KGN-loaded nano-vesicle, soluble microneedle and preparation method and application of KGN-loaded nano-vesicle and soluble microneedle

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a KGN-loaded nano vesicle, a soluble microneedle and a preparation method and application of the KGN-loaded nano vesicle and the soluble microneedle. The preparation method comprises the following steps: loading KGN into mesenchymal stem cell membrane nano vesicles by adopting a co-extrusion method to obtain KGN-atNVs; and then integrating the KGN-NVs into the dissolvable microneedle by adopting a micro-molding method to obtain the KGN-NVs-loaded dissolvable microneedle. The soluble microneedle loaded with the KGN-atNVs can effectively reduce expression of inflammatory factors such as IL-1beta and TNF-alpha, reduce joint inflammation, protect a cartilage structure, promote cartilage repair and improve joint functions. According to the KGNN-NV delivery system based on the dissolvable microneedle, the immune regulation and drug delivery advantages of the mesenchymal stem cell membrane nanovesicles and the percutaneous delivery characteristic of the dissolvable microneedle can be synergistically exerted, and an innovative and practical solution is provided for clinical treatment of arthritis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to a KGN-loaded nanovesicle, a dissolving microneedle, and a preparation method and application thereof. Background Art

[0002] Rheumatoid arthritis (RA) is a common autoimmune disease characterized by chronic inflammation and progressive cartilage damage. Its pathological process involves matrix metalloproteinase (MMP)-mediated cartilage degradation and chondrocyte degeneration, which is further exacerbated by the release of pro-inflammatory cytokines and macrophage activation. Over time, these pathological changes lead to irreversible joint structure damage and loss of function. Rheumatoid arthritis seriously affects the quality of life of patients and brings a heavy burden to the global social economy. Therefore, developing a treatment strategy that can simultaneously regulate inflammation and promote cartilage regeneration has important application prospects for the treatment of RA.

[0003] Kartogenin (KGN) is a small molecule compound and a promising candidate drug for the treatment of RA. KGN promotes the differentiation of mesenchymal stem cells (MSCs) into chondrocytes through the CBF-β-RUNX1 signaling pathway and simultaneously regulates pro-inflammatory cytokines, thereby supporting cartilage repair and improving the inflammatory microenvironment. However, the intra-articular injection of KGN is limited by the high dose requirement and rapid clearance by the body system, making it difficult to achieve the ideal therapeutic effect. Therefore, it is necessary to develop a safe and efficient drug delivery system to fully exert its therapeutic potential.

[0004] MSCs have attracted much attention because they can differentiate into osteoblasts, adipocytes, and chondrocytes, and regulate inflammation and cartilage damage in RA. However, their clinical application is restricted by challenges such as difficult isolation, in vitro senescence, and limited proliferation ability. Nanovesicles (NVs) derived from MSCs well overcome these problems. Nanovesicles derived from MSCs not only inherit the biological activity of the original stem cells but also possess strong inflammatory targeting ability, good biocompatibility, biodegradability, low immunogenicity, and can be mass-produced by methods such as the extrusion method. These characteristics make NVs promising carriers in RA drug delivery.

[0005] Dissolvable microneedles (DMNs), as an innovative transdermal drug delivery technology, have gradually attracted attention in the treatment of RA. DMNs can be made of biodegradable water-soluble polymers, enhance drug permeability by forming microchannels on the skin, and can be completely dissolved after insertion, avoiding the risk of secondary injury. Due to their minimally invasive, painless, low-cost, and easy large-scale production characteristics, DMNs provide a competitive alternative to intra-articular injection. Some existing technologies have reported drug-loaded microneedles for the treatment of rheumatoid arthritis. For example, the Chinese invention patent with the publication number CN116869916A discloses a drug-loaded hyaluronic acid microneedle and its preparation method and application. This drug-loaded hyaluronic acid microneedle carries two drugs, methotrexate and diclofenac, at the same time. Another example is that the Chinese invention patent with the publication number CN109528695B discloses a microneedle transdermal drug delivery patch for the treatment of rheumatoid arthritis and its preparation method. The active drug ingredients in this microneedle are non-steroidal anti-inflammatory drugs, polypeptide drugs, symptom-improving drugs, or natural drug extracts. Summary of the Invention

[0006] In view of this, aiming at the dual challenges of synovial inflammation and cartilage damage in RA, as well as the high-dose requirement and rapid clearance problems of KGN intra-articular injection, the present invention proposes a drug-loaded nanovesicle loaded with KGN, a dissolvable microneedle loaded with KGN@NVs, and their preparation methods and applications. The present invention loads KGN into mesenchymal stem cell membrane nanovesicles to obtain drug-loaded NVs (KGN@NVs); and combines KGN@NVs with DMNs, which can synergistically exert the immune regulation and drug delivery advantages of NVs and the transdermal delivery characteristics of DMNs, so as to achieve targeted drug delivery, enhance the therapeutic effect, reduce side effects, and improve patient compliance.

[0007] One of the purposes of the present invention is to provide a drug-loaded nanovesicle for the treatment of rheumatoid arthritis.

