A novel concept for the treatment of couperosis based on

International Journal of Pharmaceutics 510 (2016) 9–16
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
A novel concept for the treatment of couperosis based on nanocrystals
in combination with solid lipid nanoparticles (SLN)
Sung Min Pyoa,* , Martina Meinkeb , Anja F. Kleinc , Tanja C. Fischerd, Rainer H. Müllere
a
Institute of Pharmacy—Pharmaceutics, Pharmaceutical Nanotechnology & NutriCosmetics, Kelchstr. 31, 12169 Berlin, Germany
Charité-Universitätsmedizin Berlin, Department of Dermatology, Venerology and Allergology, Charitéplatz 1, 10117 Berlin, Germany
Haut- & Lasercentrum Berlin-Potsdam, Kurfürstenstraße 40, 14467 Potsdam, Germany
d
Haut- & Lasercentrum Berlin-Potsdam, Kurfürstenstraße 40, 14467 Potsdam, Germany
e
Institute of Pharmacy—Pharmaceutics, Pharmaceutical Nanotechnology & NutriCosmetics, Kelchstr. 31, 12169 Berlin, Germany
b
c
A R T I C L E I N F O
Article history:
Received 28 February 2016
Received in revised form 2 May 2016
Accepted 9 May 2016
Available online 2 June 2016
Keywords:
Couperosis treatment
Vitamin A1
Vitamin K1
Rutin
Nanocrystals
Solid lipid nanoparticles (SLN)
Optimized dermal delivery
Antioxidant activity
Penetration profile
A B S T R A C T
For the post laser treatment of couperosis a new dermal formulation was developed combining three
actives: vitamin K1, A1 and rutin, where both vitamins were incorporated into solid lipid nanoparticles
(SLN) and the poorly soluble antioxidant rutin formulated as nanocrystal. All three formulations were
stable over 6 months either on their own or after their incorporation into a hydrogel. Vitamin A1 at 0.3% in
emulsions shows local skin irritation due to very rapid release. By forming SLN, prolonged release with
less irritation potential but deeper penetration was achieved in porcine ear skin. Due to the nanosized
rutin, the new hydrogel showed clearly increased antioxidant activity, representing a stronger protection
potential against reactive oxygen species (ROS), compared to marketed anti-redness products with rutin
as raw drug powder or water-soluble derivative. In addition, rutin nanocrystals showed up to 5 times
pronounced penetration compared to mm-sized raw drug powder. The orientating in-vivo case study
revealed a three to six times faster recovery after laser treatment of couperosis by twice daily application
of the new hydrogel, regarding scabbed-over areas and erythema. Continued use of the new gel also
showed preventive properties against recurrences of veins for at least 8 month.
ã 2016 Elsevier B.V. All rights reserved.
1. Introduction
Couperosis is one of the most common skin diseases in
adulthood worldwide. It can be divided in mild to severe cases
were the typical symptoms for the mild couperosis are the
sebostatic condition of the facial skin combined with a long lasting
redness on cheeks and nose caused by hereditary weakness of the
conjunctive tissues (Crawford et al., 2004). Untreated since the
beginning of the mild stage, the fine blood capillary walls get
weakened and overstretched, losing elasticity over time. Thus, by
the next stronger blood flow the capillaries can get broken easily,
resulting in subcutaneous bleedings permanently visible as deep
purple networks. This process is accelerated by reactive oxygen
species (ROS) since ROS will negatively influence collagen
biosynthesis (Tanaka et al., 1993).
* Corresponding author.
E-mail address: [email protected] (S.M. Pyo).
http://dx.doi.org/10.1016/j.ijpharm.2016.05.017
0378-5173/ã 2016 Elsevier B.V. All rights reserved.
Using laser technology will atrophy the subcutaneous bleedings
(Raulin et al., 1997; Clark et al., 2002) leading to an immediate
discoloration of purple networks and improvement of skin
appearance. However, this technology only represents a symptomatic treatment and no final cure of couperosis. For sustainable
effects it is meaningful to treat the cause of the disease. Therefore
the development of a dermal formulation with vascular stabilizing
effects is a sensible strategy and highly desired.
Vitamin K1, also known as phytomenadione, shows those
vascular stabilizing effect. On spider veins, also a disease
characterized by subcutaneous bleedings located predominantly
on the inner site of lower legs, the vascular stabilizing effect of
vitamin K1 shows discoloration of the red spots and lines (Lewis
and Gendler, 1996). Even faster discoloration was shown on
artificially generated subcutaneous bleedings (Elson 1995) and
laser induced purpura (Shah et al., 2002) when vitamin K1 was
combined with vitamin A1 in the special ratio of 10:3 (Lou et al.,
1999), respectively. Also flavonoids are well known for their
capillary stabilizing action by reducing the permeability of blood
vessels. Comparing the permeability reducing activity of different
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S.M. Pyo et al. / International Journal of Pharmaceutics 510 (2016) 9–16
flavonoids, rutin is the most effective one with the highest antipermeability-factor (APF) of 8.5 (Muschaweck, 1950). In addition,
rutin has high antioxidant activity able to neutralize ROS.
