Design And Synthesis Of Two Analyte-responsive Fluorescent Probes And Their Application

ObjectivesThe incidence of malignant tumors is increasing year by year,which brings a serious burden to patients’ families and society.Sensitive and accurate diagnosis is essential for the early determination of tumor treatment options and the improvement of patient prognosis.Due to the excellent sensitivity,biocompatibility and high spatial-temporal resolution,fluorescent probes can be used as a powerful tool for analytical sensing and optical imaging,possessing great potential in the field of cancer research and diagnosis.EMT is a critical biological process in the malignant progression of epithelial cell-origin tumors,and is associated with drug-resistance of cancer cells.Vimentin,a cytoskeletal protein,is one of the vital markers of EMT involved in tumor growth and metastasis.Thus,Vimentin has become an attractive potential target in cancer therapy.Currently,the detection of target EMT-type tumors,especially Vimentin,is mainly performed by traditional methods such as immunohistochemistry,which is time-consuming and labor-intensive and failure to observe dynamic changes of EMT.Therefore,a better detection method for Vimentin is urgently needed to realize the early recognition of EMT.Ferroptosis is an iron-dependent form of cell death caused by excessive lipid peroxidation and engaged in the treatment resistance of various tumors.Although several methods for detecting ferroptosis exist,most of them are limited to detect a single marker,unable to observe the dynamic changes among several indicators at the same time.Therefore,this study aims to design a novel probe that can simultaneously detect NAD(P)H and mitochondrial viscosity,revealing NAD(P)H and mitochondrial changes viscosity during cell ferroptosis,and provide a new theoretical basis for the study of the mechanism of ferroptosis.Accordingly,this paper designed and synthesized two classes of responsive fluorescent probes for imaging Vimentin,NAD(P)H and mitochondrial viscosity and used them for epithelial cell-mesenchymal transition(EMT)and ferroptosis,respectively.Methods1.Vimentin-targeting AIEgen-peptide conjugates:Wash-free fluorescence detection of EMT-type cancer cells and tissues1.1 Design and synthesis of the probe.AIEgen-peptide conjugates Vim-TPE-l and Vim-TPE-2 were designed and synthesized.Their chemical structures and optical properties were confirmed by high-resolution mass spectrometry,nuclear magnetic resonance spectroscopy examination and optical analysis.1.2 Vimentin detection in living cells.The expression of Vimentin in NCI-H1975 and PC-9 cells was verified by Western blot assays.Fluorescence imaging of living cells and flow cytometry were performed to explore the specificity and sensitivity of Vim-TPE-2 to imaging cells with different levels of vimentin expression.Fluorescence imaging was further performed on the NCI-H1975 cells treated with vimentin inhibitor Withaferin A.1.3 Imaging of EMT type tissue.The xenograft tissues were obtained by subcutaneously injecting NCI-H1975 cells into BALB/c nude mice.Frozen sections of tumor tissues were prepared;the HE staining,immunohistochemical staining,and wash-free fluorescence imaging were carried out to evaluated the ability of Vim-TPE-2 probe to imaging the EMT-type tumor tissues.2.Dual-responsive fluorescent probe for imaging NAD(P)H and mitochondrial viscosity during ferroptosis in tumor cells2.1 Design and synthesis of the probe.The bifunctional fluorescent probes 3Q-1,3Q-2,6Q-1 and 6Q-2 were designed and synthesized.Chemical structures and optical properties were confirmed by high-resolution mass spectrometry,nuclear magnetic resonance spectroscopy examination and optical analysis.2.2 Cytotoxicity assays.Cytotoxicity assays were performed to evaluate the toxicity of the 3Q-2 probe in two tumor cells,PANC-1 and HT-1080.2.3 Colocalization test.Multicolor fluorescence imaging assay was performed to determine the subcellular localization of 3Q-2 probe.2.4 Evaluation of the imaging performance of 3Q-2.After treatment of PANC-1 and HT-1080 cells with nystatin,a mitochondrial viscosity inducer,cytofluorimetric imaging was conducted to determine the response of the 3Q-2 probe to changes of intracellular mitochondrial viscosity.After co-incubated with NADH,glucose and pyruvate,respectively,PANC-1 and HT-1080 cells were imaged to identify the detective ability of 3Q-2 probe to detect intracellular NADH levels and mitochondrial viscosity.2.5 Application of 3Q-2 in the cancer cell ferroptosis.Two ferroptosis HT-1080 cells models were induced by erastin,a cysteine transporter protein SLC7A11 inhibitor,and RSL3,a glutathione peroxidase 4(GPX4)inhibitor,respectively.The 3Q-2 probe detected the dynamic changes of intracellular NADH levels and mitochondrial viscosity to analyze the mechanical differences between two models.Results1.Vimentin-targeting AIEgen-peptide conjugates:Wash-free fluorescence detection of EMT-type cancer cells and tissues1.1 Optical properties