Preparation method and application of antler stem cell conditioned medium bionic nanoparticle preparation

Abstract

The invention discloses a preparation method of a bionic nanoparticle preparation of a antler stem cell conditioned culture medium, which comprises the following steps: S1, isolated culture of antler stem cells, preparation of P3 cells, and preparation of the antler stem cell conditioned culture medium ABPCs-CM from the P3 cells for later use; s2, synthesizing antler stem cell conditioned medium nanoparticles CM-NPS by adopting the antler stem cell conditioned medium ABPCs-CM obtained in the step S1; s3, preparing a neutrophile granulocyte membrane; s4, the antler stem cell conditioned medium nano-particles CM-NPS obtained in S2 and the neutrophile granulocyte membrane obtained in S3 are synthesized into the antler stem cell conditioned medium bionic nano-particle preparation, and the neutrophile granulocyte membrane bionic structure remarkably improves the targeting efficiency of the inflammation part.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for preparing a biomimetic nanoparticle formulation of deer antler stem cell conditioned medium and its application. Background Technology

[0002] Osteoarthritis (OA) is one of the most common joint diseases, characterized by joint pain and stiffness caused by degeneration of articular cartilage and subchondral bone remodeling. It often affects joints throughout the body, including large joints such as the knee and hip, and small joints such as the hand and temporomandibular joint, severely impairing patients' mobility and physical and mental health.

[0003] The pathogenesis of osteoarthritis (OA) involves both tissue repair and degradation. In the early stages of OA, the composition and structure of the cartilage matrix change, cartilage fragments detach, leading to the exposure of underlying calcified cartilage and bone. At this time, chondrocyte activity increases, attempting to repair the damage, mainly manifested as chondrocyte hypertrophy and proliferation, increased matrix synthesis, and subchondral bone remodeling. As joint damage worsens, osteoarthritis changes occur in the cartilage, including decreased chondrocyte proliferation, cartilage loss, and bone sclerosis; in severe cases, osteophyte formation and synovitis may also occur. Simultaneously, the production of reactive oxygen species within chondrocytes increases, the secretion of various pro-inflammatory factors increases, and cells begin to age.

[0004] Current treatments for osteoarthritis often only alleviate symptoms and cannot stop or reverse its progression. Drug therapy mainly includes nonsteroidal anti-inflammatory drugs (NSAIDs) and intra-articular corticosteroids. The former only relieves pain and inflammation but cannot prevent cartilage degeneration, and long-term use can lead to gastrointestinal bleeding and cardiovascular risks. While the latter is effective in the short term, repeated use accelerates cartilage matrix degradation. In severe cases, joint replacement surgery and cartilage repair techniques used clinically have unsatisfactory mechanical properties and long-term efficacy. Furthermore, stem cell therapy faces challenges such as low cell survival rates after transplantation, significant bottlenecks in clinical translation, and a certain degree of tumorigenicity.

[0005] Artificial nanomedicines are immunogenic, easily captured by the immune system, resulting in limited drug delivery to the target site, insufficient penetration of biological barriers, short clearance half-life in joint cavities, and significant burst release. Furthermore, achieving therapeutic goals often requires multiple, high-dose deliveries, which may lead to systemic toxicity. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing a biomimetic nanoparticle formulation of deer antler stem cell conditioned culture medium and its application, in order to solve the problems that existing treatments cannot cure diseases and that traditional drug delivery systems have poor circulation and targeting capabilities.

[0007] The objective of this invention is achieved as follows:

[0008] A method for preparing a biomimetic nanoparticle formulation of deer antler stem cell conditioned culture medium includes the following steps:

[0009] S1. Deer antler stem cells were isolated and cultured, and P3 cells were obtained. Deer antler stem cell conditioned medium ABPCs-CM was prepared using P3 cells for later use.

[0010] S2. Using the deer antler stem cell conditioned medium ABPCs-CM obtained in S1, deer antler stem cell conditioned medium nanoparticles CM-NPS were synthesized.

[0011] S3. Preparation of neutrophil membrane, as follows: S3.1. Neutrophils were collected from human peripheral blood using an animal peripheral blood neutrophil isolation kit; fresh anticoagulated blood was spread on the separation medium and centrifuged at 2800 rpm for 30 min using a horizontal rotor to collect the second ring cell layer. The harvested cells were washed three times with PBS and then lysed at 37°C for 10 min using erythrocyte lysis buffer; the final cells were washed three times with PBS and resuspended in serum-free cryopreservation buffer to obtain neutrophils, which were then stored at −80°C for subsequent membrane derivatization.

