Effects of Mechanical Properties of Lipid Bilayers on the Entry of Cell

Article pubs.acs.org/Langmuir

Effects of Mechanical Properties of Lipid Bilayers on the Entry of CellPenetrating Peptides into Single Vesicles Md. Zahidul Islam,† Sabrina Sharmin,† Victor Levadnyy,†,‡ Sayed Ul Alam Shibly,† and Masahito Yamazaki*,†,§,∥ †

Integrated Bioscience Section, Graduate School of Science and Technology, §Nanomaterials Research Division, Research Institute of Electronics, and ∥Department of Physics, Graduate School of Science, Shizuoka University, Shizuoka 422-8529, Japan ‡ Theoretical Problem Center of Physico-Chemical Pharmacology, Russian Academy of Sciences, Kosugina, 4, 117977, Moscow, Russia S Supporting Information *

ABSTRACT: The translocation of cell-penetrating peptides (CPPs) through plasma membranes of living cells is an important physiological phenomenon in biomembranes. To reveal the mechanism underlying the translocation of a CPP, transportan 10 (TP10), through lipid bilayers, we examined the effects of the mechanical properties of lipid bilayers on the entry of carboxyfluorescein (CF)-labeled TP10 (CF-TP10) into a giant unilamellar vesicle (GUV) using the single GUV method. First, we examined the effect of lateral tension in membranes on the entry of CFTP10 into single GUVs comprising a mixture of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) (2/8). CF-TP10 entered the GUV lumen before the membrane permeation of Alexa Fluor 647 hydrazide (AF647) from the GUV and thus before pore formation in the membrane. The fraction of entry of CF-TP10 before pore formation and the rate of membrane rupture increased with tension. The CF-TP10-induced fractional change in the membrane area increased continuously with time until membrane rupture, but it increased more slowly than did the CF-TP10 concentration in the GUV membrane. A high mole fraction of cholesterol inhibited the entry of CF-TP10 into single GUVs by suppressing the translocation of CF-TP10 from the external to the internal monolayer, although higher concentrations of CF-TP10 induced the formation of pores through which CF-TP10 rapidly translocated. Suppression of the translocation of CF-TP10 by cholesterol can be reasonably explained by the large line tension of a prepore. We discussed the role of mechanical properties in membranes on the entry of CF-TP10 into single GUVs and proposed a hypothesis of the mechanism that CF-TP10 translocates across a bilayer through transient hydrophilic prepores in the membrane.

1. INTRODUCTION It is generally believed that charged molecules cannot pass through a lipid membrane because the central core of the membrane comprises only hydrocarbon chains and thus has a low dielectric constant.1 However, a distinctive group of positively charged peptides, called cell-penetrating peptides (CPPs), can pass through the plasma membrane of living cells. This unique attribute of CPPs represents an important physiological aspect of biomembranes. CPPs are of interest to researchers conducting fundamental studies on biomembranes and also to researchers investigating the intracellular delivery of proteins, oligonucleotides, and drugs.2−7 CPPs can be categorized as arginine-rich CPPs,8−11 such as human immunodeficiency virus Tat protein-derived peptide and oligoarginine (Rn), and as amphipathic CPPs,12−16 such as penetratin, transportan (TP), and transportan 10 (TP10) (a truncated analogue of TP). There are two pathways for the entry of CPPs into cells; one is via endocytocis, and the other is via translocation through plasma membranes.2−7 In both cases, CPPs must translocate across lipid bilayers to enter a cell. However, the mechanism of the translocation of CPPs and their cargo through the plasma membrane is not clearly © 2017 American Chemical Society

revealed. Currently, it is considered that the transfer of CPPs from endosomes to the cytosol does not occur easily.17,18 Furthermore, it has been independently reported by several research groups that TP and TP10 can pass through the plasma membrane of a cell by nonendocytosis processes because several endocytosis inhibitors did not affect their translocation,12−16 although there has been one report suggesting that TP10 enters cells via the endocytotic pathway.19 Various hypotheses regarding the mechanism for the translocation of CPPs through lipid bilayers have been proposed. One model suggests that translocation occurs through the formation of inverted micelles in the bilayer20−22 and that CPPs form a neutral complex with negatively charged lipids in these inverted micelles. A second model suggests that translocation occurs through pores in the bilayer. In lipid bilayers composed of dioleoylphosphatidylserine (DOPS) and dioleoylphosphatidylethanolamine (DOPE), R6 and Tat peptide induced the formation of pores through which Received: August 22, 2016 Revised: January 21, 2017 Published: February 6, 2017 2433

