Microneedle patch for inducing immune tolerance to treat rheumatoid arthritis

Abstract

The application discloses a polymer microneedle patch co-loaded with self-antigen peptides and immunomodulators and a preparation method thereof, and studies the ability of the polymer microneedle patch to reverse immune dysfunction of rheumatoid arthritis (RA). The preparation method is as follows: a biocompatible polymer matrix material and a self-antigen are dissolved in pure water, and then an immunomodulator is added to obtain a suspension by stirring. The suspension is added to a mold to form needle tips, a backing layer solution is poured after drying, and finally the microneedle patch is demolded. Animal experiments show that the microneedle has sufficient mechanical strength to deliver drugs transdermally, effectively induces tolerogenic dendritic cells at the drug delivery site, further activates the differentiation of regulatory T cells (Treg), significantly up-regulates anti-inflammatory cytokines, down-regulates pro-inflammatory cells, antibodies and cytokines, effectively restores the immune balance of rheumatoid arthritis, and finally almost completely eliminates the symptoms and inflammatory infiltrates of rheumatoid arthritis. The microneedle preparation method is simple and rapid, and has a good clinical transformation prospect.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of transdermal administration of a microneedle patch preparation, in particular to application of a polymer microneedle co-loading self-antigens and immunomodulators in rheumatoid arthritis, and belongs to the technical field of rheumatoid arthritis treatment. BACKGROUND

[0002] Rheumatoid arthritis is a chronic synovitis characterized by erosive autoimmune disease, which is a progressive disease and recurs throughout life. This disease can cause joint swelling, pain, deformity and even varying degrees of disability, which greatly affects the quality of life of patients. According to the statistics of the World Health Organization, nearly 23 million people in the world suffer from rheumatoid arthritis, and the direct economic expenditure of each patient caused by rheumatoid arthritis is more than 15000 yuan per year. Considering that rheumatoid arthritis needs long-term medication and its development seriously affects the normal working capacity of the population, rheumatoid arthritis has brought a heavy economic burden to the country and society.

[0003] Traditional rheumatoid arthritis treatment programs mostly use oral or injection of synthetic drugs with inhibitory effects on inflammatory development, including non-steroidal anti-inflammatory drugs, anti-rheumatic drugs and glucocorticoids. These drugs mainly have anti-inflammatory and non-specific immunosuppressive effects, can temporarily relieve pain and reduce inflammatory symptoms, but cannot reverse the progression of inflammation, prevent further damage to the joints, and cannot cure the disease. Low compliance of patients to the treatment program leads to self-stopping medication, disease recurrence, and increases the difficulty of disease treatment. In recent years, new biological agents such as tumor necrosis factor-alpha (TNF-alpha) blockers can target specific pathogenic inflammatory factors and show good efficacy, but also have the risk of damaging the normal function of the immune system and causing infection, and are expensive. It is urgent to develop new rheumatoid arthritis treatment methods to target the etiology of rheumatoid arthritis, effectively repair abnormal immune responses in the body and restore immune balance.

[0004] The above drugs (chemical drugs or biological agents) are all first-line drugs for treating rheumatoid arthritis, but due to the main administration routes of oral or injection (intra-articular injection, intravenous injection, subcutaneous injection) in clinical use, the treatment is effective but still has many problems. Long-term oral administration can easily cause patients to have varying degrees of gastrointestinal reactions and renal toxic side effects, and there is a first-pass effect in liver metabolism, which can reduce the efficacy of the drug; subcutaneous and intravenous injection exert their effects through the body circulation, although the bioavailability of the drug is high, but due to poor selectivity, it can act on multiple target organs or target cells, thereby causing adverse reactions, and because of the pain, the patient's compliance is poor; intra-articular injection can directly inject the drug into the lesion site, but the operation is complex and requires special personnel to operate, and the pain is strong, so it is difficult for general patients to accept.

