International Journal of Biological Macromolecules 91 (2016) 85–91

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International Journal of Biological Macromolecules

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

Hypromellose succinate-crosslinked chitosan hydrogel films for

potential wound dressing
Qiong Jiang a,b , Wei zhou c , Jun Wang a , Rupei Tang a,c,∗ , Di Zhang c , Xin Wang a
a
Engineering Research Center for Biomedical Materials, School of Life Science, Anhui University, 111 Jiulong Road, Hefei, Anhui Province, 230601, PR China
b
College of Chemistry and Bio-Engineering, Yichun University, Yichun 336000, PR China
c
School of Pharmaceutical Science, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu Province, 214122, PR China

a r t i c l e i n f o a b s t r a c t

Article history: The objective of this study was to develop novel hydrogel films based on carboxyl-modified
Received 17 February 2016 hypromellose-crosslinked chitosan for potential wound dressing. Hypromellose (HPMC) was grafted
Received in revised form 18 May 2016 with succinic acid to yield hypromellose succinate (HPMCS), and then the reinforced hydrogel films of
Accepted 20 May 2016
HPMCS-crosslinked chitosan (HPMCS-CS) were prepared through amide bond formation using 1-ethyl-3-
Available online 21 May 2016
(3-dimethylaminopropyl)carbodiimide (EDC) and N- hydroxysuccinimide (NHS) as a catalyst. Compared
to that of blend film, mechanical properties of HPMCS-CS hydrogel films were significantly enhanced
Keywords:
both in dry and swollen state. To assess the applicability of HPMCS-CS hydrogel films as wound dress-
Chitosan
Hydrogel
ing, the swelling behavior, water vapor transmission rate (WVTR), oxygen permeability, biocompatibility
Wound dressing (cytotoxicity and hemolysis), in vitro drug release and bactericidal properties were analyzed. The results
indicated that HPMCS-CS hydrogel films with good biocompatibility possess high swelling ratio, proper
WVTR, and oxygen permeability, which might accelerate tissue regeneration. Meanwhile, gentamycin
sulfate release from drug-loaded HPMCS-CS hydrogel films were sustained, which would help to protect
wound from infection.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction common physical properties of hydrogels are very similar to that

Numerous wound dressings have been developed worldwide to
treat all kinds of epidermal damage each year. In the past, bandages,
cotton wool, lint and gauzes were used for the management of
wounds. They can absorbing wound exudates to keep a dry wound
and preventing wound infection by bacteria [1,2]. Although we are
still not fully understand the complexities of wound healing, It is
now known that a desirable wound dressing should provide a moist
environment with good biocompatibility to accelerate the tissue
regeneration [3]. An ideal wound dressing or combination of dress-
ings should also allow gaseous exchange, protect the wound from
extraneous microbes, and absorb excess wound fluids and exudates
without leakage, and be non-adherent, comfortable which could
be easily changed by fresh dressing [4–6]. Hydrogel is a gel with
water as dispersion medium [7]. In 1960s, hydrogels have been
innovated and applied in many biomedical disciplines [8]. Some
∗ Corresponding author at: Engineering Research Center for Biomedical Materials,
School of Life Science, Anhui University, 111 Jiulong Road, Hefei, Anhui Province,
230601, PR China.
E-mail address: tangrp99@iccas.ac.cn (R. Tang).
of the living tissues, such as high water content, rubbery texture,
low interfacial tension, which might attribute to an outstanding
biocompatibility and minimizes inflammatory reactions in the sur-
rounding tissues [9,10]. These excellent characteristics have made
hydrogels a promising candidate for wound dressing [11,12], which
can keep the wound moist and sterile, accelerate the healing of the
wound [13].
Chitosan (CS) is a liner polysaccharide industrially obtained by
partial N-deacetylation of chitin, which is determined as a sta-
ble, biodegradable, biocompatible and non-toxic polymer [14,15].
Moreover, it is widely used as a versatile biomaterial due to its
wound healing ability, cationicity, hemostatic property, and film-
form ability. Chitosan gives significant antibacterial activity against
a broad spectrum of pathogenic bacteria and fungi, which has
also been well documented [16–18]. However, in wet state, pure
chitosan films have a poor mechanical property due to their brit-
tleness, which limits its use as wound dressings. Hence, to improve
its strength and elasticity, blend or crosslink of chitosan with nat-
ural or synthetic polymers such as cellulose derivatives, poly(vinyl
alcohol), starch, and gelatin, have been investigated [19–23].
Cellulose is the most abundant and renewable polymer avail-
able worldwide, whose basic repeating structure of cellulose is a

