Design,Synthesis And Application Of Novel Near-infrared Photosensitizers And Fluorescence/Photoacoustic Probes

In recent years,small molecule probes have been widely used in the diagnosis and treatment of cancer due to their simple structure,easy synthesis,stable properties and good biocompatibility.For example,fluorescent probes are indispensable tools for cancer diagnosis because of their high sensitivity,simple operation,and non-invasive visualization.Organic fluorophores as photosensitizers can not only realize photodynamic therapy,but also visualize the cancer site to achieve the integration of cancer diagnosis and treatment.However,some probes are affected by short self-excitation wavelength and poor stability,resulting in large autofluorescence interference and poor penetration in vivo imaging,which limits their application in deep tissues.Near-infrared fluorescence(NIRF)probes have superior excitation wavelengths and can penetrate deep into tissues,which can be used for in vivo imaging and clinical therapy.However,NIRF still has a shallow penetration depth(< 1 mm)and lower spatial resolution.In contrast,photoacoustic imaging is a novel imaging method that combines the high contrast characteristics of optical imaging with the high penetration depth characteristics of ultrasound imaging.It can convert optical signals into acoustic signals,which in principle reduces the effect of light scattering.It also has a large penetration depth(7-10 cm)and a high spatial resolution(100 μm).These excellent properties allow photoacoustic imaging to be used for deep tissue imaging in vivo.At present,many small molecule probes have been developed for the diagnosis and treatment of cancer,but the wrong diagnosis of cancer maybe caused due to the self-background and low specificity of probes.Therefore,it is very important to develop small molecule probes with high sensitivity,low detection limit,high signal-to-noise ratio,high stability and high specificity for accurate diagnosis and treatment of cancer.Based on the advantages of small molecule probes,this thesis focuses on the design,synthesis and application of a series of small molecule probes with different functions.We have designed and synthesized a number of activated small molecule probes with high sensitivity and specificity for cancer diagnosis and treatment.The specific research content is as follows:(1)We have developed a novel membrane-tethered photosensitizer for antitumor therapy.Based on an activated fluorophore parent and heavy-atom effect,the photosensitizer is designed and can generate reactive oxygen species to kill tumor,thereby inhibiting tumor growth.Through the engineering design and screening of the parent photosensitizer,a membrane photosensitizer a PYIC4 anchored on the cell membrane by the zwitterionic lipid chain was obtained.The photosensitizer can be specifically activated by the membrane-localized enzyme APN overexpressed in cancer cells,thus achieving specific anti-tumor effects.The membrane-tethered photosensitizer could possess good membrane localization,induce efficient singlet oxygen production,and produce specific phototoxicity to tumor compared to non-membrane-localized control probe.In addition,the membrane-tethered photosensitizer exhibites prolonged blood circulation,long-lasting fluorescence imaging and sustained anti-tumor PDT in vivo.In vivo analysis shows that the photosensitizer could accurately target the tumor site,effectively inhibit the tumor growth under light conditions,and has almost no toxic side effects on other normal tissues.This work provides a very useful method for the design of photosensitizers for sustained anti-tumor therapy.(2)In order to further explain that the membrane-tethered design of photosensitizer can produce good anti-tumor effect,the killing pathway of photosensitizer on tumor is explored.The experimental results show that the cells treated with membrane-tethered photosensitizer can present the obvious characteristics of cell pyroptosis under laser irradiation.Such as the activation of caspase 1,the cleavage of GSDMD,the dilation of cell membrane,the release of cell contents LDH,the release of pro-inflammatory cytokines IL18 and IL1β,etc.It produces specific phototoxicity to tumor through pyroptosis immunogenic cell death(ICD).Furthermore,ICD can lead to the release of intracellular neoantigens and damage-associated molecular patterns(DAMPs)in cells,and creats the potential for reprogramming the tumor immune microenvironment.By activating the maturation of antigen-presenting cells and priming cytotoxic T lymphocytes,ICD can elicit powerful anti-tumor immunity for cancer ablation.Next,in vivo experiments further shows that membrane PDT could not only ablate the primary tumor,but also effectively inhibit the growth of distant tumors.Membrane PDT could make the immunogenic "cold" tumor "hot" by promoting the maturation of dendritic cells and the recruitment of cytotoxic T lymphocytes,and enhance the systemic anti-tumor immunity.This work suggests that membrane-tethered design of photosensitizer may provide a new approach to enhance systemic anti-tumor immunity and develop novel combined tumor immunotherapy.(3)We have developed a near-infrared fluorescent probe ADID based on indole regulation for the visualization of lysosomes and the tracking of lysosomal dynamic changes.The form of indole N of the probe can be regulated by p H,and the indole N will be protonated and become indole cation at acidic conditions,but it will not be protonated at neutral and basic conditions.These two forms of indole N can be converted at different p H conditions,and the process is reversible.The transformation of the morphology can lead to large changes in the optical properties of the probe,which makes it possible to design activated probe.In addition,in vitro studies have shown that ADID is viscosity sensitive and its fluorescence can only be activated under conditions of high viscosity,which creates the possibility for probe imaging in lysosomes.Cellular experiments further shows that the probe exhibited good photostability,superior selectivity and low cytotoxicity,which could be used for visual detection of lysosomes in living cells.In addition,the probe can detect the p H change process of lysosomes,which is very beneficial for the detection of the dynamic process of lysosomes in living cells.(4)We have designed and synthesized a class of near-infrared dye ROA-R based on xanthene.In vitro studies show that ROA-R has a large Stokes shift and good optical stability,and can undergo the transition of oxanthene "open" and spirocyclic "closed",and the transition is controllable.Based on this finding,we further engineered the dye parent to design two activated fluorescent and photoacoustic probes for detecting DPPⅣ activity in vitro and in vivo.It is worth noting that this is the first report of a photoacoustic probe that can be used to detect DPPⅣ activity in vitro and in vivo.In vitro experiments show that the probe undergo the "close-open" transition of spirocyclic after activation by DPPⅣ,which makes the designed fluorescence and photoacoustic probes have low cytotoxicity,high sensitivity,good specificity and high signal-to-noise ratio,and can be used for the detection of DPPIV activity levels in vitro.In vivo imaging shows that the fluorescent probe and photoacoustic probe have tumor targeting and high activation signal-to-noise ratio,which can be used for DPPⅣdetection in vivo.In conclusion,the designed xanthene probes have a wide range of application prospects due to their excellent characteristics,which provides a new platform for the construction of new fluorescence and photoacoustic probes.