The use of rutin in dermal formulations is a challenge due to its
poor solubility in both water and oil phase (Zi et al., 2007; Ling
et al., 2009). Since only dissolved drug can penetrate into the skin
and appeal its effect, rutin was used as nanocrystal. Nanocrystals
are particles made of pure cosmetic or pharmaceutical active,
having a size range from few nm to <1000 nm (Müller et al., 2011;
Du et al., 2015; Leone and Cavalli, 2015). Dermal penetration
enhancement compared to micrometer-sized powder takes place
theoretically by 3 different effects: increased concentration
gradient between dermal formulation and skin due to increased
saturation solubility of rutin in nano range, increased dissolution
velocity and high adhesion to skin (Müller et al., 2011; Keck et al.,
2008).
The limited chemical stability of both vitamins can also be a
problem for their use as dermal actives. Thus, solid lipid
nanoparticles (SLN) are an optimal delivery system as the solid
lipid is able to protect incorporated vitamins against chemical
degradation (Dingler et al., 1999). Furthermore, SLN are also
described having positive benefits for sebostatic skin (Kerscher and
Williams, 2006) since they are composed of a lipid matrix being
solid at body temperature and not melting by dermal application.
In addition, they can form a protective film barrier on the skin
(Fig. 1) (De Vringer and De Ronde, 1995; Müller and Dingler, 1998)
leading to a re-enforcement of the stratum corneum lipid film
(Müller and Dingler, 1998). The occlusivity generated by this film
also increases penetration of actives.
The aim of following study was to develop a new dermal
formulation with rutin nanocrystals and vitamin K1 and A1 SLN as
actives for a more efficient treatment of couperosis affected skin.
2. Materials and methods
2.1. Materials
Apifil was obtained from Gattefossé GmbH (Germany), cutina
CP and miranol 32 ultra from Henkel GmbH (Germany).
Plantacare1 810 UP and retinol 50C were purchased from BASF
SE (Germany). Dynasan1 118 was obtained from Sasol (Germany)
and vitamin K1 was kindly provided by Merck KGaA (Germany).
Sisterna1 PS750-C was provided by Rahn AG (Swiss) and carnauba
wax obtained from Cäsar & Lorentz GmbH (Germany). Glycerol 85%
was purchased from LS Labor-Service GmbH (Germany), euxyl1 PE
9010 from Schülke & Mayr GmbH (Germany) and hydroxypropyl
cellulose from Sigma-Aldrich (USA). Purified water was obtained
from a Milli-Q system of Merck KGaA (Germany).
2.2. Production of SLN and nanocrystal suspensions and final gel
formulation
2.2.1. Production of vitamin K1 loaded SLN suspension
The vitamin K1 loaded SLN suspension was prepared by high
pressure homogenization (HPH). Vitamin K1 at 8.0% was added into
the melted solid lipid apifil at 12.0% and the resulting hot lipid
phase was then dispersed by rotor stator stirrer into the hot
aqueous solution of Sisterna1 PS750-C at 1.0%, Plantacare1 810 UP
at 0.5% and distilled water up to 100.0%. The obtained hot preemulsion was homogenized using the Micron LAB 40 (APV
Deutschland GmbH, Unna, Germany) by applying 3 cycles at
800 bar and 85 C. The obtained nanoemulsion was cooled to room
temperature, the lipid mixture recrystallized and the emulsion
turned into a suspension.
2.2.2. Production of vitamin A1 loaded SLN suspension and
nanoemulsion
The vitamin A1 loaded SLN suspension was produced by using
the same production method as for vitamin K1. The lipid phase
consisted of 6.0% carnauba wax and 6.0% Retinol 50C corresponding to 3.0% vitamin A1 and 3.0% polysorbate 80. The aqueous phase
consisted of 2% Miranol1 32 ultra and 86% purified water. For the
production of vitamin A1 nanoemulsion the solid lipid carnauba
wax was replaced by identical amount of Miglyol 812 and only two
cycles of HPH were applied at 800 bar and 85 C.
