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dc.contributor.authorMajzoub, Ramsey N.en_US
dc.contributor.authorChan, Chia-Lingen_US
dc.contributor.authorEwert, Kai K.en_US
dc.contributor.authorSilva, Bruno F. B.en_US
dc.contributor.authorLiang, Keng S.en_US
dc.contributor.authorSafinya, Cyrus R.en_US
dc.date.accessioned2015-07-21T08:29:46Z-
dc.date.available2015-07-21T08:29:46Z-
dc.date.issued2015-06-01en_US
dc.identifier.issn0005-2736en_US
dc.identifier.urihttp://dx.doi.org/10.1016/j.bbamem.2015.03.001en_US
dc.identifier.urihttp://hdl.handle.net/11536/124630-
dc.description.abstractEndosomal entrapment is known to be a major bottleneck to successful cytoplasmic delivery of nucleic acids (NAs) using cationic liposome-NA nanopartides (NPs). Quantitative measurements of distributions of NPs within early endosomes (EEs) have proven difficult due to the sub-resolution size and short lifetime of wildtype EEs. In this study we used Rab5-GFP, a member of the large family of GTPases which cycles between the plasma membrane and early endosomes, to fluorescently label early endosomes. Using fluorescence microscopy and quantitative image analysis of cells expressing Rab5-GFP, we found that at early time points (t < 1 h), only a fraction (approximate to 35%) of RGD-tagged NPs (which target cell surface integrins) colocalize with wildtype EEs, independent of the NP\'s membrane charge density. In comparison, a GTP-hydrolysis deficient mutant, Rab5-Q79L, which extends the size and lifetime of EEs yielding giant early endosomes (GEEs), enabled us to resolve and localize individual NPs found within the GEE lumen. Remarkably, nearly all intracellular NPs are found to be trapped within GEEs implying little or no escape at early time points. The observed small degree of colocalization of NPs and wildtype Rab5 is consistent with recycling of Rab5-GDP to the plasma membrane and not indicative of NP escape from EEs.Taken together, our results show that endosomal escape of PEGylated nanoparticles occurs downstream of EEs i.e., from late endosomes/lysosomes. Our studies also suggest that Rab5-Q79L could be used in a robust imaging assay which allows for direct visualization of NP interactions with the luminal membrane of early endosomes. (C) 2015 Elsevier B.V. All rights reserved.en_US
dc.language.isoen_USen_US
dc.subjectLipid-DNA nanoparticlesen_US
dc.subjectFluorescence imagingen_US
dc.subjectParticle colocalizationen_US
dc.subjectRab GTPasesen_US
dc.subjectGene deliveryen_US
dc.subjectEarly endosomesen_US
dc.titleFluorescence microscopy colocalization of lipid-nucleic acid nanoparticles with wildtype and mutant Rab5-GFP: A platform for investigating early endosomal eventsen_US
dc.typeArticleen_US
dc.identifier.doi10.1016/j.bbamem.2015.03.001en_US
dc.identifier.journalBIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANESen_US
dc.citation.volume1848en_US
dc.citation.spage1308en_US
dc.citation.epage1318en_US
dc.contributor.department電子物理學系zh_TW
dc.contributor.departmentDepartment of Electrophysicsen_US
dc.identifier.wosnumberWOS:000353747500005en_US
dc.citation.woscount0en_US
Appears in Collections:Articles