A nanovesicle and its preparation method and application

Abstract

This invention relates to a nanovesicle, its preparation method, and its application, belonging to the field of biomedical technology. The nanovesicles are surface-modified with RVG peptides and internally encapsulate a miRNA-130a-3p mimic. The nanovesicles are derived from human placental mesenchymal stem cells. The preparation method includes: expressing RVG peptides in human placental mesenchymal stem cells using genetic engineering; obtaining RVG peptide-modified nanovesicles using an extrusion method; and loading the miRNA-130a-3p mimic into the RVG peptide-modified nanovesicles to obtain the nanovesicle product. The surface-expressed RVG peptides can improve brain targeting, thereby increasing the content of miRNAs in hypoxic-injured nerve cells and reducing the level of target genes, thus reducing the apoptosis rate, increasing cell activity, and inhibiting the release of inflammatory factors. This inhibits brain cell apoptosis in cerebral ischemia-reperfusion injury, reduces brain damage area, and promotes the recovery of neurological function.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a nanocapsule and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Ischemic stroke is the most common type of stroke. Currently, the main clinical treatment for ischemic stroke patients is to restore blood flow to ischemic tissues. However, while blood flow restoration introduces oxygen and nutrients to tissues, it can also cause more serious damage, a phenomenon known as cerebral ischemic reperfusion (CIR) injury.

[0004] Under different physiological and pathological conditions, mesenchymal stem cells (MSCs) can maintain homeostasis through multidirectional differentiation. They secrete a large number of cytokines, such as brain-derived neurotrophic factor, to transmit chemical signals between cells. However, the problems that need to be solved when MSCs are used in large-scale clinical applications include: ① The number of cells required for treatment is large and the volume is large. Infusing a large number of MSCs at one time may cause adverse reactions such as pulmonary embolism; ② Since stem cells can self-renew and differentiate in the host, the risk of abnormal differentiation or even tumor formation cannot be ruled out; ③ The cost of obtaining and transporting stem cells is high, and the storage conditions of living cells are high. The homogeneity of cells in different batches is difficult to ensure, the efficiency is low, and the effect is unstable.

[0005] Exosomes are typical extracellular vesicles with a diameter between 30 and 150 nm. These exosomes play a key role in intercellular communication by carrying a variety of bioactive components from cells, including proteins, lipids, mRNA and miRNA. Exosomes exhibit neuroprotective effects, including promoting angiogenesis, anti-inflammatory effects, promoting axonal growth and anti-oxidative stress. These effects are mediated by miRNA and signaling pathways, indicating that exosomes have certain potential as a treatment for neurological diseases.

[0006] Although mesenchymal stem cell-derived extracellular vesicles have made some progress in the treatment of central nervous system diseases in recent years, the application of extracellular vesicles is limited due to low production, insufficient targeting and limited efficacy. Summary of the invention

[0007] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a nanovesicle and a preparation method and application thereof, and to produce nanovesicles similar to extracellular vesicles by extruding mesenchymal stem cells to increase product yield, and to improve the neural targeting and therapeutic effect of the nanovesicles by composite engineering modification of the nanovesicle surface with neurotargeting peptides and internal electrotransfer of miRNA-130a-3p mimics, so as to apply the nanovesicles to the treatment of cerebral ischemia-reperfusion injury.

[0008] In order to achieve the above object, the technical solution of the present invention is:

[0009] In a first aspect, a nanovesicle is provided, the surface of which is modified with RVG peptide and the interior of which is loaded with a miRNA-130a-3p mimic, wherein the nanovesicle is derived from human placental mesenchymal stem cells.

[0010] Optionally, the particle size of the nanovesicles is 120±20 nm.

[0011] The marker proteins calnexin and GM130 of the nanovesicles of the present invention are positive, while the marker proteins calnexin and GM130 of the extracellular vesicles derived from human placental mesenchymal stem cells are negative, and thus can serve as distinguishing features between the nanovesicles of the present invention and the extracellular vesicles.

[0012] In a second aspect, the method for preparing the above-mentioned nanovesicles comprises the following steps:

[0013] S1, subculture human placental mesenchymal stem cells;

[0014] S2, using genetic engineering to make human placental mesenchymal stem cells express RVG peptide, thereby obtaining human placental mesenchymal stem cells expressing RVG peptide;

[0015] S3, treating the human placental mesenchymal stem cells expressing RVG peptide obtained in S2 by using an extrusion method to obtain RVG peptide-modified nanovesicles;

[0016] S4. Loading the miRNA-130a-3p mimic into the RVG peptide-modified nanovesicles to obtain nanovesicle products.

[0017] Optionally, in S2, the genetic engineering comprises: inserting (1-1.5)×10 8 Mesenchymal stem cells were transfected with TU / mL lentivirus at a multiplicity of infection of 80-140, and then stable transfected strains were selected using puromycin at a concentration of 2-6 μg / mL.

[0018] Optional, in S3, the extrusion method includes: diluting the cells to 1×10 in DPBS buffer. 6 ~1×107 The diluted cell suspension was squeezed through 1-0.2 μm filter membranes in the order from coarse to fine, and each specification of filter membrane was squeezed multiple times.

[0019] Optionally, in S3, the squeezed liquid is centrifuged at 4±2°C, 100,000~120,000g for 70~90min, the supernatant is discarded, and the precipitate is resuspended with PBS buffer to obtain RVG peptide-modified nanovesicles.

[0020] Alternatively, in S4, miRNA-130a-3p mimics are loaded into RVG peptide-modified nanovesicles by electroporation.

[0021] Optionally, the electroporation technique includes: electroporating the RVG peptide-modified nanovesicles and the miRNA-130a-3p mimics at 10 7 ~10 12 After mixing at a ratio of 0.1-10 μg, add electroporation buffer; set the voltage to 200-600 V for electroporation.

[0022] Optionally, during electroporation, set the capacitance to 50~200 μF and the resistance to ∞ ohms.

[0023] Optionally, after electrotransfer, the cells were incubated at 37 ± 2°C for 10-60 min, followed by centrifugation at 100,000-140,000 g for 60-90 min at 4 ± 2°C.

[0024] In a third aspect, an application of the above-mentioned nanovesicle and / or the above-mentioned method for preparing the nanovesicle in at least one of the following a1)-a6):

[0025] a1) Reducing the apoptosis rate of hypoxic damaged nerve cells or preparing products that reduce the apoptosis rate of hypoxic damaged nerve cells;

[0026] a2) Improving the activity of oxidatively damaged nerve cells or preparing products that improve the activity of oxidatively damaged nerve cells;

[0027] a3) Inhibiting the release of inflammatory factors from nerve cells or preparing products that inhibit the release of inflammatory factors from nerve cells;

[0028] a4) Preparation of products for inhibiting brain cell apoptosis caused by cerebral ischemia-reperfusion injury;

[0029] a5) Preparation of products for reducing the area of ​​brain damage caused by cerebral ischemia-reperfusion injury;

[0030] a6) Preparation of products that promote recovery of neurological function after cerebral ischemia-reperfusion injury.

[0031] The beneficial effects of the present invention are:

[0032] 1. The present invention proposes a nanovesicle modified with RVG peptide and loaded with miRNA, and the nanovesicle is derived from human placental mesenchymal stem cells. The surface-expressed RVG peptide can improve brain targeting, thereby increasing the content of miRNA-130a-3p mimics loaded in hypoxic damaged nerve cells and reducing the level of target genes, which can reduce the apoptosis rate of hypoxic damaged nerve cells, increase the activity of oxidative damaged nerve cells, and inhibit the release of inflammatory factors in nerve cells. In this way, apoptosis of brain cells injured by cerebral ischemia-reperfusion injury is inhibited, the area of ​​brain injury is reduced, and the recovery of neurological function is promoted, which has broad application prospects.

