8/10/07

Penetration of Metallic Nanoparticles in Human Full-Thickness Skin (Part Two)

This is the second part as a resume of the whole article, which can be find at
Journal of Investigative Dermatology (2007) 127

...

However, would it be realistic to envisage potential
applications of nanoparticles in the skin, as this would mean
that nanoparticles would penetrate the skin? And more
importantly, if they do penetrate, would people exposed to
such nanomaterials be accidentally contaminated and thus
exposed to a potential local and/or systemic health risk?
Indubitably, one of the functions of the skin, and more
precisely the stratum corneum (SC), is to protect the body
from the external environment (Elias, 2005). Thus, to answer
the first question, targeting one of the layers of the skin (i.e.,
SC, viable epidermis, dermis) requires first overcoming the
natural skin barrier and then retaining the delivered agent
(i.e., drug, nanoparticle, drug–nanoparticle complex) in one
specific skin layer. It is well known that only small (o600 Da)
lipophilic molecules can easily penetrate the skin passively
(Barry, 2001). Hence, over the last three decades many
strategies have been developed to modify the skin barrier
reversibly, thus enlarging the number of possible drug
candidates for systemic pathologies. These strategies enumerate
the use of chemical enhancers, novel formulations,
iontophoresis, sonophoresis, electroporation, and microneedles
(Guy, 1996; Barry, 2001; Moranti et al., 2001;
Langer, 2004). These solutions greatly improve the transdermal
delivery of a particular agent; however, retention in the
skin is still difficult to achieve. Consequently, a major part of
the literature on percutaneous absorption and delivery
suggests that particles might have great difficulty in penetrating
the skin, and that in the presence of strategies to enhance
the delivery, it would be difficult to maintain nanoparticles
on site.
Nevertheless, several articles on particle penetration have
been recently published (see details in the Discussion
session), suggesting that processes governing the penetration
of chemicals (Hadgraft, 2001) and particles might not be the
same. Although mechanisms have not yet been clarified in
the latter case, the results lead to two conclusions: (1) it may
be possible to design and produce nanoparticles for skin
applications and (2) the possibility that a person could be
accidentally contaminated with nanomaterials through the
cutaneous route might be higher than expected (Hoet et al.,
2004; Holsapple et al., 2005; Oberdo¨ rster et al., 2005)
because extensive studies on nanotoxicology and nanomaterial
safeness have recently been undertaken (Hoet et al.,
2004; Holsapple and Lehman-McKeeman, 2005; Holsapple
et al., 2005; Oberdo¨ rster et al., 2005; Thomas and Sayre,
2005).
This work, therefore, investigated whether superficially
modified iron-based nanoparticles, not designed for skin
absorption but whose dimensions are compatible with those
of skin penetration routes (Johnson et al., 1997; Tang et al.,
2001; Bouwstra and Honeywell-Nguyen, 2002; Bouwstra
et al., 2002, 2003; Cevc, 2004; Al-Amoudi et al., 2005), are
able to penetrate and perhaps permeate the skin.
RESULTS
Nanoparticle synthesis and characterization
Maghemite and iron nanoparticle syntheses were carried out
according to previously developed methods based on a
microemulsion technique (Lo´pez Quintela and Rivas, 1993).
Syntheses included a stabilization step to prevent irreversible
particle aggregation on dispersion in an aqueous medium.
Stabilization was achieved by coating the nanoparticle core
with organic molecules (tetramethylammonium hydroxide
(TMAOH), sodium bis(2-ethylhexyl) sulfosuccinate (AOT))
that were absorbed onto it. As a result, brownish/rust-colored
dispersions were obtained in both cases (Figure 1).
X-ray electron diffraction measurements revealed that
TMAOH-stabilized maghemite nanoparticles (TMAOH-NPs)
were uniform in size and as small as 6.970.9 nm. These
results were confirmed by transmission electron microscope
(TEM) visualizations (5.972.5 nm, n¼29; Figure 2a and
Table 1) and magnetic measurements (data not shown).
Nonetheless, TEM revealed that TMAOH-NPs could be found
as slightly electron-dense individual particles (Figure 2a) and
be arranged in clusters (Figure 2a) of variable sizes (up to
several hundreds of nanometers; Figure 2a and Table 1),
results that were confirmed by dynamic light scattering (DLS)
measurements (Figure 2c and Table 1), which allowed one to
(1) exclude artifacts in TEM specimens and (2) determine the
reversibility of these clusters. Consequently, the most
probable scenario is that particles may arrange in clusters,
which show an approximate relaxation time of around 50 ms,
as can be deduced from the non-diffusive peaks that appear
as ‘‘effective’’ large sizes (larger than 1 mm in Figure 2c) in the
transformed relaxation data (Blanco et al., 2000). Also, by
decreasing the pH12 of the dispersing medium, particle
clusters flocculated (Vidal Vidal J (2004). Bachelor Thesis
‘‘Preparacio´n de pinturas para apantallamento electromagne
´tico,’’ Faculty of Chemistry, University of Santiago de
Compostela), which indirectly confirmed that TMAOH ions
interact weakly with maghemite nanoparticle surfaces. Zetapotential
studies showed that the isoelectric point of
TMAOH-NPs was 6.3. Consequently, particles are respectively
negatively or positively charged when they are exposed
to a pH that is either greater or less than 6.3.
Dimensions of AOT-stabilized iron nanoparticles (AOTNPs)
were obtained by TEM and DLS measurements. TEM
showed that AOT-NP dispersions were rich in AOT and
contained highly electron-dense particles of different sizes
(Figure 2b), of which 51.1% of observed nanoparticles
(n¼43) had a diameter of 4.971.3nm (Table 1). DLS
a b c d
Figure 1. Nanoparticle dispersion macroscopic appearance. Two different
dilutions of (a, b) AOT-NP and (c, d) TMAOH-NP dispersions were
photographed with a commercial digital camera (Sony, DSC-W7) to show that
formulations are (a, d) rust-colored transparent liquids that on dilution may
modify (b; AOT-NP) or not (c; TMAOH-NP) their color. (a, d) For penetration
experiments, formulations were used without dilution.


