Artificial Digestion of Polydisperse Copper Oxide Nanoparticles: Investigation of Effects on the Human In Vitro Intestinal Co-Culture Model Caco-2/HT29-MTX

Abstract

Copper oxide nanoparticles (CuO-NP) are increasingly used in consumer-related products, which may result in increased oral ingestion. Digestion of particles can change their physicochemical properties and toxicity. Therefore, our aim was to simulate the gastrointestinal tract using a static in vitro digestion model. Toxic properties of digested and undigested CuO-NP were compared using an epithelial mono-culture (Caco-2) and a mucus-secreting co-culture model (Caco-2/HT29-MTX). Effects on intestinal barrier integrity, permeability, cell viability and apoptosis were analyzed. CuO-NP concentrations of 1, 10 and 100 µg mL^-1 were used. Particle characterization by dynamic light scattering and transmission electron microscopy showed similar mean particle sizes before and after digestion, resulting in comparable delivered particle doses in vitro. Only slight effects on barrier integrity and cell viability were detected for 100 µg mL^-1 CuO-NP, while the ion control CuCl₂ always caused significantly higher adverse effects. The utilized cell models were not significantly different. In summary, undigested and digested CuO-NP show comparable effects on the mono-/co-cultures, which are weaker than those of copper ions. Only in the highest concentration, CuO-NP showed weak effects on barrier integrity and cell viability. Nevertheless, a slightly increased apoptosis rate indicates existing cellular stress, which gives reason for further investigations.

Keywords: apoptosis; cell viability; copper oxide nanoparticles; delivered dose; intestinal barrier integrity; mono-/co-culture; static in vitro digestion.

Figure 1

Schematic illustration of the applied experimental setup. (A) Particle dispersion preparation by ultrasonication, (B) artificial digestion using a static in vitro approach, (C) dispersion characterization by transmission electron microscopy and dynamic light scattering, (D) intestinal barrier integrity measurements of transepithelial electrical resistance and permeability of FITC-dextran, (E) analysis of cell viability and induction of apoptosis by flow cytometry. The statistical differences between the samples and the NC are marked as * p < 0.05, ** p < 0.01 and *** p < 0.001, and the statistical difference between the digested/undigested samples is marked as # p < 0.05.

Figure 2

Impact of artificial digestion on the appearance of copper oxide nanoparticles (CuO-NP). Representative transmission electron microscopy images of agglomerates and/or aggregates of (A) undigested and (B) digested CuO-NP are shown. The red bars represent 250 nm.

Figure 3

Impact of digested and undigested copper oxide nanoparticles on the barrier integrity of Caco-2 mono-cultures and Caco-2/HT29-MTX co-cultures. NC: negative control (cell culture medium); PC: positive control (10 mM EGTA and 0.1% Triton X-100); DC: digestion control (10% digestive juice in culturing medium); VC: vehicle control (0.05% BSA solution). The red line represents the initial TEER value before incubation. The statistical differences relative to the TEER values before treatment are marked as: *** p < 0.001. The error bars represent the standard deviation of three independent measurements.

Figure 4

Permeability of the monolayers of Caco-2 mono-culture and Caco-2/HT29-MTX co-culture cells after incubation with digested and undigested copper oxide nanoparticles. The FITC-dextran concentrations after 24 h were normalized to the initial applied FITC-dextran concentration in the apical compartment, which was determined by incubating a Transwell insert without cells with FITC-dextran and measuring the basolateral concentration after 24 h. NC: negative control (cell culture medium); PC: positive control (10 mM EGTA and 0.1% Triton-X-100); DC: digestion control (10% digestive juice in culturing medium); VC: vehicle control (0.05% BSA solution). The statistical differences relative to NC are marked as: * p < 0.05, ** p < 0.01 and *** p < 0.001. The error bars represent the standard deviation of four independent measurements.

Figure 5

The effect of the digestion process on the cytotoxicity of copper oxide nanoparticles was determined via propidium iodide staining and flow cytometry. The cells of the Caco-2 mono-culture and the Caco-2/HT29-MTX co-culture were incubated with 10% digestive juice (DC), 0,05% BSA solution and CuO-NP/CuCl₂ for 24 h. The mean vitality values were normalized to those of the negative control (NC), represented by the red line. The statistical differences between the samples and the NC are marked as * p < 0.05, ** p < 0.01 and *** p < 0.001, and the statistical difference between the digested/undigested samples is marked as # p < 0.05. The error bars represent the standard deviation of three independent measurements.

Figure 6

Detection of apoptosis induction by flow cytometry and fluorescence microscopy. (A) Histogram of Annexin V-FITC-stained Caco-2 mono-culture cells without (black) or with copper oxide nanoparticle (CuO-NP) treatment (green: undigested, red: digested). (B) DAPI-stained Caco-2 mono-culture cells after treatment with digested CuO-NP (white arrows indicate shrunken nuclei). (C) Fold change of apoptotic cells after treatment of Caco-2 mono- and Caco-2/HT29-MTX co-cultures with undigested and digested CuO-NP (1.3 × 10⁻³ M) or copper chloride (1.3 × 10⁻³ M). The samples were normalized to the untreated negative control (red line). All samples are means of three independent replicates and were measured in triplicate. DC: digestive control (10% digestive juice).