Molecular Structure and Interactions of Lipids in the Outer Membrane of Living Cells Based on Surface-Enhanced Raman Scattering and Liposome Models.

The distribution and interaction of lipids determine the structure and function of the cellular membrane. Surface-enhanced Raman scattering (SERS) is used for selective molecular probing of the cell membrane of living fibroblast cells grown adherently on gold nanoisland substrates across their whole contact areas with the substrate, enabling mapping of the membrane's composition and interaction. From the SERS data, the localization and distribution of different lipids and their interactions, together with proteins in the outer cell membrane, are inferred. Interpretation of the spectra is mainly supported by comparison with the spectra of model liposomes composed of phosphatidylcholine, sphingomyelin, and cholesterol obtained on the same gold substrate. The interaction of the liposomes with the substrate differs from that with gold nanoparticles. The SERS maps indicate colocalization of ordered lipid domains with cholesterol in the living cells. They support the observation of ordered membrane regions of micrometer dimensions in the outer leaflet of the cell membrane that are rich in sphingomyelin. Moreover, the spectra of the living cells contain bands from the groups of the lipid heads, phosphate, choline, and ethanolamine, combined with those from membrane proteins, as indicated by signals assigned to prenyl attachment. Elucidating the composition and structure of lipid membranes in living cells can find application in many fields of research.

Chemical Mapping of Leishmania Infection in Live Cells by SERS Microscopy.

We report the direct probing of the molecular composition of Leishmania-infected macrophage cells in vitro by surface-enhanced Raman scattering (SERS). The microscopic mapping data indicate local abundance and distribution of molecular species that are very characteristic of the infection and that are observed here simultaneously. As revealed by electron microscopy, the gold nanoprobes used for SERS microspectrosopy have access to the parasitophorous vacuoles (PV) through the endosomal system. SERS nanoprobes located in the direct proximity to the parasite, in the greater volume of the PV, and in endolysosomal compartments in other cellular regions, respectively, report a characteristic chemical composition for each respective location. The data enable assessment of the distribution of ergosterol and cholesterol in the amastigote stage of the parasite and its immediate surroundings in the vacuole. Proteophosphoglycans of parasite origin, an important hallmark of the infection, are identified throughout the PV.

Relating the composition and interface interactions in the hard corona of gold nanoparticles to the induced response mechanisms in living cells.

Understanding the formation of the intracellular protein corona of nanoparticles is essential for a wide range of bio- and nanomedical applications. The innermost layer of the protein corona, the hard corona, directly interacts with the nanoparticle surface, and by shielding the surface, it has a deterministic effect on the intracellular processing of the nanoparticle. Here, we combine a direct qualitative analysis of the hard corona composition of gold nanoparticles with a detailed structural characterization of the molecules in their interaction with the nanoparticle surface and relate both to the effects they have on the ultrastructure of living cells and the processing of the gold nanoparticles. Cells from the cell lines HCT-116 and A549 were incubated with 30 nm citrate-stabilized gold nanoparticles and with their aggregates in different culture media. The combined results of mass spectrometry based proteomics, cryo soft X-ray nanotomography and surface-enhanced Raman scattering experiments together revealed different uptake mechanisms in the two cell lines and distinct levels of induced cellular stress when incubation conditions were varied. The data indicate that the different incubation conditions lead to changes in the nanoparticle processing via different protein-nanoparticle interfacial interactions. Specifically, they suggest that the protein-nanoparticle surface interactions depend mainly on the surface properties of the gold nanoparticles, that is, the ζ-potential and the resulting changes in the hydrophilicity of the nanoparticle surface, and are largely independent of the cell line, the uptake mechanism and intracellular processing, or the extent of the induced cellular stress.

Gold nanoisland substrates for SERS characterization of cultured cells.

