Carbohydrate Polymers 177 (2017) 275–283

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Research Paper

Synthesis and characterization of a multi-sensitive polysaccharide hydrogel T

for drug delivery
Wei Wei, Junjian Li, Xiaoliang Qi, Yin Zhong, Gancheng Zuo, Xihao Pan, Ting Su, Jianfa Zhang,
⁎
Wei Dong
Center for Molecular Metabolism, Nanjing University of Science & Technology, Nanjing 210094, China

A R T I C L E I N F O A B S T R A C T

Keywords: Salecan is a novel water soluble polysaccharide produced by a salt-tolerant strain Agrobacterium sp. ZX09. Poly
Hydrogels (dimethylaminoethyl methacrylate) (PDMAEMA) is a pH, thermo, and ionic strength multi-sensitive polymer
Salecan with anti-bacterial property. Here, we report a semi-interpenetrating polymer network (semi-IPN) hydrogel
Polysaccharide based on salecan and PDMAEMA. The obtained hydrogel is simultaneous sensitive to pH, ionic strength and
Stimuli-responsive
temperature: the swelling ratio maximizes at pH 1.2 and shrinks at pH value greater than 3; besides, water
Drug delivery
content of the hydrogel decreases as the ionic strength increases; in terms of temperature, the hydrogel swells/
deswells at temperatures below/above 40 °C. Cytotoxicity test shows the hydrogel is non-cytotoxic to COS-7
cells. Protein drug insulin was selected as model drug to test the in vitro release behavior of the hydrogel. Results
show the release rate increases with the swelling ratio of the hydrogel. In addition, when the temperature is
higher than the lower critical solution temperature (LCST) of PDMAEMA, the hydrogel shrinks to extrude more
drug molecules. Moreover, the release rate and release amount were higher in acid condition (pH 1.2) than at pH
7.4. In summary, this polysaccharide hydrogel is a promising material for drug delivery.

1. Introduction 2006). Oral delivery is a preferable administration route because it

Hydrogels are 3-dimensional hydrophilic polymer networks that can
absorb a large amount of water molecules while maintain their integrity
(Buwalda, Vermonden, & Hennink, 2016). Because of the high water
content, hydrogels always exhibit excellent biocompatibility so that
they have become a big class of biomaterials with great potential for
medical and biological applications (Ren et al., 2009; Zhu et al., 2016).
In recent years, stimuli-responsive hydrogels are rapidly developed and
known as smart materials (Haq, Su, & Wang, 2017). Stimuli-responsive
hydrogels can be designed into materials sensitive to temperature, pH
values, lights, and magnetic fields (Cao, Peng, Zhong, & Sun, 2014;
Cezar et al., 2016; Cheng et al., 2016; Fan et al., 2017). Materials with
stimuli-responsive ability are great candidates for drug delivery because
their release behavior can be easily controlled by the external en-
vironment (Ganguly, Chaturvedi, More, Nadagouda, & Aminabhavi,
2014; Koetting, Peters, Steichen, & Peppas, 2015; Peppas & Van
Blarcom, 2015). In drug delivery systems, the requirement to improve
patient care is always existing (Peppas, Hilt, Khademhosseini, & Langer,
avoids pain and embarrassing in injections (Gil & Hudson, 2004). In
recent years there has been increasing research on the delivery of
protein drugs (Wu, He, Wu, & Chen, 2016; Yu et al., 2016). The appli-
cation of hydrogels as protein carriers also has been widely studied
(Peppas, 2004; Vermonden, Censi, & Hennink, 2012; Vulic, Pakulska,
Sonthalia, Ramachandran, & Shoichet, 2015).
Generally, hydrogels are formed by physically or chemically cross-
linking polymers. Physical crosslinking includes interactions such as
hydrogen bond, ionic bond, and hydrophobic association (Hu,
Vatankhah-Varnoosfaderani, Zhou, Li, & Sheiko, 2015; Luo et al., 2015;
Zhang et al., 2016). On the other hand, chemical crosslinking is a kind
of stable crosslinking because of its covalent bond (Rubentheren, Ward,
Chee, & Tang, 2015). Interpenetrating polymer networks (IPNs) tech-
nique is a common method for preparing hydrogels. IPNs consist of two
or more polymer networks that physically entangled with each other
(Taylor & In Het Panhuis, 2016). Since there is no chemical bond be-
tween the networks, IPNs combine the properties of each constituent
polymer (Zhang & Peppas, 2000). Similarly, semi-IPNs are formed by

Abbreviations: DMAEMA, 2, (dimethylamino) ethyl methacrylate; PDMAEMA, poly(2-(dimethylamino) ethyl methacrylate); BIS, N,N′-methylenebis(acrylamide); APS, ammonium
persulfate; TEMED, N,N,N′,N′-tetramethylethylenediamine; ATR-FTIR, attenuated total reflectance Fourier transform infrared; XRD, X-ray diffraction; TGA, thermogravimetric analyses;
SEM, Scanning Electron Microscopy; DMEM, Dulbecco’s Modified Eagle Medium; DMSO, dimethyl sulfoxide
⁎
Corresponding author.
