Complexation chemistry for tuning release from polymer coatings.

The strategy of metal ion complexation is employed to design a delivery system for an antifouling agent (AFA) in marine paints. A poly(1-vinylimidazole-co-methyl methacrylate) copolymer (PVM), together with Cu2+ or Zn2+ formed a PVM-M2+ complex. The AFA, Medetomidine, was then coordinated into the complex. The coordination strength was investigated in solution by 1H NMR and on solid surfaces by using the Quartz Crystal Microbalance with Dissipation monitoring technique (QCM-D) and Surface Plasmon Resonance (SPR). From the 1H NMR experiments strong interactions were observed between Cu2+ and the PVM-polymer and between Medetomidine and the PVM-Cu2+ complex. From the QCM-D and SPR measurements it was shown that Cu2+, compared to Zn2+, exhibited a larger affinity for the PVM-copolymer surface that resulted in higher degree of swelling of the polymer film. Large amounts of Medetomidine were adsorbed to the PVM-Cu2+ complex resulting in low desorption rates. However, the adsorbed amount of Medetomidine was lower to the Zn2+ doped polymer and a higher desorption rate was observed. These results indicate the possibility of tuning the release of Medetomidine by altering the coordinating metal ion, which may prove to be favorable in a paint formulation.

Lipopolysaccharide removal by a peptide-functionalized surface.

Five peptides: BPI(85-109); CAP18(106-137); endotoxin inhibitor (EI); GQ33 and GQ33C, derived from lipopolysaccharide (LPS)-binding molecules were investigated for LPS-binding ability with a view to a potential use in extracorporeal therapy. The surface plasmon resonance technique (SPR) was used to monitor the interaction between LPS in solution and the surface-immobilized peptides. The peptides were covalently bound to a model dextran surface via inherent amino groups or via terminally introduced cysteine residues. The results showed that the binding efficacy and binding stability of the peptides varied greatly. The CAP18(106-137) peptide, which exhibited the highest binding efficacy and binding stability, was also immobilized on a poly(ethylene imine)-poly(ethylene glycol) (PEI-PEG) surface through maleimide-terminal PEG. The binding efficacy of the CAP18(106-137) peptide was not significantly affected by the different immobilization methods used in the attachment to a dextran or a PEI-PEG surface. LPS bound selectively to CAP18(106-137) and showed very low unspecific binding to the PEI-PEG surface layer. The EI peptide proved to have a reasonably good binding capacity but a less stable interaction with LPS. The other peptides exhibited much poorer binding efficacy. We believe that the results presented in this work can be of practical value for the development of extracorporeal treatment of patients suffering from septic shock.

Importance of substrate and photo-induced effects in Raman spectroscopy of single functional erythrocytes.

Hemoglobin (Hb) in single erythrocytes (red blood cells), adsorbed on polylysine-coated glass surfaces, was studied using resonance Raman spectroscopy and global Raman imaging. The erythrocytes were found to be sensitive to both surface adsorption and laser illumination. Substrate-dependent changes of the cell membrane shape were observed immediately after cell adsorption, while a photo-induced increase of fluorescence was observed for visible excitation (lambda=514.5 nm). Concurrent changes in Raman spectra revealed a conversion of oxy-Hb (2+) to the inactive met-Hb state (3+). These effects severely complicate the interpretation of Raman images. However, at a low accumulated photon dose, the preparation method enabled the recording of Raman spectra during the oxygenation cycle of a single erythrocyte in buffer, which illustrates the feasibility of Raman investigations of functional cells in in-vivo environments.

Acoustics of blood plasma on solid surfaces.

We have quantified surface associated coagulation of human blood plasma with a recently developed methodological system consisting of a Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), a method that measures the weight of adsorbed molecules on surfaces as a function of frequency shifts of a quartz crystal. Further, it measures the damping energy (i.e. viscoelasticity) of the adsorbed layer. Four different surfaces where studied: Heparin (Hep) surface as an active inhibitor of clot formation, titanium (Ti) surfaces that are known to activate the intrinsic pathway, polystyrene (PS) surfaces and poly(urethane urea) (PUUR) surfaces. The experiments were initiated by applying citrated human plasma at the sensor surfaces; calcium was then added toinitiate coagulation. The Hep surfaces showed no apparent indication of clot formation during one hour of incubation at room temperature. However, on Ti surfaces we observed an early and rapid change in both frequency shift and viscoelastic properties of the coagulating plasma. We inhibited the intrinsic pathway activation by using corn trypsin inhibitor (CTI), which is specific for factor FXIIa in the bulk phase, which prolonged the coagulation times for all non-heparinized surfaces. We have also found a peculiar initial plasma protein interaction phenomenon on Ti surfaces. The described methodology would be very efficient for basic studies of surface associated coagulation and as a screening method for new biomaterials.

The influence of cross-linking on protein-protein interactions in a marine adhesive: the case of two byssus plaque proteins from the blue mussel.

The interaction between two proteins, Mefp-1 and Mefp-2, from the byssal plaque of the blue mussel, Mytilus edulis, was investigated using a quartz crystal microbalance with dissipation monitoring (QCM-D) technique. The challenge in using a surface-sensitive technique to investigate the interaction between two strongly adhesive proteins was met by coupling a biotinylated version of one of the proteins (b-Mefp-1) to an inert two-dimensional arrangement of streptavidin (SA) formed on top of a biotin-doped supported phospholipid bilayer. The interaction between Mefp-1 and Mefp-2 was further investigated by addition of Mefp-2 to SA-coupled b-Mefp-1, where the latter was either in the native state or cross-linked using sodium periodate (NaIO(4)), Cu(2+), or mushroom tyrosinase. With this coupling strategy it is shown that a requirement for attraction between the two proteins is that tyrosinase is used as the cross-linking agent of b-Mefp-1. By inhibiting the enzymatic activity of tyrosinase it is also shown that enzymatic activity is required for both efficient binding of tyrosinase to SA-coupled b-Mefp-1 as well as for the subsequent binding of Mefp-2. In contrast, spontaneous adsorption of Mefp-1 to a methyl-terminated (thiolated) gold surface followed by addition of Mefp-2 results in binding of Mefp-2 for all cross-linking agents. This suggests that cross-linking of Mefp-1 adsorbed on a solid surface induces structural changes in the adsorbed protein layer, resulting in exposure of free surface patches on which Mefp-2 binds.