A ternary co-modified engineered pilose antler MSCs exosome as well as a preparation method and application thereof

Abstract

The application discloses a kind of ternary co-modified engineering velvet MSCs exosomes and preparation method and application thereof, belong to the biomedicine technical field, it includes velvet MSCs exosomes, polyarginine polypeptide-deoxyribonuclease I complex located on the surface of velvet MSCs exosome, and RGD polypeptide with Arg-Gly-Asp sequence modified on the surface of velvet MSCs exosome.The purpose is to solve the problem that the "integration" treatment mode of multiple pathological links cannot be realized when existing RA is treated, for the first time, three active components polyarginine polypeptide (P-Arg), deoxyribonuclease I (Dnase I) and RGD polypeptide are modified on the surface of velvet MSCs exosome, to form surface engineered modified exosome complex, multiple functions of immune regulation, cfDNA removal, targeted delivery and tissue protection can be realized in RA lesion, and a new, systematic treatment method is provided for rheumatoid arthritis.

Description

Technical Field

[0001] This invention relates to a ternary co-modified engineered deer antler MSCs exosome, its preparation method, and its application. Specifically, it relates to a deer antler MSCs exosome synergistically modified by three active components: polyarginine polypeptide (P-Arg), deoxyribonuclease I (DNase I), and RGD polypeptide. It also covers the preparation method of the exosome and its application in the treatment or prevention of rheumatoid arthritis (RA), belonging to the field of biomedical technology. Background Technology

[0002] Rheumatoid arthritis (RA) is a common, chronic, progressive autoimmune disease that primarily affects peripheral joints, causing synovitis, cartilage destruction, and bone erosion. Patients often experience joint redness, swelling, pain, and dysfunction; in severe cases, it can lead to joint deformities and disability. The pathogenesis of RA is complex, involving multiple factors such as immune system disorders and imbalances in bone and cartilage metabolism. Initial CD4... + Imbalance in T cell subsets plays a crucial role: inflammatory Th17 cells overdifferentiate and secrete various inflammatory factors such as IL-17, driving synovial inflammation and joint tissue destruction; while the number or function of immunosuppressive regulatory T cells (Tregs) is insufficient, failing to effectively control abnormal immune responses. An imbalanced Th17 / Treg ratio is considered a significant contributing factor to rheumatoid arthritis (RA). Furthermore, high levels of cell-free DNA (cfDNA) are frequently detected in the synovial fluid and blood of RA patients. This cfDNA originates partly from the extracellular trapping network (NET) formed by neutrophils under inflammatory stimulation and partly from the abnormal death of synovial fibroblasts and macrophages. Large amounts of unremoved cfDNA can act as endogenous danger signals (DAMPs), activating innate immune pathways such as the Toll-like receptor (TLR9), further amplifying the inflammatory response. In RA-affected joints, DNA released from NETs forms immune complexes with self-antigens, inducing the production of autoantibodies and complement activation, leading to persistent synovial inflammation and tissue damage. Studies have shown that NET-related cell-free DNA plays an important role in the pathological process of rheumatoid arthritis (RA), and timely removal of these cell-free DNAs may alleviate disease progression. However, current clinical treatments for cfDNA or NET are limited.

[0003] In the chronic inflammatory environment of the joint, chondrocytes also undergo inflammatory senescence. Senescent chondrocytes secrete large amounts of pro-inflammatory cytokines and matrix-degrading enzymes (i.e., senescence-associated secretory phenotype SASP), such as IL-6 and MMP-13, exacerbating cartilage matrix degradation and joint destruction. On the other hand, RA patients exhibit increased osteoclast production, and RANKL-mediated excessive osteoclast differentiation leads to enhanced bone resorption, which is the main cause of joint bone erosion and bone loss. In summary, the pathogenesis of RA involves multiple factors, including immune imbalance (Th17 / Treg abnormalities), persistent inflammatory response (large amounts of pro-inflammatory factors and cfDNA), premature senescence of chondrocytes, and hyperactive osteoclast activity. Existing treatments (such as methotrexate, hormones, and anti-TNF-α / IL-6 biologics) primarily target inflammation and immunity, but have limited effectiveness in protecting cartilage and improving bone destruction, and long-term use may lead to side effects such as infection. Therefore, a multi-target comprehensive therapy is urgently needed to simultaneously correct immune disorders, eliminate inflammatory inducing factors, and promote joint tissue repair.

[0004] Recent studies have revealed that mesenchymal stem cells (MSCs) are considered a potential strategy for treating autoimmune diseases such as rheumatoid arthritis (RA) due to their immunomodulatory and tissue regeneration capabilities. However, stem cell transplantation faces challenges 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, carrying various active molecules such as proteins, messenger RNA, and microRNAs, and can mediate intercellular communication. MSC-derived exosomes have been shown to exhibit similar therapeutic effects to MSCs in certain inflammation and tissue injury models, offering advantages such as ease of preservation and low immunogenicity. In RA, reports have indicated that bone marrow mesenchymal stem cell exosomes can inhibit the inflammatory response of fibroblast-like synovial cells and regulate macrophage polarization, thereby alleviating arthritis symptoms. However, the efficacy of natural exosomes remains limited, lacking the ability to actively target specific lesions. Furthermore, ordinary exosomes cannot actively clear inflammatory stimuli such as NETs. Therefore, how to engineer MSC exosomes to endow them with stronger multi-functional therapeutic capabilities is currently a hot research topic.