[0033] 2. The present invention provides a method for preparing nanovesicles modified with RVG peptide and loaded with miRNA. The prepared nanovesicles are similar to extracellular vesicles, but the yield is about 20 times that of extracellular vesicles, and the content of miRNA loaded therein is about 9570 times that of the control group, overcoming the shortcomings of low yield, insufficient targeting and limited efficacy of extracellular vesicles. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0035] Figure 1 These are morphological diagrams of human placental mesenchymal stem cells in Example 1 of the present invention, wherein (A) shows the morphological diagram of human placental mesenchymal stem cells at the 3rd passage, and (B) shows the morphological diagram of human placental mesenchymal stem cells at the 6th passage.

[0036] Figure 2 1 is a graph showing the results of the identification of surface markers of human placental mesenchymal stem cells in Example 1 of the present invention. (A) is a graph showing the results of flow cytometry of human placental mesenchymal stem cells. (B) is a statistical graph showing the positive percentage of the identification results of each surface marker of human placental mesenchymal stem cells.

[0037] Figure 3 These are graphs showing the results of surface marker identification by flow cytometry in Example 1 of the present invention, (A) is a graph showing the results of CD29 identification, (B) is a graph showing the results of CD44 identification, (C) is a graph showing the results of CD73 identification, (D) is a graph showing the results of CD90 identification, (E) is a graph showing the results of CD105 identification, (F) is a graph showing the results of CD31 identification, (G) is a graph showing the results of CD34 identification, and (H) is a graph showing the results of CD45 identification.

[0038] Figure 4 is a fluorescence microscopy image of the infection at different infection multiplicities in Example 1 of the present invention,

[0039] Figure 5 It is a statistical diagram of infection efficiency at different multiplicities of infection in Example 1 of the present invention.

[0040] Figure 6 This is a graph showing the effects of different concentrations of puromycin on the activity of human placental mesenchymal stem cells in Example 1 of the present invention.

[0041] Figure 7 This is a diagram showing the expression of Lamp2B after viral transfection of human placental mesenchymal stem cells in Example 1 of the present invention.

[0042] Figure 8 It is a transmission electron micrograph of the extracellular vesicles and various nanovesicles in Example 1 of the present invention.

[0043] Fig. 9 This is a detection diagram of marker proteins of extracellular vesicles and various nanovesicles in Example 1 of the present invention.

[0044] Fig.10 1 and 2 are graphs showing the particle size detection results in Example 1 of the present invention, wherein (A) is a graph showing the particle size distribution of extracellular vesicles, (B) is a graph showing the particle size distribution of nanovesicles, (C) is a graph showing the particle size distribution of nanovesicles modified with RVG peptide, and (D) is a graph showing the particle size distribution of nanovesicles modified with RVG peptide and loaded with miRNA.

[0045] Fig.11 This is a surface result diagram of the RVG peptide expressed in Example 1 of the present invention on the nanovesicles modified with the RVG peptide and loaded with miRNA.

[0046] Fig.12 This is a graph showing the relative content of miRNA-130a-3p in the nanovesicles modified with RVG peptide and loaded with miRNA in Example 1 of the present invention.

[0047] Fig.13 This is a comparison chart of the yields of nanovesicles modified with RVG peptide and loaded with miRNA and extracellular vesicles in Example 1 of the present invention.

[0048] Fig.14 The figures are the results of the effects of the nanovesicles modified with RVG peptide and loaded with miRNA on the levels of miRNA-130a-3p and its target gene PTEN in hypoxic damaged PC-12 cells in Example 1 of the present invention. (A) shows the effects of the nanovesicles modified with RVG peptide and loaded with miRNA on the levels of miRNA-130a-3p in hypoxic damaged PC-12 cells. (B) shows the effects of the nanovesicles modified with RVG peptide and loaded with miRNA on the levels of PTEN (phosphatase and tensin homolog, tumor suppressor) in hypoxic damaged PC-12 cells.

[0049] Fig.15These are the flow cytometry results of cell apoptosis in Example 1 of the present invention, (A) is the NC result, (B) is the sugar-oxygen stripping result, (C) is the EVs (extracellular vesicles) result, (D) is the NVs (nanovesicles) result, (E) is the NVs-RVG (RVG peptide-modified nanovesicles) result, and (F) is the NVs-RVG-130a (RVG peptide-modified and miRNA-encapsulated nanovesicles) result.

[0050] Fig.16 It is a statistical diagram of the effect on the cell apoptosis ratio in flow cytometry in Example 1 of the present invention.

[0051] Fig.17 This is a graph showing the effect of the nanovesicles modified with RVG peptide and loaded with miRNA on the activity of oxidatively damaged PC-12 cells in Example 1 of the present invention.

[0052] Fig.18 The figures are the results of the effect of the RVG peptide-modified and miRNA-encapsulated nanovesicles on the concentration of inflammatory factors in Example 1 of the present invention, (A) is the result of the effect on the concentration of TNF-α, and (B) is the result of the effect on the concentration of IL-6.

[0053] Fig.19 This is an imaging picture of the nanovesicle modified with Dir-labeled RVG peptide and loaded with miRNA in brain tissue in Example 1 of the present invention.

[0054] Fig. 20 This is a statistical diagram of the total radiation efficiency of the nanovesicles modified with the Dir-labeled RVG peptide and loaded with miRNA in brain tissue in Example 1 of the present invention.

[0055] Fig.21 This is a diagram showing the results of fluorescent TUNEL staining of brain tissue in Example 1 of the present invention.

[0056] Fig. 22 This is a graph showing the apoptosis ratio of brain cells in Example 1 of the present invention.

[0057] Fig.23 This is a diagram of TTC staining results of brain tissue in Example 1 of the present invention.

[0058] Fig.24 This is a graph showing the ratio of brain injury area in Example 1 of the present invention.

[0059] Fig.25 This is a graph showing the effect of the nanovesicles modified with RVG peptide and loaded with miRNA on neurobehavior in Example 1 of the present invention.

[0060] Fig.26 This is a diagram showing the effect of the nanovesicles modified with RVG peptide and loaded with miRNA on tissues in Example 1 of the present invention. DETAILED DESCRIPTION

[0061] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0062] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0063] The experimental methods in the following examples without specifying specific conditions are generally based on conventional conditions. All raw materials and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0064] In the following examples, miRNA refers to miRNA-130a-3p mimics, which were purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd. The sense strand is shown in SEQ ID NO.3, and the antisense strand is shown in SEQ ID NO.4.

[0065] Example 1

[0066] A method for preparing nanovesicles comprises the following steps:

[0067] S1, subculture human placental mesenchymal stem cells;

[0068] S2. Using genetic engineering to make human placental mesenchymal stem cells express the RVG peptide with an amino acid sequence as shown in SEQ ID NO.2, thereby obtaining human placental mesenchymal stem cells expressing the RVG peptide;

[0069] S3, treating the human placental mesenchymal stem cells expressing RVG peptide obtained in S2 by using an extrusion method to obtain RVG peptide-modified nanovesicles;

[0070] S4. Loading the miRNA-130a-3p mimic into the RVG peptide-modified nanovesicles to obtain nanovesicle products.

[0071] Through the above steps, human placental mesenchymal stem cells can express RVG peptides with targeted neural function, and then a large number of nanovesicles with RVG peptides expressed on the surface and similar to extracellular vesicle structures are prepared by continuous extrusion, and miRNA is loaded inside, so as to construct nanovesicles modified with RVG peptides and loaded with miRNA. The yield of the prepared nanovesicles modified with RVG peptides and loaded with miRNA is much higher than the extracellular vesicles naturally secreted by human placental mesenchymal stem cells.

[0072] In S1, human placental mesenchymal stem cells are obtained from human placental tissue, and the obtaining method includes:

[0073] The placental tissue was removed from the biosafety cabinet and washed repeatedly with saline containing 2% cyanine chain double antibody to remove residual blood.

