DNase I-loaded pilose antler stem cell exosome as well as preparation method and application thereof

Abstract

The invention discloses a DNase I-loaded pilose antler stem cell exosome and a preparation method and application thereof, and belongs to the technical field of biological medicine, DNase I enzyme is stably and efficiently loaded on the surface of the pilose antler MSCs exosome through electrostatic adsorption of poly-arginine polypeptide, and the DNase I-loaded pilose antler stem cell exosome is prepared by combining the immune regulation function and the arthritis focus targeting characteristic of the exosome. And multi-mechanism accurate treatment on inflammatory diseases such as rheumatoid arthritis is realized. According to the technology, the drug loading efficiency and the structural stability of the exosome are remarkably improved, the biological activity and the anti-inflammatory effect of the DNase I are effectively reserved, the application obstacles that a traditional protein drug is unstable in vivo, easy to remove, poor in targeting property and the like are overcome, and good treatment prospects and conversion values are achieved. Therefore, the invention has remarkable innovativeness and practicability, is suitable for functional modification of exosome drugs and treatment of various chronic inflammation related diseases, and has important industrialization and clinical popularization prospects.

Description

Technical Field

[0001] The present invention relates to antler stem cell exosomes loaded with DNase I, a preparation method and application thereof, and specifically relates to antler stem cell exosomes loaded with deoxyribonuclease I (DNase I) with high efficiency and high activity, a preparation method and application thereof, and belongs to the field of biomedicine technology. Background Art

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic synovial inflammation and joint destruction. Its pathogenesis is complex, involving abnormal activation of immune cells and excessive production of inflammatory mediators. Recent studies have revealed that cell-free DNA (cfDNA) released by neutrophil extracellular traps (NETs) may play a key role in the inflammatory response in RA. Excessive amounts of cfDNA and NETs can act as danger signals, exacerbating autoimmune responses, promoting the production of inflammatory cytokines such as IL-17, and disrupting immune homeostasis. However, traditional RA therapies (such as anti-TNF-α and biologics) primarily target specific cytokines or immune cells and lack a direct effect on clearing cfDNA accumulated in lesions.

[0003] Studies have attempted to exogenously administer DNase I to decompose NETs and cfDNA, which theoretically can reduce RA inflammatory responses and correct immune imbalances. However, free DNase I has a short half-life in the body, is easily inactivated by inhibitors (such as DNase inhibitors in serum), and is difficult to effectively enrich in inflamed joints, so the therapeutic effect is limited. AMERICA, 2023 ("Bioinspired Nanogels as Cell-Free DNA Capture and Clearance Organelles for Rheumatoid Arthritis," Proceedings of the National Academy of Sciences of the United States of America) reported a method for treating rheumatoid arthritis by conjugating deoxyribonuclease I (DNase I) to cationic peptide dendrimer nanogels. The resulting nanogels, thanks to their naturally derived peptide components, exhibit high biocompatibility. Customizable in size and surface charge density, they achieve rapid targeting, highest accumulation, prolonged duration, and excellent DNA clearance in inflamed joints. While these nanogels demonstrate significant advantages for RA treatment, potential risks and challenges remain, including: First, complex synthesis steps and poor process controllability: The synthesis of dendrimers typically involves multiple chemical modifications and precise molecular structure control, resulting in poor batch-to-batch stability and hindering scalability. Second, insufficient enzyme activity retention: DNase I in this system is often covalently coupled to the nanogels, which can affect its native conformation and lead to decreased or inactivated catalytic activity. Third, the exposure site is uncontrollable and prone to protein shielding: In a bodily fluid environment, the enzyme structure on the gel surface may be coated by serum proteins, reducing its ability to recognize and degrade cfDNA. Fourth, the delivery system is highly rigid and lacks penetration: The relatively rigid structure of the nanogel may be detrimental to tissue penetration and enrichment in inflammatory sites. Fifth, there are risks to immune safety: Long-term retention of dendrimers in the body may trigger immune responses or inflammatory stimulation, lacking the immune adaptability advantages of naturally derived carriers.

