Polymyxin B-Enriched Exogenous Lung Surfactant: Thermodynamics and Structure.

The use of an exogenous pulmonary surfactant (EPS) to deliver other relevant drugs to the lungs is a promising strategy for combined therapy. We evaluated the interaction of polymyxin B (PxB) with a clinically used EPS, the poractant alfa Curosurf (PSUR). The effect of PxB on the protein-free model system (MS) composed of four phospholipids (diC16:0PC/16:0-18:1PC/16:0-18:2PC/16:0-18:1PG) was examined in parallel to distinguish the specificity of the composition of PSUR. We used several experimental techniques (differential scanning calorimetry, small- and wide-angle X-ray scattering, small-angle neutron scattering, fluorescence spectroscopy, and electrophoretic light scattering) to characterize the binding of PxB to both EPS. Electrostatic interactions PxB-EPS are dominant. The results obtained support the concept of cationic PxB molecules lying on the surface of the PSUR bilayer, strengthening the multilamellar structure of PSUR as derived from SAXS and SANS. A protein-free MS mimics a natural EPS well but was found to be less resistant to penetration of PxB into the lipid bilayer. PxB does not affect the gel-to-fluid phase transition temperature, Tm, of PSUR, while Tm increased by ∼+ 2 °C in MS. The decrease of the thickness of the lipid bilayer (dL) of PSUR upon PxB binding is negligible. The hydrophobic tail of the PxB molecule does not penetrate the bilayer as derived from SANS data analysis and changes in lateral pressure monitored by excimer fluorescence at two depths of the hydrophobic region of the bilayer. Changes in dL of protein-free MS show a biphasic dependence on the adsorbed amount of PxB with a minimum close to the point of electroneutrality of the mixture. Our results do not discourage the concept of a combined treatment with PxB-enriched Curosurf. However, the amount of PxB must be carefully assessed (less than 5 wt % relative to the mass of the surfactant) to avoid inversion of the surface charge of the membrane.

Calcium mediated DNA binding in non-lamellar structures formed by DOPG/glycerol monooleate.

In order to test an encapsulation method of short fragmented DNA (∼ 20-300 bp), we study the solubilisation in 150 mM solution of NaCl of a cubic phase formed by glycerol monooleate (GMO) with negatively charged dioleoylphosphatidylglycerol (DOPG) up to the level of unilamellar vesicles and, subsequently, the restoration of the cubic phase using Ca2+ cations. We performed small angle X-ray and neutron scattering (SAXS and SANS) to follow structural changes in DOPG/GMO mixtures induced by increasing DOPG content. The cubic phase (Pn3m space group) is preserved up to ∼ 11 mol% of DOPG in DOPG/GMO. Above 20 mol%, the SANS curves are typical of unilamellar vesicles. The thickness of the DOPG/GMO lipid bilayer (dL) decreases slightly with increasing fraction of DOPG. The addition of 15 mM of CaCl2 solution shields the electrostatic repulsions of DOPG molecules, increases slightly dL and restores the cubic structures in the mixtures up to ∼ 37 mol% of DOPG. Zeta potential shows negative surface charge. The analysis of the data provides the radius of the water nano-channels of the formed non-lamellar structures. We discuss their dimensions with respect to DNA binding. In addition, Ca2+ mediates DNA - DOPG/GMO binding. The formed hexagonal phase, HII, binds less of DNA in comparison with cubic phases (∼ 6 wt% and ∼ 20 wt% of the total amount, respectively). The studied system can be utilized as anionic QII delivery vector for genetic material.