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Interactions of Phospholipid Vesicles with Cationic and Anionic Oligomeric Surfactants Yao Chen, Fulin Qiao, Yaxun Fan, Yuchun Han, and Yilin Wang J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b05297 • Publication Date (Web): 07 Jul 2017 Downloaded from http://pubs.acs.org on July 10, 2017

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The Journal of Physical Chemistry

Interactions of Phospholipid Vesicles with Cationic and Anionic Oligomeric Surfactants 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), CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China ‡

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

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ABSTRACT：：This work studied the interactions of 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with cationic ammonium surfactants and anionic sulfate or sulfonate surfactants of different oligomeric degrees, including cationic monomeric DTAB, dimeric C12C3C12Br2 and trimeric DDAD as well as anionic monomeric SDS, dimeric C12C3C12(SO3)2 and trimeric TED-(C10SO3Na)3. The partition coefficient P of these surfactants between the DOPC vesicles and water was determined with isothermal titration microcalorimetry (ITC) by titrating concentrated DOPC solution into the monomer solution of these surfactants. It was found that the P value increases with the increase of the surfactant oligomeric degree. Moreover, the enthalpy change and the Gibbs free energy for the transition of these surfactants from water into the DOPC bilayer become more negative with increasing the oligomeric degree. Meanwhile, the calcein release experiment proves that the surfactant with a higher oligomeric degree shows stronger ability of changing the permeability of the DOPC vesicles. Furthermore, the solubilization of the DOPC vesicles by these oligomeric surfactants was studied by ITC, turbidity and dynamic light scattering, and thus the phase boundaries for the surfactant/lipid mixtures have been determined. The critical surfactant to lipid ratios for the onset and end of the solubilization for the DOPC vesicles derived from the phase boundaries decrease remarkably with increasing the oligomeric degree. Overall, the surfactant with a larger oligomerization degree shows stronger ability in incorporating into the lipid bilayer, altering the membrane permeability and solubilizing lipid vesicles, which provides comprehensive understanding about the effects of structure and shape of oligomeric surfactant molecules on lipid-surfactant interactions.

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INTRODUCTION

Knowledge of lipid-surfactant interaction is of great importance to understand the purification and solubilization of membrane proteins and lipids, and to realize the reconstitution of a membrane protein in a native environment.1-5 Lipid-surfactant interaction strongly depends on surfactant molecular structures which determine the effective surfactant to lipid molar ratio (Re) in the interaction. When Re is very low, lipid-surfactant interaction is a partition process of surfactant monomers between lipid bilayers and aqueous phase. When Re is larger, lipid-surfactant interaction is a solubilization process of lipid vesicles by surfactants. Lichtenberg et al.6-8 proposed a three stage model to describe the interaction process: (1) At lower surfactant concentration, surfactant molecules gradually incorporate into lipid bilayer until the lipid bilayer is saturated, which may lead to a growth of the lipid vesicles; (2) At higher surfactant concentration, surfactant molecules result in membrane leaks or pores in the lipid vesicles and stabilize the hydrophobic edges with a surfactant-rich rim,9 which means the lipid bilayer is gradually disrupted and surfactant/lipid mixed micelles coexist with lipid vesicles; (3) Finally, the lipid bilayer is completely disintegrated and only surfactant/lipid mixed micelles remain. The interactions of phospholipid vesicles with conventional surfactants, such as sodium dodecyl sulfate (SDS), sodium cholate and n-alkyl polyoxyethylene ether surfactants (CnEOm), have been widely studied.10-12 Delamaza et al.10 investigated the aggregate transitions of phosphatidylcholine (PC) vesicles induced by SDS, and they found that the Re value linearly increases with the increase of the SDS concentration, which suggests that the higher surfactant concentration leads to the larger surfactant concentration in the lipid bilayers. However, the bilayer/aqueous partition coefficient K of SDS shows a 3



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maximum during the initial addition of SDS, which suggests that the SDS molecules tend to incorporate into the lipid bilayers relative to the aqueous phase. Similar results were also observed in a SDS/C12En/PC system.13 Moreover, Heerklotz et al.14 proved that the increase of the headgroup size of C12(EO)n (n = 3-8) significantly reduces the surfactant to lipid ratios for the onset and end of the lipid solubilization (Resat and Resol). With the development of surfactant structures, the understanding about the interactions of new surfactants with lipids is desired. The interactions between lipid vesicles and gemini surfactants15-17 have been studied in the past decade. Fan et al.16 studied the solubilization of DMPC vesicles by dissymmetric and symmetric cationic gemini surfactants m-6-6 (m = 12, 14 and 16) and 12-6-12. The results illustrated that gemini surfactants are more effective than the corresponding monomeric surfactant dodecyltrimethylammonium bromide (DTAB). Moreover, dissymmetric gemini surfactants m-6-6 series are more effective than symmetric 12-6-12, and the Re value decreases with the increase of m. This phenomenon can be explained by the theory proposed by Fattal et al.17 and Keller et al.,18 namely, the Re value decreases with the increase of the chain length disparity between the chains of surfactants and phospholipid hydrophobic tails. Comparing with monomeric and gemini surfactants, oligomeric surfactants have more hydrophobic tails and polar headgroups and thereby display more abundant molecular conformations. These molecular conformations endow oligomeric surfactants with more unique aggregate morphologies. However, so far the interactions between lipid vesicles and oligomeric surfactants and the resultant effects of the oligomeric topological structures on the interactions have not yet been studied. In the present work, cationic ammonium monomeric surfactant (DTAB), gemini surfactant 4


