Label‐free and dynamic monitoring of cytotoxicity to the blood–brain barrier cells treated with nanometre copper oxide

A cytotoxicity study was conducted with a primary culture of the nervous system cells, including brain microvascular endothelial cells (BMECs) and astrocytes, which are important components of the blood–brain barrier. The real‐time cell analysis (RTCA) was used to determine the cytotoxicity of copper‐oxide nanoparticles (CuO NPs). The IC₅₀ values of CuO NPs in astrocytes and BMECs were determined by the RTCA at different exposure times and were used as base values for further research. DNA damage after exposure to CuO NPs for 3 and 24 h was assessed using comet assay at the IC₅₀ obtained from RTCA. The onset time of cytotoxicity induced by CuO NPs was 2 and 2–4 h post‐exposure in BMECs and astrocytes, respectively. Furthermore, the degree of cytotoxicity induced by exposure to CuO NPs for 24–48 h in the BMECs and astrocytes was similar. Treatment with CuO NPs at 1/2*IC₅₀ and 1/5*IC₅₀ for 3 h induced genotoxicity in both cells as assessed by a measurement of DNA damage, although no cytotoxicity was observed. However, significant DNA damage was observed at all concentrations of CuO NPs used in this study, when the treatment time was 24 h.Inspec keywords: biochemistry, blood, brain, cellular biophysics, copper compounds, DNA, molecular biophysics, nanoparticles, toxicologyOther keywords: label‐free cytotoxicity monitoring, dynamic cytotoxicity monitoring, blood‐brain barrier cells, nervous system cells, brain microvascular endothelial cells, astrocytes, real‐time cell analysis, copper‐oxide nanoparticles, comet assay, genotoxicity, DNA damage measurement, time 24 h to 48 h, time 2 h to 4 h, CuO     

H2AX phosphorylation in response to DNA double-strand break formation during bystander signalling: effect of microRNA knockdown

Upon DNA double-strand break (DSB) formation, hundreds of H2AX molecules in the chromatin flanking the break site are phosphorylated on serine residue 139, termed gamma-H2AX, so that virtually every DSB site in a nucleus can be visualised within 10 min of its formation using an antibody to gamma-H2AX. One application of this sensitive assay is to examine the induction of DNA double-strand damage in subtle non-targeted cellular effects such as the bystander effect. Here whether microRNA (miRNA) serve as a primary signalling mechanism for bystander effect propagation by comparing matched human colon carcinoma cell lines with wild-type or depleted levels of mature miRNAs was investigated. No major differences were found in the levels of induced gamma-H2AX foci in the tested cell lines, indicating that though miRNAs play a role in bystander effect manifestation, they appear not to be the primary bystander signalling molecules in the formation of bystander effect-induced DSBs.     

Pendulum testing as a means of assessing the crash performance of longitudinal barrier with minor damage

Longitudinal barriers such as w-beam guardrails are subjected to a series of full-scale crash tests to determine their impact performance before being considered acceptable for use on the nation’s highways. Once longitudinal barriers are installed along a roadway, however, they often sustain minor damage in various ways. Since barriers are exclusively tested in an undamaged condition, there is very little known regarding the crash performance of barriers that have sustained minor damage. Transportation agencies responsible for deploying and maintaining these barrier systems need a better understanding of damaged barrier performance to make timely and cost-effective barrier maintenance decisions under the constraints of limited resources. This study is believed to be the first evaluation of the crash performance of strong post w-beam barrier that has sustained minor damage. A pendulum impact testing methodology was developed for the evaluation of two-post sections of strong post w-beam barrier. Pendulum tests were then conducted on barrier sections with five types of damage: (1) vertical tear, (2) horizontal tear, (3) splice damage, (4) twisted blockout, and (5) missing blockout. Based on these tests, vertical tears were found to be a significant threat to the structural adequacy of the barrier section with a high likelihood for rail rupture. A missing blockout at the splice location was found to result in marginal performance with one test resulting in a large rail tear at the splice. Mid-span horizontal tears and splice damage, with one of eight bolts lacking bearing capacity, were found to have a less significant threat on the structural adequacy of the barrier. Twisted blockout damage was found to have no effect on the structural crash performance of the strong post w-beam barrier.     

