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The evaluation of engineered nanomaterial safety has been hindered by conflicting reports demonstrating differential degrees of toxicity with the same nanoparticles. The unique properties of these materials increase the likelihood that they will interfere with analytical techniques, which may contribute to this phenomenon. We tested the potential for: 1) nanoparticle intrinsic fluorescence/absorbance, 2) interactions between nanoparticles and assay components, and 3) the effects of adding both nanoparticles and analytes to an assay, to interfere with the accurate assessment of toxicity. Silicon, cadmium selenide, titanium dioxide, and helical rosette nanotubes each affected at least one of the six assays tested, resulting in either substantial over- or under-estimations of toxicity. Simulation of realistic assay conditions revealed that interference could not be predicted solely by interactions between nanoparticles and assay components. Moreover, the nature and degree of interference cannot be predicted solely based on our current understanding of nanomaterial behaviour. A literature survey indicated that ca. 95% of papers from 2010 using biochemical techniques to assess nanotoxicity did not account for potential interference of nanoparticles, and this number had not substantially improved in 2012. We provide guidance on avoiding and/or controlling for such interference to improve the accuracy of nanotoxicity assessments. [ABSTRACT FROM AUTHOR]

In this paper, we describe the effects of the combination of topographical, mechanical, chemical and intracellular electrical stimuli on a co-culture of fibroblasts and skeletal muscle cells. The co-culture was anisotropically grown onto an engineered micro-grooved (10 µm-wide grooves) polyacrylamide substrate, showing a precisely tuned Young’s modulus (∼ 14 kPa) and a small thickness (∼ 12 µm). We enhanced the co-culture properties through intracellular stimulation produced by piezoelectric nanostructures (i.e., boron nitride nanotubes) activated by ultrasounds, thus exploiting the ability of boron nitride nanotubes to convert outer mechanical waves (such as ultrasounds) in intracellular electrical stimuli, by exploiting the direct piezoelectric effect. We demonstrated that nanotubes were internalized by muscle cells and localized in both early and late endosomes, while they were not internalized by the underneath fibroblast layer. Muscle cell differentiation benefited from the synergic combination of topographical, mechanical, chemical and nanoparticle-based stimuli, showing good myotube development and alignment towards a preferential direction, as well as high expression of genes encoding key proteins for muscle contraction (i.e., actin and myosin). We also clarified the possible role of fibroblasts in this process, highlighting their response to the above mentioned physical stimuli in terms of gene expression and cytokine production. Finally, calcium imaging-based experiments demonstrated a higher functionality of the stimulated co-cultures. [ABSTRACT FROM AUTHOR]

The issue of single-cell control has recently attracted enormous interest. However, in spite of the presently achievable intracellular-level physiological probing through bio-photonics, nano-probe-based, and some other techniques, the issue of inducing selective, single-cell-precision apoptosis, without affecting neighbouring cells remains essentially open. Here we resolve this issue and report on the effective single-cell-precision cancer cell treatment using the reactive chemistry of the localized corona-type plasma discharge around a needle-like electrode with the spot size ∼1 µm. When the electrode is positioned with the micrometer precision against a selected cell, a focused and highly-localized micro-plasma discharge induces apoptosis in the selected individual HepG2 and HeLa cancer cells only, without affecting any surrounding cells, even in small cell clusters. This is confirmed by the real-time monitoring of the morphological and structural changes at the cellular and cell nucleus levels after the plasma exposure. [ABSTRACT FROM AUTHOR]

A reusable and mediator-free cholesterol biosensor based on cholesterol oxidase (ChOx) was fabricated based on self-assembled monolayer (SAM) of thioglycolic acid (TGA) (covalent enzyme immobilization by dropping method) using bio-chips. Cholesterol was detected with modified bio-chip (Gold/Thioglycolic-acid/Cholesterol-oxidase i.e., Au/TGA/ChOx) by reliable cyclic voltammetric (CV) technique at room conditions. The Au/TGA/ChOx modified bio-chip sensor demonstrates good linearity (1.0 nM to 1.0 mM; R = 0.9935), low-detection limit (∼0.42 nM, SNR∼3), and higher sensitivity (∼74.3 µAµM−1cm−2), lowest-small sample volume (50.0 μL), good stability, and reproducibility. To the best of our knowledge, this is the first statement with a very high sensitivity, low-detection limit, and low-sample volumes are required for cholesterol biosensor using Au/TGA/ChOx-chips assembly. The result of this facile approach was investigated for the biomedical applications for real samples at room conditions with significant assembly (Au/TGA/ChOx) towards the development of selected cholesterol biosensors, which can offer analytical access to a large group of enzymes for wide range of biomedical applications in health-care fields. [ABSTRACT FROM AUTHOR]

