Self-aggregates of cholesterol-modified carboxymethyl konjac glucomannan conjugate: Preparation, characterization, and preliminary assessment as a carrier of etoposide

Article abstract of DOI:10.1016/j.carbpol.2011.04.083

Various cholesterol (CH) bearing carboxymethyl konjac glucomannan (CKGM) amphiphilic conjugates (denoted CHCKGM) were synthesized using CKGM as hydrophilic segments and CH as hydrophobic parts. Structural characteristics of these CHCKGM conjugates were in

Full text of DOI:10.1016/j.carbpol.2011.04.083

Carbohydrate Polymers 86 (2011) 513–519
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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Self-aggregates of cholesterol-modified carboxymethyl konjac glucomannan
conjugate: Preparation, characterization, and preliminary assessment as a carrier
of etoposide
Wei Haa, Hao Wua,c, Xiao-Ling Wangc, Shu-Lin Penga, Li-Sheng Dinga, Sheng Zhangb,∗, Bang-Jing Lia,∗∗
a Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China
b State Key Laboratory of Polymer Materials Engineering, Polymer Research Institute of Sichuan University, Sichuan University, Chengdu 610065, China
c Ethenic Pharmaceutic Institute, Southwest University for Nationalities, Chengdu 610041, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Various cholesterol (CH) bearing carboxymethyl konjac glucomannan (CKGM) amphiphilic conjugates
(denoted CHCKGM) were synthesized using CKGM as hydrophilic segments and CH as hydrophobic
parts. Structural characteristics of these CHCKGM conjugates were investigated using FTIR, 1H NMR and
thermogravimetric analysis (TGA). The properties of these self-aggregates were analysed by dynamic
laser light-scattering (DLS), zeta potential, transmission electron microscopy (TEM) and the fluorescence
probe technologies. The critical aggregation concentration (cac) of CHCKGM conjugates (2.59 × 10−3 to
5.89 × 10−3 mg/mL) was comparatively low, suggesting that the cholesterol fragment was very effec-
tive for forming aggregates. Etoposide was physically entrapped into the CHCKGM nanoparticles by
sonication method. The in vitro release behavior of etoposide from CHCKGM nanoparticles revealed
a sustained release property. Furthermore, these self-aggregated nanoparticles showed pH- and ionic
strength-dependent properties which caused a considerable change in their radius.
Received 15 March 2011
Received in revised form 11 April 2011
Accepted 27 April 2011
Available online 11 May 2011
Keywords:
Carboxymethyl konjac glucomannan
Cholesterol
Nanoparticles
Drug carrier
Etoposide
In vitro release
© 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Konjac glucomannan (KGM), a high molecular weight and water
soluble natural polysaccharide isolated from the tubers of the
Various polymeric amphiphiles have achieved increasing atten-
tion due to their special physicochemical and morphological
properties in water (Gaucher et al., 2005; Kataoka, Harada,
widespread plant Amorphophallus konjac in China and Japan, is
composed of ␤-(1 → 4) linked ␤-d-glucose and ␤-d-mannose in
the molar ratio 1:1.6–1:1.69 (Shimahara, Suzuki, Sugiyama, &
Nishizawa, 1975). It is well-known that KGM has several advan-
tages such as declining sugar level; promoting intestinal activity
and weight loss; reducing the risk of diabetes and heart disease
(Chen, Cheng, Liu, Liu, & Wu, 2006; Li, Xia, Wang, & Xie, 2005).
Several chemical modification techniques have been applied to
develop bio-related functional materials with better properties
compared to the initial KGM (Du et al., 2004; Xia et al., 2010). CKGM
is an anionic polymer obtained from the carboxymethylation of
KGM. CKGM has been proven as a promising biomaterial in the
drug delivery system due to its good water solubility, biocompat-
ibility, bioactivity and excellent gelation ability when mixed with
a polymer with opposite charge (Du et al., 2005a,b; Wang et al.,
2008a). However, the synthesis and characterization of modified
CKGM with hydropho-moieties and further using this amphiphile
to form self-aggregated nanoparticles have not been reported yet.