[0005] Immune tolerance strategy is an active field of research in immunology in recent years. Antigen-induced immune tolerance strategy is to induce specific immune tolerance by giving rheumatoid arthritis autoantigens, and to close the abnormal immune response of the immune system to rheumatoid arthritis autoantigens. Successful induction of immune tolerance can increase the proportion of regulatory T cells (Treg), inhibit the differentiation and proliferation of type 1 helper T cells (Th1) and type 17 helper T cells (Th17), and then down-regulate inflammatory cytokines, and finally restore immune balance. Unlike immunosuppression strategies targeting the entire immune system, antigen-specific immune tolerance strategies only target rheumatoid arthritis-related autoantigens to correct the disturbed immune system and restore immune balance.

[0006] The autoantigens of rheumatoid arthritis patients include collagen, collagen type II, filaggrin, fibrinogen, vimentin and their functional epitope peptides. Specific antibodies and cellular immune responses against the above autoantigens can be detected in rheumatoid arthritis patients. Studies have shown that oral administration of autoantigens can inhibit the occurrence of adjuvant arthritis in rats. The above results prove that autoantigens are both the cause of rheumatoid arthritis and the potential tolerogens for inducing immune tolerance to play a therapeutic role.

[0007] Another challenge of immune tolerance induction is that the use of autoantigens alone has the risk of exacerbating immune response and inflammation. Studies have shown that co-delivery of autoantigens and immunomodulators can solve this problem. Immune modulators are drugs that have regulatory functions on the immune response of the body, can inhibit the proliferation and function of cells related to immune response (T cells and B cells, etc.), and can regulate the immune response of the body. Related mechanism studies have further proved that immune modulators such as dexamethasone, cyclosporine, tacrolimus and rapamycin can regulate the degree of immune system activation, activate the differentiation and proliferation of Treg cells, and can effectively induce antigen-specific immune tolerance when combined with autoantigens.

[0008] Previous studies have mainly established immune tolerance through oral and injection strategies, which are considered to be the most effective strategies for inducing immune tolerance. However, both strategies have considerable inherent defects. For the oral strategy, the harsh acidic and alkaline environment in the gastrointestinal tract can lead to antigen degradation and inactivation. Therefore, a large dose of antigen is required, and the tolerance induction efficiency is usually low. When injected intravenously, the pain and infection risk caused by frequent injections lead to poor patient compliance.

[0009] Microneedle is a micro-needle structure with a length of 100-1000 μm made by micro-processing, and the needle body does not touch nerve tissue and blood vessels when piercing the skin keratin layer barrier, so that the microneedle delivery of drugs is almost painless and minimally invasive, and the patient has good compliance. Microneedle administration has many advantages, such as painless administration, which can greatly increase the user's compliance; microneedle administration has very low professional technical requirements, and patients can self-administer; the drug in the microneedle exists in a solid form, has good stability, and is convenient for transportation and storage. However, the existing polymer microneedle dissolves the drug and the matrix in the solvent to prepare the microneedle, which requires that the drug and the matrix have the same solubility (soluble in water or soluble in organic solvent), and when water-soluble and water-insoluble drugs need to be loaded at the same time, other complex methods such as preparation of nanoparticles, liposomes, clathrates, etc. need to be taken. These methods not only increase the difficulty of preparing microneedles, but also further reduce the drug loading capacity, and the mechanical strength of the microneedle is also easily affected. SUMMARY

[0010] In order to solve the above problems, the present application prepares a microneedle transdermal administration patch co-loading self-antigen and immunomodulator for treating rheumatoid arthritis, and studies its immune tolerance induction ability and therapeutic effect on rheumatoid arthritis. On the one hand, the present application prepares a matrix liquid by dissolving a biocompatible polymer matrix material and a self-antigen in water, and then adding an immunomodulator to directly prepare a suspension, the suspension is added to a mold to form a needle tip, and after drying, a backing layer solution is poured, and finally the microneedle patch is demolded, which simplifies the preparation steps of the microneedle; on the other hand, the transdermal delivery of self-antigen and immunomodulator simultaneously successfully induces immune tolerance in autoimmune disease model mice, and plays a significant therapeutic effect. At the same time, the use of microneedles for transdermal administration avoids the toxic side effects of traditional oral therapy and the pain and low compliance of patients associated with injection administration.

