Probing the free-state solution behavior of drugs and their tendencies to self-aggregate into nano-entities

• Published in: Nature protocols Vol. 16; no. 11; pp. 5250 - 5273
• Main Authors: LaPlante, Steven R., Roux, Valérie, Shahout, Fatma, LaPlante, Gabriela, Woo, Simon, Denk, Maria M., Larda, Sacha T., Ayotte, Yann
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.11.2021; Nature Publishing Group
• Subjects: 631/154/1435; 631/1647/2204; 631/57/2272/2276; Aggregates; Analytical Chemistry; Biological Techniques; Biomedical and Life Sciences; Chemical properties; Colloids; Colloids - chemistry; Computational Biology/Bioinformatics; Confocal microscopy; Dilution; Drugs; Life Sciences; Light scattering; Magnetic Resonance Spectroscopy - methods; Methods; Microarrays; Microscopy; Microscopy, Confocal - methods; Microscopy, Electron, Transmission - methods; Nanoparticles - chemistry; Nanostructures - chemistry; NMR; Nuclear magnetic resonance; Organic Chemistry; Pharmaceutical Preparations - chemistry; Photon correlation spectroscopy; Protocol; Scanning microscopy; Solid phases; Solutions; Transmission electron microscopy; Tumbling; Canada
• ISSN: 1754-2189; 1750-2799; 1750-2799
• DOI: 10.1038/s41596-021-00612-3

The free-state solution behaviors of drugs profoundly affect their properties. Therefore, it is critical to properly evaluate a drug’s unique multiphase equilibrium when in an aqueous enviroment, which can comprise lone molecules, self-associating aggregate states and solid phases. To date, the full range of nano-entities that drugs can adopt has been a largely unexplored phenomenon. This protocol describes how to monitor the solution behavior of drugs, revealing the nano-entities formed as a result of self-associations. The procedure begins with a simple NMR 1 H assay, and depending on the observations, subsequent NMR dilution, NMR T2-CPMG (spin-spin relaxation Carr-Purcell-Meiboom-Gill) and NMR detergent assays are used to distinguish between the existence of fast-tumbling lone drug molecules, small drug aggregates and slow-tumbling colloids. Three orthogonal techniques (dynamic light scattering, transmission electron microscopy and confocal laser scanning microscopy) are also described that can be used to further characterize any large colloids. The protocol can take a non-specialist between minutes to a few hours; thus, libraries of compounds can be evaluated within days. The solution behavior of drugs is determined by NMR, enabling the presence of fast-tumbling lone drug molecules, small drug aggregates and slow-tumbling colloids to be determined.