J. Soc.Cosmet.

Chem.,47, 185-200 (July/August1996)

Bindingof surfactantsto stratumcorneum

K. P. ANANTHAPADMANABHAN, K. K. YU,
C. L. MEYERS, AND M.P. ARONSON, UnileverResearch
U.S., Edgewater,
N.J. 07020,

Accepted
for publication
August15, 1996.

Synopsis
The bindingof surfactants to stratumcorneumproteinshasbeenimplicatedasoneof the factorsgoverning
their harshnesstowardsskin. In this paper, the binding characteristics of severalanionic surfactants,
includingrelativelyharshsurfactants suchas sodiumdodecylsulfate,TEA-sodiumlaurate,TEA-sodium
oleate, and a relatively milder one suchas sodiumlauroyl isethionate(SLI), are presented.The effect of
variablesrelevantto cleansingsuchassolutionpH, temperature,andcontacttime on surfactantbinding has
been determined.The binding of laurate,oleate,and SDS is significantlyhigher than that of SLI. The
extent of binding correlateswith their expectedirritation potentialmeasuredby the zein solubilization
techniqueas well as with the publishedresultsof irritation to skin of cleansingbars basedon these
surfactants.The reasons for the increased
bindingof surfactantsabovetheir CMC are examined.It is also
shownthat SLI bindingto skin is dependenton the solutionpH, exhibitinga minimum in binding in the
pH 7 to 9 region.

INTRODUCTION

It is well established
that sodiumdodecylsulfate(SDS)andsoapssuchassodiumlaurate
are harshtowardsskin (1-5). The harshness of anionicsurfactantswith compacthead
groupshas been suggestedto be due to their ability to bind to stratum corneum,
especiallystratumcorneumproteins(3-5). It is alsoknown that harshsurfactantssuch
as SDS denaturewater-solubleproteinssuchas BSA and lysozyme(2,6-11) and solu-
bilize water-insolubleproteins such as zein (7). In fact, the harshnessof surfactants
towardsskin hasbeencorrelatedwith their ability to solubilizezein (8,12).
Sodiumcocoylisethionateis the main ingredientin severalwidely usedpersonalwash-
ing barsthat are known to be significantlymilder to skin than SDS and soap(13,14).
The mildnessof isethionate-basedbarsis believedto be due, amongotherthings, to the
overall compositionof the bar as well as the relatively mild nature of its anionic
surfactants.Sodiumlauroylisethionate(SLI) is the main componentof sodiumcocoyl
isethionate.Severalfactorscouldbe suggested to contributeto the relativemildnessof
SLI. Theseincludeits lowercritical micelieconcentration (CMC) comparedto SDS and
its lowertendencyto bind to stratumcorneumbecause of its largerheadgroup.
The binding behaviorof SDS to solubleproteinssuchas BSA and lysozymehasbeen
185
186 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

studiedextensively(15-18). Figure 1 represents a typicalbindingisothermof SDS to

a water-solubleprotein(9,17). In the initial region(a), thereexistssomebinding to
specificsiteson the protein.This is followedby eithera plateauor a slowrisingpart of
the isotherm(b) and beyondit a third regioncorresponding to a massiveincreasein
binding because of cooperativeinteractions(c). The unfoldingof the proteinoccursin
the cooperative binding region. Beyondthe saturationpoint, the binding isotherm
showsa plateau(d), suggestingthat furtherbindingof the surfactantdoesnot occuron
theprotein.Undersaturation bindingconditions,i gramof theproteincanbeexpected
to bind asmuchas 1.5 to 2 gramsof the surfactant(7). It is importantto note that all
of this behavior occurs below the normal CMC of the surfactant.

Somedata exist on the binding of SDS to stratumcorneumand keratinousproteins

(19-28). The main differencebetweensurfactant bindingto a solubleproteinversusan
insolubleproteinis that for an insolubleprotein,the cooperative
bindingappearsto
occur at surfactant concentrations above the CMC. The limited work available on
stratumcorneumalsoindicatesthat the bindingof SDScontinues to increaseabovethe
CMC, althoughwith a slopelower than that in the pre-CMC region. This was inter-
pretedby Faucheret al. (23) to be due to the restricteddiffusionof micellesinto the
stratumcorneumcomparedto monomers.The reasons for the increasein the binding
aboveCMC itself was not discussed in terms of the thermodynamicactivity of the
monomer, which does not increaseabove the CMC. Furthermore, the data does not
adequatelycoverthe low concentrationregionin this case.
Althoughalkyl isethionate-typesurfactantsareof considerablepracticalimportancein
mild cleansing
products,their bindingbehaviorto skinhasnot beeninvestigated in the
past.This paperreportsthe resultsof bindingstudiesof soaps,SDS, andSLI to human
and porcinestratum corneumas a functionof relevantvariablessuchas surfactant
concentration,solutionpH, time of contact,and temperature.

(d)

log ([el)
Figure 1. Schematicplot of the numberof boundligandsper proteinmolecule(n) as a functionof the
logarithm of the free ligand concentration(c). Region a, specificbinding; region b, non-cooperative
binding; regionc, cooperativebinding, regiond, saturation.Reproduced from M.N. Jones,Biochem.J.,
151, 109-114 (1975).
 BINDING OF SURFACTANTS 187

EXPERIMENTAL

MATERIALS

Thematerials
usedin thesebindingexperiments
were•4C-labeled
SDSandSLI,ob-
tained from Unilever Research,Colworth Laboratory.Potassiumhydrogenphthalate
andpotassiumdihydrogenphosphate werefrom Aldrich ChemicalsCo. Sodiumborate,
TEA (triethanolamine),and sodiumhydroxideusedfor bufferpreparationswere pur-
chasedfrom FisherScientificCorp. Hairlessguineapig skin wasobtainedfrom Charles
River Laboratories
(Wilmington, MA), and the stratumcorneumwasisolatedfrom it by
a trypsinseparationprocedure.Human stratumcorneumwasalsoobtainedby trypsin
separationfrom excisedcadaverskin obtainedfrom the InternationalInstitute for the
Advancement of Science.

