Composition for treating osteoarthritis and microneedle patch loaded with composition

Abstract

The invention discloses a composition for treating osteoarthritis and a microneedle patch loaded with the composition. The composition comprises the following components: (a) an exosome derived from mesenchymal stem cells; (b) a pain-relieving and inflammation-diminishing drug; (c) a drug carrier; the mesenchymal stem cells are from bone marrow, the drug carrier comprises polycaprolactone and polydimethylsiloxane, and the pain-relieving and inflammation-diminishing drug comprises caulis sinomenii, kadsura pepper stem, angelica sinensis and safflower carthamus. The microneedle patch comprises a substrate and a microneedle which are integrally formed, the substrate is made of a matrix material, and the microneedle is made of a mixture of the matrix material and the composition for treating osteoarthritis; in the microneedle, the matrix material accounts for 10-20% of the total mass of the composition and the matrix material. The composition for treating osteoarthritis and the microneedle patch loaded with the composition are good in early-stage pain relieving and inflammation diminishing effects and good in long-term treatment effect, pain of patients with osteoarthritis is eliminated, and diseases of the patients are thoroughly and radically treated.

Description

Technical Field

[0001] The invention relates to the technical field of medicines and medicine manufacturing, and in particular to a composition for treating osteoarthritis and a microneedle patch loaded with the composition. Background Art

[0002] Microneedle patches for the treatment of osteoarthritis are an innovative transdermal drug delivery system that improves the efficiency of transdermal drug delivery by forming micropores on the skin. The matrix materials of this patch usually include biodegradable polymer materials such as sodium hyaluronate and chitosan, which not only provide the mechanical strength of the microneedles, but also help control the release of drugs. The advantages of microneedle patches are their non-invasiveness, painless operation, and ability to reduce the first-pass effect of drugs. At the same time, they can also be used to promote the repair of various tissues and organs. Research progress in recent years, including the development of bionic nanomedicines, has further enhanced the potential of microneedle patches in the treatment of osteoarthritis, providing patients with more effective and lasting treatment options through specific targeting and long-term sustained release.

[0003] Current microneedle patch products have problems such as poor initial analgesic and anti-inflammatory effects, poor long-term treatment effects, and the need to improve microneedle hemostasis and swelling. In addition, the biocompatibility is not very good. Based on this, it is urgent to develop a microneedle patch product with good initial and long-term treatment effects and good biocompatibility. Summary of the invention

[0004] The purpose of the present invention is to provide a composition for treating osteoarthritis and a microneedle patch loaded with the composition, which has good initial analgesic and anti-inflammatory effects and good long-term treatment effects, eliminates the pain of osteoarthritis patients, and allows the patients' symptoms to be completely cured.

[0005] The present invention provides a composition for treating osteoarthritis, comprising the following components:

[0006] (a) Exosomes derived from mesenchymal stem cells;

[0007] (b) analgesic and anti-inflammatory drugs;

[0008] (c) drug carrier;

[0009] Furthermore, the mesenchymal stem cells are one or more extracted from bone marrow, adipose tissue, umbilical cord blood, umbilical cord, placenta or dental pulp; the drug carrier is at least one of degradable biopolymer materials, silicone polymers, polysaccharide nanoparticles, synthetic polymer nanoparticles, silica nanoparticles, liposomes, and proteins.

[0010] Furthermore, the mesenchymal stem cells are derived from bone marrow; the degradable biopolymer material is one or more of polycaprolactone, cellulose, lignin, chitin, starch, protein, natural rubber, aliphatic polyester, polyester ether, polyphosphazene, polyorthoester, polycarbonate, polyanhydride, and polyamino acid; and the silicone polymer is one or more of polydimethylsiloxane, silicone oil, silicone resin, and silicone rubber.

[0011] Furthermore, the preparation method of exosomes derived from mesenchymal stem cells adopts one of the methods of ultracentrifugation, sucrose density gradient centrifugation, ultrafiltration, immunoaffinity capture, polymer co-precipitation, size exclusion chromatography, microfluidics technology, and drug pretreatment-based methods, and can also be prepared by other preparation methods of the prior art.

[0012] Furthermore, the drug carrier includes polycaprolactone and polydimethylsiloxane, and the mass ratio of polycaprolactone to polydimethylsiloxane is 10:1; the analgesic and anti-inflammatory drugs include Caulis Sinomenii, Caulis Piperis, Angelica sinensis, and Carthamus tinctorius, and the mass ratio of Caulis Sinomenii, Caulis Piperis, Angelica sinensis, and Carthamus tinctorius is 1:1:1:1.

[0013] Furthermore, the analgesic and anti-inflammatory drug adopts a traditional Chinese medicine formula, and the preparation method adopts the traditional Chinese medicine preparation method in the prior art.

[0014] Furthermore, the composition is a drug carrier encapsulated with exosomes derived from mesenchymal stem cells and analgesic and anti-inflammatory drugs, and the mass ratio of the exosomes derived from mesenchymal stem cells, the analgesic and anti-inflammatory drugs, and the drug carrier is (1.5-2): (0.1-0.5):1.

