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Biological interactions of quantum dot nanoparticles in skin
and in human epidermal keratinocytes
Leshuai W. Zhanga, William W. Yub, Vicki L. Colvinb, Nancy A. Monteiro-Rivierea,⁎
aCenter for Chemical Toxicology Research and Pharmacokinetics, North Carolina State University, Raleigh, NC 27606, USA
bDepartment of Chemistry, Rice University, Houston, TX 77005, USA
Received 29 October 2007; revised 6 December 2007; accepted 13 December 2007
Available online 3 January 2008
Quantum dots nanoparticles have novel optical properties for biomedical applications and electronics, but little is known about their skin
permeability and interaction with cells. QD621 are nail-shaped nanoparticles that contain a cadmium/selenide core with a cadmium sulfide shell
coated with polyethylene glycol (PEG) and are soluble in water. QD were topically applied to porcine skin flow-through diffusion cells to assess
penetration at 1 μM, 2 μM and 10 μM for 24 h. QD were also studied in human epidermal keratinocytes (HEK) to determine cellular uptake,
cytotoxicity and inflammatory potential. Confocal microscopy depicted the penetration of QD621 through the uppermost stratum corneum (SC)
layers of the epidermis and fluorescence was found primarily in the SC and near hair follicles. QD were found in the intercellular lipid bilayers of the
SC by transmission electron microscopy (TEM). Inductively coupled plasma-optical emission spectroscopy (ICP-OES) analysis for cadmium (Cd)
significantly (pb0.05)from 1.25nMto 10nMafter 24 hand 48h.There wasa significant increase inIL-6at 1.25nMto 10nM,while IL-8increased
skin were damaged allowing direct QD exposure to skin or keratinocytes, an inflammatory response could be initiated.
© 2007 Elsevier Inc. All rights reserved.
Keywords: Quantum dot; Nanoparticles; Skin penetration; Cytokines; Keratinocytes; Cellular uptake; Inflammation; Skin
Quantum dot (QD) nanoparticles are well known for their
optical characteristics which resultinstrongfluorescencewithout
photobleaching, and therefore may have potential for drug deli-
very, imaging markers, diagnostic or therapeutic applications
(Michalet et al., 2005). Streptavidin conjugated QD have been
bound to cytoskeletal elements such as tubulin and actin and
visualized with monoclonalantibodies (Wu et al.,2003). Prostate
tumors in mice were imaged with a QD-antibody conjugate that
provided a novel method of cancer labeling in vivo (Gao et al.,
2004). However, before QD can be used safely in vivo, more
information is needed about their potential for toxicity and inter-
actions with biological systems so that rational nanomaterial risk
some engineered nanomaterials with diverse chemical properties
and can serve as a portal of entry for localized or systemic
exposure to humans, especially in an occupational scenario. En-
gineered nanomaterials should be investigated for occupational
safety during manufacture, exposure scenarios likely to be en-
countered by the consumer (e.g., commercial products, medi-
cines, cosmetics), and post-use release and migration to the
environment. In addition, their biocompatibility should be
evaluated in cells and in tissues before incorporating them into
structures for biomedical devices or implants.
QD typically contain a cadmium/selenide (CdSe) core with a
zinc sulfide (ZnS) or a cadmium sulfide (CdS) shell. QD need to
be water soluble with low cytotoxicity to function as biocompa-
tible probes. It has been reported that Cd release from the core is
Available online at www.sciencedirect.com
Toxicology and Applied Pharmacology 228 (2008) 200–211
⁎Corresponding author. Center for Chemical Toxicology Research and
Pharmacokinetics North Carolina State University 4700 Hillsborough Street
Raleigh, NC 27606, USA. Fax: +1 919 513 6358.
E-mail address: Nancy_Monteiro@ncsu.edu (N.A. Monteiro-Riviere).
0041-008X/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
Author's personal copy
by oxidative attack (Tang et al., 2005). QD are unstable when
exposed to UV light, and can release Cd into the medium, a
substance that is toxic to hepatocytes (Derfus et al., 2004a). Bare
CdSe-core QD have been shown to be toxic in human neuro-
blastoma cells causing a decrease in cell viability, fragmentation
of chromosomal DNA, and loss of mitochondrial membrane
potential. However, ZnS-coated QD did not alter cells (Chan
to minimize Cd leaching into the intracellular space of MCF-7
cells (Cho et al., 2007). Surface coatings such as polyethylene
glycol (PEG) may be applied with other small groups (amino-
stability of QD in biological buffers or water (Fan et al., 2005).
Several studies have reported that the QD surface coatings and
charge can influence the toxicity of QD (Hoshino et al., 2004;
Kirchner et al., 2005). QD565 and QD655 showed a decrease in
viability in HEK (Ryman-Rasmussen et al., 2007). However, if
the coating and shell of a QD is altered, there is potential for the
QD core that usually consists of Cd to leach out into the surroun-
ding tissue and potentially be a health risk.
