Induction of Chiral Recognition with Lipid Nanodomains Produced by

Mar 3, 2017 - The adsorbed concentration (Cads) was calculated by the following equation: where Cini represents the initial concentration of S- or R-I...
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Induction of Chiral Recognition with Lipid Nanodomains Produced by Polymerization Yukihiro Okamoto, Yusuke Kishi, 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 report the induction and control of chiral recognition in liposomal membranes by the photopolymerization of diacetylenic lipids (DiynePC). The specific properties of polymerized DiynePC liposomes were characterized, and then the chiral separation performance was estimated. As the polymerization proceeds, chiral recognition to ibuprofen was induced, and its efficiency increased due to the formation of rigid nanodomains and boundary edges. Furthermore, the chiral recognition and adsorbed amount could be controlled by the ratio of rigid nanodomains, varying the composition ratio of DiynePC. Finally, the optimum condition and dominant interactions for enantioselective adsorption were clarified. Thus, our findings and results will be helpful to understand the induction of chiral recognition by polymerizable liposomes, and also become a guideline for the construction of liposomal chiral stationary phases.

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INTRODUCTION A liposome is a self-assembled vesicle composed of phospholipids and possesses unique cell membrane-like properties: fluidity,1 gel−liquid phase transition,2 and phase separation.3 In addition, liposomes can amplify molecular interaction and recognition by enabling the self-assembly of lipids and other functional molecules.4 Therefore, by designing and examining the liposome properties and by constructing the liposomal structures, improved recognition and separation have been achieved.5−8 Chiral separation is a significantly important technology, especially in pharmaceutical products,9 and requires sophisticated stationary phases due to difficulty of separation.10 For chiral separation, the stationary phase should be strictly designed so that multiple weak interactions between the chiral molecules and the stationary phase are possible.11 Previously, we had revealed that liposomes exhibit high chiral recognition via multiple weak interactions between the lipid molecules and the target molecule.12 In addition, with heterogeneous liposomes like a cell membrane model, we achieved the chiral separation of ibuprofen (IBU) and predicted the chiral recognition mechanism:13 Ordered structure of heterogeneous liposomes is necessary for efficient multiple weak interactions. Furthermore, on the surface of heterogeneous liposomes, the boundary edges of the nanodomains play a significant role in chiral recognition. At the boundary of the nanodomain, the asymmetric carbon in the glycerol region of lipids is exposed, thus enabling the chiral selective adsorption of IBU. For the emergence and enhancement of some functions, a self-assembled system with functional polymers14 and lipids15 is © XXXX American Chemical Society

among the candidates. A potential class of functional lipids is polymerizable lipids,16−18 especially photopolymerizable diacetylenic lipids.18−24 Polymerized diacetylenic (PDA) liposomes are easily prepared by photopolymerization and show stimuli responses such as color change on exposure to temperature,18 pressure,19 and biomolecules.20 Therefore, PDA liposomes have been applied in drug delivery systems21 and sensors.22 With respect to membrane properties, the PDA liposomes generate tolerance against organic solvents and form rigid and ordered nanodomains17,23 due to polymerization, which would be desirable for chiral separation and recognition. In addition, the PDA liposomes are expected to show novel functions and enhanced recognition due to polymer effects.25 As stated above, we focus on PDA liposomes for elucidation of the chiral recognition mechanism in heterogeneous liposome membranes, and for the emergence and enhancement of the chiral recognition with reference to our previous studies.12,13 First, PDA liposomes were prepared, and their membrane properties were characterized. Then, it was confirmed that the PDA liposomes induced the chiral recognition of (S)-IBU to the appropriate polymerization degree, liposomal composition, and membrane structure due to the effects of boundary edges and rigid nanodomains, while the nonpolymerized diacetylenic liposomes did not. Finally, the effect of background solution on the chiral recognition was assessed, and then dominant Received: December 14, 2016 Revised: February 21, 2017 Published: March 3, 2017 A

