Luc Villeneuve

Steady state and time-resolved fluorescence properties of metastatic and non-metastatic malignant cells from different species

Steady state and time-resolved fluorescence spectroscopy were employed to study the fluorescence from non-metastatic, metastatic and non-tumorigenic cell lines from different species. Excitations at 310 nm and 350 nm were used to monitor tryptophan and reduced nicotinamide adenine dinucleotide (NADH) fluorescence respectively. Subtle and consistent differences were observed between different categories of cell lines. It was found that the tryptophan to NADH fluorescence intensity ratio is higher in metastatic cell lines than in non-metastatic and normal cell lines. The fluorescence decay of the tryptophan residue in different cell lines was best described by triple exponential kinetics, whereas the NADH fluorescence decay was best described by mainly double and, in some cases, triple exponential kinetics. The average fluorescence lifetimes for tryptophan were in the range 2.5-3.7 ns. The average lifetime of NADH was lower (by a factor of approximately three) in metastatic cells than in non-metastatic cells and this finding is consistent for cell lines from different origins (rat or human). Correcting the fluorescence intensity for the average fluorescence lifetime of each species and for the volume of each cell line, it was shown that the concentrations of tryptophan and NADH are consistently higher in malignant metastatic cancer cells than in non-metastatic cells.

Spectroscopic and photophysical properties of some new rhodamine derivatives in cationic, anionic and neutral micelles

Abstract The spectroscopic and photophysical characterization of rhodamine 123 (dye 1 ), 4,5-dibromorhodamine methyl ester (dye 2 ) and 4,5-dibromorhodamine n-butyl ester (dye 3 ) are reported in homogeneous media like water and some alcohols and also in microheterogeneous media; anionic sodium dodecylsulfate (SDS), cationic cetyltrimethylammonium bromide (CTAB) and neutral triton X-100 (TX) micelles. The selective biodistribution of these ionic drugs in tissues and membranes strongly influence their photosentisizing properties which have been part of our earlier studies. Results suggest that the hydrogen bonding capability of the amino end group lone pair of these dyes dominates in water. All these dyes interact with anionic SDS micelles. The interaction is mainly electrostatic in nature. At low SDs concentrations (below c.m.c.), dye-SDS aggregate formation takes place. But above c.m.c. only the monomeric dye form is observed. The penetration of dye 3 in SDS is a little less compared to dyes 1 and 2 . Dyes 2 and 3 show a finite interaction with CTAB micelle unlike dye 1 . With neutral TX micelles all the dyes form strong complexes. The fluorescence quantum yield ( Φ F ) of these three dyes in TX is lower. In time-resolved fluorescence experiments, two lifetimes are observed. The effects of the TX concentration on the fluorescence decay are measured. The decay associated spectra of dye 2 in TX are obtained by global compartmental analysis. The dye-surfactant interaction mechanisms are also discussed.

Phototoxicity of Some Bromine‐Substituted Rhodamine Dyes: Synthesis, Photophysical Properties and Application as Photosensitizers

Abstract— The synthesis of some bromine‐substituted rhodamine derivatives viz., 4,5‐dibromorhodamine methyl ester (dye 2) and 4,5‐dibromorhodamine n ‐butyl ester (dye 3) are reported. These dyes were synthesized to promote a more efficient cancer cell photosensitizer for potential use in in vitro bone marrow purging in preparation for autologous bone marrow transplantation. Spectroscopic and photo‐physical characterization of these dyes together with rhodamine 123 (dye 1) are reported in water, methanol, eth‐anol and also in a microheterogeneous system, sodium dodecyl sulfate. The possible mechanism of photosensi‐tization is characterized in terms of singlet oxygen efficiency of these dyes. Singlet oxygen quantum yields for bromine‐substituted dyes are in the range of 0.3‐0.5 depending on the solvent. For dye 1 no singlet oxygen production is found. The photodynamic actions of these dyes in different cell lines are tested. It was found that dye 2 and dye 3 are efficient photosensitizers and mediate eradication of K562, EM2, myeloid cell lines (CML) and the SMF‐AI rhabdomyosarcoma line.

Ex vivo photodynamic purging in chronic myelogenous leukaemia and other neoplasias with rhodamine derivatives.

Photodynamic therapy (PDT), a cancer treatment already used early in this century, has distinctive advantages over conventional chemotherapy, namely its often observed preferential accumulation in cancer cells and its low intrinsic toxicity. Aggressive therapeutic modalities using high doses of chemotherapy and/or radiation therapy are now commonplace treatments for leukaemia, lymphoma and various non‐haematologic malignancies. These intensive approaches have often been used in association with haematopoietic‐progenitor‐cell support and have induced major responses and remissions in patients with relapsed and refractory diseases, ultimately contributing to improve the disease‐free survival of patients with high risk. This has encouraged Theratechnologies, a Montreal‐based pharmaceutical company, to develop photodynamic ex vivo purging procedures, including the development of new photosensitizers and irradiation devices for the safe eradication of neoplastic cells from autologous grafts. Our first specific objective, therefore, was to design, synthesize, purify and test photoactive rhodamine derivatives. We have also selected a gas and phosphorus coating characteristic of an efficient scanning fluorescent source for extra‐corporeal PDT using rhodamine derivatives. 4,5‐Dibromorhodamine 123 (TH9402) was selected because of its photophysical properties, low toxicity and stability. TH9402 photodynamic‐cell‐therapy process conditions recognized as safe for normal human haematopoietic stem cells and progenitors demonstrated the efficacy of the purging procedure on various leukaemias (including chronic‐myelogenous‐leukaemia as well as non‐Hodgkin‐leukaemias and metastatic‐breast‐cancer cell lines.

Spectroscopic and photophysical investigations on the nature of localization of rhodamine-123 and its dibromo derivative in different cell lines.

Steady-state and time-resolved spectroscopic properties of rhodamine-123 (rh123) and 4,5-dibromorhodamine methyl ester (dbr123) bound to different cell lines are evaluated. Studies are also performed on the dye bound to extracted mitochondria. Results are compared with those obtained in homogeneous and microheterogeneous media. Results suggest that these dyes can specifically bind only with cell mitochondria. As a result of binding, excitation and emission spectra are red shifted by 10 to 12 nm. The fluorescence decay of these dyes bound to mitochondria shows two lifetimes. Values are about 4.0 and 2.0 ns forrh123 and about 1.9 and 0.5 ns fordbr123. Detailed global analysis of emission wavelength and dye concentration dependences of the fluorescence decay is performed. Results indicate that these dyes are bound to two different binding sites at mitochondria. The decay-associated fluorescence spectrum for the species corresponding to each binding site is recovered. Species1, corresponding to the longer lifetime, is found to be more red shifted compared to species2. The fluorescence of species2 is heavily quenched. The origin of this quenching is explained in terms of resonance energy transfer between donor species2 and acceptor species1. The possible nature of the two binding sites is also discussed.