Comprehensive study of stability of copper oxide nanoparticles in complex biological media Marta J. Woźniak-Budych a, ⁎, Barbara Maciejewska a , Łucja Przysiecka a , Daria Wieczorek b , Katarzyna Staszak c , Jacek Jenczyk a , Teofil Jesionowski c , Stefan Jurga a a NanoBioMedical Centre, Adam Mickiewicz University in Poznan, Wszechnicy Piastowskiej 3, Poznan, Poland b Department of Technology and Instrumental Analysis, Faculty of Commodity Science, Poznan University of Economics and Business, Niepodległości 10, Poznan, Poland c Institute of Technology and Chemical Engineering, Faculty of Chemical Technology, Poznan University of Technology, Berdychowo 4, Poznan, Poland abstract article info Article history: Received 17 July 2020 Received in revised form 12 August 2020 Accepted 17 August 2020 Available online 21 August 2020 In response to the rapid development of nanomaterials in the biomedical field, especially in advanced cancer therapy, more comprehensive studies regarding the safety of nanomaterials are continuously required. Currently, several fundamental studies concerning the fate of nanoparticles in the biological media are needed, especially before in vivo studies. Inspired by these approaches, and the wide variation on result in the literature regarding copper (I) oxide nanoparticles (Cu 2 O NPs), in this article we modified sulfobetaine-stabilzed Cu 2 O NPs with glu- tathione (GSH) and hyaluronic acid (HA) tumor targeting ligands to investigate the effects of nanoparticles de- sign on their stability in four different biological fluids: simulated saliva, human blood plasma, gastric and intestinal fluids. In addition, we examined the kinetics of copper ions release under physiological conditions, and according to mathematical models, we proposed the most probable mechanism of mass transport. The GSH- and HA-modified, as well as unmodified sulfobetaine-stabilized Cu 2 O NPs suspended in all tested biological fluids, formed agglomerates with sizes varying from 150 to 700 nm. The highest copper ions content was ob- served for Cu 2 O NPs in simulated gastric fluid. The formation of aggregates and copper ions release in contact with biological fluids influenced the Cu 2 O NPs cytotoxicity. The results indicated that cancer cells were more sus- ceptible to HA-modified copper oxide NPs action. © 2020 Elsevier B.V. All rights reserved. Keywords: Copper (I) oxide nanoparticles Sulfobetaine Colloidal stability Copper ions release Biological studies 1. Introduction Metal nanoparticles (MNPs) exhibit great potential as a platform for drug transportation into cells [1–10]. However, the implementation of MNPs in drug delivery systems has been dominated by the use of noble nanomaterials, in particular, those based on gold and platinum [11–15]. Copper and copper-based nanomaterials show great potential for di- agnostic and therapeutic applications, such as drug delivery systems for cancer or antibiotic therapies, biosensor for virus detection, photosensi- tizer for photothermal therapy, and contrast agent for High-Resolution Magnetic Resonance Imaging [16–22]. It is also worth mentioning that copper nanoparticles and hyaluronan oligomers improve the regenera- tion of crosslinked fibrillar elastin matrixes by adult vascular smooth muscle cells [23]. Moreover, the nanocomposite containing copper nanoparticles embedded in poly(2-hydroxyethyl methacrylate) (pHEMA) matrix, due to anti-blood-clotting properties, can be applied as innovative anti-thrombogenic materials [24]. In order to assess the copper oxide-induced adverse effect but also to expand their safe use, the toxicity and stability studies in different media, especially physio- logical fluids need to be thoroughly investigated. In recently published literature a great deal of attention involves cytotoxicity, genotoxicity, and ecotoxicity of Cu and CuO NPs [25,26]. Aggregation and stability studies of CuO NPs in various media, including dissolution and possible interaction between nanoparticles and other ions, peptides and differ- ent cells have been examined as well [27–29]. However, the aspect of Cu 2 O NPs behaviour in biological media and its overall influence of ag- gregation and dissolution phenomena on nanoparticles biological activ- ity is very limited in the literature [30,31]. Even though the significant number of reports state that copper and other metal nanoparticles exhibit therapeutic potential, there are some challenges in nanomaterials cytotoxicity examination [32–34]. Despite the growing interest in nanomaterials and their biomedical application, the standardized procedures for evaluation of NPs toxicity have not yet been outlined [35]. The cellular metabolic activity can be examined via MTT, MTS, or WST-1 assay. Several inflammatory biomarkers, i.e. IL-8, IL-6, tumor necrosis factor can be used to investigate the inflammatory response induced by NPS (e.g. ELISA assay). Moreover, different biolog- ical models, based on various cancer and healthy cells (as control) can Journal of Molecular Liquids 319 (2020) 114086 ⁎ Corresponding author. E-mail address: marta.budych@amu.edu.pl (M.J. Woźniak-Budych). https://doi.org/10.1016/j.molliq.2020.114086 0167-7322/© 2020 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Journal of Molecular Liquids journal homepage: www.elsevier.com/locate/molliq