Synthesis,photophysical properties and biological evaluation of β-alkylaminoporphyrin for photodynamic therapy

A series of β-alkylaminoporphyrins conjugated with different amines at β position (D1–D3) or with electron-donating and electron-withdrawing substituents at phenyl position (D4–D6) were synthesized. Their photophysical and photochemical properties, intracellular localization, photocytotoxicities in vitro and vivo were also investigated. All target compounds exhibited no cytotoxicities in the dark and excellent photocytotoxicities against HeLa cells. Among them, D6 showed the highest phototoxicity and the lowest dark toxicity, which was more phototoxic than Hematoporphyrin monomethyl ether (HMME). In addition, D6 exhibited best photodynamic antitumor efficacy on BALB/c nude mice bearing HeLa tumor. Therefore, D6 is a powerful and promising antitumor photosensitizer for photodynamic therapy.     

Effect of Molecular Characteristics on Cellular Uptake,Subcellular Localization,and Phototoxicity of Zn(II) N-Alkylpyridylporphyrins

Tetra-cationic Zn(II) meso-tetrakis(N-alkylpyridinium-2 (or -3 or -4)-yl)porphyrins (ZnPs) with progressively increased lipophilicity were synthesized to investigate how the tri-dimensional shape and lipophilicity of the photosensitizer (PS) affect cellular uptake, subcellular distribution, and photodynamic efficacy. The effect of the tri-dimensional shape of the molecule was studied by shifting the N-alkyl substituent attached to the pyridyl nitrogen from ortho to meta and para positions. Progressive increase of lipophilicity from shorter hydrophilic (methyl) to longer amphiphilic (hexyl) alkyl chains increased the phototoxicity of the ZnP PSs. PS efficacy was also increased for all derivatives when the alkyl substituents were shifted from ortho to meta, and from meta to para positions. Both cellular uptake and subcellular distribution of the PSs were affected by the lipophilicity and the position of the alkyl chains on the periphery of the porphyrin ring. Whereas the hydrophilic ZnPs demonstrated mostly lysosomal distribution, the amphiphilic hexyl derivatives were associated with mitochondria, endoplasmic reticulum, and plasma membrane. A comparison of hexyl isomers revealed that cellular uptake and partition into membranes followed the order para > meta > ortho. Varying the position and length of the alkyl substituents affects (i) the exposure of cationic charges for electrostatic interactions with anionic biomolecules and (ii) the lipophilicity of the molecule. The charge, lipophilicity, and the tri-dimensional shape of the PS are the major factors that determine cellular uptake, subcellular distribution, and as a consequence, the phototoxicity of the PSs.     

A light in multidrug resistance: Photodynamic treatment of multidrug-resistant tumors

The major drawback of cancer chemotherapy is the development of multidrug-resistant (MDR) tumor cells, which are cross-resistant to a broad range of structurally and functionally unrelated agents, making it difficult to treat these tumors. In the last decade, a number of authors have studied the effects of photodynamic therapy (PDT), a combination of visible light with photosensitizing agents, on MDR cells. The results, although still inconclusive, have raised the possibility of treating MDR tumors by PDT. This review examines the growing literature concerning the responses of MDR cells to PDT, while stressing the need for the development of new photosensitizers that possess the necessary characteristics for the photodynamic treatment of this class of tumor.     

Cercosporin-deficient mutants by plasmid tagging in the asexual fungus<Emphasis Type="Italic"> Cercospora nicotianae</Emphasis>

We have successfully adapted plasmid insertion and restriction enzyme-mediated integration (REMI) to produce cercosporin toxin-deficient mutants in the asexual phytopathogenic fungus Cercospora nicotianae. The use of pre-linearized plasmid or restriction enzymes in the transformation procedure significantly decreased the transformation frequency, but promoted a complicated and undefined mode of plasmid integration that leads to mutations in the C. nicotianae genome. Vector DNA generally integrated in multiple copies, and no increase in single-copy insertion was observed when enzymes were added to the transformation mixture. Out of 1873 transformants tested, 39 putative cercosporin toxin biosynthesis ( ctb) mutants were recovered that showed altered levels of cercosporin production. Seven ctb mutants were recovered using pre-linearized plasmids without the addition of enzymes, and these were considered to be non-REMI mutants. The correlation between a specific insertion and a mutant phenotype was confirmed using rescued plasmids as gene disruption vectors in the wild-type strain. Six out of fifteen rescued plasmids tested yielded cercosporin-deficient transformants when re-introduced into the wild-type strain, suggesting a link between the insertion site and the cercosporin-deficient phenotype. Sequence analysis of a fragment flanking the insert site recovered from one insertion mutant showed it to be disrupted in sequences with high homology to the acyl transferase domain of polyketide synthases from other fungi. Disruption of this polyketide synthase gene ( CTB1) using a rescued plasmid resulted in mutants that were defective in cercosporin production. Thus, we provide the first molecular evidence that cercosporin is synthesized via a polyketide pathway as previously hypothesized.Communicated by E. Cerdá-Olmedo     

Unsaturated lipids protect the integral membrane peptide gramicidin A from singlet oxygen

In contrast to expectations that unsaturated fatty acids contribute to oxidative stress by providing a source of lipid peroxides, we demonstrated the protective effect of double bonds in lipids on oxidative damage to membrane proteins. Photodynamic inactivation of gramicidin channels was decreased in unsaturated lipid compared to saturated lipid bilayers. By estimating photosensitizer (boronated chlorine e₆ amide) binding to the membrane with the current relaxation technique, the decrease in gramicidin photoinactivation was attributed to singlet oxygen scavenging by double bonds in lipids rather than to the reduction in photosensitizer binding. Gramicidin protection by unsaturated lipids was also observed upon induction of oxidative stress with tert-butyl hydroperoxide.     

