Targeting Delivery Of Puerarin To Infarct Myocardium Using A Nti-cTnI Antibody Modified Liposomes

Myocardial ischemia brings great harm to people's life safety and quality of life.Long-term myocardial ischemia may lead to heart damage.Treatments of myocardial ischemic diseases of western medicine mainly rely on drug control and surgical methods.However,surgical operations put patients in a more dangerous position.Chinese traditional medicine is more acceptable to patients due to its mild nature and low toxicity.Puerarin has a significant effect on the treatment of myocardial ischemia,but the hydrophobicity of puerarin limits the application of puerarin in the treatment of myocardial ischemia.Therefore,the liposomes after modification of anti-cTnI antibody deliver puerarin directly into ischemic myocardium by targeted delivery,improving the bioavailability of puerarin greatly.The blank liposomes(LIP)and puerarin loading liposomes(Pue-LIP)were prepared by combination of the thin membrane evaporation and ultrasonic dispersion.DSPE-PEG2000-MAL and anti-cTnI antibodies were incubated in the 4-hydroxyethyl piperazine ethylsufonic acid(HEPES)solution anti-cTnI Ab-PEG2000-DSPE conjugate anti-cTnI antibody was obtained after 12h incubation.Then,Anti-cTnI Ab-Pue-LIP was obtained by incubating the anti-cTnI Ab-PEG2000-DSPE with the prepared puerarin liposomes.The morphology of the prepared liposomes was observed by transmission electron microscopy(TEM),the particle size and Zeta potential of the liposomes were measured by Nano ZS zetasizer,and the encapsulation rate was calculated after dialysis operation according to concertration of Pue.Drug toxicity was detected by MTT assay and uptake of liposomes of myocardial cells was detected by flow cytometry.Then,rat myocardial ischemiamodel was constructed.Hearts of the rats were stained with TTC to evaluate whether the rat myocardial ischemia model was successfully established.The uptake of liposomes by myocardial cells was detected after the label of fluorescence probe.The interaction of LIP,Anti-cTnI Ab-LIP with cTnI antigen was evaluated by MST,dedecting the ability of the antibody bind to the antigen.The therapeutic effects of anti-cTnI Ab-Pue-LIP were evaluated by electrocardiogram analysis and sonography.The shape of the prepared liposomes observed by electron microscopy were spherical.The results of size distribution showed that the anti-cTnI Ab-Pue-LIP had uniform distribution of particle size and the average particle size was between 100-120 nm,the liposome was negatively charged(-33.4 ± 0.79 mV).The average entrapment efficiency of anti-cTnI Ab-Pue-LIP was 72.7±1.47%and the embedding ratio was2.12±0.21%.The release rate of puerarin liposomes before modification was 47.72±2.37%at 8h,and the liposome release rate after anti-cTnI antibody modification was 46.79±2.46%at 8h.The results of the in vivo imaging tests showed that anti-cTnI Ab-LIP had the strongest fluorescence signal.The results of MTT assay showed that the toxicity of the drug to cells can be effectively reduced after being encapsulated with liposome.The results of flow cytometry revealed that the uptake of anti-cTnI Ab-LIP by cardiomyocytes was 3.6 times than that of common liposomes.The MST test results showed that the KD value of anti-cTnI Ab-LIP with the myocardial ischemia marker cardiac troponin(cTnI)was smller.Sonographic images of rats showed that the increasing of left ventricular endothelium caused by myocardial ischemia can be effectively alleviated by using Anti-cTnI Ab-LIP.The electrocardiographic results of rats showed that Anti-cTnI Ab-Pue-LIP could improve the electrocardiographic fluctuation of rats with myocardial ischemia and make them tend to the ECG of normal rats.The aboved results indicated that the liposomes made by the process of thin membrane hydration and ultrasonic dispersion were stable,small and uniform while the encapsulation efficiency is satisfactory.The drug release was not affected by the modification of anti-cTnI antibody.Cytotoxicity of puerarin to myocardial ischemic cells can be effectively reduced after encapsulation of liposome.The accumulation of Anti-cTnI Ab-LIP in the infarct heart was higher than unmodified liposomes.Moreover,the uptake of Anti-cTnI Ab-Pue-LIP by cardiomyocytes was the stronger than that of unmodified.Anti-cTnI Ab-LIP was able to improve the cinical results of heart function by sonography.Anti-cTnI Ab-Pue-LIP are more effectively heart-targeting drug delivery systems for the treatment of myocardial ischemic diseases.