METHODS

Adsorptionprocedure.
The procedure
usedto evaluatesurfactantbindingto guineapig and
humanstratumcorneumrequiredthat three8-mm punchesof SCbeplacedin 25.4-mm
(1-in) squareTeflonscreens.
Thesescreens
wereplacedin treatmentsolutionscontaining
a combination
of •4C-labeled
(1 }xCi/ml)
andnonlabeled
surfactant,
ranging
in total
concentrationfrom 1 mM to 100 mM. Beforethe addition of the SC sampleto the
treatment solution, 50 }xl of the treatment solutionwas removedto provide initial
radioactivityvalues.After the selectedtreatmenttime (for example,one hour), the
screenswere rinsed by moving them back and forth five times in a dish of distilled
water. This was to essentiallywashoff the excessbulk solutionthat would havebeen
associatedwith the corneum.Sincethe objectiveof the work was to determinethe
thermodynamic bindingof the surfactantto the corneum,excessiverinsingwasnot done
to avoiddesorption.The screens werethen blottedanddriedwith desiccation overnight.
The weight of eachindividual SC samplewas recordedusing a Perkin-Elmer AD4
Autobalance,and the samplewas placed into scintillation vials containing 0.5 ml of
distilled water. Scintiverse BD (Fisher Scientific) was added to each vial, and the
radioactivityof the stratumcorneumsampleswasdeterminedusingthe BeckmanScin-
tillation Counter. The amount of surfactant bound to the SC was calculated over the
final weightof SC. This wasparticularlyimportantsincein certainsystems
a weightloss
wasobservedupon surfactanttreatmentdue to the extractionof corneocytes and other
surfactant-soluble
components.For experimentswith soaps,it was necessary to buffer
the Na oleateand Na lauratesolutionswith 0.4 M TEA. The pH of the systemwas
around 9.5.

Stratumcorneumsamplesfor delipidizedexperimentswere preparedby treating the

Teflon screenscontainingthe 8-mm puncheswith a 2:1 chloroform/methanolsolution
for one hour and drying with nitrogen.

RESULTS

SURFACTANT BINDING TO GUINEA PIG STRATUM CORNEUM

The initial experimentsof SDS and SLI binding were done on hairlessguinea pig
stratumcorneum(GPSC).Figure2 showsthe bindingisothermof SDSandSLI to GPSC
188 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

HairlessGuineaPig StratumCorneum
pH 5.5, 1 hr TreatmentTime, RT

CMC
ß

SDS

0
0 20 40 60 80 100
SurfactantConcentration (mM)
Figure 2. Binding of SDS and SLI to hairlessguineapig stratumcorneum.

after a one-hourtreatmentat pH 5.5 and room temperature(RT). The shapeof the SDS
binding isothermto GPSCis similarto the typicalisothermgivenin Figure 1. The main
differencebetweenthe binding to skin and to water-solubleproteinsappearsto be that
in the former casethe binding continuesto occur abovethe surfactantCMC. The
amountof bindingto GPSC(1-2 mg/g) is significantlyhigherthan the reportedvalues
of surfactantbindingto keratinousproteinsuchaswool (0.4 mg/g; ref. 20).
A comparisonof SDSto SLI (one-hourtreatment,pH 5.5, RT) showsthat lessSLI binds
to GPSC than SDS overthis concentrationrange(Figure 2). Theseresultsare consistent
with the known clinicalmildnessof alkyl isethionate-basedbars(14).
Extending the treatment time to six hoursshowssomeincreasein the SDS binding,
especiallyat high concentrations
(Figure3). In contrastto this, the 24-hour binding
isothermis similar to the one-hourbinding in the low concentrationregionand slightly
lower than the one-hourbinding at 100 mM SDS. The longertreatmentsaffectedthe
physicalintegrityof the GPSC. We suspectthat the observed low bindingat 24 hours
is because of the physicalextraction/removalof partsof the corneumby the surfactant.
For this reason,further experimentswere limited to one hour or less.
Removalof lipids from the GPSC with a 2:1 chloroform/methanol solutionprior to
surfactanttreatmentresultsin a decrease
in surfactantbinding(Figure4). Theseresults
are consistentwith the reportedeffectsof delipidizationon the structureof stratum
corneum(29,30). It is suggestedthat delipidizationprimarily removesthe fluid lipids
and that upon drying, the lipids covalentlylinked to the corneocytesform a tighter
network that reducesthe surfactantpenetrationto protein regions,i.e., preventsthe
surfactantsfrom accessingthe bindingsites(29).

SURFACTANT BINDING TO HUMAN STRATUM CORNEUM

Bindingisotherms.
As with GPSC, the resultsgiven in Figure 5 showthat at pH 5.5,
 BINDING OF SURFACTANTS 189

HairlessGuineaPig Stratum
Corneum
6 hr
pH 5.5, RT
CMC

1 hr

24 hr

0 20 40 60 80 100 120
SDS (mM)
Figure 3. Influence
of equilibration
timeon SDSbindingto GPSC.

