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In this work, we shed new light on ultrasound contrast agents applied to the field of cultural heritage as an invaluable fine-tune cleaning tool for paper artworks. In this context, one of the primary and challenging issues is the removal of modern adhesives from paper artifacts. Modern adhesives are synthetic polymers whose presence enhances paper degradation and worsens its optical features. A thorough analytical and high-spatial-resolution combined study was successfully performed to test the capability of poly(vinyl alcohol)-based microbubbles stimulated by a proper noninvasive 1 MHz ultrasound field exposure in removing these adhesives from paper surfaces, in the absence of volatile invasive and toxic chemicals and without damaging paper and/or leaving residues. We demonstrate that poly(vinyl alcohol)-shelled microbubbles are suitable for interacting with paper surfaces, targeting and boosting in a few minutes the nondamaging removal of adhesive particles from paper samples thanks to their peculiar shell composition together with their ultrasound dynamics.

Significant improvement of phase-change perfluorocarbon microdroplets (MDs) in the vast theranostic scenario passes through the optimization of the MDs composition with respect to synthesis efficiency, stability, and drug delivery capability. To this aim, decafluoropentane (DFP) MDs stabilized by a shell of dimethyldioctadecylammonium bromide (DDAB) cationic surfactant were designed. A high concentration of DDAB-MDs was readily obtained within a few seconds by pulsed high-power insonation, resulting in low polydisperse 1 µm size droplets. Highly positive ζ-potential, together with a long, saturated hydrocarbon chains of the DDAB shell, are key factors to stabilize the droplet and the drug cargo therein. The high affinity of the DDAB shell with cell plasma membrane allows for localized chemotherapeutics delivery by increasing the drug concentration at the tumor cell interface and boosting the uptake. This would turn DDAB-MDs into a relevant drug delivery tool exhibiting high antitumor activity at very low drug doses. In this work, the efficacy of such an approach is shown to dramatically improve the effect of doxorubicin against 3D spheroids of mammalian tumor cells, MDA-MB-231. The use of three-dimensional (3D) cell cultures developed in the form of multicellular tumor spheroids (i.e., densely packed cells in a spherical shape) has numerous advantages compared to 2D cell cultures: in addition to have the potential to bridge the gap between conventional in vitro studies and animal testing, it will improve the ability to perform more predictive in vitro screening assays for preclinical drug development or evaluate the potential of off-label drugs and new co-targeting strategies.

Wet cleaning of paper artworks by using hydrogels is a recently proposed process in the cultural heritage field. In this contest, on one side, research has focused on more effective and tuned cleaning hydrogels; on the other, the cleaning performances of the proposed hydrogels have been studied on ancient paper and very little on modern paper. This lack of information is not negligible, since the two kinds of paper differ strongly in composition. The difference, in turn could affect the performance of cleaning gels. In this article, new chemical hydrogels were synthetized using an oxidized polyvinyl alcohol (PVA) bearing an aldehyde group at each chain end, as cross-linking agent, i.e. telechelic PVA. PVA hydrogels have been characterized as candidates for paper cleaning applications. The possibility of tuning the mechanical and retentive properties, simply by changing the ratio of two polymer concentrations, their stability and transparency, indeed, render them, potentially suitable in cultural heritage field. The cleaning capability of two hydrogels, with different PVA/telechelic PVA ratios, has been assessed both on ancient paper, than a modern one and the results have been compared. The efficacy of these hydrogels has been demonstrated characterizing the samples before and after the cleaning process by means of a multidisciplinary approach involving spectroscopic and chromatographic tests.

We realise an antibacterial nanomaterial based on the self-limited assembly of patchy plasmonic colloids, obtained by adsorption of lysozyme to gold nanoparticles. The possibility of selecting the size of the assemblies within several hundred nanometres allows for tuning their optical response in a wide range of frequencies from visible to near infrared. We also demonstrate an aggregation-dependent modulation of the catalytic activity, which results in an enhancement of the antibacterial performances for assemblies of the proper size. The gained overall control on structure, optical properties and biological activity of such nanomaterial paves the way for the development of novel antibacterial nanozymes with promising applications in treating multi drug resistant bacteria.

