In vivo photobleaching kinetics and epithelial biodistribution of hexylaminolevulinate-induced protoporphyrin IX in rat bladder cancer

In a previous paper, we showed that rat bladder instillations with 8 or 16 mM of hexyl aminolevulinate (hALA) result in diametrically opposed photodynamic therapy efficiency. Although the same fluorescent intensities were detected spectroscopically and by fluorescent microscopy in both conditions, while a given light dose resulted in tumor necrosis with an intact bladder wall after 8 mM hALA, bladders instilled with 16 mM showed total wall necrosis without impact on the tumor. The current study investigated the photobleaching and localization pattern of protoporphyrin IX (PpIX) after both hALA intravesical instillations in tumor-bearing rat bladders. The total PpIX content was evaluated by the extraction of postmortem whole bladders. Photobleaching was evaluated in vivo by fluorescent spectroscopy. Cryosections of bladders were subjected to fluorescent microscopy for cellular localization of the photosensitizer. PpIX extraction showed identical amounts of photosensitizer in tumor-bearing bladders at both concentrations. Photobleaching experiments revealed mono-exponential decay curves in both situations but with a two times faster decay constant in 16 mM bladders. Fluorescent microscopy showed an identical fluorescent pattern for normal bladders at both concentrations and tumor bladders at 8 mM with bright spots. Tumor bladders at 16 mM exhibited a more diffuse cytoplasmatic fluorescent distribution. The different response to photodynamic therapy with regard to the initial pro-drug concentration can thus be attributed to the different cellular localizations.