Material-enabled stem cell-derived exosome and preparation method and application thereof

Abstract

This invention discloses a material-empowered stem cell-derived exosome, its preparation method, and its application, belonging to the field of biomedical technology. The method involves the following steps: mesenchymal stem cells are seeded onto a substrate fused with N / VE-cadherin-Fc fusion protein and cultured to assemble into aggregates; the aggregates are dynamically cultured under staged shear stress; the culture medium is collected, and the exosomes are separated and purified. The preparation method of this invention overcomes the shortcomings of existing mesenchymal stem cell exosome preparation technologies, such as low yield and insufficient neurovascular repair function. It significantly activates exosome biogenesis-related molecular pathways, increases exosome yield by approximately four times, and is rich in miRNAs related to neurovascular repair. It can significantly improve neurological deficits in stroke mice and hindlimb motor function in rats with spinal cord injury, providing a highly efficient and stable cell-free therapeutic strategy for the treatment of central nervous system injuries.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a material-empowered stem cell-derived exosome, its preparation method, and its application. Background Technology

[0002] Central nervous system injury (including stroke, spinal cord injury, etc.) is one of the leading causes of death and permanent disability in humans, and currently there is a lack of effective neurovascular synergistic repair methods in clinical practice. Exosomes derived from mesenchymal stem cells (MSCs) have become a research hotspot for the treatment of central nervous system injury due to their low immunogenicity, ability to cross the blood-brain barrier, and rich content of active molecules such as miRNAs.

[0003] I. The essential differences between cell therapy and exosome therapy MSCs, as live-cell drugs, possess the core advantage of sensing the lesion microenvironment and dynamically regulating their secretory behavior to achieve "on-demand treatment." However, exosomes, as cell-free preparations, are essentially "static effectors"—once isolated and purified, they lose the metabolic activity and environmental sensing capabilities of their parent cells. The active molecules they carry, such as miRNAs, are "historical records" of the parent cells under specific culture conditions and cannot be dynamically adjusted according to changes in the lesion microenvironment. This fundamental difference determines that the therapeutic function of exosomes depends entirely on the culture conditions of the parent cells during their preparation. Therefore, how to precisely regulate the culture conditions of MSCs to "pre-program" them in vitro to produce exosomes with specific repair functions is a key scientific issue for clinical translation in this field.

[0004] II. Precise Control of Exosome Preparation Conditions The function of exosomes is programmable. Pre-treatment during MSC culture using physical, chemical, biological factors, or three-dimensional culture methods can endow exosomes with enhanced biological functions. Three-dimensional culture technology is one important strategy: culturing MSCs into cell aggregates can mimic the in vivo microenvironment, significantly increasing exosome yield and altering their content composition. Culture systems modified with specific basal proteins (such as cadherin-Fc fusion proteins) can further directionally regulate MSC functional differentiation and exosome contents. For example, existing technologies disclose that E / N-cad-Fc substrates can promote the anti-inflammatory and immunomodulatory functions of MSC aggregates (ZL202411802465.9); N / VE-cad-Fc substrates can promote MSC aggregate formation and effectively repair vascular damage (ZL202110254212.2); and the application of E / N-cad-Fc combined with IGF-1 to empower MSC aggregate-derived exosomes in anti-aging (CN202411802464.4). However, there are no reports on the synergistic application of neurovascular microenvironment-specific cadherin substrates with dynamic culture conditions (such as shear stress) to MSC culture and the investigation of their role in the central nervous system repair of the resulting exosomes.

[0005] III. Limitations of the Existing Technology and the Starting Point of the Invention The existing technology has the following shortcomings: (1) Most studies ignore the essential differences between cells and exosomes, directly attributing the therapeutic effect of MSCs to exosomes, and lack systematic optimization of exosome preparation conditions; (2) Existing studies mostly use single regulation methods (such as hypoxia, simple three-dimensional culture or single factor pretreatment), while there is no systematic study on the synergistic application of substrate materials and dynamic culture to directionally "pre-program" MSCs to generate exosomes with multiple nerve repair functions; (3) The repair of central nervous system injury involves multiple pathological processes such as neuronal survival, axonal regeneration, vascular reconstruction and inflammation regulation. How to make exosomes simultaneously enrich miRNAs with multiple functions such as promoting nerve regeneration, promoting angiogenesis and anti-inflammation, and achieve multi-target synergistic treatment of central nervous system injury is still a technical problem that needs to be solved in this field.

[0006] To address the aforementioned shortcomings, this invention provides a material-empowered stem cell-derived exosome, its preparation method, and its applications. Based on the scientific understanding that "exosome function is determined by preparation conditions," this invention, through the combined use of a neurovascular-specific N / VE-cad-Fc substrate and dynamic culture, synergistically regulates the composition of exosome contents. Unexpectedly, it was discovered that the exosomes prepared by this method not only significantly increased yield but were also rich in specific neurorepair-promoting miRNAs (such as miR-21 and miR-133b), achieving significant repair effects in animal models of stroke and spinal cord injury, superior to exosomes obtained by single methods disclosed in existing technologies. Summary of the Invention

[0007] The purpose of this invention is to address the technical deficiencies in the existing technology by providing a method for preparing material-empowered stem cell-derived exosomes.

[0008] Another object of the present invention is to provide materials obtained by the above preparation method to empower stem cell-derived exosomes.

[0009] Another object of the present invention is to provide the application of the above-mentioned material-empowered stem cell-derived exosomes in the preparation of drugs for treating central nervous system injury.

[0010] The technical solution adopted to achieve the purpose of this invention is: A method for preparing stem cell-derived exosomes empowered by materials includes the following steps: Step 1: Mesenchymal stem cells are seeded onto a substrate coated with N / VE-cadherin-Fc fusion protein and cultured to form N / VE-cadherin-Fc matrix-empowered mesenchymal stem cells. Step 2: The N / VE-cadherin-Fc matrix-empowered mesenchymal stem cells obtained in Step 1 are assembled to obtain cadherin-empowered mesenchymal stem cell aggregates. Step 3: Apply staged shear stress to the cadherin-enhanced mesenchymal stem cell aggregates obtained in Step 2 and perform dynamic culture. Step 4: During the dynamic culture in Step 3, the culture medium is collected, and the material-empowered stem cell-derived exosomes are isolated and purified from the culture medium.

