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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.

Cationic amphiphile DDAB (dimethyl-dioctadecyl-ammonium-bromide) can spontaneously form water-dispersed and solid supported mimicking biomembrane structures as well as valuable DNA delivery vehicles whose shape, stability and transfection efficiency can be easily optimized on varying temperature, water content and chemical composition. In this framework, disclosing the thermotropic behavior of DDAB assemblies can be considered as an essential step in conceiving and developing new non-viral vector systems. Our work has been focused primarily on understanding the mesophase structure of silicon supported DDAB thin film on varying temperature at constant relative humidity by energy dispersive X-ray diffraction (EDXD). Diffraction results have then been employed in providing a more comprehensive dynamic light scattering (DLS) analysis of corresponding thermotropic water dispersed vesicles made up of DDAB alone and in combination with helper lecithin DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) liposomes. We found that above 55 °C silicon-supported DDAB films undergo a significant thinning effect, whilst DDAB-water vesicles exhibit a reduction in size polydispersity. Upon cooling to 25 °C a distinct silicon supported DDAB mesophase, exhibiting a relative humidity-dependent spacing, has been pointed out, and modeled in terms of a lyotropic metastable gel-crystalline phase.DDAB/DOPC-water vesicles show a temperature-dependent switching in size distribution, leading to promising biomedical applications.

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

Microbubbles (MBs) are used in clinical practice as vascular ultrasound contrast agents, and are gaining popularity as a platform supporting multimodal imaging and targeted therapy, facilitating drug delivery under ultrasound exposure. Here, we report on the in vivo biological impact of newly discovered MBs with promising features as a multimodal theranostic device. The shell of the air-filled MBs is made of the poly(vinyl alcohol) (PVA), a well-established, FDA-approved polymer. Nevertheless, as size, shape and dispersity can significantly impact the biological response of particulate systems, studying their fate after administration is crucial. The safety and the biodistribution of PVA MBs were analysed in vivo and ex vivo by coupling a near infrared (NIR) fluorophore on their shell: MBs accumulated mainly in liver and spleen at 24 hours post-injection with their clearance from the spleen 7 days post-dosing. A possible way of elimination was identified in macrophages ability to engulf MBs both in vitro and in vivo. One month post-dosing, transmission electron microscopy (TEM) highlighted the lack of relevant defects and the elimination of PVA MBs by Kupffer cells. This study is the first successful attempt to fill the lack of knowledge necessary to bring PVA MBs one step closer to their possible clinical use.

Coupling near‐infrared (NIR) nanoscale absorbing materials with microbubbles (MBs) can generate a multifunctional dual imaging contrast agent. A new approach is presented for a hybrid photoacoustic/ultrasound contrast enhancer where pristine graphene is stably tethered to poly(vinyl alcohol) (PVA)‐based MBs. The main advantages of this approach are i) the preservation of optical and mechanical properties of intact graphene for an efficient photoacoustic (PA) enhancement and ii) the echogenicity and biocompatibility due to the robust anchoring of graphene to the bioinert PVA shell. PVA MBs provide ideal platforms for drug loading and ligand tethering for specific tumor targeting. One of the crucial goals toward this direction is optimizing this system in terms of balance between favorable acoustic/photoacoustic properties, immune shielding, and cytotoxicity. Such a combination strongly depends on the bridging moieties between graphene and the microbubble surface and can be easily tuned by PEGylation. The optimized graphene PVA MBs as contrast agent provide an efficient enhancement in vivo both in ultrasound and photoacoustic modes. The spectrally separable absorbance profile allows to a first demonstration of performing real‐time in vivo multiplexed photoacoustic imaging of graphene PVA MBs, and assessment of their full body biodistribution using a Vevo LAZR‐X photoacoustic imaging system.

