Accumulation of cell-penetrating peptides in large unilamellar vesicles: A straightforward screening assay for investigating the internalization mechanism.

Accumulation of Cell-Penetrating Peptides in Large Unilamellar Vesicles – A straightforward Screening Assay for Investigating the Internalization Mechanism. Jean-Marie Swiecicki1,2,3+, Margherita Di Pisa1,2,3+, Fabienne Burlina1,2,3, Pascaline Lécorché1,2,3, Christelle Mansuy1,2,3, Gérard Chassaing1,2,3, Solange Lavielle1,2,3* 1

Sorbonne Universités, UPMC Univ Paris 06, LBM, 4,Place Jussieu, 75005 Paris, France

2

Ecole Normale Supérieure – PSL research University, Département de Chimie, 24, Rue

Lhomond, 75005 Paris, France 3

CNRS, UMR 7203, LBM, 75005 Paris, France

+

equal contribution

Corresponding author: Prof. Solange Lavielle, [email protected]

Abstract The internalization of cell-penetrating peptides (CPPs) into liposomes (large unilamellar vesicles, LUVs) was studied with a rapid and robust procedure based on the quenching of a small fluorescent probe, 7-nitrobenz-2-oxa-1,3-diazole (NBD). Quenching can be achieved by reduction with dithionite or by pH jump. LUVs with different compositions of phospholipids (PLs) were used to screen the efficacy of different CPPs. In order to “validate” the composition of the membrane models, a control cationic peptide, which does not enter eukaryotic cells, was included in the study. It was found that pure DOPG or DOPG within ternary mixtures with cholesterol are the most appropriate models for studying CPP translocation. An anionic lipid, such as DOPG, is required for the adsorption of the basic peptides on the surface of LUVs. In addition, it acts as transfer agent through the lipid bilayer. A fluid phase and/or the presence of phase defects also appear mandatory for the internalization to occur. The neutralization of charges within an inverted micelle demonstrated in the case of DOPG and also proposed for a ternary mixture of PLs might not be the only mechanism for the CPP translocation. Finally, it is shown that oleic acid facilitates the entry inside LUVs in gel phase of a series of cationic peptides including CPPs and also the negative control peptide PKCi. Keywords: cell-penetrating peptides; translocation; lipid vesicles; quenching of fluorescence; This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/bip.22652 © 2015 Wiley Periodicals, Inc. This article is protected by copyright. All rights reserved.

Biopolymers: Peptide Science

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ordered and disordered phases; fatty acid

INTRODUCTION Cationic cell-penetrating peptides (CPPs) enter cells via both temperature-independent pathways (direct translocation through the lipid bilayer) and temperature-dependent routes (endocytosis). Various strategies based on model membranes such as unilamellar vesicles of different sizes (small: SUVs, large: LUVs, or giant: GUVs), suspended membranes or multilamellar vesicles have been developed to study at the molecular level the mechanisms of CPP translocation through lipid bilayers.1-15 The development of such analytical tools is crucial both to propose efficient screening strategies of peptides based on their translocation properties and to decipher the mechanisms underlying the direct translocation. Ideally, a more detailed description of the internalization pathway should give clues to rationally design new delivering agents. In this aim, the CPPs and their membrane partners (phospholipids, sphingomyelins, fatty acids, cholesterol) have to be simultaneously scrutinized. Therefore, the membrane composition plays a tremendous role in the analysis of the translocation mechanism. To quantify the internalization of peptides into LUVs, we recently reported, an assay based on the fluorescence of peptides labeled on their N-terminus with NBD (7-nitrobenz-2oxa-1,3-diazole), a small probe, which is not larger than an aromatic amino acid side chain.2,14,16,17 Treatment of the incubation medium with dithionite (DT), a non-permeant reducing agent, rapidly reduces the NBD probe, and consequently quenches its fluorescence. The residual fluorescence after DT treatment therefore corresponds to the intravesicular CPP, which is protected from the reducing agent. According to this procedure, we have studied the internalization into LUVs of several CPPs (penetratin,18 Tat,19 Arg9,20,21 and R6/W322) as well as the entry of the PKCi peptide (a protein kinase C inhibitor), which has been previously used as a cargo covalently conjugated to CPPs (Table I). This cationic peptide is not able to enter cells by itself and was used in this study as a negative control.23,24 We previously demonstrated that the direct translocation of CPPs was rapid through LUVs made of unsaturated di-oleic phospholipids (PLs), DOPG, in the fluid phase, leading within a few minutes to an apparent intravesicular concentration up to 0.5 mM, whereas the incubation concentration was 100 nM (Table II). The extent of peptide accumulation was found to depend on the CPP sequence and its extravesicular concentration. More interestingly, it was established that: i) CPPs enhanced the flipping of fluorescently labeled DOPG, and ii)

John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.

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the intravesicular CPP accumulation directly correlated with the amount of DOPG that had been transferred from the external to the internal leaflet of the LUV bilayer. The necessary neutralization of the CPP cationic charges to cross the hydrophobic core of the bilayer was achieved through interactions with the negatively charged DOPG within inverted micelles. In addition, based on the levels of CPP intravesicular accumulation and PL flip-flop measured for different Arg9 extravesicular concentrations, we proposed that these CPPs enter preferentially as dimers within inverted micelles.14 We further showed that a ternary mixture made of DOPG/DOPC/Cholesterol (3/1/6) was specifically permissive to the passage of CPPs and their conjugates. Interestingly, translocation of Arg9 or Tat concomitantly enhanced the flip-flop of the anionic DOPG when using this membrane model, thus showing that anionic PLs might again participate to a neutral and hydrophobic [(CPPp+)n(PL-)np] translocation complex.14 Here we report the internalization of CPPs into LUVs made of saturated PLs with different chain lengths (C16 for DPPG and C18 for DSPG) and of various ternary lipid mixtures containing cholesterol with either DOPC/DOPG or sphyngomyelin/DOPG, which may be considered closer to the composition of domains within biological membranes. These different membrane compositions of phospholipids were used to probe different environment for the translocation of CPPs and screen the efficacy of different CPPs. A control cationic peptide (PKCi), which does not enter eukaryotic cells, was added to the CPP list to ensure that the probed membrane composition remains selective towards CPPs (as eukaryotic membranes are). Pure DOPG or DOPG within some ternary mixtures including cholesterol were shown to be appropriate models to investigate the translocation mechanisms of CPPs. Fluid phase or disordered domains within ordered phases appear mandatory for the internalization to occur. Our data also show that the neutralization of charges within an inverted micelle, demonstrated in the case of Arg9 and DOPG LUVs, and also proposed with the ternary mixture DOPG/DOPC/Cholesterol, might not be the only mechanism for translocation. We also showed with this study that oleic acid, known as a facilitator of translocation,25 increased the entry of cationic peptides in LUVs in gel phase. However, this occurs with loss of selectivity since the negative control peptide was shown to also accumulate in LUVs.

