分析および生物分析技術ジャーナル

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The Effects of Iron Chelation and Hypoxic or Hyperoxic Oxygen Manipulation on the Accumulation/Photobleaching of Protoporphryin IX and Cytotoxcity in Human Glioma Cells

Emma Blake, James PC Allen and Alison Curnow

Background and objective: Gliomas remain the most common and problematic primary brain tumors to treat given their mobile and invasive characteristics, resulting in a 5 years survival rate of less than 5%. Recently the administration of photosensitive fluorescent drugs has been utilized to aid in the identification and resection of gliomas. In this experimental investigation of human glioma cells the effect of iron chelation and oxygenation manipulation, firstly on the accumulation of the clinically useful photosensitizer protoporphyrin IX (PpIX) and subsequently on PpIX photobleaching and cytotoxicity on irradiation, has been examined with the aim of improving fluorescence-guided resection/photodynamic therapy (PDT).
Materials and methods: Cells were incubated at concentrations of 5%, 20% or 40% oxygen for 24 hours prior to and for 3 hours following the administration of the PpIX precursors aminolevulinic acid (ALA), methyl levulinate (MAL) or hexyl aminolevulinate (HAL) with or without the iron chelator 1,2-diethyl-3-hydroxypyridin-4-one hydrochloride (CP94). PpIX levels were monitored using a fluorescence plate reader (excitation filter 400 ± 30 nm; emission filter 645 ± 40 nm). Cells were irradiated with 15 J/cm2 red lights and viability measured using the neutral red uptake assay.
Results: Manipulation of the oxygen environment and/or co administration of CP94 with PpIX precursors resulted in significant changes in both PpIX accumulation and photobleaching. Incubation with ALA/ MAL at 5% or 40% oxygen produced the greatest levels of PpIX and photobleaching respectively. Both these parameters were further elevated through co administration of CP94. The combination of hyperoxygenation and CP94 administration significantly increased photobleaching (a marker of PDT effect) but not cytotoxicity in this experimental system.
Conclusions: PpIX accumulation was greatest when the cells were grown in hypoxic (5% oxygen) conditions (mimicking the in vivo situation) with ALA/MAL+CP94. Fluorescence-guided resection of glioma may therefore be improved through the addition of the iron chelating adjuvant CP94.