[0004] Exosomes, as natural nanocarriers, possess excellent stability and biocompatibility and can be used to load drug molecules for delivery to specific tissues. Stem cell exosomes have demonstrated immunomodulatory and tissue repair effects in the treatment of diseases such as RA. However, conventional exosomes lack targets for specific pathogenic factors in the inflammatory environment of joints. Loading DNase I onto exosomes is expected to combine the delivery advantages of exosomes with the cfDNA degradation effect of DNase I, thereby providing a new solution to the pathological mechanisms of RA. Currently, there are no reports of antler MSC exosomes loaded with DNase I for the treatment of RA. The present invention provides a new and innovative approach in this area. Summary of the Invention

[0005] The present invention aims to provide DNase I-loaded antler stem cell exosomes, as well as their preparation and application. By introducing polyarginine as a mediating molecule, DNase I is effectively adsorbed and immobilized on the surface of antler stem cell exosomes, forming DNase I-functionalized exosomes. These exosomes can be used to prepare drugs for the treatment of rheumatoid arthritis. They can release DNase I activity at the site of joint inflammation, degrading excess cfDNA and NETs, ​​thereby reducing the stimulation of these pathogenic factors on the immune system. Furthermore, the bioactive molecules carried by the antler stem cell exosomes can promote immune balance, for example, by inhibiting inflammatory Th17 cells and promoting the function of regulatory T cells (Tregs), thereby alleviating RA inflammation and tissue damage at multiple levels.

[0006] The present invention is achieved through the following technical solution: a method for preparing antler stem cell exosomes loaded with DNase I, wherein polyarginine polypeptide and DNase I enzyme are dissolved in PBS buffer, and a DNase I-polyarginine complex is formed by electrostatic adsorption, and then the DNase I-polyarginine complex is mixed with antler stem cell exosomes to obtain antler stem cell exosomes loaded with DNase I. The concentration of polyarginine peptide in PBS buffer was controlled to be 1-10 mg / mL, and the concentration of DNase I enzyme was controlled to be 10-50 U / μL. The amount of exosomes from antler stem cells was controlled to be 1×10 8 ~1×10 9 Particles / ml.

[0007] The molecular weight of polyarginine polypeptide is 5000~15000Da.

[0008] Incubate at 20-25°C for 30-60 min to allow electrostatic adsorption to form a DNase I-polyarginine complex.

[0009] The DNase I-polyarginine complex was gently mixed with antler stem cell exosomes and incubated at 20-25°C for 30-60 minutes. After purification, antler stem cell exosomes loaded with DNase I were obtained.

[0010] Deer antler stem cell exosomes are obtained by digesting the deer antler tissue in digestive fluid, filtering and centrifuging to obtain deer antler mesenchymal stem cells, and then culturing and amplifying them before extracting the exosomes.

[0011] The digestion solution contains 1% type I collagenase and digestion is carried out at 37°C for 0.5 to 1 hour.

[0012] During culture, DMEM medium containing 10-15% fetal bovine serum was used and cultured at 37°C and 5% CO2 for 5-7 hours.

[0013] When extracting exosomes, the expanded cells are cultured for 12 to 24 hours, and the supernatant is collected. After impurities are removed, filtered, centrifuged, and washed, deer antler stem cell exosomes are obtained.

[0014] A DNase I-loaded antler stem cell exosome is obtained using the above preparation method.

[0015] The invention discloses a use of DNase I-loaded deer antler stem cell exosomes in the preparation of a drug for treating rheumatoid arthritis. The drug is an injection, sustained-release microspheres, a transdermal gel, or a liposome preparation, and further comprises at least one of leflunomide, methotrexate, sulfasalazine, tocilizumab, adalimumab, etanercept, or baricitinib.

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) Based on the therapeutic mechanism of DNase I, the present invention provides antler stem cell exosomes loaded with DNase I. Through the use of DNase I, free DNA in RA lesions can be degraded, thus reducing the adverse stimulation of the immune system from the source. This mechanism of action directly targets the key link in the RA inflammation amplification cycle, filling the gap in existing treatment methods that cannot clear cfDNA, making the treatment more comprehensive.

[0017] (2) The present invention solves the drawback that the existing exogenous administration of DNase I enzyme is difficult to enrich in the inflamed joint site and cannot achieve an effective therapeutic effect. It provides a process in which the DNase I enzyme is attached to the membrane surface of the exosome by electrostatic action mediated by polyarginine polypeptide to obtain DNase I-loaded exosomes. Exosomes as carriers can effectively protect the DNase I enzyme from degradation and inactivation in the body. The combination of polyarginine and exosomes can improve the stability of DNase I in the body circulation and promote its penetration into the inflamed site. Compared with the free enzyme, the DNase I loaded in the exosomes can maintain a higher concentration and longer activity in the local joint, thereby significantly enhancing the therapeutic effect.

[0018] (3) The present invention uses deer antler stem cell exosomes (deer antler MSCs exosomes). Since the microRNA and proteins carried by the exosomes can inhibit inflammatory responses and promote tissue regeneration, the DNase I-loaded exosomes prepared by the exosomes can not only play an enzymatic role in removing harmful DNA, but also the components of the deer antler MSCs exosomes can further synergistically regulate the immune system, reduce the levels of pro-inflammatory cytokines (such as TNF-α, IL-6, IL-17), increase the levels of anti-inflammatory factors (such as IL-10), and achieve the restoration of Th17 / Treg balance. This multi-target comprehensive therapeutic effect is obviously superior to therapies with a single mechanism of action.

