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

Claudia Geanina Watz,1– 4 Elena-Alina Moacă,4,5 Andreea Cioca,6 Lenuța Maria Șuta,7 Lavinia Krauss Maldea,2 Ioana Zinuca Magyari-Pavel,8,9 Mirela Nicolov,2,3 Ioan-Ovidiu Sîrbu,10,11 Felicia Loghin,12 Cristina A Dehelean4,5 1Doctoral School, “Victor Babes” University of Medicine and Pharmacy, Timisoara, 300041, Romania; 2Department of Pharmaceutical Physics, Faculty of Pharmacy, “Victor Babes” University of Medicine and Pharmacy, Timisoara, 300041, Romania; 3Center for Drug Data Analysis, Cheminformatics and the Internet of Medical Things, “Victor Babes” University of Medicine and Pharmacy, Timisoara, 300041, Romania; 4Research Center for Pharmaco-Toxicological Evaluation, “Victor Babes” University of Medicine and Pharmacy, Timisoara, 300041, Romania; 5Department of Toxicology and Drug Industry, Faculty of Pharmacy, “Victor Babes” University of Medicine and Pharmacy, Timisoara, 300041, Romania; 6Department of Pathology “Regina Maria” Health Network, Timisoara, 300645, Romania; 7Pharmaceutical Technology, Faculty of Pharmacy, “Victor Babes” University of Medicine and Pharmacy, Timisoara, 300041, Romania; 8Department of Pharmacognosy-Phytotherapy, “Victor Babes” University of Medicine and Pharmacy Timisoara, Timisoara, 300041, Romania; 9Research and Processing Center for Medicinal and Aromatic Plants, “Victor Babeș” University of Medicine and Pharmacy, Timisoara, 300041, Romania; 10Department of Biochemistry, Faculty of Medicine, “Victor Babes” University of Medicine and Pharmacy Timisoara, Timisoara, 300041, Romania; 11Complex Network Science Center, “Victor Babes” University of Medicine and Pharmacy Timisoara, Timisoara, 300041, Romania; 12Department of Toxicology, Faculty of Pharmacy, “Iuliu Hatieganu” University of Medicine and Pharmacy of Cluj-Napoca, Cluj-Napoca, 400012, RomaniaCorrespondence: Elena-Alina Moacă, “Victor Babes” University of Medicine and Pharmacy, Timisoara, 300041, Romania, Tel +40745762600, Email [email protected]: 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: cytotoxicity, magnetite NPs, 3D-microtissue, UV, HaCaT, 1BR