Anthocyanins decrease the internalization of TiO2 nanoparticles into 3D Caco-2 spheroids
Introduction
The development of nanotechnology has deeply influenced food science, and many kinds of nanoparticles (NPs) have been widely used in food products for different purposes. One of the examples is metal-based NPs, which are widely used in food industry for purposes of for instance food packaging and food additives (Cao et al., 2016, McClements et al., 2017). In addition, the development of nanomedicine also could increase oral exposure of NPs, since oral administration is more preferable during medical uses of NPs (Cao et al., 2018). Therefore, oral exposure to NPs is realistic in modern society and the interactions between NPs and intestinal cells should be evaluated. Indeed, previous studies already investigated the internalization of NPs into intestinal cells, but it is only recently that the important role of food components has been taken into consideration (Cao et al., 2016, McClements et al., 2017). Typical food components, for instance proteins, sugar and lipids, have been shown to affect the colloidal characteristics of NPs, which in turn influenced the internalization of NPs into intestinal cells (DeLoid et al., 2017, Lichtenstein et al., 2015, Zhang et al., 2019). Recently, the potential influence of phytochemicals on bio-effects of NPs has gained extensive research interests, since phytochemicals are naturally present in many food products and therefore co-exposure to phytochemicals and NPs via oral exposure route is possible in real life (Cao et al., 2017). However, the exact influence of phytochemicals on the toxicity of NPs is still in debate. While some studies showed the reduced cytotoxicity of Ag NPs by certain types of phytochemicals through the inhibition of NP-induced oxidative stress (Martirosyan et al., 2014, Martirosyan et al., 2016), we recently found reduced or even enhanced cytotoxicity of ZnO NPs by some types of phytochemicals through the modulation of signaling pathways rather than the modulation of oxidative stress (Jiang et al., 2019, Luo et al., 2018, Wu et al., 2019).
Anthocyanins are natural pigments that are widely present in almost every higher plant and many food products, but the interactions between NPs and anthocyanins as food components are less investigated. In addition, some studies also attempted using NPs to deliver phytochemicals including anthocyanins. Therefore it is necessary to understand the influence of phytochemicals on NP internalization into intestinal cells (Cao et al., 2017, Davatgaran-Taghipour et al., 2017). To the best of our knowledge, no previous study investigated the influence of anthocyanins on NP internalization into intestinal cells, but we recently showed that cyanidin (Cy) partially reduced the cytotoxicity of ZnO NPs to Caco-2 cells by altering the autophagy pathway independently of changes of oxidative stress or NP dissolution (Jiang et al., 2019).
This study used TiO2 NPs as model NPs, and attempted to figure out the main factor that might influence the internalization of NPs into 3D Caco-2 spheroids following co-exposure to TiO2 NPs and anthocyanins. TiO2 NPs were investigated because they have been used in food products for purposes of for example color development, but the potential risks associated with oral exposure to TiO2 NPs are still in debate (Warheit & Donner, 2015). Anthocyanins used in this study included Cy, delphindin (Del), malvidin (Mal), peonidin (Peo), petundin (Pet), pelargonidin (Pel), which are the most commonly distributed anthocyanins in plants (Kamiloglu, Capanoglu, Grootaert, & Van, 2015). In addition, by using different types of anthocyanins with similar structures we could investigate the possible role of chemical structures of anthocyanins (Cao et al., 2017). Anthocyanins were titrated with different concentrations of TiO2 NPs, and the binding affinity was calculated based on fluorescence quenching data. The present study developed 3D Caco-2 spheroids, because the spheroids contain multi-layered cells (Supplemental Fig. S1) and thus the model is expected to better reflect the responses of intestinal tissues compared with conventional 2D cell cultures (Zanoni, Pignatta, Arienti, Bonafe, & Tesei, 2019). 3D Caco-2 spheroids were co-exposed to TiO2 NPs and six anthocyanins for 24 h. After exposure, cytotoxicity was assessed by lactate dehydrogenase release (LDH) assay and acridine orange (AO)/4′,6-diamidino-2-phenylindole (DAPI) staining. The changes of thiols and the activation of nuclear factor erythroid-2-related factor 2 (Nrf2) were measured to indicate the antioxidant responses of 3D Caco-2 spheroids. In addition, the radical scavenging activities of anthocyanins were also evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The up-regulation of ABC transporters was investigated by real-time RT-PCR. Internalization of TiO2 NPs with or without the presence of anthocyanins was measured by using inductively coupled plasma mass spectrometry (ICP-MS). Finally, linear regression model was used to reveal the relationship between TiO2 NP internalization and binding affinity of anthocyanins and NPs.
Section snippets
Caco-2 cell culture
Caco-2 cells (ATCC, HTB-37) were cultured in DMEM/high glucose medium (Hyclone, GE Healthcare) as we described earlier (Li et al., 2018). To develop 3D spheroids, Caco-2 cells were seeded at a density of 2 × 104 per well in ultra-low attachment 24-well plates (Corning Inc., Corning, NY, USA). The cells were used for experiments at day 5 after seeding, with the cell culture medium changed at day 3. Before exposure, the diameters of 3D Caco-2 spheroids were typically>1.5 mm (Fig. S1).
TiO2 NP preparation and exposure
The TiO2 NPs
The influence of anthocyanins on colloidal properties of TiO2 NPs
As summarized in Table 1 (the distribution of hydrodynamic size and zeta potential was depicted in Supplemental Fig. S5), the suspension of TiO2 NPs had a hydrodynamic size larger than 400 nm, a PDI between 0.2 and 0.3, and a zeta potential below zero. The presence of Cy increased the hydrodynamic size while decreased PDI of TiO2 NPs, whereas the presence of Pet decreased the absolute value of zeta potential of TiO2 NPs. The rest of anthocyanins appeared to have minimal impacts on colloidal
Discussion
The present study investigated the interactions between TiO2 NPs and six typical anthocyanins, and attempted to find out the main factor to influence the internalization of TiO2 NPs into 3D Caco-2 spheroids following co-exposure to NPs and anthocyanins. The fluorescence quenching studies showed that TiO2 NPs concentration-dependently quenched the fluorescence of all tested anthocyanins (Fig. 1), which indicated the binding of anthocyanins onto NPs. Previously fluorescence quenching method has
CRediT authorship contribution statement
Junkang Wang: Investigation, Formal analysis, Writing - original draft, Writing - review & editing. Jiaqi Zhang: Investigation, Formal analysis; Writing - original draft, Writing - review & editing. Shuang Li: Investigation, Writing - review & editing. Chaobo Huang: Investigation, Writing - review & editing. Yixi Xie: Data curation, Formal analysis, Funding acquisition, Investigation, Supervision, Writing - review & editing. Yi Cao: Data curation, Formal analysis, Investigation, Supervision,
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
This work was financially supported by National Natural Science Foundation of China (31701613).
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Junkang Wang and Jiaqi Zhang contributed equally to this work.