Photodynamic inactivation of Escherichia coli immobilized on agar surfaces by a tricationic porphyrin

The photodynamic activity of 5,10,15-tris[4-(3-N,N,N-trimethylammoniumpropoxy)phenyl]-20-(4-trifluoromethylphenyl)porphyrin iodide (A3B3+) has been studied in vitro on a typical Gram-negative bacterium Escherichia coli immobilized on agar surfaces. The results obtained for the tricationic A3B3+ porphyrin were compared with those of 5,10,15,20-tetra(4-N,N,N-trimethylammoniumphenyl)porphyrin p-tosylate (TTAP4+), which is a standard active sensitizer established to eradicate E. coli in cellular suspension. The photobleaching of these porphyrins in solution was evaluated by decay in absorbance and in fluorescence. In both cases, a higher photostability was found for A3B3+ than for TTAP4+. Photodynamic inactivation capacities of these sensitizers were analyzed in E. coli cells immobilized on agar surfaces. Small colonies were treated with different amount of sensitizer (0-14 nmol) and irradiated with visible light for 3h. The light source used was either a projector or midday sun. The A3B3+ porphyrin produced a growth delay of E. coli colonies on agar surfaces. Similar result was obtained irradiating only one isolated colony through an optical fiber. Under these conditions, A3B3+ porphyrin shows a high activity to inactivate localized bacterial cells. The higher photodynamic activity of A3B3+ was confirmed by mechanical spreading of the colonies before treatment. This procedure produces complete inactivation of E. coli cells on the agar surface. Therefore, tricationic A3B3+ porphyrin is an interesting sensitizer with potential applications in photodynamic inactivation of bacteria growing as localized foci of infection.     

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.     

Photoinactivation of Escherichia coli using porphyrin derivatives with different number of cationic charges

The photodynamic effect of meso-substituted cationic porphyrins, 5-[4-(trimethylammonium)phenyl]-10,15,20-tris(2,4,6-trimethoxyphenyl)porphyrin iodide 1, 5,10-di(4-methylphenyl)-15,20-di(4-trimethylammoniumphenyl)porphyrin iodide 2 and 5-(4-trifluorophenyl)-10,15,20-tris(4-trimethylammoniumphenyl)porphyrin iodide 3, have been investigated in both homogeneous medium bearing photooxidizable substrates and in vitro on a typical gram-negative bacterium Escherichia coli. Absorption and fluorescence spectroscopic studies were compared in N,N-dimethylformamide. Fluorescence quantum yields (varphiF) of 0.10, 0.06 and 0.08 were calculated for porphyrins 1, 2 and 3, respectively. The singlet molecular oxygen, O2(1Deltag), production was evaluated using 9,10-dimethylanthracene yielding values of 0.66, 0.36 and 0.42 for porphyrins 1, 2 and 3, respectively. Guanosine 5'-monophosphate was used as biological substrate model. Similar decomposition of guanosine 5'-monophosphate was obtained using these cationic porphyrins as sensitizer. In biological medium, photosensitized inactivation of E. coli was analyzed using cells without and with one washing step. E. coli cultures were treated with sensitizer at 37 degrees C for 30 min in dark. In both procedures, a higher photoinactivation of cells (>99.999%) was found for cells treated with 10 microM of tricationic porphyrin 3 and irradiated for 5 min with visible light. Porphyrins 1 and 2 only show an important photodamage when the cells are irradiated without washing step. These results indicated that the tetracationic porphyrin 3 could be a promising sensitizer with potential applications in the photoinactivation of bacterial cells by photodynamic therapy.     

