Repeated exposure to aerosolized graphene oxide mediates autophagy inhibition and inflammation in a three-dimensional human airway model.

Hazard evaluation of engineered nanomaterials (ENMs) using real-world exposure scenario could provide better interpretation of toxicity end points for their use in the assessment of human safety and for their implications in many fields such as toxicology, nanomedicine, and so forth. However, most of the current studies, both in vivo and in vitro, do not reflect realistic conditions of human exposure to ENMs, due to the high doses implemented. Moreover, the use of cellular models cultured under submerged conditions limits their physiological relevance for lung exposure, where cells are primarily cultured at the air-liquid interface. Addressing such issues is even more challenging for emergent nanomaterials, such as graphene oxide (GO), for which little or no information on exposure is available. In this work, we studied the impact of repeated exposure of GO on a three-dimensional (3D) reconstruct of human bronchial tissue, using a nebulizer system focusing on short-term effects. The selected doses (reaching a maximum of ca. 20 â€‹µg/cm2 for a period of 4 weeks of exposure) were extrapolated from alveolar mass deposition values of a broader class of carbon-based nanomaterials, reflecting a full working lifetime of human exposure. Experimental results did not show strong toxic effects of GO in terms of viability and integrity of the lung tissue. However, since 2 weeks of treatment, repeated GO exposure elicited a proinflammatory response, moderate barrier impairment, and autophagosome accumulation, a process resulting from blockade of autophagy flux. Interestingly, the 3D airway model could recover such an effect by restoring autophagy flux at longer exposure (30 days). These findings indicate that prolonged exposure to GO produces a time window (during the 30 days of treatment set for this study) for which GO-mediated autophagy inhibition along with inflammation may potentially increase the susceptibility of exposed humans to pulmonary infections and/or lung diseases. This study also highlights the importance of using physiologically relevant in vitro models and doses derived from real-world exposure to obtain focused data for the assessment of human safety.

Correction: Ultra-efficient, widely tunable gold nanoparticles-based fiducial markers for X-ray imaging.

Correction for 'Ultra-efficient, widely tunable gold nanoparticles-based fiducial markers for X-ray imaging' by Gabriele Maiorano, et al., Nanoscale, 2016, DOI: 10.1039/c6nr07021c.

Ultra-efficient, widely tunable gold nanoparticle-based fiducial markers for X-ray imaging.

We show the development of a new class of highly efficient, biocompatible fiducial markers for X-ray imaging and radiosurgery, based on polymer shells encapsulating engineered gold nanoparticle (AuNP) suspensions. Our smart fabrication strategy enables wide tunability of the fiducial size, shape, and X-ray attenuation performance, up to record values >20 000 Hounsfield units (HU), i.e. comparable to or even higher than bulk gold. We show that the NP fiducials allow for superior imaging both in vitro and in vivo (yet requiring 2 orders of magnitude less material), with strong stability over time and the absence of classical "streak artifacts" of standard bulk fiducials. NP fiducials were probed in vivo, showing exceptional contrast efficiency, even after 2 weeks post-implant in mice.

Nanoembossed gold nanoshell with a fluorescence-like strong SERS signal.

We present a nanoembossed nanoshell with a new internal location for the formation of strong electromagnetic fields. The internally nanoembossed gold nanoshell (AuNS) is fabricated by electrostatically assembling smaller silica nanoparticles (∼15.7 nm) around the silica core (∼123.6 nm) followed by growing gold nanoseeds on the core in a wet process. FDTD calculations reveal the creation of a strong electromagnetic field (|E/Ein|max = 55 at 633 nm) at sharp edges formed by the contact between the nanoembosses and the silica core. The field formation is supported by measuring the SERS signal of Ru(bpy) encapsulated in the nanoembossing silica nanoparticles. SERS signals as strong as the corresponding fluorescence are obtained. The Raman enhancement factor (EF) is estimated to be up to 10(10) at 633 nm excitation, in addition to a comparable EF at 785 nm laser excitation. The SERS intensity of the nanoembossed nanoshell layer is sufficiently high compared to the outer or the core of the nanoshell. Finally, we fabricate all-in-one nanoparticles with all the three places where the reporter dyes are loaded and acquire the highest SERS intensity to potentially enable bio-medical applications of the nanoembossed AuNS as a sensitive and reliable labeling particle.

