European Journal of Pharmaceutical Sciences 68 (2015) 27–35

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European Journal of Pharmaceutical Sciences

journal homepage: www.elsevier.com/locate/ejps

Topical delivery of acetyl hexapeptide-8 from different

emulsions: Influence of emulsion composition and internal structure
Magdalena Hoppel a, Gottfried Reznicek b, Hanspeter Kählig a,c, Harald Kotisch d, Günter P. Resch d,e,
Claudia Valenta a,f,⇑
a
Research Platform ‘Characterisation of Drug Delivery Systems on Skin and Investigations of Involved Mechanisms’, University of Vienna, 1090 Vienna, Austria
b
Department of Pharmacognosy, University of Vienna, 1090 Vienna, Austria
c
Institute of Organic Chemistry, University of Vienna, 1090 Vienna, Austria
d
Campus Science Support Facilities GmbH, Electron Microscopy Facility, 1030 Vienna, Austria
e
Nexperion – Solutions for Electron Microscopy, 1040 Vienna, Austria
f
Department of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, 1090 Vienna, Austria

a r t i c l e i n f o a b s t r a c t

Article history: Acetyl hexapeptide-8 (AH-8) is a well-known component of anti-aging products and was recently
Received 4 August 2014 explored as a promising topical treatment of blepharospasm. Although AH-8 appears in a variety of cos-
Received in revised form 19 November 2014 metic products, its skin penetration is sparsely studied and controversially discussed. Therefore, the aim
Accepted 2 December 2014
of the present study was to investigate the influence of the vehicle type on the AH-8 delivery to the skin.
Available online 9 December 2014
Besides skin permeation experiments with Franz type diffusion cells, the spatial distribution of AH-8 in
the stratum corneum after a real in-use application was investigated by in vitro tape stripping on porcine
Keywords:
ear skin. By applying LC–MS/MS for quantification of AH-8, we demonstrated that a multiple water-
Acetyl hexapeptide-8
ATR-FTIR
in-oil-in-water (W/O/W) emulsion can significantly increase penetration of AH-8 into porcine skin
NMR compared to simple O/W and W/O emulsions. The internal structure of the developed multiple emulsion
Freeze–fracture was confirmed by electron microscopic investigations and NMR self diffusion studies. In general, a clear
LC–MS/MS superiority of water-rich W/O/W and O/W emulsions over an oil-rich W/O emulsion in terms of dermal
Multiple emulsions delivery of AH-8 was found. This enhanced delivery of AH-8 could be explained by an increased absorp-
tion of the water-rich emulsions into the skin, confirmed by combined ATR-FTIR and tape stripping
experiments.
Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction
Acetyl hexapeptide-8 (AH-8) is a topically applied hexapeptide
that has a mechanism of action similar to botulinum neurotoxin
(BoNT). It has been used effectively in anti-aging cosmetic applica-
tions and was recently explored as a promising treatment of bleph-
arospasm (Lungu et al., 2013; Gorouhi and Maibach, 2009). Up to
date, the only reliable effective treatment is a BoNT injection ther-
apy with the disadvantages of significant costs, risks of side effects
and discomfort (Lungu et al., 2013; Simpson et al., 2008).
However, topical delivery of hydrophilic macromolecules with a
molecular mass >500 Da is challenging, due to the lipophilic nature
of the skin’s uppermost layer, the stratum corneum (Gorouhi and
⇑ Corresponding author at: Department of Pharmaceutical Technology and
Biopharmaceutics, University of Vienna, Althanstraße 14, 1090 Vienna, Austria.
Tel.: +43 1 4277 55410; fax: +43 1 4277 9554.
E-mail address: claudia.valenta@univie.ac.at (C. Valenta).
Maibach, 2009; Hadgraft and Lane, 2011; Choi et al., 2012). Up to
date, the dermal delivery of large hydrophilic molecules such as
peptides is sparsely studied. The diffusion through intercellular
lipids as the classical pathway of transdermal permeation is mainly
restricted to small lipophilic drugs. Therefore, delivery of hydro-
philic molecules is attributed to two other pathways, namely to
the transport through pores in the skin as well as through hair fol-
licles and sweat ducts (Mitragotri, 2003; Hsu and Mitragotri,
2011). Many approaches for delivery of macromolecules through
the skin, including chemical enhancers and physical delivery tech-
niques, are restricted, due to skin toxicity, inconvenience or high
production costs of sophisticated drug delivery systems (Choi
et al., 2012).
Therefore, emulsions are still widely used dermal delivery
systems (Otto et al., 2009). Water-rich oil-in-water (O/W) emul-
sions are most frequently used, due to their pleasant skin feel. Con-
versely, oil-rich water-in-oil (W/O) emulsions have lower cosmetic
acceptance, caused by their greasy skin feel (Baran, 1998). More

