Effect of Citrate- and Gold-Stabilized Superparamagnetic Iron Oxide Nanoparticles on Head and Neck Tumor Cell Lines during Combination Therapy with Ionizing Radiation

Abstract

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. They are associated with alcohol and tobacco consumption, as well as infection with human papillomaviruses (HPV). Therapeutic options include radiochemotherapy, surgery or chemotherapy. Nanoparticles are becoming more and more important in medicine. They can be used diagnostically, but also therapeutically. In order to provide therapeutic alternatives in the treatment of HNSCC, the effect of citrate-coated superparamagnetic iron oxide nanoparticles (Citrate-SPIONs) and gold-coated superparamagnetic iron oxide nanoparticles (Au-SPIONs) in combination with ionizing irradiation (IR) on two HPV positive and two HPV negative HNSCC and healthy fibroblasts and keratinocytes cell lines were tested. Effects on apoptosis and necrosis were analyzed by using flow cytometry. Cell survival studies were performed with a colony formation assay. To better understand where the SPIONs interact, light microscopy images and immunofluorescence studies were performed. The HNSCC and healthy cell lines showed different responses to the investigated SPIONs. The cytotoxic effects of SPIONs, in combination with IR, are dependent on the type of SPIONs, the dose administered and the cell type treated. They are independent of HPV status. Reasons for the different cytotoxic effect are probably the different compositions of the SPIONs and the related different interaction of the SPIONs intracellularly and paramembranously, which lead to different strong formations of double strand breaks.

Keywords: citrate; gold; head and neck cancer cell lines; interactions; ionizing radiation; nanoparticles; superparamagnetic iron oxide nanoparticles.

Figure 1

(A) Illustration of citrate-stabilized superparamagnetic iron oxide nanoparticles (Citrate-SPIONs); (B) gold-coated SPIONs (Au-SPIONs) which are also stabilized using citrate ions.

Figure 2

Cal33 HNSCC cells treated with SPIONs and imaged with a light microscope after 3.5 h, 24 h, 48 h and 72 h.

Figure 3

SBLF9 fibroblasts treated with SPIONs and imaged with a light microscope after 3.5 h, 24 h, 48 h, 72 h and 96 h.

Figure 4

A colony formation assay was performed to determine the toxicity of the SPIONs. HNSCC cell lines Cal33 and UM-SCC-47 were used. Dose escalation for different concentrations of (A) Au-SPIONs and (B) Citrate-SPIONs.

Figure 5

Used methods to detect the combined effect of SPIONs and ionizing radiation. Gating strategy for cell death, colony formation assay and immunofluorescence microscopy. (A) Exemplary gating strategy of Annexin-V-APC/7AAD with UM-SCC-47 cells. (B) Colony formation assay under microscopy. Images were taken and analyzed with Biomas software. (C) Immunofluorescence microscopy of Cal33 tumor cells. Cells were stained with anti-γH2Ax for DNA double-strand breaks (green) and anti-Ki-67 for growth fraction (red). Cell nuclei were stained with DAPI (blue).

Figure 6

Induction of apoptosis and necrosis were stained with Annexin V APC and 7AAD to determine the differences in cell death of Au- and Citrate-SPIONs in different cell lines. All cell lines were treated with (A) Au-SPIONs or (B) Citrate-SPIONs. Survival decreased by apoptosis or necrosis under irradiation with 2 Gy (blue line) and a combination of irradiation and Au-SPIONs/Citrate-SPIONs (red line). The red dashed line is normalized to subtract out the cytotoxic effect of the nanoparticles to better indicate any additive effect. The graph depicts HPV+ cell lines in the top row, HPV- cells in the middle row, and healthy cells in the bottom row. Each experiment was repeated at least three times. Error bars indicate the standard deviation. * p-value ≤ 0.05.

Figure 7

Survival fraction was determined by the colony formation assay. The cells were grouped in HPV+ (UM-SCC-47 and UD-SCC-2), HPV- (HSC-4 and Cal33) and healthy cells. Irradiation was indicated in blue and Au-SPIONs/Citrate-SPIONs in combination was indicated in red. The red dashed line is normalized to subtract out the cytotoxic effect of the nanoparticles to better indicate any additive effect. (A) shows graphs for Au-SPIONs and (B) Citrate-SPIONs. The graph depicts HPV+ cell lines (UM-SCC-47 and UD-SCC-2) in the two top rows, HPV- cells in the two middle rows (HSC-4 and Cal33), and healthy cells in the two bottom rows. Each experiment was repeated at least three times. Error bars indicate the standard deviation. * p-value ≤ 0.05.

Figure 8

Anti-γH2Ax foci as DNA double-strand breaks in the presence of Au- and Citrate-SPIONs. Cal33 cells were differentiated into G0 phase and growth fraction by Ki-67. Cells were (A) treated with Au-SPIONs or (B) Citrate-SPIONs. DNA-double strand breaks were identified after a repair time of 24 h with or without 2Gy ionizing radiation. Each experiment was repeated at least three times. Error bars indicate the standard deviation.