Synthesis and Cytotoxicity Assessment of Citrate-Coated Calcium and Manganese Ferrite Nanoparticles for Magnetic Hyperthermia

Abstract

Calcium-doped manganese ferrite nanoparticles (NPs) are gaining special interest in the biomedical field due to their lower cytotoxicity compared with other ferrites, and the fact that they have improved magnetic properties. Magnetic hyperthermia (MH) is an alternative cancer treatment, in which magnetic nanoparticles promote local heating that can lead to the apoptosis of cancer cells. In this work, manganese/calcium ferrite NPs coated with citrate (Ca_xMn_1-xFe₂O₄ (x = 0, 0.2, 1), were synthesized by the sol-gel method, followed by calcination, and then characterized regarding their crystalline structure (by X-ray diffraction, XRD), size and shape (by Transmission Electron Microscopy, TEM), hydrodynamic size and zeta potential (by Dynamic Light Scattering, DLS), and heating efficiency (measuring the Specific Absorption Rate, SAR, and Intrinsic Loss Power, ILP) under an alternating magnetic field. The obtained NPs showed a particle size within the range of 10 nm to 20 nm (by TEM) with a spherical or cubic shape. Ca_0.2Mn_0.8Fe₂O₄ NPs exhibited the highest SAR value of 36.3 W/g at the lowest field frequency tested, and achieved a temperature variation of ~7 °C in 120 s, meaning that these NPs are suitable magnetic hyperthermia agents. In vitro cellular internalization and cytotoxicity experiments, performed using the human cell line HEK 293T, confirmed cytocompatibility over 0-250 µg/mL range and successful internalization after 24 h. Based on these studies, our data suggest that these manganese-calcium ferrite NPs have potential for MH application and further use in in vivo systems.

Keywords: calcium-doped manganese ferrite; cancer treatment; cellular internalization; magnetic hyperthermia; magnetic nanoparticles.

Figure 1

XRD diffraction patterns for Ca_xMn_1-xFe₂O₄ ferrites: (a) x = 0; (b) x = 0.2; (c) x = 1, and fitted patterns obtained through Rietveld refinement.

Figure 2

TEM images of MnFe₂O₄ (a), Ca_0.2Mn_0.8Fe₂O₄ (b) and CaFe₂O₄ (c) NPs and corresponding histograms of size distribution (d–f).

Figure 3

Hydrodynamic size, PDI value (a) and zeta potential (b) of the bare and citrate-functionalized Ca_xMn_1-xFe₂O₄ NPs at 0.01 mg/mL in phosphate buffer pH 7.4. Each value represents the mean ± SD (SD: standard deviation) of two or three independent measurements. Abbreviations: x = 0 + cit, x = 0.2 + cit and x = 1 + cit are citrate−functionalized MnFe₂O₄, Ca_0.2Mn_0.8Fe₂O₄ and CaFe₂O₄ nanoparticles, respectively.

Figure 4

Values of SAR (a), ILP (b) of citrate-functionalized NPs and variation of temperature (ΔT) over time (t) of MnFe₂O₄ (c), and Ca_0.2Mn_0.8Fe₂O₄ NPs (d), under an AMF at different conditions below the biological acceptance limit (H_AC × f = 2 × 10⁹ A m⁻¹ s⁻¹). Abbreviations: x = 0 + cit, x = 0.2 + cit and x = 1 + cit are citrate-functionalized MnFe₂O₄, Ca_0.2Mn_0.8Fe₂O₄, and CaFe₂O₄ nanoparticles, respectively.

Figure 5

In vitro cytotoxicity in HEK 293T cells of citrate-functionalized Ca-Mn ferrite NPs. Cell viability was assessed by MTT assay in HEK 293T cells incubated with four different concentrations of NPs (100, 150, 200, 250 µg/mL) for 24 h (a) and 48 h (b). Untreated cells at 24 h and 48 h were used as a negative control (100% viable cells). Data are expressed as the mean of three independent experiments (n = 3). Two-way ANOVA assessed by Tukey’s multiple comparison test indicates no statistically significant differences between the groups tested, denoted as ns p > 0.05. Abbreviations: x = 0 + cit, x = 0.2 + cit and x = 1 + cit are citrate-functionalized MnFe₂O₄, Ca_0.2Mn_0.8Fe₂O₄ and CaFe₂O₄ nanoparticles, respectively.

Figure 6

Bright field image of HEK 293T cells containing Ca_0.2Mn_0.8Fe₂O₄ NPs (a), cellular nucleus stained with DAPI (blue) (b), NPs coupled with CF (green) (c), and overlay of DAPI fluorescence and fluorescence of CF coupled to the NPs (d). The white arrows indicate agglomerates of NPs inside the cell. Images were obtained by fluorescence microscopy using a 40× objective. The scale bar corresponds to 20 µm and all images have the same scale.