Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide · Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide Misao Akishiba1, Toshihide Takeuchi 1,

Cytosolic antibody delivery by lipid-sensitiveendosomolytic peptideMisao Akishiba1, Toshihide Takeuchi1, Yoshimasa Kawaguchi1, Kentarou Sakamoto1, Hao-Hsin Yu1,Ikuhiko Nakase1,2, Tomoka Takatani-Nakase3, Fatemeh Madani4, Astrid Gräslund4 and Shiroh Futaki1*

One of the major obstacles in intracellular targeting using antibodies is their limited release from endosomes into thecytosol. Here we report an approach to deliver proteins, which include antibodies, into cells by using endosomolyticpeptides derived from the cationic and membrane-lytic spider venom peptide M-lycotoxin. The delivery peptides weredeveloped by introducing one or two glutamic acid residues into the hydrophobic face. One peptide with the substitutionof 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 preferentialdisruption of negatively charged membranes (endosomal membranes) over neutral membranes (plasma membranes), andthe endosomolytic peptide promotes the uptake by inducing macropinocytosis. The fidelity of this approach was confirmedthrough the intracellular delivery of a ribosome-inactivation protein (saporin), Cre recombinase and IgG delivery, whichresulted in a specific labelling of the cytosolic proteins and subsequent suppression of the glucocorticoid receptor-mediatedtranscription. We also demonstrate the L17E-mediated cytosolic delivery of exosome-encapsulated proteins.

Recent advances in molecular and cell biology have shed light onvarious key proteins in cells, which is important to develop newtherapies1. Antibodies are the supreme molecules for molecular

recognition and targeting2,3. To enable the access of antibodies to thecytosol provides a clearer understanding of the localization and behav-iour of target proteins. Modulating the molecular interplay thatinvolves these proteins also provides information on their biologicalsignificance, along with their feasibility as therapeutic targets.Although approaches to the intracellular delivery of antibodies andother bioactive proteins have been reported previously (for example,Cerrato et al.4, Ozay et al.,5 Erazo-Oliveras et al.6, Abraham et al.7,Dalkara et al.8, Kondo et al.9 and Wang et al.10), practical approachesbased on novel concepts are still needed at levels of efficacy thatachieve sufficient interactionswith cellular targetswithout cytotoxicity.

One of the major obstacles to intracellular targeting by antibodiesis their limited release from endosomes into the cytosol. Antibodiesare taken up by cells via endocytosis, which is a process that involvesthe physiological uptake of extracellular materials delivered intocells by encapsulation into vesicular compartments namedendosomes11. Without their release from vesicular compartmentsinto the cytosol, antibodies cannot interact with their targetmolecules, and may eventually be degraded in these compartments.

To overcome this issue, numerous approaches, including the use ofpH-sensitive membrane-perturbing peptides and polymers, have beenreported12–20. These molecules are designed to switch their structuresand lyse endosomal membranes, in accordance with the pH decreasein endosomal compartments. However, the current approaches areinadequate to achieve the complete release of antibodies fromendosomes2,3. This not only reduces the targeting efficacy of theantibodies, but also results in inaccurate signals during the in-cellanalysis and diagnostics. Therefore, there is a high demand for novelstrategies that achieve about a 100% release of endosome-encapsulatedantibodies into the cytosol.

We hypothesized that the reason for the incomplete endosomalescape of antibodies is an insufficient perturbation of the endosomalmembranes by the currently used endosomolytic agents. Most of thecurrently available pH-sensitive membrane-lytic peptides andpolymers are designed to perturb endosomal membranes preferen-tially by the protonation of carboxylates at an endosomal acidic pH(refs 12–15). GALA is a representative peptide in this class(WEAALAEALAEALAEHLAEALAEALEALAA-amide)12. Thispeptide has a net negative and potentially amphiphilic structure,and its hydrophilic face comprises a series of glutamate (Glu)residues. Negative charges on Glu residues in the extracellular regionprevent a peptide interaction with the membranes (no lysis). Theprotonation of Glu at an acidic pH allows a less-charged peptideto form a helical structure and interact with and perturb themembranes. However, the lytic activity of neutral (or negativelycharged) peptides is much less intense than that of cationic haemolyticpeptides21–23. In addition, cell surfaces have an overall negativecharge because of the abundance of proteoglycans and sialic acids,and the cellular uptake efficacy of negatively charged molecules islower than that of positively charged ones13. Without the incorpor-ation into endosomes, these pH-sensitive peptides do not havelytic activity.

Therefore, we employed cationic amphiphilic peptides with astrong membrane-lytic activity as the starting materials for thedesign of endosomolytic peptides (Fig. 1a). Cationic haemolyticpeptides often have basic amphiphilic helical structures. For mem-brane perturbation, the hydrophobic interaction of the peptideswith the membranes is critical21,22. The insertion of negativelycharged Glu into a hydrophobic face should effectively attenuatethe lytic activity, but still retain the ability to adsorb to the cellsurface. The facilitated entrapment of peptides into endosomesand the eventual protonation of Glu should recover the mem-brane-lytic activity of the peptides. Owing to the high cytotoxicity

1Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan. 2Nanoscience and Nanotechnology Research Center, Research Organizationfor the 21st Century, Osaka Prefecture University, Naka-ku, Sakai, Osaka 599-8570, Japan. 3School of Pharmacy and Pharmaceutical Sciences, MukogawaWomen’s University, Nishinomiya, Hyogo 663-8179, Japan. 4Department of Biochemistry and Biophysics, The Arrhenius Laboratories, Stockholm University,10691 Stockholm, Sweden. *e-mail: [email protected]

ARTICLESPUBLISHED ONLINE: 22 MAY 2017 | DOI: 10.1038/NCHEM.2779

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http://dx.doi.org/10.1038/nchem.2779

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Figure 1 | Design strategy of endosomolytic peptides and their selection. a, Design concept and working hypothesis of endosomolytic peptides. A cationic,amphiphilic peptide with a strong membrane lytic activity was selected as a design template for the endosomolytic peptides (top left). Its lytic activity wasattenuated by the insertion of negatively charged Glu residue(s) into the hydrophobic face while retaining the net positive charges of the peptide to facilitateits cell surface adsorption and endocytic uptake (top right). A reduced pH in the endosomes may make Glu more protonated and less hydrophilic.Endosomal membranes are abundant in negatively charged lipids, which increases the interaction with the cationic lytic peptides (bottom right). Thus, thepeptide preferentially perturbs the endosomal membranes to attain the cytosolic release of antibodies with sufficient efficacy to target cytosolic proteins(bottom left). Although the endosomolytic peptide obtained in this study (L17E) turned out to have a low helical structure, peptides are illustrated in thisfigure to promote an understanding of the design concept. A helical wheel projection (top right) of L17E is given. b, A marked cytosolic distribution ofAlexa488–dextran (10 kDa) facilitated by L17E indicates the fidelity of our design concept. HeLa cells were treated with Alexa488–dextran (10 kDa)(250 µg ml–1) in the presence of peptides (40 µM) in α-MEM(–) for 1 h, prior to washing with and incubation in α-MEM(+) for 3 h. The inset in eachpicture represents a magnified view of the boxed area. Scale bars, 100 µm.