Photo‐induced Leishmania DNA degradation by silver‐doped zinc oxide nanoparticle: an in‐vitro approach

Recently, the authors reported newly synthesised polyethylene glycol (PEG)ylated silver (9%)‐doped zinc oxide nanoparticle (doped semiconductor nanoparticle (DSN)) which has high potency for killing Leishmania tropica by producing reactive oxygen species on exposure to sunlight. The current report is focused on Leishmania DNA interaction and damage caused by the DSN. Here, we showed that the damage to Leishmania DNA was indirect, as the DSN was unable to interact with the DNA in intact Leishmania cell, indicating the incapability of PEGylated DSN to cross the nucleus barrier. The DNA damage was the result of high production of singlet oxygen on exposure to sunlight. The DNA damage was successfully prevented by singlet oxygen scavenger (sodium azide) confirming involvement of the highly energetic singlet oxygen in the DNA degradation process.Inspec keywords: silver, zinc compounds, nanoparticles, nanomedicine, DNA, microorganisms, cellular biophysics, biomedical engineeringOther keywords: photo‐induced Leishmania DNA degradation, PEGylated silver‐doped zinc oxide nanoparticle, Leishmania tropica, reactive oxygen species, sunlight, Leishmania DNA interaction, Leishmania cell, DNA damage, singlet oxygen scavenger, sodium azide, DNA degradation process, ZnO:Ag     

Interfacing the Cell with “Biomimetic Membrane Proteins”

Integral membrane proteins mediate a myriad of cellular processes and are the target of many therapeutic drugs. Enhancement and extension of the functional scope of membrane proteins can be realized by membrane incorporation of engineered nanoparticles designed for specific diagnostic and therapeutic applications. In contrast to hydrophobic insertion of small amphiphilic molecules, delivery and membrane incorporation of particles on the nanometric scale poses a crucial barrier for technological development. In this perspective, the transformative potential of biomimetic membrane proteins (BMPs), current state of the art, and the barriers that need to be overcome in order to advance the field are discussed.     

Elucidation of biogenic silver nanoparticles susceptibility towards Escherichia coli : an investigation on the antimicrobial mechanism

Elucidation of the role of silver nanoparticles (AgNPs) in combating bacterial infection is important for the development of new antimicrobial compounds. In this study, several key factors underlying biological effects of biogenic AgNPs were investigated on recombinant Escherichia coli (XL1‐Blue) which contains a reporter gene encoding β ‐galactosidase enzyme. Biogenic AgNPs were prepared from the tea decoction. Cytotoxicity effects were profound on the bacteria tested by the synthesised NPs. The β ‐galactosidase activity of the released intracellular proteins in the supernatant of E. coli was used as a measure of membrane damage and cellular leakage. Occurrence of a significant amount of β ‐galactosiadase activity in the supernatant of treated cells clearly demonstrated the formation of holes in the bacterial membrane. Scanning electron microscope pictures visibly indicated destruction of the membrane of the bacteria, which further confirmed membrane damage. The synthesised NPs caused damage of E. coli genomic DNA in a dose dependent manner.Inspec keywords: silver, nanoparticles, microorganisms, cellular biophysics, biomembranes, genomics, DNA, genetics, molecular biophysics, toxicology, enzymes, antibacterial activity, nanobiotechnologyOther keywords: biogenic silver nanoparticle susceptibility, bacterial infection, antimicrobial mechanism, biological effects, recombinant Escherichia coli, reporter gene encoding β‐galactosidase enzyme, tea decoction, cytotoxicity effects, intracellular proteins, membrane damage, cellular leakage, bacterial membrane, scanning electron microscopy, E. coli genomic DNA damage, Ag     

Effect of barrier layer composition and thickness on structural and optical properties of TlInGaAsN/TlGaAs(N) triple quantum wells

