Thermodynamics and Structural Evolution during a Reversible Vesicle

Article pubs.acs.org/JAFC

Thermodynamics and Structural Evolution during a Reversible Vesicle−Micelle Transition of a Vitamin-Derived Bolaamphiphile Induced by Sodium Cholate Jun-Nan Tian, Bing-Qiang Ge, Yun-Feng Shen, Yu-Xuan He, and Zhong-Xiu Chen* College of Food and Biology Engineering, Zhejiang Gongshang University, Hangzhou, Zhejiang 310018, People’s Republic of China ABSTRACT: Interaction of endogenous sodium cholate (SC) with dietary amphiphiles would induce structural evolution of the self-assembled aggregates, which inevitably affects the hydrolysis of fat in the gut. Current work mainly focused on the interaction of bile salts with classical double-layered phospholipid vesicles. In this paper, the thermodynamics and structural evolution during the interaction of SC with novel unilamellar vesicles formed from vitamin-derived zwitterionic bolaamphiphile (DDO) were characterized. It was revealed that an increased temperature and the presence of NaCl resulted in narrowed micelle−vesicle coexistence and enlarged the vesicle region. The coexistence of micelles and vesicles mainly came from the interaction of monomeric SC with DDO vesicles, whereas micellar SC contributed to the total solubilization of DDO vesicles. This research may enrich the thermodynamic mechanism behind the structure transition of the microaggregates formed by amphiphiles in the gut. It will also contribute to the design of food formulation and drug delivery system. KEYWORDS: thermodynamics, self-assembly, sodium cholate, bolaamphiphile, vesicles

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INTRODUCTION

salts, polysaccharides, phosphatidylcholine, proteins, etc. Bile salts can themselves act as surfactants in the presence of biologically active molecules. Chauhan’s group has studied systematically the interaction of bile salts with amino acid and lecithin.7−10 It was found that the aggregation of bile salt molecules has been favored in the presence of amino acids by virtue of their adsorption in bile salt micelles.7 The early changes induced in the interfacial network will influence the subsequent effects of enzymes, phospholipids, and bile salts on the biochemical reactions. Thus, the interaction between the endogenous amphiphiles and the input emulsifiers may produce varied aggregates, which affects the catalytic hydrolysis of the lipids in small intestine. Moreover, dietary emulsifiers that disrupt mucus−bacterial interactions might have the potential to promote diseases associated with gut inflammation.11 SC is an important bile salt abundant in the human body, and its micellar characteristics with classical surfactants have been extensively studied. Aggregation characteristics of SC with classical surfactants, such as anionic sodium oleate,12 cationic alkyltrimethylammonium bromides,13 non-ionic polyethylene glycol tert-octylphenyl ether, 14 zwitterionic, and 3-(3cholamidopropyl)dimethylammonio-1-propanesulfonate, 15 have been reported. Besides the work that deals with the micelle behavior of the binary surfactant system, solubilization of vesicles by bile salts has attracted considerable attention because these systems can be used to simulate biological processes and contribute to a better understanding of absorption phenomena within the small intestine. To date, thermodynamics and structural evolution of vesicles induced by bile salts were obtained mainly from classical bilayers formed from phospho-

Understanding and manipulating fat digestion are becoming increasingly important as a result of economic consequences of obesity and health concerns. According to the classic studies of Hofmann and Borgstrom,1 dietary lipids distribute between two (or three) physical states in duodenal contents: an oily or emulsion portion, a dilute, aqueous mixed micellar phase, and frequently, a precipitated “pellet”. The endogenous surfactants, bile salts, are responsible for the formation of mixed micelles, which were considered the sole vehicles for solubilization and transport of lipolytic products from the emulsion surfaces to the enterocytes.2 Furthermore, solubilization of lipolytic products within bile salt micelles has been considered a rate-limiting step in absorption of lipids.3 An early study showed that unilamellar vesicles originated in lamellar liquid crystals that form at emulsion−water interfaces in the upper small intestine during lipid hydrolysis. These unilamellar vesicles represented the primary dispersed product phase of human fat digestion. The vesicles facilitated the dissolution of lipolytic products into unsaturated mixed micelles, which produced the most favorable thermodynamic condition for maximizing lipid absorption rates from the upper small intestine.4 A recent study by Brownian dynamics simulations demonstrated that the evolution of membrane pores induced by sodium cholate (SC) differs with the size of the vesicles, which resulted in different solubilization pathways of small and large vesicles.5 Our previous research also showed that autocatalytic formation of fatty acid from the hydrolysis of fatty acid anhydrides was dependent upon the spontaneously formed vesicles.6 Therefore, the structural transition between the formed assemblies, such as vesicles and micelles, in the gut is an important and fundamental step in understanding the digestion processes. During digestion, it is inevitable that bile salts would interact with other compounds, such as dietary emulsifiers, inorganic © 2016 American Chemical Society

Received: Revised: Accepted: Published: 1977

November 22, 2015 February 1, 2016 February 9, 2016 February 9, 2016 DOI: 10.1021/acs.jafc.5b05547 J. Agric. Food Chem. 2016, 64, 1977−1988

Article

Journal of Agricultural and Food Chemistry

Figure 1. Chemical structures of (a) DDO and (b) SC.

