Masaharu Murata

Nanoparticles of Adaptive Supramolecular Networks Self-Assembled from Nucleotides and Lanthanide Ions

Amorphous nanoparticles of supramolecular coordination polymer networks are spontaneously self-assembled from nucleotides and lanthanide ions in water. They show intrinsic functions such as energy transfer from nucleobase to lanthanide ions and excellent performance as contrast enhancing agents for magnetic resonance imaging (MRI). Furthermore, adaptive inclusion properties are observed in the self-assembly process: functional materials such as fluorescent dyes, metal nanoparticles, and proteins are facilely encapsulated. Dyes in these nanoparticles fluoresce in high quantum yields with a single exponential decay, indicating that guest molecules are monomerically wrapped in the network. Gold nanoparticles and ferritin were also wrapped by the supramolecular shells. In addition, these nucleotide/lanthanide nanoparticles also serve as scaffolds for immobilizing enzymes. The adaptive nature of present supramolecular nanoparticles provides a versatile platform that can be utilized in a variety of applications ranging from material to biomedical sciences. As examples, biocompatibility and liver-directing characteristics in in vivo tissue localization experiments are demonstrated.

Gold nanoparticle-based colorimetric assay for cancer diagnosis

A novel gold nanoparticle (GNP)-based colorimetric assay was developed for cancer diagnosis. This system is based on the noncrosslinking aggregation mechanism with a cationic protein kinase C (PKC) alpha-specific peptide substrate, which is used as a coagulant of citrate-coated GNP with anionic surface charges. The phosphorylation of peptide substrates by PKCalpha suppressed GNP aggregation, resulting in a red color, but in the case of non-phosphorylation, the color of the GNP solution changed from red to blue, indicating particle aggregation. Moreover, a correlation between the color change of the GNP dispersions and the level of activated PKCalpha was identified from experiments using cancer cell lines, or xenografted mouse cancer and normal mouse tissues. When our system was applied to human breast cancers and normal human breast tissues, cancer tissue lysates became red in color, indicating GNP dispersion, while all lysates from normal tissue turned the GNP solution blue. MALDI-TOF MS analysis and Western blotting experiment confirmed that these different results between cancer and normal tissues reflected the difference in PKCalpha activity. This study is the first report on the application of the GNP-based colorimetric assay to the diagnosis of cancer.

An intracellular kinase signal-responsive gene carrier for disordered cell-specific gene therapy

We have previously reported artificial gene-regulation systems responding to cyclic AMP-dependent protein kinase (PKA) using cationic polymer. This cationic polymer (PAK) was a graft-type polymer with an oligopeptide that is a substrate for PKA and could regulate gene-expression in a cell-free system. In the present study, we carried out a detailed characterization of the PAK-DNA complex (AFM observation and DLS measurement) and tried to apply this polymer to living cells. In the unstimulated NIH 3T3 cells, transfection of the PAK-DNA complex showed no expression of the delivered gene. This means that PAK formed a stable complex with DNA in the normal cells to totally suppress gene expression. In contrast, significant expression was seen when the PAK-DNA complex was delivered to forskolin-treated cells. Thus, activated PKA disintegrates the complexes even in living cells, resulting in gene expression. Our results indicate that this type of intracellular signal-responsive polymer will be useful for the cell-specific release of genes.

Development of Human Hepatocellular Carcinoma Cell-Targeted Protein Cages

We described herein a human hepatocellular carcinoma (HCC) cell-targeted protein cage for which the HCC-binding peptide termed SP94 was modified at the surface of a naturally occurred heat shock protein (Hsp) cage. Six types of HCC-targeted Hsp cages were chemically synthesized using two types of heterobifunctional linker (SM(PEG)(n)) with different lengths and two types of SP94 peptide, which contained a unique Cys residue at the N- or C-terminus of the peptide. These Hsp cages were characterized using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF MS) analyses, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses, and dynamic light scattering (DLS) measurement. Fluorescence microscopic observations revealed that all the engineered protein cages bind selectively to HCC cells but not to the other cell lines tested (including normal liver cell). Moreover, the number of SP94 peptides on Hsp cages, conjugation site of SP94 peptide, and linker length between a Hsp cage and a SP94 peptide had important effects upon the binding of engineered Hsp cages to HCC cells. An engineered Hsp cage conjugated to the N-terminus of SP94 peptide via a longer linker molecule and containing high SP94 peptide levels showed greater binding toward HCC cells. Surprisingly, through optimization of these three factors, up to 10-fold greater affinity toward HCC cells was achieved. These results are critically important not only for the development of HCC cell-targeting devices using SP94 peptide, but also to create other cell-targeting materials that utilize other peptide ligands.

Hypoxia Inducible Factor 1 Alpha Regulates Matrigel-induced Endovascular Differentiation under Normoxia in a Human Extravillous Trophoblast Cell Line

Extravillous trophoblast (EVT) cells mimic endothelial cells during angiogenesis, inducing remodeling of the spiral arteries that increases blood flow toward the intravillous space. We have previously shown that signals involving the vascular endothelial growth factor (VEGF) axis are essential for endovascular differentiation through integrin signaling from the extracellular matrix: This was accomplished with use of the human EVT cell line TCL1, which shows tube formation that specifically recalls morphological changes in endothelial cells. To investigate endovascular differentiation in EVT further, we investigated the role of hypoxia inducible factor (HIF)1A, a subunit of HIF1 transcription factor that regulates not only adaptive responses to hypoxia, but also many cellular functions under normoxia, which was up-regulated in DNA microarray analysis during matrigel-induced endovascular differentiation under normoxia. HIF1A induces VEGF and ITGAV/ITGB3 aggregation, actions known to be important for cellular survival and endovascular differentiation in EVT. Inhibition of HIF1A up-regulation using siRNA introduction or chemical inhibition suppressed hypoxia-responsive element transcriptional activity, VEGF induction, ITGAV/ITGB3 aggregation accompanied by the inhibition of tube formation in TCL1 cells. These results suggest that HIF1A has a crucial role in regulating EVT behavior including matrigel-induced endovascular differentiation under normoxia.

