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Wild type Escherichia coli cells as well as some mutant strains lacking specific DNA repair systems are efficiently killed upon visible light-irradiation after 5 min-incubation with meso-tetra(4N-methyl-pyridyl)porphine (T4MPyP). The presence of oxygen is necessary for cell photoinactivation. The porphyrin appears to exert its phototoxic activity largely by impairing some enzymic and transport functions at the level of both the outer and cytoplasmic membrane. Thus, SDS-PAGE electrophoresis shows a gradual attenuation of some transport protein bands as the irradiation proceeds, while a complete loss of lactate and NADH dehydrogenase activities is caused by 15 min-exposure to light. On the other hand, DNA does not represent a critical target of T4MPyP photosensitization as suggested by the closely similar photosensitivity of the wild E. coli and E. coli strains defective for two different DNA repair mechanisms, as well as by the lack of any detectable alteration of the pUC19 plasmids extracted from photosensitized E. coli TG1 cells.

The phototoxicity of liposome-incorporated Zn(II)-phthalocyanine (ZnPc) and its water-soluble tetrasulphonated derivative (ZnPcTS) was studied in the tumorigenic but nonmetastatic (RE4) and the highly metastatic (4R) transformed rat embryo fibroblasts. Upon irradiation with 585–605 nm light in the presence of ZnPc, the cell survival drastically decreased, while it was unaffected by ZnPcTS. Enzymatic assays showed that ZnPc induced about a 60% decrease in the activity of the mitochondrial enzymes NADH and succinate dehydrogenase after 3 min of irradiation, while no significant reduction in the activity of lactate dehydrogenase and lysosomal N-acetyl-β-glucosaminidase was observed. The transport of thymidine, deoxyglucose and α-aminoisobutyric acid through the plasma membrane was strongly inhibited after irradiation. Similarly, the intracellular ATP content was significantly reduced. The reduction of DNA biosynthesis showed a time dependence quite similar to the photo-induced decrease in cell survival. No repair of cellular functions affected by ZnPc was observed in the 2 cell lines. These results indicate that, under our experimental conditions, hydrophobic ZnPc exerts its cytotoxic activity mainly by impairing those functions localized in the plasma membrane of the cells. Int. J. Cancer 75:412–417, 1998. © 1998 Wiley-Liss, Inc.

A highly metastatic (4R) and a nonmetastatic (RE4) transformed rat embryo fibroblast cell line were incubated with lipid-soluble Zn(II)-phthalocyanine (ZnPc) and its water-soluble tetrasulphonated derivative (ZnPcTS) and compared for phthalocyanine uptake. The hydrophobic liposome-delivered ZnPc showed a significantly greater uptake by both cell lines than did ZnPcTS. Moreover, the two phthalocyanines appear to interact with cells according to different pathways, as suggested by the different temperature-dependence of the binding process and the different inhibitory action exerted by selected serum proteins, such as lipoproteins and heavy proteins. Under all experimental conditions, the two cell lines exhibited similar interactions with ZnPc and ZnPcTS, suggesting that heterogeneity of the tumor cell population has a minor influence on the accumulation of photosensitizers.

Zinc(II) phthalocyanine (ZnPc), a potential second-generation phototherapeutic agent for tumours, has been incorporated into small unilamellar vesicles (SUVs) (diameter, 52 nm) and large unilamellar vesicles (LUVs) (diameter, 84 nm) of dipalmitoyl-phosphatidylcholine (DPPC). Absorption spectroscopy, as well as steady state and time-resolved fluorescence emission studies, indicate that ZnPc is monomeric in SUVs at a stoichiometric concentration below 0.25 μM (corresponding to an actual endoliposomal concentration of about 0.5 mM), while in LUVs it is monomeric below 2 μM. The fluorescence lifetime of the monomer is 3–3.5 ns. Upon increasing the ZnPc concentration, aggregated derivatives are formed, which are characterized by shorter fluorescence lifetimes (1.2–1.5 ns; 0.4–0.6 ns). The possible implications of these observations for the phototherapeutic efficiency of ZnPc are briefly discussed.

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.

A series of derivatives of 5,10,15,20-tetrakis-(4-N-methylpyridyl)-porphine, where one N-methyl group was replaced by a hydrocarbon chain ranging from C6 to C22, were characterized for their photophysical and photosensitizing properties. The absorption and fluorescence features of the various compounds in neutral aqueous solutions were typical of largely monomeric porphyrins, with the exception of the C22 derivative, which appeared to be extensively aggregated. This was confirmed by the very low triplet quantum yield and lifetime of the C22 derivative as compared with 0.2-0.7 quantum yields and 88-167 micros lifetimes for the other porphyrins. The photophysical properties and photosensitizing activity toward N-acetyl-L-tryptophanamide of the C22 porphyrin became comparable to those typical of the other derivatives in 2% aqueous sodium dodecyl sulfate, where the C22 compound is fully monomerized. All the porphyrin derivatives exhibited at micromolar concentrations photoinactivation activity against both Staphylococcus aureus and Escherichia coli, even though the gram-negative bacteria were markedly less photosensitive. The photosensitizing efficiency was influenced by (1) the amount of cell-bound porphyrin, which increased with increasing length of the hydrocarbon chain; and (2) the tendency to undergo partial aggregation in the cell, which seems to be especially important for the C22 derivative.

