Anionic Mixed Surfactant Aggregates with

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Interactions of Cationic/Anionic Mixed Surfactant Aggregates with Phospholipid Vesicles and Their Skin Penetration Ability Yao Chen, Fulin Qiao, Yaxun Fan, Yuchun Han, and Yilin Wang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b04093 • Publication Date (Web): 25 Dec 2016 Downloaded from http://pubs.acs.org on January 3, 2017

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Interactions of Cationic/Anionic Mixed Surfactant Aggregates with Phospholipid Vesicles and Their Skin Penetration Ability Yao Chen,†,‡ Fulin Qiao,†,‡ Yaxun Fan,† Yuchun Han,† and Yilin Wang*,†,‡ †

Key Laboratory of Colloid and Interface Science, Beijing National Laboratory for Molecular Sciences

(BNLMS), Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China ‡

University of Chinese Academy of Sciences, Beijing 100049, P. R. China

ABSTRCT: This work studied the interactions of oppositely charged surfactant mixture of oleyl bis(2hydroxyethyl) methyl ammonium bromide (OHAB) and sodium dodecyl sulfate (SDS) with 1,2-di-(9Zoctadecenoyl)-sn-glycero-3-phosphocholine (DOPC) vesicles as well as the penetration of the OHAB/SDS mixture through model skin, aimed at understanding the relationship between the ability of different surfactant aggregates in solubilizing phospholipid vesicles and their potential in irritating skin. By changing the molar fraction of OHAB (XOHAB), five kinds of aggregates are constructed: OHAB and SDS separately form cationic and anionic small micelles, while the OHAB/SDS mixtures form cationic and anionic vesicles at XOHAB = 0.30 and 0.70, respectively, and weakly charged vesicles at XOHAB = 0.50. The mixtures have much lower critical micellar concentrations (CMC) and much larger aggregates than either OHAB or SDS alone, and the CMC and the size of the OHAB/SDS vesicles decrease with the increase of XOHAB. The phase diagrams indicate that the OHAB/SDS mixtures show much stronger ability in solubilizing DOPC vesicles than individual OHAB and SDS, and decreases in the order of XOHAB = 0.30 > XOHAB = 0.50 > XOHAB = 0.70 >> XOHAB = 1.00 > XOHAB = 0. However, the ability of the surfactants in penetrating model skin decreases reversely, and the penetration of the surfactants are significantly reduced by mixing. These results indicate that the surfactant mixture with a larger

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aggregate size and a smaller CMC value displays much stronger ability in solubilizing DOPC vesicles, but much weaker ability in skin penetration.

INTRODUCTION Skin irritation elicited by surfactants is a serious problem in using detergent, soap and cosmetics.1-5 Human skin is mainly composed of two layers, the epidermis which serves as a barrier to inflection, and the dermis which provides the appendages of skin with a location place. As the outermost layer of skin, stratum corneum of mammalian epidermis consists of keratin-filled dead corneocytes and extracellular multiple crystalline lamellar lipid.6 The structure of stratum corneum shows that the keratin-filled dead corneocyte is surrounded by extracellular lipid.6-8 Therefore, for surfactant-induced skin irritation, the interaction of skin lipid with surfactants should be considered first. Researchers found that the interactions of surfactant molecules with the extracellular lamellar lipids lead many symptoms to skin, such as itching, dryness, scaling and inflammatory changes.9, 10 Charaf et al.4 developed an in vitro indicator to evaluate the skin irritation of surfactants through measuring the leakage of a fluorescent marker from phospholipid vesicles. It has been proved that the skin irritation potential of a surfactant correlates to its ability of solubilizing skin lipid.4, 9 Solubilization of lipid vesicles by surfactants experiences phase transitions from lipid vesicles to lipid/surfactant mixed vesicles and then to mixed micelles.11-14 Lichtenberg et al.12, 15 postulated a threestage model to characterize the phase transitions. In this model, the phase transition of surfactant/phospholipid mixture is determined by the effective surfactant to phospholipid molar ratio ( Re ) required for the transition. Below a certain surfactant to phospholipid ratio ( Rsat e ), surfactant molecules only incorporate into the phospholipid bilayers. Above Rsat e the phospholipid bilayers start to be disintegrated, and form phospholipid-saturated mixed micelles with surfactant molecules. While


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further increasing the surfactant to phospholipid molar ratio to Rsol e , all the lipid vesicles are disintegrated leaving only mixed micelles. This model has been widely used to characterize the solubilization of phospholipid vesicles by surfactants.11, 16-20 Generally speaking, the amount of surfactant that phospholipid vesicles can accommodate depends on the properties of phospholipid molecules, such as the nature of surfactants and the interactions between surfactants and phospholipid molecules. Among these factors the nature of the surfactants has been proved to be very important.21, 22 Goni et al.23 explored the effects of surfactant charged headgroup and hydrophobic chain length on the ability of solubilizing lipid vesicles. Phosphatidylcholine liposomes were solubilized by nine ionic surfactants, including dodecyl, tetradecyl and hexadecyl trimethylammonium bromide (DoTAB, TeTAB and HeTAB); decyl, dodecyl and tetradecyl pyridinium bromide (DePB, DoPB and TePB), and sodium decyl, dodecyl and tetradecyl sulphate (NaDeS, NaDoS and NaTeS). It was found that the Rsat e values for DoTAB, TeTAB and HeTAB decrease with increasing hydrophobic chain length, while the Rsol e values for TeTAB, TePB and NaTeS are almost the same though they have different charge properties. These results suggest that hydrophobic interaction is probably the main driving force for the formation of mixed surfactant/phospholipid micelles, whereas the headgroup charge does not play a significant role. Edwards et al.24, 25 found that in the solubilization of lecithin vesicles by nonionic surfactants of C12En series, the Rsat e values for the incorporation of the nonionic surfactant molecules into lipid bilayer is dependent on the hydrophobic interaction between the hydrophobic chains of surfactants and phospholipid, while the total solubilization of lipid bilayer (Rsol e ) is mainly controlled by the electrostatic repulsion between their headgroups. Above all, these studies were focused on the interactions of single component surfactants with lipid bilayers. Surfactant mixtures with strong synergistic intermolecular interactions are more widely applied, and thus the interactions of surfactant mixtures with lipid vesicles deserve to be well investigated.


