Bypassing multidrug resistance in human breast cancer cells with lipid/polymer particle assemblies

Abstract

Background: Multidrug resistance (MDR) mediated by the overexpression of adenosine triphosphate (ATP)-binding cassette (ABC) transporters, such as P-glycoprotein (P-gp), remains one of the major obstacles to effective cancer chemotherapy. In this study, lipid/particle assemblies named LipoParticles (LNPs), consisting of a dimethyldidodecylammonium bromide (DMAB)-modified poly(lactic-co-glycolic acid) (PLGA) nanoparticle core surrounded by a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) shell, were specially designed for anticancer drugs to bypass MDR in human breast cancer cells that overexpress P-gp.

Methods: Doxorubicin (DOX), a chemotherapy drug that is a P-gp substrate, was conjugated to PLGA and encapsulated in the self-assembled LNP structure. Physiochemical properties of the DOX-loaded LNPs were characterized in vitro. Cellular uptake, intracellular accumulation, and cytotoxicity were compared in parental Michigan Cancer Foundation (MCF)-7 cells and P-gp-overexpressing, resistant MCF-7/adriamycin (MCF-7/ADR) cells.

Results: This study found that the DOX formulated in LNPs showed a significantly increased accumulation in the nuclei of drug-resistant cells relative to the free drug, indicating that LNPs could alter intracellular traffic and bypass drug efflux. The cytotoxicity of DOX loaded-LNPs had a 30-fold lower half maximal inhibitory concentration (IC(50)) value than free DOX in MCF-7/ADR, measured by the colorimetric cell viability (MTT) assay, correlated with the strong nuclear retention of the drug.

Conclusion: The results show that this core-shell lipid/particle structure could be a promising strategy to bypass MDR.

Keywords: chemotherapy; drug delivery; multidrug resistance; polymeric nanoparticles.

Figure 1

Schematic diagram showing the structure of lipid/polymer particle assemblies. The negative phosphate charges on the hydrophilic segment of DPPC (green) can be attracted to the oppositely charged ions of the ammonium bromide ammonia headgroup of DMAB (blue), which modify the surface of DOX-loaded PLGA nanoparticles (red). Abbreviations: DMAB, dimethyldidodecylammonium bromide; DOX, doxorubicin; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; LNP, lipid/polymer particle assembly; PLGA, poly(lactic-co-glycolic) acid.

Figure 2

TEM and SEM images of LNPs formed with a core-shell supramolecular structure. A TEM image shows relatively uniform sized LNP particles (A), and a LipoParticle revealing its lipid shell thickness (B). A SEM image shows the fairly rigid surface morphology of PLGA particles by the electron-beam bombardment effect (C), and another SEM image displays the degradation and cracking of LNPs with a DPPC shell (D). Abbreviations: DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; LNPs, lipid/polymer particle assemblies; SEM, scanning electron microscopy; TEM, transmission electron microscopy.

Figure 3

In vitro drug release profiles of DOX-loaded LNPs at different pH (5.5, 6.5, and 7.4) and 37°C. Abbreviations: DOX, doxorubicin; LNPs, lipid/polymer particle assemblies.

Figure 4

P-gp expression in MCF-7 and MCF-7/ADR cells, as shown by flow cytometry analysis. Note: **P < 0.01. Abbreviations: MCF-7, Michigan Cancer Foundation-7; MCF-7/ADR, MCF-7/adriamycin; P-gp, P-glycoprotein.

Figure 5

Confocal images of the intracellular accumulation of free DOX and LNPs- DOX in MCF-7 (A) and MCF-7/ADR (B) cells. Cells were treated with 8 μg/mL free DOX or an equivalent concentration of LNPs-DOX for the different time periods as indicated. Nuclear (blue) uptake of DOX was increased dramatically by LNPs. Abbreviations: DOX, doxorubicin; free DOX, free doxorubicin; LNPs-DOX, doxorubicin loaded in lipid/polymer particle assemblies; MCF-7, Michigan Cancer Foundation-7; MCF-7/ADR, MCF-7/adriamycin.

Figure 6

Accumulation of free DOX or LNPs-DOX in MCF-7 and MCF-7/ADR cells. Cells were treated with 8 μg/mL DOX or an equivalent concentration of LNPs-DOX for 15 minutes and 1 hour. The intracellular DOX was then determined by flow cytometry analysis. Notes: *P < 0.05; **P < 0.01; ***P < 0.001. Abbreviations: DOX, doxorubicin; free DOX, free doxorubicin; h, hours; LNPs- DOX, doxorubicin loaded in lipid/polymer particle assemblies; MCF-7, Michigan Cancer Foundation-7; MCF-7/ADR, MCF-7/adriamycin; NS, no significance.

Figure 7

IC₅₀ for free DOX or LNPs-DOX in MCF-7 and MCF-7/ADR cells. Cells were treated with various concentrations of DOX or LNPs-DOX at the same dose for 24 hours. Note: ***P < 0.001. Abbreviations: Free DOX, free doxorubicin; IC₅₀, half maximal inhibitory concentration; h, hours; LNPs-DOX, doxorubicin loaded in lipid/polymer particle assemblies; MCF-7, Michigan Cancer Foundation-7; MCF-7/ADR, MCF-7/adriamycin; NS, no significance.