[0019] (4) The deer antler MSC exosomes used in this invention are autologous / homologous cell secretions with good biocompatibility and low toxic side effects. DNase I is a natural enzyme in the human body and has been used clinically to treat cystic fibrosis. Therefore, the present invention's combined treatment strategy is expected to be highly safe and does not increase the risk of significant immunosuppression. Animal experiments showed no major adverse reactions, and joint function was significantly improved, demonstrating its potential for clinical application.

[0020] (5) The DNase I-loaded deer antler stem cell exosomes of the present invention can be combined with other drugs (such as anti-inflammatory drugs) to achieve multiple treatments for RA. They can also be expanded to other autoimmune and inflammatory diseases, such as systemic lupus erythematosus and other diseases accompanied by a large amount of NETs / cfDNA. Exosomes loaded with DNase I can also be considered for treatment, thereby developing a multifunctional composite exosome therapy.

[0021] In summary, the present invention combines DNase I enzyme with exosomes for the first time, providing a method for preparing an exosome-loaded enzyme system with high stability and targeting in vivo mediated by polyarginine polypeptide, achieving efficient aggregation of DNase I enzyme at the lesion site and sustained therapeutic effect. At the same time, combined with the therapeutic efficacy of deer antler MSCs exosomes on RA, it achieves the effect of alleviating RA inflammation and tissue damage at multiple levels, making it more suitable for translational applications as a whole. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Extraction of exosomes from antler mesenchymal stem cells.

[0023] Figure 2 This is a diagram verifying the three-lineage differentiation ability of antler mesenchymal stem cells.

[0024] Figure 3 This is the basic characterization diagram of antler MSCs exosomes.

[0025] Figure 4 The graph shows the changes in the surface Zeta potential of antler MSCs exosomes modified with different concentrations of poly-arginine. In the figure, (A) is modified with 0.2 mg / ml of poly-L-arginine peptide, (B) is modified with 0.4 mg / ml of poly-L-arginine peptide, (C) is modified with 0.8 mg / ml of poly-L-arginine peptide, (D) is modified with 1 mg / ml of poly-L-arginine peptide, (E) is modified with 5 mg / ml of poly-L-arginine peptide, and (F) is modified with 10 mg / ml of poly-L-arginine peptide.

[0026] Figure 5 Figure 2 shows the particle size distribution of deer antler MSCs exosomes modified with different concentrations of poly-arginine. In the figure, (A) is a 1 mg / ml poly-L-arginine peptide, (B) is a 5 mg / ml poly-L-arginine peptide, (C) is a 10 mg / ml poly-L-arginine peptide, (D) is a 0.2 mg / ml poly-L-arginine peptide, (E) is a 0.4 mg / ml poly-L-arginine peptide, and (F) is a 0.8 mg / ml poly-L-arginine peptide.

[0027] Figure 6 Figure 2 is a graph showing the cytotoxicity evaluation of differently modified exosomes at multiple concentrations.

[0028] Figure 7 Figure 2 shows the detection of serum biochemical indicators after mice were treated with different modified exosomes. (A) shows the comparison of serum ALT (alanine aminotransferase) concentrations, (B) shows the comparison of serum AST (aspartate aminotransferase) concentrations, (C) shows the comparison of serum UREA (urea) concentrations, and (D) shows the comparison of serum CREA (creatinine) concentrations.

[0029] Figure 8 Figure 2 shows the improvement in foot morphology in RA mouse models treated with different modified exosomes.

[0030] Figure 9 Fluorescence distribution imaging of exosomes in mice under different treatment groups. DETAILED DESCRIPTION

[0031] The objectives, technical solutions and beneficial effects of the present invention are further described in detail below.

[0032] 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.

[0033] The present invention combines DNase I with antler MSCs exosomes to produce DNase I-loaded antler stem cell exosomes. This process involves isolating and culturing mesenchymal stem cells from antler tissue in vitro to extract antler MSCs exosomes, and then using a positively charged polyarginine polypeptide to electrostatically attach the DNase I enzyme to the antler MSCs exosomes to produce DNase I-loaded antler stem cell exosomes. The polyarginine polypeptide-mediated DNase I enzyme in the present invention enables efficient and stable loading of the exosomes, maintaining persistent and high concentrations of DNase I activity in localized joints, significantly enhancing its therapeutic efficacy at inflamed sites. Because antler MSCs exosomes inherently inhibit inflammatory responses and promote tissue regeneration, the exosome-enzyme system of "DNase I + antler MSCs exosomes" is not only more stable in vivo and better targeted, but also exhibits a more pronounced therapeutic effect.

