Construction Of Targeted Stepwise Drug Delivery System And Its Application In The Treatment Of Ischemic Stroke

Cerebral ischemic stroke（CIS）is characterized by its high morbidity,high mortality,high disability and high recurrence rate,thus it seriously endangers human health.The effective reversal of mitochondrial oxidative stress damage on nerve cells is essential for the protection of brain tissue and its functional recovery.However,due to the existence of the blood-brain barrier and the distribution of drugs throughout the brain,it is often difficult to achieve effective disease treatment.In this study,prodrug technology and a step-wise-targeting nano-delivery system was used to transport melatonin to the mitochondria in nerve cells,to improve the therapeutic effect of drugs,effectively protect nerve cells,and achieve precise and efficient CIS treatment.The main research contents of this paper are as follows:Objective:To achieve the purpose of accurate and efficient treatment of CIS,TGN/SHp/TPP-MLT drug-loaded micelles targeting mitochondria of nerve cells damaged by oxidative stress in brain tissue was developed.Methods:（1）Synthesis and evaluation of mitochondrial targeting antioxidant triphenylphosphine-melatonin.Antioxidant melatonin（MLT）as a model drug,the mitochondrial targeting molecule triphenylphosphine（TPP）is reacted with melatonin to synthesize triphenylphosphonium-melatonin（TPP-MLT）.TPP-MLT was obtained using column chromatography,and its structure was confirmed by nuclear magnetic resonance spectroscopy and mass spectrometry.The distribution of MLT in cell mitochondria was quantitatively determined by high performance liquid chromatography.The oxidative stress damage cell model was established by H₂O₂induction,and MTT assay as well as dead/living cell staining was used to compare cell survival rate.The fluorescence probe DCFH-DA was used to label ROS level in oxidative stress injured cells and JC-1 fluorescence probe was used to label mitochondrial membrane potential.（2）Construction and evaluation of TGN/SHp/TPP-MLT micelles.TPP-MLT as a model drug,TGN peptides with high affinity to the blood-brain barrier and SHp peptides with high affinity to glutamate receptors that were specifically up-regulated for oxidative stress-damaged nerve cells during CIS as targeted groups,polyethylene glycol-polylactic acid was used as the carrier material to construct TGN/SHp/TPP-MLT micelles.（3）The fluorescence imaging and flow cytometry were used to investigate the targeting ability of TGN/SHp/TPP-MLT step by step targeting drug-loaded micelles,the ability of crossing blood-brain barrier in vitro and the uptake.The oxidative stress damage cell model was established by H₂O₂induction,and MTT assay as well as dead/living cell staining was used to compare cell survival rate.The fluorescence probe DCFH-DA was used to label ROS level in oxidative stress injured cells.CIS animal model of transient left middle cerebral artery occlusion was made by using suture method and the neurological deficit score,TTC staining,magnetic resonance imaging imaging examination,H&E staining technique,oxidative stress index were examined respectively.Results:（1）Results confirmed that more TPP-MLT was distributed in the mitochondria and TPP-MLT was better that MLT on improving cell survival.Fluorescent imaging results demonstrated that TPP-MLT treatment effectively reduced intracellular ROS levels and improved the mitochondrial membrane potential.（2）The micelles have a particle size of 30 nm,characterized by spherical and uniform particle size distribution.The release of TPP-MLT encapsulated in nanocarriers could be maintained above 24 h.TGN/SHp/TPP-MLT micelles had no effects on the cell viability of b End.3 and PC-12 cells even at concentration of 500μg/m L.Intravenous administration of TGN/SHp/TPP-MLT micelles did not cause hemolysis,abnormal changes in body weight,as well as liver and kidney function,suggesting its good biological safety.（3）Fluorescence imaging technology and flow cytometry results confirmed that TGN/SHp nanocarriers can be specifically taken up by brain microvascular endothelial cells b End.3 cells,and have a good ability to cross the blood-brain barrier in vitro,the 2h cumulative permeability being 3.9 times than TPP-MLT.Due to the high affinity of SHp to glutamate receptors,TGN/SHp nanocarriers could also be specifically taken up by oxidative stress-damaged nerve PC-12 cells.TPP-MLT encapsulated in TGN/SHp/TPP-MLT micelles released above24 h.The biodistribution results show that the TGN/SHp/TPP-MLT micelles can significantly increase the brain tissue transport of antioxidants,and TPP-MLT was preferentially enriched in the brain tissue injury site.The in vitro pharmacodynamic results demonstrated TGN/SHp/TPP-MLT micelles could effectively improve cell survival,reduce ROS level,and reverse abnormal mitochondrial membrane potential.The CIS animal model of transient left middle cerebral artery occlusion was made.The neurological deficit score,TTC staining,magnetic resonance imaging imaging examination,H&E staining technique,oxidative stress index results demonstrated the effects of TGN/SHp/TPP-MLT micelles on CIS.Conclusion:TGN/SHp/TPP-MLT drug-loaded micelles have been successfully constructed in this project,which can be actively transported to mitochondria of nerve cells damaged by oxidative stress in brain tissue,thus improving the therapeutic effect of drugs,effectively protecting nerve cells and realizing accurate and efficient CIS treatment.