http://dx.doi.org/10.1016/j.ijbiomac.2016.05.077
0141-8130/© 2016 Elsevier B.V. All rights reserved.
86 Q. Jiang et al. / International Journal of Biological Macromolecules 91 (2016) 85–91
Fig. 1. The synthetic route of HPMCS.
␤-d-glucopyranose unit [24]. Although it is insoluble in water due
to intermolecular hydrogen bonding, solubility can be achieved
by chemical substitution, generating different cellulose deriva-
tives. Among which, hypromellose (HPMC) prepared by addition
of methyl and hydroxypropyl groups to the anhydroglucose back-
bone, possesses good film-forming capability, chemical stability,
biodegradability, and biocompatibility, which has been extensively
employed in the construction industry, food industry, and pharma-
ceutical industry [25–27]. Although HPMC has been used as a drug
delivery matrix in the pharmaceutical industry [28], the applica-
tions of HPMC as wound dressing are hampered by such obstacles
as dissolution in the physiological fluid.
Compared with blended HPMC/CS, the water solubility and
water vapor transfer rate of citric acid crosslinked HPMC/CS film
were both reported to reduce 40% [29]. Yet we not know whether
other crosslinking regent such as EDC/NHS would have the same
effect, and if so, how different ratios of HPMC and CS might affect
the properties of the products, would it lead to a much better
mechanical properties in swollen state, which is highly preferable
for materials used as wound dressing.
In this work, HPMC was first modified with succinic anhydride
to give hypromellose succinate (HPMCS), which has free carboxyl
acid groups. And then, a series of hydrogels were prepared by
crosslinking HPMCS and CS at three different ratios using EDC
and NHS as catalyst [30]. EDC is a carboxyl and amine-reactive
crosslinker. Meanwhile, NHS can make conjugation procedures
stable. EDC/NHS combination is widely used in esterification reac-
tion, which has been proved to be non-toxic, and can be easily
removed by dialysis or rinse [31]. The crosslinked hydrogel films
were characterized with Fourier transform infrared spectropho-
tometer (FTIR), and X-ray diffraction (XRD). The physical properties
of the films such as swelling ratio, mechanical properties, water
vapor and oxygen permeability were also characterized. In vitro
cytotoxicity, hemolytic potential, in vitro drug-release, and bacte-
ricidal properties were also been studied.
2. Materials and methods
2.1. Materials
HPMC was obtained from Shandong Ruitai Co. Ltd. in
China (Methoxyl: 28–30%; Hydroxypropyl: 7–12%, Mw = 8000-
10000). Chitosan (degree of deacetylation: 97%, Viscosity:
280–330 mPa s, 0.5% in 0.5% Acetic Acid at 20 ◦ C) was pur-
chased from Tokyo Chemical Industry Co. Ltd. Succinic anhydride,
1-ethyl-3-(3-dimethylaminopropyl)-carbodiiminde (EDC), N-
hydroxysuccinimide (NHS), and 4-dimethylaminopyridine (DMAP)
were purchased from Aladdin Industrial Corporation. All other
chemicals were purchased from Sinopharm Chemical Reagent Co.
Ltd.
2.2. Preparation of HPMCS
HPMC was esterificated with succinic anhydrate to give HPMCS,
as shown in Fig. 1. In brief, natural HPMC powder (5.0 g) was added
into DMF solution (80 mL), stirring until it’s dissolved. Then suc-
cinic anhydrate (7.5 g) and DMAP (0.5 g) were slowly added into
the above solution. This system was first incubated at 70 ◦ C for 7 h,
and then at room temperature to cool it down. The solution was
added slowly to stirred anhydrous diethyl ether. The precipitate
which was dried overnight in a vacuum oven was the target product
(HPMCS).
2.3. Preparation of crossslinked HPMCS-CS hydrogel films
The HPMCS (1 wt% HPMCS) and an acetic acid solution of chi-
tosan (1 wt% acetic acid, 1 wt% chitosan) were well mixed and then
