International Journal of Pharmaceutics 516 (2017) 214–224

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International Journal of Pharmaceutics

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

Co-delivery of zinc and 5-aminosalicylic acid from alginate/N-succinyl-

chitosan blend microspheres for synergistic therapy of colitis
Haogang Duana,b , Shaoyu Lüa , Hongyan Qinb , Chunmei Gaoa , Xiao Baia , Yuhui Weib ,
Xin’an Wub , Mingzhu Liua,* , Xinyu Zhangc,**, Zhen Liud
a
Department of Chemistry, Lanzhou University, Lanzhou 730000, PR China
b
Department of Pharmacy, The First Hospital of Lanzhou University, Lanzhou 730000, PR China
c
Department of Chemical Engineering, Auburn University, Auburn, AL, 36849, USA
d
Department of Physics and Engineering, Frostburg State University, Frostburg, MD 21532-2303, USA

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

Article history:
Received 15 August 2016 The present study developed novel zinc ion cross-linked alginate/N-succinyl-chitosan (NSC) blend
Received in revised form 3 November 2016 microspheres (MS) for co-delivery of zinc and 5-aminosalicylic acid (5-ASA) for synergistic therapy of
Accepted 12 November 2016 colitis. Physicochemical characterization of blend MS was assessed using scanning electron microscopy
Available online 13 November 2016 (SEM), Fourier transform infrared (FTIR) spectroscopy, and energy dispersive X-ray spectrometer (EDS).
In vitro release studies demonstrated that blend MS has a pH-dependent release property. Both 5-ASA
Keywords: and zinc have lower release in acid medium and higher release in colonic environment. The therapeutic
Alginate efficacy of zinc cross-linked blend MS was evaluated using induced-colitis rat models, and showed a
N-succinyl-chitosan
superior treatment effect in alleviating inflammation of colitis rats. No systemic toxicity was observed
Blend microspheres
after oral administration of blend MS. Therefore, zinc ion cross-linked alginate/N-succinyl-chitosan blend
Co-delivery
Colon-specific MS appeared to be a good candidate for co-delivery of zinc and 5-ASA to colon, and had great potential
application in inflammatory bowel diseases (IBD) treatment.
ã 2016 Elsevier B.V. All rights reserved.

1. Introduction gastrointestinal tract (GIT), which resulted in lower therapeutic

The inflammatory bowel disease (IBD) is a group of chronic
intestinal inflammatory diseases, mainly including Crohn's disease
(CD) and ulcerative colitis (UC). Nowadays, the prevalence and
incidence of IBD is increasing, especially in developed countries
(Moura et al., 2015). As a result of the etiology and pathogenesis of
IBD were not yet fully known, the major therapeutic strategies are
to relieve or reduce inflammatory episodes. Conventional thera-
pies for IBD treatment mainly involves aminosalicylates, cortico-
steroids, antibodies and immunomodulators (Yadav et al., 2016).
Aminosalicylates have widely been used as first-line therapy for
both the treatment of UC and maintenance of remissions (Clapper
et al., 2008). 5-ASA is the most frequently used drug in this class.
However, the rapid and extensive absorption of 5-ASA in the upper
* Corresponding author.
** Corresponding author.
E-mail addresses: duanhg13@lzu.edu.cn (H. Duan), lshy@lzu.edu.cn (S. Lü),
candyqinhy@163.com (H. Qin), gaochm@lzu.edu.cn (C. Gao), baix12@lzu.edu.cn
(X. Bai), weiyh_001@163.com (Y. Wei), xinanwu6511@163.com (X. Wu),
mzliu@lzu.edu.cn (M. Liu), xzz0004@auburn.edu (X. Zhang), zliu@frostburg.edu
(Z. Liu).
effect and the occurrence of side effects (Lichtenstein and Kamm,
2008), had limited to its application.
In recent years, colon-specific drug delivery systems (CDDS)
have been widely investigated for the effective treatment of local
colonic diseases such as IBD and colorectal cancer (Kavianinia
et al., 2016; Prudhviraj et al., 2015; Cerchiara et al., 2015; You et al.,
2015; Hua et al., 2015; Gunter and Popeyko, 2016; Seeli and
Prabaharan, 2016; Nour et al., 2016; Wang et al., 2016). CDDS could
increase the concentration of drug in colon, and also reduce many
adverse reactions from systemic absorption of the drug. Therefore,
5-ASA used to treat IBD in the form of CDDS has also drawn much
attention (Karrout et al., 2015; Omwancha et al., 2013; Mura et al.,
2011a; Wu and Yao, 2013).
Alginate (ALG) is an anionic polysaccharide that composed of
glycosidic units of b-D-mannuronic acid and a-guluronic, which has
been widely used in drug delivery systems. Due that alginate could
form gel particles in the presence of multivalent cations (e.g. Ca2+,
Zn2+, Ba2+ and Al3+) by ionic cross-linking, it has been extensively
used in CDDS. N-succinyl chitosan (NSC) is a water-soluble
derivative of chitosan that exhibits several biological properties,
has been applied as drug delivery carrier (Mura et al., 2011a, 2011b;

