Construction Of Platelet Membrane Modified PLGA-MSCs Biomimetic Nanoparticles And Its Repair Effect On Damaged Endothelium

BackgroundWith the progress of medical diagnosis and treatment technology,human beings have overcome many disease problems.However,cardiovascular disease is still the killer of human health worldwide,and the mortality rate of cardiovascular disease is still the first.For the mechanism,diagnosis,treatment and other aspects of coronary artery disease,researchers still need to continue to explore better or develop new diagnosis and treatment methods.Stem cell transplantation has shown a promising application in the treatment of ischemic heart disease.However,stem cell transplantation has problems such as immune rejection,limited origin of autologous cells,and lack of targeting.Mesenchymal stromal cells(MSCs)can srcrete soluble proteins,free nucleic acids,lipids and extracellular vesicles and so on through paracrine mechanism.These bioactive substances exist in the conditioned medium of MSCs.Studies have shown that these factors can increase the survival rate of cardiomyocytes and stimulate angiogenesis.PLGA(poly(lactic-co-glycolic acid))is one kind of copolymer with biodegradability and biocompatibility.PLGA has been used to encapsulate a variety of bioactive molecules,including proteins,genes and drugs.Platelet is one of the visible components in mammalian blood,which plays an important role in physiological and pathological processes such as hemostasis,wound healing,inflammatory response,thrombosis and organ transplantation rejection.Circulating platelets have a natural ability to target ischemic damage areas,and this targeting function is mainly played by glycoproteins on the surface of platelet membrane(PM).In recent years,biomimetic nano-drug delivery systems made of platelet membranes have gradually become a research hotspot.PurposeIn this study,PLGA-MSCs nanoparticles(NP)were constructed by combining PLGA with conditional culture medium of MSCs.PLGA-MSCs modified with platelet membrane were constructed by coating platelet membrane with nanoparticles.The characterization,time-sustained-release efficiency and biocompatibility analysis of PNP were completed.We also would explore the effect of PNP on endothelial cells in vitro.In vitro,we would mimic the vascular endothelial injury to explore the adhesion of PNP to endothelial.By establishing a rat model of internal carotid artery balloon injury and injecting PNP through tail vein,the repair effect of PNP on damaged intima was investigated in vivo.MethodsThe MSCs were cultured,and the conditioned culture medium was collected.PLGA-MSCs nanoparticles(NP)were constructed by aqueous phase and oil phase ultrasonic blending method.The characteristics of NP were identified by transmission electron microscopy(TEM),nanoparticle tracking analysis(NTA)and Atomic Force Microscopy(AFM).Enzyme-linked immuno sorbent assay(ELISA)was used to determine the ability of NP to release growth factors at different time points and to verify the effect of nano-bionic particles on human umbilical vein endothelial cells.Platelets were extracted from blood samples of volunteers,and platelet membrane(PM)was prepared by repeated freeze-thaw method.The expression of platelet specific protein CD42b before and after freeze-thaw was analyzed by Western blot.Platelet aggregation assay and ATP release assay were used to determine whether PM had the ability to activate platelet aggregation.PNP was prepared by coating PM with PLGA-MSCs nanoparticles by ultrasonic vibration method.PNP was identified by transmission electron microscopy(TEM)and atomic force microscopy(AFM),and the expression of CD42b on platelet membrane was verified by Western Blot.CCK-8 and live/dead experiments were used to detect the effect of PLGA-MSCs on the proliferation and activity of human umbilical vein endothelial cells.In vitro vascular intima injury was conducted to explore the adhesion effect of PNP onto the injured endothelium.A balloon injury model of internal carotid artery was established in rats,and PNP was injected through tail vein.The therapeutic effect of PNP on injured endothelium were further explored.Results1.The NP and PNP prepared in this experiment are tested by NTA,TEM and AFM.It can be seen that the distribution of NP is uniform and the concentration is 1×109/ml.The shape of NP can be seen under the transmission electron microscope as a smooth spherical body.The PNP image shows that the platelet membrane is successfully wrapped In the outer layer of NP,the diameters of the two are between 100-150nm,and there is no statistical difference(P>0.05);under the atomic force microscope,the morphology of NP and PNP are similar,and there is no statistical difference in diameter(P>0.05).2.Detect the VEGF,IGF-1 and HGF factors in the NP supernatant at different time points on 0 days,1 day,3 days,7 days,and 14 days by using the ELISA method.The results showed that the cumulative release percentage of growth factors of NP gradually increased,confirming PLGA has the function of slow-release growth factors.3.PM and PBS have no difference in their ability to stimulate platelet aggregation and ATP release under the same conditions(P>0.05).4.CCK-8 assay of cell activity confirmed that different concentrations of NP(10/20/40/60ug/mL)co-cultured with endothelial cells for 48 h could significantly increase the proliferation of cells(P<0.05).After 72 h,the cell activity of 40ug/ml and 60ug/mL was significantly higher than that of untreated group(P<0.001).5.After co-culture with endothelial cells with different concentrations of NP,cell death or survival staining analysis showed that NP could significantly reduce the proportion of dead cells(P<0.05).6.PNP(red fluorescence)could adhere to the injured vascular structure in the damaged surface,but NP could not adhere to the surface.In the internal carotid artery balloon injury rats model,after tail injecting PNP,we confirmed that the PNP treatment group could reduce the ratio of intima to media when compared to the groups of NP treatment group and PBS treatment group,which confirmed that the PNP could repair the damaged endothelium.ConclusionsPlatelet membrane modified PLGA-MSCs nanoparticles have the ability of adhesion to injured vascular endothelium and can promote the repair of injured endothelium through release of growth factors.