Cholesterol is an indispensable structural building block in cells
and plays a major role in many body functions. Cholesterol could
be applied to modify some biomaterials due to highly hydrophobic
sterol skeleton. It has been reported that cholesterol-bearing
chitosan as a carrier showed stronger binding for hydrophobic
guests’ molecules and higher colloidal stability compared to
&
Nagasaki, 2001; Nishikawa, Akiyoshi, & Sunamoto, 1996).
Through non-covalent association arising from intra- and/or
inter-molecular interactions among hydrophobic segments in
the aqueous medium, these polymeric amphiphiles consisting
of hydrophilic and hydrophobic segments can form micelle
or micelle-like self-aggregates with hydrophobic cores and
hydrophilic shells (Akiyoshi, 2002; Akiyoshi, Deguchi, Moriguchi,
Yamaguchi, & Sunamoto, 1993; Dalhaimer, Bermudez, & Discher,
2004; Kakizawa, Harada, & Kataoka, 2001; Mortensen, 2001;
Rotureau, Chassenieux, Dellacherie, & Durand, 2005;). Because
these self-aggregates have potential uses in biotechnology and
medicine fields due to their unique supramolecular structures,
many polymeric amphiphiles have been explored, and their physic-
ochemical properties also have been widely investigated (Benita &
Levy, 1993; Jones & Leroux, 1999; Kang & Leroux, 2004; Qiu & Bae,
2007).
∗
Corresponding author. Tel.: +86 28 85400266; fax: +86 28 85402465.
Corresponding author. Tel.: +86 28 85228831; fax: +86 28 85223843.
E-mail addresses: zslbj@163.com (S. Zhang), libj@cib.ac.cn (B.-J. Li).
∗∗
0144-8617/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2011.04.083

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W. Ha et al. / Carbohydrate Polymers 86 (2011) 513–519
DMAP, DCC, CH2Cl2, 0°C, 24 h
TFA, CH2Cl2
0°C, 3 h
HOOC
N
t
boc
HO
O
N
t
boc
O
CH2OCH2COO-
O
CH2OCH2COO-
O
CH2OCH2COO-
O
CH2OH
O
O
O
O
p
O
OH
OH
OH OH
OH
OH
m
o
n
OH
OH
Formamide/ DMF
EDCI, NHS
O
NH2
O
O
O
O
O
NHCH2C
NHCH2C
CH2OCH2COO-
O
CH2OCH2CO
CH2OCH2CO
CH2OH
O
O
O
O
O
O
O
OH
OH
OH OH
OH
OH
m
o
p
n
OH
OH
Scheme 1. Synthesis procedure of CHCKGM.
corresponding long alkyl chains (Akiyoshi, Yamaguchi,
&
3-[3-(dimethylamino)propyl]-carbodiamide (EDC), dicyclohexyl-
carbodiimide (DCC), morpholinoethane sulfonic acid (MES),
N-hydroxysuccinimide (NHS) and trifluoroacetic acid (TFA) were
purchased from Sigma Aldrich (Shanghai, China).
All other reagents were of analytical grade and were used
directly without further purification. All solvents and water were
redistilled freshly.
Sunamoto, 1991). The cholesterol modified polymeric amphiphiles
as carriers for hydrophobic drugs or genes have been developed
extensively (Furgeson, Chan, Yockman, & Kim, 2003; Wang, Liu,
Jiang, & Zhang, 2007a,b; Wang et al., 2008b; Yu, Li, Qiu, & Jin, 2008;
Yuan, Li, & Yuan, 2006).
