j. Soc.Cosmet.

Chem.,44, 123-128 (March/April1993)

I.iposomesin cosmetics:Whichkindof phospholipid?

Whichloadingmethod?
ADRIANA

MEMOLI,

LUISA G. PALERMITI,

VALTER TRAVAGLI, and FRANCO ALHAIQUE, Dipartimento

di Studidi Chimicae Tecnologia
delleSostanze
Biologicamente
Attive,
Universitadi Roma(La Sapienza),Rome(A.M., L.G.P.),
Dipartimento
FarmacoChimicoTecnologico,
Universita
di Siena,Siena
(V. T. ), and Dipartimento
FarmacoChimicoTecnologico,
Universita
di
Cagliari, Cagliari (F.A.), Italy.
Received
October
14, 1992.

Synopsis

Phospholipids
of differentorigin (eggand soya)and purity wereusedto prepareliposomesby sonication.
Loadingof thesevesicles
wasperformedby meansof two differenttechniques
usinga fluorescent
lipophilic
modelmolecule.The stabilityof the aggregated
structures
wascheckedby additionof increasing
amounts
of a surfactantto the liposomedispersion.
No remarkabledifferences
wereobserved
in eitherthe stability
in regardto surfactant-induced
breakageor the loadingcapacityof liposomesrespectivelypreparedwith
99% pure egg phosphatidylcholine
or with the vegetablephospholipid,a commercialproductthat had a
muchlowerpurity. The comparison
of the two loadingmethodsindicatedthat incorporation
of the model
moleculewithin the vesiclestructurewashigherwhenthe fluorescent
markerwasaddedbeforesonication.

INTRODUCTION

It is well known that double-chainamphiphiles,suchas phospholipids,are capableof

aggregatinginto bilayersthat assumethe form of liposomes:
closedspheres
of different
structuresand dimensionsthat canbe loadedwith activeingredients.Becauseof these
properties,liposomesare presentin severalpharmaceutical
preparationsand are largely
used in cosmetics.

The origin, the extraction,and the purificationmethod, and consequentlythe final

compositionand purity of the phospholipids
usedfor the preparationof liposomes,can
lead to dramaticallydifferentpricesand at the sametime to a great varietyof loading
capacityand stabilitystructures
(1).
The aim of this work was to comparethe behaviorof a 99% pure egg phosphatidylcholine(EPC) with that of a vegetablephospholipidthat had a muchlowerpurity and
price(P90). In this senseit is alsointerestingto pointout that, asfar asthe origin(egg
or soya)is concerned,the vegetablephospholipidappearsto be more appropriatefor
123

124

JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

topicalapplications
in cosmetics
anddermatology
because
of the high contentof polyunsaturated
fatty acids(2), like linoleicacid,whichareparticularlyvaluablein cosmetic
preparations
(1). The effectsof differentmethodsof liposomeloadingwerealsoconsidered.

Sincethe main expecteddifferences

must be relatedto the bilayerstructureof the
vesicles,1,6-diphenyl-l,3,5-hexatriene(DPH) (3) was chosenfor our studies.This
fluorescentprobe, whoseinteractionwith phospholipidvesicleswasrecentlyreviewed
(4), is localizedwithin the lipid bilayers,andthe phospholipidphasetransitionsinduce
only very smallchangesin DPH excited-state
interconversion.
Stabilityin regardto surfactant-induced
breakage
andloadingcapacitywererespectively
evaluatedby meansof turbidity measurements
and fluorescence
determinations
of the
probeincorporated
or absorbed
in the hydrophobic
bilayerof the vesicles
preparedby
sonication.

MATERIALS

99% pureL-ot-phosphatidylcholine
from eggyolk (Sigma,type III-E, hexanesolution,
100 mg/ml; and type XI-E, chloroform
solution,100 mg/ml) and90% pureenriched
soyaphosphatidylcholine
(Phospholipon
90, NattermannPhospholipids
GmbH) were
usedfor vesiclepreparation.CrystallineDPH waspurchased
from Sigma.Solutionsand
dispersions
of thismarkerwerepreparedjustbeforeuseandhandledasmuchaspossible
in the dark because
of the photosensitivity
of DPH (4).

pH 7.5 HEPESsolutions
(10- 3 M), madewithfreshly
distilled
andaleaerated
water,
were used. Cholesterol,Triton X-100, and all other productsused for the present
investigationwere of analyticalgrade. All solventswere testedfor fluorescence
at the
wavelengthof interestfor our studies.

