effect of liposomes on delipidized stratum corneum structure: an ‘in vitro’ study based on high...

Colloids and Surfaces

A: Physicochemical and Engineering Aspects 182 (2001) 35–42

Effect of liposomes on delipidized stratum corneumstructure: an ‘in vitro’ study based on high resolution low

temperature scanning electron microscopy

Olga Lopez a, Mercedes Cocera a, Paul Walther b, Ernst Wehrli b,Luisa Coderch a, Jose Luis Parra a, Alfonso de la Maza a,*

a Departamento de Tensioacti6os, Centro de In6estigacion y Desarrollo (C.I.D.),Consejo Superior de In6estigaciones Cientıficas (C.S.I.C.), C/Jordi Girona, 18-26, 08034, Barcelona, Spain

b ETH-Zentrum, Laboratory for Electron Microscopy, Uni6ersitatsstrasse 16, CH-8092, Zurich, Switzerland

Received 04 October 1999; accepted 27 November 2000

Abstract

The structural modifications induced in the delipidized stratum corneum (SC) by the addition of SC lipid liposomeswere investigated ‘in vitro’ using double-layer for high resolution low-temperature scanning electron microscopytechnique (HRLTSEM). Liposomes were prepared from a lipid mixture extracted from the SC with organic solventsand characterized by freeze-fracture electron microscopy. After delipidization and subsequent incubation withliposomes no structural modifications were detected in the SC protein domains. However, the lipid lamellar structuredetected in the native SC was lacking after delipidization. The subsequent treatment with liposomes of this delipidizedtissue led to the formation of new structures in the intercellular spaces that showed similar organization to the lipidlamellae structure of the native SC. Image analyses of the desmosomes found in the delipidized SC and of thestructures formed after treatment with liposomes demonstrated that these new structures did not correspond todesmosomes in spite of the similar appearance of both structures. Hence, the new structures could be associated witha restoration of the damaged lipid lamellae structure of the delipidized SC by the SC lipid liposomes. This effectwould involve the interaction between the lipids of the liposomes and the remaining material after delipidization.© 2001 Elsevier Science B.V. All rights reserved.

Keywords: Delipidized stratum corneum; Stratum corneum liposomes; Freeze-fracture electron microscopy; High-resolution low-temperature scanning electron microscopy

www.elsevier.nl/locate/colsurfa

Abbre6iations: FFEM, freeze-fracture electron microscopy; HRLTSEM, high-resolution low-temperature scanning electronmicroscopy; SC, pig stratum corneum; TLC-FID, thin-layer chromatography/flame ionization detection system.

* Corresponding author. Tel.: +34-93-4006161; fax: +34-93-2045904.E-mail address: [email protected] (A. de la Maza).

0927-7757/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.

PII: S0927 -7757 (00 )00823 -2

O. Lopez et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 182 (2001) 35–4236

1. Introduction

The outermost layer of the mammalian epider-mis, the stratum corneum (SC), consists of thinkeratinized cells (corneocytes) embedded in alipid-enriched intercellular matrix organized inlamellae [1,2]. The permeability barrier is locatedin this lamellar structure that mainly consists ofceramides, free fatty acids, cholesterol and choles-terol sulfate [3]. It has been demonstrated thatmixtures of these lipids are able to form bilayersin the same way that occurred with phospholipids[4,5].

The use of phospholipid liposomes as drugcarriers [6,7] or as membrane models has becomeprogressively popular to study the adsorption ofsubstances and to compare these results withthose obtained in skin studies [8,9]. Althoughvarious authors recently claim the advantages oftopical application of phospholipid liposomes[10–12], there is not consensus in that the lipidvesicles epicutaneously applied really penetrateinto the skin and which would be the cause ofsuch possible penetration. Thus, whereas someauthors suggest that liposomes are not able topenetrate deeper than SC [13,14] other researchershave reported different effects of these vesicles onthe epidermis [15,16].

