Article pubs.acs.org/molecularpharmaceutics
Iron Oxide/Hydroxide Nanoparticles with Negatively Charged Shells Show Increased Uptake in Caco-2 Cells Markus R. Jahn,† Thomas Nawroth,† Sören Fütterer,† Uwe Wolfrum,‡ Ute Kolb,§ and Peter Langguth*,† †
Department of Biopharmacy and Pharmaceutical Technology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Mainz, Germany ‡ Department of Cell and Matrix Biology, Institute of Zoology, Johannes Gutenberg University, Mainz, Germany § Institute of Physical Chemistry, Johannes Gutenberg University, Mainz, Germany ABSTRACT: The absorption of commonly used ferrous iron salts from intestinal segments at neutral to slightly alkaline pH is low, mainly because soluble ferrous iron is easily oxidized to poorly soluble ferric iron and because ferrous iron, but not ferric iron, is carried by the divalent metal transporter DMT-1. Moreover, ferrous iron frequently causes gastrointestinal side effects. Iron hydroxide nanoparticles with neutral and hydrophilic carbohydrate shells are alternatively used to ferrous salts. In these formulations gastrointestinal side effects are rare because hundreds of ferric iron atoms are safely packed in nanoscaled cores surrounded by the solubilizing shell; nevertheless, iron bioavailability is even worse compared to ferrous salts. In this study the cell uptake of iron hydroxide and iron oxide nanoparticles (FeONP) with negatively charged shells of different chemical types and sizes was compared to the uptake of those with neutral hydrophilic shells, ferrous sulfate and ferric chloride. The nanoparticle uptake was measured in Caco-2 cells with the iron detecting ferrozine method and visualized by transmission electron microscopy. The toxicity was evaluated using the MTT assay. For nanoparticles with a negatively charged shell the iron uptake was about 40 times higher compared to those with neutral hydrophilic carbohydrate shell or ferric chloride and in the same range as ferrous sulfate. However, in contrast to ferrous sulfate, nanoparticles with negatively charged shells showed no toxicity. Two different uptake mechanisms were proposed: diffusion for hydroxide nanoparticles with neutral hydrophilic shell and adsorptive endocytosis for nanoparticles with negatively charged shells. It needs to be determined whether iron hydroxide nanoparticles with negatively charged shells also show improved bioavailability in iron-deficient patients compared to iron hydroxide nanoparticles with a neutral hydrophilic shell, which exist in the market today. KEYWORDS: absorption, Caco-2 cells, iron oxide/hydroxide nanoparticles, iron deficiency, oral delivery
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radicals and to cause oxidative stress: •O2− + H2O2 → •OH + HO− + O2 (Haber-Weiss reaction). On the contrary, in the neutral and basic environment of the small intestine, soluble ferrous iron is easily oxidized to poorly soluble ferric iron. Therefore uptake via the divalent metal transporter DMT-1, the only transporter for iron molecularly identified so far, is limited (Figure 1). To improve the low tolerability and poor bioavailability of ferrous salts, different approaches have been pursued so far. For example, enteric coated dosage forms were developed primary releasing ferrous iron in the duodenum and therefore resulting in improved gastric tolerability. In another approach heme iron, which is a protoporphyrine with ferrous iron, allowed for significantly increased iron absorption taken with a meal when compared to ferrous salts.7
INTRODUCTION Although the therapy of iron deficiency with oral ferrous salts has widespread use, the bioavailability of iron from such salts is insufficient.1 Nutritional components, for example, phytate in whole grain and bran, oxalic acid in spinach, phosphates in practically all foods, or tannins from black tea and coffee may decrease the absorption of ferrous iron by forming insoluble complexes.2,3 But also drugs such as tetracyclines, gyrase inhibitors, levodopa, methyldopa, and antacids reduce the bioavailability of ferrous iron.4 The gastrointestinal side effects of ferrous salts as a consequence of oxidative stress are wellknown.4,5 In the acidic environment of the stomach ferrous salts are dissolved. Ferrous iron with weakly coordinating anions, such as FeSO4 or ferrous gluconate, represents a source of hydrogenated “free” ferrous cations, known to cause toxicity.6 In the Fenton reaction Fe2+ + H2O2 → Fe3+ + •OH + OH− ferrous iron reacts with hydrogen peroxide to form hydroxyl radicals. These radicals are highly reactive and oxidize DNA, proteins, carbohydrates, and lipids. Ferric iron may become reduced to ferrous iron: Fe3+ + •O2− → Fe2+ + O2. Catalytic amounts of iron are adequate to generate hydroxyl © 2012 American Chemical Society
Received: Revised: Accepted: Published: 1628
December 8, 2011 April 4, 2012 April 30, 2012 May 16, 2012 dx.doi.org/10.1021/mp200628u | Mol. Pharmaceutics 2012, 9, 1628−1637
Molecular Pharmaceutics
Article
Figure 1. Fate of ionic iron and FeONPs in the duodenum and possibilities for cellular uptake. For details see text. AscH2: ascorbic acid; Asc: oxidized ascorbic acid; DMT-1: divalent metal transporter 1.
nanoparticles with enterocyte membranes: One is (iv) modifying the nanoparticles with specific ligands for receptormediated endocytosis. The second approach for enhanced uptake is (iii) modifying their physicochemical characteristics for adsorptive endocytosis; in particular charge can enhance adsorption and uptake, probably via electrostatic interaction. In the present investigation the enteric uptake of FeONP with negatively charged shells in comparison to commonly used FeONP with neutral hydrophilic shells and iron salts is detailed. In addition, the effect of shell size was investigated. The uptake was studied in Caco-2 cell monolayers, which have enterocytelike characteristics as microvilli, tight junctions, and duodenal transport systems (e.g., DMT-126 or heme transport system27). The cell line is also established as an in vitro model for the uptake of nanoparticles28 as well as iron salts.29 Iron uptake was measured by the ferrozine method and visualized by transmission electron microscopy.
Iron oxide/hydroxide nanoparticles (FeONP) are an interesting alternative to ferrous salts. In theses formulations hundreds of ferric iron atoms are safely packed into nanoscaled iron oxide/hydroxide cores,8,9 resulting in less oxidative stress and less side effects, while a carbohydrate shell prevents the cores from precipitating. The iron(III)-hydroxide polymaltose complex FeONP_PM (Ferrum Hausmann Sirup, Vifor, München, Germany and Maltofer, Vifor St. Gallen, Switzerland) contains an iron(III)-hydroxide core surrounded by a polymaltose shell and has been used in Europe for more than 25 years.10 Unlike ordinary ferrous salts FeONP_PM is supposed to be absorbed by diffusion through the brush border11 and is absorbed even better when taken concomitantly with food12 and interactions of FeONP_PM with a wide range of drugs are held off.13−15 In a recently published meta-analysis of studies with iron-deficient adults, FeONP_PM has been shown to achieve similar hemoglobin levels compared to FeSO4 but is better tolerated.16 Nevertheless, doubts remain regarding the bioavailability of FeONP_PM. For example, 14 days after administration in 17 healthy men, 0.81% of the 59Fe labeled iron dose of FeONP_PM was withheld by the body, compared to 8% of a 59 Fe labeled solution of ferrous ascorbate.1 Therefore the supposed diffusion of FeONP_PM into enterocytes seems not to be very effective. According to Figure 1, the uptake of nanoparticles via the gastrointestinal mucosa can take place by (i) paracellular passageas reported for particles 20% toxic (MTT formazan generation
