Potent DNA photocleavage by zinc(II) complexes of cationic bis-porphyrins linked with aliphatic diamine

We have prepared zinc(II) complexes of cationic bis-porphyrins, as one of the attempts to improve less DNA photocleavage activities of the metal-free bis-porphyrins composed of two H(2)TMPyP-like chromophores, linked with a series of aliphatic diamines. The less activities seemed to be derived from their intermolecular self-aggregation properties in aqueous solution. The zinc(II) insertion into the metal-free cationic bis-porphyrins completely removed their self-aggregation properties, most probably due to steric hindrance between axial ligands of zinc(II) chromophores of the cationic bis-porphyrins. The DNA photocleavage activities of the zinc(II) complexes were fully enhanced, which were three times larger than that of the lead compound H(2)TMPyP. Quantitative analysis of singlet oxygen production by photosensitization of cationic bis-porphyrins was performed using 1,3-diphenylisobenzofuran, and the singlet oxygen productivities of them were found to be related to their solution properties. There is a good relationship between the activities and the productivities, which will provide insights into the further development of more effective DNA photocleavage agents.     

Lactic acid production with lactate dehydrogenase using the visible light sensitization of zinc porphyrin.

Lactic acid production with L-lactate dehydrogenase from Pig heart (LDH) and reduced methyl viologen produced by the visible light photosensitization of zinc tetrakis(4-methylpyridyl)porphyrin (ZnTMPyP) was investigated. When the sample solution containing triethanolamine as an electron-donating reagent, ZnTMPyP, methyl viologen as an electron carrier, pyruvic acid and LDH in potassium phosphate buffer (pH 7) was irradiated, lactic acid production was observed. After 240 min irradiation, the amount of lactic acid production and the yield of pyruvic acid to lactic acid were estimated to be 0.17 mmol dm(-3) and 17.0%, respectively.     

Photophysical properties and photodynamic activity in vivo of some tetrapyrroles

Some of the photophysical properties (stationary absorbance and fluorescence, fluorescence decay times and singlet oxygen quantum yields) of pheophorbide a, metal-free, ClAl-, Cu- and Mg-t-butyl-substituted phthalocyanines, metal-free, ClAl- and Cu-t-butyl-substituted naphthalocyanines and of a number of tetraphenylporphyrins (5,10,15,20-tetraphenylporphyrin, 5,10,15,20-tetra(m-hydroxyphenyl)porphyrin, 5,10,15,20-tetra(p-hydroxyphenyl)porphyrin) have been studied in comparison with hematoporphyrin IX in order to select potent photosensitizers for the photodynamic treatment of cancer. The photodynamic activity of these compounds was investigated using Lewis lung carcinoma in mice. As a consequence of the photophysical parameters (relatively short singlet state lifetimes, and high singlet oxygen quantum yields) the photodynamic activities of pheophorbide a, t-butyl-substituted ClAl-phthalocyanine and ClAl-naphthalocyanine were selected for study in greater detail. Under the conditions employed in the present study, pheophorbide a was found to be the most effective sensitizer, as judged from its strong absorption at the excitation wavelength as compared with the hematoporphyrin derivative and greater singlet oxygen quantum yield relative to the phthalocyanines and naphthalocyanines. The photodynamic activity was observed to be strongly dependent on the photophysical parameters of the compounds. The primary mechanism underlying the photodynamic activity of these sensitizers probably consists of energy transfer from the lowest triplet state of the dyes to molecular oxygen, resulting in the formation of singlet oxygen (type II of photosensitization).     

Studies on the photochemical and photocytotoxic properties of the new PDT photosensitizer aluminum sulfonated phthalocyanine.