http://dx.doi.org/10.1016/j.ejps.2014.12.006
0928-0987/Ó 2014 Elsevier B.V. All rights reserved.
28 M. Hoppel et al. / European Journal of Pharmaceutical Sciences 68 (2015) 27–35
complicated multiple water-in-oil-in-water (W/O/W) emulsions
are potential controlled release systems for dermal delivery of sen-
sitive biologicals, such as peptides. The major benefit of multiple
emulsions is a protection of the active, which can be encapsulated
in the inner water droplets. However, their use is limited by lack of
stability, caused by coalescence of the outer and inner droplets
(Hoppel et al., 2014; Olivieri et al., 2003; Patravale and
Mandawgade, 2008).
To this end, the applicability of different emulsions as dermal
carrier systems for AH-8 was investigated in this study. Due to
the skin irritation potential of conventional, synthetic surfactants
and a recently increasing attention to the environment, all emul-
sions were based on a low concentration of natural origin and bio-
degradable emulsifiers (Savić et al., 2007; Mao et al., 2012).
The skin permeation of AH-8 from a multiple W/O/W emulsion
was compared to simple O/W and W/O emulsions by Franz-type
diffusion cell experiments. Furthermore, the developed multiple
W/O/W emulsion was characterized in terms of stability, droplet
size and rheological properties. The presence of the inner water
phase in the multiple droplets was determined by electron micros-
copy and NMR self diffusion studies.
Interestingly, although AH-8 is widely used in anti-aging cos-
metic formulations, detailed investigations regarding its skin pene-
tration are still missing. In this study, the spatial distribution of AH-8
in the stratum corneum under real in-use conditions, namely after a
short finite dose application of a practically relevant AH-8 concen-
tration, was analyzed in detail. Furthermore, the stratum corneum
penetration of the emulsions themselves and their influences on
skin hydration were analyzed by combined ATR-FTIR and tape strip-
ping experiments (Hathout et al., 2010; Hoppel et al., 2014).
2. Materials and methods
2.1. Materials
Acetyl hexapeptide-8 (ArgirelineÒ powder) was kindly provided
by Lipotec GmbH (Hofheim-Wallau, Germany). Octyldodecanol &
Octyldodecyl Xyloside & PEG-30 Dipolyhydroxystearate (Easy-
novÒ) was a gift from Seppic (Cologne, Germany). Sucrose stearates
(Ryoto Sugar EsterÒ S-970 and S-1670) were kindly donated by
Mitsubishi-Kagaku Foods Corporation (Tokyo, Japan). Isopropyl
myristate (IPM) was procured from Herba Chemosan (Vienna,
Austria) and neutral oil (MiglyolÒ 812) from Dr. Temt Laboratories
(Vienna, Austria). Formic acid was obtained from Sigma Aldrich
(Vienna, Austria).
2.2. Methods
2.2.1. Formulation preparation
The composition of the different formulations is given in Table 1.
The multiple W/O/W emulsion was prepared by a one-step emul-
sification method (Hoppel et al., 2014). Briefly, both surfactants,
Table 1
Composition of the multiple W/O/W, O/W and W/O emulsions.
Excipients
Neutral oil
Isopropyl myristate
Distilled water
Octyldodecanol & Octyldodecyl Xyloside & PEG-30 Dipolyhydroxystearate (EasynovÒ)
S-1670
S-970
Acetyl hexapeptide-8 (AH-8)
EasynovÒ and sucrose stearate S-1670, were dissolved in the oil
phase under stirring and heating. AH-8 was dissolved in distilled
water at room temperature. Subsequently, the water phase was
added to the oil phase under moderate stirring (750 rpm). After-
wards, the double emulsion was stirred for 20 min at 750 rpm.
The O/W emulsion was prepared by a method previously
reported by our working group (Klang et al., 2011a). In short,
sucrose stearate S-970 was dissolved in the oil phase under stirring
at 50 °C. AH-8 was dissolved in distilled water at 40 °C. The aque-
ous phase was slowly added to the oil phase and after further stir-
ring for 10 min, the formulation was treated with an ultraturrax
(Omni 5000, Omni International, Kennesaw, USA) for 4 min at
2500 rpm.
The W/O emulsion was prepared by stirring the oil phase, con-
sisting of neutral oil and EasynovÒ, and the water phase, consisting
of AH-8 and distilled water, separately at 40 °C. Subsequently, the
water phase was added to the oil phase and the formed W/O emul-
sion was further stirred for 10 min at 750 rpm.
2.2.2. Characterization of the developed multiple W/O/W emulsion
The multiple emulsion was observed immediately after prepa-
ration and monitored over a period of 14 weeks by macroscopic
examination, in order to detect any instabilities such as creaming
or phase separation.
The droplet size of the multiple emulsion was determined using a
laser diffraction particle size analyzer (Mastersizer 3000, Malvern,
Worcestershire, UK). The instrument was operated with the Hydro
MV sample dispersion unit (Malvern, Worcestershire, UK) and soft-
ware version 2.01. Particle size distribution was calculated accord-
ing to the Mie theory. The samples were diluted with distilled
water. Droplet sizes are presented as the D(v, 0.5). This parameter
stands for the volume median diameter and marks the size where
half of the particle size of the sample is above and half of the particle
size of the sample is below this value. In addition, the D(v, 0.9) and
D(v, 0.1) were analyzed. The span was automatically calculated. This
value represents the width of the distribution based on the 10%, 50%
and 90% quantile. The droplet size measurement was carried out
