Prompt Description

**Title:** Dissecting Host Protein Interactions Mediating LNP Lysosomal Escape Using Proximity Labeling Technology **Key Scientific Questions:** 1. How do the lipid components of LNPs specifically influence their lysosomal escape efficiency? Are there key lipid structural features that determine escape efficiency? 2. Which host cell proteins functionally interact with LNPs at the critical spatiotemporal nodes of lysosomal escape? How do these interacting proteins mediate or hinder the escape process? **Research Methods:** First, a multi-component lipid library will be constructed and screened in LLC-luc cells using the Siluc reporter system to identify "high-escape" and "low-escape" LNPs with significantly different lysosomal escape efficiencies. The subcellular localization of LNPs will be dynamically tracked using confocal microscopy to precisely determine their escape time window. Subsequently, at the critical time point of escape, Ce6 photoactivated proximity labeling technology will be used to spatiotemporally label host proteins proximal to LNPs, and interacting protein groups will be identified by mass spectrometry analysis. Candidate regulatory proteins will be screened by comparing the differential interacting proteins of high and low escape LNPs. Finally, CRISPR-Cas9 gene knockout technology will be used to verify the functional role of key proteins in LNP lysosomal escape. **Expected Conclusions:** This study is expected to establish a direct correlation map between LNP lipid composition and lysosomal escape efficiency, revealing key lipid chemical features that affect escape efficiency. For the first time, the dynamic interaction network between LNPs and host proteins will be captured at the precise spatiotemporal node of lysosomal escape, and several key host factors (such as specific membrane fusion proteins, lipid transfer proteins, or ion channels) that mediate or hinder the escape process will be identified. These findings will elucidate the molecular mechanism of LNP lysosomal escape, provide a theoretical basis and new engineering targets for the rational design of efficient delivery systems, and promote the development of nucleic acid drug delivery technology.