Comparison of the efficiency and the specificity of DNA-bound and free cationic porphyrin in photodynamic virus inactivation

The risk of transmitting infections by blood transfusion has been substantially reduced. However, alternative methods for inactivation of pathogens in blood and its components are needed. Application of photoactivated cationic porphyrins can offer an approach to remove non-enveloped viruses from aqueous media. Here we tested the virus inactivation capability of meso-Tetrakis(4-N-methylpyridyl)porphyrin (TMPyP) and meso-Tri-(4-N-methylpyridyl)monophenylporphyrin (TMPyMPP) in the dark and upon irradiation. T7 bacteriophage, as a surrogate on non-enveloped viruses was selected as a test system. TMPyP and TMPyMPP reduce the viability of T7 phage already in the dark, which can be explained by their selective binding to nucleic acid. Both compounds proved to be efficient photosensitizers of virus inactivation. The binding of porphyrin to phage DNA was not a prerequisite of phage photosensitization, moreover, photoinactivation was more efficiently induced by free than by DNA bound porphyrin. As optical melting studies and agarose gel electrophoresis of T7 nucleoprotein revealed, photoreactions of TMPyP and TMPyMPP affect the structural integrity of DNA and also of viral proteins, despite their selective DNA binding.     

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

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 (phi(F)) 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, O(2)((1)Delta(g)), 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 approximately 2.9 nmol/10(6) 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 approximately 5.5 log (99.999%) decrease of cell survival, when treated with 10 microM of sensitizer. Under these conditions, a lower effect was found for porphyrin 1 (approximately 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.     

Ultraviolet inactivation of feline calicivirus,human enteric viruses and coliphages

Norwalk and Norwalk-like viruses (NLV) are major causes of food- and water-related disease in the United States. There is no host cell line in which the NLV can be tested for infectivity. Feline calicivirus (FCV) and NLV both belong to the family Caliciviridae. FCV can be assayed for infectivity in the Crandell Reese feline kidney cell line, so FCV serves as a surrogate for NLV. This study is the first report of UV inactivation of FCV and also of using the plaque technique, in contrast to the 50% tissue culture infectious dose end point technique, to determine the FCV infectivity titer. The infectivity titers (log10 plaque-forming units/mL) of UV-inactivated FCV, hepatitis A virus (HAV), poliovirus type 1 (PV1) and two small, round coliphages were plotted as a function of UV dose and analyzed by regression analysis and analysis of variance. These fitted straight-line curves represent exponential inactivation, so UV inactivation can be said to show "one-hit kinetics." The decimal inactivation doses of UV for FCV, HAV, PV1, MS2 and phiX174 were 47.85, 36.50, 24.10, 23.04 and 15.48 mW s/cm2, respectively. FCV appears to be the most UV resistant among the tested viruses.     

Photo-CIDNP evidence for charge-transfer interaction between synthetic water-soluble porphyrins and guanine

Abstract Photoreactions of aqueous solutions of synthetic water-soluble porphyrins were studied by the ¹H and ¹³C CIDNP technique. Strong polarizations, which were very sensitive to the presence of added acid, were observed on the cationic porphyrins (TMePyPH₂-TAPPH₂) when irradiated through continuous UV-visible light. They resulted from the reverse electron transfer between the semi-oxidized and the semi-reduced species of the derivative. When the experiments were carried out in the presence of nucleobases, guanine (and its derivatives) was the only residue that was polarized. This is thoroughly interpreted in terms of a reversible electron transfer reaction leading to guanine photooxidation by the porphyrin excited triplet state. It was shown to be drastically pH-dependent and was correlated to the redox potential of the porphyrin. It was not affected by the incident wavelength. The reaction proceeded through the intermediate formation of the correlated radical-ion pair: porphyrin radical anion-guanine radical cation. This study suggested that a Type I (free radical) reaction could be one of the primary processes in DNA photosensitization by porphyrins.     

Self-assembly of uncharged amphiphilic porphyrins and incorporation of C60 fullerenes in water

We synthesised an uncharged amphiphilic porphyrin, meso-tetrakis-(3,5-di-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-phenyl)-porphyrin, and investigated the supramolecular self-assembly of the porphyrins and the incorporation of C₆₀ molecules into the assembly in aqueous solutions. Spectroscopic and dynamic light scattering studies on the assembly of the amphiphilic porphyrin support that the amphiphilic porphyrins are likely held together through enhanced π–π interactions by pronounced hydrophobic effects in aqueous solutions. It was also found that C₆₀ molecules are efficiently incorporated into the assembly. The fluorescence emitted from the porphyrin ring of the porphyrin/C₆₀ co-assembly in aqueous solution is largely quenched, implying the presence of strong electronic interactions between C₆₀ and porphyrin molecules in the supramolecular assembly.     