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It has been reported that strong positive synergistic interactions exist in zwitterionic/anionic surfactant mixtures. The mixture of N-tetradecyl-N, N-dimethylbetaine (C14-Bet) and sodium dodecyl sulfate (SDS) shows the weakest ability to solubilize to phosphatidylcholine vesicles when the surfactant mixture has the lowest critical micellar concentration (CMC) at the mixing molar ratio of about 1:1.26 Similar results were obtained by Tanaka et al.27 in a mixture of SDS with zwitterionic surfactant dodecylamido propyl dimethyl aminoacetate (AD). The electrostatic repulsive force between surfactant headgroups is remarkably reduced by mixing SDS with AD, and their strong synergistic interaction leads to the lowest solubilizing ability to lipid vesicles at the AD molar fraction of 0.70. Therefore, it was concluded that the positive synergism between the surfactants weakens the lipid solubilizing ability of surfactant mixtures. Though the solubilization of lipid vesicles by surfactant mixtures has been studied, the interaction of cationic/anionic surfactant mixtures with phospholipids has not yet been well understood. Due to the strong synergism between cationic and anionic surfactants and the resulting unique functions, the interactions of cationic/anionic surfactant mixtures with phospholipids are not only important but also complicated. Benefiting from the synergism, the mixtures exhibit significantly enhanced self-assembly properties and abundant aggregation behaviors,28, 29 and thus the interactions of these different surfactant aggregates with lipid vesicles may display special situations. Understanding the interactions of cationic/anionic surfactant mixtures with lipid vesicles may provide some guidance on how to establish mild and efficient surfactant formulations. In this work, the aggregates formed by cationic surfactant oleyl bis(2-hydroxyethyl)methyl ammonium bromide (OHAB) and anionic surfactant SDS are used to solubilize 1,2-di-(9Zoctadecenoyl)-sn-glycero-3-phosphocholine (DOPC) vesicles. The molecular structures used are shown in Scheme 1. The mixtures in aqueous solutions exhibit extremely low CMC values compared with


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individual SDS or OHAB solution. Five kinds of aggregates with different charges, sizes and morphologies are obtained by changing the molar fraction of OHAB, i.e., negatively charged micelles and vesicles, positively charged micelles and vesicles, and nearly charge-neutralized vesicles. The solubilization of DOPC vesicles by these aggregates are studied by isothermal titration microcalorimetry (ITC), dynamic light scattering (DLS), turbidity and cryogenic transmission electron microscopy (CryoTEM), indicating that the OHAB/SDS mixtures need much lower surfactant to phospholipid molar ratio to solubilize the DOPC vesicles than pure SDS or OHAB solution. Furthermore, skin penetration experiments with a synthetic membrane similar to human skin show that the OHAB/SDS aggregates display much lower skin penetration ability than the OHAB or SDS micelles. Therefore the surfactant mixtures show an abnormal inverse relationship between the lipid solubilizing ability and skin penetration ability.

Scheme 1. Chemical structures of DOPC, OHAB and SDS.

EXPERIMENTAL SECTION Materials. Cationic surfactant oleyl bis(2-hydroxyethyl)methyl ammonium bromide (OHAB) was synthesized according to literatures.30-32 Sodium dodecyl sulfate (SDS) was purchased from Aldrich with purity higher than 99%. Phospholipid 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) was purchased from Sigma and used without further purification. Phosphate buffered saline (PBS) tablets


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were purchased from sigma. One PBS tablet was dissolved in 200 mL Milli-Q water to yield a solution of 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride with pH 7.4 at 25 °C. The PBS buffer was used in all the experiments. Synthetic membrane (Strat-M) was purchased from Merck KGaA. Preparation of DOPC Vesicles. DOPC powder was dissolved in chloroform, and solvent was dried under vacuum in a rotary evaporator to obtain a thin film at the bottom of the flask. The dried film was suspended in a certain volume of buffer to a desired concentration and vortexed at 25 °C for several minutes. The crude phospholipid suspension was sonicated for about 15 min by a cell sonifier at power 500 W alternating 7 s bursts and 3 s rest periods. The prepared vesicle dispersions were freshly used. Surface Tension Measurements. The surface tensions of OHAB/SDS mixtures for different XOHAB values in buffer were measured with a DCAT 11 tensiometer (Dataphy Instruments Co., Ltd.) by the Wilhelmy plate technique. All the measurements were taken at 25.00 ± 0.01 °C until successive values agreed to within 0.1 mN/m. All the surface tension curves were repeated three times or more. Dynamic Light Scattering (DLS). DLS was employed to measure the size of the OHAB/SDS aggregates at different XOHAB values and the size variation of phospholipid vesicles induced by the OHAB/SDS aggregates. Measurements were carried out at 25.00 ± 0.05 °C by using an LLS spectrometer (ALV/SP-125) with a multi-τ digital time correlater (ALV-5000). A solid-state He-Ne laser (22 mW at λ = 632.8 nm) was used as the incident beam. The scattering angle was 90° in all the measurements. All the newly prepared solutions were filtered through a 0.45 µm membrane filter of hydrophilic PVDF before the measurements. The correlation function was analyzed from the scattering data via the CONTIN method to obtain the distribution of diffusion coefficients (D) of the solutes, and then, the apparent hydrodynamic radius Rh could be calculated from the Stokes-Einstein equation Rh =


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kT/(6πηD), where k is the Boltzmann constant, T is the absolute temperature, and η is the solvent viscosity. Turbidity Measurements. Turbidity measurements were performed to determine the critical conditions for the aggregate transitions of the DOPC vesicle dispersion with the addition of OHAB, SDS or the OHAB/SDS mixtures. The experiments were carried out with a Brinkman PC920 probe colorimeter thermostated at 25.0 ± 0.1 °C, and the results are reported as 100-%T. ζ-Potential Measurements. ζ potential measurements of the OHAB/SDS mixtures in buffer were carried out at a scattering angle of 173° on a Malvern Zetasizer Nano-ZS instrument equipped with a thermostatted chamber and a 4 mW He−Ne laser (λ = 632.8 nm). The temperature was controlled at 25.0 ± 0.1 °C. Isothermal Titration Microcalorimetry (ITC). A TAM III microcalorimetric system was used to study the enthalpy changes in the micellization of the OHAB/SDS mixture and the solubilization of the DOPC vesicle dispersion by this mixture. While studying the micellization, the sample cell and the reference cell of the microcalorimeter were initially loaded with 600 µL buffer and 765 µL buffer, respectively. Concentrated OHAB/SDS mixture at different OHAB molar fractions (XOHAB) was injected consecutively into the stirred sample cell in each portion of 5 µL using a 500 µL Hamilton syringe controlled by a Thermometric 612 Lund pump until the desired concentration range was covered. While studying the solubilization process of DOPC, the sample cell and the reference cell of the microcalorimeter were initially loaded with the DOPC vesicle dispersion (600 µL) and the buffer (775 µL), respectively. The system was stirred at 60 rpm with a gold propeller, and the interval between two injections was long enough for the signal to return to baseline. The observed enthalpy change (∆Hobs) was obtained by integrating the areas of the peaks in the plot of thermal power against time. The


