Photobleaching of mono-L-aspartyl chlorin e6 (NPe6): a candidate sensitizer for the photodynamic therapy of tumors.

Most sensitizers used for the photodynamic therapy (PDT) of tumors photobleach on illumination. Thus, it is of interest to examine the photobleaching behavior of new sensitizers proposed for use in PDT. This report surveys the quantum yields and kinetics of the photobleaching of mono-L-aspartyl chlorin e6(NPe6), a hydrophilic chlorin that has many of the photoproperties desirable in a sensitizer for clinical PDT. It is a very effective sensitizer for the PDT of several types of model tumors in animals and is now in Phase I clinical trials. The quantum yield of NPe6 photobleaching in pH 7.4 phosphate buffer in air was 8.2 x 10(-4); this is greater than the yields for typical porphyrin photosensitizers. For example, the yields for hematoporphyrin and uroporphyrin are 4.7 x 10(-5) and 2.8 x 10(-5), respectively. The yield decreased significantly in organic solvents of low dielectric constant. The Sn derivative of NPe6 was more light stable than NPe6 (yield = 5.7 x 10(-6), while the Zn derivative was more sensitive (yield = 1.9 x 10(-2). Oxygen appeared to be necessary for the photobleaching of NPe6; however, bleaching was not inhibited by 100 mM azide, an efficient quencher of singlet oxygen. The photooxidizable substrates cysteine, dithiothreitol and furfuryl alcohol increased the quantum yield of photobleaching two- to four-fold, while the electron acceptor, metronidazole, increased it almost six-fold. Photobleaching yields for several other chlorins were also measured.

Photosensitizing properties of mono-L-aspartyl chlorin e6 (NPe6): a candidate sensitizer for the photodynamic therapy of tumors.

There is a large amount of interest in chlorins as photosensitizers for the photodynamic therapy of tumors because of their strong absorption in the red, where light penetration into mammalian tissues is efficient. Mono-L-aspartyl chlorin e6 (NPe6), in phosphate buffer of pH 7.4, had absorption peaks at 400 and 654 nm with molar absorption coefficients of 180,000 and 40,000 M-1 cm-1 respectively. In buffer, the NPe6 triplet had a peak at 440 nm and a lifetime under argon of approximately 300 microseconds. The triplet was efficiently quenched by ground state oxygen (kQ = 1.9 x 10(9) M-1 s-1) with the formation of singlet oxygen, as identified by its near infrared luminescence. The quantum yield of singlet oxygen production was 0.77. A number of substrates were efficiently photo-oxidized by NPe6, including furfuryl alcohol, cysteine, histidine, tryptophan and human serum albumin. These reactions were efficiently inhibited by azide (which did not quench NPe6 triplets), indicating that they are probably mediated by singlet oxygen. Thus, NPe6 has a desirable array of photoproperties for a sensitizer to be used in the clinical photodynamic therapy of tumors.

Action spectra of phthalocyanines with respect to photosensitization of cells.

Human carcinoma cells (NHIK 3025 cells) and Chinese hamster cells (V79 cells) were incubated with AlPcS1, AlPcS2 and AlPcS4, phthalocyanines with different lipophilicity but with similar photochemical properties when in monomeric solutions. The absorption- and fluorescence spectra of the dyes in the cells were recorded as well as their action spectra with respect to sensitizing cells to photoinactivation. These spectra show that under the present conditions AlPcS1 is strongly aggregated in both cell lines; AlPcS2 is aggregated in V79 cells but much less so in NHIK 3025 cells. A main finding is that the shapes of the action spectra are similar to that of the fluorescence excitation spectra, but not to the absorption spectra, indicating that the photosensitizing effects of the dyes are mainly due to their monomeric fraction in the cells. AlPcS2 and AlPcS4 localize intracellularly mainly in lysosomes while AlPcS1 was found to be more diffusely distributed in cells. As measured per quantum of fluorescence emitted, AlPcS1 and AlPcS2 are more efficient sensitizers than AlPcS4. The difference in efficiency between AlPcS2 and AlPcS4 is supposedly due to a different localization pattern on the suborganelle level.

