Fluorescence imaging of nitric oxide in living cells using o-phenylenediamine-rhodamine based polymeric nanosensors

Direct real-time measurement of nitric oxide (NO) in living cells has proven quite challenging, owing in part to the lack of tools that are selective and sensitive to measure intracellular concentrations of NO. Herein we report the synthesis and characterization of polyvinyl alcohol (PVA) based nanosensors for fluorescence imaging of cytosolic NO using an o-phenylenediamine-rhodamine (OPD-RhB) platform. More specifically, thiol-functionalized PVA incorporating RhB conjugated OPD was disulfide crosslinked to yield NO-responsive nanosensors. The polymeric nanosensors were anionic, averaged 170 nm in hydrodynamic size, and exhibited linear increases in fluorescence intensity (FLI) to micro- and nanomolar concentrations of NO in a sodium nitroprusside (SNP) concentration-dependent manner. In the presence of SNP, the engineered nanosensors demonstrated physical stability at extracellular glutathione (GSH) conditions, while favoring NO detection at cytoplasmic GSH conditions. In addition, the PVA-based nanosensors were non-cytotoxic, cell membrane-permeable and demonstrated hydrogen peroxide-dependent FL increases upon incubation with activated synoviocytes in vitro. Most notably, NO-induced cell FLIs correlated strongly with total nitrite/nitrate content of conventional Griess assays with Pearson correlation coefficients of 0.96. Comprehensively, our results show that OPD-RhB-conjugated PVA nanosensors offer real-time imaging of NO with high sensitivity in living cells that can be employed for direct quantification of NO.