Rgd tripeptide modified pilose antler mesenchymal stem cell exosome and preparation method and application thereof

Abstract

The application discloses RGD tripeptide modified Cervi Cornu Mesenchymal Stem Cell Exosome and a preparation method and application thereof, and belongs to the technical field of biological medicine, wherein RGD tripeptide containing Arg-Gly-Asp sequence is modified to the surface of Cervi Cornu MSCs exosome under mild reaction conditions, so that the exosome is endowed with good structural stability and the ability of targeting inflammatory tissues, and the anti-inflammatory effect and joint protection effect of the exosome in a RA model are significantly improved. In addition, the RGD modified exosome can be combined with various anti-rheumatic drugs to achieve better treatment effect, has good biocompatibility, safety and industrial transformation prospect, and is suitable for precise treatment of autoimmune diseases such as rheumatoid arthritis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and specifically relates to RGD tripeptide modified deer antler mesenchymal stem cell (MSCs) exosomes, a preparation method thereof and application thereof. BACKGROUND

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease mainly characterized by synovial inflammation, cartilage and bone destruction. Current treatment methods include anti-inflammatory drugs, immunosuppressive agents and biological agents, etc. These therapies can relieve symptoms but are difficult to effectively prevent joint destruction, and long-term use may produce side effects.

[0003] In recent years, stem cell exosomes have attracted attention for their potential in tissue repair and immune regulation. Exosomes are nanoscale vesicles secreted by cells, with the function of carrying proteins and nucleic acids and communicating with cells. In therapeutic applications, MSCs exosomes have been shown to have tissue repair and immune regulation effects similar to stem cells, with higher safety. For example, the deer antler stem cell exosomes reported in Chinese patent CN113197919B can significantly reduce the expression of inflammatory factors such as IL-8, IL-1β, IFN-α, CCL20, CCL2 or TNF-α, thereby achieving good anti-inflammatory effect, and can effectively improve or treat osteoarthritis, and delay the aging of human mesenchymal stem cells. For example, the miR-140 modified deer antler stem cell exosomes prepared by transfecting miR-140 lentivirus vector into deer antler stem cells reported in Chinese patent CN118703574A can significantly promote the repair of cartilage tissue damage and the inhibition of inflammation compared with unmodified deer antler stem cell exosomes. However, the existing deer antler stem cell exosomes have limited enrichment efficiency at the lesion site after administration, lack targeting to inflammatory joints, and may reduce the efficacy.

[0004] Integrins are a class of receptors present on the surface of cells, which bind to extracellular matrix proteins and play an important role in the process of synovial hyperplasia and cartilage destruction. The RGD tripeptide sequence (Arg-Gly-Asp) is a recognition ligand for integrin receptors such as αvβ3. Some drug delivery systems have used RGD peptides for targeting of tumors or inflammatory tissues, but the use of RGD for exosome targeting in arthritis treatment is novel. Deer antler is the young horn tissue of newborn antler of Cervidae such as sika deer, containing rich growth factors and stem cells. Deer antler-derived MSCs have strong proliferation and differentiation potential, and their exosomes may have unique advantages in cartilage regeneration and immune regulation. However, there is no report on the combination of deer antler MSCs exosomes with RGD targeting for RA treatment. SUMMARY

[0005] The application aims to provide an RGD tripeptide modified deer mesenchymal stem cell exosome and a preparation method and application thereof. The RGD tripeptide can specifically recognize and bind to integrin receptors, and the deer MSC exosome is rich in various immunomodulatory factors and growth factors. The RGD tripeptide is coupled to the surface of the deer MSC exosome, so that the RGD modified deer MSC exosome with high stability and good safety is obtained, the exosome is endowed with the targeted delivery capability, and the high enrichment of the exosome in the inflammatory joint part can be realized, and the concentration and action time of the effective components in the lesion are enhanced.

[0006] The application is realized by the following technical scheme: an RGD tripeptide modified deer mesenchymal stem cell exosome, in which RGD tripeptide is reacted with a suspension of deer mesenchymal stem cell exosome in the presence of a crosslinking agent to obtain the RGD tripeptide modified deer mesenchymal stem cell exosome,

[0007] The RGD tripeptide is a peptide segment containing an Arg-Gly-Asp sequence.

[0008] The crosslinking agent includes an isocyanate crosslinking agent containing a hydrophilic or lipophilic group, a carbodiimide crosslinking agent, a dialdehyde crosslinking agent or a maleimide crosslinking agent.

[0009] The crosslinking agent is EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) or EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide / N-hydroxysuccinimide).

[0010] The preparation method of the above RGD tripeptide modified deer mesenchymal stem cell exosome includes the following steps:

[0011] S1. Deer mesenchymal stem cells are separated from deer horn or deer velvet tissue after digestion, and then exosomes are extracted after culture and amplification to obtain deer mesenchymal stem cell exosomes.

[0012] S2. After the RGD tripeptide is mixed with the suspension of deer mesenchymal stem cell exosomes, the crosslinking agent is added, and the mixture is incubated at 20-25℃ for 0.5-2h, and then the free RGD peptide and the crosslinking agent are removed to obtain the RGD tripeptide modified deer mesenchymal stem cell exosome.

