effects of folic acid and polyethylene glycol coated quantum dots on toxicity and tissue uptake to...
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RESEARCH ARTICLE
Effects of folic acid and polyethylene glycol coated quantum dotson toxicity and tissue uptake to precision-cut spleen slices of rats
Md. Mamunul Haque • Hye-Yeon Im • Ji-Eun Seo • Mahbub Hasan •
Kyoungja Woo • Oh-Seung Kwon
Received: 23 April 2013 / Accepted: 14 June 2013
� The Korean Society of Pharmaceutical Sciences and Technology 2013
Abstract Nano-sized materials are increasingly used in
cosmetics, diagnosis, imaging, and drug delivery. It also
involved in specific functionality with lymphoid systems.
However, the toxicity and mechanisms of quantum dots
(QDs) uptake into mammalian cells are poorly understood.
Our study was to investigate the toxicity and tissue uptake
of polyethylene glycol-folic acid-conjugated (PEG-FA),
and only polyethylene glycol-conjugated (PEG) cadmium
selenide/cadmium sulfide (CdSe/CdS) QDs using preci-
sion-cut spleen slices of Sprague–Dawley (SD) rats. QDs
were treated with different doses (0–300 nM) to the spleen
of SD rats, and their toxic effects and tissue uptake were
examined by LDH, NADPH oxidase, and histological
analyses. No dose-dependent changes in LDH were
observed. But high uptake of the QD-PEG-FA into spleen
slices was observed by fluorescence microscopic exami-
nation in dose-dependent manner, while most of the QD-
PEG was found on the edge of the slices. The NADPH
oxidase activity was increased at high dose (300 nM) in
both QD-PEG-FA- or QD-PEG-treated spleen slices indi-
cating oxidative stresses. No damages were noticed in
histological study confirming no toxicity in both types of
QDs. Based on the above observations, we may conclude
that surface coating property is an important factor in
determining QDs uptake into mammalian cells. These
findings provide insight into the specific mechanism of
QDs uptake in cells.
Keywords CdSe/CdS quantum dots �Precision-cut spleen slice � In vitro � Toxicity �Tissue uptake
Introduction
The recent development of technology for reducing material
size has provided innovative quantum dots (QDs). QDs are
engineered structures with core size of several nanometers
(Courty et al. 2006), but can be as large as 100 nm when
organic shells and/or conjugated bio-recognition molecules
are placed onto their surface (Fischer et al. 2006). They have
unique physicochemical properties like size, shape, chemi-
cal composition, solubility, surface structure, and aggrega-
tion. QDs have been widely used in diverse field mainly
ultra-sensitive molecular sensing and imaging probes,
pharmaceutical agents, and cosmetics (Nishimori et al.
2009), targeted diagnostic cancer-imaging agents (Toku-
masu et al. 2005; Gao et al. 2004), optically guided surgery
(Kim et al. 2004), and hyperthermia therapy (Voura et al.
2004). However, the use of particle from micro- to nano-
scale not only provides benefits to diverse scientific fields,
but also poses potential risks since the responses of biolog-
ical systems to nano-sized novel materials have not been
adequately studied (Choi et al. 2009). It was also shown that
Md. M. Haque � H.-Y. Im � J.-E. Seo � M. Hasan �O.-S. Kwon (&)
Toxicology Laboratory, Korea Institute of Science and
Technology (KIST), Hwarangno 14-gil 5, Seongbuk-ku,
Seoul 136-791, Korea
e-mail: [email protected]
Md. M. Haque � J.-E. Seo � M. Hasan � O.-S. Kwon
Biological Chemistry, University of Science and Technology
(UST), Daejon 305-333, Korea
H.-Y. Im
School of Life Sciences and Biotechnology, Korea University,
Seoul 136-701, Korea
K. Woo
Nano-Materials Research Center, Korea Institute of Science and
Technology (KIST), Seoul 136-791, Korea
123
Journal of Pharmaceutical Investigation
DOI 10.1007/s40005-013-0082-3
nanoparticles cause serious effects like they can alter the
structure and function of pulmonary surfactant compared to
microparticles (Schleh et al. 2009). Additionally, nanosized
particles are toxic potential to the human health and the
environment that is not present in the bulk material (Worle-
Knirsch et al. 2007). Therefore, it is thus essential to
understand the biological activities and potential toxicity of
QDs.
