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.

Role of Common Cell Culture Media Supplements on Citrate-Stabilized Gold Nanoparticle Protein Corona Formation, Aggregation State, and the Consequent Impact on Cellular Uptake.

Sodium citrate-stabilized gold nanoparticles (AuNPs) are destabilized when dispersed in cell culture media (CCMs). This may promote their aggregation and subsequent sedimentation, or under the proper conditions, their interaction with dispersed proteins can lead to the formation of a NP-stabilizing protein corona. CCMs are ionic solutions that contain growth substances which are typically supplemented, in addition to serum, with different substances such as dyes, antioxidants, and antibiotics. In this study, the impact of phenol red, penicillin-streptomycin, l-glutamine, and ß-mercaptoethanol on the formation of the NP-protein corona in CCMs was investigated. Similar protein coronas were obtained except in the presence of antibiotics. Under these conditions, the protein corona took more time to be formed, and its density and composition were altered, as indicated by UV-vis spectroscopy, Z potential, dynamic light scattering, and liquid chromatography-mass spectrometry analyses. As a consequence of these modifications, a significantly different AuNP cellular uptake was measured, showing that NP uptake increased as did the NP aggregate formation. AuNP uptake studies performed in the presence of clathrin- and caveolin-mediated endocytosis inhibitors showed that neither clathrin receptors nor lipid rafts were significantly involved in the internalization mechanism. These results suggest that in these conditions, NP aggregation is the main mechanism responsible for their cellular uptake.

A new source of representative secondary PET nanoplastics. Obtention, characterization, and hazard evaluation.

Micro and nanoplastics (MNPLs) are emergent environmental pollutants requiring urgent information on their potential risks to human health. One of the problems associated with the evaluation of their undesirable effects is the lack of representative samples, matching those resulting from the environmental degradation of plastic wastes. To such end, we propose an easy method to obtain polyethylene terephthalate nanoplastics from water plastic bottles (PET-NPLs) but, in principle, applicable to any other plastic goods sources. An extensive characterization indicates that the proposed process produces uniform samples of PET-NPLs of around 100 nm, as determined by using AF4 and multi-angle and dynamic light scattering methodologies. An important point to be highlighted is that to avoid the metal contamination resulting from methods using metal blades/burrs for milling, trituration, or sanding, we propose to use diamond burrs to produce metal-free samples. To visualize the toxicological profile of the produced PET-NPLs we have evaluated their ability to be internalized by cells, their cytotoxicity, their ability to induce oxidative stress, and induce DNA damage. In this preliminary approach, we have detected their cellular uptake, but without the induction of significant biological effects. Thus, no relevant increases in toxicity, reactive oxygen species (ROS) induction, or DNA damage -as detected with the comet assay- have been observed. The use of representative samples, as produced in this study, will generate relevant data in the discussion about the potential health risks associated with MNPLs exposures.

Heterogeneous Rate Constant for Amorphous Silica Nanoparticle Adsorption on Phospholipid Monolayers.

The interaction of amorphous silica nanoparticles with phospholipid monolayers and bilayers has received a great deal of interest in recent years and is of importance for assessing potential cellular toxicity of such species, whether natural or synthesized for the purpose of nanomedical drug delivery and other applications. This present communication studies the rate of silica nanoparticle adsorption on to phospholipid monolayers in order to extract a heterogeneous rate constant from the data. This rate constant relates to the initial rate of growth of an adsorbed layer of nanoparticles as SiO2 on a unit area of the monolayer surface from unit concentration in dispersion. Experiments were carried out using the system of dioleoyl phosphatidylcholine (DOPC) monolayers deposited on Pt/Hg electrodes in a flow cell. Additional studies were carried out on the interaction of soluble silica with these layers. Results show that the rate constant is effectively constant with respect to silica nanoparticle size. This is interpreted as indicating that the interaction of hydrated SiO2 molecular species with phospholipid polar groups is the molecular initiating event (MIE) defined as the initial interaction of the silica particle surface with the phospholipid layer surface promoting the adsorption of silica nanoparticles on DOPC. The conclusion is consistent with the observed significant interaction of soluble SiO2 with the DOPC layer and the established properties of the silica-water interface.

Pharmacokinetics of PEGylated Gold Nanoparticles: In Vitro-In Vivo Correlation.

Data suitable for assembling a physiologically-based pharmacokinetic (PBPK) model for nanoparticles (NPs) remain relatively scarce. Therefore, there is a trend in extrapolating the results of in vitro and in silico studies to in vivo nanoparticle hazard and risk assessment. To evaluate the reliability of such approach, a pharmacokinetic study was performed using the same polyethylene glycol-coated gold nanoparticles (PEG-AuNPs) in vitro and in vivo. As in vitro models, human cell lines TH1, A549, Hep G2, and 16HBE were employed. The in vivo PEG-AuNP biodistribution was assessed in rats. The internalization and exclusion of PEG-AuNPs in vitro were modeled as first-order rate processes with the partition coefficient describing the equilibrium distribution. The pharmacokinetic parameters were obtained by fitting the model to the in vitro data and subsequently used for PBPK simulation in vivo. Notable differences were observed in the internalized amount of Au in individual cell lines compared to the corresponding tissues in vivo, with the highest found for renal TH1 cells and kidneys. The main reason for these discrepancies is the absence of natural barriers in the in vitro conditions. Therefore, caution should be exercised when extrapolating in vitro data to predict the in vivo NP burden and response to exposure.

Hepato(Geno)Toxicity Assessment of Nanoparticles in a HepG2 Liver Spheroid Model.

