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

• GTP hydrolysis-deficient Rab5 slows endosome maturation and increases their size.
• A small fraction of nanoparticles colocalize with wildtype Rab5 labeled endosomes.
• Nearly all intracellular nanoparticles colocalized with mutant early endosomes.
• The absence of Rab5-nanoparticle colocalization does not indicate endosomal escape.
• Endosomal escape occurs downstream of early endosomes.

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 < 1 h), 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.

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