Mass Spectrometry-Assisted High-Throughput Discovery of Lung-Specific Lipid Nanoparticles for <i>In Vivo</i> Protein Delivery

Precise and tissue-specific delivery of proteins remains a major barrier to realizing their therapeutic potential. Here, we report a mass spectrometry-assisted high-throughput screening (MSHTS) strategy for rapid identification of lung-targeting lipid nanoparticles (LNPs). A combinatorial library of 46 thioketal-containing biodegradable lipids was synthesized via Michael addition, enabling multiplexed biodistribution profiling in a single animal. MSHTS identified BDMPA-TK12 with preferential lung accumulation for <i>in vivo</i> protein delivery. Proteomic analysis revealed the formation of a vitronectin-enriched protein corona that promotes receptor-mediated uptake of BDMPA-TK12 by pulmonary endothelial cells. The optimized LNP efficiently delivers functional proteins, including Cre recombinase and antioxidant DJ-1, to the lung across multiple animal species. Lung-specific delivery of DJ-1 activates MAPK signaling, mitigating ferroptosis, and inflammatory injury in acute lung inflammation. Our study establishes MSHTS as a scalable strategy for discovering tissue-selective nanoparticles and significantly advances LNP-mediated delivery of proteins for diverse biomedical applications.