Fundamental Studies and Applications of Tissue Sciences

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

The role of ions in biological systems is complex: they stabilize the local structures, and ion binding can also affect the intrinsic properties of the biopolymer molecules, such as flexibility, molecular conformation, and dynamics of polyelectrolyte chains with emphasis on the valence and chemical nature of the ion that governs the polyelectrolyte counterion interactions and the overall thermodynamics of the system. Well-defined models designed to mimic the behavior of living systems, enable us to study the physicochemical foundation of these systems, which are often composed of polyelectrolyte molecules. Systematic studies made on model systems are opening new avenues to understand the molecular origin of the properties and function of tissue and tissue components. It has been demonstrated that in many systems, especially in the vicinity of ion induced phase transitions, the physical response is governed by universal scaling principles. Various physical forces and interactions are implicated in these processes, including electrostatic repulsion and attraction, hydrophobic and hydrophilic interactions, hydrogen bonding, and van der Waals forces. Polymer physics offers a powerful framework to understand and describe a myriad of biological systems and functions. Divalent cations, particularly calcium ions (Ca+2) play a key role in many biopolymer systems. In general, experiments to determine the interactions among ions and biopolymers are difficult to perform, because above a relatively low ion concentration multivalent cations cause phase separation (or precipitation) of the charged macromolecules. The addition of multivalent cations to polyelectrolyte gels swollen in nearly physiological NaCl solution can lead to a volume transition. The repulsion between the network chains is decreased as multivalent ions screen the fixed charges of the polyanions. Large numbers of condensed counterions could bring chains together since neighboring chains might share multivalent ions. Since macroscopic phase separation does not occur in gels, we often study cross-linked polymers, i.e., to extend the range of ion concentrations over which the systems remain stable. We use various new non-destructive procedures to investigate cross-linked gels of a model synthetic polymer, polyacrylic acid (PAA), and a biopolymer (DNA) to determine the size of the structural elements that govern the osmotic concentration fluctuations. We combine SANS and SAXS to estimate the osmotic modulus of HA in the presence of both monovalent and divalent counter-ions. We also study the dynamic properties (e.g., diffusion processes) of these systems by dynamic light scattering and neutron spin-echo, and determine the osmotic modulus from the relaxation response. We developed an experimental procedure to determine the distribution of counter-ions around charged biopolymer molecules using anomalous small-angle X-ray scattering (ASAXS) measurements. We analyze and compare various network elasticity models that address essential physical aspects of biopolymer systems. We are particularly interested in understanding the mechanisms of interactions of biologically important divalent cations like Ca+2 with negatively charged biopolymers (e.g., DNA). Moreover, while there is some understanding of water-ion-biopolymer interactions in low and high ionic strength regimes, there is little known about these in the intermediate physiological ionic strength regime. Because most functional biopolymers are polyelectrolytes, studying this regime is critically important for understanding biological transport processes, including transport of mass, momentum, charge, and energy. A basic feature of charged polymers is that their properties are affected by many factors, such as the intrinsic properties of the polymer (e.g., chain rigidity), polymer conformation, molecular mass, the concentration and valence of counterions, as well as inter- and intramolecular interactions (e.g., ion condensation, hydration, hydrogen bonding). To date, no satisfactory theoretical framework describing the relationship between molecular/supramolecular structure, macroscopic properties and biological function of either synthetic or natural polyelectrolyte molecules has been developed. Therefore, making systematic experimental studies on well-defined model systems under well-controlled conditions is critically important to advance our understanding of the mechanisms governing many complex biological processes. In several cases, these model systems have also become the impetus to develop novel NMR and MRI phantoms with numerous translational applications. We also focus on quantifying ion-polymer interactions in bottlebrush polymers comprised of densely grafted chains tethered to a polymer backbone. Such molecules are essential components of living systems. Typical examples are cartilage and viscoelastic mucin layers forming protective coatings in the lungs, orifices and digestive and reproductive tracts. One of our aims is to better understand the physicochemical behavior of bottlebrush molecules. We have systematically investigated solutions of synthetic polyelectrolyte bottlebrushes through a combination of measurements of solution properties (osmometry, neutron and dynamic light scattering), and molecular dynamics simulations. We found that bottlebrush polymers differ strongly from their linear polymer counterparts, not only in their architecture, but also in their physical properties that are primarily controlled by the length of the main and side-chains, the grafting density, and the steric repulsion of the side-chains. In collaboration with Prof. Yan Xia (Department of Chemistry, Stanford University) we designed and synthesized novel bottlebrush polymers and determined the physicochemical properties of these model polymer solutions (neutralized polyacrylic acid bottlebrushes) having well-controlled molecular architecture. We examined different levels of hierarchical structure of cartilage proteoglycans using complementary experimental techniques, including osmotic pressure measurements, neutron scattering and light scattering. We found that the osmotic modulus increased in the order chondroitin sulfate < aggrecan < aggrecan-hyaluronic acid complex indicating that the hierarchical bottlebrush configuration, which prevents interpenetration among the bristles of the aggrecan bottlebrushes, enhances both the mechanical properties and the osmotic resistance. We made osmotic swelling pressure and SANS measurements on DNA gels. We systematically varied the concentration of sodium and calcium ions and the pH to determine the effect of the ionic environment on the structure and thermodynamic interactions. Using anomalous small-angle X-ray scattering measurements we determined the distribution of mono- and divalent ions in the vicinity of the DNA chains. In contrast to cations very little is known about the effect of anionic counterions on negatively charged polyelectrolytes. In collaboration with Professor Matan Mussel (University of Haifa, Israel), a former post-doc, we are trying to obtain a fundamental understanding of how the structure and thermodynamic properties of biomimetic hydrogels are affected by anionic activity and composition. Our hypothesis is that changes in anionic activity will influence the chemical, structural, mechanical, and transport properties of negatively charged polymer gels by competing with the surrounding cations. For simplicity, we intend to study the effects of the “standard” Hofmeister anion series on the swelling and structural properties of polyelectrolyte gels.