Overview

Drug delivery through the skin:

molecular simulations of barrier
lipids to design more effective
noninvasive dermal and
transdermal delivery systems for
small molecules, biologics, and
cosmetics
J. Torin Huzil1,2 , Siv Sivaloganathan2, Mohammad Kohandel2
and Marianna Foldvari1
The delivery of drugs through the skin provides a convenient route of administration that is often preferable to injection because it is noninvasive and can typically
be self-administered. These two factors alone result in a significant reduction of
medical complications and improvement in patient compliance. Unfortunately, a
significant obstacle to dermal and transdermal drug delivery alike is the resilient
barrier that the epidermal layers of the skin, primarily the stratum corneum,
presents for the diffusion of exogenous chemical agents. Further advancement
of transdermal drug delivery requires the development of novel delivery systems
that are suitable for modern, macromolecular protein and nucleotide therapeutic
agents. Significant effort has already been devoted to obtain a functional understanding of the physical barrier properties imparted by the epidermis, specifically
the membrane structures of the stratum corneum. However, structural observations of membrane systems are often hindered by low resolutions, making it
difficult to resolve the molecular mechanisms related to interactions between lipids
found within the stratum corneum. Several models describing the molecular diffusion of drug molecules through the stratum corneum have now been postulated,
where chemical permeation enhancers are thought to disrupt the underlying lipid
structure, resulting in enhanced permeability. Recent investigations using biphasic
vesicles also suggested a possibility for novel mechanisms involving the formation
of complex polymorphic lipid phases. In this review, we discuss the advantages
and limitations of permeation-enhancing strategies and how computational simulations, at the atomic scale, coupled with physical observations can provide insight
into the mechanisms of diffusion through the stratum corneum. 2011 John Wiley
& Sons, Inc. WIREs Nanomed Nanobiotechnol 2011 3 449462 DOI: 10.1002/wnan.147

INTRODUCTION
Correspondence

to: foldvari@uwaterloo.ca

School of Pharmacy, University of Waterloo, Waterloo, Ontario,

Canada

2 Department

of Applied Mathematics, University of Waterloo,

Waterloo, Ontario, Canada
DOI: 10.1002/wnan.147

Vo lu me 3, September/Octo ber 2011

he choice regarding a modality used for drug

administration is dependent on several factors,
including the active substance in question, its
pharmacokinetic profile, and the desired location
of action.1 Because they are noninvasive, the
administration of drugs by mouth, inhalation, or

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Overview

directly to the skin is often preferred to those requiring

injection into the circulatory system. However,
while these delivery methods may be preferable,
they do have several disadvantages over injections.
For instance, oral administration requires efficient
absorption through the gastrointestinal (GI) tract
and a drug must therefore be resistant to the harsh
physicochemical environment present in both the
stomach and intestines. The oral delivery of drugs also
exposes them to first-pass metabolism within the liver,
often reducing their bioavailability significantly. The
inhalation of aerosol drugs eliminates both exposure
to the GI track and first-pass metabolism; however,
the difficulty to accurately meter dosage, coupled
with the requirement for complex delivery devices,
generally limits its use to drugs targeting the lungs
directly. Dermal delivery of drugs is complicated
because the skins natural role as a barrier to external
contaminants often requires the use of some method
of physical or chemical disruption.
Many next-generation therapeutic agents,
including recombinant proteins and other biologics,
such as antibodies and nucleic acids are not amenable
to oral, inhalational, or transdermal administration
because they are either susceptible to enzymatic degradation or cannot be absorbed through the epidermis
into the skin efficiently due to their molecular size.2,3
Many of these biological drugs can only be administered using injections, thereby avoiding the difficulties
associated with oral, inhalational, and transdermal
delivery, while providing immediate bioavailability at
all locations in the body. However, because injections
damage the protective layers of the epidermis, there
is a strict requirement for asepsis in order to avoid
the introduction of pathogens, such as hepatitis, or
human immunodeficiency virus (HIV). If not administered correctly, injections can also introduce contaminants, such as insoluble drug or adjuvant particles
and potentially fatal air boluses into the circulatory
system.
Numerous transdermal methodologies have now
been explored in an attempt to improve the noninvasive delivery of macromolecular drugs, including
proteins such as insulin,4 interferon- (IFN-),5 and
DNA.6 Novel, transdermal approaches to the delivery
of these macromolecules include pulmonary, buccal,
rectal, and vaginal administration methods. Intraand transdermal, sustained-release delivery of macromolecules would have many advantages compared
with administration by injection and other shortacting dosage forms. However, the lack of efficient and
safe methods for compounds with molecular weights
greater than 500 Da greatly limits the applicability of
many of these, noninvasive, methods.
450

