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Extended chain conformation of b-glucan and its

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effect on antitumor activity†

Cite this: J. Mater. Chem. B, 2017,
5, 5623
Xing Zheng, Fengzhi Lu, Xiaojuan Xu* and Lina Zhang *

Received 15th May 2017,
Accepted 21st June 2017
DOI: 10.1039/c7tb01324h
rsc.li/materials-b
Mushrooms are known as a delicacy due to their delicious taste and rich nutrition, and their b-glucans
have antitumor activity. Here, a triple helical b-glucan (THG) isolated from Lentinus edodes was
successfully fractionated into nine fractions with different weight-average molecular weights (Mw)
through ultrasonic irradiation. The Mw, radius of gyration (hRgiz), hydrodynamic radius (hRhiz), structure-sensitive
parameters (r), contour length (L), persistence length (q) and molar mass per contour length (ML) were
characterized by static light scattering (SLS), dynamic light scattering (DLS), and atomic force microscopy (AFM).
The results indicated that THG displayed an extended chain conformation with r values of 2.1  0.1 for the
nine fractions, as well as ML and q values of 2160 nm1 and 110 nm in water, which were consistent with the
data for triple helical polysaccharides. In combination with the apparent length (Lap) values visualized with AFM
and Mw, the molar mass per apparent contour length (MLap) was calculated to be 2242 nm1, which was similar
to that obtained from SLS according to the wormlike cylinder model. Thus, we established a novel method
using AFM for characterizing the chain stiffness of polysaccharides. The results from an animal assay
demonstrated that THG significantly inhibited H22 tumor growth without damage to organisms, and the THG
fractions with relatively low molecular weight and/or higher stiffness showed stronger antitumor activity,
revealing the significant molecular weight- and chain conformation-dependences of antitumor activity.
Moreover, a schematic model to describe the interaction between THG and receptors on the immune cell
membrane was proposed to illustrate these results. This work provides important information for characterizing
the chain conformation of polysaccharides and understanding the relationship between structure and antitumor
activity, which is relevant for the treatment of hepatocellular carcinoma in clinic.

1. Introduction antitumor, anti-infection, and immunomodulatory activities

Cancer has been afflicting humankind as a major threat to
public health for many years. Unfortunately, almost all of the
anticancer drugs used for conventional chemotherapy cause
serious side effects, including damage to the immune system,
which limits their application to a certain extent.1–4 Therefore,
there is an urgent requirement to investigate novel and safe
agents for the prevention and treatment of cancer. In this
context, the natural abundance and structurally complex poly-
saccharides extracted from plants, algae, and fungi have
attracted much attention due to their immunomodulatory
and antitumor activities.5–12 It has been reported that natural
polysaccharides can inhibit tumor growth by inducing tumor
cell apoptosis directly, suppressing tumor metastasis, and
enhancing the effect of immunotherapy and chemotherapy.13–17
Some promising b-glucans have been widely investigated for their
College of Chemistry & Molecule Sciences, Wuhan University, Wuhan 430072,
P. R. China. E-mail: zhangln@whu.edu.cn, xuxj@whu.edu.cn;
Fax: +86-27-68762005; Tel: +86-27-87219274
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c7tb01324h
since their first identification as potential antitumor agents in
1963.18 Moreover, b-glucans, either water-soluble or particulate,
have exhibited antitumor and immunoregulation activities in
mice.5,19,20 Water-soluble b-glucan from Grifola frondosa
enhances the cytotoxicity of NK cells and stimulates the dendritic
cell response, resulting in tumor regression.21 Curdlan, a water-
insoluble b-glucan with a triple helical chain conformation,
shows immunomodulatory activity by inducing T helper cells
and dendritic cell responses.22,23 It has been reported that these
biological activities are affected by the structure of the b-glucans
including molecular weight, degree of branching, and conformation,
which determine the interaction of these polysaccharides with
receptors on the surface of cells.24,25 For example, the binding
affinities of glucans with the CR3 receptor in a human
monocyte-like cell line (U937) are in the order of scleroglucan d
schizophyllan 4 laminarin 4 glucan phosphate 4 glucan sulfate,
of which the scleroglucan chain displays the highest stiffness.26
Furthermore, the immunoregulation activity of particulate (1,3)-
b-D-glucan from yeast can be influenced by its weight-average
molecular weight (Mw) and degree of (1,6)-linkages with a trend
for the higher Mw or (1,6)-linked (1,3)-b-D-glucans to be more