Influence of the barrier layer composition and thickness on the structural and optical properties of TlInGaAsN Triple quantum wells (TQWs) was studied. Three types of TlInGaAsN TQW structures with different barriers were grown by gas-source molecular-beam epitaxy. Strong photoluminescence emission was obtained at room temperature from the TlInGaAsN TQW samples having 10 nm-thick TlGaAsN barriers, compared with the TlInGaAsN TQWs of 26 nm-thick TlGaAs barriers and those of 30 nm-thick TlGaAsN barriers. Structural investigations revealed that the TQWs with 10 nm-thick TlGaAsN barriers have good structural qualities. On the other hand, the other two samples showed the composition modulations at the interface between the lower side of the 3rd QW layer and the barrier layer. It was found that the addition of nitrogen into barrier layers and the decrease of barrier layer thickness significantly improve the crystalline quality and in turn the luminescence properties of the TlInGaAsN TQWs.     

Spatial and temporal organization of multi-protein assemblies: achieving sensitive control in information-rich cell-regulatory systems

The regulation of cellular processes in living organisms requires signalling systems that have a high signal-to-noise ratio. This is usually achieved by transient, multi-protein complexes that assemble cooperatively. Even in the crowded environment of the cell, such assemblies are unlikely to form by chance, thereby providing a sensitive regulation of cellular processes. Furthermore, selectivity and sensitivity may be achieved by the requirement for concerted folding and binding of previously unfolded components. We illustrate these features by focusing on two essential signalling pathways of eukaryotic cells: first, the monitoring and repair of DNA damage by non-homologous end joining, and second, the mitotic spindle assembly checkpoint, which detects and corrects defective attachments of chromosomes to the kinetochore. We show that multi-protein assemblies moderate the full range of functional complexity and diversity in the two signalling systems. Deciphering the nature of the interactions is central to understanding the mechanisms that control the flow of information in cell signalling and regulation.     

Nanoscale ZnO induces cytotoxicity and DNA damage in human cell lines and rat primary neuronal cells

The toxic effects of ZnO nanoparticles (nano-ZnO) (1-100 microg/mL) suspended in DMEM were examined in human A549 cells, HepG2 cells, human skin fibroblast cells, human skin keratinocytes, and rat primary neuronal cells for 24 h. Nano-ZnO induced dose dependent cytotoxicity and damaged cell membranes. Cell death was not mediated by reactive oxygen species (ROS) or apoptosis. Nano-ZnO induced DNA damage in rat primary neuronal cells, human fibroblasts, and A549 cells. The cytotoxicity of nano-ZnO in DMEM supplemented with 10% FBS, instead of serum free DMEM, was also examined in the A549 cells, human skin fibroblast cells, and human skin keratinocytes. The levels of cytotoxicity induced were similar to those tested without FBS; in addition, ROS was observed. These results indicate that the cause of cytotoxicity is medium dependent and imply that cellular growth conditions may play a significant role in induction of cytotoxicity and DNA damage by nano-ZnO.     

Water wave scattering by a pair of thin vertical barriers with submerged gaps

The scattering of obliquely incident water waves by two thin vertical barriers with gaps at different depths has been studied assuming linear theory. Using Havelock’s expansion of water wave potential, the problem is reduced to two pairs of integral equations of the first kind, one pair involving a horizontal component of velocity across the gaps and the other pair involving the difference of potentials across each wall. These two pairs of integral equations can be solved approximately by employing a Galerkin single-term approximation technique to obtain numerical estimates for the reflection and transmission coefficients. These estimates for the reflection and transmission coefficients thus obtained are seen to satisfy the energy identity. The reflection coefficient is plotted against wave number in a number of figures for different values of various parameters involved in the problem. It is observed that the reflection coefficient vanishes at discrete frequencies when the vertical barriers are identical. For nonidentical vertical barriers the reflection coefficient never vanishes, though at some wave number it becomes close to zero. The results for a single barrier and fully submerged two barriers, and for a single barrier with a narrow gap, are also recovered as special cases.     