DERMAL AND TRANSDERMAL DRUG

DELIVERY
Delivery of drugs through the skin can be divided
into two classes, which are associated with distinct
purposes. The first class involves delivery into the skin
itself for dermatological treatment, vaccination, or
cosmetic applications, and is termed dermal delivery.
Transdermal delivery, on the other hand, while also
using the skin as the application site, introduces the
drug for transport into the circulatory system. The
delivery of drugs through the skin provides a convenient route of administration that bypasses the GI
tract, first-pass metabolism, and many of the complications associated with injectable drugs. However,
because the skin is extremely effective at protecting the
body from external pathogens and toxins, both dermal
and transdermal delivery systems must be designed to
circumvent its barrier properties.7 Despite the rapid
growth of new delivery technologies, the complications associated with noninvasive introduction of
drugs into the body are clearly evident by the fact that
as of 2008 only 20 transdermal drug formulations
had been approved by the Food and Drug Administration (FDA).8 For dermal and transdermal delivery to
become amenable for use with a wider range of nextgeneration therapeutic agents, a clear understanding
of the mechanisms associated with the barrier properties of the skin must first be developed and overcome.

SKIN STRUCTURE
The skin is composed of three primary layers: the
epidermis, dermis, and subcutaneous tissue (Figure 1).
The stratum corneum (SC) is the outmost layer
of epidermis, providing the skin with its resilient
absorption barrier properties. The SC is composed
of anywhere from 1060 layers of flattened, nonliving
corneocytes which are almost entirely made up of
cross-linked keratin (7585% of the SC is keratin)
surrounded by an intercellular matrix composed
primarily of long-chain ceramides, free fatty acids,
triglycerides, cholesterol, cholesterol sulfate, and
sterol/wax esters.9,10 In the conceptualized bricks
and mortar model of the SC, bricks correspond to
hydrophilic corneocytes and the mortar is represented
as intercellular spaces containing hydrophobic
lipids.1114 It is generally thought that absorption
may occur through (1) an intercellular route, typical
of lipophilic substances; (2) the appendages (hair
follicles and sweat ducts); or (3) an intracellular
route, more typical of hydrophilic substances. Recent
studies, describing the partitioning of lipophilic
and hydrophilic molecules with corneocytes, showed

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Skin strucutre

SAXS Pattern

Straum corneum Permeation pachways

Dermis

40 m

te
cy
eo
rn
co

Ethanol
Oleic acid
Transcutol
Proplylene glycol

Intracellular

100 nm

Intensity

Intercellular

Viable
epidermis

Disordering, fluidization
or phase separation

bare SC
6.3 nm
4.6 nm

40 nm

Effect on SC lipid

3.3 nm 2.3 nm
0.05 0.10 0.15 0.20 0.25 0.30 0.35

Lamellar-to-Lamellar
phase change

q (-1)
Crystalline

Fluid

Pn3m D-surface

bare SC
emulsion-treated SC

Intensity (A.U.)

Crystalline

liposome-treated SC
biphasic vesicle-treated SC

0.1

0.2
q (-1)

0.3

Mixed polymorphic-tocubic phase change

FIGURE 1 | Human skin structure and effect of delivery systems and permeation enhancers on permeation of drug molecules. Three-dimensional
(3D) structure of human skin (left column): first-generation approach to transdermal delivery is limited primarily by the barrier posed by skins
outermost layer called the stratum corneum, which is 1020 m thick. Underneath this layer is the viable epidermis, which measures 50100 m and
is avascular. Deeper still is the dermis, which is 12 mm thick, and contains a rich capillary bed for systemic drug absorption and nerve endings just
below the dermalepidermal junction. Chemical permeation enhancers and drug delivery systems interact with the intercellular lipids in the stratum
corneum. This interaction modifies the structural order of the lipids which are originally arranged in multiple bilayer stacks (left column) (electron
micrograph and bilayer model: Reprinted with permission from Ref 16. Copyright 2006 Elsevier). This modification, fluidization, disorder, or
rearrangement of the lipids can be monitored by small-angle X-ray scattering (SAXS)/wide-angle X-ray scattering (WAXS) (middle column).
Conventional permeation enhancers such as ethanol, oleic acid, propylene glycol and a marketed enhancer, Transcutol, cause disordering of the
lipids, but do not disrupt the bilayer configuration (right column). Among the lipid-based delivery systems, liposomes and a submicron emulsion also
have a disordering effect, whereas biphasic vesicles appear to cause rearrangement of the organization of the stratum corneum lipids into a Pn3m
cubic phase configuration (SAXS pattern: middle column, model: right column). This cubic phase could be an intercellular permeation nanopathway
that may explain the increased delivery of interferon- (IFN-) by biphasic vesicles.17

