Engineered outer vesicles based on antler bud progenitor cells as well as preparation method and application of engineered outer vesicles

Abstract

The invention discloses engineered outer vesicles based on antler bud progenitor cells, and also discloses a preparation method of the engineered outer vesicles, and the preparation method comprises the following steps: extracting the antler bud progenitor cell outer vesicles EVsABPC, and purifying through an ultracentrifugation method; and synthesizing a cell penetrating peptide ACPP, and combining the cell penetrating peptide ACPP with the EVsABPC obtained in the step 1 to prepare the engineered extracellular vesicle ACPP (at) EVsABPC modified by the cell penetrating peptide. The invention also discloses an application of the engineered outer vesicle in spinal cord injury repair. According to the method disclosed by the invention, the ACPP peptide fragment can be specifically combined with matrix metalloproteinase 2 (MMP2), so that the modified outer vesicles are accurately targeted at the spinal cord injury part. The ACPP-coated EVsABPC has excellent biocompatibility, can effectively promote nerve regeneration and reduce neuronal apoptosis, has an immune regulation effect, and is expected to be used for repairing nerve injury and treating other related diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and relates to an engineered exosome based on antler blastema progenitor cells. The present invention also relates to a preparation method and application of the engineered exosome. Background Art

[0002] Spinal cord injury is a disease that causes severe damage to sensory, motor and autonomic functions. Existing treatments, such as surgical decompression, corticosteroid therapy and rehabilitation training, have limited therapeutic effects. Studies have shown that extracellular vesicles derived from mesenchymal stem cells have the potential to promote nerve regeneration. However, traditional mesenchymal stem cells are not ideal for use in tissue regeneration due to their susceptibility to aging and limited regeneration capacity. Deer antlers are the only mammalian organ that can be fully regenerated. As a source of mesenchymal stem cells that support complete regeneration of antlers, antler blastema progenitor cells exhibit extremely strong regenerative ability and immunomodulatory properties, providing a new therapeutic approach for spinal cord injury repair. However, the direct application of antler stem cells in clinical practice will still face limitations such as ethics, safety and xenotransplantation reactions. Extracellular vesicles (EVs) based on antler blastema progenitor cells ABPC ) can effectively transmit regenerative signals originating from these cells, overcoming the limitations of direct cell application. Finally, the distribution of exosomes in the body often lacks targeting and is easy to diffuse in non-target tissues, thereby reducing its effectiveness at the site of injury. In addition, due to the complex pathological environment of the spinal cord injury site, the ability of exosomes to remain and function at the injury site is also limited. Therefore, it is necessary to design a targeting peptide-modified exosome system that can achieve efficient positioning and aggregation at the site of spinal cord injury, thereby accurately regulating the neural regeneration process, enhancing the transmission of regenerative signals, and optimizing the injury microenvironment. This is of great significance for promoting the repair of spinal cord injuries. Summary of the invention

[0003] The purpose of the present invention is to provide an engineered extracellular vesicle (EVs) based on cell-penetrating peptide (ACPP) from antler blastema progenitor cells. ABPC ), which has the characteristics of high biocompatibility, targeting inflammatory damage sites, promoting neural stem cell proliferation and promoting axon regeneration.

[0004] Another object of the present invention is to provide a method for preparing the above-mentioned engineered extracellular vesicles.

[0005] The third object of the present invention is to provide the use of the above-mentioned engineered exosomes in promoting the repair of spinal cord injury.

[0006] The technical solution adopted by the present invention is a method for preparing engineered exosomes based on antler blastema progenitor cells, which is specifically implemented according to the following steps: Step 1: Extraction of extracellular vesicles (EVs) from antler blastema progenitor cells ABPC , and purified by ultracentrifugation; Step 2: Synthesize the cell-penetrating peptide ACPP and combine it with the EVs obtained in step 1 ABPC Combined, engineered extracellular vesicles ACPP@EVsABPC modified with cell-penetrating peptides were prepared.

[0007] The present invention is also characterized in that: Step 1 is as follows: The blastema progenitor cells were extracted from the antlers of two-year-old male sika deer and cultured in a solution containing Dulbecco's modified Eagle's medium. CX43 was screened by flow cytometry. + and FGFR2 + After subculturing and expansion, extracellular vesicles (EVs) were isolated from the culture medium using ultracentrifugation. ABPC .

[0008] The ultracentrifugation step was performed at 16,000 g for 70 min at 4 °C, and the final extracellular vesicles were resuspended in pre-chilled PBS and stored at -80 °C.

[0009] Step 2 is as follows: Step 2.1, synthesize ACPP peptide using solid phase synthesis technology according to the designed sequence; Step 2.2, prepare Tris·HCl buffer; Step 2.3, dissolving the ACPP peptide synthesized in step 2.1 in the Tris·HCl buffer prepared in step 2.2 to form an ACPP solution; preparing an ethanol solution of a chemical coupling agent DSPE, then adding the ACPP solution to the ethanol solution of DSPE, and mixing and coupling in a light-proof condition; Step 2.4: EVs prepared in step 1 ABPC Add EVs to the reaction mixture obtained in step 2.3 ABPC Incubate with ACPP peptide reaction solution to allow ACPP peptide to bind to EVs ABPC surface, followed by dialysis to obtain ACPP@EVsABPC.

[0010] In step 2.1, the sequence of the ACPP peptide is: EEEEEEEE(E8)-6-aminohexanoyl-PLGLAG-RRRRRRRR(R8).

[0011] In step 2.2, the pH of the prepared Tris·HCl buffer is 6.8-7.4, and the concentration is 10-50 mM.