ARTICLES NATURE CHEMISTRY DOI: 10.1038/NCHEM.2779

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of these lytic peptides, only minor attention has been paid to theirutilization for intracellular delivery, although the targeting of cat-ionic amphiphilic peptides to tumour cells and to their mitochon-drial membranes reportedly results in tumour-cell death23.

Recent studies revealed that endosomal membranes are rich innegatively charged lipids24,25. This confers another advantage forthe cationic peptides over the neutral or anionic peptides in thatthey have a higher affinity to perturb endosomal membranes.Although this difference in the charged states of membranes pro-vides important information for designing an intracellular antibodydelivery system, very few studies have focused on this issue.

In this report, we describe the impact of our approach of usingattenuated, membrane-lytic peptides for the cytosolic targeting ofbioactive proteins including saporin (ribosome-inactivatingprotein), Cre gene recombinase and antibodies (immunoglobulinsG (IgGs)). The applicability of this system to the cytosolic deliveryof exosome-encapsulated bioactive proteins is also demonstrated.

Results and discussionDesign strategy of endosomolytic peptides and their selection.Our design consisted of simply introducing Glu into naturalmembrane-lytic peptides (Fig. 1a). (1) The perturbation activity ofthe peptides on the cell surfaces (that is, the plasma membranes)that causes cytotoxicity is abrogated by the appropriateintroduction of negatively charged Glu into the hydrophobic faceof the amphiphilic helix (Fig. 1a (top)). (2) The extent of the Glusubstitution should be as small as possible to retain the cationicfeatures. This helps the peptides to adsorb on the cell surfaces,which leads to an efficient entrapment and accumulation intoendosomes (Fig. 1a (top right)). (3) We expected that theprotonation of the Glu carboxylate, under an endosomal acidicpH, should recover the net hydrophobicity of the peptide andperturbation activity on membranes (Fig. 1a (bottom right)). (4)In addition, the cationic feature of the peptides should enhancethe electrostatic interaction with the endosomal membranes(abundant negatively charged lipids) compared with that ofplasma membranes (rich in neutral lipids) (Fig. 1a (bottomleft)). Thus, we expected that the synergistic effects of (3) and (4)will effectively perturb the endosomal membranes, and (5)thereby facilitate the escape of antibodies from the endosomesand their entry into the cytosol to target cytosolic proteins(Fig. 1a (bottom left)).

To establish the validity of this approach, nine cationic amphi-philic peptides with haemolytic activity were selected as candidatesfor the design templates (Supplementary Table 1). For conveniencein the peptide preparation, those that do not exceed 25 residues inlength were selected. The perturbation activity of these peptideson cell membranes was evaluated using theWST-1 cell-proliferationassay26 after treatment with peptides for two hours. M-lycotoxin,derived from the venom of the wolf spider Lycosa carolinensis27,showed the lowest half-maximal effective concentration (EC50) of1.36 µM. Thus, we selected it as the design template for the Glusubstitution (Fig. 1a).

To reduce the cationic charges, we introduced one or two gluta-mic acids (Glu, E) into the peptide sequence (Table 1). M-lycotoxinhas a potential amphiphilic helical structure (Fig. 1a (top right)).Five lysines in the sequence and its N-terminus amino functionshould confer cationic charges and haemolytic activity to thepeptide. Five monosubstituted analogues (L6E, L9E, A13E, L17Eand Q21E) and four disubstituted analogues (L6E/G10E,L9E/A13E, A13E/L17E and L17E/Q21E) of wild-type (WT)M-lycotoxin were designed so that one or two amino acid residuesin the hydrophobic face were replaced by Glu residues (Table 1 andSupplementary Fig. 1). This simple substitution yielded a dramaticattenuation of the plasma-membrane-perturbation activity ofthe peptides. EC50 values of these analogues, as determined using

the WST-1 assay, were >40 µM (that is, more than an about30-fold reduction in cytotoxicity), except for Q21E (3.54 µM)(Supplementary Table 2).

Next, we evaluated the ability of these peptides to promote thecytosolic release of endocytosed biomacromolecules. HeLa cellswere incubated with dextran (10 kDa) labelled with Alexa Fluor488 (Alexa488–dextran, 250 µg ml–1) as a model macromoleculein medium for one hour, and the cellular distribution of theAlexa488–dextran signal was analysed using confocal laser scanningmicroscopy (CLSM) after three hours (Fig. 1b and SupplementaryFig. 2 gives differential interference contrast images). In theabsence of peptides, Alexa488–dextran yielded only punctate, dot-like signals in the cells, indicative of the predominant encapsulationof Alexa488–dextran into the endosomes (Fig. 1b (top left)). Incontrast, one of the Glu-substituted analogues (L17E) showed asignificant stimulation of the cytosolic release of endocytosedmolecules, as intended. Almost 50% of the cells co-incubated withL17E (40 µM) showed spread signals of Alexa488–dextran in thecytosol and in the nucleus (Fig. 1b (top right)), which indicatesthe effective cytosolic release of Alexa488–dextran. Almost 20 and5% of A13E- and L9E-treated cells, respectively, had cytosolic/nuclear Alexa488–dextran signals (Fig. 1b (top middle)). For thedisubstituted peptides, ∼30% of the cells yielded cytosolic/nuclearlocalization of Alexa488–dextran signals when treated with L17E/Q21E, followed by ∼15% of cells treated with A13E/L17E (Fig. 1b(bottom right)). On the other hand, when the cells were similarlytreated with GALA, as an example of a pH-sensitive lytic peptidebearing Glu residues, no significant cytosolic Alexa488–dextransignals were observed (Fig. 1b (bottom left)).