Behavior of portable fiber reinforced concrete vehicle barriers subject to blasts from contact charges

Portable concrete barriers are commonly used to form a secure perimeter to prevent entry of terrorist vehicle borne improvised explosive devices (VBIEDs). Barrier effectiveness can be compromised when satchel charges are used to breach a protective perimeter and subsequently permit closer access to the intended target by VBIEDs. The behavior of five portable concrete vehicle barriers was tested under satchel sized contact charge explosives at the Air Force Research Labs (AFRL) test range at Tyndall Air Force Base, Florida. Four barriers representing different fiber reinforced concretes (FRCs) including two types of synthetic FRC, two steel-synthetic blend FRCs with different fiber volumes, and a traditional reinforced normal weight concrete which served as the control specimen. Each of the FRCs exhibited less material loss and surface damage compared to the control. The two steel synthetic blended concretes exhibited the least amount of damage of all barriers, with no visible difference in performance between the two fiber volumes. The control barrier had widespread spalling and limited concrete in the core of the specimen remained intact. A finite element model was created in LS-DYNA to model one FRC barrier and the control barrier to see if the models could predict the observed damage. Both models were deemed successful due to their ability to show similar patterns of damage as the tested barriers.     

Room-temperature tunnel magnetoresistance in self-assembled chemically synthesized metallic iron nanoparticles

We report on room temperature magnetoresistance in networks of chemically synthesized metallic Fe nanoparticles surrounded by two types of organic barriers. Electrical properties, featuring Coulomb blockade, and magnetotransport measurements show that this magnetoresistance arises from spin-dependent tunnelling, so the organic ligands stabilizing the nanoparticles are efficient spin-conservative tunnel barrier. These results demonstrate the feasibility of an all-chemistry approach for room temperature spintronics.     

Polyethylenimine functionalized magnetic nanoparticles as a potential non-viral vector for gene delivery

Polyethylenimine (PEI) functionalized magnetic nanoparticles were synthesized as a potential non-viral vector for gene delivery. The nanoparticles could provide the magnetic-targeting, and the cationic polymer PEI could condense DNA and avoid in vitro barriers. The magnetic nanoparticles were characterized by Fourier transform infrared spectroscopy, X-ray powder diffraction, dynamic light scattering measurements, transmission electron microscopy, vibrating sample magnetometer and atomic force microscopy. Agarose gel electrophoresis was used to asses DNA binding and perform a DNase I protection assay. The Alamar blue assay was used to evaluate negative effects on the metabolic activity of cells incubated with PEI modified magnetic nanoparticles and their complexes with DNA both in the presence or absence of an external magnetic field. Flow cytometry and fluorescent microscopy were also performed to investigate the transfection efficiency of the DNA-loaded magnetic nanoparticles in A549 and B16-F10 tumor cells with (+M) or without (?M) the magnetic field. The in vitro transfection efficiency of magnetic nanoparticles was improved obviously in a permanent magnetic field. Therefore, the magnetic nanoparticles show considerable potential as nanocarriers for gene delivery.     

Sequential Targeting in Crosslinking Nanotheranostics for Tackling the Multibarriers of Brain Tumors

The efficacy of therapeutics for brain tumors is seriously hampered by multiple barriers to drug delivery, including severe destabilizing effects in the blood circulation, the blood–brain barrier/blood–brain tumor barrier (BBB/BBTB), and limited tumor uptake. Here, a sequential targeting in crosslinking (STICK) nanodelivery strategy is presented to circumvent these important physiological barriers to improve drug delivery to brain tumors. STICK nanoparticles (STICK-NPs) can sequentially target BBB/BBTB and brain tumor cells with surface maltobionic acid (MA) and 4-carboxyphenylboronic acid (CBA), respectively, and simultaneously enhance nanoparticle stability with pH-responsive crosslinkages formed by MA and CBA in situ. STICK-NPs exhibit prolonged circulation time (17-fold higher area under curve) than the free agent, allowing increased opportunities to transpass the BBB/BBTB via glucose-transporter-mediated transcytosis by MA. The tumor acidic environment then triggers the transformation of the STICK-NPs into smaller nanoparticles and reveals a secondary CBA targeting moiety for deep tumor penetration and enhanced uptake in tumor cells. STICK-NPs significantly inhibit tumor growth and prolong the survival time with limited toxicity in mice with aggressive and chemoresistant diffuse intrinsic pontine glioma. This formulation tackles multiple physiological barriers on-demand with a simple and smart STICK design. Therefore, these features allow STICK-NPs to unleash the potential of brain tumor therapeutics to improve their treatment efficacy.     