[0015] Mesenchymal stem cell exosomes carry a variety of therapeutically active proteins and nucleic acids, which can enhance repair ability and promote osteogenesis. However, their retention effect and persistence in pathological environments are not strong. They are not only easily lost, but also easily cleared quickly.

[0016] The analgesic and anti-inflammatory drugs use pure Chinese medicine formula with little side effects; Angelica sinensis: warm in nature, pungent and sweet in taste, enters the liver, heart and spleen meridians, has the effects of nourishing blood and regulating menstruation, promoting blood circulation and relieving pain, and is mainly used for dizziness, fatigue and palpitations caused by blood deficiency, weak qi and abdominal pain due to blood deficiency, scanty menstruation, amenorrhea, dysmenorrhea; traumatic injuries, rheumatic pain, sores, swelling and pain, etc.; Safflower: warm in nature, pungent in taste, enters the heart and liver meridians, has the effects of promoting blood circulation and relieving menstruation, dispersing blood stasis and relieving pain, and is mainly used to treat amenorrhea, dysmenorrhea, lochia retention, chest pain, abdominal pain due to blood stasis, stabbing pain in the chest and ribs; traumatic injuries, sores, swelling and pain, etc.; Caulis Sinomenii: neutral in nature, bitter in taste, enters the liver and spleen meridians. It has the effects of removing rheumatism and unblocking meridians, and is mainly used to treat rheumatoid arthritis, low back pain, swollen joints, numbness of limbs, etc.; Piper mesenteriae: It is slightly warm in nature, bitter and pungent in taste, and belongs to the liver meridian. It has the effects of removing rheumatism, unblocking meridians and relieving pain. It is mainly used for wind-cold and dampness arthritis, tendon spasm, and difficulty in flexion and extension. It is a commonly used medicine for the treatment of wind-cold and dampness arthritis, and can also treat bruises, swelling and pain; The above Chinese medicine formula is used in combination to reduce inflammation and relieve pain in the early stage, and relieve the patient's early symptoms and pain.

[0017] Polydimethylsiloxane (PDMS) is a widely used silicone material. Due to its excellent biocompatibility, air permeability and transparency, it has important applications in the manufacture of microneedle patch carriers. The flexibility and biocompatibility of PDMS make it an ideal microneedle carrier material, which can easily fix microneedles on the patch for drug delivery or biosensors; polycaprolactone is a semi-crystalline linear polyester with a low melting point and glass transition temperature. It can be copolymerized with a variety of polymers and is often used as a long-term drug controlled release carrier; polydimethylsiloxane is combined with polycaprolactone to form a drug carrier. While ensuring biocompatibility and air permeability, polydimethylsiloxane can fix microneedles on the patch with its flexibility and biocompatibility, with a stable structure and long-lasting treatment; polycaprolactone is often used as a long-term drug controlled release carrier. It is a degradable biopolymer material with stable performance, which further enhances the stable release of drugs and achieves the effect of long-term treatment.

[0018] Furthermore, the preparation method of the composition is:

[0019] (1) dissolving exosomes derived from mesenchymal stem cells in PBS to prepare an exosome solution; dissolving a drug carrier in a solvent to prepare a drug carrier solution;

[0020] (2) All components of the analgesic and anti-inflammatory drug are fully crushed and blended to form a solution.

[0021] (3) The exosome solution, the analgesic and anti-inflammatory drug, and the carrier solution are respectively loaded into the gas-tight syringes at both ends of the liposome extruder, and the two are combined by repeated co-extrusion to obtain a composite.

[0022] A microneedle patch loaded with the above-mentioned composition comprises an integrally formed substrate and microneedles, wherein the substrate is composed of a matrix material, and the microneedles are composed of a mixture of the matrix material and the composition for treating osteoarthritis; in the microneedles, the matrix material accounts for 10-20% of the total mass of the composition and the matrix material.

[0023] Furthermore, the matrix material includes: one or more of RADA16, sodium hyaluronate, sodium carboxymethyl cellulose, carboxymethyl chitosan, polyvinyl alcohol, cyclodextrin, hydroxypropyl β-cyclodextrin, polyoxyethylene fatty acid ester, and pectin.

[0024] Furthermore, the matrix material is RADA16.

[0025] Furthermore, the average molecular weight of RADA16 is 1600-1700 Da.

[0026] Furthermore, the average molecular weight of RADA16 is 1650Da.

[0027] Furthermore, the D-RADA16 self-assembling peptide can be synthesized by solid-phase peptide synthesis (SPPS). RADA16 is used as an artificial extracellular matrix, and RADA16 alone can slightly increase wound closure and re-epithelialization.

[0028] Furthermore, the preparation method of the microneedle patch comprises the following steps:

[0029] (1) Preparation of microneedles;

[0030] (2) The substrate and the microneedle array are integrated into one piece.

[0031] Furthermore, the preparation method of the microneedle comprises the following steps:

[0032] (1) forming microneedles comprising a biocompatible matrix in the shape of microneedles;

[0033] (2) coating the surface of the biocompatible matrix microneedle with exosomes derived from mesenchymal stem cells and analgesic and anti-inflammatory drugs.

