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

Ramsey N Majzoub; Chia-Ling Chan; Kai K Ewert; Bruno F B Silva; Keng S Liang; Cyrus R Safinya

doi:10.1016/j.bbamem.2015.03.001

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

Biochim Biophys Acta. 2015 Jun;1848(6):1308-18. doi: 10.1016/j.bbamem.2015.03.001. Epub 2015 Mar 6.

Authors

Ramsey N Majzoub¹, Chia-Ling Chan², Kai K Ewert¹, Bruno F B Silva³, Keng S Liang⁴, Cyrus R Safinya⁵

Affiliations

¹ Department of Physics, University of California, Santa Barbara, CA 93106, USA; Department of Materials, University of California, Santa Barbara, CA 93106, USA; Molecular, Cellular and Developmental Biology Department, University of California, Santa Barbara, CA 93106, USA.
² Department of Physics, University of California, Santa Barbara, CA 93106, USA; Department of Materials, University of California, Santa Barbara, CA 93106, USA; Molecular, Cellular and Developmental Biology Department, University of California, Santa Barbara, CA 93106, USA; Institute of Physics, Academica Sinica, Taipei 11529, Taiwan; National Synchrotron Radiation Research Center, Hsinchu 30076, Taiwan.
³ Department of Physics, University of California, Santa Barbara, CA 93106, USA; Department of Materials, University of California, Santa Barbara, CA 93106, USA; Molecular, Cellular and Developmental Biology Department, University of California, Santa Barbara, CA 93106, USA; Division of Physical Chemistry, Centre for Chemistry and Chemical Engineering, Lund University, SE-221 00 Lund, Sweden.
⁴ National Synchrotron Radiation Research Center, Hsinchu 30076, Taiwan; Department of Electrophysics, National Chiao-Tung University, Hsinchu 30010, Taiwan.
⁵ Department of Physics, University of California, Santa Barbara, CA 93106, USA; Department of Materials, University of California, Santa Barbara, CA 93106, USA; Molecular, Cellular and Developmental Biology Department, University of California, Santa Barbara, CA 93106, USA. Electronic address: safinya@mrl.ucsb.edu.

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, 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.

Keywords: Early endosomes; Fluorescence imaging; Gene delivery; Lipid–DNA nanoparticles; Particle colocalization; Rab GTPases.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Animals
Cations
Cell Line
Endosomes / metabolism*
Green Fluorescent Proteins / metabolism*
Lipids / chemistry*
Liposomes
Mice
Microscopy, Fluorescence
Models, Biological
Mutant Proteins / metabolism*
Nanoparticles / chemistry*
Nucleic Acids / chemistry*
Particle Size
Polyethylene Glycols / chemistry
Recombinant Fusion Proteins / metabolism
Transfection
rab5 GTP-Binding Proteins / metabolism*

Substances

Cations
Lipids
Liposomes
Mutant Proteins
Nucleic Acids
Recombinant Fusion Proteins
Green Fluorescent Proteins
Polyethylene Glycols
rab5 GTP-Binding Proteins

Abstract

Publication types

MeSH terms

Substances

Grants and funding