Professional Documents
Culture Documents
INTRODUCTION
Nanotechnology involves the design, production, characterization, and applications of materials (molecules or devices)
whose dimensions are less than 100 nm. It has been shown
that at nanometric scale, materials acquire new properties
that can be exploited in numerous fields, including biotechnology, bioengineering, nanotechnology, and nanomedicine
(Website of the Royal Society and Royal Academy of
Engineering on Nanotechnology and Nanoscience, Final
Report at www.nanotech.org.uk/index.htm, August 2006).
Such materials are generally called nanomaterials. They can
be categorized as nanotubes, nanowires, nanoshells, nano1
particles, quantum dots, dendrimers, and biopolymers (Website of the Royal Society and Royal Academy of Engineering
on Nanotechnology and Nanoscience, Final Report at
www.nanotech.org.uk/index.htm, August 2006). Among
these, nanoparticles could play an important role in
nanomedicine.
With regard to nanoparticles, rapid advances in nanotechnology have made it possible to synthesize different
types of metallic and/or magnetic particles whose diameter is
of the order of a few nanometers and even less. In addition,
their surfaces can be modified by bioactive molecules
or imaging probes that can be adsorbed, coated, conjugated,
or linked to them. Owing to the wide applicability of
such modified systems, they have been proposed for (i) cell
labeling and targeting, (ii) tissue engineering, (iii) drug
delivery, drug targeting, and magnetic drug targeting, (iv)
magnetic resonance imaging, (v) hyperthermia, (vi) magnetofection, and (vii) analysis of biomolecules, to cite just a
few (Penn et al., 2003; Gupta and Gupta, 2005; Neuberger
et al., 2005).
Many of these applications can also be tailored to target
skin. For instance, cell labeling/targeting may help in the
early diagnosis of a skin disease, which could also be treated
with the goal of nanocarriers for drug delivery or targeting,
hyperthermia, or magnetofection. In addition, a tissue
engineering approach could be useful for skin wound healing
therapies. Furthermore, the possibility of exploiting the
magnetic properties of these particles might help in directing
and localizing these agents in a particular layer of the skin
where their action is desired.
www.jidonline.org 1701
B Baroli et al.
Skin Penetration of Metallic Nanoparticles
B Baroli et al.
Skin Penetration of Metallic Nanoparticles
200 nm
200 nm
1.0
Amplitude
0.8
0.6
0.4
0.2
= 45
= 45
= 45
= 45
= 60
= 60
= 60
= 60
= 90
= 90
= 90
= 120
= 120
= 120
= 150
= 150
= 150
1.0
0.8
Amplitude
0.6
0.4
0.2
0.0
0.0
= 90
= 90
= 90
= 90
= 90
= 60
= 60
= 60
= 45
= 45
= 45
= 120
= 120
= 120
= 150
= 150
= 150
R H (nm)
R H (nm)
Formulation
Name
Core
Coating
Vehicle
TMAOH-NP
g-Maghemite
TMAOH
H2O+TMAOH
TEM
DLS
5.972.5
48.6743.52
1238.22
13326.82
AOT-NP
Iron
AOT
H2O+AOT
4.971.3
4.870.2
12.871.5
9.672.3
23.373.6
82.6763.9
AOT, sodium bis(2-ethylhexyl) sulfosuccinate; AOT-NP, AOT-stabilized iron nanoparticle; DLS, dynamic light scattering; TEM, transmission electron
microscope; TMAOH, tetramethylammonium hydroxide; TMAOH-NP, TMAOH-stabilized maghemite nanoparticle.
Dimensions are given as mean7SD.
2
Dimensions were extrapolated from non-diffusive peaks.
1
www.jidonline.org 1703
B Baroli et al.
Skin Penetration of Metallic Nanoparticles
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
B Baroli et al.
Skin Penetration of Metallic Nanoparticles
results suggest that the surfactant properties of AOT (SigmaAldrich website. MSDS data sheet of the product AOT at http://
www.sigmaaldrich.com/cgibin/hsrun/Suite7/Suite/HAHTpage/
Suite.HsSigmaAdvancedSearch.formAction, July 2006) (Zatz
and Lee, 1997) might be responsible for the decreased SC
barrier.
