Targeting drug-loaded nano-particles for treating rheumatoid arthritis as well as preparation method and application of targeting drug-loaded nano-particles

Abstract

The invention relates to the technical field of biological pharmacy, and particularly discloses targeting drug-loaded nanoparticles for treating rheumatoid arthritis (RA) as well as a preparation method and application thereof, and the technical key points are as follows: on the basis that a GLP-1R agonist (such as liraglutide) is proved to be capable of effectively relieving rheumatoid arthritis symptoms, the targeting drug-loaded nanoparticles can be used for treating rheumatoid arthritis; gLP-1RA is wrapped by the silk fibroin nanoparticles to prepare the drug-loaded nanoparticles, and the drug-loaded nanoparticles can effectively improve the stability of GLP-1RA, prolong retention time in vivo and improve the curative effect of the drug; the drug-loaded nanoparticles and the type II collagen targeting peptide (WYRGRL) are crosslinked, so that the drug-loaded nanoparticles target the articular cartilage, the drug is gathered on the articular cartilage to be attached, the local concentration of the drug and the availability of the drug are further improved, and the curative effect of inhibiting the progress of RA is further enhanced; for lesion facet joints in RA, the treatment scheme of micro-needle administration can realize slow release of drugs, and the micro-needle is convenient to use and is suitable for patients to use at home.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biopharmaceuticals, in particular to a kind of targeted drug-loaded nanoparticles for treating rheumatoid arthritis and preparation method and application. BACKGROUND

[0002] Rheumatoid arthritis (RA) is a global disease, with high incidence and high disability rate, which brings heavy economic burden to patients and society. The existing treatment has limitations: such as traditional drugs (such as methotrexate, etc.) have large side effects, and long-term use may cause liver toxicity and bone marrow suppression; biological agents (such as TNF-α inhibitors, etc.) are expensive, and need to be injected frequently, with low patient compliance; and the drug utilization rate is low by systemic administration, and the local joint drug concentration is insufficient, which are the problems to be solved at present.

[0003] Using the anti-inflammatory potential of GLP-1RA, a new treatment option is provided: GLP-1RA (such as semaglutide, liraglutide) shows significant metabolic benefits in the treatment of diabetes and obesity, and recent studies have found that it also has strong anti-inflammatory and immunomodulatory effects (such as inhibiting pro-inflammatory cytokine production and regulating immune cell function). However, its application in RA treatment is still in the early exploration stage. It has great potential to use this "old drug" for RA treatment. However, the existing GLP-1R agonist (GLP-1RA) system has the problems of systemic side effects and insufficient drug concentration in joint lesions.

[0004] Therefore, the present application creatively designs a kind of targeted drug-loaded nanoparticles for treating rheumatoid arthritis and preparation method and application to solve the above problems. SUMMARY

[0005] The present application aims to solve the technical problems raised in the background art, and provides a kind of targeted drug-loaded nanoparticles for treating rheumatoid arthritis and preparation method and application, the scheme of the present application utilizes the potential anti-inflammatory and immunomodulatory activity of GLP-1RA, and applies it to RA treatment; the drug-loaded nanoparticles are packaged by silk fibroin nanoparticles to improve the drug stability and prolong the retention time in vivo; according to the fact that RA lesions often occur in the joint cartilage, the drug-loaded nanoparticles are modified with type II collagen targeting peptide (WYRGRL) to improve the drug targeting and the drug concentration in the joint cartilage, and achieve precise treatment of the drug; the microneedle is convenient to use, and the combination of the microneedle drug delivery system can realize continuous and convenient drug delivery in the small joint of RA, and improve the drug action time.

[0006] The above-mentioned purpose of the present application is achieved as follows: The application provides a targeting drug-loaded nanoparticle for treating rheumatoid arthritis, which is a targeting drug-loaded silk fibroin nanoparticle with a cross-linked type II collagen targeting peptide and wrapping GLP-1RA.

[0007] The application also provides a preparation method of the targeting drug-loaded nanoparticle for treating rheumatoid arthritis, which comprises the following steps: S1, preparation of the silk fibroin nanoparticle wrapping GLP-1RA: S1-1, preparation of a silk fibroin solution; S1-2, preparation of the silk fibroin nanoparticle wrapping GLP-1RA, to obtain the drug-loaded silk fibroin nanoparticle wrapping GLP-1RA; S2, preparation of the targeting drug-loaded silk fibroin nanoparticle, to obtain the targeting drug-loaded silk fibroin nanoparticle with the cross-linked type II collagen targeting peptide and wrapping GLP-1RA.

[0008] Further, the specific method of step S1-1 is as follows: (1) 5 g of silkworm cocoons are added into a 0.5-1% Na2CO3 solution with a volume of 1-2 L, and boiled for 10-15 minutes; the boiled solution is poured out, and 0.5-1% Na2CO3 solution with a volume of 1-2 L is added again, and boiled for 10-15 minutes, so that the sericin protein in the silkworm cocoons is removed completely; (2) the silk fibroin protein from which the sericin protein is removed is washed in double-distilled water for 2-3 times, so that the impurities in the silk fibroin protein are removed; (3) the washed silk fibroin protein is placed in a 37℃ oven for drying; (4) 20.19 g of lithium bromide is dissolved in 25 ml of double-distilled water, so that the concentration of LiBr is 9.3 mol / L; (5) the dried silk fibroin protein is continuously added into the 9.3 mol / L LiBr solution in small amounts, and stirred until the silk fibroin protein is no longer dissolved, and then the addition of the silk fibroin protein is stopped; (6) the silk fibroin protein solution is centrifuged at 6000 rpm / min for 10 minutes; (7) after centrifugation, the orange-yellow silk fibroin protein solution is collected and transferred into a dialysis bag with a molecular weight cut-off of 8000 Da-14000 Da, and dialyzed in double-distilled water, and the dialysis solution is changed every 6-8 hours, and the dialysis is performed for 2-3 days; (8) after the dialysis is completed, the silk fibroin protein solution is centrifuged again at 6000 rpm / min for 10 minutes; (9) after centrifugation, the silk fibroin protein solution is collected, and its concentration is measured, which is about 10-20 mg / ml.

