j. Cosmet. 53, 363-374 (November/December Sci.

, 2002)

The skin-permeation-enhancing effectof phosphatidylcholine: Caffeineas a modelactiveingredient

CHINHAN KIM, JONGWON SHIM, SANGHOON HAN, and IHSEOP CHANG, SkinResearch Institute, Pacific Co./ROD Center,

314-1, Bora-ri,Kiheung-eup, Yongin-si, Kyounggi-do, 449-900, Korea.

Accepted publication for April 29, 2002.
Synopsis

Phospholipids liposomes recognized have skin permeation or are to enhancing ability, althoughtheir mechanisms still controversial. aim of this studywasto establish methodof increasing skin are The a the permeation activeingredients, of usingphosphatidylcholine a permeation as enhancer. Caffeine wasusedas a model activeingredientand in vitroskin penetration experiments were performed usingFranz-type diffusion cellsto determine amountof absorbed the caffeine. Lipid vesicles wereprepared the microby fluidization process. encapsulation The efficiency caffeine foundto beverylow dueto the instability of was of the liposome structure the watersolubilityof caffeine. and However,the amountof absorbed caffeine was nearlyindependent the encapsulation of efficiency and the vesicle size,but increased with the increase of phosphatidylcholine concentration. Theseresults indicatedthat phosphatidylcholine couldact asa penetration enhancer, irrespective its presence vesicular of in form or solubilized form.

INTRODUCTION

Recently, many cosmeticproductshave claimed biological functions such as antiwrinkle, anti-aging,anti-acne,alepigmentation, To take real effecton the skin, the etc. biologically activeingredients shouldbe absorbed into the skin. For that reason, topical delivery activeingredients gainedconsiderable of has interestin cosmetic science (1,2). However,the stratumcorneum the skin formsan excellent of barrierto externalapplicationand it is necessary employsomepenetrationenhancers appropriate to or vehicles to increase skin permeation the activeingredients the of (3).

The useof phospholipids increase to skin permeation beenstudiedwidely. Phoshas pholipidsare used in solubilized form as penetrationenhancers (4,5) or used as a vesicular deliverysystem (6-9). The major advantage phospholipids a lowerlevel of is of the tendency towardthe inducement skin irritation, compared of with that of typical penetrationenhancers (10,11).
In this study,we investigated skin permeation the enhancing ability of phosphatidyl-

Address correspondence IhseopChang. all to

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Table I

Formulations Skin Permeation for Experiments

Vesicle

Sample

Composition

(wt%)

pH

size(Z mean)

Phosphatidylcholine
Caffeine

2.0
0.5

5.98

51 nm

Propylene glycol
Water

10
87.5

Hydrogenated phosphatidylcholine
Caffeine

2.0
0.5

6.45

141 nm

Propylene glycol
Water

10
87.5

Sorbitan oleate
Caffeine

2.0
0.5

5.50

224 nm

Propylene glycol
Water

10
87.5

Control

Caffeine

0.5

6.70

Solution

Propylene glycol
Water

10
89.5

choline,usingcaffeine a modelactiveingredient.Caffeineis a relativelypolarcomas pound with low solubility either in water (22 mg/ml) or in oil, commonlyused in cosmeticproducts.Such a property is a characteristic feature of many other natural compounds that can be usedas valuablecosmetic active ingredients. The aim of this study was to establish method of increasing a the skin permeation suchactive of ingredients.

MATERIALS
MATERIALS

AND

METHODS

Soybean phosphatidylcholine (Phosphoripon 90G, purity 93 + 3%) wasobtained from Nattermann Phospholipid GmbH (Cologne, Germany).Hydrogenated soybean phosphatidylcholine (S100-3,purity > 90%) wasprovided LipoldGmbH (Ludwigshafen, by Germany). Caffeine purchased was from Sigma.Sorbitan oleate (Arlace 80) wassup plied by Uniquema(Wilmington, DE). Propylene glycol,methanol,andacetic acidwere
all reagentgrade.
LIPID VESICLE PREPARATION

