Facile and Precise Formation of Unsymmetric Vesicles Using the Helix

Article pubs.acs.org/Langmuir

Facile and Precise Formation of Unsymmetric Vesicles Using the Helix Dipole, Stereocomplex, and Steric Effects of Peptides Akihiro Uesaka,† 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, Gokasho, Uji, Kyoto 611-0011, Japan S Supporting Information *

ABSTRACT: Unsymmetrical vesicular membranes were prepared from a binary mixture of the A3B-type and the ABtype host polypeptides, which were composed of the hydrophilic block (A) and the hydrophobic helical block (B) but with a different helix sense between the two host polypeptides. TEM and DLS revealed the formation of vesicles with ca. 100 nm diameter. The molecular assembly was driven by hydrophobic interaction, stereocomplex formation, and dipole−dipole interaction between hydrophobic helices. Furthermore, the A3B-type host polypeptide extended the hydrophilic block to the outer surface of vesicles as a result of the steric effect, resulting in the formation of unsymmetrical vesicular membranes. As a result, a functionalized AB-type guest polypeptide having the same helix sense with the A3B-type host polypeptide exposed the hydrophilic block to the outer surface. In contrast, an AB-type guest polypeptide having the same helix sense with the AB-type host polypeptide oriented the hydrophilic block to the inner surface. Functionalization of either the outer or inner surface of the binary vesicle is therefore facile to achieve when using either the right- or the left-handed helix of the functionalized guest polypeptide.

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INTRODUCTION Molecular assemblies, such as vesicles, have been prepared from various amphiphiles of lipids and copolymers and are rich in the variety of applications ranging from nanosized capsules for the drug-delivery system to model membranes.1−10 Most artificial vesicles consist of a symmetric membrane where the inner and outer surfaces have similar chemical properties. However, biological membranes are in general unsymmetric on lipid compositions between the inner and outer surfaces in which the property is closely related to the biological functions.11,12 So far, there have been several reports on preparations of unsymmetric bilayers, for example, with using bola-amphiphiles with two headgroups of different sizes,13−16 cyclodextrin delivery-based systems,17−19 an unsymmetric distribution of specific phospholipids according to the pH gradient across membranes,20−22 and the reversed micelles processed by the oil/water interface.23−26 However, precise and facile control of the unsymmetric membranes is still difficult to achieve. We have been studying self-assemblies of amphiphilic block polypeptides, which are characterized by the hydrophobic helix block to associate regularly in water. Various morphologies such as micelles, curved sheets, nanotubes, vesicles, and conjugate morphologies of nanotubes and vesicles (roundbottomed flask) were attained.27−31 The key point of these molecular assemblies is the crystalline packing of helices, which reflects the molecular surface fitting between the helices to generate molecular assemblies of curved sheets and flat sheets © 2014 American Chemical Society

depending on using the right- or left-handed helix alone and a mixture of right- and left-handed helices, respectively.29,30 In the latter case, the right- and left handed helical polypeptides were arranged alternately in the molecular assemblies because of stereocomplex formation.30 These flat sheets changed the morphology to vesicles upon heat treatment, which are named peptosomes. The vesicular membranes are considered to take an interdigitated helix structure because of antiparallel orientation on the neighboring helices as a result the favorable dipole−dipole interaction. We present here the facile preparation of unsymmetrical vesicular membranes using the interdigitated helix structure of the stereocomplex. In the stereocomplex membrane, the righthanded helix should be surrounded by the left-handed helices and vice versa.32,33 Dipole−dipole interaction between the helices will force their alignment into an antiparallel orientation.34,35 As a result, the right-handed helices should extend the hydrophilic block to the one surface of the interdigitated membrane and the left-handed helices to the other surface, leading to an unsymmetrical membrane. With a help of two kinds of the hydrophilic blocks with different sizes, the unsymmetrical membrane is expected to take a vesicular Received: February 25, 2014 Revised: March 30, 2014 Published: March 31, 2014 4273