[0005] Deer antlers are the newly grown antler tissue of sika deer and other deer species. In traditional Chinese medicine, they are believed to have the effects of "strengthening bones and tendons" and anti-aging. Modern research shows that deer antlers contain abundant growth factors and regenerative molecules, and their derived stem cells (deer antler mesenchymal stem cells) have exceptionally strong proliferative capacity and multi-lineage differentiation potential. Compared with conventional MSCs, deer antler MSCs can be passaged for more generations while maintaining viability; it has been reported that deer antler MSCs can be passaged for more than 50 generations in vitro without significant aging. Conditioned culture media for deer antler MSCs have been shown to promote skin wound healing. These characteristics make deer antler MSCs an ideal source of cells for preparing therapeutic exosomes, and they are expected to provide a stable and high-yield source of exosomes.

[0006] Currently, Chinese patent CN115386542A reports a deer antler stem cell exosome 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 multiple systemic pathological changes, there are no reports of an "integrated" treatment scheme based on deer antler MSCs exosomes. Therefore, this invention combines deer antler MSCs exosomes with modern nanomedicine technology to construct a multifunctional synergistically modified exosome therapeutic system, which has significant innovative significance and clinical application value in the field of RA treatment. Summary of the Invention

[0007] This invention aims to address the limitations of existing RA treatments in achieving an "integrated" treatment model that addresses multiple pathological stages. It proposes a ternarily co-modified engineered deer antler MSCs exosome, the first of its kind to synergistically modify the surface of deer antler MSCs exosomes with three active components: polyarginine polypeptide (P-Arg), deoxyribonuclease I (Dnase I), and RGD polypeptide. This forms a surface-engineered exosome complex that can perform multiple functions in RA lesions, including immune regulation, cfDNA clearance, targeted delivery, and tissue protection. Furthermore, this invention provides a method for preparing this exosome and its application in the preparation of drugs for treating or preventing rheumatoid arthritis, offering a novel and systemic treatment approach for the disease.

[0008] This invention is achieved through the following technical solution: a ternary co-modified engineered deer antler MSCs exosome, comprising:

[0009] A. Deer antler MSCs exosomes;

[0010] B. Polyarginine polypeptide-deoxyribonuclease I complex located on the surface of exosomes of deer antler MSCs;

[0011] C. An RGD polypeptide modified on the surface of deer antler MSCs exosomes and containing the Arg-Gly-Asp sequence.

[0012] Deoxyribonuclease I was loaded onto the surface of deer antler MSCs exosomes via electrostatic adsorption mediated by polyarginine peptides, resulting in a polyarginine peptide-deoxyribonuclease I complex located on the surface of deer antler MSCs exosomes.

[0013] The RGD peptides modified on the surface of deer antler MSCs exosomes bind to deer antler MSCs exosomes through chemical cross-linking or physical adsorption, or bind to the polyarginine peptides in the polyarginine peptide-deoxyribonuclease I complex through chemical cross-linking.

[0014] A method for preparing ternarily co-modified engineered deer antler MSCs exosomes involves using deer antler MSCs exosomes as the main body, loading deoxyribonuclease I onto the surface of deer antler MSCs exosomes via electrostatic adsorption mediated by polyarginine peptides, obtaining a polyarginine peptide-deoxyribonuclease I complex on the surface of deer antler MSCs exosomes, and then modifying the surface of deer antler MSCs exosomes with RGD peptides containing Arg-Gly-Asp sequences through chemical cross-linking or physical adsorption to form ternarily co-modified engineered exosomes.

[0015] Deer antler MSCs exosomes are obtained by cutting and digesting deer antler tissue to separate deer antler mesenchymal stem cells, and then culturing and expanding them before extracting exosomes.

[0016] Polyarginine peptides were added to the exosome solution of deer antler MSCs to obtain exosomes with polyarginine peptides on the surface. Then, deoxyribonuclease I solution was added to obtain polyarginine peptide-deoxyribonuclease I complex on the surface of deer antler MSCs exosomes.

[0017] The concentration of the deer antler MSCs exosome solution was 1×10⁻⁶. 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.

[0018] The molecular weight of polyarginine peptides is 5000–15000 Da.

[0019] Modification of the surface of deer antler MSCs exosomes involves combining RGD peptides with deer antler MSCs exosomes through chemical cross-linking or physical adsorption, or combining RGD peptides with polyarginine peptides in polyarginine peptide-deoxyribonuclease I complexes through chemical cross-linking.

[0020] The application of ternary co-modified engineered deer antler MSCs exosomes in the preparation of drugs for the treatment or prevention of rheumatoid arthritis. The ternary co-modified engineered deer antler MSCs exosomes are as described above, or prepared by the above preparation method. The drug is an injection, sustained-release microsphere, transdermal gel or liposome formulation.