[0011] The microneedle patch of the present application comprises a backing layer and a needle body, the needle body comprises a needle-forming matrix material and a drug, the needle-forming matrix material comprises a biocompatible polymer matrix material, and the drug comprises a rheumatoid arthritis self-antigen and an immunomodulator.

[0012] The biocompatible polymer matrix material includes at least one of hyaluronic acid, polyvinylpyrrolidone, collagen, fibroin, gelatin, hydroxypropyl methylcellulose, chondroitin sulfate, dextrin, polyvinyl alcohol, carboxymethyl cellulose, carboxymethyl chitosan, dextran sulfate, glycogen, amylose, dextran, hydroxypropyl cellulose, glycoside, chitosan, polyester, polyhydroxyaliphatic acid ester, hydroxybutyric acid valeric acid copolyester, high molecular polyacrylamide, polyhydroxyaliphatic acid-polyethylene glycol, poly-4-hydroxy acid, poly-α-hydroxy acid, poly-β-hydroxy acid, poly-4-hydroxybutyrate, poly-4-hydroxyvalerate, poly-4-hydroxyhexanoate, polyester amide, polycaprolactone, polylactide, polyglycolide, poly(lactic-co-glycolic acid), polydioxanone, polyortho ester, polyether ester, polyanhydride, glycolic acid-trimethylene carbonate copolymer, polyphosphate ester, polyphosphate urethane, polyamino acid, polycyanoacrylate, polytrimethylene carbonate, polyimino carbonate, polytyrosine carbonate, polycarbonate, polytyrosine aryl ester, polyalkylene oxalate, polyphosphocreatine, and derivatives of the above materials.

[0013] In some embodiments, the needle-forming matrix material is preferably one of hyaluronic acid, polyvinylpyrrolidone, and fibroin.

[0014] Further preferably, the needle-forming matrix material is preferably hyaluronic acid, and the molecular weight of the hyaluronic acid is 10-300 KDa, and the concentration is 10-100% (m / v); further preferably, the molecular weight of the hyaluronic acid is 10 KDa, and the concentration is 50-100%.

[0015] In some embodiments, the rheumatoid arthritis autoantigen includes one or more of collagen, type II collagen, filaggrin, fibrinogen, vimentin, and functional epitope peptides thereof; preferably, the rheumatoid arthritis autoantigen is further preferably a type II collagen polypeptide; further preferably, the concentration of the type II collagen polypeptide is 0.1-10% (m / v).

[0016] In some embodiments, the immunomodulator is one or more of dexamethasone, cyclosporine, tacrolimus, rapamycin, and derivatives thereof; preferably, the immunomodulator is preferably rapamycin; further preferably, the concentration of the rapamycin is 0.05-5% (m / v).

[0017] The present application also provides a preparation method of a microneedle transdermal drug delivery patch for treating rheumatoid arthritis:

[0018] (1) dissolving the rheumatoid arthritis autoantigen in water to obtain a solution;

[0019] (2) adding a biocompatible polymer matrix material into the above solution, and completely dissolving it by stirring;

[0020] (3) adding an immunomodulator, and fully stirring to obtain a suspension;

[0021] (4) adding the above suspension into a microneedle mold, using a pressurized gas to press the solution into the microneedle mold, and placing it into a drying oven for drying at room temperature to form a microneedle tip;

[0022] (5) adding a biocompatible polymer matrix solution into the mold, drying, and demolding to obtain a microneedle.

[0023] Preferably, the stirring time of step (3) is 4 h, and the drying time of step (4) and step (5) is 2 h.

[0024] Compared with the prior art, the present application has the following beneficial effects:

[0025] (1) The present application simplifies the preparation steps of the microneedle by preparing the soluble drug component into an aqueous solution and the insoluble drug component into a suspension.

