CN115025246A - Multifunctional vesicle for dual-targeting vascular repair and preparation method and application thereof - Google Patents
Multifunctional vesicle for dual-targeting vascular repair and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a multifunctional vesicle for dual-targeting vascular repair, and a preparation method and application thereof, and belongs to the technical field of biological medicines and biological materials. The multifunctional vesicle is prepared by the membrane fusion of an engineered exosome and a magnetic erythrocyte. On the basis of exosomes stimulated and secreted by mesenchymal stem cells, REDV peptide is modified on the surface of the exosomes to obtain engineered exosomes, so that the first re-targeted repair of blood vessels is realized; the magnetic nano particles are wrapped by red blood cells and fused with engineering exosomes to prepare uniform vesicles, the second targeting repair of blood vessels is realized by the magnetic response of the magnetic particles and an external magnetic field, and finally the multifunctional vesicles for dual targeting vascular repair are obtained. The multifunctional vesicle has high yield, the inclusion contains more components for promoting vascular repair (beneficial to the increase of the contents of vascular repair factors, miRNA and the like), the targeting is accurate, the first-pass effect can be overcome, the method is simple and feasible, safe and effective, and the method can be applied to the preparation of medicaments for treating cardiovascular diseases.
Description
Technical Field
The invention belongs to the technical field of biological medicines and biological materials, and particularly relates to a multifunctional vesicle for dual-targeting vascular repair, and a preparation method and application thereof.
Background
Blood vessels, which are the life pulse of the human body, are affected by various factors to cause diseases, wherein the morbidity and mortality represented by cardiovascular diseases are on a rising trend all over the world, and the health of human beings is seriously affected. Alterations in endothelial cell structure and function are a common pathological basis for many cardiovascular diseases, and are motile or promoting factors that contribute to their pathogenesis. The purpose of treating cardiovascular diseases is achieved by improving the function of endothelial cells, and the method has attracted extensive attention in preclinical research and clinical application. In recent years, with the research and the deepening of stem cells, the expectation of treating cardiovascular diseases by stem cells, particularly by stem cell exosomes, is high, and more opportunities are provided for the fields of diagnosis, treatment, prevention and the like of cardiovascular diseases.
Exosome is a membrane vesicle structure with the size of about 30-150nm secreted by cells in an exocytosis mode, and the surface lipid bilayer of the exosome contains transmembrane protein and cell cytoplasm components, and the exosome internally contains bioactive substances such as protein, mRNA and miRNA. At present, many researches on directly utilizing exosomes or taking exosomes as delivery systems exist, and the exosomes have wide acceptance on beneficial functions of promoting cell survival or inhibiting cell apoptosis, increasing angiogenesis, inhibiting inflammatory reaction, improving microenvironment, improving cardiovascular functions and the like. However, exosomes or exosomes as delivery systems still suffer from two problems: on one hand, the exosome yield of the traditional culture is low, the inclusion is complex, and the content of the effective components is not obvious; on the other hand, the natural unmodified exosomes can not avoid the first-pass effect of the body in any way when entering the body, and are difficult to accurately reach the target part and realize the enrichment at the target part.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a multifunctional vesicle for dual-targeting vascular repair, a preparation method and an application thereof, which overcome the problems that the existing exosome has low yield, the content of components for exerting effective effects of inclusion is not obvious, and the targeting site is difficult to accurately reach and realize enrichment at the target site.
In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
the invention discloses a multifunctional vesicle for dual-targeting vascular repair, which is prepared by fusing an engineered exosome and a magnetic erythrocyte through a membrane;
wherein the engineered exosome is obtained by modifying the surface of the exosome with REDV peptide.
Preferably, the particle size of the multifunctional vesicle for dual-targeting vascular repair is 127-145 nm.
Further preferably, the particle size of the multifunctional vesicle for dual-targeting vascular repair is 134 nm.
Preferably, the magnetic red blood cells are prepared from red blood cell membranes coated with magnetic nanoparticles.
Further preferably, the magnetic nanoparticles are Fe 3 O 4 And (3) nanoparticles.
Preferably, the REDV peptide is modified with palmitic acid.
Preferably, the exosomes are derived from mesenchymal stem cells.
Further preferably, the mesenchymal stem cell is an umbilical cord mesenchymal stem cell, an adipose mesenchymal stem cell or a bone marrow mesenchymal stem cell.