[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0022] (1) This invention can achieve integrated treatment of RA targeting multiple points, breaking through the limitations of traditional single-function exosomes. Traditional MSCs exosomes mainly act on a single pathway (such as anti-inflammation, anti-oxidation or macrophage polarization), and cannot simultaneously intervene in the multi-stage pathological process of RA, making it difficult to address the coexistence of immune disorders, cfDNA-mediated inflammation, cartilage aging and bone destruction. This invention achieves stable fixation of triple-function molecules on the surface of deer antler MSCs exosomes through electrostatic assembly and molecular adsorption, obtaining a three-function synergistic exosome constructed by the "P-Arg + DNase I + RGD" engineering strategy, which can achieve: inhibiting Th17, promoting Treg, and restoring Th17 / Treg immune homeostasis; surface-loaded DNase I clears cfDNA and blocks pro-inflammatory pathways such as TLR9 / NETs; delaying inflammatory senescence of chondrocytes and inhibiting excessive differentiation of osteoclasts; targeted enrichment in inflamed joints, and precise localization of multiple effects.

[0023] (2) This invention solves the problems of scalability and functional decline of traditional MSCs by constructing a novel exosome production system. Existing technologies have limitations in the in vitro passage capacity of bone marrow / adipose MSCs, which are prone to aging after 3-5 generations, resulting in low exosome yield, poor stability, and functional decline. This invention is the first to use sika deer antler mesenchymal stem cells (AMSCs) for exosome production, exhibiting strong cell passage capacity, high stability (expandable to over 50 generations), high exosome secretion, and superior biological activity.

[0024] (3) This invention endows exosomes with the ability to actively clear cfDNA by modifying the surface of exosomes with enzyme functionalization, thereby rapidly alleviating inflammation. Traditional exosomes lack endogenous nuclease activity and cannot clear a large amount of free cfDNA in the synovial fluid of RA, leading to continuous activation of immune pathways. This invention constructs a cationic interface on the surface of deer antler MSCs exosomes by modifying it with P-Arg, effectively anchoring DNase I enzyme, allowing it to retain enzyme activity and be located outside the exosome membrane, achieving efficient binding and degradation of cfDNA. Furthermore, by effectively controlling the loading ratio of DNase I, the incubation time, and the ultracentrifugation process, the dual stability of enzyme binding efficiency and biological activity is ensured.

[0025] (4) This invention can increase the targeting of exosomes and improve the delivery efficiency to joint lesions. In the prior art, natural EVs (Extracellular Vesicles) are widely distributed in the body and have no obvious tissue targeting, resulting in low therapeutic dose utilization and some even accumulating in the liver and spleen and becoming ineffective. This application grafts RGD tripeptide onto the surface of exosomes, which can bind to integrin receptors expressed by synovial cells, chondrocytes, etc., thereby enhancing the active targeting, enrichment, and retention capacity of exosomes at RA inflammatory sites. In this invention, the RGD polypeptide forms a non-covalent / covalent binding with the exosome membrane or P-Arg through Cys thiol groups. After incubation, multiple centrifugations are performed to remove free molecules, effectively preserving its complete activity and structural specificity.

[0026] (5) This invention enables a mild, standardized, and easily scalable exosome functional assembly process. In existing technologies, the preparation of some drug-loaded exosomes or functionalized EVs requires chemical cross-linking agents, dialysis, or high-temperature incubation, which carries risks of structural damage and reduced activity, hindering industrialization. This invention employs a physical method of low-temperature electrostatic adsorption combined with short-time incubation, eliminating the need for organic solvents or high-energy treatment, thus maximizing the preservation of exosome integrity and function, while also facilitating standardized scale-up under GMP (Good Manufacturing Practice) conditions. Furthermore, in this invention, by controlling the temperature throughout the process within the range of 4–37°C, combined with ultracentrifugation / particle size screening, material purification and structural consistency control can be achieved.

[0027] (6) This invention can comprehensively improve clinically relevant indicators of RA. Its efficacy has been demonstrated in animal experiments. In a CIA mouse model, after tail vein injection, the engineered deer antler MSCs exosomes with ternary co-modification can significantly reduce arthritis scores, cartilage destruction, bone erosion and inflammatory factor levels, and the Th17 / Treg ratio is significantly restored. The therapeutic effect of unmodified MSCs exosomes in RA animal experiments is limited, and there is a lack of comprehensive verification of immune cell lineage and tissue repair capacity. It can be seen that this invention uses an established mouse RA model to systematically evaluate the therapeutic effects of EV@P-Arg-DNaseI-RGD at multiple levels such as behavior, histology, molecular biology and immune lineage, and has technical advantages in RA treatment. Attached Figure Description

[0028] Figure 1 Extraction of deer antler mesenchymal stem cells.

[0029] Figure 2 This diagram demonstrates the three-lineage differentiation capacity of deer antler mesenchymal stem cells.

[0030] Figure 3 This is a basic characterization diagram of exosomes from deer antler MSCs.

[0031] Figure 4 This is a graph showing the particle size variation of different exosome materials.

[0032] Figure 5 Zeta potential diagrams for different exosome materials.

[0033] Figure 6 The effect of different concentrations of polyarginine (P-Arg) modified exosomes on cell viability is shown in the figure.

