CN118059063A - Mesenchymal stem cell bionic nano vesicle for targeted treatment of vascular endothelial cells, and preparation method and application thereof - Google Patents
Mesenchymal stem cell bionic nano vesicle for targeted treatment of vascular endothelial cells, and preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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- A61K31/713—Double-stranded nucleic acids or oligonucleotides
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
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Abstract
The invention discloses a mesenchymal stem cell bionic nano vesicle for targeted treatment of vascular endothelial cells, a preparation method thereof and application thereof in resisting ischemic cerebral apoplexy. The bionic vesicle is composed of mesenchymal stem cell membrane protein of an outer layer, hydrogenated soybean phospholipid, dioleoyl phosphatidylethanolamine, cholesterol and miR-132-3 p-protamine complex which is used as an inner core encapsulation. The mesenchymal stem cell bionic nano vesicle carrier has good biocompatibility, can evade the clearance of an immune system, has prolonged in vivo circulation time, homing to a cerebral apoplexy injury area and targeting-combining cerebral vascular endothelial cells to release miR-132-3p, plays roles of relieving oxidative stress of the vascular endothelial cells, resisting apoptosis, protecting BBB and reducing secondary brain injury, and realizes safe and efficient treatment of ischemic cerebral apoplexy.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a mesenchymal stem cell bionic nano vesicle for targeted treatment of vascular endothelial cells, a preparation method thereof and application thereof in resisting ischemic cerebral apoplexy.
Background
Stroke is a serious cerebrovascular disease that causes permanent disability and death worldwide, with ischemic stroke accounting for about 87%. Tissue-type plasminogen activator (Tissue-type plasminogen activator, t-PA) is the only drug currently approved by the U.S. food and drug administration for the treatment of acute ischemic stroke, but its use has the problems of narrow therapeutic window, bleeding risk and more contraindications. Although most preclinical studies indicate positive therapeutic effects of neuroprotective drugs in animal models of ischemic stroke, few phase III clinical trials have yielded satisfactory results due to the Blood Brain Barrier (BBB) blocking drug delivery and severe off-target toxicity. Ischemia reperfusion brain injury severely affects stroke patient survival time and quality of life. Therefore, there is an urgent need to explore safe and effective cerebral ischemia injury treatment strategies.
The BBB is located at the interface between the peripheral circulation and the brain parenchyma, regulates molecular transport between blood and brain, protects the brain from circulating pathogens, and plays the role of a dynamic "goalkeeper" which is the key to maintaining the homeostasis of the brain microenvironment. The integrity of the BBB is the basis for ensuring normal functioning of the central nervous system (CNS, central nervous system), and disruption of the BBB is closely related to the occurrence and development of a variety of brain-related diseases, such as ischemic stroke, traumatic brain injury, alzheimer's disease, parkinson's disease, and the like. When ischemic stroke occurs, cerebral blood flow is interrupted, energy supply is suddenly stopped, BBB dysfunction immediately occurs and lasts for a plurality of weeks, and liquid, toxin, various immune cells and inflammatory factors in blood enter brain parenchyma caused by the increased paracellular permeability, so that serious secondary injuries such as cerebral edema, neuroinflammation, neuronal death and the like are caused. Many preclinical and clinical studies have demonstrated that BBB damage IS closely related to the deterioration of prognosis in IS patients. Brain microvascular endothelial cells (BMECs, brain microvascular endothelial cells) line the cerebral vessels, forming the primary interface with blood. BMECs are interconnected by tight and adhesive binding proteins to form a "switch" that limits BBB permeability. When ischemic reperfusion brain injury occurs, inflammation immediately occurs in blood vessels, and BMECs is subjected to oxidative stress and apoptosis, with the destruction of tight junctions, resulting in BBB dysfunction, exacerbating brain tissue ischemic injury. Therefore BMECs is a promising therapeutic target, critical for alleviating ischemia reperfusion brain injury.
