(515d) Formation and Analysis of Tethered Bilayer Lipid Structures

Many important biological and pharmaceutical constructs such as cell membrane, exosomes and lipoplex nanoparticles are consisted of bilayer lipid structures. In this study, we varied the concentration of diphytanoylphosphatidylcholine (DPhyPC) phospholipids on a mixed self-assembled monolayer (SAM) with a thiolipid to form densely-packed tethered bilayer lipid membranes [(dp)tBLMs], loosely-packed tethered bilayer lipid membranes [(lp)tBLMs], or tethered bilayer liposome nanoparticles (tBLNs) on a planar surface. These tethered configurations were analyzed by atomic force microscopy (AFM) in the liquid medium, electrochemical impedance spectroscopy (EIS) and finite element method (FEM). The AFM graphs showed that a stable bilayer membrane with some grafted small liposomes formed when the DPhyPC concentration was higher than 5 mM. The Cole-Cole plots from the EIS measurements showed a small semicircular shape at high frequencies and a large curvy tail at low frequencies, indicating a densely packed membrane with few defects. The presence of grafted liposomes on the membrane surface did not affect the Cole-Cole plots and both AFM and EIS experiments were highly repeatable. When the DPhyPC concentration was decreased to <5 mM, the membrane became unstable. The AFM graphs showed the presence of nanopores on the membrane, caused by the tapping of the AFM tip. The pore size and location varied from case to case, indicating the transient nature of the membrane. Correspondingly, the Cole-Cole plots showed an incomplete semicircular shape at high frequencies and the shape was not repeatable experimentally. When the DPhyPC concentration was further decreased to ~0.6 mM or lower, many tethered nanoscale spherical liposomes with diameters ranging from 50 to 500 nm, instead of a lipid membrane, were observed by AFM and the small semicircular shape at high frequencies disappeared on the Cole-Cole plot. Instead, there was a large semi-circle covering a broad range of frequency with a tail near the low frequency end. The EIS measurements again became highly repeatable. The observed transition of bilayer lipid structures and Cole-Cole plots could be explained qualitatively by the finite element simulation using a 3-level circuit model by considering both the configuration and electric property (i.e. conductivity and permittivity) changes of the bilayer lipid structure. FEM modeling, together with AFM and EIS measurements, can be used as an analytical tool to study cell membrane dynamics, cell membrane-DNA interactions, cell membrane-lipoplex interactions by selecting lipid types and compositions as well as adding proper protein and sugar molecules.