[0012] S3.2 Neutrophils were thawed and washed three times with PBS, and the supernatant was removed by centrifugation; after repeated freeze-thaw cycles three times, the cells were disrupted using an ultrasonic cell disruptor to obtain a cell membrane suspension;

[0013] S3.3 Collect the cell membrane suspension after S3.2 disruption, centrifuge at 4 ℃, collect the first supernatant, centrifuge again to discard the precipitate, obtain the second supernatant, centrifuge the second supernatant again, discard the supernatant, and obtain the neutrophil membrane.

[0014] S4. Combine the CM-NPS conditioned medium nanoparticles of deer antler stem cells obtained in S2 with the neutrophil membrane obtained in S3 to synthesize a biomimetic nanoparticle formulation of conditioned medium for deer antler stem cells.

[0015] The specific operation of S1 is as follows:

[0016] S1.1 Clean the skin, disinfect, cut 1-2 cm of the tip of the deer antler, rinse repeatedly with physiological saline to remove blood, soak in PBS containing 10% penicillin-streptomycin for 5 min, rinse again with physiological saline, transfer to DMEM complete culture medium for low temperature storage, and carry out subsequent experiments in a clean bench.

[0017] S1.2 Remove the skin from the antler and take the mesenchymal part of the antler. Cut it into small pieces and place it in 0.1% type I collagenase. Digest it at 37 ℃ for 30 min. Then add complete culture medium to stop the digestion. After centrifugation, remove the supernatant and evenly disperse the obtained primary tissue blocks in T25 culture flasks. Incubate in an inverted incubator for 2 h. Add an appropriate amount of complete culture medium and culture at 37 ℃ and 5% CO2. Change the medium every 2-3 days.

[0018] S1.3 When the cells reach 80-90% confluence, trypsin digest them at 37 ℃ for 2-3 min, then stop digestion, transfer to centrifuge tubes, centrifuge at 1000 r / min for 5 min, discard the supernatant, add about 5 mL of complete culture medium, resuspend the cells, and transfer to a new culture flask to obtain P1 cells; continue passage in the same way to obtain P3 cells; the complete culture medium is DMEM medium containing 10 wt% fetal bovine serum;

[0019] S1.4 After repeatedly washing P3 cells with PBS, culture them in serum-free DMEM medium for 48 h, centrifuge to collect the supernatant, filter with a bacterial filter to obtain deer antler stem cell conditioned medium ABPCs-CM, and store at 4 ℃.

[0020] The concentration of the deer stem cell conditioned medium is as follows: 2 mg of freeze-dried deer antler stem cell conditioned medium ABPCs-CM is dissolved in 2 mL of PBS and used as the internal aqueous phase for the synthesis of deer antler stem cell conditioned medium nanoparticles CM-NPS.

[0021] The specific operation of S2 is as follows:

[0022] S2.1 PLGA microparticles loaded with deer antler stem cell conditioned medium ABPCs-CM were prepared using a water / oil / water emulsification method; the deer antler stem cell conditioned medium ABPCs-CM was used as the inner aqueous phase, and polyvinyl alcohol and PLGA-containing dichloromethane were mixed as the oil phase to obtain a mixture.

[0023] S2.2. The mixture obtained in S2.1 is subjected to ultrasonic treatment for 30 s in an ice bath, and the original emulsion and polyvinyl alcohol are introduced into water to obtain a water / oil / water emulsion.

[0024] S2.3. Emulsify the emulsion obtained in S2.2 on a high-speed homogenizer for 5 min; continuously stir the water / oil / water emulsion overnight at room temperature to promote solvent evaporation; centrifuge the solidified ABPCs-CM nanoparticles, wash them three times with water, freeze-dry them and store them in a -80 ℃ refrigerator.

[0025] The specific operation of S4 is as follows: The neutrophil membrane obtained in S3 is sonicated in an ice bath for 5 min to make it uniformly resuspended in PBS. The neutrophil membrane suspension is mixed with the deer antler stem cell conditioned medium nanoparticles CM-NPS obtained in S2 at a ratio of 1:(1-2) (v / v). The deer antler stem cell conditioned medium biomimetic nanoparticle preparation is synthesized by sonication.

[0026] The application of a biomimetic nanoparticle formulation of deer antler stem cell conditioned culture medium in the treatment of osteoarthritis.

[0027] The beneficial effects of this invention are:

[0028] 1. The biomimetic structure of the neutrophil membrane significantly improves the targeting efficiency at the site of inflammation.

[0029] Cell membrane-coated nanoparticles, as biomimetic drug carriers, can effectively evade the surveillance of the immune system, prolong drug circulation, and increase drug accumulation at the target site. Cell membrane-coated biomimetic nanocarriers utilize cell membrane coating nanotechnology to fuse the cell membrane of natural cells onto core nanoparticles, creating a biomimetic nanodelivery system. These bilayered biomimetic nanocarriers retain the physicochemical properties of the core nanocarrier while their cell membrane structure inherits biological functions similar to those of primitive cells. They can utilize cell membrane properties and the biological functions of the source cell to carry nanoparticles, loading drugs or factors to exert anti-inflammatory, wound-reducing, and bio-detoxifying effects. Therefore, neutrophil membrane-encapsulated functional substances in nanoparticles could represent a novel strategy for treating osteoarthritis.

