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ConspectusThe ligand shells of colloidal nanoparticles (NPs) can serve different purposes. In general, they provide colloidal stability by introducing steric repulsion between NPs. In the context of biological applications, the ligand shell plays a critical role in targeting, enabling NPs to achieve specific biodistributions. However, there is also another important feature of the ligand shell of NPs, namely, the creation of a local environment differing from the bulk of the solvent in which the NPs are dispersed. It is known that charged ligand shells can attract or repel ions and change the effective charge of a NP through Debye-Hückel screening. Positively charged ions, such as H+ (or H3O+) are attracted to negatively charged surfaces, whereas negatively charged ions, such as Cl- are repelled. The distribution of the ions around charged NP surfaces is a radial function of distance from the center of the NP, which is governed by a balance of electrostatic forces and entropy of ions and ligands. As a result, the ion concentration at the NP surface is different from its bulk equilibrium concentration, i.e., the charged ligand shell around the NPs has formed a distinct local environment. This not only applies to charged ligand shells but also follows a more general principle of induced condensation and depletion. Polar/apolar ligand shells, for example, result in a locally increased concentration of polar/apolar molecules. Similar effects can be seen for biocatalysts like enzymes immobilized in nanoporous host structures, which provide a special environment due to their surface chemistry and geometrical nanoconfinement. The formation of a local environment close to the ligand shell of NPs has profound implications for NP sensing applications. As a result, analyte concentrations close to the ligand shell, which are the ones that are measured, may be very different from the analyte concentrations in bulk. Based on previous work describing this effect, it will be discussed herein how such local environments, created by the choice of used ligands, may allow for tailoring the NPs' sensing properties. In general, the ligand shell around NPs can be attractive/repulsive for molecules with distinct properties and thus forms an environment that can modulate the specific response. Such local environments can also be optimized to modulate chemical reactions close to the NP surface (for example, by size filtering within pores) or to attract specific low abundance proteins. The importance hereby is that this is based on interaction with low selectivity between the ligands and the target molecules.
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Human ENP exposure is inevitable and the novel, size-dependent physicochemical properties that enable ENPs to be beneficial in innovative technologies are concomitantly causing heightened public concerns as to their potential adverse effects upon human health. This study aims to deduce the mechanisms associated with potential ENP mediated (geno)toxicity and impact upon telomere integrity, if any, of varying concentrations of both â¼16 nm (4.34 × 10-3 to 17.36 × 10-3 mg/mL) Gold (Au) and â¼14 nm (0.85 × 10-5 to 3.32 × 10-5 mg/mL) Silver (Ag) ENPs upon two commonly used lung epithelial cell lines, 16HBE14o- and A549. Following cytotoxicity analysis (via Trypan Blue and Lactate Dehydrogenase assay), two sub-lethal concentrations were selected for genotoxicity analysis using the cytokinesis-blocked micronucleus assay. Whilst both ENP types induced significant oxidative stress, Ag ENPs (1.66 × 10-5 mg/mL) did not display a significant genotoxic response in either epithelial cell lines, but Au ENPs (8.68 × 10-3 mg/mL) showed a highly significant 2.63-fold and 2.4-fold increase in micronucleus frequency in A549 and 16HBE14o- cells respectively. It is hypothesized that the DNA damage induced by acute 24-h Au ENP exposure resulted in a cell cycle stall indicated by the increased mononuclear cell fraction (>6.0-fold) and cytostasis level. Albeit insignificant, a small reduction in telomere length was observed following acute exposure to both ENPs which could indicate the potential for ENP mediated telomere attrition. Finally, from the data shown, both in vitro lung cell cultures (16HBE14o- and A549) are equally as suitable and reliable for the in vitro ENP hazard identification approach adopted in this study.
