Cutaneous Evaluation of Fe3O4 Nanoparticles: An Assessment Based on 2D and 3D Human Epidermis Models Under Standard and UV Conditions

Abstract

Purpose: The high-speed development of nanotechnology industry has fueled a plethora of engineered nanoparticles (NPs) and NP-based consumer products, further leading to massive and uncontrolled human exposure. In this regard, the researches addressing the safety assessment of NPs should be re-approached from the perspective of test parameters variety, closely simulating daily life scenarios. Therefore, the present study adopts complex in vitro models to establish the safety profile of Fe₃O₄ NPs, by using 2D and 3D human epidermis models under both standard and UV exposure conditions.

Methods: Advanced 3D human reconstructed epidermal tissues and two different monolayers of immortalized human cells (keratinocytes and fibroblasts), using series of in vitro assays were employed in the current study to evaluate multiple biological responses, as follows: i) divers protocols (skin irritation, phototoxicity assay); ii) different conditions (± UV exposure) and iii) a wide variety of quantification methods, such as: MTT, NR and LDH colorimetric tests - performed to evaluate the viability of the cells/microtissues, respectively, the cytotoxicity of the test compounds. In addition, IL-1α ELISA assay was used to quantify the inflammatory activity induced by the test samples, while immunocytochemistry analysis through fluorescent microscopy was employed to provide insightful information regarding the possible mechanism of action of test samples.

Results: The two test samples (S₁ and S₂) induced a higher cell viability decrease on immortalized human keratinocytes (HaCaT) compared to human fibroblasts (1BR3), while 3D-epidermis microtissues showed similar viabilities when treated with both samples under standard conditions (-UV rays) - for both type of evaluation protocols: skin irritation and phototoxicity. However, UV irradiation of 3D-microtissues pre-exposed to test samples led to different results between the two test samples, revealing that S₂ sample induced a significant impairment of human epidermis viability, whereas S₁ sample elicited an activity similar to the one recorded under standard conditions (-UV).

Conclusion: The present results indicate significant differences in toxicity between the two in vitro models under UV conditions, highlighting the importance of model selection and exposure parameters in assessing NP safety. Thus, our findings suggest that Fe₃O₄ NPs may pose some risks under specific environmental conditions, within the limitations of the experimental setup, and further research is needed to refine safety guidelines.

Keywords: 1BR3; 3D-microtissue; HaCaT; UV; cytotoxicity; magnetite NPs.

Figure 1

Schematic representation of Fe₃O₄ NPs synthesis through combustion method.

Figure 2

The effect induced by Fe₃O₄ test samples (S₁ and S₂) on: (A) human keratinocytes HaCaT and (B) human fibroblasts - 1BR3 at 18 h post-stimulation under standard and UV conditions, employing the NR assay. Results are presented as cell viability percentage (%) normalized to negative control cells (cells treated with medium, under standard conditions). The data represent the mean values ± standard deviation (SD) of three independent experiments. One-way ANOVA analysis was applied to determine the statistical differences followed by Dunnett post-test (*p < 0.1; **p < 0.01; ***p < 0.001; ****p < 0.0001 versus control cells -UV, considered as negative control).

Figure 3

Immortalized human keratinocytes (HaCaT) monolayer visualized by fluorescence microscopy at 18h post-exposure to S₁ (concentration of 200 µg/mL), under standard (-UV) and UV parameters. Nuclear and lysosome staining was presented both ways: i) separated (hoechst and lysosome) and combined (overlay). Red arrows present the accumulation of Fe₃O₄ NPs, preponderantly in the lysosome compartment. Control cells -UV were considered as negative control. Scale bar represents 20 µm.

Figure 4

Immortalized human keratinocytes (HaCaT) monolayer visualized by fluorescence microscopy at 18h post-exposure to S₂ test sample, under standard (-UV) and UV parameters. Nuclear and lysosome staining was presented both ways: i) separated (hoechst and lysosome) and combined (overlay). Cell nuclei alterations are indicated by white arrows and lysosomal impairment is marked by the yellow arrows. Control cells -UV were considered as negative control. Scale bar represents 20 µm.

Figure 5

Irritation (A) and phototoxicity data (B) of 3D reconstructed human epidermis after exposure to 200 µg/mL of S₁ and S₂ for a period of 18h. Results are presented as viability percentage (%) normalized to negative control = NC (microtissues treated with medium, under standard conditions -UV). The data represent the mean values ± standard deviation (SD) of three independent experiments. Positive control (PC) is represented by SDS 1%. One-way ANOVA analysis was applied to determine the statistical differences followed by Dunnett post-test (*p < 0.1; **p < 0.01; ***p < 0.001; ****p < 0.0001 versus control cells -UV, considered as NC).

Figure 6

Histopathological examination of the 3D skin models by H&E staining. Section from the untreated control microtissue, considered as negative control x40 (A); control group exposed to UV showing epidermal hyperplasia and hyperkeratosis, x10 (B). Group treated with S₁ depicted signs of epidermal hyperplasia and hyperkeratosis, x40 (C), and superficial erosion following UV exposure, x10 (D). Topical application of S₂ revealed erosion of the epithelium with less viable cells, x40 (E); with more significant erosion aspects recorded for the UV-irradiated microtissue with S₂, x40 (F).