A dnase i-loaded pilose antler stem cell exosome, and a preparation method and application thereof

Abstract

The application discloses a kind of DNase I loaded pilose antler stem cell exosome and its preparation method and application, belong to the field of biological medicine, realize DNase I enzyme stable and efficient loading on pilose MSCs exosome surface by the electrostatic adsorption of polyarginine polypeptide, and combine the immune regulation function and the characteristics of target arthritis lesion of exosome, realize the multi-mechanism precision treatment of rheumatoid arthritis and other inflammatory diseases.The technology not only significantly improves the drug-loading efficiency and structural stability of exosome, but also effectively retains the biological activity and anti-inflammatory effect of DNase I, overcoming the application barriers of traditional protein drugs, such as instability in vivo, easy clearance and poor targeting, and has good treatment prospects and conversion value.Therefore, the present application has significant innovation and practicality, is suitable for the functional modification of exosome drugs and the treatment of various chronic inflammation-related diseases, and has important industrialization and clinical promotion prospects.

Description

Technical Field

[0001] This invention relates to DNase I-loaded deer antler stem cell exosomes, their preparation method, and their applications. Specifically, it relates to a highly efficient and highly active deoxyribonuclease I (DNase I)-loaded deer antler stem cell exosome, its preparation method, and its applications, belonging to the field of biomedical technology. Background Technology

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the synovium and joint destruction. Its pathogenesis is complex, involving abnormal activation of immune cells and excessive production of inflammatory mediators. Recent studies have found that cell-free DNA (cfDNA) released during the formation of extracellular trapping networks (NETs) by neutrophils may play a crucial role in the inflammatory response of RA. Large amounts of cfDNA and NETs can act as warning signals, exacerbating the autoimmune response, promoting the production of inflammatory factors such as IL-17, and disrupting the body's immune balance. However, traditional RA therapies (such as anti-TNF-α and biologics) primarily target specific cytokines or immune cells, lacking a direct effect on clearing accumulated cfDNA at the lesion site.

[0003] Previous studies have attempted to administer exogenous DNase I to break down NETs and cfDNA, theoretically reducing the inflammatory response of rheumatoid arthritis and correcting immune imbalances. However, free DNase I has a short half-life in vivo, is easily inactivated by inhibitors (such as DNase inhibitors in serum), and is difficult to effectively accumulate in inflamed joints, thus limiting its therapeutic efficacy. The known work, "Bioinspired nanoogels as cell-free DNA trapping and scavenging organelles for rheumatoid arthritis treatment," is from the National Academy of Sciences of the United States of America. A 2023 AMERICA paper (“Bio-activated nanogels as cell-free DNA capture and clearance organelles for the treatment of rheumatoid arthritis,” Proceedings of the National Academy of Sciences) reported a method for treating RA by coupling deoxyribonuclease I (DNase I) to cationic peptide dendritic polymer nanogels. Benefiting from its naturally derived peptide components, the resulting nanogels exhibit high biocompatibility. Through customization of different sizes and surface charge densities, they can achieve the fastest targeting ability, the highest accumulation rate, the longer duration of action, and excellent DNA clearance ability in inflamed joints. This demonstrates that the nanogels possess sufficient core advantages in RA treatment. However, potential risks and challenges remain, including: 1. Complex synthesis steps and poor process controllability: The synthesis of dendritic polymers typically involves multiple chemical modifications and precise molecular structure control, resulting in poor batch-to-batch stability and difficulty in large-scale scalability. 2. Insufficient enzyme activity retention: In this system, DNase I is mostly covalently coupled to the nanogel, which can easily affect its native conformation, leading to decreased or inactivated catalytic activity. Third, uncontrollable exposure sites and susceptibility to protein masking effects: In a body fluid environment, the enzyme structure on the gel surface may be encapsulated by serum proteins, reducing its ability to recognize and degrade cfDNA. Fourth, high rigidity and insufficient penetration of the delivery system: The relatively rigid structure of nanogels may hinder tissue penetration and accumulation at inflammatory sites. Fifth, immune safety risks: Long-term retention of dendritic polymers in vivo may trigger immune responses or inflammatory stimulation, lacking the immunoadaptive advantages of naturally derived carriers.