Poly(N-isopropylacrylamide) (PNIPAM) hydrogel microparticles with different core&ndash, shell morphologies have been designed, while maintaining an unvaried chemical composition: a morphology with (i) an un-crosslinked core with a crosslinked shell of PNIPAM chains and (ii) PNIPAM chains crosslinked to form the core with a shell consisting of tethered un-crosslinked PNIPAM chains to the core. Both morphologies with two different degrees of crosslinking have been assessed by confocal microscopy and tested with respect to their temperature responsivity and deformation by applying an osmotic stress. The thermal and mechanical behavior of these architectures have been framed within a Flory&ndash, Rehner modified model in order to describe the microgel volume shrinking occurring as response to a temperature increase or an osmotic perturbation. This study provides a background for assessing to what extent the mechanical features of the microgel particle surface affect the interactions occurring at the interface of a microgel particle with a cell, in addition to the already know ligand/receptor interaction. These results have direct implications in triggering a limited phagocytosis of microdevices designed as injectable drug delivery systems.

The influence of Synthetic Organic Pigments in the degradation of Acrylem AC 33, a waterborne acrylic binder, after UVB-artificial exposure was furtherly investigated, extending the study to other pigments classes (anthraquinones, phthalocyanines, diketo-pyrrolo-pyrroles) in addition to previously studied azo-pigments. Together with Fourier Transform Infrared, both in Attenuated Reflectance and Transmission mode, and Nuclear Magnetic Resonance spectroscopies, Raman spectroscopy was also used to observe ageing processes that pigments are subjected to. Specific effects, relative to the presence of different pigments, have been observed also in this study; moreover, Raman spectroscopy highlighted photoinduced effects on some pigments, which could result useful in Cultural Heritage diagnostics, especially for street artworks.

Among different therapeutic applications of Ultrasound (US), transient membrane sonoporation (SP) - a temporary, non-lethal porosity, mechanically induced in cell membranes through US exposure - represents a compelling opportunity towards an efficient and safe drug delivery. Nevertheless, progresses in this field have been limited by an insufficient understanding of the potential cytotoxic effects of US related to the failure of the cellular repair and to the possible activation of inflammatory pathway. In this framework we studied the in vitro effects of very low-intensity US on a human keratinocyte cell line, which represents an ideal model system of skin protective barrier cells which are the first to be involved during medical US treatments. Bioeffects linked to US application at 1 MHz varying the exposure parameters were investigated by fluorescence microscopy and fluorescence activated cell sorting. Our results indicate that keratinocytes undergoing low US doses can uptake drug model molecules with size and efficiency which depend on exposure parameters. According to sub-cavitation SP models, we have identified the range of doses triggering transient membrane SP, actually with negligible biological damage. By increasing US doses we observed a reduced cells viability and an inflammatory gene overexpression enlightening novel healthy relevant strategies.

The merging of the molecular specificity of Raman spectroscopy with the extraordinary optical properties of metallic nanoarchitectures is at the heart of Surface Enhanced Raman Spectroscopy (SERS), which in the last few decades proved its worth as powerful analytical tool with detection limits pushed to the single molecule recognition. Within this frame, SERS-based nanosensors for localized pH measurements have been developed and employed for a wide range of applications. Nevertheless, to improve the performances of such nanosensors, many key issues concerning their assembling, calibration and stability, that could significantly impact on the outcome of the pH measurements, need to be clarified. Here, we report on the detailed characterization of a case study SERS-active pH nanosensor, based on the conjugation of gold nanoparticles with the pH-sensitive molecular probe 4-mercaptobenzoic acid (4MBA). We analyzed and optimized all the aspects of the synthesis procedure and of the operating conditions to preserve the sensor stability and provide the highest responsiveness to pH. Exploiting the dependence of the SERS spectrum on the protonation degree of the carboxylic group at the edge of the 4MBA molecules, we derived a calibration curve for the nanosensor. The extrapolated working point, i.e., the pH value corresponding to the highest sensitivity, falls at pH 5.6, which corresponds to the pKa value of the molecule confined at the nanoparticle surface. A shift of the pKa of 4MBA, observed on the molecules confined at the nanostructured interface respect to the bulk counterpart, unveils the opportunity to assembly a SERS-based pH nanosensor with the ability to select its working point in the sensitivity region of interest, by acting on the nanostructured surface on which the molecular probe is confined. As a proof-of-concept, the nanosensor was successfully employed to measure the extracellular pH of normal and cancer cells, demonstrating the capability to discriminate between them.