[0008] To achieve the above purpose, the present invention adopts the following technical solutions:

[0009] A drug-loaded nanovesicle for the treatment of rheumatoid arthritis, wherein kartogenin is loaded inside the drug-loaded nanovesicle, and the nanovesicle is derived from mesenchymal stem cells.

[0010] Preferably, the particle size of the drug-loaded nanovesicle is 150 - 250 nm, more preferably 180 - 220 nm.

[0011] Preferably, the particle size of the nanovesicle is 150 - 200 nm.

[0012] Another purpose of the present invention is to provide a preparation method of the aforementioned drug-loaded nanovesicle.

[0013] To achieve the above object, the present invention adopts the following technical solutions:

[0014] A method for preparing drug-loaded nanovesicles, wherein kartogenin and nanovesicles derived from cell membranes are co-extruded to obtain KGN@NVs.

[0015] Preferably, the volume ratio of kartogenin to the nanovesicles is 0.8 - 1.5:1; the concentration of kartogenin is 5 - 20 μM.

[0016] More preferably, the volume ratio of kartogenin to the nanovesicles is 1:1.

[0017] More preferably, the concentration of kartogenin is 10 μM.

[0018] Preferably, kartogenin and the nanovesicles derived from cell membranes are extruded through a polycarbonate membrane, and the number of extrusion times is 10 - 20 times.

[0019] More preferably, the pore size of the polycarbonate membrane is 200 nm.

[0020] More preferably, the number of extrusion times is 15 times.

[0021] As a preferred scheme, the nanovesicles loaded with kartogenin are prepared by the following method: the nanovesicles are mixed with a 10 μM kartogenin solution in a ratio of 1:1 and oscillated on ice; extruded 15 times through a 200 nm polycarbonate membrane to obtain KGN@NVs.

[0022] As a preferred scheme, the nanovesicles derived from cell membranes are prepared by the following method:

[0023] 1) Extract cell membranes;

[0024] 2) Mix the membrane fragments extracted in step 1) with PBS, perform ultrasonic treatment on ice, and then extrude using a polycarbonate membrane to obtain NVs.

[0025] Preferably, in step 2), the ultrasonicated solution is sequentially extruded through polycarbonate membranes with pore sizes of 400 nm and 200 nm for 12 - 20 times, and most preferably 15 times.

[0026] The third object of the present invention is to provide a dissolving microneedle loaded with KGN@NVs.

[0027] To achieve the above object, the present invention adopts the following technical solutions:

[0028] Dissolving microneedles loaded with KGN@NVs, wherein the active ingredient in the dissolving microneedles is the aforementioned drug-loaded nanovesicles.

[0029] A fourth object of the present invention is to provide a method for preparing the aforementioned dissolving microneedles loaded with KGN@NVs.

[0030] To achieve the above object, the present invention adopts the following technical solutions:

[0031] A method for preparing dissolving microneedles loaded with KGN@NVs, comprising the following steps:

[0032] (1) Prepare KGN@NVs by the aforementioned method;

[0033] (2) Dissolve hyaluronic acid in the KGN@NVs solution obtained in step (1) to obtain a KGN@NVs-HA solution;

[0034] (3) Uniformly coat the KGN@NVs-HA solution obtained in step (2) on a microneedle mold, and centrifuge to fill the solution into the tips of the microneedles; then use the HA solution to prepare the microneedle base, and after centrifugation, drying, and demolding, obtain the dissolving microneedles loaded with KGN@NVs.

[0035] Preferably, in step (3), the concentration of the HA solution is 8%-20%.

[0036] More preferably, in step (3), the concentration of the HA solution is 10%.

[0037] Preferably, in step (3), the dosage of the HA solution is 350-500 μL, preferably 400 μL.

[0038] Preferably, in step (2), the dosage ratio of HA to KGN@NVs is 1 g: 8-15 mL, more preferably 1 g: 10 mL.

[0039] Preferably, in step (2), the reaction conditions are: oscillating incubation at 35-40 °C for 3-6 hours, more preferably oscillating incubation at 37 °C for 4 hours.

[0040] Preferably, in step (3), centrifuge at 3500-5000 rpm for 1-5 minutes to fill the solution into the tips of the microneedles, more preferably centrifuge at 4200 rpm for 1 minute to fill the solution into the tips of the microneedles.

[0041] Preferably, in step (3), the drying temperature is 35-50 °C, more preferably 40 °C.

[0042] A fifth object of the present invention is to provide an application of the aforementioned drug-loaded nanovesicles and / or the aforementioned dissolving microneedles loaded with KGN@NVs in the preparation of a drug for treating rheumatoid arthritis.

[0043] The sixth object of the present invention is to provide an application of the aforementioned drug-loaded nanovesicles and / or the aforementioned soluble microneedles loaded with KGN@NVs in the preparation of drugs for anti-inflammatory, immunomodulatory, protecting articular cartilage and / or promoting cartilage repair.

[0044] To achieve the above object, the present invention adopts the following technical solutions:

[0045] The application of the aforementioned drug-loaded nanovesicles and / or the aforementioned soluble microneedles loaded with KGN@NVs in the preparation of drugs for anti-inflammatory, immunomodulatory, protecting articular cartilage and / or promoting cartilage repair.