E-mail address: weidong@njust.edu.cn (W. Dong).

http://dx.doi.org/10.1016/j.carbpol.2017.08.133
Received 16 April 2017; Received in revised form 21 August 2017; Accepted 24 August 2017
Available online 01 September 2017
0144-8617/ © 2017 Elsevier Ltd. All rights reserved.
W. Wei et al.
crosslinking one polymer network in the presence of another polymer
chain (Du et al., 2015). Semi-IPNs are widely used for fabricating hy-
drogels composed of synthetic polymer networks and polysaccharides
such as hyaluronic acid, chitosan, pullulan and cellulose (Catanzano,
D’Esposito et al., 2017; Dash, Ferri, & Chiellini, 2012; Lee, Sen, Bae,
Lee, & Webb, 2015; Nizam El-Din, Abd Alla, & El-Naggar, 2010;
Pescosolido et al., 2011; Singh et al., 2015; Singh, Saini, & Kennedy,
2008). Polysaccharide-based hydrogels have also been widely studied
in drug delivery because of their excellent biodegradability and bio-
compatibility (Xu, Huang et al., 2016).
Salecan (CAS. no. 1439905-58-4) is an extracellular polysaccharide
(EPS) produced by a salt-tolerant strain named Agrobacterium sp. ZX09.
The strain is isolated in our laboratory and the 16S rDNA sequence was
deposited in the GenBank database (accession number: GU810841).
The structure of salecan is a linear (1 → 3)-β-D-glucan consist of β-1-3-
linked glucopyranosyls with a small quantity of α-1-3-linked (Ding,
Zhang, Jiang, Xu, & Liu, 2004; Xiu et al., 2010). The abundant hydroxyl
groups in its structure make it a highly hydrophilic nature. Recently,
succinyl and pyruvyl substituent groups on salecan were identified by
enzymatic hydrolysis (Xu, Cheng et al., 2016). Moreover, salecan has
been demonstrated to be edible and can reduce adiposity and improve
glucose tolerance in high-fat diet-fed mice (Xiu, Zhan et al., 2011; Xiu,
Zhou, Zhu, Wang, & Zhang, 2011; Zhang et al., 2013; Zhou, Pu et al.,
2014; Zhou, Wang et al., 2014). These properties make it interesting to
prepare hydrogels from salecan.
Poly(2-(dimethylamino) ethyl methacrylate) (PDMAEMA) is a pH
and thermo dual responsive polymer (Li et al., 2015; Sui, Zhao et al.,
2012). Crosslinked PDMAEMA is also sensitive to ionic strength in
aqueous solution (Dragan & Cocarta, 2016). Tertiary amine groups on
PDMAEMA play an important role in this dual responsive property. At
low pH, PDMAEMA is a cationic polyelectrolyte and is water-soluble
because tertiary amine groups are protonated. While at high pH value,
tertiary amino groups are deprotonated and PDMAEMA exhibits a
thermo-responsive behavior: water-soluble at temperatures below
lower critical solution temperature (LCST) and water-insoluble at
temperatures above LCST (Han et al., 2013). Moreover, PDMAEMA was
reported to be an anti-bacterial polymer (Sui, Gao, Wang, & Gao, 2012).
Recent years have seen an increasing number of pH and thermo dual
responsive hydrogels based on PDMAEMA for drug delivery (Apopei
Loghin, Biliuta, Coseri, & Dragan, 2017; Dragan & Cocarta, 2016;
Dragan et al., 2016; Huang, Liu, Chen, Gao, & Gong, 2012).
In this paper, a series of salecan/PDMAEMA semi-IPN hydrogels
with different compositions were synthesized. To our best knowledge,
this is the first time multi-responsive hydrogels based on salecan and
PDMAEMA have been fabricated. The physical-chemical properties
were characterized by XRD, ATR-FTIR, TGA, SEM, and rheological
methods. The responsive behaviors were investigated by recording
swelling ratios under different conditions. The effect of composition
ratios on salecan/PDMAEMA semi-IPN hydrogels were also studied.
The cytotoxicity of hydrogels was investigated by MTT assay. Protein
drug insulin was selected as a model to test the drug delivery property
of the hydrogel. The in vitro release property was investigated at dif-
ferent pH values and temperatures.
