Design and Biological Evaluation of hBest1-Containing Bilayer Nanostructures

Abstract

Bestrophinopathies are a group of inherited retinal diseases caused by mutations in the BEST1 gene. The protein encoded by this gene, bestorphin-1 (hBest1), is a calcium-dependent transmembrane channel localized on the basolateral membrane of retinal pigment epithelial (RPE) cells. We have already demonstrated the surface behavior and organization of recombinant hBest1 and its interactions with membrane lipids such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), sphingomyelin (SM) and cholesterol (Chol) in models of biological membranes, which affect the hBest1 structure-function relationship. The main aim of our current investigation is to integrate pure hBest1 protein into lipid bilayer nanostructures. We synthesized and characterized various hBest1-containing nanostructures based on 1,2-Dipalmitoylphosphatidylcholine (DPPC), SM, glycerol monooleate (GMO) and Chol in different ratios and determined their cytotoxicity and incorporation into cell membranes and/or cells by immunofluorescence staining. Our results show that these newly designed nanoparticles are not cytotoxic and that their incorporation into MDCK II cell membranes (used as a model system) may provide a mechanism that could be applied to RPE cells expressing mutated hBest1 in order to restore their ion transport functions, affected by mutated and malfunctioning hBest1 molecules.

Keywords: MDCK II cells; bilayer nanostructures; hBest1.

Scheme 1

Illustration of the pentameric structure of hBest1 (in blue). Lipids are in yellow.

Scheme 2

Schematic presentation of the formation of vesicles containing hBest1.

Figure 1

Cryo-TEM images of DPPC/SM/Chol vesicles with sizes 50–250 nm, some of them multivesicular (a): average size (measured for 100 objects) of 135 nm, with size distribution in histogram below (b); and DPPC/Chol/GMO vesicles with sizes 40–380 nm, some of them multivesicular (c): average size (measured for 100 objects) of 145 nm, with size distribution in histogram below (d). Thickness of all membranes: ~5 nm. All vesicles contain 0.15 mol % of hBest1.

Figure 2

Cryo-TEM images of SM/Chol vesicles with sizes 50–200 nm, some of them multivesicular (a), with small number of particles with sizes over 200 nm (300–600 nm) and average size (measured for 100 objects) of 130 nm, with size distribution in histogram below (b); and DPPC/Chol vesicles with sizes 80–315 nm, some of them multivesicular or multilamellar (c), with small number of particles with sizes over 200 nm (300–600 nm) and average size (measured for 100 objects) of 171 nm, with size distribution in histogram below (d). Thickness of all membranes: ~5 nm. All vesicles contain 0.15 mol % of hBest1.

Figure 3

Metabolic activity defined via the MTT test after 24 h of incubation of MDCK II cells treated with different concentrations of (1) DPPC/Chol/SM, (2) DPPC/Chol/GMO, (3) SM/Chol and (4) DPPC/Chol vesicles as well as 1b, 2b, 3b and 4b vesicles containing, in addition to the same ratio of lipids, the protein hBest1. The data are presented as percentages of control cells as the mean ± SE; n = 3.

Figure 4

Immunofluorescence staining of hBest1 (green) in MDCK II cells treated for 2h with (1b) DPPC/Chol/SM and (2b) DPPC/Chol/GMO containing hBest1 protein. Nuclei are in blue; scale bar = 15 μm. The concentration of vesicles used in cell treatment was 1.5 µg/mL relative to hBest1. Negative control—MDCK II cells. Positive control—MDCK II-hBest1 cells.