The properties of photosensitization of sulfonated aluminum phthalocyanine (ALSPC), a new photosensitizer of potential use in cancer photodynamic therapy (PDT) was studied on both the molecular and cellular levels. The mechanism of ALSPC photosensitization on the molecular level was investigated by testing its efficiency of singlet oxygen (1O2) production, using the method of tryptophan degradation and that of ESR spectroscopy and observing the enhancing effect of D2O and the quenching effect of NaN3. Results of all these experiments confirmed the important role of the Type II or 1O2 mechanism in ALSPC photosensitization. In our in-vitro experiments, ALSPC's incorporation into cells and its photocytotoxic effect were investigated on a human liver cancer cell line. The cell incorporation was illustrated by the laser-excited fluorescence spectra emitted both from cell homogenate and cell monolayers incubated with ALSPC aqueous solution. The position of fluorescence peak observed, implied that ALSPC exists in the cells mainly as monomers. The efficiency of cell killing of ALSPC photosensitization was estimated by counting surviving cells with the method of trypan blue staining and by the method of radioisotope labelling. Experiments using the latter method also showed DNA damage caused by ALSPC photosensitization.     

A comparative study of the interaction of 5,10,15,20-tetrakis (N-methylpyridinium-4-yl)porphyrin and its zinc complex with DNA using fluorescence spectroscopy and topoisomerisation.

Binding of 5,10,15,20-tetrakis (N-methylpyridinium-4-yl)porphyrin (H2TMPyP4+) and its zinc complex (ZnTMPyP4+) to DNA is demonstrated by their coelectrophoresis and by absorption and fluorescence spectroscopic methods. Topoisomerisation of pBR322 DNA shows that H2TMPyP4+ unwinds DNA as efficiently as ethidium bromide showing that it intercalates at many sites. ZnTMPyP4+ may cause limited unwinding. Marked changes in the fluorescence spectra of the porphyrins are found in the presence of DNA. The fluorescence intensity of either H2TMPyP4+ or ZnTMPyP4+ is enhanced in the presence of poly (d(A-T)), whereas in the presence of poly (d(G-C] the fluorescence intensity of ZnTMPyP4+ is only slightly affected and that of H2TMPyP4+ markedly reduced. Both the porphyrins photosensitize the cleavage of DNA in aerated solution upon visible light irradiation.     

ESR evidence of the photogeneration of free radicals (GDHB*-, O2*-) and singlet oxygen ((1)O2) by 15-deacetyl-13-glycine-substituted hypocrellin B

15-Deacetyl-13-glycine-substituted hypocrellin B (GDHB) is a new type of hypocrellin derivative with an enhanced red absorption longer than 600 nm and water solubility. Visible light (> 470 nm) irradiation of an anaerobic aqueous solution of GDHB, the formation of GDHB*- was detected by an ESR method in the absence or presence of electron donor. When exposed to oxygen, superoxide anion radical and singlet oxygen were formed. The superoxide anion radical was generated by GDHB*- via electron transfer to oxygen and this process was significantly enhanced by the presence of electron donors. Singlet oxygen ((1)O2) was also formed in the photosensitization of GDHB in aerobic solution and 1,4-diazabicyclo [2,2,2] octane (DABCO), sodium azide (NaN3) and histidine inhibited the generation of (1)O2. A 9,10-diphenyl antracene (DPA)-bleaching method was used to determine the quantum yield of (1)O2 generated from GDHB photosensitization. The (1)O2 quantum yield was estimated to be 0.65. With the depletion of oxygen, the accumulation of GDHB*- would replace that of (1)O2. Evidence accumulated that the photodynamic action of GDHB may proceed via both type I and type II mechanisms and that a type II mechanism will be transformed into a type I mechanism as oxygen gets depleted.     

Genotoxicity of singlet oxygen

Singlet oxygen, ¹O₂(¹Δ_g), fulfills essential prerequisites for a genotoxic substance, like hydroxyl radicals and other oxygen radicals: it can react efficiently with DNA and it can be generated inside cells, e.g. by photosensitization and enzymatic oxidation. As might be anticipated from the non-radical character of singlet oxygen, the pattern of DNA modifications it produces is very different from that caused by hydroxyl radicals. While hydroxyl radicals produce DNA strand breaks and sites of base loss (AP sites) in high yield and react with all four bases of DNA, singlet oxygen generates predominantly modified guanine residues and few strand breaks and AP sites. There is now convincing evidence that a major product of base modification caused by singlet oxygen is 8-hydroxyguanine (7,8-dihydro-8-oxoguanine). Indeed, the recently reported miscoding properties of 8-hydroxyguanine can explain the predominant type of mutations observed when DNA modified by singlet oxygen is replicated in cells. There are also strong indications that singlet oxygen generated by photosensitization can act as an ultimate DNA modifying species inside cells. However, indirect genotoxic mechanisms involving other reactive oxygen species produced from singlet oxygen are also possible and appear to predominate in some cases. The cellular defense system against oxidants consists of effective singlet oxygen scavengers such as carotenoids. The observation that carotenoids can inhibit neoplastic cell transformation when administered not only together with but also after the application of chemical or physical carcinogens might indicate a role of singlet oxygen in tumor promotion that could be independent of the direct or indirect DNA damaging properties.     

