Evaluation Of Brain Targeting Function Of C(RGDyK) Cyclopeptide Mediated Liposomes Carrying HI-6 Antidotes

Objective:A liposome preparation of carrying oxime type reactivator（HI-6）with brain targeting function was prepared,and its brain targeting function was evaluated in vivo and in vitro.Methods:（1）In this study,HI-6 liposomes were prepared by ammonium sulfate gradient-thin film dispersion method.Using Phosphatidylcholine（PC）and Cholesterol（CHOL）as lipid material,High Performance Liquid Chromatography（HPLC）,the encapsulation rate and drug loading of HI-6liposomes were investigated in different ratios of lecithin to cholesterol,different ratios of lecithin to drug dosage,and different ultrasonic time.Combined with central point design-response surface method to optimize the prescription,the correlation was characterized by laser particle size analyzer.（2）Brain targeting peptide c（RGDyK）-PEG₂₀₀₀-CHOL was synthesized by EDC·HCl method.Based on the optimized preparation method of HI-6 liposomes,c（RGDyK）-PEG₂₀₀₀-CHOL was introduced to prepare c（RGDyK）mediated HI-6 brain-targeting liposomes,and the relevant characterization was carried out by laser particle size analyzer.（3）The primary culture and identification of brain astrocytes（AS）and brain microvascular endothelial cells（BMECs）were carried out.BMECs single culture and BMECs co-culture in vitro BBB model were prepared by Transwell chamber,and the related evaluation was carried out.（4）The cytotoxicity of free HI-6 and various liposomes containing HI-6 to BMECs was investigated by CCK-8 method.Transwell chamber was used to investigate the permeability of free HI-6 and various liposomes to BBB model in vitro.The contents of total sulfhydryl group in the whole blood and brain of SD rats exposed to DDVP were determined by ELLMAN method through intravenous administration and nasal administration.The reactivation rate of toxic enzymes in the central and whole blood of SD rats exposed to DDVP by free HI-6 and liposome was investigated.Results:In this study,c（RGDyK）-PEG₂₀₀₀-CHOL was successfully synthesized,and brain-targeting liposome(c（RGDyK）-PEG₂₀₀₀-HI-6-lipo)containing HI-6 was prepared for the first time.The prepared liposome was round and dispersed among particles,with an average encapsulation rate of（70.23±2.18）%.Theζpotential was-16.2 m V and the average particle size was 242.9 nm（PDI=0.149）.In addition,AS and BMECs were successfully extracted in this study,and the prepared liposomes loaded with HI-6 had low cytotoxicity to BMECs.The in vitro BBB model was established,and the TTER value of the model reached（54±3）Ω·cm²,which met the requirements of the model.c（RGDyK）-PEG₂₀₀₀-HI-6-Lipo has a better ability to span BBB in vitro than the unmodified Hi-6-lipo.The calculation of the reactivation rates of whole blood and central toxic enzymes in the infected rats treated by different administration routes showed that the central targeting effect of C（RGDyK）-PEG₂₀₀₀-HI-6-Lipo was stronger than that of HI-6-Lipo and free HI-6,and the intranasal route had better central targeting effect than that of intravenous route.Conclusion:In vitro and in vivo experiments of this study demonstrated that c（RGDyK）-PEG₂₀₀₀-HI-6-Lipo had a better ability to cross BBB and had a higher reactivation rate of central toxic enzymes compared with HI-6-lipo and free HI-6,indicating that liposomes modified with c（RGDyK）cyclic peptide had better central targeting.The success of this study is expected to improve the treatment level of nerve agent and reduce the harm of nerve agents to the central nervous system.