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 lines with elevated ROS levels. Together, we have developed a convenient tool to enhance NO selectively in cancer cells and allows realtime monitoring of NO release without collateral consumption of NO. Further adaption of this technology to better direct NO to cancers is possible. In addition to NO, a number of other redox active reactive species have important biological roles. Controlled generation as well as reliable detection of these species is challenging. In order to assess the generality of the method developed herein, we aimed to develop a ROS generator with a fluorescence reporter. This tool would be useful to deliver and real-time monitor ROS generation in cells without the need for secondary assays.