CN113616810A - P-selectin-targeted engineered extracellular vesicle composition and preparation method and application thereof - Google Patents
P-selectin-targeted engineered extracellular vesicle composition and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an engineered extracellular vesicle composition targeting P-selectin, which comprises extracellular vesicles, ligand molecules specifically targeting P-selectin, a molecular imaging contrast agent and endogenous or exogenous functional components carried in the extracellular vesicles; the engineered extracellular vesicles are used for specifically recognizing and combining P-selectin on the surface of damaged endothelial cells, indicating the expression quantity of the P-selectin through the signal intensity of a molecular imaging contrast agent, and delivering carried endogenous or exogenous functional components to a damaged part. The invention adopts the P-selectin-targeted engineered extracellular vesicle composition and the preparation method and application thereof, the P-selectin is taken as a target spot of the engineered extracellular vesicle to target and damage endothelial cells, the purpose of targeting and damaging related diseases of the endothelial cells is realized, and the blank that no universal technical means for targeting and diagnosing and treating the related diseases of the damaged endothelial cells exists in the prior art is filled.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to an engineered extracellular vesicle composition of a targeting P-selectin, a preparation method and application thereof.
Background
The endothelial cells are cells with a flat layer on the inner wall of the blood vessel and are positioned between blood plasma and blood vessel tissues, and not only can complete the metabolic exchange of blood plasma and tissue fluid, but also can synthesize and secrete a series of vasoactive substances to participate in various physiopathological processes. Under physiological state, endothelial cells can regulate the tension of blood vessel wall, and have protective effects of resisting inflammatory cell adhesion, resisting activation of platelet adhesion and the like. When the blood vessel is stimulated by inflammatory factors, thrombin, hyperglycemia, active oxygen and the like, endothelial cells are damaged, so that the pathological changes of vascular tone, inflammatory cell adhesion infiltration, thrombosis and the like are caused. Therefore, endothelial cell damage is closely related to the occurrence and development of tumors, cardiovascular diseases, ischemia-reperfusion related diseases, diabetes-induced microangiopathy related diseases, autoimmune-related diseases and inflammation-related diseases.
P-selectin (P-selectin) is an important adhesion molecule of the selectin family, and is mainly present in endothelial cells, namely Wolfrade bodies (Weible-Palade bodies, referred to as W-P bodies) under physiological conditions. After endothelial cell injury, P-selectin is rapidly expressed in endothelial cells and transferred to endothelial cell membranes, and participates in various pathophysiological initiation processes including inflammatory reaction and thrombosis by mediating recruitment and adhesion of inflammatory cells and platelets. These characteristics indicate that P-selectin is an indicator molecule indicating the degree of endothelial cell damage, and is also a good target for achieving targeted therapy of damaged endothelial cells. However, at present, no visual diagnosis and treatment strategy which takes P-selectin as a target point and is used for diagnosing and treating diseases related to endothelial cell injury exists.
Therefore, the design of a treatment means and a drug delivery system capable of accurately targeting the P-selectin and the realization of visual diagnosis and treatment for indicating the tissue damage degree by quantifying the expression quantity of the P-selectin have important significance.
Disclosure of Invention
The invention aims to provide an engineered extracellular vesicle composition of a targeted P-selectin and a preparation method and application thereof, the engineered extracellular vesicle takes the P-selectin as a target spot to target and damage endothelial cells, thereby realizing the purpose of targeting and diagnosing related diseases of the endothelial cells, filling the blank that no universal technical means for targeting and diagnosing related diseases of the endothelial cells exists in the prior art, and being widely applied to diagnosis and treatment of various related diseases of the endothelial cells.
In order to achieve the aim, the invention provides an engineered extracellular vesicle composition targeting P-selectin, which comprises extracellular vesicles, ligand molecules specifically targeting P-selectin, a molecular imaging contrast agent and endogenous or exogenous functional components carried in the extracellular vesicles;
the engineered extracellular vesicles are used for specifically recognizing and combining P-selectin on the surface of damaged endothelial cells, indicating the expression quantity of the P-selectin through the signal intensity of a molecular imaging contrast agent, and delivering carried endogenous or exogenous functional components to a damaged part.
Preferably, the extracellular vesicles are derived from human cells or from vesicles of platelets, including exosomes, microvesicles, platelet debris;
the human cell is a human-derived stem cell or a human-derived immune cell, and the human-derived stem cell includes but is not limited to a human placenta-derived mesenchymal stem cell, a human umbilical cord-derived mesenchymal stem cell, a human bone marrow-derived mesenchymal stem cell, a human adipose-derived mesenchymal stem cell and a human-derived induced pluripotent stem cell; human-derived immune cells, including but not limited to human T cells, human macrophages, human neutrophils, and human natural killer cells.
Preferably, the ligand molecule specifically targeting P-selectin is one or a combination of more than two of P-selectin glycoprotein ligand 1, P-selectin binding peptide, monoclonal antibody and fucoidan.
Preferably, the P-selectin binding peptide is a polypeptide comprising the core motif of the EWVDV pentapeptide that specifically binds P-selectin.
Preferably, the molecular imaging contrast agent is one or a combination of more than two of a radionuclide molecular imaging probe, an optical molecular imaging probe and a nuclear magnetic resonance molecular imaging probe.
Preferably, the optical molecular imaging probe includes, but is not limited to, a fluorescent dye, a nano fluorescent material, a quantum dot, a fluorescent protein, and luciferase.
Preferably, the functional component carried in the extracellular vesicle is a natural active molecule carried by the extracellular vesicle and derived from a donor cell thereof or a loaded exogenous functional molecule, the natural active molecule of the donor cell comprises a protein, a nucleic acid and a lipid molecule, and the loaded exogenous functional molecule is an exogenously loaded small molecule drug, a small interfering RNA, a microRNA or a protein.
A preparation method of an engineered extracellular vesicle composition targeting P-selectin comprises the following steps:
s1, separating and extracting human cells or platelet-derived extracellular vesicles;
s2, loading endogenous or exogenous functional components in the extracellular vesicles;
s3, modifying the surface of the extracellular vesicle with a ligand molecule specifically targeting P-selectin, so that the extracellular vesicle has the capability of targeting P-selectin;
s4, modifying a molecular imaging contrast agent on the extracellular vesicles or ligand molecules to enable the extracellular vesicles to have a molecular imaging function, and obtaining the engineered extracellular vesicle composition targeting the P-selectin.
