Ultrasound-Targeted Microbubble Disruption with Key Nanodroplets for Effective Ferroptosis in Triple-Negative Breast Cancer Using Animal Model

Abstract

Introduction: Triple-negative breast cancer (TNBC) is known to be the most aggressive form of breast cancer. Due to its high recurrence and mortality rates, the treatment of TNBC is a significant challenge for the medical community. Besides, ferroptosis is an emerging regulatory cell death that may provide new insights into the treatment of TNBC. As a central inhibitor of the ferroptosis process, the selenoenzyme glutathione peroxidase 4 (GPX4) is its classical therapeutic target. However, inhibition of GPX4 expression is quite detrimental to normal tissues. Ultrasound contrast agents, as an emerging visualization precision treatment, may provide a solution to the existing problem.

Methods: In this study, nanodroplets (NDs) carrying simvastatin (SIM) were constructed using the homogeneous/emulsification method. Then, the characterization of SIM-NDs was systematically evaluated. Meanwhile, in this study, the ability of SIM-NDs combined with ultrasound-targeted microbubble disruption (UTMD) to initiate ferroptosis and its respective mechanisms of ferroptosis induction were verified. Finally, the antitumor activity of SIM-NDs was investigated in vitro and in vivo using MDA-MB-231 cells and TNBC animal models.

Results: SIM-NDs exhibited excellent pH- and ultrasound-responsive drug release and noticeable ultrasonographic imaging ability, also showing good biocompatibility and biosafety. UTMD could promote increased intracellular reactive oxygen species and consume intracellular glutathione. However, SIM-NDs were efficiently internalized into cells under ultrasound irradiation, followed by the rapid release of SIM, which inhibited intracellular mevalonate production, and synergistically downregulated GPX4 expression, thereby promoting ferroptosis. Moreover, this combined treatment demonstrated strong antitumor ability in vitro and in vivo.

Conclusion: The combination of UTMD and SIM-NDs presents a promising avenue for harnessing ferroptosis in the treatment of malignant tumors.

Keywords: ferroptosis; glutathione peroxidase 4; lipid peroxidation; nanodroplets; theranostics; ultrasound-targeted microbubble disruption.

Graphical abstract

Figure 1

Characterization of SIM-NDs. (A) (SIM-) NDs synthesis diagram. (B) TEM image of SIM-NDs at pH 7.4. Scale bar: 500 nm. (C) TEM image of SIM-NDs at pH 6.5. Scale bar: 400 nm. (D) Particle size distribution of NDs. (E) Particle size distribution of SIM-NDs under various pH levels for 0.5 h. (F) Particle size distribution of SIM-NDs under various pH levels for 2 h. (G) Zeta potentials of SIM-NDs at various pH levels. (H) The drug release profiles under various pH levels. (I) The drug release profiles when exposed to ultrasound. All results represent the means ± SD (n = 3).

Figure 2

The tumor-binding ability of SIM-NDs. (A) Uptake of Dil-labeled SIM-NDs by cells under different pH conditions. Scale bar: 100μm. (B) Uptake of Dil-labeled SIM-NDs by cells under different pH conditions analyzed by FCM. (C) Uptake of Dil-labeled SIM-NDs by cells under US irradiation. Scale bar: 100μm. (D) Uptake of Dil-labeled SIM-NDs by cells under US irradiation analyzed by FCM. (E) Biodistribution of Dil-labeled SIM-NDs in BALB/c nude mice at different time intervals. (F) Quantitative analysis of the average radiant efficiency from D. Compared to the normal group (except for the liver group). All results represent the means ± SD (n = 3). ****p<0.0001.

Figure 3

CEUI of SIM-NDs. (A) CEUI of SIM-NDs in vitro. (B) CEUI of SIM-NDs in vivo. The red arrow indicates the position of the tumor.

Figure 4

UTMD combined with SIM-NDs initiates ferroptosis. (A) TEM images of TNBC cells after various therapies. The bottom figures (magnification 2X) are enlarged views of the red boxes in the above figure, respectively. The red arrows represent the mitochondria in the cells. Scale bar: 1.2 μm, 0.6 μm. (B) The relative expression level of GPX4 on MDA-MB-231 cells with various treatments. (C) Quantification of GPX4/GAPDH was performed from B using Image J. Compared to the normal group. (D) MDA levels of breast cancer cells after different treatments. (E) Lipid peroxidation levels of MDA-MB-231 cells after various treatments were quantified by FCM. (F) The percentage of positive cells was obtained from E using Flowjo. All results represent the means ± SD (n = 3). *p<0.05, **p<0.01, ***p<0.001.

Figure 5

Mechanism of ferroptosis induced by UTMD combined with SIM-NDs. (A) ROS generation in MDA-MB-231 cells after different treatments were quantified by FCM. (B) The percentage of positive cells rate was obtained from A using Flowjo. Compared to the normal group. (C) Intracellular GSH content. Compared to the normal group. (D) Fluorescence images of ROS generation in MDA-MB-231 cells after different treatments (DCFH-DA probe staining). Scale bar: 100μm. (E, F) The expression level (E) and quantitative analysis (F) of GPX4 in MDA-MB-231 cells after various treatments (Control, NDs+US, NDs+US+NAC). (G) Intracellular GSH content with various treatments (Control, NDs+US, NDs+US+NAC). (H and I) The expression level (H) and quantitative analysis (I) of GPX4 in MDA-MB-231 cells after various treatments (Control, SIM, SIM+NAC, SIM+MVA). All results represent the means ± SD (n = 3). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 6

In vitro antitumor ability of SIM-NDs upon US irradiation. (A) Fluorescence images of cell proliferation ability treated for 24 h under various treatments. Scale bar: 200μm. (B) The percentage of Edu-positive cells was obtained from A using Image J. (C) Cell viability of MDA-MB-231 cells treated for 24 h under various treatments. Compared to the normal group. (D and E) Transwell migration (D) and invasion (E) experiments for 24 h of different treatment treatments. Scale bar: 200μm. (F and G) Quantification of the migrating cells (F) and invading cells. Compared to the normal group. (G) All results represent the means ± SD (n = 3). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 7

In vivo antitumor ability of SIM-NDs upon US irradiation. (A) Photographs of isolated tumors at the end of treatment. (B) Tumor growth profiles of various groups. (C) The weight of the isolated tumors in each group. (D) Body weight changes in mice during cancer treatment. (E) HE, TUNEL, and IHC staining of isolated tumors under different therapeutic modalities. Scale bar: 100 μm. All results represent the means ± SD (n = 5). *p<0.05, **p<0.01, ***p<0.001.