Scutellarin-cyclodextrin conjugates: Synthesis, characterization and anticancer activity

Article abstract of DOI:10.1016/j.carbpol.2012.10.012

A series of scutellarin-cyclodextrin conjugates (SCU-CD conjugates), in which scutellarin was covalently bound to one of the primary hydroxyl groups of β-CD, were prepared, and their structures were determined using NMR and MS. These conjugates were further characterized by XRD and TG. The results showed that the aqueous solubility of the conjugates was much higher than that of scutellarin, and the conjugates could hardly be hydrolyzed to scutellarin in aqueous solutions. The cytotoxicity of SCU-CD conjugates on human colon cancer cell lines HT-29, SW480, Lovo and HTC116 indicated that the antitumor activities of the conjugates were better than that of scutellarin. This high antitumor activity, along with the satisfactory aqueous solubility and high stability of the conjugates, will be potentially useful for their application on human colon cancer chemotherapies.

Full text of DOI:10.1016/j.carbpol.2012.10.012

Carbohydrate Polymers 92 (2013) 1308–1314
Contents lists available at SciVerse ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Scutellarin-cyclodextrin conjugates: Synthesis, characterization and anticancer
activity
Bo Yanga,∗, Yu-Lin Zhaob, Xia Yanga, Xia-Li Liaoa, Jian Yanga, Ji-Hong Zhanga, Chuan-Zhu Gaoa
a Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, PR China
b Faculty of Chemical Engineering, Kunming University of Science and Technology, Kunming 650500, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of scutellarin-cyclodextrin conjugates (SCU-CD conjugates), in which scutellarin was covalently
bound to one of the primary hydroxyl groups of ␤-CD, were prepared, and their structures were deter-
mined using NMR and MS. These conjugates were further characterized by XRD and TG. The results
showed that the aqueous solubility of the conjugates was much higher than that of scutellarin, and the
conjugates could hardly be hydrolyzed to scutellarin in aqueous solutions. The cytotoxicity of SCU-CD
conjugates on human colon cancer cell lines HT-29, SW480, Lovo and HTC116 indicated that the antitu-
mor activities of the conjugates were better than that of scutellarin. This high antitumor activity, along
with the satisfactory aqueous solubility and high stability of the conjugates, will be potentially useful for
their application on human colon cancer chemotherapies.
Received 20 June 2012
Received in revised form 1 October 2012
Accepted 3 October 2012
Available online xxx
Keywords:
Scutellarin
␤-Cyclodextrin
Conjugate
Synthesis
Crown Copyright © 2012 Published by Elsevier Ltd. All rights reserved.
Characterization
Anticancer activity
1. Introduction
also possess the ability to include various organic and biological
substrates within its hydrophobic cavity (Szejtli, 1998). This prop-
In recent years, anticancer activities of flavonoid components
from medicinal plant extract have been reported (Himeji et al.,
2007; Li-Weber, 2009). Scutellarin (SCU, Scheme 1) is the main
effective flavonoid component of breviscapine, which is an extract
from the dried whole plant of Erigeron breviscapus (Vant.) Hand-
Mazz. It was reported to attenuate H2O2-induced cytotoxicity,
intracellular accumulation of reactive oxygen species (ROS) and
Ca2+, lipid peroxidation, and loss of mitochondrial membrane
potential (MMP) and DNA, which may represent the cellular mech-
anisms for its neuroprotective action (Hong & Liu, 2004). The
chemically standardized extract, including some scutellarin, from
Scutellaria barbata can induce cell death in the human colon can-
cer lines (Goh, Lee, & Ong, 2005). Scutellarin can also inhibit tumor
cell proliferation and migration, and regulate cell adhesion in oral
squamous cell carcinoma (OSCC) (Li et al., 2010). However, it has
poor water solubility and low antitumor activity, which inevitably
hinder its application in vivo (Li-Weber, 2009; Yang, Yang, Lin,
Chen, & Liu, 2009). Cyclodextrins (CDs) are truncated-cone polysac-
charides mainly composed of six to eight d-glucose monomers
linked by ␣-1,4-glycosidic bonds, which possess a hydrophobic cav-
ity and numerous hydroxyl groups. Simultaneously, cyclodextrins
erty may enable it to adhere to the surface of tissues or cells by
including accessible surface molecules in the cavity (Wenz, 1994).