DOI: 10.1021/acs.biomac.6b01859 Biomacromolecules XXXX, XXX, XXX−XXX

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Figure 1. Spectroscopic characterization of DiynePC/DOPC liposomes. (a) UV−vis spectra of DiynePC:DOPC (10/0) liposomes at different polymerization times. (b) CD spectra of poly DiynePC/DOPC (8/2) and DiynePC/DOPC (8/2). Experimental conditions: polymerization time, 60 min; diameter, 100 nm; temperature, 25 °C; background solution, 50 mM PB (pH = 6.7); for others, see the Experimental Section. using a fluorescence spectrophotometer (FP-8500, JASCO, Tokyo, Japan) at 510 nm excitation light. The membrane fluidity of the liposomes was evaluated by reported methods.28 The fluorescent probe DPH was added to the liposome suspension in a lipid/DPH molar ratio of 250:1; the final concentrations of the 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 after incubation at 30 °C for 30 min. The sample was excited using vertically polarized light (360 nm), and the emission intensities, both perpendicular (I⊥) and parallel (I∥) to the excitation light, were recorded at 430 nm. The polarization (P) of DPH was then calculated from the following equations:

interactions in chiral recognition were clarified for optimization of enantioselective adsorption. Thus, our findings indicate that chiral recognition ability can be induced and controlled by the liposomal membrane structures and the background solution. Therefore, our obtained knowledge will be helpful in the optimum construction of liposome membranes for various chiral separations. Moreover, our findings might be helpful in understanding chiral recognition by cells because the heterogeneous PDA membrane is similar to the cell membrane.

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EXPERIMENTAL SECTION

Materials. 1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC, Tm = −22 °C) and1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC, Tm = 42 °C) were purchased from Avanti Polar Lipid (Alabaster, AL, USA). 6-Lauroyl-2-dimethylaminonaphthalene (Laurdan) and 1,6-diphenyl-1,3,5-hexatriene (DPH) were purchased from Sigma-Aldrich (St. Louis, MO, USA). (S)-Ibuprofen (S-IBU) and (R)-ibuprofen (R-IBU) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Disodium hydrogen phosphate and sodium dihydrogen phosphate were purchased from Nacalai Tesque (Kyoto, Japan), and were used for preparation of phosphate buffer (PB). Dimethyl sulfoxide (DMSO) and urea were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Ultrapure water was prepared using Direct-Q UV3 (Merck Millipore Co., Tokyo, Japan). Liposome Preparation. Liposomes were prepared by a freeze− thaw extrusion method.26 Briefly, a chloroform solution of the lipids was dried in a round-bottomed flask under vacuum using a rotary evaporator to prepare a lipid thin film. The thin film was hydrated with ultrapure water at 60 °C, above the transition temperature (Tm), to prepare a vesicle suspension. The vesicle suspension was frozen at −80 °C and thawed at 60 °C to enhance the transformation of vesicles into multilamellar vesicles (MLVs).27 This freeze−thaw cycle was repeated five times. The unilamellar vesicles were prepared with MLVs by extruding the MLV suspension 11 times through two layers of polycarbonate membranes, with mean pore diameters of 50, 100, 200, or 400 nm, using an extruding device (Liposofast, Avestine Inc., Ottawa, ON, Canada). The obtained unilamellar vesicles were suspended in a 50 mM phosphate buffer (PB) (pH 6.7) unless otherwise noted. Polymerization of Liposomes. PDA liposomes were prepared by UV (254 nm) irradiation of the liposome suspension using a handheld UV light (UVGL-58, UVP, CA, USA) for a predetermined time at 25 °C. Characterization. Spectrometric Analysis. The spectrometric analysis of 10 mM liposome in ultrapure water was conducted using a UV−vis spectrophotometer (UV-1800, Shimazu, Kyoto, Japan). The fluorescence of 10 mM liposome in ultrapure water was measured

P=

G=

I − GI⊥ I + GI⊥

I⊥ I

where G is the correction factor. The membrane fluidity was evaluated on the basis of the reciprocal of polarization, 1/P. Laurdan is sensitive to the polarity around the molecule itself, and its fluorescence property enables the evaluation of the surface polarity of the lipid membranes. The emission spectra were measured using a fluorescence spectrophotometer at an excitation wavelength of 340 nm. The general polarization (GP340), the membrane polarity, was calculated as follows:29

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. Circular dichroism (CD) analyses were performed for the confirmation of chirality using a CD spectrophotometer (J-820W spectrometer, JASCO Co., Tokyo, Japan) under the following conditions: samples, 5 mM unpolymerized liposome or PDA liposome (DiynePC/DOPC: 8/2), prepared by 60 min UV irradiation; cell length, 0.3 cm. Dielectric dispersion analyses (DDA) were conducted for the assessment of hydration state of membrane surface30 with a network analyzer (PNA-X N5245A, Keysight Technologies, CA, USA) under following conditions: samples, 30 mM unpolymerized liposome or PDA liposome (DiynePC/DOPC: 8/2) prepared by 60 min UV irradiation; temperature, 25 °C. Differential Scanning Calorimetry (DSC). Differential scanning calorimetry data were obtained using DSC-60 (Shimazu, Kyoto, Japan) under the following conditions: samples, 50 mM PDA prepared by 60 min UV irradiation or unpolymerized liposomes suspension B

