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Toxicity and Mitochondrial Delivery of Flavonol-Based Carbon Monoxide-Releasing MoleculesEsquer Heredia, Hector Jose 01 December 2017 (has links)
Despite the reputation of carbon monoxide (CO) as a silent killer, new evidence suggests that this gaseous molecule has anti-inflammatory, anti-cancer and vasoprotective properties. Unfortunately, little is known about the role of CO in the body. However, proteins present in mitochondria are believed to be important targets. We previously synthesized a class of novel and structurally modifiable flavonol-based CO-releasing molecules (CORMs). Flavonols are commonly found in fruits and vegetables. The base structure, Flav-1, is fluorescent, exhibits low toxicity, and releases CO after exposure to visible light. Previous reports indicate that addition of a triphenylphosphonium (TPP) tail allows chemical structures to enter mitochondria. We hypothesized that addition of a TPP tail of two or eight carbons in length to Flav-1 (Mito-Flav-C2 or -C8) would facilitate targeting of mitochondria, and thus, localized light-induced CO release. Toxicity of these TPP-tailed molecules was determined in human umbilical vein endothelial cells (HUVECs) and lung epithelial carcinoma cells (A549), using standard cell viability assays. Evaluation of toxicity using the MTT assay revealed lower toxicity of Flav-1 in HUVECs compared to A549 cells, but addition of the TPP tails increased toxicity in both cell lines. However, unlike the MTT assay, Flav-1 with and without tails had similar toxicity when measured in HUVECs by the lactate dehydrogenase assay. Photo-degradation experiments were performed by exposing cells until their light emission was undetectable by using lasers in the near-UV and within the visible light spectrum. Localization of the compounds was observed using a confocal microscope by co-staining with MitoTracker Red (MTR) and Hoechst to visualize the mitochondria and nucleus, respectively. Fluorescence microscopy images of cells treated with Mito-Flav-C2 or -C8 revealed an increase in uptake, compared to Flav-1, plus co-localization with MTR, which suggests mitochondrial localization. A549 cells exposed to laser light or a full spectrum of light lost all fluorescence of Mito-Flav-C2, which indicates CO release. This work highlights the successful synthesis of the first mitochondria targeting CORMs, and that CO release is achievable using different light sources. Moreover, these TPP-tailed CORMs will allow for controlled and localized release of CO to further study its physiological targets.
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