http://dx.doi.org/10.1016/j.ijpharm.2016.11.036
0378-5173/ã 2016 Elsevier B.V. All rights reserved.
 H. Duan et al. / International Journal of Pharmaceutics 516 (2017) 214–224
Ajish et al., 2016; Kamoun, 2016; Bashir et al., 2016; Mukhopadhyay
et al., 2014; Yan et al., 2015)
As well known, alginate and/or NSC were regularly cross-linked
by Ca2+ to form microspheres, microparticles, and hydrogels for
drug release or antimicrobial (Cerchiara et al., 2015; Dai et al.,
2008; Vicini et al., 2015; Straccia et al., 2014). However, it was
reported that alginate microspheres cross-linked by Ca2+ have low
drug entrapment efficiency and the release of drug from micro-
spheres was non-controllable (Halder et al., 2005). In this study,
zinc ion was used as a cross-linker to develop alginate/NSC drug
delivery systems. It can form more compact network between zinc
ion and carboxylic groups than the most commonly used calcium
ion for more resistance to disruption in upper GIT (Das et al., 2011).
In another aspect, zinc is necessary for a variety of physiological
and biochemical functions including growth and development,
reproduction, and immunity (Fukada et al., 2011). It was reported
that zinc deficiency had been observed in IBD patients (Filippi
et al., 2006; Vagianos et al., 2007). Moreover, studies also had
demonstrated that zinc was beneficial for IBD and has protective
effects in the GIT (Di Leo et al., 2001; Luk et al., 2002; Sturniolo
et al., 2002; Lodemann et al., 2013; Sivalingam et al., 2011).
Therefore, zinc ion plays dual roles in this study. For one thing, it
was a cross-linker to form drug-delivery carriers in CDDS; for
another thing, it can be used as a ‘drug’ for treatment of IBD.
The aim of the present study is to develop zinc ion cross-linked
alginate/NSC blend MS by emulsification/gelation for colon
targeting co-delivery of zinc and 5-ASA for synergistic therapy
of colitis. The developed blend MS were characterized by SEM,
FTIR, and EDS analyses. Furthermore, in vitro release properties of
5-ASA and zinc from blend MS were studied in simulated
gastrointestinal conditions. Finally, the therapeutic efficacy of
the blend MS was evaluated in colitis rat models induced by 2, 4, 6-
trinitro-benzene-sulfonic acid (TNBS). In addition, the systemic
toxicity of blend MS was observed after oral administration for rats.
2. Materials and methods
2.1. Materials
Sodium alginate (molecular weight, MW: 68 kDa) was obtained
from Tianjin Guangfu Chemical. Chitosan (molecular weight, MW:
106 kDa, degree of deacetylation, DD: 95%) was purchased from
Sinopharm Chemical Reagent Co., Ltd., 5-ASA (purity >98.0%),
succinic anhydride and 2,4,6-trinitro-benzene-sulfonic acid
(TNBS) were obtained from J&K Scientific Ltd. All other chemicals
and reagents were analytical grade.
2.2. Synthesis of NSC
NSC was prepared according to a previous report (Mukhopad-
hyay et al., 2014). The structure of NSC was studied through FTIR
and 1H NMR (Fig. S1 in Supplementary information). Finally, the
yield of the reaction was about 86% and the substitution degree of
NSC was about 0.56 determined according to the reported method
(Mukhopadhyay et al., 2013).
2.3. Preparation of the blend MS
The blend MS was prepared using emulsification/gelation.
Briefly, NSC and sodium alginate were dissolved in 10 ml of NaOH
solution (1%, w/v), then specific amount of 5-ASA was added into
the mixture solution to form a homogeneous solution. And then
this homogeneous solution was emulsified into 40 ml of isooctane
containing Span 80 (2%, v/v) with mechanical stirring for 10 min at
800 rpm. Then 1 ml of ZnCl2 solution was added drop wise under
mechanical stirring for 30 min at 800 rpm and the bend MS formed.
215
The obtained bend MS were washed with 0.1% Tween 80 solution 3
times and finally lyophilized at
50  C. The powdered MS were
stored in sealed glass vial in vacuum desiccators.
2.4. Characterization
2.4.1. Morphology and sizes
The shape and surface morphology of the blend MS were
characterized with SEM (JOEL, Japan). The average particle size and
size distribution of the microspheres were also measured from
several SEM images.
2.4.2. Fourier transform infrared (FTIR) analysis
The chemical structure of blend MS was analyzed by FTIR
(Nicolet 670, USA). The samples were prepared by KBr pellet
method and the spectral scanning was conducted in wavelength
region between 400 and 4000 cm
1.
2.4.3. Drug loading efficiency (LE)
To determine the drug loading efficiency (LE), a specific amount
of drug-loaded blend MS were allowed to swell completely in 10 ml
of phosphate buffer solution (PBS, pH 8.0) for 48 h (37  C, 300 rpm).
The higher pH value and rotation speed were employed to prompt
break of blend MS and ensure 5-ASA complete release from the
blend MS. The mixture was centrifuged to get the polymeric debris
free of drug solution. The clear supernatant was collected and
examined with UV–vis spectrometer at 310 nm (Perkin-Elmer
Lambda 35, USA). All measurements were performed in triplicate
to calculate LE by the following formula:
Amount of 5
ASA in blend MSðmgÞ
LEð%Þ ¼  100
Mass of blend MSðmgÞ
2.4.4. Zinc ion evaluation
The chemical elements presented in blend MS were identified
by EDS (Octane Ultra, EDAX, USA). To determine the amount of zinc
in blend MS, 10 mg of blend MS were placed into 50 ml of PBS (pH
8.0) and completely swelled at 37  C for 48 h. And then the swollen
blend MS were crushed by sanitation. The amount of zinc was
determined by the complexometric titration method, which EDTA
solution (0.05 M) was used as titrant and eriochrome black-T was
regarded as indicator.
2.5. In vitro drug release
In order to investigate the release properties of 5-ASA and zinc
from blend MS, dried drug loaded blend MS (10 mg) were placed in
a dialysis bag whose molecular weight cutoff was 3500 Da, and
dispersed in 50 ml of release media with pH of 1.2 (HCl buffer), 4.5,
6.8 and 7.4 (PBS) at 100 rpm at 37  C, respectively. At predeter-
mined time interval, two samples (3.0 ml) were taken out and the
fresh buffer solution was added to maintain a constant volume.
After centrifugation at 10000 rpm for 5 min, the released amount
of 5-ASA and zinc were determined by UV spectrophotometry and
complexometric titration method, respectively. All release tests
were performed in triplicate.
2.6. Animal experiments
Male Wistar rats were obtained from laboratory animal center
of Lanzhou University, and used to evaluate in vivo treatment
efficacy and in vivo toxicity of blend MS. Animals were housed in
animal facility at a temperature of 25  C under 12 h light/dark cycle
control room. Standard feeding and drinking water were provided.
All animal experimental protocols were reviewed and approved by
216 H. Duan et al. / International Journal of Pharmaceutics 516 (2017) 214–224

