A neutrophil extracellular vesicle gene delivery system and its application in the treatment of osteoarthritis.

Abstract

This invention relates to a neutrophil extracellular vesicle gene delivery system and its application in the treatment of osteoarthritis. The extracellular vesicle gene delivery system of this invention uses highly active and pure neutrophil extracellular vesicles as gene drug carriers, constructed using electroporation to load gene drugs. This delivery system can utilize the natural chondrocyte targeting ability of neutrophil extracellular vesicles to improve the retention capacity of gene drugs in joints and the efficiency of chondrocyte delivery, thereby improving the efficacy of gene therapy; it can also avoid the pro-inflammatory effects of neutrophils themselves and synergistically treat osteoarthritis with gene drugs, ultimately improving the treatment effect of osteoarthritis. This invention, to a certain extent, solves the problems of poor joint retention capacity and inadequate chondrocyte targeting delivery efficiency of existing gene delivery carriers, and provides new strategies and new drugs for the treatment of osteoarthritis.

Description

Technical Field

[0001] This invention relates to the field of gene delivery and osteoarthritis treatment technology, specifically to the construction of a neutrophil extracellular vesicle gene delivery system and its application in the treatment of osteoarthritis. Background Technology

[0002] Osteoarthritis is a chronic joint disease with high prevalence and disability rates, characterized by irreversible damage to articular cartilage and synovial inflammation. Currently, there is no cure. The imbalance between chondrocyte synthesis and metabolism is a key factor exacerbating irreversible cartilage damage. During the progression of osteoarthritis, osteoarthritic chondrocytes gradually lose their ability to synthesize cartilage matrix; they also secrete more cartilage-degrading enzymes, such as matrix metalloproteinases and proteoglycans, further aggravating cartilage matrix damage; in addition, they produce a large number of pro-inflammatory factors and secrete damage-related molecular patterns, further aggravating synovial inflammation. Overcoming the imbalance between chondrocyte synthesis and metabolism at its root may be an effective treatment for osteoarthritis.

[0003] Gene therapy drugs can exert regulatory effects at the gene level and are potential treatments for osteoarthritis. However, due to the abundance of capillaries and lymphatic vessels in the subsynovial membrane of the joint, gene therapy drugs have difficulty staying in the joint cavity; moreover, the cartilage matrix is ​​a dense, avascular, and nerveless negatively charged three-dimensional network structure with pores only 60 nm in size, while chondrocytes are discretely distributed in the cartilage matrix, making it difficult for gene therapy drugs to penetrate the extremely dense negatively charged cartilage matrix and reach the chondrocytes.

[0004] With the development of nanomaterials technology, nanomedicine carriers can encapsulate gene drugs within nanoparticles, improving their stability and demonstrating significant advantages in disease diagnosis and treatment. Commonly used nanomedicine carriers mainly include liposomes and polymer micelles. Utilizing their small particle size and permeability, some nano-formulations can penetrate the dense cartilage matrix to reach chondrocytes. However, these traditional nanomedicine carriers suffer from problems such as easy clearance by the body and low chondrocyte targeting efficiency, failing to achieve the expected therapeutic effects. This necessitates repeated intra-articular injections, leading to poor patient compliance. Therefore, there is an urgent need to research safe and efficient gene drug delivery carriers to enhance the long-term retention of gene drugs in bone and joints and improve delivery efficiency to chondrocytes. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems of poor joint retention ability and poor chondrocyte targeted delivery efficiency of existing carriers, and to provide a novel gene drug delivery carrier, neutrophil extracellular vesicles.

[0006] Another object of the present invention is to provide a novel extraneutrophil gene delivery system using extraneutrophil vesicles as carriers.

[0007] Another object of the present invention is to provide the application of a neutrophil extracellular vesicle gene delivery system in the treatment of osteoarthritis.

[0008] A neutrophil extracellular vesicle gene delivery system, comprising neutrophil extracellular vesicles and gene drugs loaded therein.

[0009] As a preferred embodiment of the present invention, the neutrophil extracellular vesicles are cell microvesicles with a double membrane structure produced by neutrophils budding under the stimulation of inflammatory factors.

[0010] As a preferred embodiment of the present invention, the extracellular vesicles of neutrophils have a particle size in the range of 100 to 1000 nm, a potential in the range of -5 to -25 mV, and are characterized by Annexin A1 as a protein.

[0011] As a preferred embodiment of the present invention, the inflammatory factors are selected from tumor cell necrosis factor TNFα, interleukin IL-8, or phorbol ester PMA.

[0012] As a preferred embodiment of the present invention, the gene drug is selected from siRNA, miRNA, mRNA or pDNA, preferably miRNA.