significant interactions with the cell surface.15

However, a precise description of the mechanism
for drug permeation requires the detailed analysis
of molecular interactions in the skin.
Two prominent models have been developed to
explain the formation and molecular organization of
SC lipids. The Landmann model18 postulates that
lamellar granules, produced by granular cells in the
viable epidermis, extrude into the extracellular spaces
to form uninterrupted sheets in a bilayer configuration. This model was later confirmed when identical
patterns of lamellar disks (stacks of flattened liposomes), found inside granular cells, were observed in
the intercellular space.19 The bilayer structure of these
intercellular lipids is made up of two stacked lipid
bilayers composed of different ceramide molecules
(CER1CER8) in an interdigitated arrangement, having a periodicity of approximately 13 nm.20,21 More
recent studies indicate that lipids extruded by granular
cells during barrier lipid formation are also found in
other polymorphic phases, including a cubic phase,
which merges into the intercellular lamellar space.22
This is explained in the membrane folding model,
Vo lu me 3, September/Octo ber 2011

which implies that the flexibility of lipid arrangement,

through estimation of energy requirements for intercellular lipid channel formation via cubiclamellar
phase transition, is more favorable than via lamellar granule fusion.23 This model opened new areas
of research into the mechanisms of drug permeation
through the skin and how effectively lipids in the SC
can be manipulated to enhance drug delivery. As discussed in the next section, most permeation enhancers
disturb, disorder, fluidize, integrate into, or extract
lipids to various extents, which result in an increase
in absorption of some drugs.

COMPOSITION OF STRATUM
CORNEUM LIPIDS
While its composition varies depending on age and
location on the body, the SC consists primarily of
free fatty acids (mainly C22 and C24, 1525%),
long-chain ceramides (CER1CER8) (mostly C2426,
3550%, and CER1 and CER4 with C3032),
cholesterol (1525%), and cholesterol sulfate

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(510%).10,24,25 Of the ceramide species, those with

-hydroxy fatty acids, ester linked to linoleic acid, and
amide linked to sphingosine [Cer(EOS)] predominate,
and tend to be highly enriched in linoleic acid (C18:2),
constituting a minimum of 2030% of the -esterified
fatty acid.9,26 The predominant free fatty acids are
primarily saturated and range in chain lengths from
C14 to C36, with the longer chains present in higher
amounts. It is thought that the long-chain lengths and
compact stacking of the ceramides and fatty acids
are the primary determinants of the extremely stable
physical properties of the SC lipid bilayers.

density maps below a resolution of approximately

27 At this level of resolution, gross molecu10 A.
lar orientations and distinct phases of lipid packing
are distinguishable; however, atom positions are not
resolvable. Many other techniques, including infrared
(IR) spectroscopy,28,29 atomic force, confocal, and
two-photon excitation fluorescence microscopy,30
have been used to successfully study the physicochemical structure of the lipid phases in the SC and
investigate changes in their organization under differing physical parameters. Arguably, the most advantageous technique for observing the phase behavior
of lipids has been X-ray scattering including small(SAXS) and wide-angle (WAXS) methods (Figure 2).
These techniques demonstrate that SC lipids are most
often observed in a crystalline orthorhombic packing
lattice,31 composed of two distinct lamellar phases
with a repeat distance of approximately 6 and 13 nm
and a bilayer orientation that is parallel to the surface

MOLECULAR ORGANIZATION
OF THE STRATUM CORNEUM LIPIDS
Understanding the effect that each of the ceramide
and fatty acid classes play in the organization of the
SC is limited due to a lack of high-resolution electron
CCD
Camera

Scattered beam

Incident beam

Sample

(a)

Synchrotron ring

(b)

(c)

(d)

q=

4psin q

l
l
;
d=
2sin q

d=

Intensity (AU)

6.6 nm

4.6 nm
3.3 nm

2.3 nm

2p
q
0.05 0.10 0.15 0.20 0.25 0.30
q()-1

FIGURE 2 | Application of small-angle X-ray scattering (SAXS)wide-angle X-ray scattering (WAXS) for nanostructure analysis of stratum
corneum lipids. Schematic (a) and experimental setup (b) of a SAXS measurement (enlarged picture shows the multisample holder developed
in-house for the stratum corneum samples); typical scattering pattern (c); and scattering curve of human stratum corneum (d).

452

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Modeling drug delivery through the skin

of the SC.16,21 There are two types of disorder that

can be distinguished within the SC lipid regions: shortrange disorder of the alkyl chains contained within a
single lipid bilayer and long-range disorder in the
arrangement of lipids between multiple lamellae that
results in changes to the space groups for lipid packing.
The high concentration of long-chain ceramides
and fatty acids found in the SC suggests that the
bulk of barrier lipids resides in the crystalline state,
which is separated by a liquid crystalline or gel
state.32 It has been proposed that the distribution
of crystalline and gel phases of the membrane lipids
allows diffusion of small molecules across the SC and
into the dermis.33 Membranes in the liquid-crystalline
state would allow water to pass through, while
bilayers in the gel state would provide an effective
barrier to water penetration. Other models include the
formation of polar pathways across the SC through
which hydrophilic molecules may freely diffuse.34

