Aluminum-triggered structural modifications and aggregation of beta-amyloids.

We investigated the structural effects induced by Al3+ on different beta-amyloid (Abeta) fragments at pH 7.4 and T=25 degrees C, with particular attention given to the sequences 1-40 and 1-42. Al3+ caused peptide enrichment in beta sheet structure and formation of solvent-exposed hydrophobic clusters. These intermediates evolved to polymeric aggregates which organized in fibrillar forms in the case of the Al3+-Abeta(1-42) complex. Comparative studies showed that Zn2+ and Cu2+ were much less efficient than Al3+ in stimulating the spontaneous aggregation/fibrillogenesis of Abetas. Studies with liposomes as membrane models showed dramatic changes in the structural properties of the lipid bilayer in the presence of Al3+-Abeta complexes, suggesting a major role of Al3+ in Abeta-induced cell dysfunction. Al3+ effects were abolished by desferrioxamine mesylate (DFO) only in solution. We concluded that, in vivo, DFO may act as a protective agent by preventing or reverting Abeta aggregation in the extracellular spaces.

Disaccharide modulation of the mitochondrial membrane fluidity changes induced by the membrane potential.

The influence of the medium composition on the dynamic properties of mitochondrial membranes on depolarization was studied by following the fluorescence anisotropy changes of mitochondria-bound 1,6-diphenyl-1,3,5-hexatriene (DPH) and hematoporphyrin (HP) as reporters, respectively, of lipid and protein regions. On collapse of the potential, the membrane fluidity increased in NaCl-, KCl-, and monosaccharide-based media and decreased in disaccharides. Infrared spectroscopy experiments suggested that disaccharides likely change water's structure and association on the membrane surface. These results indicate that disaccharides induce membrane perturbation, which may interfere in the study of structure-function correlation in biological membranes.

The effects of singlet oxygen produced by photodynamic action on the mitochondrial permeability transition differ in accordance with the localization of the sensitizer.

We have examined whether the effects of singlet oxygen (1O2) produced by photodynamic action on the mitochondrial permeability transition (PT) can be modulated by the localization of photosensitizers in irradiated mitochondria. We have previously shown that oxidation due to 1O2 photogenerated in hematoporphyrin (HP)-loaded mitochondria can prevent opening of the PT pores, likely after degradation of some critical histidines (Salet et al, 1997, J. Biol. Chem. 272, 21938-21943). Equally, in the present study we have irradiated mitochondria in the presence of a structurally different photosensitizer producing 1O2, namely 4,5',8-trimethylpsoralen (TMP). Fluorescence studies show that TMP binds to protein sites which differ from those of HP. In sharp contrast with HP, TMP-driven photodynamic action triggers per se pore opening. Interestingly, this inducing effect is inhibited when TMP-treated mitochondria are irradiated after addition of mersalyl, a specific reagent protecting thiol groups of the inner mitochondrial membrane that are oriented toward the external hydrophilic phase. This fact suggests that 1O2-mediated thiol oxidation is responsible for TMP-photoinduced pore opening. Taken together, these findings suggest that 1O2 can activate or inactivate a cellular function such as mitochondrial PT depending on the site where it is produced in the mitochondrial membrane.

Implications of the generation of reactive oxygen species by photoactivated calcein for mitochondrial studies.

Calcein is a fluorescent probe that is widely used in studies of cell viability and mitochondrial function by microscopy fluorescence imaging. It was found to have a strong photosensitizing action that prevalently involves the generation of reactive oxygen species (ROS). The photooxidation properties of calcein in solution were studied in the presence of histidine and tryptophan as oxidizable substrates. The photodegradation of histidine was mainly mediated by singlet oxygen (1O2), as shown by the inhibitory effect of sodium azide, a specific 1O2 scavenger. On the other hand, mixed photosensitization mechanisms were present when tryptophan was used as the target of the calcein-stimulated photoprocess. In addition to 1O2, hydroxyl radicals and hydrogen peroxide were involved as reactive species, as shown by using mannitol and catalase as scavengers. The calcein-photosensitized alterations of mitochondria as a potential source of artifacts in confocal microscopy studies of cells were considered. Irradiation of isolated mitochondria with visible light (500-600 nm) in the presence of calcein induced opening of the permeability transition (PT) pore. The extent of the mitochondrial membrane photodamage, however, was modulated by the nature of the calcein environment. Thus, pore opening was triggered at short irradiation times and low dye concentrations when calcein was dissolved in the bulk medium. On the contrary, calcein concentrated in the matrix space was rather inefficient as photosensitizer even at concentrations 10 times higher than those present in the external medium.

