Delivery Order of Nanoconstructs Affects Intracellular Trafficking by Endosomes

This paper reports how the endosomal pathways of nanoparticle (NP) constructs with different surface curvatures are affected by their order of delivery. Sequential incubation of cytosine–phosphate–guanine (CpG)-conjugated spiky and spherical gold NPs with macrophages resulted in different nanoconstruct ratios at the interior edges of endosomes. Application of spiky NPs after spherical NPs accelerated the formation of late-stage endosomes and resulted in larger endosomes, and the spherical NPs were enclosed by the spiky NPs. In contrast, the reverse incubation order produced an asymmetric distribution of the two nanoconstruct shapes in smaller endosomes. Macrophages with a higher proportion of the enclosed spherical NPs as well as a larger ratio of spiky to spherical NPs at the endosomal edge showed enhanced toll-like receptor 9 activation and secretion of proinflammatory cytokines and chemokines. Our results indicate that the subcellular trafficking of targeting nanoconstructs by vesicles is affected by both the delivery order and the endosomal distribution. Our study also establishes a new approach for nanoscale monitoring of intracellular therapeutics delivery with conventional electron microscopy.