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

Abstract

The invention belongs to the technical field of biological medicine, and discloses a high-efficiency vascular endothelial cell targeting vesicle system, which is prepared from hemangioma stem cell derived exosome HemSCs-Exos and bone marrow mesenchymal stem cell derived apoptotic small body BMSCs-ABs, and is characterized in that the endothelial cell targeting vesicle system is prepared from the hemangioma stem cell derived exosome HemSCs-Exos and the bone marrow mesenchymal stem cell derived apoptotic small body BMSCs-ABs; meanwhile, the invention further discloses a preparation method of the vesicle system and application of the vesicle system in promoting proliferation and migration of endothelial cells. Different from existing targeting modification, the ingenious targeting relationship between the BMSCs-ABs and the endothelial cells belongs to non-special modification combination, and is simple and efficient; the fusion between the double EVs has the characteristic of realizing '1 + 1gt, 2', not only retains the treatment components of the HemSCs-Exos and the BMSCs-ABs, but also the fused eHEs has remarkable cell specificity distribution, and the fusion mode is simple and stable.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a high-efficiency vascular endothelial cell-targeted vesicle system and a preparation method and application thereof. Background Art

[0002] Hemangioma stem cells (HemSCs) are a specialized class of CD133+ cells isolated from infantile strawberry hemangiomas (IH). As early as 2008, HemSCs were demonstrated to have the potential to differentiate into endothelial cells, peripheral blood cells, and adipocytes. HemSCs can regulate their differentiation direction and promote endothelial cell proliferation through autocrine and paracrine factors. Recent studies have shown that exosomes (Exos) extracted from HemSC culture supernatant can significantly promote angiogenesis. Our results demonstrate that HemSC-derived Exos are superior to Exos derived from other stem cell types in promoting endothelial cell proliferation. Based on this, we hypothesize that HemSC-Exos could serve as a direct and optimal biomaterial for microcirculatory remodeling. However, natural Exos lack specific targeting to tissues and cells, resulting in disordered cell distribution and potential therapeutic risks. Therefore, the development of novel Exos therapeutic systems must address their short in vivo circulation cycle and limited cell and tissue targeting to maximize the therapeutic efficacy of Exos.

[0003] In a rat model of myocardial infarction, mesenchymal stem cell-derived apoptotic body nanovesicles (MSC-ABs) promoted endothelial cell regeneration and angiogenesis by increasing autophagy and activating the VEGF signaling pathway, thereby improving cardiac function. Apoptotic bodies (ABs) are extracellular vesicles with intact outer membrane structures that form during apoptotic cell disintegration through plasma membrane blebbing and protrusions. Compared to Exos, which can only enter cells through pinocytosis and standard endocytosis, ABs can release "Find-me" and "Eat-me" signals to attract phagocytes to migrate and phagocytose themselves. As non-professional phagocytes in the body, endothelial cells can receive "Find-me" and "Eat-me" signals to attract and phagocytose ABs. Interestingly, hypoxic conditions increase the expression of CX3CR1 on the surface of endothelial cells, which can bind to CX3CL1 on the surface of ABs, further mediating the targeted recognition and internalization of ABs by endothelial cells. This cross-linking between ABs and endothelial cells provides new insights into endothelial cell-specific targeting. ABs also offer numerous advantages, such as high yield and robust stability.

[0004] Therefore, the present invention utilizes a highly efficient endothelial cell-targeting vesicle system prepared from HemSCs and bone marrow mesenchymal stem cell-derived apoptotic body nanovesicles (BMSC-ABs), which can effectively target endothelial cells and promote their proliferation and migration. This chimeric vesicle avoids the cumbersome engineering modification of Exos while enhancing the synergistic revascularization effect of each other. Summary of the Invention

[0005] The purpose of the present invention is to provide a highly efficient vascular endothelial cell-targeted vesicle system, and providing a preparation method and application thereof is another purpose of the present invention.

[0006] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0007] A highly efficient vascular endothelial cell-targeted vesicle system is prepared by exosomes (HemSCs-Exos) derived from hemangioma stem cells and apoptotic bodies (BMSCs-ABs) derived from bone marrow mesenchymal stem cells.

