Accepted Manuscript Title: Horseradish peroxidase-catalyzed formation of hydrogels from chitosan and poly(vinyl alcohol) derivatives both possessing phenolic hydroxyl groups Author: Shinji Sakai Mehdi Khanmohammadi Ali Baradar Khoshfetrat Masahito Taya PII: DOI: Reference:
S0144-8617(14)00476-7 http://dx.doi.org/doi:10.1016/j.carbpol.2014.05.010 CARP 8873
To appear in: Received date: Revised date: Accepted date:
10-3-2014 2-5-2014 7-5-2014
Please cite this article as: Sakai, S., Khanmohammadi, M., Khoshfetrat, A. B., & Taya, M.,Horseradish peroxidase-catalyzed formation of hydrogels from chitosan and poly(vinyl alcohol) derivatives both possessing phenolic hydroxyl groups, Carbohydrate Polymers (2014), http://dx.doi.org/10.1016/j.carbpol.2014.05.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 2
Horseradish peroxidase-catalyzed formation of hydrogels from
4
chitosan and poly(vinyl alcohol) derivatives both possessing
5
phenolic hydroxyl groups
ip t
3
cr
6
8
us
7
Shinji Sakaia*, Mehdi Khanmohammadib, Ali Baradar Khoshfetratb, Masahito Tayaa
10
an
9 a
Division of Chemical Engineering, Department of Materials Science and Engineering,
Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho,
12
Toyonaka, Osaka 560-8531, Japan
Department of Chemical Engineering, Sahand University of Technology, Tabriz
51335-1996, Iran
Ac ce p
15
d
14
b
te
13
M
11
16
*Corresponding author: Shinji Sakai
17
E-mail: [email protected]
18
TEL: +81-6-6850-6252, FAX: +81-6-6850-6254
19 20 21 22
Highlights
23
•Hydrogels were obtained from chitosan and PVA derivatives both possessing Ph groups.
24
•Hydrogelation was achieved through horseradish peroxidase-catalyzed reaction
25
•Gelation time could be controlled from 20 s to 2 min.
26
•Fibroblastic cells spread on hydrogel of 3% chitosan and 1% PVA derivatives. 0 Page 1 of 25
1 2 3 4
•E. coli cell growth was inhibited on hydrogel of 3% chitosan and 1% PVA derivatives.
ip t
5 6
cr
7 8 Abstract
us
9
Horseradish peroxidase-catalyzed cross-linking was applied to prepare hydrogels
11
from aqueous solutions containing chitosan and poly(vinyl alcohol) derivatives both
12
possessing phenolic hydroxyl groups (denoted as Ph-chitosan and Ph-PVA,
13
respectively). Comparing the hydrogels prepared from the solution of 1.0% (w/v)
14
Ph-chitosan and 3.0% (w/v) Ph-PVA and that of 3.0% (w/v) Ph-chitosan and 1.0%
15
(w/v) Ph-PVA, the gelation time of the former hydrogel was 47 s, while was 10 s
16
shorter than that of the latter one. The breaking point for the former hydrogel under
17
stretching (114% strain) was approximately twice larger than that for the latter one.
18
The swelling ratio of the former hydrogel in saline was about half of the latter one.
19
Fibroblastic cells did not adhere on the former hydrogel but adhered and spread on
20
the former one. The growth of Escherichia coli cells was fully suppressed on the latter
21
hydrogel during 48 h cultivation.
M
d
te
Ac ce p
22
an
10
23
Keywords: Chitosan; Poly (vinyl alcohol); Horseradish peroxidase; Enzymatic
24
cross-linking; Hydrogel; Antibacterial activity
25 26 1 Page 2 of 25
1 2 3
ip t
4 5
cr
6 7 1. Introduction
us
8
an
9
Preparation of hydrogels by blending or conjugation of two or more different polymers
11
has been widely studied because of the unique properties of the resultant hydrogels.
12
Hydrogels composed of both chitosan and poly(vinyl alcohol) (PVA) are one such
13
material that shows unique properties that cannot be obtained using either of the
14
components alone (Koyano et al., 1998; Kweon & Kang, 1999; Liang et al., 2009;
15
Islam et al., 2012; Zhao et al., 2003). For example, Koyano et al. reported that
16
adhesion and growth of fibroblast cells on chitosan/PVA composite hydrogels can be
17
controlled by altering the ratio of chitosan and PVA (Koyano et al., 1998). PVA is a
18
water-soluble polymer that has excellent chemical resistance and physical properties.
19
High biocompatibility and durability are attractive characteristics of PVA and
20
PVA-based hydrogels when applying the materials to the biomedical field (Suciu et al.,
21
2004). Chitosan is a cationic polysaccharide, which is produced industrially by
22
deacetylation of chitin and used widely in the food industry. At present, interest is
23
growing in the use of chitosan in the medical and pharmaceutical fields because of its
24
high biocompatibility, biodegradability, and antimicrobial property (Peniche et al.,
25
2003; George & Abraham, 2006; Muzzarelli, 2009; Muzzarelli & Muzzarelli, 2009). For
Ac ce p
te
d
M
10
2 Page 3 of 25
the conjugation of chitosan and PVA, a variety of methods have been reported such
2
as irradiation by electron beam (Zhao et al., 2003) or ultraviolet light (Sabaa et al.,
3
2012) as well as chemical cross-linking with glutaraldehyde (Abdeen, 2011; Costa et
4
al., 2009). In this study, we applied horseradish peroxidase (HRP)-catalyzed
5
cross-linking to obtain hydrogels from derivatives of chitosan and PVA (Fig. 1). To
6
achieve cross-linking by HRP, chitosan and PVA derivatives possessing phenolic
7
hydroxyl (Ph) groups, denominated as Ph-chitosan and Ph-PVA, respectively, were
8
used. Cross-linking of molecules by enzymes including HRP has also attracted
9
increasing attention as a possible environmentally friendly route for polymerization of
an
us
cr
ip t
1
molecules (Muzzarelli et al., 1994; Gross et al., 2001; Kobayashi et al., 2001;
11
Rocasalbas et al., 2013). Because of the mild reaction conditions of enzymes, such
12
cross-linking systems are applicable to encapsulation of biomolecules and even living
13
cells in hydrogels for applications in pharmaceutical and biomedical fields (Lee et al.,
14
2009; Sakai et al., 2007; Ito et al., 2003). HRP is a heme protein that catalyzes the
15
conjugation of phenol and aniline derivatives in the presence of H2O2 (Kobayashi et
16
al., 2001). The major advantage of HRP, compared with other enzymes reported to be
17
effective for obtaining hydrogels such as transglutaminase and tyrosinase, is more
18
rapid hydrogelation (Teixeira et al., 2012).
20 21
d
te
Ac ce p
19
M
10
2. Materials and methods
22
2.1. Materials
23
A PVA derivative possessing carboxyl groups (PVA-COOH; AF-17, degree of
24
hydrolysis: >96.5 mol%, viscosity of 4% solution: 30 mPa s) and chitosan (Chitosan
25
LL, deacetylation: 0.80, MW: 50-100 kDa) were kindly donated by the Japan Vam & 3 Page 4 of 25
Poval Co., Ltd. (Osaka, Japan) and Yaizu Suisankagaku Ind. (Shizuoka, Japan),
2
respectively. HRP (190 U/mg), H2O2 aqueous solution (30% (w/w)),
3
N-hydroxysulfosuccinimide (NHS), lactobionic acid, 3-(p-hydroxyphenyl) propionic
4
acid, and tetramethylethylenediamine were purchased from Wako Pure Chemical
5
Industries (Osaka, Japan). Tyramine hydrochloride, 3-(4-hydroxyphenyl) propionic
6
acid, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were purchased from
7
Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) and the Peptide Institute (Osaka,
8
Japan), respectively.
us
cr
ip t
1
an
9 2.2. Synthesis of Ph-PVA and Ph-chitosan
11
Ph-PVA was synthesized based on a method described elsewhere (Sakai et al., 2013).
12
Briefly, PVA-COOH and tyramine hydrochloride were each dissolved in 100 mM
13
morpholinoethanesulfonic acid (MES) buffer (pH 6.0) at 3.1% (w/w). To this solution,
14
NHS and EDC were sequentially added at 87 and 174 mM, respectively, and then
15
stirred for 20 h at 40°C. Two Ph-chitosan specimens differing in Ph content were
16
synthesized based on a reported method (Sakai et al., 2009a). Briefly, chitosan was
17
dissolved in an 18.4 mM HCl aqueous solution at 7.6% (w/w). To this solution,
18
tetramethylethylenediamine was added at 1.1% (w/w). After adjusting pH to 4.8,
19
lactobionic acid and 3-(4-hydroxyphenyl) propionic acid were added at 0.04 and 1.5%
20
(w/w), respectively. To this solution, EDC was added at 0.3 or 1.0% (w/w), and then
21
stirred for 20 h at 40°C. The Ph-chitosan obtained by adding 0.3 and 1.0% (w/w) EDC
22
were denominated as PhL- and PhH-chitosan, respectively. A specimen obtained
23
without adding 3-(4-hydroxyphenyl) propionic acid was denominated as NPh-chitosan.
24
The resultant solution containing chitosan derivative was gradually dripped into 90%
25
(v/v) ethanol/10% (v/v) water for precipitation of the polymer. The precipitated polymer
Ac ce p
te
d
M
10
4 Page 5 of 25
was washed with 90% (v/v) ethanol/10% (v/v) water until an ultraviolet visible
2
spectrum peak at 275 nm attributed to residual tyramine was undetectable in the
3
supernatant fluid after centrifugation. The resultant polymers were then vacuum dried.