[0004] Exosomes, as natural nanocarriers, possess excellent stability and biocompatibility, making them suitable for loading drug molecules and delivering them to specific tissues. Stem cell exosomes have demonstrated immunomodulatory and tissue repair effects in the treatment of diseases such as rheumatoid arthritis (RA). However, ordinary exosomes lack targets for specific pathogenic factors in the inflammatory environment of joints. Loading DNase I onto exosomes promises to combine the delivery advantages of exosomes with the cfDNA degradation effect of DNase I, thereby providing a new solution targeting the pathological mechanisms of RA. Currently, there are no literature reports on the technology of loading DNase I onto deer antler MSCs exosomes for RA treatment; this invention provides a new and innovative approach in this field. Summary of the Invention

[0005] The purpose of this invention is to provide DNase I-loaded deer antler stem cell exosomes, their preparation method, and applications. By introducing polyarginine as a mediator molecule, DNase I enzyme is effectively adsorbed and immobilized on the surface of deer antler stem cell exosomes, forming DNase I-functionalized exosomes. These exosomes can be used to prepare drugs for treating rheumatoid arthritis (RA), enabling the release of DNase I activity at the site of joint inflammation, degrading excessive local cfDNA and NETs, ​​and reducing the stimulation of these pathogenic factors on the immune system. Simultaneously, the bioactive molecules carried by the deer antler stem cell exosomes themselves can promote the restoration of immune balance, such as inhibiting pro-inflammatory Th17 cells and promoting the function of regulatory T cells (Tregs), thereby achieving the goal of alleviating RA inflammation and tissue damage at multiple levels.

[0006] This invention is achieved through the following technical solution: a method for preparing DNase I-loaded deer antler stem cell exosomes, comprising dissolving polyarginine peptides and DNase I enzyme in PBS buffer, forming a DNase I-polyarginine complex by electrostatic adsorption, and then mixing the DNase I-polyarginine complex with deer antler stem cell exosomes to obtain DNase I-loaded deer antler stem cell exosomes.

[0007] The concentration of polyarginine peptide in the PBS buffer should be controlled at 1–10 mg / mL, and the concentration of DNase I enzyme at 10–50 U / μL.

[0008] The amount of deer antler stem cell exosomes added was controlled at 1×10 8 ~1×10 9 Particles / ml.

[0009] The molecular weight of polyarginine peptides is 5000–15000 Da.

[0010] Incubate at 20–25°C for 30–60 min to allow electrostatic adsorption and binding to form DNase I-polyarginine complex.

[0011] After gently mixing the DNase I-polyarginine complex with deer antler stem cell exosomes, the mixture was incubated at 20–25°C for 30–60 min and purified to obtain deer antler stem cell exosomes loaded with DNase I.

[0012] Deer antler stem cell exosomes are obtained by digesting deer antler tissue in a digestive solution, filtering and centrifuging to obtain deer antler mesenchymal stem cells, and then culturing and expanding them before extracting exosomes.

[0013] The digestive fluid contains 1% type I collagenase and is digested at 37°C for 0.5–1 hour.

[0014] The culture was performed using DMEM medium containing 10-15% fetal bovine serum at 37°C and 5% CO2 for 5-7 hours.

[0015] During exosome extraction, the expanded cells were cultured for 12–24 hours, and the supernatant was collected. After impurity removal, filtration, centrifugation, and washing, deer antler stem cell exosomes were obtained.

[0016] A type of DNase I-loaded deer antler stem cell exosome was obtained using the above-described preparation method.

[0017] The use of DNase I-loaded deer antler stem cell exosomes in the preparation of a medicament for treating rheumatoid arthritis, said medicament being an injection, sustained-release microsphere, transdermal gel, or liposome formulation, further comprising at least one of leflunomide, methotrexate, sulfasalazine, tocilizumab, adalimumab, etanercept, or baricitinib.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0019] (1) Based on the therapeutic mechanism of DNase I enzyme, this invention provides a deer antler stem cell exosome loaded with DNase I. Through the use of DNase I enzyme, cell-free DNA in RA lesions can be degraded, thereby reducing adverse stimulation to 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.

[0020] (2) This invention solves the drawback that existing exogenous administration of DNase I enzyme is difficult to accumulate in inflamed joints and cannot achieve effective therapeutic effects. It provides a process in which DNase I enzyme is attached to the membrane surface of exosomes by electrostatic interaction using polyarginine polypeptide as a medium to obtain DNase I loaded exosomes. Exosomes, as carriers, can effectively protect DNase I enzyme from degradation and inactivation in vivo. The binding of polyarginine to exosomes can improve the stability of DNase I in vivo circulation and promote its penetration into inflamed sites. Compared with free enzyme, DNase I loaded with exosomes can maintain a higher concentration and longer activity in the joint, thereby significantly enhancing the therapeutic effect.