In earlier papers we studied the formation ofSC lipid liposomes and the interaction of thesevesicles with surfactants [17–20]. The main objec-tive of the present work was to study ‘in vitro’ theinteraction between SC lipid liposomes and deli-pidized SC. Bearing in mind that a number ofcutaneous problems are associated with the lipiddisruption [7,11,12,15], the SC was delipidizedbefore application of liposomes to simulate a skindiseased. In connection with this, this work pro-poses the use of liposomes formed by SC lipids asa possible treatment to improve the damaged lipidstructure of the SC. To this end, double-layercoating for high-resolution low-temperature scan-ning electron microscopy (HRLTSEM) was used.The main advantage of this method over trans-mission electron microscopy freeze-fracture is thatno cleaning of replicas is needed and, conse-quently, their possible fragmentation is avoided[21]. The use of this specific technique may be

useful to know the ability of the SC lipid lipo-somes to diffuse into the intercellular spaces ofdelipidized SC and to be assembled with the lipidsof the corneocyte envelope resulting in a possiblerestoration of the SC lipid organization.

2. Materials and methods

2.1. Delipidization of SC, preparation ofliposomes and incubation with delipidized SC

The separation of hairless pig epidermis and theisolation of SC sheets were based on the methodof Wertz and Downing [22] and it has been previ-ously described in detail [20]. In order to deli-pidize the SC tissue, sheets of native samples weresuccesively extracted for 120 and 60 min withmixtures of chloroform–methanol (2:1, 1:1 and1:2, v/v). The extracted lipids were concentratedto dryness, weighed, redissolved in chloroform–methanol and then used to prepare liposomes.

In order to prepare SC lipid liposomes theextracted lipid mixture was dried under a streamof N2 at 25°C and then hydrated in a bathsonicator in TRIS buffer (pH 7.5) at 70 W, 15min at 70°C. The final lipid concentration ofliposomes (10 mg ml−1) and their compositionwere determined using thin layer chromatographycoupled to an automated flame detection system(TLC–FID) [20]. The vesicle size distribution ofliposomes was determined by dynamic light scat-tering (DLS) using a photon correlator spectrom-eter (Malvern Autosizer 4700c PS/MV, Malvern,UK) [5]. The visualization of liposomes was doneusing a freeze-fracture electron microscopy(FFEM) technique, according to the procedurepreviously described [23,24]. The replicas wereobtained by unidirectional shadowing with 2 nmof Pt/C and 20 nm of C, floated on distilled waterand then examined in a Hitachi H-600AB TEM at75 kV.

In order to study the interaction of liposomeswith delipidized SC, this tissue was incubated withliposomes at 25°C for 18 h. Afterwards, the tissuewas rinsed three times with distilled water andanalysed using HRLTSEM. Given that afterdelipidation by organic solvents the SC was also

O. Lopez et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 182 (2001) 35–42 37

dehydrated, control samples delipidized SC wereincubated in water alone to evaluate the possiblechanges induced by rehydration during incubationwith the liposome suspension.

2.2. High-resolution low-temperature scanningelectron microscopy studies

Three different samples were analyzed: nativeSC, delipidized SC and SC treated with liposomesafter delipidization. Cylindrical pieces of eachsample (diameter of about 2 mm and a length ofabout 1–2 mm) were fixed with 2% (w/v) glu-taraldehyde in 0.1 M sodium cacodylate buffer.The samples were cryoprotected with 30% (v/v)glycerol and mounted on a 3 mm aluminiumholder. Thereafter, the SC samples were frozen byplunging into liquid propane and fractured with amicrotome knife in a Balzers BAF 300 freeze-etching device (Bal-Tec., Liechtenstein) at 10−7

mbar and a temperature of −110°C. After etch-ing for 2 min, the fracture plane was coated with2 nm Pt/C (unidirectional at an angle of 45°)followed by 5–7 nm C at an angle of 90°. After-wards, the cold samples were immediately cryo-transferred on a Gatan cryo-holder into a HitachiS-900, in lens field emission scanning electronmicroscope equipped with a highly sensitive annu-lar YAG-detector for back scattered electrons[21]. Specimens were investigated at −110°C. Thebeam current was 1–3×10−11 A as measuredwith a Faraday cage. The primary acceleratingvoltage was 10 kV. Images were obtained with theback scattered electron signal and recorded digi-tally with a Gatan Digiscan 688 connected to anApple Quadra 950.

In order to study in detail the pictures obtainedby HRLTSEM, the corresponding image analyseswas performed. One-dimensional optical densityprofiles corresponding to the intensity variationsvs distance in the images (nm) were processed insix different areas. In addition, to obtain statisti-cal information the Fourier-transform of theseselected areas were computed and the array sizescorresponding to the intensity peaks distance weredetermined.

