In vitro photodynamic activity of a series of methylene blue analogues.
We have synthesized a series of symmetrical phenothiazines in which the methyl groups of methylene blue have been substituted by longer alkyl chains. Intrinsic photosensitizing ability was not altered by increasing the chain length. However, in vitro phototoxicity after 2 h incubation of RIF-1 murine fibrosarcoma cells followed the order n-propyl > n-pentyl > n-butyl > n-hexyl > ethyl > methyl, with ethyl and n-propyl analogues being 14- and 130-fold more phototoxic than methylene blue, respectively. All analogues also had an improved ratio of phototoxicity: dark toxicity (4:1 to 27:1) compared with methylene blue (3:1). Phototoxicity did not correlate with cellular phenothiazine levels, suggesting that the site of subcellular localization may be more important. After 2 h incubation of RIF-1 cells with the phototoxicity LD50 concentration, methylene blue and all analogues were observed to be localized in the lysosomes by fluorescence microscopy. On exposure to light, methylene blue relocalized to the nucleus, the ethyl analogue did not relocalize, whereas the more phototoxic n-propyl - n-hexyl analogues relocalized to the mitochondria. Relocalization to the mitochondria was associated with an octanol: buffer partition coefficient > or = 1. Therefore, the longer-chain analogues of methylene blue show significantly improved phototoxicity in vitro and, in addition, are expected to avoid the problems of mutagenicity associated with the nuclear localization of methylene blue.
Mellish
Kirste J
KJ
School of Biochemistry and Molecular Biology, Centre for Photobiology and Photodynamic Therapy, University of Leeds, UK. bmbkjm@bmb.leeds.ac.uk
Cox
Russell D
RD
Vernon
David I
DI
Griffiths
John
J
Brown
Stanley B
SB
D000818:Animals; D066298:In Vitro Techniques; D008751:Methylene Blue / Q000494:pharmacology*; D051379:Mice; D010778:Photochemotherapy; D014407:Tumor Cells, Cultured
D008751:Methylene Blue