[0034] Figure 7 Calcein-AM / PI double-stained fluorescence images of cell viability and death after treatment with different modified exosomes.

[0035] Figure 8 Immunofluorescence images showing the expression of TNF-α and COX-2 in inflammatory model cells under different exosome treatments.

[0036] Figure 9 A flow cytometry gating strategy diagram for initial CD4+ T cell sorting.

[0037] Figure 10 Flow cytometry plots to validate Th17 / Treg differentiation in in vitro experiments.

[0038] Figure 11 Comparative images of joint pathology in RA mice after treatment with exosome-based drugs.

[0039] Figure 12 This is a live fluorescence imaging image of a mouse. Detailed Implementation

[0040] The invention's objective, technical solution, and beneficial effects will be further explained in detail below.

[0041] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the claimed invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0042] This invention successfully constructed a multifunctional engineered deer antler stem cell exosome—EV@P-Arg-DNaseI-RGD—through source cell screening, structural optimization, and surface functionalization modification, and applied it to the treatment of rheumatoid arthritis (RA). This engineered exosome can simultaneously achieve immune modulation, inflammation clearance, and joint tissue repair, overcoming the limitations of existing RA treatment strategies that only target a single pathological stage. It represents the first time that an "integrated" synergistic treatment of multiple pathological stages in RA has been achieved, providing a new approach for the comprehensive treatment of RA.

[0043] The product of this invention is a surface-engineered nanoscale exosome, consisting of an exosome host and a surface functional layer. The exosome host is derived from mesenchymal stem cells (AMSCs) isolated from sika deer antler tissue, and the secreted natural exosomes are extracted after culture. The surface functional layer includes three active components: polyarginine polypeptide (P-Arg), deoxyribonuclease I (DNase I), and RGD polypeptide. The surface of the exosome host is modified with the three active components through electrostatic adsorption and enzymatic binding. Among them, P-Arg is used to provide a cationic interface to load DNase I, while improving the affinity of the exosome membrane to the cell membrane or cfDNA. DNase I has nucleic acid degradation activity and can specifically remove cfDNA in the inflammatory microenvironment. The RGD polypeptide contains the Arg-Gly-Asp sequence and has a high affinity for integrin binding, giving the exosome the ability to target synovial membrane and chondrocytes.

[0044] Specifically, the engineered exosomes involved in this invention are prepared through the following steps:

[0045] (I) Deer antler stem cell culture and exosome extraction

[0046] Fresh, healthy sika deer antler tissue was selected. Under aseptic conditions, the cartilage-cancellous bone junction area was transversely cut approximately 5 cm from the antler tip, and the tissue was cut into thin slices approximately 2 mm thick. The mesenchymal layer was then dissected under a dissecting microscope and further cut into 1 mm³ tissue blocks. The tissue blocks were placed in a digestion solution containing 0.1% type I collagenase and digested at 37°C for 2 hours. After digestion was terminated, the cells were filtered and centrifuged to collect free cells. These cells were then seeded into high-glucose DMEM medium containing 10% fetal bovine serum (FBS) and cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 90%, the cells were passaged to the third generation for further use.

[0047] To extract exosomes, P3 generation Antler Mesenchymal Stem Cells (AMSCs) were replaced with DMEM medium containing exosome-depleted FBS, and cultured for 24–48 hours before collecting the culture supernatant. Exosome extraction was performed using standard differential centrifugation. The cells were removed by centrifugation at 300×g for 10 min at 4°C, followed by centrifugation at 2000×g for 20 min to remove debris, and centrifugation at 10,000×g for 30 min to remove large vesicles. The supernatant was filtered through a 0.22 μm pore size filter, and then ultracentrifuged at 100,000×g for 70 min to collect the exosome pellet. The pellet was resuspended in PBS and washed again by ultracentrifugation to obtain a high-purity exosome sample, namely, Antler MSCs exosomes.

[0048] 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.

[0049] (ii) Surface modification P-Arg

[0050] The concentration of the extracted exosomes was adjusted to 1×10⁻⁶. 8 ~1×10 9 For particle / mL exosome solution, take 1 mL of the solution and add 25 μL of 10 mg / mL poly-L-arginine hydrochloride (molecular weight range 5000–15000 Da). Incubate the mixture at room temperature with slow rotation for 0.5–1 hour to allow P-Arg peptides to attach to the phospholipid bilayer of exosomes via electrostatic adsorption, forming positively charged exosome complexes (EV@P-Arg). Subsequently, centrifuge at 100,000 × g for 1 hour to remove unbound P-Arg peptides, and resuspend the precipitate in PBS to obtain P-Arg-modified exosomes.

[0051] It should be noted that P-Arg is a strong cationic polypeptide. Its polyguanidine groups can electrostatically adsorb onto the phosphate groups in the phospholipids on the surface of EVs. Furthermore, its flexible molecular structure endows it with excellent encapsulation and binding stability. Therefore, compared to materials such as quaternary ammonium salts or polyethyleneimine, P-Arg exhibits better biodegradability and lower cytotoxicity, making it more suitable as a functional linker for enzyme transport or peptide bonding.

