Design And Synthesis Of Near Infrared Excitated And Solid-State Fluorescent Probes And Their Application In Bioimaging And Theranostic

Fluorescence technology has made great progress due to the advances of imaging devices.Fluorescent probes,possess many advantages such as high sensitivity and selectivity,fast response and capable of in situ and real time detection,have made great progress in a wide variety of fields,such as food safety,environmental monitoring,bioimaging and disease diagnosis.Utilizing fluorescent probes to monitor intracellular biological molecules,is of great significance to understand the physiological process,early diagnosis and timely treatment of diseases.Although great improvement and wide applications have been made in fluorescent probes,several drawbacks of conventional fluorescent probes,such as photobleaching problem,autofluorescence interference in cells and tissues and shallow penetration depth,which still restricte their application in biological samples.In order to meet the increasing requirements of biomedical research and clinical diagnosis,the development of efficient fluorescent probes are still challenging.Visualizing the concentration and distribution of biological molecules in cells and in vivo is of great importance to study the physiological and pathological functions,identify biomarkers and early diagnosis of diseases.Fluorescent probe based fluorescence imaging method has become a powerful research tool for monitoring biomarkers of disease,because of its high sensitivity,the ability to achieve spatial and temporal resolution imaging,small damage to living cells and tissues.In recent years,the emergence of a new technology in the field of biomedicine and theranostic,could achieve the dual purpose of diagnosis and treatment.Moreover,theranostic approaches are particularly appealing for personalized medicine to improve cancer diagnosis and therapy.Recently reports highlighted the strategies of molecular probe design,which yielded a new generation of subcellular targeted molecular theranostic drugs with multi-function,such as cancer cell imaging,photodynamic therapy,chemotherapy,and in situ monitoring of the therapeutic effect in one go.In this thesis,we combined the advantage of two-photon fluorescence imaging,solid fluorescence and near infrared fluorescence imaging,to build a series of fluorescent probes for sensitive detection and fluorescence imaging of small molecules and enzyme that play vital roles in life activities.Based on the strategies of molecular probe design,we developed a theranostic prodrug,which could be intracellular activated by mitochondrial H2O₂.And the prodrug was used for chemo-photodynamic combination cancer therapy with satisfactory results.Details are as follows:?1?In the second chapter,we have developed a mitochondria-targeted,two-photon fluorescent probe MNAH for monitoring singlet oxygen?1O2?.The probe was reasonably designed based on the principle of photoinduced electron transfer?PET?through integration of 1O2 capturing 9-methylanthracene and a classical two-photon fluorophore naphthalimide,using triphenyl-phosphonium salt as mitochondrial-targeting group.9-methylanthracene,which was employed not only as an electron acceptor of PET process?a fluorescence quencher?for naphthalimide but also as a 1O2 trapper,was directly tethered to the amine position of naphthalimide.The probe showed great fluorescence enhancement special towards 1O2.MNAH was successfully applied for two-photon imaging of 1O2 in living cells and tissues during PDT process with deep-tissue imaging depth.MNAH could be a powerful molecular tool for studying 1O2 generation in mitochondria during PDT process.?2?The emission wavelength of most two-photon fluorescent probe is around 500 nm,which might limit their tissue-imaging depth.In the third chapter,a D-?