[0021] (3) This invention uses deer antler stem cell exosomes (deer antler MSCs exosomes). Because the microRNAs and proteins they carry can inhibit inflammatory responses and promote tissue regeneration, the DNase I-loaded exosomes prepared from them can not only exert enzymatic effects to clear harmful DNA, but the components of deer antler MSCs exosomes can also 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 restore the Th17 / Treg balance. This multi-target comprehensive therapeutic effect is obviously superior to therapies with a single mechanism of action.

[0022] (4) The deer antler MSCs exosomes used in this invention are secretions from autologous / homogeneous cells, exhibiting good biocompatibility and low toxicity. DNase I is a natural enzyme in the human body, and its clinical use in treating cystic fibrosis is well-established. Therefore, the combined treatment strategy of these two ingredients in this invention is expected to have high safety and does not increase the risk of significant immunosuppression. No major adverse reactions were observed in animal experiments, and joint function was significantly improved, demonstrating its potential for clinical application.

[0023] (5) The deer antler stem cell exosomes loaded with DNase I involved in this invention can be combined with other drugs (such as anti-inflammatory drugs) to achieve multiple treatments for RA. They can also be extended to other autoimmune and inflammatory diseases, such as systemic lupus erythematosus and other diseases with a large number of NETs / cfDNA. Exosomes loaded with DNase I can also be considered for treatment, thereby developing a multifunctional compound exosome therapy.

[0024] In summary, this invention is the first to combine DNase I enzyme with exosomes, providing a method for preparing an exosome enzyme-carrying system with high stability and targeting in vivo through polyarginine peptide-mediated preparation. This achieves efficient aggregation and sustained therapeutic effect of DNase I enzyme at the lesion site. At the same time, combined with the therapeutic efficacy of deer antler MSCs exosomes on RA, it achieves multi-level relief of RA inflammation and tissue damage, making it more suitable for translational applications. Attached Figure Description

[0025] Figure 1 Extraction of exosomes from deer antler mesenchymal stem cells.

[0026] Figure 2 This diagram demonstrates the three-lineage differentiation capacity of deer antler mesenchymal stem cells.

[0027] Figure 3 This is a basic characterization diagram of exosomes from deer antler MSCs.

[0028] Figure 4 The graph shows the surface zeta potential changes of deer antler MSCs exosomes modified with different concentrations of poly-L-arginine. In the figure, (A) is the poly-L-arginine peptide modified with 0.2 mg / ml, (B) is the poly-L-arginine peptide modified with 0.4 mg / ml, (C) is the poly-L-arginine peptide modified with 0.8 mg / ml, (D) is the poly-L-arginine peptide modified with 1 mg / ml, (E) is the poly-L-arginine peptide modified with 5 mg / ml, and (F) is the poly-L-arginine peptide modified with 10 mg / ml.

[0029] Figure 5 The particle size distribution of exosomes from deer antler MSCs modified with different concentrations of poly-L-arginine is shown in the figure. (A) is the poly-L-arginine peptide modified with 1 mg / ml, (B) is the poly-L-arginine peptide modified with 5 mg / ml, (C) is the poly-L-arginine peptide modified with 10 mg / ml, (D) is the poly-L-arginine peptide modified with 0.2 mg / ml, (E) is the poly-L-arginine peptide modified with 0.4 mg / ml, and (F) is the poly-L-arginine peptide modified with 0.8 mg / ml.

[0030] Figure 6 This is a graph showing the cytotoxicity evaluation of different modified exosomes at multiple concentrations.

[0031] Figure 7 The graph shows the detection of serum biochemical indicators in mice after treatment with different modified exosomes. In the graph, (A) is a comparison of serum ALT (alanine aminotransferase) concentration, (B) is a comparison of serum AST (aspartate aminotransferase) concentration, (C) is a comparison of serum UREA (urea) concentration, and (D) is a comparison of serum CREA (creatinine) concentration.

[0032] Figure 8 Figure showing the improvement in foot morphology in a mouse model of RA treated with different modified exosomes.

[0033] Figure 9 This is an image showing the fluorescence distribution of exosomes in mice under different treatment groups. Detailed Implementation

[0034] The invention's objective, technical solution, and beneficial effects will be further explained in detail below.

[0035] 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.

[0036] This invention combines DNase I enzyme with deer antler MSCs exosomes to prepare DNase I-loaded deer antler stem cell exosomes. The process includes isolating and culturing mesenchymal stem cells from deer antler tissue in vitro, extracting deer antler MSCs exosomes, and then using positively charged polyarginine peptides to attach DNase I enzyme to the deer antler MSCs exosomes via electrostatic interaction, resulting in DNase I-loaded deer antler stem cell exosomes. This invention utilizes polyarginine peptide-mediated DNase I enzyme, which can efficiently and stably load onto exosomes, achieving sustained and high-concentration activity of DNase I at the joint site, significantly increasing its therapeutic effect on inflamed areas. Since deer antler MSCs exosomes themselves have the function of inhibiting inflammatory responses and promoting tissue regeneration, the "DNase I + deer antler MSCs exosome" exosome enzyme loading system is not only more stable in vivo and has better targeting, but also exhibits more prominent therapeutic effects.