4
The contents of Ph groups in the resultant Ph-PVA, PhL-chitosan, and PhH-chitosan
5
measured using 1H NMR were 2.9 10-4, 2.0 10-4, and 3.9 10-4 mol-Ph/g,
6
respectively.
cr
ip t
1
7 2.3. Gelation time
9
Gelation time of polymer solutions obtained by dissolving Ph-chitosan and/or Ph-PVA
an
us
8
in Krebs Ringer Hepes-buffered saline (KRH, pH 7.4) was determined based on a
11
previously reported method (Sakai & Kawakami, 2007). A solution containing either or
12
both of Ph-chitosan and/or Ph-PVA at 5% (w/v) total polymer content was poured into
13
a 48-well plate at 400 L/well. The content of each polymer is shown in Table 1.
14
Subsequently, 100 L of phosphate-buffered saline (pH 7.4, PBS) containing HRP
15
and H2O2 was poured into each well and stirred using a magnetic stirrer bar (10-mm
16
length 4-mm diameter). When the magnetic stirring was hindered and the surface of
17
the solution swelled, this was considered to signal the formation of the gel state. The
18
final concentrations of polymer(s), HRP and H2O2 were fixed at 4% (w/v), 1 U/mL and
19
1 mM, respectively. The means and standard deviations of quadruplets were reported.
d
te
Ac ce p
20
M
10
21
2.4. Mechanical properties
22
Cuboid hydrogels, (10 mm in width 40 mm in length 3 mm in thickness), were
23
prepared for extension studies by pouring KRH containing Ph-chitosan and/or Ph-PVA,
24
HRP (1 U/mL), and H2O2 (1 mM) at the polymer concentrations shown in Table 1 into
25
molds. After 12 h of standing at room temperature in humidified air, the specimens 5 Page 6 of 25
taken from the molds were placed in a Table-Top Material Tester (EZ-Test; Shimadzu,
2
Kyoto, Japan). The specimens were cut to a 20-mm length and clamped at both ends.
3
The length between the clamps was set at 10 mm. The extension-stress profile was
4
measured by stretching a specimen at 20 mm/min until rupture. The means and
5
standard deviations were determined by the test using five specimens prepared
6
separately under each condition.
cr
ip t
1
7 2.5. Swelling study
9
Column-shaped hydrogels, 11 mm in diameter and 5 mm in thickness, were prepared
an
us
8
by pouring the polymeric solutions shown in Table 1 at 1 U/mL HRP and 1 mM H2O2
11
into molds. After 12 h of standing at room temperature in humidified air, the hydrogels
12
taken from the molds were soaked in 25-times larger volumes of PBS at room
13
temperature. The changes in diameter and height of the column-shaped hydrogels
14
were measured every day for 6 days. The swelling ratio was calculated using the
15
following equation: Swelling ratio = (volume of hydrogel after soaking in PBS)/(volume
16
of hydrogel before soaking in PBS). Five specimens prepared separately were tested
17
for each condition. The means and standard deviations were reported.
d
te
Ac ce p
18
M
10
19
2.6. Fibroblastic cell behavior
20
Hydrogels were prepared under the conditions shown in Table 1 by pouring 0.5 mL
21
solution to a 12-well tissue culture dish, so as to completely cover the bottom of a well
22
before hydrogelation. After 12 h of standing at room temperature in humidified air, 6
23
mL of Dulbecco’s modified eagles medium (DMEM, Nissui Pharmaceutical, Tokyo,
24
Japan) with 10% (v/v) fetal bovine serum (FBS) was poured into the well and allowed
25
to stand for 10 min. This step was repeated four times to remove remaining H2O2 and 6 Page 7 of 25
replace KRH with the medium. DMEM with 10% (v/v) FBS containing mouse embryo
2
fibroblast-like cell line 10T1/2 cells (Riken Cell Bank, Igaragi, Japan) was then poured
3
into each well at 2104 cells/well. At 14 h after cell seeding, adhesion of the cells on
4
hydrogels was observed using an optical microscope.
ip t
1
5 2.7. E. coli behavior
7
Hydrogels were prepared under the conditions shown in Table 1 in a plastic petri dish
8
(35 mm in diameter) by pouring 1.5 mL of mixture solution before hydrogelation. After
9
12 h of standing at room temperature in humidified air, 10 mL of Lysogeny Broth (LB)
10
medium was poured into the dish and allowed to stand for 30 min. After repeating the
11
step four times to replace the solution in hydrogel to LB medium, the dish was turned
12
out onto a sterilized paper to remove the remaining medium from the hydrogel. Fifty
13
microliters LB medium containing E. Cole MM294 cells pre-cultured for 1 day in an
14
Erlenmeyer flask (1 L) containing 200 mL medium at 37°C using a reciprocal shaker
15
(100 rpm, 4 cm amplitude) was then spread on each hydrogel using a spreader.
16
Colony development was evaluated after 48 h of incubation at 37°C.
18 19
us
an
M
d
te
Ac ce p
17
cr
6
3. Results and discussion
20
3.1. Hydrogelation
21
An attractive feature of HRP-catalyzed hydrogelation is the mild reaction conditions
22
that are suitable for mammalian cells. A potential application of Ph-chitosan/Ph-PVA
23
hydrogels obtained through a cell-friendly hydrogelation system is injectable scaffolds
24
for tissue engineering, tissue-reconstructions, including wound healing, and drug
25
delivery (Teixeira et al., 2012). For these applications, knowledge of the effect of the 7 Page 8 of 25
contents of Ph-chitosan and Ph-PVA on the time taken for hydrogelation (gelation
2
time) is important. As shown in Fig. 2, the content of each polymer influenced the
3
gelation time. At 4.0% (w/v) total polymer concentration of either or both of Ph-PVA
4
and/or PhL-chitosan, gelation time decreased with increasing PhL-chitosan content.
5
The longest and shortest gelation times were detected at 4% (w/v) Ph-PVA and 4%
6
(w/v) PhL-chitosan, respectively. Focusing on the Ph content, the trend obtained for
7
the Ph-PVA and PhL-chitosan system was that the gelation time decreased with
8
decreasing Ph group content (Fig. 2, Table 1). However, the opposite trend was
9
observed for single-component solutions of 4Ph-PVA and 2Ph-PVA in this study and
an
us
cr
ip t
1
our previous results, including Ph-chitosan (Sakai et al., 2009a, b). The independence
11
of the content of Ph groups was also shown by shorter gelation times of
12
2PhH-chitosan/2Ph-PVA solution with the higher content of Ph groups compared with
13
that of 2PhL-chitosan/2Ph-PVA. These findings indicate that the gelation time of a
14
mixed system can be estimated from the times taken for gelation of solutions
15
containing a single component, but not from the content of Ph groups in the solution.
d
te
Ac ce p
16
M
10
17
3.2. Mechanical properties and swelling in PBS
18
Blending of PVA with chitosan has been used to improve the mechanical properties of
19
matrices such as films and hydrogels containing chitosan (Liang et al., 2009; Chen et
20
al., 2013; Takei et al., 2013). We investigated the effect of conjugation by the
21
HRP-catalyzed reaction on the mechanical properties of the resultant hydrogels by
22
measuring the stresses and breaking points under tensile strain (stretching). As
23
shown in Fig. 3, the 4PhL-chitosan hydrogel broke at ca. 50% strain, while the
24
breaking point for 4Ph-PVA hydrogel occurred at ca. 150% strain. The strain at rupture
25
for PhL-chitosan/Ph-PVA conjugate hydrogels varied between the values obtained for 8 Page 9 of 25
4PhL-chitosan and 4Ph-PVA and the value increased with increasing Ph-PVA content.
2
These results indicate that conjugation through the HRP-catalyzed reaction was
3
effective for improving the brittleness of Ph-chitosan hydrogels, likewise in the case of
4
hydrogels prepared by non-enzymatic cross-linking methods (Liang et al., 2009; Chen
5
et al., 2013; Takei et al., 2013). Regarding the stress at rupture, the value for
6
1PhL-chitosan/3Ph-PVA was larger than those for 4PhL-chitosan and 4Ph-PVA. This
7
result indicates that conjugation is effective for obtaining hydrogels with superior
8
durability towards deformation, which cannot be predicted from the results for the
9
hydrogels composed of Ph-PVA or Ph-chitosan alone. Regarding the effect of the Ph
10
content in Ph-chitosan, a more brittle hydrogel was obtained from Ph-chitosan with a
11
higher content of Ph groups. This result may be explained by the hindrance of
12
deformation of the highly cross-linked network under mechanical stress, and the
13
faster gelation rate of 2PhH-chitosan/2Ph-PVA than those for 2PhL-chitosan/2Ph-PVA
14
and 2NPh-chitosan/2Ph-PVA (Fig. 2) inducing a less uniform microscopic structure.
15
The formation of hydrogels showing lower resistance to deformation under conditions
16
giving faster hydrogelation was reported for a variety of hydrogels obtained through
17
non-enzymatic cross-linking methods, such as alginate (Kuo & Ma, 2001) and
18
collagen hydrogels (Achilli & Mantovani, 2010).
cr
us
an
M
d
te
Ac ce p
19
ip t
1
For potential applications of Ph-chitosan/Ph-PVA hydrogels to the biomedical field,
20
knowledge of their swelling properties in saline is important. We investigated this
21
property by measuring the volume change of hydrogels. 4Ph-PVA hydrogel did not
22
swell markedly during 6 days of soaking in PBS (Fig. 4). In contrast, the degree of
23
swelling of 4PhL-chitosan hydrogel was the largest amongst the hydrogels examined
24
in this study. The degree of swelling decreased with increasing Ph-PVA content in 4%
25
total-polymer hydrogels. This finding may be attributed to the formation of H-bonds 9 Page 10 of 25
between the amino groups in Ph-chitosan and the OH-groups in Ph-PVA
2
(Abdel-Mohsen et al., 2011). A less homogeneous microscopic structure of the
3
hydrogel with the higher content of PhL-chitosan induced by faster hydrogelation may
4
also be a reason for this behavior (Fig. 2). The same tendency was reported for
5
chitosan/PVA hydrogels obtained through non-enzymatic cross-linking methods such
6
as repeated freezing-thawing (Abdel-Mohsen et al., 2011), ultraviolet irradiation (Kim
7
et al., 2003), and gamma ray irradiation (Tahtat et al., 2011).
