Progress in Lipid Research 42 (2003) 1–36
www.elsevier.com/locate/plipres
Review
Structure of the skin barrier and its modulation by vesicular
formulations
Joke A. Bouwstraa,*, P. Loan Honeywell-Nguyena, Gert S. Goorisa,
Maria Ponecb
a
Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden University, PO Box 9502, 2300
RA Leiden, The Netherlands
b
Department of Dermatology, Leiden University Medical Center, Leiden, The Netherlands
Abstract
The natural function of the skin is to protect the body from unwanted influences from the environment.
The main barrier of the skin is located in the outermost layer of the skin, the stratum corneum. Since the
lipids regions in the stratum corneum form the only continuous structure, substances applied onto the skin
always have to pass these regions. For this reason the organization in the lipid domains is considered to be
very important for the skin barrier function. Due to the exceptional stratum corneum lipid composition,
with long chain ceramides, free fatty acids and cholesterol as main lipid classes, the lipid phase behavior is
different from that of other biological membranes. In stratum corneum crystalline phases are predominantly present, but most probably a subpopulation of lipids forms a liquid phase. Both the crystalline
nature and the presence of a 13 nm lamellar phase are considered to be crucial for the skin barrier function.
Since it is impossible to selectively extract individual lipid classes from the stratum corneum, the lipid
organization has been studied in vitro using isolated lipid mixtures. These studies revealed that mixtures
prepared with isolated stratum corneum lipids mimic to a high extent stratum corneum lipid phase behavior. This indicates that proteins do not play an important role in the stratum corneum lipid phase behavior. Furthermore, it was noticed that mixtures prepared only with ceramides and cholesterol already form
the 13 nm lamellar phase. In the presence of free fatty acids the lattice density of the structure increases. In
stratum corneum the ceramide fraction consists of various ceramide subclasses and the formation of the 13
nm lamellar phase is also affected by the ceramide composition. Particularly the presence of ceramide 1 is
crucial. Based on these findings a molecular model has recently been proposed for the organization of the
13 nm lamellar phase, referred to as ‘‘the sandwich model’’, in which crystalline and liquid domains coexist. The major problem for topical drug delivery is the low diffusion rate of drugs across the stratum corneum. Therefore, several methods have been assessed to increase the permeation rate of drugs temporarily
and locally. One of the approaches is the application of drugs in formulations containing vesicles. In order
* Corresponding author. Tel.: +31-71-5274208; fax: +31-71-5274565.
E-mail address: [email protected] (J.A. Bouwstra).
0163-7827/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0163-7827(02)00028-0
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to unravel the mechanisms involved in increasing the drug transport across the skin, information on the
effect of vesicles on drug permeation rate, the permeation pathway and perturbations of the skin ultrastructure is of importance. In the second part of this paper the possible interactions between vesicles and
skin are described, focusing on differences between the effects of gel-state vesicles, liquid-state vesicles and
elastic vesicles.
# 2002 Elsevier Science Ltd. All rights reserved.
Contents
1. Introduction ............................................................................................................................................................... 2
2. Stratum corneum lipid composition and organization............................................................................................... 7
2.1. Lipid composition in stratum corneum .............................................................................................................7
2.2. Lipid organization in stratum corneum.............................................................................................................7
2.3. Lipid organization in stratum corneum with abnormal lipid composition ..................................................... 13
3. Relation between lipid composition and organization ............................................................................................. 14
3.1. Mixtures based on isolated pig and human ceramides .................................................................................... 14
3.1.1. The formation of stratum corneum lamellar sheets............................................................................. 14
3.1.2. The role of CER, CHOL and FFA in lipid phase behavior ............................................................... 14
3.1.3. The role of the various ceramide subclasses in the lipid phase behavior............................................. 16
3.1.4. Phase behavior in mixtures prepared from HCER[1–8]: the role of HCER1 ..................................... 17
3.1.5. Lipid mixtures based on commercially available and synthetic ceramides.......................................... 18
4. A unique molecular arrangement in the long periodicity phase............................................................................... 20
5. Extrapolation of the in vitro finding to the lipid composition and organization in diseased and in dry skin ......... 21
6. The effect of water and vesicles on the ultrastructure of the skin ............................................................................ 22
6.1. Water............................................................................................................................................................... 22
6.2. Vesicular structures ......................................................................................................................................... 23
6.2.1. Introduction ........................................................................................................................................ 23
6.2.2. Vesicles perturb the lipid organisation in stratum corneum in vitro: occlusive application ................ 25
6.2.3. Vesicles perturb the lipid organization in the stratum corneum in vitro and in vivo: non-occlusive
application ........................................................................................................................................... 26
7. Conclusions .............................................................................................................................................................. 33
References ..................................................................................................................................................................... 33
1. Introduction
Controlled delivery of drugs into the body is one of the major research topics in the pharmaceutical field. Most drugs are administered orally. However, for a variety of drugs this route of
administration is not possible, due to the high metabolic activity in the gastro-intestinal tract and
in the liver (first pass effect). This is not only a problem for very sophisticated and modern drugs,
such as peptides and proteins, but also for several more traditional drugs, such as fentanyl and
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Fig. 1. A schematic drawing of a skin cross-section. The skin is composed of a dermis and an epidermis. In the basal
layer of the epidermis cells proliferate. Upon leaving the basal layer cells start to differentiate and migrate in the
direction of the skin surface. At the interface between stratum granulosum and stratum corneum final differentiation
occurs, during which the viable cells are transformed into dead keratin filled cells (corneocytes). The corneocytes are
surrounded by a cell envelope composed of cross-linked proteins and a covalently bound lipid envelope (see arrow).
The corneocytes are embedded in lipid lamellar regions, which are orientated parallel to the corneocyte surface. Substances permeate mainly along the tortuous pathway in the intercellular lamellar regions. C=corneocyte filled with
keratin. Bar=100 nm.
estradiol. Another problem for oral drug delivery is that for some drugs a continuous delivery is
required, which is very difficult to achieve via this route. For these reasons, there is always a need
for other routes of administration. One of the alternative routes of administration is the transdermal route (via the skin). Advantages of the transdermal route are the limited metabolic activity in the skin compared to that in the liver and the possibility to achieve a continuous delivery
profile. Besides the need for transdermal administration of drugs as an alternative route for the
oral route, targeting to the skin is also an important issue, especially when considering skin diseases. However, since the natural function of the skin is to protect the body from unwanted
effects from the environment, the most important limitation in transdermal application of drugs
is the skin barrier.
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Fig. 2. The lamellar extrusion process. At the interface between stratum granulosum and stratum corneum lamellar
bodies are extruded into the intercellular regions. The lipid content is rearranged into long lipid lamellae filling the
intercellular regions in the stratum corneum. Simultaneously major changes in the lipid composition occur. (A) Schematic drawing of the lamellar body extrusion process at the stratum granulosum-stratum corneum interface (see SG/
SC). (B) An electron micrograph of the fusion of the lipid lamellae along the corneocyte. C=corneocyte; D=desmosome; LU=Landmann unit consisting of a broad-narrow-broad sequence; Ld=lipid disk; bar=100 nm.
The skin is composed of several anatomically distinct layers (Fig. 1). The protection of the skin
is provided primarily by the stratum corneum. The superficial region, which is only 10–20 mm
thick provides the primary barrier to the percutaneous absorption of compounds as well as to
water loss. Underlying the stratum corneum is the viable epidermis (50–100 mm thick), which is
responsible for generation of the stratum corneum. The dermis (1–2 mm thick) is directly adjacent
to the epidermis and provides the mechanical support for the skin. The viable epidermis is a
stratified epithelium consisting of basal, spinous and granular cell layers. Each layer is defined by
position, shape, morphology and state of differentiation of keratinocytes. The epidermis is a
dynamic, constantly self-renewing tissue, in which a loss of the cells from the surface of the
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stratum corneum (desquamation) is balanced by cell growth in lower epidermis. Upon leaving the
basal layer, the keratinocytes start to differentiate and during migration through the stratum
spinosum and stratum granulosum undergo a number of changes in both structure and composition. The keratinocyte synthesize and express numerous different structural proteins and lipids
during their maturation. The last sequences of the keratinocyte differentiation are associated with
profound changes in their structure, which result in their transformation into chemically and
physically resistant cornified squames of the stratum corneum, called corneocytes. The corneocytes are flat anucleated squamous cells packed mainly with keratin filaments, surrounded by a
cell envelope composed of cross-linked proteins and a covalently bound lipid envelope. The
corneocytes are surrounded by extracellular non-polar lipids. Interconnecting the corneocytes are
protein structures, referred to as desmosomes.
Late in the process of differentiation, characteristic organelles (lamellar bodies), appear in the
granular cells. The lamellar bodies, which play an essential role in stratum corneum formation, are
ovoid organelles enriched mainly in polar lipids and catabolic enzymes, which deliver the lipids
required for the generation of the stratum corneum. In response to a certain signal (possibly the
increase in calcium concentration), the lamellar bodies move to the apical periphery of the uppermost
granular cells, fuse with the plasma membrane and secrete their content into the intercellular spaces
by exocytosis. The lipids derived from the lamellar bodies are subsequently modified and rearranged
into intercellular lamellae orientated approximately parallel to the surface of the cells (see Fig. 2) [1–
7]. In this orientation process, most probably the lipid envelope [8–10] acts as a template.
Lamellar bodies serve as a carrier of precursors of stratum corneum barrier lipids, which consist
mainly of glycosphingolipids, free sterols and phospholipids. After the extrusion of lamellar
bodies at the stratum granulosum/stratum corneum interface, the polar lipid precursors are
enzymatically converted into nonpolar products and assembled into lamellar structures surrounding the corneocytes. Hydrolysis of glycolipids generates ceramides (CER), while phospholipids are converted into free fatty acids (FFA). The change in lipid composition and cell
structure results in the formation of a very densely packed structure in the stratum corneum. Due
to the impermeable character of the cornified envelope, the major route of penetration resides in
the tortuous pathway between the corneocytes as revealed by confocal laser scanning microscopy
and X-ray microanalysis studies [11,12]. It is for this reason that the lipids play an irreplaceable
role in the skin barrier, which makes their mutual arrangement in the lamellar domains a key
process in the formation of the skin barrier.
