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Miyata K.,Center for Disease Biology and Integrative Medicine | Nishiyama N.,Center for Disease Biology and Integrative Medicine | Kataoka K.,Center for Disease Biology and Integrative Medicine | Kataoka K.,University of Tokyo
Bioconjugate Chemistry | Year: 2012

Herein, we report a unique technique to accelerate polymer-SNA conjugation based on copper-free click chemistry: gradual freeze-thawing of the reaction solution substantially increases the conjugation rate possibly because of the reactant concentration at the microenvironment scale. This technique was applied to the conjugation between a small interfering RNA (siRNA) and PEG in an aqueous buffer at/below room temperature. © 2012 American Chemical Society.


Dong B.,University of California at Davis | Nishimura N.,University of California at Davis | Nishimura N.,Japan National Institute of Environmental Studies | Vogel C.F.,University of California at Davis | And 3 more authors.
Biochemical Pharmacology | Year: 2010

Cyclooxygenase-2 (Cox-2) plays a critical role in TCDD-induced hydronephrosis in mouse neonates. In this study we found that induction of Cox-2 by TCDD in MMDD1, a mouse macula densa cell line, is accompanied with a rapid increase in the enzymatic activity of cytosolic phospholipase A2 (cPLA2) as well as activation of protein kinases. Calcium serves as a trigger for such an action of TCDD in this cell line. These observations indicate that the basic mode of action of TCDD to induce the rapid inflammatory response in MMDD1 is remarkably similar to those mediated by the nongenomic pathway of aryl hydrocarbon receptor (AhR) found in other types of cells. Such an action of TCDD to induce Cox-2 in MMDD1 was not affected by "DRE decoy oligonucleotides" treatment or by introduction of a mutation on the DRE site of Cox-2 promoter, suggesting that this route of action of TCDD is clearly different from that mediated by the classical genomic pathway. An in vivo study with Ahrnls mouse model has shown that TCDD-induces Cox-2 and renin expression in the kidneys of the Ahrnls mice as well as Ahr+/- mice, but not in the Ahr-/- mice, indicating that this initial action of TCDD in mouse kidney does not require the translocation of AhR into the nucleus, supporting our conclusion that induction of Cox-2 by TCDD in mouse kidney is largely mediated by the nongenomic pathway of TCDD-activated AhR. © 2009 Elsevier Inc. All rights reserved.


Osada K.,University of Tokyo | Yamasaki Y.,University of Tokyo | Kataoka K.,Center for Disease Biology and Integrative Medicine | Kataoka K.,University of Tokyo
Journal of the American Chemical Society | Year: 2010

Highly regulated folding of plasmid DNA (pDNA) through polyion complexation with the synthetic block catiomer, poly(ethylene glycol)-block-poly(l-lysine) (PEG-PLys), was found to occur in such a way that rod structures are formed with a quantized length of 1/2(n + 1) of the original pDNA length folding by n times. The folding process of pDNA was elucidated with regard to rigidity of the double-stranded DNA structure and topological restriction of the supercoiled closed-circular form, and a mechanism based on Eulers buckling theory was proposed. Folded pDNA exhibited higher gene expression efficiency compared to naked pDNA in a cell-free transcription/translation assay system, indicating that the packaging of pDNA into a polyion complex core surrounded by a PEG palisade is a promising strategy for constructing nonviral gene carrier systems. Extension of this finding may provide a reasonable model to further understand the packaging mechanism of supercoiled DNA structures in nature. © 2010 American Chemical Society.


Nakagawa K.,University of Tokyo | Iwasaki A.,University of Tokyo | Oishi Y.,RIKEN | Horisaki R.,Osaka University | And 10 more authors.
Nature Photonics | Year: 2014

High-speed photography is a powerful tool for studying fast dynamics in photochemistry, spintronics, phononics, fluidics and plasma physics. Currently, the pump-probe method is the gold standard for time-resolved imaging, but it requires repetitive measurements for image construction and therefore falls short in probing non-repetitive or difficult-to-reproduce events. Here, we present a motion-picture camera that performs single-shot burst image acquisition without the need for repetitive measurements, yet with equally short frame intervals (4.4 trillion frames per second) and high pixel resolution (450×450 pixels). The principle of this method - 'motion picture femtophotography' - is all-optical mapping of the target's time-varying spatial profile onto a burst stream of sequentially timed photographs with spatial and temporal dispersion. To show the camera's broad utility we use it to capture plasma dynamics and lattice vibrational waves, both of which were previously difficult to observe with conventional methods in a single shot and in real time. © 2014 Macmillan Publishers Limited. All rights reserved.


