Lipid nanoparticles (LNPs), composed of ionizable lipids, phosphatidylcholine (PC) lipids, and cholesterol (CHOL), are the leading nonviral carriers for gene therapies. Extensive efforts have been devoted to optimizing the structure of ionizable lipids, which typically consist of an amine headgroup linked to alkyl tails to improve delivery efficacy. Even minor modifications, such as altering the branching of alkyl tails, can significantly affect the overall performance of the LNPs. However, direct observation of the nanoscopic properties of branched ionizable lipids (BILs) and their aggregates, including LNPs, remains challenging because of experimental limitations. Coarse-grained molecular dynamics (CG-MD) simulations provide valuable insights into such complex systems on relevant time and length scales. Here, we extended the SPICA force field to develop a more-accurate CG model for BILs. Using this model, we performed MD simulations of BIL-containing LNPs under various pH conditions. Our results reveal distinct morphologies of BIL-based LNPs, with double-stranded DNA (dsDNA) positioned between CHOL/DSPC domains, which serves as an effective binding site for BILs at physiological pH. Furthermore, acidification, as occurs in endosomes, induced reorganization in these domains, leading to ordering and a transformation of LNPs into spherical structures accompanied by changes in entropy. Overall, this study not only provides deeper insights into the structural and physicochemical properties of BIL-based LNPs but also establishes a framework for accelerating in silico design of BIL-based nanocarriers for gene delivery.