Preferably, the method for separating and extracting the extracellular vesicles can be differential centrifugation, density gradient centrifugation, size exclusion chromatography, ultrafiltration centrifugation, polymer-based precipitation, and immune separation technology.
Preferably, the method for loading endogenous or exogenous functional molecules in the extracellular vesicles can be a method for indirectly modifying donor cells and a method for directly modifying the extracellular vesicles.
Preferably, the method for indirectly modifying the donor cell to load the functional molecule in the extracellular vesicle can be a method for modifying the donor cell by genetic engineering, modifying the donor cell by metabolic incorporation, modifying the donor cell by bioactive materials, modifying the donor cell by hypoxic pretreatment and the like.
Preferably, the method of directly modifying the extracellular vesicles to load functional molecules into the extracellular vesicles may be electroporation, membrane surface chemical modification, hydrophobic intercalation, or co-incubation.
Preferably, the method for modifying the surface of the extracellular vesicles with the ligand molecule specifically targeting the P-selectin can be a method for inserting the ligand molecule targeting the P-selectin into the extracellular vesicle membrane through an amphiphilic compound, or a method for displaying the ligand molecule targeting the P-selectin on the surface of the extracellular vesicle membrane through genetic engineering.
Preferably, the amphiphilic compound may be dimyristoyl phosphatidylethanolamine-polyethylene glycol (DMPE-PEG), distearoyl phosphatidylethanolamine-polyethylene glycol (DSPE-PEG), dipalmitoyl phosphatidylethanolamine-polyethylene glycol (DPPE-PEG), dioleoyl phosphatidylethanolamine-polyethylene glycol (DOPE-PEG), dicaprylyl phosphatidylethanolamine-polyethylene glycol (DEPE-PEG), 1-palmitoyl-2-oleoyl phosphatidylethanolamine-polyethylene glycol (DLPE-PEG).
Preferably, the method of modifying the molecular imaging contrast agent on the extracellular vesicles or ligand molecules may be a method of modifying the imaging molecule on the extracellular vesicles and a method of modifying the imaging molecule on a ligand molecule specifically targeting P-selectin.
Preferably, the method for modifying the imaging molecule on the extracellular vesicle can be a method for modifying the imaging molecule on the extracellular vesicle by fusing and expressing a fluorescent protein or luciferase and an extracellular vesicle membrane protein by using genetic engineering, a method for labeling the extracellular vesicle by using a lipophilic fluorescent dye, a method for modifying a radioisotope on the extracellular vesicle by using metabolic incorporation, a method for adsorbing a nano fluorescent material on the surface of the extracellular vesicle membrane by using an electrostatic adsorption principle, or a method for loading a quantum dot or a nano fluorescent material into the extracellular vesicle by using co-incubation or electroporation.
Preferably, the method for modifying the imaging molecule on the ligand molecule targeting P-selectin may be a method of coupling a fluorescent dye to the side chain of an amino acid residue of the ligand molecule using click chemistry.
An application of an engineered extracellular vesicle composition of a targeting P-selectin in a reagent for diagnosing and treating diseases related to endothelial cell injury.
Preferably, the endothelial cell injury related disease can be tumor, cardiovascular disease, ischemia reperfusion related disease, diabetes induced microangiopathy related disease, autoimmune related disease, inflammation related disease.
Therefore, the engineered extracellular vesicle composition targeting the P-selectin, the preparation method and the application thereof have the following technical advantages:
in the invention, the extracellular vesicles in the engineered extracellular vesicles targeting the P-selectin are derived from human cells or platelets, and compared with the existing liposome or nano material, the extracellular vesicles consist of natural lipid and surface protein, and have the advantages of low immunogenicity, long blood half-life, high biological safety and the like; meanwhile, the production mode and the composition of the extracellular vesicles endow the extracellular vesicles with the characteristics of being easy to modify in various modes and modifying various functional groups; in addition, extracellular vesicles have a lipid membrane-encapsulated vesicle structure that enables them to load different hydrophilic functional components in the lumen like "trojan horses", or hydrophobic functional components in their phospholipid bilayers, and deliver exogenous functional components to the site of injury.
The engineering extracellular vesicle of the targeting P-selectin provided by the invention has multiple functions, and the damage degree of endothelial cells can be reflected on the basis of the quantitative expression quantity of the P-selectin by loading a molecular imaging contrast agent, so that the damage degree of tissues can be further indicated; in addition, endogenous or exogenous functional components carried in extracellular vesicles are delivered to the damaged part with high expression of P-selectin in a targeted manner, so that targeted treatment on endothelial cell damage related diseases can be realized; by combining the molecular imaging technology with the extracellular vesicle-based targeted therapy strategy, the diagnosis and treatment integration of endothelial cell injury related diseases is realized.
The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.