[0034] Furthermore, the preparation method of the microneedle specifically comprises the following steps:

[0035] (1) Filling the matrix material of the biocompatible matrix into the array cavity in the shape of microneedles;

[0036] (2) demolding the microneedle array;

[0037] (3) Exosomes derived from mesenchymal stem cells and analgesic and anti-inflammatory drugs are coated on the surface of the biocompatible matrix microneedles, and a curing agent is coated for curing.

[0038] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0039] 1. The present invention provides a composition for treating osteoarthritis and a microneedle patch loaded with the composition. The composition for treating osteoarthritis is prepared by using exosomes derived from mesenchymal stem cells, analgesic and anti-inflammatory drugs, and a drug carrier with excellent biocompatibility. The components of the composition promote each other in efficacy and stabilize each other in structure, and can simultaneously achieve anti-inflammatory and repair of the pathological environment, improve and treat the pathological environment from the root, and effectively prolong the residence and lasting effect of the composition in the pathological environment. In addition, the addition of analgesic and anti-inflammatory drugs can reduce inflammation and relieve pain in the early stage, and relieve the patient's early symptoms and pain;

[0040] 2. The present invention provides a composition for treating osteoarthritis and a microneedle patch loaded with the composition. The composition is molded and administered in the form of microneedles, which achieves the local residence and fixed-point sustained-release drug delivery effects of the composition, while also improving the penetration of the drug into deeper tissues, allowing the drug to reach the lesion site faster and achieve lasting treatment. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with the embodiments. The following embodiments are only several specific embodiments of the present invention, but the design concept of the present invention is not limited thereto, and any non-substantial changes to the present invention using this concept shall be deemed as an act of infringing the protection scope of the present invention.

[0042] Example 1

[0043] In one embodiment, a composition for treating osteoarthritis comprises the following components:

[0044] (a) Exosomes derived from mesenchymal stem cells;

[0045] (b) analgesic and anti-inflammatory drugs;

[0046] (c) drug carrier;

[0047] The mesenchymal stem cells are one or more extracted from bone marrow, adipose tissue, umbilical cord blood, umbilical cord, placenta or dental pulp; the drug carrier is at least one of degradable biopolymer materials, silicone polymers, polysaccharide nanoparticles, synthetic polymer nanoparticles, silica nanoparticles, liposomes and proteins.

[0048] In one embodiment, the mesenchymal stem cells are derived from bone marrow; the degradable biopolymer material is one or more of polycaprolactone, cellulose, lignin, chitin, starch, protein, natural rubber, aliphatic polyester, polyester ether, polyphosphazene, polyorthoester, polycarbonate, polyanhydride, and polyamino acid; the silicone polymer is one or more of polydimethylsiloxane, silicone oil, silicone resin, and silicone rubber.

[0049] In one embodiment, the drug carrier includes polycaprolactone and polydimethylsiloxane, and the mass ratio of polycaprolactone to polydimethylsiloxane is 10:1; the analgesic and anti-inflammatory drugs include Caulis Sinomenii, Caulis Piperis, Angelica sinensis, and Carthamus tinctorius, and the mass ratio of Caulis Sinomenii, Caulis Piperis, Angelica sinensis, and Carthamus tinctorius is 1:1:1.

[0050] In one embodiment, the composition is a drug carrier encapsulated with exosomes derived from mesenchymal stem cells and analgesic and anti-inflammatory drugs, and the mass ratio of the exosomes derived from mesenchymal stem cells, the analgesic and anti-inflammatory drugs, and the drug carrier is (1.5-2): (0.1-0.5):1.

[0051] In one embodiment, a microneedle patch loaded with the above-mentioned composition comprises an integrally formed substrate and microneedles, characterized in that the substrate is composed of a matrix material, and the microneedles are composed of a mixture of the matrix material and the composition for treating osteoarthritis; in the microneedles, the matrix material accounts for 10-20% of the total mass of the composition and the matrix material.

[0052] In one embodiment, the matrix material includes: one or more of RADA16, sodium hyaluronate, sodium carboxymethyl cellulose, carboxymethyl chitosan, polyvinyl alcohol, cyclodextrin, hydroxypropyl β-cyclodextrin, polyoxyethylene fatty acid ester, and pectin.

[0053] In one embodiment, the matrix material is RADA16.

[0054] In one embodiment, the average molecular weight of RADA16 is 1600-1700 Da.

[0055] In one embodiment, the average molecular weight of RADA16 is 1650 Da.

[0056] Based on the above embodiments, several groups of microneedle patches and several comparison groups and a control group were prepared, and performance tests were performed. The performance tests included SEM and TEM tests, anti-osteoarthritis effect evaluation, pathological evaluation, and sustained-release performance evaluation.

[0057] 1. SEM and TEM testing: Use SEM and TEM to observe the appearance and internal structure of each group.

