Efficient vascular endothelial cell-targeting vesicle system and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses a kind of high-efficiency vascular endothelial cell targeting vesicle system, which is prepared by hemangioma stem cell-derived exosome HemSCs-Exos and bone marrow mesenchymal stem cell-derived apoptotic body BMSCs-ABs, and the application further discloses a preparation method of the vesicle system and its application in promoting endothelial cell proliferation and migration. Unlike existing targeted modification, the ingenious targeting relationship between BMSCs-ABs and endothelial cells in the application belongs to the combination of non-special modification, which is simple and efficient. The fusion between double EVs has the characteristics of "1+1>2", which not only retains the therapeutic components of HemSCs-Exos and BMSCs-ABs, but also has a significant cell-specific distribution after fusion. The fusion method is simple and stable.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a high-efficiency vascular endothelial cell-targeting vesicle system and a preparation method and application thereof. BACKGROUND

[0002] Hemangioma stem cells (HemSCs) are a special type of CD133+ cells isolated from infantile hemangioma (IH). As early as 2008, HemSCs have been confirmed to have the potential to differentiate into endothelial cells, peripheral cells and adipocytes. HemSCs can regulate its own differentiation direction and promote the proliferation of endothelial cells through autocrine and paracrine factors. Recent studies have found that Exos extracted from HemSCs culture supernatant can significantly promote angiogenesis. Our research results show that HemSCs-derived Exos are superior to other types of stem cell-derived Exos in promoting endothelial cell proliferation. Based on this, we speculate that HemSCs-Exos can be used as a direct preferred biological functional material for microcirculation reconstruction. However, natural Exos lack specific targeting ability to tissues and cells, resulting in disordered cell distribution and potential treatment risks. Therefore, to maximize the efficacy of Exos, the problem of short biological in vivo circulation cycle and insufficient cell and tissue targeting distribution ability of the new Exos treatment system needs to be solved.

[0003] In a myocardial infarction rat model, mesenchymal stem cells-derived apoptotic body nanovesicles (MSC-ABs) can promote the regeneration of vascular endothelial cells and angiogenesis by increasing autophagy and activating the VEGF signaling pathway, thereby improving cardiac function. Apoptotic bodies (ABs) are extracellular vesicles with an intact outer membrane structure formed by plasma membrane blebbing and apoptotic membrane protrusions during the disintegration of apoptotic cells. Compared with Exos, which can only enter cells through pinocytosis and ordinary endocytosis, ABs themselves can release "Find-me" and "Eat-me" signals, and phagocytic cells actively migrate and phagocytose themselves. As a kind of non-professional phagocytic cells in vivo, vascular endothelial cells can receive "Find-me" and "Eat-me" signals, and phagocytose ABs. More interestingly, the expression of CX3CR1 on the surface of endothelial cells increases in a hypoxic environment, which can combine with CX3CL1 on the surface of ABs, further mediating the targeted recognition and endocytosis of endothelial cells to ABs. This cross-linking between ABs and endothelial cells provides us with a new idea for endothelial cell-specific targeting. In addition, ABs have many other advantages, such as high yield and strong stability.

[0004] Therefore, the present application uses a high-efficiency vascular endothelial cell targeting vesicle system prepared from HemSCs and bone marrow mesenchymal stem cells-derived apoptotic body nanovesicles (BMSC-ABs), which can achieve efficient targeting of vascular endothelial cells and promote their proliferation and migration. The chimeric vesicle avoids the cumbersome engineering modification of Exos, while enhancing the synergistic effect of revascularization. SUMMARY

[0005] The purpose of the present application is to provide a high-efficiency vascular endothelial cell targeting vesicle system, and to provide its preparation method and application, which is another purpose of the present application.

[0006] To achieve the above-mentioned purposes of the application, the following technical solutions are adopted:

[0007] A high-efficiency vascular endothelial cell targeting vesicle system, wherein the vesicle system is prepared from hemangioblast stem cell-derived exosomes HemSCs-Exos and bone marrow mesenchymal stem cell-derived apoptotic bodies BMSCs-ABs.

