Colloids and Surfaces B: Biointerfaces 81 (2010) 530536

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Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb

Characterization and preparation of coreshell type nanoparticle for

encapsulation of anticancer drug
Mi-Kyeong Jang, Young-Il Jeong, Jae-Woon Nah
Department of Polymer Science & Engineering, Sunchon National University, 315 Maegok, Suncheon, Jeonnam 540-742, Republic of Korea

a r t i c l e

i n f o

Article history:
Received 15 October 2009
Received in revised form 22 July 2010
Accepted 23 July 2010
Available online 3 August 2010
Keywords:
LMWSC
Nanoparticle
Paclitaxel
Hydrophobic anticancer drug
Tumor inhibition

a b s t r a c t
The aim of this study is to prepare delivery vehicles of paclitaxel using low molecular weight watersoluble chitosan (LMWSC) and evaluate them as an anticancer drug delivery system. LMWSC was
modied with methoxy polyethylene glycol (LMWSC-MPEG, ChitoPEG), and then it was conjugated
with cholesterol (LMWSC-MPEG-Chol). Coreshell type LMWSC-MPEG-Chol nanoparticles (LMWSC-NPs)
were prepared by the dialysis method, and the coreshell structure was conrmed by 1 H NMR analysis. To
this polymer, paclitaxel was encapsulated and coreshell type nanoparticles were prepared. The release
tests indicated that release of paclitaxel from the coreshell type nanoparticles and its transport across
the dialysis membrane was slower than dialysis of free paclitaxel. In a cytotoxicity study using CT26 cell,
the paclitaxel-encapsulated coreshell type nanoparticles (LMWSC-NPs) showed a toxicity against tumor
cells similar to paclitaxel itself. The results of a tumor inhibition test with CT26 implanted upon mouse
tumor models in vivo indicated that the application of a dose of 10 mg/kg of LMWSC-NPT showed a superior survival rate, and a slower tumor growth than when paclitaxel alone was administered, although the
tumor growth and survival rate were not signicantly changed at a dose of 2 mg/kg. The LMWSC-NPT
dose above 10 mg/kg showed a superior antitumor activity.
2010 Elsevier B.V. All rights reserved.

1. Introduction
Paclitaxel, attested by FDA in 1992, has been known to show
signicant anticancer activity to a wide range of tumors such as
refractory ovarian cancer, non-small cell lung cancer, metastatic
breast cancer, head and neck malignancies, AIDS-related Kaposis
sarcoma, etc. [15]. Despite its excellent anticancer activity, paclitaxel has serious problems including toxic side-effects and poor
solubility in the conventional aqueous injection solution. Especially, low therapeutic index of paclitaxel is attributed to its toxic
side-effects [6]. Because of low solubility of paclitaxel [7], it has to
be dispersed in a mixed solution of cremophor EL (polyethoxylated
oil) and ethanol (50:50), which is diluted 5- to 20-fold in normal
saline or in 5% dextrose solution for intravenous injection. Various
systems have been proposed to make paclitaxel formulations for
injection such as parenteral emulsion [810], mixed micelles [11],
water-soluble prodrugs [12], polymer micelles [13,14], coreshell
type nanoparticles [15], albumin-bound nanoparticles [16], and
biodegradable polymeric nanoparticles [17].
Application of nanoparticles based on biodegradable polymers
are also a useful tool for intravenous introduction of anticancer
drug into the body in order to effectively deliver the sustained

Corresponding author. Tel.: +82 61 750 3566; fax: +82 61 750 5423.
E-mail address: jwnah@sunchon.ac.kr (J.-W. Nah).
0927-7765/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2010.07.053

