Keratin Films made of Human Hair Keratin as a
Nail Substitute for Studying Drug Permeation
Lusiana, S. Reichl, C.C. Müller-Goymann
Institut für Pharmazeutische Technologie, TU Braunschweig, Mendelssohnstr. 1, D-38106 Braunschweig
Introduction
Hooves 100µm
2.0E-05
Keratin films 120µm
An early investigation showed that this model
resembled bovine hooves in terms of permeation and
binding of three tested marker substances, i.e. sodium
fluorescein (SF), fluorescein isothiocyanate-dextran MW
4000 (FD4) and rhodamine B (RB) [2].
Based on this finding, a further comprehensive
investigation was conducted to see the effect of three
nail penetration enhancers (PEs) with different
mechanisms (urea/ UR, thioglycolic acid/ TA and papain
from papaya latex/ PA) on both materials using SF, FD4
and RB as markers, which represented a water soluble,
a lipid soluble and a large molecule drug model,
respectively.
Keratin from hair was extracted under a reductive
condition with a so called Shindai method [3]. The
schematic diagram of the process is shown in Fig. 1.
Following previous study 120 µm thick keratin film was
continually used, due to its similar permeation profiles
with 100 µm thick bovine hooves [2].
Bovine Hoof Membranes Preparation
Only the sole part of bovine hoof was used in this
study. This part was cut in regular squares (2 cm x 2
cm), submerged overnight in water, and was then
sliced to a thickness of 100 µm. For permeation
purposes these slices were then punched out (Ø = 15
mm).
Permeation
Studies
and
Penetration Enhancers (PEs)
Application
of
Permeation experiments were carried out using
modified Franz diffusion cells at 32 °C. Prior to
treatment with PEs hoof membranes and keratin films
were hydrated for one hour with PBS and were then
mounted between the compartments (donor &
receiver). The receiver was filled with PBS and the PE
solution was filled into the donor compartment.
Acknowledgement
Lusiana thanks DAAD (Deutscher Akademischer
Austausch Dienst) for the scholarship.
www.pharmtech.tu-bs.de
**
*
*
P (cm/s)
*
0.0E+00
pure
Fig. 1: Schematic diagram of keratin extraction and
keratin film manufacture
1.2E-05
Keratin films 120µm
1.0E-05
urea 40% TA 5% 15h
3d
combi
urea+TA
serial
urea+TA
papain 2%
15h
Fig. 4: Permeation coefficients (cm/s) of RB before and
after treatments with PEs (n = 3-12)
260 kDa
Hooves 100µm
**
* compared to control
** hoof-keratin film comparison
p < 0.01 (t-test)
**
135 kDa
**
95 kDa
72 kDa
8.0E-06
52 kDa
42 kDa
6.0E-06
34 kDa
26 kDa
4.0E-06
17 kDa
* *
2.0E-06
* *
* *
10 kDa
*
*
0.0E+00
pure
urea 40% TA 5% 15h
combi
3d
urea+TA
serial urea papain 2%
+ TA
15h
1.4E-06
1.2E-06
P (cm/s)
1.0E-06
(a)
(b)
(c)
(d)
(e)
(f)
(g)
Fig. 5: SDS-PAGE of (a) aqueous keratin suspension
after dialysis; (b) human nail; (c) hair extract; (d), (e)
keratin film before and after curing, respectively; (f)
hoof extract (50 °C, 3d); (g) molecular weight marker;
(b)-(f) in shindai solution
Results and Discussion
Hooves 100µm
Keratin Film Manufacture
**
8.0E-06
**
Materials
Hair (blond) was dispensed from local hairdresser,
bovine hooves were purchased from online pet shop
(Edinger). Shindai solution was prepared from urea,
thiourea (Roth-Germany), 2-mercaptoethanol and
Trizma® base (Sigma-USA). A Spectra/Por membrane
(MWCO: 6-8,000 Spectrum Laboratories, Inc. Rancho
Dominiguez, Canada) was used as dialysis tubing. SF
was purchased from Fluka-Germany, FD4 from SigmaSteinheim, RB from Fluka-Swedia. UR from Roth
(Karlsruhe, Germany), TA from Merck (Hohenbrunn,
Germany) and PA from Sigma-Aldrich (Steinheim,
Germany). Phosphate buffer saline pH 7.4 (PBS) was
prepared according to Ph. Eur. 6.0 and water was used
in double distilled quality.