[0012] Step 2.3 is as follows: The ACPP peptide synthesized in step 2.1 is dissolved in the Tris·HCl buffer prepared in step 2.2, wherein the mass ratio of ACPP to Tris·HCl is 1:10-20, to form an ACPP solution; a DSPE ethanol solution with a concentration of 20-40 mg / mL is prepared; the ACPP solution is added to the DSPE ethanol solution, and Tris·HCl buffer is added to make the volume of the reaction system 4.5-5.5 mL, and then the reaction system is placed in a light-proof condition for thorough mixing and coupling.

[0013] In step 2.4, EVs ABPC The concentration was 10¹¹ particles / mL. The dialysis was performed overnight under stirring and protected from light, and the dialysate was replaced every 8-12 hours.

[0014] Another technical solution adopted by the present invention is an engineered exosome based on antler blastema progenitor cells, which is prepared by the above-mentioned preparation method.

[0015] The third technical solution adopted by the present invention is the application of the above-mentioned engineered exosomes in the repair of spinal cord injury.

[0016] The beneficial effects of the present invention are: The method of the present invention uses activated cell-penetrating peptide (ACPP) to modify the extracellular vesicles of antler blastema progenitor cells to obtain ACPP@EVs ABPC Not only does it have good targeting, it can also effectively enhance the enrichment of vesicles at the site of spinal cord injury. First, the extracellular vesicles (EVs) of antler blastema progenitor cells obtained by ultracentrifugation have good biocompatibility and regenerative signal transmission ability. Then, ACPP is modified on the surface of the vesicles using chemical coupling to generate ACPP@EVs. ABPC The modified extracellular vesicles can achieve efficient aggregation at the site of spinal cord injury through the targeting effect of ACPP, thereby significantly enhancing the local transmission of nerve regeneration signals and optimizing the damaged microenvironment. ABPC By regulating immune response, inhibiting neuronal apoptosis, and promoting neuronal proliferation and differentiation, the purpose of improving motor function recovery after spinal cord injury is finally achieved. Therefore, the present invention provides a new strategy for treating spinal cord injury with high efficiency targeting and neural regeneration potential, which has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 is an extracellular vesicle (EVs) of antler blastema progenitor cells extracted from Example 1 of the present invention. ABPC ) characterization and identification diagram, where Figure 1a TEM image showing EVs ABPC morphology, scale bar is 200 nm; Figure 1b To characterize EVs by nanoparticle tracking analysis (NTA) ABPC Quantification and size analysis; Figure 1c Western blot analysis of extracellular vesicle marker proteins (CD63, HSP90 and ANNEXIN A1) for EVs ABPC Identification of FIG. 2 is EVs extracted from Example 1 of the present invention ABPC The test results of the effect on the proliferation of neural stem cells are shown in Figure 1. Figure 2a To stain the neural stem cell markers Nestin and Sox2 to identify the extracted neural stem cells; Figure 2b For EVs ABPC Ki67 immunofluorescence staining and quantitative analysis of primary neural stem cells (NSCs) after 24 h of co-culture, scale bar, 50 μm; Figure 2c Western blot was used to detect the expression of EVs in the co-cultured cells (including control group, BMSC and EVs ABPC ) Expression levels of Nestin and Ki67 proteins in neural stem cells; FIG. 3 is an ACPP@EVs constructed in Example 2 of the present invention ABPC The identification diagram of Figure 3a The specific interactions between MMP2 and ACPP peptides, including hydrogen bonds and salt bridges, were analyzed by molecular docking simulations; Figure 3b ACPP@EVs ABPC Mass spectrometry analysis was performed, and the DIA spectrum showed the peptide sequence (EEEEEEEEEEPLGLAGR), and the error plot showed the measurement accuracy; FIG. 4 is an ACPP@EVs constructed in Example 2 of the present invention ABPC Distribution in rats with spinal cord injury, including Figure 4a ACPP@EVs ABPC Representative images and quantification of intensity distribution at the injury site; Figure 4b ACPP@EVs ABPC Distribution and quantification of various important organs in rats with spinal cord injury; FIG. 5 is an ACPP@EVs constructed in Example 2 of the present invention ABPC The test results of the effect on macrophage polarization are shown in Figure 2. Figure 5a ACPP@EVs ABPC Immunofluorescence images and quantification results after co-culture with M1 macrophages. The upper row is iNOS / F4 / 80 / DAPI; the lower row is Arg1 / F4 / 80 / DAPI; Figure 5bDifferent treatments (including control group, LPS+IFN-γ, LPS+IFN-γ+ACPP@EVs) are shown. BMSC and LPS+IFN-γ+ACPP@EVs ABPC ) The percentage of CD86+ and CD206+ cells in F4 / 80+ gated macrophages after flow cytometry sorting; Figure 6 shows ACPP@EVs ABPC Figure 2 shows the results of in vivo efficacy testing, where Figure 6a ACPP@EVs ABPC Representative images of the right hindlimb of treated rats in the crawling test; Figure 6b is the BBB (Basso, Beattie, Bresnahan) motor scoring scale for the hind limbs of rats during the 8-week treatment period; Figure 6c This is a representative footprint of a rat with spinal cord injury after treatment recorded using the Catwalk XT system; Figure 7 is ACPP@EVs ABPC The results of the test on promoting nerve regeneration and reducing neuronal apoptosis after spinal cord injury in vivo, Figure 7a are representative Nissl-stained images and quantitative analysis of the average optical density of neurons 7 days after injury (n=4), scale bar is 500µm; Figure 7b ACPP@EVs ABPC Representative immunofluorescence images and quantitative analysis of the effects on neuronal apoptosis 7 days after spinal cord injury. Neurons were stained with NeuN (green) and apoptotic cells were labeled with TUNEL (red). The scale bar is 50 µm. Figure 7c Representative immunofluorescence images of sagittal sections of the spinal cord after injury in different groups. NF (white) and GAP43 (purple) staining were used to visualize nerve fibers and regenerating axons. Figure 8 ACPP@EVs ABPC Figure 2 shows the biocompatibility test results. Representative hematoxylin and eosin (H&E) staining images of heart, liver, spleen, lung, and kidney tissues in different groups 8 weeks after surgery. DETAILED DESCRIPTION

