Beyond Iron Solubility: Particle Size as a Determinant of Cell Survival and Iron-Induced COX-2 Expression in Human Intestinal Cells

<b>Background:</b> Oral iron supplementation or food fortification is essential for managing or preventing iron deficiency but often causes gastrointestinal side effects. While solubility has traditionally been considered a requirement for iron uptake via the DMT1 transporter, recent evidence shows that insoluble iron can also be absorbed through endocytosis, raising questions about particle size and epithelial responses. <b>Methods:</b> Human intestinal cell lines (Hutu-80 and Caco-2) were exposed to physiologically relevant but elevated iron levels (0.5 mM Fe, 48 h) as ferric pyrophosphate, ferrous fumarate (both prone to precipitation), and soluble ferric EDTA. Cell survival and COX-2 protein were quantified by ELISA, solubility by ICP-OES, and particle size in cell culture medium by dynamic light scattering analyses. <b>Results:</b> Ferric pyrophosphate (0.62-3.8 μm) markedly increased COX-2 expression in Hutu-80 cells (254% ± 37%, <i>n</i> = 3, <i>p</i> = 4.11 × 10^-5) and in Caco-2 cells (78% ± 8%, <i>n</i> = 3, <i>p</i> = 0.01) compared to the control. Ferrous fumarate (237-866 nm) also induced COX-2, but only in Hutu-80 cells (62% ± 11%, <i>n</i> = 3, <i>p</i> = 0.04), whereas ferric EDTA showed no effect in either cell line. COX-2 induction was associated with larger particles in the medium (≥237 nm), whereas smaller particles (<146 nm) were not. <b>Conclusions:</b> Particle size appears to be a critical determinant of cell survival and iron-induced epithelial COX-2 expression. Iron compounds that present as both soluble and particulate forms may optimize bioavailability, but controlling aggregate size (<146 nm) could reduce inflammatory signaling. These findings may have important implications for cell culture systems and warrant <i>in vivo</i> validation in iron supplemental studies.