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(C12C3C12Br2) and trimeric surfactant (DDAD)19 as well as anionic sulfate monomeric surfactant (SDS), sulfonate gemini surfactant (C12C3C12(SO3)2)20 and trimeric surfactant (TED-(C10SO3Na)3) have been employed to investigate their interactions with the vesicles of 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in aqueous solution. The corresponding molecular structures and abbreviations are shown in Scheme 1. The lipid-surfactant interactions have been studied from thermodynamic aspect by isothermal titration microcalorimetry (ITC). The partition coefficients P of the surfactants between the lipid bilayer phase and aqueous phase have been obtained by titrating the dispersion of concentrated DOPC vesicles into the surfactant monomer solution according to the method reported.1, 18, 21 By fitting the ITC curves, the transfer enthalpy (∆Hb/w) values of the surfactant molecules from water to the lipid bilayer were obtained and the corresponding Gibbs free energy (∆Gb/w) values were also calculated.18 Meanwhile, the lipid solubilization process has been studied by separately titrating the concentrated surfactant solutions into the DOPC vesicle dispersions and the corresponding aggregate transitions during the solubilization process have also been studied by turbidity and dynamic light scattering to know the size change of the surfactant/lipid aggregates in the process. Then the phase boundaries have been obtained by fitting the critical concentrations for the aggregate transitions, and the effective surfactant to lipid ratio for the onset (Resat) and end (Resol) of solubilization and other related parameters were derived. These comprehensive studies are aimed at understanding how the oligomerization degree and charges of surfactants affect the abilities of surfactants in incorporating into lipid bilayer, permeating lipid membrane and solubilizing lipid vesicles.

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Scheme 1. Chemical structures of DTAB, C12C3C12Br2, DDAD, SDS, C12C3C12(SO3)2 and TED-(C10SO3Na)3.

EXPERIMENTAL SECTION

Materials. Cationic ammonium monomeric surfactants DTAB (98%) and anionic monomeric surfactant SDS (99%) were purchased from sigma and they were recrystallized twice before use. Cationic ammonium gemini surfactant C12C3C12Br2, cationic ammonium trimeric surfactant DDAD, and anionic sulfonate gemini surfactant C12C3C12(SO3)2 were synthesized and purified as we reported previously.19, 20 The synthesis procedure of anionic sulfonate trimeric surfactant TED-(C10SO3Na)3 was presented in Supporting Information. Phospholipid 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and calcein were purchased from Sigma and used as received. Milli-Q water of 18.2 MΩ cm was used in all experiments. Preparation of DOPC Vesicles. DOPC powder was dissolved in chloroform, and then the chloroform was dried under vacuum on a rotary evaporator to yield a thin film on the bottom of the flask. The thin film was further dried in a vacuum desiccator for at least 6 hrs. The dried film was suspended in a certain volume of water to a desired concentration and vortexed at 25 °C for several 6


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minutes until the mixture is thoroughly mixed. Unilamellar vesicles of ~ 100 nm were obtained by extruding crude vesicle suspension through a 100 nm polycarbonate membrane. These freshly prepared vesicle dispersions were directly used. Dynamic Light Scattering (DLS). Size variations of DOPC vesicles induced by the addition of different surfactants were characterized by DLS. It was 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). The calorimetric measurements were taken by a TAM III microcalorimeter system. For the solubilization of the DOPC vesicles by surfactants, concentrated surfactant solutions were titrated into the stirred sample cell which was already loaded with 600 µL water or the DOPC vesicle dispersion, while the reference cell was loaded with 765 µL water. Aliquots of concentrated surfactant solution of 5 µL were consecutively injected into the stirred sample cell using a 500 µL Hamilton syringe controlled by a Thermometric 612 Lund pump. Both the sample cell and reference cell were stirred at 70 rpm with a gold propeller, and the interval between two injections was long enough to reach equilibrium. In the case of partition experiment, the sample cell and the reference cell of the microcalorimeter were initially loaded with surfactant monomer solution (600 µL) and water (765 µL), respectively. Concentrated DOPC vesicle dispersion was consecutively titrated into the sample cell. All the titrations and the measurements of heat were done as programmed at 25.00 ± 0.01 °C. The observed enthalpy changes (∆Hobs) were obtained by integrating the areas of the peaks in the plot of thermal power against time. The reproducibility of all the experiments was within ± 4%. Turbidity Measurements. Turbidity measurements were used to study the aggregate transitions of 7



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DOPC vesicles with the addition of the surfactants. 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. Calcein Release. Calcein loaded vesicles were prepared by DOPC in water containing 20 mM calcein. The unencapsulated calcein was removed by dialysis. The vesicle solution was collected and diluted to the desired concentration. The DOPC dispersion and surfactant solution were rapidly mixed, and the fluorescence intensity of the mixtures was measured as a function of time with excitation at 495 nm and emission at 520 nm on a Hitachi model F-4700 spectrophotometer at room temperature of 25 ± 1 °C.