Figure 2. Observed enthalpy ΔHobs versus the concentration of SC in the cell for titration of different concentrations of SC to DDO at varied concentrations at 25 °C: (a and c) 40 mM SC and (b and d) 160 mM SC. Panels c and d show the ITC thermographs after subtraction of the dilution heat of SC at the same concentration.

lipids;16−22 only a few studies focused on the synthetic vesicle built by the mixture of anionic and cationic surfactants at certain conditions.23,24 We previously found that zwitterionic vitaminbased bolaamphiphiles derived from orotic acid, 1,12-diaminododecanediorotate (DDO),25 could form a stable monolayer vesicle spontaneously and guide the self-assembly of sodium caseinate.26 This bolaamphiphile represents a potential dietary surfactant and can serve as a typical monolayer vesicle. The details about how the bile salt interacts with a unilamellar synthetic vesicle and the structural evolution involved are the interests focused in this paper. We have proven that the real-time

heat release characterized by isothermal titration calorimetry (ITC) could be the indicators for the information on the structural changes of vesicles.27 In this paper, thermodynamics and the reversible vesicle−micelle transition based on DDO vesicles induced by SC were systematically studied by complementary methods. Because the preformed interfaces will be challenged by changes in the temperature, pH, and ionic strength during digestion, special attention was paid to investigate the mechanism behind the structural change of the aggregates under varied temperature and concentrations of salts. DDO is a vitamin-derived bolaamphiphile with a unique 1978

DOI: 10.1021/acs.jafc.5b05547 J. Agric. Food Chem. 2016, 64, 1977−1988


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RESULTS AND DISCUSSION Thermodynamics and Vesicle−Micelle Evolution of DDO Induced by SC at 25 °C. The molecular structures of

structure, which could be a potential dietary surfactant and represents an essential stage in the transformation of closed monolayer vesicles into solubilized micellar aggregates. Therefore, the thermodynamics of structural evolution of aggregates formed from endogenous SC and DDO could provide more information about the microstructure reconstitution for lipid solubilization as well as the synergetic self-assembly between amphiphiles in the gut. It also provides useful information for the design of food formulation and drug delivery system.

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Article

MATERIALS AND METHODS

Materials. SC (≥98%) was purchased from Aladdin. Pyrene (≥98%) was purchased from Sigma. All materials were used without further purifications. Synthesis of DDO was described in detail previously reported.25 Double-distilled water was used throughout the study. Sample Preparation. As a result of relatively poor solubility of DDO, stock solution of DDO was first prepared at the desired concentration and then was used to make mixtures with SC at the desired molar ratio. All samples were thermostated at needed temperatures for at least 24 h to reach equilibrium. Size and ζ-Potential Measurements. Average size and ζ potential of vesicles at different SC/DDO ratios with or without NaCl were measured by dynamic light scattering (DLS) using a Zetasizer Nano ZS instrument (Malvern Instruments, Ltd.). The instrument uses a laser at a wavelength of 632.8 nm and detects the scattered light at an angle of 173°. ζ potential was measured on the basis of laser Doppler electrophoresis. Solvents were filtered through 0.22 μm Millipore filters before preparing the sample solution. All measurements were performed in a temperature-controlled chamber. The experimental duration (equilibration time) was in the range of 2 min, and each test was repeated 3 or more times. Isothermal Titration Calorimetry (ITC). An isothermal titration calorimeter (VP-ITC, Microcal, Inc., Northampton, MA) was used to measure enthalpies involved in SC/DDO interactions. SC in the syringe was of 40 or 160 mM. In a typical experiment, DDO solution was placed in the 1430 μL sample cell of the calorimeter and bile salt solutions were loaded into the injection syringe. All solutions were degassed prior to use. The reference cell was filled with water. An initial injection of 1 μL was made, and then 5 or 10 μL aliquots were injected into the sample cell. The duration of each injection was 20 s, and there was an interval of 350 s to achieve complete equilibration. The solution in the titration cell was stirred at a speed of 307 revolutions min−1 throughout the experiment. Control experiments included the titration of detergent into water and water into water. Experiments were performed at five different temperatures: 25, 30.5, 37, 40, 55, and 75 °C. The results of the ITC experiments were presented in terms of the observed enthalpy change per injection (ΔHobs) or the binding enthalpy change per injection (ΔHbinding) as a function of the surfactant concentration. All experiments were repeated twice to achieve the reproducibility within ±2%. Transmission Electron Microscopy (TEM). A JEM-1230 transmission electron microscope operating at a voltage of 120 kV was used for TEM measurements. Uranium acetate (1%, w/v) was used as the staining agent. A drop of the sample solution (10 μL) was placed onto a carbon Formvarcoated copper grid (200 mesh). Filter paper was employed to absorb the excess liquid. Each experiment was repeated 2 or more times. The process of adsorption was equilibrated in a thermostatic bath at the predetermined temperature. Steady-State Fluorescence Spectral Measurements. Fluorescence measurements were performed on a Hitachi F-7000 fluorescence spectrophotometer in aqueous solutions. Pyrene (0.5 μM) was dissolved in mixed systems of bile salt and vesicle solution. Fluorescence emission spectra were recorded, employing an excitation wavelength of 335 nm with the exciting and emitting bandwidths of 5.0 and 2.5 nm, respectively, and the intensities I1 and I3 were measured at the wavelengths corresponding to the first and third vibronic peaks in the fluorescence emission spectrum of pyrene, respectively.