3.3. Adherent mammalian cells and E. coli cell behaviors
an
9
us
8
cr
ip t
1
Chitosan (Chandy & Sharma, 1990; Jarmila & Vavrikova, 2011) and PVA (Suciu et al.,
11
2004) have been investigated as promising materials for biomedical applications. To
12
evaluate the usefulness of bioconjugated Ph-chitosan/Ph-PVA hydrogels in the
13
biomedical field, we examined fibroblastic cell adhesion and antibacterial properties of
14
Ph-chitosan/Ph-PVA hydrogels prepared under different conditions. Figure 5 shows
15
10T1/2 cells 14 h after seeding onto hydrogels composed of either or both of
16
Ph-chitosan and/or Ph-PVA. The cells did not adhere onto 4Ph-PVA (Fig. 5A) or
17
1PhL-chitosan/3Ph-PVA (Fig. 5B) hydrogels and formed clusters. The cells on
18
2PhL-chitosan/2Ph-PVA adhered but did not spread (Fig. 5C). Spreading of the cells
19
was induced by increasing the content of PhL-chitosan in 3PhL-chitosan/1Ph-PVA
20
(Fig. 5D), but the degree of spreading was lower than those on a cell culture dish (Fig.
21
5G). Further increase in the content of PhL-chitosan, i.e., 4PhL-chitosan hydrogel,
22
decreased the degree of spreading (Fig. 5E). Difficulty in adhesion and spreading of
23
adherent cells has been reported for chitosan and chitosan derivative hydrogels
24
obtained through non-enzymatic cross-linking methods (Ono et al., 2000; Takei et al.,
25
2012). A greater spreading of 10T1/2 cells on 2PhL-chitosan/2Ph-PVA hydrogel than
Ac ce p
te
d
M
10
10 Page 11 of 25
those on 4PhL-chitosan hydrogel may be induced by the moderate cationic property
2
resulting from the conjugation with an appropriate amount of Ph-PVA. These results
3
suggest the possibility of controlling cell adhesion on the hydrogels by altering the
4
content of PhL-chitosan and Ph-PVA for an intended application.
5
ip t
1
Antibacterial properties are one of the attractive features of chitosan for applications in the biomedical field (Jarmila & Vavrikova, 2011; Jarry et al., 2001; Tahtat et al.,
7
2011). We evaluated the biocidal impact of Ph-chitosan/Ph-PVA hydrogels on E. coli
8
cells. Figure 6 shows the hydrogels 48 h after seeding E. coli cells. Interestingly, the
9
hydrogel containing PhL-chitosan at 2% (w/v) or more suppressed the growth of E.
an
us
cr
6
coli (Fig. 6C–E). This result can be explained by the reported mechanism of
11
antibacterial action by chitosan caused by the formation of electrostatic bonds
12
between chitosan and anionic groups on the bacterial surface membrane, which
13
would result in altered membrane permeability (Tsai & Su, 1999; Yang et al., 2008;
14
Jarmila & Vavrikova, 2011). In our specimens, it is intuitive that hydrogels with an
15
abundance of Ph-chitosan show greater cationic properties. The growth of E. coli on
16
2PhH-chitosan/2Ph-PVA (Fig. 6F) but not on 2PhL-chitosan/2Ph-PVA (Fig. 6C) can
17
also be explained by the difference in the degree of cationic strength. The degree of
18
substitution of amino groups in PhL-chitosan was less than that in PhH-chitosan.
19
These results indicate that the content and degrees of modification of Ph-chitosan in
20
Ph-chitosan/Ph-PVA hydrogels should be carefully chosen for applications in which
21
antibacterial properties are desired.
Ac ce p
te
d
M
10
22 23
4. Conclusions
24 25
Ph-chitosan/Ph-PVA hydrogels were prepared through the HRP-catalyzed 11 Page 12 of 25
cross-linking between Ph groups at different Ph-chitosan and Ph-PVA contents.
2
Gelation time was dependent on the content of each polymer, but independent of the
3
content of Ph groups in the solution. At 4.0% (w/v) total polymer concentration of
4
either or both of Ph-PVA and/or PhL-chitosan, gelation time decreased with increasing
5
Ch-PhL content. The longest and shortest gelation time detected for 4% (w/v) Ph-PVA
6
and 4% (w/v) PhL-chitosan were 73 s and 28 s, respectively. Swelling of the hydrogels
7
can be suppressed by increasing the Ph-PVA content. The swelling ratio of
8
3PhL-chitosan/1Ph-PVA hydrogel was 1.9 at 6 days of soaking in PBS. The value
9
decreased to 1.2 at 1PhL-chitosan/3Ph-PVA hydrogel. Fibroblastic cell adhesion and
an
us
cr
ip t
1
antibacterial activity studies demonstrated that these bioactivities can be controlled by
11
altering the Ph-chitosan and Ph-PVA content in the hydrogels. To obtain hydrogels
12
with greater cell adhesiveness, it was shown that an appropriate Ph-chitosan and
13
Ph-PVA content is necessary. The cells on 3PhL-chitosan/1Ph-PVA hydrogel were the
14
most spreading compared to those on 2PhL-chitosan/2Ph-PVA and 4PhL-chitosan
15
hydrogels. To obtain the hydrogels with greater antibacterial activity, it was shown that
16
a higher Ph-chitosan content is necessary. E. coli cells grew on
17
1PhL-chitosan/3Ph-PVA hydrogel but did not grow on 2PhL-Chitosan/2Ph-PVA
18
hydrogel during 48h cultivation. These results demonstrate the feasibility of
19
HRP-catalyzed hydrogelation for obtaining the hydrogels having the unique properties
20
attributed to the conjugation of Ch-chitosan and Ch-PVA which cannot be obtained
21
using either of the components alone.
Ac ce p
te
d
M
10
22 23
References
24
Abdeen, Z. (2011). Swelling and reswelling characteristics of cross-linked poly(vinyl
25
alcohol)/chitosan hydrogel film. J Disper Sci Technol, 32, 1337-1344. 12 Page 13 of 25
1
Abdel-Mohsen, A.M., Aly, A.S., Hrdina, R., Montaser, A.S., & Hebeish, A. (2011).
2
Eco-synthesis of PVA/chtiosan hydrogels for biomedical application. J Polym
3
Environ, 19, 1005-1012. Achilli, M., & Mantovani, D. (2010). Tailoring mechanical properties of collagen-based
5
scaffolds for vascular tissue engineering: The effects of pH, temperature and ionic
6
strength on gelation. Polymers, 2, 664-680.
cr
8
Chandy, T., & Sharma, C.P. (1990). Chitosan–as a biomaterial. Biomater Artif Cells Artif Organs, 18, 1-24.
us
7
ip t
4
Chen, C., Liu, L., Huang, T., Wang, Q., & Fang, Y. (2013). Bubble template fabrication
10
of chitosan/poly(vinyl alcohol) sponges for wound dressing applications. Int J Biol
11
Macromol, 62, 188-193.
M
12
an
9
Costa, E.S., Barbosa-Stancioli, E.F., Mansur, A.A.P., Vasconcelos, W.L., & Mansur, H.S. (2009). Preparation and characterization of chitosan/poly(vinyl alcohol)
14
chemically crosslinked blends for biomedical applications. Carbohyd Polym, 76,
15
472-481.
te
d
13
George, M., & Abraham, T.E. (2006). Polyionic hydrocolloids for the intestinal delivery
17
of protein drugs: Alginate and chitosan–a review. J Control Release, 114, 1-14.
18 19 20
Ac ce p
16
Gross, R.A., Kumar, A., & Kalra, B. (2001). Polymer synthesis by in vitro enzyme catalysis. Chem Rev, 101, 2097-2124. Islam, A., Yasin, T., Bano, I., & Riaz, M. (2012). Controlled release of aspirin from
21
pH-sensitive chitosan/poly(vinyl alcohol) hydrogel. J Appl Polym Sci, 124,
22
4184-4192.
23
Ito, A. et al. (2003). Transglutaminase-mediated gelatin matrices incorporating cell
24
adhesion factors as a biomaterial for tissue engineering. J Biosci Bioeng, 95,
25
196-199. 13 Page 14 of 25
2 3 4 5
Jarmila, V., & Vavrikova, E. (2011). Chitosan derivatives with antimicrobial, antitumour and antioxidant activities–a review. Curr Pharm Des, 17, 3596-3607. Jarry, C. et al. (2001). Effects of steam sterilization on thermogelling chitosan-based gels. J Biomed Mater Res, 58, 127-135.
ip t
1
Kim, S.J., Park, S.J., & Kim, S.I. (2003). Swelling behavior of interpenetrating polymer network hydrogels composed of poly(vinyl alcohol) and chitosan. React Funct
7
Polym, 55, 53-59.
us
9
Kobayashi, S., Uyama, H., & Kimura, S. (2001). Enzymatic polymerization. Chem Rev, 101, 3793-3818.
an
8
cr
6
Koyano, T., Minoura, N., Nagura, M., & Kobayashi, K. (1998). Attachment and growth
11
of cultured fibroblast cells on chitosan-blended hydrogels. J Biomed Mater Res, 39,
12
486-490.
