Chiral Selective Adsorption of Ibuprofen on a Liposome Membrane

Feb 28, 2016 - We investigated the key factors that affect enantioselective adsorption of ibuprofen (IBU) on a liposome membrane by changing its lipid...
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Chiral Selective Adsorption of Ibuprofen on a Liposome Membrane Yukihiro Okamoto, Yusuke Kishi, Takaaki Ishigami, Keishi Suga, and Hiroshi Umakoshi* Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan S Supporting Information *

ABSTRACT: We investigated the key factors that affect enantioselective adsorption of ibuprofen (IBU) on a liposome membrane by changing its lipid composition: the liposome membrane shows different membrane fluidity, surface charge, content of chiral components, and heterogeneity (nanodomain). Nonspecific interactions (hydrophobic and electrostatic) were revealed to be an important factor in enhancing the adsorbed amount of IBU, based on adsorption experiments carried out using single lipids (DPPC, DMPC, DOPC, and DLPC) and positively charged liposomes (DOTAP and liposome containing DC-Ch). Furthermore, control of the boundary edge (i.e., the nanodomain size) derived from the membrane heterogeneity was important for enantioselective adsorption; as well as multiple weak interactions between lipid molecules and IBU enantiomers. The above findings provided a good index for constructing liposomal chiral adsorbents.

1. INTRODUCTION A liposome is a self-assembled vesicle composed of phospholipids that form a bilayer membrane structure.1 Liposome membranes exhibit different phases (i.e., liquidcrystalline and gel phases), depending on the balance between lateral diffusion and hydrophobic interactions of phospholipids.2 Furthermore, the interior of heterogeneous liposome membranes possesses low-fluidity regions such as nano- and microdomains or rafts that play significant roles in the regulation of protein activity in the cell membranes.3,4 In recent years, some emergent properties have arisen from the assembly of amphiphiles, where the “ordered state” of the selfassembled surface could be key for membrane function.5 Our previous findings have shown the importance of evaluating the physicochemical membrane properties, such as membrane fluidity and polarity,6,7 for the design of liposomes used as a platform of the molecular recognition; thus, several methodologies have been developed to understand the “microscopic” environment at the lipid membrane surface. A chiral recognition function of the liposome was recently developed by investigating the adsorption behaviors of several amino acids on the liposome.8,9 Chiral recognition by the liposome was attributed to “multiple interactions” (i.e., hydrophobic and electrostatic interactions or hydrogen bonding in a hydrophobic environment) around the asymmetric carbons of the amino acid and lipid molecule.9 Here, the “ordered structure” of the lipid assembly could play a key role in the chiral recognition function.9 In addition, we discovered that the boundary edge of nanodomains on lipid membranes can potentially enhance the chiral selectivity of L-His on the liposome membrane which consists of ternary lipids (DPPC/ © XXXX American Chemical Society

DOPC/Ch) that exhibit the microphase separation. Hence, in order to achieve the chiral recognition of small molecules by liposomes, it is important to focus on both the order of the lipid-assembled surface “in” the membrane and the boundary edge of nanodomains (heterogeneity) “on” the membrane. The purpose of this study is to investigate the factors responsible for the chiral recognition of ibuprofen (IBU) by liposome membranes, and to determine possible applications in the chiral separation of pharmaceutical products such as those derived from capillary or microchip electrophoresis.10 The enantioselective adsorption behaviors of IBU on the liposome membrane and its chiral separation efficiency were investigated by changing the lipid composition, i.e., the physical state of the liposomes. The adsorption behaviors of IBU on single lipids (e.g., DPPC, DMPC, DOPC, and DLPC) and positively charged liposomes (e.g., DOTAP and liposomes containing DC-Ch) were investigated to understand the possible role of nonspecific interactions such as hydrophobic and electrostatic interactions. The role of the boundary edge in IBU adsorption (i.e., the nanodomain size), which is derived from the membrane heterogeneity of the ternary-lipid liposome, was further investigated by focusing on improving the chiral selectivity. On the basis of these results, a possible design of the liposome membrane was formulated. Received: January 26, 2016 Revised: February 25, 2016