DOI: 10.1021/acs.langmuir.6b03111 Langmuir 2017, 33, 2433−2443

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Langmuir fluorescent dyes such as AF633 leaked substantially from giant unilamellar vesicles (GUVs); following pore formation, these peptides entered the GUV lumen by diffusing through the pores.23−25 Recently, we have proposed the third model in which the translocation of CPPs occurs through transient hydrophilic prepores in the bilayer.26 In lipid bilayers in the liquid-crystalline phase, thermal fluctuations of the local lateral density of lipid membranes, which produce rarefaction and condensation, are large. As a result, various structures of rarefactions are produced transiently in the bilayers. Each area of decreased density (i.e., rarefaction) can be considered to be a prepore with an effective radius.27 When the radius of a rarefaction is above a threshold value, a hydrophilic prepore is formed, which has a toroidal structure where hydrophilic segments of lipid molecules are in contact with water at the wall of the prepore.28−33 Such prepores are unstable and hence immediately close. In the third model, we consider that CPPs can translocate across a bilayer through these hydrophilic prepores. It is reported that on application of voltage, fluctuating small electric currents were observed in a planar bilayer of interacting R9 molecules.34 Such currents are attributed to transient prepores because the currents were induced by ion translocation through the prepores. Using molecular dynamics (MD) simulation, Sun et al. indicated that R8 can prevent the closing of pores in a lipid membrane and considered that R8 can translocate across the bilayer via pores formed spontaneously by thermal fluctuations of the membrane.35 It is important to reveal the elementary processes involving the translocation of CPPs across lipid bilayers. For this purpose, a novel method has been proposed to investigate the translocation of CPPs across lipid bilayers using single GUVs.36,37 Using this single GUV method, we succeeded in observing the entry of fluorescent probe-labeled CPPs into the lumen of single GUVs and subsequent pore formation induced in the GUV membrane in real time using confocal laser scanning microscopy (CLSM). For example, it was found that a fluorescent probe, carboxyfluorescein (CF)-labeled TP10 (CFTP10), enters a GUV from aqueous solution outside the GUV by translocating across the lipid bilayer before CF-TP10induced pore formation in the lipid membrane.36 Statistical analyses of these data provided the fraction of entry of CFTP10 into a GUV before pore formation and the rate constant of CF-TP10-induced pore formation. To reveal the mechanisms of the entry of TP10 into a GUV lumen, we investigated the effects of the mechanical properties of lipid bilayers on the entry of CF-TP10 using single GUVs. First, we examined the effect of mechanical tension on the entry of CF-TP10 into GUVs composed of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) (2/8, molar ratio) and on the CF-TP10-induced membrane rupture of the GUV. These results indicated that mechanical tension (or stretching) of the membranes increased the rate of entry of CF-TP10 and also the rate constant of CF-TP10-induced membrane rupture. To elucidate the mechanism underlying these phenomena, we examined and analyzed the CF-TP10induced change in area of DOPG/DOPC (2/8) membranes over time and simultaneously the change in the CF-TP10 concentration in the membrane by analyzing the rim intensity. In contrast, it is well known that lipid bilayers containing high mole fractions of cholesterol (chol) are strong mechanically38 because the line tension of pores and prepores is larger.31,39,40 We examined the effects of a high mole fraction of cholesterol

on the entry of CF-TP10 into GUVs and on CF-TP10-induced pore formation. On the basis of these results, we discussed the elementary processes involved and the mechanism underlying the entry of CF-TP10 into GUVs.

