Maria Ada Malvindi

Effects of cell culture media on the dynamic formation of protein-nanoparticle complexes and influence on the cellular response.

The development of appropriate in vitro protocols to assess the potential toxicity of the ever expanding range of nanoparticles represents a challenging issue, because of the rapid changes of their intrinsic physicochemical properties (size, shape, reactivity, surface area, etc.) upon dispersion in biological fluids. Dynamic formation of protein coating around nanoparticles is a key molecular event, which may strongly impact the biological response in nanotoxicological tests. In this work, by using citrate-capped gold nanoparticles (AuNPs) of different sizes as a model, we show, by several spectroscopic techniques (dynamic light scattering, UV-visible, plasmon resonance light scattering), that proteins-NP interactions are differently mediated by two widely used cellular media (i.e., Dulbecco Modified Eagles medium (DMEM) and Roswell Park Memorial Institute medium (RPMI), supplemented with fetal bovine serum). We found that, while DMEM elicits the formation of a large time-dependent protein corona, RPMI shows different dynamics with reduced protein coating. Characterization of these nanobioentities was also performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectroscopy, revealing that the average composition of protein corona does not reflect the relative abundance of serum proteins. To evaluate the biological impact of such hybrid bionanostructures, several comparative viability assays onto two cell lines (HeLa and U937) were carried out in the two media, in the presence of 15 nm AuNPs. We observed that proteins/NP complexes formed in RPMI are more abundantly internalized in cells as compared to DMEM, overall exerting higher cytotoxic effects. These results show that, beyond an in-depth NPs characterization before cellular experiments, a detailed understanding of the effects elicited by cell culture media on NPs is crucial for standardized nanotoxicology tests.

A general mechanism for intracellular toxicity of metal-containing nanoparticles

We demonstrate a general mechanism for the toxicity induced by metal-containing NPs, named “lysosome-enhanced Trojan horse effect”, which provides design rules to engineer safer NPs.

Toxicity assessment of silica coated iron oxide nanoparticles and biocompatibility improvement by surface engineering.

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.

Negligible particle-specific toxicity mechanism of silver nanoparticles: the role of Ag+ ion release in the cytosol.

Toxicity of silver nanoparticles (AgNPs) is supported by many observations in literature, but no mechanism details have been proved yet. Here we confirm and quantify the toxic potential of fully characterized AgNPs in HeLa and A549 cells. Notably, through a specific fluorescent probe, we demonstrate the intracellular release of Ag(+) ions in living cells after nanoparticle internalization, showing that in-situ particle degradation is promoted by the acidic lysosomal environment. The activation of metallothioneins in response to AgNPs and the possibility to reverse the main toxic pathway by Ag(+) chelating agents demonstrate a cause/effect relationship between ions and cell death. We propose that endocytosed AgNPs are degraded in the lysosomes and the release of Ag(+) ions in the cytosol induces cell damages, while ions released in the cell culture medium play a negligible effect. These findings will be useful to develop safer-by-design nanoparticles and proper regulatory guidelines of AgNPs. From the clinical editor: The authors describe the toxic potential of silver nanoparticles (AgNP) in human cancer cell lines. Cell death following the application of AgNPs is dose-dependent, and it is mostly due to Ag+ ions. Further in vivo studies should be performed to gain a comprehensive picture of AgNP-toxicity in mammals.

Optimal Enhancement Configuration of Silica Nanoparticles for Ultrasound Imaging and Automatic Detection at Conventional Diagnostic Frequencies

Objectives:To experimentally investigate the acoustical behavior of silica nanoparticles within conventional diagnostic ultrasound fields and to determine a suitable configuration, in terms of particle size and concentration, for their employment as targetable contrast agents. We also assessed the effectiveness of a novel method for automatic detection of targeted silica nanoparticles for future tissue typing applications. Materials and Methods:Silica nanospheres of variable size (160, 330, and 660 nm in diameter) and concentration (1010–1013 part/mL) were dispersed in different custom-designed agarose-based gel samples and imaged at 7.5 MHz with a conventional echograph linked to a research platform for radiofrequency signal acquisition. Off-line analysis included evaluation of backscattered ultrasound amplitude, image brightness, and nanoparticle automatic detection through radiofrequency signal processing. Results:Amplitude of nanoparticle-backscattered signals linearly increased with particle number concentration, but image brightness did not show the same trend, because the logarithmic compression caused the reaching of a “plateau” where brightness remained almost constant for further increments in particle concentration. On the other hand, both backscatter amplitude and image brightness showed significant increments when particle diameter was increased. Taking into account particle size constraints for tumor targeting (pore size of tumor endothelium and trapping effects because of reticulo-endothelial system limit the dimension of effectively employable particles to less than 380 nm), a suitable compromise is represented by the employment of 330-nm silica nanospheres at a concentration of about 1 to 2 × 1011 part/mL. These particles, in fact, showed the best combination of number concentration and diameter value to obtain an effective enhancement on conventional echographic images. Furthermore, also the sensitivity of the developed method for automatic nanoparticle detection had a maximum (72.8%) with 330-nm particles, whereas it was lower with both bigger and smaller particles (being equal to 64.1% and 17.5%, respectively). Conclusions:Silica nanoparticles at a diameter of about 330 nm are very promising contrast agents for ultrasound imaging and specific tumor targeting at conventional diagnostic frequencies, being in particular automatically detectable with high sensitivity already at low doses. Future studies will be carried out to assess the acoustic behavior of nanoparticles with different geometries/sizes and to improve sensitivity of the automatic detection algorithm.