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reproducibility of experiments was within 4%. All the measurements were performed at 25.00 ± 0.01 °C. Cryogenic Transmission Electron Microscopy (Cryo-TEM). Cryo-TEM was used to characterize the morphologies of OHAB/SDS aggregates and the aggregate transitions of the DOPC vesicles induced by OHAB, SDS and the OHAB/SDS mixture. The samples were embedded in a thin layer of vitreous ice on freshly carbon-coated holey TEM grids by blotting the grids with filter paper, and then they were plunged into liquid ethane cooled by liquid nitrogen. Frozen hydrated specimens were imaged by using an FEI Tecnai 20 electron microscope (LaB6) operated at 200 kV with the low dose mode (about 2000 e/nm2) and the nominal magnification of 50 000. The defocus was set to 1-2µm for each specimen area. Images were recorded on Kodak SO163 films and then digitized by Nikon 9000 with a scanning step 2000 dpi corresponding to 2.54Å/pixel. Skin Penetration Experiments. Synthetic membrane (Strat-M) was used as a model membrane of skin to study the skin penetration of OHAB/SDS mixture. The skin penetration was performed using a vertical modified amber glass Franz diffusion cell (Tianjin Pharmacopoeia, Tianjin, China) with an effective diffusion area of 2.25 cm2 and a receptor volume of 16.50 mL. The synthetic membrane was sandwiched between the donor and receiver compartments, and then the two parts were fastened by a stainless steel clip. The receptor was filled with buffer and continuously stirred at 500 rpm at 25.0 ± 0.1 °C for 1 h to equilibrate the synthetic skin. After the equilibration, 2.0 mL solution of the surfactant or surfactant mixture at CT = 10 mM was added in the donor. The solution of 1 mL in the receiver compartment was withdrawn by fixed intervals and analyzed by UV-Vis spectra and total organic carbon analysis (TOC) to obtain the amount of surfactants through the model skin. To keep the volume of solution in the receptor constant, 1 mL buffer was injected into the receptor medium after each


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sampling. UV-Vis adsorption spectra were recorded in quart cuvettes (path length 1 cm) by a SHIMADZU UV 1601PC spectrometer. All the measurements were conducted at 25.0 ± 0.5 °C. The cumulative amount of surfactant permeation per unit of skin surface area (Qt/S) was calculated from equation 1: t-1 Qt = Vr Ct + ∑i=0 Vs Ci

(1)

where Ct and Ci are the surfactant concentrations of the receiver solution at each sampling time and for the ith sample, respectively, and Vr and Vs are the volumes of the receiver solution and the sample. The steady state fluxes (Jss) of surfactant permeation was calculated from the slope of the linear part of the graph where the cumulative surfactant permeation per unit of skin surface area (Qt/S) was plotted against time (t), Jss = ∆Qt ⁄∆t×S

(2)

Apparent permeability coefficients (Kp) were calculated according to the following equation, Kp = Jss ⁄Co

(3)

where Co is the surfactant concentration in the donor.

RESULTS AND DISCUSSION Aggregation Behavior of OHAB/SDS Mixtures. In previous work,33 the basic physical chemical properties and aggregation behaviors of the OHAB/SDS mixture in aqueous solution have been studied. However, herein the PBS buffer at pH 7.4 was used instead of water in order to study the interaction of the aggregates with lipid, so the aggregation behaviors of the mixtures in buffer were studied. Figure 1a shows the surface tension curves of the mixture in buffer as a function of total surfactant concentration (CT) at different molar fractions of OHAB (XOHAB). The surface tension values decrease rapidly and keep constant beyond an inflection point, which corresponds to the CMC. The derived CMC values are


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summarized in Table 1. As shown, the CMCs of SDS and OHAB in buffer are 0.78 and 0.015 mM, respectively, and the CMCs at XOHAB = 0.30, 0.50 and 0.70 are 0.0017, 0.0013 and 0.00085 mM, respectively. All the CMC values for the OHAB/SDS mixture in buffer are much lower than those in aqueous solution, because the salts in buffer can effectively reduce the electrostatic repulsion among the surfactant headgroups. Moreover, the CMCs of the OHAB/SDS mixtures are much lower than that of pure SDS solution or pure OHAB solution. It means that the mixture of oppositely charged surfactants has very strong synergistic interaction in reducing CMC. Figure 1b, 1c, and 1d present the size distribution, ζ-potential, and morphology of the OHAB/SDS aggregates beyond the CMC. As shown in Figure 1b, before mixing, the hydrodynamic radius (Rh) of the aggregates for SDS (XOHAB = 0) and OHAB (XOHAB = 1.00) are ~ 3.5 and ~ 4.0 nm, respectively, and the corresponding ζ-potential are -15 mV and 6 mV. The Cryo-TEM results in Figure 1d shows that the aggregates of either SDS or OHAB are small globular micelles of around 4 nm. After mixing, the mixed aggregates at XOHAB = 0.30, 0.50 and 0.70 show that the Rh values are ~ 300, ~ 135 and ~ 88 nm, respectively, and the corresponding zeta-potentials are -42, -10, and 25 mV. The Cryo-TEM images prove that the aggregates are large unilamellar or multilamellar vesicles. In brief, the above results indicate that five kinds of aggregates with different sizes, surface charges and morphologies are obtained by changing the XOHAB values. The specific characteristics of these aggregates are summarized in Table 1. Next, how these aggregates interact with lipid vesicles will be studied.


