The Relevance of Physico-Chemical Properties and Protein Corona for Evaluation of Nanoparticles Immunotoxicity-In Vitro Correlation Analysis on THP-1 Macrophages.

Alongside physiochemical properties (PCP), it has been suggested that the protein corona of nanoparticles (NPs) plays a crucial role in the response of immune cells to NPs. However, due to the great variety of NPs, target cells, and exposure protocols, there is still no clear relationship between PCP, protein corona composition, and the immunotoxicity of NPs. In this study, we correlated PCP and the protein corona composition of NPs to the THP-1 macrophage response, focusing on selected toxicological endpoints: cell viability, reactive oxygen species (ROS), and cytokine secretion. We analyzed seven commonly used engineered NPs (SiO2, silver, and TiO2) and magnetic NPs. We show that with the exception of silver NPs, all of the tested TiO2 types and SiO2 exhibited moderate toxicities and a transient inflammatory response that was observed as an increase in ROS, IL-8, and/or IL-1ß cytokine secretion. We observed a strong correlation between the size of the NPs in media and IL-1ß secretion. The induction of IL-1ß secretion was completely blunted in NLR family pyrin domain containing 3 (NLRP3) knockout THP-1 cells, indicating activation of the inflammasome. The correlations analysis also implicated the association of specific NP corona proteins with the induction of cytokine secretion. This study provides new insights toward a better understanding of the relationships between PCP, protein corona, and the inflammatory response of macrophages for different engineered NPs, to which we are exposed on a daily basis.

Proposing Urothelial and Muscle In Vitro Cell Models as a Novel Approach for Assessment of Long-Term Toxicity of Nanoparticles.

Many studies evaluated the short-term in vitro toxicity of nanoparticles (NPs); however, long-term effects are still not adequately understood. Here, we investigated the potential toxic effects of biomedical (polyacrylic acid and polyethylenimine coated magnetic NPs) and two industrial (SiO2 and TiO2) NPs following different short-term and long-term exposure protocols on two physiologically different in vitro models that are able to differentiate: L6 rat skeletal muscle cell line and biomimetic normal porcine urothelial (NPU) cells. We show that L6 cells are more sensitive to NP exposure then NPU cells. Transmission electron microscopy revealed an uptake of NPs into L6 cells but not NPU cells. In L6 cells, we obtained a dose-dependent reduction in cell viability and increased reactive oxygen species (ROS) formation after 24 h. Following continuous exposure, more stable TiO2 and polyacrylic acid (PAA) NPs increased levels of nuclear factor Nrf2 mRNA, suggesting an oxidative damage-associated response. Furthermore, internalized magnetic PAA and TiO2 NPs hindered the differentiation of L6 cells. We propose the use of L6 skeletal muscle cells and NPU cells as a novel approach for assessment of the potential long-term toxicity of relevant NPs that are found in the blood and/or can be secreted into the urine.

Analysis of the Direct and Indirect Effects of Nanoparticle Exposure on Microglial and Neuronal Cells In Vitro.

Environmental or biomedical exposure to nanoparticles (NPs) can results in translocation and accumulation of NPs in the brain, which can lead to health-related problems. NPs have been shown to induce toxicity to neuronal cells through several direct mechanisms, but only a few studies have also explored the indirect effects of NPs, through consequences due to the exposure of neighboring cells to NPs. In this study, we analysed possible direct and indirect effects of NPs (polyacrylic acid (PAA) coated cobalt ferrite NP, TiO2 P25 and maghemite NPs) on immortalized mouse microglial cells and differentiated CAD mouse neuronal cells in monoculture (direct toxicity) or in transwell co-culture system (indirect toxicity). We showed that although the low NP concentrations (2-25 µg/mL) did not induce changes in cell viability, cytokine secretion or NF-κB activation of microglial cells, even low NP concentrations of 10 µg/mL can affect the cells and change their secretion of protein stress mediators. These can in turn influence neuronal cells in indirect exposure model. Indirect toxicity of NPs is an important and not adequately assessed mechanism of NP toxicity, since it not only affects cells on the exposure sites, but through secretion of signaling mediators, can also affect cells that do not come in direct contact with NPs.

Increased endocytosis of magnetic nanoparticles into cancerous urothelial cells versus normal urothelial cells.

The blood-urine barrier is the tightest and most impermeable barrier in the body and as such represents a problem for intravesical drug delivery applications. Differentiation-dependent low endocytotic rate of urothelial cells has already been noted; however, the differences in endocytosis of normal and cancer urothelial cells have not been exploited yet. Here we analysed the endocytosis of rhodamine B isothiocyanate-labelled polyacrylic acid-coated cobalt ferrite nanoparticles (NPs) in biomimetic urothelial in vitro models, i.e., in highly and partially differentiated normal urothelial cells, and in cancer cells of the papillary and invasive urothelial neoplasm. We demonstrated that NPs enter papillary and invasive urothelial neoplasm cells by ruffling of the plasma membrane and engulfment of NP aggregates by macropinocytotic mechanism. Transmission electron microscopy (TEM) and spectrophotometric analyses showed that the efficacy of NPs delivery into normal urothelial cells and intercellular space is largely restricted, while it is significantly higher in cancer urothelial cells. Moreover, we showed that the quantification of fluorescent NP internalization in cells or tissues based on fluorescence detection could be misleading and overestimated without TEM analysis. Our findings contribute to the understanding of endocytosis-mediated cellular uptake of NPs in cancer urothelial cells and reveal a highly selective mechanism to distinguish cancer and normal urothelial cells.