of the probe.A pair of pure Z/E isomers of Vimentin-targeting TPE-peptide conjugates were successfully synthesized,and the ultraviolet-visible absorption and photoluminescence(PL)spectra of the two probes were measured.Vim-TPE-1 and Vim-TPE-2 both showed absorption peaks at 345 nm,while PL spectra displayed emission peaks at 490 nm and 440 nm,respectively.The fluorescence titration experiments confirm that Vim-TPE-1 and Vim-TPE-2 possess a fluorescence "turn-on" effect on Vimentin.The fluorescence response of the probes increased with the concentration of Vimentin.The fluorescence background of Vim-TPE-2 is slightly lower than Vim-TPE-1.1.2 Detection of Vimentin in living cells;Fluorescence imaging experiments demonstrated that Vim-TPE-2 could produce green hyper fluorescence in Vimentin-positive NCI-H1975 cells after co-incubation.Only weak fluorescence could be found in vimentin negative PC-9 cells;similarly,flow cytometry results indicated that NCI-H 1975 cells incubated with Vim-TPE-2 had a higher proportion of fluorescence-positive cells than PC-9 cells.Moreover,after treatment of Withaferin A,a vimentin inhibitor,NCI-H1975 cells,all cells displayed a time-and dose-dependent decrease in fluorescence intensity under fluorescence confocal microscope,suggesting that Vim-TPE-2 detects Vimentin with excellent sensitivity and specificity.1.3 Imaging EMT type tissues.Immunohistochemical staining indicated positive expression of Vimentin in the tumor tissues,and green fluorescence was observed in tissue sections after direct incubation of both Vim-TPE-1 and Vim-TPE-2 probes,confirming the potential of these probes to identify EMT-type tumor tissues.2.Dual-responsive fluorescent probe for imaging NAD(P)H and mitochondrial viscosity during ferroptosis in tumor cell2.1 Optical properties of 3Q-2.3Q-1,3Q-2,6Q-1 and 6Q-2 probes were designed and synthesized.After incubation with NAD(P)H,the 3Q-2 probe displayed an absorption peak at 595 nm and an emission peak at 670 nm.Moreover,3Q-2 featured a concentration-and time-dependent fluorescence increased response to NAD(P)H over a specific time and concentration range.In addition,the fluorescence intensity produced by 3Q-2 under 550 nm excitation light fortified with the increase of glycerol concentration.2.2 Cytotoxicity assays.Cytotoxicity assay results showed that the survival rates of PANC-1 and HT-1080 activities were not affected by 3Q-2,indicating the good biocompatibility of the probe.2.3 Colocalization test.Multicolour fluorescence imaging assay revealed that the probe could cross the cell membrane.Red fluorescence was mainly distributed in the cytoplasm;green fluorescence was mainly distributed in mitochondria,suggesting that the 3Q-2 was able to detect NAD(P)H in the cytoplasm as well as the mitochondrial viscosity.2.4 The imaging ability of 3Q-2.After treating the cells with the mitochondrial viscosity inducer nystatin,it was found that the green fluorescence intensity gradually increased with the treatment time,which confirming that the 3Q-2 probe has a high sensitivity to change of cellular mitochondrial viscosity.Fluorescence imaging results after exogenous NAD(P)H-treated cells manifested an enhanced red fluorescence intensity with increasing NAD(P)H concentration.Similarly,a positive correlation between fluorescence intensity and glucose treatment concentration was also demonstrated when the endogenous NAD(P)H was produced by promoting intracellular glycolytic pathway through co-incubation of cells with different glucose concentrations.After co-incubation of pyruvate to promote glycolysis,the decreasing intensity of fluorescence was observed as the concentration of pyruvate treatment increased.2.5 Application of 3Q-2 in the cancer cell ferroptosis.During ferroptosis of the HT-1080 cell induced by erastin,an inhibitor of cysteine transporter protein SLC7A11,intracellular NAD(P)H levels and mitochondrial viscosity significantly increased erastin treatment,and the cell membrane remained intact after 4 hours.In another ferroptosis model induced by RSL4,a glutathione peroxidase 4(GPX4)inhibitor,cells exhibited the increased intracellular NAD(P)H and mitochondrial viscosity that reaching a maximum at 2 h.The level of NAD(P)H was less elevated than that of erastin-treated HT-1080 cells.The intact form of the cells had disappeared after 4 h of incubation,and the red fluorescence produced by the probe diffused and the green fluorescence disappeared,which suggested that cell membrane and mitochondrial membrane were ruptured and ferroptosis occurred.Conclusion1.Analyte-responsive fluorescent probes targeting Vimentin were designed and synthesized.The probes were successfully used for wash-free fluorescent imaging and rapid detection of EMT-type cancer cells and tissues.2.A dual-responsive fluorescent probe that simultaneously detect the dynamic changes of NAD(P)H and mitochondrial viscosity during ferroptosis was designed and synthesized,which provides a new approach for mechanism research of cancer cell ferroptosis.