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. 2024 Aug 24;10(17):e36749.
doi: 10.1016/j.heliyon.2024.e36749. eCollection 2024 Sep 15.

Nano-scale characterization of iron-carbohydrate complexes by cryogenic scanning transmission electron microscopy: Building the bridge to biorelevant characterization

Reinaldo Digigow  1 , Michael Burgert  1 , Marco Luechinger  1 , Alla Sologubenko  2 , Andrzej J Rzepiela  2 , Stephan Handschin  2 , Amy E Barton Alston  1 , Beat Flühmann  1 , Erik Philipp  1
Affiliations

Nano-scale characterization of iron-carbohydrate complexes by cryogenic scanning transmission electron microscopy: Building the bridge to biorelevant characterization

Reinaldo Digigow et al. Heliyon. .

Abstract

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.

Keywords: Cryo-scanning transmission electron microscopy (cryo-STEM); Cryo-transmission electron microscopy (cryo-TEM); Iron deficiency anemia; Iron-carbohydrate complexes; Nanomedicines; Nanoparticles (NPs); Non-biological complex drugs (NBCDs); Physicochemical characterization of nanomedicines.

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Conflict of interest statement

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Alla Sologubenko, Andrzej J. Rzepiela, Stephan Handschin reports financial support was provided by Vifor (International) AG. Reinaldo Digigow reports a relationship with Vifor (International) AG that includes: employment. Michael Burgert reports a relationship with Vifor (International) AG that includes: employment. Marco Luechinger reports a relationship with Vifor (International) AG that includes: employment. Amy E. Barton Alston1 reports a relationship with Vifor (International) AG that includes: employment. Beat Fluhmann reports a relationship with Vifor (International) AG that includes: employment. Erik Philipp reports a relationship with Vifor (International) AG that includes: employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
The bright-field (BF) cryo-TEM (a), bright-field (BF) cryo-STEM (b), and LAADF cryo-STEM (c) images of the ferric carboxymaltose (FCM).
Fig. 2
Fig. 2
The low angle annular dark field (LAADF) cryo-STEM micrographs of (a) ferric carboxymaltose (FCM), (b) iron Sucrose (IS), (c) low molecular weight iron dextran (LMWID), (d) iron isomaltoside 1000 (IIM), (e) sodium ferric gluconate (SFG), and (f) ferumoxytol (FMX) products. The insert in the micrographs presents the data acquired at the higher magnification (630x magnification, pixel size 0.18 nm). The dark background of an about 70 nm thick amorphous layer (consisting of low-atomic number atoms and residing onto a 2 nm thick amorphous carbon substrate) provides a high contrast to the bright, crystalline clusters composed of Fe-containing nanoparticles. The background also occasionally contains foggy brighter regions (see the left bottom part of (b)) corresponding to the thickness or density fluctuations of the vitrified amorphous layer. The electron beam induced damage to the vitrified supporting layer is seen as still darker regions in the micrographs (see yellow arrows in (b, c, d, e). The micrographs are acquired in the same illumination conditions (pixel size in (a), (b), (c) and (d) is 0.49 nm, in (e) 0.7 nm and in (f) 0.35 nm) from undiluted materials.
Fig. 3
Fig. 3
Analysis of STEM micrographs with the PANDA model trained on the ferric carboxymaltose (FCM) micrographs. The mean normalized PANDA scores and standard deviations are shown for each product. Statistics are calculated over the product micrographs. Classifier scores are normalized to the mean of the FCM test set score.
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