Edgardo N. Durantini

Light-driven production of ATP catalysed by F0F1-ATP synthase in an artificial photosynthetic membrane

Energy-transducing membranes of living organisms couple spontaneous to non-spontaneous processes through the intermediacy of protonmotive force (p.m.f.) — an imbalance in electrochemical potential of protons across the membrane. In most organisms, p.m.f. is generated by redox reactions that are either photochemically driven, such as those in photosynthetic reaction centres, or intrinsically spontaneous, such as those of oxidative phosphorylation in mitochondria. Transmembrane proteins (such as the cytochromes and complexes I, III and IV in the electron-transport chain in the inner mitochondrial membrane) couple the redox reactions to proton translocation, thereby conserving a fraction of the redox chemical potential as p.m.f. Many transducer proteins couple p.m.f. to the performance of biochemical work, such as biochemical synthesis and mechanical and transport processes. Recently, an artificial photosynthetic membrane was reported in which a photocyclic process was used to transport protons across a liposomal membrane, resulting in acidification of the liposomes internal volume. If significant p.m.f. is generated in this system, then incorporating an appropriate transducer into the liposomal bilayer should make it possible to drive a non-spontaneous chemical process. Here we report the incorporation of FOF1-ATP synthase into liposomes containing the components of the proton-pumping photocycle. Irradiation of this artificial membrane with visible light results in the uncoupler- and inhibitor-sensitive synthesis of adenosine triphosphate (ATP) against an ATP chemical potential of ∼12 kcal mol−1, with a quantum yield of more than 7%. This system mimics the process by which photosynthetic bacteria convert light energy into ATP chemical potential.

Photodynamic inactivation of Escherichia coli by novel meso-substituted porphyrins by 4-(3-N,N,N-trimethylammoniumpropoxy)phenyl and 4-(trifluoromethyl)phenyl groups

The photodynamic effect of novel cationic porphyrins, with different pattern of meso-substitution by 4-(3-N,N,N-trimethylammoniumpropoxy)phenyl (A) and 4-(trifluoromethyl)phenyl (B) groups, have been studied in both solution bearing photooxidizable substrates and in vitro on a typical Gram-negative bacterium Escherichia coli. In these sensitizers, the cationic groups are separated from the macrocycle ring by a propoxy spacer. Thus, the charges have a high mobility and a minimal influence on photophysical properties of the porphyrin. These compounds produce singlet molecular oxygen, O2(1Delta(g)), with quantum yields of approximately 0.41-0.53 in N,N-dimethylformamide. In methanol, the l-tryptophan photodecomposition increases with the number of cationic charges in the sensitizer. In vitro investigations show that cationic porphyrins are rapidly bound to E. coli cells in approximately 5 min. A higher binding was found for A3B3+ porphyrin, which is tightly bound to cells still after three washing steps. Photosensitized inactivation of E. coli cellular suspensions follows the order: A3B3+ > A44+>> ABAB2+ > AB3+. Under these conditions, a negligible effect was found for 5,10,15,20-tetra(4-sulfonatophenyl)porphyrin (TPPS4(4-)) that characterizes an anionic sensitizer. Also, the results obtained for these new cationic porphyrins were compared with those of 5,10,15,20-tetra(4-N,N,N-trimethylammonium phenyl)porphyrin (TTAP4+), which is a standard active sensitizer established to eradicate E. coli. The photodynamic activity of TTAP4+ is quite similar to that produced by A4(4+). Studies in an anoxic condition indicate that oxygen is necessary for the mechanism of action of photodynamic inactivation of bacteria. The higher photodynamic activity of A3B3+ was confirmed by growth delay experiments. Photodynamic inactivation capacities of these sensitizers were also evaluated in E. coli cells immobilized on agar surfaces. Under these conditions, A3B3+ porphyrin retains a high activity to inactivate localized bacterial cells. Therefore, tricationic porphyrin A3B3+ is an interesting sensitizer with potential applications in photodynamic inactivation of bacteria in liquid suspensions or on surfaces.

