Microneedle for inducing immune tolerance as well as preparation method and application of microneedle

Abstract

The invention provides a microneedle patch for inducing immune tolerance. The patch consists of a backing layer, a substrate part and a needle body part containing immune tolerance-inducing nanoparticles. According to the invention, the backing layer, the substrate and the needle body of the microneedle are all made of water-soluble high-molecular polymer; the immune-tolerant nanoparticles comprise a polymer material, an antigen or an allergen and an immunomodulator; the immunomodulator comprises cytokines, immunomodulatory drugs and lipids, the nanoparticles capable of inducing immune tolerance are delivered to the epidermal layer and the corium layer of the skin by using a painless and minimally invasive soluble microneedle administration platform, the uptake efficiency of the antigen or the allergen is improved, and meanwhile, the immunomodulator is combined to induce the body to generate potent immune tolerance. The micro-needle for inducing immune tolerance can improve the delivery efficiency of antigens and allergens to the greatest extent, enhance the effect of inducing immune tolerance, make up for the defects of pain and the like caused by an injection way, and has wide clinical application potential.

Description

Technical Field

[0001] The present invention relates to the technical field of biological microneedles, and in particular to a microneedle for inducing immune tolerance and a preparation method thereof. Background Art

[0002] Autoimmune diseases and allergies are common diseases around the world. Autoimmune diseases are abnormal immune responses of the body's immune system to its own tissues or self-antigens; allergies are abnormal immune responses of the body's immune system to specific antigens or allergens. Currently, the main drugs for the clinical treatment of autoimmune diseases and allergies are broad-spectrum glucocorticoids or biological agents. Although these drugs can quickly control the symptoms of the disease, they cannot fundamentally solve the autoimmune disease or allergy, and long-term use of these drugs will cause varying degrees of side effects on the body's immune system. Therefore, a new treatment strategy is urgently needed to fundamentally change the course of the disease.

[0003] Currently, researchers are trying to cure autoimmune diseases or allergies by inducing immune tolerance in the body. Repeated administration of autoantigens to induce immune tolerance and then reshape the immune system homeostasis is a promising treatment for autoimmune diseases or allergies. This strategy can induce immune tolerance and alleviate the inflammatory state with the help of appropriate doses of drugs / autoantigens without significantly suppressing the body's immune system.

[0004] Antigen presenting cells play an important role in inducing immune tolerance. When dendritic cells located throughout the body come into contact with or detect foreign substances such as viral RNA or bacterial membrane lipids, they mature and undergo phenotypic transformation. Mature DCs further present captured antigens in the form of major histocompatibility complex-II)-antigen peptides on the surface of DCs and upregulate the expression of co-stimulatory molecules to activate antigen-specific T cells. Subsequently, these DCs migrate to secondary lymphoid organs, present the antigens and co-stimulatory molecule signals they collect to T cells, and initiate secondary immune responses. Once there is no co-stimulatory signal during DC-T cell interaction or the co-stimulatory signal is covered by other signals in the environment (such as CTLA4-B7 binding affects T cell activation), T cells may lose their effector function. These T cells either differentiate into regulatory T cells, which play a role in regulating or inhibiting other immune cells, or directly apoptosis.

[0005] The skin is the largest organ in the human body and the best place to regulate immunotherapy. There are a large number of APCs in the skin, including Langerhans cells), DCs, macrophages and monocytes. The active immune environment of the skin provides innate conditions for antigens and immunomodulators, making it easier to achieve the desired treatment results. Based on this, we believe that the skin is a very potential target for immune regulation.

[0006] Due to the barrier properties of the skin, the transdermal delivery of macromolecular immunoactive substances remains a major challenge. Currently, a variety of technologies have been developed to deliver macromolecular drugs into the skin for the treatment of diseases such as cancer, autoimmune diseases, allergies and inflammation. Among them, microneedles have become a very attractive method of drug delivery due to their painless, minimally invasive, localized administration, convenience and pollution-free.

[0007] Here, we invent a microneedle for inducing immune tolerance, which is a new type of microneedle patch constructed by combining nanoparticles for inducing immune tolerance and soluble microneedles, and aims to induce regulatory T cells (Treg) and induce immune tolerance antigen-specific immune tolerance by synergistically delivering autoimmune disease antigens or allergens and immunomodulators, thereby treating or preventing allergies or autoimmune diseases. Summary of the invention

[0008] One of the purposes of the present invention is to provide a microneedle patch for inducing immune tolerance, which comprises a backing layer, a substrate part and a needle body part containing immune tolerance inducing nanoparticles, wherein the matrix materials of the backing layer, substrate and needle body of the microneedle are all water-soluble high molecular polymers; the immune tolerance inducing nanoparticles use polymer materials as carrier materials, the immune tolerance inducing nanoparticles include polymer materials, antigens or allergens and immunomodulators, and the immune tolerance inducing nanoparticles are distributed at the front end of the needle body. The immune tolerance inducing microneedle patch of the present invention can achieve the co-delivery of autoantigens or allergens and immunomodulators, improve the uptake level of autoantigens or allergens on antigen presenting cells and effectively induce immune tolerance or antigen-specific immune tolerance. The immune tolerance-inducing microneedle patch prepared by the present invention can be painlessly and minimally invasively delivered to the epidermis or dermis of the skin where antigen-presenting cells are enriched; the immune tolerance-inducing microneedle patch of the present invention, which carries polymer nanoparticles containing autoantigens or allergens and immunomodulators, is mainly concentrated in the needle tip of the microneedle, forming a micro antigen reservoir, which can prolong the release time of the autoantigens or allergens and is conducive to recruiting more antigen-presenting cells.

