Theranostic pH-sensitive nanoparticles for highly efficient targeted delivery of doxorubicin for breast tumor treatment

Abstract

A multifunctional theranostic nanoplatform integrated with environmental responses has been developed rapidly over the past few years as a novel treatment strategy for several solid tumors. We synthesized pH-sensitive poly(β-thiopropionate) nanoparticles with a supermagnetic core and folic acid (FA) conjugation (FA-doxorubicin-iron oxide nanoparticles [FA-DOX@ IONPs]) to deliver an antineoplastic drug, DOX, for the treatment of folate receptor (FR)-overexpressed breast cancer. In addition to an imaging function, the nanoparticles can release their payloads in response to an environment of pH 5, such as the acidic environment found in tumors. After chemical (¹H nuclear magnetic resonance) and physical (morphology and super-magnetic) characterization, FA-DOX@IONPs were shown to demonstrate pH-dependent drug release profiles. Western blotting analysis revealed the expression of FRs in three breast cancer cell lines, MCF-7, BT549, and MD-MBA-231. The cell counting kit-8 assay and transmission electron microscopy showed that FA-DOX@IONPs had the strongest cytotoxicity against breast cancer cells, compared with free DOX and non-FR targeted nanoparticles (DOX@IONPs), and caused cellular apoptosis. The FA-DOX@IONP-mediated cellular uptake and intracellular internalization were clarified by fluorescence microscopy. FA-DOX@IONPs plus magnetic field treatment suppressed in vivo tumor growth in mice to a greater extent than either treatment alone; furthermore, the nanoparticles exerted no toxicity against healthy organs. Magnetic resonance imaging was successfully applied to monitor the nanoparticle accumulation. Our results suggest that theranostic pH-sensitive nanoparticles with dual targeting could enhance the available therapies for cancer.

Keywords: breast cancer; folate receptor targeting; magnetic nanoparticle; pH sensitivity; theranostics.

Figure 1

Synthetic route of folate-conjugated amphiphilic poly(β-thiopropionate). Abbreviations: FA, folic acid; TEA, trimethylamine; DCM, dichloromethane; NMP, N-methyl-2-pyrrolidone; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; DMAP, 4-dimethylaminopyridine; PEG, polyethylene glycol; MeOH, methanol; CY, cysteamine; TEG, tetra (ethylene glycol); CY-TEG, cysteamine capped tetra (ethylene glycol); DDA, dodecylamine; APT, 5-aminopentanol; CY-TEG-PEG-DDA-APT, poly (beta-thiopropionate) amphiphilic copolymer.

Figure 2

The characterization of synthesized nanoparticles. (A) TEM image of nanoparticles; (B) pH-dependent DOX release profiles from FA-DOX@IONPs; (C) room temperature magnetization curves of synthesized nanoparticles with and without FA coating; and (D) the size change of nanoparticles in response to pH decreasing from 7.4 to 5.0 using DLS measurement. Abbreviations: DOX, doxorubicin; TEM, transmission electron microscopy; FA, folic acid; IONP, iron oxide nanoparticle; DLS, dynamic light scattering.

Figure 3

TEM investigation of FA-DOX@IONP-mediated cell apoptosis. (A, B) Intranuclear distorted chromatin clumps are denoted by white arrows and lipid droplet by black arrow. (C, D) FA-DOX@IONPs and breast cancer cell interaction. Abbreviations: DOX, doxorubicin; TEM, transmission electron microscopy; FA, folic acid; IONP, iron oxide nanoparticle.

Figure 4

MCF-7 cells incubated with free DOX, DOX@IONPs, and FA-DOX@IONPs (at equivalent DOX concentration of 7.5 μg/mL). (A) DOX fluorescence is red (in the left panel), cell nuclei were stained with blue using DAPI, and the membranes and cytoplasm were stained with green using DIO. Merged images are shown in the right panel. (B) Fluorescence images with only DAPI and DIO staining. Scale bars =20 μm. Abbreviations: DOX, doxorubicin; FA, folic acid; IONP, iron oxide nanoparticle; DIO, 3,3′-dioctadecyloxacarbocyanine perchlorate; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 5

Viability of MCF-7 cells subjected to DOX, DOX@IONPs, and FA-DOX@IONPs, respectively, at DOX concentrations (1, 2.5, 5, 10, and 15 μg/mL) for 24 hours measured by CCK8 assay. *p<0.05 is considered to be a statistically significant difference (n=3). Abbreviations: DOX, doxorubicin; FA, folic acid; IONP, iron oxide nanoparticle; CCK8, cell counting kit-8.

Figure 6

Antitumor efficacy of targeting-active and external magnetic nanoparticles in vivo. (A) Tumor growth curve of PBS, DOX, DOX@IONPs, FA-DOX@IONPs, DOX@IONPs plus magnetic treatment, and FA-DOX@IONPs plus magnetic treatment. *p<0.05 is considered to be a statistically significant difference (n=3). (B) The body weight changes with the treatments for 24 days. (C) Tumor size on the 24th day after treatment of PBS, DOX, DOX@IONPs, FA-DOX@IONPs, and DOX@IONPs plus magnetic treatment. Abbreviations: DOX, doxorubicin; FA, folic acid; IONP, iron oxide nanoparticle.

Figure 7

H&E staining micrographs (×200) of tumor sections, heart, kidney, and liver from PBS, free DOX, DOX@IONPs, FA-DOX@IONPs, DOX@IONPs plus magnetic treatment, and FA-DOX@IONPs plus magnetic treatment groups on day 24. Scale bars =50 μm. Abbreviations: DOX, doxorubicin; FA, folic acid; IONP, iron oxide nanoparticle.

Figure 8

Prussian blue micrographs (×200) of tumor sections, heart, kidney, and liver from PBS, free DOX, DOX@IONPs, FA-DOX@IONPs, DOX@IONPs plus magnetic treatment, and FA-DOX@IONPs plus magnetic treatment groups on day 24. Scale bars =50 μm. Abbreviations: DOX, doxorubicin; FA, folic acid; IONP, iron oxide nanoparticle.

Figure 9

Coronal T2 weighted images of mice after injection at 0, 12, and 36 hours. Coronal T2 weighted images of mice after injection of (A–C) DOX@IONPs; (D–F) FA-DOX@IONPs; (G–I) DOX@IONPs plus magnetic field (magnet); and (J–L) FA-DOX@IONPs plus magnetic field. The red arrows denote the tumor site and red circles denote the liver of the mouse. Abbreviations: DOX, doxorubicin; FA, folic acid; IONP, iron oxide nanoparticle.

Figure 10

Representative micrographs of CD31 immunohistochemistry of breast cancer cells in mice treated with (A) PBS, (B) free DOX, (C) DOX@IONPs, (D) FA-DOX@IONPs, (E) DOX@IONPs plus magnetic treatment, and (F) FA-DOX@IONPs plus magnetic treatment. (G) Microvascular vessels staining and AMVD (the number of microvessels per mm²) of mice breast cancer tumors. Scale bar =100 μm. Abbreviations: DOX, doxorubicin; FA, folic acid; IONP, iron oxide nanoparticle; AMVD, average microvessel density.