Shuhei Murayama

NanoPARCEL: a method for controlling cellular behavior with external light

We developed a simple preparation procedure for the protein encapsulated nanoparticle and used the nanoparticle for spatiotemporal activity control of various proteins. We succeeded in the local protein activation within cells by light using the nanoparticle.

Gene regulation by intracellular delivery and photodegradation of nanoparticles containing small interfering RNA

Although the use of small interfering RNA (siRNA) is a promising technique for gene regulation, spatiotemporal control of the effects of siRNA must be achieved if the technique is to be safe and practical. Here, a method for spatiotemporal regulation of genes with nanoparticles containing siRNA is reported. The siRNA is encapsulated in photodegradable nanoparticles that are internalized to SKOV3-Luc cells, where the siRNA is released from the nanoparticles by UV irradiation for 30 s. The encapsulated siRNA only shows no gene-silencing effects, but release of the siRNA upon UV radiation leads to sequence-specific silencing of the luciferase gene in the cells. These results indicate that photodegradable siRNA-containing nanoparticles can be useful for time- and space-dependent regulation of gene expression in cells.

Development of a spatiotemporal method to control molecular function by using silica-based photodegradable nanoparticles

To effectively and safely use molecules, it is important to be able to control the timing and site of molecule activation. We developed a spatiotemporal method to control molecular function by using silica-based photodegradable nanoparticles that can be prepared under mild conditions. The function of various molecules, such as rhodamine B, Nile blue A, propidium iodide (PI), and rhodamine 110, bis-(N-CBZ-l-arginine amide), dihydrochloride (BZAR), was restricted by wrapping in the network structure of the nanoparticle gel. The encapsulated molecule was released from the gel by the light stimulus and its function was restored. Hence, this technique is applicable to the functional control of various molecules. The PI-encapsulated nanoparticles were internalized by the cells after being conjugated with the cell membrane permeability peptide, octaarginine, and were localized to the cytoplasm. Short-term irradiation (20 s) induced PI release from the nanoparticles and the rapid movement (less than 2 min) of the released PI to the nucleus. These nanoparticles are thus useful tools for the spatiotemporal control of various molecular functions because they permit the quick and transient release of encapsulated molecules after short-term irradiation and can be prepared under mild conditions.

Jasisoquinolines A and B, Architecturally New Isoquinolines, from a Marine Sponge Jaspis sp.

Two architecturally new isoquinolines, jasisoquinolines A and B, were isolated from a marine sponge Jaspis sp. as cathepsin B inhibitors. Their structures were determined by a combination of spectroscopic analyses and chemical methods. Both jasisoquinolines A and B inhibit cathepsin B with an IC(50) value of 10 μg/mL.

Shishicrellastatins, inhibitors of cathepsin B, from the marine sponge Crella (Yvesia) spinulata.

Two dimeric steroid derivatives, shishicrellastatin A (1) and B (2), have been isolated as cathepsin B inhibitors from the marine sponge Crella (Yvesia) spinulata. Their structures were determined by interpretation of spectroscopic data. Shishicrellastatins inhibit cathepsin B with an IC(50) value of 8 μg/mL each.

Thermal- and pH-Dependent Size Variable Radical Nanoparticles and Its Water Proton Relaxivity for Metal-Free MRI Functional Contrast Agents

For development of the metal-free MRI contrast agents, we prepared the supra-molecular organic radical, TEMPO-UBD, carrying TEMPO radical, as well as the urea, alkyl group, and phenyl ring, which demonstrate self-assembly behaviors using noncovalent bonds in an aqueous solution. In addition, TEMPO-UBD has the tertiary amine and the oligoethylene glycol chains (OEGs) for the function of pH and thermal responsiveness. By dynamic light scattering and transmission electron microscopy imaging, the resulting self-assembly was seen to form the spherical nanoparticles 10-150 nm in size. On heating, interestingly, the nanoparticles showed a lower critical solution temperature (LCST) behavior having two-step variation. This double-LCST behavior is the first such example among the supra-molecules. To evaluate of the ability as MRI contrast agents, the values of proton ((1)H) longitudinal relaxivity (r1) were determined using MRI apparatus. In conditions below and above CAC at pH 7.0, the distinguishable r1 values were estimated to be 0.17 and 0.21 mM(-1) s(1), indicating the suppression of fast tumbling motion of TEMPO moiety in a nanoparticle. Furthermore, r1 values became larger in the order of pH 7.0 > 9.0 > 5.0. Those thermal and pH dependencies indicated the possibility of metal-fee MRI functional contrast agents in the future.

