Imaging of the GI tract by QDs loaded heparin-deoxycholic acid (DOCA) nanoparticles

Article abstract of DOI:10.1016/j.carbpol.2012.07.016

This study presents an approach to deliver non invasive, near-IR imaging agent using oral delivery system. Low molecular weight heparin (LMWH)-deoxycholic acid (DOCA)/(LHD) nanoparticles formed by a self-assembly method was prepared to evaluate their physicochemical properties and oral absorption in vitro and in vivo. Near-IR QDs were prepared and loaded into LHD nanoparticles for imaging of the gastro-intestinal (GI) tract absorption. Q-LHD nanoparticles were almost spherical in shape with diameters of 194-217 nm. The size and fluorescent intensity of the Q-LHD nanoparticles were stable in 10% FBS solution and retained their fluorescent even after 5 days of incubation. Cell viability of Q-LHD nanoparticles maintained in the range of 80-95% for 24 h incubation. No damage was found in tissues or organs during animal experiments. The in vivo oral absorption of Q-LHD was observed in SKH1 mice for 3 h under different doses. From the results, we confirmed that Q-LHD was absorbed mostly into the ileum of small intestine containing intestinal bile acid transporter as observed in TEM and molecular imaging system. Our designed nanoparticles could be administered orally for bio-imaging and studying the bio-distribution of drug.

Full text of DOI:10.1016/j.carbpol.2012.07.016

Carbohydrate Polymers 90 (2012) 1461–1468
Contents lists available at SciVerse ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Imaging of the GI tract by QDs loaded heparin–deoxycholic acid (DOCA)
nanoparticles
Zehedina Khatuna, Md. Nurunnabia, Kwang Jae Chob,∗, Yong-kyu Leea,∗
a Department of Chemical and Biological Engineering, Korea National University of Transportation, Chungbuk 380-702, Republic of Korea
b Department of Otolaryngology, Head & Neck Surgery, The Catholic University of Korea, College of Medicine Uijeongbu St. Mary’s Hospital, Kyunggi-Do 480-717, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 December 2011
Accepted 5 July 2012
This study presents an approach to deliver non invasive, near-IR imaging agent using oral delivery sys-
tem. Low molecular weight heparin (LMWH)–deoxycholic acid (DOCA)/(LHD) nanoparticles formed by
a self-assembly method was prepared to evaluate their physicochemical properties and oral absorption
in vitro and in vivo. Near-IR QDs were prepared and loaded into LHD nanoparticles for imaging of the
gastro-intestinal (GI) tract absorption. Q-LHD nanoparticles were almost spherical in shape with diame-
ters of 194–217 nm. The size and fluorescent intensity of the Q-LHD nanoparticles were stable in 10% FBS
solution and retained their fluorescent even after 5 days of incubation. Cell viability of Q-LHD nanopar-
ticles maintained in the range of 80–95% for 24 h incubation. No damage was found in tissues or organs
during animal experiments. The in vivo oral absorption of Q-LHD was observed in SKH1 mice for 3 h
under different doses. From the results, we confirmed that Q-LHD was absorbed mostly into the ileum
of small intestine containing intestinal bile acid transporter as observed in TEM and molecular imag-
ing system. Our designed nanoparticles could be administered orally for bio-imaging and studying the
bio-distribution of drug.
Available online 16 July 2012
Keywords:
Heparin
Oral delivery
GI tract
In vivo imaging
Near-IR QDs
© 2012 Elsevier Ltd. All rights reserved.
1. Introduction
bile acids, such as deoxycholic acid (DOCA) and taurocholic acid
(TCA) (Lee et al., 2006, 2009). Previously, we reported that the
Low molecular weight heparin (LMWH) is a naturally occur-
ring glycosaminoglycan that is widely known as an anticoagulant
agent (Chen et al., 2009; Klement, Du, Berry, Andrew, & Chan,
2002; Lehman & Frank, 2009). LMWH is also used for initial therapy
regarding venous thromboembolism, which is a common compli-
cation of cancer patients (Lee, 2009; Streiff, 2006, 2009). Over the
last few decades, it has been found that LMWH can inhibit can-
cer progression through anti-angiogenesis and tumor metastasis.
