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Preparation of Sodium Deoxycholate (DOC) Conjugated Heparin
Derivatives for Inhibition of Angiogenesis and Cancer Cell Growth
Kwang Jae Cho, Hyun Tae Moon, Go-eun Park, Ok Chul Jeon, Youngro Byun, and Yong-kyu Lee
Bioconjugate Chem., 2008, 19 (7), 1346-1351• DOI: 10.1021/bc800173m • Publication Date (Web): 28 June 2008
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Preparation of Sodium Deoxycholate (DOC) Conjugated Heparin
Derivatives for Inhibition of Angiogenesis and Cancer Cell Growth
Kwang Jae Cho,†,‡Hyun Tae Moon,†,§Go-eun Park,‡Ok Chul Jeon,§Youngro Byun,*,|and Yong-kyu Lee*,⊥
Department of Otolaryngology, Head and Neck Surgery, The Catholic University of Korea, College of Medicine Uijeongbu, St.
Mary’s Hospital, Kyunggi-Do 480-717, Korea, Mediplex Corporation, Seoul 135-729, Korea, College of Pharmacy, Seoul
National University, San 56-1, Sillim-dong, Gwanak-gu, Seoul 151-742, Korea, and Department of Chemical and Biological
Engineering, Chungju National University, Chungbuk 380-702, Korea. Received April 28, 2008; Revised Manuscript Received
May 19, 2008
We describe new DOC (sodium deoxycholate)-heparin nanoparticles for in ViVo tumor targeting and inhibition
of angiogenesis based on chemical conjugation and the enhanced permeability and retention (EPR) effect. Heparin
has been used as a potent anticoagulant agent for 70 years, and has recently been found to inhibit the activity of
growth factors which stimulate the smooth muscle cells around tumor. From the results, DOC and heparin were
conjugated by bonding carboxyl groups of heparin with amine groups of aminated sodium deoxycholate. Larger
antitumor effects of the DOC-heparin VI (8.5 mol of DOC coupled with 1.0 mol heparin) were achieved in
animal studies, compared to heparin alone. We confirmed that the conjugated heparin retained its ability to inhibit
binding with angiogenic factor, showing a significant decrease in endothelial tubular formation. These results
provide new insights into the nontoxic anticancer drug carrier as well as the design of multifunctional bioconjugates
for targeted drug delivery.
Tumors produce a number of growth factors stimulating
angiogenesis via affinity for receptors on endothelial cells. Low
molecular weight heparin (LMWH) appears to have a greater
inhibitory effect on angiogenesis than unfractioned heparin (1–4).
It has been shown that LMWH, as a result of its smaller size
and in contrast to unfractioned heparin, can reduce binding of
growth factors to their receptors. Fragments of fewer than 18
saccharides reduce the activity of vascular endothelial growth
factor (VEGF), and fragments of fewer than 10 saccharides
inhibit the activity of basic fibroblast growth factor (bFGF) (5, 6).
Small molecular heparins (smaller than 6000 Da) are more
effective than unfractionated heparin (UFH) for inhibition of
VEGF- and bFGF-mediated angiogenesis in ViVo (3, 4).
Heparin has highly hydrophilic properties due to the nega-
tively charged group such as sulfonyl, carboxyl, and hydroxyl
within its structure (7–9). To treat tumor with heparin, we need
new structure designs to allow the use of a reduced dose to
achieve the same therapeutic response with a consequent
decrease in systemic toxicity and side reactions. Recently, a
heparin-based drug carrier has been developed to deliver an
anticancer drug to tumor sites by the EPR effect (10, 11). In
our previous study, a chemically modified unfractionated heparin
derivative for delivery of the anticancer doxorubicin was
developed and proven a safe drug carrier without the risk of
inducing hemorrhage and other side effects (11). Other studies
have developed polysaccharides such as chitosan and curdlan
as anticancer drug carriers with inhibition of tumor growth
In this study, we propose a new anticancer drug conjugate
system for the treatment of tumor and tumor vasculature by
using DOC-heparin nanoparticles. To obtain the optimized
anticancer effect of heparin, we designed DOC-heparin con-
jugates by modification of the C3-hydroxyl group of DOC with
4-nitrophenyl chloroformate (4-NPC) and ethylenediamine
(EDA). Through the conjugation method, many DOC molecules
were allowed to bind freely with one heparin structure. In
addition, the conjugated heparin retained its ability to inhibit
binding with angiogenic factors, which can induce proliferation
of smooth muscle cells. The rationale for conjugating DOC to
the polysaccharide heparin was to specifically target tumor and
tumor vasculature and to circumvent adverse reactions from drug
toxicity and bleeding by reducing the binding affinity with
anticoagulant factors such as factor Xa.
