Detection of cellular response to titanium dioxide nanoparticle agglomerates by sensor cells using heat shock protein promoter.

Nanotechnology is becoming increasingly important for products used in our daily lives, such as the masses of titanium dioxide nanoparticle agglomerates (TiO(2) NPs) used in the pharmaceutical industry, for cosmetic products, or for pigments. Meanwhile, a serious lack of detailed information concerning the interaction between the nanomaterials and cells limits their biological and medical applications. Sensing technology is very important for understanding these interactions. We have shown that TiO(2) NPs induce heat shock protein 70B' (HSP70B') mRNA [Okuda-Shimazaki et al., 2010. Int J Mol Sci 11:2383-2392]. In the current work, sensor cells for detection of cellular responses to NPs were prepared by transfecting an HSP70B' promoter-reporter plasmid. First, to find suitable cells for detection, five different mammalian cell lines were chosen as potential sensor cells. The results showed TiO(2) NP response in some cell lines, although different sensor cells had different TiO(2) NP response levels, as heat shock response ability is important for the detection. Then, we studied the TiO(2) NP time-course response and dose response. The results indicated that our sensor cells can detect TiO(2) NP cellular responses. Our work should aid in understanding the interactions between bio-nanomaterials and cells.

Effect of polyethylene glycol modification of TiO2nanoparticles 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 (TiO(2)) is a type of nanoparticle that is widely used in paints, pigments, and cosmetics; however, little is known about the impact of TiO(2) on human health and the environment. Therefore, considerable research has focused on characterizing the potential toxicity of nanoparticles such as TiO(2) and on understanding the mechanism of TiO(2) NP-induced nanotoxicity through the evaluation of biomarkers. Uncoated TiO(2) 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 TiO(2) NPs induce inflammatory and heat shock responses. In order to reduce their toxicity, we conjugated TiO(2) NPs with polyethylene glycol (PEG) to eliminate aggregation. Our findings indicate that modifying TiO(2) NPs with PEG reduces their cytotoxicity and reduces the induction of stress-related genes. Our results also suggest that TiO(2) NP-induced effects on cytotoxicity and gene expression vary depending upon the cell type and surface modification.

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 (TiO(2)), 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 TiO(2) nanoparticles (TiO(2) NPs) can cause inflammation and be harmful to humans by causing lung and brain problems. In order to evaluate the safety of TiO(2) 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 TiO(2) 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.

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 (TiO(2)) is thought to be a photocatalytic agent excited by UV light. Our aim was to investigate the photocatalytic antitumor effect of water-dispersed TiO(2) nanoparticles on C6 rat glioma cells and to evaluate the treatment responses by the spheroid models. Water-dispersed TiO(2) nanoparticles were constructed by the adsorption of chemical modified polyethylene glycol (PEG) on the TiO(2) surface (TiO(2)/PEG). Each monolayer and spheroid of C6 cells was coincubated with various concentrations of TiO(2)/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 TiO(2)/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 microg mL(-1) TiO(2)/PEG. The irradiated spheroids in the presence of TiO(2)/PEG showed growth suppression compared with control groups. In TiO(2)/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 TiO(2)/PEG have antitumoral activity. Photodynamic therapy utilizing this material can be a clue to a novel therapeutic strategy for glioma.

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.

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.

Recognition and effective degradation of 17beta-estradiol by anti-estradiol-antibody-immobilized TiO(2) nanoparticles.

Polyelectrolyte polyacrylic acid (PAA), used in the chemical modification of titanium dioxide (TiO(2)) nanoparticles, allows TiO(2) nanoparticles to remain in suspension at neutral pH. The anti-17beta-estradiol (E2) antibody was immobilized on PAA-modified TiO(2) (PAA-TiO(2)) nanoparticles via covalent bonding between the carboxylic acid of PAA and the amino group of the antibody. The anti-E2-antibody-immobilized TiO(2) (E2Ab-PAA-TiO(2)) nanoparticles can form a suspension at neutral pH, with a particle size of less than 100 nm. The E2-PAA-TiO(2) nanoparticles caused the photocatalytic degradation of a typical TiO(2) substrate, methylene blue. The anti-E2 antibody immobilized on the TiO(2) surface recognized and bound E2 in the solution, thereby improving the efficiency of E2 degradation compared with that of PAA-TiO(2) nanoparticles. These results demonstrate that the E2Ab-PAA-TiO(2) nanoparticles developed in this study can be used in water treatment technology. Furthermore, this strategy of immobilizing proteins on nanoscale TiO(2) particles creates new applications not only in the treatment of environmental waste, but also in medical and public sanitation processes.

Stable expression of a heterogeneous gene introduced via gene targeting into the HPRT locus of human fibrosarcoma cells.

To obtain a cell line that maintains stability of gene expression is important for industrial production of therapeutic proteins from recombinant cells. In this study, we attempted to improve the stability of expression of an exogenous gene by using the gene-targeting method in cultured cells. In our gene-targeting system, the green fluorescent protein (GFP) gene was used as an exogenous reporter gene targeted to the locus of the endogenous hypoxanthine phosphoribosyl transferase (HPRT) gene, which is constitutively expressed. Cell lines selected using markers of the targeting DNA were cultivated for 129 days without any drug selection, and the expression levels of GFP protein and the chromosomal structure of the gfp gene in these cell lines were evaluated. Cell lines in which gfp genes were randomly integrated into the genome showed decreased GFP expression, which resulted from loss of genes or attenuation of transcription. In contrast, cell lines in which the gfp gene was targeted to the hprt locus maintained a stable chromosomal structure and stable expression of the gfp gene, even after prolonged cultivation. These results suggest that constitutively expressed endogenous gene loci may be suitable positions for stable expression of exogenous genes, and that the gene-targeting strategy presented here may be useful for generation of cell lines for industrial protein production.