The most interesting aspect of using LMWH is that several clini-
cal applications have evidenced no severe toxicity such as bleeding
and heparin-induced thrombocytopenia (Kuderer, Ortel, & Francis,
2009; Simka & Urbanek, 2009). However, the application of LMWH
is possible only through the intravenous (IV) route to avoid its
poor absorption in the intestine; high molecular weight, nega-
tively changed structure, and high water solubility (Kim et al., 2006;
Lee, Nam, Shin, & Byun, 2001; Motlekar, Srivenugopal, Wachtel, &
Youan, 2005). Various types of approaches have been studied such
as enteric coatings, liposomes, and enhancers for the oral delivery of
heparin (Lee, Kim, & Byun, 2000; Lee et al., 2001). Some researchers
have also studied the oral delivery of LMWH by coupling with
LMWH–DOCA conjugate (LHD) has shown excellent absorption
profiles after oral administration to rodents and monkeys (Kim,
Lee, Kim, et al., 2007; Lee et al., 2007; Park, Jeon, Kim, Al-Hilal,
Jin, et al., 2010; Park, Jeon, Kim, Al-Hilal, Moon, et al., 2010; Park,
Kim, et al., 2010). The intestinal absorption of heparin derivatives
appeared due to the coupling of DOCA molecules. This DOCA could
promote intestinal absorption, increase hydrophobic properties of
heparin and interaction between heparin derivative and bile acid
transporters of intestinal membrane (Kim et al., 2005).
Highly fluorescent nano-sized quantum dots (QDs) (5–10 nm)
made of inorganic semiconductor materials (Cd, Te, and Se) possess
several unique optical properties that are ideal for in vivo imaging
(Pan & Feng, 2009; Smith & Nie, 2008). Near-infra red (near-
IR) QDs (emissions of 650–950 nm) have been found to be more
effective than visible QDs (emissions of 450–650 nm) for in vivo
(especially) deep tissue and organ imaging (Jamieson et al., 2007;
Nurunnabi, Cho, Choi, Huh, & Lee, 2010). For the biological applica-
tion of QDs, the surfaces of hydrophobic QDs have been modified by
some biocompatible polymers such as PEG (polyethylene glycol),
mPEG (methoxy polyethylene glycol), PLGA (poly(lactic-co-glycolic
acid)), and PAA (poly(acrylic acid)) (Aldeek et al., 2011; Su et al.,
2010). QDs have been used for sentinel lymph node mapping, tumor
targeting, tumor angiogenesis imaging, and metastatic cell tracking
(Kim et al., 2011; Lee et al., 2010; Zhou et al., 2010). Non-invasive
∗
Corresponding authors. Tel.: +82 43 841 5238; fax: +82 43 841 5220.
E-mail addresses: entckj@catholic.ac.kr (K.J. Cho), leeyk@ut.ac.kr (Y.-k. Lee).
0144-8617/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carbpol.2012.07.016

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Z. Khatun et al. / Carbohydrate Polymers 90 (2012) 1461–1468
imaging is the most promising technique for early diagnosis of can-
cer, angiogenesis and the bio-distribution of drugs (Gao et al., 2010;
Louie, 2010).
heparin (D2O) were: ı 5.38 [H1 of glucosamine residue (A)], ı 5.04
[H1 of iduronic acid residue (I)], ı 4.84 [I-5], ı 4.36–4.23 [A-6], ı
4.12–4.40 [I-3], ı 4.08j [I-4], ı 4.02 [A-5], ı 3.78 [I-2], ı 3.71 [A-
4], ı 3.65–3.69 [A-3], ı 3.24 [A-2]. Values for 1H NMR of aminated
DOCA (D2O) were: ı 1.2–1.9 [m, five and six rings of DOC, 1H], ı
2.1–2.3 [m, CH3 of DOC, 1H], ı 3.15 [d, 12R-OH of DOC, 2H], ı 8.0
[H of CONH]. Values of 1H NMR of LMWH–DOCA conjugates (D2O)
were: ı 1.2–1.9 [m, five and six rings of DOCA, 1H], ı 3.24–5.38 [A
or I of heparin], ı 8.0–8.2 [H of CONH of LMWH–DOCA]. The feed
mole ratio of LMWH and DOCA was controlled to 1:2.6, 1:4, 1:6.7,
and 1:13.3, respectively.