As mentioned above, the main problem in topical application of drugs is the skin barrier. In
order to enhance the drug transport across the skin, several approaches have been taken. One of
these approaches employs physical enhancement, such as the use of an electrical gradient (iontophoresis) to provide an extra driving force for drug permeation across the skin [13] or electroporation, in which short high voltage pulses make the stratum corneum more permeable [14]. The
former is most successful for charged drugs. Another approach is chemical enhancement, which
involves the application of drugs in a formulation that decreases the skin barrier by either disrupting or fluidizing the lipid lamellae and/or increases the solubility of the drug in the stratum
corneum. An example of this approach is the use of penetration enhancers [15], which are often
surface active ingredients, organic solvents or vesicular carriers such as liposomes [16].
In the first part of this paper the lipid organization in the stratum corneum of normal and diseased skin will be reviewed with emphasis on the role the various lipid classes play in stratum
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Fig. 3. Molecular structure of ceramides. (A) Ceramides in human stratum corneum (HCER) and (B) pig stratum
corneum (pigCER). Note that in the HCER mixture both CER1 and CER4 have an o-hydroxy acyl chain to which a
linoleic acid is chemically linked. In pigCER mixture only CER1 has this exceptional molecular structure.
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corneum lipid organization. As an illustration for the possible alterations caused by delivery
systems changes induced in the microstructure of the stratum corneum by water and by vesicular
formulations will be discussed in the second part of this paper.
2. Stratum corneum lipid composition and organization
2.1. Lipid composition in stratum corneum
The major lipid classes [17,18] in stratum corneum are CER, cholesterol (CHOL) and FFA.
The CER head groups are very small and contain several functional groups that can form lateral
hydrogen bonds with adjacent ceramide molecules. The acyl chain length distribution in the CER
is bimodal with the most abundant chain lengths being C24–C26. Only a small fraction of CER
has an acyl chain length of C16–C18. The chain lengths of C24 and C26 are much longer than
those in phospholipids in plasma membranes. In human stratum corneum [19,20] eight subclasses
of ceramides (HCER) have been identified (see Fig. 3). These HCER, referred to as HCER 1–8,
differ from each other by the head-group architecture (sphingosine, phytosphingosine or 6hydroxysphingosine base) linked to a fatty acid or an a-hydroxy fatty acid of varying hydrocarbon chain length. In human stratum corneum CER1 and CER4 have a very exceptional
molecular structure: a linoleic acid is linked to a o-hydroxy fatty acid with a chain length of
approximately 30–32 C-atoms. In this respect the HCER are different from ceramides isolated
from pig stratum corneum (pigCER), in which only pigCER1 has this exceptional molecular
structure [21] and pigCER5 has an unusual short chain length (acyl chain length of C16–C18). In
both species the FFA fraction consists mainly of saturated acids. The major portion of FFA has a
chain length of C22 and C24. Another important lipid in stratum corneum is cholesterol sulfate.
Although cholesterol sulfate is present in small amounts (typically 2–5% w/w), this lipid plays an
important role in the desquamation process of stratum corneum [22].
2.2. Lipid organization in stratum corneum
At the end of the 1950s and in the early 1960s, the lipid organization in human stratum corneum [23,24] has been measured using X-ray diffraction. The excellent measurements performed
with a conventional camera revealed a similar pattern as observed nowadays with the synchrotron facilities. Since at that time no information was available about the lipid organization at the
ultrastructural level, the X-ray diffraction patterns were interpreted as being from lipids organized in tubes surrounding keratin filaments. These conclusions were drawn from the similarity
between the measured and calculated diffraction curves, in which the calculated diffraction curves
were obtained from Fourier transformations of lipid associates in a geometry of hollow tubes. At
least 10 years passed before important additional information became available that provided
completely new insights in the lipid organization. Breathnach et al. [25,26] using freeze fracture
electron microscopic technique reported the presence of intercellular lipid lamellae between the
cells. This was a big step forward in understanding the structure of the stratum corneum (see Fig. 6].
For transmission electron microscopic studies the key problem in visualizing lipid lamellae was
the saturated nature of the stratum corneum lipids, which made the fixation with osmium tetroxide
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impossible. It was not before 1987, that Madison et al. used ruthenium tetroxide as a post-fixation agent to preserve the saturated lipids in the stratum corneum during the embedding procedure [27]. The subsequent electron microscopic studies revealed an unusual lamellar arrangement
of a repeating pattern with electron translucent bands in a broad-narrow-broad sequence [28–31]
(see Fig. 1). More detailed insight in the stratum corneum lipid organization has been achieved in
the mid eighties using Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction
technique. Fourier transformed infrared spectroscopy that provides information about the
mobility and the lateral packing of the lipids in the stratum corneum [32–34] made it possible to
distinguish between a hexagonal (gel-phase) and an orthorhombic sublattice. The presence of the
latter introduces a splitting of the rocking and scissoring frequencies located at approximately 720
and 1460 cm 1, respectively, due to the short-range coupling in the densely packed structure. In
addition to the formation of an orthorhombic sublattice in human stratum corneum, also a
small subpopulation of lipids is forming a liquid phase. The latter conclusion was based on the
CH2 anti-symmetric and symmetric stretching frequencies at around 2850 and 2920 cm 1,
respectively.
At the end of the eighties White et al. [35] performed X-ray diffraction studies with mouse
stratum corneum. Using small angle X-ray diffraction they observed a diffraction pattern of a
series of sharp peaks indicating the presence of a lamellar phase with a periodicity of approximately 13 nm, further referred to as the long periodicity phase (LPP). Furthermore, wide angle
X-ray diffraction studies revealed the presence of an orthorhombic sublattice [35,36] with a transition from an orthorhombic to a hexagonal subphase occurring between 20 and 40 C. However,
in these studies the presence of the hexagonal sublattice at room temperature could not be
excluded since the reflections based on the hexagonal lateral packing (strong reflection at 0.41 nm
spacing) is obscured by the reflections attributed to an orthorhombic phase (strong reflections at
0.41 and 0.37 nm spacings). Next to the orthorhombic phase, a liquid lateral packing was also
present. This conclusion was drawn from the detection of a very broad reflection at approximately 0.46 nm spacing. However, more recently it became evident that a thick layer of sebum
lipids is covering the surface of hairless mouse skin [37]. Therefore from these X-ray studies it
cannot be deduced whether the detected liquid sublattice is present in the intercellular lipid stratum corneum structures or in sebum lipids located on the skin surface. Besides the reflections that
can be attributed to the presence of crystalline phases, also other reflections have been detected.
These reflections could be attributed to the presence of hydrated crystalline CHOL in separate
domains. In a more recent publication [38] it has become clear that in mouse stratum corneum
not only the 13 nm phase is present, but also a small fraction of lipids formed a second phase with
a periodicity of approximately 6 nm, further referred to as the short periodicity phase (SPP). The
presence of LPP and SPP can be fully ascribed to the extracellular stratum corneum lipids, since
after lipid extraction only reflections attributed to proteins could be detected in the diffraction
patterns.
In an additional series of studies with human and pig stratum corneum synchrotron facilities
have been used [39,40]. Since the X-ray diffraction curves of pig and human stratum corneum
revealed the presence of very broad partly overlapping peaks additional information was required
for proper interpretation of the obtained data. This was achieved in X-ray experiments with
stratum corneum in which the lipids were re-crystallized from 120 C to room temperature. The
diffraction curves revealed the presence of a series of sharp peaks, similarly as noticed in mouse
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stratum corneum, indicating that after recrystallization the lipids in human and pig stratum corneum were organized in a LPP with a periodicity of approximately 13 nm. Comparing the peak
positions in the diffraction patterns obtained prior and after recrystallization revealed the presence of at least two lamellar phases: one lamellar phase with a periodicity of approximately 6 nm
(SPP), and the other phase with a periodicity of approximately 13 nm (LPP) [39,40]. Since the
LPP has been found to be present in all species examined until now, and has a very characteristic
molecular organization (see later), it has been suggested that the presence of this phase plays an
important role in skin barrier function.
To get more detailed information on the stratum corneum lipid organization, changes in diffraction pattern as function of temperature have also been investigated. These experiments
revealed that lipid lamellae are still present until a temperature of around 60 C, after which the
lipid lamellae disappear within a temperature range of approximately 10 C (see Fig. 4B).
Next to the lamellar organization, the lateral lipid packing in human and pig stratum corneum
has also been investigated. In human stratum corneum an orthorhombic lateral packing was
observed, which is in agreement with the earlier FTIR results [41,42]. It could not be concluded
whether a liquid phase coexisted with the orthorhombic lateral packing, since the broad reflection
of the liquid phase in the diffraction pattern was obscured by the reflections based on soft keratin
present in the corneocytes (see Fig. 5A). In addition, it could not be concluded whether a hexagonal phase is present either since the reflections of the orthorhombic phase obscure the reflections based on the hexagonal lateral packing [42], similarly to the observations made with mouse
stratum corneum. Furthermore, frequently the CHOL phase separates from the lamellar phases
[40,42]. From the orientation of the reflections in the diffraction pattern it could be deduced, that
the longest axis of the CHOL lattice is orientated perpendicularly to the basal plane of the
lamellae. Most probably the preferred orientation of CHOL crystals is dictated by the orientation
of the lipid lamellae indicating that CHOL crystals are intercalated in the lamellae between the
corneocytes. In contrast to mouse and human stratum corneum, in pig stratum corneum no
orthorhombic lateral packing was present, but a hexagonal lateral packing prevailed, which
contrasts the findings obtained with FTIR spectroscopy. Whether a liquid phase coexists with the
hexagonal sublattice was difficult to determine. However, after extraction of the lipids from pig
stratum corneum, a reduction in intensity of the broad reflection at around 0.46 nm was noticed.
