and waterbased formulations containing nanoparticles
The dermal behaviour
after topical application under in-vitro conditions (BUS-Model)
Wolfgang Pittermann 1, Olaf Lammerschop 1,
Marcel Roth 1, Michael Schmitt 1 and
1 Henkel KGaA, 40191 Duesseldorf,
2 Institute for Pharmacology,
Toxicology and Pharmacy, Veterinary School Hannover
Oil- and water-based formulations were frequently used for cosmetic
and dermatological treatments or as process chemicals. It is well known that the
specific behaviour concerning the dermal penetration and irritation potential
will be modified during and after the course of application due to supersaturated
solutions by evaporization of water and other volatile components (1). It can
also be influenced by ingredients of which the distribution coefficient is not
The aim of this in-vitro skin study was to learn the
dermal behaviour of newly developed ferrofluides (nanoparticles) dispersed in
water- and oil-based formulations under different application conditions.
Material and Methods |
|Ferrofluids are nanoparticles either magnetic or non-magnetic dis-persed
in solvent (Fig. 1, Fig. 2). Two water-based
products (product 1, 3) contain magnetite-particles coated by polyacrylic acid
and lauric acid (product 3*) respectively. The other two samples (magnetite or
zinc-ferrit) are based on the emollient dicaprylyl ether as vehicle and isostearic
acid (Cognis Deutschland) as coating material. The product 4 contains zinc-ferrit
instead of magnetite.
|To achieve long-term stability in water and organic solvents respectively,
the particle size has been adjusted to 5 - 20 nm and the particle surface has
been modified to adapt the polarity of the particles to the dispersing agent.
The core of the nanoparticles consists of non-toxic iron oxide and was synthesised
by precipitation of ferric salt precursors. A suspension of these particles is
long-term stable especially by applying a magnetic field. The surface modification
consists of fatty acids which shows a good adhesion to iron oxide.|
The isolated perfused bovine udder skin (BUS) is regarded as a viable
in-vitro model (Fig. 3). Using unimpaired
natural skin it takes into account active skin barrier properties and cutaneous
metabolic processes, so that it be used to study for both, penetration and irritation
induced by ingredients or formulations (2, 3, 4, 5).
The open application
(2 - 4 g / 100 cm2) was performed in four independent studies (Fig.
4) either by the means of glass spatula (conventional) or a intensive finger massage
(1 minute) of the skin. After the exposure periods of 1.0 h and 5.0 h the repeated
horny layer stripping method (10 times) was used (Tesa 4204, BDF, Hamburg, 19
mm × 100 mm) for studying the penetration of Fe. For the irritation assays
a short term exposure period of 0.5 h was added to the periods of 1.0 h and 5.0
h. Whole skin biopsies were prepared for the MTT-assay (cytotoxicity) and the
determination of the PGE2-tissue concentration (irritancy) compared
to untreated sites.
Additionally a new technique for a precise histochemical
determination of six different layers of the skin was introduced (Fig.
4). In contrast to the standard preparation of the whole skin biopsies this
procedure (V-max-technique, HistoServe GmbH, Münster, Germany) allows to
assay the activities of certain intracellular enzymes in freely selected regions
of epidermal or dermal layers.
On the basis of the permanent viability of
the BUS-model the time related activation or recovery of the natural skin can
be analysed and compared after topical applications or manipulation e.g. skin
A selected result (Fig. 9; product 1, conventional vs. massage application,
exposure period 1.0 h) is presented for LDH (lactate-dehydrogenase, in SG mainly)
and SDH (succinate-dehydrogenase, in SB mainly).
Calculated up-take (%)
Due to the various Fe-content in the products
a calculation of the actual up-take (percentage) into the horny layer was made
on the assumption that a fixed amount of product (2 g / 100 cm2) was
The percentual up-take was at the maximum approx. 10 % - 12 % (Fig.
5). As expected the 1st strips of all groups exhibited more Fe-content (>
1.0 %) than each of the following nine strips at both exposure periods.
the influence of the time period of exposure only the oil-based formulations (o)
showed a significant decrease of Fe analysed in the horny layer after the prolonged
A clear product differentiation between water- and oil-based
formulations (w, o) was also calculated under massage conditions for the exposure
period of 1.0 h. Higher amounts of Fe compared to conventional application were
analysed after the short exposure period for all products except the product 4.
After the long exposure period the Fe-content of the water-based formulations
remains about on a similar level. So the massage effect regarding the calculated
up-take was not generally relevant even after the prolonged exposure period.
Irritation (cytotoxicity / irritancy)
For the irritation
assay a very short exposure period of 0.5 h was included into the study design
additionally. The degree of irritation is demonstrated by the score value combined
from the relative MTT-result (cytoxicity, irreversible) and the PGE2-concentration
(irritancy, reversible) (6, 7). All columns in Fig.
6 consist of both, the singular score for irritancy on the basis and for the
cytotoxicity on the top.
Only after the very short exposure period of 0.5 h a difference between
the water- or oil-based formulations could be observed. The score value of the
oil-based formulations (product 2, 4) was more than twofold compared to the value
of the water-based formulations.
At the exposure period of 1.0 h no distinction
between the different formulations was observable any more due to the delayed
increase of the score value of the water-based products. Each formulation displayed
its own specific score profile unrelated to its individual Fe-content and type
No product differentiation could be also observed after the
prolonged exposure period of 5.0 h. The score values were significantly diminished
on the basis of the almost complete reversibility of the preinflammatory mediator
As expected intensive massage
application induces an increased score level compared to the conventional application
due the intensified release of PGE2. Under massage conditions the change
from the formulation-related profile at the exposure period of 0.5 h to the individual
product profile after additional 30 minutes was very similar to the results obtained
after the conventional application.
Generally the massage procedure also
altered the relationship between cytotoxic and irritant action of all products
independently from the type. The mechanical skin treatment by the massage induced
an increase of the PGE2-tissue concentration by about 70 %, whereas
simultaneously the cytotoxic activity of the formulations was decreased by approx.
Although the tape stripping did not result in the complete removal
of all of the stratum corneum and contents of the hair follicles the material
recovered should be treated as representative fraction of that present in the
horny layer due to the extra large size of the strips.
But regarding the
size of the nanoparticles (5 - 20 nm) a certain amount of ferrofluids could assumed
to be remained within the acro-infundibulum of the hair follicles (Fig.
|The results shown in the Figures 5 and 6 are combined in Fig.
8 for direct comparison. Clearly the penetration potential (up-take) into
the horny layer is differently profiled than the irritation potential (score).
|The results (Fig. 9, relative units) of the V-max-technique showed no
altered activity for the enzyme SDH with its main activity in SB. However, the
massage treatment induced a slight decrease of activity of LDH in SB, SS and SG,
but not in the horny and dermal layers.|
- The Fe-content and the type of formulation did
influence the results at the short term exposure period up to 1.0 h. After prolonged
exposure periods later on unspecific reactions regarding the skin irritation capability
were induced. The dermal behaviour regarding the penetration capability remains
unchanged even after 5.0 h exposure.
- Ferrofluids used as nanoparticles
do not interfere with the specific type of formulation. The actual Fe-content
and the massage effect do not influence the irritation potential specifically.
results regarding the dermal behavior of the ferrofluids indicates a high skin
compatibility even after prolonged exposure periods and intensive massage during
application. No abrasive effects on the skin could be detected.
the V-max-technique alterations of intracellular enzyme activities (SDH, LDH)
within six epidermal and dermal layers related to the type of application could
be differentiated.SDH remained unchanged even after massage application.
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