[0074] The chorionic villus tissue was separated from the placenta tissue, washed with physiological saline containing 2% blue chain double antibody, and then transferred to a 50 mL centrifuge tube containing enzyme digestion solution, ensuring that 25 mL of enzyme digestion solution was used for every 5 g of chorionic villus tissue.

[0075] The enzyme digestion solution containing the villus tissue was incubated on a shaker at 100-300 r / min and 37°C for 10-60 min. Subsequently, 5 mL of EliteGroTM-Adv serum replacement was added to terminate the digestion, and the liquid in the centrifuge tube was collected for the first time; the remaining tissue was left in the centrifuge tube and new enzyme digestion solution was added, and it was incubated on a shaker at 250 r / min and 37°C for 60 min again, and 5 mL of EliteGroTM-Adv serum replacement was added to terminate the digestion, and the liquid in the centrifuge tube was collected for the second time.

[0076] The liquids in the centrifuge tubes collected twice were mixed, passed through a 100 μm sieve, and centrifuged at 300g for 7 minutes in a centrifuge. The supernatant after centrifugation was discarded, and 5 mL of red blood cell lysis solution was added to the cell pellet, mixed evenly, and incubated for 2 minutes. Subsequently, the supernatant was discarded and the cell pellet was retained after centrifugation at 300g for 7 minutes.

[0077] Take 3 mL of complete culture medium for human placental mesenchymal stem cells, add it to the retained cell pellet to resuspend, mix evenly, then transfer to a T75 cell culture flask containing 15 mL of complete culture medium for human placental mesenchymal stem cells and culture in an incubator.

[0078] After 10 days, the cell adhesion growth status was observed under an inverted microscope, and half of the complete medium was replaced with fresh medium every 3 days. When the cell confluence reached 80%-90%, 0.25% recombinant trypsin solution was added to digest the cells and transferred to a new culture flask for culture. The culture results were as follows: Figure 1 As shown, Figure 1Middle (A) represents the third generation of human placental mesenchymal stem cells. Figure 1 Middle (B) represents the 6th generation human placental mesenchymal stem cells, which grow adherently and have a spindle-like and vortex-like morphology, meeting the morphological standards.

[0079] When the cell confluence of human placental mesenchymal stem cells reaches 80%-90%, the T25 culture flask is changed from 37°C and 5% CO to 2 Transfer the cells from the constant temperature incubator to the clean bench, discard the culture medium, add 3 mL of DPBS buffer to the culture bottle to wash the residual culture medium twice; then add 1.5 mL of 0.25% recombinant trypsin, and after 3 minutes, add 5 mL of complete culture medium to stop digestion; use a sterile Pasteur pipette to gently blow the bottom of the culture bottle, transfer the cell suspension to a 15 mL centrifuge tube, and centrifuge at 300 g for 7 minutes; discard the supernatant, then add 5 mL of fresh complete culture medium to the cell pellet, and then use a pipette to gently blow it into a single cell suspension, transfer the single cell suspension to a new culture bottle at a ratio of 1:3, shake it gently to disperse the cells evenly, and then place it in the incubator to continue culturing.

[0080] The cells can be expanded and cultured according to the method according to the experimental dosage, and the cells can be passaged into T75 or T175 culture flasks.

[0081] When the fourth generation human placental mesenchymal stem cells reached a confluence of 80% to 90%, 0.25% recombinant trypsin solution was added for digestion. After terminating the digestion, the digestion solution was centrifuged at 300g for 5 min, the supernatant was discarded, DPBS buffer was added to resuspend the cell pellet, and the cell pellet was centrifuged at 300g for 5 min again, and the supernatant was discarded; then DPBS buffer was added to resuspend the cell pellet to a cell density of 1×10 7 / mL; take 11 flow tubes and number them 1 to 11 in sequence, tubes 1 to 8 are single positive tubes, tube 9 is a negative control tube, tube 10 is a sample test tube, and tube 11 is an isotype control tube; add 100 μL of cell suspension to each tube 1 to 11, and then add 5 μL of FITC CD44 flow antibody to tube 1; add 5 μL of FITC CD45 flow antibody to tube 2; add 5 μL of FITC CD90 flow antibody to tube 3; add 5 μL of APC CD29 flow antibody to tube 4; add 5 μL of APCCD34 flow antibody to tube 5; add 5 μL of APC CD73 flow antibody to tube 6; add 5 μL of PE CD31 flow antibody to tube 7; add 5 μL of PE CD105 flow antibody to tube 8; no antibody is added to tube 9; add 5 μL of each of the above antibodies to tube 10 μL; add each isotype control antibody to tube No. 11; incubate the flow tube in the dark for 30 min, then add 300 μL of DPBS buffer to each tube, centrifuge at 300g for 5 min, discard the supernatant, add DPBS buffer to resuspend the cells, centrifuge at 300g for 5 min again, discard the supernatant, add 500 μL of DPBS buffer to resuspend the cells; finally filter with a 75 μm mesh and detect with a flow cytometer. At least 10,000 samples are collected for each sample, and the results are analyzed using FlowJo software; the results are shown in Figure 2 and Figure 3 As shown: Figure 2 (A) shows the flow cytometry results of selecting placental mesenchymal stem cells. Figure 3 Middle (A) indicates: the percentage of cells expressing CD29 is 98.4%; Figure 3 Middle (B) indicates: the percentage of cells expressing CD44 is 97.8%; Figure 3 Middle (C) indicates: the percentage of cells expressing CD73 is 99.4%; Figure 3 Middle (D) indicates: the percentage of cells expressing CD90 is 96.2%; Figure 3 Middle (E) indicates: the percentage of cells expressing CD105 is 98.8%; Figure 3 Middle (F) indicates: the percentage of cells expressing CD31 is 0.13%; Figure 3 Middle (G) indicates: the percentage of cells expressing CD34 is 0.29%; Figure 3 Middle (H) indicates that the percentage of cells expressing CD45 is 0.14%. Figure 2 The middle (B) indicates the positive percentage of each marker. Human placental mesenchymal stem cells highly express markers such as CD29, CD44, CD73, CD90 and CD105, with expression rates of more than 95%; at the same time, they lowly express CD31, CD34 and CD45, with expression rates of less than 2%. The identification results meet the standards.

[0082] In S2, genetic engineering included: using 1×10 8 Mesenchymal stem cells were transfected with TU / mL lentivirus at a multiplicity of infection of 80-140, and then stable transfected strains were selected using puromycin at a concentration of (2-6) μg / mL.

[0083] Virus-related experiments are carried out in a biosafety cabinet. The specific steps include:

[0084] (1) Lentiviral transfection.

[0085] The method for preparing a lentivirus inserted with a Lamp2B-linked RVG peptide expression gene comprises: obtaining a gene fragment inserted with a target gene (i.e., a Lamp2B-linked RVG peptide expression gene) by PCR technology, and inserting the target gene fragment into a multiple cloning site of a lentivirus expression vector by an enzyme cutting and ligation method.

[0086] The target gene is shown in SEQ ID NO.1.

[0087] The 4th generation human placental mesenchymal stem cells were digested and diluted to 8×10 4 The cell suspension was added to a 96-well plate, 100 μL per well, and placed in a 37°C, 5% CO 2 In the incubator, virus infection was performed when the cell confluence rate reached 30% to 50% on the second day;

[0088] The 1 / 2 volume infection method was used. Before infection, the prepared lentivirus with the RVG peptide expression gene inserted was taken out from the refrigerator and gradually melted in an ice bath. The culture medium was aspirated and 1 / 2 volume of fresh culture medium was added. For the 96-well plate, 50 μL of culture medium was added to each well. The infection multiplicity was set to 80, 100, 120 and 140, and the virus stock solution was added to the cells and gently mixed. After about 24 hours, the virus-containing culture medium was aspirated and replaced with fresh culture medium, and the culture was continued. After 72 hours of infection, the RVG peptide expression efficiency was preliminarily observed under an inverted fluorescence microscope. The results are shown in the figure. Figure 4 and Figure 5 As shown, Figure 4 Indicates the fluorescence expression after infection with different infection multiplicity. Figure 5 Represents the infection efficiency at different multiplicity of infection.