Photodynamic inactivation of Candida albicans sensitized by tri-and tetra-cationic porphyrin derivatives

The photodynamic action of 5-(4-trifluorophenyl)-10,15,20-tris(4-trimethylammoniumphenyl)porphyrin iodide (TFAP(3+)) and 5,10,15,20-tetra(4-N,N,N-trimethylammonium phenyl)porphyrin p-tosylate (TMAP(4+)) has been studied in vitro on Candida albicans. The results of these cationic porphyrins were compared with those of 5,10,15,20-tetra(4-sulphonatophenyl)porphyrin (TPPS(4-)), which characterizes an anionic sensitizer. In vitro investigations show that these cationic porphyrins are rapidly bound to C. albicans cells, reaching a value of approximately 1.4 nmol/10(6) cells, when the cellular suspensions were incubated with 5 microM sensitizer for 30 min. In contrast, TPPS(4-) is poorly uptaken by yeast cells. The fluorescence spectra of these sensitizers into the cells confirm this behaviour. The amount of porphyrin binds to cells is dependent on both sensitizer concentrations (1-5 microM) and cells densities (10(6)-10(8) cells/mL). Photosensitized inactivation of C. albicans cellular suspensions increases with sensitizer concentration, causing a approximately 5 log decrease of cell survival, when the cultures are treated with 5 microM of cationic porphyrin and irradiated for 30 min. However, the photocytotoxicity decreases with an increase in the cell density, according to its low binding to cells. Under these conditions, the photodynamic activity of TFAP(3+) is quite similar to that produced by TMAP(4+), whereas no important inactivation effect was found for TPPS(4)(-). The high photodynamic activity of cationic porphyrins was confirmed by growth delay experiments. Thus, C. albicans cell growth was not detected in the presence of 5 microM TFAP(3+). Photodynamic inactivation capacities of these sensitizers were also evaluated on C. albicans cells growing in colonies on agar surfaces. Cationic porphyrins produce a growth delay of C. albicans colonies and viability of cells was not observed after 3 h irradiation, indicating a complete inactivation of yeast cells. Therefore, these results indicate that these cationic porphyrins are interesting sensitizers for photodynamic inactivation of yeasts in liquid suspensions or in localized foci of infection.

Photodynamic studies and photoinactivation of Escherichia coli using meso-substituted cationic porphyrin derivatives with asymmetric charge distribution.

Abstract The photodynamic activities of novel asymmetrically meso-substituted cationic porphyrins, 5,10-di(4-methylphenyl)-15,20-di(4-trimethylammoniumphenyl)porphyrin iodide 1 and 5-(4-trifluorophenyl)-10,15,20-tris(4-trimethylammoniumphenyl)porphyrin iodide 2 and its metal complex with Pd(II) 3, have been investigated in both homogeneous medium bearing photooxidizable substrates and in vitro on a typical gram-negative bacterium Escherichia coli. The amphiphilic character of porphyrin 2 was increased by the presence of a high-lipophilic trifluoromethyl group and its photophysical properties changed by forming a complex with Pd(II). Absorption and fluorescence spectroscopic studies were compared in different media. Fluorescence quantum yields (ϕF) of 0.16 for 1 in tetrahydrofuran and 0.08 for 2 in N, N-dimethylformamide (DMF) were calculated, whereas no significant emission was detected for Pd(II) porphyrin 3. The singlet molecular oxygen, O2(1Δg), production was evaluated using 9,10-dimethylanthracene in DMF yielding relative values of 1, 0.55 and 0.47 for porphyrins 3, 2 and 1, respectively. A faster decomposition of l-tryptophan was obtained using Pd(II) porphyrin 3 as sensitizer with respect to the free-base porphyrins 1 and 2. In biological medium, the behavior of cationic porphyrins 1–3 were compared with that of 5-(4-carboxyphenyl)-10,15,20-tris(4-methylphenyl)porphyrin 4, which was used as a noncationic sensitizer. These porphyrins are rapidly bound to E. coli cells in 5 min and the amount of cell-bound sensitizer is not appreciably changed incubating the cultures for longer times. The recovered porphyrin 2 after one washing step reaches a value of ∼2.9 nmol/106 cells and this amount remains high even after three washes, indicating that this sensitizer is tightly bound to cells. Photosensitized inactivation of E. coli was analyzed using cells without and with one washing step. In both cases, a higher photoinactivation of cells was found for tricationic porphyrin 2 and 3, causing a ∼5.5 log (99.999%) decrease of cell survival, when treated with 10 μM of sensitizer. Under these conditions, a lower effect was found for porphyrin 1 (∼4 log) whereas sensitizer 4 did not produce appreciable photodamage. The results were also confirmed by growth delay experiments. These studies show that the amphiphilic tricationic porphyrin 2 and 3 bearing a trifluoromethyl group can be a promising model for phototherapeutic agents with potential applications in inactivation of bacteria by photodynamic therapy.

Porphyrin-fullerene C60 dyads with high ability to form photoinduced charge-separated state as novel sensitizers for photodynamic therapy.

