Different ways to insert carotenoids into liposomes affect structure and dynamics of the bilayer differently

We apply and quantify two techniques to incorporate carotenoids into liposomes: (i). preparation of unilamellar liposomes from mixtures of phospholipids and a carotenoid or cholesterol; (ii). insertion of carotenoids into prepared liposomes. Homogeneous liposomal fractions with a vesicle size diameter of approximately 50 nm were obtained by an extrusion method. The resulting vesicles were subjected to a three-dimensional light scattering cross-correlation measurement in order to evaluate their size distribution. The fluorescent dyes Laurdan, DiI-C(18), C(6)-NBD-PC were used to label the liposomes and to evaluate modulations of ordering, hydrophobicity and permeability to water molecules adjacent to the bilayer in the presence of carotenoids and/or cholesterol. Zeaxanthin incorporation (up to 0.1-1 mol%) attributes to the symmetric and ordered structure of the bilayer, causing both a strong hydrophobicity and a lower water permeability at the polar region of the membrane. The incorporation of lutein has similar effects, but its ordering effect is inferior in the polar region and superior in the non-polar region of the membrane. beta-Carotene, which can be incorporated at lower effective concentrations only, distributes in a more disordered way in the membrane, but locates preferentially in the non-polar region and, compared to lutein and zeaxanthin, it induces a less ordered structure, a higher hydrophobicity and a lower water permeability on the bilayer.