水杨酸键联卟啉的合成及与牛血清白蛋白的相互作用

以溴代烷氧基苯基卟啉(1)为原料与水杨酸作用,分别得到了化合物2和3,再与醋酸钴反应,得到了钴卟啉配合物4和5.新化合物分别由¹HNMR,IR,UV-Vis,MS和元素分析确证.同时,利用荧光光谱考察了金属卟啉与牛血清白蛋白的相互作用.     

Synthesis of Metal Porphyrins Tailed with Salicylic Acid and their Interaction with Bovine Serum Albumin

A synthetic method of porphyrins tailed with salicylic substituents is described.Reaction of bromoalkoxyphenyl porphyrin 1 with salicylic acid gave porphyrins 2-5. These new compounds were confirmed by ^1H NMR, IR, UV-vis, MS and elemental analysis, and observed their interaction with bovine serum albumin (BSA) in fluorescence spectrum.     

Synthesis, singlet oxygen photogeneration and DNA photocleavage of porphyrins with nitrogen heterocycle tails

Eight novel compounds were prepared by reaction of 5-(bromo- propoxyphenyl)-10,15,20-triphenylporphyrin with oxazole thiols, 1,3,4-oxadiazole thiols and 1,3,4-thiadiazole thiols, and their structures confirmed by UV-vis, IR, 1H-NMR, MS and elemental analysis. The assessment of indirectly measured 1O(2) production rates against 5,10,15,20-tetraphenyl porphyrin (H(2)TPP) were described and the relative singlet oxygen production yields were：porphyrin 5 > porphyrins 1, 3, 4, 6-8, H(2)TPP > porphyrin 2. Porphyrin 4 and porphyrin 7 showed substantial photocleavage activities toward DNA, with over 75% cleavage observed at 40 μM. It suggested that these those porphyrins with nitrogen heterocycle tails are potential photosensitive agents.     

ENHANCEMENT OF PORPHYRIN-PHOTOSENSITIZATION OF YEAST CELLS BY ETHANOL

Abstract In porphyrin photosensitization, the localization of porphyrin in the cell and the sensitizing activity have been of recent concern. Hydrophobic porphyrins are usually in a highly aggregated state in aqueous systems. This study was designed to see whether the change in the polarity of the environment by adding ethanol could modify the sensitizing effects of porphyrins using a fermentable (alcohol tolerant) yeast ( Saccharomyces cerevisiae ) cells. The results showed that (1) the addition of ethanol (˜15%) to the aqueous suspension remarkably increased inactivation and cell membrane damage both in the hematoporphyrin (HP) and protoporphyrin (PP) photosensitizations, and (2) a sharp induction of genetic changes occurred concomitantly both in HP and PP sensitized cells in the presence of ethanol. In view of the fact that the addition of ethanol modified the absorption spectra and fluorescence intensity of porphyrins in favor of deaggregation, these results may be interpreted to mean that deaggregation of porphyrins promoted by ethanol enhanced their solubility in the lipophilic environment of the cell membrane and even further inside, thereby increasing the sensitizing activities.     

Properties of polyethylene glycol supported tetraarylporphyrin in aqueous solution and its interaction with liposomal membranes

5,10,15,20-Tetrakis(4-hydroxyphenyl)porphyrin was functionalized by covalent attachment of poly(ethylene glycol) (PEG) chains of various molecular weights, 350, 2000, and 5000 Da. The properties of PEG-functionalized tetraarylporphyrins in aqueous solution and their interactions with liposomes have been studied. Electronic absorption spectroscopy, dynamic light scattering, atomic force microscopy, and fluorescence quenching were used to monitor aggregation of porphyrin chromophores and behavior of the attached PEG chains in the aqueous solution. The tendency for aggregation of porphyrin chromophores in aqueous solution and the efficiency of fluorescence quenching by KI decrease with increasing length of PEG chain linked to the porphyrin ring. The experimental results indicate that polymer clusters are present in aqueous solution of all pegylated porphyrins. The interactions between the pegylated porphyrins and phosphatidylcholine liposomes in the aqueous solution were studied using the fluorescence methods. The apparent binding constants of porphyrin chromophores to liposomes were determined. The degree of binding was found to be dependent on the molecular weight of the attached polymer.     