Phosphatidylcholine, hydrogenated or phosphatidylcholine, sorbitanoleatewere and solubilized propylene in glycolat 55C.Caffeine addedto this phosphatidylcholinewas propylene glycol solutionand stirredfor 20 minutes.Then deionized water wasadded and the mixture was homogenized using a homomixer(Mark II, F-model, Tokushu Kika, KogyoCo. Ltd., Japan)at 5000 rpm for threeminutes.This dispersion further was homogenized usingMicrofiuidizer model110 E/H (Microfluidics Newton,MA) Co., for five cycles 1000 bar (12,13). at

PHOSPHATIDYLCHOLINE

AS PENETRATION

ENHANCER

365

20 18 16
14 12

100

200

300

400

500

600

Size(nm)
Figure 1. Vesicle distribution sample (2.0% phosphatidylcholine, caffeine, size of 1 0.5% 10% propylene glycol).
DETERMINATION OF VESICLE SIZE

Samples weredilutedwith distilledwaterand the vesicle sizedistributions weredeterminedby photoncorrelation spectroscopy (Zetasizer 3000HS, MalvernInstruments Ltd,
Malvern, UK). The measurements were conductedin a CONTIN mode, and the inten-

sity-based meanvesiclesizeswere reported.

ENCAPSULATION EFFICIENCY

The encapsulation efficiency the lipid vesicles determined the spincolumngel of was by filtrationmethod(14). One gramofSephadex G50 wasswollen 10% propylene in glycol aqueous solution threehours for andwashed timeswith the same two solution. small A pieceof glass woolwasinserted the bottomof a 10-ml syringe the prepared in and gel waspoured into the syringe. separate encapsulated To the caffeine from freecaffeine in the solution,1 ml of the vesicle dispersion addedand the columnwascentrifuged was for two minutesat 2000 rpm andagainwith 1 ml of 10% propylene glycolsolution. The concentration elutedcaffeine of wasanalyzed HPLC. Free caffeine by molecules were

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18 16 14

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12

to 10
8

100

200

300

400

500

600

Size(nm)
Figure 2. Vesiclesizedistribution sample (2.0% hydrogenated of 2 phosphatidylcholine, caffeine, 0.5% 10% propylene glycol).

bound to the Sephadex and remainedin the column. The ratio of the amount of gel entrapped caffeineto the total amountof caffeinein the dispersion was calculated.
IN VITRO SKIN PERMEATION EXPERIMENT

In vitroskin permeationtestswereperformedwith abdominalskin of a femalehairless guineapig (strain IAF/HA-hrBR) using Franz diffusioncells(Lab Fine Instruments, Korea). The receptorcompartments the Franz cellswere filled with 5 ml of PBS of solution (pH 7.4) andconstantly stirredby a magnetic at 600 rpm. The excised bar skin samples weremountedwith the stratumcorneum sides facingthe donorcompartments,

witha contact of0.636cm Each i1 thelipidvesicle area 2. 300 of dispersionbetested to

wasappliedto donorcompartments, the temperature the system and of wasmaintained at 32C by a circulatingwater jacket.The entire contentof eachreceptor solutionwas withdrawn 6, 12, and24 hours at afterthe lipid dispersion applied, the receptor was and compartments wererefilledwith freshPBSsolutionto maintainthe sink condition.The receptor solutionsamples were assayed caffeine HPLC. for by

PHOSPHATIDYLCHOLINE

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ENHANCER

367

20 18 16

14
12

c:::lO
c:: 8
6 4
2

0
0 100

I
200 300 400 500 600

Size(nm)
Figure 3. Vesiclesizedistribution sample (2.0% sorbJtan of 3 oleate, 0.5% caffeine, 10% propylene glycol).

Threedifferentsets skinpermeation of experiments wereperformed this study.In the in first set, the skin-permeation-enhancing effects phosphatidylcholine, of hydrogenated phosphatidylcholine, sorbitan and oleate werecompared (Figure4). The second was set organizedto reveal the effectsof the vesiclesize (Figure 7) and the encapsulation efficiency (Figure8). The concentration effect ofphosphatidylcholine investigated was in the third set (Figure 9).