Figure 3. Size and TEM images of the SC/DDO system at different concentrations of SC at 25 °C (CDDO = 1 mM) obtained by DLS and TEM.

both DDO and SC are shown in Figure 1. Clarifying the dynamic solubilization of liposome requires real-time continuous measurements of the size, shape, and composition of the selfassemblies presented in surfactant mixtures. Figure 2 shows the ITC plots for titration of different concentrations of SC in DDO at varied concentrations. Our previous research showed that the critical vesicle concentration (cvc) of DDO is about 0.28 mM.25 Herein, we carried out the ITC measurements at the concentration range of DDO from 0.1 to 1.5 mM to ensure that both monomeric and vesicular DDO could be monitored. The concentration of SC in the syringe was already above its critical micelle concentration (cmc); thus, the syringe contained mainly SC micelles. Two initial concentrations of SC were selected: one was 40 mM, and the other was 160 mM. The concentration of SC in the syringe was chosen in such a way that, with an increasing concentration of SC in the sample cell, the cmc of SC was reached (160 mM) or not (40 mM) during the titration. Control experiments were performed by titrating SC into water at the same conditions. All of the ITC curves of the investigated surfactants are shown by the observed enthalpy (ΔHobs) against the final concentration of SC in the cell. In Figure 2a, ITC curves for titration of 40 mM SC into DDO solutions indicate that disassembling SC micelles releases heat at 25 °C. When SC micelles were titrated into DDO at either below or above the cvc, the obtained non-sigmoidal shapes of ITC curves go in a similar way and result in exothermic heat flow. An abrupt change in the heat of micellization indicates that the 1979


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Figure 4. (a) Observed enthalpy ΔHobs versus the final SC concentration in the cell for the titration of SC (160 mM) into DDO (1 mM) at different temperatures. The corresponding open symbols represent the dilution of SC (160 mM) into water at each temperature. (b) Binding enthalpy between SC (160 mM) and DDO (1 mM), which was obtained by subtracting the dilution heat of SC at the same conditions. (c) Phase diagram and typical TEM images of the SC−DDO system at different temperatures. Blue, gray, and red areas correspond to the vesicle phase, vesicle and micelle coexistence, and mixed micelle phase, respectively. The dot lines indicate phase boundaries.

of the mixture. With the increased addition of SC to the cell, SC monomers might incorporate into the vesicle layers when the concentration of DDO was above its cvc. The endothermic peak of the ITC plots in Figure 2c together with the whole exothermic heat release (Figure 2a) means that the interaction between SC and DDO monomers releases heat. From the comparison of the results of Figure 2, we can conclude that incorporation of monomeric SC into DDO vesicle solution is enthalpically favorable, whereas the incorporation of micellar SC into vesicles is an endothermic process. We observed a similar result when another anionic surfactant sodium dodecyl sulfate (SDS) was titrated into DDO.27 The transition of mixed vesicles of diethylaminobenzaldehyde (DEAB)/SDS to micelles induced by SC also led to exothermic enthalpies.23 However, ITC displayed an endothermic peak in the case of the cationic surfactant cetyltrimethylammonium bromide (CTAB).27 In Figure 2b, two points of inflection are found, which suggests the structural change of DDO vesicles. The first appears at around 0.8 mM SC, suggesting the onset of vesicle solubilization, and the second is found clearly at about 10 mM SC, indicating

association of unimers into DDO vesicles occurred at very low concentrations. However, when 160 mM SC was titrated into DDO solutions (Figure 2b), the calorimetric curves display a typical sigmoid shape, which is not found for 40 mM SC. The characteristic heat flow can essentially be ascribed to three sequential processes: demicellization of SC upon dilution, SC− DDO interactions, and surfactant-induced structural changes of DDO. We have proven the characteristic changes in ITC plot signaling interaction events with possible structural changes during the interaction between surfactants and vesicles.27 Panels c and d of Figure 2 show the ITC thermograph after subtraction the dilution heat of the SC at the same concentration. When the concentration of SC in the cell lies below its cmc, all added micelles are demicellized into monomers and the monomers are further diluted. When it is above cmc, only the micellar solution is diluted and ΔHobs drops toward zero. Figure 2 reveals that the heat release from the interaction of SC with DDO at a lower concentration and the dilution heat of the SC remains almost the same. This indicates that, at the initial titration, demicellization of SC contributes to the whole enthalpy 1980


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Figure 5. Effect of the temperature on the vesiculization of DDO vesicles (CDDO = 1 mM) upon the addition of an increased concentration of SC: (a) ITC and (b) ζ potential.

were also found in CTAB/DDO vesicles27 and in the solubilization of liposome with bile salts.16 The DLS plot is dominated by a single peak until the concentration of SC reaches 1 mM. When a higher amount of SC molecules was incorporated into DDO vesicles, a coexistence region with two kinds of particle sizes was observed: one is hundreds of nanometers, and the other is just few tens of nanometers. When the concentration of SC was above its cmc, DDO were saturated with SC micelles. As a consequence, the vesicles started to break up, changing their morphology, and fragment into mixed micelles, which are observed in DLS peaks. The cmc of SC measured by ITC is about 11.4 mM in water at 25 °C, which is similar to the reported results.12 It can be seen from Figure 3 that the vesicles could survive before the concentration of SC reaches its cmc (CSC ≤ 9.0 mM). After that, DDO vesicles could not be found in TEM images, which implies that a considerable number of vesicles had transformed into mixed micelles. This is in agreement with the second inflection point in ITC curves, which corresponds to the breakup of the vesicles to micelles (Figure 2; CSC/CDDO = 9.2:1). The transition from liposomes17,21 and DEAB/SDS catanionic vesicles23 to micelles was also found to be a smooth transformation involving a region where micelles and vesicles coexisted. Huang’ group23,24 has reported the vesicle−micelle transition of catanionic surfactant systems induced by bile salts. It was found that steric interaction between the bile salt and catanionic surfactant plays an important role in catanionic surfactant systems. In the bile salt−amino acid interaction, the dominance of electrostatic interactions at low bile salt concentrations was found, which shifted to the dominance of hydrophobic interactions at higher concentrations.9 Herein, the zwitterionic DDO bears both negative and positive charges, which is comparable to the vesicles formed from the catanionic surfactant in terms of charges bearing. The characteristics of the DDO structure are its hydrophilic head groups, which cap the ends of a saturated, C12 linear hydrocarbon hydrophobe. The end groups with a planar structure make it form a vesicle spontaneously. Our earlier work has shown that, when anionic SDS was mixed with DDO, flocculent occurred before the vesicle−micelle transition, whereas the cationic CTAB could solubilize the vesicles into micelles.27 This excludes possible contribution of electronic repulsion between the negative hydrophilic groups. Therefore,

