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

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 merocyanines in high serum or plasma environments and may expand the clinical utility of merocyanine photosensitizers beyond their traditional role in the extracorporeal purging of bone marrow grafts.

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.

Potentiation of merocyanine 540-mediated photodynamic therapy by salicylate and related drugs.

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.

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

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 (phi T) and singlet oxygen sensitization (phi delta) 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, carried out using clonogenic assays on L1210 leukemia cells, showed a good correlation with phi T and phi delta, 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.