Usually, QDs can lead to potential risks, which stem
from three basic factors. First, the toxic effects associated
with their physicochemical properties i.e., size, surface
chemistry, redox potential and so on (Li and Chen 2011;
Oberdorster 2004). Second, the breakdown of QDs can lead
to metal-induced toxicity within the cells, and this toxicity
is highly dependent on the chemical design of the QDs
(Kirchner et al. 2005; Derfus et al. 2004). The third is due
to the high surface-area-to-volume ratio of the QDs, which
provides a large available surface for enzymatic degrada-
tion and release of metallic ions (Li and Chen 2011).
Therefore, the toxicity of QDs due to the presence of heavy
metal atoms at their core, however, remains a barrier.
Although the size and charge of nanomaterials are impor-
tant when probing for toxicity, the inherent toxic elemental
composition of QDs is of primary concern (Smith et al.
2008). So, surface modification has been the major issues
in utilizing semiconductor nanocrystals (quantum dots) for
biological applications. Aggregation-free functional QDs
are of great importance for their delivery to a specific site
for biomedical diagnosis (Moon et al. 2009).
To avoid unexpected side-effects in human, appropriate
in vitro techniques that reflect in vivo situation in both men
and animals are needed. Translational research models are
emerging which begin to bridge the gap between preclin-
ical animal studies and human risk assessment. Major
breakthroughs in this area include the advancement of or-
ganotypic in vitro models (Vickers 2009).
The spleen is universally found in all vertebrates. It is
located in the left top quadrant of the abdomen in human. It
has immense functions in relation to red blood cells and the
immune system including the removal of old red blood cell
(RBC), and recycles iron. By structure, it is divided into
two distinct components, the red pulp (RP) and the white
pulp (WP). The RP consists of large numbers of sinuses
and sinusoids packed with blood, and are responsible for
spleen filtration. The WP consists of lymphoid tissue
aggregates, and is responsible for spleen immunological
function. There is one special zone in spleen named mar-
ginal zone (MZ) where most of the nanoparticles are
uptake (Demoy et al. 1997, 1999a). The uptake of nano-
particles by spleen is species variant and also depends on
nanoparticles composition (Demoy et al. 1999b) and
incubation time. Usually in vivo experiment takes long
time to induce QDs toxicity, however, Demoy et al.
(1999a) showed significant in vitro effects of QDs to spleen
slices after 3 h of incubation. In this experimental design,
the QDs dose and incubation time was optimized, and were
finalized to 300 nM and 4 h respectively.
Precision-cut slice is such type of model which means
that tissues can be cut with a consistent thickness of around
0.3 mm and with minimal cutting-induced damages (Smith
et al. 1985). So, the use of precision-cut slices is a very
good option in organotypic culture due to its architecture
(de Kanter et al. 2002).
The aim of the present study was to compare the spleen
capture of fluorescent labeled two QDs differ in their sur-
face structure, and to assess their potential toxicity in vitro.
This study mainly focused on the splenic uptake of poly-
ethylene glycol-folic acid-conjugated (PEG-FA), and only
polyethylene glycol-conjugated (PEG) cadmium selenide/
cadmium sulfide (CdSe/CdS) QDs, and whether these QDs
induce any toxicological and pathological effects in rat
spleen slices in vitro.
Materials and methods
Preparation of quantum dots
Quantum dots were prepared as *5.5 nm core of cadmium
selenide/cadmium sulfide (CdSe/CdS) with 2.5 monolayer
shell composed of Cd/S/Cd/S/Cd protected with octadec-
ylamine (QD-ODA) in Nano-Materials Research Center,
Korea Institute of Science and Technology (KIST), Seoul,
Korea (Moon et al. 2009). ODA ligands on the QDs surface
were then replaced by excess decylamine (DA) to produce
QD-DA. The polyethylene glycol (PEG, Mw = 1 kDa)
and folic acid (FA) were added sequentially in CdSe/CdS
to get CdSe/CdS-PEG-FA. The resultant QDs were red
fluorescing and hydrophilic in distilled water
(1 9 10-6 M, 10 ml; Mw & 380,000).
Experimental animal
All experiments were done in Sprague–Dawley (SD) rat
aged between 6 and 8 weeks having weighed around
250–280 g purchased from Orient Bio Inc., (Sungnam,
Kyunggi, Korea).
Equipment and chemicals
Vibratome (The Vibratome Company, St. Louis, MO,
USA), homogenizer (Wheaton overhead stirrer, Fisher
Scientific, NJ, USA), luminescence spectrometer (LS 50-B,
PerkinElmer, MA, USA), microplate reader (680, Bio-Rad
Laboratories, CA, USA), centrifuge (5415 R, Eppendorf
AG, Hamburg, Germany) and UV–vis spectrophotometer
Md. M. Haque et al.
