Ternary co-modified engineered pilose antler MSCs exosome as well as preparation method and application thereof

Abstract

The invention discloses a ternary co-modified engineered cornu cervi pantotrichum MSCs exosome and a preparation method and application thereof, and belongs to the technical field of biological medicine, the ternary co-modified engineered cornu cervi pantotrichum MSCs exosome comprises a cornu cervi pantotrichum MSCs exosome, a poly-arginine polypeptide-deoxyribonuclease I compound located on the surface of the cornu cervi pantotrichum MSCs exosome, and an RGD polypeptide modified on the surface of the cornu cervi pantotrichum MSCs exosome and containing an Arg-Gly-Asp sequence. In order to solve the problem that an integrated treatment mode of multiple pathological links cannot be realized during existing RA treatment, three active components, namely poly-arginine polypeptide (P-Arg), deoxyribonuclease I (Dnase I) and RGD polypeptide, are cooperatively modified on the surface of an exosome of pilose antler MSCs for the first time to form a surface engineering modified exosome complex; multiple functions of immunoregulation, cfDNA removal, targeted delivery and tissue protection can be realized in RA focuses, and a brand-new and systematic treatment means is provided for rheumatoid arthritis.

Description

Technical Field

[0001] The present invention relates to a ternary co-modified engineered antler MSCs exosome, a preparation method thereof, and an application thereof. The present invention specifically relates to a antler MSCs exosome synergistically modified with three active components: polyarginine polypeptide (P-Arg), deoxyribonuclease I (DNase I), and RGD polypeptide. The present invention also covers a preparation method of the exosome and its application in the treatment or prevention of rheumatoid arthritis (RA), belonging to the field of biomedicine technology. Background Art

[0002] Rheumatoid arthritis (RA) is a common, chronic, progressive autoimmune disease that primarily affects peripheral joints, causing synovial inflammation, cartilage destruction, and bone erosion. Patients often experience joint swelling, pain, and dysfunction, and in severe cases, joint deformity and disability. The pathogenesis of RA is complex, involving multiple factors such as immune system disorders and imbalanced bone and cartilage metabolism. + An imbalance in T cell subsets plays a key role: inflammatory Th17 cells overdifferentiate and secrete multiple inflammatory cytokines, including IL-17, driving synovial inflammation and joint tissue destruction. Meanwhile, immunosuppressive regulatory T cells (Tregs) are insufficient in number or function, failing to effectively control the abnormal immune response. This imbalance in the Th17 / Treg ratio is considered a key factor in the pathogenesis of RA. Furthermore, high levels of cell-free DNA (cfDNA) are often detected in the synovial fluid and blood of RA patients. This cfDNA is derived in part from extracellular traps (NETs) formed by neutrophils in response to inflammatory stimulation and in part from the abnormal cell death of synovial fibroblasts and macrophages. This large amount of uncleared cfDNA can act as endogenous danger signals (DAMPs), activating innate immune pathways such as Toll-like receptors (TLR9), further amplifying the inflammatory response. In RA-affected joints, DNA released from NETs forms immune complexes with autoantigens, inducing the production of autoantibodies and complement activation, leading to persistent synovial inflammation and tissue damage. Studies have shown that NET-associated cfDNA plays an important role in the pathological process of RA, and timely removal of this free DNA is expected to alleviate the disease progression. However, currently, there are limited clinical treatment options for cfDNA or NET.

[0003] Under the chronic inflammatory environment of the joint, chondrocytes also undergo inflammatory senescence. Senescent chondrocytes secrete large amounts of proinflammatory cytokines and matrix-degrading enzymes (known as the senescence-associated secretory phenotype (SASP)), such as IL-6 and MMP-13, which exacerbate cartilage matrix degradation and joint destruction. RA patients, on the other hand, experience increased osteoclastogenesis, and RANKL-mediated osteoclast hyperdifferentiation leads to enhanced bone resorption, a primary cause of joint bone erosion and bone loss. In summary, the pathogenesis of RA involves multiple pathways, including immune imbalance (abnormal Th17 / Treg expression), persistent inflammation (high levels of proinflammatory cytokines and cfDNA), premature chondrocyte senescence, and hyperactive osteoclast activity. Existing treatments (such as methotrexate, hormones, and biologics like anti-TNF-α / IL-6) primarily target inflammation and immunity, but have limited effects on cartilage protection and bone destruction. Furthermore, long-term use can lead to side effects such as infection. Therefore, a multi-targeted, combined approach is urgently needed to address immune dysfunction, eliminate inflammatory factors, and promote joint tissue repair.

[0004] Recent studies have shown that mesenchymal stem cells (MSCs) are considered a potential treatment for autoimmune diseases such as RA due to their immunomodulatory and tissue regeneration abilities. However, stem cell transplantation is plagued by issues such as low survival rates and potential tumorigenicity. Against this backdrop, exosomes secreted by MSCs have become a focus of attention. Exosomes are nanoscale membrane vesicles released by cells that carry a variety of active molecules, including proteins, messenger RNA, and microRNA, mediating intercellular communication. MSC-derived exosomes have been shown to exhibit therapeutic effects similar to those of MSCs in certain models of inflammation and tissue injury, offering advantages such as ease of storage and low immunogenicity. In the treatment of RA, reports have shown that exosomes derived from bone marrow mesenchymal stem cells can inhibit the inflammatory response of fibroblast-like synoviocytes and regulate macrophage polarization, thereby alleviating arthritis symptoms. However, the efficacy of natural exosomes remains limited, and they lack the ability to actively target specific lesions. Furthermore, conventional exosomes cannot actively clear inflammatory stimuli, such as NETs. Therefore, engineering MSC-derived exosomes to enhance their multi-therapeutic potential is a hot topic of research. Deer antlers are the newly grown antler tissue of cervids, such as sika deer. In traditional Chinese medicine, they are believed to have the properties of strengthening bones and tendons and anti-aging. Modern research indicates that deer antlers are rich in growth factors and regenerative molecules, and the stem cells derived from them (antler mesenchymal stem cells) possess exceptionally strong proliferation and multidirectional differentiation potential. Compared to conventional MSCs, deer antler MSCs can be passaged for many more generations and maintain their viability. It has been reported that deer antler MSCs can be passaged for over 50 generations in vitro without significant aging. Conditioned culture medium derived from deer antler MSCs has been shown to promote skin wound healing. These properties make deer antler MSCs an ideal source for the production of therapeutic exosomes, potentially providing a stable and high-yield source of exosomes.