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stimulatory.27 However, the influence of the chain conformation
and molecular weight on the biological activities of the b-glucans
is not well established yet, due to the great difficulty of both the
preparation of glucan samples and the characterization of their
conformations.
Mushrooms have been considered as a delicious, richly
nutritious food in Asia for centuries. The b-glucan isolated
from an edible mushroom, Lentinus edodes, was first found to
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act as an antitumor agent against sarcoma 180 in 1969, and has
been applied in clinical anticancer therapy in Japan.5,28–30 It is
also used as an immune adjuvant to reduce adverse reactions to
therapy in order to improve the patients’ quality of life.31–33 In
our laboratory, the b-glucan isolated from Lentinus edodes has
been confirmed to adopt a triple helical chain conformation,
but the characterization method is either complicated and
time-consuming34,35 or it is difficult to determine the degree
of chain stretch.36 However, the bioactivities of the b-glucan
isolated from Lentinus edodes are closely related to its chain
stiffness and molecular weight. It has been confirmed that this
b-glucan has molecular weight-related antitumor activity,35,37
and it activates immune responses to inhibit S-180 tumor growth
by promoting cell apoptosis and suppressing cell proliferation
through caspase-3- and p53-dependent signaling pathways
in vivo.38 Thus, our strategy is to establish a simple and feasible
method to visually characterize the morphology and size of
extended chain b-glucans, and to construct relationships between
these properties and molecular weight. The results were compared
with the outcomes from the traditional light scattering (LS)
method and the theoretical model. An evaluation of the influences
of the chain conformation and molecular weight on the antitumor
activity of b-glucan was also performed. In this study, we acquired
b-glucans from Lentinus edodes and used ultrasonic degradation in
combination with ethanol precipitation to obtain fractions with
different molecular weights. The molecular weights and chain
conformations were determined by static light scattering (SLS),
dynamic light scattering (DLS) and atomic force microscopy
(AFM). Furthermore, the chain conformation- and molecular
weight-dependences of antitumor activity of the b-glucans were
assessed in vivo. This work is important for characterizing the
apparent length and extended chain conformation of poly-
saccharides and understanding the relationship between structure
and antitumor activity.
2. Experimental
2.1 Materials and reagents
The fruiting bodies of Lentinus edodes were purchased from a
supermarket in Wuhan, China. 5-Fluorouracil (5-Fu) was purchased
from Energy Chemical (Shanghai, China). All other chemical
reagents were of analytical grade, and were purchased from Sino-
pharm Chemical Reagent Co., Ltd (Shanghai, China).
2.2 Isolation and fractionation of b-glucans
The b-glucan was isolated from fruiting bodies of Lentinus
edodes by using alkali extraction with 5% sodium hydroxide
(NaOH, w/w) and 0.05% sodium borohydride (NaBH4, w/w)
5624 | J. Mater. Chem. B, 2017, 5, 5623--5631
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according to the method established by our laboratory.36 The
extract is denoted THG herein (Fig. S1, ESI†). The chemical
structure of THG has been identified by NMR and GC-MS
analysis to be a b-1,3-D-glucan with two b-1,6-D-glucopyranoside
branches for every five b-1,3-glucopyranoside linear linkages.36 It
has been reported that ultrasonic degradation is a viable physical
method for schizophyllan (SPG) and does not cause changes in
higher-order structure.39 Briefly, the THG sample was dissolved
in water at a concentration of 2.0 mg mL1 and degraded by
using an ultrasonic crusher (JY98-IIIDN, Ningbo Scientz Bio-
technology Co., Ltd, Zhejiang, China) with a power of 600 W for
different times in an ice-water bath. Subsequently, the ultra-
sonically treated THG solutions were precipitated with ethanol
as the precipitant. Each precipitate was re-dissolved in water,
dialyzed against ultrapure water, and freeze-dried. Nine fractions
were obtained named as THG1 to THG9 according to the ultra-
sonication times. The water used in all experiments was ultrapure.
The endotoxin in all THG samples was determined to be o0.1 EU
per mg by using Endotoxin-Specific Tachypleus Amebocyte Lysate
Kit (Xiamen, China) according to the manufacturer’s instructions.
The protein in THG was measured to be o0.25% (w/w) with a BCA
protein assay kit (Beyotime Biotechnology, China).
2.3 Light scattering
Static light scattering (SLS) and dynamic light scattering (DLS)
were utilized to characterize the weight average molecular
weights (Mw) and chain conformations of the THG fractions
in water. All of the THG solutions at the desired concentrations
were made optically clean by filtration through 0.22 mm Millipore
filters (Whatman, Inc., USA). A commercial light scattering
spectrometer (ALV/SP-125, ALV, Germany) equipped with an
ALV-5000/E correlator and a He–Ne laser (at l = 632.8 nm) was
used at scattering angles y, from 301 to 1501. The angular- and
concentration-dependence of scattered intensities were analyzed
through the following equations:40
 
