(141b) Lipid Nanoparticle Permeabilization and Fusion Quantified By Two High Throughput Fluorescence Assays

Lipid nanoparticles (LNP) containing mRNA have shown success as drug delivery tools and as vaccines against infectious diseases. LNP synthesis is cost-effective and amenable to scaling, however, the LNP design space is high dimensional and it is not well known a priori how a given LNP will perform in animal or cell systems as a function of its chemical formulation. Specifically, varying the relative amount and identity of just the helper lipid and ionizable lipid have both been shown to change the efficacy of LNP delivery, cellular toxicity, and organ specificity. In this study, we develop two complementary fluorescence-based assays to quantify the degree and rate of LNP induced permeabilization as well as membrane fusion with a liposomal artificial cell target. By varying nanoparticle parameters such as LNP composition and concentration as well as environmental parameters such as temperature and pH, we probe the dependency of the cellular environment on LNP interactions.

In our work, we find that the ionizable lipid identity, helper lipid identity, target liposomal membrane composition, temperature, and LNP concentration are all controllers of cell membrane permeability. We next explore LNP activity in neutral and acidic environments to determine the effect of different ionizable lipid groups in membrane fusion. We find that the rate and extent of LNP membrane fusion is pH-sensitive, and dependent on the same variables as in the permeability study. For both the permeability and fusion assays, we are able to fit a phenomenological kinetic model to high accuracy to quantify and compare the dependencies on LNP composition, liposome composition, and environmental variables.

By collecting quantitative LNP metrics determined in vitro and relating these to in vivo results, we can better understand and inform design of targeted delivery agents for improved nanoparticle therapeutics.