Optimization of an ammonia assay based on transmembrane pH-gradient polymersomes.

Reliable ammonia quantification assays are essential for monitoring ammonemia in patients with liver diseases. In this study, we describe the development process of a microplate-based assay for accurate, precise, and robust ammonia quantification in biological fluids, following regulatory guidelines on bioanalytical method validation. The assay is based on transmembrane pH-gradient polymersomes that encapsulate a pH-sensitive ratiometric fluorophore, the fluorescence signal of which correlates with the ammonia concentration in the sample. Using a four-parameter logistic regression, the assay had a large quantification range (30-800 µM ammonia). As for selectivity, the presence of amino acids or pyruvate (up to clinically relevant concentrations) showed no assay interference. In samples with low bilirubin levels, polymersomes containing the fluorophore pyranine provided accurate ammonia quantification. In samples with high bilirubin concentrations, billirubin's optical interference was alleviated when replacing pyranine with a close to near-infrared hemicyanine fluorophore. Finally, the assay could correctly retrieve the ammonia concentration in ammonia-spiked human plasma samples, which was confirmed by comparing our measurements with the data obtained using a commercially available point-of-care device for ammonia.

Silica-coated phosphorescent nanoprobes for selective cell targeting and dynamic bioimaging of pathogen-host cell interactions.

Fluorescence in vitro bioimaging suffers from photobleaching of organic dyes, thus, functional probes with superior photostability are urgently needed. Here, we address this challenge by developing novel silica-coated nanophosphors that may serve as superior luminescent nanoprobes compatible with conventional fluorescence microscopes. We specifically explore their suitability for dynamic in vitro bioimaging of interactions between bacterial pathogens and host cells, and further demonstrate the facile surface functionalization of the amorphous silica layer with antibodies for selective cell targeting.

Safer-by-design flame-sprayed silicon dioxide nanoparticles: the role of silanol content on ROS generation, surface activity and cytotoxicity.

BACKGROUND: Amorphous silica nanoparticles (SiO2 NPs) have been regarded as relatively benign nanomaterials, however, this widely held opinion has been questioned in recent years by several reports on in vitro and in vivo toxicity. Surface chemistry, more specifically the surface silanol content, has been identified as an important toxicity modulator for SiO2 NPs. Here, quantitative relationships between the silanol content on SiO2 NPs, free radical generation and toxicity have been identified, with the purpose of synthesizing safer-by-design fumed silica nanoparticles. RESULTS: Consistent and statistically significant trends were seen between the total silanol content, cell membrane damage, and cell viability, but not with intracellular reactive oxygen species (ROS), in the macrophages RAW264.7. SiO2 NPs with lower total silanol content exhibited larger adverse cellular effects. The SAEC epithelial cell line did not show any sign of toxicity by any of the nanoparticles. Free radical generation and surface reactivity of these nanoparticles were also influenced by the temperature of combustion and total silanol content. CONCLUSION: Surface silanol content plays an important role in cellular toxicity and surface reactivity, although it might not be the sole factor influencing fumed silica NP toxicity. It was demonstrated that synthesis conditions for SiO2 NPs influence the type and quantity of free radicals, oxidative stress, nanoparticle interaction with the biological milieu they come in contact with, and determine the specific mechanisms of toxicity. We demonstrate here that it is possible to produce much less toxic fumed silicas by modulating the synthesis conditions.

Ultrabright and Stable Luminescent Labels for Correlative Cathodoluminescence Electron Microscopy Bioimaging.

The mechanistic understanding of structure-function relationships in biological systems heavily relies on imaging. While fluorescence microscopy allows the study of specific proteins following their labeling with fluorophores, electron microscopy enables holistic ultrastructural analysis based on differences in electron density. To identify specific proteins in electron microscopy, immunogold labeling has become the method of choice. However, the distinction of immunogold-based protein labels from naturally occurring electron dense granules and the identification of several different proteins in the same sample remain challenging. Correlative cathodoluminescence electron microscopy (CCLEM) bioimaging has recently been suggested to provide an attractive alternative based on labels emitting characteristic light. While luminescence excitation by an electron beam enables subdiffraction imaging, structural damage to the sample by high-energy electrons has been identified as a potential obstacle. Here, we investigate the feasibility of various commonly used luminescent labels for CCLEM bioimaging. We demonstrate that organic fluorophores and semiconductor quantum dots suffer from a considerable loss of emission intensity, even when using moderate beam voltages (2 kV) and currents (0.4 nA). Rare-earth element-doped nanocrystals, in particular Y2O3:Tb3+ and YVO4:Bi3+,Eu3+ nanoparticles with green and orange-red emission, respectively, feature remarkably high brightness and stability in the CCLEM bioimaging setting. We further illustrate how these nanocrystals can be readily differentiated from morphologically similar naturally occurring dense granules based on optical emission, making them attractive nanoparticle core materials for molecular labeling and (multi)color CCLEM.

Facile meltPEGylation of flame-made luminescent Tb3+-doped yttrium oxide particles: hemocompatibility, cellular uptake and comparison to silica.

Flame aerosol technology is a versatile method for scalable synthesis of nanoparticles. Since particles are produced and collected in a dry state, dispersibility and further functionalization could pose hurdles to their biomedical use. We report on a one-pot, scalable and robust procedure for the PEGylation of flame-made yttria and silica nanoparticles. We demonstrate improved colloidal stability, attenuated activation of blood coagulation and decreased uptake into phagocytic cells, all of which pave the way for facilitated biomedical use of flame-made oxide nanoparticles.

Mobility and settling rate of agglomerates of polydisperse nanoparticles.