Hairless
Guinea
Pig
Corneum
Stratum
CMC pH5.5,
1hrtreatment,
RT / Untreated
C

•pidized
00
i,• 20 40
I

60 ' 80
GPSC
100 120
SDS (mM)
Figure4. Effectofdelipidizarion
ofGPSCbychloroform-methanol
onSDSbinding.
one-hourtreatment,and roomtemperature,SDSbindsmorethan SLI to humanstratum
corneum(HSC). Importantly, SLI binding, unlike that of SDS, doesnot increase
significantly
aboveitsCMC. Interestingly,
bothSDSandSLIshowsignificantly lower
bindingto HSCthanto GPSC(Figure6). Forexample, SDSbindingto HSCisonly
about0.4 mg/mgof thecorneum comparedto the2 mg/mgin thecaseof GPSC.The
reasonfor this largedifference
between
GPSCandHSC is not clearat presentand
requiresfurtherstudy.Onelikelyhypothesisis that thecorneocyte
envelope
andthe
consequentresistanceto swellingare different for GPSC and HSC.
Ef•ctoftemperature.
Theeffectof temperature
onsurfactant
bindingto HSCwasalso
190 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

0.8
HSC, pH 5.5,
1 Hr treatment, RT
O.6

0.4
SDS
13.2

•
00 20 40 60
,•_3
80
SLI100
mM Surfactant
Figure 5. Binding of SDS and SLI to human stratum corneum.

investigated.Figure 7 (one-hourtreatment, pH 5.5) showsan increasein binding for

SDSby an increase
from roomtemperature to 37øC.The increase
in temperature
hasa
much lesspronouncedeffecton the binding of SLI.
Ef;•ctofpH. The effectof pH on the bindingof SLI wasinvestigated.Initial attempts
to alter the pH of treatmentsolutionsusingNaOH showedthat the pH of the system
during contactwith the HSC changedbecause of the inherentbufferingability of the
corneum.SuchpH changes dueto the bufferingability of skinhavebeenreportedin the
literature (31). Experimentswere thereforeconductedin the presentstudy by main-
tainingthe pH of the systemconstantby usinga 0.04 M buffersolution.The binding
wasdeterminedat a constantinitial SLI concentration of 20 mM. The resultsgiven in
Figure8 showthat the bindingof SLI to HSC exhibitsa minimumaroundpH 7-9, with
higher binding both at lower and higher pH values.The pH dependence resultsob-
tainedunderconstantpH conditionsclearlyindicatethat surfactants can interactwith
the skin underboth low and high pH valuesand that their interactionis the lowestin
the pH 7-9 region.

BINDING OF SOAP MOLECULES TO SKIN

Binding isothermsfor soapmoleculessuchasTEA-laurateandTEA-oleateat 37øCand

at a pH of 9.5 afteronehourof contactaregivenin Figure9. Sincetheseexperiments
weredoneusingstratumcorneumsamples fromsources differentfrom thoseusedfor the
resultsreportedin Figures2 to 8, SDS and SLI binding were repeatedusing the new
stratumcorneumsamples.A comparison of the data in Figure9 for SDS and SLI with
thosein Figure5 showsdifferencesin the absolute magnitudeof bindingof SDSandSLI
to different samples.The trends in surfactantbinding and the relative extent are,
however,essentiallythe samefor both the samples.
As can be seen, at low surfactantlevels, oleatebinds much more than all other surfac-
 BINDING OF SURFACTANTS 191

21
SDS
GPSC

SDS
HSC

LI GPSC

o
20 40 60 80 100
mM Surfactant
Figure 6. SDSand SLI bindingto humanvs guineapig stratumcomeurn.

tants.However,at higherconcentrations, the bindingof SDSandlaurateis higherthan

that of oleate. SLI appearsto bind the leastunder high-concentration conditions.In
contrastto this, lauratebinding wasfoundto increaseat about 20 mM and to become
as much as that of SDS at high concentrations. The latter is particularlyrelevantsince
the concentrations of surfactants
from cleansingbarsunderuseconditionsare no doubt
muchhigher than 20 mM. Note that the extentof surfactantbinding at a concentration
of 40 mM and the tendencyof the surfactantto irritate skin as measuredby the zein
dissolution test at the same surfactant concentration correlate well with each other (see
the inset in Figure 9).
As mentionedearlier,all the isothermsin Figure9 correspond to onehour of contactof
surfactantsolutionswith the stratumcorneum.Sincethe practicaltimesof interestfrom
a personalcleansing pointof viewareof the orderof a minuteor less,bindingisotherms
were alsodeterminedafter one minute of contact.The resultsgiven in Figure 10 show
that the binding at oneminute, asexpected,is lower than that at onehour. The overall
trend in the binding, however,appearsto be similar to that at onehour. Thus, among
the various surfactants tested, oleate binds the maximum at low concentrations. At
higher levels,the laurateappearsto bind much more than other surfactants.
192 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

0.8
pH 5.5, 1 hr
SDS 37C

0.4
SLI 37C
0.2 ß

SLI RT

ø0 20 40 60 80
mM Surfactant
Figure 7. Effectof temperatureon binding of SDS and SLI to humanSC.

0.18

0.16
0.14
0.12
0.1

0.08

0.06
rain
0.04
0.02
0 • I I , [ • [ t
2 4 6 8 10 12
pH
Figure 8. Binding of SLI to humanstratumcorneumas a functionof pH.

