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.

Lessons from surface-initiated polymerization are applied to grow cell-penetrating poly(disulfide)s directly on substrates of free choice. Reductive depolymerization after cellular uptake should then release the native substrates and minimize toxicity. In the presence of thiolated substrates, propagators containing a strained disulfide from asparagusic or, preferably, lipoic acid and a guanidinium cation polymerize into poly(disulfide)s in less than 5 min at room temperature at pH 7. Substrate-initiated polymerization of cationic poly(disulfide)s and their depolymerization with dithiothreitol causes the appearance and disappearance of transport activity in fluorogenic vesicles. The same process is further characterized by gel-permeation chromatography and fluorescence resonance energy transfer.Cell-penetrating peptides (CPPs) are short, polycationic peptides or protein domains that are used by viruses to enter cells. 1,2 Their unique ability to transport linked substrates across lipid bilayer membranes has attracted great interest in biomedical applications. Substrates of varying sizes and properties, e.g., small fluorophores to proteins and quantum dots, have been successfully transported into cells using CPPs. The mechanism of cellular uptake is under debate, currently favored are endocytosxis (i.e., macropinocytosis) or passive diffusion across the membrane, depending on conditions. Multiple, moderately hydrophobic cations seem to be all that is needed. Guanidinium cations, as in arginine, are most common, alternatives include ammonium or phosphonium cations. 1 The originally peptidic oligomer backbone has been extensively varied, covering oligocarbamates, β-peptides and several variations of synthetic polymers. 1 Currently, cell-penetrating poly(disulfide)s are emerging as the cell-penetrating molecules of the future because their cytosolic degradation liberates the substrate and eliminates toxicity, one of the key disadvantages associated with CPPs. [3][4][5] However, cell-penetrating poly-(disulfide)s have so far been used mainly in noncovalent polyplexes for gene transfection, and covalent attachment of substrates would be difficult with their preparation methods. We have found recently that poly(disulfide)s can be grown directly on solid substrates by surface-initiated ring-opening disulfide-exchange polymerization. 6 Therefore, we wondered whether the same methodology could be used to Figure 1). Probes or drugs that contain thiol group but cannot penetrate cells without assistance are the ideal substrates, which could serve as an initiator to be appended with a membrane-active poly(disulfide). Thiolated siRNA, for instance, is commercially available. The generality of this approach promises a conceptually innovative solution for a central current challenge, i.e., the noninvasive, nontoxic delivery of unmodified substrates in welldefined, covalent systems rather than complex, noncovalent formulations. In this initial report on the topic, we describe the design, synthesis and evaluation of propag...

Don't forget poly(disulfide)s. There is a rich literature pointing out the advantages of the dynamic nature of single disulfide bridges to explore self-sorting, biomolecular engineering, biomembrane analysis, and so on. Disulfide bonds between polymer chains are essential for protein folding, materials properties and the stabilization of various supramolecular architectures. However, poly(disulfide)s with disulfide bonds in the main chain are rarely used today to create interesting structures or functions. To attract attention and outline scope and limitations of poly(disulfide)s to build modern supramolecular systems, the rather eclectic recent literature on the topic is summarized. The review is moving from fascinating basic studies including photoinduced metathesis, polycatenanes and polyrotaxanes to applications in biosupramolecular systems such as micelles, membranes, tubes, gels, carriers, pores, sensors, catalysts and photosystems

The objective of the study was to explore the potential of ring tension in cyclic disulfides for thiol-mediated cellular uptake. Fluorescent probes that cannot enter cells were equipped with cyclic disulfides of gradually increasing ring tension. As demonstrated by flow cytometry experiments, uptake into HeLa Kyoto cells increased with increasing tension. Differences in carbon-sulfur-sulfur-carbon (CSSC) dihedral angles as small as 8° caused significant changes in uptake efficiency. Uptake with high ring tension was better than with inactivated or activated linear disulfides or with thiols. Conversion of thiols on the cell surface into sulfides and disulfides decreased the uptake. Reduction of exofacial disulfides into thiols increased the uptake of transporters with disulfides and inactivated controls with thiols. These results confirm the occurrence of dynamic covalent disulfide-exchange chemistry on cell surfaces. Mechanistic and colocalization studies indicate that endocytosis does not fully account for this cellular uptake with ring tension.