This study investigates the impact of exposure to human blood plasma on the physicochemical properties of two widely used intravenous (IV) iron-carbohydrate nanomedicines for treating iron deficiency and iron deficiency anaemia, ferric carboxymaltose and iron sucrose, with a particular focus on their colloidal stability and protein corona formation. Using dynamic light scattering (DLS), differential centrifugal sedimentation (DCS), size-exclusion chromatography (SEC), inductively coupled plasma mass spectrometry (ICP-MS), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and density gradient ultracentrifugation (DCS), we compared both iron-carbohydrate nanomedicines before and after plasma exposure. Both products exhibited minimal degradation and negligible protein adsorption and good colloidal stability also after exposure to blood plasma for up to 24 h. However, more pronounced changes in the surface properties of the iron sucrose NPs were observed suggesting rearrangement of the NPs' surface. These findings suggest that the high colloidal stability and low protein interaction of ferric carboxymaltose may contribute to its more sustained pharmacokinetic profile.

Intravenous iron-carbohydrate complexes are widely used nanomedicines for the treatment of iron deficiency anaemia, particularly in patients with conditions like chronic kidney disease, heart failure and inflammatory diseases. Despite the abundance of physicochemical characterization and clinical studies for these products, a clear evidence-based correlation between physicochemical properties and clinical outcome of iron-carbohydrate complexes is yet to be established. There is, nonetheless, clear evidence that the nano-bio interface determines the bio-response and mode of action. Here, the early interactions between iron-carbohydrate nanomedicines and blood components are investigated using time-resolved small-angle X-ray and neutron scattering (SAXS and SANS, respectively) under flow conditions enabled by a microfluidic device. Two clinically relevant iron-carbohydrate complexes with different carbohydrate ligand morphologies, iron sucrose (IS) and ferric carboxymaltose (FCM), were studied for their early interactions (down to minutes) with human serum albumin (HSA) and human blood serum (HBS). When mixed with HSA, IS showed rapid agglomeration behavior at the nanoscale, as evidenced by a characteristic up-turn in the low-q region of the scattering curves, whereas FCM showed much slower agglomeration. With HBS, IS displayed a similar agglomeration behavior to that observed with HSA. In contrast, FCM showed increasingly repulsive interactions amongst its clusters in blood serum, reflected by a characteristic down-turn in the low-q scattering, associated with improved colloidal stability. These results demonstrate that early nano-bio interactions are strongly formulation-dependent and governed by differences in the carbohydrate shell architecture. These findings provide a physicochemical framework for the design of future cell-based and mechanistic studies aimed at bridging physicochemical properties with clinical outcomes.

Iron-carbohydrate complexes (ICCs) are widely used nanomedicines to treat iron deficiency anemia, yet their intracellular fate and the mechanisms of action underlying their differences in treatment outcomes remain poorly understood. Here, we thus performed a comprehensive dynamic characterization of two structurally distinct ICCs - iron sucrose (IS) and ferric carboxymaltose (FCM) - in primary human macrophages, key cells to the iron metabolism. By employing innovative correlative microscopy techniques, elemental analysis, and in vitro pharmacokinetic profiling, we demonstrate that the uptake, intracellular trafficking, and biodegradation of ICCs depend on their physicochemical properties. Specifically, IS is rapidly internalized and processed within endolysosomes, resulting in fast iron release and transient cytotoxicity. Conversely, FCM is sequestered in enlarged endosomes for an extended time before its biodegradation, a phenomenon we term the Hamster Effect, which leads to slower, more sustainable iron release. These results provide unprecedented insights into the metabolic fate of ICCs, enhancing our understanding of their different pharmacokinetic and pharmacodynamic profiles in vivo.

Intravenous iron drugs are commonly used in the treatment of iron deficiency/iron deficiency anaemia. However, the cellular mechanisms underlying the uptake of these complexes remain poorly understood. This study examines the interaction of two iron complexes, iron sucrose and ferric-carboxymaltose, with murine J774A.1 and human M2a macrophages, focusing on their uptake and localization within lysosomal compartments at various time points: 45 min, 6 h, 24 h, and 5 days. We employed multiple analytical methods, including Prussian blue staining and transmission electron microscopy, to assess the intracellular iron complexes. In addition, the stability of the two complexes in cell culture medium and artificial lysosomal fluid was assessed by dynamic light scattering and transmission electron microscopy. A formation of larger aggregates for both complexes in cell culture medium was observed, likely due to interactions with serum proteins. The analysis in artificial lysosomal fluid revealed a slight, but not statistically significant, decrease in hydrodynamic diameter. Upon interaction with macrophages, our results demonstrate that iron sucrose is internalized more rapidly by both macrophage cell types compared to ferric carboxymaltose. Furthermore, the detection of ferric ions within intracellular and intralysosomal compartments occurs at a later time point following macrophage exposure to ferric carboxymaltose, suggesting slower internalization in comparison to iron sucrose. Our findings suggest that the two complexes remain intact upon reaching macrophages and, after internalization, are localized within intracellular vesicles, indicating endocytosis as the primary uptake mechanism. Iron sucrose was internalized more rapidly in comparison to ferric carboxymaltose by both macrophage types, whereas a decrease in metabolic activity was only observed for the J774A.1 macrophages in the presence of high iron sucrose concentration, i.e., 1mg/mL iron. These findings provide new insights into the dynamics of different iron-carbohydrate complexes on cellular uptake and may contribute to optimizing future drug designs.