[0034] Compared to existing DNase I enzyme treatment strategies for RA (free DNase I enzyme or DNase I-coupled nanogel), the present invention uses a novel treatment system that electrostatically adsorbs DNase I on the surface of exosomes, which has the following technical advantages: (1) Retention of enzyme activity: Through polyarginine (P-Arg)-mediated electrostatic adsorption, the damage to the active center of DNase I by traditional chemical coupling is avoided, and its natural conformation and catalytic efficiency are maintained to the greatest extent. At the same time, polyarginine (P-Arg) provides a controllable positive charge density, which helps to ensure that DNase I is exposed outward and maintains its activity.

[0035] (2) Biocompatibility and low immunogenicity: Using deer antler MSCs exosomes as a natural carrier gives the system excellent biosafety and avoids the risk of long-term retention of synthetic materials (such as dendrimers); P-Arg as a natural amino acid polymer further reduces immune stimulation and overcomes the protein adsorption and shielding problem of traditional nanogels.

[0036] (3) Excellent delivery performance: The natural homing ability and flexible membrane structure of exosomes make it easier to break through the inflammatory barrier, achieving efficient accumulation and penetration of DNase I in the joints.

[0037] (4) Synergistic treatment: The dual-functional system of "DNase I + antler MSCs exosomes" can clear inflammation-related cfDNA and block the RA disease pathway; antler MSCs exosomes can inhibit inflammatory factors (such as TNF-α, IL-6, IL-17) and promote cartilage repair, achieving the synergistic effect of "anti-inflammatory + regeneration".

[0038] (5) Long-term effect: The exosome structure can protect DNase I from enzymatic hydrolysis to maintain its activity. P-Arg-mediated electrostatic adsorption can achieve stable loading of DNase I, thereby achieving long-term high-concentration maintenance of the drug in the local joint.

[0039] (6) Transformation advantages: No complex chemical coupling process, simple electrostatic adsorption process, high batch consistency, and easier large-scale production.

[0040] Specifically, the technical solution of the present invention can be summarized as follows: Preparation of antler MSCs exosome carriers: Cultivating and extracting antler MSCs exosomes from antler tissue-derived MSCs can provide stable and efficient exosome carriers for subsequent drug loading.

[0041] First, fresh antler tissue from healthy sika deer was selected, and mesenchymal tissue was harvested under sterile conditions. The antler tissue was minced (approximately 2 mm in length) and digested in a digestion solution containing 1% type I collagenase at 37°C for 0.5–1 h, with occasional gentle shaking to release cells. After digestion, the cells were filtered through a cell strainer to obtain a single-cell suspension, centrifuged to remove the digestive enzymes, and resuspended in PBS. The cells were seeded into culture dishes and cultured in low-glucose DMEM medium supplemented with 10–15% fetal bovine serum, allowing them to adhere and grow. The culture environment was maintained at 37–38°C and 5–7% CO₂. The medium was changed after 24–72 hours to remove non-adherent cells. When the cells reached 80–90% confluence, they were passaged and expanded to obtain third- or fourth-generation antler mesenchymal stem cells (MSCs) for subsequent experiments.

[0042] Optionally, the cell surface markers (such as CD90^+, CD105^+, CD45^-, etc.) are detected by flow cytometry to confirm the mesenchymal stem cell phenotype.

[0043] Next, exosomes are extracted and purified. Well-grown antler MSCs are cultured in exosome-free medium for an additional 48 hours, and approximately several hundred milliliters of cell culture supernatant is collected. Exosomes are purified by differential centrifugation in a sequential manner: cells are cleared by centrifugation at 300 × g for 5–10 minutes; the supernatant is transferred and centrifuged at 2,000 × g for 20 minutes to remove cellular debris and large vesicles; and finally, small impurities and microvesicles are removed by centrifugation at 10,000 × g for 30 minutes. The clarified supernatant is filtered through a 0.22 μm filter and then ultracentrifuged at 100,000 × g for 70 minutes to pellet the exosomes. After carefully aspirating the supernatant, the exosome pellet is resuspended in pre-chilled PBS and centrifuged again at 100,000 × g for 70 minutes to increase purity. Finally, the exosome pellet is resuspended in an appropriate amount of PBS (typically several hundred microliters, depending on pellet size) to obtain a highly purified antler MSC exosome suspension.

[0044] The exosome concentration was determined by particle size analysis and protein detection, and successful extraction was confirmed by observing the typical cup-shaped morphology of exosomes by transmission electron microscopy and detecting exosome marker proteins (such as CD9 and CD63) by Western blot.