[0018] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0019] The present invention is based on the engineered extracellular vesicles (ACPP@EVs) of antler blastema progenitor cells. ABPC ), which is specifically implemented according to the following steps: Step 1: Extraction of exocysts (EVs) from antler blastema progenitor cells ABPC ).

[0020] Step 1.1. Extract antler blastema progenitor cells (ABPCs) from the hard horns of two-year-old male sika deer. The hard horn tissue of sika deer five days after detachment was selected for cell isolation. Cut the blastema tissue of the hard horn into small pieces and put it into a solution containing Dulbecco's modified Eagle medium (DMEM, Gibco, catalog number 11965092), and add 1% penicillin-streptomycin (Gibco, catalog number 15140122) and 0.1% mycoplasma remover to prevent cell contamination. Use trypsin to digest the tissue and prepare a single cell suspension. Sort the cells by flow cytometry to screen out double-positive cells expressing CX43 and FGFR2 (CX43 + FGFR2 + ), which is ABPCs. The sorted ABPCs cells were inoculated in a culture dish, and the culture medium was DMEM containing fetal bovine serum (FBS, Gibco, catalog number 10099141C). The cells were cultured in a constant temperature incubator at 37°C and 5% CO2. After 24 hours, the non-adherent cells were removed and only the adherent cells were retained. When the cells reached 80-90% fusion, 0.25% EDTA-containing trypsin was used for cell digestion and passage expansion. The cells were re-seeded at the same density each time they were expanded to ensure uniform growth of the cells. Passaging and experimental use: ABPCs are usually passaged to the 6th-8th generation (P6-P8) for subsequent experiments. The culture conditions should be strictly controlled during the passage process to ensure the proliferation ability and regeneration characteristics of the cells.

[0021] Step 1.2: Isolation and purification of extracellular vesicles (EVs) from antler blastema progenitor cells ABPC ): Cell viability was detected by CCK-8 kit, and only cell culture supernatant with viability greater than 90% and ABPCs of passages 6-8 (P6-P8) were used for extracellular vesicle collection. The culture medium of ABPCs was centrifuged at 750g for 20 minutes to remove cells and large impurities; and then centrifuged at 2000g for 30 minutes to remove cell debris and apoptotic bodies. Subsequently, the supernatant was centrifuged at 16000g for 70 minutes to collect the extracellular vesicle pellet. The pellet was resuspended in pre-cooled PBS and further purified by centrifugation at 16000g for 70 minutes to ensure the purity of the extracellular vesicles. All centrifugation steps were performed at 4°C to ensure the stability and functionality of the extracellular vesicles. The final extracellular vesicle pellet was resuspended in 100-200µL pre-cooled PBS and stored at -80°C.

[0022] Step 2: Preparation of ACPP-modified antler blastema progenitor extracellular vesicles (ACPP@EVs ABPC ) Step 2.1: According to the designed sequence, the ACPP peptide (EEEEEEEE(E8)-6-aminohexanoyl-PLGLAG-RRRRRRRR(R8)) was synthesized by solid phase synthesis technology. After synthesis, the peptide was purified by high performance liquid chromatography (HPLC), and the purity and molecular weight of the peptide were confirmed by mass spectrometry to ensure that it met the experimental requirements.

[0023] Step 2.2: Dissolve an appropriate amount of tris·HCl in deionized water, adjust the pH of the solution to 6.8-7.4 with 1% ammonia water, and prepare a Tris·HCl buffer with a concentration of 10-50 mM. This buffer is used for peptide dissolution and coupling reaction in subsequent reaction steps.

[0024] Step 2.3. Weigh an appropriate amount of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and dissolve it in anhydrous ethanol to prepare a DSPE solution with a concentration of 20-40 mg / mL. Dissolve the ACPP peptide synthesized in step 2.1 in the Tris·HCl buffer prepared in step 2.2, so that the mass ratio of ACPP to Tris·HCl is 1:10-20 or the molar ratio is between 1:5-10, and then add the ACPP solution to the DSPE solution. Rinse the peptide container twice with Tris·HCl buffer and add the rinse solution to the reaction system to finally make the reaction volume in the range of 2.5-3.5 mL. Add an appropriate amount of Tris·HCl buffer to make the final volume of the reaction system 4.5-5.5 mL. To avoid photolysis, wrap the reaction tube with aluminum foil and mix it on a shaker overnight in the dark to ensure that DSPE and ACPP peptide are fully coupled.

[0025] Step 2.4: EVs with a concentration of 10¹¹ particles / mL ABPC Add to the reaction mixture obtained in step 2.3 and continue incubation for a while to ensure that the peptide fragments are bound to EVs. ABPC Fully bind. After the binding reaction is completed, transfer the mixture to a dialysis membrane bag and dialyze it in sufficient deionized water (2L is recommended), and add an appropriate amount of anhydrous ethanol. During the dialysis process, stir and protect from light overnight, and replace the dialysate every 8-12 hours to ensure that the unbound reaction components are completely removed.

