Influence of Nuclear Localization Sequences on the Intracellular Fate of Gold Nanoparticles.

Directing nanoparticles to the nucleus by attachment of nuclear localization sequences (NLS) is an aim in many applications. Gold nanoparticles modified with two different NLS were studied while crossing barriers of intact cells, including uptake, endosomal escape, and nuclear translocation. By imaging of the nanoparticles and by characterization of their molecular interactions with surface-enhanced Raman scattering (SERS), it is shown that nuclear translocation strongly depends on the particular incubation conditions. After an 1 h of incubation followed by a 24 h chase time, 14 nm gold particles carrying an adenoviral NLS are localized in endosomes, in the cytoplasm, and in the nucleus of fibroblast cells. In contrast, the cells display no nanoparticles in the cytoplasm or nucleus when continuously incubated with the nanoparticles for 24 h. The ultrastructural and spectroscopic data indicate different processing of NLS-functionalized particles in endosomes compared to unmodified particles. NLS-functionalized nanoparticles form larger intraendosomal aggregates than unmodified gold nanoparticles. SERS spectra of cells with NLS-functionalized gold nanoparticles contain bands assigned to DNA and were clearly different from those with unmodified gold nanoparticles. The different processing in the presence of an NLS is influenced by a continuous exposure of the cells to nanoparticles and an ongoing nanoparticle uptake. This is supported by mass-spectrometry-based quantification that indicates enhanced uptake of NLS-functionalized nanoparticles compared to unmodified particles under the same conditions. The results contribute to the optimization of nanoparticle analysis in cells in a variety of applications, e.g., in theranostics, biotechnology, and bioanalytics.

X-ray tomography shows the varying three-dimensional morphology of gold nanoaggregates in the cellular ultrastructure.

The processing of nanoparticles inside eukaryotic cells is a key step in many wanted and unwanted nano-bio-interactions. In order to understand the effects and functions of the intracellular aggregates that are formed, their properties and their interaction with the biological matrix must be characterized. High quality synchrotron soft X-ray tomography (SXT) data were obtained from cells containing gold nanoparticles that are commonly applied as tools for optical probing or drug delivery. 3D volume rendering of both cellular organelles and the nanoparticle aggregates of different sizes in the intact cells of two cell lines reveals variation in localization, size, shape and density of the intracellular gold nanoaggregates. The dependence of such variation on incubation time and cell type, as well as on the influence of pre-aggregation of primary nanoparticles is shown. The SXT results provide a detailed picture of intracellular aggregation and will improve the design of safe and efficient nanoparticle platforms for biomedical use.

Biomolecular environment, quantification, and intracellular interaction of multifunctional magnetic SERS nanoprobes.

Multifunctional composite nanoprobes consisting of iron oxide nanoparticles linked to silver and gold nanoparticles, Ag-Magnetite and Au-Magnetite, respectively, were introduced by endocytic uptake into cultured fibroblast cells. The cells containing the non-toxic nanoprobes were shown to be displaceable in an external magnetic field and can be manipulated in microfluidic channels. The distribution of the composite nanostructures that are contained in the endosomal system is discussed on the basis of surface-enhanced Raman scattering (SERS) mapping, quantitative laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) micromapping, and cryo soft X-ray tomography (cryo soft-XRT). Cryo soft-XRT of intact, vitrified cells reveals that the composite nanoprobes form intra-endosomal aggregates. The nanoprobes provide SERS signals from the biomolecular composition of their surface in the endosomal environment. The SERS data indicate the high stability of the nanoprobes and of their plasmonic properties in the harsh environment of endosomes and lysosomes. The spectra point at the molecular composition at the surface of the Ag-Magnetite and Au-Magnetite nanostructures that is very similar to that of other composite structures, but different from the composition of pure silver and gold SERS nanoprobes used for intracellular investigations. As shown by the LA-ICP-MS data, the uptake efficiency of the magnetite composites is approximately two to three times higher than that of the pure gold and silver nanoparticles.

Relating surface-enhanced Raman scattering signals of cells to gold nanoparticle aggregation as determined by LA-ICP-MS micromapping.

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.

Specific biomolecule corona is associated with ring-shaped organization of silver nanoparticles in cells.

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.

SERS reveals the specific interaction of silver and gold nanoparticles with hemoglobin and red blood cell components.

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.