[0030] Neutrophils are the most abundant white blood cells in the human body and are crucial immune cells responsible for the innate immune response. During inflammation, they are recruited to the lesion site immediately and regulate the inflammatory process. Therapeutic microparticles with neutrophil membrane camouflage can replace neutrophils in being recruited to the inflamed area by chemokines, thus blocking the destructive effects of neutrophils on joint tissues. More importantly, these biomimetic therapeutic microparticles do not promote the secretion of corresponding inflammatory factors and chemokines by other immune cells, which could further accelerate the progression of arthritis. Previous studies have used neutrophil membrane-coated nanoparticles to treat rheumatoid arthritis and have demonstrated their ability to inhibit joint inflammation. Therefore, constructing nanoparticles with functional substances encapsulated by neutrophil membranes, while utilizing the neutrophil membrane to inhibit inflammation progression, and simultaneously using the functional substances carried in their core to promote the repair of articular cartilage, could achieve superior therapeutic effects.

[0031] 2. Antler bud progenitor cells (ABPCs) are key to antler regeneration and possess greater therapeutic potential. Stem cell therapy has always been a major research hotspot in regenerative medicine. Mesenchymal stem cells are pluripotent and self-renewing, and they respond to chemotactic signals in the body to migrate to the damaged site, directly differentiating into various cell types, such as keratinocytes, fibroblasts, and endothelial cells. Antlers are the only known mammalian organ that can regenerate entirely annually. Compared to other stem cells, antler stem cells are easier to obtain and exhibit higher proliferation and regeneration capabilities. Among them, antler bud progenitor cells are crucial for antler regeneration and possess greater therapeutic potential.

[0032] 3. The conditioned medium for deer antler bud progenitor cells is rich in various functional bioactive factors secreted by stem cells. Mesenchymal stem cells mainly exert their biological functions through the paracrine pathway. These signaling molecules can participate in tissue repair and reconstruction by inducing angiogenesis, stimulating cell proliferation, and regulating cell differentiation and migration. In this process, the conditioned medium (CM) plays an important role. CM is the supernatant obtained by culturing stem cells in DMEM medium containing fetal bovine serum (FBS) for 48 hours, and it is rich in various functional bioactive factors secreted by stem cells. ABPCs-CM can upregulate osteogenic factors, promote the proliferation, migration, and differentiation of mesenchymal stem cells into osteoblast lineage cells, thereby promoting osteogenic regeneration; at the same time, it downregulates osteoclast differentiation factors, regulates the polarization of macrophages towards the M2 phenotype, and inhibits osteoclast formation, thereby reducing bone resorption and improving the symptoms of osteoarthritis (OA). Attached Figure Description

[0033] Figure 1 The diagram shows the culture of deer antler stem cells according to the present invention; A and B are images of the tip of the deer antler; C is an image of primary deer antler stem cells; D is an image of deer antler stem cells after passage and purification.

[0034] Figure 2 Immunohistochemistry of deer antler stem cells for this invention: A is vimentin staining; B is keratin staining and osteogenic differentiation diagram; C is alkaline phosphatase staining diagram; D is alizarin red staining diagram; E is immunofluorescence identification diagram.

[0035] Figure 3 To illustrate the synthesis and concentration screening of CM-NPS nanoparticles in the conditioned medium for deer antler stem cells of this invention, A is an image stained with Alcian blue; B is an image stained with toluidine blue; C is a graph showing the results of the BCA protein quantification experiment; and D is a graph showing the results of the CCK-8 assay for different concentrations of CM-NPS.

[0036] Figure 4This is a schematic diagram illustrating the extraction of neutrophils and the preparation of cell membranes according to the present invention.

[0037] Figure 5 This invention relates to the preparation and characterization of neutrophil-inspired nanoparticles (NMMPs) in conditioned medium containing deer antler stem cells; A is a transmission electron microscope image of CM-NPS; B is a transmission electron microscope image of NMMPs; C is a particle size measurement diagram of four groups of materials; D is a statistical diagram of particle size and potential of four groups of materials.

[0038] Figure 6 For the NMMP biocompatibility test of this invention; A is a live-dead staining experiment diagram of the material co-cultured with chondrocytes; B is a diagram of its CCK-8 experiment results; C is a live-dead staining experiment diagram of the material co-cultured with macrophages; D is a diagram of the CCK-8 experiment results; E is a physical image of the hemolysis experiment; F is a statistical analysis diagram of the hemolysis rate.

