Enhanced Anti-ischemic Stroke Of ZL006 By T7-conjugated PEGylated Liposomes Drug Delivery System

ObjectiveThe treatment for ischemic stroke is one of the most challenging problems and the therapeutic effect remains unsatisfied due to the poor permeation of most drugs across the blood brain barrier(BBB).In this study,HAIYPRH(T7),a peptide that targeted to transferrin receptor(Tf R)can mediate the transport of nanocarriers across the BBB,was conjugated to liposomes for targeting and treating ischemic stroke.In addition,a novel compound named ZL006,which can specifically ameliorates the focal cerebral ischemic was entrapped in liposomes to evaluate the treatment efficacy.Methods1.The quantitative determination of ZL006 in samples was measured by HPLC.2.Liposomes loaded with ZL006,coumarin-6 and Di R were prepared respectively for different uses by the ethanol injection method,and determination of entrapment efficiency were investigated.3.Transport across the BBB in vitro was including cellular uptake by the brain capillary endothelial cells(BCECs)and MTT cell viability assay.4.Brain targeted effects in vivo were including efficiency of uptake of liposomes by the ischemic rats and biodistribution of liposomes in mice.5.In vivo pharmacodynamics was studied including evaluation of neurological deficit score and infarct area.Results1.The HPLC was calibrated with standard solutions of 1-100 ?g/ml of ZL006 dissolved in methanol(Y= 48030X-17645,correlation coefficient of R2=0.9997).The limit of quantification was 0.5ng/ml,and the coefficients of variation were all within 3.5%.2.T7-conjugated PEGylated liposomes(T7-P-LPs)loaded with ZL006 were showed satisfactory vesicle size and size distribution.The loading capacity of P-LPs/ZL006 and T7-P-LPs/ZL006 were 85.37±2.06 % and 87.89±3.58 % along with 9.40±0.45 % and 9.76±0.55 % encapsulation efficiency,respectively.3.The in vitro cellular uptake results showed that T7 modification increased liposome uptake by the brain capillary endothelial cells(BCECs),and little cytotoxicity of the targeting liposomes with or without ZL006 was observed.These results indicated that T7 played an active targeting role in T7-P-LPs uptake by BCECs and the in vitro toxicity of liposomes with or without ZL006 was evaluated on BCECs by MTT assay.4.The in vivo biodistribution and near-infrared fluorescence imaging evidenced that T7 modification rendered liposomes significantly enhanced the transport of liposomes across the BBB.5.The pharmacodynamic study suggested that,T7-P-LPs exhibited reduced infarct volume and ameliorated neurological deficit induced by ischemia reperfusion compared with unmodified liposomes or free ZL006.ConclusionIn summary,in vivo and in vitro study results indicated that T7 modification rendered liposomes significantly enhanced the transport of liposomes across the BBB and T7-modified PEGylated liposomes loaded with ZL006 exhibited reduced infarct volume and ameliorated neurological deficit compared with unmodified liposomes or free ZL006.Therefore,T7-P-LPs/ZL006 could be used as safe and potential targeted drug delivery system of ischemic stroke treatment.