Part 1: Fundamental studies of space and time-specific interactions. Rate and time-dependent effects are critically important in biomolecular and biomembrane interactions. Biological inter-actions in vivo are rarely at or near equilibrium, and indeed, nature does not intend them to be. For example, fusions events channel openings, or local recognition interactions usually occur at a certain point not only in space, but in time.
Our aims i n this continuation proposal are to study time and spatial effects in biological interactions at the molecular level (Part I), and how such sequential interactions can be controlled in vitro and in vivo (Part 2) to create new biomimetic materials for use as drug delivery vehicles or artificial tissues (Part 2). A combination of experimental techniques will be used, including the Surface Forces Apparatus (SFA), freeze-fracture and cryo-electron microscopy, and scanning tunneling (STM) and atomic force microscopy (AFM). Specific-systems to be studied are: (l) the highly specific ligand-receptor Biotin-Avidin system, (2) and the fusogenic protein Synexin. An important new part of these studies is to softly support lipid-protein bilayers on polyelectrolyte gels or on recently synthesized lipid-polymer tethers. Softly-supported membranes, in contrast to rigidly supported membranes, will allow us to do a variety of SFA experiments under conditions that are both controllable and much closer to in vivo conditions. The soft supports will allow the membranes to fluctuate thermally, allow proteins and lipids to diffuse relatively freely, and allow for larger transmembrane or membrane associated proteins to be studied with SFA. The distribution and organization within the softly supported membranes will also be examined at nanometer resolution with tapping mode AFM or a newly developed freeze-fracture STM technique. Part 2: Biomimetic materials - Self-assembling drug-delivery system. We are developing new higher order self-assembly techniques using our understanding of biological interactions to create a drug-delivery system composed of ligand-receptor tethered vesicles stabilized by an outer membrane that we call a vesosome. The vesosome can allow for a variety of individual vesicle membrane and interior compositions to provide a constant drug-release rate, or mixture of drugs in a prescribed ratio. The independent outer membrane might be tagged with proteins, enzymes or polymers for specific recognition or contain a controlled lipid composition for permeation control. Vesosomes are built up from extruded unilamellar vesicles using biotin-streptavidin linkages. The vesosomes can be sized from < l to many microns. The external membrane, which can incorporate biocompatible polyethylene glycol-lipids, is applied by reverse phase evaporation or co-extrusion with lipids. Electron microscopy will be used to characterize the vesosomes. Initial drug release studies will utilize the fluorescent probe calcein.
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