Finally, the skin resistivity of pieces exposed to PBS
remained similar to the initial values (before and after the
hydration phase) at both frequencies for 24 hours.
Skin morphology remained macroscopically similar during
penetration studies
Light transmission microscope (LTM) visualization on toluidine blue-stained (Figure 3af ) 2-mm-thick sections showed
that each skin piece used for the penetration studies
maintained good morphology throughout the experiments,
as assessed by the constant presence of 1015 SC layers
(12.775.6 mm), a compact viable epidermis (58.8719.9 mm),
and a collagen- and muscle-filled dermis (Figure 3af).
No macroscopic modification of skin architecture was
observed in PBS-exposed specimens, indicating that the
protocols used from explant to fixation did not damage
the skin, at least macroscopically (Figure 3a). No major
differences were observed in the skin samples treated with
different nanoparticle formulations or their blank solutions
(Figure 3af).
It was deduced that skin resistivity results were the direct
consequence of nanoparticle formulation exposure. In addition, considering that a decrease in skin resistivity is
www.jidonline.org 1705
B Baroli et al.
Skin Penetration of Metallic Nanoparticles
SG
c
SC
VE
emphasized that skin sections were observed with a microscope operating at 80100 kV and generally at a magnification
of 29,400 (Figure 6a and b), whereas nanoparticles were
examined with another microscope operating at 200 kV and at
magnifications up to 200,000 (Figure 2a and b). In addition,
most of the time, individual nanoparticles were less electrondense at magnifications less than 200,000. Consequently,
the discrimination between nanoparticles and structural
components of skin was extremely difficult in a gray-scale
background (Figure 6a and b). In fact, besides larger deposits
between corneocytes of desquamating SC (Figure 6a), which
are most likely made up of nanoparticles because they have
never been found in specimens used as controls (data not
shown), asserting that these electron-dense longer threadshaped structures (Figure 6b), which were found close to
the stratum granulosum, are degrading corneodesmosomes
or natural components of intercorneocyte space instead
of nanoparticle deposits, would be questionable in both
cases.
Further proof of nanoparticle penetration was obtained with an
energy dispersion spectrometry-scanning electron microscope
B Baroli et al.
Skin Penetration of Metallic Nanoparticles
Fe
3,000
Counts/second
041080 20.0 kV
150
2,500
2,000
1,500
1,000
Fe
500
200 m
10
10
keV
d
Counts/second
041089 20.0 kV
900
3,000
2,500
Fe
2,000
1,500
1,000
Fe
500
33.3 m
keV
Figure 8. Backscattered electron images of skin specimens showing iron
deposits in the SC. This figure shows (a, c) two skin specimens whose SC
(above the fracture in panels a and c) is filled with roundish aggregates of
high-density material, which are made up of iron (b and d; presence of Fe Ka1
signal, Fe highest peak). In particular, specimen (a) was treated with AOT-NPs
for 12 hours (conditions: BSE, carbon coating 10 nm, 20 kV, bar 200 mm) as
well as specimen (c) (conditions: BSE, carbon coating 10 nm, 20 kV,
bar 33.3 mm). EDS graphs (b) and (d) are the chemical analysis of specimens
(a) and (c), respectively. For details on sample preparation and data
acquisition, refer to the Materials and Methods section.
042022 20.0 kV
1.10 K
1,200
Counts/second
1,000
27.3 m
Fe
S
800
600
400
Si
Na
Fe
200
0
10
10
041460 20.0 kV
044622
20.0 kV
400
75.0 m
1.50 K
20.0 m
1,400
Counts/second
keV
1,200
Fe
1,000
800
600
Cl
S
400
Fe
200
0
keV
www.jidonline.org 1707
B Baroli et al.
