Edmondo M. Benetti

The role of the interplay between polymer architecture and bacterial surface properties on the microbial adhesion to polyoxazoline-based ultrathin films

Surface platforms were engineered from poly(L-lysine)-graft-poly(2-methyl-2-oxazoline) (PLL-g-PMOXA) copolymers to study the mechanisms involved in the non-specific adhesion of Escherichia coli (E. coli) bacteria. Copolymers with three different grafting densities α (PMOXA chains/Lysine residue of 0.09, 0.33 and 0.56) were synthesized and assembled on niobia (Nb₂O₅) surfaces. PLL-modified and bare niobia surfaces served as controls. To evaluate the impact of fimbriae expression on the bacterial adhesion, the surfaces were exposed to genetically engineered E. coli strains either lacking, or constitutively expressing type 1 fimbriae. The bacterial adhesion was strongly influenced by the presence of bacterial fimbriae. Non-fimbriated bacteria behaved like hard, charged particles whose adhesion was dependent on surface charge and ionic strength of the media. In contrast, bacteria expressing type 1 fimbriae adhered to the substrates independent of surface charge and ionic strength, and adhesion was mediated by non-specific van der Waals and hydrophobic interactions of the proteins at the fimbrial tip. Adsorbed polymer mass, average surface density of the PMOXA chains, and thickness of the copolymer films were quantified by optical waveguide lightmode spectroscopy (OWLS) and variable-angle spectroscopic ellipsometry (VASE), whereas the lateral homogeneity was probed by time-of-flight secondary ion mass spectrometry (ToF-SIMS). Streaming current measurements provided information on the charge formation of the polymer-coated and the bare niobia surfaces. The adhesion of both bacterial strains could be efficiently inhibited by the copolymer film only with a grafting density of 0.33 characterized by the highest PMOXA chain surface density and a surface potential close to zero.

Buried, Covalently Attached RGD Peptide Motifs in Poly(methacrylic acid) Brush Layers : The Effect of Brush Structure on Cell Adhesion

Iniferter-mediated surface-initiated photopolymerization was used to graft poly(methacrylic acid) (PMAA) brush layers obtained from surface-attached iniferters in self-assembled monolayers to a gold surface. The tethered chains were subsequently functionalized with the cell-adhesive arginine-glycine-aspartic acid (RGD) motif. The modified brushes were extended by reinitiating the polymerization to obtain an additional layer of PMAA, thereby burying the peptide-functionalized segments inside the brush structure. Contact angle measurements and Fourier transform infrared (FTIR) spectroscopy were employed to characterize the wettability and the chemical properties of these platforms. Time of flight secondary ion mass spectroscopy (TOF-SIMS) measurements were performed to monitor the chemical composition of the polymer layer as a function of the distance to the gold surface and obtain information concerning the depth of the RGD motifs inside the brush structure. The brush thickness was evaluated as a function of the polymerization (i.e., UV-irradiation) time with atomic force microscopy (AFM) and ellipsometry. Cell adhesion tests employing human osteoblasts were performed on substrates with the RGD peptides exposed at the surface as well as covered by a PMAA top brush layer. Immunofluorescence studies demonstrated a variation of the cell morphology as a function of the position of the peptide units along the grafted chains.

A Brush‐Gel/Metal‐Nanoparticle Hybrid Film as an Efficient Supported Catalyst in Glass Microreactors

A polymer-brush-based material was applied for the formation and in situ immobilization of silver and palladium nanoparticles, as a catalytic coating on the inner wall of glass microreactors. The brush film was grown directly on the microchannel interior by means of atom-transfer radical polymerization (ATRP), which allows control over the polymer film thickness and therefore permits the tuning of the number of nanoparticles formed on the channel walls. The wide applicability of the catalytic devices is demonstrated for the reduction of 4-nitrophenol and for the Heck reaction.

Characterization and molecular engineering of surface-grafted polymer brushes across the length scales by atomic force microscopy

With the advent of regulated, surface initiated polymerizations, specifically using controlled radical approaches, the choice of polymerizable compounds and the control over grafting chemistry have seen tremendous advancement. New analysis techniques and approaches are now needed to characterize these brushes with molecular precision. In addition, spatial structure control at the nanoscale, and tuning of thickness as well as composition of the brushes, have become feasible by utilizing recently developed enabling molecular nanofabrication approaches. Atomic force microscopy (AFM) is a powerful analytical tool for the characterization of polymer brushes, as well as for the fabrication of brush structures across the length scales. AFM has been used to investigate polymer brushes in a number of ways including imaging surface morphologies, measuring brush thickness, estimating the value of number average molar mass, observing stimulus responsive behavior and probing surface mechanical properties. In addition, AFM based methods such as nanoscratching, dip-pen nanolithography (DPN) and scanning probe oxidation (SPO) have been also employed for the nanofabrication of patterned polymer brushes. This feature article gives a short account of this field and highlights recent advances.

