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: oskwon@kist.re.kr

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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