Enhanced plasmonic processes in amino-rich plasma polymer films for applications at the biointerface.

A new plasmonic biosensor was developed in a planar chip-based format by coupling the plasmonic properties of gold nanoparticles (Au NPs) with the mechanical and bioadhesive features of unconventional organic thin films deposited from plasma, namely primary amine-based plasma polymer films (PPFs). A self-assembled layer of spherical Au NPs, 12 nm in diameter, was electrostatically immobilized onto optically transparent silanised glass. In the next step, the Au NP layer was coated with an 18 nm polymeric thick PPF layer via the simultaneous polymerization/deposition of a cyclopropylamine (CPA) precursor performed by radio frequency discharge, both in pulsed and in continuous wave modes. The CPA PFF surface plays the dual role of an adsorbent towards negatively charged chemical species as well as an enhancer of plasmonic signals. The biosensor was tested in a proof-of-concept series of experiments of human serum albumin physisorption, and chosen as a model system for blood serum. The peculiar surface features of CPA PPF, before and after the exposure to buffered solution of fluorescein isothiocyanate-labelled human serum albumin (FITC-HSA), were investigated by a multi-technique approach, including UV-visible and X-ray photoelectron spectroscopies, atomic force microscopy, scanning electron microscopy, contact angle and surface free energy measurements. The results showed the very promising potentialities from both bioanalytical and physicochemical points of view in scrutinizing the macromolecule behavior at the biointerface.

Green Light-Triggerable Chemo-Photothermal Activity of Cytarabine-Loaded Polymer Carbon Dots: Mechanism and Preliminary In Vitro Evaluation.

Carbon-based nanostructures are attracting a lot of attention because of their very low toxicity, excellent visible light-triggered optical and photothermal properties, and intriguing applications. Currently, the development of multifunctional carbon-based nanostructures for a synergistic chemo-photothermal approach is a challenging topic for the advancement of cancer treatment. Here, we report an unprecedented example of photoresponsive carbon-based polymer dots (CPDs-PNM) obtained by a one-pot thermal process from poly(N-isopropylacrylamide) (PNIPAM) without using organic solvent and additional reagents. The CPDs-PNM nanostructures were characterized by spectroscopic techniques, transmission electron microscopy, and atomic force microscopy. The CPDs-PNM exhibited high photothermal conversion efficiency, lower critical solution temperature (LCST) behavior, and good cytarabine (arabinosyl cytosine, AraC) loading capacity (62.3%). The formation of a CPDs-PNM/AraC adduct and photothermal-controlled drug release, triggered by green light excitation, were demonstrated by spectroscopic techniques, and the drug-polymer interaction and drug release mechanism were well supported by modeling simulation calculations. The cellular uptake of empty and AraC-loaded CPDs-PNM was imaged by confocal laser scanning microscopy. In vitro experiments evidenced that CPDs-PNM did not affect the viability of neuroblastoma cells, while the CPDs-PNM/AraC adduct under light irradiation exhibited significantly higher toxicity than AraC alone by a combined chemo-photothermal effect.

A nanosized photothermal responsive core-shell carbonized polymer dots based on poly(N-isopropylacrylamide) for light-triggered drug release.

Core-shell nanocomposites are one of the most important achievements in the fast-growing field of nanotechnology. The combination of multi-responsive nano-shell with luminescent and photothermal core has led to promising applications in various fields such as optics, electronics and medicine. In this work, a nanosized core-shell system composed by carbonized dots core and poly(N-isopropylacrylamide) shell was developed and the photothermal triggered release of doxorubicin was demonstrated. The system was fully characterized by H1-NMR, DLS, Z-potential, AFM, optical absorption and fluorescence measurements. A photothermal conversion efficiency (η) value of about 67.9% and a doxorubicin photo-release rate value of about 1.0% min-1 were measured. Molecular dynamic (MD) simulations data were in agreement with experimental results, at 310 K the coil-to-globule transition and a consequent desorption of doxorubicin from the polymer were observed. Both the radius of gyration and the fluctuation of the distance doxorubicin-PNIPAM pointed that the temperature above the LCST and the acid pH facilitated the polymer transition. Moreover, MD simulations and experimental data suggested an influence on the lower critical solution temperature (LCST) exerted by the number of polymer chains anchored to the carbon core.

A novel facile one-pot synthesis of photothermally responsive carbon polymer dots as promising drug nanocarriers.