[0009] Further, the specific method of step S1-2 is: (1) Take 10-20 mg / ml silk fibroin solution 100 μl, 1-2 g; take 6 mg / ml GLP-1RA 50 μl, 0.3 g, and add it to 100 μl of silk fibroin solution, so that the mass ratio of GLP-1RA to silk fibroin is between 1:5-1:10; (2) Take 2-3 ml of 4°C acetone and place it in a 15 ml centrifuge tube, and vortex on a vortexer; (3) Add the mixed solution of silk fibroin and GLP-1RA drop by drop during vortexing, and after the liquid is added dropwise, vortex for 30-60 s; (4) Centrifuge the white milky solution after vortexing at 4°C, 8000 rpm / min, for 3-5 minutes; (5) After centrifugation, pour off the supernatant and resuspend the precipitate with double distilled water; (6) Ultrasonic the resuspended nanoparticles in an ultrasonic instrument, with the following conditions: time 2 minutes, ultrasonic 2 s, pause 2 s, amplitude 20%-30%; (7) Centrifuge the nanoparticles after ultrasonic, 10000 rpm / min, 4°C, for 5 minutes; (8) After centrifugation, pour off the supernatant and add 2 ml of double distilled water to resuspend the nanoparticles, and obtain the drug-loaded silk fibroin nanoparticles wrapped with GLP-1RA.

[0010] Further, the specific method of step S2 is: (1) Take 50 mg of type II collagen targeting peptide and add it to 10 ml of double distilled water to make the concentration 5 mg / ml; (2) Take 2 ml of the prepared drug-loaded nanoparticles and place it in a 5 ml centrifuge tube; take 200 μl of the type II collagen targeting peptide solution and add it to the silk fibroin nanoparticle solution; (3) Take 5 mg of EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) and 10 mg of NHS (N-hydroxysuccinimide) and add them to the silk fibroin nanoparticle solution; perform amide reaction at room temperature, in the dark, and on a shaker (70 rpm) for about 6-12 hours; (4) After the reaction is completed, centrifuge the above solution at 4°C, 10000 rpm / min, for 10 minutes; (5) After centrifugation, pour off the supernatant, add 1 ml of double distilled water, resuspend the precipitate, and obtain the targeting drug-loaded silk fibroin nanoparticle targeting type II collagen and wrapping GLP-1RA.

[0011] The present application also provides a targeting drug-loaded microneedle containing the targeting drug-loaded nanoparticle.

[0012] The preparation method of the targeting drug-loaded microneedle comprises the following steps: (1) Preparation of HAMA solution: (A) Take 0.5 g of hyaluronic acid and add it to 50 ml of deionized water, and stir with a magnetic rotor stirrer at 400-600 rpm to completely dissolve it; (B) Add 2 ml of methacrylic anhydride to the hyaluronic acid solution, then continuously add 4 M sodium hydroxide solution, and maintain the pH value of the methacrylic anhydride / hyaluronic acid solution between 8-9, and stir overnight at 4°C; (C) The next day, dialyze the methacrylic anhydride-modified hyaluronic acid solution in a dialysis bag with a molecular weight cutoff of 8000-14000 daltons; the dialysis medium is deionized water, and the dialysis is carried out for 3 days, with the deionized water being changed every 6-12 hours; (D) After dialysis, a 0.5% HAMA solution is obtained; then the solution is placed in a 37°C oven, and after 1-2 days, a 3% HAMA solution is obtained; (2) Preparation of drug-loaded microneedle: a. Prepare a 30% polyvinyl alcohol solution: take 3 g of polyvinyl alcohol solid and add it to a 15 ml centrifuge tube, then add 10 ml of deionized water to the centrifuge tube, shake up and down, then place it in a 60°C incubator overnight to completely dissolve the polyvinyl alcohol; b. Prepare the microneedle base solution: take 2 ml of 30% polyvinyl alcohol solution in a 5 ml centrifuge tube, then add 2 ml of 3% HAMA solution, mix well, and centrifuge to remove air bubbles, and obtain the microneedle base solution; c. Needle tip air removal: take the microneedle mold, the needle type is a right square pyramid, the needle length is 1200 μm, the bottom side length is 500*500 μm, the needle spacing is 800 μm, and the array number is 15*15; immerse it in deionized water, use a 3 ml pipette to suck deionized water, and repeatedly blow the needle tip to completely fill the needle tip with deionized water, and during the process of blowing the needle tip, immerse the microneedle mold under the liquid surface to prevent air from entering the needle tip; d. After the needle tip air removal, take it out and place it on the experimental table, use a 200 μl pipette to suck off the excess liquid on the microneedle, and keep the microneedle needle tip immersed in deionized water; f. Take 1 ml of the targeted drug-loaded nanoparticles and add to 2 ml of the HAMA solution, mix well and remove the air bubbles in the liquid to obtain 3 ml of the HAMA solution containing the targeted drug-loaded nanoparticles; g. Take the HAMA solution containing the targeted drug-loaded nanoparticles and add to the microneedle mold in which the needle tip is soaked in deionized water; continuously add the solution from the middle part of the microneedle, and in the process of adding the liquid, the deionized water is extruded to the periphery of the microneedle by the HAMA solution containing the targeted drug-loaded nanoparticles, and in this process, the 200 μl pipette is used to suck the deionized water; the HAMA solution containing the targeted drug-loaded nanoparticles is fused with the deionized water at the needle tip, and the deionized water at the needle tip is replaced by the HAMA solution containing the targeted drug-loaded nanoparticles; h. Place the microneedle mold filled with liquid in a 4℃ refrigerator for 2-3 days to concentrate the HAMA solution to a state where it cannot flow; i. Take about 600 μl of the microneedle base solution and add to the microneedle mold, shake left and right and front and back to make the base solution evenly distributed in the microneedle, and continue to place in a 4℃ refrigerator for 1-2 days to make the liquid as completely volatilized as possible; j. Dry and solidify the microneedle; take the microneedle mold in the 4℃ refrigerator and place it in a 37℃ oven for 1-2 hours to dry and solidify the microneedle completely; k. Take out the completely dried microneedle mold and use tweezers to slowly take out the microneedle from the periphery of the microneedle mold.

