Tuning the Viscoelasticity of Peptide Vesicles by ... - ACS Publications

May 11, 2017 - The most frequent residue numbers of poly(sarcosine) blocks in TOF mass spectra are 28, 21, 25, and 27 for SLV, SLL, SDV, and SDL, ...
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Tuning the Viscoelasticity of Peptide Vesicles by Adjusting Hydrophobic Helical Blocks Comprising Amphiphilic Polypeptides Cheol Joo Kim,† Motoki Ueda,† Tomoya Imai,‡ Junji Sugiyama,‡ and Shunsaku Kimura*,† †

Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Kyoto-Daigaku-Katsura, Nishikyo-ku, Kyoto 615-8510, Japan ‡ Research Institute for Sustainable Humanosphere (RISH), Kyoto University, Uji, Kyoto 611-0011, Japan S Supporting Information *

ABSTRACT: Amphiphilic block polypeptides of poly(sarcosine)-b-(L-Val-Aib)6 and poly(sarcosine)-b-(L-Leu-Aib)6 and their stereoisomers were self-assembled in water. Three kinds of binary systems of poly(sarcosine)-b-(L-Leu-Aib)6 with poly(sarcosine)-b-poly(D-Leu-Aib)6, poly(sarcosine)-b-poly(LVal-Aib)6, or poly(sarcosine)-b-(D-Val-Aib)6 generated vesicles of ca. 200 nm diameter. The viscoelasticity of the vesicle membranes was evaluated by the nanoindentation method using AFM in water. The elasticity of the poly(sarcosine)-b-(LLeu-Aib)6/poly(sarcosine)-b-poly(D-Leu-Aib)6 vesicle was 11fold higher than that of the egg yolk liposome but decreased in combinations of the Leu- and Val-based amphiphilic polypeptides. The membrane elasticity is found to be adjustable by a suitable combination of helical blocks in terms of stereocomplex formation and the interdigitation of side chains among helices in the molecular assemblies.

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one morphology to the other.16,17 The concept behind the use of helical hydrophobic blocks is to avoid polymer-chain entanglements in molecular assemblies, which tend to lose the dynamic properties of membranes. A wide range of morphologies including curved and flat sheets, tubes with different lengths, vesicles with different diameters, and a chimeric round-bottomed shape have been attained by the self-assembly of amphiphilic block polypeptides comprising helical hydrophobic blocks.16−21 Unsymmetric vesicles were also prepared by binary systems of A3B- and AB-type amphiphilic polypeptides, which led to unsymmetric interdigitated monolayer formation as a result of stereocomplex formation, the antiparallel arrangement of helix dipoles, and steric hindrance of the bulky A3 unit extending outward from the vesicle.20 Furthermore, a vesicle diameter of as small as 60 nm was achieved by using nanotubes as a structural template for vesicle formation under the processes of fusion of the nanotube and sheet followed by fission.21 The amphiphilic block polypeptides having a helical hydrophobic block therefore have high potential for the preparation of various morphologies with membrane fusion and fission. For a better understanding of the molecular assemblies composed of amphiphilic block polypeptides, there are still several issues to be challenged. One such issue is in regard to membrane elasticity. Membrane physical properties are directly associate with physical stability

INTRODUCTION Amphiphilic molecules can self-assemble in water to form a variety of morphologies.1 In addition to morphology, phase separation in molecular assemblies is also known to be critical to the biological functions of membranes.2 Combinations of lipids and cholesterol have been used to shape various morphologies and to induce phase separation, resulting in tunable physical properties.3 However, lipid membranes sometimes show limitation in applications as a result of their instability for objectives.4−6 Liposome PEGylation is one way to improve the physical stability of liposomes in the bloodstream.7,8 The instability of liposomes, however, is sometimes useful and can be utilized for modifications of liposomes. For example, single unilamellar vesicles can be converted into large unilamellar vesicles by membrane fusion above the phase-transition temperature,9,10 dependent sensitively on the lipid composition and ionic strength of the medium.11 Membrane fluidity is also adjustable, for example, by mixing cholesterol, but generally with an increase in vesicle size.12 On the other hand, self-assemblies using amphiphilic copolymers generally show excellent stability due to long hydrophobic blocks forming thick membranes in water. Polymersomes, however, lack some basic properties of membrane fusion and fission, membrane fluidity, and membrane permeability, which liposomes usually show.13−15 In the aim of making polymersomes equipped with these missing dynamic properties, amphiphilic block polypeptides having a helical hydrophobic block were examined and successful in showing membrane fusion and fission to convert © XXXX American Chemical Society