Intravenous iron–carbohydrate complexes are a class of nanomedicines that are widely used globally to treat iron deficiency and iron deficiency anemia associated with a wide spectrum of disease states. Despite being widely used in clinical practice for more than seven decades, the understanding of their in vivo disposition including tissue biodistribution and kinetics of the nanoparticle degradation at the cellular level is not well-understood. Moreover, the critical quality attributes that influence in vivo pharmacokinetics have not been fully defined. In particular, the carbohydrate moiety plays an influential role in how the nanoparticulate iron–carbohydrate complex interacts with the biological system. Developing a physiologically based pharmacokinetic (PBPK) model would facilitate a deeper understating of the key nanomedicine attributes that predict in vivo performance. Because endogenous iron metabolism complicates pharmacokinetic modeling for this complex class of drugs, models of gold nanoparticles may provide a substantive roadmap to begin to build a viable PBPK model for iron–carbohydrate nanomedicines. In the future, PBPK models that integrate recent mechanistic data regarding tissue biodistribution and intracellular iron kinetics for parameterization have the potential to improve manufacturing quality and clinical use of these complex drugs.

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.

Here, we developed IronFist, a genetically encoded fluorescent reporter that enables dynamic tracking of labile ferrous ions (Fe2+) in live cells. IronFist is a bicistronic system combining the iron-sensitive hemerythrin-like (Hr) domain from the F-box and leucine-rich repeat protein 5 (FBXL5), fused to the bright fluorescent protein (FP) mNeonGreen, alongside mCherry as a reference FP signal. When labile iron levels are low, Hr-mNeonGreen undergoes ubiquitination and degradation, leading to a low green-to-red fluorescence ratio. Conversely, as cytosolic Fe2+ levels rise and Fe2+ ions bind to Hr, the green fluorescence is stabilized, increasing the IronFist ratio signal. Using IronFist for end point measurements, we observed that most cells maintain low basal labile iron levels. However, upon treatment with iron(II) sulfate or iron carbohydrate nanoparticles, we detected significant elevations in the cellular labile iron pool (LIP). Cells responded faster and more strongly to iron(II) sulfate, whereas responses to iron carbohydrate nanoparticles were slower and weaker. Time-lapse imaging further revealed substantial cell-to-cell heterogeneity in iron handling. We conclude that IronFist fills a critical methodological gap in assessing cellular iron homeostasis and related pathologies by enabling high-content tracking of iron dynamics at the single-cell level.

Intravenous (IV) iron-carbohydrate complexes are widely used nanoparticles (NPs) to treat iron deficiency anaemia, often associated with medical conditions such as chronic kidney disease, heart failure and various inflammatory conditions. Even though a plethora of physicochemical characterisation data and clinical studies are available for these products, evidence-based correlation between physicochemical properties of iron-carbohydrate complexes and clinical outcome has not fully been elucidated yet. Studies on other metal oxide NPs suggest that early interactions between NPs and blood upon IV injection are key to understanding how differences in physicochemical characteristics of iron-carbohydrate complexes cause variance in clinical outcomes. We therefore investigated the core-ligand structure of two clinically relevant iron-carbohydrate complexes, iron sucrose (IS) and ferric carboxymaltose (FCM), and their interactions with two structurally different human plasma proteins, human serum albumin (HSA) and fibrinogen, using a combination of cryo-scanning transmission electron microscopy (cryo-STEM), x-ray diffraction (XRD), small-angle x-ray scattering (SAXS) and small-angle neutron scattering (SANS). Using this orthogonal approach, we defined the nano-structure, individual building blocks and surface morphology for IS and FCM. Importantly, we revealed significant differences in the surface morphology of the iron-carbohydrate complexes. FCM shows a localised carbohydrate shell around its core, in contrast to IS, which is characterised by a diffuse and dynamic layer of carbohydrate ligand surrounding its core. We hypothesised that such differences in carbohydrate morphology determine the interaction between iron-carbohydrate complexes and proteins and therefore investigated the NPs in the presence of HSA and fibrinogen. Intriguingly, IS showed significant interaction with HSA and fibrinogen, forming NP-protein clusters, while FCM only showed significant interaction with fibrinogen. We postulate that these differences could influence bio-response of the two formulations and their clinical outcome. In conclusion, our study provides orthogonal characterisation of two clinically relevant iron-carbohydrate complexes and first hints at their interaction behaviour with proteins in the human bloodstream, setting a prerequisite towards complete understanding of the correlation between physicochemical properties and clinical outcome.