Fluorescence
measurements
werecarriedout by meansof a Perkin Elmer LS5 spectrofluorometerusingan excitationwavelengthof 350 nm and an emissionof 425 nm (slit
5/5 nm). Turbidity was evaluatedwith the sameinstrument,with excitationand
emissionwavelengths
both setat 600 nm.
Sonication
wasperformedwith a Soniprep150 apparatus
(MSE, Crowley)equippedwith
a 19-mm probe, operatingat 23 KHz and with an amplitudeof 6 m.
A phospholipids
B test kit (Wako ChemicalsGmbH) wasusedfor quantitativedeterminations of these substances.

METHODS

VesiclescontainingDPH werepreparedaccordingto two differenttechniques.

METHOD

A (MIXED

FILM)

The appropriate
amountofphospholipid
(80 mg Of P90 or 800 1 of EPCsolution),5.6

mg of cholesterol,
and222 1 of a 2 x 10-4 M methanol
solution
of DPH were
completelydissolved
in 4-5 ml of methanol.The solventwasvacuumevaporated
to

COSMETIC

LIPOSOMES

125

form a thin film of lipidsandadditivesinsidethe vessel.2.5 ml of HEPES bufferwere

added;the mixture was kept in the dark for 2 h, then gently shakenfor I h and
sonicated,
undera nitrogenstream,for40 min (8 timesfor 5 min). The temperature
was

maintained
at 15-20Cby meansof a waterbath.The liposome
dispersion
wasfinally
diluted

1:1 with

HEPES.

METHOD

B (ABSORBED FLUOROPHORE)

SUV wereprepared
according
to thesameprocedure
described
above,but nomarkerwas
addeduntil the final dilution. This 1:1 dilution at the endof the vesiclepreparationwas
performed
with a DPH dispersion
prepared
asfollows:4-5 ml of methanolwereadded
--4
to 222 Ixl of the 2 X 10

M methanolsoluuonof DPH, the solventwas vacuum

evaporated,2.5 ml of HEPES wereaddedto the residue,and the mixture wasthen

vortexedand sonicatedto obtain a homogeneous
dispersionof DPH. Unmarked liposomes
werekept in the darkovernightwith thefluorophore
dispersion.
Longertimesdid
not significantlyincrease
the amountof absorbed
DPH.
It hasbeenpointedout (5) that sonicationof phospholipiddispersions
leadsmainly to
smallunilamellarvesicles
(SUV, 10-100 nm), but according
to the aim of this study,
actualliposomesizeswerenot determined.

Liposomeseparationfrom the "free" phospholipids

and non-incorporated
DPH was
performedon 1-ml sampleswith Sephadex
G200. Columnswere elutedwith HEPES
andall the vesicles
werecollected(liposomes
wereelutedwith the void volumeand their
presence
wascheckedby meansof a turbiditytest)to reacha final volumeof 5 mi. The
phospholipids
B testwasperformed
beforeandafterthe passage
throughthe columnsin
orderto verifythe percentage
of aggregated
formwith respectto the total amountused.
All final preparations
containingthe vesicles
weretestedfor turbidity. The reproducibility of theselast measurements,
performedon the differentpreparations,indicated
that the averagedimensions
and concentration
of liposomes
were to be considered
as
constant(e.g., for all liposomaldispersions
corresponding
to a phospholipid
concentration of 0.3 mg/ml, turbidity = 71.8 + 2.1).
DPH fluorescence
was initially determinedon intact purified liposomesin order to
verifyoncemorethe reproducibility
amongthe variouspreparations
of the samekind.
The vesiclestructurewasthen brokenby dilution (1:9) with methanolfor the determination of the total amount of DPH presentin the vesicles.Quantitative DPH
determinationswere obtainedfrom appropriatecalibrationcurvesof the marker in
methanol.