Subcellular localization, redistribution and photobleaching of sulfonated aluminum phthalocyanines in a human melanoma cell line.

Intracellular localization, intracellular translocation and photobleaching following non-lethal laser microirradiation of the fluorescing derivatives of sulfonated aluminum phthalocyanines (Al-PcSn, n = 1-4) in a human melanoma cell line (LOX) were studied by means of confocal laser scanning microscopy (LSM) and image processing. Use of confocal microscopy allowed 3-dimensional information to be obtained. Both Al-PcS1 and Al-PcS2 localized diffusely in the cytoplasm of the cells, while Al-PcS3 and Al-PcS4 exhibited a granular pattern in the extranuclear fraction of the cells. None of the Al-PcSn family was observed in the nuclei of the cells except that a small fraction of fluorescence was occasionally detected in nuclei of some cells treated with Al-PcS1 and Al-PcS2. Furthermore, exactly the same granular localization patterns and positions in the same cells were found after incubation initially with Al-PcS3 (or Al-PcS4) followed by acridine orange (AO) which emits red fluorescence from lysosomes of cells. Thus, the granular fluorescence of Al-PcS3 and Al-PcS4 is confined to the lysosomes of the LOX cells. Non-lethal laser exposure of cells incubated with high concentrations of the 2 dyes resulted in a translocation of the dyes from the lysosomes to the whole cytoplasm and an increase in total intracellular fluorescence intensity. Finally, a small fraction of the dyes localized into the nuclei of the cells. The laser exposure of cells incubated with low concentrations of the lysosomally localized dyes resulted in an increase in the intracellular fluorescence intensity with no translocation of the dyes. Under all conditions, high laser exposure resulted in a decrease in the total intracellular fluorescence intensity.

Light induced relocalization of sulfonated meso-tetraphenylporphines in NHIK 3025 cells and effects of dose fractionation.

Human cervix carcinoma cells of the line NHIK 3025 were incubated for 18 h with sulfonated meso-tetraphenylporphines (TPPSn where n = 1, 2a, 2o or 4) followed by 1 h in sensitizer-free medium and then exposed to light. The fluorescing fraction of TPPS4, TPPS2o and TPPS2a has recently been shown to be located intracellularly in extracellular granules which are intracellularly localized in a similar pattern as acridine orange-stained granules, assumed to be endosomes and lysosomes (Berg, K., A. Western, J. Bommer and J. Moan. Photochem. Photobiol. 52, 481-487). Light exposure induced a relocalization of TPPS4 from its granular pattern to mainly the nuclear area while TPPS2o and TPPS2a relocalized mainly to cytoplasmic areas. After the light-induced relocalization TPPS4 became less efficient in sensitizing photoinactivation of cells as measured per fluorescing cellbound TPPS4 molecules while TPPS2a and TPPS2o became more efficient. These changes were independent of the extracellular concentration of TPPSn applied to the cells, except for cells incubated with 75 micrograms/mL TPPS4. These cells became more sensitive to light after a light exposure inactivating 20% of the cells. This increased photosensitivity seems to be related to a 2-2.5 fold increase in the amount of fluorescing cellbound TPPS4 induced by the first light exposure.

Cellular uptake and relative efficiency in cell inactivation by photoactivated sulfonated meso-tetraphenylporphines.

The cellular uptake, relative fluorescence quantum yields and photosensitizing efficiencies of meso-tetraphenylporphines sulfonated to different degrees (TPPSn) have been investigated using the human carcinoma cell line NHIK 3025. The efficiencies of these dyes in photoinactivation of cells were highly dependent on the number of sulfonate groups on the derivatives. These differences in phototoxicity were primarily due to different abilities to be taken up by cells, but were also dependent upon the cellular localization of the dyes. TPPS1 and TPPS2a were more efficiently taken up by the cells than TPPS2o and TPPS4. Plasma membrane associated TPPS4 was less efficient in cell inactivation per quantum of fluorescence emitted than intracellularly located dye. This was also to some extent the case for TPPS1 but not for TPPS2a and TPPS2o. The results presented here indicate that TPPS2a and TPPS1 are the most promising of the TPPSns for possible future use in photodynamic therapy.

