A nano-disperse ferritin-core mimetic that efficiently corrects anemia without luminal iron redox activity

Abstract

The 2-5 nm Fe(III) oxo-hydroxide core of ferritin is less ordered and readily bioavailable compared to its pure synthetic analogue, ferrihydrite. We report the facile synthesis of tartrate-modified, nano-disperse ferrihydrite of small primary particle size, but with enlarged or strained lattice structure (~2.7Å for the main Bragg peak versus 2.6Å for synthetic ferrihydrite). Analysis indicated that co-precipitation conditions can be achieved for tartrate inclusion into the developing ferrihydrite particles, retarding both growth and crystallization and favoring stabilization of the cross-linked polymeric structure. In murine models, gastrointestinal uptake was independent of luminal Fe(III) reduction to Fe(II) and, yet, absorption was equivalent to that of ferrous sulphate, efficiently correcting the induced anemia. This process may model dietary Fe(III) absorption and potentially provide a side effect-free form of cheap supplemental iron. From the clinical editor: Small size tartrate-modified, nano-disperse ferrihydrite was used for efficient gastrointestinal delivery of soluble Fe(III) without the risk for free radical generation in murine models. This method may provide a potentially side effect-free form iron supplementation.

Keywords: Bioavailability; Ferrihydrite; Iron deficiency anemia; Iron oxide; Oral iron.

Graphical abstract

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Figure 1

Formation of tartrate-modified ferrihydrite in adipate buffer as a function of pH. (A) Dispersed or colloidal (i.e. nanoparticulate) iron, determined following centrifugation and ultrafiltration to remove soluble iron (main panel), and precipitated (i.e. agglomerated) iron, determined following centrifugation (inset). Closed squares show synthetic ferrihydrite precipitated from an Fe(III) chloride solution; open triangles show modified ferrihydrite precipitated from an Fe(III) chloride solution in the presence of sodium tartrate and adipate buffer (Fe/tartrate/adipate = 1:0.5:0.5); closed circles and open diamonds show ferrihydrite precipitated from an Fe(III) chloride solution in the presence of adipate alone (Fe/adipate = 1:0.5 and 1:1 respectively). (B) Percentage of nanoparticulate iron (main panel) and precipitated iron (inset) for the synthetic (closed squares) and tartrate-modified (open triangles) ferrihydrite materials (molar ratios as above) re-suspended in the original volume of aqueous solution. (A and B) All values are expressed as a percentage of total iron in the initial solution as described in Methods. (C) Simulated gastric dissolution at pH 1.0 of dried synthetic ferrihydrite (closed squares) and tartrate-modified ferrihydrite that had been precipitated in the presence of tartrate and adipate (open triangles), at molar ratios as above, and then dried. Data were obtained following 5-min ultrafiltration (3000 Da MWCO); data are mean ± SD, n = 3.

Figure 2

Structural characterization of the ferrihydrite. (A-D), High-angle annular dark-field, aberration-corrected, scanning transmission electron microscopy (HAADF-STEM) images of tartrate-modified ferrihydrite in tryptophan (A, B) or adipate buffer (C), and of synthetic (unmodified) ferrihydrite (D). Scale bars are 50 nm (A and C) or 2 nm (B and D). (E) X-ray diffraction patterns of synthetic ferrihydrite (black) and tartrate-modified ferrihydrite (red). Both patterns have peaks characteristic of ferrihydrite, with the synthetic ferrihydrite peaks centred at 35° and 62° 2θ (2.6 Å and 1.5 Å, respectively) and the tartrate-modified ferrihydrite peaks centered at 33° and 61° 2θ (2.7 Å and 1.5 Å, respectively), indicating expansion of the ferrihydrite lattice. Sodium tartrate is shown in green. Synthetic ferrihydrite was precipitated from an Fe(III) chloride solution and tartrate-modified ferrihydrite from an Fe(III) chloride solution in the presence of tartrate and tryptophan buffer (Fe/tartrate/tryptophan = 1:0.5:0.375). Excess ligand and buffer were removed from the modified material by ultrafiltration and washing prior to drying as described in Methods.

Figure 3

Fourier transform infrared (FTIR) spectroscopy of the ferrihydrite. Synthetic (unmodified) ferrihydrite is shown in black, tryptophan in blue, sodium tartrate in green and the tartrate-modified ferrihydrite is in red. Unique tartrate but no tryptophan signatures are revealed in the modified material. Synthetic and tartrate-modified ferrihydrite were prepared as per Figure 2.

Figure 4

Electron microscopy-based characterization of tartrate-modified ferrihydrite (red) against synthetic ferrihydrite (black). (A) Background-stripped electron energy-loss spectra (EELS) for the combined C K-edges (285 eV), Ca L_2,3-edges (346 eV), O K-edges (530 eV) and Fe L_2,3-edges (709.5 eV) normalized to the continuum intensity post the Fe L_2,3-edges. (B) individual background-stripped Fe L_2,3-edges. The unaltered shape of the Fe L_2,3-edges indicates that the Fe−(O, OH)₆ octahedra remain relatively unchanged after tartrate modification: i.e. the material remains Fe(III). (C and D) Bright-field transmission electron microscopy (TEM) images of synthetic ferrihydrite (C) and tartrate-modified ferrihydrite (D) agglomerates suspended over holes in the TEM support film (see supplementary methods for details of specimen preparation). Diffraction contrast from individual crystallites produces dark regions in the images, the size of which confirms the reduced crystallite size of the modified material. Synthetic ferrihydrite was precipitated from an Fe(III) chloride solution and tartrate-modified ferrihydrite from an Fe(III) chloride solution in the presence of tartrate and adipate buffer (Fe/tartrate/adipate = 1:0.5:0.5).

Figure 5

Comparative iron absorption and bioavailability of ligand-modified ferrihydrite. (A) Effect of ferrozine on the absorption of soluble ferric iron (i.e. Fe(III) NTA) and nanoparticulate, tartrate-modified ferrihydrite (i.e. LM-Fh) in male CD1 mice. Mice were orally gavaged with ⁵⁹Fe-labeled compounds with (open bars) or without (closed bars) ferrozine treatment as detailed in Methods. Values are mean ± SD, n = 7 per group. ***P = 0.0009; ****P < 0.0001; other comparisons are not significantly different. (B) Hemoglobin levels of anemic Sprague–Dawley male rats following 2-week treatment with diets fortified with the different iron compounds: No Fe = control diet with no supplemental iron (3.1 ± 0.6 mg Fe/kg_diet); Fh = synthetic ferrihydrite (35.7 ± 0.1 mg Fe/kg_diet); LM-Fh = tartrate-modified ferrihydrite (31.4 ± 0.5 mg Fe/kg_diet); FeSO₄ = Fe(II) sulphate (35.8 ± 0.1 mg Fe/kg_diet). Tartrate-modified ferrihydrite was precipitated from an Fe(III) chloride solution in the presence of tartrate and adipate buffer (Fe/tartrate/adipate = 1:0.5:0.5). Means with a common letter are not statistically different from one another, and means labeled with **** are statistically different from the control diet (No Fe), P < 0.0001, n = 8 per group.