COLLOIDSANDSURFAC~ A

ELSEVIERColloids and Surfaces

A: Physicochemical and Engineering Aspects 186 (2001) 33-41www.clsevier.nl/locatejcolsurfa

Mechanism of mixed liposome solubilization in the presenceof sodium dodecyl sulfateNamita Deo, P. Somasundaran *

NSF IUCR Center for Advanced Studies in Novel Surfactants, Langmuir Center for Colloid and Interfaces, Columbia University,New York, NY 10027, USA

Abstract

Structural transformations of vesicles to micelles that take place during the interaction of sodium dodecyl sulfate(SOS) with individual and mixed vesicles of phosphatidyl choline (PC) and phosphatidic acid (FA), have been studiedby monitoring changes in optical density and in the concentration of free SOS monomer in the respective systems.Incorporation of the surfactant monomers (SOS) in the bilayers was found to result in an initial increase inconcentration of the mixed vesicles up to its saturation. Subsequently a progressive relaxation of these structurestogether with a simultaneous fonnation of mixed micelles was found to occur. The breakup of bilayer and thefonnation of mixed micelles were completely dependent on the structure of the individual phospholipid. Thesolubilization of the anionic phosphatidic acid vesicle was very fast in the presence of SOS, due to its simple structureand its compatibility with the SOS molecule. But solubilization of the zwitter ionic phosphatidyl choline vesicle wasvery complicated in the presence of SOS, possibly due to its complex structure and the zwitterionic nature. On theother hand, solubilization of the mixed vesicle (mixed liposome) and fonnation of mixed micelle was found to berelatively easier. This may be attributed to one of the phospholipid component preferentially coming out first throughinteraction with SOS, thereby making the overall system unstable and enhancing the micellization processes. 0 200 IElsevier Science B. V. All rights reserved.

studying solubilization of cell membranes [4- 7]and surfactants interactions with such biosystemsas skin. The interaction of surfactants with lipo-somes eventually leads to the rupture of its vesiclestructure resulting in the solubilization of phos-pholipid components. In general, there is agree-ment that growth of vesicles occurs in the initialstages, followed by the formation of a number'ofcomplex lipid-surfactant aggregates associatedwith the vesicle to micelle transformations. Theinteractions of sodium dodecyl sulfate (SDS) with

1. Introduction

The solubilization and reconstitution of biologi-cal membranes using surfactants are currently at-tracting much attention [1-3]. Surfactant-liposome systems represent good models for

. Corresponding author. Tel.: + 1-212-8542926; fax: +

212-8548362.E-mail address: ps24@co1umbia.edu (P. Somasundaran).

0927-7757/01/$ - see front matter C> 2001 Elsevier Science B.V. All rights reserved.

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N. Dec, P. Somasundaran / Colloids and Surfaces A: Physicochem. &g. Aspects 186 (2001) JJ-4j34

simplified membrane models of PC liposomes,P A liposomes and PC and P A mixed vesicleshave been investigated in this work. The abovesurfactant was chosen because of its known irri-tating action on biological surfaces [8-10].

We have characterized in detail the vesicle tomicelle structural transitions involved in the in-teraction of SDS with individual P A, PC andmixed liposomes using a combination of opticaldensity and SDS monomer measurements. TheRe (liposome surfactant molar ratio) and K(partition coefficient) values calculated using themonomer SDS concentration left in the superna-tant after interaction for I h with different lipo-somes are compared with the optical densitydata obtained.

2.2. Absorbance measurements

Absorbance measurements were made at 25°Cwith Shimadzu UV-240 spectrophotometer (A =350 urn, cell length = 5 cm). The absorbancechange of each vesicle suspension (1 mM) wasmonitored before and after interaction with SDS(1 to 12 mM). The absorbance measurementswere taken after surfactant addition and subse-quent vigorous agitation for different intervalsof time; the values were corrected taking intoaccount the dilution involved.

