International Journal of Biological Macromolecules 93 (2016) 1295–1303

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International Journal of Biological Macromolecules

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

Preparation and characterization of N-chitosan as a wound healing

accelerator
Fengling Tang a,b,1 , Lingmei Lv a,b,1 , Fei Lu a,b , Bao Rong c , Zhiquan Li c , Bitao Lu a , Kun Yu a ,
Jiawei Liu a , Fangying Dai a,b , Dayang Wu a,b , Guangqian Lan a,b,∗
a
College of Textile and Garments, Southwest University, Chongqing 400715, China
b
Chongqing Engineering Research Center of Biomaterial Fiber and Modern Textile, Chongqing 400715, China
c
The Ninth People’s Hospital of Chongqing, Chongqing 400715, China

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

Article history: Chitosan is insoluble in water due to its rigid crystalline structure, which has significantly restricted its
Received 13 July 2016 application in wound healing. The objective of this study was to synthesize a water-soluble chitosan
Received in revised form derivative, N-succinyl-chitosan (NSC), and evaluate its ability to accelerate the wound healing process.
11 September 2016
NSC was synthesized with succinic anhydride, hydrochloric acid, and alkaline chitosan under optimized
Accepted 29 September 2016
conditions, and characterized using Fourier transform infrared, proton nuclear magnetic resonance, and
Available online 30 September 2016
X-ray diffraction spectroscopy; thermal gravimetric analysis; and a solubility test. The cytotoxicity of NSC
was investigated in L929 cells, and its antibacterial activity was evaluated by the inhibition zone method
Keywords:
Wound healing
and bacterial growth curves analysis. The results showed that the solubility of NSC was substantially
N-Succinyl-chitosan improved compared to chitosan, and NSC was non-toxic with good antibacterial properties. An animal
Antibacterial activity wound healing test indicated that NSC could significantly reduce the healing time compared to chitosan.
Histopathological examination suggested that the underlying mechanisms of these effects were related
to NSC’s ability to promote the formation of granulation tissue and enhance epithelialization. Collectively,
these results demonstrate the good potential for NSC to be applied as a wound dressing material.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction Chitosan (CS) is an alkaline deacetylated product of the abun-

Skin wounds constitute a common health problem worldwide,
and a large number of patients suffer from acute trauma every
year. Traditionally, the normal wound healing process has been
divided into four connected but overlapping phases: hemostasis,
inflammation, proliferation, and remodeling [1]. Each biological
phase is crucial to wound healing. Therefore, suitable healing mate-
rials are required to facilitate the biological healing process for
providing a good and moist healing environment to help develop
resistance against infections and reduce inflammation [2,3]. Cur-
rently, the majority of wound healing materials are antibiotics, and
skin loss remains a significant challenge to clinicians. With the rise
in interest in combatting health problems, improved natural mate-
rials showing compatibility with the human body and biological
processes should be investigated.
∗ Corresponding author at: College of Textile and Garments, Southwest University,
Chongqing 400715, China.
E-mail addresses: 30353930@qq.com, j070218@swu.edu.cn (G. Lan).
1
These authors contributed equally to this work.
dant biopolymer chitin, which is composed of a linear chain of
␤-1,4-linked glucosamine [4]. As a cationic polysaccharide, CS has
many excellent properties such as biodegradability, biocompatibil-
ity, non-toxicity, antimicrobial activity, hemostatic properties, and
analgesic activities [5–8]. These advantages have therefore high-
lighted the potential of CS for applications in the wound healing
field [9,10]. However, the structure of CS results in its poor water
solubility, so that when used as a wound healing material, CS must
first be dissolved in a diluted acid, which may cause harm to the
human body. This drawback has thus far greatly limited its appli-
cation [11]. Therefore, preparation of a water-soluble CS derivative
has become a new research direction.
To best enhance its solubility and expand the potential applica-
tions of CS, hydrophilic groups are usually introduced by chemical
modification [12]. A variety of water-soluble CS derivatives have
been synthesized in the past few decades, such as O-carboxymethyl
CS [13], N-(2-carboxyethyl)-CS [14], quaternized CS [15], and PEGy-
lated CS [16]. We here focused on the development of N-succinyl
chitosan (NSC), which has recently been reported as a novel drug
delivery carrier [17,18] and adsorbent [19,20]. NSC is obtained
by introducing succinyl groups into the glucosamine units of the

