Distribution and Adsorption of Ionic Species into a Liposome

Sep 29, 2016 - *Telephone/Fax: +81-75-724-7522. ... formation constant in the BLM (Kip), and the R6G+ adsorption constant on the BLM surface (Kad). ...
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Distribution and Adsorption of Ionic Species into a Liposome Membrane and Their Dependence upon the Species and Concentration of a Coexisting Counterion Koji Murakami,† Kisho Hori,† Kohji Maeda,‡ Mao Fukuyama,‡ and Yumi Yoshida*,‡ †

Department of Chemistry and Materials Technology and ‡Faculty of Molecular Chemistry and Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo, Kyoto 606-8585, Japan S Supporting Information *

ABSTRACT: The distribution of ions into a bilayer lipid membrane (BLM) and their adsorption on the BLM are investigated by extracting a hydrophobic cation, rhodamine 6G (R6G+), into a liposome through the dialysis membrane method. R6G+ distribution mainly depends upon the concentration of the coexisting anion and its species (Cl−, Br−, BF4−, ClO4−, and picrate). On the other hand, R6G+ adsorption on the BLM surface follows the Langmuir adsorption model and is independent of the coexisting anion in the aqueous phase. We propose an extraction model of ionic species into the BLM, to explain the dependence of extraction of ionic species upon the coexisting anion. In this model, an ion is distributed with a coexisting counterion into the BLM and then forms an ion pair in the BLM. Here, the ion adsorption equilibrium on the BLM surface is independent of the species and concentration of the coexisting counterion under the same ionic strength. On the basis of this model, we estimate the distribution constant of R6G+ and anion (KD), the ion-pair formation constant in the BLM (Kip), and the R6G+ adsorption constant on the BLM surface (Kad). Even for an ultrathin membrane system, such as a BLM, R6G+ is distributed with a coexisting counterion and the distribution equilibrium of the ionic species at the water−BLM interface is analyzable similar to that at the water−organic solvent interface.

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INTRODUCTION The distribution of ionic compounds through a biological membrane is essential for ion transport into the cell. Therefore, delineating the mechanism of this distribution should allow for prediction of the direct transport of ionic species through the biological membrane, that is, permeability of ionic drugs,1 penetration of the charged peptides,2,3 or accumulation (toxicity) of an organic base or acid into the human body.4−8 Unlike a neutral species, transport of ionic species through a biological membrane is complex because the transport causes the other transfer of the coexisting ion, to maintain electroneutrality in the membrane and aqueous phases. Therefore, transport of ionic species is easily influenced by the presence of the coexisting ion. For example, the transport of penetrating peptides, such as the TAT peptide, which has positive charges in itself, is facilitated by the presence of a hydrophobic anion.9−11 Permeation of ionic drugs through a porcine membrane is also dependent upon the species of a coexisting ion.12 Even for an artificial bilayer lipid membrane (BLM), the effect of coexisting ions has been reported. Permeation of the peptide cation through a liposome membrane9,13−15 or the ion transport current in electrochemical measurement with a planar BLM16−20 was enhanced by the increase of hydrophobicity of the coexisting counterion. © 2016 American Chemical Society

The distribution constant of the ionic species between the aqueous phase and the BLM is the parameter expressing the direct transport of the ionic species through the BLM. However, it is difficult to evaluate the distribution constant experimentally, which strongly depends upon the concentration of the coexisting electrolyte21,22 or the presence of the hydrophobic coexisting ion.23 Moreover, a BLM is an ultrathin membrane of nanometer thickness and has functional groups on the membrane surface. Not only distribution into the membrane but also adsorption of ionic species on the membrane surface takes place. To evaluate the distribution constant of ionic species between the aqueous phase and the BLM, a sorption model, which takes into account the interfacial potential of the membrane surface, has been proposed,21,22,24 and many distribution constants of acid or base drugs have been estimated on the basis of this sorption model.25,26 In the sorption model, an ionic species binds to the binding site of the membrane surface, whose surface potential depends upon the concentration of the coexisting salt, according to the Gouy−Chapman theory. Basically, the sorption model differs from the classical Received: August 26, 2016 Revised: September 27, 2016 Published: September 29, 2016 10678