Photophysical characterization and photodynamic activity of metallo 5-(4-(trimethylammonium)phenyl)-10,15,20-tris(2,4,6-trimethoxyphenyl)porphyrin in homogeneous and biomimetic media

The photophysical properties and photodynamic effect of Zn(ii), Pd(ii), Cu(ii) and free-base 5-(4-(trimethylammonium)phenyl)-10,15,20-tris(2,4,6-trimethoxy phenyl)porphyrin (H2P) iodide have been studied in N,N-dimethylformamide (DMF) and in different biomimetic systems. The absorption, fluorescence, triplet state and singlet molecular oxygen production of the metal complexes were all referred to H2P. The photodynamic activity was first analyzed using 9,10-dimethylanthracene and guanosine 5'-monophosphate in N,N-dimethylformamide. The photooxidation processes were also investigated in benzene/benzyl-n-hexadecyldimethyl ammonium chloride/water reverse micelles. Photosensitization efficiency of these porphyrins was H2P approximately ZnP > PdP in homogeneous solution and ZnP > H2P > PdP in micelles, whereas no photooxidation effect was detected using the Cu(ii) complex. Human erythrocytes were used as a biological membrane model. The photohemolytic activity depended on irradiation time, sensitizer and concentration of the agent. When cells were treated with 1 microM sensitizer, the hemolytic activity was H2P > ZnP > CuP. However, it was H2P > ZnP approximately CuP using 5 microM of the respective porphyrin. Although CuP could undergo a type I photoreaction, in all cases the photohemolytic effect considerably diminishes in anoxic conditions, indicating that an oxygen atmosphere is required for the mechanism of cellular membrane damage. The behavior of these amphiphilic metallo porphyrins provides information on the photodynamic activity of these agents in biomimetic microenvironments.     

Mechanistic aspects of Escherichia coli photodynamic inactivation by cationic tetra-meso(N-methylpyridyl)porphine.

The mechanistic aspects of Escherichia coli photodynamic inactivation (PDI) have been studied in bacteria expressing the reporter protein GFP, following transfection with wild type pGFP plasmid and treatment with the hydrophilic cationic sensitizer tetra-meso(N-methyl-4-pyridyl)porphine tetratosylate (TMPyP). Cell survival and morphology during PDI were correlated with plasmid-GFP degradation in comparison to DNA and RNA strand-breaks, while photobleaching of the GFP chromophore was used to monitor protein photodamage. Singlet oxygen generated upon TMPyP photoactivation interacted with target nucleic acid polymers in a drug-and light-dose dependent manner. The hierarchy and cascade of the photodamage was in the order: genomic-DNA > total RNA > plasmid-DNA, as revealed by specific extraction and agarose electrophoresis. The notable resistance of the plasmid DNA in comparison to genomic DNA has implications for PDI of antibiotic-resistant bacteria. Re-growth of the treated cells in fresh medium showed structural features of an SOS response. Under these conditions, DNA repair machinery was initiated by typical alignment of DNA-protein co-aggregates accompanied by lateral assembly of ribosomes, apart from damaged DNA-arrays, as depicted by electron microscopy. GFP-TMPyP interactions were demonstrated by double green and red fluorescence on electrophoresis plates analyzed by spectral imaging. Photobleaching measurements revealed specific GFP photodamage directly related to PDI of the E. coli. The kinetics of both the GFP photobleaching and the K(+) efflux, representing photodamage to cytosolic proteins and membrane damage, respectively, were found to be similar. The survival curves were correlated to chromosomal degradation and ultrastructural damage. We conclude that TMPyP-dependent PDI of E. coli is primarily dependent on genomic DNA photodamage rather than on protein or membrane malfunctions.     

Efficient photodynamic inactivation of Leishmania parasites mediated by lipophilic water-soluble Zn(II) porphyrin ZnTnHex-2-PyP4+

