PHYSICOCHEMICAL DETERMINANTS OF THE SENSITIZING EFFECTIVENESS FOR PHOTOOXIDATION OF NERVE MEMBRANES BY FLUORESCEIN DERIVATIVES

Abstract— Fluorescein and 8 of its halogenated derivatives were employed as sensitizers for the photochemical block of sodium channels on giant nerve axons from lobsters. Axons were voltage clamped using a double sucrose gap technique and illuminated by filtered light from a Xe arc following exposure to sensitizer at concentrations of 5 µ M or less dissolved in the external bath. An assay for the magnitude of sensitizing effectiveness was developed consisting of the rate constant for block of channels during illumination scaled to the rate of photon capture in a thin layer of bathing solution. The kinetics for development of effectiveness during pre-illumination periods of up to 16-min duration varied widely for different derivatives despite similarities in molecular structure. At a standard 8-min pre-illumination exposure, the relative effectiveness extended over a 30,000-fold range. A model system consisting of octanol and the artificial sea-water (ASW) bathing solution was compared with the axon membrane and its environment. Octanol/ASW partition coefficients were measured for each sensitizer as well as the relative absorption in the 2 solvents at equal concentrations. Predicted values of relative sensitizing effectiveness based on the product of partition coefficient, relative absorption in octanol and mol wt of the substituents on the fluorescein skeleton were found to correlate highly with measured values. The diffusion kinetics prior to illumination correlated inversely with partition coefficients, suggesting that the more permeable sensitizers may penetrate into diffusion sinks, thus slowing the rise in concentration at sites for modification. It is concluded that sensitizers must penetrate into the membrane in order to sensitize and that the photochemistry appropriate for a low polarity environment controls the degree of membrane modification following photon capture.     

THE CONCENTRATION and FLUENCE DEPENDENCE OF DELAYED PHOTOHEMOLYSIS

Abstract— Delayed photohemolysis of human erythrocytes sensitized with the fluorescein sensitizer phloxine B has been examined. A 0.05% hematocrit suspension of cells was irradiated in the presence of sensitizer up to 2 h with fluorescent lamps. Analysis for percent hemolysis was performed at various times afterwards by measuring absorbance due to hemoglobin in the centrifuged supernatant. Lysis develops along a sigmoid time course which varies with illumination period and sensitizer concentration. The hemolytic rate was found to vary with the square of the fluence. Changing the sensitizer concentration at constant fluence showed that lytic rate varied with the 1.4 power of concentration. These results were verified in separate experiments where both concentration and dose fluence varied.     

THE POTENCY OF SENSITIZED PHOTOHEMOLYSIS DEPENDS ON THE COMPOSITION OF THE REACTION VESSEL

Abstract— Delayed photohemolysis has been shown to vary with the composition of the reaction vessel. Human erythrocytes were illuminated in the presence of sensitizer for 1 h and examined 23 h later for percent hemolysis. Four sensitizers, two cationic and two anionic, and three types of reaction vessel were studied. The cationic sensitizers were much more effective in tissue culture treated plastic than in neutral plastic or glass. All were somewhat more effective in neutral plastic than glass. Sorption of sensitizers to reaction vessels without cells present was also studied. In some cases sorption leads to virtually complete removal of sensitizer from the reaction medium. In other cases no sorption is detectable. For the highest sorbing sensitizer total replacement of the reaction medium with sensitizer-free solution did not decrease effectiveness, demonstrating that sorbed sensitizer is available to sensitize. The results suggest that photooxidation of the cell membrane occurs in two zones, one at the interface between vessel surface and cell surface, and the other where the membrane faces the bulk reaction medium. The relative importance of the interface may depend on the strength of sorption and the affinity of sensitizer for the cell membrane.     

PHOTOHEMOLYTIC LESIONS: STOICHIOMETRY OF CREATION BY PHLOXINE B

The uptake of the halogenated fluorescein sensitizers, Rose Bengal and phloxine B, by erythrocyte membranes was measured as a function of incubation time. Ghost membranes concentrate the sensitizers to levels greatly in excess of the sensitizer concentration in the suspension medium. Most of the uptake occurs in the first hour and is virtually complete by four hours. It is not significantly affected by stirring under the present conditions. The distribution of sensitizer cannot be explained as partitioning between immiscible solvents. Rather the data fits the Freundlich adsorption isotherm over a wide range of sensitizer concentrations and ghost protein values. Photohemolysis experiments were performed using sensitizer concentrations and incubation times similar to those used in the sensitizer uptake studies. The kinetics of lysis were found to vary with the 1.2 power of sensitizer concentration using PB. When combined with the uptake data for this sensitizer, these results imply that photohemolytic lesions in the red cell membrane are formed by the action of two sensitizer molecules.     

