Low molecular weight chitosan nanoparticles for CpG oligodeoxynucleotides delivery: Impact of molecular weight, degree of deacetylation, and mannosylation on intracellular uptake and cytokine induction.

Although synthetic CpG oligodeoxynucleotides (ODNs) have shown substantial potential as immunotherapeutic agents, their effective intracellular delivery remains challenging. In this work, nanoparticles prepared from low-molecular weight (LMW) chitosans were investigated as CpG ODN delivery systems. Chitosan samples with a molecular weight (M) of 5 and 15 kDa and degree of deacetylation (DDA) of 50 and 80% were prepared. Additionally, mannosylated chitosans with a substitution degree of 15% were synthesized. The impact of LMW chitosan M and DDA on nanoparticle physical properties and the associated immunostimulatory effect in RAW 264.7 cells was studied. Nanoparticles prepared with chitosan of higher DDA and larger M exhibited better CpG ODN binding ability and intracellular uptake. Nevertheless, the most efficient immunostimulatory effect was observed while using 50% acetylated and mannosylated samples. The decreased charge density on chitosan backbone resulted in the enhanced intracellular CpG ODN release, which promoted in vitro cytokine secretion. Moreover, mannose ligand grafting promoted nanoparticle uptake through receptor-mediated recognition. Overall, this research suggests that chitosan structural parameters can be modulated to prepare LMW chitosan nanoparticles that first efficiently encapsulate CpG ODN, and then release it in immune cells, thus may be used as an efficient vector for intracellular CpG ODN delivery.