Received: January 26, 2017 Revised: May 1, 2017 Published: May 11, 2017 A

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Langmuir and even cell activities to interact with. For example, mesenchymal stem cells seem to be equipped with a sensing system in the cell membrane to detect the matrix stiffness (elasticity) of the gel used for cell culturing, which leads mesenchymal stem cells to differentiate into neurons, myoblasts, and osteoblasts in accordance with the matrix stiffness.22 Another example is immune reactions. Antigen displayed on lipid membranes can trigger the immunological activity of B-cells to a certain degree, in response to the stiffness or fluidity of lipid membranes, because the association of antigen with B cell receptors is influenced by the physical property of the matrix membrane.23,24 It is therefore meaningful to find a way to tune the membrane viscoelasticity of molecular assemblies for applications of materials to interact with cells. There are various methods for measuring the physical properties of biomembranes,25−28 but the nanoindentation method using AFM is powerful for the evaluation of membrane elasticity over a wide size range from a few tens of nanometers to micrometers as reported for polymer nanoparticles,29,30 liposomes,31,32 cells,33 and polymer thin films.34 Because the amphiphilic block polypeptides used in this study self-assemble into vesicles having a diameter similar to that of liposomes, the nanoindentation method using AFM in water was employed here to evaluate the membrane elasticity in comparison to that of liposomes. With the in-depth analysis of membrane elasticity in relation to peptide structure, the physical properties of the peptide vesicles will become tunable by the suitable design and combination of the hydrophobic helical blocks of the comprising amphiphilic polypeptides.

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Figure 1. Molecular structures of amphiphilic block polypeptides having various hydrophobic helical blocks. The most frequent residue numbers of poly(sarcosine) blocks in TOF mass spectra are 28, 21, 25, and 27 for SLV, SLL, SDV, and SDL, respectively. stained with 2% uranyl acetate, followed by suctioning of the excess fluid with filter paper. Frozen-Hydrated/Cryogenic-TEM (Cryo-TEM). Cryo-TEM observations were carried out using a JEM-2000EXII transmission electron microscope (Jeol Co. Ltd., Japan) operated at 100 kV. The Leica Reichert KF-80 cryo-plunger was used to prepare cryo-TEM specimens. The dispersions were applied to a holey carbon film on a copper grid (Quantifoil from Quantifoil Micro Tools GmbH, Germany or a lacey carbon film from Ted Pella Inc., USA). The excess dispersion was removed by blotting with filter paper, and then the grids were immediately plunged into liquid ethane (−175 °C) to trap the particles in a thin film of vitreous ice by flash freezing. The excess ethane was blotted with filter paper, and then the grids were transferred into the microscope with a cryo-transfer system (model 626-DH, Gatan Inc., USA). The samples were observed at −175 °C under low-dose conditions. Dynamic Light Scattering (DLS) Measurements. The hydrodynamic diameter of molecular assemblies in Milli-Q water was analyzed by DLS-8000KS (Photal Otsuka Electronics) using a He−Ne laser. Each dispersion was filtered with a 0.80 μm PVDF (poly(vinylidene fluoride)) syringe filter (GE Healthcare UK limited) prior to measurements. Atomic Force Microscope (AFM). Substrate Modification. A dispersion of a molecular assembly in water was put on a Si-wafer substrate with a surface modified with 3-aminopropyltriethoxysilane (APTES). Si wafers were cleaned with 2% hydrofluoric acid and soaked in piranha solution for the introduction of silanol groups onto the Si-wafer surface. Then, Si wafers were treated with 1% APTES solution (toluene) at 60 °C for 10 min. The surface-modified Si wafer was confirmed to be a monolayer of APTES with a thickness of 1 nm by ellipsometry (data not shown). Imaging and Nanoindentation. Topological images and force curves of molecular assemblies were measured in Milli-Q water with an Agilent 5500 AFM (Agilent Technologies Inc., USA) in acoustic AC mode (AAC mode, so-called tapping mode) using gold-coated silicon nitride cantilevers (OMCL-TR400PB, 0.09 N/m, Olympus Corp., Tokyo, Japan). The spring constants of the cantilevers were determined by the thermal tuning method. The freshly prepared molecular assembly dispersion was incubated in a fluid liquid cell on an APTES-modified Si wafer at room temperature for 1 h. Then, the excess molecular assemblies in the fluid liquid cell were removed by replacing the Milli-Q water very slowly using a syringe. A vesicle on the Si wafer was enlarged into a maximized image for the measurement

