Shuji Sonezaki

Photoelectrochemical deoxyribonucleic acid sensing on a nanostructured TiO2 electrode

A nanostructured TiO2 electrode chemisorbed with probe deoxyribonucleic acid (DNA) can photoelectrochemically detect a dye-labeled target DNA molecule. After the hybridization between the probe and target DNA molecules, light irradiation generates electrons in the dye molecules, and these electrons are injected into the TiO2 electrode. The resulting photocurrent can be measured and corresponds to the concentration of target DNA. This sensor can quantitatively detect target DNA at lower than nanomolar concentrations. In addition, by utilizing two different dyes, different DNA sequences can be detected on the TiO2 electrode.

Sonodynamic therapy using water-dispersed TiO2-polyethylene glycol compound on glioma cells: Comparison of cytotoxic mechanism with photodynamic therapy

Sonodynamic therapy is expected to be a novel therapeutic strategy for malignant gliomas. The titanium dioxide (TiO(2)) nanoparticle, a photosensitizer, can be activated by ultrasound. In this study, by using water-dispersed TiO(2) nanoparticles, an in vitro comparison was made between the photodynamic and sonodynamic damages on U251 human glioblastoma cell lines. Water-dispersed TiO(2) nanoparticles were constructed by the adsorption of chemically modified polyethylene glycole (PEG) on the TiO(2) surface (TiO(2)/PEG). To evaluate cytotoxicity, U251 monolayer cells were incubated in culture medium including 100 μg/ml of TiO(2)/PEG for 3h and subsequently irradiated by ultraviolet light (5.0 mW/cm(2)) or 1.0MHz ultrasound (1.0 W/cm(2)). Cell survival was estimated by MTT assay 24h after irradiation. In the presence of TiO(2)/PEG, the photodynamic cytotoxic effect was not observed after 20 min of an ultraviolet light exposure, while the sonodynamic cytotoxicity effect was almost proportional to the time of sonication. In addition, photodynamic cytotoxicity of TiO(2)/PEG was almost completely inhibited by radical scavenger, while suppression of the sonodynamic cytotoxic effect was not significant. Results of various fluorescent stains showed that ultrasound-treated cells lost their viability immediately after irradiation, and cell membranes were especially damaged in comparison with ultraviolet-treated cells. These findings showed a potential application of TiO(2)/PEG to sonodynamic therapy as a new treatment of malignant gliomas and suggested that the mechanism of TiO(2)/PEG mediated sonodynamic cytotoxicity differs from that of photodynamic cytotoxicity.

Novel photodynamic therapy using water-dispersed TiO2-polyethylene glycol compound: evaluation of antitumor effect on glioma cells and spheroids in vitro.

Titanium dioxide (TiO2) is thought to be a photocatalytic agent excited by UV light. Our aim was to investigate the photocatalytic antitumor effect of water‐dispersed TiO2 nanoparticles on C6 rat glioma cells and to evaluate the treatment responses by the spheroid models. Water‐dispersed TiO2 nanoparticles were constructed by the adsorption of chemical modified polyethylene glycol (PEG) on the TiO2 surface (TiO2/PEG). Each monolayer and spheroid of C6 cells was coincubated with various concentrations of TiO2/PEG and subsequently irradiated with UV light. Damage of the cells and spheroids was evaluated sequentially by staining with the fluorescent dyes. The cytotoxic effect was correlated with the concentration of TiO2/PEG and the energy dose of UV irradiation. More than 90% of cells were killed after 13.5 J cm−2 of UV irradiation in the presence of 500 μg mL−1 TiO2/PEG. The irradiated spheroids in the presence of TiO2/PEG showed growth suppression compared with control groups. In TiO2/PEG‐treated spheroids, the number of Annexin V‐FITC‐stained cells gradually increased during the first 6 h, and subsequently propidium iodide‐stained cells appeared. The results of this study suggest that newly developed photoexcited TiO2/PEG have antitumoral activity. Photodynamic therapy utilizing this material can be a clue to a novel therapeutic strategy for glioma.