Kaida S.,Center for Disease Biology and Integrative Medicine | Kaida S.,University of Tokyo | Kaida S.,Shiga University of Medical Science | Cabral H.,Center for Disease Biology and Integrative Medicine | And 11 more authors.
Cancer Research | Year: 2010

Nanoparticle therapeutics are promising platforms for cancer therapy. However, it remains a formidable challenge to assess their distribution and clinical efficacy for therapeutic applications. Here, by using multifunctional polymeric micellar nanocarriers incorporating clinically approved gadolinium (Gd) - based magnetic resonance imaging contrast agents and platinum (Pt) anticancer drugs through reversible metal chelation of Pt, simultaneous imaging and therapy of an orthotopic animal model of intractable human pancreatic tumor was successfully performed without any serious toxicity. The strong tumor contrast enhancement achieved by the micelles correlated with the 24 times increase of r1 of the Gd chelates, the highest for the formulations using clinically approved Gd chelates reported to date. From the microsynchrotron radiation X-ray fluorescence spectrometry scanning of the lesions, we confirmed that both the Gd chelates and Pt drugs delivered by the micelles selectively colocalized in the tumor interior. Our study provides new insights for the design of theranostic micelles with high contrast enhancement and site-specific clinical potential. ©2010 AACR.


Christie R.J.,Center for Disease Biology and Integrative Medicine | Matsumoto Y.,Center for Disease Biology and Integrative Medicine | Matsumoto Y.,University of Tokyo | Matsumoto Y.,Mitsui Memorial Hospital | And 10 more authors.
ACS Nano | Year: 2012

Small interfering ribonucleic acid (siRNA) cancer therapies administered by intravenous injection require a delivery system for transport from the bloodstream into the cytoplasm of diseased cells to perform the function of gene silencing. Here we describe nanosized polymeric micelles that deliver siRNA to solid tumors and elicit a therapeutic effect. Stable multifunctional micelle structures on the order of 45 nm in size formed by spontaneous self-assembly of block copolymers with siRNA. Block copolymers used for micelle formation were designed and synthesized to contain three main features: a siRNA binding segment containing thiols, a hydrophilic nonbinding segment, and a cell-surface binding peptide. Specifically, poly(ethylene glycol)-block-poly(l-lysine) (PEG-b-PLL) comprising lysine amines modified with 2-iminothiolane (2IT) and the cyclo-Arg-Gly-Asp (cRGD) peptide on the PEG terminus was used. Modification of PEG-b-PLL with 2IT led to improved control of micelle formation and also increased stability in the blood compartment, while installation of the cRGD peptide improved biological activity. Incorporation of siRNA into stable micelle structures containing the cRGD peptide resulted in increased gene silencing ability, improved cell uptake, and broader subcellular distribution in vitro and also improved accumulation in both the tumor mass and tumor-associated blood vessels following intravenous injection into mice. Furthermore, stable and targeted micelles inhibited the growth of subcutaneous HeLa tumor models and demonstrated gene silencing in the tumor mass following treatment with antiangiogenic siRNAs. This new micellar nanomedicine could potentially expand the utility of siRNA-based therapies for cancer treatments that require intravenous injection. © 2012 American Chemical Society.


Nuhn L.,Johannes Gutenberg University Mainz | Tomcin S.,Max Planck Institute for Polymer Research | Miyata K.,Johannes Gutenberg University Mainz | Mailander V.,Max Planck Institute for Polymer Research | And 5 more authors.
Biomacromolecules | Year: 2014

To overcome the poor pharmacokinetic conditions of short double-stranded RNA molecules in RNA interference therapies, cationic nanohydrogel particles can be considered as alternative safe and stable carriers for oligonucleotide delivery. For understanding key parameters during this process, two different types of well-defined cationic nanohydrogel particles were synthesized, which provided nearly identical physicochemical properties with regards to their material composition and resulting siRNA loading characteristics. Yet, according to the manufacturing process using amphiphilic reactive ester block copolymers of pentafluorophenyl methacrylate (PFPMA) and tri(ethylene glycol)methyl ether methacrylate (MEO3MA) with similar compositions but different molecular weights, the resulting nanohydrogel particles differed in size after cross-linking with spermine (average diameter 40 vs 100 nm). This affected their knockdown potential significantly. Only the 40 nm sized cationic nanogel particles were able to generate moderate gene knockdown levels, which lasted, however, up to 3 days. Interestingly, primary cell uptake and colocalization studies with lysosomal compartments revealed that only these small sized nanogels were able to avoid acidic compartments of endolysosomal uptake pathways, which may contribute to their knockdown ability exclusively. To that respect, this size-dependent intracellular distribution behavior may be considered as an essential key parameter for tuning the knockdown potential of siRNA nanohydrogel particles, which may further contribute to the development of advanced siRNA carrier systems with improved knockdown potential. © 2014 American Chemical Society.


