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. 2024 Nov 19;14(1):28566.
doi: 10.1038/s41598-024-76621-5.

Evaluating pathological levels of intracellular cholesterol through Raman and surface-enhanced Raman spectroscopies

Enrico Baria #  1   2 , Caterina Dallari #  3   4 , Francesco Mattii  1 , Francesco Saverio Pavone  1   2   5 , Caterina Credi  1   5 , Riccardo Cicchi  1   5 , Amelia Morrone  6   7 , Claudia Capitini  1   2 , Martino Calamai  8   9
Affiliations

Evaluating pathological levels of intracellular cholesterol through Raman and surface-enhanced Raman spectroscopies

Enrico Baria et al. Sci Rep. .

Abstract

Versatile methods for the quantification of intracellular cholesterol are essential for understanding cellular physiology and for diagnosing disorders linked to cholesterol metabolism. Here we used Raman spectroscopy (RS) and surface-enhanced Raman spectroscopy (SERS) to measure changes in cholesterol after incubating human fibroblasts with increasing concentrations of cholesterol-methyl-β-cyclodextrin. RS and SERS were sensitive and accurate enough to detect high levels of cholesterol in fibroblasts from patients affected by type C Niemann-Pick disease (NPC), a lysosomal storage disorder characterized by the primary accumulation of cholesterol. Moreover, SERS was able to distinguish between fibroblasts from different NPC patients, demonstrating higher accuracy than RS and standard fluorescent labeling of cholesterol with filipin III. We show that the type of gold nanoparticles used as signal enhancer surfaces in our SERS measurements are internalized by the cells and are eventually found in lysosomes, the main site of accumulation of cholesterol in NPC fibroblasts. The higher sensitivity of SERS can thus be attributed to the specific trafficking of our gold nanoparticles into these organelles. Our results indicate that RS and SERS can be used as sensitive and accurate methods for the evaluation of intracellular cholesterol content, allowing for the potential development of an optical detection tool for the ex-vivo screening and monitoring of those diseases characterized by abnormal modification in cholesterol levels.

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Conflict of interest statement

Declarations Competing interests The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Enrichment of β-CD-chol in WT FB. Cells were analyzed in basal conditions and after 3 h of incubation with 0.2, 0.6, and 1.0 mg/mL of β-CD-chol. Cells were then probed with the fluorescent dye Filipin III and imaged with wide-field epifluorescence microscopy (a,b). Filipin III fluorescence intensity significantly increases with the increase of β-CD-chol concentration up to 0.6 mg/mL, reaching a fluorescence signal plateau between 0.6 and 1.0 mg/mL. > 30 cells were analyzed for each condition (b). Error bar S.D. Student’s t-test *** p ≤ 0.001. Pre-processing and analysis of Raman spectra. Each raw spectrum  (c) was restricted to the range between 2800 cm− 1 and 3026 cm− 1 and smoothed via Savitzky-Golay filtering, with its lowest intensity value subtracted from the whole spectrum (d). The highlighted Raman bands were assigned according to the data reported in (see also Supplementary Table T1). Finally, the overall intensities of two Raman bands centered at 2850 cm− 1 and 2898 cm− 1 were computed (e), with their ratio being used as a score for estimating lipid concentration in cells. Raman-SERS analysis of WT FB cells enriched with β-CD-chol in the range 0 − 1 mg/mL. Mean RS (f), SERS (g) and both RS and SERS (h) spectra ± standard errors (SEs) of enriched cells; ratiometric scores measured from RS (i), SERS (j) and both RS and SERS (k) spectra of enriched cells, expressed as percentage variation from the mean control (i.e. 0 mg/mL added, without NPs) value.
Fig. 2
Fig. 2
Analysis of cholesterol content in FB from NPC patients. Thawed primary cultures of FB isolated from WT control and NPC (NPCp1 and NPCp2) patients were cultured, fixed, permeabilized, labelled with Filipin III and (a) imaged with wide field epifluorescence microscopy (a). Significantly higher values of Filipin III were found analyzing NPCp1 and NPCp2 images compared to control, indicating a rise in cellular cholesterol content (b). > 30 cells were analyzed for each sample. Error bar S.D. Student’s t-test *** p ≤ 0.001. Raman-SERS analysis of FB cells from NPC patients  (c–f). Mean RS (c) and SERS (d) spectra ± SEs of WT and NPC’ cells; ratiometric scores measured from RS (e) and SERS (f) spectra of WT and NPC’ cells, expressed as percentage variation from the respective mean control values.
Fig. 3
Fig. 3
Fluorescent Rh110-AuNPs characterization. Graphical scheme depicting Rhodamine 110 conjugation to AuNPs (a). UV-Visible absorption spectra of AuNPs (black line) and Rh110 conjugated AuNPs (green line). Inset shows a magnification in the range 510–530 nm (b).TEM images of AuNPs (c). Autocorrelation curves in function of the decay time (d) and the calculated hydrodynamic diameters of AuNPs (black dot/histogram) and Rh110-AuNPs (green dot/histogram) (e). Fluorescence emission spectrum of Rhodamine 110 solution (black line) and absorbance and fluorescence spectra of Rh110-AuNPs (green line) (f).
Fig. 4
Fig. 4
Specific targeting of AuNPs in lysosomes. WT and NPCp1 fibroblasts were incubated overnight with 1 nM Rh110-AuNPs or Rh110 probe, and imaged by confocal microscopy after co-labeling with LysoTracker™ Red DND-99 and Hoechst. Scale bar 50 μm. The Pearson’s coefficient (P) values and the Manders’ coefficients M1 (fraction of Rh110-AuNPs overlapping with lysosomes) and M2 (fraction of lysosomes overlapping with Rh110-AuNPs) are also reported.
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