Marianne Krieg

Photophysical properties of 3,3'-dialkylthiacarbocyanine dyes in homogeneous solution.

The photophysical properties of 3,3′‐dialkylthiacarbocyanine iodides and chlorides were measured in various solvents. It was found that photoisomerization and fluorescence are the major contributors to the deactivation of the excited singlet state; intersystem crossing occurs with only a very low efficiency. In ethanol, a triplet yield of 0.004 and a singlet oxygen quantum yield of 0.002 were determined. The photophysical parameters of these dyes are not substantially influenced by the length of the alkyl chain or the size of the halide counterion. The substitution of an ethyl with an octadecyl‐chain only slightly hinders photoisomerization, and the replacement of the chloride with an iodide reduces only marginally the fluorescence lifetimes and fluorescence quantum yields in chloroform. A significant external heavy‐atom effect is observed using dibromoethane as a solvent: triplet and singlet oxygen yields increase7–10‐fold, and the triplet lifetime decreases from 55 μs to 15 mUs.

Photophysical properties of 3,3'-dialkylthiacarbocyanine dyes in organized media : unilamellar liposomes and thin polymer films

All symmetrical dialkylthiacarbocyanine dyes, with the exception of the diethyl derivatives, are incorporated into liposomes. Absorption and fluorescence data indicate a solubilization site close to the bilayer surface with the alkyl chains penetrating into the lipid bilayer. Incorporation into organized assemblies affects the photophysical parameters of these dyes. Photoisomerization occurring from the first excited state becomes more difficult as the restrictive effect of the solubilization site increases. As a consequence, competing deactivation processes, such as fluorescence and triplet formation, become more efficient with the result that fluorescence quantum yields, triplet yields and singlet oxygen quantum yields are larger in liposomes than in homogeneous solution. Dihexylthiacarbocyanine iodide has a fluorescence quantum yield of 0.27 and 0.10 (25 degrees C) in dimyristoylphosphatidyl-choline liposomes and ethanol, respectively, and the singlet oxygen yield increases by a factor three to 0.006 on going from ethanol to liposomes. The effect of a highly organized environment is even more pronounced in thin polymer films. In these systems, photoisomerization is completely inhibited and only triplet formation is observed in the transient absorption spectrum.

Merocyanine dyes: effect of structural modifications on photophysical properties and biological activity.

Abstract Merocyanine derivatives were prepared by structural alterations at the barbituric acid or chalcogenazole moieties. The photophysical properties of the dyes were markedly influenced by the presence of selenium rather than sulfur as a substituent at position 2 of the barbiturate. In methanol, quantum yields of both triplet state (φτ) and singlet oxygen sensitization (φΔ) were increased by over an order of magnitude, with a concomitant decrease in fluorescence, when selenium was present in the molecule. Photoisomerization, one of the dominant deactivation pathways in the sulfur‐ or oxygen‐containing analogues, was completely absent in the selenium‐containing derivatives. Efficient triplet state formation was observed for selenium‐containing derivatives incorporated into L1210 cells by diffuse reflectance laser flash photolysis. Cytotoxicity studies, camed out using clonogenic assays on L1210 leukemia cells, showed a good correlation with φτ and φΔ, measured in solution. Experimental evidence provided by this paper supports a triplet state‐, and probably singlet oxygen‐, mediated phototoxic mechanism. Photoisomerization or singlet state mechanisms can be discounted.

Effects of structural modifications on the photosensitizing properties of dialkylcarbocyanine dyes in homogeneous and heterogeneous solutions.

The photophysical characterization of structurally modified symmetric dialkylthiacarbocyanine dyes in homogeneous and biomimetic media is reported. The aim of the two specific structural modifications was to increase singlet oxygen production, hence enhancing the photosensitizing properties of these cyanine dyes. Specifically, (1) the sulfur was exchanged with selenium in order to enhance intersystem crossing via an internal heavy atom effect and (2) substituents of differing size were introduced into the meso-position of the polymethine chain to reduce photoisomerization. The result of incorporation of an internal heavy atom (selenium) into the structure of the dye yields the expected effect: this modification results in a 22-fold increase in the rate of intersystem crossing, but does not change the remaining competing deactivation rates of the first excited singlet state. As a consequence, singlet oxygen quantum yields increase from 0.001 to 0.014 in ethanol and from 0.006 to 0.08 in unilamellar liposomes. In the case of the meso-substituted thiacarbocyanine dyes, a significant reduction in photoisomerization is indeed observed. However, this modification drastically enhances internal conversion which then becomes the main deactivation pathway of the first excited singlet state. As a result, very small fluorescence and singlet oxygen quantum yields are obtained, e.g. 0.006 and 0.001, respectively, in ethanol.

Singlet oxygen production and fluorescence yields of merocyanine 540 : a comparative study in solution and model membrane systems

Singlet oxygen and fluorescence quantum yields of merocyanine 540 were measured in solution (methanol, ethanol, n-heptanol) and in model membrane systems (cationic micelles, unilamellar dimyristoyl- and dipalmitoylphosphatidylcholine vesicles). Both singlet oxygen quantum yields and fluorescence quantum yields increase with increasing viscosity/rigidity of the surrounding medium: the yield of singlet oxygen production (24 degrees C) goes from 0.002 in methanol to 0.04 in dipalmitoylphosphatidylcholine vesicles, and fluorescence yields (25 degrees C) change from 0.14 to 0.61 in the same media. The data are consistent with previous findings that photoisomerization is in direct competition with intersystem crossing and radiative relaxation. Therefore, a singlet oxygen yield close to the maximum value of 0.11 can only be achieved after both photoisomerization and internal conversion are prevented by a highly viscous environment.

