Gabriella Csík

Glycosylated derivatives of tetraphenyl porphyrin: photophysical characterization, self-aggregation and membrane binding

Abstract A series of glucose- or galactose-residue-bearing tetraphenyl porphyrins (TPPs) has recently been synthesized with the aim of studying the structural dependence of porphyrin cellular localization and efficiency in photodynamic therapy (PDT). For the present investigation, four derivatives with different (spherical or planar) configurations and/or different hydrophobicity have been selected. As a first step, singlet-state spectroscopic properties such as spectral characteristics, lifetime and quantum yield in different solvents are determined. It is found that besides the solvent composition, the configuration of the molecule critically influences the singlet-state spectroscopic properties. Possible self-aggregation processes are investigated at room temperature in phosphate buffer (pH = 7.4). Dimerization equilibrium constants are determined. These values are found to be dependent on the chemical structure of substituents on the tetraphenyl groups and only slightly on the conformation of the whole molecule. The association of non-aggregated forms of porphyrins to unilamellar liposomes, modelling the lipid bilayer of a biological membrane, is studied by fluorescence spectroscopy at neutral pH. On mixing with liposomes, amphiphilic porphyrin derivatives exhibit an increase in their fluorescence intensity and lifetime. The monomer-liposome binding constants are determined for these derivatives. The localization of liposome-bound dyes is studied by fluorescence labelling of (a) the lipid region in interaction with both lipid chains and headgroups or (b) the carbohydrate chain region of lipids. Alterations in fluorescence intensities of porphyrin derivatives in the presence of liposomes and changes in the lifetime and fluorescence excitation anisotropy of fluorescent markers in liposomes are detected only for unsymmetrically substituted amphiphilic porphyrin derivatives. Our results suggest that the presence of at least one apolar substituent on tetrapyrrolic ring is required for the localization in the lipid phase.

δ-aminolaevulinic acid-induced porphyrin synthesis and photodynamic inactivation of Escherichia coli B.

The possibility and conditions for the induction of porphyrin synthesis by exogenous delta-aminolaevulinic acid (ALA) and its applicability for the inactivation of Gram-negative bacteria Escherichia coli B. by photodynamic therapy (PDT) have been studied. The bacteria are supplemented with ALA in the log phase of growth, and are grown in a synthetic medium at 37 degrees C in the dark. The efficiency of porphyrin synthesis is detected by fluorescence spectroscopy performed on the isolated bacterial cells and the medium, respectively, and compared with results of high-performance liquid chromatography (HPLC) analysis. ALA stimulates the synthesis of protoporphyrin in the bacteria by a factor of five to six, and an increased amount of the more hydrophilic derivatives with a significant contribution of mesoporphyrin by a factor of two to three is observed in the culturing medium. The optimal conditions of ALA treatment with respect to PDT are 10-15 min of incubation of a bacterial culture of 2 x 10(7) cells ml-1 with (5-9) x 10(-3) mol l-1 ALA. The ALA-treated cells are irradiated by white light of 80 mW cm-2 under growth conditions and a decrease to 0.6% of the number of colony-forming units (CFUs ml-1) is observed after 90 min of irradiation.

Syntheses and DNA binding of new cationic porphyrin–tetrapeptide conjugates

Recently cationic porphyrin-peptide conjugates were synthesized to enhance the cellular uptake of porphyrins or deliver the peptide moiety to the close vicinity of nucleic acids. DNA binding of such compounds was not systematically studied yet. We synthesized two new porphyrin-tetrapeptide conjugates which can be considered as a typical monomer unit corresponding to the branches of porphyrin-polymeric branched chain polypeptide conjugates. Tetra-peptides were linked to the tri-cationic meso-tri(4-N-methylpyridyl)-mono-(4-carboxyphenyl)porphyrin and bi-cationic meso-5,10-bis(4-N-methylpyridyl)-15,20-di-(4-carboxyphenyl)porphyrin. DNA binding of porphyrin derivatives, and their peptide conjugates was investigated with comprehensive spectroscopic methods. Titration of porphyrin conjugates with DNA showed changes in Soret bands with bathocromic shifts and hypochromicities. Decomposition of absorption spectra suggested the formation of two populations of bound porphyrins. Evidence provided by the decomposition of absorption spectra, fluorescence decay components, fluorescence energy transfer and induced CD signals reveals that peptide conjugates of di- and tricationic porphyrins bind to DNA by two distinct binding modes which can be identified as intercalation and external binding. Tri-cationic structure and elimination of negative charges in the peptide conjugates are preferable for the binding. Our findings provide essential information for the design of DNA-targeted porphyrin-peptide conjugates.

