[Show abstract][Hide abstract] ABSTRACT: High pressure in combination with optical spectroscopy was used to gain insights into the interactions between Mg(2+), Zn(2+), and Ni(2+) ions and macrocyclic ligands of porphyrinoid type. In parallel, the central metal ion-macrocycle bonding was investigated using theoretical approaches. The symmetry properties of the orbitals participating in this bonding were analyzed, and pigment geometries and pressure/ligation effects were computed within DFT. Bacteriopheophytin a was applied as both a model chelator and a highly specific spectroscopic probe. The analysis of solvent and pressure effects on the spectral properties of the model Mg(2+), Zn(2+), and Ni(2+) complexes with bacteriopheophytin a shows that various chemical bonds are formed in the central pocket, depending on the valence configuration of the central metal ion. In addition, the character of this bonding depends on symmetry of the macrocyclic system. Since in most cases it is not coordinative bonding, these results challenge the conventional view of metal ion bonding in such complexes. In (labile) complexes with the main group metals, the metal ion-macrocycle interaction is mostly electrostatic. Significantly, water molecules are not preferred as a second axial ligand in such complexes, mainly due to the entropic constraints. The metal ions with a closed d shell may form (stable) complexes with the macrocycle via classical coordination bonds, engaging their p and s orbitals. Transition metals, due to the unfilled d shell, do form much more stable complexes, because of strong bonding via both coordination and covalent interactions. These conclusions are confirmed by DFT computations and theoretical considerations, which altogether provide the basis to propose a consistent and general mechanism of how the central metal ion and its interactions with the core nitrogens govern the physicochemical properties of metalloporphyrinoids.
[Show abstract][Hide abstract] ABSTRACT: Blood flow and pO2 changes after vascular-targeted PDT (V-PDT) or cellular-targeted PDT (C-PDT) using 5,10,15,20-tetrakis(2,6-difluoro-3-N-methylsulfamoylphenyl) bacteriochlorin (F2BMet) as photosensitizer, were investigated in DBA/2 mice with S91 Cloudman mouse melanoma, and correlated with long-term tumor responses. F2BMet generates both singlet oxygen and hydroxyl radicals under NIR irradiation, which consume oxygen. Partial oxygen pressure was lowered in PDT-treated tumors and this was ascribed both to oxygen consumption during PDT and to fluctuations in oxygen transport after PDT. Similarly, microcirculatory blood flow changes as a result of the disruption of blood vessels due to the treatment. A novel non-invasive approach combining Electron Paramagnetic Resonance oximetry and Laser Doppler blood perfusion measurements allowed longitudinal monitoring of hypoxia and vascular function changes in the same animals, after PDT. C-PDT induced parallel changes in tumor pO2 and blood flow, i.e., an initial decrease immediately after treatment, followed by a slow increase. In contrast, V-PDT led to a strong and persistent depletion of pO2, although the microcirculatory blood flow increased. Strong hypoxia after V-PDT led to a slight increase of VEGF level 24h after treatment. C-PDT caused a ca. 5-day delay in tumor growth, while V-PDT was much more efficient and led to tumor growth inhibition in 90% of animals. The tumors of 44% of mice treated with V-PDT regressed completely and did not reappear for over one year. In conclusion, mild and transient hypoxia after C-PDT led to intense pO2 compensatory effects and modest tumor inhibition but strong and persistent local hypoxia after V-PDT caused tumor growth inhibition.
