Intracellular delivery of active proteins presents an interesting approach in research and therapy. We created a protein transduction shuttle based on a new traceless click linker that combines the advantages of click reactions with implementation of reversible pH-sensitive bonds. The azidomethyl-methylmaleic anhydride (AzMMMan) linker was found compatible with different click chemistries, demonstrated in bioreversible protein modification with dyes, polyethylene glycol, or a transduction carrier. Linkages were stable at physiological pH but reversible at the mild acidic pH of endosomes or lysosomes. We show that pH-reversible attachment of a defined endosome-destabilizing three-arm oligo(ethane amino)amide carrier generates an effective shuttle for protein delivery. The cargo protein nlsEGFP, when coupled via the traceless AzMMMan linker, experiences efficient cellular uptake and endosomal escape into the cytosol, followed by import into the nucleus. In contrast, irreversible linkage to the same shuttle hampers nuclear delivery of nlsEGFP which after uptake remains trapped in the cytosol. Successful intracellular delivery of bioactive ß-galactosidase as a model enzyme was also demonstrated using the pH-controlled shuttle system. ■ INTRODUCTIONIn recent years click chemistry reactions, especially the coppercatalyzed 1,3-dipolar cycloaddition (CuAAC) and the Staudinger ligation, became useful tools for conjugating biomolecules.1−4 Both reactions have great advantages compared to other linking strategies, like high efficiency and bioorthogonality. Nevertheless, for some applications, such as drug delivery, a completely bioreversible bond between the two conjugated molecules would be favorable. Dimethylmaleic anhydride is known to form amide bonds with amines that are cleaved under mild acidic conditions. 5−10 Herein we describe the synthesis and applications of a new heterobifunctional linker based on substituted dimethylmaleic anhydride that combines the advantages of click chemistry with implementation of a pHsensitive bond between conjugated biomolecules. This linker is labile under mild acidic conditions, which are typical for tumor tissue 11 or early endosomes. 12 Moreover, this linker is cleaved off traceless, resulting in an unmodified molecule of interest. The utility in protein modification is demonstrated in examples of reversible dye labeling of proteins, pH-sensitive modification with polyethylene glycol (PEG), and intracellular transduction of proteins in bioactive form. ■ RESULTS AND DISCUSSIONThe azidomethyl-methylmaleic anhydride linker 3 (AzMMMan) was synthesized from dimethylmaleic anhydride 1 by two simple reaction steps (radical substitution with N-bromosuccinimide resulting in 2 followed by reaction with sodium azide, see Scheme 1).The heterobifunctional linker was used to introduce acidlabile azido groups into human serum albumin (HSA), EGFP, or ß-galactosidase (ß-Gal) (Scheme 2, step a) by reaction of the maleic anhydride moiety with amino groups of the proteins. The AzMMMan−HSA co...

Intracellular protein transduction technology is opening the door for a promising alternative to gene therapy. Techniques have to address all critical steps, like efficient cell uptake, endolysosomal escape, low toxicity, while maintaining full functional activity of the delivered protein.Here, we present the use of a chemically precise, structure defined threearm cationic oligomer carrier molecule for protein delivery. This carrier of exact and low molecular weight combines good cellular uptake with efficient endosomal escape and low toxicity. The protein cargo is covalently attached by a bioreversible disulfide linkage. Murine 3T3 fibroblasts could be transduced very efficiently with cargo nlsEGFP, which was tagged with a nuclear localization signal. We could show subcellular delivery of the nlsEGFP to the nucleus, confirming cytosolic delivery and expected subsequent subcellular trafficking. Transfection efficiency was concentration-dependent in a directly linear mode and 20-fold higher in comparison with HIV-TAT-nlsEGFP containing a functional TAT transduction domain. Furthermore, β-galactosidase as a model enzyme cargo, modified with the carrier oligomer, was transduced into neuroblastoma cells in enzymatically active form.

The specific transport of bioactive proteins into designated target cells is an interesting and challenging perspective for the generation of innovative biopharmaceuticals. Natural protein cytotoxins perform this task with outstanding efficacy. They enter cells with receptor-targeted specificity, respond to changing intracellular microenvironments, and by various mechanisms translocate their cytotoxic protein subunit into the cytosol. Here we imitate this toxin-based delivery strategy in an artificial setting, by bioreversible conjugation of a cytotoxic cargo protein (RNase A) with receptor-targeting PEG-folate and the pH-specific endosomolytic peptide INF7 as synthetic delivery domains. Covalent modification of the cargo protein was achieved using the pH-labile AzMMMan linker and copper-free click chemistry with DBCO-modified delivery modules. This linkage is supposed to enable traceless intracellular release of the RNase A after exposure to the endosomal weakly acidic environment. Delivery of RNase A via this polycation-free delivery strategy resulted in high cytotoxicity against receptor-positive KB tumor cells only when both PEG-folate and INF7 were attached.

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