Flavylium-Based Hypoxia-Responsive Probe for Cancer Cell Imaging

Abstract

A hypoxia-responsive probe based on a flavylium dye containing an azo group (AZO-Flav) was synthesized to detect hypoxic conditions via a reductase-catalyzed reaction in cancer cells. In in vitro enzymatic investigation, the azo group of AZO-Flav was reduced by a reductase in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) followed by fragmentation to generate a fluorescent molecule, Flav-NH2. The response of AZO-Flav to the reductase was as fast as 2 min with a limit of detection (LOD) of 0.4 μM. Moreover, AZO-Flav displayed high enzyme specificity even in the presence of high concentrations of biological interferences, such as reducing agents and biothiols. Therefore, AZO-Flav was tested to detect hypoxic and normoxic environments in cancer cells (HepG2). Compared to the normal condition, the fluorescence intensity in hypoxic conditions increased about 10-fold after 15 min. Prolonged incubation showed a 26-fold higher fluorescent intensity after 60 min. In addition, the fluorescence signal under hypoxia can be suppressed by an electron transport process inhibitor, diphenyliodonium chloride (DPIC), suggesting that reductases take part in the azo group reduction of AZO-Flav in a hypoxic environment. Therefore, this probe showed great potential application toward in vivo hypoxia detection.

Recapitulating Tumor Hypoxia in a Cleanroom-Free, Liquid-Pinning-Based Microfluidic Tumor Model

“…The hypoxia devices can be distinguished from the normoxia control by a brighter edge at the periphery of the tumor section due to light deflection (Figure A, left). After 24 h of culture, we stained the cancer cells with BioTracker 520 Green hypoxia dye, a fluorescent probe that detects hypoxia in living cells and has fluorescence intensity negatively correlated with oxygen level. − We confirmed the induction of hypoxia indicated by the centrally elevated green fluorescence in both hypoxia devices (125 and 250 μm PC films) in stark contrast to the uniform, background-level signal in the normoxia controls (Figure B). Importantly, the size of the hypoxic region under the 250 μm thick PC film was visibly larger than that under the 125 μm thick PC film.…”

Quercetin Nanoparticle-Based Hypoxia-Responsive Probe for Cancer Detection

“…Synthesis and Characterization of Flav@QCT-PEG. Flav was synthesized according to the reported protocol 26 (Figure S1). The preparation of Flav@QCT-PEG is shown in Scheme 1.…”

“…Their versatility is enhanced by their ability to switch between different species in response to external stimuli such as temperature, pH, and light . Taking advantage of their structures, flavylium compounds are good potential candidates for detecting hypoxia. − …”

Significance of Specific Oxidoreductases in the Design of Hypoxia-Activated Prodrugs and Fluorescent Turn off–on Probes for Hypoxia Imaging

“…This demonstrates the importance of POR evaluation for the effective application of prodrugs activated by this enzyme. Much recent work has focused on investigating the use of POR to bioactivate various hypoxia imaging probes [ 123 , 124 , 125 , 126 ].…”