[0011] In the above technical solution, in step 1, the N / VE-cadherin-Fc fusion protein is human nerve cell cadherin. Fc fusion protein (N-cadherin-Fc) and human vascular endothelial cell cadherin A mixture of Fc fusion proteins (VE-cadherin-Fc), wherein the concentration ratio of N-cadherin-Fc to VE-cadherin-Fc is 1:3-3:1, preferably 1:1.

[0012] In the above technical solution, in step 1, the mesenchymal stem cells are derived from umbilical cord, bone marrow, adipose tissue, or induced pluripotent stem cells (iPSCs).

[0013] In the above technical solution, in step 2, the cell assembly includes one or more of the following methods: ultra-low adhesion method, stirred culture method, microfluidic method, and scaffold culture method.

[0014] In the above technical solution, in step 3, the process of applying staged shear stress is to first apply low shear stress and then apply high shear stress. The low shear stress is 0.1-1 dyn / cm², and the dynamic culture time under low shear stress is 12-48 hours; the high shear stress is 1-3 dyn / cm², and the dynamic culture time under high shear stress is 24-96 hours.

[0015] Another aspect of the present invention includes material-empowered stem cell-derived exosomes obtained by the preparation method, wherein the content of miRNAs that promote nerve regeneration, angiogenesis and / or anti-inflammation is increased in the material-empowered stem cell-derived exosomes.

[0016] In the above technical solution, the miRNA includes miR-21, miR-126-3p and / or miR-133b.

[0017] Another aspect of the present invention includes the application of the material-empowered stem cell-derived exosomes in the preparation of drugs for treating central nervous system injury.

[0018] In the above technical solution, the central nervous system injury includes neurovascular injury from stroke and spinal cord injury.

[0019] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention provides a method for preparing exosomes by synergistically applying two techniques: substrate empowerment and dynamic shear stress culture. Compared with traditional mesenchymal stem cell exosome preparation methods, this method significantly improves the production efficiency of exosomes from mesenchymal stem cells and achieves targeted and synergistic "pre-programming" of the mother cells to obtain more exosomes with specific multiple repair functions.

[0020] 2. The material-empowered stem cell-derived exosomes of the present invention are significantly enriched with multiple functional active substances closely related to promoting nerve regeneration, angiogenesis and anti-inflammation, such as miR-21, miR-133b and miR-126-3p, thereby significantly enhancing the excellent application prospects of exosomes as drugs to promote the repair of central nervous system damage. Attached Figure Description

[0021] Figure 1 To optimize the ratio of hNFc to hVEFc, A represents the morphology and adhesion of MSCs on the matrix surface under different ratios; B represents the detection of MSC proliferation capacity under different ratios of matrix; C represents the detection of gene expression of endogenous cadherin, stemness markers, and neurotrophic factors in MSCs under different ratios of matrix; and D represents the detection of protein expression of endogenous cadherin, stemness markers, pFGFR, and pVEGFR in MSCs under different ratios of matrix.

[0022] Figure 2 The expression of endogenous cadherin and exosome biogenesis-related molecules in NV-MSCs was shown. A represents the Western Blot detection of endogenous cadherin and exosome biogenesis and secretion-related proteins; B represents the qPCR detection of exosome biogenesis and secretion-related genes.

[0023] Figure 3 Preparation and characterization of cadherin-enhanced mesenchymal stem cell aggregates, where A is a light micrograph of the NV-MAs formation process; B is the aggregate diameter statistics.

[0024] Figure 4 The expression of endogenous cadherin and stemness markers in mesenchymal stem cell aggregates was evaluated using cadherin-enhanced methods. A represents the protein expression of endogenous N-cadherin, VE-cadherin, and OCT4; B represents the gene expression of endogenous N-cadherin, VE-cadherin, and OCT4.

[0025] Figure 5 Functional evaluation of cadherin-empowered mesenchymal stem cell aggregates: A) detection of gene expression of repair-related factors; B) immunofluorescence staining of NV-MAs repair-related factors; C) Western blotting detection of proteins related to anti-apoptotic signaling pathways.

[0026] Figure 6 TEM characterization of D-NV-MAs-Exo.

[0027] Figure 7 Identification of D-NV-MAs-Exo exosome markers.

[0028] Figure 8 The efficacy of D-NV-MAs-Exo in treating ischemic stroke mice was evaluated. In this study, A represents the mNSS score on day 14, and B represents the time to paper removal experiment.

[0029] Figure 9 The efficacy of D-NV-MAs-Exo in treating spinal cord injury in rats was evaluated. A represents the BBB motor function score at week 8; B represents the grid walking test; and C represents the hot plate pain test. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0031] In the following embodiments, the human nerve cell cadherin Fc fusion protein (N-cadherin-Fc), denoted as hN-cad-Fc or abbreviated as hNFc, is a human vascular endothelial cell cadherin. The Fc fusion protein (VE-cadherin-Fc) is denoted as hVE-cad-Fc or abbreviated as hVEFc. The hN-cad-Fc is selected from the invention patent CN115040695A "Application of a Fusion Protein Active Interface Based on VE-cad-Fc / N-cad-Fc", and the sequence of hN-cad-Fc is shown in SEQ ID NO:4 of patent CN115040695A. The hVE-cad-Fc is selected from the matrix corresponding to the invention patent CN109337857A "Uses of Fusion Proteins E-cadherin-Fc, VE-cadherin-Fc and VEGF-Fc", and the sequence of hVE-cad-Fc is shown in SEQ ID NO:3 of patent CN109337857A.

[0032] The fusion protein formed by mixing hN-cad-Fc and hVE-cad-Fc is abbreviated as hNVFc.

[0033] All data are expressed as mean ± standard deviation; statistical differences among groups were assessed by one-way ANOVA. 0.05 is considered statistically significant. p 0.05, p 0.01, p 0.001, ns indicates no significant difference. Unless otherwise stated, all in vitro experimental results were from 3 independent parallel experiments, and all animal experimental results were from 5 independent parallel experiments. All results were statistically analyzed using GraphPad Prism version 8.0 software.