http://dx.doi.org/10.1016/j.ijbiomac.2016.09.101
0141-8130/© 2016 Elsevier B.V. All rights reserved.
1296 F. Tang et al. / International Journal of Biological Macromolecules 93 (2016) 1295–1303
CS N-terminal. The introduction of carboxyl groups increases the
water solubility of CS, and also improves the hygroscopicity and
humidity-retaining ability [21,22]. NSC is also reported to have
good adhesion [23] and other favorable biochemical properties
[24]. Based on the advantages listed above, we speculated that
NSC would effectively absorb the wound exudates to provide a
moist environment, and serve as a propitious material to accelerate
wound healing.
The traditional synthesis route of NSC involves use of a dimethyl
sulfoxide and acetone system [25–27]. However, this method
requires many toxic organic reagents, which may be harmful to the
human body if they cannot be completely eliminated from the final
product. Therefore, we developed a new simple and safe method
for NSC powder synthesis, using succinic anhydride and hydrochlo-
ric acid in an ethanol solution after alkaline treatment. The alkaline
treatment was applied to increase the activity of NH2 , thereby
facilitating the substitution.
The aim of the present study was to examine the effects of
NSC on wound healing and to develop a more efficient and con-
venient material for application in the field of clinical trauma.
The physicochemical characterizations, including Fourier trans-
form infrared (FTIR), nuclear magnetic resonance (NMR), and X-ray
diffraction (XRD) spectroscopy, as well as thermal characteristics
were detected. To accurately evaluate the feasibility of NSC powder
as a wound healing material, we tested its solubility, antimicrobial
properties, cytotoxicity, and rate of wound healing in an animal
model.
2. Materials and methods
2.1. Materials
The CS (MW, 100,000 Da) was purchased from Chengdu Kelong
Chemical Reagent (Chengdu, China). According to the litera-
ture [28,29], we measured the degree of deacetylation at 92.1%.
Succinic anhydride was purchased from Chongqing ChuanDong
Chemical Co., Ltd. (Chongqing, China). Staphylococcus aureus and
Escherichia coli were provided by Chongqing Science University,
Medicine and Biological Laboratories (Chongqing, China). The
murine fibroblast cell line (ACTT, CCL-1) L929 was obtained from
the Third Military Medical University, Chongqing, China. New
Zealand rabbits (20 weeks of age) were purchased from the Ani-
mal Laboratory Center of the Third Military Medical University. All
animal experiments and care were approved by the National Cen-
ter of Animal Science Experimental Teaching (ASET) at the College
of Animal Science and Technology (CAST) of Southwest University
of China, and were performed in accordance with the “Guide for the
Care and Use of Laboratory Animals” (NIH Publications No. 8023,
revised 1978). All other reagents used in this experiment were of
analytical grade.
2.2. Preparation of NSC
NSC was synthesized according to a previously reported method
[23–25] with some modifications (Fig. 1A). First, 2.0 g of CS powder
was added to 20 mL of 30% (v/v) sodium hydroxide solution, and
then 8 mL of isopropanol was added. The solution was mixed com-
pletely, and the mixture was frozen at −20 ◦ C for 10 h. When the
temperature reached room temperature, the mixture was washed
with deionized water, the pH value was adjusted to 7 with NaOH
solution (1 M), and it was vacuum-filtered. Next, 10 g of succinic
anhydride was dissolved under stirring in 200 mL of 90% (v/v)
ethanol solution, and 0.1 g of the treated CS followed by hydrochlo-
ric acid were added to the solution with stirring. The solution was
left to react for 5 h at 80 ◦ C. Finally, the precipitate was filtered to
remove the solvent, which was washed repeatedly with ethanol
and dried.
2.3. Characterization
2.3.1. FTIR
FTIR measurements were performed on the IRPrestige-21
infrared spectrum instrument (Shimadzu, Japan). The FTIR spec-
tra of CS and NSC were obtained by recording 20 scans between
4000 and 400 cm−1 at room temperature.
2.3.2. 1 H NMR spectroscopy
1 H NMR spectra were measured on the AVANCE III 400 MHz
Superconducting Fourier NMR spectrometer (Bruker, Switzerland)
at room temperature, using CD3 COOD in D2 O (2 wt%) as the solvent
to dissolve all samples. The degree of substitution of NSC was eval-
uated by calculating the relative integral values of the protons in
the 1 H NMR spectrum.
2.3.3. XRD spectrometry
The XRD spectra were obtained with the D8 Davanci X-ray