[0009] One of the purposes of the present invention is to provide a microneedle patch for inducing immune tolerance, which comprises a backing layer, a substrate and a needle body containing immune tolerance inducing nanoparticles, wherein the immune tolerance inducing nanoparticles include a polymer material, an antigen or an allergen and an immunomodulator, and the immune tolerance inducing nanoparticles are distributed at the front end of the needle body. Preferably, the polymer material is a natural or synthetic material, including: one or more of polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), polystyrene, acetylated dextran, poly-l-lysine, polyacrylamide and chitosan.

[0010] One of the purposes of the present invention is to provide a microneedle patch for inducing immune tolerance, characterized in that the antigen or allergen is selected from: glutamic acid decarboxylase (GAD), including: GAD65 and GAD67, type II collagen, myelin peptide MOG 35-55 The allergens are derived from Artemisia pollen allergens, including Artemisia vulgaris Linn. pollen allergens, Artemisia annua Linn. pollen allergens, Artemisia sieversiana Ehrhart ex Willd. pollen allergens, Artemisia argyi levl. et Van. pollen allergens, and animal dander allergens, including cat dander allergen Fe1 d1, dog dander allergen Can f1, horse dander allergens Equc c1 and Equ c2, cow dander allergens Bos d2 and Bos d5, rabbit dander allergen Ory c1, mouse dander allergen Mus M1, and allergen protein in rat dander Rat n1, one or more allergens selected from the group consisting of birch pollen allergens, in particular BETV1 and BETV4, timothy grass pollen allergens, in particular PHP2, PHP5, PHP6 and PHP7, house dust mite allergens, in particular Der P1 and Der P2, cat allergen Feld1, bee allergens, major wasp allergens, profilins, in particular Phi P12, and dust mite allergens, in particular LEP D2, chicken egg white allergen, one or more combinations of the above allergens.

[0011] As a preferred experimental scheme, the preferred antigen or allergen of the present invention is chicken ovalbumin (OVA).

[0012] One of the purposes of the present invention is to provide a microneedle patch for inducing immune tolerance, characterized in that it comprises a backing layer, a substrate portion and a needle body portion containing nanoparticles for inducing immune tolerance, wherein the nanoparticles for inducing immune tolerance include a polymer carrier, an autoantigen or an allergen.

[0013] One of the purposes of the present invention is to provide a microneedle patch for inducing immune tolerance. Preferably, the immune tolerance-inducing nanoparticles may also contain immunomodulators, which include but are not limited to: cytokines such as: one or a combination of IL-10, TGF-β; lipids: phosphatidylserine; one or a combination of rapamycin, curcumin, quercetin and Bay11-07082, vitamin D3.

[0014] As a preferred experimental scheme, the preferred immunomodulator of the present invention is rapamycin.

[0015] One of the purposes of the present invention is to provide a microneedle patch for inducing immune tolerance. Preferably, the matrix materials of the backing layer, substrate and needle body of the microneedle are all water-soluble polymers with good biocompatibility. The water-soluble polymers with good biocompatibility are selected from hyaluronic acid, polyvinyl alcohol, polyvinyl pyrrolidone, gelatin, collagen, silk fibroin, chondroitin sulfate, dermatan sulfate, heparan sulfate, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, carboxymethyl chitosan, chitosan, dextran sulfate, glycogen, amylose, dextrin, dextran, trehalose, glycosides and modified derivatives of the above materials. One or more.

[0016] As a preferred experimental scheme, the preferred water-soluble high molecular polymer of the present invention is one or more of hyaluronic acid and polyvinyl pyrrolidone.

[0017] One of the purposes of the present invention is to provide a microneedle patch for inducing immune tolerance, characterized in that it comprises a backing layer, a substrate part and a needle body part containing inducing immune tolerance, the matrix materials of the backing layer, substrate and needle body of the microneedle are all water-soluble polymers with good biocompatibility, and the water-soluble polymer is hyaluronic acid.

[0018] One of the purposes of the present invention is to provide a microneedle patch for inducing immune tolerance, characterized in that it comprises a backing layer, a substrate part and a needle body part containing inducing immune tolerance, the matrix materials of the backing layer, substrate and needle body of the microneedle are all water-soluble polymers with good biocompatibility, and the water-soluble polymer is polyvinyl pyrrolidone.

[0019] One of the purposes of the present invention is to provide a microneedle patch for inducing immune tolerance, wherein the microneedle body height is 200-1500μm, preferably 200-1000μm, more preferably 600-800μm; the width or diameter of the needle tip is 1-30μm, preferably 5-20μm, more preferably 8-12μm.

[0020] One of the purposes of the present invention is to provide a microneedle patch for inducing immune tolerance, wherein the nanoparticles for inducing immune tolerance are mainly concentrated at the front end of the soluble microneedle body, preferably accounting for 10-80% of the height of the entire microneedle body, so as to ensure that after the microneedle punctures the skin, the immune-inducing nanoparticles can be effectively released in the epidermis and dermis of the skin where antigen-presenting cells are enriched.

