Influence of Phase Change Droplet Activation and Microbubble Cavitation on the Microenvironment of Hepatocellular Carcinoma.

OBJECTIVE: Both microbubble ultrasound contrast agents and acoustic phase change droplets (APCD) have been explored in hepatocellular carcinoma (HCC). This work aimed to evaluate changes to the HCC microenvironment following either microbubble or APCD destruction in a syngeneic pre-clinical model. METHODS: Mouse RIL-175 HCC tumors were grown in the right flank of 64 immunocompetent mice. Pre-treatment, photoacoustic volumetric tumor oxygenation, and power Doppler measurements were obtained using a Vevo 3100 system (VisualSonics, Toronto, Canada). The experimental groups received a 0.1 mL bolus injection of either Definity ultrasound contrast agent (Lantheus Medical Imaging) or APCD fabricated by condensing Definity. Following injection, ultrasound destruction was performed using flash-replenishment sequences on a Sequoia with a 10L4 probe (Siemens) for the duration of enhancement. Tumor oxygenation and power Doppler measurements were then repeated immediately post-ultrasound treatment. Twenty-four hours post-treatment, animals were euthanized, and tumors were harvested and stained for CD31, Cleaved Caspase 3 and CD45. RESULTS: Imaging biomarkers demonstrated a significant reduction in percent vascularity following either microbubble or APCD destruction in the tumor microenvironment ( p < 0.022) but no significant changes in tumor oxygenation (p = 0.12). Similarly, immunohistochemistry data demonstrated a significant decrease in CD31 expression (p < 0.042) and an increase in apoptosis (p < 0.014) in tumors treated with destroyed microbubbles or APCD relative to controls. Finally, a significant increase in CD45 expression was observed in tumors treated with APCD (p = 0.046), indicating an increase in tumor immune response. CONCLUSION: Ultrasound-triggered destruction of both microbubbles and APCD reduces vascularity, increases apoptosis, and may also increase immune response in this HCC model.

In Vitro and In Vivo Evaluation of Ultrasound-Triggered Release From Novel Spinal Device.

OBJECTIVES: Bacterial infection following spinal fusion is a major clinical concern with up to 20% incidence. An ultrasound-triggered bulk-release system to combat postsurgical bacterial survival was designed and evaluated. METHODS: Polylactic acid (PLA) clips were loaded with vancomycin (VAN) and microbubbles (Sonazoid, GE HealthCare) in vitro. Stability was determined over 14 days. VAN-loaded clips were submerged in water and insonated using a Logiq E10 scanner (GE HealthCare) with a curvilinear C6 probe. Doppler-induced VAN release was quantified using spectrophotometry. For in vivo testing, clips were loaded with methylene blue (MeB) solution and Sonazoid. These clips were implanted into a rabbit along the spine at L2 and L5, as well as a pig at L1 and L3, then insonated in Doppler mode using the C6 probe. RESULTS: Sonazoid microbubbles were better preserved when incubated in VAN compared with distilled water at 4°C, 25°C, and 37°C incubation temperatures (P = .0131). Contrast enhancement was observed from both solutions when incubated at 4°C storage conditions. Insonated clips achieved average cumulative VAN release of 101.8 ± 2.8% (81.4 ± 2.8 mg) after 72 hours. Uninsonated clips had only 0.3 ± 0.1% (0.3 ± 0.1 mg) average cumulative VAN release (P < .0001). Clips retrieved from the rabbit did not rupture with insonation nor produce MeB staining of surrounding tissues. In the pig, the PLA film was visibly ruptured and MeB tissue was observed following insonation, whereas the uninsonated clip was intact. CONCLUSION: These results demonstrate ultrasound-triggered release of an encapsulated prophylactic solution and provide an important proof-of-concept for continuing large animal evaluations for translational merit.

Improved Tumor Control Following Radiosensitization with Ultrasound-Sensitive Oxygen Microbubbles and Tumor Mitochondrial Respiration Inhibitors in a Preclinical Model of Head and Neck Cancer.