drug action, reduce side-effects, facilitate extravasations into the

tumor, increase the capability to cross various physiological barriers as well as control and target the delivery [1820]. To date,
nanoparticles for formulation of anticancer agent have frequently
been prepared using poly lactic-co-glycolic acid (PLGA) [21], polylactic acid [22], poly ethylene oxide-co-poly(lactic acid) [23],
poly(-caprolactone)[24,25] and poly-propylene oxide [26]. These
polymeric nanoparticles offer a number of advantages for drug
delivery to tumor: enhanced loading efciency of drug by the
hydrophobic core, prevention of burst effects, easy modication
of targeting ligands for site specicity, and exact targeting at
the desired site by ligand. In spite of these advantages, synthetic
polymers have many obstacles such as the complexity of preparation process, the remnants of surfactant used for preparation of
nanoparticle, their own cytotoxicity in the body. Natural polymers,
however, can overcome the problems of synthetic polymers.
Chitosan has been extensively investigated as biomaterials and
carriers of anticancer drug. The structure of chitosan is a long
unbranched polysaccharide similar to that of a cellulose derivative
in which amino groups have replaced the hydroxyl group at the
C2 position [27]. It is biodegradable and non-toxic and has reactive hydroxyl and amino groups that can be modied chemically
for various applications [28]. Therefore, chitosan will emerge as an
important biomedical material of the 21st century. However, applications of chitosan are limited by the poor water solubility and the
salts introduced to solve this problem, which result in blocking the

M.-K. Jang et al. / Colloids and Surfaces B: Biointerfaces 81 (2010) 530536

reactivity of amine group. High molecular weight chitosan (HMWC)

has poor solubility in water or organic solvents, and that is one of
the severe disadvantages that are blocking its actual use.
In this study, we prepared coreshell type nanoparticles using
hydrophobically modied and PEGylated LMWSC (low molecular weight water-soluble chitosan) as an anticancer drug delivery
DS =

was repeated three times. Unreacted cholesterol chloroformate

was removed because cholesterol chloroformate is soluble in ether
while synthesized copolymer is not soluble in it. Precipitants were
obtained by ltration and then dried under vacuum for 3 days. The
substitution ratio of cholesterol using 1 H NMR was calculated using
the following equation:

Proton integration ratio of methyl group of cholosterol/3

100
(Proton integration of C1 position of chitosan + Proton integration ratio of acetyl group of chitosan)/3

system. We have previously reported on the potentials of selfassembling nanoparticles using LMWSC [29,30]. LMWSC is a
superior property that can enhance the solubility of hydrophobic anticancer drug and give passive targeting potential. MPEG
(methoxy polyethylene glycol) chains introduced as a hydrophilic
group can prevent cell adhesion by entropically driving steric
repulsion and by increasing the hydrophilicity of carrier surfaces.
Furthermore, introduction of cholesterol as a hydrophobic group
can enhance the association behavior of LMWSC, and the stable
activity of the hydrophobic drug can be enhanced by forming a
hydrophobic core. We expect that hydrophobically modied ChitoPEG (LMWSC-MPEG) may be used as carriers of hydrophobic
anticancer drug.
2. Materials and methods
2.1. Materials
Low molecular weight water-soluble chitosan (LMWSC,
18 kDa) was supplied by KITTOLIFE Co., Seoul, Korea. Methoxy
poly(ethylene glycol) N-hydroxysuccinimide (MPEG-NHS, 5 kDa)
was purchased from SunBio Co., Korea. Cholesteryl chloroformates
were purchased from Aldrich Chem. Co. Ltd., USA. Paclitaxel was
purchased from Sigma Co., USA. BALA/c mice (20 g, 5 weeks) were
purchased from Damul Science, Co. Dialysis tubing (MWCO 12,000)
was commercially obtained from Spectrum. Dimethylformamide
(DMF), dimethylsulfoxide (DMSO) was purchased from Sigma Co.,
USA and used without further purication.
2.2. Synthesis of LMWSC-MPEG copolymer
Synthesis of LMWSC-MPEG graft copolymer was performed as
follows: 100 mg of LMWSC was dissolved in 0.2 ml of deionized
water and diluted with 9.8 ml of DMSO. To this solution, MPEGNHS dissolved in 2 ml of DMSO was added and reacted for overnight
at nitrogen atmosphere. After that, the resulting solution was dialyzed extensively against deionized water for 2 days followed by
its lyophilization. The lyophilized solid was resuspended into a
plenty of DCM (dichloromethane) to remove unreacted MPEG-NHS
three times and fractionated into deionized water followed by its
lyophilization. The substitution (DS) ratio of PEG was calculated as
previously reported [31]. 1 H NMR spectra were measured to evaluate DS (data not shown). DS of PEG was found to be 10 wt.% by the
ratio of methyl group of PEG/proton of C1 position of chitosan.
DS =