the Institutional Animal Experimentation Committee of Lanzhou
University.
2.6.1. In vivo therapeutic efficacy on experimental colitis
To evaluate the therapeutic efficacy of blend MS, colitis rats
model was induced using TNBS according to our previous report
(Qin et al., 2012; Duan et al., 2016). The rats whose body weight
decreased linearly to 80–100% of the initial weight were selected as
the colitis animals (Yano et al., 2002). The healthy group received
50% ethanol instead of TNBS solution. The above rats were divided
into 5 groups (n = 6, in each group) and treated as follows: group 1
(healthy group): oral administration of 10 mlkg
1 of saline; group
2 (colitis group): oral administration of 10 mlkg
1 of saline; group
3 (blank blend MS1 group): oral administration of 21.4 mgkg
1
blend blank MS cross-linked by 2 M Zn2+; group 4 (blank blend
MS2 group): oral administration of 21.4 mgkg
1 blank blend MS
cross-linked by 4 M Zn2+; and group 5 (5-ASA loaded blend MS1
group): oral administration 21.4 mgkg
1 of 5-ASA loaded blend
MS cross-linked by 2 M Zn2+ (equivalent to 10 mgkg
1 of 5-ASA).
The dose of 5-ASA was calculated and adjusted according to the
previous report (Horvath et al., 2008) and our pre-experiment.
After administration ten days, the rats were weighed and sacrificed
by overdose of anesthesia. The distal colon segment was removed
and washed in the saline to remove contents. The visible colonic
damage was observed and the severity of inflammations was
assessed by a clinical scoring system. The ratio of distal colon wet
weight to body (C/B ratio) were calculated as another parameter
for assessment of colonic injury. And then two samples were
excised from each colon and maintained in formaldehyde (10%, v/
v) for histological evaluation.
2.6.2. In vivo toxicity study
The toxicity of developed blend MS was evaluated in adult male
Wistar rats (180–210 g). Rats were randomly divided into two
groups of 6 rats in each. Experimental group (blend MS group) was
orally given empty blend MS at a dose of 80 mgkg
1 for 21 days;
the other group (normal group) was orally administrated 0.4 ml of
saline for 21 days. All animals were fed with normal fodder and
water during the experiment, and monitored for changes in body
weight. At the end of the experiment, rats were weighted and
anesthetized to collect blood. Main blood parameters were
determined such as white blood cell (WBC), red blood cell
(RBC), hemoglobin (HGB), and platelets (PLT), mean corpuscular
volume (MCV), mean corpuscular hemoglobin (MCH), mean
platelet volume (MPV), neutrophil absolute value (NAV), and
lymphocyte absolute value (LAV). Also serum was separated to
assess biochemical parameters including aspartate transaminase
(AST), alanine transaminase (ALT), albumin (ALB), total protein
(TP), total bilirubin (TB), alkaline phosphatase (ALP), blood urea
nitrogen (BUN), and creatinine (CR). Major organ (heart, liver,
spleen, lung and kidney) were dissected from rats for histological
evaluation.
2.7. Statistics
All quantitative data are expressed as mean value  standard
deviation (SD). The differences between groups were analyzed by
the Student’s t-test and one-way ANOVA test. *p < 0.05 was
considered to be statistically significant, and extreme significance
was set at **p < 0.01.
3. Results
3.1. Preparation and formulation optimization
Table 1 shows the measured results of LE and mean particles
size of blend MS with different formulations. The LE of drug in
different formulations ranged from 4.7% to 46.7%, and the mean
particles ranged from 6.1 to 13.4 mm. According to the results of LE
and mean particles size, the formulation of F6 was selected for
further investigations.
3.2. Characterization
3.2.1. Morphology and sizes
SEM was used to study the size, shape, and surface morphology
of blend MS. Almost all of the microspheres have spherical shapes
and smooth outer surface with a few porous regions (Fig. 1). The
mean particles size of blank blend MS cross-linked by 2 M Ca2+ and
5-ASA loaded blend MS cross-linked by 2 M Ca2+ were 12.1  4.8
and 13.4  5.6 mm, respectively. Obviously decrease in particles
size were observed for blank blend MS cross-linked by 2 M Zn2+
and 5-ASA loaded blend MS cross-linked by 2 M Zn2+ (Fig. 1C and
D), which particles size were 6.8  2.6 mm and 7.4  1.6 mm,
respectively. In addition, higher zinc concentration in cross-linking
solution (4 M) led to a smaller particles size of blend MS about
6.1 1.1 mm (Fig. 1E).
3.2.2. Fourier transform infrared (FTIR) analysis
Fig. 2A shows the characteristic FTIR spectra of 5-ASA, NSC, ALG,
blank blend MS, and 5-ASA loaded blend MS. The spectrum of 5-
ASA (a), showed the stretching vibration of