The fidelity of our design concept was confirmed further usingtwo analogues of L17E. One was the D-amino acid version ofL17E (D-L17E) (Table 1). If L17E requires some protein machinery(for example, a receptor and transporter) to induce cytosolic release,its enantiomer should have less activity. However, D-L17E similarlyyielded a marked cytosolic distribution of dextran, which suggeststhat the targets of action of L17E are not proteins but membranes(Supplementary Fig. 3a (left)). The other analogue was L17D, inwhich leucine (Leu) at position 17 is substituted for aspartate(Asp) (Table 1), which also bears a carboxylate in its side chain.L17D also yielded significant cytosolic dextran signals(Supplementary Fig. 3a (right)). However, the proportion of cellsthat bore a diffuse cytosolic distribution of dextran was approxi-mately 20%. The β-COOH group in Asp has a lower pKa (3.65)than that of the γ-COOH in Glu (4.25) (ref. 28), and the sidechain of Asp is missing one methylene compared with the Gluside chain. The increase in hydrophilicity should make L17D lesseffective than L17E.

Table 1 | Sequences of M-lycotoxin and its analogues.

Peptide SequenceWT IWLTALKFLGKHAAKHLAKQQLSKL-amideL6E IWLTAEKFLGKHAAKHLAKQQLSKL-amideL9E IWLTALKFEGKHAAKHLAKQQLSKL-amideA13E IWLTALKFLGKHEAKHLAKQQLSKL-amideL17E IWLTALKFLGKHAAKHEAKQQLSKL-amideQ21E IWLTALKFLGKHAAKHLAKQELSKL-amideL6E/G10E IWLTAEKFLEKHAAKHLAKQQLSKL-amideL9E/A13E IWLTALKFEGKHEAKHLAKQQLSKL-amideA13E/L17E IWLTALKFLGKHEAKHEAKQQLSKL-amideL17E/Q21E IWLTALKFLGKHAAKHEAKQELSKL-amideD-L17E iw l t a l k f l Gk ha a kh ea kqql sk l -amideL17D IWLTALKFLGKHAAKHDAKQQLSKL-amideL17EΔ(23–25) IWLTALKFLGKHAAKHEAKQQL-amideL17EΔ(20–25) IWLTALKFLGKHAAKHEAK-amide

Lowercase letters denote D-amino acids.

NATURE CHEMISTRY DOI: 10.1038/NCHEM.2779 ARTICLES





Next, we analysed the core sequence of L17E responsible for thedextran release. The very low level of dextran cytosolic release bythe insertion of Glu at positions 6 (L6E) and 9 (L9E) suggestedthe importance of the N terminus in the membrane interaction.Thus, we prepared L17E analogues with a C-terminal deletion ofpositions 23–25 (L17EΔ(23–25)) and 20–25 (L17EΔ(20–25))(Table 1). Although the former peptide yielded a diffuse cytosolicdistribution of dextran in 15% of cells, the latter only yielded 2%(Supplementary Fig. 3b), which indicates that positions 1–22comprise the minimum sequence that exerts the proper functionof L17E. Interestingly, although there were considerable differencesin the efficacies in dextran cytosolic release between L17E and theC-terminal deleted peptides L17EΔ(23–25) and L17EΔ(20–25),the cytotoxicities of WTΔ(23–25) and WTΔ(20–25) (that is,C-terminus-truncated versions of the WT that corresponds toL17EΔ(23–25) and L17EΔ(20–25)) were almost similar (EC50,<3 µM) to that of WT (1.36 µM) (Supplementary Table 3).Therefore, the peptide chain length may play a particular role inpromoting the cytosolic release of the endosomal encapsulates.

Notably, serum only yielded marginal effects on the cytosolicrelease of dextran (cells that show cytosolic distribution in thetotal cells were ∼50 versus ∼55% in the presence and absence ofserum, respectively), although it has often been claimed that itspresence decreases the efficacy of intracellular delivery14,15

(Supplementary Fig. 3c). No considerable cytotoxicity accompaniedthe L17E treatment. The cell viability with the L17E treatment, asdetermined by the WST-1 assay, was almost 90% when the cellswere treated with 40 µM L17E in serum-free medium for onehour or serum-supplemented medium for 24 hours(Supplementary Fig. 4a,b, respectively). Prolonged treatment ofthe cells with L17E (40 µM) for four days showed no observableeffect on cell growth and proliferation (Supplementary Fig. 4c).

The applicability of L17E to cytosolic delivery was also confirmedin cells other than human cervical carcinoma HeLa cells, such ashuman colon adenocarcinoma SW480 cells, African greenmonkey kidney fibroblast-like COS-7 cells and human umbilicalvein endothelial cells, whereas only a marginal effect was observedin mouse embryonic fibroblasts (NIH3T3) (Supplementary Fig. 5).

The intracellular fate of L17E was analysed by CLSM usingC-terminal Alexa488-labelled L17E (L17E–Alexa488). HeLa cellswere treated with 20 µM L17E–Alexa488 for one hour. Afterwashing and a medium change, the signals of the internalizedL17E–Alexa488 were analysed by CLSM (Supplementary Fig. 6a(left)). Although there was some cytosolic L17E–Alexa488 labelling,punctate L17E–Alexa488 signals were observed predominantly,which suggests that a considerable amount of L17E was kept inthe endosomes. Although L17E effectively perturbs the endosomalmembranes to release the endosomal contents into the cytosol,L17E itself is rather hydrophobic and may stay with the membranesrather than penetrate into the cytosol. The L17E–Alexa488 signalsbecame less apparent over time (Supplementary Fig. 6a), whichsuggests a possible intracellular degradation of L17E. After 24hours, signals were observed mostly at the perinuclear area, coloca-lizied with lysosome marker LysoTracker (Supplementary Fig. 6b).

Additionally, based on the fluorescent intensity of Alexa488 inthe cell lysates, it was estimated that only 0.5%, or less, of thetotal L17E–Alexa488 administrated into the culture media wastaken up by the cells (Supplementary Information gives the exper-imental details). Thus, L17E is taken up into endosomes in a non-specific manner; cytosolic delivery systems that are more efficientmay be established by the enhancement of the recruitment ofL17E into the endosomal compartments.

