Novel Responsive Lipid Nanoparticles for Multidrug-Resistant Organism Therapeutics

Multidrug-resistant organisms (MDROs) are a type of microorganism that have evolved defences against many classes of antibiotics. Given the limited ways of treating MDRO-associated infections, they are now considered a threat to global health. Thus, research for the next generation of antimicrobial technologies is focusing on developing targeted therapies against which harmful bacteria cannot acquire resistance. Research on lipid nanoparticles (LNPs) with cationic lipids shows promise as treatment tools for addressing these drug resistance challenges. This project aims to develop novel LNPs with tuneable surface charges and inverse lyotropic liquid crystalline mesophases, both known to promote membrane fusion and interaction. The hypothesis is that by tuning these two properties (charge and mesophase) using different compositions of helper lipids and cationic/ionisable lipids, the formulated novel LNPs can effectively interact with specific MDROs. The lipids used in the study included monoolein (MO) as the main structure-forming lipid, which can self-assemble to a range of lyotropic liquid crystalline mesophases, including the fascinating cubic and hexagonal phases known to promote membrane fusion, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP; a cationic lipid), ALC-0315, and SM-102 (two ionisable lipids). The LNPs exhibited typical particle sizes and good dispersibility. After a month of storage, some LNPs displayed decreasing particle sizes, indicating lipid rearrangement and particle aggregation without significant deterioration. Notably, the LNPs possessed a positive surface charge under specific conditions, making them potentially interactive with negatively charged cell membranes. This positive charge was dose-dependent and remained stable over time, highlighting the potential biological significance of the size and surface charge of LNPs. The study also delved into the pH-responsive behaviour of the LNPs, showing that the chemical composition and concentration of added lipids, as well as pH conditions, influenced the internal mesophases of the LNPs. Furthermore, adding oleic acid (OA) to LNPs enhanced their interactions with microorganisms, leading to changes in particle size, surface charge, and structural transitions and potentially impacting microorganism interactions. Among the tested LNPs, SM, and DOTAP showed the most significant inhibition of E. coli growth, with other LNPs exhibiting moderate toxicity. Cellular confocal laser scanning microscopy imaging indicated that MO and ALC LNPs had similar toxicity, while DOTAP and SM LNPs showed notably stronger antimicrobial effects. The addition of OA to LNPs significantly enhanced their antimicrobial activity against E. coli; extended incubation further confirmed the impact of OA-containing LNPs on bacterial growth, highlighting their potential as effective antibacterial agents. The experiments demonstrated the effectiveness of MO, ALC, and SM LNPs in inhibiting methicillin-resistant Staphylococcus aureus (MRSA) growth. In particular, MO exhibited strong inhibitory effects on bacterial growth, surpassing positively charged LNPs containing ionisable and cationic lipids. The incorporation of OA in LNPs enhanced the inhibition of MRSA in short-term PBS treatment but not during long-term broth treatment. Notably, DOTAP LNPs, with or without OA, were less effective against MRSA than other LNPs, raising questions about the role of inverse lyotropic liquid crystalline structures in LNPs and their bacterial interactions. Finally, the interaction and inhibitory effects of LNPs were assessed on sensitive and resistant (to amphotericin B and fluconazole) strains of Cryptococcus neoformans. The determinants affecting LNP–fungi interactions are multifaceted, encompassing LNP composition, physicochemical attributes, fungal strains, and growth conditions. Despite these challenges, a consistent trend emerged, highlighting the fusogenic cubic phase, especially that of MO, as a top attribute for disrupting fungal cell walls, even though these walls are more rigid than cell membranes. Positively charged LNPs tended to have a greater disruptive effect on fungi. LNPs possess stable positive surface charges and can be further enhanced with the addition of OA to improve interactions with microorganisms. In summary, the findings indicate that LNPs with fusogenic cubic phases and positive surface charges hold promise as versatile antimicrobial agents with potential applications in addressing MDRO challenges.