MATERIALS AND METHODS

Materials. Boc-L-valine and Boc-D-valine were purchased from Tokyo Chemical Industry Co., Ltd. (Japan). Boc-L-leucine, Boc-Dleucine, 2-aminoisobutyric acid (Aib), and Z-sarcosine were purchased from Watanabe Chemical Industries, Ltd. (Japan). Egg yolk phosphatidylcholine with 99% purity was purchased from Sigma Co. (St. Louis, MO). All other reagents and solvents were purchased from commercial suppliers and used as received. Synthesis of Amphiphilic Block Polypeptides. Four kinds of amphiphilc block polypeptides (Figure 1) were synthesized. The syntheses of poly(sarcosine)-b-(L-Leu-Aib)6 and poly(sarcosine)-b-(DLeu-Aib)6 were reported previously.16 Other amphiphilic block polypeptides were synthesized similarly according to synthesis schemes S1 and S2 (Supporting Information). Circular Dichroism (CD). Circular dichroism spectra were measured with a Jasco 1600 spectropolarimeter with an optical cell of 0.1 cm path length at room temperature. The solution concentration in ethanol was adjusted to be 0.375 mM (residue). Preparation of Self-Assemblies. Each amphiphilic block polypeptide (10 mg) was dissolved in ethanol (200 μL). An aliquot of the polymer solution (20 μL, 1 mg) was injected into Milli-Q water (1 mL) under stirring at 4 °C for 30 min. The dispersions were held at 90 °C for 1 h if necessary. Egg yolk phosphatidylcoline liposome was prepared by the film hydration technique using an aqueous solution (20 mM NaCl) at 0.5 mg/mL concentration. Sonication for 2 h with a bath-type sonicator (100 W, 28 kHz, VS-100III, As One Corp., Osaka, Japan) was employed to obtain sonicated unilamellar vesicles (SUVs), and then the liposome suspension was extruded through a polycarbonate membrane (pore size of 50 nm) using a mini-extruder from Avanti Polar Lipids, Inc. (Alabaster, AL). Transmission Electron Microscope (TEM). TEM images of molecular assemblies were analyzed with a JEOL JEM-2000EXII at an accelerating voltage of 100 kV. For specimen preparation, a drop of dispersion was mounted on a carbon-coated Cu grid and negatively B

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Langmuir of force−distance curve. A small number of tapping points were employed to minimize the pressing force and to prevent the deformation of the vesicles. To calibrate the deflection sensitivity, cantilever deflection vs the z-piezo position were measured on the hard silicon substrate as a reference. The force−distance curves were fitted according to the Hertzian contact model assuming that the tip and vesicles were spherical. The Hertzian contact model using AFM is shown in the form of δ = A|d − d0|2/3,24 and F = Aδ3/2, eq 1, was employed. The radius of vesicles was calculated as the radius of a spherical cap using eq 2 because the vesicles were in the form of a spherical cap upon adsorption onto the substrate. F = Aδ 2

