title: Filter-extruded liposomes revisited: a study into size distributions and morphologies in relation to lipid-composition and process parameters.
abstract: Filter-extrusion is a widely used technique for down-sizing of phospholipid vesicles. In order to gain a detailed insight into size and size distributions of filter-extruded vesicles composed of egg phosphatidyl-choline (with varying fractions of cholesterol)--in relation to extrusion-parameters (pore-size, number of filter passages, and flow-rate), flow field-flow fractionation in conjunction with multi-angle laser light scattering (AF4-MALLS, Wyatt Technology Corp., Santa Barbara, CA) was employed. Liposome size-distributions determined by AF4-MALLS were compared with those of dynamic light scattering and correlated with cryo-transmission electron microscopy and (31)P-NMR-analysis of lamellarity. Both the mean size of liposome and the width of size distribution were found to decrease with sequential extrusion through smaller pore size filters, starting at a size range of ≈70-415 nm upon repeated extrusion through 400 nm pore-filters, eventually ending with a size range from ≈30 to 85 nm upon extrusion through 30 nm pore size filters. While for small pores sizes (50 nm), increased flow rates resulted in smaller vesicles, no significant influence of flow rate on mean vesicle size was seen with larger pores. Cholesterol at increasing mol fractions up to 0.45 yielded bigger vesicles (at identical process conditions). For a cholesterol mol fraction of 0.5 in combination with small filter pore size, a bimodal size distribution was seen indicating cholesterol micro-crystallites. Finally, a protocol is suggested to prepare large (∼ 300 nm) liposomes with rather narrow size distribution, based on the filter extrusion at defined flow-rates in combination with freeze-/thaw-cycling and bench-top centrifugation.
authors.affiliation: a Department of Physics , Chemistry and Pharmacy, University of Southern Denmark , Campusvej , Odense , Denmark .
authors.forename: Askell
authors.initials: A
authors.lastname: Hinna
authors.affiliation: b Center for Electron Microscopy of the Medical Faculty, Friedrich Schiller University Jena , Ziegelmühlenweg , Jena , Germany , and.
authors.forename: Frank
authors.initials: F
authors.lastname: Steiniger
authors.affiliation: c Aker BioPharma AS , Fjordallèen , Oslo , Norway.
authors.forename: Stefan
authors.initials: S
authors.lastname: Hupfeld
authors.affiliation: a Department of Physics , Chemistry and Pharmacy, University of Southern Denmark , Campusvej , Odense , Denmark .
authors.forename: Paul
authors.initials: P
authors.lastname: Stein
authors.affiliation: a Department of Physics , Chemistry and Pharmacy, University of Southern Denmark , Campusvej , Odense , Denmark .
authors.forename: Judith
authors.initials: J
authors.lastname: Kuntsche
authors.affiliation: a Department of Physics , Chemistry and Pharmacy, University of Southern Denmark , Campusvej , Odense , Denmark .
authors.forename: Martin
authors.initials: M
authors.lastname: Brandl
mesh_terms: D008055:Lipids / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry*; D010316:Particle Size
chemical_list: D008055:Lipids; D008081:Liposomes
keywords: Cryo transmission electron microscopy; flow field-flow fractionation; lamellarity; multi-angle light scattering