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


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 1H 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.

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Fig. 1: Drugs exist in unique multi-phase equilibria in solution.
Fig. 2: The presence of large drug colloidal aggregates can be visualized by TEM.
Fig. 3: Overview of the protocol to probe drug solution behavior.
Fig. 4: NMR dilution assay.
Fig. 5: NMR detergent assay.
Fig. 6: Probing the solution behavior of valsartan.
Fig. 7: Probing the solution behavior of methylene blue.
Fig. 8: Probing the solution behavior of candesartan cilexetil.
Fig. 9: Probing the solution behavior of lapatinib.
Fig. 10: Probing the solution behavior of lapatinib by using orthogonal techniques.

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Data availability

The NMR data that support Figs. 69 and Supplementary Figs. 15 are available in figshare (


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We thank the following agencies for helping fund this research: NSERC (Natural Sciences and Engineering Research Council of Canada), CQDM (Quebec Consortium for Drug Discovery), CFI (Canada Foundation for Innovation), Mitacs, INRS (Institut national de la recherche scientifique), Institut Pasteur, la région Auvergne-Rhône-Alpes, le ministère de l’enseignement supérieur et de la recherche (France) and NMX Research and Solutions Inc. We also thank our colleagues for their help, suggestions and encouragement: P. Bouchard, N. Girard, D. Bendahan, D. Girard, J. Tremblay and A. Nakamura.

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Authors and Affiliations



S.R.L.P. conceived the concepts described in this report. S.R.L.P. and V.R. wrote the paper. V.R., F.S., G.L.P., M.M.D., S.R.L.P., S.T.L., Y.A. and S.W. performed the experiments or helped with interpretations. Y.A. and S.T.L. implemented some of the experiments used herein.

Corresponding author

Correspondence to Steven R. LaPlante.

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The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Ulrike Holzgrabe and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 NMR 1H assay.

a, Preparation for assay in DMSO-d6. b, Preparation for assay in buffer. Shown are volumes suggested for 3-mm NMR tubes, and volumes for 5-mm tubes are in parentheses.

Extended Data Fig. 2 NMR T2-CPMG assay.

a, Preparation of the sample. b, Interpretation of the results.

Extended Data Fig. 3 Solvent solubility assay.

Preparation of the sample and interpretations.

Extended Data Fig. 4 Orthogonal assays.

ac, Preparation of the samples for DLS (a), TEM (b) and CLSM (c).

Supplementary information

Supplementary Information

Supplementary Figs. 1–6 with discussions and Supplementary Tables 1–3.

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LaPlante, S.R., Roux, V., Shahout, F. et al. Probing the free-state solution behavior of drugs and their tendencies to self-aggregate into nano-entities. Nat Protoc 16, 5250–5273 (2021).

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