Cytosolic delivery of bioactive proteins using L17E. Theapplicability of this approach for the cytosolic delivery of bioactiveproteins by L17E was confirmed using saporin (30 kDa), a

membrane-impermeable ribosome-inactivation protein (Fig. 2a)29.The applicability of this toxin conjugated with monoclonalantibodies to tumour targeting has been reported30. When HeLacells were treated with 10 µg ml–1 saporin in the presence of L17Efor one hour, a cell death of ∼80% was obtained. However, in theabsence of L17E, cell death was only observed in ∼15% of thecells (Fig. 2a (right)).

The cytosolic delivery of bioactive proteins by L17E was furtherconfirmed by a gene recombination assay using Cre protein(38 kDa), an enzyme that catalyses the recombination between thespecific DNA sequences called loxP sites31. We used cells withplasmid that encodes the loxP-DsRed-loxP-EGFP gene, in whichthe red fluorescent protein, DsRed, is constitutively expressed andthe enhanced green fluorescent protein (eGFP) expression is sup-pressed by the stop codon at the end of the DsRed gene (Fig. 2b(left)). Once Cre is introduced into the cells, the protein facilitatessite-specific gene recombination to remove the DNA regionbetween the two loxP sites, which leads to a conditional expressionof eGFP (Fig. 2b (left)). When cells were treated with extracellularlyprepared Cre protein (5 µM) in the presence of L17E (40 µM), a sig-nificant expression of eGFP was observed in the DsRed-expressingcells (Fig. 2b (bottom right)), which suggests that a recombinationreaction was induced by the L17E-mediated delivery of Cre. In con-trast, no significant eGFP signals were observed after treatment withCre in the absence of L17E (Fig. 2b (top right)). These results collec-tively indicate that L17E allows the delivery of proteins into thecytosol and preserves the protein’s bioactivity.

Cytosolic proteins targeted by intracellularly delivered antibodiesusing L17E. The hydrodynamic radii of antibodies (IgGs) areestimated to be ∼5 nm, which is about twice as large as proteinsof ∼30 kDa (ref. 32). Therefore, a more-effective perturbation ofthe endosomal membranes is needed to achieve an efficient(complete) cytosolic release of IgG from the endosomes. We werevery pleased to find that L17E allowed a marked cytosolictargeting of the antibodies (Fig. 3a (top row) and SupplementaryFig. 7a). The cytosolic distribution of Alexa488-labelled IgG(polyclonal, from human serum) was observed for almost 40% ofthe cells, which suggests the cytosolic liberation of Alexa488-labelled IgG. There was no significant diffused cytosolicappearance of Alexa488-labelled IgG in the absence of L17E(Fig. 3a (bottom)). Additionally, the use of a high-sensitivityGaAsP detector allowed the observation of a significant cytosoliclocalization of Alexa488-labelled IgG signals even at a decreasedamount of Alexa488-labelled IgG (50 µg ml–1, that is, 1/20th ofthe amount employed in Fig. 3a) for 30 minutes in the presenceof 40 µM L17E (Supplementary Fig. 7b).

Next, the ability of exogenous antibodies to target cellularproteins in the presence of L17E was demonstrated (Fig. 3b). As amodel of cytosolic targets, a monomeric DsRed fluorescentprotein that bore a His6 recognition sequence (DsRed–His6) wasexpressed in HeLa cells. The N-terminal 20 amino acid sequenceof neuromodulin (mem)33 was fused with the protein as a mem-brane localization sequence. DsRed–His6 without the memsequence showed a diffuse distribution throughout the cells(Fig. 3b (bottom row, centre)). On the other hand, distinct signalsof mem–DsRed–His6 were observed on the plasma membranes(for example, the boxed areas in Fig. 3b (top row, centre)), inaddition to punctate staining, suggestive of the anchoring ofmem–DsRed–His6 onto the cytosolic face of the plasma membranesand intracellular vesicular compartments. A significant colocaliza-tion of the monoclonal anti-His6–IgG (labelled with fluorescein iso-thiocyanate (FITC)) with mem–DsRed–His6, anchored on theplasma membranes, was observed on incubation with L17E(Fig. 3b (top row, right, boxed area)), indicative of the actual target-ing of the intracellular protein by intracellularly delivered IgG. In






the absence of L17E, anti-His6–IgG yielded punctate signals. Nosignificant anti-His6–IgG signals colocalized with mem–DsRed–His6on the plasma membranes were observed (Fig. 3b (middle row,right boxed area)), which suggests that no substantial amount ofanti-His6–IgG was released from the endosomes to interact with thetarget molecules in the cytosol. Although there were some overlapsof the anti-His6–IgG signals with the mem–DsRed–His6 signals inthe perinuclear areas, these might be caused by anti-His6–IgG insidethe endosomal membranes and mem–DsRed–His6 on the cytosolicface of the endosomal compartments and are thus deceptive. Evenwhen the amount of anti-His6–IgG was reduced to 1/20th of thatused in Fig. 3b, the same tendencies of cellular localization of theanti-His6–IgG were observed using a high-sensitivity GaAsP detector(Supplementary Fig. 8, as for Supplementary Fig. 7b). Collectively,these results further support the usefulness of this approach forintracellular targeting.

Intracellularly delivered IgG also blocked intracellular signallingand transcription. The glucocorticoid receptor (GR) is a nuclear recep-tor that is activated by steroids, including dexamethasone (Dex)(Fig. 3c). The unbound receptor resides in the cytosol. After the recep-tor is bound to ligands, the activated GR complex translocates to thenucleus and initiates the transcription of glucocorticoid-responsivegenes, which include that for metallothionein 1E (MT1E). A possiblecontribution of MT1E to tumorigenesis has been suggested34.

We used real-time quantitative PCR to analyse the messengerRNA expression level of MT1E after the stimulation of HeLa cellswith Dex. On stimulation with 100 nM Dex, the cellular mRNA

level of MT1E became 5.3-fold higher than that in unstimulatedcells (Fig. 3c, first and second bars from the left). When the cellswere treated with monoclonal anti-GR–IgG in the presence ofL17E prior to Dex stimulation, the increase in MT1E mRNAexpression decreased to <60% of that in the absence of anti-GR–IgG treatment (Fig. 3c, third bar from the left), which indicates asignificant suppression of the Dex-induced transcription by theantibody. On the other hand, on pre-treatment of the cells withnonspecific IgG (human, polyclonal) instead of GR-specific anti-GR–IgG, no significant suppression was observed (Fig. 3c, rightbar). Anti-GR–IgG successfully recognized and interacted with thecytosolic GR, and inhibited the Dex-induced nuclear translocationof GR. In the absence of L17E, anti-GR–IgG had no effect on theMT1EmRNA expression (Supplementary Fig. 9). Thus, the feasibilityof using L17E for a cytosolic antibody delivery is demonstrated.