RESULTS AND DISCUSSION

Partitioning of Surfactant Molecules into DOPC Bilayers. When surfactant molecules are initially added into the dispersion of lipid vesicles, they can insert into the vesicle bilayer spontaneously because the hydrophobic tails of surfactants tend to escape from aqueous phase and enter hydrophobic domain of lipids.22 The partition coefficient P is defined as the ratio of the surfactant molar fraction in lipid bilayer phase to the surfactant molar fraction in aqueous phase,23, 24

P=

xb xw

(1)

where xb and xw are the molar fractions of surfactant in lipid bilayer phase and aqueous phase, respectively. They can be calculated by the following equations,

xb =

Db Db + C L

xw =

Dw Dw + W

(2)

Db and Dw are the surfactant concentrations in lipid phase and aqueous phase, respectively. CL is the 8


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total concentration of lipid and W is the total concentration of water. Then Equation (1) changes to,

P=

Db Dw ÷ Db+CL Dw+W

(3)

Because the surfactant concentration is much lower than the concentration of water (55.5 mol/L), it can be assumed that W >> Dw, i.e., Dw + W ≈ W. Meanwhile, if using Dt represents the total surfactant concentration, then Dw = Dt – Db. Inserting these two expressions into Equation (3) yields the following equation,

Db=

1  2 P(Dt − C L )-W+ P 2 (Dt+CL ) − 2PW (Dt − CL )+W 2   2P 

(4)

During the titration process, the change of surfactant concentration in the bilayer (∆Db) is caused by the addition of lipid (∆CL), thus, Equation (4) can be differentiated with respect to CL, ∆Db P(Dt + CL ) + W 1 =− + ∆C L 2 2 P 2 (D + C )2 + 2 PW (C − D ) + W 2 t L L t

(5)

In this work, the dilution of surfactant concentration in the sample cell should be considered. Considering this point, the effective surfactant concentration Dte can be obtained by Equation (6),

 C L  Dte = Dt × 1 −  C L syringe   

(6)

CL syringe is the lipid concentration in the syringe. In the process of titrating lipid vesicle dispersion into surfactant monomer solution, the observed enthalpy changes (∆Hobs) obtained by ITC is mainly composed of the enthalpy change for the incorporation of surfactant molecules into lipid bilayer (∆Hb/w) and the enthalpy change for the dilution of lipid dispersion (∆Hdilution). Therefore, the relationship of ∆Hobs, ∆Hb/w and ∆Hdilution is shown by Eq. (7),

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∆H obs =

∆Db × ∆H b w + ∆H dilution ∆CL

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(7)

∆Hdilution can be obtained by titrating lipid vesicle dispersion into water, and it turns out to be very small. Combining equations (5), (6) and (7), a two parameter least square fit of the ITC curve taking P and ∆Hb/w as parameters can be obtained. Herein, Figure 1 presents the observed enthalpy changes (∆Hobs) against the DOPC concentration (CL) by titrating concentrated DOPC vesicle dispersion into different surfactant monomer solutions. All the ITC curves exhibit a similar variation tendency. The initial addition of DOPC leads to a large exothermic ∆Hobs, and then the absolute ∆Hobs value decreases continuously with increasing CL until it reaches a platform at higher CL value. During the titration process, the surfactant molecules partition between aqueous phase and lipid phase. The partition process can be divided into two steps, i.e., the surfactant molecules electrically bind to the DOPC vesicles, and then they insert into the lipid vesicles through hydrophobic interaction. These two kinds of interactions result in the exothermic enthalpy values. As more and more surfactant molecules insert into the lipid bilayers, the ∆Hobs values become smaller, and finally turn to zero when the lipid bilayers are saturated by the surfactant molecules. Comparing the ∆Hobs values for the surfactants with the same oligomerization degree but different charge property, i.e., DTAB vs SDS, C12C3C12Br2 vs C12C3C12(SO3)2, and DDAD vs TED-(C10SO3Na)3, it is found that the ∆Hobs value for the anionic surfactant is much larger than that for the corresponding cationic one, which should be attributed to that the outermost layer of the DOPC vesicles is positively charged by cationic ammonium headgroups. By fitting the ITC results (Figure 1), the partition coefficient (P) and the transfer enthalpies from water to bilayer (∆Hb/w) are obtained. In addition, the Gibbs free energy ∆Gb/w = 10


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–RTInP and the term T∆Sb/w = ∆Hb/w – ∆Gb/w for this transition process are also calculated and summarized in Table 1. Obviously, with increasing the oligomerization degree, the P values for both cationic and anionic oligomeric surfactant gradually increase. Meanwhile, the Gibbs free energy ∆Gb/w for transferring surfactant from water to bilayer becomes more negative. These results suggest that it is much easier for a surfactant with a higher oligomerization degree to insert into the lipid bilayer. On one hand, a surfactant with a higher oligomerization degree has more charged headgroups, which leads to much stronger electrostatic interactions between the surfactant and the lipid vesicles. On the other hand, a surfactant with a higher oligomerization degree has more hydrophobic tails available to insert into the lipid bilayer. Moreover, the hydrophobic tails may aggregate with each other and then insert into the lipid bilayer together. Thus the hydrophobic interaction of the surfactant with the lipid bilayer is also stronger than that of single-chain surfactant. In addition, for the oligomeric surfactants with the same oligomerization degree, the P value for anionic surfactant is larger than that for cationic one. The P values for DTAB, C12C3C12Br2 and DDAD are 4000, 17600 and 23000 respectively, while the values for SDS, C12C3C12(SO3)2 and TED-(C10SO3Na)3 are accordingly 5800, 27000 and 150000. The reason is that the anionic charges are more favorable to the outermost cationic ammonium headgroups of the DOPC vesicles, which largely increases the interaction of the anionic surfactants with the DOPC vesicles and provides more possibility for the anionic surfactants to insert into the DOPC bilayer.