Fluorescence derivatization of single‐walled carbon nanotubes for analysis by means of conventional CE‐LIF

Single-walled carbon nanotubes (SWNTs) are now widely used in many fields, and while many analytical methods for SWNTs have been reported, there are few practical analytical methods that combine the necessary levels of selectivity and sensitivity. We have developed a highly sensitive separation method for fluorescence-derivatized SWNTs by means of conventional CE with laser-induced fluorescence. First, SWNTs were dispersed using a triphenylene derivative into the water, and the excess dispersant was removed by nitric acid treatment. The dispersed SWNTs were then derivatized with a fluorescence reagent, 4-aminofluorescein. Finally, the derivatized SWNTs were analyzed using a conventional apparatus CE-LIF detection. The SWNTs migrated within 20 min. The detection sensitivity of SWNTs was improved by about 170 times with LIF detection as compared with UV detection. We anticipate that the derivatized SWNTs can also be detected with high sensitivity using LC.

Self-Assembly Behavior of Emissive Urea Benzene Derivatives Enables Heat-Induced Accumulation in Tumor Tissue

In this study we describe the construction of a system composed of thermally responsive molecules that can be induced to accumulate in tumor tissues by heating. EgX molecules consisting of an urea-benzene framework and oligoethylene glycol (OEG) functional groups with an emissive aminoquinoline formed nanoparticles (NPs) ∼10 nm in size at 23 °C with a fluorescence quantum yield of 7-10%. At higher temperatures, additional self-assembly occurred as a result of OEG dehydration, and the NPs grew to over 1000 nm in size; this was accompanied by low critical solution temperature behavior. EgXs accumulated in tumor tissues of mice at a body temperature of around 33-35 °C, an effect that was accelerated by external heating around the tumor to approximately 40 °C as a result of increased particle size and enhanced retention in tissue. These EgX NPs can serve as a tool for in vivo monitoring of tumor progression and response to treatment.

Water-Proton Relaxivities of Radical Nanoparticles Self-Assembled via Hydration or Dehydration Processes

Nanoparticles capable of accumulating in tumor tissues are promising materials for tumor imaging and therapy. In this study, two radical nanoparticles (RNPs), denoted as 1 and 2, composed of self-assembled ureabenzene derivatives possessing one or two amphiphilic side chains were demonstrated to be candidates for metal-free functional magnetic resonance imaging (MRI) contrast agents (CAs). Because of the self-assembly behavior of 1 and 2 in a saline solution, spherical RNPs of sizes ∼50-90 and ∼30-100 nm were detected. In a highly concentrated solution, RNP 1 showed considerably small water-proton relaxivity values (r1 and r2), whereas RNP 2 showed an r1 value that was around 5 times larger than that of RNP 1. These distinct r1 values might be caused by differences in the self-assembly behavior by a hydration or dehydration process. In vivo studies with RNP 2 demonstrated a slightly enhanced T1-weighted image in mice, suggesting that the RNPs can potentially be used as metal-free functional MRI CAs for T1-weighted imaging.

Fluorescence Tumor-Imaging Using a Thermo-Responsive Molecule with an Emissive Aminoquinoline Derivative

We synthesized (2,4-trifluoromethyl-7-N-bis(2,5,8,11-tetraoxatridecane-13-yl)-aminoquinoline) TFMAQ-diEg4, an emissive aminoquinoline derivative that incorporated two tetraethyleneglycol chains into an amino group. TFMAQ-diEg4 showed fluorescence and thermo-responsive properties accompanied by a lower critical solution temperature (LCST), due to the introduction of the oligoethylene glycol chain. This thermo-responsive LCST behavior occurred at the border of a cloud point. Below and above the cloud point, self-assemblies of 6-7-nm nanoparticles and ~2000-nm microparticles were observed, in vitro. In addition, TFMAQ-diEg4 showed a high solubility, over 20 mM for aqueous solution, in vivo, which not only prevented thrombosis but also allowed various examinations, such as single intravenous administration and intravenous drips. Intravenous administration of TFMAQ-diEg4, to tumor-bearing, mice led to the accumulation of the molecule in the tumor tissue, as observed by fluorescence imaging. A subset of mice was treated with local heat around their tumor tissue and an intravenous drip of TFMAQ-diEg4, which led to a high intensity of TFMAQ-diEg4 emission within the tumor tissue. Therefore, we revealed that TFMAQ-diEg4 was useful as a fluorescence probe with thermo-responsive properties.