Shedding Light on Metal‐Based Nanoparticles in Zebrafish by Computed Tomography with Micrometer Resolution

Abstract

SPIONs) which are clinically used as magnetic resonance imaging contrast agents. [4] In addition, application of an (alternating) external magnetic field for therapeutic purposes offers interesting perspectives in oncology, in that such nanoparticles can be used for a hyperthermia therapy or to target drug loaded particles to tumors. The latter is referred to as magnetic drug targeting. [5] In order to study the in vivo biodistribution of such particles, they have to be labeled by conjugation of fluorescent or radioactive agents. However, these chemical modifications can significantly change the interaction patterns with biological matter since they have a direct impact on particle size and polydispersity, electrokinetic potential (ζ-potential), and possibly the protein corona. [6][7][8][9] It is therefore recommended to carry out, whenever possible, physicochemical characterization, formulation development, pharmacokinetic studies, and hazard and safety evaluations with non-tagged nanoparticles. [10] In recent years, magnetic particle imaging (MPI), magnetic resonance imaging (MRI) or computed tomography [11] have been discussed as label-free alternative to trace engineered metal nanoparticles within a biological system. The latter technology takes advantage of their strong X-ray absorption. [12,13] In medical research, laboratory-based micro-computed tomography (μCT) has been used to monitor the biodistribution of metal-based nanoparticles in vitro or in small experimental animals. [13][14][15][16] A challenge Metal-based nanoparticles are clinically used for diagnostic and therapeutic applications. After parenteral administration, they will distribute throughout different organs. Quantification of their distribution within tissues in the 3D space, however, remains a challenge owing to the small particle diameter. In this study, synchrotron radiation-based hard X-ray tomography (SRμCT) in absorption and phase contrast modes is evaluated for the localization of superparamagnetic iron oxide nanoparticles (SPIONs) in soft tissues based on their electron density and X-ray attenuation. Biodistribution of SPIONs is studied using zebrafish embryos as a vertebrate screening model. This label-free approach gives rise to an isotropic, 3D, direct space visualization of the entire 2.5 mm-long animal with a spatial resolution of around 2 μm. High resolution image stacks are available on a dedicated internet page (http://zebrafish.pharma-te.ch). X-ray tomography is combined with physico-chemical characterization and cellular uptake studies to confirm the safety and effectiveness of protective SPION coatings. It is demonstrated that SRμCT provides unprecedented insights into the zebrafish embryo anatomy and tissue distribution of label-free metal oxide nanoparticles.

Hard X-ray microtomography of Zebrafish larvae

“…Potentially this approach could also be used to e.g. locate clusters of injected nanoparticles or other medically relevant nanomaterials, as previously shown for the synchrotron-based approach, 13 based on their strong difference in attenuation contrast with respect to the soft tissues.…”

“…Throughout the tail (Fig. 3 D/D') the notochord (9), which is flanked by the segmental muscles, the so-called myotomes (13), is very prominent and the outlines of the gut (12) can be seen in the ventral part of the embryo, anterior to the cloaca. In sagittal cross sections, shown in Figure 4, several parts of the brain, namely the telencephalon (14), diencephalon (5), metencephalon (8) (Fig.…”

“…By providing a direct comparison between our laboratory-based µCT measurements with the Skyscan 1275 system and a publicly available SRµCT dataset, 13 it can be shown which features can be readily identified with a conventional X-ray source and which additional anatomical microstructures can be revealed by employing synchrotron radiation with improved beam properties. SRµCT imaging was performed on ethanol-embedded 72 hpf zebrafish embryos at the TOMCAT X02DA beamline of the Swiss Light Source with a monochromatized beam with a photon energy of 10.25 keV and an effective voxel size of 325 nm, corresponding to a spatial resolution around 2 µm.…”

“…12 Further, a recent study has demonstrated the performance of SRµCT for the localization of injected superparamagnetic iron oxide nanoparticles in zebrafish embryos. 13 Thereby, the nanoparticle biodistribution was assessed with isotropic resolution around 2 µm, providing the full anatomical context. Other recent research efforts have successfully implemented advanced laboratory-based X-ray techniques for zebrafish embryo imaging.…”

Laboratory-based phase and absorption tomography for micro-imaging of annual layers in human tooth cementum, paraffin-embedded nerve and zebrafish embryo

“…Therefore, the zebrafish finds numerous applications including studies in pathological conditions such as kidney injury [31] and treatment such as transplantation [32], to name a few. High-resolution hard X-ray tomography was used to examine single organ-centered anatomy of the zebrafish heart [33], muscles [34], as well as nanoparticle distribution [35]. In a recent study, synchrotron radiation was applied in whole-organism histotomography, enabling extraction of cellular architectures [36].…”