an ethanolic solution of EDC/NHS was added to the mixture, and it
was made sure that they were mixed together thoroughly. HPMCS-
CS composite films were prepared by changing the weight ratio of
HPMCS to CS, such as 1:1, 1:3 and 1:5, which designated as HC-1,
HC-2, and HC-3, respectfully. The fixed molar ratio of carboxylic
acid group/EDC/NHS was 1/2/1. After 10 min of stirring, the solu-
tion was transferred to a disk, and then delivered to a vacuum oven
to evaporate the solvent. The dried films were first immersed in
NaOH solution (1 M) and then in distilled water, change the water
for several times till the pH was approximately 7. HPMCS/CS blend
film (weight ratios of HPMCS to chitosan: 1:1) prepared by the same
procedure without EDC/NHS was denoted as HC-0, and used as a
control.
2.4. Characterization of HPMCS-CS hydrogel films
2.4.1. Chemical structure
To clarify the chemical structure of the films, FTIR spectra were
recorded on a Perkin Elmer spectrophotometer using KBr pellets.
The resolution was spectrum 2 cm−1 with range from 4000 to
500 cm−1 .
2.4.2. The X-ray diffraction studies
The samples were measured by wide-angle X-ray diffraction
(XRD) with Mar 345 image plate as detector, and the source used
was Cu-K␣ (wavelength ␭ = 0.1542 nm). The scanning speed was
2.0◦ min−1 with the recorded region of 2 was 5–50◦ .
 Q. Jiang et al. / International Journal of Biological Macromolecules 91 (2016) 85–91
2.4.3. Mechanical properties
The tensile strength and elongation at break of hydrogel, neat
chitosan, and HPMCS films were measured on a universal ten-
sile tester (Yihuan Instrument Co. Ltd, Shanghai) according to
GB13022-1991 at a speed of 0.5 mm s−1 [32]. The specimens were
in rectangular shape with the dimension of 100 mm × 15 mm. A
film sample was hold between two clips and the distance between
these two grips was set as 50 mm. Five parallel tests were made for
each sample.
2.4.4. Thickness and swelling ratio
Thickness of the dried and swollen films was determined using
a micrometer (Shanghai Tool Works Co., Ltd., Shanghai, China).
Measurements were made in 6 random locations on each film.
The hydrogel water absorptivity could be described as swelling
ratio. At room temperature, the weighed dry specimens (Wswollen )
(2 cm × 2 cm) were immersed in PBS (0.05 M) for 1 h. Wipe off sur-
face water, weight again (Wdry ). The swelling ratio is defined by the
following equation [33].
swelling ratio = (Wswollen − Wdry ) × 100/ Wdry
2.4.5. Water vapor transmission rate (WVTR)
WVTR was determined by standard method E96-00 at 37 ◦ C and
a relative humidity of 85% [34]. The device used for measurement
of water vapor transmission rate (WVTR) is composed of a desicca-
tor, a number of penicillin bottles (17 mm diameter), an incubator
(37 ◦ C), and an electronic balance. The dry specimens were cut into
rounded shape, mounted on the mouth of bottles (17 mm diameter)
containing 10 mL water, and kept in a desiccator with a saturated
solution of ammonium sulfate at 37 ◦ C for 24 h. The vials were
weighted after 24 h, and WVTR (g/m2 /day) is defined by the fol-
lowing equation [35]:
WVTR = (W0 − Wf )/106 A
where A is area of the bottle mouth (mm2 ); W0 and Wf are the
weights in g of the device before and after being kept at 37 ◦ C for
24 h, respectively.
2.4.6. Oxygen permeability
Briefly, 200 mL water was added to a 250 mL flask, then the flask
mouth were mounted by the hydrogel films, and sealed tight to test
the oxygen penetration through them. After 24 h in an open envi-
ronment, the dissolved oxygen in water was analyzed by Winkler’s
method [36]. Same trail with an open flask which allow air in and
out freely was used as positive control, while flask with an airtight
plastic lid as the negative control.
2.4.7. In vitro cytotoxicity assay
