Fluorescence microscopy colocalization of lipid-nucleic acid nanoparticles with wildtype and mutant Rab5-GFP: A platform for investigating early endosomal events.
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.
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.
Ramsey N
RN
Majzoub
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.
Chia-Ling
CL
Chan
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.
Kai K
KK
Ewert
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.
Bruno F B
BF
Silva
National Synchrotron Radiation Research Center, Hsinchu 30076, Taiwan; Department of Electrophysics, National Chiao-Tung University, Hsinchu 30010, Taiwan.
Keng S
KS
Liang
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.
Cyrus R
CR
Safinya
D000818:Animals; D002412:Cations; D002460:Cell Line; D011992:Endosomes / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D008055:Lipids / Q000737:chemistry*; D008081:Liposomes; D051379:Mice; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism*; D053758:Nanoparticles / Q000737:chemistry*; D009696:Nucleic Acids / Q000737:chemistry*; D010316:Particle Size; D011092:Polyethylene Glycols / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism*
D002412:Cations; D008055:Lipids; D008081:Liposomes; D050505:Mutant Proteins; D009696:Nucleic Acids; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011092:Polyethylene Glycols; D020696:rab5 GTP-Binding Proteins
Early endosomes; Fluorescence imaging; Gene delivery; Lipid–DNA nanoparticles; Particle colocalization; Rab GTPases