Chemical enhancer induced changes in the mechanisms of transdermal
delivery of zinc oxide nanoparticles
Tsung-Rong Kuoa, Chung-Long Wua, Chih-Ting Hsub, Wen Lob, Shu-Jen Chiangc, Sung-Jan Lind,e,
Chen-Yuan Dongb,*, Chia-Chun Chena,c,**
aDepartment of Chemistry, National Taiwan Normal University, Taipei 116, Taiwan
bDepartment of Physics, National Taiwan University, Taipei 106, Taiwan
cInstitute of Atomic and Molecular Sciences, Academia Sinica, Taipei 106, Taiwan
dInstitute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei 106, Taiwan
eDepartment of Dermatology, National Taiwan University Hospital and College of Medicine, Taipei 106, Taiwan
a r t i c l e i n f o
Received 22 November 2008
Accepted 2 February 2009
Available online 20 February 2009
Second harmonic generation
a b s t r a c t
The overlapping wavelength of photoluminescence (PL) of zinc oxide nanoparticles (ZnO NPs) and
autofluorescence (AF) from the stratum corneum (SC) has for a long time held back researchers from
investigating the chemically enhanced penetration pathways of ZnO NPs into the SC lipids. However, the
non-linear polarization effect of second harmonic generation (SHG) may be used for ZnO NPs to be
distinguished from the AF of the SC. This study combined the SHG of ZnO NPs and the AF of the SC to
image the transdermal delivery of ZnO NPs under the chemical enhancer conditions of oleic acid (OA),
ethanol (EtOH) and oleic acid–ethanol (OA–EtOH). In addition to qualitative imaging, the microtransport
properties of ZnO NPs were quantified to give the enhancements of the vehicle-to-skin partition coef-
ficient (K), the SHG intensity gradient (G) and the effective diffusion path length (L). The results showed
that OA, EtOH and OA–EtOH were all capable of enhancing the transdermal delivery of ZnO NPs by
increasing the intercellular lipid fluidity or extracting lipids from the SC.
? 2009 Elsevier Ltd. All rights reserved.
At the present, one of the fastest growing scientific fields is that
of nanoscale discovery and application. Of particular concern are
the uses of nanoparticles (NPs) in applications ranging from tar-
geted fluorescent labels in the life sciences , bacterial inhibitors
, and ultraviolet radiation (UVR) protective cosmetics .
Specifically, inorganic ZnO NPs are popular active substances used
in UVR protective substances due to their broad spectral absorption
in the UVA range . Overexposure of human skin to UVR may lead
to sunburn and an increased risk of skin cancer [5–7]. To reduce
skin damages caused by UVR, many health care practitioners have
advocated UVR protective ingredients for a wide variety of
dermatological products [8,9]. Application of UVR protective
ingredients on top of the skin is indicated to penetrate ZnO NPs into
the SC of the skin. Based on inherent barrier properties, the SC
inhibits transdermal delivery of hydrophilic and large molecular
weight molecules [10,11]. The plasma membrane of the cell is
mainly composed of various types of phospholipids and membrane
proteins. The membrane proteins transport hydrophilic and larger
uncharged polar molecules (such as glucose and ions) across
membrane. However, organic compounds such as OA and EtOH are
chemical enhancers that can alter the packing structure of the SC
lipids to increase lipid fluidity and reduce skin resistance [12,13]. A
better understanding of the transdermal delivery of ZnO NPs via
chemically enhanced pathways may optimize the applications and
address biocompatibility issues of NPs in the SC.
Recently, multi-photon microscopy has been used as a non-
invasive method for the direct visualization of skin structures
[14,15]. In multi-photonimaging, fluorophore excitation is achieved
by the non-linear absorption process [16,17]. Specifically, in two-
photon microscopy, two near-infrared (near-IR) photons of
approximately half the one-photon excitation energy are simulta-
neously absorbed by the fluorescent molecule [18,19]. This
approach can be used to excite intrinsic or extrinsic fluorophores,
which are generally excited by ultraviolet (UV) or visible (VIS) light
. The near-IR radiation allows deeper penetration into the
* Corresponding author.
** Corresponding author. National Taiwan Normal University, Department of
Chemistry, 88 Sec. 4, Ting Chow Road, Taipei 116 Taiwan, Taipei 116, Taiwan. Fax:
þ886 2 8931 6363.
E-mail addresses: firstname.lastname@example.org (C.-Y. Dong), email@example.com
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/biomaterials
0142-9612/$ – see front matter ? 2009 Elsevier Ltd. All rights reserved.