In this study, we designed and synthesized a novel self-
assembled cholesterol-modified carboxymethyl konjac glucoman-
nan (CHCKGM) conjugate via introducing cholesterol moieties
(Scheme 1) into the glucose units of CKGM. By varying the feed ratio
of cholesterol, a series of CHCKGM conjugates were prepared with
different grafting ratios. The physicochemical and morphologic
characteristics of these CHCKGM aggregates were also investigated
in details. Furthermore, etoposide was chosen as a model drug to
assess the potential of CHCKGM self-aggregated nanoparticles as
a drug delivery carrier. Etoposide is an important antineoplastic
agent used against several types of tumors, including testicular
and small cell lung cancers, lymphoma, leukemia, and Kaposi’s sar-
coma (Issell, Rudolph, & Louie, 1984). However, etoposide has weak
water solubility, etoposide therapy may cause some serious side
effects such as low white blood counts, low platelet count, anemia,
hair loss, soreness of the mouth and difficulty swallowing diar-
rhea (Falkson et al., 1975). Hence, it was expected that CHCKGM
self-aggregated nanoparticles being used as a carrier for etoposide
to improve its water solubility, sustain its release and give useful
information for reducing the side effects of etoposide therapy.
CKGM was prepared as described by reference (Shimahara,
Suzuki, Sugiyama, & Nishizawa, 1975). The degree of substitu-
tion was found to be 63.7% which measured according to methods
described in the literature (Smith, 1967).
1H NMRs were measured on a 600 MHz spectrometer (Avance
Bruker-600, Switzerland). The chemical shifts of 1H NMR are
expressed in parts per million downfield relative to the inter-
nal tetramethylsilane (ı = 0 ppm) or chloroform (ı = 7.26 ppm). IR
spectra were recorded on a Perkin Elmer spectrum one FT-IR
spectrometer using KBr discs in the range of 400–4000 cm−1
region. The centrifugations were taken on TGL-20M, Saite Cen-
trifuge Co., Shanghai, China. The absorbance value of the solution at
436 nm was recorded on APL-UV-2000 spectrophotometer, Shang-
hai, China. The dynamic light scattering (DLS) was measured by
BI-9000AT, BI-200SM, Brookhaven Instruments Co., USA. The TEM
were measured by Jeol JEM-100CX, and observation was done at
an accelerating voltage of 80 kV. The fluorescence intensities were
measured by Hitachi FP-7000, Japan. Zeta potential was measured
by Malvern Nano ZSZEN 3600, England.
2. Experimental methods
2.2. Synthesis of cholesterol glycinate (CHG)
2.1. Materials and instrument
Cholesterol (1 mmol) was dissolved into 10 mL anhydrous
CH2Cl2 together with N-t-Boc-glycine (1.1 mmol) and DMAP
(0.25 mmol). Subsequently, the reaction mixture was stirred at 0 ◦C
for 24 h after the addition of 1.1 mmol of DCC. The dicyclohexylurea
(DCU) was filtered off and then filtrate was concentrated in a vac-
KGM was purchased from Chengdu Root Industry Co.,
Ltd. (Chengdu, China). Cholesterol, N-tert-butoxycarbonylglycine
(N-t-Boc-glycine), 4-(dimethylamino) pyridine (DMAP), 1-ethyl-

W. Ha et al. / Carbohydrate Polymers 86 (2011) 513–519
515
uum at room temperature. The resultant was dissolved in a minimal
amount of acetone and cooled overnight, and the precipitated DCU
was filtered off. For the removal of t-boc group, cholesterol-t-boc-
amino acid ester was dissolved in a mixture of CH2Cl2/TFA (1/1, v/v).
The reaction mixture was stirred at 0 ◦C for 3 h and then evaporated
to dryness. The deprotected derivative was dispersed in a 15% NaCl
solution, and the pH was adjusted to 5.0. The solution was filtered to
remove water insoluble tert-butyl salts. The filtrate was extracted
three times with chloroform, the organic phases were combined
and dried over Na2SO4. The Na2SO4 solid was filtered, and the sol-
vent was evaporated. The obtained oily residue was finally dried
under vacuum at room temperature.
at room temperature. The final concentration of pyrene was
1.0 × 10−6 M. Fluorescent spectra were measured using fluores-
cence spectrophotometer with a slit width of 10.0 and 2.5 nm for
excitation and emission. For fluorescence emission spectra, excita-
tion wavelength was set at 339 nm, and for fluorescence excitation
spectra, the emission wavelength was set at 390 nm.
