LETTER
Direct and Rapid Cytosolic Delivery Using Cell-Penetrating Peptides Mediated by Pyrenebutyrate Toshihide Takeuchi†, Michie Kosuge†, Akiko Tadokoro†, Yukio Sugiura†, Mayumi Nishi‡, Mitsuhiro Kawata‡, Naomi Sakai§, Stefan Matile§, and Shiroh Futaki†,¶ ,* †
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan, ‡Department of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan, §Department of Organic Chemistry, University of Geneva, Geneva, Switzerland, ¶and SORST, JST, Kawaguchi, Saitama 332-0012, Japan
A
rginine-rich peptides such as oligoarginines (1, 2) and Tat (3) have been shown in numerous studies to translocate across biomembranes. These peptides have been utilized as delivery vectors that bring exogenous proteins and various molecules into cells to modulate cellular functions. There is still dispute as to exactly how these cationic peptides enter cells, but we and others have shown that the guanidinium cations on arginine residues play crucial roles in intracellular delivery (4–8). We have also recently shown that negatively charged counteranions with high hydrophobicity, such as phosphatidylglycerol and pyrenebutyrate, can exert a great influence on the translocation behavior of arginine peptides in artificial membranes (9–11). The positively charged arginine peptides electrostatically interact with the counteranions to increase their net hydrophobicity, thus, facilitating direct translocation through the lipid bilayers. The particular effectiveness of polyaromatic tails as found in pyrenebutyrate is thought to originate from ion pair stabilization of the aromatic surface as well as accelerated translocation due to their interfacial preference (10, 11). Focusing on the concept of counteranionmediated delivery, we first investigated whether counteranions can have effects on the translocation of arginine-rich peptides through biological membranes of live cells. Pyrenebutyrate was selected as one of the
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representative counteranions, and octaarginine (R8) as a representative peptide vector (Figure 1, panel a). Preliminary quantification assay using fluorescence-activated cell sorter (FACS) has shown the significant increase in total cellular uptake of R8 into living cells in the presence of pyrenebutyrate (10). Therefore, microscopic observation was performed to examine the effect of pyrenebutyrate on the methods of internalization of R8 peptide as well as the intracellular distribution. HeLa cells were preincubated with pyrenebutyrate, followed by incubation with Alexa 488-labeled R8 peptide. The R8 peptide diffusely localized throughout the cytosol and more strongly in the nucleus (Figure 1, panels b and c). Surprisingly, this pattern of fluorescence was observed within a few minutes’ treatment of the cells with the R8 peptide after the initial 2-min preincubation with pyrenebutyrate (Figure 1, panel b). This diffuse labeling of internalized peptide was seen in almost all the cells, and very few endosome-like punctate signals were observed in this time period. Conversely, no significant R8 peptide fluorescence was observed in the cells incubated in the absence of pyrenebutyrate (Figure 1, panel d). This suggests a critical role for this compound in peptide translocation. Internalization of the R8 peptide was also observed by the treatment of the cells with the R8 peptide prior to addition of pyrenebutyrate or by the simultaneous addi-
A B S T R A C T Intracellular delivery of bioactive molecules using arginine-rich peptides, including oligoarginine and HIV-1 Tat peptides, is a recently developed technology. Here, we report a dramatic change in the methods of internalization for these peptides brought about by the presence of pyrenebutyrate, a counteranion bearing an aromatic hydrophobic moiety. In the absence of pyrenebutyrate, endocytosis plays a major role in cellular uptake. However, the addition of pyrenebutyrate results in direct membrane translocation of the peptides yielding diffuse cytosolic peptide distribution within a few minutes. Using this method, rapid and efficient cytosolic delivery of the enhanced green fluorescent protein (EGFP) was achieved in cells including rat hippocampal primary cultured neurons. Enhancement of bioactivity on the administration of an apoptosisinducing peptide is also demonstrated. Thus, coupling arginine-rich peptides with this hydrophobic anion dramatically improved their ability to translocate cellular membranes, suggesting the great impact of this approach on exploring and controlling cell function.
