Dual functionalization of chitosan/DNA complexes with mannose and hydroxyl-rich acids for enhanced gene delivery efficiency

The emergence of zoonotic diseases like COVID-19 has emphasized the pressing need for accelerated and effective vaccine development. DNA-based vaccines offer a promising platform owing to their adaptability, safety, and ease of production; nevertheless, their clinical application remains limited by poor transfection efficiency and rapid degradation. Chitosan, a biocompatible cationic polymer, has been extensively investigated as a gene delivery vehicle. Yet, its performance is often hindered by strong electrostatic interactions with nucleic acids, resulting in inefficient DNA release and suboptimal immune responses. In this study, we explored the functionalization of chitosan with hydroxyl-rich molecules, including gluconic acid/lactobionic acid and mannose, to modulate DNA binding and improve transfection efficiency. Mannose functionalization targeted antigen-presenting cells (APCs) through mannose receptors, while gluconic or lactobionic acid improved solubility, reduced electrostatic interactions, and facilitated DNA dissociation. This approach resulted in polyplexes with high transfection efficiency and effective APC internalization, partially mediated by the mannose receptor. Different chitosan derivatives: DNA ratios were tested in vitro. Dual-modified chitosan polyplexes, particularly CL-MAN 0.9-0.1 (combination of chitosan modified with lactobionic acid and mannose) and CGA-MAN 0.3-0.7 (combination of chitosan modified with gluconic acid and mannose) formulations, significantly increased TNF-α and nitric oxide production in macrophages. In human dendritic cells, mannose-targeted polyplexes enhanced surface maturation marker expression and pro-inflammatory cytokine production (TNF-α and IL-12) while avoiding IL-10 induction, essential for promoting a Th1-polarized immune response. These results indicate that this chitosan-based delivery system has strong potential as a mucosal vaccine adjuvant, offering an effective strategy for immune activation.