[0005] Currently, Chinese patent CN115386542A reports on deer antler stem cell exosomes carrying miR-143, which can significantly increase the expression level of miR-143 in osteosarcoma, thereby enhancing the inhibitory effect on osteosarcoma. However, for RA, a complex disease involving multi-system pathological changes, there have been no reports of "integrated" treatment plans based on deer antler MSC exosomes. Therefore, the present invention combines deer antler MSC exosomes with modern nanomedicine technology to construct a multifunctional, synergistically modified exosome treatment system, which has important innovative significance and clinical application value in the field of RA treatment. Summary of the Invention

[0006] This invention aims to address the current inability to achieve an "integrated" treatment model for multiple pathological components in RA treatment. The proposed ternary co-modified engineered antler MSC exosomes synergistically modify the surface of antler MSC exosomes with three active components: poly-arginine peptide (P-Arg), deoxyribonuclease I (DNase I), and RGD peptide. This creates a surface-engineered exosome complex that can achieve multiple functions in RA lesions, including immune regulation, cfDNA clearance, targeted delivery, and tissue protection. To this end, the invention also provides a method for preparing these exosomes and their use in the preparation of drugs for the treatment or prevention of rheumatoid arthritis, potentially providing a new, systemic treatment for rheumatoid arthritis.

[0007] The present invention is achieved through the following technical solution: a ternary co-modified engineered pilose antler MSCs exosome, comprising: A. Deer antler MSCs exosomes; B. Polyarginine peptide-deoxyribonuclease I complex located on the surface of velvet MSCs exosomes; C. RGD peptide containing the Arg-Gly-Asp sequence modified on the surface of antler MSCs exosomes.

[0008] Deoxyribonuclease I was loaded onto the surface of antler MSCs exosomes through electrostatic adsorption mediated by polyarginine polypeptide to obtain a polyarginine polypeptide-deoxyribonuclease I complex located on the surface of antler MSCs exosomes.

[0009] The RGD polypeptide modified on the surface of antler MSCs exosomes is combined with the antler MSCs exosomes by chemical cross-linking or physical adsorption, or is combined with the polyarginine polypeptide in the polyarginine polypeptide-deoxyribonuclease I complex by chemical cross-linking.

[0010] A method for preparing ternary co-modified engineered antler MSCs exosomes, which takes antler MSCs exosomes as the main body, loads deoxyribonuclease I on the surface of antler MSCs exosomes through polyarginine polypeptide-mediated electrostatic adsorption, obtains the polyarginine polypeptide-deoxyribonuclease I complex on the surface of antler MSCs exosomes, and then modifies the surface of antler MSCs exosomes with RGD polypeptide containing Arg-Gly-Asp sequence through chemical cross-linking or physical adsorption to form ternary co-modified engineered exosomes.

[0011] Deer antler MSCs exosomes are obtained by shearing and digesting deer antler tissue to separate deer antler mesenchymal stem cells, and then culturing and amplifying them before extracting exosomes.

[0012] Polyarginine polypeptide is added to the antler MSCs exosome solution to obtain exosomes with polyarginine polypeptide surface modification, and then deoxyribonuclease I solution is added to obtain a polyarginine polypeptide-deoxyribonuclease I complex on the surface of the antler MSCs exosomes.

[0013] The concentration of antler MSCs exosome solution was 1×10 8 ~1×10 9 particles / mL; the concentration of polyarginine polypeptide is 1-10 mg / mL, and the concentration of deoxyribonuclease I solution is 25-50 U / μL.

[0014] The molecular weight of polyarginine polypeptides is 5000 to 15000 Da.

[0015] Modification on the surface of antler MSCs exosomes is to combine the RGD polypeptide with the antler MSCs exosomes by chemical cross-linking or physical adsorption, or to combine the RGD polypeptide with the polyarginine polypeptide in the polyarginine polypeptide-deoxyribonuclease I complex by chemical cross-linking.

[0016] The use of ternary co-modified engineered antler MSCs exosomes in the preparation of a drug for treating or preventing rheumatoid arthritis, the ternary co-modified engineered antler MSCs exosomes are as described above, or are prepared by the above preparation method, and the drug is an injection, sustained-release microspheres, transdermal gel or liposome preparation.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The present invention can achieve integrated treatment of multiple targets in RA, breaking through the limitation of single function of traditional exosomes. Traditional MSCs exosomes mainly act on a single pathway (such as anti-inflammatory, anti-oxidative or macrophage polarization), and cannot intervene in the multi-link pathological process of RA at the same time, and it is difficult to deal with the coexistence of immune disorders, cfDNA-mediated inflammation, cartilage aging and bone destruction. The present invention achieves the stable fixation of triple functional molecules on the surface of deer antler MSCs exosomes through electrostatic assembly and molecular adsorption, and obtains the trifunctional synergistic exosomes constructed by the "P-Arg + DNase I + RGD" engineering strategy, which can achieve the following multiple effects: inhibiting Th17, promoting Treg, and restoring Th17 / Treg immune homeostasis; surface-loaded DNase I clears cfDNA, blocks pro-inflammatory pathways such as TLR9 / NETs; delays inflammatory aging of chondrocytes, inhibits excessive differentiation of osteoclasts; and targets and enriches in inflamed joints, with precise positioning.