Photooxidation of d(TpG) by phthalocyanines and riboflavin. Isolation and characterization of dinucleoside monophosphates containing the 4R* and 4S* diastereoisomers of 4,8-dihydro-4-hydroxy-8-oxo-2''-deoxy-guanosine.

Phthalocyanine mediated photosensitization of 2'-deoxyguanosine (dG) in oxygen saturated aqueous solution has previously been shown to result in the addition of molecular oxygen to the guanine base generating the 4R* and 4S* diastereoisomers of 4,8-dihydro-4-hydroxy-8-oxo-2'-deoxyguanosine (dO) (the asterisk denotes unambiguous assignment of the 4R and 4S diastereoisomers). The data presented here show that the same guanine modified bases are generated in a 1:1 ratio when thymidylyl-(3',5')-2'-deoxyguanosine (d(TpG)) is similarly photo-oxidized. These modified dinucleoside monophosphates, labelled d(TpO)-A and -B, have been isolated by high performance liquid chromatography and characterized by proton NMR spectrometry, fast atom bombardment mass spectrometry, and enzymatic digestions. Photosensitization in D2O instead of H2O leads to an increase in the rate of d(TpO) formation that is consistent with a type II (singlet oxygen) reaction mechanism. Three interesting properties of these modified dinucleoside monophosphates are: i) the rate of their digestion with spleen phosphodiesterase is greatly reduced relative to d(TpG), ii) they are not digested by snake venom phosphodiesterase, and iii) they are stable to 1.0 M piperidine at 90 degrees C for 30 min. The latter observation indicates that 4,8-dihydro-4-hydroxy-8-oxoguanine is not a base lesion responsible for the strand breaks observed following hot piperidine treatment of DNA exposed to type II photosensitizers or chemically generated singlet oxygen.     

Dynamic light scattering and fluorescence study of the interaction between double-stranded DNA and poly(amido amine) dendrimers

The interaction between a cationic poly(amido amine) (PAMAM) dendrimer of generation 4 and double-stranded salmon sperm DNA in 10 mM NaBr solution has been investigated using dynamic light scattering (DLS) and steady-state fluorescence spectroscopy. The structural parameters of the formed aggregates as well as the complex formation process were studied in dilute solutions. When DNA is mixed with PAMAM dendrimers, it undergoes a transition from a semiflexible coil to a more compact conformation due to the electrostatic interaction present between the cationic dendrimer and the anionic polyelectrolyte. The DLS results reveal that one salmon sperm DNA molecule forms a discrete aggregate in dilute solution with several PAMAM dendrimers with a mean apparent hydrodynamic radius of 50 nm. These discrete complexes coexist with free DNA at low molar ratios of dendrimer to DNA, which shows that cooperativity is present in the complex formation. The formation of the complexes was confirmed by agarose gel electrophoresis measurements. DNA in the complexes was also found to be significantly more protected against DNase catalyzed digestion compared to free DNA. The number of dendrimers per DNA chain in the complexes was found to be approximately 35 as determined by steady-state fluorescence spectroscopy.     

