Single step synthesis of Janus nano-composite membranes by atmospheric aerosol plasma polymerization for solvents separation.

Solvent permeation across membranes is limited due to physical resistance to diffusion from the selective layer within the membrane and to plasticizing effects generated by the solvent molecules onto the polymeric macromolecular matrix. Nano-composite thin film membranes provide promising routes to generate controlled microstructural separation materials with higher selectivities and permeabilities. Here, the fabrication of nano-composite based on octamethyl-polyhedral oligomeric silsesquioxane - hexamethyldisiloxane thin film membranes is demonstrated by aerosol assisted atmospheric plasma deposition onto pre-formed nano-porous membrane supports for the first time. Stable, atomically smooth and continuous solid films with controllable thickness down to 50 nm were achieved. The deposition process allowed for the control of the wettability of the surfaces to water and organic solvents, leading to the generation of hydrophobic but alcohol-philic surfaces. The liquid entry pressure of the films to water was found to be 8 bar from plasma polymerization as oppose to 3 bar for the bare nano-porous support only. In addition, the ideal separation selectivity for ethanol to water, up to 6.5, highlight the impact of both the surface energy and level of cross-linking of the hexamethyldisiloxane nanostructures on the diffusion mechanisms. This new atmospheric plasma deposition strategy opens-up cost-effective and environmentally friendly routes for the design of the smart Janus membrane with customizable properties and performance.

Differential uPAR recruitment in caveolar-lipid rafts by GM1 and GM3 gangliosides regulates endothelial progenitor cells angiogenesis.

Gangliosides and the urokinase plasminogen activator receptor (uPAR) tipically partition in specialized membrane microdomains called lipid-rafts. uPAR becomes functionally important in fostering angiogenesis in endothelial progenitor cells (EPCs) upon recruitment in caveolar-lipid rafts. Moreover, cell membrane enrichment with exogenous GM1 ganglioside is pro-angiogenic and opposite to the activity of GM3 ganglioside. On these basis, we first checked the interaction of uPAR with membrane models enriched with GM1 or GM3, relying on the adoption of solid-supported mobile bilayer lipid membranes with raft-like composition formed onto solid hydrophilic surfaces, and evaluated by surface plasmon resonance (SPR) the extent of uPAR recruitment. We estimated the apparent dissociation constants of uPAR-GM1/GM3 complexes. These preliminary observations, indicating that uPAR binds preferentially to GM1-enriched biomimetic membranes, were validated by identifying a pro-angiogenic activity of GM1-enriched EPCs, based on GM1-dependent uPAR recruitment in caveolar rafts. We have observed that addition of GM1 to EPCs culture medium promotes matrigel invasion and capillary morphogenesis, as opposed to the anti-angiogenesis activity of GM3. Moreover, GM1 also stimulates MAPKinases signalling pathways, typically associated with an angiogenesis program. Caveolar-raft isolation and Western blotting of uPAR showed that GM1 promotes caveolar-raft partitioning of uPAR, as opposed to control and GM3-challenged EPCs. By confocal microscopy, we have shown that in EPCs uPAR is present on the surface in at least three compartments, respectively, associated to GM1, GM3 and caveolar rafts. Following GM1 exogenous addition, the GM3 compartment is depleted of uPAR which is recruited within caveolar rafts thereby triggering angiogenesis.

Long-lasting antifog plasma modification of transparent plastics.

Antifog surfaces are necessary for any application requiring optical efficiency of transparent materials. Surface modification methods aimed toward increasing solid surface energy, even when supposed to be permanent, in fact result in a nondurable effect due to the instability in air of highly hydrophilic surfaces. We propose the strategy of combining a hydrophilic chemistry with a nanotextured topography, to tailor a long-lasting antifog modification on commercial transparent plastics. In particular, we investigated a two-step process consisting of self-masked plasma etching followed by plasma deposition of a silicon-based film. We show that the deposition of the silicon-based coatings on the flat (pristine) substrates allows a continuous variation of wettability from hydrophobic to superhydrophilic, due to a continuous reduction of carbon-containing groups, as assessed by Fourier transform infrared and X-ray photoelectron spectroscopies. By depositing these different coatings on previously nanotextured substrates, the surface wettability behavior is changed consistently, as well as the condensation phenomenon in terms of microdroplets/liquid film appearance. This variation is correlated with advancing and receding water contact angle features of the surfaces. More importantly, in the case of the superhydrophilic coating, though its surface energy decreases with time, when a nanotextured surface underlies it, the wetting behavior is maintained durably superhydrophilic, thus durably antifog.