[0034] Example 1 A method for preparing stem cell-derived exosomes empowered by materials includes the following steps: Step 1 involves seeding mesenchymal stem cells onto a substrate coated with the N / VE-cadherin-Fc fusion protein and culturing them to form N / VE-cadherin-Fc matrix-empowered mesenchymal stem cells; specifically including the following steps: Step 1.1, Preparation of hNVFc: The purified hNFc and / or hVEFc were diluted in PBS to a total protein concentration of 5 μg / mL to obtain the N / VE-cadherin-Fc fusion protein, denoted as hNVFc. The concentration ratio of hNFc to hVEFc was 1:1, 1:2, or 2:1. (Three different concentration ratios resulted in three different mixing proportions of hNVFc matrix.) Step 1.2: Add the hNVFc obtained in step 1.1 to the corresponding culture surface, incubate at 37°C for 2 hours, wash once with PBS to obtain an hNVFc matrix culture surface that can be directly used for cell culture.

[0035] Step 1.3, Preparation of hNVFc matrix-empowered mesenchymal stem cells First, MSCs are prepared according to 1×10 6 The cells were seeded at a density of 1 cell per flask into T75 cell culture flasks containing an hNVFc matrix culture surface, and expanded to the 6th generation under normal culture conditions to obtain N / VE-cadherin-Fc matrix-empowered mesenchymal stem cells, denoted as NV-MSCs.

[0036] Step 2: The N / VE-cadherin-Fc matrix-empowered mesenchymal stem cells obtained in Step 1 were cultured in DMEM / F12 complete medium until 70%-80% confluence. After trypsin digestion, an appropriate amount of medium was taken to resuspend the cells, and the cells were evenly seeded into AggreWell cell aggregate culture plates at a ratio of 100 cells / 1 aggregate. The cells were centrifuged at 1000 rpm for 5 min to induce cell sedimentation. The cells were then transferred to a cell culture incubator (5% CO2, saturated humidity, 37℃) and cultured for 18 h. The morphology of the aggregates was observed and photographed using an inverted phase-contrast microscope. The aggregates were gently detached using a pipette tip and collected in centrifuge tubes to obtain cadherin-empowered mesenchymal stem cell aggregates, denoted as NV-MAs. Step 3: The NV-MAs obtained in Step 2 are placed in serum-free medium for dynamic suspension culture (using Corning® Reusable Glass Spinner Flask), and controllable fluid shear stress is generated by stirring / fluid movement. In order to balance the adaptability of cell aggregates to the dynamic environment and exosome production efficiency, staged shear stress is applied: first, low shear stress (0.1-1 dyn / cm²) is applied as an adaptation stage to stabilize the aggregate structure, and then high shear stress (1-3 dyn / cm²) is applied as a production stage to promote exosome release and increase yield.

[0037] Step 4: Within the 12–96 h window of dynamic NV-MAs culture in Step 3, collect the supernatant of the conditioned medium at predetermined time points for subsequent processing. Purify the exosomes using differential centrifugation-ultracentrifugation: centrifuge at 300×g, 4℃ for 10 min to remove cells and collect the supernatant; centrifuge at 2000×g, 4℃ for 20 min to remove cell debris and collect the supernatant; centrifuge at 10,000×g, 4℃ for 30 min to remove apoptotic bodies and collect the supernatant; centrifuge at 100,000×g, 4℃ for 120 min, remove the supernatant, and resuspend the precipitate in PBS. This is the material-empowered stem cell-derived exosome, denoted as D-NV-MAs-Exo.

[0038] Comparative Example 1 This comparative example provides a method for preparing exosomes derived from mesenchymal stem cells, including the following steps: Step 1: MSCs are seeded onto conventional tissue culture surface (TCPS) and cultured until the 6th generation to obtain mesenchymal stem cells (MSCs).

[0039] Step 2: Mesenchymal stem cells (MSCs) are cultured using the same process as in Step 2 of Example 1 to obtain cell aggregates (MAs).

[0040] Steps 3 and 4 are the same as steps 3 and 4 in Example 1, and finally mesenchymal stem cell-derived exosomes are obtained, denoted as D-MAs-Exo.

[0041] Comparative Example 2 This comparative example provides a method for preparing exosomes derived from mesenchymal stem cells. Compared with the method in Example 1, steps 1-2 are the same, except that in steps 3-4, the exosomes obtained by static culture in T225 cell culture flasks are denoted as S-NV-MAs-Exo.

[0042] Comparative Example 3 This comparative example provides a method for preparing exosomes derived from mesenchymal stem cells. Compared with the method of Comparative Example 1, steps 1-2 are the same, except that in steps 3-4, the exosomes obtained by static culture in T225 cell culture flasks are denoted as S-MAs-Exo.

[0043] Application Example 1 like Figure 1 A and Figure 1 As shown in B, the proliferation capacity of hNVFc matrix-empowered mesenchymal stem cells obtained in Example 1 was tested for three concentration ratios: hNFc:hVEFc = 1:1, 1:2, or 2:1 (i.e., N:V = 1:1, 1:2, or 2:1). MSCs were seeded onto conventional tissue culture surfaces (TCPS) of Comparative Example 1 (corresponding to...). Figure 1In 96-well culture plates containing the three different ratios of hNVFc matrix culture surfaces obtained in Example 1 (corresponding to TC in A) and TC in A), the three different ratios of hNVFc matrix culture surfaces obtained in Example 1 (corresponding to TC in A) Figure 1 The N:V ratio in the A layer was 1:1, 1:2, or 2:1. After incubation for 4, 24, 48, and 72 h, the supernatant was discarded, and the cells were washed three times with PBS. 100 μL of CCK-8 (1:10 dilution) was added to each well, and the cells were incubated at 37°C for 4 h. The absorbance was then measured at 450 nm using a microplate reader.

[0044] To systematically evaluate the regulatory effects of hNVFc matrix obtained from different ratios of hNFc and hVEFc on MSC cell adhesion and proliferation, the results are as follows: Figure 1 A and Figure 1 As shown in B, with the increase of hNFc proportion, MSCs showed enhanced adhesion ability on the substrate culture surface in the early stage and exhibited a better proliferation trend, indicating that hNFc is beneficial to promoting cell adhesion and proliferation.