2.6. Particle size distribution and zeta potential
The sizes and size distributions of CHCKGM self-aggregated
nanoparticles in water were determined using dynamic laser light-
scattering (DLS) with a digital auto correlator at a scattering angle
of 90◦, a wavelength of 533 nm and a temperature of 25 ◦C. To inves-
tigate the relationship between the conjugates with different feed
ratio of CHG and the size of nanoparticle, a series of CHCKGM self-
aggregated nanoparticles in water with different feed ratio of CHG
were prepared. Furthermore, in order to confirm whether the size
of the CHCKGM self-aggregated nanoparticles could be affected by
the pH and the ionic strength, a series of samples with different
pH and concentrations of NaCl were prepared. The zeta poten-
tials of CHCKGM self-aggregated nanoparticles in water were also
measured.
IR (KBr, cm−1): 3434 (NH2-stretch), 2938 (C–H stretch), 1750
(C O stretch), 1676 (NH2 bending vibration), 1264 (C–N stretch),
1179 (C–O–C stretch).
1H NMR (CDCl3, ppm): 0.68 (3H, s, cholesterol 18-H3), 0.9–2.4
(28H, cholesterol l-H2, 2-H2, 4-H2, 7-H2, 8-H1, 9-H1, 11-H2, 12-H2,
14-H1, 15-H2, 16-H2, 17-H1, 20-H1, 22-H2, 23-H2, 24-H2 and 25-
H1), 0.88 (6H, d J = 6.4 Hz, cholesterol 26-H3 and 27-H3), 0.93 (3H,
dd, J = 6.24 Hz, 2.3 Hz,cholesterol 21-H3), 1.01 (3H, s, cholesterol 19-
H3), 3.98 (2H, m, OCOCH2–NH2), 4.65 (lH, m, cholesterol 3-H1), 5.37
(lH, m, cholesterol 6-H1).
2.3. Synthesis of CHCKGM
2.7. Transmission electron microscopy (TEM)
A 0.5 mmol CKGM was dissolved in 10 mL formamide solution
by water bath at 80 ◦C, then different amounts of EDC and NHS were
mixed with CKGM solution at room temperature (the molar ratio
of EDC:NHS:COO− was 1:0.5:1), followed by addition of the DMF
solution (10 mL) with different amounts of CHG. The resulting solu-
tion was stirred at room temperature under a nitrogen atmosphere
for 24 h. After reaction, the mixture was precipitated in excessive
acetone. The precipitate was carefully washed with water and THF
to remove excessive CHG and CKGM. The precipitate was dispersed
by water and dialysed by bag filter (MWCO: 8000–14000) at 25 ◦C
for 72 h, and the solvent was lyophilized to obtain white powder.
IR (KBr, cm−1): 3411 (OH-stretch), 2936 (C–H stretch), 1746
(C O stretch), 1674 (amide stretch).
To observe the morphology of CHCKGM self-aggregated
nanoparticles, sample solutions (0.5 mg/mL) were dropped onto
the carbon-coated 300 mesh copper grids. Then, the grids were
air-dried and imaged using a transmission electron microscope.
