Modulation of Membrane Microviscosity by Protein-Mediated Carotenoid Delivery as Revealed by Time-Resolved Fluorescence Anisotropy.

Carotenoids are potent antioxidants with a wide range of biomedical applications. However, their delivery into human cells is challenging and relatively inefficient. While the use of natural water-soluble carotenoproteins capable to reversibly bind carotenoids and transfer them into membranes is promising, the quantitative estimation of the delivery remains unclear. In the present work, we studied echinenone (ECN) delivery by cyanobacterial carotenoprotein AnaCTDH (C-terminal domain homolog of the Orange Carotenoid Protein from Anabaena), into liposome membranes labelled with BODIPY fluorescent probe. We observed that addition of AnaCTDH-ECN to liposomes led to the significant changes in the fast-kinetic component of the fluorescence decay curve, pointing on the dipole-dipole interactions between the probe and ECN within the membrane. It may serve as an indirect evidence of ECN delivery into membrane. To study the delivery in detail, we carried out molecular dynamics modeling of the localization of ECN within the lipid bilayer and calculate its orientation factor. Next, we exploited FRET to assess concentration of ECN delivered by AnaCTDH. Finally, we used time-resolved fluorescence anisotropy to assess changes in microviscosity of liposomal membranes. Incorporation of liposomes with ß-carotene increased membrane microviscosity while the effect of astaxanthin and its mono- and diester forms was less pronounced. At temperatures below 30 °C addition of AnaCTDH-ECN increased membrane microviscosity in a concentration-dependent manner, supporting the protein-mediated carotenoid delivery mechanism. Combining all data, we propose FRET-based analysis and assessment of membrane microviscosity as potent approaches to characterize the efficiency of carotenoids delivery into membranes.

E/Z isomerization of astaxanthin and its monoesters in vitro under the exposure to light or heat and in overilluminated Haematococcus pluvialis cells.

Thermo- and photoisomerization of astaxanthin was investigated in a model system (solutions in methanol and chloroform), and the dynamics of astaxanthin isomers and esters content was analyzed in Haematococcus pluvialis green algal cells exposed to factors inducing astaxanthin accumulation. In both systems, the astaxanthin isomerization process seems to be defined by a) the action of light (or heat), and b) the dielectric constant of the surrounding medium. Upon heating, the accumulation of Z-isomers occurred in a model system during the entire incubation period. For the first 5 h of illumination, both Z-isomers accumulated in the solutions up to 5%, and then their content decreased. The accumulated amount of the Z-isomers in the cells of H. pluvialis was found to reach 42% of the total content of astaxanthin initially, and then it decreased during the experiment. The results lead to a conclusion that both cultivation of H. pluvialis culture in specific conditions and heat treatment of the resulting extracts from it might be efficient for obtaining large amounts of economically useful astaxanthin Z-isomer.

Distribution coefficient of rifabutin in liposome/water system as measured by different methods.

Distribution coefficient (D) of rifabutin in liposome/water system was measured by phase separation and fluorescence probe quenching techniques. D values were identical suggesting that rifabutin is fully immersed into lipid bilayer. Structural studies of phospholipid bilayer employing (31)P NMR spectroscopy demonstrated that introduction of rifabutin does not alter the bilayer structure. A scheme of the rifabutin position in lipid bilayer based on the calculated size of rifabutin molecule is proposed.

Antimycobacterial activity of bacteriocins and their complexes with liposomes.

OBJECTIVES: Bacteriocins (Bcn) are natural peptides that are secreted by several taxonomically distant bacteria and exert bactericidal activity against other bacterial species. Their capacity to inhibit growth of virulent Mycobacterium tuberculosis H37Rv was evaluated in this study. METHODS: Five different Bcn were isolated and purified from bacterial culture supernatants, their amino acid sequence was determined, and activity against mycobacteria assessed in three different models: in vitro mycobacterial cultures, in vitro infection of mouse macrophages and in vivo high-dose infection of inbred mice. RESULTS: In the in vitro model, four out of five Bcn exhibited stronger antimycobacterial activity than equal concentrations of a widely used anti-TB antibiotic, rifampicin. These Bcn were non-toxic for mouse macrophages at a concentration of 0.1 mg/L (>MIC(90) of these compounds). Pure Bcn did not inhibit mycobacterial growth within murine macrophages when added at 0.01-0.1 mg/L, suggesting that at physiologically tolerable concentrations these molecules do not penetrate through the membrane of eukaryotic cells. However, when administered as a complex with phosphatidylcholine-cardiolipin liposomes, Bcn5 (selected as a model compound due to its cytotoxicity and antimycobacterial activity regular titration curves) demonstrated capacity both to inhibit intracellular growth of M. tuberculosis and to prolong survival of mice in an acute TB model. CONCLUSIONS: Given that the mechanism of Bcn bactericidal activity differs from that of all commonly used antibiotics, their possible involvement in complex TB therapies deserves further study.