We correlate the localization of silver nanoparticles inside cells with respect to the cellular architecture with the molecular information in the vicinity of the particle surface by combining nanoscale 3D cryo-soft X-ray tomography (cryo-SXT) with surface-enhanced Raman scattering (SERS). The interaction of the silver nanoparticle surface with small molecules and biopolymers was monitored by SERS in vitro over time in living cells. The spectra indicate a stable, time-independent surface composition of silver nanoparticles, despite the changing environment in the endosomal structure. Cryo-SXT reveals a characteristic ring-shaped organization of the silver nanoparticles in endosomes of different cell types. The ring-like structures inside the endosomes suggest a strong association among silver particles and with membrane structures. The comparison of the data with those obtained with gold nanoparticles suggests that the interactions between the nanoparticles and with the endosomal component are influenced by the molecular composition of the corona.

The interaction of nanoparticles with hemoglobin (Hb), a major constituent of red blood cells, is important in nanotoxicity research. We report SERS spectra of Hb using gold and silver nanoparticles at very small nanoparticle : Hb molecule ratios, that is, under conditions relevant for SERS-based nanotoxicity experiments with red blood cells at high sensitivity. We show that the structural information obtained from the experiment is highly dependent on the type of SERS substrate and the conditions under which the interaction of nanoparticles with Hb molecules takes place. In experiments with isolated red blood cells, we demonstrate that the dependence of the spectra on the type of nanoparticle used as the SERS substrate extends to whole red blood cells and red blood cell components. Regarding the applicability of SERS to red blood cells in vivo, evidence is provided that the molecular information contained in the spectra is highly dependent on the material and size of the nanoparticles. The results indicate specific interactions of gold and silver nanoparticles with Hb and the red blood cell membrane, and reflect the hemolytic activity of silver nanoparticles. The results of this study help improve our understanding of the interactions of silver and gold nanoparticles with red blood cells.

The cellular response to nanoparticle exposure is essential in various contexts, especially in nanotoxicity and nanomedicine. Here, 14-nm gold nanoparticles in 3T3 fibroblast cells are investigated in a series of pulse-chase experiments with a 30-min incubation pulse and chase times ranging from 15 min to 48 h. The gold nanoparticles and their aggregates are quantified inside the cellular ultrastructure by laser ablation inductively coupled plasma mass spectrometry micromapping and evaluated regarding the surface-enhanced Raman scattering (SERS) signals. In this way, both information about their localization at the micrometre scale and their molecular nanoenvironment, respectively, is obtained and can be related. Thus, the nanoparticle pathway from endocytotic uptake, intracellular processing, to cell division can be followed. It is shown that the ability of the intracellular nanoparticles and their accumulations and aggregates to support high SERS signals is neither directly related to nanoparticle amount nor to high local nanoparticle densities. The SERS data indicate that aggregate geometry and interparticle distances in the cell must change in the course of endosomal maturation and play a critical role for a specific gold nanoparticle type in order to act as efficient SERS nanoprobe. This finding is supported by TEM images, showing only a minor portion of aggregates that present small interparticle spacing. The SERS spectra obtained after different chase times show a changing composition and/or structure of the biomolecule corona of the gold nanoparticles as a consequence of endosomal processing.