Zn(II)-phthalocyanines as phototherapeutic agents for cutaneous diseases. Photosensitization of fibroblasts and keratinocytes

Two tetrasubstituted (RLP024 and RLP040) and one monosubstituted (MRLP101) Zn-phthalocyanines were readily accumulated by three skin-derived cell lines (HT-1080 transformed human fibroblasts, 3T3 mouse embryo fibroblasts and HaCaT human keratinocytes) upon 1 h-incubation with 0.5-5 microM phthalocyanine concentrations. The affinity was markedly larger for the tetra- as compared with the mono-substituted phthalocyanine, even though smaller phthalocyanine amounts were generally recovered from keratinocytes. As a consequence, the two tetra-substituted phthalocyanines exhibited a higher phototoxicity against all the three cell lines. Typically, the cell survival decreased by at least 80% after 1 min irradiation with 600-700 nm light at a fluence-rate of 50 mW/cm2 in the presence of 5 microM phthalocyanine. Fluorescence microscopy and caspase-3 activation studies indicate that cell death of fibroblasts largely occurred by a random-necrotic process while the keratinocytes underwent cell death predominantly via apoptosis in spite of a very similar pattern of subcellular distribution of the phthalocyanines.     

PHOTOEXCITATION OF ZINC PHTHALOCYANINE IN MOUSE MYELOMA CELLS: THE OBSERVATION OF TRIPLET STATES BUT NOT OF SINGLET OXYGEN

Abstract— Liposomes were used to deliver zinc phthalocyanine (ZnPC) to cultured mouse myeloma cells. ZnPC triplets states were observed when such systems were photoexcited. Singlet oxygen luminescence was not observed in cellular media. Solutions of ZnPC in various organic solvents, sodium dodecyl sulfate micelles and dipalmitoyl phosphatidyl choline liposomes were also prepared, and time-resolved triplet state parameters and singlet oxygen production were measured for these systems to provide comparisons for the cellular suspensions.     

PORPHYRIN-LIPOSOME INTERACTIONS: INFLUENCE OF THE PHYSICO-CHEMICAL PROPERTIES OF THE PHOSPHOLIPID BILAYER

Abstract— The quenching of the fluorescence emitted by hematoporphyrin incorporated into unilamellar liposomes of dipalmitoyl-phosphatidylcholine and dimyristoyl-phosphatidylcholine, was studied by using methylviologen, 9,10-anthraquinone-2,6-disulfonate and 9,10-anthraquinone-2-sulfonate as quenchers, in order to assess how the distribution of the porphyrin and the interaction mode of the various quenchers with the porphyrin is affected by the physico-chemical properties of the vesicles. The results obtained indicate that, below the critical temperature for the phase transition of the lipids, hematoporphyrin is preferentially distributed in the outer lipid monolayer of liposomes of dipalmitoyl-phosphatidylcholine while most hematoporphyrin molecules are located in the inner monolayer in liposomes of dimyristoyl-phosphatidylcholine. This distribution is only slightly changed when the external mean radius of liposomes increases from 26 to 50 nm. The rise of temperature above the critical value for the liquid-gel phase transition causes a shift of the hematoporphyrin molecules toward the inner phospholipid monolayer. This shift is more pronounced in liposomes of dimyristoyl-phosphatidylcholine. Studies on model systems, i.e. neutral and ionic micelles, indicate that methylviologen and anthra-quinone-type quenchers drastically differ in their interaction mechanism with hematoporphyrin. In particular, methylviologen is the only quencher which can discriminate different hematoporphyrin populations in liposomes of dimyristoyl-phosphatidylcholine and dipalmitoyl-phosphatidylcholine in both the liquid and gel phase. Anthraquinone-type quenchers interact with both hematoporphyrin populations when the lipids are in the gel phase. When the lipids are in a fluid state, the quenching occurs only on the external hematoporphyrin population in liposomes of dipalmitoyl-phosphatidylcho-line while in liposomes of dimyristoyl-phosphatidylcholine no discrimination is observed. The influence of the liposomal structure at different temperatures and of the length of the hydrocarbon chains is discussed.     

