Water-soluble heparin-PTX conjugates for cancer targeting

Article abstract of DOI:10.1016/j.polymer.2010.05.030

Targeted drug delivery to cancer cells or tumor vasculature is an attractive approach to treating cancer. We here report the synthesis of an anticancer drug conjugate composed of paclitaxel (PTX) and polysaccharide heparin through the reaction of aminated

Full text of DOI:10.1016/j.polymer.2010.05.030

Polymer 51 (2010) 3387e3393
Contents lists available at ScienceDirect
Polymer
journal homepage: www.elsevier.com/locate/polymer
Water-soluble heparinePTX conjugates for cancer targeting
Il-Kyu Park , Yu Jin Kim , Thanh Huyen Tran , Kang Moo Huh a,*, Yong-kyu Lee
a
,
1
b
,
1
a
b,
*
a
Department of Polymer Science and Engineering, Chungnam National University, Daejeon, 305-764, Republic of Korea
Department of Chemical and Biological Engineering, Chungju National University, Chungbuk 380-702, Republic of Korea
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 March 2010
Received in revised form
Targeted drug delivery to cancer cells or tumor vasculature is an attractive approach to treating cancer.
We here report the synthesis of an anticancer drug conjugate composed of paclitaxel (PTX) and poly-
saccharide heparin through the reaction of aminated PTX with the carboxyl group of heparin. The
10 May 2010
1
structure of the conjugates was identified by H NMR and FT-IR measurements. HeparinePTX conjugates
Accepted 15 May 2010
Available online 1 June 2010
have high solubility in aqueous solutions. Unlike physically encapsulated drugs, heparinePTX can self-
assemble to form spherical nanoparticles in aqueous solution as characterized by Transmission Electron
Microscopy (TEM). Size distribution of the nanoparticles as determined by Dynamic Light Scattering
Keywords:
HeparinePTX conjugate
Nanoparticles
(
DLS) was in the range of 200e400 nm depending on the coupling ratio of PTX to heparin molecules. The
anticoagulant activity of heparinePTX conjugates was decreased compared to that of heparin, thereby
reducing hemorrhagic side effects. Cellular uptake of the nanoparticles was significantly enhanced
compared to heparin as visualized by Confocal Laser Scanning Microscopy (CLSM). Furthermore, hep-
arinePTX conjugate nanoparticles exhibited higher cytotoxicity against KB cancer cells than did free PTX.
The cytotoxicity of nanoparticles was found to depend on the amount of PTX conjugated to heparin as
well as the conjugate concentration. Thus, conjugation of PTX to heparin may be useful for the solubi-
lization and targeted delivery of PTX to solid tumors.
Cancer targeting
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
anticoagulant activity [9,10]. Other studies have shown that
heparin is not just an anticoagulant, but actually a complex set of
Recent advances in tumor therapy have led to the development
of new probes, drugs, and genes for tumor targeting [1,2]. Among
these new drug targeting systems, polymeric nanoparticles have
been demonstrated to significantly improve the specificity of drug
action. This effect is mainly attributed to changes in tissue distri-
bution and pharmacokinetics [3e5]. Furthermore, it has been
demonstrated that nanoparticles can escape from the vasculature
through the leaky endothelial tissue surrounding the tumor and
accumulate in certain solid tumors. Most polymeric nanoparticles
display this enhanced permeability and retention effect (EPR
effect). Several synthetic as well as natural polymer carriers such as
poly(ethylene glycol) (PEG), N-(2-hydroxypropyl)-methacrylamide
multifunctional glycosaminoglycan molecules with many other
potential biological effects on cancer cells [11,12]. Recently, many
reports have shown that the effect of heparin on tumors is asso-
ciated with the binding of growth factors such as vascular endo-
thelial growth factor (VEGF) and basic fibroblast growth factor
(bFGF) [13,14]. Heparin is also associated with the inhibition of
heparanases enzymes that are thought to be required by tumor
cells for invasion of the vascular basement membrane [15]. These
features make heparin a promising candidate as a drug carrier for
targeted drug delivery.
