The outermost layer of mammalian skin, the stratumcorneum, by virtue of its unique architecture presents a sig-nificant barrier to the transdermal delivery of drugs. Re-cently, biophysical, morphological, and biochemical resultshave indicated that the stratum corneum forms a continuoussheath of protein-enriched corneocytes embedded in an inter-cellular matrix enriched in nonpolar lipids that are organizedas lamellar layers.1,2) Although only 10 to 15% of the stratumcorneum mass is comprised of lipids, these lipids largely dic-tate the overall skin permeability property.3) Transdermaldrug delivery promises many advantages over oral or intra-venous administration, and as a consequence it would bevery useful to reduce the skin barrier using physical ap-proaches and chemical enhancers, which would reversibly re-move the barrier resistance of the stratum corneun and thusallow the drug to penetrate into the viable tissue and enter thesystemic circulation.

There is continued interest in the development of strate-gies to alter the skin barrier to percutaneous absorption ofcompounds. Oleic acid (OA) has been studied as a skin pene-tration enhancer for drugs via its action primarily on stratumcorneum lipid structures. Studies have shown that the modesof action of OA have generated two mechanistic scenarios,which may account for the action of this enhancer: 1) lipidfluidization; and 2) lipid phase separation.4) Since the stratumcorneum lipids constitute the primary permeability barrier ofthe skin and these lipids exist in the stratum corneum inter-cellular spaces as a highly organized lamellar bilayers,1,5) it isreasonable to expect that OA applied to the skin would emul-sify or otherwise disorganize the stratum corneum lipidlamellae and thus increase skin permeability. In the presentstudy, we investigated the use of ruthenium tetroxide postfix-ation and a lanthanum tracer study to ascertain the effects ofOA on the stratum corneum lipids and epidermal permeabil-ity barrier function.

MATERIALS AND METHODS

Materials OA and propylene glycol (PG) were of analyt-

ical grade and purchased from Sigma Chemical (St. Louis,MO, U.S.A.). Ruthenium tetroxide and lanthanum nitratewere from Polysciences Inc. (Warrington, PA, U.S.A.).

Experimental Protocols Male Wistar rats weighing250—300 g were used in this study. Under general anesthesiawith 4% chloral hydrate by intraperitoneal injection, the hairfrom the back of the animals was clipped and then shavedwith an electric razor. The rats were treated on the back with10% OA/PG-soaked cotton balls. The other group serving asa control was treated with PG-soaked cotton balls. Each wasrolled gently on the skin surface for 2 h at 5-min intervals,and neither OA nor PG application produced damage to thestratum corneum.

Electron Microscopy Samples were taken from theflanks of animals for electron microscopy after either OA orPG treatment. Briefly, samples were fixed in modifiedKarnovsky’s fixative overnight at 4 °C, which contained 2%paraformaldehyde, 2% glutaraldehyde, 0.06% CaCl2, in 0.1 M

cacodylate buffer, pH 7.4. The tissues were then washed in0.1 M cacoylate buffer, postfixed in either 0.25% rutheniumtetroxide (RuO4) in 0.1 M cacodylate for 45 min in the dark atroom temperature or in 1% osmium tetroxide (OsO4) in 0.1 M

cacodylate buffer for 2 h at room temperature. The specimenswere then rinsed in buffer, dehydrated in graded ethanol solu-tions, and embedded in an Epon–epoxy resin mixture. Ultra-thin 60—80 nm sections were examined under a Jeol-1200electron microscope operated at 80 kV after staining withuranyl acetate/lead citrate.

Lanthanum Tracer Studies To evaluate the extent ofepidermal permeability barrier disruption after OA treatment,lanthanum nitrate was used to delineate the pathway and ex-tent of water permeation through the epidermis. Sampleswere soaked in equal parts of modified Karnovsky’s fixativeand 8% lanthanum nitrate solution, which contained 8% su-crose in 0.05 M Tris buffer, pH 7.6, for 1 h at room tempera-ture. Then the samples were rinsed with fixative, fixed inmodified Karnovsky’s fixative overnight at 4 °C, postfixed in1% OsO4 for 2 h, and processed as described above. Thinsections were stained with uranyl acetate only for electron

66 Biol. Pharm. Bull. 26(1) 66—68 (2003) Vol. 26, No. 1

∗ To whom correspondence should be addressed. e-mail: jiangsjbzmc@yahoo.com © 2003 Pharmaceutical Society of Japan

Examination of the Mechanism of Oleic Acid-Induced PercutaneousPenetration Enhancement: an Ultrastructural Study

Shao Jun JIANG* and Xiao Jun ZHOU

Department of Pathology, Jinling Hospital; 305 Zhong Shan Dong Lu, Nanjing, 210002, P. R. China.Received March 18, 2002; accepted June 11, 2002