[0021] One of the purposes of the present invention is to provide a method for preparing a microneedle patch for inducing immune tolerance, characterized in that it comprises the following steps:

[0022] 1) preparing immune tolerance inducing nanoparticles encapsulating antigens, allergens and / or immunomodulators;

[0023] 2) filling the immune tolerance-inducing nanoparticles prepared in step 1) into the front end of the microneedle body;

[0024] 3) Prepare a needle matrix liquid and fill it evenly into the microneedle mold. The filling can be done by at least one of a pressurization method, a decompression method or a centrifugation method. After drying and solidification, demolding is performed to obtain a microneedle vaccine patch.

[0025] Wherein, step (2) uses centrifugation to fill the immune tolerance-inducing nanoparticles into the front end of the microneedle body. The specific method is to dilute the drug-loaded dextran microparticles with sterile water for injection or a suitable sterile buffer solution, and then evenly drop the immune tolerance-inducing nanoparticles onto the microneedle mold, and then place the microneedle mold in a centrifuge for centrifugation, with a centrifugal force of 1000g-8000g, preferably 2000g-5000g; the centrifugation time is 1-20min, preferably 5min-15min; the number of centrifugation is adjusted to 1-5 times, preferably 2-3 times, according to the microneedle drug loading requirement.

[0026] In step (2), a needle matrix liquid is prepared and uniformly filled into the microneedle mold. In some embodiments, the needle matrix liquid is filled by pressurization. The specific method is that after the needle matrix liquid is spread on the microneedle mold, the microneedle mold is placed in a pressurized sealed container and pressurized. The pressurization pressure is 0.001-0.9 MPa, preferably 0.5-0.7 MPa, and the pressurization time is 1-60 min, preferably 5-35 min.

[0027] In step (2), a needle matrix liquid is prepared and uniformly filled into the microneedle mold. In some embodiments, the needle matrix liquid is filled by a reduced pressure method. The specific method is that after the needle matrix liquid is spread on the microneedle mold, the microneedle mold is placed in a reduced pressure sealed container and the vacuum pressure is reduced. The vacuum pressure is -0.01--0.1MPa, preferably -0.07--0.095MPa, and the decompression time is 1-120min, preferably 5-30min.

[0028] In step (3), a needle matrix liquid is prepared and evenly filled into the microneedle mold. In some embodiments, the needle matrix liquid is filled by centrifugation. The specific method is to spread the needle matrix liquid on the microneedle mold, and then place the microneedle mold in a centrifuge for centrifugation. The centrifugal force is 200-20000g, preferably 1000-5000g, and the centrifugation time is 1-60min, preferably 3-30min.

[0029] One of the purposes of the present invention is to provide a microneedle patch for inducing immune tolerance, which is suitable for a microneedle patch for puncturing the skin and can be used for the preparation of skin medicines.

[0030] One of the purposes of the present invention is to provide a microneedle patch for inducing immune tolerance, wherein the vaccine patch can induce regulatory T cells (Treg) or antigen-specific Treg, thereby inducing immune tolerance or antigen-specific immune tolerance.

[0031] One of the purposes of the present invention is to provide a microneedle patch for inducing immune tolerance, which can induce immune tolerance or antigen-specific immune tolerance after being administered to the skin, and treat or prevent autoimmune diseases or allergies.

[0032] Beneficial Effects

[0033] The present invention provides a nanoparticle inducing immune tolerance as a delivery carrier, which is derived from an artificial or synthetic polymer material, has good biocompatibility, and can be efficiently taken up by antigen presenting cells.

[0034] The present invention provides a nanoparticle that induces immune tolerance as a delivery carrier, which can simultaneously encapsulate autologous disease antigens, allergens and / or immunomodulators, achieve co-delivery of autologous disease antigens or allergens and immunomodulators, and improve the uptake level of autologous disease antigens or allergens in antigen presenting cells.

[0035] The present invention provides a nanoparticle that induces immune tolerance as a delivery vector. The nanoparticle can not only be used as a delivery vector, but also can realize the co-delivery of multiple immunomodulators. At the same time, the autologous disease antigens or allergens and the immunomodulators play a synergistic role, which can significantly reduce the expression of co-stimulatory molecules on antigen presenting cells, and is beneficial to inducing immune tolerance or antigen-specific immune tolerance.

[0036] The immune tolerance-inducing microneedle patch of the present invention can break through the physiological barrier of the skin stratum corneum and deliver the immune tolerance-inducing nanoparticles to the epidermis or dermis of the skin where antigen presenting cells are enriched, thereby increasing the probability of autologous disease antigens or allergens and immunomodulators being taken up by antigen presenting cells, thereby enhancing the effect of inducing immune tolerance.

[0037] The microneedle patch for inducing immune tolerance of the present invention prolongs the release time of autologous disease antigens or allergens at the administration site, is conducive to recruiting more antigen presenting cells, further improves the delivery efficiency of autologous disease antigens or allergens, and enhances the effect of inducing immune tolerance.

[0038] The invention provides a method for preparing a microneedle patch for inducing immune tolerance, which is simple and rapid to prepare, has good repeatability and is suitable for industrial production.