2.8. Preparation of etoposide-loaded nanoparticles
4 mg CHCKGM and 1 mg etoposide was dispersed in water under
gentle shaking at room temperature for 24 h, followed by sonica-
tion for 2 min. To prevent heat build-up during the sonication, the
pulse function was pulse on 2.0 s and pulse off 2.0 s. The sample
solution was centrifuged at 15,000 rpm to obtain etoposide loaded
CHCKGM nanoparticles, and the nanoparticles were washed with
deionized water three times to remove the etoposide, which was
not entrapped inside the CHCKGM nanoparticles. The amount of
etoposide was detected by UV spectrometry at 294 nm and calcu-
lated with standard curve plotted by the ꢀA294nm of the standard
versus the etoposide concentration (the standard curve was plotted
using a standard etoposide solution (Fig. S1). The etoposide encap-
sulation efficiency was calculated with the following equation:
1H NMR (DMSO, ppm): 0.67 (3H, s, cholesterol 18-H3), 0.8–2.4
(28H, cholesterol l-H2, 2-H2, 4-H2, 7-H2, 8-H1, 9-H1, 11-H2, 12-H2,
14-H1, 15-H2, 16-H2, 17-H1, 20-H1, 22-H2, 23-H2, 24-H2 and 25-
H1), 0.88 (6H, dd J = 6.4 Hz, 2.3 Hz, cholesterol 26-H3 and 27-H3),
0.93 (3H, d, J = 6.24 Hz, cholesterol 21-H3), 1.01 (3H, s, cholesterol
19-H3), 3.0–4.5 (m, glucose and mannose ring 2, 3, 4, 5, 6-H1), 3.98
(2H, m, OCOCH2–NH2), 4.63 (lH, m, cholesterol 3-H1), 5.37 (lH, m,
cholesterol 6-H1).
Encapsulation efficiency (%)
2.4. Preparation of self-aggregated CHCKGM nanoparticles
mass of etoposide used in encapsulation − mass of free etoposide
=
mass of etoposide used in encapsulation
CHCKGM self-aggregated nanoparticles were prepared by soni-
cation in aqueous medium. Briefly, CHCKGM was dispersed in water
under gentle shaking at room temperature for 24 h, followed by
sonication for 2 min. To prevent heat build-up during the sonica-
tion, the pulse function was pulse on 2.0 s and pulse off 2.0 s. The
sample solution was centrifuged at 7000 rpm to remove dust and
impurity.
×100
2.9. In vitro release studies
Release rate measurements in vitro were carried out as follows:
2 mL etoposide loaded nanoparticles solution was placed in a dial-
ysis bag (MWCO = 7000) and dialyzed against 10 mL of PBS. The
release medium was stirred at 100 rpm at 30 ◦C. At predetermined
sampling times; 4 mL medium was removed and replaced by 4 mL
fresh PBS to maintain submersed conditions. The amount of etopo-
side in the solution was determined by UV spectrometry at 294 nm
and calculated with the standard curve plotted by the ꢀA294nm
of the standard versus the etoposide concentration (the standard
curve was plotted using a standard etoposide solution, see Fig. S1).
2.5. Self-aggregation behavior of CHCKGM conjugates
The CHCKGM conjugates suspension was prepared in the same
way as the aggregate preparation. The aggregate solution was
adjusted to various CHCKGM conjugate concentration, a known
amount of pyrene in methanol was evaporated at 40 ◦C. A total
of 3 mL of various concentration of sample suspension was added
to each vial, and heated for 3 h at 65 ◦C to equilibrate the pyrene
and the nanoparticles, and remained undisturbed to cool overnight

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W. Ha et al. / Carbohydrate Polymers 86 (2011) 513–519
Fig. 1. FT-IR spectra of CHCKGM conjugates, CKGM and CHG.
Fig. 2. 1H NMR spectra for (a) CKGM in D2O, (b) CHG in CDCl3 and (c) CHCKGM-1
in DMSO.
3. Results and discussion
ilar. The decomposition temperatures of the CHCKGM conjugates
are higher than those of the pure CKGM indicating the enhanced
thermal stability of CHCKGM conjugates.
3.1. Preparation of CHCKGM conjugates
Scheme 1 shows the synthesis procedure of CHCKGM conju-
gates. An amino group was initially introduced to the cholesterol
molecule using N-tert-butoxycarbonyl glycine by a two-step reac-
tion. The CKGM was then coupled with the primary amino groups
of cholesterols with “zero length” crosslinker of EDC/NHS afforded
the final product CHCKGM.