Iron deficiency and iron deficiency anemia pose significant health challenges worldwide. Iron carbohydrate nanoparticles administered intravenously are a mainstay of treatment to deliver elemental iron safely and effectively. However, despite decades of clinical use, a complete understanding of their physical structure and the significance for their behavior, particularly at the nano-bio interface, is still lacking, underscoring the need to employ more sophisticated characterization methods. Our study used cryogenic Scanning Transmission Electron Microscopy (cryo-STEM) to examine iron carbohydrate nanoparticle morphology. This method builds upon previous research, where direct visualization of the iron cores in these complexes was achieved using cryogenic Transmission Electron Microscopy (cryo-TEM). Our study confirms that the average size of the iron cores within these nanoparticles is approximately 2 nm across all iron-based products studied. Furthermore, our investigation revealed the existence of discernible cluster-like morphologies, not only for ferumoxytol, as previously reported, but also within all the examined iron-carbohydrate products. The application of cryo-STEM for the analyses of product morphologies provides high-contrast and high-resolution images of the nanoparticles, and facilitates the characterization at liquid nitrogen temperature, thereby preserving the structural integrity of these complex samples. The findings from this study offer valuable insights into the physical structure of iron-carbohydrate nanoparticles, a crucial step towards unraveling the intricate relationship between the structure and function of this widely used drug class in treating iron deficiency. Additionally, we developed and utilized the self-supervised machine learning workflow for the image analysis of iron-carbohydrate complexes, which might be further expanded into a useful characterization tool for comparability studies.

Intravenous iron-carbohydrate nanomedicines are widely used to treat iron deficiency and iron deficiency anemia across a wide breadth of patient populations. These colloidal solutions of nanoparticles are complex drugs which inherently makes physicochemical characterization more challenging than small molecule drugs. There have been advancements in physicochemical characterization techniques such as dynamic light scattering and zeta potential measurement, that have provided a better understanding of the physical structure of these drug products in vitro. However, establishment and validation of complementary and orthogonal approaches are necessary to better understand the 3-dimensional physical structure of the iron-carbohydrate complexes, particularly with regard to their physical state in the context of the nanoparticle interaction with biological components such as whole blood (i.e. the nano-bio interface).

Iron-carbohydrate complexes are widely used to treat iron deficiencies. Macrophages play a crucial role in the uptake and fate of these nanomedicines, however, how complexed iron carbohydrates are taken up and metabolized by macrophages is still not fully understood. Using a (phospho-)proteomics approach, we assessed differences in protein expression and phosphorylation in M2 macrophages triggered by iron sucrose (IS). Our results show that IS alters the expression of multiple receptors, indicative of a complex entry mechanism. Besides, IS induced an increase in intracellular ferritin, the loss of M2 polarization, protective mechanisms against ferroptosis, and an autophagic response. These data indicate that macrophages can use IS as a source of iron for its storage and later release, however, the excess of iron can cause oxidative stress, which can be successfully regulated by the cells. When comparing IS with ferric carboxymaltose (FCM) and iron isomaltoside-1000 (IIM), complexes with a higher carbohydrate ligand stability, we observed that FCM and IIM are metabolized at a slower rate, and trigger M2 polarization loss to a lower extent. These results indicate that the surface characteristics of the iron-carbohydrate complexes may influence the cell responses. Our data show that the application of (phospho-)proteomics can lead to a better understanding of metabolic processes, including the uptake, biodegradation and bioavailability of nanomedicines.

Intravenous iron-carbohydrate complexes are nanomedicines that are commonly used to treat iron deficiency and iron deficiency anemia of various etiologies. Many challenges remain regarding these complex drugs in the context of fully understanding their pharmacokinetic parameters. Firstly, the measurement of the intact iron nanoparticles versus endogenous iron concentration fundamentally limits the availability of data for computational modeling. Secondly, the models need to include several parameters to describe the iron metabolism which is not completely defined and those identified (e.g. ferritin) exhibit considerable interpatient variability. Additionally, modeling is further complicated by the lack of traditional receptor/enzyme interactions. The known parameters of bioavailability, distribution, metabolism, and excretion for iron-carbohydrate nanomedicines will be reviewed and future challenges that currently prevent the direct application of physiologically-based pharmacokinetic or other computational modeling techniques will be discussed.

The authors wish to make the following corrections to this paper [...].