Therefore, the conjugates, which covalently linked drug molecular
to cyclodextrin, are used for drug delivery (Kamada et al., 2002; Udo
et al., 2010; Uekama, Minami, & Hirayama, 1997; Yano, Hirayama,
Kamada, Arima, & Uekama, 2002).
In this work, scutellarin-cyclodextrin conjugates (amino-SCU-
␤-CD conjugate, enamino-SCU-␤-CD conjugate and dienamino-
SCU-␤-CD conjugate, Scheme 1) were prepared, in which SCU is
covalently bound to one of the primary hydroxyl groups of ␤-CD,
and their structures were identified by 1H NMR and MS. These
conjugates were characterized by Powder X-ray diffraction and
thermal analysis. Their solubilization and stabilization effects were
evaluated as well as their anti-colon cancer activities on HT-29,
SW480, Lovo and HTC116 cell lines. These may provide a useful
approach to develop a highly effective drug candidate for human
colon cancer chemotherapies.
2. Materials and methods
2.1. Materials
Scutellarin (>99%) was obtained from Kunming Pharmaceu-
tical Corporation in Yunnan Province, PR China. ␤-Cyclodextrin
was commercially available. N,N-Dimethyl formamide (DMF) was
∗
Corresponding author. Tel.: +86 871 5920637; fax: +86 871 5920570.
E-mail address: yangbo6910@sina.com (B. Yang).
0144-8617/$ – see front matter Crown Copyright © 2012 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carbpol.2012.10.012

B. Yang et al. / Carbohydrate Polymers 92 (2013) 1308–1314
1309
Scheme 1. Chemical structure of scutellarin and scutellarin-␤-cyclodextrin conjugates.
predried over calcium hydride for 2 days and then distilled under
2.3. Preparation of
a reduced pressure prior to use. Dicyclohexylcarbodiimide (DCC)
was commercially available (Shanghai Reagent Factory) and used
without further purification. Other chemicals and solvents were of
analytical-reagent grade, and deionized double-distilled water was
used throughout the study.
mono[6-(scutellarin)ethyleneamino-6-deoxy]-ˇ-cyclodextrin
(enamino-SCU-ˇ-CD conjugate)
Mono(6-deoxy-6-ethyleneamino)-␤-CD
(en-␤-CD)
was
obtained in two steps from the parent ␤-CD (May, Kean, Easton, &
Lincoln, 1997; Petter, Salek, Sikorski, Kumaravel, & Lin, 1990). To a
solution of DMF (50 mL) containing 2.4 g (2 mmol) of en-␤-CD and
0.62 g (3 mmol) of DCC was added 0.92 g (2 mmol) of scutellarin
˚
2.2. Preparation of
in the presence of a small amount of 4 A molecular sieves. The
mono[6-(scutellarin)amino-6-deoxy]-ˇ-cyclodextrin
(amino-SCU-ˇ-CD conjugate)
reaction mixture was stirred for 2 days in an ice bath and another
2 days at room temperature, and then allowed to stand for 1 h.
The precipitate was removed by filtration and the filtrate was
poured into 300 mL of acetone. The precipitate was collected and
subsequently purified on a Sephadex G-25 column with water as
eluent. After the residue was dried in vacuo, enamino-SCU-␤-CD
conjugate was obtained in 52% yield. UV/Vis ꢀmax (H2O)/nm (log ε)
330.0 (4.04), 285.5 (4.07); 1H NMR (500 MHz, D2O): ı (ppm)
7.65–7.51 (dd, H-2ꢀ, 6ꢀ of SCU), 7.25 (t, H-3ꢀ, 5ꢀ of SCU), 6.81 (s, H-3
of SCU), 6.62 (s, H-8a of SCU), 6.42 (s, H-8b of SCU), 4.92 (s, H-1 of
en-␤-CD), 3.85–3.58 (m, H-3, 5, 6 of en-␤-CD), 3.55–3.25 (m, H-2,
4 of en-␤-CD), 3.15–2.85 (m, H of ethyl of en-␤-CD); ESI MS m/z
1621.4992 [M+H]+ (Anal. Calcd for C65H93N2O45, 1621.4972).