[0039] The microneedle of the present invention is easy and convenient to use, does not require professional medical personnel to operate, is suitable for patients to vaccinate themselves, and the vaccination process is minimally invasive and painless, which can significantly improve patient compliance. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The electron microscope images of the nanoparticles prepared in Examples 1-3.

[0041] Figure 2 Nanoparticles that induce immune tolerance for DC2.4 uptake.

[0042] Figure 3 This is the morphology of the microneedle that induces immune tolerance observed under an optical magnifying glass.

[0043] Figure 4 Laser confocal microscopy images of microneedles that induce immune tolerance.

[0044] Figure 5 Scanning electron microscopy morphology characterization of microneedles for inducing immune tolerance.

[0045] Figure 6 Effect of microneedle skin puncture in mice for inducing immune tolerance.

[0046] Figure 7 In vivo release of microneedles for inducing immune tolerance.

[0047] Figure 8 Microneedles for inducing immune tolerance reduce serum antibody levels in allergic asthma mice. Fig. 9 Statistical chart of the proportion of regulatory T cells in the draining lymph nodes of mice after microneedle induction of immune acceptance and induction of immune tolerance.

[0048] Fig.10 A diagram showing the respiratory intervals of mice after receiving microneedle induction for immune tolerance to improve allergic asthma.

[0049] Fig.11 To reduce the proportion of eosinophils in the lungs of mice with allergic asthma after receiving microneedles to induce immune tolerance.

[0050] Fig.12 Microneedle induction for immune tolerance improves lung mucus accumulation in allergic asthmatic mice. DETAILED DESCRIPTION

[0051] The following examples are further explanations of the present invention, but are by no means limitations of the scope of the present invention. The present invention will be further described in detail below with reference to the examples, but it should be understood by those skilled in the art that the present invention is not limited to these examples and the preparation methods used. Moreover, those skilled in the art may carry out equivalent substitutions, combinations, improvements or modifications to the present invention based on the description of the present invention, but these will all be included within the scope of the present invention.

[0052] Example 1 Preparation of Nanoparticles Encapsulating OVA Antigen

[0053] A certain amount of polylactic acid-co-glycolic acid was dissolved in dichloromethane and stirred to obtain 20 mg / mL of polylactic acid-co-glycolic acid. A certain amount of polyvinyl alcohol and ovalbumin were dissolved in water and stirred to obtain 25 mg / mL of polyvinyl alcohol and 10 mg / mL of ovalbumin. The aqueous solution of polyvinyl alcohol and ovalbumin was added dropwise to a dichloromethane solution of polylactic acid-co-glycolic acid copolymer at a volume ratio of 1:10 under stirring to obtain colostrum. The colostrum was added to a 50 mg / mL aqueous solution of polyvinyl alcohol at a volume ratio of 1:5 under stirring, and the dichloromethane was removed by rotary evaporation at 250W probe ultrasound. The nanoparticle solution was centrifuged twice at 7000g to obtain nanoparticles.

[0054] Example 2 Preparation of Rapamycin-loaded Nanoparticles

[0055] The polylactic acid-co-glycolic acid and rapamycin were dissolved in dichloromethane and stirred to obtain 20 mg / mL of polylactic acid-co-glycolic acid and 8 mg / mL of rapamycin. Polyvinyl alcohol was dissolved in water and stirred to obtain 25 mg / mL of polyvinyl alcohol. The polyvinyl alcohol aqueous solution was added to the polylactic acid-co-glycolic acid copolymer and rapamycin dichloromethane solution at a ratio of 1:10 under stirring to obtain colostrum. The colostrum was added to a 50 mg / mL polyvinyl alcohol aqueous solution at a ratio of 1:5 under stirring, and the dichloromethane was removed by rotary evaporation with 250W probe ultrasound. The nanoparticle solution was centrifuged twice at 7000g to obtain nanoparticles.

[0056] Example 3 Preparation of OVA and rapamycin co-loaded nanoparticles that induce immune tolerance

[0057] The polylactic acid co-glycolic acid and rapamycin were dissolved in dichloromethane and stirred to obtain 20 mg / ml of polylactic acid co-glycolic acid and 8 mg / mL of rapamycin. Polyvinyl alcohol and ovalbumin were dissolved in water and stirred to obtain 25 mg / mL of polyvinyl alcohol and 10 mg / mL of ovalbumin. The aqueous solution of polyvinyl alcohol and ovalbumin was added to the dichloromethane solution of polylactic acid co-glycolic acid and rapamycin at a ratio of 1:10 under stirring to obtain colostrum. The colostrum was added to a 50 mg / mL aqueous solution of polyvinyl alcohol at a ratio of 1:5 under stirring, and the dichloromethane was removed by rotary evaporation with 250W probe ultrasound. The nanoparticle solution was centrifuged twice at 7000g to obtain nanoparticles.

[0058] Example 4 Preparation of FITC-OVA-loaded nanoparticles

[0059] The preparation method of FITC-OVA loaded nanoparticles is similar to that of Example 1, except that OVA in Example 1 is replaced by FITC-OVA.

[0060] Example 5 Preparation of FITC-OVA and Rapamycin-loaded Nanoparticles Inducing Immune Tolerance

[0061] The preparation method of FITC-OVA loaded nanoparticles is similar to that of Example 3, except that OVA in Example 3 is replaced by FITC-OVA.

