Biocompatibility Evaluation of TiO2, Fe3O4, and TiO2/Fe3O4 Nanomaterials: Insights into Potential Toxic Effects in Erythrocytes and HepG2 Cells

Abstract

Nanomaterials such as titanium dioxide and magnetite are increasingly used in several fields, such as water remediation and agriculture. However, this has raised environmental concerns due to potential exposure to organisms like humans. Nanomaterials can cause adverse interactions depending on physicochemical characteristics, like size, morphology, and composition, when interacting with living beings. To ensure safe use and prevent the risk of exposure to nanomaterials, their biocompatibility must be assessed. In vitro cell cultures are beneficial for assessing nanomaterial-cell interactions due to their easy handling. The present study evaluated the biocompatibility of TiO₂, Fe₃O₄, and TiO₂/Fe₃O₄ nanomaterials thermally treated at 350 °C and 450 °C in erythrocytes and HepG2 cells. According to the hemolysis experiments, non-thermally treated NMs are toxic (>5% hemolysis), but their thermally treated counterparts do not present toxicity (<2%). This behavior indicates that the toxicity derives from some precursor (solvent or surfactant) used in the synthesis of the nanomaterials. All the thermally treated nanomaterials did not show hemolytic activity under different conditions, such as low-light exposure or the absence of blood plasma proteins. In contrast, non-thermally treated nanomaterials showed a high hemolytic behavior, which was reduced after the purification (washing and thermal treatment) of nanomaterials, indicating the presence of surfactant residue used during synthesis. An MTS cell viability assay shows that calcined nanomaterials do not reduce cell viability (>11%) during 24 h of exposure. On the other hand, a lactate dehydrogenase leakage assay resulted in a higher variability, indicating that several nanomaterials did not cause an increase in cell death as compared to the control. However, a holotomographic microscopy analysis reveals a high accumulation of nanomaterials in the cell structure at a low concentration (10 µg mL^-1), altering cell morphology, which could lead to cell membrane damage and cell viability reduction.

Keywords: cell viability; hemolysis assay; holotomography; nanomaterials; nanotoxicity.

Figure 1

SEM micrographs of (a) TiO₂, (b) Fe₃O₄, and (c) TiO₂/Fe₃O₄.

Figure 2

Energy dispersive X-ray elemental analysis of (a) TiO₂, (b) Fe₃O₄, and (c) TiO₂/Fe₃O₄.

Figure 3

TEM micrographs and NM diameter distributions of (a) TiO₂, (b) Fe₃O₄, and (c) TiO₂/Fe₃O₄.

Figure 4

X-ray diffraction patterns of the synthesized nanomaterials.

Figure 5

(a) Raman spectra of the synthesized nanomaterials and (b) additional vibrational modes located between 180 and 400 cm⁻¹ attributed to the brookite phase.

Figure 6

Magnetic hysteresis curves of (a) Fe₃O₄ and (b) TiO₂/Fe₃O₄ composites.

Figure 7

Photoluminescence spectra of the synthesized nanomaterials.

Figure 8

Hemolysis assay of NMs’ interaction with (a) blood and plasma in a dark environment, (b) blood and plasma under light exposure, (c) blood without plasma under a dark environment, and (d) blood without plasma under light exposure.

Figure 9

MTS cell viability assays of (a) TiO₂ at 350 °C, (b) TiO₂ at 450 °C, (c) TiO₂/Fe₃O₄ at 350 °C, (d) TiO₂/Fe₃O₄ at 450 °C, (e) Fe-TiO₂/Fe₃O₄ at 350 °C, (f) Fe-TiO₂/Fe₃O₄ at 450 °C, and (g) Fe₃O₄. Cell viability by MTS assay was determined after 24 h of exposure to increasing concentrations of NPs. Each bar represents the average value of 3 different experiments ± standard error of the mean (SEM); * p < 0.05, ** p < 0.01, *** p < 0.001 vs. untreated cells. ANOVA, Bonferroni post hoc.

Figure 10

LDH cell viability assays of (a) TiO₂ at 350 °C, (b) TiO₂ at 450 °C, (c) TiO₂/Fe₃O₄ at 350 °C, (d) TiO₂/Fe₃O₄ at 450 °C, (e) Fe-TiO₂/Fe₃O₄ at 350 °C, (f) Fe-TiO₂/Fe₃O₄ at 450 °C, and (g) Fe₃O₄. Cell viability by LDH assay was determined after 24 h of exposure to increasing concentrations of NPs. Each bar represents the average value of 3 different experiments ± SEM; * p < 0.05, *** p < 0.001 vs. untreated cells. ANOVA, Bonferroni post hoc.

Figure 11

Three-dimensional and two-dimensional holotomographic images of control cells (a–d), TiO₂ at 450 °C (e–g), and TiO₂/Fe₃O₄ at 450 °C (h–k); red arrows indicate NMs agglomerates. All images were taken at 4 μm zoom.