Grazia M. L. Messina

Cations as switches of amyloid-mediated membrane disruption mechanisms: calcium and IAPP.

Disruption of the integrity of the plasma membrane by amyloidogenic proteins is linked to the pathogenesis of a number of common age-related diseases. Although accumulating evidence suggests that adverse environmental stressors such as unbalanced levels of metal ions may trigger amyloid-mediated membrane damage, many features of the molecular mechanisms underlying these events are unknown. Using human islet amyloid polypeptide (hIAPP, aka amylin), an amyloidogenic peptide associated with β-cell death in type 2 diabetes, we demonstrate that the presence of Ca(2+) ions inhibits membrane damage occurring immediately after the interaction of freshly dissolved hIAPP with the membrane, but significantly enhances fiber-dependent membrane disruption. In particular, dye leakage, quartz crystal microbalance, atomic force microscopy, and NMR experiments show that Ca(2+) ions promote a shallow membrane insertion of hIAPP, which leads to the removal of lipids from the bilayer through a detergent-like mechanism triggered by fiber growth. Because both types of membrane-damage mechanisms are common to amyloid toxicity by most amyloidogenic proteins, it is likely that unregulated ion homeostasis, amyloid aggregation, and membrane disruption are all parts of a self-perpetuating cycle that fuels amyloid cytotoxicity.

A multitechnique study of preferential protein adsorption on hydrophobic and hydrophilic plasma-modified polymer surfaces.

The adsorption process of albumin, lysozyme and lactoferrin was investigated onto polymer surfaces, both hydrophobic and hydrophilic treated by oxygen-plasma. In particular thin films of polyhydroxymethylsiloxane (about 90 degrees of static water contact angle) were converted by oxygen plasma treatments at reduced pressure into hydrophilic SiO(x) phases (less than 10 degrees of water contact angle). The protein adsorption process was investigated in situ by quartz crystal microbalance with dissipation monitoring in terms of coverage and kinetics mechanism, while chemical structure and topography of the protein adlayers were studied ex situ by angular-resolved XPS and atomic force microscopy, respectively. The plasma surface modification of the polymer film drastically modified the adsorption process of the three proteins, both in terms of kinetics and coverage.

Zinc stabilization of prefibrillar oligomers of human islet amyloid polypeptide

The aggregation of human islet amyloid polypeptide (hIAPP) has been linked to beta-cell death in type II diabetes. Zinc present in secretory granules has been shown to affect this aggregation. A combination of EXAFS, NMR, and AFM experiments shows that the influence of zinc is most likely due to the stabilization of prefibrillar aggregates of hIAPP.

How the Surface Nanostructure of Polyethylene Affects Protein Assembly and Orientation

Protein adsorption plays a key role in the biological response to implants. We report how nanoscale topography, chemistry, crystallinity, and molecular chain anisotropy of ultrahigh molecular weight polyethylene (UHMWPE) surfaces affect the protein assembly and induce lateral orientational order. We applied ultraflat, melt drawn UHMWPE films to show that highly oriented nanocrystalline lamellae influence the conformation and aggregation into network structures of human plasma fibrinogen by atomic force microscopy with unprecedented clarity and molecular resolution. We observed a transition from random protein orientation at low concentrations to an assembly guided by the UHMWPE surface nanotopography at a close to full surface coverage on hydrophobic melt drawn UHMWPE. This assembly differs from the arrangement at a hydrophobic, on the nanoscale smooth UHMWPE reference. On plasma-modified, hydrophilic melt drawn UHMWPE surfaces that retained their original nanotopography, the influence of the nanoscale surface pattern on the protein adsorption is lost. A model based on protein-surface and protein-protein interactions is proposed. We suggest these nanostructured polymer films to be versatile model surfaces to provide unique information on protein interactions with nanoscale building blocks of implants, such as nanocrystalline UHMWPE lamellae. The current study contributes to the understanding of molecular processes at polymer biointerfaces and may support their future design and molecular scale tailoring.

Mechanisms underlying the attachment and spreading of human osteoblasts: from transient interactions to focal adhesions on vitronectin-grafted bioactive surfaces.

The features of implant devices and the reactions of bone-derived cells to foreign surfaces determine implant success during osseointegration. In an attempt to better understand the mechanisms underlying osteoblasts attachment and spreading, in this study adhesive peptides containing the fibronectin sequence motif for integrin binding (Arg-Gly-Asp, RGD) or mapping the human vitronectin protein (HVP) were grafted on glass and titanium surfaces with or without chemically induced controlled immobilization. As shown by total internal reflection fluorescence microscopy, human osteoblasts develop adhesion patches only on specifically immobilized peptides. Indeed, cells quickly develop focal adhesions on RGD-grafted surfaces, while HVP peptide promotes filopodia, structures involved in cellular spreading. As indicated by immunocytochemistry and quantitative polymerase chain reaction, focal adhesions kinase activation is delayed on HVP peptides with respect to RGD while an osteogenic phenotypic response appears within 24h on osteoblasts cultured on both peptides. Cellular pathways underlying osteoblasts attachment are, however, different. As demonstrated by adhesion blocking assays, integrins are mainly involved in osteoblast adhesion to RGD peptide, while HVP selects osteoblasts for attachment through proteoglycan-mediated interactions. Thus an interfacial layer of an endosseous device grafted with specifically immobilized HVP peptide not only selects the attachment and supports differentiation of osteoblasts but also promotes cellular migration.