The application of QD probes in biomedical imaging and
therapeutic applications has attracted much attention, but there is
limited research on their use in vivo. Studies have found that QD
can image lymph nodes and blood vessels in tissues (Kim et al.,
2004; Larson et al., 2003). QD530 have been shown to remain in
liver, lymph nodes and bone marrow of mice for one month after
tail injection despite its low affinity to cells and tissues (Ballou
et al., 2004). The injection of QD705 in mice was found in liver,
spleen and kidneys after 28days (Yang et al.,2007).Localization
potential for retention and migration to other organs.
Different types of nanoparticles have been topically applied to
have been shown to penetrate hair follicles and the stratum cor-
neum (SC) layer of the epidermis, suggesting a potential capa-
bility for nanoparticles to traverse the dermal barriers (Baroli
et al., 2007). Microfine zinc oxide (ZnO) with a mean size of
80nmand agglomeratesoftitaniumdioxide lessthan160nmdid
not penetrate the porcine SC layer in in vitro static diffusion cells
(Gamer et al., 2006). However, topical application of micronized
ZnO (26–30 nm) in a sunscreen formulation on in vitro human
skin found nanoparticles in the upper SC with minimal pene-
tration (Cross et al., 2007).
Polymeric nanoparticles coated with a 40 nm thick PEG block
copolymer layer topically applied to hairless guinea pig skin for
12 h were able to penetrate the epidermis (Shim et al., 2004).
FITC-conjugated dextran beads of 0.5 μm penetrated the SC of
human skin and reached the epidermis after 30 min of flexing
(Tinkle et al., 2003). Studies in our laboratory have shown that
fullerene amino acid-derivatized peptide nanoparticles of 3.5 nm
were capable of penetrating the dermal layers of porcine skin
flexed for 60 min and placed in flow-through diffusion cells for
8 h, while non-flexed control skin showed penetration limited to
the upper epidermal layers (Rouse et al., 2007, Monteiro-Riviere
et al., 2007). TEM found that the derivatized fullerene was
localized within the intercellular space of the stratum granulosum
layer. Additional studies with spherical QD565 and elliptical
QD655 with three different surface coatings in flow-through
diffusion cells showed penetration into porcine skin. PEG and
carboxylic acid coated QD565 were localized primarily in the
epidermis by 8 h, while the QD565 PEG-amine were localized
mainly in the dermis. QD655 coated with PEG and PEG-amine
were localized primarily within the epidermal layers after 8 h,
while the carboxylic acid-coated QD655 did not penetrate into
the epidermis until 24 h (Ryman-Rasmussen et al., 2006). Some
data suggest that QD penetration through human skin may be
conditions across investigators are difficult to control.
to months raises the concern of toxicity to surrounding tissues.
The incorporation of polyethylene glycol (PEG) as a surface
coating greatly reduces nonspecific binding to several types of
cells (Bentzen et al., 2005) and increases QD stability and so-
lubility (Yu et al., 2007). However, few studies have focused on
skin cells. The effect of surface coatings was studied in human
epidermal keratinocytes (HEK) to determine the uptake of QD,
QD cytotoxicity and inflammation potential. QD were found to
coating was the primary determinant of cytotoxicity (Ryman-
Rasmussen et al., 2007).
Another type of QD (QD621) has been studied in vivo and
in vitro, but there is limited information on its toxicity. QD621
intradermally injected in SKH-1 hairless mice migrated from the
injection site to regional lymph nodes through the lymphatic duct
system and then to the liver and other organs (Gopee et al., 2007).
The biodistribution of intra-arterially infused QD621 in perfused
skin showedthat the QD621 can migrate out of thecapillaries into
the surrounding tissue (Lee et al., 2007). QD621 with a PEG
(Chang et al., 2006). Our study used porcine skin as an in vitro
model of human skin to assess the penetration of QD621. Porcine
skin is widely used for skin penetration studies because it is
anatomically, physiologically and biochemically similar to human
skin (Monteiro-Riviere and Stromberg, 1985; Monteiro-Riviere,
1991; Monteiro-Riviere and Riviere, 1996; Simon and Maibach,
2000; Monteiro-Riviere, 2001). The objective of this study was to
assess whether QD621-PEG, which are nail-shaped with a hy-
determine cellular uptake and inflammatory potential in HEK.
Materials and methods
Quantum dots synthesis.
thesized according to the literature and the CdS shell growth temperature was
adjusted to 180 °C (Li et al., 2003; Yu et al., 2006; Yu et al., 2007). QD were
purified and stored in chloroform and the concentrations determined using the
available extinction coefficients (Yu et al., 2003). To produce amphiphilic poly-
mers, poly (maleic anhydride-alt-1-octadecene) (PMAO, Mn=30000–50000,
MW 6000; Nektar, CA) in chloroform overnight to form amphiphilic polymers
(PMAO-PEG) (molar ratios of PMAO: PEG were 1:5 and 1:30, respectively, for
negative and neutral water-soluble QD). For water-soluble nanocrystals, the QD
and PMAO-PEGwere mixed in chloroform and stirred for 1 h (molar ratioof QD:
PMAO-PEG was 1:10). Water was added in the same volume as the chloroform
solution and the chloroform was removed by rotary evaporation that resulted in a
clear and colored solution of water-soluble QD. Ultracentrifugation (Beckman
QD621 with a core/shell of CdSe/CdS were syn-
201 L.W. Zhang et al. / Toxicology and Applied Pharmacology 228 (2008) 200–211
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Coulter Optima L-80XP) was used to concentrate and purify (remove excess
visible absorption spectra of QD were measured by a Varian Cary 5000 spec-
trophotometer, which showed the first excitonic absorption peak of 615 nm.