[0013] In the step S1, the cell suspension is obtained after the deer horn or deer velvet tissue is digested, and then the deer mesenchymal stem cells are separated by density gradient centrifugation or adherent screening method.

[0014] In the step S1, the DMEM culture medium containing 10% fetal bovine serum is used for culture, and the culture is carried out at 37-38℃ and 5-7% CO2 for 24-72h.

[0015] In the step S1, the exosomes are extracted by culturing the expanded cells for 24-48 hours, taking the supernatant, and removing impurities, filtering, centrifuging and washing to obtain the Cervus elaphus L mesenchymal stem cell exosomes.

[0016] In the step S2, the mixture after incubation is dialyzed or centrifuged to remove free RGD peptide and cross-linking agent.

[0017] The RGD tripeptide modified Cervus elaphus L mesenchymal stem cell exosomes are used for preparing a medicament for treating rheumatoid arthritis, and the medicament further comprises at least one of leflunomide, methotrexate, sulfasalazine, tocilizumab, adalimumab, etanercept or baricitinib.

[0018] The medicament is an injection, a sustained-release microsphere, a transdermal gel or a liposome preparation.

[0019] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0020] (1) The present application can highly enrich the exosomes in the inflammatory joint site by specific binding of the RGD tripeptide to the integrin receptors on the surface of the synovial membrane and chondrocytes, thereby enhancing the concentration and action time of the effective components at the lesion site, significantly improving the anti-inflammatory and tissue protection effects compared with unmodified exosomes, and having the advantages of strong targeting and improved efficacy.

[0021] (2) The present application uses Cervus elaphus L MSCs exosomes, which are rich in various immune regulatory factors and growth factors, can inhibit the excessive production of pro-inflammatory cytokines (such as TNF-α, IL-6 and IL-17), and can increase the level of anti-inflammatory factors (such as IL-10); at the same time, the Cervus elaphus L MSCs exosomes can also promote the survival and matrix synthesis of chondrocytes and reduce the apoptosis of chondrocytes, thereby controlling inflammation and protecting and repairing damaged cartilage tissue, and protecting immune regulation and cartilage.

[0022] (3) The RGD tripeptide modified Cervus elaphus L MSCs exosomes involved in the present application are used as endogenous nanocarriers, have natural biocompatibility and low immunogenicity, the Cervus elaphus L MSCs derived exosomes have no tumorigenic risk, and the RGD tripeptide is a short peptide that does not cause obvious toxic side effects, so the preparation of the present application has good safety and can be repeatedly administered with low adverse reactions.

[0023] (4) The present application introduces the integrin targeting strategy into the RA exosome therapy, which can make up for the shortcomings of existing anti-rheumatic drugs in cartilage protection and local delivery, and the targeted exosome therapy can be combined with existing treatment methods, such as DMARDs (non-steroidal anti-inflammatory drugs) and biological agents, which can produce synergistic effects and improve the overall treatment effect.

[0024] To sum up, the RGD tripeptide modified Cervi Cornu MSCs exosomes provided by the application realize the targeting of integrin receptors in joint synovial membrane and cartilage tissue by introducing RGD tripeptides on the surface of Cervi Cornu MSCs exosomes, significantly improve the enrichment and effect of Cervi Cornu MSCs exosomes in the inflammatory joint, at the same time, can effectively regulate the local immune response of the joint, reduce the release of inflammatory factors, and promote cartilage protection and regeneration, so as to play a role in treating RA, and can be combined with existing treatment methods to achieve the purpose of improving the treatment effect of RA. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Extraction of Cervi Cornu mesenchymal stem cell exosomes.

[0026] Figure 2 Cervi Cornu mesenchymal stem cell three-lineage differentiation ability verification diagram.

[0027] Figure 3 Basic characterization diagram of Cervi Cornu MSCs exosomes.

[0028] Figure 4 TEM diagram of RGD modified Cervi Cornu MSCs exosomes.

[0029] Figure 5 Cervi Cornu MSCs exosome particle size distribution and Zeta potential analysis diagram.

[0030] Figure 6 RGD modified Cervi Cornu MSCs exosome particle size distribution and Zeta potential analysis diagram.

[0031] Figure 7 Routine liver and kidney function indicators of mice in each group.

[0032] Figure 8 Main organ tissue toxicity evaluation (HE staining) of mice in each group.

[0033] Figure 9 RA treatment of mice in each group.

[0034] Figure 10 Tissue distribution and joint targeting evaluation of different DID dye delivery systems in mice. DETAILED DESCRIPTION

[0035] The purposes, technical solutions and beneficial effects of the application will be further described below.

[0036] It should be pointed out that the following detailed description is exemplary and is intended to provide further description of the claimed application, unless otherwise stated, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the application belongs.

