In current times, DNA vaccines are seen as a promising approach to treat and prevent diseases, such as virus infections and cancer. Aiming at the production of a functional and effective plasmid DNA (pDNA) delivery system, four chitosan polymers, differing in the molecular weight, were studied using the design of experiments (DoE) tool. These gene delivery systems were formulated by ionotropic gelation and exploring the chitosan and TPP concentrations as DoE inputs to maximize the nanoparticle positive charge and minimize their size and polydispersity index (PDI) as DoE outputs. The obtained linear and quadratic models were statistically significant (p-value < 0.05) and non-significant lack of fit, with suitable coefficient of determination and the respective optimal points successfully validated. Furthermore, morphology, stability and cytotoxicity assays were performed to evaluate the endurance of these systems over time and their further potential for future in vitro studies. The subsequent optimization process was successful achieved for the delivery systems based on the four chitosan polymers, in which the smallest particle size was obtained for the carrier containing the 5 kDa chitosan (~82 nm), while the nanosystem prepared with the high molecular weight (HMW) chitosan displayed the highest zeta potential (~+26.8 mV). Delivery systems were stable in the formulation buffer after a month and did not exhibit toxicity for the cells. In this sense, DoE revealed to be a powerful tool to explore and tailor the characteristics of chitosan/pDNA nanosystems significantly contributing to unraveling an optimum carrier for advancing the DNA vaccines delivery field.

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 been proven to be a revolutionary technology to induce an efficient, safe and rapid response against pandemics, like the coronavirus disease (COVID-19). Minicircle DNA (mcDNA) is an innovative vector more stable than messenger RNA and more efficient in cell transfection and transgene expression than conventional plasmid DNA. This work describes the construction of a parental plasmid (PP) vector encoding the receptor-binding domain (RBD) of the S protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the use of the Design of Experiments (DoE) to optimize PP recombination into mcDNA vector in an orbital shaker. First, the results revealed that host cells should be grown at 42 °C and the Terrific Broth (TB) medium should be replaced by Luria Broth (LB) medium containing 0.01% L-arabinose for the induction step. The antibiotic concentration, the induction time, and the induction temperature were used as DoE inputs to maximize the % of recombined mcDNA. The quadratic model was statistically significant (p-value < 0.05) and presented a non-significant lack of fit (p-value > 0.05) with a suitable coefficient of determination. The optimal point was validated using 1 h of induction, at 30 °C, without the presence of antibiotics, obtaining 93.87% of recombined mcDNA. Based on these conditions, the production of mcDNA was then maximized in a mini-bioreactor platform. The most favorable condition obtained in the bioreactor was obtained by applying 60% pO₂ in the fermentation step during 5 h and 30% pO₂ in the induction step, with 0.01% L-arabinose throughout 5 h. The yield of mcDNA-RBD was increased to a concentration of 1.15 g/L, when compared to the orbital shaker studies (16.48 mg/L). These data revealed that the bioreactor application strongly incremented the host biomass yield and simultaneously improved the recombination levels of PP into mcDNA. Altogether, these results contributed to improving mcDNA-RBD biosynthesis to make the scale-up of mcDNA manufacture simpler, cost-effective, and attractive for the biotechnology industry.