This reduction in intensity is suggestive for the presence of a liquid sublattice in pig stratum corneum.
Recently using the electron diffraction technique [43] more detailed information has been
obtained on the lateral organization in human stratum corneum. Since only an area 1 mm2 is
exposed to the electron beam, it is possible to obtain reflections only from one or a few crystals.
The diffraction pattern of an orthorhombic single crystal is characterized by four strong reflections at a spacing of approximately 0.406 nm and two strong reflections at a 0.367 nm spacing.
Next to these strong reflections higher order reflections could also be detected. The strong
reflections are separated by angles that are close but not equal to 60 . A single crystal of a hexagonal sublattice is characterized by six diffraction spots separated by angles of 60 at a spacing of
0.41 nm. Since in these patterns spots are detected instead of rings, it is possible to detect whether
the hexagonal sublattice coexists with the orthorhombic one. It became clear that electron diffraction
patterns obtained from the deeper layers in the stratum corneum (at room temperature) could be
attributed uniquely to an orthorhombic packing (see Fig. 5B, C). Furthermore, very interestingly,
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Fig. 4. Small angle X-ray diffraction patterns obtained with human stratum corneum and mixtures prepared with
isolated lipids from pig stratum corneum. In the small angle X-ray diffraction (SAXD) curves the intensity is plotted as
a function of Q, the scattering vector. Q is defined as 4 p sin /l, in which is the scattering angle and l is the wavelength of the X-rays. The position of the peaks (Q1, Q2, Q3. . .Qn, n being the order of the peak) are related to the
periodicity (d) of a lamellar phase by d=2np/Qn. (A) The SAXD curve of human stratum corneum at room temperature and after recrystallization from 120 C. After recrystallization the peaks are located at equal distances strongly
indicating a lamellar phase with a periodicity of 13.4 nm. This phase is referred to as the long periodicity phase (LPP).
Comparing this curve with the curve at room temperature revealed the presence of the second lamellar phase in human
stratum corneum with a periodicity of 6.4 nm, referred to as the short periodicity phase (SPP). In (A)–(D) 1, 2, 3, 4 and
6 denotes the first, second, third, fourth and sixth order peak of the pattern based on the LPP. I and II refer to the first
and second order of the SPP. (B) The temperature-induced changes in SAXD profiles of human stratum corneum. The
heating rate was 2 C/min. Each sequential curve has been monitored during 1 minute. The lamellar phases disappear
between 60 and 75 C. The first order diffraction peak is clearly depicted in this figure. * Denotes the peak attributed to
the first order of the SPP and the second order peak of the LPP. CHOL indicates the peaks attributed to phase separated crystalline CHOL. (C) The temperature-induced changes in SAXD pattern of the equimolar CHOL:pigCER:FFA mixture. Note the formation of a new peak at 4.3 nm (close to the third order of the LPP) at around 35 C.
The lamellar phases disappear between 60 and 80 C, except for the 4.3 nm phase, which is still present at 90 C.
CHOL indicates the peaks attributed to phase separated crystalline CHOL. (D) The temperature-induced changes in
SAXD pattern of the CHOL:pigCER:FFA :cholesterol sulfate (molar ratio:1:1:1:0.06) mixture. Note the formation of
a new peak takes place at much higher temperatures and the intensity of this peak decreased strongly compared to that
in Fig. 4C. The phase behavior of this mixture resembles that shown in (B).
J.A. Bouwstra et al. / Progress in Lipid Research 42 (2003) 1–36
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Fig. 5. Lateral lipid packing in human stratum corneum and in mixtures based on lipids isolated from pig stratum
corneum. (A) WAXD pattern of human stratum corneum orientated parallel to the primary X-ray beam. The diffraction pattern is characterized by two rings indicating that the lipids are organized in an orthorhombic lateral
packing. The rings are stronger in equatorial position indicating that the lipids are orientated mainly perpendicularly
to the stratum corneum surface. Furthermore, a number of reflections can be attributed to phase separated CHOL.
The position of the reflection in the pattern indicates that CHOL crystals have a preferred orientation in a similar
direction as the lipid lamellae. The strong broad reflections at 0.46 and 0.92 nm can be attributed to soft keratin. (B)
Electron diffraction pattern of human stratum corneum grid-strip reflecting the presence of a single orthorhombic
crystal (perpendicular orientation to the electron beam). The pattern is characterized by 6 reflections at angles not
equal to 60 . Two out of the 6 reflections are located at a large distance from the beam (see arrowhead). Often also
higher order reflections can be detected. (C) Electron diffraction pattern of human stratum corneum grid-strip
reflecting the presence of three orthorhombic crystals. The position of the reflections indicate that the lattice of the unit
cell of the three crystals is rotated over an angle related to the unit cell indicating that the orientation of the various
crystals is not random but is dictated by the structure. (D) One-dimensional WAXD pattern of the equimolar
CHOL:pigCER mixture plotted as function of Q (see Fig. 4). A broad reflection at 0.415 nm spacing is attributed to
the presence of the hexagonal lattice. A large number of sharp reflections is based on phase separated CHOL. (E) Onedimensional WAXD pattern of the equimolar CHOL:pigCER:FFA mixture. Two strong sharp reflections indicate the
presence of an orthorhombic lateral packing. (F) One-dimensional WAXD pattern of the 1:1:1:0.06 CHOL:pigCER:FFA:cholesterol sulfate mixture. Two strong reflections are attributed to the orthorhombic lateral packing. The
broad reflection at 0.46 nm indicates the presence of a liquid phase. Note that due to the presence of cholesterol sulfate
the reflections attributed to CHOL disappeared.
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Fig. 6. Comparison of lipid structure and lateral organization in stratum corneum derived from healthy subjects and
from lamellar ichthyosis patients. (A) Freeze fracture micrograph of stratum corneum of normal skin. The smooth
areas indicate the lamellae located between the corneocytes. The sharp edges result from fractures across the lamellae
(see arrow). D, desmosome. Bar=500 nm. (B) Freeze fracture micrograph of stratum corneum of lamellar ichthyosis
skin. The lamellae show undulations, which are absent in normal skin. Furthermore, the fractures across the lamellae
result in less sharp edges (see arrow). Bar =500 nm. (C) Electron diffraction pattern of stratum corneum of normal
skin. The diffraction pattern shows reflections as two spacings based on various crystals in an orthorhombic packing.
(D) Electron diffraction pattern of stratum corneum of lamellar ichthyosis skin. The diffraction pattern shows 6
reflections separated by angles of 60 at the same distance from the position of the electron beam. This pattern is
characteristic for a hexagonal lateral packing. (E) Electron diffraction pattern of stratum corneum derived from
lamellar ichthyosis skin. A pattern indicative for a hexagonal lateral pattern is observed. However, next to this pattern
sharp reflections are present (see arrows) indicating that a small portion of lipids forms an orthorhombic lateral
packing.
frequently a pattern was obtained by which the orientation of three crystals was rotated over an
angle equal to that between successive reflections indicating that the orientation of these crystals
are not random, but are dictated by the long range structure.
X-ray and electron diffraction patterns obtained with stratum corneum revealed spacings corresponding to 0.406 and 0.367 nm reflections, which are slightly shorter than observed in
orthorhombic and hexagonal lattices in phospholipid based systems. Possibly, due to a strong
attractive van der Waals interaction between hydrocarbon chains of the very long hydrocarbon
chains of the CER as well as between FFA a slightly denser packing of the hydrocarbon chains is
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formed as compared to that formed in phospholipid based systems [44]. Furthermore, already
two decades ago it has been shown by Pascher [45,46] that an extensive network of hydrogen
bondings exists between the head groups of ceramides, which promotes the formation of a very
dense lattice. The surface area per acyl chain in the structure corresponding to hexagonal and
orthorhombic sublattice in human skin appears to be approximately 0.190 and 0.179 nm2,
respectively.
Changes in the lateral packing as function of temperature revealed that in human stratum corneum the transition from an orthorhombic to a hexagonal phase occurs between 35 and 40 C,
which is just above the natural skin temperature of 31–33 C. Interestingly at increasing temperature a gradual change in position of the 0.367 nm reflections was observed moving to the
positions of the 0.406 nm reflections. This indicates a gradual change in packing density. In contrast to the X-ray data using the FTIR spectroscopic technique it has also been reported that a
orthorhombic- hexagonal transition occurs at about 50 C [34]. However, very recently the FTIR
spectroscopy studies were repeated and revealed that in agreement with the diffraction methods
the orthorhombic-hexagonal transition occurs at around 35–40 C (unpublished results).
2.3. Lipid organization in stratum corneum with abnormal lipid composition
In order to determine whether an altered lipid composition results in an altered lipid phase
behavior studies have been carried out with stratum corneum derived from dry and diseased skin.
In the eighties the lipid organisation in essential fatty acid deficient (EFAD) stratum corneum
[28,47] has been elucidated. It appeared that elimination of linoleic acid from the diet resulted in
pig stratum corneum in a progressive increase in the oleate content in CER1 at the expense of the
linoleate content. This increase in CER1-oleate content was accompanied by a strong reduction
in the skin barrier. However, both electron microscopy and X-ray diffraction studies revealed no
drastic changes in the stratum corneum lipid lamellar organization in spite of there was clear
evidence that in EFAD skin the lamellar bodies contain amorphous rather than lamellar material.
In EFAD animals only a great variability in the number of intercellular lipid lamellae was reported.
It seems that other factors, such as a change in lateral packing, may also play a role in the formation of competent skin barrier (see below). The effect of the CER1 oleate/linoleate ratio on barrier
properties and lipid organization is of interest, since in normal skin this ratio decreases dramatically during the winter months [48].