Biomaterials 30 (2009) 3002–3008
imaged tissues and the point-like excitation volume greatly can
reduce overall specimenphotodamage . For skin imaging, down
to tissue depths of 100–200 mm, the AF of human skin can be
detected [22,23]. For transdermal delivery studies, two-photon
microscopy has provided three-dimensional (3-D) images detailing
the chemical enhancer- and ultrasound-induced changes in the
distributions of fluorescent probes across human skin [24–26].
Despite of the advances in the study of transdermal transport of
molecular dyes, the fluorescence of ZnO NPs and SC were almost
overlapped that held back researchers from investigating the trans-
dermal delivery of ZnO NPs with or withoutenhancers. To tackle this
issue, the current study examined the non-linear polarization effect
of SHG for ZnO NPs, based on its unique polarizable property as well
as non-centrosymmetric structure [27,28]. The generated second-
harmonic signal at twice the frequency (half the wavelength) of the
excitation sourcewas observed in ZnO NPs. Afterconfirmation of the
SC with no SHG, the combination of the SHG of ZnO NPs and AFof SC
was used to image and characterize the penetration pathway and
depth of ZnO NPs into nude mouse skin under three chemical
enhancer conditions: OA, EtOH, and OA–EtOH. In addition to quali-
tative imaging, the microtransport properties of the ZnO NPs,
including the K, G and L, were quantified relative to the control
sample (without chemical enhancers).
2. Materials and methods
2.1. Preparation of ZnO nanoparticles
ZnO NPs were prepared by adding 5.280 g of zinc acetate dehydrate (reagent
grade, Sigma–Aldrich) in 250 mL EtOH (99.5%, Showa) and the solution was heated
until it become clear . After the solution was refluxed for 1 h at 78?C,
approximate 60% of the solvent was removed by distillation and replaced by the
same amount of fresh EtOH. The solution was then placed in ice bath at 0?C and
1.392 g of lithium hydroxide (98þ%, Sigma–Aldrich) was added under ultra-
sonication until a transparent solution was obtained. After 60 min of reaction time,
the solution consisting of small ZnO NPs with around 2 nm diameter was filtered
through a 0.1 mm glass fiber filter to remove undissolved LiOH.
To avoid particle aggregation, ZnO NPs with a diameterof approximate 10 nmwas
achievedbycontinuousgrowthof2 nmZnONPs.Thesolutioncontaining2 nmparticles
was heated and mixed with 10 mL of deionized water at 60?C for about 6 min. During
the cooling process, white precipitate was formed within the solution. The precipitate
was separated from the supernatant solution by centrifugation at 3800 rpm for about
10 min. Next, the precipitatewaswashed with an EtOH-deionized watersolution(19:1
the same diameter of approximate 10 nm, and are spherical in shape. The absorption
and emission peaks of ZnO NPs are located at 370 nm and 525 nm, respectively.
2.2. Donor solution preparation
The donor vehicle solutions of ZnO NPs were consisted of EtOH, OA, and PBS
(99%, Sigma). EtOH, OA, PBS and ZnO NPs with different weight ratio were prepared
Fig.1. (a) UV–vis absorption and room-temperature PL spectra of ZnO NPs. Dual-channel two-photon AF (b) and SHG (c) images of ZnO NPs. Each image is 110 ?110 mm2. (b) and (c)
The two-photon AF (green) and SHG (blue) of ZnO NPs, respectively. (d) The TEM image of ZnO NPs with spherical shape.
T.-R. Kuo et al. / Biomaterials 30 (2009) 3002–3008 3003
for the chemical enhancer experiments. EtOH and OA were used as the chemical
enhancer in comparison with control solution of PBS. In the control donor solution,
10% (w/w) of ZnO NPs was added to the PBS solution. The OA donor solution was
prepared by adding 10% of ZnO NPs and 5% of OA (w/w) in PBS solution. The EtOH
donor solution is consisted of 10% of ZnO NPs (w/w) and 45% of ethanol (w/w) in PBS
solution. Finally, the OA–EtOH donor solution is composed of 10% of ZnO NPs, 5% of
OA and 45% of EtOH (w/w) in PBS solution.
2.3. Preparation of the skin samples of nude mice for multi-photon
dorsal region of four 10-month-old nude mice (BALB/cAnN.Cg-Foxnlnu/CrlNarl),
purchased from Laboratory Animal Center, NLAC. After the subcutaneous fat of the
skinwasremovedusing a knife,the skin specimenswere thensectioned and mounted
filled with PBS and the donor chambers were filled with 400 mL of donor solution.