Low-density lipoproteins (LDL) increase the selectivity of tumour targeting by drugs, including sensitisers for photodynamic therapy, because of the enhanced expression of specific LDL receptors in many types of transformed as compared with non-transformed cells. This investigation aims at gaining more information on the role of LDL receptors in the accumulation of photosensitizer-LDL complexes by human and rat transformed fibroblasts, and the interference of the photosensitizer with LDL recognition by the specific receptors. Both an amphiphilic hematoporphyrin IX (Hp) and a hydrophobic Zn(II)-phthalocyanine (ZnPc) photosensitizers bind to human LDL with molar ratios of 5-6:1 and 10-12:1, respectively. The hematoporphyrin-LDL complex is accumulated by human HT1080 fibroblasts mainly through the high affinity LDL receptors, while the Zn-phthalocyanine-LDL complex is internalised through non specific endocytosis because of changes in the apoB LDL structure induced by phthalocyanine association, as suggested by spectroscopic studies. The uptake of LDL-delivered hematoporphyrin, but not Zn-phthalocyanine, is about 4-fold higher in HT1080 cells stimulated for maximal expression of LDL receptors as compared with non-stimulated cells. This difference is abolished by LDL acetylation. Human LDL-bound hematoporphyrin and Zn-phthalocyanine are up taken by stimulated and non-stimulated 4R rat fibroblasts with similar efficiency. Scatchard plot analysis of human (125)I-LDL binding to 4R cells shows the presence of only low affinity receptors while 350,000 high affinity receptors are expressed per HT1080 cell. It is concluded that a careful evaluation of the lack of conformational changes of LDL is critical for guaranteeing the selectivity and efficiency of photosensitizer delivery to tumour cells.

Steady-state and time-resolved spectroscopic studies on Zn(II)-phthalocyanine (ZnPc)-low-density lipoprotein (LDL) complexes obtained through the transfer of ZnPc molecules from dipalmitoyl-phosphatidylcholine (DPPC) or palmitoyl-oleoyl-phosphatidylcholine (POPC)-dioleoyl-phosphatidylserine (OOPS) liposomes have been carried out. The number of ZnPc molecules bound to the LDL particles varies with the liposome type and the ZnPc/LDL concentration ratio in the incubation medium; the ZnPc transfer to LDL is more efficient with POPC-OOPS liposomes. Steady-state fluorescence measurements show a reduction of the ZnPc fluorescence yield as the number of ZnPc molecules bound to LDL increases (about 40% reduction is observed with a ZnPc/LDL molar ratio of 60 as compared to 1). The fluorescence decay of ZnPc is best fitted by a monoexponential decay with a lifetime of 3.8 ns at a ZnPc/LDL ratio equal to 1, while at higher ratios a shorter-living fluorescence component is detected whose amplitude increases as the ZnPc/LDL ratio is increased. This behaviour suggests the presence of ZnPc aggregates in the LDL particle.

In the separated surface-sensitizer system, a photosensitizer is physically separated from the substrate by a thin air layer under such conditions that only singlet oxygen can reach and oxidize the substrate, preventing the competition by type I photosensitized processes. This method has been used to study the reaction of singlet oxygen with Gram-positive (Streptococcus faecium) and Gram-negative (Escherichia coli) bacterial strains. Studies on cell samples exposed to singlet oxygen for different periods of time show a drastic decrease in survival for S. faecium, while E. coli becomes sensitive only when the integrity of the outer membrane is altered by treatment with CaCl2 or tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid (Tris-EDTA). Biochemical and ultrastructural analyses suggest that the cytoplasmic membrane and the genetic material are the main sites damaged by singlet oxygen.

Zn(II)-phthalocyanine (ZnPc) is a tetraazaisoindole pigment which can be prepared by chemical synthesis with a high degree of purity, and it efficiently absorbs 680 nm of light. These properties, associated with its ability to generate activated oxygen species (e.g., singlet oxygen) upon photoexcitation, make ZnPc a potential phototherapeutic agent. Actually, upon delivery of ZnPc to tumor-bearing animals after incorporation into liposomal vesicles, the dye was uptaken and retained by the tumor tissue in significant amounts and with a good degree of selectivity. Irradiation of the ZnPc-loaded tumor area with ca. 680 nm of light caused tumor necrosis to an extent which was related to the dye concentration in the tumor.© (1991) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The use of phthalocyanines as phototherapeutic agents for tumours was first proposed in 1985 (Ben-Hur and Rosenthal, 2985). A variety of cellular and animal studies, (for a review see Spikes, 1986;Tralau et al., 1987) have supported this proposal and it presently appears that some phthalocyanines are probable choices as second-generation tumour-localizers and -photosensitizers. At present, the phthalocyanines most frequently used in the experimental photodynamic therapy (PDT) of tumours include some sulfonated derivatives of the tetra-azaisoindole macrocycle (Brasseur et al., 1985;Ben-Hur and Rosenthal, 1986). Although these derivatives are relatively water-soluble, they are still a mixture of compounds with different degrees of sulfonation and/or showing structural isomerism.