[0013] The scheme of the application also provides the use of the targeted drug-loaded nanoparticles or the targeted drug-loaded microneedle for treating rheumatoid arthritis in the preparation of a product for treating rheumatoid arthritis.

[0014] The difficulty and significance of solving the technical problems of the application are: The scheme of the present application creatively proposes a new strategy for rheumatoid arthritis (RA) targeted therapy based on multifunctional integrated soluble microneedles. The strategy expands the application of mature GLP-1 receptor (GLP-1R) agonists (GLP-1RA) in the field of metabolism to RA treatment, and utilizes its potential but insufficiently recognized anti-inflammatory and immune regulation activity. In order to overcome the potential systemic side effects of GLP-1RA systemic administration and the problem of insufficient drug concentration in joint lesions, the scheme of the present application innovatively constructs a 'core-shell' structure delivery system: using biocompatible, degradable and naturally sustained-release silk fibroin nanoparticles as the shell, and efficiently loading the core drug GLP-1RA; and then through specific chemical cross-linking, the II type collagen targeting peptide (WYRGRL) is precisely modified on the surface of the nanoparticles. The targeting peptide acts as a'smart navigator', which can actively recognize and enrich in the exposed or damaged II type collagen matrix in the RA lesion joints, achieving lesion-specific targeted delivery of drugs. Finally, the functionalized nanoparticles are loaded into the soluble microneedle array. The microneedle can painlessly penetrate the stratum corneum barrier of the skin, rapidly dissolve in the interstitial fluid of the skin, release the drug-loaded targeted nanoparticles, and then the latter is efficiently targeted to the inflammatory joints after local absorption.

[0015] This 'targeted nanocarrier + transdermal microneedle + new drug use' trinity innovative design significantly improves the bioavailability of the drug at the lesion site, reduces the risk of systemic exposure, and improves patient compliance, providing a new and highly transformative solution for precise, efficient and painless treatment of RA.

[0016] Compared with the prior art, the present application has the following beneficial effects: The scheme of the present application overcomes the key clinical problems existing in the current treatment of rheumatoid arthritis (RA), and realizes safer, more effective and more convenient treatment through a new drug delivery strategy that is integrated, precise and patient-friendly. The specific problems and advantages are as follows: 1. The scheme of the present application utilizes the anti-inflammatory potential of GLP-1RA to provide a new treatment option. The scheme of the present application loads GLP-1RA and precisely delivers it to the inflammatory joints, aiming to maximize its anti-inflammatory / immune regulation effect in the local joints, and provides a potential treatment option for RA patients based on a new mechanism of action.

[0017] 2、The scheme overcomes the systemic toxic side effects of traditional drug administration. The mainstream drugs for RA (such as non-steroidal anti-inflammatory drugs, glucocorticoids, disease-modifying antirheumatic drugs DMARDs, biological agents) are mostly administered orally or by injection (subcutaneous, intravenous). These methods cause the drug to be widely distributed throughout the body, rather than specifically enriched in the affected joints. It easily causes significant systemic side effects (such as gastrointestinal damage, liver and kidney function damage, bone marrow suppression, increased risk of infection, metabolic disorders, etc.), which severely limits the increase in drug dosage and long-term use, affecting the quality of life of patients. In the present scheme, the collagen type II targeting peptide modified nanocarrier is used to achieve active targeting delivery of drugs to the RA affected joints (cartilage destruction area / synovial inflammation site), which can significantly increase the local drug concentration in the lesion, while greatly reducing the drug exposure in non-target organs, thereby reducing systemic side effects.

[0018] 3、The scheme solves the problem of insufficient drug concentration in the lesion and limited efficacy in the prior art. The drug can reach the joint, but due to factors such as synovial barrier and inflammatory microenvironment, the effective penetration and retention of the drug in the joint are often insufficient, and the drug is easily degraded, making it difficult to achieve and maintain the required concentration for treatment, which may result in unsatisfactory treatment effect, poor disease control, repeated disease onset, and continuous joint destruction. In the present scheme, the collagen type II targeting peptide guides the drug-loaded nanoparticles to precisely anchor in the damaged cartilage matrix (exposed collagen type II) or inflammatory synovial tissue of the affected joint. Moreover, the silk fibroin nanoparticles used in the present scheme not only have excellent biocompatibility and controllable degradation / sustained release characteristics, but also prevent the rapid degradation of GLP-1RA drugs in the joint after encapsulating GLP-1RA, thereby continuously releasing GLP-1RA at the lesion site, maintaining a long-term effective treatment concentration, and enhancing the efficacy.