BackgroundPhotodynamic inactivation (PDI) is emerging as a promising alternative for cutaneous leishmaniasis (CL). The chemotherapy currently used presents adverse effects and cases of drug resistance have been reported. ZnTnHex-2-PyP⁴⁺ is a porphyrin with a high potential as a photosensitizer (PS) for PDI, due to its photophysical properties, structural stability, and cationic/amphiphilic character that can enhance interaction with cells. This study aimed to investigate the photodynamic effects mediated by ZnTnHex-2-PyP⁴⁺ on Leishmania parasites.MethodsZnTnHex-2-PyP⁴⁺ stability was evaluated using accelerated solvolysis conditions. The photodynamic action on promastigotes was assessed by (i) viability assays, (ii) mitochondrial membrane potential evaluation, and (iii) morphological analysis. The PS-promastigote interaction was studied. PDI on amastigotes and the cytotoxicity on macrophages were also analyzed.ResultsZnTnHex-2-PyP⁴⁺, under submicromolar concentration, led to immediate inactivation of more than 95% of promastigotes. PDI promoted intense mitochondrial depolarization, loss of the fusiform shape, and plasma membrane wrinkling in promastigotes. Fluorescence microscopy revealed a punctate PS labeling in the parasite cytoplasm. PDI also led to reductions of ca. 64% in the number of amastigotes/macrophage and 70% in the infection index after a single treatment session. No noteworthy toxicity was observed on mammalian cells.ConclusionsZnTnHex-2-PyP⁴⁺ is stable against demetallation and more efficient as PS than the ethyl analogue ZnTE-2-PyP⁴⁺, indicating readiness for evaluation in in vivo studies as an alternative approach to CL.General significanceThis report highlighted promising photodynamic effects mediated by ZnTnHex-2-PyP⁴⁺ on Leishmania parasites, opening up perspectives for applications in CL pre-clinical assays and PDI of other microorganisms.     

Porphyrin derivatives as photosensitizers for the inactivation of Bacillus cereus endospores

Aims:  In this study, we propose (i) to study the photodynamic inactivation (PDI) efficiency of neutral and cationic porphyrin derivatives, (ii) to characterize the kinetics of the inactivation process using Bacillus cereus as a model endospore-producing bacterium and (iii) to conclude on the applicability of porphyrin derivatives in the inactivation of bacterial endospores.
Methods and Results:  The study of PDI of Bacillus cereus endospores, taken as model-endospores, using porphyrin derivatives differing in the number of positive charges and in the meso-substituent groups, showed that neutral, monocationic and dicationic porphyrins are quite ineffective, in contrast with the tri- and tetra-cationic molecules. The most effective porphyrin is a tricationic porphyrin with a meso-pentafluorophenyl group. With this photosensitizer (PS), at 0·5  μ mol l⁻¹, a reduction of 3·5 log units occurs after only 4 min of irradiation. None of the porphyrin derivatives showed toxicity in the absence of light.
Conclusions:  Some porphyrin derivatives are efficient PSs for the inactivation of bacterial endospores and should be considered in further studies. Small modifications in the substituent groups, in addition to charge, significantly improve the effectiveness of the molecule as a PS for endospore inactivation.
Significance and Impact of the Study:  Tetrapyrrolic macrocycles should be regarded as worthy to explore for the PDI of spore-producing gram-positive bacteria. The development of molecules, more selective and effective, emerges as a new objective.     

Synthesis,characterization, and photodynamic therapy activity of 5,10,15,20-Tetrakis(carboxyl)porphyrin

Water-soluble porphyrins are considered promising drug candidates for photodynamic therapy (PDT). This study investigated the PDT activity of a new water-soluble, anionic porphyrin (1-Zn), which possesses four negative charges. The photodynamic anticancer activity of 1-Zn was investigated by the MTT assay, with mTHPC as a positive control. The cellular distribution was determined by fluorescence microscopy. Holographic and phase contrast images were recorded after 1-Zn treatment with a HoloMonitor™ M3 instrument. The inhibition of A549 cell growth achieved by inducing apoptosis was investigated by flow cytometry and fluorescence microscopy. DNA damage was investigated by the comet assay. The expression of apoptosis-related proteins was also measured by western blot assays. 1-Zn had better phototoxicity against A549 cells than HeLa and HepG2 cancer cells. Interestingly, 1-Zn was clearly located almost entirely in the cell cytoplasmic region/organelles. The late apoptotic population was less than 1.0% at baseline in the untreated and only light-treated cells and increased to 40.5% after 1-Zn treatment and irradiation (P < 0.05). 1-Zn triggered significant ROS generation after irradiation, causing ΔΨm disruption (P < 0.01) and DNA damage. 1-Zn induced A549 cell apoptosis via the mitochondrial apoptosis pathway. In addition, 1-Zn bound in the groove of DNA via an outside binding mode by pi-pi stacking and hydrogen bonding. 1-Zn exhibits good photonuclease activity and might serve as a potential photosensitizer (PS) for lung cancer cells.     