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

[0009] Preferably, the BMSCs-ABs have a particle size of 200-400 nm and express the hallmark apoptosis molecule caspase 3.

[0010] The method for preparing the above-mentioned 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) Obtaining and culturing bone marrow mesenchymal stem cells (BMSCs) and inducing the production of apoptotic bodies (BMSCs-ABs) from BMSCs;

[0013] (3) Exosomes derived from hemangioma stem cells (HemSCs-Exos) and apoptotic bodies (BMSCs-ABs) derived from bone marrow mesenchymal stem cells (BMSCs-ABs) are mixed, polyethylene glycol is added, and the mixture is extruded.

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

[0015] Preferably, in step (3), after adding polyethylene glycol, the mixture is shaken at 37° C. for 30 minutes, then ultrasonicated for 5 minutes, and then extruded 10 times through a 100 nm liposome extruder.

[0016] Preferably, the extraction steps of HemSCs-Exos are:

[0017] Hemangioma specimens were first prepared into a cell suspension by enzymatic digestion, sieved, and centrifuged before nonspecific antigen blocking with FcR Blocking Reagent. The single-cell suspension was incubated with anti-CD133 antibody at room temperature for 30 minutes, and CD133+ cells, namely hemangioma stem cells (HemSCs), were sorted using a MACS separator. HemSCs were placed in a 96-well plate and cultured in a 37°C, 5% CO2 incubator for 4-6 days. They were then digested with 0.25% trypsin containing 0.02% EDTA and expanded. The culture supernatant of the HemSCs was then collected and centrifuged multiple times to remove live cells, dead cells, and cell debris. The supernatant was collected and centrifuged to purify the exosome precipitate.

[0018] Preferably, the centrifugation purification operation is as follows: first, the supernatant is ultracentrifuged at 120,000 g for 70 min to obtain an exosome precipitate, and then the precipitate is suspended in pre-cooled PBS to remove contaminating proteins and other impurities, and then centrifuged again at 120,000 g for 70 min to obtain purified exosomes HemSCs-Exos.

[0019] Preferably, the extraction steps of BMSCs-ABs are:

[0020] The rat tibia and femur with the epiphyseal ends cut open were flushed with a 1 mL syringe and cultured and expanded in α-MEM medium supplemented with 10% FBS and 1% penicillin / streptomycin mixture;

[0021] Undifferentiated BMSCs after culturing for 3 generations were seeded in 10 cm culture dishes. When the cells grew to 100% abundance, the BMSCs were induced with 250 nM staurosporine for 12 h.

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

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

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

[0025] 1. The present invention utilizes HemSCs and BMSC-ABs to prepare a highly efficient endothelial cell-targeted vesicle system, eHEs (Endothelial cell-targeted hybrid exosomes), which not only promote angiogenesis but also recruit endothelial cells by releasing "Find-me" and "Eat-me" signals. Furthermore, through CX3CL1 / CX3CR1, it can further mediate the targeted recognition and endocytosis of eHEs by endothelial cells, thereby achieving efficient targeting of endothelial cells and promoting their proliferation and migration. This chimeric vesicle avoids the tedious engineering modification of Exos while enhancing the synergistic effect of each other in revascularization.

[0026] 2. Compared with the membrane surface presentation of specific targets through molecular connection or genetic engineering on the vesicle surface, the present invention uses a simple membrane fusion strategy to cross-chimerize different vesicles, which not only can achieve the enrichment of specific targets on the vesicle surface, but also retain the active molecules in the vesicles to a certain extent.

[0027] 3. Unlike existing targeted modifications, the ingenious targeting relationship between BMSCs-ABs and endothelial cells in the present invention is a non-specially modified combination, which is simple and efficient. The fusion between the two EVs has the characteristic of achieving "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 THE DRAWINGS

[0028] Figure 1 A, Representative image of HUVECs scratch wound, B, Statistical graph of HUVECs proliferation;

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

[0030] Figure 3 AB, HUVECs scratch and statistical results, CD, immunofluorescence (IF) detection of HUVECs phagocytosis and statistical results;

[0031] Figure 4 AB and FITC-Annexin V were used to label ABs and calculate the fluorescence intensity. C and WB were used to detect the expression of Caspase3 in ABs. DE and transmission electron microscopy were used to observe ABs and calculate the particle size.