In this study, we prepared near-IR QDs loaded LHD nanoparticles
for noninvasive images of the GI tract. The Q-LHD nanoparticles
were also characterized to measure the size, morphology, stability,
and loading efficacy. Finally, the designed Q-LHD formulation was
orally administered in mice and the absorption of LHD trough the
bile acid transporter was evaluated with molecular imaging system
(KMIS), and TEM. We proposed that LHD nanoparticles can be used
as an oral imaging agent delivery for bio-imaging.
2.3. Characterization of the LHD conjugates
2. Materials and methods
The LHD conjugation was confirmed by the formation of amide
bonds between the carboxylic groups of heparin and the amine
group of DOCA using FT-IR and 1H NMR. For FT-IR, the LHD conju-
gates were scanned as a solid powder. The spectra were Fourier
transformed after 32 scans were acquired with a resolution of
2 cm−1. The chemical conjugation of LMWH and DOCA was proved
by 1H NMR of the amide bond formed between the carboxylic
groups of LMWH with the amine group of DOCA-NH2. For the
1H NMR (JNM-AL400, Jeol Ltd., Akishima, Japan) study, the LHD
conjugates (20 mg) were dissolved in D2O solvent. The size distribu-
tion, shape and morphology of the Q-LHD particles were examined
using dynamic light scattering (DLS) (ELS-Z, Otsuka Electronics
Co., Ltd., Tokyo) and a JEM-100CX TEM (JEOL, Tokyo), respec-
tively. For TEM examination, one drop of Q-LHD was placed on a
thin copper grid. The fluorescent intensities and emission spec-
tra of Q-LHD were investigated by a Varioskan flash (Thermo
Scientific, NY).
2.1. Materials
Low molecular-weight heparin (Fraxiparine or LMWH, MW:
5000 Da) was obtained from Mediplex Corp. (Seoul, Korea).
Sodium deoxycholic acid (DOCA), dimethyl formamide (DMF),
triethyl amine (TEA), 4-nitrophenylchloroformate, absolute
ethanol (EtOH), formamide, 4-methylmorpholine, ethylenedi-
amine, dimethyl sulfoxide (DMSO), N-hydroxysuccinimide (NHS),
N,Nꢀ-dicyclohexylcarbodiimide (DCC), trioctylphosphine oxide
(TOPO), hexadecylamine (HAD), cadmium oxide, selenium pow-
der, and dodecanoic acid were obtained from Sigma–Aldrich,
Co. (St. Louis, MO). Caco-2 cells were collected from the Korea
cell bank (Seoul, Korea). Cell culture reagents including fetal
bovine serum (FBS) and Minimum essential medium (MEM),
were purchased from Sigma–Aldrich, Co. (St. Louis, MO). Sodium
bicarbonate (NaHCO3) was purchased from OCI Co., Ltd. (Seoul,
Korea) N-2-hydroxylethyl-plperazine-Nꢀ-4-butanesulfonic acid
(HEPES) was purchased from Biosesang (Seoul, Korea) and
penicillin–streptomycin was purchased from Gibco BRL (Carls-
bad, CA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) was purchased from Amresco, Inc. (Solon, OH). A
Coatest Heparin FXa assay kit was purchased from Chromogenix
(Milano, Italy).
2.4. Preparation of QDs-loaded LHD nanoparticles (Q-LHD)
For loading of QDs into LHD nanoparticles, 20 mg of LHD
was dissolved in 2.5 mL of formamide; then, different amounts
of QDs solution (0.110 ␮g/mL in chloroform) was added into
the conjugate solution. The mixture was vigorously stirred to
facilitate the hydrophobic QDs to be loaded in the core of the
LHD micelles. Chloroform was evaporated due to stirring in an
open flask after overnight stirring, and the remaining chloroform
was evaporated under reduced pressure. The solution was dia-
lyzed against water for 2 days to exchange the organic solvent
with an aqueous solution. The free unloaded hydrophobic QDs
became aggregated in the aqueous medium and precipitated. The
solution was filtered by a hydrophobic filter to remove the pre-
cipitated QDs. Finally, the product was dried for 48 h by a freeze
dryer.