[0089] (2) Construction of stable lentiviral strains.

[0090] Since the lentivirus used has a puromycin resistance gene and a GFP dual screening gene, puromycin can be used to screen stable transfectants. When the cell plating confluence reaches 50%-60%, puromycin with a set concentration gradient is added. The concentration gradient is set to 0, 0.5, 1, 2, 4 and 6 μg / mL. After 48 hours of treatment, the lowest concentration that can kill more than 90% of the control cells is selected. The results are shown in the figure. Figure 6 As shown in the figure, the lowest concentration that could kill more than 90% of the control cells was 2 μg / mL. Therefore, the infected cells were treated with 2 μg / mL of puromycin to select stable transfectants.

[0091] (3) Detection of RVG peptide expression.

[0092] Western blotting was used to analyze the expression of RVG peptide (linked to Lamp2B). Human placental mesenchymal stem cells and human placental mesenchymal stem cells expressing RVG peptide were lysed using RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors. 200 μL of RIPA lysis buffer was added to each well of a 6-well plate, incubated on ice for 30 min, and then the cells were scraped off with a cell scraper. For the extracellular vesicle (derived from human placental mesenchymal stem cells) samples, nanovesicle samples, RVG peptide-modified nanovesicle samples, and RVG peptide-modified and miRNA-loaded nanovesicle sample suspensions, they were mixed with RIPA lysis buffer at a volume ratio of 1:1 and lysed on ice for 10 min; the lysate was then centrifuged at 12,000 g for 15 min, and the supernatant was the total protein solution; 5 volumes of loading buffer were added to the total protein solution, vortexed, heated at 100°C for 10 min using a metal bath (referring to a hot bath in a metal bath pot), and then centrifuged instantaneously to obtain the supernatant; after separation by 10 vt.% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the proteins in the gel were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA), sealed with 5% (w / v) skim milk powder, and reacted at room temperature for 1 minute. h, incubated with rabbit anti-human Lamp2B monoclonal antibody (diluted with antibody diluent at a volume ratio of 1:1000) and rabbit anti-human GAPDH monoclonal antibody (diluted with antibody diluent at a volume ratio of 1:1000) at 4°C overnight; washed with Tris-Buffered Saline with Tween-20 (TBST) (5 min × 3 times), then incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG secondary antibody at room temperature for 2 hours, and washed with TBST (5 min × 5 times); finally, developed with an ultrasensitive chemiluminescent solution (ECL) kit; the results are shown in Figure 2. Figure 7As shown, the expression level of Lamp2B in human placental mesenchymal stem cells transduced with lentivirus RVG-Lamp2B plasmid was significantly increased, and the expression level of Lamp2B in nanovesicles prepared by extrusion thereof was significantly increased.

[0093] In S3, the extrusion method includes: extruding the diluted cell suspension through the filter membrane in order from coarse to fine; centrifuging the extruded liquid at 100,000-120,000 g for 70-90 min at 4±2°C, discarding the supernatant, and resuspending the precipitate with PBS buffer to obtain RVG peptide-modified nanovesicles.

[0094] S4. Loading the miRNA-130a-3p mimic into the RVG peptide-modified nanovesicles to obtain nanovesicle products.

[0095] S3 and S4 specifically include the steps of: taking cultured human placental mesenchymal stem cells expressing RVG peptide from generation P4 to P10 to obtain surface RVG peptide-modified nanovesicles; culturing human placental mesenchymal stem cells expressing RVG peptide for 48 h, digesting and collecting the cells, and diluting with DPBS buffer to 5×10 6 The cells were squeezed through 1 μm, 0.4 μm and 0.2 μm filter membranes in sequence using an extruder, and each filter membrane was squeezed 11 times. The cells were centrifuged at 120,000 g for 80 min at 4°C, the supernatant was discarded, and the precipitate was resuspended in a set amount of DPBS buffer. The resuspended liquid and miRNA-130a-3p mimics were mixed at 10 9 The mixture was mixed at a ratio of 1:1 μg, and an appropriate amount of electroporation buffer was added. Then, it was transferred to a 4 mm thick electroporation cup, and the electroporation cup was placed in an electroporator, and the voltage was set to 400 V, the capacitance to 125 μF, and the resistance to ∞ ohms. After the electroporation was completed, it was transferred to a new centrifuge tube and incubated at 37°C for 30 min. Subsequently, the free miRNA-130a-3p mimics were removed by centrifugation at 140000g in an Optima XPN-100 ultracentrifuge for 70 min, and the precipitate was resuspended with an appropriate amount of DPBS buffer to obtain a certain concentration of RVG peptide-modified and miRNA-encapsulated nanovesicles, which were nanovesicle products.

[0096] (4) Identification of nanovesicle products.

[0097] 1) The negative staining method was used to observe the morphology of the nanovesicles modified with RVG peptide and loaded with miRNA by transmission electron microscopy, including the following steps: using a pipette to draw about 4.5 μL of the suspension of the nanovesicles modified with RVG peptide and loaded with miRNA, and dropping it onto the carbon film-covered side of a 300-mesh copper mesh, placing the copper mesh on a flat table and letting it stand for 3 min, so that the nanovesicles modified with RVG peptide and loaded with miRNA were precipitated and adsorbed onto the copper mesh; using medium-speed filter paper to absorb the suspension from the edge of the copper mesh, so that the copper mesh was in a semi-dry state; dropping a drop of uranyl acetate stain onto the copper mesh, quickly absorbing it from the edge of the copper mesh with filter paper, dropping another drop of uranyl acetate stain, quickly absorbing it, dropping another drop of uranyl acetate stain, standing for 30 s and then absorbing it, and leaving it at room temperature for 12 h to dry naturally; using a Talos transmission electron microscope to obtain the morphological image of the nanovesicles modified with RVG peptide and loaded with miRNA, the voltage value was 80 kV, and the exposure time was 100 ms; the results are as follows Figure 8 As shown, the morphology of the RVG peptide-modified and miRNA-loaded nanovesicles is similar to that of extracellular vesicles, which are hemispherical with a concave side and a diameter of less than 200 nm.

[0098] 3) Western blotting was used to analyze the marker proteins of RVG peptide-modified and miRNA-loaded nanovesicles, including the following steps: adding 5 volumes of loading buffer to the isolated and purified RVG peptide-modified and miRNA-loaded nanovesicles, boiling at 100°C for 5 min, and cooling to room temperature; separating by 10 vt.% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferring the proteins in the gel to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA), sealing with 5% (w / v) skim milk powder, and reacting at room temperature for 1 hr. h, incubated with rabbit anti-human CD63 monoclonal antibody (diluted with antibody diluent at a volume ratio of 1:5000), rabbit anti-human CD81 monoclonal antibody (diluted with antibody diluent at a volume ratio of 1:100), rabbit anti-human TSG101 monoclonal antibody (diluted with antibody diluent at a volume ratio of 1:200), rabbit anti-human Calnexin monoclonal antibody (diluted with antibody diluent at a volume ratio of 1:2000), and rabbit anti-human GM130 monoclonal antibody (diluted with antibody diluent at a volume ratio of 1:2000) at 4°C overnight; washed with Tris-Buffered Saline with Tween-20 (TBST) (5 min × 3 times), and then incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG secondary antibody at room temperature for 2 hours, and washed with TBST (5 min × 5 times); finally, developed using an ultrasensitive chemiluminescent solution (ECL) kit; the results are shown in Figure 2. Fig. 9As shown, similar to extracellular vesicles, nanovesicles modified with RVG peptide and loaded with miRNA highly expressed TSG101, CD63 and CD81. The difference was that the marker proteins calnexin and GM130 were positive in nanovesicles modified with RVG peptide and loaded with miRNA but negative in extracellular vesicles, thereby being able to distinguish the nanovesicle products of the present invention from extracellular vesicles.