Study on synthesis and biological activity of a galactosylated piperazinyl porphyrin

In order to obtain an carcinoma-selective drug, the synthesis and characterization of 5,10,15,20-tetra[4-(4′-galactosylpiperazinyl)phenyl]porphyrin (TGPP) is reported. The biological activity on cancer cells and the pharmacokinetics are also reported as preliminary results showing a very high liver to skin ratio and short retention time in tissues, and thus promising activity in photodynamic therapy.     

Efficient photoinactivation of methicillin-resistant Staphylococcus aureus by a novel porphyrin incorporated into a poly-cationic liposome

Antimicrobial photodynamic therapy is emerging as a promising therapeutic modality for bacterial infections. Our studies aim at identifying strategies for optimizing the antibacterial activity of porphyrin-type photosensitisers. The photoinactivation properties of a novel, positively charged meso-substituted porphyrin, namely 5-[4-(1-dodecanoylpyridinium)]-10,15,20-triphenyl-porphyrin were tested against a typically antibiotic-resistant pathogen, such as methicillin-resistant Staphylococcus aureus. This porphyrin is characterized by an unusually large quantum yield (0.95) for the generation of the hyper-reactive oxygen species, singlet oxygen. In spite of this, it exhibits a relatively low photosensitising activity against bacteria when dissolved in a homogeneous aqueous solution or incorporated into neutral lipid vesicles. On the contrary, a dramatic potentiation of the photocydal effect takes place when polycationic agents such as liposomes of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride are used as carriers. The cationic carrier primarily acts as a disorganizing agent for the native three-dimensional architecture of the bacterial wall, thereby enhancing its permeability to the photosensitiser. Consequently, the drug can deeply penetrate into the plasma membrane, and rapidly impair selected enzymic activities leading to cell death. Thus, the combination of positively charged drugs and cationic delivery systems appears to represent an innovative modality for achieving an efficient antimicrobial activity and opens new avenues for the development of this phototherapeutic application.     

Interactions of DNA with a new electron-deficient tentacle porphyrin: meso-Tetrakis[2,3,5,6-tetrafluoro-4-(2-trimethylammoniumethylamine)phenyl]porphyrin