RESULTS In Table I are reported the sequences of the NBD-labeled peptides (CPPs: penetratin,18


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R6/W322 Tat,19 Arg9,20,21 and the negative control peptide: PKCi23,24) and the abbreviations of the phospholipids (DOPG, DOPC, DPPG, DSPG), sphingomyelin and cholesterol, all used in this study.

Quantification of the Internalization into LUVs - Procedure The procedure was set up with LUVs made of 100% DOPG, as reported.14,15,17 The peptides were preincubated at 20°C for 5 min with LUVs before addition of a large excess of freshly prepared DT, as depicted in Figure 1. The decrease in fluorescence resulting from the reduction by DT of the external NBD-labeled peptides was then monitored for the following 25 min. The efficacies of accumulation inside LUVs of the different peptides were compared with the peptide extravesicular concentration set-up at 100 nM and the PLs concentration at 10 µM, corresponding to a peptide/PL ratio of 1:100.

Quantification of the Internalization – LUVs Made of Saturated PLs With the procedure set up with LUVs made of DOPG, we have quantified the internalization of different peptides into LUVs made of two saturated PLs: DPPG or DSPG. The LUVs made of DOPG (Tm = -18°C) were in fluid phase at 20°C for both the incubation with the CPP and the reduction of the NBD probe by dithionite. The melting points (Tm) of DPPG and DSPG are 41°C and 55°C, respectively, related to the length of their acyl chains (C16 and C18, respectively). Thus, to explore the role of the phase on the internalization of CPPs all the incubations with LUVs made of DPPG or DSPG were done at 37°C, and reduction with dithionite was performed at 15°C after rapid cooling of the mixture. At 37°C, the LUVs made of 100% DPPG, correspond to a mixture of fluid domains within a gel phase,26 while LUVs made of 100% DSPG are in a gel phase. With LUVs made of DPPG the accumulation of the amphiphilic peptides R6/W3 and penetratin was efficient, the percentage of internalized peptide corresponding to 39% and 52% of the incubated peptide, respectively, almost close to the thermodynamic equilibrium, after only five minutes incubation. No direct correlation between the peptide amphiphilicity and the accumulation efficacy was observed. Indeed, if R6/W3 and penetratin were structured in a α-helical structure on the LUVs surface, R6/W3, would be a pure amphiphilic peptide, compared to penetratin, the most potent. The other cationic peptides accumulated with a potency inversely related to their arginine content: PKCi (9%) > Tat (6.5%) > Arg9 (4%), containing 3, 6 and 9 arginines, respectively, (Table II). Surprisingly, the negative control


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peptide PKCi, (5 cationic residues, 3 arginines) was more efficient than the two well known CPPs Arg9 and Tat. At 37°C, during the peptide incubation, these LUVs present fluid domains in the gel phase,26 and probably phase defects at the junction of both phases, that might be responsible for the lost in selectivity. At 37°C, LUVs made of DSPG were in the gel phase. No significant difference was observed between the CPPs and the control peptide PKCi. All the peptides barely accumulated inside these LUVs (between 1.5 to 2.5%), (Table III), keeping in mind that 1% internalization corresponds to a low but significant intravesicular concentration, (around 30 µM).14

Quenching of the NBD-Fluorescence by Reduction with Dithionite or by Addition of HO- : A Comparative Study. The method of choice for the quantification of the intravesicular accumulation of a CPP is to quench the fluorescence of a CPP modified by a fluorescent probe, for example by irreversible reduction with dithionite of the NBD probe.2,14,15-17 Herein, we showed that the quenching of fluorescence could also be achieved by a rapid jump in pH (from 7 to 11), as shown in Figure 2. At pH close to 11, the fluorescence of NBD-PKCi was completely abolished, with a titration at pH 8.1. The same titration curve was also observed when NBDPKCi was incubated at 20°C with LUVs made of 100% DPPG, with these conditions the LUVs are in gel phase and PKCi was not internalized (0.9%). Noteworthy, this fluorescence quenching procedure by pH jump to 11 is totally reversible. Indeed, the addition at the end of the titration of one equivalent of H+ (vs. the amount of added OH-) led to full recovery of the initial fluorescence. Both quenching protocols (DT and HO-) were compared to measure the intravesicular accumulation of five peptides (R6/W3, Arg9, Penetratin, Tat and PKCi) after 5 min incubation at 37°C, and addition at 15°C of DT or HO-. Both quenching procedures led to similar results, (Table III). One of the advantages of HO- over DT is the obvious easiness to fully recover the fluorescence signal, by simple neutralization with H+. This observation may offer new possibilities to study kinetics in more details. However, these pH variations will have to be used with caution, checking that successive increases in the extracellular salt concentrations will not create a gradient with the lumen of the LUVs, and thus interfere with the translocation process.