Table 1
A relationship exists between the mean HCER1/CERtot and HCER4/CERtot ratios and the presence of the third order
reflection of the X-ray pattern of the LPP
HCER1/CERtot
HCER 4/CERtot
Third order reflection of LPP
0.11 0.02
0.05 0.01
0.08 0.01
0.05 0.03
Present
Absent
The X-ray diffraction profiles of stratum corneum are classified in two populations. In one population the third order
reflection of the 13.4 nm phase in the diffraction pattern of stratum corneum is absent, while in the other population
this reflection is present. The mean HCER1 and HCER4 contents of these populations are also provided. HCER1=
human ceramide 1 HCER4=human ceramide 4. CERtot=total ceramides present in human stratum corneum.
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Table 2
The positions of the peaks in the diffraction curves and the FFA/HCER, FFA/CHOL and HCER/CHOL w/w ratios
of stratum corneum from normal and lamellar ichthyosis skin
Peak postions
CHOL/HCER
FFA/CHOL
FFA/HCER
Lamellar ichthyosis
Normal
5.3–5.9 nm
0.59
0.18
0.12
6.4 nm
0.45
0.69
0.31
X-ray diffraction studies performed with dry skin and with lamellar ichthyosis skin have also
been reported. Dry skin was selected, because of its low content of HCER1 [49] and lamellar
ichthyosis skin, because of its low content of FFA [50]. The only feature in the SAXD pattern
that is uniquely related to the 13 nm phase is its third order diffraction peak (see Fig. 4A). It
appeared that in stratum corneum samples in which the third order peak was absent the contents
of HCER1 and HCER4 were also reduced (Table 1). This finding suggests that a relationship
exists between the presence of HCER1 and HCER4 and the formation of the 13 nm lamellar
phase. It should be noted that in the absence of the third order peak the reduction in HCER1
content is more pronounced than the reduction in HCER4 content.
In lamellar ichthyosis skin, in addition to a small change in CER composition, the content of
FFA is strongly reduced compared to that found in healthy stratum corneum (Table 2). SAXD
studies with stratum corneum of lamellar ichthyosis skin revealed an altered lamellar organization since the peaks were located at smaller spacings than in stratum corneum of normal skin [50].
Very recently freeze fracture electron microscopy (FFEM) and electron diffraction technique
have been used to study lipid organization in lamellar ichthyosis patients [51]. The lamellae in
lamellar ichthyosis skin showed strong undulations compared to normal skin confirming the
altered lamellar organization (see Fig. 6).
The electron diffraction technique revealed the predominant presence of a hexagonal sublattice
[51] coexisting with a small number of crystals forming an orthorhombic sublattice (Fig. 5). A
change in lamellar organisation has also been found by Ghadially et al. in stratum corneum of
autosomal recessive ichthyosis patients [52]. Whether these changes in lipid organization in diseased and dry skin can be explained by an altered lipid composition or whether other features
also play a role can be obtained by systematic studies on the phase behavior of mixtures containing major stratum corneum lipids.
3. Relation between lipid composition and organization
3.1. Mixtures based on isolated pig and human ceramides
3.1.1. The formation of stratum corneum lamellar sheets
Studies on the formation of lamellar sheets have been carried out with liposomes containing
CER isolated from pigCER [53,54]. Pig stratum corneum was selected, since it is readily available
and very closely mimics lipid phase behavior in human stratum corneum. Liposomes, prepared
J.A. Bouwstra et al. / Progress in Lipid Research 42 (2003) 1–36
15
from CHOL, pigCER, free fatty acids and cholesterol sulfate, were chosen to elucidate the role of
calcium and of acyl(glucosyl)ceramides in the formation of the broad lamellar sheets at the stratum
granulosum–stratum corneum interface. The studies clearly demonstrated that calcium as well as
acyl(glucosyl)ceramides promoted the formation of lamellar sheets at pH 7.4. Most probably calcium is shielding the negative charge of cholesterol sulfate and of the dissociated FFA. This minimizes the electrostatic repulsion forces. The absence of electrostatic repulsion facilitates the fusion
process. In case of acyl(glucosyl)ceramides it has been proposed that these lipids act as rivets holding
the outer layers of adjacent liposomes in close apposition and thus promoting the fusion process.
3.1.2. The role of CER, CHOL and FFA in lipid phase behavior
In more recent studies the lipid phase behaviour has been studied using hydrated lipid mixtures.
In CHOL:pigCER mixtures two lamellar phases with periodicities of 5.2 and 12.2 nm were
formed, mimicking lipid phase behavior in intact stratum corneum [55]. This phase behavior
was observed over a wide range of CHOL:pigCER molar ratios (between 0.4 and 2] indicating
that the formation of the lamellar phases is insensitive toward changes in CHOL:pigCER molar
ratio. Lowering a CHOL:pigCER molar ratio below 0.4, induces phase separation between
lamellar phases and formation of hydrated crystalline CHOL domains. The high insensitivity of
the lipid organization towards changes in the CHOL:pigCER molar ratio suggests that in the in
vivo situation a variation in CHOL:CER molar ratio will not lead to a substantial change in lipid
phase behavior, but that CHOL will phase separate when the amount of CHOL present in the
lipid domains exceeds the amount necessary to saturate the lamellae. The presence of the phase
separated CHOL is indeed also observed in the in vivo situation [43]. In the range of molar ratio
between 0.1 and 2, the lipids form a hexagonal lateral packing. The phase behavior in
CHOL:pigCER mixtures is different from that observed with CHOL:DPPC mixtures, in which a
hexagonal lateral packing is observed at low CHOL content, but at increased CHOL content a
phase transition from a hexagonal to an ordered fluid phase has been observed [56–58].
In addition to CHOL and CER, FFA belong to the major stratum corneum lipid classes. To
mimic the FFA composition in intact stratum corneum, a FFA mixture containing predominantly C22 and C24 fatty acids has been used. Addition of FFA to achieve equimolar
CHOL:pigCER:FFA mixture revealed the presence of two lamellar phases with periodicities of
12.8 and 5.4 nm, mimicking even more closely the lipid organization in intact stratum corneum.
Furthermore, addition of FFA induced a phase transition from a hexagonal to an orthorhombic
lattice and therefore increased the lipid density in the structure, see Fig. 5E and F [59]. This phase
transition was not observed with mixtures containing predominantly short chain (C16 and C18)
fatty acids [55]. At present it is not clear whether the liquid phase is also present. It should be
noted that the broad reflection at 0.46 nm indicative for a liquid phase might be obscured by a
large series of reflections based on phase separated crystalline CHOL, which is more profoundly
present in lipid mixtures than in stratum corneum.
Although the CHOL:pigCER:FFA mixtures mimic closely the lipid organization of intact
stratum corneum at room temperature, important differences have been observed at elevated
temperatures. As depicted in Fig. 4C, at approximately 37 C a strong increase in the intensity of
the 4.3 nm peak is observed indicating the formation of a new phase. In intact pig stratum corneum this phase has been formed only at much higher temperatures, while in human stratum
corneum this phase is almost absent.
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Besides FFA, CHOL and CER small amounts of cholesterol sulfate (typically 2–5% w/w of the
lipids) are also present in stratum corneum. Cholesterol sulfate plays an important role in the
inhibition of proteases in stratum corneum, enzymes that are important for the degradation of
the desmosomes. In the superficial stratum corneum layers cholesterol sulfate is metabolized to
cholesterol by cholesterol sulfatase, which increases the activity of proteases. These processes
promote the degradation of the desmosomes [22], which is an extremely important step in the
desquamation process. Based on the important role of cholesterol sulfate for a proper functioning
of the stratum corneum, it was decided to study the role of cholesterol sulfate as well. Addition of
only 2% m/m cholesterol sulfate to an equimolar CHOL:pigCER:FFA mixture resulted in the
reduction of the fraction of phase separated CHOL without affecting the lamellar phase behavior.
Also a fluid phase was clearly present in the CHOL:pigCER:FFA:cholesterol sulfate mixture (see
Fig. 5F). In addition, studies on the lipid phase behavior as a function of temperature revealed
[60] that in the presence of only 2% m/m cholesterol sulfate the formation of the 4.3 nm phase is
shifted to higher temperatures mimicking the lipid phase behavior of intact stratum corneum also
at elevated temperatures. It seems that cholesterol sulfate stabilizes the lipid lamellar phases
formed at room temperature. This might be due to the electrostatic interactions induced by the
presence of the negatively charged sulfate group. A stabilization of the lamellar phases after
introduction of cholesterol sulfate in the mixture has also been observed for both sphingomyeline
and phosphatidylcholine containing mixtures as well [61,62]. From the results it is clear that
cholesterol sulfate is required for a proper lipid phase behavior over a wide temperature range.
When these findings are extrapolated to the in vivo situation, it seems that cholesterol sulfate is
required to dissolve CHOL in the lamellar phases and to stabilize stratum corneum lipid organization. Therefore, a drop in cholesterol sulfate content in the superficial layers of the stratum
corneum is expected to destabilize the lipid lamellar phases and to increase the activity of the
proteases [22]. Both events will facilitate the desquamation process.
Another parameter that may affect the stratum corneum lipid behavior is pH. The pH on the
skin surface is slightly acidic (pH 5–6), while the pH in the viable epidermis is 7.4 [63,64]. At pH
7.4 the formation of the LPP in CHOL:pigCER:FFA mixtures was promoted, indicating that the
lipid organization in these mixtures is slightly sensitive to the pH. This suggests that the formation of the LPP in the lowest parts of the stratum corneum is promoted by a pH of 7.4. However,
at elevated temperature the pH had a dramatic effect on lipid phase behavior: at pH 7.4 it was
impossible to select mixtures that mimic stratum corneum lipid phase behavior over a wide temperature range. This indicates that the pH in the stratum corneum is slightly acidic confirming
results of Mauro et al. [65].
3.1.3. The role of the various ceramide subclasses in the lipid phase behavior
Since often a deviation in CER composition has been found in diseased skin [50,49,66,67] not
only the role of the varying CHOL:CER molar ratio is important, but also the role that individual CER subclasses play in stratum corneum lipid phase behavior is important. To examine this
in more detail, mixtures were prepared with pigCER(1-5), pigCER(2-6) or pigCER(1-2) [68–70].