[0018] (2) The present invention solves the problems of scalability and functional decline of traditional MSCs by constructing a new exosome production system. Among the shortcomings of the existing technology, bone marrow / adipose MSCs have limited in vitro passage capacity and are prone to aging after 3-5 generations, resulting in low exosome production, poor stability, and functional decline. The present invention is the first to use sika deer antler mesenchymal stem cells (AMSCs) for exosome production. The cells have strong passage capacity, high stability (can be expanded to more than 50 generations), high exosome secretion volume, and excellent biological activity.

[0019] (3) The present invention modifies the surface of exosomes with enzyme functionalization to give them the ability to actively clear cfDNA and quickly relieve inflammation. Traditional exosomes lack endogenous nuclease activity and are unable to clear a large amount of free cfDNA in RA synovial fluid, leading to continuous activation of immune pathways. The present invention modifies the surface of antler MSCs exosomes with P-Arg to construct a cationic interface, effectively anchoring the DNase I enzyme, allowing it to retain its enzyme activity and locate outside the exosome membrane, achieving efficient binding and degradation of cfDNA. By effectively controlling the loading ratio of DNase I, incubation time, and ultracentrifugation process, the dual stability of enzyme binding efficiency and biological activity is ensured.

[0020] (4) The present invention can increase the targeting of exosomes and improve the delivery efficiency of joint lesions. In the prior art, natural EVs (Extracellular Vesicles) are widely distributed in the body and have no obvious tissue orientation, resulting in low therapeutic dose utilization, and some even accumulate in the liver and spleen and become ineffective. The present application grafts RGD tripeptide on the surface of exosomes, which can bind to integrin receptors expressed by synovial cells, chondrocytes, etc., thereby enhancing the active targeting enrichment and retention ability of exosomes in RA inflammation sites. In the present invention, the RGD polypeptide forms a non-covalent / covalent bond with the exosome membrane or P-Arg through the Cys thiol group, and after incubation, the free molecules are removed by multiple centrifugation, which can effectively retain its complete activity and structural specificity.

[0021] (5) The present invention can achieve a mild, standardized, and easily scalable exosome functional assembly process. In the prior art, some drug-loaded exosomes or functionalized EVs require chemical crosslinking agents, dialysis, or high-temperature incubation, which poses risks of structural damage and reduced activity, making the process unfavorable for industrialization. The present invention adopts a physical method of low-temperature electrostatic adsorption + short-term incubation, which does not require organic solvents or high-energy treatment, retaining the integrity and function of exosomes to the greatest extent, while being easy to standardize and scale up under GMP (Good Manufacturing Practice) conditions. In addition, in the present invention, by controlling the temperature of the entire process within the range of 4 to 37°C, combined with ultracentrifugation / particle size screening, material purification and structural consistency control can be achieved.

[0022] (6) The present invention can comprehensively improve clinical indicators related to RA. Its efficacy has been demonstrated in animal experiments. After injection of the engineered exosomes into the CIA mouse model via the tail vein, the engineered antler MSCs exosomes modified with the three elements can significantly reduce arthritis scores, cartilage destruction, bone erosion, and inflammatory factor levels, and the Th17 / Treg ratio is significantly restored. Unmodified MSCs exosomes have limited therapeutic effects in RA animal experiments and lack comprehensive verification of immune cell lineage and tissue repair capacity. Therefore, the present invention uses the established mouse RA model to systematically evaluate the therapeutic effects of EV@P-Arg-DNaseI-RGD at multiple levels, including behavioral, histological, molecular, and immune lineage levels, and has technical advantages in the treatment of RA. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 For the extraction of deer antler mesenchymal stem cells.

[0024] Figure 2 This is a diagram verifying the three-lineage differentiation ability of antler mesenchymal stem cells.

[0025] Figure 3 This is the basic characterization diagram of antler MSCs exosomes.

[0026] Figure 4 Figure 2 is a graph showing the particle size changes of different exosome materials.

[0027] Figure 5 Zeta potential diagram of different exosome materials.

[0028] Figure 6 Graph showing the effect of exosomes modified with different concentrations of polyarginine (P-Arg) on ​​cell activity.

[0029] Figure 7 Calcein-AM / PI double-stained fluorescence images of cell activity and cell death after treatment with different modified exosomes.

[0030] Figure 8 Immunofluorescence images of TNF-α and COX-2 expression in inflammatory model cells under different exosome treatments.

[0031] Figure 9 Diagram of the flow cytometry gating strategy for sorting naive CD4+ T cells.

[0032] Figure 10 This is a flow cytometry analysis diagram for in vitro validation of Th17 / Treg differentiation.

[0033] Figure 11 This is a comparison of joint pathology in RA mice after treatment with exosome-derived drugs.

[0034] Figure 12 This is a fluorescent imaging image of a mouse in vivo. DETAILED DESCRIPTION

[0035] The objectives, technical solutions and beneficial effects of the present invention are further described in detail below.

