Application Of A Novel Fluorescent Probe Based On Dual-targeting Strategy In Imaging Of HNSCC

Research backgroundHead and neck squamous cell carcinoma(HNSCC)is the sixth most common malignant tumor all over the world.The clinical imaging diagnosis of HNSCC is mainly based on conventional imaging methods,and the treatment of it is mainly based on surgery,radiotherapy and chemotherapy.Surgical treatment for HNSCC still depends on the combination of inspection and palpation,which relies on the clinical experience of the surgeon.Such a procedure has great subjectivity and individual differences,with high technical sensitivity.Besides,it is also difficult to identify tumor margins.It is important to improve the optical technology of the identification of tumor tissue and normal tissue.Utilizing the imaging tools such as fluorescent probes may provide a real-time feedback of fluorescence imaging to assist preoperative imaging diagnosis and guide tumor resection during surgery,which might become a breakthrough to solve the clinical problems above.Choosing an appropriate targeting strategy and designing a novel fluorescent probe with more prominent tumor specificity can enhance the targeting ability and selectivity of the fluorescent probe,which may also effectively increase the responsiveness of the tumor tissue and the identification ability of the tumor margin.Based on the existing clinical problems such as the high sensitivity of HNSCC surgical techniques and the difficulty in identifying tumor margins,this project uses bioinformatics technology to select new targeting strategies to optimize the targeting performance of fluorescent probes and strengthen tumor specificity.We confirmed the high expression of Sodium-dependent multivitamin transporter(SMVT)expression in HNSCC,and utilized the aberrant high peroxynitrite(ONOO-)expression in the tumor microenvironment to design probe RB-PN.This probe has dual-targeting strategies:The SMVT active targeting on the cell membrane surface and a passive response strategy of ONOO-tumor microenvironment.RBPN showed the ability of imaging tumor cells and could distinguish tumors cells from normal cells.In vivo models were also used to verify its in vivo imaging function and biosafety performance to identify its future clinical application potential.Since the dual-targeting strategy of using SMVT and ONOO’ fluorescent probes has not been reported before,and the application of fluorescent probe visualization in HNSCC is still in its infancy,the development and application of RB-PN has a strong innovative and significant research value.Research methods1.Probe targeting strategy selection,chemical synthesis and characterization test.The bioinformatics databases were used to analyze the SMVT subcellular expression site and its expression in tumor and paracancerous tissues of HNSCC.Immunohistochemistry and immunofluorescence techniques were used to verify the differences in expression between tumors and paracancerous tissues in pathological sections of patients.After that,we designed an SMVT-targeted,ONOO-responsive small molecule fluorescent probe RB-PN,and use fluorescence titration experiments to confirm the chemical structure and characterization.2.Application of dual targeting probe RB-PN in imaging and identification of HNSCC cells.We used CCK-8 assay to verify the biological safety of RB-PN in HNSCC cells and normal cells,and used HOECHST staining to confirm whether the probe is involved in cell apoptosis.We then used exogenous ONOO-responsiveness and endogenous ONOOresponsiveness test to confirm probe ONOO-responsiveness.We also used competitive blocking experiment to confirm SMVT targeting strategy.We confirmed RB-PN’s fluorescence imaging real-time feedback and fluorescence stability.Tumor cell screening experiment was performed to confirm probe’s HNSCC cell recognition function.3.Application of dual targeting probe RB-PN in 3D tumor mass and in vivo fluorescence imaging.We established a 3D tumor micromass model,and pretreated with IL-2 and IFN-γ,to perform three-dimensional imaging and reconstruction through RB-PN to expand the potential application of visualization of the efficacy of immunotherapy.We then constructed a zebrafish biological model and evaluated in vivo imaging performance.After that,we constructed a HNSCC tumor-bearing mouse model,and evaluate the safety of RB-PN in vivo application and the biological toxicity to important organs.4.Prediction of potential sites for targeting strategies for HNSCC.On the basis of the design of the dual targeting probe RB-PN for HNSCC targeting strategy,we further expanded the targeting strategy for HNSCC,and summarized the biomarkers used by the fluorescent probe designed for HNSCC.We also analyzed the potential biological targets