lipid and constrained interlayer waters via some convoluted path. This transport pathway is highly influenced by the structure and solubility of the molecule
as it passes through the heterogeneous lipid bilayers. Many different methods can be used to enhance
the permeation of drugs across the skin (Figure 3).
Physically bypassing the SC is the simplest technique,
creating both macroscopic and microscopic pathways
through which molecules can penetrate. Techniques
such as electroporation35,36 and iontophoresis37 utilize voltage gradients to introduce a disruption of
the SC. Pretreatment of the skin in this way is
thought to enhance the passage of large, polar
molecules, such as peptides through the SC. Currently, the most successful application of iontophoresis is the intradermal administration of lidocaine
as a local anesthetic prior to dermatological procedures and administration of intravenous drugs.38
Unfortunately, the equipment required for these techniques are large, limiting their availability to clinical
applications.
Alternatively, technologies that puncture or
abrade the outer layers of the epidermis include
microneedles (10100 m in length), where the drug
is coated on the microneedle surface to aid in rapid
absorption,39 dermal abrasion,40 and needle-free

DIFFUSION ACROSS THE STRATUM

CORNEUM
It is generally accepted that molecular diffusion
through the SC requires flow through the intercellular
Skin permeation
enhancer
strategies

Stratum corneum
manipulation

Stratum corneum
bypass

Removal by stripping,
abrading or depilatories

Physical methods

Electroporation

Photomechanical
wave

Optimization of drug
and/or vehicle

Hydration

Prodrug approach

Liposomes

Solvent extraction of
lipids

Supersaturation
(concentration gradient)

Micro- and
nanoemulsions

Eutectic mixtures

Nanoparticles

Chemical permeation
enhancers

Combination
approaches

Delivery systems

Device and
formulation

Complex formulations

Synergistic
enhancers

Specialized
patches

Micro- and
nanoemulsions

Inkjet/
microneedle

Physical method
and delivery
system

Microneedles

Ultrasound

Ion pairs

Polymeric systems

Biphasic vesicles

Needle-free and
ballistic injections

Iontophoresis

Conjugation with
hydrophobic moieties

Targeting systems to
hair follicles

Pheroid particles

Thermoporation

Magnetophoresis

FIGURE 3 | An overview of skin permeation enhancer strategies. A representative flowchart illustrating several skin permeation enhancer
strategies. Subcategories include stratum corneum (SC) bypass, SC manipulation, drug vehicle optimization, delivery systems, and combination
approaches. Methods in bold under SC manipulation are also considered physical methods.

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high-pressure injection.41 Yet other technologies use

magnetophoresis, sonophoresis, and photomechanical
waves42 to apply electromagnetic, ultrasonic, or
mechanical energy to the skin; however, these
techniques have met with limited success as methods
for transdermal drug delivery43 and are often used
as complementary techniques to existing, passive
enhancement systems.

CHEMICAL PENETRATION
ENHANCERS
Chemical penetration enhancers (CPEs) are a group
of pharmacologically inactive compounds, which
reversibly alter the barrier properties of the skin.
Current application of chemical enhancers for drug
permeation enhancement is based mostly on in vitro
or in vivo screening studies that quantitatively measure the extent of enhancement. More than 300
chemical enhancers can be classified into three essential groups based on their mechanism of permeation
enhancement.44,45 Group 1 enhancers extract skin
lipids or damage the SC, thereby weakening the barrier. Examples include solvents (e.g., ethanol) and
organic acids (e.g., salicylic acid). Group 2 enhancers
increase drug solubility within the skin. Examples
include polyols (e.g., propylene glycol). Group 3
enhancers disorder intercellular lipids. Examples
include terpenes, surfactants, fatty acids, fatty acid
esters, Azone (1-dodecylazacycloheptan-2-one) and
its derivatives, amides (e.g., dimethylformamide), and
sulfoxides [e.g., dimethylsulfoxide (DMSO)]. The
Azone-like enhancer mechanism of action has been
most extensively characterized, showing how the partitioning of this molecule into the skin creates a
disturbance of the lipid headgroups of skin ceramides,
thereby enhancing permeation for many small drug
molecules.46 Surfactants also tend to penetrate into
intercellular spaces, increasing lipid phase fluidity and
decreasing the resistance to permeation.4749 In general, absorption enhancement through lipid channels
depends on the ability of the enhancer to integrate with
the existing lipids and create a perturbed microenvironment based on mechanisms, such as (1) alteration
of lipid phase fluidity, (2) enhancement of solubility characteristics of the skin for the drug to be
delivered, (3) creation of a disordering effect among
the alkyl chains of skin lipids, and (4) localized
separation of lipid domains to create hydrophilic
pores.5052 One of the first-generation transdermal
delivery mechanisms used liposomes to encapsulate
drugs.53,54 Liposome-encapsulated drugs are able to
merge with skin lipids, facilitating the delivery of their
payloads into the skin.14 Recently developed delivery
454

systems including deformable liposomes,55 synergistic

combinations of enhancers, nanoparticles,56 biphasic
vesicles, and pheroids5658 (Figure 3). These systems
show increasing potential for delivering a wider selection of drugs including drugs with higher molecular
weights.
As drugs are primarily transported through the
intercellular lipid regions of the SC, the structural
mechanisms of lamellar disruption in the presence
of CPEs and delivery systems are now of great
interest for the development of rational strategies to
enhance drug penetration through the skin. However,
as the overall barrier properties of the skin are not
fundamentally changed during the use of CPEs, they
are commonly utilized to offer improvement to factors
such as dose control, and not to widen the overall
applicability of transdermal drug delivery. While the
molecular size of potentially deliverable drugs is likely
to increase with further research, larger compounds,
such as those used in vaccines and gene therapy,
continue to be problematic when used in combination
with CPEs.