First, the phase behavior of equimolar mixtures was examined. These studies revealed that in
equimolar mixtures prepared with pigCER[1-5] or pigCER[1-2] the lipids were organized in the
LPP and the SPP with periodicities of approximately 12–13 nm and 5–6 nm, respectively, similarly as observed in intact stratum corneum (Fig. 7). The exception was found with an equimolar
J.A. Bouwstra et al. / Progress in Lipid Research 42 (2003) 1–36
17
Fig. 7. Schematic presentation of the phase behavior of various CHOL:pigCER mixtures as function of CER composition and of CHOL:CER molar ratio. Mixtures were prepared with full spectrum of CER [CER(1-6)], with CER 1
and 2 [CER(1,2)], or with CER mixture in which CER1 is absent [CER(2-6)]. In addition, the phase behavior of the
equimolar CHOL:CER:FFA mixture and human stratum corneum (SC) is depicted. The equimolar CHOL:CER(16):FFA mixture mimics most closely the phase behavior in human stratum corneum.
CHOL:pigCER[2-6] mixture, in which the LPP was only weakly present [69], indicating that the
CER1 plays a crucial role in the formation of the LPP. Similar observations have been made
when the long-chain FFA were incorporated into CHOL:CER mixtures. FFA hardly affected the
lamellar lipid organization. Again, only CER1 has been found to play a crucial role in the formation of the LPP. While in mixtures containing full spectrum of CER, changes in CHOL:CER
molar ratio over a wide range had no effect on the formation of LPP, the situation changed in
mixtures containing only some ceramide subclasses. This was most clearly observed in mixtures
prepared from CHOL and CER[1-2]: the LPP was only weakly present at a CHOL:CER[1-2]
molar ratio of 0.6 and absent at lower molar ratios. This is in contrast to the CHOL:CER[1-6]
mixtures in which this phase was formed even at a molar ratio of 0.2.
3.1.4. Phase behavior in mixtures prepared from HCER[1-8]: the role of HCER1
The lipid phase behavior of mixtures prepared with HCER [71,72] slightly differed from that
observed with mixtures prepared from pigCER. Namely, in CHOL:HCER mixtures the LPP was
dominantly present and only a small fraction of lipids formed the SPP. Furthermore, addition of
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FFA promoted the presence of the SPP. Similarly as in pigCER containing mixtures, the addition
of FFA resulted in a transition from a hexagonal to an orthorhombic phase. However, in mixtures prepared with HCER next to the orthorhombic phase, a liquid phase was clearly present.
This phase was noticed in pigCER containing mixtures only in the presence of cholesterol sulfate.
Similarly as in pigCER containing mixtures, in the absence of HCER1 only a small population of
lipids formed the LPP, despite the presence of HCER4 with a similar molecular structure (see
Fig. 3) as HCER1. To elucidate the role of fatty acid linked to o-hydroxyacid, natural HCER1
was replaced by either synthetic CER1-linoleate (CER1-lin), by CER1-oleate (CER1-ol) or by
CER1-stearate (CER1-ste). The following changes in lipid phase behavior were noticed. (i) No
liquid phase could be detected when HCER was substituted by CER1-ste. (ii) Substitution of
HCER1 by either CER1-ol or CER1-lin revealed the presence of the liquid phase. This phase was
less prominent in mixtures prepared with CER1-lin than with CER1-ol. (iii) As far as the lamellar
phases are concerned, the LPP was not present in HCER:CHOL mixtures in which HCER1 was
substituted for CER1-ste. (iv) The 12–13 nm lamellar phase was dominantly present in mixtures
in which HCER1 was substituted by either HCER1-lin or HCER1-ol and (v) addition of FFA to
the HCER:CHOL mixtures in which HCER1 was replaced by CER1-ol promoted the formation
of the SPP. The results of these studies indicate that for the formation of the LPP a certain (optimal) fraction of lipids has to form a liquid phase.
3.1.5. Lipid mixtures based on commercially available and synthetic ceramides
In the past various studies have been performed with mixtures based on commercially available
ceramides, like bovine-brain CER. Several studies used model membranes prepared with palmitic
acid, CHOL and bovine-brain CER. The choice for palmitic acid was based on a study by Lampe
et al. [73], in which it was stated that the main population of fatty acids in stratum corneum has a
chain of C16 and C18:1. However, in more recent studies it became clear that this was not a
correct choice and that mainly fatty acids with chain length C22 and C24 are present in human
stratum corneum [18,74]. NMR studies carried out with equimolar mixtures of CHOL and
bovine brain CERIII in the absence and presence of palmitic acid revealed that the main population of lipids forms a crystalline phase, but that also a small proportion of lipids form a more
mobile phase [75,76]. Formation of a crystalline phase in the presence of a substantial amount of
CHOL is a very exceptional observation for lipid membranes. For example, in phospholipid
containing mixtures an increasing amount of CHOL results in a transition from a crystalline to a
liquid-ordered phase [57,58]. The difference in phase behavior might be ascribed to either differences in the head group size or differences in chain length between phospholipids and ceramides.
The lateral packing was strongly dependent on the size of the head-group. For example, equimolar cholesterol:sphingomyelin mixtures form a liquid lateral packing, while equimolar cholesterol:bovine-brain ceramide III mixtures having the same chain length distribution form a
crystalline phase [77]. Furthermore, in the CHOL:bovine-brain CERIII:palmitic acid mixtures
more than 80% of the lipids formed a crystalline phase, while a small portion formed a more
mobile phase [76]. This mixture formed a liquid phase at around 50 C. As far as the lateral
packing is concerned these lipids mimic stratum corneum lipid organisation quite closely. However, as shown more recently using the X-ray diffraction technique, the CHOL:bovine-brain
CERIII:palmitic acid does not form the characteristic LPP [78,79]. It seems that bovine-brain
CERIII based mixtures differ in lipid phase behavior from that observed in intact stratum cor-
J.A. Bouwstra et al. / Progress in Lipid Research 42 (2003) 1–36
19
Fig. 8. Model for molecular arrangement of the long periodicity phase (LPP). The electron density profile calculated
from the electron diffraction profile of the LPP indicates the presence of a broad-narrow-broad sequence in the
repeating unit of the LPP (arrows) (left panel). This is in agreement with the broad-narrow-broad pattern found in
RuO4 fixed stratum corneum (right panel). Based on these and other (see text) observations a molecular model is presented (middle panel), in which CER1 plays an important role in dictating the broad–narrow–broad sequence. Furthermore, the fluid phase is located in the central narrow band. In adjacent regions the crystallinity is gradually
increasing from the central layer. Even in the presence of thecentral fluid layer the barrier function is retained while
deformation as a consequence of shear stresses is facilitated. The latter might be of importance for the elastic properties
of the skin.
neum. There is one report on the formation of a 10.5 nm lamellar phase in CHOL:bovine-brain
CER mixtures interpreted as a two layer structure. This structure, however, differs from the LPP
phase present in stratum corneum [80]. In several studies Fourier Transformed Infrared (FTIR)
spectroscopy measurements were carried out using mixtures prepared from either bovine-brain
CERIII, synthetic CER2 or synthetic CER5 [81,82], CHOL and palmitic acid. At physiological
temperature the CD2 scissoring mode of palmitic acid and the CH2 scissoring mode of ceramides
are each split into two components, indicating that these molecules are located in different lattices. Measurements at elevated temperature confirmed that the components were not properly
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mixed in one orthorhombic lattice, but phase separated in different lattices. A similar behavior
was found when mixing fatty acids with either synthetic CER2 or synthetic CER5 [81]. Similarly,
using FT-Raman spectroscopy also phase separation was observed between CER and saturated
fatty acid [83]. It should be noted that the components had a uniform chain length. Recently we
have observed that C22 fatty acid did not mix properly with isolated pigCER and CHOL
[Bouwstra et al., unpublished results]. However, in earlier publications [59,84] it was reported that
after addition of FFA with a chain length distribution between 16 and 24 to the CHOL:pigCER
no phase separation occurred. The latter was observed by X-ray diffraction and by electron diffraction. Therefore, to achieve proper mixing of FFA and CER the use of mixtures of FFA
containing varying instead of uniform chain length is essential.
In several studies liposomes were also used as model systems [85–87]. Although these systems
are excellent models for studying bilayer permeability, some fundamental problems are
encountered when using liposomes as a stratum corneum lipid model. Namely when vesicles were
prepared at a pH value ranging between 5 and 7, which approximates the pH in the stratum corneum [88–90] the vesicles show a tendency to fuse. This is probably due to the fact that at pH 5 FFA,
CER and CHOL are not charged and at pH 7.4, FFA are most probably only partially charged.