Mono(6-deoxy-6-amino)-␤-CD (amino-␤-CD) was obtained in
three steps from the parent ␤-CD and purified by Sephadex chro-
matography with aqueous solution (Belanger & Perly, 1992; Croft
& Bartsch, 1983). A solution of amino-␤-CD (2.2 g, 2 mmol), scutel-
larin (SCU, 1.1 g, 2.4 mmol), DCC (0.56 g, 3 mmol) and a small
˚
amount of 4 A molecular sieves in DMF (50 mL) were stirred for
1 h in an ice bath and subsequently for 24 h at room temperature.
The insoluble materials were removed by filtration. The filtrate was
poured into 300 mL of acetone. The precipitate was collected and
subsequently purified on a Sephadex G-25 column with water as
eluent. The residue was dried in vacuo, and amino-␤-CD conjugate
was obtained in yield of 46%. UV/Vis ꢀmax (H2O)/nm (log ε) 332.0
(4.10), 284.5 (3.95); 1H NMR (500 MHz, D2O): ı (ppm) 7.68–7.53 (m,
H-2ꢀ, 6ꢀ of SCU), 7.35–7.25 (m, H-3ꢀ, 5ꢀ of SCU), 7.08 (s, H-3 of SCU),
6.37 (s, H-8 of SCU), 4.95 (s, H-1 of amino-␤-CD), 3.88–3.62 (m, H-3,
5, 6 of amino-␤-CD), 3.62–3.28 (m, H-2, 4 of amino-␤-CD); ESI MS
m/z 1577.4407 [M+H]+ (Anal. Calcd for C63H88NO45, 1577.4550).
2.4. Preparation of
mono[6-(scutellarin)diethyleneamino-6-deoxy]-ˇ-cyclodextrin
(dienamino-SCU-ˇ-CD conjugate)
Mono(6-deoxy-6-diethyleneamino)-␤-CD (dien-␤-CD) was
obtained in two steps from the parent ␤-CD (May et al., 1997;

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B. Yang et al. / Carbohydrate Polymers 92 (2013) 1308–1314
Table 1
Petter et al., 1990). A solution of dien-␤-CD (2.4 g, 2 mmol), scutel-
Some physicochemical properties of SCU and SCU-␤-CD conjugates.
larin (1.1 g, 2.4 mmol), DCC (0.62 g, 3 mmol) and a small amount
of 4 A molecular sieves in DMF (50 mL) were stirred for 1 h in
Solubility (mmol/L)a
˚
Compound
MW
an ice bath and subsequently for 24 h at room temperature. The
insoluble materials were removed by filtration. The filtrate was
poured into 300 mL of acetone. The precipitate was collected and
subsequently purified on a Sephadex G-25 column with water as
eluent. After the residue was dried in vacuo, dienamino-SCU-␤-CD
conjugate was obtained in a yield of 54%. UV/Vis ꢀmax (H2O)/nm
(log ε) 333.0 (4.25), 292.5 (4.17); 1H NMR (500 MHz, DMSO-d6): ı
(ppm) 7.68–7.45 (dd, H-2ꢀ, 6ꢀ of SCU), 7.20–7.12 (t, H-3ꢀ, 5ꢀ of SCU),
6.95 (s, H-3 of SCU), 6.80 (s, H-8 of SCU), 4.83 (s, H-1 of dien-␤-CD),
3.71–3.52 (m, H-3, 5, 6 of dien-␤-CD), 3.48–3.25 (m, H-2, 4 of
dien-␤-CD), 2.85–2.55 (m, H of ethyl of dien-␤-CD); ESI MS m/z
1662.5052 [M−H]− (Anal. Calcd for C67H92N3O45, 1662.5394).