2.3. SDS concentration in bilayer

The SOS concentration in lipid bilayers wasdetermined after interactions of individual vesi-cles with SOS for 1 h. The above mixtures werethen filtered through an Amicon filter (Molecu-lar wt cutoff 3000) in order to separate SOSmonomers from vesicles and micelles in thebulk. The monomer concentration in eachfiltrate was determined by a two-phase titrationmethod [12]. The SDS concentration in each bi-layer was calculated by subtracting themonomer concentration in the filtrate from theinitial concentration. The partition coefficient(K) and effective surfactant to phospholipid mo-lar ratio (Re) were calculated by using the equa-tion described below.

2. Experimental

SDS was obtained from Fluka, which is WO/opure, and used as such. Surface tension datagive an indirect measure of purity of the SDSsample. We have measured the surface tensionof SDS in triple distilled water as well as inphosphate butTer (data is not shown) but nominimum was observed in the surface tensioncurve. This indicates the SDS sample is pureand without any further purification we used itfor our experimental purposes. Phosphatidicacid (PA) from egg yolk lecithin andphophatidyl choline (PC) also from egg lecithinwas purchased from Sigma. The butTer used wasphosphate butTer at pH 7.0.

2.4. Solubilization parameters

2.1. Liposome preparation

Unilamellar liposomes of uniform size wereprepared following the method of Helenius [II).A lipidic film was formed by removing the or-ganic solvent from the individual lipid (1 mM),and lipid mixtures (lipid compositions PC/P A1: I molar ratio) by rotary evaporation. Eachlipid film was dispersed in the phosphate bufferand sonicated in a bath sonication unit at 40°Cfor I h. The resulting liposomes were sizedthrough a polycarbonate filter of 0.2 ~ poreSize.

The perturbation of the phospholipid (PL) bi-layer produced by the surfactants leads to thesolubilization of the lipid components via mixedmicelle formation [13). This solubilization resultsin changes in the optical density of the system.which was monitored during the solubilizationprocess.

When defining the parameters related to thesolubilization of liposomes, it is essential to con-sider the fact that mixing of lipids and SDS isnot ideal because of specific interactions betweenboth components [14,15). To evaluate the alter-ations of lipid bilayers caused by SDS, the effec-tive SDS\PL molar ratio, Re, in an aggregate(liposome or micelle) is defined as follows [13).

N. Deo, P. Somasundaran / Colloids and surfaces A: Physicochem. Eng. Aspects 186 (2001) 33-41

(rotal surfactant) - (surfactant momoner) S I . .w = tota monomer concentration Inaqueous mediumSPL = total SDS concentration in lipid bilayerThe partition coefficients of SDS in liposome

have been determined by applying Eq. (4). Thepartition coefficient during solubilization may beregarded as a dynamic equilibrium between thedifferent stages of transition from lipid bilayer tomixed micelles.

the~ Re= c.(Total PL) - (pL monomer)

(It

The second term of the denominator is negligi-ble due to the low solubility of PL in water. It isgenerally believed that partition equilibrium ofsurfactants between the bilayer and the aqueousmedium governs the incorporation of surfactantsinto liposomes, thereby producing saturation andsolubilization of these structures.

Lichtenberg [13] and Almong et al. [16] haveshown that for mixing of lipids and surfactant, indilute aqueous media, the distribution of surfac-tant between lipid bilayers and aqueous media isgiven by a partition coefficient K.