2. MATERIALS AND METHODS 2.1. Chemicals. DOPC and DOPG were purchased from Avanti Polar Lipids Inc. (Alabaster, AL). Bovine serum albumin (BSA) was purchased from Wako Pure Chemical Industry Ltd. (Osaka, Japan). Cholesterol, biotin-labeled BSA, and streptavidin were purchased from Sigma-Aldrich Co. (St. Louis, MO). Alexa Fluor 647 hydrazide (AF647), 5-(and 6)-carboxyfluorescein succinimidylester, and N-((6(biotinoyl) amino)-hexanoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (biotin-X-DHPE, referred to as biotin-lipid) were purchased from Invitrogen Inc. (Carlsbad, CA). TP10 was synthesized by the FastMoc method using a 433A peptide synthesizer (PE Applied Biosystems, Foster City, CA).36 Synthesis of the carboxyfluorescein-labeled TP10 (CF-TP10) fluorescence probe, which has one CF fluorophore at the N-terminus of the peptide, and the methods for peptide cleavage, purification, and identification were described previously.36 2.2. GUV Preparation and Observation. GUVs of DOPG/ DOPC/biotin-lipid (molar ratio 20/79/1; hereafter abbreviated PG/ PC (2/8)-GUVs) and GUVs of DOPG/DOPC/chol/biotin-lipid (molar ratio 20/59/40/1; cholesterol mole fraction 0.33; hereafter abbreviated PG/PC/chol (2/6/4)-GUVs) were prepared in buffer H (10 mM HEPES, pH 7.5, 100 mM NaCl, and 1 mM EGTA) containing 0.1 M sucrose and 6 μM AF647 by the natural swelling method.41 The preparation method of GUVs containing small vesicles in the GUV lumens was described previously.36 Untrapped fluorescent probes were removed by using the membrane filtering method.42 After the purification, a GUV suspension (0.1 M sucrose in buffer H as the internal solution, 0.1 M glucose in buffer H as the external solution) was transferred into a handmade microchamber, formed on a glass slide by placing a U-shaped silicone-rubber spacer between the glass slide and a coverslip (for experiments using a single micropipette)43 or two parallel-deposited bar-shaped silicon-rubber spacers between the glass slide and a coverslip (for experiments using two micropipettes).44 To prevent strong interaction between the glass surface and the GUVs, the inside of the microchamber was coated with 0.10% (w/v) BSA in the same buffer as used for the experiments.43 Single GUVs were fixed on the slide glass or the coverslip in the chamber using the strong association between biotin and streptavidin.36 For experiments using CF-TP10, the GUVs were observed using CLSM (FV-1000, Olympus, Tokyo, Japan) at 25 ± 1 °C using a stage thermocontrol system (Thermoplate, Tokai Hit, Shizuoka, Japan).36 For experiments using TP10, GUVs were observed using an inverted fluorescence, differential interference contrast (DIC) microscope (IX71, Olympus) at 25 ± 1 °C controlled by the stage thermocontrol system. 2.3. Effect of Tension on the Entry of CF-TP10 into PG/PC (2/ 8)-GUVs and on CF-TP10-Induced Membrane Rupture. First, a single GUV containing small vesicles was held at the tip of a micropipette (called micropipette A). The micropipette had a diameter of ∼10 μm and was coated using 0.50% (w/v) BSA dissolved in buffer H containing 0.10 M glucose. The single GUV was held for several minutes using a small aspiration pressure that provided a membrane tension of 0.20 or 0.50 mN/m. Then, to apply a specific tension to the GUV membrane, the GUV was rapidly (in ∼10 s) aspirated by applying an aspiration pressure of ΔP (= Pout − Pin), where Pout and Pin are the pressures of the outside and the inside of a micropipette, respectively.27,45 The tension of the GUV membrane, σ, can be controlled by ΔP as follows46

σ=

ΔPdP 4(1 − dP/D V )

(1)

where dp is the inner diameter of the micropipette and DV is the diameter of the spherical part of the aspirated GUV outside the micropipette. After maintaining this tension for a few minutes, a 2434

DOI: 10.1021/acs.langmuir.6b03111 Langmuir 2017, 33, 2433−2443

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Langmuir

Figure 1. Effects of tension on the entry of CF-TP10 into single PG/PC (2/8)-GUVs containing small vesicles. (A) CLSM images of a GUV obtained using the fluorescence emitted by (1) AF647 and (2) CF-TP10. σ = 1.0 mN/m. The numbers above each image show the time (s) after which we started to add 0.50 μM CF-TP10. The bar is 20 μm. (B) (1) DIC image of a GUV fixed at the tip of micropipette A using a small aspiration pressure. (2) CF-TP10 solution was continuously added from micropipette B into the neighborhood of the GUV shown in (B1). dp is the inner diameter of the micropipette, and DV and DV0 are the diameters of the spherical part of the aspirated GUV outside the micropipette after and before the interaction with CF-TP10, respectively. The bar is 20 μm. (C) The normalized rim intensity of the GUV due to CF-TP10 shown in (A) as a function of time. The red data points and green squares correspond to FI of AF647 in the GUV lumen and of CF-TP10 in the rim of the GUV, respectively. (D) Tension dependence of the fraction of entry of CF-TP10 before pore formation at 6 min, Pentry (6 min). 0.60 μM (green ○), 0.50 μM (red □), and 0.30 μM (Δ) CF-TP10. 2.5. Interaction of PG/PC/chol (2/6/4)-GUVs with CF-TP10. We examined the interaction of CF-TP10 with single PG/PC/chol (2/ 6/4)-GUVs containing AF647 and small vesicles (or containing AF647 but not small vesicles) using a CLSM controlled at 25 ± 1 °C by a stage thermocontrol system.36 The CF-TP10 solution (in buffer H containing 0.1 M glucose) was continuously provided to the neighborhood of a GUV through a micropipette with a diameter of ∼20 μm. The distance between the GUV and the tip of the micropipette was ∼60 μm, and the ΔP of the micropipette was −30 Pa. The fluorescence intensities were obtained as described in Section 2.3.