531

In this equation, each peak was as follows: methyl group of

cholesterol, 2 ppm; C1 position of chitosan, 6.16.5 ppm of Fig. 2(a);
acetyl peak of LMWSC, 1.92.0 ppm of Fig. 2(b) [2931].
The result indicated that the DS value of cholesterol is 3.8 wt.%.
2.4. Preparation of coreshell type LMWSC nanoparticles
(LMWSC-NPs)
LMWSC-MPEG-Chol (25 mg) was dispersed in 10 ml of PBS (pH
7.4) and magnetically stirred for 30 min. Paclitaxel (4 mg) was dissolved in 2 ml of ethanol, and it was slowly dropped into the above
solution while sonication was performed using a bath type sonicator. This solution was sonicated again using a bar type sonicator
for 20 s (2 s 10 times). This solution was introduced into dialysis
tube (dialysis membrane, molecular weight cut-off: 12,000 g/mol).
Dialysis was performed in PBS (pH 7.4). To harvest paclitaxelencapsulated LMWSC nanoparticles (LMWSC-NPTs), dialysis was
performed in distilled water for 12 h, followed by centrifugation.
Then, LMWSC-NPTs were redistributed into deionized water
and ltered by 0.45 m syringe lter. After that, LMWSC-NPT solution was lyophilized and analyzed. Experimental drug contents
were 10% (w/w) (theoretical drug contents: 13.8% (w/w) and loading efciency was 69.4% (w/w).
Drug contents =

Amount of drug in the nanoparticles

100
Total weight of nanoparticles

Loading efciency =

Residual amount of drug in the nanoparticles

100
Feeding amount of drug

2.5. Characterization of coreshell type LMWSC-NPs and

LMWSC-NPT
To characterize paclitaxel and LMWSC-NP, paclitaxelencapsulated LMWSC nanoparticles (LMWSC-NPT) were quantied
by high performance liquid chromatography (HPLC) using phenomenex sphereclone 5 micro ODS(2) 250 mm 4.6 mm column
and 75% methanol as a moving phase. At this point, the ow
rate was set to 1.5 ml/min, and the temperature at 50 C. The UV
detector (226 nm) was used for the measurement. To prepare the
sample for the measurement of HPLC, paclitaxel was dissolved in
ethanol, making 20 l for injection.
For the drug release test, 10 mg of LMWSC-NPT was reconstituted in 5 ml of phosphate-buffered saline (PBS, pH 7.4,
0.1 M), and then this solution was introduced to dialysis bag

Proton integration ratio of methyl group of MPEG/3

100
(Proton integration of C1 position of chitosan + Proton integration ratio of acetyl group of chitosan)/3

2.3. Synthesis of LMWSC-MPEG-Cholesterol copolymer

Synthesis of LMWSC-MPEG-Cholesterol (LMWSC-MPEG-Chol)
copolymer was performed as follows [30]: cholesterol chloroformate and LMWSC-MPEG copolymer was dissolved in DMSO and
stirred magnetically for 6 h at room temperature (25 C). After that,
the resulting solution was precipitated to cold ether. This process

(molecular weight cut-off (MWCO): 12,000 g/mol). This dialysis

bag was introduced into a bottle with 95 ml of PBSSDS (pH 7.4,
0.1 M, SDS 2.0% (w/v)). For comparison, 1.0 mg of paclitaxel was
suspended in 5 ml of PBSSDS (pH 7.4, 0.1 M, SDS 2.0% (w/v)) and
magnetically stirred until paclitaxel was completely dissolved. This
was introduced into dialysis bag, and then the dialysis bag was
introduced into a bottle with 95 ml of PBSSDS. At predetermined
time intervals, the whole media was taken and replaced with fresh

