Characterization and analytical applications of dye-encapsulated zwitterionic liposomes

Abstract

The aim of this project was to use marker encapsulated liposomes as biomembrane mimicking entities in order to study membrane properties like permeability and to better understand the interaction of biological lipid bilayers with membrane-active molecules, like beta blocker drugs and antimicrobial peptides (AMP). The physical characteristics of liposomes, such as size, surface charge and encapsulation capacity were also studied using electrophoretic, fluorescence and light scattering techniques. In addition, marker-encapsulated and self-lysing liposomes were used to study antigen-antibody binding. The immunoassay application of these self-lysing liposomes was also investigated. The first area of research is focused on investigating the effect of the liposome lipid composition on the size and the electrical properties of zwitterionic liposomes. The cholesterol composition of phosphatidylcholine (PC) and sphingomyelin (Sph) liposomes is varied and the effect on their size, zeta potential and electrophoretic mobility is monitored using dynamic light scattering (DLS), laser doppler velocimetry (LDV), and capillary zone electrophoresis (CZE) techniques, respectively. In addition, the permeability and the encapsulation capacity of large unilamellar vesicles (LUV), or liposomes that are made by extrusion, were compared as their lipid and cholesterol composition varied. The size and electrophoretic mobility of zwitterionic liposomes was found to increase with the cholesterol composition. The interaction of indolicidin, a 13-mer cationic AMP, with (dye-encapsulated) liposomes that were made of different lipid and cholesterol composition was investigated by DLS, fluorescence and capillary electrophoresis (CE) methods. DLS results show a change in liposome size, and size distribution index (PI), after indolicidin interaction. Fluorescence leakage experiments show the extent of membrane perturbation caused by the AMP and the AMP’s innate tryptophan fluorescence provided qualitative information regarding the type (polar/non-polar) and nature of the liposome-AMP interaction, as lipid composition of the liposomes varied. In addition, CZE and liposome electrokinetic chromatography (LEKC) techniques were also used to further probe the (polar/non-polar/electrostatic) nature of this interaction. The immunoassay application of the marker encapsulated liposomes was investigated using a combination of fluorescence, DLS, and CE-LIF (capillary electrophoresis with laser induced fluorescence detector) techniques. The liposomes were made from a non-lamellar lipid DOPE (dioleoylphosphatidylethanolamine) that was stabilized with a 20% bilayer lipid DPPC (dipalmitoylphosphatidylcholine) and a 1% hapten-attached DPPE lipid. Small hapten molecules, like biotin and DNP (dinitrophenyl), were attached to the liposome surface via the DPPE lipid, and used to detect their conjugate molecules (avidin and anti-DNP antibody) in a homogeneous solution. The biotin-attached DOPE liposomes aggregate and leaked their marker content in standard avidin solution. The extent of liposome aggregation and the fluorescence intensity of the leaked dye are dependent on the concentration of avidin present in solution. The different parameters that affect the quality of the assay were also investigated.