The Skin-permeation-Enhancinegff Ect Of

8/2/2019 The Skin-permeation-Enhancinegff Ect Of

http://slidepdf.com/reader/full/the-skin-permeation-enhancinegff-ect-of 1/12

j. Cosmet.ci., 53, 363-374 (November/December002)

The skin-permeation-enhancingffect of

phosphatidylcholine:affeineas a modelactive ngredient

CHINHAN KIM, JONGWON SHIM, SANGHOON HAN, and

IHSEOP CHANG, SkinResearchnstitute, acificCo./ROD Center,

314-1, Bora-ri,Kiheung-eup,ongin-si, younggi-do,49-900, Korea.

Acceptedor publication pril 29, 2002.

Synopsis

Phospholipidsr liposomes re recognizedo have skin permeation nhancing bility, although heirmechanismsre still controversial.he aim of this studywas o establish methodof increasinghe skinpermeation f active ngredients, singphosphatidylcholinesa permeation nhancer. affeinewasusedas

a model active ngredientand in vitroskin penetration xperiments ere performed sing Franz-typediffusion ells o determine he amountof absorbed affeine. ipid vesicles ereprepared y the micro-fluidizationprocess. he encapsulationfficiency f caffeinewas ound o be very ow due to the instabilityof the liposome tructure nd he watersolubilityof caffeine. owever, he amountof absorbed affeinewas

nearly ndependent f the encapsulationfficiency nd the vesiclesize,but increased ith the increase f

phosphatidylcholineoncentration.hese esultsndicated hat phosphatidylcholineouldact asa penetra-tion enhancer,rrespective f its presencen vesicular orm or solubilized orm.

INTRODUCTION

Recently, many cosmeticproductshave claimed biological functions such as anti-wrinkle, anti-aging, anti-acne,alepigmentation,tc. To take real effecton the skin, the

biologically ctive ngredients houldbe absorbednto the skin. For that reason, opical

deliveryof active ngredients asgainedconsiderablenterest n cosmetic cience1,2).However, he stratumcorneumof the skin formsan excellentbarrier o externalappli-cationand it is necessaryo employsomepenetrationenhancers r appropriate ehiclesto increasehe skin permeation f the active ngredients 3).

The useof phospholipidso increase kin permeation as beenstudiedwidely. Phos-pholipidsare used in solubilized orm as penetrationenhancers4,5) or used as avesicular eliverysystem 6-9). The major advantage f phospholipidss a lower evelof the tendency oward he inducement f skin irritation, compared ith that of typical

penetrationenhancers10,11).

In this study,we investigatedhe skin permeation nhancing bility of phosphatidyl-

Address ll correspondenceo IhseopChang.

363



364 JOURNAL OF COSMETIC SCIENCE

Table I

Formulations or Skin Permeation xperiments

Vesicle

Sample Composition (wt%) pH size Z mean)

1 Phosphatidylcholine 2.0 5.98 51 nmCaffeine 0.5

Propylene lycol 10Water 87.5

2 Hydrogenated hosphatidylcholine 2.0 6.45 141 nmCaffeine 0.5

Propyleneglycol 10Water 87.5

3 Sorbitan oleate 2.0 5.50 224 nm

Caffeine 0.5


Control Caffeine 0.5 6.70 Solution


choline,usingcaffeine s a model active ngredient.Caffeine s a relativelypolar com-pound with low solubility either in water (22 mg/ml) or in oil, commonlyused in

cosmeticproducts.Such a property is a characteristiceature of many other natural

compoundshat can be used as valuablecosmetic ctive ingredients.The aim of thisstudy was to establisha method of increasinghe skin permeationof suchactiveingredients.

MATERIALS AND METHODS

MATERIALS

Soybean hosphatidylcholinePhosphoripon©0G, purity 93 + 3%) wasobtainedrom

Nattermann PhospholipidGmbH (Cologne,Germany).Hydrogenated oybean hos-phatidylcholineS100-3,purity > 90%) wasprovided y LipoldGmbH (Ludwigshafen,Germany).Caffeinewaspurchasedrom Sigma.Sorbitan leate Arlace 80) wassup-plied by Uniquema Wilmington, DE). Propylene lycol,methanol,and aceticacidwereall reagent grade.

