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A03 HAMADA, Tsutomu |Proposed Research Projects (2014-2015)

Paper | Original Paper

2015

Hiroki Himeno, Hiroaki Ito, Yuji Higuchi, Tsutomu Hamada, *Naofumi Shimokawa, Masahiro Takagi,
Coupling between pore formation and phase separation in charged lipid membranes,
Physical Review E 92, 62713 (2015).

[Summary] We investigated the effect of charge on the membrane morphology of giant unilamellar vesicles (GUVs) composed of various mixtures containing charged lipids.

Thapakorn Tree-udom, Jiraporn Seemork, Kazuki Shigyou, Tsutomu Hamada, Naunpun Sangphech, Tanapat Palaga, Numpon Insin, Porntip Pan-In, and *Supason Pattanaargson Wanichwecharungruang ,
Shape Effect on Particle-Lipid Bilayer Membrane Association, Cellular Uptake and Cytotoxicity,
ACS Applied Materials & Interfaces 7, 23993–24000 (2015).

[Summary] We study the effect of particle shape on the particle–membrane interaction by monitoring the association between particles of various shapes and lipid bilayer membrane of artificial cell-sized liposomes.

*Tsutomu Hamada, Rie Fujimoto, Shunsuke F. Shimobayashi, Masatoshi Ichikawa, *Masahiro Takagi,
Molecular behavior of DNA in a cell-sized compartment coated by lipids,
Physical Review E 91, 62717 (2015).

[Summary] The behavior of long DNA molecules in a cell-sized confined space was investigated. Microscopic observation revealed that the adsorption of coiled DNA onto the membrane surface depended on the size of the vesicular space.

Sunatda Arayachukeat, Jiraporn Seemork, Porntip Pan-In, Kittima Amornwachirabodee, Naunpun Sangphech, Titiporn Sansureerungsikul, Kamonluck Sathornsantikun, Chotima Vilaivan, Kazuki Shigyou, Prompong Pienpinijtham, Tirayut Vilaivan, Tanapat Palaga, Wijit Banlunara, Tsutomu Hamada, and *Supason Pattanaargson Wanichwecharungruang,
Bringing macromolecules into cells and evading endosomes by oxidized carbon nanoparticles,
Nano Letters 15(5), 3370-3376 (2015).

[Summary] A great challenge exists in finding safe, simple, and effective delivery strategies to bring matters across cell membrane. Popular methods such as viral vectors, positively charged particles and cell penetrating peptides possess some of the following drawbacks: safety issues, lysosome trapping, limited loading capacity, and toxicity, whereas electroporation produces severe damages on both cargoes and cells. Here, we show that a serendipitously discovered, relatively nontoxic, water dispersible, stable, negatively charged, oxidized carbon nanoparticle, prepared from graphite, could deliver macromolecules into cells, without getting trapped in a lysosome. The ability of the particles to induce transient pores on lipid bilayer membranes of cell-sized liposomes was demonstrated. Delivering 12-base-long pyrrolidinyl peptide nucleic acids with d-prolyl-(1S,2S)-2-aminocyclopentanecarboxylic acid backbone (acpcPNA) complementary to the antisense strand of the NF-κB binding site in the promoter region of the Il6 gene into the macrophage cell line, RAW 264.7, by our particles resulted in an obvious accumulation of the acpcPNAs in the nucleus and decreased Il6 mRNA and IL-6 protein levels upon stimulation. We anticipate this work to be a starting point in a new drug delivery strategy, which involves the nanoparticle that can induce a transient pore on the lipid bilayer membrane.

Mari Takahashi, Priyank Mohan, Akiko Nakade, Koichi Higashimine, Derek Mott, Tsutomu Hamada, Kazuaki Matsumura, Tomohiko Taguchi, and *Shinya Maenosono,
Ag/FeCo/Ag Core/shell/shell Magnetic Nanoparticles with Plasmonic Imaging Capability,
Langmuir 31, 2228–2236 (2015).

[Summary] Magnetic nanoparticles (NPs) have been used to separate various species such as bacteria, cells, and proteins. In this study, we synthesized Ag/FeCo/Ag core/shell/shell NPs designed for magnetic separation of subcellular components like intracellular vesicles. A benefit of these NPs is that their silver metal content allows plasmon scattering to be used as a tool to observe detection by the NPs easily and semipermanently. Therefore, these NPs are considered a potential alternative to existing fluorescent probes like dye molecules and colloidal quantum dots. In addition, the Ag core inside the NPs suppresses the oxidation of FeCo because of electron transfer from the Ag core to the FeCo shell, even though FeCo is typically susceptible to oxidation. The surfaces of the Ag/FeCo/Ag NPs were functionalized with ε-poly-l-lysine-based hydrophilic polymers to make them water-soluble and biocompatible. The imaging capability of the polymer-functionalized NPs induced by plasmon scattering from the Ag core was investigated. The response of the NPs to a magnetic field using liposomes as platforms and applying a magnetic field during observation by confocal laser scanning microscopy was assessed. The results of the magnetophoresis experiments of liposomes allowed us to calculate the magnetic force to which each liposome was subjected.

