An in vitro lipid-mixing assay to investigate the fusion between small extracellular vesicles and endosome.

Small extracellular vesicles (sEVs) such as exosomes can efficiently deliver nucleic acids into the cytosol of recipient cells. However, the molecular mechanism of the subsequent fusion with an endosome is not well understood. In this study, we developed an in vitro lipid-mixing assay using an endosomal-mimicking anionic liposome to investigate the fusion between sEVs and endosomes. We observed that the particle number ratio between the sEVs and the anionic liposomes, the diameter of the liposomes, and the buffer pH were all important for fusion activity. Furthermore, we optimized the liposomal lipid composition and demonstrated that incorporating the anionic lipid bis(monooleoylglycero) phosphate and cholesterol was important for efficient and reliable fusion. Our in vitro assay suggested that a decrease in pH increased the fusion activity. Additionally, it was suggested that this pH-dependent increase in the fusion activity was predominantly due to a change in the sEVs. sEVs possess a larger fusion activity than artificial liposomes that mimic the physicochemical properties of exosomes. These results are consistent with those of previous in vivo studies, supporting the physiological relevance of our system. This study provides an important platform for further research to clarify the molecular mechanisms of fusion between sEVs and endosomes.

Enhancing the Endocytosis of Phosphatidylserine-Containing Liposomes through Tim4 by Modulation of Membrane Fluidity.

Phosphatidylserine (PS) is a unique lipid that is recognized by the endogenetic receptor, T-cell immunoglobulin mucin protein 4 (Tim4), and PS-containing liposomes have potential use in therapeutic applications. We prepared PS-containing liposomes of various lipid compositions and examined how lipid membrane fluidity affects PS recognition by Tim4 and the resulting endocytosis efficiency into Hela cells. Surface plasmon resonance and laurdan studies showed that increasing lipid membrane fluidity increased the stability of the PS-Tim4 interaction but hampered the entry of liposomes into cells. These results show that endocytosis efficiency is determined by balancing opposing forces induced by membrane fluidity. We found that inclusion of the zwitterionic helper lipid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, into liposomes ensured efficient cellular internalization because the presence of this lipid provides an ideal balance of lipid fluidity and Tim4 affinity. The results showed that PS recognition by Tim4 and the resulting endocytosis efficiency can be maximized by modulating the membrane fluidity of liposomes by selecting a zwitterionic helper lipid. This study improves our understanding of how to rationally optimize nanotechnology for targeted drug delivery.

Effect of Sample Concentration on Nanoparticle Tracking Analysis of Small Extracellular Vesicles and Liposomes Mimicking the Physicochemical Properties of Exosomes.

For quantitative analysis, data should be obtained at a sample concentration that is within the range of linearity. We examined the effect of sample concentration on nanoparticle tracking analysis (NTA) of small extracellular vesicles (sEVs), including exosomes, by comparing NTA results of sEVs with those obtained for polystyrene nanoparticles (PSN) and liposomes, which mimic lipid composition and physicochemical properties of exosomes. Initially, NTA of PSN at different concentrations was performed and the particle sizes determined were validated by dynamic light scattering. The major peak maxima for PSN mixtures of different sizes at the higher particle numbers were similar, with some fluctuation of the minor peak maxima observed at the lower particle number, which was also observed for sEVs. Sample concentration is critical for obtaining reproducible data for liposomes and exosomes and increasing the sample concentration caused an increase in data variability because of particle interactions. The inter-day repeatability of particles sizes and concentration for sEVs were 7.47 and 4.51%, respectively. Analysis of the linearity range revealed that this was narrower for sEVs when compared with that of liposomes. Owing to the use of liposomes that mimic the lipid composition and physicochemical properties of exosomes and proteinase-treated sEVs, it was demonstrated that these different analytical results could be possibly caused by the protein corona of sEVs. Consideration of the sample concentration and linearity range is important for obtaining reproducible and reliable data of sEVs.

Acidic pH-induced changes in lipid nanoparticle membrane packing.

