Novel Photoaffinity-based NIR Probes For Tumor Imaging And Therapy

Background: At present,near infrared fluorescence(NIRF)imaging technology as a non-invasive imaging tool with high sensitivity,great spatial resolution and deep penetration depth has been widely applied for tumor imaging.The indocyanine green(ICG)as a near infrared dye has been approved by FDA(Food and Drug Administration)for tumor imaging.Under NIR laser irradiation,these dyes(such as Cypate)with good stability and optical properties can not only generate near infrared fluorescence but also produce thermal effect to kill tumor cells,which can realize the integration of tumor diagnosis and treatment.Although these small molecular probes can effectively accumulate the tumor site so as to achieve tumor imaging and therapy,it is not possible to maintain a long circulation time in the body resulting from cell or tissue metabolism,which has seriously affected the tumor imaging and therapy.Therefore,it is of great significance for tumor diagnosis and treatment to develop a theranostic molecular probe that can prolong the retention time at the tumor siteObjective: Through introducing a photo-crosslinker diazirine into the fluorescent probes,we will develop a photoaffinity-based NIR molecular probe.Upon 405 nm irradiation,the probe can be photocrosslinked to the cells or tissues based on the carbine insertion,which greatly improves the tumor imaging and photothermal therapy effects.Method:(1)The photoaffinity-based probe DACF consisting of cypate dye,folic acid and diazirine group,was synthesized by the classical amide condensation and click reactions.All the intermediate products were characterized by 1H-NMR,13C-NMR and HR-MS;(2)The absorption and emission properties of DACF were measured by UV-vis and fluorescence spectroscopies,respectively.The photoacoustic(PA)imaging in vitro was performed with a multispectral optoacoustic tomography scanner.The photothermal effect and temperature increment were evaluated under 808 nm laser irradiation;(3)MTT assay was used to estimate the in vitro cytotoxicity of DACF on 4T1 cells;(4)Confocal laser scanning microscopy(CLSM)was employed to evaluate the target and retention effect of DACF in 4T1 cells;(5)The photothermal ablation effect of DACF trapped in 4T1 cells was characterized through MTT,live/dead staining and flow cytometry;(6)The in vivo NIRF imaging of 4T1 tumor-bearing female nude mice was performed to evaluate the tumor-targeting and retaining capabilities of DACF;(7)The PA imaging in vivo was performed to evaluate the retention time of DACF in tumor;(8)The photothermal therapeutic effect of DACF for tumors was investigated in vivo through analyzing the tumor change,survival rate,tumor metastasis and tissue slices.Result: Firstly DACF can effectively target and internalize the tumor cells.In comparison with 3T3 cells,the cell uptake of DACF was apparently higher in 4T1 cells.In vitro cell experiments indicate that DACF can be covalently crosslinked with endogenous macromoleculars or proteins upon 405 nm laser irradiation,resulting in prolonging retention time,which might be very useful for long-time tracking and effective photothermal therapy of tumors.In vivo NIRF and PA imagings,DACF could not only specifically target the tumors,but also enhance the imaging time upon 405 nm laser irradiation.Additonally,DACF could effectively kill the tumor cells based on the photothermal therapy and prevent its recurrence.Conclusion:We have designed and synthesized a novel photoaffinity-based NIR probe containing a photo-crosslinker diazirine.Both in vitro and in vivo experiments demonstrated that the probe could not only specifically image the tumors with longer retention time,but also dramatically enhance the photothermal therapeutic efficacy upon 405 nm laser irradiation.