diffractometer (Bruker, Switzerland) with Cu K␣ radiation applied
in the range of 5–70◦ (2␪) at 40 kV and 40 mA.
2.3.4. Thermal gravimetric analysis (TGA)
TGA has been widely used to study the thermal stability and
characteristics of the thermal decomposition of polymers. In this
study, TGA was carried out using the TG 209 F3 Tarsus thermogravi-
metric analyzer (NETZSCH, Germany) in the temperature range of
40–800 ◦ C at a heating rate of 10 ◦ C/min in nitrogen.
2.3.5. Solubility test
To evaluate the solubility of NSC and CS at different pH values,
each sample (100 mg) was dispersed in 20 mL H2 O. The pH of the
solution was adjusted with 1.0 mol/L HCl and NaOH. The transmit-
tance of each solution was measured at 600 nm using an ultraviolet
(UV)–visible spectrophotometer (UV-2550, HITACHI, Japan). The
sample was considered to be insoluble when the transmittance of
the polymer solution was less than 50% of the transmittance for
aqueous media at different pH [30].
The maximum solubility of NSC and CS in deionized water was
quantitatively estimated by dispersing NSC and CS in deionized
water, followed by shaking at 37 ◦ C for 24 h to ensure that the solu-
tions were basically saturated with the dissolved samples. Finally,
the mixtures were filtered through a 0.45-mm filter paper. Solubil-
ity was estimated from the change in filter paper weight [31].
2.4. Cytotoxicity assay in vitro
The cytotoxicity of NSC was evaluated by the 3-(4,5-
dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide blue
(MTT; an indicator dye) method using L929 cells. First, L929 cells
were cultured in Dulbecco’s modified Eagle medium supplemented
with 1% antimycotic solution and 1.2% glutamine at 37 ◦ C in atmo-
spheric humidity (5% CO2 , 95% air). The NSC and CS powders were
UV-sterilized for 30 min, and 0.01 g NSC or CS was immersed in
5 mL of serum-containing medium (2 mg/mL) for 12 h at 37 ◦ C.
The samples were centrifuged at 550 × g for 5 min to obtain the
supernatant. The cells were seeded 1 × 104 cells/well in 96-well
plates and pre-incubated in atmospheric humidity (5% CO2 , 95%
air, 37 ◦ C). After 24 h, the cells were treated with the NSC and CS
supernatants, respectively; untreated cells were used as controls.
The plates were then incubated for 24, 48, or 72 h, and the MTT
assay was conducted. Ten microliters of MTT solution (5 mg/mL)
was added to each well and the plates were incubated for another
4 h. Then, the medium was removed, and the formazan crystals
 F. Tang et al. / International Journal of Biological Macromolecules 93 (2016) 1295–1303
Fig. 1. (A) Schematic illustration of the synthesis of NSC. (B) FTIR spectra of CS (a) and NSC (b). (C) 1 H NMR spectra of CS (a) and NSC (b).
formed in the living cells were solubilized in 100 ␮L dimethyl
sulfoxide. The absorbance of each well was measured at 490 nm
on a microplate reader (Multiskan MK3) and measurements were
replicated three times at each time point. The cell viability (%) of
the treated cells was calculated as follows:
A490(treated)
Cell viability (%) = × 100%
A490(untreated)
where A490(treated) is the absorbance value of CS or NSC, and
A490(untreated ) is the absorbance of the control.
2.5. Antimicrobial activity
The in vitro antimicrobial activities of NSC and CS were evalu-
ated by the inhibition zone method and evaluation of the bacterial
growth curves. For this purpose, the gram-positive bacterium S.
aureus and the gram-negative bacterium E. coli were used. E. coli
and S. aureus (approximately 1 × 107 –108 colony forming units/mL)
were cultured in the sterilized nutrient broth for these tests.
2.5.1. Inhibition zone method
An inhibition zone refers to the sterile field formed on a solid
medium as a result of killing or preventing the growth of bacteria.
The inhibition zone method is one of the most common qualitative
methods to detect the antibacterial activity of an organic antibac-
terial agent, and the size of the inhibition zone can be used to
characterize whether or not an antibacterial effect is remarkable.
CS and NSC powders (0.01 g) were UV-sterilized for 30 min, and
then added to 10 mL deionized water, respectively. Nutrient agar
medium was sterilized by autoclaving. The bacterial suspensions
were diluted and spread over the agar plates evenly. Sterilized fil-
ter papers (1 cm in diameter) were soaked in the NSC solution, CS
solution, or deionized water for 5 min, and placed on the center
of the agar plates, respectively, which were incubated for 24 h at