4. A unique molecular arrangement in the long periodicity phase
In 1989 a model that describes the molecular arrangement in the 13 LPP was proposed for the
first time [91]. Based on the broad-narrow-broad pattern obtained after fixation with ruthenium
tetroxide a trilayer model was proposed. In this model the CER are arranged in a planar
arrangement and the linoleic moiety of CER1 is not located in the narrow layer, but is randomly
distributed in the two broad layers adjacent on both sides of the narrow layer. Furthermore, the
CHOL interfacial area is assumed to be similar to that of the CER in planar alignement. Based
on more recent knowledge about the lipid phase behavior of CHOL:pigCER and CHOL:HCER
mixtures another trilayer model has been proposed for the molecular arrangement of the LPP [72]
(see Fig. 8). In this model the ceramides are either partly interdigitating (the broad low-electron
density layers) or fully interdigitating (the narrow low-electron density layer in the center of the
lamellae) and are arranged tale to tale in a double arrangement in the lipid layers. The two broad
low-electron density regions are formed by ceramides with the long-chain fatty acids (predominantly C24–C26) linked to the (phyto)sphingosine backbone and by CHOL, while the narrow low-electron density region is formed by CHOL and the unsaturated C18 acyl chain linked to
the o-hydroxy fatty acid. The proposed molecular model is based on the following findings: (a)
the electron density profile of the repeating unit calculated from the intensities of the 1st to 10th
order of the lamellar pattern attributed to the LPP in CHOL:pigCER mixtures [70] resulted in a
sequence of broad–narrow–broad low electron density regions with in between the higher electron density regions corresponding to the head groups, (b) the crucial role CER1 plays in the
formation of the long periodicity phase and (c) the bimodal fatty acid chain-length distribution of
the ceramides [18,74], and (d) the observed phase separation in a mixture containing ceramides
with long acyl chains and short acyl chains [74]. The LPP consists of three different regions in
which the liquid sublattice is located in the central lipid layer. There, mainly unsaturated linoleic
acid and CHOL are present. Adjacent to this central layer two regions are located in which a
J.A. Bouwstra et al. / Progress in Lipid Research 42 (2003) 1–36
21
gradual change in lipid mobility occurs in the direction perpendicular to the central plane. The
decreased mobility in these adjacent layers can be attributed to the presence of less mobile long
saturated hydrocarbon chains (see Fig. 3). Since only a small fraction of lipids forms a fluid phase
in the stratum corneum, it is assumed that this fluid phase in the central lipid layer is not a continuous one. Because the lipid lamellae are mainly orientated parallel to the surface of the corneocytes, substances always have to pass the crystalline lipid lamellar region and partly diffuse
through the less densely packed lipid regions parallel to these regions. In this way an excellent
barrier is maintained, even when a fluid phase is present. When comparing our model with the
molecular model proposed by Swarzendruber et al. [91], our model suggests an approximately
equal interfacial area of CHOL and of ceramides having a tail to tail arrangement, while Swarzendruber’s model suggests an equal interfacial area of CHOL and ceramides in a planar alignment. Dahler and Pascher reported an interfacial area of ceramides in planar arrangement of
approximately 0.25 nm2 [92]. This is different from that of CHOL (0.37 nm2). The interfacial area
of ceramides having a tail to tail double arrangement is predicted to be approximately 0.40 nm2
[92], which is indeed a value close to that of CHOL. Furthermore, in their trilayer model the presence of a liquid layer is not possible, due to the random distribution of the linoleate of CER1. It is
a task for future studies to validate whether the proposed ‘‘sandwich model’’ correctly represents
the lipid organization of the in the stratum corneum. In 1993, another model was proposed by
Forslind [93] for the presence of liquid phases in the stratum corneum. This model postulated the
presence of a continuous liquid phase from the superficial layers of the stratum corneum down to
the viable epidermis, the so-called mosaic model. Although this was the first model including the
presence of a liquid phase in stratum corneum lipid structures, until now no experimental data are
available to verify this model.
5. Extrapolation of the in vitro finding to the lipid composition and organization in diseased
and in dry skin
In atopic dermatitis patients and in dry skin [49,94] a reduced HCER1 content alters the organization of lamellar phases [95], which reflects the observations made with lipid mixtures. There,
in the absence of HCER1 the formation of the 12–13 nm lamellar phase was reduced, while the
formation of the 5–6 nm lamellar phase was strongly promoted. In addition, in dry skin, in skin
during the winter season and in essential fatty acid deficient skin the HCER1-ol content is
increased at the expense of HCER1-lin. One can expect that changes in lipid phase behavior
might also occur [48], since in HCER1-ol containing lipid mixtures the presence of the liquid
phase is increased as compared to the HCER1-lin containing mixtures. In fact, in essential fatty
acid deficient and dry skin a reduced skin barrier has been observed, while the ultrastructural
appearance of the lipid lamellae was still similar to that in normal skin [28,47]. It can be speculated that fraction of lipids forming a fluid phase increases. Although the presence of a subpopulation of lipids forming the fluid phase might be required for a proper functioning of the
stratum corneum, an excessive presence of a fluid phase may lead to the reduction of the barrier
function. Furthermore, we have also noticed that in CHOL:HCER and CHOL:pigCER mixtures
a hexagonal lateral packing is formed and that the presence of FFA facilitates transformation of
the hexagonal into an orthorhombic lateral packing. The prominent presence of the hexagonal
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lateral packing in lamellar ichthyosis skin [51] might be caused by the observed reduced content
of FFA in this skin disease. Finally, cholesterol sulfate was found to promote the formation of a
fluid phase [59]. An increase in cholesterol sulfate content, as observed in recessive x-linked ichthyosis skin [96], may further lead to reduction of the lattice density and consequently to an
increase in the stratum corneum permeability and in this way can account for the observed
reduction of barrier function in this skin disease.
6. The effect of water and vesicles on the ultrastructure of the skin
6.1. Water
Under normal conditions, the stratum corneum is a relatively dry tissue. Its water content
amounts about 20% w/w. Increased hydration of the stratum corneum, which can be achieved by
Fig. 9. Hydration of stratum corneum. In fully hydrated stratum corneum the water is taken up by corneocytes. In
addition, in the intercellular regions water is also present in separate domains (W). This is shown by the freeze fracture
technique (a) and by cryo-scanning electron microscopy in combination with cryo-planing (b). After pretreatment with
water, domains of rough structures are also present in the intercellular lipid regions indicating that the lipid structure is
locally affected by water. Thick arrow: stratum corneum/stratum granulosum interface, IL=intercellular lipids,
RS=rough structure, W=water pool, SS=smooth surfaces, D=desmosome, C=corneocyte, Bar=1000 nm.
J.A. Bouwstra et al. / Progress in Lipid Research 42 (2003) 1–36
23
occlusion of the skin, generally increases transdermal delivery of topically applied drugs. Because
stratum corneum lipids play an important role in the skin barrier function, the information on the
effect of hydration on stratum corneum lipid organization is of great importance. Water has been
found to exert only a minor effect on the temperatures of lipid transitions in the stratum corneum, in contrast to that observed for phospholipid membranes [97]. X-ray diffraction [28,38–40]
as well as FTIR spectroscopy [34] revealed that water did not affect lipid alkyl chain order at
room temperature, while electron spin resonance studies revealed that increased water content
increased the mobility of the hydrocarbon chains [98]. Furthermore, X-ray diffraction studies
reported that the lamellae do not swell upon increasing the water content in stratum corneum,
which again contrasts the finding in phospholipid membranes [99]. It has been suggested that
increased alkyl chain mobility might be limited to certain domains in the intercellular regions.
This is in agreement with observations made with freeze fracture electron microscopy [100].
Using this technique, in untreated stratum corneum samples the lipid lamellae were visualized
as smooth areas at the plane of fracture. However, after extensive treatment with PBS, next to
these smooth regions areas with a rough surface appeared in the intercellular domains indicating
changes in lipid structure. Furthermore, water domains were detected not only in the corneocytes,
but also in the intercellular regions. A similar phenomenon was observed using cryo-planing in
combination with cryo-scanning electron microscopy: uptake of water by corneocytes and presence of water domains in the intercellular regions at high water content (unpublished results, see
Fig. 9B). The presence of water domains strongly indicates that phase separation occurs between
the lipid lamellae and the water. This is not surprising because the lipids present in the intercellular domains are rather lipophilic, especially at pH values at which FFA are not dissociated.
Continuous water channels across the stratum corneum would create a large interface between
lipid domains and water. This is considered energetically unfavorable and therefore it is not
expected to occur.
6.2. Vesicular structures
6.2.1. Introduction
To increase drug transport across the skin penetration enhancers as well as other chemical
methods have been used. One of the most controversial methods to increase drug transport across
the skin is the use of vesicles. Although it has been generally accepted that the use of vesicles with
appropriate composition should result in increased drug transport across the skin, many questions arise about the mechanism of action of these vesicular formulations. It is for this reason that
in this paper we intend to summarize the present knowledge on the mechanism by which vesicular
structures can alter drug transport. The use of vesicles for transdermal drug delivery has been
introduced by Mezei and Gulasekharam in 1980 [101,102]. These authors claimed that intact
liposomes penetrate across the skin. This statement received a lot of scepticism. Later on various
reports appeared in literature, in which it was concluded that penetration of intact vesicles [103–
105] does not occur. Furthermore, it was found that gel-state vesicles are less effective in
increasing drug permeation across the skin than liquid-state vesicles [106–109]. Gel-state vesicles
can even inhibit drug permeation across the skin. Furthermore, variation in other physical characteristics of the vesicles, such as size, number of bilayers and charge, exerted a less pronounced
effect on the drug permeation rate [110]. In a few studies occlusive application was compared to
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J.A. Bouwstra et al. / Progress in Lipid Research 42 (2003) 1–36
non-occlusive application. These studies revealed that occlusive application of vesicle suspension
was less effective than the non-occlusive one [111,112]. These results were somewhat unexpected,
since water has been reported to be an effective permeation enhancer [113]. In the beginning of
the nineties a new physical parameter was introduced, namely the elasticity of the vesicles bilayers
[111]. According to Cevc and Blume [111] the water gradient is an important driving force for
elastic vesicles. These authors claim an intact vesicle penetration through the skin, as long as
elastic vesicles, the Transfersomes1, are used. As a result of the hydration force in the skin,
elastic vesicles are claimed to ‘squeeze’ through stratum corneum lipid lamellar regions [111].