Kuo, C.K., & Ma, P.X. (2001). Ionically crosslinked alginate hydrogels as scaffolds for
d
13
M
10
tissue engineering: part 1. Structure, gelation rate and mechanical properties.
15
Biomaterials, 22, 511-521.
17 18
Kweon, D.K., & Kang, D.W. (1999). Drug-release behavior of chitosan-g-poly(vinyl
Ac ce p
16
te
14
alcohol) copolymer matrix. J Appl Polym Sci, 74, 458-464. Lee, F., Chung, J.E., & Kurisawa, M. (2009). An injectable hyaluronic acid-tyramine
19
hydrogel system for protein delivery. J Control Release, 134, 186-193.
20
Liang, S., Liu, L., Huang, Q., & Yam, K.T. (2009). Preparation of single or
21
double-network chitosan/poly(vinyl alcohol) gel films through selectively
22
cross-linking method. Carbohyd Polym, 77, 718-724.
23 24 25
Muzzarelli, R.A.A. (2009). Chitins and chitosans for the repair of wounded skin, nerve, cartilage and bone. Carbohyd Polym, 76, 167-182. Muzzarelli, R.A.A., Ilari, P., Xia, W., Pinotti, M., & Tomasetti, M. (1994). 14 Page 15 of 25
1
Tyrosinase-mediated quinone tanning of chitinous materials. Carbohyd Polym, 24,
2
295-300.
3
Muzzarelli, R.A.A., & Muzzarelli, C. (2009). Chitin and chitosan hydrogels. In G.O. Phillips, and P.A. Williams (Eds.), Handbook of Hydrocolloids. (2nd ed.)
5
(pp.849-888). Cambridge UK. Woodhead Publishing Ltd.
7
Ono, K. et al. (2000). Photocrosslinkable chitosan as a biological adhesive. J Biomed
cr
6
ip t
4
Mater Res, 49, 289-295.
Peniche, C., Arguelles-Monal, W., Peniche, H., & Acosta, N. (2003). Chitosan: An
9
attractive biocompatible polymer for microencapsulation. Macromol Biosci, 3,
12 13
an
Rocasalbas, G. et al. (2013). Laccase-assisted formation of bioactive chitosan/gelatin
M
11
511-520.
hydrogel stabilized with plant polyphenols. Carbohyd Polym, 92, 989-996. Sabaa, M.W., Mohamed, R.R., Eltaweel, S.H., & Seoudi, R.S. (2012). Crosslinked
d
10
us
8
poly(vinyl alcohol)/carboxymethyl chitosan hydrogels for removal of metal ions and
15
dyestuff from aqueous solutions. J Appl Polym Sci, 123, 3459-3469. Sakai, S., Hashimoto, I., Ogushi, Y., & Kawakami, K. (2007). Peroxidase-catalyzed
Ac ce p
16
te
14
17
cell-encapsulation in subsieve-size capsules of alginate with phenol moieties in
18
water-immiscible fluid dissolving H2O2. Biomacromolecules, 8, 2622-2626.
19
Sakai, S., Hirose, K., Taguchi, K., Ogushi, Y., & Kawakami, K. (2009b). An injectable,
20
in situ enzymatically gellable, gelatin derivative for drug delivery and tissue
21
engineering. Biomaterials, 30, 3371-3377.
22 23
Sakai, S., & Kawakami, K. (2007). Synthesis and characterization of both ionically and enzymatically crosslinkable alginate. Acta Biomater, 3, 495-501.
24
Sakai, S. et al. (2013). Polyvinyl alcohol-based hydrogel dressing gellable on-wound
25
via a co-enzymatic reaction triggered by glucose in the wound exudate. J Mater 15 Page 16 of 25
1 2
Chem B, 1, 5067-5075. Sakai, S., Yamada, Y., Zenke, T., & Kawakami, K. (2009a). Novel chitosan derivative soluble at neutral pH and in-situ gellable via peroxidase-catalyzed enzymatic
4
reaction. J Mater Chem, 19, 230-235.
5
ip t
3
Suciu, A.N., Iwatsubo, T., Matsuda, M., & Nishino, T. (2004). A study upon durability of the artificial knee joint with PVA hydrogel cartilage. JSME C, 47, 199-208.
7
Tahtat, D. et al. (2011). Influence of some factors affecting antibacterial activity of
cr
6
PVA/chitosan based hydrogels synthesized by gamma irradiation. J Mater Sci
9
Mater Med, 22, 2505-2512.
an
10
us
8
Takei, T., Nakahara, H., Ijima, H., & Kawakami, K. (2012). Synthesis of a chitosan derivative soluble at neutral pH and gellable by freeze-thawing, and its application
12
in wound care. Acta Biomater, 8, 686-693.
M
11
Takei, T. et al. (2013). Effect of chitosan-gluconic acid conjugate/poly(vinyl alcohol)
14
cryogels as wound dressing on partial-thickness wounds in diabetic rats. J Mater
15
Sci Mater Med, 24, 2479-2487.
te
Teixeira, L.S., Feijen, J., van Blitterswijk, C.A., Dijkstra, P.J., & Karperien, M. (2012).
Ac ce p
16
d
13
17
Enzyme-catalyzed crosslinkable hydrogels: Emerging strategies for tissue
18
engineering. Biomaterials, 33, 1281-1290.
19 20 21
Tsai, G.J., & Su, W.H. (1999). Antibacterial activity of shrimp chitosan against Escherichia coli. J Food Prot, 62, 239-243. Yang, X., Liu, Q., Chen, X., Yu, F., & Zhu, Z. (2008). Investigation of PVA/ws-chitosan
22
hydrogels prepared by combined γ-irradiation and freeze-thawing. Carbohyd
23
Polym, 73, 401-408.
24 25
Zhao, L. et al. (2003). Synthesis of antibacterial PVA/CM-chitosan blend hydrogels with electron beam irradiation. Carbohyd Polym, 53, 439-446. 16 Page 17 of 25
1 2 3
ip t
4 5
cr
6 7 Figure captions
us
8
10
an
9
Fig. 1. Scheme for the formation of Ph-chitosan/Ph-PVA.
M
11
Fig. 2. Gelation times for polymeric solutions of differing composition. Values
13
represent the mean (n = 4) with standard deviation.
te
14
d
12
Fig. 3. Strain-stress profiles of hydrogels obtained from polymeric solutions of differing
16
composition. Values represent the mean (n = 5) with standard deviation.
17
Ac ce p
15
18
Fig. 4. Swelling profiles of hydrogels obtained from polymeric solutions of differing
19
composition. Values represent the mean (n = 5) with standard deviation.
20 21
Fig. 5. Microphotographs of 10T1/2 cells 14 h after seeding on hydrogels obtained
22
from polymeric solutions of differing composition. Bars: 100 m.
23 24
Fig. 6. Photographs of hydrogels obtained from polymeric solutions of differing
25
composition 48 h after seeding E. coli cells. Bars: 1 cm. 17 Page 18 of 25
1 2 3
Table 1. Hydrogel compositions prepared through the HRP-catalyzed reaction.
4 3 2 1 2 2 2
1 2 3 4 -
4 2 -
2 -
1.16 1.07 0.98 0.89 0.80 1.56 1.36 0.58 0.58
us
4Ph-PVA 1PhL-chitosan/3Ph-PVA 2PhL-chitosan/2Ph-PVA 3PhL-chitosan/1Ph-PVA 4PhL-chitosan 4PhH-chitosan 2PhH-chitosan/2Ph-PVA 2NPh-chitosan/2Ph-PVA 2Ph-PVA
Ph content [10-5 mol-Ph/mL]
cr
Concentration [% (w/v)] Chitosan derivative Ph-PVA PhL- PhH- NPh-
6
an
5
ip t
4
Ac ce p
te
d
M
HRP and H2O2 concentrations were fixed at 1 U/mL and 1 mM, respectively.