2.2. Synthesis of the LHD conjugates
The chemical conjugates of LMWH and DOCA were synthesized
by conjugating the hydroxyl group of DOCA with the carboxylic
group of LMWH according to our previous method (Kim et al.,
2006; Lee et al., 2001; Motlekar et al., 2005). For amination of
deoxycholic acid (DOCA, 0.77 mmol) in 5 mL DMSO was reacted
with 4-nitrophenyl chloroformate (4-NPC, 3.86 mmol) and triethy-
lamine (4.63 mmol) for 6 h at room temperature. After reaction,
the precipitant was removed by 0.45 ␮m filter membrane. The fil-
trate was extracted with 25 mL ethyl acetate and 25 mL water.
The crude product from aqueous solution was washed with ethyl
acetate three times, and then DOCA carbonate was obtained as a
powder type after freeze-drying. To obtain aminated DOCA, DOCA
carbonate was reacted with 4-methylmorpholine (1.10 mmol) and
ethylenediamine (0.05 mmol) overnight at room temperature. The
product was concentrated by rotary evaporation and then precipi-
tated by adding acetonitrile.
For preparation of the LMWH–DOCA conjugate, LMWH
(0.02 mmol) was dissolved in water and adjusted to pH 5.0 by
adding 0.1 M HCl solution. The solution was mixed with EDAC
(0.04 mmol), NHS (0.04 mmol), and aminated DOCA (0.044 mmol).
After 30 min, the mixture was dialyzed (MWCO: 2000) against
water to remove unreacted NHS, EDAC, and aminated DOCA. The
final product, LMWH–DOCA, was obtained and stored at 4 ◦C after
freeze-drying. The dried LMWH–DOCA conjugate was analyzed
by 1H NMR and FT-IR (Bruker, Germany). Values for 1H NMR of
2.5. Stability of LHD and Q-LHD
To investigate the stability of LHD and Q-LHD nanoparticles,
we measured the changes in the fluorescent intensity and aver-
age size of Q-LHD nanoparticles in an aqueous solution and serum
over time at three different pH (5, 7 and 9) in 0.1 M PBS buffer,
respectively. LHD and Q-LHD nanoparticles were separately dis-
persed into each pH buffer solution (1 mg/mL) for 2 h. The average
diameter of the LHD and Q-LHD nanoparticles was then measured
using a DLS (ELS-Z, Otsuka Electronics Co., Ltd., Tokyo). The stability
of LHD and Q-LHD nanoparticles in the presence of serum (10% (v/v)
in PBS and FBS) was also measured to evaluate non-specific binding
with proteins. To minimize interference by large molecules in FBS,
the serum solution was filtered using a 0.45 ␮m filter membrane.
The LHD and Q-LHD nanoparticles were then incubated in the FBS
solution for five days, and any change in size was monitored.

Z. Khatun et al. / Carbohydrate Polymers 90 (2012) 1461–1468
1463
2.6. In vitro cytotoxicity study
hydroxyl groups in DOC structure, C3 OH > C12 OH (Geldern et al.,
2004). The coupling ratio of DOCA molecules with LMWH was var-
ied according to the feed mole ratio of DOCA and LMWH. We found
that five moles of DOCA were coupled with LMWH with maximum
(Table 1). The purities of each conjugates were analyzed by liq-
uid chromatography (LC). All products were found over 79% purity
and 43% yield. Among various conjugates, LHD6.7 was selected
for loading of QDs due to its highest yield and purity. LHD conju-
gates formed self-assembled nanoparticles in an aqueous medium
because of their lipophilic property. The average particle size of the
nanoparticles was about 185 nm in diameter. The average size of
the LHD particles decreased with an increasing amount of conju-
gated DOCA due to the increased hydrophobic interaction among
the DOCA molecules in aqueous medium. Critical micelles concen-
tration (CMC) also varied from 0.001 to 0.005 mg/mL among the
different formulations due to variation in the number of coupled
DOCA molecules (the data are not shown). The CMC data has shown
that the CMC decreased according to increasing coupling amount
of DOCA.