MATERIALS AND METHODS
Materials. Fraxiparin (101 IU/mg, heparin) of average
molecular weight ca. 5000 Da was purchased from GlaxoSmith-
Kline (Brentford, Middlesex, UK). Sodium deoxycholate (DOC),
4-nitrophenyl chloroformate, triethylamine, dicyclohexylcarbo-
diimide (DCC), hydroxysuccinimide (HOSu), 4-methylmorpho-
line, ethylene diamine, dimethyl sulfoxide (DMSO), and ethyl
acetate were purchased from Sigma Chemical Co. (St. Louis,
MO). Formamide was obtained from Merck (Darmstadt, Ger-
many). Coatest Factor Xa assay kits were from Chromogenix
Tel.: +82-2-880-7866, Fax: +82-2-872-7864 (Y. Byun). Tel.: +82-
43-841-5224, Fax: +82-43-841-5220 (Y. Lee).
†Dr. Cho and Dr. Moon are equal contributors to this article.
‡The Catholic University of Korea.
|Seoul National University.
⊥Chungju National University.
Bioconjugate Chem. 2008, 19, 1346–1351
2008 American Chemical Society
Published on Web 06/28/2008
(Milano, Italy). All reagents were of analytical grade and were
used without further purification.
Preparation of DOC-Heparin Conjugates (Figure 1). For
amination of sodium deoxycholate (DOC), DOC (0.93 mmol)
in 5 mL DMSO was reacted with 4-nitrophenyl chloroformate
(4-NPC, 4.65 mmol) and triethylamine (5.58 mmol) for 6 h at
room temperature. After reaction, the precipitant was removed
by 0.45 µm filter membrane. The filtrate 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 DOC carbonate was obtained as a powder type after
freeze-drying. To obtain aminated DOC, DOC carbonate was
reacted with 4-methylmorpholine (1.42 mmol) and ethylenedi-
amine (0.071 mmol) overnight at room temperature. The product
was concentrated by rotary evaporation and then precipitated
by adding acetonitrile. The precipitated product was dried under
vacuum for 24 h.
For preparation of the DOC-heparin conjugate, heparin (0.01
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 DOC (0.044 mmol).
After 30 min, the mixture was dialyzed (MWCO: 2000) against
water to remove unreacted NHS, EDAC, and aminated DOC.
The final product, DOC-heparin, was obtained and stored at 4
°C after freeze-drying. The dried DOC-heparin conjugate was
analyzed by1H NMR and FT-IR (Bruker, Germany). Values
for1H NMR of heparin (D2O) were: δ 5.38 [H1 of glucosamine
residue (A)], δ 5.04 [H1 of iduronic acid residue (I)], δ 4.84
[I-5], δ 4.36-4.23 [A-6], δ 4.12-4.40 [I-3], δ 4.08j[I-4], δ
4.02 [A-5], δ 3.78 [I-2], δ 3.71 [A-4], δ 3.65-3.69 [A-3], δ
3.24 [A-2]. Values for1H NMR of aminated DOC (D2O) were:
δ 1.2-1.9 [m, five and six rings of DOC, 1H], δ 2.1-2.3 [m,
CH3of DOC, 1H], δ 3.15[d, 12R-OH of DOC, 2H], δ 8.0 [H
of CONH]. Values of 1H-NMR of DOC-heparin conjugates
(D2O) were: δ 1.2-1.9 [m, five and six rings of DOC, 1H], δ
3.24-5.38 [A or I of heparin], δ 8.0-8.2 [H of CONH of
Ninhydrin Colorimetric Method. For the coupling ratio of
DOC in the DOC-heparin conjugate, the ninhydrin colorimetric
method was used as described previously (14). In brief, 80 µL
of the ninhydrin solution was added to 300 µL aminated DOC
and the test tube covered with a piece of paraffin film to avoid
the loss of solvent due to evaporation. With gentle stirring, the
solution was heated for 5 min at 100 °C. After cooling to room
temperature in a cold water bath, the absorbance was recorded
with a spectrophotometer at 570 nm in wavelength. With the
standard curves of aminated DOC, the coupling ratios of
DOC-heparin conjugates were determined by subtracting the
OD values of remaining aminated DOC.
