Optical Nanosensing of Lipid Accumulation due to Enzyme Inhibition

Subscriber access provided by UNIV OF SOUTHERN INDIANA

Article

Optical Nanosensing of Lipid Accumulation due to Enzyme Inhibition in Live Cells Vesna Živanovi#, Stephan Seifert, Daniela Drescher, Petra Schrade, Stephan Werner, Peter Guttmann, Gergo Peter Szekeres, Sebastian Bachmann, Gerd Schneider, Christoph Arenz, and Janina Kneipp ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.9b04001 • Publication Date (Web): 17 Jul 2019 Downloaded from pubs.acs.org on July 18, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

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

Vesna Živanović†‡, Stephan Seifert§, Daniela Drescher†, Petra Schrade⊥, Stephan Werner∥, Peter Guttmann∥, Gergo Peter Szekeres†‡, Sebastian Bachmann⊥, Gerd Schneider∥, Christoph Arenz†‡, Janina Kneipp†‡*

†Department

of Chemistry, Humboldt-Universität zu Berlin, Brook-Taylor-Str. 2, 12489

Berlin, Germany

‡School

of Analytical Sciences Adlershof SALSA, Humboldt-Universität zu Berlin, Albert-

Einstein-Str. 5 -9, 12489 Berlin, Germany

§Institute

of Medical Informatics and Statistics, Kiel University, University Hospital

Schleswig-Holstein, 24105 Kiel, Germany

⊥Department

of Anatomy, Charité Universitätsmedizin Berlin, Berlin, Germany

ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

∥Helmholtz-Zentrum

Page 2 of 61

Berlin für Materialien und Energie, BESSY II, Albert-Einstein-Str. 15,

12489 Berlin, Germany

* To whom correspondence should be addressed: [email protected]

2 ACS Paragon Plus Environment

Page 3 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

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 surfaceenhanced 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, respectively. The vibrational spectra elucidate to a 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 (RF) based approaches, in particular by minimal depth (MD) 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


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 61

the identification of molecular co-localization 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.

Keywords

lipid accumulation, SERS, tricyclic antidepressants, random forest, gold nanoparticles, sphingomyelin, lipidosis

Lipids are a very important class of molecules in eukaryotic cells. Apart from being an important means of energy storage, they make up the extracellular membrane and all membranes that define the different cellular compartments, where they comprise the environment for membrane-bound and transmembrane proteins and for a large part of the enzyme machinery of a cell. The lipid metabolism of an animal cell is controlled by a body


Page 5 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

of highly specialized enzymes that act under defined conditions in the endolysosomal system. Impairment of proper enzyme function due to genetic defects1,

2

or due to an

inhibition of the enzymes by drugs leads to lipid storage disorders.3 For example, in Niemann-Pick disease type A and type B, the activity of the enzyme acid sphingomyelinase

(ASM)

that

degrades

sphingomyelin

into

ceramide

and

phosphorylcholine is dramatically reduced,4, 5 and as a consequence, large amounts of sphingomyelin accumulate in the lysosomes.6 ASM also takes part in lysosomal permeation and stability, and recent reports suggest that lysosomal ASM could be a potential target in cancer therapy.3, 7 Over 300 drugs can induce an accumulation of lipids in lysosomes,8 and therefore drug-induced lipidosis is a major concern in drug development.9, 10

So far, lysosomal lipid accumulation has been detected by transmission electron microscopy (TEM) in fixed cells. However, elucidating the composition and structure of the accumulated lipids as well as the cellular events connected to lipidosis with high specificity and without extensive labeling at the subcellular level of living cells is a major


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 61

challenge and has been achieved only indirectly so far, e.g., by modulation of the emission of label structures.11 Here, we study lipid accumulation directly in the lysosomes as a consequence of drug-induced enzyme inhibition in living cells in cell cultures using surface-enhanced Raman scattering (SERS). SERS provides detailed vibrational information on the molecular structure and composition in cells,12-14 as it makes use of the strongly increased Raman signals in the local optical fields of plasmonic nanostructures.15 Gold nanoparticles can reach the cellular interior by endocytosis, therefore, SERS can specifically report on the biochemical environment of such optical nanoprobes in the endolysosomal system.16-18 In our experiments, we study the influence of three different tricyclic antidepressants (TCA) on the activity of the lysosomal ASM. Based on previous work using model systems19 and indirect studies with labeled cells20-22 it has been proposed that in the presence of TCA, the interaction between ASM and its docking site at the lysosomal membrane is diminished, and ASM is rendered to rapid proteolytic degradation.19


Page 7 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

As will be discussed here, lipid composition and structure upon delivery of the TCA desipramine (Des), imipramine (Imi), and amitriptyline (Ami) can be characterized with SERS nanoprobes in our cellular model. The influence of each TCA is studied for its delivery before, after, and together with the SERS nanoprobes, respectively. Since the lipid SERS signals are not as high as in pure lipid models that were studied previously,14, 23, 24

random forest (RF)25, that is, machine learning based, approaches are applied. They

demonstrate to be particularly useful in the identification of the weaker lipid spectral signatures and their correlation with spectral contributions of the TCA and proteins in the lysosomes. The data show a high abundance of lipids when cultured cells are exposed to the TCA, depending on the conditions and duration of incubation of the cells with the drug molecules. More specifically, in accord with a lack of ASM when a TCA is present, an abundance of sphingomyelin is detected, since it cannot be degraded by enzyme molecules. The analyses of the SERS data with the RF based approach surrogate minimal depth (SMD)26 detail the interactions of the drugs and the lysosomal membranes. Ultrastructural data from TEM and cryo X-ray tomography (cryo-XT) substantiate the


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 61

accumulation of the lipids and reveal differences in the processing and localization of the gold nanoprobes due to the action of the antidepressants.

Results and discussion

TCA cause myelin figures and change the properties of gold nanoaggregates in the lysosome

In our experiments, the gold nanoparticles that serve as optical nanoprobes are taken up into the cells by endocytosis. In order to determine the most efficient way of observing the interactions and influence of TCA molecules in the cells by SERS, different sequences of incubation of the cell cultures with the gold nanoprobes and each TCA, were applied (Scheme 1).


Page 9 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Scheme 1. Representation of the incubation of the cells with the TCA and the gold nanoparticles that were used as SERS probes. (A) Cells were incubated with TCA molecules for 24 h prior to the incubation with gold nanoparticles for 6h. (B) Cells were incubated with gold nanoparticles for 6h prior to the incubation with TCA molecules for 24 h. (C) Cells were incubated with TCA molecules and gold nanoparticles for 24 h simultaneously.

In order to study the localization of the gold nanoparticles and the formation of nanoaggregate and the overall state of the cells after their incubation with gold nanoparticles and/or TCA, cryo X-ray tomography (cryo-XT) was employed for the 3D imaging of whole, intact cells in a nearly native state27 and offers the possibility to characterize in detail the behavior of intracellular nanoaggregates.28 Furthermore, due to 9 ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 61

the slightly higher spatial resolution, TEM imaging was used to verify potential lipid accumulation that is suggested to occur as consequence of drug action.29 In Figure 1A and 1B, TEM micrographs of cells that were incubated with Des for 24 h prior to the incubation with gold nanoparticles for 6 h (according to Scheme 1A) show several vesicles that contain multilamellar membranes, so-called myelin figures. The myelin figures are a clear evidence of lipid accumulation, and agree with observations made previously in other cells incubated with Des.29 When the gold nanoparticles have entered the cells after incubation with Des, they are associated with the myelin figures (Figure 1A and 1B). The formation of the myelin figures and their association with gold nanoparticles can also be seen after the simultaneous incubation of Des and gold nanoparticles for 6 h, as evidenced by cryo-XT (Figure 2C to 2F). X-ray images of cells incubated simultaneously with Des and gold nanoparticles for 24 h (according to Scheme 1C) shown in Figure S1 also show lipid accumulation and gold nanoparticle aggregates. In contrast, in the control samples no lipid accumulation is observed (Figure 1E and 1F and Figure 2A). Moreover, the gold nanoparticles form smaller aggregates in the control cells compared to the cells treated with Des (compare Figures 1E and 1F with Figures 1A to 1D and Figure 2A with 10 ACS Paragon Plus Environment