[0058] 2. Evaluation of anti-osteoarthritis effect: An animal model of knee osteoarthritis (OA) was established by performing anterior cruciate ligament transection (ACLT) on SD rats, and the therapeutic effect of microneedles on OA in different test cases was evaluated. Specifically, Sprague-Dawley (SD) rats weighing 180-220g were selected (purchased and bred at Tongji University, and the experimental protocol has been approved by the Animal Care and Use Committee of Tongji University). Under an appropriate amount of isoflurane anesthesia, the anterior cruciate ligament of the right leg of the SD rats was exposed and cut. In the sham operation group, the anterior cruciate ligament was exposed but not cut; the microneedle patches prepared in different embodiments were given 2 weeks after the operation, once every 2 days for 2 weeks; 8 weeks after the ACLT operation, the rats were sacrificed for further experiments. The micro CT images of the sagittal and coronal planes of the rat tibial plateau revealed the changes in the bone morphology of the lower joint in different groups. Tb BV / TV refers to the ratio of trabecular volume to total volume, and is an indicator for evaluating the number of trabeculae. After osteoarthritis modeling and treatment, Tb BV / TV increases, which means an increase in the number of trabeculae or an increase in bone tissue density, indicating an improvement in bone quality. Tb.SP refers to the trabecular spacing, which is an indicator for evaluating the tightness of trabecular arrangement. Tb.PF is the trabecular morphological factor, which is an indicator for evaluating the morphology of trabecular arrangement. In osteoarthritis, trabeculae may undergo abnormal fractures, sparse or irregular arrangement, and other pathological changes, leading to an increase in Tb.PF.

[0059] 3. Pathological evaluation: Pathological evaluation of rats: The joint structure and tissue pathology of different groups were observed by hematoxylin-eosin staining, toluidine blue staining and safranin fast green staining.

[0060] 4. Evaluation of sustained-release performance: In order to visualize the skin penetration of the drug, a microneedle patch containing FITC (fluorescent dye) was prepared. The microneedle patch was inserted into the skin of the miniature pig and kept for 3 minutes. The green fluorescence signal of FITC in the skin tissue was observed by confocal microscopy, and skin tissue images at different depths were collected by longitudinal sectioning.

[0061] Several groups of microneedle patches and several comparison groups and a control group were prepared, and the specific preparation conditions of each group were as described in the experimental example.

[0062] Test Example 1

[0063] This embodiment provides a composition for treating osteoarthritis, the composition comprising the following components:

[0064] (a) Exosomes derived from mesenchymal stem cells;

[0065] (b) analgesic and anti-inflammatory drugs;

[0066] (c) drug carrier;

[0067] The composition is a drug carrier encapsulated by exosomes derived from mesenchymal stem cells and analgesic and anti-inflammatory drugs, and the preparation method thereof is as follows:

[0068] (1) Preparation of exosomes from mesenchymal stem cells: S1. Cultivate bone marrow mesenchymal stem cells to 80% confluence and collect the culture medium; S2. Centrifuge the culture medium: first at 300g for 10 min, then at 2000g for 10 min, and then at 10000g for 30 min, and collect the supernatant; S3. Filter the supernatant using a 0.22 μm filter membrane, and then centrifuge at 100000g for 2 h, collect the precipitate to obtain exosomes;

[0069] (2) Preparation of analgesic and anti-inflammatory drugs: Weigh 100 g each of Caulis Sinomenii, Caulis Piperis, Angelica Sinensis, and Carthamus Tinctorius, fully grind and blend, and mix to prepare a 95% solution;

[0070] (3) Preparation of drug carrier: polycaprolactone and polydimethylsiloxane solution were mixed and stirred at a mass ratio of 10:1 at room temperature for 5 h, and then the liquid was centrifuged at 10000 rpm for 20 min, and the precipitate was collected;

[0071] (4) Preparation of the composition: The exosomes derived from mesenchymal stem cells are resuspended in sterile PBS to prepare a 1 mg / mL exosome solution derived from mesenchymal stem cells; the analgesic and anti-inflammatory drug is resuspended in sterile PBS to prepare a 1 mg / mL analgesic and anti-inflammatory drug solution; the drug carrier is suspended in ultrapure water to prepare a 1 mg / mL drug carrier solution; the exosome solution, the analgesic and anti-inflammatory drug solution, and the drug carrier solution are respectively loaded into the airtight syringes at both ends of the liposome extruder according to a volume ratio of 1.8:0.3:1, and a polycarbonate membrane with a pore size of 0.2 μm is set in the middle, and the two are repeatedly co-extruded through the liposome extruder to combine and obtain the composition.

[0072] This embodiment also provides a microneedle patch loaded with the composition; the microneedle patch comprises an integrally formed substrate and microneedles, the substrate is composed of a matrix material, and the microneedles are composed of a mixture of the matrix material and the composition for treating osteoarthritis; in the microneedles, the matrix material accounts for 15% of the total mass of the composition and the matrix material, and the total mass is 1000 g.

[0073] The matrix material is RADA16, and the average molecular weight of RADA16 is 1650Da;

[0074] The preparation method of the microneedle patch comprises the following steps:

[0075] (1) ultrasonically mixing the matrix material and 10 mL of the composition according to the corresponding mass fractions, and removing bubbles from the mixed solution by ultrasound;

[0076] (2) The mixed liquid is cast on the PDMS mold and vacuumed for 3 times, each time for 3 minutes. After the mixed liquid fills the mold cavity and forms a needle body, the matrix material is added to the mold to form a substrate, and then placed in a dryer at 25°C for 24 hours to form a needle.

