Atsuko Aoi

Low‐intensity ultrasound and microbubbles enhance the antitumor effect of cisplatin

Cell permeabilization using microbubbles (MB) and low‐intensity ultrasound (US) have the potential for delivering molecules into the cytoplasm. The collapsing MB and cavitation bubbles created by this collapse generate impulsive pressures that cause transient membrane permeability, allowing exogenous molecules to enter the cells. To evaluate this methodology in vitro and in vivo, we investigated the effects of low‐intensity 1‐MHz pulsed US and MB combined with cis‐diamminedichloroplatinum (II) (CDDP) on two cell lines (Colon 26 murine colon carcinoma and EMT6 murine mammary carcinoma) in vitro and in vivo on severe combined immunodeficient mice inoculated with HT29‐luc human colon carcinoma. To investigate in vitro the efficiency of molecular delivery by the US and MB method, calcein molecules with a molecular weight in the same range as that of CDDP were used as fluorescent markers. Fluorescence measurement revealed that approximately 106–107 calcein molecules per cell were internalized. US–MB‐mediated delivery of CDDP in Colon 26 and EMT6 cells increased cytotoxicity in a dose‐dependent manner and induced apoptosis (nuclear condensation and fragmentation, and increase in caspase‐3 activity). In vivo experiments with xenografts (HT29‐luc) revealed a very significant reduction in tumor volume in mice treated with CDDP + US + MB compared with those in the US + CDDP groups for two different concentrations of CDDP. This finding suggests that the US–MB method combined with chemotherapy has clinical potential in cancer therapy. (Cancer Sci 2008; 99: 2525–2531)

EVALUATION OF TRANSFECTION EFFICIENCY IN SKELETAL MUSCLE USING NANO/MICROBUBBLES AND ULTRASOUND

Recent studies have revealed that ultrasound contrast agents with low-intensity ultrasound, namely, sonoporation, can noninvasively deliver therapeutic molecules into target sites. However, the efficiency of molecular delivery is relatively low and the methodology requires optimization. Here, we investigated three types of nano/microbubbles (NMBs)-human albumin shell bubbles, lipid bubbles and acoustic liposomes-to evaluate the efficiency of gene expression in skeletal muscle as a function of their physicochemical properties and the number of bubbles in solution. We found that acoustic liposomes showed the highest transfection and gene expression efficiency among the three types of NMBs under ultrasound-optimized conditions. Liposome transfection efficiency increased with bubble volume concentration; however, neither bubble volume concentration nor their physicochemical properties were related to the tissue damage detected in the skeletal muscle, which was primarily caused by needle injection.

A non-invasive tissue-specific molecular delivery method of cancer gene therapy.

A Japanese word, monozukuri (literally translated “making things”) is the philosophy of first having the idea and then the faith in the technical expertise and experience to accomplish the result. We believe that the concept of engineering is monozukuri. Through the process of monozukuri, engineered natural science based on mathematics and physics has been developed. Medicine is the field of study which has been developed for maintaining daily healthy life with diagnosis, treatment, examination, and protection. Biomedical engineering is the interdisciplinary study of engineering and medicine, and should be developed based on monozukuri. In this particular research, we have developed a physical molecular delivery method for cancer gene therapy using nano/microbubbles and ultrasound. First, the behavior of cavitation bubbles and subsequent shock wave phenomena involved in the mechanism of molecular delivery were analyzed, combining theory and computer simulation. In a second step, the methodology was optimized in vitro and in vivo. Finally, the therapeutic potential of the method in pre‐clinical models was evaluated using transgenes relevant to cancer gene therapy instead of reporter genes, and whole body, non‐invasive imaging using single photon emission computed tomography (SPECT/CT) was used to evaluate the selectivity of gene delivery in vivo.

Antitumor effectiveness of cisplatin with ultrasound and nanobubbles

The potentiation of antitumor effect of cis‐diamminedichloroplatinum (II), cisplatin, with ultrasound (1 MHz, 0.6 MPa) and lipid‐shelled nanobubbles in vitro (EMT6, C26, MCF7, A549) and in vivo on s.c. tumor in mice (HT29‐expressing luciferase) were evaluated. In vitro and in vivo antitumor effects were measured by an MTT assay and a real‐time in vivo imaging, respectively. The effective antitumor effect was seen both in vitro and in vivo when ultrasound and nanobubbles were used, while other treatment groups with cisplatin with ultrasound did not show the effectiveness. The antitumor effect was not attributed to necrosis but apoptosis, which was confirmed by increase in the activity of the pro‐apoptosis signal caspase‐3 and Bax. In conclusion, the combination of ultrasound and nanobubbles with cisplatin is an effective chemotherapy of solid tumors and may prove useful in clinical application.