Correlated light and electron microscopy (CLEM) has become essential in life sciences due to advancements in imaging resolution, sensitivity, and sample preservation. In nanotoxicology─specifically, studying the health effects of particulate matter exposure─CLEM can enable molecular-level structural as well as functional analysis of nanoparticle interactions with lung tissue, which is key for the understanding of modes of action. In our study, we implement an integrated high-resolution fluorescence lifetime imaging microscopy (FLIM) and hyperspectral fluorescence imaging (fHSI), scanning electron microscopy (SEM), ultrahigh resolution helium ion microscopy (HIM) and synchrotron micro X-ray fluorescence (SR μXRF), to characterize the nanobio interface and to better elucidate the modes of action of lung epithelial cells response to known inflammatory titanium dioxide nanotubes (TiO2 NTs). Morpho-functional assessment uncovered several mechanisms associated with extensive DNA, essential minerals, and iron accumulation, cellular surface immobilization, and the localized formation of fibrous structures, all confirming immunomodulatory responses. These findings advance our understanding of the early cellular processes leading to inflammation development after lung epithelium exposure to these high-aspect-ratio nanoparticles. Our high-resolution experimental approach, exploiting light, ion, and electron sources, provides a robust framework for future research into nanoparticle toxicity and its impact on human health.

• Keywords
• TiO2 nanotubes, correlated light and electron microscopy, helium ion microscopy, lung epithelium inflammation, nanobio interface, synchrotron micro X-ray fluorescence,
• MeSH
• Epithelial Cells * drug effects   pathology   MeSH
• Microscopy, Fluorescence MeSH
• Humans MeSH
• Nanoparticles * chemistry   toxicity   MeSH
• Nanotubes chemistry   MeSH
• Lung * drug effects   pathology   cytology   MeSH
• Titanium * chemistry   toxicity   MeSH
• Particle Size MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Titanium * MeSH
• titanium dioxide MeSH Browser

Investigating the electrical double layer (EDL) structure has been a long-standing challenge and has seen the emergence of several sophisticated techniques able to probe selectively the few molecular layers of a solid/water interface. While a qualitative estimation of the thickness of the EDL can be obtained using simple theoretical models, following experimentally its evolution is not straightforward and can be even more complicated in nano- or microscale systems, particularly when changing the ionic concentration by several orders of magnitude. Here, we bring insight into the structure of the EDL of SiO2 nanoparticle suspensions and its evolution with increasing ionic concentration using angle-resolved second harmonic scattering (AR-SHS). Below millimolar salt concentrations, we can successively characterize inner-sphere adsorption, diffuse layer formation, and outer-sphere adsorption. Moreover, we show for the first time that, by appropriately selecting the nanoparticle size, it is possible to retrieve information also in the millimolar range. There, we observe a decrease in the magnitude of the surface potential corresponding to a compression in the EDL thickness, which agrees with the results of several other electroanalytical and optical techniques. Molecular dynamics simulations suggest that the EDL compression mainly results from the diffuse layer compression rather than outer-sphere ions (Stern plane) moving closer to the surface.

• Publication type
• Journal Article MeSH

The routinely employed periodic boundary conditions complicate molecular simulations of physiologically relevant asymmetric lipid membranes together with their distinct solvent environments. Therefore, separating the extracellular fluid from its cytosolic counterpart has often been performed using a costly double-bilayer setup. Here, we demonstrate that the lipid membrane and solvent asymmetry can be efficiently modeled with a single lipid bilayer by applying an inverted flat-bottom potential to ions and other solute molecules, thereby restraining them to only interact with the relevant leaflet. We carefully optimized the parameters of the suggested method so that the results obtained using the flat-bottom and double-bilayer approaches become mutually indistinguishable. Then, we apply the flat-bottom approach to lipid bilayers with various compositions and solvent environments, covering ions and cationic peptides to validate the approach in a realistic use case. We also discuss the possible limitations of the method as well as its computational efficiency and provide a step-by-step guide on how to set up such simulations in a straightforward manner.

• MeSH
• Lipid Bilayers * chemistry   MeSH
• Solvents MeSH
• Molecular Dynamics Simulation * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Lipid Bilayers * MeSH
• Solvents MeSH

Ion-specific effects play a crucial role in controlling the stability of colloidal systems and regulating interfacial processes. Although mechanistic pictures have been developed to explain the electrostatic structure of solid/water colloidal interfaces, ion-specific effects remain poorly understood. Here we quantify the average interfacial water orientation and the electrostatic surface potential around 100 nm SiO2 and TiO2 colloidal particles in the presence of NaCl, RbCl, and CaCl2 using polarimetric angle-resolved second harmonic scattering. We show that these two parameters can be used to establish the ion adsorption mechanism in a low ionic strength regime (<1 mM added salt). The relative differences between salts as a function of the ionic strength demonstrate cation- and surface-specific preferences for inner- vs outer-sphere adsorption. Compared to monovalent Rb+ and Na+, Ca2+ is found to be preferentially adsorbed as outer-sphere on SiO2 surfaces, while a dominant inner-sphere adsorption is observed for Ca2+ on TiO2. Molecular dynamics simulations performed on crystalline SiO2 and TiO2 surfaces support the experimental conclusions. This work contributes to the understanding of the electrostatic environment around colloidal nanoparticles on a molecular level by providing insight into ion-specific effects with micromolar sensitivity.

• Publication type
• Journal Article MeSH