[0037] The present application aims to solve the problems of poor targeting of existing common exosomes after administration, limited enrichment efficiency, and reduced therapeutic effect, especially for rheumatoid arthritis (RA) with synovial inflammation, cartilage and bone destruction, etc. Although existing treatment drugs (such as leflunomide, methotrexate, etc.) can alleviate the inflammatory symptoms of RA, they lack targeting of inflammatory joints and have limited therapeutic effect. Therefore, the present application provides a RGD tripeptide modified deer horn MSCs exosome. The deer horn MSCs exosome is obtained by obtaining and culturing mesenchymal stem cells from deer antler / deer horn tissue and then extracting exosomes, ensuring that high-purity and high-activity deer horn MSCs exosomes are obtained. Then, the RGD tripeptide functional molecule is directly modified on the surface of the deer horn MSCs exosome by chemical coupling, etc., which endows the deer horn MSCs exosome with targeting ability for synovial and cartilage integrin receptors. It can be used for preparing drugs for treating RA. In the treatment of RA, the exosome is combined with integrin in the diseased joint tissue to achieve targeted delivery of the exosome, play an immune regulation and cartilage protection role, and achieve targeted treatment of RA.

[0038] Therefore, the present application covalently couples RGD tripeptide and carboxyl on the surface of deer horn MSCs exosome to construct an exosome delivery system with natural regeneration / immune regulation function and integrin targeting ability. The core innovation lies in:

[0039] (1) RGD-mediated active targeting: through specific binding of RGD tripeptide and integrin highly expressed in the lesion site, precise delivery is achieved, and the enrichment efficiency of exosomes in diseased tissues is significantly improved.

[0040] (2) Specific adhesion of RGD modification: through covalent coupling (not physical adsorption), RGD is stably modified on the surface of the exosome, which improves the targeting and prolongs the residence time of the exosome in the lesion site, thereby enhancing the therapeutic effect.

[0041] (3) Innovation of treatment strategy: traditional methods rely on exosome drug loading or in vitro functional enhancement, while the present application realizes "drug-free treatment" through targeted modification, avoiding the complexity and potential toxicity of drug loading process. It can be used alone or in combination with existing RA drugs (such as methotrexate), to maximize the therapeutic effect through synergistic effect and provide a new treatment mode for RA.

[0042] The specific steps for preparing the RGD tripeptide modified deer horn MSCs exosome are as follows:

[0043] (I) Acquisition and culture of deer horn MSCs

[0044] Newborn antler or pilose antler tissues of healthy adult sika deer are selected, and the cartilage-like tissue part is taken under sterile conditions. The tissue is cut into pieces (about 2 mm in size) and digested with collagenase (37°C for 0.5-1 h) to obtain a suspension of free cells. Mesenchymal stem cells (MSCs) are isolated by density gradient centrifugation (for example, the cell suspension obtained by collagenase digestion is slowly added to the upper layer of 1.073 g / mL Percoll solution, avoiding disturbing the interface, and a centrifugal speed of 400-800 x g is used for 20-30 minutes) or adherent screening (for example, after inoculation, the culture bottle is left standing for 12-24 hours, and then the non-adherent cells are removed and the adherent cells are cultured). The obtained pilose antler MSCs are inoculated in a culture bottle and cultured in a DMEM medium containing 10% fetal bovine serum at 37-38°C and 5-7% CO2 for 24-72 h and then subcultured and expanded. The cell morphology and proliferation are monitored during the culture to ensure that a sufficient number of third-generation pilose antler MSCs are obtained for exosome extraction.

[0045] (ii) Extraction of exosomes

[0046] The pilose antler MSCs cultured above are replaced with exosome-free feeding medium (such as exosome-removed serum medium) and cultured for 24-48 h. The culture supernatant is collected, the cells are removed by centrifugation at 300 x g for 5 minutes, the cell debris is removed by centrifugation at 2000 x g for 20 minutes, and then the large particle impurities are removed by centrifugation at 10,000 x g for 30 minutes. The supernatant is filtered through a 0.22 μm filter membrane, and the exosomes are precipitated by ultracentrifugation at 100,000 x g for 70 minutes. The supernatant is discarded, the exosome precipitate is resuspended in an appropriate amount of sterile PBS, and centrifuged at 100,000 x g for 70 minutes for washing, to obtain pilose antler mesenchymal stem cell exosomes (pilose antler MSC exosomes), which are resuspended in PBS for storage.

[0047] The obtained exosome particles can be confirmed by nanoparticle size analysis (NTA) or transmission electron microscopy to have a particle size in the range of 50-150 nm, and identified by Western Blot of exosome markers (such as CD63, TSG101) to determine its source.

[0048] (iii) RGD tripeptide modification

[0049] The RGD tripeptide is modified on the surface of the exosome by chemical coupling. Specifically, a peptide segment containing the Arg-Gly-Asp sequence is selected. The purified exosome suspension is mixed with a solution containing the RGD tripeptide, and a crosslinking agent is added under mild conditions (pH 6.5-7.4, PBS buffer system, and the reaction process should avoid vigorous shaking to maintain the integrity and activity of the exosome). The amino group of the RGD peptide covalently bonds with the carboxyl group on the surface of the exosome membrane. The reaction is incubated at room temperature for a certain period of time (e.g., 20-25°C for 0.5-2h), and gentle shaking is performed during the reaction to ensure full reaction. After the reaction is completed, the mixture is placed in a dialysis bag and dialyzed against PBS buffer, or free RGD peptide and reagents are removed by ultracentrifugation to obtain RGD tripeptide-modified Cervus elaphus Linnaeus MSCs exosomes.