1297
37 ◦ C. The antimicrobial effects of the NSC, CS, and acid solution
were qualitatively evaluated through observing and comparing the
size of the inhibition zones formed on the bacterial agar plates.
2.5.2. Bacterial growth curves
According to the definition of absorbance, the absorbance value
is proportional to the concentration of the light-absorbing sub-
stance in solution. At a certain range, the concentration of the
bacterial suspension solution is inversely proportional to transmit-
tance and is proportional to the optical density (OD) value. Thus,
the OD value was used to represent the bacteria concentration of
the sample solution in this study. NSC solutions with a series of con-
centrations were prepared in beef extract peptone liquid medium,
and 1 mL of each of these solutions was added into aseptic test
tubes. Then, 100 ␮L of the pre-prepared bacterial (E. coli or S. aureus)
suspension solution, diluted 100 times, and nutrient broth were
added to set the final concentrations of NSC to 0.5, 1, 2, 4, 6, and
8 mg/mL. Non-treated liquid medium with bacteria was used as a
control. These mixtures were oscillated at a constant temperature
of 37 ◦ C. At different incubation times (0, 6, 12, 24, and 48 h), the
OD value of each mixture was read at ␭ = 610 nm [32,33]. All mea-
surements were repeated three times. The bacteria inhibition ratio
of NSC at various concentrations was calculated after 48 h using the
following equation [34]:
At − A0
Inhibition ratio (%) = 100 − 100 ×
Actrl − A0
Where A0 is the OD value of the bacterial broth medium before
incubation, and At and Actrl are the OD values of NSC and the control
group at different incubation time points, respectively.
1298 F. Tang et al. / International Journal of Biological Macromolecules 93 (2016) 1295–1303
2.6. Wound healing assay
A wound healing assay was carried out using a full-
thickness wound model in rabbits. The New Zealand rabbits were
anaesthetized using xylazine hydrochloride injection (0.2 mL/kg).
Subsequently, the fur on the dorsal side of the rabbits was shaved,
and the skin of the anaesthetized rabbits was lightly swabbed with
alcohol and air-dried. On each rabbit, three full-thickness skin exci-
sion wounds of 3 cm in diameter were created on the dorsum. The
first wound was the control and was not treated, the second wound
was sprinkled with 1 g of NSC powder, and the third wound was
sprinkled with 1 g of CS powder. After surgery, each rabbit was
housed in a single cage at room temperature and the conditions
of the wound surface on the dorsum of the rabbits were recorded
daily. The wounds were photographed at 3, 7, 10, and 14 days after
the surgery. All images were adjusted to be the same size and res-
olution, and the pixel method was used to measure the wound
closure rate. For each of the days above, the extent of wound closure
was calculated according to the following equation:
P0 − Pt
Wound closure (%) = × 100%
P0
where P0 is the pixel value of the original wound area, and Pt is the
pixel value of the wound area at day t.
On days 3, 7, 10, and 14 after treatment, the rabbits were euth-
anized. At each time point, tissue specimens were collected for
histological analysis. The excised tissues were preserved in 10%
buffered formalin for 24 h, and embedded in paraffin. All sam-
ples were vertically sectioned (5-mm thickness) and stained with
hematoxylin and eosin. These sections were observed under a DXM
1200F microscope (Nikon H600L, Germany) and analyzed.
2.7. Statistical analysis
All data are expressed as mean ± standard deviation (SD). Dif-
ferences between means were analyzed for statistical significance
using the Student’s t-test or one-way analysis of variance (ANOVA).
P-values less than 0.05 indicated statistical significance.
3. Results and discussion
3.1. Characterization of NSC and CS
3.1.1. FTIR spectroscopy
Fig. 1B shows the FTIR spectra of CS (a) and NSC (b), indicat-
ing a marked difference between them. In the FTIR spectrum of CS,
the characteristic absorption bands appeared at 3300–3500 cm−1
(stretching vibration of the OH and NH2 groups), 1660 cm−1
(Amide I), 1600 cm−1 (–NH2 bending), and 1385 cm−1 (Amide III).
The absorption bands at 1156 cm−1 (asymmetric stretching of the
C O C bridge), and at 1082 and 1013 cm−1 (skeletal vibration
involving the C O stretching) are characteristics of the sugar struc-
ture [35,36]. Compared with the FTIR spectrum of CS, the absorption
bands of the stretching vibrations of OH and NH (3442 cm−1 )