Preferably, the exosome is obtained by inducing, culturing and secreting mesenchymal stem cells through cobalt chloride.
Preferably, the concentration of the cobalt chloride induction culture is 100-200 mu mol/L, and the time is 12-48 h.
The invention also discloses a preparation method of the multifunctional vesicle for double-targeting vascular repair, which comprises the steps of blending the engineered exosome and the palmitic acid modified REDV peptide to prepare the REDV peptide modified exosome; and finally, uniformly mixing and extruding the magnetic erythrocytes and the exosomes modified by the REDV peptide according to the mass ratio of 1: 1-1: 3 to prepare the double-targeting vascular repair multifunctional vesicle.
The invention also discloses application of the double-targeting vascular repair multifunctional vesicle in preparation of a medicament for treating cardiovascular diseases.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a multifunctional vesicle for dual-targeting vascular repair, which is prepared by taking a stem cell to stimulate secretion of an exosome, modifying the exosome by REDV peptide, and fusing the engineered exosome with an erythrocyte containing a magnetic nanoparticle. The functional vesicle has excellent endothelial targeting property and magnetic responsiveness, can be doubly targeted and enriched at a vascular injury part, is beneficial to enrichment and detention at the vascular injury part, realizes vascular injury repair, not only is the targeting capacity improved, but also the enrichment of intelligent particles at a target position is increased by combining physical response, the delivery efficiency and the controlled release of bioactive substances are increased, a more intelligent delivery system is created, the accurate targeting and enrichment of the target part are realized, better vascular repair and treatment effects are achieved, the application potential of cardiovascular treatment is realized, and the functional vesicle can be applied to the preparation of medicaments for treating cardiovascular diseases.
Furthermore, the exosome is separated by using the stem cells induced by the cobalt chloride, so that the operation is simple, the exosome is easy to control, the prepared exosome is high in yield, contains more functional components such as cytokines and miRNA which are beneficial to vascular repair, and can efficiently repair vascular injury.
The preparation method of the multifunctional vesicle for dual-targeting vascular repair provided by the invention is characterized in that based on exosomes stimulated and secreted by mesenchymal stem cells, REDV peptide is modified on the surface of the exosomes to obtain engineered exosomes, so that the first re-targeting repair of blood vessels is realized; wrapping magnetic nanoparticles with erythrocytes, fusing with engineering exosomes to obtain uniform vesicles, and preparing the magnetic nanoparticlesAnd the magnetic response of the magnetic particles is combined with an external magnetic field to realize second targeted repair of the blood vessel, and finally the multifunctional vesicle for double targeted repair of the blood vessel is obtained. In the method, the stem cells are induced and cultured, so that the stem cells can be promoted to secrete more cytokines, miRNA and the like which are beneficial to vascular repair, the protein concentration of the exosome obtained by separation is higher, and the exosome has a stronger vascular repair effect; by modification with REDV peptides, specific binding to tetrapeptides with specific targeting to endothelial cells, i.e., integrin alpha 4 β 1 The endothelial cells are specifically adsorbed, the proliferation and adhesion of the endothelial cells are promoted, the endothelialization and vascularization are improved, and the repair of damaged vascular tissues is further promoted; compared with the common exosome modification method, the method comprises the following steps: the method is simple, feasible, safe, effective and easy to industrialize, can selectively deliver the multifunctional vesicle to a target site, and realizes the precise treatment of the exosome.