2.2.3. Production of rutin nanocrystal suspension
The rutin nanosuspension was produced by wet bead milling
combined with high pressure homogenization. First, a rutin
nanosuspension concentrate consisting of 18.0% rutin, 2.0%
polysorbate 80, 1.0% euxyl1 PE 9010 and water for injection up
to 100% was prepared by wet bead milling using a PML-2 (Bühler
AG, Switzerland) at 2000 rpm and pump capacity of 10% with 0.4–
0.6 mm yttria oxide stabilized zirconium oxide beads (Hosokawa
Alpine, Germany). This concentrate was further diluted to the final
concentration of 5.0% rutin, 2.0% polysorbate 80, 5.0% glycerol
85.0%, 1.0% euxyl1 PE 9010 and processed by two cycles HPH at
300 bar using an EmulsiFlex-C50 (Avestin Europe GmbH,
Germany).
2.2.4. Production of couperosis gel formulation
For dermal application the three aforementioned suspensions
were combined into one gel formulation. The gel base was
produced by dispersing 5.0% hydroxypropyl cellulose in 65.0% hot
purified water at 85 C in an ointment bowl and stirred gently with
a pestle until room temperature was reached. The evaporated
amount of water was complemented and 2.5% glycerol and 1.0%
Fig. 1. Model illustration of couperosis affected sebostatic skin. The protection barrier of the skin is disordered. Water loss and skin irritation potential are increased (left).
Mechanism of action of dermal applied SLN for couperosis skin: Protection of the skin from irritation due to the restoring of the distorted skin barrier by repairing the
endogenous protective lipid film with an occlusive film, which also leads to minimized water loss (right) and increased penetration.
S.M. Pyo et al. / International Journal of Pharmaceutics 510 (2016) 9–16
11
euxyl1 PE 9010 were added dropwise for improved spreading
properties and preservation, respectively. Into this gel base 12.5%
vitamin K1 SLN suspension, 10.0% vitamin A1 SLN suspension and
4.0% rutin nanocrystal suspension were incorporated by gently
stirring with the pestle, corresponding to 1.0% vitamin K1, 0.3%
vitamin A1 and 0.2% rutin.
supernatant was added into 1.5 ml of a methanolic DPPH solution
and the discoloration of DPPH was measured at a wavelength of
517 nm using a PharmaSpec UV-1700 photometer (Shimadzu
Corporation, Japan) over 60 min. As zero adjustment 75 ml of
Miglyol in 1.5 ml methanolic DPPH solution was used. The
antioxidant activities of all samples were investigated triplicate.
2.3. Characterization of the nanosuspensions
2.5. Ex-vivo penetration study on porcine ear skin
2.3.1. Photon correlation spectroscopy (PCS)
Z-averages (intensity weighted mean diameter of the bulk
population) and polydispersity indices (related to the width of size
distribution) of produced SLN and nanocrystal suspensions were
analyzed by PCS using a Zetasizer Nano ZS (Malvern Instruments,
UK). The samples were analyzed after dilution (10 ml nanosuspension in 5.0 ml purified water). For each sample, 10
measurement runs were performed and the average calculated.
To analyze the particle sizes of actives incorporated into gel, 50 mg
of the prepared active gel was dissolved in 100 ml purified water
and 1 ml of this solution was diluted with purified water up to
100 ml.
For the ex-vivo penetration study, porcine ears were used on the
day of slaughter. The hair was cut using a scissor. Shaving was
avoided, since the upper layers of the skin may be injured and thus
can influence the results. Cut hair and other pollutions on the
surface were removed by washing the porcine ear with 18–20 C
cold water avoiding the use of alcohol or surfactant. The clean
porcine ear was then patted dry with a soft handkerchief. Only
intact skin without any injuries and skin changes were selected as
an investigation area with a size of 2 cm x 4 cm 20 mg of each
sample were applied on this area homogenously by spreading
avoiding massaging and were allowed to penetrate for a period of
20 and 60 min. After the penetration time an adhesive tape (tesa
Film No. 5529, Beiersdorf, Germany) was used to remove a thin
layer of the stratum corneum. One investigation area was stripped
for 30 times. The obtained tapes were fixed on slide frames and the
amount of corneocytes on 1 cm x 1 cm on 1 cm x 1 cm of the tapes
was investigated by UV analysis (Lambda 650, PerkinElmer,
Waltham, USA) at a wavelength of 800 nm. Followed, the tapes
were cut out from the frame to the size of 1.9 cm x 3.5 cm and
transferred into a vial and the actives were quantitatively extracted
with 2.0 ml of acetonitrile and dimethyl sulfoxide in a ratio of 1:1
using a shaker (Edmund Bühler Swip KS-10, Hechingen, Germany)
for 1 h at 150 rpm. The concentrations of the active were analyzed
using high-performance liquid chromatography (Kontron Instruments GmbH, Germany).