Tumor hypoxia (oxygen deficiency) is a major contributor to radiotherapy resistance. Ultrasound-sensitive microbubbles containing oxygen have been explored as a mechanism for overcoming tumor hypoxia locally prior to radiotherapy. Previously, our group demonstrated the ability to encapsulate and deliver a pharmacological inhibitor of tumor mitochondrial respiration (lonidamine (LND)), which resulted in ultrasound-sensitive microbubbles loaded with O2 and LND providing prolonged oxygenation relative to oxygenated microbubbles alone. This follow-up study aimed to evaluate the therapeutic response to radiation following the administration of oxygen microbubbles combined with tumor mitochondrial respiration inhibitors in a head and neck squamous cell carcinoma (HNSCC) tumor model. The influences of different radiation dose rates and treatment combinations were also explored. The results demonstrated that the co-delivery of O2 and LND successfully sensitized HNSCC tumors to radiation, and this was also enhanced with oral metformin, significantly slowing tumor growth relative to unsensitized controls (p < 0.01). Microbubble sensitization was also shown to improve overall animal survival. Importantly, effects were found to be radiation dose-rate-dependent, reflecting the transient nature of tumor oxygenation.

Tumoral oxygenation and biodistribution of Lonidamine oxygen microbubbles following localized ultrasound-triggered delivery.

Prior work has shown that microbubble-assisted delivery of oxygen improves tumor oxygenation and radiosensitivity, albeit over a limited duration. Lonidamine (LND) has been investigated because of its ability to stimulate glycolysis, lactate production, inhibit mitochondrial respiration, and inhibit oxygen consumption rates in tumors but suffers from poor bioavailability. The goal of this work was to characterize LND-loaded oxygen microbubbles and assess their ability to oxygenate a human head and neck squamous cell carcinoma (HNSCC) tumor model, while also assessing LND biodistribution. In tumors treated with surfactant-shelled microbubbles with oxygen core (SE61O2) and ultrasound, pO2 levels increased to a peak 19.5 ± 9.7 mmHg, 50 s after injection and returning to baseline after 120 s. In comparison, in tumors treated with SE61O2/LND and ultrasound, pO2 levels showed a peak increase of 29.0 ± 8.3 mmHg, which was achieved 70 s after injection returning to baseline after 300 s (p < 0.001). The co-delivery of O2andLNDvia SE61 also showed an improvement of LND biodistribution in both plasma and tumor tissues (p < 0.001). In summary, ultrasound-sensitive microbubbles loaded with O2 and LND provided prolonged oxygenation relative to oxygenated microbubbles alone, as well as provided an ability to locally deliver LND, making them more appropriate for clinical translation.

Development of a Dual Drug-Loaded, Surfactant-Stabilized Contrast Agent Containing Oxygen.

Co-delivery of cancer therapeutics improves efficacy and encourages synergy, but delivery faces challenges, including multidrug resistance and spatiotemporal distribution of therapeutics. To address these, we added paclitaxel to previously developed acoustically labile, oxygen-core, surfactant-stabilized microbubbles encapsulating lonidamine, with the aim of developing an agent containing both a therapeutic gas and two drugs acting in combination. Upon comparison of unloaded, single-loaded, and dual-loaded microbubbles, size (~1.7 µm) and yield (~2 × 109 microbubbles/mL) (~1.7) were not statistically different, nor were acoustic properties (maximum in vitro enhancements roughly 18 dB, in vitro enhancements roughly 18 dB). Both drugs encapsulated above required doses calculated for head and neck squamous cell carcinoma, the cancer of choice. Interestingly, paclitaxel encapsulation efficiency increased from 1.66% to 3.48% when lonidamine was included. During preparation, the combination of single drug-loaded micelles gave higher encapsulation (µg drug/g microbubbles) than micelles loaded with either drug alone (lonidamine, 104.85 ± 22.87 vs. 87.54 ± 16.41), paclitaxel (187.35 ± 8.38 vs. 136.51 ± 30.66). In vivo intravenous microbubbles produced prompt ultrasound enhancement within tumors lasting 3-5 min, indicating penetration into tumor vasculature. The ability to locally destroy the microbubble within the tumor vasculature was confirmed using a series of higher intensity ultrasound pulses. This ability to locally destroy microbubbles shows therapeutic promise that warrants further investigation.