Effect of delivery system on the pharmacokinetic and phototherapeutic properties of bis(methyloxyethyleneoxy) silicon-phthalocyanine in tumor-bearing mice.

A Si(IV)-phthalocyanine bearing two methoxyethyleneglycol axial ligands bound to the central metal ion (SiPc) has been prepared by chemical synthesis and analyzed for its phototherapeutic activity after administration in a Cremophor or liposome formulation to C57B1/6 mice bearing a subcutaneously transplanted Lewis lung carcinoma (LLC). The maximum drug accumulation in the tumor is found at 24 h after intraperitoneal injection, independent of the delivery system. However, the tumor concentration of SiPc in the Cremophor formulation is about two-fold higher, while the drug concentration in liver and skin shows similar trends with the two delivery systems. The drug accumulation and retention in the brain is much larger when using Cremophor emulsion. Photodynamic therapy (672 nm, 370 mW m-2, 360 J cm-2) at 24 h after the injection of Cremophor emulsion- or DPPC liposome-formulated SiPc causes a very efficient and similar response for the LLC (approximately 8 versus 22 mm mean tumor diameter for the control groups at 21 days after phototreatment). These very promising effects, obtained both at higher and lower tumor drug concentrations, clearly demonstrate the potential phototherapeutical activity of the newly synthesized SiPc.

Changes of the fluidity of mitochondrial membranes induced by the permeability transition.

The dynamic properties of protein and lipid regions of mitochondrial membranes during the permeability transition (PT) process were studied by following the anisotropy changes of hematoporphyrin (HP) and 1,6-diphenyl-1,3,5-hexatriene (DPH), respectively. We show that opening of the PT pore is accompanied by a remarkable increase of mitochondrial membrane fluidity which is specifically localized to protein sites, while lipid domains are unaffected. The increased membrane fluidity is not related to the collapse of transmembrane potential that follows the PT, as demonstrated by a comparison between the anisotropy properties of permeabilized mitochondria and impermeable, depolarized organelles. Parameters such as osmotic swelling and temperature, which are shown to affect the mitochondrial membrane dynamics in the absence of permeability transition, cannot alone account for the pore dynamical properties. We suggest that the observed increase in fluidity is mainly due to a conformational change of pore-forming protein(s) during the "assembly" of the PT pore.

Photodynamic action of porphyrin on Ca2+ influx in endoplasmic reticulum: a comparison with mitochondria.

We have studied the distribution properties of haematoporphyrin (HP) and protoporphyrin (PP) in mitochondria and endoplasmic reticulum after isolation from rat liver. The photosensitizing efficiency of porphyrin on the Ca2+ influx function of microsomes has been compared with that obtained on Ca2+ uptake in mitochondria. HP and PP are accumulated in microsomes to a greater extent than in mitochondria, both porphyrins binding to membrane protein sites. The Ca2+ influx functions of mitochondria and microsomes, before and after irradiation in the presence of HP or PP, were studied by following the changes in the free Ca2+ concentration in the medium as revealed by the variations in fluorescence intensity of the Ca2+ indicator Calcium Green-1. For the same amount of incorporated porphyrin, the Ca2+ influx function of microsomes is degraded by irradiation more rapidly than that of mitochondria. The protective effect of dithiothreitol suggests that thiol groups in the Ca2+-transporting enzyme are the preferential targets of the photodynamic effect. These results suggest that intracellular Ca2+ movements are altered primarily by the endoplasmic reticulum rather than by mitochondrial damage, in good agreement with other observations made in porphyrin-loaded irradiated cells.