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Figure 1. Chemical structures of host polypeptides of Sm3L and S26D and guest polypeptides of lipo-S26L and lipo-S26D. membrane (Avanti Polar Lipids) at 28 °C to obtain uniformly sized liposomes (1 mg/mL). Transmission Electron Microscopy (TEM). TEM images were taken by using a JEOL JEM-2000EXII at an acceleration voltage of 100 kV. Peptide aqueous solutions were applied to a carbon-coated Cu grid, and the samples were negatively stained with 2% uranyl acetate, followed by suctioning the excess fluid with filter paper. Frozen-Hydrated/Cryogenic-TEM (Cryo-TEM). The peptide dispersions were frozen quickly in liquid ethane that was cooled with liquid nitrogen. The samples were examined at a 100 kV acceleration voltage at liquid-nitrogen temperature. Dynamic Light Scattering (DLS). The DLS measurements were made using DLS-8000KS (Photal Otsuka Electronics) with a He−Ne laser at 25 °C. Before DLS measurement, each prepared sample was filtered with a 0.20 μm PVDF (poly(vinylidene fluoride)) syringe filter (GE Healthcare U.K. Ltd/). Adhesion of AuNPs to Vesicles. Preliminarily, peptides with lipoic acid were mixed with amphiphilic peptides in ethanol, and the molecular assemblies were prepared from the solutions according to a previously described procedure. The peptide aqueous solutions were applied to a carbon-coated Cu grid, and then AuNPs of 10 nm diameter were added to the grid. After excess AuNPs were washed with dispersion media, the samples were negatively stained with 2% uranyl acetate, followed by suctioning of the excess fluid with filter paper. Fluorescence Analysis. The fluorescence spectra of the peptide dispersions were obtained using a JASCO FP-6600 spectrofluorometer at an optional temperature with a transmission cell. The membrane leakage was examined with fluorescein-labeled dextran (FITC-dextran, Mw = 4000 (Sigma-Aldrich Japan, Tokyo, Japan, excitation wavelength 470 nm, monitoring wavelength 520 nm) as a nonionic encapsulating agent. The S253L + S26D and S703L + S26D vesicles loading FITC−dextran were prepared by the ethanol injection method using 10 mg/mL FITC−dextran solution as a dispersion medium. The dispersion was held at 90 °C for 1 h, followed by elution through a Sephacryl S-100 column (1.5 cm × 30 cm, GE Healthcare Bio-Sciences) using Milli-Q as an eluent (Figure S3). Fractions 8−10 contained vesicles loading FITC−dextran, which was confirmed by TEM observation (Figure S4). The ninth fraction (100 μL) was diluted with 100 mM acrylamide aqueous solution (2.0 mL), which quenched the fluorescence of the leaked FITC−dextran and/or the loaded FITC−dextran by penetration into the vesicle with heat treatment or with the addition of Triton X-100. Circular Dichroism (CD). CD spectra were obtained with a JASCO J-1500 circular dichroism spectrometer with an optical cell of 0.2 cm optical path length at room temperature. The concentrations were set at 5.02−26.6 μM.

structure upon exposing the bulky hydrophilic block to the outer surface to relieve the steric hindrance. To obtain this type of unsymmetric vesicle, we designed and synthesized two host polypeptides, [(Sar)m]3-b-(L-Leu-Aib)6 (Sm3L, A3B type) (m = 25 and 70) and (Sar)26-b-(D-LeuAib)6 (S26D, AB type) (Figure 1). It is expected that these two host polypeptides should form vesicles with the interdigitated helix membrane of the stereocomplex, and the inner surface is covered with the hydrophilic blocks of the AB-type peptides and the outer surface is covered with those of the A3B-type peptides. Furthermore, the functionalization of one of the unsymmetric membranes was examined by the insertion of the functionalized guest polypeptide into the unsymmetric binary vesicle. Two kinds of AB-type guest polypeptides with lipoic acid at the terminal of poly(sarcosine) block (lipo-S26L and lipo-S26D) were prepared for surface modification. The hydrophobic part of a (CH2)4 chain in lipoic acid did not affect the hydrophilic property of the poly(sarcosine) chain.32 The guest polypeptides of lipo-S26L and lipo-S26D are expected to be inserted into the binary unsymmetric membrane in places of host polypeptides of Sm3L and S26D, respectively, according to their helix sense. The unsymmetrical surface in terms of the lipoic acid distribution is evaluated here by visualization with the adhesion of gold nanoparticles (AuNPs) to lipoic acid groups by transmission electron microscope (TEM) images.32