Table 1Lipid composition (wt%) of the material extracted from stra-tum corneum forming liposomes

%wtLipids

Ceramides 3627Cholesterol

Free fatty acids 24Cholesteryl esters 4

3Triglycerides5Cholesteryl sulfate

3. Results and discussion

The lipids extracted from the SC were able toform liposomes (lipid composition is shown inTable 1). The dynamic light scattering techniqueshowed for these vesicles a bimodal distributioncurve, in which the size varied in a range between80 and 150 nm. FFEM was a useful technique tovisualize these bilayers, in agreement with previ-ous studies [4,24]. Fig. 1 shows some of thesevesicles with sizes around 100 nm of diameter inaccordance with the dynamic light scatteringmeasurements.

The use of HRLTSEM was suitable to visualizeSC samples according to a method previouslydescribed [21]. This improved SEM techniqueshowed similar resolution than the conventionalfreeze-fracture TEM [25] and has one additionaladvantage: The replica-cleaning process is not

Fig. 1. Freeze-fracture electron micrograph corresponding toSC lipids liposomes. The magnifications are given in themicrograph.


Fig. 2. Low-temperature scanning electron micrograph of thenative SC, overview (A) and detailed (B). The different struc-tures are indicated as follows: corneocytes as arrow, fracturealong the lipid lamellae as arrow heads and fracture acrosslipid lamellae as open arrows.

intercellular space, however, the fracture planegoes along the lamellae of the multilayered lipidorganization resulting in a nice view on the verysmooth and relatively flat surface of the SC lipids(arrow heads). Occasionally the bilayers werefractured straightacross, resulting in sharp edges(open arrows) [21]. This observation, that is moreclearly noted in the more enhanced micrograph ofFig. 2(B), indicates that lipid matrix was formedby multiple layers.

Fig. 3. Low-temperature scanning electron micrograph of deli-pidized SC, overview (A) and detailed (B). The corneocytes areindicated with arrows and the lipid region with arrow heads.

necessary and, consequently, the risk that replicastend to fall into small pieces is avoided.

The micrographs obtained for native SC areshown in Fig. 2(A) and (B). The picture of Fig.2(A) corresponds with an overview in which canbe seen corneocytes (arrows), characterized by theparticular granular pattern of keratins entirelyfilling their interior and by the absence of cellorganelles. Mostly, the fracture plane in the cor-neocyte lies vertically to the skin surface. In the


Fig. 4. Low-temperature scanning electron micrograph of deli-pidized SC after incubation with water alone.

Fig. 5. Low-temperature scanning electron micrograph of deli-pidized SC after incubation with liposomes. The new struc-tures are indicated with arrows.

that these structures consisted in irregular shapes,smooth steps (arrow) and slightly granular frac-ture planes. The fact that these structures werenot detected in either native or delipidized SC(even after incubation with water alone) indicatesthat they were due to the treatment with SC lipidliposomes. Probably the lipids from the liposomeswere able to pass through the intercellular spacesof the delipidized SC and there they form alamellar organization similar to that present in thenative SC. This reorganization of the lipids would

Two micrographs of delipidized SC at differentmagnifications are shown in Fig. 3(A) and (B).These pictures reveal that the granular appearanceof the fracture plane corresponding to the cor-neocytes (keratin tonofilaments, arrows) remainedunaltered after the extraction with organic sol-vents. It is interesting to note that no steps associ-ated with the multilayered lipid organization wereobserved in the lipid domains, although thesmooth fracture plane visualized between the cor-neocytes indicates that some lipids (possibly thoselinked with the corneocyte envelopes) persistedafter delipidization (arrow heads). The disappear-ance of the steps indicates that an important partof the intercellular lipids were extracted by theorganic solvents as it was reported in a previouswork [26]. The control samples of delipidized SCincubated in water alone were also examined byHRLTSEM Fig. 4 and no differences were foundwith respect to the SC delipidized without waterincubation Fig. 3(B). Hence, structural changesinduced by rehydration were rejected.

Fig. 5 shows delipidized SC treated with lipo-somes. Although no changes were found inprotein domains, structural changes in the lamel-lar lipid regions were clearly detected with respectto the delipidized SC. In order to observe in detailthese new structures a more enhanced magnifica-tion of Fig. 5 is shown in Fig. 6. It may be seen

Fig. 6. Higher magnification of an area of micrograph shownin Fig. 5 where the new structures formed after incubationwith liposomes are observed.


