Article pubs.acs.org/Langmuir
Systematical Characterization of Phase Behaviors and Membrane Properties of Fatty Acid/Didecyldimethylammonium Bromide Vesicles Keishi Suga,† Tomoya Yokoi,† Dai Kondo,† Keita Hayashi,‡ Seiichi Morita,§ Yukihiro Okamoto,† Toshinori Shimanouchi,∥ and Hiroshi Umakoshi*,† †
Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyamacho, Toyonaka, Osaka 560-8531, Japan ‡ Department of Chemical Engineering, National Institute of Technology, Nara College, 22 Yata-cho, Yamatokoriyama, Nara 639-1080, Japan § Department of Materials Science, National Institute of Technology, Wakayama College, 77 Noshima, Nada, Gobo, Wakayama 644-0023, Japan ∥ Graduate School of Environmental and Life Science, Okayama University, 3-1-1 Tsushima-naka, kita-ku, Okayama, Okayama 700-8530, Japan S Supporting Information *
ABSTRACT: Fatty acids (FAs) are known to form vesicle structures, depending on the surrounding pH conditions. In this study, we prepared vesicles by mixing FAs and a cationic surfactant, and then investigated their physicochemical properties using fluorescence spectroscopy and dielectric dispersion analysis (DDA). The assemblies formed from oleic acid (OA) and linoleic acid (LA) were modified by adding didecyldimethylammonium bromide (DDAB). The phase state of FA/DDAB mixtures was investigated with pH titration curves and turbidity measurements. The trigonal diagram of FA/ionized FA/DDAB was successfully drawn to understand the phase behaviors of FA/DDAB systems. The analysis of fluidities in the interior of the membrane with use of 1,6-diphenyl-1,3,5-hexatriene (DPH) indicated that the membrane fluidities of OA/DDAB and LA/DDAB at pH 8.5 slightly decreased in proportion to the molar ratio of DDAB in FA/DDAB systems. The fluorescent probe 6-lauroyl-2-dimethylamino naphthalene (Laurdan) indicated that the LA vesicle possessed a dehydrated surface, while the OA vesicle surface was hydrated. Modification of LA vesicles with DDAB induced the hydration of membrane surfaces, whereas modification of OA vesicles by DDAB had the opposite effect. DDA analysis indicated that the membrane surfaces were hydrated in the presence of DDAB, suggesting that the surface properties of FA vesicles are tunable by DDAB modification.
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FA assemblies can be utilized as an enclosed vesicle (nano capsule) as effectively as liposomes. The environmental pH is one of the essential factors that define the aggregate state of FA molecules. Micelles are the dominant aggregation species at higher pH regions (ionized FA > protonated FA), while oil droplets are formed in the lower pH region (pH < pKa).3,4,9,10 In diluted systems (>95 wt % water), transitions from one type of FA/ soap structure to another can be easily induced by changing the pH.11 When the pH of the system is maintained near the pKa of the FAs, favoring the balance between its ionized and protonated states, a stable vesicle can be obtained. The pH range of the vesicular phase can be extended in mixed systems, such as short-chain fatty acids, alcohols,10 and cationic surfactants.12 Because the formation and stability of FA vesicles can be modulated by their compositions and surrounding conditions, such as pH and temperature, not only the morphological properties (e.g., diameter), but also the physicochemical properties
INTRODUCTION
Fatty acid (FA) vesicles are colloidal suspensions of closed lipid bilayers, composed of FAs and their ionized species (soap).1 The formation of FA vesicles is restricted to a narrow pH range (ca. 7−9), where approximately half of the carboxylic groups are ionized.2 Fatty acid vesicles are observed in a small region within the FA−soap−water ternary phase diagram above the chain-melting temperature of the corresponding fatty acid−soap mixture.3 Compared with diacylglycerophospholipid vesicles (conventional liposomes), FA vesicles, which are composed of single-chain amphiphilic molecules, have some different properties depending on the surrounding pH,4 such as dynamic nature, self-growth, and transition of their assemblies. A vesicle bilayer structure can act as a soft interface for the functionalization of biomolecules.5 In our previous studies, the physicochemical properties of lipid membranes were found to play important roles: recognition of the peptide fragment of oxidized superoxide dismutase,6 enzyme-like activities of porphyrin,7 in vitro regulation of cell-free gene expression systems, and other functions of the biomolecules.8 It is therefore expected that the bilayer structure of © 2014 American Chemical Society
Received: February 28, 2014 Revised: October 1, 2014 Published: October 8, 2014 12721