R=

3/2

⎤1/2 ⎡ Eves 2R tipR ves ⎥ δ 3/2 = 1.335⎢ ⎢⎣ (R tip + R ves)(1 − vves 2)2 ⎥⎦

(1)

2

r +h 2h

(2)

E is the elastic modulus of the vesicle, Rtip is the radius of the tip, υ is Poisson’s ratio of the vesicle, and δ is the deformation of the sample from the tip−sample contact point (indentation depth). (Poisson’s ratio, 0.5; radius of the tip, 30 nm).

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RESULTS AND DISCUSSION CD Analysis. Amphiphilic block polypeptides of poly(sarcosine)-b-(L-Val-Aib)6 (SLV) and poly(sarcosine)-b-(D-ValAib)6 (SDV), where the block lengths of poly(sarcosine) determined from the signal intensity ratio of N-CH3 of sarcosine residues compared to CH3 of Val resides in NMR spectra were 28 and 25, respectively, for SLV and SDV, were synthesized. Conformations of these peptides were analyzed by CD spectroscopy (Figure 2). In ethanol, a double-minima

Figure 3. Transmission electron microscopy (TEM) images of selfassemblies stained by uranyl acetate: (a, b) SLV, (c, d) SDV, (e, f) a mixture of SLV and SDV (SLVSDV, 1:1 mixture) (black arrow; small sheet, white arrow; wormlike micelles). The self-assemblies were held at 90 °C for 1 h (b, d, f).

small self-assemblies grew into larger sheets upon heating at 90 °C for 1 h. On the other hand, in the case of poly(sarcosine)-b(L-Leu-Aib)6 (in which the block length of poly(sarcosine) was estimated to be 21 residues, SLL), curved sheets were identified just after injection into water and were converted into nanotubes upon heating.37 Because the curving of the sheet is ascribed to the tight packing of helices leading to consecutive helix tilting in the molecular assembly, the planarity of the sheet prepared by SLV or SDV suggests that the (Val-Aib)6 blocks have an inferior helix-packing ability compared to that of the (Leu-Aib) 6 blocks upon molecular assembly. Dimethyl substitution at the β-carbon of Val may cause severe steric hindrance so as to prevent the helices from their tight association in water. Molecular assemblies composed of a mixture of the rightand left-handed helices were analyzed (Figure 3e,f). As previously reported, the binary system of poly(sarcosine)-b(L-Leu-Aib)6 and poly(sarcosine)-b-(D-Leu-Aib)6 (SLLSDL) yielded planar sheets just after injection into water, which were then converted into vesicles upon heating as a result of stereocomplex formation between the right- and left-handed helices (Figure 4a,b).16 The planar sheet was shown to be composed of an interdigitated monolayer owing to the tight helix packing.16,38 On the other hand, the binary system of SLV and SDV (SLVSDV) generated only small sheets, which grew into larger sheets only upon heating, but vesicle formation failed (Figure 3e,f). Taken together, the (Val-Aib)6 blocks are considered to have less potential for molecular assembly with regard to helix association in water than do the (Leu-Aib) blocks. When SLL was mixed with SDV or SLV (SLLSDV or SLLSLV) followed by injection into water, distorted small sheets (SLLSDV) or twisted ribbons (SLLSLV) were observed

Figure 2. Circular dichroism spectra of amphiphilic block polypeptides in ethanol: (blue) SLV and (orange) SDV.

