Correlation between the Efficiency of Singlet Oxygen Generation and the Luminescence Parameters of Photosensitizers

Optics and Spectroscopy - The quantum yield of interconversion to triplet states for free-base 2,3,7,8,12,13,17,18-octaethylporphyrin and 5,10,15,20-tetraphenylporphyrin, as well as of...     

Regularities of Bonding of Chlorin e 6 to the Oligomeric Enzyme Lactate Dehydrogenase

Using differential-spectrophotometry, spectral-luminescence, and polarization methods, we have investigated regularities of complexing of a promising photodynamic sensitizer — chlorin e ₆ — with a key glycolytic enzyme — lactate dehydrogenase (LDH). The parameters of the dye–enzyme complex have been estimated by the difference between the spectral characteristics of the free dye and the dye bonded to the enzyme. It is shown that the tetrameric LDH molecule forms an equilibrium complex with four chlorin molecules and the sensitizer is bonded independently to each subunit entering into the composition of the tetramer. It has been established that the spectral characteristics of chlorin bonded to LDH are sensitive to the structure transformations arising in the active center of the enzyme as a result of the formation of an unproductive enzyme–coenzyme–substrate complex, which allows the conclusion that the dye is localized in the neighborhood of the active center of LDH.     

Comparative Effect of Low-intensity Laser Radiation in Green and Red Spectral Regions on Functional Characteristics of Sturgeon Sperm

A comparative study of the effect of low-intensity laser radiation in green (λ = 532 nm) and red (λ = 632.8 nm) spectral regions at equal average irradiance (3 mW cm⁻²) on functional characteristics of Siberian sturgeon spermatozoa is carried out. Confirmation of the photobiomodulation effect of the radiation is obtained by analyzing spermatozoa motility, percentage of motile spermatozoa and fertilization rate. It is shown that, depending on the energy dose, the laser radiation in red and green spectral regions can have both stimulatory and inhibitory effects on spermatozoa motility. Contrary to popular belief that the short-wavelength radiation has great prospects in reproductive biotechnologies (due to more efficient absorption of radiation by cellular chromophores and increased generation of ROS), convincing evidence of a more pronounced stimulatory effect of radiation in the red spectral region was obtained. For the first time, metal-free porphyrins capable of acting as endogenous photosensitizers generating ROS were detected and identified in animal sperm. Using luminol-dependent chemiluminescence, it is shown that the increased production of ROS capable of exerting an inhibitory effect on biological systems at high concentrations is among the possible reasons for reduction in the stimulatory effect of radiation when moving from red to green spectral region.     

Antimicrobial photodynamic therapy: The impact of laser radiation on mucous tissue stained with photosensitizer methylene blue

We have calculated the spatial distribution of absorbed light energy within layers of a mucous tissue that contains photosensitizer methylene blue. Under irradiation regimes that are typical of antimicrobial photodynamic therapy (laser radiation wavelength, 670 nm; power density, 150–300 mW/cm²; dose, 9–18 J/cm²), we have analyzed conditions that are necessary for the light penetration and delivery of methylene blue to a nidus of infection on a mucosal surface. We have performed a computer simulation of thermal fields, estimated maximal heating temperatures, and considered the degree at which the heating affects the trans-mission of light through a stained tissue.     

Modeling laser irradiation conditions for mucosal tissues in antimicrobial photodynamic therapy

We use computer modeling to analzye empirically selected conditions for antimicrobial photodynamic therapy of mucosal tissues. We calculate the optical and thermal fields for experimental conditions for low-intensity (cold) laser irradiation used in treatment of lesions in mucosal tissues stained by methylene blue: λ = 670 nm, power density 150–300 mW/cm², doses 9–18 J/cm²; λ = 632.8 nm, 15 mW/cm², dose 4.5 J/cm². For numerical estimates, we used the optical characteristics of methylene blue and three layers of mucosal tissues at the laser radiation wavelengths, and also the thermal characteristics of the tissues. The experimental conditions were optimized using the ratio of the tissue penetration depth for the absorbed optical energy and the penetration depth of methylene blue into the lesion, while maintaining safe tissue heating temperatures.     