Luminescent and photothermic carbon polymer dots (CPDs-PNM), composed of a carbonized core and cross-linked chains of poly(N-isopropylacrylamide), were synthetized by a novel, simple, solvent- and reagent-free method. The formation of CPDs-PNM was controlled by both temperature and heating time. The CPDs-PNM exhibited LCST behaviour, high photothermal conversion efficiency, curcumin loading capacity and no toxicity to eukaryotic cells. Proof of concept experiments confirmed an excellent thermally induced drug release activity to be used for photothermally controlled drug release.

Specific, Surface-Driven, and High-Affinity Interactions of Fluorescent Hyaluronan with PEGylated Nanomaterials.

Hybrid nanomaterials are a subject of extensive research in nanomedicine, and their clinical application is reasonably envisaged in the near future. However, the fate of nanomaterials in biological environments poses serious limitations to their application; therefore, schemes to monitor them and gain control on their toxicity could be of great help for the development of the field. Here, we propose a probe for PEGylated nanosurfaces based on hyaluronic acid (HA) functionalized with rhodamine B (RB). We show that the high-affinity interaction of this fluorogenic hyaluronan (HA-RB) with nanoparticles exposing PEGylated surfaces results in their sensing, labeling for super-resolution imaging, and synergistic cellular internalization. HA-RB forms nanogels that interact with high affinity-down to the picomolar range-with silica nanoparticles, selectively when their surface is covered by a soft and amphiphilic layer. This surface-driven interaction triggers the enhancement of the luminescence intensity of the dyes, otherwise self-quenched in HA-RB nanogels. The sensitive labeling of specific nanosurfaces also allowed us to obtain their super-resolution imaging via binding-activated localization microscopy (BALM). Finally, we show how this high-affinity interaction activates a synergistic cellular uptake of silica nanoparticles and HA-RB nanogels, followed by a differential fate of the two partner nanomaterials inside cells.

The curious case of opossum prion: a physicochemical study on copper(ii) binding to the bis-decarepeat fragment from the protein N-terminal domain.

The opossum is a peculiar model of immunity to prion diseases. Here we scrutinised the bis-decarepeat peptide sequence of the opossum prion (Op_bis-deca) protein by a multitechnique approach, with a combined experimental (potentiometry, UV-visible, circular dichroism, NMR and EPR spectroscopy, quartz crystal microbalance with dissipation monitoring and confocal microscopy) and simulation (DFT calculations) approach. Results showed that the macrochelate structures formed upon the binding to Cu(ii) by the analogous bis-octarepeat peptide sequence of human prion (Hu_bis-octa) are not found in the case of Op_bis-deca. At physiological pH and equimolar amount of copper ions, the [CuLH-2] is the major species formed by Op_bis-deca. In this species one imidazole and two amide nitrogen atoms are involved in metal coordination and its stability constant value is lower than that of the analogous species formed by Hu_bis-octa, due to the presence of an extra proline residue. Moreover, the study on the interaction of the peptides or the peptide/Cu(ii) complexes with the model cell membranes made of supported lipid bilayers disclosed different levels of interaction, monitored by the viscoelastic changes of the membranes, which exhibited a similar viscoelastic response at the interface of the two complexes, while in the absence of Cu(ii), the Hu_bis-octa/SLB interface was more viscoelastic than the Op_bis-deca one.

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.

Enhancement of fibroblastic proliferation on chitosan surfaces by immobilized epidermal growth factor.

Chitosan membranes were modified with mouse epidermal growth factor (EGF) by a photochemical technique. Photochemical immobilization was performed via a two-step process, in which EGF was first reacted with a heterobifunctional cross-linker sulfo-SANPAH (sulfosuccinimidyl 6(4'-azido-2'-nitrophenyl-amino)hexanoate) and then immobilized on the chitosan membrane by UV irradiation. The success of immobilization process was checked by Fourier transform infrared attenuated total reflection spectroscopy and X-ray photoelectron spectroscopy. Atomic force microscopy was used to evaluate the surface topography. The mitogenic effect of the EGF-modified chitosan membrane was investigated using mouse fibroblasts (L929 cell line), and cell proliferation was investigated by MTT and crystal violet assays. The results obtained from cell culture experiments showed that immobilized EGF stimulated fibroblast growth on chitosan membranes, and a considerable difference in cell proliferation was detected on EGF-modified chitosan membranes.

Expression of cell adhesion receptors in human osteoblasts cultured on biofunctionalized poly-(epsilon-caprolactone) surfaces.