Fibrous wound dressings encapsulating essential oils as natural antimicrobial agents.

Preventing infections is one of the main focuses of wound care. The colonisation of wounds by microorganisms can in fact have negative consequences on the healing process, delaying it. Here, we propose the use of essential oils as natural antimicrobial agents for cellulose-based fibrous dressings. We demonstrate the production of composite electrospun fibres that effectively encapsulate three different types of essential oils (cinnamon, lemongrass and peppermint). The fibrous scaffolds are able to inhibit the growth of Escherichia coli, even when small amounts of essential oils were used. At the same time, they are not cytotoxic, as proved by biocompatibility assays on skin cell models. The created dressings are promising as advanced biomedical devices for topical treatments.

In vivo assessment of CdSe-ZnS quantum dots: coating dependent bioaccumulation and genotoxicity.

Semiconductor nanocrystals, or Quantum Dots (QDs), have gained considerable attention due to their unique size-dependent optical and electronic properties that make them attractive for a wide range of applications, including biology and nanomedicine. Their widespread use, however, poses urgent questions about their potential toxicity, especially because of their heavy metal composition that could cause harmful effects to human health and environment. In this work, we evaluated in vivo the long-term toxicity of CdSe-ZnS QDs with different surface coatings, probing oral administration in the model system Drosophila melanogaster. In particular, we found that all the differently coated QDs significantly affect the lifespan of treated Drosophila populations and induce a marked increase in reactive oxygen species (ROS) levels. Furthermore, we observed that these QDs induce severe genotoxic effects and increased rate of apoptosis in Drosophila haemocytes. These toxic effects were found to be mainly related to the in vivo degradation of QDs with consequent release of Cd(2+) ions, while the coating of QDs can modulate their bioaccumulation in the organism, partly decreasing their overall toxicity.

Physical assessment of toxicology at nanoscale: nano dose-metrics and toxicity factor.

In this work, we propose a systematic and reproducible evaluation of nanoparticles (NPs) toxicology in living systems, based on a physical assessment and quantification of the toxic effects of NPs by the experimental determination of the key parameter affecting the toxicity outcome (i.e., the number of NPs) and of the NPs "toxicity factor". Such a strategy was applied to a well determined scenario, i.e., the ingestion of citrate-capped gold NPs (AuNPs) of different sizes by the model system Drosophila melanogaster. Using these AuNPs as a reference toxicity standard, we were able to define different regions in the multiparametric space of toxicity, enabling the classification of the toxic levels of other nanomaterials, such as quantum dots and pegylated AuNPs. This approach may pave the way to a systematic classification of nanomaterials, leading to important developments in risk assessment and regulatory approval, as well as in a wide range of nanomedicine applications.

Neurons sense nanoscale roughness with nanometer sensitivity.

The interaction between cells and nanostructured materials is attracting increasing interest, because of the possibility to open up novel concepts for the design of smart nanobiomaterials with active biological functionalities. In this frame we investigated the response of human neuroblastoma cell line (SH-SY5Y) to gold surfaces with different levels of nanoroughness. To achieve a precise control of the nanoroughness with nanometer resolution, we exploited a wet chemistry approach based on spontaneous galvanic displacement reaction. We demonstrated that neurons sense and actively respond to the surface nanotopography, with a surprising sensitivity to variations of few nanometers. We showed that focal adhesion complexes, which allow cellular sensing, are strongly affected by nanostructured surfaces, leading to a marked decrease in cell adhesion. Moreover, cells adherent on nanorough surfaces exhibit loss of neuron polarity, Golgi apparatus fragmentation, nuclear condensation, and actin cytoskeleton that is not functionally organized. Apoptosis/necrosis assays established that nanoscale features induce cell death by necrosis, with a trend directly related to roughness values. Finally, by seeding SH-SY5Y cells onto micropatterned flat and nanorough gold surfaces, we demonstrated the possibility to realize substrates with cytophilic or cytophobic behavior, simply by fine-tuning their surface topography at nanometer scale. Specific and functional adhesion of cells occurred only onto flat gold stripes, with a clear self-alignment of neurons, delivering a simple and elegant approach for the design and development of biomaterials with precise nanostructure-triggered biological responses.

Modular plastic chip for one-shot human papillomavirus diagnostic analysis.