Advances in Free Radical Biology & Medicine 05/2014;
[Show abstract][Hide abstract] ABSTRACT: Progress in the photodynamic therapy (PDT) of cancer should benefit from a rationale to predict the most efficient of a series of photosensitizers that strongly absorb light in the phototherapeutic window (650-800 nm) and efficiently generate reactive oxygen species (ROS=singlet oxygen and oxygen-centered radicals). We show that the ratios between the triplet photosensitizer-O2 interaction rate constant (kD ) and the photosensitizer decomposition rate constant (kd ), kD /kd , determine the relative photodynamic activities of photosensitizers against various cancer cells. The same efficacy trend is observed in vivo with DBA/2 mice bearing S91 melanoma tumors. The PDT efficacy intimately depends on the dynamics of photosensitizer-oxygen interactions: charge transfer to molecular oxygen with generation of both singlet oxygen and superoxide ion (high kD ) must be tempered by photostability (low kd ). These properties depend on the oxidation potential of the photosensitizer and are suitably combined in a new fluorinated sulfonamide bacteriochlorin, motivated by the rationale.
Chemistry - A European Journal 03/2014; · 5.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The previously published method allowing the separation of non-ferric (iron-free) and ferric (iron-saturated) forms of human serum transferrin via capillary electrophoresis has been further developed. Using a surface response methodology and a three-factorial Doehlert design we have established a new optimized running buffer composition: 50mM Tris-HCl, pH 8.5, 22.5% (v/v) methanol, 17.5mM SDS. As a result, two previously unobserved monoferric forms of protein have been separated and identified, moreover, the loss of ferric ions from transferrin during electrophoretic separation has been considerably reduced by methanol, and the method selectivity has been yet increased resulting in a total separation of proteins exerting only subtle or none difference in mass-to-charge ratio. The new method has allowed us to monitor the gradual iron saturation of transferrin by mixing the iron-free form of protein with the buffers with different concentrations of ferric ions. It revealed continuously changing contribution of monoferric forms, characterized by different affinities of two existing iron binding sites on N- and C-lobes of protein, respectively. Afterwards, the similar experiment has been conducted on-line, i.e. inside the capillary, comparing the effectiveness of two possible modes of the reactant zones mixing: diffusion mediated and electrophoretically mediated ones. Finally, the total time of separation has been decreased down to 4min, taking the advantage from a short-end injection strategy and maintaining excellent selectivity.
Journal of Chromatography A 03/2014; · 4.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The interference of NAMI-A ([ImH][RuCl4(Im)(DMSO)], Im – imidazole, DMSO – dimethyl sulfoxide) with the metabolism of nitric oxide (NO) has been proposed as one of the possible pathways of the antimetastatic activity of this complex. With regard to this observation we present herein detailed spectrophotometric studies on interaction of the NAMI-A complex with NO. The reactivity of NAMI-A toward NO has been studied in aqueous solution under physiological-like conditions (pH = 7.4, [NaCl] = 0.1 M, T = 37 °C). The ability of NAMI-A as well as its hydrolytic products to bind NO has been confirmed spectrophotometrically and separation of reaction products was performed with application of the HPLC technique. The relatively slow NO binding requires opening up a coordination site in the parent NAMI-A complex via simultaneously occurring hydrolysis. The studies in the presence of albumin showed that NO can coordinate to NAMI-A–albumin adducts. The capability of nitrosyl derivatives (Ru2+–NO+) to undergo reduction of the NO+ moiety in the presence of ascorbic acid, glutathione and dithionite has been studied with application of the NO sensor. The obtained results showed that under selected conditions, nitrosyl complexes cannot liberate nitric oxide via one electron reduction using applied reductants. This is due to the relatively low reduction potential of the NO+ group bound to Ru(II) (−0.69 V), determined in electrochemical studies.