[0013] As a preferred embodiment of the present invention, the neutrophil extracellular vesicle gene delivery system delivers gene drugs into neutrophil extracellular vesicles via electroporation.

[0014] As a preferred embodiment of the present invention, the ratio of the gene drug to the drug load of neutrophil extracellular vesicles is 100-1500 pmol / μg, preferably 500-1000 pmol / μg.

[0015] As a preferred embodiment of the present invention, the neutrophil extracellular vesicle gene delivery system has a particle size in the range of 100 to 1000 nm, a potential in the range of -5 to -25 mV, and an encapsulation efficiency in the range of 40 to 90%.

[0016] The application of neutrophil extracellular vesicles as gene delivery vectors as described in this invention.

[0017] The application of the neutrophil extracellular vesicle gene delivery system described in this invention in the preparation of drugs for osteoarthritis.

[0018] This invention utilizes the inflammatory joint tropism of neutrophils retained in extracellular vesicles to enhance the enrichment and retention of gene-based drugs in inflamed joints. Simultaneously, it overcomes the pro-inflammatory effect of neutrophils themselves, allowing them to synergistically exert a therapeutic effect on osteoarthritis with gene-based drugs. This invention will fill the current gap in the lack of curative drugs for osteoarthritis, providing a novel delivery carrier for gene-based drugs and offering new strategies and drugs for the treatment of osteoarthritis.

[0019] This invention discloses a technical solution for preparing high-yield, highly active extracellular vesicles of neutrophils, the specific steps of which are as follows:

[0020] (1) Extraction and isolation of neutrophils: Femurs and tibias of SPF-grade C57BL6 mice were extracted and isolated. Bone marrow was expelled using a syringe filled with 1640 medium, and erythrocyte lysis buffer was added. The mixture was incubated at 4°C for 3-10 min, centrifuged at 200-500 g, and resuspended in 1640 medium to obtain a cell suspension. A series of equal volumes of Percoll layering solutions with varying concentrations (50-60%, 60-70%, and 70-80%) were then spread. Finally, the cell suspension was centrifuged at 500-1500 g for 20-60 min. Neutrophils were obtained from the junction of the 50-60% and 60-70% Percoll layering solutions.

[0021] (2) Add 20-100 ng / mL of inflammatory factors such as tumor cell necrosis factor TNFα, interleukin IL-8, or phorbol ester PMA to the isolated neutrophils (1×10⁻⁶ cells / mL). 7 The inflammatory factor concentration (number of cells / mL) was co-incubated at 37°C for 10-120 min. The preferred concentration of inflammatory factors was 40-70 ng / mL, and more preferably 40-55 ng / mL.

[0022] (3) Collect the cell supernatant obtained in (2), centrifuge at 500-1500g for 1-20 min at 4℃, and collect the supernatant; centrifuge again at 10000-15000g for 1-20 min at 4℃, and collect the supernatant; finally, centrifuge at 20000-25000g for 0.5-4 h at 4℃. Collect the precipitate, wash it three times with PBS, and obtain the desired extracellular vesicles of neutrophils.

[0023] The extraction and separation method for extracellular vesicles of neutrophils disclosed in this invention can be used to produce large quantities of high-purity extracellular vesicles of neutrophils that highly express the functional protein Annexin A1.

[0024] This invention discloses a method for preparing a neutrophil extracellular vesicle gene delivery system. Gene drugs are loaded into the aforementioned neutrophil extracellular vesicles using electroporation, thereby constructing the neutrophil extracellular vesicle gene delivery system. The specific steps are as follows:

[0025] (1) Preparation of pre-transfection solution: Mix the gene drug with neutrophil extracellular vesicles using PBS or culture medium. Place in an electroporation plate to obtain the pre-transfection solution; wherein, the gene drug includes, but is not limited to, siRNA, miRNA, mRNA and pDNA, preferably miRNA; the mixing ratio of gene drug to neutrophil extracellular vesicles is 100-1500 pmol / μg, preferably 500-1000 pmol / μg.

[0026] (2) Electroporation: The pre-transfection solution described in step (1) is subjected to electroporation treatment, wherein the electroporation method is CA-137, DS-150, CM-138, DS-120, CM-137, EH-100, CM-150, EO-100, DN-100, EN-138, DS-138, EN-150, DS-137, EW-113, DS-130 (electroporation instrument: LONZA 4D-Nucleofector).

[0027] (3) Collection of neutrophil extracellular vesicle gene delivery system by ultra-high speed centrifugation: Collect the liquid described in (2), centrifuge at 20000-25000g for 0.5-4h at 4℃. Collect the precipitate, wash it three times with PBS, and obtain the neutrophil extracellular vesicle gene delivery system.