*To whom correspondence should be addressed. E-mail:
[email protected]. Received for review March 18, 2006 and accepted May 18, 2006 Published online June 16, 2006 10.1021/cb600127m CCC: $33.50 © 2006 by American Chemical Society
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Figure 1. Direct and facile translocation of the R8 peptide into the cyotsol in the presence of pyrenebutyrate without using endocytosis. a) A schematic representation of pyrenebutyrate (1-pyrenebutyric acid)-mediated delivery of the octaarginine (R8) vector into cells. b) A time-course observation of peptide internalization. HeLa cells were preincubated with pyrenebutyrate in PBS for 2 min prior to the addition of R8-Alexa in PBS (see Methods) at 37 °C and analyzed by confocal microscopy without washing the cells. c) Prominent cytosolic diffusion of the R8 peptide in the presence of pyrenebutyrate in PBS. HeLa cells were incubated for 4 min with R8-Alexa in the presence of pyrenebutyrate after pretreatment with pyrenebutyrate as in panel b. d) In the absence of pyrenebutyrate, no significant signals of R8-Alexa were observed under the same conditions. e) When HeLa cells were incubated with R8Alexa (10 M) in a serum-containing medium for 1 h at 37 °C, punctate signals of the peptide suggestive of endocytic uptake were predominantly observed. Scale bar, 20 m (panels c– e). f) Peptide concentration-dependent increase in cell-associated fluorescence in pyrenebutyrate-treated cells. g) No significant effect of the pyrenebutyrate treatment on cell membrane integrity (LDH release assay). h) No significant pyrenebutyrate-induced cytotoxicity. Error bars represent the mean ⴞ standard deviation (SD) of three samples (panels f– h).
tions of the R8 peptide and pyrenebutyrate. However, the internalization efficiency of the peptide in the latter two cases seems to be slightly lower than in the former case, presumably due to the more prominent aggregation of pyrenebutyrate with R8 peptide (Supplementary Figure 1). Therefore, later experiments were conducted by incubating cells with pyrenebutyrate prior to addition of peptide. The quantity of internalized peptide in the presence of 50 M pyrenebutyrate increased linearly with increasing peptide concentration, and no significant threshold in the peptide concentration was observed for the cellular uptake under the given conditions (Figure 1, panel f). Damage to the plasma membranes by this pyrenebutyrate/R8 treatment would 300
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yield nuclear staining with propidium iodide. However, no nuclear staining was observed, suggesting that this cytosolic peptide labeling was not due to membrane disruption (data not shown). The integrity of the membranes and the absence of cytotoxicity by this treatment were further confirmed by the lactate dehydrogenase (LDH) release assay and MTT [⫽3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-2Htetrazolium bromide] assay (Figure 1, panels g and h, respectively). This efficient cytosolic delivery of the R8 peptide in the presence of pyrenebutyrate was similarly observed in other cell lines such as COS-7, PC12, CHO-K1, and RAW264.7 (data not shown) and was also observed with other membrane-permeable peptides derived TAKEUCHI ET AL.
from HIV-1 Tat (12), HIV-1 Rev (4), and Antennapedia (Penetratin) (13) (Supplementary Figure 2), indicating the wide applicability of this concept. The ultimate goal of this technology should be the delivery of large molecular weight proteins into the cytosol. To assess the effectiveness of this approach to cytosolic protein delivery, an enhanced green fluorescent protein (EGFP) bearing an R8 segment on its N-terminus (R8-EGFP, molecular mass ⬃30 kDa) was prepared. Surprisingly, a similarly diffuse R8-EGFP fluorescence was observed in more than 70% of the HeLa cells (Figure 2, panel a, left and middle panels). As in the case of the R8 peptide (Figure 1, panel c), R8-EGFP was distributed diffusely in the cytosol www.acschemicalbiology.org
LETTER but also strongly in the nucleus (Figure 2, panel a, middle panel), which is very similar to that of the corresponding protein intracellularly expressed by transfection (Figure 2, panel b). The efficiency of obtaining EGFP fluorescent cells was much higher compared to transfection. In contrast, when the cells were similarly incubated with EGFP lacking the R8 segment in the presence of pyrenebutyrate, no significant signals of EGFP were observed (Figure 2, panel a, right panel). Transfection of nondividing cells such as neuronal primary cultures is difficult. We therefore determined whether the effects of pyrenebutyrate were also observed in rat hippocampal primary cultured neurons. Confocal microscopic observation of the cells revealed that these cells stained strongly for R8-EGFP (Figure 2, panel c). Interestingly, it was often observed that neurites were as effectively stained as the cell bodies. At this