Real time monitoring of nitric oxide release

Abstract

Nitric oxide NO is an endogenously produced gaseous signalling molecule with cancer therapeutic potential Since NO is unstable under ambient conditions it is difficult to generate as well as reliably detect this gas Although a number of methodologies for enhancement as well as detection of NO are available these events are typically mutually exclusive The most common problem during NO delivery and concomitant detection is consumption of NO Thus a strategy where a small molecule can generate NO in a controlled manner to the cancer cells selectively along with a fluorescence reporter for NO would be useful for NO based cancer therapy As a proof of concept we first designed and synthesized FLUORO NO a new class of triggerable NO donors with an in built fluorescence reporter Upon activation by an esterase enzyme the compound produces NO as well as a fluorescence signal simultaneously without NO consumption Cellular studies with a FLUORO NO derivative revealed a dose dependent enhancement of NO as well as fluorescence Next in order to deliver NO selectively to cancer cells a second stimulus for activation was chosen hydrogen peroxide H2O2 a reactive oxygen species ROS As ROS is frequently found to be elevated in rapidly dividing cells such as cancers H2O2 has been previously used to specifically activate prodrugs and latent fluorophores as imaging agents in cancers Boronate ester is known to react with H2O2 to produce an alcohol hence this functional group was chosen as the metabolic stimulus We designed and synthesized a series of arylboronate ester based diazeniumdiolates BORO NO Having established that BORO NO derivatives are capable of generating NO when triggered by H2O2 next we synthesized Thera NO a H2O2 activated NO donor with fluorescence reporter Upon activation by H2O2 in buffer a nearly quantitative correlation between fluorescence signal and NO generation is observed When encountered with cellular situations with varying ROS levels Thera NO is observed to preferentially generate NO in cell