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DOI: 10.1021/acs.jpcb.6b00840 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B

2. EXPERIMENTAL METHODS 2.1. Materials. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, Tm = 41 °C), 1,2-dimyristoyl-sn-glycero-3phosphocholine (DMPC, Tm = 24 °C), 1,2-dilauroyl-snglycero-3-phosphocholine (DLPC, Tm = 0 °C), 1,2-dioleoyl3-trimethylammonium-propane (DOTAP, Tm = below −20 °C), and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC, Tm = −22 °C), Cholesterol (Ch), DC-cholesterol (DC-Ch) and porcine brain sphingomyelin (SM, Tm = 45 °C) were purchased from Avanti Polar Lipid (Alabaster, AL). 6Lauroyl-2-dimethylaminonaphthalene (Laurdan) and 1,6-diphenyl-1,3,5-hexatriene (DPH) were purchased from SigmaAldrich (St. Louis, MO). (S)-ibuprofen (S-IBU) and (R)ibuprofen (R-IBU) were purchased from Santa Cruz Biotechnology (Dallas, TX). Disodium hydrogen phosphate and sodium dihydrogen phosphate were purchased from Nacalai Tesque (Kyoto, Japan). Chemical structures of these lipids and dyes are written in supporting figure (Figure S1). The locations of lipid and dye were predicted from the log P values, which were calculated using ChemBioDraw 12.0.2 (CambridgeSoft Corporation, Cambridge, MA). 2.2. Liposome Preparation. Liposomes were prepared using a freeze−thaw extrusion method.11 Briefly, a chloroform solution containing lipids was dried in a round-bottomed flask under vacuum with a rotary evaporator to prepare a lipid thin film. The thin film was hydrated with ultrapure water at and above the transition temperature (Tm) to prepare a vesicle suspension. The vesicle suspension was frozen at −80 °C and thawed at 50 °C to enhance the transformation of small vesicles to large multilamellar vesicles (MLVs); this freeze−thaw cycle was performed five times. The MLVs were used to prepare smaller unilamellar vesicles by extruding the MLV suspension 11 times through two layers of polycarbonate membranes, with mean pore diameters of 100 nm using an extruding device (Liposofast; Avestin Inc., Ottawa, ON, Canada). The obtained unilamellar vesicles were suspended in 50 mM phosphate buffer (pH 6.7) because we clarified that the adsorption amount of IBU on liposome surface was highest under this condition. 2.3. Evaluation of Ibuprofen Adsorption on the Liposomal Membranes. The adsorbed amount of S or R form IBU was estimated by similar method reported in literature.7,9 The liposome suspensions (lipid concentration: 1.25 mM) was mixed with the S- or R-form IBU (1.25 mM) and incubated at 25 °C for 24 h. After incubation, DOPC or DOTAP liposome was removed with an ultrafiltration membrane (molecular cut off: 50 kDa, Toyo Roshi Kaisha, Ltd., Tokyo, Japan), and the other liposomes by ultracentrifuge method at a speed of 55000 rpm for 2 h (Micro Ultracentrifuge himac CS100FNX; Hitachi Koki Co., Ltd., Tokyo, Japan). The concentration of S- or R-form IBU in supernatant (Csup) was measured with ultraviolet spectrophotometer (xMark Microplate Spectrophotometer, Bio-Rad Laboratories, Inc., Hercules, CA) by using a calibration curve at 263 nm. The adsorbed concentration (Cads) was calculated by the following equations:

where Cads(S form) and Cads(R form) are the concentration of S- and R-form IBU adsorbed on the liposomes, respectively. 2.4. Evaluation of Membrane Polarity with Laurdan. Laurdan is sensitive to the polarity around the molecule itself, and its fluorescence property enables to evaluate the surface polarity of lipid membranes. The Laurdan emission spectra exhibit a red shift caused by dipolar relaxation. The emission spectra were measured using a fluorescence spectrophotometer (FP-8500, JASCO, Tokyo, Japan) at an excitation wavelength of 340 nm, and the general polarization (GP340), the membrane polarity, was calculated as follows:12 GP340 = (I440 − I490)/(I440 + I490)