over an observation period of 14 weeks. The developed formulation
was prepared and analyzed in triplicate (n = 3). The formulations
were stored at room temperature (25 ± 5 °C).
2.2.3. Skin permeation studies using Franz-type diffusion cells
In vitro skin permeation studies were performed using Franz-
type diffusion cells (Permegear, Hellertown, USA). Porcine skin
was cut with a dermatome (GB 228R, Aesculap, Center Valley,
USA) set at 700 lm. The skin samples were stored at 24 °C for
not longer than 6 months. Appropriately cut skin pieces were
clamped between the donor and the receptor chamber of the diffu-
sion cells having a permeation area of 0.95 cm2. The diffusion cells
were thermostatted at 32 °C to maintain skin surface temperature
and were continuously stirred with magnetic bars. The acceptor
compartment was filled with 2 ml of 0.1% aqueous formic acid. An
Formulation code
W/O/W O/W W/O
Formulation composition (%, w/w)
– 20 76
20 – –
75.99 75.99 19.99
1.5 – 4
2.5 – –
– 4 –
0.01 0.01 0.01
 M. Hoppel et al. / European Journal of Pharmaceutical Sciences 68 (2015) 27–35
infinite dose of 250 mg cm 2 of formulation was applied onto the
skin in the donor chamber. Samples for the analysis of permeated
AH-8 by LC–MS/MS were taken after 2, 4, 6 and 8 h by replacing
the whole acceptor medium (2 ml) with fresh 0.1% aqueous formic
acid. Five parallel experiments were performed for each formulation
(n = 5).
2.2.4. In vitro tape stripping
Full-thickness porcine ear skin was used for the tape stripping
experiments. Pig ears were purchased from a local abattoir (Hol-
labrunn, Austria) and stored at 24 °C up to a maximum of
6 months. Four experiments for each formulation were performed
(n P 4). At the beginning of the tape stripping procedure, the pig
ears were defrosted, cleaned with cold water and freed from hair
with scissors. The transepidermal water loss (TEWL) of the skin
was measured (AquaFluxÒ, Biox Ltd., London, UK), in order to con-
firm an intact skin barrier function and to monitor the defrosting
process. An amount of 5 mg cm 2 of the respective formulation
was applied and distributed with a saturated glove finger. After
an exposure time of 1 h, the tape stripping procedure was started.
The skin surface was cleaned with a dry tissue, before application
of the first tape strip (CorneofixÒ, Courage + Khazaka electronic
GmbH, Cologne, Germany). The outlines of the tape strip were
marked with a permanent marker to ensure that the following
strips were placed on the exact same location. Twenty sequential
strips were removed and the corneocytes on the tape strips were
quantified by NIR densitometry (SquameScanÒ 850A, Heiland elec-
tronic GmbH, Wetzlar, Germany) (Klang et al., 2011b; Franzen
et al., 2012). A mean stratum corneum thickness of 16.24 lm of
the used porcine ears was calculated by removing corneocytes
with tape strips until the detection limit of the NIR densitometer
was reached (n = 7). After each removed tape strip, the drug con-
tent of each strip was analyzed by LC–MS/MS after extracting
AH-8 from the strip with 0.1% aqueous formic acid.
2.2.5. Quantification of AH-8
The samples (5 ll) were analyzed using liquid chromatography/
mass spectrometry (LC–MS/MS) on an Ultimate 3000 RSLC-series
system (Dionex, Germering, Germany) coupled to a triple quadru-
pole mass spectrometer (AB Sciex Instruments API 4000) equipped
with an orthogonal ESI source operated in positive mode.
LC separation was performed on an Acclaim RSLC 120 C18 col-
umn (3 lm, 100  2.1 mm I.D., Thermo Fisher Scientific, Waltham,
USA), preceded by an Acclaim 120 C18 guard cartridge (5 lm,
10  2 mm I.D., Thermo Fisher Scientific, Waltham, USA), at a flow
rate of 0.4 ml min 1 and a column temperature of 30 °C. The
mobile phase consisted of a linear gradient mixed from 0.1% aque-
ous formic acid (mobile phase A) and acetonitrile (mobile phase B).
The gradient ranged from 5% B at 0 min to 17% B in 8 min, purging
with 95% B for 2 min, then again 5% B to equilibrate the column for
5 min before application of the next sample (total analysis time
15 min). AH-8 eluted at 3.95 min. Selective and sensitive detection
and quantification was carried out using MS/MS fragmentation of
AH-8 giving a quasimolecular ion at m/z 445 [M+2]2+. MRM m/z
445/144 was used for calibration curves with external standard
AH-8 (injection volume 5 ll) to give a linear concentration range
from 0.1 ng ml 1 to 5000 ng ml 1 (correlation coefficient 0.9991).
The triple quadrupole mass spectrometer operated with the fol-
lowing parameters: ESI pos., IS 5500, EP 10, CUR 10, GS1 40, GS2
40, TEM 500 °C, CAD 4, CEM 2100, DF -25, DP 76, CE 39, CXP 12,
dwell 400 ms.
2.2.6. Combined ATR-FTIR and tape stripping experiments
A recently validated combination of ATR-FTIR and tape stripping
experiments was used to investigate the stratum corneum penetra-
tion of the emulsions (Hoppel et al., 2014). In short, 80 ll of the for-
29
mulations were applied on full-thickness porcine ear skin samples
(2  7.5 cm) and incubated for 2 h at the skin surface temperature
of 32 °C. After removal of the formulation from the skin surface
with a soft tissue, a strip of adhesive film was placed on the skin
sample and removed in one continuous movement. Ten sequential
tape strips were removed in this manner. ATR-FTIR spectra were
recorded from the skin surface prior to the tape stripping procedure
as well as after each removed tape strip. Experiments were per-