MiR-132-3p, also known as angiomiRs, is a key regulator of angiogenesis, and has been shown to play a role in endothelial dysfunction, angiogenesis and cardiovascular disease. The p120 Ras-GTPase activator protein (p 120RasGAP, also designated as RASA 1) is a direct acting target of miR-132-3p, and is deactivated by enhancing the inherent GTPase activity of Ras, and is a key negative regulator of vascular development and reconstruction. Research shows that miR-132-3p can inhibit RASA1 expression in mice BMECs, increase Ras activity, induce protein phosphorylation of downstream PI3K/Akt pathway, effectively reduce ROS generation and apoptosis level in damaged BMECs, increase expression of tight junction protein, restore BBB barrier function, and have neurovascular protection effect in acute phase of ischemic cerebral apoplexy. However, in vivo delivery of mirnas still faces a number of difficulties. First, mirnas are easily degraded by widely distributed nucleases in body fluids, resulting in fewer mirnas actually reaching the treatment site. Studies have shown that naked RNA is almost completely degraded a few seconds after entering human plasma. Second, exogenous mirnas are immunogenic and are readily cleared by the mononuclear phagocyte system (mononuclear phagocytosis system, MPS). In addition, since there is charge repulsion between miRNA negatively charged and also negatively charged cell membranes, poor cellular uptake results. Finally, off-target side effects and poor lysosomal escape capacity, etc., are all obstacles to current miRNA applications. Therefore, it is important to develop a targeted drug delivery system for treating ischemic stroke by miRNA.
Disclosure of Invention
The invention aims to solve the problem that miRNA in vivo delivery is hindered, and constructs a mesenchymal stem cell bionic nano vesicle for targeted treatment of vascular endothelial cells. The bionic nano vesicle has good biocompatibility and prolonged in vivo circulation time, can actively target BMECs of ischemic cerebral apoplexy injury, realizes lysosome escape through the transformation of inverted Hexagonal phase (Hexagonal II, H II) of DOPE phospholipid after being ingested by BMECs, releases miR-132-3p to inhibit the transcription of RASA1 genes, and plays roles of resisting apoptosis, relieving oxidative stress and BBB protection by regulating and controlling downstream Ras/PI3K/Akt signal paths, thereby realizing the effect of safely and efficiently treating the ischemic cerebral apoplexy.
The aim of the invention is achieved by the following technical scheme:
A mesenchymal stem cell bionic nano vesicle for targeted treatment of vascular endothelial cells is characterized in that mesenchymal stem cell membrane proteins are inserted into a phospholipid film formed by Hydrogenated Soybean Phospholipid (HSPC), dioleoyl phosphatidylethanolamine (DOPE) and cholesterol through hydrophobic interaction to form a bionic vesicle outer layer, and miR-132-3 p-protamine complex is loaded in the vesicle. The nano vesicle prepared by the invention has a uniform spherical structure with a lipid bilayer, and the average particle size is about 120 nm.
In some examples, in the mesenchymal stem cell biomimetic nano-vesicles for targeted treatment of vascular endothelial cells, the molar ratio of Hydrogenated Soybean Phospholipid (HSPC), dioleoyl phosphatidylethanolamine (DOPE) and cholesterol in the phospholipid film is (2-3): 1: (1-2), preferably in a molar ratio of 5:2:3.
In some examples, in the mesenchymal stem cell bionic nano-vesicle for targeted therapy of vascular endothelial cells, the mass ratio of mesenchymal stem cell membrane protein to phospholipid film is 1: (80-120), preferably the mass ratio is 1:100.
The mesenchymal stem cell membrane protein is obtained by extracting mesenchymal stem cells by using a membrane protein extraction kit, for example, the mesenchymal stem cell membrane protein is obtained by extracting the mesenchymal stem cell membrane protein by using a ProteoExtract Native Membrane protein extraction kit membrane protein extraction kit.
The mesenchymal stem cells of the invention can be obtained by purchase or extraction, for example, from bone marrow of a healthy male C57BL/6 mouse with the age of 4 weeks, and the obtained mesenchymal stem cells can be cultured by a conventional method, for example, the whole bone marrow adherence method is used for purification and expansion.