[0052] (III) Loading DNase I enzyme

[0053] Add 0.5 μL of DNase I solution with a concentration of 25–50 U / μL (i.e., total enzyme activity of 12.5–25 U) to the above EV@P-Arg solution, mix slowly, and incubate at 37°C for 0.5–2 hours. This allows DNase I to form a stable binding with the P-Arg peptide through electrostatic interaction and anchor on the surface of the exosome, forming the EV@P-Arg-DNase I complex. Then, centrifuge at 100,000 × g for 1 hour to remove unbound enzyme molecules, and resuspend in PBS to obtain functionalized exosomes carrying active DNase I on their surface.

[0054] 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 assays showed that the engineered exosomes still possessed significant cfDNA degradation capabilities.

[0055] (iv) Grafted RGD polypeptide

[0056] 70 μL of pre-prepared RGD tripeptide (GRGDNP type, 10 mM, dissolved in sterile water) was added to the EV@P-Arg-DNase I complex system and incubated at room temperature for 1–2 hours. The C-terminal thiol groups in the RGD sequence formed stable covalent bonds with the thiol groups on the surface of the P-Arg peptide or exosome membrane via disulfide bonds or nucleophilic reactions, or attached to the surface through hydrophobic adsorption, thus completing the modification of the RGD-targeting peptide. Finally, the free RGD peptide was removed by ultracentrifugation, and the resulting exosome was resuspended in PBS to obtain the final engineered exosome EV@P-Arg-DNaseI-RGD.

[0057] Zeta potential analysis showed that the surface potential of exosomes increased from the original -30 mV to +20-30 mV, indicating that the P-Arg and RGD modification was successful. TEM observation showed that the exosomes were morphologically intact and did not aggregate or structurally damaged. The average particle size measured by NTA was about 120-130 nm, and the functional assembly process did not significantly affect the stability of its nanostructure.

[0058] During RGD modification, RGD peptides containing the Arg-Gly-Asp sequence are used. The C-terminal thiol group (-SH) in the RGD peptide can bind with the thiol group (-SH) on the surface of the P-Arg peptide or exosome membrane to form a stable disulfide bond (-SS-) under oxidizing conditions. Under mild oxidizing conditions, the C-terminal thiol group (-SH) in the RGD peptide undergoes a nucleophilic reaction with the active thiol group to form a disulfide covalent bond, thus completing the stable grafting of the RGD peptide. Under moderate hydration conditions, the RGD peptide can bind to exosome membrane proteins through hydrogen bonding and hydrophobic adsorption, attaching to the surface of the exosome and achieving stable binding of the RGD targeting peptide.

[0059] The engineered exosomes obtained based on the above preparation method can achieve the following functions in RA lesions through synergistic modification of three active ingredients (P-Arg, DNase I, and RGD):

[0060] (1) Immune regulation: Deer antler MSCs exosomes contain a variety of immunomodulatory miRNAs, which can inhibit the differentiation of CD4+ T cells into Th17 cells, promote the generation of Treg cells, and restore immune homeostasis.

[0061] (2) cfDNA clearance: Studies have shown that cell-free DNA (cfDNA) in the synovial fluid of rheumatoid arthritis patients mainly originates from neutrophil extracellular traps (NETs) and synovial cell necrosis. As endogenous DAMPs, cfDNA can activate the TLR9 and STING signaling pathways, further stimulating Th17 cell polarization and inflammatory factor expression. This invention achieves cfDNA clearance by surface loading of DNase I, which can weaken the chronic inflammatory cycle of RA from the source and block pro-inflammatory signal sources; targeted delivery.

[0062] (3) RGD peptides can bind to integrins (such as αvβ3) 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), and these integrins are upregulated in synovial vascular endothelial cells, fibroblast-like synovial cells, and chondrocytes of RA patients. By grafting RGD peptides onto the surface, EVs can actively approach cells at the site of inflammation, achieving targeted delivery to the lesion and improving bioavailability.

[0063] (4) Tissue protection: Exosomes contain a variety of repair-promoting factors, which can slow down the inflammatory aging of chondrocytes and inhibit osteoclast differentiation, thus preventing the destruction of cartilage and bone structures.

[0064] Furthermore, the engineered exosomes prepared by this invention have the following advantages that distinguish them from the prior art:

[0065] (1) Uniqueness of source cells: For the first time, deer antler MSCs are used as the source cells for exosomes, which solves the problems of poor batch stability and activity decay of exosomes from traditional MSCs.

[0066] (2) Multifunctional assembly design: The EV@P-Arg-DNaseI-RGD constructed in this invention has four mechanisms: targeting, clearing, regulating and protecting, which is significantly superior to natural exosomes that only have immune regulation or repair promotion functions.

[0067] (3) The surface modification strategy is simple and efficient: electrostatic adsorption and controllable covalent connection are used to achieve efficient encapsulation of exosome functional components and retain their activity without affecting the integrity of the membrane structure.

[0068] (4) Mild and controllable process conditions: The entire process operates at low temperatures (4-37℃) and for short durations (1-2 h), avoiding protein denaturation or exosome aggregation, making it suitable for industrial scale-up.