formed in triplicate (n = 3). The amount of removed corneocytes
on each tape strip was analyzed by NIR densitometry.
ATR-FTIR spectra of incubated porcine ear skin samples were
obtained by placing the skin samples on the ZnSe ATR crystal of
an ATR-FTIR spectrometer (Tensor 27, Bio-ATR I tool, Bruker Optics,
Ettlingen, Germany) equipped with a liquid nitrogen cooled mer-
cury cadmium telluride (MCT) detector. Spectra were recorded in
double sided mode at the skin surface temperature of 32 °C as
the average of 60 scans in the frequency range 4000–870 cm 1 at
a resolution of 4 cm 1 using Blackman-Harris 3-term apodization
and a zero filling factor of 8.
ATR-FTIR spectra of the formulations were recorded under the
same conditions as the incubated skin samples by direct applica-
tion of formulation on the ZnSe crystal of the same tool.
Data analyses were performed using OPUS 5.5 software (Bruker
Optics, Germany). Peak areas were calculated by OPUS integration
method B where a straight line is drawn between two defined fre-
quency limits of the measured absorbance and the area above this
line is then integrated. To account for variability in degree of con-
tact between the crystal and the sample, the amide II absorbance
was used as an internal standard (Hoppel et al., 2014; Saad et al.,
2012). The frequency limits for integration were (1) 1669–
1600 cm 1 for the amide I, (2) 1568–1508 cm 1 for the amide II
and (3) 1125–1089 cm 1 for the C–O stretching absorbance.
The following features were analyzed to investigate the effects
of the formulations on the stratum corneum: (1) the ratio of the
amide I/amide II absorbance, which reflects stratum corneum
hydration and (2) the intensities of the C–O stretching vibration
1100 cm 1, which report of the presence of vehicle components
in the stratum corneum (Hathout et al., 2010).
Most characteristic bands of the formulation components were
overlapping with typical bands origin from the skin. Therefore,
spectra of control samples incubated with water and tape stripped
in the same manner were subtracted from the treated samples at
the respective stratum corneum depth. As a control, spectra of
untreated samples were subtracted from other untreated skin sam-
ples to ensure that no remaining absorbances in the observed
region falsified the results (n = 10) Hoppel et al., 2014.
In order to relate the penetrated amount of vehicle components
to the respective applied formulation, a further normalization
procedure was applied. Due to differing absorption coefficients of
the C–O stretching absorbances, caused by different oil types and
amounts, the measured C–O stretching absorbances were addition-
ally related to the corresponding C–O stretching absorbances of the
respective formulation.
2.2.7. Rheological characterization
Rheological properties of all emulsions were determined by
measuring the dynamic viscosity. Flow curves were established
with increasing shear rates from 1 to 100 s 1. Experiments were
carried out on a MCR Modular Compact Rheometer (Anton Paar,
Graz, Austria) with a cone and plate device (diameter 50 mm, cone
angle 1°). The temperature was maintained at 23 ± 0.2 °C. All
measurements were performed in triplicate (n = 3).
2.2.8. Electron microscopy
Freeze–fracture experiments were performed according to
Severs (2007) on a BAF-400 freeze–fracture machine (Balzers,
30 M. Hoppel et al. / European Journal of Pharmaceutical Sciences 68 (2015) 27–35
Liechtenstein): A small amount of the suspension was pipetted
onto a freeze-fracture carrier and plunged rapidly into supercooled
liquid nitrogen slush. The frozen samples were transferred to the
pre-cooled freeze–fracture device.
After reaching a vacuum of 2  10 7 mbar, a series of tungsten
knife microtome sweeps was taken until the desired fracture result
had been achieved. The sample was freeze etched at 100 °C for
2 min. To protect the specimen from contamination by condensa-
tion, the cold microtome assembly was left stationary directly
above the specimen.
For platinum coating of the frozen, rapidly rotating sample at an
angle of 45°, the gun was switched to 80–100 mA, 1.6 kV. Once
coating commenced, the knife was quickly retracted to the rear
position. Coating was terminated at a thickness of 7 nm. Subse-
quently, the rotating sample was coated with carbon at 100–
120 mA, 2.0 kV at an angle of 90° to a thickness of 20 nm.
The finished replicas were removed from the machine and
floated off in a dish on 70% ethanol. They were picked up with
400 mesh grids, air dried and examined with an FEI Morgagni
268D TEM operated at 80 kV. Digital images were acquired using
an 11 megapixel Morada CCD camera.
2.2.9. NMR experiments
The NMR experiments were performed on an Avance III
600 MHz spectrometer (Bruker BioSpin, Rheinstetten, Germany)
using a 5 mm broadband observe probe (BBFO SMART) at 25 °C.
The diffusion data were derived from the signal attenuation in a
series of 32 stimulated echo spectra with increasing gradient
amplitudes g from 2% to 95% of maximum power (0.5 T m 1) using
the pulse sequence from Wu et al. (1995). The gradient amplifier
was calibrated using a doped 1% water in D2O sample (Bruker Bio-
Spin, Rheinstetten, Germany) to give a self diffusion coefficient for
the water of 1.91  10 9 m2 s 1. The duration of the gradient
pulses d was set to 2 ms, the diffusion delay D was varied between
50 and 250 ms.
2.2.10. Statistical data analysis