[0026] Step 2.5: Purified ACPP-modified EVs ABPC (ACPP@EVs ABPC ) and stored in an ultra-low temperature freezer at -80°C to ensure its activity and stability. In subsequent experiments or clinical applications, it can be diluted appropriately after thawing and used directly to obtain the best therapeutic effect.

[0027] In the present invention, ACPP@EVs ABPC ) in the preparation method: The purpose of step 1 is to extract the exocysts (EVs) of antler blastema progenitor cells. ABPC ). Antler blastema progenitor cells (ABPCs) were extracted from two-year-old male sika deer and CX43 was screened by flow cytometry. + FGFR2 + Cells are then centrifuged to separate extracellular vesicles (EVs) from the culture medium. ABPC The obtained extracellular vesicles were characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) for particle size and morphology. Its advantages are: it can efficiently and accurately separate and purify extracellular vesicles (EVs ABPC ), flow cytometry is used to ensure that the selected cells have specific regenerative capacity. The centrifugation step can remove impurities to the maximum extent and ensure the purity of the extracellular vesicles. In addition, the extracellular vesicles have good biocompatibility and regenerative potential, and have broad application prospects in the fields of regenerative medicine and neural repair.

[0028] The purpose of step 2 is to prepare cell-penetrating peptide (ACPP)-modified antler blastema progenitor cell extracellular vesicles (ACPP@EVs ABPC ), so that the exosomes have targeting and cell penetration capabilities, thereby enhancing their therapeutic effects at the site of injury. The key steps and principles involved are: ACPP has cell penetration function and can specifically bind to matrix metalloproteinases 2 / 9 (MMP2 / 9), which are highly expressed in the microenvironment of damaged tissues, especially in inflammation and injury sites. ACPP can be activated in damaged tissues rich in matrix metalloproteinases, making the modified exosomes (ACPP@EVs ABPC ) precisely targets the damaged part, thus improving its therapeutic efficiency. This specific binding enables the extracellular vesicles to not only have cell penetration ability, but also achieve directional delivery in specific pathological environments, maximizing their regeneration and repair effects. The advantages of this modification are: ACPP@EVs ABPC By binding to MMP2 / 9 in a targeted manner, it can accumulate at the site of disease or injury, reduce the distribution of drugs in normal tissues, and reduce side effects. At the same time, this modification also enhances the cell penetration ability of the exosomes, making it easier for them to enter the cell interior, further enhancing the therapeutic effect.

[0029] Example 1 Step 1. Extract ABPCs cells from the hard horns of two-year-old male sika deer, and select the hard horn blastema tissue five days after horn removal for cell isolation. Cut the blastema tissue into small pieces of about 1 mm³. Place the cut tissue blocks in a culture medium containing Dulbecco's modified Eagle medium, and add 1% penicillin-streptomycin and 0.1% mycoplasma remover to prevent cell contamination. Incubate the blastema tissue in a constant temperature incubator at 37°C and 5% CO2 for 1 hour, then add trypsin (0.25%, containing EDTA) to digest the tissue, and continue digestion for about 30 minutes until the tissue blocks are completely dissociated. Use trypsin-free DMEM to neutralize the digestion solution, and then filter through a cell strainer (70μm pore size) to obtain a single cell suspension. Cells were sorted by flow cytometry to screen out double-positive cells expressing CX43 and FGFR2 (CX43 + FGFR2 + ), which are antler blastema progenitor cells (ABPCs). The sorted ABPCs cells were divided into 2×10 5 The cells were inoculated at a density of 10 cells / cm² in a 10 cm dish in DMEM containing fetal bovine serum. The cells were cultured in an incubator at 37°C and 5% CO2 for 24 hours, and the non-adherent cells were removed, leaving only the adherent cells. When the cells reached 80-90% confluence, the cells were digested with 0.25% trypsin and amplified by subculture, and re-inoculated at the same density to ensure uniform cell growth. ABPCs are usually subcultured to passage 6-8 (P6-P8) for subsequent experiments. The culture conditions must be strictly controlled during the subculture process to ensure cell proliferation ability and regeneration characteristics.

[0030] Step 2. Use CCK-8 kit to detect cell viability. Only culture medium with viability greater than 90% and ABPCs of passages 6-8 (P6-P8) were used for the isolation and collection of extracellular vesicles. The culture medium of ABPCs was centrifuged at 750g for 20 minutes to remove cells and large impurities. The supernatant was transferred to a new centrifuge tube and centrifuged at 2000g for 30 minutes to remove cell debris and apoptotic bodies. The supernatant was transferred again and centrifuged at 16000g for 70 minutes to collect the extracellular vesicle pellet. The pellet was resuspended in pre-cooled PBS and then centrifuged at 16000g for 70 minutes for further purification to ensure the purity of the extracellular vesicles. All centrifugation steps were performed at 4°C to ensure the stability and functionality of the extracellular vesicles. The final extracellular vesicle pellet was resuspended in 100µL pre-cooled PBS and stored in an ultra-low temperature refrigerator at -80°C for subsequent experiments.

[0031] Example 2 Based on Example 1, EVs prepared in Example 1 were used. ABPC Preparation of ACPP@EVs ABPC : Step 1. According to the designed sequence (EEEEEEEE(E8)-6-aminohexanoyl-PLGLAG-RRRRRRRR(R8)), the ACPP peptide was synthesized by solid phase synthesis technology. After synthesis, the peptide was purified by high performance liquid chromatography (HPLC) to ensure its high purity. The molecular weight of the peptide was confirmed by mass spectrometry to ensure that it met the experimental requirements.