123
(Cary 1E, Varian, CA, USA) were used for biological
experiments.
b-Nicotinamide adenine dinucleotide phosphate hydro-
gen (b-NADPH), nicotinamide adenine dinucleotide
hydrogen (NADH), sodium pyruvate, dihydroethidium
(DHE), ethidium bromide, salmon testes DNA, bicinch-
oninic acid (BCA) and copper(II) sulfate were purchased
from Sigma Chemicals (St. Louis, MO, USA). Paraffin
(McCormick Scientific, St. Louis, MO, USA), HPLC grade
xylene (J.T. Baker, Philipsberg, NJ, USA) and ethanol
(Merck KGaA, Darmstadt, Germany) were used for his-
tology. Dulbecco’s modified eagle’s medium (DMEM)
with some modifications were used as incubating media
(Demoy et al. 1999a).
Preparation of precision-cut spleen slices
Animals were sacrificed by decapitation and spleen was
collected after trimming fat and connective tissues and kept
in cold Krebs–Henseleit (KHS) buffer (pH 7.4). Cylindrical
tissue cores (5 mm) were made by pushing rotating thin-
wall sharpened tubing through the tissue. The obtained
tissue cores were then sliced using a vibratome at 250 lm
thickness. Before experiments, tissue slices were gently
flushed out by a constantly stream of buffer and incubated
in DMEM media with gentle rocking for 1 h.
In vitro QDs treatment
Spleen slices were taken in a 24 well plate having one slice
per well containing 1 ml DMEM media (with 10 % fetal
bovine serum, FBS). Slices were treated with various
concentrations (0–300 nM) of CdSe/CdS-PEG-FA, and
CdSe/CdS-PEG QDs and kept in incubator (5 % CO2 and
37 �C) with gentle agitation on a rotating table for 4 h.
Tissue homogenization
The incubated slices were then homogenized by following
a modified method described elsewhere (Faddah et al.
2007). Briefly, slices were homogenized by using a
homogenizer in a buffer containing 5 mM Tris–Cl (pH 7.4)
and 150 mM NaCl. Homogenates were centrifuged at
15,0009g for 30 min at 4 �C, and collected supernatants
were stored at -80 �C for different biochemical assays.
Protein assay
Total protein concentrations were measured by BCA assay
with bovine serum albumin (BSA) as a standard. Samples
were mixed with freshly prepared assay solution (BCA:
CuSO4�5H2O = 50:1) for the colorimetric quantitation of
total protein concentration. After 30 min of incubation at
37 �C in dark, absorbance was taken at 540 nm by a
microplate reader.
LDH assay
LDH activity of the incubated media from QDs-treated
slices were measured based on a modified method reported
elsewhere (Legrand et al. 1992). Samples were mixed with
LDH substrate mixture containing 50 mM phosphate buf-
fer (pH 7.4) and 0.18 mM NADH supplemented with
0.6 mM sodium pyruvate. The excitation was done at
340 nm for 3 min by using a spectrophotometer. The mean
extinction diminution per min was divided by molar
absorptivity of NADH (6317 l/mol cm at 340 nm and
25 �C (McComb et al. 1976) to determine the activity of
LDH in each sample. All standards and samples were run
in duplicates and the LDH oxidase activity was quantified
in comparison with a standard curve.
NADPH oxidase assay
A fluorescent method was used using DHE as substrate for
measuring enzymatic activity of nicotinamide adenine
dinucleotide phosphate (NADPH) oxidase (Cao et al.
2007). In Brief, the homogenates were incubated with
salmon testes DNA (0.5 mg/ml), b-NADPH (0.1 mmol/l)
and DHE (10 lmol/l), at 37 �C for 30 min in the dark
chamber. NADPH oxidase enzyme results in superoxide
(O2•-) formation by using b-NADPH as a substrate. The
resultant superoxide then oxidizes DHE and yields fluo-
rescent ethidium. Then ethidium intercalate with DNA to
further amplify the fluorescent signal whose intensity is
proportional to O2•- production. The reactions were stop-
ped by adding 50 mM NaHCO3 (pH 10.6) and fluorescence
were measured at an excitation of 485 nm and emission of
590 nm with a luminescence spectrometer using FL Win-
LabTM software (version 3.4.00.02, PerkinElmer, MA,
USA). All standards and samples were run in duplicates
and the NADPH oxidase activity was quantified in com-
parison with a standard curve.