Kc 1 1
¼ 1 þ Rg2 z q2 þ 2A2 c (1)
Ry Mw 3
K = 4p2n2(dn/dc)2/(NAl04) (2)
where q = (4pn/l0) sin(y/2), and NA, n, l0, hRgiz, A2 and c
respectively represent Avogadro’s number, the refractive index
of the solvent, the wavelength of light in a vacuum, the z-mean
radius of gyration, the second viral coefficient and the concen-
tration of the polymer solution. The specific refractive index
increment (dn/dc) of THG in water is 0.140 mL g1.41 A Zimm
plot was used to calculate the values of Mw and hRgiz.
The z-mean hydrodynamic radius (hRhiz) of THG in water at
25 1C was calculated by using the Stokes–Einstein relation:
kB T
hR h iz ¼ (3)
6pZ0 Dz
where kB is the Boltzmann constant, T is the temperature in
units of K, Z0 is the solvent viscosity, and Dz represents the
translational diffusion coefficient.
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Journal of Materials Chemistry B
2.4 Atomic force microscopy
THG samples were dissolved in ultrapure water at concentrations
of 1 mg mL1, and then diluted to the desired concentrations. A
drop of 5 mL diluted THG solution was placed on freshly cleaved
mica and allowed to dry in air at room temperature. Topographic
images of THG samples in water were recorded using a commercial
atomic force microscope (Cypher ES, Asylum Research, Oxford, USA)
in AC mode at 35 1C. Silicon probes with a spring constant of
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2 N m1 and resonance frequency of 70 kHz (ASYELEC-01, Asylum
Research) were employed. The average radius of the tip was 28 nm.
All data from the images were analyzed using the AFM accessory
software, and the images presented were flattened only when
necessary.
2.5 Antitumor activity in vivo
The mouse hepatoma carcinoma cell line (H22) was purchased
from the China Center for Type Culture Collection (CCTCC,
Wuhan, China). The cell was cultured in the Roswell Park
Memorial Institute 1640 medium (RPMI 1640, HyClone) with
penicillin (100 U mL1), streptomycin (100 mg mL1) and 10%
heat-inactivated (56 1C, 30 min) fetal bovine serum (FBS, Gibco)
at 37 1C under a humidified atmosphere of 95% air and 5%
CO2. Six-week-old BALB/c mice (male, weighing 20  1 g) were
purchased from the Center for Animal Experiment ABSL-III
Laboratory of Wuhan University (Hubei, China). Animal study
protocols were approved by the Institutional Animal Care and
Use Committee (IACUC) of Wuhan University. The mouse
subcutaneous (s.c.) tumor transplantation models were used
to evaluate the antitumor activity of THG samples. Briefly,
BALB/c mice were injected subcutaneously with H22 tumor
cells (1  106 per mouse) in 0.1 mL 0.9% NaCl into the right
armpit. After inoculation for 24 h, mice were randomly
assigned to different treatment groups and treated daily with
an intraperitoneal (i.p.) injection of saline (negative control),
5-Fu (20 mg kg1, positive control), THG (0.5 mg kg1, 1 mg kg1,
5 mg kg1, 10 mg kg1) or THG fractions (from THG1 to THG9,
5 mg kg1) for 20 days. Then all mice were weighed and
sacrificed. All the tumors and spleens were removed and
weighed. According to the mean weight of the tumors, the tumor
inhibition rates were calculated as follows: tumor inhibition rate
(%) = [(Average tumor weight of the control group  Average
tumor weight of the test group)/(Average tumor weight of the
control group)]  100%. The spleen index was expressed as the
spleen weight relative to the body weight. For a survival assay, tumor-
bearing mice were treated as above and observed over 60 days.
2.6 Apoptosis analysis in vivo
Tumor tissues were fixed with 4% paraformaldehyde for 24 h,
and then sliced. The slices were stained with hematoxylin and
eosin (HE) for histology observation under a light microscope.
For the terminal deoxynucleotidyl transferase-mediated deoxyuridine
triphosphate nick-end labelling (TUNEL) assay, slides were treated
with an ‘‘In Situ Cell Death Detection Kit’’ (Fluorescein, Reche,
Germany) according to the manufacturer’s instructions, and
observed under a fluorescence microscope (Leica DMi8,
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Germany). For immunohistochemical analysis, slides were
incubated with primary antibodies including anti-Bcl-2, anti-
Bax, and anti-caspase-3 (Servicebio, each at 1 : 100 dilution),
and observed under a light microscope.
2.7 Apoptosis analysis and cell viability assay in vitro
Apoptosis analysis was measured by using the Annexin V-FITC/
PI double staining apoptosis detection kit (KeyGEN BioTECH).
H22 cells were cultured in 6-well plates and treated with 0, 50,
100 or 200 mg mL1 THG for 72 h. Cells were collected and
washed twice with PBS, then incubated with Annexin V-FITC/PI
for 15 min in the dark at room temperature following the
manufacturer’s protocol. Samples were then analyzed within
1 h by flow cytometry (BD Biosciences). The cell viability of H22
cells was detected using a Live/Dead Cell Double Staining Kit
(Sigma, USA). H22 cells were cultured in 6-well plates and treated
with 0, 50, 100 or 200 mg mL1 THG. After 72 h incubation,
collected cells were washed twice with PBS and stained with
Calcein-AM/PI at 37 1C for 15 min following the manufacturer’s
protocol. The cells were visualized with a fluorescence micro-
scope (Leica DMi8, Germany).
Cell viability was determined by using a standard 3-(4,5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT)
assay. Different kinds of cells (H22, HepG2, HeLa, MCF-7, MNK-
45, SH-sy5y, L02 and RAW264.7, purchased from CCTCC) were
seeded in 96-well plates at a density of 5  103 cells per well in final
volumes of 200 mL and incubated for 12 h. The cells were then
incubated with THG at final concentrations of 0, 50, 100, 150 or
200 mg mL1. After 72 h incubation, 20 mL of MTT (5 mg mL1,
Sigma) solution was added to each well and incubated for 4 h at
37 1C with 5% CO2. The supernatant was then removed care-
fully, and the cells were treated with 150 mL of dimethyl
sulfoxide (DMSO, Sinopharm, China), followed by gentle shaking.
The plates were analyzed at a wavelength of 492 nm on a microplate
reader (BMG LABTECH, FLUOstar OPTIMA, Germany). The data are
presented as cell viability (%) = [(Experimental group  Blank
group)/(Negative control group  Blank group)]  100%.
2.8 Statistical analysis
All data are presented as the mean  S.E. from at least three
independent experiments unless specified otherwise. The student’s
t test was performed, and the differences were considered
statistically significant at p o 0.05. Survival curves were created
using the Kaplan–Meier method, and statistical analyses of
survival curves used a log-rank test.
3. Results and discussion
3.1 Molecular weight and chain stiffness of THG
SLS and DLS measurements of THG fractions were performed
to obtain molecular weights and chain conformation parameters.
The Mw values of the THG fractions were rapidly reduced within
5 min by ultrasonic irradiation, and then slowed down gradually
(Fig. S2, ESI†). The apparent hydrodynamic radius distributions
f (Rh) of the THG fractions in water with concentrations of
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For THG, the values of ML, q and contour length per main-chain
residue (h) can be calculated using the molar mass of the THG
repeating unit (M0 = 1134) according to the following
equations:34,35

(Mw2/12hRg2iz)2/3 = ML4/3 + (2/15)(ML1/3/q)Mw (4)

h = (M0/5)/(ML/3) (5)

The relationship between Mw and hRgiz of the degraded THG

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fractions established by using eqn (4) is shown in Fig. 1b, and