[0039] Figure 7 Micro-CT images of rat knee joints after treatment with different nanomaterials;

[0040] Figure 8 HE and Masson sections of rat knee joints after 4 weeks of treatment with different nanomaterials;

[0041] Figure 9 HE and Masson sections of rat knee joints after 8 weeks of treatment with different nanomaterials. Detailed Implementation

[0042] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0043] Example 1: Isolation and culture of deer antler stem cells (ABPCs) and extraction of deer antler stem cell conditioned medium (ABPCs-CM)

[0044] S1.1 Clean the skin, disinfect, and cut 1-2 cm of tissue from the tip of the deer antler. Figure 1 (AB) The blood was repeatedly rinsed with physiological saline, soaked in PBS solution containing 10% penicillin-streptomycin for 5 min, rinsed again with physiological saline, and then transferred to DMEM complete culture medium for low-temperature storage. Subsequent experiments were carried out in a clean bench.

[0045] S1.2. Remove the antler skin and collect the mesenchymal portion. After thoroughly chopping, place it in 0.1% type I collagenase and digest at 37°C for 30 min. Then add complete culture medium to stop digestion. Centrifuge and remove the supernatant. Disperse the obtained primary tissue blocks evenly in T25 culture flasks, invert the incubator for 2 hours, add an appropriate amount of complete culture medium, and culture at 37°C with 5% CO2. Change the medium every 2-3 days. Figure 1 C);

[0046] S1.3 When the cells reach 80-90% confluence, trypsin digest them at 37 ℃ for 2-3 min, then stop digestion, transfer to centrifuge tubes, centrifuge at 1000 r / min for 5 min, discard the supernatant, add an appropriate amount of complete culture medium, resuspend the cells, and transfer to a new culture flask to obtain P1 ( Figure 1 D). P3 cells were obtained by passage using the same method. P3 cells were used throughout this experiment.

[0047] S1.4 After repeatedly washing P3 cells with PBS, culture them in serum-free DMEM medium for 48 h, centrifuge to collect the supernatant, filter with a bacterial filter, store at 4 ℃, and use promptly.

[0048] The deer antlers mentioned were provided by Huamao Deer Industry in Gulin County, Luzhou City, Sichuan Province. Male sika deer aged two years, approximately 10 days after the antlers began to grow (before the two antlers branched), were selected.

[0049] The complete culture medium is DMEM medium containing 10 wt% fetal bovine serum.

[0050] Example 2: Immunohistochemistry, immunofluorescence, and osteogenic differentiation identification of ABPCs

[0051] Immunohistochemical experiment

[0052] S1. Place cell crawling sheets into six-well plates, seed P3 generation ABPCs, and culture for 24 hours. Fix with PBS three times, 4% paraformaldehyde for 30 min, incubate with 3% H2O2 at room temperature in the dark for 20 min, then wash three times with PBS on a shaker for 5 min each time. Incubate with 3% BSA for 15 min. Add rabbit anti-rat vimentin primary antibody or rabbit anti-rat cytokeratin primary antibody, and incubate overnight at 4°C in a humidified chamber.

[0053] S2. The next day, wash three times with PBS for 5 minutes each time. Add the corresponding secondary antibody to the primary antibody at room temperature and incubate for 30 minutes. Repeat the PBS washing. Add freshly prepared DAB staining solution and observe under a microscope. After staining, rinse thoroughly with tap water for 5 minutes. Counterstain with hematoxylin for 2 minutes, rinse with running water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with running water, re-blue with hematoxylin blue solution, rinse with running water, dehydrate, and mount. Detect the expression of vimentin and keratin in ABPCs.

[0054] The results are as follows Figure 2 As shown, the cells expressed positive vimentin (…). Figure 2 A), negative for keratin expression ( Figure 2B) indicates that ABPCs belong to mesenchymal stem cells.

[0055] Osteogenic Differentiation Identification Experiment

[0056] 1. Third-generation ABPCs were cultured using osteogenic induction medium;

[0057] 2. After 7 days of induction culture, staining was performed using the BCIP / NBT alkaline phosphatase kit;

[0058] 3. After 21 days of induction culture, the osteogenic differentiation effect of ABPCs was detected using alizarin red staining.

[0059] The osteogenic induction medium consisted of 50 mL of complete medium supplemented with 500 μL of glutamine, 500 μL of sodium β-glycerophosphate, 100 μL of ascorbic acid, and 5 μL of dexamethasone.

[0060] The results are as follows Figure 2 As shown, alkaline phosphatase staining occurs ( Figure 2 C); Calcium nodules were observed ( Figure 2 (D) indicates that ABPCs can be induced to differentiate into osteopathic forms, proving their stem nature.

[0061] Immunofluorescence assay

[0062] 1. Prepare and fix cell slides as described above. Add 0.5% Triton solution to the cell slides at room temperature and incubate for 10 min. Add PBS and wash three times on a shaker, 10 min each time. Block with 0.5% goat serum at 37 ℃ for 1 h, wash three times with PBS, 5 min each time. Add rabbit anti-rat CD73, CD90, and CD105 primary antibodies respectively, and incubate overnight at 4 ℃ in a humidified chamber.

