Yuya Sato

Suspension cell lines of hematopoietic origin are important model systems for immunology and hematologic cancer research because they provide self-renewing, accessible, and experimentally tractable...

Tat-PEG-lipid is added into cell suspension to induce cell fusion.

The advent of intracranial stents has revolutionized the endovascular treatment of cerebral aneurysms. The utilization of stents has rendered numerous cerebral aneurysm amenable to endovascular treatment, thereby obviating the need for otherwise invasive open surgical options. Stent placement has become a mainstream approach because of its safety and efficacy. However, further improvements are required for clinically approved devices to avoid the frequent occurrence of thrombotic complications. Therefore, controlling the thrombotic complications associated with the use of devices is of significant importance. Our group has developed a unique stent coated with a 2-methacryloyloxyethyl phosphorylcholine (MPC)-based polymer. In this study, the surface characteristics of the polymer coating were verified using X-ray photoelectron spectroscopy and atomic force microscopy. Subsequently, the antithrombotic properties of the coating were evaluated by measuring platelet count and thrombin–antithrombin complex levels of whole human blood after 3 h of incubation in a Chandler loop model. Scanning electron microscopy was utilized to examine thrombus formation on the stent surface. We observed that MPC polymer-coated stents significantly reduced thrombus formation as compared to bare stents and several clinically approved devices. Finally, the coated stents were further analyzed by implanting them in the internal thoracic arteries of pigs. Angiographic imaging and histopathological examinations that were performed one week after implantation revealed that the vascular lumen was well maintained and coated stents were integrated within the vascular endothelium without inducing adverse effects. Thus, we demonstrated the efficacy of MPC polymer coating as a viable strategy for avoiding the thrombotic risks associated with neurovascular stents.

Cell fusion has several biotechnological applications. We previously reported cell surface modification with interactive ligands to induce cell–cell attachment, resulting in higher cell fusion efficiency when combined with a polyethylene glycol (PEG)-induced method. However, spontaneous cell fusion never occurred, even though cell–cell contact was strongly induced by ligand modification. The interactive ligands placed between the two membranes might obstruct spontaneous cell fusion during lipid exchange between the two cellular membranes. We hypothesized that two distinct cell membranes must be brought together to realize efficient membrane fusion by temporal ligand modification. Here, we aimed to induce spontaneous membrane fusion with single-polymer conjugates using liposomes and cells. We synthesized poly(ethylene glycol)-lipids (PEG-lipids) carrying the Tat peptide (Tat-PEG-lipid), a representative cell-penetrating peptide, and used it for cell–cell anchoring. We found that Tat-PEG-lipid interacted with the bilayer membrane via the Tat peptide and lipid domain and induced membrane fusion when the lipid was nonanoyl (C9) or dodecanoyl (C12) and the PEG chain was 5 kDa, as assessed by quartz crystal microbalance with dissipation monitoring, dynamic light scattering, and fluorescence resonance energy transfer measurements, transmission electron microscopy, and confocal microscopy. It was found that Tat-PEG-lipid could induce membrane attachment and fusion of cells as well as liposomes, but it did not remain stable on the membrane. Thus, membrane fusion could be effectively promoted by the attachment of lipid membranes induced by transient surface modification with a Tat-PEG-lipid containing a nonanoyl or dodecanoyl lipid chain and a 5 kDa PEG chain.

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.

The use of amphiphilic molecules such as poly(ethylene glycol)-conjugated phospholipid (PEG-lipid) enables incorporation into liposome surfaces by exogenous addition as a result of the self-assembly with lipids. This technique can be applicable for manipulation of both liposomes and cells. In this study, we aimed to characterize Tat peptide (YGRKKRRQRRR)-conjugated PEG-lipids when used to exogenously surface modify liposomes (size: ca. 100 nm). We earlier reported that cells, which were surface modified with Tat peptides conjugated to PEG-lipids could attach spontaneously to material surfaces without any chemical modification. Here, we synthesized different types of Tat-PEG-lipids by combining PEG of different molecular weights (5 and 40 kDa) with different lipids with three acyl chains (myristoyl, palmitoyl, and stearoyl, respectively) and then studied the spontaneous adsorption of modified liposomes onto a substrate surface induced by the different Tat-PEG-lipids. The amount of adsorbed liposomes strongly depended on the number of incorporated Tat-PEG-lipid moieties: a decrease in both the PEG and the acyl chain lengths led to adsorption of higher amounts of liposomes. Furthermore, when a collagenase-cleavable amino acid sequence was inserted between the Tat sequence and the PEG segment, adsorbed liposomes could be harvested from the substrate by collagenase treatment with no difference in desorption efficiency between the different Tat-PEG-lipids. Thus, Tat-PEG-lipid can be a suitable tool for the manipulation of liposomes and cells.