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PBSSDS media. The released amount of paclitaxel from nanoparticles was evaluated by HPLC method described above.
2.6. Analysis of coreshell type LMWSC-NPs
Particle size of polymeric micelle was measured with a dynamic
laser scattering spectrophotometer (DLS-7000, Otsuka Electronics
Co., Japan). A sample solution prepared by the dialysis method was
used for particle size measurement (concentration: 0.1 wt.%).
The 1 H nuclear magnetic resonance (NMR) spectra of the copolymer and coreshell type nanoparticles were measured in D2 O
or D2 O + pyridine using a 400 MHz NMR spectrometer (AVANCE
400FT-NMR 400 MHz, Bruker).
2.7. Cytotoxicity of paclitaxel and LMWSC-NP against tumor cell
line in vitro
To test the anti-proliferation effect of empty LMWSC-NP,
paclitaxel and LMWSC-NPT, CT26 colon carcinoma cells were maintained under 5% CO2 incubator at 37 C. The effect of empty
LMWSC-NP, free paclitaxel and LMWSC-NPT on the tumor cell proliferation was determined using the MTT cell proliferation assay.
Empty LMWSC-NP was reconstituted in DMEM (supplemented
with 10% FBS) with a concentration of 1 mg/ml and then diluted it
with DMEM (10% FBS) for an appropriate concentration. Paclitaxel
was dissolved in 100% DMSO and diluted 100 times using DMEM
(supplemented with 10% serum) and diluted it to an appropriate
concentration. LMWSC-NPTs were dissolved in DMEM (supplemented with 10% serum) and diluted to adjust the equivalent
concentration of the free paclitaxel. The tumor cell lines were
seeded at a density of 5 103 per well in 96-well plates using 100 l
of DMEM supplemented with 10% serum in a CO2 incubator (5%
CO2 at 37 C) for 12 h. After that, 100 l of DMEM (supplemented
with 10% serum) containing empty LMWSC-NP or free paclitaxel
or LMWSC-NPT was added. After 1 or 2 days of incubation, MTT
was added to the 96 wells and incubated for 4 h in a CO2 incubator
(5% CO2 at 37 C). Then, the supernatant was discarded, and 100 l
of DMSO was added to each of the 96 wells. The absorbance was
measured at 560 nm using a microtiter plate reader (Thermomax
microplate reader, Molecular Devices).
2.8. Antitumor activity of LMWSC-NPT at in vivo with CT26
mouse tumor models
CT26 murine tumor cells (5 104 cells/mice) were implanted
subcutaneously into the back of BALB/c mice (Female, average
body weight was 20 g). When tumors were grown to approximately 3 mm 3 mm (approximately day 14), the animals were
divided into treatment and control groups. Each group consisted of
8 tumor-bearing mice that were ear-tagged and followed-up individually throughout the study. The intravenous administration of
drugs or vehicle began on day 15. For this in vivo test, the paclitaxel dissolved in cremophor vehicle and LMWSC-NPT dissolved in
distilled water were used. Each drug was administered at doses of
2 mg/kg as a low dose and 10 mg/kg as a high dose 4 times for 12
days at intervals of 3 days. The control group received the vehicle
(cremophor:dehydrated ethyl alcohol, 1:1, v/v). The mortality was
monitored daily, and the tumor growth was measured at two or
three day intervals by calliper measurement. Tumor volume was
calculated using the following formula:
length width2
Tumor volume (mm ) =
2
3

8 mice were used for each group.

Fig. 1. Chemical structure of LMWSC-MPEG-Chol (a) and schematic illustration of

coreshell type LMWSC-NP (b).

