Co-assembly of oligo-urethane nanoparticles with defined lipid additives to tailor RNA delivery into cells

Developing safe and effective biomaterials to deliver RNA into cells has grown in importance over recent years and has enabled the clinical translation of several gene therapies. Self-assembled oligo-urethane nanoparticles (PNPs) have several advantageous properties, such as (1) mitigating immune cell response in vivo, (2) low cytotoxicity in a broad range of cells, and (3) capable of delivering oligonucleotides or proteins into cells. Here, we were interested in defining unique configurations of PNPs to assess their relative cytotoxicity to human cells, comparing them to a popular commercial lipid system MessengerMax, as some cationic lipids have been found to have associated toxicity, instigating the search for less toxic counterparts. This was then followed by demonstrating the ability of enhanced green fluorescent protein (EGFP) mRNA to be loaded onto the PNPs, and to establish methods to enable the efficient transgene expression in a number of cell types, including Caco-2 and C₂C₁₂ cells which are known to be difficult-to-transfect cells. The addition of small amounts of PEGylated lipid to PNP significantly increased EGFP expression in HEK293T cells, Caco-2 cells and C₂C₁₂ cells at an effective mRNA/PNPs ratio (wt/wt) of 1:40, while the addition of the ionizable cationic lipid, significantly increased EGFP expression in C₂C₁₂ cells and differentiated C₂C₁₂ cells (myofibers) at mRNA/PNPs ratio (wt/wt) of 1:125, while keeping PNPs mass constant to 62.5 μg and changing the mRNA mass. Our study demonstrated that lipid-assisted, PNP-mediated mRNA delivery achieved a high transfection efficiency in terminally differentiated myofibers, paving the way for potential innovative therapeutics, e.g. targeted to muscular dystrophies. STATEMENT OF SIGNIFICANCE: There are reports of diverse delivery agents for RNA, but there is still unmet need for more versatile, and safe delivery vehicles. In this study we demonstrated that when compared to MessengerMax (a lipid-based mRNA transfection reagent), self-assembled oligo-urethane nanoparticle (PNP) showed favorable cell viability. The scope of PNP as a RNA delivery vehicle was extended beyond HEK293T cells by showing transfections into a number of cell types. The addition of select lipids to PNPs, significantly increased EGFP expression in difficult-to-transfect cells: Caco-2 (2.9-fold), C₂C₁₂ (1.4-fold) and differentiated C₂C₁₂ (1.28-fold) cells when compared to PNPs. These findings indicate that select lipid incorporation on PNPs provide a promising strategy to improve the efficacy of RNA therapeutics for diseases in clinical applications.