Co-localization of fluorescent labeled lipid nanoparticles with specifically tagged subcellular compartments by single particle tracking at low nanoparticle to cell ratios.
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.
a Department of Bioengineering, Therapeutic Sciences and Pharmaceutical Chemistry , University of California , San Francisco , CA , USA.|b Department of Pediatrics and Genetics , Stanford University School of Medicine , Stanford , CA , USA.
Matthew
M
Tiffany
a Department of Bioengineering, Therapeutic Sciences and Pharmaceutical Chemistry , University of California , San Francisco , CA , USA.
Francis C
FC
Szoka
D001692:Biological Transport / Q000502:physiology; D002784:Cholesterol; D016503:Drug Delivery Systems; D005229:Fatty Acids, Monounsaturated; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006801:Humans; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry*; D008156:Luciferases; D053758:Nanoparticles / Q000737:chemistry* / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D011412:Propanolamines / Q000737:chemistry; D000644:Quaternary Ammonium Compounds; D034741:RNA, Small Interfering; D014280:Triglycerides / Q000737:chemistry
C000626006:1,2-dilinoleyloxy-3-dimethylaminopropane; D005229:Fatty Acids, Monounsaturated; D005456:Fluorescent Dyes; D008055:Lipids; D010714:Phosphatidylethanolamines; D011412:Propanolamines; D000644:Quaternary Ammonium Compounds; D034741:RNA, Small Interfering; D014280:Triglycerides; C094877:dioleoyl phosphatidylethanolamine; D011092:Polyethylene Glycols; D002784:Cholesterol; C000626005:DMG-PEG 2000; D008156:Luciferases; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane
Endocytosis; acidification; nucleic acid delivery