The uptake and more importantly the subcellular distribution of photosensitizers are major determinants of their efficacy. In this paper, the cellular internalization of chlorin e6 (Ce6), a photosensitizer bearing three carboxylic chains, is considered with emphasize on pH effects. Small unilamellar vesicles are used as models to investigate the dynamics of interactions of Ce6 with membranes. The entrance and exit steps from the outer lipid hemileaflet are very fast (~ms). A slow transfer of Ce6 through the membrane was observed only for thin bilayers made of dimyristoleoyl-phosphatidylcholine. Ce6 did not permeate through bilayers consisting of longer phospholipids more representative of biological membranes. These results along with previous data on the interactions of Ce6 with low-density lipoproteins (LDL) are correlated with cellular studies. After 15 min incubation of HS68 human fibroblasts with Ce6, fluorescence microscopy revealed labeling of the plasma membrane and cytosolic vesicles different from lysosomes. When vectorized by LDL, Ce6 was mainly localized in lysosomes but absent from the plasma membrane. Internalization of LDL bound photosensitizer via ApoB/E receptor mediated pathway was demonstrated by overexpression experiments. A pH decrease from 7.4 to 6.9 did not affect the intracellular distribution of Ce6, but significantly increased its overall cellular uptake.

The photosensitizing properties of three chlorins, meso-tetra(3-hydroxyphenyl)chlorin (m-THPC), chlorin e6 (Ce6) and meso-tetraphenylchlorin substituted by two adjacent sulfonated groups (TPCS(2a)) are compared in solution and when incorporated in dioleoyl-sn-phosphatidylcholine (DOPC) liposomes. In solution, the three chlorins possess a similar efficacy to generate singlet oxygen (quantum yield approximately 0.65). The formation of conjugated dienes was used to determine their ability to induce the peroxidation of methyl linoleate as a target of singlet oxygen. In ethanol solution, the apparent quantum yield for this process is the same for the three chlorins and its value agrees with that expected from the known rates for the decay of singlet oxygen and its reaction with methyl linoleate. When incorporated in liposomes, the order of efficacy is m-THPC > TPCS(2a) > Ce6. This order is tentatively assigned to the relative embedment of the photosensitizer within the lipidic bilayer, TPCS(2a) and Ce6 being anchored by their negative chains nearer to the water-lipid interface. The photoinduced permeation of the lipidic bilayer by these chlorins was investigated by measuring the release of carboxyfluorescein entrapped into DOPC liposomes. The charged chlorins, in particular TPCS(2a), are the most efficient, a result discussed in relation with the technology of photochemical internalization, PCI.