[0045] Polyarginine-mediated DNase I loading: Using positively charged polyarginine peptides, DNase I is attached to the exosome surface via electrostatic interactions, thereby preparing functionalized DNase I exosomes. This method is simple and gentle, maintains DNase I enzymatic activity, and achieves high loading rates. The specific preparation steps are as follows: (1) Material preparation: Obtain DNase I enzyme (molecular weight approximately 37 kDa) and poly-L-arginine peptide. Poly-arginine is rich in positively charged amino acid residues and can form complexes with negatively charged membrane surfaces and enzyme proteins.

[0046] (2) Complex formation: A certain amount of DNase I enzyme (final concentration 10-50 U / mL) and poly-L-arginine peptide (molecular weight 5000-5000 Da, concentration 1-10 mg / ml) were dissolved in PBS buffer and mixed, and incubated at room temperature for 10-20 min to allow poly-arginine to form a stable bond with DNase I. Subsequently, the resulting DNase I-poly-arginine complex was added to the exosome suspension. According to a certain ratio of exosomes to DNase I enzyme protein (for example, 1×10 exosomes), the concentration of poly-arginine peptide was 100-100 mg / ml. 8Add 50 U / ml of DNase I (50 U / ml of DNase I) to ensure that sufficient enzyme molecules are evenly attached to each exosome. Gently mix and incubate at 4°C for 30 minutes to 1 hour to allow polyarginine to adsorb DNase I to the exosome membrane.

[0047] (3) Purification: After incubation, the DNase I-loaded exosomes were precipitated by ultracentrifugation (100,000 × g for 70 min) to remove unbound free DNase I and peptides. The precipitate was resuspended in PBS and gently pipetted to mix, thereby obtaining the DNase I-loaded antler MSCs exosome preparation. To verify the loading effect, a portion of the sample can be analyzed: for example, the activity of exosome-bound DNase I (by adding a known concentration of DNA substrate and measuring its degradation products) or the presence of DNase I on the exosomes can be detected by Western blotting.

[0048] (4) Preparation and storage: Based on the therapeutic needs, the exosomes loaded with DNase I are prepared into a preparation that can be used for in vivo administration. The exosome suspension can be mixed with an appropriate buffer solution (such as normal saline) in a certain proportion to prepare an injection and stored at low temperature (4°C for short-term storage or -80°C for long-term storage) to maintain enzyme activity and exosome stability. Sterile operation should be maintained during the preparation process, and excipients can be added as needed to improve the stability of the preparation.

[0049] In some specific implementation cases, the DNase I-loaded antler stem cell exosomes obtained in the above manner can be prepared into injections, sustained-release microspheres, transdermal gels or liposome preparations for the treatment of RA. They can also be used in combination with existing RA treatment drugs, such as leflunomide, methotrexate, sulfasalazine, tocilizumab, adalimumab, etanercept or baricitinib, to make the RA treatment effect more prominent.

[0050] In summary, this invention combines the unique biological properties of velvet MSC exosomes with nuclease therapy, achieving the first-ever use of exosomes to deliver DNase I for the treatment of RA. This innovative approach distinguishes it from traditional anti-inflammatory drugs and biologics. By applying velvet MSC exosomes loaded with DNase I for the prevention and treatment of rheumatoid arthritis, it can enhance the release of DNase I at the inflamed joint site to clear cfDNA, reduce inflammatory stimulation, and leverage the exosomes' own components to regulate immune balance (reducing Th17 responses and enhancing Treg function), significantly enhancing the therapeutic effect.

[0051] 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.

[0052] Example 1: Deer antler tissue was minced (approximately 2 mm in strips) and digested in a digestion solution containing 0.1% type I collagenase at 37°C for 1 hour, with occasional gentle shaking to release cells. After digestion, the single-cell suspension was filtered through a cell strainer, centrifuged to remove the digestive enzymes, and resuspended in PBS. The cells were seeded into a culture dish and cultured in low-glucose DMEM medium containing 10% fetal bovine serum to allow adherence and growth. The culture environment was 37°C and 5% CO2. After 24 hours of culture, the medium was changed to remove non-adherent cells. When the cells reached 80-90% confluence, they were passaged and expanded to obtain third-generation deer antler MSCs for subsequent experiments.