Hypothesis One of the main assets of crosslinked polymer-shelled microbubbles (MBs) as ultrasound-active theranostic agents is the robustness of the shells, combined with the chemical versatility in modifying the surface with ligands and/or drugs. Despite the long shelf-life, subtle modifications occur in the MB shells involving shifts in acoustic, mechanical and structural properties. Experiments We carried out a long-term morphological and acoustic evolution analysis on elastomeric polyvinyl-alcohol (PVA)-shelled MBs, a novel platform accomplishing good acoustic and surface performances in one agent. Confocal laser scanning microscopy, acoustic spectroscopy and AFM nanomechanics were integrated to understand the mechanism of PVA MBs ageing. The changes in the MB acoustic properties were framed in terms of shell thickness and viscoelasticity using a linearised oscillation theory, and compared to MB morphology and to nanomechanical analysis. Findings We enlightened a novel, intriguing ageing time evolution of the PVA MBs with double behaviour with respect to a crossover time of ∼50 days. Before, significant changes occur in MB stiffness and shell thickness, mainly due to a massive release of entangled PVA chains. Then, the MB resonance frequency increases together with shell thickening and softening. Our benchmark study is of general interest for emerging viscoelastomeric bubbles towards personalised medicine.

Phase change contrast agents for ultrasound (US) imaging consist of nanodroplets (NDs) with a perfluorocarbon (PFC) liquid core stabilized with a lipid or a polymer shell. Liquid ↔ gas transition, occurring in the core, can be triggered by US to produce acoustically active microbubbles (MBs) in a process named acoustic droplet vaporization (ADV). MB shells containing polymerized diacetylene moiety were considered as a good trade off between the lipid MBs, showing optimal attenuation, and the polymeric ones, displaying enhanced stability. This work reports on novel perfluoropentane and perfluorobutane NDs stabilized with a monolayer of an amphiphilic fatty acid, i.e. 10,12-pentacosadiynoic acid (PCDA), cured with ultraviolet (UV) irradiation. The photopolymerization of the diacetylene groups, evidenced by the appearance of a blue color due to the conjugation of ene-yne sequences, exhibits a chromatic transition from the nonfluorescent blue color to a fluorescent red color when the NDs are heated or the pH of the suspension is basic. An estimate of the molecular weights reached by the polymerized PCDA in the shell, poly(PCDA), has been obtained using gel permeation chromatography and MALDI-TOF mass spectrometry. The poly(PCDA)/PFC NDs show good biocompatibility with fibroblast cells. ADV efficiency and acoustic properties before and after the transition were tested using a 1 MHz probe, revealing a resonance frequency between 1 and 2 MHz similar to other lipidic MBs. The surface of PCDA shelled NDs can be easily modified without influencing the stability and the acoustic performances of droplets. As a proof of concept we report on the conjugation of cyclic RGD and PEG chains of the particles to support targeting ability toward endothelial cells. ispartof: LANGMUIR vol:35 issue:31 pages:10116-10127 ispartof: location:United States status: published

In this study, The behavior of Acrylem AC 33–a waterborne paint binder, chemically a ethyl acrylate-methyl methacrylate copolymer-after an UVB-artificial ageing was evaluated. In particular, it has been investigated its behavior in presence of three different azo-pigments – Pigment Red 9, Pigment Yellow 3 and Pigment Yellow 74-, in order to evaluate the effects of these pigments on photodegradation processes of the medium. For this aim, Fourier Transformed InfraRed, both in Attenuated Reflectance and transmission mode, and Nuclear Magnetic Resonance Spectroscopies were used. Thanks to this multianalytical approach, the chemical variations, both on the surface and on the bulk of the pictorial film, have been assessed and the degradation products of surfactant present in the binder were identified. Furthermore, specific effects of these synthetic organic pigments on the photodegradation of the matrix and its additives were observed. The information obtained from this study can be employed in the characterization of differential ageing phenomena in artworks, especially in case of outdoor exposition as it occurs in street-art conditions.