[0008] Preferably, the HemSCs-Exos has a particle size of 50-70nm, and expresses typical membrane surface proteins CD9 and TSG101, but does not express cytoplasmic protein Calnexin.

[0009] Preferably, the BMSCs-ABs has a particle size of 200-400nm, and expresses the marker apoptosis molecule caspase3.

[0010] The preparation method of the above high-efficiency vascular endothelial cell-targeting vesicle system comprises the following steps:

[0011] (1) Obtain and culture hemangioma stem cells HemSCs, and extract exosomes HemSCs-Exo from the hemangioma stem cells HemSCs;

[0012] (2) Obtain and culture bone marrow mesenchymal stem cells BMSCs, and induce the bone marrow mesenchymal stem cells BMSCs to produce apoptotic bodies BMSCs-ABs;

[0013] (3) Mix the hemangioma stem cell-derived exosomes HemSCs-Exos and the bone marrow mesenchymal stem cell-derived apoptotic bodies BMSCs-ABs, add polyethylene glycol, and perform extrusion treatment to obtain the high-efficiency vascular endothelial cell-targeting vesicle system.

[0014] Preferably, in step (3), the mixing weight ratio of HemSCs-Exos and BMSCs-ABs is 1:1, and the added amount of polyethylene glycol is 5% of the total weight of HemSCs-Exos and BMSCs-ABs.

[0015] Preferably, in step (3), after adding polyethylene glycol, first oscillate at 37℃ for 30 minutes, then perform ultrasonic treatment for 5 minutes, and then perform extrusion treatment for 10 times through a 100nm liposome extruder.

[0016] Preferably, the extraction step of HemSCs-Exos is as follows:

[0017] First, the hemangioma sample is prepared into a cell suspension by enzyme digestion method, screened, centrifuged, and then subjected to non-specific antigen blocking with FcR Blocking Reagent, and the single cell suspension is incubated with anti-CD133 antibody at room temperature for 30 min, and CD133+ cells, i.e., hemangioma stem cells HemSCs, are sorted out by MACS Separator; the HemSCs are placed in a 96-well plate and cultured at 37 DEG C in a 5% CO2 incubator for 4-6 days, then digested with 0.25% trypsin containing 0.02% EDTA and subjected to expansion culture; then the hemangioma stem cell HemSCs culture supernatant is collected, subjected to multiple centrifugations, and successively removed of living cells, dead cells and cell debris, and the supernatant is collected and centrifuged to obtain exosome precipitate.

[0018] Preferably, the centrifugal purification operation is that the supernatant is subjected to ultracentrifugation at 120,000g for 70 min to obtain exosome precipitate, and then the precipitate is suspended with pre-cooled PBS to remove contaminated proteins and other impurities, and then subjected to 120,000g centrifugation again for 70 min to obtain purified exosomes HemSCs-Exos.

[0019] Preferably, the extraction step of the BMSCs-ABs is as follows:

[0020] The cut rat tibia and femur epiphysis are flushed with a 1 mL syringe, and cultured and expanded in an alpha-MEM culture medium added with 10% FBS and 1% penicillin / streptomycin mixture;

[0021] The undifferentiated BMSCs after 3 generations of culture are inoculated in a 10 cm culture dish, and when the cells grow to 100% abundance, the BMSCs cells are induced and treated with 250 nM of staurosporine for 12 h.

[0022] After successful induction of BMSCs apoptosis, the cell supernatant is collected, centrifuged at 800g for 10 min, and then centrifuged at 16,000g for 30 min to obtain BMSCs-ABs.

[0023] The application further discloses application of the high-efficiency vascular endothelial cell targeted vesicle system in promoting endothelial cell proliferation and migration.

[0024] Compared with the prior art, the application has the following beneficial effects:

[0025] 1、The application utilizes HemSCs and BMSC-ABs to prepare an efficient endothelial cell-targeted hybrid exosome system eHEs (Endothelial cell-targeted hybrid exosomes), which not only has the effect of promoting angiogenesis, but also recruits endothelial cells by releasing "Find-me" and "Eat-me" signals, and can further mediate the target recognition and endocytosis of endothelial cells to eHEs via CX3CL1 / CX3CR1, so as to realize efficient targeting of endothelial cells and promote the proliferation and migration of the endothelial cells. The chimeric vesicle avoids the cumbersome engineering modification of Exos, and at the same time enhances the synergistic effect of revascularization.