[0032] Step 2: Dissolve 0.44 g Tris·HCl in 45 mL deionized water and adjust the pH to 6.8-7.4 with 1% ammonia water to prepare Tris·HCl buffer.

[0033] Weigh 5 mg of DSPE and dissolve it in 200 µL of anhydrous ethanol. Dissolve the synthesized ACPP peptide in 1 mL of Tris·HCl buffer and add it to the DSPE solution. Then rinse the peptide container twice with Tris·HCl buffer and add the rinse solution to the reaction system to finally bring the reaction volume to 3.2 mL. Add 1.8 mL of Tris·HCl buffer to bring the final reaction volume to 5 mL. Wrap the reaction tube in aluminum foil to protect it from light and mix it on a shaker overnight to ensure that the ACPP peptide is fully coupled to DSPE.

[0034] 1×10¹¹ particles / mL of EVs ABPC The exosomes were added to the coupling reaction mixture and incubated for 2 hours to ensure that the ACPP peptide was fully bound to the exosomes. The reaction mixture was transferred to a dialysis membrane bag and dialyzed in 2L of deionized water, while adding an appropriate amount of anhydrous ethanol. The dialyzate was stirred overnight in the dark and the dialysate was replaced every 12 hours to ensure that the unbound peptide was completely removed.

[0035] Step 3: After dialysis, the purified ACPP-modified extracellular vesicles (ACPP@EV) were measured using NTA. sABPC ) particle concentration, the final particle concentration is about 1×10 10 Particles / mL. Morphological analysis by TEM ensured that ACPP@EVs ABPC The particle size and morphology met the requirements, and the markers of extracellular vesicles were analyzed by Western Blot to verify the modification effect.

[0036] Step 4: Purified ACPP@EVs ABPC After the samples are packaged, they are stored in an ultra-low temperature freezer at -80°C. In subsequent experiments or clinical applications, the samples are thawed and appropriately diluted before direct use to achieve the best therapeutic effect.

[0037] Example 3 EVs prepared in Example 1 ABPC Perform performance testing, including: Transmission electron microscopy (TEM) characterization: The isolated extracellular vesicles (EVs) were characterized using transmission electron microscopy (TEM; TECNAI Spirit, FEI, USA). ABPC ) for morphological analysis. The sample was dropped onto a copper grid for negative staining to observe EVs ABPC TEM was used to confirm the spherical structure of EVs and their integrity, and representative images were taken.

[0038] Nanoparticle Tracking Analysis (NTA): EVs were measured using a nanoparticle tracking analyzer (NTA; ZetaView, ParticleMetrix, Germany). ABPC Size distribution and concentration of EVs ABPC The suspension is tested by the NTA system, which records the Brownian motion trajectory of the particles in the liquid and calculates the diameter of each particle. A detailed particle size distribution diagram is generated to ensure that the obtained EVs ABPC The particle size was within the desired range.

[0039] To confirm EVs ABPC The marker expression of EVs was further characterized by Western Blot. First, 2 μg of ABPCs cell lysate and 2 μg of EVs were collected. ABPC Samples are loaded into each gel lane. After protein separation by electrophoresis, they are transferred to a membrane and detected using specific antibodies against extracellular vesicle markers (such as CD63, HSP90) and small extracellular vesicles (such as ANNEXIN A1). The nature and purity of the samples are further verified.

[0040] The test results are as follows: (1) EVs ABPC Characterization: like Figure 1a As shown, the prepared EVs ABPC Observation by transmission electron microscopy (TEM) showed that it has a spherical structure with an average size of about 150-200nm (the scale bar in the figure is 200nm) and a uniform morphology. Figure 1b As shown, EVs were analyzed by nanoparticle tracking analysis (NTA). ABPC Quantification and size analysis further confirmed that EVs ABPC The average diameter of the vesicles was 182.4 ± 1.7 nm, indicating that the size of the vesicles was uniform. Figure 1c As shown, Western Blot results showed that EVs ABPCThe extracellular vesicle-specific markers, including CD63, HSP90, and ANNEXIN A1, were successfully expressed in the culture medium, verifying the purity and functional integrity of the extracellular vesicles. These results indicate that functional extracellular vesicles were successfully isolated and purified, and have the potential for further application.

[0041] (2) EVs ABPC Performance test of the effect on neural stem cell proliferation: The following three experimental schemes were designed to verify EVs ABPC Effects on neural stem cells (NSCs): Control group: No extracellular vesicles (EVs) were applied, and the proliferation and function of NSCs were observed without treatment. BMSC group (EVs BMSC ): Using extracellular vesicles (EVs) derived from bone marrow mesenchymal stem cells (BMSCs) BMSC ), and evaluate its effect on NSCs proliferation and function as the basic experimental group. ABPC group (EVs ABPC ): Using EVs ABPC , and observed its effects on the proliferation and function of NSCs. As shown in Figure 2, the extracted EVs ABPC It has a significant effect on the proliferation of neural stem cells (NSCs). Figure 2a (Merge in the figure refers to the image after superimposing different channels) Immunofluorescence staining of neural stem cell markers Nestin and Sox2 is shown, and DAPI is used to stain the cell nucleus, proving that the extracted primary cells are neural stem cells. Figure 2b Shown with EVs ABPC After 24 hours of co-culture, the results of Ki67 immunofluorescence staining of NSCs are shown in the figure. The scale bar is 50 μm. Ki67 is a marker of cell proliferation. The position of the cell nucleus can be observed by DAPI staining. BMSC EVs ABPC ), EVs ABPC The treated group showed more Ki67-positive cells, indicating that EVs ABPC It can significantly promote the proliferation of neural stem cells. Figure 2c Western blot results show that in different treatment groups (control group, EVs BMSC Group, EVs ABPC The expression levels of Nestin and Ki67 proteins in neural stem cells after co-culture (group 1). ABPC The expression of Nestin and Ki67 in the treatment group was significantly higher than that in other groups, further verifying that EVs ABPC Promoting effect on the proliferation of neural stem cells.