After 12 h, the skin samples were removed from Franz diffusion cells and excess
formulationonthe skin surfacewas washed 3–4 times withPBS.The circularregion of
the skin exposed to the donor solutionwas removed using a knife and then placed on
a microscope slide. The skin specimen was then sealed with a glass coverslip for
viewing. Note that the conditions of steady state of ZnO NPs diffusion were verified in
calculating the parameters of ZnO NPs penetration. The SHG intensity profiles were
measured for the diffusion times of 10 and 12 h.It was found thatthe gradientprofiles
of SHG intensity of ZnO NPs were similar under the same donor solution condition.
In this study, the diffusion time of 12 h was selected for the experiment.
2.4. Dual-channel multi-photon microscopy
A home-built two-photon microscope based on a commercial upright
microscope (E800; Nikon, Japan) was used in this study. The output of
a titanium-sapphire pulse laser (Tsunami TM, Spectra Physics, Mountain View,
CA) pumped by a diode-pumped, solid state laser (Millennia TM X, Spectra
Physics, Mountain View, CA) operating at the wavelength of 780 nm was used as
the excitation source. Upon reflection by the short-pass, main dichroic mirror
(700dcspruv-3p, Chroma Technology, Rockingham, VT), circularly polarized laser
source was then focused by an oil-immersion objective (S Fluor, NA 1.3; Nikon,
Melville, NY). After the signals were generated, the broadband AF and SHG were
separated by a secondary dichroic mirror (435 dcxr, Chroma Technology) and
filtered respectively by two band-pass filters (MF: E435lp-700sp, SHG: HQ390/
20,Chroma Technology) forthe respective
(435–700 nm) and SHG signals (380–400 nm). Single-photon counting photo-
multiplier tubes (R7400P, Hamamatsu, Japan) were used in this system. Each
optical scan was composed of 512 ?512 pixels. For large-area scans of the skin
specimens, a two-dimensional stage scanning system (H101; Prior Scientific
Instruments, Cambridge, UK) was used for specimen translation, after each x–y
scan of the sample.
detection ofbroadband AF
2.5. Data collection and analysis
ImageJ software (National Institutes of Health, Bethesda, MD) was utilized to
process images. For each skin sample, four sites were examined and each of the sites
was 330 ?330 mm2in size. The z¼0 mm position was defined by the imaging depth
where the highest average SHG count was detected. At each depth, ImageJ averages
the SHG counts associated with the pixels in the 512 by 512 pixel image of the
imaged field. For each site analyzed by ImageJ, a plot of the average SHG count
versus the corresponding skin depth was used to generate the site-specific average
SHG spatial distribution profile (SSDP).
The values for EK, EGand EL, were obtained following previously published
methods [22–24]. The EKvalue was evaluated from the average SHG intensity on the
surface of each of the four sites of sample skins. Assuming that the SHG intensity of
the ZnO NPs permeant, I, is directly proportional to its concentration in the skin, EK
Fig. 2. Two-dimensional (2-D) dual-channel two-photon AF and SHG images of ZnO NPs in nude mouse skin samples under the four chemical enhancer conditions. Each image is
330?330 mm2. The ZnO NPs SHG (blue) and the SC AF (green) were merged at the same imaging depth. The locations of representative corneocytes in the SC were marked with
a yellow arrow.
T.-R. Kuo et al. / Biomaterials 30 (2009) 3002–3008 3004
can be approximated from the average SHG intensity profiles using the following
EK ¼IEnhancerðz ¼ 0Þ
IControlðz ¼ 0Þ
where IEnhancer(z¼0) is the average value of the SHG intensity of ZnO NPs on the
surface of the chemically enhanced skin samples and IControl(z¼0) is the average
value of the SHG intensity of ZnO NPs on the surface of the control (PBS) skin
The SHG intensity gradient, G, in the SC was evaluated based on the average
intensity values and calculated by performing a linear regression for the data points
comprising the first 11 axial scans of the skin. In calculating the enhancement in the
ZnO NPs permeant intensity gradient, EG, the following equation was used:
where (dI/dz)Enhancerand (dI/dz)Controlare the ZnO NPs permeant intensity gradients
corresponding to the intensity profiles of the enhanced skin samples and that of the
control skin samples, respectively.
The enhancement in the effective diffusion path length, EL, for enhanced skin
samples relative to the control skin samples was evaluated from the values of EKand
where the values of EKand EGwere evaluated from the average SHG intensity
profiles using the two-photon microscopy.