[0019] 4、The scheme can improve patient compliance and has the convenience of administration. Because RA is a chronic disease, it needs long-term or even lifelong treatment, and frequent subcutaneous injection (such as biological agents) brings pain, fear, and adverse reactions at the injection site to patients, which burdens the patients. The present scheme can significantly improve treatment compliance (poor compliance directly affects treatment effect and disease control). In the present scheme, soluble microneedles are used as drug delivery carriers, and the microneedle array can painlessly penetrate the stratum corneum barrier of the skin and rapidly dissolve in the interstitial fluid to release the drug, which is simple to operate (similar to a patch). This non-invasive / minimally invasive, self-managed drug delivery method greatly improves patient acceptance and long-term medication compliance. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a mode diagram for preparing the targeted drug-loaded silk nanoparticles in the embodiments of the present application; Figure 2SEM, particle size, Fourier infrared spectrum and release rate detection of different types of silk fibroin nanoparticles prepared in the embodiments of the present application; Figure 3 High performance liquid chromatography detection, encapsulation rate analysis and drug release rate detection and analysis of drug-loaded nanoparticles in the embodiments of the present application; Figure 4 Targeted distribution of rhodamine b isothiocyanate (RBITC) labeled non-targeted nanoparticles and targeted nanoparticles on chondrocytes and articular cartilage tissue in the embodiments of the present application; Figure 5 Preparation of drug-loaded microneedles, fluorescence and scanning electron microscope detection of drug-loaded microneedles in the embodiments of the present application; Figure 6 Dissolution experiment and transdermal experiment of drug-loaded microneedles in the embodiments of the present application; Figure 7 Mechanical detection of microneedles and release experiment of nanoparticles in microneedles in the embodiments of the present application; Figure 8 Comparison of the therapeutic effect of GLP-1RA or MNs / GLP-1RA@NPs-WYRGRL on RA in the embodiments of the present application. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in combination with the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application.

[0022] The implementation of the present application is described in detail below in combination with specific embodiments.

[0023] Referring to Figures 1-8 , a preferred embodiment provided by the present application is shown.

[0024] The present application provides a kind of targeted drug-loaded nanoparticles for treating rheumatoid arthritis and preparation method, and targeted drug-loaded microneedle comprising targeted drug-loaded nanoparticles, and its application in treating rheumatoid arthritis.The present application aims to overcome the problems of systemic toxicity, low patient compliance, insufficient drug concentration in lesion and limited efficacy in the treatment of rheumatoid arthritis in the prior art.The specific implementation of the present application is as follows. EMBODIMENT

[0025] 1, emulsion preparation of induced rheumatoid arthritis rats 1.1, First prepare 0.01M acetic acid, then filter the acetic acid with a 0.22μm filter. Dilute 10mg of bovine type II collagen with 5ml of 0.01M acetic acid to a concentration of 2mg / ml. Incubate at 4°C overnight until the collagen is completely dissolved.

[0026] 1.2, Take 5ml of Freund's complete adjuvant and add it to 5ml of bovine type II collagen solution. Move the mixed solution to a 50ml centrifuge tube and vortex it on ice at a speed of 10000rmp / min or higher using a handheld small electric mixer until an emulsion is formed. After the emulsion is dropped into water, the emulsion does not break up within 1min, and an emulsion with a final concentration of 1mg / ml bovine type II collagen is formed.

[0027] 2, Primary immunization Take 6-8 week old SD female rats (about 150g) and anesthetize them. Disinfect the right footpads of the rats with 75% alcohol, then inject about 200ul of the bovine type II collagen emulsion into the subcutaneous tissue of the foot sole using a 1ml syringe. Press the injection site with a cotton swab for 1min after injection.

[0028] 3, Secondary immunization On the 21st day after primary immunization, use a 1ml syringe to inject about 200ul of the bovine type II collagen emulsion into the tail base of the rats using a multi-point injection method. After secondary immunization, observe and photograph the symptoms of the rats' hind feet every week, and calculate the changes in clinical scores.

[0029] 4, Treatment of rheumatoid arthritis (RA) rats 4.1, On the 28th day, divide the rats into 4 groups: NC group: normal control rats, from the 4th week, assess the clinical symptoms and joint scores of the rats' hind feet every week, and on the 16th week, section and HE and safranin O fast green stain the rats' hind feet; RA group: collagen-induced rheumatoid arthritis rats, treated from the 4th week, assess the clinical symptoms and joint scores of the rats' hind feet every week, and on the 16th week, section and HE and safranin O fast green stain the rats' hind feet; RA+GLP-1RA group: collagen-induced rheumatoid arthritis rats treated with GLP-1RA after the disease; RA+MNs / GLP-1RA@NPs-WYRGRL group: collagen-induced rheumatoid arthritis rats treated with MNs / GLP-1RA@NPs-WYRGRL after the disease.

[0030] 4.2, RA+GLP-1RA group of rats treatment: the RA+GLP-1RA group of rats were anesthetized, insulin needle 50 μl of 6 mg / ml of GLP-1RA drug injection, injection site with cotton swab press 1 min. From the 4th week of treatment, 1-2 times a week, to the 16th week of treatment, the rats were evaluated every week for clinical symptoms and joint scores. After treatment, the rats were sectioned and stained with HE and Safranin O fast green.