Anticoagulant Activity of DOC-Heparin Conjugates.
DOC-heparin conjugate (100 µL) was mixed with 100 µL of
antithrombin III (ATIII) solution to make DOC-heparin
conjugate-ATIII complexes, where ATIII concentration was
in excess of the DOC-heparin conjugate concentration. The
solution was incubated at 37 °C for 3 min, and 100 µL of FXa
was added to the solution. The resulting solution was then
incubated for an additional 30 s. The concentration of FXa was
also in excess of the DOC-heparin conjugate concentration.
The substrate (200 µL, 0.8 µmol/mL) was then added and
incubated at 37 °C for 3 min. The reaction was terminated by
adding 300 µL of 20% acetic acid. The bioactivity and the
concentration of DOC-heparin conjugate were calculated from
the absorbance at 405 nm.
Endothelial Tubular Formation. Human endothelial cells
were resuspended at 4 × 105cells/mL phenol red-free RPMI
containing glutamine (2 mM). Then, 100 µL growth factor-free
Matrigel with or without SDF-1R (200 ng/mL) was plated into
96-well plates (Costar, Corning, NY) and incubated at 37 °C
for 30 min for gelation. Thereafter, cells were seeded in gelated
Matrigel in the presence of different stimuli. Plates were
Figure 1. Synthesis and schematic structure of DOC-heparin conjugate.
DOC-Heparin DerivativesBioconjugate Chem., Vol. 19, No. 7, 2008 1347
incubated for 24 h, and then tubular formation was analyzed
after 6 h of incubation. For inhibition experiments, heparin or
DOC-heparin conjugates were used at 10, 50, and 250 µg/mL
and added together with the cells before seeding the cells on
Matrigel. After 6 h of incubation, cell growth and three-
dimensional organization were observed through a reverse-
phase-contrast photomicroscope (Olympus 1 × 71), and the
results were expressed as the mean number of junctions/5 fields
at × 100 original magnification. Tubular formation and inhibi-
tion of tubular formation experiments were performed in
duplicate and repeated at least 3 times.
Human Tumor Xenograft. Four- to six-week-old Athymic
BALB/c-nu/nu female nude mice (14-18 g) were purchased
from SLC Inc. (Japan) and maintained under specific pathogen-
free conditions. All experiments were approved by the institu-
tional guidelines of the Institutional Animal Care and Use
Committee (IACUC) of the Catholic University of Korea
College of medicine in accordance with the NIH Guidelines.
Cultured KB cells (ATCC, Rockville, MD) were trypsinized,
washed twice with serum-free EMEM medium (ATCC), and
suspended at 1 × 107cells/mL PBS buffer. 50 µL of the
suspended cells was subcutaneously injected into the back of
the mice. On day 12-15 after tumor injection, the resulting
tumors reached a volume of 45-55 mm3. According to body
weight and tumor size, the animals were divided into four
experimental groups of five mice each: groups A, B, C, and D,
respectively, received through the tail vein injections of 100
µL of saline as control (Group A, n ) 5), heparin (10 mg/kg,
Group B, n ) 5), DOC-heparin VI (5 mg/kg, Group C, n )
5), and DOC-heparin VI (10 mg/kg, Group D, n ) 5). Each
drug was administered twice a week for four weeks after tumor
inoculation. Data are expressed as means ( SE. One-way
ANOVA was used to compare groups, where P values of <0.05
were considered significant.
