Results and Discussion

Fluorescence measurement of PpIX induced by ALA-PEG acid esters

In order to define the rate of PpIX formation, we investigated the effect of concentration and duration of exposure of ALA and its derivatives, using different cell lines and direct fluorescence measurements.

Figure 1


Figure 2

Once inside the cell, ALA esters are rapidly hydrolysed by cellular esterases into free ALA acid which can then enter the metabolic pathway leading to the production of PpIX [4]. As displayed on figures 1 and 2, ALA hexylester resulted in the highest relatives fluorescence values whereas ALA-PEG-esters 4 and 5 are less efficient for the production of PpIX.

However, we carried out a premilary study of the ALA-PEG-esters toxicity and first results demonstrated that these derivatives seem to be less cytotoxic at high concentrations than corresponding alkyl esters.

Fluorescence measurement of PpIX induced by ALA derivatives containing peptide

9N-termnial protected peptide derivatives 8 was first studied and directly used as a precursor of PPIX. However, we did not observe any formation of PpIX. Using N-terminal deprotected peptide 9, the production of PPIX was observed (Figure 3).

Figure 3

The intensity of fluorescence mesured is weaker than the fluorescence measured using the ALA-methylester as a reference, possibly because two steps are necessary for formation of free ALA. First, the cleavage of the ALA-peptide bond by peptidases at the cell surface. Second, the intracellular hydrolysis of the ester group.

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