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.
Keywords: Cellular uptake; Extracellular vesicle; Lipid nanoparticle; Microfluidics; Physical characteristic.