DISCUSSION

The binding behaviorof SDS, SLI, and soapmoleculespresentedhere clearlycorrelates

with their expectedharshness toward skin as determinedby the zein dissolutiontech-
nique(seeinsetin Figure9). Thesefindingsarealsoconsistent with the higherharshness
of soapscomparedto syndetbarscontainingcocoylisethionateas the main active(14).
The binding of SLI at all the surfactantconcentrations is lower than that of SDS. The
higherbinding of oleatethan that of othersurfactants at low concentrations is consistent
with the highersurfaceactivity of oleatebecauseof its longerhydrocarbon chainlength.
 BINDING OF SURFACTANTS 193

0.5
Surfactant % Zein
(40 mM) Dissolved
SL! 62%
-,- 0.4 SDS 80%

Na/TEA Laurate 86% SDS

Na/TEA Oleate 92%
0.3
Na/TEA Laurate

Na/TEA Oleate
0.2

SLI

0
0 10 20 30 40 50
Surfactant Concentration, mM
Figure 9. Binding of surfactants
to HSC, one-hourtreatment,37C. Zein solubilizationvaluesat 40 mM
surfactants are also included.

Interestingly,laurateand SDSbind almostto the sameextentat higherconcentrations.

All theseobservations are in agreementwith the acceptednotion that surfactantswith
compacthead groupsinteract much more stronglywith the stratum corneumproteins
than thosewith large head groups.
The observationthat in most casesthe surfactantbinding increasesabove the CMC
suggests that miceliesmay havea rolein controllingthe binding behaviorof surfactants.
For example,Figures2-7 showthat both SDS and SLI binding exhibit a nearplateau
or a slowlyrising regionjust abovethe CMC. However,in the caseof SDS at concen-
trationsabove60 mM, a sharplyrisingregionis observed.This is especiallyvisible in
the caseof the GPSC. A plateauin the binding isothermabovethe CMC would indicate
that only the surfactantmonomeris involvedin this phaseof the binding process.From
a practicalpoint of view, theseresultssuggestthat the initial "plateaubinding" can be
lowered by lowering the surfactantCMC, sincemonomeractivity doesnot increase
appreciablyabovethe surfactantCMC. Thus, if micellizationis mademorefavorableby
lowering the CMC, then binding of the surfactantto the corneumat any given con-
centrationshouldbe effectivelyreduced.This is consistentwith the conventionalwis-
dom of increasingthe mildnessof anionic surfactantsby using nonionic surfactant
coactives to lower their CMC.

ROLE OF MICELLES

If we acceptthe abovehypothesis
that monomeractivity controlsthe surfactantbinding
194 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

0.03
f
0.025
Na/TEA

0.02- pte
o.o15

,
O.Ol
0.005
o
o lO 20 30 40 50 60
Surfactant Concentration, mra
Figure 10. Binding of surfactants
to HSC, one-minutetreatmentat 37C.

to the stratumcorneum,then the obviousquestionis: Why doesthe binding increase

aboveCMC in somecases(seeFigures2-7)? In this regard,a brief discussion on the
structureof stratumcorneumitselfwill be helpful. Accordingto the currentlyaccepted
model, stratum corneumhasa biphasicstructure,with protein-richcorneocytes em-
beddedin a matrix of lipids. This is analogousto a brick-and-mortarstructure,with the
corneocytes and the barrierlipids representing the bricksand the mortar, respectively
(32). The lipids consistof both fluid and rigid lipids. The corneocyteenvelopealso
consistsof membraneproteinsandlipidstogetherwith somecovalentlyattachedsurface
lipids,whichmakesthemcompatible with thesurrounding matrixlipids(30). Thus, for
the surfactantmoleculesto interactwith the deeperprotein-richregionsin a normal
corneum,they must diffusethroughthe lipid regions.It is believedthat the network
structure of the stratum corneum and the conformational features of the membrane
proteinslimit the accessibilityof the surfactantto all the potentialbindingsiteson the
keratinousproteins.This may involvea thermodynamiccomponentarisingfrom the
cohesiveforcesholdingthe networktogether,includingthosefrom the conformational
featuresof the proteins.In addition, a kinetic componentrelatedto the slowdiffusion
of surfactantto the bindingsites,especiallyat the corneocyte-lipid matrix interface,also
may be affectingthe bindingbehavior.Miceliesmaycontributeto surfactantbinding in
severalwaysthat include:i) solubilizationof fluid lipids leadingto exposure of binding
regionsthat were otherwiseunexposed,ii) abstractionof calciumor other multivalent
ionsto reducecorneocyteadhesions,iii) introducingosmoticstresses on the membrane
leadingto its breakdown,and iv) contributingto favorablefree-energychangesthat
accompanythe cooperative bindingprocessleadingto protein unfolding.The latter is
equivalentto direct bindingof surfactantaggregates, suchasthe conventional micelies
or premicellarclusters(33), to proteins.Processes i and ii enhancethe accessibility
of the
proteins in the lower regionsof the stratum corneumand are essentiallykinetic in
 BINDING OF SURFACTANTS 195

character.Processesiii and iv areintrinsiceffectsof miceliesto the binding equilibrium

of insolubleproteinsand are, thus, thermodynamic in nature.Thesecontributionsare
discussed below.

