Construction Of Novel NO Fluorescent Probes And Applications In Distinguishing Tumor-associated Macrophage Phenotypes

Nitric oxide（NO）is a key messenger molecule in the body and plays important physiological functions in cardiovascular,immune,reproductive and other systems.However,excess NO can trigger cardiovascular disease,neurological diseases,diabetes,cancer,etc.Therefore,real-time and accurate monitoring of the production and distribution of NO in biological systems is of great significance for the exploration of its physiological and pathological functions.Fluorescent probes have the characteristics of visualization,high sensitivity,high selectivity,and real-time detection,and are currently the most widely used cell biology tools.In 2019,our group constructed an aromatic secondary amine type NO fluorescent probe,which used the significant difference in NO synthase content for the first time to achieve high-contrast fluorescence differentiation of M1 and M2 macrophages.However,the developed NO fluorescent probe was visible light absorption and emission,which is not conducive to biological applications.The developed NO fluorescent probe also lacked tumor targeting,which is not conducive to the fluorescence differentiation of M1 and M2 macrophages in tumors.To solve the above problems,the following research work was carried out in this thesis:（1）An aromatic secondary amine-type NO fluorescent probe CRSA was constructed using C-rhodamine in the near-infrared region as the fluorescent dye platform and N-benzyl-4-methoxyaniline as the identification group.The probe could simultaneously and rapidly detect the two main downstream products of NO,nitrogen trioxide（N₂O₃）and peroxynitrite anion（ONOO^-）,without interference from ascorbic acid/dehydroascorbic acid/methylglyoxal（AA/DHA/MGO）,showing high specificity and sensitivity for NO detection（limit of detection of 7 n M）.Moreover,subcellular fluorescence localization experiments showed that the probe overlapped almost completely with commercial mitochondrial probes（Pearson’s overlap coefficient is 0.96）,indicating that it could specifically localize mitochondria and detect NO in mitochondria.The intracellular NO sensing experiment showed that the probe could image not only exogenous NO but also endogenous NO.The probe had been successfully applied to the fluorescence differentiation of tumor related macrophage phenotypes（M1 and M2）,and is expected to become an imaging tool for screening immune anticancer drugs.（2）Although the NO fluorescent probe designed in the above work could distinguish the phenotype of tumor-associated macrophages,it lacked tumor targeting and could not accurately achieve fluorescence screening of immune anticancer drugs in solid tumors.In this work,the aromatic probe primary amine type NO fluorescent probe CRCA was constructed using C-rhodamine fluorescent dye as the platform and p-methoxyaniline as the identification group,and demonstrated its high specificity and sensitivity for NO sensing in vitro and in vivo.Taking advantage of the characteristics of overexpression of azo reductase in cancer cells,the azo group was introduced into the amino group of the recognition group of CRCA,and the tumor-targeted and activatable NO fluorescent probe CRCA-Azo was constructed.High performance liquid chromatography-mass spectrometry（HPLC-MS）experiments showed that CRCA-Azo could successfully release the recognition group of NO,p-methoxyaniline,under the catalysis of azo reductase,to generate the NO fluorescent probe CRCA.It was proved that CRCA-Azo had tumor targeting and activability,and could avoid the interference of inflammatory tissues on imaging tumors.Cell experiments showed that after co-incubation of CRCA-Azo-loaded He La cancer cells and M1 macrophages,the fluorescence intensity was much greater than that of any of the above types of cells loaded with CRCA-Azo.Therefore,CRCA-Azo had the ability to specifically image tumors and distinguish the phenotype of macrophages,and is expected to achieve accurate screening of immune drugs in solid tumors.