The epidermal permeability barrier appears to be regulated primarily by the lamellar arrangement of lipidbilayers between coneocytes of the stratum corneum and presents a significant barrier to the transdermal deliv-ery of drugs. The aim of the present study was to investigate the effects of oleic acid on the ultrastructure of stra-tum corneum lipids in rat skin. Wistar rats were treated topically with 10% oleic acid/propylene glycol for 2 h,the structure of stratum corneum was examined by electron microscopy using osmium tetroxide or rutheniumtetroxide postfixation, and the epidermal barrier function was evaluated in a lanthanum tracer study. Ultrastruc-tural examination revealed that there was a marked alteration in the stratum corneum and the tracer penetratedinto the intercellular spaces of the stratum corneum after application of oleic acid. These results suggest thatruthenium tetroxide postfixation is a powerful tool for the study of the stratum corneum lipid structure. Oleicacid might increase the epidermal permeability through a mechanism involving the perturbation of stratumcorneum lipid bilayers and lacunae formation to enhance transdermal drug delivery.

Key words stratum corneum lipid; penetration enhancement; oleic acid; ruthenium tetroxide; lanthanum tracer study

microscopy.

RESULTS AND DISCUSSION

Ultrastructural Observations Figure 1 shows that aftertreatment with either 10% OA/PG or PG alone using OsO4

postfixation. As shown in Fig. 1A, the corneocytes of thestratum corneum were cohesive in PG-treated epidermis.However, the stratum corneum was considerably disrupted by2-h OA treatment. As illustrated in Fig. 1B, the corneocyteswere widely separated and most of them were removed fromthe upper stratum corneum. This finding may reflect the sim-plest approach to enhance physically the percutaneous ab-sorption of a compound across the skin by stripping off theoutermost layers of the skin, the stratum corneum. A sequen-tial increase in transepidermal water loss is observed as thestratum corneum is progressively removed, indicating that theepidermal permeability is increased.6)

To gain insight into the mechanism by which OA affectsthe epidermal permeability barrier, we next examined the ve-hicle-treated and OA-treated epidermis using RuO4 postfixa-tion. In vehicle-treated epidermis, the intercellular lipid bi-

layers of the stratum corneum were tightly packed, and therewas no deviation from the normal (Fig. 2A, normal notshown). As shown in Fig. 2B, after 2-h application of OA,the intercellular lipid bilayers had altered drastically, and theintercellular domains of the stratum corneum revealed an ab-sence of normal appearance; instead, elongated lacunae ap-peared within the widened interstices of the stratumcorneum. This result demonstrates that the application of OAcauses distinct alterations in the stratum corneum domains.

The organization of the stratum corneum resembles as abrick wall, with the bricks representing the corneocytes andthe mortar representing the intercellular lipids. The lipids(mortar) are thought to be the major element underlying bar-rier function, particularly to water and other hydrophilic ma-terials.1,2) The ability of OA to enhance percutaneous absorp-tion has long been known.3,7,8) It appears that the stratumcorneum lipids constitute the epidermal permeability barrier,and that disruption of this stratum corneum lipid structurewill put the skin barrier at risk. Recent results obtained withattenuated total reflectance infrared spectroscopy suggest thatthe action of OA could be due to both mechanisms, i.e., lipidfluidity and lipid phase separation.4) Another theory suggeststhat the penetration enhancement effect of OA primarily dis-orders the alkyl chain near the polar region of the lipid bilay-ers, and lipid phase separation can result in substantially en-hanced permeability in mammalian skin.7) However, anychanges in the lipid matrix, especially increased fluidity ofthe hydrocarbon chains of the lipids or lipid phase separa-tion, would result in higher permeability.9)

Lanthanum Tracer Studies To delineate further the ef-fect of OA-induced skin permeation enhancement, we em-ployed the lanthanum tracer method. Because the intact stra-tum corneum is impermeable and therefore excludes water-soluble lanthanum nitrate, the identification of tracer withinthe stratum corneum indicates the presence of a defect in thepermeability barrier. As shown in Fig. 3A, the tracer perme-ated through the intercellular spaces of the stratum granulo-sum but not the stratum corneum in vehicle-treated epider-mis. However, in OA-treated epidermis, the tracer extended

January 2003 67

Fig. 1. Electron Micrograph of PG or 10% OA-Treated Rat Skin Fixedwith OsO4

After exposure to PG for 2 h, the majority of corneocyes of the stratum corneumwere tightly cohesive (1A). The application of OA resulted in corneocyte separationand most were removed from the upper stratum corneum (1B). Scale bar�1 mm.

Fig. 2. Electron Microscopic Findings in the Stratum Corneum of RatSkin Treated with OA Using RuO4 Fixation

Note the preservation of the intercellular lipid bilayers in PG-treated epidermis (A,arrows). After 2-h OA treatment, the expansion of lacunae (asterisks, B) was observedin the widened intercellular spaces of the stratum corneum. Scale bar�100 nm.

Fig. 3. Lanthanum Nitrate Was Employed as a Tracer to Evaluate the Bar-rier Function

The tracer is found in the intercellular spaces of the granular layers (A, arrowheads),but not in the stratum corneum in PG-treated epidermis. In contrast, after 2-h OA treat-ment, abundant tracer was present within the stratum corneum (B, arrowheads). Scalebar, A�500 nm; B�1 mm.

into the intercellular spaces of the stratum corneum (Fig.3B). This indicates that the increased permeability to wateroccurs within the epidermis with OA treatment.