[0026] (2) Rheumatoid arthritis is a long-term chronic autoimmune disease, and currently, the treatment is mainly based on the strategy of inhibiting inflammation, which needs to be taken for a lifetime and has the risk of repeated course of disease. The microneedle patch prepared by the present application can deliver the self-antigen and the immunomodulator simultaneously, successfully induce immune tolerance in the autoimmune disease model mice, and treat rheumatoid arthritis by targeting the pathological mechanism. At the same time, the in vivo experiment shows that the microneedle patch for treating rheumatoid arthritis of the present application can reverse the progress of collagen-induced arthritis in mice, and has the prospect of new drug development.

[0027] (3) The present application uses the microneedle patch to treat rheumatoid arthritis patients who need long-term medication, and solves the problems of toxic side effects of traditional oral treatment and pain and low compliance of patients accompanied by injection administration. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The microneedle co-loaded with type II collagen and rapamycin prepared for Example 1 of the present application.

[0029] Figure 2 The skin puncture diagram of the microneedle patch of Example 1 of the present application.

[0030] Figure 3 The joint thickness and clinical score of the collagen-induced arthritis mice treated by the microneedle patch of Example 2 of the present application.

[0031] Figure 4 The paw photos of the collagen-induced arthritis mice treated by the microneedle patch of Example 2 of the present application.

[0032] Figure 5 The pathological sections of the collagen-induced arthritis mice treated by the microneedle patch of Example 2 of the present application.

[0033] Figure 6 The microneedle co-loaded with the type II collagen polypeptide and rapamycin prepared in Example 3 of the present application.

[0034] Figure 7 The skin puncture graph of the microneedle patch of Example 3 of the present application.

[0035] Figure 8 The joint thickness and clinical score of the collagen-induced arthritis mice treated by the microneedle patch of Example 2 of the present application.

[0036] Figure 9 The paw photos of the collagen-induced arthritis mice treated by the microneedle patch of Example 4 of the present application.

[0037] Figure 10 The pathological sections of the collagen-induced arthritis mice treated by the microneedle patch of Example 4 of the present application.

[0038] Figure 11 The effect of the microneedle patch of Example 4 of the present application on remodeling immune balance of the collagen-induced arthritis mice.

[0039] Figure 12 The effect of the microneedle patch of Example 5 of the present application on inducing tolerogenic dendritic cells of the collagen-induced arthritis mice.

[0040] Figure 13 The effect of the microneedle patch of Example 5 of the present application on inducing immune tolerance of the collagen-induced arthritis mice. DETAILED DESCRIPTION

[0041] The following examples are further illustrations of the present application and those skilled in the art can understand the advantages of the present application according to the content of the present application. However, those skilled in the art should understand that the protection scope of the present application is not limited to the following specific embodiments. The terms used in the examples of the present application are for describing the specific embodiments and are not intended to limit the protection scope of the present application.

[0042] The present application does not limit the source of the raw materials used, and if not specifically stated, the raw materials used in the present application are all ordinary commercially available products in the technical field.

[0043] Example 1

[0044] The type II collagen was dissolved in water to form a 0.3% (m / v) aqueous solution, and then hyaluronic acid was added to form a 50% (m / v) aqueous solution, and rapamycin was added, and stirred for 4 h to form a uniform suspension (0.05%, m / v).

[0045] The above suspension was added to the microneedle mold, and a pressurized gas was used to press the solution into the microneedle mold, and then placed in a drying oven at room temperature for 2 h to form the microneedle tips.

[0046] 30 μl of the hyaluronic acid solution (50%, m / v) was added to the above microneedle mold, and dried for 2 h to finally obtain the microneedle patch.

[0047] The prepared microneedle is shown in Figure 1 The microneedle fracture strength was 51.68 ± 1.65 MPa, and the Young's modulus was 0.48 ± 0.03 GPa (as shown in Table 1), and the microneedle skin puncture effect is shown in Figure 2 , which shows that the prepared microneedle can effectively puncture the skin; the microneedle can load collagen 30.07 ± 6.96 μg, and rapamycin 21.45 ± 2.40 μg.