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Figure 2. (a) Cartesian diagram prepared from fluidity and polarity values of each liposomes before polymerization. (b) Fluorescence spectra of Laurdan in each liposome before polymerization. Experimental conditions: diameter, 100 nm; temperature, 25 °C; background solution, 50 mM PB (pH = 6.7); for others, see the Experimental Section.

Figure 3. Variation of (a) GP340 values and (b) Laurdan spectra of DiynePC/DOPC (8/2) liposomes prepared by different UV-irradiation times. Experimental conditions: diameter, 100 nm; temperature, 25 °C; background solution, 50 mM PB (pH = 6.7); for others, see the Experimental Section. Error bars represent standard deviations (n = 3).

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sealed in an alumina hermetic pan; heating rate, 2 °C/min between 30 and 60 °C. Evaluation of Ibuprofen Adsorption on the Liposomal Membranes. The adsorbed amount of S- or R-IBU was estimated by a similar method.13 100 μL liposome suspension (lipid concentration: 10 mM) was mixed with 300 μL PB and 100 μL S-or R-IBU (10 mM), and incubated at 25 °C for a predetermined time. After incubation, the liposomes were removed using an ultrafiltration membrane (molecular cutoff: 50 kDa, Toyo Roshi Kaisha, Ltd., Tokyo, Japan), or by ultracentrifugation at a speed of 55 000 rpm for 2 h (Micro Ultracentrifuge himac CS100FNX, Hitachi Koki Co., Ltd., Tokyo, Japan). The concentration of S- or R-IBU in the supernatant (Csup) was measured using an ultraviolet spectrophotometer (xMark Microplate Spectrophotometer, Bio-Rad Laboratories, Inc., Hercules, CA, USA) by plotting a calibration curve of IBU at 263 nm. The adsorbed concentration (Cads) was calculated by the following equation:

RESULTS Characterization of the PDA Liposome. As it is known that the PDA liposomes show red color, we evaluated the polymerization of the PDA liposomes at 470 nm. Figure 1a shows that the absorbance at 470 nm increased as the polymerization proceeded, and this increment stopped 60 min after the onset of the reaction. In addition to these data, the fluorescence spectrum was also recorded for each of the PDA liposomes. SI Figure S1 indicates that at the first stage of polymerization, the fluorescence is observed at 610 nm (Ex = 510 nm). While this fluorescence gradually disappeared, the fluorescence at 540 nm (Ex = 510 nm) increased by the end of the polymerization. These phenomena agree with the previous reports,31 and indicate that the polymerization proceeded and stopped 60 min after the onset of the reaction under our experimental conditions. The CD analyses demonstrated the confirmation of the PDA liposomes. Figure 1b shows that, after polymerization, the intensity of the CD spectra increased, which implies increased chirality, especially chiral ordering of the headgroup, by polymerization.32 To study the membrane state, the membrane fluidity and membrane polarity of the liposomes (at 25 °C) are plotted as a Cartesian plot in Figure 2a.33 Only the DiynePC liposome was found to exist as a gel (s) phase with a lower fluidity and a

Cads = C ini − Csup where Cini represents the initial concentration of S- or R-IBU. The separation parameter (SS/R) was calculated by the following equation:

SS/R = Cads(S‐IBU)/Cads(R‐IBU) where Cads (S‑IBU) and Cads (R‑IBU) are the concentrations of S-IBU and R-IBU adsorbed on the liposomes, respectively. C

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Biomacromolecules Table 1. DSC Data of Each DiynePC/DOPC Liposomea DiynePC:DOPC

CDiynePC

Tm

ΔHm

ΔT

[molar ratio]

[mM]

[°C]

[kJ/mol]

[°C]

[%]

50 50 40 40 30 30

42.13 42.03 40.22 39.90 39.63 39.55

30.93 21.53 39.73 27.11 37.50 22.32

3.68 3.85 5.51 7.73 6.07 7.04

30.19 31.76 40.48

[10:0] [10:0] [8:2] [8:2] [6:4] [6:4]