[0045] Application Example 2 Example 1: Detection of endogenous cadherin and stem cell marker protein expression in hNVFc matrix-empowered mesenchymal stem cells with hNFc:hVEFc ratios of 1:1, 1:2, or 2:1. ① Extraction of total cellular protein: MSCs were seeded onto the hNVFc substrate culture surface of Example 1 and TCPS of Comparative Example 1, respectively, and cultured. The culture medium was discarded and the cells were washed with PBS. 100 μL of pre-chilled RIPA lysis buffer was added to each well and incubated on ice for 10 min. Cells were collected thoroughly with a cell scraper and the lysis buffer was transferred to a centrifuge tube. The tubes were centrifuged at 4°C and 13,000 rpm for 15 min. The supernatant was collected to obtain the sample corresponding to Comparative Example 1 and three samples corresponding to different proportions of Example 1. The protein concentration in each sample was determined according to the BCA kit instructions; finally, loading buffer (1:5 dilution) was added to the supernatant of each tube, and the tubes were boiled for 10 min, aliquoted, and stored in an ultra-low temperature freezer at -80℃.

[0046] ② Western Blotting: Each sample was added to a 10% SDS-polyacrylamide gel for electrophoresis. Electrophoresis was stopped after the proteins were separated to their appropriate positions according to their molecular weight. The protein was then transferred to a PVDF membrane following standard transfer procedures. After transfer, the PVDF membrane was blocked with 5% BSA at room temperature for 2 h. The membrane was then incubated with the following antibody conditions: mouse anti-human VE-cadherin antibody (VE-cadherin) (intracellular domain, 1:2000 dilution), rabbit anti-human N-cadherin antibody (N-cadherin) (intracellular domain, 1:2000 dilution), rabbit anti-human OCT4 protein antibody (1:2000 dilution), and mouse anti-human β-actin (1:5000 dilution). Incubation was performed at 37°C for 1 h or 4°C overnight. The membrane was washed three times with TBST solution at room temperature for 5 min each time. HRP-labeled goat anti-rabbit / goat anti-mouse IgG (H+L) antibody (1:2000 dilution) was added and incubated at room temperature for 2 h. Washing was repeated three times with TBST solution. Finally, an appropriate amount of chemiluminescent imaging reagent was added, and the membrane was observed and photographed using a Western blotting instrument. The images were then captured using ImageDevice. J software was used for semi-quantitative analysis of the bands, with β-actin as an internal reference for normalization.

[0047] Example 1: Detection of endogenous cadherin and stem cell marker gene expression in hNVFc matrix-empowered mesenchymal stem cells with hNFc:hVEFc ratios of 1:1, 1:2, or 2:1. ① mRNA extraction: MSCs were seeded onto the hNVFc substrate culture surface of Example 1 and TCPS of Comparative Example 1, respectively, and cultured. After discarding the culture medium, the cells were washed three times with PBS. 1 mL of Trizol reagent was added to each well to lyse the cells. After thorough mixing, the cells were transferred to 1.5 mL centrifuge tubes, 200 μL of chloroform was added, the mixture was vigorously shaken for 15 s, incubated at room temperature for 5 min, and centrifuged at 12000g for 15 min at 4°C. The supernatant was aspirated into a new tube, 500 μL of isopropanol was added, and the mixture was incubated at room temperature for 10 min. After centrifugation at 12000g for 10 min at 4°C, the supernatant was discarded, and the precipitate was resuspended in 1 mL of 75% ethanol. The mixture was centrifuged at 7500g for 5 min at 4°C, the supernatant was discarded, and the cells were air-dried at room temperature. 20 μL of ddH2O was added, and the mRNA was fully dissolved in a 55°C water bath for 10 min. The mRNA was then aliquoted and stored at -80°C.

[0048] ② Preparation of cDNA: Take a new RNase-free PCR tube, add 4 μL of RNase-free water, 4 μL of gDNA wiper Mix, and 8 μL of RNA to each tube, mix well, centrifuge briefly for 5 s, place in a PCR instrument and remove genomic DNA according to the program of 42℃ for 2 min and 4℃ ∞; then add 4 μL of 5×HiScript II qRT SuperMix II, and reverse transcribe according to the program of 50℃ for 15 min, 85℃ for 5 s and 4℃ ∞; after appropriate dilution, store the cDNA obtained by reverse transcription at -20℃.

[0049] ③ Quantitative Real-Time PCR: Add 2 μL of cDNA as template to each tube, followed by 1 μL of N-cadherin, VE-cadherin, OCT4 primer, 10 μL of 5×ChamQ Universal SYBR qPCR Master Mix, and 7 μL of RNase-free H2O. After mixing, centrifuge briefly for 5 s, place in a real-time PCR instrument, and run according to the following program: 95℃ 30 s, 95℃ 3 s, 60℃ 10 s, 40 cycles; 95℃ 15 s, 60℃ 60 s, 95℃ 15 s. Use β-actin as an internal control for each group. Set up three replicates for each sample, and use 2^... The relative expression level was calculated using the ΔΔCt method and statistical analysis was performed.

[0050] Example 1 showed the following: detection of the secretion capacity of hNVFc matrix-empowered mesenchymal stem cells for neural / vascular trophic factors and related signaling pathways in three hNFc:hVEFc ratios of 1:1, 1:2, or 2:1. To assess the impact of changes in endogenous cadherin expression on related cell signaling pathways and secreted factors, total protein and total RNA were extracted from NV-MSCs cultured in different proportions of hNVFc substrate in Example 1, according to the methods described in this application example. Simultaneously, total protein and total RNA were extracted from MSCs in Comparative Example 1. Gene expression of VEGF, HGF, ANG, GDNF, and BDNF was detected by quantitative real-time PCR. Protein expression of FGFR, pFGFR, VEGFR, and pVEGFR was detected by Western blotting. Primer sequences for the relevant genes are shown in Table 1.

[0051] Table 1 Primer sequences of relevant genes The above test results are as follows Figure 1 C and Figure 1 As shown in D, by Figure 1As can be seen from D in Example 1, compared with the MSCs of Comparative Example 1, the NV-MSCs obtained in Example 1 showed higher levels of endogenous N-cadherin and VE-cadherin expression, indicating that the hNVFc matrix can induce and maintain the neurovascular-associated adhesion phenotype of cells and enhance intercellular interactions.