[0050] In some possible embodiments, in addition to containing the Arg-Gly-Asp sequence, a functional group facilitating coupling can be introduced at the N- or C-terminus of the peptide according to the structure of the RGD tripeptide. The optional functional groups include, for example, a cysteine residue (Cys), a thiol group, an aminothiol group (-SH), a carboxyl group (-COOH), an azido group (-N3), an alkyne group (-C≡CH), a biotin group, etc. These groups can be introduced by, for example, chemical modification, click chemistry, or enzyme-catalyzed coupling. In addition, the crosslinking agent can be, for example, an isocyanate crosslinking agent containing a hydrophilic or lipophilic group, a carbodiimide crosslinking agent, a dialdehyde crosslinking agent, or a maleimide crosslinking agent. The isocyanate crosslinking agent containing a hydrophilic or lipophilic group can be, for example, isocyanate-NCO. The carbodiimide crosslinking agent can be, for example, EDC (14mM). EDC / NHS or EDC / NHS-activated polycarboxyl polymers (such as polyglutamic acid or hyaluronic acid) can also be used. The dialdehyde crosslinking agent can be, for example, glutaraldehyde. The maleimide crosslinking agent can be, for example, maleimide-PEG-NHS or maleimide-PEG-COOH.

[0051] The specific embodiments of the application are described below with reference to the following examples, but the scope of protection of the application is not limited to the following examples.

[0052] Example 1: Cervus elaphus Linnaeus MSCs exosomes

[0053] The new-born velvet antler tissue of healthy adult Cervus nippon was selected, and the cartilage-like tissue part was taken under sterile conditions. The tissue was cut into 2 mm pieces, digested with collagenase at 37°C for 1 h to obtain a free cell suspension. Mesenchymal stem cells (MSCs) were separated by density gradient centrifugation. The obtained velvet antler MSCs were inoculated in a culture bottle, and DMEM medium containing 10% fetal bovine serum was used for culture, which was cultured at 37°C, 5% CO2 for 24 h and subcultured for expansion. The cell morphology and proliferation were monitored during the culture process, and 1×10 8 ~ 1×10 9 velvet antler MSCs were obtained.

[0054] The above velvet antler MSCs were continuously added to the culture medium containing exosome-depleted FBS and DMEM high-sugar medium for continuous culture for 48 h. The culture supernatant was collected, and the cells were removed by centrifugation at 300×g for 5 min, the cell debris was removed by centrifugation at 2000×g for 20 min, and then the large particle impurities were removed by centrifugation at 10,000×g for 30 min. The supernatant was filtered through a 0.22µm filter membrane, and the exosomes were precipitated by ultracentrifugation at 100,000×g for 70 min. The supernatant was discarded, and the exosome precipitate was resuspended in an appropriate amount of sterile PBS and centrifuged at 100,000×g for 70 min for washing. The velvet antler MSCs exosomes were obtained and resuspended in PBS for storage.

[0055] Example 2: Velvet antler MSCs exosomes

[0056] The new-born velvet antler tissue of healthy adult Cervus nippon was selected, and the cartilage-like tissue part was taken under sterile conditions. The tissue was cut into 1 cm, digested with collagenase at 37°C for 0.5 h to obtain a free cell suspension. Mesenchymal stem cells (MSCs) were separated by density gradient centrifugation. The obtained velvet antler MSCs were inoculated in a culture bottle, and DMEM medium containing 10% fetal bovine serum was used for culture, which was cultured at 37°C, 5% CO2 for 72 h and subcultured for expansion. The cell morphology and proliferation were monitored during the culture process, and 1×10 7 ~ 1×10 8 velvet antler MSCs were obtained.

[0057] The above Cervi Cornu Pantotrichum MSCs were continuously added to the high-sugar DMEM medium containing exosome-depleted FBS and DMEM, and were continuously cultured for 48 h. The culture supernatant was collected, and cells were removed by centrifugation at 300 x g for 5 min, cell debris was removed by centrifugation at 2000 x g for 20 min, and large particulate impurities were removed by centrifugation at 10,000 x g for 30 min. After the supernatant was filtered through a 0.22 μm filter membrane, the exosomes were precipitated by ultracentrifugation at 100,000 x g for 70 min. The supernatant was discarded, and the exosome precipitate was resuspended in an appropriate amount of sterile PBS, and was centrifuged at 100,000 x g for 70 min for washing, to obtain Cervi Cornu Pantotrichum MSCs exosomes, which were resuspended in PBS for storage.

[0058] Example 3: RGD tripeptide-modified Cervi Cornu Pantotrichum MSCs exosomes

[0059] The RGD peptide segment with the amino acid sequence Arg-Gly-Asp was used.

