Exosomes (diameter 30-200 nm) are a subtype of extracellular vesicles secreted by cells containing DNA, microRNA (miRNA), and proteins. Exosomes are expected to be valuable as a means of delivering drugs or functional miRNAs in treatment of diseases. However, the delivery of exosomes is not sufficiently effective, even though exosomes have intrinsic delivery functions. Cell-penetrating peptides (CPPs) are short peptide families that facilitate cellular intake of molecules and vesicles. We previously reported that the modification of cells, and liposomes with CPP-conjugated-lipids, CPPs conjugated with poly (ethylene glycol)-conjugated phospholipids (PEG-lipid), that induce adhesion by CPPs, can be useful for cell-based assays and harvesting liposomes. In this study, we aimed to modulate the exosome surface using Tat peptide (YGRKKRRQRRR)-PEG-lipids to improve intracellular delivery to endothelial cells. We isolated and characterized exosomes from the medium of HEK 293 T cell cultures. Tat conjugated PEG-lipids with different spacer molecular weights and lipid types were incorporated into exosomes using fluorescein isothiocyanate labeling to optimize the number of Tat-PEG-lipids immobilized on the exosome surface. The exosomes modified with Tat-PEG-lipids were incubated with human umbilical vein endothelial cells (HUVECs) to study the interaction. Tat conjugated with 5 kDa PEG and C16 lipids incorporated on the exosome surface were highly detected inside HUVECs by flow cytometry. Fluorescence was negligible in HUVECs for control groups. Thus, Tat-PEG-lipids can be modified on the exosome surface, improving the intracellular delivery of exosomes.

Extracellular vesicles (EVs) are natural carriers for intercellular communication within the human body. Mimicking and utilizing EVs by combining them with artificial nanocarriers such as liposomes for drug delivery has garnered considerable attention. However, current technologies for manipulating EVs to facilitate their fusion with liposomes are limited; the existing technique of polyethylene glycol (PEG)-induced fusion is highly inefficient for fusion. In our previous study, we demonstrated that membrane fusion could be induced by Tat peptide (YGRKKRRQRRR)-conjugated poly(ethylene glycol)-phospholipids (Tat-PEG-lipids), in which the Tat peptide and lipid domain facilitate membrane attachment and subsequent fusion between cells and liposomes. This approach is promising for forming EV and liposomal hybrids. In this study, we aim to fuse EVs and liposomes using Tat-PEG-lipids. We isolated and characterized EVs derived from HEK293T cell culture medium and treated a mixture of EVs and liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and cholesterol (1:1, molar ratio), with Tat-PEG-lipids with different lipid chain lengths. Here, we used nonanoyl (C9), dodecanoyl (C12), and myristoyl (C14) groups as lipid anchors with 5 kDa PEG chains. Dynamic light scattering analysis revealed a large increase in the apparent size of mixture of EVs and liposomes by adding Tat-PEG-lipids (especially C14, C12, followed by C9). Fluorescence resonance energy transfer, confocal laser scanning microscopy, and transmission electron microscopy, used to analyze the reaction process, revealed that the membrane fusion occurred between EVs and liposomes but not their aggregates. The short lipid domain of Tat-PEG-lipids effectively induced membrane fusion and the formation of hybrid EVs and liposomes. Thus, Tat-PEG-lipids (C9 and C12) could be promising candidates for inducing membrane fusion to fabricate EV-liposome hybrids.

PEG is frequently used for liposomal surface modification. However, the PEGylation can cause immune response. Here we synthesized PMPC-lipid for liposome coating. We found that PMPC-lipid can be alternative to PEG-lipid for liposome coating.

Liposome surface coating has been studied to avoid the immunological responses caused by the complement system, and alternative materials to poly(ethylene glycol) (PEG) have been explored recently since the production of anti-PEG IgM antibodies has been found in human. We previously reported a liposome coating with poly(2-methacryloyloxyethyl phosphorylcholine) (poly(MPC))-conjugated lipids (PMPC-lipids) and demonstrated its protective effect on blood protein interactions. Here, we attempted to modify the liposome surface by exogenously adding PMPC-lipids, which were spontaneously incorporated into the outer membrane via hydrophobic interactions. The polymerization degree of the PMPC segment was regulated from 10–100. The incorporated ratio of PMPC-lipid increased with a decrease in the degree of PMPC polymerization. Due to surface modification with PMPC-lipids, increase in the length of the PMPC-chain increased the size of the liposomes. The modified liposomes were kept stable for 14 d in terms of their size, polydispersity, and surface properties, where approximately 70% of PMPC-lipids were incorporated into the liposome surface. We demonstrated that liposome surface modification with PMPC-lipids can inhibit protein adsorption when exposed to serum, regardless of the degree of polymerization of PMPC. In addition, the PMPC-lipid modified surface was not recognized by the anti-PEG IgM antibody, whereas PEG-lipid was recognized by the antibody. Thus, we successfully fabricated an inert liposome surface via spontaneous modification with PMPC-lipids, where only outer bilayer surface was modified. This technique can be available for full loading of water-soluble active pharmaceutical ingredient inside the modified liposome.