The mole of the protamine and miR-132-3p in the miR-132-3 p-protamine compound disclosed by the invention is 2:1, the preparation method of which can be according to the conventional method in the art, in one embodiment, a specific preparation method is provided: miR-132-3p and protamine are respectively dissolved in pre-cooled DEPC water, so that protamine is used for: miR-132-3p is 2:1, and standing for 5 minutes.
The invention also provides a preparation method of the mesenchymal stem cell bionic nano vesicle for targeted treatment of vascular endothelial cells, which comprises the following steps:
(1) Dissolving soybean phosphatide, dioleoyl phosphatidylethanolamine and cholesterol in absolute ethyl alcohol according to a molar ratio, and evaporating by a rotary evaporator to form a phosphatide film;
(2) Dispersing miR-132-3 p-protamine complex in PBS solution, ultrasonically hydrating a phospholipid film through water bath, and stirring for 30 minutes by using a magnet to form a phospholipid film suspension;
(3) The mesenchymal stem cell membrane protein and the phospholipid film are 1: (80-120) adding mesenchymal stem cell membrane protein into the phospholipid film suspension liquid, and extruding 10 times through cellulose acetate membranes with apertures of 800nm, 400nm and 200nm respectively to obtain mesenchymal stem cell bionic vesicle solution; centrifuging to obtain mesenchymal stem cell bionic vesicle.
Centrifugation in the methods of the present invention may be performed in a manner conventional in the art, for example, centrifugation at 16,000g for 30 minutes. The mesenchymal stem cell bionic vesicle obtained by the invention can be resuspended by PBS and stored at 4 ℃.
In the method of the present invention, the molar ratio of hydrogenated soybean phospholipid, dioleoyl phosphatidylethanolamine and cholesterol is (2-3): 1: (1-2), preferably in a molar ratio of 5:2:3; in one example, the mass ratio of the mesenchymal stem cell membrane protein to the phospholipid film is 1:100; the pore size of the microporous filter membrane of the liposome extruder is 800nm, 400nm and 200nm.
The invention also provides application of the mesenchymal stem cell bionic nano vesicle for targeted treatment of vascular endothelial cells in preparation of medicines for treating ischemic cerebral apoplexy.
In the invention, the mesenchymal stem cell bionic nano vesicle finally formed by the mesenchymal stem cell membrane proteins inserted into the phospholipid bilayer through hydrophobic interaction has the following advantages compared with the prior art:
(1) Can evade the elimination of MPS and prolong the internal circulation time;
(2) The homing of the damaged part of the bionic vesicle brain is started in response to the excessive secreted SDF-1 chemotactic factor in the damaged part of the ischemic brain;
(3) The bionic vesicle can be selectively absorbed and acted by the damaged endothelial cells by combining with VCAM-1 which is up-regulated on the surface of the damaged endothelial cells;
(4) After the bionic vesicle is taken up by BMECs, DOPE phospholipid is subjected to H Ⅱ phase transformation in a lysosome acidic environment to release miR-132-3p to activate a RASA1/Ras/PI3K/Akt signal pathway at the downstream, so that BMECs oxidative stress and apoptosis level are reduced, BBB and cerebral vessels are protected, and secondary brain injury is reduced.
(5) The mesenchymal stem cell bionic nano vesicle for targeted treatment of vascular endothelial cells prepared by the invention is subjected to in vitro migration experiments, in vitro lysosome escape experiments, in vitro blood brain barrier protection experiments, in vivo targeting research and in vivo pharmacodynamics research. The result shows that the use of the mesenchymal stem cell bionic vesicle-entrapped miR-132-3p can prolong the in-vivo circulation time, avoid degradation by nuclease, and realize targeted delivery into injured endothelial cells, reduce BBB permeability, reduce secondary brain injury and play a role in resisting ischemic brain injury.