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

[0033] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of this application. The experimental methods in the embodiments are conventional methods unless otherwise specified, and the materials, reagents, etc. in the embodiments are all commercially available unless otherwise specified.

[0034] Example 1 Construction, characterization analysis and in vitro cell function testing 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 specimen was prepared into a cell suspension by enzymatic digestion, sieved, centrifuged and then non-specific antigen blocked with FcR Blocking Reagent. The single cell suspension was then incubated with anti-CD133 antibody at room temperature for 30 minutes, and CD133+ cells, which were hemangioma stem cells HemSCs, were sorted out by MACS Separator. HemSCs were placed in a 96-well plate and cultured in an incubator at 37°C and 5% CO2 (volume percentage). After culturing for 4-6 days, 0.25% trypsin containing 0.02% EDTA was used for digestion, and subsequent amplification culture was performed. Among them, 0.02% EDTA (ethylenediaminetetraacetic acid) refers to the mass percentage, that is, 0.02 grams of EDTA are contained in every 100 grams of solution. 0.25% trypsin is a mass percentage, which means that 0.25 grams of trypsin are contained in every 100 grams of solution.

[0038] 2) Identification of HemSCs

[0039] HemSCs were cultured for 3 generations and identified by flow cytometry. HemSCs were reselected with flow cytometry staining buffer and the cell suspension concentration was adjusted to 3.0×10 6 Nonspecific antigens were blocked using FcR Blocking Reagent. HemSCs were incubated with fluorescein-labeled anti-CD29, CD44, CD90, CD105, CD31, CD34, CD45, and CD144 antibodies at room temperature for 30 minutes. The cells were washed three times with staining buffer and resuspended for analysis using flow cytometry.

[0040] CD105 (also known as endoglin), CD90, CD29 (integrin β1), CD133 (stem cell marker), CD44, and CD144 cell surface antigens are positive in hemangioma stem cells (HemSCs), while hematopoietic antigens such as CD45, CD34, and CD14 are negative.

[0041] 3) HemSCs differentiation study

[0042] a. Study on osteogenic differentiation of HemSCs

[0043] HemSCs after three generations of culture were seeded in 6-well plates. When the cell abundance grew to 80%, mesenchymal stem cell osteogenic differentiation 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 induction of HemSCs into bone; dark red or reddish-brown plaques (mineralized nodules) appeared in the extracellular matrix, with irregular morphology and mostly distributed in the cell cluster area. Uninduced HemSCs showed only very weak or no red staining.

[0044] b. Study on adipogenic differentiation of HemSCs

[0045] HemSCs after passage 3 were seeded in 6-well plates. When cell confluence reached 90%-100%, medium A, a mesenchymal stem cell adipogenic differentiation medium, was added to the plates and replaced with medium B after three days of induction. After one day of induction with medium B, medium A was switched back to for another three days. After four cycles of induction, the cells were cultured in medium B for 5-7 days, until lipid droplets became sufficiently large and round (i.e., 30% of lipid droplets were present in the entire field of view). Induction was then terminated by Oil Red O staining to confirm adipogenic differentiation of HemSCs. Bright red or orange-red, round lipid droplets of varying sizes appeared within the cells and could fuse into larger vacuoles. Lipid droplets were primarily located in the cytoplasm, and nuclei were often counterstained blue with hematoxylin, creating a red-blue contrast. Uninduced HemSCs lacked prominent red lipid droplets and displayed a uniform, pale red background.

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

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

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

[0049] Exos were isolated using ultracentrifugation. The supernatant of HemSCs culture was collected from a large number of cultures and centrifuged multiple times to remove live cells, dead cells, and cell debris. The supernatant was then collected and centrifuged at 120,000 g for 70 minutes to obtain the exosome precipitate. The precipitate was gently suspended in pre-chilled PBS to remove contaminating proteins and other impurities, followed by another round of centrifugation at 120,000 g for 70 minutes 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. Figure 5 shown.