2. Materials and methods
2.1. Materials
Salecan (Cas.No.1439905-58-4, molecular weight 2 × 106 Da) was
supplied by Center for Molecular Metabolism, Nanjing University of
Science & Technology. DMAEMA was obtained from Energy Chemical
Technology (Shanghai) Co. Ltd. N,N′-Methylenebis(acrylamide) (BIS),
ammonium persulfate (APS), and N,N,N′,N′-tetra-
methylethylenediamine (TEMED) were purchased from Aladdin
Reagent Co. Ltd., Shanghai, China. MTT cell proliferation and cyto-
toxicity detection kit was purchased from Nanjing KeyGen Biotech Co.,
276
Carbohydrate Polymers 177 (2017) 275–283
Table 1
Feed ratios for salecan/PDMAEMA hydrogels.
Sample codes Salecan (mL) DMAEMA with BIS Final polymer
(mL) concentrations
S5D5 5 5 6 wt%
S4D6 4 6 6.8 wt%
S3D7 3 7 7.6 wt%
S2D8 2 8 8.4 wt%
S1D9 1 9 9.2 wt%
PDMAEMA 0 10 10 wt%
LTD, China. Insulin was purchased from Dalian Meilun Biology
Technology Co., Ltd (Dalian, China).
2.2. Preparation of salecan solution
Salecan was prepared as 2% w/v aqueous solution for further use.
Firstly, 2 g of salecan powder was added to 100 mL deionized water.
The mixture was stirred by a magnetic stirrer at room temperature for
24 h. Finally, a transparent and uniform solution was obtained. Salecan
solution was stored in 4 °C for further use.
2.3. Fabrication of salecan/PDMAEMA semi-IPN hydrogels
Hydrogels described in this work were synthesized by polymerizing
and crosslinking DMAEMA monomer in the presence of salecan. The
method was used according to our previous work (Wei et al., 2015; Wei
et al., 2016). Briefly, aqueous solution of 10 wt% DMAEMA having
0.4 wt% BIS were prepared and mixed with salecan solution. The
composition ratios were listed in Table 1. The mixture was stirred under
an argon atmosphere for 1 h. After that, 25 μL of TEMED and 8 mg of
APS were added to initiate the polymerization. The solution was stirred
continuously for 5 min. Then the solution was poured into a mold and
sealed immediately. The polymerization could be allowed to proceed
for overnight at room temperature. After the polymerization, the hy-
drogels were moved into deionized water carefully. During the next
7 days, the deionized water was refreshed several times every day to
completely eliminate unreacted reagents. Finally, the hydrogels were
lyophilized and stored in desiccator.
2.4. Characterization
2.4.1. Stability of salecan in semi-IPN hydrogels
The stability of salecan in the semi-IPNs was determined by mea-
suring the amount of salecan in the hydrogel washing solution.
Salecan/PDMAEMA hydrogels were immersed in deionized water for
7 days, then the concentrations of salecan in the washing solutions were
determined by the phenol-sulfuric acid method (DuBois, Gilles,
Hamilton, Rebers, & Smith, 1956; Hu, Feng et al., 2014). Briefly, for
each sample, 2 mL of washing solution was added to a tube. Then 1 mL
of 6% (w/w) phenol aqueous solution was added, followed by 5 mL of
concentrated H2SO4 added carefully drop wise. The tube was shaken for
10 min to get the mixture uniform. Finally, the concentration of salecan
was measured by a UV–vis spectrometer at 490 nm with standard curve
(y = 10.993x + 0.0785, R2 = 0.9992, 0.008–0.032 mg/mL). All the
samples were performed in duplicate.
2.4.2. Attenuated total reflectance-Fourier transform infrared spectra
(ATR-FTIR)
ATR-FTIR spectroscopy of hydrogels were acquired through a
Nicolet iS10 FTIR instrument (Thermo Fisher Scientific, USA). The
conditions were wavenumber range 500–4000 cm−1, 32 repeated
scans, and 4 cm−1 scan resolution. Background measurements were
performed prior to sample testing and subtracted automatically from
the sample readings.
W. Wei et al.
2.4.3. X-ray diffraction (XRD)
The XRD patterns were obtained through a XRD instrument (DMAX-
2200) with Cu Kα radiation (λ = 0.154 nm). Data were recorded from
2θ = 10–60° at 30 kV and 20 mA.
2.4.4. Thermogravimetric analyses (TGA)
Thermogravimetric analyses (TGA) was performed using a TA in-
strument (Model Q600 thermal gravimetric analyzer). The xerogel
powder and salecan powder were heated from 25 °C to 600 °C under a
nitrogen atmosphere at a heating rate of 10 °C/min.
2.4.5. Rheology
The mechanical property of hydrogels was tested using a MCR 101
rheometer (Anton Paar) with a parallel-plate (diameter 50 mm).