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EXPERIMENTAL SECTION

Materials. Amphiphilic peptides S253L and S703L were synthesized according to Scheme S1 (Supporting Information). The synthesis details are described in the Supporting Information. S26D, lipo-S26L, and lipo-S26D were obtained as previously reported.36 Au nanoparticles (AuNP) were purchased from BBInternational (U.K.) with an average diameter of 10 nm. All reagents and solvents were purchased commercially and were used as received unless otherwise noted. Sarcosine N-carboxy anhydride (Sar NCA) monomer was purified just before polymerization according to conventional procedures. Preparation of Molecular Assembly. A solution of polypeptides in ethanol (0.05 mg/μL, 5.0 μL) was injected into 10 mM Trisbuffered saline (TBS: pH 7.4) and Milli-Q water with stirring at 4 °C. After 30 min, the dispersion was heated with an aluminum heat source if necessary, following cooling at room temperature for measurements. Preparation of DMPC Liposomes. Dimyristoylphosphatidylcholine (DMPC) liposomes were prepared by a film hydration technique. The DMPC liposomes were extruded 20 times using a miniextruder (Avanti Polar Lipids) through a 100 nm pore size polycarbonate

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RESULTS AND DISCUSSION Morphology Analysis. When a mixture of host polypeptides S253L and S26D (1/1 mol/mol) was dispersed in 4274

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Figure 2. TEM images (negatively stained with 2% uranyl acetate) of molecular assemblies prepared from S253L + S26D in (a, b) TBS and (c, d) Milli-Q water (a, c) before and (b, d) after heating to 90 °C for 1 h. (e) Cryo-TEM image of molecular assemblies prepared from S253L + S26D in Milli-Q water upon heat treatment at 90 °C for 1 h. (f) Histogram of the diameter of the vesicles observed by cryo-TEM.

Figure 4. (a) Hydrodynamic diameters and (b) PDI of molecular assemblies prepared by varying the feed molecular ratios of S703L/ S26D. The vesicles were prepared from S703L + S26D in Milli-Q water upon heating to 90 °C for 1 h.

Tris-buffered saline (TBS, pH 7.4), planar circular sheets of ca. 430 nm diameter were observed by TEM (Figure 2a). The planar sheet formation suggests that the right-handed and the left-handed helices should form a stereocomplex in the membrane; otherwise, curved sheets should have been obtained if the single component would have self-assembled as previously reported.32 Upon subsequently holding the dispersion at 90 °C for 1 h, homogeneous vesicles were obtained (Figure 2b). The dynamic process from the planar circular sheet to the vesicle transition is considered in which the hydrophobic edge of the planar sheet should be concealed by sticking the edge upon heating. The process of this type of morphology change has been visualized by TEM with the help of gold nanoparticles in a previous paper.36 The DLS measurements revealed that the diameter of this vesicle was ca. 170 nm with a narrow size distribution (PDI = 0.084). Furthermore, the diameter did not change by additional heating to 90 °C for 1 h, indicating that the vesicles should be a thermodynamically stable morphology. In the case of using pure water as the dispersion medium, these peptides assembled into planar sheets of ca. 58 nm, which were transformed into homogeneous vesicles with treatment at 90 °C for 1 h (Figure 2c−f). The diameter of this vesicle was 91 nm, and the PDI was 0.073 by DLS measurements. Notably, the binary system afforded homogeneous vesicles, but the diameter in pure water became half of that in a buffered saline. It is suggested that the smaller vesicle should be formed upon the extension of the bulky hydrophilic A3 block of the A3B-type host polypeptide, leading to the large curvature. However, the larger vesicle should have symmetrical membranes resulting from the

Figure 3. TEM images (negative stained with 2% uranyl acetate) of molecular assemblies prepared from S703L + S26D in (a, b) TBS and (c, d) Milli-Q water (a, c) before and (b, d) after heating to 90 °C for 1 h. (e) Cryo-TEM image of molecular assemblies prepared from S703L + S26D in Milli-Q water upon heat treatment at 90 °C for 1 h. (f) Histogram of the vesicle diameters observed by cryo-TEM.

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Figure 5. TEM images (negatively stained with 2% uranyl acetate) of molecular assemblies prepared from (a−d) S253L + S26D and (e−h) S703L + S26D in (a, b, e, and f) TBS and (c, d, g, and h) Milli-Q water upon heat treatment at 90 °C for 1 h. These molecular assemblies contained (a, c, e and g) lipo-S26L and (b, d, f and h) lipo-S26D. An aliquot of a 10 nm AuNP suspension was added to the molecular assemblies on the TEM grid. (i) The number of AuNPs per unit area. Each value represents the mean ± SD (n > 20). The p value: *** p < 0.001.

