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Absolute Quantification of Drug Vector Delivery to the Cytosol
Author(s) -
Lucchino Marco,
Billet Anne,
Bai SiauKun,
Dransart Estelle,
Hadjerci Justine,
Schmidt Frédéric,
Wunder Christian,
Johannes Ludger
Publication year - 2021
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.202102332
Subject(s) - cytosol , endosome , drug delivery , chemistry , biophysics , drug discovery , biochemistry , biology , cell , enzyme , organic chemistry
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.