Revealing composition and conformation of liposomes using integrated methods of HPLC/AF4, UV-Vis absorption, Refractive Index, MALLS, DLS, and SAXS-WAXS

Full text: Liposomes are increasingly better developed as efficient drug carriers. Structural characterization of the functional liposomes with and without drug-uptake and the consequent drug transport and conditional drug delivery, is of interest in biomedical applications. Here, we report an established combined methods using high-performance liquid chromatography (HPLC), asymmetric flow field- flow fractionation (AF4), UV-Vis absorption, refractive index (RI), multi-angle laser light scattering (MALLS), dynamic light scattering (DLS), and small and wide angle X-ray scattering for structural characterization of liposome solutions. We demonstrate an example of using the integrated system to successfully determine hydrodynamic radius and its distribution, molecular mass, lipid aggregation number, of a model liposome system of unilamellar vesicles (ULV). The radius of gyration Rg of 50 nm, hydrodynamic radius Rh of 50 nm, bilayer thickness, and lipid aggregation number of the ULV, with and without drug molecule loading, are determined using simultaneous small and wide angle X-ray scattering, incorporated with HPLC/UV-vis/RI, at the high-flux 13A BioSAXS undulator beamline of the 3.0 GeV Taiwan Photon Source. Moreover, a chain-chain packing peak of the ULV bilayers could be observed at q = 1.488Å-1 (d = 4.28 Å); reversible changes of the peak position and peak broadening with temperatures between 20 – 50C, providing a direct evidence of the correlation of the lipid chain-chain packing with the commonly observed gel-ripple-fluid phase transitions of multilamellar vesicles. (author)