bioRxiv : the preprint server for biologyOct 1, 2026

How lipid nanoparticles may disrupt cell recycling compartments

mRNA Technology Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Oct 2, 2026

Abstract

LP01 LNP shows maximal membrane engagement at pH 5.8, while BiP20 LNP requires pH 5.1.

Both LP01 and BiP20 LNPs have an apparent pKa of 6.6 under the tested conditions.
Electrostatic attraction and clustering precede membrane disruption when LNPs interact with the endosomal membrane.
LNP engagement causes membrane disruption by creating highly curved structures, ultimately leading to membrane rupture.
The release of mRNA cargo occurs only after the complete lysis of the endosomal membrane.
Membrane destabilization and cargo release are distinct mechanistic steps in the process of endosomal escape.

Simplified

Endosomal escape remains the central bottleneck limiting lipid nanoparticle (LNP)-mediated mRNA delivery. In this study, we use giant unilamellar vesicles (GUVs) mimicking the endosomal membrane to directly visualize the interaction and membrane disruptions caused by two model LNPs constituted by two different ionizable lipids: LP01 and BiP-20 (a Branched ionizable Phospholipid) that are both benchmark ionizable lipids for in vivo gene editing in liver. Despite identical TNS-measured apparent pKa (6.6) for both lipids in our conditions, LP01 LNP showed maximal membrane engagement at pH 5.8, while BiP20 LNP required pH 5.1, demonstrating that bulk pKa alone cannot predict the pH at which functional membrane interaction occurs. Using confocal microscopy, we show that the LNP engagement with the endosomal membrane proceeds through first electrostatic attraction, clustering, membrane disruption by forming highly curved morphologies, and finally leading to the rupture of the whole endosomal mimicking membrane. Notably, the mRNA cargo is released only upon complete lysis of the membrane. This indicates that the membrane destabilization and cargo release are mechanistically distinct steps. These findings establish a cell-free platform for dissecting individual sub-steps of endosomal escape.

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How lipid nanoparticles may disrupt cell recycling compartments

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