fluorescence spectrophotometer with a peak position of 621 nm. TEM specimens
of the QD621 were prepared by evaporating one drop (∼10 μl) of nanocrystal
solution on a carbon coated copper grid. The TEM micrographs were taken on a
data wasobtainedby countingmore than1000 individualnanocrystallineparticles
the inorganic CdSe/CdS was 8.40×5.78 nm (Fig. 1). The overall hydrodynamic
size of the water-soluble QD was 39–40 nm, on which the PEG molecules
completely covered the structure (Yu et al., 2007).
Flow-through diffusion cells.
Chang and Riviere, 1991). Porcine skin was obtained from the back of female
weanling Yorkshire pigs. Pig skin was clipped 24 h prior to dosing. The pig was
mm steel circular punch was used to provide a dosing area of 0.64 cm2. Each
circular piece of dermatomed skin was placed epidermal side up into each of the
at 37 °C with a Brinkmann constant-temperature circulator. The dermal side of the
2 ml/h by a multi-channel peristaltic cassette pump. The perfusate was adjusted to
pH 7.3–7.5 during the experiments. Diffusion cells were equilibrated in perfusate
treatment) were topically dosed on the skin with a positive displacement pipette.
Diffusion cell perfusate was collected for fluorescence and inductively coupled
plasma-optical emission spectroscopy (ICP-OES) detection at 1, 2, 3, 4, 5, 6, 7, 8,
from the cells. Half of the skin was placed in Trump's fixative for TEM and the
other half immediately frozen at −80 °C for laser scanning confocal microscopy.
skin using a Leica TCS SP1 confocal laser scanner interfaced to an inverted Leica
was equipped with a confocal–differential interference contrast (DIC)system. QD
were excited with a UV laser (351 nm and 364 nm) and an Argon laser (488 nm)
with emission channels of 610–632 nm. Images were captured using LCS Lite
Jung) at 20 μm in thickness. Tissue samples were cut perpendicular to the knife
QD621 were imaged in the epidermal and dermal layers of
with the skin standing upright so that sectioning is conducted from the bottom of
the dermis up through the epidermal layers. This prevents any contamination that
couldbedueto draggingtheQDfromthesurface oftheknifeto thedermallayers.
All skin sections were analyzed and all imaging data are representative of the
individual diffusion cell experiments.
TEM of skin sections.
minced into1mm3piecesandplacedin7ml glassvials.Sampleswerethenrinsed
in 0.1 M phosphate buffer (pH 7.2) and post-fixed in 1% osmium tetroxide
(Polysciences, Inc., Warrington, PA) in 0.1 M sodium phosphate buffer (pH 7.2),
embedded in Spurr's resin. Unstained sections (600–800 Å) were mounted on
copper grids and then examined on a Philips EM208S TEM. Unstained sections
provided better visualization.
Skin samples fixed in Trump's at 4 °C for 24 h were
Fluorescence quantification of perfusate.
perfusate sample collected from the flow-through diffusion cells at different time
points was transferred to black 96-well black plates with microclear bottoms
(Corning/Costar USA) and assayed on a tunable Molecular Dynamics Gemini
EM™. The excitation wavelength was 360 nm and the emission wavelength at
621 nm with a cut off 610 nm. Background fluorescence was the perfusate without
QD to determine the sensitivity of the fluorometer.
To quantitate QDfluorescence,each
Inductively coupled plasma-optical emission spectroscopy (ICP-OES).
Perfusate samples were also evaluated for Cd by ICP-OES. Perfusate (200 μl) was
placedina40mlscrewcapcentrifugetubeand500 μlof concentratedoptimagrade
nitric acid was added. 6 h later the samples were refluxed 30 min in a boiling water
bath and then mixed with the deionized water, and transferred to 15 ml PVC
centrifuge tubes for analysis in a Perkin Elmer Model 2100DV ICP-Emission
generated with spiked recovery and the slope of the most sensitive analytical line.
Cell culture and viability assay.
the cells were exposed to QD nanoparticles in KGM-2. The control wells consisted
was prepared with KGM-2 to provide the serial dilution with the concentration of
0.3125 nM, 0.625 nM, 1.25 nM, 2.5 nM, 5 nM and 10 nM. HEK at 24 h and 48 h
Neonatal HEK (Cambrex, Corp, Walkersville,
Fig. 1. High resolution TEM image of QD621. The QD are nail shaped and have
a mean dimension of 8.40 nm in length and 5.78 nm in width.
Fig. 2. DIC image of normal porcine skin treated with water (control) for 24 h.
Note the stratum corneum (SC), epidermis (E) and dermis (D). The epidermis is
clearly demarcated by a dotted yellow and green lines. A cross-section of a hair
follicle (H) is in the dermis.