of NSC became narrower and shifted to a lower wave number.
No absorption bands appeared at 1720–1750 cm−1 . The peaks at
1024 cm−1 (primary hydroxyl group) and at 1071 cm−1 (secondary
hydroxyl group) showed certain changes after the treatment. The
peaks at 1244 cm−1 and 1317 cm−1 for OH groups showed only
a slight shift. These results indicated that the succinyl derivation
reaction took place at the N-position [27,37]. Furthermore, new
absorption bands could be observed at 1416 cm−1 , corresponding
to symmetric stretching of the COO group, and at 1557 cm−1 (Amide
II). The peak at 1654 cm−1 (Amide I) increased and the peak at
1591 cm−1 (–NH2 bending) decreased greatly [38,39]. These results
could further suggest that the amino groups of CS were substituted,
and that NH CO groups were formed in NSC.
3.1.2. 1 H NMR spectroscopy
Fig. 1C shows the 1 H NMR spectra of CS (a) and NSC (b)
in CH3 COOD/D2 O. The native CS showed chemical shifts at
1.89–2.01 ppm, corresponding to protons of the residual acetyl
group (H-a). The peak at 3.07 ppm was attributed to the anomeric
protons of glucosamine (H-2), and the signals from 3.35 to 4.09 ppm
were assigned to the non-anomeric protons of the CS backbone
(H-1, 3, 4, 5, 6) [14,40–42]. Compared to that of CS, there were
large differences in the 1 H NMR spectrum of NSC, and two addi-
tional peaks were observed. The new chemical shifts from 2.40
to 2.71 ppm were attributed to methene protons of the succinyl
group ( COCH2 CH2 COOH , H-c), and the small signal at 4.50 ppm
belongs to the amide proton (-NH-COCH2 CH2 COOH, H-b). In addi-
tion, some other peaks (H-2, H-1, 3, 4, 5, 6) were slightly shifted
and the intensities changed as a result of the chemical grafting
[26,36,43,44]. These results confirmed the successful substitution
of the succinyl group onto the CS backbone. The 1 H NMR spec-
troscopy results were consistent with the FTIR results.
The determination of the degree of substitution (DS) of NSC was
calculated to be 67.30% using the following equation [45,46]:
Integrated area at 2.40 − 2.71 ppm
DS (%) = × 100
4 × Integrated area at 3.10 ppm
3.1.3. XRD spectrometry
Fig. 2A shows the XRD patterns of CS (a) and NSC (b). There were
two major characteristic peaks in the diffractogram of CS at 11.0◦
and 20.1◦ , which are the typical fingerprints of semi-crystalline CS.
The reflection decline at 2␪ = 11.0◦ was assigned to crystal form I,
and the strongest reflection appeared at 2␪ = 20.8◦ , corresponding
to crystal form II [47]. Because of the presence of several strong
intermolecular and intramolecular hydrogen bonds, CS forms crys-
talline regions easily [48]. In contrast, the XRD spectrum of NSC
showed only one broad peak at around 2␪ = 20.8◦ , and the peak
was obviously weakened. The crystal form I peak in the spectrum
had disappeared, suggesting that compared with CS, the capacity
of NSC to form intermolecular hydrogen bonds (H-bonds) might be
greatly decreased after the introduction of succinyl groups onto
the CS backbone [38]. Consequently, this phenomenon resulted
in destruction of the crystal forms in NSC macromolecules, and a
larger fraction of the amorphous phase formed. Furthermore, the
broad peak observed at around 2␪ = 20.8◦ indicated that there was
a small fraction of the crystalline phase in NSC remaining due to
the weak intramolecular H-bonds [30].
3.1.4. TGA
The TGA and derivative thermogravimetric (DTG) curves of CS
and NSC are displayed in Fig. 2 B and Fig. 2C, respectively, indicat-
ing two distinct stages of weight loss in the degradation of CS and
NSC. The first stage was mainly attributed to the loss of free water
and bound water in the polymers. The second stage of degradation
started at nearly 180 ◦ C for NSC and at 250 ◦ C for CS, which corre-
sponded to pyrogenic decomposition and the cleavage of the main
chain; the main weight loss occurred at this stage. The total weight
loss of CS was 62.56% up to a heating temperature of 792.55 ◦ C,
which was less than that of NSC, at 69.56%. In addition, the DTG
curves shown in Fig. 2C indicated that the maximum weight loss
rate temperature (310.02 ◦ C) of CS was slightly higher than that
(218.08 ◦ C) of NSC. These results demonstrate that the thermal sta-
bility of NSC is worse than that of CS, which is attributed to the lower
number of hydrogen bonds and the decrease in the crystallinity as
a result of the loaded succinyl groups.
 F. Tang et al. / International Journal of Biological Macromolecules 93 (2016) 1295–1303 1299