Paclitaxel (Taxol) is an effective anticancer drug that has shown
significant antineoplastic activity against various human cancers,
including breast and ovarian tumors [16,17]. However, the clinical
use of PTX is limited due to its poor solubility and toxicity.
Currently, available PTX formulations include a combination of
Cremophor EL and ethanol for solubilization. However, Cremophor
EL is toxic and has side effects such as hypersensitivity, nephro-
toxicity and neurotoxicity [17]. In order to overcome these limita-
tions, polymeric carriers such as PG, polyethylene glycol (PEG) and
albumin have been widely used to conjugate to or encapsulate PTX
copolymer (HPMA) and poly(L-glutamic acid) (PG) have been
successfully used in improving cancer chemotherapy [6e8].
Heparin is a natural polysaccharide held together by glycosidic
linkages. The covalently linked sulfate, hydroxyl and carboxyl
groups of heparin are the main functional groups contributing to its
*
Corresponding authors.
[18]. These carriers produced desirable pharmacokinetics,
E-mail addresses: khuh@cnu.ac.kr (K.M. Huh), leeyk@cjnu.ac.kr (Y.-k. Lee).
Park and Kim equally contributed to this article as co-first authors.
1
enhanced anti-tumor activity and low toxicity. To prepare a stable
0
032-3861/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.polymer.2010.05.030

3388
I.-K. Park et al. / Polymer 51 (2010) 3387e3393
Fig. 1. Synthesis and schematic structure of heparinePTX conjugate.
water-soluble conjugate exerting anti-tumor effects, we selected
the natural polysaccharide heparin due to its following advantages:
Samyang Genex Co. (Daejeon, Korea). 4-nitrophenyl chloroformate
(4-NPC), triethylamine, N-(3-dimethylaminopropyl)-N -ethylcar-
0
(
a) it is highly hydrophilic due to the presence of sulfate, hydroxyl
boimide hydrochloride (EDC), 4-methylmorpholine, N-hydrox-
ysuccinimide (NHS), ethylenediamine, dimethyl sulfoxide (DMSO),
methanol, and n-hexane were purchased from Sigma Chemical CO.
(St. Louis, MO). Penicillin-streptomycin, fetal bovine serum (FBS),
0.25% (w/v) trypsine0.03% (w/v) EDTA solution, and EMGM
medium were purchased from American Type Culture Collection
(Rockville, MD). RPMI-1640 medium (without FA) was obtained
from Invitrogen (Carlsbad, CA). All reagents were analytical grade
and used as received without further purification.
and carboxyl groups; (b) it is non-toxic in vivo and readily
neutralized by antagonists; and (c) it acts as an anticancer agent.
Several strategies for the conjugation of drugs to polymer
carriers have been developed to promote cancer cell targeting. In
these approaches, drugs are directly conjugated to polymer carriers
or to a linker for cleavage by lysosomal enzymes. This strategy can
endow derivatives with enhanced water-solubility and chemical
stability, an improved pharmacokinetic and distribution profile,
and reduced side effects [4,5,18]. In this paper, we report the
synthesis, characterization and in vitro experiment of heparinePTX
conjugate nanoparticles. We expect that our strategy will be
applied to passive targeted delivery of therapeutic agents to tumor
tissues.