This study was aimed to investigate whether the activation of poly-(epsilon-caprolactone) (PCL) surface by low-energy irradiation and/or the biofunctionalization by absorption of arginine-glycine-aspartic sequences (RGD), can modify the expression of integrins closely related to the osteoblast activity. For this purpose, we analysed the physicochemical changes induced by irradiation and RGD immobilization, the consequences on cell adhesion and spreading, and the effects on integrin expression. PCL irradiated with 5 x 10(15)He(+)/cm(2) (10 keV energy) (irr-PCL) showed an altered surface layer with a partial loss of carboxyl species and the formation of carbonyl groups. Moreover, irr-PCL showed a small smoothening effect and a less polar character in comparison to the pristine ones. The RGD immobilization was observed only on irr-PCL (surface coverage: 7.0 pmol/cm(2)). Human osteoblasts (hOB) were cultured on untreated PCL (ut-PCL), ut-PCL+RGD, irr-PCL, and irr-PCL+RGD. After 24h, ut-PCL hindered the cell adhesion, while a discrete layer of hOB with a good cytoskeleton organization was detected on irr-PCL and irr-PCL+RGD. Before seeding, the single hOB suspension expressed alpha1, alpha2, alpha3, alpha5, beta1, and alphaVbeta3; after 24h, cells cultured on tissue-plastic expressed high levels of beta1 and alphaVbeta3, while alpha1 showed a low intensity and alpha2, alpha3, and alpha5 were negative. beta1 and alphaVbeta3 were selected to evaluate the interaction between cells and PCL samples. The beta1 expression was higher in hOB cultured on irr-PCL than on the other samples. A significant increase in alphaVbeta3 expression was observed only in irr-PCL+RGD, and confirmed by the gene expression analysis. In conclusion, ion irradiation and RGD adsorption on PCL surfaces modulate the expression of integrin involved in hOB growth and function, indicating the effectiveness of biomimetic surfaces in promoting cell adhesion. Ultimately, the study of integrin expression may suggest proper changes to the surface structure in order to improve the osteoconductivity of selected materials.

Evaluation of L929 fibroblast attachment and proliferation on Arg-Gly-Asp-Ser (RGDS)-immobilized chitosan in serum-containing/serum-free cultures.

In this study, chitosan membranes prepared by the solvent casting method were modified with the Arg-Gly-Asp-Ser (RGDS) sequence of fibronectin using the photochemical immobilization technique. The results obtained from attenuated total reflection-Fourier transform infrared spectra and X-ray photoelectron spectroscopy studies confirmed the successful immobilization of RGDS on chitosan membranes. The immobilized peptide concentration was determined by ninhydrin analysis on the order of 10(-7) mol/cm(2). In vitro cell culture studies were performed with L929 mouse fibroblasts to investigate the effect of biomodification on fibroblast cell behaviour in serum-free and 10% serum-containing media. The results obtained from cell culture studies pointed out the specific interactions between biosignal RGDS molecules and fibroblast cells. A triggered cell attachment and proliferation were observed on RGDS-modified chitosan membranes that were more distinguishable in serum-free medium. In addition, the photochemical immobilization technique was realized in the presence of a photomask that was used to immobilize the RGDS molecules in a defined micropattern. L929 mouse fibroblasts attached on the RGDS-micropatterned areas indicating integrin-mediated interactions.

Surface tailoring of polyacrylate-grafted graphene oxide for controlled interactions at the biointerface.

The actual surface termination and lateral size of a nanomaterial is crucial in its interaction with biomolecules at the aqueous interface. Graphene oxide (GO) nanosheets have been demonstrated as promising nanoplatform for both diagnostic and therapeutic applications. To this respect, 'smart' GO nanocarriers have been obtained by the surface functionalisation with polymers sensitive, e.g., to pH, as the polyacrylate (PAA) case. In this work, hybrid GO/PAA samples prepared respectively at low (GOPAAthin) or high (GOPAAthick) monomer grafting ratio, were scrutinised both theoretically, by molecular dynamic calculations, and experimentally by a multitechnique approach, including spectroscopic (UV-visible, fluorescence, Raman, Attenuated-total reflectance-Fourier transformed infrared and X-ray photoelectron spectroscopies), spectrometric (time-of-flight secondary ion and electrospray ionisation mass spectrometries) and microscopic (atomic force and confocal microscopies) methods. The actual surface termination, evaluated in terms of the relative ratio between polar and dispersive groups at the surface of the GO/polymer systems, was found to correlate with the average orientation of hydrophilic/hydrophobic domains of albumin, used as model protein. Moreover, the comparison among GO, GO-PAAthin and GO-PAAthick in the optical response at the interface with aqueous solutions, both at acid and at physiological pH, showed that the hybrid GO-polymer platform could be suitable not only to exploit a pH-triggered drug release but also for a modulation of the GO intrinsic emission properties. Energy transfer experiments on the GO/polymer oxide/fluorescein-labelled albumin/doxorubicin assembly showed significant differences for GO and GO-PAA samples, thus demonstrating the occurrence of different electronic processes at the hybrid nano-bio-interfaces. Confocal microscopy studies of cellular uptake in neuroblastoma cells confirmed the promising potentialities of the developed nanoplatform for applications at the biointerface.