Results are expressed as mean values ± standard deviation
(S.D.) of at least three experiments. Statistical data analyses were
performed using the GraphPadPrism3 program (GraphPad Soft-
ware, San Diego, USA). Student’s t-test or ANOVA with p < 0.05 as
level of significance were employed for the analysis of parametric
data. Non-parametric were analyzed using the Mann–Whitney or
Kruskal–Wallis test with p < 0.05 as level of significance.
3. Results
3.1. Characterization of the multiple W/O/W emulsion
It was possible to prepare a stable natural surfactant based mul-
tiple W/O/W emulsion by a recently reported one-step emulsifica-
tion method (Hoppel et al., 2014; Morais et al., 2008). The
alkylpolyxyloside containing emulsifier EasynovÒ was used as
hydrophobic W/O and sucrose stearate S-1670 as the hydrophilic
O/W emulsifier. The overall surfactant content was kept low at
4%. After preliminary studies, 1.5% of EasynovÒ and 2.5% of sucrose
stearate S-1670 was found to be the most suitable blend to stabi-
lize the multiple emulsion.
The developed multiple emulsion was characterized in terms of
droplet size, structure, stability and rheological properties. Over
the whole observation period of 14 weeks, the multiple emulsion
remained macroscopically stable with no observable creaming or
phase separation.
Laser diffraction measurements revealed a volume median
mean of 570 ± 50 nm after preparation and 710 ± 100 nm after
14 weeks of storage. The calculated span value which describes
the width of the particle size distribution varied between
1.10 ± 0.05 after preparation and 1.55 ± 0.49 at the end of the
observation period.
Due to the small droplet size of the outer oil droplets, conven-
tional methods such as light microscopy were inapplicable to dis-
tinguish between a multiple and simple emulsion. Therefore, the
presence of the inner water phase was initially confirmed by elec-
tron microscopic investigations. For this purpose, freeze–fracture
sample preparation was used to visualize the inner water droplets.
Fig. 1 shows the water containing oil droplets of the multiple emul-
sion. The frequency of multiple droplet appearance was deter-
mined by visual estimation. From the electron microscopic
pictures, it could be assessed that approximately 90% of the oil
droplets contained one single large water droplet.
The presence of the inner water phase and the incorporation of
AH-8 was further investigated by non-destructive NMR self diffu-
sion studies. In Fig. 2 the echo attenuation of the residual water
signal (HDO) by increasing the gradient amplitude g is shown as
a function of different diffusion delays D. The chosen representa-
tion of the signal intensities as ln(I/I0) against the square of the gra-
dient amplitude g2 gave an almost linear decay at a short diffusion
delay D = 50 ms resulting in a diffusion coefficient of 1.6  10 9 -
m2 s 1. This corresponds to the free unrestricted diffusion of water
in the outer compartments. By increasing the diffusion delay up to
250 ms, more and more deviations of the mono exponential echo
decay gave a non linear curve. Compared to the free displacement
in a bulk fluid a second diffusion behavior up to 3 orders of magni-
tude reduced could be estimated. This slow diffusion could be
assigned to the water in the inner compartment where the move-
ment of the molecules is restricted by the volume of the droplets.
Within the W/O/W emulsion, it is also possible to find 1H NMR
signals for the peptide, not covered by the other components of the
formulation and therefore suitable for self diffusion analysis. The
free self diffusion of AH-8, solely in D2O solution, was measured
as 2.57  10 10 m2 s 1. The observed signal decay in the self diffu-
sion experiments for AH-8 in the formulation was found to be at
least 2 orders of magnitude lower.
In order to avoid effects of different emulsifiers on the dermal
delivery of AH-8, sugar based surfactants at the same concentra-
tion were used to stabilize the W/O and O/W emulsions (Table 1).
The viscosity of the multiple W/O/W emulsion was compared to
the prepared simple O/W and W/O emulsions at a shear rate of
10 s 1 and 23 °C. While the multiple W/O/W and the W/O emul-
sion with a measured viscosity of 5.89 ± 1.76 mPa s and
48.0 ± 2.4 mPa s, respectively, represented fluid formulations, the
preparation of the O/W emulsion led to a semi-solid formulation
with a viscosity of 1630 ± 14 mPa s (Klang et al., 2011a).
3.2. In vitro skin permeation: Franz-type diffusion cells
3.2.1. Validation of LC–MS/MS for quantification of AH-8
The method of LC–MS/MS was validated regarding specificity of
AH-8 quantification from the acceptor phase in Franz-type diffu-
sion cell experiments with porcine skin. For this purpose, a blank
multiple emulsion was applied on the porcine ear skin in the
Franz-type diffusion cells and the acceptor chamber was filled with
2 ml of 0.1% aqueous formic acid. Due to the most reliable quanti-
fication results, 0.1% formic acid was used as acceptor medium.
Samples of the acceptor medium were taken after 6 h. The drawn
samples were analyzed in order to confirm that neither skin nor
formulation compounds in the acceptor medium interfere with
the AH-8 quantification. Moreover, after addition of a known
amount of AH-8 to these drawn samples, a recovery of
99.4 ± 2.1% of AH-8 was obtained.
 M. Hoppel et al. / European Journal of Pharmaceutical Sciences 68 (2015) 27–35 31