[0060] The Cervi Cornu Pantotrichum MSCs exosome suspension (0.5 ml) of Example 1 was mixed with the above RGD peptide segment (0.1 ml) solution, 1 mg EDC was added under appropriate mild conditions, and incubation was performed at 25°C for 2 h, with gentle shaking to ensure full reaction. After the reaction was completed, the mixture was placed in a dialysis bag for dialysis against PBS buffer, to obtain RGD tripeptide-modified Cervi Cornu Pantotrichum MSCs exosomes.

[0061] Example 4: RGD tripeptide-modified Cervi Cornu Pantotrichum MSCs exosomes

[0062] The RGD peptide segment with the amino acid sequence Arg-Gly-Asp was used.

[0063] The Cervi Cornu Pantotrichum MSCs exosome suspension (1.0 ml) of Example 2 was mixed with the above RGD peptide segment (200 ml) solution, 2 mg EDC / NHS was added under appropriate mild conditions, and incubation was performed at 22°C for 1.5 h, with gentle shaking to ensure full reaction. After the reaction was completed, the mixture was placed in a dialysis bag for dialysis against PBS buffer, to obtain RGD tripeptide-modified Cervi Cornu Pantotrichum MSCs exosomes.

[0064] Example 5: RGD tripeptide-modified Cervi Cornu Pantotrichum MSCs exosomes

[0065] The RGD peptide segment with the amino acid sequence Arg-Gly-Asp was used.

[0066] The suspension of Cervi Cornu MSCs exosomes of Example 1 (0.75 ml) was mixed with the solution of the above RGD peptide segment (0.15 ml), 1.5 mg of EDC was added under suitable mild conditions, and the reaction was allowed to proceed fully by incubation at 20°C for 1 h with gentle shaking. After the reaction was completed, the mixture was placed in a dialysis bag for dialysis against PBS buffer to obtain RGD tripeptide-modified Cervi Cornu MSCs exosomes.

[0067] Example 6: RGD tripeptide-modified Cervi Cornu MSCs exosomes

[0068] The RGD peptide segment with Biotin- at the N-terminal was used, and its amino acid sequence was: Arg-Gly-Asp.

[0069] The suspension of Cervi Cornu MSCs exosomes of Example 2 (0.6 ml) was mixed with the solution of the above RGD peptide segment (0.12 ml), 1 mg of NHS was added under mild conditions, and the reaction was allowed to proceed fully by incubation at 23°C for 0.5 h with gentle shaking. After the reaction was completed, the mixture was placed in a dialysis bag for dialysis against PBS buffer to obtain RGD tripeptide-modified Cervi Cornu MSCs exosomes.

[0070] Example 7: RA treatment drug

[0071] The RGD tripeptide-modified Cervi Cornu MSCs exosomes of Example 3 were mixed with Methotrexate at a mass ratio of 1:5 to prepare an RA treatment drug.

[0072] Example 8: RA treatment drug

[0073] The RGD tripeptide-modified Cervi Cornu MSCs exosomes of Example 5 were mixed with Tocilizumab at a mass ratio of 1:2 to prepare an RA treatment drug.

[0074] The products of the above Examples 1 to 8 were respectively subjected to the following detection or experiment.

[0075] (I) Verification of extraction of Cervi Cornu MSCs

[0076] The free cell suspension obtained in Example 1 was filtered using a cell filter (70 μm) to collect the cells. Subsequently, the MSCs were purified by Percoll density gradient centrifugation or directly subjected to adherent screening. The main equipment used included: a super-clean workbench, a carbon dioxide incubator, an inverted phase contrast microscope, a constant-temperature water bath, a centrifuge, a filter, and an electron transmission microscope (TEM), etc.

[0077] The results are shown in Figure 1

[0078] Figure 1 ​Extraction of exosomes from deer antler mesenchymal stem cells. Figure 1 (A) shows the mesenchymal layer of deer antler; (B) shows primary mesenchymal stem cells emerging from the surrounding tissue; (C) is a TEM image of deer antler mesenchymal stem cells. Figure 1 It can be demonstrated that mesenchymal stem cells with typical morphological characteristics were successfully isolated from deer antler cartilage-like tissue. The cells have the ability to adhere to the wall and are arranged in a spindle or whorl shape, which is consistent with the morphological characteristics of MSCs. Furthermore, under TEM, the cytoplasm contains abundant organelles, which supports their stem cell identity.

[0079] (II) Verification of the differentiation capacity of deer antler MSCs

[0080] Third-generation deer antler mesenchymal stem cells (the deer antler MSCs of Example 1) were seeded into 24-well plates. After the cell confluence reached 70-80%, the medium was replaced with osteogenic induction medium, adipogenic induction medium, and chondrogenic induction medium, respectively, and cultured for 21 days. The following staining was then performed: Alizarin Red S staining for osteogenic differentiation, Oil Red O staining for adipogenic differentiation, and Alcian Blue staining for chondrogenic differentiation. The staining was observed under a microscope to verify the three-lineage differentiation capacity.

[0081] See results Figure 2 As shown.