Stimulation of the cytosolic delivery of exosomal contents usingL17E. Exosomes have been considered a promising vehicle for theintracellular delivery of biomacromolecules, including bioactiveproteins and nucleic acids. However, the low efficacy of thecytosolic release of exosome-encapsulated molecules is an obstacleto achieving this goal35. We herein show that L17E may have theability to promote the endocytic uptake and cytosolic delivery ofexosome-encapsulated proteins.

First, the stability of exosomes on L17E treatment was confirmedin terms of FITC–dextran leakage from exosomes. Exosomes thatencapsulate FITC–dextran (4 kDa) were incubated with L17E

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Figure 2 | Cytosolic delivery of bioactive proteins using L17E. a, Enhanced cytosolic effect of a ribosome inactivation protein (saporin) by co-administrationwith L17E. HeLa cells were treated with saporin (10 µg ml–1) in the presence of L17E (40 µM) in α-MEM(–) for 1 h, prior to washing with and incubation inα-MEM(+) for 6 h. Then, the cell viability was analysed using the WST-1 assay (right). The results are presented as the mean ± s.e.m. (n = 3). b, Cre/loxPrecombination assay system and successful gene targeting via the intracellular delivery of Cre recombinase with the help of L17E. Cells were treated with Creprotein (5 µM) in the presence of L17E peptide (40 µM) for 1 h prior to protein washout and incubation for another 24 h to be subjected to confocalmicroscopic observation (right).






(40 µM) for one hour at 37 °C. The release of FITC–dextran intoPBS was analysed after separating the exosomes by ultracentrifuga-tion (Supplementary Table 4). The results showed that in both thepresence and absence of L17E, only ∼4% of FITC–dextran wasreleased. This confirms the absence of a marked perturbationeffect of L17E for exosomal membranes.

After confirming that L17E is unable to perturb exosomemembranes, the ability of L17E to release exosome-encapsulatedbiomacromolecules into the cytosol was then evaluated. HeLa cells

were treated with FITC–dextran-encapsulated exosomes in thepresence of L17E (40 µM) for two hours. CLSM analysis showeda significant cytosolic and nuclear distribution of dextran(Supplementary Fig. 10). This showed the effectiveness of theL17E-mediated cytosolic delivery of exosomal content. We alsoconfirmed the incorporation of exosomes into the endosomesthrough the CLSM analysis of exosomes that bore the CD63–GFPfusion protein (CD63–GFP–exosomes). CD63 is an exosome-associated membrane protein. CD63–GFP–exosomes were isolated

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Figure 3 | Cytosolic proteins targeted via intracellularly delivered antibodies using L17E. a,b, Efficient cytosolic release of IgGs (a) and the targeting ofmembrane-anchored proteins (b) in the presence of L17E. a, Cells were treated with Alexa488-labelled human IgG (1 mg ml–1) in the presence of L17E(40 µM) in α-MEM(–) for 1 h. After a cell wash, the cells were incubated further in α-MEM(+) for 30 min prior to CLSM analysis. b, A significantcolocalization of the monoclonal anti-His6–IgG (labelled with FITC) with mem–DsRed–His6, anchored on plasma membranes, was observed on incubationwith L17E (top row). The successful targeting of the cytosolic protein by IgG suggests that a significant fraction of the IgG retained its functional structureeven in a reducing cytosolic environment rich in glutathione47 (also see Goto and Hamaguchi48), although IgG is composed of four peptide chainscrosslinked via disulfide bridges. In the absence of L17E, anti-His6–IgG yielded punctate signals, and no significant anti-His6–IgG signals were observedon plasma membranes colocalized with mem–DsRed–His6 (middle row). Anti-His6–IgG incubated with cells that expressed DsRed–His6(without bearing a mem sequence) showed a diffuse appearance of anti-His6–IgG throughout the cells without localizing on membranes (bottom row).c, Blocked signalling and transcription by an intracellularly delivered antibody in the presence of L17E. The cells were incubated with 300 µg ml–1

anti-GR antibody in the presence of L17E (40 µM) in PBS(+) for 30 min at 37 °C. Then, the mRNA levels of MT1E mRNA on stimulation with Dex wereanalysed. The results are presented as the mean ± s.e.m. (n = 9). ***, P < 0.001; N/S, not significant (one-way analysis of variance (ANOVA) followed byDunnett’s post hoc test). d, L17E also enhances the efficacy of exosome-mediated intracellular delivery. HeLa cells were treated with exosomes that containedsaporin for 1 h. Then, FBS was added to the sample and incubated for 48 h. The results are presented as the mean ± s.e.m. (n = 4).






from the cells that express CD63–GFP fusion proteins. Treatment ofthe cells with CD63–GFP–exosomes in the presence of L17E yieldedsignificant signals of the CD63–GFP–exosomes in the cells, whichshows colocalization with the lysosome marker (that is,LysoTracker) (Supplementary Fig. 11). Taken together, exosome-encapsulated cargoes were released into the cytosol from the endo-somal compartment in the presence of L17E.

The feasibility of L17E for the intracellular delivery of exosome-encapsulated bioactive proteins was further demonstrated usingsaporin. Exosomes that contained saporin were treated with L17E(40 µM) in the medium for one hour, and the cytotoxicity wasevaluated using the WST-1 assay after 48 hours (Fig. 3d (left)).Although the treatment with saporin-encapsulated exosomes inthe presence of L17E led to significant cell death, the exosomeswere not toxic to the cells in its absence, which indicates the potentialof L17E in exosome-mediated intracellular delivery (Fig. 3d (right)).

Biophysical study on the perturbation of membranes by L17E: noapparent pH-sensitive membrane perturbation observed forL17E. We inserted a negatively charged Glu into the potentially

hydrophobic face of WT M-lycotoxin to attenuate the membraneinteraction of the peptide and membrane-lytic activity. Thepossible protonation of the Glu residues under a reduced pH inthe endosomes should achieve hydrophobicity and positivecharges of the peptides, by which the selective perturbation ofendosomal membranes would be expected. To validate our designstrategy, we conducted dye-leakage assays using artificialmembrane systems (Fig. 4a)36.