Dynamic strain sweep was performed to determine the linear viscoe-
lasticity region. Frequency sweep measurement (0.01% strain) was
used to test the storage modulus G’ and loss modulus G”, as a function
of frequency, at 25 °C.
2.5. Swelling behavior
The swelling behavior of hydrogels in this work were studied by a
gravimetric method. Freeze-dried hydrogels were immersed into deio-
nized water to determine the swelling kinetics at 25 °C. The samples
were weighed at prearranged time points, after wiping out the excess
water on the surface by moistened filter paper. The Water uptake (WU)
was calculated by Eq. (1):
WU = (Ws − Wd)/Wd (1)
Here, Ws is the mass of the swelling sample at time t and Wd is the mass
of dried sample.
For temperature sensitive test, hydrogels were allowed to swell to
equilibrium in deionized water at different temperature in the range of
25–60 °C. Temperature was controlled by a thermostated water bath
with a resolution of 0.1 °C.The weight of equilibrium swollen hydrogels
were recorded after wiping off the surface water with moistened filter
paper. Equilibrium water uptake (EWU) was evaluated by Eq. (2):
EWU = (We − Wd)/Wd (2)
Here, We is the mass of swollen gel at the equilibrium state.
To test the pH sensitivity, hydrogels were immersed in PBS with
different pH values. 0.1 M of HCl acid and NaOH solution was used to
make PBS to the desired pH value and keep the ionic strength constant.
After equilibrium soaking, samples were wiped and then weighed. EWU
was calculated by Eq. (2).
Similarly, ionic strength sensitive test was performed by calculate
EWU of hydrogels in NaCl solution with different ionic strength. NaCl
solution was prepared by dissolve NaCl powder in deionized water. All
the experiments were done in triplicate.
2.6. Morphology
Scanning Electron Microscopy (SEM) (JEOLJSM-6380LV) was ap-
plied to freeze-dried hydrogels to observe the internal cross-sections.
The samples were sputter-coated with gold for 20 min to enhance
conductive for SEM observation. SEM images were acquired at an op-
erating voltage of 20 kV.
2.7. Cytotoxicity
The cytotoxicity of hydrogels was characterized by the MTT assay.
In this work, COS-7 cells were used to determine the cytotoxicity of
these hydrogels and the method was similar to the literature (Hu, Feng
et al., 2014). Firstly, COS-7 cells were cultured in a 96-well cell culture
plate in Dulbecco’s modified Eagle medium (DMEM) containing 10%
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Carbohydrate Polymers 177 (2017) 275–283
fetal bovine serum (FBS) at 37 °C and 5% CO2. After 24 h, cells were
divided into six groups and culture medium was replaced by hydrogel
extracting liquid (DMEM containing serum contacted to hydrogels at
37 °C for 7 days). Cells were cultured for a continuous 24 h, and cell
adhesion was observed using a phase-contrast optical microscope
(OLYMPUS, Japan). Then, 50 μL of MTT solution was dropped to each
well and incubated for 4 h. Finally, 150 μL of dimethyl sulfoxide
(DMSO) was added to each well, forming an MTT formazan solution.
The optical density was tested at 570 nm with a microplate reader. The
negative control was normal DMEM.
2.8. Drug delivery
Insulin was chosen as a model drug for in vitro release experiments.
For drug loading, hydrogels were immersed in 0.5 mg/mL insulin
aqueous solution (20 mL) until equilibrium. Loading amount (mL) was
calculated as follows:
mL = V0c0 − V1c1 (3)
here V0 and c0 are the volume (20 mL) and concentration (0.5 mg/mL)
of loading solution, respectively. V1 is the volume of drug solution after
loading which can be measured by a measuring cylinder. c1 is the
concentration of insulin solution after loading. This concentration was
determined by the Bradford method with a microplate reader at
595 nm.
To follow the in vitro release process, insulin-loaded hydrogels were
transferred into a tube containing 20 mL PBS solution (pH 1.2 or pH
7.4, ionic strength = 0.1 M) at 37 °C or 42 °C. The tubes were shaken
with a rotating speed of 150 rpm. At predetermined time intervals,
1 mL of release medium was removed and 1 mL fresh medium was
added to keep the volume. The cumulative percent insulin release (Er
%) was calculated by the following equation:
n−1
V0 Cn + Vd ∑ Ci
1
Er % = × 100%
mL (4)
here V0 is the volume of release medium (20 mL). Cn represents the
concentration of release drug after nth taken. Vd is the volume of release
medium removed (1 mL). mL is the loading amount of insulin. Similar
equation can be found in reference (Dragan & Cocarta, 2016).
2.9. Statistical analyses
Statistical analysis was performed using SPSS 19.0 software (SPSS
Inc., Chicago, IL). Two-tailed Student’s t-test and ANOVA were used to
identify statistical comparisons between experimental groups.