Obi N.,University of Tsukuba | Momotake A.,University of Tsukuba | Kanemoto Y.,Center for Disease Biology and Integrative Medicine | Matsuzaki M.,Center for Disease Biology and Integrative Medicine | And 3 more authors.
Tetrahedron Letters | Year: 2010

The synthesis, photochemistry, and biological application of 1-acyl-5-methoxy-8-nitro-1,2-dihydroquinoline (MNDQ-caged carboxylic acid) are described. Optimization experiments were carried out on three acetyl derivatives (3a-c), and the most appropriate analogue for application to the caging of glutamate was determined to be 3c. Thus, a MNDQ-caged glutamate (MNDQ-Glu) was synthesized, and the photochemical release of glutamate by uncaging of MNDQ-Glu was confirmed by NMR, MS, and HPLC analysis. When MNDQ-Glu was tested with pyramidal neurons in hippocampal slices, whole-field UV illumination resulted in a large inward current due to the release of l-glutamate. A short two-photon uncaging of MNDQ-Glu at single dendritic spines induced a transient current that exhibited similar kinetic properties to miniature excitatory postsynaptic currents (mEPSC). © 2010 Elsevier Ltd. All rights reserved.


Itaka K.,Center for Disease Biology and Integrative Medicine | Itaka K.,University of Tokyo | Ishii T.,University of Tokyo | Hasegawa Y.,Center for Disease Biology and Integrative Medicine | And 3 more authors.
Biomaterials | Year: 2010

Gene delivery using cationic polymers has attracted much attention due to their potential advantages, such as large DNA loading capacity, ease of large-scale production, and reduced immunogenicity. We recently reported that polyplexes from poly[N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide] (P[Asp(DET)]), having an efficient endosomal escape due to pH-selective membrane destabilization, showed high transfection efficiency with minimal toxicity. Pharmacogenomic analysis demonstrated that P[Asp(DET)] also provided long-term security after transfection. We hypothesized that the biodegradability of P[Asp(DET)] played a significant role in achieving effective transfection. Gel permeation chromatography (GPC) and electrospray ionization mass spectrometry (ESI-MS) measurements of P[Asp(DET)] revealed their ability to undergo rapid degradation. In contrast, a derivative polycation, N-substituted polyglutamide (P[Glu(DET)]), showed no degradability, indicating that the degradation of P[Asp(DET)] was induced by a specific self-catalytic reaction between the PAsp backbone and the side-chain amide nitrogen. Degradation products of P[Asp(DET)] caused no cytotoxicity, even at high concentrations in the culture medium. Repeated transfection by administering the polyplexes for every 24 h showed that biodegradable P[Asp(DET)] provided a continuous increase in transgene expression, while non-degradable P[Glu(DET)] showed a decrease in transgene expression after 48 h, coupled with fluctuations in expression profiles of endogenous genes. In vivo intraperitoneal injection of P[Asp(DET)] induced minimal inflammatory cytokine induction to a level comparable to that of normal saline. These results indicate that the biodegradability of P[Asp(DET)] played a key role in achieving safe and sustained transgene expression, by minimizing cumulative toxicity caused by polycations remaining in cells or in the body. © 2009 Elsevier Ltd. All rights reserved.


Ohsako S.,Center for Disease Biology and Integrative Medicine
Genes and Environment | Year: 2011

Many researchers propose that invisible internal alterations that occur through exposure to environmental factors during fetal or neonatal stages affect the risk of cancer, hypertension, and diabetes after maturation. Barker's hypothesis, which states that reduced fetal growth is strongly associated with metabolic syndromes including cardiovascular disease and diabetes, has now been widely accepted and expanded into the Developmental Origins of Health and Disease (DOHaD). Potential molecular mechanisms underlying this phenomenon include the alteration and persistence of epigenomic programming. Clear biochemical evidence has not yet been obtained in human studies; however, in laboratory animals, the fetal environment including physical and chemical factors altered epigenomic states such as DNA methylation and histone modification, and persistent changes affected specific gene expression regulation, resulting in disease susceptibility. Furthermore, in recent studies, environmental chemical exposure during pregnancy altered sperm DNA methylation patterns of male offspring, and the altered status and resulting phenotypes were inherited in the next generation. Challenging and eccentric studies focusing on epigenetic transgenerational effects are currently being conducted to demonstrate the existence of Lamarckian inheritance. © The Japanese Environmental Mutagen Society.

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