(6) The invention aims at key components BMECs of the first barrier BBB of ischemic cerebral apoplexy to treat, reduce the oxidative stress and apoptosis level of the cerebral apoplexy and recover tight connection, thereby reducing the permeability of the BBB, avoiding inflammatory molecules, immune cells and the like from entering the brain parenchyma in peripheral circulation and exacerbating brain injury. An effective targeted BMECs drug delivery strategy is provided, and a basis is provided for researching the key therapeutic effects of BMECs and BBB in ischemia reperfusion brain injury.
(7) The mesenchymal stem cell membrane protein is inserted into the phospholipid bilayer to construct the mesenchymal stem cell bionic nano vesicle, the preparation steps are simple, the membrane protein is convenient to store, and good biological activity is maintained, so that the nano vesicle has good biocompatibility, long circulation characteristic and BMECs targeting characteristic.
In conclusion, the mesenchymal stem cell membrane protein is inserted into the phospholipid bilayer to construct the mesenchymal stem cell bionic nano vesicle, so that the mesenchymal stem cell bionic nano vesicle has great application potential for targeted delivery of drugs to vascular endothelial cells damaged by ischemic stroke.
Drawings
FIG. 1 is a transmission electron microscope image of mesenchymal stem cell bionic nanovesicles targeted for treatment of vascular endothelial cells;
FIG. 2 is a laser particle sizer measurement of nanovesicle particle size;
FIG. 3 example 2 sodium dodecyl sulfate-polyacrylamide gel electrophoresis of mesenchymal stem cell biomimetic nanovesicles targeted for treatment of vascular endothelial cells;
FIG. 4 is a western blot of mesenchymal stem cell biomimetic nanovesicles targeted for treatment of vascular endothelial cells of example 3;
FIG. 5 is an agarose gel electrophoresis diagram of the biomimetic vesicle inner core miR-132-3p and protamine complex of example 4 in different ratios.
FIG. 6 is an in vitro SDF-1 trend migration pattern of mesenchymal stem cell biomimetic nanovesicles targeted for treatment of vascular endothelial cells according to example 5.
Fig. 7 is an in vitro blood brain barrier permeability profile of mesenchymal stem cell biomimetic nanovesicles targeted for treatment of vascular endothelial cells of example 6.
Fig. 8 is an in vivo fluorescence profile of mesenchymal stem cell biomimetic nanovesicles targeted for treatment of vascular endothelial cells of example 7 (left) and semi-quantitative graph thereof (right) for treatment of ischemic stroke.
Fig. 9 is an Evans blue staining chart (left) and a quantification chart (right) of a brain slice for treating ischemic stroke by using mesenchymal stem cell bionic nano-vesicles targeted for treating vascular endothelial cells in example 8.
Fig. 10 is a TTC staining chart of brain sections of example 9 for treatment of ischemic stroke with mesenchymal stem cell biomimetic nanovesicles targeted for treatment of vascular endothelial cells.
Fig. 11 is a graph showing the behavioral score of mesenchymal stem cell biomimetic nanovesicles targeted for treatment of vascular endothelial cells for treatment of ischemic stroke in example 10.
Detailed Description
The invention is further illustrated below with reference to specific examples and figures, which are carried out under the preferred conditions of the invention. The methods are conventional methods unless otherwise specified, and the starting materials are commercially available from the public sources unless otherwise specified.
Example 1
Preparation and characterization of mesenchymal stem cell bionic nano-vesicle for targeted treatment of vascular endothelial cells
(1) Preparation of mesenchymal stem cell membrane protein
The extraction method of the mesenchymal stem cells comprises the following steps: healthy male C57BL/6 mice of 4 weeks old were sacrificed, 75% ethanol was soaked for 10min, the feet were subtracted in an ultra clean bench, the shin and femur were separated, the connection of the shin and femur was further sheared, the muscles and fat attached to them were removed, and care was taken to maintain the integrity of the medullary cavity. Rinsing 3 times with PBS solution containing green-chain double antibody, shearing the tibia and femur, sucking 1mL DMEM complete culture medium, and flushing the bone marrow cavity content into the culture dish. The cell suspension was blown off, filtered with a 70 μm sieve, transferred to a 15mL centrifuge tube, centrifuged 300g, the supernatant was discarded, resuspended in DMEM complete medium, transferred to a T25 cell flask, cultured in a 5% co 2 cell incubator at 37 ℃, and after 3 days, the liquid was changed for the first time, and the culture was continued for purification and expansion. Extracting mesenchymal stem cell membrane protein by adopting ProteoExtract Native Membrane protein extraction kit membrane protein extraction kit.