[0037] Compared to existing treatment strategies for DNase I enzymes in RA (free DNase I enzymes or DNase I-coupled nanogels), this invention employs a novel treatment system that uses electrostatic adsorption of DNase I on the surface of exosomes, offering the following technical advantages:

[0038] (1) Preservation of enzyme activity: Through electrostatic adsorption mediated by polyarginine (P-Arg), the destruction of the active site of DNase I by traditional chemical coupling is avoided, and its native conformation and catalytic efficiency are maintained to the greatest extent. At the same time, the controllable positive charge density provided by polyarginine (P-Arg) helps to ensure that DNase I is exposed to the outside and maintains its activity.

[0039] (2) Biocompatibility and low immunogenicity: Using deer antler MSCs exosomes as a natural carrier gives the system excellent biocompatibility and avoids the risk of long-term retention of synthetic materials (such as dendritic polymers); P-Arg, as a natural amino acid polymer, further reduces immune stimulation and overcomes the protein adsorption and shielding problem of traditional nanogels.

[0040] (3) Excellent delivery performance: The natural homing ability and flexible membrane structure of exosomes make it easier to break through the inflammatory barrier and achieve efficient accumulation and penetration of DNase I in the joint.

[0041] (4) Synergistic treatment: The dual-function system of "DNase I + deer antler MSCs exosomes" can clear inflammation-related cfDNA and block the RA treatment pathway; deer 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".

[0042] (5) Long-lasting effect: The exosome structure can protect DNase I from enzymatic degradation to maintain its activity. P-Arg-mediated electrostatic adsorption can achieve stable loading of DNase I, thereby achieving long-lasting high concentration of drug in the joint.

[0043] (6) Conversion advantages: No complex chemical coupling process, simple electrostatic adsorption process, high batch-to-batch consistency, and easier to scale up production.

[0044] Specifically, the technical solution of the present invention can be summarized as follows:

[0045] Preparation of exosome vectors from deer antler MSCs: Deer antler MSCs exosomes were cultured and extracted using deer antler tissue-derived MSCs, which can provide stable and efficient exosome vectors for subsequent drug loading.

[0046] First, fresh antler tissue from healthy sika deer was selected, and mesenchymal tissue was obtained under aseptic conditions. The antler tissue was cut into small pieces (approximately 2mm in size) and placed in a digestive solution containing 1% type I collagenase at 37°C for 0.5–1 hour, with gentle agitation occasionally to release cells. After digestion, a single-cell suspension was obtained by filtration through a cell filter, centrifugation was used to remove the digestive enzymes, and the cells were resuspended in PBS. Cells were seeded in culture dishes and cultured in low-glucose DMEM medium containing 10–15% fetal bovine serum to encourage adhesion and growth. The culture environment was 37–38°C and 5–7% CO2. After 24–72 hours of culture, the medium was replaced to remove non-adherent cells. When the cells reached 80–90% confluence, they were passaged to obtain third or fourth generation antler mesenchymal stem cells (antler MSCs) for subsequent experiments.

[0047] Optionally, the obtained cell surface markers (such as CD90^+, CD105^+, CD45^-, etc.) can be detected by flow cytometry to confirm the mesenchymal stem cell phenotype.

[0048] Next, exosomes were extracted and purified. Healthy antler MSCs were cultured for 48 hours in exosome-free medium, and the cell culture supernatant was collected in several hundred milliliters. The exosomes were purified by differential centrifugation: cells were removed by centrifugation at 300×g for 5–10 minutes; the supernatant was transferred and centrifuged at 2,000×g for 20 minutes to remove cell debris and large vesicles; then centrifuged at 10,000×g for 30 minutes to remove small impurities and microvesicles. The clarified supernatant was filtered through a 0.22 μm filter membrane and then centrifuged at 100,000×g for 70 minutes to precipitate the exosomes. After carefully discarding the supernatant, the exosome precipitate was resuspended in pre-cooled PBS and centrifuged again at 100,000×g for 70 minutes to improve purity. Finally, the exosome precipitate was resuspended in an appropriate amount of PBS (volume depends on the precipitate size, generally several hundred microliters) to obtain a high-purity antler MSCs exosome suspension.