Agglomerate settling impacts nanotoxicology and nanomedicine as well as the stability of engineered nanofluids. Here, the mobility of nanostructured fractal-like SiO2 agglomerates in water is investigated and their settling rate in infinitely dilute suspensions is calculated by a Brownian dynamics algorithm tracking the agglomerate translational and rotational motion. The corresponding friction matrices are obtained using the HYDRO++ algorithm [J. G. de la Torre, G. del Rio Echenique, and A. Ortega, J. Phys. Chem. B 111, 955 (2007)] from the Kirkwood-Riseman theory accounting for hydrodynamic interactions of primary particles (PPs) through the Rotne-Prager-Yamakawa tensor, properly modified for polydisperse PPs. Agglomerates are generated by an event-driven method and have constant mass fractal dimension but varying PP size distribution, mass, and relative shape anisotropy. The calculated diffusion coefficient from HYDRO++ is used to obtain the agglomerate mobility diameter dm and is compared with that from scaling laws for fractal-like agglomerates. The ratio dm/dg of the mobility diameter to the gyration diameter of the agglomerate decreases with increasing relative shape anisotropy. For constant dm and mean dp, the agglomerate settling rate, us, increases with increasing PP geometric standard deviation σp,g (polydispersity). A linear relationship between us and agglomerate mass to dm ratio, m/dm, is revealed and attributed to the fast Brownian rotation of such small and light nanoparticle agglomerates. An analytical expression for the us of agglomerates consisting of polydisperse PPs is then derived, us=1-ρfρpg3πµmdm (ρf is the density of the fluid, ρp is the density of PPs, µ is the viscosity of the fluid, and g is the acceleration of gravity), valid for agglomerates for which the characteristic rotational time is considerably shorter than their settling time. Our calculations demonstrate that the commonly made assumption of monodisperse PPs underestimates us by a fraction depending on σp,g and agglomerate mass mobility exponent. Simulations are in excellent agreement with deposition rate measurements of fumed SiO2 agglomerates in water.

The silanol content and in vitro cytolytic activity of flame-made silica.

HYPOTHESIS: The surface chemistry of synthetic amorphous silicas is essential for their applicational performance and for understanding their interactions with biological matter. Synthesis of silica by flame spray pyrolysis (FSP) allows to control the content and type of hydroxyl groups which also affects the cytolytic activity. EXPERIMENTS: By controlling the FSP process variables, silica nanoparticles with the same specific surface area but different surface chemistry and content of internal silanols are prepared by combustion of hexamethyldisiloxane sprays, as characterized by Raman and infrared spectroscopy, thermogravimetric analysis, and titration with lithium alanate. Cytolytic activity is assessed in terms of membrane damage in human blood monocytes in vitro. FINDINGS: Unlike commercial fumed silica, FSP-made silicas contain a significant amount of internal silanol groups and a high surface hydroxyl density, up to ∼8OH/nm2, similar to silicas made by wet-chemistry. Increasing the residence time of particles at high temperature during their synthesis reduces the internal and surface hydroxyl content and increases the relative amount of isolated silanols. This suggests incomplete oxidation of the silica matrix especially in short and "cold" flames and indicates that the silica particle formation pathway involves Si(OH)4. The surface chemistry differences translate into lower cytolytic activity for "cold-" than "hot-flame" silicas.

Quantitative analysis of the deposited nanoparticle dose on cell cultures by optical absorption spectroscopy.

AIM: The delivered nanoparticle dose to cells in vitro may depend on nanoparticle sedimentation rate. Here, the conditions under which optical absorption spectroscopy can be used to quantify the deposited nanoparticle dose in vitro are investigated. MATERIALS & METHODS: Nanoparticle cytotoxicity in both upright and inverted cell culture orientations is studied in the presence and absence of serum. RESULTS: Dissolvable nanoparticles, such as ZnO, exhibit no difference in upright and inverted cultures due to dissolved Zn(2+) ions that dominate cytotoxicity. Insoluble nanoparticles, however, exhibit different sedimentation rates and deposited doses that are linked to the observed cytotoxicity. CONCLUSION: The combined use of upright-inverted cell orientations and optical absorption spectroscopy can provide a simple experimental approach to interpret in vitro nano-biointeractions.

Shape-switching microrobots for medical applications: the influence of shape in drug delivery and locomotion.

The effect of dynamic shape switching of hydrogel bilayers on the performance of self-folding microrobots is investigated for navigation in body orifices and drug release on demand. Tubular microrobots are fabricated by coupling a thermoresponsive hydrogel nanocomposite with a poly(ethylene glycol)diacrylate (PEGDA) layer, to achieve spontaneous and reversible folding from a planar rectangular structure. Graphene oxide (GO) or silica-coated superparamagnetic iron oxide nanoparticles are dispersed in the thermoresponsive hydrogel matrix to provide near-infrared (NIR) light sensitivity or magnetic actuation, respectively. The NIR light-responsive microstructures are fabricated for triggered drug delivery while magnetic nanocomposite-based microrobots are used to analyze the role of shape in locomotion. Experimental analysis and computational simulations of tubular structures show that drug release and motility can be optimized through controlled shape change. These concepts are finally applied to helical microrobots to show a possible way to achieve autonomous behavior.

Plasmonic biocompatible silver-gold alloyed nanoparticles.

The addition of Au during scalable synthesis of nanosilver drastically minimizes its surface oxidation and leaching of toxic Ag(+) ions. These biocompatible and inexpensive silver-gold nanoalloyed particles exhibit superior plasmonic performance than commonly used pure Au nanoparticles, and as such these nanoalloys have great potential in theranostic applications.