3a). Upon subsequent heating to 90 °C for 1 h, vesicles were obtained via stereocomplex formation (Figure 3b). The DLS measurements revealed that this vesicle diameter was 125 nm with a narrow size distribution (PDI = 0.020). In the case of using pure water as the dispersion medium, these peptides assembled into planar sheets of ca. 61 nm, which transformed the morphology into homogeneous vesicles at 90 °C for 1 h (Figure 3c−f). This vesicle diameter was 112 nm, and the PDI was 0.008 by DLS measurements. The S703L + S26D vesicle thus became slightly smaller in pure water than in TBS. However, the diameter change with changing ionic strength was not as clearly shown in comparison with the case of the S253L + S26D vesicle. A mixture of S25L and S26D afforded vesicles upon heating to 90 °C for 1 h (Figure S5). The vesicle diameters (PDI) were 235 nm (0.064) and 207 nm (0.077) in pure water and TBS, respectively. The vesicle diameters decrease in the order of S25L + S26D (207 nm) > S253L + S26D (170 nm) > S703L + S26D (125 nm) in TBS, which does not correspond to the decreasing order of S25L + S26D (235 nm) > S703L + S26D (112 nm) > S253L + S26D (91 nm) in pure water. However, these vesicles can be roughly classified into two groups, one with ca. 200 nm diameter and the other with ca. 100 nm diameter. When we focus our attention on the effect of the bulky A3 moiety on the vesicle size, S253L + S26D and S703L + S26D vesicles in either TBS or pure water belong to the latter

random molecular orientation of the A3B polypeptide where the bulky hydrophilic A3 blocks are sticking outward from or inward toward the vesicle evenly. We interpret the size change as a result of the dipole−dipole interaction between helices operating in pure water to prefer strictly the antiparallel orientation between the neighboring helices, leading to the localization of the bulky hydrophilic A3 blocks at the outer surface in water. This consideration is further discussed later. Because the binary system can afford two types of vesicles depending on the medium’s ionic strength, we checked the physical stability of the smaller vesicle at a high salt concentration. The binary vesicles prepared in pure water (250 μL) were mixed in a 300 mM NaCl aqueous solution (250 μL). After incubation at 4 °C for 2 weeks, the vesicles were found to remain as they were in water by TEM and DLS measurements (a hydrodynamic diameter of 89 nm with a PDI of 0.088). It is therefore suggested that once the peptides selfassembled into the small vesicles in pure water they maintain their morphology regardless of the medium’s ionic strength probably because the helices should be stabilized by stereocomplex formation and the dipole−dipole interaction, which should strongly inhibit the flip-flop motion of the peptides in the membrane. When another mixture of the host polypeptides of S703L + S26D (1/1 mol/mol) was dispersed in pH 7.4 TBS, planar sheets of ca. 100 nm diameter were observed by TEM (Figure 4276

