Imaging of hypoxia, oxygen consumption and recovery in vivo during ALA-photodynamic therapy using delayed fluorescence of Protoporphyrin IX

Background: Hypoxic lesions often respond poorly to cancer therapies. Particularly, photodynamic therapy (PDT) consumes oxygen in treated tissues, which in turn lowers its efficacy. Tools for online monitoring of intracellular pO₂ are desirable.

Methods: The pO₂ changes were tracked during photodynamic therapy (PDT) with δ-aminolevulinic acid (ALA) in mouse skin, xenograft tumors, and human skin. ALA was applied either topically as Ameluz cream or systemically by injection. Mitochondrial pO₂ was quantified by time-gated lifetime-based imaging of delayed fluorescence (DF) of protoporphyrin IX (PpIX).

Results: pO₂-weighted images were obtained with capture-times of several seconds, radiant exposures near 10 mJ/cm², spatial resolution of 0.3 mm, and a broad dynamic range 1-50 mmHg, corresponding to DF lifetimes ≈20-2000 μs. The dose-rate effect on oxygen consumption was investigated in mouse skin. A fluence rate of 1.2 mW/cm² did not cause any appreciable oxygen depletion, whereas 6 mW/cm² and 12 mW/cm² caused severe oxygen depletion after radiant exposures of only 0.4-0.8 J/cm² and <0.2 J/cm², respectively. Reoxygenation after PDT was studied too. With a 5 J/cm² radiant exposure, the recovery times were 10-60 min, whereas with 2 J/cm² they were only 1-6 min. pO₂ distribution was spatially non-uniform at (sub)-millimeter scale, which underlines the necessity of tracking pO₂ changes by imaging rather than point-detection.

Conclusions: Time-gated imaging of PpIX DF seems to be a unique tool for direct online monitoring of pO₂ changes during PDT with a promising potential for research purposes as well as for comparatively easy clinical translation to improve efficacy in PDT treatment.