[0046] Preferably, the drug regulates the inflammatory microenvironment of RA by inhibiting the expression of IL-1β and TNF-α, relieves the inflammatory response, and blocks cartilage damage caused by inflammation while doing so.

[0047] The beneficial effects of the present invention are as follows:

[0048] 1. The present invention proposes an innovative treatment strategy that uses soluble microneedles to deliver mesenchymal stem cell membrane nanovesicles loaded with KGN to achieve targeted treatment of rheumatoid arthritis. KGN is a small molecule known to promote chondrogenesis, and nanovesicles derived from stem cell membranes have the advantages of enhancing drug delivery efficiency and biocompatibility. The present invention first loads KGN using nanovesicles derived from mesenchymal stem cell membranes, and then integrates KGN@NVs into soluble microneedles to achieve transdermal delivery. This method has the advantages of minimally invasive and painless, and significantly improves the treatment effect. This dual-functional delivery system co-regulates the inflammatory microenvironment by inhibiting pro-inflammatory cytokines, and at the same time promotes chondrogenesis, effectively relieving synovial inflammation and cartilage damage caused by RA.

[0049] 2. The present invention uses the co-extrusion method to prepare KGN@NVs, showing high drug loading efficiency, excellent encapsulation rate and long-term stability. Research shows that the average particle size of KGN@NVs is about 220 nm, with a stable zeta potential, and the stability of KGN@NVs under physiological conditions exceeds 4 days, ensuring continuous drug release.

[0050] 3. The in vitro and in vivo experimental results show that KGN@NV-MN can effectively reduce the expression of inflammatory factors (IL-1β and TNF-α), reduce joint inflammation, protect cartilage structure, promote cartilage repair, and improve joint function. The KGN@NV delivery system based on soluble microneedles exhibits efficient drug release and excellent treatment effects, has good biocompatibility and patient compliance, and is expected to become a powerful tool for the treatment of RA in the future. Description of the Drawings

[0051] Figure 1 Schematic diagram of using a hyaluronic acid microneedle patch to deliver kartogenin-loaded nanovesicles for the treatment of rheumatoid arthritis.

[0052] Figure 2 Schematic diagram of preparing nanovesicles by membrane extraction and extrusion method.

[0053] Figure 3 Morphological characterization results of mesenchymal stem cell-derived nanovesicles; among them, Figure 3 -A is the fluorescence microscope image of MSC cell membrane stained with Dil, and the scale bar is 100 nm; Figure 3 -B is the transmission electron microscope image of MSC cell membrane, and the scale bar is 100 nm; Figure 3 -C is the confocal laser scanning microscope image, and the scale bar is 2 μm; Figure 3 -D is the TEM image of nanovesicles, and the scale bar is 100 nm.

[0054] Figure 4 Schematic diagram of the preparation of KGN@NVs.

[0055] Figure 5 Characterization results of KGN@NVs; among them, Figure 5 -A is the Zeta potential analysis result graph of NVs and KGN@NVs; Figure 5 -B is the hydrodynamic diameter distribution result graph of NVs and KGN@NVs measured by dynamic light scattering; Figure 5 -C is the stability test result graph of KGN@NVs in PBS.

[0056] Figure 6 Drug loading performance test results of nanovesicles at different concentrations of KGN; among them, Figure 6 -A is the graph of the maximum drug loading amount of nanovesicles at different concentrations of KGN; Figure 6 -B is the encapsulation efficiency of nanovesicles at different concentrations of KGN.

[0057] Figure 7 Schematic diagram of preparing microneedles by micro-molding method.

[0058] Figure 8 Overall optical photograph and scanning electron microscope image of KGN@NV-MN; among them, Figure 8 -A is the overall optical photograph of KGN@NV-MN; Figure 8 -B is the SEM image of KGN@NV-MN.

[0059] Figure 9 Fluorescence microscope image, drug release amount and dissolution performance analysis results of KGN@NV-MN; among them, Figure 9-A is the fluorescence microscope image of KGN@NV-MN; Figure 9 -B is the graph of the drug release amount of KGN@NV-MN at different time points (15 and 30 minutes) in PBS; Figure 9 -C is the statistical analysis result graph of the microneedle dissolution performance.

[0060] Figure 10 is the dissolution process graph of the microneedle morphology; among them, Figure 10 -A is the microneedle morphology graph at 0 min; Figure 10 -B is the microneedle morphology graph at 1 min; Figure 10 -C is the microneedle morphology graph at 3 min; Figure 10 -D is the microneedle morphology graph at 5 min; Figure 10 -E is the microneedle morphology graph at 7 min; Figure 10 -F is the microneedle morphology graph at 9 min.

[0061] Figure 11 is the schematic diagram of the experimental procedure for studying the therapeutic effect of KGN@NV-MN.

[0062] Figure 12 are the representative images of the paws of rats in each group.

[0063] Figure 13 are the graphs of the changes in the foot thickness of rats in each group during the treatment process. The results are expressed as mean ± standard deviation, n = 5, *p < 0.05, **p < 0.01, compared with the Control group.

[0064] Figure 14 are the graphs of the clinical scores of rats in each group. The results are expressed as mean ± standard deviation, n = 5, *p < 0.05, **p < 0.01, compared with the Control group.