A new electron-deficient tentacle porphyrin meso-tetrakis[2,3,5,6-tetrafluoro-4-(2-trimethylammoniumethylamine)phenyl]porphyrin (TθF₄TAP) has been synthesized. The binding interactions of TθF₄TAP with DNA polymers were studied for comparison to those of an electron-deficient tentacle porphyrin and an electron-rich tentacle porphyrin; these previously studied porphyrins bind to DNA primarily by intercalative and outside-binding modes, respectively. The three tentacle porphyrins have similar size and shape. The basicity of TθF₄TAP indicated that it has electronic characteristics similar to those of the intercalating electron-deficient tentacle porphyrin. However, TθF₄TAP binds to calf thymus DNA, [poly(dA-dT)]₂, and [poly(dG-dC)]₂ in a self-stacking, outside-binding manner under all conditions. Evidence for this binding mode included a significant hypochromicity of the Soret band, a conservative induced CD spectrum, and the absence of an increase in DNA solution viscosity. As found previously for the electron-rich porphyrin, the results suggest that combinations of closely related self-stacked forms coexist. The mix of forms depended on the DNA and the solution conditions. There are probably differences in the detailed features of the self-stacking adducts for the two types of tentacle porphyrins, especially at high R (ratio of porphyrin to DNA). At low R values, the induced CD signal of TθF₄TAP/CT DNA resembled that of TθF₄TAP/[poly(dA-dT)]₂, suggesting that TθF₄TAP binds preferentially at AT regions. Competitive binding experiments gave evidence that TθF₄TAP binds preferentially to [poly(dA-dT)]₂ over [poly(dG-dC)]₂. Thus, despite the long, positively charged, flexible substituents on the porphyrin, the binding of TθF₄TAP is significantly affected by base-pair composition. Similar characteristics were found previously for the electron-rich tentacle porphyrin. Thus, significant changes in electron richness have relatively minor effects on this outside binding selectivity for AT regions. TθF₄TAP is the first porphyrin with electron deficiency and shape similar to intercalating porphyrins that does not appear to intercalate. All porphyrins reported to intercalate have had pyridinium substituents. Thus, the electronic distribution in the porphyrin ring, not just the overall electron richness, may play a role in facilitating intercalation. © 1997 John Wiley & Sons, Inc. Biopoly 42: 203–217, 1997     

Photobiological activity of exogenous and endogenous porphyrin derivatives in Escherichia coli and Enterococcus hirae cells

Photodynamic treatment, the combined application of a photosensitiser and visible light, represents a new and promising approach for the inactivation of microorganisms. The photosensitising potentials of exogenous zinc-phthalocyanine-tetrasulphonate (ZnPsTS), tetraphenylporphyrins (TPPs) and endogenous porphyrin derivatives were tested and compared on Gram-negative and Gram-positive bacteria, Escherichia coli B. and Enterococcus hirae, respectively. The synthesis of endogenous porphyrins was induced by 5-aminolevulinic acid (δ-ALA). The porphyrin- or δ-ALA-treated cells were irradiated with white light. The photosensitising efficiency of endogenous derivatives on both types of bacteria is ZnPcTS < TP(4-OGluOH)₃P < TP(4-OGluOH)₄P. However, neither exogenous derivatives exhibit appreciable photosensitising activity for disinfection application. ALA-induced photodynamic treatment showed good potential for the inactivation of Escherichia coli cells, but not towards Enterococcus hirae cells. The failure of photosensitisation of the Enterococcus hirae strain selected indicates that apart from the Gram-positive character, other structural elements of the membrane can influence the result of photodynamic treatments. Received: 13 October 1999 / Accepted: 1 January 2001     

Transformation and mutagenic potential of porphyrin photodynamic therapy in mammalian cells

The transformation and mutagenic potential of porphyrin photodynamic therapy has been examined in mammalian cells. The mutagenic frequency in Chinese hamster cells at the Na+/K+ ATPase locus was measured by resistance to ouabain following treatment with either photodynamic therapy (PDT) or UV irradiation. The C3H 10T 1/2 mouse embryo cell system was used to document the transformation frequency following PDT, UV irradiation, gamma irradiation or exposure to 3-methylcholanthrene (MCA). Treatments with UV irradiation were effective in producing mutants resistant to ouabain, and treatments with UV irradiation, gamma irradiation and MCA generated transformants at frequencies comparable to those which are reported in the literature. However, PDT treatment conditions (which produced a full range of cytotoxicity) did not induce any mutagenic or transformation activity above background levels.     