Drawings
FIG. 1 is a diagram of the expression of a surface marker protein of an exosome detected by Western Blot according to the present invention;
FIG. 2 is a graph of the exosome particle size distribution of the present invention;
FIG. 3 is a graph showing the particle size distribution of the exosomes of the present invention after being modified with REDV peptide;
FIG. 4 is a transmission electron microscope observation appearance of exosomes of the present invention;
FIG. 5 is a transmission electron microscope observation appearance of the exosomes of the present invention after being modified with REDV peptide;
FIG. 6 shows Fe of the present invention 3 O 4 The particle size distribution map of the nanoparticles;
FIG. 7 shows Fe of the present invention 3 O 4 Observing an appearance image of the nano particles by a transmission electron microscope;
FIG. 8 is a transmission electron microscope observation appearance image of the multifunctional vesicle for double-targeting vascular repair of the present invention;
fig. 9 is a particle size distribution diagram of the multifunctional vesicle for dual-targeted vascular repair of the present invention;
FIG. 10 is a statistical graph of cell proliferation at different culture times according to the present invention;
FIG. 11 is a diagram of the uptake of cells observed by a confocal microscope according to the present invention; wherein, A is an exosome modified by REDV peptide, and B is a multifunctional vesicle for dual-targeting vascular repair;
FIG. 12 is a graph of the test of cell migration in a scratch test according to the present invention;
FIG. 13 is a graph of the cell mobility of each group of the statistical analysis of the present invention.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It is noted that the terms "comprises" and "comprising," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The invention is described in further detail below with reference to the accompanying drawings:
the invention provides a multifunctional vesicle for dual-targeting vascular repair, which is prepared by fusing an engineered exosome and a magnetic erythrocyte through a membrane;
wherein the engineered exosome is obtained by modifying an exosome derived from a pluripotent stem cell, and the pluripotent stem cell is preferably a mesenchymal stem cell; further, the modification is carried out by modifying with palmitic acidA decorated REDV peptide; the mesenchymal stem cells are umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells or bone marrow mesenchymal stem cells; the magnetic nanoparticles are Fe 3 O 4 And (3) nanoparticles.
The invention provides a preparation method of a multifunctional vesicle for double-targeting vascular repair, which comprises the steps of firstly separating a pluripotent stem cell to obtain an exosome, and then blending the obtained exosome and an REDV peptide modified by palmitic acid to prepare the REDV peptide modified exosome; finally, uniformly mixing and extruding the magnetic erythrocytes and the exosomes modified by the REDV peptide according to the mass ratio of 1: 1-1: 3 to prepare the multifunctional vesicle for dual-targeting vascular repair;
wherein the exosome is obtained by performing cobalt chloride induced culture and secretion on the mesenchymal stem cells, the concentration of the cobalt chloride induced culture is 100-200 mu mol/L, and the time is 12-48 h; the magnetic red blood cells are prepared from a red blood cell membrane wrapped with magnetic nanoparticles.
Example 1
1. Stem cell culture
Configuring the P3-P6 generation human umbilical cord-derived mesenchymal stem cells to have a concentration of 4-7 × 10 by using an exosome-free culture medium 4 The cell suspension of each/mL is inoculated into a culture vessel and then placed at 37 ℃ and 5% CO 2 The cells were cultured in a saturated humidity incubator for 1d, and whether the cells had a characteristic morphology of umbilical cord mesenchymal stem cells was observed under an inverted microscope (purchased from Nikang), and whether the cell state was good or not was observed. Then, the culture medium is discarded, the induction medium is added for treatment for 24h, the cell morphology is observed under an inverted microscope, and whether the morphology and the viability state of the cells are changed after the induction medium is added is observed. Then replacing the culture medium with a serum-free and exosome-free culture medium for continuous culture;
wherein the exosome-free culture medium is prepared by adding fetal bovine serum (purchased from Gibco) with the volume fraction of 10% into an alpha-MEM culture medium (purchased from Gibco), uniformly mixing, centrifuging 110000g by 12h, and removing lower-layer precipitates; the induction culture medium is prepared by adding fetal calf serum with volume fraction of 5% and 150 μmol/L cobalt chloride (purchased from national drug group chemical reagent Co., Ltd.) solution into alpha-MEM culture medium, centrifuging for 110000g 12h, and discarding the lower layer precipitate. The serum-free and exosome-free culture medium is prepared by centrifuging 110000g for 12h in an alpha-MEM culture medium and then removing a lower-layer precipitate.
2. Stem cell exosome (Exos) isolation
Collecting the serum-free and exosome-free culture medium in the step 1, centrifuging for 5min to remove dead cells under the conditions of 300g and 4 ℃, filtering by using a 0.22-micron filter to remove cell fragments and other particles with larger particle sizes, finally performing ultrafiltration concentration by using a 300KD ultrafiltration membrane to separate exosomes, identifying, storing for later use (within 4 months) in a refrigerator at minus 80 ℃ for a long time, diluting to the required concentration by using a sterile PBS buffer solution during use, ensuring the use as soon as possible, and avoiding repeated freeze thawing;
wherein, the identification steps are as follows: protein concentration of the extracted exosomes was confirmed by BCA protein quantitative assay kit (purchased from solibao (PC0020)), and 0.2mg/mL was used as storage concentration; meanwhile, nanoparticle tracking analysis (purchased from NanoFCM) is used for measuring the particle size distribution of the extracted exosomes, a transmission electron microscope (purchased from JEOL) is used for observing the morphological characteristics of the extracted exosomes, and Western blot is used for identifying the expression conditions of the marker proteins CD9 and CD63 of the exosomes.