[0042] Example 4 ACPP@EVs prepared in Example 2 ABPC The performance test results are as follows: (3) ACPP@EVs ABPC Conduct performance testing like Figure 3a As shown in the figure, the interaction between matrix metalloproteinase 2 (MMP2) and ACPP peptide was analyzed by molecular docking simulation. The figure shows the specific binding site between MMP2 and ACPP peptide, which contains multiple hydrogen bonds and salt bridge interactions, proving that ACPP peptide can specifically target MMP2, thereby making the modified extracellular vesicles (ACPP@EVs ABPC ) can precisely target the site of injury. Figure 3b ACPP@EVs ABPC The mass spectrometry analysis results show the deconvoluted DIA (Data-Independent Acquisition) mass spectrum, and the peptide sequence is EEEEEEEEEEEPLGLAGR. The spectrum clearly shows the ion peaks of each amino acid residue of the peptide, further confirming the sequence composition of the ACPP peptide. The error graph below shows that the measurement errors are all within 0.05Da, indicating that the mass spectrometry analysis results are highly accurate. These data further verify the ACPP@EVs ABPC The structural and functional characteristics of the protein ensure its good targeting and functional integrity.

[0043] Example 5 (4) ACPP@EVs ABPC Testing of distribution in rats with spinal cord injury As shown in Figure 4, ACPP@EVs were detected by in vivo fluorescence imaging. ABPC In vivo distribution in a rat model of spinal cord injury. Figure 4a ACPP@EVs ABPC Distribution intensity at the site of spinal cord injury. EVs labeled with the fluorescent tracer Dir ABPC and ACPP@EVs ABPC The in vivo imaging results show that ACPP@EVs ABPC The fluorescence intensity at the damaged site was significantly higher than that of unmodified EVs ABPC The results of quantitative analysis further showed that ACPP@EVs ABPC The fluorescence intensity at the site of spinal cord injury was significantly higher than that in the control group, which was statistically significant (p < 0.01). Figure 4b ACPP@EVs ABPCThe distribution of ACPP@EVs in the main organs of rats with spinal cord injury. ABPC It is distributed in the spinal cord, spleen, liver and other organs to a certain extent, but its enrichment in the spinal cord is significantly higher than that in other organs. ABPC Group, ACPP@EVs ABPC The fluorescence intensity in the spinal cord increased significantly, while the distribution in the liver and spleen decreased, indicating that ACPP modification can reduce the nonspecific distribution of extracellular vesicles in non-targeted organs. ABPC The fluorescence intensity in different organs and its enrichment in the spinal cord were significantly higher than those in the control group (p<0.01), while its distribution in organs such as the spleen and liver was significantly reduced (p<0.001).

[0044] (5) ACPP@EVs ABPC Testing the impact of the inflammatory environment Three experimental schemes were designed to verify EVs ABPC The effects on macrophages were as follows: control group: no extracellular vesicles (EVs) were given. BMSC group (ACPP@EVs BMSC ): Using EVs derived from ACPP peptide modification BMSC (ACPP@EVs BMSC ABPC group (ACPP@EVs ABPC ): EVs modified with ACPP peptide ABPC Figure 5 shows the ACPP@EVs ABPC Effects on macrophage polarization. Figure 5a As shown, ACPP@EVs ABPC Immunofluorescence images and quantitative analysis after co-culture with M1 macrophages. The upper row shows iNOS (M1 macrophage marker) with F4 / 80 and DAPI staining, showing the distribution of M1 macrophages in different treatment groups. The lower row shows Arg1 (M2 macrophage marker) with F4 / 80 and DAPI staining, showing the distribution of M2 macrophages. The quantitative analysis results show that ACPP@EVs ABPC Treatment group and ACPP@EVs BMSC Compared with the control group, the fluorescence intensity of iNOS was significantly reduced (p < 0.01), while the fluorescence intensity of Arg1 was significantly increased (p < 0.05), indicating that ACPP@EVs ABPC It can promote the polarization of M1 to M2 macrophages. Figure 5b The F4 / 80 in different treatment groups is shown. + CD86 in macrophage subsets + (M1 macrophage marker) and CD206+ Flow cytometry analysis results of M2 macrophage markers. In the LPS+IFN-γ treatment group, the proportion of M1 macrophages increased significantly (CD86 + ), while ACPP@EVs ABPC The treated group showed CD206 + The proportion of M2 macrophages was significantly increased (p<0.05) compared with the control group and other treatment groups. ABPC It more effectively promoted the polarization of M2 macrophages and had a significant immunomodulatory effect. By comparing the results of these three groups of experiments, we can systematically evaluate the ACPP-modified ABPC extracellular vesicles (ACPP@EVs ABPC ) and ordinary extracellular vesicles (ACPP@EVs BMSC ) and the control group, verifying its potential application in regulating the immune microenvironment.

