# Dimensions, stability, and deformability of DOPC-cholesterol giant unilamellar vesicles formed by droplet transfer

## TL;DR

This study explores how cholesterol affects the size, stability, and flexibility of artificial cell-like structures called GUVs, using a common production method.

## Contribution

The study reveals how varying cholesterol content in GUVs influences their properties using the droplet transfer method, offering insights for synthetic biology and microrobotics.

## Key findings

- Cholesterol increases the median diameter and dimensional distribution of GUVs.
- Cholesterol reduces GUV stability over time but does not significantly affect deformability.
- Adjusting cholesterol in the oil phase allows precise tuning of GUV properties.

## Abstract

Understanding cell membrane-like lipid bilayers is crucial for studying fundamental biological mechanisms. Giant Unilamellar Vesicles (GUVs) are key tools for this investigation and have applications in both synthetic biology and, more recently, in microrobotics. The effects of cholesterol, a key component of cellular membranes, on synthetic phospholipid membrane models like GUVs are however not fully understood, as they may vary with lipid composition and production method.

We examined the size distribution, temporal stability and deformability of GUVs prepared with the droplet transfer method using different Dioleoylphosphatidylcholine (DOPC) to cholesterol ratios in the oil phase (namely 100:0, 85:15, 71:29, 60:40). Phase-contrast microscopy assessed size and stability, while deformability was tested by loading the GUVs with an aqueous ferrofluid and applying a uniform magnetic field to induce their elongation. Image analysis was conducted using Fiji and a custom Julia script.

The median diameters increased with the content of cholesterol, together with the dimensional distribution. In terms of stability, cholesterol generally reduced GUV median diameter over time, while it varyingly influenced the number of vesicles. As for deformability, beyond the expected elongation dependent on the intensity of the applied magnetic field, there were no statistically significant differences in GUV deformability in the presence or absence of cholesterol.

Our findings suggest that cholesterol can lead to increased average diameter of GUVs made with DOPC through droplet transfer, while varyingly affecting their time-stability and not affecting their deformability. This study shows how small adjustments on a straightforward protocol like the droplet transfer method, provide a simple and effective way of tailoring GUV properties. Edits in the oil phase enable precise tuning of GUV membranes providing a tool for both fundamental studies and applications such as artificial cells or microrobots.

Cell membranes play a key role in the overall functioning of cells and their environmental interactions. For a better understanding of their mechanisms, a common tool is represented by Giant Unilamellar Vesicles (GUVs). These micrometric vesicles are produced through the supramolecular assembly of phospholipids, the main molecule of cell membranes, and used to explore basic biological processes, build synthetic cells, and even design microrobots. Here we study the effects that cholesterol, a key component of natural cell membranes, has on GUVs’ membrane properties. While these can vary with the production method and the types of lipids, in this study we focus on one of the most widely adopted methods (known as droplet transfer) and one of the most common phospholipids (dioleoylphosphatidylcholine or DOPC). Our results suggest that the effect of cholesterol on the size and stability of GUVs depends on its concentration, while their flexibility seems not to be affected. By exploring these properties, we aim to deepen our understanding of cell membranes and improve the design of synthetic systems for future applications.