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Figure 1. (a) Surface tension curves of the OHAB/SDS mixtures plotted against total surfactant concentration CT at different XOHAB values. (b) Size distributions, (c) ζ-potentials, and (d) Cryo-TEM images of 2.0 mM OHAB/SDS mixtures in buffer at different XOHAB values.


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Table 1. The CMC, size and ζ potential values of the OHAB/SDS mixtures at different XOHAB values. The effective surfactant to lipid ratio for the onset (Resat) and completion (Resol) of solubilization of DOPC vesicles. Dwsat and Dwsol are the equilibrium surfactant monomer concentrations at the phase boundaries for the surfactant/DOPC system. XOHAB

CMC (mM)

Morphology

Rh (nm)

ζ-potential (mV)

Resat

Resol

Dwsat(mM)

Dwsol (mM)

0

0.78

Micelle

3.5

-15

1.22

1.76

0.99

2.36

0.30

0.0017

Vesicle

300

-42

0.21

0.37

0.01

0.04

0.50

0.0013

Vesicle

135

-10

0.25

0.46

0.02

0.06

0.70

0.00086

Vesicle

88

25

0.31

0.59

0.04

0.09

1.00

0.015

Micelle

4.0

6.0

0.80

2.61

0.07

1.73

Solubilization of DOPC Vesicles. A typical phase diagram14 for the solubilization of lipid vesicles by surfactants is shown in Figure 2. The two solid lines depict the required surfactant concentrations for the onset (Dtsat) and the completion (Dtsol) of the solubilization process. The Dtsat and Dtsol values are plotted against lipid concentration (L) and the two phase boundaries separate the phase diagram into three parts, namely, mixed vesicles, vesicle/micelle coexistence region and mixed micelles. The slopes of the boundaries correspond to the surfactant to lipid ratios for the onset (Resat) and the end (Resol) of solubilization. Surfactant monomers (Dw) coexist with the mixed aggregates in all the regions of the phase diagram. The relationship between these parameters is shown in the following equations, Dtsat = Dwsat + ResatL

(4)

Dtsol = Dwsol + ResolL

(5)

Dt

(Dt) Surfactant concentration

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Mixed micelles sol

Dt sol

Re sol

Dw

Coexistence

sat

sat

Re sat

Dw

0

Dt

Mixed vesicles

(L) Lipid Concentration

L

Figure 2. Schematic phase diagram of a lipid-surfactant mixture in solution.


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Isothermal titration microcalorimetry (ITC) is a powerful method to detect the solubilization of lipid vesicles by surfactants17, 18, 34, 35 and can be used to determine the lipid-surfactant phase boundaries, because the phase transitions of lipids from vesicles to micelles are always accompanied by the release or consumption of heat. In the present work, in order to obtain the lipid-surfactant phase boundaries, ITC is used to monitor the process of titrating concentrated SDS, OHAB and the OHAB/SDS mixture (CT = 40 mM) into the DOPC vesicles of different concentrations, which reflects the solubilization of the DOPC vesicles by the surfactant mixtures. Figure 3 shows the ITC curves and the curves can be divided into three groups according to their shapes and varying tendency. Group 1 only includes the curves of SDS (XOHAB = 0), group 2 includes the curves of the OHAB/SDS mixtures (XOHAB = 0.30, 0.50 and 0.70), and group 3 are the curves of OHAB (XOHAB = 1.00). In order to understand the phase transitions of DOPC vesicles reflected in the ITC curves so as to know how the critical concentrations for the phase transitions are determined from the ITC curves, three typical ITC curves from the three groups are further studied as representatives. Figure 4 presents the curves of the ∆Hobs against the total surfactant concentration CT for separately titrating 40 mM pure SDS solution (XOHAB = 0), pure OHAB solution (XOHAB = 1.00) or OHAB/SDS mixture at XOHAB = 0.70 into 2.0 mM DOPC vesicle dispersion, as well as the turbidity and scattering intensity curves in the corresponding processes. The Cryo-TEM images of the aggregates in the different stages of these curves are shown in Figure 5. These results indicate that all the interaction processes between the surfactants and DOPC clearly display three regions (I, II and III) as labeled in Figure 4.


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3 XOHAB = 0

0 -3

CDOPC

-6

0 mM 1 mM 2 mM 3 mM 4 mM 5 mM

-9 -12 2 0 -2 -4

XOHAB = 0.30

-6 -8 0.5 0.0

∆ Hobs (kJ/mol)

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-0.5 -1.0

XOHAB = 0.50

-1.5 -2.0 -2.5 1 0 -1 XOHAB = 0.70

-2 -3 -4 2 1 0 -1

XOHAB = 1.00

-2 -3 0

3

6

9

12

15

18

CT (mM)

Figure 3. The observed enthalpy changes ∆Hobs against the total surfactant concentration CT by titrating concentrated OHAB/SDS mixtures of different XOHAB values into the solution of the DOPC vesicles of different concentrations (CDOPC). The DOPC concentration in the sample cell increases from 1.0 mM to 5.0 mM. The total surfactant concentration titrated is fixed at 40 mM, and the volume of each injection is adjusted to guarantee the best signal-to-noise ratio for different DOPC concentrations.


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Dt

sol

Dt

c

-2 -4 III

II

I

-6 90

∆ Hobs (kJ/mol)

ITC

sol

Dt

ITC

-2

1 0

-3

I

II

III

0 ITC

I

II

III

-1

0

Turbidity

Turbidity

60

40

40 120

Size

100

150

40

0 2

4

6

CT (mM)

8

81 78 75

Size

90 60

100 90

0 0

15

110

30

20

12

Turbidity

Size

Intensity

Intensity

60

9

72 120

120

80

6

84

100-%T

100-%T

50

3

87

80

60

-2

-4

100

70

-1

-3

-4

80

100-%T

sat

Dt

ITC

2

0

∆ Hobs (kJ/mol)

sat

Dt

b

∆ Hobs (kJ/mol)

sol

sat

Dt

a

Intensity

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80

0

2

4

6

8

10 12 14 16

CT (mM)

0

1

2

3

4

CT (mM)

Figure 4. The observed enthalpy changes ∆Hobs, turbidity and scattering intensity versus the total surfactant concentration CT for titrating (a) SDS, (b) OHAB and (c) OHAB/SDS mixture at XOHAB = 0.70 into 2 mM DOPC vesicle dispersion.


