The influence of storage conditions on delta amino levulinic acid induced toxicity and phototoxicity in vitro

Summary 

Delta amino levulinic acid photodynamic therapy (ALA-PDT) represents one of the most prominent advances in PDT. ALA itself or its derivatives are marketed for a variety of clinical indications. Despite the development of clinical applications, experimental ALA results are very heterogeneous and experimentally used parameters are still not standardized. This suggests that some problems remain unsolved that are likely to impair experiments to be performed but also that clinical results obtained could be greatly improved. Frequently unmentioned or imprecise data concern solvents, pH of ALA solutions, storage time, ALA degradation or ALA efficacy. In addition, diversity of experimental model is huge while capabilities of ALA transformation into PpIX are known to vary from one cell to the other. Thus, the aim of the present paper was to quantify the level of ALA degradation or changes in ALA efficacy using one single cell line without presuming of the mechanisms and determine the conditions of storage inducing the best transformation into PpIX and/or cell phototoxicity. We added ALA diluted in water, PBS or RPMI to C6 cells, a murine brain tumour cell line that can be used in vivo as an orthotopic graft. We measured in cells used as tools for final bio efficacy estimation, both the induced fluorescence and phototoxicity in various conditions of storage before use chosen to be as close as possible to the real lab conditions. Water had been found to better preserve ALA than, respectively, PBS and RPMI and this for any temperature or storage durations. The lowest temperature and the shortest duration for storage used had also been shown to better preserve ALA-induced fluorescence and phototoxicity. The fact that these properties were found to be better preserved in 7.4 buffered solvent could be in relationship with a fast ALA condensation occurring at neutral or lightly acidic pH modifying its availability for an optimal transformation into PpIX.

Keywords: ALA, Photodynamic therapy, PDT, Protoporphyrin IX, Glioma cells

Abbreviations: ALA, delta amino levulinic acid, PpIX, protoporphyrin IX, LD, lethal dose, MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide, PDT, photodynamic therapy, PBS, Phosphate Buffer Saline, RPMI, Roswell Park Memorial Institute