[0069] In summary, the engineered deer antler MSCs exosomes provided by this invention have significant innovations in structural design, functional integration, source cell screening, and preparation methods, and have clear conditions for industrialization, providing a novel and systematic treatment for rheumatoid arthritis.

[0070] In specific implementation cases, the engineered exosomes described above can be formulated into injectable preparations for the treatment of rheumatoid arthritis (RA) via tail vein injection or intra-articular injection. Animal experiments have demonstrated that the optimal dosage is 1 × 10⁻⁶ exosomes. 8 The medication is administered in granules / vials, injected once every 3-5 days, with a total of 4 or more injections required to achieve significant therapeutic effects. In other possible implementation cases, the engineered exosomes described above can also be formulated into sustained-release microspheres, transdermal gels, or liposomes for the treatment of rheumatoid arthritis (RA).

[0071] The specific implementation of the present invention will be described below with reference to the embodiments. Of course, the scope of protection of the present invention is not limited to the following embodiments.

[0072] Example 1:

[0073] Fresh, healthy sika deer antler tissue was selected. Under aseptic conditions, the cartilage-cancellous bone junction area was transversely cut approximately 5 cm from the antler tip, and the tissue was cut into thin slices approximately 2 mm thick. The mesenchymal layer was then dissected under a dissecting microscope and further cut into 1 mm³ tissue blocks. The tissue blocks were placed in a solution containing 1% type I collagenase and digested at 37°C for 1 hour. After digestion, the cells were filtered and centrifuged to collect free cells, which were then seeded in high-glucose DMEM medium containing 10% fetal bovine serum (FBS) and cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 90%, the cells were passaged to expand to the third generation for use. To extract exosomes, the P3 generation antler mesenchymal stem cells were replaced with DMEM medium containing exosome-depleted FBS and cultured for another 48 hours before collecting the culture supernatant. Exosomes were extracted using standard differential centrifugation. Cells were removed by centrifugation at 300×g for 10 min at 4°C, debris was removed by centrifugation at 2000×g for 20 min, and large vesicles were removed by centrifugation at 10,000×g for 30 min. The supernatant was filtered through a 0.22 μm pore size filter membrane and then collected by ultracentrifugation at 100,000×g for 70 min. The exosome precipitate was resuspended in PBS and washed by ultracentrifugation again to obtain high-purity deer antler MSCs.

[0074] The concentration of the extracted exosomes was adjusted to 1×10⁻⁶. 8 For particle / mL exosome solution, take 1 mL of exosome solution and add 25 μL of 10 mg / mL poly-L-arginine hydrochloride. Incubate the mixture at 4°C with slow rotation for 1 hour to allow P-Arg peptides to attach to the phospholipid bilayer of exosomes via electrostatic adsorption, forming positively charged exosome complexes (EV@P-Arg). Subsequently, centrifuge at 100,000 × g for 1 hour to remove unbound P-Arg peptides, and resuspend the precipitate in PBS to obtain P-Arg-modified exosomes.

[0075] Add 0.5 μL of DNase I solution with a concentration of 50 U / μL (i.e., total enzyme activity of 62.5 U) to the above EV@P-Arg solution, mix slowly, and incubate at 37°C for 1 hour. This allows DNase I to form a stable binding with the P-Arg peptide through electrostatic interaction and anchor on the surface of the exosome, forming the EV@P-Arg-DNase I complex. Subsequently, centrifuge at 100,000×g for 1 hour to remove unbound enzyme molecules, and resuspend in PBS to obtain functionalized exosomes carrying active DNase I on their surface.

[0076] 50 μL of pre-prepared RGD tripeptide was added to the EV@P-Arg-DNase I complex system, and the mixture was incubated at 4 °C for 1 hour. The C-terminal thiol group in the RGD sequence formed a stable covalent bond with the thiol group on the surface of the P-Arg peptide or exosome membrane via disulfide bonding or nucleophilic reaction, completing the modification of the RGD-targeting peptide. Finally, the free RGD peptide was removed by ultracentrifugation, and the resulting engineered exosome EV@P-Arg-DNaseI-RGD was obtained after resuspending in PBS.

[0077] The product from Example 1 above was subjected to the following tests or experiments.

[0078] (I) Validation of extraction of MSCs from deer antler

[0079] The free cell suspension obtained in Example 1 was filtered through a cell filter (70 μm) to collect the cells. MSCs were then purified by Percoll density gradient centrifugation or directly screened for adhesion. 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).

[0080] See results Figure 1 As shown.

[0081] Figure 1 Extraction of deer antler mesenchymal stem cells. Figure 1 (A) is a photograph of a fresh deer antler tissue section. The image shows a distinct mesenchymal layer region in the deer 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 (B) shows an optical microscope observation of deer antler mesenchymal stem cells. In the figure, primary cultured deer antler mesenchymal stem cells can be seen crawling out of the tissue block from the wall and arranged in a spindle or spiral shape. The arrow symbol in the figure indicates the deer antler mesenchymal stem cells that grow adherently. Figure 1 Image C is a TEM image of deer antler mesenchymal stem cells. It can be seen that deer antler mesenchymal stem cells can secrete vesicle-like exosomes with uniform size distribution and typical morphology. The arrow symbol in the image indicates the exosome granular structure.

