The characteristics, cellular uptake and intracellular trafficking of nanoparticles made of hydrophobically-modified chitosan

Abstract

It has been reported that nanoparticles (NPs) prepared by hydrophobically-modified polymers could accumulate passively in the tumor tissue; however, their cellular uptake mechanism and intercellular trafficking pathway have never been understood. This study was designed to address these concerns, using NPs prepared by a hydrophobically-modified chitosan (N-palmitoyl chitosan, NPCS). Molecular dynamic simulations found that a degree of substitution (DS) of 5% of palmitoyl groups on its backbone was sufficient to allow NPCS to form NPs, due to a significant increase in the intra- and intermolecular hydrophobic interactions. With an increase of DS, there were more palmitoyl groups present on the surface of NPs which were then able to interact with the cell membranes. A greater extent of cellular uptake of NPCS NPs was observed with increasing the DS on NPCS. The internalization of NPCS NPs was clearly related with the lipid raft-mediated routes; with increasing the DS on NPCS, the caveolae-mediated endocytosis became more important. The results obtained in the intracellular trafficking study showed that NPCS NPs entered cells via caveolae and transiently localized to caveosomes before trafficking to the endosomal pathway. These results suggest that the prepared NCPS NPs may serve as a carrier for intracellular delivery of therapeutic agents.

Introduction

Chitosan (CS), a natural-origin polysaccharide, is biodegradable, non-toxic and soft-tissue compatible, and thus has been used extensively in biomedical applications [1]. It is known that the pKa of CS is approximately 6.5 [1], [2]. In aqueous media at pH 7.4, CS was found to form dissociated precipitates due to deprotonation of its amine groups. To enhance its intermolecular contact, we conjugated a hydrophobic palmitoyl group onto the free amine groups of CS in a previous study [3]. The synthesized N-palmitoyl CS (NPCS) in an aqueous environment was demonstrated to exhibit a rapid nanostructure transformation within a narrow pH range through a proper balance between charge repulsion and hydrophobic interaction. Subcutaneous injection of aqueous NPCS into a rat model resulted in rapid formation of a massive hydrogel at the location of injection.

In dilute aqueous media, we found that NPCS polymers were able to self-assemble into nanoparticles (NPs), due to the hydrophobic interaction between their conjugated palmitoyl groups. Hydrophobically-modified polymers have been used to fabricate NPs as a drug-delivery vehicle for therapeutic applications [4], [5], [6]. Previous studies have shown that these NPs could accumulate passively in the tumor tissue [4], [5]. The cellular uptake mechanism and intracellular fate of 5β-cholanic-acid conjugated glycol CS, a hydrophobic glycol CS (HGC), has been reported by Nam et al. [7]; interestingly, several distinct uptake pathways were identified to be involved in the internalization of HGC with a single degree of substitution (DS). Whether different DS (or hydrophobicity) could affect the endocytosis of hydrophobically-modified polymers remains to be understood. In the present study, we prepared NPs of NPCS with different DS; their cellular uptake and the potential endocytosis and intracellular trafficking pathways were investigated herein.

A few synthetic methods have been developed to conjugate hydrophobic side chains on the CS backbone. Nevertheless, these synthetic methods led to either a relatively low DS of 1–5% [8], [9], [10], [11], [12] or caused an uncontrollable DS and produced undesired by-products [13]. In this study, we employed a method which was able to produce NPCS with relatively high and controllable DS (up to 20% DS). The synthesis was accomplished in a single-step reaction which had been used in forming amide bonds in the synthesis of peptides [3], [14].

The NPs prepared by the synthesized NPCS polymers with different DS were characterized using dynamic light scattering (DLS) and molecular dynamic (MD) simulations. The cytotoxicity of NPs was evaluated by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay as well as the lactate dehydrogenase (LDH) assay. The cellular internalization efficiency, potential endocytosis mechanism and intracellular trafficking pathway of test NPs were investigated via a flow cytometer and a confocal laser scanning microscope (CLSM).