Location of Polyelectrolytes in Swollen Lipid Oligobilayers.

Osteoarthritis is caused by degeneration of the cartilage, which covers the bone ends of the joints and is decorated with an oligolamellar phospholipid (PL) bilayer. The gap between the bone ends is filled with synovial fluid mainly containing hyaluronic acid (HA). HA and PLs are supposed to reduce friction and protect the cartilage from wear in joint movement. However, a detailed understanding of the molecular mechanisms of joint lubrication is still missing. Previously, we found that aqueous solutions of HA and poly(allylamine hydrochloride) (PAH), the latter serving as a polymeric analogue to HA, adsorb onto the headgroups of surface-bound 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) oligobilayers and significantly enhance their stability with respect to shear forces, typically occurring in joint movement. We now investigated the precise location of PAH chains across the lipid films in neutron reflectivity measurements, as bridging of the oligobilayers by polyelectrolytes (PEs) might be the cause for their improved mechanical stability. In a first set of experiments, we used hydrogenated PAH and chain-deuterated DMPC (DMPC-d54) to improve the contrast between the lipids and potentially intruding PAH. However, due to difficulties in distinguishing between incorporation of water and PAH, penetration into the lipid chain region could hardly be proven quantitatively. Therefore, we designed a more elaborate experiment based on mixed films of DMPC-d54 and hydrogenated DMPC, which is insensitive to water penetration into the films. Beside facilitating a detailed structural characterization of the oligolamellar system, this elaborate approach showed that PAH adsorbs to the DMPC heads and penetrates the lipid tail strata. No PAH was found in the lipid head strata, which excludes bridging of several lipid bilayers by the PE chains. The data are consistent with the assumption that PAH bridges are formed between the headgroups of two adjacent bilayers and contribute to the enhanced mechanical stability.

Drastic Swelling of Lipid Oligobilayers by Polyelectrolytes: A Potential Molecular Model for the Internal Structure of Lubricating Films in Mammalian Joints.

Osteoarthritis is the most common arthropathy in western civilization. It is primarily caused by the degeneration of lipid-coated cartilage, leading to increased friction in joints. Hyaluronic acid (HA), a negatively charged polysaccharide and the main component of the synovial fluid, is held responsible for joint lubrication. It is believed that HA, adsorbed to the lipid-coated cartilage, forms a protective layer against wear. Studies have shown that the concentration and molecular weight (MW) of HA are reduced in joints suffering from osteoarthritis. On the basis of these observations, local joint injections of HA or mixtures of HA and surface-active phospholipids (SAPLs) have been applied as medical cures to restore the functionality of the joints in a procedure called viscosupplementation. However, this cure is still disputed, and no consensus has been reached with respect to optimum HA concentration and MW. To provide detailed insight in the structural rearrangement of lipid films upon contact with HA or polymeric analogues, we studied the interaction of the polyelectrolyte poly(allylamine hydrochloride) (PAH) with surface-bound oligobilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) by neutron reflectivity (NR) and ellipsometry. Using this model system, we found a drastic swelling of the lipid films as a function of PAH concentration, whose strength compares to that in previous studies on HA incubation. In contrast, no significant dependence of film thickness on PAH MW was observed. A detailed picture of the film architecture was developed which inter alia shows that charged PAH is adsorbed to the lipid headgroups, leading to electrostatic repulsion. The swelling behavior is well explained by the equilibrium of Coulomb and van der Waals interactions in a DLVO-based model. Our detailed structural analysis of the PAH/lipid interfacial layer may help to elucidate the mechanisms of viscosupplementation and derive a structure-function relationship for the lubricating interface in mammalian joints.

Surface-Active Lipid Linings under Shear Load--A Combined in-Situ Neutron Reflectivity and ATR-FTIR Study.

We study shear effects in solid-supported lipid membrane stacks by simultaneous combined in-situ neutron reflectivity (NR) and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). The stacks mimic the terminal surface-active phospholipid (SAPL) coatings on cartilage in mammalian joints. Piles of 11 bilayer membranes of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) are immobilized at the interface of the solid silicon support and the liquid D2O backing phase. We replace the natural hyaluronic acid (HA) component of synovial fluid by a synthetic substitute, namely, poly(allylamine hydrochloride) (PAH), at identical concentration. We find the oligolamellar DMPC bilayer films strongly interacting with PAH resulting in a drastic increase of the membranes d spacing (by a factor of ∼5). Onset of shear causes a buckling-like deformation of the DMPC bilayers perpendicular to the applied shear field. With increasing shear rate we observe substantially enhanced water fractions in the membrane slabs which we attribute to increasing fragmentation caused by Kelvin-Helmholtz-like instabilities parallel to the applied shear field. Both effects are in line with recent theoretical predictions on shear-induced instabilities of lipid bilayer membranes in water (Hanasaki, I.; Walther, J. H.; Kawano, S.; Koumoutsakos, P. Phys. Rev. E 2010, 82, 051602). With the applied shear the interfacial lipid linings transform from their gel state Pß' to their fluid state Lα. Although in chain-molten state with reduced bending rigidity the lipid layers do not detach from their solid support. We hold steric bridging of the fragmented lipid bilayer membranes by PAH molecules responsible for the unexpected mechanical stability of the DMPC linings.

A TRLFS study on the complexation of novel BTP type ligands with Cm(III).

Two BTP-type N-donor ligands with different numbers of aromatic nitrogen atoms (2,6-bis(4-ethyl-pyridazin-1-yl)pyridine, Et-BDP and 2,6-bis(4-(n)propyl-2,3,5,6-tetrazine-1-yl)pyridine, (n)Pr-Tetrazine) have been synthesized and characterized by NMR and MS techniques. The complexation with Cm(III) in 2-propanol-water (1 : 1, vol.) is studied for both ligands using time resolved laser-induced fluorescence spectroscopy (TRLFS) and the complexation properties are compared to (n)Pr-BTP. With increasing the ligand concentration three different species, the 1 : 1-, 1 : 2- and 1 : 3-complex, were found. Log ß3 values of 7.6 for the formation of Cm(Et-BDP)3 and 9.2 for the formation of Cm((n)Pr-Tetrazine)3 are determined. The complexation with (n)Pr-Tetrazine shows slow kinetics. Thermodynamic data of the complexation reactions are determined in a temperature range of 25 °C-60 °C. The complexation with Et-BDP is exothermic (ΔH = -16.3 ± 1.2 kJ mol(-1)) and exergonic (ΔG = -43.8 ± 2.6 kJ mol(-1)) whereas the complexation with (n)Pr-Tetrazine is endothermic (ΔH = 43.9 ± 3.1 kJ mol(-1)) and exergonic (ΔG = -51.7 ± 2.2 kJ mol(-1)). In the case of the latter the complexation is driven by a highly positive reaction entropy change (ΔS = 320.6 ± 15.4 J mol(-1) K(-1)). In comparison to (n)Pr-BTP, less negative ΔG values were found for the complexation of Cm(III) with both ligands.