Overcoming anticancer resistance by photodynamic therapy-related efflux pump deactivation and ultrasound-mediated improved drug delivery efficiency

Abstract

One of the major obstacles to successful chemotherapy is multi-drug resistance (MDR). A multi-drug resistant cancerous cell abnormally overexpresses membrane transporters that pump anticancer drugs out of the cell, resulting in low anticancer drug delivery efficiency. To overcome the limitation, many attempts have been performed to inhibit the abilities of efflux receptors chemically or genetically or to increase the delivery efficiency of anticancer drugs. However, the results have not yet been satisfactory. In this study, we developed nanoparticle-microbubble complexes (DOX-NPs/Ce6-MBs) by conjugating doxorubicin loaded human serum albumin nanoparticles (DOX-NPs) onto the surface of Chlorin e6 encapsulated microbubbles (Ce6-MBs) in order to maximize anticancer efficiency by overcoming MDR. Under the ultrasound irradiation, DOX-NPs and Ce6 encapsulating self-assembled liposomes or micelles were effectively delivered into the cells due to the sonoporation effect caused by the microbubble cavitation. At the same time, reactive oxygen (ROS) generated from intracellularly delivered Ce6 by laser irradiation arrested the activity of ABCG2 efflux receptor overexpressed in doxorubicin-resistant breast cancer cells (MCF-7/ADR), resulting in increased the chemotherapy efficacy. In addition, the total number of side population cells that exhibit the properties of cancer stem-like cells were also reduced by the combination of photodynamic therapy and chemotherapy. In conclusion, DOX-NPs/Ce6-MBs will provide a platform for simultaneously overcoming MDR and increasing drug delivery and therefore, treatment efficiency, under ultrasound irradiation.

Keywords: Efflux pump; Microbubble; Multidrug resistance; Nanomedicine; Side population; Sonoporation; Ultrasound.

Scheme 1

Schematic illustration of this study demonstrating mechanisms to overcome anticancer resistance of MCF-7/ADR cells using DOX-NPs/Ce6-MBs with US and laser irradiation in MCF-7/ADR cells

Fig. 1

Characteristics of the DOX-NPs/Ce6-MBs complex. a Size distribution of the DOX-NPs. b Scanning electron microscopy (SEM) image of DOX-NPs. Scale bar represents 100 nm. c In vitro release profile of DOX from DOX-NPs. d Size distribution of the Ce6-MBs and DOX-NPs/Ce6-MBs

Fig. 2

Confirmation of efflux pump receptor expression in MCF-7/ADR cells. a Qualitative and quantitative mRNA expression analysis of three types of efflux pump receptors in MCF-7 and MCF-7/ADR cells by reverse transcriptase (RT)-PCR and by real-time PCR, respectively. b Immunocytochemical analysis of the expression of P-glycoprotein receptor in MCF-7 and MCF-7/ADR cells (scale bar = 20 µm)

Fig. 3

Confocal microscopy images showing intracellular uptake of DOX and Ce6 in MCF-7/ADR cells with or without US irradiation. The blue color represents cell nuclei. Red and green channels represent DOX and Ce6, respectively (scale bar = 20 µm.)

Fig. 4

Intracellular ROS generation. The reactive oxygen species (ROS) generation in MCF-7/ADR cells was determined with or without laser irradiation after DOX-NPs/Ce6-MBs treatment with US irradiation (0.2 W/cm², 50% duty cycle, 30 s)

Fig. 5

Intracellular retention of DOX in MCF-7/ADR cells. a Fluorescence images showing DOX retention inside MCF-7/ADR cells after DOX-NPs/Ce6-MBs treatment and subsequent US irradiation (0.2 W/cm², 50% duty cycle, 30 s) every 30 min (scale bar = 20 µm). b Quantitative flow cytometry data showing MCF-7/ADR cell percentages without DOX because of pumping out

Fig. 6

Side population analysis. Image cytometry data showing percentage of side population MCF-7/ADR cells after treatment with DOX-NPs/Ce6-MBs, followed by US or US + Laser irradiation

Fig. 7

Cell viability assay. Cytotoxicity of DOX-NPs/Ce6-MBs complex and its incomplete comparison groups with and without US-, Laser-treated combination therapy. 671 nm laser was irradiated with 1.0 J/cm², 50 mW per well. US was applied with a power of 0.2 W/cm² and duty cycle of 50% for 5 s per well. Statistical analysis was performed using Student’s t-test (* P < 0.05, ** P < 0.01)