Ultrasound-stimulated Microbubbles for Treatment of Pancreatic Cancer Cells with Radiation and Nanoparticles: In vitro Study

Abstract

Purpose: This study aims to investigate the radiation enhancement effects of ultrasound-stimulated microbubbles (USMB) with X-rays and nanoparticles on pancreatic cancer cells in vitro.

Methods: Sonazoid™ microbubbles were used for USMB treatment with a commercially available ultrasound unit. The characterization of the microbubbles before and after ultrasound exposure with different mechanical parameters was evaluated microscopically. Two pancreatic cancer cell lines, MIAPaCa-2 and PANC-1, were treated with different concentrations of microbubbles in combination with 150 kVp X-rays and hydrogen peroxide-modified titanium dioxide nanoparticles. Cell viability was evaluated using a water-soluble tetrazolium dye and a colony formation assay. In addition, intracellular reactive oxygen species (ROS) induced by the combined treatment were assessed.

Results: The number of burst microbubbles increased with ultrasound's higher mechanical index and the exposure time. A significant radiation enhancement effect with a significant increase in ROS levels was observed in MIAPaCa-2 cells treated with USMB and 6 Gy X-rays, whereas it was not significant in PANC-1 cells treated with the same. When a higher concentration of USMB was applied with X-rays, no radiation enhancement effects were observed in either cell line. Moreover, there was no radiation enhancement effect by USMB between cells treated with and without nanoparticles.

Conclusions: The results indicate that USMB treatment can additively enhance the therapeutic efficacy of radiation therapy on pancreatic cancer cells, while the synergistic enhancement effects are likely to be cell type and microbubble concentration dependent. In addition, USMB did not improve the efficacy of nanoparticle-induced radiosensitization in the current setting.

Keywords: Microbubbles; nanoparticles; radiation therapy; radiosensitizer; ultrasound.

Figure 1

(a) Image of the ultrasound exposure setup for samples in a 24-well flat bottom plate. (b) The color of the microbubble solution was changed from milky white to transparent by ultrasound exposure. US: Ultrasound

Figure 2

(a) A representative microscopy image of Sonazoid™ microbubbles. (b) Size distribution was obtained from 120 single microbubbles measured using microscopy images

Figure 3

Number of microbubbles burst by ultrasound at different exposure parameters. (a) Different frequencies and mechanical indexes (MIs) with an exposure time of 3 min, and (b) different exposure times with 4.0 MHz and an MI of 1.0. Microbubbles were counted using a microscope before and after exposure. Data are represented as the means ± standard deviations obtained from nine independent microscopy images for each exposure setting. MI: Mechanical index

Figure 4

Effect of ultrasound-stimulated microbubbles on the viability of (a) MIAPaCa-2 and (b) PANC-1 cells. Cells were treated with 0%–0.4% v/v microbubbles ± ultrasound. Cell viability was measured 24 h after treatment using a water-soluble tetrazolium assay. Each data are normalized to the untreated control samples (cells without both microbubbles and ultrasound) and represented as the means ± standard deviations obtained from at least three independent measurements. *P < 0.05 compared to cells treated with ultrasound alone. (c) Detection of dead cells by trypan blue staining for MIAPaCa-2 cells treated with 0.01% and 0.1% v/v microbubbles and ultrasound. MB: Microbubbles; US: Ultrasound

Figure 5

Effect of ultrasound-stimulated microbubbles (USMB) on the viability and survival curve of (a and c) MIAPaCa-2 and (b and d) PANC-1 cells treated with 0–6 Gy X-ray irradiation. Cells were treated with 0.01% or 0.4% v/v USMB before X-ray irradiation. Cell viability was evaluated using a colony formation assay. Each data of cell viability are normalized to the untreated control samples (cells without both USMB and X-rays) (a and b), and each survival curve was obtained by normalizing the viability to the unirradiated control samples for each USMB treatment (c and d). Data are presented as the means ± standard deviations obtained from at least three independent measurements. *P < 0.05 compared to cells treated with X-rays alone for each radiation dose, and **P < 0.05 compared to cells treated with 0.01% or 0.4% v/v USMB for each radiation dose. USMB: Ultrasound-stimulated microbubbles

Figure 6

Reactive oxygen species generation in (a) MIAPaCa-2 and (b) PANC-1 cells treated with 0.01% v/v ultrasound-stimulated microbubbles and 6 Gy of X-rays. Each fluorescence intensity of carboxy 2’,7’-dichlorodihydrofluorescein diacetate is normalized to the untreated control samples (cells without both microbubbles and ultrasound) as 1.0 and represented as the means ± standard deviations obtained from three independent measurements. *P < 0.05. CTL: Control; USMB: Ultrasound-stimulated microbubbles; RT: Radiation

Figure 7

Effect of ultrasound-stimulated microbubbles (USMB) on the survival of (a) MIAPaCa-2 and (b) PANC-1 cells treated with titanium dioxide nanoparticles (TiO_xNPs) and 0–6 Gy X-ray irradiation. Cells were treated with 0.01% v/v USMB and 1.0 mg/mL TiO_xNPs before X-ray irradiation. The survival rate was evaluated using a colony formation assay and presented as the means ± standard deviations obtained from at least three independent measurements. *P < 0.05

Figure 8

Difference in suspended cell state between MIAPaCa-2 and PANC-1 cells. Microscopy images of cells were obtained after cell detachment using trypsin