Direct Measurement And Computation Of Forces Between Mem

Child Health And Human Development

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Abstract

Our current purview includes polymer-coated lipids used in drug delivery, DNA/lipid assemblies for gene therapy; DNA assemblies such as are seen in viral capsids and in vitro; polypeptides and polysaccharides in suspension; and lipid/water liquid-crystals. In all these systems we simultaneously observe the structure of packing as well as measure intermolecular forces or interaction energies. We would like to characterize and codify measured forces in order to build a practical molecular physics of living systems. Our undertaking is strengthened by its strong connection with physical theory, in particular statistical mechanics applied to liquid-crystals and to complex fluids. Acting in liquids these forces depend on temperature, salts, solutes, and specific ligands; we speak of them as "thermodynamic" forces whose strengths and specificities are exquisitely controlled by cellular and solutions conditions. Their changes with water chemical potential reveal hydration; their dependence on solute chemical potentials indicate "excess" or "deficit" due to solute attraction or repulsion. We can think of them as "direct", e.g., electrostatic, van der Waals, or hydration forces, or as "indirect", acting through changes in the translation and configuration free energy of concentrated macromolecules. Normally in the domain of physical field theory, primarily recognized in chemical engineering, van der Waals or "charge-fluctuation" forces act in striking but often neglected ways among biological materials. As the dominant force between hydrocarbons they cohere the molecules that composer membranes as well as contribute to the powerful surface tension at membrane interfaces. At the next level, interactions between membranes, van der Waals forces are then dominant -- perhaps sole -- attraction that creates membrane multilayers or allows membranes to adhere to artificial surfaces. Similarly in proteins, van der Waals forces are locally weak attractions that sum in their ubiquity to cohere and stabilize protein. Because of the difficulty of the underlying physical theory, van der Waals forces have not been considered treated rigorously in most cases where Nature uses them. More than that, it has been difficult to design measurements that test their strength and consequence. Our work during the past year has ranged from the theoretical physics of more general mathematical equations to compute van der Waals interactions as well as wet chemistry of making preparations under which these forces can be systematically observed. (One unexpected bonus has been the beginning of a collaboration with engineers using our equations to design production procedures for thin-film resistors in computer chips. We expect the collaboration to work to our benefit by providing us with experimental data that are used to compute van der Waals forces.) People usually think of ions as particles whose primary properties are determined by their electric charge. In fact because ions vary widely in their effects on biological materials, ion "specificity" beyond simple charge properties is a major issue in biology. One overlooked property of ions is the polarizability, the ability of the charge to shift or fluctuate, a property seen in charge fluctuation forces. During the past year we have been using ion polarizability to explain the difference between chloride, bromide, iodide, nitrate and other negative ions that seem to bind to membranes. Our collaborators in France have seen that positively charged membranes will often collapse in the presence of the larger ions that adhere to them. Normally ions do not go near the poor solvent interiors of membranes. The reason for the extra attraction seems to be van der Waals forces that are bigger with the larger ion. Here at the NIH we have begun to see how the attraction between membranes varies when salts of different ions chloride vs. bromide are dissolved in the intervening water. Membrane multilayers will swell by 50% with bromide but not with chloride. Again at least one important factor in this ion discrimination is the neglected van der Waals force. From such membrane measurements we are following a new strategy to understand puzzling properties of ions with many kinds of biological materials. Our theoretical work has been to reconsider from their foundations van der Waals interactions as they act in liquids and solids. Most earlier work treated interacting bodies simplistically. For example, a membrane was treated as though its interface with surrounding water was a sudden step. We have recently generalized the theory so that the important polarizability of the material can be more realistically described as the spatially varying property that it in fact is. That ability to handle spatially varying properties was what attracted our computer-chip collaborators. In a related theoretical formulation, we have developed formulae to compute interactions within multilayered systems. These are being applied to measurements where the behavior of membranes is driven by their proximity to confining surfaces. We can now model membrane surfaces more realistically, even those coated with polymers as used in drug-delivery preparations (see below). From glycosylated cell surfaces to sterically stabilized liposomes polymers attached to membranes attract biological and therapeutic interest. To what extent does the protective sponginess of polymers bound to membrane surfaces resemble the spongy character of polymers in free solution? We have found remarkably simple conditions under which the resemblance can be predicted and used in the design of polymer-stabilized membrane systems. We compared the forces between polyethyleneglycol (PEG) -coated bilayers, obtained by our laboratory's Osmotic Stress method, with the osmotic pressure of PEG solutions. The criterion for a connection is that the polymer density and size in the "brush" on the membrane by high enough that in a bulk solution of equivalent weight concentration the polymer osmotic pressure is independent of polymer molecular weight. (Technically the latter is referred to as the des Cloiseaux semi-dilute regime of bulk polymer solutions.) In the design of liposomal drug delivery systems, drugs packaged into lipid-membrane vesicles, there is latitude to adjust the distance between PEG's grafted to the lipids as well as to choose PEG molecular weight. It is know that in order to shield the membrane surface from proteins that might destroy the vesicle, the PEG density must be that of a brush, molecules spreading out to the degree of strong overlap where they cover the surface to a degree that fits the criterion of a polymer "brush."