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Quantification of the Internalization – LUVs Made of Binary or Ternary Mixtures of Lipids. As for LUVs made of DOPG, the incubation and the reduction were both performed at 20°C, and three peptides were used: R6/W3 (amphiphilic peptides family), Arg9 (pure cationic peptides family) and PKCi (a negative control peptide). Arg9, and to a lesser extent R6/W3, accumulated in LUVs made of DOPG (internalization 15.7% and 9.7%, respectively), but not in LUVs made of the corresponding neutral unsaturated PLs DOPC, as we recently reported.14,15 To further analyze the effect of the membrane charge density, we have systematically varied the incorporation of cholesterol into LUVs made of DOPG (binary mixture) or DOPG/DOPC (ternary mixture), (Table IV). After finding that the best DOPG/Cholesterol ratio was 4:6, with an internalization reaching 11.2% for Arg9, we investigated the accumulation of three peptides using different ratios of DOPG/DOPC with 60% mol of cholesterol, (Table IV). We were delighted to observe that the incorporation of DOPC to DOPG, in a 3:1 ratio for DOPG/DOPC (and 60%mol Chol), increased the internalization of Arg9 from 11.2% to 26% after 5 min incubation, as we already reported.14 This serendipitous discovery led us to further explore ternary mixtures of lipids. Herein, we established that in fact this increase was not linear, but a bell-shaped curve was obtained with a maximum for DOPG/DOPC/Chol (3:1:6). Both a decrease and an increase of DOPC in DOPG led to a noticeable decrease in the internalization of Arg9 (Table IV). The internalization of Arg9 was analyzed as a function of the incubation time, since this phenomenon could have been an artifact related to different kinetics of internalization in these ternary mixtures. The same bell-shaped curve for the accumulation was observed depending on the DOPG/DOPC ratio (Table V, and Figure 3). The internalization increased (1.6- to 2.0-fold) by increasing the incubation times (from 5 to 120 min). This enhancement was significantly more important than the one previously observed with LUVs made of pure DOPG (only 1.25-fold increase, with 19.7 % peptide internalized after 120 min compared to 15.7 % after 5 min incubation).14 Here, for LUVs made of DOPG/DOPC/Chol (3:1:6) and (2.5:1.5:6) a plateau at 43.8% and 35%, respectively, was attained in less than 60 minutes for the best initial internalizations, i.e. 26% and 19.5% (Table V). In both cases, the burst of internalization occurred within the first 15 min of incubation. For the other DOPG/DOPC ratios (with Chol fixed at 60% mol), the internalizations levels doubled between 5 min and 120 min incubation, with a slight but significant increase between 15 and 120 min.


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Quantification of the Flip-flopping of Labeled DOPG in LUVs Made of DOPG/Sphingomyelin/Cholesterol. We then analyzed the flip-flopping of C6-NBD-PG incorporated in the external leaflet of LUVs made of DOPG/bSM/Chol (3:1:6) in the presence of unlabeled Arg9 or Tat (i.e. Acetyl-Arg9 and Acetyl-Tat), as described previously.14 The flipping of the labeled DOPG in the internal leaflet of these LUVs was enhanced, but, the number of DOPG molecules to flip per LUV was not directly proportional to the number of Arg9 or Tat molecules that were internalized, in contrast to what has been observed with DOPG and DOPG/DOPC/Chol (3:1:6) compositions. The % of DOPG flipping for Acetyl-Arg9 and Acetyl-Tat was about one third of the expected values for the internalization of neutral CPP/PL complexes and taking into account the % of CPP internalization, (Arg9: found 5.3 ± 1.3%, expected 13.6 ± 1.9%; Tat: found 2.5 ± 0.8% expected 7.74 ± 0.42).

Internalization of CPPs in the Presence of Oleic Acid Very recently Herce et al. hypothesized that fatty acids, such as oleic acid, might carry on arginine-rich peptides towards the interior of cells and mediate their translocation.25 Consequently, the internalization of the five peptides (R6/W3, Arg9, PKCi, penetratin, Tat) were quantified with our assay, in the presence of one unsaturated fatty acid, either oleic acid ((9Z)-octadec-9-enoic acid), or linoleic acid ((9Z,12Z)-9,12-Octadecadienoic acid), with LUVs made of DSPG, that is in the gel phase at 37°C and only allowed a weak transfer of these cationic peptides from the inner to the outer leaflet (vide supra, and Table VI). But, in contrast to Herce et al.’s experiments, the pH inside LUVs and in the incubation medium was the same, since the addition of the fatty acid in the incubation medium at the concentration used did not change the pH value. The chosen ratio Peptide/Oleic acid/DSPG varied from 1:0.054:100 to 1:3.3:100 (i.e. from catalytic to stoichiometric concentrations of oleic acid). As reported by Herce et al.,25 with their partition system and also with cells, we indeed observed an increase in the accumulation within LUVs made of DSPG in the gel phase. With oleic acid the increase was drastic, ranging from 6.0-fold for penetratin to 28.7-fold for PKCi (with 3.3 eq. of fatty acid relative to the peptide, Table VI). The % of accumulation was in the 30% range for all peptides, except for penetratin, whose internalization was the less affected by addition of oleic acid, increasing from 1.7% to about 10%. This improvement was dependent on the oleic acid concentration. For example for R6/W3, the accumulation increased from 2.5 ± 0.5% to 5.6 ± 1.3, 38.0 ± 2.5, 38.3 ± 4.1 and 33.0 ± 4.1%, with


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increasing concentrations of oleic from 0 to 5.4, 22, 82.5 and 330 nM, respectively, with the CPPs fixed at 100 nM and PLs at 10 µM. The increase in internalization efficiency occurs for Peptide/Oleic acid ratio varying from 1:0.054 to 1:0.22, with a plateau reached at 1:0.22 (or below). This improvement was better with oleic acid compared to linoleic acid, which whatever the peptide only doubled - tripled the internalization compared to pure DSPG.