The impact of silver nanoparticles (NPs) and microparticles (MPs) on bacterial and fungal assemblages was studied in soils collected from a low arctic site. Two different concentrations (0.066% and 6.6%) of Ag NPs and Ag MPs were tested in microcosms that were exposed to temperatures mimicking a winter to summer transition. Toxicity was monitored by differential respiration, phospholipid fatty acid analysis, polymerase chain reaction-denaturing gradient gel electrophoresis and DNA sequencing. Notwithstanding the effect of Ag MPs, nanosilver had an obvious, additional impact on the microbial community, underscoring the importance of particle size in toxicity. This impact was evidenced by levels of differential respiration in 0.066% Ag NP-treated soil that were only half that of control soils, a decrease in signature bacterial fatty acids, and changes in both richness and evenness in bacterial and fungal DNA sequence assemblages. Prominent after Ag NP-treatment were Hypocreales fungi, which increased to 70%, from only 1% of fungal sequences under control conditions. Genera within this Order known for their antioxidant properties (Cordyceps/Isaria) dominated the fungal assemblage after NP addition. In contrast, sequences attributed to the nitrogen-fixing Rhizobiales bacteria appeared vulnerable to Ag NP-mediated toxicity. This combination of physiological, biochemical and molecular studies clearly demonstrate that Ag NPs can severely disrupt the natural seasonal progression of tundra assemblages. [ABSTRACT FROM AUTHOR]

Novel hybrid magnetic cross-linked enzyme aggregates of phenylalanine ammonia lyase (HM-PAL-CLEAs) were developed by co-aggregation of enzyme aggregates with magnetite nanoparticles and subsequent crosslinking with glutaraldehyde. The HM-PAL-CLEAs can be easily separated from the reaction mixture by using an external magnetic field. Analysis by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) indicated that PAL-CLEAs were inlayed in nanoparticle aggregates. The HM-PAL-CLEAs revealed a broader limit in optimal pH compared to free enzyme and PAL-CLEAs. Although there is no big difference in Km of enzyme in CLEAs and HM-PAL-CLEAs, Vmax of HM-PAL-CLEAs is about 1.75 times higher than that of CLEAs. Compared with free enzyme and PAL-CLEAs, the HM-PAL-CLEAs also exhibited the highest thermal stability, denaturant stability and storage stability. The HM-PAL-CLEAs retained 30% initial activity even after 11 cycles of reuse, whereas PAL-CLEAs retained 35% of its initial activity only after 7 cycles. These results indicated that hybrid magnetic CLEAs technology might be used as a feasible and efficient solution for improving properties of immobilized enzyme in industrial application. [ABSTRACT FROM AUTHOR]

The primary objective of this study was to fabricate a TiO2 nanotubular surface, which could maintain hydrophilicity over time (resist aging). In order to achieve non-aging hydrophilic surfaces, anodization and annealing conditions were optimized. This is the first study to show that anodization and annealing condition affect the stability of surface hydrophilicity. Our results indicate that maintenance of hydrophilicity of the obtained TiO2 nanotubes was affected by anodization voltage and annealing temperature. Annealing sharply decreased the water contact angle (WCA) of the as-synthesized TiO2 nanotubular surface, which was correlated to improved hydrophilicity. TiO2 nanotubular surfaces are transformed to hydrophilic surfaces after annealing, regardless of annealing and anodization conditions; however, WCA measurements during aging demonstrate that surface hydrophilicity of non-anodized and 20 V anodized samples decreased after only 11 days of aging, while the 60 V anodized samples maintained their hydrophilicity over the same time period. The nanotubes obtained by 60 V anodization followed by 600 °C annealing maintained their hydrophilicity significantly longer than nanotubes which were obtained by 60 V anodization followed by 300 °C annealing. [ABSTRACT FROM AUTHOR]

