Synthesis and Characterization of Mannosylated Formulations to Deliver a Minicircle DNA Vaccine

Abstract

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.

Design of Experiments to Achieve an Efficient Chitosan-Based DNA Vaccine Delivery System

“…Furthermore, the nanoparticles stability was also evaluated following their incubation in the cell culture medium, with or without FBS supplementation, to simulate the in vitro cell transfection and in vivo extracellular conditions, to ensure the success of cellular transfection of these chitosan nanoparticles [ 13 , 35 ]. Thus, all chitosan nanoparticles were resuspended and incubated for 6 h at 37 °C in two different mediums, MEM-α (normally used for dendritic cell culture) and DMEM-F12 (normally used for human fibroblast culture) with and without supplements.…”

“…As demonstrated in Figure 3 , for both media, the different chitosan nanoparticles remained stable since it was not possible to find pDNA at the first moment of resuspension (0 h) or after 6 h of the incubation process, due to the absence of nucleic acid bands in the electrophoretic mobility profile. It is noteworthy that the presence of FBS in the medium does not have any destabilizing effect on the nanoparticles, being a good indicator of the resilience of the nanoparticles in the extracellular environment [ 13 , 35 ]. The effect observed in the agarose gels of Figure 3 C,D is due to the culture medium, since lane 2 corresponds only the sample medium and presents the same aspect.…”

“…The mutated E7 gene was cloned into pMC.CMV-MCS-EF1-GFP-SV40polyA parental minicircle cloning vector, next to CMV7 promoter, as described previously [ 13 ]. Thus, the plasmid DNA containing the mutated E7 gene (pDNA-E7mut) was amplified in ZYCY10P3S2T Escherichia coli host strain.…”

“…The statistical analysis was performed with two-way ANOVA followed by Tukey test (GraphPad Prism 9 software, San Diego, CA, USA). The second assay aimed to evaluate the nanoparticle’s stability upon their incubation in the cell culture medium, with and without FBS supplementation, to mimic the in vitro cell transfection conditions and extracellular environment of human body, to ensure the success of cellular transfection of these chitosan nanoparticles [ 13 , 35 ]. Thus, nanoparticles were centrifuged and resuspended in 50 μL of DMEM-F12 or MEM alpha medium supplemented with 10% FBS or without this supplementation.…”

“…However, the transfection efficiency of eukaryotic cells with naked nucleic acids, such as plasmid DNA, is disfavored by the negative repulsion of cell membrane. In this field, several biomaterials based on cationic liposomes [9,10], peptides [11,12] and polymers [13,14] have been shown to be able to efficiently condense, protect, carry, and deliver nucleic acids to eukaryotic cells. For instance, chitosan and chitosan derivatives have been used as DNA delivery systems due to their cationic charge, biocompatibility, biodegradability, mucoadhesiveness and permeability-enhancing properties [15][16][17][18][19].…”