[0036] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the claimed invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.

[0037] Through source cell screening, structural optimization, and surface functionalization, this study successfully constructed a multifunctional engineered exosome derived from antler stem cells—EV@P-Arg-DNaseI-RGD—for the treatment of rheumatoid arthritis (RA). These engineered exosomes simultaneously modulate immune function, clear inflammation, and repair joint tissue, transcending the limitations of existing RA treatment strategies that target only a single pathological component. For the first time, they achieve an integrated, synergistic approach to treating multiple RA pathologies, offering new insights into the comprehensive treatment of RA.

[0038] The product of the present invention is a surface-engineered nano-scale exosome composed of an exosome main body and a surface functional layer. The exosome main body is derived from mesenchymal stem cells (AMSCs) isolated from sika deer antler tissue, and the natural exosomes secreted by the exosomes are extracted after culture. The surface functional layer includes three active components: polyarginine peptide (P-Arg), deoxyribonuclease I (DNase I), and RGD peptide. The three active components are modified on the surface of the exosome main body through electrostatic adsorption and enzyme binding. P-Arg is used to provide a cationic interface for loading DNase I, while also increasing the affinity of the exosome membrane with cell membranes or cfDNA. DNase I has nucleic acid degradation activity and can specifically eliminate cfDNA in the inflammatory microenvironment. The RGD peptide contains an Arg-Gly-Asp sequence and has high-affinity integrin binding ability, which enables the exosomes to target synovium and chondrocytes.

[0039] Specifically, the engineered exosomes involved in the present invention are prepared by the following steps: (1) Deer antler stem cell culture and exosome extraction Fresh, healthy sika deer antler tissue was obtained. Under sterile conditions, the junction of cartilage and cancellous bone was transversely dissected approximately 5 cm from the antler tip. Thin slices approximately 2 mm thick were cut, and the mesenchymal layer was excised under a dissecting microscope. The slices were then cut into 1 mm³ blocks. The blocks were digested in a solution containing 0.1% type I collagenase at 37°C for 2 hours. After digestion, free cells were collected by filtration and centrifugation and seeded in high-glucose DMEM medium supplemented with 10% fetal bovine serum (FBS). The cells were cultured in a 37°C, 5% CO2 incubator. Cells were passaged when they reached 90% confluency and expanded to the third passage for later use.

[0040] To extract exosomes, P3 antler mesenchymal stem cells (AMSCs) were cultured in DMEM supplemented with exosome-depleted FBS for 24 to 48 hours, and the supernatant was collected. Exosomes were extracted using standard differential centrifugation: cells were removed by centrifugation at 300 × g for 10 minutes, debris was removed by centrifugation at 2000 × g for 20 minutes, and large vesicles were removed by centrifugation at 10,000 × g for 30 minutes at 4°C. The supernatant was then filtered through a 0.22 μm pore size filter and ultracentrifuged at 100,000 × g for 70 minutes to collect the exosome pellet. The pellet was resuspended in PBS and washed by ultracentrifugation again to obtain a highly purified exosome sample, i.e., antler MSC exosomes.

[0041] Transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and Western blot were used to detect exosome marker proteins such as CD9, CD63 and CD81 to confirm the structure and purity of exosomes.

[0042] (II) Surface modification with P-Arg The concentration of the exosomes extracted above was adjusted to 1×10 8 ~1×10 9 To prepare 1 ml of exosome solution containing 100 μg of poly-L-arginine hydrochloride (molecular weight range, 5,000–15,000 Da), add 25 μL of 10 mg / mL poly-L-arginine hydrochloride (molecular weight range, 5,000–15,000 Da). The mixture was incubated at room temperature with gentle rotation for 0.5–1 hour to allow the P-Arg peptide to electrostatically attach to the surface of the exosome phospholipid bilayer, forming a positively charged exosome complex (EV@P-Arg). Subsequently, the mixture was centrifuged at 100,000 × g for 1 hour to remove free unbound P-Arg peptide, and the pellet was resuspended in PBS to obtain exosomes surface-modified with P-Arg.

[0043] It should be noted that P-Arg is a strongly cationic peptide whose polyguanidine groups electrostatically adsorb to phosphate groups in phospholipids on the surface of EVs. Its flexible molecular structure also provides it with excellent coating and binding stability. Therefore, compared with materials such as quaternary ammonium salts or polyethyleneimine, P-Arg has better biodegradability and lower cytotoxicity, making it more suitable as a functional linker for enzyme loading or peptide grafting.

[0044] (III) Loading DNase I enzyme Add 0.5 μL of a DNase I solution at a concentration of 25–50 U / μL (i.e., a total enzyme activity of 12.5–25 U) to the EV@P-Arg solution. Gently mix thoroughly and incubate at 37°C for 0.5–2 hours to allow DNase I to stably bind to the P-Arg polypeptide through electrostatic interactions and become anchored on the exosome surface, forming the EV@P-Arg-DNase I complex. Unbound enzyme molecules were then removed by centrifugation at 100,000 × g for 1 hour. Functionalized exosomes carrying active DNase I were then obtained after resuspending in PBS.

[0045] SDS-PAGE electrophoresis and Coomassie brilliant blue staining confirmed the presence of a DNase I-specific band of approximately 31 kDa in the exosomes, and enzyme activity detection showed that the engineered exosomes still had significant cfDNA degradation ability.