The cytotoxicities of the LHD conjugates, water-soluble QDs, and
Q-LHD nanoparticles were examined through human colon cancer
cell line of Caco-2 cells (Korea cell bank). The Caco-2 cells were cul-
tured at 37 ◦C in a humidified atmosphere containing 5% CO2 in a
MEM medium with 10% fetal calf serum. The cells (5 × 104 cells/mL)
grown as a monolayer were harvested by 0.25% trypsin–0.03% EDTA
solution. The cells (200 ␮L) in their respective media were seeded
in a 96-well plate and pre-incubated for 24 h before the assay. The
cell viability of the Caco-2 cell lines was evaluated in the different
groups by the MTT colorimetric assay kit. The Caco-2 cells were
placed in the 96-well plates and incubated for 24 h. After suction,
the complete medium and the sample were placed and again incu-
bated in the referred condition. MTT solution aliquots at 5 mg/mL in
PBS were prepared followed by culture incubation with this solu-
tion at 5% in the culture medium for 4 h in an incubator with a moist
atmosphere of 5% CO2 and 95% air at 37 ◦C. Afterwards, 100 ␮L of the
MTT solubilizing solution was added to each well. After smooth and
slow shaking for 15 min, the MTT colorimetric assay absorbance
was measured at a 570 nm wavelength with a Varioskan flash and
was directly proportional to the cell viability. The cell viability
was expressed as a percentage. It was calculated by the following
equation:
3.2. Loading of near-IR QDs into LHD conjugates
Near-IR QDs has been synthesis according to our previous
methods (Kim et al., 2011). After the conjugation and charac-
terization of LHD conjugates, hydrophobic, highly luminescent
near-IR QDs (wavelength of 661 nm) was loaded into the nanopar-
ticles by solid dispersion methods. In brief, the LDH6.7 conjugate
was dissolved in formamide solution and stirred until the solu-
tion become clear. Chloroform containing QDs solution was added
with the LHD6.7 solution and stirred overnight at ambient condi-
tions. The mixture was dialyzed again DI water for 48 h to remove
the organic solvents. The loaded amount of hydrophobic near-
IR QDs and the florescent intensity of QD-loaded LHD (Q-LHD)
nanoparticles were varied by varying the amount of near-IR QDs
added to the solution. The loading efficiency of Q-LHD series was
changed from 56% to 81%, indicating that strong hydrophobic
interaction between near-IR QDs and conjugated DOCA in LHD.
The result also indicated that the encapsulated QDs endow the
increased hydrophobic property and dense nanoparticle structure
as shown in TEM (Fig. 2A). The QDs loaded LHD nanoparticles with
spherical shape and uniform size distribution were obtained as
shown in ELS data (Table 1). After loading of QDs in LHD6.7, the
average size distribution was varied from 194 nm to 217 nm in
diameter.
ꢀ
ꢁ
sample absorbance
control absorbance
cell viability (%) =
× 100
2.7. Oral absorption and imaging study
To observe the absorption sites of the Q-LHD nanoparti-
cles, SKH1 mice were used, each weighing about 20–25 mg.
After 12 h of fasting, near-IR QD-loaded LHD nanoparticles were
administered at different doses (2.5 mg/kg and 5 mg/kg) by oral
gavage. After 8 h of administration, the mice were dissected and
their tissues of the small intestinal parts were collected and
washed in PBS buffer for 30 min. The tissue from each part was
homogenized with a homogenizer for 3 min and centrifuged at
4000 rpm for 5 min. The supernatant part of the extracts was
collected and the fluorescence was measured by a Varioskan
flash.
To measure the anti-Factor Xa (FXa) activity, the blood
samples were diluted with human normal plasma (100 ␮L),
anti-thrombin III (ATIII) solution (100 ␮L), and DI water and
incubated at 37 ◦C for 3 min. Further, FXa (100 ␮L) was added
to the solution, and the solution was again incubated for 30 s.