[0077] The microneedle patch (1cm*1cm square sheet) is composed of 45 microneedles, the microneedles have a height of 1000±10μm, a base width of 900±10μm, a center space spacing of adjacent microneedles of 573±5μm, and a needle tip length of 800μm.

[0078] Test Example 2

[0079] This embodiment provides a composition for treating osteoarthritis, the composition comprising the following components:

[0080] (a) Exosomes derived from mesenchymal stem cells;

[0081] (b) analgesic and anti-inflammatory drugs;

[0082] (c) drug carrier;

[0083] The composition is a drug carrier encapsulated by exosomes derived from mesenchymal stem cells and analgesic and anti-inflammatory drugs, and the preparation method thereof is as follows:

[0084] (1) Preparation of exosomes from mesenchymal stem cells: S1. Cultivate bone marrow mesenchymal stem cells to 80% confluence and collect the culture medium; S2. Centrifuge the culture medium: first at 300g for 10 min, then at 2000g for 10 min, and then at 10000g for 30 min, and collect the supernatant; S3. Filter the supernatant using a 0.22 μm filter membrane, and then centrifuge at 100000g for 2 h, collect the precipitate to obtain exosomes;

[0085] (2) Preparation of analgesic and anti-inflammatory drugs: Weigh 100 g each of Caulis Sinomenii, Caulis Piperis, Angelica Sinensis, and Carthamus Tinctorius, fully grind and blend, and mix to prepare a 95% solution;

[0086] (3) Preparation of drug carrier: polycaprolactone and polydimethylsiloxane solution were mixed and stirred at a mass ratio of 10:1 at room temperature for 5 h, and then the liquid was centrifuged at 10000 rpm for 20 min, and the precipitate was collected;

[0087] (4) Preparation of the composition: The exosomes derived from mesenchymal stem cells are resuspended in sterile PBS to prepare a 1 mg / mL mesenchymal stem cell-derived exosome solution; the analgesic and anti-inflammatory drug is resuspended in sterile PBS to prepare a 1 mg / mL analgesic and anti-inflammatory drug solution; the drug carrier is suspended in ultrapure water to prepare a 1 mg / mL drug carrier solution; the exosome solution, the analgesic and anti-inflammatory drug solution, and the drug carrier solution are respectively loaded into the airtight syringes at both ends of a liposome extruder in a volume ratio of 1.5:0.1:1, a polycarbonate membrane with a pore size of 0.2 μm is set in the middle, and the two are repeatedly co-extruded through the liposome extruder to combine and obtain the composition.

[0088] This embodiment also provides a microneedle patch loaded with the composition; the microneedle patch comprises an integrally formed substrate and microneedles, the substrate is composed of a matrix material, and the microneedles are composed of a mixture of the matrix material and the composition for treating osteoarthritis; in the microneedles, the matrix material accounts for 15% of the total mass of the composition and the matrix material, and the total mass is 1000 g.

[0089] The matrix material is RADA16, and the average molecular weight of RADA16 is 1650Da;

[0090] The preparation method of the microneedle patch comprises the following steps:

[0091] (1) ultrasonically mixing the matrix material and 10 mL of the composition according to the corresponding mass fractions, and removing bubbles from the mixed solution by ultrasound;

[0092] (2) The mixed liquid is cast on the PDMS mold and vacuumed for 3 times, each time for 3 minutes. After the mixed liquid fills the mold cavity and forms a needle body, the matrix material is added to the mold to form a substrate, and then placed in a dryer at 25°C for 24 hours to form a needle.

[0093] The microneedle patch (1cm*1cm square sheet) is composed of 45 microneedles, the microneedles have a height of 1000±10μm, a base width of 900±10μm, a center space spacing of adjacent microneedles of 573±5μm, and a needle tip length of 800μm.

[0094] Test Example 3

[0095] This embodiment provides a composition for treating osteoarthritis, the composition comprising the following components:

[0096] (a) Exosomes derived from mesenchymal stem cells;

[0097] (b) analgesic and anti-inflammatory drugs;

[0098] (c) drug carrier;

[0099] The composition is a drug carrier encapsulated by exosomes derived from mesenchymal stem cells and analgesic and anti-inflammatory drugs, and the preparation method thereof is as follows:

[0100] (1) Preparation of exosomes from mesenchymal stem cells: S1. Cultivate bone marrow mesenchymal stem cells to 80% confluence and collect the culture medium; S2. Centrifuge the culture medium: first at 300g for 10 min, then at 2000g for 10 min, and then at 10000g for 30 min, and collect the supernatant; S3. Filter the supernatant using a 0.22 μm filter membrane, and then centrifuge at 100000g for 2 h, collect the precipitate to obtain exosomes;

[0101] (2) Preparation of analgesic and anti-inflammatory drugs: Weigh 100 g each of Caulis Sinomenii, Caulis Piperis, Angelica Sinensis, and Carthamus Tinctorius, fully grind and blend, and mix to prepare a 95% solution;

[0102] (3) Preparation of drug carrier: polycaprolactone and polydimethylsiloxane solution were mixed and stirred at a mass ratio of 10:1 at room temperature for 5 h, and then the liquid was centrifuged at 10000 rpm for 20 min, and the precipitate was collected;