Histological analysis
Histological observations were performed according to the
standard laboratory procedures. After 4 h incubation, QDs
treated slices were fixed in 10 % formalin, embedded in a
paraffin block and sliced into 5 lm thickness by using a
microtome. The slices were placed onto glass slides and
visualized under a fluorescence microscope (Olympus
BX51, Olympus Corporation, Tokyo, Japan) for the
localization of the QDs. Later, the same slices were stained
with hematoxylin–eosin (H–E) and subsequently processed
Effects of folic acid and polyethylene glycol
123
for histopathological examination under a light microscope
(Nikon Eclipse 50i, Nikon Corporation, Tokyo, Japan).
Statistical analysis
Each of the experimental value was compared with the
control. Data were presented as mean ± standard error of
the mean (SEM). Statistical significances between the
QDs-treated and control group were assessed by one way
analysis of variance (ANOVA) using the generalized linear
model (GLM) procedure of SAS (SAS Institute Inc., Cary,
NC, USA) to evaluate the differences of mean among
multi-group data. Duncan’s multiple range tests was per-
formed for comparing the datasets between groups. The
value of p \ 0.05 was considered statistically significant.
Results and discussion
The main reason for developing nanotechnology is to
extend the limits of sustainable development at the nano-
scale with less consumption of energy, water and materials.
Current approaches strongly suggest that consequences of
nanotechnology are best addressed within the existing
system applications such as biology, chemistry or elec-
tronics. Meanwhile, potential public and occupational
exposures of manufactured nanomaterials have been
increased dramatically because of their improved quality
and performances in many consumer products as well as
medical therapies. Therefore, information regarding the
toxicity of manufactured nanomaterials must be explored.
To reduce the use of experimental animals as well as to
reduce the costs of drug development, and also to get more
insight of the action and fate of novel compounds in
human, the use of in vitro slicing systems is a promising
approach. Although in vitro systems are powerful, it will
never replace in vivo studies completely because of several
complex interactions are missing during handling in vitro
(de Kanter et al. 2002) as cells are cultured without natural
embedding and cellular environment, in the absence of
blood flow, lack of transporter proteins, and deficiency of
intrinsic catalytic activity etc.
Viability of cells in spleen slices
Protein is the most vital biochemical constituent in all
biological systems. It plays a crucial role in the synthesis of
many enzymes and helps maintain a stable condition in the
animal body. Protein synthesis is one of the core functions
of cells, and up-down regulation of genes in response to
extra- and intra-cellular signals represents one of the main
areas of study when evaluating toxicity. After treatment of
some drugs, QDs or any other foreign substances to cells,
they show some chemical reactions. The dramatic changes
can be detected in the protein levels.
In our study, Table 1 indicates that total protein con-
centrations of QDs-treated slices and incubated media did
not changed significantly with control even at high con-
centration (300 nM) of QDs. But changes in protein pro-
duction do not linked with just one system alone.
Variations in protein synthesis due to QDs interactions can
occur in subcellular components such as the nucleus,
endoplasmic reticulum, ribosomes, cytoskeleton and cyto-
plasm. Therefore, measurements of protein concentrations
are not enough for the complete understanding of the toxic
effects of QDs on cells.
Another important change can be observed on cell via-
bility. Lactate dehydrogenase (LDH) is an essential oxido-
reductase enzyme found in mitochondria, and its activity
increases in case of loss of the mitochondrial function.
LDH catalyzes the inter-conversion of lactate and pyruvate
with concomitant interconversion of NADH and NAD?.
When disease, injury or toxic material damages tissues,
LDH is released upon the damages of plasma membranes.
So, LDH activity level often serves as an index of cellular
damages (Choi and Lee 2004; Nemmiche et al. 2007).
The incubated media of QDs-treated spleen slices were
mixed with LDH substrate mixtures and the excitation was
detected. The changes of LDH activity in QDs-treated
slices are shown in Fig. 1 and it was found that no statis-
tically significant differences were observed between QDs-
treated slices and control slice.
So, protein concentration assay (Table 1), and LDH
assay (Fig. 1) did not show any significant changes in
different concentrations of QDs indicating CdSe/CdS QDs
may have no or very low toxicity whether it is coated by
PEG-FA, or only by PEG.
In vitro localization and spleen uptake of QDs
The outer surfaces of QDs are important in organ-specific
distribution after administration into mice. QDs were varied
in their uptake into the liver, spleen, lung, kidneys, and bone
marrow on the basis of their surface moieties (Fischer et al.