[0082] Figure 2 This diagram demonstrates the three-lineage differentiation capacity of deer antler mesenchymal stem cells. Figure 2 Image (A) shows osteogenic differentiation (Alizarin Red S staining), image (B) shows adipogenic differentiation (Oil Red O staining), and image (C) shows chondrogenic differentiation (Alcian Blue staining). Figure 2 It is evident that deer antler mesenchymal stem cells possess excellent osteogenic, adipogenic, and chondrogenic differentiation capabilities, meeting the criteria for the three-lineage differentiation of MSCs.

[0083] (III) Structural characterization of exosomes from deer antler MSCs

[0084] The ultrastructure morphology of deer antler MSCs exosomes (Example 1) was observed using transmission electron microscopy (TEM, JEM-1400, JEOL); the particle size distribution of deer antler MSCs exosomes (Example 1) was detected using a nanoparticle tracking analyzer (NTA, ZetaView PMX120, Particle Metrix); and the surface potential of deer antler MSCs exosomes (Example 1) was measured using a Zeta potential analyzer (Zetasizer Nano ZS90, Malvern).

[0085] See results Figure 3 As shown.

[0086] Figure 3 This is a basic characterization diagram of exosomes from deer antler MSCs. Figure 3 In the diagram, (A) is a TEM morphology image, (B) is a magnified TEM image, (C) is a particle size distribution map (NTA), and (D) is a zeta potential map. Figure 3 As shown, the exosomes of deer antler MSCs exhibit a typical cup-shaped vesicle structure with a particle size concentrated at 100 nm and a Zeta potential of -31.86 ± 1.62 mV, which meets the internationally recognized standards for the biophysical characteristics of exosomes.

[0087] (iv) Structural stability of RGD-modified antler MSCs exosomes

[0088] The morphology of RGD-modified antler MSCs exosomes (Example 3) was observed using transmission electron microscopy (TEM); the particle size distribution of antler MSCs exosomes (Example 1) and RGD-modified antler MSCs exosomes (Example 3) was detected using a nanoparticle tracking analyzer (NTA); and the surface potential of antler MSCs exosomes (Example 1) and RGD-modified antler MSCs exosomes (Example 3) was determined using a Zeta potential analyzer.

[0089] See results Figure 4 to Figure 6 As shown.

[0090] Figure 4 TEM images of exosomes from RGD-modified antler MSCs. Figure 4 Typical exosome morphological characteristics were observed, including: 1) Morphological characteristics: Exosomes exhibited spherical or cup-shaped structures with clear edges and intact structures, consistent with typical ultrastructural morphology of exosomes; 2) Size range: The main diameters were concentrated between 30 and 150 nm, with the target particle in the figure being approximately 100 nm, falling within the typical exosome particle size range; 3) Surface coating: The particle surface possessed a distinct phospholipid bilayer membrane structure, demonstrating good membrane structural integrity; 4) Distribution: The figure showed a single exosome particle at the center, surrounded by several smaller particles, possibly exosome aggregates or small vesicles, indicating relatively uniform exosomes in the preparation system; 5) Structural homogeneity: No obvious fragments or broken particles were observed. Figure 4 The results showed that the RGD-modified deer antler MSCs exosomes prepared in Example 3 of the present invention had good purity and stability, and no structural abnormalities were observed due to RGD coupling.

[0091] Figure 5 Figure 1 shows the particle size distribution and zeta potential analysis of exosomes from deer antler MSCs. Figure 5Figure 2 shows the particle size distribution of the Cervi Cornu Pantotrichum MSCs exosomes (Example 1). The particle size is concentrated in the range of 100-130 nm, with a main peak of about 117 nm. The overall particle size distribution is narrow, with a clear peak, indicating good uniformity of the particle size. This result shows that the Cervi Cornu Pantotrichum MSCs exosomes before RGD modification are typical nanoscale particles, which meet the standard of natural exosomes. Figure 5 Figure 2 shows the particle size distribution of the Cervi Cornu Pantotrichum MSCs exosomes (Example 1). The particle size is concentrated in the range of 100-130 nm, with a main peak of about 117 nm. The overall particle size distribution is narrow, with a clear peak, indicating good uniformity of the particle size. This result shows that the Cervi Cornu Pantotrichum MSCs exosomes before RGD modification are typical nanoscale particles, which meet the standard of natural exosomes.

[0092] Figure 6 Figure 2 shows the particle size distribution of the Cervi Cornu Pantotrichum MSCs exosomes (Example 1). The particle size is concentrated in the range of 100-130 nm, with a main peak of about 117 nm. The overall particle size distribution is narrow, with a clear peak, indicating good uniformity of the particle size. This result shows that the Cervi Cornu Pantotrichum MSCs exosomes before RGD modification are typical nanoscale particles, which meet the standard of natural exosomes. Figure 6 Figure 2 shows the particle size distribution of the Cervi Cornu Pantotrichum MSCs exosomes (Example 1). The particle size is concentrated in the range of 100-130 nm, with a main peak of about 117 nm. The overall particle size distribution is narrow, with a clear peak, indicating good uniformity of the particle size. This result shows that the Cervi Cornu Pantotrichum MSCs exosomes before RGD modification are typical nanoscale particles, which meet the standard of natural exosomes. Figure 6 Figure 2 shows the particle size distribution of the Cervi Cornu Pantotrichum MSCs exosomes (Example 1). The particle size is concentrated in the range of 100-130 nm, with a main peak of about 117 nm. The overall particle size distribution is narrow, with a clear peak, indicating good uniformity of the particle size. This result shows that the Cervi Cornu Pantotrichum MSCs exosomes before RGD modification are typical nanoscale particles, which meet the standard of natural exosomes.