**
1.2E-05
4.0E-06
Fig. 2: Permeation coefficients (cm/s) of SF before and
after treatments with PEs (n = 3-6)
Experimental Setup
* compared to control
** hoof-keratin film comparison
p < 0.01 (t-test)
1.6E-05
P (cm/s)
Nail as a part of the body offers not only a protective
function, but is also a barrier for drugs intended for nail
diseases’ treatment. Despite of the need of human nails
for studying ungual drug preparation, unfortunately,
their availability is somehow limited. Bovine hoof is up
to now an accepted model for human nail but
significant differences between them have been
reported [1]. These differences could be related to
hooves’ ability in retaining more water and thus making
them more permeable, although human nail, hair and
bovine hoof are composed of similar keratin proteins
(α−form). Therefore a novel nail model made of human
hair was developed and its physical properties were
then compared with bovine hooves.
Keratin films 120µm
* compared to control
** hoof-keratin film comparison
p < 0.01 (t-test)
**
The permeation coefficients of three markers were
overall higher on keratin films (p<0.01), compared to
hooves, after treatments with PEs. Exceptions could be
seen in the case of SF after treatment with 40% UR
(Fig. 2) and for RB after treatments with 5% TA (Fig.
4).
**
8.0E-07
6.0E-07
*
4.0E-07
2.0E-07
*
*
*
* *
* *
urea 40% TA 5% 15h
combi
3d
urea+TA
serial
urea+TA
0.0E+00
pure
papain 2%
15h
Fig. 3: Permeation coefficients (cm/s) of FD4 before
and after treatments with PEs (n = 3-10)
The duration of the treatment and the concentration
used were adjusted for every PE. All the PEs were
dissolved in water (except for PA in PBS), with the
concentrations (all w/w) of 40% for UR (for 3d), 5% for
TA (for 15h) and 2% for PA (for 15h). A combination of
UR and TA (for 15h) as well as a serial treatment (UR
for 3d + TA 15h) was also examined. The cells were
kept at room temperature during treatments; except
for PA (32 °C). After treatments, both compartments
were then rinsed twice with double distilled water and
PBS.
Receivers were filled with PBS and donors with marker
solutions in PBS (SF 500 µg/mL, FD4 1000 µg/mL and
RB 250 µg/mL). Samples (100 µL) were taken from the
receivers for 7-30h and replaced by the same amount
of fresh buffer. The fluorescence intensities of the
samples were analysed using fluorescence plate reader
(Tecan, Switzerland) with λ excitation at 485 nm, λ
emission at 535 nm filters for SF and FD4; λ excitation
at 535 nm, λ emission at 590 nm for RB.
While the permeability of bovine hooves was increased
by the presence of the different PEs in the case of SF,
RB permeation across hooves was increased (p<0.01)
after TA treatment only.
For the large molecule model compound FD4, PEs
surprisingly proved efficient in combination or serial
application only. These different responses on PEs could
be due to the different ability of membranes in
retaining water (thus PEs solutions). Hoof retained
more water (up to 45% of its weight) than keratin films
(only up to 5%).
This ability could influence membranes’ pH value and as
a consequence affected the amount of permeated
markers. For example, RB, a basic dye, could be
expected to give a higher flux in an acidic environment,
such as in 5% TA solution (pH 1.6) (p<0.01) instead of
40% urea solution (pH 9.2).
Conclusions
Although the main protein components of keratin films
are closely similar to those of human nail (Fig. 5), the
use of keratin films as a human nail model has to be
limited for selected substances due to the result of the
present study. A conclusion must be carefully deducted,
especially when penetration enhancers are applied.
References
SDS polyacrylamide gel electrophoresis (SDSPAGE)
[1] Khengar R. H., et al., Pharm. Res., 2007; 24(12):
2207-12.
SDS-PAGE was performed to separate the proteins of
the keratinous materials under study according to their
molecular weights. The stacking and resolving gel were
prepared from 12% and 10% polyacrylamide,
respectively. The electrophoresis was run at 180 V, 80
mA and 25 W. Afterwards the gel was stained with
coomassie brilliant blue.
[2] Lusiana, Reichl, S. and Müller-Goymann, C. C., 2nd
PharmSciFair, 2009.
[3] Nakamura A., et al., Biol. Pharm. Bull., 2002;
25(5): 569-72.
7th World Meeting on Pharmaceutics, Biophamaceutics
and Pharmaceutical Technology, Malta, 08.–11.3.2010