Sensitizing effect of <Emphasis Type="Italic">Z,Z</Emphasis>-bilirubin IXα and its photoproducts on enzymes in model solutions

In model systems, we have studied side effects which may be induced by light during phototherapy of hyperbilirubinemia (jaundice) in newborn infants, with the aim of reducing the Z,Z-bilirubin IXα (Z,Z-BR IXα) level. We have shown that the sensitizing effect of Z,Z-BR IXα, localized at strong binding sites of the human serum albumin (HSA) macromolecule, is primarily directed at the amino acid residues of the carrier protein and does not involve the molecules of the enzyme (lactate dehydrogenase (LDH)) present in the buffer solution. The detected photodynamic damage to LDH is due to sensitization by bilirubin photoisomers, characterized by lower HSA association constants and located (in contrast to native Z,Z-BR IXα) on the surface of the HSA protein globule. Based on study of the spectral characteristics of the photoproducts of Z,Z-BR IXα and comparison of their accumulation kinetics in solution and the enzyme photo-inactivation kinetics, we concluded that the determining role in sensitized damage to LDH is played by lumirubin. The photosensitization effect depends on the wavelength of the radiation used for photoconversion of bilirubin. When (at the beginning of exposure) we make sure that identical numbers of photons are absorbed by the pigment in the different spectral ranges, the side effect is minimal for radiation corresponding to the long-wavelength edge of the bilirubin absorption band. We have shown that for a bilirubin/HSA concentration ratio >2 (when some of the pigment molecules are sorbed on the surface of the protein globule), the bilirubin can act as a photosensitizing agent for the enzyme present in solution. We discuss methods for reducing unfavorable side effects of light on the body of newborn infants during phototherapy of hyperbilirubinemia. __________ Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 75, No. 3, pp. 383–394, May–June, 2008.     

Spectral fluorescence and polarization characteristics of <Emphasis Type="Italic">Z,Z</Emphasis>-bilirubin IXα

We have studied the spectral fluorescence and polarization characteristics of Z,Z-bilirubin IXα, at room temperature in chloroform and in aqueous buffer medium, within an equilibrium complex with human serum albumin (HSA), and also under low temperature conditions (T = −100°C) in isobutyl alcohol. We have observed a bathochromic shift of the fluorescence spectra, which is most pronounced for the bilirubin-albumin complex. The following are considered as possible reasons for the observed dependence of the position of the fluorescence (fluorescence excitation) spectra on the excitation (detection) wavelength: structural and spectral differences between the chromophores making up the bilirubin molecule; conformational heterogeneity of the pigment in solution; a contribution to the fluorescence from molecules which have not completed the vibrational relaxation process; inhomogeneous orientational broadening of the levels; heterogeneity of the microenvironment of the chromophores in the protein matrix. We show that polarized fluorescence of bilirubin occurs at room temperature, due to the anomalously short fluorescence lifetime τ (picosecond or subpicosecond ranges). Despite such a short τ, the absorption and emission polarization spectra suggest the presence of intramolecular nonradiative singlet-singlet energy transfer when bilirubin is excited to high vibrational sublevels of the S₁ state (degree of polarization p = 0.11–0.12). When fluorescence is excited on the long-wavelength slope of the absorption band, no transfer occurs: the degree of polarization (p = 0.46−0.47) is close to the limiting value (p = 0.50). We discuss the question of the role played by exciton interactions between chromophores in the bilirubin molecule when it is excited. __________ Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 74, No. 1, pp. 108–119, January–February, 2007.     

Formation of an Equilibrium Complex of Lactate Dehydrogenase with Chlorin e 6

Using spectrophotometric, spectral-luminescent, and polarization methods, we have detected the formation of strong equilibrium complexes of a key glycolytic enzyme — lactate dehydrogenase — with a promising photodynamic sensitizer — chlorin e ₆. It has been established that enzymes serve as the most sensitive targets destroying tumor cells subjected to photodynamic therapy.