Scutellarin
462
1576
1620
1663
0.35 0.01
16 0. 5
20 0.5
Amino-SCU-␤-CD conjugate
Enamino-SCU-␤-CD conjugate
Dienamino-SCU-␤-CD conjugate
25 0.5
a
In water at 25 ◦C.
2.9. Measurement of cytotoxicity
The cytotoxic activities of scutellarin and its conjugates were
evaluated as cell survival after treatment. Cell viability was
evaluated by a microculture tetrazolium reduction assay using
MTT (3-(4,5-dimethyltriazol-2-yl) 2,5-diphenyltetrazolium bro-
mide) assay. Briefly, 50 mL of MTT stock solution (2 mg/mL in PBS)
was added to 150 mL cell cultures in 96-microwell flat-bottom
plates for 72 h incubation at 37 ◦C. Plates were then centrifuged and
MTT containing culture medium were removed. Precipitated form-
azan was dissolved in 150 mL of DMSO. Results were read with
15 min in a spectrometer at 490 nm, and the means of triplicates
were calculated. Cell inhibition rate is expressed as percentage of
control samples.
2.5. Measurement of aqueous solubility
The aqueous solubility of the conjugates was assessed by prepa-
ration of its saturated aqueous solution (Montassier, Duchêne, &
Poelman, 1997). An excess amount of conjugate was put in 5 mL of
water (pH ca. 7), and the mixture was stirred for 1 h. After remov-
ing the insoluble substance by filtration, the filtrate was evaporated
under reduced pressure to dryness and the residue was dosed by
weighing method.
3. Results and discussion
3.1. Chemistry
2.6. Characterization of the conjugates
Mono(6-deoxy-6-amino)-␤-CD (amino-␤-CD) was prepared in
three steps according to the method of previous study (Fig. 1)
(Belanger & Perly, 1992; Croft & Bartsch, 1983). The mono [6-O-(p-
toluentsulfonyl)]-␤-CD (6-OTs-␤-CD) was converted to mono(6-
azido-6-deoxy)-␤-CD by the reaction with sodium azide in water.
The compound was reduced by treatments with triphenylphos-
phine in DMF and then with concentrated ammonium hydroxide to
obtain amino-␤-CD. Mono(6-deoxy-6-ethyleneamino)-␤-CD (en-
␤-CD) and mono(6-deoxy-6-diethyleneamino)-␤-CD (dien-␤-CD)
were prepared in two steps according to the method of previ-
ous study (Fig. 1) (May et al., 1997; Petter et al., 1990). Firstly,
one of the primary alcohol group of ␤-CD was tosylated using
p-toluenesulfonyl chloride in alkaline aqueous solution. Then,
6-OTs-␤-CD was converted to mono en-␤-CD or dien-␤-CD on
heating in excess ethylenediamine or diethylenetriamine. The
amino-␤-CD, en-␤-CD and dien-␤-CD were coupled to the scutel-
larin carboxyl group which was first activated using DCC in the
UV/Vis spectra were performed on a Shimadzu UV 3600 spec-
trophotometer, and pH 7.2 buffer solution was used in the spectral
measurements.
1H NMR experiment was performed on a Bruker Avance DRX500
spectrometer at 298 K in a D2O or DMSO-d6. Samples were kept at
least 24 h before measurement for equilibration.
Electrospray ionization mass spectra (ESI MS) were recorded on
an Agilent 6210 TOF mass spectrometer in D2O.
Powder X-ray diffraction (XRD) was measured in a D/max-3B
˚
diffractometer using Cu-␬␣ (k = 15460 A) with 30 mA, 40 kV, and a
scanning rate of 5◦/min. Powder samples were mounted on a sam-
ple holder and scanned with a step size of 2Â = 0.02◦ between 2Â = 3◦
and 70◦.
Thermal analyses (TG) were recorded on a NETZSCH STA449F3
instrument, with a 10 ◦C/min heating rate from room temperature
to 500 ◦C under N2 flow (100 mL/min).