Surface immobilization of fibronectin-derived PHSRN peptide on functionalized polymer films--effects on fibroblast spreading.

The Pro-His-Ser-Arg-Asn (PHSRN) sequence in fibronectin is a second cell-binding site that synergistically affects Arg-Gly-Asp (RGD). The PHSRN peptide also induces cell invasion and accelerates wound healing. We report on the surface immobilization of PHSRN by spontaneous adsorption on polysiloxane thin films which have different surface free energy characteristics. Low-surface energy (hydrophobic) polysiloxane and the corresponding high-surface energy (hydrophilic) surfaces obtained by UV-ozone treatments were used as adsorbing substrates. The peptide adsorption process was investigated by quartz crystal microbalance with dissipation monitoring and atomic force microscopy. Both adsorption kinetics and peptide rearrangement dynamics at the solid interface were significantly different on the surface-modified films compared to the untreated ones. Fibroblast cells cultures at short times and in a simplified environment, i.e., a medium-free solution, were prepared to distinguish interaction events at the interface between cell membrane and surface-immobilized peptide for the two cases. It turned out that the cell-adhesive effect of immobilized PHSRN was different for hydrophobic compared to hydrophilic ones. Early signatures of cell spreading were only observed on the hydrophilic substrates. These effects are explained in terms of different spatial arrangements of PHSRN molecules immobilized on the two types of surfaces.

Confined protein adsorption into nanopore arrays fabricated by colloidal-assisted polymer patterning

A combination of plasma surface modification of polymer thin films and colloidal nanosphere lithography was used to fabricate two-dimensional nanopore arrays as protein nanocontainers.

Electrospun Scaffolds for Osteoblast Cells: Peptide-Induced Concentration-Dependent Improvements of Polycaprolactone

The design of hybrid poly-ε-caprolactone (PCL)-self-assembling peptides (SAPs) matrices represents a simple method for the surface functionalization of synthetic scaffolds, which is essential for cell compatibility. This study investigates the influence of increasing concentrations (2.5%, 5%, 10% and 15% w/w SAP compared to PCL) of three different SAPs on the physico-chemical/mechanical and biological properties of PCL fibers. We demonstrated that physico-chemical surface characteristics were slightly improved at increasing SAP concentrations: the fiber diameter increased; surface wettability increased with the first SAP addition (2.5%) and slightly less for the following ones; SAP-surface density increased but no change in the conformation was registered. These results could allow engineering matrices with structural characteristics and desired wettability according to the needs and the cell system used. The biological and mechanical characteristics of these scaffolds showed a particular trend at increasing SAP concentrations suggesting a prevailing correlation between cell behavior and mechanical features of the matrices. As compared with bare PCL, SAP enrichment increased the number of metabolic active h-osteoblast cells, fostered the expression of specific osteoblast-related mRNA transcripts, and guided calcium deposition, revealing the potential application of PCL-SAP scaffolds for the maintenance of osteoblast phenotype.

Novel pH responsive calix[8]arene hydrogelators: self-organization processes at a nanometric scale

Water soluble amphiphilic calix[8]arenes have been found to form pH-responsive and reversible supramolecular hydrogels at sub-millimolar concentration. Depending on their upper rim functionalization, pH variations trigger hydrogelation under basic or acidic conditions. Studies on self-organization processes by atomic force microscopy (AFM) measurements on differently charged surfaces are reported.

Aminofunctionalization and sub-micrometer patterning on silicon through silane doped agarose hydrogels

Agarose based hydrogels doped with amino-propyl-triethoxy-silane have been prepared and used for the biofunctionalization of hydroxylated Si (100) surfaces. The doped hydrogels consist of two independent polysaccharide and siloxane networks. The confluent colloidal layer coverage of the substrate from the transferring hydrogels is illustrated by various techniques. The line shape of the N 1s and C 1s X-ray photoemission spectra is shown to depend strongly on the polar angle of the photoelectron emission. This effect is explained in terms of different indepth distributions of N and C species and the dominant presence of surface oriented amino groups. The surface modification induces an alteration of wettability, but the intrinsic nanoscale topography of the transferred films is the determinant factor of the interaction with water. Furthermore, it is found that the transferred functionalization is compatible with the formation of sub-micrometer patterns by exploiting as masks polystyrene based colloidal monolayers. Both nanodots and nanoporous wells with a hexagonal lattice were observed by atomic force microscopy and their biofunctionality demonstrated by fluorescence microscopy after fluorophore labelled IgG immobilization.