Accepted Manuscript

Intestine-targeted delivery potency of the O-carboxymethyl

chitosan–gum Arabic coacervate: Effects of coacervation acidity
and possible mechanism

Guo-Qing Huang, Li-Na Liu, Xiao-Na Han, Jun-Xia Xiao

PII: S0928-4931(16)31917-8
DOI: doi: 10.1016/j.msec.2017.05.074
Reference: MSC 8056
To appear in: Materials Science & Engineering C
Received date: 26 October 2016
Revised date: 2 May 2017
Accepted date: 13 May 2017

Please cite this article as: Guo-Qing Huang, Li-Na Liu, Xiao-Na Han, Jun-Xia Xiao
, Intestine-targeted delivery potency of the O-carboxymethyl chitosan–gum Arabic
coacervate: Effects of coacervation acidity and possible mechanism, Materials Science &
Engineering C (2017), doi: 10.1016/j.msec.2017.05.074

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 ACCEPTED MANUSCRIPT

Intestine-targeted delivery potency of the O-carboxymethyl

chitosan – gum Arabic coacervate: Effects of coacervation
acidity and possible mechanism
Guo-Qing Huanga, Li-Na Liub, Xiao-Na Hana, Jun-Xia Xiaoa,*
a
College of Food Science and Engineering, Qingdao Agricultural University,

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Qingdao 266109, China
b
Institute of Agro-Food Science and Technology, Shandong Academy of Agricultural

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Sciences, Jinan 250100, China
Abstract: The potential of the glutaraldehyde-crosslinked O-carboxymethyl chitosan

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(O-CMC) – gum Arabic (GA) coacervate as an intestine-targeted delivery system for
hydrophobic compounds was concerned. OCMC – GA coacervates and emulsified
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bovine serum albumin (BSA)-loaded microcapsules were prepared in pH 3.0, 4.5, and
6.0 and their swelling behaviors as well as BSA release profiles in simulated
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gastrointestinal fluids were measured. All the coacervates showed higher swelling
ratios in the simulated gastric solution than in the simulated intestine and colon
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solutions and the values increased as the coacervation pH increased from 3.0 to 6.0,
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but a reversed trend was observed for the BSA release profile. SEM, TEM, and
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CLSM analysis revealed that the coacervation acidity influenced the swelling and
BSA release behavior by changing the matrix density and O-CMC content of the
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coacervates. It was concluded that the O-CMC – GA coacervate could be used to

deliver hydrophobic compounds to the intestine and its delivery performance could be
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tailored by selecting appropriate coacervation acidity and crosslinking degree.

Keywords: O-carboxymethyl chitosan; gum Arabic; coacervate; intestine-targeted
delivery; microstructure.
1. Introduction
Complex coacervation is the electrostatic interaction between two oppositely
charged biopolymers. This reaction and its resultant products, namely coacervates,

*
Corresponding author.
E-mail address: xjxfood@qau.edu.cn (J.X. Xiao)
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have been widely used in the food industry for microencapsulation or as food
ingredients and in the pharmaceutical industry as biomaterials [1]. Recently, the
potential of coacervates as delivery systems for proteins and small molecule drugs
have attracted great attentions. Compared with other common vehicles such as
hydrogels and microparticles, coacervates have high loading capacity and can be
formed quickly in aqueous medium without using organic solvents [2]. Due to these

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advantages, coacervates have been recognized as an exciting new class of drug
delivery vehicles [3].

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As a rare naturally-occurring cationic polysaccharide, chitosan is one of the most
popular biopolymers for the development of pH-sensitive drug delivery systems due