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0.0

0.0

0

a

c

b

DTAB

C12C3C12Br2

-0.2

∆ Hobs (kJ/mol)

-1

DDAD

-0.5

-0.4

-1.0 -2

-0.6

-1.5 -3

-0.8 -2.0 -4

-1.0 0

3

6

9

0

12

3

6

9

12

0

3

6

9

12

CL (mM) 0

0

0.0

e

SDS

d ∆ Hobs (kJ/mol)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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-1

-2

-2

-4

f

C12C3C12(SO3)2

TED-(C10SO3Na)3

-0.3 -0.6 -0.9

-6

-3

-1.2 -1.5

-8 -4 0

3

6

9

12

0

1

2

3

0

3

6

9

12

CL (mM)

Figure 1. The observed enthalpy (∆Hobs) against the final DOPC concentration (CL) from ITC by titrating the dispersion of DOPC vesicles into different solutions of surfactant monomers. (a) Titrating 30 mM DOPC into 3 mM DTAB, (b) titrating 30 mM DOPC into 0.5 mM C12C3C12Br2, (c) titrating 10 mM DOPC into 0.05 mM DDAD, (d) titrating 30 mM DOPC into 3 mM SDS, (e) titrating 5 mM DOPC into 0.04 mM C12C3C12(SO3)2, and (f) titrating 15 mM DOPC into 0.05 mM TED-(C10SO3Na)3. The red solid lines are calculated by Equation (7), combining with Equations (5) and (6), and taking P and ∆Hb/w as parameters.

Table 1. The Partition Coefficient P and Thermodynamic Parameters for the Partition of the Surfactants between the DOPC Bilayer and Water Surfactants

P×104

∆Hb/w (kJ/mol)

∆Gb/w (kJ/mol)

T∆Sb/w (kJ/mol)

DTAB C12C3C12Br2 DDAD

0.41 1.76 4.30

-2.10 -23.8 -24.8

-21.5 -24.2 -26.4

19.4 0.50 1.60

SDS C12C3C12(SO3)2 TED-(C10SO3Na)3

0.58 2.70 15.1

-22.6 -23.5 -25.4

-21.5 -25.5 -29.5

-1.10 2.00 4.10

12


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Calcein Release. As known, the permeability of lipid vesicles can be changed by surfactants,25, 26 which leads to release of dyes solubilized in lipid vesicles. Therefore, calcein release experiments were performed to study how oligomeric surfactants impact the permeability of DOPC vesicles. As shown in Figure 2, for either anionic oligomeric surfactant series or cationic oligomeric surfactant series, all the curves change in the same manner. The fluorescence intensity increases sharply upon the initial addition of surfactants, suggesting that the calcein dye is released from the vesicles. Then the fluorescence intensity reaches a maximum at higher surfactant concentration, and further increasing surfactant concentration leads to a decrease in fluorescence intensity. The surfactant concentration needed for the maximum calcein release (Cmax) is obtained from the curves. For DTAB, C12C3C12Br2 and DDAD, the Cmax values are 1.15 mM, 0.19 mM and 0.06 mM, respectively. As to SDS, C12C3C12(SO3)2 and TED-(C10SO3Na)3, the Cmax values are 0.91 mM, 0.11 mM and 0.04 mM, respectively. The Cmax value decreases with the increasing of oligomerization degree. This means that the surfactant with a higher oligomerization degree shows much stronger power to enhance calcein release from the lipid vesicles. The release of dyes from lipid vesicles has been thought to be affected by surfactants from two aspects.27 Firstly, surfactants can change the permeability of lipid vesicles through perturbing membrane, but not disintegrating lipid membrane. Secondly, surfactants can disintegrate lipid vesicles and in turn significantly increase the permeability until lipid vesicles are completely broken down. Herein, the concentrations of all the surfactants used for the calcein release experiments are much lower than their CMCs, where the disruption of lipid vesicles has not started yet. Thus, the increase of the permeability of the lipid vesicles should be caused by the incorporation of the surfactant molecules into the vesicle 13



bilayers and the resultant disturbance of the surfactants to the ordered DOPC bilayers. The calcein release results suggest that the surfactant with a higher oligomerization degree has much stronger ability to incorporate into the lipid bilayer, i.e, has a large partition coefficient. 100

180

DTAB C12C3C12Br2

a

DDAD

150 120 90 60 30 0 0.01

0.1

1

Fluorescence Intensity (a.u.)

Fluorescence Intensity (a.u.)

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b 90

TED-(C10SO3Na)3

80

70

60 0.01

10

SDS C12C3C12(SO3)2

0.1

1

10

CS (mM)

CS (mM)

Figure 2. The variation of calcein release from 1 mM DOPC vesicles with increasing surfactant concentration (Cs).

Solubilization of DOPC Vesicles by Surfactants with Different Oligomeric Degrees. As discussed above, the partition of the surfactants between the DOPC bilayers and water phase has been studied at lower surfactant to lipid ratio (Re). To further understand the lipid-surfactant interaction, the solubilization of the DOPC vesicles at higher Re values are investigated as follows. ITC measurement has been used to study the solubilization of lipid vesicles by surfactants because the lipid-surfactant interaction is always accompanied by the uptake or release of heat.21, 28 Figure 3 depicts the variation of the observed enthalpy changes (∆Hobs) against the final surfactant concentration (Cs) in the process of titrating concentrated surfactant solutions into the DOPC vesicles of various concentrations.