THE HEMOLYSIS OF ERYTHROCYTES BY SINGLET OXYGEN GENERATED IN THE GAS PHASE

Many sensitizers cause photodynamic hemolysis of erythrocytes. As these sensitizers usually participate in Type I as well as Type II processes, the determination of the mechanism(s) of photosensitized hemolysis is always ambiguous. Here, human erythrocytes were proved to hemolyze upon treatment with singlet oxygen (1 delta g) generated with fluoranthene in the gas phase. These conditions rigorously exclude the participation of superoxide anion. The standard diagnostic tests for singlet oxygen (enhanced effect in D2O and protection by NaN3) gave the anticipated results when the erythrocytes were treated with 1O2 generated in the gas phase. When the erythrocytes were irradiated in a buffer solution containing fluoranthene, the results of the diagnostic tests depended on the sensitizer concentration.     

Influence of substitutions on asymmetric dihydroxychlorins with regard to intracellular uptake, subcellular localization and photosensitization of Jurkat cells

The search for new efficient sensitizers for photodynamic therapy (PDT) points to improve photophysical properties like absorption in the red region and singlet oxygen quantum yield as well as to control the localization of the sensitizer within the tumour cell. Depending on their physicochemical properties and their uptake mechanism, sensitizers can reach different intracellular concentrations and localize in different subcellular compartments. Moreover, the preferential localization of a sensitizer in target organelles, like mitochondria or lysosomes, could determine the cell death mechanism after PDT. This study aimed to investigate the influence of substitutions on dihydroxychlorins with regard to intracellular uptake, subcellular localization and cell death pathway. Moreover, the effect of a liposome-based delivery system was tested. The intracellular uptake was found to be strictly dependent on the sensitizer molecular structure and the means of its delivery. The most polar sensitizer in this study (compound 3) had, depending on incubation time, an intracellular concentration 2-8 times higher than the unsubstituted chlorin 1. All investigated photosensitizers localize predominantly in lysosomes but after longer incubation times weak fluorescence intensity was also detected in mitochondria and Golgi apparatus. The cell death pathway was found to be influenced by the sensitizer intracellular concentration and the applied light doses. In general, the increasing amphiphilicity of the sensitizer molecules is correlated with an increased sensitizer uptake and an increased rate of necrotic cells after irradiation.     

A hand-held fiber-optic implement for the site-specific delivery of photosensitizer and singlet oxygen

We have constructed a fiber optic device that internally flows triplet oxygen and externally produces singlet oxygen, causing a reaction at the (Z)-1,2-dialkoxyethene spacer group, freeing a pheophorbide sensitizer upon the fragmentation of a reactive dioxetane intermediate. The device can be operated and sensitizer photorelease observed using absorption and fluorescence spectroscopy. We demonstrate the preference of sensitizer photorelease when the probe tip is in contact with octanol or lipophilic media. A first-order photocleavage rate constant of 1.13 h(-1) was measured in octanol where dye desorption was not accompanied by readsorption. When the probe tip contacts aqueous solution, the photorelease was inefficient because most of the dye adsorbed on the probe tip, even after the covalent ethene spacer bonds have been broken. The observed stability of the free sensitizer in lipophilic media is reasonable even though it is a pyropheophorbide-a derivative that carries a p-formylbenzylic alcohol substituent at the carboxylic acid group. In octanol or lipid systems, we found that the dye was not susceptible to hydrolysis to pyropheophorbide-a, otherwise a pH effect was observed in a binary methanol-water system (9:1) at pH below 2 or above 8.     

KINETIC FACTORS GOVERNING SENSITIZED PHOTOOXIDATION OF EXCITABLE CELL MEMBRANES

Abstract— The kinetic factors which determine the rate at which Na⁺ channels in nerve membranes become photochemically modified were studied on giant axons from lobsters using the double sucrose gap voltage clamp technique. Axons were bathed in artificial sea water containing sensitizing dyes and illuminated from a Xe are source with light in the visible region while being repetitively step depolarized. Successive values of peak Na⁺ current and time-to-peak were monitored and rate constants for their change served as the assay for magnitude of modification. Action spectra for four sensitizers in the fluorescein series exhibited red shifts of roughly 17nm demonstrating that sensitizing species are not simply free in solution. Eosin Y diffuses to its sensitization sites with a half time of 70s indicating the existence of a major diffusion barrier which may mean that dye must penetrate to the interior of the membrane to be effective. Eosin Y is removed from sensitization sites by rinse with the same half time but shows two fractions: a faster fraction comprising 80% of sensitizing effectiveness and a slower fraction comprising 20%. The concentration dependence for Eosin Y is linear below 10 μ M and shows a progressive saturation at higher values, where the relationship is difficult to determine because of shielding. Different sensitizers vary in their ability to sensitize block of channels vs disruption of inactivation, demonstrating separate processes for the two modifications. It is suggested that both modifications proceed from single photon absorption events by individual sensitizer molecules bound or located close to the modification sites on the channels.     