˚
presence of a small amount of 4 A molecular sieves. The scutellarin-
cyclodextrin conjugates were obtained by purifying on a Sephadex
column with water as eluent. The chemical structure of the
enamino-SCU-␤-CD conjugate was determined by MS and NMR
spectra as shown in Fig. 2.
In the high resolution mass spectrum, the enamino-SCU-␤-CD
conjugate gave a quasi-molecular ion of 1621.4992 [M+H]+. The
formation of a 1:1 conjugation could be confirmed by the 7:6 ratio
between the peak areas of H1 proton of ␤-CD (7 protons) and aro-
matic proton (6 protons) of SCU in the 1H NMR spectrum. The result
indicates that SCU is introduced to one of the primary hydroxyl
groups of ␤-CD.
2.7. Hydrolysis of SCU-ˇ-CD conjugate
The conjugates in aqueous solution were added to phosphate
buffer solutions of pH 1.5 or 7.2 at 37 ◦C. The final concentration of
the conjugates was 4.0 × 10−4 M. At appropriate intervals, 200 ␮L
of the reaction solutions were collected, and 60 ␮L of which was
subjected to HPLC analysis under the following conditions: Lichro-
spher C18 HPLC column (Hanbon, 5 ␮m, 150 mm × 4.6 mm, CHA),
a flow rate of 0.5 mL/min, a mobile phase of 1.67% acetic acid
solution–acetonitrile (7:3, v/v), a detection at 335 nm.
From Table 1, it is of interest to note that the conjugates were
highly soluble (16–25 mmol/L) in water, the solubility being >40
times, in molar concentration, than that of SCU. This increase in sol-
ubility is attributable to the phase change from a crystalline state
of SCU with lower solubility to an amorphous state of the conju-
gates with higher solubility. The suggestion was supported by the
powder XRD patterns (Fig. 3). This amorphous state occurred prob-
ably because the conjugate was formed to self-inclusion complex.
2.8. Cell culture and treatments
Cells were cultured at 5 × 105 mL−1 in RPMI 1640 supplemented
with 10% heat-inactivated fetal bovine serum at 37 ◦C in a humidi-
fied atmosphere of 5% CO2 in air. They were seeded at 5 × 104 mL−1
and treated with the indicated amounts of scutellarin and its con-
jugates.

B. Yang et al. / Carbohydrate Polymers 92 (2013) 1308–1314
1311
Fig. 1. Preparation of SCU-␤-CD conjugate. Reagents: (a) p-toluenesulfonyl chloride; (b) ethylenediamine or diethylenediamine; (c) sodium azide; (d) triphenylphosphine,
ammonium hydroxide; (e) scutellarin.
As reported previously, the conjugates are due to the formation
of the self-inclusion complexes, which inhibits the intermolec-
ular stacking association of the conjugates to form less soluble
crystals (Yano, Hirayama, Arima, & Uekama, 2001). The supposi-
tion was proved by the experiments with two-dimensional (2D)
NMR spectroscopic study, which part of the enamino-SCU-␤-CD
conjugate and dienamino-SCU-␤-CD conjugate were intramolecu-
larly included in the CD cavity, forming a self-inclusion complex.
With short amine linkage, the amino-SCU-␤-CD conjugate could
not be showed the formation of the self-inclusion complex (see
Supporting Information). This is the probably reason with the dif-
ference of aqueous solubility of three conjugates.
3.2. X-ray diffraction of the conjugate
Powder XRD patterns allow examination of the medium and
long range ordering of materials (de Araújo et al., 2008). In contrast
Fig. 2. ESIMS (A), and 1H NMR (B) spectra of enamino-SCU-␤-CD conjugate.

1312
B. Yang et al. / Carbohydrate Polymers 92 (2013) 1308–1314
point of ca. 220 ◦C and a decomposition temperature of ca. 360 ◦C
(Fig. 4a).