LIPID VESICLE PREPARATION

Phosphatidylcholine,r hydrogenated hosphatidylcholine,nd sorbitanoleatewere

solubilizedn propylene lycolat 55øC.Caffeinewasadded o this phosphatidylcholine-propyleneglycol solutionand stirred or 20 minutes.Then deionizedwater was addedand the mixture was homogenized sing a homomixer Mark II, F-model, Tokushu

Kika, KogyoCo. Ltd., Japan)at 5000 rpm for threeminutes.This dispersion as urtherhomogenizedsingMicrofiuidizer model110 E/H (Microfluidics o., Newton,MA)for five cycles t 1000 bar (12,13).



PHOSPHATIDYLCHOLINE AS PENETRATION ENHANCER 365

20 -

18

16

14

12

0 500 60000 200 300 400

Size(nm)Figure 1. Vesicle izedistribution f sample (2.0% phosphatidylcholine,.5% caffeine, 0% propyleneglycol).

DETERMINATION OF VESICLE SIZE

Samples erediluted with distilledwaterand the vesicle izedistributionsweredeter-minedby photoncorrelation pectroscopyZetasizer 000HS, Malvern nstruments td,Malvern, UK). The measurementswere conducted n a CONTIN mode, and the inten-

sity-basedmean vesiclesizeswere reported.

ENCAPSULATION EFFICIENCY

The encapsulationfficiency f the lipid vesicles asdetermined y the spincolumngelfiltrationmethod 14). One gram ofSephadex 50 wasswollen n 10% propylene lycol

aqueous olution or threehours ndwashedwo timeswith the same olution.A smallpieceof glasswoolwas nsertedn the bottomof a 10-ml syringe ndthe prepared elwaspoured nto the syringe.To separatehe encapsulatedaffeinerom free caffeinenthe solution,1 ml of the vesicledispersion asaddedand the columnwas centrifugedfor two minutesat 2000 rpm andagainwith 1 ml of 10% propylene lycolsolution.Theconcentrationf eluted caffeinewas analyzedby HPLC. Free caffeinemoleculeswere




20

18

16

14

12

to 10

• 8

o 60000 200 300 400 500

Size(nm)Figure 2. Vesiclesizedistributionof sample (2.0% hydrogenatedhosphatidylcholine,.5% caffeine,10% propyleneglycol).

bound to the Sephadex el and remained n the column. The ratio of the amount of

entrappedcaffeine o the total amountof caffeine n the dispersionwas calculated.

IN VITRO SKIN PERMEATION EXPERIMENT

In vitro skin permeation estswere performedwith abdominalskin of a femalehairless

guineapig (strain IAF/HA-hrBR) using Franz diffusioncells (Lab Fine Instruments,Korea). The receptorcompartments f the Franz cells were filled with 5 ml of PBSsolution pH 7.4) andconstantly tirredby a magneticbarat 600 rpm. The excised kinsamplesweremountedwith the stratumcorneumsides acing he donorcompartments,

witha contactrea f0.636cm .Each 00 •1of the ipidvesicleispersiono be estedwasapplied o donorcompartments,nd the temperature f the systemwasmaintainedat 32øC by a circulatingwater jacket.The entire contentof each eceptor olutionwaswithdrawn t 6, 12, and24 hours fter he ipid dispersion asapplied, nd he receptorcompartments ererefilledwith freshPBS solution o maintain he sink condition.The

receptor olutionsampleswere assayedor caffeineby HPLC.




20

18

16

14

12

c::: O

c:: 8

6

4

2

0 I0 100 200 300 400 500 600

Size(nm)Figure 3. Vesiclesizedistributionof sample (2.0% sorbJtan leate,0.5% caffeine, 0% propylene lycol).

Threedifferentsetsof skinpermeation xperiments ereperformedn this study. n the

first set, the skin-permeation-enhancingffectsof phosphatidylcholine,ydrogenatedphosphatidylcholine,ndsorbitan leatewerecomparedFigure4). The second etwasorganized o reveal the effectsof the vesiclesize (Figure 7) and the encapsulation

efficiencyFigure8). The concentrationffectofphosphatidylcholineas nvestigatednthe third set (Figure 9).