Transfersomes1 have been successfully used as drug carriers for hydrocortisone, inulin, estradiol
[114], lidocaine and tetracaine [115] and very large molecules such as bovine serum albumin and
insulin [116–119] in vivo. Recently, El Maghraby et al [114,120] studied the transport of estradiol
across human skin applied in Transfersomes1 in vitro. Although they observed an increase in
estradiol flux, the effect was less dramatic than reported by the group of Cevc for the in vivo
situation. Although there is no doubt that Transfersomes1 have advantages with respect to
increasing the transport of active agents across the skin, it was also claimed that the intact elastic
Transfersomes1 penetrated through the stratum corneum and through the underlying viable skin
into the blood circulation. The latter was met with much scepticism. Based on the principle that
edge activators and membrane forming agents form elastic bilayers, another approach has been
used in studies of van den Bergh et al. [121]. These authors prepared elastic vesicles with sucrose
ester laurate (L-595] and octaethyleneglycol laurate ester (PEG-8-laurate) and studied the effect
of the edge activator on the vesicle morphology. Both FFEM and cryo-transmission electron
microscopy revealed that vesicles containing 30 mol% PEG-8-L form unilamellar spherical
bilayers. Increasing the PEG-8-laurate content to 70 mol% within bilayers composed of either
L-595 led to an increase in vesicle size and to a formation of less spherical perforated vesicles
coexisting with threadlike micelles. This has been observed in the absence and in the presence of
stabilizing agents such as cholesterol sulfate. Recent studies from our laboratory revealed that
when vesicles prepared from surfactants are used the flux of drugs incorporated into vesicles significantly increases when the bilayers became elastic (unpublished results), similarly as observed
by El Maghraby et al. [120].
Table 3
The phospholipid content of the NAT50, NAT89 and NAT106 liposomes (% is weight percentage of the total amount
of lipids)
PC with LPC
PE
PA and N-acyl PE
PI and PE
N acyl PE
PI
NAT 50 (%)
NAT 89 (%)
NAT 106 (%)
28
2
10
25
85
10
5
11
32
4
20
PA=phosphatidic acid, PC=phosphatidylcholine, PE=phosphatidyl ethanol amine, PI=phosphatidylinositol,
LPC=lysophosphatidylcholine.
J.A. Bouwstra et al. / Progress in Lipid Research 42 (2003) 1–36
25
Fig. 10. Effect of topical treatment with liquid-state vesicles prepared from septaoxyethylenelaurylether and CHOL on
lipid organization in human stratum corneum. Water pools (W) are shown in which vesicle (see arrows) are located.
This has been observed in the uppermost 4–5 layers in the stratum corneum. Bar=200 nm.
In the next section we will describe the interactions between vesicles and skin, focusing on two
aspects, namely the changes in skin ultrastructure and in penetration pathway of (model) drugs
across the skin when applied in vesicles.
6.2.2. Vesicles perturb the lipid organisation in stratum corneum in vitro: occlusive application
One of the first studies in which vesicle-skin interactions were visualized has been performed
with isolated human stratum corneum incubated for 48 h with vesicles prepared from CHOL and
polyoxyethylenealkylether surfactants. Hofland et al. [122] reported that after this long incubation time liquid as well as gel-state vesicles fused at the superficial layer of the stratum corneum, but that only liquid-state vesicles induced perturbations in lipid organization and
formation of water pools within the stratum corneum. In these water pools vesicular structures
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J.A. Bouwstra et al. / Progress in Lipid Research 42 (2003) 1–36
were observed (see Fig. 10). Because the frequency of appearance of water pools was not determined and in a recent study water pools have also been observed after pretreatment with a
phosphate buffer saline solution (100; see Fig. 9), it is not clear whether vesicles indeed promoted
the formation of water pools. In addition, recently liquid-state vesicles prepared from phospholipids have been compared with gel-state vesicles based on either ceramides or distearylphosphatidylcholine [123]. Gel-state vesicles formed stacks of lamellae on the surface of the stratum
corneum and only occasionally changes in the lipid organization were observed in superficial
stratum corneum layers. Liquid-state vesicles induced perturbations in the lipid organization in
deeper layers of the stratum corneum. These observations confirmed the results obtained with the
surfactant-based vesicles.
In another study the effect of composition of liquid-state liposomes on stratum corneum lipid
organisation was studied [124] with three different phospholipid vesicles prepared according to
Gehring [125]. The compositions of the vesicles referred to as NAT106, NAT50 and NAT89 are
provided in Table 3. In these formulations the amount of phosphatidylcholine varied dramatically.
Furthermore, in the phosphatidylcholine a fraction lysophosphatidylcholine (single-chain phospholipid) was also present. NAT50 (low phosphatidycholine content) liposomes only fused on the
stratum corneum surface and did not cause perturbations in stratum corneum lipid organization,
See Fig. 11. When the stratum corneum was treated with the NAT89 liposomes (medium phosphatidylcholine content), rough structures were formed in the outermost four layers of the stratum corneum, indicating either intrusion of rough ultrastructures formed by fusion of vesicles or
alteration of the lipid lamellae. Up to the fourth corneocyte layer structures resembling vesicles
were observed. The third formulation, NAT106, containing a high fraction of phosphatidylcholine induced marked changes in the stratum corneum ultrastructure. The corneocytes were considerably swollen as compared to untreated skin. The ultrastructure of intercellular lipid lamellae
showed flattened spherical structures, indicating that a substantial amount of liposome material
was incorporated in the stratum corneum intercellular regions. These changes were present
throughout the stratum corneum. The reason for this strong interaction is not clear yet. A possible explanation is the presence of lysophosphatidylcholine in the phosphatidylcholine fraction.
Since this is a single chain lipid, it might act as an edge activator making the vesicles elastic (see
later).
The results described above clearly illustrate that liquid-state vesicles might act not only in
superficial stratum corneum layers, but may also induce lipid perturbations in deeper layers of the
stratum corneum, while gel-state vesicles interact only with the outermost layers in the stratum
corneum. This might explain the difference in drug permeation enhancement between gel-state
and liquid-state vesicles. In addition, fusion of gel-state vesicles on top of the stratum corneum might also act as an additional barrier for diffusion of drugs and therefore inhibit skin
permeation.
6.2.3. Vesicles perturb the lipid organization in the stratum corneum in vitro and in vivo: nonocclusive application
Since it has been postulated that elastic vesicles require a hydration gradient to exert a maximal
effect on the stratum corneum ultrastucture several studies have been performed in which the
effect of elasticity of vesicle bilayers on either penetration pathway or ultrastructure of the skin
was investigated. Focused on the comparison between Transfersomes1 and conventional lipo-
J.A. Bouwstra et al. / Progress in Lipid Research 42 (2003) 1–36
27
Fig. 11. Liposome-induced changes in lipid organization in human stratum corneum. (A) Freeze fracture electron
micrograph of human stratum corneum treated with NAT50 liposomes. No changes in stratum corneum lipid organization have been observed. Vesicle formulations are present on the surface of the skin (V). The vesicles fuse (see arrow)
and form lipid lamellae. This fusion process is clearly detected. Bar=100 nm. (B) Freeze fracture electron micrograph
of human stratum corneum treated with NAT89 liposomes. Rough lipid structures (RS) have been observed in the
outermost 4–5 corneocyte layers indicating that either ingredients of the NAT89 liposomes fuse resulting in the formation of rough structures or the NAT89 liposomes induce perturbations in the SC lipid lamellar structure. Vesicles
(V) are still present. (C) Freeze fracture electron micrograph of human stratum corneum treated with NAT106 liposomes. The ultrastructure of the stratum corneum changed dramatically. Extremely swollen corneocytes have been
visualized between which flat lipid islands are located (see arrows). No smooth areas indicating intact lipid lamellae
could be visualized. The perturbations were visualized throughout the stratum corneum. C=corneocytes. Bar=100
nm. (D) A high magnification of the lipid organization in by NAT106 liposome-treated human stratum corneum. Flat
lipid islands (see arrows) are clearly present. It seems that treatment with NAT106 liposomes results in lipid domain
forming. C=corneocyte. Bar=100 nm.
28
J.A. Bouwstra et al. / Progress in Lipid Research 42 (2003) 1–36
somes, recently studies have been performed in which the penetration of fluorescent labels across
mice skin was visualized. For this purpose Schätzlein and Cevc [118,126] intercalated Rhodamine-DHPE [1,2-dihexadecanoyl-sn-glycero-3-phosphatidyl-ethanolamine-N-Lissamine rhodamine B sulfonyl, triethylamonium salt) in the bilayers of Transfersomes# prepared from soya
phosphatidylcholine and sodium cholate. The suspensions were applied onto mice skin for 4–12
h. Thereafter, the skin was examined ex vivo using confocal laser scanning microscopy. The
confocal images revealed the existence of an inter-cluster pathway between groups of cells (see
Fig. 12).
These pathways appeared as a high fluorescence intensity pathway in the intercellular lipid
lamellar regions. The authors interpret these regions as being virtual pores between the corneocytes through which vesicles can penetrate. However, one should realize that confocal laser
scanning microscopy is a technique that cannot be used to visualize the transport of vesicles as
intact entities, however, it can be excellently used to visualize the transport of the label which can
be considered as a model drug. Very recently the interaction between skin and elastic vesicles
prepared from L-595 and PEG-8-L has been studied [127]. The vesicles were applied onto human
skin in vitro and mouse skin in vivo [121,128] and the ultrastructure of the skin was visualized
using RuO4 post-fixation in combination with transmission electron microscopy.
One hour non-occlusive application of elastic vesicles onto mice skin in vivo revealed striking
changes in the lipid organization, namely islands of lamellar stacks were observed in the inter-
Fig. 12. Penetration of fluorescent label applied in Transfersomes across murine skin. Note the hexagonal shape of the
stratum corneum cells is clearly visible. With permission [126].
J.A. Bouwstra et al. / Progress in Lipid Research 42 (2003) 1–36
29
cellular regions throughout the stratum corneum, as depicted in Fig. 13. The pattern of the
lamellar stacks was different from that of the naturally formed lamellae by the stratum corneum
lipids indicating a fast partitioning of vesicle ingredients into deeper layers of the stratum corneum. This was not observed with rigid vesicles. However, since mice skin is more permeable than
human skin and in addition is more sensitive to formulations applied onto the skin, it is essential
to perform the studies on human skin.