Intracellular localization of sulfonated meso-tetraphenylporphines in a human carcinoma cell line.

The intracellular localization of meso-tetraphenylporphines sulfonated to different degrees (TPPSn), in a human cervix carcinoma cell line (NHIK 3025), was studied by fluorescence microscopy and fluorescence spectroscopy. After an 18 h incubation, TPPS4, TPPS2a and TPPS2o were localized in extranuclear granules. Studies of cells stained with both TPPS4, and acridine orange, which is known to fluoresce red in lysosomes, indicated that these granules were lysosomes. In addition, a fraction of the cellbound TPPS4, TPPS2a and TPPS2o seems to be associated with the plasma membrane. Fluorescence quenching studies of cells doublestained with acridine orange and TPPS4 indicated that TPPS4 is also localized in the nucleus and in the extralysosomal cytoplasm. The intracellular location of TPPS1 differed from that of the other TPPSns studied: In 6 out of 9 experiments fluorescing extranuclear granules were found. A diffuse fluorescence extending from the perinuclear area was also observed.

Cellular inhibition of microtubule assembly by photoactivated sulphonated meso-tetraphenylporphines.

This work relates to studies on modes of phototoxicity by sulphonated mesotetraphenylporphines on cultured cells. Toxicity appears to be related to inhibition of microtubule function. Treatment of human cervix carcinoma cells of the line NHIK 3025 incubated for 18 h with meso-tetraphenylporphine sulphonates (TPPSn where n = 2a, 2o or 4) and exposed to light, inhibits multiplication for the first hours after light exposure, a significant fraction of the cells accumulating in mitosis. The maximal number of cells in mitosis after treatment (approximately 20%) is dependent on the fluence but is similar for all three photosensitizers. For the first hours after treatment the mitotic cells were always mainly in metaphase; mainly seen as c-metaphases and three-group metaphases. During this time anaphase and telophase cells were absent or greatly reduced in number. Indirect immunofluorescence staining of beta-tubulin showed that the spindle apparatus of mitotic cells was perturbed in all cases. Results are presented which indicate that photoactivation of TPPSn located on the plasma membrane destroys microtubules in interphase cells and leads to arrest of the cells in mitosis. The localization of the dye which sensitizes the photoinduced perturbation of microtubules is further discussed.

Evaluation of sulfonated aluminum phthalocyanines for use in photochemotherapy. A study on the relative efficiencies of photoinactivation.

The cellular photosensitivity caused by aluminum phthalocyanines sulfonated to different degrees (AlPcSn) has been investigated. The phototoxic effect increased with decreasing number of sulfonate groups on the macrocycle, with the exception of AlPcS1 which was less phototoxic than AlPcS2 but more phototoxic than AlPcS3 and AlPcS4. The tendency of the AlPcSns to aggregate in our cellular system increased with increasing lipophilicity of the sensitizers. The aggregates had little or no photosensitizing activity. The low efficiency of cell inactivation caused by AlPcS1 can be explained by the highly aggregated state of this sensitizer in the cells. AlPcS2 and AlPcS3 induced a lower degree of cell inactivation per fluorescing quantum and per quantum absorbed by monomeric species than did AlPcS2 and AlPcS1. AlPcS4 and AlPcS3 are therefore suggested to be in different intracellular locations than AlPcS2 and AlPcS1.

Evaluation of sulfonated aluminum phthalocyanines for use in photochemotherapy. Cellular uptake studies.