PDA PDA PDA

Polymerization degree

Experimental conditions: lipid concentration, 50 mM; polymerization time, 60 min; diameter, 100 nm; incubation temperature, 25 °C; incubation time, 24 h; background solution, 50 mM PB (pH = 6.7); for others, see Experimental Section.

a

Figure 4. Dependence of adsorbed amount and selectivity of S/R-IBU on (a) poly DiynePC/DOPC and (b) nonpolymerized DiynePC/DOPC liposomes. Experimental conditions: polymerization time, 60 min; diameter, 100 nm; incubation temperature, 25 °C; incubation time, 24 h; background solution, 50 mM PB (pH = 6.7); for others, see the Experimental Section. Error bars represent standard deviations (n = 3).

higher GP340 value than those of the other liposomes. Only the DiynePC/DOPC (2/8) liposomes showed a liquid (l) phase, the highest fluidity, and a low GP340 value. The DiynePC/ DOPC liposome mixtures (8/2, 6/4, 4/6) were possibly clustered in the region of the heterogeneous (s+l) membranes. On the other hand, this characterization method could not be applied to the case of polymerized liposomes because of the strong autofluorescence from the PDA liposomes except the GP340 measurements. In the PDA liposome system (DiynePC/ DOPC: 8/2), the GP340 values, which are an index of the hydrophobicity of the inner membrane, increased after irradiation for 45 min, as shown in Figure 3. This increment means that polymerization produced highly ordered gel phase regions.34 In addition to the hydrophobization of the inner membrane, the hydration state of the surface membrane was assessed after polymerization by dielectric dispersion analysis (DDA). DDA demonstrated that dehydration was also caused by polymerization30 (SI Figure S4). Thus, polymerization caused dehydration and hydrophobization in inner and surface regions of the liposomal membrane. For evaluating the phase of the PDA liposomes, DSC measurements were recorded and the results are shown in Table 1. The phase transition temperature (Tm) decreased as the ratio of DOPC was increased, because the Tm of DOPC is −22 °C. In addition, the enthalpy (ΔHm) of the PDA liposomes became smaller as compared to that of the unpolymerized liposomes, because the stability of the liposome membranes increased after polymerization.34 The peak width (ΔT) increased for PDA liposomes and was the maximum for the PDA liposome (Diyne PC/DOPC: 8/2). This peak

broadening for PDA liposomes implies a more rigid domain formation in PDA liposomes than in unpolymerized liposomes.22 The polymerization degree of the PDA liposomes was estimated according to Temprana et al.’s report,34 as shown in Table 1. Each PDA liposome contains a nonpolymerized lipid (30−40% polymerization degree), and thus, a variety of domains were observed in liposome membranes, such as DiynePC containing polyDiynePC and polyDiynePC containing DiynePC, as well as DiynePC, DOPC, and polyDiynePC domains. Thus, the GP340 measurements, DDA and DSC analyses indicate that a larger number of rigid nanodomains were produced in the PDA liposomes compared to in the unpolymerized liposomes. According to our previous results,13 which describe the importance of domain edges in the enantioselective adsorption, this increase in rigid nanodomain formation is favorable for the enantioselective adsorption of IBU. From these characterizations, we can conclude that we successfully prepared PDA liposomes that form various rigid nanodomains in the PDA liposomal membrane. Chiral Separation of IBU. The enantioselective adsorption of IBU on the PDA and the unpolymerized liposomes was assessed after 24 h incubation. Figure 4 indicates that the unpolymerized liposome did not show any large enantioselective adsorption of IBU (SS/R = 3.0), while the PDA liposomes (DiynePC/DOPC: 8/2) exhibited the highest enantioselective adsorption (SS/R = 18.9). Furthermore, as the ratio of DiynePC was increased (DiynePC/DOPC: 9/1, 10/0), the selectivity decreased. Subsequently, we attempted to evaluate the polymerization time and the polymerization degree required for the chiral D

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Figure 5. Relationship between polymerization time of DiynePC/DOPC (8:2) liposome and adsorbed amount and selectivity of S/R-IBU. Conditions: diameter, 100 nm; incubation temperature, 25 °C; incubation time, 24 h; background solution, 50 mM PB (pH = 6.7); for others, see the Experimental Section. Error bars represent standard deviations (n = 3).