Effects of photodynamic action on respiration in nonphosphorylating mitochondria.

We have studied the effects of singlet oxygen produced by photodynamic action on respiration in nonphosphorylating mitochondria (state 4). Isolated rat liver mitochondria were incubated with 3 microM hematoporphyrin and irradiated at 365 nm with a fluence rate of 25 W/m2. After short durations of irradiation, state 4 respiration with beta-hydroxybutyrate as substrate increases while respiration with succinate is negligibly affected. When mitochondria have been uncoupled with carbonylcyanide-p-trifluoromethoxyphenyl hydrazone before irradiation, no change occurs in beta-hydroxybutyrate-driven respiration, while succinate-driven respiration strongly decreases. Stimulation of state 4 NADH respiration cannot be explained by slippage of the NADH ubiquinone oxidoreductase because the stoichiometry of the redox pump was found insensitive to photodynamic action. In the light of the metabolite theory for linear enzymatic chains applied to state 4 respiration (Brand et al., Biochem. J. 255, 535-539, 1988), these results suggest that stimulation of NADH respiration is simply due to an increase of membrane leaks which occurs after irradiation. In the case of succinate-driven respiration, a strong inhibition of succinate dehydrogenase activity has been demonstrated after irradiation. It can be suggested that this inhibition introduces a negative control coefficient over state 4 respiration, counterbalancing the effects due to leakage.

Photophysical properties of porphyrin planar aggregates in liposomes.

This paper describes studies of some photophysical properties of non-covalent planar aggregates of hematoporphyrin and protoporphyrin. This porphyrin species has been recently discovered and can be generated in lipid bilayers such as liposomes and inner mitochondrial membranes. The relative weight of this species in different media, as compared to porphyrin monomers and stacked aggregates, has been deduced by fluorescence decay studies. In contrast with what is observed for stacked aggregates, promotion of planar suprastructures can occur both in aqueous and lipid environments. The spectroscopic properties are very similar to those of the corresponding monomers, in particular as regards the shape of the absorption and emission spectra. The fluorescence decay times are generally higher than those of the monomers, and depend on the medium in which the planar aggregates are formed. The photooxidation properties of porphyrin planar aggregates, as revealed by oxygen consumption and histidine photodegradation upon irradiation at 365 nm, were compared to those of the monomers. The extent of the photooxidation process is nearly 20-30% higher in planar aggregates than in the monomers. In contrast, it is well known that cofacial aggregates are photochemically inert and only monomeric species of porphyrin are efficient photosensitizers. The biological relevance of these findings is discussed.

Singlet oxygen produced by photodynamic action causes inactivation of the mitochondrial permeability transition pore.

We have studied the effects of singlet oxygen produced by photodynamic action on the cyclosporin A-sensitive permeability transition (PT) in isolated rat liver mitochondria. Mitochondria were incubated with 3 microM hematoporphyrin and irradiated at 365 nm with a fluence rate of 25 watts/m2. For short durations of irradiation (60 s) the adenine nucleotide translocase was inactivated, but mitochondria retained their ability to form a proton electrochemical gradient and accumulated Ca2+ and Pi at the same rate as non-irradiated controls. Strikingly, however, the oxidative effects of photodynamic action prevented opening of the PT pore which is normally induced by Ca2+ plus Pi or by treatment with diethyl pyrocarbonate (a histidine reagent) or diamide (a thiol oxidant). We show that the most likely targets for photodynamic action are critical histidines that undergo degradation. Irradiated, hematoporphyrin-loaded mitochondria treated with diethyl pyrocarbonate or diamide still undergo the PT when treated with phenylarsine oxide, which reacts with a critical dithiol involved in pore modulation (Petronilli, V., Costantini, P., Scorrano, L., Colonna, R., Passamonti, S., and Bernardi, P. (1994) J. Biol. Chem. 269, 16638-16642). These data suggest (i) that the dithiol cysteines are not oxidized by photodynamic action, but rather became inaccessible to oxidants; and (ii) that irradiation of hematoporphyrin-loaded mitochondria does not lead to pore denaturation, but rather to site-selective inactivation of discrete pore functional domains.