[0062] Example 6 Preparation of Cy5-OVA and Rapamycin-loaded Nanoparticles Inducing Immune Tolerance

[0063] The preparation method of Cy5-OVA loaded nanoparticles is similar to that of Example 1, except that OVA in Example 1 is replaced by Cy5-OVA.

[0064] Example 7 Preparation of FITC-OVA and DiD-loaded Nanoparticles

[0065] The preparation method of FITC-OVA and DiD loaded nanoparticles is similar to that of Example 3, except that OVA in Example 3 is replaced by FITC-OVA and rapamycin is replaced by DiD.

[0066] Example 8 Particle size, potential and PDI determination of Examples 1-3

[0067] Zetasizer Nano ZS90 laser particle size analyzer was used to measure the particle size, potential and PDI of the nanoparticles inducing immune tolerance in Examples 1-3; Micro-BCA method and high performance liquid chromatography were used to respectively measure the encapsulation efficiency of OVA and rapamycin in the 3 nanoparticles inducing immune tolerance. As shown in Table 1, the particle size of the nanoparticles prepared in Example 1 was 254.6±1.7nm, the potential was -14.5±0.4mV, and the encapsulation efficiency of OVA was 79.5±0.3%. The particle size of the nanoparticles prepared in Example 2 was smaller than that in Example 1, with a particle size of 254.6±1.7nm, a potential of -13.2±0.6mV, and an encapsulation efficiency of rapamycin of 76.5±0.2%. The particle size of the nanoparticles in Example 3 was 308.3±4.6 nm, the potential was -14.2±0.7 mV, and the encapsulation efficiency of OVA and rapamycin was 78.8±0.5% and 71.1±0.3%, respectively.

[0068] Table 1 Particle size, potential and encapsulation efficiency of Examples 1-3

[0069] Example 1 Example 2 Example 3 Average particle size (nm) 268.8±0.8 254.6±1.7 308.3±4.6 PDI 0.08±0.02 0.04±0.07 0.16±0.01 Zeta potential (mV) -14.5±0.4 -13.2±0.6 -14.2±0.7 OVA encapsulation rate (%) 79.5±0.3 - 78.8±0.5 Rapa encapsulation efficiency (%) - 76.5±0.2 71.1±0.3

[0070] Example 9 Electron microscopic characterization of co-loaded OVA and rapamycin

[0071] A small amount of the nanoparticles of Examples 1-3 was taken with a capillary tube and dropped onto a copper mesh. After the sample dried naturally, a 1% phosphotungstic acid solution was added for dyeing for 2 minutes. The filter paper absorbed the excess dye solution and the sample was dried before being inspected and observed. Figure 1 As shown, the prepared nanoparticles are uniform in size and are spherical particles.

[0072] Example 10 Preparation of microneedle patch for inducing immune tolerance:

[0073] 1) Filling the front end of the microneedle body with immune tolerance-inducing nanoparticles containing antigen OVA and immunomodulator rapamycin: Prepare the immune tolerance-inducing nanoparticles containing antigen OVA and immunomodulator rapamycin according to the steps of Example 3, take 40 μL of the nanoparticle solution and evenly drip it onto the microneedle mold, and then place the microneedle mold in a centrifuge for centrifugation (3040 g, 10 min). After the centrifugation, evenly drip 40 μL of the nanoparticle solution again.

[0074] μL of dextran microparticle solution was added to the microneedle mold, centrifuged (3040g, 10min), and the mold was centrifuged again (3040g, 5min), and the immune tolerance-inducing nanoparticles containing the antigen OVA and the immunomodulator rapamycin were filled into the front end of the microneedle body. 2) Preparation of needle matrix liquid: Sodium hyaluronate (8.2KDa): injection water was mixed and dissolved at a mass ratio of 1:2 (w / w) to obtain a soluble microneedle needle matrix liquid.

[0075] 3) Pressurized perfusion: 60 μL of the above needle matrix solution was drawn and applied to the microneedle mold. The microneedle mold was placed in a pressurized chamber and the pressure was adjusted to 0.3 MPa for 20 min.

[0076] min.

[0077] 4) Curing and demoulding: Place the pressurized microneedle mold as a whole in a desiccator and dry it at room temperature for 5 hours. After peeling, store it in a desiccator or use it directly.

[0078] Example 11 Preparation of OVA-loaded nanoparticle microneedles

[0079] Preparation of antigen-loaded OVA nanoparticle microneedles The preparation method is similar to that of Example 10, except that the immune tolerance-inducing nanoparticles in step 1 of Example 10 are replaced by the nanoparticles in Example 1, and the remaining steps are the same.

[0080] Example 12 Preparation of Rapamycin-loaded Nanoparticle Microneedles

[0081] Preparation of microneedles loaded with rapamycin nanoparticles The preparation method is similar to that of Example 10, except that the immune tolerance-inducing nanoparticles in step 1 of Example 10 are replaced by the nanoparticles in Example 2, and the remaining steps are the same.

[0082] Example 13 Determination of drug loading on the needle tip of the microneedle patch for inducing immune tolerance

[0083] All the microneedle tips of the microneedle patch of Example 10 were stripped, shaken and ultrasonicated in a water bath to fully dissolve in water for injection, and the amount of antigen protein carried by the microneedle patch was determined by Bradford method to be 5.5±0.5 μg. The amount of rapamycin carried by the microneedle patch was determined by high performance liquid chromatography to be 15.9±0.6 μg.