DOI: 10.1021/acs.langmuir.6b03162 Langmuir 2016, 32, 10678−10684

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prepared from KH2PO4 and NaOH. All aqueous solutions were prepared using distilled water. Preparation of the Liposome Solution. The solution of multilamellar PC vesicles was prepared as follows. The chloroform solution containing 18.2 mg of PC in a 100 mL recovery flask was evaporated at 65 °C under reduced pressure (15 kPa). The recovery flask with a PC layer was ultrasonicated after the addition of an aqueous solution (7 cm−3) containing 0.1 M (M = mol dm−3) phosphate buffer (pH 7) and various concentrations of NaX (X = Cl−, Br−, BF4−, ClO4−, and Pic−) into the recovery flask. The vesicles were converted into unilamellar liposomes by extrusion of the multilamellar vesicles through polycarbonate membranes with 0.1 μm pore size (610005, Avanti Polar Lipids, Inc., Alabaster, AL, U.S.A.) using an extruder (Avanti Mini-Extruder, Avanti Polar Lipids, Inc., Alabaster, AL, U.S.A.) and a 1000 μL syringe. The extrusion was repeated more than 11 times at 50 °C, which is 10 °C higher than the phase transition temperature of PC. Evaluation of the Prepared Liposome. The diameter of the prepared liposome was evaluated to be 200 ± 90 nm by the dynamic light scattering technique (Nicomp 380, Particle Sizing Systems Japan Co., Ltd., Tokyo, Japan). It was confirmed that the liposome diameter did not change, even after the extraction experiments. The PC concentration in the liposome solution was determined by an in vitro assay kit especially designed for this purpose, which is based on the choline oxidase−N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS) method.41 The number of liposomes in the liposome solution was calculated from the PC concentration (M), the molecular surface area (0.456 nm2/lipid42,43), and the liposome diameter (200 ± 90 nm), assuming a membrane thickness of 5 nm, and was estimated to be 550 000 lipids/liposome. For the liposome solution with 3.3 × 10−3 M PC, the number of liposomes was 1.6 × 1013 dm−3. Extraction Procedure with the Liposome. The extraction of R6G+ with various anions into liposomes was carried out based on the dialysis membrane method.23,24,44 The aqueous solution was separated using a dialysis tube of regenerated cellulose (diameter of 16 mm, thickness of 20.3 μm, pore size of 5 nm, and 14 000 Da molecular weight cutoff, UC 20-32-100, Viskase Companies, Inc., Lombard, IL, U.S.A.). The dialysis tube including the aqueous inner solution (1 cm−3), whose top and bottom were tightly tied with a Nylon line (Nasuly N-Walker Nylon W-DMV, YGK Yoz-Ami Co., Ltd., Naruto, Japan) to avoid inner solution leakage, was soaked in a test glass tube (height of 180 mm and internal diameter of 15 mm) filled with the outer solution (5 cm−3). The inner solution contained 0.10 M phosphate buffer (pH 7), various concentrations of NaX (X = Cl−, Br−, BF4−, ClO4−, or Pic−), and 1.0 × 10−6 M R6GCl, whereas the outer solution contained 0.10 M phosphate buffer (pH 7), various concentrations of NaX (X = Cl−, Br−, BF4−, ClO4−, or Pic−), and liposomes consisting from 0.9 × 10−3 to 3.5 × 10−3 M PC. The ionic strength of aqueous solution is mainly determined by 0.10 M phosphate buffer, and every extraction experiment was under the same ionic strength. The test glass tubes with the outer and inner solutions were shaken for 15 h at 20 °C in a reciprocal shaker (Taiyo incubator personal, Taiyo Kagakukogyo Co., Tokyo, Japan). We confirmed that the extraction time of 15 h was enough to attain extraction equilibrium by measuring the R6G+ concentrations in the inner and outer solutions, where their concentration after extraction indicate same value. To avoid R6G+ adsorption, the dialysis tube and glass tube required pretreatment. The dialysis tube was soaked in an aqueous solution of the same composition as that of the inner solution for over 24 h before the extraction process. The surface of the glass tube was cleaned by soaking in a piranha solution [3:7 H2O2(30%)/H2SO4] silanized as a cationic surface by infusing an ethanol solution of 17% (v/v) 3aminopropyltriethoxysilane into the glass tube, holding it for 2 h, and washing with methanol.45 For the extraction, two sets of test glass tubes were prepared for each experiment: one in the presence of liposomes (the measurement cell) and the other in their absence (the reference cell). The amount of extracted R6G+ was estimated from the difference between the R6G+ concentration in the inner solution of the measurement cell,