Significance was determined at a p value < 0.05.
3. Results and discussion
3.1. Preparation of salecan/PDMAEMA semi-IPN hydrogels
The preparation of salecan/PDMAEMA semi-IPN hydrogels is simple
and convenient. The process is presented in Fig. 1. Free-radical poly-
merization was used in this work. Actually, the first step of poly-
merization was the reaction between APS and TEMED: Free radicals
were produced by homolytic scission of APS moieties, accelerated by
TEMED (Feng, Guo, & Qiu, 1988; Guilherme, da Silva, Rubira,
Geuskens, & Muniz, 2004). Then DMAEMA monomer and BIS cross-
linker was initiated by these sulfate free radicals to allow the process of
polymerization and crosslinking to begin. Salecan chains were physi-
cally entangled with PDMAEMA networks along with process of poly-
merization and crosslinking. Finally, a semi-IPN hydrogel is obtained.
The advantage for semi-IPNs is that each polymer keeps its own prop-
erty since no chemical reactions happen between each other
(Domingues et al., 2013). The stability of salecan in the hydrogel was
W. Wei et al.
Fig. 2. (A) FTIR spectra, (B) XRD patterns and (C) TGA curves of salecan, PDMAEMA and
salecan/PDMAEMA semi-IPN hydrogel.
characterized by phenol sulfuric acid method and the data are shown in
Table S1. Results show very little salecan was detected in the washing
solution, indicating most of salecan was well incorporate into the
PDMAEMA network.
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Carbohydrate Polymers 177 (2017) 275–283
Fig. 1. Schematics of preparation of salecan/PDMAEMA semi-IPN hy-
drogel.
3.2. ATR-FTIR
The components of the semi-IPN hydrogel were examined by ATR-
FTIR spectroscopy, and the spectrum was compared with those of
salecan and PDMAEMA in Fig. 2A. In the spectrum of salecan, the broad
peak at 3297 cm−1 was attributed to eOH stretching vibration. The
peak at 1046 cm−1 represented CeO stretching vibration in the glu-
copyranose ring. The peaks at 893 cm−1 and 814 cm−1 were attributed
to D-glucopyranose group: 893 cm−1 refers to β-configuration and
814 cm−1 refers to α- configuration, respectively. For the spectrum of
PDMAEMA, peaks at 2946, 2821, and 2771 cm−1 were assigned to the
eCH3 groups. The characteristic absorption of ester groups appeared at
1722 cm−1 (Guo, Yuan, Yao, & Gao, 2006). The peaks at 1455 and
1386 cm−1 were attributed to CH2 groups.
In the spectrum of salecan/PDMAEMA semi-IPNs, characteristic
peaks of both salecan and PDMAEMA were observed. The broad peak at
3237 cm−1 was assigned to eOH of salecan. Peaks at 2946, 2821, and
2771 cm−1 were attributed to eCH3 groups of PDMAEMA. Meanwhile,
the characteristic band of PDMAEMA at 1722 cm−1 was also appeared.
Specifically, the absorptions at 1556 cm−1 and 1040 cm−1 were no-
tably enhanced, which could be caused by the presence of salecan.
These results demonstrated that the hydrogel was composed of salecan
and PDMAEMA.
3.3. X-ray diffraction (XRD)
Fig. 2B shows XRD patterns of salecan, salecan/PDMAEMA, and
PDMAEMA hydrogels. In the XRD pattern of salecan, the peak at
2θ = 21° was assigned to the crystalline regions of polysaccharide
structure (Hu, Feng et al., 2014). However, in the XRD pattern of
salecan/PDMAEMA, there was only a weaker and broader halo. This
result hinted that the crystallinity of salecan decreased in the semi-IPN
hydrogels. The intra and inter-molecular hydrogen bonds of salecan
could disappear after the interpenetrating process.
3.4. Thermogravimetric analysis (TGA)
The thermal stability of materials was studied by TGA and results
were shown in Fig. 2C. The thermal degradation of salecan was char-
acterized by our previous work (Wei et al., 2015). Briefly, salecan lost
12 wt% of weight before 150 °C because of the loss of moisture. Next, in
the range of 250–600 °C, the weight of salecan decrease continuously
because the molecule skeleton was thermo-degraded. For PDMAEMA,
the weight loss before 100 °C was the removal of water molecules. The
main degradation of PDMAEMA exhibits two stages which occurs at
270–330 °C and 350–450 °C, respectively. The first stage were caused
by removal of the dimethylaminoethyl group and the second one was
ascribed to elimination of CO, CO2 and carbonization processes (Roy
et al., 2013). The TGA curve of salecan/PDMAEMA hydrogel showed
the same trend of PDMAEMA. However, some notable difference can be
found. Salecan/PDMAEMA hydrogel lost more water before 100 °C
suggested a more hydrophilic property at room temperature. For the
main degradation, the decomposition salecan/PDMAEMA hydrogel
happened earlier than PDMAEMA. This indicates the thermal stability
of semi-IPNs was weaker than that of pure PDMAEMA. Besides, the
stages of main degradation were not further divided, suggesting that
W. Wei et al.
Fig. 3. (A) Storage modulus and (B) loss modulus of the hydrogels in frequency range of
0.1–10 Hz.