[0045] (6) ACPP@EVs ABPC In vivo efficacy testing: A rat spinal cord injury (SCI) model was established and ACPP@EVs were administered three times a week using tail vein injection. ABPC As shown in Figure 6, ACPP@EVs ABPC The results of in vivo efficacy tests showed its significant therapeutic effect on rats with spinal cord injury. Figure 6a Representative images of the right hind limbs of rats in different treatment groups in the crawling test are shown. Compared with the untreated SCI group, ACPP@EVs ABPC The rats in the treated group showed better motor function, and their crawling posture was close to that of the normal group (sham operation group), while ACPP@EVs BMSC The recovery degree of rats in the ACPP@EVs group was slightly lower than that in the ABPC Group. Figure 6b The BBB (Basso, Beattie, Bresnahan) motor scoring scale results of rats during the 8-week treatment period. The BBB score is used to evaluate the recovery of hind limb motor function in rats after spinal cord injury. Compared with the spinal cord injury group, ACPP@EVs ABPC The scores of the treatment group were significantly improved at each time point and were close to normal levels at 8 weeks. BMSC The group also showed significant improvement, but the effect was not as good as ACPP@EVs ABPC Group. Figure 6c Representative footprints of rats in different treatment groups recorded using the Catwalk XT system are shown. Compared with the gait abnormalities of the SCI group, ACPP@EVs ABPC The gait of the treated group was significantly improved, and the gait pattern was closer to that of the normal group of rats. Footprint images show that ACPP@EVsABPC The limb coordination and movement trajectory of rats in the treatment group were significantly improved, while ACPP@EVs BMSC The group also showed some improvement, but not as good as ACPP@EVs ABPC These results show that ACPP@EVs ABPC It has a significant therapeutic effect in the recovery of motor function after spinal cord injury, which is superior to other treatment groups. It can effectively promote the recovery of motor function and improve gait coordination in rats.

[0046] (7) ACPP@EVs ABPC Test on the efficacy of promoting nerve regeneration in rats with spinal cord injury: As shown in Figure 7, ACPP@EVs ABPC It can promote nerve regeneration and reduce neuronal apoptosis after spinal cord injury in vivo. Figure 7a Representative Nissl staining images are shown to assess neuronal survival after spinal cord injury. ACPP@EVs ABPC The treated group showed a higher neuronal density compared with the spinal cord injury group and ACPP@EVs BMSC Compared with the ACPP@EVs ABPC The Nissl staining optical density of the treated group increased significantly, indicating that ACPP@EVs ABPC It can effectively promote the survival and regeneration of neurons. The quantitative analysis results on the right show that ACPP@EVs ABPC The mean optical density of the group was significantly higher than that of the other groups (p<0.001). Figure 7b The immunofluorescence images and quantitative analysis of neuronal apoptosis. NeuN labeled neurons (green), and TUNEL labeled apoptotic cells (red). Compared with the spinal cord injury group, ACPP@EVs ABPC Treatment significantly reduced the number of TUNEL-positive cells, indicating that it has a protective effect on neurons. The quantitative results on the right show that ACPP@EVs ABPC The proportion of apoptotic cells in the SCI group was significantly lower than that in the ACPP@EVs group. BMSC group (p<0.05). Figure 7c Immunofluorescence images of nerve regeneration after spinal cord injury in different treatment groups are shown. NF (white) is used to mark nerve fibers, and GAP43 (purple) is used to mark regenerating axons. ACPP@EVs ABPC The treated group showed more nerve fiber extension and regeneration, compared with the spinal cord injury group and ACPP@EVs BMSC The length of nerve fibers was significantly increased compared with the control group. The quantitative analysis on the right shows that ACPP@EVs ABPCThe nerve fiber length of the treated group was significantly longer than that of the other treated groups (p<0.001). These results indicate that ACPP@EVsABPC can significantly promote neuronal survival after spinal cord injury, reduce apoptosis, and accelerate the regeneration and repair of nerve fibers, with good neuroprotection and regeneration-promoting effects.

[0047] Example 6 (8) ACPP@EVs ABPC Biocompatibility testing: like Figure 8 As shown, ACPP@EVs ABPC The biocompatibility test results of the ACPP@EVs group were obtained by hematoxylin and eosin (H&E) staining, showing the tissue morphology of the heart, liver, spleen, lung, and kidney tissues in different groups 8 weeks after surgery. Heart: The myocardial tissue structure of each group remained intact, and no obvious pathological changes were observed. BMSC Group and ACPP@EVs ABPC There was no significant difference in the myocardial tissue morphology between the two groups, indicating that ACPP@EVs ABPC There was no obvious toxicity to heart tissue. Liver: The liver tissues of each group showed complete liver lobule structure, and no obvious necrosis or inflammatory response was observed. The spinal cord injury group showed slight signs of tissue damage, but ACPP@EVs ABPC Group and ACPP@EVs BMSC The liver tissue structure of the sham operation group and ACPP@EVs group was close to normal, with no significant lesions. ABPC The spleen tissue of the spinal cord injury group showed no abnormalities, while the spinal cord injury group showed slight inflammatory cell infiltration, but the ACPP@EVs treatment group showed some improvement. Lung: In the lung tissue, the spinal cord injury group showed some abnormal expansion of the bubble structure, accompanied by a certain degree of inflammatory response, while ACPP@EVs ABPC and ACPP@EVs BMSC The lung tissue morphology of the group was relatively normal, indicating that ACPP@EVs ABPC It has good biocompatibility with lung tissue. Kidney: The tubular and glomerular structures of kidney tissues in each group remained intact, with no obvious pathological changes. ACPP@EVs ABPC Group and ACPP@EVs BMSC The renal tissue of the group showed close to normal, and no toxic reaction was observed. In summary, the H&E staining results showed that ACPP@EVs ABPC It showed good biocompatibility to major organs (heart, liver, spleen, lung, and kidney) within 8 weeks after surgery and did not cause obvious tissue toxicity or pathological changes, further verifying its safety in vivo application.

