Riboflavin-citrate conjugate multicore SPIONs with enhanced magnetic responses and cellular uptake in breast cancer cells

Abstract

Breast cancer accounts for up to 10% of the newly diagnosed cancer cases worldwide, making it the most common cancer found in women. The use of superparamagnetic iron oxide nanoparticles (SPIONs) has been beneficial in the advancement of contrast agents and magnetic hyperthermia (MH) for the diagnosis and treatment of cancers. To achieve delivery of SPIONs to cancer cells, surface functionalization with specific ligands are required. Riboflavin carrier protein (RCP) has been identified as an alternative target for breast cancer cells. Here, we report a novel riboflavin (Rf)-based ligand that provides SPIONs with enhanced colloidal stability and high uptake potential in breast cancer cells. This is achieved by synthesizing an Rf-citrate ligand. The ligand was tested in a multicore SPION system, and affinity to RCP was assessed by isothermal titration calorimetry which showed a specific, entropy-driven binding. MRI and MH responses of the coated Rf-SPIONs were tested to evaluate the suitability of this system as a theranostic platform. Finally, interaction of the Rf-SPIONs with breast cancer cells was evaluated by in vitro cellular uptake in MCF-7 breast cancer cells. The overall characterization of the Rf-SPIONs highlighted the excellent performance of this platform for theranostic applications in breast cancer.

Fig. 1. (A) Synthesis scheme of Rf-citrate. (B and C) HPLC chromatogram of Rf and the Rf-citrate crude product, respectively. (D) Mass spectrum of the Rf-citrate crude product with a major peak at m/z = 573.1444.

Fig. 2. (A) Cryo-TEM image of Rf-SPIONs showing clustered SPION cores. The inset shows the SAED image, indexed to an inverse spinel structure of iron oxide. (B) Hydrodynamic diameter distribution of Rf-SPIONs in water. The inset shows the physical appearance of the dispersion. (C) Histogram of core size distribution of Rf-SPIONs (n = 200), showing a mean size of 10 nm. (D) XRD pattern of Rf-SPIONs compared to the standard profile of magnetite (JCPDS no. 00-001-1111). (E) Field-dependent magnetization plots of Rf-SPIONs, measured at 10 and 300 K. The inset shows magnetization curves at the low field. (F) Zero-field and field cooled magnetization (ZFC/FC) versus temperature curves recorded with an applied field of 50 Oe.

Fig. 3. (A) HRTEM image of a cluster of Rf-SPIONs. (B–D) Fourier transform of selected cores, indexed to the zone axes of the magnetite crystals. (E–G) Filtered Fourier transform. Areas with similar crystallographic orientation are highlighted in the same colours. (H) Fourier transform of the whole cluster. (I) Filtered Fourier transform of the cluster.

Fig. 4. (A) Fluorescence spectra (λ_ex = 440 nm) of free Rf (yellow line), C-SPIONS (red line) and Rf-SPIONs (blue line). (B) Thermogravimetric curves of SPIONs (black line) and Rf-SPIONs (blue line). (C) Rf-SPION and (D) C-SPION DLS stability measurements.

Fig. 5. Raw ITC data (A and E), integrated heat plots (B, F), and thermodynamic parameters (C and G) of the binding between RCP with Rf-SPIONs (blue plots) and C-SPIONs (red plots). (D) Schematic representation of the proposed interaction of the Rf-citrate ligand with SPION. (H) Interaction between free Rf with key amino acid residues in the binding site of RCP.

Fig. 6. (A) T₂ weighted MR phantom image of the SPIONs. (B) Plot of T₂⁻¹versus Fe concentration for Rf-SPIONs (blue) and C-SPIONs (red) in water. (C) SAR values as a function of the applied magnetic field at a frequency of 763 kHz for the SPIONs. (D) SAR values of the SPIONs were measured at frequencies of 163 and 491 kHz under a constant magnetic field strength of 24 kA m⁻¹.

Fig. 7. (A) Cell viability of MCF-7 cells after being treated with different concentrations of Rf-SPIONs for 4, 12, and 24 h. The viability was evaluated by MTT assay with Triton-X as a positive control. (B) Relative side scattering signals of the cells under different incubation conditions. Statistical significance was analyzed by a one-way ANOVA with p < 0.01 (**) and p < 0.001 (***). (C) Uptake observation by Prussian blue staining. Blue pigments represent iron while cells are counterstained in pink by neutral red.