[0046] (IV) Grafted RGD peptide 70 μL of pre-prepared RGD tripeptide (GRGDNP, 10 mM, dissolved in sterile water) was added to the EV@P-Arg-DNase I complex. The complex was incubated at room temperature for 1–2 hours. The C-terminal thiol group of the RGD sequence formed a stable covalent bond with the P-Arg polypeptide or the exosome membrane surface thiol group through disulfide or nucleophilic reactions, or adhered to the surface through hydrophobic adsorption, completing the modification of the RGD targeting peptide. Finally, free RGD peptide was removed by ultracentrifugation, and the engineered exosomes, EV@P-Arg-DNaseI-RGD, were resuspended in PBS.

[0047] Zeta potential analysis showed that the surface potential of exosomes increased from the original -30 mV to +20-30 mV, indicating that P-Arg and RGD modification was successful; TEM observation showed that the exosomes were intact in morphology without aggregation or structural damage. NTA measured the average particle size to be approximately 120-130 nm, and the functional assembly process did not significantly affect the stability of its nanostructure.

[0048] During the RGD modification process, an RGD polypeptide containing the Arg-Gly-Asp sequence is used. The C-terminal thiol (-SH) in the RGD polypeptide can combine with the P-Arg polypeptide or the thiol (-SH) on the surface of the exosome membrane under oxidative conditions to form a stable disulfide bond (-SS-). Under mild oxidative conditions, the C-terminal thiol (-SH) in the RGD polypeptide undergoes a nucleophilic reaction with the thiol active group to form a disulfide covalent bond, completing the stable grafting of the RGD polypeptide. Under moderate hydration conditions, the RGD polypeptide can bind to the exosome membrane protein through hydrogen bonding and hydrophobic adsorption, attach to the exosome surface, and achieve stable binding of the RGD targeting peptide.

[0049] The engineered exosomes obtained based on the above preparation method can achieve the following functions in RA lesions through the synergistic modification of three active ingredients (P-Arg, DNase I and RGD): (1) Immune regulation: Deer antler MSCs exosomes contain a variety of immune regulatory miRNAs, which can inhibit the differentiation of CD4+ T cells into Th17, promote the generation of Tregs, and restore immune homeostasis.

[0050] (2) cfDNA clearance: Studies have shown that cell-free DNA (cfDNA) in the synovial fluid of patients with rheumatoid arthritis is primarily derived from neutrophil extracellular traps (NETs) and synovial cell necrosis. cfDNA, as endogenous DAMPs, can activate the TLR9 and STING signaling pathways, further stimulating Th17 cell polarization and inflammatory cytokine expression. The present invention achieves cfDNA clearance by surface-loading DNase I, which can weaken the chronic inflammatory cycle of RA at the source and block the source of pro-inflammatory signals; targeted delivery.

[0051] (3) RGD peptides can bind to integrins (such as αvβ3) that are highly expressed in synovial and cartilage tissues, achieving drug enrichment at the site of inflammation. Specifically, the RGD (Arg-Gly-Asp) sequence can specifically bind to multiple integrin subtypes (such as αvβ3 and α5β1), which are upregulated in synovial vascular endothelial cells, fibroblast-like synoviocytes, and chondrocytes of RA patients. By grafting RGD peptides on the surface, EVs can actively move toward cells at the site of inflammation, achieving lesion-targeted delivery and improving bioavailability.

[0052] (4) Tissue protection: Exosomes themselves contain a variety of pro-repair factors that can slow down the inflammatory aging of chondrocytes and inhibit osteoclast differentiation, preventing the destruction of cartilage and bone structures.

[0053] Furthermore, the engineered exosomes prepared by the present invention have the following advantages over the prior art: (1) Uniqueness of source cells: For the first time, deer antler MSCs were used as the source cells for exosomes, solving the problems of poor batch stability and activity attenuation of exosomes derived from traditional MSCs.

[0054] (2) Multifunctional assembly design: The EV@P-Arg-DNaseI-RGD constructed in the present invention has four mechanisms: targeting, clearance, regulation, and protection, which are significantly superior to natural exosomes that only have immune regulation or repair-promoting functions.

[0055] (3) The surface modification strategy is simple and efficient: electrostatic adsorption + controlled covalent connection is used to achieve efficient encapsulation of exosome functional components while retaining their activity without affecting the integrity of the membrane structure.

[0056] (4) The process conditions are mild and controllable: the whole process has a low operating temperature (4-37°C) and a short operation time (1-2 h), which avoids protein denaturation or exosome aggregation and is suitable for industrial scale-up.

[0057] In summary, the engineered antler MSCs exosomes provided by the present invention have significant innovations in structural design, functional integration, source cell screening and preparation methods, have clear conditions for industrialization, and can provide a new and systematic treatment for rheumatoid arthritis.

[0058] In a specific implementation case, the engineered exosomes can be made into an injectable preparation and injected into the tail vein or intra-articular cavity for the treatment of RA. Animal experiments have shown that the dosage of exosomes is 1×10 8 Particles / unit, injected once every 3 to 5 days, and a significant therapeutic effect can be seen after taking the drug for more than 4 times. In other possible implementation cases, the above-mentioned engineered exosomes can also be made into sustained-release microspheres, transdermal gels or liposome preparations for the treatment of RA.

[0059] The specific implementation of the present invention is described below with reference to the following examples. Of course, the protection scope of the present invention is not limited to the following examples.