where I440 and I490 represent the fluorescence intensity of Laurdan at 440 and 490 nm, respectively. The total concentrations of lipid and Laurdan were 100 and 1 μM, respectively. 2.5. Evaluation of Membrane Fluidity by Using DPH. The inner membrane fluidity of the liposomes was evaluated by previous reports.13 Fluorescent probe DPH was added to the liposome suspension in a molar ratio of 250/1 lipid/DPH; the final concentrations of lipid and DPH were 100 and 0.4 μM, respectively. The fluorescence polarization of DPH (Ex = 360 nm, Em = 430 nm) was measured using a fluorescence spectrophotometer (FP-8500, JASCO, Tokyo, Japan) after incubation at 30 °C for 30 min. The sample was excited with vertically polarized light (360 nm), and emission intensities both perpendicular (I⊥) (0°, 0°) and parallel (I||) (0°, 90°) to the excited light were recorded at 430 nm. The polarization (P) of DPH was then calculated from the following equations: P=

G=

I − GI⊥ I + GI⊥

I⊥ I

i⊥ and i|| are emission intensities perpendicular to the horizontally polarized light (90°, 0°) and parallel to the horizontally polarized light (90°, 90°), respectively, and G is the correction factor. The membrane fluidity was evaluated on the basis of the reciprocal of polarization, 1/P.

3. RESULTS AND DISCUSSION 3.1. Adsorption of S-/R-Ibuprofen on Various Liposomes. The possibility of chiral selective adsorption of IBU on the liposome was investigated by selecting various liposomes with different phase transition temperatures. Figure 1 shows the adsorption behaviors of S-/R-IBU on each liposome. The DPPC liposome did not enantioselectively adsorb IBU, as its adsorbed amount was less than 10%. On the other hand, the DMPC and DOPC liposomes adsorbed a 2-fold amount of IBU when compared to that adsorbed by the DPPC liposome. Furthermore, the DMPC and DOPC liposomes displayed miniscule and zero chiral recognition, respectively. DLPC liposomes adsorbed the maximum amount of IBU (∼30%), despite a marginal improvement in its chiral selectivity. Almost all liposomes exhibited low chiral selectivity toward S-/R-IBU. IBU is a rather hydrophobic molecule; hence, it can distribute easily within the hydrophobic environment of the lipid membrane.14 Thus, it is possible to introduce variations in the liposome membrane surface after IBU adsorption. In order to investigate the actual state of the lipid membranes upon IBU

Cads = C ini − Csup

Here Cini represents an initial concentration of S- or R-form IBU. The separation parameter (SS/R) was calculated by the following equations: SS / R = Cads(S form)/Cads(R form) B

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internal hydrophobic environment of liposomes with that have a higher fluidity can be easily exposed to the bulk aqueous environment, which makes hydrophobic molecular interactions more facile.17 Therefore, with respect to adsorption, the DLPC is the most suitable membrane among the liposomes considered in this work. The values of both the fluidity and polarity of these liposomes after S- or R-IBU adsorption are plotted in Figure 2. Although a significant change was not observed even in the presence of IBU, the membrane fluidity of DOPC liposomes decreased after IBU adsorption, implying that the membrane surface with higher fluidity was stabilized through the insertion of the IBU molecule. Therefore, we believe that the IBU molecule inserts into the membrane environment of liposomes that exhibit a higher fluidity via a hydrophobic interaction. The membrane fluidity reflects the characteristics of the membrane interior at a deeper region (hydrophobic region), since a nonpolar hydrophobic molecule (DPH) was in general used as a molecular probe. The results above reveal that IBU could interact with the membrane in the deeper hydrophobic region. In our previous report, multiple interactions between chiral molecules and the surface region of the lipid membrane were extremely important for inducing chiral selectivity in amino acid adsorption.9 Thus, poor chiral selectivity was thought to result from the location of the IBU molecules in the interior of the liposome membrane. 3.2. Chiral Selective Adsorption of S-/R-Ibuprofen on Ternary Lipid Liposomes. On the basis of these findings, modification of the chiral condition at the deeper hydrophobic region could be useful for enhancing the chiral selective adsorption of IBU. Possible candidates for membrane building blocks are sphingomyelin (SM) and cholesterol (Ch), since