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Nanopartículas del Metal , Nanopartículas , Daño del ADN , Células Epiteliales , Oro/química , Humanos , Pulmón/química , Nanopartículas del Metal/química , Nanopartículas del Metal/toxicidad , Nanopartículas/toxicidad , Plata/químicaRESUMEN
We report a simple and direct fluorimetric vesicle-based method for measuring the transport rate of the light-driven ions pumps as specifically applied to the chloride pump, halorhodopsin, from Natronomonas pharaonis (pHR). Previous measurements were cell-based and methods to determine average single channel permeability challenging. We used a water-in-oil emulsion method for directional pHR reconstitution into two different types of vesicles: lipid vesicles and asymmetric lipid-block copolymer vesicles. We then used stopped-flow experiments combined with fluorescence correlation spectroscopy to determine per protein Cl- transport rates. We obtained a Cl- transport rate of 442 (±17.7) Cl-/protein/s in egg phosphatidyl choline (PC) lipid vesicles and 413 (±26) Cl-/protein/s in hybrid block copolymer/lipid (BCP/PC) vesicles with polybutadine-polyethylene oxide (PB12PEO8) on the outer leaflet and PC in the inner leaflet at a photon flux of 1450 photons/protein/s. Normalizing to a per photon basis, this corresponds to 0.30 (±0.07) Cl-/photon and 0.28 (±0.04) Cl-/photon for pure PC and BCP/PC hybrid vesicles respectively, both of which are in agreement with recently reported turnover of ~500 Cl-/protein/s from flash photolysis experiments and with voltage-clamp measurements of 0.35 (±0.16) Cl-/photon in pHR-expressing oocytes as well as with a pHR quantum efficiency of ~30%.
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Cloruros/metabolismo , Halorrodopsinas/química , Transporte Iónico/genética , Liposomas/química , Cloruros/química , Cloruros/efectos de la radiación , Halobacteriaceae/química , Halobacteriaceae/genética , Halorrodopsinas/genética , Cinética , Luz , Liposomas/metabolismo , Liposomas/efectos de la radiaciónRESUMEN
The authors apologized for the unfortunate error in figure during publication of the article and they also explained that some of the solid grey graphs in Fig. 5 are intentionally based on the same data. For 8 different surface makers (CD14, CD73, CD34, CD105, CD19, CD90, CD45, HA-DR) in accordance to the guidelines of the manufacturer a panel of 4 different isotype controls were used, corresponding to 4 different fluorescence channels.
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Despite growing interest in light-driven ion pumps for use in optogenetics, current estimates of their transport rates span two orders of magnitude due to challenges in measuring slow transport processes and determining protein concentration and/or orientation in membranes in vitro. In this study, we report, to our knowledge, the first direct quantitative measurement of light-driven Cl- transport rates of the anion pump halorohodopsin from Natronomonas pharaonis (NpHR). We used light-interfaced voltage clamp measurements on NpHR-expressing oocytes to obtain a transport rate of 219 (± 98) Cl-/protein/s for a photon flux of 630 photons/protein/s. The measurement is consistent with the literature-reported quantum efficiency of â¼30% for NpHR, i.e., 0.3 isomerizations per photon absorbed. To reconcile our measurements with an earlier-reported 20 ms rate-limiting step, or 35 turnovers/protein/s, we conducted, to our knowledge, novel consecutive single-turnover flash experiments that demonstrate that under continuous illumination, NpHR bypasses this step in the photocycle.
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Cloruros/metabolismo , Halorrodopsinas/metabolismo , Luz , Halobacteriaceae , Transporte Iónico/efectos de la radiación , CinéticaRESUMEN
BACKGROUND: Mesenchymal stromal cells (MSCs) have an inherent migratory capacity towards tumor tissue in vivo. With the future objective to quantify the tumor homing efficacy of MSCs, as first step in this direction we investigated the use of inorganic nanoparticles (NPs), in particular ca. 4 nm-sized Au NPs, for MSC labeling. Time dependent uptake efficiencies of NPs at different exposure concentrations and times were determined via inductively coupled plasma mass spectrometry (ICP-MS). RESULTS: The labeling efficiency of the MSCs was determined in terms of the amount of exocytosed NPs versus the amount of initially endocytosed NPs, demonstrating that at high concentrations the internalized Au NPs were exocytosed over time, leading to continuous exhaustion. While exposure to NPs did not significantly impair cell viability or expression of surface markers, even at high dose levels, MSCs were significantly affected in their proliferation and migration potential. These results demonstrate that proliferation or migration assays are more suitable to evaluate whether labeling of MSCs with certain amounts of NPs exerts distress on cells. However, despite optimized conditions the labeling efficiency varied considerably in MSC lots from different donors, indicating cell specific loading capacities for NPs. Finally, we determined the detection limits of Au NP-labeled MSCs within murine tissue employing ICP-MS and demonstrate the distribution and homing of NP labeled MSCs in vivo. CONCLUSION: Although large amounts of NPs improve contrast for imaging, duration and extend of labeling needs to be adjusted carefully to avoid functional deficits in MSCs. We established an optimized labeling strategy for human MSCs with Au NPs that preserves their migratory capacity in vivo.