Page 11 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 2B to F). The segmentation images obtained after the reconstruction of tomograms from cryo-X ray micrographs (Figure 2A and 2B), as well as the slices of the reconstructed tomograms (Figure 2C to 2E, Movie S1) and projection image (Figure 2F) showed clearly that the formed nanoaggregates are larger and differ in morphology in the presence of Des (compare 2A and 2B). The presence of a significantly larger amount of lipids in the lysosomes of the Des-treated cells could also have an indirect influence on the formation and assembly of the gold nanoaggregates. Nevertheless, we have shown previously that also in the absence of cells, gold nanoparticles form aggregates in the presence of Des alone, and that these aggregates change only slightly when culture medium is added.30 Therefore, we conclude that different nanoparticle aggregate size and morphology in the Des-treated cells must be caused by Des itself. The slices of the tomographic reconstruction obtained from cells treated with gold nanoparticles for 6 h prior to the treatment with Des for 24 h (according to Scheme 1B) show no single nanoparticles in the cells, but small and large nanoaggregates (Figure 2C to 2F). Comparing the amount of nanoparticles in the reconstructed cryo-XT data as described previously in ref. 28, we find that the uptake of nanoparticles in the different incubation schemes is not affected (not 11 ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 61

detailed here), but their organization in the vesicles is. As we discussed in previous work, the arrangement of nanoparticles inside the endo-lysosomal system can greatly affect the SERS enhancement that is obtained from them.31 All nanoparticles are associated with the vesicular structures that appear darker compared to the rest of the vesicles. The darker vesicles are carbon or nitrogen-rich organelles32 and point towards an accumulation of lipids. Their association with the gold nanoparticles is in agreement with the observations made by TEM (Figure 1A to 1D) and shows that in principle probing by SERS nanosensors will enable us to study the lipid accumulation in more detail.


Page 13 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 1. TEM micrographs of 3T3 fibroblast cells incubated with: (A-B) desipramine for 24 h prior to the incubation with gold nanoparticles for 6 h, (C-D) desipramine and gold nanoparticles simultaneously for 6 h, and (E-F) gold nanoparticles for 6 h (controls). Scale bars: A, B, E, F, 100 nm; C, and D, 250 nm.

Figure 2. Gold nanoparticles visualized after tomogram segmentation in cells treated with: (A) gold nanoparticles for 6 h, and (B) gold nanoparticles and desipramine simultaneously for 6 h. (C-F) Slices of tomographic reconstructions of cryo-X ray images obtained from cells treated with gold nanoparticles for 6 h prior to treatment with desipramine for 24 h. Scale bars: 1 µm.


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 61

The average SERS spectra vary for different incubation conditions

While the TCAs are expected to be mainly entrapped in the lysosomes,20, 33 a continuous exposure of the cells to gold nanoparticles directly before the acquisition of the SERS spectra, i.e., following the protocols shown in Scheme 1A and 1C, leads to a distribution of gold nanoparticles in all vesicles of the endocytic pathway, in accord with previous observations.31 This is evidenced by the TEM micrographs and cryo-XT data (Figure 1, Figure 2, Figure S1), where the vesicles that are observed range from the early endosomal to the lysosomal stage, since the cells take up nanoparticles continuously over a course of 6 h (Figure 1, Figure 2) and 24 h (Figure S1), respectively, and some of them had enough time to reach lysosomes, while others may have been just taken up and therefore reside in early endosomes. When the sequence is reversed, and the cells are first incubated with gold nanoparticles and then with the antidepressant (following Scheme 1B), most of the nanoparticles must be located in lysosomes, rendering the SERS probing more selective for those vesicles where the accumulation of the lipids is expected to occur.


Page 15 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 3 shows average SERS spectra of cells treated with Ami (blue traces), Des (red traces), and Imi (black traces) for the different incubation conditions as denoted, together with spectra of control cells that were treated only with gold nanoparticles according to each respective protocol (green traces). The spectra of the cells treated with TCA vary for the different incubation conditions (compare Figures 3A, 3B, and 3C). Nevertheless, for TCA delivery prior to incubation of gold nanoparticles (according to Scheme 1A) and for simultaneous delivery of TCAs and gold nanoparticles (according to Scheme 1C), the average SERS spectra of individual cells treated with the three different antidepressants exhibit a high degree of similarity (compare black, red and blue traces both of the panels 3A and 3C). This similarity in the spectra, suggesting a similarity in the molecular composition of the endolysosomal compartment, would be in agreement with the similar effects of the three antidepressant molecules and the activity of ASM in the lysosomes. The spectra of the TCA-treated cells differ from the average spectra of the control cells (green traces in all panels of Figure 3), in particular in the cases of separate delivery of gold nanoparticles and TCA. Moreover, as exemplified by the cells chosen here, the average SERS spectra of different cells under the same respective TCA exposure and 15 ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 61

incubation are similar, indicating very low cell-to-cell variability (compare spectra of the same color in Figure 3A and 3C), although greater differences between average spectra of individual cells as well as between incubation with different antidepressants are found for incubation scheme B (Figure 3B).

Figure 3. Averages of SERS spectra extracted from the mapping data of cells treated with imipramine (Imi, black traces), desipramine (Des, red traces), amitriptyline (Ami, blue traces) (A) for 24 h prior to 6 h treatment with gold nanoparticles (cf. Scheme 1A), and,


Page 17 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

(B) for 24 h after incubation with gold nanoparticles for 6 h (cf. Scheme 1B) (green trace), (C) for 24 h simultaneously to an incubation with gold nanoparticles (cf. Scheme 1C). The green traces represent averages from control cells treated with (A) gold nanoparticles for 6 h, (B) gold nanoparticles for 6 h followed by chase period in culture media for 24 h, and (C) gold nanoparticles for 24 h. Each spectrum is an average of all SERS spectra measured in one individual cell. Examples of two cells per incubation condition (one per control) were chosen randomly. Excitation wavelength: 785 nm, acquisition time for the single spectra: 1 s, excitation intensity: 2 × 105 W cm–2. All scale bars: 20 cps.

When cells are incubated with antidepressants for 24 h prior to incubation with gold nanoparticles for 6 h (Figure 3A), the TCA-treated and control samples display a number of spectral differences. Several bands around 1000 cm-1, mainly assigned to phenylalanine and tyrosine34 (for assignments see Table S1) contribute more to the SERS spectra of treated cells. This suggests that proteins and/or protein fragments are abundant in the endolysosomes of the TCA-treated cells. Treated and non-treated cells also share many bands assigned to protein vibrations, e.g., around 500, 650 and 830 cm-1.35, 36 The


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 61

band around 1310 cm-1, assigned to the CH3/CH2 twisting mode of lipids,34, 37 changes its relative intensity compared to the band around 1350 cm-1, which is attributed to a CH deformation vibration mainly from proteins (Figure 3A).34 The intensification of signals assigned to lipids suggests their abundance in the endolysosomal vesicles when the cells are exposed to any of the three TCA. Further spectral contributions from lipids include the band at 415 cm-1, assigned to sterols14, 38 and the signal of a vibration around 1130 cm-1, due to the C–C stretching modes of long lipid chains,37 proving a strong contribution of lipids to the SERS spectra of the TCA-treated cells. In contrast, the strong deformation mode of the CH2 group of lipid molecules around 1440 cm-1 decreases in relative intensity in the spectra of the treated cells. Even though a high concentration of TCA molecules is expected in the lysosomes after exposure to the drugs for 24 h, the characteristic SERS signature as a whole of the respective TCA molecules30 was not found in the spectra of the antidepressant-treated cells. Only some vibrations that are particularly strong, e.g., of the rings of the TCA molecules can be identified, in particular at 1030 cm-1 (Figure 3A). Although we observed a strong interaction of Ami, Des and Imi with gold nanoparticles outside cells,30 the data here suggest that due to the hydrophobic nature of the TCA 18 ACS Paragon Plus Environment

Page 19 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

molecules and their interaction with the lysosomal membrane, the interaction with the gold nanoparticles is diminished, and only some vibrational modes can be observed.

