Beruflich Dokumente
Kultur Dokumente
MEMOLI,
LUISA G. PALERMITI,
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
124
topicalapplications
in cosmetics
anddermatology
because
of the high contentof polyunsaturated
fatty acids(2), like linoleicacid,whichareparticularlyvaluablein cosmetic
preparations
(1). The effectsof differentmethodsof liposomeloadingwerealsoconsidered.
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
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
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
RESULTS
AND
DISCUSSION
126
Fluorescence
Valuesof DPH-LoadedLiposomes
Prepared
With Phospholipids
of DifferentType and
Fluorescence Determined
Phospholipid
Methanol
MethodA
MethodB
EPC
170.4 + 8.5
148.5 + 8.0
47.9 + 2.5
160.0 -+ 8.2
45.5 + 2.5
34.7 + 2.0
144.3 + 8.0
33.7 + 2.0
[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.
COSMETIC
LIPOSOMES
Table
127
II
% DPH in liposomes
Phospholipid
EPC
P90
MethodA
MethodB
74.9 + 3.6
71.2 + 3.2
54.1 - 3.0
52.6 + 3.1
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
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