(1) Protein content determination by BCA kit method
The protein concentration was determined according to the procedure of the BCA protein quantitative determination kit. And diluting the extracted exosomes to different times, and determining according to the kit operation protocol. The experimental results show that: for exosome extracted without adding an induction culture medium, about 1.2L of exosome suspension can be obtained by separating 3L of human umbilical cord mesenchymal stem cell supernatant, and the protein concentration of the exosome suspension is 0.64 g/L; and the exosome extracted after the culture of the induction culture medium can be separated into about 1.5L of exosome suspension by 3L of human umbilical cord mesenchymal stem cell supernatant, and the measured protein concentration is 0.8g/L, so the exosome is extracted by selecting the mode of culture of the induction culture medium.
(2) Western blot identification of exosome membrane protein expression
The cells were lysed with RIPA buffer containing protease inhibitors (purchased from petun clouds), protein samples were collected and electrophoresed in 10% sodium dodecyl sulfate polyacrylamide gels, the electrophoresed proteins were transferred to polyvinylidene fluoride membranes and blocked with 5% skim milk at room temperature, GAPDH, CD9, CD63 (1: 1000) primary antibody (both purchased from Abcam) were added and incubated overnight at 4 ℃, the blots were washed and then incubated with horseradish peroxidase-labeled goat-anti-rabbit secondary antibody (1: 3000) at room temperature for 1h, and protein expression was detected by exposure to a chemiluminescent substrate on a gel imager. As shown in figure 1, according to the Western blot detection result, the obtained exosomes are all expressed by characteristic membrane proteins CD9 and CD 63.
3. Targeted modification of exosomes
Taking the exosome with the concentration of 0.2mg/mL obtained by the separation in the step 2 and the palmitic acid modified REDV peptide (synthesized by Nanjing peptide industry Co., Ltd.) to be mixed to prepare the REDV peptide modified exosome, namely REDV-Exos; and simultaneously, measuring the particle size distribution of the modified exosome by using nanoparticle tracking analysis, and observing the morphological characteristics of the modified exosome by using a transmission electron microscope.
Wherein, (1) the specific steps for detecting the particle size of the exosome before and after modification are as follows: taking a 1.5mL EP tube, diluting the extracted exosomes to 0.02mg/mL by using a PBS solution, then adding the diluted exosomes into a particle size measurement sample cell for nanoparticle tracking analysis, and detecting the particle sizes of the exosomes before and after modification as shown in figures 2 and 3, wherein the results show that the particle sizes of the unmodified exosomes are mainly distributed between 70-150 nm, the peak value of the particle size distribution of the exosomes before modification is 89nm, and the peak value of the particle size distribution is about 102.3nm after the REDV peptide modification, which indicates that the modification is successful. (2) The transmission electron microscope observation comprises the following specific steps: 1.5mL of EP tube was used, and the exosomes before and after modification were each diluted to 0.02mg/mL with PBS solution. The 200 mesh copper mesh with carbon support membrane is placed in a clean container (e.g. petri dish) and 10 μ L each of the diluted exosome suspension is pipetted onto the copper mesh with support membrane, left to stand at room temperature for 5min, and then excess liquid is carefully blotted off the edges with a strip of filter paper. After the materials are slightly dried, 3% of tungsten phosphate negative staining solution is respectively dripped to carry out standing staining for 3min, then, redundant staining solution is sucked, and observation and photographing are carried out under a transmission electron microscope after natural drying. As shown in fig. 4 and fig. 5, transmission electron micrographs show that the shape of the exosomes after the REDV peptide modification is substantially intact, and the structures of the exosomes before and after the modification are similar, presenting a typical disc-like vesicle.
4. Magnetic nanoparticles
The magnetic nanoparticles in the invention are selected from Fe 3 O 4 And (3) nanoparticles.