intermolecular interaction such as hydrogen bond existed between
salecan and PDMAEMA (Wang, Zhou, & Xiao, 2013). These results hint
salecan/PDMAEMA semi-IPNs possess high hydrophilicity and can be
thermo stable at temperature lower than 100 °C.
3.5. Rheological properties
Rheological tests have been widely used to investigate the me-
chanical property of hydrogels (Sathaye et al., 2015; Ziv et al., 2014). In
this work, the storage modulus (G’) and loss modulus (G”) in the fre-
quency range of 0.1–10 Hz were tested and data were depicted in
Fig. 3A and B. Generally, storage modulus represents elastic property
while loss modulus represents viscosity. In Fig. 3A, G’ values were
found to be independent of frequency. Moreover, the values of G’ were
much larger than those of G”, suggesting hydrogels were elastic solid-
like (Calvet, Wong, & Giasson, 2004; Diba, Wang, Kodger,
Parsa, & Leeuwenburgh, 2017). Besides, from Fig. 3A, G’ values in-
creased with the increasing of PDMAEMA content in hydrogels. This is
because the flexibility of polymer was weakened when polymer density
increased. Higher polymer density enhances polymer chains entangle-
ments and reduce the flexibility of chains. Therefore, higher storage
modulus was obtained (Espinosa-Garcı́a et al., 2007).
3.6. Swelling behavior
The swelling kinetics of hydrogels immersed in deionized water at
25 °C was shown in Fig. 4A. Hydrogels began to absorb water when
they contacted with deionized water, and their swelling ratio increased
along with the time until equilibrium. As shown in Fig. 4A, semi-IPN
hydrogels exhibited a faster swelling rate than pure PDMAEMA hy-
drogel. Moreover, the equilibrium swelling ratios of semi-IPN hydrogels
279
Carbohydrate Polymers 177 (2017) 275–283
were also larger than the pure one. And the higher the content of
salecan, the higher swelling ratio was. As reported before, swelling
ratios of hydrogels could be improved by increasing the overall hy-
drophilicity of hydrogel system (Nistor, Chiriac, Vasile,
Verestiuc, & Nita, 2011). In this work, salecan is a highly hydrophilic
polysaccharide which was interpenetrated into the PDMAEMA net-
work. As a result, the hydrophilicity of hydrogels was enhanced so that
the ability of swelling water was strengthened. Besides, the PDMAEMA
content increased from S5D5 to S1D9. In other word, the polymer
density of S1D9 was higher than that of S5D5. Generally, higher
polymer density lead to impact network structure which is dis-
advantage for absorbing water (Wei et al., 2015). Consequently, swel-
ling ratios of salecan/PDMAEMA hydrogels in deionized water at 25 °C
can be designed by controlling the ratio of salecan to DMAEMA.
PDMAEMA is a thermo, pH, and ionic strength multi-sensitive
polymer (Li et al., 2015; Sui, Zhao et al., 2012; You & Auguste, 2008).
By preparing a semi-IPN network, some of the original properties of
PDMAEMA and salecan can be maintained. Fig. 4B shows equilibrium
swelling ratios of hydrogels in deionized water at different temperature
points range from 25 to 60 °C. The LCST of PDMAEMA was reported at
around 40 °C (Han et al., 2013). As shown in Fig. 4B, the swelling status
of hydrogel did not changed very much before 30 °C. When temperature
rise to 40–45 °C, the water content of hydrogels sharply decreased.
After 50 °C, the samples showed no significant different (p > 0.05) in
swelling ratios, suggesting the hydrogels collapsed into similar net-
works.
To test the pH sensitive property of our hydrogels, samples was
allowed to swell in PBS solutions (ion strength = 0.1 M) with different
pH values. The data was displayed in Fig. 4C. With the pH value in-
creased from 1.2 to 7.4, swelling ratios of hydrogels decreased con-
tinuously. In other words, hydrogels shrink in a neutral environment
when compared with their status in an acid condition. This is because
PDMAEMA is a weak polycation, which will be protonated in acid en-
vironment. The electrostatic interactions between protonated tertiary
amino group make the network expanded. While in neutral pH, the free
electron pair of amino groups in DMAEMA unit could conjugate with
the carbonyl (as shown in Fig. 4C) (Li, Xu, Zhai, Peng, & Li, 2011). This
led to hydrogel shrinkage in a neutral PBS solution.
