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Fluorescence microscopy colocalization of lipid-nucleic acid nanoparticles with wildtype and mutant Rab5-GFP: A platform for investigating early endosomal events.

Majzoub, Ramsey N; Chan, Chia-Ling; Ewert, Kai K; Silva, Bruno LU ; Liang, Keng S and Safinya, Cyrus R (2015) In Biochimica et Biophysica Acta 1848(6). p.1308-1318
Abstract
Endosomal entrapment is known to be a major bottleneck to successful cytoplasmic delivery of nucleic acids (NAs) using cationic liposome-NA nanoparticles (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<1h), only a fraction (≈35%) of RGD-tagged NPs (which target cell surface integrins) colocalize with wildtype EEs,... (More)
Endosomal entrapment is known to be a major bottleneck to successful cytoplasmic delivery of nucleic acids (NAs) using cationic liposome-NA nanoparticles (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<1h), only a fraction (≈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. (Less)
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organization
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type
Contribution to journal
publication status
published
subject
in
Biochimica et Biophysica Acta
volume
1848
issue
6
pages
1308 - 1318
publisher
Elsevier
external identifiers
  • pmid:25753113
  • wos:000353747500005
  • scopus:84925067809
ISSN
0006-3002
DOI
10.1016/j.bbamem.2015.03.001
language
English
LU publication?
yes
id
b241a0c0-b33b-48cf-9da3-589598a07e7a (old id 5264970)
date added to LUP
2015-04-16 13:58:33
date last changed
2017-10-01 04:24:43
@article{b241a0c0-b33b-48cf-9da3-589598a07e7a,
  abstract     = {Endosomal entrapment is known to be a major bottleneck to successful cytoplasmic delivery of nucleic acids (NAs) using cationic liposome-NA nanoparticles (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&lt;1h), only a fraction (≈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.},
  author       = {Majzoub, Ramsey N and Chan, Chia-Ling and Ewert, Kai K and Silva, Bruno and Liang, Keng S and Safinya, Cyrus R},
  issn         = {0006-3002},
  language     = {eng},
  number       = {6},
  pages        = {1308--1318},
  publisher    = {Elsevier},
  series       = {Biochimica et Biophysica Acta},
  title        = {Fluorescence microscopy colocalization of lipid-nucleic acid nanoparticles with wildtype and mutant Rab5-GFP: A platform for investigating early endosomal events.},
  url          = {http://dx.doi.org/10.1016/j.bbamem.2015.03.001},
  volume       = {1848},
  year         = {2015},
}