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25

a

DTAB/DOPC

∆ Hobs (kJ/mol)

2.0 CDOPC 0 mM 1 mM 2 mM 3 mM 4 mM

1.5 1.0 0.5

b

C12C3C12Br2/ DOPC

20

c

DDAD/ DOPC

20 CDOPC 0 mM 1 mM 2 mM 3 mM 4 mM

15 10 5

10


-10

0.0 0 -20

-0.5 0

10

20

30

40

-5 0.1

50

1

10

0.1

1

CS (mM)

d

SDS/DOPC

5

e

C12C3C12(SO3)2/ DOPC

8

f

TriSDoS/DOPC

1

∆ Hobs (kJ/mol)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



-1

-2

0

6

-5

4 CDOPC

-10

0 mM 1 mM 2 mM 3 mM 4 mM

-15 -20

CDOPC 0 mM 1 mM 2 mM 3 mM 4 mM

2 0 -2 -4

-25 1

10

0.1

CS (mM)

1

0.1

1

Figure 3. Observed enthalpy changes ∆Hobs against the final surfactant concentration (Cs) for separately titrating the solution of (a) DTAB, (b) C12C3C12Br2, (c) DDAD, (d) SDS, (e) C12C3C12(SO3)2 or (f) TED-(C10SO3Na)3 into the solution of the DOPC vesicles of different concentrations marked in the figures.

Figure 3a, b and c present the ∆Hobs values against Cs for titrating concentrated cationic oligomeric surfactants DTAB, C12C3C12Br2 and DDAD into pure water or the DOPC vesicles. When these surfactants are titrated into water, the ITC curves reflect the demicellization processes of these surfactants, a large and almost constant endothermic ∆Hobs is observed at first, and followed by an abrupt decrease beyond a certain surfactant concentration, i.e, CMC. The CMCs of these surfactants obtained from the ITC curves are listed in Table 2. When the surfactant solutions are titrated into the DOPC vesicles, the situations are different. For DTAB and C12C3C12Br2, the ITC curves exhibit a similar changing tendency. The initial addition of the surfactants into the DOPC vesicles leads to a slight 15



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changing endothermic ∆Hobs, which is smaller than the corresponding demicellization enthalpy. In this process, the surfactant micelles demicellize into monomers and thereafter the monomers electrically bind to the lipid vesicles and the hydrophobic chains insert into the DOPC bilayers. The exothermic electrostatic interaction may be stronger than endothermic hydrophobic interaction, thus the ∆Hobs for the initial titration of the surfactants into the DOPC vesicles is less endothermic than that for the demicellization of the surfactants. Further adding DTAB or C12C3C12Br2 induces a sudden drop of ∆Hobs, which even changes to exothermic. The remarkable change should be the beginning of the phase transition from the lipid bilayers to the surfactant/lipid mixed micelles. When the surfactant concentration keeps on increasing, the surfactant/lipid bilayers transfer to the surfactant/lipid mixed micelles. Finally the ∆Hobs values return back to zero or very small endothermic values, which refer to the end of lipid vesicle solubilization. Thus two critical surfactant concentrations for the onset (Dtsat) and end (Dtsol) of lipid solubilization can be obtained from the ITC curves. As to trimeric cationic surfactant DDAD (Figure 3c), the initial addition of DDAD leads to a large constant exothermic enthalpy, which is quite different from the endothermic enthalpy for monomeric DTAB and gemini C12C3C12Br2. As mentioned above, the initial ∆Hobs is resulted from the enthalpy change for the demicellization of DDAD micelles into monomers, and the electrostatic and hydrophobic interactions of the DDAD molecules with the DOPC vesicles. Comparing with DTAB and C12C3C12Br2, DDAD has three polar head groups and three hydrophobic tails. Therefore, the electrostatic and hydrophobic interactions of DDAD with the DOPC vesicles should be much stronger than those of the DOPC vesicles with DTAB or C12C3C12Br2, and leads to large exothermic enthalpy changes. Moreover, the amide groups in DDAD molecule may form hydrogen bonds with the oxygen atom of the DOPC molecule, which is also an exothermic process. 16


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These factors result in the large exothermic ∆Hobs value for the beginning of titrating DDAD into the DOPC vesicles. Later, increasing DDAD concentration induces a sudden increase and then decrease in ∆Hobs. Finally, the curves merge with the demicellization curve of DDAD into water and the ∆Hobs is close to zero. These transitions correspond to the start and end of the lipid vesicle solubilization by DDAD, respectively. Figure 3d, e and f present the ITC results by titrating anionic surfactants SDS, C12C3C12(SO3)2 and TED-(C10SO3Na)3 into the DOPC vesicles, respectively. For these three surfactants, the ITC curves exhibit the similar shape. The ∆Hobs values are exothermic at the beginning of titration, which is quite different from that of DTAB and C12C3C12Br2. The ∆Hobs is also composed of the enthalpy change for the demicellization of DDAD micelles, and the electrostatic interaction and hydrophobic interaction between the surfactant molecules and the DOPC vesicles. Comparing with cationic surfactants, anionic surfactants have much stronger electrostatic interaction with the DOPC vesicle because the outer layer of the DOPC vesicle is positively charged. As a result, the ∆Hobs value for the initial titration of anionic surfactant into the DOPC vesicles is exothermic. When more surfactant molecules are added into the vesicle dispersion a large endothermic broad peak appears, which should result from the solubilization of DOPC vesicles by the surfactants. The beginning and the end of the endothermic peak represent the onset and the end of the solubilization, respectively. Obviously, either anionic oligomeric surfactants or cationic oligomeric surfactants experience the similar process in solubilizing the DOPC lipid vesicles. However, the concrete situations for the surfactant-lipid interactions are different. By analyzing the inflexion points in the above ITC curves, the critical surfactant concentrations (Dtsat and Dtsol) for the lipid solubilization can be obtained, which will 17



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be confirmed by the following turbidity and DLS measurements.

