The cellular uptake of nanoparticles (NPs) represents a critical step in nanomedicine and a crucial point for understanding the interaction of nanomaterials with biological systems. No specific mechanism of uptake has been identified so far, as the NPs are generally incorporated by the cells through one of the few well-known endocytotic mechanisms. Here, an alternative internalization route mediated by microvilli adhesion is demonstrated. This microvillus-mediated adhesion (MMA) has been observed using ceria and magnetite NPs with a dimension of <40 nm functionalized with polyacrylic acid but not using NPs with a neutral or positive functionalization. Such an adhesion was not cell specific, as it was demonstrated in three different cell lines. MMA was also reduced by modifications of the microvillus lipid rafts, obtained by depleting cholesterol and altering synthesis of sphingolipids. We found a direct relationship between MAA, cell cycle, and density of microvilli. The evidence suggests that MMA differs from the commonly described uptake mechanisms and might represent an interesting alternative approach for selective NP delivery.

CeO₂ nanoparticles (CNPs) have been investigated as promising antioxidant agents with significant activity in the therapy of diseases involving free radicals or oxidative stress. However, the exact mechanism responsible for CNP activity has not been completely elucidated. In particular, in situ evidence of modification of the oxidative state of CNPs in human cells and their evolution during cell internalization and subsequent intracellular distribution has never been presented. In this study we investigated modification of the Ce(III)/Ce(IV) ratio following internalization in human cells by X-ray absorption near edge spectroscopy (XANES). From this analysis on cell pellets, we observed that CNPs incubated for 24 h showed a significant increase in Ce(III). By coupling on individual cells synchrotron micro-X-ray fluorescence (μXRF) with micro-XANES (μXANES) we demonstrated that the Ce(III)/Ce(IV) ratio is also dependent on CNP intracellular localization. The regions with the highest CNP concentrations, suggested to be endolysosomes by transmission electron microscopy, were characterized by Ce atoms in the Ce(IV) oxidation state, while a higher Ce(III) content was observed in regions surrounding these areas. These observations suggest that the interaction of CNPs with cells involves a complex mechanism in which different cellular areas play different roles.

Discoidal nanoparticles showed highest blood-brain barrier permeability and transfer efficiency via tunneling nanotubes in glioblastoma cells, offering a promising strategy for nanoparticles delivery and intercellular distribution within tumors.

Vascular oxidative stress is considered a worsening factor in the progression of Alzheimer’s disease (AD). Increased reactive oxygen species (ROS) levels promote the accumulation of amyloid-β peptide (Aβ), one of the main hallmarks of AD. In turn, Aβ is a potent inducer of oxidative stress. In early stages of AD, the concomitant action of oxidative stress and Aβ on brain capillary endothelial cells was observed to compromise the blood–brain barrier functionality. In this context, antioxidant compounds might provide therapeutic benefits. To this aim, we investigated the antioxidant activity of cerium oxide nanoparticles (CNP) in human cerebral microvascular endothelial cells (hCMEC/D3) exposed to Aβ oligomers. Treatment with CNP (13.9 ± 0.7 nm in diameter) restored basal ROS levels in hCMEC/D3 cells, both after acute or prolonged exposure to Aβ. Moreover, we found that the extent of CNP uptake by hCMEC/D3 was +43% higher in the presence of Aβ. Scanning electron microscopy and western blot analysis suggested that changes in microvilli structures on the cell surface, under pro-oxidant stimuli (Aβ or H₂O₂), might be involved in the enhancement of CNP uptake. This finding opens the possibility to exploit the modulation of endothelial microvilli pattern to improve the uptake of anti-oxidant particles designed to counteract ROS-mediated cerebrovascular dysfunctions.

Some aquaporins (AQPs) allow the diffusion of hydrogen peroxide (H₂O₂), the most abundant ROS, through the cell membranes. Therefore, the possibility of regulating the AQP-mediated permeability to H₂O₂, and thus ROS scavenging, appears particularly important for controlling the redox state of cells in physiological and pathophysiological conditions. Several compounds have been screened and characterized for this purpose. This study aimed to analyze the effect of cerium oxide nanoparticles (CNPs) presenting antioxidant activity on AQP functioning. HeLa cells express AQP3, 6, 8, and 11, able to facilitate H₂O₂. AQP3, 6, and 8 are expressed in the plasma membrane and intracellularly, while AQP11 resides only in intracellular structures. CNPs but not cerium ions treatment significantly increased the water and H₂O₂ permeability by interacting with AQP3, 6, and especially with AQP8. CNPs increased considerably the AQP-mediated water diffusion in cells with oxidative stress. Functional experiments with silenced HeLa cells revealed that CNPs increased the H₂O₂ diffusion mainly by modulating the AQP8 permeability but also the AQP3 and AQP6, even if to a lesser extent. Current findings suggest that CNPs represent a promising pharmaceutical agent that might potentially be used in numerous pathologies involving oxidative stress as tumors and neurodegenerative diseases.

Cytoplasmic structures showing a selective concentration of both polyubiquitinated proteins and proteasome have been described in various epithelial, hematopoietic, mesenchymal and neural cells in vitro or in fetal tissues, as well as in chronically-infected, mutated preneoplastic and neoplastic tissues. These cytoplasmic structures differ from other ubiquitin-reactive cytoplasmic bodies, like sequestosomes, aggresome-like-induced structures in dendritic cells (DALIS)/non-dendritic cells (ALIS) and aggresomes in showing distinctive ultrastructural organization (particle-rich cytoplasmic structure or PaCS), a cytochemical pattern and a functional profile. Their formation can be induced in vitro in dendritic or natural killer cells by trophic factors and interleukin treatment. They originate in close connection with ribosomes, while, as a result of their growth, the cytoskeleton and other surrounding organelles are usually dislocated outside their core. Interestingly, these particulate cytoplasmic structures are often found to fill cytoplasmic blebs forming proteasome- and polyubiquitinated protein-discharging vesicles, called ectosomes, which are found to detach from the cell and freely float in the extracellular space. To clearly point out the importance of the polyubiquitinated proteins and proteasome containing cytoplasmic structures, their role in cell biology and pathology has been carefully analyzed.