Cellular Uptake of Substrate-Initiated Cell-Penetrating Poly(disulfide)s

Abstract

Substrate-initiated, self-inactivating, cell-penetrating poly(disulfide)s (siCPDs) are introduced as general transporters for the covalent delivery of unmodified substrates of free choice. With ring-opening disulfide-exchange polymerization, we show that guanidinium-rich siCPDs grow on fluorescent substrates within minutes under the mildest conditions. The most active siCPD transporters reach the cytosol of HeLa cells within 5 min and depolymerize in less than 1 min to release the native substrate. Depolymerized right after use, the best siCPDs are nontoxic under conditions where cell-penetrating peptides (CPPs) are cytotoxic. Intracellular localization (cytosol, nucleoli, endosomes) is independent of the substrate and can be varied on demand, through choice of polymer composition. Insensitivity to endocytosis inhibitors and classical structural variations (hydrophobicity, aromaticity, branching, boronic acids) suggest that the best siCPDs act differently. Supported by experimental evidence, a unique combination of the counterion-mediated translocation of CPPs with the underexplored, thiol-mediated covalent translocation is considered to account for this decisive difference.

Interfacing Functional Systems

“…[6,20,21] Alternatively, streptavidin has been equipped with transporters such as cellpenetrating poly(disulfide)s (CPDs). [4,22] The resulting primary systems 10 turned out to enter cells as easily as expected. Understood structural modifications of the CPDs have been applied to direct the streptavidin to specific intracellular targets such as the nucleoli.…”

Epidithiodiketopiperazines: Strain-Promoted Thiol-Mediated Cellular Uptake at the Highest Tension

“…11−14 Disulfides in general are increasingly recognized to enter cells by thiol-mediated uptake, i.e., covalent attachment by disulfide exchange with exofacial thiols followed by efficient uptake via diverse, to a good part unknown mechanisms. 11−23 The emergence of thiol-mediated uptake called for the application of ring tension. 11 Uptake efficiencies were found to increase with ring tension from relaxed disulfides 3 with θ ≈ 90° to lipoic acid derivatives 4 with θ = 35° and asparagusic acid derivatives 5 with θ = 27°.…”

“…11−23 The emergence of thiol-mediated uptake called for the application of ring tension. 11 Uptake efficiencies were found to increase with ring tension from relaxed disulfides 3 with θ ≈ 90° to lipoic acid derivatives 4 with θ = 35° and asparagusic acid derivatives 5 with θ = 27°. 12,13 The most efficient “AspA tag” as in 5 allowed the delivery of functional peptides, 14 liposomes and polymersomes 13 into cells, and the transferrin receptor (TFRC) has been identified as one of the targets.…”

Cell-Penetrating, Guanidinium-Rich Oligophosphoesters: Effective and Versatile Molecular Transporters for Drug and Probe Delivery

“…Noteworthy contributions from many other groups have also been reported, including guanidinium-rich modified peptides, 10–12 cyclic peptides, 13,14 peptide nucleic acids, 15 and transporters resulting from the oligomerization of guanidinium-containing monomers such as norbornenes, 16,17 methacrylamides, 18 and cyclic disulfides. 19 These transporters have been shown to enable or enhance the passage of numerous cargos including small molecules, 20–22 peptides and proteins, 23–26 and oligonucleotides, 27–31 across multiple biological barriers 1,2,32,33 including the cell wall of algae. 34 Here, we report a new class of molecular transporters, guanidinium-rich oligophosphoesters, which exhibit increased delivery efficacy and offer several advantages over previously reported systems including our original oligoarginines and more recent oligocarbonates (Figure 1).…”