Lanthanum is an electron-dense trivalent cation materialthat can be used as a tracer for delineating extracellularspaces and intercellular junctions. It can also be used as atracer in studying the permeability barrier.10) The localizationof the barrier defect to the stratum corneum by the applica-tion of OA is supported by the results of lanthanum perfusionstudies, which have shown that the tracer percolates throughthe stratum corneum interstices in OA-treated, but not controlepidermis. A similar situation occurs in the inhibition ofHMG CoA reductase, which results in defective barrier func-tion that correlates with enhanced tracer permeabilitythrough the stratum corneum.11)

The present ultrastructural studies provide clear insightinto the potential mechanism of permeability barrier alter-ations in the skin. Under basal conditions, the stratumcorneum lipids comprise broad compact sheets, organized asa lamellar structure.12) In contrast, the formation of lacunaeand distinct disorganization of the stratum corneum lipid bi-layer structure appeared in OA-treated epidermis. It is rea-sonable to propose a mechanism of percutaneous penetrationenhancement: the formation of lacunae could be assumed tobe one type of permeable defect in the stratum corneum,which reduces the diffusional resistance to enhance water-soluble molecule transport through the stratum corneum. It ispossible that with a penetration enhancer, the lacunae are en-larged and the size of the lacunar domain is increased; thesespaces eventually coalesce, forming a continuous tubuloretic-ular network. As a result, the penetration threshold is eventu-ally breached and forms a pathway for molecule transport.13)

The use of saturated and unsaturated fatty acids such asstearic acid, OA, linoleic acid, and linolenic acid for drugpermeation enhancement is of interest in the area of topicaland transdermal drug delivery systems research. These fattyacids have been shown to be effective penetration enhancersfor a variety drugs.14,15) Selective perturbation of the intercel-lular lipid bilayers in the stratum corneum appears to be themajor route of enhancing the activity of fatty acids. Thecombined infrared spectroscopy and DSC results suggest thatcertain exogenously applied fatty acids can disrupt the stra-

tum corneum lipid structure and increase motional freedomor fluidity of the lipids.8,16) It is not clear from these results,however, whether structural differences seen among the vari-ous acids are due to their relative disrupting power or simplyreflect varied uptake of these compounds by the stratumcorneum. Therefore further studies need to be conducted toclarify this.

In summary, we utilized the RuO4 postfixation and lan-thanum perfusion methods to evaluate the effects of OA-in-duced skin penetration enhancement. The results presentedhere demonstrate that topical application of OA induces stra-tum corneum lipid structure disorder in vivo. OA may en-hance percutaneous penetration mainly through a dual mech-anism involving stratum corneum lipid bilayer perturbationand lacunae formation.

REFERENCES

1) Elias P. M., Menon G. K., Adv. Lipid Res., 24, 1—6 (1991).2) Elias P. M., J. Invest. Dermatol., 80, 44—49 (1983).3) Francoeur K. L., Golden G. M., Potts R. O., Pharm. Res., 7, 621—627

(1990).4) Naik A., Pechtold L. A. R. M., Potts R. O., Guy R. H., J. Control. Re-

lease, 37, 299—306 (1995).5) Swartzendruber D. C., Wertz P. W., Kitko D. J., Madison K. C., Down-

ing D. T., J. Invest. Dermatol., 92, 251—257 (1989).6) Bommannan D., Potts R. O., Guy R. H., J. Invest. Dermatol., 95,

403—408 (1990).7) Ongpipattanakul B., Burnette R. R., Potts R. O., Francoeur M. L., J.

Pharm. Res., 8, 350—354 (1991).8) Takeuchi Y., Yasuwaka H., Yamaoka Y., Takahashi N., Tamura C., Mo-

romoto Y., Fukushima S., Vasavada R. C., Chem. Pharm. Bull., 41,1434—1437 (1993).

9) Bhatia K. S., Gao S., Singh J., J. Control. Release, 47, 81—89 (1997).10) Shaklai M., Tavassoli M., J. Histochem. Cytochem., 30, 1325—1330

(1982).11) Menon G. K., Feingold K. R., Mao-Qiang M., Schaude M., Elias P.

M., J. Invest. Dermatol., 98, 209—219 (1992).12) Hou S. Y. E., Mitra A. K., Whiter S. H., Menon G. K., Gadially R.,

Elias P. M., J. Invest. Dermatol., 96, 215—223 (1991).13) Menon G. K., Elias P. M., Skin Pharmacol., 10, 235—246 (1997).14) Kuljit S. B., Jagdish S., J. Pharm. Sci., 87, 462—468 (1997).15) Aungst B. J., Blake J. A., Hussain M. A., Pharm. Res., 7, 712—718

(1990).16) Golden G. M., McKie J. E., Potts R. O., J. Pharm. Sci., 76, 25—28

(1987).

68 Vol. 26, No. 1