[0060] Example 1: Fresh, healthy sika deer antler tissue was obtained. Under sterile conditions, the junction of cartilage and cancellous bone was transversely dissected approximately 5 cm from the antler tip. The tissue slices were cut into approximately 2 mm thick slices. The mesenchymal layer was excised under a dissecting microscope and further cut into 1 mm³ blocks. The blocks were digested in 1% type I collagenase at 37°C for 1 hour. After digestion, free cells were collected by filtration and centrifugation and seeded in high-glucose DMEM medium supplemented with 10% fetal bovine serum (FBS). The cells were cultured at 37°C in a 5% CO2 incubator. When the cell confluence reached 90%, they were passaged and expanded to the third passage for future use. To extract exosomes, P3 antler mesenchymal stem cells were replaced with DMEM medium containing exosome-depleted FBS. After 48 hours of culture, the supernatant was collected. Exosomes were extracted using standard differential centrifugation, with cells removed by centrifugation at 300 × g for 10 min, debris removed by centrifugation at 2000 × g for 20 min, and large vesicles removed by centrifugation at 10,000 × g for 30 min at 4°C. The supernatant was then filtered through a 0.22 μm pore size filter membrane and ultracentrifuged at 100,000 × g for 70 min to collect the exosome precipitate. The precipitate was resuspended in PBS and washed by ultracentrifugation again to obtain high-purity antler MSCs.

[0061] The concentration of the exosomes extracted above was adjusted to 1×10 8 For 1000 μg / mL of exosome solution, add 25 μL of 10 mg / mL poly-L-arginine hydrochloride to 1 mL of the exosome solution. Incubate the mixture at 4°C with gentle rotation for 1 hour to allow the P-Arg peptide to electrostatically attach to the surface of the exosome phospholipid bilayer, forming a positively charged exosome complex (EV@P-Arg). Subsequently, centrifuge at 100,000 × g for 1 hour to remove free unbound P-Arg peptide, and resuspend the pellet in PBS to obtain exosomes surface-modified with P-Arg.

[0062] To the EV@P-Arg solution, add 0.5 μL of a 50 U / μL DNase I solution (total enzyme activity of 62.5 U). Mix gently and incubate at 37°C for 1 hour. This allows DNase I to stably bind to the P-Arg polypeptide through electrostatic interactions and become anchored on the exosome surface, forming the EV@P-Arg-DNase I complex. Unbound enzyme molecules are then removed by centrifugation at 100,000 × g for 1 hour. Functionalized exosomes carrying active DNase I are then obtained after resuspending in PBS.

[0063] 50 μL of the pre-prepared RGD tripeptide was added to the EV@P-Arg-DNase I complex and incubated at 4°C for 1 hour. The C-terminal thiol group of the RGD sequence formed a stable covalent bond with the thiol groups on the P-Arg polypeptide or the exosome membrane surface through disulfide bonds or nucleophilic reactions, completing the modification of the RGD targeting peptide. Finally, free RGD peptide was removed by ultracentrifugation, and the engineered exosomes, EV@P-Arg-DNaseI-RGD, were resuspended in PBS.

[0064] The product of Example 1 was used to perform the following tests or experiments.

[0065] (1) Extraction and verification of antler MSCs The free cell suspension obtained in Example 1 was filtered through a 70 μm cell strainer to collect the cells. MSCs were then isolated and purified by Percoll density gradient centrifugation or directly subjected to adherence screening. The main equipment used included a clean bench, a CO2 incubator, an inverted phase-contrast microscope, a constant-temperature water bath, a centrifuge, filters, and a transmission electron microscope (TEM). See the results Figure 1 shown.

[0066] Figure 1 Extraction of antler mesenchymal stem cells. Figure 1 Middle (A) is a fresh antler tissue slice. In the figure, there is a clear mesenchymal layer in the antler tissue. The tissue is dense and rich in blood vessels. The arrow symbol indicates the mesenchymal layer (i.e. the sampling area; Figure 1 Middle (B) shows optical microscopic observation of antler mesenchymal stem cells. In the figure, primary cultured antler mesenchymal stem cells can be seen adhering to the wall around the tissue block and crawling out, arranging in a spindle or vortex shape. The arrows in the figure indicate antler mesenchymal stem cells growing on the wall. Figure 1 Middle (C) is a TEM image of antler mesenchymal stem cells. The image shows that antler mesenchymal stem cells can secrete vesicular exosomes with uniform size distribution and typical morphology. The arrow symbol in the image indicates the exosome particle structure.

[0067] Depend on Figure 1 It can be proved that mesenchymal stem cells with typical morphological characteristics were successfully isolated from antler cartilage-like tissue. The cells have the ability to grow adherently and are arranged in a spindle or spiral shape, which is consistent with the morphological characteristics of MSCs. Under TEM, it can be seen that the cytoplasm contains rich organelles, supporting their stem cell identity.

[0068] (II) Verification of the differentiation ability of antler MSCs Third-generation antler mesenchymal stem cells (MSCs from Example 1) were seeded into 24-well plates. After reaching 70-80% confluency, the cells were cultured in osteogenic, adipogenic, and chondrogenic induction media, respectively, for 21 days each. The cells were then stained for osteogenic differentiation using Alizarin Red S, adipogenic differentiation using Oil Red O, and chondrogenic differentiation using Alcian Blue. The staining was observed microscopically to verify trilineage differentiation capacity.

[0069] See the results Figure 2 shown.

[0070] Figure 2 This is a diagram to verify the three-line differentiation ability of antler mesenchymal stem cells. Figure 2 Middle (A) is the image of osteogenic differentiation (Alizarin Red S staining), Figure 2 Middle (B) is the image of adipogenic differentiation (Oil Red O staining), Figure 2 Middle (C) is the image of chondrogenic differentiation (Alcian Blue staining). Figure 2 It can be seen that deer antler mesenchymal stem cells have good osteogenic, adipogenic and chondrogenic differentiation capabilities, have typical mesenchymal stem cell characteristics, and meet the three-lineage differentiation identification standards of MSCs.