Effect of Polyethylene Glycol Modification of TiO2 Nanoparticles on Cytotoxicity and Gene Expressions in Human Cell Lines

Nanoparticles (NPs) are tiny materials used in a wide range of industrial and medical applications. Titanium dioxide (TiO2) is a type of nanoparticle that is widely used in paints, pigments, and cosmetics; however, little is known about the impact of TiO2 on human health and the environment. Therefore, considerable research has focused on characterizing the potential toxicity of nanoparticles such as TiO2 and on understanding the mechanism of TiO2 NP-induced nanotoxicity through the evaluation of biomarkers. Uncoated TiO2 NPs tend to aggregate in aqueous media, and these aggregates decrease cell viability and induce expression of stress-related genes, such as those encoding interleukin-6 (IL-6) and heat shock protein 70B’ (HSP70B’), indicating that TiO2 NPs induce inflammatory and heat shock responses. In order to reduce their toxicity, we conjugated TiO2 NPs with polyethylene glycol (PEG) to eliminate aggregation. Our findings indicate that modifying TiO2 NPs with PEG reduces their cytotoxicity and reduces the induction of stress-related genes. Our results also suggest that TiO2 NP-induced effects on cytotoxicity and gene expression vary depending upon the cell type and surface modification.

Properties of TiO2-polyacrylic acid dispersions with potential for molecular recognition.

Titanium dioxide (TiO2)/polyacrylic acid (PAA) (TiO2/PAA) particles were formed by mixing PAA and an acidic solution of TiO2 nanoparticles in dimethylformamide (DMF) followed by heat treatment. TEM and particle analysis showed that the resulting particles had a narrow size distribution. The colloid was very stable and aggregation was not observed over a wide pH range (3-9) or at high salt concentration. The residual carboxylic acid of PAA could be modified via EDC/NHS activation to form an amide bond with a protein. An antibody was attached to the hybrid nanoparticle and specific binding to antigen was monitored by surface plasmon resonance. The results suggest that TiO2/PAA nanoparticles are candidates as the base component of a photocatalytic system with potential for substrate selectivity.

Dye-sensitized biosystem sensing using macroporous semiconducting metal oxide films

Transparent semiconducting oxide (TiO2, SnO2 and ZnO) films having highly interconnected pores in the range of 30–200 nm were fabricated in one-pot using polystyrene-block-poly(ethylene oxide) (PS-b-PEO) diblock copolymer. Such porous structures were helpful for efficient transport of bulky guest species such as proteins. Biosensing protocols were constructed in the films by using bulky biomolecules, namely, adsorbed DNA, adsorbed antibody and adsorbed antigen–antibody (immunoassay). In the DNA-based system, the macroporous TiO2 film was the most useful among porous anatase films such as mesoporous and mesoporous/PS beads derived macroporous ones, leading to significant detection of 125 times higher photocurrent generated from a dye (Cy5) attached to DNA than those observed for the others. Macroporous SnO2 and ZnO films were also available for the construction of similar biomolecule immobilized electrodes. Extremely complicated immunoassay reactions involving bulky proteins (antigen, antibody, etc.) were also established using the macroporous TiO2 and SnO2 films. Especially photocurrent for electron transfer from Cy5 attached to the second antibody was successfully detected by using the SnO2 film for exceptionally selective sensing applications.

Development of sensor cells using NF-κB pathway activation for detection of nanoparticle-induced inflammation.

The increasing use of nanomaterials in consumer and industrial products has aroused concerns regarding their fate in biological systems. An effective detection method to evaluate the safety of bio-nanomaterials is therefore very important. Titanium dioxide (TiO2), which is manufactured worldwide in large quantities for use in a wide range of applications, including pigment and cosmetic manufacturing, was once thought to be an inert material, but recently, more and more studies have indicated that TiO2 nanoparticles (TiO2 NPs) can cause inflammation and be harmful to humans by causing lung and brain problems. In order to evaluate the safety of TiO2 NPs for the environment and for humans, sensor cells for inflammation detection were developed, and these were transfected with the Toll-like receptor 4 (TLR4) gene and Nuclear Factor Kappa B (NF-κB) reporter gene. NF-κB as a primary cause of inflammation has received a lot of attention, and it can be activated by a wide variety of external stimuli. Our data show that TiO2 NPs-induced inflammation can be detected by our sensor cells through NF-κB pathway activation. This may lead to our sensor cells being used for bio-nanomaterial safety evaluation.

Analysis of the interaction between monoclonal antibodies and human hemoglobin (native and cross-linked) using a surface plasmon resonance (SPR) biosensor.

