Marco Indrieri

The effect of surface nanometre-scale morphology on protein adsorption.

BACKGROUND Protein adsorption is the first of a complex series of events that regulates many phenomena at the nano-bio interface, e.g. cell adhesion and differentiation, in vivo inflammatory responses and protein crystallization. A quantitative understanding of how nanoscale morphology influences protein adsorption is strategic for providing insight into all of these processes, however this understanding has been lacking until now. METHODOLOGY/PRINCIPAL FINDINGS Here we introduce novel methods for quantitative high-throughput characterization of protein-surface interaction and we apply them in an integrated experimental strategy, to study the adsorption of a panel of proteins on nanostructured surfaces. We show that the increase of nanoscale roughness (from 15 nm to 30 nm) induces a decrease of protein binding affinity (<or=90%) and a relevant increase in adsorbed proteins (<or=500%) beyond the corresponding increase of specific area. We demonstrate that these effects are caused by protein nucleation on the surface, which is promoted by surface nanoscale pores. CONCLUSIONS/SIGNIFICANCE These results show that the adsorption of proteins depends significantly on surface nanostructure and that the relevant morphological parameter regulating the protein adsorption process is the nanometric pore shape. These new findings improve our understanding of the role of nanostructures as a biomaterial design parameter and they have important implications for the general understanding of cell behavior on nanostructured surfaces.

Direct Microfabrication of Topographical and Chemical Cues for the Guided Growth of Neural Cell Networks on Polyamidoamine Hydrogels

Cell patterning is an important tool for organizing cells in surfaces and to reproduce in a simple way the tissue hierarchy and complexity of pluri-cellular life. The control of cell growth, proliferation and differentiation on solid surfaces is consequently important for prosthetics, biosensors, cell-based arrays, stem cell therapy and cell-based drug discovery concepts. We present a new electron beam lithography method for the direct and simultaneous fabrication of sub-micron topographical and chemical patterns, on a biocompatible and biodegradable PAA hydrogel. The localized e-beam modification of a hydrogel surface makes the pattern able to adsorb proteins in contrast with the anti-fouling surface. By also exploiting the selective attachment, growth and differentiation of PC12 cells, we fabricated a neural network of single cells connected by neuritis extending along microchannels. E-beam microlithography on PAA hydrogels opens up the opportunity of producing multifunctional microdevices incorporating complex topographies, allowing precise control of the growth and organization of individual cells.

Biomimetic poly(amidoamine) hydrogels as synthetic materials for cell culture

BackgroundPoly(amidoamine)s (PAAs) are synthetic polymers endowed with many biologically interesting properties, being highly biocompatible, non toxic and biodegradable. Hydrogels based on PAAs can be easily modified during the synthesis by the introduction of functional co-monomers. Aim of this work is the development and testing of novel amphoteric nanosized poly(amidoamine) hydrogel film incorporating 4-aminobutylguanidine (agmatine) moieties to create RGD-mimicking repeating units for promoting cell adhesion.ResultsA systematic comparative study of the response of an epithelial cell line was performed on hydrogels with agmatine and on non-functionalized amphoteric poly(amidoamine) hydrogels and tissue culture plastic substrates. The cell adhesion on the agmatine containing substrates was comparable to that on plastic substrates and significantly enhanced with respect to the non-functionalized controls. Interestingly, spreading and proliferation on the functionalized supports are slower than on plastic exhibiting the possibility of an easier control of the cell growth kinetics. In order to favor the handling of the samples, a procedure for the production of bi-layered constructs was also developed by means the deposition via spin coating of a thin layer of hydrogel on a pre-treated cover slip.ConclusionThe obtained results reveal that PAAs hydrogels can be profitably functionalized and, in general, undergo physical and chemical modifications to meet specific requirements. In particular the incorporation of agmatine warrants good potential in the field of cell culturing and the development of supported functionalized hydrogels on cover glass are very promising substrates for applications in cell screening devices.

Adhesive-free colloidal probes for nanoscale force measurements: Production and characterization

We describe novel approaches for the production and characterization of epoxy- and adhesive-free colloidal probes for atomic force microscopy (AFM). Borosilicate glass microspheres are strongly attached to commercial AFM cantilevers exploiting the capillary adhesion force due to the formation of a water meniscus, and then a thermal annealing of the sphere-cantilever system at a temperature slightly below the softening point of borosilicate glass. Controlling the wettability of the surfaces involved turned out to be a crucial element for the control of surface adhesion and for the implementation of a completely adhesive-free production method of colloidal probes. Moreover, we present a statistical characterization protocol of the probe dimensions and roughness based on the AFM inverse imaging of colloidal probes on spiked gratings. We have assessed the influence of defects of the grating on the characterization of the probe, and discussed the accuracy of our characterization technique in comparison to the methods based on scanning electron or optical microscopy, or on the manual analysis of AFM inverse images.

