Co-localization of fluorescent labeled lipid nanoparticles with specifically tagged subcellular compartments by single particle tracking at low nanoparticle to cell ratios

Abstract

We utilized quantitative high-resolution single particle tracking to study the internalization and endosomal sorting of lipid nanoparticles (LNPs) by HeLa cells in vitro to gain a better understanding of how cells process LNPs that are used for siRNA delivery. We compared the trafficking of three formulations that have been demonstrated to deliver siRNA into cells. They were composed of either a tritratable anionic lipid, formulation of cholesterol hemisuccinate (CHEMS), or a titratatable cationic lipid formulation of 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA) or a non-titratable cationic formulation lipid formulation of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). They also contained either a substantial percentage of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol and 5 mole percent 1,2-dimyristoyl-sn-glycerol-[methoxy(polyethylene glycol)-2000 (PEG-DMG). We optically measured the endosomal pH experienced by individual LNPs, observed the internalization pathways used and tracked the particles as they co-localized with fluorescent protein tags on compartment-specific proteins, during endosomal sorting to the lysosome. The data revealed significant differences in the accumulation in subcellular compartments among the three formulations, which help to explain the observed effects LNP composition exerts on in vitro delivery efficiency.

Keywords:

• Endocytosis
• acidification
• nucleic acid delivery

Acknowledgements

We gratefully acknowledge Dr. Kurt Thorn and the UCSF Nikon Imaging center for use of the microscopes.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Funding

This work was supported by the National Institute of Health [R01EB003008] (FCS) and the NIH training grant T32 GM007175. Dr. Tiffany was partially supported by grant [R613-CR11] from the Cystic Fibrosis Foundation, 10.13039/100006501 (A. Verkman Principal Investigator).