[0049] Exosome concentration was determined by particle size analysis and protein detection. The typical cup-shaped morphology of exosomes could be observed by transmission electron microscopy, and exosome marker proteins (such as CD9 and CD63) could be detected by Western blotting to confirm successful extraction.

[0050] Preparation of DNase I loading via polyarginine: A positively charged polyarginine peptide is used to attach DNase I to the surface of exosomes via electrostatic interactions, thereby preparing functionalized DNase I exosomes. This method is simple and mild, maintains the enzyme activity of DNase I, and achieves a high loading rate. The specific preparation steps are as follows:

[0051] (1) Material preparation: DNase I enzyme (molecular weight approximately 37 kDa) and poly-L-arginine polypeptide were obtained. Poly-arginine is rich in positively charged amino acid residues, which can form a complex with the negatively charged membrane surface and enzyme protein.

[0052] (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. The mixture was incubated at room temperature for 10-20 min to allow polyarginine to form a stable binding with DNase I. Subsequently, the resulting DNase I-polyarginine complex was added to the exosome suspension. The ratio of exosomes to DNase I enzyme protein was adjusted according to a certain ratio (e.g., 1×10⁻⁶ exosomes). 8Add DNase I at a rate of 50 U / ml (particles / ml) to ensure that each exosome has an average amount of enzyme molecules attached. After gentle mixing, incubate at 4°C for 30 min to 1 h to allow polyarginine to adsorb DNase I onto the exosome membrane.

[0053] (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 mixed by pipetting to obtain the DNase I-loaded deer antler MSCs exosome preparation. To verify the loading effect, a portion of the sample can be analyzed: for example, the DNase I activity bound to the exosomes can be measured (by adding a known concentration of DNA substrate and measuring its degradation products), or Western blotting can be performed to detect the presence of DNase I on the exosomes.

[0054] (4) Formulation preparation and storage: According to the therapeutic needs, DNase I-loaded exosomes are prepared into formulations suitable for in vivo administration. The exosome suspension can be mixed with an appropriate buffer solution (such as physiological saline) in a certain proportion to prepare an injection, and stored at low temperatures (short-term storage at 4°C or long-term freezing at -80°C) to maintain enzyme activity and exosome stability. Aseptic techniques are maintained during formulation preparation, and excipients can be added as needed to improve formulation stability.

[0055] In some specific implementation cases, the DNase I-loaded deer antler stem cell exosomes obtained by the above method can be prepared into injections, sustained-release microspheres, transdermal gels or liposomes 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.

[0056] In summary, this invention combines the unique biological characteristics of deer antler MSCs exosomes with nuclease therapy, achieving for the first time a protocol for delivering DNase I via exosomes for the treatment of rheumatoid arthritis (RA), which is innovative and distinct from traditional anti-inflammatory drugs and biologics. By applying DNase I-loaded deer antler MSCs exosomes to the prevention and treatment of RA, it is possible to enhance the release of DNase I at the site of joint inflammation to clear cfDNA, reduce inflammatory stimulation, and regulate immune balance (reducing Th17 response and enhancing Treg function) based on the components of the exosomes themselves, significantly enhancing the therapeutic effect.

[0057] 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.

[0058] Example 1:

[0059] Deer antler tissue was cut into small pieces (approximately 2 mm in size) and placed in a digestive solution containing 0.1% type I collagenase at 37°C for 1 hour, with gentle agitation occasionally to release cells. After digestion, a single-cell suspension was obtained by filtration through a cell filter, centrifugation was used to remove the digestive enzymes, and the cells were resuspended in PBS. The cells were seeded in culture dishes and cultured in low-glucose DMEM medium containing 10% fetal bovine serum to encourage adhesion and growth. The culture environment was 37°C and 5% CO2. After 24 hours of culture, the medium was replaced to remove non-adherent cells. When the cells reached 80-90% confluence, they were passaged to obtain third-generation deer antler MSCs for subsequent experiments.

[0060] Healthy antler MSCs were cultured for 48 hours in exosome-free medium, and the cell culture supernatant was collected in approximately several hundred milliliters. The exosomes were purified by differential centrifugation: cells were removed by centrifugation at 300×g for 5 min; the supernatant was transferred and centrifuged at 2,000×g for 20 min to remove cell debris and large vesicles; then centrifuged at 10,000×g for 30 min to remove minute impurities and microvesicles. The clarified supernatant was filtered through a 0.22 μm filter and then centrifuged at 100,000×g for 70 min to precipitate the exosomes. The supernatant was carefully discarded, and the exosome precipitate was resuspended in pre-cooled PBS and centrifuged again at 100,000×g for 70 min to improve purity. Finally, the exosome precipitate was resuspended in an appropriate amount of PBS (volume depends on the precipitate size, generally several hundred microliters) to obtain a high-purity antler MSCs exosome suspension.

