Construction Of Novel Near-infrared Fluorescent Probes And Their Applications In Biosensing And Tumor Theranostics

Objective:Near-infrared fluorescent probes are used in the fields of biosensing and tumor theranostics because they can avoid the interference from biological background fluorescence and exhibit strong tissue penetration as well as low light damage.Nitroreductase（NTR）is overexpressed in tumor hypoxia microenvironment and regarded as an important tumor marker,which can act as a potential target for tumor diagnosis and treatment.Therefore,developing NTR-activated near-infrared fluorescent probe is of great significance for the diagnosis and treatment of malignant tumor.Fluoride ion（F^-）has benificial and detrimental effects to human health.Insufficient intake of F^-can lead to dental caries and osteoporosis.However,prolonged excessive intake can cause fluorosis,resulting in a series of diseases.Therefore,simple and rapid determination of F^-is very important.Aiming at developing NTR and F^--responsive near-infrared fluorescent probes and studying their biological applications,the main work in this thesis is as follows:1.A novel fluorescent probe named as NDXPI was developed for detecting NTR.The probe was based on the hemicyanine skeleton ADXPI,which exhibits good fluorescent and photosensitive performance.The response of NDXPI to NTR was studied,followed by investigating the fluorescence imaging and photodynamic therapy in cells and in vivo.This work may provide new technical means for the diagnosis and treatment of malignant tumors.2.A near-infrared fluorescent probe PHXPI for detecting F^-was constructed using the well-performing hemicyanine skeleton HXPI as the fluorophore and dimethyl thiophosphoryl group as the recognition unit.The fluorescence response and applications of PHXPI to detecting F^-in real samples was studied.Methods:1.Using IR-780,m-nitrophenol,K₂CO₃,Sn Cl₂as raw materials,the near-infrared fluorophore ADXPI was synthesized via two steps.Then the amino group in ADXPI was oxidized into nitro group by trifluoroacetic anhydride and H₂O₂to obtain the near-infrared fluorescent probe NDXPI.NDXPI was characterized by means of high resolution mass spectrometry,¹H NMR and ¹³C NMR.The performance of NDXPI for detecting NTR and photosensitive capacity in vitro were investigated by ultraviolet-visible（UV-vis）absorption spectroscopy and fluorescence spectroscopy.High performance liquid chromatography（HPLC）was performed to verify the sensing mechanism of NDXPI to NTR.The imaging and sensing capabilities of NDXPI for NTR at the cellular level were studied by laser confocal imaging and flow cytometry.The photodynamic killing ability was evaluated by means of MTT assay and Calcein-AM/PI staining.The fluorescence imaging in vivo was conducted using small animal fluorescence imaging technology.The therapy ability of tumor and biological safety of NDXPI were evaluated by recording tumor volume and mouse weight,testing liver and kidney function as well as pathological slices assay.2.The near-infrared fluorophore HXPI was synthesized with IR-780,resorcinol and K₂CO₃as raw materials.Then HXPI was reacted with dimethyl thiophosphoryl chloride in CH₂Cl₂with triethylamine as the acid-binding agent to obtain the probe PHXPI.PHXPI was characterized by means of high resolution mass spectrometry,¹H NMR and ¹³C NMR.The performance of PDXPI for detecting F^-was investigated by UV-vis absorption spectroscopy and fluorescence spectroscopy.HPLC and mass spectrometry were conducted to confirm the detection mechanism.The application of PHXPI to sensing F^-in actual samples（water samples and toothpaste）was investigated.The cytotoxicity of PHXPI was evaluated by MTT method.And the availability of PHXPI in imaging intracellular F^-was studied by laser confocal imaging.Results:1.The near-infrared fluorescent probe NDXPI shows good fluorescent enhanced response to NTR,with a detection limit of 2.10 ng/m L.NDXPI can thus be used for the sensitive determination of NTR in complex samples.Cellular experiments show that NDXPI can image the over-expressed NTR in anoxic A549 cells.In addition,the probe displays significant photodynamic killing ability after 660 nm laser irradiation.In vivo studies reveal that NDXPI can visualizing tiny tumor lesions in tumor-bearing nude mice.Besides,NDXPI can significantly inhibit the tumor growth with no damage on major organs,liver and kidney functions.2.The near-infrared fluorescent probe PHXPI shows good fluorescent off-on response to F^-,with a detection limit of 4.28μM.More importantly,the response time of PHXPI to F^-is only 10 s,which is the fastest fluorescent probe for F^-so far.Satisfactory results have been obtained in determining F^-in tap water and toothpaste by PHXPI.Confocal laser experiments show that PHXPI can image F^-in cells.Conclusion:In this thesis,near-infrared fluorescent probes for NTR and F^-were developed based on the near-infrared hemicyanine skeleton,respectively.These probes can be used in the biosensing and imaging of NTR and F^-.Besides,the NTR-responsive near-infrared fluorescent probe can also be used for tumor imaging and photodynamic therapy,which has potential value in the fields of early tumor diagnosis and tumor theranostics.