ion-pair partition model, which has been adopted in the water− octanol system.27 The sorption model can express the effect of the ionic strength on the distribution but cannot satisfactorily explain the enhancement12 and depression23 of the ionic species distribution by the coexisting hydrophobic ion under the same ionic strength. One approach to explain the effect of the coexisting counterion is through a partition model of the target ion and the coexisting counterion, where both of these ions are simultaneously distributed into the membrane to maintain electroneutrality in each phase and form the ion pair in the membrane. This model has been adopted to express the ionic distribution equilibrium between water and a rather high-polar organic solvent, such as nitrobenzene.28−32 The partition model of ions is thermodynamically equivalent to the classical partition model of the ion pair formed in the aqueous phase, which has been extensively employed for analyzing the water− octanol system.27 However, unlike the ion-pair partition model, the partition model of ions makes it possible to evaluate the Gibbs free energy for the ion transfer,33 the multiple distribution of ions, and the distribution of ions accompanied by a side reaction (ion-pair formation or complex formation).34,35 The model has been applied to the distribution of drugs in the water−organic solvent system accompanied by an acid−base reaction.36−38 On the basis of the partition model of ions, we have also analyzed the dependence of the distribution ratio upon the concentration of coexisting ions at the water− organic solvent system and evaluated the distribution of alkylammonium ion, alkylsulfonate,39 alkali metal ion facilitated by the complex formation,39 and the multiple distribution of actinide ions.40 Therefore, if distribution of the ion in a BLM system is achieved in the same way as that between water and an organic solvent, the distribution equilibrium of ions in the BLM system can be analyzed on the basis of the dependence of the distribution ratio upon the concentration of the coexisting counterion. The present paper aims to analyze the distribution of ions between the aqueous phase and the BLM based on the partition model of ions and to evaluate the distribution constant of ions from the dependence of the distribution ratio upon the concentration of the coexisting counterion under a constant ionic strength. We use rhodamine 6G (R6G+) as the target cation and examine the dependence of the distribution ratio of R6G+ between water and the BLM upon concentration of a coexisting anion and its species (Cl−, Br−, BF4−, ClO4−, and picrate) in the dialysis method with a liposome. In the analysis, we also demonstrate that the R6G+ adsorption equilibrium on the BLM surface is not influenced by the coexisting anion under a constant ionic strength. The distribution constant of ions, the ion-pair formation constant in the BLM, and the adsorption constant of R6G+ on the BLM surface are evaluated.

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EXPERIMENTAL SECTION

Chemicals. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (PC, >99%) was obtained from Sigma-Aldrich (Tokyo, Japan). The chloride salt of rhodamine 6G (R6GCl, practical grade), picric acid (98%), an in vitro assay kit for determination of PC (Wako Phospholipids C), and other reagents, such as NaCl, NaBr, NaBF4, NaClO4, NaOH, KH2PO4, chloroform, ethanol, and methanol (analytical grade), were purchased from Wako Pure Chemical Industries (Osaka, Japan). 3-Aminopropyltriethoxysilane (>96%) was obtained from Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan). Sodium picrate (NaPic) solution was prepared by titration of picric acid with NaOH. Phosphate buffer was 10679

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Langmuir [R6G+]mea, and that of the reference cell, [R6G+]ref. The concentration of R6G+ in the solution was determined by fluorescence spectrometry (FP6200, JASCO Co., Tokyo, Japan). Determination of the Apparent Distribution Ratio. The apparent distribution ratio (R) of R6G+ between the aqueous phase (W) and the liposome membrane (lip) was defined as the ratio of the concentration of R6G+ in lip ([R6G+]Tlip) to the concentration of R6G+ in W ([R6G+]W)