2.3.2. Laser diffractometry (LD)
LD measurement was performed by using a Mastersizer 2000
(Malvern Instruments, UK) to detect lager particles which cannot
be detected by PCS. The dispersion medium was purified water and
the optical parameters used were 1.456 for the real refractive index
and 0.01 for the imaginary refractive index. The obscuration was
adjusted from 4 to 6%. Stirring speed was set to 750 rpm and no
sonication was used. As characteristic parameters LD volume
weighted diameters LD 50%, 90% and 99% were obtained.
2.3.3. Light microscopy (LM)
To confirm the results obtained from LD measurements, LM was
performed using an Orthoplan Leitz (Wetzlar, Germany). The
microscope was connected to a camera CMEX 3200 (Arnhem,
Netherlands) and magnifications of 160, 400, 630 and 1000-fold
were used.
2.3.4. Zeta potential (ZP)
The charge of the particle surface was investigated using a
Zetasizer Nano ZS (Malvern Instruments, UK). Two different media
were used, the original dispersion medium of each suspension and
purified water (adjusted to 50 mS/cm with NaCl solution and pH of
5.5). The Helmholtz-Smoluchowski equation was used to convert
the measured electrophoretic mobility into zeta potential.
2.4. In-vitro antioxidant activity
The antioxidant activity of the rutin nanocrystal gel (Section 2.2.4) was compared with other marketed anti-redness
products, having rutin or its water-soluble derivatives disodium
rutinyl disulfate and troxerutin as active compound in combination
with one or even two of the following antioxidants: resveratrol,
tocopherol, tocopheryl acetate, sodium ascorbyl phosphate. The
official selling prices of these products varied from 8.00 to 97.00
Euro. The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay was performed with a methanolic DPPH solution. Its absorbance at a
wavelength of 517 nm was adjusted to 1. For the preparation of the
methanolic sample solution 0.5 g sample was added to 2.5 g
methanol. This mixture was shaken for one hour at 150 rpm and
20 C using an Edmund Bühler Swip KS-10 (Hechingen, Germany)
and then centrifuged at 15,000 rpm (corresponding to 20,627 G)
and 20 C using an Eppendorf Centrifuge 5451C (Hamburg,
Germany) to remove undissolved particles which can lead to
erroneous measurements by UV light scattering. 75 ml of the clear
2.6. High-performance liquid chromatography (HPLC)
2.6.1. HPLC analysis of rutin
The concentrations of the active rutin were determined using a
KromaSystem 2000 version 1.7 (Kontron Instruments GmbH,
Germany), a solvent delivery pump equipped with a 20 ml loop, an
auto sampler (model 560) and an UV detector model 430 (Kontron
Instruments SpA, Italy) which measured at 255 nm. The analytical
column was an Eurospere1 C18 RS (250 4.6 mm). As solvent
system acetic buffer (pH 4.8) and acetonitrile in a ratio of 8:2 (v/v)
were used. Each sample was measured in duplicate.
2.6.2. HPLC analysis of vitamin A1
The concentrations of the active vitamin A1 were measured as
described in 2.6.1. Only the wavelength was changed to 325 nm.
The analytical column was a Lichrospher1 60 RP (250 4 mm). As
solvent system acetonitrile and purified water in a ratio of 8:2 (v/v)
were used with 1 ml ortho-phosphoric acid per 1 L solvent system.
The samples were measured in duplicate.
2.7. In-vivo human case study
An in-vivo case study on one male volunteer was performed to
investigate the redness reducing effects of couperosis gel
(Section 2.2.4) on affected skin areas. After an initial laser
treatment, the gel was applied twice daily for a period of 10 days.
The VISIA Complexion Analysis system (Canfield Imaging Systems,
Fairfield, New Jersey) was used to evaluate the vascular structure of
the volunteer left nasal wing visually with high-resolution
pictures. Prior to the measurement, the volunteer had to be
acclimated for at least 20 min in a room with controlled
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S.M. Pyo et al. / International Journal of Pharmaceutics 510 (2016) 9–16
temperature of 20–22 C and constant relative air humidity of 40–
50%. For taking the pictures, the volunteer had to sit up straight,
rested chin and forehead in a defined position, facing a mirror in
the instrument with a neutral facial mimic and closed eyes. The
visual evaluation was performed on day 0, 1, 2, 5 and 10 after the
end of laser treatment. To prevent the reoccurrence of couperosis,
treatment was continued with a gel whereat the concentrations of
vitamin K1 and A1 were reduced from 1.0 to 0.5% and 0.3–0.15%,
respectively. After 8 month of twice daily application the skin was
re-analyzed.
