Construction,Imaging And Tumor Therapy Of Near-infrared Pyridine Salt And Onium Salt Photodiagnostic Agents

Currently,the clinical cancer therapy that relied on surgical resection,radiotherapy and chemotherapy,which inevitably suffered from the risks of damaged normal tissues,destroyed immune system and increased recurrence rate.Multimodal imaging-guided phototherapy exhibited promising applications in cancer treatment due to its low invasiveness,high temporal and spatial selectivity,and the ability to maintain positive therapeutic effects as well as low side effects.However,the developed photosensitizers（PSs:as an important component of phototherapy）generally possessed the advantages of high oxygen dependence,low tissue penetration ability,and even unclear action mechanism with biomolecules,which were not conducive to the treatment of deep solid tumors and the construction of novel high-efficiency phototherapeutic agents.To address these problems,in this work,series of organic small molecular photosensitizers with D-π-A structure were constructed by regulating the structures and types of central linkage groups（conjugated chain length,flexible chain length,benzene ring,thiophene）,where 4-diethylamine coumarin moiety with good planarity and luminescence were as the chromophore and electron donor（D）,cationic pyridine salts,onium salts were as the electron acceptor（A）and subcellular organelle targeting group.Further,optimizing the intramolecular charge transfer（ICT）process and molecular stacking orientation to:（i）decrease energy level(ΔE_st)between the first excited singlet state（S₁）and the first excited triplet state（T₁）,improve the efficiency and species of reactive oxygen species（ROS）,which could efficiently alleviate the oxygen dependence;（ii）locate on specific subcellular organelles,control the generated ROS within specifically targeted sites to achieve precise killing and efficient treatment;（iii）transform single-modality imaging guided phototherapy system to multi-modality imaging guided phototherapy system by introducing effective thermal effect groups on the basis of traditional fluorescence PSs;（iv）change the indirect action objects（mainly utilizing the oxidability of ROS）of excited-state PSs（PSs*）,select water or other biomolecules such as genetic material（DNA/RNA）,amino acids,reducing coenzymes,etc.,increase the oxidative damage degree of PSs to tumor cells（electron transfer process）;（v）image cell damage in virtue of commercial dyes,and analyze the cell death mechanism.Finally,summarizing the rules among the structure of PSs,ROS release and cell death pathway to further guide the design and development of new phototherapeutic agents.Specific studies were as follows.1.Tracing the kinetic changes of plasma membrane mediated by coumarin-based pyridine salt based on lifetime imagingThe organic fluorescence sensor（MO）with D-π-A structure was prepared by Knoevenagel condensation reaction,where 4-diethylamine coumarin was used as electron donor（D）,cationic pyridine salt was used as electron acceptor（A）,connected with flexible alkoxy chain.Due to the suitable amphiphilic and electrostatic interactions,MO can efficiently locate on plasma membrane in dark atmosphere,which was be known as an ideal membrane marker.Besides,MO exhibiting sensitive response to liposome with improved fluorescence lifetime would lay a solid foundation for fluorescence lifetime imaging（FLIM）in vitro and cellular level.Finally,with the regulation of ROS,the fluorescence sensor MO enabled real-time in situ monitor the damage and heterogeneity of plasma membrane.2.Alkyl chain regulated Type-I/Type-II coumarin-based pyridine salts derivatives for photodynamic treatmentBased on previous study,a series of organic small molecular photosensitizers（EBD-1～EBD-5）with D-π-A structure were developed successfully via adjusting the length of alkyl chain,where 4-diethylamine coumarin and pyridine salt were as electron donor（D）and electron acceptor（A）,respectively.Cell imaging indicated that the prepared photosensitizer possessed excellent anchoring ability on plasma membrane of cancer cells due to the suitable amphipathy.Whereas,EBD-1 with the shortest chain showed superior two-photon properties and free radical generation ability,which could damage plasma membrane and self-report cell viability.In vivo experiments indicated that under 808 nm laser radiation,EBD-1 inhibited tumor growth significantly.3.Subcellular organelle targeting and synergistic therapy by Type-I onium salt derivatives with extended conjugate chainsA series of the diagnosis and treatment platforms with D-A structure were developed（DOBY、CMTP-1、CMTP-2 and CMTP-3）using 4-diethylamine coumarin as electron donor（D）and cation onium salt as electron acceptor（A）.Specifically,the dynamic distribution of intracellular signal transduction pathway including its escaping from endosome/lysosome and aggregating in mitochondria were revealed by CMTP-2 for the first time.With NIR-I light irradiation,CMTP-2 produced highly toxic free radicals and heat,activating mitochondrial autophagy and cancer cells apoptosis.In-vivo experiments presented that CMTP-2 realized complete tumors ablation under 808 nm irradiation,exhibiting great potential for synergistic phototherapy.4.Self-assembly,mitochondrial targeting and photocatalytic treatment ofπ-bridge-regulated onium salt derivativesTwo organic small molecule photocatalysts d-C_T and d-S_T with D-π-A structure were prepared by introducing cation onium salt groups as well as regulating centralπbridge（phenyl-thiophene）.Interestingly,the C_2vsymmetry and larger molecular dipole moment enabled d-S_T directionally assemble into helical nanofibers in aqueous solution,which greatly improved the contact area between the photocatalysts and substrates and thus accelerated the photo-reaction rate.In addition,the helical d-S_Texhibited fine surface potential and crystallinity,providing more possibilities for photocatalytic H₂O splitting to produce hydrogen free radicals（H·）.Cell imaging presented that d-S_T with positive charge could easily locate on mitochondria,redox coenzymes NADPH,block ATP synthesis,induce cell apoptosis and ferroptosis.