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
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
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.
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
Regarding 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 exposure period.
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 /
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 of formulation.
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 prostaglandin E2-concentration.
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.
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. 50 %.
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. 7).
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
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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