2014

Hiroki Himeno, Naofumi Shimokawa, Shigeyuki Komura, David Andelman, *Tsutomu Hamada, Masahiro Takagi,
Charge-induced phase separation in lipid membranes,
Soft Matter 10, 7959-7967 (2014).

[Summary] Phase separation in lipid bilayers that include negatively charged lipids is examined experimentally. We observed phase-separated structures and determined the membrane miscibility temperatures in several binary and ternary lipid mixtures of unsaturated neutral lipid, dioleoylphosphatidylcholine (DOPC), saturated neutral lipid, dipalmitoylphosphatidylcholine (DPPC), unsaturated charged lipid, dioleoylphosphatidylglycerol (DOPG(−)), saturated charged lipid, dipalmitoylphosphatidylglycerol (DPPG(−)), and cholesterol. In binary mixtures of saturated and unsaturated charged lipids, the combination of the charged head with the saturation of the hydrocarbon tail is a dominant factor in the stability of membrane phase separation. DPPG(−) enhances phase separation, while DOPG(−) suppresses it. Furthermore, the addition of DPPG(−) to a binary mixture of DPPC/cholesterol induces phase separation between DPPG(−)-rich and cholesterol-rich phases. This indicates that cholesterol localization depends strongly on the electric charge on the hydrophilic head group rather than on the ordering of the hydrocarbon tails. Finally, when DPPG(−) was added to a neutral ternary system of DOPC/DPPC/cholesterol (a conventional model of membrane rafts), a three-phase coexistence was produced. We conclude by discussing some qualitative features of the phase behaviour in charged membranes using a free energy approach.

Shunchi Kawasaki, *Takahiro Muraoka, Haruki Obara, Takerou Ishii, Tsutomu Hamada, *Kazushi kinbara,
Thermodriven Micrometer-Scale Aqueous-Phase Separation of Amphiphilic Oligoethylene Glycol Analogues,
Chemistry-An Asian Journal 9, 2778-2788 (2014).

[Summary] Thermoresponsive materials with a lower critical solution temperature (LCST) are receiving growing attention, of which examples of non-polymeric small molecules are limited. Monodisperse oligoethylene glycol amphiphiles that contain aromatic units with a LCST in water have been developed and applied to peptide extraction. Concentration-dependent hysteretic transmittance changes were observed in response to temperature elevation and reduction. Dynamic light scattering measurements and phase contrast microscopy revealed the formation of micrometer-sized aggregates upon heating at a concentration above 5.0 mM; these aggregates self-assembled to form larger aggregates upon cooling before dissolution. The “interaggregate” interactions are likely to cause the hysteretic behavior. As an application of this thermodriven phase separation, selective extraction of peptide fragments containing high percentages of hydrophobic and aromatic amino acid residues was successfully demonstrated.

Tatsuya Shima, Takahiro Muraoka, Tsutomu Hamada, Masamune Morita, Masahiro Takagi, Hajime Fukuoka, Yuichi Inoue, Takashi Sagawa, Akihiko Ishijima, Yuki Omata, Takashi Yamashita, *Kazushi Kinbara,
Micrometer-Size Vesicle Formation Triggered by UV Light,
Langmuir 30, 7289-7295 (2014).

[Summary] Vesicle formation is a fundamental kinetic process related to the vesicle budding and endocytosis in a cell. In the vesicle formation by artificial means, transformation of lamellar lipid aggregates into spherical architectures is a key process and known to be prompted by e.g. heat, infrared irradiation, and alternating electric field induction. Here we report UV-light-driven formation of vesicles from particles consisting of crumpled phospholipid multilayer membranes involving a photoactive amphiphilic compound composed of 1,4-bis(4-phenylethynyl)benzene (BPEB) units. The particles can readily be prepared from a mixture of these components, which is casted on the glass surface followed by addition of water under ultrasonic radiation. Interestingly, upon irradiation with UV light, micrometer-size vesicles were generated from the particles. Neither infrared light irradiation nor heating prompted the vesicle formation. Taking advantage of the benefits of light, we successfully demonstrated micrometer-scale spatiotemporal control of single vesicle formation. It is also revealed that the BPEB units in the amphiphile are essential for this phenomenon.

Masamune Morita, *Tsutomu Hamada, Mun’delanji C. Vestergaard, Masahiro Takagi,
Endo- and Exocytic Budding Transformation of Slow-Diffusing Membrane Domains Induced by Alzheimer’s Amyloid Beta,
Physical Chemistry Chemical Physics 16, 8773-8777 (2014).

[Summary] Cell-sized liposomes are a powerful tool for clarifying physicochemical mechanisms that govern molecular interactions. Herein, budding transformation of membrane domains was induced by amyloid beta peptides. The peptides increased the membrane viscosity as demonstrated by the Brownian motion of membrane domains. These results could aid in understanding the physicochemical mechanism of Alzheimer's disease.