Sada-atsu Mukai

Engineering hybrid exosomes by membrane fusion with liposomes.

Exosomes are a valuable biomaterial for the development of novel nanocarriers as functionally advanced drug delivery systems. To control and modify the performance of exosomal nanocarriers, we developed hybrid exosomes by fusing their membranes with liposomes using the freeze–thaw method. Exosomes embedded with a specific membrane protein isolated from genetically modified cells were fused with various liposomes, confirming that membrane engineering methods can be combined with genetic modification techniques. Cellular uptake studies performed using the hybrid exosomes revealed that the interactions between the developed exosomes and cells could be modified by changing the lipid composition or the properties of the exogenous lipids. These results suggest that the membrane-engineering approach reported here offers a new strategy for developing rationally designed exosomes as hybrid nanocarriers for use in advanced drug delivery systems.

Exosomes as nanocarriers for systemic delivery of the Helicobacter pylori virulence factor CagA

CagA, encoded by cytotoxin-associated gene A (cagA), is a major virulence factor of Helicobacter pylori, a gastric pathogen involved in the development of upper gastrointestinal diseases. Infection with cagA-positive H. pylori may also be associated with diseases outside the stomach, although the mechanisms through which H. pylori infection promotes extragastric diseases remain unknown. Here, we report that CagA is present in serum-derived extracellular vesicles, known as exosomes, in patients infected with cagA-positive H. pylori (n = 4). We also found that gastric epithelial cells inducibly expressing CagA secrete exosomes containing CagA. Addition of purified CagA-containing exosomes to gastric epithelial cells induced an elongated cell shape, indicating that the exosomes deliver functional CagA into cells. These findings indicated that exosomes secreted from CagA-expressing gastric epithelial cells may enter into circulation, delivering CagA to distant organs and tissues. Thus, CagA-containing exosomes may be involved in the development of extragastric disorders associated with cagA-positive H. pylori infection.

Microbial growth at hyperaccelerations up to 403,627 × g

It is well known that prokaryotic life can withstand extremes of temperature, pH, pressure, and radiation. Little is known about the proliferation of prokaryotic life under conditions of hyperacceleration attributable to extreme gravity, however. We found that living organisms can be surprisingly proliferative during hyperacceleration. In tests reported here, a variety of microorganisms, including Gram-negative Escherichia coli, Paracoccus denitrificans, and Shewanella amazonensis; Gram-positive Lactobacillus delbrueckii; and eukaryotic Saccharomyces cerevisiae, were cultured while being subjected to hyperaccelerative conditions. We observed and quantified robust cellular growth in these cultures across a wide range of hyperacceleration values. Most notably, the organisms P. denitrificans and E. coli were able to proliferate even at 403,627 × g. Analysis shows that the small size of prokaryotic cells is essential for their proliferation under conditions of hyperacceleration. Our results indicate that microorganisms cannot only survive during hyperacceleration but can display such robust proliferative behavior that the habitability of extraterrestrial environments must not be limited by gravity.

Liquid/liquid dynamic phase separation induced by a focused laser

We found that a focused laser can generate microscopic phase separation in an oil/water system. An oil droplet emerges and grows at the focus of the laser in a water-rich homogeneous medium. In contrast, in an oil-rich homogeneous phase, water droplets spring out in a successive manner from the focus of the laser, move away, and disappear in the surroundings, forming a flower-like pattern. The mechanism of this dynamic phase separation is discussed under the framework of the mean field theory.

Cycloamylose-nanogel drug delivery system-mediated intratumor silencing of the vascular endothelial growth factor regulates neovascularization in tumor microenvironment

RNAi enables potent and specific gene silencing, potentially offering useful means for treatment of cancers. However, safe and efficient drug delivery systems (DDS) that are appropriate for intra‐tumor delivery of siRNA or shRNA have rarely been established, hindering clinical application of RNAi technology to cancer therapy. We have devised hydrogel polymer nanoparticles, or nanogel, and shown its validity as a novel DDS for various molecules. Here we examined the potential of self‐assembled nanogel of cholesterol‐bearing cycloamylose with spermine group (CH‐CA‐Spe) to deliver vascular endothelial growth factor (VEGF)‐specific short interfering RNA (siVEGF) into tumor cells. The siVEGF/nanogel complex was engulfed by renal cell carcinoma (RCC) cells through the endocytotic pathway, resulting in efficient knockdown of VEGF. Intra‐tumor injections of the complex significantly suppressed neovascularization and growth of RCC in mice. The treatment also inhibited induction of myeloid‐derived suppressor cells, while it decreased interleukin‐17A production. Therefore, the CH‐CA‐Spe nanogel may be a feasible DDS for intra‐tumor delivery of therapeutic siRNA. The results also suggest that local suppression of VEGF may have a positive impact on systemic immune responses against malignancies.