the values of ML and q were calculated as 2160 nm1 and
110 nm, respectively. Thus, h was estimated to be 0.32 nm
according to eqn (5), which lies in the range of 0.29–0.33 nm
calculated from X-ray diffraction.44 Moreover, these parameters
were consistent with those of triple helical SPG.45,46 Therefore,
Fig. 1 Chain conformation of THG in water. The apparent hydrodynamic
it could be concluded that THG and its degraded fractions
radius distributions of THG fractions in water at concentrations of adopted a triple helix conformation in water, and the ultrasonic
0.1 mg mL1 at 25 1C (scattering angle y = 601) (a). The relationship of degradation hardly changed this triple helical structure. The
Mw and hRgiz (b). hRgiz for THG fractions in water compared with the obtained ML and q values were then used to fit the Kratky–Porod
theoretical curves for the unperturbed wormlike chain (the solid line) with
wormlike chain using the Mw and hRgiz data; the agreement with
q = 110 nm and ML = 2160 nm1. The dotted line represents the relation-
ship of hRgiz vs. Mw for THG fractions of low molecular weight (c).
the theoretical solid curve was highly satisfactory, as shown in
Fig. 1c. Clearly, the solid curve deviated from the dotted line and
slightly bent down at high molecular weight. As reported, eqn (4)
0.1 mg mL1 at 25 1C (scattering angle y = 601) are shown in
was established at Kuhn segment number nk (Mw/2qML) o 2.
Fig. 1a. All fractions displayed only one symmetrical peak,
According to the ML and q values, the nk of the THG fractions was
showing their good dispersions at the molecular level in the
estimated to lie in the range 0.63–3.70, as shown in Table 1. When
dilute solutions. Additionally, the peak values of the THG fractions
Mw was higher than 1.0  106 for samples THG1–THG4, nk was
decreased sequentially with increasing ultrasonic irradiation time,
slightly bigger than 2. In the relatively low molecular weight range
demonstrating that THG was successfully degraded into fractions
of 3.01  105–9.18  105 (THG5–THG9), especially at Mw o 5.8 
with different molecular sizes.
105 of THG7–THG9, the exponent n of hRgiz B Mnw was 0.9,
Mw and hRgiz values were calculated by Zimm plots (Fig. S3,
indicating the inherent rod-like rigidity of THG, which decreased
ESI†) according to eqn (1) and (2). The hRhiz values were
to 0.7 with increasing molecular weight. It has been reported
determined according to the Stokes–Einstein relation (eqn (3))
that SPG adopts a rod-like chain conformation with n of 1.0 at
from DLS. The experimental results of Mw, hRgiz, and hRhiz of the
Mw o 5.0  105.45,46 These results further confirmed that THG
THG samples are summarized in Table 1. The structure-
had a similar chain stiffness to the triple helical SPG.
sensitive dimensionless parameter (r) defined as hRgiz/hRhiz is
a qualitative measure of the macromolecular chain conformation 3.2 Visualized patterns of extended THG chains in water
of polymers. Generally, the r value for a flexible linear polymer
To visually understand the extended chain stiffness of THG via
lies in the range of 1.5–1.7, and that for a stiff chain is greater
a simple method, topographic images of the THG fractions
than 2.42,43 The r values of the THG fractions were estimated to
dissolved in water were recorded with AFM. The AFM images of
be 2.1  0.1 by using hRgiz and hRhiz data (Table 1), suggesting a
nine THG fractions in water at concentrations of 2 mg mL1
stiff chain conformation of THG in water.
were obtained, and six of them are shown in Fig. 2 (the others
The stiffness of a wormlike chain is defined by the molar
are shown in Fig. S4, ESI†). As expected, all nine fractions
mass per unit contour length (ML) and the persistence length (q).
mainly displayed a kind of linearly extended morphology with
few circular structures. Their apparent average contour lengths
Table 1 Mw, hRgiz, hRhiz, r, Lap, and nk values for THG fractions in water
(Lap) were estimated by counting 100 chains (see for example,
determined by SLS, DLS, and AFM
Fig. 2g for THG1) and are summarized in Table 1. Fig. 2h shows
Mw  104 hRgiz hRhiz Lap the Mw dependence of Lap; there is a linear relationship,
Samples (g mol1) (nm) (nm) r (nm) nk suggesting the validity of the Lap values obtained. Therefore,
THG1 176 142 66 2.2 752 3.70 it is feasible to characterize chain stiffness of THG fractions by
THG2 139 126 60 2.1 617 2.93 using AFM. The MLap defined by MLap = Mw/Lap was then
THG3 116 107 51 2.1 512 2.44
THG4 101 97 47 2.1 437 2.31 estimated to be 2242 nm1 from the slope of the plot, close to
THG5 91.8 89 44 2.0 396 1.93 the ML value (2160 nm1) calculated from the Mw and hRgiz values
THG6 88.8 87 43 2.0 357 1.87 obtained from SLS according to eqn (4). This further confirmed
THG7 57.9 64 30 2.1 232 1.22
THG8 43.3 49 24 2.0 186 0.91 that AFM was a feasible method for visual characterization of
THG9 30.1 35 16 2.2 120 0.63 the stiffness of the extended molecular chain. Moreover, these