We demonstrate a simple approach for fabricating cell-compatible SERS substrates, using repeated gold deposition and thermal annealing. The substrates exhibit SERS enhancement up to six orders of magnitude and high uniformity. We have carried out Raman imaging of fixed mesenchymal stromal cells cultured directly on the substrates. Results of viability assays confirm that the substrates are highly biocompatible and Raman imaging confirms that cell attachment to the substrates is sufficient to realize significant SERS enhancement of cellular components. Using the SERS substrates as an in vitro sensing platform allowed us to identify multiple characteristic molecular fingerprints of the cells, providing a promising avenue towards non-invasive chemical characterization of biological samples.

Optical Nanosensing of Lipid Accumulation due to Enzyme Inhibition in Live Cells.

Drugs that influence enzymes of lipid metabolism can cause pathological accumulation of lipids in animal cells. Here, gold nanoparticles, acting as nanosensors that deliver surface-enhanced Raman scattering (SERS) spectra from living cells provide molecular evidence of lipid accumulation in lysosomes after treatment of cultured cells with the three tricyclic antidepressants (TCA) desipramine, amitryptiline, and imipramine. The vibrational spectra elucidate to great detail and with very high sensitivity the composition of the drug-induced lipid accumulations, also observed in fixed samples by electron microscopy and X-ray nanotomography. The nanoprobes show that mostly sphingomyelin is accumulated in the lysosomes but also other lipids, in particular, cholesterol. The observation of sphingomyelin accumulation supports the impairment of the enzyme acid sphingomyelinase. The SERS data were analyzed by random forest based approaches, in particular, by minimal depth variable selection and surrogate minimal depth (SMD), shown here to be particularly useful machine learning tools for the analysis of the lipid signals that contribute only weakly to SERS spectra of cells. SMD is used for the identification of molecular colocalization and interactions of the drug molecules with lipid membranes and for discriminating between the biochemical effects of the three different TCA molecules, in agreement with their different activity. The spectra also indicate that the protein composition is significantly changed in cells treated with the drugs.

SERS and Cryo-EM Directly Reveal Different Liposome Structures during Interaction with Gold Nanoparticles.

The combination of gold nanoparticles with liposomes is important for nano- and biotechnology. Here, we present direct, label-free characterization of liposome structure and composition at the site of its interaction with citrate-stabilized gold nanoparticles by surface-enhanced Raman scattering (SERS) and cryogenic electron microscopy (cryo-EM). Evidenced by the vibrational spectra and cryo-EM, the gold nanoparticles destroy the bilayer structure of interacting liposomes in the presence of a high amount of citrate, while at lower citrate concentration the nanoparticles interact with the surface of the intact liposomes. The spectra of phosphatidylcholine and phosphatidylcholine/sphingomyelin liposomes show that at the site of interaction the lipid chains are in the gel phase. The SERS spectra indicate that cholesterol has strong effects on the contacts of the vesicles with the nanoparticles. By combining cryo-EM and SERS, the structure and properties of lipid-nanoparticle composites could be tailored for the development of drug delivery systems.

Surface-enhanced hyper Raman hyperspectral imaging and probing in animal cells.

Hyper Raman scattering, that is, spontaneous, two-photon excited Raman scattering, of organic molecules becomes strong when it occurs as surface-enhanced hyper Raman scattering (SEHRS), in the proximity of plasmonic nanostructures. Its advantages over one-photon excited surface-enhanced Raman scattering (SERS) include complementary vibrational information resulting from different selection rules, probing of very small focal volumes, and beneficial excitation with long wavelengths. Here, imaging of macrophage cells by SEHRS is demonstrated, using SEHRS labels consisting of silver nanoparticles and two different molecules, 2-naphthalenethiol and para-mercaptobenzoic acid, that are excited off-resonance. The vibrational signatures of the molecules are discriminated using hyperspectral analysis and provide information about the subcellular localization of the SEHRS probes. The SEHRS based hyperspectral imaging approach presented here uses principal component analysis (PCA) to localize the reporter molecules inside the cells and is augmented by hierarchical cluster analysis (HCA). The high sensitivity of SEHRS spectra with respect to small environmental changes can be utilized for mapping of physiological parameters in the endosomal system of the cells. This is illustrated by discussing the spatial distribution of endosomes of varying pH inside the cytosol.