Construction Of Enzyme-Activatable Photoacoustic Probes For Radiation Monitoring And Tumor Theranostic Application

Background:Photoacoustic(PA)imaging is a newly emerged imaging modality in recent years that combines the high sensitivity of fluorescence imaging with the high spatial resolution and deep tissue penetration of ultrasound imaging,and has great potential in biomedical research.In recent years,a large number of PA imaging probes have been reported for disease diagnosis,therapeutic evaluation and disease status assessment in vivo.Activatable PA probes can selectively switch PA signal "on"from an "off" state upon interaction in the critical biomolecular or special biological microenvironment.These probes can effectively reduce background interference and improve detection sensitivity,which is conducive to high sensitivity real-time imaging in vivo.More importantly,if the ratio of PA signals of two different bands is used as the signal output,the problem that the output signals of traditional molecular probes are easily interfered by external factors can be effectively avoided,which greatly improves the accuracy of detection and imaging.In addition,theranostics PA probes that combine diagnostic information with therapeutic outcomes hold great potential for accurate diagnosis and personalized medicine.Objective:This paper was focuses on the essential scientific problem of early diagnosis and effective treatment of tumor.Two new types of enzyme-activatable PA imaging probes are designed and synthesized.Taking advantages of high accuracy of quantitative analysis of ratiometric photoacoustic in vivo,we performed near-infrared(NIR)fluorescence and photoacoustic(FL/PA)bimodal imaging of tumor cells and their apoptosis process,which realizing accurate quantitative analysis of biological enzymes in vivo,and providing a new strategy for accurate estimation of tumor radiation doses and efficient theranostics.Methods:In the first part,a new type of caspase-3 activatable ratiometric PA probe AcDEVD-Cy-RGD was designed and synthesized to study the tumor apoptosis in vivo by using near-infrared FL and PA imaging.This fluorogenic probe is comprised by a cyclic peptide(cRGD)targeting tumor cells,near-infrared dye(CyNH2),a caspase-3-cleavable peptide substrate(Ac-DEVD).Our probe can be specifically recognized and cleaved by the caspase-3 enzyme,while generating in situ bright NIR fluorescence.Moreover,this probe also emitted ratiometric photoacoustic(PA710/PA680)signals in a caspase-3 concentration-dependent manner,allowing for sensitive and quantitative detection of caspase-3 activity in vivo.More importantly,by quantitatively monitoring the activity of caspase-3 in radiation-treated breast tumors,we provide a new method for an accurate estimation of the radiation doses imparted to the tumors in vivo.In the second part,we report a new type of ALP-activatable photoacoustic imaging probe f-RCP.The probe consists of a tumor-targeting group(cRGD),an activatable photosensitizer(CyOP)and a 1O2-sensitive furan for cross-linking RNA.In ALP-positive tumor tissues,the probe dephosphorylates and releases strong near infrared fluorescence accompanied by changes in ratiometric PA(PA685/PA785)signal.Taking advantages of high accuracy of quantitative analysis of ratiometric photoacoustic in vivo,we can perform in situ real-time and highly sensitive imaging and quantitative analysis of ALP in living tumors.In addition,ALP can activate the photodynamic ability of photosensitizer,and generate a large amount of 1O2 under the irradiation of red light,leading to the cycloaddition reaction between the probe and cytoplasmic RNAs,which resulting in improved enrichment and prolonged residence time in tumors.More excitingly,this process can also induce severe apoptosis of tumor cells,realizing tumor theranostics.Results:In the first part,we first studied the optical properties of the probe AcDEVD-Cy-RGD,and found that it could be specifically recognized and cleaved by caspase-3 and released strong near-infrared fluorescence,which successfully monitor cell apoptosis in chemotherapeutic drugs or radiation-treated tumors.Then,we studied the PA properties of the probe and found that it had excellent ratiometric PA responsiveness to caspase-3,which can be used for noninvasive and quantitative detection of the caspase-3 expression level as well as cell apoptosis in vivo.Further taking advantage of the deep tissue penetration of the ratiometric PA imaging,we implemented the real-time visualization of the tumor response to chemo/radiotherapy by using this probe and,for the first time,achieve the accurate assessment of radiation doses delivered to tumors in living mice.In the second part,the probe f-RCP can be cleaved specifically by the ALP to produce activatable NIR fluorescence and show excellent ratiometric PA responsiveness,while its 1O2 generation ability is significantly enhanced.Through RNA gel electrophoresis experiments,it was found that the activatable photosensitizers generated 1O2 under red light irradiation could trigger the covalent cross-linking reaction between f-RCP and RNA.In cell experiments,it was observed through cell imaging and gel electrophoresis experiments that f-RCP could successfully label RNAs in the cytoplasm and showed a prolonged retention in tumor cells.In animal experiments,we found that the retention time of the probe was nearly 24 h longer than that of conventional probe in FL imaging and ratiometric PA imaging.Our further study found that the cross-linking of probe f-RCP to RNAs could cause mitochondrial damage,leading to severe apoptosis of tumor cells and remarkable tumor suppression.Conclusion:This thesis designed and constructed two new types of enzyme-activatable PA imaging probes AcDEVD-Cy-RGD and f-RCP to carry out sensitive imaging of tumor apoptosis in vivo,and tumor theranostics endowed by enzyme-light dual-regulated covalent modification of cytoplasmic RNA,achieving an accurate quantitative assessment of the radiation doses imparted to the tumors and precise tumor theranostics.