Intracellular sorting of differently charged chitosan derivatives and chitosan-based nanoparticles

Chitosan (Chi) is a biodegradable nontoxic polycation with multiple reactive groups that is easily used to obtain derivatives with a desired charge and hydrophobic properties. The aim of this work was to study the intracellular traffic of positively charged hexanoyl-chitosan (HC) or HC-based nanoparticles (HCNPs) and negatively charged succinoyl-chitosan (SC) and SCNPs in epithelial and macrophage cell lines. By using flow cytometry we demonstrated that positively charged HC adhered to cell membranes quicker and more efficiently than negatively charged SC or NPs. However confocal studies showed that SC and SCNPs penetrated cells much more efficiently than HC while HCNPs did not enter the epithelial cells. Macrophages also phagocyted better negatively charged material but were able to engulf both HC and HCNPs. Upon entering the cells, SC and SCNPs were co-localized with endosomes and lysosomes while HC was found in mitochondria and, to a lesser extent, in lysosomes of epithelial cells. Macrophages, RAW264.7, more efficiently transported all Chi samples to the lysosomal compartment while some positively charged material was still found in mitochondria. Incubation of Chi derivatives and ChiNPs at pH specific to mitochondria (8.0) and lysosomes (4.5) demonstrated the neutralization of Chi charge. We concluded that epithelial cells and, to a lesser extent, macrophages sort charged material to the organelles neutralizing Chi charge.