Photodynamic damages induced by a monocationic porphyrin derivative in a human carcinoma cell line

The photokilling activity of 5-(4-trimethylammoniumphenyl)-10,15,20-tris(2,4,6-trimethoxyphenyl)porphyrin (CP) was evaluated on a Hep-2 human larynx-carcinoma cell line. Cell treatment was carried out with 5 μM CP incorporated into liposomal vesicles. Under violet-blue exciting light, the red fluorescence of CP was mainly detected as a filamentous pattern characteristic of mitochondrial localization. Similar pattern was also observed using rhodamine 123 in Hep-2 cells. No dark cytotoxicity was observed using 5 μM CP concentration and long incubation time (24 h). Using Hoechst-33258 and caspase-3 immunostaining methods, cell cultures treated for 24 h with CP and exposed to light for 7.5 min (27 J/cm²) showed a great amount of apoptotic cells (40%). In contrast, necrotic cells (58%) were observed using the same drug concentration but irradiated for 15 min (54 J/cm²). The results show that CP can induce different mechanisms of cell death depending on irradiation doses in the photodynamic treatments, which makes this agent an interesting sensitizer with potential application in photodynamic tumor therapy.     

Efficient photosensitization of malignant human cells in vitro by liposome-bound porphyrins

Comparative kinetics of porphyrin uptake and release by HeLa cells, incubated with equivalent concentrations of either hematoporphyrin (Hp) in aqueous solution or Hp and its dimethylester (HpDME) bound to unilamellar liposomes, show that liposomal porphyrins are bound at a higher rate and in considerably larger amounts. Moreover, the release of cell-bound porphyrins into the medium is remarkably reduced and slowered after cell loading with liposome-bound porphyrins. The presence of 1% bovine or human serum albumin (but not serum globulins) in the medium has no effect on uptake and release of liposome-bound porphyrins by HeLa cells, whereas it remarkably decreases the uptake of aqueous Hp. Parallel studies of cell photodamages under known concentrations of cell-bound porphyrin unequivocally demonstrate that the photodynamic effect is strictly related to the porphyrin load. As a consequence a dramatic increase of cell-photosensitizing efficiency is obtained by binding Hp (and even more HpDME) with liposomes. Electron microscopy investigations on cell damages caused by loading with liposome-bound porphyrins and subsequent illumination show that the plasmatic membrane is one important cell site of porphyrin interaction and photodynamic effect.     

Photodynamic Effects Induced bymeso-Tetrakis[4-(carboxymethyleneoxy)phenyl]porphyrin Using Rat Hepatic Microsomes as Model Membranes

Porphyrins, in combination with light, offer an alternate approach to the treatment of cancer, in the form of photodynamic therapy (PDT). With a view to locate new porphyrins for use in PDT, we evaluated the ability of a novel water-soluble porphyrin,meso-tetrakis[4-(carboxymethyleneoxy)phenyl]porphyrin (T4CPP) to induce photodamage in membranes, using rat hepatic microsomes as a model system. Hepatic microsomes treated with T4CPP and exposed to visible light showed significant lipid peroxidation, as assessed by the formation of conjugated dienes, lipid hydroperoxides, and thiobarbituric acid-reactive substances. The peroxidation induced was both time- and concentration-dependent. T4CPP plus light also resulted in the destruction of the microsomal enzymes adenosine triphosphatase and glucose-6-phosphatase. Analysis of the products of peroxidation and selective inhibition by specific inhibitors showed that the oxidative damage induced was mainly due to singlet oxygen and partly due to hydroxyl radical. The porphyrin T4CPP was efficiently labeled with^99mTc. When this^99mTc-labeled porphyrin was injected into a mammary-tumor-bearing rat, it accumulated in the tumor. Our studies suggest that T4CPP, due to its potential to localize in tumors and to induce membrane damage as exemplified by alteration in rat liver microsomes, may have possible applications in this new modality of cancer treatment.     

