A study of the stability of tri(glucosyloxyphenyl)chlorin, a sensitizer for photodynamic therapy, in human colon tumoural cells: a liquid chromatography and MALDI-TOF mass spectrometry analysis.
Asymmetrical glycoconjugated tetrapyrrolic macrocycles are under study as efficient sensitizers for photodynamic therapy (PDT). In this context, tri(meta-O-beta-glucopyranosyloxyphenyl)chlorin [TPC(m-O-Glu)(3)] 2a/3a was found to be four times more photoactive in vitro than Foscan. In a further study of this interesting glycoconjugate, its metabolism by cellular glycosidases in HT29 cells has to be explored. Cellular extracts of HT29 cells incubated with TPC(m-O-Glu)(3) (24h, 6microM) were analyzed by MALDI-TOF mass spectrometry and high performance liquid chromatography (HPLC). In MALDI-TOF mass spectra, the presence of compounds distinct from TPC(m-O-Glu)(3) (m/z 1151) were observed at m/z 989, 827 and 665 corresponding to the loss of one, two or three glucose units (162u) and were be ascribed to TPC(m-OH)(m-O-Glu)(2) 2/3b,b',b", TPC(m-OH)(2)(m-O-Glu) 2/3c,c',c" and TPC(m-OH)(3) isomers 2d/3d, respectively. The porphyrins resulting from chlorin oxidation TPP(m-O-Glu)(3) 4a, TPP(m-OH)(m-O-Glu)(2) 4b,b", TPP(m-OH)(2)(m-O-Glu) 4c,c" and TPP(m-OH)(3) 4d were also observed. The HPLC profile (lambda(anal)=420 nm) showed eight peaks consistent with mass spectra. The kinetics of deglucosylation was studied from HPLC profiles between 1 and 48h incubation. The concentration of triglucoconjugated and diglucoconjugated molecules was maximum around 3 and 8h incubation, respectively, whereas, totally deglucosylated species appeared only after incubation for more than 10h. The fully deglycosylated porphyrin TPP(m-OH)(3) is the final metabolite, being observed at a concentration 15 times higher than that of the remaining TPC(m-O-Glu)(3) 2a/3a. Compared to the photobiological activity of the parent molecule [TPC(m-O-Glu)(3)], a three times higher TPP(m-OH)(3) concentration was necessary to observe a similar in vitro photoactivity.
Laville
I
I
LPBC, UMR CNRS 7033, Université Pierre et Marie Curie, 4 place Jussieu, case 138, 75252 Paris Cedex 05, France.
Pigaglio
S
S
Blais
J-C
JC
Loock
B
B
Maillard
Ph
P
Grierson
D S
DS
Blais
J
J
D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D002851:Chromatography, High Pressure Liquid; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000532:radiotherapy*; D005947:Glucose / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D005959:Glucosidases / Q000378:metabolism; D019073:HT29 Cells; D006801:Humans; D007700:Kinetics; D015394:Molecular Structure; D010778:Photochemotherapy; D017319:Photosensitizing Agents / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D011166:Porphyrins / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D017319:Photosensitizing Agents; D011166:Porphyrins; C494930:tri(glucosyloxyphenyl)chlorin; C006969:chlorin; D005959:Glucosidases; D005947:Glucose