[0048] Table 1: Mechanical strength of microneedle patch co-loaded with type II collagen and rapamycin

[0049]

[0050] Example 2

[0051] 0.6 ml of type II collagen solution (2 mg / ml) and 0.6 ml of complete Freund's adjuvant were loaded into a syringe, and 1 ml of emulsion was prepared by repeatedly pressing the microporous filter film. A 7-week-old DBA / 1 mouse was selected, and 100 μl of the above emulsion was subcutaneously injected at the base of the tail, and on the 21st day, the complete Freund's adjuvant was replaced with incomplete Freund's adjuvant to prepare the emulsion, and the above operation was repeated to prepare a collagen-induced rheumatoid arthritis (CIA) model.

[0052] Mice were randomly divided into five groups: a healthy group, a control group (model group), a microneedle group loaded with type II collagen, a microneedle group loaded with rapamycin, and a microneedle group co-loaded with type II collagen and rapamycin. The healthy group mice received no treatment, while the other four groups were CIA model mice. Starting on day 20 after modeling, each group received microneedle treatment with type II collagen (30 μg type II collagen), microneedles loaded with rapamycin (20 μg rapamycin), or microneedles co-loaded with type II collagen and rapamycin (30 μg type II collagen peptide, 20 μg rapamycin), respectively. Administration was performed via microneedle injection into the back of the mouse, once every other day for a total of six administrations.

[0053] After administration, the thickness of the mouse foot was measured daily using calipers, and the degree of joint swelling in each group of rats was scored. Figure 3 The results showed that the plantar thickness and inflammatory index of the microneedle group co-loaded with type II collagen and rapamycin were significantly lower than those of the model group, the microneedle-type II collagen group and the microneedle-rapamycin group, indicating that type II collagen and rapamycin have a synergistic effect.

[0054] On day 46, photos of the mouse's paws were taken, such as... Figure 4 As shown, microneedle treatment with co-loaded type II collagen and rapamycin significantly alleviated the redness and swelling of the mouse paws, and was significantly better than the microneedle-type II collagen group and the microneedle-rapamycin group, indicating that type II collagen and rapamycin have a synergistic effect and have a better therapeutic effect on rheumatoid arthritis.

[0055] After euthanizing the mice, their paws were removed for HE staining. Figure 5 The results showed that the CIA model group mice had severe joint inflammation. The microneedles co-loaded with type II collagen and rapamycin had a significantly better ability to inhibit joint inflammation than the microneedles-type II collagen group and the microneedles-rapamycin group, demonstrating that type II collagen and rapamycin have a synergistic effect and the treatment effect is better than using the two drugs alone.

[0056] Example 3

[0057] Type II collagen peptides were dissolved in water to prepare a 0.3% (m / v) aqueous solution. Hyaluronic acid was then added to prepare a 50% (m / v) aqueous solution. Rapamycin was added, and the mixture was stirred for 4 hours to prepare a homogeneous suspension (0.05%, m / v).

[0058] The above suspension was added into a microneedle mold, and the solution was forced into the microneedle mold using pressurized gas. The mold was then placed in a drying oven and dried at room temperature for 2 hours to form microneedle tips.

[0059] Add 30 μl of hyaluronic acid solution (50%, m / v) to the microneedle mold and dry for 2 hours to finally peel off the microneedle patch.

[0060] The prepared microneedles were observed Figure 6 It can be seen that the prepared microneedles co-loaded with collagen type II polypeptide and rapamycin have regular shape and complete needle tip; the breaking pressure is 55.64±1.43 MPa, and the Young's modulus is 0.44±0.03 GPa (as shown in Table 2), and the skin puncture effect of the microneedles is shown in Figure 7 , indicating that the prepared microneedles can effectively puncture the skin; the microneedles can load collagen type II polypeptide 31.21±0.60 μg and rapamycin 5.88±0.96 μg.

