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

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.

Enhancing the enthalpic contribution of hydrogen bonds by solvent shielding.

In biological systems, polar interactions are heavily burdened by high desolvation penalties resulting from strong solute-solvent interactions. As a consequence thereof, enthalpic contributions of hydrogen bonds to the free energy of binding are severely diminished. However, this effect is strongly attenuated for interactions within solvent-shielded areas of proteins. In microcalorimetric experiments, we show that the bacterial lectin FimH utilizes conformational adaptions to effectively shield its binding site from solvent. The transition into a lower dielectric environment results in an enthalpic benefit of approximately -13 kJ mol-1 for mannoside binding. However, this effect can be abrogated, if the hydrogen bond network within the binding site is disturbed by deoxygenation of the ligand. Conformational adaption leading to reduced local dielectric constants could represent a general mechanism for proteins to enable enthalpy-driven recognition of polar ligands.

Preclinical hazard evaluation strategy for nanomedicines.

The increasing nanomedicine usage has raised concerns about their possible impact on human health. Present evaluation strategies for nanomaterials rely on a case-by-case hazard assessment. They take into account material properties, biological interactions, and toxicological responses. Authorities have also emphasized that exposure route and intended use should be considered in the safety assessment of nanotherapeutics. In contrast to an individual assessment of nanomaterial hazards, we propose in the present work a novel and unique evaluation strategy designed to uncover potential adverse effects of such materials. We specifically focus on spherical engineered nanoparticles used as parenterally administered nanomedicines. Standardized assay protocols from the US Nanotechnology Characterization Laboratory as well as the EU Nanomedicine Characterisation Laboratory can be used for experimental data generation. We focus on both cellular uptake and intracellular persistence as main indicators for nanoparticle hazard potentials. Based on existing regulatory specifications defined by authorities such as the European Medicines Agency and the United States Food and Drug Administration, we provide a robust framework for application-oriented classification paired with intuitive decision making. The Hazard Evaluation Strategy (HES) for injectable nanoparticles is a three-tiered concept covering physicochemical characterization, nanoparticle (bio)interactions, and hazard assessment. It is cost-effective and can assist in the design and optimization of nanoparticles intended for therapeutic use. Furthermore, this concept is designed to be adaptable for alternative exposure and application scenarios. To the knowledge of the authors, the HES is unique in its methodology based on exclusion criteria. It is the first hazard evaluation strategy designed for nanotherapeutics.

Immobilization of Enzymes on PLGA Sub-Micrometer Particles by Crosslinked Layer-by-Layer Deposition.

Enzyme immobilization is of high interest for industrial applications. However, immobilization may compromise enzyme activity or stability due to the harsh conditions which have to be applied. The authors therefore present a new and improved crosslinked layer-by-layer (cLbL) approach. Two different model enzymes (acid phosphatase and ß-galactosidase) are immobilized under mild conditions on biocompatible, monodisperse, sub-micrometer poly(lactide-co-glycolide) (PLGA) particles. The resulting PLGA enzyme systems are characterized regarding their size, surface charge, enzyme activity, storage stability, reusability, and stability under various conditions such as changing pH and temperature. The developed and characterized cLbL protocol can be easily adapted to different enzymes. Potential future uses of the technology for biomedical applications are discussed. PLGA-enzyme particles are therefore injected into the blood circulation of zebrafish embryos in order to demonstrate the in vivo stability and activity of the designed system.