Skin Penetration of Metallic Nanoparticles
1,200
Fe
Counts/second
1,000
800
600
400
Fe
200
041116 20.0 kV
600
50.0 m
10
130
041069 20.0 kV
231 m
041072 20.0 kV
900
33.3 m
keV
Counts/second
041093 20.0 kV
350
85.7 m
c
3,000
3,000
Counts/second
2,500
Fe
2,000
1,500
1,000
500
0
2,000
Fe
1,500
1,000
500
Fe
2
2,500
10
keV
Fe
2
10
keV
DISCUSSION
The results of this study showed that rigid nanoparticles
smaller than 10 nm, whose aqueous dispersions are formed
by individual particles and their reversible clusters (ranging
up to several micrometers; Table 1), are able to passively
penetrate the skin through the SC lipidic matrix and hair
follicle orifices, reaching the deepest layers of the SC, the
stratum granulosum, and hair follicles. In some exceptional
cases, nanoparticles were also found in the viable epidermis.
These results are important because, to our knowledge, it is
the first time that such findings have been reported.
1708 Journal of Investigative Dermatology (2007), Volume 127
B Baroli et al.
Skin Penetration of Metallic Nanoparticles
B Baroli et al.
Skin Penetration of Metallic Nanoparticles
DLS measurements
For both nanoparticles, DLS measurements were performed using an
ALV SP-86 goniometer, an ALV 5000 Multi-tau correlator, and a
Coherent DPSS 532 laser operating at a wavelength of 532 nm and a
power of 100 mW. The correlation functions were accumulated for
2 minutes and analyzed using the ALV-5000 WIN software based on
the CONTIN algorithm adapted to the specific correlator noise.
Temperature was fixed at 201C. The logarithmically sampled
relaxation time spectra (amplitude vs log(t)), which were the result
of the CONTIN inversion of the normalized correlation functions,
were transformed using the StokesEinstein relation, the electrolyte
solvent viscosity Z0, and the refractive index n at the actual
temperature T to yield the hydrodynamic radius RH (kTq2t)/6pZ0,
where k is the Boltzmann constant and q (4pn/l)sin(y/2) is the
scattering vector as a function of wavelength in vacuum, l, and
scattering angle y. Measurements were performed at angles of 45,
60, 90, 120, and 1501.
Skin preparation
Full-thickness human skin was obtained from female donors
undergoing surgical reduction of the abdomen. Anesthesia was
induced by Diprivan and maintained with a mixture of Sevorane
(sevoflurane) and Remifentanil. Excided skin was then cleaned of fats
using a surgical scissors and stored at 231C until use. Experimental
protocols and skin donation were approved by the ethical committee
of Azienda Ospedaliera G. Brotzu (Cagliari, IT; protocols no. 133
and no. 135, respectively issued on 22 December 2003 and 26
January 2004). Nevertheless, patient consensus remained obligatory
for specimen donation. Experiments were conducted according to
the Declaration of Helsinki Principles (http://www.wma.net).
Penetration experiments
Nanoparticle penetration experiments (n: 3, 4) were carried out
using handmade vertical diffusion cells (Rofarma Italia S.r.l., IT;
diameter: 9.0570.46 mm, area: 0.6470.03 cm2), which were
1710 Journal of Investigative Dermatology (2007), Volume 127
Nanoparticle quantification
Nanoparticle concentration in receptor suspensions was quantified
using an ICP-optical emission spectrometer (VISTA-MPX, Varian
Inc., Palo Alto, CA) spectrometer. Before analysis, samples were
diluted with a 1% HNO3 (Fluka; cat. no. 84385) solution.
Statistical analysis
Data subjected to statistical analysis were evaluated by the unpaired
two-population Students t-test, where Pp0.05 was considered to be
significant.
B Baroli et al.
Skin Penetration of Metallic Nanoparticles
ACKNOWLEDGMENTS
We thank Professor P. Cabras (UniCa, IT) for kindly allowing free access to the
ICP, and S. Bernardini and S. Collu (UniCa, IT) for histological specimen
preparation assistance. This research was partially funded by UniCa (Local
Research Program), MIUR (Research Program of National Interest: protocol
no. 2001037727_003), and the Spanish Ministry of Science and Technology
(project no. MAT2002-00824). This work was performed in Cagliari, Sardinia,
Italy; particles were produced and characterized in Santiago de Compostela,
Galicia, Spain.