[0031] 4.3, RA+MNs / GLP-1RA@NPs-WYRGRL group: The RA+MNs / GLP-1RA@NPs-WYRGRL group of rats were anesthetized, and 50 μl of 6 mg / ml of GLP-1RA drug was prepared into a targeted drug-loaded microneedle, and the microneedle was used to treat the RA rats. The implementation is as follows: 4.3.1, preparation of silk fibroin solution 4.3.1.1, take 5g of domestic silkworm cocoon, add it to 0.5-1% Na2CO3 solution, the volume of Na2CO3 solution is about 1-2L, and boil for 10-15 minutes. Pour off the boiled solution, add 0.5-1% Na2CO3 solution 1-2L again, boil for 10-15 minutes, and remove the sericin protein in the cocoon completely.

[0032] 4.3.1.2, wash the silk fibroin protein without sericin protein in double distilled water, 2-3 times, and remove as much impurities as possible from the silk fibroin protein. The washed silk fibroin protein is placed in a 37°C oven for drying.

[0033] 4.3.1.3, take lithium bromide (LiBr) 20.19g, dissolve in 25ml double distilled water, so that the concentration of LiBr is 9.3mol / L. Take the dried silk fibroin protein, add it to the 9.3mol / L LiBr solution in small amounts continuously, stir until the silk fibroin protein no longer dissolves, and stop adding the silk fibroin protein.

[0034] 4.3.1.4, centrifuge the silk fibroin protein solution at 6000rpm / min for 10 minutes. After centrifugation, collect the orange-yellow silk fibroin protein solution and transfer it to a dialysis bag with a molecular weight cutoff of 8000Da-14000Da, dialyze in double distilled water, change the dialysate every 6-8 hours, and dialyze for 2-3 days. After dialysis, the silk fibroin protein solution is centrifuged again at 6000rpm / min for 10 minutes. After centrifugation, the silk fibroin protein solution is collected and its concentration is measured, which is about 10-20mg / ml.

[0035] 4.3.2, preparation of silk fibroin nanoparticles coated with GLP-1RA 4.3.2.1, Take 10-20 mg / ml silk fibroin solution 100 μl, about 1-2 g; take 6 mg / ml GLP-1RA 50 μl, about 0.3 g, add it to 100 μl silk fibroin solution, so that the mass ratio of GLP-1RA to silk fibroin is between 1:5-1:10.

[0036] 4.3.2.2, Take 2-3 ml of acetone at 4°C and put it in a 15 ml centrifuge tube, and vortex on a vortexer. Add the mixed solution of silk fibroin and GLP-1RA drop by drop during vortexing. After the liquid is added dropwise, vortex for 30-60 s. Centrifuge the milky white solution after vortexing at 4°C, 8000 rpm / min, for 3-5 minutes.

[0037] 4.3.2.3, After centrifugation, pour off the supernatant and resuspend the precipitate with double distilled water. Ultrasonic the resuspended nanoparticles in an ultrasonic instrument, with the following conditions: time 2 minutes, ultrasonic 2 s, pause 2 s, amplitude 20%-30%.

[0038] 4.3.2.4, Centrifuge the nanoparticles after ultrasonic, 10000 rpm / min at 4°C, for 5 minutes. After centrifugation, pour off the supernatant and add 2 ml double distilled water to resuspend the nanoparticles, and obtain the drug-loaded silk fibroin nanoparticles wrapped with GLP-1RA (GLP-1RA@NPs).

[0039] 4.3.3, Preparation of targeted drug-loaded silk fibroin nanoparticles 4.3.3.1, Take 50 mg of type II collagen targeting peptide (WYRGRL) and add it to 10 ml double distilled water to make the concentration 5 mg / ml.

[0040] 4.3.3.2, Take 2 ml of the prepared GLP-1RA@NPs solution and place it in a 5 ml centrifuge tube. Take 200 μl of type II collagen targeting peptide solution and add it to the GLP-1RA@NPs solution.

[0041] 4.3.3.3, Take 5 mg of EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) and 10 mg of NHS (N-hydroxysuccinimide) and add them to the GLP-1RA@NPs solution. Perform amide reaction at room temperature, in the dark, and on a shaker (70 rpm) for about 6-12 hours.

[0042] 4.3.3.4, After the reaction is completed, the above solution is centrifuged at 4°C, 10000 rpm / min, for 10 minutes. After centrifugation, the supernatant is discarded, 1 ml of double distilled water is added, and the precipitate is resuspended to obtain the targeting drug-loaded silk fibroin nanoparticle (GLP-1RA@NPs-WYRGRL) targeting type II collagen peptide and wrapping GLP-1RA.

[0043] 4.3.4, Preparation of targeted drug-loaded microneedles 4.3.4.1, Preparation of HAMA solution. Take 0.5 g of hyaluronic acid and add it to 50 ml of deionized water, and stir with a magnetic rotor stirrer at 400-600 rpm until it is completely dissolved. Add 2 ml of methacrylic anhydride to the hyaluronic acid solution, then continuously add 4 M sodium hydroxide solution to maintain the pH of the methacrylic anhydride / hyaluronic acid solution between 8-9, and stir overnight at 4°C. The next day, the methacrylic anhydride-modified hyaluronic acid solution is dialyzed in a dialysis bag with a molecular weight cutoff of 8000-14000 daltons. The dialysis medium is deionized water, and the dialysis is carried out for 3 days, with the deionized water being replaced every 6-12 hours. After dialysis, a 0.5% or so HAMA solution is obtained. Then the solution is placed in a 37°C oven, and after 1-2 days a 3% or so HAMA solution is obtained.