3. Results and discussion
3.1. Characterization of the nanosuspensions and the couperosis gel
Directly after the production, the vitamin K1 loaded SLN
suspension had a z-average of 135 nm and an LD diameter 95% of
219 nm (Fig. 2). The polydispersity index of 0.127 represents a
narrow size distributed system and the zeta potential of 40.9 mV
indicates a good physical stabilization. Indeed, after storage time of
3 month the particle size and polydispersity index did not change
distinctly. Compared to the vitamin K1 SLN suspension, the
suspension with vitamin A1 loaded SLN showed smaller z-average,
polydispersity index and LD diameter 95% of 105 nm, 0.170 and
210 nm (Fig. 2), but the values are still in the desired range. The zeta
potential with 52.1 mV is greater than |–30 mV| and suggests also
a physically well stabilized system. With a z-average of 107 nm
after 3 month storage at 25 C no growth or agglomeration of the
particles could be observed. Rutin nanocrystal suspension
possessed a PCS and LD diameter 50% of 285 nm and 240 nm with
a polydispersity index of 0.215 after production. The particle size
increased over the storage time of 3 month at 25 C to a PCS and LD
diameter 50% of 312 nm and 302 nm. The polydispersity index
remained constant.
Nanosuspensions are highly dispersed systems with high
interfacial energy:
E¼gA
(g interfacial tension, A interfacial area of particles). Thus in
principle they are prone to aggregation. The PCS diameter
increased by 27 nm. By considering a usual standard deviation
of PCS of about 1%, corresponding to about 10 nm, this is no
significant increase; the bulk population is stable. The LD diameter
increased by about 60 nm. LD has a larger measuring range than
PCS, thus also detecting particles above about 5 mm, which are
outside the range of PCS. The increase indicates a very slight
formation of aggregates, potentially also caused by bridging of the
Fig. 2. Particle size diameters and polydispersity indices of vitamin A1 and K1
loaded SLN suspensions directly after production and after 3 months storage at
25 C.
chains of the gel forming agent. However, overall the stability is
very good for a dermal formulation.
Based on this data, all three suspensions on their own possessed
a good physical stability prerequisite for incorporation into a gel
or cream.
To determine whether a possible particle growth was induced
by mixing the three suspensions, or potentially induced by the
incorporation into the gel base, the pure mixture of the three
suspensions without the gel base was also investigated via PCS, LD
and light microscopy. The mixture showed a z-average of 212 nm
with a polydispersity index of 0.216. The increase of the
polydispersity index can be explained by the different particle
sizes of each suspension in the mixture. The LD90 and LD99
diameters of the mixture were 278 nm and 460 nm could be
obtained immediately after mixing together the three suspensions.
The storage at 25 C did not influence the values significantly. Thus,
it could be ensured that mixing together the three suspensions
does not lead to an increase in the particle size. A fresh prepared
mixture was then incorporated into the prepared gel base and the
particle size and its distribution were analyzed. The same PCS and
LD diameters could be obtained as the incorporated mixture. So
there was no influence of the gel base given to the particle size.
Addition of particles to a gel base can affect the rheological
behavior. In case of addition of very small particles such as
nanocrystals in very low concentration, the viscosity decreases but
only negligible or minor. The pseudoplastic flow curve in the shear
stress/shear rate diagram only slightly decreases to lower values,
with no impact on the spreading behavior onto the skin. In case
exactly the same viscosity as the gel base is desired, a slightly
higher gel former concentration can be used.
3.2. In-vitro antioxidant activity study
UV exposure of the skin causes the formation of free radicals,
also known as reactive oxygen species (ROS), which promote the
accelerated degradation of tissue stabilizing collagen (Müller at al.,
2011). In addition, the accumulated fragments of the decomposed
collagen lead to an inhibition of the synthesis of new collagen
(Rittié and Fisher, 2002). Thus, radical exposure is always
associated with a progressive reduction of the extracellular matrix
of the skin tissue (Fisher et al., 2002; Jenkins, 2002). Due to the fact
that couperosis is caused by the weakness of connective tissue, the
main key aspect for an efficient treatment on molecular level
should be the protection of the skin from reactive oxygen species.
Antioxidants are able to neutralize the reactive oxygen species into
harmless molecules. Ideally, the treatment should include a
formulation with a potent antioxidant activity to avoid new
damages and allow a more efficient causal therapy of couperosis. In
order to assess the antioxidant activity of the couperosis gel
(Section 2.2.4), the DPPH assay was performed and the result was
then compared with the antioxidant activities of 8 anti-redness
products available on the market containing rutin or its watersoluble derivative in combination with one or even two of the
following antioxidants: resveratrol, tocopherol, tocopheryl acetate,
sodium ascorbyl phosphate.