The effect of irradiation modification and RGD sequence adsorption on the response of human osteoblasts to polycaprolactone.

Using techniques of tissue engineering, synthetic substitutes can be applied for the repair and regeneration of damaged bone. It has been found that material surface properties are crucial for cell adhesion and spreading, i.e. cell activities that are related directly to the ability of osteoblasts to proliferate. This fact has promoted the strategy of creating an ECM-like layer onto materials, so as to influence the cell response. In this study human bone-derived osteoblasts have been used to test the effects of surface modification by low energy ion beams of a poly epsilon-caprolactone (PCL) substrate and subsequent RGD adsorption. Osteoblasts were seeded and grown onto untreated and irradiated poly epsilon-caprolactone films, with or without RGD-adsorption step, and viability, morphology, and spreading of the osteoblasts were studied at different time endpoints. Differences were observed in the organization of cytoskeleton within cells: stress fibers were more evident in irradiated samples vs. untreated and total cell adhesion was higher. Surface characterization by X-ray Photoelectron Spectroscopy, Atomic Force Microscopy, and surface free energy measurements showed that the polar character of PCL, i.e., the acid-base term, was increased following irradiation treatment. Moreover the irradiated PCL had a nano-sized topography, which also could improve osteoblasts adhesion. We found that the treatment of the surface with ion beam is per se improving osteoblasts adhesion and spreading onto PCL. Furthermore, also if a significant RGD adsorption was obtained for irradiated PCL surfaces, it was found that in the investigated conditions it seems to have only a minor effect on the cell response. This study suggests that new strategies involving irradiation-based treatments can be adopted to promote the initial steps of bone deposition onto synthetic surfaces, exploiting the surface-induced reorganization of the ECM matrix.

Fast exopolysaccharide secretion of Pseudomonas aeruginosa on polar polymer surfaces.

The influence of the surface chemical structure and related physicochemical properties on the adhesion of P. aeruginosa has been studied for moderately hydrophobic polymers and for hydrophilic surfaces obtained by O2-plasma treatments and 50 keV Ar+ beam irradiation of poly(hydroxymethylsiloxane) and poly(ethyleneterephthalate). The surface chemical structure has been obtained by X-ray photoelectron spectroscopy, the roughness was measured by atomic force microscopy, and the surface free energy was evaluated from contact angle measurements for all the polymer substrates before and after the irradiation treatments. It is shown that a massive and unusually fast secretion of exopolysaccharides onto highly polar surfaces, corresponding to the formation of complex three-dimensional multilayers (i.e., biofilm-like structures), occurs already after 2 h of incubation. It is suggested that such highly polar surfaces can operate either by promoting, by means of a still unknown biomolecular mechanism, an early gene expression process or by mimicking the P. aeruginosa cellular walls.

Ultrathin and nanostructured ZnO-based films for fluorescence biosensing applications.

The fluorescence-based sensing capability of ultrathin ZnO-SiO(2) nanoplatforms, deposited by an integrated approach of colloidal lithography and metal organic chemical vapor deposition, has been investigated upon adsorption of fluorescein-labeled albumin, used as model analyte biomolecule. The protein immobilization process after spontaneous adsorption/desorption significantly enhances the green emission of the different ZnO-based films, as evidenced by scanning confocal microscopy, corresponding to a comparable protein coverage detected by X-ray photoelectron spectroscopy. Moreover, experiments of fluorescence recovery after photobleaching evidence that the protein lateral diffusion at the biointerface is affected by the chemical and/or topographical patterning of hybrid ZnO-SiO(2) surfaces. The used approach is very promising for biomolecular detection applications of these ZnO-SiO(2) nanoplatforms, by simple sizing of the 2D vs. 3D patterning design, which in turn is accomplished by the fine tuning of the integrated colloidal lithography-chemical vapor deposition processes.

Microcapillary-like structures prompted by phospholipase A2 activation in endothelial cells and pericytes co-cultures on a polyhydroxymethylsiloxane thin film.

