4.8 Article

Absolute Quantification of Drug Vector Delivery to the Cytosol

Journal

ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
Volume 60, Issue 27, Pages 14824-14830

Publisher

WILEY-V C H VERLAG GMBH
DOI: 10.1002/anie.202102332

Keywords

cell-penetrating peptides; drug delivery; endosomal escape; membranes; Shiga toxin B-subunit

Funding

  1. Cell and Tissue Imaging (PICT-IBiSA) and Nikon Imaging Centre, Institut Curie [ANR10-INBS-04]
  2. Institut National du Cancer [2019-1-PLBIO-05-1]
  3. Agence National de la Recherche [ANR-16-CE23-0005-02, ANR-19-CE13-0001-01]
  4. Fondation pour la Recherche Medicale [EQU202103012926]
  5. Mizutani Foundation for Glycosciences [200014]
  6. Institut Carnot
  7. French Ministry of Higher Education and Research (AMX funding)
  8. La Ligue Nationale Contre le Cancer
  9. Frontieres de l'Innovation en Recherche et Education (FIRE) Doctoral School-Bettencourt Program
  10. European Union [H2020-MSCA-ITN-2015]
  11. Fondation pour la Recherche Medicale (FRM)
  12. [ANR-11-LABX-0038]
  13. [ANR-10-IDEX-0001-02]
  14. Agence Nationale de la Recherche (ANR) [ANR-19-CE13-0001] Funding Source: Agence Nationale de la Recherche (ANR)

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A sensitive and robust assay has been developed for quantifying the relative or absolute numbers of macromolecular drugs in the cytosol, facilitating the optimization of drug delivery vectors and enhancing understanding of the translocation process.
Macromolecular drugs inefficiently cross membranes to reach their cytosolic targets. They require drug delivery vectors to facilitate their translocation across the plasma membrane or escape from endosomes. Optimization of these vectors has however been hindered by the difficulty to accurately measure cytosolic arrival. We have developed an exceptionally sensitive and robust assay for the relative or absolute quantification of this step. The assay is based on benzylguanine and biotin modifications on a drug delivery vector of interest, which allow, respectively, for selective covalent capture in the cytosol with a SNAP-tag fusion protein and for quantification at picomolar sensitivity. The assay was validated by determining the absolute numbers of cytosolic molecules for two drug delivery vectors: the B-subunit of Shiga toxin and the cell-penetrating peptide TAT. We expect this assay to favor delivery vector optimization and the understanding of the enigmatic translocation process.

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