Although medical ultrasound offers compelling opportunities to improve therapy in principle, progress in the field has been limited because of an insufficient understanding of the potential genotoxic and cytotoxic effects of ultrasound on biological systems. This paper is mainly focused on an in vitro study of effects with respect to genotoxicity and viability induced by 1- and 3-MHz medical ultrasound in murine fibroblasts (NIH-3T3) at low-intensity exposure (spatial peak temporal average intensity Ita

In this paper we report on the study of the interface of hybrid shell droplets encapsulating decafluoropentane (DFP), which exhibit interesting potentialities for ultrasound (US) imaging. The fabrication of the droplets is based on the deposition of a dextran methacrylate layer onto the surface of surfactants. The droplets have been stabilized against coalescence by UV curing, introducing crosslinks in the polymer layer and transforming the shell into an elastomeric membrane with a thickness of about 300 nm with viscoelastic behaviour. US irradiation induces the evaporation of the DFP core of the droplets transforming the particles into microbubbles (MBs). The presence of a robust crosslinked polymer shell introduces an unusual stability of the droplets also during the core phase transition and allows the recovery of the initial droplet state after a few minutes from switching off US. The interfacial tension of the droplets has been investigated by two approaches, the pendant drop method and an indirect method, based on the determination of the liquid ↔ gas transition point of DFP confined in the droplet core. The re-condensation process has been followed by capturing images of single MBs by confocal microscopy. The time evolution of MB relaxation to droplets was analysed in terms of a modified Church model to account for the structural complexity of the MB shell, i.e. a crosslinked polymer layer over a layer of surfactants. In this way the microrheology parameters of the shell were determined. In a previous paper (Chem. Commun., 2013, 49, 5763–5765) we showed that these systems could be used as ultrasound contrast agents (UCAs). In this work we substantiate this view assessing some key features offered by the viscoelastic nature of the droplet shell.

A multichannel glass-PDMS microfluidic chip functionalized with a novel aptasensor has been developed for the detection of Ochratoxin A (OTA). The aptasensor is built on a glass substrate covered with a poly(2-hydroxyethtyl methacrylate) brush layer, which is subsequently functionalized with an aptamer having high affinity towards OTA. The assay relies on an aptamer-linked immobilized sorbent assay (ALISA) using two different OTA aptamers, which, in presence of OTA, assemble to form a sandwich-like structure generating a chemiluminescent signal. The chip is combined with an array of amorphous silicon photosensors that transduce the chemiluminescent signal into an electrical signal that can be processed to detect and quantify the OTA in the sample. The successful detection of OTA has been demonstrated in standard solutions and in beer samples spiked with OTA. The results shows that the values measured for OTA, applying the ALISA assay, are comparable to those measured by the reference methods. The sensitivity of the proposed analytical method is 0.32 pA L/mg, while the LOD and the LOQ are 0.82 and 2.5 mg/L, respectively. Taking into account both the LOD of the ALISA and the method applied for OTA extraction from beer samples, we extrapolated that 1.1 g of beer sample is sufficient to detect OTA contamination under the regulatory limits.

We realized and tested a strategy for developing reproducible and stable two-dimensional gold nanoparticle cluster arrays arranged on silicon substrates, to be used for surface-enhanced Raman spectroscopy. We combined electron beam lithography and molecular functionalization to finely control the shape of the nanoparticle assemblies. Atomic force microscopy analysis allowed to optimize the procedure of nanoparticle cluster array fabrication in terms of electron beam dose and chemical protocol, enabling to achieve a good regularity in spacing and size (i.e. area and layer number) of the clusters. MicroRaman space resolved measurements were undertaken on large 2D arrays (100 × 100 μm2) made up of regularly microsized clusters. The high surface enhanced Raman signal measured on the structures highlights the full correspondence of the spatial and optical periodicity achieved. This standardized procedure represent the basis to realize versatile platforms for nano-optical investigation and towards an efficient high-sensitive multiplex sensing.