Effects of Photofrin photodynamic action on mitochondrial respiration and superoxide radical generation.

Cyanide-resistant respiration increases after irradiation of isolated mitochondria in the presence of Photofrin. This suggests an enhancement of electron leakage which has been evaluated by measuring superoxide radical formation in submitochondrial particles incubated with 6 micrograms/ml Photofrin in the medium and irradiated with increasing doses of light at 365 nm. After a dose of 4.5 kJ/m2 has been delivered, superoxide generation increases by a factor of approximately 2.5 at the level of NADH dehydrogenase and by a factor approximately 1.5 in the cyt bc1 region. These effects have been compared with changes observed in NADH-, succinate- and ascorbate-driven respiration and their implications discussed.

Fluence rate effects on photodynamic therapy of B16 pigmented melanoma.

In vivo experiments were performed to evaluate the effect of fluence rate on the efficiency of Zn(II)-2,3 naphthalocyanine (ZnNc) photosensitization of B16 pigmented melanoma subcutaneously transplanted in C57B1/6 mice. The tumour was irradiated with 774 nm light at 24 h after an injection of liposome--which incorporated ZnNc (0.5 mg kg-1 b.w.). A total light dose of 360 J cm-2 was delivered at fluence rates of 260, 320, 380, 440 and 500 mW cm-2. Separate groups of mice utilized to monitor tumour temperature changes during irradiation without or after anaesthesia. Tumour response was determined by measuring the mean tumour diameter of the treated towards the untreated animals for a period of 21 days following PDT, as well as the percentage of cured animals. The most promising result (40% complete tumour response) was obtained with anaesthetized mice following 380 mW cm-2. Higher dose rates (440 and 500 mW cm-2) led to less promising results for both anaesthetized and non anaesthetized mice. These results outline the potential of PDT with longer wavelengths for the treatment of highly pigmented tumour tissues. The optimal fluence rate for tumour treatment should be chosen in order to avoid inflammatory effects (tissue swelling) and oxygen suppression with sublethal injury to the tumour cells.

Si(IV)-methoxyethylene-glycol-naphthalocyanine: synthesis and pharmacokinetic and photosensitizing properties in different tumour models.

A Si(IV)-naphthalocyanine bearing two methoxyethylenglycol axial ligands to the centrally coordinated metal ion (SiNc) was prepared by chemical synthesis and assayed for the phototherapeutic activity after administration in a Cremophor formulation to C57BI/6 mice bearing a subcutaneously transplanted Lewis lung carcinoma or B16 pigmented melanoma. Pharmacokinetic studies indicate that the maximal accumulation in the tumour occurs at 24 h after intraperitoneal injection of 0.5 mg kg-1 of SiNc, although the naphthalocyanine concentration in the Lewis lung carcinoma (0.70 microgram g-1) is significantly larger than that in the B16 pigmented melanoma (0.15 microgram g-1). This results in a higher selectivity of tumour targeting in the case of the lung carcinoma. Photodynamic therapy (782 nm, 370 mW cm-2, 360 J cm-2) at 24 h after SiNc injection causes an efficient tumour response for Lewis lung carcinoma (50% lower tumour diameter on day 19 post-treatment as compared to untreated controls) while the pigmented melanoma shows only a minor response regarding the rate of tumour growth.

Temperature-induced changes in fluorescence properties as a probe of porphyrin microenvironment in lipid membranes. 2. The partition of hematoporphyrin and protoporphyrin in mitochondria.

