Functionalized drug delivery systems have been investigated to improve the targetability and intracellular translocation of therapeutic drugs. We developed high functionality and quality lipids that met unique requirements, focusing on the quality of functional lipids for the preparation of targeted nanoparticles using microfluidic devices. While searching for a lipid with high solubility and dispersibility in solvents, which is one of the requirements, we noted that KK-(EK) 4 -lipid imparts nonspecific cellular association to polyethylene glycol (PEG)-modified (PEGylated) liposomes, such as cell-penetrating peptides (CPPs). We investigated whether KK-(EK) 4 -lipid, which has a near-neutral charge, is a novel CPP-modified lipid that enhances the intracellular translocation of nanoparticles. However, the cellular association mechanism of KK-(EK) 4 -lipid is unknown. Therefore, we synthesized (EK) n -lipid derivatives based on the sequence of KK-(EK) 4 -lipid and determined the sequence sites involved in cellular association. In addition, KK-(EK) 4 -lipid was applied to extracellular vesicles (EVs) and mRNA encapsulated lipid nanoparticles (mRNA-LNPs). KK-(EK) 4 -lipid-modified EVs and mRNA-LNPs showed higher cellular association and in vitro protein expression, respectively, compared to unmodified ones. We elucidated KK-(EK) 4 -lipid to have potential for applicability in the intracellular delivery of liposomes, EVs, and mRNA-LNPs.

Targeted liposomes using ligand peptides have been applied to deliver therapeutic agents to the target sites. The postinsertion method is commonly used because targeted liposomes can be prepared by simple mixing of ligand peptide-lipid and liposomes. A large-scale preparation method is required for the clinical application of ligand-peptide-modified liposomes. Largescale preparation involves an increase in volume and a change in the preparation conditions. Therefore, the physicochemical properties of liposomes may change owing to large alterations in the preparation conditions. To address this issue, we focused on a microfluidic device and developed a novel ligand peptide modification method, the microfluidic post-insertion method. Methods: We used integrin αvβ3-targeted GRGDS (RGD) and cyclic RGDfK (cRGD)-modified high functionality and quality (HFQ) lipids, which we had previously developed. First, the preparation conditions of the total flow rate in the microfluidic device for modifying HFQ lipids to polyethylene glycol (PEG)-modified (PEGylated) liposomes were optimized by evaluating the physicochemical properties of the liposomes. The targeting ability of integrin αvβ3-expressing colon 26 murine colorectal carcinoma cells was evaluated by comparing the cellular association properties of the liposomes prepared by the conventional post-insertion method. Results: When the RGD-HFQ lipid was modified into PEGylated liposomes by varying the total flow rate (1, 6, and 12 mL/min) of the microfluidic device, as the total flow rate increased, the polydispersity index also increased, whereas the particle size did not change. Furthermore, the RGD-and cRGD-modified PEGylated liposomes prepared at a total flow rate of 1 mL/min showed high cellular association properties equivalent to those prepared by the conventional post-insertion method. Conclusion: Microfluidic post-insertion method of HFQ lipids might be useful for clinical application and large-scale preparation of targeted liposomes.