Figure 1. Adsorption of S-/R-ibuprofen and its chiral recognition behavior in several liposomes. The adsorption amount of ibuprofen was measured after 24 h incubation. The chiral selectivity of the adsorption was determined as a ratio of the percentage of adsorption of S-/R-ibuprofen.

adsorption, the membrane properties were further evaluated using a previously reported methodology.6 In Figure 2, the membrane fluidity and membrane polarity of the liposomes (at 30 °C) are plotted as a Cartesian plot, where the phase state of the liposome membrane can be classified based on the GP340 and 1/P values. The DPPC liposome was found to exist as a solid-ordered (so) phase with a lower fluidity and higher GP340 value than those of other liposomes. The DMPC and DOPC were found near the boundary between the liquid-ordered (lo) phase and liquid-disordered (ld) phase, showing that they can be regarded as a disordered phase that is partially mixed with an ordered phase. On the contrary, the DLPC showed the highest fluidity despite a low GP340 value. In general, the surrounding environment affects the membrane properties of the liposome.15,16 In particular, the

Figure 2. Transition of membrane properties before and after ibuprofen adsorption. (Left) Cartesian diagram analyzed by Laurdan and DPH before or after adsorption of ibuprofen. (Right) Parts a−d show the closed-up plot of the Cartesian diagram. C

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The Journal of Physical Chemistry B both of them have additional asymmetric carbons at the interface of the glycerol and hydrophobic regions18,19 (Figure S1). However, the SM liposome membrane exhibited an so phase similar to that of the DPPC liposome20 and did not show any adsorption, because the interior hydrophobic region could not be exposed. One solution to this problem is the preparation of liposomes that mix SM and other lipids. Several studies have reported on the physicochemical properties and phase diagram of liposome membranes prepared by DOPC/SM/Ch;21,22 the phase diagram of DOPC/SM/Ch is depicted in Figure 3. For

Figure 4. Adsorption of S-/R-ibuprofen and its chiral recognition behavior in various liposomes. The liposomes were prepared by using DOPC and SM, together with cholesterol (Ch) or positively charged cholesterol (DC-Ch). DOTAP liposome was also used as a reference of positively charged liposome. The adsorption amount of ibuprofen was measured 24 h incubation after. The chiral selectivity of the adsorption was determined as a ratio of the percentage of S-/Ribuprofen adsorption.

liposomes adsorbed more S-IBU and showed a higher chiral selectivity (SS/R ∼ 3) than composition C liposomes. Thus, modification of the nanophase-separated lipid membrane with SM or Ch was found to improve the chiral selectivity with IBU. (2). Effect of Electrostatic Interaction on the DOPC/SM/ DC-Ch Liposome. Since the electrostatic interactions between the IBU and liposome surface are considered key to increasing both the amount adsorbed and chiral selectivity,23 their effect was investigated by using positively charged DC-Ch in place of Ch. It is hypothesized that DOPC/SM/Ch phase diagram could be applied in the case of the DOPC/SM/DC-Ch system, because the steroid structure is common to both Ch and DCCh. As shown in Figure 4, DOPC/SM/DC-Ch liposomes of compositions B and C exhibited a markedly enhanced adsorption of S-IBU, in contrast to the liposomes of composition D (B, C, and D of Figure 3). The adsorption of R-IBU was also enhanced, similar to S-IBU. Therefore, the DOPC/SM/DC-Ch and DOPC/SM/Ch liposomes of compositions B and C had almost the same SS/R values. In the case of composition D, the adsorbed amounts of S and R-IBU increased slightly, while no improvement was seen in the SS/R value of composition D in DOPC/SM/Ch. These results indicate that the electrostatic interaction enhances the adsorption by retaining the enantioselective power. Subsequently, to resolve the role of domain and positive charge, the IBU adsorption on a positively charged DOTAP liposome was also investigated. Figure 4 indicates that ∼50−60% of S-and RIBU was adsorbed with low chiral selectivity (SS/R ∼ 0.9). These results show the significance of the positive charge in the liposome membrane, especially at the local domain of the lo phase and with cholesterol, in the chiral selective adsorption of IBU enantiomers. (3). Membrane Properties after Ibuprofen Adsorption. As previously described, the membrane properties of the liposomes were analyzed by measuring both membrane fluidity and polarity before and after IBU adsorption (Figure S2). Almost all the data were clustered in the region of the heterogeneous membranes (i.e., lo + ld or so + ld). In contrast to the findings from single-lipid liposomes, the variation of the properties before and after IBU adsorption was not as distinct. This could be a result of the heterogeneity of the membrane surface, where IBU can accumulate in an ordered phase14 and