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to its high biocompatibility, excellent biodegradability, low toxicity, abundant
availability, and acceptable production cost [4]. Its combination with sodium alginate
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or tri-polyphosphate has been broadly used to realize the intestine-targeted delivery of
probiotics and hydrophilic compounds [5-7]. To extend the application of
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chitosan-based coacervates in the delivery of hydrophobic compounds, a polyanion

with emulsifying capability, such as gelatin [8], soybean protein isolate [9], and gum
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Arabic (GA) [10], could be introduced into the coacervation system. Among these
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combinations, the chitosan – GA pair has drawn the most interests. Its coacervation
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conditions as well as the characteristics, composition, and application of the resultant

coacervates have been exploited in detail [11-17]. These researches provide valuable
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information for the practical application of the chitosan – GA coacervation system and
reveal that the system could be used for the controlled release of hydrophobic
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compounds.
However, chitosan is soluble only in acidic solutions, which may limit its
utilization in the delivery of liable compounds. Derivation is an effective way to
generate desirable solubility for chitosan. O-carboxymethyl chitosan (O-CMC) is an
important derivative of chitosan and possesses many outstanding properties including
water solubility, non-toxicity, biodegradability, biocompatibility, and excellent pH
sensitivity [20]. O-CMC has been used to construct intestine-targeted delivery
systems for hydrophilic drugs through coacervation with other oppositely-charged
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polyelectrolytes [21, 22].

Coacervation acidity is a critical factor that affects the performance of coacervate
delivery systems. It has been reported able to change the microcapsule layer thickness
[23], drug release dynamics [24], and layer mass of the multilayers [25] of
chitosan-based coacervate delivery systems. Hence, the purpose of this work is to
investigate the potency of glutaraldehyde-crosslinked O-CMC – GA coacervate

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separated in different acidities as a pH-sensitive delivery vehicle. Meanwhile, the
possible mechanism of the different delivery behavior caused by coacervation acidity

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is also concerned from the microstructure point of view. This work is expected to
provide valuable information for designing tailor-made O-CMC – GA coacervate as

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an intestine-targeted delivery system for hydrophobic compounds.
2. Materials and methods
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2.1. Materials
O-CMC with degree of substitution 0.35 was synthesized in the same method as
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a previous work [26]. GA was a gift from Guangzhou Darong Trading Company
(Guangzhou, China). All other reagents were of analytical grade.
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2.2. O-CMC – GA coacervate preparation and glutaraldehyde crosslinking

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The O-CMC – GA coacervates were prepared in the optimum temperature,

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biopolymer ratio, and total biopolymer concentration according to a previous work

[26]. The stock solutions of O-CMC (10 mg/mL in distilled water) and GA (40
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mg/mL in distilled water) were mixed well in the same volume ratio. Then, the
solution pH was adjusted to 3.0, 4.5, or 6.0 with 0.1 mol/L HCl and the reaction was
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allowed at 25 °C under mild magnetic agitation. One hour later, the condensed phase
was separated by centrifugation at 1800 g for 10 min, collected, and added to the 0.5
mg/mL glutaraldehyde solution of the same pH as the corresponding coacervate was
prepared in. Several hours later, the crosslinking products were collected, washed
with deinoized water, and freeze-dried for characterization.
2.3. Swelling behavior in simulated gastrointestinal fluids
Freeze-dried O-CMC – GA coacervates of known weigh (W0) were separately
immersed in simulated gastric (pH 1.2 HCl solution), intestine (pH 6.8 PBS solution),
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and colon (pH 7.4 PBS solution) fluids at 37 °C for 3 h. At an interval of 30 min, the
swollen complexes were taken out, blotted on filter paper, and weighed (Wt). The
swelling ratio (SR) was then calculated according to the following equation:
𝑊𝑡 − 𝑊0
SR (%) =
𝑊0
2.4. Model drug release profile in simulated gastrointestinal fluids
The W/O emulsion of bovine serum albumin (BSA) was used as a model

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hydrophobic drug to evaluate the intestine-targeted delivery potency of the O-CMC –
GA coacervates.