DISCUSSION The translocation potency of fluorescent peptides trough LUVs can be quantified by quenching the fluorescence of the NBD probe either by reduction with dithionite or by pH jump. The necessary fluorescent probe NBD (7-nitrobenz-2-oxa-1,3-diazole) should only marginally perturb the intrinsic properties of the parent peptide, since it is not larger than the side chain of an aromatic amino acid such as tryptophan. With this procedure based on fluorescence quenching, the translocation of cationic peptides into LUVs of different compositions has been studied, either in binary or ternary mixtures, to try to decipher the internalization process(es) of cell penetrating peptides. For studying these model membranes, a cationic peptide PKCi, which does not enter cells, has always been added, as a negative control among all cationic peptides capable to enter cells. Comparison of the data with DOPG (fluid phase), DPPG (fluid domains in gel phase),26 and DSPG (gel phase) showed that the accumulation of cationic peptides in LUVs is dependent on: i) the physical phase of bilayer (fluid vs gel) or ii) on the existence of phase defects at the junction of nanodomains between two phases, iii) evidently on the peptide sequence. We believe that the PL spontaneous flip-flop rate (slow for saturated vs. rapid for unsaturated, and slower with PL having the longest acyl chains) might be one key to understand the efficiency of the direct translocation process. Nevertheless, it is not sufficient to rationalize the different internalization yields among the CPPs. The cationic peptide PKCi, which does not enter cells unless conjugated to a CPP, translocates neither into LUVs made of DOPG in fluid phase14 nor into LUVs made of DSPG in gel phase. In contrast PKCi was shown to accumulate even more efficiently than Tat and Arg9 inside LUVs made of DPPG, containing fluid domains in gel phase, inferring the importance of nanodomains with phase defects in the translocation process. DOPG, with its permeability to all studied CPPs and its selectivity profile can be viewed as an appropriate


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model for screening the entry of cationic CPPs into LUVs as a model system for CPP translocation into cells. The internalization showed a different profile of selectivity with ternary mixtures of PLs containing cholesterol compared to what has been observed with LUVs made of 100% DOPG and of saturated PLs (DPPG or DSPG). As with LUVs made of DOPG, the cationic negative

control

peptide

PKCi

was

barely

internalized

into

LUVs

made

of

DOPG/DOPC/Chol, showing the selectivity and suggesting that this composition of lipids can also be considered as a relevant model of membranes to study CPP translocation. However, with the best ternary composition, DOPG/DOPC/Chol (3:1:6) corresponding to a disordered ld phase,27 Tat, was readily internalized, being almost as efficient as Arg9, while with LUVs made of 100% DOPG Tat and R6/W3 were less efficient than Arg9. With this ternary mixture DOPG/DOPC/Chol (3:1:6), R6/W3, the most amphiphilic CPPs, was only poorly internalized. The replacement of DOPC by another neutral lipid, sphingomyelin (from egg or brain sources), also led to interesting results. Sphingomyelin has a high affinity for cholesterol with a tight packing into liquid-ordered lo domains within a disordered liquid phase, yielding a model for “lipid rafts”.28 Neither the negative control PKCi, nor the amphiphilic peptide R6/W3, was able to enter LUVs in the two studied ternary mixtures incorporating sphingomyelin. The internalization of the cationic Arg9 was dramatically increased (22.6%) in DOPG/eSM/Chol (3:1:6) compared to DOPG/eSM/Chol (1.5:2.5:6), which was almost impermeable to Arg9 (< 0.1%). This percent of internalization was reached in the first five minutes, with minor changes after two hours. The internalizations were rather similar with sphingomyelin from either egg or brain sources, which differs mainly in the preponderant species: N-(hexadecanoyl)- versus N-(octadecanoyl)-sphing-4-enine-1-phosphocholine (C16 vs C18), respectively. Additionally, the internalization of another cationic peptide, Tat, was quantified in LUVs made of DOPG/bSM/Chol (3:1:6). With this composition, Tat was shown to accumulate (12.9%). Nevertheless, the internalization mechanism in that case did not seem to only rely on the flipping of DOPG. It might be that with this ternary mixture, defects serve as nucleation sites for small-scale phase-separated lipid domains. These defects could allow the incorporation and transfer of CPPs. It is also plausible that within the domains, the C6NBD-DOPG labeled PLs used to quantify the flip-flop, is not distributed uniformly as in fluid DOPG membranes and a model of statistical recruitment of PLs (DOPG vs. NBD-labeled DOPG). Consequently, the neutral complex within the inverted micelle might also sequester unlabeled DOPG molecules (over the statistical recruitment) for the neutralization of the CPP


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cationic charges. This contribution would affect the number of C6-NBD-DOPG, which flipped from the external to the internal leaflet and are measured in our assay. What can be proposed on the mechanism of the direct translocation of these highly charged cationic peptides through a bilayer? With LUVs made of 100% DOPG, it was previously established that the translocation involved DOPG flip-flop, R6/W3 and Arg9 entering LUVs as a neutral complex. In the cases of Arg9 and R6/W3 it was further established that the complex was made of a dimer of CPPs, the guanidinium charges being neutralized by the phosphates of DOPG.14,15 DOPG represents only a minor constituent of eukaryotic membrane bilayer. One way to compensate for a lower proportion of anionic PLs is to use LUVs with ternary lipid composition with phase defects, and/or yielding models of “raft domains”, as DOPG/DOPC/Chol or DOPG/eSM/Chol and DOPG/bSM/Chol. Indeed, some of the studied cationic peptides enter selectively into these ternary mixtures, but the efficacies strictly depend on the ratio of DOPG and on the peptide sequence, these compositions being permissive to certain cationic peptides and not to cationic amphiphilic ones like R6/W3. In addition, the internalization of Arg9 and Tat correlated also to the flipflop of the DOPG. Thus, DOPG appears to be important for two steps. Firstly, it is involved in the adsorption of the peptide on the LUV surface, replacing the negatively charged glycosaminoglycans (GAGs) present on cells. GAGs participate in the accumulation of CPPs from the extracellular medium to the cell surface.22,29-32 On model LUV systems, DOPG “compensates” the absence of GAGs or mimics the negative surrounding. Secondly, DOPG is involved in the translocation per se, where only a few molecules of DOPG must be required for the transfer of neutral species made of DOPG and charged peptides, such as within an inverted micelle. What is more intriguing regarding the entry mechanism is the efficacy of oleic acid, which even with PLs in gel phase allows a rapid and efficient transfer of cationic peptides. The effect of oleic acid reported by Herce et al.25 was reproduced here with LUVs, even in the absence of a pH gradient. Thus, even tightly organized PLs in a gel phase may be “disturbed” by the presence of oleic acid to allow the passage of cationic species. However, this “assisted” translocation is no longer selective since a negative control cationic peptide was efficiently transferred as Arg9 in the presence of oleic acid. These preliminary data require further studies to unveil the specific role of oleic acid, which might not be just a transfer agent, which could recycle depending on its protonation state.25 How is the organization of the membrane perturbed by a fatty acid, could it be by the formation of anionic pores? Does the fatty acid recruit cationic peptides to form a neutral complex? If so,


