0153] The MMA microneedle group (G6) was treated with 40 mg of 5% imiquimod ointment daily. On the second day (Day 2), 4 hours after the ointment was applied, the MMA microneedle patch obtained in Comparative Example 2 was applied to the back of the mice and wrapped with bio-adhesive tape. The microneedle group (G7) was treated with 40 mg of 5% imiquimod ointment daily. On the second day (Day 2), 4 hours after the ointment was applied, the core-shell microneedle patch of Example 1 was applied to the back of the mice and wrapped with bio-adhesive tape.

[0154] The purpose of wrapping the bio-adhesive tape with the mice was to secure the microneedle patch and prevent it from being rubbed off or peeled off due to the mice's free movement. All tapes and microneedles were left on for 6 hours.

[0155] Every 24 hours, photographs were taken of the skin on the back of the most representative mouse in each group. The results are as follows: Figure 8 As shown. Two researchers visually assessed the severity of psoriasis lesions using the PASI (Pain and Severity Index, categorized as erythema, induration, and desquamation), and the scores were averaged to obtain the final score. Figure 9 PASI change curves. Figures 8 and 9 show that the uninduced G1 group remained healthy, while the untreated G2 group experienced severe keratinization and erythema. The G3 group did not experience remission, indicating that fatty acid release alone cannot reverse the progression of psoriasis. The G4 group received a systemic injection of regulatory T cells compared to the G7 group, using a higher cell count than the clinical reference (4 × 10⁻⁶). 7 The G5 group (cells / kg mice) also failed to effectively alleviate psoriasis, with its weak effect possibly due to low cell enrichment and altered cell function observed in previous studies. Intradermal injection of regulatory T cells in the G5 group also showed some efficacy, but because the area of ​​cell action after intradermal injection was relatively small, it did not achieve the same level of effect as G7. The advantage of G7 (which can degrade to fatty acids) over G6 (which cannot degrade to fatty acids) is that G7 can reduce skin erythema to a greater extent.

[0156] and Figure 8 The experimental methods were the same, the difference being that on days 3, 4, and 5, three mice from groups G7 and G6 were selected respectively to take photographs of their back skin. The results are as follows: Figure 10 As shown. Figure 10 The results showed that the three mice from G7 had better skin condition and less redness than the three mice from G6, demonstrating that the core-shell microneedles of this invention have better efficacy in treating skin diseases such as psoriasis.

[0157] 7. Cell viability experiment with direct addition of fatty acids

[0158] Instead of using core-shell microneedles to slowly degrade fatty acids, fatty acids were directly added to the cell solution to test cell viability. The specific method is as follows: sterile sodium propionate (1M) was added to 100 μL of 10... 6 Cells were incubated at different final concentrations (0 mM, 60 mM, 120 mM, 180 mM) in 96-well plates at a concentration of 1 / mL for 2 hours, and then cell morphology was observed. The results are as follows: Figure 11 As shown. From Figure 11 As can be seen, with the increase of sodium propionate concentration, the cell morphology changed significantly, and the appearance changed from round to wrinkled (red arrow). This proves that the direct addition of excessive fatty acids to the cell solution will generate a huge osmotic pressure, which will cause the cells to lose water, wrinkle and die.

[0159] 8. Effect of in vitro culture time on the functional expression of regulatory T cells

[0160] Mouse spleen was processed using the EasySep kit. TM Mouse CD4+CD25+Regulatory T CellIsolation Kit II(Stemcell TM Regulatory T cells extracted by Technologies were added to complete T cell culture medium at a concentration of 10... 6 Cells were cultured at a concentration of [number] cells / mL in 96-well plates. The complete T-cell culture medium consisted of 10% (v / v) heat-inactivated fetal bovine serum, 100 IU / mL penicillin and streptomycin, 1 mM sodium pyruvate, 1X non-essential amino acids, 20 mM HEPES buffer, 50 μM β-mercaptoethanol (all purchased from Gibco), and 1500 IU / mL interleukin-2 (Peprotech, catalog number 200-02). Cell counts were determined at different in vitro culture times: cells were pipetted to evenly disperse the settled cells in the culture medium, and 10 μL of the cell dispersion was diluted with Countess... TM 3. (Thermo Fisher Scientific) Counting. Determination of Foxp3 expression: Regulatory T cells cultured for different time periods were analyzed using flow cytometry with the fixed viability stain 510 (BDHorizon) antibody. TM ,564406), BV421 Rat Anti-Mouse CD4 (BD Horizon TM (GK1.5,562891) and Alexa 647Rat anti-Mouse Foxp3(BD Pharmingen TMCells labeled with MF23,560401 were analyzed using a Beckman CytoFlex S flow cytometer to determine the percentage of CD4+Foxp3+ cells among viable cells. Results are as follows: Figure 12 As shown in the figure. The results indicate that although the number of cells gradually increases with the increase of culture time, the expression of Foxp3, which determines the function of regulatory T cells, will gradually decrease. The number of cells and the expression level of Foxp3 are negatively correlated. Therefore, the regulatory T cells of this application are not cultured in vitro, but are extracted and used immediately.