measurements then revealed that the average size of larger
particles, which according to TEM calculation represent
4.6%, was 82.6763.9nm (Figure 2d and Table 1). Zetapotential
studies on AOT-NPs were not conducted because
these particles were an intermediate product. However,
considering that AOT is a sulfonate (a sodium salt of a
sulfonic acid) and that AOT-NPs were dispersed in water, it
can be reasonably deduced that particles might be surrounded
by a double shell of AOT molecules and that the
outermost layer of AOT provides a negatively charged surface
that stabilizes particle aqueous dispersion. In addition, TEM
results allow one to assume that AOT-NP dispersion is
composed of AOT-NPs and AOT-reversed micelles.
Both nanoparticles had superparamagnetic properties at
room temperature and could be classified as hydrophilic
nanoparticles (Lo´pez Pe´rez et al., 1997; Lo´pez-Quintela
et al., 1997). However, nanoparticle superparamagnetic
properties were not intentionally exploited to investigate
their penetration ability merely as a function of size and
superficial properties.
Possible routes of skin penetration
Nanoparticle dimensions are considered the most important
parameters because chemical penetration into the skin can
occur through pilosebaceous pores (diameter: 10–70 mm)
(Lauer et al., 1996), sweat gland pores (diameter: 60–80 mm)
(Roberts MS (2004). Skin structure and function. Proceedings
of the preconference course ‘‘Fundamentals of percutaneous
penetration’’. Perspective in percutaneous penetration, La
Grand Motte, 2004), and most commonly through the lipidic
matrix that fills a gap of 75 nm, in air-dried conditions
(Johnson et al., 1997), between the SC dead corneocytes,
cementing them. This lipidic matrix is composed of a mixture
of lipids (Elias, 2005) which arrange in a head–head tail–tail
configuration, thus forming a supramolecular structure of
parallel and repeating lipidic bilayers (Bouwstra and Honeywell-
Nguyen, 2002; Bouwstra et al., 2002, 2003). The
presence of (1) aqueous pores of ca. 2.871.3nm (Tang
et al., 2001), most likely located in the head–head regions
and (2) fluid lipophilic areas (42.37 nm), within the tail–tail
region that measures 12.8nm in total (long periodicity phase)
and comprises a central fluid area (2.37 nm) and two adjacent
areas (4.57nm each) whose fluidity gradually decreases by
moving away from both sides of the central area (Bouwstra
and Honeywell-Nguyen, 2002; Bouwstra et al., 2002, 2003),
has been demonstrated.

