Study And Exploration Of Activatable Photoacoustic Probes In Atherosclerotic Plaque Imaging

Atherosclerotic cardiovascular and cerebrovascular disease is the main cause of death of Chinese residents.With the increasing level of population aging in China,the incidence and mortality of cardiovascular diseases continue to rise,seriously affecting the implementation of the"healthy China"strategy.In the process of occurrence and development of atherosclerosis,there are two factors are important.On the one hand,oxidative stress,as the initial mechanism of atherosclerosis,runs through the whole process of atherosclerosis and is closely related to the occurrence and development of a variety of cardiovascular and cerebrovascular diseases.On the other hand,plaque rupture caused by proteolytic enzymes is the important pathological basis of ischemic cardio-cerebrovascular events and death in patients.Therefore,accurate assessment of the level of oxidative stress in plaques and the potrease enzymatic activity is of great significance for the early diagnosis and intervention of acute cardio-cerebrovascular events.However,at present,the clinical medical imaging techniques for plaque detection mainly include ultrasound,computed tomography imaging and magnetic resonance imaging,which mainly detect the structural information of plaques with organic lesions and the degree of vascular stenosis.It is difficult to reflect the early molecular level information related to plaque oxidative stress level and rupture risk.In recent years,molecular imaging technology can be used to detect disease-related biomarkers at cell,tissue,and in vivo levels.it is expected to provide a reliable imaging tool for early diagnosis of atherosclerosis,risk of plaque rupture and evaluation of therapeutic effect.Among many molecular imaging technologies,photoacoustic imaging combines the advantages of high resolution of optical imaging and high penetration depth of ultrasonic imaging,and can achieve high resolution imaging and detection of molecular level information in deep tissue.However,at present,there are still many problems in photoacoustic imaging probes used to evaluate plaque vulnerability,such as low signal intensity of photoacoustic contrast agent,poor imaging reliability,few activated photoacoustic imaging probes and so on.It limits the prospect of its clinical application.In view of the current challenges in imaging the intraplaque oxidative stress levels and rupture risk of atherosclerotic plaques,a series of new activatable molecular imaging probes is designed in this paper,which can specifically detect molecular biomarkers related to oxidative stress and plaque rupture.Besides,their feasibility is verified by solution,in vitro and in vivo experiments.Therefore,these probes provide a new way for early diagnosis and intervention of vulnerable atherosclerotic plaques.The details are as follows:1)In chapter 2,to accurately detect the superoxide anion(O₂^·-)that is produced in the early stage of oxidative stress,we developed a novel kind of ratiometric semiconducting polymer nanoparticle（RSPN）for photoacoustic imaging of vulnerable plaque in atherosclerotic mice complicated with pneumonia.Specifically,RSPN can react with O₂^·-and exhibit enhanced photoacoustic signals at about 690 nm,while 800 nm is regarded as an internal photoacoustic reference.As a result,RSPN can provide a reliable determination of O₂^·-within aortic atherosclerosis by analyzing the ratios of photoacoustic signals,which can successfully reflect the oxidative stress level in vulnerable plaque.Besides,RSPN enables to specifically distinguish plaque-bearing mice and plaque-bearing mice complicated with pneumonia from healthy mice,which provides a reliable tool to predict the vulnerability of plaque for reducing the mortality of the atherosclerotic-induced cardiovascular disease.2)In chapter 3,malondialdehyde（MDA）,the product of oxidative stress,is used as the target analyte.In order to solve the problem of absorption/emission wave length of traditional malondialdehyde probe,we use molecular design strategy to develop the first near-infrared malondialdehyde responsive probe（MRM）,which can be used to accurately detect the level of malondialdehyde in the disease site in vivo.Firstly,we synthesized a stable photoacoustic contrast agent（NIR-2）with strong photoacoustic signal,and further obtained MRM through one-step reaction.Under acidic conditions,MRM can react specifically with malondialdehyde,which increases the conjugated system of the probe and shows"on"photoacoustic and fluorescence signals.In order to improve the biocompatibility of MRM,we designed a malondialdehyde activated nanoprobe（NPs）,which can reliably image the dynamic changes of malondialdehyde at the cell level and in mice.Therefore,the probe shows a great application prospect in the diagnosis of inflammatory diseases.3)In chapter 4,in order to comprehensively evaluate the level of oxidative stress in atherosclerosis,based on the malondialdehyde probe developed in chapter 3,we further designed a multi-functional diagnostic tool with both urinary malondialdehyde detection ability and in vivo photoacoustic imaging for the simultaneous detection of systemic（urine）and local（plaque）oxidative stress.Specifically,we developed a highly sensitive and selective method for the detection of urinary malondialdehyde by optimizing the reaction conditions and testing methods of probe MRM with malondialdehyde,and its reliability was fully verified by blind and clinical experiments.In addition,we designed a ratio photoacoustic nanoprobe co-activated by acid and malondialdehyde to reliably detect the level of malondialdehyde in plaques.By combining urine detection with in vivo photoacoustic imaging,our multi-functional diagnostic tool can image the levels of systemic and local oxidative stress in different stages of atherosclerosis,during pneumonia infection and during drug treatment.it has important clinical significance for auxiliary diagnosis,risk stratification and efficacy monitoring of atherosclerosis.4)In chapter 5,using cathepsin B（CTB）,a biomarker of advanced atherosclerotic plaque rupture,as the target analyte,we developed a lipid unlocking CTB responsive probe（L-CRP）based on molecular design strategy,which can reliably reflect the activity of CTB in lipid-rich plaques in vivo.In addition,L-CRP can be enriched in atherosclerotic plaques and exhibit activatable photoacoustic signals that can be used for deep tissue imaging.Importantly,the L-CRP can be used for risk stratification in atherosclerotic mice and can well distinguish atherosclerotic plaque areas from normal blood vessels in human samples in vitro.Therefore,the activatable photoacoustic probe is expected to achieve high sensitivity detection of proteolytic enzyme activity in plaques.5)In chapter 6,to simultaneously image the level of oxidative stress and the risk of plaque rupture in atherosclerotic plaques,we developed a multifactor activatable ratio photoacoustic nanoprobe（MNPs）that can simultaneously detect the content of MDA and cathepsin B to evaluate the level of oxidative stress and the activity of proteolytic enzymes in atherosclerotic plaques.The nanoprobe can detect the content of MDA and CTB at the same time and show non-interference photoacoustic and fluorescence signals.In addition,MNPs can well target atherosclerotic plaques and achieve multi-channel imaging of multiple markers in atherosclerotic plaques,which is of great significance for the further study of molecular pathological changes in atherosclerotic plaques.