Mechanisms Of Extracellular Vesicles Uptake By Endothelial Cells Under Low And Oscillatory Shear Stress

Extracellular vesicles（EVs）,including exosomes,microvesicles,and other subtypes of cell-derived vesicles,have become an important mediator of intercellular communication and a potential therapeutic carrier,and have a wide application prospect.EVs are usually administered through systemic injection,which exposes them to a dynamic environment in the blood.The injected EVs spread in the blood and experienced a wide range of flow velocities,resulting in different vascular shear rates.The endothelial cells covering the inner surface of blood vessels are susceptible to the shear stress of blood flow.Hemodynamics caused by blood flow generates multiple mechanical forces that directly act on the endothelial cells on the surface of vessel walls.The distribution of EVs in blood vessels under different shear stress（flow field）and the effect of vascular endothelial cells on the phagocytosis of EVs is of great significance to guide the application and development of EVs in drug delivery carriers.As far as we know,few studies demonstrate the interplay of EVs and endothelial cells.Thus,the phagocytosis of EVs by vascular endothelial cells during blood flow needs to be elucidated.In this study,red blood cells derived extracellular vesicles（RBCEVs）were taken as the research object to study the effects of endothelial cells on the uptake of RBCEVs under different shear stress in vitro,the distribution of RBCEVs in blood vessels,and the function and mechanism of RBCEVs uptake by endothelial cells in different blood flow pattern in vivo.In detail,the main contents and conclusions of this study are as follows:（1）Preparation and characterization of RBCEVs.RBCEVs were prepared by the method of hypotonic and co-extruded.The sizes,surface zeta potential,morphology,transmission electron microscopy detection of RBCEVs were determined.Subsequently,RBCEVs were characterized in vitro by stability test,protein composition analysis,protein characterization.The results indicated that the RBCEVs can be successfully fabricated by the method of co-extrusion.RBCEVs had a typical hollow vesicle structure,good dispersibility,and good stability in an aqueous solution.In addition,RBCEVs retained the protein composition and key function proteins（CD47）on the surface of the nanodrugs.The successful preparation of RBCEVs creates the basic conditions for the subsequent experiments.（2）Low and oscillatory shear stress（LSS and OSS）enhanced the uptake of RBCEVs by endothelial cells in vitro.To verify whether the uptake of RBCEVs by endothelial cells can be affected by different flow shears,the orbital shaker system was used to simulate various flow shear conditions.Compared with normal shear stress（NSS）and high shear stress（HSS）,LSS promotes endothelial cells to uptake more RBCEVs.In addition,the parallel-plate flow chamber showed that more RBCEVs were internalized by endothelial cells under OSS compared with NSS.（3）Low and oscillatory shear stress enhanced the uptake of RBCEVs by endothelial cells in vivo.We further established three animal models,including the visual zebrafish model,mouse carotid artery ligation model,and Apoe^-/-mouse model,to investigate the RBCEVs uptake by endothelial cells in vivo.In the zebrafish model,the laser confocal real-time imaging and manual calculation showed that the velocity of PCV in the tail was only a third of that in DA,which was consistent with previous reports.Subsequently,the uptake of fluorescent-labeled RBCEVs was observed by a laser confocal microscope.The results showed that RBCEVs were prone to be internalized by endothelial cells in slow-flow regions（LSS regions）.Moreover,based on the mouse carotid artery ligation model,which was previously built to research atherosclerosis formation on the basis of OSS in the vessel,we found that a large number of RBCEVs were deposited and uptaken by endothelial cells in established OSS regions.In the atherosclerosis mouse model,we confirmed that RBCEVs were prone to uptake by endothelial cells in the lesion areas.In short,these results demonstrate that RBCEVs were preferentially internalized by endothelial cells in vivo,which may be due to the LSS or OSS increased the contact time of RBCEVs between endothelial cells.In addition to RBCEVs,and we also verified the uptake of endothelial cells to solid nanoparticles such as gold nanoparticles and PLGA in vivo.The results showed that both LSS and OSS could promote the uptake of nanoparticles by endothelial cells.We believe that the uptake of all kinds of same nano-size particles could be promoted by OSS and LSS.（4）Calcium ion inhibits phagocytosis of endothelial cells.Calcium ion involved in the uptake RBCEVs by endothelial cells was screened out.LSS was capable of promoting the engulfment of RBCEVs by endothelial cells,but the promoting effect could be suppressed by the addition of calcium ions.In contrast,BAPTA-AM,a specific calcium chelating agent,was able to facilitate the engulfment effect caused by LSS and relieved the suppression effect induced by HSS.Furthermore,it was found that both LSS and HSS can reduction of endothelial cells’membrane fluidity with probe Laurdan.In term of molecular mechanisms,calcium-mediated membrane fluidity is the key to the uptake of endothelial cells.These data directly demonstrate that calcium signals can mediate the engulfment of RBCEVs by endothelial cells by inhibiting the fluidity of the cell membrane.Taken together,LSS and OSS can increase the uptake of RBCEVs by endothelial cells in vivo and in vitro.The mechanism of uptake is mainly regulated by the calcium-mediated membrane fluidity of endothelial cells.The results provide a theoretical basis for the regulation and utilization of vesicle drug delivery systems in endothelial cells.