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bromide] for viability as previously described (Mosmann, 1983). The absorbance,
directly proportional to cell viability, was determined spectrophotometrically at
from this study provided the range of QD concentrations not cytotoxic to HEK.
Cytokine release assay.
Hercules, CA). This system utilized multiplexing to simultaneously assay for
which is specific to each cytokine and possessing a unique spectral address were
were rinsed and then incubated with a fluorescent-labeled reporter molecule that
specifically binds to the analyte. The contents of each well were analyzed in the
Bio-Plex array reader. As the beads flow into the reader, one laser identifies the
quantify the specific cytokine relative to the standard curve. The average
concentration (pg/mL) of each cytokine for each treatment and time point was
calculated and normalized using the corresponding viability data. The limit of
detection for the cytokines is as follows: IL-1β (0.8 pg/ml), IL-6 (1.1 pg/ml), IL-8
(0.5 pg/ml), IL-10 (0.9 pg/ml), and TNF-α (3.0 pg/ml).
Human cytokinesIL-6, IL-8, IL-10, TNF-α andIL-1β
TEM of QD621 in HEK.
calization of QD621. HEK were seeded in cell culture flasks (25 cm2; ~100,000
cells) and grownto 70% confluency at 37 °C in a humidified 5% CO2environment.
TEM was conducted on HEK to determine the lo-
Hank's Balanced Salt Solution (HBSS), and fixed in Trump's fixative at 4 °C. Cells
were then rinsed in 0.1 M phosphate buffer (pH 7.2), pelleted in a microcentrifuge
tetroxide and processed as previously described. Unstained thin sections on copper
grids were examined on a Philips EM208 S transmission electron microscope.
centration (normalized to viability) for each treatment were calculated and the
significant differences (pb0.05) determined using ANOVA (SAS 9.1 for Win-
dows; SAS Institute, Cary, NC). When significant differences were found, a
pairwise comparison among different treatments were conducted within each
exposure length and sampling time using the t-test (LSD).
The mean values for HEK viability and cytokine con-
TEM of QD
The shape of the QD621 can be seen by TEM (Fig. 1). The
mean width of the QD621 is 5.78±0.97 nm and length is 8.40±
1.9 nm. QD621 with a CdSe core and CdS shell coated with a
PEG polymer coils has a hydrodynamic size of 39±1 nm in
Fig. 3. Confocal image of skin treated with QD621 for 24 h. A) 1 μM dose. B, C, D 10 μM dose. Top row across: confocal-DIC images depicting the skin section.
Middle row across: fluorescence indicating QD621 only in the skin. Bottom row across: Overlay of DIC and fluorescence depicting 1 μM and 10 μm doses of QD621
localized in the SC.
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diameter from size-exclusion chromatography. The QD in an
aqueous solution appeared dark red and showed a strong red
fluorescence under the 360 nm-UV light. QD621 are very stable
inwaterandphysiologicalbufferswithina widepHrange (4–10)
as well as in 1 M NaCl (Yu et al., 2007).
Laser scanning confocal microscopy of normal skin
Skin treated with water in the flow-through diffusion cells
served as the vehicle control. At 24 h, confocal-DIC images
depicted normal porcine skin morphology showing an intact
SC, epidermis and dermal layers (Fig. 2). The compact SC cell
layers as well as the stratum granulosum, stratum spinosum and
stratum basale layers were intact. Cross-section of a hair follicle
as depicted in Fig. 2 was obvious in most of the confocal
images. The photomultiplier tube (PMT) was used to control the
signal intensity (gain) to minimize the noise/background but
maintained a bright QD621 signal. The autofluorescence signal
in porcine skin was minimal.
Laser scanning confocal microscopy of QD621-treated skin
All confocal images depicted normal intact SC layers in all
treatments. At the lowest concentration of 1 μM, QD621 were
located primarily in the SC layers of the skin (Fig. 3A). No
QD621 fluorescence was detected in the stratum granulosum,
stratum spinosum, or stratum basale layers of the epidermis.
In one sample of the 2 μM dose, a small amount of fluorescence
was detected in the upper epidermal layers, but most of the QD
remained in the SC or in between the stratum granulosum–
stratum corneum interface (data not shown). At the highest
in the SC layers. In some instances, QD were seen in the upper
epidermis and outer root sheath of the hair follicle (Fig. 3C, D).
TEM of QD621 in skin
TEM was used to assess the precise localization and dis-
tribution of QD621 in the skin. The QD621 were localized
within the intercellular lipid bilayers of the upper most layers of
the SC. Greater amounts of QD621 were found in the superficial
layers of the SC with a decrease in QD concentration deeper
into the SC layers (Fig. 4A). The characteristic nail shape of
QD621 is clearly seen in Fig. 4B.
Fluorescence measurements of perfusate
any time point or concentration. To test the sensitivity of fluo-
rometer, the stock solution of QD621 was diluted into 20 nM,
5 nM, 2 nM, 0.5 nM. The resulting fluorescence intensity vs
concentration plot is proportional (R2=0.9972). When the per-
fusate was spiked with the 0.5 nM QD (20,000 times dilution of
thedetection limit offluorescence was below this sensitivity level.