[0065] Figure 15 are the hematological parameters of rats on the 20th day after KGN@NV-MN treatment; among them, Figure 15 -A is the graph of the red blood cell (RBC) count result; Figure 15 -B is the graph of the white blood cell (WBC) count result; Figure 15 -C is the graph of the detection result of the platelet (PLT) level; Figure 15 -D is the graph of the detection result of the granulocyte level; Figure 15 -E is the graph of the detection result of the lymphocyte ratio; Figure 15 -F is the analysis result graph of the monocyte ratio; The results are expressed as mean ± standard deviation, n = 5, ns: no significant difference, *p < 0.05, **p < 0.01.

[0066] Figure 16 are the H&E staining images of the rat paw tissues, and the scale bar is 100 μm.

[0067] Figure 17 It is a safranin O-fast green (SO / FG) staining image of rat paw tissues, and the scale bar is 100 μm.

[0068] Figure 18 It is a graph showing the ELISA test results of the IL-1β levels in the sera of rats in each group. The results are expressed as mean ± standard deviation, n = 5, ns: no significant difference, *p < 0.05, **p < 0.01.

[0069] Figure 19 It is a graph showing the ELISA test results of the TNF-α levels in the sera of rats in each group. The results are expressed as mean ± standard deviation, n = 5, ns: no significant difference, *p < 0.05, **p < 0.01. Detailed implementation manners

[0070] The technical solutions of the present invention will be further clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts fall within the protection scope of the present invention.

[0071] In view of the dual challenges of synovial inflammation and cartilage damage in RA, the present invention designs a dissolvable microneedle patch, which encapsulates MSC-derived nanovesicles loaded with kartogenin (KGN@NVs). As Figure 1 shown, the degradable microneedle patch realizes the percutaneous delivery of kartogenin-loaded nanovesicles into the joint cavity. This microneedle system integrates the immunomodulatory and drug delivery functions of MSC-derived NVs with the efficient percutaneous delivery ability of DMNs. And through in vitro and in vivo experiments, its efficacy in regulating inflammation and promoting cartilage repair is verified. The controlled release of nanovesicles not only regulates the inflammatory microenvironment, but also enhances the chondrogenic activity, promoting cartilage regeneration while inhibiting inflammation. The present invention provides a new treatment paradigm for the treatment of RA.

[0072] In the embodiments of the present invention, the methods for blood analysis and serum cytokine measurement: After the experiment, blood (500 μL per portion) is collected and placed in an anticoagulant tube, and a blood analyzer is used to analyze red blood cells, white blood cells, platelets and white blood cell subsets (lymphocytes, neutrophils and monocytes). ELISA kits are used to detect the levels of pro-inflammatory cytokines TNF-α and IL-1β in the sera, and the operations follow the manufacturer's protocols.

[0073] Example 1. Preparation and morphological characterization of mesenchymal stem cell-derived nanovesicles

[0074] 1. Extraction of cell membrane

[0075] Culture mesenchymal stem cells (MSCs) in a cell culture flask until about 90% confluence. Use a cell scraper to separate the MSCs and centrifuge the cells at 2000 rpm for 3 minutes to collect the cells. Lyse the cells on ice for 10 - 15 minutes using a membrane protein extraction reagent containing PMSF. Sonicate the cells on ice and centrifuge at 4°C at 1000 g for 10 minutes to remove cell debris. Carefully collect the supernatant and centrifuge at 14,000 g for 30 minutes to isolate the membrane fragments. Analyze the morphology and structure of the MSC membrane using a fluorescence microscope and a transmission electron microscope (TEM).

[0076] 2. Preparation of nanovesicles (NVs)

[0077] Mix the extracted membrane fragments with phosphate - buffered saline (PBS) and sonicate on ice using a sonicator for 1 minute. Extrude the solution 15 times through polycarbonate membranes with pore sizes of 400 nm and 200 nm successively to obtain NVs and store them at 4°C for later use.

[0078] Results and Discussion:

[0079] To prepare mesenchymal stem cell - derived nanovesicles, the present invention first extracts the cell membrane and processes it through a liposome extruder to obtain nanovesicles with predictable sizes, as specifically Figure 2 shown. Then, the prepared mesenchymal stem cell - derived nanovesicles are characterized. Under the characterization of a fluorescence microscope and a transmission electron microscope, the morphological characteristics of the cell membrane are successfully observed. The fluorescence microscope image of the Dil - stained MSC cell membrane is as shown in Figure 3 -A, where the MSC membrane shows a fragmented state under the fluorescence microscope; the TEM image is as shown in Figure 3 -B, and this result further confirms the structural integrity of the membrane. These results indicate that the MSC membrane is successfully extracted and shows a fragmented structure.

[0080] The NVs prepared by the extrusion method are further characterized using a laser confocal microscope and TEM. The confocal laser scanning microscope image is as shown in Figure 3 -C, showing that the NVs are evenly distributed and have a relatively uniform particle size. The TEM image is as shown in Figure 3 -D, where the NVs are in an elliptical shape, have a transparent core, and the particle size is within 200 nm. The nanoscale size helps to improve the efficiency of the drug delivery system, can promote cell uptake, and enhance tissue permeability.

