Preparation Of A Drug Carrier Based On Brain Microvascular Endothelial Cell-derived Exosomes And Its Therapeutic Effect On AD Mice

ObjectiveThe accumulation ofβ-Amyloid（Aβ）in the brain is one of the major factors in the pathogenesis and progression of Alzheimer’s disease（AD）.P-glycoprotein（p-gp）,as one of the members of the ATP transport family,plays a role in clearning Aβin the treatment of alzheimer’s disease.The expression of P-gp in the pathological blood-brain barrier（BBB）was lower than the normal level,which hindered the clearance of A.Exosome（Ex）is a tiny vesicle secreted by cells.Functional proteins and RNA carried by Ex from different sources play a role in the treatment of AD and other diseases.As a free radical scavenger,Edaravone（Eda）plays an active role in the treatment of neurological diseases such as stroke and AD.In this study,Ex is intended to be developed into a new drug carrier,and the exosomes derived from brain microvascular endothelial cells（HBMVECs-Ex）are obtained by means of ultra-fast centrifugation.Investigate loading capacity of HBMVECs-Ex on the model drug edaravone,and the influence of HBMVECs-Ex on the expression of P-gp and the effect of Aβon the transport efficiency is analyzed.To explore the effects of HBMVECs-Ex on the drug-loading capacity of Eda,the learning and memory of AD mice and its mechanism of action,to achieve the dual therapeutic effect of carrier and drug.MethodsHBMVECs-Ex were extracted by ultracentrifugation,and the specific proteins,particle size and surface morphology of HBMVECs-Ex were characterized by Western Blot,potentiometric nano-particle size molecular weight analyzer,and atomic force microscope（AFM）.Monolayer HBMVECs cells were used to simulate the blood-brain barrier to explore the effect of HBMVECs-Ex on Aβclearance efficiency;Western Blot experiments were used to explore the interaction between Aβand P-gp,and the effect of HBMVECs-Ex on p-gp in HBMVECs after pretreatment of Aβ.Using a confocal microscope,observe the uptake and mechanism of Di I-labeled HBMVECs-Ex by HBMVECs cells.Lateral ventricular injection Aβ_1-42was used to induce the establishment of AD mouse model;The Morris Water Maze test（MWM）was used to analyze the differences of learning and memory ability between different groups of mice;The neuroprotective effect of HBMVECs-Ex was investigated by nils staining;Immunofluorescence assay was used to investigate the effect of HBMVECs-Ex on p-gp and A in AD model mice.The HBMVECs-Ex loaded with Eda（Eda-HBMVECs-Ex）was prepared by incubation method.The loading efficiency of HBMVECs-Ex on edaravone was investigated by ultraviolet spectrophotometer.HPLC was used to analyze the Eda content in the brain of mice.MWM was used to investigate whether Eda-HBMVECs-Ex could further improve the learning and memory ability of AD mice.ResultsIn this paper,HBMVECs-Ex was successfully separated by hypercentrifugation,and the obtained HBMVECs-Ex had good dispersion and uniform particle size.In vitro experiments,Aβ_1-42was used as a drug tool to establishe the blood-brain barrier model of dementia to study the protective effect of HBMVECs-Ex on BBB.The results showed that HBMVECs-Ex had the function of restoring P-gp expression in HBMVECs after pretreatment with Aβ_1-42.In the Western Blot experiment and vitro penetration experiment of Aβ,compared with the Aβ_1-42pretreated cells,the P-gp level was significantly improved when treated with HBMVECs-Ex and the permeability of FITC-Aβ₄₂in vitro BBB model was significantly improved.In the uptake experiment,HBMVECs showed a large uptake of HBMVECs-Ex.In vivo experiments,Aβ_1-42was used as a drug tool to establish an AD mouse model by lateral ventricular injection to study the neuroprotective effect of HBMVECs-Ex on model mice.The results showed that the injection of HBMVECs-Ex into tail vein could regulate the expression of P-gp in mice,enhance the clearance of Aβ,and improve the learning and memory ability of AD mice.In the Morris water maze experiment,HBMVECs-Ex reduced the escape latency of AD mice,increased the number of times of crossing the platform and the percentage of the quadrant where the platform was located.In the results of Nissl staining,HBMVECs-Ex had a certain protective effect on hippocampal neurons.In immunofluorescence experiments,HBMVECs-Ex can significantly increase the expression of P-gp and enhance the scavenging efficiency of Aβ.Eda-HBMVECs-Ex was successfully prepared,and the encapsulation efficiency of HBMVECs-Ex to edaravone was quantitatively examined by an ultraviolet spectrophotometer.The results showed that the encapsulation efficiency was more than 20%.The contents of edaravone in the brain of mice were analyzed by HPLC,and the results showed that HBMVECs-Ex could increase the contents of edaravone in the brain.MWM was used to analyze the learning and memory ability of mice,and the results showed that Eda-HBMVECs-Ex significantly reduces the escape latency and increases the Number of mice crossing platforms on day 6.ConclusionsHBMVECs-Ex is a nano-sized vesicle with good dispersion and uniform particle size,which has the function of improving the learning and memory ability of AD mice induced by Aβ.The mechanism may be to regulate the expression of P-gp on BBB and improve the clearance efficiency of Aβin the brain to protect the function of neurons.Eda-HBMVECs-Ex gives full play to the function of HBMVECs-Ex as a nano-carrier.Loading Eda assists it to enter the brain of mice,allows Eda to accumulate in the brain,and further exerts its brain protective effect,achieving the dual role of Eda and HBMVECs-Ex protective treatment can improve the learning and memory ability of AD mice.