[0044] 4.3.4.2, Preparation of drug-loaded microneedles. A 30% polyvinyl alcohol solution is prepared. Take 3 g of polyvinyl alcohol solid and add it to a 15 ml centrifuge tube, then add 10 ml of deionized water to the centrifuge tube, shake up and down, then place it in a 60°C incubator overnight to completely dissolve the polyvinyl alcohol.

[0045] 4.3.4.3, Preparation of microneedle base solution. Take 2 ml of 30% polyvinyl alcohol solution in a 5 ml centrifuge tube, then add 2 ml of 3% HAMA solution, mix well, and centrifuge to remove air bubbles, to obtain the microneedle base solution.

[0046] 4.3.4.4, Microneedle tip degassing. Take the microneedle mold, the needle type is a right square pyramid, the needle length is 1200 μm, the bottom side length is 500*500 μm, the needle spacing is 800 μm, and the array number is 15*15. Soak it in deionized water, use a 3 ml pipette to suck deionized water, and repeatedly blow the needle tip to completely fill the needle tip with deionized water. During the process of blowing the needle tip, the microneedle mold is kept under the liquid surface to prevent air from entering the needle tip. After degassing the microneedle tip, take it out and place it on the experimental table, use a 200 μl pipette to suck off the excess liquid on the microneedle, and keep the microneedle tip immersed in deionized water.

[0047] 4.3.4.5, Take the targeted drug-loaded nanoparticles (GLP-1RA@NPs-WYRGRL) 1 ml, add to 2 ml of HAMA solution, mix evenly, and then remove the air bubbles in the liquid to obtain 3 ml of HAMA solution containing the targeted drug-loaded nanoparticles.

[0048] 4.3.4.6, Take the HAMA solution containing the targeted drug-loaded nanoparticles, and add it to the microneedle mold in which the needle tip is soaked in deionized water. Continuously add the solution from the middle of the microneedle, and in the process of adding the liquid, the deionized water is squeezed to the periphery of the microneedle by the HAMA solution containing the targeted drug-loaded nanoparticles. In this process, 200 μl of a pipette is used to suck away the deionized water. The HAMA solution containing the targeted drug-loaded nanoparticles will merge with the deionized water at the needle tip, thereby replacing the deionized water at the needle tip with the HAMA solution containing the targeted drug-loaded nanoparticles.

[0049] 4.3.4.7, Place the microneedle mold filled with liquid in a 4°C refrigerator for 2-3 days to concentrate the HAMA solution to a state where it cannot flow. Take about 600 μl of the microneedle base solution and add it to the microneedle mold. Shake left and right and front and back to distribute the base solution evenly in the microneedle. Continue to place it in a 4°C refrigerator for 1-2 days to allow the liquid to evaporate as completely as possible.

[0050] 4.3.4.8, Dry and solidify the microneedle. Take the microneedle mold from the 4°C refrigerator and place it in a 37°C oven for 1-2 hours to completely dry and solidify the microneedle. Take out the completely dried microneedle mold and use tweezers to gently remove the targeted drug-loaded nanoparticle microneedle (MNs / GLP-1RA@NPs-WYRGRL) from the periphery of the microneedle mold.

[0051] 4.3.5, Treatment of RA rats with targeted drug-loaded nanoparticle microneedles Anesthetize the rats in the RA+MNs / GLP-1RA@NPs-WYRGRL group, disinfect the foot sole with 75% alcohol, and then insert the microneedle prepared with 50 μl of 6 mg / ml GLP-1RA into the rat foot skin, press for about 1 min, and then spray deionized water on the surface of the microneedle to allow the microneedle to fully combine with the deionized water. After about 5 min, the microneedle base falls off and the needle tip remains in the intradermal of the rat foot sole. Start treatment from the 4th week, treat 1-2 times per week, and end treatment at the 16th week. Evaluate the clinical symptoms and joint scores of the rat hind feet every week. After treatment, perform sectioning and HE and Safranin O fast green staining on the rat hind feet.

[0052] In summary, in the above-mentioned embodiments, the nanoparticles loaded with GLP-1RA and modified by the type II collagen targeting peptide are used to target the diseased joints, and the emerging microneedle (MN) transdermal drug delivery system is used as a drug carrier, which can be used for subcutaneous administration in the diseased joints, penetrate the stratum corneum to create microchannels and deliver drugs in a minimally invasive manner. The role of the GLP-1RA-loaded targeting silk fibroin nanoparticles in the treatment of RA is analyzed by rat arthritis score, HE staining, immunohistochemistry, immunofluorescence staining and western blotting. In the collagen-induced arthritis (CIA) rat model, the GLP-1RA-loaded targeting silk fibroin nanoparticles are administered by microneedles at the diseased joints, which significantly reduces synovial inflammation and cartilage destruction. Histological analysis confirms the effect of the present application, indicating that the joint structure is significantly improved and the disease progression is delayed.

[0053] Through the above-mentioned embodiments of the present application, the present application provides a new treatment mechanism, i.e. the new application of GLP-1RA in the treatment of RA, i.e. the medical use of GLP-1RA (such as semaglutide and liraglutide) in the treatment of rheumatoid arthritis ("old drugs for new use"). In addition, a targeted delivery system is designed, i.e. silk fibroin nanoparticles modified by type II collagen targeting peptide as a carrier. Moreover, the drug delivery system has a sustained-release property: the method of loading GLP-1RA on silk fibroin nanoparticles. At the same time, the present application designs the integration and optimization of the microneedle platform, i.e. the process of embedding the drug-loaded targeting nanoparticles into the soluble microneedle matrix. The present application provides the synergistic effect of the multi-level delivery mechanism: the synergistic effect of the three-level delivery system of the protection logic: microneedle→nanocarrier→targeting peptide.