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Figure 5. The Cryo-TEM micrographs of the samples locating at different surfactant concentration regions in Figure 4: (A) Pure DOPC vesicles (2 mM), (B) DOPC/SDS (region I), (C) DOPC/SDS (region II), (D) DOPC/SDS (region III), (E) DOPC/OHAB (region I), (F) DOPC/OHAB (region II), (G) DOPC/OHAB (region III), (H) DOPC/OHAB/SDS (region I), (I) DOPC/OHAB/SDS (region II), DOPC/OHAB/SDS (region III).

For SDS (Figure 4a), when the SDS concentration locates in region I, the initial additions of SDS lead to a large exothermic value and sharp decrease in ∆Hobs. The large exothermic ∆Hobs value suggests that the negatively charged dodecyl sulfate ion (DS-) electrostatically bind to the quaternary ammonium headgroups of DOPC, and the decrease of ∆Hobs means that the DOPC vesicle is gradually saturated by DS-. When the DOPC vesicle is electrostatically saturated by DS-, further added SDS molecules insert


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into the vesicle bilayers through hydrophobic interaction. The two effects may basically cancel each other out, and thus ∆Hobs levels off at the end of region I. In addition, both the turbidity and the scattering intensity show a slight decrease with the initial addition of SDS in this region. The Cryo-TEM image (Figure 5B) proves that, in this region, only the outer layers of the multilamellar DOPC vesicles are disintegrated by the low concentration of SDS, and the size of the DOPC/SDS vesicles gets smaller than that of the DOPC vesicles (Figure 5A). Similar phenomenon has also been observed in other surfactant systems.17, 24, 25 When the SDS concentration falls in region II, the exothermic ∆Hobs value sharply decreases first and then keeps constant. Meanwhile, the turbidity and scattering intensity also decrease significantly. The Cryo-TEM image (Figure 5C) shows that, in this region, the amount of vesicles decreases markedly and rod-like micelles are formed, which means that the solubilization occurs and the rod-like micelles should be the aggregates of the surfactants with DOPC. Therefore, the sudden change of ∆Hobs at the beginning of region II is defined as the onset of DOPC solubilization (Dtsat), while the constant ∆Hobs value and the continuous decrease of the turbidity and scattering intensity later in this region suggest that the mixed DOPC/SDS bilayers are in equilibrium with the DOPC/SDS rod-like micelles and the bilayer phase gradually transfers into micellar phase. When the SDS concentration is in region III, the exothermic ∆Hobs value starts to further decrease at first and becomes very close to zero in final. Meanwhile both the turbidity and scattering intensity continue to decrease, but the decreasing magnitude becomes smaller than that in region II, and finally reaches a lower constant value. The Cryo-TEM image (Figure 5D) presents small globular mixed micelles in this region. These results imply that the beginning of region III corresponds to the end of DOPC solubilization (Dtsol), where DOPC vesicles have been completely disintegrated, and further added SDS micelles only mix with the existing mixed DOPC/SDS micelles.


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For OHAB (Figure 4b), when the OHAB concentration is in region I, the ∆Hobs keeps constant and small exothermic values for the first few injections of OHAB, which is quite different from the result of SDS. This means that the electrostatic interaction of cationic surfactant OHAB with DOPC vesicles is much weaker than that of anionic surfactant SDS with DOPC vesicle because of the less favorable charge match between OHAB and DOPC. However the OHAB molecules prefer to insert into the DOPC bilayers due to their much longer hydrophobic tails compared with SDS. The offset of these two effects results in the platform in the ∆Hobs curve. Moreover, in this region, both the turbidity and scattering intensity increase with the addition of OHAB, and the Cryo-TEM image (Figure 5E) shows that large unilamellar vesicles are formed and the size is larger than pure DOPC vesicles. Thus the OHAB molecules insert into the DOPC vesicles and enlarge the vesicles, similar to the situations in literatures,36, 37

but do not induce phase transition. Further increasing the OHAB concentration to region II, the ∆Hobs

value sharply changes from significant exothermic to endothermic at first, and then the endothermic value decreases slightly. Correspondingly, the turbidity and scattering intensity of the vesicle dispersion decrease significantly. The Cryo-TEM image (Figure 5F) shows that the number of vesicles changes less and rod-like micelles are formed. Combining these results, the sudden change of ∆Hobs should be the beginning (Dtsat) of the solubilization process of the DOPC vesicles. With the increase of OHAB concentration in region III, the ∆Hobs value increases sharply to a large endothermic value firstly, and then decays to zero. Meanwhile, the turbidity and scattering intensity decrease at a slower rate. The Cryo-TEM image (Figure 5G) shows that all the vesicles and rod-like micelles have transferred into wormlike micelles. Therefore, in region III, all the DOPC vesicles have been disintegrated into rod-like micelles at Dtsol, the further added OHAB molecules induce the micellar growth from rod-like into wormlike, and finally all the micelles transfer into wormlike micelles. The wormlike micelle solution


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exhibits a substantial viscosity by shaking. The formation of wormlike micelles can be attributed to stronger hydrophobic interaction between OHAB and DOPC than that between SDS and DOPC. As to the OHAB/SDS mixture at XOHAB = 0.70 (Figure 4c), when the total surfactant concentration is in region I, ∆Hobs displays a large exothermic value, and the turbidity and scattering intensity increase sharply with the increase of the surfactant concentration. The Cryo-TEM image (Figure 5H) shows large DOPC vesicles in this region. Therefore, the solubilization of DOPC vesicles does not begin in this region. The increase of turbidity and scattering intensity should be resulted from the incorporation of the OHAB/SDS molecules into the DOPC lipid bilayer and the resultant growth of the vesicles. When the surfactant concentration increases to region II, a large endothermic peak is found in the ITC curve, meanwhile, the turbidity and scattering intensity start to decrease significantly at first and then reaches lower and nearly constant values. The Cryo-TEM image (Figure 5I) proves that small vesicles are formed in this region. The beginning and end of the endothermic peak correlate well with the transition points in the turbidity and the scattering intensity curves, which can be defined as the onset (Dtsat) and end (Dtsol) of DOPC solubilization. When the surfactant concentration is in region III, the ∆Hobs value gradually decays to zero with the increasing of surfactant concentration, while the turbidity and scattering intensity of the mixture increase continuously, and the Cryo-TEM image (Figure 5J) shows larger and multiple-layer vesicles. Unlike transparent pure SDS or OHAB micellar solution, the OHAB/SDS mixture forms vesicles by itself. Therefore, the DOPC vesicles are solubilized by the OHAB/SDS vesicles and they form mixed vesicles finally. The increase of turbidity and scattering intensity in the later region should be resulted from the accumulation of the turbid OHAB/SDS vesicles. In brief, the above results illustrate that the critical surfactant concentrations for the solubilization of the DOPC vesicles by SDS, OHAB and OHAB/SDS mixture can be obtained by the combination of ITC,