[0051] 5) Isolation and identification of BMSCs

[0052] Rat BMSCs were derived from rat tibia and femur. The epiphyseal ends of rat tibia and femur were flushed with a 1 mL syringe and 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 to every 100 mL of culture medium). BMSCs after three generations were used for subsequent flow cytometry identification and cell experiments.

[0053] 6) Identification of BMSCs

[0054] BMSCs were cultured for 3 generations and identified by flow cytometry. BMSCs were reselected with flow cytometry staining buffer and the cell suspension concentration was adjusted to 3.0×10 6 Nonspecific antigens were blocked using FcR Blocking Reagent. BMSCs were incubated with fluorescein-labeled anti-CD29, CD44, CD73, CD34, CD271, and CD45 antibodies for 30 min at room temperature. The cells were washed three times with staining buffer and resuspended for analysis using flow cytometry.

[0055] Test results:

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

[0057] Negative: CD34, CD45

[0058] 7) BMSCs differentiation study

[0059] a. Study on osteogenic differentiation of BMSCs

[0060] BMSCs after three generations of culture were seeded in 6-well plates. When the cell abundance grew to 80%, mesenchymal stem cell osteogenic differentiation 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. Finally, Alizarin red staining was used to analyze the induction of BMSCs into bone; dark red or reddish-brown plaques (mineralized nodules) appeared in the extracellular matrix with irregular morphology and were mostly distributed in the cell cluster area. Uninduced BMSCs showed only very weak or no red staining.

[0061] b. Study on adipogenic differentiation of BMSCs

[0062] BMSCs after three passages of culture are seeded in 6-well plates. When the cell abundance reaches 90%-100%, medium A, a mesenchymal stem cell adipogenic differentiation medium, is added to the plates and replaced with medium B after three days of induction. After one day of induction with medium B, medium A is switched back to for another three days. After four cycles of induction, medium B is used to maintain the culture for 5-7 days until the lipid droplets become sufficiently large and round. After induction, Oil Red O staining is performed to determine the adipogenic differentiation of BMSCs: bright red or orange-red, round lipid droplets appear within the cells. The droplets vary in size and can fuse into larger vacuoles. Lipid droplets are primarily located in the cytoplasm, and the nuclei are often counterstained blue with hematoxylin, creating a red-blue contrast. Uninduced BMSCs lack distinct red lipid droplets and display a uniform, light red background.

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

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

[0065] a. Induction of BMSCs-ABs:

[0066] Undifferentiated BMSCs after culturing for 3 generations were seeded in 10 cm culture dishes. When the cells grew to 100% abundance, the BMSCs were treated with 250 nM 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, the cell supernatant was collected and BMSCs-ABs were obtained by centrifugation at 800g for 10 min and 16,000g for 30 min. The protein concentration of BMSCs-ABs was determined by BCA kit, and the particle size and potential distribution of BMSCs-ABs were detected by DLS. The morphological characteristics of BMSCs-ABs were observed by TEM. The expression of caspase3 protein in BMSCs-ABs was detected by Western blot. At the same time, Annexin-V-FITC was used to fluorescently label BMSCs-ABs, and the fluorescence distribution of BMSCs-ABs was observed by confocal microscopy, and the fluorescence intensity of FITC was analyzed using a nanoflow cytometer. The results are shown in Figure 2. Figure 4 shown.

[0069] 9) Preparation and characterization of eHEs

[0070] HemSCs-Exos and BMSCs-ABs were mixed in an equal weight ratio and 5% polyethylene glycol (PEG) was added. The mixture was shaken at 37°C for 30 minutes. The mixture was then sonicated for 5 minutes and extruded 10 times through a 100 nm liposome extruder. The size and zeta potential of the eHEs were determined by DLS. The morphology of the eHEs was observed by TEM.

[0071] 10) Detection of membrane fusion

[0072] First, fluorescence resonance energy transfer (FRET) was used to detect membrane fusion, and the fluorescence intensity within the wavelength range of 520 to 620 nm was collected by a multifunctional microplate reader. To further confirm that HemSCs-Exos and BMSCs-ABs had fused, laser confocal microscopy 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 with lysis buffer, centrifuged, and the supernatant collected. Protein concentration in each group was measured using a BCA kit, and total protein enrichment in each group was determined by Western blot.