In vitro cytotoxicity assay of HPMCS-CS hydrogel films against
NIH3T3 fibroblast cells was measured by MTT assay [37]. In brief,
NIH3T3 fibroblast cells were seeded into a 96-well plate at density
of 1 × 104 cells/well in DMEM supplemented with 10% FBS, peni-
cillin (100 U/mL), and streptomycin (100 ␮g/mL), and incubated for
24 h in 5% CO2 at 37 ◦ C. To get the leach liquor, the hydrogel films
were immersed in DMEM culture media overnight. The leach liquor
was added to 96-well plate culturing NIH3T3 cells. After 1, 3, 5 days,
the cell viabilities were evaluated by MTT assay.
2.4.8. Hemolytic evaluation
The hemolytic tests were performed by a method from Amer-
ican Society for Testing and Materials [38]. The hydrogel films
(2 cm × 2 cm) were first immersed in 7 mL of PBS, and kept at 37 ◦ C
for 24 h. Then 1 mL of diluted acid-citrate-dextrose (ACD) blood
was added and incubated at 37 ◦ C for another 3 h. Negative and
87
positive controls were prepared by adding blood to 7 mL of PBS
and water, respectively, and also incubated at 37 ◦ C for 3 h. After
incubation, the fluids were collected and centrifuged at 8000 rpm
for 15 min. Optical density (OD) of the supernatants was measured
at 540 nm. The percentage of hemolysis was calculated according
to the following formula.
Hemolysis(%) = (ODs − OD(−) )/(OD(+) − OD(−) ) × 100
where ODs, OD(−) and OD(+)is the optical density of the sample,
negative control and positive control, respectively.
2.4.9. Antibiotic-release testing
Gentamycin sulfate was used as a model antibacterial drug to
evaluate the antibiotic loading and in vitro release of the hydrogel
films [39]. To load drug, a dried film (6 g) was immersed in 50 mL
of gentamicin sulfate solution (30 mg/mL) for 24 h, then dried in a
vacuum oven. So we supposed all drug were loaded in films. Under
the current experimental condition, the drug loading efficiency
was 20%, and encapsulation efficiency was 100%. The gentamycin-
loaded film was then immersed in 500 mL of PBS (pH = 7.4) under
constant stirring at 37 ◦ C. 600 ␮L aliquot samples were taken at
specific time points, and gentamycin concentration in it was mea-
sured by UV spectrophotometer at 355 nm. Equal amount of fresh
PBS was added after sampling each time.
2.4.10. Bactericidal properties
Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC
25922) were used for measurement of bactericidal properties. The
two strains were cultured for 24 h, and then diluted to 1 × 106
CFUs/mL [40]. Free and drug-loaded hydrogel films (500 U/cm2 )
were cut to round pieces (d = 6 mm), and placed in 24-well plate,
respectively. Then, 1 mL of bacterial suspension per well was added
and incubated at 37 ◦ C. A MTT proliferation assay kit was used to
measure the absorption at 6, 12, and 24 h [41].
2.5. Statistical analysis
All tests were performed at least three times and data were
presented as mean ± standard deviation. Statistics were conducted
using the analysis of Variance (ANOVA) procedure with SAS soft-
ware (Version 9.1, Statistical Analysis System Inc., Cary, NC, USA).
Differences were considered statistically significant at p < 0.05 by
two-tailed unpaired t-test.
3. Results and discussion
3.1. Synthesis of HPMCS
HPMC was modified with carboxylic acid groups to form HPMCS.
The synthetic pathway of HPMCS is shown in Fig. 1. The chemi-
cal structure of the starting materials and product was confirmed
by FTIR spectroscopy, as shown in Fig. 2A. The peak attributed to
HPMC was observed at 3470 cm−1 , which corresponded to OH
bond stretching [42]. In the spectra of HPMCS, the characteristic
peaks of carboxyl groups were obviously observed at 1742 cm−1
while there were no peaks at the same location in spectra of HPMC,
which confirmed the grafting of succinic anhydride onto HPMC