Drawings
FIG. 1 is a flow diagram of the preparation of engineered extracellular vesicles targeting P-selectin;
FIG. 2 is a scheme of the synthesis of dimyristoylphosphatidylethanolamine-polyethylene glycol-P-selectin binding peptide (DMPE-PEG-PBP, DPP) in example IV;
FIG. 3 shows the results of NMR detection of one-dimensional H spectra of DPP synthesized in example four;
FIG. 4 is a schematic diagram of the preparation process of P-selectin-binding peptide-modified engineered extracellular vesicles (PBP-EVs) according to example four;
FIG. 5 is a schematic representation of the different states of extracellular vesicles of examples one, four, five and six, wherein A is Extracellular Vesicles (EVs), B is PBP-EVs, C is Gaussia luciferase (Gluc) -modified PBP-EVs, and D is Cyanine5.5 fluorescent dye (Cy5.5) -modified PBP-EVs;
FIG. 6 is the results of characterization and identification of prepared PBP-EVs in example seven, A is the flow cytometry detection results of the labeling efficiency of the synthesized PBP-EVs, B is the results of the particle size distribution of EVs and PBP-EVs, C is the results of the membrane potential detection of EVs and PBP-EVs, D is the transmission electron microscopy pictures (scale: 100nm) of EVs and PBP-EVs, E is the protein immunoblot strip of extracellular vesicle marker proteins Alix, TSG101 and CD63 in the mesenchymal stem cell lysate, the EVs extracted by separation and the prepared PBP-EVs;
FIG. 7 shows the stability test results of PBP-EVs prepared in example eight after storage at 4 ℃ for 1,3 and 7 days;
fig. 8 is an image of PBP-EVs ability to target damaged endothelial cells using Gluc bioluminescence imaging and Gluc signal intensity statistics for the ninth example, indicating P <0.05 compared to the time points corresponding to the EVs-injected group;
fig. 9 is an image of PBP-EVs ability to target damaged endothelial cells using cy5.5 fluorescence imaging and cy5.5 signal intensity statistics as shown in example nine, indicating P <0.05 compared to the time points corresponding to the EVs injected group;
fig. 10 is an image of PBP-EVs ability to target ischemia reperfusion injury kidney using Gluc bioluminescence imaging and Gluc signal intensity statistics for the tenth example, indicating P <0.05 compared to time points corresponding to the EVs injected group;
fig. 11 is an image of PBP-EVs ability to target ischemia reperfusion injury to kidney in example ten using cy5.5 fluorescence imaging and cy5.5 signal intensity statistics, indicating P <0.05 compared to time points corresponding to the EVs injected group;
FIG. 12 is an image of PBP-EVs signal intensity indicating different degrees of renal injury detected by Gluc bioluminescence imaging and a statistic of Gluc signal intensity indicating P <0.05 in example eleven;
fig. 13 is a test of kidney function and tissue structure after treatment with EVs and PBP-EVs in the eleventh example, a is serum creatinine and urea nitrogen levels on days 3 and 7 after injury in unilateral ischemia reperfusion kidney injury mice, indicating P <0.05 compared to PBS group; # denotes P <0.05 compared to EVs group; B-C is a hematoxylin & eosin staining picture of the ischemia reperfusion injury kidney tissue on the 3 rd day after the injury and statistics on the number of protein tube type and brush border lost tubules in the picture, and the scale is 100 mu m; denotes P <0.05 compared to PBS group; # indicates that P was <0.05 compared to EVs group.
Detailed Description
The technical solution of the present invention is further illustrated by the accompanying drawings and examples. It is understood that the described embodiments are a few, but not all embodiments of the invention and that the invention can be made by any method known or later developed by those skilled in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any mentioned methods may be performed in the order of events mentioned or in any other order that is logically possible. 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.
Unless defined otherwise, technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs.
The technical solution of the present invention is further described in detail with reference to several preferred embodiments, the implementation conditions adopted in the following embodiments can be further adjusted according to actual needs, the implementation conditions not mentioned are generally the conditions in routine experiments, and all the reagents can be obtained commercially. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Example one
An extraction method of extracellular vesicles secreted by human placenta-derived mesenchymal stem cells comprises the following steps:
1) prior to the experiment, ordinary Fetal Bovine Serum (FBS) was centrifuged at 120,000g for 18 hours at 4 ℃ to remove Extracellular Vesicles (EVs) in fetal bovine serum, and filtered through a 0.22 μm needle filter to obtain EVs-removed fetal bovine serum (EV-free FBS); and EV-free complete medium (containing 10% EV-free FBS, 1% diabody, 1% glutamine, 1% nonessential amino acids, 87% DMEM/F12 medium) was prepared using the above EV-free FBS for culturing human placenta-derived mesenchymal stem cells (hP-MSCs);
2) when the culture is normally carried out at 75cm2When the confluence degree of hP-MSCs in the cell culture bottle reaches 80%, discarding the old culture medium, adding 10mL of EV-free complete culture medium, continuing to culture for 24h, and then collecting the conditioned medium for gradient centrifugal separation of EVs;
3) centrifuging the collected 40mL of conditioned medium containing hP-MSCs-derived EVs at 500g for 10min to remove cell debris; collecting supernatant, centrifuging at 10,000g for 30min, and removing vesicles and apoptotic bodies with larger diameters; collecting the supernatant again, transferring to an ultracentrifuge tube for centrifugation at 100,000g for 70min, and precipitating hP-MSCs source EVs;
4) discarding the supernatant, washing the hP-MSCs-derived EVs in the tube once with 40mL sterile PBS, centrifuging for 120min at 100,000g, and finally precipitating to obtain hP-MSCs-derived EVs (as shown in the schematic diagram of FIG. 5A);
5) all steps and the whole process of extracting the EVs from the hP-MSCs are under the aseptic condition of 4 ℃;
6) confirming the concentration of the extracted EVs by using a BCA protein quantitative kit; the particle size distribution of the extracted EVs was confirmed by a dynamic light scattering instrument, and the morphological characteristics of the extracted EVs were confirmed by a transmission electron microscope.
Example two
The method for culturing the human placenta-derived mesenchymal stem cells by using the nitric oxide hydrogel and loading endogenous functional components in extracellular vesicles comprises the following steps:
1) coating nitric oxide hydrogel with concentration of 2 μ g/μ L at 75cm2Placing the bottom of a cell culture bottle in a cell culture box at 37 ℃ for more than 2 h;
2) culturing hP-MSCs in a cell culture flask coated with nitric oxide hydrogel, and separating and extracting EVs secreted by the hP-MSCs cultured on the nitric oxide hydrogel according to the extraction method in the embodiment 1;
3) and detecting and confirming the endogenous functional components loaded in the separated EVs by ELISA and real-time fluorescent quantitative PCR technology.
EXAMPLE III
The method for loading exogenous functional component adriamycin in the extracellular vesicles through co-incubation is as follows:
1) collecting the conditioned medium of the MDA-MB-231 cells, and separating and extracting EVs secreted by the MDA-MB-231 cells according to the extraction method in the first embodiment;
2) adding 1mmol/L adriamycin (Dox) into separated and extracted EVs, uniformly mixing, and then placing in a 37 ℃ cell culture box for incubation for 30 min;
3) transferring the co-incubated EVs/Dox mixture into an ultracentrifuge tube, adding 40mL of sterile PBS (phosphate buffer solution) for washing once, centrifuging for 120min at 100,000g, and finally precipitating to obtain Dox-loaded MDA-MB-231 cell-derived EVs;
4) the loading efficiency of Dox in EVs was confirmed by detecting the fluorescence signal of Dox at 590nm wavelength.