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

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

[0077] 13) Study on the phagocytosis of eHEs by HUVECs

[0078] HemSCs-Exos (20 μg / mL), BMSCs-ABs (20 μg / mL), and eHEs (20 μg / mL) were labeled with DiO and co-cultured with HUVECs for 12 hours. Cells were washed three times with phosphate-buffered saline (PBS) and fixed with 4% PFA for 30 minutes. Cells were washed three times with PBS and counterstained with DAPI for nuclei. Representative images were captured using confocal microscopy, and fluorescence intensity was analyzed by flow cytometry.

[0079] 14) Detection of the 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 using an optical microscope at 0 h, 12 h, 24 h, and 36 h.

[0081] 15) Effects of eHEs on HUVECs migration ability

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

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

[0084] HemSCs-Exos have a more significant effect on promoting endothelial cell proliferation than BMSC-Exos. Through endothelial cell scratch assay, we compared the effect of HemSCs-Exos on promoting endothelial cell proliferation under the same exosome dose (20μg / mL) stimulation conditions. Figure 1 shown.

[0085] HemSCs-Exos and BMSC-ABs were able to achieve vesicle fusion after ultrasound and extrusion. We confirmed the fusion between the two vesicles by fluorescence resonance energy transfer (FRET) and immunofluorescence, respectively. Figure 2 shown.

[0086] After fusion, eHEs showed enhanced endothelial cell targeting and endothelial cell proliferation-promoting effects. Through immunofluorescence experiments, we observed that eHEs had more intraendothelial cell distribution than HemSCs-Exos in the same period of time, and eHEs played the best role in promoting endothelial cell proliferation, such as Figure 3 shown. Figure 3 Figures AB and HUVEC scratch analysis and statistical analysis showed that eHEs significantly promoted HUVEC proliferation and migration compared with PBS and HemSCs-Exos. Figures CD and immunofluorescence (IF) analysis showed that HUVEC phagocytosis of eHEs was statistically analyzed. To investigate the phagocytosis of eHEs by HUVECs in vitro, HemSCs-Exos (20 μg / mL) and eHEs (20 μg / mL) were labeled with DiO. Excess fluorescent dye was removed using Amicon Ultra-0.5 ultrafiltration centrifuge tubes, and the samples were rinsed twice with PBS. To verify the removal of unbound DiO, the supernatant of stained eHEs was used as a negative control (NC). The two vesicles were then co-cultured with HUVECs for 12 hours. The cells were washed three times with PBS and fixed with 4% PFA for 30 minutes. The cells were washed again three times with PBS, and the nuclei were counterstained with DAPI. Representative images were captured using confocal microscopy, and fluorescence intensity was analyzed by flow cytometry. The results showed that HUVECs significantly phagocytized eHEs.

[0087] The successful induction of BMSCs was observed by fluorescence detection and WB detection, and the extracted ABs expressed the hallmark apoptosis molecule caspase 3. At the same time, the ABs were observed by transmission electron microscopy and the particle size was detected, and it was found that they had a typical vesicle-like structure with a particle size mainly distributed between 200-400nm. Figure 4 shown.

[0088] HemSCs-Exos were observed by transmission electron microscopy and their particle size was detected. It was found that they had a typical vesicle-like structure with a particle size mainly distributed between 50-70nm. At the same time, HemSCs-Exos were detected to express typical membrane surface proteins CD9 and TSG101, but not cytoplasmic protein Calnexin. Figure 5As shown. In the experiment, a multifunctional microplate reader collected the fluorescence intensity in the wavelength range of 520 to 620 nm. The detection of FRET signals showed 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 had fused. DiO and DiD marked the membrane structures of HemSCs-Exos and BMSCs-ABs, respectively. Under a confocal microscope, if green and red fluorescence overlapped at the same position (i.e., yellow fluorescence), it indicated that the two membrane structures had fused.

Claims

1. A highly efficient vascular endothelial cell targeting vesicle system, characterized in that: The vesicle system is prepared by exosomes HemSCs-Exos derived from hemangioma stem cells and apoptotic bodies BMSCs-ABs derived from bone marrow mesenchymal stem cells.