[0161] 9. Effects of fatty acids on Treg function

[0162] Different concentrations (molar concentrations of sodium propionate in the mixed cell solution) of sodium propionate were co-incubated with regulatory T cells for 0–3 days, and Foxp3 expression levels were tested (the test method was the same as the Foxp3 expression test method described above). Results are as follows: Figure 13 As shown in the left figure. From Figure 13 As shown in the left figure, sodium propionate has an increasing effect on the expression level of Foxp3 as the culture time is extended, but at the same time point, the effect of different concentrations of sodium propionate on the expression level of Foxp3 is not significantly different.

[0163] Simultaneously, 2 mM sodium propionate was co-incubated with regulatory T cells (Treg) and conventional T cells (Tcon). Specifically, mouse spleen cells were incubated using the Mouse T Cell Isolation Kit (Stemcell). TM Tcon was obtained from Technologies. The Tcon was first stained with 5 μM CFSE (as described above), and then stained with 10 μM CFSE. 6 Cells / mL were cultured in 96-well plates of complete T-cell culture medium (the culture medium was basically the same as in Part 8, "Effect of In Vitro Culture Time on Regulatory T-cell Functional Expression," except that the concentration of interleukin-2 was 50 IU / mL). Different amounts of freshly extracted Tregs and different volumes of 1M sodium propionate solution were then added to achieve different Tcon:Treg ratios and a final sodium propionate concentration of 2mM. The proliferation coefficient was obtained by dividing the number of expanded cells by the number of unexpanded cells. The results are shown below. Figure 13 As shown in the right figure. From Figure 13The right-hand figure shows that the proliferation coefficient of conventional T cells (the proliferation coefficient is calculated by dividing the number of expanded cells by the number of unexpanded cells) is significantly inhibited by Treg cells, and the presence of sodium propionate can enhance this inhibition. This inhibition is evident when the ratio of Tcon to Treg is much higher (4:1, 8:1). In summary, propionate can enhance the inhibitory function of Treg, primarily by maintaining the expression of the characteristic transcription factor Foxp3 and inhibiting Tcon.

[0164] 10. Effects of core-shell microneedle degradation of fatty acids on Treg function

[0165] The microneedle shells obtained in S1 and S2 of Example 1 were placed in an esterase solution prepared with 1 mL of 100 IU / mL RPMI 1640 medium and reacted for 24 hours. The released solution was ultrafiltered to remove protein. A small amount of the filtrate was analyzed for propionate concentration using liquid chromatography-tandem mass spectrometry. 10% (v / v) fetal bovine serum was added to the remaining liquid to prepare culture media with different propionate concentrations. These media were co-cultured with Tregs for a period of time to test Foxp3 expression. The results are as follows: Figure 14 As shown in the left figure. From Figure 14 The left figure shows that the fatty acid salts degraded by microneedles can react with directly added fatty acid salts ( Figure 13 This achieves a significant effect, namely, increasing the expression of Foxp3 in Treg.

[0166] The propionate derived from the degradation of the microneedle shells obtained in S1 and S2 of Example 1 was co-cultured with different proportions of Treg and Tcon for a period of time (using the same method). Figure 13 The experimental method shown in the right figure is as follows: Figure 14 As shown in the right figure, the fatty acid salts degraded from the microneedle shell can also enhance the inhibitory effect of Treg on Tcon: the proliferation coefficient of conventional T cells was significantly inhibited by Treg cells at all ratios. Figure 13 Comparative analysis shows that the propionate produced by the degradation of the microneedle shell in esterase can achieve essentially the same effect as the chemical propionate in enhancing the expression of Foxp3 in Treg and enhancing the inhibitory effect of Treg on Tcon.

[0167] 11. Microneedle diagram after esterase degradation

[0168] Scanning electron micrographs of microneedles removed from mice after animal experiments, such as... Figure 15 As shown, the overall structure of the microneedles remains intact and is not left on the skin. However, some structural changes indicate that the mechanical properties have been altered after the outer shell degraded.

Claims

1. A core-shell microneedle, characterized in that, It includes a microneedle shell with perforations on the sidewalls and a core filling the cavity enclosed by the microneedle shell, wherein the compound of the microneedle shell is a polymer; the pore size of the perforations is larger than the outer diameter of one cell; The polymer is a copolymer of methyl methacrylate and vinyl propionate; in the copolymer of methyl methacrylate and vinyl propionate, the molar ratio of methyl methacrylate to vinyl propionate is (3:7) to (4:6); the copolymer of methyl methacrylate and vinyl propionate is obtained by polymerization reaction of methyl methacrylate, vinyl propionate and thermal initiator; The core comprises a hydrogel and cells dispersed in the hydrogel; the cells are regulatory T cells.

2. The core-shell microneedle as described in claim 1, characterized in that, The thermal initiator is selected from 2,2'-azobis(isobutyronitrile) and / or benzoyl peroxide.