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(e.g., fluidity and polarity) must be investigated. In previous studies, the organization of the hydrophobic interior of FA vesicles has been compared with micelles in the case of the decanoic acid/sodium decanoate system, using fluorescence lifetime and anisotropy measurements of the incorporated chromophore perylene.13 The comparison between vesicles and micelles is also important to clarify the thermodynamic and kinetic aspects of various aggregation states. On the basis of dialysis experiments, it has been suggested that the formation of FA vesicles may require a much higher energy barrier than the formation of (ionized) FA micelles.9 It is also important to develop methods to prepare stable FA vesicles and investigate their possible applications. Oleic acid is a typical amphiphilic molecule that forms a bilayer vesicle with a hydrogen bond network of carboxylic acid groups at the surface of oleic acid and oleate.14 The chemical structure of oleic acid has an unsaturated CC bond in its hydrocarbon chain, while the structure of linoleic acid has two unsaturated CC bonds (see the Supporting Information, Figure S1). FAs have a variety of roles in biological systems, acting as antioxidants15 and structural components of membranes.16 It has been reported that vesicles composed of single-chain amphiphilic molecules can be stabilized by modification with cationic surfactants, alcohols, and other molecules.17−21 The FAs have been drawing attention not only as membrane components, but also functional molecules in the membrane, resulting in pH-responsible membrane fusion.22 Conventionally, the evaluation of vesicle formation has been performed with electro-microscopic analysis,3 but the physicochemical properties, such as membrane fluidity and polarity, have not been systematically clarified. In the present study, FA assemblies modified with the cationic surfactant didecyldimethylammonium bromide (DDAB) were prepared for investigating the physicochemical properties of their vesicular membranes. The pH titration curve of each FA/DDAB system was determined, together with the variation of the turbidity of the solution.10 Based on the Henderson−Hasselbalch equation, the molar fraction of the protonated FA was calculated, and the phase states of the FA/DDAB systems in aqueous solutions were summarized in a trigonal diagram as a function of ionized FA/ protonated FA/DDAB. Based on the previous studies for the characterization of phospholipid vesicles,23 the membrane characteristics, i.e., membrane fluidity, polarity, and head group mobility, were evaluated. Focusing on the hydration of membranes, the effect of cationic surfactant on FA vesicles was discussed.
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state.24 Based on the Henderson−Hasselbalch equation,10 the molar fraction of the protonated FA (PD) was calculated as follows: [HA] + [A−] + [DDAB] = 1 pH = pK a + log 10 pH − pKa =
[A−] [HA]
1 − [DDAB] − [HA] [HA]
PD = [HA] =
1 − [DDAB] 1 + 10 pH − pKa
where [HA], [A−], and [DDAB] represent the molar fraction of protonated FA, ionized FA, and DDAB, respectively. Measurement of Membrane Fluidity. The fluidities in the interior and exterior of the FA/DDAB membrane were evaluated by measuring the fluorescence anisotropy of the DPH and TMA-DPH incorporated in the vesicles, respectively, using the fluorescence spectrophotometer FP-6500 (JASCO, Tokyo, Japan).23,25 A sample of 10 μL of 100 μM DPH or TMA-DPH in ethanol was added into 1 mL of 0.25 mM vesicle suspension. The samples were incubated at least 30 min in the dark. The samples were excited with vertically polarized light (360 nm), and emission intensities both perpendicular (I⊥) and parallel (I∥) to the excited light were recorded at 430 nm. The polarization (P) of DPH was then calculated by using the following equations:
P = (I − GI⊥)/(I + GI⊥)
G = i⊥/i where i⊥ and i∥ are emission intensities perpendicular and parallel to the horizontally polarized light, respectively, and G is the correction factor. The membrane fluidities were evaluated based on the reciprocal of polarization, 1/P. The membrane fluidities were measured at room temperature. Evaluation of the Polarity of the Membrane Surface Using Laurdan. The fluorescent probe Laurdan is sensitive to the polarity around itself, which allows the surface polarity of lipid membranes to be determined.25,26 Laurdan emission spectra exhibit a red shift caused by dielectric relaxation. Thus, emission spectra were calculated by measuring the general polarization (GP340) for each emission wavelength as follows:
MATERIALS AND METHODS
GP340 = (I440 − I490)/(I440 + I490)
Materials. Oleic acid (OA), linoleic acid (LA), and palmitoleic acid (PA) were purchased from Wako Pure Chemical Industries (Osaka, Japan). γ-Linoleic acid (γ-LA), 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-(4-trimethylamoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMADPH), and 6-lauroyl-2-dimethylamino naphthalene (Laurdan) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Didecyldimethylammonium bromide (DDAB) and other chemicals were from Tokyo Chemical Industry (Tokyo, Japan). Chemical structures of amphiphilic molecules are shown in the Supporting Information (Figure S1). These chemicals were used without further purification. Preparation of FA Assemblies. Fatty acids (FAs) were added to distilled water. After pH adjustment with 88 mM of NaOH aq (pH >11), the sample solutions were titrated by 100 mM of HCl, with stirring at room temperature. FA/DDAB suspensions were prepared based on the same method shown above. The total concentration of amphiphilic agents (FA + DDAB) was 80 mM. The size distribution analysis was performed with the Dynamic Light Scattering method by LB-500 (HORIBA, Kyoto, Japan). For the analysis of membrane properties (fluidity, polarity, and dielectric dispersion analysis), the FA/DDAB samples were prepared with a 50 mM bicine buffer at pH 8.5, where the sample solutions were once saponified with NaOH (88 mM) and titrated to pH 8.5 by adding HCl (100 mM). The prepared FA/DDAB samples were extruded through a 100 nm polycarbonate membrane filter (purchased from AVESTIN). The pKa values of OA and LA were defined as the pH where half of the FA was in the ionized
