I. Morphological and Nanomechanical Studies of Lipid Bilayers Composed of Polymerizable and Non-Polymerizable Lipids II. Fluidity Studies of Platelet Plasma Membranes

Abstract

Two main projects are discussed in this dissertation. The first project: Morphological and nanomechanical studies of planar supported lipid bilayers (PSLB) composed of polymerizable and non-polymerizable lipids as potential platforms for biosensors, is discussed in Chapters 2 through 4. Chapters 2 and 3 focus on PSLBs composed of the polymerizable lipid, bis-SorbPC and Chapter 4 focuses on bis-DenPC16,16. These studies are important because PSLBs are widely studied as platforms for receptor-based biosensors. PSLBs composed of fluid lipids lack the stability necessary for many technological applications due to the relatively weak non-covalent interactions between lipid molecules. Lipid polymerization enhances bilayer stability, but greatly reduces lipid diffusion and membrane fluidity. In an effort to enhance bilayer stability while maintaining fluidity, PSLBs composed of mixtures of polymerizable lipids and fluid lipids were prepared and characterized. Fluidity studies of these bilayers showed that considerable fluidity is retained even when the polymer fraction is substantial, which suggests that these bilayers are phase segregated, composed of polymerized and fluid domains. However, domains had not been observed previously. Chapter 2 of this dissertation describes the work done with atomic force microscopy (AFM) to study the phase segregation of mixed PSLBs composed of the polymerizable lipid bis-SorbPC and the fluid lipid DPhPC. This work provided direct evidence for polymerization-induced phase segregation of these mixed PSLBs, forming membranes composed of fluid and poly(lipid) domains. In these mixed bilayers, DPhPC formed a semi-continuous phase of greater height surrounding island-like domains of poly(bis-SorbPC) of lesser height. Numerous studies demonstrate that retention of bioactivity upon reconstitution of transmembrane proteins typically requires both membrane fluidity and elasticity. Thus, AFM force mapping was employed to study the nanomechanical properties of lipid bilayers, which is described in Chapter 3. This is the first study done to quantify the nanoscale mechanical properties of bis-SorbPC before and after polymerization, and mixed bilayer composed of bis-SorbPC and DPhPC. The results showed that the resistance to rupture and elastic modulus of bis-SorbPC increased upon polymerization. In addition, the results showed that the breakthrough force and the elastic modulus of DPhPC in mixed bilayers were different to pure bilayers due to the size (interface/edge effects) and the purity of the domains in mixed PSLBs. Findings similar to Chapters 2 and 3 are discussed in Chapter 4, with a different polymerizable lipid; bis-DenPC16,16. Comparing the results of bis-SorbPC (Chapter 3) and bis-DenPC (Chapter 4) showed that the position of the polymerizable moiety significantly changed the nanomechanical properties of PSLBs. In addition, no direct evidence of phase segregation was observed in mixed PSLBs composed of bis-DenPC and DPhPC during sub-micron scale morphological and nanomechanical studies. The second project: Fluidity studies of platelet plasma membranes, is the focus of Chapter 5. Therein, the feasibility of employing fluorescence recovery after photobleaching (FRAP) to determine the diffusion coefficient of platelet plasma membranes in response to lipophilic molecules is investigated. Mechanical circulatory devices, used in patients with heart failure to restore blood flow, cause thrombosis due to the abnormal flow of blood and supra-physiologic shear on blood platelets when blood passes through these devices, known as shear-mediated platelet activation (SMPA). It has been hypothesized that the membrane fluidity plays a role in treating SMPA and that the fluidity can be modulated with lipophilic molecules. Accordingly, the possibility of employing FRAP to study the lateral diffusion coefficient of platelet plasma membranes was investigated and a protocol was developed. The results showed that FRAP of platelet membranes is a suitable technique to determine the lateral fluidity of platelets before and after treating with exogenous lipophilic molecules. Therefore, the protocol established here will be helpful to study the fluidity of shear-activated platelets allowing to test the hypothesis. Further, this protocol will be beneficial to investigate the fluidity of platelet plasma membranes in the development of treatment methodologies targeting the material properties of platelets to reduce SMPA.