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

Abstract

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.

Intracellular optical probing with gold nanostars

“…The high abundance of a signal at 1123 cm -1 indicates the presence of lipids in the local environment of the nanostars obtained at a HEPES concentration of 50 mM ( Figure 5E), in accordance with the expected uptake of the nanoparticles into endolysosomal structures where they can interact with the endolysosomal membrane. 42 In these cells, a few spectra also show signals at the typical frequencies of the lipid ester C=O stretching vibration around 1740 cm -1 , and most bands associated with C-H deformation vibrations have a Raman shift of 1432 cm -1 or of 1440 cm -1 characteristic of CH 2 groups that could be part of long lipid chains ( Figure 5E). 67 The interaction of the nanostars with a lipid rich environment is also visible in the characteristic Raman shift of the CH 2 deformation of lipids that occurs around 1365 cm -1 ( Figure 5F) or the =C-H deformation around 1273 cm -1 .…”

“…37 The average spectra with the nanostars synthesized at different HEPES concentration differ ( Figure 5A-5C). To account for the known variability between individual spectra from the same cell culture batch that is expected due to (i) the biological variation, (ii) the particular local composition in the environment of the many different nanostars within each probed cell, and (iii) the variation that is inherent to each SERS experiment, 42,65 the occurrence of bands in each individual spectrum was analyzed regardless of their absolute or relative intensities ( Figure 5D-5F). The simultaneous analysis of the average spectra and the band occurrences is helpful to draw conclusions on the interactions between biomolecules and particular types of nanoparticles, here the nanostars of different preparations, and their effect on cellular processing.…”

“…SERS can give information on the interaction of the molecules at the immediate surface of gold nanostructures and was recently shown to indicate details on the structure and interactions of and within the hard corona of plasmonic nanoparticles in living cells. 38,41 Adding the localization in the cellular ultrastructure by cryo soft X-ray nanotomography (cryo-SXT) of vitrified cell samples, a powerful tool to study the interaction of gold nanoparticles with the cellular ultrastructure and with each other, 37,38,42 we link the interactions at the gold nanostar surface to their fate inside the cells. Of the known surfactant-free synthesis routes, 11,[43][44][45] generation of the nanostars in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer for reducing, as capping agent and growth director is used here to ensure compatibility with the cell culture conditions.…”

Application of random forest based approaches to surface-enhanced Raman scattering data

“…Since the single spectra represent different types of SERS information that individually co-occur in the SERS data sets it is obvious that this analysis is capable to distinguish between co-occuring SERS signals, especially when they are causal for the analyzed outcome. In a first application to experimental SERS data it has been demonstrated that this information is very useful to illuminate complex biological processes in cells like the interaction of antidepressants and lipids 35 .…”

Bio‐probing with nonresonant surface‐enhanced hyper‐Raman scattering excited at 1,550 nm

“…[ 13 ] Reporters can also be multifunctional; as important examples, drugs that are delivered by gold nanoparticles can be followed by their own SERS spectrum, [ 14 ] and their interaction with the biological environment can be monitored. [ 15 ]…”