A thin film of poly(hydroxymethylsiloxane) (PHMS) has been deposited on glass dishes and tested as artificial support material for vascularization from mixed cultures of endothelial cells (EC) and pericytes (PC). The EC/PC co-cultures adhered massively on PHMS, with the formation of net-like microcapillary structures. Such evidence was not found on control glass substrates in the same co-culture conditions neither on PHMS for EC and PC in monocultures. The physicochemical characterization of PHMS and control glass surface by time-of-flight secondary ion mass spectrometry, X-ray photoelectron spectroscopy, water contact angle and atomic force microscopy, pointed to the main role of the polymer hydrophobilicy to explain the observed cellular behavior. Moreover, enhanced intercellular cross-talk was evidenced by the up-regulation and activation of cytoplasmic and Ca(2+)-independent phospholipase A(2) (cPLA(2) and iPLA(2)) expression and cPLA(2) phosphorylation, leading to the cell proliferation and microcapillary formation on the PHMS surface, as evidenced by confocal microscopy analyses. Co-cultures, established with growth-arrested PCs by treatment with mitomycin C, showed an increase in EC proliferation on PHMS. AACOCF(3) or co-transfection with cPLA(2) and iPLA(2)siRNA reduced cell proliferation. The results highlight the major role played by EC/PC cross-talk as well as the hydrophobic character of the substrate surface, to promote microcapillary formation. Our findings suggest an attractive strategy for vascular tissue engineering and provide new details on the interplay of artificial substrates and capillary formation.

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.

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.

UV-O3-treated and protein-coated polymer surfaces facilitate endothelial cell adhesion and proliferation mediated by the PKCalpha/ERK/cPLA2 pathway.

We examined the adhesion and proliferation of immortalized endothelial cells GP8.39 (ECs) onto polyethyleneterephtalate (PET) and polyhydroxymethylsiloxane (PHMS) thin films, functionalized by UV-O(3) treatment and/or protein immobilization. The modified surface topography showed partial oxidation for both polymers, a slight increase in wettability and monopolar basic character for PET, and a hydrophilic bipolar acid-base behaviour for PHMS. UV-O(3) treatment did not induce significant roughness changes (under 1 nm) as shown by atomic force spectroscopy measurements (AFM). The EC adhesion and spreading onto untreated and modified surfaces were investigated both before and after immobilization of collagen (CA) and fibronectin (FN) adlayers. AFM analyses showed an open-weave protein layer on both untreated polymers which became a tight-woven net after UV-O(3) irradiation of underlying films. On day 5 after seeding, cell count analyses on irradiated PET surfaces, CA/FN-coated or not, showed EC adhesion and proliferation significantly greater than those on untreated polymers, indicating that UV-O(3) irradiation promoted fast endothelialization. A less pronounced EC spreading behaviour on treated PHMS was observed. In ECs grown on irradiated and CA- or FN-coated PET, the levels of phospho-protein kinase Calpha (p-PKCalpha, phospho-ERK1/2, and phospho-cytosolic phospholipase A(2) (p-cPLA(2)), all enzymes taken as signaling markers of cell adhesion and proliferation, decreased in comparison to those in CA- or FN-coated untreated PET. In contrast, in ECs grown on UV-O(3)-treated PHMS, Western blot analyses showed increased levels of p-PKCalpha, p-ERK1/2 and p-cPLA(2) in comparison with cells grown onto untreated polymer. The growth response of ECs to the substrates was related to the changes of polarity properties of UV-O(3)-treated polymer films, from hydrophobic/neutral towards hydrophilic/charged layers, and the signaling pathway remodelling to the cell proliferation degree.

Pericyte adhesion and growth onto polyhydroxymethylsiloxane surfaces nanostructured by plasma treatment and ion irradiation.

The study deals with the adhesion and proliferation of bovine retina pericytes onto surfaces of poly(hydroxymethylsiloxane) (PHMS) modified either by cold plasma or by low-energy ion beams. The surface treatment was able to convert the original polymer matrix into SiO2-like phases for O2-plasma or ion-mixed SiCxOy(Hz) phases for ion irradiation, respectively, with different modification levels of the surface free energy (SFE) and related surface wettability. Pericytes exhibited a negligible adhesion and proliferation onto untreated PHMS, an enhanced adhesion but not proliferation on plasma-treated PHMS, and great adhesion and proliferation to full confluence on ion-irradiated PHMS, as measured by X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), quartz crystal microbalance, and optical microscopy. On the other hand, the adhesion and proliferation of GP8.39 endothelial cells (EC), which are strongly associated with pericytes in microvasculature, were very scarce onto both untreated and surface-modified PHMS. The surface-selective pericytal response was related to changes of physicochemical properties of PHMS film, from hydrophobic/neutral towards hydrophilic/negatively charged polymer layers, as well as to short- and long-time events of cell-surface interaction. We propose that surface properties can mediate and modulate cell-polymer matrix adhesion through the establishment of stereospecific chemical interactions and/or electrostatic repulsion, which can also explain the different behavior of pericytes compared to EC.