where I440 and I490 are the emission intensities of Laurdan excited with 340 nm light at room temperature. The fluorescent spectrum of each sample was normalized. The total concentrations of amphiphilic agent (FA + DDAB) and Laurdan in the test solution were 100 and 1 μM, respectively. Dielectric Dispersion Analysis. The sample solutions were diluted to 30 mM in distilled water. The dielectric permittivity (ε′) and dielectric loss (ε′′) were measured by a network analyzer (Agilent, PNA-X N5245A, 10 MHz to 50 GHz), according to the method described in the previous report.27 The dielectric spectra of the relative permittivity (ε′)/the dielectric loss (ε′′) for FA/DDAB vesicles were evaluated. The relaxation frequency ( f i: i = 1−3) and the amplitude of relaxation (εi: i = 1−3) were analyzed with third-type Debye’s equations. 3
ε′ − ε′h =
∑ i=1
3
ε ′′ =
∑ i=1
Δεi 1 + (f /fc 2 )2
Δεi(f /fci ) 1 + (f /fc 2 )2
where f and C0 represent the measuring frequency and cell constant, respectively. ε′h is the limit of the relative permittivity at higher frequency for the FA/DDAB suspension. Of the observed relaxations, 12722
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Figure 1. pH titration curves and variation of turbidity of FA systems. The FA suspensions in distilled water were obtained by adding NaOH (88 mM). The pH titration curves of OA (A) and LA (B) were obtained by adding HCl (100 mM) at room temperature. The turbidities of the sample solution (OD400) for OA (C) or LA (D) systems were measured by using UV−vis spectroscopy. For the pH titration and turbidity measurement, the total concentration of amphiphilic agent (FA + DDAB) was 80 mM. The captions (i), (ii), (iii), and (iv) indicate the inflection point in the titration curves or turbidity curves. The captions (Micelle, Vesicle, and OW) indicate the dominant assembly within the pH region described by arrows. The pH(i) and pH(iii) were determined on the basis of turbidity measurements. the third relaxation observed at around 25 GHz is assigned to the mobility of water, according to the previous reports.25,28 Langmuir Monolayer Analysis of FA. The π-A isotherms were measured with a KSV film balance (trough dimensions: width 150 mm, length 475 mm). The surface pressure was measured with use of a platinum plate as a pressure probe. Monolayers were prepared by spreading an aliquot (8 μL) of the mixture of FA/DDAB dissolved in chloroform (total concentration of amphiphilic agent (FA + DDAB): 3 mM) onto an aqueous subphase. Monolayers of the FA/DDAB mixture were spread over an initial surface of 273 cm2. Film compression was started 30 min after spreading, at the rate of 100 cm2/min. Experiments were repeated at least three times at room temperature.
determined by using the pH titration method and the turbidity analysis: Figure 1 for OA and LA; Figure S2, Supporting Information, for palmitoleic acid (PA) and γ-linoleic acid (γLA); Figures S3 and S4, Supporting Information, for OA/ DDAB; and Figures S5 and S6, Supporting Information, for LA/DDAB. As shown in Figure 1, the four inflection points were observed at different pH values as a function of the HCl amount, and were labeled as (i), (ii), (iii), and (iv). It has been reported that the inflection points in the pH titration curve indicate the phase transition pH of the FA assemblies.9 An increase of turbidity in the sample solution was observed at two distinct pH points: at the higher pH for the phase transition from micelle to vesicle, and the lower pH for the phase transition from vesicle to oil-in-water emulsion.10 These results suggest the existence of five pH regions in FA−water systems, which are identified as follows: micelle, pH > pH(i); vesicle− micelle coexistence, pH(i) < pH < pH(ii); vesicle, pH(ii) < pH < pH(iii); vesicle−oil-in-water (O/W) emulsion coexistence, pH(iii) < pH < pH(iv); and O/W emulsion, pH < pH(iv).3,9,11 The same tendencies were also observed in the case of other FAs systems: LA (Figure 1B,D); PA and γ-LA (Figure S2, Supporting Information); OA/DDAB (Figures S3 and S4, Supporting Information); LA/DDAB (Figures S5 and S6, Supporting Information); OA/2-decanoic acid = 2/1 (data not
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RESULTS AND DISCUSSION Preparation of FA Vesicles by the pH Titration Method. Ionized FAs are known to form micelle structures in NaOH solution at pH 12,3 where the FA (RCOOH) transforms into sodium salt (RCOO−−Na+). The structure of a sodium/FA salt is cone-like, and its hydrophilic head group occupies a large volume compared with its monoalkyl tail, resulting in micelle structures at higher pH regions. The FA vesicle solutions show higher turbidities than micelle solutions because FA vesicles are usually larger (>100 nm) than micelles (