Example four
A method for modifying a ligand molecule P-selectin binding peptide specifically targeting P-selectin on extracellular vesicles by using a hydrophobic insertion method comprises the following steps:
1) a P-selectin binding peptide (PBP) is synthesized having the amino acid sequence: CDAEWVDVS, molecular weight 1028Da, purity more than 99%;
2) preparing an amphiphilic compound dimyristoyl phosphatidylethanolamine-polyethylene glycol-maleimide (DMPE-PEG-MAL) with the molecular weight of 5 kDa;
3) 30mg of DMPE-PEG-MAL and 30mg of PBP were dissolved in 2mL of dimethylformamide;
4) adjusting the pH of the solution to 8.0 by triethylamine;
5) placing the reaction system in N2Stirring at room temperature for 24h (reaction formula shown in figure 2) to obtain reaction product DMPE-PEG-PBP (DPP);
6) putting the reaction product into a dialysis bag with the molecular weight cutoff of 3kDa, stirring and dialyzing for 3 days at 4 ℃ by using deionized water, and removing impurities introduced in the reaction process;
7) freeze-drying the dialyzed reaction product to obtain DPP powder;
8) 0.5mg of DPP powder was dissolved in 150. mu. L D2In O, after the sample is completely dissolved, moving the sample into a nuclear magnetic tube, performing nuclear magnetic resonance one-dimensional hydrogen spectrum detection at 25 ℃ by taking deuterated acetone as an internal standard, confirming the components of a reaction product, and showing that a characteristic peak (7.0-7.6ppm area) of an indole ring contained in PBP exists in the reaction product, which shows that the PBP is coupled to DMPE-PEG to synthesize DPP (shown in figure 3) according to the experimental steps;
9) 3mg of the above-mentioned DPP (molecular weight 6028Da) lyophilized powder was dissolved in 1mL of sterile PBS to obtain a DPP stock solution with a concentration of 500. mu.M;
10) adding DPP storage solution into EVs solution with the concentration of 1 mug/muL according to the proportion of 100:1 to ensure that the final concentration of DPP is 5 muM;
11) mixing the above systems, standing at room temperature (25 deg.C) and incubating for 30 min;
12) transferring the incubated system into a 100kDa ultrafiltration centrifuge tube, and centrifuging at 13,000rpm for 20min at room temperature to remove unbound DPP;
13) the liquid remaining after centrifugation and the pellet were resuspended in PBS to give a PBP-modified engineered EVs (PBP-EVs) suspension (fig. 4 and fig. 5B schematic).
EXAMPLE five
A method for labeling molecular imaging contrast agent luciferase on extracellular vesicles comprises the following steps:
1) packaging a lentivirus expressing Gaussia luciferase (Gluc) -Lactadherin (Lactadherin) fusion protein (Gluc-lac):
one day before packaging virus, inoculating 1X 106293T cells in 10cm cell culture dishes;
② adding 45 mu L Lipo2000 gently into 1.5mL Opti-MEM culture medium, reversing the upper part and the lower part, mixing evenly, and incubating for 5min at room temperature; meanwhile, 9 mu g of the target gene plasmid pLV-Gluc-lac and the packaging plasmid pMDLg/pRRE (4.5 mu g), pRSV-REV (1.8 mu g) and pMD2.G (2.7 mu g) are added into another 1.5mL of Opti-MEM culture medium and are gently blown and beaten and mixed evenly; the plasmid solution is gently dripped into the Lipo2000 solution, the mixture is gently inverted and uniformly mixed, the blowing and sucking with a pipette are avoided, and the mixture is incubated at room temperature for 20min to obtain a mixture of the plasmid and the Lipo 2000;
thirdly, taking out 293T cells paved one day in advance, discarding the old culture medium, gently adding 5mL of culture medium without double antibody, gently dripping the mixture of the plasmid and Lipo2000 into the culture medium, and uniformly mixing; the whole process needs to be kept soft, and 293T cells are prevented from falling off from the bottom of the culture dish;
fourthly, after 12 hours of incubation in a cell culture box at 37 ℃, the culture medium containing the plasmids is discarded, 10mL of fresh DMEM complete culture medium is added into 293T cells, and the cells are placed at 37 ℃ and 5 percent CO2Continuously culturing for 48h in a cell culture box with saturated humidity;
collecting 293T culture medium containing virus particles, transferring the medium into a centrifuge tube, centrifuging at 1,300rpm for 5min to remove cell debris to obtain virus liquid containing virus particles for expressing Gluc-lac;
2) preparing EV donor cells stably expressing Gluc-lac:
firstly, preparing hP-MSCs with the cell confluence ratio of 50% in a six-hole plate, discarding an old culture medium, adding 1mL of a culture medium without double antibody, then adding 1mL of the culture medium containing virus particles and 2 muL of Polybrene with the concentration of 8mg/mL, and gently mixing;
secondly, sealing the pore plate by using a sealing film, placing the sealed pore plate in a centrifuge, centrifuging the sealed pore plate at the temperature of 1,600rpm and 37 ℃ for 60min, then removing the sealing film, and placing the pore plate in an incubator for incubation for 3-6 h;
thirdly, removing the culture medium containing the virus particles, replacing the culture medium with a fresh complete culture medium, placing the culture medium in a cell culture box for continuous culture, and observing the cell state at any time;
fourthly, after the cells are cultured for 48 hours, 2 mug/mL puromycin is added into the cells for screening; setting wild cells and adding the same amount of puromycin as a control, and after the wild cells are completely dead, remaining transfected cells are hP-MSCs for stably expressing Gluc-lac;
3) isolation of luciferase Gluc-labeled EVs:
culturing the hP-MSCs for stably expressing Gluc-lac in 75cm2In a cell culture flask;
secondly, separating and extracting EVs in the conditioned medium according to the experimental method in the first embodiment to obtain the EVs which are luciferase Gluc marked EVs;
4) DPP at a working concentration of 5. mu.M was added to Gluc-labeled EVs at a concentration of 1. mu.g/. mu.L as in example four, and the mixture was incubated at room temperature for 30min, followed by washing free DPP by ultrafiltration centrifugation and resuspension with PBS to obtain luciferase Gluc-labeled PBP-EVs (as shown in FIG. 5C schematic).