[0103] (4) Preparation of the composition: The exosomes derived from mesenchymal stem cells are resuspended in sterile PBS to prepare a 1 mg / mL exosome solution derived from mesenchymal stem cells; the analgesic and anti-inflammatory drug is resuspended in sterile PBS to prepare a 1 mg / mL analgesic and anti-inflammatory drug solution; the drug carrier is suspended in ultrapure water to prepare a 1 mg / mL drug carrier solution; the exosome solution, the analgesic and anti-inflammatory drug solution, and the drug carrier solution are respectively loaded into the airtight syringes at both ends of the liposome extruder in a volume ratio of 2:0.5:1, and a polycarbonate membrane with a pore size of 0.2 μm is set in the middle, and the two are repeatedly co-extruded through the liposome extruder to combine and obtain the composition.

[0104] This embodiment also provides a microneedle patch loaded with the composition; the microneedle patch comprises an integrally formed substrate and microneedles, the substrate is composed of a matrix material, and the microneedles are composed of a mixture of the matrix material and the composition for treating osteoarthritis; in the microneedles, the matrix material accounts for 15% of the total mass of the composition and the matrix material, and the total mass is 1000 g.

[0105] The matrix material is RADA16, and the average molecular weight of RADA16 is 1650Da;

[0106] The preparation method of the microneedle patch comprises the following steps:

[0107] (1) ultrasonically mixing the matrix material and 10 mL of the composition according to the corresponding mass fractions, and removing bubbles from the mixed solution by ultrasound;

[0108] (2) The mixed liquid is cast on the PDMS mold and vacuumed for 3 times, each time for 3 minutes. After the mixed liquid fills the mold cavity and forms a needle body, the matrix material is added to the mold to form a substrate, and then placed in a dryer at 25°C for 24 hours to form a needle.

[0109] The microneedle patch (1cm*1cm square sheet) is composed of 45 microneedles, the microneedles have a height of 1000±10μm, a base width of 900±10μm, a center space spacing of adjacent microneedles of 573±5μm, and a needle tip length of 800μm.

[0110] Test Example 4

[0111] This embodiment provides a composition for treating osteoarthritis, the composition comprising the following components:

[0112] (a) Exosomes derived from mesenchymal stem cells;

[0113] (b) analgesic and anti-inflammatory drugs;

[0114] (c) drug carrier;

[0115] The composition is a drug carrier encapsulated by exosomes derived from mesenchymal stem cells and analgesic and anti-inflammatory drugs, and the preparation method thereof is as follows:

[0116] (1) Preparation of exosomes from mesenchymal stem cells: S1. Cultivate bone marrow mesenchymal stem cells to 80% confluence and collect the culture medium; S2. Centrifuge the culture medium: first at 300g for 10 min, then at 2000g for 10 min, and then at 10000g for 30 min, and collect the supernatant; S3. Filter the supernatant using a 0.22 μm filter membrane, and then centrifuge at 100000g for 2 h, collect the precipitate to obtain exosomes;

[0117] (2) Preparation of analgesic and anti-inflammatory drugs: Weigh 100 g each of Caulis Sinomenii, Caulis Piperis, Angelica Sinensis, and Carthamus Tinctorius, fully grind and blend, and mix to prepare a 95% solution;

[0118] (3) Preparation of drug carrier: polycaprolactone and polydimethylsiloxane solution were mixed and stirred at a mass ratio of 10:1 at room temperature for 5 h, and then the liquid was centrifuged at 10000 rpm for 20 min, and the precipitate was collected;

[0119] (4) Preparation of the composition: The exosomes derived from mesenchymal stem cells are resuspended in sterile PBS to prepare a 1 mg / mL exosome solution derived from mesenchymal stem cells; the analgesic and anti-inflammatory drug is resuspended in sterile PBS to prepare a 1 mg / mL analgesic and anti-inflammatory drug solution; the drug carrier is suspended in ultrapure water to prepare a 1 mg / mL drug carrier solution; the exosome solution, the analgesic and anti-inflammatory drug solution, and the drug carrier solution are respectively loaded into the airtight syringes at both ends of the liposome extruder according to a volume ratio of 1.8:0.3:1, and a polycarbonate membrane with a pore size of 0.2 μm is set in the middle, and the two are repeatedly co-extruded through the liposome extruder to combine and obtain the composition.

[0120] This embodiment also provides a microneedle patch loaded with the composition; the microneedle patch comprises an integrally formed substrate and microneedles, the substrate is composed of a matrix material, and the microneedles are composed of a mixture of the matrix material and the composition for treating osteoarthritis; in the microneedles, the matrix material accounts for 10% of the total mass of the composition and the matrix material, and the total mass is 1000 g.

[0121] The matrix material is RADA16, and the average molecular weight of RADA16 is 1650Da;

[0122] The preparation method of the microneedle patch comprises the following steps:

[0123] (1) ultrasonically mixing the matrix material and 10 mL of the composition according to the corresponding mass fractions, and removing bubbles from the mixed solution by ultrasound;

[0124] (2) The mixed liquid is cast on the PDMS mold and vacuumed for 3 times, each time for 3 minutes. After the mixed liquid fills the mold cavity and forms a needle body, the matrix material is added to the mold to form a substrate, and then placed in a dryer at 25°C for 24 hours to form a needle.