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Rastreo Celular , Oro/química , Células Madre Mesenquimatosas/citología , Nanopartículas del Metal/química , Animales , Diferenciación Celular , Movimiento Celular , Supervivencia Celular , Células Cultivadas , Endocitosis , Exocitosis , Humanos , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos BALB C , Tamaño de la PartículaRESUMEN
The use of nanotechnologies involving nano- and microparticles has increased tremendously in the recent past. There are various beneficial characteristics that make particles attractive for a wide range of technologies. However, colloidal particles on the other hand can potentially be harmful for humans and environment. Today, complete understanding of the interaction of colloidal particles with biological systems still remains a challenge. Indeed, their uptake, effects, and final cell cycle including their life span fate and degradation in biological systems are not fully understood. This is mainly due to the complexity of multiple parameters which need to be taken in consideration to perform the nanosafety research. Therefore, we will provide an overview of the common denominators and ideas to achieve universal metrics to assess their safety. The review discusses aspects including how biological media could change the physicochemical properties of colloids, how colloids are endocytosed by cells, how to distinguish between internalized versus membrane-attached colloids, possible correlation of cellular uptake of colloids with their physicochemical properties, and how the colloidal stability of colloids may vary upon cell internalization. In conclusion three main statements are given. First, in typically exposure scenarios only part of the colloids associated with cells are internalized while a significant part remain outside cells attached to their membrane. For quantitative uptake studies false positive counts in the form of only adherent but not internalized colloids have to be avoided. pH sensitive fluorophores attached to the colloids, which can discriminate between acidic endosomal/lysosomal and neutral extracellular environment around colloids offer a possible solution. Second, the metrics selected for uptake studies is of utmost importance. Counting the internalized colloids by number or by volume may lead to significantly different results. Third, colloids may change their physicochemical properties along their life cycle, and appropriate characterization is required during the different stages.
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Membrana Celular/metabolismo , Coloides/análisis , Endocitosis/fisiología , Nanopartículas/análisis , Técnicas de Cultivo de Célula , Células Cultivadas , Coloides/química , Coloides/metabolismo , Humanos , Nanopartículas/química , Nanopartículas/metabolismo , Propiedades de SuperficieRESUMEN
Protein adsorption to nanoparticles is described as a chemical reaction in which proteins attach to binding sites on the nanoparticle surface. This process is defined by a dissociation coefficient, which tells how many proteins are adsorbed per nanoparticle in dependence of the protein concentration. Different techniques to experimentally determine dissociation coefficients of protein adsorption to nanoparticles are reviewed. Results of more than 130 experiments in which dissociation coefficients have been determined are compared. Data show that different methods, nanoparticle systems, and proteins can lead to significantly different dissociation coefficients. However, we observed a clear tendency of smaller dissociation coefficients upon less negative towards more positive zeta potentials of the nanoparticles. The zeta potential thus is a key parameter influencing protein adsorption to the surface of nanoparticles. Our analysis highlights the importance of the characterization of the parameters governing protein-nanoparticle interaction for quantitative evaluation and objective literature comparison.
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Técnicas de Química Analítica/métodos , Nanopartículas/química , Corona de Proteínas/química , Adsorción , Animales , HumanosRESUMEN
The dependence of the ionic conductance of ultra-thin polyelectrolyte multilayer (PEM) films on the temperature and the number of bilayers has been investigated by the recently developed low energy bombardment induced ion transport (BIIT) method. To this end multilayers of alternating poly(sodium 4-styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) layers were deposited on a metal electrode and subsequently bombarded by a low energy potassium ion beam. Ions are transported through the film according to the laws of electro-diffusion towards a grounded backside electrode. They are neutralized at the interface between the polymer film and the metal electrode. The detected neutralization current scales linearly with the acceleration potential of the ion beam indicating Ohmic behavior for the (PAH/PSS)x multilayer, where x denotes the number of bilayers. The conductance exhibits a non-monotonic dependence on the number of bilayers, x. For 2 ≤ x ≤ 8 the conductance increases non-linearly with the number of bilayers. For x ≥ 8 the conductance decreases with increasing number of bilayers. The variation of the conductance is rationalized by a model accounting for the structure dependence of the conductivity. The thinnest sample for which the conductance has been measured is the single bilayer reflecting properties dominated by the interface. The activation energy for the ion transport is 0.49 eV.