[0063] 2. The following day, after washing with PBS, add the corresponding secondary antibody for the primary antibody species, incubate in the dark for 1 h, wash with PBS 3 times, 5 min each time. Add ready-to-use DAPI working solution, incubate at 37 ℃ in the dark for 15 min, wash with PBS 3 times, 5 min each time. Mount with 10% glycerol, observe under an upright fluorescence microscope, and detect the expression of CD73, CD90, and CD105 of ABPCs by immunofluorescence.

[0064] The results are as follows Figure 2 As shown in Figure E, CD73 and CD90 expression were positive, while CD73 and CD90 expression was negative, indicating that ABPCs are mesenchymal stem cells.

[0065] Example 3: Synthesis of CM-NPS conditioned medium nanoparticles for deer antler stem cells and screening of the concentration of conditioned medium for deer antler stem cells in the nanoparticles.

[0066] Synthesis of CM-NPS nanoparticles in conditioned medium for deer antler stem cells

[0067] S2.1 PLGA microparticles loaded with ABPCs-CM were prepared using a water / oil / water (w / o / w) emulsification method. ABPCs-CM was used as the inner aqueous phase (PBS was used as the inner aqueous phase in the NM-CON-NPS group), and polyvinyl alcohol (0.1% w / v) was mixed with dichloromethane (DCM) containing PLGA as the oil phase.

[0068] S2.2. The mixture was sonicated in an ice bath for 30 seconds. Immediately afterward, the original emulsion and polyvinyl alcohol (0.7% w / v) were introduced into water to obtain a w / o / w emulsion.

[0069] S2.3. Emulsify the secondary emulsion on a high-speed homogenizer for 5 min. Stir the w / o / w emulsion continuously overnight at room temperature to promote solvent evaporation. Centrifuge the solidified ABPCs-CM nanoparticles, wash them three times with water, freeze-dry them, and store them at -80 ℃.

[0070] S2.4 Screening of the concentration of conditioned medium for deer antler stem cells in nanoparticles

[0071] S2.4.1 The collected ABPCs-CM were freeze-dried using a vacuum freeze dryer and weighed. They were then dissolved in 2 mL of PBS at concentrations of 0, 0.5, 1, 1.5, 2, and 3 mg respectively as the internal aqueous phase for the synthesis of CM-NPS, resulting in six different concentrations of CM-NPS.

[0072] S2.4.2 The protein concentrations in the remaining supernatant after synthesis and in CM-NPS after soaking in PBS for 24 h were detected using the BCA protein quantification kit.

[0073] S2.4.3. Chondrocytes were seeded in 96-well plates, and six different concentrations of CM-NPS were added to complete culture medium for chondrocyte culture. Cell proliferation was detected using a CCK-8 assay kit at 12h, 24h, and 48h. The control group received no treatment.

[0074] S2.4.4. Chondrocytes were seeded in 6-well plates and cultured for 72 h as described above. The chondrogenic activity of the chondrocytes was then detected using an alcine blue staining kit and a toluidine blue staining kit, respectively. The appropriate concentration of conditioned medium was determined by combining the CCK-8 results and the staining results.

[0075] The results are as follows Figure 3 As shown, in Alcian blue staining ( Figure 3 A) and toluidine blue staining ( Figure 3In B), group 5 showed better chondrogenic effects. However, in BCA protein quantification (… Figure 3 The results (C) showed that the protein concentrations of CM-NPS in groups 5 and 6 were similar after dissolving in PBS. However, the CCK-8 results ( Figure 3 D) The results showed that the cell viability of chondrocytes in groups 5 and 6 was similar at 24 h and 48 h after treatment, and further increasing the CM concentration had little effect on cell viability. Therefore, this study ultimately selected the CM concentration used in group 5 as the final experimental concentration.

[0076] Example 4: Extraction of neutrophils and preparation of cell membranes

[0077] S3. Extraction and identification of neutrophils

[0078] S3.1 Neutrophils were collected from human peripheral blood (aged 18-45 years, with negative results for all four pre-transfusion tests) using an animal peripheral blood neutrophil isolation kit. Fresh anticoagulated blood was spread on the separation medium and centrifuged at 2800 rpm for 30 min using a horizontal rotor to collect the second ring of cells. The harvested cells were washed three times with PBS and then lysed at 37°C for 10 min using erythrocyte lysis buffer. The resulting cells were washed three times with PBS, resuspended in serum-free cryopreservation buffer, and stored at −80°C for subsequent membrane derivatization.

