The long term goal of the research proposed here is to understand the molecular mechanisms involved in electric field induced permeabilization and fusion of artificial and natural membranes. This long term goal will be achieved by accomplishing the following specific aims: i) measure the thermomechanical properties (in particular the level of cohesion) of reconstituted lipid membranes and relate these properties to the electric field strengths required to cause membrane permeabilization, ii) measure and evaluate the role of intimacy of contact and forces of adhesion (in addition to membrane cohesion) in the electrofusion of reconstituted lipid membranes, iii) relate cohesive and adhesive properties of RBCs, parental myeloma and hybridoma cells to the electric field strengths required to cause permeabilization and fusion. The threshold field strengths for electropermeabilization and electrofusion are expected to be strongly dependent on membrane composition, interbilayer separation and forces of membrane- membrane interaction. This proposal addresses these issues by selectively reconstituting lipid, cholesterol, polypeptide and surface polymer into cell-size artificial bilayer vesicles. Membrane cohesive properties and adhesion will be measured with a micropipet manipulation technique and interbilayer separation will be determined by x-ray diffraction. Electropermeabilization of individual vesicles manipulated by a micropipet will be observed directly with an interference contrast video-microscope. Similarly, the assay for electrofusion of two isolated vesicles will be direct observation. Based on these measurements current theories of membrane stability will be critically evaluated and methods for increasing electrofusion yield will be developed; e.g., by the action of non-adsorbing polymers which promote strong intermembrane adhesion and close contact. Studies on parental myeloma and genetic hybrid cells (hybridomas) will also be made in order to extend this work from selectively reconstituted systems to cells of actual interest in applications of the electroporation and electrofusion technique. These fundamental studies will find direct application in newly developing areas of biomedical and biochemical technologies, such as drug and hemoglobin encapsulation, antibody loading and genetic hybridization.
| Bagatolli, Luis A; Needham, David (2014) Quantitative optical microscopy and micromanipulation studies on the lipid bilayer membranes of giant unilamellar vesicles. Chem Phys Lipids 181:99-120 |
| Yarmolenko, Pavel S; Zhao, Yulin; Landon, Chelsea et al. (2010) Comparative effects of thermosensitive doxorubicin-containing liposomes and hyperthermia in human and murine tumours. Int J Hyperthermia 26:485-98 |
| Needham, D; Nunn, R S (1990) Elastic deformation and failure of lipid bilayer membranes containing cholesterol. Biophys J 58:997-1009 |