Fig. 7. Low-temperature scanning electron micrograph of azone of delipidized SC shown desmosomes.

corneosomes were between 6 and 10 nm (averagedistance of 8 nm and standard deviation of 1.055).The Fourier-transform of digitized images of thecorneosomes that provides more statistical infor-mation also indicated that the peak distance was7.8 nm. This distance corresponded to the diame-ter previously reported for the filaments in desmo-somes [29]. The fact that this distance was notdetected in the new structures and the poor peri-odicity in the profiles demonstrated that thesestructures were not desmosomes.

Hence, we may assume that the new structureswere the result of the interaction of the lipidsforming liposomes with the lipids of the cor-neocyte envelope remaining in the intercellularspaces after delipidization. Although we have notexperimental evidence to explain how the newstructures were formed, their presence in the inter-cellular space requires a previous diffusion of thelipids from the liposomes to the intercellularspaces. In any case the appearance of the newstructures seems to involve a mechanism of re-as-

give rise to the new structures in the fractureplane. Despite this argument, the similarity of thenew structures and the corneosomes (modifieddesmosomes in the SC) found in the delipidizedsample and previously described [27,28] made nec-essary additional analyses of the micrographs.

Thus, a comparison of the corneosomes and thenew structures was performed by means of theimage analysis of digitized SEM pictures. To thisend, a profile of the new structures Fig. 6 wasanalyzed and compared with that shown in Fig. 7.This new figure present corneosomes found in thedelipidized SC, which exhibited the typical granu-lar appearance due to their intermediate filaments[27].

Two density optical profiles performed for thecorneosomes and for the new structures areshown in Fig. 8(A) and (B), respectively. Theright part of each profile corresponds to the back-ground, which was similar in both cases, the slightdifferences observed are due to the fact that mi-crographs of Figs. 6 and 7 come from differentfracture experiments. In the left part of the profi-les, corresponding to the structures studied, cleardifferences could be detected. Thus, the cor-neosome profile Fig. 8(A) shows higher both peri-odicity and number of peaks than that for thenew structures Fig. 8(B). The most frequent dis-tance between peaks in the six profiles of the

Fig. 8. Optical density profiles of representative areas corre-sponding to the new structures presents in Fig. 6 (A) and todesmosomes in Fig. 7 (B). The analyzed areas are indicated ineach micrograph as a continuous line.


sembly of the lipid molecules with the lipidslinked to the outer face of the envelope. Thisre-assembly could be associated with the possiblerestoration of the damaged SC lipid structures bythe action of the incubation with SC lipid lipo-somes. In fact, comparison of pictures of Figs. 2and 5 shows a certain similarity between the lipidareas of both the native and the treated withliposome samples. In any case this possibilityshould be confirmed in ‘in vivo tests’ and corrob-orated in clinical assays.

The effect of lipid and surfactant vesicles on SChas been studied by a number of authors[7,11,12,15,30,31]. In these works different struc-tural SC changes that disturb the intercellularlipids organization have been reported i.e. forma-tion of water pools and vesicles, deposition ofindividual molecules, etc. However, no similarstructures than those detected in our work havebeen reported to date. The main reasons could bethe following: The need of liposomes formed bylipids directly extracted from SC, which may facil-itate the lipid assembly in a similar way that theoriginal arrangement. The use of delipidized SC,which not only could simulate a diseased skin butalso allow the observation of the changes pro-duced in the intercellular regions. Finally, theapplication of the new tecnique of double layercoating for HRLTSEM suitable for obtaining im-ages with the advantages of the FFEM but with-out its drawbacks. This point represents animprovement in the scientific field of the cryotech-niques, which may be useful in future skin studies.

From these findings, it can be establised thatthe SC lipid liposomes induce modifications in thedelipidized SC. These modifications could be as-sociated with a restoration of the damaged inter-cellular lipid structure. This fact suggests apotential therapeutic application of these vesicles,in which the container appears to be as importantas the contents (encapsulated drugs).

Acknowledgements

We thank G. von Knorring for his expert tech-nical assistance. This work was supported by

funds from DGICYT (Prog. no. PB94-0043),Spain.

References

[1] N.D. Lazo, J.G. Meine, D.T. Downing, J. Invest. Derma-tol. 105 (1995) 296.

[2] H. Schaefer, T.E. Redelmeier, in: S. Karger (Ed.), SkinBarrier. Principles of Percutaneous Absorption, Switzer-land, 1996, pp. 55–77.

