Synthesis and characterization of fullerols C₆₀(OH)_n; the use as cytotoxic agent

ARTICLES

- Photo-oxidative stress in the presence of a water-soluble derivative of C₆₀: ESR and AFM assays, Bertrand Vileno, Andrzej Sienkiewicz, Malgorzata Lekka, Pierre R. Marcoux and László Forró, Supramolecular Structure and Function 9, Springer Netherlands, 2008, pages 153-180. (download chapter )

Keywords: Hydroxylated C₆₀; Fullerol; Singlet Oxygen; Oxidative Stress; Protein denaturation; ESR

Abstract: We report on the electron spin resonance (ESR) study of the photo-oxidative stress-mediated protein conformation changes in the spin-labelled protein staphylococcal nuclease (SNase). The photo-oxidative stress was brought on by photosensitization of singlet oxygen (¹D_g) in the presence of a novel photosensitizer, water-soluble fullerol C₆₀(OH)₁₉(ONa)₁₇, and resulted in partial protein denaturation. This process was monitored via ESR measurements performed for a spin-labelled SNase Thr-62-Cys mutant, with MTSSL spin label (SL) attached to the cysteine 62 residue (SNase T62C-SL). Prior to ESR measurements of the oxidative stress-induced alterations in protein conformations, the efficiency of C₆₀(OH)₁₉(ONa)₁₇ for 1 g-generation was confirmed by three different techniques: (i) selective reactive scavenging of 1 g and ESR detection of the resulting paramagnetic product, (ii) 1 g-mediated photo-oxidative loss of tryptophan and (iii) by measuring the characteristic nearinfrared phosphorescence of singlet oxygen at 1270 nm. The observed evolution of the ESR spectra of SNase T62C-SL as a function of exposure to the photooxidative stress points to marked changes in protein conformation due to the deleterious action of ¹D_g.

- Spectroscopic and photophysical properties of a highly derivatised C₆₀ fullerol, Bertrand Vileno*, Pierre R. Marcoux, Malgorzata Lekka, Andrzej Sienkiewicz, Titusz Feher, László Forró, Advanced Functional Materials, 2006, 16, 120-128. (download article )

Keywords: Hydroxylated C₆₀; Polyacide; Singlet Oxygen; Oxidative Stress.

Abstract: Hydroxylated C₆₀, also called fullerols, are a class of water-soluble fullerenes. Here we report the synthesis through acidic conditions of a highly derivatized fullerol (up to 36 carbons per C₆₀ were oxidized). Spectroscopic investigations (X-ray photoelectron spectroscopy and infrared absorption) highlighted the coexistence of both acidic and basic forms for the hydroxyl addends of derivatized C₆₀. A pH titrimetry revealed that, at millimolar concentrations, only ten protons per one fullerol molecule are labile. Such a low value, as compared to 36 hydroxyl groups, is explained by the formation of clusters.  The UV-visible absorption study performed over a large range of concentrations also points to the aggregation phenomenon. Moreover, this study shows that the clusters of fullerols appear at relatively low (micromolar) concentrations. The ESR study, based on the attack of singlet oxygen (¹D_g) on 2,2,6,6-tetramethyl-4-piperidinol (TMP-OH), proved the potential of hydroxylated C₆₀ for performing efficient generation of ¹D_g in aqueous solutions. ESR measurements, which allowed for detection and quantification of ¹D_g, revealed also generation of reactive oxygen species (ROS). The yield of generation of ¹D_g and ROS was found to be strongly correlated with the concentration of fullerol, thus also pointing to aggregation of fullerol molecules. Exposing glioblastoma cells to the oxidative stress in the presence of hydroxylated C₆₀ and visible light was also performed. AFM technique was used to monitor the relevant diminishment of the Young modulus values for cells exposed to the oxidative stress. These results point to a possible application field of fullerols for performing biooxidations.

AFM (error mode) image of glioblastoma cells in phosphate buffered saline (PBS). Inset: Schematic illustration of measurements of local elastic properties of cell with the use of AFM.

- Singlet oxygen-mediated oxidation of cellular and subcellular components: ESR and AFM assays, Bertrand Vileno*, Malgorzata Lekka, Andrzej Sienkiewicz, Pierre R. Marcoux, Andrzej J. Kulik, Sandor Kasas, Stefan Catsicas, Alfreda Graczyk, László Forró, Journal of Physics Condensed Matter, 2005, 17, 1-12. (download article )

Keywords: Hydroxylated C₆₀; local elastic properties; Singlet Oxygen; Oxidative Stress

Abstract: Singlet molecular oxygen (¹D_g) is known to be a very reactive chemical intermediate in photooxygenation reactions. We report a comprehensive in vitro study of ¹D_g-generation in the presence of a fullerene-based water-soluble sensitizer also called fullerol: C₆₀(OH)_n. Efficiencies of the photodynamic generation of ¹D_g in the presence of our fullerol, were measured using Electron Spin Resonance (ESR). Subsequently, ESR were used to monitor structural damage to a doubly spin labeled protein caused by the photooxidative stress. Also Atomic Force Microscopy (AFM) experiments were performed to measure changes in the local elastic properties of living and glutaraldehyde-fixed neurons exposed to the toxic action of ¹D_g. Remarkably, the Young’s modulus values obtained for four different cell lines revealed a pronounced drop as a function of exposure to ¹D_g. Thus, our ESR and AFM results bring the evidence that even upon covalent multi-functionalization fullerene-based compounds are still efficient ¹D_g-generators in aqueous media and might be implemented as oxidizing agents in biological systems.

Jablonski Kasha diagram showing type II mechanism, responsible for the singlet oxygen generation

CONFERENCES

- Singlet oxygen mediated oxidation of cellular and subcellular components: ESR and AFM assays (poster), B. Vileno, Malgorzata Lekka, Andrzej Sienkiewicz, Pierre R. Marcoux, Andrzej J. Kulik, Sandor Casas, Alfreda Graczyk, László Forró, Workshop on structure and function of biomolecules, Bedelwo (Poland),13-15 May 2004.