recognition of IBU. Figure 5 shows that after a reaction time of 45 min, the chiral recognition was induced in the polyDiynePC/DOPC mixture with 24% polymerization degree, which was prepared with DiynePC/DOPC (8/2). Finally, we attempted to improve the adsorption performance of the liposomes. As the ratio of DOPC was increased, the adsorbed amount of both S- and R-IBU increased, as shown in Figure 4, while the selectivity decreased. In addition to the composition ratio, the effects of liposomal diameter and incubation temperature on the chiral recognition were investigated, as shown in SI Figures S2 and S3. The liposomal diameter did not have any notable effect on the selectivity, except for the smallest diameter liposomes (50 nm). The adsorbed amount of R-IBU increased at an incubation temperature above 50 °C and selectivity was decreased. The optimum incubation time of IBU and the PDA liposomes (DiynePC/DOPC: 8/2) was also elucidated for chiral selective adsorption. Figure 6 clearly shows that 1 h of incubation was sufficient for the chiral separation of IBU. The effect of background solutions should also be considered for higher chiral selectivity and clarification of chiral recognition mechanism. Figure 7 demonstrated the effect of background solutions on the enantioselective adsorption: Higher ionic strength (1 M PB) affected the selectivity of S,R-IBU. Increment of DMSO and urea, which causes dehydration in lipid bilayer35 and cleaves hydrogen bonding,36 respectively, increased adsorption amount and reduced enantioselectivity. Higher pH PB solution (pH = 11) decreased adsorption amounts of S,R-IBU (Cads (S‑IBU) = 2.1%, Cads (R‑IBU) = 0.52%, SS/R = 3.6) compared to these at pH 6.7.

Figure 6. Effect of incubation time on adsorbed amount and selectivity of S/R-IBU by PDA liposomes (DiynePC/DOPC: 8/2). Conditions: polymerization time, 60 min; diameter, 100 nm; incubation temperature, 25 °C; background solution; 50 mM PB (pH = 6.7); for others, see the Experimental Section. Error bars represent standard deviations (n = 3).

E

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Figure 7. Effect of background solutions on the adsorption amount of S/R-IBU and enantioselectivity by PDA liposomes (DiynePC/DOPC: 8/2). Conditions: polymerization time, 60 min; diameter, 100 nm; incubation temperature, 25 °C; incubation time, 24 h; for others, see the Experimental Section. Error bars represent standard deviations (n = 3).

Figure 8. Schematic illustration of chiral recognition mechanism by PDA liposomes.

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DISCUSSION The characterization of PDA liposomes indicates that a variety of PDA liposomes, i.e., liposomes of different compositions and polymerization degrees were successfully prepared by UV light irradiation. On the other hand, we could not obtain higher polymerization degrees because the diacetylenic lipids require a highly regular crystalline state (gel phase) for polymerization.37 Therefore, in spherical liposomes, a larger curvature would not allow higher polymerization degree. However, a low polymerization degree is favorable for the chiral recognition. The reason for this is described in the following section. In the chiral recognition of IBU, the PDA liposomes (DiynePC/DOPC: 8/2) showed the highest selectivity and increment in selectivity with longer polymerization time. In our previous paper, we predicted that the boundary edge of the nanodomain in the heterogeneous membrane is one of the important factors for the following reasons: at the boundary edge, the chiral recognition site, i.e., the asymmetric carbon in the glycerol region of lipids, is exposed, and thus chiral recognition can be efficiently attained. Indeed, some researchers

reported height differences among the nanodomains and deformation of the lipid membrane at the boundary of nanodomains.38,39 This deformation at the boundary edge would expose the asymmetric carbon and allow effective chiral selective interaction. In the PDA liposome systems prepared with the composition DiynePC/DOPC (8/2), a variety of nanodomains such as lone DiynePC, DOPC, and polyDiynePC domains were produced in addition to DiynePC containing polyDiynePC domains, polyDiynePC containing DiynePC domains, and so forth, because of the pre-existing nanodomains and a moderate polymerization degree (31.76%). Therefore, these complex nanodomains induced many boundary edges in especially PDA liposome (DiynePC/DOPC: 8/2), exposed chiral recognition sites, and enabled chiral recognition. On the other hand, if 100% polymerization degree was attained, or an almost homogeneous membrane was formed by polyDiynePC/ DOPC (10/0, 9/1), the complex nanodomains would not have been formed. Therefore, chiral recognition cannot be induced in 100% polymerized systems and in almost F