Fig. 2. (A) XRD spectra of CS (a) and NSC (b). (B) TGA thermograms of CS (a) and NSC (b). (C) DTG curves for CS (a) and NSC (b).

Table 1 3.3. Antimicrobial activity

Solubility of CS and NSC.

Sample Solubilitya Solubilityb (g/L) 3.3.1. Inhibition zone test

As shown in Fig. 4, no inhibition zone formed on the solid plates
pH: 2 4 6 8 10 12
of the control and CS groups. This is due to the fact that CS is insol-
CS 䊉 䊉 䊉 × × × 0
uble in water. By contrast, clear zones surrounding the NSC filter
NSC 䊉 䊉 䊉 䊉 䊉 䊉 30.49 ± 0.46
papers were observed. In addition, the size of the inhibition zone
a
Each sample (100 mg) was dispersed in 20 mL solvent, and the pH of the solution against E. coli was slightly larger than that formed against S. aureus.
was adjusted with 1.0 mol/L HCl and NaOH. 䊉, soluble; ×, insoluble.
b
Maximum solubility of the samples in deionized water.
This phenomenon indicated that the diffusion of antimicrobial sub-
stances from the NSC sample generated growth inhibition, and the
aqueous solution of NSC had superior antibacterial effects against
S. aureus and E. coli compared to CS.

3.1.5. Solubility test

The solubility of NSC and CS was assessed at various pH values.
As shown in Table 1, CS could only dissolve in the acidic medium.
By contrast, NSC could dissolve in neutral, acidic, and alkaline solu-
tions. These results indicate that after introducing the succinyl
group into the N-position of CS, the new product had better solubil-
ity over a much wider pH range. In addition, the saturated solubility
of NSC in deionized water was obtained (Table 1). Therefore, com-
pared to NSC, CS was essentially insoluble. As previously reported,
polymers with lower crystallinity tend to dissolve more easily
[49]. Because of the presence of several strong intermolecular and
intramolecular H-bonds, CS is known to be insoluble in water [50].
The succinylation of CS destroyed the CS structure regularity mak-
ing it amorphous, and the introduction of the hydrophilic COOH
group effectively improved the water solubility of CS, allowing it to
dissolve in acidic, neutral, and alkaline aqueous solutions.
3.3.2. Bacterial growth inhibition curves
As shown in Fig. 5A and B, when the concentration of NSC was
increased from 0.5 mg/L, 1 mg/L, to 2 mg/L, the OD values stabilized
after incubating for 24 h. When the concentration was increased
from 4 mg/L, 6 mg/L, to 8 mg/L, the OD value showed little change
after 12 h. The inhibition ratios of NSC after incubating for 48 h are
shown in Fig. 5C. Therefore, with an increase in NSC concentration,
the OD value decreased gradually, growth inhibition was enhanced,
and the duration of bacterial inhibition increased. Effective inhibi-
tion could be maintained for at least 48 h. Furthermore, comparison
of the OD values showed that NSC exhibits stronger growth inhibi-
tion against E. coli than against S. aureus, which is consistent with
the results of the inhibition zone test. These results revealed that
NSC has excellent and sustained bacteriostatic action.

3.2. Cytotoxicity assay in vitro

The cytotoxicity of CS and the NSC was investigated in vitro using

the L929 cell line with the MTT assay, and the absorbance was mea-
sured to evaluate cell viability. Fig. 3 shows the viability of L929
cells after 24 h, 48 h, and 72 h of incubation with CS or NSC. CS is
generally considered to be a safe polymer; indeed, the absorbance
values surpassed 80% of those of the non-treated control, which is
consistent with a previous report showing that CS had low cyto-
toxicity [51]. After 24 h, the cytotoxicity of CS was slightly higher
than that of NSC, and there were no significant differences among
the samples at 48 h. However, at the end of 72 h, the absorbance
of NSC was maintained at around 130%, which was significantly
higher than that of CS, showing the much better biocompatibility
of NSC. This phenomenon suggests that NSC has no significant cyto-
toxicity and has the potential to promote the proliferation of L929
cells to a certain extent, demonstrating good biocompatibility for Fig. 3. Cell proliferation after incubation with CS and NSC for 24 h, 48 h, and 72 h
the wound. **P < 0.01.
1300 F. Tang et al. / International Journal of Biological Macromolecules 93 (2016) 1295–1303

Fig. 4. Digital photographs of the inhibition zone formed in antibacterial testing of the untreated control (Ctl), CS, and NSC against S. aureus and E. coli.