Aggregate Transitions of DOPC Vesicles Induced by Surfactants. In order to confirm and understand the surfactant-induced phase transitions of the DOPC vesicles reflected in the ITC curves above, turbidity and DLS measurements have been performed under almost the same conditions as the ITC experiments. Similar studies have been carried out in our previous work for other surfactant/lipid systems.29 Figure 4 presents the turbidity curves of the DOPC vesicles with the increasing of surfactant concentration and the corresponding ITC curves for comparison. The red dashed lines are the Dtsat and Dtsol values obtained from the ITC curves, and they are also consistent with the transition points observed in the turbidity curves. The dash lines, i.e., the transition points divide the phase transitions into three regions for all the systems. The aggregate size in each region characterized by DLS is shown in Figure 5. In the following text, Figure 4a is taken as a representative to discuss the phase transitions as well as the surfactant-lipid interactions in the three regions.

18


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sol

sat

a

sat

Dt

Dt

70

DTAB/DOPC

Turbidity ITC

60

30

b

1.5

sat

sol

Dt

Dt

c

C12C3C12Br2/ DOPC

sol

Dt

35

Dt

DDAD/ DOPC

8

12 30

25

100-% T

50 8

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40

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sol

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Dt

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Dt

SO3Na)3/DOPC

2

50 1

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0.4

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-4

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


10 0.01

0.1

1

30 -25 0.01

-3 0.1

1

CS (mM)

Figure 4. Turbidity (black) and observed enthalpy changes ∆Hobs (blue) by titrating (a) DTAB to 2 mM DOPC, (b) C12C3C12Br2 to 1 mM DOPC, (c) DDAD to 1 mM DOPC, (d) SDS to 2 mM DOPC, (e) C12C3C12(SO3)2 to 1 mM DOPC and (f) TED-(C10SO3Na)3 to 2 mM DOPC.

19



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a

DTAB/DOPC

c

C12C3C12Br2/DOPC

b

37 mM

0.60 mM

III

III

32 mM

4.0 mM

0.40 mM

25 mM

3.0 mM

0.30 mM

II

18 mM

I

10

10

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2

3

I

0.05 mM

DOPC

DOPC 4

10

0.10 mM

0.5 mM

DOPC 1

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7 mM

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13 mM

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6.0 mM

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0

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20 mM

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0.50 mM

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III 17 mM

0.60 mM

14 mM

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0.25 mM

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I

I

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3

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0.05 mM

0.05 mM

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TED-(C10SO3Na)3/DOPC

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DOPC 4

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10

10

1

10

2

3

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4

10

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Size (nm)

Figure 5. Size variation of the DOPC vesicles by adding (a) DTAB, (b) C12C3C12Br2, (c) DDAD, (d) SDS, (e) C12C3C12(SO3)2 or (f) TED-(C10SO3Na)3 into 2 mM DOPC vesicle solution.

In region I, with the addition of surfactant, the turbidity decreases sharply at first, and then it reaches a short platform. However, the mean vesicle size of the mixture (Figure 5) are ~ 100 nm and nearly does not change and only the size distribution gets much broader until the DTAB concentration increases to 18 mM. This suggests that the lipid vesicle solubilization does not take place in this region. The reduction of turbidity should be caused by the dilution of the DOPC vesicle dispersion and the added 20


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surfactant molecules may separate the small clusters of the lipid vesicles due to electrostatic repulsion force.30 When the surfactant concentration locates in region II, the turbidity decreases rapidly until it reaches a lower constant value. The sudden drop of turbidity means that the large lipid vesicles start to be disintegrated, and the final lower turbidity value of the mixture means that almost all the DOPC vesicles are disintegrated into small aggregates. Meanwhile, the DLS curve (Figure 5) becomes a bimodal curve with a new peak at ~ 6 nm, indicating that mixed surfactant/lipid micelles are formed in region II. Although the intensity of the small size distribution increases with the increase of surfactant concentration, the large peak at ~ 100 nm does not disappear even at larger surfactant concentrations. The possible reason is that although small aggregates are the dominating aggregates in the mixture, a very small amount of large aggregates contribute to the light scattering intensity in a very large extent.31 In region III, the turbidity keeps a very small and almost unchanged value, however, the amount of small aggregates from DLS significantly increases as indicated by the enhancement of the left peak in Figure 5a, which suggests that the large aggregates have transferred into small ones. Above all, the turbidity and the size distribution from DLS confirm the solubilization process reflected in the ITC curves. The critical surfactant concentrations for the onset (Dtsat) and end (Dtsol) of solubilization are obtained from the beginning and end of region II from the turbidity curves and they are in good agreement with those from the ITC curves. Thus the phase boundaries for the lipid-surfactant mixtures are plotted by the Dtsat and Dtsol values against the DOPC concentration as shown in Figure 6, which will be discussed next.