[0028] The neutrophil extracellular vesicle gene delivery system of this invention has a particle size between 100-1000 nm and a potential between -5 and 25 mV. The encapsulation efficiency is 40%-90%.

[0029] The neutrophil extracellular vesicle gene delivery system described in this invention can maintain the inflammatory joint targeting of neutrophils. Furthermore, it has been demonstrated that it can effectively remain in inflamed joints and penetrate and target chondrocytes in cartilage tissue, while the neutrophil extracellular vesicles themselves also possess certain anti-inflammatory capabilities.

[0030] The application of the neutrophil extracellular vesicle gene delivery system described in this invention in the treatment of osteoarthritis.

[0031] Beneficial effects:

[0032] This invention develops neutrophil extracellular vesicles as a novel gene drug delivery carrier. Gene drugs are loaded into neutrophil extracellular vesicles using electroporation, resulting in a corresponding neutrophil extracellular vesicle gene delivery system. This system demonstrates significantly better therapeutic effects in osteoarthritis than single neutrophil extracellular vesicles or free gene drugs. This novel gene delivery carrier, neutrophil extracellular vesicles, lacks enzyme-rich organelles and exhibits low immunogenicity, enabling stable gene drug loading. Furthermore, neutrophil extracellular vesicles possess chondrocyte tropism, cartilage tissue penetration, and anti-inflammatory capabilities, allowing for effective retention at the osteoarthritis site and demonstrating safe and efficient delivery. They also synergistically enhance the therapeutic effect of gene drugs on osteoarthritis, making them an excellent gene drug delivery carrier for osteoarthritis.

[0033] The neutrophil extracellular vesicle extraction and separation technology disclosed in this invention can successfully extract high-purity, high-yield, and high-stability neutrophil extracellular vesicles (Examples 1-8). The method for constructing a neutrophil extracellular vesicle gene delivery system disclosed in this invention is simple, rapid, and versatile, and can be used for loading various gene drugs, including siRNA (Example 9), miRNA (Example 10), mRNA (Example 11), and pDNA (Example 12). The successfully constructed neutrophil extracellular vesicle delivery system (Examples 13-15) has good encapsulation efficiency (Example 16) and drug loading capacity (Example 17), and good in vitro stability (Example 18). Furthermore, gene drug loading does not affect the expression of Annexin A1 in neutrophil extracellular vesicles (Example 19), and demonstrates good joint retention (Example 20) and chondrocyte targeting ability (Example 21). This provides a new technical platform for gene drug delivery and has broad application prospects.

[0034] The neutrophil extracellular vesicle gene delivery system disclosed in this invention has the best therapeutic effect compared with single neutrophil extracellular vesicles or gene drugs (Example 22), providing a new carrier and new drug for the treatment of osteoarthritis. Attached Figure Description

[0035] Figure 1 This is a transmission electron microscope image of extracellular vesicles of neutrophils in this invention (scale bar: 100 nm).

[0036] Figure 2 This is a diagram showing the expression results of Annexin A1 and Alix in extracellular vesicles of neutrophils in this invention.

[0037] Figure 3 This is a graph showing the particle size and potential of extracellular vesicles of neutrophils stored in PBS at 4°C for 10 days according to the present invention.

[0038] Figure 4 Transmission electron microscopy results of the extracellular vesicle gene delivery system of this invention (scale bar: 100nm).

[0039] Figure 5 The graph shows the particle size and potential results of the extracellular vesicles and extracellular vesicle gene delivery system of the present invention after storage at 4°C for 10 days.

[0040] Figure 6 This is a diagram showing the expression results of Annexin A1 in extracellular vesicles of neutrophils before and after drug loading in this invention.

[0041] Figure 7 This describes the long-term expression of the extracellular vesicle gene delivery system of this invention at the joints of mice.

[0042] Figure 8 This is a fluorescence intensity diagram of Cy5 at the joint of a mouse using the extracellular vesicle gene delivery system of the present invention.

[0043] Figure 9 This is a fluorescence intensity diagram of DIR at the joint of a mouse using the extracellular vesicle gene delivery system of the present invention.

[0044] Figure 10 This image shows the penetration of the neutrophil extracellular vesicle gene delivery system of this invention into mouse cartilage tissue.

[0045] Figure 11 The expression level of the gene delivered to the joint by the extracellular vesicle gene delivery system of this invention is in ns. There is no significant difference, ****P<0.0001, one-way ANOVA.

[0046] Figure 12 This is a diagram showing the results of the chondrogenic effect of intra-articular injection of a neutrophil extracellular vesicle gene delivery system in mice with osteoarthritis, using safranin-fast green staining technology (scale bar: 100 μm).