[0099] 4) Determination of the particle size of RVG peptide-modified and miRNA-loaded nanovesicles by nanoparticle tracking analyzer: The RVG peptide-modified and miRNA-loaded nanovesicles were diluted to 1×10 7 The particle size was measured using a nanoparticle tracking analyzer. Fig.10 As shown: Fig.10 Middle (A) shows the size distribution of extracellular vesicles; Fig.10 Middle (B) shows the particle size distribution of nanovesicles; Fig.10 Middle (C) shows the particle size distribution of RVG peptide-modified nanovesicles; Fig.10 Middle (D) shows the particle size distribution of RVG peptide-modified and miRNA-encapsulated nanovesicles. It can be seen that the particle size of RVG peptide-modified and miRNA-encapsulated nanovesicles is similar to that of extracellular vesicles, which is distributed around 120 nm.

[0100] 5) Identification of the position of RVG peptide in nanovesicles modified with RVG peptide and loaded with miRNA: Since there is no suitable antibody for RVG peptide, the inventors designed to replace RVG peptide with Flag peptide bound to an antibody, and prepared nanovesicles expressing Flag peptide and loading miRNA according to the same method as in this example; 50 μL of anti-Flag magnetic agarose and 450 μL of PBS buffer were measured and added to a 1.5 mL centrifuge tube in sequence, and gently vortexed to mix; the centrifuge tube was placed in a magnetic stand, the magnetic agarose was collected on the side of the centrifuge tube, and the supernatant was discarded; the magnetic agarose was washed, 500 μL of PBS buffer was measured and added to the centrifuge tube, and gently vortexed to mix, the centrifuge tube was placed in a magnetic stand, the magnetic agarose was collected on the side of the centrifuge tube, and the supernatant was discarded; the washing was repeated once, for a total of two washes; 300 μL of the nanovesicle suspension expressing Flag peptide and loading miRNA was measured and added to the centrifuge tube containing magnetic agarose, gently vortexed to mix, and incubated at room temperature for 20 min. The centrifuge tube was placed in a magnetic stand, the magnetic agarose was collected on the side of the centrifuge tube, and the supernatant was discarded; 50 μL of PBS buffer was measured and added to the centrifuge tube, and the mixture was gently vortexed to quantify the protein concentration. 5× loading buffer was added, vortexed and mixed, and heated at 100°C for 10 min using a metal bath; the centrifuge tube was placed in a magnetic stand, the magnetic agarose was collected on the side of the centrifuge tube, and the supernatant was aspirated to detect the level of Lamp2B linked to Flag; the control group was a nanovesicle that did not express the Flag peptide and only loaded with miRNA-130a-3p mimics, and the treatment method was the same; the results are shown in Fig.11 As shown, after the nanovesicles that did not express Flag peptide and loaded with miRNA-130a-3p mimics were incubated with anti-Flag magnetic agarose, no nanovesicles that loaded with miRNA-130a-3p mimics were detected, indicating that the nanovesicles themselves did not bind to the anti-Flag magnetic agarose, while the anti-Flag magnetic agarose successfully bound to the Flag on the surface of the nanovesicles modified with Flag peptide and loaded with miRNA-130a-3p mimics and enriched the nanovesicles modified with Flag peptide and loaded with miRNA-130a-3p mimics, indicating that the Flag peptide was located on the surface of the nanovesicles, indirectly proving that the RVG peptide in the nanovesicles modified with RVG peptide and loaded with miRNA-130a-3p mimics constructed by the same method was also located on the surface.

[0101] 6) Detection of miRNA-130a-3p content: Use QIAGEN's miRNeasy Mini kit to extract miRNA; the concentration and purity of the extracted total RNA are measured by spectrophotometer; the content is detected by Ruibo Bio's miRNA RT-qPCR kit; the results are as follows Fig.12As shown, the content of miRNA-130a-3p in the nanovesicles modified with RVG peptide and loaded with miRNA is about 9570 times that in the nanovesicles modified with RVG peptide.

[0102] (5) Comparison with extracellular vesicles.

[0103] 1) Comparison of yield: Extracellular vesicle suspension was prepared by differential centrifugation; the cell supernatant was filtered through a 0.22 μm filter membrane. The cell supernatant was concentrated using a tangential flow ultrafiltration system (100 kDa polyethersulfone membrane package); the concentrated cell supernatant was centrifuged at 300g for 10 min at 4°C, the supernatant was collected, and the precipitate was discarded; the supernatant was centrifuged at 2000g for 10 min at 4°C, the supernatant was collected, and the precipitate was discarded. The supernatant was centrifuged at 12000g for 30 min at 4°C, the supernatant was collected, and the precipitate was discarded. The supernatant was centrifuged at 120,000 g for 70 min at 4°C, the supernatant was discarded, and the precipitate was resuspended with DPBS buffer; after resuspension, the precipitate was transferred to a new ultracentrifuge tube, centrifuged at 120,000 g for 70 min at 4°C, the supernatant was discarded, and the precipitate was resuspended with a set volume of DPBS buffer to obtain extracellular vesicles of a set concentration; the nanovesicles and extracellular vesicles modified with RVG peptide and loaded with miRNA obtained in this example were used to detect the particle concentration using nanoparticle tracking analysis, and the yield was calculated according to the following formula:

[0104] Yield (particles / cell) = particle concentration (particles / mL) ÷ cell number (cell / mL).

[0105] The results are as follows Fig.13 As shown, the yield of nanovesicles modified with RVG peptide and loaded with miRNA was 20 times that of extracellular vesicles.

[0106] 2) Comparison of particle-protein ratio: The exosome protein lysis buffer was melted and mixed at 4°C and then placed on ice. The RVG peptide-modified and miRNA-loaded nanovesicle suspension and the extracellular vesicle suspension were mixed with the lysis buffer at a volume ratio of 1:1 and then lysed on ice for 10 min. The supernatant was centrifuged at 12,000 g for 5 min at 4°C and added to a 96-well plate. The plate was filled to 20 μL with PBS buffer solution. BCA working solution was added, 200 μL per well, and incubated at 37°C for 20 min. After detection by an enzyme-labeled instrument, the protein concentration of the sample was calculated according to the standard curve. The particle concentration was detected using nanoparticle tracking analysis. The particle-protein ratio was calculated according to the following formula:

[0107] Particle-protein ratio = particle concentration (particles / mL) ÷ protein concentration (μg / mL).

[0108] To ensure the accuracy of the results, three groups of extracellular vesicles prepared by the same method were used for testing. The average particle-protein ratio of the three groups of RVG peptide-modified and miRNA-loaded nanovesicles was calculated to be 4.81×10 9 / μg, in line with the standards of the "Specifications for the Preparation and Inspection of Exosomes from Human Mesenchymal Stem Cells" issued by the China Food and Drug Enterprise Quality and Safety Promotion Association in 2024 (no less than 1×10 8 / μg).

[0109] The application of the nanovesicles in this embodiment and / or the method for preparing the nanovesicles includes cell level experiments and animal experiments.

[0110] (1) Experiments at the cellular level.

[0111] In the experiment at the cellular level, the method for establishing the hypoxic injury model includes: culturing PC-12 cells derived from the cell line of pheochromocytoma of the adrenal medulla of adult rats, digesting and collecting the cells when the confluence reaches 80%-90%, and resuspending them to 1.5×10 in RPMI 1640 medium containing 10% FBS and 1% blue chain double antibody. 5 2 mL of cell suspension was added to each well of a 6-well plate and placed at 37°C and 5% CO 2 Culture in an incubator for 12 h, replace with RPMI 1640 medium (without glucose), and place in a 37°C, 95% N 2 , 5%CO 2 The cells were cultured in an incubator for 12 h to obtain the hypoxic injury model.