New Journal of Chemistry 02/2014; · 3.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A novel long-lifetime highly luminescent ruthenium polypyridyl complex containing 2-nitroimidazole moiety [Ru(dip)2(bpy-2-nitroIm)]Cl2 (dip=4,7-diphenyl-1,10-phenanthroline, bpy-2-nitroIm=4-[3-(2-nitro-1H-imidazol-1-yl)propyl]-2,2'-bipyridine) has been designed cancer treatment and imaging. The luminescence properties of the synthesized compound strongly depend on the oxygen concentration. Under oxygen-free conditions quantum yield of luminescence and the average lifetime of emission were found to be 0.034 and 1.9μs, respectively, which is ca. three times higher in comparison to values obtained in air-equilibrated solution. The binding properties of the investigated ruthenium complex to human serum albumin have been studied and the apparent binding constant for the formation of the protein-ruthenium adduct was determined to be 1.1×10(5)M(-1). The quantum yield and the average lifetime of emission are greatly enhanced upon binding of ruthenium compound to the protein. The DNA binding studies revealed two distinguished binding modes which lead to a decrease in luminescence intensity of ruthenium complex up to 60% for [DNA]/[Ru]<2, and enhancement of emission for [DNA]/[Ru]>80. Preliminary biological studies confirmed fast and efficient accumulation of the ruthenium complex inside cells. Furthermore, the ruthenium complex was found to be relatively cytotoxic with LD50 of 12 and 13μM for A549 and CT26 cell lines, respectively, under normoxic conditions. The retention and cellular uptake of ruthenium complex is enhanced under hypoxic conditions and its LD50 decreases to 8μM for A549 cell line.
Journal of inorganic biochemistry 02/2014; 134C:83-91. · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: High-valent iron-oxo species have been invoked as reactive intermediates in catalytic cycles of heme and nonheme enzymes. The studies presented herein are devoted to the formation of compound II model complexes, with the application of a water soluble (TMPS)Fe(III) (OH) porphyrin ([meso-tetrakis(2,4,6-trimethyl-3-sulfonatophenyl)porphinato]iron(III) hydroxide) and hydrogen peroxide as oxidant, and their reactivity toward selected organic substrates. The kinetics of the reaction of H2 O2 with (TMPS)Fe(III) (OH) was studied as a function of temperature and pressure. The negative values of the activation entropy and activation volume for the formation of (TMPS)Fe(IV) O(OH) point to the overall associative nature of the process. A pH-dependence study on the formation of (TMPS)Fe(IV) O(OH) revealed a very high reactivity of OOH(-) toward (TMPS)Fe(III) (OH) in comparison to H2 O2 . The influence of N-methylimidazole (N-MeIm) ligation on both the formation of iron(IV)-oxo species and their oxidising properties in the reactions with 4-methoxybenzyl alcohol or 4-methoxybenzaldehyde, was investigated in detail. Combined experimental and theoretical studies revealed that among the studied complexes, (TMPS)Fe(III) (H2 O)(N-MeIm) is highly reactive toward H2 O2 to form the iron(IV)-oxo species, (TMPS)Fe(IV) O(N-MeIm). The latter species can also be formed in the reaction of (TMPS)Fe(III) (N-MeIm)2 with H2 O2 or in the direct reaction of (TMPS)Fe(IV) O(OH) with N-MeIm. Interestingly, the kinetic studies involving substrate oxidation by (TMPS)Fe(IV) O(OH) and (TMPS)Fe(IV) O(N-MeIm) do not display a pronounced effect of the N-MeIm axial ligand on the reactivity of the compound II mimic in comparison to the OH(-) substituted analogue. Similarly, DFT computations revealed that the presence of an axial ligand (OH(-) or N-MeIm) in the trans position to the oxo group in the iron(IV)-oxo species does not significantly affect the activation barriers calculated for CH dehydrogenation of the selected organic substrates.
