Although we have reported our attempts to actively control microbubbles in flow using acoustic radiation force for future drug delivery systems, the microbubbles we used are not applicable for in vivo experiments. Thus, we examined two types of nanobubble with a drug-retaining function. Because the nanobubbles are invisible in a conventional optical observation, we observed the behavior of nanobubbles using ultrasound images (echograms). First, we found the optimal settings of echography to guarantee the relationship between the brightness variation and lipid concentration of nanobubbles. Then, we derived the destructive coefficient using two types of path under continuous ultrasound exposure of 5 MHz. Results indicate that the controllability is related to the construction of nanobubbles and the spatial distribution of the ultrasound field. We realized that the design of the ultrasound field is important with Bubble A, whereas the frequency of ultrasound emission needs to be discussed with Bubble B.

We have developed a support system using our ultrasound diagnosis robot, which is able to support manual handling of ultrasound probe in echography to alleviate fatigue of examiner. This system realizes a coordinated motion according to the motion of the probe, which is hold by the robot and is moved by an examiner. We have established four kinds of situations, which are initial fixation, coordinate motions with/without contact on the body surface, and automatic chase motion of an internal organ. The system recognizes when the examiner grasps the ultrasound probe by 6-axis force sensor and touches it on body surface by processing echograms. Not only unskilled examiners but also a professional sonographer have evaluated the performance of the system after elucidating multiple parameters for compliance control and self-weight and moment compensation of the probe. As the results, this system has the potential to be able to support advanced diagnosis for conventional echography.

We are now developing an in vivo cell delivery system, which is realized by producing aggregates in which bubbles attach onto the surface of cells with acoustic radiation force. We confirmed the controllability of the aggregation of cells with bubbles compared with those without bubbles. However, because of the destruction of bubbles under continuous ultrasound exposure, there is a compatibility problem between controllability and duration of aggregation. Therefore, we introduced the use of a standing wave of an acoustic field to trap the aggregates in the nodes, in which the aggregates are not directly exposed to a high-pressure sound, and manupilation by varying the acoustic field. We prepared the travelling and oscillation motions of the ultrasound standing wave and investigated the mobility of aggregates that were trapped in the nodes of the standing wave to be translated two-dimensionally. Regarding travelling motion, we found that the node velocities of 10 and 20 µm/s showed better performance than that of 40 µm/s. Regarding oscillation motion, we succeeded in the continuous oscillation of aggregates for 36 s. Also, we found the problem of immobile cells, which was considered to be due to the destruction of bubbles.

Although immunotherapy has been recognized as a new therapeutic method, there is a problem of low cell concentration because of cell dispersion in the bloodstream. We previously developed methods of active induction of cells applicable to future therapeutic methods by using aggregations of bubbles containing Colon-26 cells. However, Colon-26 cells are not appropriate for therapeutic applications. Furthermore, the controllability of aggregation under ultrasound exposure has been confirmed only in still water, not in a flow. Therefore, we utilized T-cell hybridoma (CD8-OVA1.3) and induced the aggregates of bubbles and cells by using acoustic radiation force with the motion of travelling nodes. We improved our observation system including its digital camera for the active induction of aggregates containing therapeutic cells in a flow under ultrasound exposure. We found that the induction performance of bubble-surrounded cells (BSCs) was enhanced with an ultrasound frequency of 5 MHz rather than 3 or 7 MHz. Furthermore, we found that a node velocity of 5 mm/s shows the best performance compared with 1 or 10 mm/s, at which the flow velocity was 10 mm/s. We believe that the parameters realized in this study indicate effective in vivo applications in the future.

We previously reported methods of the active control of the cells by forming bubble-surrounded cells (BSCs), in which bubbles were attached to the surface of the cells, to enhance the controllability of the cells under ultrasound exposure. However, mechanical or biological damage to the cells contained in BSCs according to the conditions of ultrasound exposure has not been clarified. Therefore, we carried out the viability validation of the cells in BSCs versus various conditions of ultrasound exposure. First, we verified the reliability of cell viability by introducing two methods of CCK-8 assay and lactate dehydrogenase assay. Then, we confirmed that the surrounded amount of bubbles decreases the cell viability under continuous wave exposure rather than burst wave. We optimized the parameters to produce BSCs with the concentration of bubbles of 0.3 mg ml ⁻¹ and the concentration of the cells of 1.0 × 10 ⁵ ml ⁻¹ , to maintain the cell viability under ultrasound exposure. Also, using the maximum sound pressure lower than 300 kPa-pp, and low duty ratio less than 50%, the cell viability would be maintained more than 75% within the emission duration of 60 s.