Quinacrine enhances vesicular stomatitis virus inactivation and diminishes hemolysis of dimethylmethylene blue-phototreated red cells

Several photodynamic methods for virus inactivation in red blood cell (RBC) suspensions have resulted in unwanted hemolysis during extended 1-6 degrees C storage. To explore the possibility that hemolysis may be mediated by a membrane-bound dye, a molecule similar in structure to yet different in light absorption properties from the photosensitizer was used as an inhibitor for RBC membrane binding in virus photoinactivation and photohemolysis studies. The addition of 500 pM quinacrine to oxygenated RBC before treatment with 3.6 microM dimethylmethylene blue (DMMB) and 219 mJ/cm2 red light resulted in an increased extracellular concentration of the sensitizer, increased extracelluar viral inactivation kinetics, and decreased hemolysis during 1-6 degrees C storage without alteration of quinacrine absorption properties. These results collectively suggest that despite its recognized affinity for viral nucleic acid, DMMB also binds to RBC membranes and that the bound dye is, in part, responsible for photoinduced hemolysis.     

A NEW HYPOTHESIS FOR THE TARGET IN PHOTOHEMOLYSIS: DIMERS OF THE BAND 3 PROTEIN

Abstract— Photohemolysis has been investigated extensively during the past 20 years, but the membrane target has eluded identification. It is proposed here that the target is the dimer of the band 3 anion exchange protein. This new hypothesis is based on three observations from the literature: (1) the kinetics of photohemolysis sensitized by exogenous sensitizers or induced by UV light obey a power dependence with light dose, with an exponent near two. From target theory this implies a two-component target. (2) Band 3 molecules exist in the erythrocyte membrane as dimers. (3) Binding of eosin to band 3 via an isothiocyano group markedly increases its potency to sensitize hemolysis. Experiments with eosin isothiocyanate bound to band 3 show that lytic rate varies as the 1.45 power of light dose, and that the bound sensitizer bleaches considerably, thereby blunting the effectiveness of higher light doses.     

Panchromatic ruthenium sensitizer based on electron-rich heteroarylvinylene π-conjugated quaterpyridine for dye-sensitized solar cells

The first example of a heteroarylvinylene π-conjugated quaterpyridine Ru(II) sensitizer (N1044) was synthesized and used in dye-sensitized solar cells; the dye has an effective panchromatic absorption band, covering the entire visible spectrum up to the NIR region, and superior electrochemical characteristics (HOMO/LUMO and bandgap energies) with respect to previous representative Ru(II) bi- and quaterpyridine sensitizers. A record IPCE curve ranging from 360 to 920 nm has been measured with a maximum of 65% at 646 nm and still 33% efficiency at 800 nm; this leads to substantially increased photocurrent (19.2 mA cm(-2)) when compared to the prototype N719 Ru(II) sensitizer.     

Determination of octanol/water partition coefficients by flow injection analysis

Determination of octanol/water partition coefficients by batch-extraction methods is currently the only acceptable procedure for application to structure/activity relationships. Work on the development of flow-injection systems to determine these partition coefficients is reported. It is shown that it is not necessary to know the concentration of the analytic in either phase; the partition coefficient should be equal to the differences in the octanol and water peak widths divided. by the system residence time. For a limited number of compounds, this is shown to be valid. Several systems were developed, including a flow-through dialyzer which was 5 times more efficient than the best commercial unit.     

Comparison of two simulation methods to compute solvation free energies and partition coefficients

