Development, validation and quantitative assessment of an enzymatic assay suitable for small molecule screening and profiling: A case-study

The successful discovery and subsequent development of small molecule inhibitors of drug targets relies on the establishment of robust, cost-effective, quantitative, and physiologically relevant in vitro assays that can support prolonged screening and optimization campaigns. The current study illustrates the process of developing and validating an enzymatic assay for the discovery of small molecule inhibitors using alkaline phosphatase from bovine intestine as model target. The assay development workflow includes an initial phase of optimization of assay materials, reagents, and conditions, continues with a process of miniaturization and automation, and concludes with validation by quantitative measurement of assay performance and signal variability. The assay is further evaluated for dose–response and mechanism-of-action studies required to support structure–activity-relationship studies. Emphasis is placed on the most critical aspects of assay optimization and other relevant considerations, including the technology, assay materials, buffer constituents, reaction conditions, liquid handling equipment, analytical instrumentation, and quantitative assessments. Examples of bottlenecks encountered during assay development and strategies to address them are provided

Biocompatibility and internalization assessment of bare and functionalised mesoporous silica nanoparticles

[EN] We report herein an evaluation of the effect of several mesoporous silica nanoparticles (MSNs) on the cellular uptake and in vitro cytotoxicity in human cells. Bare MSNs and MSNs functionalized with polyethylene glycol or hyaluronic acid are employed to evaluate uptake efficiency and mechanisms of endocytosis in cancer (MDA-MB-231) and non-cancer (MCF10A) cells. Moreover, changes in viability, cell cycle, oxidative stress, and mitochondrial membrane potential are evaluated. Our results confirm that MSNs are internalized efficiently by human cells and that uptake mechanisms differ for cell types and particles. We also confirm that MSNs are biocompatible materials that do not induce ROS/RNS production, nor changes on mitochondrial membrane potential or cell cycle.The authors want to thank the Spanish Government RTI2018-100910-B-C41 (MCUI/AEI/FEDER, UE) and PI18/01219 (ISCIII), the Generalitat Valenciana (PROMETEO/2018/024 and ACIF/2016/030), and CIBER-BBN (CB07/01/2012) and CIBER-ONC (CB16/12/00481) for support.Garrido-Cano, I.; Candela-Noguera, V.; Herrera, G.; Cejalvo, JM.; Lluch, A.; Marcos Martínez, MD.; Sancenón Galarza, F.... (2021). Biocompatibility and internalization assessment of bare and functionalised mesoporous silica nanoparticles. Microporous and Mesoporous Materials. 310:1-12. https://doi.org/10.1016/j.micromeso.2020.110593S112310Contado, C. (2015). Nanomaterials in consumer products: a challenging analytical problem. Frontiers in Chemistry, 3. doi:10.3389/fchem.2015.00048Wang, Y., Zhao, Q., Han, N., Bai, L., Li, J., Liu, J., … Wang, S. (2015). 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