[0093] (Five) Biocompatibility and safety

[0094] The Cervi Cornu Pantotrichum MSCs exosomes (EV) of Example 1 and the RGD tripeptide modified Cervi Cornu Pantotrichum MSCs exosomes (EV@RGD) of Example 3 were injected subcutaneously and into the tail vein of mice, respectively. The blank group and the PBS group (pH 7.4 phosphate buffered saline) were used as controls. The following indicators in the serum of mice in each group were measured: ALT (alanine aminotransferase), AST (aspartate aminotransferase), UREA (urea), and CREA (creatinine), to evaluate the in vivo biological safety of EV and EV@RGD. The skin at the injection site and the overall state of the animals were also observed. After 7 days of continuous observation, the skin at the injection site and the main organs (heart, liver, spleen, lungs, and kidneys) were taken for HE staining to evaluate the in vivo safety and tissue compatibility of EV and EV@RGD.

[0095] The results are shown in Figure 7 andFigure 8 The results are shown in Table 1.

[0096] Figure 7 The routine liver and kidney function indicators of each group of mice were determined. Figure 7 Figure 2 shows the serum ALT levels of mice. It can be seen that the ALT levels of the four groups were not significantly increased, and there was no significant difference between the EV@RGD group and the Control group (ns), indicating that it did not cause obvious liver cell damage. Figure 7 Figure 2 shows the serum ALT levels of mice. It can be seen that the ALT levels of the four groups were not significantly increased, and there was no significant difference between the EV@RGD group and the Control group (ns), indicating that it did not cause obvious liver cell damage. Figure 7 Figure 2 shows the serum ALT levels of mice. It can be seen that the ALT levels of the four groups were not significantly increased, and there was no significant difference between the EV@RGD group and the Control group (ns), indicating that it did not cause obvious liver cell damage. Figure 7 Figure 2 shows the serum ALT levels of mice. It can be seen that the ALT levels of the four groups were not significantly increased, and there was no significant difference between the EV@RGD group and the Control group (ns), indicating that it did not cause obvious liver cell damage.

[0097] Figure 8 Figure 3 shows the main organ and tissue toxicity evaluation (HE staining) of each group of mice. It can be observed from the figure whether there are pathological changes in the morphology of each tissue. For heart tissue, the myocardial fibers of each group are arranged in order, and there is no inflammatory cell infiltration, among which the morphology of the EV@RGD group is consistent with that of the control group, indicating that there is no toxicity to the heart. For liver tissue, the liver cord of each group is arranged clearly, and there is no obvious inflammation, edema or necrosis, among which the EV@RGD group has no signs of tissue damage, indicating no damage to the liver tissue. For spleen tissue, the white pulp and red pulp structure of each group is complete, and there is no obvious hyperplasia or hemorrhagic necrosis, and the tissue morphology of each group is consistent, indicating no toxicity to the spleen. For lung tissue, the PBS group and part of the treatment group show mild interstitial widening, the alveolar structure of the EV@RGD group is clear, the ventilation cavity is complete, and there is no obvious hemorrhage or edema, showing good lung tissue compatibility. For kidney tissue, the glomerular structure of each group is complete, and the renal tubules are arranged in order. The EV@RGD group has no glomerular necrosis or renal interstitial abnormalities, indicating no nephrotoxicity.

[0098] (6) RA treatment effect

[0099] A collagen-induced RA mouse model was constructed, which was randomly divided into groups and then injected with PBS (pH 7.4 phosphate buffered saline), Example 1 (EV), Example 3 (EV@RGD) and Example 8 (EV@RGD + tocilizumab) preparations, respectively, and injected into the tail vein at the 1st, 3rd and 6th day of treatment, with a dose of 100 μL (about 1 × 10 9

[0100] The results are shown in Table 1. Figure 9 The results are shown in Table 1.​