3.3. Stabilities of Q-LHD nanoparticles
A
substrate (200 ␮L) was then added to the solution, and
again the solution was incubated for 3 min. Finally, the reac-
tion was ended by adding 300 ␮L of 20% acetic acid. The oral
absorption of Q-LHD was calculated from the absorbance at
405 nm.
Statistics. Statistical analysis was done using ANOVA. p < 0.05
was accepted as statistically significant. Error bars represent stan-
dard deviation.
The stabilities of LHD6.7 and Q-LHD5 against 10% serum and pH
changes were analyzed by measuring the change in the size and flu-
orescent intensity of LHD6.7 and Q-LHD5 nanoparticles (Fig. 2B–D).
After each LHD6.7 and Q-LHD5 was dispersed in PBS buffer with
different pH (5, 7 and 9) for 2 h, the sizes were measured by using
ELS. Our results demonstrated that the pH change had a minimal
effect on the stability of LHD6.7 and Q-LHD5. For example, the mean
diameter of Q-LHD5 was around 195, 220 and 225 nm in pH 5,
6 and 7, respectively. We then examined the change of the size
distribution and fluorescent intensity of Q-LHD5 for 5 days of incu-
bation in the 10% serum (FBS). As shown in Fig. 2D, the average
size was increased a little to 230 nm in diameter, indicating a rel-
ative stability in 10% serum for 5 days. This result may indicate no
severe leakage of QDs from LHD5 nanoparticle. We believe that LHD
nanoparticles have excellent solubility and stability, suggesting a
role as a probe in biological systems imaging. To the best of our
knowledge, this is the first method for delivery of imaging agent
through orally in combined with LHD nanoparticles.
3. Results and discussion
3.1. Synthesis and characterization of LHD
We synthesized LMWH–DOCA conjugates for oral absorption
imaging by linking the LMWH with DOCA (Fig. 1). Conjugation
between the carboxyl groups of LMWH and the amine groups of
DOCA was confirmed by the presence of signals at ı 8.0–8.2 ppm
in the 1H NMR spectrum. The selective modification of C3 OH was
conducted by a suitable protected side chain of C12 OH group.
It also took advantage of the known reactivity order of the two

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Z. Khatun et al. / Carbohydrate Polymers 90 (2012) 1461–1468
Fig. 1. Conjugation of LMWH and DOCA. An orally active LHD conjugate was synthesized by forming an amide bond between the aminated group of DOCA and the carboxyl
group of LMWH.
3.4. In vitro cytotoxicity study
on the cell viability and metabolic activities was not numerically
significant.
To assess the cytotoxicity of Q-LHD5, a Caco-2 cell line was
used. Blank LHD nanoparticles, water-soluble QDs, and Q-LHD5
nanoparticles were incubated with cells with different concentra-
tions (0.1 ␮g/mL, 1 ␮g/mL, 10 ␮g/mL, 50 ␮g/mL and 100 ␮g/mL)
for different time durations (24 h and 48 h). Overall, MTT assay
indicates that cytotoxic effects were not significant because cell
viability was still in the range from 80% to 95% for 24 h incuba-
tion. After 48 h incubation, the cell viability of the Caco-2 cells
was still maintained over 60% after treating of Q-LHD5, no observ-
ing IC50 values in the range of 0.1–100 ␮g/mL (Fig. 3A and B).
Also, according to statistics, the differences among the groups
were not significant because p-values among the groups were
greater than 0.05. Therefore, we believe that the effect of Q-LHD5
3.5. Imaging of oral absorption
Blood samples were collected by retro orbital pulses using
capillary tube at 30 min, 60 min, 120 min, and 180 min after
the oral administration of Q-LHD5 nanoparticles (2.5 mg/kg and
5 mg/kg). After collecting plasma samples, we measured plasma
concentration and fluorescent intensity of Q-LHD5 nanoparticles.
The results showed that the Q-LHD5 nanoparticles were rapidly
absorbed in the intestine and found in the blood circulation at both
doses (Fig. 4A). The maximum plasma concentrations (Cmax) were
0.82 0.19 and 0.95 0.3 IU/mL for 2.5 mg/kg and 5 mg/kg, respec-
tively, where as Tmax was 120 min for both dosages. The effective
Table 1
Characterization of LHD and Q-LHD to measure the coupling ratio, loaded QDs conc., yield, particle size and purity.