Determination of singlet oxygen quantum yields with 1,3-diphenylisobenzofuran in model membrane systems.

The oxidation of 1,3-diphenylisobenzofuran by singlet oxygen was investigated in methanol and in two different types of liposomes. It was found that at high concentrations of scavenger 1,3-diphenylisobenzofuran, e.g., > 100 microM in methanol, the 1:1 oxidation stoichiometry is lost and more than one scavenger molecule per molecule of singlet oxygen is consumed. In model membrane systems, where local scavenger concentrations are high due to compartmentalization, correct singlet oxygen quantum yields with 1,3-diphenylisobenzofuran are only determined if the increased oxidation is taken into account.

Structurally modified trimethine thiacarbocyanine dyes : Effect of N-alkyl substituents on antineoplastic behavior

The effect of dye localization and dye distribution on the antineoplastic behavior of photosensitizers was investigated with a homologous series of trimethine thiacarbocyanine dyes in L1210 leukemia and A549 lung carcinoma cells. These dyes were synthesized with N-alkyl groups of different sizes (ethyl to octadecyl) to vary their lipophilic properties without compromising their photophysics. While dyes with smaller N-alkyl groups (ethyl to decyl) were already cytotoxic in the dark, longer chain cyanines exhibited antineoplastic activity only after exposure to light. Results from this study indicate that the switch from dark cytotoxicity to phototoxicity occurred when dyes, due to a decrease in cationic character with increasing size of alkyl substituents, were no longer able to cross the plasma membrane. Dark cytotoxicity decreased with increasing size of N-alkyl groups and was cell-line independent. On the other hand, photodynamic damage varied by several orders of magnitude depending on the cell line and the length of the alkyl substituents. The most effective photosensitizer was the dioctadecyl dye which achieved a 4- to 5-log reduction of leukemia cells, although it had very modest triplet and singlet oxygen quantum yields of 0.008 and 0.006, respectively. This study also showed that photobiological performance can be improved greatly by optimizing dye binding properties via structural modifications.

Elemental Selenium Generated by the Photobleaching of Seleno-Merocyanine Photosensitizers Forms Conjugates with Serum Macro-Molecules That are Toxic to Tumor Cells

Abstract Elemental selenium generated by the photobleaching of selenomerocyanine dyes forms conjugates with serum albumin and serum lipoproteins that are toxic to leukemia and selected solid tumor cells but well tolerated by normal CD34-positive hematopoietic stem and progenitor cells. Serum albumin and lipoproteins act as Trojan horses that deliver the cytotoxic entity (elemental selenium) to tumor cells as part of a physiological process. They exploit the fact that many tumors have an increased demand for albumin and/or low-density lipoprotein. Se(0)-protein conjugates are more toxic than selenium dioxide, sodium selenite, selenomethionine, or selenocystine. They are only minimally affected by a drug resistance mechanism, and they potentiate the cytotoxic effect of ionizing radiation and several standard chemotherapeutic agents. The cytotoxic mechanism of Se(0)-protein conjugates is not yet fully understood. Currently available data are consistent with the notion that Se(0)-protein conjugates act as air oxidation catalysts that cause a rapid depletion of intracellular glutathione and induce apoptosis. Drugs modeled after our Se(0)-protein conjugates may prove useful for the local and/or systemic therapy of cancer.

Inactivation of photosensitizing merocyanine dyes by plasma, serum and serum components.

Abstract— Merocyanine dyes with an oxygen in the electron donor heterocycle were rapidly degraded by plasma, serum and serum components. Replacement of the oxygen by a sulfur or selenium atom rendered the dyes refractory to degradation. The degradation of labile merocyanine dyes was temperature dependent and oxygen independent. The plasma component that was responsible for the degradation of merocyanine dyes was sensitive to heat and detergent, suggesting an enzymatic process. The identification of the structural requirements for sensitivity/resistance to degradation provides the experimenter with a simple means to manipulate the stability of mer‐ocyanines in high serum or plasma environments and may expand the clinical utility of merocyanine photosen‐sitizers beyond their traditional role in the extracorporeal purging of bone marrow grafts.

POTENTIATION OF MEROCYANINE 540-MEDIATED PHOTODYNAMIC THERAPY BY SALICYLATE AND RELATED DRUGS

Abstract— Simultaneous exposure to merocyanine 540 (MC540) and light of a suitable wavelength kills leukemia, lymphoma and neuroblastoma cells but is relatively well tolerated by normal pluripotent hematopoietic stem cells. This differential phototoxic effect has been exploited in preclinical models and a phase I clinical trial for the extracorporeal purging of autologous bone marrow grafts. Salicylate is known to potentiate the MC540‐mediated photokilling of tumor cells. Assuming that salicylate induces a change in the plasma membrane of tumor cells (but not normal hematopoietic stem cells) that enhances the binding of dye molecules it has been suggested that salicylate may provide a simple and effective means of improving the therapeutic index of MC540‐mediated photodynamic therapy. We report here on a direct test of this hypothesis in a murine model of bone marrow transplantation as well as in clonal cultures of normal murine hematopoietic progenitor cells. In both systems, salicylate enhanced the MC540‐sensitized photoinactivation of leukemia cells and normal bone marrow cells to a similar extent and thus failed to improve the therapeutic index of MC540 significantly. On the basis of a series of dye‐binding studies, we offer an alternative explanation for the potentiating effect of salicylate. Rather than invoking a salicylate‐induced change in the plasma membrane of tumor cells, we propose that salicylate displaces dye molecules from serum albumin, thereby enhancing the concentration of free (active) dye available for binding to tumor as well as normal hematopoietic stem cells.