Figure 6. Observed enthalpy ΔHobs versus the total concentration of SC in the cell for the titration of SC (160 mM) into DDO (1 mM) in the presence of varied concentrations of NaCl at 25 °C. The solid symbols indicate the titration curves, and the corresponding open symbols represent the dilution of SC (160 mM) in brines at 25 °C.

significant structural changes of DDO vesicles. The CMC of SC in the presence of DDO is reasonably less than that in water (about 11.4 mM) as a result of the synergism effect. The absence of the second inflection point in panels b and d of Figure 2 implies that significant structural changes of DDO vesicles happened only when added SC reached to its cmc. The combination of calorimetric and structural data may provide a unique possibility to make both molecular and quantitative interpretations of the processes involved in SC− DDO interactions. We measured the hydrodynamic diameter distribution of the SC/DDO system (CDDO = 1 mM) with an increased concentration of SC by DLS to find the structural changes implied by the inflections on the ITC curves. The results are shown in Figure 3. Only one hydrodynamic diameter distribution at about 410 nm is found, which is for individual DDO assembly. In the presence of very low amounts of SC, enlarged vesicles were formed as a result of the incorporation of SC into the layer of the DDO membrane. The enlarged vesicles 1981


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Figure 7. Phase diagrams of the SC/DDO system in (a) 0.1 M, (b) 0.4 M, (c) 0.6 M, and (d) 0.8 M NaCl solution. Blue, gray, and red areas correspond to the vesicle phase, vesicle and micelle coexistence, and mixed micelle phase, respectively. The dot lines indicate phase boundaries.

micelles to unimers (demicellization). Because the transfer enthalpy of a surfactant from water to a micelle is positive at a low temperature and negative at a high temperature, an increase in the temperature favors the endothermic reverse reaction. As shown in Figure 4a, at 25 °C, the interaction of SC unimers with DDO vesicles is exothermic and dilution of the SC micelle solution releases more heat. At 30.5 °C, the titration calorimetric curves present approximately the same enthalpy in both cases. However, when the temperature increased to or over 37 °C, the observed enthalpy of both the interaction between SC and DDO and corresponding dilution process becomes positive. The higher the temperature is, the more heat adsorbed. With the increase of the temperature, an increase of the cmc of SC is observed, which is evidenced by the inflection point. In comparison to the dilution of SC, the interaction of SC and DDO is less endothermic, shown by the dilution line located above the titration line on ITC plots. The binding enthalpy ΔHbinding is thus equal to the difference between the total measured enthalpy change and the enthalpy change for SC dilution in water at the same conditions. The obtained results are

the planar structure of the end groups of DDO makes it interact rather though hydrophobic interaction. The facial amphiphilic structure of SC and the steric interaction with DDO may promote the vesicle−micelle transition. Temperature Dependence of the Reversible Vesicle− Micelle Transition of DDO Induced by SC. The above results showed the aggregation information on the dynamic disassembly of DDO vesicles in the presence of SC at 25 °C. To obtain a better understanding of the temperature dependence of the interaction of SC with DDO, we extended the ambient titration to higher temperatures, including 37 °C. Special attention was addressed to the structure evolution of DDO induced by SC. The concentration of DDO was set at 1 mM. The phase diagrams of SC/DDO mixtures were obtained from a number of solubilization and reconstitution experiments performed at different temperatures. The ITC results and the phase diagrams are summarized in Figure 4. It can be seen that the demicellization enthalpy at temperatures below 30.5 °C is exothermic and then becomes endothermic as the temperature rises. The observed enthalpy corresponds to the breakup of the 1982


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Figure 8. TEM images of aggregates formed in SC/DDO mixtures (CDDO = 1 mM) in 0.4 M NaCl solutions: (a) CSC = 0 mM and 25 °C, (b) CSC = 2 mM and 25 °C, (c) CSC = 3.5 mM and 25 °C, (d) CSC = 6 mM and 25 °C, (e) CSC = 3.5 mM and 55 °C, and (f) CSC = 6 mM and 75 °C.

implying the increased difficulty for totally destroyed DDO vesicles. The partitioning of SC unimers into DDO layers was also delayed. This is confirmed by Figure 4c, in which the coexistence region disappears at 40−70 °C. To find the detailed information about the thermodynamic mechanism behind the structural evolution of DDO vesicles, we investigated the effect of the temperature on the vesiculization of DDO using ITC and the change of the ζ potential of DDO vesicles upon the addition of an increased concentration of SC. The results are shown in Figure 5. Figure 5a depicts that dilution of vesicle solution is exothermic at the covered temperatures, which implies that the formation of DDO vesicles is an endothermic process. This is different from the demicellization of SC solution at higher temperatures, where endothermic curves were observed and lots of heat is needed for the disassociation of SC micelles (curves with open symbols in Figure 4a). It is well-known that the entropic contribution, which dominates the hydrophobic effect at room temperature, is reduced at higher temperatures. The association of classical ionic and non-ionic surfactants is becoming more and more enthalpydriven at elevated temperatures.29 Micellization of bile salts is entropy-controlled at low temperatures and enthalpy-controlled at high temperatures in the presence of amino acids.10 Herein, the vesiculization of DDO is totally different, in which it adsorbs heat at a high temperature during association. The interaction of

