•Ultrasound could facilitate the bacteria-mediated gene delivery (bactofection).•Escherichia coli were used as delivery vehicle of GFP gene into cancer cell MCF-7.•Bactofection efficiency was 0.5% when E. coli cell surface was modified with avidin.•Bactofection efficiency was 2% when ultrasound was irradiated.•Bactofection efficiency became 8% by using both avidin-modification and ultrasound.The present study demonstrates that ultrasound irradiation can facilitate bacteria-mediated gene delivery (bactofection). Escherichia coli modified with avidin were employed as a vehicle for delivery of the green fluorescent protein (GFP) gene, a model heterologous gene, into the breast cancer cell line MCF-7. Avidin-mediated binding of E. coli to MCF-7 cells enhanced the internalization of E. coli by approximately 17%, irrespective of the use of ultrasound irradiation. Furthermore, the use of ultrasound irradiation increased the internalization by approximately 5%, irrespective of the presence of avidin on the E. coli cell surface. The percentages of GFP-expressing MCF-7 cells at 24 h after bactofection were below 0.5% and 2% for the case with only avidin-modification of E. coli cell surface and only ultrasound irradiation, respectively. However, combining avidin modification with the ultrasound treatment increased this value to 8%. Thus, the use of avidin-modified bacteria in conjunction with ultrasound irradiation has potential as an effective strategy for tumor-targeted bactofection.

•Dual-frequency ultrasound (US) with TiO2 nanoparticles (NPs) enhanced OH radicals.•0.5 MHz US at 0.8 W/cm2 and 1 MHz US at 0.4 W/cm2 with TiO2 NPs was optimum.•Targeting protein-immobilized TiO2 NPs were used for its uptake by cancer cells.•“Targeted-TiO2/dual-US treatment” for 60 s suppressed cancer cell growth by 46%.The present study demonstrated the enhanced hydroxyl (OH) radical generation by combined use of dual-frequency (0.5 MHz and 1 MHz) ultrasound (US) and titanium dioxide (TiO2) nanoparticles (NPs) as sonocatalyst. The OH radical generation became the maximum, when 0.5 MHz US was irradiated at an intensity of 0.8 W/cm2 and 1 MHz US was irradiated at intensities at 0.4 W/cm2 in the presence of TiO2 NPs under the examined conditions. After incorporation of TiO2 NPs modified with targeting protein pre-S1/S2, HepG2 cancer cells were subjected to the dual-frequency US at optimum irradiation intensities (“targeted-TiO2/dual-US treatment”). Growth of the HepG2 cells was reduced by 46% compared with the control condition after irradiation of dual-frequency US for 60 s with TiO2 NPs incorporation. In contrast, HepG2 cell growth was almost the same as that in the control condition when cells were irradiated with either 0.5 MHz or 1 MHz ultrasound alone without TiO2 NP incorporation.

The present study demonstrated that the combined use of the sonocatalytic reaction (using ultrasound and titanium dioxide) and the Fenton reaction exhibited synergistically enhanced hydroxyl (OH) radical generation. Dihydroxybenzoic acid (DHBA) concentration as index of OH radical generation was 13 and 115 μM at 10 min in the sonocatalytic reaction and Fenton reaction, respectively. On the other hand, the DHBA concentration was 378 μM at 10 min in the sonocatalytic–Fenton reaction. The sonocatalytic–Fenton reaction was used for degradation of lignin. The lignin degradation ratio was 1.8%, 49.9%, and 60.0% at 180 min in the sonocatalytic reaction, Fenton reaction, and sonocatalytic–Fenton reaction, respectively. Moreover, the sonocatalytic–Fenton reaction was applied to pretreatment of lignocellulosic biomass to enhance subsequent enzymatic saccharification. The cellulose saccharification ratio was 11%, 14%, 16% and 25% at 360 min of pretreatment by control reaction, the sonocatalytic reaction, Fenton reaction, and sonocatalytic–Fenton reaction, respectively.Highlights► This is the first study to combine sonocatalytic and Fenton reactions. ► Sonocatalytic–Fenton reaction showed synergistically enhanced OH radical generation. ► The OH radical generation was applied to lignin degradation and biomass pretreatment. ► Lignin degradation ratio by sonocatalytic–Fenton reaction was 60.0% at 180 min. ► Cellulose saccharification ratio by sonocatalytic–Fenton reaction was 25% at 360 min.

