Pathologic Tissue Injury and Inflammation in Mice Immunized with Plasmid DNA-Encapsulated DOTAP-Based Lipid Nanoparticles.

Abstract

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