title: Real-time analysis of endogenous protoporphyrin IX fluorescence from δ-aminolevulinic acid and its derivatives reveals distinct time- and dose-dependent characteristics in vitro.
abstract: Photodynamic therapy (PDT) and photodiagnosis based on the intracellular production of the photosensitizer protoporphyrin IX (PPIX) by administration of its metabolic precursor -aminolevulinic acid (ALA) achieved their breakthrough upon the clinical approval of MAL (ALA methyl ester) and HAL (ALA hexyl ester). For newly developed ALA derivatives or application in new tumor types, in vitro determination of PPIX formation involves multiparametric experiments covering variable pro-drug concentrations, medium composition, time points of analysis, and cell type(s). This study uses a fluorescence microplate reader with a built-in temperature and atmosphere control to investigate the high-resolution long-term kinetics (72 h) of cellular PPIX fueled by administration of either ALA, MAL, or HAL for each 10 different concentrations. For simultaneous proliferation correction, A431 cells were stably transfected with green fluorescent protein. The results indicate that the peak PPIX level is a function of both, incubation concentration and period: maximal PPIX is generated with 1 to 2-mM ALA/MAL or 0.125-mM HAL; also, the PPIX peak shifts to longer incubation periods with increasing pro-drug concentrations. The results underline the need for detailed temporal analysis of PPIX formation to optimize ALA (derivative)-based PDT or photodiagnosis and highlight the value of environment-controlled microplate readers for automated in vitro analysis.
authors.affiliation: Paracelsus Medical University, Institute of Physiology and Pathophysiology, Strubergasse 21, Salzburg A-5020, AustriabParacelsus Medical University/Salzburger Landeskliniken, Department of Internal Medicine I, Muellner Hauptstrasse 48, Salzburg A-5020, Au.
authors.forename: Tobias
authors.initials: T
authors.lastname: Kiesslich
authors.affiliation: Norwegian University of Science and Technology, Department of Cancer Research and Molecular Medicine, Erling Skjalgssons gate 1, N-7491 Trondheim, Norway.
authors.forename: Linda
authors.initials: L
authors.lastname: Helander
authors.affiliation: Paracelsus Medical University/Salzburger Landeskliniken, Institute of Pathology, Muellner Hauptstrasse 48, Salzburg A-5020, Austria.
authors.forename: Romana
authors.initials: R
authors.lastname: Illig
authors.affiliation: Tecan Austria, Untersbergstrasse 1, Grödig A-5082, Austria.
authors.forename: Christian
authors.initials: C
authors.lastname: Oberdanner
authors.affiliation: Paracelsus Medical University/Salzburger Landeskliniken, Department of Internal Medicine I, Muellner Hauptstrasse 48, Salzburg A-5020, Austria.
authors.forename: Andrej
authors.initials: A
authors.lastname: Wagner
authors.affiliation: University of Salzburg, Department of Materials Science and Physics, Division of Physics and Biophysics, Hellbrunnerstraße 34, Salzburg A-5020, Austria.
authors.forename: Herbert
authors.initials: H
authors.lastname: Lettner
authors.affiliation: Paracelsus Medical University, Institute of Physiology and Pathophysiology, Strubergasse 21, Salzburg A-5020, Austria.
authors.forename: Martin
authors.initials: M
authors.lastname: Jakab
authors.affiliation: University of Salzburg, Department of Materials Science and Physics, Laboratory of Photodynamic Inactivation of Microorganisms, Hellbrunnerstraße 34, Salzburg A-5020, Austria.
authors.forename: Kristjan
authors.initials: K
authors.lastname: Plaetzer
mesh_terms: D000622:Aminolevulinic Acid / Q000008:administration & dosage* / Q000493:pharmacokinetics*; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003199:Computer Systems; D004305:Dose-Response Relationship, Drug; D006801:Humans; D009374:Neoplasms, Experimental / Q000188:drug therapy* / Q000378:metabolism*; D010778:Photochemotherapy / Q000379:methods*; D011524:Protoporphyrins / Q000378:metabolism*; D011838:Radiation-Sensitizing Agents / Q000008:administration & dosage / Q000493:pharmacokinetics; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D011524:Protoporphyrins; D011838:Radiation-Sensitizing Agents; D000622:Aminolevulinic Acid; C028025:protoporphyrin IX