SERS spectra of cells treated with gold nanoparticles for 6 h prior to the treatment with the respective antidepressant for 24 h (Figure 3B) show several additional modes, e.g., at 680 cm-1, 725 cm-1, 811 cm-1, 1202 cm-1, 1225 cm-1, and 1530 cm-1 compared to the average spectra of the control sample (Figure 3B, green trace) and the samples that are exposed to the TCA molecules prior to the gold nanoparticles (Figure 3A). Some of them are assigned to vibrations of the antidepressant molecules, e.g., at 1202 cm-1 and 1530 cm-1.30 Moreover, also here, significant contributions come from lipid molecules, e.g., from the C–C stretching of the acyl chains at 1090 cm-1 and1140 cm-1, and the CH2 deformation vibrations of the acyl chains around 1440 cm-1.37, 39, 40 A band that can be assigned to the choline part of the polar head groups around 725 cm-1 is present in all spectra (Figure 3B). Considering the relatively low cross-sections of the typical vibrations of lipid molecules compared to those of proteins,41 the strong contribution of lipid vibrational modes to the spectra implies a high local concentration of lipids in the gold nanoparticle containing


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 61

vesicles. The contributions of the vibrational bands assigned to the TCA differ for the individual antidepressants (compare black, red and blue traces in Figure 3B). As an example, the band around 790 cm-1 can be seen in the spectra of cells treated with Des and Ami, but not in the spectra of Imi-treated cells, while the signal at 890 cm-1 is present in the Imi and Des-treated cells, but not in Ami-treated cells. The presence of very characteristic lipid signatures in the average SERS spectra evidences a high abundance of the lipids in the close proximity of the gold nanoparticles. Furthermore, the almost exclusive probing of lysosomes (as discussed above) lowers variability in the SERS spectra due to the absence of earlier-stage vesicles, and the contribution of the TCA molecules becomes more pronounced, enabling observation of differences in structure and interaction of the three different TCA in the SERS spectra.

When the cells are treated with gold nanoparticles and antidepressants simultaneously for 24 h, their average SERS spectra do not differ much from those of the respective control samples (Figure 3C). Differences between the SERS spectra of the treated and control cells (compare black, blue, and red traces with green trace) are seen in the relative


Page 21 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

intensity of the band around 500 cm-1 and are assigned to S–S stretching from the disulfide bridges and to other signals from proteins. The SERS signatures of the respective TCA in the cells are not pronounced (Figure 3C), although it is known that during the delivery in the cell culture medium, the interactions between the gold nanoparticles and the TCA molecules are still unaltered.30 In principle, the SERS fingerprints of Ami, Des, and Imi could be expected to be preserved inside the cell as well in the case of co-delivery, similar to other molecules in live cell SERS.13 An explanation for the absence of TCA signals here could be the strong affinity of the TCA molecules for the lysosomal membrane and lipids,42 and their separation from the nanoparticles as soon as they come into the proximity of the membranes.

The high sensitivity of SERS in the dynamic and complex environment of the druginfluenced endolysosomal compartment leads to fingerprint-like spectral data. Although the average spectra, combining the molecular information from all different spots and endolysosomal structures of a particular cell are very reproducible for a particular incubation and TCA (Figure 3), all individual spectra must be analyzed in order to observe


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 61

a particular molecular composition in defined subcellular locations. Different signal strengths observed for the different molecular species complicate the extraction of clear spectral signatures from the SERS data further. In order to learn about the influence of the antidepressants and their localization and interactions in the live cells from the SERS spectra, random forest (RF) analysis was applied to all individual spectra. In order to verify that RF analysis is capable of the differentiation, the data were classified according to their origin from treated cells and control samples, respectively, and classification shows high sensitivity and specificity for all incubation conditions (Table S2). Subsequently, minimal depth (MD) variable selection43 and surrogate minimal depth (SMD)26 are applied here to analyze particular spectral variables regarding their importance, as basis for a distinction of the different groups of spectra, and regarding their relations, respectively. The analyses of the importance and of the relations of the spectral bands give information about the molecular groups and their interactions, respectively, that are relevant for the distinction between TCA-treated and control samples. Thereby, the molecular effects of exposure of the cells to TCA can be identified and localized.


Page 23 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Molecular characterization after nanoparticle application to TCA treated cells

The bands that are selected by MD as important variables for SERS spectra of cells treated with a TCA for 24 h prior to treatment with gold nanoparticles for 6 h (according to Scheme 1A) are shown in Table 1 for all three TCA. Each variable is considered a spectral feature in the SERS spectrum, and all variables can be assigned to a specific vibration or particular molecule (for tentative assignments see Table S1). The important bands can be assigned to the respective antidepressant, to lipids and to proteins. They are in very good agreement with the bands that frequently appear in the SERS spectra of cells (compare Table 1 to Figure 3A). Nevertheless, also bands that do not have a particularly high intensity in the average spectra are selected, e.g., at 818 cm-1, 1018 cm-1, 1061 cm-1, and 1450 cm-1. These bands appear almost exclusively due to the different vibrations of lipid molecules. Their presence in the several hundreds of individual spectra clearly provides evidence of a lipid rich environment in the lysosomes of the live cells treated with TCA and confirms the observations made by TEM and cryo-XT of the fixed cells.


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 61

Table 1. Important bands that were selected by minimal depth applied to the SERS spectra of cells treated with antidepressants for 24 h prior to 6 h incubation with gold nanoparticles. The bands are assigned to the respective antidepressant, lipids and proteins. Ami Ami

961

1028

1537

Lipids

746

1018

1061

505

833

1130

Des

780

871

1034

1538

Lipids

818

1013

1049

1457

505

1505

1610

Imi

1032

1532

Lipids

1014

1151

1459

505

1125

1548

Protein s

1381

1458

Des

Protein s Imi

Protein s

1487 1607

Despite the high similarities in the average SERS spectra of cells treated with different antidepressants (compare blue, red, and black traces in Figure 3A), differences in the effects of exposure to the three different antidepressant molecules are found by the analysis of the individual spectra, as indicated by the important bands in Table 1. These differences are mostly found for the vibrations of lipids, e.g., at 746 cm-1, ~1018 cm-1, 1061 cm-1, and 1381 cm-1 that are selected only in Ami-treated cells, while the band at 818 cm-1 is exclusively selected for Des-treated cells. In studies of lipid models, we have 24 ACS Paragon Plus Environment

Page 25 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

shown previously that the bands at 818 cm-1 and 1018 cm-1 are characteristic of liposomes containing only sphingomyelin or liposomes containing sphingomyelin together with other lipids.23 The band at 1018 cm-1 is the strongest band in the spectrum of sphingomyelin when it interacts with gold nanoparticles and is assigned to a C–N stretching vibration of the choline part of the lipid polar heads.23 The selection of the sphingomyelin bands suggests that the content of sphingomyelin in the vesicles of the treated cells is important for the separation of treated cells and controls. We conclude that the sphingomyelin must accumulate in the TCA-treated cells, which would be in agreement with the known lack of the ASM enzyme that is induced by the TCA.20, 21

The results show that selected bands also can be assigned to the TCA molecules themselves (Table 1), and that the important bands for the experiments with the different antidepressants partly overlap. The TCA bands at 1030 cm-1 and 1537 cm-1 are selected for the separation of cells incubated with each of the three TCA. In the absence of cells, the band at 1030 cm-1 is the strongest band in the SERS spectrum of the antidepressant molecules,30 and the signal strength of the band at 1537 cm-1 was found to change with


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 61

pH.30 In addition, in the Des-treated cells, the bands at 780 cm-1 and 870 cm-1, assigned to different vibrations of the rings,30 are identified, confirming accumulation of the TCA in the lysosomes.29