[0052] Depend on Figure 1 As shown in C, when the ratio of hVEFc increases, the expression levels of pro-angiogenic factors secreted by cells are all enhanced. Moreover, under the condition of hNFc:hVEFc=1:1, the expression levels of neurotrophic factors (such as GDNF, BDNF, etc.) are higher, indicating that this ratio has advantages in providing bidirectional neurotrophic support. Considering the comprehensive evaluation of cell adhesion / proliferation, stemness maintenance, neurotrophic factor secretion levels, and FGFR and VEGFR phosphorylation activation in Example 1, the hNVFc obtained with hNFc:hVEFc=1:1 yields the best effect for subsequent steps, and is denoted as the optimal ratio group.

[0053] Application Example 3 Detection of endogenous cadherin expression and exosome production related molecules in NV-MSCs Protein expression detection: Extraction of total cellular protein: Total protein was extracted from the NV-MSCs obtained in the optimal ratio in Example 1 and the MSCs obtained in Comparative Example 1 using the method in Example 2, resulting in two groups of total protein.

[0054] Western Blotting: Electrophoresis, electroporation, and blocking were performed using the method described in Example 2. The PVDF membrane was incubated with the following antibody conditions: mouse anti-human VE-cadherin antibody (intracellular domain, 1:2000 dilution), rabbit anti-human N-cadherin antibody (intracellular domain, 1:2000 dilution), rabbit anti-human TSG101 protein antibody (1:2000 dilution), rabbit anti-human CD81 protein antibody (1:2000 dilution), rabbit anti-human RAB27A protein antibody (1:2000 dilution), and mouse anti-human β-actin (1:5000 dilution) at 37°C for 1 h or overnight at 4°C. The membrane was washed three times with TBST solution at room temperature for 5 min each time. HRP-labeled goat anti-rabbit / mouse IgG (H+L) antibody (1:2000 dilution) was added and incubated at room temperature for 2 h. The membrane was washed three times with TBST solution. Finally, an appropriate amount of chemiluminescent imaging reagent was added, and the membrane was observed and photographed using a Western blotting instrument. The images were then analyzed using ImageDevice. J software was used for semi-quantitative analysis of the bands, with β-actin as an internal reference for normalization, such as... Figure 2 As shown in A in the diagram.

[0055] Gene expression detection: mRNA extraction: mRNA was extracted from NV-MSCs obtained in the optimal ratio in Example 1 and MSCs obtained in Comparative Example 1 using the method in Example 2, resulting in two groups of mRNAs.

[0056] cDNA preparation: cDNA was prepared from the NV-MSCs obtained in the optimal ratio in Example 1 and the MSCs obtained in Comparative Example 1 using the method in Example 2, resulting in two sets of cDNA.

[0057] Real-time PCR: After reverse transcription, 2 μL of cDNA from each group was added to each tube as a template, along with 1 μL of N-cadherin, VE-cadherin, and primers for STAM1, HGS, TSG101, ALIX, ATG5, RAB27A, RAB27B, and SNAP23, 10 μL of 5×ChamQ Universal SYBR qPCR Master Mix, and 7 μL of RNase-free H2O. After thorough mixing, the mixture was briefly centrifuged for 5 s and then placed in a real-time PCR instrument for amplification according to the procedure described in section 3.2. The obtained data used β-actin expression as an internal control, with three replicates for each sample. The results were analyzed using 2^... The relative expression level was calculated using the ΔΔCt method and statistically processed. The results are as follows: Figure 2 As shown in B, the relevant gene primer sequences are shown in Table 2: Table 2 Primer sequences of related genes To further elucidate the effects of hNVFc matrix on the exosome biogenesis and secretion capabilities of mesenchymal stem cells, [the study was conducted]. Figure 2 As shown in A, compared with the MSCs of Comparative Example 1, the expression of several key proteins related to exosome synthesis, sorting, and secretion processes in the NV-MSCs of Example 1 was significantly upregulated, indicating that the hNVFc matrix of Example 1 can promote the establishment of the molecular basis for exosome production and release, giving NV-MSCs a stronger exosome generation potential. Figure 2 As shown in section B, the expression levels of genes closely related to exosome formation and secretion, such as TSG101, CD81, and RAB27A, in NV-MSCs were significantly higher than those in Comparative Example 1 MSCs, indicating that the vesicle biogenesis process of NV-MSCs in Example 1 was in a more active functional state. Furthermore, the expression levels of endogenous N-cadherin and VE-cadherin in NV-MSCs of Example 1 were significantly increased, exhibiting a more prominent neurovascular phenotype. This phenotype provides a cellular and molecular basis for the exosome-mediated neurovascular repair by NV-MSCs of Example 1.

[0058] Application Example 4 Preparation of Cadmin-Enhanced Mesenchymal Stem Cell Aggregates (NV-MAs) in Example 1 and Characterization of Endogenous Cadmin and Stem Markers: From Figure 3 As shown in A, after 18 hours of culture in AggreWell plates, both the MSCs of Comparative Example 1 and the NV-MSCs of Example 1 formed structurally complete and clearly defined cell aggregates, indicating that the aggregate preparation method described in this invention has good spheroidization consistency and reproducibility. Figure 3 As shown in B, the average diameters of aggregates MAs and NV-MAs formed by 100 cells are 77 μm and 71 μm, respectively.

[0059] Using the gene expression and protein expression detection procedures described in Application Example 2, the transcription and protein levels of endogenous N-cadherin, VE-cadherin, and the stemness marker OCT4 in MSCs of Comparative Example 1, NV-MSCs of Example 1, MAs of Comparative Example 1, and NV-MAs of Example 1 were compared. Statistical analysis was performed using ImageJ software, and the results are as follows: Figure 4 As shown, qPCR and WB results indicated that the transcription and expression levels of endogenous N-cadherin and VE-cadherin were significantly increased in the NV-MAs group, and the expression of the stem cell marker OCT4 was enhanced. This suggests that NV-MAs further enhanced the neurovascular-associated adhesion molecular phenotype of NV-MSCs, thereby helping to maintain or enhance the functional state of cells.

