Long-Term Intracellular Tracking of Label-Free Nanoparticles in Live Cells and Tissues with Confocal Microscopy.

The label-free imaging of inorganic nanoparticles (NPs) using confocal laser scanning microscopy (CLSM) provides a powerful and versatile tool for studying interactions between NPs and biological systems. Without the need for exogenous labels or markers, it simply benefits from the differential scattering of visible photons between biomaterials and inorganic NPs. Validation experiments conducted on fixed and living cells in real-time, as well as mouse tissue sections following parenteral administration of NPs. Additionally, by incorporating reporter fluorophores and utilizing both reflectance and fluorescence imaging modalities, the method enables high-resolution multiplex imaging of cellular structures and NPs. Different sizes and concentrations of Au NPs are tested as for Ag, Fe3O4, and CeO2 NPs, all with biological interest. Overall, the comprehensive study of NP imaging by confocal microscopy in reflectance mode provides valuable insights and tools for researchers interested in monitoring the nano-bio interactions.

MOF-Beads Containing Inorganic Nanoparticles for the Simultaneous Removal of Multiple Heavy Metals from Water.

Pollution of water with heavy metals is a global environmental problem whose impact is especially severe in developing countries. Among water-purification methods, adsorption of heavy metals has proven to be simple, versatile, and cost-effective. However, there is still a need to develop adsorbents with a capacity to remove multiple metal pollutants from the same water sample. Herein, we report the complementary adsorption capacities of metal-organic frameworks (here, UiO-66 and UiO-66-(SH)2) and inorganic nanoparticles (iNPs; here, cerium-oxide NPs) into composite materials. These adsorbents, which are spherical microbeads generated in one step by continuous-flow spray-drying, efficiently and simultaneously remove multiple heavy metals from water, including As(III and V), Cd(II), Cr(III and VI), Cu(II), Pb(II), and Hg(II). We further show that these microbeads can be used as a packing material in a prototype of a continuous-flow water treatment system, in which they retain their metal-removal capacities upon regeneration with a gentle acidic treatment. As proof-of-concept, we evaluated these adsorbents for purification of laboratory water samples prepared to independently recapitulate each of two strongly polluted rivers: the Bone (Indonesia) and Buringanga (Bangladesh) rivers. In both cases, our microbeads reduced the levels of all the metal contaminants to below the corresponding permissible limits established by the World Health Organization (WHO). Moreover, we demonstrated the capacity of these microbeads to lower levels of Cr(VI) in a water sample collected from the Sarno River (Italy). Finally, to create adsorbents that could be magnetically recovered following their use in water purification, we extended our spray-drying technique to simultaneously incorporate two types of iNPs (CeO2 and Fe3O4) into UiO-66-(SH)2, obtaining CeO2/Fe3O4@UiO-66-(SH)2 microbeads that adsorb heavy metals and are magnetically responsive.

Characterization of Nanoparticle Batch-To-Batch Variability.

A central challenge for the safe design of nanomaterials (NMs) is the inherent variability of NM properties, both as produced and as they interact with and evolve in, their surroundings. This has led to uncertainty in the literature regarding whether the biological and toxicological effects reported for NMs are related to specific NM properties themselves, or rather to the presence of impurities or physical effects such as agglomeration of particles. Thus, there is a strong need for systematic evaluation of the synthesis and processing parameters that lead to potential variability of different NM batches and the reproducible production of commonly utilized NMs. The work described here represents over three years of effort across 14 European laboratories to assess the reproducibility of nanoparticle properties produced by the same and modified synthesis routes for four of the OECD priority NMs (silica dioxide, zinc oxide, cerium dioxide and titanium dioxide) as well as amine-modified polystyrene NMs, which are frequently employed as positive controls for nanotoxicity studies. For 46 different batches of the selected NMs, all physicochemical descriptors as prioritized by the OECD have been fully characterized. The study represents the most complete assessment of NMs batch-to-batch variability performed to date and provides numerous important insights into the potential sources of variability of NMs and how these might be reduced.