2.2. Preparation of aminated PTX
The synthetic methods for aminated PTX and heparinePTX
conjugates were similar to the method described in a previous
report [19]. For amination of PTX, PTX (0.026 mmol) in 5 mL of
DMSO was reacted with triethylamine (0.174 mmol) and 4-nitro-
phenyl chloroformate (4-NPC, 0.174 mmol) for 12 h at room
temperature. The reactant was then extracted with 20 mL of
methanol and 20 mL of n-hexane. The crude product was washed
with 10 mL of n-hexane for 3 times to obtain PTX carbonate. To
obtain aminated PTX, PTX carbonate was reacted with 4-methyl-
morpholine (1.73 mmol) and ethylenediamine (86.4 mmol) over-
night at room temperature. The reactant was concentrated by
2
. Materials and methods
2
.1. Materials
Low molecular weight heparin (114.1 IU/mg, heparin) of average
molecular weight ca. 5000 Da was purchased from Glaxosmithkline
Brentford, Middlesex, UK). Paclitaxel (PTX) was provided by
(

I.-K. Park et al. / Polymer 51 (2010) 3387e3393
3389
Fig. 2. 1H NMR spectra of heparin in D
O (A), heparinePTX in D O (B) and heparinePTX in D O/DMSO (0.7/0.3, v/v) (C) at 25 C.
ꢀ
2 2 2
evaporation and unreacted chemicals were removed by adding
shapes of heparinePTX conjugate nanoparticles were analyzed
using Electronic Light Scattering (ELS, Otsuka Electronics, ELS-Z
series) and a Transmission Electron Microscope (TEM, JEM 1010,
JEOL, Japan).
2
0 mL of n-hexane. The final product was dried under vacuum for
2
4 h.
2.3. Preparation of heparinePTX conjugates
2.5. Anticoagulant activity of heparinePTX conjugates
HeparinePTX conjugates were synthesized by coupling reaction
of heparin with aminated PTX. For example, heparin (0.01 mmol)
was dissolved in water after which the pH was adjusted to pH
To obtain mixtures of heparinePTX conjugateseATIII com-
plexes, heparinePTX conjugates (100 mL) at 100 mg/mL were mixed
5
.0e6.0 by the addition of 0.1 M HCl solution. EDC (0.1 mmol) was
with 100
m
L of anti-thrombin III (ATIII) solution (1 IU/mL). The
ꢀ
mixed and stirred with heparin solution, followed by the addition
of NHS (0.024 mmol) and aminated PTX (0.026 mmol). After
reaction at room temperature for 12 h, the solution was dialyzed
mixture solution was incubated at 37 C for 5 min, after which FXa
(100
m
L) was added to the mixture solution which was incubated at
ꢀ
37 C for 1 min. The substrate (200
mL) was then added, followed by
ꢀ
(
MWCO: 2000) against water to remove unreacted EDC, NHS, and
incubation at 37 C for 3 min. The reaction was terminated by the
aminated PTX. HeparinePTX was obtained after freeze-drying. The
product was confirmed by H NMR (JNM-AL400, Jeol Ltd, Akishima,
addition of 300 mL of 20% acetic acid. The anticoagulant activity of
heparinePTX conjugates were calculated from the absorbance at
405 nm using UVeVis spectrometry.
1
1
17
Japan). The values for H NMR of PTX (CDCl
3
) were:
], 2.2 [m, OAc], 2.4 [m,
, PG], 4.3 [d,
3
d 1.11 [s, CH ],
16
19
18
1
.20 [s, CH
3
], 1.69 [s, CH
3
], 1.97 [s, CH
3
3
20
OAc], 3.78 [d, CH], 4.17 [d, CH
2
], 4.27 ppm [H-
a
2.6. Confocal microscopy study
2
2
0
7
5
2’
CH
CH]. The values for H NMR of heparin (D
5.04 [H1 of iduronic acid residue(I)],
4.36e4.23 [A-6], 4.12e4.40 [I-3], 4.08 [I-4], 4.02
3.78 [I-2], 3.71 [A-4], 3.65e3.69 [A-3], 3.24 [A-2]. The
values of H NMR of heparinePTX (D O) were 1.11e2.4 [CH or
OAc of PTX], 3.24e5.38 [A or I of heparin].
