Nuclear Targeting of Chlorin e6 Enhances Its Photosensitizing Activity

Abstract

Although photosensitizers, molecules that produce active oxygen species upon activation by visible light, are being extensively used in photodynamic therapy to treat cancer and other clinical conditions, problems include normal cell and tissue damage and associated side effects, which are attributable in part to the fact that cytotoxic effects are largely restricted to the plasma membrane. We have previously shown that the photosensitizer chlorin e 6 has significantly higher photosensitizing activity when present in conjugates containing specific ligands and thus able to be internalized by receptor-expressing cells. In this study we use insulincontaining conjugates to which variants of the simian virus SV40 large tumor antigen nuclear localization signal (NLS) were linked to target chlorin e 6 to the nucleus, a hypersensitive site for active oxygen species-induced damage. NLSs were either included as peptides crosslinked to the carrier bovine serum albumin or encoded within the sequence of a ␤-galactosidase fusion protein carrier. The results for photosensitization demonstrate clearly for the first time that NLSs increase the photosensitizing activity of chlorin e 6 , maximally reducing the EC 50 by a factor of over 2000-fold. This has widereaching implications for achieving efficient cell typespecific photodynamic therapy.Photosensitizers such as porphyrins are molecules that produce active oxygen species upon activation by visible light and are currently being extensively used in photodynamic therapy to treat cancer and other clinical conditions (1-3). Because normal cells are able to accumulate porphyrins, however, and porphyrins are only excreted slowly from the body, prolonged skin photosensitization as well as other effects can be a problem (4, 5), leading to normal cell and tissue damage (6). A high priority with respect to photodynamic therapy is accordingly to increase the specificity of the uptake of photosensitizers in particular target cells, thereby enabling the active dose of porphyrins administered to patients to be reduced. We have previously shown that the photosensitizer chlorin e 6 has significantly higher photosensitizing activity when present in conjugates containing specific ligands such as insulin or concanavalin A and thus is able to be internalized by receptorexpressing cells (7-9). Photosensitization could be competed by incubating cells in the presence of an excess of unconjugated ligand (7-9), indicating that cellular uptake was receptor-dependent. Because only cells expressing specific receptors are targeted in this approach (see Refs. 7-9), it is clear that it enables selectivity in terms of the cell types targeted for photosensitization.Due to the fact that injury induced by singlet oxygen comprising 80% of all of the active oxygen species generated upon porphyrin activation is localized within less than 0.1 m of the site of its production, the effect of photosensitizers is integrally dependent on their site of cellular accumulation (1). Although most porphyrins such as chlorin e 6...

Mutual exclusivity of DNA binding and nuclear localization signal recognition by the yeast transcription factor GAL4: implications for nonviral DNA delivery

“…35,36 Specific recognition sequences for the DNAbinding protein can be included within the plasmid to be expressed in the target cell, to enable it to be recognized by the protein and thus be cotransported into the target cells. GAL4 is particularly interesting in this respect since its DNA binding domain, which recognizes a specific 17 bp sequence, 26 appears to include or overlap with an efficient NLS.…”

Radiosensitizing effect of 5-aminolevulinic acid-induced protoporphyrin IX in glioma cells in�vitro

“…Intracellular ROS production was detected using the oxidant-sensitive fluorescent probe DCFD with a confocal laser scanning microscope (17,18). Cells were seeded into 35-mm glass-bottomed dishes in fresh medium containing 1 mM 5-ALA and incubated in the dark at 37˚C for 4 h. After the incubation, cells were washed with PBS twice, incubated with 10 µM DCFD for 10 min, washed with PBS and exposed to 10 Gy of ionizing irradiation.…”

Targeted intracellular delivery of photosensitizers to enhance photodynamic efficiency

“…[3][4][5] That uneven intracellular distribution of PS can lead to differences in toxicity has been shown using laser microbeam irradiation. 6 In contrast to cell membranes and other cytoplasmic organelles, the cell nucleus [7][8][9] is known to be a very sensitive target for reactive oxygen species. In order to reduce the dose of PS administered to patients and hence minimize the harmful side-effects of PDT, new approaches have been devised to increase the effectiveness of tumour-cell killing through targeted delivery of PS to hypersensitive subcellular sites.…”

“…69 2′,7′-Dichlorofluorescin diacetate penetrates into living cells and is deacetylated by intracellular esterases; because reactive oxygen species react with 2′,7′-dichlorofluorescin to yield fluorescent 2′,7′-dichlorofluorescein, this can be used to visualize the sites of intracellular production of reactive oxygen species and sites of PS subcellular location, as well as being an indicator of intracellular photodynamic activity in situ. 8,9,69,86 The insulin-BSA-chlorin e6 conjugate at low concentrations suppresses the propagation of hepatoma cells (half-maximal effect at about 1 nmol/L). Other ligands that have been used in internalizable PS conjugates include transferrin, 87 epidermal growth factor 88 and maleylated BSA, a ligand for 'scavenger' receptors.…”