Fig. 5. Bacterial growth curves; OD values at 610 nm of the control and different concentrations of NSC against (A) E. coli and (B) S. aureus. (C) Inhibition ratios of NSC after
incubating for 48 h.

Wound infection can greatly delay the time of wound heal- pathogens causing surgical wound infections. NSC showed an out-
ing; therefore, a wound healing material must also have excellent standing antimicrobial effect and durability of this effect against
antibacterial property. S. aureus and E. coli are the common S. aureus and E. coli. The antibacterial mechanism of CS is gener-
 F. Tang et al. / International Journal of Biological Macromolecules 93 (2016) 1295–1303 1301

ally considered to involve its positively charged amino groups in

an acidic condition, which allow CS to react with the negatively
charged cell wall of bacteria, thereby disturbing the structure or
changing the permeability of the cell membrane, causing leakage
of intracellular components leading to cell death [50–54,]. Because
most biochemical reactions need to be carried out in water, this
antibacterial property of natural CS is hindered since it cannot dis-
solve in water. The introduction of the hydrophilic carboxyl group
was here shown to improve the water solubility of CS, leading to
ionization of hydrogen ions when dissolved in water, which can
enhance the cations of the NH3+ group. Furthermore, the suc-
cessful substitution of the succinyl group led to a decrease of the
intermolecular H-bonds of CS, which in turn decreased the embed-
ding degree of the amino group, resulting in an increase of the
NH2 concentration. In addition, after chemical modification, NSC
contained many reactive functional groups, an amino group, car-
boxyl group, as well as a hydroxyl group. When dissolved in water,
these functional groups allow it to chelate with various metal ions
[55]. Therefore, NSC may inhibit the uptake of trace elements and
the combination of essential nutrients for bacterial growth, which Fig. 6. Wound closure ratios of the control (open triangles), CS (closed circles), and
improves the antibacterial property [56,57]. Moreover, NSC showed NSC (open diamonds) groups at different times.
greater growth inhibition against E. coli as compared to S. aureus,
which is attributed to the fact that S. aureus, as a gram-positive bac- 3.4. Wound healing assay
terium, has a thicker cell wall and is hence more resistant to NSC
than E. coli [58]. 3.4.1. Wound closure
To assess and compare the wound healing abilities of CS and NSC,
full-thickness wounds were created on the dorsum of each rabbit.

