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mRNA-Based FRET-FLIM Imaging Platform for Quantifying Lipid Nanoparticle Endosomal Escape and Membrane Damage.

Yian Fang^1,2, Bin Ma^1,2, Zhiqiang Zhao^1,2,3

¹State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China.

Journal of the American Chemical Society

|August 15, 2025

Summary

Researchers developed a new method, FRET-lifetime imaging for mRNA-LNP tracking (FLINT), to measure how effectively mRNA exits endosomes. This tool tracks mRNA release and endosomal damage, aiding in the development of better lipid nanoparticle (LNP) delivery systems.

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Published on: October 18, 2024

Area of Science:

Biotechnology
Cell Biology
Drug Delivery

Background:

Efficient endosomal escape and membrane damage are critical for mRNA-lipid nanoparticle (LNP) delivery, yet quantifying these processes is difficult.
Current methods lack the dynamic resolution to track mRNA release and endosomal integrity simultaneously in live cells.

Purpose of the Study:

To develop and validate a novel platform, FRET-lifetime imaging for mRNA-LNP tracking (FLINT), for real-time monitoring of mRNA release kinetics and endosomal damage.
To investigate the impact of LNP composition and endosomal interactions on mRNA delivery efficiency.

Main Methods:

Developed FLINT, an intramolecular Förster Resonance Energy Transfer-Fluorescence Lifetime Imaging Microscopy (FRET-FLIM) platform utilizing disulfide-linked fluorophores on mRNA.
Leveraged the differential glutathione (GSH) concentration between the cytosol and endosomes for GSH-activated signal amplification (20-50×) to track cytosolic mRNA.
Applied FLINT to analyze mRNA release and endosomal damage for different LNP formulations in live cells.

Main Results:

FLINT successfully tracked mRNA release and endosomal damage dynamics in live cells.
LNPs with escape-favorable lipids showed rapid cytosolic mRNA release, while uptake-optimized LNPs accumulated in endosomes.
Observed GSH influx during endosomal damage and correlated membrane rupture with mRNA expression.
Demonstrated that lipid organization within LNPs, alongside endosomal damage, influences mRNA delivery efficiency.

Conclusions:

FLINT provides a powerful tool to decouple uptake, escape, and structural factors influencing LNP delivery efficiency.
The platform enables mechanistic studies and optimization of LNPs for enhanced mRNA delivery in vitro and in vivo.
FLINT reveals critical insights into the complex interplay between LNPs, endosomes, and cytosolic mRNA delivery.