concentration, internalization of the R8-EGFP was observed for about 40% of the hippocampal cells. When the concentration of R8-EGFP was raised to 20 M, this increased to ⬎80% (Figure 2, panel d). No significant toxicity was observed under these treatments. It is important to determine whether these pyrenebutyrate effects can be expanded to show a biological effect due to increased translocation of a delivery vector; the results of quantification of cell-associated peptides and the eventual biological effects are not universally parallel (14). We therefore applied this counteranion-mediated delivery to investigate whether this compound would enhance the effects of an apoptosis-inducing peptide (pro-apoptotic domain peptide, PAD) (15) that mediates its effect in the cytosol. PAD is an amphiphilic basic peptide including 14 residues of D-amino acids, D-(KLAKLAK)2. It has been reported that cytosolic delivery of the PAD peptide leads to mitochondrial membrane disruption and eventual apoptosis (14–16). On the other www.acschemicalbiology.org
hand, the peptide is nontoxic without being internalized into cells. A dramatic effect of the addition of pyrenebutyrate was confirmed by the observation of mitochondrial depolarization using a mitochondrial membrane-potentialsensitive dye, JC-1. This marker exhibits a potential-dependent accumulation in mitochondria (J-aggregates), and they are observed as red fluorescence. When the mitochondrial membrane is depolarized, the fluorescence changes to green (17). When HeLa cells were treated with R8-PAD in the absence of pyrenebutyrate, red fluorescent aggregation was predominantly observed (Figure 3, panel a, left and center panels). In contrast, when the cells were pretreated with pyrenebutyrate, green fluorescence was observed in substantially all of the cells (Figure 3, panel a, right panel) only after this treatment with R8-PAD. This
suggests that effective mitochondrial depolarization was accomplished by the accelerated internalization of R8-PAD in the presence of pyrenebutyrate. The biological effect was also assessed on the basis of cell death induction mediated by the intracellularly delivered apoptosis-inducing peptide (R8-PAD) using MTT assays. Cell viability was approximately 50% (Figure 3, panel b). However, preincubation of cells in pyrenebutyrate (10 min) before addition of the R8-PAD peptide reduced cell viability, and the enhancement of cell death was dependent on the concentration of pyrenebutyrate. The highest activity (⬍20% cell viability) was observed in cells treated in the presence of 50 M pyrenebutyrate. These results suggest that not only cellular uptake but also the biological effect can be enhanced by this counteranion-mediated delivery.
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Figure 2. Efficient cytosolic delivery of EGFP protein attained. a) Internalization of the enhanced green fluorescent protein (EGFP) bearing the R8 peptide (R8-EGFP) into HeLa cells. Cells were treated with R8-EGFP (10 M) in PBS in the presence of pyrenebutyrate as described in Figure 1, panel c (left and middle panels). EGFP without the R8 segment (10 M) showed very little internalization even in the presence of 50 M pyrenebutyrate (right panel). b) A similar cellular localization pattern as in panel a was observed in cells transfected with the plasmid coding R8-EGFP (pR8-EGFP). c) Internalization of R8-EGFP into neurites including cell bodies of hippocampal primary cultured neuronal cells. The cells were treated as in panel a. d) Diffuse labeling was observed in more than 80% of the cells within 4 min after treatment with pyrenebutyrate (50 M) and R8-EGFP (20 M) in PBS (left panel). No significant internalization of R8-EGFP was observed for the cells similarly treated in the absence of pyrenebutyrate (right panel). Scale bars, 20 m (panel a– c) and 50 m (panel d). VOL.1 NO.5 • 299–303 • 2006
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Figure 3. Reinforcement of cytosolic activity by the proapoptotic domain (PAD) peptide intracellularly delivered in the presence of pyrenebutyrate. a) Accelerated cancellation of the mitochondrial membrane potential by the R8-PAD peptide in the presence of pyrenebutyrate. As controls, HeLa cells were treated with R8-PAD (5 M, 4 min) in serum containing ␣-MEM (left panel) or PBS (middle panel) in the absence of pyrenebutyrate. Alternatively, the cells were treated with pyrenebutyrate (2 min) prior to incubation with R8-PAD (4 min) (final concentration of pyrenebutyrate and R8-PAD, 50 and 5 M, respectively) (right panel). The effect of the PAD peptide on the respective cells was then analyzed by confocal microscopic observation after washing the cells twice with PBS and incubating with a mitochondrial membrane potential-sensitive marker, JC-1 (5 M) at 37 °C for 30 min. Scale bar, 20 m. b) Enhanced cytosolic activity of the PAD peptide by pyrenebutyrate. Error bars represent the mean ⴞ SD of six samples.