[0112] 1) Detection of miRNA-130a-3p content in cells: The hypoxic injury model was cultured in different culture media. The RVG peptide-modified nanovesicle group contained RVG peptide-modified nanovesicles (1×10 9 The RVG peptide-modified and miRNA-loaded nanovesicle group contained nanovesicles (1×10 9 The cells were cultured in RPMI 1640 medium at 37°C and 5% CO 2 The cells were cultured in a cell culture incubator. After 24 h, miRNA was extracted using QIAGEN's miRNeasy Mini kit. The extracted total miRNA was detected using Ruibo Bio's miRNA RT-qPCR kit to detect the level of miRNA-130a-3p, with U6 as the internal reference. The results are shown in Fig.14 As shown in (A), RVG peptide-modified and miRNA-loaded nanovesicles significantly increased the level of miRNA-130a-3p in hypoxic PC-12 cells.

[0113] 2) Detection of PTEN content in cells: After the hypoxic injury model was established, different culture media were replaced according to the groups: the glucose-oxygen deprivation group was RPMI 1640 culture medium; the RVG peptide-modified nanovesicle group was nanovesicles containing RVG peptide-modified nanovesicles (1×10 9 The RVG peptide-modified and miRNA-encapsulated nanovesicle group contained nanovesicles (1×10 9 The cells were cultured in RPMI 1640 medium at 37°C and 5% CO 2 The cells were cultured in a cell culture incubator. Total RNA was extracted using an omega RNA extraction kit 24 hours later. The PTEN level was detected using a Takara RT-qPCR kit using the extracted total RNA. The results are shown in Fig.14 As shown in (B), RVG peptide-modified and miRNA-loaded nanovesicles significantly reduced the level of PTEN in hypoxic-injured PC-12 cells.

[0114] 3) Effects of RVG peptide-modified and miRNA-loaded nanovesicles on apoptosis of hypoxic damaged cells: except for the negative control group, which was cultured under normal conditions, the other groups established hypoxic damage models; different culture media were replaced according to the groups: the negative control group was cultured with RPMI1640 medium; the glucose-oxygen deprivation group was cultured with RPMI 1640 medium; the extracellular vesicle group was cultured with extracellular vesicles (1×10 9 The nanovesicle group was cultured in RPMI 1640 medium containing nanovesicles (1×10 9 The RVG peptide-modified nanovesicle group contained 1×10 9 1×10 10 / mL) of RPMI 1640 medium; the RVG peptide-modified and miRNA-encapsulated nanovesicle group contained RVG peptide-modified and miRNA-encapsulated nanovesicles (1×10 9 The cells were cultured in RPMI 1640 medium at 37°C and 5% (v / v) CO 2 The cells were cultured in a cell culture incubator; after 24 h, the cells were washed with 4°C precooled PBS buffer and digested with trypsin (EDTA-free, phenol red-free) to collect the cells; the cells were washed twice with 4°C precooled PBS buffer and the PBS buffer was discarded; 1 volume of Annexin V binding solution was added to make a final concentration of 1×10 6 / mL cell suspension; Take 100 μL of cell suspension and add it to a new tube, add 5 μL of Annexin V-633 conjugate to the cell suspension, then add 5 μL of PI dye solution, and incubate at room temperature in the dark for 15 min. Add 400 μL of 1-fold volume of Annexin V Binding Solution, pass through a 75 μm mesh, and use a flow cytometer to detect within 1 hour; the apoptosis ratio is calculated according to the following formula:

[0115] Apoptosis ratio (%) = Q2 quadrant ratio (%) + Q3 quadrant ratio (%).

[0116] The results are as follows Fig.15 As shown: PI is propidium iodide, which can stain late-stage apoptotic cells. Annexin-APC is a phospholipid-binding protein labeled with allophycocyanin dye, which can stain early-stage apoptotic cells. Fig.15 Middle (A) is the NC result graph in flow cytometry, showing that the total proportion of early apoptotic and late apoptotic cells is 11.81%; Fig.15 (B) The result of glucose-oxygen stripping in flow cytometry shows that the total proportion of early apoptotic and late apoptotic cells is 20.55%; Fig.15 Middle (C) is the result of flow cytometry EVs, showing that the total proportion of early apoptotic and late apoptotic cells is 12.99%; Fig.15 Middle (D) is the flow cytometry NVs result of cell apoptosis, showing that the total proportion of early apoptotic and late apoptotic cells is 10.35%; Fig.15 Middle (E) is the result of flow cytometry NVs-RVG, showing that the total proportion of early apoptotic and late apoptotic cells is 13.77%; Fig.15 Middle (F) Flow cytometry results of NVs-RVG-130a, showing that the total proportion of early apoptotic and late apoptotic cells was 10.72%. Fig.16 The results show the effects of extracellular vesicles and various nanovesicles on the apoptosis rate of PC-12 cells damaged by hypoxia; it shows that nanovesicles modified with RVG peptide and loaded with miRNA can significantly inhibit cell apoptosis after cell hypoxia damage.

[0117] 4) Effects of RVG peptide-modified and miRNA-loaded nanovesicles on the activity of oxidatively damaged cells: PC-12 cells were cultured at 1×10 5 The cells were inoculated at a density of 100 μL / mL in a 96-well plate and placed in a 37°C, 5% (v / v) CO 2 The cells were cultured in a cell culture incubator; after 12 h, different culture media were replaced according to the groups: the control group was cultured with H 2 O 2 (0.5 mol / L) RPMI 1640 medium; the extracellular vesicle group was H2 O 2 (0.5 mol / L) and extracellular vesicles (1×10 9 The nanovesicle group was cultured in RPMI 1640 medium containing H 2 O 2 (0.5 mol / L) and nanovesicles (1×10 9 RVG peptide-modified nanovesicles were H 2 O 2 (0.5 mol / L) and RVG peptide-modified nanovesicles (1×10 9 RPMI1640 medium (cells / mL); the RVG peptide-modified and miRNA-encapsulated nanovesicle group contained H 2 O 2 (0.5 mol / L) and RVG peptide-modified and miRNA-loaded nanovesicles (1×10 9 The cells were cultured in RPMI 1640 medium at 37°C and 5% (v / v) CO 2 After 24 h, the cells were cultured in a cell culture incubator; the RPMI 1640 medium containing 10 vt.% CCK-8 reagent was replaced at 37°C and 5% (v / v) CO 2 Culture in a cell culture incubator; after 3 h, measure the absorbance at 450 nm using an ELISA reader; calculate cell activity according to the following formula:

[0118] Cell activity (%) = (OD experimental group-OD blank group) / (OD control group-OD blank group) × 100%.

[0119] like Fig.17 As shown, RVG peptide-modified and miRNA-loaded nanovesicles can inhibit oxidative damage in PC-12 cells.

[0120] 5) Effects of RVG peptide-modified and miRNA-loaded nanovesicles on inflammatory factors: BV2 cells were cultured at 3×10 5 The cells were inoculated into 6-well plates at a density of 100 / mL, with 2 mL per well, and incubated at 37°C and 5% CO 2 The cells were cultured in a cell culture incubator; after 24 h, different culture media were replaced according to the groups: LPS was used for inflammatory injury first, and the negative control group was DMEM culture medium; the control group was DMEM culture medium containing LPS (1 μg / mL); the extracellular vesicle group was DMEM culture medium containing LPS (1 μg / mL) and extracellular vesicles (1×10 9 The nanovesicle group was cultured in DMEM medium containing LPS (1 μg / mL) and nanovesicles (1×10 9The RVG peptide-modified nanovesicle group contained LPS (1 μg / mL) and RVG peptide-modified nanovesicles (1×10 9 The RVG peptide-modified and miRNA-loaded nanovesicle group contained LPS (1 μg / mL) and RVG peptide-modified and miRNA-loaded nanovesicles (1×10 9 The cells were cultured in DMEM medium at 37°C and 5% CO 2 The cells were cultured in a cell culture incubator; after 24 h, the cell culture medium was collected and centrifuged at 2-8°C and 1000×g for 20 min to remove impurities and cell debris; the supernatant was taken and the content of inflammatory factors TNF-α and IL-6 was detected using an enzyme linked immunosorbent assay (ELISA) kit.