[0026] 2、Compared with the specific target membrane surface presentation realized by molecular connection or genetic engineering modification on the surface of the vesicle, the application realizes cross-chimerization between different vesicles through a simple membrane fusion strategy, which can not only realize the enrichment of specific targets on the surface of the vesicle, but also retain the active molecules in the vesicle to a certain extent.

[0027] 3、Unlike existing targeted modification, the ingenious targeted relationship between BMSCs-ABs and endothelial cells in the application belongs to the combination of non-specific modification, which is simple and efficient; the fusion between double EVs has the characteristic of "1+1>2", which not only retains the therapeutic components of HemSCs-Exos and BMSCs-ABs, but also the fused eHEs have a significant cell-specific distribution, and the fusion method is simple and stable. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 A, HUVECs scratch representative graph, B, HUVECs proliferation statistical graph;

[0029] Figure 2 A, fluorescence resonance energy transfer (FRET) detection, B, immunofluorescence (IF) detection of co-labeling;

[0030] Figure 3 A-B, HUVECs scratch and statistical results, C-D, immunofluorescence (IF) detection of HUVCEs phagocytosis and statistical results;

[0031] Figure 4 A-B, FITC-AnnexinV labeled ABs and fluorescence intensity statistics, C, WB detection of Caspase3 expression in ABs, D-E, ABs transmission electron microscopy observation and particle size statistics;

[0032] Figure 5 A-B, HemSCs-Exos transmission electron microscopy observation and particle size detection, C, WB detection of HemSCs-Exos surface results. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative effort fall within the scope of the present application. The experimental methods in the embodiments are conventional methods, and the materials, reagents and the like in the embodiments are commercially available unless otherwise specified.

[0034] Example 1e Construction, characterization analysis and in vitro cell function test of eHEs

[0035] The construction method of eHEs includes the following steps:

[0036] 1) In vitro acquisition, culture and expansion of HemSCs

[0037] The hemangioma sample is prepared into a cell suspension by enzyme digestion, screened, centrifuged, and then subjected to non-specific antigen blocking with FcR Blocking Reagent. Then the single cell suspension is incubated with anti-CD133 antibody at room temperature for 30 min, and CD133+ cells are sorted out by MACS Separator, which are hemangioma stem cells HemSCs. HemSCs are placed in a 96-well plate and cultured in a 37°C, 5% CO2(v / v) incubator. After 4-6 days of culture, 0.25% trypsin containing 0.02% EDTA is used for digestion, and subsequent expansion culture is carried out. 0.02% EDTA (ethylenediaminetetraacetic acid) refers to the mass percentage, i.e. 0.02 grams of EDTA per 100 grams of solution. 0.25% trypsin is the mass percentage, which means that there are 0.25 grams of trypsin per 100 grams of solution.

[0038] 2) Identification of HemSCs

[0039] The HemSCs after 3 generations of culture are identified by flow cytometry. HemSCs are reselected with flow staining buffer, and the cell suspension concentration is adjusted to 3.0x10 6 Cells / ml. Non-specific antigen blocking is performed with FcR Blocking Reagent, and fluorescein-labeled anti-CD29, CD44, CD90, CD105, CD31, CD34, CD45 and CD144 antibodies are incubated with HemSCs at room temperature for 30 min, washed with staining buffer for 3 times, and the cells are resuspended and detected using a flow cytometer.

[0040] CD105 (also known as Endoglin), CD90, CD29 (integrin beta 1), CD133 (stem cell marker), CD44, CD144 cell surface antigens are positive in HemSCs, while hematopoietic antigens such as CD45, CD34, CD14, etc. are negative.

[0041] 3) HemSCs induced differentiation study

[0042] a. HemSCs osteogenic induced differentiation study

[0043] HemSCs after 3 generations of culture were inoculated in 6-well plates, when the cell abundance grew to 80%, mesenchymal stem cell osteogenic induction medium was added to the well plate, and the medium was changed every 3 days. After 2-4 weeks of induction, the cell morphological changes and growth were observed, and finally alizarin red staining was used to analyze the osteogenic induction of HemSCs; the extracellular matrix showed dark red or red-brown patches (mineralized nodules), irregular in shape, mostly distributed in cell cluster areas. Uninduced HemSCs showed only very weak or no red staining.