3. Results
3.1. Characterization of paclitaxel-encapsulated coreshell type
LMWSC nanoparticles (LMWSC-NPTs)
Coreshell type LMWSC nanoparticles (LMWSC-NPs) have a
unique chemical structure as shown in Fig. 1(a), and we presume,
after NMR analysis, that it forms a coreshell structure in the
aqueous environment as shown in Fig. 1(b), i.e., the cholesterol is
conjugated in the LMWSC main chain and forms a hydrophobic
core of the coreshell, and PEG has formed a hydrated outer shell.
To prove this hypothesis, the coreshell type nanoparticles were
prepared by sonication and the dialysis method, and its structure
was conrmed using 1 H NMR as shown in Fig. 2. Specic peaks
of LMWSC (16), MPEG (1115), and cholesterol (ad) were conrmed by D2 O + pyridine solution. When these nanoparticles were
in D2 O, the specic peaks of cholesterol disappeared while the
peaks of MPEG and LMWSC were still shown. These results proved
that cholesterol formed the hydrophobic inner core and the MPEG
formed the hydrated outer shell.
As an anticancer drug, paclitaxel was encapsulated into
coreshell type LMWSC-NPs. Drug content was about 10% (w/w).
The size of empty LMWSC-NP was around 270 nm, and the particle size of LMWSC-NPT was not signicantly changed compared to
empty nanoparticles as shown in Table 1. However, LMWSC-NPT
showed a slightly broad distribution pattern compared to empty
nanoparticles as shown in Fig. 3. Release of paclitaxel was monitored by HPLC as shown in Fig. 4(b). The result of HPLC revealed
that the retention time (RT) of paclitaxel is 3.4 min. Paclitaxel in the
LMWSC-NPT was released over 1 month continuously while paclitaxel itself was released fast in several days (Fig. 4(a)). These results
indicated that LMWSC-NPs are acceptable vehicles for sustained
release of paclitaxel. Furthermore, the release rate of paclitaxel was
slower than that we had expected. It was suggested that the low
release rate was due to the strong hydrophobicity of paclitaxel.
3.2. Cytotoxicity of paclitaxel-encapsulated coreshell type
nanoparticles
To examine the cytotoxicity of LMWSC-NPT, paclitaxel and
LMWSC-NP were treated to CT26 colon carcinoma cells. Fig. 5
shows cell survivability against drug concentration. Survivability of tumor cells gradually decreased as the drug concentration
increased with paclitaxel treatment. Treatment of LMWSC-NPT

M.-K. Jang et al. / Colloids and Surfaces B: Biointerfaces 81 (2010) 530536

Fig. 2.

533

H NMR spectra of coreshell type LMWSC-NP. Coreshell type nanoparticles in D2 O + pyridine (a) and D2 O (b).

Table 1
Characterization of coreshell type nanoparticles.
Drug contents (%, w/w)

Empty LMWSC-NP
Paclitaxel-encapsulated nanoparticles (LMWSC-NPT)

10.0

Particle size (nm)

Intensity

Volume

Number

304.9 56.3
330.7 82.3

274.6 49.9
274.6 68.8

254.0 40.5
234.8 51.4

Drug contents = [drug weight in the nanoparticle/total weight of nanoparticle] 100.

showed an almost similar trend of cell survivability in proportion to

the drug concentration. As can be seen in Fig. 5, the empty LMWSCNP did not have signicant cytotoxicity against tumor cells, i.e.,
higher than 80% of tumor cells survived at the highest concentration of nanoparticles (1000 g/ml). These results indicate that
paclitaxel-encapsulated coreshell type LMWSC-NP have a potency
similar to that of paclitaxel itself.

3.3. In vivo, antitumor activity of LMWSC-NPT

Fig. 3. Particle size distribution of empty (a) and paclitaxel-encapsulated coreshell

type nanoparticles (b).

For in vivo test of paclitaxel carriers, paclitaxel was dissolved

in cremophor vehicle, but LMWSC-NPT was dispersed in distilled
water because of the enhanced water solubility. Approximately
2 weeks after tumor implantation into the back of the mice, an
advanced tumor appeared (approximately 35 mm in diameter),
the mice received a vehicle (control group) or drug via tail vein.
Fig. 6(a) shows the survival rate of the mice treated with paclitaxel and LMWSC-NPT. The control group showed the fastest death
rate among all the groups. At a high dose of paclitaxel 10 mg/kg,
the mice died more rapidly than other three groups until 40 days.
The survival rate of the groups of paclitaxel 2 mg/kg, 10 mg/kg, and
LMWSC-NPT 2 mg/kg were higher than the control group. Practically, the survival rate between the groups of paclitaxel 2 mg/kg,
10 mg/kg, and LMWSC-NPT 2 mg/kg were not signicantly different. At a dose of 10 mg/kg of LMWSC-NPT, the highest survival rate
was maintained throughout all the experimental period among all
of the test groups.
The in vivo antitumor activity of paclitaxel and LMWSC-NPT
against CT26 murine tumor was tested for tumor volume as shown