In order to learn more about the molecular interactions and co-localization that take place in the treated cells, we investigated the relations of the selected variables utilizing SMD. SMD was developed recently to analyze the relations of variables including their mutual causality and to improve variable selection by including variable relationships in the selection process.26 Here, SMD was applied to learn which other bands co-occur with the important bands in the SERS spectra. Figure 4A shows as examples the bands related to several important bands from cells incubated with Des and Imi according to Scheme 1A. Supporting information Table S3, Table S4, and Table S5 contain the relations of all important bands for the experiments with all three antidepressants using Scheme 1A, sorted according to the assignment of the important bands to the TCA, to lipids, and to proteins, respectively. The important bands assigned to the drug molecules differ in the Des and Imi-treated cells. However, all bands of the TCA molecules that are selected as


Page 27 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

important have related bands assigned to the respective TCA as well (Figure 4A, red labels). This is in full agreement with the detection of several different vibrational modes of the same molecular species, a known hallmark of any vibrational spectroscopy, including SERS. All the related TCA bands can be associated with different ring modes of the TCA molecules.30 More interestingly, the band of Des at 780 cm-1 is related to vibrational modes assigned to lipids at 720 cm-1and 1273 cm-1 23 (Figure 4A, yellow labels in first row). The band around 720 cm-1 is assigned to the C–N stretching of the choline part of phospholipids23, 44, while the band at 1273 cm-1 is assigned to a =CH deformation of the lipid chain.23 In Figure S2A, single spectra from cells treated with Des are shown as examples that contain some of the important and related bands, e.g., 505 cm-1, 818 cm-1, ~1130 cm-1, ~1450 cm-1, as discussed above. The relations between the modes of the TCA and the lipid bands indicate that both species must be present in the small probed volumes, and possibly interact in the lysosomes. An interaction between antidepressants and lysosomal membranes can be based on electrostatic interactions of the negatively charged lipids in the inner lysosomal membrane with the positively charged amino group of the drug.19 However, also the ring part of the TCA molecules could interact with the 27 ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 61

hydrophobic part of the lysosomal membrane.42 The correlation of bands related to the TCA (780 cm-1, 960 cm-1, 1030 cm-1), the CH2 deformation (1446 cm-1, 1460 cm-1, 1480 cm-1), and the C–C stretching vibrations (1055 cm-1, 1135 cm-1) of the hydrophobic part of the membrane (see Table S3 for the full list of related lipid bands) supports such an interaction here as well.


Page 29 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 61

Figure 4. Bands related to the important bands (left) selected by SMD analysis of the SERS spectra from cells treated with desipramine and imipramine, respectively, for 24 h (A) prior to 6 h incubation with gold nanoparticles (cf. Scheme 1A), (B) after 6 h incubation with gold nanoparticles, and (C) simultaneously with gold nanoparticles for 24 h (cf. Scheme 1C). Variables (bands) assigned to antidepressants are marked in red, to lipids in yellow, and to proteins in green. Supporting information tables S3 to S11 show the complete relations for important bands from all classes of molecules with all three TCA.

The selected bands that are assigned to sphingomyelin at 818 cm-1 and 1018 cm-1

23

discussed above, appear in Des- and Imi-treated cells, and both are related to similar bands of lipids and, moreover, to proteins. In the examples of single SERS spectra from Imi-treated cells (Figure S2B), the band at 1018 cm-1 appears together with related lipid bands. The relation of lipid bands with each other confirms that they originate from vesicles with a high local concentration of lipids. The influence of the protein bands in the separation of the spectra from TCA-treated and control cells suggest that in the lipid-rich environment, the composition and interactions of proteins is significantly changed.


Page 31 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Molecular characterization after TCA treatment of nanoparticle containing cells

RF analyses was also carried out for the data sets obtained after an incubation of the cells with the TCA molecules according to Scheme 1B, comprising an incubation with gold nanoparticles for 6h prior to treatment with the respective TCA molecule. The important bands are shown in Table 2. The important bands that are assigned to the TCA display overlap with the selection for the reverse sequence of drug/nanoparticle incubation (compare with Table 1). However, here, the important bands vary for cells treated with the three different molecules. Several characteristic bands of the antidepressant molecules are selected in the classification of the Ami-treated cells and controls, while in the case of the Des and Imi treated cells, only a few bands are selected (Table 2). These data indicate a different interaction of Ami with gold nanoparticles when compared to Imi and Des, also known from SERS spectra in the absence of cells.30


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 61

Table 2. Important bands that were selected by minimal depth applied to the SERS spectra of cells treated with gold nanoparticles for 6 h, prior to 24 h treatment with antidepressants. The bands are assigned to the respective antidepressant, lipids and proteins.

Ami Ami

620

691

783

869

960

1028

Lipids

540

610

886

721

1082

1090

1138

1165

640

669

829

853

1253

1607

1618

1340

Des

1036

1533

Lipids

750

1277

1325

1380

Protein s

1180

1276

1420

Des

Protein s

837

1237

1505

1432

1437

1458

1468

1477

1554

Imi Imi

1028

1111

Lipids

1093

1087

1098

The differences in the important bands assigned to lipids and proteins point toward a different influence of each of the antidepressant molecules on the molecular composition and interactions in the lysosomes. As examples, the number of lipid and protein bands is particularly high when the cells are incubated with Ami (Table 2, first rows), while none of the important bands that distinguish the spectra from the Imi-treated cells is assigned to proteins at all (Table 2, last rows). It is known that the activity of different antidepressants is dose dependent, and Ami shows a higher activity in cells compared to Des and Imi.3, 45 The fewer spectral features, pointing out fewer biochemical changes induced by the 32 ACS Paragon Plus Environment

Page 33 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

respective drug (at the same concentration) are in agreement with a lower activity of Des and Imi compared to Ami.3, 45 The important bands that are assigned to lipids in the Ami and Imi-treated cells are mainly due to C–C stretching modes. In addition, in Ami-treated cells, bands assigned to the vibrations of the polar head at 721 cm-1 and 1180 cm-1 are selected. In the classification of data from the Des-treated cells, the CH2 deformation vibrations of the lipid chains have a significant influence. It has been proposed that the aromatic ring system of the TCA intercalates between the acyl chains, and the polar part of the TCA is located close to the polar head of the lipids.42 The selection of bands assigned to the Des rings and to the acyl chains implies that exactly these parts of the molecules undergo an interaction, and that the proposed intercalation indeed occurs when Des is present in the lysosomes. Interestingly, almost all lipid bands selected as important in the Ami treated cells (Table 2) were found in the SERS spectra of a liposomenanoparticles model system consisting of phosphatidylcholine, sphingomyelin, and cholesterol.23 We infer that in addition to sphingomyelin, also other lipid molecules are present in the lysosomal lipid accumulations.


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 61

Similarly as discussed for the incubation experiments discussed before, lipid signals were selected here as bands that are related to the important bands. Figure 4B shows examples of such related bands (for complete tables showing the relations for all classes of molecules with all three TCA cf. Table S6, Table S7, and Table S8), in addition, examples of SERS spectra of Des- and Imi-treated cells are shown in Figure S3. Important bands assigned to lipid vibrations are those of the CH2 deformation modes at 1433 cm-1, 1440 cm-1, and 1475 cm-1, and they are related to other vibrations of the lipid chains, e.g., the C–C stretching mode around 1086 cm-1 (Figure S3). The appearance of the CH2 deformation modes at slightly different Raman shifts can be caused by a different packing of the lipids in the lysosomes, but it can also indicate different types of interaction with gold nanoparticles.23 In the case of the Imi-treated cells, several C–C stretching modes of the lipid chains (1086, 1096 cm-1) are selected as important and are also found in the examples of single spectra (Figure S3). The band at 1086 cm-1 is related only to other vibrational modes of lipids (1170 cm-1, 1191 cm-1, and 1415 cm-1 , see also Figure S3B, bottom trace).


Page 35 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

The selection of important bands was used as a basis for chemical mapping, and allowed an assessment of the spatial distribution of those lysosomes from which the respective SERS signals originate. Figure 5 shows the distribution of important bands in the maps of cells exposed to Ami following incubation according to Scheme 1B. Since the selection of the bands is motivated by the distinction between TCA-treated and non-treated cells, the maps can selectively show the molecular changes related to the influence of the TCA. This is different from previous mapping of SERS signals from lipids and other molecular species in a disease model14 and also allows co-localization of drug-induced molecular differences in the lysosomes.