R=

Figure 2a shows the dependence of [R6G+]mea and [R6G+]ref upon the concentration of the coexisting anion in the presence and absence of liposomes. During the extraction procedure, R6G+ might not only be captured by liposomes but could also remain on the surface of the glass tube or the dialysis membrane. To compensate for these effects, [R6G+]mea was subtracted from [R6G+]ref. [R6G+]ref was independent of the concentration of the coexisting anion (Cl− or ClO4−), whereas [R6G+]mea decreased when the anion concentration increased to more than 10−2 M for Cl− or 10−3 M for ClO4−. The degree of decrease in [R6G+]mea for ClO4− was larger than that for Cl−. Therefore, the extraction of R6G+ by liposomes depended upon the concentration and the species of the anion.

[R6G+]Tlip [R6G+]W

(1)

where [A]B indicates the mole concentration (M) of A in phase B. We assumed the ion-pair formation in W to be negligible and the concentration of R6G+ in W to be equal to [R6G+]W, which was experimentally estimated as [R6G+]mea. [R6G+]Tlip was estimated as the apparent concentration of R6G+ in lip based on the decrease in the R6G+ concentration of the inner solution caused by the addition of the liposome, ([R6G+]ref − [R6G+]mea)

[R6G+]Tlip = ([R6G+]ref − [R6G+]mea )

(Vout + Vin) Vlip

(2)

where Vin and Vout are the volumes of the inner and outer solutions in the dialysis tube, respectively, and Vlip is the volume of the BLM phase of all liposomes, which was calculated from the PC concentration, [PC] (M), determined using an in vitro assay kit, the thickness of the BLM (x = 5 nm), and the molecular area of the PC (A = 0.456 nm2/ molecule42,43) according to eq 3 Vlip = [PC]VoutNAAx /2

(3)

where NA is the Avogadro constant. In the calculation of R, the concentration of R6G+ in the internal aqueous phase of the liposome was assumed to be [R6G+]W. Even if the amount of R6G+ transferring into the internal aqueous phase of the liposome is small, the effect on R was considered negligible because the volume of the internal aqueous phase was about 1% of the total volume of the outer and inner solutions.

Figure 2. Dependence of [R6G+]W in the presence of PC (●, [R6G+]mea) and in the absence of PC (○, [R6G+]ref) upon the concentration of the coexisting (a) Cl− or (b) ClO4−. Original composition of the aqueous solution: 1.0 × 10−1 M phosphate buffer (pH 7.0), 1.8 × 10−7 M R6GCl, and x M NaX. The outer solution of the dialysis tube contained 3.3 × 10−3 M PC (added as a liposome).

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RESULTS AND DISCUSSION Extraction of R6G+ by Liposomes. The extraction of R6G+ by liposomes is shown in Figure 1. In the presence of liposomes, [R6G+]mea decreased with the increase of [PC] and reached 23% of the initial concentration without liposomes. After the extraction procedure, the size of the liposomes, measured by dynamic light scattering, did not change as a result of the extraction. Extraction of R6G+ by liposomes was attributed to the R6G+ distribution in the membrane phase of the liposomes or to R6G+ adsorption on their surfaces.

Extraction Equilibria of R6G+ by Liposomes. We modeled R6G+ extraction, as shown in Figure 3. The extraction of R6G+ by liposomes is expressed by the following equilibria: the adsorption equilibrium on the BLM surface, the ion distribution equilibrium in the BLM, and the ion-pair formation equilibrium in the BLM. The adsorption equilibrium was assumed to be independent of the ion distribution equilibrium, although Escher et al. assumed that the ionic species were first adsorbed on the BLM surface and then distributed into the membrane phase.23,24,44 The concentration of the ion pair, R6G+X−, in the aqueous phase is negligible compared to [R6G+]W. The distribution

Figure 1. Extraction of R6G+ by liposomes as a function of [PC] in outer solution of the dialysis membrane. Aqueous solution: 2.2 × 10−7 M R6GCl, 1.0 × 10−3 M NaCl, and 0.10 M phosphate buffer. Diameter of the liposome: 200 nm (550 000 lipids/liposome).

