Boris M. Dzhagarov

Toward understanding the high PDT efficacy of chlorin e6-polyvinylpyrrolidone formulations: Photophysical and molecular aspects of photosensitizer-polymer interaction in vitro

It is recognized that chlorin e6-polyvinylpyrrolidone (Ce6-PVP) formulations are characterized by a high efficacy in photodynamic therapy of malignant tumors. Currently, a commercially available formulation of this type is Photolon (Fotolon) with Ce6:PVP=1:1 (w/w) and the weight-average molecular weight of PVP is 1.2x10(4). To gain a better understanding of the role played by PVP in Ce6-PVP formulations, we carry out experiments on IR and UV-VIS absorption, steady-state and time-resolved fluorescence, time-resolved triplet-triplet absorption, octanol-water partitioning, and solubility of chlorin e6 in buffer solutions at pH 6.3, 7.4, and 8.5 in presence of PVP with Ce6:PVP ratios ranging from 1:0 to 1:1000 (w/w) for PVP samples with weight-average molecular weights of 8x10(3), 1.2x10(4), and 4.2x10(4). We show that Ce6 interacts with PVP by forming molecular complexes via hydrophobic interactions and determine the Ce6-PVP binding constant, as well as the mean number of PVP monomers per binding site. We find that complexation of Ce6 with PVP prevents Ce6 aggregation in aqueous media and leads to an enhancement of Ce6 fluorescence quantum yield, while keeping the quantum yield of the intersystem crossing essentially unchanged. Possible scenarios of how the presence of PVP can favorably affect the PDT efficacy of chlorin e6 in Ce6-PVP formulations are discussed.

Photophysics of cationic 5,10,15,20-tetrakis-(4-N-methylpyridyl) porphyrin bound to DNA, [poly(dA-dT)]2 and [poly(dG-dC)]2: on a possible charge transfer process between guanine and porphyrin in its excited singlet state

Abstract The photophysical parameters (fluorescence quantum yield and lifetime, triplet state formation and lifetime) of a cationic prophyrin, i.e. 5,10,15,20-tetrakis(4-N-methylpyridyl) porphyrin (H2(TMpy-P4)), were determined directly in phosphate buffer solution in the native state and in complexes with calf thymus DNA, [poly(dA-dT)]2 and [poly(dG-dC)]2. In [poly(dG-dC)]2-porphyrin complexes (mostly intercalative), interaction induced an efficient fluorescence quenching and decrease in the triplet state quantum yield. No such effects were observed in [poly(dA-dT)]2-pophyrin complexes (mostly non-intercalative). The case of DNA was intermediate between the other two. On the basis of the redox properties of the various nucleic bases, it was shown that these observations can be interpreted in terms of fast reversible intermolecular electron transfer from the guanine moiety to the porphyrin in the excited state, when the porphyrin is intercalated with an appropriate geometry within the DNA duplex. These observations and interpretations support a type I (electron transfer) mechanism as the primary event leading to the photodynamic activity of cationic free base porphyrins used as photosensitizers in the process of photoinduced DNA strand breaks.

Time-resolved fluorescence reveals two binding sites of 1,8-ANS in intact human oxyhemoglobin

Time-resolved fluorescence of 1,8-anilinonaphthalene sulfonate (1,8-ANS) fluorescent probe bound to intact human oxyhemoglobin (HbO2) is investigated. Fluorescence emission spectra of 1,8-ANS in a potassium buffer solution (pH 7.4) of HbO2 undergo a substantial blue shift during first 6 ns after pulsed optical excitation at 337.1 nm. Nonexponential fluorescence kinetics of 1,8-ANS in the HbO2 solution are studied by the decay time distribution and conventional multiexponential analyses for a set of emission wavelength range of lambdaem = 455-600 nm. These fluorescence decays contain components with mean decay times of <0.5 ns, 3.1-5.5 ns, and 12.4-15.1 ns with spectrally-dependent relative contributions. The shortest decay component is assigned to free 1,8-ANS molecules in the bulk buffer environment, whereas the two longer decay components are assigned to two types of binding sites of 1,8-ANS in the HbO2 molecule presumably differing by polarity and accessibility to water molecules. The results represent the first experimental evidence of heterogeneous binding of 1,8-ANS to intact human oxyhemoglobin.