[0053] Well-growing antler MSCs were cultured in exosome-free medium for an additional 48 hours. Approximately several hundred milliliters of cell culture supernatant were collected. Exosomes were purified by differential centrifugation: cells were cleared by centrifugation at 300 × g for 5 minutes; the supernatant was transferred and centrifuged at 2,000 × g for 20 minutes to remove cellular debris and large vesicles; and finally, small impurities and microvesicles were removed by centrifugation at 10,000 × g for 30 minutes. The clarified supernatant was filtered through a 0.22 μm filter and then ultracentrifuged at 100,000 × g for 70 minutes to pellet the exosomes. After carefully aspirating the supernatant, the exosome pellet was resuspended in pre-chilled PBS and centrifuged again at 100,000 × g for 70 minutes to increase purity. Finally, the exosome pellet was resuspended in an appropriate amount of PBS (typically several hundred microliters, depending on the pellet size) to obtain a highly purified antler MSC exosome suspension.

[0054] Dissolve DNase I and poly-L-arginine peptide (molecular weight 5000-15000Da) in PBS buffer and mix. In each ml of PBS buffer, the content of poly-L-arginine peptide is 1mg / ml and the content of DNase I is 10U / ml. Incubate at room temperature for 20 minutes to allow poly-arginine to form a stable bond with DNase I. Then, add the resulting DNase I-polyarginine complex to the exosome suspension. Add according to the ratio: 1×10 exosomes 8 The concentration of particles / ml corresponds to 50 U / mL of DNase I to ensure that sufficient enzyme molecules are evenly attached to each exosome. After gentle mixing, incubate at 4°C for 30 minutes to 1 hour to allow polyarginine to adsorb DNase I to the exosome membrane. Following incubation, pellet the DNase I-loaded exosomes by ultracentrifugation (100,000 × g for 70 minutes) to remove unbound free DNase I and peptides. Resuspend the pellet in PBS and gently pipette to mix. This provides the DNase I-loaded antler MSC exosome preparation.

[0055] Example 2: The only difference between this example and Example 1 is that the amounts of poly-L-arginine polypeptide, DNase I enzyme, and exosomes are different, and the remaining steps and related parameter conditions are consistent with Example 1.

[0056] Specifically, DNase I enzyme and poly-L-arginine polypeptide were dissolved in PBS buffer and mixed so that the content of poly-L-arginine polypeptide was 5 mg / ml and the content of DNase I enzyme was 25 U / ml per 1 ml of PBS buffer. Further, when the obtained DNase I-polyarginine complex was added to the exosome suspension, the addition ratio was: 1×10 exosomes 8 The amount of particles / ml corresponds to 25 U / ml of DNase I enzyme.

[0057] Example 3: The only difference between this example and Example 1 is that the amounts of poly-L-arginine polypeptide, DNase I enzyme, and exosomes are different, and the remaining steps and related parameter conditions are consistent with Example 1.

[0058] Specifically, DNase I enzyme and poly-L-arginine polypeptide were dissolved in PBS buffer and mixed so that the content of poly-L-arginine polypeptide was 10 mg / ml and the content of DNase I enzyme was 50 U / ml per 1 ml of PBS buffer. Further, the obtained DNase I-polyarginine complex was added to the exosome suspension at a ratio of: 1×10 exosomes 8 The amount of particles / ml corresponds to 50 U / ml of DNase I enzyme.

[0059] Comparative Example 1: The only difference between this example and Example 1 is that the amounts of poly-L-arginine polypeptide, DNase I enzyme, and exosomes are different, and the remaining steps and related parameter conditions are consistent with Example 1.

[0060] Specifically, DNase I enzyme and poly-L-arginine polypeptide were dissolved in PBS buffer and mixed so that the content of poly-L-arginine polypeptide was 0.2 mg / ml and the content of DNase I enzyme was 5 U / ml per 1 ml of PBS buffer. Further, when the obtained DNase I-polyarginine complex was added to the exosome suspension, the addition ratio was: 1×10 exosomes 8 The amount of particles / ml corresponds to 5 U / mL of DNase I enzyme.

[0061] Comparative Example 2: The only difference between this example and Example 1 is that the amounts of poly-L-arginine polypeptide, DNase I enzyme, and exosomes are different, and the remaining steps and related parameter conditions are consistent with Example 1.

[0062] Specifically, DNase I enzyme and poly-L-arginine polypeptide were dissolved in PBS buffer and mixed so that the content of poly-L-arginine polypeptide was 0.4 mg / ml and the content of DNase I enzyme was 8 U / ml per 1 ml of PBS buffer. Further, when the obtained DNase I-polyarginine complex was added to the exosome suspension, the addition ratio was: 1×10 exosomes 8 The amount of particles / ml corresponds to 8 U / ml of DNase I enzyme.

[0063] Comparative Example 3: The only difference between this example and Example 1 is that the amounts of poly-L-arginine polypeptide, DNase I enzyme, and exosomes are different, and the remaining steps and related parameter conditions are consistent with Example 1.

