The Detection Of Atherosclerotic Plaques Via Dual-channel Fluorescence Imaging Of Biomarkers

Atherosclerosis is a disease in which atherosclerotic plaque is deposited on the blood vessel wall,resulting in artery narrowing.Cardiovascular diseases caused by atherosclerosis are one of the most lethal diseases in the world.The pathogenesis of atherosclerosis mainly includes activation of pro-inflammatory signaling pathway,overexpression of cytokines/chemokines and increase of oxidative stress.Oxidative stress is a key mechanism in the formation of atherosclerosis.When the artery wall in blood vessels is damaged,endothelial cells will undergo oxidative stress and produce large amounts of reactive oxygen species(ROS)and active nitrogen(RNS)to break the balance of the organism’s original antioxidant defense system,thus causing a series of inflammatory reactions.For example,bromate(HBrO)and nitrosyl peroxide(ONOO-)is active species with strong oxidation ability,can with proteins,lipids,cholesterol and rapid response such as DNA,During atherosclerosis,these strongly oxidizing substances promote the oxidation of lipids and cholesterol,thus accelerating the process of plaque formation.In order to neutralize the oxidative stress that occurs during inflammation,large amounts of reducing substances(such as glutathione)need to be produced in the cell to maintain normal cell function.Gamma-glutamyltransferase(GGT)can decompose extracellular glutathione into cysteine-glycine and glutamic acid.These downstream products pass through the cell membrane and then enter the cell for glutathione synthesis,in order to maintain the REDOX balance in the cell.Therefore,gamma-glutamyltransferase activity plays an important role in controlling glutathione level and maintaining REDOX balance in cells.In order to study the changes of reactive oxygen species and REDOX related enzymes in cells during disease progression,two fluorescent probes were designed and synthesized for simultaneous imaging of hypobromic acid and gamma-glutamyltransferase,hypobromic acid and nitrous oxide,respectively.These probes are highly sensitive,highly selective and low cytotoxic,and can be used for real-time,in situ fluorescence imaging of reactive oxygen species in cells and during atherosclerosis.The research content of this paper is as follows:1.A double-detection fluorescent probe C-HBrO-GGT,which can be sequentially activated by gamma-glutamyltransferase and subbromic acid,was designed and synthesized to mark the site of early atherosclerotic plaque.The probe was composed of 4-amino-7-trifluoromethyl coumarin as the parent,which was connected with γ-glutamine(γ-Glu)and 2-methylthiophenyl at coumarin 4 and 3,respectively.C-hbro-gamma-glutamyltransferase is hydrolyzed by gammaglutamyltransferase in solution,exposing the amino group of coumarin.The 2methylthiothiphenyl and amino group were then cyclized with subbromic acid and the probe fluoresced brightly yellow at 550 nm.The probe has good photophysical properties and low cytotoxicity,and can be used to detect γ-glutamyltransferase and subbromic acid effectively in vitro.At the cellular level,the probe was successfully used to distinguish normal cells from cancer cells,and detected elevated levels of γ-glutamyltransferase and subbromic acid in cells exposed to drug-stimulated oxidative stress.At the same time,the probe was successfully targeted to the plaque site of atherosclerotic mice,providing an effective tool to predict the location of plaque formation.2.A two-photon fluorescence probe(N-HBrO-ONOO-)was designed and synthesized for simultaneous imaging of hypobromic acid and nitrous oxide.The probe N-HBrO-ONOO-consists of methyl p-nitrobenzoate as the recognition group of nitrite peroxide,masked amino and S-methyl groups as the recognition group of hypobromic acid and 1,8-naphthalimide as fluorophones.The probe was chemically tested,and the experimental results showed that the probe N-HBrO-ONOOhad a good ability to simultaneously image hypobromic acid and nitrous oxide.The selectivity and stability experiments show that the probe can perform stable fluorescence imaging on subbromic acid and nitrous oxide for a long time,which lays a good foundation for the next cell imaging and in vivo imaging.