illustrated in Figure 4b. Incorporation of SC into DDO vesicles changed from endothermic to exothermic with the increase of the temperature, and the binding enthalpy became more negative. Increasing the temperature causes the structure of water to break down. As a result, the positive effect related to destruction of structured water surrounding alkyl chains is dominated by the negative effect connected with the condensation of hydrophobic chains inside the micelle and the interaction enthalpy becomes more exothermic upon increasing temperature. The phase transition obtained by detailed examinations of structure evolution of DDO vesicles is clearly shown in Figure 4c. The solubilization of DDO vesicles by SC in the ITC cell consisted of consecutive steps, such as demicellization of the SC, followed by the interaction of SC with DDO and the breaking of the DDO vesicles. At 25−37 °C, three parts of the ITC isotherms can be assigned clearly according to the “three-stage” model of surfactant-induced vesicle solubilization proposed by Helenius and Simons.28 As mentioned above, the beginning inflection point on the ITC plot in Figure 2b (1 mM DDO) is supposed to signal the onset of the solubilization process. The curve before the emergence of the maximum peak indicates the coexistence of vesicles and micelles. The last break point corresponds well to the solubilization of vesicles. It can be seen from Figure 4a that the maximum peak appears later at higher temperatures, 1983


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Figure 9. Observed enthalpy ΔHobs versus the total SC concentration in the cell for the titration of SC (160 mM) into 1 mM DDO in (a and c) 0.1 M NaCl and (b and d) 0.8 M NaCl solutions at different temperatures. The corresponding open symbols represent the dilution of SC (160 mM) in brines at each temperature.

Figure 10. Change of I1/I3 in the fluorescence spectra of pyrene dissolved in (a) 1 mM DDO solution with varying concentrations of SC at 25 °C and (b) SC/DDO mixture as a function of the temperature (CSC = 15 mM).

anionic surfactant SC. However, the ζ potential of DDO vesicles at higher temperatures was less negative than that at an ambient temperature, which indicates a weaker interaction between SC and DDO induced by the elevated temperature. We postulate that the coexistence of micelles and vesicles depends upon the

SC with DDO is exothermic (Figure 4b), which means that an elevated temperature is not helpful for the binding. This is confirmed by the results of the change of the ζ potential of DDO upon the addition of SC, shown in Figure 5b. It is reasonable for DDO vesicles to bear more negative charges when binding with 1984


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Journal of Agricultural and Food Chemistry Table 1. cmc, ΔHdemic, and ΔCpdemic of SC in Water and Aqueous NaCl Solutions at Varied Temperaturesa 0.0 M NaCl

0.1 M NaCl

T (°C)

cmc (mM)

ΔHdemic (kJ mol−1)

ΔCpdemic (kJ mol−1 K −1)

25 30.5 37 40 55 75

11.14 12.66 16.27 16.75 17.27 23.04

17.19 23.71 19.31 4.78 7.37 14.52

1.19 0.25 −2.76 −2.34 0.27 0.36

a

cmc (mM) 7.53 7.85 10.35 11.34 12.87 15.25

0.8 M NaCl



cmc (mM)



5.59 19.20 29.18 15.85 7.57 8.55

2.47 2.01 −1.45 −2.50 −0.25 0.049

4.33 4.52 6.14 6.27 6.91 7.84

5.77 21.06 21.63 5.49 5.73 6.36

2.78 1.43 −2.65 −2.68 0.024 0.031

cmc values were estimated by ITC, and ΔHdemic and ΔCpdemic values were calculated from the van’t Hoff equation.

Table 2. cmc, ΔHdemic, and ΔCpdemic of the SC/DDO (1 mM) Mixture in Water and Aqueous NaCl Solutions at Varied Temperaturesa 0.0 M NaCl

0.1 M NaCl

0.8 M NaCl

T (°C)

cmc (mM)



cmc (mM)



cmc (mM)



25 30.5 37 40 55 75

7.04 8.33 8.49 8.66 8.70 11.13

9.21 18.38 16.04 2.11 6.43 13.91

−1.77 −1.58 −0.86 −0.07 0.26 0.34

6.91 7.40 9.32 9.45 9.53 12.56

9.21 18.38 16.04 2.11 6.43 13.91

1.67 0.65 −2.50 −2.18 0.33 0.37

4.36 6.15 6.13 6.19 6.97 8.60

46.22 23.78 1.10 4.55 8.25 10.59

−4.08 −3.79 −1.17 0.70 0.18 0.12

a

cmc values were estimated by ITC, and ΔHdemic and ΔCpdemic values were calculated from the van’t Hoff equation.