PHASE TRANSITIONS IN THE

STRATUM CORNEUM
Like the phospholipid bilayer surrounding a living
cell, the membranes found in the SC must possess dynamic properties, otherwise they would be
impenetrable barriers across which not even water
could pass. While phase changes in membrane bilayers composed of phospholipids have been extensively
studied, the phase behaviors of lipids found in the
SC have only been studied since the late 1970s.59 A
great deal of what we now know about the molecular organization and structure of lipid bilayers and
how they respond to the introduction of exogenous
chemicals comes from the development of synthetic
systems such as liposomal bilayers.13 The most common phases found in hydrated phospholipid and
ceramide artificial membrane systems include four
liquid-crystalline and crystalline lamellar phases and
several nonlamellar phases. The nonlamellar phases
include both hexagonal and cubic phases, where
hexagonal phases are formed by tubular aggregates
and are either normal or reverse hexagonal liquidcrystalline phases and cubic phases, including cubic,
rhombic, and tetragonal, that are formed by the interaction of curved bilayers. Recent studies have shown
that lipids extruded by granular cells during barrier
lipid formation in the epidermis may also exist in other
polymorphic states such as a cubic phase, which then
merge into the lamellar intercellular space.22,23 These
observations support the idea that a transition from

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lamellar to these nonlamellar phases could also occur

within the SC lipids themselves, producing aqueous
channels through which drug molecules could freely
diffuse.
We have recently described a novel formulation consisting of biphasic vesicles designed for the
delivery of IFN-, a 19-kDa protein for the topical
treatment of human papillomavirus infections.60 As
part of the mechanistic studies related to these systems, we have conducted SAXS and WAXS analysis
of the SC lipids structural arrangement after treatment with biphasic vesicles.17 These studies showed
a previously unobserved effect on the structural order
of the SC lipids, namely the induction of a polymorphic rearrangement of the lipids as a result of
biphasic vesicle interaction (Figure 1). SAXS and
WAXS studies showed that biphasic vesicles induced
the formation of a three-dimensional bicontinuous
Pn3m cubic phase in the SC lipids. Formation of
this cubic phase seems to be unique to biphasic
vesicles, as their subcomponents, liposomes and submicron emulsion, or commonly used CPEs do not
induce such a polymorphic phase change (Figure 1).
The cubic phase is a complex three-dimensional network of independent aqueous channels within a lipid
matrix on an infinite periodic minimal surface. The
interaction of biphasic vesicles also induces the conversion of the more commonly observed orthorhombic
lipid-packing configuration (which provides a strong
barrier to permeation of substances) to a more permeable hexagonal configuration or liquid state. The
combination of these two effects may be responsible for increasing the permeability of IFN- across
the SC.
Ultimately, the presence of a particular space
group in the supramolecular organization of the SC
depends on several factors: (1) the chemical structure
and amount of lipid present, (2) the water content
of the system, (3) the presence of other solutes and
(4) temperature. Because of the relationship between
lipid structure and aggregate form, it is not surprising that complex lipid structures behave in unusual
ways. The tendency to produce these larger transitions
also depends on the local curvature of the membrane, which ultimately depends on the local lipid
composition and fluidity of the membrane. Altering
individual physical parameters such as temperature,
ceramide composition, and cholesterol or fatty acid
concentrations results in the generation of many of
these complex phases in SC lipid lamellae.17,23,61
For instance, the addition of cholesterol to a fluidphase bilayer results in its intercalation between
lipid molecules, filling in free space and decreasing the flexibility of surrounding lipid chains.62
Vo lu me 3, September/Octo ber 2011

Unfortunately, owing to its relatively disordered

nature, a lack of available molecular detail related
to the physical properties of the SC makes developing models and understanding the diffusion process
difficult.

