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.

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.

We report two-photon excited nonresonant surface-enhanced hyper Raman scattering (SEHRS) spectra of tricyclic antidepressant (TCA) molecules during their interaction with biocompatible gold nanostructures and with silver nanostructures. The SEHRS spectra of amitriptyline, desipramine, and imipramine are compared with surface-enhanced Raman scattering (SERS) spectra on both kinds of nanoparticles, obtained with excitation at 532 and 785 nm. The SEHRS spectra of the TCA molecules show several intense contributions by infrared-active vibrations. Combining SEHRS with SERS therefore enables a comprehensive vibrational characterization of the interaction of the molecules with the nanostructures. SEHRS and SERS data indicate that the molecules interact with the silver nanostructures mainly via their ring moiety. In contrast, in the interaction with gold, the methylaminopropyl side chain plays a very important role, along with parts of the ring system. It is possible to obtain the spectra of the molecules with near-infrared excitation and with gold nanoparticles in cell culture media. The spectral signatures of the drug molecules collected at low pH values characteristic of late endosomal stages or of acidified tissues are very stable and show only small changes in the interaction of the TCA with the gold nanoparticles. The results will help to develop tools for the characterization of new nanoparticle-based drug delivery platforms in real biological environments.

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.

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 offresonance. 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.