Inhibition of prostate cancer growth using doxorubicin assisted by ultrasound-targeted nanobubble destruction

Abstract

Ultrasound (US)-targeted microbubble destruction has been widely used as an effective drug-delivery system. However, nanobubbles (NBs) have better stability and stronger penetration than microbubbles, and drug delivery assisted by US-targeted NB destruction (UTND) still needs to be investigated. Our aim was to investigate the effect of doxorubicin (DOX) on the inhibition of prostate cancer growth under UTND. Contrast-enhanced US imaging of transplanted PC3 prostate cancer in mice showed that under a combination of 1 W/cm(2) US power and a 100 Hz intermittent pulse with a "5 seconds on, 5 seconds off" mode, NBs with an average size of (485.7±33) nm were effectively destroyed within 15 minutes in the tumor location. PC3 cells and 20 tumor-bearing mice were divided into four groups: a DOX group, a DOX + NB group, a DOX + US group, and a DOX + NB + US group. The cell growth-inhibition rate and DOX concentration of xenografts in the DOX + NB + US group were highest. Based on another control group and these four groups, another 25 tumor-bearing mice were used to observe the treatment effect of nine DOX injections under UTND. The xenografts in the DOX + NB + US group decreased more obviously and had more cellular apoptosis than other groups. Finally, electron microscopy was used to estimate the cavitation effect of NBs under US irradiation in the control group, NB group, US group, and NB + US group. The results of scanning electron microscopy showed that PC3 cells in the DOX + NB + US group had more holes and significantly increased cell-surface folds. Meanwhile, transmission electric microscopy confirmed that more lanthanum nitrate particles entered the parenchymal cells in xenografts in the NB + US group compared with the other groups. This study suggested that UTND technology could be an effective method to promote drugs to function in US-irradiated sites, and the underlying mechanism may be associated with a cavitation effect.

Keywords: cavitation effect; nanobubble; prostate cancer; ultrasound therapy.

Figure 1

Morphology and size distribution of NBs. Notes: (A) NBs observed under TEM after negative staining; (B) NBs observed under LSCM; (C) size-distribution curve of NBs from dynamic light-scattering test in a Malvern detector. Abbreviations: LSCM, laser-scanning confocal microscopy; NBs, nanobubbles; SD, standard deviation; TEM, transmission electron microscopy; PdI, polydispersity index.

Figure 2

Growth-inhibition assay (CCK-8) for PC3 cells in different treatment groups. Note: *P<0.05. Abbreviations: DOX, doxorubicin; NB, nanobubble; US, ultrasound.

Figure 3

Destruction of NBs in tumor xenografts at different ultrasonic power intensities. Notes: (A) Imaging of NBs in tumor xenografts under conventional contrast-enhanced grayscale ultrasound. (B–D) Destruction and waveform conditions of NBs in tumor-xenograft regions under three intensities of ultrasonic power: 1, 1.75, 2.5 W/cm², respectively. Abbreviations: NBs, nanobubbles; M, mass.

Figure 4

Distribution of DOX in a variety of tissues in the four groups. Note: *P<0.05. Abbreviations: DOX, doxorubicin; NB, nanobubble; US, ultrasound.

Figure 5

Changes in tumor growth in the different groups. Notes: (A) Therapeutic and monitoring schedule of tumor xenografts. (B) Conditions of tumor-bearing mice after respective treatments. Red circles indicate regions of tumor xenografts. (C) Status of isolated tumor xenografts in all five groups after treatments. (D) CEUS imaging of maximum sections of tumor xenografts in the five groups before and after treatment. Blue circles indicate tumor-xenograft regions. (E) Changes in tumor-xenograft volume with days of intervention measures in the five groups. (F) Changes in the body weight of tumor-bearing mice with days of intervention measures in the five groups. (G) Changes in the ultrasonic signal intensity of the maximum sections of xenografts in the five groups. (H) Mass and density of final isolated tumor xenografts in all five groups. All scale bars 2 cm. Abbreviations: CEUS, contrast-enhanced US; DOX, doxorubicin; NB, nanobubble; US, ultrasound.

Figure 6

H&E and TUNEL staining of myocardial tissues and xenograft tissues of nude mice in all groups. Notes: (A–E) H&E staining of myocardial tissues of all groups; (F–J) TUNEL staining of myocardial tissues of all groups; (K–O) H&E staining of xenograft tissues of all groups; and (P–T) TUNEL staining of xenograft tissues of all groups. Scale bars 50 µm. Abbreviations: DOX, doxorubicin; H&E, hematoxylin–eosin; NB, nanobubble; TUNEL, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling; US, ultrasound.

Figure 7

Cavitation effect of US burst of NBs on cells and tumor xenografts. Notes: (A–D) Effects of the four types of treatment on the cell surface visualized under SEM. White arrows indicate pores on the cell surface. (E–H) Condition of lanthanum nitrate particles in tumor xenografts of all treatment groups visualized under TEM after electron staining. Black arrows indicate the locations of lanthanum nitrate-particle deposition. (I–L) Observation of the status of lanthanum nitrate particles in tumor xenografts under TEM without electron staining. Black arrows indicate the locations of lanthanum nitrate-particle deposition. Abbreviations: NBs, nanobubbles; SEM, scanning electron microscopy; TEM, transmission electron microscopy; US, ultrasonic irradiation.