Azurin, a bacterial protein, can be internalized in cancer cells and induce apoptosis. Such anticancer effect is coupled to the formation of a complex with the tumour-suppressor p53. The mechanism by which azurin stabilizes p53 and the binding sites of their complex are still under investigation. It is also known that the predominant mechanism for p53 down-regulation implies its association to Mdm2, the main ubiquitin ligase affecting its stability. However, the p53/Mdm2 interaction, occurring at the level of both their N-terminal domains, has been characterized so far by experiments involving only partial domains of these proteins. The relevance of the p53/Mdm2 complex as a possible target of the anticancer therapies requires a deeper study of this complex as made up of the two entire proteins. Moreover, the apparent antagonist action of azurin against Mdm2, with respect of p53 regulation, might suggest the possibility that azurin binds p53 at the same site of Mdm2, preventing in such a way p53 and Mdm2 from association and thus p53 from degradation. By following the interaction of the two entire proteins by atomic force spectroscopy, we have assessed the formation of a specific complex between p53 and Mdm2. We found for it a binding strength and a dissociation rate constant typical of dynamical protein-protein interactions and we observed that azurin, even if capable to bind p53, does not compete with Mdm2 for the same binding site on p53. The formation of the p53/Mdm2/azurin ternary complex might suggest an alternative anti-cancer mechanism adopted by azurin.

Drugs can be delivered by a temperature change-driven shrinking of the nanocarrier followed by the cargo release. This paper describes a different structural response to temperature, performed by nanoparticles of poly(N-isopropylacrylamide) and hyaluronic acid. Around 35 °C, the hydrophobicity of the vinyl polymer drives a core-shell rearrangement with the acrylamide chains confined in the core and the polysaccharide moiety forming the shell. In this arrangement, the nanoparticles enable the active targeting of tumor cells, due to the specific interaction of hyaluronic acid with the CD44 receptors. When doxorubicin-loaded nanoparticles are up-taken, the polysaccharide part degrades in the cytoplasm and the cytotoxic effect of the anticancer drug in colon adenocarcinoma cells has a 2-fold increase with respect to healthy fibroblasts. These core-shell particles have hyaluronic acid as the key factor for the specific targeting of tumor cells and drug release with poly(N-isopropylacrylamide) driving the transition.

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).

The coherent oscillations of the surface electron gas, known as surface plasmons, in metal nanostructures can give rise to the localization of intense electromagnetic fields at the metal-dielectric interface. These strong fields are exploited in surface enhanced spectroscopies, such as Surface Enhanced Raman Scattering (SERS), for the detection and characterization of molecules at very low concentration. Still, the implementation of SERS-based biosensors requires a high level of reproducibility, combined with cheap and simple fabrication methods. For this purpose, SERS substrates based on self-assembled aggregates of commercial metallic nanoparticles (Nps) can meet all the above requests. Following this line, we report on a combined micro-Raman and Atomic Force Microscopy (AFM) analysis of the SERS efficiency of micrometric silver Np aggregates (enhancement factors up to 109) obtained by self-assembly. Despite the intrinsic disordered nature of these Np clusters, we were able to sort out some general rules relating the specific aggregate morphology to its plasmonic response. We found strong evidences of cooperative effects among the NPs within the cluster and namely a clear dependence of the SERS-efficiency on both the cluster area (basically linear) and the number of stacked NPs layers. A cooperative action among the superimposed layers has been proved also by electromagnetic simulations performed on simplified nanostructures consisting of stacking planes of ordered Nps. Being clear the potentialities of these disordered self-assembled clusters, in terms of both easy fabrication and signal enhancement, we developed a specific nanofabrication protocol, based on electron beam lithography and molecular functionalization, that allowed for a fine control of the Np assemblies into designed shapes fixing their area and height. In particular, we fabricated 2D ordered arrays of disordered clusters choosing gold Nps owing to their high stability. AFM measurements confirmed the regularity in spacing and size (i.e. area and layer number) of the aggregates. Preliminary SERS measurements confirm the high signal enhancement and demonstrate a quite good reproducibility over large number of aggregates within 100×100 µm2 2D super-structure. The availability of such a multisensor could allow a careful statistical analysis of the SERS response, thus leading to a reliable quantitative estimate of the presence of relevant molecular species even at ultra-low concentration.