2
], 4.39 [dd, CH], 4.96 [d, CH], 4.78 and 5.63 [d, CH], 5.67 [d,
1
2
O) were: d 5.38 [H1 of
To visualize the cellular uptake and intracellular distribution
of FITC-labeled heparinePTX conjugates, confocal laser scanning
glucosamine residue(A)],
4.84 [I-5],
A-5],
d
d
d
d
d
d
microscopy (Zeiss LSM510, Germany) was performed on KB cells
Ò
[
d
d
d
d
grown on a Lab-Tek II chamber slide (Nalge Nunc, Napevillem,
1
2
d
3
d
Table 1
Coupling ratio and bioactivity of heparinePTX conjugates.
2.4. Characterization of heparinePTX conjugates
HeparinePTXI
HeparinePTXII
HeparinePTXIII
Heparin (mol)
Feed mole ratio of
PTX (mol)
Coupling ratio of
PTX (mol)
1
1
1
2.6
1
5
UVeVis absorption spectra were analyzed on a Sinco PDA
UVeVis spectrometer. The content of PTX conjugated to heparin
was calculated from UVeVis measurements based on a standard
0.8
1.7
4.1
curve of PTX (
gates were analyzed by Fourier Transform infrared spectroscopy
FT-IR) using a MAGNA 560 spectrometer after which samples were
made as KBr pellets. The average size, size distribution and particle
l
¼ 228 nm). The IR spectra of heparinePTX conju-
Anticoagulant
activity (IU/mg)
107.9
104.8
95.2
(
Anticoagulant activity of heparin is 114.1 IU/mg.

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I.-K. Park et al. / Polymer 51 (2010) 3387e3393
Fig. 3. FT-IR spectra of heparin (A), PTX (B) and heparinePTX (C).
IL). FITC (64 mg) was conjugated with heparin or heparinePTX
were seeded at 100 000 cells/well in a 96-well plate in RPMI-
ꢀ
ꢀ
(
200 mg) in sodium bicarbonate buffer (pH 8.5) for 6 h at 25 C.
1640 and preincubated at 37 C for 24 h before the assay. MTT
ꢀ
HeparineFITC or heparinePTXeFITC were precipitated by
assay was performed on KB cells by incubation at 37 C for 1 day
ethanol and then dialyzed against water for 4 days. The cells
of different concentrations of each compound in quadruplicate.
Control was incubated at 37 C for 1 day without any drug. This
ꢀ
were incubated with 1
m
g/mL FITC-labeled heparin or 1
m
g/mL
FITC-labeled heparinePTX conjugates in RPMI-1640 medium for
assay is based on the reduction of yellow tetrazolium compo-
nent (MTT) to insoluble purple-colored formazan produced by
ꢀ
3
h at 37 C and 5% CO
2
atmosphere. The cells were then washed
L of 4%
three times with PBS buffer (pH 7.4) and finally 200
m
the mitochondria of viable cells. After 48-h of incubation, 100
of medium containing 20 L of MTT solution was added to each
well and the plate was incubated for an additional 4 h, followed
by the addition of 100 L of MTT solubilization solution (10%
mL
formaldehyde in phosphate buffer saline was added. Cell images
were taken under low power magnification (10ꢁ lens) with
conventional fluorescence microscopy and a FITC filter. The FITC-
labeled areas were then scanned with a confocal microscope
m
m
Triton X-100 plus 0.1 N HCl in anhydrous isopropanol, Sigma,
Milwaukee, WI) to each well. The solution was gently mixed to
dissolve the MTT formazan crystals. The absorbance of each well
was read with a microplate reader at a wavelength of 570 nm.
The background absorbance of the wells at 690 nm was
measured and subtracted from the 570 nm measurement. The
results are expressed as % cell viability, and were obtained by
dividing the optical density values (OD) of the treated groups
(T) by the OD of the controls (C) ([T/C ꢁ 100%]). Statistical
analysis was done using ANOVA. p < 0.01 was accepted as
statistically significant. Error bars represent standard error of
mean (SEM).
(
excitation/emission wavelengths: 488 nm and 510 nm) at higher
magnification (63ꢁ lens).
2.7. Cytotoxicity of heparinePTX conjugates
KB cells (human nasopharyngeal epidermoid cancer cells)
were used to observe the cytotoxicity of PTX or heparinePTX.