Goldstein JI, Newbury DE, Echlin P, Joy DC, Fiori C, Lifshin E (1981) Scanning
electron microscopy and X-ray microanalysis. New York: Plenum Press,
53121
Gupta AK, Gupta M (2005) Synthesis and surface engineering of iron oxide
nanoparticles for biomedical applications. Biomaterials 26:39954021
Guy RH (1996) Current status and future prospects of transdermal drug
delivery. Pharm Res 13:17659
Hadgraft J (2001) Skin, the final frontier. Int J Pharm 224:118
Higaki K, Amnuaikit C, Kimura T (2003) Strategies for overcoming the stratum
corneum: chemical and physical approaches. Am J Drug Del 1:187214
Hoet PHM, Bruske-Hohlfield I, Salata OV (2004) Nanoparticles known and
unknown health risks. J Nanobiotechnol 2:12
Holsapple MP, Farland WH, Landry TD, Monteiro-Riviere NA, Carter JM,
Walker NJ et al. (2005) Research strategies for safety evaluation of
nanomaterials, part II: toxicological and safety evaluation of nanomaterials, current challenges and data needs. Toxicol Sci 88:127
Holsapple MP, Lehman-McKeeman LD (2005) Forum series: research
strategies for safety evaluation of nanomaterials. Toxicol Sci 87:315
Honeywell-Nguyen PL, Gooris GS, Bouwstra JA (2004) Quantitative
assessment of the transport of elastic and rigid vesicle components and
a model drug from these vesicle formulations into human skin in vivo.
J Invest Dermatol 123:90210
Inada H, Ghanem AH, Higuchi WI (1994) Studies on the effects of applied
voltage and duration on human epidermal membrane alteration/recovery
and the resultant effects upon iontophoresis. Pharm Res 11:68797
Johnson ME, Blankschtein D, Langer R (1997) Evaluation of solute permeation
through the stratum corneum: lateral bilayer diffusion as the primary
transport mechanism. J Pharm Sci 86:116272
Kalia YN, Guy RH (1995) The electrical characteristics of human skin in vivo.
Pharm Res 12:160513
Kierszenbaum AL (2002) Histology and cell biology. St Loius: Mosby Inc., 305
REFERENCES
Al-Amoudi A, Dubochet J, Norlen L (2005) Nanostructure of the epidermal
extracellular space as observed by cryo-electron microscopy of vitreous
sections of human skin. J Invest Dermatol 124:76477
Alvarez-Roman R, Naik A, Kalia YN, Guy RH, Fessi H (2004) Skin penetration
and distribution of polymeric nanoparticles. J Control Release 99:
5362
Barry BW (2001) Novel mechanisms and devices to enable successful
transdermal drug delivery. Eur J Pharm Sci 14:10114
Bartek MJ, LaBudde JA, Maibach HI (1972) Skin permeability in vivo:
comparison in rat, rabbit, pig and man. J Invest Dermatol 58:11423
Blanco MC, Leisner D, Vazquez C, Lopez-Quintela MA (2000) Dynamic light
scattering in transient reversible gels. Langmuir 16:858594
Bouwstra JA, Gooris GS, Dubbelaar FER, Ponec M (2002) Phase behavior of
stratum corneum lipid mixtures based on human ceramides: the role of
natural and synthetic ceramide 1. J Invest Dermatol 118:60617
Bouwstra JA, Honeywell-Nguyen PL (2002) Skin structure and mode of action
of vesicles. Adv Drug Deliv Rev 54(S1):S4155
Bouwstra JA, Honeywell-Nguyen PL, Gooris GS, Ponec M (2003) Structure of
the skin barrier and its modulation by vesicular formulations. Prog Lipid
Res 42:136
Burnette RD, DeNuzzio JD (1997) Impedance spectroscopy: application to
human skin. In: Mechanism of transdermal drug delivery (Potts RO, Guy
RH, eds), New York: Marcel Dekker Inc., 21530
Cevc G (2004) Lipid vesicles and other colloids as drug carriers on the skin.