3. Results and discussion
3.1. Optical properties, dual-channel two-photon and electron
microscopic images of ZnO NPs
The optical properties of ZnO NPs were first examined by UV–vis
absorption and PL spectroscopy. The absorption onset of 380 nm
andthebroadblue-greenemissionbandatw571 nmwere observed
(Fig. 1a). They were agreed with the previous reports [30–32].
Because the fluorescence of ZnO NPs and SC were almost over-
lapped, in this work, we utilized thenon-linearoptical characteristic
of ZnO NPs to observe their transport with chemical enhancers in
SC. Fig. 1b and c shows the dual-channel two-photon images with
The weight ratios of the four donor vehicle solutions consisting of EtOH, OA, OA–
EtOH and PBS.
Donor solution ZnO NPsPBS Oleic acidEthanol
Fig. 3. The magnified images of selected regions of interest in Fig. 2. (a), (b), (c) and (d) represent the donor solution conditions of control, OA, EtOH and OA–EtOH respectively.
T.-R. Kuo et al. / Biomaterials 30 (2009) 3002–30083005
the size (110?100 mm2) of ZnO NPs. Displayed in Fig. 1b and c are,
respectively, the two-photon AF (bandwidth 435 to 700 nm) and
SHG (bandwidth 380–400 nm) images of ZnO NPs. The dual-
channel two-photon images show that ZnO NPs can be effectively
imaged by two-photon microscopy. Besides the optical images, the
ZnO NPs were also characterized by transmission electron micros-
copy (TEM). The electron microscopic image (Fig.1d) indicated that
the averaged diameter of the NPs was w10 nm.
3.2. Imaging the chemical enhanced transdermal delivery of ZnO NPs
To analyze the transdermal transport of ZnO NPs with different
chemical enhancers, the representative dual-channel two-photon
images of ZnO NPs distribution in the nude mouse SC are shown in
Fig. 2, while the formulations of four donor solutions, control and
enhancers OA, EtOH and OA–EtOH, are presented in Table 1. In
Fig. 2, the SHG images (blue pseudocolor) show the distributions of
ZnO NPs and AF (green pseudocolor) is used to indicate the SC of
the skin. Where appropriate, the locations of representative cor-
neocytes in the SC are markedwith ayellowarrowin each image, as
the corneocytes can be clearly identified. Moreover, Fig. 3 shows
the magnified images of selected regions of interest as marked in
Fig. 2. The SHG of ZnO NPs and the AF of SC are merged at the same
imaging depth. The images are presented in column at z¼0 mm
(the skin surface), z¼15 mm and z¼30 mm, to provide qualitative
information on the amount and locations of ZnO NPs with chemical
From the dual-channel two-photon images, the ZnO NPs are
observed to transport between the intercellular domains of the SC
and distribute heterogeneously within the SC. In addition, the
majority of the second harmonic generation of ZnO NPs is found to
locate at the uppermost layers of the skin at approximate depths
around 30 mm, with insignificant SHG detected beyond that depth.
The result suggests that the SC acts as the primary barrier to the
transdermal transport of ZnO NPs and it is through the multi-
lamellar lipid regions between the corneocytes that the primary
ZnO NPs transport across the mouse skins. Furthermore, the
tendency of ZnO NPs to distribute in the intercellular domains
suggests that the intracellular pathways are not effective for ZnO
NPs delivery. These suggestions correspond to the accumulation of
ZnO NPs on the surface of human skin and around corneocytes
presented by electron micrographs .
3.3. 3-D SHG images of ZnO NPs distribution in the SC under
different chemical enhancer conditions
To illustratethe extentof ZnO NPs penetration, axial-scans using
SHG microscopy was applied to show the 3-D distribution of ZnO
Fig. 4. 3-D SHG images of ZnO NPs in the upper 60 mm of the nude mouse skin samples. Each 3-D image corresponds to a field size of 330?300 mm2and a depth of 60 mm from the
surface of the nude mouse skin. The color scale is a relative indication of the SHG intensity, where regions in white (corresponding to 255) reflect high SHG intensity and regions in
black (corresponding to 0) reflect low SHG intensity. (a–d) illustrate the ZnO NPs spatial distributions under the four donor solution conditions (Table 1). The white arrow indicates
the penetration pathway of ZnO NPs into the SC.