3000
2000
1000
0
3000
2000
1000
0
d
3.4. Release behavior of conjugates in aqueous solutions
c
In order to reach the colon as an intact form, the colon-targeting
drugs should survive the passage through stomach and small intes-
tine. The physiological factors which affect on drug release from
drugs are pH values, secretion of esterase and bile acids, intestinal
contents and microflora. Herein, we investigated the effects of pH
values on drug release behavior of the conjugate. Fig. 5 shows the
hydrolysis rate-pH profile of the conjugate in aqueous solutions.
The conjugates were markedly stable under physiological condi-
tions of pH 1.5 or 7.2 at 37 ◦C. The results suggest that the conjugates
may not be significantly subject to the chemical degradation until
they reach to the colon.
3000
2000
1000
0
b
10000
5000
0
a
10
20
30
40
50
60
70
2
(degrees)
Fig. 3. Powder X-ray diffractograms (Cu-␬␣) for: (a) SCU, (b) amino-SCU-␤-CD con-
jugate, (c) enamino-SCU-␤-CD conjugate, (d) dienamino-SCU-␤-CD conjugate.
3.5. In vitro cytotoxicity studies
110
100
The cytotoxicity tests for the conjugates were evaluated in vitro
for antiproliferative activity against human cancer cell lines HT-29,
SW480, Lovo and HTC116 by the MTT assay using camptothecin as
reference compound. The IC50 values were calculated as showed
in Table 2. As expected, these conjugates presents more satis-
factory antiproliferative activity than free scutellarin; Excitingly,
the IC50 values of enamino-SCU-␤-CD conjugate and dienamino-
SCU-␤-CD conjugate were 7.7 × 10−5–7.6 × 10−5 mol/dm3 and
4.8 × 10−5–4.6 × 10−5 mol/dm3 respectively, which were much
lower than that of free scutellarin (3 × 10−4 mol/dm3 and
2.1 × 10−4 mol/dm3) in HT-29 and SW480 colon cancer cell. On
control tests, the IC50 values of amino-␤-CD, enamino-␤-CD and
dienamino-␤-CD exceed 5 mM in HT-29, SW480, Lovo and HTC116
colon cancer cell, indicating that these derivatives of ␤-CD were
very nontoxic to all cell lines tested. The mixtures of scutellarin
and amino-␤-CD, enamino-␤-CD and dienamino-␤-CD showed
almost similar IC50 values to free scutellarin (see Supporting
Information). These results proved that the increase in cytotoxicity
were attributable to the formation of conjugates (SCU-CD conju-
gates), in which scutellarin were covalently bound to one of the
primary hydroxyl groups of ␤-CD.
a
b
90
80
70
c
60
d
50
40
30
20
0
100
200
T (oC)
300
400
500
Fig. 4. TG curves for: (a) SCU, (b) amino-SCU-␤-CD conjugate, (c) enamino-SCU-␤-
CD conjugate, (d) dienamino-SCU-␤-CD conjugate.
to the crystalline character of SCU (Fig. 3a), All the conjugates have
amorphous structures (Fig. 3b–d).
However, the exact mechanisms remain unconfirmed. Never-
theless, the plausible explanation for these data could be that the
conjugates were likely induce their passive accumulation on colon
cancer cell lines tested in results of increase in cytotoxicity. In
previous reported, the ␤-CD cavities of the conjugates, in which
a molecule were covalently bound to one of the hydroxyl groups
of ␤-CD, had the capability of including cholesterol or lecithin of
the damaged cell membranes (Liu, Zhang, Chen, & Wang, 2007;
3.3. Thermal analysis of the conjugate
The thermal properties of the SCU-␤-CD conjugates were inves-
tigated by thermogravimetric analysis (TG). The TG curves showed
that the three conjugates decompose at ca. 305 ◦C (Fig. 4b–d).
However, the SCU had different thermal stability with a melting
100
90
100
95
80
90
70
85
60
B
A
50
80
0
5
10
15
20
25
0
5
10
15
20
25
Time (h)
Time (h)
Fig. 5. Time courses for disappearance of SCU-␤-CD conjugate and appearances of scutellarin in phosphate buffer at 37 ◦C. (A) at pH 1.5; (B) at pH 7.2. Key: (ꢀ) amino-SCU-␤-CD
conjugate; (᭹) enamino-SCU-␤-CD conjugate; (ꢁ) dienamino-SCU-␤-CD conjugate. Each point represents the mean SE of 3 experiments.