[0101] Figure 9 The image shows the RA treatment status of mice in each group. The visual assessment of metatarsal and phalangeal joint inflammation and swelling in each group of mice can be observed. The Control group served as a normal control group; mice showed no inflammatory stimulation, and their paw joints appeared normal, without swelling, redness, or deformity. This group served as a baseline reference, representing the state of healthy joints. The PBS group served as a disease model control group; mice in this group showed significant redness, swelling, and deformity of the joints, exhibiting typical inflammatory joint lesions, indicating successful model establishment and no therapeutic effect from PBS. In the EV group, treatment with deer antler MSCs exosomes reduced joint swelling, but the degree of redness and swelling remained significant, indicating that exosomes alone had some effect on inflammation, but the effect was limited. In the EV@RGD group... Treatment with RGD-modified deer antler MSCs exosomes resulted in significant reduction of paw swelling and a near-normal appearance in mice after treatment. This indicates that RGD modification enhances the directionality and cellular uptake efficiency of deer antler MSCs exosomes, leading to a more significant improvement in inflammation and demonstrating the key role of RGD in targeted delivery. In the EV@RGD+tocilizumab group, combined treatment with RGD-modified deer antler MSCs exosomes and tocilizumab resulted in almost complete recovery of paws in mice after treatment, with no obvious swelling or erythema. This suggests that RGD-modified exosomes combined with tocilizumab can synergistically exert anti-inflammatory effects, achieving dual regulation of targeted delivery and immunosuppression, demonstrating superior therapeutic efficacy compared to single-drug therapy.

[0102] (vii) In vivo stability study

[0103] Rheumatoid arthritis (RA) mouse models were randomly divided into groups, and the distribution of three different DID dye delivery systems in major tissues and organs of RA mice was observed. Fluorescence imaging was used to observe the localization and enrichment levels 72 hours after injection, and the in vivo stability and joint targeting were compared. The three different DID dye delivery systems were: free dye (Free DID), antler MSCs exosome-loaded dye (EV-DID), and RGD-modified antler MSCs exosome-loaded dye (EV@RGD-DID).

[0104] See results Figure 10 As shown.

[0105] Figure 10 To evaluate the tissue distribution and joint targeting of different DID dye delivery systems in mice. Figure 10It can be seen that the fluorescence signal distribution of the Free DID group is the most dispersed, mainly concentrated in the liver and spleen, indicating that free DID is easy to be cleared in the body, lacks stability and targeting; the fluorescence signal of the EV-DID group is more concentrated than that of the Free DID, mainly accumulated in the liver and part of the inflammatory joints (Paws), indicating that the exosome has certain immune escape and delivery capacity, but still has part of non-targeted distribution; the EV@RGD-DID group appears the strongest fluorescence signal in the joint part of the foot (Paws), and the signal still exists obviously after 72 hours, showing excellent targeted enrichment capacity; the signal in other organs (such as liver, spleen) is weak, indicating that its in vivo distribution is more selective, at the same time, the group retains a certain signal in the serum, suggesting that the material exists stably in the circulation and is not easy to be quickly cleared. It can be shown that the RGD modified deer horn MSCs exosome prepared by the application has stronger in vivo stability and joint targeting.

[0106] The above is only a preferred embodiment of the present application, and does not limit the present application in any form. Any simple modification or equivalent change made according to the technical essence of the present application to the above embodiment falls within the protection scope of the present application.

Claims

1. A method for preparing RGD tripeptide-modified deer antler mesenchymal stem cell exosomes with high targeting and long residence time, characterized in that: Includes the following steps: S1. After digesting deer antler or deer velvet tissue, deer velvet mesenchymal stem cells are isolated, and after culturing and expanding, exosomes are extracted to obtain deer velvet mesenchymal stem cell exosomes; S2. After mixing the suspension of RGD tripeptide with deer antler mesenchymal stem cell exosomes, the pH was adjusted to 6.5–7.4, and a cross-linking agent was added. The mixture was incubated at 20–25°C for 0.5–2 hours to remove free RGD peptide and cross-linking agent, thereby obtaining RGD tripeptide-modified deer antler mesenchymal stem cell exosomes. The RGD tripeptide is a peptide segment with an Arg-Gly-Asp sequence. The crosslinking agent is EDC or EDC / NHS.

2. The preparation method according to claim 1, characterized in that: In step S1, after digesting the antler or deer velvet tissue, a cell suspension is obtained, and then deer velvet mesenchymal stem cells are separated by density gradient centrifugation or adherence screening.

3. The preparation method according to claim 1, characterized in that: In step S1, the culture is carried out using DMEM medium containing 10% fetal bovine serum at 37–38°C and 5–7% CO2 for 24–72 hours.

4. The preparation method according to claim 1, characterized in that: In step S1, during exosome extraction, the expanded cells are cultured for 24–48 hours, and the supernatant is collected. After impurity removal, filtration, centrifugation, and washing, deer antler mesenchymal stem cell exosomes are obtained.

5. The preparation method according to claim 1, characterized in that: In step S2, the incubated mixture is dialyzed or centrifuged to remove free RGD peptides and cross-linking agents.

6. An RGD tripeptide-modified deer antler mesenchymal stem cell exosome with high targeting and long residence time, characterized in that: It is prepared by any one of the methods of claims 1 to 5.

7. The use of RGD tripeptide-modified deer antler mesenchymal stem cell exosomes as described in claim 1 in the preparation of a medicament for treating rheumatoid arthritis, characterized in that: The drugs also include at least one of leflunomide, methotrexate, sulfasalazine, tocilizumab, adalimumab, etanercept, or baricitinib.

8. The application according to claim 7, characterized in that: The drug is an injection, sustained-release microsphere, transdermal gel, or liposome preparation.

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