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Fig. 2 AFM images of THG1 (a), THG3 (b), THG5 (c), THG7 (d), THG8 (e),
and THG9 (f) in water at concentrations of 2 mg mL1. Apparent contour
length distributions f (Lap) of THG1 (g). Dependence of Mw on Lap for THG
fractions (h).
conformation parameters were in agreement with those of
ML = 2150  150 nm1, q = 200  30 nm and d = 2.6  0.4 nm
for triple helical SPG in water.45,46 Thus, a simple method using
AFM to characterize morphology and contour length of the
extended chain was established. Furthermore, with a decrease of
molecular weight, the THG chains changed from long extended
chains to short rods as shown in the AFM images. Thus, the chain
conformation and stiffness of polysaccharides could be visualized
via AFM.
3.3 Influences of chain conformation and molecular weight
on antitumor activity of THG
Hepatocellular carcinoma (HCC), with increasing worldwide
incidence, is one of the most common and lethal malignancies
in the world.47,48 Dosage-dependence is a very important factor
of any drug in clinical application. Here, murine hepatocellular
carcinoma H22 cell suspensions were injected subcutaneously
into the right flank of groups of mice to develop tumor-bearing
mice, which were treated daily with i.p. injections of THG at
dosages of 0.5 mg kg1, 1 mg kg1, 5 mg kg1, or 10 mg kg1,
according to group. As shown in Fig. 3a and b, the mean tumor
weight was effectively inhibited by THG with values ranging
from 1.11 g (the saline control group) to 0.50 g (5 and 1 mg kg1
THG groups; inhibition of 55% after 20 days treatment, Fig. 3c).
In contrast to the saline control group, the tumor growth of the
positive control group (5-Fu, 20 mg kg1) was significantly
depressed. However, the body weight and spleen index were
seriously decreased with 5-Fu (Fig. 3d and e), and no such side
effects occurred in THG groups, indicating the good safety of
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Fig. 3 THG showed significant dosage-dependent antitumor activity in
H22 bearing mice. H22 tumor-bearing mice were intraperitoneally
injected with or without THG for 20 days, and then weighed and sacrificed.
The weights of extracted tumors (a). The photos of extracted tumors (b).
The tumor inhibition rate of THG (c). The body weights of tumor-bearing
mice (d). The spleen index of tumor-bearing mice (e). Survival curves of
tumor-bearing mice after 60 days treatment (f). The bars represent the
S.E., *p o 0.05. Survival curves were created using the Kaplan–Meier
method, and statistical analyses of survival curves used a log-rank test.
THG treatment. Interestingly, THG exhibited weak antitumor
activity at either high (10 mg kg1) or low (0.5 mg kg1) dosage,
showing a different dosage-dependence to that seen with
chemical drugs. The mice survival rates also showed similar
dosage-dependence after 60 days treatment (Fig. 3f), namely,
the dosages of 5 and 1 mg kg1 led to the highest survival rate
of 80%, while the dosages of 10 and 0.5 mg kg1 only slightly
prolonged the life of mice compared with the negative control.
In general, the therapeutic effect of chemical drugs increases
with increasing dosage. The different dosage-dependence of
THG for antitumor activity suggested that THG acted in a
different way to 5-Fu. In our previous work, we have demonstrated
that the b-glucan from Lentinus edodes inhibits S-180 tumor growth
through activating immune cells,38 and it has been reported that
the binding of polysaccharides to receptors on the surface of
immune cells initiates the immune responses.24,25 As is widely
known, polysaccharides easily self-aggregate due to the strong
hydrogen bonding between hydroxyls in polymer chains.49,50 It is
thus speculated that aggregation may reduce the binding of
polysaccharide chains to receptors, leading to weak or even no
activation of immune responses. DLS was then performed to study
whether or not aggregation of THG occurred within the used
dosage range. The apparent hydrodynamic radius distribution
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f (Rh) of THG samples with different concentrations (Fig. S5,
ESI†) displayed only one symmetric peak with the same
peak position in dilute solutions at concentrations lower than
1 mg mL1, demonstrating that only isolated THG chains
(individuals) existed in these solutions. Two peaks occurred at
the concentration of 1 mg mL1, corresponding to individual
chains at the lower position and THG aggregates at the higher
position. The aggregation of THG was further evidenced by AFM
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images (Fig. 4). THG existed as single chains (individuals) in
the extremely dilute solution with an average height of 0.83 
0.14 nm (Fig. 4a and b). This result is in agreement with the
chain height of triple helical scleroglucan (0.92  0.27 nm)
measured by tapping mode AFM,51 but a little lower than those
of SPG and scleroglucan, which were respectively characterized
to be 1.08  0.27 nm and 1.14  0.30 nm using noncontact
mode AFM.52,53 These slight differences in the chain height of
triple helical glucans are partly due to the diverse measuring modes,
tips and scanning parameters.54,55 When the concentration of THG
slightly increased from 1 to 5 mg mL1, the single chains self-
aggregated with each other through intermolecular interactions,
leading to the formation of loose networks (Fig. 4c). The average
heights of the chains and the connection points were 0.83 
0.14 nm and 1.32  0.21 nm, respectively (Fig. 4d). With a
Fig. 4 AFM images of the dilute THG solution at concentrations of 1 mg mL1
(a), 5 mg mL1 (c), 8 mg mL1 (e). (b, d, and f) Are the heights of the THG chains
at the blue lines in (a, c, and e), respectively.
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continued increase in concentration, the chains of THG over-
lapped into intricate networks (Fig. 4e), similar to those reported
for triple helical SPG and stiff polysaccharide AF1.56,57 The
average height of overlapped chains was 1.89  0.26 nm
(Fig. 4f), confirming that the stiff chain conformation of THG
easily self-aggregated with increased concentration. It is worth
noting that the superimposition of different strands makes the
evaluation of heights difficult when a polymer network is present.
THG strongly self-aggregated at a concentration of 1 mg mL1 or
higher. In this work, the used dosages of 0.5, 1, 5 and 10 mg kg1
corresponded to the injected THG concentrations of 0.05,
0.1, 0.5 and 1 mg mL1. Thus, THG aggregated at the dosage
of 10 mg kg1, which reduced the binding of THG to immune
cells, leading to a lower antitumor effect. Whether the immune
responses are reduced or not by THG aggregation will be further
studied in our future work. Taken together, THG possessed
potent antitumor activity against murine H22 hepatocellular
carcinoma with significant chain conformation-dependence
and low toxicity side effects.
To further explore the effect of molecular weight upon
antitumor activity of THG in vivo, daily i.p. injections of the
nine THG fractions were administered into H22 tumor-bearing
mice at a dosage of 5 mg kg1 for 20 days. Then all mice were
weighed and sacrificed, and tumors and spleens were isolated
and weighed. The representative results are shown in Fig. 5.
The mean tumor weight was significantly decreased from 1.13 g
to 0.30 g in a molecular weight-dependent manner by THG
treatment (Fig. 5a), with the inhibition rate reaching 73%,
which is much higher than that for the 5-Fu group (Fig. 5b).
Moreover, there was no significant change in the body weight
Fig. 5 The molecular weight-dependent antitumor activity of THG
against H22 cells. The weights of extracted tumors from tumor-bearing
mice after treatment with THG fractions (5 mg kg1 per day) for 20 days (a).
The tumor inhibition rates after treatment with THG fractions (b). The body
weights of mice after THG treatment for 20 days (c). Survival curves of
tumor-bearing mice after treatment with THG fractions (5 mg kg1 per
day) for 60 days (d). The bars represent the S.E., *p o 0.05, **p o 0.05 vs.
control and ***p o 0.05 vs. 5-Fu. Survival curves were created using the
Kaplan–Meier method, and statistical analyses of survival curves used a
log-rank test.
This journal is © The Royal Society of Chemistry 2017