Surface plasmon resonance as detection tool for lipids lateral mobility in biomimetic membranes.

A procedure based on surface plasmon resonance (SPR) is proposed to monitor the lateral mobility of lipid molecules in solid-supported bilayer lipid membranes (ssBLMs), an essential prerequisite for the formation of important microdomains called lipid rafts (LRs). The procedure relies on the marked tendency of the ganglioside GM1 to be recruited by LRs and to act as a specific receptor of the beta-subunit of the cholera toxin (ChTB). In the presence of both GM1 and ChTB, spontaneous formation of lipid rafts domains in mobile ssBLMs is accompanied by an appreciable increase in the amount of adsorbed ChTB, as monitored by SPR.

Cell adhesion on nanotextured slippery superhydrophobic substrates.

In this work, the response of Saos2 cells to polymeric surfaces with different roughness/density of nanometric dots produced by a tailored plasma-etching process has been studied. Topographical features have been evaluated by atomic force microscopy, while wetting behavior, in terms of water-surface adhesion energy, has been evaluated by measurements of drop sliding angle. Saos2 cytocompatibility has been investigated by scanning electron microscopy, fluorescent microscopy, and optical microscopy. The similarity in outer chemical composition has allowed isolation of the impact of the topographical features on cellular behavior. The results indicate that Saos2 cells respond differently to surfaces with different nanoscale topographical features, clearly showing a certain inhibition in cell adhesion when the nanoscale is particularly small. This effect appears to be attenuated in surfaces with relatively bigger nanofeatures, though these express a more pronounced slippery/dry wetting character.

Osteoblast-like cell behavior on plasma deposited micro/nanopatterned coatings.

The behavior of cells in terms of cell-substrate and cell-cell interaction is dramatically affected by topographical characteristics as shape, height, and distance, encountered in their physiological environment. The combination of chemistry and topography of a biomaterial surface influences in turns, important biological responses as inflammatory events at tissue-implant interface, angiogenesis, and differentiation of cells. By disentangling the effect of material chemistry from the topographical one, the possibility of controlling the cell behavior can be provided. In this paper, surfaces with different roughness and morphology were produced by radiofrequency (RF, 13.56 MHz) glow discharges, fed with hexafluoropropylene oxide (C(3)F(6)O), in a single process. Coatings with different micro/nanopatterns and the same uppermost chemical composition were produced by combining two plasma deposition processes, with C(3)F(6)O and tetrafluoroethylene (C(2)F(4)), respectively. The behavior of osteoblast-like cells toward these substrates clearly shows a strict dependence of cell adhesion and proliferation on surface roughness and morphology.

Influence of chemistry on wetting dynamics of nanotextured hydrophobic surfaces.

In this work, the role of a chemical parameter, such as the degree of fluorination, on the wetting behavior of nanotextured hydrophobic surfaces is investigated. Texture and chemistry tuning of the surfaces has been accomplished with single batch radiofrequency low-pressure plasma processes. Polystyrene substrates have been textured by CF(4) plasma etching and subsequently covered by thin films with a tunable F-to-C ratio, obtained in discharges fed with C(4)F(8)-C(2)H(4). Measurements of wetting dynamics reveal a regime transition from adhesive-hydrophobic to slippery-superhydrophobic, i.e., from wet to non wet states, as the F-to-C rises at constant topography. Such achievements are strengthened by calculation of the solid fraction of surface water contact area applying Cassie-Baxter advancing and receding equations to water contact angle data of textured and flat reference surfaces.

Increasing cell adhesion on plasma deposited fluorocarbon coatings by changing the surface topography.

