mRNA-Based FRET-FLIM Imaging Platform for Quantifying Lipid Nanoparticle Endosomal Escape and Membrane Damage
Efficient endosomal escape and membrane damage are crucial for mRNA-lipid nanoparticle (LNPs) delivery but remain challenging to quantify. Here, we develop FRET-lifetime imaging for mRNA-LNP tracking (FLINT), an intramolecular FRET-FLIM platform to dynamically monitor mRNA release kinetics and endosomal damage in live cells. Capitalizing on the 50- to 100-fold higher concentration of glutathione (GSH) in the cytosol relative to endocytic vesicles, the FLINT employs disulfide-linked fluorophores conjugated to mRNA uridines, enabling precise tracking of cytosolic mRNA via GSH-activated signal amplification (20-50×). Using FLINT, we find that LNPs with escape-favorable lipid compositions (e.g., C12-200) exhibit rapid FRET signal diffusion, while uptake-driven LNPs (e.g., MC3) accumulate in endosomes with limited release. FLINT further reveals GSH influx during endosomal damage and correlates membrane rupture patterns with mRNA expression. Additionally, FLINT uncovers the influence of lipid organization within LNPs─in addition to endosomal membrane damages─on mRNA delivery efficiency. This FLINT platform decouples uptake, escape, and structural determinants of delivery efficiency, offering a transformative tool for LNP optimization and mechanistic studies both in vivo and in vitro.
Authors
- Chuanshu Huang
- Yian Fang (ORCID: https://orcid.org/0000-0002-0906-7434)
- Yiguang Wang (ORCID: https://orcid.org/0000-0002-1676-4211)
- Zhenyu Xuan (ORCID: https://orcid.org/0000-0001-9344-8493)
- Chenzhong Liao (ORCID: https://orcid.org/0000-0001-7080-1768)
- Xinglin Yang (ORCID: https://orcid.org/0000-0002-1431-2836)
- Yifan Ge (ORCID: https://orcid.org/0000-0001-9135-9569)
- Jiajia Guo (ORCID: https://orcid.org/0000-0002-6118-8241)
- Wei You (ORCID: https://orcid.org/0000-0002-8698-309X)
- Lei Zhou (ORCID: https://orcid.org/0000-0001-8056-9464)
- Lei Miao (ORCID: https://orcid.org/0009-0004-1647-1445)
- Bin Ma (ORCID: https://orcid.org/0000-0002-3913-0545)
- Zhiqiang Zhao
- Mi Zhang (ORCID: https://orcid.org/0000-0003-4032-7502)
- Kailong Li
- Yao Feng
- Yu Wang (ORCID: https://orcid.org/0000-0001-6351-6467)
Institutions
- Shenyang Pharmaceutical University (CN)
- Horiba (Japan) (JP)
- China Pharmaceutical University (CN)
- Hefei University of Technology (CN)
- Capital Medical University (CN)
- Chinese Academy of Sciences (CN)
- Peking University (CN)
- CHI Health Creighton University Medical Center - Bergan Mercy (US)
- Chinese PLA General Hospital (CN)
- Centrum Kopernika Badań Interdyscyplinarnych (PL)
- Shanghai Institute of Organic Chemistry (CN)
- Beijing National Laboratory for Molecular Sciences (CN)
- Evive Biotech (China) (CN)
- Peking University International Hospital (CN)
- Shenzhen Institutes of Advanced Technology (CN)
- Horiba (United Kingdom) (GB)
- Viva Biotech (China) (CN)
- State Key Laboratory of Natural and Biomimetic Drugs (CN)
- Academia Sinica (TW)
Publication Details
- Journal
- Journal of the American Chemical Society
- Published
- 2025-08-15
- DOI
- https://doi.org/10.1021/jacs.5c07897
- Citations
- 5
- Primary Topic
- Force Microscopy Techniques and Applications
- Type
- article
- Field-Weighted Citation Impact
- 6.23
Funders
- National Natural Science Foundation of China
- Beijing Municipal Science and Technology Commission
- Beijing Municipal Natural Science Foundation
- National Key Research and Development Program of China
- Anhui Provincial Key Research and Development Plan