Nanometer sized polymer based Schottky junctions

The aim of this work was to realize and characterize rectifying junctions with nanometer thickness based on conducting polymers. Various monolayer films were deposited onto different flat graphite electrodes in order to obtain the Schottky interfaces. The second electrical contact was realized by approaching the monolayer film with sharp tip electrode at nanometric distances, in order to create tunneling barriers. A couple of hundred of junctions were realized following this procedure. The investigated junctions have shown rectifying behavior on the current/voltage characteristics in the 96% of the cases. The analysis of the current/voltage characteristics revealed the typical behavior of Schottky barriers. The ideality factor and the Schottky barrier of the junctions were calculated. Moreover, a linear relationship between the threshold voltage and the tunneling barrier width was revealed. In our knowledge, this is the first report of a Schottky junction realized with a monolayer of polymer film.     

Effect of ionic strength solution on the stability of chitosan–DNA nanoparticles

Chitosan–DNA nanoparticles employed in gene therapy protocols consist of a neutralised, stoichiometric core and a shell of the excess of chitosan which stabilises the particles against further coagulation. At low ionic strength, these nanoparticles possess a high stability; however, as the ionic strength increases, it weakens the electrostatic repulsion which can play a decisive part in the formation of highly aggregated particles. In this study, new results about the effect of ionic strength on the colloidal stability of chitosan–DNA nanoparticles were obtained by studying the interaction between chitosans of increasing molecular weights (5, 10, 16, 29, 57 and 150?kDa) and calf thymus DNA. The physicochemical properties of polyplexes were investigated by means of dynamic light scattering, static fluorescence spectroscopy, optic microscopy, transmission electronic microscopy and gel electrophoresis. After subsequent addition of salt to the nanoparticles solution, secondary aggregation increased the size of the polyplexes. The nanoparticles stability decreased drastically at the ionic strengths 150 and 500?mM, which caused the corresponding decrease in the thickness of the stabilising shell. The morphologies of chitosan/DNA nanoparticles at those ionic strengths were a mixture of large spherical aggregates, toroids and rods. The results indicated that to obtain stable chitosan–DNA nanoparticles, besides molecular weight and N/P ratio, it is quite important to control the ionic strength of the solution.     

A Trojan-Horse-Like Biomimetic Nano-NK to Elicit an Immunostimulatory Tumor Microenvironment for Enhanced GBM Chemo-Immunotherapy

Although the chemo- and immuno-therapies have obtained good responses for several solid tumors, including those with brain metastasis, their clinical efficacy in glioblastoma (GBM) is disappointing. The lack of safe and effective delivery systems across the blood-brain barrier (BBB) and the immunosuppressive tumor microenvironment (TME) are two main hurdles for GBM therapy. Herein, a Trojan-horse-like nanoparticle system is designed, which encapsulates biocompatible PLGA-coated temozolomide (TMZ) and IL-15 nanoparticles (NPs) with cRGD-decorated NK cell membrane (R-NKm@NP), to elicit the immunostimulatory TME for GBM chemo-immunotherapy. Taking advantage of the outer NK cell membrane cooperating with cRGD, the R-NKm@NPs effectively traversed across the BBB and targeted GBM. In addition, the R-NKm@NPs exhibited good antitumor ability and prolonged the median survival of GBM-bearing mice. Notably, after R-NKm@NPs treatment, the locally released TMZ and IL-15 synergistically stimulated the proliferation and activation of NK cells, leading to the maturation of dendritic cells and infiltration of CD8⁺ cytotoxic T cells, eliciting an immunostimulatory TME. Lastly, the R-NKm@NPs not only effectively prolonged the metabolic cycling time of the drugs in vivo, but also has no noticeable side effects. This study may offer valuable insights for developing biomimetic nanoparticles to potentiate GBM chemo- and immuno-therapies in the future.     