[3] M. Fartasch, Microsc. Res. Tech. 38 (1997) 361.[4] W. Abraham, P.W. Wertz, D.T. Downing, Biochim. Bio-

phys. Acta 939 (1988) 403.[5] A. De la Maza, A.M. Manich, L. Coderch, P. Bosch, J.L.

Parra, Colloids Surf. A 101 (1995) 9.[6] J. Lasch, W. Wohlrab, Biomed. Biochim. Acta 45 (1986)

1295.[7] H. Schreier, J. Bouwstra, J. Controlled Release 30 (1994)

1.[8] L. Gallois, M. Fiallo, A. Laigle, W. Priebe, A. Garnier-

Suillerot, Eur. J. Biochem. 241 (1996) 879.[9] K. Miyajima, S. Tanikawa, M. Asano, K. Matsuzaki,

Chem. Pharm. Bull. 42 (1994) 1345.[10] M. Schaller, R. Steinle, H.C. Korting, Acta Derm.

Venereol (Stockh) 77 (1997) 122.[11] B.A.I. Van den Bergh, I. Salomons-de Vries, J.A. Bouw-

stra, Int. J. Pharm. 167 (1998) 56.[12] E.M.J. Van Kuijk-Meuwissen, H.E. Junginger, J.A.

Bouwstra, Biochim. Biophys. Acta 137 (1998) 31.[13] H.C. Korting, W. Stolz, M.H. Schmid, G. Maierhofer,

Br. J. Dermatol. 132 (1995) 571.[14] J. Lasch, R. Laub, W. Wohlrab, J. Controlled Release 18

(1991) 55.[15] H.E.J. Hofland, J.A. Bouwstra, H.E. Bodde, F. Spies,

H.E. Junginger, Br. J. Dermatol. 132 (1995) 853.[16] M. Kirjavainen, A. Urtti, I. Jaaskelainen, T. Marjukka

Suhonen, P. Paronen, R. Valjakka-Koskela, J. Kiesvaara,J. Monkkonen, Biochim. Biophys. Acta 1304 (1996) 179.

[17] A. De la Maza, O. Lopez, M. Cocera, L. Coderch, J.L.Parra, Colloids Surf. A 147 (1999) 341.

[18] A. De la Maza, O. Lopez, L. Coderch, J.L. Parra, Col-loids Surf. A 145 (1998) 83.

[19] A. De la Maza, O. Lopez, M. Cocera, L. Coderch, J.L.Parra, Chem. Phys. Lipids 94 (1998) 181.

[20] O. Lopez, A. De la Maza, L. Coderch, J.L. Parra, J. Am.Oil Chem. Soc. 73 (1996) 443.

[21] P. Walther, E. Wehrli, R. Hermann, M. Muller, J. Mi-crosc. 179 (1995) 229.

[22] P.W. Wertz, D.T. Downing, J. Lipid Res. 24 (1983) 753.[23] S.U. Egelhaaf, E. Wehrli, M. Muller, M. Adrian, P.

Schurtenberger, J. Microsc. 184 (1996) 214.[24] O. Lopez, A. De la Maza, L. Coderch, C. Lopez-Iglesias,

E. Wehrli, J.L. Parra, FEBS lett. 426 (1998) 314.


[25] W.H.M. Craane-Van Hinsberg, J.C. Verhoef, F. Spies,J.A. Bouwstra, G.S. Gooris, H.E. Junginger, H.E. Bodde,Microsc. Res. Tech. 37 (1997) 200.

[26] O. Lopez, A. De la Maza, L. Coderch, J.L. Parra,Colloids Surf. A 123–124 (1997) 415.

[27] H.E. Bodde, B. Holman, F. Spies, A. Weerheim, J.Kempenaar, M. Mommaas, M. Ponec, J. Invest. Derma-tol. 95 (1990) 108.

[28] S.J. Chapman, A. Walsh, Arch. Dermatol. Res. 282(1990) 304.

[29] P.P. Joazeiro, G.S. Montes, J. Anat. 175 (1991) 27.[30] S. Zellmer, W. Pfeil, J. Lasch, Biochim. Biophys. Acta

1237 (1995) 176.[31] H.E.J. Hofland, J.A. Bouwstra, F. Spies, H.E. Bodde,

F.J. Nagelkerke, C. Cullander, H.E.J. Junginger, Lipo-some Res. 5 (1995) 241.

effect of liposomes on delipidized stratum corneum structure: an ‘in vitro’ study based on high...

Documents