CC BY
10B-O-4
BIOMEDICAL PHOTONICS
Protein-mediated carotenoid delivery into liposomes affects local microviscosity of the membranes as revealed by fluorescence anisotropy
A.N. Semenov1, D.A. Gvozdev1, A.R. Hashimova1, Eu.Yu. Parshina1, D.V. Zlenko1, A.A. Bayzhumanov1, N.Y. Lotosh2, A.A. Selishcheva12, N.N. Sluchanko13, Eu.G. Maksimov13
1-M.V. Lomonosov Moscow State University, 1 Leninskie Gory St., Moscow, 119991, Russia 2- National Research Center "Kurchatov Institute", 1 Acad. Kurchatov Sq., Moscow, 123182, Russia 3- Federal Research Center of Biotechnology of the Russian Academy of Sciences, 33 Leninskyprospect, Moscow, 119071, Russia semenov@physics.msu.ru
Carotenoids are natural substances with a range of biomedical applications due to their profound antioxidant properties. Anti-inflammation and oxidative protection effects of carotenoids significantly improve the efficiency of treatment of neurodegenerative pathologies, cancer, hematologic and cardiovascular deceases. Since carotenoids are not synthesized in human, there should be a constant supply of carotenoid-rich nutrients or specialized supplements and drugs. However, the delivery of carotenoids is limited by their lipophilic properties, which limits their efficiency in water solutions and hence biocompatibility. A possible means to overcome this obstacle is the use of special water-soluble proteins capable of reversibly binding carotenoids and transferring them into the cell membrane. In particular, the AnaCT-DH protein based on the water-soluble carotenoprotein isolated from the cyanobacterium Anabaena sp. PCC 7120 [1] proved efficient in delivering carotenoid echinenone into membranes of cultured human cells of a different type. The aim of the present work was to study the interactions between AnaCTDH and the membrane of model mono-lamellar liposomes by assessing membrane microviscosity to characterize the efficiency of protein-mediated delivery of carotenoids.
Liposomes were made from soy lipid S75 (phospholipid with 70 % phosphatidylcholine and 20% phosphatidyleth-anolamine, Lipoid GmbH, Germany) as described earlier [2]. The produced liposomes were homogeneous with a mean diameter of 100 nm. Liposomes were labeled by fluorescent probe 1,3,5,7-tetramethyl-BODIPY which steadily emits fluorescence only being bounded within membrane and its fluorescence intensity and lifetime depend on the local surroundings. After the labeling, the solution of liposomes was supplemented with echinenone-carrying AnaCTDH. The membrane microviscosity was measured by assessing anisotropy of fluorescence of BODIPY in the membrane. The excitation was performed by picosecond optical pulse generator (InTop, Russia) with excitation at 500 nm and detection at 560 nm. Two fluorescence decay curves, corresponding to vertical and horizontal polarization of detected fluorescence, were measured using TCSPC system with hybrid photodetector GaAsP (Becker&Hickl GmbH, Germany). Kinetics were comprised to each other to determine the rotational correlation time (0) of the BODIPY molecule in the membrane. Since 0 is proportional to membrane viscosity n [3], we were able to estimate changes in microviscosity of liposomes membranes after the incubation with AnaCTDH at different concentrations.
Addition of AnaCTDH to liposomes increased 0 only in the temperature range 10o-30oC. The effect was concentration-dependent: at 20oC with AnaCTDH concentration 720 nM 0=0.429±0.007 ns, at 1440 nM 0=0.492±0.010 ns (in control intact liposomes 0=0.370±0.013 ns). It means echinenone is delivered into liposomes membrane. At higher temperatures (30o-40oC), the correlation time 0 does not change with an addition of AnaCTDH, demonstrating that echinenone does not change microviscosity. It means that at physiological temperatures incorporation of liposomes with echinenone does not erupt viscoelasticity and biomechanics of membrane and does not affect its stability and integrity.
The present work was supported by Russian Scientific Foundation grant № 22-25-00183.
[1] E. G. Maksimov, E. Yu. Parshina, N. N. Sluchanko et al. Soluble cyanobacterial carotenoprotein as a robust antioxidant nanocar-rier and delivery module, Antioxidants, Vol. 9(869), PP. 1-23 (2020).
[2] V. A. Shchelkonogov, N.Y Lotosh, A.A. Selishcheva et al. Lipoic acid nanoforms based on phosphatidylcholine: production and characteristics, European Biophysics Journal, Vol. 49, PP. 95-103 (2020).
[3] J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Springer), Fluorescence Anisotropy (1999).
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