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interactions could be considered key interactions according to the literature.44−46 Indeed, hydrogen bonding played a significant role in chiral recognition of IBU enantiomers as shown in Figure 7a. The increment of urea concentration resulted in a decrease of enantioselectivity because urea would weaken the hydrogen bonding between IBU and lipids as well as lipids and lipid molecules or water molecules, and increase hydrophobicity. In addition, adequate hydrophobic interaction, which is between the phenyl and isobutyl group of IBU and the acyl chain of DiynePC, was also important in chiral recognition. In the polymerized rigid domain, hydrophobicity was increased compared to that of unpolymerized domain, and this hydrophobization induces enantioselective adsorption. On the other hand, excess hydrophobicity of the lipid membrane due to dehydration by DMSO reduced enantioselectivity as shown in Figure 7b. Similarly, excess IBU hydrophilicity also inhibited the interaction between lipid membranes, and thus decreased adsorption and enantioselectivity at higher pH (pH = 11), where IBU is deprotonated and hydrated.47 In addition to the hydrophobic interaction and hydrogen bonding, the electrostatic interaction, which is between negatively charged carboxyl groups of IBU and positively charged head trimethylammonium groups of DiynePC, was also a dominant force in chiral recognition as shown in Figure 7c. In higher PB concentration (1 M), the electrostatic interaction was reduced, and thus enantioselectivity was also reduced. In conclusion, in rigidordered domains, enantioselective adsorption would be caused by different strengths of hydrophobic interaction, hydrogen bonding, and electrostatic interaction between S,R-IBU and lipid molecules. On the other hand, in disordered domains, both S,R-IBU can be inserted into the acyl chains of lipid molecules, and thus hydrophobic interaction is considered a dominant force. Therefore, chiral recognition cannot be attained. With respect to application for enantioselective adsorption, an alternate and similar material used for chiral separation is a molecular imprinting material (MIP).48 As opposed to our method, the use of MIP requires skillful techniques for the preparation of the chiral recognition materials and the chiral recognition ability cannot be easily modified. On the other hand, in our system, the polymerized liposomes can be easily prepared and the recognition ability can be modified by changing the composition ratio of the polymerizable lipids, the polymerization conditions, and the surrounding environment as well as by adding chiral recognition molecules such as membrane proteins.49 In addition to these features, the polymerized lipid is stable against organic solvent and often spontaneously forms helical structures,50 which are favorable for chiral separations.51 Therefore, with respect to the application, our method could be used for a chiral stationary phase in high performance liquid chromatography, capillary, or microchip electrophoresis. With regard to academic points, our results may reveal the chiral recognition mechanism by the cell membranes. The chiral recognition by cell membranes can be attributed to both membrane proteins49 and the lipid assembly.52 In addition to these factors, our results can propose that the raft domains53 are also important factors in chiral recognition. The domains in PDA liposomes are similar to raft domains in the cells, which show ordered phase and are composed of sphingolipids, cholesterols, and membrane proteins. Therefore, chiral recognition by the cell membrane could be attributed to the membrane proteins, lipid assembly, raft domains, and