Magnetically Guided Protein Transduction by Hybrid Nanogel Chaperones with Iron Oxide Nanoparticles

Protein pharmaceuticals show great therapeutic promise, but effective intracellular delivery remains challenging. To address the need for efficient protein transduction systems, we used a magnetic nanogel chaperone (MC): a hybrid of a polysaccharide nanogel, a protein carrier with molecular chaperone-like properties, and iron oxide nanoparticles, enabling magnetically guided delivery. The MC complexed with model proteins, such as BSA and insulin, and was not cytotoxic. Cargo proteins were delivered to the target HeLa cell cytosol using a magnetic field to promote movement of the protein complex toward the cells. Delivery was confirmed by fluorescence microscopy and flow cytometry. Delivered β-galactosidase, inactive within the MC complex, became enzymatically active within cells to convert a prodrug. Thus, cargo proteins were released from MC complexes through exchange interactions with cytosolic proteins. The MC is a promising tool for realizing the therapeutic potential of proteins.

Non-Engineered Nanoparticles of C 60

We discovered that rubbing bulk solids of C60 between fingertips generates nanoparticles including the ones smaller than 20 nm. Considering the difficulties usually associated with nanoparticle production by pulverisation, formation of nanoparticles by such a mundane method is unprecedented and noteworthy. We also found that nanoparticles of C60 could be generated from bulk solids incidentally without deliberate engineering of any sort. Our findings imply that there exist highly unusual human exposure routes to nanoparticles of C60, and elucidating formation mechanisms of nanoparticles is crucial in assessing their environmental impacts.

Phase behaviors of agarose gel

We present evidence for the existence of phase separation in the gel state of agarose having the mixture of water and methanol as the gel solvent. Firstly, the sol-gel transition line and the cloud point line are determined independently as a function of the concentration of agarose as well as the concentration of methanol in the mixed solvent by the quasi-equilibrium cooling of the solutions. Then the spinodal line is determined by quenching the solutions below the sol-gel transition line. We find that the spinodal line appears below the cloud point line and both lines are entirely buried below the sol-gel transition line in the aqueous agarose system. The concentration fluctuations are, therefore, frozen into the polymer network of agarose gel that promotes the opacity of the resultant gel. The structure of agarose gel is observed by the confocal laser scanning microscope (CLSM) imaging technique that reveals that the density fluctuations are grown up to micrometer scale in space. The phase separation boundary is found to shift to the higher temperature region than the sol-gel transition line when the concentration of methanol in the mixed solvent is increased. The results indicate that the position of the phase separation boundary in relative to the sol-gel transition line varies with the quality of solvent. These results are in agreement with the theory of the sol-gel transition in which both the divergence of the connectivity and the thermodynamic instability are taken into account.

Amylose-Based Cationic Star Polymers for siRNA Delivery

A new siRNA delivery system using a cationic glyco-star polymer is described. Spermine-modified 8-arm amylose star polymer (with a degree of polymerization of approximately 60 per arm) was synthesized by chemoenzymatic methods. The cationic star polymer effectively bound to siRNA and formed spherical complexes with an average hydrodynamic diameter of 230 nm. The cationic 8-arm star polymer complexes showed superior cellular uptake characteristics and higher gene silencing effects than a cationic 1-arm polymer. These results suggest that amylose-based star polymers are a promising nanoplatform for glycobiomaterials.

Anomalously stable dispersions of graphite in water/acetone mixtures.

Highly stable dispersions of graphite are obtained in water/acetone mixtures, while graphite precipitates promptly in water and in acetone. We found that acetone facilitates disintegration of the graphite particles and their aggregates during a sonication process. Fine graphite particles are stabilised by the electrostatic repulsion due to negative charges on the graphite particle surfaces.