Current medically relevant metals for prosthetic reconstructions enjoy a relatively good success rate, but their performance drops significantly in patients with compromised health status, and post-surgical infections still remain an important challenge. To address these problems, different nanotechnology-based strategies have been exploited to create implantable metals with an enhanced bioactivity and antibacterial capacities. Among these, oxidative nanopatterning has emerged as a very effective approach to engender nanoporous surfaces that stimulate and guide the activity of adhering cells. The resulting nanoporosity is also attractive because it offers nanoconfined volumes that can be exploited to load bioactive compounds and modulate their release over time. Such extended elution is needed since a single exposure to growth factors and/or antibiotics, for instance, may not be adequate to further sustain bone regeneration and/or to counteract bacterial colonization. In this article, we assessed the capacities of nanoporous titanium surfaces generated by oxidative nanopatterning to provide controlled and sustained elution of proteins and antibiotic molecules. To this end, we have selected bovine serum albumin (BSA) and vancomycin to reflect commonly used compounds, and investigated their adsorption and elution by Fourier-transform infrared (FT-IR) and ultraviolet–visible (UV-VIS) spectroscopy. Our results demonstrate that while the elution of albumin is not significantly affected by the nanoporosity, in the case of vancomycin, nanoporous surfaces provided an extended release. These findings were successively correlated to the establishment of interactions with the surface and physical-entrapment effects exerted by the nanopores, ultimately highlighting their synergistic contribution to the release profiles and thus their importance in the design of nanostructured eluting platforms for applications in medicine. [ABSTRACT FROM AUTHOR]

We describe the existence and structure of large fullerenes in terms of symmetry breaking of the molecule. Specifically, we describe the existence of in terms of breaking of the icosahedral symmetry of by the insertion into its middle of an additional decagon. The surface of is formed by 12 regular pentagons and 25 regular hexagons. All 105 edges of are of the same length. It should be noted that the structure of the molecules is described in exact coordinates relative to the non-orthogonal icosahedral bases. This symmetry breaking process can be readily applied, and could account for and describe other larger cage cluster fullerene molecules, as well as more complex higher structures such as nanotubes. [ABSTRACT FROM AUTHOR]

Cancer can be easily treated when found early. A probe capable of detecting cell transformation may increase the success rate of early diagnosis of cancer. In this report we have tested the ability of a fluorescent, quadruplex DNA binding probe, 3,6-bis(1-methyl-4- vinylpyridinium) carbazole diiodide (BMVC), to detect cell transformation in vitro. BMVC was applied to living cells in several different models of cell transformation, and the fluorescence signals of BMVC were measured. The degrees of cell transformation in these models were characterized by alterations in cellular morphological phenotype and subcellular organization. When BMVC probes were applied, the number of BMVC-positive cells increased in accordance with the degree of transformation. BMVC was capable of significantly detecting formation of foci, increased cellular motility, cell proliferation, cell apoptosis, anchorage-independent growth, and increased invasiveness of transformed cells. These results demonstrate the ability of BMVC probes to detect cell transformation and indicate that BMVC is of promise for use as a probe in early cancer detection. [ABSTRACT FROM AUTHOR]

Investigating the interactions between nanoscale materials and microorganisms is crucial to provide a comprehensive, proactive understanding of nanomaterial toxicity and explore the potential for novel applications. It is well known that nanomaterial behavior is governed by the size and composition of the particles, though the effects of small differences in size toward biological cells have not been well investigated. Palladium nanoparticles (Pd NPs) have gained significant interest as catalysts for important carbon-carbon and carbon-heteroatom reactions and are increasingly used in the chemical industry, however, few other applications of Pd NPs have been investigated. In the present study, we examined the antimicrobial capacity of Pd NPs, which provides both an indication of their usefulness as target antimicrobial compounds, as well as their potency as potential environmental pollutants. We synthesized Pd NPs of three different well-constrained sizes, 2.0±0.1 nm, 2.5±0.2 nm and 3.1±0.2 nm. We examined the inhibitory effects of the Pd NPs and Pd2+ ions toward gram negative Escherichia coli (E. coli) and gram positive Staphylococcus aureus (S. aureus) bacterial cultures throughout a 24 hour period. Inhibitory growth effects of six concentrations of Pd NPs and Pd2+ ions (2.5×10−4, 10−5, 10−6, 10−7, 10−8, and 10−9 M) were examined. Our results indicate that Pd NPs are generally much more inhibitory toward S. aureus than toward E. coli, though all sizes are toxic at ≥10−5 M to both organisms. We observed a significant difference in size-dependence of antimicrobial activity, which differed based on the microorganism tested. Our work shows that Pd NPs are highly antimicrobial, and that fine-scale (<1 nm) differences in size can alter antimicrobial activity. [ABSTRACT FROM AUTHOR]