III-Nitride Heterostructure Layered Tunnel Barriers For a Tunable Hyperspectral Detector

We report on the fabrication and characterization of Ill-nitride layered tunnel barriers with applications for a new type of tunable hyperspectral imaging detector with intrinsically hyperspectral pixels. This would enable each pixel to be individually tunable in real-time through a range of wavelengths, with the number and width of spectral channels being dynamically adjustable. Shape-engineered electron barriers fabricated from III-nitride heterostructures allow barrier height to be varied by application of a voltage. A spectroscopy of photon wavelength is enabled via the collection of photoexcited electrons across this barrier. The device is envisioned for tunable detection of ultraviolet through infrared wavelengths.     

Cellular Association and Assembly of a Multistage Delivery System

The realization that blood‐borne delivery systems must overcome a multiplicity of biological barriers has led to the fabrication of a multistage delivery system (MDS) designed to temporally release successive stages of particles or agents to conquer sequential barriers, with the goal of enhancing delivery of therapeutic and diagnostic agents to the target site. In its simplest form, the MDS comprises stage‐one porous silicon microparticles that function as carriers of second‐stage nanoparticles. Cellular uptake of nontargeted discoidal silicon microparticles by macrophages is confirmed by electron and atomic force microscopy (AFM). Using superparamagnetic iron oxide nanoparticles (SPIONs) as a model of secondary nanoparticles, successful loading of the porous matrix of silicon microparticles is achieved, and retention of the nanoparticles is enhanced by aminosilylation of the loaded microparticles with 3‐aminopropyltriethoxysilane. The impact of silane concentration and reaction time on the nature of the silane polymer on porous silicon is investigated by AFM and X‐ray photoelectron microscopy. Tissue samples from mice intravenously administered the MDS support co‐localization of silicon microparticles and SPIONs across various tissues with enhanced SPION release in spleen, compared to liver and lungs, and enhanced retention of SPIONs following silane capping of the MDS. Phantom models of the SPION‐loaded MDS display negative contrast in magnetic resonance images. In addition to forming a cap over the silicon pores, the silane polymer provides free amines for antibody conjugation to the microparticles, with both VEGFR‐2‐ and PECAM‐specific antibodies leading to enhanced endothelial association. This study demonstrates the assembly and cellular association of a multiparticle delivery system that is biomolecularly targeted and has potential for applications in biological imaging.     

Non-targeted effects of radiation: bystander responses in cell and tissue models

The standard paradigm for radiation effects in cellular systems has involved direct damage to DNA and in particular, DNA double strand breaks as the triggering lesions leading to mutation, cell death and transformation. Recently, however, a growing body of evidence has reported non-targeted effects, which are not a direct consequence of the initial lesions produced in cellular DNA. These have included bystander responses, genomic instability, gene induction, adaptive responses and low dose hypersensitivity. A common observation of these responses is that they dominate at low doses and saturate with increasing dose. Non-targeted effects may therefore have consequences for extrapolation of risk estimates to low doses if these are important in vivo. A range of experimental techniques is being used to study non-targeted responses, including microbeam approaches. Microbeams have considerable advantages in that they allow individual cells and subcellular targets to be selected within populations with precise low doses and, if required, exact dose rates. Recent advances also allow targeting of 3-D cell systems. The mechanisms underlying non-targeted responses appear to involve production of reactive oxygen species and direct cell-to-cell signalling via gap junctional intercellular communication although significant differences exist in different cell types. The triggering lesions for these responses remain unclear however. Some non-targeted responses may be inter-related, for example in the case of bystander responses and instability and may be part of a general stress response system in irradiated populations. Some non-targeted effects may also act as protective mechanisms; if they lead to the removal of potentially damaged cells from the population.