Chemistry - A European Journal 01/2014; · 5.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The presented results cover a comparative mechanistic study on the reactivity of compound (Cpd) I and II mimics of a water-soluble iron(III) porphyrin, [meso-tetrakis(2,4,6-trimethyl-3-sulfonatophenyl)porphinato]iron(III), Fe(III)(TMPS). The acidity of the aqueous medium strongly controls the chemical nature and stability of the high-valent iron(IV) oxo species. Reactivity studies were performed at pH 5 and 10, where the Cpd I and II mimics are stabilized as the sole oxidizing species, respectively. The contributions of ΔH(⧧) and ΔS(⧧) to the free energy of activation (ΔG(⧧)) for the oxidation of 4-methoxybenzaldehyde (4-MB-ald), 4-methoxybenzyl alcohol (4-MB-alc), and 1-phenylethanol (1-PhEtOH) by the Cpd I and II mimics were determined. The relatively large contribution of the ΔH(⧧) term in comparison to the -TΔS(⧧) term to ΔG(⧧) for reactions involving the Cpd II mimic indicates that the oxidation of selected substrates by this oxidizing species is clearly an enthalpy-controlled process. In contrast, different results were found for reactions with application of the Cpd I mimic. Depending on the nature of the substrate, the reaction at room temperature can be entropy-controlled, as found for the oxidation of 4-MB-alc, or enthalpy-controlled, as found for 1-PhEtOH. Importantly, for the first time, activation volumes (ΔV(⧧)) for the oxidation of selected substrates by both reactive intermediates could be determined. Positive values of ΔV(⧧) were found for reactions with the Cpd II mimic and slightly negative ones for reactions with the Cpd II mimic. The results are discussed in the context of the oxidation mechanism conducted by the Cpd I and II mimics.
[Show abstract][Hide abstract] ABSTRACT: A capillary electrophoresis-based method for the cost-effective and high efficient separation of iron-free and iron-saturated forms of two members of transferrin family: transferrin and lactoferrin has been developed. The proposed qualitative method relying on the SDS application allowed us to separate iron-free and iron-saturated forms of these proteins, as well as human serum albumin, used as an internal standard. Owing to the distinct migration times under established conditions, the combination of transferrin and lactoferrin assays within a single analytical procedure was feasible. The performance of the method using a fused-silica capillary has been compared with the results obtained using the same method but performed with the use of a neutral capillary of the same dimensions. Neutral capillary has been used as an alternative, since the comparable resolution has been achieved with a concomitant reduction of the electroosmotic flow. Despite of this fact, the migration of analytes occurred with similar velocity but in opposite order, due to the reverse polarity application. A quantitative method employing fused-silica capillary for iron saturation study has been also developed, to evaluate the iron saturation in commercial preparations of lactoferrin.
Journal of Chromatography A 10/2013; · 4.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Our previous study has shown a prolonged retention and accumulation of Zn-pheophorbide a, a water-soluble derivative of chlorophyll a, in tumor tissue (Szczygiel et al. ). This prompted us to further evaluate the phototherapeutic potential of this photosensitizer of excellent physicochemical properties.
Cellular uptake of Zn-pheophorbide, its localization in cells, cytotoxicity, phototoxicity and cell death mechanisms were studied in human adenocarcinoma cell lines: A549, MCF-7 and LoVo. The PDT efficacy was tested against A549 tumors growing in nude mice.
Zn-pheophorbide a even at very low concentrations (∼1×10(-6)M) and at low light doses (5J/cm(2)) causes a strong photodynamic effect, leading to 100% cell mortality. Confocal microscopy showed that in contrast to most derivatives of chlorophyll, Zn-pheophorbide a does not localize to mitochondria. The photodynamic effects and the cell death mechanisms of Zn-pheophorbide a, its Mg analog (chlorophyllide a) and Photofrin were compared on the A549 cells. Zn-pheophorbide a showed the strongest photodynamic effect, at low dose killing all A549 cells via apoptosis and necrosis. The very high anti-cancer potential of Zn-pheophorbide was confirmed in a photodynamic treatment of the A549 tumors. They either regressed or were markedly inhibited for up to 4 months after the treatment, resulting, on average, in a 5-fold decrease in tumor volume.
These results show that Zn-pheophorbide a is a very promising low-cost, synthetically easily accessible, second generation photosensitizer against human cancer.
Photodiagnosis and photodynamic therapy 09/2013; 10(3):266-77.
