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Iron deficiency anaemia (IDA), the most widespread nutritional disorder, is a persistent global health issue affecting millions, especially in resource-limited geographies. Oral iron supplementation is usually the first choice for exogenous iron administration owing to its convenience, effectiveness and low cost. However, commercially available iron supplementations are often associated with oxidative stress, gastrointestinal side effects, infections and solubility issues. Herein, we aim to address these limitations by employing ferritin proteins—self-assembled nanocaged architectures functioning as a soluble cellular iron repository—as a non-toxic and biocompatible alternative. Our in vitro studies based on PAGE and TEM indicate that bare ferritin proteins are resistant to gastric conditions but their cage integrity is compromised under longer incubation periods and at higher concentrations of pepsin, which is a critical component of gastric juice. To ensure the safe delivery of encapsulated iron cargo, with minimal cage disintegration/degradation and iron leakage along the gastrointestinal tract, we fabricated the surface of ferritin with chitosan. Further, the stoichiometry and absorptivity of iron-chelator complexes at both gastric and circumneutral pH were estimated using Job's plot. Unlike bipyridyl, deferiprone exhibited pH dependency. In vitro kinetics was studied to evaluate iron release from bare and chitosan-fabricated ferritins employing both reductive (in the presence of ascorbate and bipyridyl) and non-reductive (direct chelation by deferiprone) pathways to determine their bio-mineral stabilities. Chitosan-decorated ferritin displayed superior cage integrity and iron retention capability over bare ferritin in simulated gastric fluid. The ability of ferritins to naturally facilitate controlled iron release in conjugation with enteric coating provided by chitosan may mitigate the aforementioned side effects and enhance iron absorption in the intestine. The results of the current study could pave the way for the development of an oral formulation based on ferritin-caged iron bio-mineral that can be a promising alternative for the treatment of IDA, offering better therapeutic outcomes. [ABSTRACT FROM AUTHOR]

The self-assembled ferritin nanocages, nature's solution to iron toxicity and its low solubility, scavenge free iron to synthesize hydrated ferric oxyhydroxide mineral inside their central cavity by protein-mediated ferroxidase and hydrolytic/nucleation reactions. These complex processes in ferritin commence with the rapid influx of Fe 2+ ions via the inter-subunit contact points (i.e., pores/channels). Investigation of these pores as Fe 2+ uptake routes in ferritins remains a subject of intense research, in iron metabolism, toxicity, and bacterial pathogenesis, which are yet to be established in the bacterioferritin (BfrA) from Mycobacterium tuberculosis ( Mtb ). The electrostatic properties of this protein indicate that the 4-fold and B-pores might serve as potential Fe 2+ entry routes. Therefore, in the current work, electrostatics at/along these pores was altered by site-directed mutagenesis to establish their role in Fe 2+ uptake/oxidation (ferroxidase activity) in Mtb BfrA. Despite forming self-assembled protein nanocompartment, these 4-fold and B-pore variants exhibited partial loss of ferroxidase activity and lower accumulation of transient species, which not only indicated their role in Fe 2+ entry but also suggested the existence of multiple pathways. Although the B-pore variants inhibited the rapid ferroxidase activity to a larger extent, they had minimal impact on their cage stability. The current work revealed the relative contribution of these pores toward rapid Fe 2+ uptake/oxidation and cage stability, possibly as consequences of their differential symmetry, number of modified residues (at each pore), and heme content. Therefore, these findings may help to understand the role of these pores in iron acquisition and Mtb proliferation under iron-limiting conditions to control its pathogenesis.