[0125] The microneedle patch (1cm*1cm square sheet) is composed of 45 microneedles, the microneedles have a height of 1000±10μm, a base width of 900±10μm, a center space spacing of adjacent microneedles of 573±5μm, and a needle tip length of 800μm.

[0126] Test Example 5

[0127] This embodiment provides a composition for treating osteoarthritis, the composition comprising the following components:

[0128] (a) Exosomes derived from mesenchymal stem cells;

[0129] (b) analgesic and anti-inflammatory drugs;

[0130] (c) drug carrier;

[0131] The composition is a drug carrier encapsulated by exosomes derived from mesenchymal stem cells and analgesic and anti-inflammatory drugs, and the preparation method thereof is as follows:

[0132] (1) Preparation of exosomes from mesenchymal stem cells: S1. Cultivate bone marrow mesenchymal stem cells to 80% confluence and collect the culture medium; S2. Centrifuge the culture medium: first at 300g for 10 min, then at 2000g for 10 min, and then at 10000g for 30 min, and collect the supernatant; S3. Filter the supernatant using a 0.22 μm filter membrane, and then centrifuge at 100000g for 2 h, collect the precipitate to obtain exosomes;

[0133] (2) Preparation of analgesic and anti-inflammatory drugs: Weigh 100 g each of Caulis Sinomenii, Caulis Piperis, Angelica Sinensis, and Carthamus Tinctorius, fully grind and blend, and mix to prepare a 95% solution;

[0134] (3) Preparation of drug carrier: polycaprolactone and polydimethylsiloxane solution were mixed and stirred at a mass ratio of 10:1 at room temperature for 5 h, and then the liquid was centrifuged at 10000 rpm for 20 min, and the precipitate was collected;

[0135] (4) Preparation of the composition: The exosomes derived from mesenchymal stem cells are resuspended in sterile PBS to prepare a 1 mg / mL exosome solution derived from mesenchymal stem cells; the analgesic and anti-inflammatory drug is resuspended in sterile PBS to prepare a 1 mg / mL analgesic and anti-inflammatory drug solution; the drug carrier is suspended in ultrapure water to prepare a 1 mg / mL drug carrier solution; the exosome solution, the analgesic and anti-inflammatory drug solution, and the drug carrier solution are respectively loaded into the airtight syringes at both ends of the liposome extruder according to a volume ratio of 1.8:0.3:1, and a polycarbonate membrane with a pore size of 0.2 μm is set in the middle, and the two are repeatedly co-extruded through the liposome extruder to combine and obtain the composition.

[0136] This embodiment also provides a microneedle patch loaded with the composition; the microneedle patch comprises an integrally formed substrate and microneedles, the substrate is composed of a matrix material, and the microneedles are composed of a mixture of the matrix material and the composition for treating osteoarthritis; in the microneedles, the matrix material accounts for 20% of the total mass of the composition and the matrix material, and the total mass is 1000g.

[0137] The matrix material is RADA16, and the average molecular weight of RADA16 is 1650Da;

[0138] The preparation method of the microneedle patch comprises the following steps:

[0139] (1) ultrasonically mixing the matrix material and 10 mL of the composition according to the corresponding mass fractions, and removing bubbles from the mixed solution by ultrasound;

[0140] (2) The mixed liquid is cast on the PDMS mold and vacuumed for 3 times, each time for 3 minutes. After the mixed liquid fills the mold cavity and forms a needle body, the matrix material is added to the mold to form a substrate, and then placed in a dryer at 25°C for 24 hours to form a needle.

[0141] The microneedle patch (1cm*1cm square sheet) is composed of 45 microneedles, the microneedles have a height of 1000±10μm, a base width of 900±10μm, a center space spacing of adjacent microneedles of 573±5μm, and a needle tip length of 800μm.

[0142] Comparison group 1

[0143] The control group 1 is exactly the same as the test group 1, with the only difference being that in the microneedles of the control group 1, the composition for treating osteoarthritis contains only exosomes derived from mesenchymal stem cells, and the total mass of the composition for treating osteoarthritis is consistent with that of the test group 1.

[0144] Comparison group 2

[0145] The control group 2 is exactly the same as the test group 1, with the only difference being that among the microneedles in the control group 1, the composition for treating osteoarthritis only contains analgesic and anti-inflammatory drugs, and the total mass of the composition for treating osteoarthritis is consistent with that of the test group 1.

[0146] Control group 1

[0147] The difference between the control group 1 and the test group 1 is that the microneedles of the control group 1 are not added with the composition for treating osteoarthritis.

[0148] The above test group, comparison group and control group were subjected to performance tests, and the results were analyzed as follows:

[0149] 1. SEM and TEM tests: The results of SEM tests showed that the microneedles in the test groups 1-5 and the control groups 1-2 were conical in shape, uniform in size and sharp in tip; the TEM results showed that the microneedles in the test groups 1-5 and the control groups 1-2 were spherical and irregular in shape as a whole, with external substances wrapping the internal spheres, indicating that the drug carriers were wrapped by analgesic and anti-inflammatory drugs and / or exosomes derived from mesenchymal stem cells.

