R. Ragheb, J. Bolze, G. Taylor, N. Tamaddoni, D. Griffiths, U. Nobbmann
Keywords: liposomes, nanoparticle tracking analysis, dynamic light scattering, X-ray scattering, size, concentration, fluorescence, zeta potential
Summary:Liposomes are spherical vesicles with an aqueous core enclosed by one or more phospholipid bilayers or lamellae and are frequently classified based on their size and number of bilayers . They are often used in studies of model biological membranes, phase transition and spacing, targeted drug delivery to specific areas of a human body, etc . The biophysical characterization of these systems and their payloads is critical to understanding and optimizing their fabrication and function. This study looks at optimal conditions for extruding liposomes as well as their stability under different conditions. We highlight the limit of detection for fluorescently labeled liposomes. The intricacies of liposome formation and characterization were measured by Nanoparticle Tracking Analysis (NTA) using a NanoSight NS300 and Dynamic (DLS) and Electrophoretic (ELS) Light Scattering using a Zetasizer ZSP, as well as by small-and wide-angle X-ray scattering (SAXS/WAXS) using an Empyrean Nano edition. DOPC, DOPC-Cholesterol, and Rh-DPPE incorporated lipids were used to prepare MLV suspensions. They were repeatedly frozen/thawed 5x, unless otherwise noted, and stored at -20°C until extrusion and Small Unilamellar Vesicle (SUV) formation. SUVs were formed by extruding thawed MLV suspensions through hand-held NanoSizer mini-extruders housing polycarbonate track-etched membranes of various pore-sizes including 50 nm, 100 nm, 200 nm, and 400 nm. The single-use NanoSizer kits (extruders, syringes and needles) and reusable extruder heating block were obtained from T&T Scientific (Knoxville, TN). NTA and DLS confirmed a number of metrics. Eleven passes were needed to reach target pore sizes. Starting lipid concentration (2 vs 20 mg/mL) had minimal effect on size freeze-thaw cycles on size and concentration, Nanosizer Extruders proved robust with stable size and concentration across 20 mL (11 passes/mL). There was a noticeable difference in resulting concentrations of liposomes subjected to step-down extrusion vs extrusion directly at different pore sizes. This provided milder conditions and higher retention of liposomes. Increasing the percentage of cholesterol in DOPC further displaced head groups in the lipid bilayer causing an increase in the zeta potential. Fluorescence measurements on Rh-DPPE incorporated into DOPC showed that lower percentages could be further measured to determine the true limit of detection. Small-angle X-ray scattering (SAXS) allowed to investigate the bilayer stacking in MLVs and from wide-angle X-ray scattering (WAXS) data the order of the alkyl chains (gel phase vs. liquid phase) could be studied as a function of sample temperature. References:  Patil YP, Jadhav S. Chemistry and Physics of Lipids 177 (2014) 8-18.  Chibowski E, Szczes A. Adsorption 22 (2016) 755-765.