as a reference for the design of targeting strategies for HNSCC in the future development of fluorescent probes.Research results1.RB-PN based on the dual-targeting strategy was successfully prepared and tested for characterizations.Bioinformatics analysis was performed on the expression of SMVT in HNSCC.based on which further verification was carried out using tissue sections and immunological techniques,and it was confirmed that it can be used as a strategic choice for HNSCC-targeted fluorescent probes.RB-PN was constructed,and the chemical structure of the RB-PN probe was confirmed by H-NMR and C-NMR spectra.The chemical response time of RB-PN was further tested by spectral testing experiments.The fluorescence of the reaction system at 575 nm was significantly enhanced.The fluorescence signal can be observed after 5 seconds,and the fluorescence intensity reached the maximum value about 1 minute after detection and remained unchanged.The limit of detection reached 7 nM,confirming the good chemical properties of the probe.2.The fluorescent probe RB-PN has good biological safety and fluorescence imaging performance in HNSCC cells,and has good SMVT active targeting strategy and ONOOpassive targeting strategy.After 12 hours of treatment with different concentrations of RBPN,the cell survival rate was still greater than 80%.HOECHST staining experiments confirmed that the probe did not induce or participate in cell apoptosis.Exogenous and endogenous ONOO-experiments found that the probe was can effectively respond to ONOO-in HNSCC cells,and the SMVT competitive blocking experiment also showed that the fluorescence intensity of the blocking group is significantly lower than that of the control group.In the tumor screening experiment,the fluorescence intensity of RB-PN in HNSCC cells was significantly higher,and HNSCC was successfully screened out.Immediate imaging experiments and fluorescence stability experiments confirmed that RB-PN had fast response rate and stable fluorescence imaging performance in living cells,and stable fluorescence signals could be observed within 180 minutes.3.RB-PN had good fluorescence imaging performance in HNSCC tumor micromass and in vivo,and has good safety in vivo application.We constructed HNSCC tumor micromass,and compared with the blank control group.The relative fluorescence intensity increased by 1.77 times after adding the probe.After the intervention of cytokines IFN-y and IL-2,the fluorescence signals were respectively increased to those of the blank control group.(2.32 times and 2.85 times respectively).In zebrafish models,RB-PN successfully achieved fluorescence imaging in vivo,and showed good response performance.In HNSCC tumorbearing mouse model,after 12 hours injection,Hematoxylin and eosin staining(H&E staining)of organs including liver,kidney,heart,spleen,lung,stomach and intestinal tract was performed to confirm that RB-PN had no obvious damage to important organs,which means RB-PN had good safety in vivo application.4.The authors summarized and evaluated the HNSCC targeting strategies of fluorescent probes in current clinical research and preclinical research and predicted potential targeting strategies.Based on the optimization of the targeting strategy designed by RB-PN for HNSCC,and in order to explore more effective HNSCC targeting measures,the authors summarized the HNSCC targeting strategies of fluorescent probes in current clinical researches and preclinical researches.Through technical analysis,38 genes(70 proteins with different lengths)were predicted to be overexpressed in HNSCC tissues,and their corresponding protein subcellular localization can be expressed on the cell membrane surface,which could be used as fluorescent probe targets specific for HNSCC in the future.Research conclusionBioinformatics technology was used to analyze the expression of SMVT receptor subcellular-expression sites and expression levels in HNSCC and adjacent normal tissues.After the further verification by immunological technology,an SMVT-targeted,ONOO-responsive small molecule fluorescent probe RB-PN was designed.We confirmed the chemical structure and characterization of RB-PN.In cell level,we distinguished HNSCC from normal cells,and confirmed that it had good fluorescence imaging functions of HNSCC cells and tumor micro mass.In vivo models were confirmed to confirm its in vivo imaging function and biosafety performance.Furthermore,we explored the tumor targeting strategy of HNSCC through databases searches and bioinformatics analysis,including the existing clinical trials and preclinical studies.The potential targeting strategy of fluorescent probe were predicted,providing theoretical basis and data support for future designing of fluorescent probes which are specific for HNSCC.