Effects of Systematic Variation in Size and Surface Coating of Silver Nanoparticles on Their In Vitro Toxicity to Macrophage RAW 264.7 Cells.

In literature, varying and sometimes conflicting effects of physicochemical properties of nanoparticles (NPs) are reported on their uptake and effects in organisms. To address this, small- and medium-sized (20 and 50 nm) silver nanoparticles (AgNPs) with specified different surface coating/charges were synthesized and used to systematically assess effects of NP-properties on their uptake and effects in vitro. Silver nanoparticles were fully characterized for charge and size distribution in both water and test media. Macrophage cells (RAW 264.7) were exposed to these AgNPs at different concentrations (0-200 µg/ml). Uptake dynamics, cell viability, induction of tumor necrosis factor (TNF)-α, ATP production, and reactive oxygen species (ROS) generation were assessed. Microscopic imaging of living exposed cells showed rapid uptake and subcellular cytoplasmic accumulation of AgNPs. Exposure to the tested AgNPs resulted in reduced overall viability. Influence of both size and surface coating (charge) was demonstrated, with the 20-nm-sized AgNPs and bovine serum albumin (BSA)-coated (negatively charged) AgNPs being slightly more toxic. On specific mechanisms of toxicity (TNF-α and ROS production) however, the AgNPs differed to a larger extent. The highest induction of TNF-α was found in cells exposed to the negatively charged AgNP_BSA, both sizes (80× higher than control). Reactive oxygen species induction was only significant with the 20 nm positively charged AgNP_Chit.

Corrigendum: Intrinsic and Extrinsic Properties Affecting Innate Immune Responses to Nanoparticles: The Case of Cerium Oxide.

[This corrects the article on p. 970 in vol. 8, PMID: 28855907.].

Formation of the Protein Corona: The Interface between Nanoparticles and the Immune System.

The interaction of inorganic nanoparticles and many biological fluids often withstands the formation of a Protein Corona enveloping the nanoparticle. This Protein Corona provides the biological identity to the nanoparticle that the immune system will detect. The formation of this Protein Corona depends not only on the composition of the nanoparticle, its size, shape, surface state and exposure time, but also on the type of media, nanoparticle to protein ratio and the presence of ions and other molecular species that interfere in the interaction between proteins and nanoparticles. This has important implications on immune safety, biocompatibility and the use of nanoparticles in medicine.

Intrinsic and Extrinsic Properties Affecting Innate Immune Responses to Nanoparticles: The Case of Cerium Oxide.

We review the apparent discrepancies between studies that report anti-inflammatory effects of cerium oxide nanoparticles (CeO2 NPs) through their reactive oxygen species-chelating properties and immunological studies highlighting their toxicity. We observe that several underappreciated parameters, such as aggregation size and degree of impurity, are critical determinants that need to be carefully addressed to better understand the NP biological effects in order to unleash their potential clinical benefits. This is because NPs can evolve toward different states, depending on the environment where they have been dispersed and how they have been dispersed. As a consequence, final characteristics of NPs can be very different from what was initially designed and produced in the laboratory. Thus, aggregation, corrosion, and interaction with extracellular matrix proteins critically modify NP features and fate. These modifications depend to a large extent on the characteristics of the biological media in which the NPs are dispersed. As a consequence, when reviewing the scientific literature, it seems that the aggregation state of NPs, which depends on the characteristics of the dispersing media, may be more significant than the composition or original size of the NPs. In this work, we focus on CeO2 NPs, which are reported sometimes to be protective and anti-inflammatory, and sometimes toxic and pro-inflammatory.

Size-Dependent Protein-Nanoparticle Interactions in Citrate-Stabilized Gold Nanoparticles: The Emergence of the Protein Corona.