Activation of Phase Change Contrast Agents Using Ionizing Radiation.

The feasibility of activating phase change contrast agents (PCCA) made from Definity (Lantheus Medical Imaging) using X-rays was investigated. A 10 mL of Definity PCCA (0.1 mL PCCA/mL) were injected into gelatin phantoms and irradiated using doses of 0, 30, 50, and 100 Gy. Size distribution and PCCA activation were measured after irradiation. Definity PCCAs were activated at a threshold of 50 Gy. Changes were visible with microscopy, visual inspection of T-flasks, and ultrasound imaging of gelatin phantoms. Moreover, increasing the radiation dose above 50 Gy appeared to further activate PCCA. These results indicate Definity PCCAs may be useful for ultrasound-based radiation dosimetry.

Shaping the synthesis of surfactant-stabilized oxygen microbubbles to accommodate encapsulated drug.

We have developed oxygen filled microbubbles, SE61O2, for localized, ultrasound-triggered oxygen delivery to hypoxic tumors prior to radiation therapy. Microbubbles, created by sonication, have a shell composed of D-α-Tocopherol polyethylene glycol 1000 succinate (TPGS) and sorbitan monostearate. Preliminary studies in mice with breast tumor xenographs showed that increases in oxygen partial pressure levels lasted less than 3 min, which is insufficient for most clinical applications. Hence, we investigated the potential of incorporating a hydrophobic antiglycolytic drug, modeled with Nile red. A new fabrication method was developed by first creating drug-loaded TPGS micelles. The resulting microbubbles had similar shell compositions, physical size, morphology, and acoustic properties as the original method. However, microbubble yield was more than doubled, resulting in twice the encapsulation efficiency. For the TPGS micelle method these include similar shell compositions (94.4 ± 0.6 % Montane 60), physical size post freeze-drying and reconstitution (1.57 ± 0.42 µm), morphology (spherical), and acoustic properties (maximum enhancement 19.92 ± 0.55 dB). However, microbubble yield was more than doubled, resulting in twice the encapsulation efficiency (up to 10.49 %). We propose that a nonideal mixture is formed when the surfactants are combined by the standard method, resulting in the formation of mixed micelles that are more stable, making microbubble creation more difficult during the sonication step.

Emerging Applications of Ultrasound-Contrast Agents in Radiation Therapy.

Radiation therapy (RT) causes DNA damage through ionization, leading to double-strand breaks. In addition, it generates reactive oxygen species (ROS), which are toxic to tumor cells and the vasculature. However, hypoxic regions in the tumor have been shown to not only decrease treatment response but also increase the likelihood of recurrence and metastasis. Ultrasound-sensitive micro-bubbles are emerging as a useful diagnostic and therapeutic tool within RT. Contrast-enhanced ultrasound (CEUS) has shown great promise in early prediction of tumor response to RT. Ultrasound-triggered micro-bubble cavitation has also been shown to induce bio-effects that can sensitize angiogenic tumor vessels to RT. Additionally, ultrasound can trigger the release of drugs from micro-bubble carriers via localized micro-bubble destruction. This approach has numerous applications in RT, including targeted oxygen delivery before radiotherapy. Furthermore, micro-bubbles can be used to locally create ROS without radiation. Sonodynamic therapy uses focused ultrasound and a sonosensitizer to selectively produce ROS in the tumor region and has been explored as a treatment option for cancer. This review summarizes emerging applications of ultrasound contrast agents in RT and ROS augmentation.