ꢀ
The KB cell lines were cultured at 37 C in a humidified atmo-
2
sphere containing 5% CO in FA-deficient medium RPMI-1640
containing 10% fetal calf serum. The cells grown as a monolayer
were harvested by 0.25% trypsine0.03% EDTA solution. The cells
Fig. 4. Size distribution (A) and shape (B) of heparinePTXII conjugates using ELS and TEM, respectively. Samples were measured at a concentration of 10 wt% in water.

I.-K. Park et al. / Polymer 51 (2010) 3387e3393
3391
3
. Results and discussion
the heparin molecule (molecular weight: 5000 Da). Thus, the
modification of 5 COOH groups by PTX may produce water-soluble
heparinePTX (Fig. 2).
3.1. Synthesis and characterization of heparinePTX conjugates
Anticoagulant activities of heparinePTX conjugates were
reduced as the amount of PTX in heparinePTX increased as shown
in Table 1. Anticoagulant activity of heparin and heparinePTXIII
conjugate measured by chromogenic assay were 114.1 and 95.2 IU/
mg, respectively. This might be attributed to the introduction of
PTX, leading to a conformational change in heparin structure that
decreases the affinity to ATIII and affects the anticoagulant mech-
anism [22]. Although some clinical trials have indicated beneficial
effects of heparin in cancer patients, its application is still limited
due to high anticoagulant activity leading to hemorrhagic compli-
cations. It was reported that modification of the carboxyl group of
heparin by chemical conjugation diminishes its affinity to factor Xa
and/or anti-thrombin and thus its anticoagulant activity. However,
PTXeheparin conjugates maintain the sulfate groups of heparin,
which bind endothelial cells or block FGF-heparan sulfate binding,
and may therefore decrease FGF activity. Thus, the anti-adhesion,
inhibition of angiogenesis, metastasis and tumor growth may be
still conserved [23e25]. Conjugated heparin holds several advan-
tages as an anticancer drug carrier: i) More heparinePTX conju-
gates can access cancer cells with higher water-solubility than can
free PTX ii) Conjugation of an anticancer drug to heparin reduces
the number of negative charges of heparin, thereby decreasing side
effects such as thrombocytopenia (HIT) or bleeding that arise from
the charge and size of heparin [26]. iii) Through the intact sulfate
group, PTX conjugated heparin retains its ability to bind with
We synthesized heparinePTX nanoparticles for cancer targeting
by conjugating the polysaccharide heparin with PTX. To introduce
an amine group to PTX, ethylenediamine (EDA) was covalently
linked to the hydroxyl group of PTX in the presence of EDC and NHS
as shown in Fig. 1. The presence of an amine group on the PTX
structure was confirmed by characteristic peaks at 2.0 ppm in the
2
NMR spectrum of NH ePTX. However, the specific conjugation site
in PTX could not be identified. Cavallaro et al. also reported that the
0
succinylation of PTX takes place preferentially at the 2 -OH position
under mild conditions [20]. Thus, the amidation of PTX might
0
preferentially occur at the 2 -hydroxyl group over the 7-hydroxyl
group due to less steric hindrance [21]. The amide linkage between
heparin and aminated PTX was confirmed by the presence of
signals at
d 1.5e3 ppm. The presence of CeN in primary amide
conjugates was further confirmed by
420e1400 cm in the FT-IR spectra, indicating the conjugation
a specific band at
ꢂ1
1
between heparin and PTX (Fig. 3). The weak intensity of C]C
ꢂ1
stretching of PTX at 710 cm in the heparinePTX spectrum might
result from the interference of the stretching of functional groups in
heparin. The coupling ratio of PTX to heparin was determined by
UV-spectroscopy at 228 nm using 1% (w/w) heparinePTX conju-
gate. As shown in Table 1, the amount of PTX conjugated to heparin
was increased (from 0.8 to 4.1 mol) by increasing the feed molar
ratio of PTX to heparin (from 1 to 5). There are 10 COOH groups in
Fig. 5. Confocal microscopy image on KB cell lines after treating with (A) HeparineFITC, (B) HeparinePTXIeFITC, (C) HeparinePTXIIeFITC, (D) HeparinePTXIIIeFITC conjugates.