In orderto studyand comparethe resistance

of liposomes,the changein turbidity by
progressive
additionof a surfactant
(Triton X-100) wasmeasured
(6,7).

RESULTS

AND

DISCUSSION

In Table I the fluorescences

of DPH in the vesicledispersionare reportedfor the
differenttypesof phospholipids
andfor the two methodsof vesicleloading.In the same
tablethe fluorescence
measured
whenvesicles
werebrokenwith methanolis alsogiven.

126

JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

Table

Fluorescence
Valuesof DPH-LoadedLiposomes
Prepared
With Phospholipids
of DifferentType and
Fluorescence Determined

Phospholipid

on Vesicles Broken With

Methanol

MethodA

MethodB

EPC

170.4 + 8.5

148.5 + 8.0

EPC (broken SUV)

P90
P90 (broken SUV)

47.9 + 2.5
160.0 -+ 8.2
45.5 + 2.5

34.7 + 2.0
144.3 + 8.0
33.7 + 2.0

Reportedresultsrepresentthe meanvaluesobtainedfrom five separateexperiments.

Reproducibilitycanbe evaluatedby the low rangeof fluorescence
fluctuationsin the
differentpreparations("5%). As it is possibleto observe,only a small differencein
DPH fluorescence
betweenEPC and P90 liposomeshasbeendetected.When compared
with intact vesicles,brokenliposomesin methanolalwaysgave a much lower fluorescencebecause
of the presence
of the organicsolvent(4). From the fluorescence
values
reportedin this table, it is alsopossibleto observethat lessDPH waspresentin the
"Method B" formulationsbecauseof the smalleramount of the probe that can be
liposomallyincorporated
by meansof thistechnique.The markeractuallypresentin the
vesicleswascalculatedfrom the valuesdeterminedin methanol(i. e., afterthe breakage
of the aggregated
structure),wherefluorescence
is linearlydependenton DPH concentration. In this sense,it must alsobe pointed out that surfactantsare often usedto
disaggregate
liposomestructures,but their presence
leadsto higherfluorescence
values
in water dispersions,gives non-linearcalibrationcurves,and can inducefluorescence
changesin the fluorophore(8-10) that canyield uncorrector misleadingresults.
The phospholipidtest indicatedthat over 95% of the initial amountof phospholipids
was recoveredas SUV after the passagethroughSephadex;nevertheless,
for a correct
comparison
amongthe differentpreparations,
theseminorvariationshavebeenconsideredand the percentage
of entrappedor absorbed
DPH wascalculated
accordingto the
followingexpression:
[DPH]a
% DPH

[DPH]b

x K x

100

wherethesubscripts
a andb indicate
theDPH concentrations
(mmoles
x ml-) after
and beforepurification,respectively,
and K is the ratio betweenphospholipidconcentrations (mg/ml) beforeand after the passagethrough Sephadex.The coefficientK
allowscomparisonof the differentpreparations
by consideringthe small lossof phospholipid during purificationand by correctingat the sametime the dilution factor.

In TableII thepercentage
of directlyentrapped
(MethodA) or absorbed
DPH onempty
vesicles(MethodB) is givenfor both EPC and P90. No variationsbetweentype III-E
andXI-E EPC weredetected.Reportedresultsarethe averagevaluesobtainedfrom five
separateexperiments.

As it canbe observedfrom obtainedresults,the differencein loadingcapacitybetween

EPC and P90 vesicles,althoughdetectable,is alwaysbelow4%.

In orderto comparethe stabilityof EPC and P90 liposomes,

the changes
in turbidity
of the vesicledispersionby addition of increasingamountsof Triton X-100 were

COSMETIC

LIPOSOMES

Table

127

II

Effectof the Type of Phospholipidand of the LoadingTechniqueon the Percentageof DPH in

the Vesicles

% DPH in liposomes

Phospholipid
EPC
P90

MethodA

MethodB

74.9 + 3.6
71.2 + 3.2

54.1 - 3.0
52.6 + 3.1

evaluated.The turbidity initially increased,indicatingthat surfactantmoleculeswere

incorporated
by the vesicles;
then it decreased
almostlinearlybecause
of the formation
of mixedmicelies(9). The trendof thesecurvescanbe directlyrelatedto the stability
of the aggregatedstructurein the form of vesicles(7). Resultsreportedin Figure 1,
which refer to severaldifferentpreparationsand phospholipidconcentrations,indicate
that no differencewasobservedbetweenthe two typesof liposomes.