HPLC

ASSAY

All samples werefilteredwith a 13-mm diskfilter (poresize0.45 pm) before injection. The analysiswas performedwith an Agilent 1100 series(Agilent Technologies) equipped with a diodearraydetector. ZorbaxSB-C18 (Agilent Technologies) A column wasusedfor the stationary phase, the mobilephase and wasa mixtureof 60% methanol,
39% water, and 1% acetic acid at a flow rate of 0.8 ml/min. Caffeine was detectedat a

wavelength 272 nm. of

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PC

200

HydrogenatedPC
Sorbitan Oleate Control

15o

D_ 100

50

10

15

20

25

Time(hr)
Figure 4. Cumulativeamountsof caffeinepermeated through excised guinea pig skin from different formulations (seeTable I). PC, phosphatidylcholine.
RESULTS
LIPID

AND

DISCUSSION

VESICLE

PREPARATION

Three differentformulations were testedfor the skin permeation caffeine of (Table I). The first contained 2% phosphatidylcholine, second the contained 2% hydrogenated phosphatidylcholine, the third contained sorbitan and 2% oleate.All the formulations

except control the wereprepared a Microfiuidizer with the same by operating conditions (1000 bar, five cycles).However, their vesiclesizesvaried with their composition (TableI). Sample1, whichconsisted phosphatidylcholine, of showed smallest the vesicle size,with the appearance a yellowish, of translucent liquid (Figure 1). Sample showed 2 a similarappearance, its average but vesicle was141 nm (Figure2). Sorbitan size oleate formedan opaque, milky white dispersion whichthe average in vesicle wasabove size 200 nm (Figure 3).
IN VITRO SKIN PERMEATION OF CAFFEINE

The samples weretested the in vitroskinpermeation caffeine. remove effect for of To the of the concentration gradient,the caffeine propylene and glycolcontents werefixedat

PHOSPHATIDYLCHOLINE

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369

40

35

30

25

20
15

10

2000

4000

6000

8000

10000

Size(nm)
Figure5. Vesicle distribution sampleafter size of 1 homogenization (2.0%phosphatidylcholine, step 0.5%
caffeine,10% propylene glycol).

0.5% and 10%, respectively, all the formulations. cumulative in The amounts perof meated caffeine the receptor in compartment plotted were versus time (Figure and 4), the values 24 hours at werecompared assess differences, to the usinga one-tailed t-test with a 95% confidence level. Sample1 (phosphatidylcholine) showed significant a increase the skinpermeation caffeine in of compared othersamples the control with and (P-value between samples and 2: 0.036; P-valuebetween 1 samples and 3: 0.012). 2 Sample (hydrogenated 2 phosphatidylcholine) sample (sorbitan and 3 oleate) alsoincreased permeation skin compared the control, theireffects with but werenotsostrong
as in sample 1.

The resultof thisskinpermeation experiment suggested phosphatidylcholine that with an unsaturated fatty acid chainwould have the most effectiveskin-permeation-

enhancing ability. Although there some are different explanations themechanism about of action (7-9, 15,16),it iswidelyaccepted unsaturated that phospholipids be used can
to increase permeation, this agrees skin and with our experimental result.
EFFECT OF VESICLE SIZE

Viewed fromthepointof thevesicular of the samples, canbeinterpreted the size it that above experimental indicates thesmaller result that vesicles would themore be effective

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18 16
14

8
6 4
2

100

200

300

400

500

600

Size(nm)
Figure 6. Vesiclesizedistributionof sample1 after microfluidization step (2.0% phosphatidylcholJne, 0.5% caffeine,10% propylene glycol).

in skin permeation. investigatethe effectof size,phosphatidylcholine To vesicles of different size were preparedand tested for the permeationof caffeine.The large-size vesicles were collectedat the homogenization step, and the small-sizevesicles were prepared the final microfiuidization by process, described Materialsand Methods. as in The sizeof the vesicles collected the homogenization showed at step broadranges 700 of nm-8 pm (Figure5) with two peaks.The sizeof the vesicles obtained after the microfiuidization stepwassmaller than 400 nm (Figure6). The skinpermeation experimental resultis shown Figure7. Therewasno significant in differencebetweenthe large- and small-sizevesicles preparations, indicating that the permeationmechanism caffeineis not relatedto vesiculardelivery.In a vesicular of transdermal deliverysystem, drug or an activematerialis encapsulated the vesicles a in and thesevesicles penetrate skin to a certaindepthwith their structure the intact (7). In this case, smallervesicles the havean advantage penetration. in However,contradictory resultsabout the relation between the size of the vesicles and the skin permeationof drugswerereported Theseimply that therecouldbe othermechanisms actionby (8). of which phosphatidylcholine increases permeation. skin