Fig. 4D showed the influence of ionic strength on the swelling be-
havior of salecan/PDMAEMA hydrogels which was evaluated by mea-
suring equilibrium swelling ratios as a function of NaCl concentration.
As seen from the figure, EWU of hydrogels decreased with the increase
of NaCl concentration. NaCl concentration is an easy way to control the
ionic strength of the solution. For all the hydrogel samples, the water
content declined sharply when NaCl concentration increased from
0.01 mol/L to 0.3 mol/L. The reason for this phenomenon is that the
electrostatic repulsion between polycation can be screened by excessive
counterions in solution (Ghimici, Dragan, & Popescu, 1997). As a result,
the swelling ability of hydrogel was weakened. The result indicates
salecan/PDMAEMA hydrogels are ion strength sensitive. In conclusion,
salecan/PDMAEMA semi-IPNs is a thermo, pH, and ionic strength
multi-sensitive hydrogel.
3.7. Morphology
The internal structure of freeze-dried hydrogels was studied by SEM
and the images were shown in Fig. 5. The hydrogels fabricated in this
work exhibited porous structures with continuous boundaries. These
porous structures were formed by the sublimation of water molecules
during lyophilization (Guo et al., 2015). Therefore, the pore-size of
hydrogel may relate directly to its swelling ability: more water content
result to larger ice crystal aggregates, which were eventually sub-
limated to form larger pores. From the images in Fig. 5, the pore size
decreased with the increase of PDMAEMA content. The pore sizes for
S5D5, S4D6, S3D7, S2D8, S1D9, PDMAEMA are 249 ± 9.3 μm,
187 ± 8.5 μm, 160 ± 5.7 μm, 127 ± 14 μm, and 93 ± 13 μm,
W. Wei et al.
Fig. 4. Swelling behavior of the hydrogels: (A) Swelling kinetic curves in deionized water at 25 °C. (B) Swelling ratio values in deionized water at 25–60 °C. (C) Swelling ratios values in
PBS with pH values range from 1.2–7.4 at 25 °C. (D) Swelling ratios values in NaCl solutions with concentration from 0.01–1 M at 25 °C.
respectively. This trend is the same as that of swelling ratio in deionized
water. The result demonstrates that the pore size of hydrogel is de-
pendent on its swelling ability.
3.8. Cytotoxicity
To evaluate the feasibility of salecan/PDMAEMA semi-IPNs as a
drug carrier, the cytotoxicity against COS-7 cells was carried out in vitro
by MTT assay. The results of MTT assay are plotted in Fig. 6. According
to the data, cell viabilities of all samples were over 85% compared to
control after 24 h incubation, suggesting hydrogels did not show sig-
nificant cytotoxicity to COS-7 cells (Catanzano, Docking,
Schofield, & Boateng, 2017). In addition, the optical images of COS-7
cells incubated with these hydrogel extracts are exhibited in Fig. S1.
The majority of cells incubated with hydrogel extracts showed a bipolar
and flattened morphology, which was similar to the control group.
These results indicated the salecan/PDMAEMA hydrogels were non-
cytotoxic and could be used for drug delivery.
3.9. In vitro drug delivery
The common protein drug insulin was chosen as a model water-
soluble drug to study the in vitro drug delivery ability of hydrogels.
Because of the stimuli-response property of these hydrogels, different
release conditions were selected to test the release profiles.
Firstly, hydrogels with different salecan content were tested in
physiological condition (pH 7.4, 37 °C) and the result was present in
Fig. 7A. As Fig. 7A shows, all the selected samples (S5D5, S3D7,
PDMAEMA) exhibit burst release at the beginning. This is a common
phenomenon in hydrogel drug delivery system, because drug molecules
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Carbohydrate Polymers 177 (2017) 275–283
near to hydrogel surface could be easily diffused to the release medium
(Bai et al., 2012; Hu, Darcos, Monge, Li, Zhou, & Su, 2014). After that,
the release rate slowed down and become more sustained. Besides, the
release rate of insulin from S5D5 hydrogel was faster than S3D7 hy-
drogel, and PDMAEMA hydrogel shows the lowest release rate and
amount. This trend is similar to the swelling ratio discussed above. It is
reported that the release behavior of the hydrogel is indeed related to
its swelling properties: the increase in the swelling ratio leads to a
larger pore size, which is favorable for the drug molecule diffuse out
from the hydrogel (Guo et al., 2006; Huh, Zhao, & Kim, 2015). There-
fore, the release rate and cumulative drug release amount are related to
the salecan content.