Interaction of meso-tetrakis (4-N-methylpyridyl) porphyrin in its free base and as a Zn(II) derivative with large unilamellar phospholipid vesicles

Our aim was to investigate the interaction of the cationic meso-tetrakis (4-N-methylpyridyl) porphyrin, a photosensitizer used for photodynamic therapy, in its free base form (TMPyP) and complexed with Zn(II) (ZnTMPyP), with large unilamellar vesicles (LUVs), as a model for the gram-negative bacterial cell wall. Mixtures of the zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) phospholipids, at different molar percentages, were used as LUVs. A significant increase of porphyrin affinity at higher POPG molar concentrations was observed from the binding constant values, K _b, estimated by optical absorption and steady-state fluorescence. Besides, as demonstrated by time-resolved fluorescence, this affinity increase is also followed by a higher fraction of vesicle-bound porphyrin in the LUVs. Moreover, based on the K _b values, we have observed a higher affinity of the ZnTMPyP to the POPG containing LUVs as compared to the TMPyP. Steady-state fluorescence quenching and zeta potential studies revealed that both porphyrins are possibly located at the LUVs Stern layer region. Therefore, the electrostatic attraction between the positively charged porphyrin peripheral groups and the negatively charged outer surface of the LUVs plays an important role in porphyrin association and localization. Our results have improved the understanding of the successful application of cationic porphyrins on the photo-inactivation of gram-negative bacteria. Since a higher accumulation of the ZnTMPyP in the bacterial cell wall would be expected, this porphyrin could be a more efficient therapeutic drug for this treatment.     

Electrostatic binding of substituted metal phthalocyanines to enterobacterial cells: Its role in photodynamic inactivation

The effect of ionic substituents in zinc and aluminum phthalocyanine molecules and of membrane surface charge on the interaction of dyes with artificial membranes and enterobacterial cells, as well as on photosensitization efficiency was studied. It has been shown that increasing the number of positively charged substituents enhances the extent of phthalocyanine binding to Escherichia coli cells. This, along with the high quantum yield of singlet oxygen generation, determines efficient photodynamic inactivation of Gram-negative bacteria by zinc and aluminum octacationic phthalocyanines. The effect of Ca²⁺ and Mg²⁺ cations and pH on photodynamic inactivation of enterobacteria in the presence of octacationic zinc phthalocyanine has been studied. It has been shown that effects resulting in lowering negative charge on outer membrane protect bacteria against photoinactivation, which confirms the crucial role in this process of the electrostatic interaction of the photosensitizer with the cell wall. Electrostatic nature of binding is consistent with mainly electrostatic character of dye interactions with artificial membranes of different composition. Lower sensitivity of Proteus mirabilis to photodynamic inactivation, compared to that of E. coli and Salmonella enteritidis, due to low affinity of the cationic dye to the cells of this species, was found.     

Singlet oxygen photosensitization by EGFP and its chromophore HBDI

The photosensitization of reactive oxygen species and, in particular, singlet oxygen by proteins from the green fluorescent protein (GFP) family influences important processes such as photobleaching and genetically targeted chromophore-assisted light inactivation. In this article, we report an investigation of singlet oxygen photoproduction by GFPs using time-resolved detection of the NIR phosphorescence of singlet oxygen at 1275 nm. We have detected singlet oxygen generated by enhanced (E)GFP, and measured a lifetime of 4 μs in deuterated solution. By comparison with the model compound of the EGFP fluorophore 4-hydroxybenzylidene-1,2-dimethylimidazoline (HBDI), our results confirm that the β-can of EGFP provides shielding of the fluorophore and reduces the production of this reactive oxygen species. In addition, our results yield new information about the triplet state of these proteins. The quantum yield for singlet oxygen photosensitization by the model chromophore HBDI is 0.004.     

Morphology of Cationic Liposome/DNA Complexes in Relation to Their Chemical Composition

