Construction And Biological Application Of Organelle-targetable Two-photon Fluorescent Probes

In recent years,fluorescent probes have been extensively used as ideal detection tools because of their unique advantages(e.g.,high sensitivity,strong operability,and suitability for in vivo bioimaging)over other sensors.Two-photon fluorescent probe possesses several advantages,such as deeper imaging depth,less photobleaching and higher spatial resolution.It has been widely applied to the research areas of biology and medicine.Stroke,as one of the main lethal and disabling factors of global diseases,has ranked among the top three major mortality rates of diseases in China over the past two decades.The only effective measure for the clinical treatment of ischemic stroke is to use tissue plasminogen activator(t-PA)for thrombolysis.However,its time window is narrow(3–4.5 h)so that it is applicable only to 3%–5%of patients.Therefore,it is of great significance to explore the pathogenesis of cerebral ischemia and identify new treatment measures.In this dissertation,a series of two-photon fluorescent probes were designed and synthesized by exploiting the occurrence of inflammation during a stroke.The probes'fluorescence properties and application in the early diagnosis of stroke were preliminarily investigated.(1)A two-photon pH probe Mito-BNO with mitochondria-targeting function was designed and synthesized.This probe can recognize H~+very sensitively,and was characterized by ultra-fast reaction speed and reversible reaction,thereby enabling the real-time detection of pH changes in cells and in vivo.By coupling the mitochondria-targeting group triphenylphosphine,the detection sensitivity and accuracy were effectively increased.Application of the probe Mito-BNO in a cell model of oxygen glucose deprivation(OGD)and a mouse stroke model revealed that it can achieve the in situ detection and early diagnosis of stroke.(2)A two-photon NO probe Lyso-TP-NO with lysosome-targeting function was designed and synthesized.The N-nitrosation of an aromatic secondary amine was utilized to effectively reduce pH and other interferences and thus increase the reliability of the probe in detecting inflammation.Application of this probe to a cell model of oxygen glucose deprivation/reoxygenation(OGD/R)and a mouse stroke model proved that cerebral ischemia-reperfusion injury can induce inflammatory reactions in cells.By adding cycleanine,we found that the degree of cell death was significantly reduced,indicating that the drug can be used to effectively monitor the surgical treatment of secondary brain damage caused by stroke.(3)A two-photon GSH probe ER-GSH with endoplasmic reticulum-targeting function was also designed and synthesized.The probe responded well to GSH,and the fluorescence intensity increased 303.5-fold upon reacting with GSH for 40minutes in the one-photon mode.The probe was applied to a mouse stroke model,and the effects of the anti-inflammatory drugs cycleanine and NS-398 on the recovery system of post-stroke mice were evaluated.It was found that this class of anti-inflammatory drugs exerted a very good effect on the recovery of the reduction system in mice after stroke.