Fe 3 O 4 The nano-particles are synthesized by a hydrothermal method, and the specific method comprises the following steps: weighing FeCl 3 -6H 2 0.675g of O solid is added into 20mL of ethanol and placed on a magnetic stirrer to be fully stirred until a transparent light yellow solution is obtained, and the rotating speed of the magnetic stirrer is controlled at 800 rpm. Then, 1.8g of sodium acetate and 0.5g of polyethylene glycol were added, followed by further stirring for 40min to obtain a transparent solution. And after stirring, transferring all the liquid into a stainless steel high-pressure hydrothermal reaction kettle with a polytetrafluoroethylene lining, covering and screwing the hydrothermal reaction kettle, putting the hydrothermal reaction kettle into an oven, and setting the temperature of the oven at 200 ℃ for 12 hours to allow the hydrothermal reaction kettle to react fully. After the reaction is finished, the drying oven is closed, the reaction kettle is naturally cooled to room temperature, the reaction kettle is taken out, liquid in the reaction kettle is transferred to a centrifugal tube for centrifugation, centrifugation is carried out for 10min at 5000rpm, supernatant is discarded after the centrifugation is finished, the lower-layer black precipitate is washed by adding 3 times of ethanol, the mixture is mixed and then centrifuged again, the centrifugation is carried out for 10min at 5000rpm, the mixture is washed by pure water after 3 times of the centrifugation, the washing is carried out for 3 times, the black precipitate is dried in the drying oven after the washing is finished, the temperature of the drying oven is set to be 60 ℃, and the drying time is 6h, so that Fe can be obtained 3 O 4 And (3) nanoparticles.
Fe 3 O 4 The nano-particles can also be directly purchased Fe with the particle size of about 10-50 nm 3 O 4 And (3) nanoparticles.
Fe was measured using a dynamic light scattering particle sizer (Malvern (Nano ZS)) 3 O 4 The particle size distribution of the nanoparticles is observed by using a Transmission Electron Microscope (TEM) 3 O 4 Morphological characteristics of the nanoparticles.
(1) Particle size measurement by nanometer particle sizer
Weighing the prepared Fe 3 O 4 0.5mg of nano particles are dissolved in 2mL of pure water, the ultrasonic dispersion is uniform, and the ultrasonic power is high800w, performing ultrasonic treatment for 1min to obtain uniform dispersion, transferring the uniform dispersion to a sample cell of a nanometer particle analyzer, and measuring the particle size distribution. As shown in FIG. 6, Fe was measured 3 O 4 The particle size of the nano particles is 70-80 nm, and the particle size is larger than the result of electron microscope measurement due to slight agglomeration of the magnetic particles in an aqueous solution.
(2) Transmission electron microscope
The method is the same as the previous method. As shown in FIG. 7, the results show that Fe was produced 3 O 4 The nano particles are spherical and have the particle size of 20-40 nm.
5. Erythrocyte membrane separation
Extracting erythrocyte membrane by hypotonic method. Collecting blood (autoblood or chicken blood) in an EDTA blood collection tube, centrifuging at 3000rpm for 4min in a centrifuge, discarding the supernatant, then resuspending with 3 times of volume of PBS buffer solution with pH value of 7.4, repeating for 3 times, then resuspending the precipitate with PBS, centrifuging at 11000rpm at high speed after ice bath for 2h for 15min after ice bath, carefully discarding the supernatant after centrifugation, then resuspending the precipitate with PBS, and repeating for 3 times to obtain the erythrocyte membrane.
6. Magnetic red blood cell preparation
Resuspending the erythrocyte membrane prepared in step 5 in PBS, and adding the Fe prepared in step 4 into the solution 3 O 4 Nanoparticles, erythrocyte membranes and Fe 3 O 4 The mass ratio of the nano particles is 2:1, the nano particles are uniformly mixed by repeated oscillation, magnetic red blood cells are formed by low-power ultrasound, then the magnetic red blood cells are repeatedly extruded for 8 times by a liposome extruder through acetate fiber membranes of 400nm, 200nm and 100nm in sequence, and then the magnetic red blood cells are separated by an external magnet.