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turbidity, scattering intensity and Cryo-TEM. According to the method, the phase diagrams for the solubilization of DOPC vesicles are obtained and presented in Figure 6, which will be discussed below. Phase Boundaries for the Solubilization of DOPC Vesicles by OHAB/SDS Mixtures. Figure 6 shows the phase boundaries for the phase transitions of the DOPC vesicles induced by the OHAB/SDS mixtures with different molar fractions of OHAB. The Dtsat and Dtsol values are plotted against the DOPC concentration, and two straight lines are separately obtained for Dtsat and Dtsol in each plot. The slopes of the two lines are the effective surfactant to lipid molar ratio for the onset (Resat) and completion (Resol) of the DOPC solubilization. The intercept values of the line (Dwsat and Dwsol) are the equilibrium surfactant monomer concentrations at the onset and end of solubilization. All these parameters are listed in Table 1. The Resat and Resol values represent the ability of a surfactant in solubilizing lipid vesicles, i.e., the lower the Re values of a surfactant, the stronger its ability to solubilize lipid vesicles. As shown in Table 1, both the Resat and Resol values for the OHAB/SDS mixtures at XOHAB = 0.30, 0.50 and 0.70 are much lower than those for pure OHAB (XOHAB = 1.0) and pure SDS (XOHAB = 0) solutions. This is quite different from the results observed in many reports,26, 27, 38, 39 where the Resat and Resol values for surfactant mixtures are much larger than single component surfactants. Moreover, herein the Resat and Resol values for the OHAB/SDS mixtures increase with the increase of the OHAB fraction, i.e., in the order of XOHAB = 0.30 < XOHAB = 0.50 < XOHAB = 0.70. Specially, the Resat value of pure OHAB solution is smaller than that of pure SDS solution, while the Resol value of pure OHAB solution is larger than that of pure SDS solution. On one hand, the OHAB/SDS mixtures have much lower CMCs than either OHAB or SDS solution, which suggests strong hydrophobic interaction and strong electrostatic bind between OHAB and SDS molecules. Therefore, the mixture may interact with the DOPC vesicle in the form of OHAB/SDS pair. The OHAB/SDS pair has much larger headgroup and more hydrophobic tails, thus, it can easily incorporate into the DOPC vesicle bilayers and disintegrate the vesicles. On the


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other hand, as mentioned above, the Resat and Resol values for the mixtures slightly increase with the increase of XOHAB. Although the CMC values also slightly decrease with the increase of XOHAB, this may not be the main reason for the variation of the Re values. As shown in Figure 1c, the ζ-potentials of the OHAB/SDS mixtures increase from -42 mV to -12 mV and then 15 mV with the increase of XOHAB. According to the molecular structure of DOPC, the outer layer of the DOPC vesicle is positively charged. So the electrostatic interaction of the OHAB/SDS mixture with the DOPC vesicle decreases in the order of XOHAB = 0.30 > XOHAB = 0.50 > XOHAB = 0.70. Thus, the interaction of the mixture at XOHAB = 0.30 with DOPC vesicle should be the strongest, which finally leads to the lowest Resat and Resol values. As to pure SDS and OHAB, OHAB has much longer hydrophobic tails than SDS. The hydrophobic interaction between OHAB and DOPC is much stronger than that between SDS and DOPC. As a consequence, the Resat value of OHAB is much lower than that of SDS. However, the Resol value of OHAB is larger than that of SDS. This can be explained by the model proposed by Fattal et al.,40 namely, the surfactant to lipid ratio required for completely solubilizing lipid vesicles decreases with the increase in chain length disparity between the surfactant and phospholipid molecules. In the present system, the disparity between SDS and DOPC is larger than that between OHAB and DOPC. The OHAB molecule has a C18 hydrophobic tail, much longer than the C12 tail of SDS. SDS has a cone shaped structure with a smaller packing parameter, while OHAB has a cylindrical structure with a larger packing parameter. Therefore, the SDS molecules can significantly impact the curvature of the lipid bilayer, and in turn make the lipid bilayer unstable. Although OHAB starts to solubilize the DOPC vesicles at a lower concentration than SDS, it needs a higher concentration to complete the solubilization of the DOPC vesicles. In brief, the ability of the OHAB/SDS systems in solubilizing DOPC vesicles decreases in the order of XOHAB = 0.30 > XOHAB = 0.50 > XOHAB = 0.70 > XOHAB = 1.00 > XOHAB = 0.


21

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15

XOHAB = 0

12 9

sol

Re

6

sat

Re

3 0 15 XOHAB = 0.30 12 9

Total OHAB/SDS Concentration (mM)

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6 3

0 15 XOHAB = 0.50 12 9 6 3

0 15

XOHAB = 0.70

12 9 6 3 0

16

XOHAB = 1.00

12 8 4 0 0

1

2 3 4 CDOPC(mM)

5

6

Figure 6. The phase boundaries for the transitions of the DOPC vesicles induced by the OHAB/SDS mixtures with different molar fractions of OHAB. Open squares and solid squares represent the data points of Dtsat and Dtsol, respectively.


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Penetration Profiles of OHAB/SDS Mixtures at Different XOHAB Values. The performances of the OHAB/SDS mixtures in skin penetration were studied using a synthetic membrane, which is considered as a standard model for in vitro transdermal diffusion tests replacing human or animal skin.41 Figure 7 shows the permeation profiles of the OHAB/SDS mixtures at different XOHAB values. The permeation flux (Jss) and permeability coefficient (Kp) are summarized in Table 2. The Jss and Kp values stand for the ability of the OHAB/SDS solution to penetrate skin. XOHAB = 0

400

(a) OHAB+SDS

XOHAB = 0.30 XOHAB = 0.50 XOHAB = 0.70

300

XOHAB = 1.00

Cumulative Permeated Surfactant Amount (µg/cm2)

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200 100 0 400

(b) OHAB

300 200 100 0 400

(c) SDS

300 200 100 0 0

5

10

15

20

25

30

35

Time (h)

Figure 7. Permeation profiles of OHAB/SDS mixture through a synthetic membrane similar to human skin. (a) Total amount of OHAB and SDS, (b) the amount of OHAB, and (c) the amount of SDS penetrated through the model skin at different XOHAB values. For all the curves above, (■) XOHAB = 0, (●) XOHAB = 0.30, (□) XOHAB = 0.50, (▼) XOHAB = 0.70, and (〇) XOHAB = 1.00.