[0061] Table 2: Mechanical strength of microneedle patches co-loaded with collagen type II polypeptide and rapamycin

[0062]

[0063] Example 4

[0064] 0.6 ml of collagen type II solution (2 mg / ml) and 0.6 ml of complete Freund's adjuvant were loaded into a syringe, and 1 ml of emulsion was prepared by repeatedly extruding the microporous filter membrane. A 7-week-old DBA / 1 mouse was selected, and 100 μL of the above emulsion was subcutaneously injected at the base of the tail. On the 21st day, the complete Freund's adjuvant was replaced with incomplete Freund's adjuvant to prepare an emulsion, and the above operation was repeated to prepare a collagen-induced rheumatoid arthritis (CIA) model.

[0065] The mice were randomly divided into five groups, namely, a healthy group, a control group (model group), a microneedle group co-loaded with collagen type II polypeptide (MN-CII), a microneedle group co-loaded with rapamycin (MN-Rapa), and a microneedle group co-loaded with collagen type II polypeptide and rapamycin (MN-CII-Rapa) (i.e., the microneedle for treating rheumatoid arthritis according to the present application). The healthy group of mice was not treated, and the other four groups were CIA model mice. On the 20th day after modeling, the MN-CII group, the MN-Rapa group, and the MN-CII-Rapa group were treated with microneedles co-loaded with collagen type II polypeptide (30 μg of collagen type II polypeptide), microneedles co-loaded with rapamycin (5 μg of rapamycin), and microneedles co-loaded with collagen type II polypeptide and rapamycin (30 μg of collagen type II polypeptide and 5 μg of rapamycin), respectively. The administration mode was to administer the microneedles on the back of the mouse, and the administration was performed every other day, for a total of 6 times.

[0066] The thickness of the mouse paw was measured every other day with a vernier caliper after the start of administration, and the joint swelling degree of the CIA mice in each group was scored. Figure 8The results show that the thickness of the sole and the inflammation score of the microneedle group co-loaded with type II collagen polypeptide and rapamycin are significantly lower than those of the model group, the microneedle-type II collagen polypeptide group and the microneedle-rapamycin group, and are basically consistent with those of the healthy group. It can be seen that the type II collagen polypeptide and rapamycin in the microneedle co-loaded with the type II collagen polypeptide and rapamycin have a synergistic effect, and the treatment effect on rheumatoid arthritis is excellent.

[0067] On the 46th day, the mouse sole was photographed, Figure 9 The results show that the ability of the microneedle co-loaded with type II collagen polypeptide and rapamycin to improve the swelling phenomenon of the mouse sole caused by inflammation is significantly better than that of the microneedle-type II collagen polypeptide group and the microneedle-rapamycin group, and is basically consistent with that of the healthy group. It can be seen that the type II collagen polypeptide and rapamycin have a synergistic effect, and the treatment effect on rheumatoid arthritis is excellent.

[0068] After the mice were sacrificed, the mouse soles were taken out for HE staining and ponceau solid green staining, Figure 10 The results prove that the CIA model group of mice has severe arthritis inflammation and damaged cartilage structure, and the ability of the microneedle co-loaded with type II collagen polypeptide and rapamycin to inhibit arthritis inflammation and protect cartilage structure is significantly better than that of the microneedle-type II collagen polypeptide group and the microneedle-rapamycin group, proving that the type II collagen polypeptide and rapamycin have a synergistic effect, and the treatment effect is better than that of using the two drugs alone.

[0069] After the mice were sacrificed, the mouse soles were ground and centrifuged to obtain the supernatant, which was the mouse joint extract. The contents of IL-4, TNF-α, IL-17A and TGF-β in the joint extract were detected by enzyme-linked immunosorbent assay (ELISA). As shown in Figure 11 The results show that the type II collagen polypeptide and rapamycin have a synergistic effect, and the treatment effect on rheumatoid arthritis is excellent.