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why is the internalization 3-times less efficient for penetratin compared to the four other cationic peptides, including PKCi, and why with different CPP/oleic acid ratios varying from 1:0.22 to 1:3.3 the internalization of Arg9 is reaching the same level? Whatever the mechanism, which needs further investigation, it should be recalled that in mammalian cells the concentration of fatty acids is very low and their addition in the culture medium is associated with cytotoxicity via pore formation or detergent effect.

CONCLUSIONS The internalization of cationic peptides into LUVs can be studied with a rapid and robust procedure based on the quenching of the fluorescence of the small NBD probe. Different compositions of phospholipids can be used to screen the efficacy of different CPPs, but as shown herein a negative control peptide, that is a peptide, which has been shown not to enter in cells, should be added to “validate” the composition. Pure DOPG or DOPG within ternary mixture including cholesterol were shown to be the most appropriate compositions of PLs. A fluid phase and/or the presence of disordered domains within ordered phases appear mandatory for the internalization to occur. An anionic lipid, such as DOPG, is required, for the adsorption of the CPPs on the surface of LUVs, (to “compensate” the adsorption mediated by GAGs in cells), and more specifically as a transfer agent through the bilayer, since the translocation occurs without loss of membrane integrity. The neutralization of charges within an inverted micelle made of CPPs and PLs molecules, demonstrated in the cases of Arg9, Tat and R6/W3, and also proposed with ternary mixtures might not be the only mechanism. Herein we showed that this strict correlation was not observed with ternary mixtures incorporating sphingomyelin, but this discrepancy might also be due to the experiment per se. However, the data with oleic acid as a facilitator of CPPs crossing raise new questions on the constitution of a neutral complex. Indeed, oleic acid facilitates the entry of all studied cationic peptides inside LUVs in gel phase with a priori no disorder and no defect. These intriguing results need further investigation, especially in the context of cells translocation. The identification of negatively charged molecules involved in the neutralization of cationic peptides and in a selective translocation of CPPs in mammalian cells remains a challenge, could it be lysophosphatic acid, gangliosides?33-35


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MATERIALS AND METHODS Synthesis and characterization of the studied peptides General procedure for solid-phase peptide synthesis: Standard Boc- and Fmoc-amino acids, MBHA resin (0.51 or 0.43mmolg-1), Boc-Glycine-PAM resin (0.64 mmol g-1), Boc-Serine(Bzl)-PAM resin (0.49 mmol g-1), 1-hydroxybenzotriazole (HOBt), dicyclohexylcarbodiimide (DCC) and O(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) were purchased from Iris Biotech, GmbH or Novabiochem. 4-Chloro-7-nitrobenz-2-oxa-1,3-diazole (NBD-Cl), was obtained from Sigma–Aldrich and Boc-Cys(NPys)-OH from Bachem. Acetylated or fluorescently labeled peptides were synthesized by solid-phase methodology with an ABI 433A peptide synthesizer and use of Boc (Arg9, R6/W3, Tat, and penetratin) or Fmoc (PKCi) strategies, starting from a MBHA resin. For the different couplings, activation was accomplished with DCC/HOBt in NMP (Boc strategy) or HBTU/DIEA in NMP (Fmoc strategy). Coupling of NBD-Cl at the N-termini of these peptides was performed manually. Coupling of NBD-Cl to the peptidyl resin: NBD-Cl has been successfully added to the Ntermini of all the peptides. We would like to emphasize the sensitivity of NBD to piperidine. As a consequence, NBD has to be added at the N-terminus of a peptide after Fmoc removal and deformylation of the tryptophan residues. The coupling to NBD was achieved by addition of NBD-Cl (80 mg, 0.4 mmol, 4.0 equiv) and DIEA (172 mL, 1 mmol, 10 equiv) in DMF (2 mL) to the peptidyl resin (0.1 mmol). The solution was stirred overnight and then rinsed with DMF, CH2Cl2 and MeOH (5 mL in each case) and dried under vacuum. Cleavage of the peptides: At the end of the synthesis, peptides obtained by the Boc strategy were cleaved from the resin by treatment with HF (2 h, 0°C) with use of anisole (1.5 mL g-1 peptidylresin) and methylsulfide (0.25 mL g-1 peptidyl-resin) as scavengers. Each peptide was precipitated in cold diethyl ether, and the scavengers were removed by filtration. The crude peptide was dissolved in a solution (10%, v/v) of acetic acid in water and lyophilized. At the end of the synthesis, the peptidylresins obtained by the Fmoc strategy were cleaved from their corresponding resin in TFA with use of water (5%, v/v) and triisopropylsilane (TIS, 5%, v/v) as scavenger. The crude peptides obtained by precipitation in cold ether were lyophilized. Purification and characterization of the peptides: The peptides were analyzed and purified by RP-HPLC with use of solvents A (0.1% TFA in H2O) and B (0.1% TFA in acetonitrile) in a Waters System with detection at 220 and 280 nm (340 nm for peptides containing an NBD-Tag). Analytical RP-HPLC was performed with an Ace 5 C8–300 column (4.6 x 250 mm) at a flow rate of 1.0 mL min1

. Preparative RP-HPLC was performed with a SymmetryPrep C8 column (7.8 x 300 mm) at a flow

rate of 5.0 mL min-1 with the optimized gradients. The peptides, (over 95% purity), were characterized by MALDI-TOF MS with α-cyano-4-hydroxycinnamic acid (HCCA) as the matrix.