18 Page 19 of 25
Ac ce p
te
d
M
an
us
cr
ip t
Figure 1
HO
HO
Ph-PVA
Ph-PVA OH
OH
HRP/H2O2 OH
O
HO
HO HO
HO
Ph-chitosan
Page 20 of 25
Ph-chitosan
Ac ce p
te
d
M
an
us
cr
ip t
Figure 2
h 4P
h
1P
C L-
A V P
-
h
ito
sa
2
h 3P / n h
Ph
c L-
A V -P
ito
sa
3
h 2P / n h
Ph
c L-
A V P
-
ito
sa
h 1P / n
A V P
-
4
L Ph
ch
sa o it
-
4
n
H Ph
2
ch
sa o it
-
a
h
Ph
n
c H-
s ito 2
h 2P / n
NP
-
h
c h-
A V P a
s ito
h 2P / n
-
A V P
h 2P
A V P
-
Page 21 of 25
an
us
cr
ip t
Figure 3
M
0.18 㻌4Ph-PVA 㻌1PhL-chitosan/3Ph-PVA 㻌2PhL-chitosan/2Ph-PVA 㻌3PhL-chitosan/1Ph-PVA 㻌4PhL-chitosan 㻌2PhH-chitosan/2Ph-PVA 2NPh-chitosan/2Ph-PVA 㻌
te
d
0.15
Ac ce p
Stress (N)
0.12 0.09 0.06 0.03 0.00 0
40
80
120
160 Page 22 of 25
Strain (%)
2NPh-chitosan/2Ph-PVA
us
3PhL-chitosan/1Ph-PVA 4PhL-chitosan 2PhH-chitosan/2Ph-PVA
Ac ce p
te
d
M
an
4Ph-PVA 1PhL-chitosan/3Ph-PVA 2PhL-chitosan/2Ph-PVA
cr
ip t
Figure 4
d
Page 23 of 25
Ac
ce
pt
ed
M
an
us
cr
i
Figure 5
Page 24 of 25
Ac ce p
te
d
M
an
us
cr
ip t
Figure 6
Page 25 of 25
S0144-8617(14)00476-7 http://dx.doi.org/doi:10.1016/j.carbpol.2014.05.010 CARP 8873
To appear in: Received date: Revised date: Accepted date:
10-3-2014 2-5-2014 7-5-2014
Please cite this article as: Sakai, S., Khanmohammadi, M., Khoshfetrat, A. B., & Taya, M.,Horseradish peroxidase-catalyzed formation of hydrogels from chitosan and poly(vinyl alcohol) derivatives both possessing phenolic hydroxyl groups, Carbohydrate Polymers (2014), http://dx.doi.org/10.1016/j.carbpol.2014.05.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 2
Horseradish peroxidase-catalyzed formation of hydrogels from
4
chitosan and poly(vinyl alcohol) derivatives both possessing
5
phenolic hydroxyl groups
ip t
3
cr
6
8
us
7
Shinji Sakaia*, Mehdi Khanmohammadib, Ali Baradar Khoshfetratb, Masahito Tayaa
10
an
9 a
Division of Chemical Engineering, Department of Materials Science and Engineering,
Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho,
12
Toyonaka, Osaka 560-8531, Japan
Department of Chemical Engineering, Sahand University of Technology, Tabriz
51335-1996, Iran
Ac ce p
15
d
14
b
te
13
M
11
16
*Corresponding author: Shinji Sakai
17
E-mail: [email protected]
18
TEL: +81-6-6850-6252, FAX: +81-6-6850-6254
19 20 21 22
Highlights
23
•Hydrogels were obtained from chitosan and PVA derivatives both possessing Ph groups.
24
•Hydrogelation was achieved through horseradish peroxidase-catalyzed reaction
25
•Gelation time could be controlled from 20 s to 2 min.
26
•Fibroblastic cells spread on hydrogel of 3% chitosan and 1% PVA derivatives. 0 Page 1 of 25
1 2 3 4
•E. coli cell growth was inhibited on hydrogel of 3% chitosan and 1% PVA derivatives.
ip t
5 6
cr
7 8 Abstract
us
9
Horseradish peroxidase-catalyzed cross-linking was applied to prepare hydrogels
11
from aqueous solutions containing chitosan and poly(vinyl alcohol) derivatives both
12
possessing phenolic hydroxyl groups (denoted as Ph-chitosan and Ph-PVA,
13
respectively). Comparing the hydrogels prepared from the solution of 1.0% (w/v)
14
Ph-chitosan and 3.0% (w/v) Ph-PVA and that of 3.0% (w/v) Ph-chitosan and 1.0%
15
(w/v) Ph-PVA, the gelation time of the former hydrogel was 47 s, while was 10 s
16
shorter than that of the latter one. The breaking point for the former hydrogel under
17
stretching (114% strain) was approximately twice larger than that for the latter one.
18
The swelling ratio of the former hydrogel in saline was about half of the latter one.
19
Fibroblastic cells did not adhere on the former hydrogel but adhered and spread on
20
the former one. The growth of Escherichia coli cells was fully suppressed on the latter
21
hydrogel during 48 h cultivation.
M
d
te
Ac ce p
22
an
10
23
Keywords: Chitosan; Poly (vinyl alcohol); Horseradish peroxidase; Enzymatic
24
cross-linking; Hydrogel; Antibacterial activity
25 26 1 Page 2 of 25
1 2 3
ip t
4 5
cr
6 7 1. Introduction
us
8
an
9
Preparation of hydrogels by blending or conjugation of two or more different polymers
11
has been widely studied because of the unique properties of the resultant hydrogels.
12
Hydrogels composed of both chitosan and poly(vinyl alcohol) (PVA) are one such
13
material that shows unique properties that cannot be obtained using either of the
14
components alone (Koyano et al., 1998; Kweon & Kang, 1999; Liang et al., 2009;
15
Islam et al., 2012; Zhao et al., 2003). For example, Koyano et al. reported that
16
adhesion and growth of fibroblast cells on chitosan/PVA composite hydrogels can be
17
controlled by altering the ratio of chitosan and PVA (Koyano et al., 1998). PVA is a
18
water-soluble polymer that has excellent chemical resistance and physical properties.
19
High biocompatibility and durability are attractive characteristics of PVA and
20
PVA-based hydrogels when applying the materials to the biomedical field (Suciu et al.,
21
2004). Chitosan is a cationic polysaccharide, which is produced industrially by
22
deacetylation of chitin and used widely in the food industry. At present, interest is
23
growing in the use of chitosan in the medical and pharmaceutical fields because of its
24
high biocompatibility, biodegradability, and antimicrobial property (Peniche et al.,
25
2003; George & Abraham, 2006; Muzzarelli, 2009; Muzzarelli & Muzzarelli, 2009). For
Ac ce p
te
d
M
10
2 Page 3 of 25
the conjugation of chitosan and PVA, a variety of methods have been reported such
2
as irradiation by electron beam (Zhao et al., 2003) or ultraviolet light (Sabaa et al.,
3
2012) as well as chemical cross-linking with glutaraldehyde (Abdeen, 2011; Costa et
4
al., 2009). In this study, we applied horseradish peroxidase (HRP)-catalyzed
5
cross-linking to obtain hydrogels from derivatives of chitosan and PVA (Fig. 1). To
6
achieve cross-linking by HRP, chitosan and PVA derivatives possessing phenolic
7
hydroxyl (Ph) groups, denominated as Ph-chitosan and Ph-PVA, respectively, were
8
used. Cross-linking of molecules by enzymes including HRP has also attracted
9
increasing attention as a possible environmentally friendly route for polymerization of
an
us
cr
ip t
1
molecules (Muzzarelli et al., 1994; Gross et al., 2001; Kobayashi et al., 2001;
11
Rocasalbas et al., 2013). Because of the mild reaction conditions of enzymes, such
12
cross-linking systems are applicable to encapsulation of biomolecules and even living
13
cells in hydrogels for applications in pharmaceutical and biomedical fields (Lee et al.,
14
2009; Sakai et al., 2007; Ito et al., 2003). HRP is a heme protein that catalyzes the
15
conjugation of phenol and aniline derivatives in the presence of H2O2 (Kobayashi et
16
al., 2001). The major advantage of HRP, compared with other enzymes reported to be
17
effective for obtaining hydrogels such as transglutaminase and tyrosinase, is more
18
rapid hydrogelation (Teixeira et al., 2012).
20 21
d
te
Ac ce p
19
M
10
2. Materials and methods
22
2.1. Materials
23
A PVA derivative possessing carboxyl groups (PVA-COOH; AF-17, degree of
24
hydrolysis: >96.5 mol%, viscosity of 4% solution: 30 mPa s) and chitosan (Chitosan
25
LL, deacetylation: 0.80, MW: 50-100 kDa) were kindly donated by the Japan Vam & 3 Page 4 of 25
Poval Co., Ltd. (Osaka, Japan) and Yaizu Suisankagaku Ind. (Shizuoka, Japan),
2
respectively. HRP (190 U/mg), H2O2 aqueous solution (30% (w/w)),
3
N-hydroxysulfosuccinimide (NHS), lactobionic acid, 3-(p-hydroxyphenyl) propionic
4
acid, and tetramethylethylenediamine were purchased from Wako Pure Chemical
5
Industries (Osaka, Japan). Tyramine hydrochloride, 3-(4-hydroxyphenyl) propionic
6
acid, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were purchased from
7
Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) and the Peptide Institute (Osaka,
8
Japan), respectively.
us
cr
ip t
1
an
9 2.2. Synthesis of Ph-PVA and Ph-chitosan
11
Ph-PVA was synthesized based on a method described elsewhere (Sakai et al., 2013).
12
Briefly, PVA-COOH and tyramine hydrochloride were each dissolved in 100 mM
13
morpholinoethanesulfonic acid (MES) buffer (pH 6.0) at 3.1% (w/w). To this solution,
14
NHS and EDC were sequentially added at 87 and 174 mM, respectively, and then
15
stirred for 20 h at 40°C. Two Ph-chitosan specimens differing in Ph content were
16
synthesized based on a reported method (Sakai et al., 2009a). Briefly, chitosan was
17
dissolved in an 18.4 mM HCl aqueous solution at 7.6% (w/w). To this solution,
18
tetramethylethylenediamine was added at 1.1% (w/w). After adjusting pH to 4.8,
19
lactobionic acid and 3-(4-hydroxyphenyl) propionic acid were added at 0.04 and 1.5%
20
(w/w), respectively. To this solution, EDC was added at 0.3 or 1.0% (w/w), and then
21
stirred for 20 h at 40°C. The Ph-chitosan obtained by adding 0.3 and 1.0% (w/w) EDC
22
were denominated as PhL- and PhH-chitosan, respectively. A specimen obtained
23
without adding 3-(4-hydroxyphenyl) propionic acid was denominated as NPh-chitosan.
24
The resultant solution containing chitosan derivative was gradually dripped into 90%
25
(v/v) ethanol/10% (v/v) water for precipitation of the polymer. The precipitated polymer
Ac ce p
te
d
M
10
4 Page 5 of 25
was washed with 90% (v/v) ethanol/10% (v/v) water until an ultraviolet visible
2
spectrum peak at 275 nm attributed to residual tyramine was undetectable in the
3
supernatant fluid after centrifugation. The resultant polymers were then vacuum dried.