[0079] S3.2 At room temperature, fix neutrophils in paraformaldehyde for 30 min, wash three times with PBS, and prepare cell smears. After air-drying, stain with Swiss stain for 10 min, drain, and then stain again with Giemsa stain for 4 min. After staining, slowly rinse the smears with running water to remove excess stain; after air-drying, observe under a microscope.

[0080] The results are as follows Figure 4 As shown, the cell nuclei exhibit diverse morphologies, the cell bodies range from round to oval, and the cytoplasm is pale pink with numerous small, diffusely distributed light red or light purple granules, confirming that the extracted cells are neutrophils.

[0081] Preparation of neutrophil cell membrane

[0082] S3.3 Neutrophils were thawed and washed three times with PBS, then centrifuged (800 rpm, 2 min) to remove the supernatant. After three freeze-thaw cycles, the cells were disrupted using an ultrasonic cell disruptor (30 w, 3 min).

[0083] S3.4. Collect the cell membrane suspension after disruption, centrifuge at 4 ℃ (400 rpm, 10 min), collect the supernatant, centrifuge at 13000 rpm, 20 min, discard the precipitate, centrifuge the supernatant at 100000 rpm, 30 min, discard the supernatant, and collect the neutrophil membrane.

[0084] Example 5

[0085] Preparation and characterization of neutrophil-inspired nanoparticles (NMMPs) in conditioned medium containing deer antler stem cells

[0086] Preparation of S4 and NMMP

[0087] The harvested neutrophil membranes were sonicated on ice for 5 min to resuspend them uniformly in PBS. The cell membranes and nanoparticles were then mixed at a ratio of 1:(1-2) (v / v) and NMMPs were synthesized using an ultrasonic method.

[0088] Electron microscopy of NMMP

[0089] After negative staining with sodium phosphotungstenate solution, the morphology of NMMPs was observed using a transmission electron microscope.

[0090] NMMP particle size and potential measurement

[0091] The particle size and potential of NMMP and CM-NPS were characterized using a Malvern laser particle size analyzer.

[0092] The results are as follows Figure 5 As shown, A is a transmission electron microscope (TEM) image of CM-NPS, B is a TEM image of NMMP (scale bar: 200 nm), C is a particle size measurement diagram of the four materials, and D is a statistical diagram of particle size and potential of the four materials. TEM revealed a clear cell membrane structure on the surface of NMMP. Detection showed that the particle size of CM-NPS was 195 nm, which increased to 229 nm after being coated with a neutrophil membrane, while the potential slightly decreased. All these results indicate that biomimetic therapeutic microparticles loaded with antler bud progenitor cell conditioned medium and coated with neutrophils were successfully prepared.

[0093] Example 6: Biocompatibility testing of neutrophil-inspired nanoparticles (NMMPs) in conditioned medium containing deer antler stem cells

[0094] 1. NMMP Live / dead® Viability Kit Detection

[0095] Macrophages and chondrocytes were cultured for 1, 3, and 5 days, respectively, and cell death was detected using a Calcein-AM / PI double staining kit. The control group received no treatment.

[0096] The results are as follows Figure 6 As shown, A represents the live / dead staining experiment of the material co-cultured with chondrocytes, and B represents its CCK-8 assay results; C represents the live / dead staining experiment of the material co-cultured with macrophages, and D represents its CCK-8 assay results. With increasing co-incubation time with each group of nanomaterials, the fluorescence density of live cells in each group significantly increased, and only a small number of erythrocytes were observed in each group. This indicates that the prepared NMMP has a certain degree of biosafety and can be used for subsequent animal experiments.

[0097] 2. NMMP hemolysis test

[0098] 1. Preparation of 5% red blood cell suspension: Take 5 mL of fresh anticoagulated rabbit blood, dilute it with physiological saline, centrifuge at 2000 rpm for 10 min, discard the supernatant, repeat 3 times, and finally collect the precipitated red blood cells and prepare a 5% red blood cell suspension with physiological saline.

[0099] 2. Mix the suspensions of nanomaterials in each group with an equal volume of red blood cell suspension. Use 0.1% Triton as a positive control and PBS as a negative control. Incubate at 37 °C for 1 h. After centrifugation, take pictures to record the condition of each group and collect the supernatant. Measure the absorbance A at 540 nm.

[0100] The formula for calculating the hemolysis ratio (HR) is: HR(%) = (At - AN) / (AP - AN) × 100%

[0101] In the formula, At represents the absorbance of each group of nanomaterials at the predetermined sampling time point, and AN and AP represent the absorbance of the negative control group and the positive control group, respectively. This experiment was repeated three times.

[0102] The results are as follows Figure 6 As shown, E is a photograph of the hemolysis experiment, and F is the statistical analysis of the hemolysis rate. In the Triton X-100 positive control group, after co-incubation with the red blood cell suspension, the liquid turned bright red with no red blood cell precipitation, and under the microscope, the red blood cells were found to be largely ruptured. The CM-NPS, NM-Con-NPS, and NMMP groups were similar to the negative control group, with no obvious hemolysis observed, and no significant changes were seen in the cells under the microscope. Statistical analysis revealed that the hemolysis rate of each material group was less than 5%, indicating that the material has sufficient safety.