Background Experimental studies of antimicrobial peptides interacting with lipid membranes recently attracted growing interest due to their numerous biomedical applications. However, the influence of such peptides on the structural organisation of lipid membranes in connection with the actual cell response still remains an elusive issue. Methods X-ray diffraction was employed on detecting the sensitivity of the periodical spacing of dipalmitoyl-phosphatidyl-choline stacked as solid-supported bilayers to the presence of varying amounts of the peptide alamethicin in a wide range of peptide-to-lipid molar ratios. These results were then correlated with the effects of alamethicin on biological membranes in vitro as observed by optical microscopy and microculture tetrazolium assay on the tumour cells HeLa to provide a comprehensive and quantitative analysis of these effects, based on a dose–response relationship. Results The experiments allowed correlating the periodical spacing and the peptide-to-lipid molar ratio on alamethicin-dipalmitoyl-phosphatidyl-choline samples. Two different trends of periodical spacing vs . peptide-to-lipid molar ratio clearly appeared at low and high hydration levels, showing intriguing non-linear profiles. Unexpected correspondences were observed between the peptide-to-lipid molar ratio range where the changes in dipalmitoyl-phosphatidyl-choline structure occur and the alamethicin doses which alter the viability and the plasma membrane morphology of HeLa . Conclusions Alamethicin might induce either mechanical or phase changes on dipalmitoyl-phosphatidyl-choline bilayers. Such easily accessible ordering information was well-calibrated to predict the alamethicin doses necessary to trigger cell death through plasma membrane alterations. General significance This benchmark combined study may be valuable to predict bioeffects of several antimicrobial peptides of biomedical relevance.

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 development of ultrasensitive and rapid approaches to detect tumor markers at very low concentrations even in a physiological environment represents a challenge in nano-medicine. The p53 protein is at the center of the cellular network that protects organisms against the insurgence of tumors, most of which are related to alteration of p53 expression. Therefore p53 is regarded as a valuable prognostic marker whose detection at high sensitivity may considerably contribute to early diagnosis of cancers. In this work we have applied an analytical method based on surface enhanced Raman spectroscopy with high sensitivity and rapidity to improve traditional bioaffinity techniques. The Raman reporter bifunctional linker 4-aminothiophenol (4-ATP) first assembled onto 50 nm gold nanoparticles (Nps) has then been azotated to bind low concentration wild-type and two mutated forms of p53 proteins. The Raman signal enhancement of the resulting p53-(4-ATP-Np) systems has been used to identify the p53 molecules captured on a recognition substrate constituted by the azurin (Az) protein monolayer. Az has shown a strong association for both wild-type and mutated p53 proteins, allowing us to selectively detect these proteins at concentrations as low as 500 fM, in a human serum environment.

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.

The ex situ chemical solution deposition route for YBa 2 Cu 3 O 7-δ (YBCO) epitaxial nanocomposite films' growth is one of the most promising approaches for low-cost and large-scale production of coated conductors for high magnetic field applications. A key advantage of this method is that the nanoparticle (NP) dimension, concentration, and composition can be finely controlled and defined before the deposition process. However, in order to scale up the ex situ process, important and challenging problems remain to be solved, because NPs coalescence, precipitation, and reactivity can cause film-quality degradation. With the aim of overcoming these issues, the possibility of improving YBCO properties by the use of nanodiamond (ND) as artificial pinning center (APC) has been investigated. ND and, in general, carbon-based nanomaterials (CBN) are certainly among the most promising materials developed in the recent years due to their unique mechanical and electronic properties. Among the many proposed applications for CBN, their use in the field of high critical temperature superconductors has not still been fully developed, despite the undoubted advantages in terms of stability and size control of NP. In the present work, the effect of ND addition to YBCO precursor solution on structural and morphological film properties has been estimated and preliminary results have been used to understand if ND can be considered a reliable new tool for APC introduction.