T.-R. Kuo et al. / Biomaterials 30 (2009) 3002–30083006
NPs. In Fig. 4, each 3-D image stack represents a field of view
330?300 mm2in size and at a depth of 60 mm from the surface of
the skin. Fig. 4a–d illustrates the SHG spatial distributions of ZnO
NPs across the skin sample (60 mm) with four donor solutions,
control and enhancers OA, EtOH and OA–EtOH, respectively. The
attached color scales were used for these images.
The penetration pathways of ZnO NPs are visible in the 3-D
images of Fig. 4. A significant quantity of ZnO NPs accumulation
indicated by white arrows reveals the transdermal pathway of ZnO
NPs with chemical enhancers. Such result correlates with the
previous discussion that ZnO NPs with chemical enhancers trans-
port through intercellular pathway to penetrate into SC and do not
enter the corneocytes. Moreover, the axial distribution of ZnO NPs
with OA enhancer appears to increase at channel-like area,
compared with control solution. The increase in axial distribution
reflects the increased intensity of SHG signals. For EtOH enhancer,
the SHG signal intensity of ZnO NPs is apparently spread out across
the SC. The essence of the axial distribution for ZnO NPs with
OA–EtOH enhancer is closer to the axial distribution of ZnO NPs
with EtOH enhancer, rather than that of ZnO NPs with OA enhancer.
3.4. Axial SHG intensity profiles of ZnO NPs
In addition to qualitative imaging, we also determined the
average ZnO SHG intensity profiles under different chemical
enhancer conditions and the results are shown in Fig. 5. The
average SHG intensity of each donor solution was plotted on the y
axis and the depth (z) from the skin surface (at z¼0 mm) was
plotted on the x axis in mm scale. The error bars represent standard
deviation (SD) of the average value of the ZnO NPs SHG intensity.
From the profiles, the SHG intensities of ZnO NPs with OA, EtOH
and OA–EtOH enhancers on the skin surface appear to be higher
than that of ZnO NPs with control donor solution. As the depth of
the skin increases, the SHG signals of ZnO NPs with OA enhancer
become to decay slower than those of ZnO NPs with the individual
EtOH and OA–EtOH enhancers. In regard to EtOH and OA–EtOH
enhancers, the increases in the SHG intensity gradient are almost
the same. With noise signals defined by less than one-tenth of the
SHG intensity, the penetration depths of ZnO NPs with control,
enhancers OA, EtOH and OA–EtOH were 10, 30, 25 and 20 mm from
the skin surface.
3.5. Chemical enhancements in the vehicle-to-skin partition
coefficient, the intensity gradient and the effective diffusion path
length for ZnO NPs
To quantify our imaging results, we calculated from the average
SHG intensity profiles in Fig. 5 and listed, in Table 2, the EKvalues of
the ZnO NPs at the skin surface, I(z¼0), forenhancers OA, EtOH and
OA–EtOH, respectively. To calculate the EG, values, the intensity
gradients were determined from the average SHG intensity profiles
and the results are ?3.26?1.73, ?2.02?1.36, ?4.74?3.04 and
?4.81?3.67, for control, enhancers OA, EtOH and OA–EtOH
respectively. Linear regression yields
enhancers OA, EtOH and OA–EtOH to be 0.92, 0.94, 0.99 and 0.99,
respectively. Based on the slope values determined, the EGvalues
for enhancers OA, EtOH and OA–EtOH are 0.62?0.86, 1.45?0.83
and 1.48?0.93, respectively. Utilizing the equation EL¼EK/EG, the
values of enhancers OA, EtOH and OA–EtOH are 1.85?1.44,
enhancement values calculated for sulforhodamine B (SRB) with
oleic acid enhancer are: EK¼4.56?2.05, EL¼3.66?1.67, and
From the EKvalue of 1.15?0.46 for OA enhancer, the accumu-
lationof ZnO NPs on the skin surface is slightlyincreased by15%. OA
is capable of altering SC lipid ordering to change the transdermal
permeability of the permeant. The increase of ZnO NPs on the skin
surface can thus be explained by the formation of separate domains
of OA pools within the SC lipids. Such explanation corresponds to
EG¼0.62?0.86 for OA enhancer. EG(0.62?0.86)<1 reflects the
fact that the delivery of ZnO NPs into SC is through the phase-
separatedOAdomains. After ZnO NPs accumulate intoOA pools, the
decreasing rate of the SHG intensity gradient is 62% for OA
enhancer, compared with the control. The observed increase in
EL¼1.85?1.44 for OA enhancer verifies the creation of a micro-
transport environment in the SC.