Fig. 1. Electron microscopic images of water containing oil droplets of the W/O/W emulsion after freeze–fracture sample preparation. The magnification is illustrated by the
black scale bar.

1000
Cumulative amount of AH-8 permeated

0.0 W/O/W
50 ms
-0.5 800 O/W
100 ms
-1.0 150 ms W/O
-1.5 Increasing 600
200 ms
[ng cm-2]
ln(I/I0)

-2.0 250 ms
-2.5 400
-3.0
-3.5 200

-4.0
-4.5 0
0 0.05 0.1 0.15 0.2 0.25 0 2 4 6 8
g 2 [T 2 m-2 ] Time [hours]

Fig. 2. 1H NMR echo attenuation at increasing diffusion delay D. The echo Fig. 3. Effect of the emulsion type on the in vitro skin permeation of AH-8 across
attenuation of the HDO signal by increasing the gradient amplitude g is shown as porcine skin at several time points up to 8 h. Values are presented as the cumulative
a function of different diffusion delays D. The signal intensities as ln(I/I0) are plotted drug amounts (ng cm 2). Five experiments were performed for each formulation
against the square of the gradient amplitude g2. (n = 5) using Franz-type diffusion cells. Indicated values are means ± SD.

3.2.2. Effect of the formulation on the in vitro skin permeation of AH-8
The in vitro skin permeation of AH-8 from the developed multi-
ple W/O/W emulsion was compared to a simple O/W and W/O
emulsion. The results of these experiments are given in Fig. 3. A
comparison of the cumulative drug amounts clearly showed that
AH-8 permeated more rapidly and to a significantly higher extent
from the multiple W/O/W and the O/W emulsion. In contrast, skin
permeation of AH-8 from the W/O emulsion was not detectable.
After 8 h the skin permeation could be ranked in the order
W/O/W > O/W > W/O. A significant difference between the cumu-
lative permeated amount of AH-8 after 8 h from the multiple
W/O/W emulsion (755 ± 149 ng cm 2) and the O/W emulsion
(456 ± 120 ng cm 2) was found (two tailed and unpaired Student’s
t-test; p < 0.01).
3.3. In vitro skin penetration: tape stripping
The skin penetration of AH-8 from the different emulsions was
determined by in vitro tape stripping on porcine ear skin. Despite
interindividual differences in stratum corneum thickness, a larger
number of tape stripping experiments performed by our working
group revealed that a large part of the barrier of the porcine ear
stratum corneum is removed after 20 tape strips (Klang et al.,
2011b). The detected amounts of AH-8 in different layers of the
stratum corneum are presented in Fig. 4. According to these
results, the penetration of AH-8 from the different formulations
was in the following order: W/O/W > O/W > W/O.
This rank order was confirmed even more clearly after calcula-
tion of the total amount of AH-8 that entered the stratum corneum.
32
50
Amount of AH-8 [ng cm -2 ]
40
30
20
10
0 ties of the C–O stretching vibration 1100 cm 1 mainly originate
Strips 1-5 Strips 6-10
Fig. 4. Concentration of AH-8 extracted from tape strips 1–5, 6–10, 11–15 and 16–
20. The presented values are means ± SD of four individual experiments for each
formulation (n = 4).
Amount of AH-8 that entered the stratum
60
50
corneum [ng cm-2 ]
40
30
20
10
0 the formulation treated skin samples were additionally calculated
W/O/W
Fig. 5. Penetration of AH-8 from different emulsions into the stratum corneum of
porcine ear skin. Values are presented as the total amount of AH-8 detected on 20
removed tape strips. Four individual experiments were performed for each
formulation (n = 4).
The results showed that the multiple emulsion significantly
enhanced the skin penetration of AH-8 (Fig. 5). After the residence
time of 1 h, 46.7 ± 6.2 ng cm 2 of applied AH-8 penetrated into the
stratum corneum from the multiple emulsion. In comparison,
24.7 ± 4.9 ng cm 2 and 9.5 ± 2.1 ng cm 2 entered the stratum cor-
neum from the O/W and the W/O emulsion, respectively. Thus,
the multiple W/O/W emulsion led to 4.91 ± 0.66-fold and
1.89 ± 0.25-fold higher skin deposition of AH-8 than the W/O and
O/W emulsion, respectively. The O/W emulsion showed
2.61 ± 0.52-fold increased skin penetration of AH-8 in comparison
to the W/O emulsion. Statistical analysis revealed significant differ-
ences in the penetrated AH-8 amounts between all formulations
(One-way ANOVA with post hoc Tukey test; p < 0.01).
3.4. Combined ATR-FTIR and tape stripping experiments
Combined with a tape stripping procedure, the ATR-FTIR tech-
nique can be employed to detect exogenous substances in different
layers of the stratum corneum. The absorbance of the C–O stretch-
ing vibration 1100 cm 1 which report of the presence of surfac-
tant and oily component of the applied formulation was used to
investigate the skin penetration of the different formulations
(Hathout et al., 2010).
A method for tracking exogenous substances semi-quantita-
tively, even if bands are overlapping with features arising from
M. Hoppel et al. / European Journal of Pharmaceutical Sciences 68 (2015) 27–35
the skin itself, was recently validated by our working group
W/O/W (Hoppel et al., 2014). Spectral subtraction of untreated from trea-
ted skin samples was applied to overcome the problem of overlap-
O/W
ping absorbances and to ease the analysis of the observed
W/O absorption band in the finger print region of the spectra. After
spectral subtraction, the obtained spectra were equal to the spectra
of the corresponding formulations themselves, thus allowing a
comparison of the different formulations (Fig. 6). As seen in
Fig. 7, the W/O emulsion led to the highest incorporation of vehicle
components into the initial layers of the stratum corneum,
whereas smaller amounts of vehicle components were detected
after incubation with the W/O/W and the O/W emulsion.
These findings are not unexpected, since the observed intensi-
Strips 11-15 Strips 16-20
from the incorporation of the oily component into the stratum cor-
neum. However, although the content of the oily component was
significantly higher in the W/O emulsion (76% vs. 24%), all formu-
lations penetrated into a similar stratum corneum depth and were
only detectable in the initial layers of the stratum corneum.
Due to the fact that these findings only provided information
about the absolute amount of vehicle components incorporated
into the stratum corneum, the relative incorporated amount of
applied formulation was calculated in a further step. To account
for variations of type and content of the oily component, the mea-
sured absorbances of the C–O stretching vibration were correlated
to the corresponding absorbances of the applied formulation
(Fig. 8).
Taking the higher oil content of the W/O emulsion in account, a
significantly lower amount of the applied W/O emulsion was
absorbed into the initial layers of the stratum corneum compared
to the multiple W/O/W and the O/W emulsion.
Since skin hydration also plays an important role in terms of
skin penetration enhancement, the amide I to amide II ratios of
O/W W/O and compared. Interestingly, no significant difference in skin
hydration level after incubation with the different formulation
was found.
4. Discussion
To create stable multiple W/O/W emulsions, two emulsifiers
with different HLB values are necessary to stabilize the two present
interfaces. Improvement can be achieved by a sophisticated combi-
nation of the hydrophilic and lipophilic surfactant (Morais et al.,
2008; Frenkel et al., 1983; Garti and Bisperink, 1998). Moreover,
care has to be taken to avoid extreme temperatures during the pro-
duction process, in order to increase the stability of the thermally
sensitive peptide (Ruiz et al., 2006).
The results of the present study showed that the use of two nat-
ural surfactants in a low concentration led to the formation of a
multiple W/O/W emulsion with good long-term stability. An
increased median mean of the oil droplets and an increased span
value indicated increasing polydispersity of the formulations at
the end of the observation period. However, no macroscopic insta-
bilities, such as creaming, were found over the whole observation
period. Especially for W/O/W multiple emulsions, creaming is a
valuable indicator for potential steps toward rupture of the inter-
nal water droplets entrapped in the outer oil droplets (Jiao and
Burgess, 2003).
Due to the fact that laser diffraction measurements only pro-
vided information regarding the size of the outer oil droplets, the
presence of the inner water phase was confirmed by electron
microscopic investigations. The detected structure of the multiple
droplets is in accordance with a previous work (Hoppel et al.,
2014). A similar shape with oil droplets that contained one large
 M. Hoppel et al. / European Journal of Pharmaceutical Sciences 68 (2015) 27–35 33