Abstract The photodynamic activities of a porphyrin-C60 dyad (P-C60) and its metal complex with Zn(II) (ZnP-C60) were compared with 5-(4-acetamidophenyl)-10,15,20-tris(4-methoxyphenyl)porphyrin (P), both in homogeneous medium-bearing photooxidizable substrates and in vitro on the Hep-2-human-larynx–carcinoma cell line. This study represents the first evaluation of dyads, with a high capacity to form a photoinduced charge-separated state, to act as agents to inactivate cells by photodynamic therapy (PDT). Absorption and fluorescence spectroscopic studies were performed in toluene and N,N-dimethylformamide (DMF). The emission of the porphyrin moiety in the dyads is strongly quenched by the attached fullerene C60 moiety. The singlet molecular oxygen, O2(1Δg), productions (ΦΔ) were determined using 9,10-dimethylanthracene (DMA). The values of ΦΔ were strongly dependent on the solvents polarity. Comparable ΦΔ values were found for dyads and P in toluene, while O2(1Δg) production was significantly diminished for the dyads in DMF. In more polar solvent, the stabilization of charge-transfer state takes place, decreasing the efficiency of porphyrin triplet-state formation. Also, both dyads photosensitize the decomposition of L-tryptophan in DMF. In biological medium, no dark cytotoxicity was observed using sensitizer concentrations ≤1 μM and 24 h of incubation. The uptake of sensitizers into Hep-2 was studied using 1 μM of sensitizer and different times of incubation. Under these conditions, a value of ∼1.5 nmol/106 cells was found between 4 and 24 h of incubation. The cell survival after irradiation of the cells with visible light was dependent upon light-exposure level. A higher photocytotoxic effect was observed for P-C60, which inactivates 80% of cells after 15 min of irradiation. Moreover, both dyads keep a high photoactivity even under argon atmosphere. Thus, depending on the microenvironment where the sensitizer is localized, these compounds could produce biological photodamage through either an O2(1Δg)-mediated photoreaction process or a free-radicals mechanism under low oxygen concentration. These results show that molecular dyads, which can form a photoinduced charge-separated state, are a promising model for phototherapeutic agents, with potential applications in cell inactivation by PDT.

Photodynamic Studies of Metallo 5,10,15,20-Tetrakis(4-methoxyphenyl) porphyrin: Photochemical Characterization and Biological Consequences in a Human Carcinoma Cell Line¶

The photodynamic activities of the free‐base 5,10,15,20‐tetrakis(4‐methoxyphenyl)porphyrin (TMP) and their metal complexes with zinc(II) (ZnTMP), copper(II) (CuTMP) and cadmium(II) (CdTMP) have been compared in two systems: reverse micelle of n‐heptane/sodium bis(2‐ethylhexyl)sulfosuccinate/water bearing photooxidizable substrates and Hep‐2 human larynx carcinoma cell line. The quantum yields of singlet molecular oxygen, O2(1Δg), production (ΦΔ) of TMP, ZnTMP and CdTMP in tetrahydrofuran, were determined yielding values of 0.65, 0.73 and 0.73, respectively, while O2(1Δg) formation was not detected for CuTMP. In the reverse micellar system, the amino acid l‐tryptophan (Trp) was used as biological substrate to analyze the O2(1Δg)‐mediated photooxidation. The observed rate constants for Trp photooxidation (kobsTrp) were proportional to the sensitizer quantum yield of O2(1Δg). A value of ∼2 × 107 s−1M−1 was found for the second‐order rate constant of Trp (krTry) in this system. The response of Hep‐2 cells to cytotoxicity photoinduced by these agents in a biological medium was studied. The Hep‐2 cultures were treated with 1 μM of porphyrin for 24 h at 37°C and the cells exposed to visible light. The cell survival at different light exposure levels was dependent on ΦΔ. Under these conditions, the cytotoxic effect increases in the order: CuTMP ≪ TMP < ZnTMP ∼ CdTMP, correlating with the production of O2(1Δg). A similar behavior was observed in both the chemical and biological media indicating that the O2(1Δg) mediation appears to be mainly responsible for the cell inactivation.

