Cetuximab-Coated Thermo-Sensitive Liposomes Loaded with Magnetic Nanoparticles and Doxorubicin for Targeted EGFR-Expressing Breast Cancer Combined Therapy

Abstract

Background: One major limitation of cancer chemotherapy is a failure to specifically target a tumor, potentially leading to side effects such as systemic cytotoxicity. In this case, we have generated a cancer cell-targeting nanoparticle-liposome drug delivery system that can be activated by near-infrared laser light to enable local photo-thermal therapy and the release of chemotherapeutic agents, which could achieve combined therapeutic efficiency.

Methods: To exploit the magnetic potential of iron oxide, we prepared and characterized citric acid-coated iron oxide magnetic nanoparticles (CMNPs) and encapsulated them into thermo-sensitive liposomes (TSLs). The chemotherapeutic drug, doxorubicin (DOX), was then loaded into the CMNP-TSLs, which were coated with an antibody against the epidermal growth factor receptor (EGFR), cetuximab (CET), to target EGFR-expressing breast cancer cells in vitro and in vivo studies in mouse model.

Results: The resulting CET-DOX-CMNP-TSLs were stable with an average diameter of approximately 120 nm. First, the uptake of TSLs into breast cancer cells increased by the addition of the CET coating. Next, the viability of breast cancer cells treated with CET-CMNP-TSLs and CET-DOX-CMNP-TSLs was reduced by the addition of photo-thermal therapy using near-infrared (NIR) laser irradiation. What is more, the viability of breast cancer cells treated with CMNP-TSLs plus NIR was reduced by the addition of DOX to the CMNP-TSLs. Finally, photo-thermal therapy studies on tumor-bearing mice subjected to NIR laser irradiation showed that treatment with CMNP-TSLs or CET-CMNP-TSLs led to an increase in tumor surface temperature to 44.7°C and 48.7°C, respectively, compared with saline-treated mice body temperature ie, 35.2°C. Further, the hemolysis study shows that these nanocarriers are safe for systemic delivery.

Conclusion: Our studies revealed that a combined therapy of photo-thermal therapy and targeted chemotherapy in thermo-sensitive nano-carriers represents a promising therapeutic strategy against breast cancer.

Keywords: breast cancer; cetuximab; combined therapy; doxorubicin; epidermal growth factor receptors; iron oxide magnetic nanoparticles.

Figure 1

Schematic illustration of NIR-triggered DOX release from CET-DOX-CMNP-TSLs.

Figure 2

Characterization of CMNPs, TSLs, CMNPs–TSLs, and CET-DOX-CMNP-TSLs. (A) TEM images and size distributions as measured by DLS; (B) Normalized field-dependent magnetization curve for the CMNPs and CMNP-TSLs; (C) T₂-Weighted MR images of CMNP –TSLs aqueous solutions with various Fe concentrations; (D) Plot of 1/T₂ over Fe ion concentration (mM) of the CMNP–TSLs aqueous solution, the slope indicates the specific relaxivity (r₂); (E) Chromatogram for CET-TSLs, Free CET and Free TSLs; and (F) SDS-Page electrophoresis profile of the (1) Protein ladder, (2) CET and (3) CET-TSLs.

Figure 3

Effect of NIR laser irradiation on DOX release from DOX-TSLs and DOX-CMNPs-TSLs: (A1) Infrared thermal images showing the photo-thermal effect of NIR laser irradiation on water and CMNP–TSLs at different irradiation times (λ_max 808 nm, 2 W/cm⁻²) and CMNP concentration; (A2) Photo-thermal heating curves of TSLs solution and CMNP–TSLs at various CMNP concentrations and various irradiation times (λ_max 808 nm, 2 W/cm⁻²); and DOX release from different formulations at three different pH levels (B1) pH 7.4, (B2) pH 6.8, and (B3) pH 5.5 with and without NIR irradiation (λ_max 808 nm, 2 W/cm⁻², t=5min) (*p<0.05, **p<0.01).

Figure 4

Cancer cell viability and uptake following treatment with CET-DOX-CMNP-TSLs. (A and B) Qualitative cellular uptake of TSLs coated with and without CET by SKBR-3 and MCF-7 cells imaged by fluorescence microscopy (scale bar = 50 µm) and the quantitative analysis of the same analyzed by flow cytometry (n=3, ***p<0.001); (C and D) Relative cell viability of SKBR-3 and MCF-7 cells incubated for 24 h with different concentrations of CMNP (as a function of Fe₃O₄) in CET-CMNP-TSLs with or without NIR λ_max 808 nm laser irradiation (2 W/cm² for 5 min) (n=5) (*p <0.05, **p<0.01); (E and F) Cell viability of SKBR-3 and MCF-7 cells incubated for 24 h with CET-DOX-CMNP-TSLs (as a combination function of DOX and CMNP at a ratio of 1:15) at the same concentration with and without NIR irradiation at λ_max 808 nm and 2 W/cm² for 5 min (n=5) (*p <0.05, **p <0.01,***p <0.001).

Figure 5

Photo-thermal effect of CMNP-TSLs treatment plus NIR laser irradiation on tumor temperature; (A) Thermographic images and (B) quantification of tumor temperatures in tumor-bearing mice following NIR laser irradiation for different time periods plus treatment with normal saline, CMNP-TSLs and CET-CMNP-TSLs. (*p <0.05, **p <0.01, ***p <0.001). The white circle represents the tumor site.

Figure 6

The hemolytic effect of CMNP in all formulations at different concentrations (10, 20, 30, 40, and 50 µg/mL). (A) Visually observed photographs of rabbit RBCs exposed with different concentrations of CMNPs, normal saline (-Ve control) and deionized water (+Ve control), followed after centrifugation. (B) Quantitative hemolysis rate of CMNPs at concentration ranges from 10–50µg/mL as measured by a microplate reader (BioTek, USA) at 570 nm λ_max.