| Cancer is a major problem that threatens human health.Although a variety of diagnostic methods have been developed and used in clinical practice,their accuracy is still limited,which makes early cancer difficult to detect.The patients cannot receive timely and effective treatment.In recent years,a large number of new methods and tools for cancer diagnosis have been reported,but there are still many challenges and difficulties,such as false positive signals,off-target effects and so on.Single biomarker detection cannot achieve accurate diagnosis of the disease.So,adopting dual/multibiomarkers cascade activation probes to improve the accuracy of the examination may realize early and accurate diagnosis of cancer.Cutaneous melanoma is the most serious skin cancer worldwide with characteristics of high mortality and strong metastases.According to the US Cancer Institute statistics,the 5-year survival rate of skin melanoma in the early stage is 99%,while that in the late stage drops to 30%.So,there is an urgent need to develop tools that can be used to diagnose melanoma at an early stage.Considering that melanoma cells have overexpressed tyrosinase and 10 times higher content of reactive oxygen species in cancer cells than normal cells and overexpressed tyrosinase in melanoma cells,we designed and synthesized the target compound MB-m-Borate using methylene blue as a fluorophore.This H2O2-tyrosinase cascade-activated derivative can precisely identify melanoma.To our knowledge,this is the first time that hydrogen peroxide-tyrosinase has been introduced into a dual activation probe design for precise identification of melanocytes and melanoma.In this thesis,we investigated the in vitro/in vivo activation mechanism of the target compound MB-m-Borate,and used MB-m-Borate to accurately image melanoma in a tumor-bearing mouse model.The specific research contents are as follows:1.Using methylene blue as the starting material,the compound MB-mBorate containing aryl boron esters was designed and synthesized,and its chemical structure was characterized by 1H NMR,13C NMR and MS.2.The activation mechanism of the compound in vitro was validated.First,it was verified by high performance liquid chromatography that hydrogen peroxide can activate the compound and convert it into an intermediate,and then release the fluorescent group-methylene blue after the addition of tyrosinase.The experiment results showed that the compound can release the fluorophore only under the sequential activation of hydrogen peroxide and tyrosinase.3.MB-m-Borate is able to specifically recognize melanoma cells.Cytotoxicity experiments showed that MB-m-Borate had no obvious toxicity to both melanoma cells and non-melanoma cells,which proved that MB-mBorate has good biocompatibility and can be used for in vivo bioimaging.The probe MB-m-Borate was activated stepwise by hydrogen peroxide and tyrosinase in melanoma cells.MB-m-Borate could distinguish melanoma cells from non-melanoma cells,especially normal melanocytes.4.MB-m-Borate was applied to tumor-bearing mice for in vivo bioimaging of melanoma.Imaging experiments in vivo showed that MB-mBorate can exhibit significant fluorescence enhancement at the tumor site.In experiments with mice pretreated with N-acetyl-L-cysteine(NAC,an antioxidant)or kojic acid(Kojic acid,a tyrosinase inhibitor),we observed that the fluorescence intensity of MB-m-Borate decreased significantly.These data indicate that the probe MB-m-Borate has a specific response to melanomas with high expression of hydrogen peroxide and tyrosinase,and the probe is expected to be a tool for melanoma diagnosis.In conclusion,the strategy of dual biomarkers cascade activation can well improve the accuracy of early diagnosis of melanoma,and provides a powerful molecular tool for the precise diagnosis of melanoma.At the same time,this dual biomarker activation strategy may provide a new idea for the development of precise diagnosis methods for other cancers. |
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