[0060] Functional evaluation of cadherin-enhanced mesenchymal stem cell aggregates To simulate the injury / inflammatory microenvironment, 10 ng / mL TNF-α was added to the complete culture medium. Four groups of samples—MSCs (Comparative Example 1), NV-MSCs (Example 1), MAs (Comparative Example 1), and NV-MAs (Example 1)—were used as research subjects. The responses of the four groups to TNF-α stimulation were compared using the following indicators: transcriptional and secretion levels of repair-related factors, anti-apoptotic and anti-inflammatory capabilities, to comprehensively evaluate the functional advantages of cadherin-empowered mesenchymal stem cell aggregates. Figure 5 A- Figure 5 As shown in C.

[0061] Immunofluorescence staining method: NV-MAs were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 solution for 10 min, blocked with 3% BSA solution for 1 hour, incubated overnight with primary antibody, washed three times with PBS, incubated overnight with fluorescent secondary antibody, unbound secondary antibody was washed away, DAPI solution containing anti-fluorescence quencher was added, and the results were observed and photographed using a fluorescence confocal microscope. Figure 5 As shown in B in the diagram.

[0062] Depend on Figure 5 A and Figure 5 As shown in C, under inflammatory stimulation with 10 ng / mL TNF-α, NV-MAs exhibited higher expression levels of repair-related genes compared to other groups, including significantly increased levels of pro-angiogenic factors and neurotrophic factors. Furthermore, NV-MAs displayed stronger anti-inflammatory and anti-apoptotic properties under inflammatory conditions, with a significant downregulation of pro-inflammatory gene expression. Western blot analysis revealed that NV-MAs significantly enhanced the phosphorylation levels of AKT and mTOR, further upregulating the expression of the anti-apoptotic protein Bcl-2 and inhibiting the expression of the pro-apoptotic protein Bax. Figure 5 The B-value in the study showed that NV-MAs can express a large number of anti-inflammatory and neurovascular repair factors. These results indicate that NV-MAs enhance their potential therapeutic efficacy in damage repair by activating the PI3K / AKT / mTOR signaling pathway, thereby improving their survival and functional output in the inflammatory microenvironment.

[0063] Application Example 5 The exosomes D-NV-MAs-Exo obtained in Example 1 were purified and subjected to quality control and characterization: "triple positive and one negative" biomarkers were detected; the exosome solution was diluted with ultrapure water to 10... 6 -10 9 After determining the concentration range ( / mL), the particle size was analyzed using a nanoparticle tracking analyzer (NTA), and the particle concentration was recorded. 10 μL of exosome solution resuspended in PBS was dropped onto a copper grid and allowed to stand at room temperature for 1-2 minutes. Excess liquid was absorbed along the outside of the copper grid with filter paper. 10 μL of 20 mg / mL phosphotungstic acid was added for negative staining for 1-2 minutes. After drying, the sample was observed and photographed under a transmission electron microscope (TEM). The results are as follows: Figure 6 As shown.

[0064] Depend on Figure 6 It can be seen that the D-NV-MAs-Exo of Example 1 has a clearly defined, nearly circular membrane vesicle structure, which is cup-shaped in shape, and its particle size is in the range of 30-150 nm for exosomes.

[0065] Application Example 6 Comparison of exosome production from four groups obtained in Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3: Particle concentration was determined by NTA and normalized to the initial cell number (particles / 10). ^6The yield differences of exosomes in four groups (D-NV-MAs-Exo from Example 1, D-MAs-Exo from Comparative Example 1, S-NV-MAs-Exo from Comparative Example 2, and S-MAs-Exo from Comparative Example 3) were quantitatively compared per unit cell to evaluate the effect of cadherin-enhanced combined with dynamic culture on improving the production efficiency of exosomes derived from mesenchymal stem cell aggregates and the advantages of the process, as shown in Table 3.

[0066] Table 3 Comparison of exosome yields using different preparation methods Table 3 shows that dynamic culture resulted in an average exosome production efficiency 1-2 times higher than static culture. In the S-MAs group, the production efficiency was significantly higher per 10... 6 One cell can yield 1.44 × 10⁶ exosomes. 9 One, while D-NV-MAs group has 10 6 Each cell can yield 5.68 × 10⁶ cells. 9 The production efficiency of exosomes increased by approximately four times. This indicates that cadherin-enhanced dynamic culture can significantly improve the production efficiency of exosomes derived from MSC aggregates.

[0067] Application Example 7 Identification and functional evaluation of exosome markers: 1. Extraction of exosome proteins: The purified exosomes D-NV-MAs-Exo from Example 1 were transferred to sterile centrifuge tubes, pre-cooled at -20°C for 12 hours, sealed with sealing film while retaining the vent, and placed in a pre-cooled lyophilizer at -80°C for 12 hours to remove moisture and enrich the sample. Pre-cooled RIPA lysis buffer was added to the lyophilized exosome powder, gently mixed, and lysed on ice for 15 minutes. The mixture was then centrifuged at 14,000g for 15 minutes at 4°C, and the supernatant was collected as the exosome protein. Protein concentration was determined using the BCA method, and the expression levels of exosome-related markers were detected by Western blotting. The results are as follows: Figure 7 As shown.

[0068] 2. Characterization of functional miRNAs: The four groups of exosomes obtained from Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were purified and resuspended in 200 μL of lysis buffer. After vortexing and incubation at room temperature for 5 min for complete lysis, an equal volume of binding buffer was added, and the mixture was gently mixed and transferred to an adsorption column. The column was centrifuged at 12000 g for 1 min to remove the filtrate. 500 μL of pre-chilled wash buffer (containing anhydrous ethanol) was added to the adsorption column, and the column was centrifuged at 12000 g for 1 min to discard the filtrate. The washing step was repeated once. Finally, the adsorption column was transferred to a new collection tube, and 30-50 μL of pre-warmed elution buffer (65°C) was added to the center of the column. After incubation at room temperature for 2 min, the column was centrifuged at 12000 g for 1 min to collect the eluent, which was the exosomal miRNA. The concentration and purity (A260 / A280 ratio) were detected using a micro spectrophotometer. The entire operation was performed under RNase-free conditions. RNase-free PCR tubes were used to remove gDNA and reverse transcribe it to obtain cDNA, as described in Application Example 2. The expression of specific miRNAs was detected by quantitative real-time PCR. The primer sequences of the relevant genes are shown in Table 4, and the results are shown in Table 5.