ICP-OES of perfusate
ICP-OESwas alsousedtodetectCdpresenceinthe perfusate.
timepoints.These results support the fluorescencemeasurements
above that there was no evidence of absorption in the flow-
through diffusion cells.
Fig. 4. TEM of QD621 in the SC. A) QD621 in the intercellular lipid bilayers of the SC cell layers. B) Higher magnification of the enlarged area in Panel A showing
individual nail-shaped QD621 (arrows) and some small agglomerates.
Fig. 5. MTT viability of QD621. Mean viability (±SEM) at 24 h and 48 h.
Histogram with different letters (A, B, C, and D for 24 h; a, b, c, d for 48 h)
denote mean values that are statistically different at pb0.05.
204 L.W. Zhang et al. / Toxicology and Applied Pharmacology 228 (2008) 200–211
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The MTT assay was used to assess the viability of HEK
treated with 0.3125 nM, 0.625 nM, 1.25 nM, 2.5 nM, 5 nM and
10 nM of QD621. There was a statistically significant decrease
(pb0.05) in viability at 1.25 nM, 2.5 nM, 5 nM and 10 nM at
both 24 h and 48 h relative to the controls, which was dose
dependent (Fig. 5). The 0.3125 nM and 0.625 nM of QD621 did
not show a significant decrease in viability at 24 h and 48 h
relative to controls.
Normalized cytokine release from HEK
The expression of cytokines IL-6, IL-8, IL-10, TNF-α and IL-
1β for inflammation was assessed at 24 h and 48 h. The level of
IL-8 in the control averaged 350 pg/ml at 24 h and 439 pg/ml at
the 2.5 nM, 5 nM and 10 nM concentrations compared to control
IL-6 expression for controls was 24 pg/ml at 24 h and 48 h
(Fig. 6B). A statistically significant increase in IL-6 expression
was dose dependent. IL-6 release by HEK treated with the 10 nM
QD621 was elevated 2.4 fold compared to controls after 24 h.
Fig. 6. CytokinereleaseinHEKtreatedwithQD621.A)IL-8expression.B)IL-6
expression. Histogram with different letters (A–D for 24 h; a–c for 48 h) denote
mean values that are statistically different at pb0.05. Data represent the means±
Fig. 7. TEM of HEK treated with QD621 at 24 h. A) Control. B) HEK dosed with 2 nM QD621. C) HEK dosed with 10 nM QD621.D) QD uptake or on the peripheral
in HEK treated with10nM dose (arrows).
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The expression of TNF-α, IL-1β and IL-10 was minimal and
below the detection limit and considered as background noise.
TEM of QD in HEK
At 24 h, HEK control depicted normal morphology with few
vacuoles in the cytoplasm (Fig. 7A). HEK dosed with 2 nM
(Fig. 7B) and 10nM (Fig. 7C) of QD621 showed no apoptotic
cells. Treatment of the 10 nM of QD621 showed uptake of QD
as dark granules agglomerates distributed throughout the cells
(Fig. 7D). Often, QD agglomerates were seen in focal cyto-
plasmic vacuoles (Fig. 8A). At times, individual QD were
visible in the vacuoles accompanied by agglomerates (Fig. 8B).
Higher magnification of the insert in Fig. 8B verified the
QD621 with their characteristic nail shape (arrows, Fig. 8C),
while some QD agglomerates were amorphous in shape. In
addition to being present in vacuoles, QD also showeda random
distribution in the cytoplasm of HEK (Fig. 9A). QD located
around the cell membranes (arrow heads, Fig. 9A) retained their
shape while aggregates were seen freely (small arrows, Fig. 9A)
in the cytoplasm or in the vacuoles (large arrows, Fig. 9A). QD
were noted in the extended cell membrane processes that could
suggest endocytosis as a mechanism of uptake (Fig. 9B). In
general, it appeared that individual QD were localized on the
surface of the cell while they appeared as agglomerates within
Fig. 9. TEM of HEK dosed with 10 nM of QD621 at 24 h showing different patterns of QD621. A) Numerous individual QD were present in vacuoles (large arrow), in
the cytoplasm (small arrows) and around the periphery of the cell membrane (arrow head). B) Higher magnification of the insert in Panel A depicting individual
QD621 on the cell membrane process.
Fig. 8. TEM of HEK dosed with 10 nM of QD621 at 24 h showingboth agglomerates and nail-shaped individual A) TEM of HEK treated with QD621 at 24 h showing
agglomerates in cytoplasmic vacuoles. QD621. B) Low magnification of HEK showing several QD scattered throughout the cytoplasm. C) High magnification of the
rectangular insert in Panel A with individual QD621 (arrows) and agglomerates.