[0081] The above image analysis results indicate that MSC-derived nanovesicles with uniform size and morphology can be successfully prepared by the liposome extrusion method. The transparent and stable characteristics of NVs shown by TEM prove that they maintain good structural integrity during the preparation process, further indicating the potential of NVs as drug delivery carriers.

[0082] Example 2. Preparation and Characterization of Kartogenin-Loaded Nanovesicles (KGN@NVs)

[0083] 1. Preparation of KGN-Loaded Nanovesicles (KGN@NVs)

[0084] KGN@NVs were prepared by the co-extrusion method, specifically including: mixing NVs with a 10 μM KGN solution in a 1:1 ratio, oscillating on ice for 2 minutes, and then extruding through a 200 nm polycarbonate membrane 15 times to obtain KGN@NVs.

[0085] The preparation process of KGN@NVs is as Figure 4 shown. In the present invention, KGN and cell membrane fragments were co-extruded through a liposome extruder to achieve the successful encapsulation of KGN, making full use of the structural flexibility of cell membrane-derived nanovesicles.

[0086] 2. Characterization of KGN@NVs

[0087] The particle size and zeta potential of NVs and KGN@NVs were measured at room temperature using a nanoparticle size analyzer and a zeta potential analyzer. The nanoparticle size was measured by a laser nanoparticle size analyzer equipped with a 10 mV helium-neon laser light source, and the zeta potential was measured using standard electrophoresis techniques. All experiments were repeated three times.

[0088] The particle size and zeta potential of NVs and KGN@NVs were measured by dynamic light scattering (DLS). As Figure 5 shown in -B, the average particle size of NVs was 164.53 ± 6.00 nm, while the average particle size of KGN@NVs increased to 202.17 ± 17.64 nm. This increase in particle size is consistent with the successful encapsulation of KGN, and the loading of the drug increased the overall mass and hydrodynamic diameter of NVs. In addition, the zeta potential analysis results showed a significant difference between NVs and KGN@NVs, as detailed in Figure 5 -A. The average zeta potential of NVs was -8.325 ± 0.79 mV, while the zeta potential of KGN@NVs was -4.23 ± 0.0051 mV. This decrease in negative potential may be due to the encapsulation of KGN changing the surface charge density of NVs. These results indicate that KGN@NVs were successfully assembled, and the encapsulation of KGN effectively changed the surface properties of the nanovesicles.

[0089] 3. Stability of KGN@NVs

[0090] To evaluate the stability of KGN@NVs, the particles were suspended in PBS and stored at 4 °C. The particle size changes were measured using a nanoparticle size analyzer on days 0, 2, 4, 6, and 8 to evaluate the stability.

[0091] The long-term stability of drug delivery systems is a key parameter to ensure therapeutic efficacy and prevent drug leakage. In this invention, the stability of KGN@NVs was evaluated by monitoring the particle size changes of KGN@NVs in PBS solution. As Figure 5 shown in -C, within the first 4 days, the average particle size of KGN@NVs remained at approximately 200 nm; on days 6 and 8, the particle size slightly decreased to approximately 180 nm, which may be due to slight structural rearrangement or compression of the nanovesicles. However, there was no significant particle size fluctuation overall, indicating that KGN@NVs maintained good stability over a long period and had the ability to maintain drug encapsulation and structural integrity.

[0092] 4. Drug Loading Performance of KGN@NVs

[0093] KGN solutions with different concentrations (0.1, 0.5, 1, 2, 5, and 10 μM) were prepared separately. An appropriate amount of NVs was mixed with these KGN solutions in a 1:1 ratio, and KGN@NVs were prepared according to the aforementioned method. The samples were centrifuged at 15,000 g for 10 minutes, and the KGN content in the supernatant was detected using an enzyme-linked immunosorbent assay (ELISA) at λ = 278 nm.

[0094] The drug loading efficiency (LE) was calculated according to the following formula:[[]]

[0095] LE = M 1 / (M 2 + M 3 )

[0096] where LE: drug loading efficiency, representing the amount of drug loaded per unit mass of NVs;

[0097] M 1 : the amount of KGN in NVs;

[0098] M 2 : the mass of KGN;

[0099] M 3 : the mass of NVs.

[0100] The encapsulation efficiency (EE%) was calculated according to the following formula:[[]]

[0101] EE% = M 1 / M 4 × 100%

[0102] Among them, EE%: encapsulation efficiency, indicating the proportion of the drug encapsulated in the system;

[0103] M 4 : The total mass of KGN in the system.

[0104] To evaluate the drug-loading performance of KGN@NVs, the present invention tested the loading amount and encapsulation efficiency of different concentrations of KGN. As Figure 6 shown in -A, when the KGN concentration was 10 μM, the maximum drug-loading amount of NVs was 15.91 μg. At the same time, the encapsulation efficiency reached 45%, as detailed in Figure 6 -B. These results provided an effective drug dose basis for subsequent experiments to ensure the optimal therapeutic dose of KGN in the treatment of rheumatoid arthritis.

[0105] The above characterization results of KGN@NVs showed excellent drug-loading ability, adjustable particle size characteristics and stable physicochemical properties, further demonstrating its application potential as a drug delivery platform for RA targeted therapy.