Binding of the cationic 5-coordinate Zn(II)-5,10,15,20-tetrakis(4-N-methylpyridyl)porphyrin to DNA and model polynucleotides: Ionic-strength dependent intercalation in [poly(dG-dC)]2

Interactions of water-soluble cationic porphyrins and their metal- derivatives with DNA attract interest of investigators as for fundamental studies (for example, the use of photoexcited porphyrins as probes of DNA local structure and dynamics [1]) and for applied biomedical investigations as well (for example, the photodestruction of viral nucleic acids in blood and blood components by using visible light [2]). In spite of fairly well elaborated formal classification of the types of porphyrin-DNA interactions, the relative selectivity of GC vs AT binding, the nature of the charge interactions between the porphyrins and the polymers, the exact geometries of the porphyrin-polymer complexes and at last the porphyrin-induced DNA structural distortions are not yet fully understood and are subjects to very active discussions [3].

Photosensitized Singlet Oxygen Luminescence from the Protein Matrix of Zn-Substituted Myoglobin

A nanosecond laser near-infrared spectrometer was used to study singlet oxygen ((1)O2) emission in a protein matrix. Myoglobin in which the intact heme is substituted by Zn-protoporphyrin IX (ZnPP) was employed. Every collision of ground state molecular oxygen with ZnPP in the excited triplet state results in (1)O2 generation within the protein matrix. The quantum yield of (1)O2 generation was found to be equal to 0.9 ± 0.1. On the average, six from every 10 (1)O2 molecules succeed in escaping from the protein matrix into the solvent. A kinetic model for (1)O2 generation within the protein matrix and for a subsequent (1)O2 deactivation was introduced and discussed. Rate constants for radiative and nonradiative (1)O2 deactivation within the protein were determined. The first-order radiative rate constant for (1)O2 deactivation within the protein was found to be 8.1 ± 1.3 times larger than the one in aqueous solutions, indicating the strong influence of the protein matrix on the radiative (1)O2 deactivation. Collisions of singlet oxygen with each protein amino acid and ZnPP were assumed to contribute independently to the observed radiative as well as nonradiative rate constants.

Photophysical properties and singlet oxygen generation efficiencies of water-soluble fullerene nanoparticles.

As various fullerene derivatives have been developed, it is necessary to explore their photophysical properties for potential use in photoelectronics and medicine. Here, we address the photophysical properties of newly synthesized water‐soluble fullerene‐based nanoparticles and polyhydroxylated fullerene as a representative water‐soluble fullerene derivative. They show broad emission band arising from a wide‐range of excitation energies. It is attributed to the optical transitions from disorder‐induced states, which decay in the nanosecond time range. We determine the kinetic properties of the singlet oxygen (1O2) luminescence generated by the fullerene nanoparticles and polyhydroxylated fullerene to consider the potential as photodynamic agents. Triplet state decay of the nanoparticles was longer than 1O2 lifetime in water. Singlet oxygen quantum yield of a series of the fullerene nanoparticles is comparably higher ranging from 0.15 to 0.2 than that of polyhydroxylated fullerene, which is about 0.06.