[0103] Example 7: Establishment of a rat model of knee arthritis

[0104] 1. Rats were randomly divided into five groups (sham-operated group, OA group, CM-NPS group, NM-Con-NPS group, and NMMP group). They were then anesthetized by isoflurane inhalation, and the right knee joint was shaved and sterilized with povidone-iodine. The skin and muscles of the knee joint were separated, the anterior cruciate ligament was removed, and the results were examined using the drawer test.

[0105] 2. After cleaning and suturing, the rats were injected with penicillin to prevent infection. The sham-operated group was exposed to the knee joint cavity by separating the skin and muscles, while the OA rats were injected into the knee joint cavity three times a week with 100 uL of normal saline or nanomaterials (CM-NPS, NM-Con-NPS, or NMMP).

[0106] 3. After 1 and 2 months of treatment, all animals were euthanized and knee joint samples were collected.

[0107] Preferably, the rats are 8-week-old SPF-grade SD rats, provided by the Experimental Animal Center of Southwest Medical University, and uniformly housed in the central animal facility of Southwest Medical University, where the housing temperature is 22-26 ℃, the relative humidity is 40%-60%, and they are continuously exposed to a 12-hour light / dark cycle. All rats can eat and drink freely, and the rat bedding is changed every 3 days.

[0108] Example 8: Imaging analysis of rat knee joints

[0109] Micro-CT scans of rat knee joints were performed at 4 and 8 weeks of treatment, and statistical analysis was conducted to assess the degree of damage to the rat knee joints.

[0110] The results are as follows Figure 7 As shown, after osteoarthritis modeling surgery, numerous osteophytes were observed in the knee joint, with rough joint edges, severe damage, and significant swelling. The damage worsened after 8 weeks. In the CM-NPS treatment group, osteophytes in the knee joint were only slightly reduced, and swelling remained slightly higher than in the normal group. In the NM-Con-NPS group, joint swelling was not significant, osteophytes were fewer, but cartilage defects were observed. In the NMMP group, due to the combined cartilage-promoting effect of ABPCs-CM and neutrophil membrane camouflage, excellent osteoarthritis treatment was observed, with no significant osteophyte formation or cartilage defects, and the joint surface was relatively smooth, almost indistinguishable from the sham surgery group. This indicates that NMMP has a good therapeutic effect on osteoarthritis.

[0111] Example 9: HE and Masson section analysis of rat knee joints after nanomaterial treatment

[0112] The results are as follows Figure 8As shown, after 4 weeks of treatment, HE and Masson sections of the rat knee joints revealed that the sham-operated group had a smooth joint surface, continuous cartilage, and normal morphology; while the OA group showed cartilage erosion, cartilage matrix degradation, and severe joint damage. After 4 weeks of nanomaterial treatment, the joint damage was improved to varying degrees, and cartilage degradation was alleviated. The CM-NPS treatment group showed uneven cartilage thickening, but the joint surface was not smooth. In the NM-Con-NPS treatment group, the joint surface smoothness was improved compared to the OA group, but significant degradation of the cartilage matrix still occurred. The NMMP group showed a smooth and continuous joint surface; although the cartilage matrix was slightly less than that of the sham-operated group, it showed a good overall treatment effect.

[0113] The results are as follows Figure 9 As shown, after 8 weeks of treatment, the trend was basically the same as that at 4 weeks. However, with the increase in treatment time, the NMMP group showed better treatment results, and the cartilage matrix was thickened compared to 4 weeks, basically recovering to a state similar to that of the sham surgery group.

Claims

1. A method for preparing a biomimetic nanoparticle formulation of deer antler stem cell conditioned culture medium, characterized in that, Includes the following steps: S1. Deer antler stem cells were isolated and cultured, and P3 cells were obtained. Deer antler stem cell conditioned medium ABPCs-CM was prepared using P3 cells for later use. S2. Using the deer antler stem cell conditioned medium ABPCs-CM obtained in S1, deer antler stem cell conditioned medium nanoparticles CM-NPS were synthesized. S3. Preparation of neutrophil membrane, as follows: S3.