MOLECULAR MODELING
To advance our understanding of the physicochemical properties related to drug diffusion, including
the effect of both drug and vehicle on permeation
across the SC, it is essential to first determine the predominant mechanisms for drug penetration through
the SC. How this knowledge is obtained depends on
several factors. Technological advancements in molecular imaging will undoubtedly increase the overall
resolution at which researchers can observe lipids in
these systems. However, obtaining data related to the
atomic structures of these systems requires technology
not yet available. Because molecules are dynamic, it
is often difficult to extrapolate atomic motions from
experimentally derived structures. This applies to the
movement of membrane leaflets, including motions
between leaflets, the bulk motion of lipid molecules,
and motion of atoms within the lipid itself.63 One
possible way of observing these interactions is the
development of computational models where physical parameters available in the literature can provide
insight into the intrinsic molecular behavior of these
complex lipid systems.64 Motions of each atom within
the system affect the energy of the molecules, which
can be calculated at a given time, given the relative
atom positions using classical simulation techniques
such as molecular mechanics (MM). Molecular modeling allows for the observation of molecular conformations at timescales that are difficult to obtain
through experimental analysis alone. The principal
simulation technique used to examine realistic molecular systems is known as molecular dynamics (MD).
MD has its underpinnings in statistical mechanics,
where it is assumed that statistical ensemble averages are identical to time averages of the system. By
utilizing several, well-established, approximations, a
real-time depiction of atomic motion in these systems can be collected. Once a statistically adequate
sample has been obtained, detailed atomic interactions within the system can be evaluated and energies
calculated.
Modeling lipid interactions within SC membranes is the first step toward obtaining a clearer
understanding of the free-energy landscapes contributing to both the stabilization and destabilization
of these macromolecular complexes. The acquisition
of simulation data relating to phase transitions

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associated with various lipids and penetration

enhancers will provide data required for the development of structure activity relationships (SARs) relating
the structure of CPEs to their role in the formation of
complex phase transitions.

MODELING MEMBRANE SYSTEMS

To date, the majority of simulations examining
lipids are used to study the structure, dynamics, and
interactions of membrane proteins and peptides.65,66
A significant number of models have been proposed
that attempt to describe the behavior of hydrophobic drug molecules that enter the cell through passive
diffusion across the phospholipid membrane.6769 So,
what can researchers expect to obtain by performing MD simulations targeted toward these types of
membranes? In principle, through the use of statistical mechanics, such simulations can provide a
complete picture describing the motion of lipids and
other molecules in the system, as well as access to
thermodynamic properties.
It is not obvious how one relates bulk thermodynamic partitioning experiments to partitioning
into bilayers, but detailed computer simulations can
be used to interpret these experiments, elucidate the
relative importance of entropic and enthalpy contributions, and relate these to properties such as
chain ordering, free volume distribution, polarity, and
shape of the small molecules being investigated.70 It
is possible to obtain sufficiently accurate experimental information to validate simulations by comparing
average properties to experimentally measured values. Simulating the aggregation of randomly dissolved
phospholipids into bilayers takes on the order of tens
of nanoseconds, whereas the formation of small phospholipid vesicles has been observed in near-atomic
detail using simulations only 100 ns in length.71 Cubic
lipid phases have been studied by MD, and a phase
transition between a cubic and an inverted hexagonal
phase have been observed.72 Similar methods have
also been applied to understanding the detailed mechanisms of ion permeation through ion channels,7375
proton exclusion in aquaporin,76,77 and the mechanisms of bilayer fusion.78
There are technical limitations related to the size
and accuracy of the systems that can be simulated, and
difficulties with accurately incorporating important
variables such as pH, transmembrane potential differences, and low concentrations of ions. In addition,
because there is limited data related to the starting
configurations of many of these lipid simulations, the
choice of initial conditions may also bias results in
undesirable ways.
456

UMBRELLA AND REPLICA EXCHANGE

SAMPLING OF MEMBRANE SYSTEMS
A significant challenge when designing simulations
is the observation of conformational changes that
are too slow to detect on a timescale of only tens
to hundreds of nanoseconds. Fortunately, extensions
to the basic simulation methods make it possible
to calculate free-energy differences from these types
of simulations.79,80 Techniques including umbrella
sampling and replica exchange molecular dynamics
(REMD)81 are ideally suited for the study of
membrane systems and allow for the simulation
of molecular processes that normally occur on
long timescales. However, these techniques require
previous knowledge of a specific reaction coordinate
and the convergence of all motions that are not part
of the reaction coordinate.
In umbrella sampling, a biasing potential is used
to restrict a system to sample phase space within
a specified region. By placing windows along the
reaction coordinate, one can generate a free-energy
profile (also called potential of mean force), which
quantitatively describes why one region of space is
more favorable than another (Figure 4). By restraining
a molecule at different depths within the bilayer
(effectively forcing the drug molecule to spend time
in unfavorable regions of the system), an energy
distribution can be calculated that depends both on
the restraining potential and the actual distribution
without the restraining potential. By utilizing a set
of such simulations to reconstruct the distribution
throughout the entire bilayer, it is possible to correct
for the addition of restraining potentials. The result is
an accurate sampling of the available conformational
space, even for cases where there is a very large
difference in free energy between the least and the
most favorable positions.
REMD, also referred to as parallel tempering,
is a simulation method aimed at improving the
dynamic properties of Monte Carlo (MC) method
simulations of physical systems. Typically, an MC
simulation consists of a stochastic step that evaluates
the energy of the system and accepts/rejects updates
based on the temperature (T).82,83 If two identical
simulations are performed at temperatures separated
by T, one would observe that if T is small
enough, the energy histograms obtained by collecting
values of energies over a set of n MC steps will
create two distributions that overlap. We can interpret
this overlap by assuming that system configurations
sampled at temperature T1 are likely to appear during
a simulation at T2 . Because the Markov chain has no
memory of its past, we can create a new update for the
system composed of the two systems at T1 and T2 . At