[0044] b. HemSCs adipogenic induced differentiation study

[0045] HemSCs after 3 generations of culture were inoculated in 6-well plates, when the cell abundance grew to 90%-100%, mesenchymal stem cell adipogenic induction medium A was added to the well plate, and after 3 days of induction, B was replaced. After 1 day of B induction, continue to replace A for 3 days of induction. After 4 cycles of such induction, continue to maintain culture with B for 5-7 days until the lipid droplets become large and round enough, i.e. when there are 30% lipid droplets in the entire field of view, the induction is ended, and then oil red O staining is performed to analyze the adipogenic induction of HemSCs; intracellular bright red or orange-red round lipid droplets appear, with varying sizes and can fuse into larger vacuoles. Lipid droplets are mainly located in the cytoplasm, and the nucleus is often stained blue with hematoxylin, forming a red-blue contrast. Uninduced HemSCs have no obvious red lipid droplets, only showing a uniform light red background.

[0046] Medium A composition: add 10% fetal bovine serum, penicillin (100 U / ml) and streptomycin (100 μg / ml) to the α-MEM basal medium.

[0047] Medium B composition: add final concentration of 0.1 μM dexamethasone, 0.45 mM IBMX, 1 μg / ml insulin and 0.2 mM indomethacin to 100 ml of growth medium. Optionally, 1 μM rosiglitazone can be added to accelerate the differentiation process.

[0048] 4) Extraction and characterization analysis of HemSCs-Exo

[0049] Exos were isolated using ultracentrifugation method. The culture supernatant of HemSCs was collected and centrifuged several times to remove live cells, dead cells, cell debris, and then the supernatant was collected and centrifuged (120,000g, 70min) to obtain the exosome precipitate. The precipitate was suspended with pre-cooled PBS to remove the contaminated proteins and other impurities, and then another round of centrifugation (120,000g, 70min) was performed to collect the purified exosomes.

[0050] The particle size of HemSCs-Exo was analyzed by NTA, and the morphology of exosomes was observed by TEM. The specific surface markers CD9 and TSG101 of exosomes were identified by immunoblotting, and the negative marker Calnexin was detected to determine the purity of HemSCs-Exo. The results are shown in Figure 5

[0051] 5) Isolation and identification of BMSCs

[0052] Rat BMSCs were derived from rat tibia and femur, and the cut end of rat tibia and femur was flushed with a 1 mL syringe, and then cultured and expanded in α-MEM complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin mixture (i.e. 10 mL FBS and 1 mL penicillin / streptomycin mixture were added per 100 mL of culture medium). The 3rd passage of BMSCs was used for subsequent flow identification and cell experiments.

[0053] 6) Identification of BMSCs

[0054] The 3rd passage of BMSCs was identified by flow cytometry. The BMSCs were reselected with flow staining buffer, and the cell suspension concentration was adjusted to 3.0x10 6 Cell / ml. Non-specific antigen blocking was performed with FcR Blocking Reagent, and fluorescein-labeled anti-CD29, CD44, CD73, CD34, CD271 and CD45 antibodies were incubated with BMSCs at room temperature for 30 min, washed with staining buffer for 3 times, and the cells were resuspended and detected by flow cytometry.

[0055] Detection results:

[0056] Positive: CD29, CD44, CD73, CD271

[0057] Negative: CD34, CD45

[0058] 7) BMSCs differentiation induction study

[0059] ​a. Study on osteogenic induction differentiation of BMSCs

[0060] After 3 generations of culture, BMSCs were inoculated in 6-well plates. When the cell abundance grew to 80%, mesenchymal stem cell osteogenic induction medium was added to the well plate, and the medium was changed every 3 days. After 2-4 weeks of induction, the cell morphological changes and growth were observed, and finally alizarin red staining was used to analyze the osteogenic induction differentiation of BMSCs; dark red or red-brown patches (mineralized nodules) in the extracellular matrix, irregular in shape, were mainly distributed in the cell cluster area. Uninduced BMSCs showed only very weak or no red staining.