The temperature dependence of hematoporphyrin and protoporphyrin fluorescence quantum yields (phi F) was studied after delivery to whole mitochondria or isolated inner (IMM) and outer (OMM) mitochondrial membranes, obtained from liver of Wistar rats. These studies are very sensitive to variations of the porphyrin lipid environment. Before incorporation, the porphyrins were dissolved in 0.01 M sodium phosphate, 0.15 M NaCl, pH 7.4) NaCl/Pi (only hematoporphyrin) or dispersed into liposomes of dipalmitoylphosphoglycerocholine (Pam2GroPCho), sometimes enriched with cholesterol or cardiolipin. Whole mitochondria show higher incorporation capacity of hematoporphyrin and protoporphyrin than isolated IMM and OMM, probably because additional, energy-sensitive transport mechanisms for the porphyrin uptake occur in intact organelles. A small decrease in protoporphyrin uptake is observed in OMM in comparison with IMM; in contrast, the decrease in hematoporphyrin uptake by OMM is rather significant. A comparison between the results obtained with IMM, OMM and whole mitochondria show that both porphyrins, when released to the intact organelles, preferentially localize in the IMM, irrespective of the lipid carrier used. NaCl/Pi-dissolved hematoporphyrin probably interacts with some membrane proteins, due to the similarity of the Arrhenius plots with those obtained for liposome-entrapped human serum albumin/hematoporphyrin complexes which were used as models to mimic hematoporphyrin-membrane protein binding sites. Liposomal hematoporphyrin and protoporphyrin bind to lipid domains. Hematoporphyrin accumulates in specific, localized lipid regions, perhaps in the boundary lipids area surrounding some inner-mitochondrial carriers; protoporphyrin accomodates in more rigid, lipid areas. On these bases, the higher photoactivity of hematoporphyrin, previously observed in mitochondria, in comparison with protoporphyrin, can be easily explained. Formation of linear dimers/aggregates, endowed with higher phi F than that of the monomers, are postulated to occur for both porphyrins only in the inner mitochondrial membrane.

Temperature-induced changes in fluorescence properties as a probe of porphyrin microenvironment in lipid membranes. 1. The partition of hematoporphyrin and protoporphyrin in liposomes.

Temperature-induced fluorescence changes were studied for hematoporphyrin and protoporphyrin, incorporated into liposomes of dipalmitoylphosphoglycerocholine (Pam2GroPCho) or dimiristoylphosphoglycerocholine (Myr2GroPCho). In some cases, cholesterol or cardiolipin were added to the vesicles for better mimicking the lipid composition of biological membranes. The experimental conditions were appropriately chosen in order to reproduce different possible configurations of the porphyrin molecule in lipid membranes: namely, at the polar water/headgroups, headgroups/lipid and lipid/lipid interfaces. A peculiar feature observed in some of the above liposomal systems was the appearance of discontinuities in the Arrhenius plots of the fluorescence quantum yields, with relevant changes of the values of activation energies. These discontinuities were due to an increase of the fluorescence signal in a temperature range corresponding to the transition of the different lipids from the gel-to-liquid crystal state. The observed phenomena are consistent with the formation of non-covalent linear dimers or linear higher aggregates of the porphyrin molecules. The intermolecular contacts required for the formation of these species are favoured by at least three situations: disruption of the ordered lipid structure during the gel-to-liquid crystal phase transition; competition of other polar groups (e.g., the -OH group of cholesterol) with the porphyrin carboxylate groups for the polar phospholipid headgroups; and steric constraints due to overcrowding of porphyrin molecules in a restricted space.

Photophysical properties of porphyrins in biological membranes.

This review illustrates the photophysical properties of some porphyrins, especially those used for biomedical applications, in relation to their photosensitizing efficiency in biological membranes. Porphyrin absorption and luminescence properties are mainly examined. The factors influencing the affinity of porphyrins for biological membranes, including the dye hydrophobicity, the charge and aggregation state, the pH of the medium and the physicochemical properties of the dye environment, are discussed. These factors determine the differences in the photophysical properties of porphyrins in biological membranes. Particular attention is paid to the porphyrin aggregation state: only monomeric species and possibly planar end-to-end aggregates are endowed with significant photosensitizing ability. Many conclusions presented are based on data obtained on membrane model systems such as micelles or liposomes which can mimic specific situations occurring in cells.

Interaction of phthalocyanines with lipid membranes: a spectroscopic and functional study on isolated rat liver mitochondria.