The biocompatibility of amino functionalized CdSe/ZnS quantum-dot-Doped SiO2 nanoparticles with primary neural cells and their gene carrying performance⋆

Nanoparticles have an enormous potential for the development of applications in biomedicine such as gene or drug delivery. We developed and characterized NH(2) functionalized CdSe/ZnS quantum dot (QD)-doped SiO(2) nanoparticles (NPs) with both imaging and gene carrier capabilities. We show that QD-doped SiO(2) NPs are internalized by primary cortical neural cells without inducing cell death in vitro and in vivo. Moreover, the ability to bind, transport and release DNA into the cell allows GFP-plasmid transfection of NIH-3T3 and human neuroblastoma SH-SY5Y cell lines. QD-doped SiO(2) NPs properties make them a valuable tool for future nanomedicine application.

Monodispersed and size-controlled multibranched gold nanoparticles with nanoscale tuning of surface morphology

A novel seed-mediated synthetic route to produce multibranched gold nanoparticles is reported, in which it is possible to precisely tune both their size and nanostructuration, while maintaining an accurate level of monodispersion. The nanoscale control of surface nanoroughness/branching, ranging from small bud-like features to elongated spikes, allows to obtain fine tuning of the nanoparticle optical properties, up to the red and near-IR region of the spectrum. Such anisotropic nanostructures were demonstrated to be excellent candidates for SERS applications, showing significantly higher signals with respect to the standard spherical nanoparticles.

Fluorescent Nanocrystals Reveal Regulated Portals of Entry into and Between the Cells of Hydra

Initially viewed as innovative carriers for biomedical applications, with unique photophysical properties and great versatility to be decorated at their surface with suitable molecules, nanoparticles can also play active roles in mediating biological effects, suggesting the need to deeply investigate the mechanisms underlying cell-nanoparticle interaction and to identify the molecular players. Here we show that the cell uptake of fluorescent CdSe/CdS quantum rods (QRs) by Hydra vulgaris, a simple model organism at the base of metazoan evolution, can be tuned by modifying nanoparticle surface charge. At acidic pH, amino-PEG coated QRs, showing positive surface charge, are actively internalized by tentacle and body ectodermal cells, while negatively charged nanoparticles are not uptaken. In order to identify the molecular factors underlying QR uptake at acidic pH, we provide functional evidence of annexins involvement and explain the QR uptake as the combined result of QR positive charge and annexin membrane insertion. Moreover, tracking QR labelled cells during development and regeneration allowed us to uncover novel intercellular trafficking and cell dynamics underlying the remarkable plasticity of this ancient organism.

Rod-Shaped Nanocrystals Elicit Neuronal Activity In Vivo**

The development of novel nanomaterials has raised great interest in efforts to evaluate their effect on biological systems, ranging from single cells to whole animals. In particular, there exists an open question regarding whether nanoparticles per se can elicit biological responses, which could interfere with the phenomena they are intended to measure. Here it is reported that challenging the small cnidaria Hydra vulgaris in vivo with rod-shaped semiconductor nanoparticles, also known as quantum rods (QRs), results in an unexpected tentacle-writhing behavior, which is Ca(2+) dependent and relies on the presence of tentacle neurons. Due to the absence of surface functionalization of the QRs with specific ligands, and considering that spherical nanoparticles with same composition as the QRs fail to induce any in vivo behavior on the same experimental model, it is suggested that unique shape-tunable electrical properties of the QRs may account for the neuronal stimulation. This model system may represent a widely applicable tool for screening neuronal response to nanoparticles in vivo.

Impact of Amorphous SiO2 Nanoparticles on a Living Organism: Morphological, Behavioral, and Molecular Biology Implications.

It is generally accepted that silica (SiO2) is not toxic. But the increasing use of silica nanoparticles (SiO2NPs) in many different industrial fields has prompted the careful investigation of their toxicity in biological systems. In this report, we describe the effects elicited by SiO2NPs on animal and cell physiology. Stable and monodisperse amorphous silica nanoparticles, 25 nM in diameter, were administered to living Hydra vulgaris (Cnidaria). The dose-related effects were defined by morphological and behavioral assays. The results revealed an all-or-nothing lethal toxicity with a rather high threshold (35 nM NPs) and a LT50 of 38 h. At sub lethal doses, the morphophysiological effects included: animal morphology alterations, paralysis of the gastric region, disorganization and depletion of tentacle specialized cells, increase of apoptotic and collapsed cells, and reduction of the epithelial cell proliferation rate. Transcriptome analysis (RNAseq) revealed 45 differentially expressed genes, mostly involved in stress response and cuticle renovation. Our results show that Hydra reacts to SiO2NPs, is able to rebalance the animal homeostasis up to a relatively high doses of SiO2NPs, and that the physiological modifications are transduced to gene expression modulation.