Oxidative damage induced by a novel porphyrin on rat brain mitochondria and its possible implications in therapy

Summary

Free radical-induced oxidative damage is involved in several pathological disorders. On the other hand, selective induction of peroxidation in diseased tissue is a promising approach to the treatment of cancer by photodynamic therapy. In this study we have used rat brain mitochondria as a model to evaluate the ability of a new water soluble porphyrin, 5,10,15,20-tetrakis[4-(carboxymethyleneoxy)phenyl]porphyrin (T4CPP), to induce peroxidative damage during photosensitization. Peroxidation in mitochondria, one of the crucial targets of the photodynamic effect, was assessed from the formation of thiobarbituric acid reactive substances and lipid hydroperoxides. The effect on mitochondrial function was estimated from the loss of a mitochondrial marker enzyme, succinate dehydrogenase (SDH). The photodamage was observed to be time- and concentration-dependent of T4CPP. Inhibition studies suggested involvement of singlet oxygen (¹O₂) and, to a lesser extent, of hydroxyl (OH), peroxyl (ROO^?) and superoxide radicals (O₂^?) in the photodamage. The addition of γ-linolenic acid (a promoter of lipid peroxidation) to the system led to an enhancement of the T4CPP-induced peroxidative damage. Thus, our study indicated that the combination of γ-linolenic acid and T4CPP could enhance the photodynamic effect and has potential applications in photodynamic therapy.     

Interactions of meso-tetra-(4-N-oxyethylpyridyl) porphyrin, its 3-N analog and their metallocomplexes with duplex DNA

Interactions of meso-tetra-(4-N-oxyethylpyridyl) porphyrin (TOEPyP(4)), its 3-N analog (TOEPyP(3)) and their Co, Cu, Ni, Zn metallocomplexes with duplex DNA have been investigated by uv/visible absorbance and circular dichrosim spectroscopies. Results reveal the interactions of these complexes with duplex DNA are of two types. (1) External binding of duplex DNA by metalloporphyrins containing Zn and Co, and (2) Binding of duplex DNA both externally and internally (by intercalation) by porphyrins not containing metals, and metalloporphyrins containing Cu and Ni. Results indicate that (4N-oxyethylpyridyl) porphyrins intercalate more preferably in the structure of duplex DNA and have weaker external binding than 3N-porphyrins.     

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.     

Synthesis, properties, and photodynamic inactivation of Escherichia coli using a cationic and a noncharged Zn(II) pyridyloxyphthalocyanine derivatives

The photodynamic effect of a cationic Zn(II) N-methylpyridyloxyphthalocyanine (ZnPc 2) and a noncharged Zn(II) pyridyloxyphthalocyanine (ZnPc 1) has been compared in both homogeneous media bearing photooxidizable substrates and in vitro using a typical Gram-negative bacterium Escherichia coli. Absorption and fluorescence spectroscopic studies were analyzed in different media. Fluorescence quantum yields (varphiF) of 0.23 for ZnPc 1 and 0.22 for ZnPc 2 were calculated in N,N-dimethylformamide (DMF). The singlet molecular oxygen, O2(1Deltag), production was evaluated using 9,10-dimethylanthracene (DMA) in DMF yielding values of PhiDelta=0.56 for ZnPc 1 and 0.59 for ZnPc 2. A faster decomposition of L-tryptophan (Trp), which was used as biological substrate model, was obtained using ZnPc 2 as a sensitizer with respect to ZnPc 1. In biological medium, the E. coli cultures were treated with 10 microM of sensitizer for different times at 37 degrees C in the dark. Both ZnPcs 1 and 2 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 ZnPc 2 after one washing step is approximately 3 times higher than 1, reaching a value of approximately 3 nmol/10(6) cells. After irradiation with visible light, a higher photoinactivation of cells was found for ZnPc 2. Thus, a approximately 4.5 log (99.997%) decrease of cell survival was obtained after 30 min of irradiation. On the other hand, a very low photodamage was found for cells treated with ZnPc 1 (approximately 0.5 log). Also, these results were established by stopping of growth curves for E. coli. In the structure of ZnPc 2, the cationic centers are isolated from the phthalocyanine ring by an ether bridge, which also provides a higher mobility of the charges facilitating the interaction with the outer membrane of the Gram-negative bacteria. These studies show that cationic ZnPc 2 is an efficient phototherapeutic agent with potential applications in photodynamic inactivation of bacteria.