3392
I.-K. Park et al. / Polymer 51 (2010) 3387e3393
angiogenic factors as well as anti-heparanase activity [27]. Conse-
quently, heparinePTXIII conjugate with reduced anticoagulant
activity could be a safe and effective prodrug that warrants further
investigation.
and retention of PTX and heparin in tumor tissue might occur in
vivo via formation of the water-soluble conjugate.
3.4. Cell viability of heparinePTX conjugates
3.2. Size and morphology of heparinePTX nanoparticles
To compare the cytotoxicities between free PTX and hep-
arinePTX nanoparticles, we performed MTT assay using the KB cell
line. Fig. 6 showed the cell viability of KB cells treated with different
drug and conjugate concentrations after 24 h and 48 h. After 24 h of
incubation, cell viability was decreased from w80%ew50% when
the concentration of heparinePTX nanoparticles was increased
from 0.01% to 1% (w/w). At the same concentration, cell viability
was slightly diminished when the coupling ratio of PTX to heparin
was increased. Decreasing cell viability is in the following order:
heparinePTXI > heparin e PTXII > heparinePTXIII (Fig. 6A).
Treatment for 48 h decreased cell viability to 25% at 1% (w/w) and
We found that heparinePTX conjugates are soluble in water,
indicating that the water-solubility of PTX was significantly
enhanced through conjugation with water-soluble heparin.
Importantly, heparinePTX conjugates readily self-assembled in
aqueous solution to form nanoparticles due to hydrophobic inter-
actions between PTX molecules conjugated to the heparin back-
bone. Fig. 4 shows the size distribution and morphology of
heparinePTX self-assembled nanoparticles. The mean diameter of
heparinePTX ranged from 200 to 400 nm and decreased with
increasing PTX coupling ratio due to the formation of more
compact hydrophobic inner core (Fig. 4A). TEM micrograph of
heparinePTX nanoparticles shows uniform spherical shapes with
diameters of 200 nm, which is smaller than the size measured by
DLS (Fig. 4B). This was mainly due to the process involved in the
preparation of samples. In the case of the TEM method, TEM images
depicts the size at the dried state of the sample, whereas DLS
method involves the measurement of size in the hydrated state. In
other words, the size determined by TEM is an actual diameter (dry
state) of the nanoparticles, whereas the size measured by the laser
light scattering method is a hydrodynamic diameter (hydrated
state), and therefore the nanoparticles will have a larger hydrody-
namic volume due to solvent effect in the hydrated state; hence, the
size measured by DLS method was larger than the size observed by
TEM method. HeparinePTX conjugate nanoparticles likely have
a core/shell structure composed of a hydrophobic inner core con-
taining aggregated PTX molecules and a hydrophilic heparin shell
layer. Thus, heparinePTX nanoparticles (200e400 nm in diameter)
are considered to be suitable for passive delivery of PTX to targeted
tumors due to the Enhanced Permeability Retention (EPR) effect
50% at 0.01% (w/w) of heparinePTXIII (Fig. 6B). HeparinePTXIII
showed similar cytotoxicity to free PTX at the same concentrations
despite lower PTX content. Thus, heparinePTX nanoparticles are
more potent for killing cancer cells than free PTX, suggesting that
heparinePTX can provide more efficient and less toxic effects for
treating cancer. PTX is supposed to be transported into cells by
passive diffusion, but heparinePTX nanoparticles are likely to be
internalized within cells via endocytosis. The higher toxicity of
heparinePTX conjugate nanoparticles might be attributed to its
greater uptake and accumulation in cancer cells compared to free
[28,29].