2006). Addition of PEG polymer to QDs surface was dem-
onstrated to prolong particle circulation in the blood and
significantly decrease their uptake by spleen and liver resi-
dent phagocytes. PEG effectively prevents plasma proteins
from adhering to the particle surface by creating a steric
shield around the particle, and thus avoids uptake by
mononuclear phagocytes (Dobrovolskaia et al. 2008). The
incorporation of PEG as QDs surface coating reduced their
non-specific binding to several types of cells (Zhang et al.
2008), also PEG-coated QDs produces differential tissue and
organ deposition in mice in a time- and size-dependent
manner (Ballou et al. 2004). Focusing this issue, QDs-treated
Md. M. Haque et al.
123
thin tissue sections (5 lm) were observed under fluorescence
microscope. Image analysis study (Fig. 2) showed high
uptake of CdSe/CdS-PEG-FA QDs by the spleen tissue in a
dose-dependent manner (Fig. 2g, h) while CdSe/CdS-PEG
was found on the periphery of the spleen (Fig. 2d). Anti-
bodies (Gao et al. 2004), peptides (Akerman et al. 2002),
folic acid (Meng et al. 2011), or small-molecule ligands are
attached to QDs for targeting specific proteins on cells.
Folate receptors (FR) can bind to FA derivatives and medi-
ates their delivery to the interior of cells. Folate receptor beta
is a protein that is encoded by FOLR2 gene, and this protein
is detected in the spleen (Kawasaki and Fearnhead 1975).
Many other FR are also identified in the spleen (Shen et al.
1994). So, FA coated CdSe/CdS QDs are capable of inter-
nalizing into spleen, and this endocytosis process could be
carrier dependent. This study suggests the physicochemical
parameters may affect QDs uptake into cells, but the precise
mechanism of QDs endocytosis and internalization into the
cellular organelles may require extensive investigation with
receptor recognition.
Measurement of NADPH oxidase activity
In a normal healthy body, the generation of pro-oxidants
in the form of reactive oxygen species (ROS) and reactive
nitrogen species (RNS) is effectively checked by various
antioxidants defense levels. However, when a body gets
exposed to adverse toxic materials, ROS levels are
increased dramatically resulting in oxidative stresses
(Devasagayam et al. 2004). Moreover, ROS hyper-pro-
duction by neutrophils causes direct tissue damages in a
broad range of inflammatory diseases (Lambeth 2007).
The secretion of ROS is one of the main responses when
macrophages are contacting with foreign bodies such as
the QDs. As macrophages and their responses to QDs are
important to understand their toxicity, the secretion of
ROS is a key area of active research (Pulskamp et al.
2007; Shukla et al. 2005). So, ROS are considered to be
important factors in the pathogenicity of QDs. ROS are
produced by amino-catalyzed reactions during the acti-
vation of phagocytic NADPH oxidase (Halliwell and
Gutteridge 1985).
Tissue homogenates were incubated with NADPH oxi-
dase assay mixture and the formation of O2•- was detected
in the reaction mixture. From Fig. 3, it was clear that
NADPH oxidase activity was increased at high concen-
tration (300 nM) of both the QDs compared with control
indicating that oxidative stresses were. Though CdSe/CdS-
PEG do not uptake into spleen cells yet it can induce
oxidative stress. From this point, it can be mentioned that
core/shell element is the crucial factor rather than coatings
of the QDs for persuading oxidative stresses.
Table 1 Protein concentration
of control and QDs-treated
precision-cut rat spleen slices
Data are presented as
mean ± SEM (n = 5 slice per
group)
Quantum dots
concentration
Experimental QDs
CdSe/CdS-PEG (lg/ml) CdSe/CdS-PEG-FA (lg/ml)
Slice Media Slice Media
0 nM 598.3 ± 23.4 3395.0 ± 22.5 630.2 ± 23.4 3406.1 ± 21.3
3 nM 595.4 ± 51.9 3416.8 ± 27.5 675.6 ± 30.0 3352.7 ± 20.3
30 nM 530.6 ± 41.1 3388.5 ± 14.4 597.1 ± 24.4 3407.4 ± 25.6
300 nM 572.1 ± 31.6 3372.8 ± 26.8 605.1 ± 12.8 3335.2 ± 17.8
Fig. 1 Effects of CdSe/CdS-
PEG, and CdSe/CdS-PEG-FA
QDs on protein concentration.