The DPPH assay is a well known method for screening the
antioxidant properties of molecules. The DPPH molecule is
characterized as a stable free radical due to the strong delocalization of the spare electron over the whole molecule, so that it does
not dimerise as it would be the case with the most other free
radicals. The free DPPH radical shows a strong absorption band
centered at about 517 nm, causing the characteristic deep violet
color of its methanolic solution. By the reduction of the DPPH
radical to its neutral DPPH
H molecule, which occurs with the
presence of an antioxidant, the intensity of the absorption at
517 nm decreases, turning the solution to pale yellow. This
S.M. Pyo et al. / International Journal of Pharmaceutics 510 (2016) 9–16
of 97.00 Euro. Also no correlation could be made from the used
active agent to the antioxidant activity of the related product. For
example the products B, C, F and H had disodium rutinyl disulfate
incorporated as active agent but B and C belongs to the antioxidant
activity class II where F and H counts to class III. The same applies
to troxerutin, which was a component of product D (class II) and G
(class III). The product E had rutin unchanged as mm-sized powder
and A rutin combined with troxerutin as active agent combination.
To obtain an improvement in the solubility of rutin, most
marketed products are working with chemical derivative of rutin,
such as disodium rutinyl disulfate, troxerutin and rutin-glucoside.
However the change in structure will reduce the bioactivity, since
the antioxidant activity depends on structural features, such as
hydroxyl bound dissociation energy, resonance delocalization of
phenol radicals and steric hindrance derived from groups
substituting the hydrogen in the aromatic ring (Craft et al.,
2012). These features get definitely changed by derivatisation as it
has been previously shown in the patent of Petersen. The
antioxidant capacity of rutin nanocrystals and rutin-glucoside
was assessed by measuring the sun protection factor (SPF) in a
human in vivo study. Compared to the water soluble rutinglucoside, dissolved rutin from nanocrystals at only 1/500
concentration showed doubled SPF. Simplified, it means an
increase of the bioactivity of the factor 1000. This significant
increase in bioactivity was attributed to the skin permeability of
the original molecule compared to the water soluble derivative,
which prefers to stay in the hydrophilic environment of the cream
instead of penetrating through lipophilic parts of the skin.
To conclude, this experiment proved the superiority of the
developed couperosis gel formulation (Section 2.2.4) compared to
commercial anti-redness products with respect to its antioxidant
activity and thus supports its use in the treatment of couperosis.
property allows the simple visual monitoring of antioxidant
activities. A faster and stronger reduction of the violet color
represents a more effective antioxidant activity. One rutin
molecule reacts with two DPPH radicals as reducing agent and
get oxidized itself at the catechol 30 , 40 -dihydroxyl group.
Not only the antioxidants but also the ambient air is able to
reduce the methanolic DPPH solution. In order to discern whether
the discoloration of the methanolic DPPH solution was really
caused by the antioxidant or only accidently by ambient air, a
control was measured, consisting of 1.5 ml methanolic DPPH
solution without the addition of an antioxidant but with 75 ml
Miglyol 812 instead. So, the real antioxidant activity was expressed
by the inhibition of DPPH activity in percentage and calculated
according to the following equation, where A(control) is the
absorbance of the control and A(sample) the absorbance of the
sample.
inhibition of DPPH activity½% ¼
13
AðcontrolÞ AðsampleÞ
100%
AðcontrolÞ
The effect of nanonization on the antioxidant activity of
cosmetic products can be clearly seen on Fig. 3. The couperosis
gel formulation with rutin incorporated as nanocrystals shows
superior antioxidant potential compared to all eight marketed
products having rutin as mm-sized powder or hydrophilic rutin
derivatives. While all tested marketed anti-redness products were
able to inhibit the DPPH activity at maximum 60% even with one or
two more antioxidants incorporated, the couperosis gel was able to
inhibit the DPPH activity for more than 85% within the identical
reaction time.
Generally the products could be classified into three different
antioxidant activity classes. Class I showed the highest antioxidant
activity with DPPH inhibition above 85%. Only representative of
this group was the couperosis gel (Section 2.2.4). Class II
represented by the products A–E had a medium antioxidant
potential, with DPPH inhibitions between 20 and 60%. The
products F–H showed almost no antioxidant potential with DPPH
inhibition lower than 10% and thus were classified as class III.
No inferences could be drawn from the price of a product to its
antioxidant activity. Product A had an official selling price of 10.99
Euro were product H could be purchased for an official selling price
3.3. Ex-vivo penetration study on porcine ear skin
3.3.1. Penetration profile of rutin raw drug powder vs. nanocrystal
The antioxidant effectiveness of the final product not only
depends on the bioactivity of rutin, but also on its bioavailability.
Only the rutin that penetrates deep enough into the skin is
bioactive. Therefore, in addition to the DPPH assay, an ex-vivo
anoxidant acvity of an-redness products
100
90
80
inhibion of DPPH acvity [%]
70
couperosis gel
60
product A
product B
50
product C
product D
40
product E
product F
30
product G
product H
20
10
0
0
-10
10
20
30
40
50
60
70
me [min]
Fig. 3. Comparison of the antioxidant activity with DPPH assay of couperosis final gel formulation with eight marketed anti-redness products, having rutin or its watersoluble derivatives as main active in combination with one or two additional antioxidants.