To enable the release of the encapsulated nucleic acids into the cytosol of targeted cells, the interaction of lipid nanoparticles (LNPs) with endosomes is critical. We investigated changes in the physicochemical properties of LNPs containing ionizable cationic lipids that were induced by acidic pH, which reflects the conditions in the maturation of endosomes. We prepared a LNP containing an ionizable cationic lipid. The laurdan generalized polarization values, which are related to the hydration degree of the lipid membrane interface and are often used as an indicator of membrane packing, decreased with a decrease in pH value, showing that the membrane packing was decreased under acidic conditions. Furthermore, the pH-induced variation increased with an increasing percentage of ionizable cationic lipids in the LNPs. These results indicated that electrostatic repulsion between lipid molecules at acidic pH decreased the packing density of the lipids in the LNP membrane. Reducing the order of lipids could be a trigger to form a non-bilayer structure and allow fusion of the LNPs with the membrane of maturing endosomes in an acidic environment. The LNPs were used to incorporate and transport small interfering RNA (siRNA) into cells for knockdown of the expression of ß-galactosidase. The knockdown efficiency of siRNA encapsulated in LNPs tended to increase with the ratio of KC2. These results, which demonstrate the underlying phenomena for the fusion of membranes, will help clarify the mechanism of the release of encapsulated nucleic acids.

Programmed multiple complexation for the creation of helical structures from acyclic phenol-bipyridine oligomer ligands.

Two new multidentate ligands, H3L(1) and H4L(2), possessing bipyridine-phenol repetitive units were designed so that the multi-metal complexation could produce a single-helical structure in a pre-programmed fashion. The ligands were synthesized by successive palladium-catalyzed coupling reactions. The complexation of H3L(1) with zinc(II) and nickel(II) acetate afforded [L(1)Zn2(OAc)] and [(L(1))2Ni4](OAc)2, respectively. Each of the ligand moieties in these complexes formed a one-turn single helix. The zinc(II) complex [L(1)Zn2(OAc)] underwent a helix compression-extension motion in solution. The complexation of the H3L(1) ligand with iron(III) chloride gave a dinuclear complex [(HL(1))2Fe2Cl2] with a non-helical dimeric structure. The longer ligand H4L(2) afforded a trinuclear complex [L(2)Zn3(OAc)2] with a 1.5-turn single-helical structure upon complexation with zinc(II) acetate. The reaction of the H4L(2) ligand with cobalt(II) acetate under aerobic conditions gave a mixed valence complex [L(2)Co3(OAc)3(OMe)], which had two trivalent and one divalent cobalt ions. The structural features of the trinuclear complexes significantly depended on the metals; [L(2)Co3(OAc)3(OMe)] had a helical pitch of 7.6 Å, which was almost twice that of [L(2)Zn3(OAc)2] (4.0 Å).

Hierarchical helix of helix in the crystal: formation of variable-pitch helical π-stacked array of single-helical dinuclear metal complexes.

Unique helix of helix structures were formed via intermolecular π-stacking and metal-metal interactions in the crystal of single-helical dinuclear complexes [L(2)M(2)] (M = Pd, Ni) having an acyclic bis(N(2)O(2))-type ligand. The difference in the helical winding angle of the constituents (401.7° for [L(2)Pd(2)]; 421.3° for [L(2)Ni(2)]) led to variation of the helical pitches of the helical array (7(2) helix for [L(2)Pd(2)]; 6(2) helix for [L(2)Ni(2)]).

Highly cooperative double metalation of a bis(N2O2) ligand based on bipyridine-phenol framework driven by intramolecular π-stacking of square planar nickel(II) complex moieties.

Highly cooperative double metalation took place when a novel ligand based on a bipyridine-phenol framework was allowed to react with nickel(II) acetate. The π-stacking of the square planar metal complex moieties is responsible for the highly cooperative double metalation judging from the X-ray crystal structure in which two complex moieties stack on top of each other in a parallel fashion.