HPLC ASSAY

All samples ere ilteredwith a 13-mm diskfilter (poresize0.45 pm) before njection.The analysiswas performed with an Agilent 1100 series Agilent Technologies)equippedwith a diodearraydetector.A ZorbaxSB-C18 (Agilent Technologies)olumn

wasused or the stationary hase, nd the mobilephasewasa mixtureof 60% methanol,39% water, and 1% acetic acid at a flow rate of 0.8 ml/min. Caffeine was detected at a

wavelength f 272 nm.




250

• 200E

o 15o

E

D_ 100

o• 50

PC

HydrogenatedPCSorbitan Oleate

Control

i i I

0 5 10 15 20 25

Time(hr)Figure 4. Cumulativeamountsof caffeinepermeated hrough excisedguinea pig skin from differentformulations seeTable I). PC, phosphatidylcholine.

RESULTS AND DISCUSSION

LIPID VESICLE PREPARATION

Three different ormulationswere tested or the skin permeationof caffeine Table I).The first contained % phosphatidylcholine,he second ontained2% hydrogenatedphosphatidylcholine,nd the third contained % sorbitanoleate.All the formulationsexcept he controlwereprepared y a Microfiuidizer with the sameoperating ondi-tions (1000 bar, five cycles).However, their vesiclesizesvaried with their composition

(Table ). Sample1, which consisted f phosphatidylcholine,howedhe smallest esiclesize,with the appearancef a yellowish, ranslucentiquid (Figure 1). Sample showeda similarappearance,ut its average esicle izewas 141 nm (Figure2). Sorbitan leateformedan opaque,milky white dispersionn which he average esicle izewasabove

200 nm (Figure 3).

IN VITRO SKIN PERMEATION OF CAFFEINE

The samples ere ested or the in vitroskinpermeation f caffeine. o remove he effectof the concentrationradient, he caffeine nd propylene lycolcontentswere ixed at




40

35

30

25

• 20

m 15

10

0

0 2000 4000 6000 8000 10000

Size(nm)

Figure5. Vesicle ize istributionfsample afterhomogenizationtep2.0%phosphatidylcholine,.5%caffeine,10% propyleneglycol).

0.5% and 10%, respectively,n all the formulations.he cumulativemounts f per-meated affeinen the receptorompartmentereplottedversusime (Figure ), andthe values t 24 hourswerecomparedo assesshe differences,singa one-tailed-testwith a 95% confidenceevel. Sample1 (phosphatidylcholine)howed significantincreasen the skinpermeationf caffeine ompared ith othersamplesnd he control(P-valuebetween amples and 2: 0.036; P-valuebetween amples and 3: 0.012).Sample (hydrogenatedhosphatidylcholine)nd sample (sorbitan leate) lso n-creasedkinpermeationompared ith the control, ut theireffects erenot sostrongas in sample 1.

The resultof thisskinpermeationxperiment uggestedhat phosphatidylcholineithan unsaturatedatty acid chain would have the most effectiveskin-permeation-enhancingbility.Althoughhere resome ifferent xplanationsbout hemechanismof action 7-9, 15,16), t is widelyacceptedhat unsaturatedhospholipidsanbe usedto increase kin permeation, nd this agreeswith our experimentalesult.

EFFECT OF VESICLE SIZE

Viewed rom hepointof thevesicularizeof the samples,t canbe nterpretedhat theabove xperimentalesultndicateshat hesmalleresiclesouldbe hemore ffective




20,

18

16

14

• 8

6

4

2

0

0 100 200 300 400 500 600

Size(nm)Figure 6. Vesiclesize distributionof sample1 after microfluidization tep (2.0% phosphatidylcholJne,

0.5% caffeine,10% propyleneglycol).

in skin permeation.To investigate he effect of size, phosphatidylcholineesicles f

different size were preparedand tested for the permeationof caffeine.The large-sizevesicleswere collectedat the homogenization tep, and the small-sizevesicleswerepreparedby the final microfiuidization rocess, s describedn Materialsand Methods.The sizeof the vesicles ollected t the homogenizationtepshowed road anges f 700

nm-8 pm (Figure5) with two peaks.The sizeof the vesicles btainedafter the micro-fiuidization tepwassmaller han 400 nm (Figure6).