[Show abstract][Hide abstract] ABSTRACT: Lactoferrin is considered as a part of the innate immune system that plays a crucial role in preventing bacterial growth, mostly via an iron sequestration mechanism. Recent data show that bovine lactoferrin prevents late-onset sepsis in preterm very low birth weight neonates by serving as an iron chelator for some bacterial strains; thus, it is very important to control the iron saturation level during diet supplementation. An accurate estimation of lactoferrin iron saturation is essential not only because of its clinical applications but also for a wide range of biochemical experiments. A comprehensive method for the quantification of iron saturation in lactoferrin preparations was developed to obtain a calibration curve enabling the determination of iron saturation levels relying exclusively on the defined ratio of absorbances at 280 and 466 nm (A 280/466). To achieve this goal, selected techniques such as spectrophotometry, ELISA, and ICP-MS were combined. The ability to obtain samples of lactoferrin with determination of its iron content in a simple and fast way has been proven to be very useful. Furthermore, a similar approach could easily be implemented to facilitate the determination of iron saturation level for other metalloproteins in which metal binding results in the appearance of a distinct band in the visible part of the spectrum.
Analytical and Bioanalytical Chemistry 04/2013; · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Titanium dioxide photosensitization may be achieved in various ways, involving surface modification with appropriate species. The photosensitization process requires a visible light-induced electron or hole injection into conduction or valence band, respectively. Efficiency of this process depends on electronic interaction between the photosensitizer moiety (surface complex) and TiO2 particle. At least two types of the charge injection mechanisms may be distinguished—in the first, charge is transferred from the excited state of the sensitizer molecule to the conduction or valence band while the second mechanism involves a direct molecule-to-band charge transfer (MBCT). The MBCT process can be realized by surface titanium(IV) complexes with various organic and sometimes inorganic ligands. Catechol, phthalic acid or salicylic acid derivatives, as well as cyanometallate anions, upon chemisorption at TiO2 surface constitute an especially interesting group of ligands to yield various titanium(IV) surface complexes. Geometry of these complexes, electronic structures and possibility of their use as photosensitizers of TiO2 are discussed on the basis of experimental data and quantum-chemical modeling. Also prospective applications of photoinduced electron transfer and photocatalytic activity of such systems are presented.
[Show abstract][Hide abstract] ABSTRACT: A photoinduced electron injection from the excited photosensitizer to the conduction band is usually a key step of photosensitization of wide bandgap semiconductors. The significance of this process has been recognized especially in the case of titanium dioxide, when applied as a visible light active photocatalyst or as an anode material in dye-sensitized solar cells. An alternative mechanism of the semiconductor sensitization may involve a photoinduced hole injection into the valence band. This paper reviews semiconductor systems (NiO, copper compounds, doped diamond, TiO2) photosensitized by coordination compounds capable of a photoinduced hole injection into the valence band. The applicability of such systems for photocatalysis and photovoltaics is presented.
[Show abstract][Hide abstract] ABSTRACT: In the Ni-substituted chlorophylls, an ultrafast (<60fs) deactivation channel is created, which is not present in Ni-porphyrins. This observation prompted us to investigate in detail the mechanism of excitation-to-heat conversion in Ni-substituted chlorophylls, experimentally, using time-resolved laser-induced optoacoustic spectroscopy, and theoretically, using group theory approach. The Ni-substituted chlorophylls show exceptional photostability and the optoacoustic measurements confirm the prompt and very efficient (100%) excitation-into-heat conversion in these complexes. Considering their excellent spectral properties and the loss-free excitation-into-heat conversion they are likely to become a new class of versatile photocalorimetric references. The curious features of the Ni-substituted chlorophylls originate from the symmetry of a ligand field created in the central cavity. The central NNi(2+) bonds, formed via the donation of two electrons from each of the sp(2) orbitals of two central nitrogens to an empty [Formula: see text] hybrid centered on Ni(2+), have a considerable covalent character. The extreme rate of excited state relaxation is then not due to a ladder of the metal centered d-states, often invoked in metalloporphyrins, but seems to result from a peculiar topology of the potential energy surface (a saddle-shaped crossing) due to the covalent character of the NNi(2+) bonds. This is confirmed by a strong 0→0 character of electronic transitions in these complexes indicating a similarity of their equilibrium geometries in the ground (S(0)) and the excited states (both Q(X) and Q(Y)). The excitation energy is very efficiently converted into molecular vibrations and dissipated as heat, involving the central Ni(2+). These Ni-substituted pigments pose a fine exemplification of symmetry control over properties of excited states of transition metal complexes.