1. Neutrophils were collected from human peripheral blood using an animal peripheral blood neutrophil isolation kit; fresh anticoagulated blood was spread on the separation medium and centrifuged at 2800 rpm for 30 min using a horizontal rotor to collect the second ring cell layer. The harvested cells were washed three times with PBS and then lysed at 37°C for 10 min using erythrocyte lysis buffer; the final cells were washed three times with PBS and resuspended in serum-free cryopreservation buffer to obtain neutrophils, which were then stored at -80°C for subsequent membrane derivatization. S3.2 Neutrophils were thawed and washed three times with PBS, and the supernatant was removed by centrifugation; after repeated freeze-thaw cycles three times, the cells were disrupted using an ultrasonic cell disruptor to obtain a cell membrane suspension; S3.3 Collect the cell membrane suspension after S3.2 disruption, centrifuge at 4 ℃, collect the first supernatant, centrifuge again to discard the precipitate, obtain the second supernatant, centrifuge the second supernatant again, discard the supernatant, and obtain the neutrophil membrane. S4. Combine the CM-NPS conditioned medium nanoparticles of deer antler stem cells obtained in S2 with the neutrophil membrane obtained in S3 to synthesize a biomimetic nanoparticle formulation of conditioned medium for deer antler stem cells.

2. The method for preparing a biomimetic nanoparticle formulation of deer antler stem cell conditioned culture medium according to claim 1, characterized in that, The specific operation of S1 is as follows: S1.1 Clean the skin, disinfect, cut 1-2 cm of the tip of the deer antler, rinse repeatedly with physiological saline to remove blood, soak in PBS containing 10% penicillin-streptomycin for 5 min, rinse again with physiological saline, transfer to DMEM complete culture medium for low temperature storage, and carry out subsequent experiments in a clean bench. S1.2 Remove the skin from the antler and take the mesenchymal part of the antler. Cut it into small pieces and place it in 0.1% type I collagenase. Digest it at 37 ℃ for 30 min. Then add complete culture medium to stop the digestion. After centrifugation, remove the supernatant and evenly disperse the obtained primary tissue blocks in T25 culture flasks. Incubate in an inverted incubator for 2 h. Add an appropriate amount of complete culture medium and culture at 37 ℃ and 5% CO2. Change the medium every 2-3 days. S1.3 When the cells reach 80-90% confluence, trypsin digest them at 37 ℃ for 2-3 min, then stop digestion, transfer to centrifuge tubes, centrifuge at 1000 r / min for 5 min, discard the supernatant, add about 5 mL of complete culture medium, resuspend the cells, and transfer to a new culture flask to obtain P1 cells; continue passage in the same way to obtain P3 cells; the complete culture medium is DMEM medium containing 10 wt% fetal bovine serum; S1.4 After repeatedly washing P3 cells with PBS, culture them in serum-free DMEM medium for 48 h, centrifuge to collect the supernatant, filter with a bacterial filter to obtain deer antler stem cell conditioned medium ABPCs-CM, and store at 4 ℃.

3. The method for preparing a biomimetic nanoparticle formulation of deer antler stem cell conditioned culture medium according to claim 1, characterized in that, The concentration of the conditioned medium for deer antler stem cells is as follows: 2 mg of freeze-dried deer antler stem cell conditioned medium ABPCs-CM is weighed and dissolved in 2 mL of PBS, which is used as the internal aqueous phase for the synthesis of deer antler stem cell conditioned medium nanoparticles CM-NPS.

4. The method for preparing a biomimetic nanoparticle formulation of deer antler stem cell conditioned culture medium according to claim 1, characterized in that, The specific operation of S2 is as follows: S2.1 PLGA microparticles loaded with deer antler stem cell conditioned medium ABPCs-CM were prepared using a water / oil / water emulsification method; the deer antler stem cell conditioned medium ABPCs-CM was used as the inner aqueous phase, and polyvinyl alcohol and PLGA-containing dichloromethane were mixed as the oil phase to obtain a mixture. S2.

2. The mixture obtained in S2.1 is subjected to ultrasonic treatment for 30 s in an ice bath, and the original emulsion and polyvinyl alcohol are introduced into water to obtain a water / oil / water emulsion. S2.

3. Emulsify the emulsion obtained in S2.2 on a high-speed homogenizer for 5 min; continuously stir the water / oil / water emulsion overnight at room temperature to promote solvent evaporation; centrifuge the solidified ABPCs-CM nanoparticles, wash them three times with water, freeze-dry them and store them in a -80℃ refrigerator.

5. The method for preparing a biomimetic nanoparticle formulation of deer antler stem cell conditioned culture medium according to claim 1, characterized in that, The specific operation of S4 is as follows: The neutrophil membrane obtained in S3 is sonicated in an ice bath for 5 min to make it uniformly resuspended in PBS. The neutrophil membrane suspension is mixed with the deer antler stem cell conditioned medium nanoparticles CM-NPS obtained in S2 at a ratio of 1:(1-2) (v / v). The deer antler stem cell conditioned medium biomimetic nanoparticle preparation is synthesized by sonication.

6. The application of a biomimetic nanoparticle formulation of deer antler stem cell conditioned culture medium as described in any one of claims 1-5, characterized in that, Application in the treatment of osteoarthritis.

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