Large unilamellar vesicles (LUVs) that contain 8-aminonaphtha-lene-1,3,6-trisulfonic acid (ANTS) and its quencher, p-xylene-bis-pyridinium bromide (DPX), were treated with peptides at varyingconcentrations. Increases in fluorescence intensity by the liberationof ANTS from LUVs were analysed to evaluate the membrane-lyticactivity36. Given that the outer leaflet of the cell surface membranes(plasma membranes) is predominantly composed of neutral lipidsand that endosomal membranes are rich in negatively chargedlipids24,25, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC) was employed as a plasma membrane model lipid.Combinations of lipids of POPC and a negatively charged lipid,1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), were

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Figure 4 | Biophysical study on the perturbation of membranes by L17E. a,b, Preferable perturbation of negatively charged membranes by L17E (left),regardless of the pH (liposome leakage assay). WT M-lycotoxin (right) also shows a preferential perturbation of the negatively charged membranes,but produces a greater perturbation at a neutral pH than at an acidic pH. The membrane-perturbation ability of the WT peptide against respectivemembranes was in the order: POPC/POPG (3:1) LUVs at pH 7.4 (filled squares, [P/L]50= 3.3 × 10−4) > POPC/POPG (3:1) LUVs at pH 5.0 (open square,[P/L]50= 1.8 × 10−3) > POPC LUVs at pH 7.4 (filled circles, [P/L]50= 7.9 × 10−3) >> POPC LUVs at pH 5.0 (open circles, [P/L]50 > 0.3). The [P/L]50 value ofL17E against POPC/POPG (3:1) LUVs at pH 5.0 (1.2 × 10−2) (open circles, left graph) was comparable to that of WT against POPC LUVs at pH 7.4([P/L]50= 7.9 × 10−3) (filled circles, right graph), which suggests that L17E has a successful perturbation ability in the endosomes and leads to the cytosolicrelease of endosomal encapsulates. The results are presented as the mean ± s.e.m. (n = 3). b, CD spectra of M-lycotoxin (open circles) and L17E (filled circles)in 10 mM MES that contains 150 mM NaCl in the presence of 2.67 mM POPC/POPG (3:1) LUVs at pH 5.0 (left) and 7.4 (middle) (P/L ratio, 1.5 × 10−2) and1 mM POPC (100%) at pH 7.4 (right) (P/L ratio, 2.0 × 10−2). CD spectra of L17E suggest that the peptide has only a very weak helical structure. CD spectraof WT, Q21E, L17E, L9E and L17E/Q21E in the presence of POPC/POPG (3:1) LUVs at pH 5.0 and 7.4 are also given in Supplementary Fig. 15b.






employed as endosome model membranes (Fig. 4a)37. L17E inducedlittle dye leakage from POPC LUVs at pH 7.4 with a half-maximaleffective peptide/lipid ratio ([P/L]50) > 0.3 (Fig. 4a (left, filledcircles)). On the other hand, L17E showed a >25 times higher

perturbation activity against POPC/POPG (3:1) LUVs at pH 5.0,to yield a [P/L]50 value of 1.2 × 10−2 (Fig. 4a (right, opensquares)). This difference in perturbation activity supported thatendosome-selective perturbation of L17E stimulated the cytosolic

(4) Efficient cytosolic release ofbiomacromolecules

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Figure 5 | Inhibitor studies: L17E has a physiological effect that induces macropinocytosis and promotes cellular uptake. a, The absence of a diffusecytosolic appearance of Alexa488–dextran (10 kDa) when cells were treated at 4 °C for 30 min in the presence of L17E suggests the involvement ofendocytosis in L17E-mediated cytosolic delivery. b, A decrease in the numbers of cells that show diffuse cytosolic Alexa488–dextran signals by the treatmentof metabolic (NaN3) (left) and macropinocytosis (EIPA) (right) inhibitors. The results are presented as the mean ± s.e.m. (n = 3). *, P < 0.05; ***, P < 0.001(left, unpaired Student’s t-test; right, unpaired Student’s t-test with Welch’s correction). c, Stimulated Alexa488–dextran (10 kDa) uptake by the treatment ofcells with L17E in serum-free (left) and serum-supplemented (right) α-MEM for 1 h. The results are presented as the mean ± s.e.m. (n = 3). *, P <0.05;**, P < 0.01; ***, P < 0.001 (one-way ANOVA followed by Dunnett’s post hoc test). d, Increased exosome uptake by L17E. HeLa cells were treated withexosomes that contained FITC-labelled dextran in α-MEM(–) for 1 h and then in α-MEM(+) for 11 h. The results are presented as the mean ± s.e.m. (n = 3).**, P < 0.01; ***, P < 0.001 (one-way ANOVA followed by Dunnett’s post hoc test). e, Proposed mechanism of action of L17E: (1) Insertion of a Glu residuein the M-lycotoxin sequence led to the attenuation of the membrane-lytic activity. (2) The induction of macropinocytosis by the peptide led to an enhancedcellular uptake of L17E and biomacromolecules (for example, antibodies). (3) L17E showed a lipid-sensitive disruption of endosomal membranes. However,unlike conventional endosomolytic peptides and polymers, L17E had no apparent pH sensitivity in membrane lysis. (4) Antibodies and other proteins wereeffectively released into the cytosol with the help of L17E to have their bioactivities and functions.






release of endocytosed biomacromolecules very well. However, sur-prisingly, L17E had a perturbation activity against POPC/POPG(3:1) LUVs at pH 7.4 (Fig. 4a (left, filled squares, [P/L]50 = 1.0 ×10−2)) comparable to that at pH 5.0 (see above). Therefore, L17Ehad little pH dependence, but had a specific perturbation ability tonegatively charged membranes, which yields a marked contrast toconventional pH-dependent endosomolytic peptides and polymers.