Figure 2. Nanoparticle characterization. Nanoparticle TEM micrographs are
shown in (a) and (b). A drop of each nanoparticle dispersion was deposited on
formvar-coated grids, air-dried, and observed using a TEM operating at
200 kV. (a) TMAOH-NPs and (b) AOT-NPs respectively appeared as
individual slightly (a; gray) or highly (b; dark) electron-dense dots depending
on particle density. (a) TMAOH-NP formulation did not appear
homogeneously dispersed, and particles that were found very close together
(cluster of particles) seemed to be larger and more electron dense. (b) Excess
of AOT in AOT-NP dispersion determined gray shadows in the micrograph
background. Bar¼200 nm. Transformed relaxation data of DLS
measurements of (c) TAMOH-NP and (d) AOT-NP aqueous dispersions
showed the dominant presence of (c) non-diffusive and (d) diffusive peaks,
which are associated with the presence of reversible (c) clusters of particles
and (d) individual particles whose hydrodynamic radius (RH) may vary from a
few nanometers to several nanometers. Refer to Table 1 for actual dimensions.



By comparing the dimensions of skin openings and
possible routes of entrance with nanoparticle sizes, it was
hypothesized that individual nanoparticles might be small
enough to potentially penetrate the skin. However, the extent
of penetration, as well as the route of entrance, was
considered to depend strongly on all the interactions that
could occur between nanoparticles and skin components
and/or structures.
Also, a lipophilic–hydrophilic gradient and a pH gradient
(Wagner et al., 2003; Elias, 2005) exist in the skin. Although
the lipophilic–hydrophilic gradient is easily identified to be
localized between SC (lipophilic) and viable epidermis
(hydrophilic), little has been written about the pH gradient
of the skin in research articles on skin absorption. However,
we would like to emphasize that the isoelectric point of the
skin has been known since early in the 20th century. It is now
recognized to be between 3.5 and 4.8 (Wilkerson, 1935;
Higaki et al., 2003), which means that skin is negatively
charged under physiological conditions. In addition, the pH
of the skin surface varies with gender, anatomical sites, and
experimental setting. Wagner et al. (2003) have found
that superficial skin pH ranges between 4.7 and 5.5 in vivo,
and between 5.8 and 6.0 in vitro (frozen skin). They have
also demonstrated that the pH of the buffer in contact with
the dermis (in the receiving chamber) may further influence
the pH gradient across SC, viable epidermis, and dermis
(Wagner et al., 2003). Using a neutral buffer (pH 7.4) in
the receiving chamber and frozen skin, Wagner et al. (2003)
found the following values of acidity after 3 hours of
buffer incubation: upper SC: pH 6–6.3; deeper SC and
outermost viable epidermis: pH 6.5; and deeper viable
epidermis: pH 7–7.3.
Consequently, this background may lead one to hypothesize
that penetration of nanoparticles, whose dimensions
are compatible with skin absorption routes, might be further
influenced by their superficial charges.
To verify our hypothesis, we performed some studies of
skin penetration.
Penetration experiment set-up
To avoid skin variability among species (Bartek et al., 1972;
Feldman and Maibach, 1974), penetration experiments were
conducted ex vivo using full-thickness skin pieces of healthy
human female donors who did not have a dermatological
disease history. The age of donors did not appear to influence
skin barrier properties, because specific skin electrical
resistivities calculated before starting the penetration experiments
(see below) were in accordance with normal skin
values (12–120 kOcm2, direct current) (Inada et al., 1994)
and of the order of 73.72723.67 kOcm2 when values were