K= SPL m

3. Result.. and discus...ions

-- (PL + SpJSw ,-,

Where SPL is the concentration of SDS in thebilayers and Sw is the SOS concentration inaqueous medium. For PL »SPL, the definition ofK, given by Schurtenberger [17] applies:

K= SPL_~ (1)-- (PL.Sw) - Sw ,-,

whereas Re is the above mentioned ratio of sur-factant to PL in the vesicle bilayer (Re = SpJPL).Under any other conditions, Eq. (2) has to beemployed to define K:

ReK=

SW<1 + Re)

The overall solubilization process of PL bilay-ers by SDS can be characterized by two parame-ters, termed Resat and Resol, corresponding to theSDSjlipid molar ratios. These parameters corre-spond to the Re at which SDS (i) saturates theliposome; and (ii) leads to total solubilization ofthe liposomes. The determination of theseparameters was carried out by measuring SDSmonomer concentration in the solution with lipo-some after 24 h of interactions. Thus, in anyliposome sample the necessary SDS concentrationneeded to saturate the bilayer is given by:

(4)

Solubilization of liposomes made up of I mMphosphatidyl choline only by sodium dodecyl sul-fate as measured by optical density is given in Fig.I. Here, the dependence of liposome solubilizationon SDS concentration for different interactiontimes is shown. After 15 min. of interaction,about 35% of the liposome has been solubilizedby 12 mM SDS. As the interaction was allowed tocontinue the solubilization increased, with com-plete solubilization observed after 12 h of interac-tion even with a lower (9 mM) amount of SOS.Optical density data for the solubilization of PAliposome by SDS is given in Fig. 2 for differentintervals of time. It is obvious from this figurethat solubilization of P A liposome takes placemuch more rapidly than PC liposome and about80 to 9()O/o solubilization was observed in thepresence of I mM and 2 mM of SOS after only 15min of interaction. Almost complete solubilizationof the above liposome was observed in the pres-ence of 6 mM of SOS after 15 min of interaction.As the interaction time increased, solubilization ofthe above liposome increased rapidly and almostcomplete solubilization was observed in the pres-ence of I mM of SDS after 12 h of interaction.Thus SDS can completely solubilize P A liposomeat I mM, but it takes longer, whereas at higherconcentrations of SDS the solubilization takesplace much more rapidly.

Fig. 3 illustrates solubilization of I mM ofliposome (IPA:IPC) by SDS at different intervalsof time. Mter 15 min of interaction, in the pres-ence of I mM of SOS, an increase in opticaldensity from 1.2 to 1.25 was observed. This in-crease in the optical density is attributed to the

Total surfactant = Sw + SPL (5)

Where:

N. Dec, P. Somasundaran / Colloids and SlUfaces A: Physicochem. Eng. Aspects 186 (.2001) 3.3-4136

concentration of surfactant molecules. As the in-teraction time increased at I roM of SDS theoptical density passed through a maximum andthen gradually decreased indicating that solubi-lization is occurring. Complete solubilization of ImM of liposome by 10 mM of SDS was observed

adsorption of SDS onto the lipid vesicle and theresultant increase in the size of the vesicle. Athigher concentrations of SDS also, the same pro-cesses may be occurring, but it is difficult toobserve the intermediate process because the reac-tion is very fast due to the availability of the high

Solubilization of phosphatidyl choline liposome (1 roM) by sodium dodecyl sulfate at different intervals of time.F~

Fig. 2. Solubilization of phosphatidic acid liposome (I roM) by SDS at different intervals of time.

N. Deo, P. Somasundaran / Colloids and surfaces A: Physicochem. Eng. Aspects 186 (2/XJ1) 33-41 37

Fig. 3. Solubilization of I mM of Iiposome(IPA:IPC) as a function of SDS concentration at different intervals of time.

Fig. 4. Solubilization of I mM of different liposomes by SDS after 15 min of interaction.

after 15 min of interaction whereas in the presenceof 4 mM of SDS, complete solubilization wasobserved after 6 h. This indicates that solubiliza-tion of liposome depends on both the concentra-tion of the surfactant as well as time ofinteraction with liposome. More interaction timeis needed at low surfactant concentrations. for

complete solubilization.The solubilization of I mM of different lipo-

somes by SDS, after 15 min interaction time, isshown in Fig. 4. It is evident that solubilization ofP A liposome is very fast and complete solubiliza-tion takes place at 6 mM of SDS during the aboveinterval. Whereas complete solubilization of ImM

N. Deo. P. Somasundaran / Colloids and Surfaces A: Physicochem. Eng. Aspects 186 (2001) 33-4138

solubilization of PC liposome is not completeduring the I h of interaction with different SOSconcentrations, suggesting that the system did notyet reach mixed CMC under these conditions.

