Executive Summary : | Rationale of research: Under hypoxic conditions, overexpression of DT-diphorase, azoreductase and nitroreductase (NTR) was observed and this was used to measure hypoxia. It has been witnessed that, NTR-catalysed one electron reduction is influenced by oxygen concentration in tumor cells in presence of NADH or NADPH as one electron donor. Nitroaromatic compounds possess high stability under physiological conditions and are used as substrates for nitroreductase (NTR) in hypoxic cells. Therefore, we aim to utilize NTR as a biomarker to monitor hypoxia by utilizing its ability to reduce nitro groups present on the compounds and Cu(II) as ROS generator. This two-pronged approach will help in imaging, quantitation and treatment of hypoxic tumor cells. Scientific Objective: we propose to actually construct a device using our complexes as marker to probe hypoxia Hypothesis to be tested: Whether quantitation of NTR can be directly correlated to detection of tumors using metal-based fluorogenic probes. Main experiments to be carried out: (i) The proposed ligands will be synthesized according to the procedure standardized in our lab. After establishing their identities by NMR and HRMS, the corresponding Cu(II) and Fe(III) complexes will be synthesized and characterized. (ii) After studying the absorption-emission profiles of the synthesized compounds, fluorescence changes after treatment with increasing concentration of NTR and fixed NADH concentration will be monitored and the % of hypoxia will be calculated. (iii) Cancer cells will be grown under controlled oxygen conditions and will be imaged. The accumulation of the compounds in solid tumors will also be studied by growing tumors in petri dish to mimic the exact environment inside the tumor. (iv) Cytotoxicity profile will be tested. (v) Based on NTR activity, a serological test will be developed for quantitation of hypoxia alongside prototype design and validation. Estimate of significance to the field of research: a- The low-cost point-of-care diagnostics for early detection of diseases is the need of the hour as evidenced by current pandemic. Early detection of cancer, a silent killer with expensive therapy, can save lives and money of many people. Developing an affordable diagnostic tool aids in identifying location of hypoxic tissues that will immensely help in defining the course of therapy. b- In this regard, we propose use of enzyme NTR as a biomarker in developing a diagnostic tool using metal-based fluorogenic probes designed in our laboratory. |