[0061] DNase I enzyme and poly-L-arginine peptide (molecular weight 5000–15000 Da) were dissolved in PBS buffer, with each 1 ml of PBS buffer containing 1 mg / ml of poly-L-arginine peptide and 10 U / ml of DNase I enzyme. The mixture was incubated at room temperature for 20 min to allow the poly-arginine to form a stable binding with DNase I. Subsequently, the resulting DNase I-polyarginine complex was added to the exosome suspension. The ratio of exosomes added was 1 × 10⁻⁶. 8 The particle / ml corresponds to 50 U / ml of DNase I enzyme to ensure sufficient enzyme molecules are evenly attached to each exosome. After gentle mixing, incubate at 4°C for 30 min to 1 h to allow polyarginine to adsorb DNase I onto the exosome membrane. After incubation, DNase I-loaded exosomes are precipitated by ultracentrifugation (100,000 × g for 70 min) to remove unbound free DNase I and peptides. Resuspend the precipitate in PBS and gently pipette to mix, thus obtaining the DNase I-loaded deer antler MSCs exosome preparation.

[0062] Example 2:

[0063] The only difference between this embodiment and Example 1 is the amount of poly-L-arginine polypeptide, DNase I enzyme, and exosomes used. All other steps and related parameters are the same as in Example 1.

[0064] Specifically, DNase I enzyme and poly-L-arginine peptide were dissolved in PBS buffer and mixed to achieve a concentration of 5 mg / ml poly-L-arginine peptide and 25 U / ml DNase I enzyme per 1 ml of PBS buffer. Further, when the resulting DNase I-polyarginine complex was added to the exosome suspension, the addition ratio was 1 × 10⁻⁶ exosomes. 8 The amount of DNase I enzyme at 25 U / ml corresponds to granules / ml.

[0065] Example 3:

[0066] The only difference between this embodiment and Example 1 is the amount of poly-L-arginine polypeptide, DNase I enzyme, and exosomes used. All other steps and related parameters are the same as in Example 1.

[0067] Specifically, DNase I enzyme and poly-L-arginine peptide were dissolved in PBS buffer and mixed to ensure that the concentration of poly-L-arginine peptide was 10 mg / ml and the concentration of DNase I enzyme was 50 U / ml per 1 ml of PBS buffer. Further, when the resulting DNase I-polyarginine complex was added to the exosome suspension, the addition ratio was 1 × 10⁻⁶ exosomes. 8 The amount of DNase I enzyme at 50 U / ml corresponds to granules / ml.

[0068] Comparative Example 1:

[0069] The only difference between this embodiment and Example 1 is the amount of poly-L-arginine polypeptide, DNase I enzyme, and exosomes used. All other steps and related parameters are the same as in Example 1.

[0070] Specifically, DNase I enzyme and poly-L-arginine peptide were dissolved in PBS buffer and mixed to achieve a concentration of 0.2 mg / ml poly-L-arginine peptide and 5 U / ml DNase I enzyme per 1 ml of PBS buffer. Further, when the resulting DNase I-polyarginine complex was added to the exosome suspension, the addition ratio was 1 × 10⁻⁶ exosomes. 8 The amount of DNase I enzyme at 5 U / mL corresponds to granules / ml.

[0071] Comparative Example 2:

[0072] The only difference between this embodiment and Example 1 is the amount of poly-L-arginine polypeptide, DNase I enzyme, and exosomes used. All other steps and related parameters are the same as in Example 1.

[0073] Specifically, DNase I enzyme and poly-L-arginine peptide were dissolved in PBS buffer and mixed to achieve a concentration of 0.4 mg / ml poly-L-arginine peptide and 8 U / ml DNase I enzyme per 1 ml of PBS buffer. Further, when the resulting DNase I-polyarginine complex was added to the exosome suspension, the addition ratio was 1 × 10⁻⁶ exosomes. 8 The amount of DNase I enzyme at 8 U / ml corresponds to granules / ml.

[0074] Comparative Example 3:

[0075] The only difference between this embodiment and Example 1 is the amount of poly-L-arginine polypeptide, DNase I enzyme, and exosomes used. All other steps and related parameters are the same as in Example 1.

[0076] Specifically, DNase I enzyme and poly-L-arginine peptide were dissolved in PBS buffer and mixed to achieve a concentration of 0.8 mg / ml poly-L-arginine peptide and 10 U / ml DNase I enzyme per 1 ml of PBS buffer. Further, when the resulting DNase I-polyarginine complex was added to the exosome suspension, the addition ratio was 1 × 10⁻⁶ exosomes. 8 The particle / ml corresponds to a DNase I concentration of 10 U / mL.