backbone.
3.2. Preparation and characterization of the crosslinked
HPMCS-CS hydrogel films
HPMCS and chitosan could be homogeneously mixed, and
form stable aqueous solution with certain viscosity. HPMCS was
88 Q. Jiang et al. / International Journal of Biological Macromolecules 91 (2016) 85–91
Fig. 2. (A) FTIR spectra of HPMC and HPMCS; (B) HPMCS, neat chitosan (CS), crosslinked HPMCS-CS hydrogel, and blend HPMCS-CS films (HC-0); (C) X-ray diffraction patterns
of HPMCS, CS, crosslinked HPMCS-CS hydrogel films.
subsequently crosslinked with chitosan to give HPMCS-CS hydro-
gels. Fig. 2B showed FTIR spectra of HPMCS, neat chitosan (CS),
crosslinked HPMCS-CS hydrogel films, and blend HPMCS-CS hydro-
gel film (HC-0). The characteristic peak of CS were observed at
1590 cm-1, which corresponding to NH bending [43]. The peak at
1740 cm-1 were observed from the spectra of HPMCS, which corre-
sponding to CO stretching. These peaks (illustrated as dotted line)
observed in HPMCS and CS, respectively, did not exist in crosslinked
hydrogel films, but were observed in spectra of blend film (HC-
0). This indicated that free amines of CS and carboxy group of
HPMCS were consumed during the crosslinking reaction. Further-
more, the peaks at 1640 cm−1 and 1560 cm−1 were assigned to the
amide linkage of the crosslinked hydrogel films, which indicated
that crosslinked HPMCS-CS hydrogels were successfully prepared.
3.3. The X-ray diffraction
The XRD patterns of HPMCS, CS, and crosslinked HPMCS-CS
hydrogel films were shown in Fig. 2C. On account of the generally
amorphous state of HPMCS and CS films, all of films exhibited one
broad peak at 10◦ . Furthermore, the diffraction angles of HPMCS
exhibited at 19.5◦ , while CS showed a broad peak at 22◦ . The XRD
patterns of crosslinked HPMCS-CS hydrogel films showed that they
had the same peak position with HPMCS and CS, but the diffraction
intensity became stronger with the higher and sharper peak pat-
tern. This implied a slightly higher crystallinity of the crosslinked
HPMCS-CS hydrogel films [44].
3.4. Mechanical properties
The tensile strength and elongation at break of HPMCS, CS,
crosslinked HPMCS-CS hydrogel, and blend HPMCS-CS film (HC-
0) were investigated, and the results were shown in Table 1.
Tensile strength represented to the maximum tension that a mate-
rial can withstand without tearing, while elongation at break
was used to evaluate the malleability of materials [45]. In dry
state, crosslinked HPMCS-CS hydrogel films have greater tensile
strength than HPMCS, and a more outstanding malleability than
pure HPMCS and CS films. These results were probably attributed
to the crosslinked network and disrupted crystallinity of CS hydro-
gel films. Furthermore, compared to those of blend films, the tensile
strength and elongation at break of crosslinked HPMCS-CS hydrogel
films were obviously improved. In swollen states, the mechanical
property of HPMCS could not be measured, because HPMCS film
was huddled as soon as it immersed in PBS solution, and the ten-
sile strength of pure CS decreased to merely 3.15 ± 0.36 MPa, while
the maximum tensile strength of crosslinked PVA-COOH/CS films
reached up to 7.84 ± 1.30 MPa (HC-2). This suggested that the addi-
tion of HPMCS and crosslinking with CS could effectively strengthen
the composites, especially in swollen states.
3.5. Thickness and swelling ratio
The photographs of the dried and swollen films were presented
in Fig. 1S (Supporting Information). The thickness increased sub-
stantially as the dried films became swollen, and all crosslinked
films possessed a higher thickness compared with neat CS film
 Q. Jiang et al. / International Journal of Biological Macromolecules 91 (2016) 85–91 89