PHOSPHATIDYLCHOLINE

AS PENETRATION

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250

200 -

Microfluidized

............ e Homogenized
--o-Control

150

100

50

10

15

20

25

Time(hr)
Figure7. Effect vesicle on the skinpermeation caffeine. of size of Microfluidized, vesicle smaller size than 400 nm; homogenized, vesicle 700 nm-8 Im. Bothsamples size contained 0.5% caffeine, 2.0% phosphatidylcholine, 10% propylene and glycol.Controlwas0.5% caffeine 10% propylene and glycolsolution.
ENCAPSULATION EFFICIENCY

To investigate the lack of the contributionof vesiculardelivery,the encapsulation efficiency caffeine of wasdetermined the spin columngel filtration method.For by

freshly prepared phosphatidylcholine liposomes, encapsulation the efficiency 34%, was

but three daysafter the preparation (storedat room temperature) encapsulated the caffeine wasreduced 3.3%. It is the rapid breakdown the liposome to of structure and the water solubilityof caffeine (22 mg/ml) that lead to the release encapsulated of caffeine. Thusit isveryhardto expect caffeine that wouldpenetrate skinin theform the
of an intact vesicle.

Thisconcept also was validated a skinpermeation by experiment using emptyvesicle. an Dispersions emptyphosphatidylcholine of vesicles wereprepared the same by method, usinga Microfiuidizer andpre-solubilized , caffeine added this dispersion was to just before skin permeation the experiment reduce spontaneous to the insertion caffeine of into the vesicles. This preparation yieldednearlyidenticalresults comparison in with
caffeine-loaded vesicles (Figure 8).

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Caffeine

loaded vesicle

200

Empty vesicle + Caffeine solution

Control

150

Q_ 100

5O

10

15

2o

25

Tirne(hr)
Figure 8. Relationship between encapsulation the efficiency the skinpermeation caffeine. and of Caffeineloaded vesicles thesame were preparation sample asin TableI. The empty of 1 vesicles prepared were by thesame method, except caffeine added that was afterthemicrofluidization Control 0.5% caffeine step. was
and 10% propylene glycol solution.
EFFECT OF PHOSPHOLIPIDS

Accordingto the aboveresults,neither the structurenor the size of the vesicles an is

important factorin the skinpermeation caffeine. possible of phospholipids of The role in enhancing permeation to disrupt stratum skin is the corneum lipid composition and increase fluidity (17). This effect expected be concentration-dependent,so its was to and threekindsof formulations differing phosphatidylcholine in concentration prewere
paredandtestedfor the skinpermeation caffeine. of The resultshowed that the increase

in phosphatidylcholine concentration induced increase caffeine the of permeation (Figure 9).

CONCLUSION

In our study,we foundthat phosphatidylcholine enhanced skin permeation caffeine of irrespective its vesicular of characteristics, as sizeand encapsulation such efficiency. It

PHOSPHATIDYLCHOLINE

AS PENETRATION

ENHANCER

373

3OO

250

= 1% PC ......... 2% PC .... + 4%PC

200

"

150

E
Q100

50

10

15

20

25

Time(hr)
Figure 9. Effectof phosphatidylcholine concentration the skin permeation caffeine. on of

canbe interpreted that phosphatidylcholine itself canact asa permeation enhancer, its mechanism more closely connected with modulatingskin barrierfunctionthan with vesicular delivery.In fact,mostof the caffeine molecules the formulation in werelocated in the continuous phase and not in the vesicles because caffeine's of water solubilityand leakage fromthevesicles. Oil-soluble materials mayleadto different results because they tend to remain in the vesicles. However,phosphatidylcholine a reliable,mild, skin is permeation enhancer that canbe usedin cosmetic products.

REFERENCES

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(4) Y. YokomizoandH. Sagitani, Effects phospholipids the percutaneous of on penetration indomethof acinthroughthe dorsal skinof guinea pigsin vitro, Contr. J. ReL,38, 267-274 (1996). (5) Y. Yokomizo and H. Sagitani,The effectsof phospholipids the percutaneous on penetration of indomethacin throughthe dorsal skinof guineapig in vitro.2. The effects the hydrophobic of group in phospholipids a comparison general and with enhancers, Contr. J. ReL,42, 37-46 (1996).

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