Phase Boundaries for DOPC-Surfactant Mixtures. As shown in Figure 6, the phase boundaries of the Dtsat and Dtsol values against the DOPC concentration CL are fitted by a liner least square method. 21



The intercepts, Dwsat and Dwsol, are the equilibrium surfactant monomer concentrations at the boundaries. The slopes of the phase boundaries are the effective surfactant to lipid molar ratio for the onset (Resat) and end (Resol) of solubilization, which are used to evaluate the lipid solubilizing power of a surfactant, i.e., a surfactant with a lower Re value exhibits a stronger ability to solubilize lipid vesicles. According to the model proposed by Lichtenberg et al.,6-8 the Dwsat and Dwsol values should be the same because of the constant chemical potential of surfactant monomers in the vesicle/micelle coexistence range. However, the present Dwsat values are not the same as the Dwsol values for each surfactant. The difference should be caused by experimental errors. The obtained Resat, Resol, Dwsat and Dwsol values are listed in Table 2 for further discussing. 50

50

a

CS (mM)

2.5

b

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40 30

sol

c

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2.0

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CS (mM)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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sol

Re

30 20 sol Dw

10

sat

Re

sat Dw

0 0

1

2

3

4

0.0 5 0

1

2

3

4

5

0

1

2

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CL (mM)

Figure 6. Phase diagrams for the surfactant/DOPC mixtures. The data points on the phase boundaries were obtained from the Dtsat and Dtsol values as indicated in Figure 4.

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Table 2. The Effective Surfactant to Lipid Molar Ratio for the Onset (Resat) and End (Resol) of DOPC Solubilization, and the Surfactant Monomer Concentration (Dwsat and Dwsol) at the Phase Boundaries for the Interaction of Oligomeric Surfactant with DOPC Vesicles Surfactant

Resat

Resol

Dwsat

Dwsol

CMC (mM)

DTAB C12C3C12Br2 DDAD

2.33 0.35 0.05

6.41 1.30 0.21

10.0 0.68 0.03

13.9 6.40 0.21

14.1 1.10 0.21

SDS C12C3C12(SO3)2 TED-(C10SO3Na)3

1.84 0.07 0.04

9.2 0.22 0.16

5.88 0.06 0.03

7.61 0.72 0.24

8.40 0.08 0.16

Clearly, Figure 6 and Table 2 indicate that the Re values decrease with the increase of oligomerization degree for either anionic or cationic oligomeric surfactants. Therefore, the ability of these oligomeric surfactants to solubilize DOPC vesicles increases with the oligomerization degree. The reasons for these results will be comprehensively discussed in the next section. In addition, not all the Dwsol values for the surfactants are lower than their CMCs. For SDS and DTAB, both the Dwsat and Dwsol values are lower than their CMCs, which is in accordance with former studies.32, 33

However, for gemini and trimeric surfactants, the Dwsol values are larger than their CMCs. The reason

is that the CMC values of the gemini and trimeric surfactants are very low, and thus the amount of the gemini or trimeric surfactants needed for completing of the lipid solubilization may exceed their CMCs even if they have stronger ability to solubilize the lipid vesicles.

Impact of Surfactant Molecular Shape on Partition Coefficient between DOPC Bilayer and Water and on Solubilizing Lipid Ability. According to all the results above, the oligomerization degree shows similar effects on the partition coefficient between the DOPC bilayer and water as well as on the solubilizing ability to lipid vesicles, no matter if the surfactants are cationic or anionic. 23



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First of all, see the partition of the surfactants between the DOPC vesicle bilayers and water. For either anionic surfactants or cationic surfactants, the partition coefficient P between the lipid bilayer and water increases with the oligomerization degree. For examples, the P values of SDS, C12C3C12(SO3)2 and TED-(C10SO3Na)3 are 5800, 27000 and 151000, respectively. According to the definition of P, TED-(C10SO3Na)3 has the strongest power to insert into the lipid bilayer, while SDS is the weakest. It is known that, for the partition process, surfactant molecules electrically bind to the lipid vesicle at first, and then insert into lipid bilayer through hydrophobic interactions between surfactant molecules and lipid molecules. For the gemini and trimeric surfactants, they have much stronger electrostatic interaction with the lipid vesicles than monomeric surfactants, because they have more charged headgroups. Additionally, the hydrophobic tails for each surfactant molecule may not insert into the lipid bilayer independently. The hydrophobic tails may entangle with each other through hydrophobic interaction and then insert into the lipid bilayer as a whole. The entangled alky tails have much stronger hydrophobicity than single-chain surfactant, which largely increases the hydrophobic interaction of the oligomeric surfactants with the lipid bilayer. Consequently, the surfactant with a higher oligomerization degree inserts into the DOPC bilayer more easily. The oligomerization degree also significantly affects the solubilizing ability of the surfactants to the lipid vesicles. Previous studies14, 17, 18 proved that the molecular shape of surfactants greatly affects their solubilizing ability to lipid vesicles. It is known that phospholipid molecule has a cylindrical shape because of the large volume of its hydrophobic tail. However, normally the surfactant molecule has a cone-like shape because the amphiphilic headgroup needs more space than the cross section area of the hydrophobic tail. When the cone shaped surfactants interact with lipid bilayers, the hydrophobic tails of 24