[0106] Example 3. Preparation and characterization of dissolvable microneedles loaded with KGN@NV

[0107] 1. Preparation of microneedles (MN)

[0108] The hyaluronic acid (HA) solution was used to prepare the tips of the microneedles and was evenly coated on a polydimethylsiloxane (PDMS) microneedle mold (10×10 array, 121 microneedles, height: 1000 μm, base: 400 μm×400 μm, center spacing: 750 μm). The mold was centrifuged at 2000 rpm for 1 minute to ensure that the needle cavities were completely filled. Subsequently, 400 μL of the HA solution was added to prepare the microneedle base. After two centrifugation treatments, the mold was placed in an oven at 40 °C to be completely dried and finally demolded. The microneedles were stored in a desiccator.

[0109] 2. Preparation and drug loading of dissolvable microneedles loaded with KGN@NV (KGN@NV-MN)

[0110] 0.1 g of HA was dissolved in 1 mL of the KGN@NVs solution and incubated with shaking at 37 °C for 4 hours. Subsequently, the KGN@NVs-HA solution was evenly coated on the microneedle mold and centrifuged at 4200 rpm for 1 minute to fill the tips of the microneedles. After removing the excess solution, 400 μL of a 10% HA solution was added. After two centrifugation treatments, the mold was placed in an oven at 40 °C to be completely dried and finally demolded to obtain KGN@NV-MN. After staining with Dil, the microneedle imaging was analyzed by fluorescence microscopy. By dissolving KGN@NV-MN in PBS and shaking at a constant temperature for 15 - 30 minutes, and then centrifuging at 15,000 g for 10 minutes, the absorbance of the supernatant was measured to calculate the drug-loading amount.

[0111] 3. Dissolution Performance of KGN@NV-MN

[0112] To evaluate the dissolution performance of the microneedles, the tips of the microneedles were immersed in PBS in a Petri dish. Dissolution times of 1, 3, 5, 7, and 9 minutes were set, and images were taken using a 4× microscope. The change in the height of the microneedles was analyzed using ImageJ software.

[0113] Results and Discussion:

[0114] A schematic diagram of the preparation of KGN@NV-MN by micro-molding is shown as Figure 7 follows. This method effectively integrates KGN@NVs into the hyaluronic acid-based microneedle matrix. Visual observation of the microneedles showed that the microneedle patches were neatly arranged and no obvious defects were seen, as detailed in Figure 8 -A. Scanning electron microscope (SEM) images further confirmed that the microneedles had a complete conical geometry, were precisely arranged, had a smooth surface, and sharp tips, as detailed in Figure 8 -B. This structure is crucial for effective skin penetration and helps to achieve minimal trauma and efficient drug delivery.

[0115] Fluorescence microscopy analysis further confirmed the successful integration of KGN@NVs, as shown in Figure 9 -A. Red fluorescence was observed at the tip of the microneedles, indicating that KGN@NVs were concentrated in the tip region, which is of great significance for achieving targeted drug delivery. The overall results showed that the micro-molding process effectively encapsulated KGN@NVs while maintaining the integrity and physical properties of the microneedle structure.

[0116] We simulated the in vivo environment in PBS solution to evaluate the dissolution performance of KGN@NV-MN. The results are shown in Figure 10 -C. By observing the morphological changes of the microneedles over time, the dissolution process of the microneedles was recorded: the tip began to dissolve at 1 minute, the microneedles dissolved to the middle at 3 - 5 minutes, only a small amount of the needle base remained at 7 minutes, and they were completely dissolved at 9 minutes. By quantitatively analyzing the change in the height of the microneedles, it was found that approximately 90% of the needle body was completely dissolved after 9 minutes, indicating that the microneedles had rapid and efficient dissolution performance, as detailed in Figure 9 -C.

[0117] Meanwhile, the drug release curve of KGN@NV-MN was also analyzed. The results are shown in Figure 9 -B. The release amount of KGN was 1.59 ± 0.04 mg at 15 minutes of dissolution and reached 2.01 ± 0.17 mg at 30 minutes of complete release. This indicates that the microneedles can achieve controlled and sustained drug release during dissolution, providing a reliable mechanism for effective drug delivery.

[0118] The successful preparation of KGN@NV-MN demonstrates the application potential of micro-molding technology in constructing structurally stable and functionally efficient dissolvable microneedles. As a microneedle matrix, hyaluronic acid has good biocompatibility and degradability, meeting the requirements of transdermal therapeutic applications. The sharp geometric structure of the microneedles significantly improves the drug transdermal delivery efficiency while ensuring minimal trauma. The property of KGN@NV-MN to rapidly dissolve and release drugs within 9 minutes has significant advantages for acute conditions such as rheumatoid arthritis. In addition, the local high-concentration release of KGN can synergistically regulate the inflammatory microenvironment and promote cartilage regeneration.

[0119] In summary, the performance of KGN@NV-MN in terms of efficient preparation, excellent dissolution properties, and controllable drug release lays the foundation for its further clinical application in the treatment of RA.

