Pathologic\nTissue Injury and Inflammation in Mice\nImmunized with Plasmid DNA-Encapsulated DOTAP-Based Lipid Nanoparticles

Ionizable cationic lipids have been developed to mitigate\nthe toxicity\nof quaternary ammonium lipids, such as DOTAP. Despite its toxicity,\nDOTAP can promote localization of lipid nanoparticles (LNPs) in target\ntissues, serving as one of the ionizable cationic helper lipids. Notably,\nDOTAP-based nanoadjuvants prepared via microfluidic methods showed\na better T-cell response. Previous studies showed that DOTAP-based\nLNPs prepared by the lipid-film method resulted in obvious adverse\nevents. Therefore, our research focused on evaluating the tissue localization\nand adverse toxicity of a DOTAP-based delivery system prepared through\nmicrofluidic techniques. We assessed the delivery efficacy, biodistribution,\ninflammatory response, and pathological injury in various tissues.\nIn our study, the plasmid DNA encoding the receptor-binding domain\n(RBD) of SARS-CoV-2 was encapsulated using a mixture of lipids that\nincluded DOTAP, DOPE, cholesterol, and DMG-PEG2000 via microfluidic\nmixing. The LNP-RBDs were smaller than those prepared via the traditional\nlipid membrane system. We found that LNP-DNA complexes can be effectively\ndelivered and expressed in muscle tissue, with specific antibodies\nin serum induced postimmunization. Initial distribution of the liposomes\nwas observed in the muscle and liver. Interestingly, both LNPs and\nDNA showed sustained presence in the lungs and spleen in the group\nimmunized with DNA-encapsulated DOTAP-based LNPs, whereas lower amounts\nof DNA were detected in the group immunized with dissociative DNA.\nWe detected obvious inflammatory responses and pathological injuries\nin the muscle, heart, and liver, and the side effects decreased when\nthe immunization dose decreased. These findings suggest that DOTAP-based\nLNPs have obvious advantages for targeting the lungs and spleen. Additionally,\ninflammatory responses and pathological injuries occur in a dose-dependent\nmanner in the muscles, heart, and liver. In conclusion, these findings\ncontribute to the development of an LNP delivery system with DOTAP,\nhighlighting its potential to enhance tissue localization and promote\nhigh levels of expression when coordinated with ionizable lipids.