In this article, we report the design and development of a plastic modular chip suitable for one-shot human papillomavirus (HPV) diagnostics, namely detection of the viral presence and relative genotyping, by two sequential steps performed directly on the same device. The device is composed of two modular and disposable plastic units that can be assembled or used separately. The first module is represented by a polydimethylsiloxane (PDMS) microreactor that is exploited for real-time polymerase chain reaction (PCR) and, thus, is suitable for detecting the presence of virus. The second unit is a PDMS microwell array that allows virus genotyping by a colorimetric assay, based on DNA hybridization technology developed on plastic, requiring simple inspection by the naked eye. The two modules can be easily coupled to reusable hardware, enabling the heating/cooling processes and the real-time detection of HPV. By coupling real-time assay and colorimetric genotyping on the same chip, the assembled device may provide a low-cost tool for HPV diagnostics, thereby favoring the prediction of cancer risk in patients.

Disposable plastic microreactors for genomic analyses.

We show the design, development and assessment of disposable, biocompatible, fully plastic microreactors, which are demonstrated to be highly efficient for genomic analyses, such as amplification of DNA, quantitative analyses in real time, multiplex PCR (both in terms of efficiency and selectivity), as compared to conventional laboratory equipment for PCR. The plastic microreactors can easily be coupled to reusable hardware, enabling heating/cooling processes and, in the case of qPCR applications, the real-time detection of the signal from a suitable fluorescent reporter present in the reaction mixture during the analysis. The low cost production of these polymeric microreactors, along with their applicability to a wide range of biochemical targets, may open new perspectives towards practical applications of biochips for point of care diagnostics.

Single-molecule mechanical unfolding of amyloidogenic beta2-microglobulin: the force-spectroscopy approach.

The recombinant production of a novel chimeric polyprotein is described. The new protein contains either wild-type beta(2)-microglobulin (beta(2)m) or its truncated variant (DeltaN6 beta(2)m) (see picture). Structural characterization is achieved by means of single-molecule force spectroscopy studies of specific beta(2)m regions which could be involved in amyloidogenesis.

Patterned structures of in situ size controlled CdS nanocrystals in a polymer matrix under UV irradiation.

A method of in situ formation of patterns of size controlled CdS nanocrystals in a polymer matrix by pulsed UV irradiation is presented. The films consist of Cd thiolate precursors with different carbon chain lengths embedded in TOPAS polymer matrices. Under UV irradiation the precursors are photolyzed, driving to the formation of CdS nanocrystals in the quantum size regime, with size and concentration defined by the number of incident UV pulses, while the host polymer remains macroscopically/microscopically unaffected. The emission of the formed nanocomposite materials strongly depends on the dimensions of the CdS nanocrystals, thus, their growth at the different phases of the irradiation is monitored using spatially resolved photoluminescence by means of a confocal microscope. X-ray diffraction measurements verified the existence of the CdS nanocrystals, and defined their crystal structure for all the studied cases. The results are reinforced by transmission electron microscopy. It is proved that the selection of the precursor determines the efficiency of the procedure, and the quality of the formed nanocrystals. Moreover it is demonstrated that there is the possibility of laser induced formation of well-defined patterns of CdS nanocrystals, opening up new perspectives in the development of nanodevices.

Microscale patterning of hydrophobic/hydrophilic surfaces by spatially controlled galvanic displacement reactions.

In this letter, we report the design and fabrication of different metal patterns for the realization of spatially controlled hydrophobic/hydrophilic regions with micrometer resolution. The fabrication procedure, based on a combination of lithographic techniques and wet-chemistry reactions (namely, spontaneous Galvanic displacement reactions) is reliable, undemanding, and highly versatile, allowing the achievement of precise spatial control along with the use of a wide variety of different materials.

Exploring local flexibility/rigidity in psychrophilic and mesophilic carbonic anhydrases.

Molecular flexibility and rigidity are required to determine the function and specificity of protein molecules. Some psychrophilic enzymes demonstrate a higher catalytic efficiency at low temperatures, compared to the efficiency demonstrated by their meso/thermophilic homologous. The emerging picture suggests that such enzymes have an improved flexibility of the structural catalytic components, whereas other protein regions far from functional sites may be even more rigid than those of their mesophilic counterparts. To gain a deeper insight in the analysis of the activity-flexibility/rigidity relationship in protein structure, psychrophilic carbonic anhydrase of the Antarctic teleost Chionodraco hamatus has been compared with carbonic anhydrase II of Bos taurus through fluorescence studies, three-dimensional modeling, and activity analyses. Data demonstrated that the cold-adapted enzyme exhibits an increased catalytic efficiency at low and moderate temperatures and, more interestingly, a local flexibility in the region that controls the correct folding of the catalytic architecture, as well as a rigidity in the hydrophobic core. The opposite result was observed in the mesophilic counterpart. These results suggest a clear relationship between the activity and the presence of flexible and rigid protein substructures that may be useful in rational molecular and drug design of a class of enzymes playing a key role in pathologic processes.