Journal of Materials Chemistry B
or spleen index for THF treated groups, compared with the
saline control (Fig. 5c and Fig. S6, ESI†). Furthermore, the
survival rates of the mice treated with THG fractions were
significantly higher than those of the saline control and 5-Fu
groups (Fig. 5d), suggesting the good therapeutic effect and
safety of the THG fractions. Interestingly, the THG fractions
with lower molecular weights (3.01  105–1.01  106) exhibited
larger inhibitory effects than those with higher molecular
weights (1.16  106–1.76  106) against H22 tumor growth,
Published on 23 June 2017. Downloaded by Universidade Federal do Parana on 16/03/2018 13:08:44.
and were significantly greater than that of 5-Fu treatment.
Therefore, the antitumor activity of THG was also molecular
weight-dependent. As reported, the binding of polysaccharides
to receptors increases with chain stiffness.26 In view of the
discussion above, THG samples with molecular weights lower
than 1.01  106 and Kuhn segment numbers smaller than 2
exhibited higher chain stiffness. In addition, with a decrease of
molecular weight, the THG chains changed from long extended
chains to short rods (as seen in AFM images). These results
suggest that a high level of chain stiffness is beneficial to the
antitumor activity of b-glucan. It could be explained that the THG
samples with lower molecular weight and higher stiffness exhibited
stronger binding to receptors on the surface of immune cells due to
the occurrence of cross-link receptors,27 finally leading to higher
antitumor activity. In our recent work, the stiff polysaccharide AF1
with molecular weight lower than 1.0  106 isolated from Auricularia
auricula-judae has also been observed to show higher tumor
inhibition due to stronger immune responses stimulated by lower
molecular weight AF1.58
3.4 THG promotes apoptosis of H22 tumor cells in vivo
To test whether THG promotes apoptosis in vivo, the tumor
damage in H22 tumor-bearing mice was determined by HE
Fig. 6 THG promoted apoptosis of H22 tumor cells in vivo. HE staining
assay (a) and TUNEL assay (b) of tumor tissues in tumor-bearing mice
treated with saline (control), 5-Fu or THG (5 mg kg1 per day).
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Paper
staining and TUNEL staining assays. In the HE staining assay, a
distinct irregular arrangement of karyomorphism and nuclear
rupture were observed in THG-treated and 5-Fu-treated mice
but not in the saline control (Fig. 6a). Compared with the
control group, cell apoptosis happened in THG-treated tumor
tissues as well as in 5-Fu-treated mice (Fig. 6b). Moreover, THG
down-regulated the level of the anti-apoptotic protein Bcl-2,
and up-regulated the level of the pro-apoptotic proteins Bax and
caspase-3 in tumor tissues (Fig. S7, ESI†), further confirming
the tumor cell apoptosis in THG-treated mice. These results
demonstrated that THG inhibited H22 tumor growth by promoting
tumor cell apoptosis in vivo.
Apoptosis analysis in vitro was used to investigate whether or
not THG induced tumor cell apoptosis directly. The results
showed that THG (50–200 mg mL1) did not trigger H22 cell
apoptosis directly in vitro (Fig. 7a) while the result of the live/
dead cell double staining assay confirmed that THG did not
show obvious toxicity against H22 cells in vitro. Similarly, the
cell viabilities of other tumor cells (including HepG2, HeLa,
MCF-7, MNK-45 and SH-sy5y cells) and normal cells (such as
RAW264.7 and L02) were not reduced by THG in vitro (Fig. 7c).
The results indicated that THG did not directly induce tumor
cell apoptosis or death, and has a good safety profile in normal
cells. It has been reported that b-glucan, a biological response
modifier, is recognized by receptors (such as dectin-1 and CR3)
on immune cells, leading to natural and adaptive host immune
responses against cancer.59–61 Our previous studies have
demonstrated that b-glucan from Lentinus edodes displays
immune activity through MAPK signaling pathways.62,63 There-
fore, the facts that THG did not directly induce H22 tumor cells
apoptosis or death in vitro but promoted apoptosis of H22
tumor cells in vivo, suggest that the inhibition of H22 tumor
growth in mice by THG could be mainly attributed to its
Fig. 7 THG induced no H22 cell apoptosis in vitro and showed no
cytotoxicity to various kinds of cell lines. H22 cells were treated with 0,
50, 100 or 200 mg mL1 THG for 72 h, then stained with Annexin V-FITC/PI
for flow cytometry (a) or stained with Calcein-AM/PI (Live/Dead Cell
Double Staining Kit; green: viable cells, red: dead cells) for fluorescence
microscope observation (b). Cell viability was determined by the MTT
method after THG treatment for 72 h (c).
J. Mater. Chem. B, 2017, 5, 5623--5631 | 5629