[0064] Specifically, DNase I enzyme and poly-L-arginine polypeptide were dissolved in PBS buffer and mixed so that the content of poly-L-arginine polypeptide was 0.8 mg / ml and the content of DNase I enzyme was 10 U / ml per 1 ml of PBS buffer. Further, when the obtained DNase I-polyarginine complex was added to the exosome suspension, the addition ratio was: 1×10 exosomes 8 Particles / ml corresponds to the amount of DNase I 10 U / mL.

[0065] The following experiments were conducted on the products of Examples 1 to 3 and Comparative Examples 1 to 3, respectively: (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 (A) is the antler mesenchymal layer; (B) is the primary mesenchymal stem cells crawling out from the surrounding tissue; (C) is the TEM image of antler mesenchymal stem cells. 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.

[0067] (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.

[0068] See the results Figure 2 shown.

[0069] Figure 2 This is a diagram to verify the three-line differentiation ability of antler mesenchymal stem cells. Figure 2 (A) is the image of osteogenic differentiation (Alizarin Red S staining), (B) is the image of adipogenic differentiation (Oil Red O staining), and (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 abilities, which meet the three-lineage differentiation identification criteria of MSCs.

[0070] (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).

[0071] See the results Figure 3 shown.

[0072] Figure 3 This is the basic characterization diagram of antler MSCs exosomes. Figure 3 (A) is the TEM morphology diagram, (B) is the TEM magnification diagram, (C) is the particle size distribution diagram (NTA), and (D) is the Zeta potential diagram. Figure 3 As shown, antler MSCs exosomes present a typical cup-shaped vesicle structure, with a particle size concentrated at 100 nm and a Zeta potential of -31.86±1.62 mV, which meets the internationally recognized standards for exosome biophysical characteristics.

[0073] (IV) Load effect test The antler MSCs exosome preparations loaded with DNase I in Examples 1 to 3 and Comparative Examples 1 to 3 were taken, and the loading amount of DNase I enzyme was determined by the BCA protein concentration method, and the stability of the exosome preparations was determined by transmission electron microscopy (TEM) combined with nanoparticle tracking analysis (NTA).

[0074] See the results Figure 4 and Figure 5 .

[0075] Figure 4 The graph of the surface Zeta potential changes of antler MSCs exosomes modified with different concentrations of polyarginine is shown in Figure 2. Figure 4 It can be seen that the DNase I loading amount ( Figure 4 D to F) are higher than those in Comparative Examples 1 to 3 ( Figure 4 A to C in the figure), and the loading efficiency was further improved with the increase of polyarginine concentration.

[0076] Figure 5 The particle size distribution of antler MSCs exosomes modified with different concentrations of polyarginine is shown in Figure 2. Figure 5 It can be seen that compared with Comparative Examples 1 to 3 ( Figure 5 D to F), Examples 1 to 3 ( Figure 5 The particle morphology of (A to C) is stable and the particle size changes little. (V) Biocompatibility and safety The DNase I-loaded antler MSCs exosome preparation (EV@PD) prepared in Example 3 was used as a reference, and the biocompatibility and safety evaluations were conducted at the cellular and animal levels, respectively: (1) In vitro cytotoxicity assays were performed using mouse macrophages (RAW264.7). (2) Animal level: The preparations were injected into the tail vein of DBA / 1 mice, and the blank group and PBS group (phosphate buffered saline with a pH of 7.4) were used as controls. The physiological status and pathological changes of major organs and tissues were observed within 14 days.

[0077] See the results Figure 6 and Figure 7 shown.

[0078] Figure 6 The cytotoxicity evaluation diagram of different modified exosomes at multiple concentrations is shown in Figure 2. Figure 6 It can be seen that the cells in the EV@PD group had normal morphology and good proliferation, and there was no significant difference in cell viability between the EV group and the EV@PD group.

[0079] Figure 7 The serum biochemical indexes of mice treated with different modified exosomes are shown in Figure 2. Figure 7 It can be seen that there were no abnormal pathological changes in the main liver and kidney function tests of mice in the EV@PD group.

[0080] This demonstrates that the DNase I-loaded antler MSCs exosome preparation prepared by the method of the present invention has good biocompatibility and in vivo safety, and is suitable for further use in disease treatment research.

[0081] 6. RA treatment RA model mice were treated with PBS (phosphate buffered saline, pH 7.4), Example 1 (EV), Example 3 (EV@PD), and Example 8 (EV@PD + tocilizumab, with a mass ratio of EV@PD to tocilizumab of 1:1), respectively. Each RA model mouse was injected with the corresponding preparation via the tail vein at a dose of 200 μL, once every two days, for 21 consecutive days.

[0082] See the results Figure 8 shown.