(2) Preparation of miR-132-3 p-protamine complex
The preparation method comprises the following steps: miR-132-3p and protamine are respectively dissolved in pre-cooled DEPC water, so that protamine is used for: miR-132-3p is 2:1, and standing for 5 minutes.
(3) The preparation method comprises the steps of dissolving HSPC, DOPE and cholesterol in ethanol in a molar ratio of 5:2:3, and evaporating by a rotary evaporator to form a phospholipid film. The miR-132-3 p-protamine complex is dispersed in PBS solution, the phospholipid film is hydrated by water bath ultrasonic wave, and the mixture is stirred for 30 minutes by a magnet. The membrane protein: the phospholipid film was 1:100, adding membrane proteins into the phospholipid membrane suspension, and respectively extruding through cellulose acetate membranes with apertures of 800nm, 400nm and 200nm for 10 times to obtain the mesenchymal stem cell bionic vesicle solution. After centrifugation at 16,000g for 30min, the liposome pellet was resuspended in PBS and stored at 4 ℃. The morphology of the bionic nano-vesicles is characterized by a transmission electron microscope, and the nano-vesicles can be observed to be in a uniform spherical structure with a lipid bilayer in figure 1, and the average particle size is about 120 nm. The particle size of the nano vesicles measured by a laser particle sizer is shown in figure 2.
The mesenchymal stem cell biomimetic nanovesicles used in the following examples are all vesicles prepared in this example unless otherwise specified.
Example 2
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiment of mesenchymal stem cell bionic nano vesicle for targeted treatment of vascular endothelial cells
Protein expression profiles of mesenchymal stem cell total protein (Total cell protein), membrane protein (Memberne protein), mesenchymal stem cell biomimetic vesicles (MSCosome) prepared in example 1 and naked liposomes (Liposome) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE). After protein concentration of each group was determined with BCA, each group was diluted to the same concentration with RIPA lysate, mixed well with 5 Xloading buffer, heated at 100℃for 10 minutes, and samples were added to loading wells of 4-20% Bis-Tris protein gel, each well loading volume being 20. Mu.L, containing 60. Mu.g protein. After electrophoresis is completed, the gel is dyed by using coomassie brilliant blue staining solution, and the dyed protein expression band diagram shows that the mesenchymal stem cell bionic vesicle has an expression profile similar to that of mesenchymal stem cell membrane proteins, which indicates that the mesenchymal stem cell membrane proteins are successfully integrated into the bionic nano vesicle. The results are shown in FIG. 3.
Example 3
Western immunoblotting experiment of mesenchymal stem cell bionic nano vesicle for targeted treatment of vascular endothelial cells
Each set of samples was lysed with RIPA solution, centrifuged at 12,000g for 20 min, the supernatant collected and the protein concentration quantified. After separation by SDS-PAGE, the proteins were transferred to PVDF membrane with a pore size of 0.22. Mu.m. The PVDF membrane is blocked by TBST solution containing 5% skimmed milk powder, and is incubated for 1 hour at room temperature with shaking; incubation with primary antibody solutions against CD47 (1:1000), CXCR4 (1:500), VLA-4 (1:1000) and GADPH (1:1000) overnight with shaking at 4 ℃; finally, the HRP-conjugated IgG secondary antibody (1:5000) was incubated for 1 hour at room temperature. The strips were soaked with ECL luminophore and exposed in Tanon 4600,4630 series full-automatic chemiluminescence image analysis system. The results are shown in fig. 4, which demonstrate that the mesenchymal stem cell key membrane proteins CD47, CXCR4 and VLA-4 can successfully transfer to the surface of the biomimetic nanovesicle.
