Nobuki Kudo

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.

Prophylactic immunization with Bubble liposomes and ultrasound-treated dendritic cells provided a four-fold decrease in the frequency of melanoma lung metastasis

Melanoma has an early tendency to metastasize, and the majority of the resulting deaths are caused by metastatic melanoma. It is therefore important to develop effective therapies for metastasis. Dendritic cell (DC)-based cancer immunotherapy has been proposed as an effective therapeutic strategy for metastasis and recurrence due to prime tumor-specific cytotoxic T lymphocytes. In this therapy, it is important that DCs present peptides derived from tumor-associated antigens on MHC class I molecules. Previously, we developed an innovative approach capable of directly delivering exogenous antigens into the cytosol of DCs using perfluoropropane gas-entrapping liposomes (Bubble liposomes, BLs) and ultrasound. In the present study, we investigated the prevention of melanoma lung metastasis via DC-based immunotherapy. Specifically, antigens were extracted from melanoma cells and used to treat DCs by BL and ultrasound. Delivery into the DCs by this route did not require the endocytic pathway. The delivery efficiency was approximately 74.1%. DCs treated with melanoma-derived antigens were assessed for in vivo efficacy in a mouse model of lung metastasis. Prophylactic immunization with BL/ultrasound-treated DCs provided a four-fold decrease in the frequency of melanoma lung metastases. These in vitro and in vivo results demonstrate that the combination of BLs and ultrasound is a promising method for antigen delivery system into DCs.

ACTIVATION OF MICROBUBBLES BY SHORT-PULSED ULTRASOUND ENHANCES THE CYTOTOXIC EFFECT OF CIS-DIAMMINEDICHLOROPLATINUM (II) IN A CANINE THYROID ADENOCARCINOMA CELL LINE IN VITRO

Ultrasound targeted microbubble destruction has succeeded in delivering drugs and genes. This study was designed to explore characteristics of ultrasound targeted microbubble destruction using short-pulsed diagnostic ultrasound. Canine thyroid adenocarcinoma cells were exposed to short-pulsed diagnostic ultrasound in the presence of cis-diamminedichloroplatinum (II) (cisplatin) and ultrasound contrast agent Sonazoid(®) microbubbles. The cytotoxic effect of cisplatin was enhanced by short-pulsed diagnostic ultrasound and microbubbles. Incubation time with microbubbles influenced the cytotoxic effect of cisplatin. However, exposure duration did not affect the cytotoxic effect of cisplatin. Therefore, short-pulsed diagnostic ultrasound may activate microbubbles near cells and deliver cisplatin into cells. In addition, activation of microbubbles may be concluded in a short time. Our results suggest that short exposure duration could be potentially sufficient to induce efficient drug delivery by ultrasound targeted microbubble destruction using short-pulsed diagnostic ultrasound.

A Simple Technique for Visualizing Ultrasound Fields Without Schlieren Optics

A simple technique designed for visualization of ultrasound fields without Schlieren optics is introduced. An optical system of direct shadowgraphy with diverging light, which consists of a point light source and a shadow screen, constituted the basic system, but the screen was replaced by focusing optics: a camera that makes a virtual screen at its focus plane. The proposed technique visualizes displacement of light deflected by ultrasound, and the use of focusing optics enables flexible settings of the virtual screen position and optical magnification. Insufficient sensitivity of shadowgraphy was overcome by elimination of non-deflecting light using image subtraction of shadowgrams taken with and without ultrasound exposure. A 1-MHz focused transducer for ultrasound therapy and a 20-MHz miniature transducer for intravascular imaging were used for experiments, and alternate pressure change in short-pulsed ultrasound was visualized, indicating the usefulness of the proposed technique for evaluation of medical ultrasound fields.

Optical methods for visualization of ultrasound fields

A brief review of optical techniques for visualizing ultrasound fields is given. Acousto-optic interaction is discussed as a basic principle for visualizing inhomogeneity in refractive index fields. Light diffraction and deflection are also discussed as important phenomena, especially for visualization of ultrasound fields. Three techniques, Schlieren, phase contrast, and shadowgraph techniques, used for visualization of ultrasound fields are reviewed with discussion of the differences in optical systems and visualized field images. A newly developed focused shadowgraph technique that achieves sensitive detection with simple optics is introduced, and its usefulness for visualization of very short pulses of diagnostic ultrasound equipment is shown.