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M.-K. Jang et al. / Colloids and Surfaces B: Biointerfaces 81 (2010) 530536

Fig. 4. Release of paclitaxel from coreshell type LMWSC-NP (a). For comparison, 1.0 mg of paclitaxel distributed in PBSSDS (pH 7.4, 0.1 M, SDS 2.0% (w/w)) was introduced
into dialysis bag and 10 mg of LMWSC-NPT (equivalent concentration of 1.0 mg paclitaxel) was introduced into dialysis bag. Typical HPLC characteristic peak of the quantied
paclitaxel (b).

in Fig. 6(b). Tumor volume (mm3 ) was measured from 15 days after
the tumor cell implantation. When paclitaxel and LMWSC-NPT are
injected at a low dose of 2 mg/kg, mice did not show any signicant
changes against control group as a tumor volume.
At high doses of 10 mg/kg, paclitaxel showed a decreased tumor
growth compared to the control group, paclitaxel (2 mg/kg), and
LMWSC-NPT (2 mg/kg). At a dose of 10 mg/kg of paclitaxel, the
tumor growth was favorably suppressed, but the tumor growth
restarted after 30 days. As shown in Fig. 6(b), LMWSC-NPT at a
dose of 10 mg/kg showed superior antitumor activity among all
the groups. In the case of LMWSC-NPT, the tumor growth was well
suppressed until 38 days and restarted after 40 days. As shown in
Fig. 6(c), the body weight changes among control groups, paclitaxel
(2 mg/kg), and LMWSC-NPT (2 mg/kg) were not signicantly different. At high doses of 10 mg/kg, LMWSC-NPT showed decreased
body weight changes. These results indicated that tumor volume of
LMWSC-NPT at 10 mg/kg was smaller than the other group, inducing decreased body weight changes.
Although tumor growth and survival rate did not signicantly
change at low dose of 2 mg/kg of LMWSC-NPT, LMWSC-NPT at
doses of 10 mg/kg showed a superior survival rate and a decreased
tumor growth compared to paclitaxel. LMWSC-NPT at the dose of
10 mg/kg seems to offer a superior antitumor activity.

4. Discussion
Chitosan, a (14) linked-2-amino-2-deoxy--d glucan, is
biodegradable, non-cytotoxic, and has some biologically active
characteristics. For its cationic properties, chitosan is extensively
investigated as a gene or drug carriers, and its amine group
may support easy modication for drug delivery vehicles [3234].
Especially, unlike HMWC (high molecular weight chitosan) chitooligosaccharide itself such as LMWSC are known to have an
metastatic activity to tumor cells [35]. Furthermore, LMWSC is
characterized as water-soluble chitosan having low molecular
weight and high density of free-amine group. Enhanced mucosal
delivery and enhanced efciency of transfection are the advantages
of LMWSC which give the increased free-amine group and cationic
properties [36,37].