(1) In compliance with the 3Rs policy to reduce, refine and replace animal experiments, the development of advanced in vitro models is needed for nanotoxicity assessment. Cells cultivated in 3D resemble organ structures better than 2D cultures. This study aims to compare cytotoxic and genotoxic responses induced by titanium dioxide (TiO2), silver (Ag) and zinc oxide (ZnO) nanoparticles (NPs) in 2D monolayer and 3D spheroid cultures of HepG2 human liver cells. (2) NPs were characterized by electron microscopy, dynamic light scattering, laser Doppler anemometry, UV-vis spectroscopy and mass spectrometry. Cytotoxicity was investigated by the alamarBlue assay and confocal microscopy in HepG2 monolayer and spheroid cultures after 24 h of NP exposure. DNA damage (strand breaks and oxidized base lesions) was measured by the comet assay. (3) Ag-NPs were aggregated at 24 h, and a substantial part of the ZnO-NPs was dissolved in culture medium. Ag-NPs induced stronger cytotoxicity in 2D cultures (EC50 3.8 µg/cm2) than in 3D cultures (EC50 > 30 µg/cm2), and ZnO-NPs induced cytotoxicity to a similar extent in both models (EC50 10.1-16.2 µg/cm2). Ag- and ZnO-NPs showed a concentration-dependent genotoxic effect, but the effect was not statistically significant. TiO2-NPs showed no toxicity (EC50 > 75 µg/cm2). (4) This study shows that the HepG2 spheroid model is a promising advanced in vitro model for toxicity assessment of NPs.

Dynamic Equilibrium in the Cetyltrimethylammonium Bromide-Au Nanoparticle Bilayer, and the Consequent Impact on the Formation of the Nanoparticle Protein Corona.

Nanoparticles in ionic solutions are usually surrounded by stabilizing molecules that avoid aggregation and determine their surface properties, which strongly influence their behavior. The present work aims to shed light on the static vs dynamic nature of the cetyltrimethylammonium bromide (CTAB) bilayer on gold nanoparticles and to understand its effects on nanoparticle evolution in biological systems. A systematic study of the CTAB bilayer of Au nanorods and nanospheres was carried out, exploring the role of excess free surfactant in solution on the surface properties of nanoparticles and their colloidal stability. The results indicated the presence of a CTAB bilayer in which the external layer was in rapid dynamic equilibrium with the free surfactant in solution. The internal surfactant layer of the gold nanospheres was also found to be in dynamic equilibrium. Conversely, the gold nanorods had a permanent internal layer. Consequently, the CTAB-nanoparticle dispersions always contained free CTAB in excess to maintain the colloidal stability of the NPs. In contrast, decreasing the free CTAB concentration resulted in nanoparticle aggregation. The impact of the dynamic equilibrium on the exposure of particles to biological fluids and on the formation of the nanoparticle protein corona was studied, revealing the different fates of the nanoparticles, which depended on the amount of free CTAB in solution.

Mechanomodulation of Lipid Membranes by Weakly Aggregating Silver Nanoparticles.

Silver nanoparticles (AgNPs) have wide-ranging applications, including as additives in consumer products and in medical diagnostics and therapy. Therefore, understanding how AgNPs interact with biological systems is important for ascertaining any potential health risks due to the likelihood of high levels of human exposure. Besides any severe, acute effects, it is desirable to understand more subtle interactions that could lead to milder, chronic health impacts. Nanoparticles are small enough to be able to enter biological cells and interfere with their internal biochemistry. The initial contact between the nanoparticle and cell is at the plasma membrane. To gain fundamental mechanistic insight into AgNP-membrane interactions, we investigate these phenomena in minimal model systems using a wide range of biophysical approaches applied to lipid vesicles. We find a strong dependence on the medium composition, where colloidally stable AgNPs in a glucose buffer have a negligible effect on the membrane. However, at physiological salt concentrations, the AgNPs start to weakly aggregate and sporadic but significant membrane perturbation events are observed. Under these latter conditions, transient poration and structural remodeling of some vesicle membranes are observed. We observe that the fluidity of giant vesicle membranes universally decreases by an average of 16% across all vesicles. However, we observe a small population of vesicles that display a significant change in their mechanical properties with lower bending rigidity and higher membrane tension. Therefore, we argue that the isolated occurrences of membrane perturbation by AgNPs are due to low-probability mechanomodulation by AgNP aggregation at the membrane.

Assessment of iron oxide nanoparticle ecotoxicity on regeneration and homeostasis in the replacement model system Schmidtea mediterranea.

Iron oxide nanoparticles (IONs) are used in a number of applications, from food to cosmetics, from medical applications to magnetic storage. In spite of the 550 tons produced each year in Europe alone, no effective dose limit recommendations are established and the overall risks connected to IONs are still debated. The incorporation of IONs in daily life raises a concern about their effects on the environment, on living organisms, and on human health. In this study, we used freshwater planarians to assess the nanoecotoxicity of IONs. Planarians are free-living invertebrates known for their astonishing regenerative ability. Because of their sensitivity to toxicants, they are often used to determine the effects of toxic, genotoxic and carcinogenic environmental compounds with an approach in line with the 3Rs (Reduce, Refine, Replace) principle. Planarians were exposed to IONs at concentrations up to 1 mg/mL and their effects were evaluated at the behavioral, morphofunctional and molecular levels, with a special emphasis on the regeneration process. Our results indicate that IONs did not affect the stem cell population dynamics, nor did they induce substantial changes in either homeostatic or regenerating planarians. As positive controls, gold nanoparticles coated with the pro-apoptotic anti-cancer drug hexadecylmethylammonium bromide, silver nanoparticles and highly concentrated polystyrene nanoparticles were used. These all elicited toxic effects. Therefore, we conclude that IONs at environmental concentrations are safe for planarians, and that the planarian is a powerful model system that can replace vertebrate animal models in nanoecotoxicology research and for nanoecotoxicology studies.