Surface modifications of highly monodisperse citrate-stabilized gold nanoparticles (AuNPs) with sizes ranging from 3.5 to 150 nm after their exposure to cell culture media supplemented with fetal bovine serum were studied and characterized by the combined use of UV-vis spectroscopy, dynamic light scattering, and zeta potential measurements. In all the tested AuNPs, a dynamic process of protein adsorption was observed, evolving toward the formation of an irreversible hard protein coating known as Protein Corona. Interestingly, the thickness and density of this protein coating were strongly dependent on the particle size, making it possible to identify different transition regimes as the size of the particles increased: (i) NP-protein complexes (or incomplete corona), (ii) the formation of a near-single dense protein corona layer, and (iii) the formation of a multilayer corona. In addition, the different temporal patterns in the evolution of the protein coating came about more quickly for small particles than for the larger ones, further revealing the significant role that size plays in the kinetics of this process. Since the biological identity of the NPs is ultimately determined by the protein corona and different NP-biological interactions take place at different time scales, these results are relevant to biological and toxicological studies.

Interaction of gold nanoparticles and nickel(II) sulfate affects dendritic cell maturation.

Despite many investigations have focused on the pristine toxicity of gold nanoparticles (GNPs), little is known about the outcome of co-exposure and interaction of GNPs with heavy metals which can possibly detoxify or potentiate them. Here, the combined exposure of nickel (II) sulfate (NiSO4) and GNPs on the maturation response of dendritic cells (DCs) was explored. Exposure to GNPs or NiSO4 separately induced cell activation. When cells were exposed to a mixture of both, however, the observed cell activation pattern indicated a competitive rather than an additive effect of both inducers with levels similar to those induced by NiSO4 alone. Quantification of the GNP uptake by DCs demonstrated a significant decrease in intracellular gold content during co-incubation with NiSO4. An extensive physiochemical characterization was performed to determine the interaction between GNPs and NiSO4 in the complex physiological media using nanoparticle tracking analyses, disc centrifugation, UV-visible spectroscopy, ICP-MS analyses, zeta potential measurements, electron microscopy, and proteomics. Although GNPs and NiSO4 did not directly interact with each other, the presence of NiSO4 in the physiological media resulted in changes in GNPs' charge and their associated protein corona (content and composition), which may contribute to a decreased cellular uptake of GNPs and sustaining the nickel-induced DC maturation. The presented results provide new insights in the interaction of heavy metals and NPs in complex physiological media. Moreover, this study highlights the necessity of mixture toxicology, since these combined exposures are highly relevant for human subjection to NPs and risk assessment of nanomaterials.

Properties of silver nanoparticles influencing their uptake in and toxicity to the earthworm Lumbricus rubellus following exposure in soil.

Physicochemical properties of nanoparticles influence their environmental fate and toxicity, and studies investigating this are vital for a holistic approach towards a comprehensive and adequate environmental risk assessment. In this study, we investigated the effects of size, surface coating (charge) of silver nanoparticles (AgNPs) - a most commonly-used nanoparticle-type, on the bioaccumulation in, and toxicity (survival, growth, cocoon production) to the earthworm Lumbricus rubellus. AgNPs were synthesized in three sizes: 20, 35 and 50 nm. Surface-coating with bovine serum albumin (AgNP_BSA), chitosan (AgNP_Chit), or polyvinylpyrrolidone (AgNP_PVP) produced negative, positive and neutral particles respectively. In a 28-day sub-chronic reproduction toxicity test, earthworms were exposed to these AgNPs in soil (0-250 mg Ag/kg soil DW). Earthworms were also exposed to AgNO3 at concentrations below known EC50. Total Ag tissue concentration indicated uptake by earthworms was generally highest for the AgNP_BSA especially at the lower exposure concentration ranges, and seems to reach a plateau level between 50 and 100 mg Ag/kg soil DW. Reproduction was impaired at high concentrations of all AgNPs tested, with AgNP_BSA particles being the most toxic. The EC50 for the 20 nm AgNP_BSA was 66.8 mg Ag/kg soil, with exposure to <60 mg Ag/kg soil already showing a decrease in the cocoon production. Thus, based on reproductive toxicity, the particles ranked: AgNP_BSA (negative) > AgNP_PVP (neutral) > Chitosan (positive). Size had an influence on uptake and toxicity of the AgNP_PVP, but not for AgNP_BSA nor AgNP_Chit. This study provides essential information on the role of physicochemical properties of AgNPs in influencing uptake by a terrestrial organism L. rubellus under environmentally relevant conditions. It also provides evidence of the influence of surface coating (charge) and the limited effect of size in the range of 20-50 nm, in driving uptake and toxicity of the AgNPs tested.

