Multi-stage-mixing to control the supramolecular structure of lipid nanoparticles, thereby creating a core-then-shell arrangement that improves performance by orders of magnitude

As they became the dominant gene therapy platform, lipid nanoparticles (LNPs) experienced nearly all their innovation in varying the structure of individual molecules in LNPs. This ignored control of the spatial arrangement of molecules, which is suboptimal because supramolecular structure determines function in biology. To control LNPs’ supramolecular structure, we introduce multi-stage-mixing (MSM) to successively add different molecules to LNPs. We first utilize MSM to create a core-then-shell (CTS) synthesis. CTS-LNPs display a clear core-shell structure, vastly lower frequency of LNPs containing no detectable mRNA, and improved mRNA-LNP expression. With DNA-loaded LNPs, which for decades lagged behind mRNA-LNPs due to low expression, CTS improved DNA-LNPs’ protein expression by 2-3 orders of magnitude, bringing it within range of mRNA-LNPs. These results show that supramolecular arrangement is critical to LNP performance and can be controlled by mixing methodology. Further, MSM/CTS have finally made DNA-LNPs into a practical platform for long-term gene expression. Competing Interest Statement The authors have declared no competing interest. Footnotes

revised to clarify the core concept of using multi-staging system to build lipid nanoparticles with internal supramolecular nanostructure; Figures 1-4 reorganized to emphasize the material aspects of the multi-staging system lipid nanoparticles