3.2. Self-aggregation behavior of CHCKGM conjugates
The fluorescence probe technique was applied to study the
self-aggregation behavior of CHCKGM conjugates on a molecular
level. Pyrene was chosen as the fluorescent probe due its photo-
physical properties (Wilhelm et al., 1991). In a polar environment
(water), pyrene shows only small fluorescence intensity due to its
poor solubility and self-quenching, however, once the hydropho-
bic micro-domains in an aqueous solution was formed, pyrene will
emits extensive radiation because its preferential locating inside
or close to the hydrophobic micro-domains of micells. Therefore,
pyrene is commonly used as a fluorescence probe to monitor the
self-aggregation behavior of surfactants and/or polymers.
In low concentration range of CHCKGM conjugates, as shown
in Fig. S2, the fluorescence excitation spectra of pyrene incor-
porated into various concentrations of CHCKGM self-assembled
nanoparticles shows no significant change in the total fluorescence
intensity. With the concentration increasing, however, the fluores-
cence intensity increased remarkably, reflecting the partitioning of
pyrene into the hydrophobic microdomains of self-aggregates. The
partitioning of pyrene led to the shift of the main peak, (0, 0) band,
from 332 to 337 nm.
Fig. 1 shows the IR spectra of CKGM and CHCKGMs with increas-
ing feed ratio of CHG. The spectra of CKGM (Fig. 1a) show the
following selected peaks: 3435 (O–H stretch), 2923 (C–H stretch),
1602 (C O stretch of carboxyl methyl), 1059 (C–O–C stretch)
(cm−1). In Fig. 1b, the following selected IR peaks for CHCKGMs are
shown: 3434 (OH stretch), 2938 (C–H stretch), 1750 (C O stretch
of ester bond), 1675 (NH2 bending vibration), 1139–1264 (C–N
stretch) (cm−1). With increasing feed ration of CHG, the IR peaks
of CHCKGMs in the region of 1668–1674 cm−1 (amide stretch)
increased significantly, which confirmed the formation of an amide
linkage between the primary amines of cholesterol and the car-
boxylates of CKGM. It can be seen that the intensity of amide
stretch (1668–1674 cm−1) is increasing with the feed ratios of
cholesterol–glycine increased, which indicates the DS is increasing.
The 1H NMR spectra of CKGM, CHCKGM and CHG are shown
in Fig. 2, the proton assignment of CHG and CHCKGM are listed in
experimental section. Compared with the proton signals of CKGM
(Fig. 2a) and CHG (Fig. 2b), the more intensive –CH3 peaks (ı
0.5–1.0) and double bond signal (ı 5.3) of CHG confirm the presence
of cholesterol moieties (Fig. 2c). The characteristic peaks of CKGM
appear between 3.0 and 4.8 ppm.
The TGA thermograms of CKGM, CHG and CHCKGM conjugates
are shown in Fig. 3. The curves show that both CKGM and CHG
involve two steps weight losses. The first slight weight loss at
low temperatures can be attributed to the dryness procedure dur-
ing heating and the subsequent second major weight loss can be
attributed to the decomposition of CKGM (277 ◦C) or CHG (310 ◦C).
In the cases of CHCKGM conjugates, three main stages of degrada-
tion could be observed: a moisture loss at a temperature around
100 ◦C, followed by a major weight loss of the CKGM component
at about 270 ◦C and another subsequent weight loss of the CHG
component around 300 ◦C. However, the weight loss of both CKGM
and CHG component overlapped to one major weight loss stage
because the decomposition temperature of CKGM and CHG is sim-
Fig. 3. TGA thermograms of CKGM, CHCKGMs and CHG.

W. Ha et al. / Carbohydrate Polymers 86 (2011) 513–519
517
is ascribed to the hydrophobic interaction between hydrophobic
moieties. It seemed that the cholesterol segments offered strong
binding sites and was very efficient for the formation of aggre-
gates. Therefore, the cholesterol modified CKGM conjugates are the
promising alternatives to encapsulate substances via hydrophobic
interaction.