-A structured two-photon naphthalene fluorophore and a red-emitting BODIPY was conjugated by using through-bond energy transfer to construct a novel two-photon fluorescent probe platform.It displayed highly energy transfer efficiency,large two-photon active absorption cross section and red fluorescence emission,which are highly desirable for bioimaging applications.By integrating the platform and a thiophenol-responsive group,thiophenol could be detected as low as 4.9 nM in tube.Experimental results exhibited that thiophenol could be imaging in cells and tissues with tissue-imaging depths of 90-220 ?m.?3?Near-infrared?NIR?light has the advantage of deep tissue penetration and reduced interference from auto-fluorescence in living animals.In the fivth chapter,we reported a NIR fluorescent probe NALP for turn-on trapping of ALP activity in living cancer cells and tumors.NALP was composed of a NIR-emitting fluorophore as a reporter and phosphate as a triggered moiety.Phosphate group is directly tethered to the hydroxyl group of fluorophore,which prohibited the fluorescence.The probe exhibited high selectivity and remarkable fluorescence turn-on response to ALP in aqueous solutions with a detection limit of 0.28 U/L.Benefiting from NIR excitation and emission,high contrast on the imaging signal could be achieved in response to endogenous ALP activity.Impressively,we successfully used NALP for imaging of endogenous ALP activity in cancer cells,and applied it for fluorescence imaging of ALP in tumor tissues and living tumor xenograft in nude mice for the first time.?4?Subcellular targeted cancer therapy and fluorescence monitoring of therapeutic effect are highly desirable for clinical applications.Photodynamic therapy?PDT?is driven by activating photosensitizers?PSs?to generate reactive oxygen species,generally singlet oxygen for effective cancer cell killing,is considered to be a safe,minimally invasive treatment.In Chapter 6,we developed a novel NIR PS,NPS,with maximum excitation and emission wavelength at 680 nm and 710 nm,respectively.Based on this novel NIR PS,we designed and synthesized a mitochondria-targeting antitumor theranostic prodrug PNPS,which contained three components.The first was a NIR fluorophore,NPS,that could provide fluorescence monitor drug release upon activation and act as a NIR PS.NPS is a fluorophore that is preferably localized in mitochondria,due to its lipophilic quaternary ammonium salt structure;it is no fluorescence and phototoxicity when the hydroxyl group is protected.The second was bisboronate group acted as H2O₂ reaction site.The third component was an anticancer drug,5'-deoxy-5-fluorouridine?5'-DFUR?.When H2O₂ reacted with PNPS,released the 5'-DFUR and turned the bisboronate group into a hydroxyl group,the formed p-hydroxybenzyl alcohol group could undergo a 1,6-rearrangement elimination reaction,which released free NPS and resulted in turn-on fluorescence and activated phototoxicity.PNPS was applied for imaging-guided chemo-photodynamic combination cancer therapy with satisfactory results.?5?Solid fluorescence dyes,such as tetraphenylethene,show di?erent emission mechanism from traditional dyes,which are nonemissive when molecularly dissolved but highly emissive when aggregated.However,most TPE derivatives have maximum excitation wavelength at 350 nm in the UV region,UV-light shows much biological damage and confocal laser-scanning microscopy typically use longer-wavelength at 405 nm,which may limit their application in biological system.In the fourth chapter,we developed a novel solid fluorescence fluorophore,HTPQ,based on excited-state intramolecular proton transfer?ESIPT?mechanism.HTPQ showed strong solid fluorescence with maximum excitation wavelength at 410 nm and maximum emission wavelength at 550 nm.HPTQ was completely water insoluble,showed strongly fluorescence in the solid state.It exhibited good photophysical properties,such as large stokes shifts,intense fluorescence and significant photostability.The large stokes shift of HPTQ could contribute two additional desired advantages:an increase in sensitivity and minimization of background fluorescence.What's more,both its strong insolubility and fluorescence can be controlled by the internal hydrogen bond between the phenolic hydrogen and the imine nitrogen,because the ESIPT process of HPTQ was prohibited.Therefore,HPTQ based probes might show almost no background fluorescence in living system fluorescence imaging.We further developed a novel activity-localized fluorescent probe,HTPQA for alkaline phosphatase?ALP?based on HTPQ.When triggered with ALP,the probe released a precipitating fluorophore,HTPQ,that showed bright solid-state fluorescence in aqueous media with more than 100-fold fluorescence enhancement,thereby producing a localizable fluorescence signal that afforded in situ and spatiotemporal imaging of ALP activity in live cells.Further more,HTPQA was successfully applied for imaging ALP activity in osteosarcoma cells and osteosarcoma tissues with satisfactory results,indicated its promising use in disease diagnosis.