The specific method comprises the following steps: 5mg of erythrocyte membrane is weighed into 100mL of PBS buffer, and 2.5mg of Fe is weighed 3 O 4 And adding the nano particles into the liquid, and shaking and uniformly mixing. Placing the mixture in an ultrasonic cleaner after mixing, setting ultrasonic power at 60w, performing ultrasonic treatment for 30min, controlling the temperature at 20 ℃ in the whole process, and under the auxiliary action of ultrasonic treatment, forming a spherical object spontaneously after the lipid bilayer of the erythrocyte membrane reaches a certain concentration to minimize the surface area of the spherical object, and rapidly wrapping the magnetic nanoparticles when encountering the nanoparticles,so that the membrane-coated magnetic red blood cells can be formed centering on the nanoparticles. Then, repeatedly extruding for 8-12 times by a liposome extruder through 400nm, 200nm and 100nm acetate fiber membranes in sequence, transferring the obtained product into a clean small beaker, placing an external magnet on one side of the wall of the beaker, and attracting magnetic red blood cells to the side so as to collect the magnetic red blood cells to obtain the magnetic artificial red blood cells. After the collection, the cells were diluted to 4% concentration in PBS buffer and stored in a refrigerator at 4 ℃ for further use.
7. Preparation of double-targeting vascular repair multifunctional vesicles (REDV-Exos + R-NPs)
Mixing the magnetic red blood cells prepared in the step 6 and the REDV peptide modified exosomes prepared in the step 3 according to the mass ratio of 1:1, and then placing the mixture in an ultrasonic cell disruptor (BRANSO (550)) to perform ice bath ultrasonic mixing under the ultrasonic condition of the ultrasonic power of 80W, the ultrasonic time of 1min and the ultrasonic interval of 6s for 4 s. Then, a liposome extruder (Morgec (Liposoeasy LE-1)) is used for repeatedly extruding for 8 times through acetate fiber membranes with the diameters of 400nm, 200nm and 100nm sequentially to finally obtain the multifunctional vesicle for double-targeting vascular repair. From fig. 8, it can be seen that some black fine particles are wrapped in the membrane structure, which primarily indicates that the complex vesicle is successfully prepared. As can be seen from FIG. 9, due to the influence of the magnetic particles, the prepared dual-targeting vesicle has a particle size of 127-145 nm and a peak value of 134nm, which is slightly increased.
8. In vitro validation of cell proliferation
The proliferation effect of the REDV peptide modified exosome (REDV-Exos) and REDV-Exos + R-NPs on human umbilical vein vascular endothelial cells (HUVECs) is detected by an MTT method. Cells were seeded at 8000 cells/well in 96-well plates, and the cells were treated with the different drugs described above, with 6 replicate wells per group, while a control group without drug treatment and a blank group without cells and drug were set. After further incubation of the cells for 1d, 3d and 5d, the plates were removed, 20. mu.L of MTT solution (MTT dissolved in PBS at a concentration of 5 mg/mL) was added to each well, and after 4h of incubation, the cell viability was calculated by reading the absorbance values of each well and substituting them into the formula on a fully automatic enzyme scale (purchased from Thermo scientific) using a 490nm wavelength.
Cell survival (%) ═ OD (drug group) -OD (blank group)/OD (control group) -OD (blank group)
As shown in fig. 10, it was experimentally observed that both groups of vesicles were non-toxic to vascular endothelial cells and were able to promote cell proliferation, and this proliferation promoting effect was time-dependent, i.e., the proliferation promoting ability of cells was increased with the increase of incubation time. After 24h of co-incubation, the proliferation rate of the cells in the REDV-Exos group reaches 115%, and the proliferation rate of the cells in the functional vesicle group reaches 130%.
9. In vitro cell experiment verifies its cell uptake
(1) Fluorescent markers REDV-Exos and REDV-Exos + R-NPs
The REDV modified exosomes and the double targeting vesicles described above were incubated with 1, 1-dioctadecyl-3, 3,3, 3-tetramethylindodicarbocyanine perchloro (DiD) tracer (5. mu.M; Sigma-Aldrich) for 4 minutes, respectively, treated with 0.5% BSA/PBS to neutralize excess dye, and the dye was removed by centrifugation to give fluorescently labeled REDV-Exos and REDV-Exos + R-NPs.
(2) Confocal detection of cellular uptake
For internalization analysis, HUVECs cells were plated at 2X 10 5 The concentration per dish was inoculated in 35mm confocal culture dishes and treated with 20. mu.g/mL of DiD-labeled REDV-Exos and REDV-Exos + R-NPs. After 18 hours of incubation, cells were washed twice with PBS, fixed with 4% paraformaldehyde (ex solibao) for 10min, and the nuclei were stained with 4, 6-diamino-2-phenylindole (DAPI staining solution, ex muron). Cells were observed for uptake of REDV-Exos and REDV-Exos + R-NPs using a confocal laser microscope (purchased from Nikang).