Table 1
The mechanical properties of HPMCS, CS, crosslinked HPMCS-CS, and blend HPMCS-CS films. Data are presented as the mean ± SD (n = 6, p < 0.05).

Sample Dry state Swollen state

Tensile strength [MPa] Elongation at break [%] Tensile strength [MPa] Elongation at break [%]

HPMCS 12.97 ± 1.60 13.31 ± 0.47 – –

CS 76.49 ± 3.08 5.59 ± 0.47 3.15 ± 0.36 50.92 ± 7.57
HC-0 36.05 ± 2.13 20.92 ± 1.53 1.28 ± 0.43 38.42 ± 3.65
HC-1 52.22 ± 2.06 27.94 ± 1.91 1.99 ± 0.26 46.91 ± 0.81
HC-2 51.06 ± 1.30 25.04 ± 1.45 7.84 ± 1.30 100.69 ± 9.53
HC-3 51.86 ± 2.47 24.35 ± 1.07 6.67 ± 1.30 82.65 ± 10.14

100 B 500
A 12h 24h 36h 48h 60h
90 450
80 400

Water vapor loss (mg)

70 350
Swelling ratio(%)

60 300
50 250
40 200
CS
30 150
HC-0
20 HC-1 100
HC-2
10 HC-3 50
0 0
0 5 10 15 20 25 blank HC-1 HC-2 HC-3
Time(h)

C 12.5 D 150
Oxygen permeability(mg/mL)

0.6% phenol CS HC-1 HC-2 HC-3

12.0
Cell Viability(%)

100
11.5

11.0
50

10.5

10.0 0
negative control HC-1 HC-2 HC-3 positive control 1 day 3 day 5 day

Fig. 3. (A) Swelling ratio of CS, crosslinked HPMCS-CS hydrogel, and blend HPMCS-CS films (HC-0) at a function of the incubation times; (B) Water vapor transmission loss
from crosslinked HPMCS-CS hydrogel films at 12, 24, 36, 48 and 60 h intervals; (C) Oxygen permeability of the crosslinked HPMCS-CS hydrogel films; (D) Cell viability of
NIH3T3 fibroblasts cultured with extract of crosslinked HPMCS-CS hydrogel and pure CS films for 1, 3, 5 day. Data are presented as the mean ± SD (n = 6, p < 0.05).

(Supporting Information, Table S1). The swelling ratio of CS, was 1100.88 g/m2 /day. Therefore, as a wound dressing material,
crosslinked HPMCS-CS, and blended HPMCS-CS films (HC-0) were HC-1, HC-2, and HC-3 could reduce the evaporative water loss by
presented in Fig. 3A. The swelling ratio of blended film was 35.64%, 21.29% and 20.48% to yield a moist environment.
increased rapidly within two hours and reached the maximum
value, then began to reduce rapidly to a relatively low level. All 3.7. Oxygen permeability
crosslinked films possessed a higher swelling ratio compared with
neat CS film. Moreover, the swelling ratio of crosslinked films The results of oxygen permeability across crosslinked HPMCS-
increased gradually with the increasing amount of CS, and the CS hydrogel films were shown in Fig. 3C. In present work, the
equilibrium swelling ratio were in the range of 75% to 83%. These dissolved oxygen in water of negative control and positive con-
phenomena were probably attributed to the increased molecu- trol was 10.96 and 12.09 mg/L, respectively. Meanwhile, the oxygen
lar spacing and weakened hydrogen bonding resulted from the concentration in water of flasks covered with HC-1, HC-2, and HC-
crosslinking reaction between HPMCS and chitosan in crosslinked 3 films were 11.32, 11.60, 11.83 mg/L. The results indicated that
films [46]. crosslinked HPMCS-CS hydrogel films allow a certain amount of
oxygen to penetrate through, which is suitable for cell regeneration,
3.6. Water vapor permeability and helpful to accelerate the healing process.

The water vapor loss of crosslinked HPMCS-CS hydrogel films 3.8. In vitro cytotoxicity assay
were presented in Fig. 3B. The WVTRs of HC-1, HC-2, and HC-3
were 708.70, 866.51 and 875.45 g/m2 /day, respectively. Mean- In vitro cytotoxicity of crosslinked HPMCS-CS hydrogel films
while, WVTR of blank sample obtained from a water-free surface were evaluated by examining the proliferation of NIH3T3 fibrob-
90 Q. Jiang et al. / International Journal of Biological Macromolecules 91 (2016) 85–91

100
A 100 B
90
80

Cumulative Release(%)
80
70
Hemolysis rate(%)

60
60
50
40 40
CS
30 HC-0
HC-1
20 20 HC-2
HC-3
10
0
0
HPMCS chitosan HC-1 HC-2 HC-3 control 0 5 10 15 20 25 30 35 40 45 50
Time(h)

C 3.0 D 3.5

2.5 Control 3.0 Control

Hydrogel Hydrogel
Drug-hydrogel 2.5 Drug-hydrogel
2.0
Relative OD

Relative OD
2.0
1.5
1.5
1.0
1.0

0.5
0.5

0.0 0.0
6h 12h 24h 6h 12h 24h

Fig. 4. Hemolytic potential of crosslinked HPMCS-CS hydrogel and their parent polymer films (A); The accumulated amount of gentamycin sulfate released from CS, crosslinked
HPMCS-CS hydrogel, and blend HPMCS-CS films (B); Bactericidal test against Staphylococcus aureus(C) and Escherichia coli (D); n = 6, p < 0.05.

