Nanoparticles Based on Natural Lipids Reveal Extent of Impacts of Designed Physical Characteristics on Biological Functions

Nanoparticles based on lipids (LNPs) are essential in pharmaceuticals and intercellular communication, and their design parameters span a diverse range of molecules and assemblies. In bridging the gap in insight between extracellular vesicles (EVs) and synthetic LNPs, one challenge is understanding their in-cell/in-body behavior when simultaneously assessing more than one physical characteristic. Herein, we demonstrate comprehensive evaluation of LNP behavior by using LNPs based on natural lipids (N-LNPs) with designed physical characteristics: size tuned using microfluidic methods, surface fluidity designed based on EV components, and stiffness tuned using biomolecules. We produce 12 types of N-LNPs having different physical characteristics─two sizes, three membrane fluidities, and two stiffnesses for in vitro evaluation─and evaluate cellular uptake vitality and endocytic pathways of N-LNPs based on the physical characteristics of N-LNPs. To reveal the extent of the impact of the predesigned physical characteristics of N-LNPs on cellular uptakes in vivo, we also carried out animal experiments with four types of N-LNPs having different sizes and fluidities. The use of N-LNPs has helped to clarify the extent of the impact of inextricably related, designed physical characteristics on transportation and provided a bidirectional guidepost for the streamlined design and understanding of the biological functions of LNPs.