[0061] b. Study on adipogenic induction differentiation of BMSCs

[0062] After 3 generations of culture, BMSCs were inoculated in 6-well plates. When the cell abundance grew to 80%, mesenchymal stem cell osteogenic induction medium was added to the well plate, and the medium was changed every 3 days. After 2-4 weeks of induction, the cell morphological changes and growth were observed, and finally alizarin red staining was used to analyze the osteogenic induction differentiation of BMSCs; dark red or red-brown patches (mineralized nodules) in the extracellular matrix, irregular in shape, were mainly distributed in the cell cluster area. Uninduced BMSCs showed only very weak or no red staining.

[0063] In this operation, the composition of medium A and B is the same as above.

[0064] 8) Induction, extraction and characterization identification of BMSCs-ABs

[0065] a. Induction of BMSCs-ABs:

[0066] After 3 generations of culture, undifferentiated BMSCs were inoculated in 10 cm culture dishes. When the cell abundance grew to 100%, the BMSCs cells were treated with 250 nM of staurosporine for 12 h, and the degree of apoptosis of BMSCs was detected by TUNEL staining and flow cytometry.

[0067] b. Extraction and identification of BMSCs-ABs

[0068] After successful induction of BMSCs apoptosis, collect cell supernatant, centrifuge at 800g for 10min, 16,000g for 30min to obtain BMSCs-ABs. BCA kit was used to determine the protein concentration of BMSCs-ABs. DLS was used to detect the particle size and potential distribution of BMSCs-ABs. TEM was used to observe the morphological characteristics of BMSCs-ABs. Western blot was used to detect the expression of caspase3 protein in BMSCs-ABs. At the same time, Annexin-V-FITC was used to label BMSCs-ABs, and the fluorescence distribution of BMSCs-ABs was observed by confocal microscope. The fluorescence intensity of FITC was analyzed by nano flow meter. The results are shown in Figure 4 .

[0069] 9) Preparation and characterization of eHEs

[0070] HemSCs-Exos and BMSCs-ABs were mixed in equal mass ratio, and 5% polyethylene glycol (PEG) was added, and oscillated at 37°C for 30min. Then ultrasonic treatment for 5min, extruded 10 times by 100nm liposome extruder. DLS was used to detect the size and Zeta potential of eHEs. TEM was used to observe the morphology of eHEs.

[0071] 10) Detection of membrane fusion

[0072] Firstly, fluorescence resonance energy transfer (FRET) was used to detect membrane fusion, and the fluorescence intensity within 520 to 620nm was collected by multifunctional enzyme label instrument. To further confirm the fusion of HemSCs-Exos and BMSCs-ABs, laser confocal microscope was used to observe the fluorescence co-labeling of DiO and DiD. The results are shown in Figure 5 .

[0073] 11) Protein enrichment analysis of eHEs

[0074] HemSCs-Exos, BMSCs-ABs and eHEs were lysed by lysis solution, centrifuged, and the supernatant was taken. BCA kit was used to measure the protein concentration of each group, and Western blot was used to detect the enrichment of total protein in each group.

[0075] 12) Western blot detection of CX3CL1 / CX3CR1 expression

[0076] The hypoxic microenvironment can increase the expression of CX3CR1 on the surface of ECs, thereby promoting the binding with ABs expressing CX3CL1. The levels of CX3CR1 and CX3CL1 were detected by Western blot.

[0077] 13) HUVECs phagocytosis of eHEs

[0078] HemSCs-Exos (20 pg / mL), BMSCs-ABs (20 pg / mL) and eHEs (20 pg / mL) were labeled with DiO and co-cultured with HUVECs for 12 h. Phosphate-buffered saline (PBS) was used to wash the cells three times, and the cells were fixed with 4% PFA for 30 min. The cells were washed with PBS again three times and counterstained with DAPI. Representative images were taken using a confocal microscope, and the fluorescence intensity was analyzed using a flow cytometer.