Figure 3. Phase diagram of liposomes prepared by ternary-lipids (DOPC, SM and Ch). The data of solubility curve and tie-lines are plotted based on the previous findings.24 A, B, C, C(ld), C(lo), D, D(ld) and D(lo) show the composition of the liposomes that were used for the experiment of ibuprofen adsorption described in Figure 4 and Figure 5.

example, the liposomes of a mixture of SM and DOPC, together with Ch at specific compositions, can induce nanophase separation between the l0 phase (mainly by SM) and the ld phase (mainly by DOPC). In addition, recent findings on the chiral recognition function of liposomes against amino acids have revealed the importance of the boundary edge of nanophase separation in inducing a higher chiral selectivity. Thus, the liposome membrane that comprises of ternary lipids should exhibit a higher adsorption surface and higher chiral selective function. (1). DOPC/SM/Ch Liposome. The adsorption behaviors of IBU on the liposome were investigated using liposomes prepared by ternary lipids: compositions at A, B, C, and D are illustrated in Figure 3. Figure 4 shows the adsorption behaviors of S-/R-IBU on these liposomes. As previously described, the DOPC liposome (A, Figure 3) did not exhibit a high adsorption yield and chiral selectivity. However, when the DOPC liposome was modified with Ch (B, Figure 3), the resultant DOPC/Ch liposome adsorbed a 2-fold amount of SIBU when compared to that adsorbed by DOPC liposomes, and it showed a higher chiral selectivity (SS/R ∼ 2). However, both the amount of S-IBU adsorbed and chiral selectivity were slightly reduced by increasing the SM ratio (C, Figure 3), in contrast to the results observed with composition B. On the basis of this observation, it can be said that SM inhibits IBU adsorption because of its solid phase. Composition D D

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mixture. In our previous work,9 the chiral selective adsorption of amino acid on the DPPC liposome was achieved via an induced change of the membrane properties, followed by a weak interaction between lipid molecules and amino acids at the initial stage of the adsorption process. In addition, the boundary edge of the separated nanodomains is important in adsorption and recognition,26,27 and nanodomains of the DPPC/DOPC/Ch liposome play an important role in enhancing the chiral selectivity.10 Hence, the adsorption behavior on these compositions of liposomes can be considered as follows: in the compositions of C(ld) and D(ld), both S- and R-IBU can easily interact with lipid molecules because of their partially loose structures. In contrast, in the compositions of C(lo) and D(lo), the interactions of IBU between lipid molecules are suppressed due to their moderately packed state. This suppression resulted in the decreased adsorption of IBU in C(lo) and D(lo) over C(ld) and D(ld). However, the formation of nanodomains that expose chiral recognition sites (i.e., around the asymmetric carbons) enhances the chiral selective adsorption. The above results indicate that the chiral selectivity in IBU adsorption could also be improved if the heterogeneous surface of the ternary lipid liposome, in particular the boundary compositions, is considered.

