Ultrasound-Mediated Cavitation Enhances EGFR-Targeting PLGA-PEG Nano-Micelle Delivery for Triple-Negative Breast Cancer Treatment

Abstract

Triple-negative breast cancer (TNBC) is highly recurring and metastatic breast cancer with overexpressing epidermal growth factor receptor (EGFR). Herein, a series of in vitro and in vivo analyses were used to explore the therapeutic effect of EGFR-targeting nano-micelles (PLGA-PEG/DOX@anti-EGFR) combined with ultrasound-mediated cavitation (UMC). The prepared nano-micelle drug carriers have good biocompatibility and can greatly increase the drug accumulation in tumor regions, thereby reducing off-target toxicity while enhancing anti-tumor efficacy. Moreover, an in vivo analysis of the practical utility of this treatment modality was conducted by using SonoVue^TM microbubbles to achieve cavitation under different power intensity levels, with an ultrasonic power intensity of 0.5 W/cm² maximizing the intra-tumoral blood perfusion. Relative to PLGA-PEG@DOX/anti-EGFR nano-micelles treatment alone, the combination with UMC was better able to suppress tumor growth even at low concentrations. As such, combining actively targeted drug-carrier molecules with UMC represents an effective approach to enhancing therapeutic efficacy while reducing the adverse, systemic effects associated with DOX and other chemotherapeutic drugs, and it can be considered as a promising clinical prospect in the treatment of TNBC.

Keywords: DOX; EGFR-targeting; PLGA nano-micelle; SonoVueTM; triple-negative breast cancer; ultrasound-mediated cavitation.

Scheme 1

The synthesis of PLGA-PEG@DOX/anti-EGFR nano-micelles and schematic illustration of their subsequent application in UMC combined with chemotherapy.

Figure 1

(a) PLGA-PEG@DOX/anti-EGFR nano-micelle synthesis strategy. (b) PLGA-PEG@DOX/anti-EGFR nano-micelles were assessed via TEM. (c) DLS was used to assess PLGA-PEG@DOX and PLGA-PEG@DOX/anti-EGFR nano-micelle size distributions. (d) PLGA-PEG@DOX and PLGA-PEG@DOX/anti-EGFR nano-micelle zeta potential values. (e) Cumulative DOX release from PLGA-PEG@DOX/anti-EGFR (pH = 7.4 or 6.3) in vitro over the course of 24 h in a 10% FBS solution at 37 °C. Samples were assessed in triplicate.

Figure 2

Ultrasonic cavitation-induced vascular permeability enhances DOX delivery to tumor cells. (a) Experimental overview: (1) free DOX, PLGA-PEG@DOX, or PLGA-PEG@DOX/anti-EGFR nano-micelles were added, after which (2) UMC treatment of the vascular barrier was performed, and (3) excess drug or nano-micelles were removed, after which the levels of these agents that had penetrated the vascular barrier was assessed, with (4) a CCK-8 assay being used to examine the impact of PBS, free DOX, PLGA-PEG@DOX, and PLGA-PEG@DOX/anti-EGFR nano-micelles on MDA-MB-468 cell viability. (b) UMC treatment promoted the penetration of free DOX, PLGA-PEG@DOX, and PLGA-PEG@DOX/anti-EGFR nano-micelles across the vascular barrier and (c,d) thereby significantly enhanced the cytotoxicity of these treatments towards MDA-MB-468 tumor cells owing to their ability to more efficiently cross the vascular layer (n = 3). * p < 0.05, ** p < 0.01, **** p < 0.0001. UMC(+) = with ultrasound-mediated cavitation, UMC(−) = without ultrasound-mediated cavitation.

Figure 3

Flow cytometry (a) and corresponding mean fluorescence intensity values (b) for MDA-MB-468 cells following treatment with PLGA-PEG@DOX or PLGA-PEG@DOX/anti-EGFR nano-micelles (5 µg/mL DOX equivalent dose) for the indicated amounts of time (n = 3). *** p < 0.001, **** p < 0.0001. (c) MDA-MB-468 cells were assessed via confocal microscope following an 8 h treatment with PLGA-PEG@DOX/anti-EGFR nano-micelles (5 µg/mL DOX equivalent dose). Scale bar: 10 µm. (d) MDA-MB-468 cell viability was assessed following the indicated treatments with a range of DOX doses (2.5–20 µg/mL) for 24 h. Data are means ± standard deviation (n = 5). ^# PLGA-PEG@DOX+UMC vs. PLGA-PEG@DOX/anti-EGFR+UMC. ^# p < 0.05, ^## p < 0.01. * PLGA-PEG@DOX/anti-EGFR vs. PLGA-PEG@DOX/anti-EGFR+UMC. ** p < 0.01, *** p < 0.001. (e) MDA-MB-468 cells fluorescence was assessed following the indicated treatment. Scale bar: 50 µm. UMC(+) = with ultrasound-mediated cavitation, UMC(−) = without ultrasound-mediated cavitation.

Figure 4

Assessment of UMC-induced changes in tumor perfusion. (a) The intra-tumoral perfusion of MDA-MB-468 tumor-bearing mice was assessed via CEUS pre- and post-treatment. (b) PI values from pre- and post-treatment CEUS analyses. Data are means ± standard deviation (n = 5). ** p < 0.01, **** p < 0.0001. Scale bar: 5 mm. UMC(+) = with ultrasound-mediated cavitation, UMC(−) = without ultrasound-mediated cavitation.

Figure 5

Biodistribution studies of free DOX, PLGA-PEG@DOX, and PLGA-PEG@DOX/anti-EGFR nano-micelles. MBA-MD-468 tumor-bearing mice were intravenously administered appropriate nano-micelles or free drug. Ex vivo fluorescence images of tumor (1) and major organs, including the heart (2), liver (3), spleen (4), lungs (5), and kidneys (6), in these mice were assessed at 2, 4, 8, 12, 24, and 48 h following free DOX (a), PLGA-PEG@DOX (b), or PLGA-PEG@DOX/anti-EGFR nano-micelle (c) administration. UMC(+) = with ultrasound-mediated cavitation, UMC(−) = without ultrasound-mediated cavitation.

Figure 6

(a) Representative images of MBA-MD-468 tumor-bearing mice in the indicated treatment groups. (b) Relative tumor volume and (c) murine survival rate over time were assessed in the indicated treatment groups. Data are means ± standard deviation (n = 5). ** p < 0.01. UMC(+) = with ultrasound-mediated cavitation, UMC(−) = without ultrasound-mediated cavitation.

Figure 7

(a) DOX intra-tumoral penetration following intravenous PLGA-PEG@DOX/anti-EGFR injection with or without UMC. (b) H&E and TUNEL staining of MBA-MD-468 tumor samples in the indicated treatment groups. Scale bar: 100 µm. UMC(+) = with ultrasound-mediated cavitation, UMC(−) = without ultrasound-mediated cavitation.