For EtOH enhancer, the EK value of 1.81?0.56 shows that
chemical enhancer of EtOH can enhance the accumulation of ZnO
NPs on the skin surface by 81%. EtOH is a potent solvent for both
polar and non-polar species. Therefore, it is possible that the
presence of EtOH in the donor solution is capable of leaching
significant quantities of non-covalently bound amphiphilic SC
lipids, causing the modulation of the skin barrier. However, the
enhancement of EG value for EtOH enhancer is 1.45?0.83>1,
indicating that the decreasing rate of the SHG intensity gradient is
faster than control by 145%. Therefore, the effect of the modulation
of skin barrier caused by EtOH enhancer is constrained, allowing
ZnO NPs to accumulate merely on the skin surface with a minimum
penetration. The ELvalue of the EtOH enhancer,1.25?0.65, reflects
the accumulation of ZnO NPs on the skin surface associated with
the fast decreasing rate of the SHG intensity gradient.
As regard to OA–EtOH enhancer, the EK value of 1.93?0.62
demonstrates the significant ZnO accumulation on the skin surface
by 93%. Compared with EtOH donor solution, the additional pres-
ence of OA in OA–EtOH donor solution appears to increase the ZnO
NPs accumulation by only 12%. Whereas, compared to OA solution,
adding EtOH enhancer to OA donor solution, the accumulation of
ZnO NPs increases less than 81%. This phenomenon suggests the
and 1.30? 0.93,respectively. For reference, the
Fig. 5. Axial (z) SHG intensity profiles of ZnO NPs in nude mouse skins (skin surface at
z ¼0 mm). The average SHG intensity profiles of ZnO NPs under the four chemical
enhancer conditions (Table 1) are shown.
Calculated values of EK, EG, and EL, for transdermal delivery of ZnO NPs.
Donor solution I(z¼0)
1.15?0.46 0.62?0.86 1.85?1.44
T.-R. Kuo et al. / Biomaterials 30 (2009) 3002–30083007
mutual interference of OA and EtOH effects on the SC lipids. The Download full-text
phase-separated domains formed by OA enhancer to decrease the
skin barrier are disturbed by the effect of EtOH enhancer in
modulating the SC lipid fluidity. The decreasing rate of the SHG
intensity gradient for OA–EtOH enhancer is shown by the EGvalue
of 1.48 ?0.93>1, with 86% faster than OA enhancer and with 3%
faster than EtOH enhancer. Consequently, the ELvalue of 1.30? 0.71
for OA–EtOH enhancer indicates that the SC lipid fluidity increased
by OA enhancer is offset by the leaching effect of EtOH enhancer.
In this work, we succeed in combining the second harmonic
generation of ZnO nanoparticles and autofluorescence of the
stratum corneum to image the transdermal pathway of ZnO
nanoparticles with oleic acid, ethanol and oleic acid–ethanol
enhancers, using two-photon microscopy. To quantify dual-channel
two-photon microscopy images, the enhancement values of
vehicle-to-skin partition coefficient and second harmonic genera-
tion intensity gradient were calculated from the second harmonic
generation signal intensity of ZnO nanoparticles. The ratio of the
enhancement values of vehicle-to-skin partition coefficient and
second harmonic generation intensity gradient reflects the
enhancement values of effective diffusion path length. The results
suggested that the multilamellar lipid regions between the cor-
neocytes were the pathways for ZnO nanoparticles delivery. With
oleic acid enhancer, the transport of ZnO nanoparticles into the
stratum corneum is endorsed by the phase-separated oleic acid
domains, while ethanol enhancer leaches significant amount of
non-covalently bound amphiphilic stratum corneum lipids to
modulate the skin barrier. As regards oleic acid–ethanol donor
solution, the increase of stratum corneum lipid fluidity associated
with oleic acid enhancer was offset by the effect of ethanol in
loosening the stratum corneum lipid structure. Among our choices
of the different chemical enhancer conditions, the oleic acid–
ethanol combination can be regarded as the most effective donor
solution in transdermal ZnO nanoparticles into the stratum
This study was financially supported by the National Science
Council (NSC), Taiwan (NSC-96-3112-B-002-025, NSC-95-3112-B-
002-019, NSC-94-3112-B-002015Y, and NSC-96-2120-M003-001),
and National Taiwan University Hospital (975-842). The two-
photon experiments and data analysis were performed at the
Optical Molecular Imaging Microscopy Core Facility (A5) of the
National Research Program for Genomic Medicine of NSC inTaiwan.
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