A nanocarrier based on a genetically engineered protein cage to deliver doxorubicin to human hepatocellular carcinoma cells

Herein, we report the preparation of genetically engineered protein cages (HspG41C-SP94), taken up selectively by human hepatocellular carcinoma (HCC) cells. An engineered protein cage-doxorubicin (DOX) conjugate was as cytotoxic as free DOX against HCC cells but much less cytotoxic against normal hepatocytes.

Liver cell specific targeting by the preS1 domain of hepatitis B virus surface antigen displayed on protein nanocages

Protein nanocages are self-organized complexes of oligomers whose three-dimensional architecture can been determined in detail. These structures possess nanoscale inner cavities into which a variety of molecules, including therapeutic or diagnostic agents, can be encapsulated. These properties yield these particles suitable for a new class of drug delivery carrier, or as a bioimaging reagent that might respond to biochemical signals in many different cellular processes. We report here the design, synthesis, and biological characterization of a hepatocyte-specific nanocage carrying small heat-shock protein. These nanoscale protein cages, with a targeting peptide composed of a preS1 derivative from the hepatitis B virus on their surfaces, were prepared by genetic engineering techniques. PreS1-carrying nanocages showed lower cytotoxicity and significantly higher specificity for human hepatocyte cell lines than other cell lines in vitro. These results suggested that small heat-shock protein-based nanocages present great potential for the development of effective targeted delivery of various agents to specific cells.

Oxidative Stress Produced by Xanthine Oxidase Induces Apoptosis in Human Extravillous Trophoblast Cells

Abstract Oxidative stress has been recognized as an important factor in the pathophysiology of preeclampsia. It has been reported that the expression of xanthine oxidase (XO) in the cytotrophoblast and plasma hydrogen peroxide (H2O2) level are significantly higher in preeclamptics than in control women. The aim of this study was to clarify the biological influence of reactive oxygen species (ROS) produced by XO on extravillous trophoblast (EVT) cells. TCL1 cells, a human immortalized EVT cell line, were incubated with xanthine and XO (X/XO). We then measured the cell number, urate level of the culture media and the apoptotic cell ratio. Similar experiments were performed with additional administration of allopurinol, catalase, L-NAME or D-NAME, and with administration of H2O2 in substitution for X/XO. We assessed the effects of H2O2 on invasion ability, tube-like formation and protein expression of HIF1A and ITGAV of TCL1. Finally, the apoptotic cell ratio using primary cultured trophoblasts was measured following exposure to H2O2. X/XO decreased the relative cell number and increased the urate level and apoptotic cell ratio significantly. Elevation of the urate level and apoptotic cell ratio was attenuated by allopurinol and catalase, respectively. L-NAME and D-NAME had no influence on these effects. H2O2 also decreased the relative cell number. Pretreatment with H2O2 significantly inhibited the invasion ability, tube-like formation and HIF1A and ITGAV of TCL1. H2O2 also induced apoptosis in primary cultured trophoblasts. In conclusion, ROS produced by XO induced apoptosis and affected EVT function including invasion and differentiation.

Biological evaluation of protein nanocapsules containing doxorubicin

This study describes the applications of a naturally occurring small heat shock protein (Hsp) that forms a cage-like structure to act as a drug carrier. Mutant Hsp cages (HspG41C) were expressed in Escherichia coli by substituting glycine 41 located inside the cage with a cysteine residue to allow conjugation with a fluorophore or a drug. The HspG41C cages were taken up by various cancer cell lines, mainly through clathrin-mediated endocytosis. The cages were detected in acidic organelles (endosomes/lysosomes) for at least 48 hours, but none were detected in the mitochondria or nuclei. To generate HspG41C cages carrying doxorubicin (DOX), an anticancer agent, the HspG41C cages and DOX were conjugated using acid-labile hydrazone linkers. The release of DOX from HspG41C cages was accelerated at pH 5.0, but was negligible at pH 7.2. The cytotoxic effects of HspG41C–DOX against Suit-2 and HepG2 cells were slightly weaker than those of free DOX, but the effects were almost identical in Huh-7 cells. Considering the relatively low release of DOX from HspG41C–DOX, HspG41C–DOX exhibited comparable activity towards HepG2 and Suit-2 cells and slightly stronger cytotoxicity towards Huh-7 cells than free DOX. Hsp cages offer good biocompatibility, are easy to prepare, and are easy to modify; these properties facilitate their use as nanoplatforms in drug delivery systems and in other biomedical applications.

Basic fibroblast growth factor-treated adipose tissue-derived mesenchymal stem cell infusion to ameliorate liver cirrhosis via paracrine hepatocyte growth factor

Recent studies show that adipose tissue‐derived mesenchymal stem cells have potential clinical applications. However, the mechanism has not been fully elucidated yet. Here, we investigated the effect of basic fibroblast growth factor‐treated adipose tissue‐derived mesenchymal stem cells infusion on a liver fibrosis rat model and elucidated the underlying mechanism.