[0054] Compared with the prior art, the scheme of the present application overcomes the problems of large drug side effects and low patient compliance in traditional treatment methods. The silk fibroin nanoparticles (NPs) in the scheme of the present application have good biocompatibility and degradability, can protect GLP-1RA from degradation, and improve drug stability. Moreover, the microneedles (MNs) containing GLP-1RA@NPs as a new drug delivery system can deliver GLP-1RA@NPs directly to the surrounding of the diseased joint, improve the local drug concentration, and reduce the systemic side effects. In addition, the silk fibroin nanoparticles (NPs) can achieve sustained release of GLP-1RA, prolong the drug action time, and reduce the drug administration frequency. At the same time, the microneedles designed in the present application can penetrate the stratum corneum of the skin and deliver the drug to the dermis, avoiding the pain and discomfort of traditional injection. The microneedles are easy to use and suitable for patients to use at home, improving the treatment compliance. By combining the anti-inflammatory effect of GLP-1RA and the advantages of the microneedle delivery system, the microneedles containing NPs@GLP-1RA show significant characteristics and innovations in the treatment of rheumatoid arthritis, and are expected to become an efficient and safe treatment method, providing a new idea and method for RA treatment.

[0055] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A targeted drug-loaded nanoparticle for treating rheumatoid arthritis, characterized in that, The targeted drug-loaded nanoparticles are cross-linked type II collagen targeting peptides and targeted drug-loaded silk fibroin nanoparticles encapsulating GLP-1R agonists.

2. The method for preparing targeted drug-loaded nanoparticles for treating rheumatoid arthritis according to claim 1, characterized in that, Includes the following steps: S1. Preparation of silk fibroin nanoparticles encapsulating GLP-1RA: S1-1, Preparation of silk fibroin solution; S1-2, Preparation of GLP-1RA-encapsulated silk fibroin nanoparticles to obtain drug-loaded silk fibroin nanoparticles encapsulated with GLP-1RA. S2. Preparation of targeted drug-loaded silk fibroin nanoparticles: cross-linked type II collagen targeting peptides and GLP-1RA-encapsulated targeted drug-loaded silk fibroin nanoparticles were obtained.

3. The method for preparing targeted drug-loaded nanoparticles for treating rheumatoid arthritis according to claim 2, characterized in that, The specific method for step S1-1 is as follows: (1) Take 5g of silkworm cocoons and add them to a 0.5-1% Na2CO3 solution with a volume of 1-2L. Boil for 10-15 minutes. Discard the boiled solution and add another 1-2L of 0.5-1% Na2CO3 solution. Boil for 10-15 minutes to remove the sericin from the silkworm cocoons. (2) Wash the silk fibroin after removing the sericin in double-distilled water 2-3 times to remove impurities from the silk fibroin; (3) Place the washed silk fibroin in a 37°C oven to dry; (4) Take 20.19 g of lithium bromide and dissolve it in 25 ml of double-distilled water to make the concentration of LiBr 9.3 mol / L; (5) Take the dried silk fibroin and add it in small amounts continuously to a 9.3 mol / L LiBr solution. Stir until the silk fibroin no longer dissolves, then stop adding silk fibroin. (6) Centrifuge the silk fibroin solution at 6000 rpm for 10 minutes; (7) After centrifugation, collect the orange-yellow silk fibroin solution and transfer it to a dialysis bag with a molecular weight cutoff of 8000Da-14000Da. Dialyze it in double-distilled water, changing the dialysis solution every 6-8 hours for 2-3 days. (8) After dialysis, the silk fibroin solution was centrifuged again at 6000 rpm for 10 minutes. (9) After centrifugation, collect the silk fibroin solution and measure its concentration, which is approximately 10-20 mg / ml.

4. The method for preparing targeted drug-loaded nanoparticles for treating rheumatoid arthritis according to claim 3, characterized in that, The specific method for step S1-2 is as follows: (1) Take 100 μl of 10-20 mg / ml silk fibroin solution, 1-2 g; take 50 μl of 6 mg / ml GLP-1RA, 0.3 g, and add it to 100 μl of silk fibroin solution so that the mass ratio of GLP-1RA to silk fibroin is between 1:5 and 1:

10. (2) Take 2-3 ml of acetone at 4°C and place it in a 15 ml centrifuge tube, and vortex it on a vortex mixer; (3) Add the mixed solution of silk fibroin and GLP-1RA drop by drop during the vortexing process. After the liquid is added, vortex for 30-60 seconds. (4) Centrifuge the vortexed milky white solution at 4℃, 8000 rpm / min, for 3-5 minutes; (5) After centrifugation, discard the supernatant and resuspend the precipitate in double-distilled water; (6) The suspended nanoparticles were sonicated in an ultrasonic instrument under the following conditions: time 2 minutes, sonication 2 seconds, pause 2 seconds, amplitude 20%-30%; (7) After sonication, the nanoparticles were centrifuged again at 10,000 rpm / min for 5 minutes at 4°C. (8) After centrifugation, discard the supernatant, add 2 ml of double-distilled water, and resuspend the nanoparticles to obtain drug-loaded silk fibroin nanoparticles encapsulating GLP-1RA.

