A rapid and reliable strategy for identification of LNP and protein corona in vitro and in vivo

TL;DR

This paper introduces a fast and reliable method to detect lipid nanoparticles and their protein corona in both lab and living conditions.

Contribution

The study combines SDS-PAGE, CBB staining, and MS techniques for visual verification and quantification of LNPs and their protein corona.

Findings

SDS-PAGE with CBB staining provides rapid visual verification and semi-quantification of LNPs with high linearity (R2 = 0.992).

LC-MS/MS and GC–MS accurately detect and quantify key lipids in LNPs with excellent linearity (R2 > 0.90).

In vivo and in vitro protein coronas differ significantly, with in vivo coronas enriched in opsonins like immunoglobulins.

Abstract

This study develops an integrated analytical method for visual verification and subsequent analysis of lipids and protein coronas of lipid nanoparticles (LNPs) in vitro and in vivo. In this strategy, SDS-PAGE combined with Coomassie brilliant blue (CBB) staining enabled rapid and reliable visual verification and semi-quantification of LNPs/lipids in solutions, yielding a linear standard curve of R2 = 0.992. LC-MS/MS and GC–MS provided specific detection and quantification of key lipids (SM-102, DSPC, DMG-PEG2000, cholesterol), with excellent linearity for all four lipids (R2 > 0.90). DMG-PEG2000 was quantifiable in the original LNP formulation but could not be reliably quantified after gel extraction due to the sample processing workflow. LNPs and associated protein corona were separated by SDS-PAGE and visualized by CBB after isolation from bulk solutions by size-exclusion chromatography (SEC) or sucrose density gradient centrifugation (S-DGC). LC-MS/MS and GC–MS detected SM-102, DSPC and cholesterol respectively in the forefront of SDS-PAGE gel loaded with factions eluted in the first peak of SEC chromatogram or the pellet layer of S-DGC. TEM confirmed the presence of intact LNPs in these fractions. We found that samples collected from both SEC and S-DGC yielded highly consistent protein coronas as measured by protein MS, enriched in apolipoproteins, immune-related proteins. In addition, pathways such as complement/coagulation and cholesterol metabolism were enriched. Importantly, SEC/SDS-PAGE/CBB combined with protein MS were successfully used to analyze in vivo LNP/protein corona, which revealed stark differences in composition between in vivo and in vitro protein corona, with opsonins such as immunoglobulins enriched in the former. Furthermore, we demonstrated that SDS-PAGE/CBB could be effectively used to indirectly assess LNP uptake by cells. In summary, a rapid and reliable analytical metheod for simultaneous detection of LNP and protein corona with visual verification is validated, which will improve quality control in study of LNP protein corona.

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