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surfactants insert into the lipid bilayers and their headgroups interact with the lipid polar fragments. Fattal et al.19 and Keller et al.18, 34 found that, with increasing the disparity between the hydrophobic chain lengths of phospholipid and surfactant, the curvature of lipid-surfactant aggregates increases. As a result, the cone shaped surfactant generates strong perturbation to the close packed lipid bilayers, which finally leads to a decrease in Resat and Resol. In this work, the Resat and Resol values of the oligomeric surfactants decrease with the increase of oligomerization degree. However, the hydrophobic chain disparity between these surfactants and DOPC keeps unchanged, because all the surfactants use din this work have the same hydrophobic chain length (C12). Thus, the lower Re value for the surfactant with a higher oligomerization degree can be understood from the following two aspects. On one hand, gemini and trimeric surfactants have more hydrophobic tails than monomeric surfactants. Trimeric surfactant can be regarded as three monomeric surfactants at least, and gemini surfactant can be regarded as two monomeric surfactant. According to the definition of Re, it represents the surfactant to lipid molar ratio for the occurrence of the phase transitions. Therefore, at the same lipid concentration, the surfactant concentration needed to reach the phase boundaries of the DOPC/surfactant mixtures decreases with the increase of the oligomerization degree. On another hand, the surfactant molecules with different oligomerization degrees have different shapes. For examples, comparing the Re values for DTAB (Resat = 2.33), C12C3C12Br2 (Resat = 0.35) and DDAD (Resat = 0.05), it can be found that the Re values do not decay in the ratio of 1 : 1/2 : 1/3. The Re values for the gemini and trimeric surfactants are much smaller than assumed on the base of the numbers of the hydrophobic chains and headgroups. The nonlinear decrease of Re indicates that the gemini and trimeric surfactants may interact with the lipid bilayer in the following mechanism. Take the gemini 25



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surfactant C12C3C12Br2 as an example, the hydrophobic tails get close to each other due to the hydrophobic interaction, thus the hydrophobic chain volume of C12C3C12Br2 (V2) should be smaller than that of two DTAB molecules (2V1). Meanwhile, the two quaternary ammonium headgroups are separated not only by the spacer, but also by the electrostatic repulsion force. So the headgroup area of C12C3C12Br2 (a2) is much larger than that of two DTAB molecules (2a1). Therefore, the packing parameter of C12C3C12Br2 (P2 = V2/a2l) should be much smaller than that of DTAB (P1 = V1/a1l) since V2 is much smaller than 2V1 while a2 is much larger than 2a1. Similarly, these factors also result in the lowest packing parameter value for the trimeric surfactant DDAD (P3). In all, the molecular packing parameters of the oligomeric surfactants decrease with the increase of the oligomerization degree, i.e. P1 > P2 > P3. The surfactant with a smaller packing parameter will generate a more significant disturbance to the lipid bilayer, and in turn performs a stronger ability in disrupting and solubilizing lipid vesicles. As shown in Figure 7, the trimeric surfactant has the lowest P3 value. The cone-shaped trimeric surfactant has the largest curvature, so significantly impact the curvature of the lipid bilayer and make the lipid bilayer unstable, which finally leads to the disintegration of lipid vesicle.

Figure 7. Illustration of the molecular packing of oligomeric surfactants and their interactions with lipid 26


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bilayers.

CONCLUSIONS In summary, the interactions of cationic and anionic oligomeric surfactants with the DOPC vesicles have been studied with respect to different surfactant to lipid molar ratios (Re). At lower Re values, the partition experiments illustrate that the partition coefficient P of the surfactants between the lipid bilayer and water significantly increases with increasing the oligomerization degree. Meanwhile, the ∆Hb/w and ∆Gb/w values for transferring surfactant molecules from water to bilayer become more negative. Moreover, calcein release experiments indicate that the surfactant with a higher oligomerization degree enhances the permeability of the lipid membrane, so promote the release of the dyes from the DOPC vesicle. At larger Re values, the phase diagrams for the solubilization of the DOPC vesicles by the oligomeric surfactants demonstrate that the surfactant with a higher oligomerization degree is more effective in solubilizing lipid vesicles. These results can be understood from the molecular structure and shape of the surfactants. Firstly, the surfactant with a higher oligomerization degree has more charged headgroups and more hydrophobic tails, and thus exhibits stronger electrostatic and hydrophobic interactions with the lipid vesicles, leading to stronger ability in incorporating into the lipid bilayer, i.e, a larger partition coefficient between the lipid bilayer and water phase. Moreover, the hydrophobic tails of the oligomeric surfactants can aggregate with each other, forming a more compactly packed cone-shape with a smaller packing parameter. This kind of molecular packing and molecular shape have more free volume and are very different from the situation of the lipid molecules, and thus exert a more significant 27



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disturbance to the lipid bilayer, displaying a stronger ability in disrupting and solubilizing lipid vesicles. Therefore the surfactant with a larger oligomerization degree shows stronger ability in improving lipid membrane permeability and solubilizing lipid vesicles. This work provides comprehensive understanding about the effects of structure and shape of surfactants on lipid-surfactant interactions and the resultant phase behavior of lipid vesicles.

SUPPORTING INFORMATION Synthesis and characterization of TED-(C10SO3Na)3. Raw ITC data for the titrations of the surfactant solutions into the DOPC dispersions. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (Y. L. W.).

ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (21327003, 21633002) and the ISF/NSFC joint project.

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