# Quantification of cholesterol incorporation in giant unilamellar vesicles produced by a modified cDICE method

## TL;DR

This paper introduces a method to measure and increase cholesterol in model cell membranes using fluorescent sensors, improving their use in synthetic biology and membrane studies.

## Contribution

A non-disruptive method to quantify and modulate cholesterol content in emulsion-based GUVs using fluorescent biosensors.

## Key findings

- Cholesterol content in GUVs can be increased to at least 40 mol% using MβCD–CL complexes.
- Fluorescent biosensors like NR12A and Flipper-TR effectively quantify cholesterol incorporation.
- Cholesterol addition triggers liquid-ordered domain formation in DOPC:DMPC vesicles.

## Abstract

Cholesterol is an essential component of eukaryotic cell membranes, influencing membrane packing, fluidity, and domain formation. Replicating these properties in model membranes is critical for reconstitution studies, but common emulsion-based methods for producing giant unilamellar vesicles (GUVs) fail to incorporate cholesterol efficiently. Here, we use methyl-β-cyclodextrin–cholesterol (MβCD–CL) complexes to deliver cholesterol into GUVs produced by the emulsion droplet interface crossing encapsulation (eDICE) method and demonstrate a convenient way to quantify the degree of cholesterol incorporation using fluorescent membrane biosensors. Spectral imaging of NR12A as well as fluorescence lifetime imaging of Flipper-TR revealed dose-dependent increases in cholesterol content for DOPC GUVs upon MβCD–CL addition, consistent with increased membrane order. By calibrating these effects against GUVs with defined cholesterol contents prepared via gel-assisted swelling, we found that the cholesterol content of eDICE vesicles can be increased to at least 40 mol%. Binary mixtures of DOPC with saturated lipids (DMPC and PC (18 : 0–14 : 0)) showed a similar trend as pure DOPC GUVs. Interestingly, we could trigger liquid-ordered domain formation by adding cholesterol to DOPC : DMPC vesicles. Our findings provide a quantitative and non-disruptive method to modulate and assess cholesterol content in emulsion-based GUVs, advancing their use in bottom-up synthetic biology and membrane biophysics.

MβCD-mediated cholesterol incorporation into emulsion-based GUVs quantified in situ using environment-sensitive fluorescent dyes.