Photodynamic effect of novel chlorin e6 derivatives on a single nerve cell

Chlorin e(6) and its derivatives are promising sensitizers for photodynamic therapy (PDT). In order to compare the photodynamic effects of 8 novel derivatives of chlorin e(6) and to explore some mechanisms of their effects at the cellular level, we studied PDT-induced changes in bioelectric activity of crayfish mechanoreceptor neuron that was used as a sensitive experimental model. Neurons were insensitive to red laser irradiation (632.8 nm; 0.3 W/cm(2)) or to photosensitizers alone, but changed firing rate and died under the photodynamic effect of nanomolar concentrations of sensitizers. The dynamics of neuron responses depended on photosensitizer type and concentration. The dependence of neuron lifetime on photosensitizer concentration allowed comparing efficiencies of different photosensitizers. Radachlorin was the most potent photosensitizer comparable with mTHPC. High photodynamic efficiency of some chlorin e(6) derivatives was related to weak dependence of neuron lifetime on sensitizer concentration, indicating to the initiation of 2-3 secondary processes such as free radical membrane damage by one absorbed photon. Photodynamic efficiency of sensitizers depended on amphiphilicity influencing their intracellular localization.     

Synthesis, structure, and characterization of two polyoxometalate-photosensitizer hybrid materials

Two hybrid materials based on the tris(bipyridine)ruthenium(II), [Ru(bpy)₃]²⁺ and Keggin-type polyoxometalates, [PW₁₁O₃₉]⁷⁻ and [PW₁₂O₄₀]³⁻, namely, [Ru(bpy)₃][K₅PW₁₁O₃₉] (1) and [Ru(bpy)₃][KPW₁₂O₄₀] (2) were synthesized. X-ray crystallographic study of the red-colored complex, 2, shows that it crystallizes in the orthorhombic space group P_bcn and the polyanions are associated with the [Ru(bpy)₃]²⁺ counterions by Coulombic forces and supramolecular interactions. The molecular complex is further connected and forms a three-dimensional framework through C-H?O_POM and other weak interactions. These complexes were further characterized by FT-IR, UV-Vis, ¹H and ³¹P NMR, luminescent spectra and computational studies. Significantly, these combined spectroscopic studies show that these polyoxometalate-dye hybrids have strong electronic interactions between the cationic dye and polyanion units.     

Photosensitized cross-linking of erythrocyte membrane proteins. Evidence against participation of amino groups in the reaction

Exposure of human erythrocyte ghosts (pH 8, 10°C) to visible light in the presence of the photosensitizer, methylene blue, results in a relatively rapid loss of spectrin (bands 1 and 2 on sodium dodecyl sulfate gel electropherograms) and the appearance of high molecular weight cross-linked derivatives. Isolated spectrin also undergoes photosensitized cross-linking, indicating that the reaction is not lipid-dependent.Extensive cross-linking was neither reversed by dithiothreitol nor prevented by prior blocking of SH groups with $\text{N-}\text{ethylmaleimide}$, suggesting that cysteine residues are not crucial bridging sites. The possible requirement for NH₂ groups, as suggested by previous model studies (Dubbelman, T.M.A.R., de Goeij, A.F.P.M. and van Steveninck, J. (1978) Biochim. Biophys. Acta 511, 141–151), was tested. Succinylation of spectrin protected against cross-linking, but this effect is attributed to the disruption of quaternary structure, as deduced from sedimentation measurements. However, virtually complete blocking of NH₂ groups by amidination perturbed overall structure relatively little, and had no effect on cross-linking. Moreover, exogenous amines such as ethylamine, added in large excess to spectrin prior to irradiation, did not interfere with cross-link formation. These results suggest that NH₂ groups are not involved in the reaction.     

Synthesis of 2-morpholinetetraphenylporphyrins and their photodynamic activities

A series of 2-morpholinetetraphenylporphyrins functionalized with various substituents (Cl, Me, MeO group) at 4-phenyl position were prepared via nucleophilic substitution of 2-nitroporphyrin copper derivatives with morpholine by refluxing under a nitrogen atmosphere and then demetalization. Their basic photophysical properties, intracellular localization, cytotoxicities in vitro and in vivo were also investigated. All synthesized photosensitizers exhibited longer maxima absorption wavelengths than Hematoporphyrin monomethyl ether (HMME). They showed low dark cytotoxicity compared with that of HMME and were more phototoxic than HMME against Eca-109 cells in vitro. M3 also exhibited better photodynamic antitumor efficacy on BALB/c nude mice at a lower concentration. Therefore, M3 is a promising antitumor photosensitizer in photodynamic therapy application.