Dynamics of pH-dependent self-association and membrane binding of a dicarboxylic porphyrin: a study with small unilamellar vesicles

AbstractSteady-state and stopped-flow measurements of the absorbance and fluorescence of aqueous solutions were performed to characterize the pH-dependent ionization and aggregation states of deuteroporphyrin. Porphyrin self-association promoted by neutralization of the carboxylic groups takes place within a few milliseconds impeding characterization of the monomer ionization states. Extrapolation at infinite dilution of the values obtained from steady-state measurements yielded the pKs of the carboxylic groups (6.6, 5.3) and inner nitrogens (4.1, 2.3). The kinetics of interactions of the porphyrin with unilamellar fluid state dioleoylphosphatidylcholine vesicles was examined in a large pH range, with focus on the entry step. From alkaline pH to a value of 6.5, the entrance rate is maximal (1.69×106 M−1 s−1 versus phospholipid concentration). It decreases to 2.07×105 M−1 s−1 at lower pH with an apparent pK of 5.39. This effect appears to be related to the formation of porphyrin dimer rather than to the protonation of inner nitrogen. In keeping with previous data, these results support the concept of a pH-mediated selectivity of carboxylic porphyrins for tumor. They also indicate that the propensity of these molecules to self-associate at low pH could yield to some retention in acidic intracellular vesicles of the endosome/lysosome compartment

A new UV-visible confocal laser scanning microspectrofluorometer designed for spectral cellular imaging.

International audienceWith the aim to perform spectroscopic studies and spectral images inside living cells, a microspectrofluorometer has been designed for two-dimensional spectral imaging in the visible and in the near-UV region. The main advantage of the device relies on its ability to scan the laser beam along one direction of the sample. This scanning is optically coupled with one direction of a bidimensional detector, allowing an instantaneous recording of a one-dimensional spectral image. The overall scanning of the sample is achieved by means of submicrometric displacements of the stage in the perpendicular direction. The main characteristics and performances of the microspectrofluorometer in terms of sensitivity (detection of a few molecules), spatial resolution (0.5 x 0.5 x 1 microm), and spectral resolution (1 nm) are presented. Finally, applications of this new apparatus concerning in situ localization and spectral characterization of two dyes are shown with Drosophila salivary glands (ethidium bromide) and T47D tumor cells (Hoechst 33342)

Confocal raman microspectroscopy and imaging study of theraphthal in living cancer cells.

Binary systems combining a transition metal complex and ascorbate have been proposed recently for catalytic therapy of malignant tumors. The killing effect on tumor cells is achieved by production of free radicals in the course of accelerated oxidation of ascorbate by dioxygen in the presence of transition metal complexes. Further progress in the development of binary catalytic systems (BCSs) requires a special method for their investigation in cells and tissues, because neither component of BCSs fluoresces. Here a resonance Raman confocal spectral imaging (RR CSI) technique was introduced as a unique approach to monitor quantitatively the transition metal complexes within living cells. Intracellular accumulation, localization, and retention of theraphthal (TP), a catalyst of the advanced TP/ascorbate BCS, were investigated in A549 cells with the RR CSI technique. The cellular analysis was complemented with the detailed study of molecular interactions of TP in solution and environmental factors affecting the RR spectrum of TP. TP does not penetrate into membranes, it binds very weakly to DNA and RNA, but it readily forms complexes with proteins. Binding with Ca(2+) cations and decreasing pH below 6 induce aggregation of TP. By analyzing RR spectra recorded from every point within a TP-treated cell, three states of the agent were discriminated, namely, monomeric TP in polar environment, TP bound to proteins, and aggregated TP. Their cytoplasmic and nuclear distributions were mapped at different stages of uptake and efflux. By introducing organelle-selective fluorescent probes into drug-treated cells and measuring intracellular localization of both the probe and the drug, compartmentation of TP was revealed. Cell growth suppression by the TP/ascorbate system was measured, and probable molecular and organelle targets of radical damage were characterized