14
S.M. Pyo et al. / International Journal of Pharmaceutics 510 (2016) 9–16
penetration study on porcine ear skin was performed to assess the
penetration depth and amount of penetrated rutin within the
stratum corneum. For investigating the influence on the skin
penetration by reducing the particle size, the penetration profile of
the gel containing nanocrystals (LD diameter 50% of 256 nm) was
compared to the profile of a reference gel, which contained rutin as
mm-sized powder (LD diameter 50% of 32,2 mm).
An improved penetration of a dermal applied active into the
skin is achieved when the total penetrated amount is higher or
when the active can be found in higher amounts in deeper layers.
Both of these cases apply to the formulation with rutin nanocrystals. The cumulative amount for rutin applied as nanocrystal
was 48 mg and is 2.5 times higher compared to the raw drug
powder formulation with only 18 mg total drug amount. That
means, even if the same quantity of drug was applied to the
identical skin area, 2.5 fold higher amount of rutin permeates into
the skin when formulated as nanocrystals. Comparing the 30
stripped tapes one by one, the amount of rutin was always superior
for the nanocrystal formulation. In average, the nanocrystal gel
tapes showed 2.5 times higher active amount. The biggest
difference between the formulations could be observed for the
8th tape, which showed 7-fold higher concentration of rutin for the
nanocrystal gel (Table 1). In summary, rutin applied as nanocrystal
gel shows a 2.5-fold increased quantity of active penetration into
the skin compared to the raw drug powder gel. These higher
amounts did not only reach the upper layers of the stratum
corneum but also all layers of the examined stratum corneum,
including deeper layers.
Hence, the performed tape stripping test on porcine ear skin
shows clearly the improved penetration when rutin was applied as
nanocrystal. The increase in skin penetration in combination with
the superior antioxidant activity results in a promising improved
bioactivity when dermally applied. This leads to a more effective
protection of the couperosis skin from reactive oxygen species.
3.3.2. Penetration profile of vitamin A1 nanoemulsion vs. SLN
Couperosis is characterized by the weakness of the connective
tissues. Topically applied vitamin A1 allows a causal therapy due to
its new collagen forming properties. This is the reason why most of
the anti-redness products on the market contain this active agent.
Very common is the usage of vitamin A1 in emulsions but this form
of delivery system shows a strong local skin irritation such as
erythema as well as an increased sensitivity to sunlight. One
explanation for these side effects is the strong initial release of the
active from the nanoemulsion. By incorporating the active in the
new dermal delivery system SLN, a prolonged release with less
irritation potential was expected. So both systems were compared
in their penetration profiles after a short exposure time of 20 min
(initial release) and after a long penetration time of 60 min
(prolonged release) with the aim to identify the better-suited
Table 1
The absolute amount of rutin in different tape strip numbers
after the application of suspensions with raw drug powder
(RDP) and nanocrystals (NC).
delivery system for achieving prolonged release with higher
concentrations in deeper skin layers.
Since the lipid content can take influence on skin penetration,
the liquid lipid Miglyol 812 was used to replace the solid lipid of the
SLN formulation in the nanoemulsion. Also the particle size can be
a sensitive factor for the penetration rate and had to be equalized.
Therefore, great care has been given during the production steps to
obtain almost same particle size distributions. Directly before
starting the penetration study, the emulsion showed LD diameter
95% of 0.227 mm, 99% of 0.274 mm and 100% of 0.345 mm. SLN
suspension possessed almost similar particle size distribution with
LD diameter 95% of 0.210 mm, 99% of 0.250 mm and 100% of
0.305 mm.
The relative concentrations of the penetrated vitamin A1 from
nanoemulsion and SLN suspension in respective layers of the
stratum corneum (SC) after 20 min penetration time were shown
on the left side of Fig. 4. In the depth of first 3% of the SC a high
amount of 46% of the active agent could be found from the
nanoemulsion whereas just 3% of active agent reached the same
depth applied as SLN. Also in the depth of 12% of the SC an
obviously higher relative concentration of vitamin A1 could be
found from the nanoemulsion with 4.7% compared to the SLN
suspension with 0.5% only. Hence, after 20 min a tenfold stronger
penetration of vitamin A1 could be obtained when applied as
nanoemulsion.
According to the results of the penetration profiles after 20 min,
the initial release of vitamin A1 from of the nanoemulsion could be
proven. By contrast, the SLN released the active tenfold lower,
implying the required low skin irritation potency.