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The role of QD nanoparticles has received a great deal of
attention due to the increase in imaging and therapeutic appli-
cations. Based on their unique physicochemical parameters, QD
are best known for their optical characteristics that emit strong
fluorescence and have potential for biomedical imaging and in
the electronics industries (Hardman, 2006). However, the toxi-
city of QD in skin needs to be investigated since exposure could
occur during the production process and in consumers during
biological or medical applications, either via topical exposure or
by distribution to skin after systemic exposure. The QD core
consists of heavy metals and their shell, surface coatings, and
size are important determinants of toxicity. If their shell or
surface coating is altered in any way, the heavy metal core could
leach and be a potential health risk. The penetration, loca-
lization, and toxicity of QD in skin and skin cells have become a
critical issue as the uses for QD increases.
Our study demonstrated that topically applied PEG coated
QD621 with a hydrodynamic diameter (DH) of 39±1 nm were
capable of penetrating only the uppermost layers of the porcine
SC 24 h after exposure. Previous studies in our laboratory with
PEG coated QD655 and QD565 of 45 nm and 35 nm (DH)
showed penetration through the epidermal layers at 8 h (Ryman-
Rasmussen et al., 2007). QD synthesized with the same core/
shell and with similar surface coatings may have similar hydro-
dynamic diameters but different penetration rates. Spherical
carboxylic acid-coated QD565 (14 nm) penetrated primarily
through SC and localized within the epidermal layers by 8 h,
while the elliptical QD655 (18 nm) was observed in the epi-
dermis at 24 h but not after 8 h (Ryman-Rasmussen et al., 2007).
QD penetration of skin not only depends on the type, shape and/
or size, but also their surface charge. Laser scanning confocal
microscopy showed that QD621 primarily remained on the
surface of the skin and sometimes near hair follicles. TEM
showed penetration of the QD621 only in the uppermost SC
QD621-PEG was dissolved in water, while QD565-PEG and
QD655-PEG were in a borate buffer which has a similar visco-
sity as water but a higher pH. QD565 and QD655 penetrated
QD655 had a pH of 8.3, while the water diluted QD621 had a
neutral pH. The SC layers remained intact and no other mor-
phologicalchangeswerenoted byeitherconfocalmicroscopy or
TEM due to pH effects that possibly could alter the skin barrier
formation or morphology that would allow for penetration.
Therefore, the QD penetration of porcine SC seen by QD565,
655 and 621 is independent of the vehicle or pH.
These three QD have the same chemical composition inclu-
ding a “rigid” core and a “soft” surface coating. Penetration may
not only be determined by size and charge, but also by the shape
of the rigid core and durability of the coating. It has been
reported that elastic particles were able to distribute through the
epidermis faster, while rigid particles were found to remain on
the surface of the upper SC (Honeywell-Nguyen et al., 2004).
The most common route of penetration in skin is via the inter-
cellular spaces between the corneocytes. Our lab has shown the
diameter of porcine corneocytes to be 32 μm and the vertical
and lateral gaps between corneocytesare 19 nm (Van der Merwe
et al., 2006). Therefore, the QD could potentially pass through
the corneocytes lateral intercellular spaces since the QD621 has
a rigid core length of 8.4 nm and width of 5.8 nm but overall size
of 39–40 nm. It is theoretically possible that the outer PEG
through the intercellular space and remain lodged within the SC
lipid bilayers. QD penetration may be limited through the epi-
dermis due to their large size and irregular configuration and
this fact could explain different behavior between the spherical
QD565 (4.6 nm core) and elliptical QD655 (6 nm for minor axis,
12 nm for major axis of the core). Therefore, the 1 μM QD621
and QD655 (smaller and more regular in shape) would have less
difficulty with skin penetration. Species differences alluded to
earlier may be a function of intercellular lipid structure or hair
follicle density which could modify these penetration processes
outer root sheath of hair follicles. This is similar to other reports
that showed TiO2microparticles and polystyrene nanoparticles
had distributed near orifices in human hair follicles (Lademann
et al., 1999; Alvarez-Roman et al., 2004). Other studies with
the SC and hair follicles (Baroli et al., 2007). The transappenda-
geal route has been previously reported as a potential route of
exposure. Unlike QD565 and 655, we noted that QD621 re-
mained primarily on the surface of the SC layer after 24 h, but at
the highest concentration of 10 μM were found in some hair
the dermis. This raised the possibility of contamination of the
knife during the sectioning process. However, our tissue samples
were cut perpendicular to the knife with the skin standing upright
so that sectioning is conducted from the bottom of the dermis up
through the epidermal layers. This prevents any contamination
that could be due to dragging the QD from the surface to the
dermal layers. In addition, confocal microscopy can evaluate the
optical section within the tissue (Z-stacks) to distinguish if QD is
on the surface of sections or within the sections.
Nanomaterials such as TiO2and ZnO are key ingredients that
are added to sunscreens to protect the skin from UV induced
damage. A study by Cross et al. (2007) reported that most mic-
ronized transparent zinc oxide nanoparticles of 26–30 nm in oil/
water formulations topically applied to human skin in in vitro
static cells for 24 h remained on the surface of the SC. The use of
oils as a vehicle may prevent the partitioning of the nanoparticle
from the oil into the SC bilipid layers, therefore potentially
reducing penetration. The authors also detected low levels of zinc
inthereceptorfluidandattributed it tonormalzincconcentrations
inskin,as wellasdue tooneoftheformulations thatdissolvedthe
particles and allowed the penetration of elemental zinc to diffuse
titanium through human skin with microfine TiO2, while studies
with microfine zinc and TiO2particles applied to porcine skin did
not show penetration (Gamer et al., 2006). In contrast, unpub-
lished observations for QD (Bronaugh, personal communication)
207L.W. Zhang et al. / Toxicology and Applied Pharmacology 228 (2008) 200–211
Author's personal copy
underlying the importance of studying penetration on an indi-
vidual nanoparticle basis.