Synthesis of porphyrin dyads with potential use in solar energy conversion

A convenient procedure for the synthesis of porphyrin derivative dyads is described. The dyads consist of a free base porphyrin covalently linked to a zinc porphyrin or ferrocene by an amide bond. 5-(4-Substituted phenyl)-10,15,20-tris(4-methylphenyl) porphyrins were synthesized from meso-(4-methylphenyl) dipyrromethanes 1, which was obtained with appreciable yield (83%). The reaction of dipyrromethane 1 with a mixture of two appropriate substituted benzaldehydes affords the desired meso-substituted porphyrins, which can be easily separated by flash chromatography. These porphyrins bearing either one 4-acetamidophenyl group 2 or 4-carboxymethylphenyl group 3, and three 4-methylphenyl peripheral functional groups, were prepared with notable yields (15–17%) in a two-step one-flask reaction. Basic hydrolysis of the porphyrins 2 and 3 yielded amino 4 and acid porphyrin 5, respectively. Treatment of 5 with zinc acetate afforded the corresponding metal complex Zn-acid porphyrin 6. The dyads 7 and 8 were obtained by the coupling reaction between the acid chloride derivatives of either Zn-acid porphyrin 6 or ferroceneacetic acid and amino porphyrins 4, respectively. The present strategy may be easily used for preparation of other similar dyad derivatives. These compounds could have interesting applications in electronic materials. Preliminary studies of light energy conversion by SnO2 electrodes coated with porphyrin dyads 7 and 8 were performed. The results show that dyads 7 and 8 may be suitable for solar energy conversion devices.

Mechanistic insight of the photodynamic inactivation of Escherichia coli by a tetracationic zinc(II) phthalocyanine derivative

Photodynamic inactivation (PDI) of Escherichia coli has been studied in cultures treated with zinc(II) 2,9,16,23-tetrakis[4-(N-methylpyridyloxy)]phthalocyanine (ZnPPc(+4)) to obtain insight about the mechanism of damage. This phthalocyanine is rapidly bound to cells, reaching a value of approximately 0.8 nmol/10(6) cells when the cultures were incubated with 2 microM sensitizer. After 30 min of irradiation, a 4 log decrease of E. coli survival was observed. The photocytotoxic action was investigated in plasmid and genomic DNA by electrophoretic analysis. Absorption spectroscopic studies showed that this cationic phthalocyanine interacts strongly with DNA (K(DNA)=4.7 x 10(6)M(-1)). Photocleavage of calf thymus DNA sensitized by ZnPPc(+)4 was not found even after long irradiation periods. Similar results were also observed in genomic DNA extracted from E. coli cells after PDI treatment. Modifications of plasmid DNA isolated from bacteria were only observed after long irradiation periods. However, under these conditions transmission electron microscopy of the PDI bacteria revealed an aggregation of cytoplasmic macromolecules and irregularities in cell barriers. Also, scanning electron microscopy showed a shrunken appearance in cells after PDI. Even so, release of intracellular biopolymers was not detected by absorption. On the other hand, outer and inner membranes permeabilization assays showed an increase in the permeability. Consequently, alterations in the cell membrane functionality induced by ZnPPc(+4) appear to be the major cause of E. coli inactivation upon PDI.

Near-IR sensitization of wide band gap oxide semiconductor by axially anchored Si-naphthalocyanines

Near-IR dye sensitized solar cells are very interesting due to their potential applications in panchromatic cells, semi-transparent windows and in tandem cells. In this work we show the utilization of axially anchored Si-naphthalocyanine dye in the spectral sensitization of TiO2 nanostructured photoelectrodes. We report the first successful evaluation of a naphthalocyanine in the production of sensitized photocurrent with maximum incident photon to current efficiency (IPCE) at λ ∼ 790 nm.

Photodynamic activity of cationic and non-charged Zn(II) tetrapyridinoporphyrazine derivatives: biological consequences in human erythrocytes and Escherichia coli

The photodynamic activity of a cationic Zn(II) tetramethyltetrapyridinoporphyrazinium salt (ZnPc ) was compared with that of a non-charged Zn(II) tetrapyridinoporphyrazine (ZnPc 1), both in vitro using human red blood (HRB) cells and a typical Gram-negative bacterium Escherichia coli. Absorption and fluorescence spectroscopic studies were analyzed in different media. Fluorescence quantum yields (phi(F)) of 0.35 for ZnPc 1 and 0.30 for ZnPc 2 were calculated in N,N-dimethylformamide (DMF). The singlet molecular oxygen, O(2)((1)Delta(g)), production was evaluated using 9,10-dimethylanthracene (DMA) in DMF yielding values of Phi(Delta)= 0.56 for ZnPc 1 and 0.50 for ZnPc 2. In biological medium, the photodynamic effect was first evaluated in HRB cells. Both phthalocyanines produce similar photohemolysis of HRB cells, reaching values >90% of lysis after 5 min of irradiation with visible light. The photodynamic effect is accompanied by an increase in the membrane fluidity of HRB cells. However, these studies on E. coli cells showed that the cationic ZnPc 2 produces a higher photoinactivation of Gram-negative bacteria than ZnPc 1. Also, these results were established by stopped of growth curves for E. coli. Therefore the studies show that cationic ZnPc 2 is an efficient phototherapeutic agent with potential applications in tumor cell and Gram-negative bacteria inactivation by photodynamic therapy.