The effect of temperature on the release of insulin from hydrogels
was investigated by immersing drug-loaded hydrogels (S5D5) in PBS
solution at different temperatures. As the LCST of PDMAEMA is around
40 °C, 37 °C (below LCST) and 42 °C (above LCST) were selected for this
experiment. The release curve is plotted in Fig. 7B. As observed from
the figure, the release at 42 °C was more rapid than the release at 37 °C.
The reason is that the hydrogel exhibits a partial shrinkage at 42 °C
environment according to the relationship between the swelling prop-
erties and the temperature. In this situation, the hydrostatic pressure in
the hydrogel accumulates and then leads to a rapid release (Ngadaonye,
Geever, Cloonan, & Higginbotham, 2012). On the other hand, at 37 °C,
the swelling ratio of the hydrogel did not change so much that the drug
molecules were still released through free diffusion into the external
environment. Thus, the salecan/PDMAEMA hydrogel has a certain
temperature-controlled drug release behavior that higher temperature
triggers a faster release of drugs. Similar results were also reported in
other literatures (Chen & Wang, 2014; Cirillo et al., 2015).
The effect of pH on drug release is shown in Fig. 7C. Simulated
W. Wei et al.
Fig. 6. Cell viability of COS-7 cells after treatment with hydrogel extracts.
gastric fluid (pH 1.2) and simulated intestinal fluid (pH 7.4) were se-
lected as the release medium. The release behavior of the S5D5 hy-
drogel in these mediums was investigated. The release curve showed
that the release of the drug in pH 1.2 environment was significantly
higher than that in pH 7.4. As the insulin was loaded into the hydrogel
in a neutral environment, the status of the hydrogel didn’t change very
much when moved to simulated intestinal fluid. So the drug molecules
spread stably into the release liquid as previously. However, in acidic
condition (pH 1.2), the tertiary amino group is protonated and the
electrostatic interaction makes the hydrogel network expand, which
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Carbohydrate Polymers 177 (2017) 275–283
Fig. 5. SEM images of (A) S5D5, (B) S4D6, (C) S3D7, (D)
S2D8, (E) S1D9, (F) PDMAEMA.
causes the hydrogel to continue to absorb water and the pore size is
further increased. As a result, insulin molecules could be released more
easily from the expanded hydrogel networks. Similar results were ob-
served in other pH-responsive drug release systems research (Guo et al.,
2006; Wu et al., 2011). Drugs released in stomach can be further moved
to intestine and functioned. This strategy could be used as long lasting
oral delivery as reported in other literature (Bellinger et al., 2016).
4. Conclusion
Here, we report a multi stimuli-responsive hydrogel based on
salecan and PDMAEMA semi-IPNs for insulin controlled delivery. The
structure of this novel hydrogel was confirmed by ATR-FTIR and XRD.
Meanwhile, the thermo-stability was demonstrated by TGA that the
hydrogel was stable below 100 °C. The solid-elastic property of this
hydrogel was affirmed by dynamic frequency sweep rheological mea-
surement. Swelling behavior in different conditions shows that the
hydrogel is thermo, pH, and ionic strength sensitive. The swelling ratio
and rate of the hydrogels in deionized water is accelerated by in-
creasing the content of salecan. Besides, the swelling ratio rapidly de-
clines between 40 and 45 °C, reflecting the temperature sensitivity.
Because of protonation of the tertiary amino group, the hydrogel shows
a higher swelling ratio in acidic environment. Moreover, the swelling
ratio decreases with the increase of environmental ionic strength. SEM
images of hydrogel show the network formation with porous structure,
and the pore size range from 93 to 249 μm according to salecan content.
What’s more, the hydrogel was demonstrated non-cytotoxicity to COS-7
cells. In vitro drug release from salecan/PDMAEMA hydrogel showed a
W. Wei et al.
Fig. 7. (A) Insulin release curve of hydrogels of different composition (S5D5, S3D7, and
PDMAEMA) in pH 7.4 PBS (solid symbols with different colors), and insulin release curve
of hydrogel (S5D5) in PBS with different pH values (black line, pH 7.4-solid square, pH
1.2-opened square); (B) Insulin release curve of S5D5 in PBS (pH 7.4) at 37 °C and 42 °C.
controlled release profile. The release rate could be regulated de-
pending upon the salecan content, temperature, and pH values. In
conclusion, this kind of smart hydrogel can release drugs in both pH 1.2
and pH 7.4 condition at 37 °C, indicating potential as an oral drug
delivery carrier.
Conflict of interest
The authors declare no competing financial interest.
Acknowledgments
Wei Wei and Junjian Li contributed equally to this work. This work
was supported by the National Natural Science Foundation of China
under Grant 51573078.
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.2017.08.133.
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