In FIG. 11, the red color is represented by DID-labeled REDV-Exos and REDV-Exos + R-NPs, and the blue color is DAPI staining of the cell nuclei, indicating that the group of REDV-Exos + R-NPs have more red color concentrated around the blue color (i.e., the double-targeted functional vesicles are more concentrated around the cells), indicating that they are more taken up by the cells.
10. In vitro validation of cell migration
The effect of REDV-Exos and REDV-Exos + R-NPs on the migration of HUVECs was evaluated using a scratch test. Taking a six-hole plate, firstly using a ruler and a marking pen to position the six holesThe back of the plate is drawn with straight lines, one line is drawn at intervals of 0.5 cm-1.0 cm, and at least five lines are ensured to pass through each hole. Then taking HUVECs with logarithmic growth period according to 1.2X 10 4 Cells/well were seeded in six-well plates and cultured overnight to allow adherent growth of cells as a monolayer, then parallel lines were drawn perpendicular to the marker line using a 10 μ L tip, after which the cells were gently washed 3 times with PBS, the drawn cells were removed, leaving a cell-free space, and each well was filled with fresh serum-free medium containing REDV-Exos and REDV-Exos + R-NPs. Images of the scratched area were obtained after 0, 6 and 12 hours and the migrated area was measured using Image-Pro Plus 6.0 software as follows: mobility ═ a0-An)/a0 × 100, where a0 represents the initial void area (t ═ 0h) and An represents the residual void area when measured (t ═ n h).
The results of fig. 12 and 13 show that: the migration speed and migration rate of cells were significantly increased for the double-targeted vesicles (REDV-Exos + R-NPs) compared to the REDV-Exos alone, indicating that the double-targeted vesicles are better able to promote cell migration, thereby accelerating vascular repair.
Example 2
1. Stem cell culture
Configuring the P3-P6 generation adipose-derived mesenchymal stem cells into a concentration of 4-7 × 10 by using an exosome-free culture medium 4 The cell suspension of each/mL is inoculated into a culture vessel and then placed at 37 ℃ and 5% CO 2 The culture was carried out in a saturated humidity incubator for 1 day, and observed under an inverted microscope. Subsequently, the medium was discarded, and the induction medium was added for treatment for 24 hours and observed under an inverted microscope. Then replacing the culture medium with a serum-free and exosome-free culture medium for continuous culture;
wherein, the exosome-free culture medium is prepared by adding fetal bovine serum (purchased from Gibco) with the volume fraction of 10 percent into an alpha-MEM culture medium (purchased from Gibco), uniformly mixing, centrifuging for 110000g by 12h, and removing a lower layer of sediment; the induction culture medium is prepared by adding fetal calf serum with volume fraction of 5% and 200 μmol/L cobalt chloride (purchased from national drug group chemical reagent Co., Ltd.) solution into alpha-MEM culture medium, centrifuging for 110000g 12h, and discarding the lower layer precipitate. The serum-free and exosome-free culture medium is prepared by centrifuging 110000g of alpha-MEM culture medium for 12h, and then removing a lower precipitate.
2. Stem cell exosome (Exos) isolation
The stem cell exosome isolation method was the same as in example 1.
3. Targeted modification of exosomes
The targeted modification method of exosomes is the same as in example 1.
4. Erythrocyte membrane separation
The erythrocyte membrane separation method is the same as in example 1.
5. Magnetic red blood cell preparation
The magnetic red blood cells were prepared in the same manner as in example 1.
6. Preparation of multifunctional vesicle for dual-targeting vascular repair
And (3) mixing the magnetic red blood cells prepared in the step (5) with the REDV peptide modified exosomes prepared in the step (3) according to the mass ratio of 1:3, and then placing the mixture in an ultrasonic cell disruptor to perform ice-bath ultrasonic mixing under the ultrasonic condition that the ultrasonic power is 80W, the ultrasonic time is 1min, and the ultrasonic time is 4s and 6 s. Then, a liposome extrusion instrument is used for repeatedly extruding for 10 times through 400nm, 200nm and 100nm cellulose acetate membranes in sequence, and finally the multifunctional vesicle for dual-targeting vascular repair is obtained.