Leaving both formulations penetrate for an extended exposure
time of 60 min, the penetration profiles behaves inversely (Fig. 4,
right) In the first 22% of the SC only 2.5% of active could be detected
from nanoemulsion showing almost no change compared to the
penetrated amount after 20 min exposure time whereas a fivefold
higher concentration (12%) reached the same depth as SLN
suspension. Considering the SC in the depth of 36% and 50%,
more than three times higher concentrations of active could be
detected from SLN (2.4% and 1.8%) compared to nanoemulsion
(0.8% and 0.5%).
Thus, compared to the established vitamin A1 nanoemulsion,
the SLN shows a better suited prolonged release into the skin,
promising less skin irritation, without an adverse influence on
efficacy. Already after 60 min exposure time, the SLN show notably
higher concentrations of the active in stratum corneum and deeper
penetration depth. Therefore, SLN as delivery system promises an
improvement in couperosis treatment with new collagen forming
action and fewer side effects such as irritation.
3.4. In-vivo human case study
Although both in-vitro (Section 3.2) and ex-vivo (Section 3.3)
data indicate improved bioactivity of the new developed
couperosis gel, the final proof of its superior efficacy can only be
shown by a human in-vivo study. Therefore, an orientating study
with one male subject was performed. Prior to the combined laser
and gel treatment, a high-resolution picture of the couperosis
affected skin (left nasal wing, Fig. 5A) was taken as a negative
control for the visual evaluation. On picture A of Fig. 5 it can be
clearly seen, that the patient already possesses the advanced third
stage of the disease, due to the dark purple color of the capillaries,
the high quantity of visible lines and the characteristic reticulate
association to it.
The laser treatment was performed in three sections by using a
long pulsed cynosure Nd: YAG laser with an average energy fluence
of 75–95 J/cm2 and a pulse of 5 mm/20 msec. Since the laser
treatment acts by burning the affected capillaries, the body reacts
S.M. Pyo et al. / International Journal of Pharmaceutics 510 (2016) 9–16
penetraon profiles of NE and SLN
aer 60 min exposure me
penetraon profiles of NE and SLN
aer 20 min exposure me
0
10
rel. amount of vitamin A1 [%]
20
30
40
50
0
60
0
0
1
11
5
20
8
27
12
31
10
rel. amount of vitamin A1 [%]
20
30
40
50
60
36
15
39
20
horny layer thickness [%]
horny layer thickness [%]
15
30
37
41
45
50
53
58
44
50
53
56
60
63
66
68
62
71
66
74
71
77
75
80
79
82
82
NE
NE
SLN
SLN
Fig. 4. Penetration profiles of vitamin A1 nanoemulsion (blue) and SLN suspension (red) on porcine ear skin after 20 (left) and 60 min (right) application time. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Visual evaluation of treated skin area during 8 months after laser treatment combined with twice a day application of couperosis gel.
with immune response, especially with scabbing, slight swelling
and irritated red skin around the treated spots (Fig. 5B). Typically,
the scabbed-over areas last for 5–7 days and the erythema for 12–
14 days. By twice daily application of the couperosis gel, a
pronounced faster recovery of the scabbed-over areas and
erythema could be observed compared to experiences from other
couperosis patients. Only after two days of couperosis gel
application, both the swelling and scabbing have completely
regressed (Fig. 5C). Thus, the healing time could be reduced to its
half compared to standard treatment experiences. Not only the
swelling and scabbing but also the erythema disappeared faster by
applying the couperosis gel. Picture D of Fig. 5 shows the left nasal
wing after 9 days of regularly gel application. A complete
normalized skin surface could be observed. In summary, the
combination of laser and couperosis gel treatment optimize the
therapy of couperosis affected skin by reducing the healing time.
To avoid the reappearance of telangiectasia that is commonly
observed over time, the administration of the couperosis gel was
continued with reduced concentrations of active (0.15% vitamin A1
and 0.5% vitamin K1 and 0.2% rutin). The prophylactic application
ensured the preservation of improved skin appearance for at least 8
months (Fig. 5E).
4. Conclusion
Physically stable vitamin A1 and K1 SLN and rutin nanocrystal
suspensions were successfully produced, they remained stable
after their incorporation into a couperosis gel pre-requisite for a
product for patients. With a DPPH inhibition of more than 85%, the
new developed couperosis gel was superior in its antioxidative
activity compared to commercial anti-redness products with DPPH
inhibitions always lower than 60%. Also a better suited prolonged
release into the skin could be observed for vitamin A1 SLN
compared to the standard nanoemulsion, which could reduce
additional flushing (side effect of vitamin A1) after application to
couperosis skin. Also looking at the results of the orientating in
16
S.M. Pyo et al. / International Journal of Pharmaceutics 510 (2016) 9–16
vivo case study, the combination of SLN and nanocrystals is a
promising novel formulation with improved antioxidant activity
and use in couperosis.
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