[0121] The results are as follows Fig.18 As shown, Fig.18 (A) shows the effect of RVG peptide-modified and miRNA-loaded nanovesicles on TNF-α concentration. Fig.18 Middle (B) shows the effect of RVG peptide-modified and miRNA-loaded nanovesicles on IL-6 concentration, indicating that RVG peptide-modified and miRNA-loaded nanovesicles can inhibit the expression of inflammatory factors and have a good anti-inflammatory effect.

[0122] (2) Animal experiments

[0123] In animal experiments, the method for establishing a rat model includes: raising SD rats in an SPF-grade mouse house for one week to adapt them to the environment, using Zotai 50 anesthetic (50 mg / kg) in a clean bench for intramuscular anesthesia; after anesthesia, the rats were fixed on the operating table in a supine position, and a warming blanket was used to maintain body temperature; the neck hair was removed with an electric clipper, the skin was incised along the midline of the neck (the incision length was about 2 cm), the glandular tissue and fascia were bluntly separated, the common carotid artery was exposed, the vagus nerve was separated, and a 5-0 suture was passed under the common carotid artery and a slipknot was tied; the tissue around the common carotid artery was separated along the common carotid artery, the internal carotid artery, the external carotid artery and the bifurcation were exposed and separated, and a 5-0 suture was passed under the external carotid artery, with a dead knot tied at the distal end; a slipknot was tied at the proximal end, and a slipknot was tied at the internal carotid artery for later use. Use ophthalmic surgical scissors to make an incision between the knot point and the slipknot of the external carotid artery, quickly insert the marked thread plug into the incision, and fix the thread plug with the slipknot; gently pull the distal node with the left hand to make the blood vessels of the external carotid artery and the internal carotid artery nearly straight, pull the slipknot of the fixed thread plug with the right hand, fold it into the internal carotid artery, untie the slipknot of the internal carotid artery, and slowly push the thread plug into the internal carotid artery; the bifurcation point of the internal carotid artery and the external carotid artery is 1-2 mm away from the thread plug mark, tighten the slipknot of the external carotid artery to fix the thread plug, and open the slipknot of the common carotid artery. After 2 hours, reperfusion was performed and the thread plug was gently withdrawn; when the obvious resistance disappeared, it indicated that the head end had been withdrawn from the internal carotid artery and entered the stump of the external carotid artery, cut the thread plug outside the blood vessel, and sutured the skin. A rat model of cerebral ischemia-reperfusion injury was obtained.

[0124] 1) Targeting of RVG peptide-modified and miRNA-loaded nanovesicles.

[0125] 15 min after reperfusion in rat models, drugs labeled with Dir dye were injected through the tail vein (the dye working solution with a concentration of 100 μmol / L was prepared by diluting 10 times with PBS buffer solution; the drug suspension: dye working solution = 10:1 (v:v) was mixed and incubated at 37°C for 30 min; 10 mL of PBS buffer was added to the mixed liquid after incubation and mixed evenly, and the excess dye solution was removed after centrifugation at 100000×g for 1.5 h, and the bottom precipitate was resuspended with PBS buffer). The groups were as follows: extracellular vesicle group: 1.5 mL of extracellular vesicles (1×10 10 Nanovesicle group: injected with 1.5 mL of nanovesicles (1×10 10 RVG peptide-modified nanovesicle group: 1.5 mL (1×10 10 RVG peptide-modified and miRNA-encapsulated nanovesicle group: 1.5 mL (1×10 10After the injection of the drug, the animals were put back into the cage after they woke up.

[0126] The brain tissue was dissected after 24 h, and images were collected using a small animal in vivo imaging system (excitation wavelength 750 nm; emission wavelength 780 nm).

[0127] The results are as follows Fig.19 and Fig. 20 As shown, Fig.19 An image showing the Dir-labeled RVG peptide-modified and miRNA-loaded nanovesicles in brain tissue. Fig. 20 It represents the total radiation efficiency of Dir-labeled RVG peptide-modified and miRNA-loaded nanovesicles in brain tissue, indicating that the RVG-modified nanovesicles have certain brain targeting properties.

[0128] 2) Effects of RVG peptide-modified and miRNA-loaded nanovesicles on brain cell apoptosis.

[0129] The rats were injected with drugs through the tail vein 15 min after reperfusion. The rats were divided into the following groups: negative control group: injected with 1.5 mL of normal saline; model group (middle cerebral artery occlusion model): injected with 1.5 mL of normal saline; extracellular vesicle group: injected with 1.5 mL of extracellular vesicles (1×10 10 Nanovesicle group: injected with 1.5 mL of nanovesicles (1×10 10 RVG peptide-modified nanovesicle group: 1.5 mL (1×10 10 RVG peptide-modified and miRNA-encapsulated nanovesicle group: 1.5 mL (1×10 10 / mL), and the animals were returned to the cage after the injection of the drug was completed and awakened. Thus, the rat models of negative control group, model group, extracellular vesicle group, nanovesicle group, RVG peptide-modified nanovesicle group, and RVG peptide-modified and miRNA-encapsulated nanovesicle group were obtained.

[0130] After 24 hours, tissue samples were collected, fixed, embedded, and paraffin sliced. The slices were placed in the following order: xylene I, 10 min; xylene II, 10 min; xylene III, 10 min; anhydrous ethanol I, 5 min; anhydrous ethanol II, 5 min; anhydrous ethanol III, 5 min; and washed with distilled water. (The dewaxing time should be appropriately extended in winter). After the slices were slightly dried, a circle was drawn around the tissue with a tissue pen (to prevent the liquid from flowing away), and proteinase K working solution was dripped into the circle to cover the tissue, and incubated in a 37°C incubator for 22 min. The slides were placed in PBS buffer and washed on a decolorizing shaker for 3 times, each time for 5 min. (Proteinase K working solution preparation method, original solution: PBS buffer = 1:9). After the slices were slightly dried, a membrane breaking working solution was dripped into the circle to cover the tissue, and incubated at room temperature for 20 min. The slides were placed in PBS buffer and washed on a decolorizing shaker for 3 times, each time for 5 min. (The membrane permeabilization solution is 0.1% triton solvent, triron stock solution: PBS buffer = 1:1000v / v). After the slices are slightly dried, the buffer is added to the circle to cover the tissue, and the buffer is incubated at room temperature for 10 min. According to the number of slices and tissue size, take the appropriate amount of TDT enzyme, deoxyuridine triphosphate reagent (dUTP), and buffer in the TUNEL kit in a ratio of 1:5:50, add to the circle to cover the tissue, and lay the slices flat in a humidified box and incubate in a 37℃ constant temperature box for 1 h. A small amount of water is added to the humidified box to maintain humidity. The slices are washed 3 times with PBS buffer, 5 min each time. After removing the PBS buffer, DAPI staining solution is added to the circle and incubated at room temperature for 10 min away from light. The slides are placed in PBS buffer and shaken on a decolorizing shaker to wash 3 times, 5 min each time. After the slices are slightly dried, they are sealed with anti-fluorescence quenching sealing agent. The sections were observed and images were collected under a fluorescence microscope (DAPI ultraviolet excitation wavelength 330-380 nm, emission wavelength 420 nm, blue light; CY3 excitation wavelength 510-561 nm, emission wavelength 590 nm, red light). The apoptosis ratio was calculated as follows: apoptosis ratio (%) = number of apoptotic cells ÷ total number of cells × 100%.

[0131] The results are as follows Fig.21 and Fig. 22 As shown, Fig.21 Indicates the results of brain tissue fluorescent TUNEL staining, including DAPI staining and Cy3 staining and the combined results of the two. Fig. 22 It represents the apoptosis rate of brain cells, indicating that nanovesicles modified with RVG peptide and loaded with miRNA can inhibit the apoptosis of brain cells.