brane and cytosol to reach the membrane dure, these results should open new opporAs for mechanisms that enable this efficient internalization, a direct membrane compartments. No significant differences in tunities to the utilization of this approach to the studies in cell biology. This approach translocation without a requirement for cellular distribution were observed by the can also be applicable to the introduction of endocytosis is postulated (Figure 1, treatment of the cells with the R8 peptide. panels b and c). As support of this, a very Therefore, pyrenebutyrate acts “like a trans- chemically modified proteins, e.g., with fluorescent moieties and cross-linking agents similar cellular distribution, as well as location catalyst” to accelerate the transloincrease in cellular uptake, was observed in cation of the peptides. In addition, once the into cells, as well as natural bioactive the presence of pyrenebutyrate even when R8 peptide was delivered into cells, pyrene- proteins. High-throughput screening systems may be established by using this the cells were treated with R8-Alexa at 4 °C, butyrate could be immediately removed system. Great possibility in the interface of where endocytosis does not work (Supplefrom cells simply by washing in PBS, and chemistry and biology will come up to mentary Figure 3). This yields a marked this would prevent possible damage to the explore and control cell function. difference from observations of cells treated cells that may be induced by a prolonged This approach has a limitation in that it is with 10 M R8 peptide at 37 °C in the incubation with pyrenebutyrate (Figure 4). not applicable in the presence of a medium absence of pyrenebutyrate (Figure 1, As shown above, the pyrenebutyrateor serum; the competition with various ionic panel e); the punctate signals observed in mediated translocation of arginine-rich species in the medium would hamper the the perinuclear area suggested that the peptides achieves direct and efficient interaction of the arginine vectors with majority of the peptide in this case was delivery of bioactive proteins bearing argipyrenebutyrate. Screening efforts to find taken up and was trapped in endocytic nine vectors into the cytosol. Importantly, serum-insensitive counteranion activators vesicles (14, 18). In addition, the importhis increase in translocation was shown to are ongoing. However, the high efficiency of tance of the membrane potential is manifest in a biological response. As there this approach would outweigh this limitaproposed as a driving force in this was no toxicity associated with this procetion. A few minutes’ treatment of the cells counteranion-mediated translocation with pyrenebutyrate/peptides in PBS (Supplementary Figure 4), as was is sufficient for enhancing translocaobserved for the internalization of Pyrenebutyrate Pyrenebutyrate + R8 PBS wash tion, and the incubation medium arginine-rich peptides into liposomes could then be replaced with a more (19) and suspension cells (20) in the complete medium for longer incubaabsence of pyrenebutyrate. tions. For therapeutic applications, Interestingly, the fluorescence an ‘ex-vivo’-like approach may be microscopic images of the pyreneemployable, where isolated cells are butyrate indicated that the majority treated with target proteins and then of the pyrenebutyrate stayed in the Figure 4. PBS wash of the cells yields immediate removal incorporated into the body again. membrane compartments in the cells, of pyrenebutyrate from cells. HeLa cells were treated with Overall, considering these especially those in the perinuclear pyrenebutyrate for 2 min (left panel), followed by nonfluorescently labeled R8 peptide (final concentration of outstanding features, the region (Figure 4). The intracellular pyrenebutyrate and R8, 50 and 10 M, respectively) for pyrenebutyrate-mediated delivery distribution of pyrenebutyrate was 4 min, and immediately analyzed by fluorescence using arginine-rich peptides will observed after only 2 min incubation microscopy without washing (middle panel) or, provide a new concept in membrane with the cells; this molecule freely alternatively, washed 3ⴛ with PBS prior to microscopic translocation. diffuses through the plasma memobservation (right panel). Scale bar, 20 m. 302