Sample name
Feed molar ratio
(heparin:DOCA)
Coupling molar ratio
(heparin:DOCA)
Loaded QDs conc.
(␮g/mL)
Yield (%)
Particle size (nm)
Purity (%)
LHD2.6
LHD4.0
LHD6.7
LHD13.3
Q-LHD1
Q-LHD2
Q-LHD3
Q-LHD4
Q-LHD5
1:2.6
1:4
1:1.20
1:2.18
1:4.28
1:5.79
1:4.28
1:4.28
1:4.28
1:4.28
1:4.28
–
–
–
–
43.1
54.5
64.0
43.5
56.2
60.4
69.0
77.1
81.0
205.5 40
186.9 35
187.6 32
176.2 23
194.1 33
199.4 37
206.1 42
212.6 48
217.1 43
91.87
79.24
97.49
82.25
–
–
–
–
–
1:6.7
1:13.3
1:6.7
1:6.7
1:6.7
1:6.7
1:6.7
0.110
0.165
0.220
0.275
0.330

Z. Khatun et al. / Carbohydrate Polymers 90 (2012) 1461–1468
1465
Fig. 2. TEM image and stabilities of Q-LHD5 nanoparticles. TEM observation (A) of Q-LHD5 and average particle size distribution of LHD (ꢀ) and Q-LHD5 (ꢁ) at different pH
conditions (B). Average particle size distribution (C) and fluorescent intensity (D) of Q-LHD5 in 10% FBS solutions for 5 days.
area under the curve (AUC) was 24.49 6.0 and 37.38 11.0,
respectively. AUC also varied as the dose varied. In our previous
et al., 2010). The data on the oral absorption of Q-LHD5 showed a
similar trend, which indicated that the loaded QDs had no effect on
the oral absorption (Fig. 4B). In our previous study, it was proved
that a higher coupling ratio of DOCA with LMWH increased the oral
study, we found that the effective maximum concentration (Emax
)
increased as the quantity of conjugated DOCA increased (Park, Kim,
100
A 100
80
B
80
60
40
20
60
40
20
0
0
0.1
0.1
1
10
100
50
100
1
10
50
Concentration of Samples (μg/mL)
Concentration of Samples (μg/mL)
Fig. 3. Cell viability of Caco-2 cells after treating LHD6.7 (ꢀ), water-soluble QDs (ꢁ) and Q-LHD5 ( ) nanoparticles for 24 and 48 h, respectively. The data are plotted as
mean SD (n = 3).

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Z. Khatun et al. / Carbohydrate Polymers 90 (2012) 1461–1468
A 0.5
B
1200
0.4
1000
800
600
400
200
0.3
0.2
0.1
0.0
0
30
60
180
30
120
60
120
180
Time (min)
Time (min)
Fig. 4. Plasma concentration (A) and fluorescent intensity (B) after oral administration of Q-LHD5 nanoparticles in mice. The dosage of Q-LHD5 nanoparticles in mice was
controlled to 2.5 (ꢀ) and 5 mg/kg (᭹).
Fig. 5. (A) Ex vivo fluorescent images of the GI tract and organs isolated at 8 h post oral administration of Q-LHD5 nanoparticles. (B) Quantification of the ex vivo fluorescent
intensity of Q-LHD5 nanoparticles. The dosage of Q-LHD5 nanoparticles was controlled to 2.5 (ꢀ) and 5 mg/kg (ꢁ). The mice treated with only PBS buffer set as a control
(n = 3).

Z. Khatun et al. / Carbohydrate Polymers 90 (2012) 1461–1468
1467
Fig. 6. TEM images of isolated (A) stomach, (B) duodenum, (C) jejunum and (D) ileum after oral administration of Q-LHD5 nanoparticles. White arrows indicate the presence
of Q-LHD5 nanoparticles in the tissues of the ileum and jejunum. Scale bar represents 100 nm.
absorption of LHD compared with a lower coupling ratio of DOCA
(Park, Jeon, Kim, Al-Hilal, Jin, et al., 2010; Park, Jeon, Kim, Al-Hilal,
Moon, et al., 2010).
duodenum (1.5–3% of the total absorbed Q-LHD5 nanoparticles).