Abstract

Electron microscopy is used to show the morphology of liposome/DNA complexes as related to their cationic component, the molar ratio of the helper lipid (usually DOPE¹), the nature of the DNA-component, as well as the composition of the media. Liposomes made of monovalent cationic amphiphiles adhere and fuse during interaction with negatively charged DNA thereby complexing the DNA. The size of the resulting complexes is depending upon charge neutralization and is smallest at a slightly positive net charge. At molar ratios of DOPE, to the cationic component of ≥ 1.5, hexagonal lipid tubules are formed, especially in media containing high salt concentrations, and even in the control lipid mixture, not interacting with any DNA or oligonucleotide. Complexes, made of plasmid-DNA, monovalent cationic amphiphiles, and DOPE at a lower molar ratio, show additionally to the semifused or fused liposomes a new structure, called spaghetti-like structure, representing a bilayer-coated, supercoiled DNA. Single-strand and short oligonucleotides seem not to form such structures during interaction with monovalent cationic liposomes. Neither fusion nor spaghetti formation is observed during interaction of DNA with liposomes made of polyvalent cationic amphiphiles. In general, small complexes consisting of some few semifused liposomes bearing the self-encapsulated nucleic acid and additionally the spaghetti-like structure, free or connected with these complexes, seem to be candidates for the transfectionactive structure rather than large extended H_II¹-lipid arrangements.     

The depth of porphyrin in a membrane and the membrane's physical properties affect the photosensitizing efficiency

Photosensitized biological processes, as applied in photodynamic therapy, are based on light-triggered generation of molecular singlet oxygen by a membrane-residing sensitizer. Most of the sensitizers currently used are hydrophobic or amphiphilic porphyrins and their analogs. The possible activity of the short-lived singlet oxygen is limited to the time it is diffusing in the membrane, before it emerges into the aqueous environment. In this paper we demonstrate the enhancement of the photosensitization process that is obtained by newly synthesized protoporphyrin derivatives, which insert their tetrapyrrole chromophore deeper into the lipid bilayer of liposomes. The insertion was measured by fluorescence quenching by iodide and the photosensitization efficiency was measured with 9,10-dimethylanthracene, a fluorescent chemical target for singlet oxygen. We also show that when the bilayer undergoes a melting phase transition, or when it is fluidized by benzyl alcohol, the sensitization efficiency decreases because of the enhanced diffusion of singlet oxygen. The addition of cholesterol or of dimyristoyl phosphatydilcholine to the bilayer moves the porphyrin deeper into the bilayer; however, the ensuing effect on the sensitization efficiency is different in these two cases. These results could possibly define an additional criterion for the choice and design of hydrophobic, membrane-bound photosensitizers.     

Structural analysis and sheep pituitary receptor binding of GnRH and its complexes with metal ions

Binding of GnRH and its metal complexes to a sheep pituitary receptor have been investigated showing that Cu(II)-GnRH complex is more effectively bound to the receptor than the metal-free ligand, while Ni(II) and Co(II) complexes are less effective than the metal-free GnRH. Earlier studies have explained reasonably well the complex formation with cupric ion, while in this work extensive 1H NMR measurements have been performed for free gonadotropin-releasing hormone (GnRH) and its complexes with Ni(II) in DMSO (dimethyl sulfoxide) solution. This study shows the high order of organization of the metal-free peptide in DMSO solution with two structured 'domains' whose relative orientation is modulated by the mobility of the central glycine. Furthermore, theoretical calculations were performed for the Ni(II)-GnRH complex. The data obtained in this work supports previous studies on the co-ordination of Ni(II) ions with GnRH in aqueous solutions at high pH [J. Inorg. Biochem. 33 (1988) 11] and suggest an experimental procedure to reproduce high pH in DMSO solution. In the Ni(II) complex, the metal ion was found to co-ordinate with four nitrogen atoms inducing a well definite arrangement of aromatic side-chains and a rigid backbone structure.     

The complexation of aqueous metal ions relevant to biological applications: 2. Evaluation of simultaneous equilibria of poorly soluble zinc salts with select amino acids

Abstract

Simultaneous equilibria calculations were completed for seven aqueous zinc-ligand systems: zinc citrate plus either glycine, alanine, or serine, and zinc succinate plus either glycine, alanine, or serine, and zinc oxalate plus glycine. Mixed-ligand complexes were predicted for all but the zinc citrate-glycine system, and the proportion tends to peak around 5 molar equivalents of amino acid. Potential bioavailability of zinc appears to be increased by the inclusion of amino acids in solution, roughly in parallel with the increase in solubility of the zinc salt. Therefore, measurement of the change in solubility caused by addition of amino acids to aqueous solution gives qualitative insight to the potential increase in bioavailability of the metal ion, and mixed-ligand complexes are a significant proportion of the complexes present in solution.     