NH and

OH at
3448 cm
1, the C

O stretching vibration at 1656 cm
1, the NH
bending vibration at 1616 cm
1, and the C

N stretch with the peak
at 1353 cm
1 and the

COOH symmetric stretching vibration at
1388 cm
1. The FTIR spectrum of ALG (b) exhibited the absorption

Table 1
Loading efficiency (LE) and mean particles of blend MS in different formulations (n = 3)

Formulation NCS /mg ALG /mg Zn2+ /moll
1 Ca2+ /moll
1 5-ASA /mg LE /% Mean particle size /mm
F1 10 30 2 – 10 17.6  3.2 10.6  1.4
F2 10 20 2 – 10 13.5  1.6 9.8  2.1
F3 10 10 2 – 10 10.5  1.2 7.4  1.6
F4 20 10 2 – 10 11.2  3.2 8.1  1.5
F5 30 10 2 – 10 11.1  2.5 8.6  3.1
F6 10 10 2 – 20 46.7  4.6 8.3  2.5
F7 10 10 2 – 16 32.4  3.8 7.8  1.7
F8 10 10 2 – 4 4.7  2.4 7.1  3.3
F9 10 10 4 – 10 19.8  2.8 6.1  1.1
F10 10 10 1 – 10 5.8  1.4 8.8  2.7
F11 10 10 0.5 – 10 4.4  0.8 9.3  1.5
F12 10 10 2 – – – 6.8  2.6
F13 10 10 – 2 10 6.4  1.1 13.4  5.6
F14 10 10 – 2 – – 12.1  4.8
 H. Duan et al. / International Journal of Pharmaceutics 516 (2017) 214–224
Fig. 1. SEM micrographs of blank blend MS cross-linked by 2 M Ca2+ (A), 5-ASA loaded blend MS cross-linked by 2 M Ca2+ (B), blank blend MS cross-linked by 2 M Zn2+ (C), 5-
ASA loaded blend MS cross-linked by 2 M Zn2+ (D), and 5-ASA loaded blend MS cross-linked by 4 M Zn2+ (E) (scale bar = 50 mm).
peaks of O

H and C

H stretching vibrations at 3394 cm
1 and
2927 cm , respectively; the absorption peaks at 1624 cm
1 and

1
1415 cm
1 assigned to asymmetric and symmetric stretching
vibrations of carboxyl anion, respectively. In the case of NSC, it
exhibited stretching vibration peaks of

OH and

NH2 at
3425 cm
1, the C¼O stretching of amide I band at 1664 cm
1,
and the amide II at 1585 cm
1, the symmetric stretching of the


COO
at 1408 cm
1 (c). For the blank blend MS, the absorption
band at around 3392 cm
1 concerned with
OH stretching
vibration for ALG shifted to a high wave number at 3425 cm
1.

COO
at 1415cm
1 of ALG and at
The symmetric stretching of the

1408 cm of NSC shifted to a higher wave number at 1429 cm
1.

