Design, characterization, and intracellular trafficking of biofunctionalized chitosan nanomicelles

The hydrophobically modified glycol chitosan (HGC) nanomicelle has received increasing attention as a promising platform for the deliveryof chemotherapeutic drugs. To improve the tumor selectivity of HGC, here an avidin and biotin functionalization strategy was applied. Thehydrodynamic diameter of the biotin-avidin-functionalized HGC (cy5.5-HGC-B4F) was observed to be 104.7 nm, and the surface chargewas +3.1 mV. Confocal and structured illumination microscopy showed that at 0.1 mg/ml, cy5.5-HGC-B4F nanomicelles were distributedthroughout the cytoplasm of MDA-MB-231 breast cancer cells after 2 h of exposure without significant cytotoxicity. To better understandthe intracellular fate of the nanomicelles, entrapment studies were performed and demonstrated that some cy5.5-HGC-B4F nanomicelleswere capable of escaping endocytic vesicles, likely via the proton sponge effect. Quantitative analysis of the movements of endosomes inliving cells revealed that the addition of HGC greatly enhanced the motility of endosomal compartments, and the nanomicelles weretransported by early and late endosomes from cell periphery to the perinuclear region. Our results validate the importance of using live-cellimaging to quantitatively assess the dynamics and mechanisms underlying the complex endocytic pathways of nanosized drug carriers.