Micro/Nanoscale Parallel Patterning of Functional Biomolecules, Organic Fluorophores and Colloidal Nanocrystals.

We describe the design and optimization of a reliable strategy that combines self-assembly and lithographic techniques, leading to very precise micro-/nanopositioning of biomolecules for the realization of micro- and nanoarrays of functional DNA and antibodies. Moreover, based on the covalent immobilization of stable and versatile SAMs of programmable chemical reactivity, this approach constitutes a general platform for the parallel site-specific deposition of a wide range of molecules such as organic fluorophores and water-soluble colloidal nanocrystals. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11671-009-9386-7) contains supplementary material, which is available to authorized users.

Real-time PCR in a plastic chip based on solid state FRET.

We show a novel solid state optical detection platform, integrated in a plastic biochip, based on colloidal nanocrystal FRET donors. The approach exploits a "smart" polymeric layer with both optical and biorecognition properties that allows real-time monitoring of biomolecular interactions and quantitative analyses of real-time PCR. The proposed strategy, demonstrated here for DNA detection, may open interesting perspectives for a wide range of applications, such as for proteomic studies.

Charge transport in disordered films of non-redox proteins.

Electrical conduction in solid state disordered multilayers of non-redox proteins is demonstrated by two-terminal transport experiments at the nanoscale and by scanning tunneling microscopy (STM/STS experiments). We also show that the conduction of the biomolecular films can be modulated by means of a gate field. These results may lead to the implementation of protein-based three-terminal nanodevices and open important new perspectives for a wide range of bioelectronic/biosensing applications.

Metal-enhanced fluorescence of colloidal nanocrystals with nanoscale control.

Engineering the spectral properties of fluorophores, such as the enhancement of luminescence intensity, can be achieved through coupling with surface plasmons in metallic nanostructures. This process, referred to as metal-enhanced fluorescence, offers promise for a range of applications, including LEDs, sensor technology, microarrays and single-molecule studies. It becomes even more appealing when applied to colloidal semiconductor nanocrystals, which exhibit size-dependent optical properties, have high photochemical stability, and are characterized by broad excitation spectra and narrow emission bands. Other approaches have relied upon the coupling of fluorophores (typically organic dyes) to random distributions of metallic nanoparticles or nanoscale roughness in metallic films. Here, we develop a new strategy based on the highly reproducible fabrication of ordered arrays of gold nanostructures coupled to CdSe/ZnS nanocrystals dispersed in a polymer blend. We demonstrate the possibility of obtaining precise control and a high spatial selectivity of the fluorescence enhancement process.

Formation and characterization of glutamate dehydrogenase monolayers on silicon supports.

In this paper we have tested two different procedures (the "three-step" and the "four-step" procedures) for the covalent immobilization of glutamate dehydrogenase (GDH) onto silicon supports. Atomic force microscopy (AFM), Fourier-transform infrared spectroscopy (FT-IR), fluorescence spectroscopy and an enzymatic assay were used to probe the structure and activity of the immobilized enzyme. Our results demonstrate that coupling through the "three-step" procedure does not significantly affect either the fold pattern or the activity of the enzyme, suggesting that this method could be ideally suited to the development of high quality monolayers for use in enzyme-based planar biosensors.

Retention of nativelike conformation by proteins embedded in high external electric fields.

In this Communication, we show that proteins embedded in high external electric fields are capable of retaining a nativelike fold pattern. We have tested the metalloprotein azurin, immobilized onto SiO2 substrates in air with proper electrode configuration, by applying static fields up to 10(6)-10(7) Vm. The effects on the conformational properties of protein molecules have been determined by means of intrinsic fluorescence measurements. Experimental results indicate that no significant field-induced conformational alteration occurs. Such results are also discussed and supported by theoretical predictions of the inner protein fields.