新型NO供体的合成及其体外释放NO性能

新型NO供体的合成及其体外释放NO性能;金属卟啉;NO载体;合成;体外释NO作用     

Factors Affecting Virus Photoinactivation by a Series of Phenothiazine Dyes

A series of four phenothiazine dyes, including methylene blue (MB), were previously tested for their ability to photoinactivate viruses in red cell suspensions. One of the dyes, 1,9-dimethyl-3-dimethylamino-7-dimethylaminophenothiazine (1,9-dimethylmethylene blue), exhibited good intracellular and extracellular virucidal activity for several RNA and DNA viruses under conditions that minimally affected red cell properties. In order to understand why the virucidal specificity of 1, 9-dhnethylmethylene blue was greater than other phenothiazines tested, the physical and chemical properties of the dye were compared to three other closely related analogues (MB, 1,9-dimethyl-3-diethylamino-7-dlbutylaminophenothiazine [compound 4-140], 1,9-dimethyl-3-dimethylamino-7-diethylaminophenothiazine [compound 6-136]). All compounds required light and oxygen for virucidal activity and had relatively high singlet oxygen yields (>0.5), but 1,9-dimethylmethylene blue had a singlet oxygen yield approximately 50% greater than that of MB. In addition, the hydrophobicity/hydophilicity of the compounds varied, with the partition coefficients (2-octanol : water) ranging from 0.11 for MB to 3560 for compound 4-140. The dyes had the following affinities for DNA: 1,9-dimethylmethylene blue > compound 6-136 > MB ～ compound 4-140. This order was similar to the order of activities for photoinactivation of the nonenveloped bacteriophage, R17, by the four compounds. Results with the most hydrophobic compound, 4-140, contrasted with those obtained with 1,9-dimethylmethylene blue. Compound 4-140 had a high affinity for protein and a low affinity for DNA. Although compound 4-140 and light inactivated the nonenveloped bacteriophage R17 poorly, the dye readily photoinactivated enveloped viruses in buffer. However, unlike results with 1,9-dimethylmethylene blue, viral inactivation of enveloped viruses by compound 4-140 was completely inhibited by the presence of red cells and plasma. Thus, the high affinity of 1,9-di-methyymethylene blue for DNA and the dye's efficient singlet oxygen yield suggest viral nucleic acid as a potential target, which could explain the photosensitizer's ability to inactivate viruses without adversely affecting anuclete red cells.     

EVALUATION OF PORPHYRIN CHARACTERISTICS REQUIRED FOR PHOTODYNAMIC THERAPY

The cytotoxicity (in the dark), phototoxicity (red light) and subcellular localization (using confocal laser scanning microscopy) were determined for 15 porphyrins (1-15) in C6 glioma cells. The partition coefficient in 2-octanol was also determined for each porphyrin at pH 7.4. The cytotoxicity increased with pi (log of partition coefficient) up to pi values of +2. The 7 porphyrins with cationic side chains exhibited a classical parabolic correlation between phototoxicity and pi, with maximal activity at a pi value of approximately 1.0. There was also a significant correlation between subcellular localization and degree of phototoxicity, with the three most photosensitive porphyrins all possessing cationic side chains, and all three localizing in mitochondria.     

Synthesis of Novel Porphyrin Derivatives and Their Cytotoxic Activities against A431 Cells

Three novel porphyrins, including two Schiff‐bases porphyrins, 5,10,15‐triphenyl‐20‐[4‐(2‐(4‐formyl)phenoxy)ethoxy]phenyl porphyrin ( H₂Pp ( 1 )), 5,10,15‐triphenyl‐20‐[4‐(2‐(4‐hydroxyimino)phenoxy)ethoxy]phenyl porphyrin ( H₂Pp ( 2 )) and 5,10,15‐triphenyl‐20‐[4‐(2‐(4‐m‐hydroxyanilinodeneformyl)phenoxy)ethoxy]phenyl porphyrin ( H₂Pp ( 3 )), as well as three metalloporphyrins ( CuPp ( 1a ), ZnPp ( 1b ), and CoPp ( 1c )) of porphyrin H₂Pp ( 1 ) were synthesized. Their molecular structures were characterized by ¹H‐NMR, MS, UV/VIS, and FT‐IR spectra. Furthermore, they were evaluated by their cytotoxicities against human epidermal squamous cell carcinoma cell (A431) and normal human horn cells (HaCaT) in vitro with MTT assay. Interestingly, these porphyrins and metalloporphyrins, which had a negligible cytotoxicity to HaCaT cells, showed highly cytotoxicity against A431 cells with IC₅₀ values in the range of 6.6–9.8 μM , and metalloporphyrins exhibited higher cytotoxicity than that of metal‐free porphyrins.     

