An Orthogonal Quantitative NMR-FRET Approach for Integrity-Resolved Analysis of Lipid Vesicle Dynamics

Abstract While lipid-based vesicles gain increasing attention as biomembrane models, artificial cells, and drug carriers, analytical investigation of their composition and integrity remains a major challenge, particularly for monitoring changes during processing and membrane transport. To address this challenge, we present an orthogonal analytical platform that combines 1H nuclear magnetic resonance (NMR) and Förster resonance energy transfer (FRET) spectroscopy to enable integrity-resolved quantification of lipid vesicles. While 1H NMR provides chemically selective calibration-based quantification of individual lipid species, FRET-based assays enable direct monitoring of intact vesicle translocation across membranes, allowing for discrimination between free lipids and vesicle-associated lipids. We investigated vesicles composed of different phospholipid mixtures, including their formation and integrity as well as their membrane transport, and revealed remarkable composition-dependent differences. While vesicles composed of phosphatidylcholine and cholesterol exhibit instability, compositional loss, and limited membrane transport, inclusion of anionic lipids, such as phosphatidylglycerol and phosphatidylserine, significantly improves structural integrity and increases the fraction of lipids transported in vesicular form. These results demonstrate that orthogonal quantification of free and vesicle-associated lipids provides mechanistic insights into vesicle dynamics during processing and membrane transport, supporting the rational design of vesicular systems for applications in artificial cell research and lipid-based nanomedicines, where vesicle composition directly governs mechanical stability as well as biological interactions.