EXAMPLE six
The method for labeling the fluorescent dye of the molecular imaging contrast agent on the ligand molecule targeting the P-selectin comprises the following steps:
1) synthesis of fluorescent dye cy5.5-labeled DPP:
firstly, 1mg of Cy5.5-NHS powder is dissolved in 39.23 mu L of DMSO to obtain a Cy5.5-NHS solution;
② dissolving 4mg DPP powder in 162 mul deionized water to obtain DPP solution, and using 1MNaHCO3Adjusting the pH to 8.5;
③ adding 19.5 mu L of Cy5.5-NHS solution into the DPP solution, stirring and reacting for 24h at 4 ℃ to obtain a reaction product Cy5.5 marked DPP (Cy5.5-DPP);
adding the reaction product into a dialysis bag with the molecular weight cutoff of 3kDa, stirring and dialyzing for 3 days at 4 ℃ by using deionized water, and freeze-drying to obtain Cy5.5-DPP powder;
2) according to the experimental method of example four, Cy5.5-DPP of 5 μ M and EVs with concentration of 1 μ g/μ L were incubated at room temperature for 30min, unbound DPP was removed by ultrafiltration centrifuge tube, and the fluorescent dye Cy5.5-labeled PBP-EVs was obtained after resuspension (as shown in FIG. 5D schematic).
EXAMPLE seven
Method for identifying and characterizing PBP-EVs
1. Flow cytometry for detecting labeling efficiency of Cy5.5-labeled PBP-EVs
1) 200. mu.L of EVs sample at a concentration of 1. mu.g/. mu.L was added to a 1.5mL EP tube, followed by 2. mu.L of Cy5.5-DPP stock at a concentration of 500. mu.M in the tube, and incubation was carried out at room temperature for 30 min; free Cy5.5-labeled DPP was then washed away by ultrafiltration centrifugation and the remaining liquid and pellet were resuspended in 100. mu.L PBS to yield Cy5.5-labeled PBP-EVs;
2) performing flow detection on Cy5.5-labeled PBP-EVs under an APC channel by taking unmodified EVs as a blank control; the results showed that the labeling efficiency of Cy5.5-labeled PBP-EVs was about 98.6% (FIG. 6A).
2. Nano-particle size potential analyzer for detecting PBP-EVs particle size distribution
1) 300. mu.L of EVs sample at a concentration of 0.1. mu.g/. mu.L was added to a 1.5mL EP tube, followed by 3. mu.L of DPP stock at a concentration of 500. mu.M in the tube, and incubation was carried out at room temperature for 30 min;
2) subsequently, free DPP was washed off by ultrafiltration centrifugation and resuspended in 1mL PBS to give a PBP-EVs sample, which was placed on ice;
3) after a 0.2-micron filter membrane is used for dedusting a sample, the sample is added into a sample cup of a nanometer particle size potential analyzer, and the particle sizes of EVs and PBP-EVs samples are measured, the result shows that the particle sizes of the unmodified EVs and PBP-EVs are mainly distributed between 30 nm and 200nm, and the peak value (78.81nm) of the EVs particle size distribution slightly moves to the right after PBP engineering modification, and is about 97.33nm (shown in figure 6B).
3. Detection of PBP-EVs membrane potential by nano-particle size potential analyzer
1) 300. mu.L of EVs sample at a concentration of 1. mu.g/. mu.L was added to a 1.5mL EP tube, followed by 3. mu.L of DPP stock at a concentration of 500. mu.M in the tube, and incubation was carried out at room temperature for 30 min;
2) free DPP is washed away by ultrafiltration centrifugation and resuspended by 0.5mL PBS to obtain a PBP-EVs sample, which is placed on ice;
3) EVs and DPP-EVs samples are added into a U-shaped sample cup and inserted into a sample groove of a nanometer particle size potential analyzer, zeta potentials of the EVs and PBP-EVs samples are measured, and the result shows that the membrane potential of the EVs is not changed by DPP modification, and the membrane potentials of the PBP-EVs and the EVs are both negatively charged (shown in figure 6C).
4. Transmission electron microscope detection of PBP-EVs morphology
1) 200. mu.L of EVs sample at a concentration of 1. mu.g/. mu.L was added to a 1.5mL EP tube, followed by 2. mu.L of DPP stock at a concentration of 500. mu.M in the tube, and incubation was carried out at room temperature for 30 min; free DPP is washed away by ultrafiltration centrifugation, and PBP-EVs are resuspended by 100 mu L PBS to obtain a PBP-EVs sample which is placed on ice;
2) placing a 200-mesh copper net with a carbon support film on a sealing film, sucking 10 μ L of EVs or PBP-EVs sample, dripping the sample on the copper net, standing at room temperature for 5min, and sucking excessive liquid from the edge of the liquid drop by using filter paper;
3) dropping 2% phosphotungstic acid dye solution on a copper net loaded with EVs and PBP-EVs samples, dyeing for 1min, sucking the redundant dye solution by using filter paper, airing at room temperature, observing under a transmission electron microscope, and displaying Transmission Electron Microscope (TEM) pictures of EVs and PBP-EVs, wherein the PBP-EVs have the same morphological structure as the EVs and are both classical cup-shaped vesicle structures (figure 6D).
5. Identification of PBP-EVs marker protein by protein immunoblotting experiment
1) 200. mu.L of EVs sample at a concentration of 1. mu.g/. mu.L was added to a 1.5mL EP tube, followed by 2. mu.L of DPP stock at a concentration of 500. mu.M in the tube, and incubation was carried out at room temperature for 30 min; free DPP is washed away by ultrafiltration centrifugation, and PBP-EVs are resuspended by 100 mu L PBS to obtain a PBP-EVs sample which is placed on ice;
2) the concentrations of hP-MSC lysate, EVs and PBP-EVs are determined by using a BCA method, an SDS-PAGE sample buffer solution is added into a boiling water bath for 10min to prepare hP-MSC lysate, EVs and PBP-EVs protein samples, EV marker proteins Alix, TSG101 and CD63 in the hP-MSC lysate, EVs and PBP-EVs are detected by a protein immunoblotting experiment, protein components in the PBP-EVs are similar to those of the EVs, and the PBP-EVs are still rich in the EV marker proteins (figure 6E).