Enhanced detection with spectral imaging fluorescence microscopy reveals tissue- and cell-type-specific compartmentalization of surface-modified polystyrene nanoparticles.

BACKGROUND: Precisely targeted nanoparticle delivery is critically important for therapeutic applications. However, our knowledge on how the distinct physical and chemical properties of nanoparticles determine tissue penetration through physiological barriers, accumulation in specific cells and tissues, and clearance from selected organs has remained rather limited. In the recent study, spectral imaging fluorescence microscopy was exploited for precise and rapid monitoring of tissue- and cell-type-specific distribution of fluorescent polystyrene nanoparticles with chemically distinct surface compositions. METHODS: Fluorescent polystyrene nanoparticles with 50-90 nm diameter and with carboxylated- or polyethylene glycol-modified (PEGylated) surfaces were delivered into adult male and pregnant female mice with a single intravenous injection. The precise anatomical distribution of the particles was investigated by confocal microscopy after a short-term (5 min) or long-term (4 days) distribution period. In order to distinguish particle-fluorescence from tissue autofluorescence and to enhance the detection-efficiency, fluorescence spectral detection was applied during image acquisition and a post hoc full spectrum analysis was performed on the final images. RESULTS: Spectral imaging fluorescence microscopy allowed distinguishing particle-fluorescence from tissue-fluorescence in all examined organs (brain, kidney, liver, spleen and placenta) in NP-treated slice preparations. In short-time distribution following in vivo NP-administration, all organs contained carboxylated-nanoparticles, while PEGylated-nanoparticles were not detected in the brain and the placenta. Importantly, nanoparticles were not found in any embryonic tissues or in the barrier-protected brain parenchyma. Four days after the administration, particles were completely cleared from both the brain and the placenta, while PEGylated-, but not carboxylated-nanoparticles, were stuck in the kidney glomerular interstitium. In the spleen, macrophages accumulated large amount of carboxylated and PEGylated nanoparticles, with detectable redistribution from the marginal zone to the white pulp during the 4-day survival period. CONCLUSIONS: Spectral imaging fluorescence microscopy allowed detecting the tissue- and cell-type-specific accumulation and barrier-penetration of polystyrene nanoparticles with equal size but chemically distinct surfaces. The data revealed that polystyrene nanoparticles are retained by the reticuloendothelial system regardless of surface functionalization. Taken together with the increasing production and use of nanoparticles, the results highlight the necessity of long-term distribution studies to estimate the potential health-risks implanted by tissue-specific nanoparticle accumulation and clearance.

Quantifying the Sensitivity of Multipolar (Dipolar, Quadrupolar, and Octapolar) Surface Plasmon Resonances in Silver Nanoparticles: The Effect of Size, Composition, and Surface Coating.

The effect of composition, size, and surface coating on the sensitivity of localized multipolar surface plasmon resonances has been spectroscopically investigated in high-quality silver colloidal solutions with precisely controlled sizes from 10 to 220 nm and well-defined surface chemistry. Surface plasmon resonance modes have been intensively characterized, identifying the size-dependence of dipolar, quadrupolar, and octapolar modes. Modifications of the NP's surface chemistry revealed the higher sensitivity of large sizes, long molecules, thiol groups, and low-order resonance modes. We also extend this study to gold nanoparticles, aiming to compare the sensitivity of both materials, quantifying the higher sensitivity of silver.