Adv Drug Deliv Rev 56:675711
Elias PM (2005) Stratum corneum defensive functions: an integrated view.
J Invest Dermatol 125:183200
Feldman RJ, Maibach HI (1974) Percutaneous penetration of some pesticides
and herbicides in man. Toxicol Appl Pharmacol 28:12632
Golden GM, Guzek DB, Kennedy AH, McKie JE, Potts RO (1987) Stratum
corneum lipid phase transitions and water barrier properties. Biochemistry 26:23828
Kontturi K, Murtomaki L, Hirvonen J, Paronen P, Urtii A (1993) Electrochemical characterization of human skin by impedance spectroscopy:
the effect of penetration enhancers. Pharm Res 10:3815
Langer R (2004) Transdermal drug delivery: past progress, current status, and
future prospects. Adv Drug Deliv Rev 56:5578
Lauer AC, Ramachandran C, Lieb LM, Niemiec S, Weiner ND (1996)
Targeted delivery to the pilosebaceous unit via liposomes. Adv Drug
Deliv Rev 18:31124
Lopez Quintela MA, Rivas J (1993) Chemical reactions in microemulsions: a
powerful method to obtain ultrafine particles. J Colloid Interface Sci
158:44651
Lopez Perez JA, Lopez-Quintela MA, Rivas J, Mira J, Charles SW (1997)
Advances in the preparation of magnetic nanoparticles by the microemulsion method. J Phys Chem B 101:80457
Lopez-Quintela MA, Quiben J, Rivas J (1997) Use of microemulsions in the
production of nanostructured materials. In: Industrial applications of
microemulsions (Solans C, Kunieda H, eds), New York: Marcel Dekker
Inc., 24765
Martinsen G, Grimnes S, Haug E (1999) Measuring depth depends on
frequency in electrical skin impedance measurements. Skin Res Technol
5:17981
Meidan VM, Bonner MC, Michniak BB (2005) Transfollicular drug delivery
is it a reality? Int J Pharm 306:114
Moranti P, Rocco E, Wolf R, Rocco V (2001) Percutaneous absorption and
delivery systems. Clin Dermatol 19:489501
Neuberger T, Schopf B, Hofmann H, Hofmann M, von Rechenberg B (2005)
Superparamagnetic nanoparticles for biomedical applications: possibilities and limitations of a new drug delivery system. J Magn Magn Mat
293:48396
Oberdorster G, Oberdorster E, Oberdorster J (2005) Nanotoxicology: an
emerging discipline evolving from studies of ultrafine particles. Environ
Health Perspect 113:82339
Penn SG, He L, Natan MJ (2003) Nanoparticles for bioanalysis. Curr Opin
Chem Biol 7:60915
www.jidonline.org 1711
B Baroli et al.
Skin Penetration of Metallic Nanoparticles
Tsuji JS, Maynard AD, Howard PC, James JT, Lam C, Warheit DB et al. (2006)
Research strategies for safety evaluation of nanomaterials, Part IV: risk
assessment of nanoparticles. Toxicol Sci 89:4250
van den Bergh BA, Bouwstra JA, Junginger HE, Wertz PW (1999a) Elasticity of
vesicles affects hairless mouse skin structure and permeability. J Control
Release 62:36779
van den Bergh BA, Vroom J, Gerritsen H, Junginger HE, Bouwstra JA (1999b)
Interactions of elastic and rigid vesicles with human skin in vitro:
electron microscopy and two-photon excitation microscopy. Biochim
Biophys Acta 1461:15573
Tinkle SS, Antonini JM, Rich BA, Roberts JR, Salmen R, DePree K et al. (2003)
Skin as a route of exposure and sensitization in chronic beryllium
disease. Environ Health Perspect 111:12028