Fig. 7. The effect of NSC on wound healing. (A) Photographs of wounds treated with CS and NSC, or not treated (Ctrl) at day 0, 3, 7, 10, and 14 post-operation. (B) Representative
histopathological profiles of skin wounds of the control (Ctrl), CS, and NSC groups at day 0, 3, 7, 10, and 14.
1302 F. Tang et al. / International Journal of Biological Macromolecules 93 (2016) 1295–1303
Each wound was observed at 0, 3, 7, 10, and 14 days after surgery. As
shown in Fig. 6, the rate of macroscopic wound healing increased in
the following order: NSC > CS > control. The macroscopic observa-
tions of wound closure with each material are shown in Fig. 7A. On
day 0, the surfaces of wounds were covered with different samples.
On day 3, there was an obvious difference in the NSC group, in that
the wounds covered with NSC powder showed conspicuous con-
traction. On day 7, all wounds showed marked scab formation, and
the untreated wounds were still slightly red and swollen. Compared
with the control and CS groups, the NSC-treated wounds were sig-
nificantly reduced in size, with 80.22% wound closure (Fig. 6). After
day 10, all wounds were contracted from the edges and part of the
scab formation fell off. As compared to the CS and control groups,
the scab formation of the NSC group was substantially reduced, and
the wound area in the NSC group was significantly decreased with
almost complete wound closure. On day 14, some scab formation
could still be observed on the wound surface of the control group,
whereas the entire scab of the NSC group had fallen off and the
CS group had only minor scab formation. The wounds of the NSC
group became clean and pinkish in color, showing almost complete
healing.
Wound healing is a dynamic biological process, including the
phases of homeostasis, inflammation, proliferation, and remod-
eling. The process is often accompanied by inflammatory cell
migration, neovascularization, fibroblast proliferation, epithelial-
ization, and the production of extracellular matrix proteins [59].
Maintaining a moist wound environment is beneficial to the regen-
eration and epithelialization, which could accelerate the wound
healing process [60]. After the chemical modification, the molecular
entanglements of NSC greatly decreased and NSC contained many
active functional groups. The presence of the many hydrophilic car-
boxyl groups conferred better water retention property to NSC,
allowing it to create a moist environment in the wound bed [61–63].
Furthermore, intermolecular and intramolecular H-bonds in the
polymer were destroyed and more amino groups were exposed,
so that part of the NSC powders could dissolve and provide some
favorable antibacterial effects. In the early stage of wound forma-
tion, NSC powders could absorb and dissolve in the wound exudates
quickly, and then formed a layer of film to isolate the wound from
the outside world. This phenomenon suggests that NSC shows good
potential to provide a suitable moist and antimicrobial healing
environment. By contrast, CS cannot act directly on the wound and
impedes the formation of a scab due to its poor solubility. Indeed,
in the in vivo wound healing experiment, the NSC group showed a
better wound contraction rate and shorter healing time.
3.4.2. Histological examination
The histological results of the wounds at different days are
shown in Fig. 7B. Microscopic observations on day 3 showed little
difference between the groups, and scab formation was observed
on the epidermis of all groups. We noted some pink and homo-
geneous gelatiniform material in the NSC group, which was likely
caused by absorption of the NSC powder in the wound exudate
that formed a colloid on the wound surface. At day 7 after the oper-
ation, all wounds showed an increase in inflammatory cells and
granulation generation. The dermis of the NSC group showed more
abundant fibroblasts and better re-epithelialization and dermal re-
modeling compared to the CS and control wounds. At day 10 after
the operation, there was still substantial scar formation observed
on the wounds of the control and CS groups. In contrast, the dermis
of the NSC wounds showed very few inflammatory cells, and faster
proliferation of microvessels and collagen tissues in the granulation
tissues was observed. Moreover, the epithelium of the NSC-treated
wounds was more mature and showed substantial improvement
in wound healing. After 14 days, all scabs fell off and regenera-
tion of new epithelium was observed in all wounds. The increased
deposition of collagen and the stratum corneum were observed in
all wounds. In addition, compared to the control and CS groups,
the boundary layer between the epidermis and dermis was more
clear and orderly in the NSC group. Furthermore, the NSC-treated
wounds showed a more well-organized superficial epithelium and
were nearly completely repaired. The histological examination of
the NSC group showed that there were more fibroblasts, neovascu-
larization, and collagen tissues compared to the CS-treated wounds.
This phenomenon confirmed that NSC can accelerate the regener-
ation of new epithelium and promote wound healing.
4. Conclusion
In this study, a dissolvable CS derivative was successfully pre-
pared by reacting CS with succinic anhydride and hydrochloric acid
in alcohol. The chemical structures and physical properties of NSC
were characterized by FTIR, 1 H NMR, XRD, and TGA. The results
confirmed the successful modification of the amino group, and the
substitution degree of NSC was calculated to be 67.30%. The change
of the CS structure greatly improved the water solubility of NSC,
which was 30.49 ± 0.46 g/L. NSC showed excellent biocompatibility
and antibacterial properties. For E. coli and S. aureus, the inhibition
ratios of 8 mg/mL NSC after incubating for 48 h were 92.16 ± 0.82%
and 86.56 ± 0.31%, respectively. These results provide support for
applying NSC to dermal wound healing. In an experimental study
of rabbit skin wound healing, NSC was found to reduce bacterial
contamination, promote granulation tissue formation and epithe-
lialization, and significantly shorten the healing time compared to
CS. Collectively, these results indicate that NSC has great potential
to be used as a novel and desirable wound healing material. Addi-
tional studies are needed to further verify the clinical efficacy of
NSC.
Acknowledgements
This work was supported by National Undergraduate Training
Programs for Innovation and Entrepreneurship (201510635048).
This work was also funded by a Hi-Tech Research and Development
863 Program of China grant (No. 2013AA102507).
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