In designing new biomaterials, it is of outstanding importance to consider how cells respond to specific chemical and topographical features on the material surface. The behavior of most cell types in vivo is strictly related to specific chemical and topographical cues that characterize the extra cellular environment. In particular, during their lives cells react to topographical patterns such as those of the extracellular matrix (ECM), of micro and/or nanometric dimensions. The production of micrometric and/or nanometric features on artificial materials usually involves expensive and time-consuming methods of manufacturing, such as electron beam and colloidal lithography. In this article, different "Teflon-like" structured surfaces were deposited from tetrafluoroethylene (C(2)F(4))-fed plasmas, for the study of cell adhesion and growth. The reaction of different cell lines to different topographical features was evaluated and compared with cell behavior on flat samples with the same chemical composition. Cell adhesion was calculated from area covered by cells at different time of culture. Beside this, cell proliferation was determined with the MTT test. Cell morphology and filopodia interaction with the nanofeatures were also estimated by optical and scanning electron microscopy. A dramatic difference both in adhesion and growth was found between cells seeded on flat and rough surfaces with the density and spreading of adhered cells varying as a function of the roughness of coatings.

Nanotexturing of polystyrene surface in fluorocarbon plasmas: from sticky to slippery superhydrophobicity.

In this work plasma etching processes have been studied to roughen and fluorinate polystyrene surface as an easy method to achieve a superhydrophobic slippery character. Radiofrequency discharges have been fed with CF(4)/O(2) mixtures and the effect of the O(2):CF(4) ratio, the input power, and the treatment duration have been investigated in terms of wettability, with focus on sliding performances. For this purpose, surface morphological variations, evaluated by means of scanning electron microscopy and atomic force microscopy, together with the chemical assessment by X-ray photoelectron spectroscopy, have been correlated with water contact angle hysteresis and volume resolved sliding angle measurements. Results indicate that by increasing the height and decreasing the density of the structures formed by etching, within a tailored range, a transition from sticky to slippery superhydrophobicity occurs. A short treatment time (5 min) is sufficient to obtain such an effect, provided that a high power input is utilized. Optimized surfaces show a unaltered transparency to visible light according to the low roughness produced.

Fetuin-A gene expression, synthesis and release in primary human hepatocytes cultured in a galactosylated membrane bioreactor.

This paper reports on human hepatocytes cultured in a galactosylated membrane bioreactor in order to explore the modulation of the effects of a pro-inflammatory cytokine, Interleukin-6 (IL-6) on the liver cells at molecular level. In particular the role of IL-6 on gene expression and production of a glycoprotein, fetuin-A produced by hepatocytes, was investigated by culturing hepatocytes in the membrane bioreactor, both in the absence and presence of IL-6 (300 pg/ml). IL-6 modulated the fetuin-A gene expression, synthesis and release by primary human hepatocytes cultured in the bioreactor. A 75% IL-6-induced reduction of fetuin-A concentration in the medium was associated with a 60% increase of C-reactive protein in the same samples. Real-time-PCR demonstrated an 8-fold IL-6-induced reduction of fetuin-A gene expression. These results demonstrate that the hepatocyte galactosylated membrane bioreactor is a valuable tool to study IL-6 effects and gave evidence, for the first time, that IL-6 down-regulates the gene expression and synthesis of fetuin-A by primary human hepatocytes. The human hepatocyte bioreactor behaves like the in vivo liver, reproducing the same hepatic acute-phase response that occurs during the inflammatory process.

Novel membranes and surface modification able to activate specific cellular responses.

The design of new polymeric biomaterials together with new strategies to modify membrane surface are crucial to optimise cell-biomaterial interactions in vivo and in vitro biohybrid systems. In this study we report on the novel semipermeable membranes synthesised from a polymeric blend of modified polyetheretherketone and polyurethane able to support the long-term maintenance and differentiation of human liver cells and on the surface modification of polyethersulfone membranes by plasma polymerisation of acrylic acid monomers and by immobilization of arginine-glycine-aspartic acid (RGD) peptide through a hydrophilic "spacer arm" molecule. The performance of the modified and unmodified membranes was tested by evaluation of the liver function expression of primary human hepatocytes in terms of albumin production, protein secretion and drug biotransformation.

Galactose derivative immobilized glow discharge processed polyethersulfone membranes maintain the liver cell metabolic activity.