Single-stranded DNA aptamers recognizing human hepatocarcinoma were isolated by means of a systematic evolution of ligands by exponential enrichment using whole cells as targets (cell-SELEX). After 11 rounds of cell-SELEX procedure using human hepatoma HepG2 cells as targets and human normal hepatocyte cells as counterparts, 12 independent DNA aptamer candidate sequences were obtained. The specific interaction between selected DNA aptamers and targeted cell was confirmed. Dissociation constants of the 12 sequences obtained were also estimated in the range of 19–450 nM. Moreover, the consensus secondary structure was found in the isolated aptamers, which was responsible to the recognition of HepG2 cells.Single-stranded DNA aptamers recognizing human liver cancer cells (HepG2) were isolated by means of a systematic evolution of ligands by exponential enrichment using whole cells as targets (cell-SELEX). The selected DNA aptamers specifically binds to human hepatocarcinoma, and its dissociation constant was 19 nM. The consensus secondary structure was found in the isolated aptamers, which was responsible to the recognition of HepG2 cells.

Our previous study suggested new sonodynamic therapy for cancer cells based on the delivery of titanium dioxide (TiO2) nanoparticles (NPs) modified with a protein specifically recognizing target cells and subsequent generation of hydroxyl radicals from TiO2 NPs activated by external ultrasound irradiation (called TiO2/US treatment). The present study first examined the uptake behavior of TiO2 NPs modified with pre-S1/S2 (model protein-recognizing hepatocytes) by HepG2 cells for 24 h. It took 6 h for sufficient uptake of the TiO2 NPs by the cells. Next, the effect of the TiO2/US treatment on HepG2 cell growth was examined for 96 h after the 1 MHz ultrasound was irradiated (0.1 W/cm2, 30 s) to the cells which incorporated the TiO2 NPs. Apoptosis was observed at 6 h after the TiO2/US treatment. Although no apparent cell-injury was observed until 24 h after the treatment, the viable cell concentration had deteriorated to 46% of the control at 96 h. Finally, the TiO2/US treatment was applied to a mouse xenograft model. The pre-S1/S2-immobilized TiO2 (0.1 mg) was directly injected into tumors, followed by 1 MHz ultrasound irradiation at 1.0 W/cm2 for 60 s. As a result of the treatment repeated five times within 13 days, tumor growth could be hampered up to 28 days compared with the control conditions.Highlights► New sonodynamic therapy using titanium dioxide (TiO2) nanoparticles was suggested. ► TiO2 nanoparticles were modified with a targeting protein recognizing specific cells. ► Hydroxyl radical was generated from TiO2 nanoparticles under ultrasound (US) irradiation. ► TiO2/US treatment could prevent the growth of cancer cells in vitro. ► TiO2/US treatment could prevent tumor growth of the xenograft model.

A non-woven titanium dioxide (TiO2) fabric was applied to disinfection by ultrasound (US) irradiation, and the disinfection efficiency and lipid peroxidation of Escherichia coli (E. coli) cell membrane were evaluated to investigate the killing process. The addition of non-woven TiO2 fabric enhanced hydroxyl (OH) radical generation and disinfection efficiency. Judging from the disinfection experiments using glutathione or t-butanol as a radical scavenger, the OH radical played a major role in cell killing in sonodynamic disinfection with non-woven TiO2 fabric. Moreover, to understand the detailed killing process, damage to cell membrane was also evaluated using a diphenyl-1-pyrenylphosphine (DPPP) fluorescent probe, which detects the membrane’s lipid peroxidation. The addition of non-woven TiO2 fabric aggravated this peroxidation. This aggravation was caused by the OH radical according to an assay using a radical scavenger. From these results, it was concluded that non-woven TiO2 fabric as a sonocatalyst promoted peroxidation of the polyunsaturated phospholipid component of the lipid membrane initially and induced a major disorder in the E. coli cell membrane under US irradiation.

Recently, our group discovered an alternative titanium dioxide (TiO2) activation method that uses ultrasound irradiation (US/TiO2) instead of ultraviolet irradiation. The pre-S1/S2 protein from hepatitis B virus, which recognizes liver cells, was immobilized to the surface of TiO2 nanoparticles using an amino-coupling method. The ability of the protein-modified TiO2 nanoparticles to recognize liver cells was confirmed by surface plasmon resonance analysis and immuno-staining analyses. After uptake of TiO2 nanoparticles by HepG2 cancer cells, the cells were injured using this US/TiO2 method; significant cell injury was observed at an ultrasound irradiation intensity of 0.4 W/cm2. Together with these results, this strategy could be applied to new cell injuring systems that use ultrasound irradiation in place of photodynamic therapy in the near future.