To develop a stable immuno-assay system for quantification of human hemoglobin (Hb), the interaction between various antibodies and Hb was studied using a surface plasmon resonance (SPR) biosensor in the BIAcore equipment (Amersham Pharmacia Biotech) with an immobilized anti-Hb antibody sensor chip. When polyclonal antibodies were used, the immuno-reactivity of purified and commercially available Hb decreased drastically with incubation times up to 14 h. This instability of immuno-reactivity of Hb is attributable to the conformational changes in Hb induced by oxidation. On the other hand, of the sixteen monoclonal antibodies tested, four antibodies (MSU-102, -103, -106 and -115) were found to maintain their immuno-reactivities at least up to 24 h. During long-term storage, however, the immuno-reactivity of Hb with these monoclonal antibodies decreased significantly. The chemical betabeta-cross-linking of Hb was effectively able to stabilize the structure of Hb and immuno-reactivity with monoclonal antibodies such as MSU-103 for periods at least up to 70 days. Therefore, the combination of specific monoclonal antibodies such as MSU-103 and a betabeta-cross-linked Hb standard could be used for the quantification of Hb.

Mismatch-induced lethality due to a defect in Escherichia coli RecQ helicase in exonuclease-deficient background: Dependence on MutS and UvrD functions.

Escherichia coli DNA-unwinding protein RecQ has roles in the regulation of general recombination and the processing of stalled replication forks. In this study, we found that knockout of the recQ gene in combination with xonA xseA recJ mutations, which inhibit methyl-directed mismatch repair (MMR), caused about 100-fold increase in sensitivity to a purine analog 2-aminopurine (2AP). Intriguingly, inactivation of a MMR initiator due to the either mutation mutS or uvrD completely suppressed the 2AP sensitivity caused by recQ xonA xseA recJ mutations, suggesting that RecQ helicase might act on the DNA structures that are generated by the processing of DNA by the MutSLH complex and UvrD helicase. Moreover, the recQ gene knockout in combination with xonA xseA recJ mutations enhanced 2AP-induced filament formation, and increased by twofold the rate of spontaneous forward mutations in the thyA locus but did not increase the rate of rifampicin-resistant mutations. We discuss about the possible interplay between E. coli RecQ helicase and mismatch recognition factors.

Characterization of the Erythropoietin (EPO) Protein Production in the Recombinant Human Cultured Cells, in which the Exogenous EPO Gene was Introduced into Hypoxanthine Phosphoribosyl Transferase (HPRT) Gene Locus

The fields of biological industry need to develop stable and efficient protein production systems. We previously reported that green fluorescent protein (GFP) gene, integrated into hypoxanthine phosphoribosyl transferase (HPRT) gene locus, was expressed at the constant level throughout long-term cultivation without any drug selection. In this study, we developed and characterized recombinant human erythropoietin (EPO) producer cell lines. Three kinds of hprt gene-targeting vectors with a different EPO expression cassette were constructed. The one kind of the vector, phprt-IVS-GT-EPO #52 (#52), has both EPO cDNA sequence and intron in the expression cassette. The #52 gene-targeting vector was introduced into HT1080 cell by electroporation, and both gene-targeted and randomly-integrated clones were selected by G418/6-thioguanine (6TG) or G418 drug screening, respectively. Three gene-targeted clones and four randomly-integrated clones were picked up arbitrarily and evaluated for specific EPO production rates. Average and standard deviation values of three gene-targeted clones were 0.32 ± 0.05 pg cell–1 day–1 (CV = 14.6%), and the values of four randomly-integrated clones were 0.38 ± 0.22 pg cell–1 day–1 (CV = 59.1%). The low CV value of gene-targeted clones was also observed in the other two kinds of the targeting-vector (CV = 15.2% and 16.1%). The results show that the producer cell lines developed by gene-targeting into hprt locus are uniformity with respect to the specific production rates of a recombinant protein. This character could make it easier to select producer cell lines among many candidate cell lines with great diversity of production level. The gene-targeted clones maintained the specific EPO production rates during long-term cultivation, at least 60 days, without any selection drugs (G418 and/or 6TG). While the specific EPO production rates decreased to 50–60% of the initial values during 60 days cultivation without any drug selection in the randomly-integrated clones. The results show that the long-term stability of recombinant protein production rates achieved by the targeted integration into hprt locus is also applicable to the secreted EPO protein.