Fabio Domenici, Anna Rita Bizzarri, Salvatore Cannistraro Biophysics and Nanoscience Centre, Faculty of Science, Università della Tuscia, Viterbo, Italy Background: One of the main challenges in biomedicine is improvement of detection sensitivity to achieve tumor marker recognition at a very low concentration when the disease is not significantly advanced. A pivotal role in cancer defense is played by the p53 tumor suppressor, therefore its detection with high sensitivity may contribute considerably to early diagnosis of cancer. In this work, we present a new analytical method based on surface-enhanced Raman spectroscopy which could significantly increase the sensitivity of traditional bioaffinity techniques. p53 molecules were anchored to gold nanoparticles by means of the bifunctional linker 4-aminothiophenol (4-ATP). The characteristic vibrational bands of the p53-4-ATP nanoparticle system were then used to identify the p53 molecules when they were captured by a recognition substrate comprising a monolayer of azurin in molecules possessing significant affinity for this tumor suppressor. The Raman signal enhancement achieved by 4-ATP-mediated crosslinking of p53 to 50 nm gold nanoparticles enabled detect of this protein at a concentration down to 5 × 10-13 M. Keywords: surface-enhanced Raman spectroscopy, p53, ultrasensitive detection, atomic force microscopy

In this work we employed micrometric subwavelength hole arrays on thin metal films as surface plasmon-based biochemical sensors. This allowed for determining, from a single mid-infrared measurement, both the thickness and the absorption spectrum of liposome adsorbed phospholipid monolayers and trilayers. Improved sensitivity is observed due to anti-crossing behavior when mid-infrared molecular vibrations frequency matches surface plasmon mode resonance. The development of optical sensors of few molecular layers is attracting considerable interest because they ensure non-contact interaction of the probing system with the target and portability for on-site biodiagnostics. Surface Plasmon Polaritons (SPP) propagating at the interface between a low-loss metal film and a dielectric medium have been exploited for increasing the sensitivity to few-molecule-thick layers, as the probing electric field is strongly confined by SPPs in the near-field region of the interface (Adato et al., P Natl Acad Sci U S A 106(46):19227–19232, 2009; Wu et al., Nat Mater 11(1):69–75, 2012; Limaj et al., Appl Phys Lett 98(9):091902, 2011).

This work is focused on the in vitro study of the effects induced by medical ultrasound (US) in murine fibroblast cells (NIH-3T3) at a low-intensity of exposure (spatial peak temporal average intensity Ita

A study of the Surface Enhanced Raman Scattering (SERS) from micrometric metallic nanoparticle aggregates is presented. The sample is obtained from the self-assembly on glass slides of micro-clusters of silver nanoparticles (60 and 100 nm diameter), functionalized with the organic molecule 4-aminothiophenol in water solution. For nanoparticle clusters at the micron scale, a maximum enhancement factor of 10(9) is estimated from the SERS over the Raman intensity ratio normalized to the single molecule contribution. Atomic force microscopy, correlated to spatially resolved Raman measurements, allows highlighting the connection between morphology and efficiency of the plasmonic system. The correlation between geometric features and SERS response of the metallic structures reveals a linear trend of the cluster maximum scattered intensity as a function of the surface area of the aggregate. On given clusters, the intensity turns out to be also influenced by the number of stacking planes of the aggregate, thus suggesting a plasmonic waveguide effect. The linear dependence results weakened for the largest area clusters, suggesting 30 mu m(2) as the upper limit for exploiting the coherence over large scale of the plasmonic response. (C) 2014 AIP Publishing LLC.

Extraordinary optical transmission (EOT) peaks mediated by plasmonic excitations can be observed in a variety of subwavelength patterned metallic surfaces. In this paper, we have fabricated and spectroscopically character- ized plasmon devices exhibiting EOT peaks at terahertz (THz) frequencies. These devices, which resonate with intermediate and collective modes of macromolecules, can be used for detection of materials of biological interest and their performances have been experimentally determined by measuring the variation of the EOT frequencies for thin sub-micrometric organic layers deposited onto the device surface.