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Table 2. The permeation flux (Jss) and permeability coefficient (Kp) of the OHAB/SDS mixtures at different XOHAB values through the synthetic membrane. XOHAB

Jss (µg/cm2 h)

Kp (×102 cm/h)

0

11.498 ± 0.590

0.399 ± 0.020

0.30

4.342 ± 0.576

0.129 ± 0.017

0.50

4.795 ± 0.606

0.130 ± 0.016

0.70

5.831 ± 0.598

0.145 ± 0.014

1.00

8.026 ± 0.414

0.178 ± 0.010

Figure 7a shows the cumulative amount of OHAB and SDS permeated through the model skin, and Figure 7b and 7c present the cumulative penetrated amount of OHAB and SDS, respectively. Before mixing, the cumulative permeated amount of individual OHAB quickly increases to ~ 100 µg/cm2 during the first 6 h, while it gradually increases to ~ 270 µg/cm2 from 6 h to 33 h (Figure 7b). However, the cumulative permeated amount of individual SDS steeply increases to ~ 200 µg/cm2 in the first 6 h, and then increases to ~ 390 µg/cm2 from 6 h to 33 h (Figure 7c). Obviously SDS shows much stronger permeating ability through the model skin than OHAB, possibly because the SDS molecules are smaller than the OHAB molecules. In particular, after mixing SDS with OHAB, the permeating ability of the surfactants is significantly reduced. The OHAB molecules in the OHAB/SDS mixtures almost do not penetrate through the model skin in the first 10 h, and then the cumulative permeated amount of OHAB increases gradually to a smaller extent, 30 ~ 110 µg/cm2. The cumulative permeated amount of SDS in the OHAB/SDS mixtures 60 ~ 100 µg/cm2 in the first 6 h and then almost keeps invariant. These phenomena indicate that in the mixtures, SDS penetrates through the model skin more quickly than OHAB. Besides the reason that the SDS molecules are smaller than the OHAB molecules, another important reason is that OHAB has much stronger self-assembly ability than SDS, which means that almost all the OHAB molecules have joined the mixed OHAB/SDS vesicles, and thus show weaker and slower penetration ability. When the


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OHAB/SDS mixture penetrates the model skin, free SDS monomers may penetrate firstly. With the decrease of the SDS concentration in the donor, more and more OHAB molecules may be released from the mixed vesicles, leading to the increase of the free monomeric OHAB concentration in the donor and the resulting enhanced penetration OHAB after 6 h. By comparing the final cumulative amounts of OHAB and SDS permeated through the model skin (Figure 7a), it can be found that the cumulative permeated amounts of the surfactants increase in the order of XOHAB = 0.30 < XOHAB = 0.50 < XOHAB = 0.70 < XOHAB = 1.00 < XOHAB = 0. As shown in Table 2, both individual SDS and OHAB, i.e., at XOHAB = 0 and XOHAB =1.00, have much larger Jss and Kp values than those for the OHAB/SDS mixtures at XOHAB = 0.30, 0.50 and 0.70. The results can be explained from two aspects. On one hand, the aggregates for individual SDS and OHAB are globular micelles with a Rh of ~ 3.5 and 4.0 nm (Table 1), which are much smaller than the aggregates of the mixtures at XOHAB = 0.30, 0.50 and 0.70. So the results suggest that small aggregates are much easier to penetrate skin. Very similar results have also been reported.39, 42 On another hand, either SDS or OHAB has a much larger CMC value than the OHAB/SDS mixtures, so their monomer concentration in the SDS or OHAB solution should be much larger than that in the mixtures. The surfactant monomers can penetrate the model skin much easily than micelles and vesicles. Therefore, the Jss and Kp values of SDS (XOHAB = 0) are larger than OHAB (XOHAB = 1.00) because the CMC of SDS (0.78 mM) is much larger than that of OHAB (0.015 mM). It is concluded that the surfactant system with larger CMC and smaller aggregate size has stronger ability of skin penetration. In addition, the Jss and Kp values for the OHAB/SDS mixture slightly decreases in the order of XOHAB = 0.30 < XOHAB = 0.50 < XOHAB = 0.70. Here the CMC should not be the main reason given that the CMC values of these three mixtures are all very low. As shown in Table 1, the aggregate size of the mixture decreases in the order of XOHAB = 0.30


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< XOHAB = 0.50 < XOHAB = 0.70. That is to say, the mixture with a larger aggregate size always exhibits weaker skin penetration ability. In all, the skin penetration ability of the OHAB/SDS mixture increases in the order of XOHAB = 0.30 < XOHAB = 0.50 < XOHAB = 0.70 XOHAB = 0.50 > XOHAB = 0.70 >> XOHAB = 1.00 > XOHAB = 0, and the mixtures show much stronger ability than individual OHAB and SDS. However, the skin penetration experiment illustrates that the ability of the OHAB/SDS mixtures in penetrating model skin decreases in the order of XOHAB = 0 > XOHAB = 1.00 >> XOHAB = 0.70 > XOHAB = 0.50 > XOHAB = 0.30. These results imply that the surfactant mixture with a larger aggregate size and a smaller CMC value displays much stronger ability in