Example 4
Compression ratio investigation of protamine to miR-132-3p in mesenchymal stem cell bionic nano vesicle kernel for targeted treatment of vascular endothelial cells
The inner core of the mesenchymal stem cell biomimetic nanovesicle uses protamine to compress miR-132-3p to neutralize the negative charge of miRNA and form a nanoscale complex that is easily encapsulated. To determine the optimal compression ratio of protamine to miR-132-3p, protamine and miRNA were mixed in molar ratios of 0:1, 0.4:1, 0.8:1, 1:1, 1.2:1, 1.6:1, and 2:1, respectively, and incubated for 5 minutes at 4 ℃.1, the method comprises the following steps: 5, diluting the complex with red fluorescence loading buffer, adding 10 mu L of the complex containing 200ng miRNA into a loading hole of 2% agarose gel, carrying out 100V electrophoresis for 20 minutes in a Tanon HE-120 multifunctional horizontal electrophoresis tank, and finally exposing in a Tanon 1600-series multifunctional gel image analysis system. The electrophoresis results in fig. 5 show that protamine is able to fully compress miRNA when the molar ratio of protamine to miRNA is 2:1.
Example 5
In vitro SDF-1 trend investigation of mesenchymal stem cell bionic nano-vesicles for targeted treatment of vascular endothelial cells
The important role of SDF-1/CXCR4 axis in the migration of the bionic nano-vesicle to the brain injury site is verified by a Transwell experiment. Firstly, establishing a Transwell monolayer cell model: the bEnd.3 cells were plated at a density of 5X 10 4 cells/well in a Transwell cell culture chamber (pore size 1.0 μm, surface area 0.33cm 2), placed in 24 well plates and cultured routinely for 7 days, and the transendothelial resistance (TEER) of the cell monolayer was measured using a Millicell-ERS voltmeter (Millicell-ERS 2, millipore, USA), and monolayer cells with TEER values higher than 200Ω.cm 2 were used as BBB experimental models. After stimulating cells with 100ng/mL TNF- α for 12 hours, diO-labeled biomimetic nanovesicle solution diluted with 0.25mg/mL DMEM medium and control formulation were added to the upper chamber of the Transwell chamber at a loading volume of 800. Mu.L and 200. Mu.L of DMEM medium with or without SDF-1 was added to the lower chamber. After incubation at 37 ℃ for 6 hours, the lower chamber medium was collected and the fluorescence signal intensity was detected by a full-band multifunctional microplate reader. The results are shown in fig. 6, and the results show that the mesenchymal stem cell bionic nano-vesicles can home to the cerebral ischemia injury site through the enriched SDF-1 cytokines of the CXCR4 protein on the surface towards the cerebral ischemia injury site.
Example 6
In vitro blood brain barrier protection effect investigation of mesenchymal stem cell bionic nano vesicle for targeted treatment of vascular endothelial cells
An in-vitro blood brain barrier injury model is established, and the protection effect of the bionic nano vesicle on the blood brain barrier is examined. The bEnd.3 cells were seeded at a density of 5X 10 4 cells/well in the upper chamber of a Transwell cell culture chamber and placed in a 24-well plate for conventional culture for 7 days to form a dense single cell layer. Cells were stimulated with 100ng/mL of TNF- α solution diluted in DMEM medium for 12h, followed by treatment with different formulations for 12h, with normal cells as controls. After adding 1mg/mL of 10kDa FITC-dextran solution into the upper chamber of the Transwell chamber and co-culturing for 4 hours, the solution of the lower chamber was collected, fluorescence signal intensity was measured with a full-band multifunctional microplate reader, and paracellular permeability was examined with the flux of FITC-dextran across a monolayer of cells. The apparent permeability coefficient (Papp) of FITC-dextran to cells was calculated as:
dQ/dt is the transport amount of FITC per minute (ng/min), A is the surface area of the filter membrane (cm 2),C0 is the initial concentration of FITC (ng/mL), 60 is the conversion of minutes to seconds, the result is shown in FIG. 7, and the result shows that the mesenchymal stem cell bionic nano-vesicle can target and repair damaged vascular endothelial cells at the cell level, thereby restoring the function of the blood brain barrier.