1
The absorption band at 1664 cm
1 of NSC shifted to 1629 cm
1, the
amide II peak (1585 cm
1) of NSC disappeared in blank blend MS.
The FTIR of 5-ASA loaded blend MS was similar to those of blank
blend MS (e).
3.2.3. Drug loading efficiency (LE)
The amount of 5-ASA encapsulated in different formulations
were measured by an UV method, and the results were showed in
Table 1. Increasing of ALG: NSC ratio could improve the content of
5-ASA in blend MS, which might be due to more three-dimension
network, resulting from increased cross-linking sites. Also, the
amount of 5-ASA in initial NSC solution could lead to an obvious
increase of LE in blend MS. Finally, the concentration of cross-
linker (Zn2+) could influence on the LE of 5-ASA in blend, i.e.,
decreasing concentration of Zn2+ could reduce the content of 5-
ASA in blend MS, which is attributed to lower cross-linking density.
3.2.4. Zinc ion evaluation in blend MS
The chemical element distribution of drug loaded blend MS
cross-linked by Ca2+ and Zn2+ is evaluated by EDS, and the results
were shown in Fig. 2B. It can clearly be seen that calcium is mainly
present in the Ca2+ cross-linked blend MS, whereas, the presence of
zinc was primary in Zn2+ cross-linked blend MS. The amount of
zinc in blend MS was shown in Fig. 2C. The concentration of zinc in
cross-linking solution has obviously influenced the amount of zinc
in blend MS. Increasing the zinc concentration from 0.5 M to 4 M in
cross-linking solution resulted in an increasing of zinc content in
blend MS, and the amount of zinc reached the maximum (196.2 mg
217
of zinc per mg of the blend MS) at the cross-linker concentration of
4 M.
3.3. In vitro release properties
In vitro release experiments were performed by dialysis
diffusion method at different release media (the value of pH
was 1.2, 4.5, 6.8 and 7.4, respectively) mimicking the in vivo GIT
conditions at 37  C. Both 5-ASA and zinc were pH-sensitive release
and the cumulative release amount of 5-ASA and zinc was
increased with the improvement of the pH of medium. Less than
5% and 20% of encapsulated 5-ASA were released from blend MS
over 12 h at pH of 1.2 and 4.5, respectively. However, the released 5-
ASA from blend MS was obviously increased at pH 6.8 and pH 7.4 in
a sustained way. About 60.2% and 71.5% of entrapped 5-ASA was
released from blend MS over 8 h at pH 6.8 and pH 7.4, respectively
(Fig. 3A). The effect of the Zn2+ concentration on 5-ASA release
showed that increasing Zn2+ concentration in cross-linking
solution led to slower release of 5-ASA from blend MS (Fig. 3B).
Also, the release properties of 5-ASA and zinc from blend MS were
evaluated in a pH progression medium simulating the conditions
of different parts of a real GIT (pH 1.2 HCl buffer for releasing at the
first hour, then pH 4.5 PBS for the next 2 h, and pH 6.8 PBS for the
2 h, finally pH 7.4 PBS until the end). The results indicated that 5-
ASA and zinc were rarely released at the condition of pH 1.2, and on
overpass 5% of 5-ASA was released in pH 4.5 PBS. Only small
amount of 5-ASA and zinc (about 10%) was released from blend MS
in the SIF at pH 6.8 over 2 h. However, the released drug from blend
MS was significant increased at pH 7.4 and maintained a sustaining
release characteristics in this condition.
The release behavior of zinc from blend MS was similar to 5-ASA
release, and also exhibited a pH-sensitive release, which cumula-
tive release of zinc from blend MS was increased with pH
increasing (Fig. 3C and D).
3.4. In vivo treatment effects on experimental colitis
The therapeutic effect of this colon-specific co-delivery system
for treatment IBD was evaluated on a TNBS-induced colitis rats
after oral administration. As can be seen from Fig. 4A, healthy rat
218 H. Duan et al. / International Journal of Pharmaceutics 516 (2017) 214–224

Fig. 2. The FTIR of blend MS (A), the EDS spectrums of blend MS (B), and the amount of Zn2+ in blend MS (C).

colon was intact and looked pink, and showed no macroscopic
damage, however, the colon from colitis rats showed on the
formation of ulcers and macroscopic edematous inflammation,
with a thickening, hyperemia and rigidity in the intestinal wall and
a consequent reduction in length. Internally, colon exhibited
congestion, necrotic and edema of the colon mucosa. In contrast,
colon from the rats treated with two blank blend MS showed an
obvious amelioration than those of colitis rats, and only very small
ulcer spots were observed in internal walls of colon. On the other
hand, the severity of the inflammation was significantly decreased
upon administration 5-ASA loaded MS1. The colons from this group
rats presented small slight hyperemia, and were very similar to
those of healthy rats. Barely there were necrotic areas in the
internal walls of this colon.
 H. Duan et al. / International Journal of Pharmaceutics 516 (2017) 214–224 219

Fig. 3. In vitro release profiles of drug from blend MS: (A) release profiles of 5-ASA from blend MS cross-linked by 2 M Zn2+ in HCl buffer at pH 1.2, PBS at pH 4.5, pH 6.8 and pH
7.4; (B) release profiles of 5-ASA from blend MS cross-linked different levels Zn2+ in PBS at pH 7.4; (C) release profiles of 5-ASA and zinc from blend MS in gradually pH-
changing buffers; (D) release profiles of zinc from blend MS cross-linked 2 M Zn2+ in HCl buffer at pH 1.2, PBS at pH 4.5, pH 6.8 and pH 7.4 (The values are mean  SD, n = 3).