2. The highly efficient vascular endothelial cell targeting vesicle system according to claim 1, characterized in that: The particle size of the HemSCs-Exos is 50-70 nm, and the HemSCs-Exos express typical membrane surface proteins CD9 and TSG101, but do not express the cytoplasmic protein Calnexin.

3. The high-efficiency vascular endothelial cell-targeting vesicle system according to claim 1, characterized in that: The BMSCs-ABs have a particle size of 200-400 nm and express the hallmark apoptosis molecule caspase 3.

4. The method for preparing the high-efficiency vascular endothelial cell-targeting vesicle system according to claim 1, characterized in that: The following steps are involved: (1) Obtain and culture hemangioma stem cells (HemSCs), and extract exosomes (HemSCs-Exo) from the hemangioma stem cells (HemSCs); (2) Obtaining and culturing bone marrow mesenchymal stem cells (BMSCs) and inducing the production of apoptotic bodies (BMSCs-ABs) from BMSCs; (3) Exosomes derived from hemangioma stem cells (HemSCs-Exos) and apoptotic bodies (BMSCs-ABs) derived from bone marrow mesenchymal stem cells (BMSCs-ABs) are mixed, polyethylene glycol is added, and the mixture is extruded.

5. The method for preparing the high-efficiency vascular endothelial cell-targeting vesicle system according to claim 4, characterized in that: In step (3), the mixing weight ratio of the HemSCs-Exos and BMSCs-ABs is 1:1, and the amount of polyethylene glycol added is 5% of the total weight of the HemSCs-Exos and BMSCs-ABs.

6. The method for preparing the high-efficiency vascular endothelial cell-targeting vesicle system according to claim 4, characterized in that: In step (3), after adding polyethylene glycol, the mixture is shaken at 37°C for 30 minutes, then ultrasonicated for 5 minutes, and then extruded 10 times through a 100nm liposome extruder.

7. The method for preparing the high-efficiency vascular endothelial cell-targeting vesicle system according to claim 4, characterized in that: The extraction steps of HemSCs-Exos are as follows: Hemangioma specimens were first prepared into a cell suspension by enzymatic digestion, sieved, and centrifuged before nonspecific antigen blocking with FcR Blocking Reagent. The single-cell suspension was incubated with anti-CD133 antibody at room temperature for 30 minutes, and CD133+ cells, namely hemangioma stem cells (HemSCs), were sorted using a MACS separator. HemSCs were placed in a 96-well plate and cultured in a 37°C, 5% CO2 incubator for 4-6 days. They were then digested with 0.25% trypsin containing 0.02% EDTA and expanded. The culture supernatant of HemSCs was then collected and centrifuged multiple times to remove live cells, dead cells, and cell debris. The supernatant was collected and centrifuged to purify the exosome precipitate.

8. The method for preparing the high-efficiency vascular endothelial cell-targeting vesicle system according to claim 7, characterized in that: The centrifugation purification operation is as follows: the supernatant is first ultracentrifuged at 120,000g for 70 minutes to obtain the exosome precipitate, and then the precipitate is suspended in pre-cooled PBS to remove contaminating proteins and other impurities, and then centrifuged again at 120,000g for 70 minutes to obtain purified exosomes HemSCs-Exos.

9. The method for preparing a high-efficiency vascular endothelial cell-targeting vesicle system according to claim 4, characterized in that: The extraction steps of the BMSCs-ABs are as follows: The rat tibia and femur with the epiphyseal ends cut open were flushed with a 1 mL syringe and cultured and expanded in α-MEM medium supplemented with 10% FBS and 1% penicillin / streptomycin mixture; Undifferentiated BMSCs after culturing for 3 generations were seeded in 10 cm culture dishes. When the cells grew to 100% abundance, the BMSCs were induced with 250 nM staurosporine for 12 h. After successful induction of BMSCs apoptosis, the cell supernatant was collected and centrifuged at 800 g for 10 min and then at 16,000 g for 30 min to obtain BMSCs-ABs.

10. Use of the high-efficiency vascular endothelial cell-targeting vesicle system according to any one of claims 1 to 3 in promoting endothelial cell proliferation and migration.

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