[0047] Figure 13 This is a diagram showing the results of detecting the anti-inflammatory effect of an intra-articular injection of a neutrophil extracellular vesicle gene delivery system in mice with osteoarthritis using H&E staining technology (scale bar: 100 μm). Detailed Implementation

[0048] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but these embodiments do not constitute any limitation on the present invention.

[0049] Example 1: Preparation and characterization of neutrophils

[0050] Bone marrow was extracted from the femur and tibia of SPF-grade C57BL6 mice using a syringe to aspirate 1640 medium. The cells were centrifuged (500g, 3 min), the supernatant was discarded, 1 mL of erythrocyte lysis buffer was added, and the mixture was incubated at 4℃ for 3 min. The cells were then centrifuged again (500g, 3 min), the supernatant was discarded, and the cells were resuspended in 1 mL of 1640 medium to obtain a cell suspension. Equal volumes of a series of Percoll concentration gradients (55%, 65%, and 75%) were spread. Finally, the cell suspension was centrifuged at 1000g for 30 min. Cells at the boundary between the 55% and 65% Percoll layers were collected and washed three times with PBS to obtain neutrophils. The neutrophil purity was 91.6%, and the total apoptosis rate was only 8.1%, indicating that the neutrophils extracted using this method have high purity and good viability, and can, to some extent, avoid the formation of apoptotic bodies, which is beneficial for the subsequent extraction and separation of extracellular vesicles from neutrophils.

[0051] Example 2: Preparation of extracellular vesicles of neutrophils

[0052] Neutrophils were resuspended in PBS to a concentration of 1×10⁻⁶. 7 Add 50 ng / mL of tumor cell necrosis factor TNFα. Incubate at 37°C for 1 h, and collect the cell supernatant. Centrifuge at 500g for 3 min at 4°C, centrifuge at 13000g for 3 min at 4°C, collect the supernatant, centrifuge at 20000g for 1 h at 4°C, collect the precipitate, and wash three times with PBS to obtain neutrophil extracellular vesicles.

[0053] Example 3: Morphological characterization of extracellular vesicles of neutrophils

[0054] The PBS suspension of neutrophil extracellular vesicles prepared in Example 2 was dropped onto a copper grid coated with a carbon membrane and allowed to stand at room temperature. Excess solution was absorbed with filter paper. The mixture was negatively stained with 0.1% sodium phosphotungstenate solution for 5 min, and excess stain was absorbed with filter paper. After drying at room temperature, the mixture was observed and photographed using an HT-7700 transmission electron microscope (100 kV). The transmission electron microscope images are shown below. Figure 1 The results showed that the extracellular vesicles of neutrophils had a typical extracellular vesicle "cashier-like" structure with a particle size of about 100 nm.

[0055] Example 4: Characterization of particle size and concentration of extracellular vesicles of neutrophils

[0056] The neutrophil extracellular vesicle PBS suspension prepared in Example 2 was analyzed using a nanoparticle tracking analyzer. The instrument sensitivity was set to 80.0, the frame rate to 30.0, and the shutter speed to 100. The results showed that the hydrated particle size of the neutrophil extracellular vesicles was 142 nm, and the particle size was approximately 10 nm per 100 nm. 7 Approximately 1 × 10⁹ neutrophils can be extracted. 9 1 neutrophil extracellular vesicle.

[0057] Example 5: Potential characterization of extracellular vesicles of neutrophils

[0058] The potential of the neutrophil extracellular vesicle PBS suspension prepared in Example 2 was measured using a potentiometer. The results showed that the average potential of the neutrophil extracellular vesicles was -15 mV.

[0059] Example 6: Protein characterization of extracellular vesicles of neutrophils

[0060] The protein concentration of extracellular vesicles of neutrophils was determined using a BCA protein quantification kit, and the results showed that 10 7 Each neutrophil can produce extracellular vesicles containing 4 μg of protein.

[0061] Example 7: Purity of extracellular vesicles of neutrophils

[0062] Annexin A1 is a marker for extraneutrophil vesicles, and Alix is ​​a marker for exosomes. Therefore, the expression of Annexin A1 and Alix on the extraneutrophil vesicles prepared in Example 2 was detected using the Wes automated protein expression quantification system. The results are as follows: Figure 2 As shown, the expression of Annexin A1 on extracellular vesicles of neutrophils stimulated with 50 ng / mL TNFα for 1 h was high, while the expression of Alix was low compared to exosomes, indicating that the extracellular vesicles of neutrophils had good activity and high purity at this time.

