In vitro assay of singlet oxygen generation in the presence of water-soluble derivatives of C 60

Abstract

Singlet oxygen ( 1 Δ g ) is a very efficient oxidizing agent that targets numerous molecules in living cells. We report a comprehensive in vitro study of 1 Δ g -generation in the presence of two fullerene-based water-soluble sensitizers, fullerol C 60(OH) n and fullerene-core star-like polymer, C 60 [>N-CH 2CH 2(OCH 2CH 2) 2OCH 3] 6. Efficiencies of the photodynamic generation of 1 Δ g in the presence of C 60-derivatives and a reference sensitizer, the amino acid protoporphyrin IX-derivative, PP(Ala) 2(Arg) 2, were measured using electron spin resonance (ESR). Subsequently, atomic force microscopy (AFM) experiments were performed to measure the local elastic properties of living and glutaraldehyde-fixed cells exposed to the toxic action of 1 Δ g . Remarkably, the Young's modulus values obtained for four different cell lines revealed a pronounced drop as a function of exposure to 1 Δ g . Thus, our ESR and AFM results bring the evidence that the multi-functionalized fullerene-based compounds are efficient 1 Δ g -generators in aqueous media and might be implemented as oxidizing agents in biological systems. Moreover, we also show that AFM can be used as a sensitive tool for detecting early changes occurring to the subcellular structures under exposure to the oxidative stress.