Sulfide silver autometallography to differentiate the ultrastructural localization of iron-carbohydrate complexes inside macrophages

Abstract

Iron is an essential element for numerous physiological processes in the human body, and maintaining proper iron homeostasis is critical for health. Iron imbalance can lead to conditions like iron deficiency anemia (IDA). Intravenous (IV) iron drugs, including iron-carbohydrate complexes such as iron sucrose (IS) and ferric carboxymaltose (FCM), are commonly used in the treatment of IDA. However, the cellular mechanisms underlying the uptake and intracellular fate of these complexes remain poorly understood despite over seven decades of clinical use. This study introduces a novel application of sulfide silver autometallography (ssAMG) to track iron inside macrophages treated with two widely used intravenous iron products, IS and FCM. Using this technique, the ultrastructural localization of iron in macrophages was visualized in comparison to ferric ion (Fe³⁺). ssAMG improved the visualization of IS and FCM, revealed as crystalline, cluster-like particles, i.e. silver precipitates, localized inside intracellular vesicles. Interestingly, for both iron-carbohydrate complexes, the silver precipitates were observed exclusively inside the cells and not on the cell surface as seen for ferric ion. These results suggest that IS and FCM are not internalized by macrophages as ferric iron bound to transferrin and provide new insights into the cellular processing of iron-carbohydrate complexes to advance the understanding of iron homeostasis.

Keywords: Biodistribution; Drug delivery; Electron microscopy; Ferric carboxymaltose; Iron sucrose; Sulfide silver autometallography.

Fig. 1

Detection of iron in macrophages using sulfide silver autometallography. (A) Scheme of workflow including three main steps: iron exposure, in which the cells (J774A.1) are subjected to a treatment with iron solution; sulfidation, in which the cells are incubated in a sulfur solution allowing iron sulfide (Fe–S) to form; and silver precipitation, in which silver atoms (Ag) reduced from silver ions (Ag+) accumulate on Fe–S AMG nanocrystals. Gradient staining of J774A.1 cells treated with increasing concentrations of iron using ssAMG (B) and Prussian blue (C).

Fig. 2

Visualization and ultrastructural localization of ferric iron in macrophages. (A) Macrophages treated with ferric iron and imaged using ssAMG (first column on the left) showed high-contrast silver precipitates (black clusters), whereas the three other controls did not. Representative whole-cell images are shown on the top row, and each corresponding zoom-in is in the bottom row. (B) ssAMG significantly improves the visualization of iron and shows two ultrastructural localizations of iron inside cellular vesicles or at the cellular surface (bottom row (+)). “Mag.” stands for “Magnification”.

Fig. 3

Representative whole-cell images of macrophages taken iron up from different sources. (Left to right) Representative images of macrophages treated with 120 µM of ferric iron (Fe³⁺), lower dose of IS (0.1 mg/ml), and higher dose of FCM (1 mg/ml).

Fig. 4

Visualization and ultrastructural localization of IS. Compared to the control of macrophages treated with the lower dose (0.1 mg/ml) of IS (A), the use of ssAMG (B) clearly revealed the presence of iron indicated by high-contrast silver precipitates (black clusters) at the subcellular level. “Mag.” stands for “Magnification”.

Fig. 5

Visualization and ultrastructural localization of FCM. Compared to the control of macrophages treated with the higher dose (1 mg/ml) of FCM (A), the use of ssAMG (B) clearly revealed the presence of iron indicated by high-contrast silver precipitates (black clusters) at the subcellular level. “Mag.” stands for “Magnification”.