[0069] Table 4. Primer sequences for relevant genes: Table 5. Characterization of functional miRNAs in different exosomes Depend on Figure 7 It can be seen that, compared with the total cellular protein extracted by MSC in Application Example 2, the D-NV-MAs-Exo of Example 1 showed typical positive markers CD9, CD63 and CD81, and did not express the negative marker Calnexin, which meets the ISEV exosome marker identification criteria. Furthermore, as shown in Table 5, compared with other exosomes, the D-NV-MAs-Exo of Example 1 carried higher levels of neuroprotective and angiogenesis-related miRNAs (e.g. miR-21, miR-126-3p, miR-133b), indicating that cadherin-enhanced combined with dynamic culture can enhance the therapeutic potential of D-NV-MAs-Exo in neurovascular repair-related applications.

[0070] Application Example 8 Application of D-NV-MAs-Exo in the treatment of central nervous system diseases in Example 1: 1. Application of D-NV-MAs-Exo in the treatment of stroke 1.1. Establishment of a mouse model of ischemic stroke (PTS) and exosome implantation 10 mg of Rose Bengal reagent was dissolved in 1 mL of physiological saline, and 150 μL was injected intraperitoneally into each mouse 10 min before the operation. The hair on the mouse's head was removed, and the area was disinfected with an alcohol swab. Using straight forceps, the skin on the top of the mouse's head was gently pulled back, and a sagittal incision of approximately 0.8 cm was made along the right side of the midline of the head to expose the skull. A perforated black adhesive pad was placed on the exposed skull, and the skull was irradiated with a cold light source for 15 minutes. A mouse model of ischemic stroke (denoted as the stroke group) was successfully constructed. S-MAs-Exo (Comparative Example 3), S-NV-MAs-Exo (Comparative Example 2), EN-IGF1-3D-Exo (exosomes from patent CN119639663A), D-MAs-Exo (Comparative Example 1), and D-NV-MAs-Exo (Examination Example 1) were injected via the tail vein to obtain the Stroke+S-MAs-Exo group, Stroke+S-NV-MAs-Exo group, Stroke+EN-IGF1-3D-Exo group, Stroke+D-MAs-Exo group, and Stroke+D-NV-MAs-Exo group, respectively. Each of the five groups of mice was implanted with 30 μg of Exo. The sham group and the Stroke group were implanted with an equal volume of PBS.

[0071] 1.2 Behavioral evaluation of neurological function in mice Five groups of mice were treated with exosomes for 14 days. The degree of neurological damage and sensory and motor function were assessed using the modified neurological deficit score (mNSS, as shown in Table 6) and the adhesive-removal test. A higher mNSS score indicated more severe neurological impairment. The adhesive-removal test evaluated sensory and motor function in the forelimbs of the mice. A circular adhesive label with a diameter of 5 cm was attached to the relatively hairless palms of both hands of the mice. The mice were then returned to their cages, and the time it took for them to remove the label was recorded. If the label was not removed within 5 minutes, it was recorded as 5 minutes.

[0072] Table 6 mNSS Scoring Table

[0073] 2. D-NV-MAs-Exo promotes recovery in mice with ischemic stroke (Adhesive-removal test) To evaluate the therapeutic effect of D-NV-MAs-Exo on stroke mice, the mNSS score and paper-tear test were used to assess neurological function 14 days after treatment. The results are as follows: Figure 8As shown, compared with the stroke group, the Stroke+S-MAs-Exo group, the Stroke+S-NV-MAs-Exo group, the Stroke+EN-IGF1-3D-Exo group and the Stroke+D-MAs-Exo group, the mice in the Stroke+D-NV-MAs-Exo group showed greater improvement in mobility and nerve damage recovery.

[0074] Depend on Figure 8 As shown in A, on day 14 post-stroke, the Stroke+D-NV-MAs-Exo group had the lowest mNSS score compared to other groups; Figure 8 As indicated by B in the diagram, the paper-peeling experiment showed that D-NV-MAs-Exo transplantation significantly improved the sensitivity and motor function of the forefoot in a mouse model of ischemic stroke. These results demonstrate that D-NV-MAs-Exo treatment can effectively improve neurological deficits and motor-sensory function in PTS mice, promoting stroke recovery. Compared to EN-IGF1-3D-Exo in patent CN119639663A, D-NV-MAs-Exo exhibits stronger damage repair capabilities due to its neurovascular-specific cadherin-enhanced properties.

[0075] 3. Application of D-NV-MAs-Exo in the treatment of spinal cord injury in rats 3.1 Establishment of a spinal cord injury (SCI) model and exosome implantation Rats were anesthetized by isoflurane inhalation. The fur on their backs was cleaned, centered on the T10 segment, and the skin was incised approximately 1 cm along the midline of the spine. The dorsal muscles were bluntly dissected layer by layer to expose the T9–T11 vertebral structures. A laminectomy was performed on the T10 segment to expose the spinal cord. Subsequently, using the NYU Impactor-III device, a 10 g metal rod was dropped vertically from a height of 25 mm to deliver a momentary impact to the T10 segment, establishing a moderate spinal cord injury model. Immediately after the impact, the animals exhibited hind limb twitching and abnormal tail wagging, consistent with the behavioral characteristics of a successful model. After the injury was established, the incision was sutured in layers, and the rats were placed on a heated pad to recover. The sham-operated group only exposed the spinal cord without the impact treatment. After modeling, cefuroxime sodium was administered intramuscularly for 3 consecutive days to prevent infection, and bladder emptying was performed twice daily for the following 14 days. Two weeks after injury, 50 μg of PBS, S-MAs-Exo, S-NV-MAs-Exo, EN-IGF1-3D-Exo, D-MAs-Exo, and D-NV-MAs-Exo were injected in situ to obtain six models: SCI group, SCI+S-MAs-Exo group, SCI+S-NV-MAs-Exo group, SCI+EN-IGF1-3D-Exo group, SCI+D-MAs-Exo group, and SCI+D-NV-MAs-Exo group. The sham surgery group (Sham) served as a negative control, and the SCI group served as a positive control.