The spleen slices were treated
with different concentrations
(0–300 nM) of CdSe/CdS-PEG,
and CdSe/CdS-PEG-FA QDs
for 4 h at 37 �C. Protein
contents of CdSe/CdS-PEG (a),
and CdSe/CdS-PEG-FA (b)-
treated slices are illustrated.
Values are represented as
mean ± SEM (n = 5 slice per
group)
Effects of folic acid and polyethylene glycol
123
Histopathological analysis
Histological analysis was performed on the QDs-treated
tissue slices to assess the signs of potential toxicities and
tissue abnormalities. Hematoxylin–Eosin staining is the
standard histological staining method that gives an overview
of the tissue structure. Slices were fixed in formalin (10 %),
paraffin embedding, sectioned and stained with H–E for
Fig. 2 Tissue distribution of
CdSe/CdS-MPA QDs in various
organs of mice. After 4 h
incubation with CdSe/CdS-PEG
(a–d), and CdSe/CdS-PEG-FA
(e–h) at different doses
(0–300 nM), 5 lm sections
were observed under
fluorescence microscope
(4 9 magnification and scale
bar of 5 lm). Panels I and J are
the magnified micro-
photographs of insets of (d, h)
respectively
(40 9 magnification and scale
bar of 1 lm). Arrows indicate
QD particles observed in the
microphotograph
Fig. 3 Slices were treated with different concentrations (0–300 nM)
of CdSe/CdS-PEG (a), and CdSe/CdS-PEG-FA (b). After 4 h of slice
incubation with QDs, NADPH oxidase assay was performed, using b-
NADPH as substrate. Results represent NADPH oxidase activity in
control and QDs-treated groups. Mean ± SEM values are the average
of duplicate runs of each slice (n = 5 slice per group). Histogram
with asterisk (*) denotes mean value that is statistically different at
p \ 0.05. Statistical was assessed by one-way ANOVA using the
GLM procedure of SAS
Md. M. Haque et al.
123
nuclear and cytoplasmic staining of cells. No clear tissue/
cellular damages or abnormalities were observed in both the
QDs treatment (Fig. 4). Here we only showed the control and
300 nM QDs-treated slices as QDs were mostly found in that
dose. The remaining results of other dosed groups (3 and
30 nM) were identical, therefore, the data are not shown. As
no damages were noticed in histological study (Fig. 4), we
can conclude that CdSe/CdS QDs coated with PEG alone or
PEG-FA is not toxic to spleen tissue of SD rat in vitro.
Conclusions
From the above discussions, we may conclude that PEG
coated CdSe/CdS QDs do not uptake by spleen in SD rat
whereas CdSe/CdS QDs when coated with FA in combi-
nation with PEG, can easily uptake. So, surface coating
properties of QDs are very much crucial factors in deter-
mining its interaction with mammalian cells. The NADPH
oxidase activity was increased at high dose (300 nM) of
both the QDs indicating both QDs can induce oxidative
stresses. On the other hand, no abnormal alterations in the
tissue structure were observed even at high dose of both the
QDs. This study might be help to gain a better under-
standing of the complex interactions between spleen tissue
and CdSe/CdS QDs in accordance with FA and polyeth-
ylene glycol coatings.
Further studies of relationship between toxicity, and
variety of sizes, shapes, concentrations and chemical
modifications on the surface of QDs are needed, and the
Fig. 4 Histological analysis of QDs-treated rat spleen slices. CdSe/
CdS-PEG, and CdSe/CdS-PEG-FA with different concentrations
(0–300 nM) were treated to rat spleen slices. After 4 h of QDs
incubation, 250 lm slices were fixed with 10 % formalin, paraffi-
nized. Then the deparaffinized tissue sections (5 lm) were stained
with H-E solution, and observed under light microscope. Micro-
photographs represent control group (a, d), CdSe/CdS-PEG (b, e), and
CdSe/CdS-PEG-FA (c, f); magnified scales of a, b, c (10 9 magni-
fication and scale bar of 1 lm), and d, e, f (40 9 magnification and
scale bar of 500 nm). Histological photographs of the control and
300 nM QDs-treated slices are illustrated here as this group contained
large number of QDs
Effects of folic acid and polyethylene glycol
123
potential studies based on these data will provide very
useful information about development of drug delivery
systems using nano-sized materials.
Acknowledgments This work was supported by the project of
Korea Institute of Science and Technology (Project No. 2V02150).
Md. M. Haque, HY Im, JE Seo, M Hasan, K Woo and OS Kwon
declare that they have no conflict of the interest.
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