Influence of the Molecular Weight and the Presence of Calcium Ions on the Molecular Interaction of Hyaluronan and DPPC

Abstract

Hyaluronan is an essential physiological bio macromolecule with different functions. One prominent area is the synovial fluid which exhibits remarkable lubrication properties. However, the synovial fluid is a multi-component system where different macromolecules interact in a synergetic fashion. Within this study we focus on the interaction of hyaluronan and phospholipids, which are thought to play a key role for lubrication. We investigate how the interactions and the association structures formed by hyaluronan (HA) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) are influenced by the molecular weight of the bio polymer and the ionic composition of the solution. We combine techniques allowing us to investigate the phase behavior of lipids (differential scanning calorimetry, zeta potential and electrophoretic mobility) with structural investigation (dynamic light scattering, small angle scattering) and theoretical simulations (molecular dynamics). The interaction of hyaluronan and phospholipids depends on the molecular weight, where hyaluronan with lower molecular weight has the strongest interaction. Furthermore, the interaction is increased by the presence of calcium ions. Our simulations show that calcium ions are located close to the carboxylate groups of HA and, by this, reduce the number of formed hydrogen bonds between HA and DPPC. The observed change in the DPPC phase behavior can be attributed to a local charge inversion by calcium ions binding to the carboxylate groups as the binding distribution of hyaluronan and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine is not changed.

Keywords: binding distribution; hyaluronan; molecular interaction; phase behavior; phospholipid.

Figure 1

Electrophoretic mobility of DPPC vesicles, hyaluronan and their mixture as a function of CaCl₂ concentration at 25 °C. All solutions contained 155 mM NaCl.

Figure 2

DSC measurements of DPPC vesicles with HA of varying molecular weights. (Top): 150 mM sodium chloride solutions. The inset shows a magnification of the pre-transition. (Bottom): 150 mM sodium chloride with 10 mM calcium chloride solutions. The inset shows a magnification of the pre-transition.

Figure 3

DLS data of the vesicle-HA aggregates as a function of the MW of HA. (Left) sodium chloride solutions (150 mM) and (right) sodium chloride with calcium chloride (150 mM/10 mM).

Figure 4

Mean size of the size populations of DPPC vesicles with and without HA as measured by DLS. Diamonds: 150 mM NaCl solutions. Circles: 150 mM NaCl and 10 mM CaCl₂ solutions. We note that the small size population for NaCl and NaCl/CaCl₂ coincide for no HA and the two points lie on top of each other.

Figure 5

Electron density profiles obtained by the model fitting of the SAXS data on vesicles of DPPC with HA with an MW of 250 kDa (dashed lines) and without HA (solid lines). The curves were measured at different temperatures corresponding to the L_β0 (25 °C), P_β0 (37 °C) and L_α (50 °C) phase. (Top): 150 mM sodium chloride solutions. (Bottom): 150 mM sodium chloride with 10 mM calcium chloride solutions.

Figure 6

SANS scattering curves of DPPC (solid line), DPPC and HA with MW of 1500 kDa (dashed line) and DPPC with HA with MW of 10 kDa (dotted line) in 150 mM NaCl/D₂O. Scattering data of DPPC with HA of 1500 kDa and DPPC with HA of 10 kDa have been shifted vertically (multiplied by 2 and 4, respectively) for better comparison.

Figure 7

Snapshot from the simulation box: (a) initial position, (b) final structure. Green polymer represents HA. DPPC lipids are colored in the following way: hydrogen (white), carbon (turquoise), oxygen (red), nitrogen (blue) and phosphorus (yellow). (c) Enlarged snapshot from the simulation box at the final state. Phospholipids are represented in line connected structure, HA and Ca²⁺ as beads. In the picture we see simulation results in the presence of CaCl₂ at 37 °C, and some calcium ions are found within the black circles in panel C.

Figure 8

Electron density profile of the simulated DPPC bilayer with adsorbed HA.

Figure 9

Map showing the number of HA-phospholipid H-bonds between different atom classes in HA. In the graph, HA (y-axis) and DPPC (x-axis) in NaCl solution at 37 °C are shown. The color scale indicates the number of H-bonds.

Figure 10

Distribution of distances between the DPPC O4 atom in the phosphate moiety and HA atoms in aqueous: (a) NaCl, (b) CaCl₂ solutions at 37 °C. This oxygen class of DPPC was chosen since it frequently participates in H-bonds with the classes of atoms in HA as shown in Figure 8. Additionally, carboxyl group atoms (O5 and O6) are presented as calcium ions cause a shift toward the DPPC bilayer.

Figure 11

Ratios between the number of specific interactions found in CaCl₂ and NaCl solutions as a function of temperature. The hydrogen bonds between DPPC and HA are denoted by H-bond, the hydrophobic contacts between HA and DPPC are denoted by HP. The hydrogen bonds between water and HA are denoted by HA-H₂O.

Figure 12

Distribution of distances between DPPC-O4 atom class and: (a) sodium ions, (b) calcium ions. Distribution of distances between: (c) sodium ions, (d) calcium ions and the HA-O5 atom class.