Ultrasound image-guided therapy enhances antitumor effect of cisplatin

PurposeThe aim of this study was to clarify whether ultrasound image-guided cisplatin delivery with an intratumor microbubble injection enhances the antitumor effect in a xenograft mouse model.MethodsCanine thyroid adenocarcinoma cells were used for all experiments. Before in vivo experiments, the cisplatin and microbubble concentration and ultrasound exposure time were optimized in vitro. For in vivo experiments, cells were implanted into the back of nude mice. Observed by a diagnostic ultrasound machine, a mixture of cisplatin and ultrasound contrast agent, Sonazoid, microbubbles was injected directly into tumors. The amount of injected cisplatin and microbubbles was 1xa0μg/tumor and 1.2xa0×xa0107 microbubbles/tumor, respectively, with a total injected volume of 20xa0μl. Using the same diagnostic machine, tumors were exposed to ultrasound for 15xa0s. The treatment was repeated four times.ResultsThe combination of cisplatin, microbubbles, and ultrasound significantly delayed tumor growth as compared with no treatment (after 18xa0days, 157xa0±xa055 vs. 398xa0±xa049xa0mm3, Pxa0=xa00.049). Neither cisplatin alone nor the combination of cisplatin and ultrasound delayed tumor growth. The treatment did not decrease the body weight of mice.ConclusionUltrasound image-guided anticancer drug delivery may enhance the antitumor effects of drugs without obvious side effects.

Effects of cell culture scaffold stiffness on cell membrane damage induced by sonoporation.

PurposeAs basic studies to realize in vivo sonoporation, rates of cell membrane damage during sonoporation were evaluated using monolayer cells cultured on scaffolds with different degrees of stiffness.MethodsFour types of scaffolds, constructed using collagen gel, 10 and 30xa0% acrylamide gels, and a coverslip, were used for cultivation of monolayer cells. Young’s moduli measured using an atomic force microscope were in the range 0.09–8.6xa0kPa for the gel scaffolds, whereas Young’s modulus for living cells was 4.5xa0kPa. Cells with attached microbubbles were exposed to one-shot pulsed ultrasound of 8.0/−1.3xa0MPa in peak positive/negative pressures with durations of 3, 100, and 10,000 cycles.ResultsCell membrane damage was visualized by fluorescence microscopy using propidium iodide. The 3-cycle ultrasound pulse had no significant effect; however, the rates of damage caused by 100-cycle and 10,000-cycle pulses showed a strong tendency for higher rates of damage with a higher Young’s modulus.ConclusionThe experimental results indicate that the stiffness of the underlying layer of adherent cells should be considered as an essential parameter of the sonoporation condition and that the optimum exposure conditions for in vivo sonoporation should be determined with consideration of the physical properties of underlying tissues.

Basic studies on sonoporation with size- and position-controlled microbubbles adjacent to cells

We have been studying a sonoporation technique that uses one-shot exposure of short-pulsed ultrasound to cells with attached microbubbles. Here, we introduce a new observation system developed for elucidation of sonoporation mechanisms. An inverted-type microscope was equipped with optical tweezers that enable position control of a microbubble. A Laguerre-Gaussian (LG) beam, a Gaussian beam with a dark spot at the center, was used to trap a microbubble with a refractive index lower than that of the surrounding water. The LG beam was produced using a phase-only spatial light modulator and 1,065-nm laser light. Results of basic experiments showed that the tweezers can trap bubbles of 1-30 μm in diameter suspended in water, and the minimum optical power for trapping bubbles of 5 μm in diameter was around 10 mW. Sonoporation experiments were carried out using size- and position-controlled bubbles placed on cell membranes or placed at separated positions from cells, and the results indicated the importance of the newly developed system for elucidation of sonoporation mechanisms.

Development of in vivo measurement system for temperature rise in animal tissue under exposure to ultrasound with acoustic radiation force

Acoustic radiation force (ARF) has been recently used for the tissue elasticity measurement and imaging. On the other hand, it is predicted that the higher temperature rises occur. In vivo measurement of temperature rise in animal experiments is important, whereas the measurement using thermocouples have some problems such as position mismatch of a temperature measuring junction of thermocouple and a focal point of ultrasound and so on. Therefore, in vivo measurement system for solving the above problems was developed in this study. The feasibility of the developed system was verified by the experiments using a tissue mimicking materials (TMM), TMM with a bone mode, an extracted porcine liver and a bone of chicken. Moreover, relations between conditions of ultrasound irradiation and temperature rises were investigated using the system. These results showed that temperature rises at focus on the surface of bone may exceed an allowable temperature rise which WFUMB guideline recommends, even though the acoustic intensity is within the limits of acoustic output regulation in diagnostic ultrasound devices.

Temperature elevation of biological tissue model exposed by focused ultrasound with acoustic radiation force

Focused ultrasound with acoustic radiation force (ARF) is beginning to be used for imaging and measuring tissue elasticity. On the other hand, it was suggested that the temperature elevation near bone at focus may be significant within the limits of acoustic output regulation in diagnostic ultrasound devices (Herman; 2002). In this study, with the aim of obtaining the relationships between temperature elevations and parameters of ultrasound exposure with ARF, temperature elevations in two kinds of tissue models with or without bone were numerically evaluated. The results showed that the temperature elevation at focus on the surface of bone may exceed an allowable temperature elevation which WFUMB guideline recommends, even though the acoustic intensity is within the limits of acoustic output regulation in diagnostic ultrasound devices.