the variation of the averaged properties of membranes is not very significant. For liposomes in the presence of IBU, a larger increase in the membrane fluidity (1/P, x-axis) was observed for DOPC/SM/DC-Ch with composition C, although such an increase was not observed in DOPC/SM/Ch with the same composition. These results imply that the molecular packing of the ordered phase domain relaxed with the insertion of IBU. In summary, efficient and chiral-selective adsorption can be obtained by mixing different types of lipids. The ordered phase contributes to chiral selectivity (SM and Ch) and the disordered phase leads to higher adsorption (DOPC and DCCh) on a heterogeneous membrane. 3.3. Effect of Different Domain Boundary Edges on the Chiral Selective Adsorption of S-/R-Ibuprofen on Ternary Lipid Liposomes. For further improvement of the chiral selectivity and adsorption, other compositions near the boundary between the binary and homogeneous phases were investigated. Many studies have reported on the phase diagram of ternary lipid liposomes that improve the understanding of the behaviors of microphase separation on lipid bilayer membranes.21,22 The tie-lines in the phase diagram of the DOPC/SM/Ch liposome membrane have been previously reported by determining the area of different phase regions through the fluorescence microscopic analysis.24 The data reported in the previous paper25 are replotted in Figure 3, where the lipid composition of two separated microphases in the ternary-lipid liposomes remains unchanged on the same tieline, despite different ratios of the area of the microphases. The liposomes of compositions C and D contain both lo and ld domains. For C, the lipid compositions at phases lo and ld are denoted as C(lo) and C(ld), respectively, while those of D are denoted as D(lo) and D(ld) respectively. Figure 5 shows the

4. CONCLUSIONS The key factors for the enantioselective adsorption of IBU with liposomes were investigated by comparing various compositions of liposomes. The results of the adsorption experiments with single lipids (e.g., DPPC, DMPC, DOPC, and DLPC) and positively charged liposomes (e.g., DOTAP and DC-Ch) indicate that nonspecific interactions (hydrophobic and electrostatic) and membrane fluidity are critical for adsorption increments. We further demonstrated by using heterogeneous composition liposomes that the control of the boundary edge (i.e., nanodomain formation), which is derived from membrane heterogeneity, is an important factor for enantioselective adsorption of IBU. Additional factors are also vital, such as the multiple weak interactions between lipid molecules and IBU enantiomers. Boundary edges (nanodomains) expose the asymmetric carbon in the glycerol region of lipids and enhance the chiral selective adsorption of IBU. Thus, although further investigation is needed, we demonstrated that appropriate design of the liposome surface could shed light on the chiral selective separation of small enantiomers.

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Figure 5. Adsorption of S-/R-ibuprofen and its chiral recognition behavior in the liposomes prepared by ternary-lipids.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b00840. Chemical structures of lipids and dyes and membrane fluidity and polarity of the heterogeneous liposomes before and after IBU adsorption (PDF)

adsorption behaviors of IBU on the ternary lipid liposomes of composition C, with C(lo) and C(ld), and of composition D, with D(lo) and D(ld). A higher chiral selectivity was observed in the cases of C(lo) (SS/R ∼ 5) and D(lo) (SS/R ∼ 13) than those of C(ld) and D(ld). On the other hand, a lesser amount of IBU was adsorbed for C(lo) and D(lo) than in the mixed (C and D) and disordered phase liposomes (C(ld) and D(ld)). The area ratio of two different domains on the liposome membrane can be estimated from the ratio of the tie-line length.26 At C(lo) and D(lo), the liposome membrane is therefore composed of the lo phase and a minute amount of the ld phase. In addition, the liposomes at C(lo) and D(lo) could possess a moderately packed membrane because of the presence of the ld phase in the

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AUTHOR INFORMATION

Corresponding Author

*(H.U.) Telephone: +81-(0)6-6850-6287. Fax: +81-(0)6-68506286. E-mail: [email protected]. Notes

The authors declare no competing financial interest. E

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ACKNOWLEDGMENTS

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REFERENCES

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This research was supported by the Funding Program for NextGeneration World-Leading Researchers of the Council for Science and Technology Policy (CSTP) (GR066), Grant-inAid for Scientific Research A (26249116), Grant-in-Aid for Exploratory Research (T15K142350 and T15K142040), JSPS Grant-in-Aid for JSPS Fellows (13J03878). Y.O. acknowledges the Multidisciplinary Research Laboratory System in Osaka University.

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