The kinetics of molecular oxygen migration in the isolated α chains of human hemoglobin as revealed by molecular dynamics simulations and laser kinetic spectroscopy

Bimolecular and germinate molecular oxygen (O(2)) rebinding to isolated α chains of human adult hemoglobin in solutions is analyzed. Multiple extended molecular dynamics (MD) simulations of the O(2) migration within the protein after dissociation are described. Computational modeling is exploited to identify hydrophobic pockets within the αchains and internal O(2) migration pathways associated with the experimentally observed ligand rebinding kinetics. To initiate dissociation, trajectories of the liganded protein are interrupted, the iron-dioxygen bond is broken, and the parameters of the iron-nitrogen bonds are simultaneously altered to produce a deoxyheme conformation. MD simulations provide 140 essentially independent trajectories (up to 25-ns long) of the O(2) migration in the protein. The time dependence of cavities occupancy, obtained by the MD simulations, and the kinetics of O(2) rebinding, measured by flash-photolysis techniques, allow us to obtain the kinetics of the entire O(2) migration process within the nanosecond time range and construct an explicit kinetic model of the O(2) migration and rebinding process. The amino acids that have the most pronounced effect on the ligand migration within the α chain matrix are predicted.

Dynamics and efficiency of the photosensitized singlet oxygen formation by chlorin e6: The effects of the solution pH and polyvinylpyrrolidone

The effects of the solution pH and polyvinylpyrrolidone on the dynamics and efficiency of the formation of singlet oxygen 1O2 in buffer media (pH 6.3–8.5) photosensitized by chlorin e6 are studied. It is demonstrated that the quantum yield of the 1O2 formation photosensitized by chlorin e6 decreases with decreasing solution pH due to the aggregation of photosensitizer molecules. Polyvinylpyrrolidone facilitates the disaggregation of chlorin e6, thus increasing its photosensitizing ability. For a complex of chlorin e6 with this polymer, the luminescence kinetics of singlet oxygen is inverted, which should be taken into account in the determination of the lifetime of 1O2 in real biological systems.

Does photodissociation of molecular oxygen from myoglobin and hemoglobin yield singlet oxygen

Time-resolved luminescence measurements in the near-infrared region indicate that photodissociation of molecular oxygen from myoglobin and hemoglobin does not produce detectable quantities of singlet oxygen. A simple and highly sensitive method of luminescence quantification is developed and used to determine the upper limit for the quantum yield of singlet oxygen production. The proposed method was preliminarily evaluated using model data sets and confirmed with experimental data for aqueous solutions of 5,10,15,20-tetrakis(4-N-methylpyridyl) porphyrin. A general procedure for error estimation is suggested. The method is shown to provide a determination of the integral luminescence intensity in a wide range of values even for kinetics with extremely low signal-to-noise ratio. The present experimental data do not deny the possibility of singlet oxygen generation during the photodissociation of molecular oxygen from myoglobin and hemoglobin. However, the photodissociation is not efficient to yield singlet oxygen escaped from the proteins into the surrounding medium. The upper limits for the quantum yields of singlet oxygen production in the surrounding medium after the photodissociation for oxyhemoglobin and oxymyoglobin do not exceed 3.4×10(-3) and 2.3×10(-3), respectively. On the average, no more than one molecule of singlet oxygen from every hundred photodissociated oxygen molecules can succeed in escaping from the protein matrix.

Mutual effects of proton and sodium chloride on oxygenation of liganded human hemoglobin.

The different effects of pH and NaCl on individual O2‐binding properties of α and β subunits within liganded tetramer and dimer of human hemoglobin (HbA) were examined in a number of laser time‐resolved spectroscopic measurements. A previously proposed approach [Dzhagarov BM & Lepeshkevich SV (2004) Chem Phys Lett390, 59–64] was used to determine the extent of subunit dissociation rate constant difference and subunit affinity difference from a single flash photolysis experiment. To investigate the effect of NaCl concentration on the association and dissociation rate constants we carried out a series of experiments at four different concentrations (0.1, 0.5, 1.0 and 2.0 m NaCl) over the pH range of the alkaline Bohr effect. As the data suggest, the individual properties of the α and β subunits within the completely liganded tetrameric hemoglobin did not depend on pH under salt‐free conditions. However, different effects NaCl on the individual kinetic properties of the α and β subunits were revealed. Regulation of the O2‐binding properties of the α and β subunits within the liganded tetramer is proposed to be attained in two quite different ways.