Example eight
The method for detecting the stability of the PBP-EVs comprises the following steps:
1) 200 μ L of EVs sample at a concentration of 1 μ g/μ L was added to a 1.5mL EP tube, followed by 2 μ L of Cy5.5-labeled DPP stock at a concentration of 500 μ M in the tube, and incubation at room temperature for 30 min; free Cy5.5-labeled DPP was then washed away by ultrafiltration centrifugation and Cy5.5-labeled PBP-EVs were resuspended in 100. mu.L PBS to give a sample of Cy5.5-labeled PBP-EVs;
2) standing the prepared Cy5.5-labeled PBP-EVs sample at 4 ℃, respectively preserving for 1,3 and 7 days, and performing flow detection on each group of EVs sample by taking unmodified EVs as blank control under an APC channel; the result shows that the PBP-EVs stored at 4 ℃ for one week can still maintain about 99% of Cy5.5 positive rate, and the PBP-EVs can be stably stored in the environment of 4 ℃ for more than 1 week (shown in figure 7).
Example nine
Two methods for detecting endothelial cell targeted injury capacity of PBP-EVs
Gluc bioluminescence detection of targeting ability of PBP-EVs to damaged endothelial cells
1) Preparing Gluc-labeled PBP-EVs according to the experimental method described in the fifth embodiment, wherein the Gluc-EVs are used as a control and stored at 4 ℃ for later use;
2) inoculating Human Umbilical Vein Endothelial Cells (HUVECs) into a 24-well plate, and performing anoxic treatment for 6H/reoxygenation for 12H (H/R) injury treatment on 3 wells of the 24-well plate when the cell confluency reaches about 60%, so as to induce the HUVECs to express P-selectin; normal culture of cells in another 3 wells;
3) 500. mu.L of fresh medium was replaced for each well of cells and 100. mu.g/mL of Gluc-labeled EVs and PBP-EVs were added to 2-well normal-cultured and 2-well H/R injury-treated HUVECs, respectively; adding PBS with the same volume as the control into the HUVECs which are normally cultured in the other 1 hole and are subjected to H/R injury treatment in the 1 hole, and placing the HUVECs in a cell culture box at 37 ℃ for continuous culture for 2 hours;
4) unbound Gluc-labeled EVs and PBP-EVs are washed away by PBS, 500 mu L of PBS and 100ng/mL coelenterazine are added into cells of each hole, the cells are immediately placed in a small animal imager for bioluminescence imaging, internalized EVs and PBP-EVs in each hole are quantified through Gluc signal intensity, and the result shows that the PBP-EVs can identify P-selectin on the surface of vascular endothelial cells, are more bound and enter H/R damaged HUVECs, and show obvious targeting to damaged endothelial cells highly expressing the P-selectin (figure 8).
Cy5.5 fluorescence imaging detection of PBP-EV Targeted Capacity to injured endothelial cells
1) According to the experimental procedure described in example six, 200. mu.L of each EVs sample at a concentration of 1. mu.g/. mu.L was added to 2 1.5mL EP tubes, followed by 2. mu.L of Cy5.5-DPP stock solution at a concentration of 500. mu.M in one tube and 2. mu.L of DMPE-PEG-Cy5.5 stock solution at a concentration of 500. mu.M in the other tube, and incubation was carried out at room temperature for 30 min; then free Cy5.5-DPP and DMPE-PEG-Cy5.5 are washed away by ultrafiltration centrifugation, and EVs samples in 2 tubes are resuspended by 100 microliter PBS to obtain Cy5.5 marked EVs and PBP-EVs samples, and the samples are preserved at 4 ℃ in a closed light for standby;
2) spreading the cell slide in a 24-well plate, inoculating HUVECs, performing H/R injury treatment on cells in 3 wells when the cell confluence is about 60%, and normally culturing the cells in the other 3 wells;
3) changing 500 mu L of fresh culture medium for each hole of cells, adding 50 mu L of prepared Cy5.5 labeled EVs or PBP-EVs (the final concentration is 100 mu g/mL) into 2-hole normal culture and 2-hole H/R injury treated HUVECs culture medium respectively, adding PBS with the same volume as the control into the rest 2-hole cells, and placing the cells in a 37 ℃ cell culture box for further culture for 2H;
4) then washing unbound Cy5.5-labeled EVs and PBP-EVs with PBS, adding 500 μ L of 4% paraformaldehyde, fixing at room temperature for 10min, and washing with PBS for 2 times; then adding 200 mu L TritonX-100 with the concentration of 1% for membrane rupture, and incubating for 10min at room temperature;
5) taking out the cell slide, placing on a glass slide, dripping 10 mu L of prepared Alexa Fluor 488-phalloidin (1:500 dilution) and DAPI (1:2000 dilution) counterstain solution on the slide, and staining for 30min at room temperature;
6) PBS (phosphate buffer solution) is used for washing out redundant staining solution, anti-fluorescence attenuation blocking tablets are added dropwise for blocking, pictures are collected by a laser confocal scanning microscope, confocal pictures show that compared with other treatment groups, H/R (human/animal) damaged HUVECs internalize a large amount of PBP-EVs, the PBP-EVs have excellent targeting capacity on damaged endothelial cells, and the PBP-EVs can be adhered to the damaged endothelial cells through high-expression P-selectin and are phagocytosed and internalized by the damaged endothelial cells (figure 9).
Example ten
Taking mouse unilateral renal ischemia reperfusion injury as an example, two methods for detecting the capacity of PBP-EVs to target endothelial injury tissues are provided.