The thermodynamic integration (TI) and expanded ensemble (EE) methods are used here to calculate the hydration free energy in water, the solvation free energy in 1‐octanol, and the octanol‐water partition coefficient for a six compounds of varying functionality using the optimized potentials for liquid simulations (OPLS) all‐atom (AA) force field parameters and atomic charges. Both methods use the molecular dynamics algorithm as a primary component of the simulation protocol, and both have found wide applications in fields such as the calculation of activity coefficients, phase behavior, and partition coefficients. Both methods result in solvation free energies and 1‐octanol/water partition coefficients with average absolute deviations (AAD) from experimental data to within 4 kJ/mol and 0.5 log units, respectively. Here, we find that in simulations the OPLS‐AA force field parameters (with fixed charges) can reproduce solvation free energies of solutes in 1‐octanol with AAD of about half that for the solute hydration free energies using a extended simple point charge (SPC/E) model of water. The computational efficiency of the two simulation methods are compared based on the time (in nanoseconds) required to obtain similar standard deviations in the solvation free energies and 1‐octanol/water partition coefficients. By this analysis, the EE method is found to be a factor of nine more efficient than the TI algorithm. For both methods, solvation free energy calculations in 1‐octanol consume roughly an order of magnitude more CPU hours than the hydration free energy calculations. © 2012 Wiley Periodicals, Inc.     

Slow cation transfer follows sensitizer regeneration at anatase TiO2 interfaces

After rapid photoinduced electron injection into TiO2 and regeneration by a donor, D, such as iodide or phenothiazine, sensitizers are present in an environment distinctly different from that prior to light absorption. Significantly, the absorption spectrum of the Ru(II) sensitizer in this new environment is one that is known to be less favorable for excited-state electron injection. The transient absorption features were found to report on photoinduced variations in the local electronic environment of the Ru(II) sensitizer-TiO2 interface that were induced by ion transfer. The data demonstrate that slow (micros to ms) cation transfer follows regeneration to yield the sensitizer that was initially photoexcited.     

ADMET evaluation in drug discovery. 11. PharmacoKinetics Knowledge Base (PKKB): a comprehensive database of pharmacokinetic and toxic properties for drugs

Good and extensive experimental ADMET (absorption, distribution, metabolism, excretion, and toxicity) data is critical for developing reliable in silico ADMET models. Here we develop a PharmacoKinetics Knowledge Base (PKKB) to compile comprehensive information about ADMET properties into a single electronic repository. We incorporate more than 10?000 experimental ADMET measurements of 1685 drugs into the PKKB. The ADMET properties in the PKKB include octanol/water partition coefficient, solubility, dissociation constant, intestinal absorption, Caco-2 permeability, human bioavailability, plasma protein binding, blood-plasma partitioning ratio, volume of distribution, metabolism, half-life, excretion, urinary excretion, clearance, toxicity, half lethal dose in rat or mouse, etc. The PKKB provides the most extensive collection of freely available data for ADMET properties up to date. All these ADMET properties, as well as the pharmacological information and the calculated physiochemical properties are integrated into a web-based information system. Eleven separated data sets for octanol/water partition coefficient, solubility, blood-brain partitioning, intestinal absorption, Caco-2 permeability, human oral bioavailability, and P-glycoprotein inhibitors have been provided for free download and can be used directly for ADMET modeling. The PKKB is available online at http://cadd.suda.edu.cn/admet.     

酞菁化合物光敏化氧化-还原反应的研究——Ⅰ.吸收光谱的研究

本文报道了几种金属酞菁化合物的吸收光谱及其特征参数。它们在可见区域内单分子吸收光谱基本是相似的,其最低电子跃迁特征吸收峰λ_max在650—700nm范围,其值随溶剂极性增加而略向红移,取代基团不同,对λ_max值影响不大,络合金属不同,λ_max值略有差别。某些取代酞菁化合物在极性小的溶剂中形成二聚体。它们是无光活性的,不适宜作光敏化剂。本文探讨了有关二聚体的形成与溶剂极性、溶液浓度、取代基团性质及络合金属原子的关系。结果表明二聚体的形成取决于取代基团的性质以及溶剂的介电常数与溶液浓度。最后计算锌酞菁磺酰胺在氯仿与乙醇中,单分子与二聚体的平衡常数K_eq=C_D/C_M²约为～10⁶l·mol^-1。     

Microscopic structure and solvation in dry and wet octanol

Octanol-water partition coefficients are extraordinarily successful for correlating and predicting numerous processes of pharmacological and environmental importance. The amphiphilic nature of the 1-octanol molecules allows the octanol phase to mimic the complexities of many different environments ranging from biomembranes to soil. However, the structural details of the octanol phase and whether its structure is altered upon water saturation are not yet fully understood. Configurational-bias Monte Carlo simulations in the Gibbs ensemble demonstrate that a diverse spectrum of hydrogen-bonded aggregates exists in dry and wet 1-octanol, and that water saturation substantially alters the 1-octanol environment from predominantly linear aggregates in dry octanol to larger cylindrical micelles with water cores in wet octanol. These simulation results are able to reconcile the conflicting views (chain-like or water-centered aggregates vs spherical micelles) of the 1-octanol structure inferred from thermodynamic arguments, spectroscopic measurements, and diffraction experiments. Calculated partition constants quantify the influence of water saturation on the solubility characteristics of the dry and wet octanol phases.     