The clinical scores of colon for the colitis rats are presented in
Fig. 5A (about 7.8), and markedly increased compared with healthy
rats (p < 0.01). The clinical score of colon from rats treated with
two blank blend MS (about 6.1 and 5.2) obviously decreased
compared with colitis rats. Especially, the noticeably difference of
clinical score was observed between rats treated blank blend MS2
and colitis rats (p < 0.01). After treated with 5-ASA loaded blend
MS1, the clinical score of colon was sharply declined (p < 0.01), and
reached the minimum (only 2.4).
Similar results were observed in the results of the C/B ratio
compared with those of clinical score systems (Fig. 5B). The basic
value of the C/B ratio of healthy rats was 4.46 mgg
1, however, for
the colitis rats, it was about 14.41 mgg
1 and observably increased
compared to those of healthy rats (p < 0.01). After treated with
blank blend MS (2 M Zn2+ cross-linked MS and 4 M Zn2+ cross-
linked MS), the C/B ratio of rats were markedly decreased (10.78
and 7.93 mgg
1). Especially, rats treated with 5-ASA loaded blend
MS1 showed lower C/B ratio of 5.84 mgg
1 compared with those
of colitis rats (p < 0.01).
Histological evaluation could provide an effective support for
the results of macroscopic observation and the C/B ratio. The
histological results were showed in Fig. 4B. For rats of health group,
it exhibited normal colon structure with normal enterocytes,
goblet cells, muscularis mucosae, and normal submucosa. The
colon from colitis rats showed mucosal damage and necrosis of
granulation tissue, which was accompanied by destroying of the
normal architecture of the colon and severe inflammatory
infiltration throughout the mucosa and submucosa. The micro-
scopic of colon from rats treated with two blank blend MS showed
an obviously improvement of inflammatory process with a few of
inflammatory cell infiltration. In addition, the colon from the rats
treated with blank MS2 showed obvious amelioration in inflam-
matory status. In particular, the colon from the rats treated with 5-
ASA loaded MS1 presented largely normal mucosal architecture
with slight presence of inflammatory infiltration.
3.5. In vivo toxicity
After oral administration of blend MS1 at the dose of 80 mg
kg
1 for 21 days, blend MS treated rats did not show any weight
loss, and no difference in body weight was observed compared to
normal group rats (Fig. 6A). The main blood parameters after oral
administration of blend MS1 are shown in Table 2. The values of
WBC, RBC, HGB, and PLT from blend MS treated rats were 8.82109
l
1, 8.631012 l
1, 153.90 gl
1, and 1017.60109 l
1, respectively.
Slight variations but no statistical significance was observed in
blood parameters compared to those in normal rats. In another
aspect, the levels of AST and ALT in blend MS treated rats were
131.40 and 69.30 Ul
1, respectively, and somewhat elevated
compared to normal rats (119.70 and 58.60 Ul
1, respectively).
However, no statistical changes were observed compared to
normal rats (p > 0.05). A mild increase but no significant difference
in serum creatinine and albumin were obtained in blend MS
treated rats (9.89  0.73 and 0.28  0.04 mmoll
1, respectively)
220 H. Duan et al. / International Journal of Pharmaceutics 516 (2017) 214–224

Fig. 4. Photographs (A) and histological evaluation (B) (magnification 10) of the colon from health rats, colitis rats, blank blend MS1 treated rats, blank blend MS2 treated
rats, and 5-ASA loaded blend MS1 treated rats.

compared to those of normal rats (9.62  1.35 and 0.23  0.06,
respectively).
The histological evaluation of main tissues (heart, liver, and
spleen, lung, and kidney) from blend MS1 treated rats was
presented in Fig. 6B. No pathological changes were observed in
blend MS treated rats, and the appearance of all tissues in blend MS
treated rats was similar to those of normal rats.
4. Discussion
For colonic diseases such as IBD and colorectal cancer, to
maintain a therapeutic level of drug at the disease site and avoid
toxic or side effects of drug is crucial. However, most of the oral
drugs were extensively absorbed in upper part of the GIT, and the
drug level achieved to the colon site for therapy is not sufficient
after oral administration. Therefore, CDDS was developed to solve
this problem. In CDDS, pH-dependent delivery systems composed
of natural polysaccharide and its derivatives have drawn large
attention. Especially, microspheres or microparticles systems have
been developed and exhibited superior property of colon-specific
delivery.
In the present study, a novel zinc ion cross-linked blend MS
composed of ALG and NSC was developed to co-deliver 5-ASA and
zinc in colon for colitis treatment. The emulsification/gelation
method was successfully used to prepare different formulations of
blend MS. In this process, the negatively-charged carboxylic groups
 H. Duan et al. / International Journal of Pharmaceutics 516 (2017) 214–224 221

Fig. 5. The clinical activity scores of colon from rats treated with different blend MS (A), and the C/B ratio of colitis rats after treatment with different blend MS (B) (* p < 0.05
and ** p < 0.01, vs colitis, # p < 0.05, vs healthy).