Biochimica et Biophysica Acta 09/2012; 1827(1):30-37. · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: NAMI-A i.e. (ImH)[trans-RuCl(4)(DMSO)(Im)] (where Im is imidazole) is a ruthenium(III) complex with promising antimetastatic activity, which has been classified for II phase clinical trial. In this study, its binding properties toward apo-transferrin (apo-Tf) with regard to its hydrolytic and redox behavior are systematically investigated by the use of fluorescence spectroscopy. The reaction of NAMI-A and its reduced form with apo-Tf is proceeded by formation of aqua derivatives and the presence of at least one labile aqua ligand is sufficient to form adducts. It is found that presence of bicarbonate is not necessary for interaction of studied ruthenium complexes with apo-Tf. The calculated association constants for both NAMI-A and its reduced form are very similar with the values of 1.28×10(4)M(-1) and 1.36×10(4)M(-1) at 37°C, respectively however, the reduced derivatives reach the equilibrium ca. 8-10 times slower. The percentage of ruthenium content in protein fractions separated from protein-unbounded ruthenium by using FPLC (fast protein liquid chromatography) method is rather high and depends on redox state of the complex, for most samples is found higher for reduced species.
Journal of inorganic biochemistry 07/2012; 116C:11-18. · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sulfonamides of halogenated bacteriochlorins bearing Cl or F substituents in the ortho positions of the phenyl rings have adequate properties for photodynamic therapy, including strong absorption in the near-infrared (λ(max) ≈ 750 nm, ε ≈ 10(5) M(-1) cm(-1)), controlled photodecomposition, large cellular uptake, intracellular localization in the endoplasmic reticulum, low cytotoxicity, and high phototoxicity against A549 and S91 cells. The roles of type I and type II photochemical processes are assessed by singlet oxygen luminescence and intracellular hydroxyl radical detection. Phototoxicity of halogenated sulfonamide bacteriochlorins does not correlate with singlet oxygen quantum yields and must be mediated both by electron transfer (superoxide ion, hydroxyl radicals) and by energy transfer (singlet oxygen). The photodynamic efficacy is enhanced when cellular death is induced by both singlet oxygen and hydroxyl radicals.
Free Radical Biology and Medicine 04/2012; 52(7):1188-200. · 5.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chlorins have intense red absorptions and high tumor affinities that make them interesting candidates for photodynamic therapy (PDT) of cancer. This paper reports cytotoxicity, phototoxicity, in vitro cellular uptake, and in vivo biodistribution and PDT efficacy of a synthetic chlorin derivative (TCPCSO₃H) towards Cloudman melanoma cells (S91). No cytotoxic effects were observed in vitro at concentrations up to 20 μm, and no toxicity was observed in vivo in DBA mice with doses up to 2 mg kg⁻¹. Pharmacokinetics and biodistribution of TCPCSO₃H were evaluated in vivo in DBA mice bearing S91 tumors. TCPCSO₃H demonstrated preferential accumulation in S91 mouse melanoma, with tumor-to-normal tissue ratios of 5 and 11 for muscle and skin, respectively, 24 h after intravenous injection of 2 mg kg⁻¹. Photodynamic therapy performed under these conditions with 70 mW cm⁻² diode laser irradiation at 655 nm for 25 min (total light dose=105 J cm⁻²) resulted in scab formation, followed by temporary or permanent (>60 days) tumor remission. According to the Kaplan-Meier analysis, the median survival time of the control group was 9 days, whereas that of the treated group was 38 days.