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Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide

Nature Chemistry volume 9, pages 751–761 (2017)Cite this article

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Abstract

One of the major obstacles in intracellular targeting using antibodies is their limited release from endosomes into the cytosol. Here we report an approach to deliver proteins, which include antibodies, into cells by using endosomolytic peptides derived from the cationic and membrane-lytic spider venom peptide M-lycotoxin. The delivery peptides were developed by introducing one or two glutamic acid residues into the hydrophobic face. One peptide with the substitution of leucine by glutamic acid (L17E) was shown to enable a marked cytosolic liberation of antibodies (immunoglobulins G (IgGs)) from endosomes. The predominant membrane-perturbation mechanism of this peptide is the preferential disruption of negatively charged membranes (endosomal membranes) over neutral membranes (plasma membranes), and the endosomolytic peptide promotes the uptake by inducing macropinocytosis. The fidelity of this approach was confirmed through the intracellular delivery of a ribosome-inactivation protein (saporin), Cre recombinase and IgG delivery, which resulted in a specific labelling of the cytosolic proteins and subsequent suppression of the glucocorticoid receptor-mediated transcription. We also demonstrate the L17E-mediated cytosolic delivery of exosome-encapsulated proteins.

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Figure 1: Design strategy of endosomolytic peptides and their selection.
Figure 2: Cytosolic delivery of bioactive proteins using L17E.
Figure 3: Cytosolic proteins targeted via intracellularly delivered antibodies using L17E.
Figure 4: Biophysical study on the perturbation of membranes by L17E.
Figure 5: Inhibitor studies: L17E has a physiological effect that induces macropinocytosis and promotes cellular uptake.

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Acknowledgements

This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant nos JP15H02497 and JP16H01145. This work was also supported by the Collaborative Research Program of the Institute for Chemical Research, Kyoto University. M.A. and Y.K. are grateful for the JSPS Research Fellowship for Young Scientists.

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Authors and Affiliations

  1. Institute for Chemical Research, Kyoto University, Uji, 611-0011, Kyoto, Japan

    Misao Akishiba, Toshihide Takeuchi, Yoshimasa Kawaguchi, Kentarou Sakamoto, Hao-Hsin Yu, Ikuhiko Nakase & Shiroh Futaki

  2. Nanoscience and Nanotechnology Research Center, Research Organization for the 21st Century, Osaka Prefecture University, Naka-ku, Sakai, 599-8570, Osaka, Japan

    Ikuhiko Nakase

  3. School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya, 663-8179, Hyogo, Japan

    Tomoka Takatani-Nakase

  4. Department of Biochemistry and Biophysics, The Arrhenius Laboratories, Stockholm University, Stockholm, 10691, Sweden

    Fatemeh Madani & Astrid Gräslund

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Contributions

M.A. designed and executed most of the experiments. H.-H.Y., F.M. and A.G. were involved in the peptide design, evaluation of the endosomal escape and discussion of the peptide–membrane interaction. Y.K. and I.N. were involved in the experiments using saporin and exosomes, respectively. T.T.-N. was involved in the statistical analysis of the experimental data. K.S. was involved in the peptide synthesis and the biophysical studies. S.F. directed the project, designed the experiments, analysed the data and wrote the paper with T.T.

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Correspondence to Shiroh Futaki.

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Akishiba, M., Takeuchi, T., Kawaguchi, Y. et al. Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide. Nature Chem 9, 751–761 (2017). https://doi.org/10.1038/nchem.2779

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