Therefore, the fate of elastic vesicles after application onto excised human skin was also examined. Four types of interactions were noticed (see Fig. 14) (i) The presence of spherical lipid
structures containing or surrounded by electron dense spots, indicative for the presence of vesicle
material both on the surface of the skin and in between the upper 3–4 cell layers. (ii) Oligolamellar vesicles were observed between the 2–3 upper corneocyte layers. (iii) Large areas containing
lipids, surfactants and electron dense spots and vesicle bilayers were observed deeper down into the
stratum corneum. (iv) Large areas of lamellar stacks were present throughout the entire stratum
corneum. Occasionally it was observed that these lamellar stacks disorganized the intercellular skin
lipid bilayers stacks similarly as observed in mice stratum corneum in vivo. The bilayers in these
stacks were frequently orientated perpendicularly to the lamellae of the stratum corneum (Fig. 14C).
Only when the surfactant ingredients accumulated in large regions, the lamellar stacks were orientated randomly (Fig. 14D). The formation of islands of lamellar stacks was similar to that observed
frequently in elastic vesicles treated stratum corneum of hairless mouse skin [128]. Treatment with
conventional rigid gel-state vesicles affected the most apical corneocytes only to some extent.
None of the vesicle formulations affected the viable epidermis or dermis indicating that the
vesicle ingredients remained mainly in the stratum corneum. In addition to these extraordinary
Fig. 13. Effect of surfactant-based vesicles on lipid structure in mouse stratum corneum. Transmission electron
micrograph of mouse skin treated non-occlusively with surfactant-based elastic vesicles for 3 h. In the intercellular
regions isolated domains are found consisting of an array of narrow small lamellae. The pattern of these domains is
different from that of the stratum corneum lipid lamellae. The stacks are orientated perpendicular to the basal plane of
the stratum corneum lipid lamellae (SL). Black arrows indicate the stratum corneum lipid lamellae. Black-white arrow
refers to the lamellar stacks formed by the vesicle ingredients. Bar=100 nm.
30
J.A. Bouwstra et al. / Progress in Lipid Research 42 (2003) 1–36
Fig. 14. (a) Elastic vesicles prepared from L-595 and PEG-8-L changes the ultrastructure of human stratum corneum
in vitro. In the electron micrographs typical examples of the ultrastructural changes are depicted. Transmission electron micrograph of human skin treated non-occlusively for 16 h in vitro withphosphate buffer saline. Between the
corneocytes (C) the intercellular lipid lamellae (icl) are located. Bar=100 nm. (b) Surfactant-based elastic vesicles . In
the 4–5 outermost layers in the stratum corneum the lipid organization has locally been perturbed (dicl). Frequently
single lipid lamellae are visualized most probably originating from the vesicles. Fusion of vesicles has also been observed.
C=corneocyte. Bar =100 nm. (c) Surfactant-based elastic vesicles. Single lamellae and dark spots are observed (see
arrow) between the lamellae not observed in the control. C=corneocyte. Bar=100 nm. (d) Surfactant-based elastic
vesicles. In the intercellular regions characteristic domains are found consisting of an array of narrow small lamellae (see
arrows). The stacks are orientated perpendicular to the basal plane of the stratum corneum lamellae similarly as in mouse
stratum corneum. The arrowhead indicates the presence of a Landman unit characteristic for the skin lipid lamellae.
Bar=100 nm. (e) Surfactant-based elastic vesicles. A large region of aggregated isolated domains consisting of narrow
small lamellae (see arrows) has been visualized. The orientation of the lamellar stacks is randomly. Bar=100 nm.
features observed in human and mice skin, the penetration pathway of a fluorescent label intercalated in the bilayers of elastic or rigid vesicles was visualized using two photon excitation
microscopy. Again the results were very unexpected. When the label was intercalated in the
bilayers of the elastic vesicles, an inhomogeneous label distribution was observed (see Fig. 15).
Thread-like channels were visualized that might serve as penetration pathways for the dyes.
However, without changing the settings of the microscope no dye could be detected in the viable
J.A. Bouwstra et al. / Progress in Lipid Research 42 (2003) 1–36
31
Fig. 15. Penetration of diacylphosphatidylethanolamine across human stratum corneum. Fluorescein diacylphosphatidylethanolamine was applied onto human skin in either rigid vesicles (A), elastic vesicles (B) or micelles (C) after
which the stratum corneum has been the penetration of the label was monitored by two photon excitation fluorescence
spectroscopy. The XY-images (9595 mm) have been taken approximately parallelly to the skin surface. Application in
rigid vesicles and micelles (A,C) results in a homogenous distribution of the fluorescent label in the intercellular space
resulting in the visualization of the hexagonal shape of the cells in stratum corneum. In contrast application of the
fluorescent label in elastic vesicles (B) results in an inhomogeneous label distribution: thread-like channels are visualized indicating that the penetration pathway is localized in channels.
epidermis. The distribution of the label in stratum corneum was different from that observed with
Transfersomes1 (see Fig. 12) indicating that these elastic vesicles exert another interaction with
the stratum corneum than Transfersomes1 do. Intercalation of the label in the bilayers of rigid
vesicles resulted in an almost homogeneous label distribution in the intercellular regions, which
can be deduced from the appearance of the hexagonal corneocytes orientated approximately
parallel to the stratum corneum surface (see Fig. 15). Furthermore, the label could only be
detected in the superficial stratum corneum layers. In case of micelles a similar label distribution
was observed as seen with rigid vesicles. These results clearly demonstrate that elastic vesicles
induce another penetration pathway for substances through the stratum corneum than the conventional vesicles do and that it is not caused by an increased concentration of the single-chain
PEG-8-L (the only component of the micelles) present in elastic vesicles. Based on the results
from fluorescent spectroscopy and transmission electron microscopy, it became clear that there is
no evidence that material strongly associated with vesicles or the vesicle ingredients themselves
penetrate fast into the viable epidermis. According to Cevc and Blume [111], the osmotic force
resulting from the hydration gradient in the skin is the dominant force for partitioning of vesicles
into the stratum corneum. However, recently we have noticed that even in a fully hydrated state
the water content in the lowest stratum corneum layers close to the viable epidermis is much
lower than in the central regions of the stratum corneum. Therefore, it is expected that as a result
of the osmotic force, the vesicle ingredients will not penetrate beyond the level of the lowest layers
in the stratum corneum. This is exactly what we have noticed in our visualization studies.
Whether the interactions observed in vitro with excised human skin can be extrapolated to the
in vivo situation has recently been studied [129] using FFEM. Elastic and rigid vesicles were
applied non-occlusively onto human skin for one hour after which the stratum corneum was
stripped sequentially. The stripped stratum corneum was fractured approximately parallel to the
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J.A. Bouwstra et al. / Progress in Lipid Research 42 (2003) 1–36
corneocytes along the lipid lamellae. Surprisingly, the presence of vesicles was noticed up to the
ninth strip in the stratum corneum in channel like regions (see Fig. 16). These channel-like
regions have a very similar appearance as the thread-like channels observed with two photon
excitation fluorescent spectroscopy (see Fig. 15). This indicates that the label distribution in vitro
is similar to the vesicle distribution in vivo. Furthermore, when substances remain strongly associated with the vesicles, elastic vesicles can be used to transfer substances rapidly into the deeper
layers of the stratum corneum, after which the substances can permeate into the viable epidermis.
Although these are very encouraging results, still many questions remain to be solved. For
example, both in vitro in excised human skin and in vivo in mouse skin, islands of lamellar stacks
are detected in the stratum corneum with transmission electron microscopy (see Figs. 13 and 14).
However, when examining the vesicular structures in human skin after in vivo application, we
have never noticed that the vesicular structures were composed of lamellar stacks. This suggests
that in human stratum corneum in vivo we deal with another structure of the penetrating material
than in mouse stratum corneum in vivo and human stratum corneum in vitro. Whether this is due
to subtle differences in treatment or to differences in hydration gradient remains subject for further studies.
Fig. 16. Effect of elastic vesicles prepared from L-595 and PEG-8-L on human stratum corneum lipid organization.
(A) Phosphate buffer saline treated human skin in vivo. Freeze fracture electron micrograph of human stratum corneum using the stripping method. Shown is the fracture orientated parallel to the skin surface along the lipid lamellae.
Smooth areas with desmosomes have been visualized (asteriks). Occasionally a fracture across the lipid lamellae is
depicted (arrow). (B) Human skin treated in vivo for 1 h with elastic vesicles. Freeze fracture electron micrograph of
human stratum corneum using the stripping method. Shown is the stratum corneum lipid organization of the ninth
strip. Channel-like regions are observed with vesicular structures (V). This strongly suggests that vesicles are present in
the deeper layers of the stratum corneum.
J.A. Bouwstra et al. / Progress in Lipid Research 42 (2003) 1–36
33
7. Conclusions
One of the problems in studying stratum corneum lipid organization is the complexity of the
tissue with its unusual lipid composition. Since it appeared to be impossible to selectively extract
lipids from the stratum corneum, the role the various lipid classes play in lipid phase behavior
could only be studied with isolated lipid mixtures. These studies revealed that mixtures prepared
with isolated CER, CHOL, FFA and cholesterol sulfate mimic stratum corneum lipid organization very closely. Furthermore, so far deviation in lipid organization in diseased and dry skin
could often be explained by the results obtained with the isolated lipid mixtures illustrating an
excellent in vitro-in vivo correlation. Since proteins are not present in the isolated lipid mixtures,
lipid phase behavior studies strongly suggest that proteins do not play an important role in stratum
corneum lipid organization. Although substantial progress has been made in elucidating the stratum corneum lipid phase behavior, many parameters are still unknown. As an example, no
information is available about the distribution of the various lipid classes and ceramide subclasses
between the crystalline LPP and SPP. Furthermore, only limited information is available about
the abnormal lipid organization and composition in diseased human skin. Information on this
tissue is hampered by its limited availability.
Despite the presence of crystalline lipid phases in stratum corneum a proper design of the
delivery system as illustrated for vesicular formulations will contribute to a wider variety of drugs
that can be given transdermally. An important challenge in the next years will be to design
delivery systems that will make it possible to transport large proteins across this barrier.
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