Figure 5. Distribution of bands selected as important variables in random forest analysis in a cell treated with gold nanoparticles for 6 h prior to treatment with amitriptyline for 24


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 61

h, representing lipids (green) and antidepressants (red) species together with bright field images of the cell. The chemical images are generated by mapping the intensity of the bands at 620 cm-1, 780 cm-1, 960 cm-1 and 1030 cm-1 assigned to amitriptyline, 1140 cm1

assigned to lipid alkyl chains, 1280 cm-1 and 1458 cm-1 to the CH2 deformation vibration

in lipids, and the overall intensity obtained from the full spectral range of 300 - 1800 cm-1 to reveal the distribution of the SERS probes in the cell. Scale bars: 5 µm.

Molecular characterization after simultaneous incubation with TCA and nanoparticles

The RF classification of SERS spectra obtained from cells treated with antidepressants and gold nanoparticles simultaneously (according to Scheme 1C) was achieved with high sensitivity (Table S2), even though the differences observed in the average spectra of individual cells treated with the different TCA (Figure 3C) were not pronounced. Common important bands included those assigned to vibrations of all three antidepressants around 380 cm-1, 1037 cm-1, and 1205 cm-1 (Table 3). The band at 380 cm-1 is assigned to the N–C modes from rings and aliphatic parts of the TCA,46 the mode around 1037 cm-1 is also assigned to the aliphatic part of the antidepressants,30 and the vibration at 1205 cm36 ACS Paragon Plus Environment

Page 37 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

1

is due to modes of the ring moiety.30 Several important bands assigned to the TCA

overlap between the different sequences. Exceptions are the bands at 380 cm-1, 446 cm1,

591 cm-1, and 1205 cm-1, that are selected only in the case of simultaneous delivery of

the drugs and the gold nanoprobes. The reason for the appearance of new antidepressant modes is probably a different interaction of the TCA with the gold nanoparticles when they are co-delivered.

Table 3. Important bands that were selected by minimal depth applied to the SERS spectra of cells treated with antidepressants and gold nanoparticles for 24 h. The bands are assigned to the respective antidepressant, lipids and proteins. Ami Ami

384

868

1037

1205

Lipids

532

1060

1084

1151

1167

Proteins

826

933

948

1101

1213

1511

1561

Des

446

1037

1205

Lipids

1061

1082

1090

1160

Proteins

1007

1591

Imi

384

591

1037

1111

1205

1207

1544

Lipids

410

532

919

940

952

1060

1084

Proteins

948

1248

1516

1560

1591

1576

Des

Imi

1095

116

117

146

148

7

8

3

8

Similar bands assigned to lipids are selected for Ami and Des-treated cells (Table 3) and mainly belong to C–C stretching vibrations. In contrast, in the Imi-treated cells the list of


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 61

important bands also contains those of the CH2 deformation vibrations. It should be noted that although most of the bands represent typical vibrations of phospho- and sphingolipids, in the Des treated cells, the bands around 410 cm-1 and 532 cm-1, and in Ami treated cells the band at around 532 cm-1 are assigned to cholesterol39 (Table 3). This suggests that cholesterol must be present in the lysosomes containing the nanoprobes. The appearance of cholesterol bands in the spectra is in accordance with a previous study that discusses the possibility of a cholesterol accumulation due to an inhibition of ASM.3 The contribution of selected bands that are assigned to proteins is major in the case of Imi and Ami-treated cells, while in Des-treated cells only two protein bands are selected.

Figure 4C shows examples of signals that are related to the important bands from the TCA and lipid vibrations in the Des and Imi treated cells. The relations for all classes of molecules with all three TCA for the simultaneous incubation (according to Scheme 1C) are shown in Table S9, Table S10, and Table S11. As an example, in Des-treated cells, the C–C stretching vibration of the aliphatic chain of Des at 1037 cm-1 is related to the vibrations of the lipid polar head around 720 cm-1, and the C–C stretching vibration of the


Page 39 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

lipids at 1090 cm-1 is related to the vibrations of the rings of Des (1200 cm-1) (Figure 4C), both in accordance with an interaction of the TCA molecules and lipids.42 Similarly, in the Imi-treated cells, the C–C stretching modes of the lipids at 1037 cm-1 are related to ring modes of Imi at 685 and 965 cm-1 (Figure 4C). Also the CH2 deformation vibrations of the lipid chains around 1460 cm-1 are related to the ring modes of Imi, in particular to the bands around 770 cm-1 and 1537 cm-1. Examples of single SERS spectra from cells treated with the different antidepressants do not show a high variability (Figure S4), in agreement with the observations made for the average spectra (Figure 3C). The relation of the TCA with lipid bands is similar to those in the other incubation conditions (compare Figure 4C with 4A and 4B). However, the relatively strong bands of the CH2 deformation modes that appear in the SERS spectra (Figure S4) are specific and probably due to the more hydrophobic surface of the gold nanoparticles when they are co-delivered with the TCA. Based on these SERS data, as well as on the properties of the nanoaggregates in the cells, the simultaneous delivery of nanoparticles and antidepressants differs greatly from the other two incubation sequences. A possible reason for this could be different properties of the nanoparticle aggregates and the altered surface properties of the 39 ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 61

nanoparticles in the presence of the antidepressants. Nevertheless, the RF analyses, supported by the example spectra indicate a lipid-rich environment, and confirm the initial observation of lysosomal lipid accumulations.

Conclusions

The SERS data from live fibroblast cells indicate changes in the biochemical composition of the lysosomes in cell samples that are treated with one of the three antidepressants Ami, Des, and Imi. This is independent of the sequence of incubation of the cell cultures with the drugs and the SERS nanoprobes. Ultrastructural data from TEM and X-ray nanotomography show an enrichment of lipids in the TCA treated cells compared to untreated controls, and verify that the presence of the drug molecules leads to the accumulation of lipids in the lysosomes of the living cells. Average spectra from each individual cells, show some contributions from vibrational modes of lipids, as well as the intense bands of the respective TCA molecules and support this general observation of lipid accumulation.


Page 41 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

The analysis of all individual spectra by the random forest based approaches minimal depth (MD) and surrogate minimal depth (SMD) reveals the biochemical changes due to drug action for a particular incubation sequence and TCA to much greater detail, and with very high sensitivity. As was shown here, the selection of important bands that distinguish drug-treated from control lysosomes is feasible regardless of the varying signal strength of different molecular species and the lower signals of the lipids in the individual spectra, and was used for their spatial localization in chemical maps. In cells incubated with TCA prior to exposure to the SERS nanoprobes, it shows that bands assigned to antidepressants and lipids are important for the distinction between treated cells and controls. More specifically, the spectral data suggest the accumulation of the lipid sphingomyelin, in agreement with a drug-induced lack of ASM.20-22 The SMD analysis that identifies bands that are related to signals from the TCA molecules confirms a previous proposition19 that the TCA interact with the lysosomal membrane, probably via electrostatic interactions. The spectra also indicate that the composition and interactions of proteins is significantly changed in cells treated with the drugs.


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 61

The most informative results are obtained from the cells incubated first with nanoparticles and then with antidepressants. There, almost exclusive probing of lysosomal structures, the target of the TCA, is ensured. The analyses reveal clearly a different influence of the three different TCA molecules on the molecular composition and interactions, in agreement with their different activity that has been reported.3,

45

Furthermore, as

illustrated by the verification of the proposed42 intercalation of the aromatic rings in the Des molecule with the acyl chains of the lipids, the in vivo SERS data from the lysosomes enable us to better understand the interaction of the TCA molecules with lipid membranes in the cellular model. The SERS nanoprobes also show that in addition to sphingomyelin other lipids are accumulated. In particular, cholesterol accumulation is observed, in agreement with previous discussions.3 When the cells are treated with antidepressants and gold nanoparticles simultaneously, the gold nanoparticles form aggregates in the presence of the drug molecules,30 thereby slightly altering the conditions for the optical probing. Nevertheless, the SERS data confirm the lipids and TCA spectral contributions as main discriminators between TCA treated and control cells. At the same time, this illustrates the robustness of the probing approach that we have used here. 42 ACS Paragon Plus Environment

Page 43 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

The successful application of RF based machine learning on the SERS data demonstrates that also cellular constituents with comparably weak spectral contributions can be used for reliable spectral classification and imaging. From the methodological perspective, the analysis of bands that are related to a specific discriminator (here: the important bands identified by the MD approach) is particularly useful in harnessing spectra from mixtures of different molecular species and interacting components, and in samples that are heterogeneous at the microscopic level. In this regard, the utilization of RF analysis in live cell SERS is an important step towards several applications of the approach in cellular biochemistry.