钌卟啉轴向配合物的合成与结构表征

采用一种简单的合成方法, 以钌羰基卟啉(1)和二氧钌卟啉(2)为原料, 与不同的氨基酸酯作用, 得到系列单氨基酸酯羰基和双氨基酸酯为轴向配体的钌卟啉配合物(4a~4c和5a~5d). 所有化合物均经过红外光谱、 核磁共振谱和质谱等表征手段确证为目标化合物, 并用X射线单晶衍射测定了化合物4a的分子结构, 这对研究钌卟啉与生物大分子的相互作用具有重要的意义.     

THE ROLE OF DNA DAMAGE IN PM2 VIRAL INACTIVATION BY METHYLENE BLUE PHOTOSENSITIZATION

Abstract— This study investigates the importance of DNA damage in viral inactivation by phenothiazines and light. Phenothiazines, including methylene blue (MB), toluidine blue and azure B are of particular interest because of their ability to bind to nucleic acids in vitro. Initial studies employing phages T7, MS2 and PM2 indicated that both DNA and RNA phages as well as enveloped and nonenveloped phages can be inactivated by phenothiazine photosensiti-zation. PM2, which contains a lipid-protein bilayer and supercoiled DNA, was used for the mechanistic studies to model blood-borne viruses. Viral DNA damage was assessed following treatment of phage to known levels of viral inactivation by extracting the DNA and analyzing for both direct and piperidine-catalyzed strand cleavage by gel electrophoresis. DNA strand cleavage was found to be both sensitizer concentration and light dose dependent. Both viral inactivation and DNA damage were found to be oxygen-dependent events. In parallel experiments, strand cleavage of isolated PM2 DNA treated with MB and light was also found to be oxygen dependent, in contrast to some previous reports. Transfection studies, which measure the infectivity of the extracted viral DNA, indicated that DNA from MB-treated phage was just as capable of generating progeny virus as the untreated controls. It was therefore concluded that the observed DNA damage is not correlated with loss of phage infectivity.     

Doubly charged porphyrin ion tandem mass spectrometry: Implications for structure elucidation

Under electron ionization (EI) conditions, porphyrins yield unusually high intensities of doubly charged molecular and fragment ions. These doubly charged ions offer unique opportunities for the structure elucidation of porphyrins by tandem mass spectrometry (MS/MS). First, they fragment to a greater extent than the corresponding singly charged ions under both EI/MS and EI/MS/MS conditions. Second, doubly and singly charged porphyrin ions often fragment via different pathways, and can therefore yield different structural information. This paper describes several ways in which analyses of doubly charged porphyrin ions with a triple quadrupole tandem mass spectrometer can be useful in structure elucidation of porphyrins. The effect of the metal atom on the fragmentation of metalloporphyrins in an EI source is demonstrated by correlating the extent of doubly charged fragment ion formation to a stability index. Doubly charged porphyrin ions are shown to yield predominantly doubly charged daughter ions upon collisionally activated dissociation (CAD), and are also shown to fragment to a greater extent than corresponding singly charged porphyrin ions. Advantages and disadvantages of doubly charged porphyrin ion MS/MS for structure elucidation are discussed.     