[0048] In summary, the present invention designs and uses ACPP@EVsABPC , successfully achieved targeted treatment of spinal cord injury and nerve regeneration. ACPP@EVs ABPC By specifically binding to matrix metalloproteinase 2 (MMP2), ACPP@EVs precisely targets the site of spinal cord injury, promotes the proliferation of neural stem cells, and effectively reduces neuronal apoptosis. ABPC It showed significant immunomodulatory effects, which could promote the polarization of M1 macrophages to M2 macrophages and optimize the damaged microenvironment. At the same time, in vivo experiments proved that ACPP@EVs ABPC It can significantly improve the motor function recovery of rats with spinal cord injury and effectively promote nerve fiber regeneration in vivo. Biocompatibility tests show that ACPP@EVs ABPC No obvious toxic reactions were caused in major organs, and it has good biosafety. In summary, this invention proposes an innovative extracellular vesicle therapy strategy that combines targeted therapy and immune regulation functions, demonstrating that ACPP@EVs ABPC ACPP@EVs have great potential in promoting the repair of spinal cord injury. This method can not only be used to treat spinal cord injury, but also has broad application prospects in other neurodegenerative diseases. Future research will further explore the potential of ACPP@EVs ABPC The efficacy of the drug in other degenerative disease models will be further studied, and its mechanism of action and potential for clinical application will be further investigated.

Claims

1. A method for preparing engineered exosomes based on antler blastema progenitor cells, characterized in that: Follow the steps below to implement it: Step 1: Extraction of extracellular vesicles (EVs) from antler blastema progenitor cells ABPC , and purified by ultracentrifugation; Step 2: Synthesize the cell-penetrating peptide ACPP and combine it with the EVs obtained in step 1 ABPC Combined, engineered extracellular vesicles ACPP@EVsABPC modified with cell-penetrating peptides were prepared.

2. The method for preparing engineered exosomes based on antler blastema progenitor cells according to claim 1, characterized in that: Step 1 is as follows: The blastema progenitor cells were extracted from the antlers of two-year-old male sika deer and cultured in a solution containing Dulbecco's modified Eagle's medium. CX43 was screened by flow cytometry. + and FGFR2 + After subculturing and expansion, extracellular vesicles (EVs) were isolated from the culture medium using ultracentrifugation. ABPC .

3. The method for preparing engineered exosomes based on antler blastema progenitor cells according to claim 2, characterized in that: The ultracentrifugation step was performed at 16,000 g for 70 min at 4 °C, and the final extracellular vesicles were resuspended in pre-chilled PBS and stored at -80 °C.

4. The method for preparing engineered exosomes based on antler blastema progenitor cells according to claim 1, characterized in that: Step 2 is as follows: Step 2.1, synthesize ACPP peptide using solid phase synthesis technology according to the designed sequence; Step 2.2, prepare Tris·HCl buffer; Step 2.3, dissolving the ACPP peptide synthesized in step 2.1 in the Tris·HCl buffer prepared in step 2.2 to form an ACPP solution; preparing an ethanol solution of a chemical coupling agent DSPE, then adding the ACPP solution to the ethanol solution of DSPE, and mixing and coupling in a light-proof condition; Step 2.4: EVs prepared in step 1 ABPC Add EVs to the reaction mixture obtained in step 2.3 ABPC Incubate with ACPP peptide reaction solution to allow ACPP peptide to bind to EVs ABPC surface, followed by dialysis to obtain ACPP@EVsABPC.

5. The method for preparing engineered exosomes based on antler blastema progenitor cells according to claim 4, characterized in that: In step 2.1, the sequence of the ACPP peptide segment is: EEEEEEEE(E8)-6-aminohexanoyl-PLGLAG-RRRRRRRR(R8).

6. The method for preparing engineered exosomes based on antler blastema progenitor cells according to claim 4, characterized in that: In step 2.2, the pH of the prepared Tris·HCl buffer is 6.8-7.4, and the concentration is 10-50 mM.

7. The method for preparing engineered exosomes based on antler blastema progenitor cells according to claim 4, characterized in that: Step 2.3 is as follows: The ACPP peptide synthesized in step 2.1 is dissolved in the Tris·HCl buffer prepared in step 2.2, wherein the mass ratio of ACPP to Tris·HCl is 1:10-20, to form an ACPP solution; a DSPE ethanol solution with a concentration of 20-40 mg / mL is prepared; the ACPP solution is added to the DSPE ethanol solution, and Tris·HCl buffer is added to make the volume of the reaction system 4.5-5.5 mL, and then the reaction system is placed in a light-proof condition for thorough mixing and coupling.

8. The method for preparing engineered exosomes based on antler blastema progenitor cells according to claim 4, characterized in that: In step 2.4, EVs ABPC The concentration was 10¹¹ particles / mL. The dialysis was performed overnight under stirring and protected from light, and the dialysate was replaced every 8-12 hours.

9. An engineered exosome based on antler blastema progenitor cells, characterized in that: The compound is prepared by the preparation method described in any one of claims 1 to 8.

10. Use of the engineered exosomes according to any one of claims 1-8 in repairing spinal cord injury.

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