3. The core-shell microneedle as described in claim 2, characterized in that, The thermal initiator is 2,2'-azobis(isobutyronitrile).

4. The core-shell microneedle as described in claim 1, characterized in that, The perforation is circular or polygonal in shape; And / or, the thickness of the microneedle shell is 80~150μm; And / or, the outer surface shape of the microneedle shell is conical or pyramidal; And / or, the volume of the cavity in each microneedle is 0.05~0.5μL.

5. The core-shell microneedle as described in claim 4, characterized in that, The polygon is a triangle, a quadrilateral, or a pentagon; And / or, the outer surface of the microneedle shell is conical in shape; And / or, the volume of the cavity in each microneedle is 0.1~0.2 μL.

6. The core-shell microneedle as described in claim 5, characterized in that, The diameter of the perforation is 50-200 μm; And / or, the volume of the cavity in each microneedle is 0.148 μL.

7. The core-shell microneedle as described in claim 4, characterized in that, The length of the cone is 1000~1500μm, and the length is the vertical distance from the tip of the cone to the bottom.

8. The core-shell microneedle as described in claim 4, characterized in that, The diameter of the base of the cone is 300-800 μm, and the base is the side opposite to the tip of the cone.

9. The core-shell microneedle as described in claim 1, characterized in that, The core also includes cell nutrient solution; And / or, the concentration of the cells in the kernel is 10. 6 ~10 8 / mL; And / or, the cells are obtained by magnetic bead adsorption extraction; And / or, the cells are cells that have not undergone any biological treatment after extraction; And / or, the hydrogel is one or more of gelatin, methacrylic anhydride gelatin, matrix protein gel, sodium alginate gel and chitosan gel; And / or, the core fills the opening of the cavity; And / or, the number of said cells loaded in each core-shell microneedle is 1000 to 5000.

10. The core-shell microneedle as described in claim 9, characterized in that, The cell nutrient solution is a cell culture medium or a glucose solution; And / or, the concentration of the cells in the kernel is 10. 7 ~5x10 7 / mL; And / or, the hydrogel is gelatin or methacrylic anhydride gelatin; And / or, the number of said cells loaded in each core-shell microneedle is 2000 to 4000.

11. The core-shell microneedle as described in claim 10, characterized in that, The cell culture medium was RPMI 1640 medium; And / or, the concentration of the cells in the kernel is 2 x 102 7 / mL; And / or, the number of said cells loaded in each core-shell microneedle is 2500, 2960, or 3000.

12. The core-shell microneedle as described in claim 9, characterized in that, The concentration of the hydrogel in the cell nutrient solution is 0.01~0.03 g / mL.

13. The core-shell microneedle as described in claim 9, characterized in that, The concentration of the hydrogel in the cell nutrient solution is 0.02 g / mL.

14. A method for preparing core-shell microneedles as described in any one of claims 1 to 13, characterized in that, It includes the following steps: A1: Prepare the microneedle shell with perforated sidewalls; A2: Apply the suspension of the cells and the hydrogel into the cavity of the microneedle shell and solidify the suspension.

15. The method for preparing core-shell microneedles as described in claim 14, characterized in that, The method for preparing the microneedle shell includes the following steps: S1: Fill the cavity of the first female mold with the liquid of the compound of the microneedle shell; S2: Press the first male mold into the cavity filled with the liquid. The end of the first male mold abuts against the side wall of the first female mold to form the perforation. After the compound has solidified, the first male mold can be pulled out.

16. The method for preparing core-shell microneedles as described in claim 15, characterized in that, The first female mold and the first male mold are made of PDMS.

17. The method for preparing core-shell microneedles as described in claim 15, characterized in that, The first female mold and the first male mold are respectively made from the second male mold and the second female mold, and the second male mold and the second female mold are both made of metal.

18. The method for preparing core-shell microneedles as described in claim 17, characterized in that, The second male mold and the second female mold were obtained by 3D printing technology.

19. The method for preparing core-shell microneedles as described in claim 15, characterized in that, In S2, the pressing method is to first wait for the liquid of the compound in S1 to solidify, then invert the first male mold onto the solid of the compound, and then heat it to liquefy the solid of the compound, and the first male mold sinks into the cavity by itself.

20. A microneedle patch, characterized in that, It includes a plurality of core-shell microneedles as described in any one of claims 1 to 13 and a substrate connecting the core-shell microneedles.

21. The microneedle patch as described in claim 20, characterized in that, The substrate is made of the same material as the microneedle shell.

22. The microneedle patch as described in claim 20, characterized in that, The microneedles are arranged in an array.

23. The microneedle patch as described in claim 22, characterized in that, The microneedles are arranged in an 8x8 array.

24. A method for preparing a microneedle patch as described in any one of claims 20-23, comprising the method for preparing core-shell microneedles as described in any one of claims 14-19, characterized in that, The compound of the microneedle shell is cured at the junction between the microneedles of the first negative mold to form the substrate.

25. The use of a microneedle as described in any one of claims 1-13 or a microneedle patch as described in any one of claims 20-23 in the preparation of a medical device for treating psoriasis.

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