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

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.