Paper
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Fig. 8 Schematic diagram of interactions between triple helical THG and
receptors on an immune cell.
immune modulation. In summary, THG possessed potent
antitumor activity through its immune modulation.
In view of these results, a schematic model to describe the
interactions between THG and receptors on the immune cell
membrane is proposed in Fig. 8. As a result of the many active
hydroxyl groups on the THG chains, strong interactions
between THG and receptors occurred on immune cells, resulting
in the indirect antitumor activity. The isolated extended triple
helical chains of THG had a greater probability of interaction
with receptors, finally leading to a stronger antitumor effect. The
aggregated chains led to a lower probability of interaction with
receptors and a weaker effect. Therefore, the interaction of THG
with immune cells was affected by the chain conformation and
molecular weight. The chances of interaction between THG and
receptors were reduced by the aggregation of THG at high
concentration. Moreover, lower molecular weight THG with
relatively stiff chains interacted more strongly with the receptors
than higher molecular weight THG.
4. Conclusions
The triple helical b-glucan (THG) extracted from Lentinus edodes
was successfully degraded into a series of fractions with different
molecular weights through ultrasonic irradiation. This degradation
method was demonstrated to decrease only the molecular weight;
the triple helical structure was retained. The data of molecular
weights, structure-sensitive parameters, contour lengths, and molar
mass per contour length of the THG fractions demonstrated that
THG displayed a stiff triple helical chain conformation in water. The
morphology and size of this stiff chain were visualized with AFM,
and the results were consistent with those calculated from SLS and
the triple helix theory model. Thus, we established a new method
using AFM to characterize the chain stiffness of polysaccharides,
which is important for investigating the relationship of structure
with bioactivity. THG was found to possess potent antitumor activity
against murine hepatocellular carcinoma by promoting tumor cell
apoptosis without damage to the organisms in vivo, resulting in a
significant improvement in the survival rate of tumor-bearing mice.
The inhibition rate reached 73%, which is much higher than that
obtained by using 5-Fu, and there was no significant change in the
5630 | J. Mater. Chem. B, 2017, 5, 5623--5631
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Journal of Materials Chemistry B
body weight and spleen index from the THG-treated mice compared
with the saline control. This showed the good therapeutic effect and
safety of THG. Moreover, the antitumor activity of THG was affected
seriously by its Mw and chain conformation, revealing the significant
molecular weight- and chain conformation-dependences of
antitumor activity. In our findings, the higher antitumor activity
was a result of relatively stronger interactions of THG fractions
having more isolated extended triple helical chains with receptors
on the immune cell membrane. The work provided important
information concerning chain stiffness and molecular weight, and
their correlation to antitumor activity, which could be useful for
the treatment of hepatocellular carcinoma in clinic.
Acknowledgements
This work was supported by the Major Program of the National
Natural Science Foundation of China (21334005), the Major
International Joint Research Project (21620102004), National
Natural Science Foundation of China (21574102 and 21274114),
National Key R&D Program of China (2016YFD0400202), and
the New Century Excellent Talents Program of the Education
Ministry (NCET-13-0442). X. Zheng gratefully acknowledges the
financial support from the Fundamental Research Funds for
the Central Universities (2015203020205).
References
1 S. T. Sonis, L. S. Elting, D. Keefe, D. E. Peterson, M. Schubert,
M. Hauer-Jensen, B. N. Bekele, J. Raber-Durlacher, J. P. Donnelly
and E. B. Rubenstein, Cancer, 2004, 100, 1995–2025.
2 N. C. Miltenburg and W. Boogerd, Cancer Treat. Rev., 2014,
40, 872–882.
3 Q. Yi, J. Ma, K. Kang and Z. Gu, J. Mater. Chem. B, 2016, 4,
4922–4933.
4 A. Shakeri-Zadeh, S. Khoee, M.-B. Shiran, A. M. Sharifi and
S. Khoei, J. Mater. Chem. B, 2015, 3, 1879–1887.
5 G. Chihara, Y. Maeda, J. Hamuro, T. Sasaki and F. Fukuoka,
Nature, 1969, 222, 687–688.
6 J. Alper, Science, 2001, 291, 2338–2343.
7 J. Finkelstein, Nature, 2007, 446, 999.
8 G. W. Hart and R. J. Copeland, Cell, 2010, 143, 672–676.
9 D. B. Werz and P. H. Seeberger, Chem. – Eur. J., 2005, 11,
3194–3206.
10 T. Lipinski, A. Fitieh, J. St. Pierre, H. L. Ostergaard, D. R. Bundle
and N. Touret, J. Immunol., 2013, 190, 4116–4128.
11 S. Wawra, P. Fesel, H. Widmer, M. Timm, J. Seibel, L. Leson,
L. Kesseler, R. Nostadt, M. Hilbert, G. Langen and
A. Zuccaro, Nat. Commun., 2016, 7, 13188.
12 X. Huang, S. Nie, H. Cai, G. Zhang, S. W. Cui, M. Xie and
G. O. Phillips, J. Funct. Foods, 2015, 15, 525–532.
13 B. Wu, J. Cui, C. Zhang and Z. Li, Int. J. Biol. Macromol.,
2012, 50, 1116–1120.
14 J.-s. Bae, K.-h. Jang and H. K. Jin, Cancer Lett., 2006, 235, 60–68.
15 G. Liu, S. Kuang, S. Wu, W. Jin and C. Sun, Sci. Rep., 2016,
6, 26722.
This journal is © The Royal Society of Chemistry 2017

