Covalent conjugation of a
photosensitizer to a
ligand that specifically recognized and internalized by a
cell-surface receptor may be a way of improving the selectivity of
photodynamic therapy (
PDT). The class A Type-I
scavenger receptor of macrophages, which among other
ligands recognizes maleylated
serum albumin and has a high capacity is a good candidate for testing this approach.
Chlorin(e6) was covalently attached to
bovine serum albumin to give conjugates with molar substitution ratios of 1:1 and 3:1 (
dye to
protein), and these conjugates could then be further modified by maleylation. A novel way of purifying the conjugates by
acetone precipitation was developed in order to remove traces of unbound
dye that could not be accomplished by size-exclusion chromatography. Conjugates were characterized by
polyacrylamide gel electrophoresis and thin-layer chromatography.
Photosensitizer uptake was measured by target J774 murine macrophage-like cells and nontarget OVCAR-5 human
ovarian cancer cells, and
phototoxicity was examined after illumination by a 660 nm
diode laser by a tetrazolium assay. All of the purified conjugates were taken up by and after illumination killed J774 cells while there was only small uptake and no
phototoxicity toward OVCAR-5 cells. The higher
dye:
protein ratio and maleylation of the conjugates both produced higher uptakes and lower survival ratios in J774 cells. The uptake and
phototoxicity by J774 cells were decreased after incubation at 4 degrees C demonstrating internalization, and confocal microscopy with organelle-specific green
fluorescent probes showed largely lysosomal localization. Uptake and
phototoxicity by J774 cells could both be competed by addition of the
scavenger receptor ligand maleylated
albumin. These data show that
scavenger receptor-targeted
PDT gives a high degree of specificity toward macrophages and may have applications in the treatment of
tumors and
atherosclerosis.