Table 1.

Efficacy of sonodynamic therapy in vivo.

In vivo parameters	In vivo efficacy	Class of sonosensitizer	Primary mechanism of sonosensitizer
Doxorubicin-loaded microbubbles (DOX-MBs) were administered intravenously in Lewis rats while one of the two tumors (pancreatic carcinomas) was exposed to ultrasound (1.3 MHz; mechanical index 1.6). DOX tissue concentration was measured in tumors and control organs after the experiment [101].	All rats survived the DOX-MB administration without any sign of embolisation/occlusion of the pulmonary vasculature. Ultrasound targeted destruction of DOX-MBs resulted in a 12-fold higher tissue concentration of DOX and a significantly lower tumor growth in the target tumor compared to the contralateral control tumor.	DOX: anthracycline, ROS agent	It intercalates DNA, preventing DNA replication and protein synthesis, has been shown to reverse drug resistance in drug resistant K562/A02 leukemia cells [85], and produce ROS after ultrasound activation [102]
Metronomic cyclophosphamide (MCTX) was employed and administered through drinking water to athymic mice that harbored MDA-MB-231 breast cancer tumors. Ultrasound stimulated microbubble treatments were conducted at 1 MHz employing short bursts (0.00024 duty cycles) at 1.6 MPa in combination with the commercial microbubble agent Definity [103].	The USMB induced an acute reduction of blood flow as confirmed with US contrast imaging and DiOC7 perfusion staining. Longitudinal experiments demonstrated that significant growth inhibition occurred in MCTX-only and USMB-only treatment groups relative to control tumors. The combined USMB and MCTX treatment group showed significant growth inhibition and survival prolongation relative to the USMB-only and MCTX-only treatment groups.	MCTX: vascular disrupting agent, alkylating agent, Definity: echo contrast agent (increases microbubble concentration)	MCTX alkylates guanine nucleotides, inhibiting DNA replication and protein synthesis; the agent is converted in the liver to an active form for chemotherapeutic effects; and increased microbubbles from Definity, thereby amplifying inertial cavitation
A novel porphyrin-derived sonosensitizer designated DEG (7,12-bis(1-(2-(2-hydroxyethoxy)ethoxy)ethyl)-3,8,13,17-tetramethylporphyrin-2,18 dipropionatomanganese) was injected into SCID mice xenograft models with MKN-74 gastric cancer cells, followed by ultrasound (1.0 MHz, 1.0 W/cm2 output intensity, and 10% duty cycle for 1-2 min) [104].	SDT with DEG three times a week for 2 weeks potently inhibited tumor growth compared to ultrasound only or no treatment. It was shown that ROS are generated and mediate sonotoxicity of ultrasound with DEG on MKN-74 cells.	DEG: ROS agent, porphyrin	It generates ROS after excitation from sonoluminescent light that disrupts mitochondrial membrane potential, loss of electrochemical gradient, causes cristae to fragment, and induces apoptotic cascade to trigger caspase proteases
Epirubicin hydrochloride (EPI) inhibition on tumor growth by ultrasound was tested using five-week-old male nude mice injected s.c. with HL-60 human promyelocytic leukemia cells. 1-MHz ultrasound and 3 W/cm2 output power density were applied through aquasonic coupling gel for 30 s to the tumor region of a mouse [86].	Ultrasound applied locally to the tumor resulted in a substantially increased drug uptake in tumor cells. The inhibition on tumor growth depended on the position of drug injection and phospholipid-based microbubble (PMB) application. Artificial sonoporation nuclei significantly enhanced transient pore formation on cell membranes which facilitates outside drugs entry into the cells.	EPI: ROS agent, anthracycline	It generates ROS after excitation from sonoluminescent light that disrupts mitochondrial membrane potential, loss of electrochemical gradient, causes cristae to fragment, and induces apoptotic cascade to trigger caspase proteases
The taxane docetaxel (Taxotere) was used for evaluating SDT as it has previously been shown to have potent antitumor effects when combined with small molecule vascular disrupting agents. Experiments were conducted on PC3 human prostate cancer cell tumors implanted in athymic mice. USMB treatments were performed at a frequency of 1 MHz employing sequences of 50 ms bursts (0.00024 duty cycles) at 1.65 MPa. USMB treatments were administered on a weekly basis for 4 weeks with docetaxel (DTX) being given intravenously at a dose level of 5 mg/kg [105].	The USMB treatments, either alone or in combination with DTX, induced an acute reduction in tumor perfusion, accompanied by significantly enhanced necrosis and apoptosis after 24 hours. Longitudinal experiments showed a modest prolongation in survival but no significant growth inhibition occurred in DTX-only and USMB-only treatment groups relative to control tumors. The combined USMB-DTX treatment group produced tumor shrinkage in weeks 4–6 and significant growth inhibition and survival prolongation relative to the control, USMB-only, and DTX-only treatment groups.	DTX: cytoskeleton agent, taxane	It stabilizes GDP-bound tubulin polymers, thereby inhibiting mitosis