Fig. 6. In vivo toxicity of blend MS after oral administration of blank blend MS1 at a dose of 80 mgg
1 for 21 days: (A) the bodyweight changes of rats, (B) histological images of
main tissues from rats (magnification 10).
222 H. Duan et al. / International Journal of Pharmaceutics 516 (2017) 214–224

Table 2 cross-linking density. The FTIR of blend MS was used to identify the
Qualitative analysis of blood parameters and serum biochemical parameters in rats
interactions between polymers and Zn2+, which indicated the
after oral administration of blend MS1 at a dose 80 mgg
1 for 21 days (n = 6).
formation of intermolecular hydrogen bonds between ALG and
Different parameters Groups NSC molecules, and cross-linked networks between Zn2+ and the
Normal Blend MS

COO
of ALG and NSC. In addition, the characteristic absorption
Blood WBC (  109 l
1) 9.40  1.08 8.82  0.82
of 5-ASA could not be observed in 5-ASA loaded blend MS,
RBC (  1012 l
1) 9.12  0.65 8.63  0.74 suggesting that 5-ASA was incorporated in network of blend MS
HGB (gl
1) 155.60  1.82 153.90  2.13 during the preparation procedure.
MCV (fl) 57.42  1.02 56.82  0.68 The enhanced in vitro release drug from blend MS at higher pH
MCH (pg) 18.33  0.51 17.86  0.84
(6.8 and 7.4) could be explained with the protonation effect of
PLT (  109 l
1) 995.60  54.80 1017.60  80.70
MPV (fl) 8.48  0.43 8.14  0.21 carboxyl groups (