We have studied in vitro toxicity of iron oxide nanoparticles (NPs) coated with a thin silica shell (Fe3O4/SiO2 NPs) on A549 and HeLa cells. We compared bare and surface passivated Fe3O4/SiO2 NPs to evaluate the effects of the coating on the particle stability and toxicity. NPs cytotoxicity was investigated by cell viability, membrane integrity, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) assays, and their genotoxicity by comet assay. Our results show that NPs surface passivation reduces the oxidative stress and alteration of iron homeostasis and, consequently, the overall toxicity, despite bare and passivated NPs show similar cell internalization efficiency. We found that the higher toxicity of bare NPs is due to their stronger in-situ degradation, with larger intracellular release of iron ions, as compared to surface passivated NPs. Our results indicate that surface engineering of Fe3O4/SiO2 NPs plays a key role in improving particles stability in biological environments reducing both cytotoxic and genotoxic effects. [ABSTRACT FROM AUTHOR]

With increasing production and applications of nanostructured zinc oxide, e.g., for biomedical and consumer products, the question of safety is getting more and more important. Different morphologies of zinc oxide structures have been synthesized and accordingly investigated. In this study, we have particularly focused on nano-micro ZnO tetrapods (ZnO-T), because their large scale fabrication has been made possible by a newly introduced flame transport synthesis approach which will probably lead to several new applications. Moreover, ZnO-T provide a completely different morphology then classical spherical ZnO nanoparticles. To get a better understanding of parameters that affect the interactions between ZnO-T and mammalian cells, and thus their biocompatibility, we have examined the impact of cell culture conditions as well as of material properties on cytotoxicity. Our results demonstrate that the cell density of fibroblasts in culture along with their age, i.e., the number of preceding cell divisions, strongly affect the cytotoxic potency of ZnO-T. Concerning the material properties, the toxic potency of ZnO-T is found to be significantly lower than that of spherical ZnO nanoparticles. Furthermore, the morphology of the ZnO-T influenced cellular toxicity in contrast to surface charges modified by UV illumination or O2 treatment and to the material age. Finally, we have observed that direct contact between tetrapods and cells increases their toxicity compared to transwell culture models which allow only an indirect effect via released zinc ions. The results reveal several parameters that can be of importance for the assessment of ZnO-T toxicity in cell cultures and for particle development. [ABSTRACT FROM AUTHOR]

The development of safe and efficient gene carriers is the key to the clinical success of gene therapy. The present study was designed to develop and evaluate the chitosan-graft-polyethylenimine (CP)/DNA nanoparticles as novel non-viral gene vectors for gene therapy of osteoarthritis. The CP/DNA nanoparticles were produced through a complex coacervation of the cationic polymers with pEGFP after grafting chitosan (CS) with a low molecular weight (Mw) PEI (Mw = 1.8 kDa). Particle size and zeta potential were related to the weight ratio of CP:DNA, where decreases in nanoparticle size and increases in surface charge were observed as CP content increased. The buffering capacity of CP was significantly greater than that of CS. The transfection efficiency of CP/DNA nanoparticles was similar with that of the Lipofectamine™ 2000, and significantly higher than that of CS/DNA and PEI (25 kDa)/DNA nanoparticles. The transfection efficiency of the CP/DNA nanoparticles was dependent on the weight ratio of CP:DNA (w/w). The average cell viability after the treatment with CP/DNA nanoparticles was over 90% in both chondrocytes and synoviocytes, which was much higher than that of PEI (25 kDa)/DNA nanoparticles. The CP copolymers efficiently carried the pDNA inside chondrocytes and synoviocytes, and the pDNA was detected entering into nucleus. These results suggest that CP/DNA nanoparticles with improved transfection efficiency and low cytotoxicity might be a safe and efficient non-viral vector for gene delivery to both chondrocytes and synoviocytes. [ABSTRACT FROM AUTHOR]

