Ultrasound-Sensitive Microbubble Approaches for Overcoming Tumor Hypoxia in Head and Neck Squamous Cell Carcinoma
Authors:   , Lacerda Quezia R. , Eisenbrey John Robert , Wheatley Margaret A. 
Published: May 2023

Several studies have shown that tumor hypoxia is directly associated with a poor prognosis in patients with advanced head and neck squamous cell carcinoma (HNSCC). Hypoxic and/or anoxic areas have an oxygen partial pressure (pO2) ranging from 0 to 10 mmHg. A pO2 <10 mmHg in HNSCC patients is directly linked to poor local tumor control, establishing disease-free status, and overall survival, whether treated by radiation alone or combined with surgery or systemic chemotherapy. Increasing the oxygen tension of HNSCC would significantly improve its sensitivity to radiation, which is especially important as nearly 75% of all HNSCC patients receive radiation therapy. As a means to mitigate tumor hypoxia before radiation, several treatment modifications have been investigated, including the use of ultrasound-sensitive microbubbles containing oxygen. Our group has developed an O2 microbubbles stabilized by sorbitan monostearate and water-soluble vitamin E, Tocopheryl polyethylene glycol succinate (TPGS) (termed SE61O2) that provides enhancement under ultrasound interrogation in vitro for over 10 minutes. These bubbles are readily imaged within hypoxic tumors and can be noninvasively destroyed within the tumor vasculature via ultrasound. Prior work demonstrated the feasibility of disrupting oxygen bubbles within a murine breast tumor model and a model of metastatic breast cancer in the brain was capable of raising the mean tumor pO2 to as much as 20 mmHg, and this increase in oxygenation significantly improved both tumor control and animal survival. However, the duration of oxygenation using this platform was limited, lasting only 2 minutes in vivo. The scientific premise of this work aimed to overcome the translational limitations of our group's current O2 microbubble platform by combining the localized delivery of O2 with a pharmacological inhibitor of tumor mitochondrial respiration (lonidamine (LND)) to prolong oxygenation. The potential of this combined approach was validated in an HNSCC tumor model to access the ability to provide sustained oxygenation and improve tumor control and survival by further sensitizing tumors to radiotherapy. In this thesis, SE61O2 microbubbles loaded with LND (SE61O2/LND) were fabricated by modifying our previously reported methods. The LND was first entrapped in TPGS micelles by incubation methods to extend the drug/micelle interaction and increase the drug loading in SE61 microbubbles. Microbubbles loaded with LND had an average encapsulation of 25.7 ± 1.5 [mu]g/mL microbubble, surpassing the minimum required drug loading of at least 4.8 [mu]g/mL, assuming 20% delivery. Notably, despite the inclusion of LND, these agents retained their echogenicity and stability, showing a maximum enhancement of 18.7 ± 0.8 dB, and a mean diameter of 1.65 [mu]m with a bubble concentration of (2.7 ± 0.2)x109 MB/mL. Experiments in vivo showed that co-delivery of O2 and LND via ultrasound-sensitive microbubbles provided sustained oxygenation (300 seconds) relative to oxygenated microbubbles alone (120 seconds), as well as showing an improvement of LND pharmacokinetics in both the plasma and tumor tissue illustrating the platform's ability to locally deliver LND. Furthermore, therapy experiments delivering 5 Gy of radiation demonstrated that SE61O2/LND administered with metformin pre-treatment improved radiosensitivity and animal survival in an HNSCC model.