The skin permeation xperimental esult s shown n Figure 7. There wasno significantdifferencebetween he large- and small-sizevesicles reparations, ndicating that the

permeationmechanism f caffeine s not related o vesiculardelivery. n a vesiculartransdermal eliverysystem, drug or an activematerial s encapsulatedn the vesiclesand thesevesicles enetrate he skin to a certaindepth with their structure ntact (7). In

this case, he smallervesicles avean advantagen penetration.However,contradictoryresultsabout the relation between the size of the vesicles nd the skin permeationof

drugswere eported 8). These mply that therecouldbe othermechanismsf actionbywhich phosphatidylcholinencreaseskin permeation.




250

200 -

150

100

• 50

• Microfluidized

............ omogenized--o-- Control

! I I i

0 5 10 15 20 25

Time(hr)Figure 7. Effectof vesicle izeon the skinpermeationf caffeine. icrofluidized,esicle izesmaller han400 nm; homogenized,esicle ize700 nm-8 I•m. Both samples ontained .5% caffeine, .0% phospha-tidylcholine, nd 10% propylene lycol.Controlwas0.5% caffeine nd 10% propylene lycolsolution.

ENCAPSULATION EFFICIENCY

To investigate he lack of the contributionof vesiculardelivery, the encapsulationefficiency f caffeinewasdetermined y the spin columngel filtration method.Forfreshly reparedhosphatidylcholineiposomes,he encapsulationfficiency as34%,but three daysafter the preparationstoredat room temperature)he encapsulatedcaffeinewas reduced o 3.3%. It is the rapid breakdown f the liposome tructureand

the water solubilityof caffeine 22 mg/ml) that lead to the release f encapsulatedcaffeine.hus t isveryhard o expecthat caffeine ouldpenetratehe skin n the formof an intact vesicle.

This concept asalso alidated y a skinpermeation xperiment singan emptyvesicle.

Dispersionsf emptyphosphatidylcholineesicles ereprepared y the samemethod,usinga Microfiuidizer, andpre-solubilizedaffeine asadded o this dispersionustbefore he skin permeation xperimento reduce he spontaneousnsertionof caffeineinto the vesicles. his preparation ieldednearly dentical esults n comparison ithcaffeine-loaded esicles Figure 8).




250

• 200E

o 150

E

Q_ 100

• 5Oo

Caffeine loaded vesicle

Empty vesicle + Caffeine solutionControl

i I I

0 5 10 15 2o 25

Tirne(hr)Figure 8. Relationshipetweenhe encapsulationfficiency nd the skinpermeationf caffeine. affeine-

loaded esicles ere he same reparationf sample as n Table . The empty esicles ereprepared ythe samemethod, xcepthat caffeine asadded fter hemicrofluidizationtep.Controlwas0.5% caffeineand 10% propyleneglycol solution.

EFFECT OF PHOSPHOLIPIDS

According o the aboveresults,neither the structurenor the size of the vesicles s an

important actor n the skinpermeationf caffeine. he possibleoleof phospholipidsin enhancingkinpermeations to disrupt he stratum orneumipid compositionndincreasets fluidity (17). This effectwasexpectedo be concentration-dependent,ndsothreekindsof formulationsiffering n phosphatidylcholineoncentrationerepre-paredand tested or the skin permeation f caffeine. he resultshowedhat the increase

in phosphatidylcholineoncentrationnduced he increase f caffeine ermeationFig-ure 9).

CONCLUSION

In our study,we found hat phosphatidylcholinenhanced kin permeation f caffeineirrespectivef its vesicular haracteristics,uchas sizeand encapsulationfficiency.t




3OO

250

•- 200

o

"• 150

E

Q- 100

o• 50

= 1% PC

............. 2% PC

+ 4%PC

0 5 10 15 20 25

Time(hr)Figure 9. Effectof phosphatidylcholineoncentrationn the skin permeation f caffeine.

can be interpreted hat phosphatidylcholinetself canact as a permeation nhancer,tsmechanismmore closely onnected ith modulatingskin barrier function han withvesicular elivery. n fact,mostof the caffeinemoleculesn the formulationwere ocatedin the continuous hase nd not in the vesicles ecause f caffeine's ater solubilityandleakagerom the vesicles. il-solublematerialsmay ead o different esults ecauseheytend to remain in the vesicles. owever,phosphatidylcholines a reliable,mild, skin

permeation nhancerhat canbe used n cosmetic roducts.