[0077] The following experiments were conducted on the relevant products of Examples 1 to 3 and Comparative Examples 1 to 3, respectively:

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

[0082] (II) Verification of the differentiation capacity of deer antler MSCs

[0083] 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.

[0084] See results Figure 2 As shown.

[0085] 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), image (B) shows adipogenic differentiation (Oil Red O staining), and image (C) shows chondrogenic differentiation (Alcian Blue staining). Figure 2 It is evident that deer antler mesenchymal stem cells possess excellent osteogenic, adipogenic, and chondrogenic differentiation capabilities, meeting the criteria for the three-lineage differentiation of MSCs.

[0086] (III) Structural characterization of exosomes from deer antler MSCs

[0087] 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).

[0088] See results Figure 3 As shown.

[0089] Figure 3 This is a basic characterization diagram of exosomes from deer antler MSCs. Figure 3 In the diagram, (A) is a TEM morphology image, (B) is a magnified TEM image, (C) is a particle size distribution map (NTA), and (D) is a zeta potential map. Figure 3 As shown, the exosomes of deer antler MSCs exhibit a typical cup-shaped vesicle structure with a particle size concentrated at 100 nm and a Zeta potential of -31.86 ± 1.62 mV, which meets the internationally recognized standards for the biophysical characteristics of exosomes.

[0090] (iv) Load effect test

[0091] The DNase I-loaded deer antler MSCs exosome preparations from Examples 1 to 3 and Comparative Examples 1 to 3 were used. The DNase I enzyme loading 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).

[0092] See results Figure 4 and Figure 5 .

[0093] Figure 4 The graph shows the surface zeta potential changes of deer antler MSCs exosomes modified with different concentrations of polyarginine. Figure 4 It can be seen that the DNase I loading in Examples 1 to 3 ( Figure 4 The values ​​of D to F in the middle were all higher than those in comparative examples 1 to 3. Figure 4 (From A to C), and the loading efficiency is further improved with the increase of polyarginine concentration.

[0094] Figure 5 The particle size distribution of exosomes from deer antler MSCs modified with different concentrations of polyarginine is shown in the figure. Figure 5 It can be seen that, compared with comparative examples 1 to 3 ( Figure 5 Regarding (D to F), Examples 1 to 3 ( Figure 5 The particle morphology of particles A to C is stable, and the particle size variation is small.

[0095] (v) Biocompatibility and safety

[0096] The DNase I-loaded deer antler MSCs exosome formulation (EV@PD) prepared in Example 3 was used as a reference. Biocompatibility and safety were evaluated at the cellular and animal levels.

[0097] (1) In vitro cytotoxicity was detected at the cellular level using mouse macrophages (RAW264.7);

[0098] (2) At the animal level, the preparation was injected into the tail vein of DBA / 1 mice, with the blank group and PBS group (phosphate buffered saline with pH 7.4) as controls, and the physiological status and pathological changes of major organs and tissues were observed within 14 days.

[0099] See results Figure 6 and Figure 7 As shown.

[0100] Figure 6 This is a graph evaluating the cytotoxicity of different modified exosomes at multiple concentrations, by... Figure 6 It is evident that the cells in the EV@PD group had normal morphology, good proliferation, and no significant difference in cell viability compared to the EV group and the EV@PD group.

[0101] Figure 7 The graph shows the detection of serum biochemical parameters in mice after treatment with different modified exosomes. Figure 7 It is evident that no abnormal pathological changes were observed in the main liver and kidney function tests of the EV@PD group mice.

[0102] This demonstrates that the DNase I-loaded deer antler MSCs exosome preparation prepared by the method of the present invention has good biocompatibility and in vivo safety, and is suitable for further research on disease treatment.

[0103] (vi) Treatment of RA

[0104] RA model mice were treated with PBS (phosphate-buffered saline with 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). Each RA model mouse was injected with the corresponding preparation via the tail vein at a dose of 200 μL, once every 2 days, for 21 consecutive days.

[0105] See results Figure 8 As shown.

[0106] Figure 8 Images showing the improvement in foot morphology in a mouse model of RA treated with different modified exosomes. Figure 8 As can be seen, compared with normal mice (control), mice treated with EV@PD and EV@PD+tocilizumab showed significant relief of joint redness and swelling, improved mobility, and significantly reduced inflammation scores. The other groups (PBS and EV) showed no significant improvement, and the condition of the PBS group continued to deteriorate.