Methods

Materials

Desipramine hydrochloride, imipramine hydrochloride, amitriptyline hydrochloride, and gold(III) chloride hydrate were purchased from Sigma-Aldrich. Trisodium citrate dihydrate was obtained from Th. Geyer. 3T3 mouse fibroblast cells were purchased from DSMZ, Braunschweig, Germany. Fetal calf serum (FCS) and Dulbecco’s Modified Eagle Medium 43 ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 61

(DMEM), cell shield, PBS buffer, PBS buffer without Ca and Mg salts, trypsin/EDTA solution, DMSO were purchased from Biochrom. Epon for TEM preparation was purchased from SERVA, Heidelberg, Germany.

Gold nanoparticle synthesis

Gold nanoparticles were obtained by the reduction of gold(III) chloride hydrate with sodium citrate according to the protocol reported in ref

47.

Briefly, gold(III) chloride hydrate is

dissolved in Millipore water and boiled at 80 °C. This step was followed by addition of sodium citrate. The gold nanoparticles have an average size of 30 nm and a concentration of 10–10 M.

Cell culture and incubation with TCA and gold nanoprobes

3T3 mouse fibroblast cells were seeded at a density of 2.5 × 103 to 4.0 × 103 cells/cm2 and grown as monolayers in a cell culture flask (75 cm2) in DMEM supplemented with 10 % FBS and 1 % of Zell Shield. After 2-3 days, the cells were sub-cultured. For SERS experiments, the cells were seeded on sterilized glass slides (1.5 cm × 1.5 cm) in 6-well


Page 45 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

plates at a density of 2.5 × 102 to 4.0 × 102 cells/cm2. The nanoparticles were mixed with the culture medium (DMEM with 10 % FBS) in a 1:9 molar ratio and incubated for several different periods as shown in Scheme 1. In the first experiment, the cells were first incubated with the respective antidepressant for 24 h and then with the gold nanoparticles for 6 h (Scheme 1A). In the second experiment, this sequence was reversed, and the cells were incubated first with nanoparticles for 6 h and then with the respective antidepressant for 24 h (Scheme 1B). In the third experiment, the cells were incubated simultaneously with the respective antidepressant and gold nanoparticles for 24 h (Scheme 1C). The controls consisted of cells incubated with nanoparticles for the same time period as in each experiment with the TCA, and the incubation with culture medium containing TCAs was replaced by an incubation in standard culture medium without TCA.

Incubation of antidepressants, separately and together with gold nanoparticles was done in the cell culture medium. Solutions of desipramine hydrochloride, imipramine hydrochloride, and amitriptyline hydrochloride were prepared at a concentration of 1 × 10−5 M using sterilized MilliQ water and added to cell culture medium. When incubated


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 46 of 61

with gold nanoparticles together, the antidepressants were mixed with the gold nanoparticles prior to adding them to the culture medium. The final concentration of TCA molecules was 1 × 10-6 M.

For X-ray microscopy, the cells were grown as a monolayer on Formvar-coated grids (HZB2 R2/2, Quantifoil, Grossloebichau, Germany) under standard conditions for 24 h. After 24 h, the cells were incubated with gold nanoparticles for 6 h as control samples, and according to sequence in scheme 1B and 1C.

Transmission electron microscopy of cells

For TEM imaging, the cells were fixed using 2.5% glutaraldehyde in cacodylate buffer. The fixed cell suspension was embedded in agarose. Further steps included drainage by an ascending alcohol series. The samples were further immersed in propylene oxide as an intermediate medium (2 × 15 min) and a mixture of propylene oxide and Epon in the ratio 2:1 for 1 hour, 1:1 for 1 hour, and 1:2 for 1 hour, followed by overnight immersion with pure Epon. Semi-thin microtome (Leica, Bensheim, Germany) sections, 0.5 μm to 1 μm thick, were prepared. The sections were further stained with Richardson staining 46 ACS Paragon Plus Environment

Page 47 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

solution, and ultrathin sections of 70 nm were cut with an Ultracut S (Leica, Bensheim, Germany). Sections were deposited on copper grids and stained with uranyl acetate and lead citrate. Images of the sections were obtained using an EM 906 electron microscope (Zeiss, Oberkochen, Germany) operated at 80 kV.

Cryo X-ray tomography experiments

For vitrification, the Formvar-coated grids with the cells were washed with PBS buffer. Excess of PBS buffer was blotted with filter paper, followed by plunge-freezing in liquid ethane. X-ray microscopy was performed at beamline U41-XM equipped with a cryo-stage at the electron storage ring BESSY II (Helmholtz-Zentrum Berlin für Materialien und Energie, Berlin) at a photon energy of 510 eV. Tilt series of up to 131 images were acquired from individual cells at different angles in increments of 1° at a pixel size of 9.8 nm (25 nm zone plate objective). Depending on the sample thickness, the exposure time was adjusted for each tilt angle series and varied between 1 s and 4 s.

The projection images were first pre-processed by flat-field correction (average from 10 flat-field images obtained under the same experimental conditions) and then normalized 47 ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 48 of 61

in order to correct for different beam current and longer exposure times at higher tilt angles. Tomograms were obtained by alignment of the corrected tilt series and tomographic reconstruction using the software eTomo IMOD,(Colorado, USA). The intracellular gold nanoparticles were used as fiducial markers to align the images of a tilt series. Reconstruction of the sample volume was carried out by backprojection. For improved contrast, some tomograms were binned by a factor of two, no binning was applied to projection images. To generate 3D views of the particle distribution in cells, surface rendering of the particles and their aggregates was performed using Amira software (Thermo Fisher Scientific, Oregon, USA) on the basis of the reconstructed data.

SERS experiments

After the incubation with the gold nanoparticles and the respective TCA, the culture medium was removed, the cells were thoroughly washed with PBS buffer, and SERS spectra were obtained. The single-stage spectrograph (Horiba, Munich, Germany) was equipped with a CCD detector and a diode laser (Toptica, Munich, Germany) operating at 785 nm and an intensity of 2.0 × 105 W/cm2. Use of a 60× water immersion objective 48 ACS Paragon Plus Environment

Page 49 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

(Olympus, Hamburg, Germany) resulted in a laser spot size of 1 μm. Raman microspectra were recorded from 300 cm−1 to 1900 cm−1 at a resolution of ∼5–8 cm−1 considering the whole spectral range in raster scans on the sample, with an acquisition time of 1 s per spectrum. The spectra were frequency calibrated using a spectrum of a tolueneacetonitrile mixture (1:1).

Random forest analysis of SERS spectra

Random forest (RF) analyses43 were applied to the SERS mapping data from cells treated with the antidepressants and their respective controls for each of the three incubation conditions. Data pre-processing included de-spiking, interpolation in the range from 351.2 cm-1 to 1900 cm-1 with a distance of 1.6 cm-1, resulting in 969 spectral variables p, and vector-normalization. Furthermore, we utilized the package ranger48 in R software to obtain

RF

classification

models

and

the

package

SMD

(https://github.com/StephanSeifert/SurrogateMinimalDepth) to select important and related variables with minimal depth (MD) variable importance and the mean adjusted agreement of surrogate minimal depth (SMD), respectively.26 49 ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 50 of 61

In order to separate the SERS spectra that contain SERS signals from spectra that do not contain vibrational information in the data sets, an RF classification model was trained on 48 empty spectra and 52 spectra containing SERS spectral fingerprints that were manually selected from the data from all incubation conditions. In the model, the number of variables to possible split at each node mtry equal to p1/2 was 31, the number of trees

ntree was 10000, and the minimal node size min.node.size was 1. Subsequently, the spectra of all the SERS mapping data sets were classified based on this model, and only those spectra that were classified as containing SERS signals were further used, ranging from ~200 to ~1700 spectra for a particular incubation experiment and a respective TCA molecule (Table S12).