[0084] Experimental Example 14 DC2.4 uptake induced immune tolerance nanoparticles

[0085] DC2.4 cells were collected at 1×10 6cells / well were inoculated in a 12-well plate. When the cells grew to 80%, the culture medium was removed, the cells were washed once with PBS, 1 mL of blood-free and anti-antibody-free culture medium was added, and free FITC-labeled OVA, as well as FITC-OVA nanoparticles prepared in Example 4 and FITC-OVA and rapamycin-induced immune tolerance nanoparticles prepared in Example 5 were added. The dosage was 10 μg / well of FITC-OVA. After incubation at 37°C for 1 hour, the cell surface was gently rinsed once with PBS, and then directly blown with PBS, centrifuged (2000 rpm×4 min) and washed once, and finally resuspended with 400 μL of PBS solution and detected by flow cytometry. The results are shown in Figure 2 As shown, compared with free OVA, the uptake of immune tolerance-inducing nanoparticles on DC2.4 cells was significantly increased, with a significant difference (****, i.e., p<0.0001). Figure 2 In the figure, Free FITC-OVA represents the free OVA group, Free FITC-OVA / Rapa represents the mixture of free FITC-OVA and rapamycin, PLGA@FITC-OVA is the nanoparticle of Example 4, and PLGA@FITC-OVA-Rapa is the nanoparticle of Example 5.

[0086] Example 15 Characterization of microneedle patch co-loaded with FITC-OVA and DiD nanoparticles

[0087] Prepare the co-loaded FITC-OVA and DiD nanoparticles according to Example 7, and then prepare the microneedle patch according to the method of Example 10. Place the microneedle on a horizontal table, adjust the angle of the optical magnifier, and observe the morphology of the microneedle. Figure 3 As shown, the optical photograph of the microneedle shows a yellow needle body on the needle tip, which indicates that the co-loaded FITC-OVA and DiD nanoparticles can be successfully loaded into the microneedle tip and mainly accumulate at the needle tip.

[0088] Experimental Example 16 Characterization of microneedles co-loaded with FITC-OVA and DiD nanoparticles by laser confocal microscopy.

[0089] The microneedles prepared in Example 15 were characterized by placing the microneedles prepared in Example 15 under a laser confocal microscope to photograph and record the microneedle tip morphology and fluorescence distribution. Figure 4 As shown, the results of laser confocal microscopy showed that the fluorescence of each can be clearly observed at the tip of the microneedle of the nanoparticles loaded with DiD and FITC-OVA, indicating that DiD and FITC-OVA can be successfully loaded. The drug is loaded by centrifugation, and the fluorescence of DiD and FITC is mainly distributed at the tip of the microneedle tip, accounting for about 60% of the height of the entire microneedle body.

[0090] Example 17 Characterization of Microneedles Inducing Immune Tolerance by Scanning Electron Microscopy

[0091] The microneedles for inducing immune tolerance were prepared according to the method of Example 10. The microneedles prepared in Example 10 were vacuum-plated with gold. After the surface of the microneedles was fully coated with Au film, the prepared samples were placed on the stage and the morphology of the microneedles was observed by scanning electron microscopy. Figure 5 As shown, the microneedle tips have no breakage, good morphology, clear and obvious arrangement of the tips, and uniform spacing.

[0092] Example 18 Effect of microneedle patch puncturing the skin to induce immune tolerance

[0093] According to the method of Example 10, microneedles for inducing immune tolerance were prepared. The microneedle patch was inserted into the depilated skin on the back of Balb / c female mice and pressure was applied for 3 minutes. Then, the microneedle base was immediately removed, and a small amount of 0.4% trypan blue solution was taken and dripped onto the medicated skin site for staining for 1 minute. The medicated site was then washed three times with saline. After the excess dye was washed off, the mouse was killed, and the skin at the medicated site was removed and placed under an optical magnifying glass to observe the holes on the skin surface. Because microneedle puncture will produce an array of holes on the skin surface, the trypan blue dye solution will penetrate into the holes to form obvious blue spots. If the spots are blurred, it means that the puncture effect is not good. The results are as follows: Figure 6 As shown, after applying a 10×10 microneedle patch on the back skin of mice, a neat and obvious array of puncture holes can be seen, which indicates that the induced immune tolerance microneedle can break through the stratum corneum barrier and successfully puncture the skin. It can be seen that the induced immune tolerance microneedle tip has a good puncture effect.

[0094] Example 19 Inducing immune tolerance microneedle patch in vivo release

[0095] Nanoparticles were prepared using the Cy5-labeled antigen OVA protein according to the method of Example 6, and then a microneedle patch was prepared according to Example 10. The microneedle patch was inserted into the depilated skin on the back of Balb / c female mice and pressurized for 3 minutes. The dosage was 5 μg Cy5-OVA, and the microneedle base was immediately removed. The mice were anesthetized by intraperitoneal injection of 4% (w / v) chloral hydrate, and then placed in a living imager to observe the fluorescence intensity. The excitation wavelength was set to 650nm and the emission wavelength was set to 670nm. The fluorescence brightness of the back skin after different times of administration was recorded to investigate the release of the microneedle vaccine in vivo. The results are shown in the figure. Figure 7 As shown, within 96 hours, the induced immune tolerance microneedle patch had a higher fluorescence intensity than the free Cy5-OVA / Rapa MN, which means that its sustained release effect was better. The two had significant differences to varying degrees at each time point, indicating that the induced immune tolerance microneedle patch had a better sustained release effect.