Intravenous (IV) iron nanoparticle preparations are widely used to treat iron deficiency. The mechanism of mononuclear phagocyte system-mediated clearance of IV iron nanoparticles is unknown. The early uptake and homeostasis of iron after injection of ferric carboxymaltose (FCM) in mice was studied. An increase in serum iron was observed at 2.5 h followed by a return to baseline by 24 h. An increase in circulating monocytes was observed, particularly Ly6Chi and Ly6Clow. FCM was also associated with a time-dependent decrease in liver Kupffer cells (KCs) and increase in liver monocytes. The increase in liver monocytes suggests an influx of iron-rich blood monocytes, while some KCs underwent apoptosis. Adoptive transfer experiments demonstrated that following liver infiltration, blood monocytes differentiated to KCs. KCs were also critical for IV iron uptake and biodegradation. Indeed, anti-Colony Stimulating Factor 1 Receptor (CSF1R)-mediated depletion of KCs resulted in elevated serum iron levels and impaired iron uptake by the liver. Gene expression profiling indicated that C-C chemokine receptor type 5 (CCR5) might be involved in monocyte recruitment to the liver, confirmed by pharmaceutical inhibition of CCR5. Liver KCs play a pivotal role in the clearance and storage of IV iron and KCs appear to be supported by the expanded blood monocyte population.

Un-complexed polynuclear ferric oxyhydroxide cannot be administered safely or effectively to patients. When polynuclear iron cores are formed with carbohydrates of various structures, stable complexes with surface carbohydrates driven by multiple interacting sites and forces are formed. These complexes deliver iron in a usable form to the body while avoiding the serious adverse effects of un-complexed forms of iron, such as polynuclear ferric oxyhydroxide. The rate and extent of plasma clearance and tissue biodistribution is variable among the commercially available iron–carbohydrate complexes and is driven principally by the surface characteristics of the complexes which dictate macrophage opsonization. The surface chemistry differences between the iron–carbohydrate complexes results in significant differences in in vivo pharmacokinetic and pharmacodynamic profiles as well as adverse event profiles, demonstrating that the entire iron–carbohydrate complex furnishes the pharmacologic action for these complex products. Currently available physicochemical characterization methods have limitations in biorelevant matrices resulting in challenges in defining critical quality attributes for surface characteristics for this class of complex nanomedicines.

Cellular iron supply is required for various biochemical processes. Measuring bioavailable iron in cells aids in obtaining a better understanding of its biochemical activities but is technically challenging. Existing techniques have several constraints that make precise localization difficult, and the lack of a functional readout makes it unclear whether the tested labile iron is available for metalloproteins. Here, we use geNOps; a ferrous iron-dependent genetically encoded fluorescent nitric oxide (NO) biosensor, to measure available iron in cellular locales. We exploited the nitrosylation-dependent fluorescence quenching of geNOps as a direct readout for cellular iron absorption, distribution, and availability. Our findings show that, in addition to ferrous iron salts, the complex of iron (III) with N,N'-bis (2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED) can activate the iron (II)-dependent NO probe within intact cells. Cell treatment for only 20 min with iron sucrose was also sufficient to activate the biosensor in the cytosol and mitochondria significantly; however, ferric carboxymaltose failed to functionalize the probe, even after 2 h of cell treatment. Our findings show that the geNOps approach detects available iron (II) in cultured cells and can be applied to assay functional iron (II) at the (sub)cellular level.

The emerging landscape of nanomedicine includes a wide variety of active pharmaceutical ingredients and drug formulations. Their design provides nanomedicines with unique features leading to improved pharmacokinetics and pharmacodynamics. They are manufactured using conventional or biotechnological manufacturing processes. Their physical characteristics are vastly different from traditional small-molecule drugs. Pharmacists are important members of the multi-disciplinary team of scientists involved in their development and clinical application. Consequently, their training should lead to an understanding of the complexities associated with the production and evaluation of nanomedicines. Therefore, student pharmacists, post-doctoral researchers, and trainees should be given more exposure to this rapidly evolving class of therapeutics. This commentary will provide an overview of nanomedicine education within the selection of pharmacy programs globally, discuss the current regulatory challenges, and describe different approaches to incorporate nanomedicine science in pharmacy programs around the world.

The field of nanomedicine is a rapidly growing scientific domain. Nanomedicine encompasses a diverse number of active pharmaceutical ingredients. Submissions of Investigational New Drugs and New Drug Applications have risen dramatically over the last decade. There are over 50 nanomedicines approved for use by the US Food and Drug Administration (FDA). Because of the fundamental role pharmacists will play in therapeutic and administrative decisions regarding nanomedicines, it is imperative for future pharmacists to gain exposure early in their training to this rapidly evolving class of drugs. This commentary describes nanomedicines, discusses current regulatory challenges, and provides recommendations for judicious incorporation of nanomedicine topics into the Doctor of Pharmacy curriculum based on emerging pharmaceutical and clinical science applications.