[0063] Example 8: In vitro stability characterization of extracellular vesicles of neutrophils

[0064] The neutrophil extracellular vesicle PBS suspension prepared in Example 2 was stored at 4°C, and its particle size and potential were measured daily (e.g., ...). Figure 3 As shown in the figure, the results indicate that the extracellular vesicles of neutrophils in PBS at 4°C showed no significant difference in particle size and potential over 10 days, indicating good storage stability.

[0065] Example 9: Preparation of a neutrophil extracellular vesicle gene delivery system (siRNA@MV)

[0066] 2 μg of extracellular neutrophil vesicles and 1600 pmol of fluorescent FAM-labeled nonfunctional siRNA (FAM-siRNA) were added to an electroporation plate and mixed thoroughly with PBS to a final volume of 20 μL. Electroporation was performed using the DS137 program of a LONZA 4D-Nucleofector. The samples were then recovered from the plate, centrifuged at 20,000 g for 1 h at 4 °C, the supernatant was discarded, the precipitate was washed three times with PBS, and resuspended in PBS to obtain the extracellular vesicle gene delivery system, i.e., siRNA@MV.

[0067] Example 10: Preparation of a neutrophil extracellular vesicle miRNA delivery system (miRNA@MV)

[0068] 2 μg of extracellular neutrophil vesicles and 1600 pmol of miRNA-140 were added to an electroporation plate and mixed thoroughly with PBS to a final volume of 20 μL. Electroporation was performed using the DS137 program of a LONZA 4D-Nucleofector. The samples were then recovered from the plate, centrifuged at 20,000 g for 1 h at 4 °C, the supernatant was discarded, the precipitate was washed three times with PBS, and resuspended in PBS to obtain the extracellular vesicle gene delivery system, miR140@MV.

[0069] Example 11: Preparation of a neutrophil extracellular vesicle mRNA delivery system (mRNA@MV)

[0070] 2 μg of extracellular neutrophil vesicles and 1600 pmol of green fluorescent protein (GFP) mRNA were added to an electroporation plate and mixed thoroughly with PBS to a final volume of 20 μL. Electroporation was performed using the DS137 program of a LONZA 4D-Nucleofector. The samples were then recovered from the plate, centrifuged at 20,000 g for 1 h at 4 °C, the supernatant was discarded, the precipitate was washed three times with PBS, and resuspended in PBS to obtain the extracellular vesicle gene delivery system, i.e., mRNA@MV.

[0071] Example 12: Preparation of a neutrophil extracellular vesicle pDNA delivery system (pDNA@MV)

[0072] Add 2 μg of extracellular neutrophil vesicles and 1600 pmol of pDNA (pmaxGFP) to an electroporation plate, mix thoroughly with PBS to a final volume of 20 μL, and perform electroporation using the DS137 program of a LONZA 4D-Nucleofector. The sample from the plate is then recovered, centrifuged at 20000g for 1 h at 4 °C, the supernatant is discarded, the precipitate is washed three times with PBS, and resuspended in PBS to obtain the extracellular vesicle gene delivery system, pDNA@MV.

[0073] Example 13: Morphological characterization of extraneutrophil vesicle gene delivery system

[0074] The PBS suspension of the neutrophil extracellular vesicle gene delivery system (miR140@MV) obtained in Example 10 was added dropwise onto a copper grid coated with a carbon membrane. After standing at room temperature, excess solution was absorbed with filter paper. The sample was negatively stained with 0.1% sodium phosphotungstenate solution for 5 min, excess stain was absorbed with filter paper, and the sample was dried at room temperature. The sample was then observed and photographed using an HT-7700 transmission electron microscope (100 kV). The transmission electron microscope images are shown below. Figure 4 The results showed that the extracellular vesicle gene delivery system consisted of spherical vesicles with a double membrane structure. Compared with the unloaded system, the extracellular vesicles after drug loading showed a significant increase in particle size of about 50 nm and exhibited a more plump spherical structure.

[0075] Example 14: Particle size and concentration characterization of extraneutrophil vesicle gene delivery system

[0076] The PBS suspension of the extracellular vesicle gene delivery system (miR140@MV) obtained in Example 10 was analyzed using a nanoparticle tracking analyzer. The instrument sensitivity was set to 80.0, the frame rate to 30.0, and the shutter speed to 100. The results showed that the hydrated particle size of the extracellular vesicle gene delivery system was 200 nm. 7 Extracellular vesicles extracted from neutrophils can be used to prepare 1.0 × 10⁻⁶ cells. 9 Neutrophil extracellular vesicle gene delivery system.