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The BSA microcapsules were prepared according to a reported method with

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minor modifications [27]. The aqueous solution of BSA (10%, w/v) was blended with
4-fold volumes of food-grade soybean oil and emulsified at 10000 rpm and 35 °C for
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15 min in the presence of Span as emulsifier (1.6% of the total volume, v/v). The
primary W/O emulsion was transferred to four-fold volumes of the stock GA solution
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and further homogenized for 3 min at 3000 rpm. Subsequently, the stock O-CMC
solution was slowly added to the W/O/W emulsion until the weight ratio of O-CMC
to GA reached 1:4. Then, the solution pH was adjusted to 3.0, 4.5, or 6.0 and left at
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25 °C with mild magnetic agitation. One hour later, glutaraldehyde was added until its
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final concentration reached 0.5 mg/mL and the solution was maintained at 30 °C for
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2 h, 6 h, or 12 h. Finally, the microcapsules were collected and lyophilized for BSA

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release analysis.
Pre-weighed dried BSA microcapsules were immersed in the simulated gastric,
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intestine, and colon solutions at 37 °C for 3 h respectively in sequence. After the

incubation in one simulated fluid completed, the swollen microcapsules were taken
out, blotted on filter paper, and then transferred to the next solution. At an interval of
1 h, 100 μL release medium was taken out and the BSA content was measured by
Bradford protein assay at 595 nm using a spectrophotometer. The percentage of
cumulative amount of released BSA was determined from standard calibration curve.
2.5. Microstructure observation
Both scanning (SEM) and transmission electron micrography (TEM) were

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performed to reveal the microstructure of the O-CMC – GA coacervates. In SEM, the

freeze-dried powders were mounted on circular aluminum stubs with double-sided
sticky tape, coated with gold, then examined and photographed in a scanning electron
microscope (JEOL-7500F, Jeol, Japan) at an accelerating voltage of 2 kV.
In TEM, suspensions of the O-CMC – GA coacervates crosslinked by 0.5 mg/mL
glutaraldehyde for 6 h were dropped onto Formvar-coated copper grids. After

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complete drying, the samples were stained using 2% (w/v) phosphotungstic acid. The
morphological analysis was then performed using a transmission electron microscope

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(HT7700, Hitachi, Japan).
2.6. Viscoelasticity measurement

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The viscoelasticity of the crosslinked O-CMC – GA coacervates were measured
with a MCR30 rheometer (Paar Physica, Messtechnik, Stuttgart, Germany) at 25 °C.
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A truncated cone–plate geometry (2°, 6 cm diameter) was used and the distance
between the flat surfaces of both elements was 0.047 mm. After the plateau got
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contacted with the cone, the exposed surface of sample was covered with a thin layer
of silicone oil to prevent evaporation during measurement. Frequency sweeps (0.1 –
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50 Hz) were carried out for a strain of 1.0%, which was in the linear regime as
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checked by strain sweep measurements (data not shown).

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2.7. Confocal laser scanning microscopy (CLSM)

O-CMC and GA were labeled with fluorescein isothiocyanate (FITC) and
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rhodamine B isothiocyanate (RBITC) respectively in the same method as reported by

Lamprecht et al. [24]. After the remaining dyes were washed away with 9.5% ethanol,
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the labeled polyelectrolytes were lyophilized and then used to prepare the O-CMC –
GA coacervate in the same conditions mentioned above. The CLSM images of the
samples were taken by a LEICA TCS SP5 II system (Leica Microsystems, Solms,
Germany).
3. Results and discussion
3.1. pH-responsive behavior in simulated gastrointestinal fluids
The swelling behavior of the O-CMC – GA coacervates prepared in pH 3.0, 4.5,
and 6.0 as well as crosslinked by 0.5 mg/mL glutaraldehyde for different durations
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were illustrated in Fig. 1, 2, and 3 respectively. It could be seen that the SRs of the
coacervates increased with incubation proceeding and decreased with crosslinking
duration elongation. All the O-CMC – GA coacervates showed certain pH-sensitivity,
that is, they displayed higher swelling degree in the simulated gastric solution (pH 1.2)
than in the simulated intestine (pH 6.8) and colon solutions (pH 7.4). This
pH-responsive behavior was contrary to that of the glutaraldehyde-crosslinked
O-CMC microsphere [29] and the Ca2+-crosslinked-O-CMC – carbopol beads [22],

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which swelled more severely in the neutral media than in the pH 1.2 solution,

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indicating that the electrostatic interaction with GA changed the pH-dependent
hydrogen bonding of O-CMC with water and the property of polyanion and/or