Direct observation of singlet oxygen production by merocyanine 540 associated with phosphatidylcholine liposomes

The production of singlet molecular oxygen (1O2) by the photosensitizing dye merocyanine 540 (MC540) bound to phosphatidylcholine liposomes has been demonstrated by direct detection of 1O2 luminescence at 1268 nm. 1O2 phosphorescence emission was enhanced in deuterated buffer and upon saturation of the sample with oxygen and could be quenched by the addition of sodium azide to the external medium. No 1O2 luminescence was detected in nitrogen-saturated samples, in the absence of dye, or with MC540 in aqueous solution. Photobleaching of liposome-bound MC540 was also observed to be dependent on oxygen concentration. These studies are consistent with 1O2 intermediacy in the mechanism of MC540-mediated photosensitization.     

Metal binding and antibacterial activity of ciprofloxacin complexes

Four novel cobalt(II), copper(II), nickel(II) and zinc(II) complexes of the fluoroquinolone antibiotic ciprofloxacin have been prepared. The compounds were characterized by IR, UV-Visible, molar conductivity and elemental analyses. In all of the complexes, the drug ligand, ciprofloxacin (CFL) was coordinated through two carbonyl oxygen atoms. Octahedral and square-planar geometries have been proposed for the cobalt(II), nickel(II) and zinc(II), and copper(II) complexes, respectively. In vitro tests of susceptibility to these metal complexes showed stronger activity than that of ciprofloxacin against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Bacillus dysenteriae.     

Optical absorption and fluorescence spectroscopy studies of ground state melanin-cationic porphyrins complexes.

Optical absorption and fluorescence spectroscopies were employed in the study of the interaction between synthetic L-dopa (dihydroxyphenylalanine) melanin and the cationic porphyrins tetrakis(4-N-methylpyridyl) porphyrin (TMPyP), tetrakis(4-N-benzylpyridyl)porphyrin (TBzPyP), zinc tetrakis(4-N-methylpyridyl)porphyrin (ZnTMPyP) and zinc tetrakis (4-N-benzylpyridyl)porphyrin (ZnTBzPyP). Optical absorption and fluorescence properties of the porphyrins were dependent on the symmetry of the central ring. No evidence was found for dimerization of the porphyrins in phosphate buffer, pH 7, in the concentration range between 4 x 10(-8) to 5 x 10(-5) M. Addition of L-dopa melanin red shifted the optical absorption spectra of porphyrins, concomitant to broadening and reduction in intensity of the bands. L-Dopa melanin also strongly quenched the fluorescence of the porphyrins. Time resolution of the fluorescence decay of porphyrins showed at least two lifetimes that were only slightly modified in the presence of melanin. The interaction between melanin and porphyrin resulted in the formation of non-fluorescent ground state complexes. It was found that there are two different classes of binding sites in melanin for complexation with cationic porphyrins and the values of dissociation constants are of the order of 10(-8) M. These values and the number of binding sites are dependent on the nature of the porphyrins. It was shown that the binding has electrostatic origin, but it is also affected by metal coordination and hydrophobic interaction.     

Gene transfer mediated by YKS-220 cationic particles: convenient and efficient gene delivery reagent.

A monocationic lipid, YKS-220, with a symmetrical and biodegradable structure can be used as an effective gene transfer vector in a cationic particle form (not a cationic liposome form), and is obtained by diluting an ethanol solution of YKS-220 and DOPE (1:5, molar ratio) with an aqueous medium. This preparation method is more convenient than that for cationic liposomes. YKS-220 cationic particles showed a heterogeneous large mean diameter of 4.4 microm. An obvious size change was not observed when plasmid DNA was added. The transfection activity of YKS-220 cationic particles was comparable to those of YKS-220 liposomes and DOSPA liposomes (LipofectAMINE), and even higher than that of DOGS (TRNSFECTAM). Interestingly, the YKS-220 cationic particle/DNA complexes were resistant to the neutralizing effect of serum. All of these findings indicate that YKS-220 cationic particles are a convenient and efficient gene delivery reagent.