The GI tract was then observed by TEM to observe the presence of
Q-LHD5 nanoparticles in each region. The data confirmed that the
maximum number of particles was absorbed by the ileum (Fig. 6D)
(Kim, Lee, Lee, et al., 2007). Few numbers of particles were found in
the jejunum (Fig. 6C), and no particles were found in the stomach
and duodenum (Fig. 6A and B). Quantitative analysis of the fluores-
cence intensity indicated that the amount of Q-LHD5 accumulated
at the absorption site was approximately 6–10 folds higher than
that of other organs. These results are in good agreement with TEM
data (Fig. 6).
The effective use of nanoparticles for biomedical imaging would
be visualized when in vivo biodistribution is clearly understood.
From our results, the fluorescent signals at the specific area were
much stronger than in the other organs, which allow for a clear dis-
crimination between organs. This high specific absorption of Q-LHD
nanoparticles might be due to a combination of bile acid transporter
mediated absorption as well as adequate carrier system. Overall,
these results demonstrated that Q-LHD nanoparticles are absorbed
at ileum of small intestine and show potential for use as an imaging
agent for oral absorption study.
The mice were also dissected after the oral administration of dif-
ferent doses of Q-LHD5 nanoparticles to get images of the isolated
GI tract and organs such as the liver, heart, and kidneys by KMIS
for visualizing the absorption site. The KMIS images showed the
strongest fluorescent signal in the ileum, indicating that nanopar-
ticles were absorbed mainly through the ileum due to the presence
of large numbers of the intestinal bile acid transporter (IBAT) (Kim,
Lee, Lee, et al., 2007)). On the contrary, few amounts of nanoparti-
cles were absorbed in the stomach, duodenum, and jejunum where
the fluorescent signal was weaker than in the ileum (Fig. 5A), and
sensitive organs such as the liver and kidneys contained higher flu-
orescence as compared to the heart. The absorption percentages
were measured by a Variskan flash after extraction of the organs
to get the exact, absorbed quantities of Q-LHD5 nanoparticles at
different parts of the small intestine and stomach. The fluores-
cent intensities of the Q-LHD5 nanoparticles were 62% and 75% for
the 2.5 mg/kg dose, and 65% and 90% for the 5 mg/kg dose in the
jejunum and ileum, respectively (Fig. 5B). The fluorescent inten-
sities of the nanoparticles in the stomach and duodenum were
negligible, viz., below 1% and 2% for 2.5 mg/kg and below 1.5% and
3% for 5 mg/kg, respectively. The maximum absorption amounts
of Q-LHD5 nanoparticles were found in the jejunum and ileum
(62–90% of the total absorbed Q-LHD5 nanoparticles) in the case of
both the 2.5 mg/kg and 5 mg/kg doses, whereas a negligible amount
of Q-LHD5 nanoparticle absorption was found in the stomach and
4. Conclusions
Near-IR QDs loaded LHD nanoparticles was successfully pre-
pared for noninvasive images of the GI tract. The designed Q-LHD
formulation was orally administered in mice and the absorp-
tion of LHD trough the bile acid transporter was evaluated with

1468
Z. Khatun et al. / Carbohydrate Polymers 90 (2012) 1461–1468
molecular imaging system (KMIS) and TEM. Q-LHD nanoparticles
were absorbed at ileum of small intestine and show potential for
use as an imaging agent for oral absorption study. The designed
carrier for oral delivery imaging has been proven to be safe and
effective for imaging GI absorption with a constant oral absorption
and pharmacokinetics profile.
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Acknowledgements
Lee, D. Y., Kim, S. Y., Kim, Y. S., Son, D. H., Nam, J. H., Kim, I. S., et al. (2007). Suppression
of angiogenesis and tumor growth by orally active deoxycholic acid–heparin
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This research was supported by Basic Science Research Pro-
gram through the National Research Foundation of Korea (NRF)
funded by the Ministry of Education, Science and Technology
(2010-0021427).
Lee, Y.-k., Nam, J. H., Shin, H.-c., & Byun, Y. (2001). Conjugation of low-molecular-
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