Inour study,greater amounts ofQD621werefoundwithinthe
intercellular spaces of the outermost SC layers as depicted in
Fig.4A.Higher magnification ofthisarea showed that individual
QD621 retained their characteristic nail shape (Fig. 4B) but often
they appeared as agglomerates. Penetration of these nanocrystals
into the intercellular bilipid layer of the SC suggests that QD621
could potentially enter the skin via the intercellular bilipid path-
low contrast, but they provide better visualization of the QD
within the intercellular space of the SC. If routine staining for
membranes with lead citrate and uranyl acetate is used, then the
small size of QD would be impossible to distinguish from the
black cellular membrane staining.
If skin has been damaged in any way that perturbs the SC
barrier, then QD621 will have direct contact with the keratino-
cytes. In a few instances, we have seen QD localized in the upper
epidermal layers with the 2 μM dose. The rare instances that
QD621 were noted in the epidermal layers by confocal micro-
viable epidermis. In addition to the flow-through diffusion cell
study,wealsoinvestigatedthe effectofQDonHEK todetermine
(Lee et al., 2007; Yang et al., 2007) have shown that different
into the surrounding tissues. This raises the concern for cyto-
toxicity or irritation potential to the skin cells after systemic
QD toxicity clearly depends on a variety of physicochemical
properties such as size, shape and the chemical structure of the
core, shell and surface coatings. The cytotoxicity of surface mo-
dified QD with and without PEG were studied in human breast
cancer cells which showed that changes in cytotoxicity and
et al., 2006). When a ZnS coating was added to the QD, no cell
toxicity was detected by MTTat a 300 nM concentration for 24 h
(Chan et al., 2006). Other investigators have shown that a PEG
coating provides an inert coat that minimizes endocytic uptake
and reduces the toxicity compared with non-PEG coated QD
(Chan and Nie, 1998). Moreover, the cellular uptake of QD was
2005).N-acetylcysteine (NAC) modified QDreduces the amount
NAC anti-oxidative capability (Choi et al., 2007). Recently, Mn
or Cu doped ZnSe QD nanocrystals manufactured without heavy
metal cores were less cytotoxic, and may be acceptable alter-
natives to the higher quantum yield Cadmium containing mate-
heavy metal core, with a ZnS shell and PEG surface coating may
minimize the toxicity to cells.
In this aspect of the study, we assessed the cytotoxic and
inflammatory potential of QD621 in HEK. MTT viability and
the inflammatory cytokines IL-1β, IL-6, IL-8, IL-10, and TNFα
were assessed at 24 and 48 h post treatment. The MTT viability
assay in our study showed a dose dependent decrease in via-
bility from 0.3125 nM to 10 nM dose that was statistically
significant at 1.25 nM–10 nM compared to the controls. The
cytotoxic and inflammatory effects (IL-8, IL-6) were dose and
time dependent. TEM verified that the QD localized within the
cells and along the periphery of the cell membrane after 24 h.
Keratinocytes produce cytokines that serve as mediators for
inflammatory and immunologic reactions in skin exposed to
irritants (Allen et al., 2000, 2001a,b; Corsini and Galli, 2000;
Monteiro-Riviere et al., 2003; Barker et al., 1991; Nickoloff et al.,
inflammatory cells, have systemic effects on the immune system,
influence keratinocyte proliferation and differentiation, and affect
the production of other cytokines (Grone, 2002). The proinflam-
matory cytokines IL-8, IL-6, TNF-α, and IL-1β have been very
well studied and characterized and are regularly used as indicators
ofinflammation(Grone,2002;Barker etal.,1991;Nickoloff etal.,
1991). Although different toxicants may elicit different responses
in HEK, studies in our laboratory have shown cytokine release by
HEK in response to jet fuel exposure (Allen et al., 2000, 2001a,b;
Chou et al., 2003; Monteiro-Riviere et al., 2003).
Other nanomaterials like multi-walled carbon nanotubes
(MWCNT) (Monteiro-Riviere et al., 2005), 6-aminohexanoic
acid-functionalized single-walled carbon nanotubes (SWCNT)
(Zhang et al., 2007), and fullerenes (Rouse et al., 2006) also
caused an increase in IL-8 in HEK. We have also studied the
inflammatory response of HEK to different QD (QD565 and
655) of different sizes and surface coatings. Only the carboxylic
acid coated QD at the 20nM concentration depicted a significant
increase in the release of IL-1β, IL-6, and IL-8 at 24 h (Ryman-
Rasmussen et al., 2007, Monteiro-Riviere et al., 2007).