The generation of hydroxyl (OH) radicals was investigated during ultrasonic irradiation and in the presence of TiO2. The effect of TiO2 on an ultrasonic system’s oxidation power was evaluated by examining the oxidation of salicylic acid. The generation of the salicylic acid derivatives, 2,3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA, was measured by high-performance liquid chromatography coupled with electrochemical detection under different experimental conditions. The presence of TiO2 enhanced the generation of DHBA during ultrasonic irradiation, thus indicating a higher oxidation power in the ultrasonic system. Al2O3 also increased the generation of DHBA during irradiation; however, the effect of TiO2 was found to be higher than that of Al2O3. The addition of OH radical scavengers such as dimethylsulfoxide (DMSO), methanol and mannitol significantly suppressed the production of DHBA, and DMSO was found to have the highest suppressive effect among all scavengers. The effects of dissolved gases on the generation of OH radicals were further studied, and their power was found to be in the order Xe > Ar > O2 > N2. The degassing of the irradiation solution completely suppressed the generation of OH radicals. These results indicate that the presence of TiO2 accelerates the generation of OH radicals during ultrasonic irradiation, and that the process may be mediated through the induction of cavitation bubbles in irradiating solutions.

A series of experiments were carried out to study the degradation of methylene blue by the irradiation of ultrasound onto TiO2 in aqueous solution. A statistically significant decrease in the concentration of methylene blue was observed after 60 min irradiation. While the reduction was 22% of the initial concentration without H2O2, addition of H2O2 significantly enhanced the degradation of methylene blue for the TiO2 containing system (85% reduction of the initial concentration). The addition of H2O2 had no effect on the methylene blue degradation when the system contained Al2O3. The degradation ratio of methylene blue was dependent on the amount of TiO2 and also the specific surface area of TiO2 in the solution. The effects of radical scavenging agents on the degradation of methylene blue were also investigated for the system with TiO2. It was found that the radical scavenging agents dimethyl sulfoxide (DMSO), methanol, and mannitol suppressed the degradation, with DMSO being the most effective. The effect of pH on the degradation of methylene blue was further investigated. An U-shaped change in the concentration of methylene blue in the presence of TiO2 was observed along with the change in pH values (pH 3–12), and the highest degradation ratio was observed at around pH 7. In conclusion, ultrasound irradiation of TiO2 in aqueous solution resulted in significant generation of hydroxyl radicals, and this process may have potential for the treatment of organic dyes in wastewater.

•TiO2 nanoparticles injure the culture cell membrane in a dose and time-dependent manner.•The cell death induced by TiO2 is accompanied by condensed nuclear and genomic DNA fragmentation.•The apoptotic enzyme caspase-3 is also activated by TiO2.•In addition, cell death could not be prevented by the endocytosis inhibitor cytochalasin D.•Thus, even contact of TiO2 to the cell membrane could induce apoptotic cell death.We investigated the effects of nanosized TiO2 particles on the death of mouse leukemia L1210 cells. TiO2 particles suppressed proliferation and induced cell death, as measured by lactate dehydrogenase (LDH) release into the culture medium. Chromatin condensation, which is typical of the initiation of cell death, was observed in approximately 14% cells cultured with titanium dioxide (TiO2) particles for 12 h. Furthermore, giant DNA fragments of approximately 2 Mbp and high-molecular-weight DNA fragments between 100 kbp and 1 Mbp were observed in cells cultured for 18 h with TiO2 particles. These giant and high-molecular-weight DNA fragments were further degraded into smaller DNA fragments, appearing as DNA ladders. Corresponding to the generation of DNA fragments, caspase-3 activity increased in cells treated with TiO2 particles. TiO2 particle-induced LDH release was not inhibited by cytochalasin D, an inhibitor of endocytosis. These results suggest that nanosized TiO2 particles can induce apoptosis associated with DNA fragmentation and caspase-3 activation and that TiO2 particle-induced apoptosis is not caused by endocytosis but is associated with contact of the particles with the cell surface.

An image analyzing method was developed to evaluate in situ bioluminescence expression, without exposing the culture sample to the ambient oxygen atmosphere. Using this method, we investigated the effect of dissolved oxygen concentration on bioluminescence from an obligate anaerobe Bifidobacterium longum expressing bacterial luciferase which catalyzes an oxygen-requiring bioluminescent reaction.

This is the first study to demonstrate that Microcystis aeruginosa, a typical algal bloom-forming cyanobacterium, can be effectively inactivated by ultrasound (US) irradiation in the presence of titanium dioxide (TiO2) particles as a sonocatalyst. When a culture broth of M. aeruginosa was ultrasonically irradiated for 15 min in the presence of 0.5 g/mL of TiO2 particles 2 mm in diameter, the cell survival ratio was 0.13, which was significantly lower than that in the case of US irradiation alone (0.87). Moreover, regrowth of M. aeruginosa in the culture was also inhibited for 10 days following ultrasonic disinfection in the presence of TiO2 particles for 15 min.