Synthesis, properties and photodynamic inactivation of Escherichia coli by novel cationic fullerene C60 derivatives.

A novel N,N-dimethyl-2-(4'-N,N,N-trimethylaminophenyl)fulleropyrrolidinium iodide (DTC(60)(2+)) has been synthesized by 1,3-dipolar cycloaddition using 4-(N,N-dimethylamino) benzaldehyde, N-methylglycine and fullerene C(60). This approach produced an N-methyl-2-(4'-N,N-dimethylaminophenyl)fulleropyrrolidine with 38% yield. Exhaustive methylation of this fullerene derivative with methyl iodide yielded 95% of dicationic DTC(60)(2+). The spectroscopic and photodynamic properties of the DTC(60)(2+) were compared with a non-charged N-methyl-2-(4'-acetamidophenyl)fulleropyrrolidine (MAC(60)) and a monocationic N,N-dimethyl-2-(4'-acetamidophenyl)fulleropyrrolidinium iodide (DAC(60)(+)). The dicationic DTC(60)(2+) is essentially aggregated in solution of different solvents and it is partially dissolved as monomer in benzene/benzyl-n-hexadecyldimethyl ammonium chloride (BHDC) 0.1M/water (W(0)=10) reverse micelles. The singlet molecular oxygen, O(2) ((1)Delta(g)), production was evaluated using 1,3-diphenylisobenzofuran. The photodynamic effect was strongly dependent on the medium, diminishes when the sensitizer is aggregated and increases in an appropriately surrounded microenvironment. The photodynamic inactivation produced by these fullerene derivatives was investigated in vitro on a typical Gram-negative bacterium, Escherichia coli. Photosensitized inactivation of E. coli cellular suspensions by DTC(60)(2+) exhibits a approximately 3.5 log decrease of cell survival (99.97% of cellular inactivation), when the cultures are treated with 1 microM of sensitizer and irradiated for 30 min. This photosensitized inactivation remains high even after one washing step. Also, the photodynamic activity was confirmed by growth delay of E. coli cultures. The growth was arrested when E. coli was exposed to 2 microM of cationic fullerene and irradiated, whereas a negligible effect was found for the non-charged MAC(60). These studies indicate that dicationic DTC(60)(2+) is an interesting agent with potential applications in photodynamic inactivation of bacteria.