3.3. Fluorescence images of heparineFITC, HeparinePTXeFITC
conjugates
To evaluate the extent of cellular uptake of heparinePTX
nanoparticles, FITC was conjugated with heparin and heparinePTX
conjugate and confocal laser microscopy was used. Fig. 5 shows KB
cancer cells viewed by confocal laser scanning microscopy after 3 h
incubation with heparineFITC, heparinePTXIeFITC, heparin-
ePTXIIeFITC, and heparinePTXIIIeFITC at 1
mg/mL FITC. We
observed that heparinePTXeFITC was highly internalized. The
green fluorescence coming from the nanoparticles was detected
within the cell cytoplasm, whereas weak fluorescence intensity
was observed in KB cells treated with heparineFITC. Confocal
microscopy clearly indicates that heparinePTX underwent greater
cellular uptake than heparin, whereas heparineFITC was hardly
taken-up by the cells. Their different physicochemical properties
offer different cellular uptake capacity. The increase in cellular
uptake of heparinePTX might also result from the possibility that
the nanoparticles interact with the cell membrane to a greater
extent and thereby experience endocytosis. On the other hands, it is
difficult for heparin to interact with the cell membrane because of
its highly negative charge and large size [30]. Cellular uptake is an
important factor in any nanoparticle targeting system since circu-
lating nanoparticles can diffuse into tumor tissue through the leaky
vessel structure without being uptaken and therefore can easily
diffuse back into the main bloodstream without accumulating.
Thus, the result suggests that more efficient cellular internalization
Fig. 6. In vitro cytotoxicity of free PTX and heparinePTX nanoparticles against KB
cancer cells after incubation for 24 h (A) and 48 h (B).

I.-K. Park et al. / Polymer 51 (2010) 3387e3393
3393
PTX. Heparin is highly hydrophilic due to negatively charged
groups such as sulfonyl, carboxyl and hydroxyl groups within its
structure [23,31]. Therefore, to treat a tumor with heparin, new
structure designs that achieve the same therapeutic response with
a reduced dose in order to decrease systemic toxicity and side
reactions are needed. In the previous study, we found that heparin
conjugated with a hydrophobic moiety retained its ability to inhibit
binding with angiogenic factor, showing a significant decrease in
endothelial tubular formation [16,32]. Furthermore, heparinePTX
conjugates showed improved water-solubility. PTX is a microtu-
bule-stabilizing agent that promotes polymerization of tubulin,
causing cell death by disrupting the dynamics necessary for cell
division. It is currently used for treatment of ovarian, metastatic
breast, lung, head, and neck cancer [33]. Thus, nanosized self-
assembled heparinePTX conjugate nanoparticles could be an effi-
cient all-in-one carrier not only for the solubilization of PTX, but
also for its anti-angiogenesis and passive targeted delivery to tumor
tissues.
Foundation Grant funded by the Korean Government (KRF-
D00289), and by BioImaging Research center at GIST.
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Paclitaxel was aminated to conjugate with heparin via an amide
2
linkage. The conjugation of PTX with heparin significantly
increased the water-solubility of PTX. A higher coupling ratio of PTX
to the heparin backbone could be obtained by increasing the PTX
feed ratio. HeparinePTX conjugates readily self-assembled to form
spherical nanoparticles (200 nm) in aqueous solution. Hep-
arinePTX conjugate nanoparticles demonstrated greater cytotox-
icity to KB cancer cells than did free PTX. Furthermore, the
enhanced cellular uptake and reduced anticoagulant activity of
heparin were achieved by conjugation with PTX. This simple
synthetic strategy with increased drug solubility and enhanced cell
cytotoxicity suggests that heparinePTX conjugates could be an
efficient system for cancer treatment. Further studies in animals
should be carried out to confirm the anti-tumor effect of hep-
arinePTX in vivo.
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This work was supported by a grant from the Fundamental R&D
Program for Core Technology of Materials funded by the Ministry of
Knowledge Economy, Republic of Korea, by the Korea Research
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