[0077] Example 15: Potential characterization of extraneutrophil extracellular vesicle gene delivery system

[0078] The potential of the PBS suspension of the extraneutrophil extraneutrophil gene delivery system was measured using a potentiometer. The average potentials of the extraneutrophil extraneutrophil gene delivery systems prepared in Examples 9-12 are shown in the table below:

[0079] Table 1. Mean potential of extraneutrophil vesicle gene delivery system

[0080]

[0081] Example 16: Determination of encapsulation efficiency of extraneutrophil extracellular vesicle gene delivery system

[0082] Taking Cy3-labeled miR140 (Cy3-miR140) as an example, Cy3-miR140@MV was prepared according to the method in Example 10. The amount of MV was 2 μg, the amount of miRNA was 1600 pmol, and the electroporation method was DS137. The electroporated sample was centrifuged at 20000g for 1 h at 4℃, and the supernatant was collected. The fluorescence intensity of the supernatant was detected using a microplate reader with an excitation wavelength of 532 nm and an emission wavelength of 580 nm. A fluorescence standard curve of Cy3-miR140 was plotted, and the Cy3-miR140 content in the supernatant was calculated according to the fitting formula. The encapsulation efficiency was calculated according to the following formula: Encapsulation efficiency (%) = (Total molar amount of Cy3-miR140 - Molar amount of Cy3-miR140 in the supernatant) / Total molar amount of Cy3-miR140 × 100%. The results showed that the encapsulation efficiency of the extracellular vesicle gene delivery system (Cy3-miR140@MV) was 56%.

[0083] Example 17: Determination of drug loading capacity in extraneutrophil extracellular vesicle gene delivery system

[0084] Taking Cy3-labeled miR140 (Cy3-miR140) as an example, Cy3-miR140@MV was prepared according to the method in Example 10. The amount of MV was 2 μg, the amount of miRNA was 1600 pmol, and the electroporation method was DS137. The electroporated sample was centrifuged at 20000g for 1 h at 4℃, and the precipitate was collected. The weight of the outer tube of the blank ultrafiltration tube was recorded. The extracellular vesicle precipitate obtained by centrifugation was transferred to an ultrafiltration tube (molecular weight cutoff of 3000 Da) for desalting. An ultrafiltration tube with an equal volume of PBS was added served as a control. The preparation in the ultrafiltration tube was recovered and lyophilized, and the weight of the lyophilized ultrafiltration tube was recorded. Each tube was reconstituted with 100 μL of PBS containing 1% Triton X-100, then transferred to a 96-well plate and the fluorescence intensity was measured using a microplate reader. The mass of Cy3-miR140 in the lyophilized Cy3-miR140@MV was calculated, and the drug loading was calculated using the following formula: Drug loading (%) = Mass of Cy3-miR140 in the lyophilized Cy3-miR140@MV / (Weight of the lyophilized ultrafiltration tube - Weight of the blank ultrafiltration tube) × 100%. The results showed that the drug loading of the neutrophil extracellular vesicle gene delivery system was 0.1%.

[0085] Example 18: In vitro stability of extraneutrophil vesicle gene delivery system

[0086] The PBS suspension of the extracellular vesicle gene delivery system (miR140@MV) obtained in Example 10 was stored at 4°C, and its particle size and potential were measured daily. Figure 5 As shown in the figure, the results indicate that the particle size and potential of the extracellular vesicle gene delivery system for neutrophils did not differ significantly within 10 days in PBS at 4°C, demonstrating good storage stability.

[0087] Example 19: Determination of the relative expression level of Annexin A1 protein in a neutrophil extracellular vesicle gene delivery system

[0088] The expression of Annexin A1 in neutrophil extracellular vesicles (miR140@MV) obtained in Example 10 before and after drug loading was detected using the Wes fully automated protein expression quantification system. Figure 6 As shown, the results indicate that there was no significant difference in the expression level of Annexin A1 on extracellular vesicles of neutrophils before and after drug loading, suggesting that electroporation does not affect the content of Annexin A1 protein on extracellular vesicles.

[0089] Example 20: Retention capacity of neutrophil extracellular vesicle gene delivery system in joints

[0090] SPF-grade male C57BL6 mice at 8 weeks gestation were selected. Under isoflurane anesthesia, the anterior cruciate ligament of the right knee joint of the mice was transcribed. After 4 weeks, the osteoarthritis model was established. The osteoarthritis mice were divided into the following groups: Normal (normal group), Sham (sham operation group), Single Saline (single saline injection group), Multiple miR140@MV (1.5 μg miR140@MV injected into the knee joint once a week for a total of four times), and Single miR140@MV (1.5 μg miR140@MV injected into the knee joint once a week). Different drug treatments were administered according to the group. Knee joint tissues of the mice were collected on days 1, 2, 3, 7, 9, 14, 19, 21, 24, and 28. After soaking in liquid nitrogen, the tissues were ground, and total RNA was extracted from the joints using an RNA extraction kit. After reverse transcription, the amplification was performed using a qPCR instrument. Normalized treatment was performed with Normal as the control group. The relative miR140 expression of each group at different time points was examined, with U6 expression used as an internal reference.