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Modeling drug delivery through the skin

E
y position

2
1

4
4
5

FIGURE 4 | Schematic representation of the umbrella sampling technique. To perform umbrella sampling, one must first generate a series of
configurations along a predetermined reaction coordinate. In this example, the reaction coordinate is defined by applying a constant force (y ) to a
permeation enhancer and pulling it through the (x , z ) plane of a membrane composed of ceramides, cholesterol and free fatty acids (dashed arrow).
Configurations generated in this way serve as the starting points for the umbrella sampling windows, which are run in independent simulations.
Configurations of the system which are generated during the pulling step are extracted after the initial simulation is complete. The middle image
corresponds to the independent simulations conducted within each sampling window, with the center of mass of the permeation enhancer restrained
in that particular window by an umbrella biasing potential. The right panel illustrates an ideal histogram of configurations, with neighboring windows
overlapping such that a continuous energy function with respect to the passage of the permeation enhancer through the bilayer can later be derived
from these simulations.

any given MC step, we can update the global system

by swapping the configuration of the two systems or
alternatively trading the two temperatures. Through
the careful choice of temperatures and number of
replicas, we can achieve an improvement in the
sampling properties of an MC simulation that greatly
exceeds the additional computational cost of running
parallel simulations.

MULTISCALE AND CONTINUUM

SIMULATIONS
Through the use of a hierarchical approach, based on
parameters obtained from both experimental and MD
simulation, it is also possible to create multiscale simulations that can represent extremely large membrane
Vo lu me 3, September/Octo ber 2011

systems at timescales not amenable to the previously

mentioned atomistic simulations.84,85 Multiscale simulations are generally based on a three-tiered modeling
approach consisting of the generation of (1) atomistic
MD simulations, (2) coarse-grained mathematical
models, and (3) validation through correlation with
experimental data. The continuum approach represents biochemical systems as sets of differential equations where the concentrations of chemical species are assumed to vary continuously and
smoothly across the reaction space. This approach
draws on the rather extensive foundation of mathematical and engineering theory that exists to solve
such systems, but does not deal with molecular
species explicitly. For instance, the utilization of
MD simulations to obtain the microscopic diffusion

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Overview

coefficient within SC lipid bilayers has been coupled

with a course-grained approach to obtain effective
macroscopic diffusion coefficients that are in good
agreement with experimental data.86,87 Molecular
modeling procedures such as those described here
will allow (1) the elucidation of mechanisms of interaction between carrier molecules and drugs and
(2) extraction of molecular energetic and structural
parameters to be employed in the formulation of
mathematical models of diffusion through the SC.

Upper hydration
shell

Upper bilayer
composed of
ceramide and
cholesterol
Intermembrane
waters

MODELING THE STRATUM

CORNEUM
The systematic simulation of lipids observed in the SC
is limited, in part, because of the limited structural data
describing the primary lipid composition and their
respective role in the barrier properties of the skin. Preliminary MD simulations have been targeted at studying components of the SC, and generally consider free
fatty acids and cholesterol, but contained no ceramide
molecules due to a lack of adequate parameters.88
Because there is no universal atomic force field containing parameters for simulating all proteins, nucleic
acids, lipids, carbohydrates, and arbitrary small
molecules, selection of correct atomic parameters is
essential. The most widely used parameters for the
lipid component of typical membrane protein simulations in simulation packages such as GROMACS are
known as the Berger lipids.89 Because ceramides lack
the phosphate headgroup found in standard phospholipid parameters, new parameters need to be tested for
their ability to replicate experiment results adequately.
Initial parameters for ceramides were first introduced
in simulations of single homogeneous ceramide bilayers composed of C16:0CER[NS].90 Here, the partial
charges for atoms within the ceramide headgroup
were taken from the side chain atoms of serine.91
Atomistic simulations of SC bilayers are now progressing quickly, the first simulations involving CPEs
examined DMSO on a hydrated gel-phase bilayer
composed of C24:0CER[NS].92 In these simulations,
the authors showed that DMSO accumulated in the
ceramide headgroup region, weakening lateral forces
between them. At high concentrations of DMSO,
ceramide bilayers were observed going through a
phase transition from the gel phase to the liquidcrystalline phase. Because the liquid-crystalline phase
is thought to be markedly more permeable to solutes
than the gel phase, these simulations correlated well
with experimental results showing that high concentrations of DMSO fluidize the SC lipids, thereby
enhancing permeability. Simulations involving fully
hydrated bilayers composed of ceramide 24:0[NS],
458