Journal of Materials Chemistry B
16 H. Lu, Y. Yang, E. Gad, C. A. Wenner, A. Chang, E. R. Larson,
Y. Dang, M. Martzen, L. J. Standish and M. L. Disis, Clin.
Cancer Res., 2011, 17, 67–76.
17 F. Hong, J. Yan, J. T. Baran, D. J. Allendorf, R. D. Hansen,
G. R. Ostroff, P. X. Xing, N.-K. V. Cheung and G. D. Ross,
J. Immunol., 2004, 173, 797–806.
18 I. C. Diller, Z. T. Mankowski and M. E. Fisher, Cancer Res.,
1963, 23, 201–208.
Published on 23 June 2017. Downloaded by Universidade Federal do Parana on 16/03/2018 13:08:44.
19 R. Zhang and K. J. Edgar, Biomacromolecules, 2014, 15,
1079–1096.
20 Y. Masuda, H. Inoue, H. Ohta, A. Miyake, M. Konishi and
H. Nanba, Int. J. Cancer, 2013, 133, 108–119.
21 Y. Masuda, D. Nawa, Y. Nakayama, M. Konishi and
H. Nanba, J. Leukocyte Biol., 2015, 98, 1015–1025.
22 S. LeibundGut-Landmann, O. Grosz, M. J. Robinson,
F. Osorio, E. C. Slack, S. V. Tsoni, E. Schweighoffer,
V. Tybulewicz, G. D. Brown, J. Ruland and C. Reis e Sousa,
Nat. Immunol., 2007, 8, 630–638.
23 S. LeibundGut-Landmann, F. Osorio, G. D. Brown and
C. Reis e Sousa, Blood, 2008, 112, 4971–4980.
24 G. Chan, W. K. Chan and D. Sze, J. Hematol. Oncol., 2009,
2, 25.
25 H. Huang, G. R. Ostroff, C. K. Lee, S. Agarwal, S. Ram,
P. A. Rice, C. A. Specht and S. M. Levitz, J. Immunol., 2012,
189, 312–317.
26 A. Mueller, J. Raptis, P. J. Rice, J. H. Kalbfleisch, R. D. Stout,
H. E. Ensley, W. Browder and D. L. Williams, Glycobiology,
2000, 10, 339–346.
27 J. A. Cleary, G. E. Kelly and A. J. Husband, Immunol. Cell
Biol., 1999, 77, 395–403.
28 K. Oba, M. Kobayashi, T. Matsui, Y. Kodera and J. Junichi,
Anticancer Res., 2009, 29, 2739–2745.
29 S. Yoshino, T. Tabata, S. Hazama, N. Iizuka, K. Yamamoto,
M. Hirayama, A. Tangoku and M. Oka, Anticancer Res., 2000,
20, 4707–4711.
30 Y. Y. Maeda and G. Chihara, Nature, 1971, 229, 634.
31 S. Yoshino, S. Watanabe, M. Imano, T. Suga, S. Nakazawa,
S. Hazama and M. Oka, Hepato-Gastroenterology, 2010, 57,
172–177.
32 D. Higashi, K. Seki, Y. Ishibashi, Y. Egawa, M. Koga,
T. Sasaki, K. Hirano, K. Mikami, K. Futami, T. Maekawa
and M. Sudo, Anticancer Res., 2012, 32, 2365–2368.
33 H. Kataoka, T. Shimura, T. Mizoshita, E. Kubota, Y. Mori,
T. Mizushima, T. Wada, N. Ogasawara, S. Tanida, M. Sasaki,
S. Togawa, H. Sano, Y. Hirata, M. Ikai, H. Mochizuki, K. Seno,
S. Itoh, T. Kawai and T. Joh, Hepato-Gastroenterology, 2009, 56,
547–550.
34 L. N. Zhang, X. F. Zhang, Q. Zhou, P. Y. Zhang, M. Zhang
and X. L. Li, Polym. J., 2001, 33, 317–321.
35 L. Zhang, X. Li, X. Xu and F. Zeng, Carbohydr. Res., 2005,
340, 1515–1521.
36 S. Li, Y. Huang, S. Wang, X. Xu and L. Zhang, J. Phys. Chem. B,
2014, 118, 668–675.
This journal is © The Royal Society of Chemistry 2017
View Article Online
Paper
37 U. Surenjav, L. Zhang, X. Xu, X. Zhang and F. Zeng, Carbohydr.
Polym., 2006, 63, 97–104.
38 H. Xu, S. Zou, X. Xu and L. Zhang, Sci. Rep., 2016, 6, 28802.
39 K. Zhong, Q. Zhang, L. Tong, L. Liu, X. Zhou and S. Zhou,
Ultrason. Sonochem., 2015, 23, 75–80.
40 B. H. Zimm, J. Chem. Phys., 1948, 16, 1093–1099.
41 Y. Zhang, X. Xu, J. Xu and L. Zhang, Polymer, 2007, 48,
6681–6690.
42 M. Sletmoen, E. Geissler and B. T. Stokke, Biomacro-
molecules, 2006, 7, 858–865.
43 L. Yu, X. Xu, J. Zhou, G. Lv and J. Chen, Food Hydrocolloids,
2017, 65, 165–174.
44 T. L. Bluhm and A. Sarko, Can. J. Chem., 1977, 55, 293–299.
45 T. Yanaki, T. Norisuye and H. Fujita, Macromolecules, 1980,
13, 1462–1466.
46 Y. Kashiwagi, T. Norisuye and H. Fujita, Macromolecules,
1981, 14, 1220–1225.
47 A. Arzumanyan, H. M. G. P. V. Reis and M. A. Feitelson, Nat.
Rev. Cancer, 2013, 13, 123–135.
48 R. L. Siegel, K. D. Miller and A. Jemal, Ca-Cancer J. Clin.,
2016, 66, 7–30.
49 S. Xu, Y. Lin, J. Huang, Z. Li, X. Xu and L. Zhang, J. Mater.
Chem. A, 2013, 1, 4198–4206.
50 J.-Y. Yin, S.-P. Nie, Q.-B. Guo, Q. Wang, S. W. Cui and
M.-Y. Xie, Carbohydr. Polym., 2015, 124, 331–336.
51 A. K. Vuppu, A. A. Garcia and C. Vernia, Biopolymers, 1997,
42, 89–100.
52 T. M. McIntire and D. A. Brant, J. Am. Chem. Soc., 1998, 120,
6909–6919.
53 T. M. McIntire and D. A. Brant, Biopolymers, 1997, 42, 133–146.
54 T. M. McIntire, R. M. Penner and D. A. Brant, Macromolecules,
1995, 28, 6375–6377.
55 L. Schefer, I. Usov and R. Mezzenga, Biomacromolecules,
2015, 16, 985–991.
56 C. Li, M. Numata, A.-H. Bae, K. Sakurai and S. Shinkai,
J. Am. Chem. Soc., 2005, 127, 4548–4549.
57 Y. Meng, S. Zou, M. Jiang, X. Xu, B. Z. Tang and L. Zhang,
J. Mater. Chem. B, 2017, 5, 2616–2624.
58 Z. Ping, H. Xu, T. Liu, J. Huang, Y. Meng, X. Xu, W. Li and
L. Zhang, J. Mater. Chem. B, 2016, 4, 4565–4573.
59 C. Qi, Y. Cai, L. Gunn, C. Ding, B. Li, G. Kloecker, K. Qian,
J. Vasilakos, S. Saijo, Y. Iwakura, J. R. Yannelli and J. Yan,
Blood, 2011, 117, 6825–6836.
60 J. Tian, J. Ma, K. Ma, H. Guo, S. E. Baidoo, Y. Zhang, J. Yan,
L. Lu, H. Xu and S. Wang, Eur. J. Immunol., 2013, 43,
1220–1230.
61 Y. Ning, D. Xu, X. Zhang, Y. Bai, J. Ding, T. Feng, S. Wang,
N. Xu, K. Qian, Y. Wang and C. Qi, Int. J. Cancer, 2016, 138,
2713–2723.
62 X. Xu, C. Pan, L. Zhang and H. Ashida, J. Biol. Chem., 2011,
286, 31194–31198.
63 X. Xu, M. Yasuda, S. Nakamura-Tsuruta, M. Mizuno and
H. Ashida, J. Biol. Chem., 2012, 287, 871–878.
J. Mater. Chem. B, 2017, 5, 5623--5631 | 5631