Example 7
In vivo distribution and targeting investigation of mesenchymal stem cell bionic nano vesicles for targeted treatment of vascular endothelial cells
C57B6/J healthy male mice were randomly divided into three groups: targeted drug delivery system group (MSCo/miR-132-3 p group), non-targeted drug delivery system group (Lipo/miR-132-3 p group) and Sham operation group (Sham) group. Each group was injected with Cy7 fluorescent-labeled vehicle of the corresponding group, respectively, in the tail vein after cerebral ischemia reperfusion injury, respectively. At 2h, 6h and 12h post-dose, the distribution of each group of formulations In the brains of tMCAO/R mice was observed using a small animal biopsy imager (In Vivo IMAGING SYSTEM, IVIS), see left figure 8. Semi-quantitative analysis was performed by Region-Of-Interest (ROI) analysis, see right in FIG. 8. From the graph, at different time points, the fluorescence signals of the MSCo/miR-132-3p group are obviously increased compared with those of the Lipo/miR-132-3p group and the Sham group, the fluorescence intensities of the Lipo/miR-132-3p group and the Sham group are gradually weakened along with the duration of time, and the MSCo/miR-132-3p group still has stronger fluorescence intensity, which indicates that the mesenchymal stem cell membrane protein is integrated into a phospholipid bilayer, so that the bionic vesicle has prolonged in vivo circulation time and can be targeted and enriched in a diseased brain region.
Example 8
Investigation of in vivo blood brain barrier protection effect of mesenchymal stem cell bionic nano vesicle for targeted treatment of vascular endothelial cells
Evans blue staining was used to assess permeability of the blood brain barrier in vivo. the tMCAO mice were re-perfused for 1h and then injected with different formulations by tail vein, and after 48 hours of in vivo circulation, the mice were injected with 4% Evans blue solution at a concentration of 4mL/kg, and after 2 hours were perfused by heart with PBS and 4% paraformaldehyde. Mice were sacrificed and their brains were isolated, cut into 1mm thick coronal sections, photographed with a camera, see fig. 9, and the results demonstrate that mesenchymal stem cell biomimetic nanovesicles can reduce blood brain barrier permeability in an animal model of ischemic stroke.
Example 9
In vivo pharmacodynamics research of mesenchymal stem cell bionic nano vesicle for targeted treatment of vascular endothelial cells
After tMCAO mice are reperfusion for 1 hour, PBS, naked miR-132-3p, mesenchymal stem cell bionic vesicles (MSCosome) without miR-132-3p entrapped, non-targeting liposomes (Lipo/miR-132-3 p) without mesenchymal stem cell membrane proteins and mesenchymal stem cell bionic nano vesicles (MSCo/miR-132-3 p) are respectively injected into tail veins, and healthy mice are used as controls. After 48 hours of internal circulation, the brain is taken and frozen for 20 minutes at the temperature of minus 20 ℃, and the coronal surface is rapidly cut into 1 mm-wide coronal slices from front to back, and 5 slices are taken. The sections were placed in 1% (w/v) TTC staining solution and incubated at 37℃for about 20min until white brain tissue was observed in the infarcted area and bright red brain tissue was observed, and the stained sections were gently placed on a white plate and photographed with a camera. TTC staining results are shown in figure 10, wherein the rose part is normal brain tissue, the white part is infarct area, PBS, naked miR-132-3p and MSCosome administration groups have larger damage, the Lipo/miR-132-3p administration group can only slightly relieve brain damage due to lack of targeting, and the infarct area of MSCo/miR-132-3p can be obviously reduced, which indicates that the mesenchymal stem cell bionic nano vesicle has good protection effect on ischemic brain damage.
Mice were given the same formulation as described above by tail vein injection 1 hour after cerebral ischemia reperfusion injury, followed by administration every other day for a total of 7 days, after which post-operative treated mice were scored for neurological function according to the five-level quartering method established by Zea-Longa for evaluation of neurological deficit. The results of the neural function scoring are shown in fig. 11, and it can be seen that the mesenchymal stem cell bionic nano vesicle MSCo/miR-132-3P treatment can obviously reduce the neural function damage of the tMCAO mouse, and the statistical difference is obvious (P < 0.001).