specific concentration of CF-TP10 solution was continuously added to the neighborhood of the GUV from a second, wider micropipette (called micropipette B, diameter of ∼20 μm). The detailed experimental methods were described previously.44 The changes in the fluorescence intensity of the rim and the inside of the GUV over time during the interaction of CF-TP10 were measured as described previously.36 During the interaction with CF-TP10, the GUV was suddenly aspirated completely into the micropipette as a result of the rupture of the GUV membrane. The time of rupture was determined as the time when the GUV was completely aspirated with a time resolution of δ1). In contrast, in the interaction of CF-TP10 with single PG/PC (2/8)-GUVs, the rim intensity increases more rapidly than does δ (Figure 2B). We consider that this is related to the translocation of CF-TP10 from the external to the internal monolayer, but currently we cannot reasonably explain this different time course quantitatively. As described in Section 3.2, kFF is an important factor influencing the rate of entry of CF-TP10 into the GUV lumen. On the basis of the above discussion, we next elucidate which factor influences the value of kFF in GUV membranes. It is generally believed that lipid bilayers in the Lα phase undergo thermal fluctuation, resulting in transient decreases or increases in the lateral density of lipid membranes in some local areas.27 When the size of an area of decreased density reaches a critical value, this area is converted to a hydrophilic prepore with an effective radius r.33 The probability of the production of a prepore is larger in a stretched membrane or when tension is applied. On the basis of the theory of tension-induced pore formation,28,55−57 the production of a prepore in the bilayer changes the total free energy of the system by a term of the free energy of a prepore U(r) consisting of two factors: one term (−πr2σ) is associated with lateral tension, σ, and favors an increase in the size of the prepore, and the other term (2πrΓ) is due to the line tension, Γ, (i.e., the line free energy per unit length) of the prepore edge and favors a decrease in the size of the prepore (Figure S5). Hence, U(r) can be described as U (r ) = 2πr Γ − πr 2σ

closing39,58 and the characteristics of rupture of a GUV induced by tension.27,31−33,45 On the basis of this thermal fluctuation of lipid bilayers, we propose the following hypothesis for the mechanism of entry of CF-TP10 into the PG/PC (2/8)-GUV lumen. Initially, the imbalance in CF-TP10 concentration in the two monolayers (COM > CIM) induces a stretching of the internal monolayer. This induces an applied tension in the internal monolayer, which decreases Umax and hence increases the formation of prepores in the membrane. It is considered that the structure of a prepore is a toroidal-like structure. Therefore, CF-TP10 molecules bound to negatively charged lipids in the external monolayer can diffuse to the wall of a toroidal prepore and then into the internal monolayer. Recently, Sun et al. also conducted MD simulations and observed that R8 can prevent the closing of membrane pores and considered that R8 can pass through naturally occurring thermal pores.35 These pores correspond to the prepores mentioned above. Therefore, we can consider that CF-TP10 can prevent the closing of prepores and can thus transfer from the external to the internal monolayer by passing through the prepores. We can reasonably assume the additivity of the tension σex due to aspiration by the micropipette and the tension due to the imbalance in the CF-TP10 concentration in the two monolayers, σTP10, and hence the total tension, σt, can be expressed by σt = σex + σTP10. (It is noted that the additivity of tension due to the aspiration of a GUV and the tension due to osmotic pressure was experimentally proved recently.48) Because the rate of prepore formation is determined by σt, the application of external tension can increase the rate of prepore formation, which can reasonably explain the result that external tension increased the rate of entry of CF-TP10 into a GUV (Figure 1D). Figure 6 shows that Umax of the free energy of a prepore decreases with an increase in σ by 1.5 mN/m (curves A and B). The initial slope of U(r) decreases with a decrease in Umax, and hence the rate of prepore formation increases with increasing σ. In contrast, it is established that high mole fractions of cholesterol increase the line tension of a pore, Γ, thereby suppressing thermal fluctuation of the lipid bilayer; for example, in DOPC/chol-GUVs, Γ increases with increasing mole fraction of cholesterol, Xchol, at low concentrations of Xchol (0 ≤ Xchol ≤ 0.20; e.g., at Xchol = 0.20, the increase in Γ was 5.3 pN).39 On the basis of this data, we can consider that the Γ value for PG/PC/chol (2/6/4) is larger than that for PG/PC (2/8) by ∼5 pN (curve C). Figure 6 shows that the initial slope of U(r) of curve C (corresponding to PG/PC/chol (2/6/4)) is greater than that of curve B (corresponding to PG/PC (2/8)). We can therefore reasonably consider that in PG/PC/chol (2/ 6/4)-GUVs the rate of prepore formation is much lower and the prepores are too small for the entry of CF-TP10, thereby suppressing the entry of CF-TP10 into PG/PC/chol (2/6/4)GUVs. This analysis indicates that the applied tension and the line tension greatly affect the radius of prepores and the rate of prepore formation. As a result, the rate of entry of CF-TP10 into the GUV lumen is highly dependent on the external tension and the presence of cholesterol. Our results clearly show that the entry of CF-TP10 into single PG/PC (2/8)-GUVs occurred before pore formation, which eliminates the second model (translocation through pores) described in the Introduction. The experimental evidence available to support the first model (inverted micelle formation) is that negatively charged lipids were transferred from the external to the internal monolayer during the translocation of CPPs such as TAT and oligoarginine and