[0082] Depend on 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.

[0083] (II) Verification of the differentiation capacity of deer antler MSCs

[0084] 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.

[0085] See results Figure 2 As shown.

[0086] Figure 2 This diagram serves as a validation of the three-lineage differentiation capacity of deer antler mesenchymal stem cells. Figure 2 Image (A) shows osteogenic differentiation (Alizarin Red S staining). Figure 2 Image B shows adipogenic differentiation (Oil Red O staining). Figure 2 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, exhibiting typical mesenchymal stem cell characteristics and meeting the trilineage differentiation identification criteria for MSCs.

[0087] (III) Structural characterization of exosomes from deer antler MSCs

[0088] 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).

[0089] See results Figure 3 As shown.

[0090] Figure 3 This is a basic characterization diagram of exosomes from deer antler MSCs. Figure 3 (A) and (B) are TEM morphology images and magnified TEM images, respectively. The extracted exosomes show typical cup-shaped or sac-shaped membranous structures, with the arrows pointing to the exosome particles. Figure 3 The middle (C) diagram shows the particle size distribution (NTA). The diagram reveals that the average particle size of the exosomes is approximately 122.4 nm, exhibiting a unimodal distribution, indicating uniform particle size. Figure 3Figure (D) shows the Zeta potential diagram, where the Zeta potential of the exosomes is -31.86 ± 1.62 mV, indicating good colloidal stability. This demonstrates that the deer antler MSCs exosomes prepared in this invention meet internationally recognized standards for exosome biophysical characteristics.

[0091] (iv) Structural characterization of engineered exosomes

[0092] The particle size and zeta potential of engineered exosomes (Example 1, EV@PDR) were determined using nanoparticle tracking analysis (NTA) and zeta potential analyzer, and were compared with antler MSCs (EV), exosomes with P-Arg surface modification (EV@P), and functionalized exosomes with active DNase I on the surface (EV@PD).

[0093] See results Figure 4 and Figure 5 As shown.

[0094] Figure 4 This is a graph showing the particle size variation of different exosome materials. Figure 5 Zeta potential maps of different exosome materials, by 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 be neutral and the particle size increases slightly, indicating that the engineering modification is successful and maintains the nanoscale.

[0095] (v) Biocompatibility and safety

[0096] Different modified exosome groups were collected: EV, EV@P, EV@PD and EV@PDR. The relative viability of cells after treatment with different modified exosomes was detected by CCK-8 assay. After co-incubating the different modified exosome groups with cells for 24 hours, the distribution of live cells (green) and dead cells (red) was observed under a fluorescence microscope by Calcein-AM / PI double staining.

[0097] See results Figure 6 and Figure 7 As shown.

[0098] Figure 6 The figure shows the effect of different concentrations of polyarginine (P-Arg) modified exosomes on cell viability. As can be seen from the figure, within the range of P-Arg concentration not exceeding 40 μg / mL, various modified exosomes did not significantly inhibit cell viability, indicating that polyarginine has good biocompatibility in in vitro cell experiments at this concentration; however, this safe dose is only for cell experiments and cannot be directly used as a dosage reference for animal experiments.

[0099] Figure 7Calcein-AM / PI double-stained fluorescence images show cell viability and cell death after treatment with different modified exosomes. As can be seen from the images, none of the exosome groups (EV, EV@P, EV@PD, EV@PDR) caused significant cell death, and the cells were in good condition, indicating that the different modified exosomes have good biocompatibility and low toxicity at the cell level.

[0100] (vi) Anti-inflammatory mechanism

[0101] Different modified exosomes, namely 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 at pH 7.4) were used as controls. After 24 hours, the expression of TNF-α (green) and COX-2 (red) proteins was detected by immunofluorescence staining, and the cell nuclei were labeled with DAPI (blue). The cells were photographed and analyzed by fluorescence microscopy.

[0102] See results Figure 8 As shown.

[0103] Figure 8 Immunofluorescence images showing the expression of TNF-α and COX-2 in inflammatory model cells under different exosome treatments. As can be seen from the images, LPS stimulation (PBS group) significantly upregulated the expression of TNF-α and COX-2; while after treatment with different engineered exosomes, especially the EV@PD and EV@PDR groups, the expression of inflammatory factors was significantly reduced, indicating that they have good anti-inflammatory effects.

[0104] In vitro isolation of mouse initial CD4 + T cells were screened using flow cytometry combined with staining for surface marker antibodies such as CD45, CD3, CD4, and CD62L / CD44, and naïve CD4 cells were selected according to a predefined gating strategy. + T cells. See also Figure 9 As shown, high-purity initial CD4 was successfully obtained through a rigorous gating process. + T cell population (CD45) + CD3 + CD4 + CD62L + CD44 - Its purity reaches over 97%, and it can be used for subsequent functional analysis or immune experiments.

[0105] Differentiation was induced under Th17 or Treg polarization conditions, and different exosome groups (Control, PBS, EV, EV@P, EV@PD, and EV@PDR) were treated. IL-17A was detected by flow cytometry. + CD4+ (Th17) and CD25 + Foxp3 + CD4 + (Treg) cell proportion.

