Methodological needs in the quality and safety characterisation of nanotechnology-based health products: Priorities for method development and standardisation.

Nanotechnology-based health products are providing innovative solutions in health technologies and the pharmaceutical field, responding to unmet clinical needs. However, suitable standardised methods need to be available for quality and safety assessments of these innovative products prior to their translation into the clinic and for monitoring their performance when manufacturing processes are changed. The question arises which technological solutions are currently available within the scientific community to support the requested characterisation of nanotechnology-based products, and which methodological developments should be prioritized to support product developers in their regulatory assessment. To this end, the work presented here explored the state-of-the-art methods to identify methodological gaps associated with the preclinical characterisation of nanotechnology-based medicinal products and medical devices. The regulatory information needs, as expressed by regulatory authorities, were extracted from the guidance documents released so far for nanotechnology-based health products and mapped against available methods, thus allowing an analysis of methodological gaps and needs. In the first step, only standardised methods were considered, leading to the identification of methodological needs in five areas of characterisation, including: (i) surface properties, (ii) drug loading and release, (iii) kinetic properties in complex biological media, (iv) ADME (absorption, distribution, metabolism and excretion) parameters and (v) interaction with blood and the immune system. In the second step, a detailed gap analysis included analytical approaches in earlier stages of development, and standardised test methods from outside of the nanotechnology field that could address the identified areas of gaps. Based on this analysis, three categories of methodological needs were identified, including (i) method optimisation/adaptation to nanotechnological platforms, (ii) method validation/standardisation and (iii) method development for those areas where no technological solutions currently exist. The results of the analysis presented in this work should raise awareness within the scientific community on existing and emerging methodological needs, setting priorities for the development and standardisation of relevant analytical and toxicological methods allowing the development of a robust testing strategy for nanotechnology-based health products.

A tanscriptomics study to elucidate the toxicological mechanism of methylmercury chloride in a human stem cell based in vitro test.

Traditional approaches in evaluating the hazard of drug candidates on the developing offspring are often time-consuming and cost-intensive. Moreover, variations in the toxicological response of different animal species to the tested substance cause severe problems when extrapolating safety dosages for humans. Therefore, more predictive and relevant toxicological systems based on human cell models are required. In the presented study the environmental toxicant methylmercury chloride (MeHgCl), known to cause structural developmental abnormalities in the brain, was used as reference compound to develop a concept contributing to a mechanistic understanding of the toxicity of an investigated substance. Despite the fact, that there are significant data available from animal studies and from poisonings in Japan and Iraq, uncertainties on the mechanism of MeHgCl during human development are still remaining and qualify the substance for further analysis. Transcriptomics analysis in combination with a human cell based in vitro model has been used in order to elucidate the toxicity of MeHgCl at molecular level. Differentiating neural precursor cells that have been exposed continuously to non- and low-cytotoxic concentrations of MeHgCl were investigated. Quantitative change in the mRNA expression profiles of selected genes demonstrated the sensitivity of the cell model and its qualification for a transcriptomics study screening changes in the expression profile of the complete human genome of MeHgCl-treated human neural cells. Potential biomarkers were identified and these candidate marker genes as well as their involvement in a possible toxic mechanism of MeHgCl during the human neurulation process are hereby introduced. The study confirmed the hypothesis that a cellular model based on a human stem cell line can be applied for elucidating unknown mode of actions of developmental toxicants.

MicroRNA profiling as a tool for pathway analysis in a human in vitro model for neural development.

MicroRNAs (miRNA) are a recently recognised class of small, non-coding RNAs involved in the post-transcriptional regulation of gene expression and with crucial implication for mammalian development. In particular, they play key roles in neuronal development, from early neurogenesis to neuronal differentiation and synaptic development, and also in in vitro systems. The detection of embryotoxic hazards in the preclinical phase is still a challenge, often due to species-species variations. In this study we analysed whether miRNA expression profiles in a human pluripotent cell model can be a helpful tool for a more mechanistic approach to pharmacology and toxicology. Differentiating human pluripotent cells were repeatedly treated with non-cytotoxic doses of methylmercury chloride (MeHgCl), a well known brain developmental toxicant. The expression of proteins, mRNA and miRNAs were used to monitor successful neural differentiation. Significant changes in the expression of 12 miRNAs were detected. By using available bioinformatics tools, we obtained validated and predicted targets for the identified miRNAs, on which we performed functional clustering analysis. Through this approach, we identified several terms and functional clusters associated with neural development, together with indicators of general toxic effect, such as apoptosis or stress response-related genes. Interestingly, our results also suggest a previously undiscovered association between MeHgCl and the ubiquitin-proteasome protein degradation pathway. Although further investigations are needed, our results suggest that miRNA expression analysis is a powerful tool in pathway-oriented toxicity and could improve early-phase hazard assessments.