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LETTER METHODS Peptides and Proteins. All the peptides used in this report were chemically synthesized by Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis as reported (16). Actual sequences of the synthesized peptides: R8-Alexa, RRRRRRRRGC(Alexa)-amide; Tat-Alexa, GRKKRRQRRRPPQC(Alexa)-amide; Rev-Alexa, TRQARRNRRRRWRERQRGC(Alexa)-amide; Penetratin-Alexa, RQIKIWFQNRRMKWKKGC(Alexa)-amide; R8-PAD (16), RRRRRRRRGG-D-(KLAKLAK)2-amide. The EGFP protein bearing (His)6 and (Arg)8 segments and a thrombin cleavage site on its N-terminus (R8-EGFP) and the EGFP bearing (His)6 and thrombin cleavage site (lacking R8) were prepared as previously reported (16). Details in preparation of the peptides and protein were described (see Supporting Information). Confocal Microscopy. 2 ⫻ 105 cells were plated into 35-mm glass-bottomed dishes (Iwaki) and cultured for 48 h. After removing the medium, the cells were washed twice with PBS. The cells were first incubated with pyrenebutyrate (1-pyrenebutyric acid) (67 M) in PBS for 2 min, and then the peptide solution in PBS (40 M) was added to yield the final concentration of pyrenebutyrate and peptides (50 and 10 M, respectively). A 20 M peptide solution in PBS was employed to obtain a final peptide concentration of 5 M. After incubation for 4 min, the cells were washed with PBS (⫻5). Distribution of the fluorescently labeled peptides was analyzed without fixing using a confocal scanning laser microscope (Olympus FV300) equipped with a 40⫻ objective lens. For the observation of hipocampal primary cultures, a Zeiss LSM510 equipped with a 63⫻ objective lens was employed. For 4 °C experiments, cells were preincubated in a refrigerator (4 °C) for 1 h. Washing and incubation of the cells were then conducted using cold PBS and the 4 °C-refrigerator, respectively, prior to observation of the cells in cold PBS. For the experiments with the mitochondrial membrane potential probe JC-1, the cells were preincubated with pyrenebutyrate (2 min) prior to R8-PAD treatment (4 min) (final concentration of pyrenebutyrate and R8-PAD, 50 and 5 M, respectively). After washing twice with PBS and incubation with JC-1 (5 M) for 30 min in ␣-MEM containing 10% (v/v) calf serum, the cells were analyzed with confocal microscopy. Fluorescence Microscopy. The cells were treated as stated above using nonfluorescently labeled R8 peptide instead of R8-alexa. Distribution of pyrenebutyrate was analyzed by a fluorescence microscope (Olympus IX-50) equipped with a 20⫻ objective lens using Hg lamp as a light source (excitation, 330 –385 nm; emission, ⬎420 nm). Quantitation of Peptide Uptake. HeLa cells were preincubated with pyrenebutyrate in PBS for 5 min prior to the addition of increasing concentrations of R8-Alexa in PBS at 37 °C; final concentration of pyrenebutyrate was fixed at 50 M. After incubation for 15 min, the internalized peptide was analyzed by FACS. LDH Release Assay. HeLa cells were incubated with pyrenebutyrate in PBS for 5 min and then with peptide in the presence of pyrenebutyrate for
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30 min. LDH release was calculated from the percentage of the released LDH in pyrenebutyratetreated cells relative to that of cells treated with 1% (v/v) Tween 20 under the same conditions. MTT Assay. HeLa cells were treated with 50 M pyrenebutyrate for 10 min followed by 5 M R8 or Tat in the continued presence of pyrenebutyrate for 20 min in PBS. Following 2⫻ PBS washes, the cells were further incubated in the absence of pyrenebutyrate and peptides for 24 h in ␣-MEM containing 10% (v/v) calf serum. Cell viability was then analyzed by MTT assay. HeLa cells were pretreated with pyrenebutyrate for 10 min, followed by incubation with R8-PAD (5 M) for 20 min in PBS. The cells were washed with PBS and incubated in ␣-MEM containing 10% (v/v) calf serum for 24 h. Cell viability was then analyzed by the MTT assay. Acknowledgment: This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology o Japan, Institute of Sustainability Science, Kyoto University (SF), and the Swiss NSF (S.M.). T.T. is grateful for a JSPS Research Fellowship for Young Scientists. Supporting Information Available: This material is available free of charge via the Internet.
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