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.

Interaction of glycosylated tetraphenyl porphyrins with model lipid membranes of different compositions

Abstract Porphyrin derivatives are one of the major groups of compounds applied in photodynamic therapy. Their effect and interaction with different cell organelles and macromolecules is dependent on their chemical structure.We have examined the effect of the molecular structure of porphyrin derivatives on binding to liposomes without net charge and with negative charge. In this study a series of substituted tetraphenyl porphyrins (TPPs) has been used. Two of them, TP(4-OGluOH)4P and TP(4-OXylOH)4P, are symmetrically substituted derivatives having four carbohydrate — glucose or xylose — moieties linked to the macrocycle. The other two derivatives, TP(4-OGluOH)3P and TPF5(4-OGalOH)3P, were selected as asymmetrically substituted amphiphilic molecules with slightly different hydrophobic character due to the different ligand moieties. Association constants (KL) of derivatives to liposomes and rate constants of liposome binding have been determined and also the gel-to-liquid-crystalline phase-transition parameters have been monitored in order to determine the type and strength of interaction. According to our measurements, the KL values follow the order TP(4-OGluOH)3P>TPF5(4-OGalOH)3P>TP(4-OXylOH)4P for both types of liposomes. TP(4-OGluOH)4P is able to bind only to vesicles holding negative charges on their surface. At 18°C, the measured data are best fitted by two exponentials for three TPP derivatives. The binding of TP(4-OGluOH)4P follows single-exponential kinetics. At 37°C, TP(4-OGluOH)3P binds to DMPC liposomes according to biexponential kinetics, but all the other binding processes follow monoexponential kinetics under similar conditions. The strongest effect on phase-transition parameters is caused by TP(4-OGluOH)3P. From the results it can be concluded, that the binding process of TPPs, especially that of the symmetrically substituted derivatives, is drastically influenced by the surface charges of the model membranes. According to present data, the negative surface charges of DMPC/DMPG liposomes can facilitate the association of the symmetrically substituted hydrophilic porphyrins to the liposomes. However, the asymmetrically substituted derivative TP(4-OGluOH)3P, which is the most lipophilic compound in the present investigation, still has the largest association constant to both neutral and negatively charged liposomes. In general, in both types of liposomes the order of the association constant follows the order of the lipophilicity of the porphyrin derivatives.

New trends in photobiology. Phage nucleoprotein-psoralen interaction: quantitative characterization of dark and photoreactions

The irradiation of the phage T7 system containing psoralen as photosensitizer causes many processes, each of them leading to phage inactivation. These processes include the UV-induced photoreactions in the phage nucleic acid, and photoreactions in the nucleic acid sensitized by either psoralen or psoralen photobreakdown products. In addition the intercalation of the psoralen molecule itself in the phage nucleic acid as well as the psoralen photobreakdown products cause phage inactivation. Under appropriate experimental conditions these reactions can be studied and characterized separately. The quantitative characteristics (e.g. inactivation cross-section, action spectra and index for dark genotoxicity) are demonstrated for different linear and angular psoralens. Some theoretical and practical consequences of the results obtained are discussed.