Preparation and characterization of macromolecular “hedge” brushes grafted from Au nanowires

Linear polymer brush structures of poly(methacrylic acid) chains, exhibiting a width from several hundred to 20–30 nm and a controllable height (nano-“hedge”), were grafted from designer substrates by photopolymerization using thiol-based iniferters assembled on Au nano-lines. The Au nano-lines were obtained using H-terminated silicon substrates by AFM tip assisted (“dip-pen”) nanolithography of HAuCl4 which yielded Au nano-wires upon reduction in contact with the substrate. The polymeric nano-“hedge” structures were characterized by AFM.

pH Responsive Polymeric Brush Nanostructures: Preparation and Characterization by Scanning Probe Oxidation and Surface Initiated Polymerization

pH-responsive PHEMA-based polymeric nanostructures were grown in a controlled manner by ATRP-based surface-initiated polymerization. Initiator nanopatterns were obtained on silicon wafers covered with OTS resists made by AFM scanning probe oxidation lithography. AFM images confirmed isolated grafting of stimuli-responsive hedge and dot brush structures exhibiting dimensions corresponding to a few tens of chains.

Thin Polymer Brush Decouples Biomaterial’s Micro-/Nanotopology and Stem Cell Adhesion

Surface morphology and chemistry of polymers used as biomaterials, such as tissue engineering scaffolds, have a strong influence on the adhesion and behavior of human mesenchymal stem cells. Here we studied semicrystalline poly(ε-caprolactone) (PCL) substrate scaffolds, which exhibited a variation of surface morphologies and roughness originating from different spherulitic superstructures. Substrates were obtained by varying the parameters of the thermal processing, that is, crystallization conditions. The cells attached to these polymer substrates adopted different morphologies responding to variations in spherulite density and size. In order to decouple substrate topology effects on the cells, sub-100 nm bioadhesive polymer brush coatings of oligo(ethylene glycol) methacrylates were grafted from PCL and functionalized with fibronectin. On surfaces featuring different surface textures, dense and sub-100 nm thick brush coatings determined the response of cells, irrespective to the underlying topology. Thus, polymer brushes decouple substrate micro-/nanoscale surface topology and the adhesion of stem cells.

Next-Generation Polymer Shells for Inorganic Nanoparticles are Highly Compact, Ultra-Dense, and Long-Lasting Cyclic Brushes

Cyclic poly-2-ethyl-2-oxazoline (PEOXA) ligands for superparamagnetic Fe3 O4 nanoparticles (NPs) generate ultra-dense and highly compact shells, providing enhanced colloidal stability and bio-inertness in physiological media. When linear brush shells fail in providing colloidal stabilization to NPs, the cyclic ones assure long lasting dispersions. While the thermally induced dehydration of linear PEOXA shells cause irreversible aggregation of the NPs, the collapse and subsequent rehydration of similarly grafted cyclic brushes allow the full recovery of individually dispersed NPs. Although linear ligands are densely grafted onto Fe3 O4 cores, a small plasma protein such as bovine serum albumin (BSA) still physisorbs within their shells. In contrast, the impenetrable entropic shield provided by cyclic brushes efficiently prevents nonspecific interaction with proteins.

Creeping Proteins in Microporous Structures: Polymer Brush-Assisted Fabrication of 3D Gradients for Tissue Engineering

Coupling of rapid prototyping techniques and surface-confined polymerizations allows the fabrication of 3D multidirectional gradients of biomolecules within microporous scaffolds. The compositional gradients can be tailored by polymer-brush-assisted diffusion of protein solutions. This technique allows spatial control over stem cells manipulation within 3D environments.

Mimicking natural cell environments: design, fabrication and application of bio-chemical gradients on polymeric biomaterial substrates

Gradients of biomolecules on synthetic, solid substrates can efficiently mimic the natural, graded variation of properties of the extracellular matrix (ECM). Such gradients represent accessible study platforms for the understanding of cellular activities, and they also provide functional supports for tissue engineering (TE). This review describes the most relevant methods to produce 2-dimensional (2D) as well as 3-dimensional (3D) gradient supports for cell manipulations, and also addresses the response of cells from different origins when seeded on these constructs. The fabrication strategies summarized encompass the combination of polymer and surface chemistries, micro- and nano-engineering construction strategies and biotechnological approaches. This multidisciplinary scheme has enabled the design and realization of diverse, synthetic supports as cellular environments, spanning from the first gradient self-assembled monolayer (SAM) to multilayers, and hybrid constructs mimicking the complexity of natural tissue environments. The standing challenge is bringing these advances in the fabrication of supports to a dynamic functioning in space and time, towards the successful imitation of the most complex bio-chemical system ever studied: our body.