PHOTOBLEACHING OF MONO-l-ASPARTYL CHLORIN e6 (NPe6): A CANDIDATE SENSITIZER FOR THE PHOTODYNAMIC THERAPY OF TUMORS

Abstract— Most sensitizers used for the photodynamic therapy (PDT) of tumors photobleach on illumination. Thus, it is of interest to examine the photobleaching behavior of new sensitizers proposed for use in PDT. This report surveys the quantum yields and kinetics of the photobleaching of mono- l -aspartyl chlorin e₆ (NPe6), a hydrophilic chlorin that has many of the photoproperties desirable in a sensitizer for clinical PDT. It is a very effective sensitizer for the PDT of several types of model tumors in animals and is now in Phase I clinical trials. The quantum yield of NPe6 photobleaching in pH 7.4 phosphate buffer in air was 8.2 × 10⁻⁴; this is greater than the yields for typical porphyrin photosensitizers. For example, the yields for hematoporphyrin and uroporphyrin are 4.7 × 10 ⁵ and 2.8 × 10⁻⁵, respectively. The yield decreased significantly in organic solvents of low dielectric constant. The Sn derivative of NPe6 was more light stable than NPe6 (yield = 5.7 × 10 ⁻⁶), while the Zn derivative was more sensitive (yield = 1.9 × 10⁻²). Oxygen appeared to be necessary for the photobleaching of NPe6; however, bleaching was not inhibited by 100 mM azide, an efficient quencher of singlet oxygen. The photooxidizable substrates cysteine, dithiothreitol and furfuryl alcohol increased the quantum yield of photoblcaching two- to four-fold, while the electron acceptor, met-ronidazole, increased it almost six-fold. Photobleaching yields for several other chlorins were also measured.     

Synthesis and photovoltaic properties of organic sensitizers incorporating a thieno[3,4-c]pyrrole-4,6-dione moiety

Novel organic sensitizers containing a thieno[3,4-c]pyrrole-4,6-dione (TPD) moiety with triphenylamine or julolidine as the electron donor have been designed and synthesized for quasi-solid-state dye-sensitized solar cells (DSSCs). For comparison, two organic dyes based on a terthiophene spacer have also been synthesized. The absorption, electrochemical and photovoltaic properties of all sensitizers have been systematically investigated. We found that the incorporation of TPD is highly beneficial to broaden the absorption spectra of the organic sensitizers and prevent the intermolecular interaction. Therefore, the charge recombination possibility is reduced, which is revealed by the controlled intensity modulated photovoltage spectroscopy. A quasi-solid-state DSSC based on sensitizer FNE38 with TPD and triphenylamine moieties demonstrates a solar energy conversion efficiency of 4.71% under standard AM 1.5G sunlight without the use of coadsorbant agents.     

SKIN PHOTOSENSITIVITY AND PHOTODESTRUCTION OF SEVERAL POTENTIAL PHOTODYNAMIC SENSITIZERS

The major side effect associated with porphyrins (Photofrin II) in clinical photodynamic therapy is skin photosensitivity. In order to avoid this deleterious reaction, patients must remain out of the sunlight for approximately 1 month. A possible procedure to reduce the amount of skin photosensitivity is to photodegrade (photobleach) the compound in the skin. In this study, we report a series of experiments describing the photodegradation rates of two photosensitizers currently receiving attention due to their potential for use in PDT (mono L-aspartyl chlorin e6 and chloroaluminum sulfonated phthalocyanine). These compounds are compared to Photofrin II (PfII). Experiments consisted of determining photodegradation rates and efficiencies of the sensitizers in (i) phosphate buffered saline (PBS), (ii) PBS with fetal calf serum (to enhance absorption and simulate cellular binding or deaggregation), (iii) Chinese Hamster Ovary cells, and (iv) Balb/c mice. We performed two standardized skin sensitivity assays using the Hartely albino guinea pig irradiated with a UV blue point lamp and Balb/c mice irradiated with the therapeutic wavelength of each sensitizer. In addition, we performed a cell clonogenicity assay comparing photodegraded and fresh PfII on CHO cells. The photodegraded PfII exhibited significant phototoxicity, although the fluorescence was bleached by more than 70%. The results show that PfII causes major skin photosensitization and that the other compounds produce no substantial skin sensitivity. Our studies suggest that photodegradation of PfII with 630 nm light has little influence on the phototoxicity of the compound. In addition, skin sensitivity was not alleviated with prior photobleaching with 405 nm light.