4
The contents of Ph groups in the resultant Ph-PVA, PhL-chitosan, and PhH-chitosan
5
measured using 1H NMR were 2.9 10-4, 2.0 10-4, and 3.9 10-4 mol-Ph/g,
6
respectively.
cr
ip t
1
7 2.3. Gelation time
9
Gelation time of polymer solutions obtained by dissolving Ph-chitosan and/or Ph-PVA
an
us
8
in Krebs Ringer Hepes-buffered saline (KRH, pH 7.4) was determined based on a
11
previously reported method (Sakai & Kawakami, 2007). A solution containing either or
12
both of Ph-chitosan and/or Ph-PVA at 5% (w/v) total polymer content was poured into
13
a 48-well plate at 400 L/well. The content of each polymer is shown in Table 1.
14
Subsequently, 100 L of phosphate-buffered saline (pH 7.4, PBS) containing HRP
15
and H2O2 was poured into each well and stirred using a magnetic stirrer bar (10-mm
16
length 4-mm diameter). When the magnetic stirring was hindered and the surface of
17
the solution swelled, this was considered to signal the formation of the gel state. The
18
final concentrations of polymer(s), HRP and H2O2 were fixed at 4% (w/v), 1 U/mL and
19
1 mM, respectively. The means and standard deviations of quadruplets were reported.
d
te
Ac ce p
20
M
10
21
2.4. Mechanical properties
22
Cuboid hydrogels, (10 mm in width 40 mm in length 3 mm in thickness), were
23
prepared for extension studies by pouring KRH containing Ph-chitosan and/or Ph-PVA,
24
HRP (1 U/mL), and H2O2 (1 mM) at the polymer concentrations shown in Table 1 into
25
molds. After 12 h of standing at room temperature in humidified air, the specimens 5 Page 6 of 25
taken from the molds were placed in a Table-Top Material Tester (EZ-Test; Shimadzu,
2
Kyoto, Japan). The specimens were cut to a 20-mm length and clamped at both ends.
3
The length between the clamps was set at 10 mm. The extension-stress profile was
4
measured by stretching a specimen at 20 mm/min until rupture. The means and
5
standard deviations were determined by the test using five specimens prepared
6
separately under each condition.
cr
ip t
1
7 2.5. Swelling study
9
Column-shaped hydrogels, 11 mm in diameter and 5 mm in thickness, were prepared
an
us
8
by pouring the polymeric solutions shown in Table 1 at 1 U/mL HRP and 1 mM H2O2
11
into molds. After 12 h of standing at room temperature in humidified air, the hydrogels
12
taken from the molds were soaked in 25-times larger volumes of PBS at room
13
temperature. The changes in diameter and height of the column-shaped hydrogels
14
were measured every day for 6 days. The swelling ratio was calculated using the
15
following equation: Swelling ratio = (volume of hydrogel after soaking in PBS)/(volume
16
of hydrogel before soaking in PBS). Five specimens prepared separately were tested
17
for each condition. The means and standard deviations were reported.
d
te
Ac ce p
18
M
10
19
2.6. Fibroblastic cell behavior
20
Hydrogels were prepared under the conditions shown in Table 1 by pouring 0.5 mL
21
solution to a 12-well tissue culture dish, so as to completely cover the bottom of a well
22
before hydrogelation. After 12 h of standing at room temperature in humidified air, 6
23
mL of Dulbecco’s modified eagles medium (DMEM, Nissui Pharmaceutical, Tokyo,
24
Japan) with 10% (v/v) fetal bovine serum (FBS) was poured into the well and allowed
25
to stand for 10 min. This step was repeated four times to remove remaining H2O2 and 6 Page 7 of 25
replace KRH with the medium. DMEM with 10% (v/v) FBS containing mouse embryo
2
fibroblast-like cell line 10T1/2 cells (Riken Cell Bank, Igaragi, Japan) was then poured
3
into each well at 2104 cells/well. At 14 h after cell seeding, adhesion of the cells on
4
hydrogels was observed using an optical microscope.
ip t
1
5 2.7. E. coli behavior
7
Hydrogels were prepared under the conditions shown in Table 1 in a plastic petri dish
8
(35 mm in diameter) by pouring 1.5 mL of mixture solution before hydrogelation. After
9
12 h of standing at room temperature in humidified air, 10 mL of Lysogeny Broth (LB)
10
medium was poured into the dish and allowed to stand for 30 min. After repeating the
11
step four times to replace the solution in hydrogel to LB medium, the dish was turned
12
out onto a sterilized paper to remove the remaining medium from the hydrogel. Fifty
13
microliters LB medium containing E. Cole MM294 cells pre-cultured for 1 day in an
14
Erlenmeyer flask (1 L) containing 200 mL medium at 37°C using a reciprocal shaker
15
(100 rpm, 4 cm amplitude) was then spread on each hydrogel using a spreader.
16
Colony development was evaluated after 48 h of incubation at 37°C.
18 19
us
an
M
d
te
Ac ce p
17
cr
6
3. Results and discussion
20
3.1. Hydrogelation
21
An attractive feature of HRP-catalyzed hydrogelation is the mild reaction conditions
22
that are suitable for mammalian cells. A potential application of Ph-chitosan/Ph-PVA
23
hydrogels obtained through a cell-friendly hydrogelation system is injectable scaffolds
24
for tissue engineering, tissue-reconstructions, including wound healing, and drug
25
delivery (Teixeira et al., 2012). For these applications, knowledge of the effect of the 7 Page 8 of 25
contents of Ph-chitosan and Ph-PVA on the time taken for hydrogelation (gelation
2
time) is important. As shown in Fig. 2, the content of each polymer influenced the
3
gelation time. At 4.0% (w/v) total polymer concentration of either or both of Ph-PVA
4
and/or PhL-chitosan, gelation time decreased with increasing PhL-chitosan content.
5
The longest and shortest gelation times were detected at 4% (w/v) Ph-PVA and 4%
6
(w/v) PhL-chitosan, respectively. Focusing on the Ph content, the trend obtained for
7
the Ph-PVA and PhL-chitosan system was that the gelation time decreased with
8
decreasing Ph group content (Fig. 2, Table 1). However, the opposite trend was
9
observed for single-component solutions of 4Ph-PVA and 2Ph-PVA in this study and
an
us
cr
ip t
1
our previous results, including Ph-chitosan (Sakai et al., 2009a, b). The independence
11
of the content of Ph groups was also shown by shorter gelation times of
12
2PhH-chitosan/2Ph-PVA solution with the higher content of Ph groups compared with
13
that of 2PhL-chitosan/2Ph-PVA. These findings indicate that the gelation time of a
14
mixed system can be estimated from the times taken for gelation of solutions
15
containing a single component, but not from the content of Ph groups in the solution.
d
te
Ac ce p
16
M
10
17
3.2. Mechanical properties and swelling in PBS
18
Blending of PVA with chitosan has been used to improve the mechanical properties of
19
matrices such as films and hydrogels containing chitosan (Liang et al., 2009; Chen et
20
al., 2013; Takei et al., 2013). We investigated the effect of conjugation by the
21
HRP-catalyzed reaction on the mechanical properties of the resultant hydrogels by
22
measuring the stresses and breaking points under tensile strain (stretching). As
23
shown in Fig. 3, the 4PhL-chitosan hydrogel broke at ca. 50% strain, while the
24
breaking point for 4Ph-PVA hydrogel occurred at ca. 150% strain. The strain at rupture
25
for PhL-chitosan/Ph-PVA conjugate hydrogels varied between the values obtained for 8 Page 9 of 25
4PhL-chitosan and 4Ph-PVA and the value increased with increasing Ph-PVA content.
2
These results indicate that conjugation through the HRP-catalyzed reaction was
3
effective for improving the brittleness of Ph-chitosan hydrogels, likewise in the case of
4
hydrogels prepared by non-enzymatic cross-linking methods (Liang et al., 2009; Chen
5
et al., 2013; Takei et al., 2013). Regarding the stress at rupture, the value for
6
1PhL-chitosan/3Ph-PVA was larger than those for 4PhL-chitosan and 4Ph-PVA. This
7
result indicates that conjugation is effective for obtaining hydrogels with superior
8
durability towards deformation, which cannot be predicted from the results for the
9
hydrogels composed of Ph-PVA or Ph-chitosan alone. Regarding the effect of the Ph
10
content in Ph-chitosan, a more brittle hydrogel was obtained from Ph-chitosan with a
11
higher content of Ph groups. This result may be explained by the hindrance of
12
deformation of the highly cross-linked network under mechanical stress, and the
13
faster gelation rate of 2PhH-chitosan/2Ph-PVA than those for 2PhL-chitosan/2Ph-PVA
14
and 2NPh-chitosan/2Ph-PVA (Fig. 2) inducing a less uniform microscopic structure.
15
The formation of hydrogels showing lower resistance to deformation under conditions
16
giving faster hydrogelation was reported for a variety of hydrogels obtained through
17
non-enzymatic cross-linking methods, such as alginate (Kuo & Ma, 2001) and
18
collagen hydrogels (Achilli & Mantovani, 2010).
cr
us
an
M
d
te
Ac ce p
19
ip t
1
For potential applications of Ph-chitosan/Ph-PVA hydrogels to the biomedical field,
20
knowledge of their swelling properties in saline is important. We investigated this
21
property by measuring the volume change of hydrogels. 4Ph-PVA hydrogel did not
22
swell markedly during 6 days of soaking in PBS (Fig. 4). In contrast, the degree of
23
swelling of 4PhL-chitosan hydrogel was the largest amongst the hydrogels examined
24
in this study. The degree of swelling decreased with increasing Ph-PVA content in 4%
25
total-polymer hydrogels. This finding may be attributed to the formation of H-bonds 9 Page 10 of 25
between the amino groups in Ph-chitosan and the OH-groups in Ph-PVA
2
(Abdel-Mohsen et al., 2011). A less homogeneous microscopic structure of the
3
hydrogel with the higher content of PhL-chitosan induced by faster hydrogelation may
4
also be a reason for this behavior (Fig. 2). The same tendency was reported for
5
chitosan/PVA hydrogels obtained through non-enzymatic cross-linking methods such
6
as repeated freezing-thawing (Abdel-Mohsen et al., 2011), ultraviolet irradiation (Kim
7
et al., 2003), and gamma ray irradiation (Tahtat et al., 2011).