lasts in the extract of sterile hydrogel. Cytotoxic effects caused by
the extract of crosslinked HPMCS-CS hydrogels and their parent
polymers on NIH3T3 fibroblasts were assessed by a MTT assay [47],
as shown in Fig. 3D. Cell viability of crosslinked HPMCS-CS hydro-
gel and neat CS films were more than 92% after 1, 3, and 5 days
culture. This result indicated that crosslinked HPMCS-CS hydrogels
and their parent polymers were non-toxic to NIH3T3 fibroblasts.
3.9. Hemolytic potential
It is imperative that new biomaterials that directly contact with
wound should have excellent biocompatibility. Hemolysis is the
rupturing of erythrocytes and the release of their contents into
plasma. The hemolytic potential of crosslinked HPMCS-CS hydrogel
films and parent polymers were investigated, and the results were
presented in Fig. 4A. Hemolytic percentage of HC-1, HC-2, and HC-3
had been observed as 1.8%, 1.5%, and 1.6%, respectively. Compared
to control group, crosslinked HPMCS-CS hydrogel films and their
parent polymers shown very low hemolysis rates. According to
the requirements of International Organization for Standardization
(ISO) 10993-4, the hemolytic percentage of non-hemolytic mate-
rial should be below 2%. So the result indicated that crosslinked
HPMCS-CS hydrogel films were blood-compatible materials.
3.10. In vitro drug-release
Wound dressing is actually a drug release system by releas-
ing a drug for a prolonged period of time. Gentamycin sulfate was
first absorbed by hydrogels, and then the drug released was inves-
tigated. As shown in Fig. 4B, the drug released very fast in the
beginning. About 50% of gentamycin sulfate was released in solu-
tion after 6 h, which coincided with the swelling time. Then the drug
release rate reduced gradually, and equilibrium reached at about
24 h. The cumulative release of gentamycin sulfate from hydrogel
films was higher than that of neat CS and blend film. These results
indicated that more drugs could be absorbed by crosslinked hydro-
gel films, which proven the potential to be used as a wound dressing
as well as a carrier or drug delivery system.
3.11. Bactericidal properties
Fig. 4C and Fig. 4D presented results of bactericidal proper-
ties of gentamycin sulfate-loaded hydrogel film (HC-2) against
S. aureus and E. coli, respectively. Obviously, the growth of both
strains cultured with gentamycin sulfate-impregnated hydrogels
was obviously restrained, while both strains grew normally with-
out drug. Interestingly, growth of these bacteria cultured with
hydrogel films without drug were also somewhat slower than con-
trol, which indicated that HPMCS-CS hydrogel films had certain
antibacterial performance. This result may be associated with the
ammonium ion on CS. Furthermore, the bioactivity of antibiotics
(gentamycin sulfate) in hydrogel films containing antibiotics had
not been affected, and could suppress the bacterial proliferation
effectively.
4. Conclusions
A series of novel carboxyl-modified hypromellose-crosslinked
chitosan hydrogel films (HPMCS-CS) were successfully prepared.
Addition of HPMCS substantially increases the swelling ratio and
mechanical properties in swollen state of chitosan films, which
might provide excellent ability for its use as a potential wound
dressing. The moderate WVTR and oxygen permeability might help
to keep a moist wound environment, and accelerate tissue regener-
 Q. Jiang et al. / International Journal of Biological Macromolecules 91 (2016) 85–91
ation. The crosslinked hydrogel films could absorb more drugs than
their parent polymers, and undergo sustained drug release. The
antibacterial experiment indicated that gentamycin sulfate-loaded
crosslinked hydrogel films could effectively suppress growth of
both S. aureus and E. coli. These studies revealed that the crosslinked
HPMCS-CS hydrogel films might be used as potential wound dress-
ing materials.
Acknowledgments
This work is financially supported by the Chinese Program for
New Century Excellent Talents in Universities (No. NCET-11-0661),
the National Natural Science Foundation of China (No. 21174054,
21004030), the Scientific Research Foundation for Returned Schol-
ars from Ministry of Education of China, the Natural Science
Foundation of Anhui Province of China (No. 1408085MB26), and the
Academic and Technology Introduction Project of Anhui University
of China (AU02303203).
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.ijbiomac.2016.
05.077.
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