B. Yang et al. / Carbohydrate Polymers 92 (2013) 1308–1314
1313
Table 2
IC50 (mM) of SCU and SCU-␤-CD conjugates in various colon cancer cell lines.
Cell line
Scutellarin (mM)
Amino-SCU-␤-CD
Enamino-SCU-␤-CD
Dienamino-SCU-␤-CD
conjugate (mM)
conjugate (mM)
conjugate (mM)
HT-29
SW480
Lovo
0.30
0.21
0.077
0.078
0.16
0.15
0.052
0.045
0.076
0.048
0.039
0.034
0.077
0.046
0.057
0.048
HTC116
Zhang, Chen, Yu, & Liu, 2009). It has been known that during tumor
angiogenesis, the nascent capillaries supplying nutrients to the
tumors possess large gaps in between vascular endothelial cells
when compared to healthy tissues (Matsumura & Maeda, 1986).
The junction gap in the cancer cell membrane was loose, which
allowed conjugates to include cholesterols of the cell membrane.
So the conjugates tend to collect and accumulate in the interstitial
space of the tumor. With long amine linkage, the enamino-SCU-␤-
CD and dienamino-SCU-␤-CD conjugate could be inclined to insert
into the hydrophobic regions of the bilayers in result of easy anchor
at the loose bilayer of the cancer cell membrane. In the case of the
short amine linkage, amino-SCU-␤-CD conjugate could not insert
into the hydrophobic regions of the bilayers. So the amino-SCU-␤-
CD conjugate showed lower cytotoxicity than enamino-SCU-␤-CD
and dienamino-SCU-␤-CD conjugate.
Foundation of Yunnan Province (No. 2009ZC045M), which are
gratefully acknowledged.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.carbpol.2012.10.012.
References
Belanger, N., & Perly, B. (1992). NMR investigations of the conformation of a new
cyclodextrin-based amphiphilic transporter for hydrophobic drugs: Molecular
lollipops. Journal of Molecular Structure, 273, 215–226.
Croft, A. P., & Bartsch, R. A. (1983). Synthesis of chemically modified cyclodextrins.
Tetrahedron, 39, 1417–1474.
This supposition was also supported by the control experiments
with stained yeast cells by benzenesulfonamidoquinolino-␤-
cyclodextrin (HQAS-1-CD and HQAS-2-CD, Fig. S8 a and b). As
yeast cells have many features analogous to mammalian cells, the
pre-cultured yeast cells were transferred to a solution of HQAS-
1-CD and HQAS-2-CD, and fluorescence microscopic images were
recorded after ca. 30 min with HQAS-2-CD, some damaged cells
which had loose junction gap in the cell membrane (including
splinter and dead cells) exhibited weak fluorescence at cell divi-
sion domains (Fig. S9 a and b), but most of the yeast cells did not
exhibit any appreciable fluorescence (see Supporting Information).
In addition, HQAS-1-CD showed poorer stained yeast cell pro-
gram than HQAS-2-CD (Liu et al., 2007; Zhang et al., 2009). This
phenomenon pointed that healthy cells which possess a compact
and integrated cell membrane had the ability to exclude conju-
gate molecule of cyclodextrin. The self-included conformation of
HQAS-2-CD also restricted the its interaction with cell membranes.
Therefore, HQAS-2-CD gave no response with healthy cells. How-
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increased, which allowed the ␤-CD cavity of HQAS-2-CD to include
competitively exposed molecules of the cell membrane and anchor
in the loose bilayer of the cell. With long amine linkage, HQAS-2-CD
could be easy to insert into the hydrophobic regions of the bilayers
than HQAS-1-CD.
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Acknowledgments
This work was supported by National Natural Science Foun-
dation of China (NNSFC) (No. 21062009) and the Natural Science

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