## 3 Results  
The range of concentrations used in this study (60-600 \(\mathrm{\SIUnitSymbolMicro m}^{-1}\)) was based on reports from other studies on various TiO\({}_{2}\) samples [1; 16] as well as our preliminary tests. A 24-h exposure of Caco-2 cells to the tested substances did not cause a significant decrease in cell viability (XTT test). Only in the case of the S4 sample at the two highest concentrations (600 and 300 \(\mathrm{\SIUnitSymbolMicro m}^{-1}\)) did the studied parameter visibly drop (Figure 1A). Because of the lack of visible signs of cytotoxicity of the S1-S3 samples towards the cells after 24 h of incubation, we extended the exposure time to 72 h. The prolonged contact of the epithelial cells with the tested samples clearly revealed the decrease in the proliferation of Caco-2 cells in the presence of the S1-S3 samples (Figure 1A vs. Figure 1B). Figure 1: Effect of the tested substances (S1–S4) on the proliferation of Caco-2 cells after 24-h (**A**) and 72-h (**B**) exposures measured with the XTT assay. Ctr neg—negative control, Ctr pos—positive control (mean \(\pm\) SD, * significantly different from the negative control at \(p\) < 0.05). The 72h-IC\({}_{50}\) values are given in Table 1. The lowest cytotoxicity, with an IC\({}_{50}\) of above 1 mg mL\({}^{-1}\), was observed for the S1 sample. This value was several magnitudes higher than the concentrations obtained in natural conditions in human intestines [3]. At the same time, the impact of S2 and S3 was stronger, with IC\({}_{50}\) values of 343.7 and 353.6 ug mL\({}^{-1}\), respectively. The highest cytotoxicity was observed in case of S4, where 72h-IC\({}_{50}\) was estimated at 194.7 ug mL\({}^{-1}\). These differences most likely reflect discrepancies in the composition of the tested samples, as manufacturers use different technologies to produce the substances, which results in significant differences in the final formulations. Likewise, the quantitative determination of ATP (adenosine triphosphate), based on the number of metabolically active cells, revealed that exposure to the highest concentrations of the tested substances, S2 and S4, for 24 h led to a noticeable decrease in the measured parameter (Figure 2A). After 72 h of exposure, the metabolic activity of Caco-2 cells was significantly reduced at concentrations of 300 and 600 ug mL\({}^{-1}\) for all tested substances (Figure 2B). The observed decline in intracellular ATP concentration could be attributed to disruptions in cell morphology and increased cell membrane permeabil  
Figure 2: Effect of the tested substances (S1–S4) on the metabolic activity of Caco-2 cells after 24 h (**A**) and 72 h (**B**) exposures assessed by measuring cellular ATP content. Values are expressed as mean \(\pm\) SE. The asterisk represents a statistical difference at \(p<0.05\). studies of the cytosolic LDH enzyme, which is not released into the external environment under physiological conditions. While a 24-h exposure to the tested substances resulted in negligible toxicity (Figure 3A), elevated release of LDH was observed after 72 h of Caco-2 cell incubation with S1-S4 (Figure 3B). To capture the differences and complexity in the responses of individual nanoparticles to the evaluated parameters (ATP, LDH, cytokine release) after a 72-h incubation of Caco-2 cells with the tested substances S1-S4, we applied the four-parameter logistic (4 PL) model, which enables a more accurate and detailed analysis of dose-response relationships (Supplementary Materials Figures S2 and S3). This approach allowed us to extract key parameters, such as EC50 values and response amplitudes, thereby providing a comprehensive understanding of the cytotoxic effects and biological impact of the tested nanoparticles.