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crosslinking agent determined the swelling behavior of O-CMC-based coacervates to
a large extent.
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The coacervation acidity mainly affected the SRs of the O-CMC – GA
complexes in the simulated gastric solution. In the case of same glutaraldehyde
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crosslinking duration, the coacervates separated in pH 6.0 had the highest degree of
swelling in the pH 1.2 solution, followed by those separated in pH 4.5 and 3.0 in
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sequence. For example, after crosslinking by 0.5 mg/mL glutaraldehyde for 2 h, the
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SRs of the coacervates separated in pH 6.0 at the end of the 3-h incubation in the
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simulated gastric, intestine, and colon solutions were 7.64, 3.03, and 3.16, while those
of the coacervates separated in pH 4.5 and 3.0 were 7.08, 3.66, 4.70 and 3.68, 2.78,
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3.43 respectively. As the glutaraldehyde crosslinking duration increased to 12 h or

higher, the pH-sensitivity of the coacervates declined obviously regardless of the
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coacervation acidity. In crosslinking duration 12 h, the SRs of the coacervates

separated in pH 6.0, 4.5, and 3.0 in the three simulated solutions decreased to only
3.48, 2.38, 2.36; 3.89, 2.40, 2.65; and 2.41, 2.08, 2.41 respectively; when the
crosslinking period further increased to 24 h, the SRs values were became very close
to 2.86, 2.04, 2.19; 3.55, 2.29, 2.36; and 2.25, 2.06, 2.29 respectively. This
phenomenon was consistent with the finding that excessive crosslinking could
eliminate the pH-sensitivity of chitosan hydrogel [30].

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4.0 2h 4h 6h 12h 24h pH1.2

3.5
3.0
2.5
2.0
1.5
3.0 pH6.8
Swelling ratio

2.5
2.0
1.5

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1.0

4.0 pH7.4
3.5
3.0

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2.5
2.0
1.5

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1.0
0.5 1.0 1.5 2.0 2.5 3.0
Glutaraldehyde crosslinking duration (h)
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Fig. 1. Swelling behaviors of the O-CMC – GA coacervates prepared in pH 3.0 and
crosslinked by 0.5 mg/L glutaraldehyde for different durations in the simulated gastric
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(top), intestine (middle), and colon (bottom) solutions.

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8 2h 4h 6h 12h 24h pH1.2

7
6
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5
4
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3
2

pH6.8
Swelling ratio

4
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3
2
1
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6 pH7.4
5
4
3
2
1
0.5 1.0 1.5 2.0 2.5 3.0
Glutaraldehyde crosslinking duration (h)

Fig. 2. Swelling behaviors of the O-CMC – GA coacervates prepared in pH 4.5 and

crosslinked by 0.5 mg/L glutaraldehyde for different durations in the simulated gastric
(top), intestine (middle), and colon (bottom) solutions.
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9 2h 4h 6h 12h 24h pH1.2

8
7
6
5
4
3
2
1
3.5 pH6.8
Swelling ratio

3.0
2.5
2.0
1.5

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1.0

pH7.4
3.5
3.0

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2.5
2.0
1.5

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1.0
0.5 1.0 1.5 2.0 2.5 3.0

Glutaraldehyde crosslinking duration (h)

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Fig. 3. Swelling behaviors of the O-CMC – GA coacervates prepared in pH 4.5 and
crosslinked by 0.5 mg/L glutaraldehyde for different durations in the simulated gastric
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(top), intestine (middle), and colon (bottom) solutions.

3.2. Model drug release behavior in simulated gastric solutions
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The W/O BSA emulsion was used as a model drug to investigate the
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intestine-targeted delivery potency of the O-CMC – GA coacervates for hydrophobic

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compounds and the results were given in Fig. 4.