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Preparation of LUV suspensions DOPG, DOPC, DPPG, DSPG, Egg Chicken Sphingomyelin (eSM), Brain Porcine Sphingomyelin (bSM) and C6-NBD-PG were purchased from Avanti Polar Lipids, and cholesterol from Sigma–Aldrich as a solution in chloroform.

General procedure for preparation of symmetric LUVs Depending on the desired LUV composition, the appropriate amount of phospholipids, sphingolipids and cholesterol were placed in a round-bottomed flask and chloroform was slowly evaporated under vacuum at 40 °C using a rotary evaporator and then placed 1 h under high-vacuum. The lipids were hydrated using PBS buffer. Depending on the phase transition temperature of the phospholipids, the buffers were pre-warmed or not for the gentle hydration step. For high temperature transition phospholipids, the extruder was heated using a thermocontrolled circulation bath (DPPG: 50°C, DSPG: 65°C). The turbid suspension of multilamellar vesicles was subsequently extruded 7 times through a 200 nm polycarbonate track-etch membrane and 10 times through a 100 nm polycarbonate track-etch membrane (Whatman) using a 10 mL Thermobarrel extruder (Lipex Biomembranes). The LUVs solutions were used from the day after their preparation and within one week. LUVs made of low temperature transition phospholipids (DOPG, DOPC) were stored at 4 °C, whereas LUVs made of high temperature transition phospholipids or made of mixtures of lipids were stored at room temperature. General procedure for preparation of LUVs labeled selectively on the outer leaflet by C6-NBD-PG For the preparation of LUVs labeled on the outer leaflet, the C6-NBD-PG solution in chloroform was dried in vacuo and re-dissolved in DMSO. To this solution was added a suspension of LUVs prepared as described above, to yield LUVs labeled with NBD quasi-exclusively on the outer leaflet (1% C6- NBD-PG for LUVs made of 100% DOPG, 0.3% C6-NBD-PG for LUVs made of DOPG/bSM/cholesterol in a ratio of 3:1:6). The amount of DMSO was 0.02%. The LUVs solutions were stored at 4°C and used only on the day after their preparation. DSPG LUVs containing oleic and linoleic acid where obtaining by adding a suspension of LUVs prepared as described above, to the calculated amounts of fatty acids re-suspended in 4 µL of DMSO.

Fluorescence experiments Sample preparation: when a Trp residue was present in the sequence the concentration was determined by UV (ε = 5550 L mol-1 cm-1), otherwise the peptide was weighed, the presence of the trifluoroacetate ions being taken into account; that is, nine trifluoroacetate ions for the Arg9 CPP. Fluorescence experiment: emission spectra and time-course fluorescence measurements were recorded with a Jasco Fluorescence Spectrophotometer at 20°C (controlled with a Jasco MCB-100 mini circulation bath). As an excitation source a xenon lamp was used, and the excitation and emission


13


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slide widths were set at 5 nm. To avoid peptide adsorption and subsequent fluorescence quenching, polystyrene cuvettes were used. Experiments with labeled LUVs were more sensitive and required the use of quartz cuvettes coated for at least 30 min with BSA in PBS buffer (10 mg mL-1) and rinsed thoroughly with PBS buffer. In polystyrene or in quartz cuvettes coated with BSA, the internalization experiments yielded equivalent results. During the whole measurement the solution was stirred at 800 rpm. Internalization experiments: unlabeled LUVs were used with NBD-labeled CPPs. The excitation wavelength was fixed at 460 nm, and the emission wavelength was fixed at 555 nm. For the flip-flopping experiments, C6-NBD-labeled LUVs were used. The excitation wavelength was fixed at 465 nm, and the emission wavelength was fixed at 547 nm.

Expected flip-flopping percentage from the internalization percentage Expected flip-flopping: Let n be the number of moles of peptide initially introduced, p the fraction of peptide internalized, and z the expected formal electrical charge of the considered peptide. If it is assumed that one peptide induced the flipping of z phospholipid units upon its internalization into LUVs, the expected flipped fraction should be equal to npz. Determination of the expected flipped C6-NBD-PG: For the example of Arg9, the solution (3 mL) of LUVs made of DOPG/bSM/cholesterol (3:1:6) placed in the fluorimeter cuvette at a total concentration of 10 mm contains 9.0 nmol of DOPG. As a consequence, the quantity of phospholipids present on the outer leaflet is equal to 4.5 nmol. Of the Arg9, 22.6 % was found located inside the vesicles, meaning that n p z = 0.61 nmol of phospholipids should have flipped. This corresponds to 13.56 % of the DOPG.

ACKNOWLEDGMENTS This work was supported in part by the ANR # J12R139 « ELIPTIC », Dr. M. Di Pisa being a post-doctoral fellow. The authors wish to thank for fruitful discussions Dr. Eric LABBÉ, Ecole Normale Supérieure – PSL research University, Département de Chimie, 24, Rue Lhomond, 75005 Paris, France; Sorbonne Universités, UPMC Univ Paris 06, LBM, 4,Place Jussieu, 75005 Paris, France; CNRS, UMR 7203, LBM 75005 Paris France. In addition, the Institute for Advanced Studies of University of Cergy-Pontoise is gratefully acknowledged for the financial and organizational support of the “Peptides in Paris Symposium 2014, PIPS 2014”, held from October 5-10, 2014.