The variation in SDS monomer concentration(Sw) versus SDS\phospholipid molar ratio (Re)for different liposomes is illustrated in Fig. 6. It isclear from the figure that the monomer concentra-tion left in the supernatant increases with increasein surfactant concentration. Here a preferentialincorporation of surfactant species into liposomegoverns the initial stages of this interaction, lead-ing to the beginning of bilayer saturation at sur-factant concentrations lower than its CMC.Additional amounts of surfactant increases thefree surfactant until the surfactant CMC isreached and solubilization starts to occur at thispoint. Thus, the Re saturation parameter corre-sponds to the surfactant/phospholipid molar rationeeded for the initiation of the bilayer solubiliza-tion via mixed micelle formation. This surfactantconcentration may be considered to be the CMCof the new surfactant/phospholipid mixed system.The phosphatidic acid vesicle and mixed lipo-somes reach the Re saturation at 6 and 10 mM

liposome (IPA:lPC) was observed in the presenceof 10 mM of SDS, only 35% solubilization of PCliposome was observed under the same condi-tions. This suggests that the PC liposome struc-ture is more rigid so the interaction of SDS andPC liposome is hindered. The solubilization rateof liposome (IPA:IPC) is faster than that of PCeven though it is composed of both PC and P A.This may be due to the exiting of one of thephospholipid components preferentially first dur-ing surfactant liposome interaction, which candestabilize the system and enhance the solubiliza-tion process.

Fig. 5 represents the SDS monomer present inthe supernatant after I h of interaction with dif-ferent liposomes. In the case of phosphatidic acidliposome, the monomer concentration of SDSremains constant after interaction with 6 mM ofSDS whereas in the case of the mixed liposomethe same effect was observed at around 10 mM ofSDS. This indicates complete solubilization ofliposome and onset of formation of micelles. Incontrast to the above, in the case of PC liposomethe monomer concentration of SDS continues toincrease in the concentration range studied. Thus

1.2

~EIi:0

~..c.4)c04)...E0

§E

UIcUI

0.8

0.6

0.4

0.2

00 2 4 10 12 146 8

SOS concentration, mM

Fig. 5. SDS monomer concentration in the supernatant after I h of interactions with I mM of different liposomes.

N. Dec, P. Somasundaran / Colloids 0IId Surfaces 14.: Physiccchem. £IIg. Aspects 186 (2001) 33-41 39

1.2

0.8

i"E 06'i'U)

0-4

0.2

00 2 . 6

Re

108 12

Fig. 6. Variation of SDS monomer con~ntration (Sw) with SDS\Phospholipid molar ratio (Re) for different liposomes

expected due to coulombic repulsion. On theother hand. the hydrophobic dodecyl group caneasily interact with the hydrophobic chain of thephosphatidic acid to form a mixed micelle. This isconsidered to be the reason for the faster rate ofsolubilization of P A liposome in the presence ofSOS. In contrast to P A, the molecular structureof phosphatidyl choline is complex as it containstwo types of charge groups. When SOS moleculeinteracts with PC liposomes it can be expected toexperience the coulombic attraction due to thepresence of N+(CH3h but since the nitrogen isbonded to three bulky (CH3) groups, the interac-tion between SO~ - and N+(CH3h may not be

very strong. On the other hand, the hydrophobicchain is not compatible with the aqueous mediumand hence association can be expected betweenthe hydrophobic chains as shown in Fig. 7b, butthis may not be energetically feasible due to thebulky structure of the phosphotidyl cholinemolecule. SOS and PC under these conditions willinteract to arrive at a compatible rearrangementamong them selves. Another possible way of in-teraction of SOS with PC molecule is throughhydrophobic chain-chain interaction as shown inFig. 7c. Since the PC molecule does carry a