[0096] Example 20 Investigation of serum antibody production by microneedles induced by immune tolerance

[0097] Female Balb / c mice aged 6-8 weeks were randomly divided into 7 groups, including Naive, Untreated, subcutaneously injected with nanoparticles (SC) of Example 3, microneedles loaded with free OVA and rapamycin, and microneedles prepared in Example 11, Example 12, and Example 10. The mice were administered on days -21, -7, and 0, respectively. The microneedles in different groups were administered subcutaneously on the back for 3 minutes each time. After the administration, the microneedle base was removed. On days 0 and 14, 2mg Alum and 20μg OVA were injected intraperitoneally to establish the model. On days 21-23, 50μg OVA was administered by nasal drops. The mice were killed on day 25. Dosage: OVA 5.5μg / mouse, rapamycin 15.5μg / mouse. As Figure 8 a is the immunization schedule.

[0098] Blood was collected from mice sacrificed 25 days after the above immunization protocol, and serum was separated. ELISA was used to detect the level of specific anti-OVA antibody IgG in the mouse serum. Figure 8 As shown in b, the IgG antibodies induced by the microneedles that induced immune tolerance were the lowest, indicating that the immune response produced in the body by the microneedles that induced immune tolerance was the lowest, and compared with the model mice, it could inhibit the production of antibodies to allergens.

[0099] Example 21 Treg levels in inguinal lymph nodes of mice receiving microneedle-induced immune tolerance

[0100] Treg cells are a type of T cell subset that controls the body's autoimmune reactivity. They are one of the important factors in maintaining the body's immune tolerance and are of great significance for maintaining peripheral tolerance. The mice were grouped and treated according to the experimental plan of Example 20. On the 25th day, the mice were killed, the inguinal lymph nodes were taken, and a single cell suspension was prepared. The staining buffer of anti-mouse CD11c, CD40, and CD86 flow cytometry antibodies was blown evenly and placed at 4°C in the dark for 40 minutes. After the staining was completed, PBS was added to terminate the staining, centrifuged at 500g for 4 minutes, and the cell pellet was resuspended with PBS and tested on the machine. The results are as follows Fig. 9 As shown, the microneedles prepared in Example 10 induced the highest proportion of Treg in the inguinal lymph nodes, indicating that the microneedles for inducing immune tolerance have the ability to induce tolerance.

[0101] Example 22: Breathing Interval Study of Mice Receiving Microneedle Induction of Immune Tolerance

[0102] Airway reactivity measurement is very important for the diagnosis, treatment and efficacy evaluation of allergic asthma. There are many methods for measuring mouse airway reactivity, which are mainly divided into non-invasive and invasive detection. The most commonly used method for non-invasive measurement of mouse airway reactivity is to measure Penh, which is calculated by volume plethysmography. Specifically, the mouse is placed in a specific container, and the changes in the gas pressure in the container are recorded to obtain the mouse's respiratory curve, and Penh can be calculated from this respiratory curve. The mice were grouped and treated according to the experimental plan of Example 20. On day 25, each group of mice was placed in the nebulizer chamber of the respiratory curve instrument of the whole-body volume plethysmography system, and different concentrations of methacholine were introduced to observe and record the breathing intervals of the mice. The results are shown in the figure. Fig.10 As shown, at a concentration of 25 mg / mL of methacholine, the mice treated with the microneedles prepared in Example 10 had the lowest respiratory interval value, indicating that the microneedles prepared in Example 10 can effectively relieve the symptoms of allergic asthma.

[0103] Example 23 Detection of eosinophils in bronchoalveolar lavage fluid of mice receiving induced immune tolerance microneedle

[0104] Eosinophil infiltration in the airways of patients with allergic asthma leads to induced immune airway inflammation. The mice were divided into groups and treated according to the experimental plan of Example 20. The alveolar lavage fluid of mice receiving induced immune tolerance microneedle eosinophil detection was performed. The mice were killed on the 25th day, and the lungs of the mice were lavaged with 1 mL of PBS. The cell pellets in the lavage fluid were collected and stained with anti-mouse CD11c, Siglec-F and CD45 flow cytometry antibody staining buffer. After blowing evenly, the cells were placed at 4°C in the dark for 40 minutes. After the staining was completed, PBS was added to terminate the staining, and the cells were centrifuged at 500g for 4 minutes. The cell pellets were resuspended with PBS and tested on the machine. The results are as follows. Fig.11 As shown, the microneedles prepared in Example 10 produced the least eosinophils in the lungs, indicating that the microneedles induced immune tolerance had the lowest lung allergy symptoms.

[0105] Example 24 Detection of lung mucus in mice receiving induced immune tolerance microneedle

[0106] According to Example 20 Figure 8 a Planned grouping and treatment, on day 25, the mice were killed, and the lung tissues of each group were taken out for periodic acid-Schiff staining to detect the mucus in the mice's lungs. Fig.12 As shown, the mice treated with the microneedles prepared in Example 10 had the least positive area in the lungs, indicating that the microneedles induced immune tolerance had the least collagen accumulation in the lungs and had the best therapeutic effect.