[0070] The bilateral inguinal lymph nodes and spleens of the mice were taken out at the same time, and the lymphocyte suspension was obtained by density gradient centrifugation after grinding. Treg cells (CD4+CD25+FoxP3+) and Th1 cells (IL-4+CD4+) were labeled respectively to detect the phenotype of lymphocytes in the main lymphatic organs of the mice, Figure 11The results show that after the microneedle co-loaded with collagen II polypeptide and rapamycin treatment, the Treg cells in the main lymphatic organs of the mice increase, the Th1 cells significantly decrease, and the Treg / Th1 balance is biased to the Treg cells, and the immune balance of the arthritic mice is reshaped. In summary, the collagen II polypeptide and rapamycin have a synergistic effect, and the combination of the two can better induce immune tolerance and reshape the immune balance than the single use.

[0071] Example 5

[0072] 0.6 ml of collagen II solution (2 mg / ml) and 0.6 ml of complete Freund's adjuvant were loaded into a syringe, and 1 ml of emulsion was prepared by repeatedly extruding the microporous filter membrane. 7-week-old DBA / 1 mice were selected, and 100 μl of the above emulsion was subcutaneously injected at the base of the tail. On the 21st day, the complete Freund's adjuvant was replaced with incomplete Freund's adjuvant to prepare the emulsion, and the above operation was repeated to prepare a collagen-induced rheumatoid arthritis (CIA) model.

[0073] The mice were randomly divided into five groups, namely, a healthy group, a control group (model group), a microneedle loaded with collagen II polypeptide group (MN-CII), a microneedle loaded with rapamycin group (MN-Rapa), and a microneedle co-loaded with collagen II polypeptide and rapamycin group (MN-CII-Rapa) (i.e., the microneedle for treating rheumatoid arthritis of the application). The healthy group of mice was not treated, and the other groups were CIA model mice. On the 20th day after modeling, the MN-CII group, the MN-Rapa group, and the MN-CII-Rapa group received microneedles loaded with collagen II polypeptide (30 μg of collagen II polypeptide), microneedles loaded with rapamycin (5 μg of rapamycin), and microneedles co-loaded with collagen II polypeptide and rapamycin (30 μg of collagen II polypeptide and 5 μg of rapamycin), respectively. The administration mode was microneedle administration on the back of the mouse, once every other day, for a total of 6 times.

[0074] On the 33rd day, the mice were sacrificed, and a piece of 1 cm*1 cm back skin was cut and crushed into 1 ml of phosphate buffer containing collagenase. After 37°C shaking for 30 min, the skin cell suspension was obtained, and the cell surface CD11C, CD40, and CD80 molecules were stained with flow cytometry antibodies to mark the DC cells. The results show that the proportion of CD11C+ cells in the skin increases after modeling, but the expression of the second signal molecules CD40 and CD80 decreases in the microneedle co-loaded with collagen II polypeptide and rapamycin group, indicating that it effectively induces the generation of tolerogenic DC in the skin, i.e., it can express the first signal molecule (CD11C), but lacks the second signal (CD40, CD80), as shown in Figure 12 .

[0075] After the mice are sacrificed, the spleen is taken out, grinded and the lymphocyte suspension is obtained by density gradient centrifugation, 500 μl of the lymphocyte suspension is added to each well of a 12-well plate, and 500 μl of the type II collagen polypeptide (10 μg / ml) is added, and after 72 h of stimulation, centrifugation is performed, and the supernatant is used for detecting the contents of IL-6, TNF-α and IL-10 by using an ELISA kit. The results prove that the spleen lymphocytes of the microneedle group co-loaded with the type II collagen polypeptide and rapamycin secrete significantly reduced pro-inflammatory cytokines IL-6 and TNF-α, but the secretion amount of the anti-inflammatory cytokine IL-10 is increased, see Figure 13 It can be seen that the type II collagen polypeptide and rapamycin have a synergistic effect, and the microneedle co-loaded with the type II collagen polypeptide and rapamycin can remodel the immune balance in the main lymphatic organs and induce antigen-specific immune tolerance.