COOH) on ALG and NSC at lower pH, which led
LAV (  109 l
1) 7.75  0.76 7.32  0.48 to the shrinkage of blend MS, and limited the release of 5-ASA from
NAV (fl) 1.72  0.53 1.46  0.37 blend MS (Fig. 3). When the pH changed to 6.8 and 7.4, which
corresponds to the pH of ileocecal and colon region of the GIT, a
Serum biochemical AST (Ul
1) 119.70  21.50 131.40  18.10
ALT (Ul
1) 58.60  5.16 69.30  6.76
maintained drug release was obtained due to the stretching and
TP (gl
1) 56.70  4.52 60.65  3.84 deprotonation of carboxyl groups in ALG and NSC, leading to
ALB (gl
1) 35.64  2.84 37.15  3.65 swelling and disruption of blend MS. High Zn2+ concentration
TB (mmoll
1) 2.68  0.69 2.85  0.46 could reduce the release of drug, attributing to that high Zn2+
ALP (Ul
1) 168.70  52.60 179.40  61.40
concentration led to the high cross-linking density between zinc
BUN (mmoll
1) 9.62  1.35 9.89 0.73
CR (mmoll
1) 0.23  0.06 0.28  0.04 and polymers, and formed more rigid and compact polymer
network structure. The high cross-linking density could reduce the
swelling of the blend MS and might slower the drug release from
of ALG and NSC were cross-linked by Zn2+ and the blend MS was the blend MS (Straccia et al., 2014; Sinha et al., 2015; Pillay et al.,
formed. Zinc ion was used as a cross-linker to prepare the blend MS 2005). Furthermore, the rigid and compact polymer network
for more resistance to disruption in upper GIT, which attributed to structure could reduce the penetration of water molecule, which
the formation of more compact network between zinc and could decrease the leakage and diffusion of drug molecules from
carboxylic groups than the most commonly used calcium (Das gel network (Banerjee et al., 2013).
et al., 2011). In vivo treatment efficacy of blend MS after oral administration
Increasing of ALG significantly increases the LE of 5-ASA was further evaluated using TNBS-induced colitis rats. The C/B
following with the increased particles size of the blend MS ratio and macroscopic observation of colon were used as important
(Table 1). However, there is no difference in LE, and only slight parameters for monitoring the severity of ulcerative colitis, which
increase of particles size of blend MS has been observed with the can visually evaluate the inflammatory degree of colon and judge
increase of NSC. Increasing the amount of 5-ASA actually led to an the treatment efficacy according to the real inflammatory status of
obvious increase of LE of blend MS, and has no noticeable effect on the colon (Karrout et al., 2015). The clinical scores of macroscopic
the particles size of blend MS. In addition, the concentration of zinc observations were regarded as another assessment index, and
also has influence on LE and the particles size. Increased LE of drug quantified using the “Wallace score system” (Wallace et al., 1989).
in blend MS and decreased particles size of blend MS are observed Higher clinical score represents more severe situation of the
with the increasing of Zn2+ concentration. The possible reason is disease. The blend MS cross-linked by different Zn2+ concentration
that increased Zn2+ will enhance the formation of network for drug showed a favorable treatment efficacy for colitis rats, which
entrapment, which is due to greater level of cross-linking between attributed to the release of zinc ions in inflamed colon for
Zn2+ and polymers. On the other hand, the blend MS prepared treatment of colitis (Luk et al., 2002; Sturniolo et al., 2002).
using low Zn2+ concentration might have large pores because of Furthermore, Zn2+ could improve intestinal barrier function and
insufficiency in cross-linking degree and/or drug leaching through protect against intestine damage (Lodemann et al., 2013;
the pores, which led to lower drug encapsulation (Das et al., 2014). Sivalingam et al., 2011; Chen et al., 1999; Hering and Schulzke,
In addition, the decrease of the particle size of blend MS could be 2009). Rats treated with 5-ASA loaded blend MS1 obtained best
explained by the formation of more compact polymer network, therapeutic effect, which could be attributed to the synergistic
causing physical retention of 5-ASA inside the blend MS. Increasing effects of targeting delivery of 5-ASA and zinc ion to inflamed colon
the 5-ASA content also results in an obvious improvement of drug for colitis treatment.
LE in blend MS. Compared to Ca2+ cross-linked blend MS, blend MS In vivo toxicity study was assessed by monitoring body weight
cross-linked by Zn2+ generally had smaller mean size (6.8 mm), changes, determination of blood parameters and serum biochemi-
Because Zn2+ radius was smaller than Ca2+ radius in a single cal parameters, and histological evaluation of main tissues. No
coordination number, and the blend MS cross-linked by Zn2+ have a mortal animal was observed during the experiment, indicating
more compact “egg box” structure than those of cross-linked by that blend MS has lower toxicity after oral administration. Blood
Ca2+, which led to a smaller volume occupied by Zn2+ in the blend parameters have no significant difference after oral administration
MS. Moreover, Zn2+ has stronger affinity with ALG because of the for 21 days at the dose of 80 mgkg
1, which demonstrated the
bond of guluronic and manuronic acids, while calcium binds only lower hematotoxicity of blend MS. In fact, serum biochemical
to guluronic acid chains (Wasastjerna, 1923; Costa Cuozzo et al., parameters such as AST and ALT have a direct correlation with the
2014). This might result in a smaller particle size of blend MS cross- damages of liver parenchyma (Mukhopadhyay et al., 2015). The
linked by Zn2+. No significant difference in particle size of 5-ASA determined level of AST and ALT after oral administration of blend
loaded blend MS cross-linked by Zn2+ were observed compared MS suggested that blend MS did not produce hepatotoxicity.
with the zinc cross-linked blank blend MS (Fig. 1D). According to Furthermore, serum creatinine and albumin were most commonly
this result, it seemed that the drug loading process did not highly measured to evaluate the nephrotoxicity and renal disease. A rapid
affect to the size of blend MS cross-linked by Zn2+. Furthermore, an increase in the serum creatinine and albumin level is an index of
increase in concentration of Zn2+ significantly decreased the mean renal injury (Mukhopadhyay et al., 2015). A slight increase in
particle size of the blend MS (Fig. 1E), which attributed to the serum creatinine and albumin indicated no nephrotoxicity of blend
formation of more compact network resulted from increased MS compared to those of normal rats (Table 2). Again, the
 H. Duan et al. / International Journal of Pharmaceutics 516 (2017) 214–224
histological structure of main tissues from rats treated with blend
MS displayed the relative normal histological morphologies,
suggesting that blend MS exhibited excellent security and
biocompatibility. So, it can be interpreted from in vivo toxicity
study that blend MS could be a safe drug delivery carrier for oral
administration of 5-ASA.
5. Conclusions
In summary, a blend MS cross-linked by zinc ion for colon-
specific co-delivery of 5-ASA and zinc has been successfully
fabricated using alginate and N-succinyl chitosan by emulsifi-
cation/gelation. In vitro drug release studies confirmed that blend
MS showed the property of pH-dependent release for 5-ASA and
zinc, and it was able to release most of 5-ASA at colonic pH value.
Moreover, the blend MS system markedly alleviated the colonic
inflammation and improved the efficacy of 5-ASA in healing of
colitis rats by co-delivery zinc to colon. In vivo toxicity study
demonstrated the excellent safety of blend MS after oral
administration. Therefore, the described drug co-delivery system
might be employed as a safe and effective carrier for clinical
treatment of IBD.
Acknowledgements
The authors gratefully acknowledge the financial support of the
National Natural Science Foundation of China (grant no. 51541304,
51273086, 51503091), Special Doctorial Program Fund from the
Ministry of Education of China (grant no. 20130211110017), and the
Fundamental Research Funds for the Central Universities (grant no.
lzujbky-2016-41).
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.
ijpharm.2016.11.036.
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