[0132] 3) The effect of RVG peptide-modified and miRNA-loaded nanovesicles on the area of ​​brain injury.

[0133] According to the method of part 2) of (2) animal experiment, rat models of negative control group, model group, extracellular vesicle group, nanovesicle group, RVG peptide-modified nanovesicle group and RVG peptide-modified and miRNA-encapsulated nanovesicle group were obtained.

[0134] After 7 days, the animals were killed and the brain tissue was quickly removed (within 10 minutes) and placed in a -20°C refrigerator for 30 minutes. Brain slices were cut to a thickness of 2 mm and placed in TTC staining solution in a 37°C light-proof water bath for 30 minutes. The container was gently shaken every 5 minutes to ensure sufficient staining. The brain slices were removed and fixed with a universal tissue fixative, and images were collected 24 hours later. The formula for calculating the damage area ratio is as follows: Damage area ratio (%) = damage area ÷ total area × 100%. The results are shown in Fig.23 and Fig.24 As shown, Fig.23 Represents the results of TTC staining of brain tissue. Fig.24 It represents the ratio of brain injury area, indicating that nanovesicles modified with RVG peptide and loaded with miRNA can significantly reduce the injury area of ​​cerebral ischemia-reperfusion injury.

[0135] 4) Effects of RVG peptide-modified and miRNA-loaded nanovesicles on neurobehavioral evaluation.

[0136] According to the method of part 2) of (2) animal experiment, rat models of negative control group, model group, extracellular vesicle group, nanovesicle group, RVG peptide-modified nanovesicle group and RVG peptide-modified and miRNA-encapsulated nanovesicle group were obtained.

[0137] After 7 days, neurobehavioral scores were performed according to the Longa scoring standard. In the Longa scoring standard, the higher the score, the more severe the animal's behavioral disorder. Fig.25 As shown, RVG peptide-modified and miRNA-loaded nanovesicles can improve neurobehavior after cerebral ischemia-reperfusion injury.

[0138] 5) Biosafety of RVG peptide-modified and miRNA-loaded nanovesicles.

[0139] According to the method of part 2) of (2) animal experiment, rat models of negative control group, model group, extracellular vesicle group, nanovesicle group, RVG peptide-modified nanovesicle group and RVG peptide-modified and miRNA-encapsulated nanovesicle group were obtained.

[0140] After 7 days, tissue samples were collected, fixed, embedded, and paraffin sliced. The slices were placed in the following order: environmentally friendly dewaxing solution I, 20 min; environmentally friendly dewaxing solution II, 20 min; anhydrous ethanol I, 5 min; anhydrous ethanol II, 5 min; 75% alcohol, 5 min; washed with tap water. The slices were treated with high-definition constant staining pretreatment solution for 1 min. The slices were dyed with hematoxylin staining solution for 3-5 min, washed with tap water, differentiated with differentiation solution, washed with tap water, blued with blue reversion solution, and rinsed with running water. The slices were dehydrated in 95% alcohol for 1 min and stained in eosin staining solution for 15 s. The slices were placed in the following order: anhydrous ethanol I, 2 min; anhydrous ethanol II, 2 min; anhydrous ethanol III, 2 min; n-butanol I, 2 min; n-butanol II, 2 min; xylene I, 2 min; xylene II, 2 min; transparent and neutral gum sealing. Microscope examination, image acquisition and analysis.

[0141] The results are as follows Fig.26 As shown, no obvious histopathological abnormalities or lesions were found in the tissues and organs of each group, indicating that the RVG peptide-modified and miRNA-loaded nanovesicles have good biosafety in vivo.

[0142] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A nanovesicle, characterized in that: The surface of the nanovesicles is modified with RVG peptide, and the mimic of miRNA-130a-3p is loaded inside, and the nanovesicles are derived from human placental mesenchymal stem cells; The particle size of the nanovesicles is 120 ± 20 nm; The amino acid sequence of the RVG peptide is shown in SEQ ID NO.2; The sense strand of the miRNA-130a-3p mimic is shown in SEQ ID NO.3, and the antisense strand is shown in SEQ ID NO.

4.

2. A method for preparing nanovesicles according to claim 1, characterized in that: Includes steps: S1, subculture human placental mesenchymal stem cells; S2, using genetic engineering to make human placental mesenchymal stem cells express RVG peptide, thereby obtaining human placental mesenchymal stem cells expressing RVG peptide; S3, treating the human placental mesenchymal stem cells expressing RVG peptide obtained in S2 by using an extrusion method to obtain RVG peptide-modified nanovesicles; S4. Loading the miRNA-130a-3p mimic into the RVG peptide-modified nanovesicles to obtain nanovesicle products.

3. The method for preparing nanovesicles according to claim 2, characterized in that: In S1, the subculture method includes: obtaining human placental mesenchymal stem cells from human placental tissue, discarding the culture medium, and washing the residual culture medium with 3-7 mL of DPBS buffer, then adding 1-2 mL of 0.25% recombinant trypsin, adding 3-10 mL of complete culture medium after 1-5 min to stop digestion, and obtaining a cell suspension by pipetting; centrifuging the cell suspension at 200-400g for 3-10 min, then discarding the supernatant and retaining the cell pellet; then adding 3-10 mL of fresh complete culture medium, pipetting into a single cell suspension, and transferring the single cell suspension to a new culture bottle at a ratio of 1:3 for continued culture.

4. The method for preparing nanovesicles according to claim 2, characterized in that: In S2, the genetic engineering includes: inserting (1-1.5)×10 8 Mesenchymal stem cells were transfected with TU / mL lentivirus at a multiplicity of infection of 80-140, and then stable transfected strains were selected using puromycin at a concentration of 2-6μg / mL.

5. The method for preparing nanovesicles according to claim 4, characterized in that: The gene fragment inserted into the target gene was obtained by PCR technology, the target gene was Lamp2B connected to RVG peptide expression gene, and the target gene fragment was inserted into the multiple cloning site of the lentiviral expression vector by enzyme ligation method; the gene sequence of the expressed Lamp2B connected to RVG peptide is shown in SEQ ID NO.

1.

6. The method for preparing nanovesicles according to claim 2, characterized in that: In S3, the squeezing method includes: diluting the cells to 1×10 in DPBS buffer. 6 ~1×10 7 The diluted cell suspension was squeezed through 1-0.2 μm filter membranes in the order from coarse to fine, and each specification of filter membrane was squeezed multiple times.

7. The method for preparing nanovesicles according to claim 6, characterized in that: In S3, the squeezed liquid is centrifuged at 100,000-120,000 g for 70-90 min at 4±2° C., the supernatant is discarded, and the precipitate is resuspended in PBS buffer to obtain RVG peptide-modified nanovesicles.

8. The method for preparing nanovesicles according to claim 2, characterized in that: In S4, miRNA-130a-3p mimics were loaded into RVG peptide-modified nanovesicles by electroporation; The electroporation technique includes: RVG peptide-modified nanovesicles and miRNA-130a-3p mimics are electroporated at 10 7 ~10 12 After mixing at a ratio of 0.1-10 μg, add electroporation buffer; set the voltage to 200-600 V for electroporation.

9. Use of the nanovesicle according to claim 1 in at least one of the following a1) to a6): a1) preparing products for reducing the apoptosis rate of hypoxic damaged nerve cells; a2) Preparation of products for increasing the activity of oxidatively damaged nerve cells; a3) Preparation of products for inhibiting the release of inflammatory factors in nerve cells; a4) Preparation of products for inhibiting brain cell apoptosis caused by cerebral ischemia-reperfusion injury; a5) Preparation of products for reducing the area of ​​brain damage caused by cerebral ischemia-reperfusion injury; a6) Preparation of products that promote recovery of neurological function after cerebral ischemia-reperfusion injury.

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