The random forest based methods MD and SMD are applied here to select important variables and to analyse the relations of those spectral variables, that is, spectral bands, that separate cells treated with the antidepressants from their respective controls. Since this analysis is only reasonable when the RF models are capable of the separation, an RF classification model (mtry = 31, ntree = 10000, min.node.size =1) was trained for each


Page 51 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

incubation experiment first, using a subsample of each data set as training data and the remaining samples as test data. The number of spectra in the respective training and test sets are shown in Table S13 in the Supporting Information. From the prediction results of the test data, sensitivity and selectivity were calculated for each data set (Table S2). Sensitivity was obtained by dividing the number of correctly classified spectra to the total number of spectra observed in a TCA treated cell. Selectivity was calculated as the fraction of correctly classified control spectra among all control spectra obtained for a specific incubation condition.

Subsequently,

MD

with

the

parameters

mtry = p3/4 = 173, ntree = 10000, and

min.node.size = 1 was applied in classification mode to select the spectral bands that are important for the separation. Examples of MD graphs are shown in Supporting Information Figure S5. The peaks of the important bands were further analyzed by SMD26 with the same parameters as MD and 100 surrogate variables s to identify those variables that are related to them, that is, bands that are capable to frequently replace the important bands


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 52 of 61

in random forests (see Figure S6 for examples). Variables that feature a mean adjusted agreement value above a threshold26 are identified as related variables.

Acknowledgment V.Ž. and G.P.S. gratefully acknowledge funding by a fellowship of the School of Analytical Sciences Adlershof (DFG GSC 1013), J.K. by ERC grant no. 259432, C.A. by the Deutsche Forschungsgemeinschaft, DFG (AR-376/12-2), S.S. by the German Federal Ministry of Education and Research (BMBF) within the framework of the e:Med research and funding concept (01Zx1510). We thank HZB for the allocation of synchrotron radiation beam time.

Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/

Assignments of important bands in the SERS spectra, sensitivity and specificity of the random forest classification, tables with the important bands and their related variables for all experimental conditions, examples of single SERS spectra for all experimental conditions, results of RF classification of SERS spectra. 52 ACS Paragon Plus Environment

Page 53 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 54 of 61

References 1.

H. Schulze, and K. Sandhoff, Lysosomal Lipid Storage Diseases, Cold Spring Harb

Perspect Biol., 2011, 3, 1-19. 2.

A. H. Futerman, and G. van Meer, The Cell Biology of Lysosomal Storage Disorders, Nat.

Rev. Mol. Cell Bio., 2004, 5, 554-565. 3.

N. H. Petersen, O. D. Olsen, L. Groth-Pedersen, A. M. Ellegaard, M. Bilgin, S. Redmer, M. S. Ostenfeld, D. Ulanet, T. H. Dovmark, A. Lonborg, S. D. Vindelov, D. Hanahan, C. Arenz, C. S. Ejsing, T. Kirkegaard, M. Rohde, J. Nylandsted, and M. Jaattela, TransformationAssociated Changes in Sphingolipid Metabolism Sensitize Cells to Lysosomal Cell Death Induced by Inhibitors of Acid Sphingomyelinase, Cancer Cell, 2013, 24, 379-393.

4.

R. O. Brady, J. N. Kanfer, M. B. Mock, and D. S. Fredrickson, The Metabolism of Sphingomyelin. Ii. Evidence of an Enzymatic Deficiency in Niemann-Pick Diseae, Proc.

Natl. Acad. Sci. U. S. A., 1966, 55, 366-369. 5.

K. Horinouchi, S. Erlich, D. P. Perl, K. Ferlinz, C. L. Bisgaier, K. Sandhoff, R. J. Desnick, C. L. Stewart, and E. H. Schuchman, Acid Sphingomyelinase Deficient Mice: A Model of Types a and B Niemann–Pick Disease, Nat. Genet., 1995, 10, 288-293.

6.

E. F. Neufeld, Lysosomal Storage Diseases, Annu. Rev. Biochem., 1991, 60, 257-280.

7.

T. Kirkegaard, A. G. Roth, N. H. Petersen, A. K. Mahalka, O. D. Olsen, I. Moilanen, A. Zylicz, J. Knudsen, K. Sandhoff, and C. Arenz, Hsp70 Stabilizes Lysosomes and Reverts Niemann–Pick Disease-Associated Lysosomal Pathology, Nature, 2010, 463, 549-554.


Page 55 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

8.

N. Liu, E. A. Tengstrand, L. Chourb, and F. Hsieh, Di-22: 6-Bis (Monoacylglycerol) Phosphate: A Clinical Biomarker of Drug-Induced Phospholipidosis for Drug Development and Safety Assessment, Toxicol. Appl. Pharmacol., 2014, 279, 467-476.

9.

M. J. Reasor, K. L. Hastings, and R. G. Ulrich, Drug-Induced Phospholipidosis: Issues and Future Directions, Expert. Opin. Drug. Saf., 2006, 5, 567-583.

10.

J. A. Shayman, and A. Abe, Drug Induced Phospholipidosis: An Acquired Lysosomal Storage Disorder, Biochim. Biophys. Acta, Mol. Cell. Biol. Lipids, 2013, 1831, 602-611.

11.

P. V. Jena, D. Roxbury, T. V. Galassi, L. Akkari, C. P. Horoszko, D. B. Iaea, J. BudhathokiUprety, N. Pipalia, A. S. Haka, J. D. Harvey, J. Mittal, F. R. Maxfield, J. A. Joyce, and D. A. Heller, A Carbon Nanotube Optical Reporter Maps Endolysosomal Lipid Flux, ACS

Nano, 2017, 11, 10689-10703. 12.

J. Kneipp, H. Kneipp, M. McLaughlin, D. Brown, and K. Kneipp, In Vivo Molecular Probing of Cellular Compartments with Gold Nanoparticles and Nanoaggregates, Nano Lett., 2006, 6, 2225-2231.

13.

A. Matschulat, D. Drescher, and J. Kneipp, Surface-Enhanced Raman Scattering Hybrid Nanoprobe Multiplexing and Imaging in Biological Systems, ACS Nano, 2010, 4, 32593269.

14.

V. Živanović, G. Semini, M. Laue, D. Drescher, T. Aebischer, and J. Kneipp, Chemical Mapping of Leishmania Infection in Live Cells by SERS Microscopy, Anal. Chem., 2018, 90, 8154-8161.

15.

J. Kneipp, H. Kneipp, and K. Kneipp, SERS--a Single-Molecule and Nanoscale Tool for Bioanalytics, Chem. Soc. Rev., 2008, 37, 1052-1060.


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

16.

Page 56 of 61

J. Kneipp, H. Kneipp, B. Wittig, and K. Kneipp, Following the Dynamics of pH in Endosomes of Live Cells with Sers Nanosensors, J. Phys. Chem. C, 2010, 114, 74217426.

17.

K. Bando, N. I. Smith, J. Ando, K. Fujita, and S. Kawata, Analysis of Dynamic SERS Spectra Measured with a Nanoparticle During Intracellular Transportation in 3d, J. Opt., 2015, 17, 1-13.

18.

D. Drescher, and J. Kneipp, Nanomaterials in Complex Biological Systems: Insights from Raman Spectroscopy, Chem. Soc. Rev., 2012, 41, 5780-5799.

19.

M. Kölzer, N. Werth, and K. Sandhoff, Interactions of Acid Sphingomyelinase and Lipid Bilayers in the Presence of the Tricyclic Antidepressant Desipramine, FEBS Lett., 2004, 559, 96-98.

20.

S. Albouz, M. Vanier, J. Hauw, F. Le Saux, J. Boutry, and N. Baumann, Effect of Tricyclic Antidepressants on Sphingomyelinase and Other Sphingolipid Hydrolases in C6 Cultured Glioma Cells, Neurosci. Lett., 1983, 36, 311-315.