[0071] (III) Structural characterization of antler MSCs exosomes The ultrastructural morphology of the antler MSCs exosomes (Example 1) was observed using a transmission electron microscope (TEM, JEM-1400, JEOL). The particle size distribution of the antler MSCs exosomes (Example 1) was detected using a nanoparticle tracking analyzer (NTA, ZetaView PMX120, Particle Metrix). The surface potential of the antler MSCs exosomes (Example 1) was measured using a Zeta potential analyzer (Zetasizer Nano ZS90, Malvern).

[0072] See the results Figure 3 shown.

[0073] Figure 3 This is the basic characterization diagram of antler MSCs exosomes. Figure 3 (A) and (B) are TEM morphology images and TEM magnified images, respectively. The extracted exosomes are shown to have typical cup-shaped or sac-shaped membrane structures, and the arrows point to exosome particles. Figure 3 Middle (C) is the particle size distribution diagram (NTA), in which the average particle size of exosomes is about 122.4 nm, showing a single peak distribution, indicating that the particle size is uniform. Figure 3Middle (D) shows the zeta potential of the exosomes, which is -31.86±1.62 mV, indicating good colloidal stability. This indicates that the antler MSC exosomes prepared in this invention meet internationally recognized standards for exosome biophysical characteristics.

[0074] (IV) Structural characterization of engineered exosomes Nanoparticle tracking analysis (NTA) and Zeta potential analyzer were used to measure the particle size and Zeta potential of the engineered exosomes (Example 1, EV@PDR), and compared with those of antler MSCs (EV), exosomes modified with P-Arg on the surface (EV@P), and functionalized exosomes carrying active DNase I on the surface (EV@PD).

[0075] See the results Figure 4 and Figure 5 shown.

[0076] Figure 4 The particle size change diagram of different exosome materials is shown in Figure 2. Figure 5 is the Zeta potential diagram of different exosome materials, Figure 4 and Figure 5 It can be seen that with the gradual superposition of exosome functional modifications (P-Arg, DNase-I, RGD), its Zeta potential gradually tends to neutral and the particle size increases slightly, indicating that the engineering modification is successful and maintains the nanoscale.

[0077] (V) Biocompatibility and safety Different modified exosome groups were taken: EV, EV@P, EV@PD and EV@PDR, and the relative survival rate of cells after treatment with different modified exosomes was detected by CCK-8 method; after incubating the different modified exosome groups with cells for 24 hours, Calcein-AM / PI double staining was used, and the distribution of live cells (green) and dead cells (red) was observed under a fluorescence microscope.

[0078] See the results Figure 6 and Figure 7 shown.

[0079] Figure 6 The figure shows the effect of exosomes modified with different concentrations of polyarginine (P-Arg) on ​​cell activity. As can be seen from the figure, when the P-Arg concentration is not higher than 40μg / mL, various modified exosomes have no significant inhibitory effect on cell activity, indicating that polyarginine has good biocompatibility in in vitro cell experiments at this concentration; however, this safe dose is limited to cell experiments and cannot be directly used as a reference for dosage in animal experiments.

[0080] Figure 7Calcein-AM / PI double-stained fluorescence images of cell activity and cell death after treatment with different modified exosomes. As can be seen in the figure, all exosome groups (EV, EV@P, EV@PD, EV@PDR) did not cause obvious cell death, and the cells were in good condition, indicating that different modified exosomes have good biocompatibility and low toxicity at the cellular level.

[0081] (6) Anti-inflammatory mechanism Different modified exosomes, including EV, EV@P, EV@PD, and EV@PDR, were added to LPS-induced inflammatory model cells. A blank group and a PBS group (phosphate-buffered saline, pH 7.4) were used as controls. After 24 hours, immunofluorescence staining was used to detect the expression of TNF-α (green) and COX-2 (red) proteins. DAPI was used to label the cell nucleus (blue), and fluorescence microscopy was used for photography and analysis.

[0082] See the results Figure 8 shown.

[0083] Figure 8 These are immunofluorescence images of TNF-α and COX-2 expression in inflammatory model cells under different exosome treatments. As can be seen in the figure, LPS stimulation (PBS group) significantly upregulated the expression of TNF-α and COX-2; after treatment with different engineered exosomes, especially in the EV@PD and EV@PDR groups, the expression of inflammatory factors was significantly weakened, indicating that it has a good anti-inflammatory effect.

[0084] In vitro isolation of mouse naive CD4 + T cells were screened by flow cytometry combined with surface marker antibody staining such as CD45, CD3, CD4, and CD62L / CD44, and the naïve CD4 T cells were screened according to the set gating strategy. + T cells. Figure 9 As shown, through a strict gating process, high-purity initial CD4 + T cell population (CD45 + 、CD3 + 、CD4 + 、CD62L + 、CD44 - ), with a purity of over 97%, which can be used for subsequent functional analysis or immune experiments.

[0085] Different exosome groups (Control, PBS, EV, EV@P, EV@PD, and EV@PDR) were treated with Th17 or Treg polarization conditions, and IL-17A was detected by flow cytometry. + 、CD4 +(Th17) and CD25 + 、Foxp3 + 、CD4 + (Treg) cell ratio.

[0086] See the results Figure 10 shown.