[0120] Example 4. Therapeutic effect of KGN@NV-MN in the AIA rat model

[0121] 1. Construction of the RA animal model

[0122] All animal experiments were conducted following aseptic procedures and approved by the Animal Care and Use Committee of Chongqing University of Science and Technology. Male rats aged 6 - 8 weeks and weighing 180 - 200 g were selected and adaptively fed for 1 week. The right hind paw was disinfected with iodophor, and 0.1 mL of complete Freund's adjuvant (CFA) was subcutaneously injected into the plantar region.

[0123] The rats were divided into five groups (n = 5 per group): Native group: not induced with RA and not treated; Control group: induced with RA but not treated; MN group: after induction of RA, treated with a blank microneedle patch for 10 minutes and fixed with medical tape for 30 minutes; SC group (SC Injection): after induction of RA, subcutaneously injected with 0.1 mL of KGN@NVs solution; KGN@NV-MN group: after induction of RA, treated with a KGN@NV-MN patch for 10 minutes and fixed with medical tape for 30 minutes.

[0124] The experiment lasted for 22 days. RA treatment started on the 4th day, and the drug was administered once every 3 days for a total of 6 times. Blood was collected 2 days after the last treatment, the rats were sacrificed, and the ankle joints were taken for hematoxylin-eosin (H&E) and safranin O-fast green cartilage staining.

[0125] 2. Measurement of foot swelling degree and clinical score

[0126] The foot swelling was monitored using a vernier caliper on days 0, 5, and every 3 days thereafter. The clinical score was evaluated based on the appearance of the foot, with a score range of 0 - 4 (0: no erythema or swelling; 1: mild erythema / swelling limited to the ankle joint; 2: mild erythema / swelling extending to the tarsus; 3: moderate erythema / swelling extending to the metatarsus; 4: severe erythema / swelling involving the entire foot).

[0127] Results and Discussion:

[0128] The adjuvant-induced arthritis (AIA) rat model induced by complete Freund's adjuvant (CFA) is widely used in the evaluation of treatment strategies due to its high similarity to human RA in immunology and pathology. In this study, the anti-inflammatory effect of KGN@NV-MN was evaluated through a 6-dose regimen, with an intervention every two days. The experimental groups included an untreated group (Control), a blank microneedle group (MN), a subcutaneous injection group (SC), and a KGN@NV-MN group, as detailed in Figure 11 .

[0129] As Figure 13 shown, the foot swelling of the rats in the Control group gradually worsened and reached a peak in the later stage of the experiment. In contrast, the foot swelling of the rats treated with KGN@NV-MN was significantly reduced, approximately 30% less than that of the Control group (p < 0.01). The clinical score results further supported this observation. As Figure 14 shown, the score of the KGN@NV-MN group was significantly lower than that of the Control group and the MN group, showing an effective inhibitory effect on joint inflammation (p < 0.01). Representative images of the rat paws visually showed that the swelling and deformity in the KGN@NV-MN treatment group were significantly reduced, as detailed in Figure 12 .

[0130] The significant improvement in foot swelling and clinical score indicates that KGN@NV-MN has potent anti-inflammatory properties. This efficacy is mainly attributed to the sustained release of kartogenin loaded in the nanovesicles. KGN has been proven to promote chondrocyte differentiation and matrix synthesis and play a synergistic role in inflammation regulation.

[0131] Example 5. Hematological Safety and Immunomodulatory Effect of KGN@NV-MN

[0132] To evaluate the hematological safety and systemic immune response of KGN@NV-MN treatment, the blood parameters of rats in each group were detected on day 20, and the results are as Figure 15 shown. There was no significant difference in red blood cell (RBC) count among the experimental groups, indicating that KGN@NV and soluble microneedles have good blood compatibility and do not cause hemolytic effects, as detailed in Figure 15 -A.

[0133] Unlike RBC, the white blood cell (WBC) count showed significant changes, as shown in Figure 15 -B. The WBC levels of rats in the Control group and the MN group were significantly increased, which might be related to the inflammatory state during the establishment of the AIA model. After treatment with KGN@NV-MN, the WBC count was significantly decreased and lower than that of the SC group (p<0.01), showing a stronger anti-inflammatory effect. In addition, the change trend of granulocyte level was consistent with that of WBC. KGN@NV-MN significantly inhibited the granulocytosis caused by RA and restored it to near the normal level, showing excellent immunomodulatory effects, as shown in Figure 15 -D. There were no obvious abnormal changes in platelet levels, as shown in Figure 15 -C.

[0134] The results of lymphocyte analysis were as shown in Figure 15 -E. Compared with the Native group, the lymphocyte ratios in the Control group and the MN group were significantly decreased, which might reflect the immune dysregulation caused by RA. Both KGN@NV-MN and SC treatments effectively restored the lymphocyte ratio, and the restoration effect in the KGN@NV-MN group was more significant. There were no significant differences in monocyte ratio analysis among groups, as shown in Figure 15 -F.

[0135] The above hematological results demonstrated that KGN@NV-MN not only had good biocompatibility but also showed excellent anti-inflammatory and immunomodulatory effects. This systematic immune regulation helped RA patients restore immune homeostasis, improve disease progression, and reduce potential side effects of treatment simultaneously.