Figure 3. Extraction equilibria of R6G+ at the interface between W and the BLM of the liposome. 10680

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Langmuir constant (KD), the ion-pair formation constant in the BLM (Kip), and the adsorption constant (Kad), are defined by eqs 4−6, respectively KD =

Table 1. Equilibrium Constants Evaluated from Figure 4 X− −

Cl Br− BF4− ClO4− Pic−

[R6G+]lip [X−]lip [R6G+]W [X−]W

(4)

+ −

K ip =

K ad =

[R6G X ]lip [R6G+]lip [X−]lip

(5)

(6)

where NR6G+,ads and Nsite,ads are the mole numbers per unit area of occupied and unoccupied sites on the surface of the liposome, respectively, and the total mole number per unit area of sites for adsorption, NTads, is defined as (NR6G+,ads + Nsite,ads). The parameter R in eq 1 is defined as ⎛ KD ⎞1/2 − 1/2 R = K ipKD[X−]W + ⎜ ⎟ ([X ]W ) + ⎝ [R6G ]W ⎠ +

T 2Nads K ad x(1 + K ad[R6G+]W )

KD

Kad (mol−1 dm3)

ND 2800 2300 15000 76000

ND 0.12 3.0 18 40

16000 13000 11000 13000 14000

The above analysis for distribution of R6G+ into the liposomes and their adsorption on the liposome surface is based on the assumption that R6G+ adsorption is not influenced by the concentration and species of the coexisting anion. In the Supporting Information, we confirm this hypothesis by investigating the effect of the coexisting anion on R6G+ adsorption on the surface of the PC membrane, studying a PC monolayer adsorbed at the water−decane interface (Figure S2 of the Supporting Information). A significant influence of the coexisting anion on R6G + adsorption was not observed. To illustrate the dependence of R on [X−]W clearly, R was replotted against log[X−]W, as shown in Figure 5. The lines in Figure 5 are theoretical curves calculated on the basis of eq 7. In eq 7, the contribution of each term, i.e., the ion pair formed in the BLM (K ip K D [X − ] W ), the ion distribution ((K D / [R6G+]W)1/2([X−]W)1/2), and the R6G+ adsorption on the membrane surface (2NTadsKad/x(1 + Kad[R6G+]W)), to the total R is calculated on the basis of equilibrium constants shown in Table 1. The contribution is shown in Figure S3 of the Supporting Information. There are two concentration ranges of [X−]W in Figure 5. In the low concentration range, R is assumed to be a constant value independent of log[X−]W. Thus, in this range, R showed the same value regardless of the species of X−, indicating that X− hardly participated in the extraction of R6G+ by liposomes. As shown in Figure S3 of the Supporting Information, the extraction of R6G+ by liposomes was predominately caused by the adsorption of R6G+ on their surfaces, which was not influenced by X−. On the other hand, with increasing [X−]W, R gradually increased and the curve shifted to a lower value of log[X−]W with increasing hydrophobicity of X−. In this region, not only adsorption of R6G+ on the liposome surface but also their distribution into the liposome membrane, which was influenced by [X−]W, as shown by eq 4, was involved in the extraction of R6G+ by liposomes (Figure S3 of the Supporting Information). When the distributions of R6G+ and X− were significant, ion-pair formation was induced in the BLM and R increased further. These results suggest that the distribution equilibrium of R6G+ and X− and the adsorption equilibrium on the liposome surface are independent of each other and that the two equilibria can be distinguished by plotting, as shown in Figure 5. R6G+ Adsorption on the Liposome Surface. To confirm the adsorption equilibrium of R6G+ on the liposome surface, R in the presence of Cl− as a counterion was analyzed by changing [R6G+]W from 1.4 × 10−7 to 2.4 × 10−4 M. This condition indicates that R is not dependent upon [Cl−]W of lower than 10−2 M and R6G+ was not distributed into the liposome membrane. We assumed that the decrease in [R6G+]W by adding liposomes corresponded to R6G+ad and plotted NR6G+,ads against [R6G+]W, as shown in Figure 6a. NR6G+,ads increased with the increase of [R6G+]W and reached the saturation adsorption amount of 1.9 × 10−7 mol m−2 at [R6G+]W of 1.4 × 10−4 M. Then, NTads was assumed to be 1.9 ×

NR6G+,ads [R6G+]W Nsite,ads

Kip (mol−1 dm3)

(7)

Detail of derivation of eq 7 is shown in the Supporting Information. Equation 7 is expressed as a quadratic function of ([X−]W)1/2. According to eqs 1 and 2, we calculated R from ([R6G+]ref − [R6G+]mea) of Figure 2 and plotted it against ([X−]W)1/2 in Figure 4. The estimated R indicated large dependence upon ([X−]W)1/2. A high concentration of the hydrophobic anion produced a greater increase in R with increasing ([X−]W)1/2. For low ([X−]W)1/2, the R of all anions was extrapolated to be almost 1000.