5. The method for preparing targeted drug-loaded nanoparticles for treating rheumatoid arthritis according to claim 4, characterized in that, The specific method for step S2 is as follows: (1) Take 50 mg of type II collagen targeting peptide and add it to 10 ml of double-distilled water to make its concentration 5 mg / ml; (2) Take 2 ml of the prepared drug-loaded nanoparticles and place them in a 5 ml centrifuge tube; take 200 μl of type II collagen-targeting peptide solution and add it to the silk fibroin nanoparticle solution; (3) Take 5 mg of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and 10 mg of NHS (N-hydroxysuccinimide) and add them to the silk fibroin nanoparticle solution; carry out the amide reaction at room temperature, in the dark and on a shaker (70 rpm) for about 6-12 hours. (4) After the reaction is complete, the above solution is centrifuged at 4°C, 10000 rpm / min for 10 minutes. (5) After centrifugation, discard the supernatant, add 1 ml of double-distilled water, and resuspend the precipitate to obtain cross-linked type II collagen targeting peptide and GLP-1RA-encapsulated targeted drug-loaded silk fibroin nanoparticles.

6. A targeted drug-loaded microneedle (MNs) comprising the targeted drug-loaded nanoparticles of claim 1 or the targeted drug-loaded nanoparticles prepared by any one of claims 2-5.

7. The method for preparing targeted drug-loaded microneedles according to claim 6, characterized in that, Includes the following steps: (1) Preparation of HAMA solution: (A) Take 0.5g of hyaluronic acid, add it to 50 ml of deionized water, and stir with a magnetic stirrer at 400-600 rpm until it is completely dissolved; (B) Add 2 ml of methacrylic anhydride to the hyaluronic acid solution, and then continuously add 4 M sodium hydroxide solution to maintain the pH value of the methacrylic anhydride / hyaluronic acid solution between 8 and 9, and stir overnight at 4°C; (C) On the second day, the methacrylic anhydride-modified hyaluronic acid solution was dialyzed in a dialysis bag with a molecular weight cutoff of 8,000-14,000 Daltons; the dialysis medium was deionized water, and the dialysis was performed for 3 days, with the deionized water being changed every 6-12 hours. (D) After dialysis, a 0.5% HAMA solution was obtained; then the solution was placed in an oven at 37°C, and after 1-2 days, a 3% HAMA solution was obtained. (2) Preparation of drug-loaded microneedles: a. Prepare a 30% polyvinyl alcohol solution: Add 3g of solid polyvinyl alcohol to a 15ml centrifuge tube, then add 10ml of deionized water to the centrifuge tube, shake it up and down, and then place it in a 60℃ incubator overnight to allow the polyvinyl alcohol to dissolve completely. b. Preparation of microneedle base solution: Take 2 ml of 30% polyvinyl alcohol solution into a 5 ml centrifuge tube, then add 2 ml of 3% HAMA solution, mix thoroughly, and centrifuge to remove air bubbles to obtain the microneedle base solution. c. Exhausting air from the microneedle tip: Take a microneedle mold with a regular square pyramid shape, a needle length of 1200μm, a base side length of 500*500μm, a needle spacing of 800μm, and an array number of 15*15; immerse it in deionized water, use a 3ml pipette to draw up the deionized water, and repeatedly blow on the needle tip to completely fill the needle tip with deionized water. During the blowing process, keep the microneedle mold below the liquid surface to prevent air from entering the needle tip area; d. After venting the microneedle tip, remove it and place it on the lab bench. Use a 200μl pipette to remove excess liquid from the microneedle, keeping the microneedle tip immersed in deionized water. f. Take 1 ml of targeted drug-loaded nanoparticles and add them to 2 ml of HAMA solution. After mixing evenly, remove the air bubbles in the liquid to obtain 3 ml of HAMA solution containing targeted drug-loaded nanoparticles. g. Take a HAMA solution containing targeted drug-loaded nanoparticles and add it to a microneedle mold in which the needle tip is immersed in deionized water; continuously add the solution from the middle of the microneedle. During the liquid addition process, the deionized water is squeezed to the periphery of the microneedle by the HAMA solution containing targeted drug-loaded nanoparticles. During this process, use a 200μl pipette to remove the deionized water; the HAMA solution containing targeted drug-loaded nanoparticles merges with the deionized water at the needle tip, replacing the deionized water at the needle tip with the HAMA solution containing targeted drug-loaded nanoparticles. h. Place the microneedle mold filled with liquid in a 4°C refrigerator for 2-3 days to concentrate the HAMA solution to a non-flowing state. i. Take about 600 μl of microneedle base solution and add it to the microneedle mold. Shake it left and right and back and forth to make the base solution evenly distributed in the microneedles. Continue to place it in a refrigerator at 4°C for 1-2 days to allow the liquid to evaporate as completely as possible. j. Drying and curing the microneedles: Take the microneedle mold out of the 4℃ refrigerator and place it in a 37℃ oven to continue drying for 1-2 hours to ensure that the microneedles are completely dry and cured; k. Take out the completely dried microneedle mold and use tweezers to slowly remove the microneedles from around the mold.

8. The application of the targeted drug-loaded nanoparticle for treating rheumatoid arthritis according to claim 1, or the targeted drug-loaded nanoparticle prepared by the preparation method according to any one of claims 2-5, or the targeted drug-loaded microneedle according to claim 6, or the targeted drug-loaded microneedle prepared by the preparation method according to claim 7, in the preparation of products for treating rheumatoid arthritis.

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