Ferritin-protein levels in Caco-2 cells following exposure to LM Fe(III) poly oxo-hydroxide (nano Fe), Fe(III) maltol (FeM) or Fe(II) sulphate-ascorbate (FeSO₄ + AA).

<p><b>A</b>, Ferritin-protein regulation in differentiated and undifferentiated cells. ***, <i>p</i>=0.0003. Cells were incubated for 1 h with 200 μM Fe plus a further 23 h in fresh, non-supplemented MEM to allow for ferritin formation. <b>B</b>, Phase distribution of Fe in the BSS uptake medium: i.e. fractional percentage of microparticulate (black bars), nanoparticulate (red bars) and soluble Fe (open bars) for each Fe material. Values are mean ± s.d. of 3 independent experiments. <b>C</b>, Effect of LM Fe(III) poly oxo-hydroxide particle dispersion (in BSS medium, closed bars) or agglomeration (in MEM medium, open bars) on ferritin-protein levels in differentiated cells: the LM Fe(III) poly oxo-hydroxide was dispersed in its nano-form (99 ± 2% nano) using BSS or agglomerated (97 ± 2% microparticulate) using MEM. Data are mean of 3 independent experiments (each experiment with 3 replicate wells). FeM: soluble iron control, Fe(III) maltol. ***, <i>p</i>=0.0002 for the comparison between BSS and MEM. <b>D</b>, TEER changes in differentiated Caco-2 cell monolayer at different time points during incubation with BSS supplemented with LM Fe(III) poly oxo-hydroxide (open circles) or non-supplemented BSS control (closed inverted triangles). Incubations were for 3 h with 200 μM Fe (measurements at 1, 2 & 3 h) plus a further 21 h in fresh, non-supplemented MEM (24-h). Values are expressed as a percentage of the initial measurement and are shown as mean ± s.d. of 3 independent experiments (each experiment with 3 replicate wells). Experimental points are connected with a solid line to aid visualization and not because a linear relationship is assumed between time and TEER measurement. Detailed methodology is available in the Methods Section and in Methods S1.</p

Lysososmal dissolution of LM Fe(III) poly oxo-hydroxide.

<p><b>A</b>, Solubility in simulated lysosomal conditions at pH 5.0 with 10 mM citric acid and 0.15 M NaCl. Soluble Fe was measured by ICP-OES following 5 min ultrafiltration (3000 Da MWCO) for the LM Fe(III) poly oxo-hydroxide (black) and for un-modified Fe(III) poly oxo-hydroxide (solid blue). Nanoparticulate Fe was obtained from the Fe in the supernatant following centrifugation excluding the soluble (ultrafilterable) Fe, and is shown for LM Fe(III) poly oxo-hydroxide (red) and for un-modified Fe(III) poly oxo-hydroxide (dotted blue). Values are plotted as mean ± s.d. of 3 independent experiments (each experiment with 3 replicates). <b>B</b>, Effect of inhibition of lysosomal acidification using monensin on Fe utilization by differentiated Caco-2 cells. Data are shown as a percentage of the control (without monensin) at 24 h: i.e. 1 h exposure to 200 µM nanoparticulate LM Fe(III) poly oxo-hydroxide (open circles) or Fe(III) maltol (closed squares) ± 5-30 µM monensin followed by 23 h in non-supplemented MEM. Results are means ± s.d. of 3 independent experiments (each experiment with 3 replicate wells). **, <i>p</i><0.005; ***, <i>p</i><0.001 in relation to the soluble Fe control (Fe(III)maltol). <b>C</b>, Change in TEER in the Caco-2 cell monolayer following 1 h exposure to 10 μM monensin (closed squares), 30 μM monensin (open diamonds) or non-supplemented BSS control (closed inverted triangles) and with 23 h further incubation in fresh MEM (24 h in total). Values are expressed as a percentage of the initial measurement at the start of the exposure time (corresponding to 0 h) and are shown as mean ± s.d. of 2 independent experiments (each experiment with 3 replicate wells). Experimental points are connected with a solid line to aid visualization and not because a linear relationship is assumed between time and TEER measurement. ***, <i>p</i>=0.0003; ****, <i>p</i><0.0001 in relation to the non-supplemented BSS control.</p

Characterisation of hydrolysed Fe(III) with simulated digestion and of aquated LM Fe(III) poly oxo-hydroxide.

<p><b>A</b>, Transmission Electron Microscopy (TEM) images collected from a drop of suspension after simulated digestion of 1 mM Fe(III) chloride in the presence of 2 g/L mucin and low molecular weight ligands. The boxed regions are shown magnified below and highlight the presence of fine, poorly crystalline nanoparticles dispersed in an amorphous gel. Crystallinity is indicated by the spots in the inset diffractograms (fast Fourier transforms) in the boxed regions and lattice spacings are discussed in the main text. Scale bar represents 5 nm. <b>B</b>, Whole area EDX analysis of a particle agglomerate similar to those in ‘A’ shows elemental compositions (the specimen support film and grid produce the background C and Cu signals respectively). <b>C</b>, Hydrodynamic size distribution of nanoparticulate 500 µM LM Fe(III) poly oxo-hydroxide in balanced salt solution (BSS) measured by Dynamic Light Scattering (DLS). Values are expressed as mean diameter ± s.d. (3 independent measurements) on a log₁₀ scale.</p

Caco-2 cell acquisition of dietary iron(III) invokes a nanoparticulate endocytic pathway

Dietary non-heme iron contains ferrous [Fe(II)] and ferric [Fe(III)] iron fractions and the latter should hydrolyze, forming Fe(III) oxo-hydroxide particles, on passing from the acidic stomach to less acidic duodenum. Using conditions to mimic the in vivo hydrolytic environment we confirmed the formation of nanodisperse fine ferrihydrite- like particles. Synthetic analogues of these (~ 10 nm hydrodynamic diameter) were readily adherent to the cell membrane of differentiated Caco-2 cells and internalization was visualized using transmission electron microscopy. Moreover, Caco-2 exposure to these nanoparticles led to ferritin formation (i.e., iron utilization) by the cells, which, unlike for soluble forms of iron, was reduced ( p =0.02) by inhibition of clathrin-mediated endocytosis. Simulated lysosomal digestion indicated that the nanoparticles are readily dissolved under mildly acidic conditions with the lysosomal ligand, citrate. This was confirmed in cell culture as monensin inhibited Caco-2 utilization of iron from this source in a dose dependent fashion ( p <0.05) whilet soluble iron was again unaffected. Our findings reveal the possibility of an endocytic pathway for acquisition of dietary Fe(III) by the small intestinal epithelium, which would complement the established DMT-1 pathway for soluble Fe(II