(a)

0.16
0.14
0.12
Absorbance Units
0.10
0.08
(1)
0.06

(2)
0.04

(3)
0.02

1125 1120 1115 1110 1105 1100 1095 1090

Wavenumber cm-1

(b)
0.12
0.10

(1)
Absorbance Units
0.08
0.06

(2)
0.04

(3)
0.02

1125 1120 1115 1110 1105 1100 1095 1090

Wavenumber cm-1

(c)
0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40
Absorbance Units

(1)

(2)

(3)
1125 1120 1115 1110 1105 1100 1095 1090
Wavenumber cm-1

Fig. 6. Comparison of sample ATR-FTIR spectra of emulsion treated porcine skin. Each figure shows (1) an ATR-FTIR spectrum of the respective emulsion itself, (2) difference
spectra of emulsion treated skin with decreasing absorbances of the C–O vibrational mode during the removal of ten tape strips and (3) difference spectra of two untreated
skin samples during the removal of ten tape strips. The latter served as a control that no remaining absorbances arising from inhomogeneity of different skin samples falsified
the results. (a) Multiple W/O/W emulsion, (b) O/W emulsion and (c) W/O emulsion.
34 M. Hoppel et al. / European Journal of Pharmaceutical Sciences 68 (2015) 27–35
0.35
W/O/W
0.30
Area ratio: C-O/Amide II
O/W
0.25
W/O
0.20
0.15
0.10
0.05
0.00
0 10 20 30 40
Stratum corneum depth [%]
Fig. 7. Ratio of the C–O stretching absorbances to the amide II absorbances as a
function of stratum corneum depth. Peak areas of the C–O absorbances were related
to the amide II absorbance of the respective ATR-FTIR spectra to account for
variability of contact between the ATR crystal and the sample. Each point represents
absorbance ratios calculated from ATR-FTIR spectra recorded from the skin surface
and after removal of each tape strip. Indicated values are means ± SD of three
individual experiments for each formulation (n = 3).
0.12
Relative concentration [a.u.]
0.10
0.08
0.06
0.04
0.02
0.00
0 10 20 30 40
Stratum corneum depth [%]
Fig. 8. Calculated relative concentrations of incorporated vehicle components as a
function of stratum corneum depth. Peak areas of the C–O stretching absorbances
were related (1) to the amide II absorbance of the respective ATR-FTIR spectra to
account for variability of contact between the ATR crystal and the sample and (2) to
the corresponding C–O stretching absorbance of the respective formulation.
Indicated values are means ± SD of three individual experiments for each formu-
lation (n = 3).
internal water droplet was reported with the use of IPM as oily
component. Moreover, NMR self diffusion experiments revealed
differences in diffusion behavior of free and incorporated water,
indicating the W/O/W structure of the emulsion (Hindmarsh
et al., 2005).
A precise quantification of the AH-8 distribution in the different
water phases was challenging, due to a tendency of the inner drop-
lets to rupture under application of centrifugation or filtration.
Although it was not possible to localize the peptide precisely, the
observed slow displacement of AH-8 in NMR self diffusion studies
might be caused by a restricted movement in the W/O/W system.
However, due to the chosen one-step emulsification method, it is
likely that the peptide is present in the inner as well as in the outer
phase. The low displacement might also be caused by a bond of the
peptide to the surface of the emulsions. Therefore, further investi-
gations are necessary in order to make precise conclusions on the
exact location of the peptide within the multiple emulsion.
The influences of the vehicle composition and emulsion type on
the dermal delivery of AH-8 were investigated by in vitro skin
studies. Porcine skin was chosen as an in vitro model for human
skin, due to its similarities in epidermal thickness, lipid composi-
tion and permeability (Sekkat et al., 2002).
The superiority of the W/O/W and O/W emulsions might be a
consequence of their high water content which might support
the skin permeation of AH-8. The non-existing skin permeation
of AH-8 from the W/O emulsion could therefore be explained by
the oil deposit on the skin surface which provided an additional
barrier for the hydrophilic peptide (Williams, 2003).
Although, Franz-type diffusion cell experiments are still a useful
and meaningful in vitro screening tool for choosing between sev-
eral prospective formulations (Russell and Guy, 2009; Pantelic
et al., 2013), the used experimental setup with 0.1% formic acid
50 as acceptor medium did not reflect real in-use conditions. How-
ever, solubility consideration are an important factor in Franz type
diffusion cells (Ng et al., 2010). Due to the isoelectric point of AH-8
which is near the physiological pH of 7.4 AH-8 carries no net-
charge and exhibits lowest solubility at physiological pH. There-
