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

Abstract

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 β-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.

Probing Red Blood Cell Membrane Microviscosity Using Fluorescence Anisotropy Decay Curves of the Lipophilic Dye PKH26

“…The membrane microviscosity of fresh egg yolk lecithin liposomes was higher than the microviscosity of RBC membranes and slightly higher (Table 1 and Supplementary Figure S2) than the membrane fluidity microviscosity of typical lecithin liposomes [46]. We previously obtained similar results of high membrane microviscosity of fresh egg yolk lecithin-made liposomes in comparison with liposomes prepared from different phospholipids [47]. Such a result can be explained by the abundance of additional components, which, along with phosphatidylcholine (80.5%) and phosphatidylethanolamine (11.7%) as main constituents, are presented in fresh egg yolk lecithin, and include cholesterol, lysophosphatidylcholine, sphingomyelin, and several minor neutral lipids [48].…”

BODIPY Dye Derivative for Irreversible Fluorescent Labeling of Eukaryotic Cells and Their Simultaneous Cytometric Analysis

“…The absorption and fluorescence emission spectra were measured using a Cary50 Bio spectrophotometer (Varian) and a Cary Eclipse spectrofluorometer (Varian), respectively. The fluorescence quantum yield was measured using a homologous derivative of BODIPY, 8-decene-1,3,5,7-tetramethyl-BODIPY [ 3 , 4 , 5 ], as a standard (the quantum yield of the standard in DMSO was considered to be 0.99).…”

“…Спектры поглощения и эмиссии флуоресценции измеряли с использованием спектрофотометра Cary50 Bio (Varian) и спектрофлуориметра Cary Eclipse (Varian) соответственно. Квантовый выход флуоресценции измеряли с использованием гомологичного производного BODIPY: 8-децен-1,3,5,7-тетраметил-BODIPY [3][4][5], в качестве стандарта (квантовый выход стандарта в DMSO считали равным 0.99).…”