We previously reported on self-assembling chitosan nanoparticles having a coreshell structure [30]. Self-assembled LMWSC
nanoparticles are composed of cholesterol-conjugated LMWSC
backbone and MPEG side chain. They have a coreshell structure
in aqueous environment, i.e., the cholesterol that makes up the
hydrophobic inner core is conjugated with the MPEG that makes
up the hydrophilic outer shell to form the LMWSC nanoparticle.
Hydrophobic inner core is composed of cholesterol-conjugated
LMWSC, and the outer shell of the nanoparticles is composed of
MPEG. These nanoparticles have particle size around 30150 nm
according to the composition ratio [30]. After lyophilization of
these nanoparticles, they can easily reconstitute into aqueous
solution such as distilled water or phosphate-buffered saline (pH
7.4, 0.1 M) (data not shown). Miwa et al. [38] have reported
that paclitaxel-entrapped micelle carriers are prepared from Nlauryl-carboxymethyl-chitosan with a maximum of 2.37 mg/ml
and their particle sizes were less than 100 nm. However, their
micellar solution was maintained in an aqueous solution rather
than in lyophilized form. For long-term storage, lyophilized form of
micellar solution or nanoparticle solution is preferred to aqueous
solution because the lyophilized form has some advantages such as
maintenance of peculiar properties of drug, avoidance of aggregation or precipitation of carriers and therapeutical activity of drug
when compared to aqueous solution [3941]. At this point, reconstitution of lyophilization is an important factor for clinical usage.
As shown in Fig. 3, nanoparticles with empty LMWSC-NP and the
LMWSC-NPT are completely reconstituted in the aqueous solution,
and no precipitants or large aggregates were observed. Hydrophobically modied chitosan as a carrier of paclitaxel has been reported
by Kim et al. [32] and their self-assembled nanoparticles showed
0.949.0% (w/w) of loading contents. Also, they reported that the
size of their nanoparticles increased up to 400 nm according to the
drug contents. However, their carriers of paclitaxel have drawbacks
in the surface properties. The surfaces of our nanoparticles are
covered with PEG, which gave the barrier properties of nanoparticulate, but their nanoparticles are not covered with PEG. That is why
their nanoparticles gave no protection. As shown in Fig. 2, strong
PEG peaks were observed at D2 O while the peaks of cholesterol moiety were not observed. However, when the nanoparticle broke up
in D2 O + pyridine, the peaks of cholesterol moiety were observed.

M.-K. Jang et al. / Colloids and Surfaces B: Biointerfaces 81 (2010) 530536

Fig. 5. Cell cytotoxicity of LMWSC-NPT against CT26 tumor cells. CT26 cells (5 103
cells/well) were treated with paclitaxel and LMWSC-NPT for 1 day (top) and 2 day
(middle). Empty LMWSC-NP was also employed for comparison of cell cytotoxicity
(bottom). Empty LMWSC-NP was treated to CT26 cells (5 103 cells/well).

These results indicate that the outer shell of nanoparticles is composed of PEG.
Currently, nanoparticles as colloidal drug carriers have been
extensively investigated for tumor targeting applications [18,42].
During the past decade, the coreshell type nanoparticles or polymeric micelles have been in the spotlight for drug targeting to
tumor since their coreshell structure is suitable to solubilize
hydrophobic anticancer drug [12,14,43]. Furthermore, their stealth
properties by surface-enriched PEG encourage long-circulation
of drug vehicles, avoidance of reticuloendotherial system (RES)

535

Fig. 6. Antitumor activity of LMWSC-NPT against CT26 colon carcinoma xenograft

model. Each drug was administered at doses of 2 mg/kg as a low dose and
10 mg/kg as a high dose for 4 times for 12 days at intervals of 3 days (arrows).
The control group received the vehicle (cremophor:dehydrated ethyl alcohol, 1:1,
v/v). 8 mice were used for each group: (a) Survival ratios of mice were monitored daily. LMWSC-NPT (paclitaxel-encapsulated coreshell type nanoparticles,
10 mg/kg) showed increased survival time compared to all other treatment (P < 0.01
vs. paclitaxel 10 mg/kg); (b) Tumor growth after drug injection was measured at
two or three day intervals. LMWSC-NPT (paclitaxel-encapsulated coreshell type
nanoparticles, 10 mg/kg) effectively suppressed tumor growth (P < 0.01 vs. paclitaxel
10 mg/kg); and (c) Body weight changes after tumor implantation following drug
administration. LMWSC-NPT (paclitaxel-encapsulated coreshell type nanoparticles, 10 mg/kg) showed decrease to all other treatment (P < 0.01 vs. paclitaxel
10 mg/kg).