It hasbeensuggestedthat surfactants abovetheir CMC causesomedelipidizationof the

stratum corneum(5,34), even though no consensus existson this topic (35-37). An
alternativeview is that surfactants
canremoveselectivelycertaincomponentssuchasthe
ceramides and in the processcausechanges in the lipid composition (38). It is possible
that the reasonsfor the discrepancies
existingin the literatureare essentiallydue to the
differencesin the protocolsusedin variousstudies,especiallythe surfactantconcentra-
tion and time and frequencyof exposure.Our unpublishedresultsof an ongoingTEM
studyof porcinestratumcorneumtreatedwith SDSfor onehour, similarto the con-
ditionsof the binding experiments,indicatethat SDS at levelsabovethe CMC cause
significantdelipidization(39). Similarly, our ultrastructuralstudiesof human skin
subjectedto an in vitroarm-washprotocolreportedelsewhere showedthat soapscaused
significantlipid removalandproteindamage(40). Suchremovalof lipidsby surfactant
miceliesmay play an importantrolein enhancingthe diffusionof surfactantmonomers
andmicellesto the corneocyte-matrixinterface.Therefore,continuedbindingabovethe
surfactantCMC is not unreasonable, evenwhen only monomersare involvedin the
binding process.Note that the resultsof surfactantbinding to delipidizedcorneum
discussedearlier(seeFigure4) may appearto be contradictory to the proposedhypoth-
esisof miceliesenhancingthe monomerbinding by enhancingthe surfactantdiffusion
by removingfree lipids. The apparentcontradictioncan, however,be reconciledby
noting that the delipidizationstudydiscussed
earlierwasdoneusingsolventsand that
subsequently the skinwasdriedbeforecontactingwith the surfactantsolution.During
dryingthe covalentlylinked lipidswouldhaveformeda stronghydrophobic network
that would be much more impenetrableto the surfactants
than the normal corneum
containingboth the free lipids and the covalentlybondedlipids.
Yet anothermechanismby whichanionicmiceliescaninfluencethe bindingprocess
may
be by sequestering
metallicionssuchascalcium.Eventhoughthe roleof ionssuchas
calcium in stratum corneumcohesionis not well understood,it is known that addition
of EDTA, a well-known metal ion chelator, enhancesthe surfactant-inducedreleaseof
corneocytes
(41-42). If the underlyingreasonfor the effectof EDTA is metal ion
complexation,onecanexpectmiceliesalsoto play a similarroleand reducethe cohesion
betweencorneocytes,
which in turn canenhancethe penetrationof surfactantsto surface
regionsof corneocytes.
Once the surfactantmoleculesobtain accessto protein-rich corneocyteregions, the
extent of binding and especiallycooperativebinding in the postmicellarregion is
primarilydeterminedby theinherenttendency of the surfactant
monomerto bind to the
proteinsin the corneocyte membrane.The cooperative binding in thesesystemsmay
well inducethe changesin the conformationof the keratinousproteinspresentin the
corneocytemembrane,which in turn may lead to damageto the corneum.This may
requiresomethresholdlevelof bindingto overcome the cohesiveforcesarisingfrom the
conformationof the proteinaswell asthe lipids holdingthe membranetogether.This
is similar to Breuer'sconceptof the "elasticenergy"holdingthe keratinousnetwork
together(26). Surfactantbindingobviouslyinducesrepulsiveelectrostatic forcesthat at
somecriticalbindingshouldovercome"cohesive forces"andleadto an unfoldingof the
protein.Thus, whileall micelies,includingnonionicsurfactant micelies,cancausesome
196 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

removalof fluid lipidsfromthe matrixlipid, significantdamageto theproteinitselfwill

still dependupon the ability of the surfactantmonomerto bind to the corneocyte
membraneproteins.This is consistent with the reportedobservationsthat nonionic
surfactantssuchasTriton X-100, in combinationwith somemechanicalagitation,can
releasecorneocytesrfrom the lipid matrix without damagingthe membraneenvelope
(43). In contrastto this, treatments with harshsurfactants
suchasSDSor soapsresult
in damageto lipids and corneocyteproteins(38,39).
In addition to the abovemechanisms, micellesmay be contributingdirectly to the
unfoldingprocessby creatingosmoticstresses on the corneocyte envelope.It is well
knownthat corneocytes swellwhenexposed to waterand aqueoussurfactantsolutions
(44). In general,the extentof corneumswellingby anionicsurfactants increaseswith
their harshnesstowardskin(44). It hasalsobeenreportedthat theextentof swellingas
a function of SDS concentrationattains a plateau value near the CMC of SDS (44),
suggestingthat monomersthat bind to the corneum,includingthe accessible siteson
the membrane,may be enhancingcorneocyte swelling.This is consistentwith the
presentobservation that initial surfactantbindingto thecorneumexhibitsa nearplateau
or a slowly rising region aroundthe surfactantCMC. Interestingly, it has also been
reported(44) that the surfactant-induced swellingprocess is reversibleup to about35
mM (1%) SDS, but is not reversible aboveabout70 mM (2%), indicatingirreversible
damageat suchlevels.The sharprisein the SDSbinding beyondthe CMC alsooccurs
around60 mM, suggesting that thisriseis responsible
for the irreversible
damageto the
corneum.

The origin of the sharpincrease

in SDSbindingoccurringaround60 mM may involve
contributions from miceliar exclusion and the associated excluded volume effects. Be-
yondthe CMC, the corneocyte
envelope
mayactasa semipermeable
membranethat does
not allow the micellesto penetrateinto the cells. This is similar to Middleton's(45,46)
conceptthat the corneocyte
membraneactsas a semipermeable envelopeholdingthe
water-absorbingNMFs (naturalmoisturizing
factors)
within the cellandallowingwater
to move across the membrane. Exclusion of micelles will indeed result in an osmotic
effectdue to an increasein the concentration of micelles,leadingto mechanicalstresses
on the membrane.In normal systems,one would have expecteddeswellingof the
corneumbecause of osmoticstresses. Interestingly,the reportedresultsdo not indicate
any deswellingabovethe surfactant CMC (44). This may be because the waterrespon-
siblefor the swellingis associated with the hydrationof the surfactant-bound proteins
and the NMFs, and the removalof the boundwater may requirea critical pressure
(energy)that is greaterthan that whichcanbe achievedby micellarexclusion.However,
anotherpathwayis availableto releasethe osmoticstress.This may involveunfolding
of the membraneproteinsthat arealreadyweakenedby surfactantbinding, leadingto
a physicalbreakdown of themembrane structure.The unfoldedproteins,in turn, expose
moresitesfor surfactant binding.This phenomenon canalsobeviewedin the following
manner:When surfactantbindsto water-swollen proteinsin a cooperative fashion,
micellesare essentiallytransferredfrom solutionto the "solid" protein phase.This
process leadsto a decrease in the chemicalpotentialassociated with the micelles.If this
chemicalpotentialdifference,coupledwith the free energyof cooperative surfactant-
proteinbinding,is greaterthanthemolecularinteractions holdingthe proteinnetwork
intact, the protein shouldunfold to accommodate boundmicelles.Thus an increasein
 BINDING OF SURFACTANTS 197