Figure 4. Dependence of R estimated from ([R6G+]ref − [R6G+]mea) upon the concentration of the coexisting anion, [X−]W. X− = Pic− (▲), ClO4− (□), BF4− (■), Br− (○), or Cl− (●). Lines (- - -) indicate the approximate curves analyzed according to eq 7. The experimental conditions are the same as in Figure 2.

Assuming that the equilibria for R6G+ extraction are as shown in Figure 3, the obtained R was analyzed on the basis of eq 7 by quadratic curve approximation and KD, Kip, and Kad were evaluated as shown in Table 1. In evaluating Kad from experimental values corresponding to the third term on the right-hand side of eq 7, we used 1.9 × 10−7 mol m−2 as the T value of Nads , which was estimated in other adsorption experiments (see later the R6G+ Adsorption on the Liposome Surface section). 10681

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Figure 5. R versus log[X−]W plot replotted from Figure 4. X− = Pic− (▲), ClO4 − (□), BF4− (■), Br− (○), or Cl− (●). Lines (- - -) indicate the approximate curves calculated using the equilibrium constants of Table 1 and eq 7. The experimental conditions are the same as in Figure 2.

10−7 mol m−2. The adsorption equilibrium, which is defined by eq 6, is expressed by eq 8 based on the Langmuir adsorption model θ (8) 1−θ where θ is the fractional occupancy of the adsorption sites and is defined as NR6G+,ads/NTads. Figure 6b shows the plot of log[θ/ (1 − θ)] against log[R6G+]W. The linear relationship (slope = 0.962) is clearly observed in a wide concentration range. These results indicate that the adsorption of R6G+ on the BLM surface is explainable with the Langmuir adsorption model, although aggregation of R6G+ molecules is expected in this concentration.46,47 This linear relationship existed in both lower and higher concentrations of R6G+. Hence, we considered that the aggregation of R6G+ does not significantly influence the plot of log[θ/(1 − θ)] against log[R6G+]W. On the basis of the intercept in Figure 6b, Kad was estimated to be 16 200, which is similar to the value derived through the quadratic curve approximation of the R versus ([X−]W)1/2 plot in Figure 4 (16 000 in Table 1). Therefore, Kad estimated by curve approximation of the R versus ([X−]W)1/2 plot is valid. Distribution Equilibrium of R6G+ and Anions. If the estimated value of KD truly results from the distribution equilibrium of R6G+ and X− at the W−BLM interface, KD can be converted to the Gibbs free energy for the ion transfer from W to BLM, ΔGtr°, and can be expressed as follows: K ad[R6G+]W =

ln KD = −(ΔGtr° ,R6G+ + ΔGtr° ,X−)/RT

Figure 6. (a) Equilibrium isotherm for the adsorption of R6G+ on the surface of a liposome and (b) linear dependence of log[θ/(1 − θ)] on log[R6G+]W. Aqueous solution: 0.1 M phosphate buffer (pH 7) and 1.0 × 10−3 M NaCl. Liposomes: 3.3 × 10−3 M PC (added as liposomes).