3.3. Preparation and characterization of CHCKGM
self-aggregated nanoparticles
Self-aggregated nanoparticles were formed by sonication when
CHCKGM conjugates were dispersed in water at the concentration
of 0.5 mg/mL. Increasing the feed ratio of cholesterol moiety results
in the smaller size of the nanoparticles (Table 1), although there is
tiny difference in CHCKGM-2 and CHGKGM-3. These results sug-
gest that the higher grafting density of the hydrophobic cholesterol
grafts favor to smaller aggregates. This trend of the size variation
with graft density is consistent with the micells of graft copolymers
in selective solvent (Hu et al., 2008).
Fig. 4. Plot of the intensity ratio I337/I332 (from pyrene excitation spectra) as a func-
tion of log C (CHCKGM-1, cycles; CHCKGM-2, triangles; CHCKGM-3, squares).
The morphology of self-aggregates was investigated by TEM
(Fig. 5). As shown in Fig. 5a–c, those nanoparticles are almost
sphere. From the TEM images, the size of these nanoparticles
decreased from 400 nm to less than 100 nm with feed ratio of
cholesterol moiety increasing from 0.3 to 1. However, as shown
in Table 1, the sizes of the self-aggregated nanoparticles measured
by DLS are in the range from 1033 to 391 nm, which were obvi-
ously larger than the sizes determined by TEM. We believe this
may result from the shrinkage of particles during the process of
the solvent evaporation in the samples preparation.
As shown in Fig. 5d, the zeta potential of CHCKGM-1 self-
aggregated nanoparticles in water were negative, and the absolute
value of zeta potential of CHCKGM-1 self-aggregated nanopar-
ticles was 30.8 1 mV, suggesting that the negative charged
carboxymethyl group in CKGM molecules were mainly distributed
on the surface of self-aggregated nanoparticles due to their
hydrophilic property.
Fig. 4 demonstrates the intensity ratio (I337/I332) of the pyrene
excitation spectra versus the logarithm of the concentration of
CHCKGM. Critical aggregation concentration (cac), the threshold
concentration of self-aggregate formation by intra- or intermolec-
ular association, was determined from the crossover point in the
low concentration ranges. The cac values (Table 1) of the CHCKGM
conjugates are in the range of 2.59 × 10−3 to 5.89 × 10−3 mg/mL,
which are one order of magnitude lower than those of low molecu-
lar weight surfactants (Nagasaki et al., 1998), and even more lower
than those of other polymeric amphiphiles (Kim et al., 2000; Kim,
Lee, Kwon, Chung, & Jeong, 2002), including other cholesterol-
modified aggregates (Wang, Liu, Jiang, & Zhang, 2007a; Yu, Li,
Qiu, & Jin, 2008). The higher feed ratio of the cholesterol moi-
ety induces the lower cac. The low cac value implies that the
self-aggregates can be formed easily by modified CKGM and the
corresponding aggregates maintain the stability in dilute condi-
tion. The major driving force for the aggregation of amphiphiles
Fig. 5. (a) TEM image of CHCKGM-1, (b) TEM image of CHCKGM-2, (c) TEM image of CHCKGM-3, (d) zeta potential of CHCKGM-1.

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W. Ha et al. / Carbohydrate Polymers 86 (2011) 513–519
Table 1
Characterization of CHCKGM self-aggregated nanoparticles.
Samplesa
Feed mole ratiob
CAC × 10−3 (mg/mL)
Diameter (nm)
Polydispersity index
CHCKGM-1
CHCKGM-2
CHCKGM-3
0.3:1
0.5:1
1:1
5.89
3.89
2.59
1033 47
477 30
391 23
0.223 0.047
0.126 0.027
0.246 0.002
a
CHCKGM conjugates, where the number indicates the increasing feed ratio of CH.
Mole ratio of CH: the sugar units of CKGM.
b
3.4. Drug loading and in vitro drug release study
Etoposide was the first agent recognised as a topoisomerase II
inhibiting anticancer drug. Research on etoposide helps the under-
standing of mechanisms by which the drugs poison topoisomerase
II. With the demonstrated antineoplastic activity of the drug, FDA
approval was granted for etoposide in 1983. Up to now, the work
to improve the therapeutic index of etoposide mostly focused
on dose-regimens and pharmaceutical preparation (Hande, 1998).