[0079] 14) Effect of eHEs on endothelial cell proliferation

[0080] HUVECs were seeded in a 6-well plate, and a straight line was drawn from top to bottom along the center of the 6-well plate using a flat pipette tip. The distribution of endothelial cells at the scratch site was recorded at 0 h, 12 h, 24 h and 36 h by optical microscopy.

[0081] 15) Effect of eHEs on the migration ability of HUVECs

[0082] HUVECs were seeded in the upper layer of a Transwell chamber, and HemSCs-Exos, BMSCs-ABs and eHEs or pure culture medium were added to the lower chamber. The cells were then incubated in an incubator for 24 h. Then the chamber was removed, the culture medium was removed, and the cells were washed with PBS three times and fixed with formaldehyde for 20 min. The cells were stained with 0.1% crystal violet for 20 min, and then the bottom surface of the chamber was gently washed with PBS, and the distribution of HUVECs in the lower layer of the chamber was observed under an optical microscope.

[0083] The experimental results are shown in Figures 1-5 .

[0084] HemSCs-Exos had a more significant effect on promoting endothelial cell proliferation than BMSC-Exos. Through the endothelial cell scratch experiment, we compared the effect of HemSCs-Exos on promoting vascular endothelial cell proliferation compared to other stem cell-derived Exos under the same exosome dose (20 pg / mL) stimulation, as shown in Figure 1 .

[0085] HemSCs-Exos and BMSC-ABs can fuse with each other after ultrasound and extrusion. We confirmed the fusion between the two vesicles by fluorescence resonance energy transfer (FRET) and immunofluorescence, respectively, as shown in Figure 2 .

[0086] The eHEs after fusion exhibit enhanced endothelial cell targeting and pro-endothelial cell proliferation effects. We observed by immunofluorescence experiments that eHEs have more distribution in endothelial cells than HemSCs-Exos in the same time, and eHEs exert the optimal pro-endothelial cell proliferation effect, as shown in Figure 3 . Figure 3 , A-B, HUVECs scratch and statistical results, detection shows that compared with PBS, HemSCs-Exos, eHEs significantly promote HUVECs proliferation and migration; C-D, immunofluorescence (IF) detection of HUVCEs phagocytosis and statistical results. To study the phagocytosis of HUVECs to eHEs in vitro, HemSCs-Exos (20 μg / mL) and eHEs (20 μg / mL) were first labeled with DiO. Excess fluorescent dye was removed by Amicon Ultra-0.5 ultrafiltration centrifugal tube, and the sample was washed twice with PBS. To verify that the unbound DiO was removed, the supernatant of the stained eHEs was used as a negative control (NC), and then the above two vesicles were co-cultured with HUVECs for 12 h. PBS was washed three times, and fixed with 4% PFA for 30 min. Again, PBS was washed three times, and DAPI was used for cell nucleus re-staining. Representative images were taken using a confocal microscope, and fluorescence intensity was analyzed using a flow cytometer. The results show that HUVCEs significantly phagocytose eHEs.

[0087] BMSCs induction was observed by fluorescence detection and WB detection, respectively, and the extracted ABs expressed the typical apoptosis molecule caspase 3. Transmission electron microscopy observation and particle size detection of ABs were also performed, and it was found that they had typical vesicular structures, and the particle size was mainly distributed between 200-400 nm, as shown in Figure 4 .

[0088] Transmission electron microscopy observation and particle size detection of HemSCs-Exos were performed, and it was found that they had typical vesicular structures, and the particle size was mainly distributed between 50-70 nm. HemSCs-Exos were also detected to express typical membrane surface proteins CD9 and TSG101, and not to express cytoplasmic protein Calnexin, as shown in Figure 5The FRET signal was detected by a multifunctional microplate reader collecting the fluorescence intensity in the wavelength range of 520 to 620 nm. The detection of FRET signal indicated that the distance between the fluorescent molecules of DiO and DiD was close enough, suggesting that the membrane structures of HemSCs-Exos and BMSCs-ABs were fused. DiO and DiD labeled the membrane structures of HemSCs-Exos and BMSCs-ABs, respectively. Under the confocal microscope, if green and red fluorescence were observed overlapping at the same location (i.e., yellow fluorescence), it indicated that the two membrane structures were fused.