In vitro assessment of potential bladder papillary neoplasm treatment with functionalized polyethyleneimine coated magnetic nanoparticles.

Normal porcine urothelial cells have been shown to have a much lower rate of endocytosis than urothelial papillary neoplasm cells. This could be used as a mechanism for selective delivery of toxic compounds, such as polyethyleneimine coated nanoparticles (NPs). However, these NPs induce nonselective toxicity through direct membrane disruption. This toxicity can be reduced by functionalization of NPs with L-glutathione reduced or bovine serum albumin by reducing their surface charge. Functionalization was confirmed with Fourier Transform Infrared Spectroscopy, Dynamic Light Scattering and zeta potential measurements. Viability assays showed that bovine serum albumin coating reduced NPs cytotoxicity immediately after 3 h exposure and that such NPs were more toxic to urothelial papillary neoplasm cells compared to normal porcine urothelial cells at 50 µg/ml NPs concentration. However, 24 h after exposure, bovine serum albumin functionalized NPs had similar effect on viability of both cell lines. NPs showed some selective toxicity towards urothelial papillary neoplasm cells compared to normal cells after 3 h, however this was not confirmed after 24 h.

The Effect of Different Types of Nanoparticles on FUS and TDP-43 Solubility and Subcellular Localization.

Increased environmental pollution has been suggested as one of the possible causes for increased incidence of neurodegenerative and developmental disorders. Through the environmental pollution, everyday consumer products and nanomedical applications, we are also exposed to various nanoparticles (NPs). Specific types of NPs have been shown to be able to cause neural damage in vivo through processes such as disruption of the blood-brain barrier, induction of neuroinflammation, increase in oxidative stress and protein aggregation. In this study, we analysed the influence of PEI-coated magnetic NPs designed for biotechnological applications and industrial SiO2, TiO2 N and TiO2 P25 NPs on intracellular localization and solubility of fused in farcoma (FUS) and TAR-DNA binding protein 43 (TDP-43) that are important pathological hallmarks of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). SH-SY5Y neuroblastoma cells and B16 mouse melanoma cells were exposed to NPs for 24 h and analysed using confocal microscopy and Western blot. Exposure to 50 µg/ml TiO2 N and 4 µg/ml PEI NPs in SH-SY5Y cells caused cell toxicity-induced changes in expression in different biochemical/cellular fractions for both FUS and TDP-43 proteins. TiO2 N induced a drop in nuclear levels of TDP-43 and increase in cytoplasmic levels of FUS, while PEI NPs increased nuclear levels of FUS. Furthermore, TiO2 N and PEI induced a reduction of FUS and TDP-43 quantity in the less soluble urea fraction. No formation of stress granules was observed. These results demonstrate that TiO2 N and PEI NPs can affect the behaviour of FUS and TDP-43 proteins; however, the changes were relatively minor compared to pathological changes even for the high NP concentrations (50 µg/ml) used in this study.

Effect of different parameters used for in vitro gene electrotransfer on gene expression efficiency, cell viability and visualization of plasmid DNA at the membrane level.

BACKGROUND: Gene electrotransfer is a nonviral method used for DNA delivery into cells. Several steps are involved. One of them is the interaction of DNA with the cell membrane, which is crucial before DNA can enter the cell. We analysed the level of DNA-membrane interaction in relation to electrotransfer efficiency and the importance of the electrophoretic accumulation of DNA at the cell membrane. Systematic comparison of long-duration, short-duration and combinations of electropermeabilizing short (high-voltage; HV) and electrophoretic long (low-voltage; LV) pulses were performed. The effect of Mg(2+) ion concentrations on electrotransfer and their effect on DNase activity were explored. METHODS: To visualize the DNA-membrane interaction, TOTO-1 labeled DNA was used. Transfection efficiency was assessed with plasmid DNA coding for green fluorescent protein. RESULTS: Higher relative electrotransfer efficiency was obtained by using longer pulses, whereas shorter pulses preserved cell viability. Short-duration pulses enabled higher (24%) overall transfection yield compared to long-duration pulses (12%), although a higher DNA-membrane interaction was observed. No significant difference in transfection was obtained between different HV-LV pulsing protocols, although the highest DNA-membrane interaction was observed with HV + LV pulses. The formation of the DNA-membrane complex depended on the Mg(2+) concentration, whereas DNase inhibitor did not affect gene expression. CONCLUSIONS: Gene electrotransfer is a complex phenomenon, where many factors mutually affect the process and the DNA-membrane interaction only comprises the first step. We showed that longer electric pulses are optimal for higher transfection efficiency but reduce viability, whereas shorter pulses enable moderate transfection efficiency and preserve viability. Thus, each application needs a careful choice of pulsing protocol.