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REFERENCES 1. Thoren, P. E. G.; Persson, D.; Karlsson, M.; Norden, B. FEBS Lett. 2000, 482, 265–268. 2. Drin, G.; Mazel, M.; Clair, P.; Mathieu, D.; Kaczorek, M.; Temsamani, J. Eur J Biochem 2001, 268, 1304−1314. 3. Magzoub, M.; Kilk, K.; Eriksson, L. E. G.; Langel, Ü.; Gräslund, A. Biochim Biophys Acta Biomembr 2001, 1512, 77–89. 4. Thorén, P. E. G.; Persson, D.; Esbj.rner, E. K.; Goksör, M.; Lincoln, P.; Nordén, B. Biochemistry 2004, 43, 3471–3489. 5. Barany-Wallje, E.; Keller, S.; Serowy, S.; Geibel, S.; Pohl, P.; Bienert, M.; Dathe, M. Biophys J 2005, 89, 2513 –2521. 6. Magzoub, M., Pramanik, A., and Gräslund, A. Biochemistry 2005, 44, 14890–14897. 7. Lamazière, C. Wolf, O. Lambert, G. Chassaing, G. Trugnan, J. Ayala-Sanmartin, PLoS One 2008, 3, e1938. 8. Ziegler, A. Adv Drug Deliv Rev 2008, 60, 580–597. 9. Alves, I. D.; Bechara, C.; Walrant, A.; Zaltsman, Y.; Jiao, C.-Y.; Sagan, S. PLoS ONE 2011, 6, e24096. 10. Marks, J. R.; Placone, J.; Hristova, K.; Wimley, W. C. J Am Chem Soc 2011, 133, 8995–9004. 11. Säälik, P.; Niinep, A.; Pae, J.; Hansen, M.; Lubenets, D.; Langel, Ü.; Pooga, M. J Controlled Release 2011, 153, 117–125. 12. Walrant, A.; Matheron, L.; Cribier, S.; Chaignepain, S.; Jobin, M.-L.; Sagan, S.; Alves, I. D. Anal Biochem 2013, 438, 1–10. 13. Wheaten, S. A.; Ablan, F. D. O.; Spaller, B. L.; Trieu, J. M.; Almeida, P. F. J Am Chem Soc 2013, 135, 16517–16525. 14. Swiecicki, J.-M.; Bartsch, A.; Tailhades, J.; Di Pisa, M.; Heller, B.; Chassaing, G.; Mansuy, C.; Burlina, F.; Lavielle, S. ChemBioChem 2014, 15, 884–891. 15. Di Pisa, M.; Chassaing, G.; Swiecicki, J.-M. Biochemistry, DOI: 10.1021/bi501392n 16. McIntyre, J. C.; Sleight, R. G. Biochemistry, 1991, 30, 11819–11827. 17. Terrone, D.; Sang, S. L. W.; Roudaia, L.; Silvius, J. R. Biochemistry 2003, 42, 13787–13799. 18. Derossi, D.; Calvet, S.; Trembleau, A.; Brunissen, A.; Chassaing, G.; Prochiantz, A. J Biol Chem 1996, 271, 18188 – 18193. 19. Vives, E.; Brodin, P.; Lebleu, B. J Biol Chem 1997, 272, 16010 –16017. 20. Mitchell, D. J.; Steinman, L.; Kim, D. T.; Fathman, C. G.; Rothbard, J. B. J Pept Res 2000, 56, 318– 325. 21. Stanzl, E. G. B.; Trantow, M.; Vargas, J. R.; Wender, P. A. Acc Chem Res 2013, 46, 2944 –2954. 22. Jiao, C.-Y.; Delaroche, D.; Burlina, F.; Alves, I. D.; Chassaing, G.; Sagan, S. J Biol Chem 2009, 284, 33957–33965.


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23. Aussedat, B.; Sagan, S.; Chassaing, G.; Bolbach, G.; Burlina, F. Biochim et Biophys Acta Biomembr 2006, 1758, 375-383. 24. Burlina, F.; Sagan, S.; Bolbach, G.; Chassaing, G. Angew Chem Int Ed 2005, 44, 4244–4247. 25. Herce, H. D.; Garcia, A. E.; Cardoso, M. C. J Am Chem Soc 2014, 136, 17459-67. 26. Armstrong, C. L.; Barrett, M. A.; Toppozini, L.; Kucerka, N.; Yamani, Z.; Katsaras, J.; Fragneto, G.; Rheinstädter, M. C. Soft Matter 2012, 8, 4687–4694. 27. London, E.; Biochim Biophys Acta – Mol Cell Res 2005, 1746, 203–220. 28. Bezlyepkina, N.; Gracià, R. S.; Shchelokovskyy, P.; Lipowsky, R.; Dimova, R. Biophys J 2013, 104, 1456–1464. 29. Belting, M. Trends Biochem Sci 2003, 28, 145–151. 30. Ziegler, A.; Seelig, J. Biophys J 2004, 86, 254–263. 31. Gump, J. M.; June, R. K.; Dowdy, S. F. J Biol Chem 2010, 285, 1500–1507. 32. Ziegler, A.; Seelig, J. Biochemistry 2011, 50, 4650–4664. 33. Davidsen, J.; Mouritsen, O.G.; Jørgensen, K.; Biochim Biophys Acta 2002, 1564, 256e262. 34. Mills, J.K.; Needham, D. Biochim Biophys Acta 2005, 1716, 77–96. 35. Jespersen, H.; Andersen, J. H.; Ditzel, H. J.; Mouritsen, O. G. Biochimie 2012, 94, 2e10.


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Table I

Peptides and Lipids Used in This

Study Peptidesa and Lipids Used in this Study

Referred in Text as

RQIKIWFQNRRMKWKK-NH2 RRWWRRWRR-NH2 YGRKKRRQRRR-NH2 RRRRRRRRR-NH2 RFARKGALRQKNV-NH2

Penetratin R6/W3 Tat Arg9 PKCi

1,2-Oleoyl-sn-glycero-3phosphoglycerol

DOPG

1,2-Oleoyl-sn-glycero-3phosphocholine

DOPC

1,2-Palmitoyl-sn-glycero-3phosphoglycerol

DPPG

1,2-Sterayl-sn-glycero-3phosphoglycerol

DSPG

Sphingomyelin (egg; brain)

eSM; bSM

Cholesterol

Chol

a

The NBD probe, (7-nitrobenz-2-oxa-1,3-diazole), was incorporated at the N-terminus of the peptide, leading to the corresponding fluorescent peptides.