RESULTS AND DISCUSSION
We synthesized DOC-heparin conjugates for cancer targeting
and angiogenesis inhibition by linking the polysaccharide
heparin (about 5000 Da) with sodium deoxycholate (DOC).
Conjugation between the carboxyl groups of polysaccharide
heparin and the amine groups of DOC was confirmed by the
presence of signals at δ 8.0-8.2 ppm in the1H 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 hydroxyl
groups in DOC structure, C3-OH > C12-OH (15). We also
found a sharp peak at δ 2.0 ppm, indicating the presence of a
hydroxyl group at the C12-OH position. The amount of DOC
conjugated to polysaccharide heparin estimated by the ninhydrin
colorimetric method was maximized to 8.5 mol based on 1 mol
of heparin (DOC-heparin VI) as shown in Table 1. By changing
feed mole ratio of heparin, EDAC, HOSu, and aminated DOC,
we controlled the coupling ratio between DOC and heparin.
When dissolved in water, the DOC-heparin conjugates pro-
duced a clear solution at a concentration of 50 mg/mL. AFM
and size measurement showed that the particles formed uniform
spheres with a narrow size distribution as shown in Figure 2.
The mean diameter of the nanoparticles (DOC-heparin IV) was
185 nm with a standard deviation of 2.8 nm, as determined by
dynamic light scattering. A nanoparticle size of about 100-300
nm is considered to be suitable for passive targeted delivery
utilizing the EPR effect. Several research groups using liposomes
and other macromolecules have shown that particles with small
size (less than 400 nm and ideally less than 200 nm) are more
efficient in cell targeting than larger particles (16).
Table 1. Reaction Condition and Coupled Ratio of DOC in DOC-Heparin Conjugates
Aminated DOC (mol) 1.3
Figure 2. Size distribution and AMF image of DOC-heparin conjugate. (a) Size distribution of DOC-heparin conjugates by dynamic light scattering.
(b) AFM image of DOC-heparin nanoparticles.
1348 Bioconjugate Chem., Vol. 19, No. 7, 2008Cho et al.
Biological Activity. Antifactor Xa activity of DOC-heparin
conjugates measured by chromogenic assay decreased with the
increase of coupled DOC in DOC-heparin as shown in Table
2. When the carboxyl group of heparin is modified by conjuga-
tion, its affinity to factor Xa and/or antithrombin and thus its
anticoagulant activity diminishes (17–19). The conjugated
heparin presents several advantages as an anticancer drug carrier:
(i) More DOC-heparin conjugates can access cancer cells,
bypassing the coagulation cascade. (ii) Conjugation to DOC in
DOC-heparin reduces the amount of negative charges of
heparin, decreasing side effects such as heparin-induced throm-
bocytopenia (HIT) or bleeding that arise from the charge and
size of heparin (20, 21). (iii) Through the intact sulfate group,
the conjugated heparin retains its ability to inhibit binding with
angiogenic factors, which can induce proliferation of smooth
muscle cells. It is widely known that a high degree of sulfation
and optimum saccharide chain length are essential for recogni-
All of the advantages suit our purpose to develop a biocom-
patible novel drug carrier that acts as an efficient antitumor
agent, and is free of undesired interactions with blood and vessel
DOC-Heparin Conjugates Inhibit Tumor Vasculature
Table 2. Bioactivity of DOC-Heparin Conjugates
Figure 3. Inhibition of tubular formation by heparin and DOC-heparin conjugates at different concentrations.
Figure 4. Antitumor effects of DOC-heparin conjugates. (a) Shrinkage in tumor volume and (b) changes in body weight after treatment with saline
(0), 10 mg/kg heparin (•), 5 mg/kg of DOC-heparin VI (1), and 10 mg/kg of DOC-heparin VI (∆), respectively. All data represent mean (
DOC-Heparin Derivatives Bioconjugate Chem., Vol. 19, No. 7, 2008 1349