All the microcapsules released BSA very quickly in the three simulated solutions,
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implying that BSA was liberated in burst mode. Both the coacervation acidity and
glutaraldehyde crosslinking duration affected the BSA release profile. Contrary to the
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swelling behavior, the microcapsules prepared in pH 3.0 released the highest

percentage of BSA after consecutive incubation in each simulated gastrointestinal
fluid for 3 h and the cumulative releases were 41.16%, 72.38%, and 96.37%
respectively in crosslinking duration 2 h, whereas those of the coacervates separated
in pH 4.5 and 6.0 were as low as 16.92%, 39.08%, 55.20% and 21.24%, 42.47%,
60.92% respectively. As the glutaraldehyde crosslinking duration increased to 6 h and
12 h, the percentage of released BSA decreased markedly. At the end of the
consecutive incubation in the three simulated fluids, the cumulative BSA releases of
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the microcapsules separated in pH 3.0 were 67.60% and 38.34% respectively for the
two crosslinking durations, and those of the microcapsules prepared in pH 4.5 and 6.0
were only 30.40%, 28.69% and 24.43%, 24.50% respectively. These results indicated
that the BSA release profile in the simulated gastrointestinal fluids could be tailored
by varying the coacervation acidity or crosslinking degree, but excessive crosslinking
could greatly reduce the liberation of BSA to the intestine, though it effectively

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inhibited BSA leakage to the simulated gastric solution.
The swelling behavior – drug release profile relationship observed in the O-CMC

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– GA coacervates was inconsistent with the finding that higher SR often led to more
drug release [22, 31], though such inconsistency has also been reported in the folate –

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chitosan nanogels [32]. NU
pH3.0
100 2h 6h 12h
80
60
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40
20
Cumulative BSA release (%)

60
pH4.5
50
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40
30
20
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10
0
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70 pH6.0
60
50
40
30
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20
10
0
0 2 4 6 8 10
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Incubation duration (h)

Fig. 4. Release profile of BSA from the O-CMC – GA microcapsules prepared in pH

3.0 (top), 4.5 (middle), and 6.0 (bottom) as well as crosslinked by 0.5 mg/mL
glutaraldehyde for 2 h, 6 h, or 12 h.
3.3. SEM
To explore the possible mechanism to the different pH-responsive and drug
release behavior caused by coacervation acidity and crosslinking degree,
microstructural observations were carried out. The SEM micrographs of the O-CMC –
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GA coacervates crosslinked by 0.5 mg/mL glutaraldehyde for 2 h, 6 h, and 12 h were

illustrated in Fig. 5. It could be seen that all the coacervates were highly porous and
the network structure of the coacervates separated in pH 6.0 and 4.5 were more
compact than that of pH 3.0; besides, as the glutaraldehyde crosslinking duration
increased to 12 h, the network structures became much more intensified, especially
regarding that prepared in pH 6.0.

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2h 6h 12 h

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pH 3.0

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pH 4.5
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pH 6.0
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Fig. 5. SEM micrographs of the O-CMC – GA coacervates separated in pH 3.0 (top

row), 4.5 (middle row), and 6.0 (bottom row) after crosslinking by 0.5 mg/mL
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glutaraldehyde for 2 h (left column), 6 h (middle column), and 12 h (right column).

3.4. TEM
The coacervation acidity could influence the water content and consequently the
structure compactness of coacervates [33]. The O-CMC – GA coacervates crosslinked
by 0.5 mg/mL glutaraldehyde for 2 h were selected as representatives for TEM
observation and the results were given in Fig. 6. It could be clearly seen that as the
coacervation pH increased from 3.0 to 6.0, the size of dark area in the image increased
obviously. The dark and light areas represented the matrix and cavity in the
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coacervates respectively. Hence, the complexes prepared in pH 3.0 had the lowest
matrix density, followed by pH 4.5 and 6.0 in sequence. This result was consistent
with a previous finding that the O-CMC – GA coacervates separated in pH 3.0, 4.5,
and 6.0 were watery, gel-like, and elastic respectively [34]. Higher matrix density
implied more intensified network structure and effective protection from drug leakage.
This was possibly a main reason why more BSA was released from the microcapsules

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separated in pH 3.0.

a b c

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Fig. 6. SEM micrographs of the O-CMC – GA coacervates separated in pH 3.0 (a),

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4.5 (b), and 6.0 (c) after crosslinking by 0.5 mg/mL glutaraldehyde for 2 h.
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3.5. Viscoelasticity
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The viscoelasticity measurement results (Fig. 7) agreed very well with the SEM
(Fig. 5) and TEM (Fig. 6) observations. All the coacervates exhibited mainly the
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elastic behavior as evidenced by higher G′ than G′′ and the modulus values increased
with glutaraldehyde crosslinking duration elongation. Coacervation in pH 6.0 led to
the highest G′&G′′ values, followed by pH 4.5 and 3.0 sequence. High elasticity could
hinder the migration of BSA from the inside of the coacervates and consequently the
BSA release behavior as a function of coacervation acidity mentioned above occurred.