Cotton effect of SLV and a mirror image of SDV were observed to indicate the α-helical conformation of the (Val-Aib)6 blocks. In general, Val is known as a helix breaker in protein structures, but it can constitute the α-helical structure when involved in the Aib-rich sequence of the α-helix preference.35 In water, these spectrum shapes were retained but upon intensifying the signal strength at 222 nm, suggesting that these peptides assembled in water with forming helix bundles.36 Morphology of Molecular Assemblies. The morphologies of the molecular assemblies of these amphiphilic polypeptides in water were analyzed by TEM observation (Figure 3a−d). SLV or SDV assembled into small planar sheets and wormlike micelles just after injection into cold water. The C

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Figure 4. TEM images of self-assemblies stained by uranyl acetate: (a, b) a mixture of SLL and SDL (SLLSDL, 1:1 mixture); (d, e) a mixture of SLL and SDV (SLLSDV, 1:1 mixture); and (g, h) a mixture of SLL and SLV (SLLSLV, 1:1 mixture). The self-assemblies were held at 90 °C for 1 h (b, e, h). The size distribution of each vesicle (c, f, and i) was obtained by a dynamic light scattering measurement.

Figure 5. (a) Atomic force microscopy (AFM) in the water image of SLLSDL vesicles on an APTES-modified Si wafer. (b−g) AFM in water images of one vesicle (magnified) and height profiles along the lines in b, d, and f for (b, c) SLLSDL, (d, e) SLLSDV, and (f, g) SLLSLV vesicles. (h) Average width and height of each vesicle.

(Figure 4e,h). With the help of the tight helix association ability of the (Leu-Aib)6 block, the (Val-Aib)6-base peptides are taken in vesicular assemblies. As a result, three kinds of vesicles can be prepared from the current binary systems. DLS measurements revealed that the average diameters of the vesicles were similar to each other at 203 nm (PDI = 0.15), 221 nm (PDI = 0.03), and 204 nm (PDI = 0.07), respectively, for SLLSDL, SLLSDV, and SLLSLV vesicles (Figure 4c,f,i). The small PDI values indicate that the formed vesicles have a homogeneous size distribution. Cryo-TEM images of molecular assemblies of SLLSDL, SLLSDV, and SLLSLV also confirmed the vesicle formation (Figure S2), and the sizes and their size distributions are consistent with the DLS data. The vesicle size was reported to be dependent on the hydrophobic helix length.18 In the

(Figure 4d,g). The difference in morphology between SLLSDL and SLLSDV, a planar sheet and a distorted small sheet (Figure 4a,d), suggests that the interdigitation of the isobutyl groups of stereocomplex helices (SLLSDL) should be so tight and symmetric as to lead to a planar sheet, but the combination of the isobutyl and isopropyl groups in the case of SLLSDV is asymmetric enough to cause a distortion in the self-assembled sheet. Similarly, twisted ribbons of SLLSLV should be generated by the unfavorable packing of (L-Leu-Aib)6 and (LVal-Aib)6 helices as a result of the different side chains to prevent the molecular assembly from growing into the curved sheet as observed with poly(sarcosine)25-b-(L-Leu-Aib)6. Upon holding the temperature at 90 °C for 1 h, the morphology was converted to vesicles in either binary system D

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Figure 6. (a) Typical force−distance curve of the SLLSDV vesicle measured by AFM in water. (b) Curve fitting of section C in the force−distance curve according to the Hertzian contact model (F = Aδ1.5). (c) Schematic illustration of the vesicle distortion in relation to the tip positions of the five sections from A to E.