REFERENCES

(1) J. L. Zatz, Optimizing skin delivery,Cosmet.oiletr.115, 31-35 (2000).(2) J. W. Wiechers,Avoiding ransdermalosmetic elivery,Cosmet.oilerr. 15, 39-46 (2000).(3) S. Magdassi nd E. Touitou,NovelCosmeticelivery ystemsMarcelDekker, New York, 1999), pp.

1-97.

(4) Y. Yokomizoand H. Sagitani,Effects f phospholipidsn the percutaneousenetration f indometh-acin hrough he dorsal kinof guineapigs n vitro, . Contr.ReL,38, 267-274 (1996).

(5) Y. Yokomizo and H. Sagitani,The effectsof phospholipids n the percutaneousenetrationofindomethacinhrough he dorsal kin of guineapig in vitro.2. The effects f the hydrophobic roupin phospholipidsnd a comparisonith general nhancers,. Contr.ReL,42, 37-46 (1996).




(6)

(7)

(8)

(9)

(10)

(11)

(12)

(13)

(14)

(15)

(16)

(17)

H. Schreier, ndJ. Bouwstra, iposomes nd niosomess opicaldrug carriers:Dermal and ransdermal

drug delivery, . Contr.Rel., 30, 1-15 (1994).G. Cevcand G. Blume, Lipid vesicles enetrate nto intact skin owing to the transdermal smoticgradientand hydration orce,Biochim. iophys. cta, 1104, 226-232 (1992).

G. M. E1Maghraby, . C. Williams, andB. W. Barry,Skindeliveryof oestradiolromdeformablendtraditionaliposomes:echanistictudies,. Pharm.harmacol.,1,1123-11341999).G. M. E1Maghraby,A. C. Williams, andB. W. Barry,Skindeliveryof oestradiolrom ipid vesicles:Importance f liposome tructure,nt.J. Pharm., 04, 159-169 (2000).H. Sasaki,M. Kojima,J. Nakamura, ndJ. Shibasaki, cute oxicityandskin rritationof pyrrolidonederivatives s transdermal enetrationenhancer,Chem.Pharm.Bull., 38, 2308-2310 (1990).

T. K. Ghosh,W. R. Pfister,and S. I. Yum, Transdermalnd Topical rug Delivery ystemInterpharmPress,BuffaloGrove, ll., 1997), pp. 191-214.

E. Mayhew,R. Lazo,W. J. Vail, J. King, and A.M. Green,Characterizationf liposomes reparedusinga microemulsifier, iochim. iophys.cta, 775, 169-174 (1984).J. C. Vuillemard, Recent advancesn the large-scale roductionof lipid vesiclesor use in food

products:Microfluidization, . Microencapsul.,, 547-562 (1991).

D. W. Fry, J. C. White and I. D. Goldman,Rapid separation f low molecularweight solutesromliposomes ithout dilution,J. Anal. Biochem.,0, 809-815 (1978).

M. Kirjavainen,A. Urtti, R. Valjakka-Koskela, . Kiesvaara, nd J. M6nkk6nen,Liposome-skininteractionsnd heir effects n the skinpermeation f drugs,Eur.J. Pharm.Sci.,58, 207-217 (1999).F. P. Bonnia,L. Montenegro,N. Scrofani, t al., Effects f phospholipid ased ormulations n in vitro

and n vivopercutaneousbsorption f methyl nicotinate,. Contr.Rel., 34, 53-63 (1995).M. Kirjavainen, . M6nkk6nen, M. Saukkosaari, . Valjakka-Koskela, . Kiesvaara, nd A. Urtti,Phospholipidsffectstratumcorneum ipid bilayer luidity and drug portioning nto the bilayers, .Contr.Rel., 58, 207-214 (1999).

Documents

The Skin-permeation-Enhancinegff Ect Of