Assessing the In Vitro Activity of Selected Porphyrins in Human Colorectal Cancer Cells

Standard in vitro analyses determining the activity of different compounds included in the chemotherapy of colon cancer are currently insufficient. New ideas, such as photodynamic therapy (PDT), may bring tangible benefits. The aim of this study was to show that the biological activity of selected free-base and manganese (III) metallated porphyrins differs in the limitation of colon cancer cell growth in vitro. White light irradiation was also hypothesized to initiate a photodynamic effect on tested porphyrins. Manganese porphyrin (>1 μM) significantly decreased the viability of the colon tumor and normal colon epithelial cells, both in light/lack of light conditions, while decreasing a free-base porphyrin after only 3 min of white light irradiation. Both porphyrins interacted with cytostatics in an antagonistic manner. The manganese porphyrin mainly induced apoptosis and necrosis in the tumor, and apoptosis in the normal cells, regardless of light exposure conditions. The free-base porphyrin conducted mainly apoptosis and autophagy. Normal and tumor cells released low levels of IL-1β and IL-10. Tumor cells released a low level of IL-6. Light conditions and porphyrins were influenced at the cytokine level. Tested manganese (III) metallated and free-base porphyrins differ in their activity against human colon cancer cells. The first showed no photodynamic, but a toxic activity, whereas the second expressed high photodynamic action. White light use may induce a photodynamic effect associated with porphyrins.     

THE EFFECTS OF PORPHYRIN STRUCTURE AND AGGREGATION STATE ON PHOTOSENSITIZED PROCESSES IN AQUEOUS AND MICELLAR MEDIA

The efficiency of several porphyrins at 10 μM and 83 μM as sensitizers of the photooxidation of 0.1 mM tryptophan and histidine via a singlet oxygen-mechanism was studied in pH 7.4-buffered aqueous solutions and in aqueous dispersions of Triton X-100 micelles. The porphyrins were either solubilized in the bulk aqueous medium or associated with the micellar phase, whereas the amino acids were always located in the aqueous phase. With those porphyrins, such as uroporphyrin I, meso-tetra (4-sulfonatophenyl)porphine, meso-tetra(4-carboxyphenyl)porphine and meso-tetra)N,N,N-trimethylanilinium)porphine, which are > 98% monomeric in both media, the efficiency of histidine photooxidation was independent of the site of O₂(¹Δ_g) generation, as shown by the closely similar values for the photooxidation rate constant and oxygen-consumption quantum yield in the presence and absence of Triton micelles; the same indications were provided by photokinetic experiments with tryptophan. Actually, laser flash photolysis studies showed that the micelle-incorporation of the above mentioned porphyrins brought about only minor changes in their photophysical properties, including the relative yield of O₂(¹Δ_g) generation. On the other hand, hematoporphyrin IX, its Zn²⁺-complex, and coproporphyrin III are largely aggregated in homogeneous aqueous solution; their incorporation into Triton micelles caused an increase of the triplet quantum yield and an enhancement of the oxygen-consumption quantum yield and photooxidation rate constant for both histidine and tryptophan. The lower photosensitizing efficiency of aggregated porphyrin species in comparison with the corresponding monomeric porphyrin was confirmed by measuring the initial rate and quantum yield of oxygen consumption upon irradiation of 1 mM histidine and tryptophan in the presence of different hematoporphyrin concentrations within the 0.3-100μM range.     

The 5-aminolevulinic acid-induced porphyrin biosynthesis in benign and malignant cells of the skin.

In fluorescence diagnosis and photodynamic therapy of neoplastic tissues 5-aminolevulinic acid is used to synthesize endogenous porphyrins as photosensitizers. The efficacy of neoplastic tissues to fluorescence diagnosis and photodynamic therapy is thought to be dependent on the total level of intralesional formed porphyrins. The available profiles of porphyrin metabolites in normal and in neoplastic cell lines after administration of 5-aminolevulinic acid vary considerably. Thus, this is the first in-vitro study which compares the porphyrin biosynthesis in normal skin cells (HaCaT, fibroblasts) with melanoma cells (Bro, SKMel-23, SKMel-28). After incubation with 1 mM 5-aminolevulinic acid, kinetics of porphyrin levels and metabolites were determined in the cells and the corresponding supernatants. Exogenous 5-aminolevulinic acid induced porphyrin formation in all cells with maximum values after an incubation period of 16-36 h. Increase of porphyrin levels varied from 10- to 80-fold (SKMel-28>HaCaT>fibroblasts>SKMel-23>Bro) with minimum 1.5 times higher levels of porphyrins in the supernatants than in the cells. In cells and supernatants protoporphyrin and coproporphyrin were the predominantly formed porphyrin metabolites. Metastatic melanoma cells (SKMel-23, SKMel-28) accumulated much higher porphyrin levels than primary melanoma cells (Bro). In conclusion, by optimizing the treatment modalities, especially the light source, topical photodynamic therapy (PDT) could become a treatment alternative of melanoma metastases in progressive disease.