New strategies aimed to surface modification of polymeric membranes are crucial to optimise cell-biomaterial interactions in vivo and in vitro biohybrid systems. In this paper, we investigated the surface modification of Polyethersulfone (PES) membranes by plasma polymerisation of acrylic acid monomers (PES-pdAA) and by immobilization of galactonic acid through a hydrophilic "spacer arm" molecule (PES-pdAA-SA-GAL). The modification steps were characterised by high resolution X-ray photoelectron spectroscopy. The performance of modified and unmodified membranes was evaluated by assessing the expression of liver specific biotransformation functions of pig and human hepatocytes. Human liver cells cultured on PES-pdAA-SA-GAL membranes displayed an enhanced albumin production, urea synthesis and protein secretion for 24 days of culture. The immobilisation of galactose derivative units on the membrane allowed specific interactions with hepatocytes biomimicking the cellular microenvironment and produced an improvement of the long-term maintenance and differentiation of human hepatocytes.

Adhesion and proliferation of fibroblasts on RF plasma-deposited nanostructured fluorocarbon coatings: evidence of FAK activation.

We used combined plasma-deposition process to deposit smooth and nanostructured fluorocarbon coatings on polyethylenethereftalate (PET) substrates, to obtain surfaces with identical chemical composition and different roughness, and investigate the effect of surface nanostructures on adhesion and proliferation of 3T3 Swiss Albino Mouse fibroblasts. Untreated PET and polystyrene (PS) were used as controls for cell culture. We have found that the statistically significant increase of cell proliferation rate and FAK (a nonreceptor tyrosine kinase) activation detected on ROUGH fluorocarbon surfaces is due to the presence of nanostructures. Changes in cytoskeletal organization and phospho FAK (tyr 397) localization were evident after 60 min on cells adhering to ROUGH surfaces. This change was characterized by the formation of actin stress fibers along lamellar membrane protrusion instead of usual focal contacts. Also the morphology of the adhering fibroblasts (60 min) adhering on ROUGH surfaces was found quite different compared to cells adhering on smooth ones.

Proliferative and re-defferentiative effects of photo-immobilized micro-patterned hyaluronan surfaces on chondrocyte cells.

A photo-immobilisation procedure was utilised to create two different micro-patterned surfaces (tracks 25 and 5 microm wide) of hyaluronan (Hyal) on polyethylene-terephthalate (PET) previously plasma activated. Aim of the study was to investigate the proliferation and re-differentiation capacity of articular chondrocytes cultured on micro-patterned Hyal, compared to homogeneous Hyal and plain plasma-treated (pt-)PET substrates. Cytotoxicity, cell proliferation, activation and differentiation of articular knee cartilage chondrocytes (Mongrel sheep) were evaluated after 14 days of culture. It was found that micro-patterned Hyal surfaces induced the adhesion, migration and alignment of chondrocytes, as shown by light and scanning electron microscopy. Furthermore, the same surfaces induced chondrocyte differentiation, with a significant increase of aggrecan and collagen type II production, while homogeneous Hyal and pt-PET surfaces did not.

Biotransformation and liver-specific functions of human hepatocytes in culture on RGD-immobilized plasma-processed membranes.

In this paper we report on the metabolic response of human hepatocytes grown on polyethersulfone membranes surface modified with a plasma-deposited acrylic acid coating and RGD peptide covalently immobilized through a "spacer arm" molecule. The modified surfaces were characterized by means of X-ray photoelectron spectroscopy and water contact angle measurements. The performance of modified and unmodified membranes was evaluated by assessing the expression of liver specific and biotransformation functions of human hepatocytes. Diclofenac, a non-steroidal anti-inflammatory drug, was used to investigate the biotransformation functions. Surface-modified membranes elicit specific cellular responses and induce hepatocytes to enhance the synthesis rate of albumin and urea, particularly in the presence of diclofenac. Also the biotransformation functions were expressed at high levels.

Stable plasma-deposited acrylic acid surfaces for cell culture applications.

Continuous and modulated glow discharges were used to deposit thin films from acrylic acid vapors. Different deposition regimes were investigated, and their effect on chemical composition, morphology and homogeneity of the coatings, as well as on their stability in water and resistance to sterilization. Stable films were utilized in cell adhesion experiments with human fibroblasts.