Fundamental Studies and Applications of Tissue Sciences

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

Linked publications, trials & patents

Abstract

Ion-biopolymer interactions play a central role in a variety of biological processes. The action of ions is complex: they stabilize the local structures, and ion binding can also affect the intrinsic properties of the biopolymer molecules, such as flexibility, electrostatic interactions and the overall thermodynamics of the system. Well-defined model systems designed to mimic the behavior of living systems, enable us to study the physicochemical foundation of these systems, which are often composed of polyelectrolytes whose characteristics are largely determined by the properties of water hydrating the polyelectrolyte molecules, the counter-ions present in the solution, and the interaction between and among the components (polyelectrolytes, water molecules and counter-ions). 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, that 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. The important role that polymer physics plays in the behavior of biological systems offers a powerful framework to identify variables required to describe certain biological functions. Divalent cations, particularly calcium ions (Ca+2) play a key role in many biopolymer systems. In general, experiments to determine the interactions between 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. Since macroscopic phase separation does not occur in gels, we often study cross-linked polymers, i.e., extending 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 different biopolymers such as DNA and hyaluronic acid (HA) 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 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 critical 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 complex biological processes. In many cases, these model systems become the basis of novel translational NMR and MRI phantoms, and non-invasive methods to measure and map ECM properties. We 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 tract of humans and animals. Our aim 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. 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. Despite the tremendous potential of bottlebrush polymers in both biology and materials science, the effects of the charged bottlebrush topology on the physicochemical behavior of their solutions have not yet been fully elucidated. For example, the effect of charges on the conformation of bottlebrush molecules remains poorly understood. The effect of ions is particularly important in the biological milieu where both mono- and divalent counter-ions are present. Elucidating these basic physical properties is of fundamental importance to fully understand the basic molecular physics underlying numerous biological functions. Knowledge of the molecular origin of tissue properties is essential for developing successful tissue engineered regenerative medicine strategies and understanding many normal and pathological processes. With Dr. Matan Mussel, we developed a multicomponent model that imposes conservation laws and constitutive relations for polymer chains, water, and ions to predict swelling behavior of polyelectrolyte materials. We have demonstrated that understanding the organization of charged macromolecules in near-physiological environment may shed light on the mechanism of structure formation in living systems in which the complexity of the structure and interactions makes it difficult to perform conclusive experiments under well-controlled conditions. 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. We found that that (i) the monovalent ion cloud was only weakly influenced by the addition of divalent ions, and (ii) the divalent counter-ion cloud tightly followed the contour of the polymer chains.