Abstract
Fluorescent sterol probes, comprising a fluorophore connected to a sterol backbone by means of a linker, are promising tools for enabling high-resolution imaging of intracellular cholesterol. In this study, we evaluated how the size of the linker, site of its attachment and nature of the fluorophore, affect the localization and trafficking properties of fluorescent sterol probes. Varying lengths of linker using the same fluorophore affected cell penetration and retention in specific cell compartments. A C-4 linker was confirmed as optimal. Derivatives of heterocyclic sterol precursors attached with identical C-4 linker to different fluorophores at diverse positions also showed significant differences in their binding properties to various intracellular compartments and kinetics of trafficking. Two novel red-emitting probes with good cell permeability, fast intracellular labelling and slightly different distribution displayed very promising characteristics for sterol probes. These probes also strongly labelled endo/lysosomal compartment in cells with pharmacologically disrupted cholesterol transport, or with a genetic mutation of cholesterol transporting protein NPC1, that overlapped with filipin staining of cholesterol. Overall, the present study demonstrates that the physicochemical properties of the fluorophore/linker pairing determine the kinetics of uptake and distribution and subsequently influence the applicability of final probes.
Highlights
Fluorescent sterol probes, comprising a fluorophore connected to a sterol backbone by means of a linker, are promising tools for enabling high-resolution imaging of intracellular cholesterol
In our previous study[8] we reported fluorescent probes based on heterocyclic sterol derivatives with various fluorophores attached to different regions on a sterol scaffold and revealed striking differences in their efficacy
We previously demonstrated the utility of the green-excited fluorophore BODIPY-abiraterone acetate, FP-58
Summary
Fluorescent sterol probes, comprising a fluorophore connected to a sterol backbone by means of a linker, are promising tools for enabling high-resolution imaging of intracellular cholesterol. Derivatives of heterocyclic sterol precursors attached with identical C-4 linker to different fluorophores at diverse positions showed significant differences in their binding properties to various intracellular compartments and kinetics of trafficking. Two novel red-emitting probes with good cell permeability, fast intracellular labelling and slightly different distribution displayed very promising characteristics for sterol probes. A very promising probe was a conjugate of the green-emitting BODIPY fluorophore with sterol P1, designated as FP-5 This probe showed high cholesterol- and cholesteryl acetate-binding specificity in spectroscopic studies and fast labelling of cholesterol-rich compartments in cellular studies. Design of fluorescently labelled sterol-like molecules, we have developed probes that are able to detect Chol in various intracellular compartments
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