[0107] This demonstrates that the deer antler MSCs exosome preparation loaded with Dnase I provided by this invention exhibits good anti-inflammatory therapeutic effects in a mouse model of RA, superior to free enzyme or unloaded exosomes, indicating its synergistic therapeutic advantage. Furthermore, combining the Dnase I-loaded deer antler MSCs exosome preparation with other drugs can significantly enhance the therapeutic effect of RA.

[0108] (vii) Long-term efficacy of RA treatment

[0109] To further evaluate the sustained effect of deer antler MSCs exosomes loaded with multifunctional molecules (P-Arg, DNase I) in the treatment of RA, mice were injected via tail vein with a DiD fluorescently labeled formulation (EV@PD-DiD), with a control group consisting of Free DiD. The fluorescence distribution in mice was detected by imaging at 12 h, 48 h, and 72 h after administration.

[0110] Figure 9 This is an imaging map showing the fluorescence distribution of exosomes in mice from different treatment groups. Figure 9 As can be seen, the Free DiD group signal was mainly concentrated in organs of the reticuloendothelial system such as the liver, and was rapidly cleared after 12 hours. By 48 hours and 72 hours, there was almost no obvious residual fluorescence signal. In contrast, the EV@PD-DiD group showed obvious fluorescence signals in the joints of the limbs 12 hours after injection and maintained them until 72 hours, demonstrating good joint targeting ability and in vivo stability, suggesting that it has excellent long-acting delivery characteristics and can achieve sustained efficacy.

[0111] The results indicate that the deer antler MSCs exosome preparation loaded with Dnase I constructed in this invention has excellent biodistribution characteristics and can prolong the retention time of the active ingredient in the joint, thereby achieving a long-term therapeutic effect. This is of great significance for improving treatment compliance and reducing the frequency of administration in rheumatoid arthritis.

[0112] 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 deer antler stem cell exosome loaded with DNase I for sustained targeted therapy of rheumatoid arthritis, characterized in that: The exosomes were prepared using the following steps: S1. Poly-L-arginine peptides with a molecular weight of 5000–15000 Da were dissolved in PBS buffer, and DNase I was dissolved in the PBS buffer. The concentration of poly-L-arginine peptides in the PBS buffer was controlled to be 1–10 mg / mL, and the concentration of DNase I was controlled to be 10–50 U / μL. The DNase I-polyarginine complex was formed by electrostatic adsorption. S2. Mix the DNase I-polyarginine complex with deer antler stem cell exosomes, controlling the amount of deer antler stem cell exosomes added to be 1×10⁻⁶. 8 ~1×10 9 Particles / ml were used to obtain DNase I-loaded deer antler stem cell exosomes.

2. The deer antler stem cell exosomes loaded with DNase I according to claim 1, characterized in that: Incubate at 20–25°C for 30–60 min to allow electrostatic adsorption and binding to form DNase I-polyarginine complex.

3. The deer antler stem cell exosomes loaded with DNase I according to claim 1, characterized in that: After gently mixing the DNase I-polyarginine complex with deer antler stem cell exosomes, the mixture was incubated at 20–25°C for 30–60 min and purified to obtain deer antler stem cell exosomes loaded with DNase I.

4. The DNase I-loaded deer antler stem cell exosomes according to claim 1, characterized in that: Deer antler stem cell exosomes are obtained by digesting deer antler tissue in a digestive solution, filtering and centrifuging to obtain deer antler mesenchymal stem cells, and then culturing and expanding them before extracting exosomes.

5. The DNase I-loaded deer antler stem cell exosomes according to claim 4, characterized in that: The digestive fluid contains 1% type I collagenase and is digested at 37°C for 0.5–1 hour.

6. The DNase I-loaded deer antler stem cell exosomes according to claim 4, characterized in that: The culture was performed using DMEM medium containing 10-15% fetal bovine serum at 37-38°C and 5-7% CO2 for 24-72 hours.

7. The DNase I-loaded deer antler stem cell exosomes according to claim 4, characterized in that: During exosome extraction, the expanded cells were cultured for 24–48 hours. The supernatant was then removed, filtered, centrifuged, and washed to obtain deer antler stem cell exosomes.

8. The use of DNase I-loaded deer antler stem cell exosomes as described in any one of claims 1 to 7 in the preparation of a medicament for sustained targeted treatment of rheumatoid arthritis, characterized in that: The drug is an injection, sustained-release microsphere, transdermal gel or liposome formulation, and also includes at least one of leflunomide, methotrexate, sulfasalazine, tocilizumab, adalimumab, etanercept or baricitinib.

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