3.3. Adherent mammalian cells and E. coli cell behaviors
an
9
us
8
cr
ip t
1
Chitosan (Chandy & Sharma, 1990; Jarmila & Vavrikova, 2011) and PVA (Suciu et al.,
11
2004) have been investigated as promising materials for biomedical applications. To
12
evaluate the usefulness of bioconjugated Ph-chitosan/Ph-PVA hydrogels in the
13
biomedical field, we examined fibroblastic cell adhesion and antibacterial properties of
14
Ph-chitosan/Ph-PVA hydrogels prepared under different conditions. Figure 5 shows
15
10T1/2 cells 14 h after seeding onto hydrogels composed of either or both of
16
Ph-chitosan and/or Ph-PVA. The cells did not adhere onto 4Ph-PVA (Fig. 5A) or
17
1PhL-chitosan/3Ph-PVA (Fig. 5B) hydrogels and formed clusters. The cells on
18
2PhL-chitosan/2Ph-PVA adhered but did not spread (Fig. 5C). Spreading of the cells
19
was induced by increasing the content of PhL-chitosan in 3PhL-chitosan/1Ph-PVA
20
(Fig. 5D), but the degree of spreading was lower than those on a cell culture dish (Fig.
21
5G). Further increase in the content of PhL-chitosan, i.e., 4PhL-chitosan hydrogel,
22
decreased the degree of spreading (Fig. 5E). Difficulty in adhesion and spreading of
23
adherent cells has been reported for chitosan and chitosan derivative hydrogels
24
obtained through non-enzymatic cross-linking methods (Ono et al., 2000; Takei et al.,
25
2012). A greater spreading of 10T1/2 cells on 2PhL-chitosan/2Ph-PVA hydrogel than
Ac ce p
te
d
M
10
10 Page 11 of 25
those on 4PhL-chitosan hydrogel may be induced by the moderate cationic property
2
resulting from the conjugation with an appropriate amount of Ph-PVA. These results
3
suggest the possibility of controlling cell adhesion on the hydrogels by altering the
4
content of PhL-chitosan and Ph-PVA for an intended application.
5
ip t
1
Antibacterial properties are one of the attractive features of chitosan for applications in the biomedical field (Jarmila & Vavrikova, 2011; Jarry et al., 2001; Tahtat et al.,
7
2011). We evaluated the biocidal impact of Ph-chitosan/Ph-PVA hydrogels on E. coli
8
cells. Figure 6 shows the hydrogels 48 h after seeding E. coli cells. Interestingly, the
9
hydrogel containing PhL-chitosan at 2% (w/v) or more suppressed the growth of E.
an
us
cr
6
coli (Fig. 6C–E). This result can be explained by the reported mechanism of
11
antibacterial action by chitosan caused by the formation of electrostatic bonds
12
between chitosan and anionic groups on the bacterial surface membrane, which
13
would result in altered membrane permeability (Tsai & Su, 1999; Yang et al., 2008;
14
Jarmila & Vavrikova, 2011). In our specimens, it is intuitive that hydrogels with an
15
abundance of Ph-chitosan show greater cationic properties. The growth of E. coli on
16
2PhH-chitosan/2Ph-PVA (Fig. 6F) but not on 2PhL-chitosan/2Ph-PVA (Fig. 6C) can
17
also be explained by the difference in the degree of cationic strength. The degree of
18
substitution of amino groups in PhL-chitosan was less than that in PhH-chitosan.
19
These results indicate that the content and degrees of modification of Ph-chitosan in
20
Ph-chitosan/Ph-PVA hydrogels should be carefully chosen for applications in which
21
antibacterial properties are desired.
Ac ce p
te
d
M
10
22 23
4. Conclusions
24 25
Ph-chitosan/Ph-PVA hydrogels were prepared through the HRP-catalyzed 11 Page 12 of 25
cross-linking between Ph groups at different Ph-chitosan and Ph-PVA contents.
2
Gelation time was dependent on the content of each polymer, but independent of the
3
content of Ph groups in the solution. At 4.0% (w/v) total polymer concentration of
4
either or both of Ph-PVA and/or PhL-chitosan, gelation time decreased with increasing
5
Ch-PhL content. The longest and shortest gelation time detected for 4% (w/v) Ph-PVA
6
and 4% (w/v) PhL-chitosan were 73 s and 28 s, respectively. Swelling of the hydrogels
7
can be suppressed by increasing the Ph-PVA content. The swelling ratio of
8
3PhL-chitosan/1Ph-PVA hydrogel was 1.9 at 6 days of soaking in PBS. The value
9
decreased to 1.2 at 1PhL-chitosan/3Ph-PVA hydrogel. Fibroblastic cell adhesion and
an
us
cr
ip t
1
antibacterial activity studies demonstrated that these bioactivities can be controlled by
11
altering the Ph-chitosan and Ph-PVA content in the hydrogels. To obtain hydrogels
12
with greater cell adhesiveness, it was shown that an appropriate Ph-chitosan and
13
Ph-PVA content is necessary. The cells on 3PhL-chitosan/1Ph-PVA hydrogel were the
14
most spreading compared to those on 2PhL-chitosan/2Ph-PVA and 4PhL-chitosan
15
hydrogels. To obtain the hydrogels with greater antibacterial activity, it was shown that
16
a higher Ph-chitosan content is necessary. E. coli cells grew on
17
1PhL-chitosan/3Ph-PVA hydrogel but did not grow on 2PhL-Chitosan/2Ph-PVA
18
hydrogel during 48h cultivation. These results demonstrate the feasibility of
19
HRP-catalyzed hydrogelation for obtaining the hydrogels having the unique properties
20
attributed to the conjugation of Ch-chitosan and Ch-PVA which cannot be obtained
21
using either of the components alone.
Ac ce p
te
d
M
10
22 23
References
24
Abdeen, Z. (2011). Swelling and reswelling characteristics of cross-linked poly(vinyl
25
alcohol)/chitosan hydrogel film. J Disper Sci Technol, 32, 1337-1344. 12 Page 13 of 25
1
Abdel-Mohsen, A.M., Aly, A.S., Hrdina, R., Montaser, A.S., & Hebeish, A. (2011).
2
Eco-synthesis of PVA/chtiosan hydrogels for biomedical application. J Polym
3
Environ, 19, 1005-1012. Achilli, M., & Mantovani, D. (2010). Tailoring mechanical properties of collagen-based
5
scaffolds for vascular tissue engineering: The effects of pH, temperature and ionic
6
strength on gelation. Polymers, 2, 664-680.
cr
8
Chandy, T., & Sharma, C.P. (1990). Chitosan–as a biomaterial. Biomater Artif Cells Artif Organs, 18, 1-24.
us
7
ip t
4
Chen, C., Liu, L., Huang, T., Wang, Q., & Fang, Y. (2013). Bubble template fabrication
10
of chitosan/poly(vinyl alcohol) sponges for wound dressing applications. Int J Biol
11
Macromol, 62, 188-193.
M
12
an
9
Costa, E.S., Barbosa-Stancioli, E.F., Mansur, A.A.P., Vasconcelos, W.L., & Mansur, H.S. (2009). Preparation and characterization of chitosan/poly(vinyl alcohol)
14
chemically crosslinked blends for biomedical applications. Carbohyd Polym, 76,
15
472-481.
te
d
13
George, M., & Abraham, T.E. (2006). Polyionic hydrocolloids for the intestinal delivery
17
of protein drugs: Alginate and chitosan–a review. J Control Release, 114, 1-14.
18 19 20
Ac ce p
16
Gross, R.A., Kumar, A., & Kalra, B. (2001). Polymer synthesis by in vitro enzyme catalysis. Chem Rev, 101, 2097-2124. Islam, A., Yasin, T., Bano, I., & Riaz, M. (2012). Controlled release of aspirin from
21
pH-sensitive chitosan/poly(vinyl alcohol) hydrogel. J Appl Polym Sci, 124,
22
4184-4192.
23
Ito, A. et al. (2003). Transglutaminase-mediated gelatin matrices incorporating cell
24
adhesion factors as a biomaterial for tissue engineering. J Biosci Bioeng, 95,
25
196-199. 13 Page 14 of 25
2 3 4 5
Jarmila, V., & Vavrikova, E. (2011). Chitosan derivatives with antimicrobial, antitumour and antioxidant activities–a review. Curr Pharm Des, 17, 3596-3607. Jarry, C. et al. (2001). Effects of steam sterilization on thermogelling chitosan-based gels. J Biomed Mater Res, 58, 127-135.
ip t
1
Kim, S.J., Park, S.J., & Kim, S.I. (2003). Swelling behavior of interpenetrating polymer network hydrogels composed of poly(vinyl alcohol) and chitosan. React Funct
7
Polym, 55, 53-59.
us
9
Kobayashi, S., Uyama, H., & Kimura, S. (2001). Enzymatic polymerization. Chem Rev, 101, 3793-3818.
an
8
cr
6
Koyano, T., Minoura, N., Nagura, M., & Kobayashi, K. (1998). Attachment and growth
11
of cultured fibroblast cells on chitosan-blended hydrogels. J Biomed Mater Res, 39,
12
486-490.