present system, the same helix length was thus adopted to result in a similar size among the three kinds of vesicles despite different helix association abilities. Dimensions of Vesicles by AFM in Water. The vesicles were carefully adsorbed on silicon wafer substrates with surfaces modified by 3-aminopropyltriethoxysilane (APTES). The adsorbed vesicles on the silicon wafer were observed via AFM in water (Figure 5) by measuring the force−distance curves at the maximally magnified image (Figure 6). The AFM tapping points were limited to 32 × 32 points over about a 400 × 400 nm2 area to avoid the deformation or breakdown of the vesicles. The shape of the vesicles was found to be a spherical cap as a result of adsorption onto the APTES-modified surface. The dimensions of the width and height are similar among the three kinds of the vesicles (Figure 5h). Using these dimensions, we calculated the membrane curvatures according to eq 2 in the experimental section to be 134, 118, and 127 nm, respectively, for SLLSDL, SLLSDV, and SLLSLV vesicles on the silicon wafer. Membrane Elasticity of Vesicles. The nanoindentation method was employed to evaluate the membrane elasticity of the vesicles. First, the topology of the vesicle was measured to put the AFM tip on the set point, where the tip just contacts the vesicle. From this point, the AFM tip was pushed into the vesicle about 10 nm followed by retraction by 100 nm at a constant velocity of 10 nm/s. The typical force curve of SLLSDV vesicles is shown in Figure 6a. With the approach of the AFM tip to the vesicle, a slight repulsive force appeared (A region), followed by the attractive force (B region). Further indentation turned the force from attraction to repulsion. The force increase (C region) corresponds to the pushing process of the vesicle by about 10 nm. The force curve of the C region was curve-fitted according to the Hertzian contact model to obtain the elasticity of the vesicle membrane (Figure 6b). About 40 force−distance curves were obtained with each vesicle. The elastic modulus values of SLLSDL, SLLSDV, and SLLSLV vesicles were evaluated, and their distributions were curve-fitted to obtain the medians of 11.9, 4.3, and 4.3 MPa, respectively (Figure 7). Even though the distribution of the elastic modulus values is large especially with SLLSDL vesicles, it can be concluded confidently that the elastic modulus values decrease in the order

Figure 7. Elastic modulus distribution and average elastic modulus of each vesicle calculated by the Hertzian contact model: (red) SLLSDL vesicles, (green) SLLSDV vesicles, and (blue) SLLSLV vesicles.

of SLLSDL vesicle > SLLSDV vesicle ≈ SLLSLV vesicle. The 2.8-fold-higher elasticity of SLLSDL vesicles compared to that of others can be explained by various factors of (i) stereocomplex formation between right- and left-handed helices,16 (ii) the interdigitation of isobutyl groups similar to the Leu zipper, and (iii) the higher hydrophobicity of isobutyl compared to that of the isopropyl group. Therefore, when Valbased peptides were incorporated into Leu-based peptides, the helix-packing strength was lowered, as shown by the morphologies of the distorted planar sheets of SLLSDV and twisted ribbons of SLLSLV compared to the large planar sheet of SLLSDL prior to heat treatment (Figure 4). The differences in physical properties among the vesicles can also be evaluated by viscoelastic damping or plastic deformation during the process of the unloading of the AFM tip from the vesicle in the force−distance curves.39,40 Figure 8a shows the schematic illustration of the hysteresis patterns in force− distance curves. When the loading−unloading curve of the AFM tip against a vesicle moves along OAO, it is fully elastic. On the other hand, the response curve along OAB corresponds to permanent plastic deformation. In general, viscoelastic E

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liposome. Another important point of view on the elasticity is the thin hydrophobic layer of these membranes. The hydrophobic helical blocks are composed of 12 residues, making the helices 1.8 nm long, which is about half of the liposomes and far thinner than polymersomes. Despite the thin hydrophobic layer, these vesicles show elasticity as high as that of egg yolk liposome and poly(dimethylsiloxane)68-b-poly(2methlyoxazoline)11 polymersome. The interdigitation of the side chains between helices should tighten the membrane very much. Upon choosing a proper amino acid for the helix block, a variety of vesicles with varying membrane stiffness are thus available, which may be useful for applications, especially those for biomembrane systems.

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CONCLUSIONS Leu- and Val-based helical blocks were used for amphiphilic block polypeptides. The helical block length was fixed at 12 residues, and this length is insufficient for any single component to self-assemble into vesicle. Three kinds of binary systems, however, can generate vesicles upon heating. The membrane elasticity became the highest with the combination of the (L-Leu-Aib)6 and (D-Leu-Aib)6 blocks owing to stereocomplex formation and the interdigitation of isobutyl groups between helices. The combination of the Leu-based helical block with the Val-based helical block, however, softened the membrane to be as flexible as egg yolk liposome. The membrane stiffness is therefore tunable by a suitable choice of amino acids and their combinations.