extrapolated from current measures at 10 Hz (n¼80). This
frequency was chosen to minimize inductive and capacitive
contributions of viable epidermis on skin resistivity values,
whose modifications, if any, could then be considered as the
reflection of SC barrier alteration (Burnette and DeNuzzio,
1997; Martinsen et al., 1999). In addition, skin electrical
resistivity was monitored at 1 kHz because standard deviation
decreases at this frequency (Rosell et al., 1988) and
differences among groups can be better evaluated. Nevertheless,
if a major alteration occurs in the SC barrier, it should
normally be visible at both frequencies.
As initial resistivity measurements confirmed the presence
of an adequate SC barrier, we proceeded with the experiments,
which were conducted in static conditions to avoid
potential penetration enhancements due to the magnetic field
that is generated during electro-magnetic stirring, and which
would have activated nanoparticle magnetic properties.
Once skin pieces were clamped on vertical cells, they were
equilibrated with phosphate-buffered saline (PBS) for 1 hour
(hydration phase) before exposure to nanoparticle dispersions
for 3, 6, 12, or 24 hours.
Nanoparticle formulation may alter skin barrier properties
As a first result, penetration experiments showed that the
dispersing medium of a nanoparticle formulation might be
responsible for altering, to different extents, the skin barrier
properties. In each experiment, we compared the resistivity of
skin pieces exposed to PBS, a particular nanoparticle
formulation, and its blank solution (obtained by centrifuging
an aliquot of the formulation under examination). Results
showed that both nanoparticle dispersions and their blank
solutions were able to diminish skin barrier. However, these
modifications should be considered minor, because decreases
in skin resistivity measurements could be generally
noticed only at 1 kHz.
In particular, the TMAOH-NP dispersion caused a decrease
in skin resistivity of 2 kOcm2 (1 kHz; Po0.05) after 12 hours of
skin contact. This barrier perturbation is very mild when
compared with the abrupt decrease (ca. 6 kOcm2 at 1 kHz and
ca. 44 kOcm2 at 10 Hz; in both cases Po0.05) in skin
resistivity caused by a basic TMAOH control solution, which
was visible at both frequencies already after 3 hours. This result
was not expected because either TMAOH-NP dispersion or
TMAOH control solution should have been at pH 12. It was
then discovered that the slow adsorption of CO2 decreased the
basicity of TMAOH-NP dispersion down to pH 7 without
flocculating particle clusters, which in turn explained the
observed discrepancy in skin resistivity. It has been hypothesized
that CO2 adsorption might have sequestrated hydroxyl
ion excess in TMAOH-NP dispersion, limiting its destructive
action on epithelia (Sigma-Aldrich website. MSDS data sheet of
TMAOH at
http://www.sigmaaldrich.com/cgibin/hsrun/Suite7/
Suite/HAHTpage/Suite.HsSigmaAdvancedSearch.formAction,
July 2006). In fact, using another batch of TMAOH-NPs that
was instead basic, no differences in skin resistivity were
observed in skin specimens exposed to nanoparticle dispersion
or its blank solution at both frequencies. Nevertheless, the
TMAOH-NP formulation used throughout this study was the
neutral one.
AOT-NP dispersion and its blank solution produced a
significant decrease in skin resistivity (ca. 5 kOcm2 at 1 kHz
and ca. 60 kOcm2 at 10 Hz) after 24 hours when compared
with PBS. However, during the experiment, the decrease in
skin resistivity was only visible at 1 kHz and after 3 hours
(4 kOcm2; Po0.05). No differences were observed between
AOT-NP dispersions and its blank within 24 hours.

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