Claims

1. A highly efficient vascular endothelial cell targeting vesicular system, characterized in that, The vesicle system is prepared by hemangioma stem cell-derived exosomes HemSCs-Exos and bone marrow mesenchymal stem cell-derived apoptotic bodies BMSCs-ABs; specifically, HemSCs-Exos and BMSCs-ABs are mixed in an equal mass ratio, 5% polyethylene glycol is added, oscillation is carried out at 37°C for 30 min, then ultrasonic treatment is carried out for 5 min, and extrusion is carried out 10 times through a 100 nm liposome extruder.

2. The highly efficient vascular endothelial cell targeting vesicular system according to claim 1, wherein, The particle size of HemSCs-Exos is 50-70 nm, and HemSCs-Exos expresses typical membrane surface proteins CD9 and TSG101, and does not express cytoplasmic protein Calnexin.

3. The highly efficient vascular endothelial cell targeting vesicular system according to claim 1, wherein, The particle size of BMSCs-ABs is 200-400 nm, and BMSCs-ABs express the marker apoptosis molecule caspase 3.

4. The method of producing a highly efficient vascular endothelial cell targeting vesicular system according to claim 1, wherein The method comprises the following steps: (1) obtaining and culturing hemangioma stem cells HemSCs, and extracting exosomes HemSCs-Exos from the hemangioma stem cells HemSCs; (2) obtaining and culturing bone marrow mesenchymal stem cells BMSCs, and inducing the bone marrow mesenchymal stem cells BMSCs to produce apoptotic bodies BMSCs-ABs; (3) mixing hemangioma stem cell-derived exosomes HemSCs-Exos and bone marrow mesenchymal stem cell-derived apoptotic bodies BMSCs-ABs in an equal mass ratio, adding 5% polyethylene glycol, oscillating at 37°C for 30 min, then ultrasonic treatment for 5 min, and extruding 10 times through a 100 nm liposome extruder, thereby obtaining the vesicle system.

5. The method for preparing the highly efficient vascular endothelial cell-targeting vesicle system according to claim 4, characterized in that, The extraction step of HemSCs-Exos is as follows: First, the hemangioma sample is prepared into a cell suspension by enzyme digestion, screened, centrifuged, and then subjected to non-specific antigen blocking with FcR Blocking Reagent, and the single-cell suspension is incubated at room temperature for 30 min with an anti-CD133 antibody, and CD133+ cells are sorted out by MACS Separator, which are hemangioma stem cells HemSCs; the HemSCs are placed in a 96-well plate and cultured in a 37°C, 5% CO2 incubator for 4-6 days, then digested with 0.25% trypsin containing 0.02% EDTA, and expanded and cultured; then the hemangioma stem cell HemSCs culture supernatant is collected, subjected to multiple centrifugations, and sequentially removes live cells, dead cells, and cell debris, collects the supernatant, and centrifugally purifies to obtain exosome precipitate.

6. The method for preparing the highly efficient vascular endothelial cell-targeting vesicle system according to claim 5, characterized in that, The centrifugal purification operation is as follows: first, the supernatant is ultracentrifuged at 120,000 g for 70 min to obtain exosome precipitate, then the precipitate is suspended with pre-cooled PBS to remove contaminated proteins and other impurities, and then the purified exosomes HemSCs-Exos are obtained by centrifugation at 120,000 g for 70 min.

7. The method for preparing the highly efficient vascular endothelial cell-targeting vesicle system according to claim 4, characterized in that, The extraction step of BMSCs-ABs is as follows: The cut-opened epiphysis of rat tibia and femur is rinsed with a 1 mL syringe, and cultured and expanded in an α-MEM culture medium added with 10% FBS and 1% penicillin / streptomycin mixture; After 3 generations of culture, undifferentiated BMSCs were inoculated in 10 cm culture dishes. When the cells grew to 100% abundance, 250 nM of star-shaped spore bacteria was used to induce BMSCs for 12 h; After successful induction of BMSCs apoptosis, the cell supernatant was collected, centrifuged at 800 g for 10 min, and then centrifuged at 16,000 g for 30 min to obtain BMSCs-ABs.

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