(6)

The maximum in U(r) (i.e., Umax) equals U(r*) = πΓ /σ at r = r* (= Γ/σ) (Figure 6). The radius of prepores, r, varies with time because of thermal fluctuation. When r is less than r*, it closes quickly (i.e., no pore formation), but when r is above r*, pore formation occurs. Several phenomena can be reasonably explained by this theory, such as the dynamics of pore 2

Figure 6. Free-energy profile of a prepore, U(r), for various tensions and line tensions: (A) σ = 5.5 mN/m and Γ = 12 pN, (B) σ = 4.0 mN/ m and Γ = 12 pN, and (C) σ = 3.0 mN/m and Γ = 18 pN. U(r) was determined on the basis of eq 6. 2441

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Langmuir Present Address

that there was a defined stoichiometry of CPPs and charged lipids during the transfer.22 The former part of this evidence can be explained by the third model (translocation through prepores); during the translocation of CPPs through a prepore, CPP molecules bound to negatively charged lipids in the external monolayer can diffuse into the internal monolayer through the wall of the toroidal prepore structure,44 inducing the transfer of negatively charged lipids. However, the latter part of this evidence (i.e., the stoichiometry) cannot be explained by the third model. As described above, the results of the effects of mechanical properties of lipid membranes on the entry of CF-TP10 into single GUVs supports the third model (translocation through prepores), although the first model (inverted micelle formation) cannot be ruled out.

(Md.Z.I.) Department of Biotechnology and Genetic Engineering, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid for Scientific Research (B) (no. 15H04361) from the Japan Society for the Promotion of Science (JSPS) to M.Y.