[0106] See results Figure 10 As shown.

[0107] Figure 10 The flow cytometry analysis was used to verify the Th17 / Treg differentiation in vitro. As shown in the figure, the PBS treatment significantly induced an increase in the proportion of Th17 cells and a decrease in the proportion of Treg cells, while the EV@PDR group effectively inhibited Th17 differentiation and upregulated the proportion of Treg cells, suggesting that it has the function of regulating the Th17 / Treg balance and playing an anti-inflammatory and immunomodulatory role.

[0108] (vii) Treatment efficacy of RA

[0109] After establishing the RA mouse model, mice were treated with PBS, methotrexate (MTX), or different types of exosome drugs. After a certain period of continuous administration, images of the mouse feet were taken to assess the degree of relief of joint redness and swelling.

[0110] See results Figure 11 As shown.

[0111] Figure 11 The images show a comparison of joint pathology in RA mice after treatment with exosome materials. As can be seen from the images, compared with normal mice (Control), the joints of mice in the PBS group were significantly red and swollen, and the pathological condition was severe. After treatment with EV@PD and EV@PDR, the redness and swelling of the joints were significantly relieved, especially EV@PDR, indicating that it has a good anti-inflammatory therapeutic effect.

[0112] (viii) Long-term effectiveness

[0113] DID dyes were injected into mice in free form (Free DID), loaded onto EVs (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 a small animal in vivo fluorescence imaging system (IVIS) to assess the in vivo distribution and targeting homing ability of exosomes.

[0114] See results Figure 12 As shown.

[0115] Figure 12The image shows a live fluorescence image of a mouse. As can be seen from the image, 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, demonstrating that engineered exosomes have good potential as delivery vectors.

[0116] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications or equivalent changes made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A ternarily co-modified engineered deer antler MSCs exosome, characterized in that: Include: A. Deer antler MSCs exosomes; B. Polyarginine polypeptide-deoxyribonuclease I complex located on the surface of exosomes of deer antler MSCs; C. An RGD polypeptide modified on the surface of deer antler MSCs exosomes and containing the Arg-Gly-Asp sequence. The polyarginine polypeptide-deoxyribonuclease I complex located on the surface of deer antler MSCs exosomes is at a concentration of 1×10 8 ~1×10 9 A polyarginine peptide with a concentration of 1–10 mg / mL and a molecular weight of 5000–15000 Da was added to a deer antler MSCs exosome solution with a particle / mL ratio to obtain exosomes with a surface modified with polyarginine peptide. Then, a deoxyribonuclease I solution with a concentration of 25–50 U / μL was added to load deoxyribonuclease I onto the surface of deer antler MSCs exosomes through electrostatic adsorption mediated by polyarginine peptide.

2. The engineered deer antler MSCs exosomes with ternary co-modification according to claim 1, characterized in that: The RGD peptides modified on the surface of deer antler MSCs exosomes bind to deer antler MSCs exosomes through chemical cross-linking or physical adsorption, or bind to the polyarginine peptides in the polyarginine peptide-deoxyribonuclease I complex through chemical cross-linking.

3. A method for preparing engineered deer antler MSCs exosomes with ternary co-modification, characterized in that: Includes the following steps: S1. Preparation of deer antler MSCs exosomes; S2. At a concentration of 1×10 8 ~1×10 9 A polyarginine polypeptide with a concentration of 1–10 mg / mL and a molecular weight of 5000–15000 Da was added to a deer antler MSCs exosome solution with a particle size of 1 / mL to obtain exosomes with a surface modified with polyarginine polypeptide. Then, a deoxyribonuclease I solution with a concentration of 25–50 U / μL was added to load deoxyribonuclease I onto the surface of deer antler MSCs exosomes through electrostatic adsorption mediated by polyarginine polypeptide, thus preparing a polyarginine polypeptide-deoxyribonuclease I complex. S3. RGD peptides containing the Arg-Gly-Asp sequence were modified onto the surface of deer antler MSCs exosomes by chemical cross-linking or physical adsorption to form ternary co-modified engineered exosomes.

4. The method for preparing engineered deer antler MSCs exosomes with ternary co-modification according to claim 3, characterized in that: Deer antler MSCs exosomes are obtained by cutting and digesting deer antler tissue to separate deer antler mesenchymal stem cells, and then culturing and expanding them before extracting exosomes.

5. The method for preparing engineered deer antler MSCs exosomes with ternary co-modification according to claim 3, characterized in that: Modification of the surface of deer antler MSCs exosomes involves combining RGD peptides with deer antler MSCs exosomes through chemical cross-linking or physical adsorption, or combining RGD peptides with polyarginine peptides in polyarginine peptide-deoxyribonuclease I complexes through chemical cross-linking.

6. The application of ternarily co-modified engineered deer antler MSCs exosomes in the preparation of drugs for the treatment or prevention of rheumatoid arthritis, characterized in that: The engineered deer antler MSCs exosomes co-modified by the ternary method are as described in any one of claims 1 or 2, or prepared by any one of claims 3 to 5, wherein the drug is an injection, sustained-release microsphere, transdermal gel or liposome formulation.

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