SDS, respectively, whereas in the case of phos-phatidyicholine, the Re saturation has not yetbeen reached. Thus the phosphatidyl choline re-quires more SDS for complete solubilization, eventhough the molar concentrations of both phos-phatidic acid and phosphatidyl choline are thesame. This is attributed to the rigidity of thephosphatidyl choline structure. Even though themolar concentration of mixed (PA:PC) liposomeis almost double than the individual phospholipidliposomes the Re saturation of the mixed lipo-some is reached at around 9 to 10 mM of SDS.As indicated earlier this is attributed to the prefer-ential exiting of one of the liposome componentsduring the interaction with SDS and the resultantdestabilization, which enhances the solubilizationprocesses. This is also possibly the reason as towhy the Resat of liposome (IPA:IPC) lies be-tween that of phosphatidic acid and phosphatidylcholine liposome.

The proposed mechanism of interaction of SDSwith individual phospholipids is shown in Fig. 7.The interaction of phosphatidic acid with SDS isstraightforward due to its simpler molecular struc-ture (Fig. 7a). Because of the anionic nature ofboth SDS and P A, head on interaction is not

40 N. Deo, P. Somasundaran I Colloids and Surfaces A: Physicochem. Eng. Aspects 186 (2001) 33-41

solubilization of phosphatidic acid liposome isvery fast in presence of sodium dodecyl sulfate,while solubilization of zwitterionic phosphatidyl-choline liposome is very slow under similar con-ditions. In the initial interaction stages a directrelationship was established between the growthof vesicles (PC liposome and mixed liposome),and monolayer formation, which is directly de-pendent on the liposome:SOS molar ratio. How-ever, under similar conditions completebreakdown of PA liposome was observed.Mixed micelles begin to form only when thephospholipid bilayer becomes saturated withSOS, which is clear from the monomer study.

positive charge group, it will definitely try toaggregate with some anionic molecules and thiscomplicates the process, leading to slower solu-bilization than in the case of P A liposome. Dueto the above structural complications the PCmolecule may not be able to form mixed mi-celles with SDS molecules easily. In contrast,phosphatidic acid does not have such structuralproblems, the rnicellization and solubilizationhence being faster.

4. Conclusions

From these findings we may conclude that the

.Q(A) C~o.co.R,

~ H 0

~-~

b

jqao-S-O-C1zH25II ~.OOO-.-0

---OHR1o R2- 14 to 15

(8)

9(C)0 I

. II R1-C-O-?H2~-S-O-C1~ !

II R2-C-O-9-H0 n i.

0 I :--

~II-ICH3

0

!HzC -O-,.-O-~--~u-~N . -.. ~'Z .

Fig. 7. Probable mechanisms of interaction of sodium dodecyl sulfate with different lipids.

N. »eo, P. Somasundaran / Colloids and Surfaces A: Physicochem. Eng. Aspects 186 (.2001) 33-41

The monomer concentrations of SOS in the su-pernatant increases until complete solubilizationof liposome, after which no further increase inSOS monomer was observed with further in-crease in SOS concentration. This confirmedthat solubilization of liposome by SOS is a mi-ceUization process. The monomer measurementin the supernatant gives direct evidence of Licht-enberg's three-stage model. The anionic chargeon SOS does not affect its adsorption onto an-ionic liposome surfaces; indicating that hydro-phobic and Vanderwals forces play the majorrole in the SOSjliposome interaction processes.

Ackno"'ledgements

Authors acknowledge the support of the Na-tional Science Foundation (Grant Number9804618. Industrial/University cooperation re-search center for adsorption studies in novelsurfactants). The financial support of UnileverResearch laboratory, US, is thankfullyacknowledged.

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