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Amphiphilic sensitizers self-associate in aqueous environments and form aggregated species that exhibit no or only negligible photodynamic activity. However, amphiphilic photosensitizers number among the most potent agents of photodynamic therapy. The processes by which these sensitizers are internalized into tumor cells have yet to be fully elucidated and thus remain the subject of debate. In this study the uptake of photosensitizer aggregates into tumor cells was examined directly using subcellular time-resolved fluorescence spectroscopy with a high temporal resolution (20-30 ps) and high sensitivity (time-correlated single-photon counting). The investigations were performed on selected sensitizers that exhibit short fluorescence decay times (< 50 ps) in aggregated form. Derivatives of pyropheophorbide-a ether and chlorin e6 with varying lipophilicity were used for the study. The characteristic fluorescence decay times and spectroscopic features of the sensitizer aggregates measured in aqueous solution also could be observed in A431 human endothelial carcinoma cells administered with these photosensitizers. This shows that tumor cells can internalize sensitizers in aggregated form. Uptake of aggregates and their monomerization inside cells were demonstrated directly for the first time by means of fluorescence lifetime imaging with a high temporal resolution. Internalization of the aggregates seems to be endocytosis mediated. The degree of their monomerization in tumor cells is strongly influenced by the lipophilicity of the compounds.

Spectroscopic studies of aqueous solutions of haematoporphyrin-type sensitisers reveal that photobleaching during eposure to light is followed by the formation of stable red-absorbing photoproducts. Experiments in model systems (sensitisers bound to human serum albumin or in a suspension of resealed erythrocyte 'ghosts') and in tumour tissue show that similar photomodification takes place in all investigated environments. Loss of total absorption and emission intensities is accompanied by an increase of absorption in the red spectral region (630-650 nm) which is used for the treatment of tumours because of the deeper penetration of light into tissues. This should be taken into account when the duration of illumination is chosen to reach an appropriate photodynamic dose using Hp-type sensitisers in the photodynamic treatment of tumours.

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