[0076] In conclusion, the matrix solution is prepared by preparing the soluble component into a solution and the insoluble component into a suspension, the suspension is added into a mold to form a needle tip, the backing layer solution is poured after drying, and finally the microneedle patch is obtained by demolding, which simplifies the preparation steps of the microneedle. Further, the microneedle patch can transdermally deliver the self-antigen and the immunomodulator at the same time, effectively induces the generation of tolerogenic dendritic cells in the skin, reduces the inflammatory T cells in the main lymphatic organs, increases the proportion of regulatory T cells, reverses the abnormally activated immune system, reduces the pro-inflammatory cytokines in the joint part, increases the anti-inflammatory cytokines, successfully induces the immune tolerance, remodels the immune balance, significantly relieves the joint swelling (i.e. the thickness of the sole) and the inflammatory performance, and restores them to the level of the healthy group. It can be seen that the present application exerts an unexpected excellent therapeutic effect on rheumatoid arthritis. In addition, the microneedle patch is used for drug delivery for the rheumatoid arthritis patients who need long-term medication, and solves the problems of the toxic and side effects of the traditional oral treatment and the pain and low compliance of the patients accompanied by injection drug delivery.

[0077] The above only describes the preferred embodiments of the present application and does not limit the present application in any form. Although the present application has been disclosed as the preferred embodiments, it is not intended to limit the present application. Any person skilled in the art can make some changes or modifications to the above disclosed technical contents without departing from the scope of the technical solution of the present application, and any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present application still belong to the scope of the technical solution of the present application.

Claims

1. A microneedle transdermal drug delivery patch for treating rheumatoid arthritis, characterized in that, The microneedle transdermal drug delivery patch includes a backing layer and a needle body. The needle body includes a needle-forming matrix material and a drug. The needle-forming matrix material is hyaluronic acid, and the drug includes rheumatoid arthritis autoantigen and an immunomodulator. The rheumatoid arthritis autoantigen is collagen type II (CII), and the immunomodulator is rapamycin. The concentration of CII collagen in the microneedle is 0.1%-10% (m / v), and the concentration of rapamycin is 0.05%-5% (m / v). The preparation method of the microneedle includes the following steps: 1) Dissolving the rheumatoid arthritis autoantigen in water to obtain a transparent solution; 2) Adding hyaluronic acid to the above solution and stirring until completely dissolved; 3) Adding the immunomodulator and stirring thoroughly to obtain a suspension; 4) Adding the above suspension to a microneedle mold, using pressurized gas to press the solution into the microneedle mold, and drying it at room temperature for 2 hours in a drying autoclave to form microneedle tips. 5) Add hyaluronic acid solution to the mold, dry for 2 hours to form a microneedle base, and demold to obtain microneedles.

2. A method for preparing a microneedle transdermal drug delivery patch for treating rheumatoid arthritis according to claim 1, characterized in that, The preparation method of the microneedles includes the following steps: 1) Dissolving rheumatoid arthritis autoantigen in water to obtain a transparent solution; 2) Adding hyaluronic acid to the above solution and stirring to completely dissolve it; 3) Adding an immunomodulator and stirring thoroughly to obtain a suspension; 4) Adding the above suspension to a microneedle mold, using pressurized gas to press the solution into the microneedle mold, and drying it in a drying oven at room temperature for 2 hours to form microneedle tips; 5) Adding hyaluronic acid solution to the mold, drying for 2 hours to form a microneedle base, and demolding to obtain the microneedles.

3. The method for preparing the microneedle transdermal drug delivery patch for treating rheumatoid arthritis according to claim 2, characterized in that, The concentration of the autoantigen in the rheumatoid arthritis autoantigen solution is 0.1%-10% (m / v), and the concentration of the immunomodulator is 0.05%-5% (m / v).

4. The microneedle transdermal drug delivery patch for treating rheumatoid arthritis according to claim 1, or the microneedle transdermal drug delivery patch prepared by any one of the preparation methods of claims 2-3, for use in the preparation of drugs for inducing immune tolerance, regulating immune balance, and / or preparing drugs for treating autoimmune diseases.

5. The application according to claim 4, characterized in that, The autoimmune disease mentioned is rheumatoid arthritis.

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