binding occursabovethe CMC. Obviouslythe situationis complex,and a quantitative

model is requiredto sort out the details.
All of the four factors discussed earlier are in line with the results obtained for the
kineticsof SDS binding to humanandguineapig stratumcorneum.In both thesecases,
the binding wasfound to increasewith increasein time up to about six hours. Super-
imposedoverthis increase,thereexistsa sharplyrisingregionat SDS levelswell above
the CMC. As mentionedearlier, the relativelymild SLI, on the other hand, doesnot
exhibit sucha marked increasein binding aboveits CMC.
The relativemagnitudes of differentmiceliarcontributions
to enhancedsurfactantbind-
ing abovethe CMC arenot clearat present.Bindingexperiments with already-dispersed
corneocytesaswell asthosewith barriersthataredamaged by techniquessuchasthermal
pretreatmentor sonicationmay providebetterinsightinto the relativecontributions.

EFFECT OF pH ON SLI BINDING

As mentionedearlier, SLI binding wasfoundto exhibit a minimum aroundpH 7-9,

with an increasein both the acidicand alkalineregions.This behaviorcanbe explained
in termsof the dependence of propertiesof the stratumcorneumproteinsand lipids on
pH. An underlyingassumption in the followinganalysisis that "true equilibrium"is
difficult to attain in a complexsystemsuchas stratumcorneumand that the reported
bindingat differentpH valuesmaybeat differentstagesof equilibriumdependingupon
suchfactorsasthermodynamic bindingpotential,accessibility to differentbindingsites,
etc. An increasein pH canbe expectedto decrease the binding of the anionicsurfactant
to keratin.This is because keratinhasisoelectric pointsat aboutpH 5 (47). Abovethis
pH, the net negativechargeon the proteincanbe expectedto increasewith increasein
pH. This, in turn, will lower the thermodynamic driving forcefor surfactantbinding
to the protein. The experimentallyobserved decreasein binding with increasein pH up
to abouta pH of 9.0 is consistent with this expectation.However,the binding appears
to increaseabovepH 9. This is possiblydue to the effectof pH on matrix lipids aswell
asthe membranelipids. In general,the aqueoussolubilityof the fatty acids,which are
a significantcomponentof the bilayerlipidsin the stratumcorneum(5), canbe expected
to increasewith increasein pH, especiallyat pH valuesabove8 or 9. Even though the
ionizationof the carboxylicacidgroupcanoccurat pH valuesabove5, formationof an
acid-soapcomplexlimits the solubilityof the soapbelow pH 9. Above this pH, a
markedincreasein solubilitywith pH canbe expected.Thus, the delipidizationprocess
itselfmay be morefavorableat pH valuesabove9. Furthermore,the negativechargeon
the proteinstructureitselfcanbe expectedto increase with increase in pH, andthis may
reducethe conformational stabilityof the foldedprotein. Thus the overallaccessibility
and vulnerabilityof the structureto surfactants canbe expectedto increaseeventually
with increasein pH. This cutoffseemsto be aroundpH 9 and becomes pronouncedat
pH values > 10.
The effectof pH on the mildnessof formulatedskin cleansingcompositions is complex
andstill a matterof debate(seereference
48 for a reviewof the subject).For formulations
containingpotentiallyionizablecomponents, Murahataand Aronsonrecentlyshowed
that increasein pH canhavea pronounced effecton mildness(48). The abovediscussion
198 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

and the resultsin Figure8 indicatethat an alkalinepH canalsoincreasethe suscepti-

bility of the stratumcorneumto increased binding of anionicsurfactants,which can in
turn lead to protein unfolding. This effect is thus likely to also contributeto the
harshness of alkalinecompositions
to the skin.

SUMMARY AND CONCLUSIONS

1. TEA-oleatebindsto stratumcorneumin higheramountsthan all other surfactants

at low surfactantconcentrations. This is consistentwith the expectedhigher surface
activityof longer-chainsoapscomparedto lower-chain-length soapsandsurfactants.
At concentrations aboveabout20 mM (for example,at a concentration of 40 mM),
the surfactantbinding to HSC is about the same for oleate, laurate, and SDS.
Importantly, the binding of SLI is significantlylower than that of the other three
surfactants.The extent of binding correlateswith the irritation potential of these
surfactantsmeasuredby the zein dissolutiontest as well as with the well-known
clinical irritation of cleansingbarsbasedon thesesurfactants.
2. Binding of SDS, TEA/Na oleate,and TEA/Na laurateto HSC increases abovethe
surfactantCMC. Similarly, the bindingof SDSto guineapig stratumcorneumshows
a sharpincreaseat concentrations well aboveCMC. The reasonsfor the increasein
surfactantbinding abovetheir CMC are not clearat present.The role of micellesin
removinglipids and increasingthe accessibility of membraneproteinsto surfactant
monomersmay be implicatedin the enhancedsurfactantbinding at concentrations
above the CMC.
3. Increasingthe temperaturefromroomtemperature to 37øCresultsin an increase
in
binding of SDS to HSC, with no measurable
effecton SLI.
4. The SLI exhibitsa pH-dependentbindingbehaviorto HSC, with a minimum around
the pH 7-9 region.