Example 3
1. Stem cell culture
Configuring the P3-P6 generation mesenchymal stem cells into 4-7 × 10 concentration by using an exosome-free culture medium 4 The cell suspension of each/mL is inoculated into a culture vessel and then placed at 37 ℃ and 5% CO 2 The culture was carried out in a saturated humidity incubator for 1 day, and observed under an inverted microscope. Subsequently, the medium was discarded, and the induction medium was added for treatment for 24 hours and observed under an inverted microscope. Then replacing the culture medium with a serum-free and exosome-free culture medium for continuous culture;
wherein the exosome-free culture medium is prepared by adding fetal bovine serum (purchased from Gibco) with the volume fraction of 10% into an alpha-MEM culture medium (purchased from Gibco), uniformly mixing, centrifuging 110000g by 12h, and removing lower-layer precipitates; the induction culture medium is prepared by adding fetal calf serum with volume fraction of 5% and 100 μmol/L cobalt chloride (purchased from national drug group chemical reagent Co., Ltd.) solution into alpha-MEM culture medium, centrifuging for 110000g 12h, and discarding the lower layer precipitate. The serum-free and exosome-free culture medium is prepared by centrifuging 110000g for 12h in an alpha-MEM culture medium and then removing a lower-layer precipitate.
2. Stem cell exosome (Exos) isolation
The stem cell exosome isolation method was the same as in example 1.
3. Targeted modification of exosomes
The targeted modification method of exosomes is the same as in example 1.
4. Erythrocyte membrane separation
The erythrocyte membrane separation method is the same as in example 1.
5. Magnetic red blood cell preparation
The magnetic red blood cells were prepared in the same manner as in example 1.
6. Preparation of multifunctional vesicle for dual-targeting vascular repair
And (3) mixing the magnetic red blood cells prepared in the step (5) with the REDV peptide modified exosomes prepared in the step (3) according to the mass ratio of 1:2, and then placing the mixture in an ultrasonic cell disruptor to perform ice-bath ultrasonic mixing under the ultrasonic condition that the ultrasonic power is 80W, the ultrasonic time is 1min, and the ultrasonic time is 4s and 6 s. Then, a liposome extrusion instrument is used for repeatedly extruding for 12 times through 400nm, 200nm and 100nm cellulose acetate membranes in sequence, and finally the multifunctional vesicle for dual-targeting vascular repair is obtained.
The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Claims (10)
1. The multifunctional vesicle for dual-targeting vascular repair is characterized in that the multifunctional vesicle is prepared by fusing an engineered exosome and a magnetic erythrocyte through a membrane;
wherein the engineered exosome is obtained by modifying REDV peptide on the surface of the exosome.
2. The multifunctional vesicle for dual-targeted vascular repair of claim 1, wherein the multifunctional vesicle for dual-targeted vascular repair has a particle size of 127-145 nm.
3. The multifunctional vesicle for dual-targeted vascular repair of claim 1, wherein the magnetic red blood cells are prepared from a red blood cell membrane coated with magnetic nanoparticles.
4. The multifunctional vesicle for dual-targeted vascular repair of claim 1, wherein the REDV peptide is modified with palmitic acid.
5. The multifunctional vesicle for dual-targeted vascular repair of claim 1, wherein the exosomes are derived from mesenchymal stem cells.
6. The multifunctional vesicle for dual-targeting vascular repair of claim 5, wherein the mesenchymal stem cell is an umbilical cord mesenchymal stem cell, an adipose mesenchymal stem cell or a bone marrow mesenchymal stem cell.
7. The multifunctional vesicle for dual-targeting vascular repair of claim 1, wherein the exosome is secreted by mesenchymal stem cells through cobalt chloride-induced culture.
8. The multifunctional vesicle for dual-targeting vascular repair of claim 7, wherein the concentration of the cobalt chloride induction culture is 100-200 μmol/L, and the time is 12-48 h.
9. The method for preparing the multifunctional vesicle for dual-targeting vascular repair according to claim 1, wherein the engineered exosome is blended with the palmitic acid modified REDV peptide to prepare the REDV peptide modified exosome; and finally, uniformly mixing and extruding the magnetic erythrocytes and the exosomes modified by the REDV peptide according to the mass ratio of 1: 1-1: 3 to prepare the double-targeting vascular repair multifunctional vesicle.
10. The use of the multifunctional vesicle for dual-targeting vascular repair of any one of claims 1-8 in the preparation of a medicament for treating cardiovascular diseases.
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