[0076] 3.2 BBB Motor Function Score The Basso-Beattie-Bresnahan (BBB) ​​scoring system was used to quantitatively analyze the hindlimb motor function of rats. BBB scores were assessed weekly for 8 weeks after modeling. The BBB score ranged from 0 to 21, where 0 represented complete absence of hindlimb movement, a score below 8 indicated only joint movement, 8–13 suggested some coordinated gait, 14–20 indicated relatively stable movement, and 21 reflected normal motor function. Before each assessment, rats were allowed free movement in an open space for 5 minutes, followed by a comprehensive evaluation of their motor performance over 3 minutes by three researchers using the scoring criteria.

[0077] 3.3 Grid walking experiment The grid crawling test was used to assess the fine motor control of the hind limbs in rats following spinal cord injury. Eight weeks after modeling (six weeks of exosome treatment), rats were placed in the center of a metal grid platform 50 cm high, 45 × 45 cm in area, and with 1.5 × 1.5 cm apertures. No prior training was required. After the animal firmly grasped the grid with all four limbs and released its tail, an observer recorded the total number of steps taken by both hind limbs within 120–150 steps and counted the number of times the hind limbs missed a step and fell. The hind limb error rate was calculated using the formula: Hind limb error rate (%) = Number of missed steps / Total number of hind limb steps × 100%.

[0078] 3.4 Hot plate pain response test The temperature of the hot plate was stabilized at 52 °C, and a transparent cylinder was placed above it to restrict the animals' movement. Eight weeks after stem cell transplantation, six groups of rat models were placed in the device sequentially, and timing began when both hind limbs were fully in contact with the hot plate. Avoidance behaviors such as jumping or licking the hind paws were identified as pain responses, and timing was immediately stopped and the latency of the response was recorded. Results are as follows: Figure 9 As shown.

[0079] 4. D-NV-MAs-Exo promotes recovery in rats with spinal cord injury. One day after successful SCI modeling, the BBB scores of all rats dropped to 0. Within 14 days, there was some recovery, but all scores remained below 8, with only joint movement observed and no weight-bearing on the feet. After exosome treatment, the scores of the treatment groups significantly improved, and the SCI+D-NV-MAs-Exo group recovered faster than the other treatment groups. After 3 weeks of treatment, the SCI+D-NV-MAs-Exo group was the first to reach 12 points, indicating that the rats in this group could perform relatively stable and accurate movements. After 8 weeks of treatment, the average score reached 15 points (e.g., ...). Figure 9 The scores (A) in the D-NV-MAs-Exo group significantly exceeded those of the SCI+S-MAs-Exo group (8-10 points), indicating that D-NV-MAs-Exo has superior therapeutic efficacy. Figure 9 As shown in B, in the grid crawling experiment, the SCI+D-NV-MAs-Exo group had a lower walking error rate compared to other groups, indicating that it had a stronger ability to control the placement of its hind paws, demonstrating good fine motor function and limb coordination; Figure 9 As indicated by C in the figure, the hot plate test results showed that after 8 weeks of exosome treatment, the SCI+D-NV-MAs-Exo group had a higher pain threshold, stronger pain tolerance, and longer reaction time to thermal stimulation compared to other treatment groups, indicating that the SCI+D-NV-MAs-Exo group had a lower degree of thermal hypersensitivity than other treatment groups. In summary, these results suggest that D-NV-MAs-Exo treatment can effectively improve neurological deficits and motor performance in SCI rats and promote spinal cord injury recovery.

[0080] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing stem cell-derived exosomes with material-empowered technology, characterized in that, Includes the following steps: Step 1: Mesenchymal stem cells are seeded onto a substrate coated with N / VE-cadherin-Fc fusion protein and cultured to form N / VE-cadherin-Fc matrix-empowered mesenchymal stem cells. Step 2: The N / VE-cadherin-Fc matrix-empowered mesenchymal stem cells obtained in Step 1 are assembled to obtain cadherin-empowered mesenchymal stem cell aggregates. Step 3: Apply staged shear stress to the cadherin-enhanced mesenchymal stem cell aggregates obtained in Step 2 and perform dynamic culture. Step 4: During the dynamic culture in Step 3, the culture medium is collected, and the material-empowered stem cell-derived exosomes are isolated and purified from the culture medium.

2. The preparation method according to claim 1, characterized in that, In step 1, the N / VE-cadherin-Fc fusion protein is human nerve cell cadherin. Fc fusion protein and human vascular endothelial cell cadherin A mixture of Fc fusion proteins.

3. The preparation method according to claim 2, characterized in that, The human nerve cell cadherin Fc fusion protein and human vascular endothelial cell cadherin The concentration ratio of Fc fusion protein is 1:3-3:1, preferably 1:

1.

4. The preparation method according to claim 1, characterized in that, In step 1, the mesenchymal stem cells are derived from umbilical cord, bone marrow, adipose tissue, or induced pluripotent stem cells.

5. The preparation method according to claim 1, characterized in that, In step 2, the cell assembly includes one or more of the following methods: ultra-low adhesion method, stirred culture method, microfluidic method, and scaffold culture method.

6. The preparation method according to claim 1, characterized in that, In step 3, the process of applying staged shear stress involves first applying low shear stress and then applying high shear stress. The low shear stress is 0.1-1 dyn / cm², and the dynamic culture time under low shear stress is 12-48 hours. The high shear stress is 1-3 dyn / cm², and the dynamic culture time under high shear stress is 24-96 hours.

7. The material-empowered stem cell-derived exosomes obtained by the preparation method according to any one of claims 1 to 6, characterized in that, The material enhances the content of miRNAs that promote nerve regeneration, angiogenesis, and / or anti-inflammation in stem cell-derived exosomes.

8. The material-empowered stem cell-derived exosome according to claim 7, characterized in that, The miRNAs include miR-21, miR-126-3p, and / or miR-133b.

9. The use of the material described in claim 7 or 8 to empower stem cell-derived exosomes in the preparation of drugs for treating central nervous system injury.

10. The application according to claim 9, characterized in that, The central nervous system injury includes neurovascular damage from stroke and spinal cord injury.

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