critical for the total solubilization of DDO vesicles. Because the aggregation of anionic SC is strongly dependent upon the ionic strength, we speculate that the total solubilization of DDO vesicles will be enhanced because NaCl favors the formation of SC micelles. Figure 6 is the ITC thermograph titration of SC (160 mM) into DDO (1 mM) in the presence of different concentrations of NaCl at 25 °C. The solid curves shown in Figure 6 indicate the observed enthalpy versus SC concentration in the cell during the titration. The curves with open symbols are the titration curves of SC into NaCl solution at the same condition, which gives the dilution heat of SC in different concentrations of NaCl. It can be seen that the heat in both the interaction of SC and DDO and the dilution of SC changes from exothermic to endothermic. In addition, the enthalpy values for the interaction become more positive. Moreover, the cmc values of SC under all investigated conditions strongly depend upon the concentration of the added salt. Evidently, the cmc decreases when the concentrations of salt increase. It is well-known that an increasing ionic strength reduces the electrostatic repulsion of negatively charged molecules, favoring the aggregation process and the stabilization of the micelles. We speculate that the saturation and solubilization of vesicles are likely to occur at lower concentrations of SC compared to that in aqueous solution. Therefore, it is reasonable to deduce that total solubilization of DDO vesicles must be easier in the presence of NaCl. ITC curves in Figure 6 illustrate that heat flow gradually reaches the maximum earlier with the increased concentration of NaCl. To visualize the structural change of the vesicles solubilized by SC with the help of NaCl at different temperatures, phase diagrams are shown in Figure 7. At 25 °C, in the presence of 0.1 M NaCl, the coexistence region appears when the concentration of SC is at 1.5 M (Figure 7a), whereas the same region occurs when the concentration of SC is about 1.0 M in the absence of NaCl (Figure 4c). An increased concentration of NaCl at 0.4 and

interaction of SC unimers with DDO vesicles. Only when the concentration of SC is high enough for considerable SC micelles to interrupt the membrane layer of DDO are the vesicles then broken up into mixed micelles. An increased cmc, along with the weak interaction between the SC and DDO vesicles at higher temperatures, could be responsible for the delay of the inflection points that appeared on the ITC curves and the changes on the phase diagram. On the other hand, the narrowed or disappeared coexistence region is mainly compensated by the vesicle region but not by the mixed micelle region, suggesting that DDO vesicles are more stable when the temperature increases. We speculate that heating might afford a repairing effect, which could be attributed to the endothermic process of the formation of DDO vesicles. To test our hypothesis, the broken DDO vesicles that were solubilized by SC at ambient temperature were subjected to heating. When the temperature was above 70 °C, the peak assigned to the mixed micelles disappeared in the DLS plot, leaving the peak of vesicles. The repaired vesicles would return to mixed micelles if they were cooled, exhibiting the characteristic repairing effect and reversible transition. This heating-induced micelle−vesicle transition was also observed in the vesicle solution containing the cationic surfactant and SC.24 An increase in the cmc of SC promotes the heating-induced micelle−vesicle transition. For the SC/DDO mixture, the increased temperature will make SC move from the aggregates to water, partially “‘undoing’” the original SC-induced vesicle−micelle transition at room temperature and contributing to the observed heatinginduced reversible transition. A similar reversible micellar− vesicular/lamellar transition were also found in dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC)/SC systems: upon heating, mixed micelles would turn into vesicles or lamellar structures; on the contrary, the solubilization of membranes occurred upon cooling.17 Vesicle−Micelle Transition of DDO Induced by SC in NaCl Solution. The above results show that SC micelles are 1985


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changed the phase boundaries but affected the steady-state size of cholate-containing PC vesicles.18 These results are not in agreement with the SC/DDO systems, in which the presence of NaCl narrowed the width of the vesicle and micelle coexistence region. Investigation of the Change of Micropolarity and Hydrophobic Area during the Structural Evolution of DDO Vesicles Induced by SC. Because structural evolution between vesicles and micelles may be accompanied by the changes of micropolarity of the membranes, we continued our research to characterize the micropolarity of aggregates using pyrene as a fluorescence probe. Figure 10a shows the change of I1/I3 when the surfactant at varying concentrations was added into 1 mM DDO in the presence of NaCl. A decrease of the I1/I3 ratio was observed when an increased concentration of SC was introduced into the DDO solution. This suggests that, in the binary system, pyrene resides in a more hydrophobic environment. In the initial stage of the solubilization, the insertion of the surfactant into the vesicle led to more dense layers with less micropolarity as a result of the strengthened hydrophobic interaction. On the other hand, large vesicles that turned into small micelles would provide more nonpolar areas. Both results contributed to the decrease of I1/I3. In comparison to SC solution alone, the interaction of SC and DDO provided a more hydrophobic place for accommodation of pyrene. This was expressed in Figure 9a, where the curve for SC is on the top of the group. The increased concentration of NaCl enhanced the solubilization process, resulting in a gradually decreased ratio of I1/I3, which implies the formation of a new aggregate with less micropolarity. The minimum value of the I1/ I3 ratio was obtained when DDO vesicles totally turned into micelles at a high concentration of salt. Figure 10b shows that, upon heating, the value of I1/I3 increases, indicating that fluorescein was reversibly capsulated into the vesicles. Because the value of I1/I3 of pyrene decreased with the elevated temperature in water,27 it is reasonable to deduce that increased I1/I3 comes from the structural transition between the assemblies. In comparison to vesicles, micelles with a smaller size provide more hydrophobic areas for pyrene; therefore, the increased value of I1/I3 suggests that some of the micelles might turn back into vesicles, which is consistent with the repairing effect contributed by heating in the reversible micelle−vesicle transition. The knowledge of demicellization enthalpy as a function of the temperature makes it possible to calculate the value of isobaric heat capacity of demicellization. The positive ΔCpdemic suggests that hydrophobic molecules are transferred from a nonpolar medium to water, and the absolute value of ΔCpdemic is a linear function of the hydrophobic surface area that becomes exposed to water during this process.31,32 Table 1 lists the cmc, enthalpies of demicellization (ΔHdemic), and heat capacity change (ΔCpdemic) of pure SC in water and aqueous NaCl solutions at varied temperatures. The ΔCpdemic was calculated from the van’t Hoff equation. It can be seen that the cmc decreases with the increased concentration of NaCl. An increased temperature results in a larger value of cmc. Table 2 shows the cmc, ΔHdemic, and ΔCpdemic for SC mixed with DDO in water and aqueous NaCl solutions at varied temperatures. In comparison to pure SC solution, the SC/ DDO mixture displayed a smaller cmc at the same conditions. The change of the cmc along with the concentration of NaCl and temperature can be explained by the classical physiochemical