Table 2. Gibbs Free Energy for the Ion Transfer from W to BLM, ΔG°tr, Evaluated from KD in Table 1 ΔG°tr,X− − ΔG°tr,Pic− (kJ mol−1) X− −

Cl Br− BF4− ClO4− Pic−

W−BLM

W−DCE30,32,48

ND 14 8.3 6.4 0.0

45.6 38.8 12.4 9.0 0.0

Adsorption and Distribution of R6G+ to a Liposome. The amount of R6G+ located in or on the BLM of a liposome is calculated by employing the equilibrium constants given in Table 1. Taking into account the liposome diameter (200 nm) and the molecular surface area of PC (0.456 nm2/lipid), a liposome is composed of 550 000 molecules and the volume of its BLM phase is 0.63 × 10−18 dm−3/liposome. When 10−6 M R6GCl in W containing 0.03 M NaClO4 and 0.1 M phosphate buffer (pH 7) is extracted by a liposome of 3.5 × 10−3 M PC (3.8 × 1015 liposomes/dm−3), the bulk concentration of R6G+ in W decreases to 1.6% of the R6G+ concentration without the liposome. The molecular numbers of the adsorbed R6G+ on the BLM surface, the dissociated R6G+ in the BLM, and the associated R6GClO4 in the BLM are 8, 4, and 13 molecules/ liposome, respectively. These values seem to be very small but not negligible from the viewpoint of the concentration in the BLM as pointed out by Kakiuchi et al.49 Considering the small volume of the BLM, the total concentration of R6G+ in the BLM is estimated at 6.9 × 10−5 M, 24% of which is present as dissociated R6G+. The dissociated ion amount of the 10−5 M order in the BLM is considered enough to induce large ionic conductivity in the BLM.16,17

(9)

According to eq 9, (ΔG°tr,R6G+ + ΔG°tr,X−) is calculated from the KD given in Table 1. To compare the experimental values to those reported in the W−1,2-dichloroethane (DCE) system,30,32,48 ΔGtr° of X− is expressed as (ΔG°tr,X− − ΔG°tr,Pic−) with reference to that of Pic−. The result shows a clear correlation between the ΔG°tr values estimated in the W−BLM and W−DCE systems (Table 2). ΔG°tr,X− for a hydrophobic ion, such as BF4− or ClO4−, for the W−BLM system is a rather similar value to that for the W−DCE system, but ΔGtr,X ° − for a hydrophilic ion, such as Br−, is smaller than that for the W− DCE system. These results suggest that the inner phase of the BLM has physicochemical properties similar to those of a hydrophobic solvent, such as DCE, but has higher affinity for a hydrophilic anion, as compared to DCE. The same tendency was also observed by comparing the ΔG°tr of organic weak acid anions estimated at the W−BLM system and that estimated for the W−octanol system.23 10682

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CONCLUSION In the present study, the extraction of R6G+ by liposomes was examined for different species and concentrations of the coexisting counterion under the same ionic strength. The apparent distribution ratio showed two types of dependence upon the concentration of the counterion. In the first case, the apparent distribution ratio was independent of the counterion concentration and mainly resulted from the adsorption equilibrium of R6G+ on the liposome surface. In the second case, the apparent distribution ratio was strongly dependent upon species of the counterion and its concentration, indicating that two equilibria related with the counterion occurred: the distribution of R6G+ and the counterion at the W−BLM interface and the ion-pair formation in the BLM. We analyzed the apparent distribution ratio based on the partition model of ions and evaluated the three equilibrium constants of R6G+ adsorption on the BLM surface, the distribution of ions into the BLM, and the ion-pair formation in the BLM. Equilibria existing in the W−BLM system are similar to those in W− organic solvent system, even if the membrane is quite thin. The proposed analysis in this study makes the quantitative evaluation of the distribution and adsorption in the W−BLM system of an ionic compound. Therefore, it is possible to predict the transport mechanism of ionic species through a biomembrane, because adsorption and distribution are closely related to the transport mechanism.3 In addition, the viewpoint obtained in the present study is valuable for the development of penetration enhancers in drug delivery.9−11

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b03162. Derivation of eq 7, effect of a coexisting anion on adsorption of R6G+ on the surface of BLM, and contribution of equilibria to the apparent distribution ratio (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Telephone/Fax: +81-75-724-7522. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Prof. Tokuzo Kawase of the Kyoto Institute of Technology for his helpful support regarding the light scattering measurement of the liposome. This study was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grants 22550073 and 26410150.

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DOI: 10.1021/acs.langmuir.6b03162 Langmuir 2016, 32, 10678−10684

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DOI: 10.1021/acs.langmuir.6b03162 Langmuir 2016, 32, 10678−10684