A novel lipopolyplex platform for dual mRNA delivery via core- and surface-loading

The approval of Onpattro® (2018) and Comirnaty (2020) has driven interest in nanoparticulate nucleotide delivery. Newer concepts in gene therapy however, require not only the delivery of one, but multiple nucleotides. Examples are CRISPR/Cas9 gene editing and cancer immunotherapy. However, the current gold standard for nucleotide delivery - lipid nanoparticles - faces significant challenges, including limitations for co-encapsulation and nucleotide-nucleotide interactions. Aim of this study was to design a core-shell system featuring separate encapsulation of two nucleotides via a two-step formulation process. Six distinct cationic polymers were combined with three anionic polymers, resulting in 18 core compositions. Screening of these formulations identified three potent lipopolyplexes (LPPs), which were further evaluated and compared in terms of transfection efficiency, expression kinetics, storage stability, and nebulization performance. Among them, the combination of poly-L-arginine and poly-L-glutamic acid demonstrated the highest overall performance. Our systems enabled precise co-delivery of two model mRNAs in a controlled ratio, demonstrating potential for advanced therapeutic applications. Additionally, the role of mRNA localization within the LPP was investigated. Surface-loaded mRNA demonstrated superior transfection efficiency and shear resistance compared to core-loaded mRNA, which lost functionality under nebulization.