In cell signal transduction experiments, NF-κB was known to
activate IL-6 expression in human myeloma cells (Xiao et al.,
2004). Keratinocytes can express pro-inflammatory genes inclu-
Ithas beenshown thatsinglewalledcarbonnanotubes (SWCNT)
induces IL-6 expression by NF-κB initiated by TNF-α. TNF-α
was not detected in our studies.
In other studies, QD without a shell induced apoptosis by
inhibited Ras/ERK survival signaling (Chan et al., 2006). The
Fas receptor was also activated by QD due to oxidative stress
(Choi et al., 2007). However, our QD621 had a shell of CdS that
prevented the leakage of the Cd and therefore, no apoptotic cells
were evident in any of our treatments.
Other studies with silica-coated QD have shown activation
of 50 genes detected by high throughput gene expression
analysis (Zhang et al., 2006). Genes involved with intracellular
vesicle localization and vesicular proteins and cell membrane
associated proteins were abundant. Proteomic studies with
MWCNT in HEK also showed altered protein expression of 36
proteins by 24 h and 106 proteins by 48 h by two-dimensional
gel electrophoresis and mass spectrometry (Witzmann and
Monteiro-Riviere 2006). Proteins associated with metabolism,
cell signaling, stress, cytoskeletal elements, and vesicular traf-
ficking were affected but classic marker proteins associated
with apoptosis were absent.
208L.W. Zhang et al. / Toxicology and Applied Pharmacology 228 (2008) 200–211
Author's personal copy
Our work depicted the localization of QD621 in both the free
cytoplasm and intracellular cytoplasmic vacuoles in HEK treated
with 10 nM QD621. Our previous studies showed individual
MWCNT in the intracytoplasmic vacuoles of HEK (Monteiro-
Riviere et al., 2005). AHA-SWNT was localized within the
intracytoplasmic vacuoles of HEK as aggregates (Zhang et al.,
2007). Fullerene-based amino acid nanoparticles were also found
in vacuoles probably by the mechanism of endocytosis (Rouse
et al., 2006). QD565 and QD655 showed uptake of agglomerated
QD in HEK (Ryman-Rasmussen et al., 2007). The QD621
nanoparticles that accumulated in cytoplasmic vacuoles were
heterogeneous with small agglomerates and were distributed
randomly throughout the cytoplasm. Single QD having the cha-
racteristic QD621 nail shape were localized around the periphery
of the cell membrane and in free cytoplasm or cytoplasmic va-
cuoles. Other reports have stated that the fluorescence of PEG-
coated QD were not detected in the cytoplasm of HeLa cells, but
cationic cadmium telluride (CdTe) QD around cell membranes
were also confirmed by membrane staining with diaminofluor-
escein as a specific green fluorescent dye showing co-localization
TEM for cellular localization or organelle localization. Our TEM
results show the localization on the cellular membrane and within
vacuoles in the cytoplasm. After QD uptake by cells, they formed
lost their basic optical properties due to alterations in their surface
week by assessing the fluorescent signal of QD in perfusate
(unpublished observations). This suggests that QD may degrade
over time in certain buffers or biological media. A blue shift of
in oxidative environments to simulate the biological degradation
even within lysosomes and peroxisomes (Derfus et al., 2004a;
Chang et al., 2006). If the CdS shell degrades and cadmium
leaching from thecoreoccurs,then this could lead to safetyissues
due to the toxic effect of free cadmium to cells and tissues.
The concentration of Cd in 20 μM (where “molarity” here
38,000 μM (Gopee et al., 2007). This is consistent with the
3000 atoms of Cd per quantum dot. After QD was diluted from
stock solution to 0.5 nM, the Cd concentration was 0.95 μM,
which can be detected spectrophotometrically with the signal
greater than background (perfusate buffer). For ICP-OES, the
detection limit is 0.026 μg/ml (0.23 μM), which is four times
more sensitive than fluorescence. Using both techniques, Cd was
not detected in the perfusate.
In summary, this study provides information on QD621
penetration and distribution in skin and toxicity in HEK. Pe-
netration of QD621 into skin is minimal and limited to the
uppermost SC layers and areas near hair follicles. We did not
detect any Cd in the perfusate by ICP-OES or QD by fluore-
scence indicating lack of dermal absorption. QD621 were
localized and cytotoxic at 1.25nM in HEK. Our findings also
demonstrated the irritation potential for inflammation in HEK
posed by QD621. For safety and biomedical applications, the
core, shell, surface coatings, shape, size and charge should be
tested in skin and HEK to minimize QD dermal toxicity or
perturbations to the skin such as an open wound, cut, or alte-
ration to this skin barrier could expose QD to viable skin cells.
Additional tests such as tape stripping or abrasion should be
conducted to determine ifpenetrationto this barrier wouldallow
an enhancement of absorption of QD. Also, if QD are available
for use in biological applications, the lowest concentration of
QD with low toxicity showing a high fluorescence intensity
would be optimal for use in biomedical applications.
The authors would like to thank Dr. Wayne Robarge for
conducting the ICP-OES analysis, and Mr. Alfred Inman for his
helpful suggestions. This research was supported by the U.S.
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