[0136] Example 6. Protective effect of KGN@NV-MN on articular cartilage

[0137] To further evaluate the therapeutic effect of KGN@NV-MN, the present invention used hematoxylin-eosin (H&E) staining and safranin O-fast green (SO / FG) staining to perform histological analysis on rat paw tissues to observe the integrity of cartilage structure and the degree of inflammatory cell infiltration. The results were as shown in Figures 16 - 17 shown.

[0138] The results of H&E staining were as shown in Figure 16 shown. The cartilage of rats in the Control group and the MN group was severely eroded, accompanied by a large number of inflammatory cell infiltrations, which were typical features of late-stage RA. While the KGN@NV-MN group and the SC group significantly alleviated this pathological progression, the cartilage surface was smooth and the inflammatory cell infiltration was significantly reduced. The results of SO / FG staining were as shown in Figure 17As shown, the results were consistent with the trend of H&E staining. The cartilage matrix staining in the Control group and the MN group was weak, showing a significant reduction in glycosaminoglycan content, indicating severe damage to the cartilage matrix. In contrast, the KGN@NV-MN group and the SC group retained strong red staining, suggesting effective protection of chondrocyte and matrix integrity.

[0139] These histological evidences indicate that KGN@NV-MN has a dual role in alleviating inflammation and protecting the structure of articular cartilage. The realization of this effect is closely related to the property of KGN in promoting chondrocyte differentiation and matrix repair, while the nanovesicle delivery system ensures the targeted release and long-term action of the drug, further enhancing the therapeutic effect.

[0140] Example 7. Regulatory effect of KGN@NV-MN on pro-inflammatory cytokines

[0141] Pro-inflammatory cytokines such as IL-1β and TNF-α play a key role in synovial inflammation and cartilage destruction in RA. These cytokines are secreted by activated immune cells, which can stimulate abnormal proliferation of synoviocytes, recruit neutrophil infiltration into the joint cavity, and exacerbate the destruction of cartilage and bone. In this invention, the levels of IL-1β and TNF-α in the sera of rats in each group were detected by ELISA to evaluate the anti-inflammatory effect of KGN@NV-MN. The results are as Figure 18 、 Figure 19 shown. Compared with the Native group, the levels of IL-1β and TNF-α in the Control group and the MN group were significantly increased, indicating the systemic inflammatory response induced by RA. While the levels of IL-1β and TNF-α in the KGN@NV-MN treatment group were significantly decreased, returning to near the Native group level, showing an effective anti-inflammatory regulatory effect (p<0.01). The effect of KGN@NV-MN in reducing pro-inflammatory cytokines was not significantly different from that of the SC treatment group, but KGN@NV-MN achieved non-invasive delivery through the microneedle system, providing a more patient-friendly alternative.

[0142] The above results indicate that KGN@NV-MN successfully regulated the inflammatory microenvironment of RA by significantly inhibiting the expression of IL-1β and TNF-α. This multi-target anti-inflammatory mechanism can not only alleviate the inflammatory response but also block the cartilage destruction caused by inflammation, providing new ideas for the treatment of RA.

Claims

1. A drug-loaded nanovesicle for treating rheumatoid arthritis, characterized in that: The drug-loaded nanovesicles are loaded with kartogenin, and the nanovesicles are derived from mesenchymal stem cells.

2. The nanovesicle according to claim 1, characterized in that The particle size of the drug-loaded nanovesicles is 150-250nm.

3. The method for preparing the drug-loaded nanovesicles according to any one of claims 1 to 2, characterized in that: KGN@NVs were prepared by co-extrusion of kartogenin and cell membrane-derived nanovesicles.

4. The method according to claim 3, characterized in that The volume ratio of the kartogenin to the nanovesicle is 0.8-1.5:1; the concentration of the kartogenin is 5-20 μM.

5. The method according to claim 3, characterized in that: The kartogenin and the cell membrane-derived nanovesicles are extruded through a polycarbonate membrane, and the number of extrusions is 10 to 20 times.

6. Dissolving microneedles loaded with KGN@NVs, characterized in that: The active ingredient in the dissolving microneedle is the drug-loaded nanovesicle according to claim 1.

7. The method for preparing the KGN@NVs-loaded dissolving microneedles according to claim 6, characterized in that: The steps include: (1) preparing KGN@NVs by the method described in any one of claims 3 to 5; (2) dissolving hyaluronic acid in the KGN@NVs solution obtained in step (1) to obtain a KGN@NVs-HA solution; (3) The KGN@NVs-HA solution obtained in step (2) is uniformly coated on the microneedle mold, and the solution is filled into the tip of the microneedle by centrifugation; then, the HA solution is used to prepare a microneedle substrate, and after centrifugation, drying, and demolding, a soluble microneedle loaded with KGN@NVs is obtained.

8. The method according to claim 7, characterized in that In step (2), the reaction conditions are: incubation with shaking at 35-40°C for 3-6 hours.

9. Use of the drug-loaded nanovesicles according to any one of claims 1 to 2 and / or the KGN@NVs-loaded dissolving microneedles according to claim 6 in the preparation of a drug for treating rheumatoid arthritis.

10. Use of the drug-loaded nanovesicles according to any one of claims 1 to 2 and / or the KGN@NVs-loaded soluble microneedles according to claim 6 in the preparation of drugs for anti-inflammation, immunomodulation, protection of articular cartilage and / or promotion of cartilage repair.

Images