Development and characterization of fluorescent cholesteryl probes with enhanced solvatochromic and pH-sensitive properties for live-cell imaging

Abstract

We present novel fluorescent cholesteryl probes (CNDs) with a modular design based on the solvatochromic 1,8-phthalimide scaffold. We have explored how different modules-linkers and head groups-affect the ability of these probes to integrate into lipid membranes and how they distribute intracellularly in mouse astrocytes and fibroblasts targeting lysosomes and lipid droplets. Each compound was assessed for its solvatochromic behavior in organic solvents and model membranes. Molecular dynamics simulations and lipid partitioning using giant unilamellar vesicles showed how these analogs behave in model membranes compared to cholesterol. Live-cell imaging demonstrated distinct staining patterns and cellular uptake behaviors, further validating the utility of these probes in biological systems. We compared the empirical results with those of BODIPY-cholesterol, a well-regarded fluorescent cholesterol analog. The internalization efficiency of fluorescent CND probes varies in different cell types and is affected mainly by the head groups. Our results demonstrate that the modular design significantly simplifies the creation of fluorescent cholesteryl probes bearing distinct spectral, biophysical, and cellular targeting features. It is a valuable toolkit for imaging in live cells, measuring cellular membrane dynamics, and studying cholesterol-related processes.

Keywords: Cholesterol; Fluorescence microscopy; Fluorescent reporter; Lipid droplets; Lipid rafts; Lysosomes.; Membrane; pH-sensitivity.

Fig. 1

Structural modularity of CND probes. Variation of head groups and linkers used to make CND1-CND10.

Fig. 2

Emission spectra of CND2-CND4 in 1% octyl glucoside micellar solution in Sorensen’s buffer at various pH (λ_Ex = 405 nm) demonstrating pH sensitivity of these analogs (top row). The fluorescence in the 470–650 nm range was used to calculate area under the curve to determine the relative fluorescence change (mean ± SEM, bottom row). The fluorescence increases 2-3-fold with pH drop from 7 to 5.

Fig. 3

Partitioning of CNDs between L_d/L_o phases in phase-separated GUVs. A, representative fluorescent confocal image of CND3 and Ld marker DiD. To determine the partitioning, the fluorescence intensity around the vesicle perimeter was measured using the GUV-AP plugin for ImageJ (see supplementary info). Scale bar, 10 μm. B, Fraction of probe partitioning into L_o phase for all tested CNDs.

Fig. 4

Characterization of probes using molecular dynamics simulations. A, analysis of the tilt angle of cholesterol. C10-C13 atoms were selected as selection (inset). B, analysis of the depth of probe immersion in the membrane in SM: Chol: POPC system. C, analysis of hydrogen bond patterns between cholesterol, probes and surrounding water, POPC, and sphingomyelin (SM).

Fig. 5

Live cell imaging of CND probes in cultured astrocytes. A, sample images for analogs that are neutral (CND1, 8), positively charged (CND3), and negatively charged (CND10) compared with Bchol (average Z-stacks). Overlay images show co-labeled LysoView (red) overlapping with selected CNDs better than Bchol. Scale bar, 10 μm. B, Manders’ (MCC) and Pearson’s (PCC) correlation coefficients between probe/LysoView at different time points and cell types. C, relative probe uptake and accumulation measured by particle count at different time points in astrocytes and 3T3 fibroblasts. Significance was calculated using a Wilcoxon matched-pairs signed rank. Differences were considered statistically significant at P ≤ 0.05 (*), P ≤ 0.01 (**), and P ≤ 0.001 (***); ns – not significant.

Fig. 6

CND3 fluorescence changes during pH manipulation in astrocytes. A, sample images of CND3 co-loaded with Transferrin-Alexa568 conjugate in astrocytes 1 h after incubation and washing. When astrocytes were perfused with normal (pH 7.3), acidic (pH 5.5), and NH₄Cl-containing Tyrode’s solutions, CND3 fluorescence changes were observed. Scale bar, 20 μm. B, average fluorescence change of CND3 and Transferrin-Alexa568 in astrocytes during 1-minute perfusion of the three solutions. Error bars are standard errors of means.