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The possibility of the flip-flop motion in the S703L + S26D vesicle was also examined. The S703L + S26D vesicle in pure water (250 μL) was mixed with a 300 mM NaCl aqueous solution (250 μL), and the solution was incubated at 4 °C for 2 weeks. The TEM and the DLS measurements revealed that the vesicle retained the original vesicle structure and retained the dimensions (a hydrodynamic diameter of 123 nm with a PDI of 0.019). These results also show that the vesicles prepared from S703L + S26D (1/1 mol/mol) in pure water were stable in the same way as the vesicles prepared from S253L + S26D in pure water. These vesicles have membranes with a thickness of ca. 9 nm, meaning that the outer surface area is larger than the inner surface area by ca. 21% in the case of vesicles with a 100 nm diameter. The number of components in the outer surface is then generally larger than that in the inner surface in the case of the bilayer membranes under the assumption of taking the same occupying area of the outer surface component as for the inner surface component. In the present vesicles, the membranes are considered to be composed of the interdigitated helices as previously discussed,27 and the situation differs from that of the bilayer membranes, but we studied the vesicle size variation upon changing the mixing ratio of S703L + S26D. The hydrodynamic diameters of the vesicles show a minimum value of 98 nm for a feed molar ratio of 1.25:1 (S703L/S26D) (Figure 4). It is therefore considered that S703L peptides are extruding the bulky hydrophilic A3 block to the outer surface (outer surface component) in pure water and that S703L peptides can be accommodated in the outer surface more than S26D (inner surface component) in the inner surface for geometrical reasons involving the vesicles. We also have confirmed that these hydrophobic blocks took an α-helical conformation in these vesicles by CD measurements. CD spectra of the molecular assemblies prepared from S703L, S26D, and S703L + S26D (1.25:1) showed that the hydrophobic dodecapeptide blocks took the right-handed, the left-handed, and the right-handed helices, respectively (Figure S6). In the case of the right-handed helix pattern of the S703L + S26D (1.25:1) vesicle, the intensity of the Cotton effect corresponds well to the amount of excess S703L against S26D, and the rest of S703L and S26D canceled out the Cotton effect. Adsorption of AuNPs to Vesicles. To functionalize either the outer or inner surface of the unsymmetric vesicles, the guest polypeptides of lipo-S26L or lipo-S26D having different helix senses with respect to each other were incorporated into S253L + S26D and S703L + S26D vesicles, and the distribution of lipoS26L or lipo-S26D in the membranes was visualized by the adsorbed AuNPs via the lipoic acid moiety of lipo-S26L or lipoS26D. The guest polypeptides of lipo-S26L and lipo-S26D have a hydrophobic helix block with the same helix sense as the host polypeptides of Sm3L and S26D, respectively. When the stereocomplex formation and the dipole−dipole interaction are operating dominantly for the molecular assembly, the distribution of guest lipo-S26L in the membrane should be the same as that of host S253L or S703L, and that of guest lipoS26D should be the same as that of host S26D because of the same helix sense. First, S253L + lipo-S26L + S26D (0.8/0.2/1.0 mol/mol/ mol) and S253L + S26D + lipo-S26D (1.0/0.8/0.2 mol/mol/ mol), were assembled into vesicles in both TBS and pure water (Figure 5a−h). These ternary mixtures gave the same morphology of vesicles as the binary mixtures. The number of the AuNPs adsorbed onto the vesicle surface was examined

Figure 6. Time-lapse fluorescence intensity at 40 (blue), 50 (green), and 60 °C (red) of the vesicles prepared from (a) S253L + S26D and (b) S703L + S26D in the medium containing 100 mM acrylamide and 150 mM NaCl. (c) Effect of Triton X-100 addition to the vesicles prepared from S253L + S26D (orange squares) and S703L + S26D (blue circles).

small vesicle except for the S253L + S26D vesicle in TBS. However, this exception can be reasonably explained by the shielding effect of the high ionic strength on the dipole−dipole interaction as described before. If so, the true exception is the S703L + S26D vesicles in TBS, which adopted the small vesicle even in the high-salt buffer. S703L should construct a highdensity polymer brush structure on the vesicle surface as a result of the collection of long polysarcosine chains, which may diminish the salt concentration at the interface between poly(sarcosine) and (L-Leu-Aib)6, resulting in the strict antiparallel orientation between the adjacent helices as a result of the effective dipole−dipole interaction operating between helices. This consideration, however, remains to be studied. 4277

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The leaked FITC−dextran was quenched by acrylamide in the medium. The emission intensity of both vesicles prepared from S253L + S26D and S703L + S26D remained unchanged for 50 min in the quenching medium even at 60 °C, suggesting that these vesicles encapsulated FITC−dextran stably (Figure 6a,b). With the addition of over ca. 1 wt % Triton X-100, the fluorescence was quenched by the structural change in the membrane (Figure 6c). This critical concentration is similar to the peptosome prepared from the PEG-gramicidin A conjugate and 1 order higher than that of dimyristoylphosphatidylcholine liposome as previously reported.27 Because there are no differences between the two kinds of vesicles having different sizes of the A3 moiety, the membrane robustness should be preserved even with the bulky A3 moiety.