CONCLUSIONS

Froman overallcomparison
betweenEPC andP90 liposomes,
reportedresultsindicate
that the differences,
althoughdetectable
in somecases,
areneversuchthat theysupport
the useof the 99% pureandmuchmoreexpensive
productfor largescaleor commercial
preparations.

1,5

1,0

0,5

0,0

10.

15

20

TritonX-100(Mx 104)
Figure 1. Effectof increasing
surfactant
concentration
on the turbidityof liposomedispersions.
Turbidity
changes
are expressed
asthe ratio betweenthe valueobserved
in the presence
of Triton X-100 and that of
the referencewithout surfactant.Reportedexperiments
refer to EPC and P90 liposomes.Phospholipid
concentrations
were 0.30 mg/ml and 0.90 mg/ml. For the higherphospholipid
concentration,
abscissa
valuesmust be multiplied by 3.0.

128

JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

Experimentalresultsindicatealsothat the amountof lipophilicprobein the liposome

structureis affectedby the loadingmethod;consequently,
the possibilityof incorporating hydrophobic
substances
in emptyliposomes
(that canbe founddirectlyon the
market)can lead to a productthat is differentfrom that obtainedwhenliposomes
are
preparedfrom a co-precipitated
film.

REFERENCES

(1) H. Lautenschliiger,
J. RiSding,and M. Ghyczy,The useof liposomes
from soyaphospholipids
in
cosmetics,
SOFW, 114, 531-534 (1988).
(2) J. Pikul and F. A. Kummerow,Thiobarbituricacid reactivesubstance
formationas affectedby
distributionof polyenoicfatty acidsin individualphospholipids,
J. Agric.Food.Chem.,39, 451-457
(1991).

(3) B. J. LitmanandY. Barenholz,

Fluorescent
probe:Diphenyhexatriene,
Methods
Enzymol.,
81, 678685 (1982).

(4) T. Parasassi,
G. De Stasio,R. M. Rush,andE. Gratton,A photophysical
modelfor diphenylhexatrienefluorescence
decayin solventsand in phospholipid
vesicles,Biophys.
J., 59, 466-475
(1991).

(5) H. Ringsdorf,B. Schlarb,andJ. Venzmer,Moleculararchitecture

andfunctionof polymericoriented
systems:
Modelsfor the studyof organization,surfacerecognitionand dynamicsof biomembranes,
Angew.Chem.Int. Ed. Engl., 27, 113-158 (1988).
(6) K. Anzai, H. Utsumi, K. Inoue, S. Nojima, and T. Kwan, Electronspinresonance
studieson the
interactionbetweenliposomalmembraneand Triton X-100, Chem.Pharm.Bull., 28, 1762-1767
(1980).

(7) G. Strauss,
F. Alhaique,A. Memoli,E. Santucci,andF. M. Riccieri,The stabilityofdrugocarrying
liposomes
in the presence
of detergents,Polymer
Preprints,
27, 48 (1986).
(8) N.J. Turro, M. Griitzel,andA.M. Braun,Photophysical
andphotochemical
processes
in micellar
systems,Angew.Chem.Int. Ed. Engl., 19, 675-696 (1980).
(9) A. K. Mathur, C. Agarwal, B. S. Pangtey,A. Sing, and B. N. Gupta, Surfactant-induced
fluorescencechangesin fluorescein
dye, Int. J. Cosmet.
Sci., 10, 213-218 (1988).
(10) Y. H. PaikandS.C. Shim,Photophysical
properties
of psoralens
in micellarsolutions,
J. Photochem.
Photobiol.
A, 56, 349-358 (1991).