0.6 M results in a delayed appearance of the coexistence region, which is found when the concentration of SC was 2.5 mM (Figure 7b) and 4.5 mM (Figure 7c), respectively. In other words, the starting points of the coexistence region are shifted to a higher SC concentration with an increased concentration of NaCl. However, the mixed micelle region, which signals the total solubilization of DDO vesicles, appears earlier with the increased addition of NaCl. This region starts at 10, 5.5, 4.5, 4.2, and 3 mM SC when the corresponding concentration of NaCl is at 0, 0.1, 0.4, 0.6, and 0.8 M, respectively. The results about the structural evolution shown in Figure 7 support our previous hypothesis that the coexistence of micelles and vesicles mainly come from the interaction of SC unimers with DDO vesicles and SC micelles contribute more to the solubilization of DDO vesicles than to mixed micelles. The presence of NaCl promotes the micellization of amphiphilic SC, thus delaying the formation of SC unimers. A higher ionic strength causes a better shielding of the polar head groups of the bile salt molecules and, thus, shifts the phase boundaries to lower detergent concentrations. Therefore, an increased addition of NaCl results in the delay until the disappearance of the coexistence region. Typical TEM images of SC/DDO mixtures (CDDO = 1 mM) in 0.4 M NaCl solution are shown in Figure 8. At an ambient temperature (25 °C), the aggregates are mainly spherical vesicles when CSC is 2 mM (Figure 8b). As CSC increases (3.5 mM), vesicles (Figure 8c) become smaller and their edges are irregular, implying that solubilization of DDO vesicles occurred. As CSC reaches 6 mM, the spherical vesicles totally disappear and worm-like mixed micelles are observed (Figure 8d). When the temperature increases to 55 or 75 °C, the images of vesicles are observed again at CSC = 3.5 or 6 mM (panels e and f of Figure 8). These results confirm that the vesicles could be totally solubilized at a lower SC concentration in the presence of NaCl and a higher temperature induced a reversible micelle−vesicle transition. It is noteworthy that most of the shrunken coexistence region was compensated by the enlargement of the vesicle region when the temperature increased. This result can be easily explained taking into consideration the previously discussed repairing effect of heating. To further test our hypothesis, heat flow of titrations of SC (160 mM) into 1 mM DDO in the presence of 0.1 and 0.8 M NaCl at different temperatures was monitored by ITC. The results are shown in Figure 9. Panels a and b of Figure 9 depict that the titration heat reaches a maximum earlier at higher temperatures in the presence of NaCl, which implies an enhanced solubilization of DDO vesicles by SC micelles. Panels c and d of Figure 9 show that binding of SC to DDO vesicles is enthalpically favorable at the elevated temperature. A higher concentration of NaCl results in less heat release during the binding process. The broken DDO vesicles that were solubilized by SC in the presence of NaCl were also subjected to heating. It is noteworthy that most of the vesicle−micelle transitions were reversible, except when the concentration of NaCl was above 0.6 M and that of SC was more than 10 mM. This may be attributed to the enhanced solubilization of DDO vesicles by easily formed SC micelles in the presence of salts, which offset the repairing effect of heating. The effect of NaCl on the vesicle−micelle transition by SC has been extensively investigated. Walter et al.30 reported that NaCl could enhance the stability of the octylglucoside-saturated bilayer and lipid (egg phosphatidylcholine)-saturated mixed micellar structures, delay the saturation of the bilayer and the formation of mixed micelles, and increase the width of the coexistence region. The ionic strength slightly 1986