Photochemical and pharmacokinetic properties of selected flavins

Some photochemical and pharmacokinetic properties of riboflavin, lumiflavin and the 2',3',4',5' tetraacetyl, tetrapropionyl, tetrabutiryl and tetrapalmitoyl esters of riboflavin have been studied. The esters are considerably more photostable than riboflavin but less so than lumiflavin, and appear to be photosensitizers with a behaviour similar to that of riboflavin, promoting photoreactions of biological targets even in the absence of molecular oxygen. The various flavins display important differences in their pharmacokinetic behaviour. Riboflavin, lumiflavin and the short-chain esters (the acetyl and propionyl esters) are rapidly cleared from serum, and recovered in comparable amounts from the liver and kidneys. These results are in agreement with their hydrophilic or moderately hydrophobic character. In contrast, the longer-chain butiryl and palmitoyl esters exhibit a prolonged retention in serum and undergo a significantly larger accumulation in the liver as compared with the kidneys; they are also found in the spleen. In all cases the tissue uptake of these esters becomes appreciable only after 24 h. These results are coherent with the highly hydrophobic character of these esters, which induce a slow release from serum lipoproteins and have a preferential clearance via the bile-gut pathway, showing affinity for the components of the reticuloendothelial system. These long-chain riboflavin esters will probably have a greater and more persistent risk of photoinduced hepatotoxicity than riboflavin, lumiflavin and the short-chain esters.     

PHOTOKINETIC AND PHOTOPHYSICAL MEASUREMENTS OF THE SENSITIZED PHOTOOXIDATION OF THE TRYPTOPHYL RESIDUE IN N-ACETYL TRYPTOPHANAMIDE AND IN HUMAN SERUM ALBUMIN

Abstract— The photosensitized oxidation of 10–100 μ M N -acetyl-L-tryptophanamide (NATA) in neutral aqueous solution and in the presence of various dyes proceeds by a pure O₂(¹Δ_g)-involving mechanism. Incorporation of the tryptophyl (Trp) residue into the polypeptide chain of human serum albumin (HSA) has no influence on the mechanism and efficiency of Trp photooxidation when sensitized either by methylene blue, a non-binding dye, or by rose bengal, a dye that gives non-covalent 1: 1 complexes with HSA. This is due to the location of the Trp residue in close proximity of the protein surface and, in the case of rose bengal, to the coincidence of the photophysical properties (including the quantum yield of O₂(¹Δ_g) generation) for the free and HSA-bound dye. Hematoporphyrin also binds to HSA with 1: 1 stoichiometry, although at a different site from rose bengal. Bound Hp again displays photophysical properties very similar with those of free Hp; however, the efficiency of Trp photo-oxidation in HSA is about 5-fold higher than in NATA owing to a limited rearrangement of the protein structure, induced by Hp binding, which enhances the probability of chemical quenching of O₂(¹Δ_g) by the indole ring.     

Synthesis of tetrasubstituted Zn(II)-phthalocyanines carrying four carboranyl-units as potential BNCT and PDT agents

The synthesis of two tetrasubstituted zinc(II)phthalocyanines carrying four carbon-carbon linked o-carboranyl-units (40 boron atoms, 27.5% boron by weight) is presented. In an in vitro model test the new compounds showed a good photosensitizing efficiency, that encourages further studies for their evaluation as possible BNCT-PDT sensitizers.     

STUDIES ON THE MECHANISM OF THE HEMATOPORPHYRIN-SENSITIZED PHOTOOXIDATION OF 1,3-DIPHENYLISOBENZOFURAN IN ETHANOL and UNILAMELLAR LIPOSOMES

The 5 microM hematoporphyrin-sensitized photooxidation of 1,3-diphenylisobenzofuran (DPBF) was studied in homogeneous ethanolic solutions and in aqueous dispersions of unilamellar liposomes of dipalmitoylphosphatidylcholine; both the porphyrin and DPBF are embedded in the phospholipid bilayer. The rate and quantum yield of DPBF photooxidation were found to increase upon increasing the substrate concentration and were higher in the liposome system, while they were unaffected by the fluidity of the phospholipid bilayer. Time-resolved spectroscopic measurements showed that the photooxidation of DPBF in ethanol solution proceeds by a type II O2(1 delta g)-involving mechanism. In the liposomal vesicles the high local concentration of hematoporphyrin (Hp) and DPBF in the phospholipid bilayer (ca 2000-fold higher than the stoichiometric concentration) enhances the probability of energy transfer from triplet Hp to DPBF with generation of triplet DPBF; hence O2 (1 delta g) formation can be promoted by both triplet Hp and triplet DPBF. A minor fraction of triplet DPBF quenchings appears to generate radical species which propagate DPBF damage by chain reaction.