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(1) Hille, B. Ionic Channels of Excitable Membranes, 2nd ed.; Sinauer Associates, Inc.: Sunderland, MA, 1992. (2) Magzoub, M.; Gräslund, A. Cell-penetrating peptides: small from inception to application. Q. Rev. Biophys. 1999, 37, 147−195. (3) Zorko, M.; Langel, Ű . Cell-penetrating peptides: mechanism and kinetics of cargo delivery. Adv. Drug Delivery Rev. 2005, 57, 529−545. (4) Madani, F.; Lindberg, S.; Langel, Ű .; Futaki, S.; Gräslund, A. Mechanisms of cellular uptake of cell-penetrating peptides. J. Biophys. 2011, 2011, 414729. (5) Bechara, C.; Sagan, S. Cell-penetrating peptides: 20 years later, where do we stand? FEBS Lett. 2013, 587, 1693−1702. (6) Stanzl, E. G.; Trantow, B. M.; Vargas, J. R.; Wender, P. A. Fifteen years of cell-penetrating, guanidinium-rich molecular transporters: basic science, research tools, and clinical applications. Acc. Chem. Res. 2013, 46, 2944−2954. (7) Di Pisa, M.; Chassaing, G.; Swiecicki, J.-M. Translocation mechanism(s) of cell-penetrating peptides: biophysical studies using artificial membrane bilayers. Biochemistry 2015, 54, 194−207. (8) Green, M.; Loewenstein, P. M. Autonomous functional domains of chemically synthesized human immunodeficiency virus tat transactivator protein. Cell 1988, 55, 1179−1188. (9) Vivès, E.; Brodin, P.; Lebleu, B. A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J. Biol. Chem. 1997, 272, 16010− 16017. (10) Richard, J. P.; Melikov, K.; Vivès, E.; Ramos, C.; Verbeure, B.; Gait, M. J.; Chernomordik, L. V.; Lebleu, B. Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake. J. Biol. Chem. 2003, 278, 585−590. (11) Futaki, S.; Suzuki, T.; Ohashi, W.; Yagami, T.; Tanaka, S.; Ueda, K.; Sugiura, Y. Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery. J. Biol. Chem. 2001, 276, 5836−5840. (12) Pooga, M.; Hällbrink, M.; Zorko, M.; Langel, Ű . Cell penetration by transportan. FASEB. J. 1998, 12, 67−77. (13) Pooga, M.; Kut, C.; Kihlmark, M.; Hällbrink, M.; Fernaeus, S.; Raid, R.; Land, T.; Hallberg, E.; Bartfai, T. M.; Langel, Ű . Cellular translocation of proteins by transportan. FASEB. J. 2001, 15, 1451− 1453. (14) Soomets, U.; Lindgren, M.; Gallet, X.; Pooga, M.; Hällbrink, M.; Elmquist, A.; Balaspiri, L.; Zorko, M.; Pooga, M.; Brasseur, R.; Langel, Ű . Deletion analogues of transportan. Biochim. Biophys. Acta, Biomembr. 2000, 1467, 165−176. (15) EL-Andaloussi, S.; Johansson, H.; Magnusdottir, A.; Järver, P.; Lundberg, P.; Langel, Ű . TP10, a delivery vector for decoy oligonucleotides targeting the Myc protein. J. Controlled Release 2005, 110, 189−201. (16) Tints, K.; Prink, M.; Neuman, T.; Palm, K. LXXLL peptide convert transportan 10 to a potent inducer of apoptosis in breast cancer cells. Int. J. Mol. Sci. 2014, 15, 5680−5698. (17) El-Sayed, A.; Futaki, S.; Harashima, H. Delivery of macromolecules using arginine-rich cell-penetrating peptides: ways to overcome endosomal entrapment. AAPS J. 2009, 11, 13−21.

5. CONCLUSIONS The application of mechanical tension by an external force on the lipid membrane increased the rate of entry of CF-TP10 into the PG/PC (2/8)-GUV lumen without pore formation. To the best of our knowledge, this is the first experimental data to show that mechanical tension increases the membrane permeability of substances such as CPPs without pore formation. Stretching of the membrane due to an applied tension increases its thermal fluctuations and hence increases the transient prepore formation, which is the main factor of the increase in the translocation of CF-TP10 from the external to the internal monolayer, resulting in the increase in the entry of CF-TP10. In contrast, the presence of a high mole fraction of cholesterol inhibited the entry of CF-TP10 into single GUVs as a result of the suppression of the translocation of CF-TP10 from the external to the internal monolayer at low concentrations. However, at higher concentrations CF-TP10 induced stochastic pore formation in the membranes, through which the rapid translocation of CF-TP10 occurred. Suppression of the translocation of CF-TP10 by cholesterol can be reasonably explained by the decrease in thermal fluctuations in the membrane due to the large line tension of prepores. On the basis of these results, we have proposed a hypothesis that the imbalance in CF-TP10 concentration in the two monolayers induces stretching of the internal monolayer, which increases prepore formation, and CF-TP10 translocates from the external to the internal monolayer due to diffusion via the wall of hydrophilic prepores with a toroidal-like structure.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b03111. Relationship between the rim intensity and the CF-TP10 concentration in a GUV membrane, a schematic diagram for the elementary processes for the entry of CF-TP10, time course of the CF-TP10 concentration in DOPG/ DOPC/chol-GUV, and a scheme of a prepore in a lipid bilayer (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: 81-54-238-4741. E-mail: yamazaki.masahito@ shizuoka.ac.jp. ORCID

Masahito Yamazaki: 0000-0002-5959-9914 2442

DOI: 10.1021/acs.langmuir.6b03111 Langmuir 2017, 33, 2433−2443

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DOI: 10.1021/acs.langmuir.6b03111 Langmuir 2017, 33, 2433−2443