Cellular uptake of aluminum phthalocyanine sulfonated to different degree was studied by means of fluorescence measurements and HPLC chromatography. These results were correlated to the lipophilic property of each drug measured as the distribution of the drug between a lipophilic phase (Triton X-114) and an aqueous phase. All the sulfonated aluminum phthalocyanines were taken up into cells to a higher extent than porphyrins of a similar lipophilicity. The cellular uptake of monosulfonated aluminum phthalocyanine was 10-fold higher than the cellular uptake of tetrasulfonated aluminum phthalocyanine and at least 50% higher than tetra(3-hydroxy-phenyl)porphin which is so far the porphyrin shown to be taken up into cells to the highest extent.

Strategies for selective cancer photochemotherapy: antibody-targeted and selective carcinoma cell photolysis.

A principle objective in chemotherapy is the development of modalities capable of selectively destroying malignant cells while sparing normal tissues. One new approach to selective photochemotherapy, antibody-targeted photolysis (ATPL) uses photosensitizers (PS) coupled to monoclonal antibodies (MAbs) which bind to cell surface antigens on malignant cells. Selective destruction of human T leukemia cells (HBP-ALL) was accomplished by coupling the efficient PS chlorin e(6) to an anti-T cell MAb using dextran carriers. Conjugates with chlorin: MAb ratios of 30:1 retained > 85% MA b binding activity, and had a quantum yield for singlet oxygen production of 0.7 +/- 0.1, the same as that of free chlorin e(6). Cell killing was dependent on the doses of both MAb-PS and 630-670 nm light and occurred only in target cell populations which bound the MAb. On the order of 10(10) singlet oxygen molecules were necessary to kill a cell. A second approach to specific photochemotherapy, selective carcinoma cell photolysis (SCCP), relies on preferential accumulation of certain cationic PS by carcinoma cell mitochondria. We have evaluated several classes of cationic dyes, and in the case of N,N'-bis-(2-ethyl-1,3-dioxolane)-kryptocyanine (EDKC) and some of its analogs, have demonstrated highly selective killing of human squamous cell, bladder and colon carcinoma cells in vitro. In isolated mitochondria, EDKC uptake and fluorescence depended on membrane potential, and the dye specifically photosensitized damage to Complex I in the electron transport chain. N,N'-bis-(2-ethyl-1,3-dioxolane)-kryptocyanine and some of its analogs accumulated within subcutaneous xenografts of human tumors in nude mice with tumor:skin ratios > 8. Photoirradiation caused significant inhibition of tumor growth, without cutaneous phototoxicity.

Antibody-targeted photolysis: selective photodestruction of human T-cell leukemia cells using monoclonal antibody-chlorin e6 conjugates.

Selective in vitro photodestruction of HPB-ALL human T-cell leukemia cells was accomplished using the photosensitizer chlorin e6 coupled through dextran molecules to an anti-T-cell monoclonal antibody (mAb), anti-Leu-1. Conjugates with mAb/chlorin molar ratios as high as 1:36 retained mAb binding activity, and the absorption spectrum and quantum efficiency for singlet oxygen production of bound chlorin (0.7 +/- 0.2) were unchanged from that of the free photosensitizer. Phototoxicity, as measured by a clonogenic assay and by uptake of ethidium bromide, was dependent on the doses of both mAb-chlorin and 630- to 670-nm light, was enhanced by 2H2O, and was observed only in target populations that bound the mAb. Similarly, free chlorin e6 in solution had no photodynamic effect in amounts 100 times more than that carried by the mAb. For this antibody-targeted system, approximately 10(10) molecules of singlet oxygen were necessary to kill a cell.

Zinc tetrasulphophthalocyanine as a photodynamic sensitizer for biomolecules.

Monomeric zinc tetrasulphophthalocyanine in aqueous buffer is an effective sensitizer for the photo-oxidation of amino acids and nucleic acid bases and for the photodynamic inactivation of the enzyme, lysozyme; these reactions appear to be mediated by singlet oxygen.