21.

R. Hurwitz, K. Ferlinz, and K. J. B. c. H.-S. Sandhoff, The Tricyclic Antidepressant Desipramine Causes Proteolytic Degradation of Lysosomal Sphingomyelinase in Human Fibroblasts, Biol. Chem. Hoppe-Seyler,1994, 375, 447-450.

22.

A. Gulbins, F. Schumacher, K. A. Becker, B. Wilker, M. Soddemann, F. Boldrin, C. P. Müller, M. J. Edwards, M. Goodman, C. C. Caldwell, B. Kleuser, J. Kornhuber, I. Szabo, and E. Gulbins, Antidepressants Act by Inducing Autophagy Controlled by Sphingomyelin– Ceramide, Mol. Psychiatry, 2018, 23, 2324-2346.


Page 57 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

23.

V. Živanović, Z. Kochovski, C. Arenz, Y. Lu, and J. Kneipp, Sers and Cryo-Em Directly Reveal Different Liposome Structures During Interaction with Gold Nanoparticles, J. Phys.

Chem. Lett., 2018, 9, 6767-6772. 24.

B. Moody, C. M. Haslauer, E. Kirk, A. Kannan, E. G. Loboa, and G. S. McCarty, In Situ Monitoring of Adipogenesis with Human-Adipose-Derived Stem Cells Using SurfaceEnhanced Raman Spectroscopy, Appl. Specctroc., 2010, 64, 1227-1233.

25.

L. Breiman, Random Forests, Mach. Learn., 2001, 45, 5-32.

26.

S. Seifert, S. Gundlach, and S. Szymczak, Surrogate Minimal Depth as an Importance Measure for Variables in Random Forests, Bioinformatics, 2019, 1-9.

27.

G. Schneider, P. Guttmann, S. Heim, S. Rehbein, F. Mueller, K. Nagashima, J. B. Heymann, W. G. Müller, and J. G. McNally, Three-Dimensional Cellular Ultrastructure Resolved by X-Ray Microscopy, Nat. Methods, 2010, 7, 985-988.

28.

D. Drescher, T. Büchner, P. Guttmann, S. Werner, G. Schneider, and J. Kneipp, X-Ray Tomography Shows the Varying Three-Dimensional Morphology of Gold Nanoaggregates in the Cellular Ultrastructure, Nanoscale Adv., 2019, DOI: 10.1039/C9NA00198K.

29.

Z. Xia, G. Ying, A. L. Hansson, H. Karlsson, Y. Xie, A. Bergstrand, J. W. DePierre, and L. Nässberger, Antidepressant-Induced Lipidosis with Special Reference to Tricyclic Compounds, Prog. Neurobiol., 2000, 60, 501-512.

30.

V. Živanović, F. Madzharova, Z. Heiner, C. Arenz, and J. Kneipp, Specific Interaction of Tricyclic Antidepressants with Gold and Silver Nanostructures as Revealed by Combined One-and Two-Photon Vibrational Spectroscopy, J. Phys. Chem. C, 2017, 121, 2295822968.


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

31.

Page 58 of 61

T. Büchner, D. Drescher, H. Traub, P. Schrade, S. Bachmann, N. Jakubowski, and J. Kneipp, Relating Surface-Enhanced Raman Scattering Signals of Cells to Gold Nanoparticle Aggregation as Determined by LA-ICP-MS Micromapping, Anal. Bioanal.

Chem., 2014, 406, 7003-7014. 32.

Y. Wang, C. Jacobsen, J. Maser, and A. Osanna, Soft X-Ray Microscopy with a Cryo Scanning Transmission X-Ray Microscope: Ii. Tomography, J. Microsc., 2000, 197, 80-93.

33.

S. Albouz, B. Tocque, J. Hauw, J. Boutry, F. Le Saux, R. Bourdon, and N. Baumann, Tricyclic Antidepressant Desipramine Induces Stereospecific Opiate Binding and Lipid Modifications in Rat Glioma C6 Cells, Life Sci., 1982, 31, 2549-2554.

34.

Z. Movasaghi, S. Rehman, and I. U. Rehman, Raman Spectroscopy of Biological Tissues,

Appl. Spectrosc. Rev., 2007, 42, 493-541. 35.

M. Aioub, and M. A. El-Sayed, A Real-Time Surface Enhanced Raman Spectroscopy Study of Plasmonic Photothermal Cell Death Using Targeted Gold Nanoparticles, J. Am.

Chem. Soc., 2016, 138, 1258-1264. 36.

L. A. Austin, B. Kang, and M. A. El-Sayed, Probing Molecular Cell Event Dynamics at the Single-Cell Level with Targeted Plasmonic Gold Nanoparticles: A Review, Nano Today, 2015, 10, 542-558.

37.

J. L. Lippert, and W. L. Peticolas, Laser Raman Investigation of the Effect of Cholesterol on Conformational Changes in Dipalmitoyl Lecithin Multilayers, Proc. Natl. Acad. Sci. U.

S. A., 1971, 68, 1572-1576. 38.

H. Wu, J. V. Volponi, A. E. Oliver, A. N. Parikh, B. A. Simmons, and S. Singh, In Vivo Lipidomics Using Single-Cell Raman Spectroscopy, Proc. Natl. Acad. Sci. U. S. A.,2011, 108, 3809-3814. 58 ACS Paragon Plus Environment

Page 59 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

39.

K. Czamara, K. Majzner, M. Z. Pacia, K. Kochan, A. Kaczor, and M. Baranska, Raman Spectroscopy of Lipids: A Review, J. Raman Spectrosc., 2015, 46, 4-20.

40.

I. W. Levin, E. Keihn, and W. C. Harris, A Raman Spectroscopic Study on the Effect of Cholesterol on Lipid Packing in Diether Phosphatidylcholine Bilayer Dispersions, Biochim.

Biophys. Acta, Mol. Cell. Biol. Lipids, 1985, 820, 40-47. 41.

R. Manoharan, J. J. Baraga, M. S. Feld, and R. P. Rava, Quantitative Histochemical Analysis of Human Artery Using Raman Spectroscopy, J. Photochem. Photobiol. B, Biol., 1992, 16, 211-233.

42.

C. B. Fox, J. M. Harris, a. Brillouin, and R. Scattering, Confocal Raman Microscopy for Simultaneous Monitoring of Partitioning and Disordering of Tricyclic Antidepressants in Phospholipid Vesicle Membranes, J. Raman Spectrosc., 2010, 41, 498-507.

43.

H. Ishwaran, U. B. Kogalur, E. Z. Gorodeski, A. J. Minn, and M. S. Lauer, High-Dimensional Variable Selection for Survival Data, J. Am. Stat. Assoc., 2010, 105, 205-217.

44.

H. Akutsu, Direct Determination by Raman Scattering of the Conformation of the Choline Group in Phospholipid Bilayers, Biochemistry, 1981, 20, 7359-7366.

45.

N. Beckmann, D. Sharma, E. Gulbins, K. A. Becker, and B. Edelmann, Inhibition of Acid Sphingomyelinase by Tricyclic Antidepressants and Analogons, Fronti. Physiol., 2014, 5, 1-14.

46.

A. Jaworska, and K. Malek, A Comparison between Adsorption Mechanism of Tricyclic Antidepressants on Silver Nanoparticles and Binding Modes on Receptors. SurfaceEnhanced Raman Spectroscopy Studies, J. Colloid Interface Sci., 2014, 431, 117-124.

47.

P. C. Lee, and D. Meisel, Adsorption and Surface-Enhanced Raman of Dyes on Silver and Gold Sols, J. Phys. Chem.,1982, 86, 3391-3395. 59 ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

48.

Page 60 of 61

M. N. Wright, and A. Ziegler, A Fast Implementation of Random Forests for High Dimensional Data in C++ and R, J. Stat. Softw., 2017, 77, 1-17.


Page 61 of 61 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

For Table of Contents Only


Optical Nanosensing of Lipid Accumulation due to Enzyme Inhibition

Recommend Documents