Preparation Of Plasma Exosomes As Drug Carriers And Their Protective Mechanism Against Cerebral Ischemia-reperfusion Injury

ObjectiveIschemic stroke is the leading cause of severe disability and death worldwide.Ischemic Reperfusion(I/R)triggers severe oxidative stress,resulting in massive neuronal apoptosis due to the production of large amounts of reactive oxygen species(ROS)in the mitochondria and activation of the mitochondria-mediated apoptotic pathway.The accumulation of ROS leads to the degradation of Tight Junction Protein(TJP),which plays a protective role in brain microvascular endothelial cells,and impairs the integrity of the blood-brain barrier(BBB),further aggravating brain injury.In ischemic stroke treatment studies,most drugs have difficulty crossing the BBB to achieve their therapeutic effects.Exosome(Exo)has been favored by many researchers because of its advantages in particle size,biocompatibility,and immunological properties.Plasma Exosomes(Pla-Exo)are easy to be extracted in large quantities and are less likely to induce immune responses and abnormal proliferation of cancer cells in the body.Therefore,in this project,we will prepare plasma exosomes and examine their drug loading capacity,investigate the protective effect and mechanism of Pla-Exo against I/R injury,and further explore the dual therapeutic effect of Pla-Exo and drugs.MethodsIn this experiment,Pla-Exo was obtained from fresh plasma by orbital blood sampling and ultracentrifuged,and the morphology,particle size and protein expression of Pla-Exo were characterized by atomic force microscopy,particle size analysis and Western Blot.endothelial cells(HBMECs)were used to explore the possible pathways and mechanisms of Pla-Exo through BBB;the uptake of Pla-Exo in brain tissue was observed by laser confocal microscopy,and the tissue distribution of Pla-Exo was examined;the uptake mechanism of Pla-Exo was investigated by in vitro BBB penetration assay.The intracellular ROS accumulation in the ischemic area was examined by the intracellular ROS assay to investigate the mechanism of its neuroprotective effect;the mitochondrial membrane potential changes in the ischemic brain tissue were examined by the mitochondrial membrane potential assay to determine the mitochondrial damage;the mitochondrial membrane integrity was examined by the Western Blot assay.In the neurofunctional experiments,the middle cerebral artery occlusion ischemia/reperfusion(MCAO/R)model and Pla-Exo administration model were established using C57BL/6 mice,and neurofunctional scoring was performed by Zea-Longa score and Ludmila Belayev score principles;2,3,5-triphenyltetrazolium chloride(TTC)assay was used to detect the infarct ratio in brain tissue.The immunofluorescence staining assay was performed to investigate the improvement of neuronal(Neu N)damage in the ischemic brain region in the MCAO/R and Pla-Exo groups;the Evans Blue(EB)assay was performed to detect BBB integrity;the immunofluorescence assay was performed to observe the endothelial cell marker CD34;the neuroprotective effect of drugs and the recovery of cerebral edema were measured by calculating the cerebral edema ratio;the Western Blot assay and the Western Blot assay were performed to detect the neurological effects of drugs.Western Blot assay and immunofluorescence assay were performed to detect the breakage and expression level of TJP including Occludin and Clandin-5.Finally,the mechanism was verified by Western Blot assay on ischemic and non-ischemic tissues.The expression of HSP70 in Pla-Exo was inhibited using VER155008(VER)as an inhibitor of heat shock protein 70(HSP70);Western Blot assay was performed to detect HSP70 protein levels in brain;then TTC staining assay,intracellular ROS assay,mitochondrial membrane potential assay,Zea-Longa score and Ludmila Belayev score was performed to verify the key role of HSP70 in the neuroprotective effect of Pla-Exo.The incubation method was applied to prepare Pla-Exo-Met loaded with metformin(Met);the loading efficiency of Pla-Exo-Met was investigated by UV spectrophotometry;the in vivo and ex vivo uptake assays were performed to investigate whether Pla-Exo-Met could be taken up by brain tissues;the neuroprotective effect of Pla-Exo-Met was further investigated by TTC staining.ResultsIn this paper,the plasma exosomes extracted by ultracentrifugation met the purity requirements.Pla-Exo had a particle size of about 100 nm,relatively uniform morphology and structural integrity,and the same operation was ensured for each extraction to extract Pla-Exo with uniform particle size and morphology.In the penetration experiments using HBMECs to mimic BBB,Pla-Exo had good penetration and improved penetration in the Lipopolysaccharide(LPS)-induced inflammatory environment with improved penetration.The BBB permeability of Pla-Exo was achieved through HSP70 and endothelial Toll like receptor 4(TLR4)mediated cytokinesis.In uptake studies,Pla-Exo can be taken up by brain tissue and thus exert therapeutic effects.In immunofluorescence assays and Western Blot experiments,Pla-Exo inhibited ROS accumulation in ischemic brain regions,elevated the damaged mitochondrial membrane potential,and maintained the integrity of mitochondrial membranes.In neurofunctional assay,Pla-Exo reduced the area of brain infarction and improved the neural injury in mice with I/R by TTC staining assay,neurofunctional scoring,and immunofluorescence assay.EB assay,Western Blot assay and immunofluorescence assay all found that Pla-Exo reduced TJP breakdown and enhanced the integrity and permeability of BBB after I/R.Statistically Pla-Exo caused a significant decrease in the rate of brain edema in mice after I/R.Western Blot experiments verified that Pla-Exo increased the level of Bcl-2 protein and inhibited the expression of apoptotic factors Cleaved Caspase-9,Cleaved Caspase-3 and Bax in ischemic brain regions of mice after I/R.Inhibition of HSP70 expression in Pla-Exo resulted in more ROS accumulation,lower mitochondrial membrane potential,higher cerebral infarction rate,and higher neurological scores in the Pla-Exo+VER group,and the therapeutic effects of Pla-Exo were reversed,validating the key role of HSP70 in the neuroprotective effects of Pla-Exo.Pla-Exo-Met was successfully prepared,and the encapsulation rate was above 48%.Pla-Exo-Met could cross the blood-brain barrier,allowing Met to accumulate in the brain and thus play a synergistic therapeutic role in reducing the volume of cerebral infarction and improving neurological impairment in mice.ConclusionsPla-Exo has good stability.pla-Exo allows Pla-Exo to enter brain tissue through specific binding between its own carried HSP70 and TLR4 receptors on the surface of HBMECs,achieving brain targeting and BBB penetration.pla-Exo increases the expression of HSP70 in the ischemic region after I/R,reverses mitochondrial damage due to abnormal increase in ROS by inhibiting damage,reversed the abnormal decrease of mitochondrial membrane potential,alleviated mitochondrial dysfunction,attenuated BBB damage,and inhibited the expression of apoptotic factors,thus exerting a neuroprotective effect to ameliorate I/R injury.pla-Exo,as a good nanocarrier,loaded with Met assisted its entry into the brain,which in turn exerted a dual protective therapeutic effect to restore neurological damage.