Photoinduced Inactivation of T7 Phage Sensitized by Symmetrically and Asymmetrically Substituted Tetraphenyl Porphyrin: Comparison of Efficiency and Mechanism of Action¶

Abstract We investigated the efficiency and the mechanism of action of two—one symmetrically and one asymmetrically substituted—glycoconjugated tetraphenyl porphyrins in their photoreaction with T7 phage as a model of nucleoprotein (NP) complexes. A correlation was found between the dark inactivation of T7 and the binding of porphyrins determined by fluorescence spectroscopy. Both types of porphyrin sensitized the photoinactivation of T7, but the slopes of inactivation kinetics were markedly different. There was no correlation between the dark binding and the photosensitizing efficacy of the two derivatives. Inactivation was moderated by 1,3-diphenylisobenzofuran and 1,3-dimethyl-2-thiourea; however, neither of them inhibited T7 inactivation completely. This result suggests that both Type-I and Type-II reactions play a role in the virus inactivation. Optical melting studies revealed structural changes in the protein part but not in the DNA of the photochemically treated NP complex. Polymerase chain reaction analysis of a 555 bp segment of gene 1 and a 3826 bp segment of genes 3 and 4 failed to demonstrate any DNA damage.

Interaction of hydro- or lipophilic phthalocyanines with cells of different metastatic potential.

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

New daunomycin-oligoarginine conjugates: synthesis, characterization, and effect on human leukemia and human hepatoma cells.

In this article, the synthesis, a novel chromatographic procedure and characteristics of a new class of daunomycin (Dau)–oligoarginine conjugates are described. In these compounds oligoarginine with 6 or 8 residues (Argn, n = 6, 8) is attached to Dau by different covalent bond: squaric amide (Dau‐□‐Argn), oxime (DauNOCH2COArgn), or hydrazone (HGlu(Argn)NHNDau). Conjugates were characterized by RP‐HPLC and mass spectrometry. We report also on our findings concerning chemical and biological properties of Dau‐conjugates as a function of covalent linkage, site of conjugation and length of the oligoarginine moiety. Stability, fluorescent properties as well as cytostatic effect and cellular uptake of these compounds were studied. Dau‐conjugates with squaric amide or oxime linkage were stable, but continuous release of free Dau was observed from the hydrazone conjugate in solution. We found that some spectral characteristics (e.g., the amplitude of the emission spectrum) of conjugates could be sensitive for the site of coupling (amino vs. oxo function). Cytostasis and cellular uptake of conjugates were investigated both on human leukemia (HL‐60) and human hepatoma (HepG2) cell lines by MTT assay and flow cytometry We found that cytostatic effect and uptake properties of Dau‐conjugates were dependent on the acid stability of the linkage (hydrazone vs. oxime/amide) applied and more markedly on the cell line studied.

A New Daunomycin–Peptide Conjugate: Synthesis, Characterization and the Effect on the Protein Expression Profile of HL-60 Cells in Vitro

Daunomycin (Dau) is a DNA-binding antineoplastic agent in the treatment of various types of cancer, such as osteosarcomas and acute myeloid leukemia. One approach to improve its selectivity and to decrease the side effects is the conjugation of Dau with oligopeptide carriers, which might alter the drug uptake and intracellular fate. Here, we report on the synthesis, characterization, and in vitro biological properties of a novel conjugate in which Dau is attached, via an oxime bond, to one of the cancer specific small peptides (LTVSPWY) selected from a random phage peptide library. The in vitro cytostatic effect and cellular uptake of Dau═Aoa-LTVSPWY-NH(2) conjugate were studied on various human cancer cell lines expressing different levels of ErbB2 receptor which could be targeted by the peptide. We found that the new daunomycin-peptide conjugate is highly cytostatic and could be taken up efficiently by the human cancer cells studied. However, the conjugate was less effective than the free drug itself. RP-HPLC data indicate that the conjugate is stable at least for 24 h in the pH 2.5-7.0 range of buffers, as well as in cell culture medium. The conjugate in the presence of rat liver lysosomal homogenate, as indicated by LC-MS analysis, could be degraded. The smallest, Dau-containing metabolite (Dau═Aoa-Leu-OH) identified and prepared expresses DNA-binding ability. In order to get insight on the potential mechanism of action, we compared the protein expression profile of HL-60 human leukemia cells after treatment with the free and peptide conjugated daunomycin. Proteomic analysis suggests that the expression of several proteins has been altered. This includes three proteins, whose expression was lower (tubulin β chain) or markedly higher (proliferating cell nuclear antigen and protein kinase C inhibitor protein 1) after administration of cells with Dau-conjugate vs free drug.