[0087] Figure 10 This is a flow cytometric analysis diagram for in vitro experimental verification of Th17 / Treg differentiation. As can be seen from the figure, the PBS group significantly induced an increase in the proportion of Th17 cells and a decrease in the proportion of Treg cells when treated, while the EV@PDR group could effectively inhibit Th17 differentiation and increase the proportion of Treg cells, suggesting that it has the effect of regulating Th17 / Treg balance and exerting anti-inflammatory immune regulatory effects.

[0088] (VII) RA treatment effect After establishing the RA mouse model, the mice were treated with PBS, methotrexate (MTX), or different types of exosome drugs. After a certain period of continuous drug administration, the appearance of the mouse feet was photographed to evaluate the degree of relief of joint redness and swelling.

[0089] See the results Figure 11 shown.

[0090] Figure 11 This is a comparison of joint pathology in RA mice after treatment with exosome-derived drugs. As can be seen from the figure, compared with normal mice (Control), the joints of mice in the PBS group were obviously red and swollen, and the pathological state was serious. After treatment with EV@PD and EV@PDR, the joint redness and swelling were significantly alleviated, especially with EV@PDR, indicating that it has a good anti-inflammatory therapeutic effect.

[0091] (8) Long-term effect DID dye was injected into mice in free form (Free DID), loaded in EV (EV-DID) or engineered EV@PDR (EV@PDR-DID). Dynamic fluorescence imaging was performed at multiple time points (2h, 6h, 12h, 24h, 48h and 72h) using the small animal in vivo fluorescence imaging system (IVIS) to evaluate the in vivo distribution and targeted homing ability of exosomes.

[0092] See the results Figure 12 shown.

[0093] Figure 12 This is a fluorescent imaging image of a mouse in vivo. As can be seen from the figure, compared with Free DID and ordinary EV-DID, EV@PDR-DID exhibits stronger fluorescence signal intensity and longer retention time in vivo, indicating that it has superior in vivo targeting and enrichment capabilities, indicating that engineered exosomes have good potential as a delivery carrier.

[0094] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the scope of protection of the present invention.

Claims

1. A ternary co-modified engineered pilose antler MSCs exosome, characterized by: Include: A. Deer antler MSCs exosomes; B. Polyarginine peptide-deoxyribonuclease I complex located on the surface of velvet MSCs exosomes; C. RGD peptide containing the Arg-Gly-Asp sequence modified on the surface of antler MSCs exosomes.

2. The ternary co-modified engineered pilose antler MSCs exosomes according to claim 1, characterized in that: Deoxyribonuclease I was loaded onto the surface of antler MSCs exosomes through electrostatic adsorption mediated by polyarginine polypeptide to obtain a polyarginine polypeptide-deoxyribonuclease I complex located on the surface of antler MSCs exosomes.

3. The ternary co-modified engineered pilose antler MSCs exosomes according to claim 2, characterized in that: The RGD polypeptide modified on the surface of antler MSCs exosomes is combined with the antler MSCs exosomes by chemical cross-linking or physical adsorption, or is combined with the polyarginine polypeptide in the polyarginine polypeptide-deoxyribonuclease I complex by chemical cross-linking.

4. A method for preparing ternary co-modified engineered pilose antler MSCs exosomes, characterized by: Taking antler MSCs exosomes as the main body, deoxyribonuclease I was loaded on the surface of antler MSCs exosomes through polyarginine peptide-mediated electrostatic adsorption. After the polyarginine peptide-deoxyribonuclease I complex was obtained on the surface of antler MSCs exosomes, RGD polypeptide containing the Arg-Gly-Asp sequence was modified on the surface of antler MSCs exosomes through chemical cross-linking or physical adsorption to form ternary co-modified engineered exosomes.

5. The method for preparing the ternary co-modified engineered pilose antler MSCs exosomes according to claim 4, characterized in that: Deer antler MSCs exosomes are obtained by shearing and digesting deer antler tissue to separate deer antler mesenchymal stem cells, and then culturing and amplifying them before extracting exosomes.

6. The method for preparing ternary co-modified engineered pilose antler MSCs exosomes according to claim 4, characterized in that: Polyarginine polypeptide is added to the antler MSCs exosome solution to obtain exosomes with polyarginine polypeptide surface modification, and then deoxyribonuclease I solution is added to obtain a polyarginine polypeptide-deoxyribonuclease I complex on the surface of the antler MSCs exosomes.

7. The method for preparing ternary co-modified engineered pilose antler MSCs exosomes according to claim 6, characterized in that: The concentration of antler MSCs exosome solution was 1×10 8 ~1×10 9 particles / mL; the concentration of polyarginine polypeptide is 1-10 mg / mL, and the concentration of deoxyribonuclease I solution is 25-50 U / μL.

8. The method for preparing ternary co-modified engineered pilose antler MSCs exosomes according to claim 6, characterized in that: The molecular weight of polyarginine polypeptides is 5000 to 15000 Da.

9. The method for preparing ternary co-modified engineered pilose antler MSCs exosomes according to claim 4, characterized in that: Modification on the surface of antler MSCs exosomes is to combine the RGD polypeptide with the antler MSCs exosomes by chemical cross-linking or physical adsorption, or to combine the RGD polypeptide with the polyarginine polypeptide in the polyarginine polypeptide-deoxyribonuclease I complex by chemical cross-linking.

10. Use of ternary co-modified engineered pilose antler MSCs exosomes in the preparation of a drug for treating or preventing rheumatoid arthritis, characterized by: The ternary co-modified engineered pilose antler MSCs exosomes are as described in claims 1 to 3, or are prepared by the preparation method of claims 4 to 9, and the drug is an injection, sustained-release microspheres, transdermal gel or liposome preparation.

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