REFERENCES

(1) C. Prottey, Factorswhich determinethe skin irritation potentialof soapsand detergents,

J. Soc.
Cosmet.Chem., 26, 29-46 (1975).
(2) G. Imokawa, K. Sumura, and M. Katsumi, Study on skin roughnesscausedby surfactants:II.
Correlationbetweenproteindenaturation
and skin roughness,
J. Am. Oil. Chem.Soc.,52, 484-489
(1975).
(3) G. Imokawaand T. Takeuchi,Surfactants and skin-roughness, Cosmet.Toiletr., 91, 32-46 (1976).
(4) L. D. Rhein and F. A. Simion, "SurfactantInteractionswith Skin," in InterfacialPhenomena in
BiologicalSystems,SurfactantScienceSeries,Vol. 39, Max Bender,Ed. (Marcel Dekker, New York,
1991), pp.33-49.
(5) W. Matthies, "DermatologicalObservations (Humans)," in AnionicSurfactants, Their Biochemistry,
ToxicologyandDermatology, 2nd ed., Surfactant ScienceSeries,Vol. 43, C. Gloxhuberand K. Kun-
stler, Eds. (Marcel Dekker, New York, 1992), pp. 291-329.
(6) J. SteinhardtandJ. A. Reynolds,MultipleEquilibriain Proteins (AcademicPress,New York, 1969),
pp. 234-301.
(7) C. Tanford, The Hydrophobic Effect.'
Formationof Micelles
and BiologicalMembranes, 2nd ed. (Wiley-
Interscience, New York, 1980), pp. 146-164.
(8) M. J. Schwugerand F. G. Bartnik, "Interactionof Anionic Surfactantswith Proteins,Enzymesand
Membranes,"in AnionicSurfactants, SurfactantScienceSeries,Vol. 10, C. Gloxhuber, Ed. (Marcel
Dekker, New York, 1980), pp. 1-49.
 BINDING OF SURFACTANTS 199

(9) M. N. Jones,Surfactantinteractions
with biomembranes
andproteins,Chem.Soc.Rev., 21, 127-136
(1992).
(10) I. D. Robb, "Polymer-Surfactant Interactions,"in AnionicSurfactants, Physical
Chemistry
ofSurfactant
Action, SurfactantScienceSeries,Vol. 10, Lucassen-Reyenders, Ed. (Marcel Dekker, New York,
1981), pp. 109-142.
(11) K. P. Ananthapadmanabhan, "Protein-Surfactant Interactions,"in Interactions
ofSurfactants
with ?oly-
mersand Proteins,E. D. Goddardand K. P. Ananthapadmanabhan, Eds. (CRC Press,Boca Raton,
Florida, 1993), pp. 319-366.
(12) E. Gotte, Skin compatibilityof tensidesmeasuredby their capacityfor dissolvingzein, in Proc.4th
Int. Cong.SurfaceActiveSubs.,Brussels,
3, 83-90 (1964).
(13) F. Swanson,Clinical evaluationof a new neutraldetergentbar,J. Am. Med. Assoc.,162, 459-461
(1956).
(14) D. D. Strube, S. W. Koontz, R. I. Murahata, and R. F. Theiler, The flex washtest: A test method
for evaluatingthe mildnessof personal
washingproducts,J.Soc.Cosmet. Chem.,40, 297-306 (1989).
(15) O. LaurieandJ. Oakes,Protein-surfactant interactions:
Spinlabel studyof interactionof BSA with
SDS,J. Chem.Soc.FaradayTrans.I, 72, 1324-1328 (1976).
(16) J. Oakes,Protein-surfactantinteractions:
NMR andbindingstudiesof interactions betweenBSAand
SDS,J. Chem.Soc.FaradayTrans.I, 70, 2200-2209 (1974).
(17) M. N. Jones,Biochem. J., 151, 109-114 (1975).
(18) K. P.M. Ananthapadmanabhan, M. Aronson,N.J. Turro, and X. Lei, A spectroscopic study of
interactionof SDS with bovineserumalbumin, Langmuir,11, 2525-2533 (1995).
(19) K. Kerr, Surfactant-proteininteractions,Unpublishedresults,Unilever Research,Port Sunlight,
1990.
(20) J. C. Griffith andA. E. Alexander,Equilibriumadsorptionisothermsfor wooldetergentsystems, J.
Coll. InterfaceSci., 25, 311-316 (1967); seealsoJ. ColloidlnterfaceSci.
25, 317-321 (1967).
(21) S. P. Harrold and B. A. Pethica,Thermodynamics of the adsorption
of smallmoleculesby proteins,
Trans.FaradaySoc.,54, 1876-1884 (1958).
(22) I. H. Blank and E. Gould, Penetrationof anionicsurfactantsinto skin, J. Invest.Dermatol.,33,
327-336 (1959).
(23) J. A. Faucherand E. D. Goddard,Interactionof keratinoussubstrateswith sodiumlauryl sulfate:
Sorption,J. Soc.Cosmet.
Chem.,29, 323-338, (1978).
(24) R. D. Kulkarni and E. D. Goddard, "Destructionof the ElectrophysiologicalPotentialof Excised
Frog Skin by Surfactants,"in Advances
in Bioelectrochemistry.'
Ions,Surfaces,Membranes, Adv. Chem.
Series,no. 188, M. Blank, Ed. (ACS, Washington,D.C., 1976)pp. 445-459.
(25) G. Imokawaand T. Takeuchi,Surfactants andskin-roughness,Cosmet.Toiletr.,91, 32-46 (1976); G.
Imokawa, Comparativestudyon the mechanismof irritation by sulfateand phosphatetype anionic
surfactants,"J.Soc.Cosmet.Chem.,31, 45456 (1980).
(26) M. M. Breuer,The interactionbetweensurfactantsand keratinoustissues,
J. Soc.Cosmet. Chem.,30,
41-64 (1979).
(27) J. G. Dominguez,J. L. Parra, R. M. Infante, F. Pelejero,F. Balaguer,and T. A. Sastre,New
approachto the theoryof adsorptionand permeabilityof surfactants on keratinicproteins:Specific
behaviorof certainhydrophobicchains,J. Soc.Cosmet. Chem.,28, 165-182 (1977).
(28) A. Conradsand H. Zahn, A studyof the interactionof sodiumdodecylsulfatewith proteinsof human
heelstratumcorneum,Int. J. Cosmet. Sci., 9, 29-46 (1976).
(29) P. D. Wertz, D. C. Swartzendruber,D. J. Kitko, C. Kathi, M. D. Madison,andT. Downing, The
role of the corneocyte
lipid envelopein cohesionof the stratumcorneum,J. Invest.Dermatol.,89,
169-172 (1987).
(30) D.C. Swartzendruber,P. W. Wertz, M.D. Madison, and D. T. Downing, Evidence that the
corneocytehasa chemicallyboundlipid envelope,J. Invest.Dermatol.,88, 709-713 (1987).
(31) R. I. Murahata, R. Toton-Quinn,and M. Finkey, Effectof pH on the productionof irritation in a
chamberirritation test,J. Am. Acad.Dermatol.,18, 62456 (1988).
(32) P.M. Elias, Epidermallipids, barrierfunctionanddesquamation,
J. Invest.Dermatol.,80, 44s-49s
(1983).
(33) R. L. Hill andL. D. Rhein,Characterization
of the sub-micellar
speciesin mixedsurfactantsolutions.
I: Mixtures of sodiumdodecylsulfateand an alkyl polyethoxysulfate,J. Dispersion
Sci. Tech.,9(3),
269-308 (1988).
200 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