1. Establishing mouse unilateral renal ischemia-reperfusion injury (IRI) model
1) Injecting 2.5% avermectin (240mg/kg) into abdominal cavity to anaesthetize mouse, smearing eye ointment on eyeball of mouse to keep it moist, removing hair in back operation area of mouse, and sterilizing with iodophor;
2) making a longitudinal incision on the skin of the left kidney area on the back of the mouse, cutting the peritoneum, performing intermittent separation on the fat tissue behind the kidney, and exposing the left kidney;
3) separating connective tissues around the renal pedicle by using an ophthalmological forceps, exposing the renal artery and vein, clamping the renal artery and vein by using a microscopic vascular clamp to cause injury ischemia, turning the kidney into purple black, loosening the vascular clamp after 45min of ischemia, and recovering red color of the kidney within 1min to indicate that the reperfusion is finished;
4) sequentially suturing peritoneum and skin with 6-0 suture, placing animal in cage, and placing on heating pad until reviving;
5) randomly dividing IRI mice into PBS group and EVs treatment group, making incision and suturing only on the skin and peritoneum in the kidney area, and using the mice without ischemia reperfusion injury as a Sham operation group (Sham);
6) two groups of mice were injected with 100. mu.g/200. mu.L of EVs or PBP-EVs 3 consecutive times through the tail vein, respectively, and an equal volume of PBS was injected as a control 3 days after IRI.
Detection of targeting ability of PBP-EVs to ischemia reperfusion injury kidney by Gluc bioluminescence imaging
1) After 12h of mouse renal ischemia reperfusion, 100 μ g of Gluc-labeled EVs or PBP-EVs was injected via tail vein;
2) respectively carrying out Gluc living imaging on the two groups of mice 2, 6, 12 and 24 hours after injection, and starting and initializing a small animal living imaging system before imaging; meanwhile, Abamectin (240mg/kg) is injected into the abdominal cavity to anaesthetize the mice;
3) after injecting Gluc substrate coelenterazine (5mg/kg) into the abdominal cavity, immediately placing the mouse into a small animal imager cabin to enable the mouse to be in a prone position and adjust the position of the mouse, then carrying out bioluminescence imaging, collecting pictures, and imaging for multiple times until signals begin to weaken;
4) at the corresponding time points, the mice were bioluminescent imaged as per this procedure and the images were analyzed using the Living Image software.
5) The results are shown in fig. 10, after EVs were injected via tail vein, the Gluc signal in the damaged kidney region reached the peak value 6h after injection, and then gradually decreased; and the Gluc signal of the damaged kidney region of the mice injected with the PBP-EVs is gradually increased after injection until the peak value is reached at 12h, and the signal intensity is higher than that of the mice injected with the EVs within 6-24h after injection, so that the PBP-EVs have good damaged kidney targeting capability.
Cy5.5 fluorescence imaging detection of PBP-EVs Targeted ability to ischemia reperfusion injury kidney
1) After 12h of mouse renal ischemia reperfusion, 100 μ g of Cy5.5-labeled EVs or PBP-EVs was injected via tail vein;
2) at the corresponding time point, an excessive amount of Avermectin deeply anesthetized mouse is injected into the abdominal cavity, 40mL precooled PBS is used for perfusion, and free EVs and PBP-EVs in circulation are removed; taking out the kidney and other major organs at the injured side, and immersing in 4% paraformaldehyde for fixation;
3) after all specimens are obtained, an Imaging System IVIS Lumina Imaging System is opened and initialized, an organ to be detected is placed in a small animal imager for fluorescence Imaging, exciting light is 640nm, emitted light is Cy5.5, pictures are collected and analyzed by Living Image software.
4) The results show that the Cy5.5 signal intensity of the damaged kidney of the PBP-EVs injection group is higher than that of the EVs group (figure 11) at 6, 12 and 24h after injection, and the PBP-EVs can target the damaged kidney and are specifically enriched in the damaged kidney.
EXAMPLE eleven
By taking unilateral renal ischemia-reperfusion injury with different degrees as an example, the application of PBP-EVs in diagnosing and treating the ischemia-reperfusion renal injury is provided.
1. Establishing unilateral renal ischemia-reperfusion injury (IRI) models with different injury degrees
Establishing unilateral kidney IRI models with different damage degrees, specifically, as described in example ten, clamping left side kidney artery and vein of a mouse to ensure that the kidney is ischemic for different time and is reperfused, so as to obtain mouse unilateral kidney IRI models with different damage degrees; wherein unilateral renal ischemia 15 min/reperfusion is mild IRI model, ischemia 30 min/reperfusion is moderate IRI model, and ischemia 45 min/reperfusion is severe IRI model.
Diagnosis of degree of renal ischemia reperfusion injury by bioluminescence imaging of Gluc-labeled PBP-EVs
1) Establishing mouse models of three different degrees of renal injury (mild IRI, moderate IRI and severe IRI), and injecting 100 mu g of Gluc-labeled EVs or PBP-EVs through tail vein after reperfusion for 12 h;
2) after 12h of injection, detecting the Gluc signal intensity of the damaged kidney region of 2 groups of mice through bioluminescence imaging, and starting and initializing a small animal living body imaging system before imaging; meanwhile, Abamectin (240mg/kg) is injected into the abdominal cavity to anaesthetize the mice;
3) after injecting Gluc substrate coelenterazine (5mg/kg) into the abdominal cavity, immediately placing the mouse into a small animal imager cabin to enable the mouse to be in a prone position and adjust the position of the mouse, then carrying out bioluminescence imaging, collecting pictures, and imaging for multiple times until signals begin to weaken;
4) at the corresponding time points, the mice were bioluminescent imaged as per this procedure and the images were analyzed using the Living Image software.
5) The Gluc imaging result shows that the accumulation amount of the PBP-EVs in the damaged kidney is increased along with the increase of the damage degree of the kidney, and Gluc signals which are positively correlated with the damage degree are released; in contrast, in the EVs-injected group, only the damaged kidney region of the severely kidney-damaged mice showed increased Gluc signal (fig. 12). The result shows that the contrast agent for molecular imaging is loaded on the PBP-EVs, so that the degree of the kidney injury can be judged by detecting the signal intensity of the contrast agent accumulated in the damaged kidney PBP-EVs, and a reference index is provided for early diagnosis of acute kidney injury.
