Salinomycin-Loaded Iron Oxide Nanoparticles for Glioblastoma Therapy

Abstract

Salinomycin is an antibiotic introduced recently as a new and effective anticancer drug. In this study, magnetic iron oxide nanoparticles (IONPs) were utilized as a drug carrier for salinomycin for potential use in glioblastoma (GBM) chemotherapy. The biocompatible polyethylenimine (PEI)-polyethylene glycol (PEG)-IONPs (PEI-PEG-IONPs) exhibited an efficient uptake in both mouse brain-derived microvessel endothelial (bEnd.3) and human U251 GBM cell lines. The salinomycin (Sali)-loaded PEI-PEG-IONPs (Sali-PEI-PEG-IONPs) released salinomycin over 4 days, with an initial release of 44% ± 3% that increased to 66% ± 5% in acidic pH. The Sali-IONPs inhibited U251 cell proliferation and decreased their viability (by approximately 70% within 48 h), and the nanoparticles were found to be effective in reactive oxygen species-mediated GBM cell death. Gene studies revealed significant activation of caspases in U251 cells upon treatment with Sali-IONPs. Furthermore, the upregulation of tumor suppressors (i.e., p53, Rbl2, Gas5) was observed, while TopII, Ku70, CyclinD1, and Wnt1 were concomitantly downregulated. When examined in an in vitro blood-brain barrier (BBB)-GBM co-culture model, Sali-IONPs had limited penetration (1.0% ± 0.08%) through the bEnd.3 monolayer and resulted in 60% viability of U251 cells. However, hyperosmotic disruption coupled with an applied external magnetic field significantly enhanced the permeability of Sali-IONPs across bEnd.3 monolayers (3.2% ± 0.1%) and reduced the viability of U251 cells to 38%. These findings suggest that Sali-IONPs combined with penetration enhancers, such as hyperosmotic mannitol and external magnetic fields, can potentially provide effective and site-specific magnetic targeting for GBM chemotherapy.

Keywords: GBM; blood–brain barrier; drug delivery; external magnetic field; iron oxide nanoparticles; salinomycin.

Figure 1

Characterization of polyethylenimine polyethylene glycol iron oxide nanoparticles (PEI-PEG-IONPs) and release of salinomycin from salinomycin-loaded (Sali)-PEI-PEG-IONPs: (a) transmission electron microscopy (TEM) images of PEI-PEG-IONPs, with core size measurement of the nanoparticles; (b) size distribution histogram; (c) Fourier transform infrared (FTIR) spectrum; (d) release of salinomycin from the nanoparticles in pH 7.4 (physiological pH) and pH 4.5 (pH of acidic intracellular compartments such as endosomes).

Figure 2

Biocompatibility of different concentrations of PEI-PEG-IONPs (termed IONPs) on mouse brain-derived microvessel endothelial (bEnd.3) and U251 cell lines after 48 h treatment using MTT assay (n = 5). The Y-axis represents cell viability compared to the control.

Figure 3

Uptake of IONPs and Sali-IONPs by (a) bEnd.3 and (b) U251 after 4 h treatment. Note: * indicates a significant difference at p < 0.05. Data was presented as mean ± standard deviation (SD); n = 3.

Figure 4

TEM images of (a) IONP and (b) Sali-IONP uptake by U251 cells after 4 h of the treatment.

Figure 5

Cytotoxicity evaluation of salinomycin and Sali-IONPs on U251 after 48 h treatment. Note: * indicates a significant difference compared to the control group at p <0.05. Data was presented as mean ± SD; n = 6.

Figure 6

Cell apoptosis/necrosis of U251 upon treatment, stained with FITC-Annexin V and PI: (a) control, (b) IONPs (c) salinomycin, and (d) Sali-IONPs, with (Q4) live, (Q3) early apoptotic, (Q2) late apoptotic, and (Q1) necrotic cells.

Figure 6

Cell apoptosis/necrosis of U251 upon treatment, stained with FITC-Annexin V and PI: (a) control, (b) IONPs (c) salinomycin, and (d) Sali-IONPs, with (Q4) live, (Q3) early apoptotic, (Q2) late apoptotic, and (Q1) necrotic cells.

Figure 7

Cell proliferation analysis of carboxyfluorescein succinimidyl ester (CFSE)-labelled U251 upon treatment with IONPs, salinomycin, and Sali-IONPs: (a) CFSE flow cytometry graph and (b) the relative cell proliferation inhibition by mean CFSE_control/mean CFSE_treated. Note: * indicates a significant difference compared to the control group at p < 0.05.

Figure 8

Fluorescence microscopy images of U251 treated with either salinomycin or Sali-IONPs after 48 h of the treatment. Red and blue fluorescence colors represent Alexa Fluor@ 488 phalloidin-stained F-actin and DAPI-stained cell nuclei, respectively.

Figure 9

Reactive oxygen species (ROS) generation in U251 treated with either IONPs, salinomycin, or Sali-IONPs at different time-points. Note: * indicates a significant difference compared to the control group at p < 0.05. Data was presented as mean ± SD; n = 5.

Figure 10

Relative gene expression of U251 cell treated with either IONPs, salinomycin, or Sali-IONPs after 48 h of the treatment. Note: * and ** indicate a significant difference compared to the control and salinomycin groups, respectively, at p < 0.05. Data was presented as mean ± SD; n = 5.

Figure 11

Evaluation of anticancer efficacy of Sali-IONPs compared to the free salinomycin on a BBB-brain tumor model in vitro: (a) Sali-IONPs permeability across the bEnd.3 monolayer with or without magnet and mannitol; (b) cytotoxicity of each formulation on U251 cells after penetrating the bEnd.3 monolayer. Note: * indicates a significant difference at p < 0.05 with the other treated groups. Data was presented as mean ± SD; n = 3.

Figure 11

Evaluation of anticancer efficacy of Sali-IONPs compared to the free salinomycin on a BBB-brain tumor model in vitro: (a) Sali-IONPs permeability across the bEnd.3 monolayer with or without magnet and mannitol; (b) cytotoxicity of each formulation on U251 cells after penetrating the bEnd.3 monolayer. Note: * indicates a significant difference at p < 0.05 with the other treated groups. Data was presented as mean ± SD; n = 3.