To determine the reason for this lack of pH dependence,methods of membrane perturbation were analysed using WTpeptide (Fig. 4a (right)). The WT peptide also showed a higher per-turbation for negatively charged membranes than for neutral mem-branes. However, a certain pH dependence was also observed for theWT peptide, which showed a higher perturbation activity at pH 7.4than at 5.0, against POPC/POPG (3:1) and POPC (100%) LUVs(Fig. 4a (right)). Thus, the apparent lack of pH dependence in themembrane-perturbation ability of L17E may be explained asfollows. Glu is in its charged state at pH 7.4, and insertion of thischarged residue into the WT sequence should yield a considerabledecrease in the membrane-perturbation activity. Alternatively, WThas lower membrane-lytic activity at pH 5.0 (Fig. 4a (right)), andthe partial protonation of Glu at pH 5.0 (which makes the peptidemore hydrophobic) should yield a smaller effect of Glu insertionon the membrane perturbation than at pH 7.4. L17E may thusachieve a similar membrane perturbation at pH 5.0 to that at pH7.4, to yield a pH-independent method of membrane perturbationof L17E (Supplementary Fig. 12a). The same explanation wouldbe applicable to L17E/Q21E and Q21E peptides (SupplementaryFig. 13). Thus, L17E can be considered to have an attenuated mem-brane-lytic activity compared with WT. One might wonder whethera similar endosomolytic effect could be obtained using a lower con-centration of WT. Cell-penetrating peptides may also be able tostimulate the endosomal escape of biomacromolecules. However,treatment with either 1 µM WT or 40 µM R8 (a representativecell-penetrating peptide) yielded no significant cytosolic appearanceof Alexa488–dextran (Supplementary Fig. 14).

Compared with L17E, L17D had an inferior ability to releaseendosome-trapped Alexa488–dextran into the cytosol(Supplementary Fig. 3a). This parallels the dye-leakage assayresults (Supplementary Fig. 12b). L17D induced dye leakagefrom POPC/POPG (3:1) LUVs at pH 5.0 with [P/L]50 > 0.3(Supplementary Fig. 12b, open squares), which is considerablylower than that of L17E ([P/L]50 = 1.2 × 10−2). As described above,Asp is more hydrophilic than Glu, and therefore the substitutionof Leu to Asp on the WT peptide results in a weaker membrane-perturbation activity compared to that with Glu substitution(Supplementary Fig. 12b).

As amphiphilic helical structures are the canonical conformationthat yields membrane perturbation, many conventional pH-sensi-tive endosomolytic peptides have been designed to have anenhanced helical structure at a reduced pH. However, differentfrom these peptides, no enhanced helical structure was observedfor L17E at a reduced pH (Fig. 4b). Although the WT peptideshowed a typical helical structure, the circular dichroism (CD)spectrum of L17E in the presence of POPC/POPG (3:1) LUVs atpH 5.0 was suggestive of a very weak helical structure (Fig. 4b(left)), even though this peptide showed considerable lytic activityunder these conditions. Analysed by molecular ellipticity at 222nm ([θ]222) as a measure of helical content38, a rather small decreasein helical content was observed at the reduced pH ([θ]222 at pH 7.4and 5.0 were –6.1 × 103 and –4.9 × 103 deg cm2 dmol−1, respect-ively) (Fig. 4b (centre)). No significant concentration effects wereobserved on the secondary structure of L17E, regardless ofwhether the peptide concentration induced membrane lysis (P/Lratio of 0.5–2.0 × 10−2) (Supplementary Fig. 15). L17E also had avery low level of helical structure in the presence of POPC LUVsat pH 7.4 (Fig. 4b, right). Therefore, the L17E membrane-

perturbation effects could be a result of the electrostatic interactionwith negatively charged membranes, without the need for amphi-philic helical structure formation. The insertion of a single Gluresidue into the hydrophobic face effectively acts as a safety catchof the membrane-perturbation ability of the peptide.

Inhibitor studies: L17E has a physiological effect that inducesmacropinocytosis and so promotes cellular uptake. To evaluatefurther the fidelity of our design strategy, the involvement ofendocytosis in the L17E-mediated cytosolic release was confirmedby the treatment of cells at 4 °C (at this temperature, endocytosisdoes not occur)39 (Fig. 5a and Supplementary Fig. 16). Nosignificant Alexa488–dextran signal was observed in the cellsunder this treatment. Sodium azide treatment leaves the cells inan ATP-depleted state and thus inhibits endocytosis40. Thistreatment also diminished the number of cells with a diffuseappearance of the cytosolic dextran, which further supports theinvolvement of endocytosis in the L17E-mediated cytosolic releaseof biomacromolecules (Fig. 5b (left)).

More-detailed studies suggested the involvement of macropino-cytosis in this process (Fig. 5b (right)). Macropinocytosis is afluid-phase form of endocytosis driven by actin organization41,and often plays a role in the cellular entry of viruses and cationiccell-penetrating peptides42,43. Significant suppression in the L17E-mediated cytosolic diffusion of Alexa488–dextran was observed bytreatment with EIPA (5-(N-ethyl-N-isopropyl)amiloride), a repre-sentative macropinocytosis inhibitor (Fig. 5b (right))39. L17E mayalso be able to induce macropinocytosis. An increased uptake ofdextran (also employed as an extracellular fluid marker) is acriterion that defines macropinocytosis. In fact, about a 40%increase in Alexa488–dextran uptake was attained by L17E treat-ment regardless of the absence or presence of serum (Fig. 5c (leftand right, respectively)). The enhanced uptake of exosomes wasalso observed in the presence of L17E (Fig. 5d). L17E has a positivelycharged structure even after the insertion of Glu. The cationicfeature of this peptide not only helps L17E to adsorb on cell surfaces,but may also help to induce macropinocytosis, as in the case of cat-ionic cell-penetrating peptides39,43. This eventually leads to anenhanced cellular uptake of the molecular cargo, together withL17E, to accelerate their cytosolic targeting. Treatment with cla-thrin-mediated endocytosis inhibitors, which include phenylarsineoxide44, monodansylcadaverine45 and dynasore46, did not preventthe cytosolic distribution of dextran, which suggests that clathrin-mediated endocytosis does not significantly contribute to thisprocess (Supplementary Fig. 17).

