Background/Objectives: Cervical cancer is a leading cause of cancer-related mortality among women, primarily driven by persistent infections with high-risk human papillomavirus (HPV), particularly HPV-16. Vaccines based on plasmid DNA encoding the viral oncogenes E6 and E7 represent a promising immunotherapeutic strategy, but their efficacy remains limited due to poor cellular uptake. Cell-penetrating peptides such as RALA improve intracellular delivery, and functionalization with octa-arginine peptide conjugated to mannose (R8M) further enhances targeting of antigen-presenting cells (APCs). This study aimed to obtain the minicircle DNA (mcDNA) encoding mutant HPV-16 E6 and/or E7 antigens, and optimize its complexation with mannosylated RALA-based nanoparticles to improve vector delivery and consequently antigen presentation. Methods: Nanoparticles were formulated at different concentrations of RALA, with and without R8M functionalization. Their characterization included hydrodynamic diameter, polydispersity index, zeta potential, complexation efficiency (CE), stability, morphology, and Fourier-Transform Infrared Spectroscopy. In vitro assays in JAWS II dendritic cells (DCs) assessed biocompatibility, transfection efficiency and target gene expression. Results: Optimal conditions were obtained at 72.5 µg/mL of RALA, producing nanoparticles smaller than 150 nm with high CE (>97%) and uniform size distribution. Functionalization with R8M at 58 µg/mL preserved these characteristics when complexed with all mcDNA vectors. The formulations were biocompatible and effectively transfected DCs. Mannosylated formulations enhanced antigenic expression compared to non-mannosylated counterparts, evidencing a mannose-receptor-mediated uptake, while increasing the production of pro-inflammatory cytokines. Conclusions: Nanoparticles based on the RALA peptide and functionalized with R8M significantly improved mcDNA transfection and gene expression in APCs. These findings support further investigation of this system as a targeted DNA vector delivery platform against HPV-16.