We report here on the fabrication and the characterization of a new stationary phase for ultrathin layer chromatography (UTLC) based on a brush-gel polymer film obtained by cross-linking poly(glycidyl methacrylate) and di(ethylene glycole)dimethacrylate. The use of polymer brushes to design ultra thin layer chromatography plates opens interesting perspectives. Very thin polymer films can be covalently grafted on a glass surface with virtually any chemical functionality. By selecting and/or chemically modifying suitable monomers, stationary phases characterized by high affinity for specific substances can be accurately tailored. The structure of the brush gel formed on glass substrates was characterized by Fourier Transform Infrared Spectroscopy (FT-IR), Atomic Force Microscopy (AFM) and Field-Emission Scanning Electron Microscopy (FESEM). The efficiency of poly(GMA-co-DEGDMA)-NH2 UTLC stationary phase compares very well with that of silica-gel TLC and other UTLC devices. However, the separation of a fluorescent dyes mixture with our UTLC device occurs faster and with less solvent compared to typical TLC methods. In addition the same plate can be re-used several times for multiple analyses without any reduction of the separation efficiency.

The tumour suppressor p53 plays a crucial role in cell stress response and its anticancer activity is mainly down-regulated by the oncoprotein Mdm2 that, upon binding to p53, blocks its transcriptional activity and promotes its ubiquitin-dependent degradation. Targeting Mdm2-p53 interaction is believed to be the most direct of all p53-activating strategies to treat tumours in which p53 has retained its wild-type function. The bacterial protein Azurin has been shown to bind p53, inhibiting cancer cell proliferation likely through a post-translational increasing of the p53 level. This apparent antagonist action with respect to the Mdm2-p53 functional interaction suggests that binding of Azurin to p53 might interfere with the Mdm2-p53 association and, thus, preventing p53 from degradation. Toward this end, a detailed kinetic characterization of the binding interaction of these three proteins has been performed by surface plasmon resonance. The occurrence of specific binary interactions of both Azurin and Mdm2 with p53, as investigated more appropriately in their full-length conformation, is ascertained and the corresponding association and dissociation rate constants are measured. Interestingly enough, the three proteins are likely engaged in a ternary interaction, whose kinetics points out that binding of Azurin to p53 causes a significant decrease of the Mdm2-p53 association rate constant and binding affinity, without hindering the accessibility of Mdm2 to the binding pocket of p53. The Azurin-induced p53 conformational change, as demonstrated by circular dichroism, suggests that the protein may affect Mdm2-p53 association through an allosteric mechanism, which could give an useful insight into designing new anticancer drugs.

A detailed knowledge of the interaction between bacterial membranes and antibiotics provides important information to prevent high levels of antibiotic resistance exhibited by pathogenic strains. We investigated by energy dispersive X-ray diffraction (EDXD) the structure ordering of dioleoyl-phosphatidylcholine (DOPC) lipid interacting with antimicrobial peptide alamethicin, varying the lipid/peptide (L/P) molar ratio under two different hydration levels. In conditions of full hydration (100%) we found that the bilayer thickness is constant between L/P = 20 and L/P = 80 indicating that in this range, the system has reached the threshold value for the channel formation, while at the relative hydration of 45% a linear decrease of the bilayer thickness as function of L/P was revealed. The kinetic study of the complex alamethicin–DOPC at different L/P values, shows that the Bragg peak energy variation versus the hydration time has a biexponential behavior characterized by two different time constants.

Self-assembling of amphipathic lipid films on solid support allows the structural investigation of important biological model systems, such as the vectorlike lipid membranes, in order to improve DNA transfection in nonviral gene therapy. We present a neutron reflectivity study of a binary lipid system composed of dioleoylphosphatidylcholine (DOPC) and dimethyldioctadecylammonium bromide (DDAB) deposited on [100] silicon support by means of spin coating technique. We underline their lyotropic behavior under saturated deuterium oxide (D2O) vapor thus pointing out that the lipid mixture is organized in ordered domains composed of plane lamellar bilayers of noninteractive DOPC and DDAB.