ConclusionsIn this report, we present a novel concept for the design of endoso-molytic peptides that effectively deliver biomacromolecules, includ-ing antibodies, into the cytosol (summarized in Fig. 5e). Here theintroduction of a single negatively charged Glu residue into thepotentially hydrophobic face of M-lycotoxin, a cationic mem-brane-lytic peptide, effectively acted as a safety catch to modulatethe lytic activity of this peptide, and selectively perturb the endoso-mal membranes. The membrane-lytic activity is induced by theelectrostatic interaction of the positively charged peptide with thenegatively charged endosomal membranes. Unlike the currentpH-sensitive lytic peptides, L17E does not have a significant pH-dependent membrane lytic activity. In contrast with the WTpeptide, no enhanced helical structure is needed for L17E-mediatedmembrane lysis. This cationic feature of L17E not only facilitates thecytosolic release of endocytosed biomacromolecules in terms ofphysicochemical peptide–membrane interactions, but should alsostimulate the physiological uptake of the cargo via the inductionof macropinocytosis. Although many endosome-lytic peptides andpolymers have been reported, to the best of our knowledge, this is






the first demonstration of peptides that can achieve an enhanced cel-lular uptake via the induction of macropinocytosis, which leads toan efficient cytosolic delivery. Thus, our approach should beuseful to design systems that achieve an effective cytosolic deliveryof biomacromolecules.

In our approach, biomacromolecules are endocytosed into cellsin nonspecific manners, without using specific machineries tocapture and accumulate these biomacromolecules into endosomes.Improvements of the system should be achievable by a greaterrecruitment of the cargoes of interests together with endosomolyticpeptides into endosomal compartments. To develop peptides with ahigher endosomolytic activity that achieve a similar extent of endo-somolytic activity with less of the peptide is also a practicalapproach, which is under study in our laboratory.

MethodsCellular uptake of 10 kDa dextran and microscopic observation. HeLa cells wereseeded into 35 mm glass-bottom dishes (Iwaki), and the cells were allowed to reach80–90% confluence in 24 h. These cells were washed twice with serum-free α-minimum essential medium (α-MEM(–)), and then incubated with 250 µg ml–1

dextran (10 kDa) labelled with Alexa Fluor 488 (Alexa488–dextran) (Invitrogen) inthe presence of a peptide (40 µM) in α-MEM(–) for 1 h at 37 °C. Cells were washedwith α-MEM(–) and incubated in serum-supplemented α-MEM (α-MEM(+)) for 3h at 37 °C. The cellular uptake of dextran was analysed in live cells using anFV1000 Olympus confocal laser scanning microscope. The numbers of thecells that show a diffuse cytosolic signal of dextran among 250–700 cells werecounted and the percentages were used as a measure of the endosomolyticactivity for each sample.

Recognition of membrane-anchored DsRed–His6 proteins by intracellularlydelivered anti-His6 antibody. HeLa cells plated on 35 mm glass-bottom dishes(1.5 × 105 cells) were transfected with expression vectors for DsRed–His6 andmem–DsRed–His6 (DsRed–His6–pCI and mem–DsRed–His6–pCI, respectively)using Lipofectamine LTX (Invitrogen), in accordance with the manufacturer’sinstructions. After incubation for 46 h, the cells were treated with anti-6× His tagantibody (FITC) (ab1206, Abcam) (500 µg ml–1) in the presence or absenceof L17E (40 µM) in α-MEM(–) for 1 h at 37 °C, followed by incubation foranother 30 min in α-MEM(+). The localization of the fluorescently labelledantibody in live cells was analysed by CLSM. In addition, the ability of theanti-6× His tag antibody (FITC) to recognize correctly the His-tagged proteins in thecells was confirmed by CLSM, for which the cells were fixed in 4% paraformaldehydeand permeabilized with 0.5% Triton X-100 prior to staining with the antibody(Supplementary Fig. 18).

Inhibition of MT1E mRNA induction by intracellularly delivered anti-GRantibody. HeLa cells were seeded in a 24-well plate (Iwaki) in Dulbecco’sModified Eagle Medium without phenol red (DMEM(–)) supplemented with 10%charcoal-stripped fetal bovine serum (FBS) (Wako) (DMEM(+)) so that the cellswere 80–90% confluent in 24 h. Cells were washed twice with PBS and thenincubated with 300 µg ml–1 anti-GR antibody (BuGR2) (Invitrogen) in thepresence of L17E (40 µM) in PBS supplemented with 1 mMMgCl2 and 1 mMCaCl2(PBS(+)) for 30 min at 37 °C. Cells were washed twice with PBS and thenincubated with 100 nM Dex in serum-free medium for 60 min. After incubation,cells were washed twice and additionally incubated for 2 h in DMEM(+).Cells were washed with PBS twice and total RNA was extracted using theRNeasy Mini Kit (Qiagen), in accordance with the manufacturer’s instructions.cDNA was synthesized from total RNA using a PrimeScript RT reagent kit(Takara Bio), and real-time PCR was performed with a 7300 Real-Time PCR System(Applied Biosystems) using PowerUp SYBR Green PCR Master Mix(Applied Biosystems).

ANTS/DPX leakage assay. Peptides were incubated for 1 h at 37 °C with 134 µMLUVs in 10 mM MES buffer that contained 150 mM NaCl (pH 7.4 or 5.0). Thefluorescence intensity was measured at an excitation wavelength of 355 nm andemission of 535 nm using a Wallac 1420 Victor2 Microplate Reader (PerkinElmer).The percentage of ANTS released was calculated as follows: % ANTSreleased = 100 × ([Fobs–F0]/[Fmax–F0]), where Fobs represents the observedfluorescence intensity, Fmax is the maximum fluorescence intensity after addingTriton X-100 at a final concentration of 0.1% (v/v) and F0 represents the initialfluorescence intensity.

Data availability statements. All the available data can be obtained by contactingthe corresponding author.

Received 21 April 2016; accepted 11 April 2017;published online 22 May 2017

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AcknowledgementsThis work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHIGrant nos JP15H02497 and JP16H01145. This work was also supported by theCollaborative 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.

Author contributionsM.A. designed and executedmost of the experiments. H.-H.Y., F.M. and A.G. were involvedin 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 andexosomes, respectively. T.T.-N. was involved in the statistical analysis of the experimentaldata. K.S. was involved in the peptide synthesis and the biophysical studies. S.F. directed theproject, designed the experiments, analysed the data and wrote the paper with T.T.

Additional informationSupplementary information is available in the online version of the paper. Reprints andpermissions information is available online at www.nature.com/reprints. Publisher’s note:Springer Nature remains neutral with regard to jurisdictional claims in published maps andinstitutional affiliations. Correspondence and requests for materials should be addressed to S.F.

Competing financial interestsThe authors declare no competing financial interests.





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