[0091] The results show (e.g.) Figure 7 As shown in the figure, in osteoarthritis mice, miR140 expression was significantly reduced in the single-dose saline group, approximately 10% of the miR140 level in the normal group. The joint miR140 levels in the single-dose and multiple-dose groups were approximately 3-4 times higher than those in the normal group, and significantly higher than those in the saline group. This indicates effective delivery of miR140@MV, significantly increasing miR140 expression at the joint. Notably, a single dose can maintain miR140 expression at the joint for up to 28 days, demonstrating the long-term retention effect of miR140@MV in the joint, which is beneficial for improving patient compliance.

[0092] Example 21: Chondrocyte targeting capability of extraneutrophil vesicle gene delivery system

[0093] SPF-grade male 8-week-old C57BL6 mice were selected, and the anterior cruciate ligament was transectomized in the right knee joint of the mice under isoflurane anesthesia. After 4 weeks, the mouse osteoarthritis model was established.

[0094] Taking Cy5-labeled miR140 (Cy5-miR140) as an example, Cy5-miR140@DIR-MV was prepared by staining MV with DIR according to the method in Example 10. Free drug Cy5-miR140 and blank extracellular vesicle DIR-MV were used as controls. The drug was injected intra-articularly into the right knee joint of mice at a dose of 1.5 μg per mouse. Cy5 (e.g., Cy5-miR140@DIR-MV) in the right knee joint of the experimental mice was observed using a small animal in vivo imaging system. Figure 8 (as shown) and DIR (as shown) Figure 9The fluorescence intensity of the extracellular vesicle gene delivery system (as shown in the figure) changes over time. The results indicate that the system can remain in vivo for up to 48 hours, which is beneficial for its therapeutic effect.

[0095] Articular cartilage was extracted from each group of mice 48 hours after drug administration. Sections were prepared using a cryostat, with a section thickness of 8 μm. After the tissue sections were adsorbed onto glass slides, they were fixed in 4% paraformaldehyde for 1 hour. The slides were mounted with 90% glycerol, and the distribution of the formulation was observed under a confocal microscope (e.g., [missing information]). Figure 10 (As shown in the image). The results showed that 48 hours after administration, no Cy5-miR140 was present in the cartilage tissue of the Cy5-miR140 group, indicating that Cy5-miR140 was completely cleared from the joints of these mice. DIR-MV was observed distributed in the cartilage tissue of DIR-MV mice, indicating that MV can penetrate the dense cartilage matrix. Colocalized fluorescence signals distributed deep within the cartilage were observed in the cartilage tissue of the Cy5-miR140@DIR-MV group mice, indicating that Cy5-miR140@DIR-MV can penetrate the dense cartilage matrix in vivo. Given that only chondrocytes are present in the cartilage tissue, this further demonstrates that Cy5-miR140@DIR-MV has chondrocyte targeting capabilities.

[0096] Example 22: Therapeutic effect of neutrophil extracellular vesicle gene delivery system on osteoarthritis

[0097] Using miR140 as a gene drug, a neutrophil extracellular vesicle gene delivery system (miR140@MV) was constructed according to Example 10.

[0098] SPF-grade male C57BL6 mice, 8 weeks old, underwent anterior cruciate ligament transection of the right knee joint under isoflurane anesthesia. After 4 weeks, the osteoarthritis model was established. The mice were randomly divided into 5 groups of 5 mice each, as follows: saline, free miR140 (700 pmol / mouse), blank extracellular vesicles (MV, 1.5 μg), non-functional extracellular vesicle gene delivery system (miRNC@MV, 1.5 μg), and extracellular vesicle gene delivery system (miR140@MV, 1.5 μg, equivalent to 700 pmol miR140). Healthy mice (Normal) purchased at the same time as the model mice served as the control group. Additionally, a sham-operated group (Sham) with only the joint capsule transection was used for comparison to avoid the influence of model construction procedures on the experiment. Administered medication every 7 days for a total of four times, with all indicators measured 4 days after the last administration.