(34) G. Imokawa,S. Akasaki,Y. Minematsu,andM. Kawai, Importanceof intercellularlipidsin water-

retentionpropertiesof the stratumcorneum:Inductionandrecovery
studyof surfactantdry skin,Arch.
Dermatol. Res., 281, 45-51 (1989).
(35) A. W. Fulmer and G. J. Kramer, Stratumcorneumlipid abnormalitiesin surfactant-induced
dry
scalyskin,J. Invest.Dermatol.,86, 598-602 (1986).
(36) M. Fartasch,T. L. Diepgen, and O. P. Hornstein,Morphologicalchangesof epidermallipid layers
of stratumcorneumin sodiumlaurylsulfateinduceddry skin:A functionalandultrastructural study,
J. Invest.Dermatol.,96, 617 (1991).
(37) M. Rieger, Skin lipids and their importanceto cosmeticscience,Cosmet.
Toiletr., 102(7), 36-49
(1987).
(38) A. W. Rawlings,A. Watkinson,J. Rogers,H. J. Mayo, and I. R. Scott,Abnormalitiesin stratum
corneumstructure,lipid composition,and desmosome
degradationin soap-induced
winter xerosis,J.
Soc.Cosmet.Chem.,45, 203-220 (1994).
(39) K. P. Ananthapadmanabhan, M. Misra, K. K. Yu, and M.P. Aronson,Unpublishedresults,Uni-
lever Research, 1996.
(40) S. Mukherjee,S. Prowell, K. Hoyberg,R. Gursky,M. Davies,C. L. Meyers,K. P. Ananthapad-
manabhan,and M. Aronson,Cleanserinducedstructuralchangesin corneum.Presentedat the 53rd
Annual Meeting of the AmericanAcademyof DermatologyNew Orleans,February1995.
(41) M. Takahashi,M. Aizawa,K. Miyazawa,andY. Machida,Effectsof surfaceactiveagentson stratum
corneumcell adhesion,"J. Soc.Cosmet.
Chem.,38, 21-28 (1987).
(42) T. Egelrudand A. Lundstrom,The dependence of detergentinducedcell dissociation
in non-palmo-
plantarstratumcorneumon endogenous proteolysis,
J. Invest.Dermatol.,90, 456-459 (1992).
(43) C. S. King, P. J. Dykes,and R. Marks, Preparationand characterization
of the nonionicdetergent
solubleproteinsof human stratum comeum,J. Invest.Dermatol.,79, 297-302 (1982).
(44) L. D. Rhein, C. R. Robbins,K. Fernee,and R. Cantore,Surfactantstructureeffectson swellingof
isolatedhuman stratumcorneum,J. Soc.Cosmet. Chem.,37, 125-139 (1986).
(45) J. D. Middleton, The mechanismof water binding in stratum corneum, Brit. J. Dermatol.,80,
437-450 (1968).
(46) J. D. Middleton, The mechanismof actionof surfactants
on the water binding propertiesof isolated
stratum corneum,J. Soc.Cosmet.
Chem.,20, 399-412 (1969).
(47) E. D. Goddard,Substantivitythroughcationicsubstitution,Cosmet.
Toiletr., 102, 71-78 (1987).
(48) R. I. MurahataandM. P. Aronson,The relationshipbetweensolutionpH and clinicalirritancyfor
carboxylic
acid-based
personal
washingproducts,
J. Soc.Cosmet.
Chem.,45, 239-246 (1994).