A Robust Oxygen Microbubble Radiosensitizer for Iodine‐125 Brachytherapy

Abstract Iodine‐125 (125I) brachytherapy, a promising form of radiotherapy, is increasingly applied in the clinical treatment of a wide range of solid tumors. However, the extremely hypoxic microenvironment in solid tumors can cause hypoxia‐induced radioresistance to 125I brachytherapy, resulting in therapeutic inefficacy. In this study, the aim is to sensitize hypoxic areas in solid tumors using ultrasound‐activated oxygen microbubbles for 125I brachytherapy. A modified emulsion freeze‐drying method is developed to prepare microbubbles that can be lyophilized for storage and easily reconstituted in situ before administration. The filling gas of the microbubbles is modified by the addition of sulfur hexafluoride to oxygen such that the obtained O2/SF6 microbubbles (OS MBs) achieve a much longer half‐life (>3×) than that of oxygen microbubbles. The OS MBs are tested in nasopharyngeal carcinoma (CNE2) tumor‐bearing mice and oxygen delivery by the OS MBs induced by ultrasound irradiation relieve hypoxia instantly. The post‐treatment results of brachytherapy combined with the ultrasound‐triggered OS MBs show a greatly improved therapeutic efficacy compared with brachytherapy alone, illustrating ultrasound‐mediated oxygen delivery with the developed OS MBs as a promising strategy to improve the therapeutic outcome of 125I brachytherapy in hypoxic tumors.


Cytotoxicity of MBs.
The biocompatibility of MBs was evaluated in CNE2 cells. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37 °C under 5% CO 2 . CNE2 cells (100 µL) (~60,000 cells/ml) were seeded in each well of a 96-well plate and cultured for 24 h.
The culture medium was replaced with fresh one containing the OS MBs at different concentrations from 0 to ~3.2 × 10 9 MBs/mL. The cells were subsequently cultured for 24 h and 48 h, respectively. The cell viability was evaluated by an MTT assay. Twenty milliliters of MTT solution was added to each well and the plate was incubated at 37 °C for another 3 h. After the supernatant of each well removed, 200 L of DMSO was added. The absorbance value of each well at 570/690 nm was recorded by a plate reader (xMark, Bio-Rad, USA) to evaluate the cell viability at each concentration.

Ultrasound Characterization of MBs
The acoustic property and stability of the MBs were investigated using a diagnostic ultrasound system (Acuson S2000, Siemens, Germany) in a gel phantom, made of a 2% (w/v) agarose solution. The diluted OX MBs, OS MBs and SF MBs (~2.0 × 10 7 MBs/mL, 1 mL) were loaded into the sample wells in the gel phantom, submerged in the water. Ultrasound images of the MBs in the gel phantom were acquired by the ultrasound system at a frequency of 9 MHz and a mechanical index (MI) of 0.06 at specific time points. The ultrasound images were analyzed by the Image J software to obtain average grey values as the intensity of the ultrasound signal of the MBs.

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The CNE2 cells and LM6 cells were first treated in a 125 I seed irradiation model. [1] Briefly, 500 µL of CNE2 and LM6 cells (~1.0 × 10 4 cells) were seeded in the wells of a 24-well plate respectively and divided into four groups: Group 1, Control; Group 2, OS MBs; Group 3, BT alone; and Group 4, BT + OS MBs. For groups with the addition of OS MBs, 20 µL of the OS microbubble solution (~2.0 × 10 8 MBs/mL) was added into the culture medium and PBS was added for the control group. As for groups with BT, a transwell containing the 125 I seeds (~2,600 μCi) was inserted into the culture well. All groups were incubated in a hypoxia incubator chamber (1% O 2 ) for 48 h. The cells were collected and suspended in PBS at a concentration of ~1.0 × 10 6 cells/mL. An comet assay was performed to analyze the radiation-induced DNA damage of cells with different treatments, following a protocol. [2] A 0.8% agarose solution (100 L) at 60 was dropped on a microscope glass slide and a cover slip was placed on the top of the gel. After the gelation and the removal of the cover slip, the freshly prepared mixture of the cell suspension (10 µL) and a 1.

Animal Model
Female BALB/c Nude mice in the study were purchased from Vital River Laboratories (Beijing, China) and treated in accordance with the protocol approved by The Institutional Animal Care and Use Committee of Sun Yat-sen University Cancer Center and the IACUC approval number is L102042018000A. The mice were anesthetized using pentobarbital and 50 µL of PBS containing CNE2 cells (~1.0 × 10 6 cells/mL) was subcutaneously injected into the hind leg of each mouse. After two weeks, the volume of the xenograft tumors reached ~200 mm 3 . The tumor size was measured by the ultrasound imaging using a portable ultrasonic system (Logic E, GE, USA) and the tumor volume was calculated using the modified ellipsoid formula (i.e., 0.5 × (Length × Width 2 )) based on the ultrasound images.

Intratumroal Oxygen Level Measurement
The intratumoral oxygen level at the center of the tumor was directly measured using a

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intratumoral pO 2 was recorded at the specific time intervals for twenty minutes. The PBS was used as the control.

Histological Analysis
After the in vivo brachytherapy, three tumors from each group were dissected to make frozen formalin-fixed sections, stained with hematoxylin and eosin (H&E) and antibodies specific for cytokeratin AE1/AE3, respectively, in order to assess the apoptosis and necrosis of the tumors with different treatments. Furthermore, the excised major organs including the heart, liver, spleen, lung, and kidney were stained with H&E to evaluate the safety of the OS MBs. For the hypoxia-related biomarker HIF-1 analysis, three tumors from mice before/after the treatment of the brachytherapy in combination of OS MBs were sampled and stained with anti-HIF-1 antibody to assess the expression level of HIF-1 . Finally, the excised tissue slices were examined with a fluorescence microscope (DM250, Leica, Germany) for the histological analysis.  The HIF-1 appears as a brown color stained with the 3,3′-diaminobenzidine and the nucleus appears as a blue color stained with the hematoxylin and eosin. Figure S4. Hematoxylin and eosin stained tissue sections of the major organs including heart, liver, spleen, lung, and kidney of the healthy mice and mice treated with the brachytherapy in combination of OS MBs for twelve days.