Not yet reviewed by Pith; the record is open.

Lipid nanoparticles (LNPs) are a leading platform in the delivery of RNA-based therapeutics, playing a pivotal role in the clinical success of mRNA vaccines and other nucleic acid drugs. Their performance in RNA encapsulation and delivery is critically governed by the molecular structure of ionizable lipids and the overall formulation composition. However, mechanistic insight into how these factors govern LNP architecture and function remains limited, primarily owing to the challenges of capturing nanoscale assembly and organization using experimental techniques. Here, we employ coarse-grained molecular dynamics simulations to systematically investigate how ionizable lipid chemistry influences LNP self-assembly, internal organization, and surface properties. We further explore the effects of formulation ratios and pH-dependent deprotonation on both the internal structure and surface morphology of LNPs. Leveraging these insights, we demonstrate how in silico structural characteristics can inform the rational design of novel ionizable lipids and optimization of formulation ratios, supported with experimental validations. Our findings offer a molecular-level understanding of LNP assembly dynamics and architecture, thereby establishing a computational framework linking lipid chemistry and LNP formulation to the structure and performance of LNP, to advance the rational design of novel LNP delivery systems.