fore, an acidic medium was used in order to provide perfect sink
conditions.
However, to avoid interactions between the skin samples and
the acidic acceptor medium, additional tape stripping experiments
as a more practically orientated experimental setup were
performed. These experiments are of great importance in case of
multiple emulsions, since the shear stress, usually applied by the
W/O/W user, might lead to bursting of the internal water droplets and
O/W release of the active matter (Olivieri et al., 2003). The tape strip-
W/O ping technique is a well-known, simple and inexpensive method
for skin absorption assessment. A recent study by our working
group showed that penetration into porcine ear skin was highly
comparable to the in vivo situation on human skin, confirming
the suitability of the porcine ear skin model for in vitro skin
penetration assessment (Klang et al., 2012). Interestingly, there is
a surprising lack of comparative studies, which deal with AH-8
containing vehicles applied in a non-occluded and finite dose
application, although this experimental set up reflects real in-use
conditions of topical formulations (Pantelic et al., 2013; Narkar,
50
2010).
The results of the in vitro tape stripping experiments showed
the same trend as the Franz-type diffusion cells. The superiority
of the W/O/W and O/W emulsion is in accordance with data from
the literatures (Hoppel et al., 2014; Ferreira et al., 1995, 1994). Due
to the lower viscosity, the W/O/W emulsion showed a good spread-
ability on the skin. The increased osmotic pressure gradients
caused by water evaporation could therefore lead to bursting of
the inner water droplets, thus releasing AH-8 from its encapsula-
tion (Ferreira et al., 1995). However, influence of viscosity on the
dermal delivery of actives is controversially discussed in the
literature. In some cases, a link between low viscosity of a vehicle
and faster drug delivery is reported (Kogan and Garti, 2006). In
contrast, other studies revealed no correlation between a formula-
tion’s viscosity and the skin permeation of the incorporated active
(Klang et al., 2011a; Fang et al., 2008).
According to the combined ATR-FTIR and tape stripping exper-
iments, a significantly lower amount of the applied W/O emulsion
was absorbed into the initial layers of the stratum corneum com-
pared to the multiple W/O/W and the O/W emulsion. This may
be caused by the fact that W/O emulsions are more occlusive by
forming a protective oily layer on the skin surface after evaporation
of the water from the applied formulation (Otto et al., 2009;
Williams, 2003).
Another reason for enhanced skin penetration of AH-8 from the
water-rich emulsions might be an increase in hydration level of the
stratum corneum caused by exposure to the external aqueous
phase (Otto et al., 2009). Interestingly, no significant difference in
skin hydration level after incubation with the different formulation
was found. This could be explained by the fact that topically
 M. Hoppel et al. / European Journal of Pharmaceutical Sciences 68 (2015) 27–35
applied formulations may increase skin hydration by either occlu-
sion (W/O) or by providing water from the vehicle to the stratum
corneum (W/O/W and O/W) Otto et al., 2009. However, consider-
ing that these studies were performed ex vivo and excised porcine
ears lack an active circulation, in vivo studies on human volunteers
would be more meaningful.
5. Conclusion
Although peptides are widely used in cosmetics, their skin pen-
etration is controversially discussed and mainly studied through
indirect measurements like anti-wrinkle tests. The first compari-
son of AH-8 delivery into the skin from different emulsions showed
that emulsion type and composition have a strong impact on the
dermal delivery of AH-8. Due to significantly enhanced delivery
of AH-8 from the multiple W/O/W emulsion compared to simple
O/W and W/O emulsions, natural surfactant based multiple emul-
sion can be assumed as promising skin-friendly vehicles for topical
delivery of AH-8. Future studies should focus on the distribution of
AH-8 in different layers of the skin and detailed investigations of
skin hydration effects of multiple W/O/W emulsions in vivo would
be of further interest.
Acknowledgements
The authors would like to thank the research platform ‘‘Charac-
terisation of drug delivery systems on skin and investigations of
involved mechanism’’ (University of Vienna, Austria) for financing
this Project. We would also express our gratitude to Lipotec GmbH
(Germany) for kindly providing acetyl hexapeptide-8 and Evelyn
Holper for technical assistance.
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