[0099] (1) Gene delivery efficiency: At the study endpoint, mice were euthanized, mouse joints were collected, and RNA was extracted. Using U6 expression as an internal control, the expression level of miR140 in the knee joints of each group of mice was detected by RT-qPCR (e.g., ...). Figure 11 (As shown in the figure). The results showed that miR140@MV could significantly increase the level of miR140 at the joint, approximately twice that of free miR140. This indicates that the miR140@MV prepared in this invention can significantly improve the in vivo delivery efficiency of miR140, antagonize the decrease in joint miR140 caused by osteoarthritis, and synergistically work with MV to upregulate miR140, ultimately significantly increasing the level of miR140 at the joint. The RT-qPCR primers used are shown in Table 2:

[0100] Table 2. List of primers for RT-qPCR detection

[0101]

[0102]

[0103] (2) Staining of cartilage tissue from mouse joints: At the study endpoint, mice were euthanized, and their joints were collected and fixed in 4% paraformaldehyde at room temperature for 48 hours. Paraffin sections of the joints were prepared and stained with safranin O-fast green. The sections were then observed using an upright fluorescence microscope. The results are as follows: Figure 12 As shown, the saline group exhibited severe cartilage damage, resulting in loss of Safranin O staining. In contrast, the miR140, MV, miRNC@MV, and miR140@MV groups all reduced cartilage wear to some extent. The miR140@MV group showed a larger area of ​​Safranin O staining, indicating that the cartilage remained largely intact. This suggests that miR140@MV possesses cartilage repair capabilities. Furthermore, significant osteophytes were observed in the saline and miRNC@MV groups, while significant synovial invasion was observed in the miR140 and MV groups, indicating that free miR140 and blank MV have limited efficacy in treating osteoarthritis. The miR140@MV group, however, showed smooth cartilage edges, with no observed osteophytes or synovial invasion, demonstrating excellent therapeutic effects for osteoarthritis, significantly superior to the miR140 and MV groups.

[0104] (3) Staining of synovial tissue from mouse joints: At the end of the study, mouse joints were collected and fixed in 4% paraformaldehyde at room temperature for 48 h. After paraffin embedding, the tissue was sectioned using a paraffin microtome, with a section thickness of 4 μm. The synovial tissue sections of the knee joint were analyzed by hematoxylin and eosin staining, and the sections were observed using an upright fluorescence microscope. The results are as follows: Figure 13As shown, compared to the single-layer synovial lining cells in the normal and sham-operated groups, the saline group exhibited significantly thickened synovium, a significantly increased cell number, and dense, band-like inflammatory infiltration. While the degree of synovial inflammatory infiltration in the miR140, blank MV, and miRNC@MV groups was somewhat alleviated, the synovium still showed some degree of thickening and invasion into the meniscus and cartilage. However, the miR140@MV group showed a single-layer synovial lining cell, without significant inflammatory infiltration or synovial invasion, and no obvious inflammation was observed in the synovium. Its anti-inflammatory effect was significantly better than that of the miR140 and MV groups.

[0105] The above embodiments are merely illustrative examples of the technical solutions and effects of the present invention and should not be construed as limiting the scope of protection of the present invention. The present invention also utilizes IL-8 and PMA to prepare miRNA-140@MV, which exhibits effects very similar to those in the above embodiments and demonstrates good therapeutic efficacy against osteoarthritis.

Claims

1. A neutrophil extracellular vesicle gene delivery system, characterized in that... It consists of extracellular vesicles of neutrophils and the gene-loaded drug therein. The extracellular vesicles of neutrophils are cell microvesicles with a double membrane structure that are produced by budding of neutrophils under stimulation by inflammatory factors for 60-120 min. The inflammatory factors are selected from tumor cell necrosis factor TNFα, interleukin IL-8, or phorbol ester PMA. The extracellular vesicles of neutrophils have a particle size in the range of 100-1000 nm, a potential in the range of -5 to -25 mV, and are characterized by Annexin A1 as a protein.

2. The extracellular vesicle gene delivery system for neutrophils according to claim 1, characterized in that... Gene drugs were introduced into extracellular vesicles of neutrophils using electroporation.

3. The extracellular vesicle gene delivery system for neutrophils according to claim 2, characterized in that... The drug loading ratio of the gene therapy to the extracellular vesicles of neutrophils is 100-1500 pmol / μg.

4. The extracellular vesicle gene delivery system for neutrophils according to claim 3, characterized in that... The drug loading ratio of the gene drug to neutrophil extracellular vesicles is 500-1000 pmol / μg.

5. The extracellular vesicle gene delivery system for neutrophils according to claim 1, characterized in that... The encapsulation efficiency of the neutrophil extracellular vesicle gene delivery system is 40-90%.

6. The use of the extracellular vesicle gene delivery system of any one of claims 1 to 5 in the preparation of a medicament for treating osteoarthritis.

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