Small angle X-ray scattering (SAXS)

SAXS measurements were performed on a SAXSLAB GANESHA 300 XL SAXS system (Denmark) equipped with a GeniX 3D Cu ultra low divergence micro focus sealed tube source (France) that produced X-rays with a wavelength of λ=1.54 Å. A sample-to-detector distance of 713 mm was used to access a q-range of 0.15 ≤ q ≤ 4.47 nm⁻¹ with q=4π/λ(sin θ), where 2θ was the angle between the incident X-ray beam and the detector measuring the scattered intensity. Extruded lipid suspensions of DOPC/DPPC/cholesterol (23:47:30 molar ratio; 40 mM) with and without 10 mol% of artepillin C were placed in 2-mm quartz capillaries (Germany). The sample temperature was kept at 30°C with the aid of a Julabo temperature controller (Germany). The acquisition time of the SAXS data was 6 hours for each sample, and the background signal (scattering of a capillary filled with water) was subtracted from the obtained profiles. The experimental SAXS diagrams were fitted by using the Global Analysis program (GAP) version 1.3, provided by Dr. Georg Pabst of the Austrian Academy of Sciences – Graz. Herewith, we obtained the electron density profile of the polar head group, of the acyl chain regions of the lipid bilayers, and of the fraction of the resulting unilamellar vesicles (32,33) by using the function:

where NUV is the fraction number of positionally non-correlated particles (i.e., unilamellar vesicles), S(q) is the structure factor (inter-particle interaction), and F(q) is the form factor, which gives the electron density profile. From the parameters that describe the head group regions, it was possible to calculate the thickness of the membrane (dB) through the following equation (34):

where z_H is the headgroup position measured from the center of the bilayer, and σ_H is the width of the Gaussian of the electron-dense distribution over the headgroup region.