in TBS and pure water (Figure 5a,b). In TBS, the high ionic strength should screen and diminish the dipole−dipole interaction in the molecular assembly process. In either vesicle, the adsorbed AuNPs were identified, indicating that the distribution of lipo-S26L or lipo-S26D in the membrane should be random. Similarly, AuNPs also adsorbed onto the vesicle surfaces of both S703L + lipo-S26L + S26D (0.8/0.2/1.0 mol/ mol/mol) and S703L + S26D + lipo-S26D (1.0/0.8/0.2 mol/ mol/mol) support no unsymmetric membranes being formed in TBS as described previously (Figure 5e,f). When S253L + lipo-S26L + S26D (0.8/0.2/1.0 mol/mol/ mol) and S253L + S26D + lipo-S26D (1.0/0.8/0.2 mol/mol/ mol) vesicles were prepared in pure water, AuNPs adsorbed onto only the S253L + lipo-S26L + S26D vesicle, and no AuNPs adsorbed onto the S253L + S26D + lipo-S26D vesicle (Figure 5c,d). The selective adsorption of AuNPs onto the S703L + lipo-S26L + S26D vesicle but not onto the S703L + S26D + lipo-S26D vesicle was similarly observed (Figure 5g,h). These observations clearly confirmed our consideration that the unsymmetric vesicles were formed in pure water with help of the stereocomplex formation and the dipole−dipole interaction, and the guest polypeptides were incorporated into the unsymmetric vesicles according to the rule of stereocomplex formation (side-by-side arrangement of the right- and lefthanded helices). Although the guest polypeptide of lipo-S26L has a smaller hydrophilic block than the host polypeptide of S253L or S703L, the mixture comprised only 10% of the guest polypeptide in the ternary mixture and did not affect the molecular assembly because of the strong dipole−dipole interaction in pure water. Therefore, guest lipo-S26L exposed the hydrophilic chains to the outer surface because of the same helix sense with host Sm3L. The numbers of AuNPs adsorbed onto the vesicle surfaces are summarized in Figure 5i. The selective adsorption of AuNPs onto the surfaces of the S253L + lipo-S26L + S26D vesicle and the S703L + lipo-S26L + S26D vesicle was clearly shown by a p value of less than 0.001. It is therefore concluded that lipo-S26L and lipo-S26D were incorporated into the vesicles in an opposite way about the location in the membrane, extending the lipoic acid group outward or inward. Because the structural difference between lipo-S26L and lipo-S26D is only the helix sense of the hydrophobic block, the different molecular orientation in the membrane is ascribed to the stereocomplex formation taking place quantitatively in these vesicles. Taken together, a mixture of host polypeptides Sm3L and S26D afforded the unsymmetric vesicles in pure water, having A3 blocks on the outer surface and A blocks on the inner surface. The functionalization of either the outer surface or the inner surface could be achieved by using the functionalized guest polypeptide according to the helix sense. The formation of the unsymmetric membranes is ascribed to the dipole− dipole interaction in addition to stereocomplex formation. Stability of Vesicles. The robustness of the vesicle membranes composed of A3B-type vesicles was examined by membrane leakage with heat treatment or with the addition of Triton X-100. Triton X-100 is a typical surfactant used to disassemble molecular assemblies. In this report, Triton X-100 was inserted into the membrane and solubilized vesicles. FITC−dextran in vesicles was released into aqueous acrylamide to quench the fluorescence of the leaked FITC-dextran. When the peptide vesicles were disassembled by Triton X-100, FITC−dextran, which was entrapped in the vesicles, leaked out.

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CONCLUSIONS Unsymmetric vesicles were prepared from the binary system of the A3B-type right-handed helix peptide and the AB-type lefthanded helix peptide as a result of tight helix packing using the dipole−dipole interaction and stereocomplex formation. Either the outer or inner vesicle surface can be functionalized with the directed insertion of the functionalized AB-type right- or lefthanded helix peptide. These vesicles are therefore facile for modifying one surface of the vesicles just by changing the helix sense of the functionalized peptide. The discrimination of the vesicle surface was strict with this method. Furthermore, the vesicles were very homogeneous in size and also robust. The application of the vesicles to DDS is currently underway.

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ASSOCIATED CONTENT

S Supporting Information *

Synthesis schemes. Details of Sm3L matrix-assisted laser desorption ionization−time-of-flight (MALDI-TOF) mass data of the compounds. Elution profile of the vesicles loading FITC−dextran through a Sephacryl S-100 column. Additional TEM images. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: (+81)75-383-2401. Tel: (+81)75-383-2400. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study is part of a joint research project focusing on the development of the basis of technology for establishing COE for nanomedicine, carried out through the Kyoto City Collaboration of Regional Entities for Advancing Technology Excellence (CREATE), the Japan Science and Technology Agency, part of the project titled ”Development of PET Probe for Imaging Solid Tumors Using Nanocarriers” under the contract of Innovation Plaza Kyoto, the Japan Science and Technology Agency, and part of the Innovative Techno-Hub for Integrated Medical Bio-Imaging of the Project for Developing Innovation Systems, from the Ministry of Education, Culture, Sports, Science and Technology (MEXT). 4278

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dx.doi.org/10.1021/la500752x | Langmuir 2014, 30, 4273−4279