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(3) Thomson, A. B. R.; Keelan, M.; Garg, M. L.; Clandinin, M. T. Intestinal aspects of lipid absorption: in review. Can. J. Physiol. Pharmacol. 1989, 67, 179−191. (4) Hernell, O.; Staggers, J. E.; Carey, M. C. Physical-chemical behavior of dietary and biliary lipids during intestinal digestion and absorption. 2. Phase analysis and aggregation states of luminal lipids during duodenal fat digestion in healthy adult human beings. Biochemistry 1990, 29, 2041−2056. (5) Haustein, M.; Wahab, M.; Mögel, H. J.; Schiller, P. Vesicle Solubilization by bile salts: comparison of macroscopic theory and simulation. Langmuir 2015, 31, 4078−4086. (6) Cao, C.; Wang, Q. B.; Tang, L. J.; Ge, B. Q.; Chen, Z. X.; Deng, S. P. Chain-length-dependent autocatalytic hydrolysis of fatty acid anhydrides in polyethylene glycol. J. Phys. Chem. B 2014, 118, 3461−3468. (7) Kumar, K.; Chauhan, S. Surface tension and UV−visible investigations of aggregation and adsorption behavior of NaC and NaDC in water−amino acid mixtures. Fluid Phase Equilib. 2015, 394, 165−174. (8) Chauhan, S.; Kumar, K.; Chauhan, M. S.; Rana, D. S.; Umar, A. Acoustical and volumetric studies of proline in ethanolic solutions of lecithin at different temperatures. Adv. Sci., Eng. Med. 2013, 5, 991−997. (9) Kumar, K.; Chauhan, S. Volumetric, compressibility and viscometric studies on sodium cholate/sodium deoxycholate−amino acid interactions in aqueous medium. Thermochim. Acta 2015, 606, 12− 24. (10) Kumar, K.; Patial, B. S.; Chauhan, S. Conductivity and fluorescence studies on the micellization properties of sodium cholate and sodium deoxycholate in aqueous medium at different temperatures: effect of selected amino acids. J. Chem. Thermodyn. 2015, 82, 25−33. (11) Chassaing, B.; Koren, O.; Goodrich, J. K.; Poole, A. C.; Srinivasan, S.; Ley, R. E.; Gewirtz, A. T. Dietary emulsifiers impact the mouse gut microbiota promoting colitis and metabolic syndrome. Nature 2015, 519, 92−96. (12) Hildebrand, A.; Garidel, P.; Neubert, R.; Blume, A. Thermodynamics of demicellization of mixed micelles composed of sodium oleate and bile salts. Langmuir 2004, 20, 320−328. (13) Manna, K.; Chang, C. H.; Panda, A. K. Physicochemical studies on the catanionics of alkyltrimethylammonium bromides and bile salts in aqueous media. Colloids Surf., A 2012, 415, 10−21. (14) Patel, V.; Bharatiya, B.; Ray, D.; Aswal, V. K.; Bahadur, P. Investigations on microstructural changes in pH responsive mixed micelles of Triton X-100 and bile salt. J. Colloid Interface Sci. 2015, 441, 106−112. (15) Naskar, B.; Mondal, S.; Moulik, S. P. Amphiphilic activities of anionic sodium cholate (NaC), zwitterionic 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and their mixtures: A comparative study. Colloids Surf., B 2013, 112, 155−164. (16) Walter, A.; Vinson, P. K.; Kaplun, A.; Talmon, Y. Intermediate structures in the cholate-phosphatidylcholine vesicle-micelle transition. Biophys. J. 1991, 60, 1315−1325. (17) Polozova, A. I.; Dubachev, G. E.; Simonova, T. N.; Barsukov, L. I. Temperature-induced micellar-lamellar transformation in binary mixtures of saturated phosphatidylcholines with sodium cholate. FEBS Lett. 1995, 358, 17−22. (18) Meyuhas, D.; Bor, A.; Pinchuk, I.; Kaplun, A.; Talmon, Y.; Kozlov, M. M.; Lichtenberg, D. Effect of ionic strength on the self-assembly in mixtures of phosphatidylcholine and sodium cholate. J. Colloid Interface Sci. 1997, 188, 351−362. (19) Lesieur, P.; Kiselev, M. A.; Barsukov, L. I.; Lombardo, D. Temperature-induced micelle to vesicle transition: kinetic effects in the DMPC/NaC system. J. Appl. Crystallogr. 2000, 33, 623−627. (20) Ollila, F.; Slotte, J. P. A thermodynamic study of bile salt interactions with phosphatidylcholine and sphingomyelin unilamellar vesicles. Langmuir 2001, 17, 2835−2840. (21) Hildebrand, A.; Neubert, R.; Garidel, P.; Blume, A. Bile salt induced solubilization of synthetic phosphatidylcholine vesicles studied by isothermal titration calorimetry. Langmuir 2002, 18, 2836−2847. (22) Coreta-Gomes, F. M.; Martins, P. T.; Velázquez-Campoy, A.; Vaz, W. L.; Geraldes, C. F.; Moreno, M. J. Interaction of bile salts with model

theory of surfactant. It is noteworthy that the isobaric heat capacity ΔCpdemic is found negative for most SC/DDO systems at a lower temperature, which implies that a hydrophobic interaction was involved in most cases. At a higher temperature, ΔCpdemic becomes positive, suggesting that some molecules were transferred from a nonpolar hydrophobic area to water. These results are in agreement with the change of micropolarity, shown in Figure 10. In summary, a vitamin-derived bolaamphiphilic DDO was used to form unilamellar vesicles and complementary methods were applied to characterize the thermodynamics and structural evolution of the vesicles induced by SC. It was found that, with increasing the temperature, the observed enthalpy change of the interaction of SC with DDO changed from exothermic to endothermic. However, incorporation of SC into DDO vesicles changed from endothermic to exothermic with the increase of the temperature, suggesting that the tendency of SC molecules to interact with DDO at the elevated temperature is enthalpically favorable. Three regions for vesicles, micelle−vesicle coexistence, and mixed micelles were observed on the phase diagram during the interaction of SC with DDO, which were also displayed by the break points on the ITC thermographs. An increased temperature and the presence of NaCl resulted in a narrowed or disappeared micelle−vesicle coexistence region accompanied by the enlarged vesicular region. A detailed investigation of the mechanism behind the structural evolution of DDO vesicles by the measurement of the ζ potential and micropolarity revealed that the coexistence of micelles and vesicles mainly comes from the interaction of monomeric SC with DDO vesicles, whereas micellar SC contributes more to the total solubilization of DDO vesicles to mixed micelles. The current research provides a detailed investigation of thermodynamics and structural revolution of the controllable self-assembly of novel zwitterionic unilamellar vesicles in the presence of SC. This research may also stimulate new effective routes to understand the determinants of the microstructure and macroscopic phase transition during the interaction of endogenous bile salts with dietary amphiphiles and enrich the thermodynamic mechanism behind solubilization of membranes and digestion of lipids.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Funding

The authors are grateful to the Zhejiang Provincial Top Key Discipline of Food Science and Biotechnology for financial support (JYTSP20141012). This work was partly supported by the Natural Science Foundation of Zhejiang Province (LY13C200001). Notes

The authors declare no competing financial interest.

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REFERENCES

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Thermodynamics and Structural Evolution during a Reversible Vesicle

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