Nanomanufacturing of titania interfaces with controlled structural and functional properties by supersonic cluster beam deposition

Great emphasis is placed on the development of integrated approaches for the synthesis and the characterization of ad hoc nanostructured platforms, to be used as templates with controlled morphology and chemical properties for the investigation of specific phenomena of great relevance in interdisciplinary fields such as biotechnology, medicine, and advanced materials. Here, we discuss the crucial role and the advantages of thin film deposition strategies based on cluster-assembling from supersonic cluster beams. We select cluster-assembled nanostructured titania (ns-TiO2) as a case study to demonstrate that accurate control over morphological parameters can be routinely achieved, and consequently, over several relevant interfacial properties and phenomena, like surface charging in a liquid electrolyte, and proteins and nanoparticles adsorption. In particular, we show that the very good control of nanoscale morphology is obtained by taking advantage of simple scaling laws governing the ballistic deposition regime of low-energy, mass-dispersed clusters with reduced surface mobility.

ATP-dependent looping of DNA by ISWI

Snf2 related chromatin remodelling enzymes possess an ATPase subunit similar to that of the SF-II helicases which hydrolyzes ATP to track along DNA. Translocation and any resulting torque in the DNA could drive chromatin remodeling. To determine whether the ISWI protein can translocate and generate torque, tethered particle motion experiments and atomic force microscopy have been performed using recombinant ISWI expressed in E. coli. In the absence of ATP, ISWI bound to and wrapped DNA thereby shortening the overall contour length measured in atomic force micrographs. Although naked DNA only weakly stimulates ATP hydrolysis by ISWI, both atomic force microscopy and tethered particle motion data indicate that the protein generated loops in the presence of ATP. The duration of the looped state of the DNA measured using tethered particle motion was ATP-dependent. Finally, ISWI relaxed positively supercoiled plasmids visualized by atomic force microscopy. While other chromatin remodeling ATPases catalyze either DNA wrapping or looping, both are catalyzed by ISWI.

Nanoscale Roughness Affects the Activity of Enzymes Adsorbed on Cluster-Assembled Titania Films

In this study, we investigated how the adsorption properties governed by the nanometer-scale surface morphology of cluster-assembled titanium oxide films influence the catalytic activity of immobilized serine-protease trypsin. We developed an activity assay for the parallel detection of physisorbed enzyme activity and mass density of the adsorbed proteins in microarray format. The method combines a microarray-based technique and advanced quantitative confocal microscopy approaches based on fluorescent labeling of enzymes and covalent labeling of active sites of surface-bound enzymes. The observed diminishing trypsin binding affinity with increasing roughness, as opposed to the steep rise in its saturation uptake, was interpreted as heterogeneous nucleation-driven adsorption of trypsin at the rough nanoporous titania surface. The increase in relative activity of adsorbed trypsin is proportional to the fractional saturation of titania surfaces, expressed as percentage of saturation uptake. In turn, the specific activity, that is, the ratio of active proteins to the absolute number of adsorbed proteins, drops with growing saturation uptake and surface roughness, witnessing a reduction in the accessibility of enzyme active sites. Both geometrical constraints of titania nanopores and the clusterwise adsorption of trypsin were identified as the key factors underpinning the steric hindrance of the immobilized enzyme. These findings are relevant for the optimization of rough nanoporous surfaces as carriers of immobilized enzymes. The proposed activity assay is particularly advantageous in the screening of candidate materials for enzyme immobilization.

Linearized texture of three-dimensional extracellular matrix is mandatory for bladder cancer cell invasion

In the fields of biomaterials and tissue engineering simulating the native microenvironment is of utmost importance. As a major component of the microenvironment, the extracellular matrix (ECM) contributes to tissue homeostasis, whereas modifications of native features are associated with pathological conditions. Furthermore, three-dimensional (3D) geometry is an important feature of synthetic scaffolds favoring cell stemness, maintenance and differentiation. We analyzed the 3D structure, geometrical measurements and anisotropy of the ECM isolated from (i) human bladder mucosa (basal lamina and lamina propria) and muscularis propria; and, (ii) bladder carcinoma (BC). Next, binding and invasion of bladder metastatic cell line was observed on synthetic scaffold recapitulating anisotropy of tumoral ECM, but not on scaffold with disorganized texture typical of non-neoplastic lamina propria. This study provided information regarding the ultrastructure and geometry of healthy human bladder and BC ECMs. Likewise, using synthetic scaffolds we identified linearization of the texture as a mandatory feature for BC cell invasion. Integrating microstructure and geometry with biochemical and mechanical factors could support the development of an innovative synthetic bladder substitute or a tumoral scaffold predictive of chemotherapy outcomes.

Quantitative Investigation by Atomic Force Microscopy of Supported Phospholipid Layers and Nanostructures on Cholesterol-Functionalized Glass Surfaces

Understanding the interaction mechanisms of phospholipids with surfaces is crucial for the exploitation of lipid bilayers as models of the cell membrane as well as templates for biosensors. Moreover, controlling and manipulating lipid nanoparticles for the investigation of their properties by means of single-particle sensitive surface techniques require the ability to tailor the chemical properties of surfaces to achieve a stable and sparse binding of lipid particles, while keeping them from aggregating, or denaturing. Here we present a quantitative morphological and structural investigation by atomic force microscopy of supported phospholipid layers and nanostructures on cholesterol-functionalized glass surfaces, in comparison with other surfaces with different interfacial properties. We show that the functionalization of glass coverslips with cholesterol groups is a viable route for the production of optically transparent, scanning probe microscopy-compatible clean substrates for the effective immobilization of both extended single lipid bilayers and lipid nanoparticles.