homogeneous PDA liposome membranes prepared by only DiynePC. In addition to the exposure of chiral centers, S-IBU would preferentially interact with the chiral centers,40,41 especially with the exposed chiral center at the boundary, for the reduction of interfacial free energy.42 Thus, we can conclude that a large number of complex nanodomains and boundary edges are important for the chiral recognition of IBU and that these factors can be easily controlled by the lipid composition and polymerization degree. As for the effect of phase state and its properties, it was predicted that the ordered phase in the heterogeneous membrane is favorable for the chiral recognition of IBU, while the disordered phase is favorable for increment in the IBU adsorption. In the PDA liposome system (DiynePC/ DOPC: 8/2) showing the highest selectivity, the GP340 values increased after 45 min of irradiation (polymerization), and relative permittivity was decreased after polymerization, as shown in Figure 3 and SI Figure S4. This GP340 increment and decrease of change in relative permittivity (3~5 GHz) as well as the CD results indicate that polymerization caused highly chiral ordering of the headgroup, hydrophobization, and dehydration of membrane. This high ordering permits multiple interactions between the S-IBU and the lipid molecules for chiral recognition. On the other hand, as the DOPC ratio increases, the adsorbed amount of IBU was increased, because increment of the disordered phase enables the hydrophobic interaction between the lipid molecules and S- or R-IBU at the acyl chains.43 In addition to these results, SI Figure S3 also supports this conclusion and indicates that the incubation temperature affects the chiral adsorption of IBU on the PDA liposomes. At temperatures (50 °C) above Tm, the packing density of the PDA liposome system (DiynePC/DOPC: 8/2) would decrease and the rigid domains would loosen compared to those of the PDA liposome system (DiynePC/DOPC: 8/2) at room temperature. This change accelerated the hydrophobic interactions at the acyl chains and increased the adsorbed amount of R-IBU. Furthermore, for optimization of the chiral recognition performance, the effect of the liposomal diameter on the chiral recognition was also investigated. SI Figure S2 shows no significant effect of the liposomal diameter on the chiral recognition except for the 50-nm-diameter liposomes. In the case of 50 nm PDA liposomes, the high curvature resulted in a low lipid packing density and low polymerization degree (18.21%), which failed to produce a variety of nanodomains. Therefore, the 50 nm PDA liposomes showed lower chiral recognition than the larger diameter PDA liposomes. From these results, we can conclude that for the chiral recognition of IBU, highly chirally ordered nanodomains and boundary edges are significantly important as shown in Figure 8, while the adsorbed amount is affected by the disordered phase. In other words, for the control of chiral selectivity and the adsorbed amount, a balance between the highly ordered nanodomains, boundary edges, and the disordered phase should be considered. With respect to enantioselective interaction between S,R-IBU and lipid molecules, the chiral recognition mechanism is considered as follows: In the rigid domain, ordered lipid domains and stiff structure of IBU prevent IBU from being inserted into the acyl chain region of the lipid membrane and resulted in low adsorption. Furthermore, this suppression makes IBU and lipids interact in the surface region of liposomes. In the surface region of the liposomal membrane, electrostatic force, hydrogen bonding, and hydrophobic G

DOI: 10.1021/acs.biomac.6b01859 Biomacromolecules XXXX, XXX, XXX−XXX

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CD, circular dichroism; DSC, differential scanning calorimetry; MLVs, multilamellar vesicles

membrane proteins in raft domains, as well as other factors not specified here.

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CONCLUSION We successfully induced chiral recognition power by polymerization of lipids, and clarified dominant factors and interactions forces for chiral recognition. In the chiral recognition of IBU with PDA liposomes, the formation of rigid nanodomain and boundary edge was of significant importance for multiple interactions, and these could be controlled by changing the composition of liposomes and polymerization conditions. Thus, our findings will be helpful in the optimum construction of liposome membranes for various chiral separations. Moreover, our findings might be helpful in understanding chiral recognition by cells because the heterogeneous PDA membrane is similar to the cell membrane.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.6b01859. Fluorescence spectra of DiynePC/DOPC (10/0) liposomes in different polymerization times; effect of the diameter of PDA liposomes (DiynePC/DOPC: 8/2) on the adsorbed amount and selectivity of S/R ibuprofen; effect of incubation temperature on the adsorbed amount and selectivity of S/R ibuprofen by PDA liposomes (DiynePC/DOPC: 8/2); hydration state of DiynePC/ DOPC (8/2) liposomes before and after polymerization by dielectric dispersion analyses (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Hiroshi Umakoshi: 0000-0002-9241-853X Author Contributions

Y. Okamoto and H. Umakoshi planned this research and designed experiments. Y. Okamoto, Y. Kishi, K. Suga, and H. Umakoshi performed these experiments and analyzed the data. Y. Okamoto and H. Umakoshi wrote this manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS 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). Y. O. acknowledges Asahi glass foundation and Shimadzu Science Foundation.

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ABBREVIATIONS IBU, ibuprofen; PDA, polymerized diacetylene; DOPC, 1,2dioleoyl -sn-glycero-3-phosphocholine; DiynePC, 1,2-bis(10,12tricosadiynoyl)-sn-glycero-3-phosphocholine; DPH, 1,6-diphenyl-1,3,5-hexatriene; Laurdan, 6-lauroyl-2-dimethylamino naphthalene; PB, phosphate buffer; DMSO, dimethyl sulfoxide; DDA, dielectric dispersion analysis; MIP, molecular imprinting; H

DOI: 10.1021/acs.biomac.6b01859 Biomacromolecules XXXX, XXX, XXX−XXX

Article

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DOI: 10.1021/acs.biomac.6b01859 Biomacromolecules XXXX, XXX, XXX−XXX