Claims (10)
1. A mesenchymal stem cell bionic nano vesicle for targeted treatment of vascular endothelial cells is characterized in that the vesicle is formed by inserting mesenchymal stem cell membrane proteins into a phospholipid film formed by hydrogenated soybean phospholipid, dioleoyl phosphatidylethanolamine and cholesterol through hydrophobic interaction to form a bionic vesicle outer layer, and miR-132-3 p-protamine complex is loaded in the vesicle.
2. The mesenchymal stem cell bionic nano-vesicle targeted for treating vascular endothelial cells according to claim 1, wherein the mesenchymal stem cell bionic nano-vesicle has an average particle size of 123.30 ±0.78nm.
3. The mesenchymal stem cell biomimetic nano-vesicle targeted for the treatment of vascular endothelial cells according to claim 1, wherein the molar ratio of hydrogenated soybean phospholipid, dioleoyl phosphatidylethanolamine and cholesterol in the phospholipid membrane is (2-3): 1: (1-2), preferably in a molar ratio of 5:2:3.
4. The mesenchymal stem cell bionic nanovesicle for targeted therapy of vascular endothelial cells according to claim 1, wherein the mass ratio of mesenchymal stem cell membrane protein to phospholipid membrane is 1: (80-120), preferably the mass ratio is 1:100.
5. The mesenchymal stem cell bionic nanovesicle for targeted therapy of vascular endothelial cells according to claim 1, wherein the mesenchymal stem cell membrane protein is extracted from the mesenchymal stem cells by using a membrane protein extraction kit.
6. The mesenchymal stem cell biomimetic nanovesicle targeted for the treatment of vascular endothelial cells according to claim 1, wherein the molar ratio of protamine to miR-132-3p in the miR-132-3 p-protamine complex is 2:1, a step of; preferably, the preparation method comprises the following steps: miR-132-3p and protamine are respectively dissolved in pre-cooled DEPC water, so that protamine is used for: miR-132-3p is 2:1, and standing for 5 minutes.
7. The method for preparing the mesenchymal stem cell bionic nano-vesicle for targeted therapy of vascular endothelial cells according to any one of claims 1 to 6, comprising the following steps:
(1) Dissolving soybean phosphatide, dioleoyl phosphatidylethanolamine and cholesterol in absolute ethyl alcohol according to a molar ratio, and evaporating by a rotary evaporator to form a phosphatide film;
(2) Dispersing miR-132-3 p-protamine complex in PBS solution, ultrasonically hydrating a phospholipid film through water bath, and stirring for 30 minutes by using a magnet to form a phospholipid film suspension;
(3) The mesenchymal stem cell membrane protein and the phospholipid film are 1: (80-120) adding mesenchymal stem cell membrane protein into the phospholipid film suspension liquid, and extruding 10 times through cellulose acetate membranes with apertures of 800nm, 400nm and 200nm respectively to obtain mesenchymal stem cell bionic vesicle solution; centrifuging to obtain mesenchymal stem cell bionic vesicle.
8. The method of claim 7, wherein the centrifugation is at 16,000g for 30 minutes; preferably, the obtained mesenchymal stem cell biomimetic vesicles can be resuspended with PBS and stored at 4 ℃.
9. The method of claim 7, wherein the molar ratio of hydrogenated soybean phospholipid, dioleoyl phosphatidylethanolamine and cholesterol is (2-3): 1: (1-2), preferably in a molar ratio of 5:2:3; preferably, the mass ratio of the mesenchymal stem cell membrane protein to the phospholipid film is 1:100; the pore size of the microporous filter membrane of the liposome extruder is 800nm, 400nm and 200nm.
10. Use of the mesenchymal stem cell bionic nano-vesicle for targeted treatment of vascular endothelial cells according to any one of claims 1-6 in preparation of a medicament for treating ischemic stroke.
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