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solubilizing DOPC vesicles, but much weaker ability in skin penetration. Moreover, in the surfactant mixture, the component with lower self-assembly ability penetrates the model skin more quickly. This work suggests that the ability of a surfactant mixture to solubilize lipid vesicle does not always correlate to its skin penetration power. Utilizing mixtures of oppositely changed surfactants can simultaneously enhance the mildness of surfactant formulations and their ability in solubilizing lipid vesicles.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (Y. L. W.). ACKNOWLEDGMENT This work was supported by the Chinese Academy of Sciences and National Natural Science Foundation of China (21327003, 21361140353). We greatly appreciate for the helps on Cryo-TEM from Dr. Ji Gang in Center for Biological Electron Microscopy, Institute of Biophysics, in Chinese Academy of Sciences. REFERENCES (1) Blank, I. H. Action of Soap on Skin. Arch. Dermatol. 1939, 39, 811-824. (2) Effendy, I.; Maibach, H. I. Detergent and Skin Irritation. Clin. Dermatol. 1996, 14, 15-21. (3) Hall-Manning, T. J.; Holland, G. H.; Rennie, G.; Revell, P.; Hines, J.; Barratt, M. D.; Basketter, D. A. Skin Irritation Potential of Mixed Surfactant Systems. Food. Chem. Toxicol. 1998, 36, 233-238. (4) Charaf, U. K.; Hart, G. L. Phospholipid Liposomes Surfactant Interactions as Predictors of Skin Irritation. J. Soc. Cosmet. Chem. 1991, 42, 71-85. (5) Wilhelm, K. P.; Surber, C.; Maibach, H. I. Effect of Sodium Lauryl Sulfate Induced Skin Irritation on In Vivo Percutaneous Penetration of Four Drugs. J. Investig. Dermatol. 1991, 97, 927-932. (6) Polakowska, R. R.; Goldsmith, L. A. The cell envelope and transglutaminases. In Physiology,


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Biochemistry, and Molecular Biology of the Skin, 2 ed.; Goldsmith, L. A., Ed. Oxford University Press: New York, 1991; pp 168-201. (7) Barbieri, J. S.; Wanat, K.; Seykora, J. Skin: Basic Structure and Function. In Pathobiology of Human Disease, Mitchell, L. M. M. N., Ed. Academic Press: San Diego, 2014; pp 1134-1144. (8) Montagna, W.; Parakkal, P. F. The Structure and Function of Skin. Academic Press: New York, 1974. (9) Downing, D. T.; Abraham, W.; Wegner, B. K.; Willman, K. W.; Marshall, J. L. Partition of Sodium Dodecyl Sulfate into Stratum Corneum Lipid Liposomes. Arch. Dermatol. Res. 1993, 285, 151-157. (10) Womack, M. D.; Kendall, D. A.; MacDonald, R. C. Detergent Effects on EnzymeActivity and Solubilization of Lipid Bilayer Membranes. Biochim. Biophys. Acta 1983, 733, 210-215. (11) Helenius, A.; Simons, K. Solubilization of Membranes by Detergents. Biochim. Biophys. Acta 1975, 415, 29-79. (12) Lichtenberg, D. Characterization of the Solubilization of Lipid Bilayers by Surfactants. Biochim. Biophys. Acta 1985, 821, 470-478. (13) Lichtenberg, D.; Ahyayauch, H.; Alonso, A.; Goñi, F. M. Detergent Solubilization of Lipid Bilayers: a Balance of Driving Forces. Trends Biochemi. Sci. 2013, 38, 85-93. (14) Lichtenberg, D.; Opatowski, E.; Kozlov, M. M. Phase Boundaries in Mixtures of MembraneForming Amphiphiles and Micelle-Forming Amphiphiles. Biochim. Biophys. Acta 2000, 1508, 1-19. (15) Lichtenberg, D.; Robson, R. J.; Dennis, E. A. Solubilization of Phospholipids by Detergents Structural and Kinetic Aspects. Biochim. Biophys. Acta 1983, 737, 285-304. (16) Almog, S.; Litman, B. J.; Wimley, W.; Cohen, J.; Wachtel, E. J.; Barenholz, Y.; Ben-Shaul, A.; Lichtenberg, D. States of Aggregation and Phase Transformations in Mixtures of Phosphatidylcholine and Octyl Glucoside. Biochemistry 1990, 29, 4582-4592. (17) Fan, Y.; Li, Y.; Cao, M.; Wang, J.; Wang, Y. L.; Thomas, R. K., Micellization of Dissymmetric


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Cationic Gemini Surfactants and Their Interaction with Dimyristoylphosphatidylcholine Vesicles. Langmuir 2007, 23, 11458-11464. (18) Heerklotz, H.; Lantzsch, G.; Binder, H.; Klose, G.; Blume, A. Thermodynamic Characterization of Dilute Aqueous Lipid/Detergent Mixtures of POPC and C12EO8 by Means of Isothermal Titration Calorimetry. J. Phys. Chem. 1996, 100, 6764-6774. (19) Heerklotz, H.; Seelig, J. Titration Calorimetry of Surfactant–Membrane Partitioning and Membrane Solubilization. Biochim. Biophys. Acta 2000, 1508, 69-85. (20) Heller, M.; Greenzaid, P.; Lichtenberg, D. The Activity of Phospholipase D on Aggregates of Phosphatidylcholine, Dodecylsulfate and Ca2+. Adv. Exp. Med. Biol. 1978, 101, 213-220 (21) Delamaza, A.; Parra, J. L. Solubilization of Unilamellar Liposomes Caused by Quaternary Ammonium Surfactants. J. Control. Release. 1995, 37, 33-42. (22) Delamaza, A.; Parra, J. L. Solubilization of Unilamellar Liposomes by Betaine-Type Zwitterionic Anionic Surfactant Systems. J. Am. Oil. Chem. Soc. 1995, 72, 131-136. (23) Urbaneja, M. A.; Alonso, A.; Gonzalezmanas, J. M.; Goni, F. M.; Partearroyo, M. A.; Tribout, M.; Paredes, S. Detergent Solubilization of Phospholipid Vesicles-Effect of Electric Charge. Biochem. J. 1990, 270, 305-308. (24) Edwards, K.; Almgren, M. Surfactant-Induced Leakage and Structural Change of Lecithin Vesicles: Effect of Surfactant Headgroup Size. Langmuir 1992, 8, 824-832. (25) Edwards, K.; Almgren, M.; Bellare, J.; Brown, W. Effects of Triton X-100 on Sonicated Lecithin Vesicles. Langmuir 1989, 5, 473-478. (26) Delamaza, A.; Parra, J. L. Solubilization of Phospholipid-Bilayers by C-14 Alkyl Betaine Anionic Mixed Surfactant Systems. Colloid Polym. Sci. 1995, 273, 331-338. (27) Tanaka, K.; Takeda, T.; Nakamura, M.; Yamamura, S.; Miyajima, K. Interactions of Mixed


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Anionic Mixed Surfactant Aggregates with

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