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uptake, and passive targeting of tumor [12,15,43]. Especially, the

passive targeting of these drug vehicles can be described by EPR
(enhanced permeation and retention) effect to solid tumor [38]. Its
reduced particle size (10200 nm) enables intravenous injection of
hydrophobic drug and passive targeting to a specic organ [16,17].
The loading of paclitaxel in the coreshell type nanoparticles
of LMWSC-MPEG-Chol was identied using HPLC (Fig. 4(b)). The
result of HPLC revealed that the retention time (RT) of paclitaxel is
3.4 min. As shown in Fig. 4(a), paclitaxel release from the coreshell
type LMWSC-NPT continued over 1 month while paclitaxel released
itself from the dialysis membrane for 10 days.
Feng and Huang have reported the paclitaxel-encapsulated
nanospheres [22]. In their report, nanospheres released paclitaxel
less than 60% in 80 days. They reported that the duration of diffusion was 2 months. Cho et al., reported that sodium salicylate is
affected to the dissolution and release of paclitaxel from the polymeric micelle [44]. We used SDS to increase the dissolution and
release rate of paclitaxel from nanoparticles. However, paclitaxel
release took longer than expected. There are some experimental
factors that affect the release rate of paclitaxel from the nanoparticles. For example, dialysis membrane itself can act as a barrier of
drug transportation. Other researchers have also reported that free
paclitaxel was released very slow [44]. Furthermore, the hydrophobic core environment can increase the duration of drug release.
The antitumor efcacy of coreshell type LMWSC-NPT is shown
in Fig. 6(a)(c). The coreshell type LMWSC-NPT was most effective to suppress tumor growth at 10 mg/kg paclitaxel dose while
paclitaxel at equivalent dose was less effective to suppress tumor
growth. Although the tumor growth of both coreshell type and
paclitaxel (LMWSC-NPT) treatment at 2 mg/kg paclitaxel dose
was not signicantly different, the coreshell type LMWSC-NPT
at 10 mg/kg paclitaxel dose showed a higher survival rate up
to 50 days as shown in Fig. 6(a). Furthermore, body weight
changes were also smaller than paclitaxel treatment at 10 mg/kg
paclitaxel dose. These results indicated that the coreshell type
LMWSC-NPT is suitable paclitaxel carriers for inhibition of tumor
growth in vivo with minimized side-effects. Antitumor effects of
nanoparticulate-paclitaxel were also reported by other researchers
[32,45]. Polymeric micelle based on PEG-polylactide copolymer
also showed effectiveness in inhibiting tumor growth but their targeting mechanism was not fully understood [45]. Hydrophobically
modied glycol chitosan was also available to treat solid tumor
[32].
Our results demonstrated that the coreshell type LMWSC-NPT
is a useful vehicle to increase the solubility of paclitaxel and delivery
to the tumor since the coreshell type LMWSC-NPT showed excellent antitumor activity against CT26-bearing mice. Furthermore,
the coreshell type of LMWSC-NPT has an effective targeting property to tumor, thanks to its enhanced antitumor activity whereas
paclitaxel itself has no selectivity.

[11]
[12]
[13]

5. Conclusion

[35]
[36]
[37]

LMWSC was modied with MPEG (LMWSC-MPEG) and

then cholesterol was conjugated to them (LMWSC-MPEG-Chol).
Coreshell type LMWSC-NPs were prepared by the dialysis method,
and the coreshell structure was conrmed from the results of
1 H NMR. To this polymer, paclitaxel was encapsulated and the
coreshell types LMWSC-NPs were prepared. The release test indicated that paclitaxel in the LMWSC-NPT was sustained release from
nanoparticles but paclitaxel itself was more rapidly released out. A
cytotoxicity study using colon carcinoma cells, CT26, paclitaxelencapsulated coreshell type LMWSC-NPT showed an almost
similar toxicity against tumor cells as paclitaxel itself. The results
of the tumor inhibition test with CT26 mouse tumor models in vivo

indicated that the LMWSC-NPT at a dose of 10 mg/kg has a superior

survival rate, and decreasing the tumor growth to the paclitaxel;
however, the tumor growth and survival rate were not signicantly
different from each other at a dose of 2 mg/kg of LMWSC-NPT.
LMWSC-NPT above the dose of 10 mg/kg seems to have a superior
antitumor activity.
Acknowledgment
This work was partially supported by KITTOLIFE Co. and
Research Foundation of Engineering College, Sunchon National University.
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