Lower bilayer
composed of
ceramide and
cholesterol

Lower hydration
shell

FIGURE 5 | Construction of two ceramide bilayers separated by a

thin layer of water. To observe the ability of a permeation enhancer to
cross the stratum corneum and interactions at the intermembrane
interface, a bilayer assembly was created using the CHARMM c33b1
package. To aid computational efficiency, a segment having a small
cross-sectional area of 50 50 A was chosen. For the initial selection
of the parameters required for ceramide equilibration, a ratio of 2:1
ceramide C15:0CER[NS] to cholesterol was used. These initial
parameters produced an upper and lower bilayer containing 72
ceramide molecules (green sticks) and 36 cholesterol molecules (blue
sticks) each. The two bilayers are separated by a 5 A layer of water
and are surrounded by a 12 A layer of water on either side of the
leaflet.

lignoceric acid C24:0, and cholesterol (Figure 5) have

been performed to address the effect of these different SC components on its structural properties.93,94
Information collected from these simulations showed
that at physiological temperatures, the lipids were in
the gel phase with ordered lipid tails. Interestingly,
the large asymmetry in the tail lengths of ceramide
molecules resulted in a fluid-like environment at the
bilayer midplane, a detail proposed in earlier models
of SC permeability.95 These results have now laid the
groundwork for the simulation of multicomponent
SC bilayers in the presence of libraries of CPEs.
Techniques such as umbrella sampling and REMD
will undoubtedly provide an image of the structure and
functional relationships between all the components
of the SC lipid bilayers and their interplay during
phase changes, facilitating the rational design of new,
passive transdermal delivery modalities for a wider
range of macromolecules than is currently available
today (Figure 6).

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WIREs Nanomedicine and Nanobiotechnology

Modeling drug delivery through the skin

Computational

Obtain samples of stratum

corneum from skin or
create synthetic systems

Using data collected from

experiment, construct
model stratum corneum
bilayers at varying ratios

Assay samples and apply

penetration enhancer
or delivery system

Equilibrate model systems

and introduce penetration
enhancers to the models

Slow
process

Fast reversible
process

ck

ba

ed

Fe

Experimental

Molecular dynamics

nt

me

eri

xp

oe

Waxs

int

Saxs

Obtain physical parameters

for lipid phases (cubic,
lamellar and hexagonal)

Observe correlations
between empirial and
computational paramenters

Develop a penetration
enhancement structure
activity relationship library

Develop an atomic scale

model of lipid/ enhancer
interactions

FIGURE 6 | Synergistic feedback of computational simulations into

predictive enhancement of the experimental observation of permeation
enhancer effects. Using computational techniques it is possible to
simulate the effects that drugs and penetration enhancers may have on
components of the stratum corneum. Several parameters can be
introduced and a predictive library can be constructed. This library can
then be used to guide subsequent experimental design in an effort to
eliminate unproductive results and speed combinatorial screening of
large chemical libraries.

CONCLUSIONS
Research into the life sciences cannot achieve its
full social, economic, and scientific potential until it

is able to explore the underlying structural design

principles of life and how they impact an entire
organism. Predicting cellular behavior by computational means must become an overarching and
unifying concept that integrates the recent advances
in technology that include proteomics, genomics,
bioinformatics, and nanotechnology as necessary
components. Today, researchers have the advantage
that the chemical/physical properties of biomolecules
can be rigorously described, quantified, and simulated computationally. This advantage also extends
to the simulation of structures such as membranes
and lipid mixtures, for which atomic resolution data
are not yet available. Simulations can bridge the gap
that exists between available structures and atomic
details relating to functional relationships in lipid
systems.
An understanding of the mechanisms associated with phase transitions in membranes of the
SC will undoubtedly aid in the design of new delivery systems. The creation of large databases of lipid
interactions with penetration enhancers and their nary mixtures will support both computational and
experimental model development by limiting the
overall search space required. There is a unique
opportunity to work with existing developmental
stage drugs that had previously been shelved due
to excessive difficulties when administered orally.
We believe that new noninvasive transdermal technologies suitable for difficult-to-deliver molecules
can be designed and that we are approaching a
new frontier, where the next generation of delivery systems will provide enhancement to a broad
range of compounds through a structured reordering
of the SC.

ACKNOWLEDGMENTS
This article was supported by grants from the Canadian Institutes of Health Research and the Natural Sciences
and Engineering Research Council of Canada (NSERC). Research involving SAXS and WAXS was conducted
at the National Synchrotron Light Source, Brookhaven National Laboratory, Upton, NY, which is supported
by the U.S. Department of Energy, Division of Materials Sciences and Division of Chemical Sciences, under
Contract No. DE-AC02-98CH10886. The generous support of the Canada Research Chairs Program, the
Canada Foundation for Innovation, and the Ontario Research Fund for M. Foldvari is gratefully acknowledged.

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