Hyaluronan is a biopolymer that is essential for many biological processes in the human body, like the regulation of tissue lubrication and inflammatory responses. Here, we study the behavior of hyaluronan at aqueous surfaces using heterodyne-detected vibrational sum-frequency generation spectroscopy (HD-VSFG). Low-molecular-weight hyaluronan (∼150 kDa) gradually covers the water-air interface within hours, leading to a negatively charged surface and a reorientation of interfacial water molecules. The rate of surface accumulation strongly increases when the bulk concentration of low-molecular-weight hyaluronan is increased. In contrast, high-molecular-weight hyaluronan (>1 MDa) cannot be detected at the surface, even hours after the addition of the polymer to the aqueous solution. The strong dependence on the polymer molecular weight can be explained by entanglements of the hyaluronan polymers. We also find that for low-molecular-weight hyaluronan the migration kinetics of hyaluronan in aqueous media shows an anomalous dependence on the pH of the solution, which can be explained from the interplay of hydrogen bonding and electrostatic interactions of hyaluronan polymers.

The biological functions of natural polyelectrolytes are strongly influenced by the presence of ions, which bind to the polymer chains and thereby modify their properties. Although the biological impact of such modifications is well recognized, a detailed molecular picture of the binding process and of the mechanisms that drive the subsequent structural changes in the polymer is lacking. Here, we study the molecular mechanism of the condensation of calcium, a divalent cation, on hyaluronan, a ubiquitous polymer in human tissues. By combining two-dimensional infrared spectroscopy experiments with molecular dynamics simulations, we find that calcium specifically binds to hyaluronan at millimolar concentrations. Because of its large size and charge, the calcium cation can bind simultaneously to the negatively charged carboxylate group and the amide group of adjacent saccharide units. Molecular dynamics simulations and single-chain force spectroscopy measurements provide evidence that the binding of the calcium ions weakens the intramolecular hydrogen-bond network of hyaluronan, increasing the flexibility of the polymer chain. We also observe that the binding of calcium to hyaluronan saturates at a maximum binding fraction of ∼10-15 mol %. This saturation indicates that the binding of Ca2+ strongly reduces the probability of subsequent binding of Ca2+ at neighboring binding sites, possibly as a result of enhanced conformational fluctuations and/or electrostatic repulsion effects. Our findings provide a detailed molecular picture of ion condensation and reveal the severe effect of a few, selective and localized electrostatic interactions on the rigidity of a polyelectrolyte chain.

The molecular configuration and chemistry at the zinc/zinc oxide-polyester interface were studied by using two complementary spectroscopic techniques: attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and sum-frequency generation (SFG) spectroscopy. It was shown that ATR-FTIR should be considered as a (3D) interphase-sensitive technique with probing depths of 250-400 nm in the headgroup region (2000-1200 cm^-1). On the other hand, SFG is known to be a (2D) interface-sensitive technique. The ATR-FTIR measurements showed that carboxylate groups are formed within the near-interface region of the polyester phase. SFG measurements showed that the carboxylic acid groups are stable at the polymer-zinc/zinc oxide interface. In addition, in situ ATR-FTIR and SFG measurements have been conducted when exposing the polyester-zinc/zinc oxide system to D₂O. The exposure to D₂O is observed to lead to an additional conversion of ester and carboxylic acid groups to carboxylate groups. The comparison of the SFG and ATR-FTIR measurements shows that this conversion occurs much slower at the polyester-zinc/zinc oxide interface than in the bulk of the polyester. Finally, the strengths and limitations as well as the complementarity of both techniques are discussed.

We study the relation between the macroscopic viscoelastic properties of aqueous hyaluronan polymer solutions and the molecular-scale dynamics of water using rheology measurements, differential dynamic microscopy, and polarization-resolved infrared pump-probe spectroscopy. We observe that the addition of hyaluronan to water leads to a slowing down of the reorientation of a fraction of the water molecules. Near pH 2.4, the viscosity of the hyaluronan solution reaches a maximum, while the number of slowed down water molecules reaches a minimum. This implies that the water molecules become on average more mobile when the solution becomes more viscous. This observation indicates that the increase in viscosity involves the expulsion of hydration water from the surfaces of the hyaluronan polymers, and a bundling of the hyaluronan polymer chains.

Many biopolymer hydrogels are environmentally responsive because they are held together by physical associations that depend on pH and temperature. Here, we investigate how the pH and temperature responses of the rheology of hyaluronan hydrogels are connected to the underlying molecular interactions. Hyaluronan is an essential structural biopolymer in the human body with many applications in biomedicine. Using two-dimensional infrared spectroscopy, we show that hyaluronan chains become connected by hydrogen bonds when the pH is changed from 7.0 to 2.5 and that the bond density at pH 2.5 is independent of temperature. Temperature-dependent rheology measurements show that because of this hydrogen bonding the stress relaxation at pH 2.5 is strongly slowed down in comparison to pH 7.0, consistent with the sticky reptation model of associative polymers. From the flow activation energy, we conclude that each polymer is cross-linked by multiple (5-15) hydrogen bonds to others, causing slow macroscopic stress relaxation, despite the short time scale of breaking and reformation of each individual hydrogen bond. Our findings can aid the design of stimuli-responsive hydrogels with tailored viscoelastic properties for biomedical applications.