The phototransformation Pr→Pfr of 124-kDa phytochrome from etiolated Avena was further studied using two approaches. One was the monitoring of the P fr appearance after excitation of Pr with ms pulses. The absorbance increase in the region 720–750 nm after ca. 10 ms of excitation occurs in two steps (as already reported by Pratt, et al., 1982), with lifetimes (percentages) in the range of 30 ms (ca. 50%) and 1 s (ca. 50%) at 275 K. Both rate constants were only slightly affected by temperature in the range 275–293 K. In contrast, the relative amplitude of the two components of the kinetics was influenced by temperature. The small D/H isotope effect on the time constants of P fr appearance and on the relative percentage of the components make it difficult to decide whether the isotope substitution affects the last or the previous to last, or both steps of Pfr appearance. The results indicate that a solvent-assisted activated step occurs previous to the last, while the last step is better interpreted as a conformational change without proton exchange. Parallel ways leading from Pr to Pfr include an initial isomerization of the chromophore and solvent-assisted chromophore and protein conformational changes. The results show that the solvent-assisted processes involve only changes in hydrogen bridges rather than full proton transfer. These data supplement those from ps fluorescence (Brock, et al., 1987) and µs transient absorbance (Aramendia, et al., 1987). A simplified picture of the transformation is shown in the scheme.

Escherichia coli, as most Gram-negative bacteria, is insensitive to the photosensitizing action of both lipid-soluble Zinc-phthalocyanine (Zn-Pc) and water-soluble Zinc-mono/disulfonated phthalocyanine (Zn-PcS). Photosensitivity can be induced by alteration of the outer membrane, as obtained by either induction of competence or treatment with Tris-EDTA. Both phthalocyanines largely bind at the level of the cytoplasmic membrane; however, Zn-PcS shows a superior photosensitizing activity as compared with Zn-Pc. Biochemical analyses performed on irradiated cells suggest that the cytoplasmic membrane is an important target of the photoprocess, while DNA is not involved.

Porphycene (a porphyrin isomer) has been incorporated into small unilamellar vesicles (diameter about 52 nm), and its first excited singlet and triplet states have been investigated under these conditions. Below a local concentration of about 4 mM in the vesicle, the photophysical properties of porphycene are as in organic solvents. Above this critical local concentration a second shorter-lived component appears in the fluorescence and triplet decays, and the fluorescence yield and lifetime and population of the longer-lived triplet are lower. Also, the rate constant of triplet quenching by oxygen is somewhat higher, and the yields of porphycene- and 2,7,12,17-tetra-n-propylporphycene-sensitized singlet molecular oxygen formation in the vesicles drop as well. Furthermore, the absorbance and fluorescence yield change with temperature when the porphycene concentration is greater than the critical limit: at just below the phase transition temperature of the lipid the two values rise steeply from low plateaus to plateaus about twice as high upon warming. The inherently less sensitive Beer—Lambert absorption plot curves downwards starting at a somewhat higher value than the critical local concentration (10 mM). The findings are discussed on the basis of (i) aggregation of porphycene at high local concentrations and (ii) localization at two distinct sites within the vesicle.

The absorption and fluorescence properties of Zn2+-phthalocyanine (Zn-Pc) have been characterized in homogeneous and microheterogeneous media. By our preparation procedure, the phthalocyanine can be associated in a monomeric state with cationic micelles, unilamellar liposomes, and low-density lipoproteins; the distribution of Zn-Pc in the hydrophobic phases appears to be controlled by the nature of the lipid environment. The potential use of liposome-bound Zn-Pc for photosensitization studies in aqueous media and for phototherapeutic applications in vivo is briefly discussed.

— Zn(II)phthalocyanine (ZnPc) generates O2(1Δg) with a quantum yield of ca. 0.4 upon photocxcitation at 354 or 600 nm in ethanolic solution as determined by time-resolved phosphorescence studies at 1270 nm and photooxidation experiments using 1,3-diphenylisobenzofuran (DPBF) as substrate. The quantum yield of photooxidation slightly increases upon incorporation of ZnPc into unilamellar liposomes of dipalmitoylphosphatidylcholine. Under our irradiation conditions (600 nm, 18°C, and short light exposure times), DPBF(5–50 μM) undergoes photooxidation by a pure Type II mechanism; the rate constant for the O2(1Δg) + DPBF reaction is (1.1 ±0.1) x 109 M-1 s_1 in ethanol solution and determined to be about two orders of magnitude smaller when both ZnPc and DPBF are embedded into liposomes.