Koki Kanehira

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.

Effects of Titanium Dioxide Nanoparticle Aggregate Size on Gene Expression

Titanium dioxide (titania) nanoparticle aggregation is an important factor in understanding cytotoxicity. However, the effect of the aggregate size of nanoparticles on cells is unclear. We prepared two sizes of titania aggregate particles and investigated their biological activity by analyzing biomarker expression based on mRNA expression analysis. The aggregate particle sizes of small and large aggregated titania were 166 nm (PDI = 0.291) and 596 nm (PDI = 0.417), respectively. These two size groups were separated by centrifugation from the same initial nanoparticle sample. We analyzed the gene expression of biomarkers focused on stress, inflammation, and cytotoxicity. Large titania aggregates show a larger effect on cell viability and gene expression when compared with the small aggregates. This suggests that particle aggregate size is related to cellular effects.

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.

Molecular mechanism of DNA damage induced by titanium dioxide nanoparticles in toll-like receptor 3 or 4 expressing human hepatocarcinoma cell lines

BackgroundTitanium dioxide nanoparticles (TiO2 NPs) are widely used in the biological sciences. The increasing use of TiO2 NPs increases the risk of humans and the environment being exposed to NPs. We previously showed that toll-like receptors (TLRs) play an important role in the interactions between NPs and cells. Our previous results indicated that TLR4 increased the DNA damage response induced by TiO2 NPs, due to enhanced NP uptake into the cytoplasm, whereas TLR3 expression decreased the DNA damage response induced by TiO2 NPs because of NP retention in the endosome. In this study, we explored the molecular mechanism of the DNA damage response induced by TiO2 NPs using TLR3 or TLR4 transfected cells. We examined the effect of TLR3 or TLR4 over-expression on oxidative stress and the effect of DNA damage induced by TiO2 NPs on gene expression levels.ResultsOur results showed evidence for elevated oxidative stress, including the generation of reactive oxygen species (ROS), with increased hydrogen peroxide levels, decreased glutathione peroxidase, and reduced glutathione and activated caspase-3 levels in cells exposed for 48 h to 10 μg/ml TiO2 NPs. These effects were enhanced by TLR4 and reduced by TLR3 over-expression. Seventeen genes related to DNA double-strand breaks and apoptosis were induced, particularly IP6K3 and ATM.ConclusionOur results indicated that TiO2 NPs induced ROS, and the above molecules are implicated in the genotoxicity induced by TiO2 NPs.

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.

Role of toll-like receptors 3, 4 and 7 in cellular uptake and response to titanium dioxide nanoparticles

Abstract Innate immune response is believed to be among the earliest provisional cellular responses, and mediates the interactions between microbes and cells. Toll-like receptors (TLRs) are critical to these interactions. We hypothesize that TLRs also play an important role in interactions between nanoparticles (NPs) and cells, although little information has been reported concerning such an interaction. In this study, we investigated the role of TLR3, TLR4 and TLR7 in cellular uptake of titanium dioxide NP (TiO2 NP) agglomerates and the resulting inflammatory responses to these NPs. Our data indicate that TLR4 is involved in the uptake of TiO2 NPs and promotes the associated inflammatory responses. The data also suggest that TLR3, which has a subcellular location distinct from that of TLR4, inhibits the denaturation of cellular protein caused by TiO2 NPs. In contrast, the unique cellular localization of TLR7 has middle-ground functional roles in cellular response after TiO2 NP exposure. These findings are important for understanding the molecular interaction mechanisms between NPs and cells.

Effects of Toll-like Receptors 3 and 4 Induced by Titanium Dioxide Nanoparticles in DNA Damage-Detecting Sensor Cells

Live cell-based sensor reporter systems (so-called sensor cells) were employed to detect host defense systems, including DNA damage response, stimulated by nanoparticles (NPs). Our previous work established the use of DNA damage-detecting sensor cells containing the B-cell translocation gene 2(BTG2) promoter-reporter plasmid and showed that Toll-like receptors (TLRs) are involved in the cellular response and uptake of TiO2 NPs. These results suggested that TLRs could be involved in many cellular responses. However, the effect of TLRs on DNA damage induced by TiO2 NPs is unknown. Here we investigated the role of TLR 3 and 4 in DNA damage induced by PEG- 2 NPs reduces DNA damage response compared to unmodified TiO2 NPs. The overexpression of TLR3 reduces DNA damage mediated by both TiO2 and PEG-TiO2 NPs. In contrast, overexpression of TLR4 increases the DNA damage response induced by TiO2 NPs. The results indicate that co-transfection of TRL4 expression vector affects the sensitivity of DNA damage response, but does not affect the detection limit of the DNA damage response. These finding will aid in understanding the molecular interaction mechanisms between NPs and cells.