BACKGROUND: Nucleic acids are increasingly being recognized for their potential as therapeutic agents for the treatment of a variety of pathologies, such as genetic diseases, viral infections, and cancer. However, the safe delivery of these negatively charged macromolecules to their intended sites of action remains a major challenge. PURPOSE: This study aimed to design and characterize cationic particles for use as nonviral vectors for nucleic acid delivery; another primary objective was to evaluate the biocompatibility between the particles and DNA. METHODS: The particles were synthesized via a polyaddition process between isophorone diisocyanate and a mixture of polyethylene glycol and polycaprolactone diol. The structural characteristics were assayed using a variety of techniques, including measurements of pH, refractive index, and Zetasizer, drug release and penetration through an artificial membrane, SEM, FTIR and Raman spectroscopy, thermal analyses, cell viability, and in vivo evaluation of skin parameters. RESULTS: The findings revealed that nearly neutral-pH particles were successfully synthesized, displaying a broad size distribution ranging from 400–900 nm, a prolonged release profile, and an encapsulation efficacy of 72.5%. Thermal analyses demonstrated that the samples remained stable at temperatures up to 200 °C, and the results of the spectroscopy, cell assay, and evaluations on mouse skin suggest that the obtained particles are safe for use as DNA carriers. CONCLUSION: Cationic polyurethane carriers present a potential alternative to the more established polyethylenimine. However, additional studies are necessary to fully assess the therapeutic effectiveness of these formulations.