| Ischemic stroke can cause oxidative stress and produce large amounts of reactive oxygen species(ROS).ROS can cause brain endothelial cells and neurons to die by oxidizing and destroying brain microvascular endothelial cell membranes and neurons,thereby exacerbating brain damage and cerebral infarction.Edaravone(Edvone,Edv)as a strong free radical scavenger eliminates oxygen free radicals,inhibits brain cell peroxidation,and reduces tissue damage,so it is clinically used in the treatment of patients with ischemic stroke.However,Edv has a short half-life and low bioavailability,so a higher dose of Edv(30 mg,2 times/day)must be injected,which can cause complications such as renal dysfunction.In addition,due to the poor stability of Edv,it needs temporary preparation and complete intravenous injection within half an hour.These problems affect the effective use of Edv in clinical treatment of ischemic stroke.Therefore,it is important to find new strategies to improve the safety and availability of Edv in order to better treat ischemic stroke.Blood Exosome(Exo)is a natural drug carrier with natural brain targeting and good biocompatibility.It can efficiently penetrate the blood-brain barrier(BBB).Therefore,in this study,plasma exosomes with natural brain targeting were selected as a drug carrier,and Edv was loaded by incubation and drug loading to improve the stability and brain targeting of Edv.In vivo and in vitro uptake experiments were used to explore the brain targeting of edaravone-containing plasma exosomes(Exo-Edv);H&E staining experiments and hemolysis experiments were used to detect the in vivo safety of Exo-Edv;neurological scores,TTC staining,Nissl staining and other experiments to explore the neuroprotective effect of Exo-Edv on ischemic stroke rats.MethodsIn this study,a BCA protein quantification method for Exo content was established,and the iconic protein,particle size and morphology of Exo were investigated;the drug concentration of Edv was detected by the ultraviolet-visible spectrophotometer(UV)method,and its linearity,Methodology such as precision,repeatability,recovery rate;screening the optimal method for preparing Exo-Edv through orthogonal experiment;preparing Exo-Edv through4 ℃ incubation method,and verifying the morphology,particle size,and landmark protein of Exo-Edv The in vivo safety of Exo-Edv was investigated through hemolysis experiment and H&E staining experiment;the HPLC method was established to investigate the in vivo pharmacokinetics of Exo+DEV,and the pharmacokinetics of Exo+DEV were investigated.The tissue distribution was investigated;in vivo and in vitro uptake experiments were used to evaluate the brain targeting of Exo-Edv;the effects of Exo-Edv on permanent ischemic stroke rats were studied through TTC staining,neurological function score,and Nissl staining experiments.Neuroprotection.ResultsThe BCA quantification method of Exo content established in this study,the measured Exo concentration is 52.8 mg/m L,the particle size is about 150 nm,the shape is uniform and small,and the marker protein of exosomes is highly expressed,indicating that the ultracentrifugation extraction Obtained are plasma exosomes with higher purity;the linearity,precision,repeatability,and recovery methodologies of the established Edv UV quantitative analysis method meet the requirements.The optimal preparation method for preparing Exo-Edv was screened by orthogonal experiment: Plasma exosomes and Edv were incubated at 4 ℃ for 24 h,the concentration of Edv incubation was 1 mg/m L,and the concentration of plasma exosomes was 1 mg/ m L.The prepared Exo-Edv has a complete structure,a small sphere with regular morphology,uniform particle size,and good stability.The results of in vivo and in vitro uptake experiments show that Exo-Edv can target brain microvascular endothelial cells in the ischemic penumbra,and is compatible with The neuronal cells in the penumbra area are well co-localized.The results of pharmacokinetic studies show that Exo-Edv can prolong the half-life of DEV,reduce the clearance rate of Edv,and improve the stability of Edv in vivo.The results of TTC staining and neurological function score show that Exo-Edv can reduce the brain of PMCAO rats The infarct volume and the improvement of its neurological score indicate that Exo-Edv has a good protective effect on brain damage in rats with ischemic stroke.The results of Nissl staining show that Exo-Edv can inhibit the damage of Nissl bodies caused by cerebral ischemia,reduce the enlargement of Nissl bodies caused by ischemia,increase the number of Nissl bodies,and improve the vitality of neurons.Explains the neuroprotective effect of Exo-Edv on brain injury in rats with ischemic stroke.The results of hemolytic experiments and H&E staining experiments show that Exo-Edv will not produce hemolysis,nor will it cause pathological changes in various organs,which proves that Exo-Edv has good biological safety.ConclusionsThe plasma exosomes containing edaravone prepared in this study have brain-targeting properties,which can be targeted to the infarct area on the ischemic side of the brain through the principle of combining transferrin and transferrin receptor,and can reduce the brain Infarct volume,improve neurological score,increase the number of Nissl bodies,increase the number of positive neurons,and ultimately achieve neuroprotection.These research results provide a certain experimental basis for the potential clinical treatment of Exo-Edv to restore ischemic stroke injury. |