Table II

Internalization of Peptides in LUVs Made of a Single Type of PL % Internalization*

Lipids R6/W3

Arg9 =

PKCi =

Penetratin =

(5+/3Arg)

(3+/3Arg)

=

Tat (8+/6Arg)=

(6+/6Arg)

(9+/9Arg)

DOPGa

9.7 ± 0.6

15.7 ± 2.2

< 0.1

2.2 ± 0.3

9.3 ± 0.9

DPPG

39 ± 3

4.0 ± 0.2

9±3

52 ± 1

6.5 ± 1.5

DSPG

2.5 ± 0.5

2.7 ± 1.0

0.9 ± 0.6

1.7 ± 0.5

1.5 ± 0.5

LUVs (100 nm, 10 µM PLs), with the indicated PLs, were incubated for 300 s with the NBD-labeled peptide (100 nM) at 20°C for DOPG and 37°C for the other LUVs. For the LUVs made of DPPG or DSPG the temperature was rapidly decreased to 15°C and dithionite (DT, final concentration 10 mM) was added to reduce at 15°C the external NBD-labeled peptide. The fluorescence quenching was then recorded for 700 s. The residual fluorescence corresponded to the NBD-labeled peptide that had been internalized into the LUVs. * The % of internalized CPP was determined by plotting the asymptote of the reduction curve after DT addition, and by considering the intersection between this asymptote and the y-axis. = net positive charges of the peptide and number of arginines. All data correspond to 2 to 4 independent experiments ± s.e.m. a ChemBioChem14, both the 300 s incubation and the reduction by dithionite were done at 20°C.


17


Table III

Page 18 of 26

Accumulation of Peptides in LUVs Made of a Single Type of PL - Quenching

of the Fluorescence by Reduction (DT) or by Addition of HOLipids

% Internalization*

Quenching Procedure R6/W3

Arg9 =

PKCi =

Penetratin =

=

Tat

(6+/6Arg)

(9+/9Arg)

(5+/3Arg)

(3+/3Arg)

(8+/6Arg)=

39 ± 3

4.0 ± 0.2

9.0 ± 3.0

52 ± 1

6.5 ± 1.5

DPPG

DT

DPPG

HO-&

28.7 ± 3.4

9±3

9.5 ± 6.5

62 ± 1

13.5 ± 1.5

DSPG

DT

2.5 ± 0.5

2.7 ± 1.0

0.9 ± 0.6

1.7 ± 0.5

1.5 ± 0.5

4.2 ± 0.2

3.8 ± 0.2

1.2 ± 0.3

3.9 ± 0.1

2.3 ± 0.3

DSPG

HO

-&

LUVs (100 nm, 10 µM PLs), with the indicated PLs, were incubated for 300 s with the NBD-labeled peptide (100 nM) at 37°C. Then, the temperature was decreased (Peltier cooler) to 15°C, and either dithionite (DT, final concentration 10 mM) was added to reduce the external NBD-labeled peptide or HO- was added, (NaOH 30 µL, 1M), leading to an increase of the pH to 11.2. The fluorescence quenching was then recorded for 700 s. The residual fluorescence corresponded to the NBD-labeled peptide that had been internalized into the LUVs. *: The % of internalized CPP was determined by plotting the asymptote of the reduction curve after DT addition, and by considering the intersection between this asymptote and the y-axis. &: At the end of the experiment, HCl was added (one equivalent vs. added HO-) both the initial pH and the initial fluorescence were recovered. All data correspond to 2 to 4 independent experiments ± s.e.m.


18

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Table IV

Internalization of Peptides in LUVs Made of DOPG, DOPC, Sphingomyelin

and Cholesterol % Internalization*≠

Lipids Composition R6/W3

Arg9 =

PKCi

(6+/6Arg)

(9+/9Arg)

(5+/3Arg)=

9.7 ± 0.6

15.7 ± 2.2

< 0.0001

n.d.

< 0.1

n.d.

DOPC/Chol (1:1)

< 0.1

< 0.1

< 0.1

DOPG/Chol (9:1)

2.5 ± 0.2

5.1 ± 0.3

0.2

DOPG/Chol (5:5)

0.7±0.2

5.7 ± 0.8

< 0.1

DOPG/Chol (4:6)

0.8 ± 0.1

11.2 ± 1.0

< 0.1

0.80 ± 0.05

10.7 ± 1.1

0.2 ± 0.1

DOPG/DOPC/Chol (3:1:6)

1.8 ± 0.3

26.0 ± 1.5

0.22 ± 0.01

DOPG/DOPC/Chol (2.5:1.5:6)

2.8 ± 0.5

19.5 ± 1.6

< 0.1

DOPG/DOPC/Chol (2:2:6)

1.2 ± 0.1

16.3 ± 1.2

< 0.1

DOPG/DOPC/Chol (1.5:2.5:6)

1.8 ± 0.3

6.7 ± 1.8

0.5 ± 0.1

DOPG/eSM/Chol (1.5:2.5:6)

0.9 ± 0.5

0.08 ± 0.02

0.1 ± 0.05

DOPG/eSM/Chol (3:1:6)

1.0 ± 0.2

18.6 ± 2.6

0.16 ± 0.08

DOPG/bSM/Chol (3:1:6) ≠

0.10 ± 0.05

22.6 ± 3.2

0.1

DOPGa DOPC

a

DOPG/DOPC/Chol (3.5:0.5:6) a

=

LUVs (100 nm, 10 µM PLs), with the indicated composition were incubated for 5 min at 20°C with the NBDlabeled peptide (100 nM). Dithionite (DT, final concentration 10 mM) was then added to reduce the external NBD-labeled peptide, and the fluorescence quenching was recorded for 25 min. The residual fluorescence corresponded to the NBD-labeled peptide that had been internalized into the LUVs. *: The % of internalized CPP was determined by plotting the asymptote of the reduction curve after DT addition, and by considering the intersection between this asymptote and the y-axis. a ChemBioChem14. ≠ With this composition, DOPG/bSM/Chol (3:1:6), Tat was internalized (12.9 ± 0.7%), about 2-times less than with eSM, as already reported,14 whereas Penetratin did not enter within these LUVs (

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