Absorption and emission spectroscopic studies on Zn(II)-phthalocyanine (ZnPc) incorporated into unilamellar liposomes of dipalmitoylphosphatidylcholine, sometimes added with cholesterol or cardiolipin, and released to rat liver mitochondria via the three types of liposomal vesicles indicated that: (a) ZnPc predominantly dissolves in all lipid domains of biological membranes with the exception of cardiolipin-containing regions; a partial localization of ZnPc in protein binding sites is also postulated; (b) the spectroscopic properties of ZnPc, although mainly determined by the aggregation state of the dye, are somewhat influenced by the physico-chemical characteristics of the lipid environment; (c) ZnPc-binding lipid domains in mitochondria are mainly localized in the outer membrane; this conclusion is clearly deduced from the trends of Arrhenius plots of the ZnPc fluorescence quantum yield in whole mitochondria and isolated inner or outer membrane in the temperature range -10 degrees C-(+)45 degrees C; (d) the nature of the ZnPc-binding site in mitochondria is not dependent on the chemical composition of the liposome carrier, contrary to what observed for other hydrophobic dyes, such as porphyrins. This has been also confirmed by photosensitization experiments. Actually, illumination of ZnPc-loaded mitochondria by 600-700 nm light causes a decline of the respiratory control ratio, which is essentially dependent on the amount of incorporated photosensitizer, irrespective of the composition of the carrier.

Hematoporphyrin derivative (Photofrin) photodynamic action on Ca2+ transport in monkey kidney cells (CV-1).

After 24 h incubation with Photofrin (PF), photodynamic action has been studied on Ca2+ transport in CV-1 cells. A moderate increase of the cytosolic free Ca2+ concentration [Ca2+]i is observed immediately after a dose of irradiation which yields a survival rate of less than 5% at 48 h. In parallel, studies on digitonin-permeabilized cells indicate that such a treatment inhibits endoplasmic reticulum Ca2+ uptake with few alterations of this process in mitochondria. In contrast, ADP-stimulated respiration is impeded and intracellular ATP level decreases. It is suggested that direct damage to endoplasmic reticulum as well as mitochondrial disturbance are the primary mechanisms responsible for a nontransient elevation of [Ca2+]i preceding cell death.

Photosensitization of mitochondria by liposome-bound porphyrins.

We have compared the photodynamic activities of hematoporphyrin (HP) and protoporphyrin (PP) on isolated rat liver mitochondria by measuring the decline of the respiratory control ratio (RCR) after irradiation at 365 nm. Before addition to the respiratory medium, the dyes were dissolved in phosphate-buffered saline (PBS) or incorporated into unilamellar liposomes of dipalmitoyl-phosphatidylcholine (DPPC), sometimes enriched with cholesterol (Chol) or cardiolipin (Card), which are naturally present in mitochondrial membranes. Chol and especially Card strongly increase the porphyrin uptake by mitochondria. In all experimental conditions, PP is taken up by mitochondria to a higher extent than HP. Nevertheless, under conditions giving the same amount of mitochondria-bound dye, HP is a more efficient photosensitizer than PP. As the efficiency of singlet oxygen production has been shown to be equivalent for the two porphyrins in monomeric state, the resulting photobiological effects are explained in terms of different localization of HP and PP in the mitochondrial membranes. In particular, HP preferentially localizes in the protein-rich polar domains of the inner mitochondrial membrane, whereas PP dissolves in the lipid regions of the membranes.

Comparative pharmacokinetic and photodynamic studies with zinc(II) phthalocyanine in hamsters bearing an induced or transplanted rhabdomyosarcoma.

Comparative pharmacokinetic studies in hamsters bearing an induced or first-generation transplanted rhabdomyosarcoma that were injected with liposome-incorporated zinc(II) phthalocyanine (ZnPc) show a higher drug concentration in the induced tumour. The selectivity of tumour targeting is underlined by the fact that, 24 h after injection, larger amounts of ZnPc are found in the tumour than in the liver. Photodynamic therapy investigations were carried out using 673 nm light from an argon-dye laser. On the basis of different assessment criteria (changes in mean tumour diameter with time, tumour mass regression, survival time of the treated groups of animals, and histological determination of the necrotic tissue) the photosensitizing effect of ZnPc appears to be comparable for both kinds of tumour in spite of the higher uptake of photosensitizer by the induced tumour.