PBP-EVs for promoting repair of kidney function and structure of severe ischemia-reperfusion injury
1) Establishing a severe IRI kidney injury model, and injecting 100 mu g EVs or PBP-EVs through tail vein after 12h of reperfusion, wherein PBS is used as a control;
2) on 3 rd and 7 th days after the severe IRI, taking a mouse serum sample, and detecting the creatinine and urea nitrogen content in the mouse serum sample, wherein the result shows that the creatinine and urea nitrogen level in the mouse serum is increased sharply and then is slightly reduced on the 3 rd day after the injury; EVs treatment can reduce creatinine and urea nitrogen levels in serum of severe IRI mice; after the PBP-EVs targeted therapy, the serum creatinine and urea nitrogen levels of the severe IRI mice are remarkably reduced (figure 13A), which shows that the PBP-EVs promote the repair of the damaged kidney function;
3) hematoxylin & eosin (H & E) staining was performed on the kidney tissue sections on day 3 after severe IRI, and the repair of the damaged kidney structure was observed. Tissue section staining results show that after severe IRI, a large number of tubular lesions in the kidney necrose, manifesting as a large number of protein casts and brush border abscission; after EVs treatment, the number of protein cast and brush border shedding is reduced; after treatment with PBP-EVs, only a small fraction of the tubules appeared damaged (fig. 13B-C), suggesting that PBP-EVs promoted repair of damaged renal structures.
Therefore, the engineered extracellular vesicle composition targeting the P-selectin, the preparation method and the application thereof are adopted, the P-selectin is taken as a target spot of the engineered extracellular vesicle to target and damage endothelial cells, the purpose of targeting and damaging related diseases of the endothelial cells is realized, the blank that no universal technical means for targeting and diagnosing and treating the related diseases of the damaged endothelial cells exists in the prior art is filled, and the engineered extracellular vesicle composition can be widely applied to diagnosis and treatment of various related diseases of the damaged endothelial cells.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting the same, and although the present invention is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that: modifications and equivalents may be made to the invention without departing from the spirit and scope of the invention.
Claims (10)
1. An engineered extracellular vesicle composition targeting P-selectin, characterized by: comprises extracellular vesicles, ligand molecules specifically targeting P-selectin, molecular imaging contrast agents and endogenous or exogenous functional components carried in the extracellular vesicles;
the engineered extracellular vesicles are used for specifically recognizing and combining P-selectin on the surface of damaged endothelial cells, indicating the expression quantity of the P-selectin through the signal intensity of a molecular imaging contrast agent, and delivering carried endogenous or exogenous functional components to a damaged part.
2. The P-selectin-targeting engineered extracellular vesicle composition of claim 1, wherein: the extracellular vesicles are derived from human cells or from vesicles of platelets, including exosomes, microvesicles, platelet debris;
the human cell is a human-derived stem cell or a human-derived immune cell, and the human-derived stem cell includes but is not limited to a human placenta-derived mesenchymal stem cell, a human umbilical cord-derived mesenchymal stem cell, a human bone marrow-derived mesenchymal stem cell, a human adipose-derived mesenchymal stem cell and a human-derived induced pluripotent stem cell; human-derived immune cells, including but not limited to human T cells, human macrophages, human neutrophils, and human natural killer cells.
3. The P-selectin-targeting engineered extracellular vesicle composition of claim 1, wherein: the ligand molecule specifically targeting P-selectin is one or the combination of more than two of P-selectin glycoprotein ligand 1, P-selectin binding peptide, monoclonal antibody and fucoidin.
4. The P-selectin-targeting engineered extracellular vesicle composition of claim 3, wherein: the P-selectin binding peptide is a polypeptide containing an EWVDV pentapeptide core motif and specifically binds to P-selectin.
5. The P-selectin-targeting engineered extracellular vesicle composition of claim 1, wherein: the molecular imaging contrast agent is one or the combination of more than two of a radionuclide molecular imaging probe, an optical molecular imaging probe and a nuclear magnetic resonance molecular imaging probe.
6. The P-selectin-targeting engineered extracellular vesicle composition of claim 4, wherein: the optical molecular imaging probe comprises but is not limited to fluorescent dye, nano fluorescent material, quantum dot, fluorescent protein and luciferase.
7. The P-selectin-targeting engineered extracellular vesicle composition of claim 1, wherein: the functional components carried in the extracellular vesicles are natural active molecules or loaded exogenous functional molecules carried by the extracellular vesicles and derived from donor cells of the extracellular vesicles, the natural active molecules of the donor cells comprise proteins, nucleic acids and lipid molecules, and the loaded exogenous functional molecules are small molecule drugs, small interfering RNAs, microRNAs or proteins loaded exogenously.
8. A preparation method of an engineered extracellular vesicle composition targeting P-selectin is characterized by comprising the following steps:
s1, separating and extracting human cells or platelet-derived extracellular vesicles;
s2, loading endogenous or exogenous functional components in the extracellular vesicles;
s3, modifying the surface of the extracellular vesicle with a ligand molecule specifically targeting P-selectin, so that the extracellular vesicle has the capability of targeting P-selectin;
s4, modifying a molecular imaging contrast agent on the extracellular vesicles or ligand molecules to enable the extracellular vesicles to have a molecular imaging function, and obtaining the engineered extracellular vesicle composition targeting the P-selectin.
9. Use of an engineered extracellular vesicle composition targeting P-selectin, characterized in that: the application in the diagnosis and treatment of endothelial cell injury related diseases.
10. The use of an engineered extracellular vesicle composition targeting P-selectin according to claim 9, wherein: the related diseases of endothelial cell injury include but are not limited to tumors, cardiovascular diseases, ischemia-reperfusion related diseases, diabetes-induced microangiopathy related diseases, autoimmune related diseases, and inflammation related diseases.
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| CN114848608A (en) * | 2022-05-17 | 2022-08-05 | 东南大学 | Protein or polypeptide delivery carrier and preparation method and application thereof |
| CN114848608B (en) * | 2022-05-17 | 2024-01-30 | 东南大学 | Protein or polypeptide delivery carrier and preparation method and application thereof |
| CN116574676A (en) * | 2023-05-15 | 2023-08-11 | 南开大学 | Extracellular vesicle/engineering extracellular vesicle, and preparation method and application thereof |
| CN116574676B (en) * | 2023-05-15 | 2024-03-05 | 南开大学 | Extracellular vesicle/engineering extracellular vesicle, and preparation method and application thereof |
| CN117257975A (en) * | 2023-11-22 | 2023-12-22 | 四川大学华西医院 | Multifunctional extracellular vesicle and preparation method and application thereof |
| CN117257975B (en) * | 2023-11-22 | 2024-03-19 | 四川大学华西医院 | Multifunctional extracellular vesicle and preparation method and application thereof |
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