The sonodynamically induced antitumor effect of porfimer sodium (PF) was evaluated on a chemically induced mammary tumor in Sprague-Dawley rats. The timing of 24 hours after the administration of PF was chosen for the ultrasonic exposure, based on pharmacokinetic analysis of the PF concentrations in the tumor, plasma, skin, and muscle. The rats were exposed to ultrasound (3 W/cm2) for 15 min [106].	The synergistic effect between PF administration and ultrasonic exposure on the tumor growth inhibition was significant. The ultrasonic intensity showed a relatively sharp threshold for the synergistic antitumor effect, which is typical of an ultrasonic effect mediated by acoustic cavitation. Therefore, a marked synergistic effect between PF administration and ultrasonic exposure on the tumor growth inhibition was observed at a PF dose of 2.5 mg/kg and at a free-field ultrasonic intensity of 3 W/cm2.	PF: ROS agent, hematoporphyrin derivative	It generates ROS after excitation from sonoluminescent light that disrupts mitochondrial membrane potential, loss of electrochemical gradient, causes cristae to fragment, and induces apoptotic cascade to trigger caspase proteases
5-Aminolevulinic acid (ALA), a precursor to the ROS agent protoporphyrin IX (PpIX) was investigated for its antiangiogenic potency in vivo. SAS human oral cancer cell suspensions were injected s.c. into the flanks of BALB/c mice. ALA was intraperitoneally injected into mice in the ALA and ultrasound + ALA groups at a dose of 250 mg/kg body weight. After 4 hours of administration of ALA, the mice were placed on a plexiglass plate with the tumor immersed in degassed water. Tumors were irradiated by ultrasound (1.1 MHz, 2 W/cm2, 50% duty cycle) for 5 min [107].	Ultrasound treatment significantly decreased microvessel density (MVD) compared with control, and the reduction of MVD was more prominent in the ultrasound + ALA group. Accordingly, the expression level of VEGF, a critical proangiogenic factor, was reduced in tumors treated with ultrasound irradiation. Ultrasound plus ALA induced more significant decrease in VEGF expression than ultrasound alone. It also inhibited the secretion of VEGF in SAS cells more significantly in the presence of ALA.	ALA: ROS agent, precursor to hematoporphyrin derivative	It generates ROS after excitation from sonoluminescent light that disrupts mitochondrial membrane potential, loss of electrochemical gradient, causes cristae to fragment, and induces apoptotic cascade to trigger caspase proteases
Reversal of DOX resistance was investigated in a study of low intensity ultrasound. Athymic nude mice were inoculated with HepG2 multidrug resistant hepatocellular carcinoma cells. Ultrasound with pulsed irradiation (0.5 W/cm2) was administered for 10 min to both ultrasound/DOX and ultrasound only groups [96].	Ultrasonic treatment resulted in an average 62% reduction in tumor volume a month later. The relative levels of MDR1 and MRP were dramatically reduced in ultrasound/DOX groups, suggesting a reversal of drug resistance.	DOX: anthracycline, ROS agent	It intercalates DNA, preventing DNA replication and protein synthesis, ROS agent
The study was conducted on CT26 colon carcinoma tumors in BALB/c mice. In the respective groups, protoporphyrin IX (PpIX) or the gold nanoparticle-protoporphyrin IX conjugate was injected into the tumors. Ultrasound irradiation (1.1 MHz, 2 W/cm2, 3 min) was performed on the tumors 24 hours after injection [108].	A significant difference in the average relative volumes of the tumors 13 days after treatment was found between the ultrasound + gold nanoparticle-protoporphyrin IX group and the other groups. The longest doubling and 5  folding times were observed in the ultrasound + gold nanoparticle-protoporphyrin IX and ultrasound + protoporphyrin IX groups.	PpIX: ROS agent hematoporphyrin derivative	It generates ROS after excitation from sonoluminescent light that disrupts mitochondrial membrane potential, loss of electrochemical gradient, causes cristae to fragment, and induces apoptotic cascade to trigger caspase proteases
C57BL/6J female mice were inoculated s.c. with Hepa1-6 hepatocellular carcinoma cells. Herpes simplex virus thymidine kinase under the control of kinase domain-containing receptor (KDR, angiogenic growth factor's corresponding receptor) promoter was used for targeted gene therapy. Plasmid DNA with or without microbubble contrast agent of SonoVue was intravenously injected. Ultrasound (1 MHz, 2 W/cm 2 , 5 min) was delivered to hepatic carcinomas in mice. The KDR-tk gene transfer was followed by ganciclovir injection for 10 days and then the diameters of tumors were measured every 4 days for 28 days [109].	Compared with the group treated by ultrasound alone, KDR-tk gene   treatment   by ultrasound combined with SonoVue restrained tumor growth and increased survival time of tumor-bearing mice; microvessel density in group mediated by ultrasound and SonoVue was significantly lower than that in ultrasound alone group. An apoptosis index increased in the group treated by ultrasound and SonoVue compared with the group treated by ultrasound alone, whereas there was no significant difference between group mediated by SonoVue alone and phosphate-buffered saline alone group.	SonoVue: echo contrast agent	It increases microbubbles in systemic circulation to enhance effects of inertial cavitation, substantially increasing the efficacy of viral gene transfer

Bold print refers to studies that demonstrated efficacy only through improved drug delivery and not ultrasound-mediated activation of chemotherapeutic agents.