[0150] 2. Evaluation of anti-osteoarthritis effect: (1) In the sham operation group, the lower joint bones of the osteoarthritis model rats underwent severe bone resorption, showing sparse trabeculae; while in Test Examples 1-5 and Comparative Group 1, the morphology of the lower joint bones was improved and almost restored to the level of normal rats in the sham operation group; (2) After osteoarthritis modeling and treatment, in Test Examples 1-5 and Comparative Group 1, TbBV / TV increased to a level close to that of the control group, which means an increase in the number of trabeculae or an increase in bone tissue density, indicating an improvement in bone quality; Tb.SP was significantly increased in the osteoarthritis positive control group, indicating osteophyte formation, and in Test Examples 1-5 and Comparative Group 1, after the osteoarthritis model was treated with microneedles, Tb.SP was significantly reduced compared with other groups, indicating a reduction in osteophyte formation, and Tb.Pf was reduced in this group, indicating that the microneedles of the composition delayed the progression of osteoarthritis. It is worth noting that the control group 1 had obvious irritability in the first week, while the control group 2 did not achieve the above effect, but like test examples 1-5, there was no obvious irritability, indicating that the analgesic and anti-inflammatory drugs in the composition played a role.

[0151] In summary, it was observed in the rat osteoarthritis model that the microneedles using the composition for treating osteoarthritis of the present invention had a good therapeutic effect, effectively alleviated the progression of osteoarthritis, and helped to restore normal bone morphology.

[0152] 3. Pathological evaluation: The sham operation group showed normal joint structure and tissue, while the control group 1 showed pathological characteristics of irregular articular cartilage, bone hyperplasia and inflammatory cell infiltration. In contrast, the knee joints of rats in test examples 1-5 and control group 1 showed varying degrees of pathological improvement, among which test example 1 had the best effect.

[0153] 4. Sustained-release performance evaluation: The diffusion depth of FITC in the microneedle patch of Experimental Examples 1-5 in the skin exceeds 150 microns, showing an excellent transdermal penetration effect, indicating that the microneedle patch of Experimental Examples 1-5 can accurately deliver effective substances to the bone joints for treatment. In addition, the continuous fluorescence signal also shows that the microneedle patch has an excellent sustained-release effect.

[0154] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A composition for treating osteoarthritis, characterized in that: The composition comprises the following components: (a) Exosomes derived from mesenchymal stem cells; (b) analgesic and anti-inflammatory drugs; (c) drug carrier; The mesenchymal stem cells are one or more extracted from bone marrow, adipose tissue, umbilical cord blood, umbilical cord, placenta or dental pulp; the drug carrier is at least one of degradable biopolymer materials, silicone polymers, polysaccharide nanoparticles, synthetic polymer nanoparticles, silica nanoparticles, liposomes and proteins.

2. The composition according to claim 1, characterized in that The mesenchymal stem cells come from bone marrow; the degradable biopolymer material is one or more of polycaprolactone, cellulose, lignin, chitin, starch, protein, natural rubber, aliphatic polyester, polyester ether, polyphosphazene, polyorthoester, polycarbonate, polyanhydride, and polyamino acid; the silicone polymer is one or more of polydimethylsiloxane, silicone oil, silicone resin, and silicone rubber.

3. The composition according to claim 2, characterized in that The drug carrier includes polycaprolactone and polydimethylsiloxane, and the mass ratio of polycaprolactone to polydimethylsiloxane is 10:1; the analgesic and anti-inflammatory drugs include Caulis Sinomenii, Caulis Piperis, Chinese Angelica, and Carthamus tinctorius, and the mass ratio of Caulis Sinomenii, Caulis Piperis, Chinese Angelica, and Carthamus tinctorius is 1:1:1:

1.

4. The composition according to claim 3, characterized in that The composition is a drug carrier encapsulated by exosomes derived from mesenchymal stem cells and analgesic and anti-inflammatory drugs, and the mass ratio of the exosomes derived from mesenchymal stem cells, the analgesic and anti-inflammatory drugs, and the drug carrier is (1.5-2): (0.1-0.5):

1.

5. A microneedle patch loaded with the composition of any one of claims 1 to 4, comprising an integrally formed substrate and microneedles, characterized in that: The substrate is composed of a matrix material, and the microneedles are composed of a mixture of the matrix material and the composition for treating osteoarthritis; in the microneedles, the matrix material accounts for 10-20% of the total mass of the composition and the matrix material.

6. The microneedle patch according to claim 5, characterized in that: The matrix material comprises: one or more of RADA16, sodium hyaluronate, sodium carboxymethyl cellulose, carboxymethyl chitosan, polyvinyl alcohol, cyclodextrin, hydroxypropyl β-cyclodextrin, polyoxyethylene fatty acid ester and pectin.

7. The microneedle patch according to claim 6, characterized in that: The matrix material is RADA16.

8. The microneedle patch according to claim 7, characterized in that: The average molecular weight of RADA16 is 1600-1700 Da.

9. The microneedle patch according to claim 8, characterized in that: The average molecular weight of RADA16 is 1650 Da.