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Figure 8. (a) Schematic illustration of force−distance curves for viscoelastic material (OAC) and plastic material (OAB). (b) Histogram of the plasticity index calculated from each force−distance curve: (red) SLLSDL, (green) SLLSDV, and (blue) SLLSLV vesicles.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.7b00289. Experimental details on the syntheses and characterizations of the amphiphilic block polypeptides, cryogenic TEM images, and atomic force microscopy in a water image of an egg yolk liposome (PDF)

materials show the curve along OAC, and the plasticity index, η, is defined by η = 1 − SCAB/SOAB. (SCAB and SOAB represent the areas of the shapes delineated by the subscripts.) The plasticity indices of the vesicles are shown in Figure 8b. The indices, which correspond to the energy dissipation upon the loading− unloading process, are clearly smaller with SLLSDL vesicle than with SLLSDV or SLLSLV vesicles. This result is consistent with the SLLSDL vesicle being the most elastic among the vesicles as described before. The elasticities of various vesicles were evaluated by the nanoindentation method to be reported as follows: liposomes; egg yolk (Ld phase) 2 MPa,31 DPPC (So phase) 81 MPa,41 polymersomes; polystyrene115-b-poly(acrylic acid)15 of 61 MPa,29 poly(dimethylsiloxane)68-b-poly(2-methlyoxazoline)11 of 17 MPa,30 other vesicles; and Boc-Phe-Phe-OH of 275 GPa42 and poly(L-lactic acid) of 4 GPa.43 The elasticity values of amphiphilic polypeptide vesicles appear to be close to that of the egg yolk liposomes. To confirm the elasticity of egg yolk liposomes by our own measuring system, the elastic modulus of egg yolk liposomes was evaluated (Figure S3) to be 1.08 ± 0.42 MPa for liposomes that were 63 nm in diameter, which is slightly smaller than the reported value of 1.93 MPa for an egg yolk liposome of 50 nm diameter.31 The deviation is considered to be due to the differences in the liposome size and AFM environments of the types of cantilevers, equipment, and substrates. It is therefore notable that the elasticity of SLLSDL vesicles composed of the Leu-based polypeptides is nearly 11 times higher than that of the egg yolk liposome, but SLLSDV and SLLSLV vesicles are softened by the Val-based polypeptides down to the elasticity closer to the egg yolk

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AUTHOR INFORMATION

ORCID

Shunsaku Kimura: 0000-0003-0777-9697 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partially supported by a Grant-in-Aid for Scientific Research (A) of JSPS KAKENHI (grant number JP16H02279) from the Japan Society for the Promotion of Science.

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REFERENCES

(1) Discher, D. E.; Eisenberg, A. Science 2002, 297, 967. (2) Lingwood, D.; Simons, K. Science 2010, 327, 46. (3) Hullin-Matsuda, F.; Ishitsuka, R.; Takahashi, M.; Kobayashi, T. Methods Mol. Biol. 2009, 580, 203. (4) Lasic, D.; Papahadjopoulos, D. Science 1995, 267, 1275. (5) Liu, D. Z.; Chen, W. Y.; Tasi, L. M.; Yang, S. P. Colloids Surf., A 2000, 172, 57. (6) Presti, F. T.; Pace, R. J.; Chan, S. I. Biochemistry 1982, 21, 3831. (7) Klibanov, A. L.; Maruyama, K.; Torchilin, V. P.; Huang, L. FEBS Lett. 1990, 268, 235.

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DOI: 10.1021/acs.langmuir.7b00289 Langmuir XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.langmuir.7b00289 Langmuir XXXX, XXX, XXX−XXX