DNA vaccines still represent an emergent area of research, giving rise to continuous progress towards several biomedicine demands. The formulation of delivery systems to specifically target mannose receptors, which are overexpressed on antigen presenting cells (APCs), is considered a suitable strategy to improve the DNA vaccine immunogenicity. The present study developed binary and ternary carriers, based on polyethylenimine (PEI), octa-arginine peptide (R8), and mannose ligands, to specifically deliver a minicircle DNA (mcDNA) vaccine to APCs. Systems were prepared at various nitrogen to phosphate group (N/P) ratios and characterized in terms of their morphology, size, surface charge, and complexation capacity. In vitro studies were conducted to assess the biocompatibility, cell internalization ability, and gene expression of formulated carriers. The high charge density and condensing capacity of both PEI and R8 enhance the interaction with the mcDNA, leading to the formation of smaller particles. The addition of PEI polymer to the R8-mannose/mcDNA binary system reduces the size and increases the zeta potential and system stability. Confocal microscopy studies confirmed intracellular localization of targeting systems, resulting in sustained mcDNA uptake. Furthermore, the efficiency of in vitro transfection can be influenced by the presence of R8-mannose, with great implications for gene expression. R8-mannose/PEI/mcDNA ternary systems can be considered valuable tools to instigate further research, aiming for advances in the DNA vaccine field.

Nucleic acid vaccines have become a revolutionary technology to give a fast, safe, cost-effective and efficient response against viral infections, such as SARS-CoV-2 or Human papillomavirus (HPV). However, to ensure their effectiveness, the development of adequate methods to protect, carry, and deliver nucleic acids is fundamental. In this work, nanoparticles (NPs) of chitosan (CS)-tripolyphosphate (TPP)-plasmid DNA (pDNA) were thoroughly modulated and characterized, by measuring the charge and size through dynamic light scattering (DLS) and morphology by scanning electron microscopy (SEM). Stability, cytotoxicity and cellular uptake of NPs were also evaluated. Finally, the effect of polyplexes on the expression of HPV E7 antigen in human fibroblast and RAW cells was investigated through polymerase chain reaction (PCR) and real-time PCR. The results showed NPs with a spherical/oval shape, narrow size distribution <180 nm and positive zeta potentials (>20 mV) and good stability after one month of storage at 4 °C in formulation buffer or when incubated in culture medium and trypsin. In vitro studies of NPs cytotoxicity revealed that the elimination of formulation buffers led to an improvement in the rate of cell viability. The E7 antigen transcription was also increased for NPs obtained with high pDNA concentration (60 μg/mL). The analyzed CS-TPP-pDNA polyplexes can offer a promising vehicle for nucleic acid vaccines, not only in the prevention or treatment of viral infections, but also to fight emergent and future pathogens.