[0083] Figure 8 Figure 2 shows the improvement of foot morphology in RA mouse models treated with different modified exosomes. Figure 8 It can be seen that compared with normal mice (control), the joint redness and swelling of mice treated with EV@PD and EV@PD+tocilizumab were significantly relieved, the range of motion was improved, and the inflammation score was significantly reduced. The improvement effects in the other groups (PBS and EV) were not obvious, and the condition of the PBS group continued to worsen.

[0084] This demonstrates that the DNase I-loaded antler MSCs exosome preparation provided by the present invention has a favorable anti-inflammatory therapeutic effect in an RA mouse model, superior to free enzyme or unloaded exosomes, demonstrating its synergistic therapeutic advantage. Furthermore, combining the DNase I-loaded antler MSCs exosome preparation with other drugs significantly enhanced the therapeutic effect of RA.

[0085] (VII) Long-term effectiveness of RA treatment To further evaluate the sustained effect of antler MSCs exosomes loaded with multifunctional molecules (P-Arg, DNase I) in the treatment of RA, DiD fluorescently labeled preparations (EV@PD-DiD) were injected into the tail vein of mice. Free DiD (free probe) was set as the control group. Imaging of fluorescence distribution in mice was performed at 12 h, 48 h, and 72 h after administration.

[0086] Figure 9The fluorescence distribution images of exosomes in mice under different treatment groups are shown in Figure 2. Figure 9 As can be seen, the signal in the Free DiD group was primarily concentrated in organs of the reticuloendothelial system, such as the liver, and was rapidly cleared after 12 hours. By 48 and 72 hours, almost no significant fluorescent signal remained. In contrast, the EV@PD-DiD group displayed a clear fluorescent signal in the joints of the limbs 12 hours after injection and maintained it for up to 72 hours, demonstrating good joint targeting and in vivo stability, suggesting its excellent long-term delivery properties and sustained drug efficacy.

[0087] The results indicate that the DNase I-loaded deer antler MSCs exosome preparation constructed by the present invention has excellent biodistribution characteristics, can prolong the local retention time of the active ingredients in the joints, thereby achieving a long-term therapeutic effect, which is of great significance for improving the treatment compliance of rheumatoid arthritis and reducing the frequency of administration.

[0088] 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 method for preparing antler stem cell exosomes loaded with DNase I, characterized by: The polyarginine peptide and DNase I enzyme were dissolved in PBS buffer and combined by electrostatic adsorption to form a DNase I-polyarginine complex. The DNase I-polyarginine complex was then mixed with antler stem cell exosomes to obtain antler stem cell exosomes loaded with DNase I. The concentration of polyarginine peptide in PBS buffer was controlled to be 1-10 mg / mL, and the concentration of DNase I enzyme was controlled to be 10-50 U / μL. The amount of exosomes from antler stem cells was controlled to be 1×10 8 ~1×10 9 Particles / ml.

2. The preparation method according to claim 1, wherein: The molecular weight of polyarginine polypeptide is 5000~15000Da.

3. The preparation method according to claim 1, wherein: Incubate at 20-25°C for 30-60 min to allow electrostatic adsorption to form a DNase I-polyarginine complex.

4. The preparation method according to claim 1, wherein: The DNase I-polyarginine complex was gently mixed with antler stem cell exosomes and incubated at 20-25°C for 30-60 minutes. After purification, antler stem cell exosomes loaded with DNase I were obtained.

5. The preparation method according to claim 1, wherein: Deer antler stem cell exosomes are obtained by digesting the deer antler tissue in digestive fluid, filtering and centrifuging to obtain deer antler mesenchymal stem cells, and then culturing and amplifying them before extracting the exosomes.

6. The preparation method according to claim 5, characterized in that: The digestion solution contains 1% type I collagenase and digestion is carried out at 37°C for 0.5 to 1 hour.

7. The preparation method according to claim 5, characterized in that: During culture, DMEM medium containing 10-15% fetal bovine serum was used and cultured at 37-38°C and 5-7% CO2 for 24-72 hours.

8. The preparation method according to claim 5, characterized in that: When extracting exosomes, the expanded cells are cultured for 24 to 48 hours, and the supernatant is collected. After impurities are removed, filtered, centrifuged, and washed, deer antler stem cell exosomes are obtained.

9. A DNase I-loaded antler stem cell exosome, characterized by: The method is obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the DNase I-loaded deer antler stem cell exosomes as claimed in claim 9 in the preparation of a medicament for treating rheumatoid arthritis, characterized in that: The drug is an injection, sustained-release microspheres, transdermal gel or liposome preparation, and also includes at least one of leflunomide, methotrexate, sulfasalazine, tocilizumab, adalimumab, etanercept or baricitinib.

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