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2 h, G' 2 h, G'' pH3.0

102
3 6 h, G' 6 h, G''
8x102 12h, G' 12h, G''
6x10
2
4x10
2
2x10

pH4.5
G'&G'' (Pa)

3
10

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3
pH6.0
5x10
3
4x10

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3
3x10
3
2x10
3
1x10

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1
10
Frequency (Hz)
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Fig. 7. Viscoelasticity of the O-CMC – GA coacervates separated in pH 3.0 (a), 4.5
(b), and 6.0 (c) after crosslinking by 0.5 mg/mL glutaraldehyde for 2 h, 6 h, and 12 h.
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3.6. CLSM
CLSM has been proved to be an effective tool to visualize and quantify polymer
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distribution in coacervates [35]. The coacervates crosslinked by 0.5 mg/mL

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glutaraldehyde for 2 h were selected to see their difference in coacervate composition.

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According to Fig. 8, we can see that as the coacervation acidity increased from 3.0 to
6.0, the intensity of the green fluorescence, corresponding to the O-CMC content,
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increased accordingly. The inconsistency between the swelling behavior and BSA
release profile could possibly be ascribed to the different O-CMC contents in the
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coacervates. The water adsorption of the O-CMC – GA coacervates was mainly

contributed by O-CMC [22] and consequently the coacervates separated in pH 6.0 had
the greatest swelling due to its highest O-CMC content; nevertheless, the crosslinking
reaction also occurred to O-CMC and hence the coacervates should have the most
compact network structure, which could be confirmed by the SEM, TEM, and
viscoelasticity analysis results. The crosslinked network structure could effectively
trap BSA in the microcapsules, leading to the reversed relationship between the
swelling behavior and BSA release profile
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a b c

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Fig. 8. Merged CLSM images of the O-CMC – GA coacervates separated in pH 3.0
(a), 4.5 (b), and 6.0 (c). Green and red fluorescence represented O-CMC and GA

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respectively.

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4. Conclusion
The potency of the glutaraldehyde-crosslinked O-CMC – GA coacervates as an
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intestine-targeted delivery system was investigated. The coacervates displayed
different swelling ratios in simulated gastrointestinal fluids and could release most
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model drug to the simulated intestine and colon solutions under appropriate
conditions. The coacervation acidities and crosslinking degree greatly affected the
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swelling and BSA release profile. As the coacervation acidity increased from pH 6.0
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to 3.0, the swelling ratios of the coacervates in the simulated gastrointestinal fluids
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decreased accordingly, but the accumulative BSA release increased in contrast. The
mechanism for such differences was explored by microstructural analysis. SEM, TEM,
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and CLSM revealed that the different matrix density and O-CMC content were
possibly the major reasons for the swelling and BSA release behaviors observed in the
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three coacervation acidities. It was concluded that the OCMC – GA coacervates could
be used for the intestine-targeted delivery of hydrophobic compounds and the
required drug release profile can be designed by selecting an appropriate coacervation
acidity and crosslinking agent/degree.
Conflict of interest
None declared.
Acknowledgements
We gratefully acknowledge the financial support from the National Natural
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Science Foundation of China (grant number 31571890) and the Shandong Provincial
Natural Science Foundation (grant number ZR2015CM037).
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Highlights

 Crosslinked O-CMC – GA coacervates swell more severely in the gastric fluid.

 Higher coacervation acidity leads to greater swelling degree but less BSA
release.
 Coacervation acidity affects the matrix density and composition of coacervates.
 O-CMC – GA coacervates can deliver hydrophobic compounds to the intestine.

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 The delivery potency of O-CMC coacervates varies with coacervation acidity.

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