A novel supersaturated self-emulsifying drug delivery system (Super-SEDDS) loaded with scutellarin-phospholipid complex (SPC) was developed. The system aimed to address the limitations presented by conventional SEDDS as delivery carriers for drugs with poor water-solubility, low liposolubility and high dose. As an intermediate, SPC was first prepared based on the response surface design. The presence of amorphous scutellarin was demonstrated through differential scanning calorimetry (DSC) and X-ray diffraction (XRD), while enhanced liposolubility was confirmed through comparison with scutellarin powder via an octanol/water distribution test. On the basis of the solubility study and ternary phase diagram, Super-SEDDS containing SPC of up to 200% equilibrium solubility (Seq) was designed, which composed of ethyl oleate, Cremophor RH40 and Transcutol HP with a ratio of 60∶25∶15 (w/w%). The subsequent in vitro lipolysis study and ex vivo intestinal absorption test indicated that Super-SEDDS enhanced the cumulative dissolution from 70% to 100% and improved the intestinal absorption from 0.04 to 0.12 µg/cm2 compared with scutellarin powder. Furthermore, an in vivo study demonstrated that Super-SEDDS achieved the AUC0-t of scutellarin up to approximate 1.7-fold as scutellarin powder. It was also proved superior to SPC and the conventional SEDDS. Super-SEDDS showed great potential for expanding the usage of SEDDS and could act as an alternative to conventional SEDDS. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the acute toxicity of intravenously administrated amorphous silica nanoparticles (SNPs) in mice. The lethal dose, 50 (LD50), of intravenously administrated SNPs was calculated in mice using Dixon's up-and-down method (262.45±33.78 mg/kg). The acute toxicity was evaluated at 14 d after intravenous injection of SNPs at 29.5, 103.5 and 177.5 mg/kg in mice. A silicon content analysis using ICP-OES found that SNPs mainly distributed in the resident macrophages of the liver (10.24%ID/g), spleen (34.78%ID/g) and lung (1.96%ID/g). TEM imaging showed only a small amount in the hepatocytes of the liver and in the capillary endothelial cells of the lung and kidney. The levels of serum LDH, AST and ALT were all elevated in the SNP treated groups. A histological examination showed lymphocytic infiltration, granuloma formation, and hydropic degeneration in liver hepatocytes; megakaryocyte hyperplasia in the spleen; and pneumonemia and pulmonary interstitial thickening in the lung of the SNP treated groups. A CD68 immunohistochemistry stain indicated SNPs induced macrophage proliferation in the liver and spleen. The results suggest injuries induced by the SNPs in the liver, spleen and lungs. Mononuclear phagocytic cells played an important role in the injury process. [ABSTRACT FROM AUTHOR]

Nanostructured three-dimensional constructs combining layer-by-layer technology (LbL) and template leaching were processed and evaluated as possible support structures for cartilage tissue engineering. Multilayered constructs were formed by depositing the polyelectrolytes chitosan (CHT) and chondroitin sulphate (CS) on either bidimensional glass surfaces or 3D packet of paraffin spheres. 2D CHT/CS multi-layered constructs proved to support the attachment and proliferation of bovine chondrocytes (BCH). The technology was transposed to 3D level and CHT/CS multi-layered hierarchical scaffolds were retrieved after paraffin leaching. The obtained nanostructured 3D constructs had a high porosity and water uptake capacity of about 300%. Dynamical mechanical analysis (DMA) showed the viscoelastic nature of the scaffolds. Cellular tests were performed with the culture of BCH and multipotent bone marrow derived stromal cells (hMSCs) up to 21 days in chondrogenic differentiation media. Together with scanning electronic microscopy analysis, viability tests and DNA quantification, our results clearly showed that cells attached, proliferated and were metabolically active over the entire scaffold. Cartilaginous extracellular matrix (ECM) formation was further assessed and results showed that GAG secretion occurred indicating the maintenance of the chondrogenic phenotype and the chondrogenic differentiation of hMSCs. [ABSTRACT FROM AUTHOR]