[0107] In summary, the microneedles for inducing immune tolerance that co-load antigens and immunomodulators prepared in Example 10 have sufficient mechanical strength, meet the requirements of skin puncture, and can encapsulate effective amounts of antigens and immunomodulators, and have a sustained release effect. At the same time, the nanoparticles for inducing immune tolerance are uniform in size and are spherical particles, which can be taken up in large quantities by immune cells, can significantly inhibit the antibody level in the serum of allergic asthma model mice, and induce the production of regulatory T cells in lymph nodes, indicating that they have the ability to induce tolerance, improve the respiratory interval of allergic asthma mice, reduce the infiltration of eosinophils in the lungs and the accumulation of mucus, and significantly enhance the induction of immune tolerance to treat allergic asthma.

[0108] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modify the technical contents disclosed above into equivalent embodiments of equivalent changes without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A microneedle patch for inducing immune tolerance, characterized in that: The invention comprises a backing layer, a base part and a needle body containing immune tolerance inducing nanoparticles. The immune tolerance inducing nanoparticles comprise polymer materials, antigens or allergens and immunomodulators. The immune tolerance inducing nanoparticles are distributed at the front end of the needle body.

2. The immune tolerance inducing nanoparticle according to claim 1, characterized in that: The polymer material is a natural and synthetic polymer material, including one or more of polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PLGA), polystyrene, acetylated dextran, poly-L-lysine, polyacrylamide and chitosan.

3. The microneedle for inducing immune tolerance according to any one of claims 1-2, characterized in that: The antigen or allergen is selected from glutamate decarboxylase (GAD), including GAD65 and GAD67, type II collagen, myelin peptide MOG 35-55 The allergens include pollen allergens derived from Artemisia, including Artemisia vulgaris Linn. pollen allergens, Artemisia annua Linn. pollen allergens, Artemisia sieversiana Ehrhart ex Willd. pollen allergens, Artemisia argyi levl. et Van. pollen allergens, and animal dander allergens, including cat dander allergen Fe1 d1, dog dander allergen Can f1, horse dander allergens Equc c1 and Equ c2, cow dander allergens Bos d2 and Bos d5, rabbit dander allergen Ory c1, mouse dander allergen Mus M1, and allergen protein Rat in rat dander. n1, one or more allergens selected from the group consisting of birch pollen allergens, in particular BETV1 and BETV4, timothy grass pollen allergens, in particular PHP2, PHP5, PHP6 and PHP7, house dust mite allergens, in particular Der P1 and Der P2, cat allergen Feld1, bee allergens, major wasp allergens, profilins, in particular Phi P12, and dust mite allergens, in particular LEP D2, chicken egg white allergen, one or more combinations of the above allergens.

4. The microneedle for inducing immune tolerance according to any one of claims 1-2, characterized in that: The immunomodulators include cytokines: IL-10, TGF-β; lipids: phosphatidylserine; one or a combination of more than one of rapamycin, curcumin, quercetin, Bay11-07082 and vitamin D3.

5. The microneedle patch for inducing immune tolerance according to any one of claims 1-2, characterized in that: The matrix material of the backing layer, substrate and needle body of the microneedle is a water-soluble polymer, and the water-soluble polymer is selected from hyaluronic acid, polyvinyl alcohol, polyvinyl pyrrolidone, gelatin, collagen, silk fibroin, chondroitin sulfate, dermatan sulfate, heparan sulfate, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, carboxymethyl chitosan, chitosan, dextran sulfate, glycogen, amylose, dextrin, dextran, trehalose, glycoside and one or more of the modified derivatives of the above materials.

6. The microneedle patch for inducing immune tolerance according to any one of claims 1-2, characterized in that: The polymer nanoparticles loaded with antigens or allergens and immunomodulators are concentrated at the front end of the microneedle body and account for 10-80% of the total length of the entire soluble microneedle body.

7. A method for preparing a microneedle patch for inducing immune tolerance according to any one of claims 1 to 6, comprising the following steps: 1) preparing immune tolerance inducing polymer nanoparticles encapsulating antigens, allergens and / or immunomodulators; 2) filling the immune tolerance-inducing nanoparticles prepared in step 1) into the front end of the microneedle body; 3) Prepare a needle matrix liquid and fill it evenly into the microneedle mold. The filling can be done by at least one of a pressurization method, a decompression method or a centrifugation method. After drying and solidification, demolding is performed to obtain a microneedle vaccine patch.

8. Use of the microneedle patch for inducing immune tolerance according to any one of claims 1 to 6 or the microneedle patch for inducing immune tolerance prepared by the preparation method according to claim 7 in preparing drugs for administration to the skin.

9. The microneedle patch for inducing immune tolerance according to any one of claims 1 to 6, characterized in that It can induce immune tolerance or antigen-specific immune tolerance through transcutaneous immunization.

10. Use of the microneedle patch for inducing immune tolerance according to any one of claims 1 to 6 in the preparation of a drug for treating or preventing autoimmune diseases or allergic diseases, characterized in that The microneedle can induce immune tolerance or antigen-specific immune tolerance through transcutaneous immunization.

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