Therefore, we developed a new method by using a carrier for load-
ing etoposide, which aimed at improving its therapeutic index.
The incorporation of etoposide into the CHCKGM-2 particles
occurred simultaneously during sonication and the encapsulation
efficiency achieved to 39.4%. The in vitro release profile of etopo-
side from etoposide/CHCKGM-2 particles was presented in Fig. 6.
The etoposide appeared to be released in a biphasic way, which
characterized by an initial rapid release period followed by a step
of slower release. The burst effect was observed in 2 h, in which 55%
of the drug was released from the nanoparticles. After this initial
effect, etoposide was released in a continuous way for up to 23 h,
reaching to close 96% of cumulative release.
Fig. 7. Average hydrodynamic diameter ꢀDhꢁ of CHCKGM-1 self-aggregated spheres
as a function of pH.
3.5. pH and ionic strength effects on CHCKGM nanoparticles
mental pH is adjusted to 8 or higher, the radius of the particles
tends towards to a saturation value. It is worth noting that this
change is completely reversible. When the pH is adjusted from
12 to 3, similar results have also been observed. The extension of
this expansion depends on ionic strength of the aqueous solution
at given pH value. The effect of varying salt (NaCl) concentra-
tion is shown in Fig. 8, as can directly be seen that the swelling
of the particles increases with increasing salt concentration due
to more intense electrostatic shielding effects. Similar observa-
tions have been made with other polyelectrolytes based particles
(Liu & Armes, 2003).
The conformation of polyelectrolytes is very sensitive towards
the physicochemical conditions of their surrounding medium
(e.g., ionic strength, pH, or presence of multivalent ions). With
the pH increasing, the carboxyl groups of the CKGM segment
are progressively neutralized corresponding to more hydrophilic
–COO− groups formation. As a result of the associated electro-
static repulsion between the increasing numbers of identically
charged carboxylate anions along the CKGM backbone, the shells
of such particles should swell considerably upon increasing the
pH. As shown in Fig. 7, the particles radius increases from 660 nm
to 880 nm as the pH increases from 4 to 8. When the environ-
Fig. 6. Etoposide release from etoposide/CHCKGM-2 nanoparticles at 30 ◦C in PBS
(pH 7.4).
Fig. 8. Average hydrodynamic diameter ꢀDhꢁ of CHCKGM-1 self-aggregated spheres
as a function of NaCl concentrations.

W. Ha et al. / Carbohydrate Polymers 86 (2011) 513–519
519
4. Conclusion
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In summary, novel carboxymethyl konjac glucomannan con-
jugates with cholesterol conjugates were synthesized and char-
acterised. These polymeric amphiphiles can form self-aggregates
in aqueous media. The cac values of conjugates are smaller than
5.89 × 10−3 mg/mL, indicating that the cholesterol residue is very
effective to form aggregates. Those self-aggregates exhibit a pH-
and ionic strength-dependent properties causing a considerable
increase (decrease) of their radius. Hydrophobic drug etoposide
was successfully entrapped into the CHCKGM nanoparticles and its
release behavior in vitro exhibited a sustained release. These pre-
liminary results indicated that CHCKGM self-aggregates could be a
potential drug carrier for the drug delivery.
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Acknowledgements
This project is supported by National Natural Science Foun-
dation of China (NSFC Grant Nos. 21074138, 51073107 and
50703025), CAS Innovation Program (Grant No. KSCX2-EW-J-22)
and the Opening Project of State Key Laboratory of Polymer Mate-
rials Engineering (Sichuan University) (KF201003).
Liu, S. Y.,
ior of
&
a
Armes, S. P. (2003). Synthesis and aqueous solution behav-
pH-responsive schizophrenic diblock copolymer. Langmuir, 19,
Appendix A. Supplementary data
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between bovine serum albumin and the self-assembled hydrogel nanoparticle
of hydrophobized polysaccharides. Journal of the American Chemical Society, 118,
6110–6115.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carbpol.2011.04.083.
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