Kuo, C.K., & Ma, P.X. (2001). Ionically crosslinked alginate hydrogels as scaffolds for
d
13
M
10
tissue engineering: part 1. Structure, gelation rate and mechanical properties.
15
Biomaterials, 22, 511-521.
17 18
Kweon, D.K., & Kang, D.W. (1999). Drug-release behavior of chitosan-g-poly(vinyl
Ac ce p
16
te
14
alcohol) copolymer matrix. J Appl Polym Sci, 74, 458-464. Lee, F., Chung, J.E., & Kurisawa, M. (2009). An injectable hyaluronic acid-tyramine
19
hydrogel system for protein delivery. J Control Release, 134, 186-193.
20
Liang, S., Liu, L., Huang, Q., & Yam, K.T. (2009). Preparation of single or
21
double-network chitosan/poly(vinyl alcohol) gel films through selectively
22
cross-linking method. Carbohyd Polym, 77, 718-724.
23 24 25
Muzzarelli, R.A.A. (2009). Chitins and chitosans for the repair of wounded skin, nerve, cartilage and bone. Carbohyd Polym, 76, 167-182. Muzzarelli, R.A.A., Ilari, P., Xia, W., Pinotti, M., & Tomasetti, M. (1994). 14 Page 15 of 25
1
Tyrosinase-mediated quinone tanning of chitinous materials. Carbohyd Polym, 24,
2
295-300.
3
Muzzarelli, R.A.A., & Muzzarelli, C. (2009). Chitin and chitosan hydrogels. In G.O. Phillips, and P.A. Williams (Eds.), Handbook of Hydrocolloids. (2nd ed.)
5
(pp.849-888). Cambridge UK. Woodhead Publishing Ltd.
7
Ono, K. et al. (2000). Photocrosslinkable chitosan as a biological adhesive. J Biomed
cr
6
ip t
4
Mater Res, 49, 289-295.
Peniche, C., Arguelles-Monal, W., Peniche, H., & Acosta, N. (2003). Chitosan: An
9
attractive biocompatible polymer for microencapsulation. Macromol Biosci, 3,
12 13
an
Rocasalbas, G. et al. (2013). Laccase-assisted formation of bioactive chitosan/gelatin
M
11
511-520.
hydrogel stabilized with plant polyphenols. Carbohyd Polym, 92, 989-996. Sabaa, M.W., Mohamed, R.R., Eltaweel, S.H., & Seoudi, R.S. (2012). Crosslinked
d
10
us
8
poly(vinyl alcohol)/carboxymethyl chitosan hydrogels for removal of metal ions and
15
dyestuff from aqueous solutions. J Appl Polym Sci, 123, 3459-3469. Sakai, S., Hashimoto, I., Ogushi, Y., & Kawakami, K. (2007). Peroxidase-catalyzed
Ac ce p
16
te
14
17
cell-encapsulation in subsieve-size capsules of alginate with phenol moieties in
18
water-immiscible fluid dissolving H2O2. Biomacromolecules, 8, 2622-2626.
19
Sakai, S., Hirose, K., Taguchi, K., Ogushi, Y., & Kawakami, K. (2009b). An injectable,
20
in situ enzymatically gellable, gelatin derivative for drug delivery and tissue
21
engineering. Biomaterials, 30, 3371-3377.
22 23
Sakai, S., & Kawakami, K. (2007). Synthesis and characterization of both ionically and enzymatically crosslinkable alginate. Acta Biomater, 3, 495-501.
24
Sakai, S. et al. (2013). Polyvinyl alcohol-based hydrogel dressing gellable on-wound
25
via a co-enzymatic reaction triggered by glucose in the wound exudate. J Mater 15 Page 16 of 25
1 2
Chem B, 1, 5067-5075. Sakai, S., Yamada, Y., Zenke, T., & Kawakami, K. (2009a). Novel chitosan derivative soluble at neutral pH and in-situ gellable via peroxidase-catalyzed enzymatic
4
reaction. J Mater Chem, 19, 230-235.
5
ip t
3
Suciu, A.N., Iwatsubo, T., Matsuda, M., & Nishino, T. (2004). A study upon durability of the artificial knee joint with PVA hydrogel cartilage. JSME C, 47, 199-208.
7
Tahtat, D. et al. (2011). Influence of some factors affecting antibacterial activity of
cr
6
PVA/chitosan based hydrogels synthesized by gamma irradiation. J Mater Sci
9
Mater Med, 22, 2505-2512.
an
10
us
8
Takei, T., Nakahara, H., Ijima, H., & Kawakami, K. (2012). Synthesis of a chitosan derivative soluble at neutral pH and gellable by freeze-thawing, and its application
12
in wound care. Acta Biomater, 8, 686-693.
M
11
Takei, T. et al. (2013). Effect of chitosan-gluconic acid conjugate/poly(vinyl alcohol)
14
cryogels as wound dressing on partial-thickness wounds in diabetic rats. J Mater
15
Sci Mater Med, 24, 2479-2487.
te
Teixeira, L.S., Feijen, J., van Blitterswijk, C.A., Dijkstra, P.J., & Karperien, M. (2012).
Ac ce p
16
d
13
17
Enzyme-catalyzed crosslinkable hydrogels: Emerging strategies for tissue
18
engineering. Biomaterials, 33, 1281-1290.
19 20 21
Tsai, G.J., & Su, W.H. (1999). Antibacterial activity of shrimp chitosan against Escherichia coli. J Food Prot, 62, 239-243. Yang, X., Liu, Q., Chen, X., Yu, F., & Zhu, Z. (2008). Investigation of PVA/ws-chitosan
22
hydrogels prepared by combined γ-irradiation and freeze-thawing. Carbohyd
23
Polym, 73, 401-408.
24 25
Zhao, L. et al. (2003). Synthesis of antibacterial PVA/CM-chitosan blend hydrogels with electron beam irradiation. Carbohyd Polym, 53, 439-446. 16 Page 17 of 25
1 2 3
ip t
4 5
cr
6 7 Figure captions
us
8
10
an
9
Fig. 1. Scheme for the formation of Ph-chitosan/Ph-PVA.
M
11
Fig. 2. Gelation times for polymeric solutions of differing composition. Values
13
represent the mean (n = 4) with standard deviation.
te
14
d
12
Fig. 3. Strain-stress profiles of hydrogels obtained from polymeric solutions of differing
16
composition. Values represent the mean (n = 5) with standard deviation.
17
Ac ce p
15
18
Fig. 4. Swelling profiles of hydrogels obtained from polymeric solutions of differing
19
composition. Values represent the mean (n = 5) with standard deviation.
20 21
Fig. 5. Microphotographs of 10T1/2 cells 14 h after seeding on hydrogels obtained
22
from polymeric solutions of differing composition. Bars: 100 m.
23 24
Fig. 6. Photographs of hydrogels obtained from polymeric solutions of differing
25
composition 48 h after seeding E. coli cells. Bars: 1 cm. 17 Page 18 of 25
1 2 3
Table 1. Hydrogel compositions prepared through the HRP-catalyzed reaction.
4 3 2 1 2 2 2
1 2 3 4 -
4 2 -
2 -
1.16 1.07 0.98 0.89 0.80 1.56 1.36 0.58 0.58
us
4Ph-PVA 1PhL-chitosan/3Ph-PVA 2PhL-chitosan/2Ph-PVA 3PhL-chitosan/1Ph-PVA 4PhL-chitosan 4PhH-chitosan 2PhH-chitosan/2Ph-PVA 2NPh-chitosan/2Ph-PVA 2Ph-PVA
Ph content [10-5 mol-Ph/mL]
cr
Concentration [% (w/v)] Chitosan derivative Ph-PVA PhL- PhH- NPh-
6
an
5
ip t
4
Ac ce p
te
d
M
HRP and H2O2 concentrations were fixed at 1 U/mL and 1 mM, respectively.
18 Page 19 of 25
Ac ce p
te
d
M
an
us
cr
ip t
Figure 1
HO
HO
Ph-PVA
Ph-PVA OH
OH
HRP/H2O2 OH
O
HO
HO HO
HO
Ph-chitosan
Page 20 of 25
Ph-chitosan
Ac ce p
te
d
M
an
us
cr
ip t
Figure 2
h 4P
h
1P
C L-
A V P
-
h
ito
sa
2
h 3P / n h
Ph
c L-
A V -P
ito
sa
3
h 2P / n h
Ph
c L-
A V P
-
ito
sa
h 1P / n
A V P
-
4
L Ph
ch
sa o it
-
4
n
H Ph
2
ch
sa o it
-
a
h
Ph
n
c H-
s ito 2
h 2P / n
NP
-
h
c h-
A V P a
s ito
h 2P / n
-
A V P
h 2P
A V P
-
Page 21 of 25
an
us
cr
ip t
Figure 3
M
0.18 㻌4Ph-PVA 㻌1PhL-chitosan/3Ph-PVA 㻌2PhL-chitosan/2Ph-PVA 㻌3PhL-chitosan/1Ph-PVA 㻌4PhL-chitosan 㻌2PhH-chitosan/2Ph-PVA 2NPh-chitosan/2Ph-PVA 㻌
te
d
0.15
Ac ce p
Stress (N)
0.12 0.09 0.06 0.03 0.00 0
40
80
120
160 Page 22 of 25
Strain (%)
2NPh-chitosan/2Ph-PVA
us
3PhL-chitosan/1Ph-PVA 4PhL-chitosan 2PhH-chitosan/2Ph-PVA
Ac ce p
te
d
M
an
4Ph-PVA 1PhL-chitosan/3Ph-PVA 2PhL-chitosan/2Ph-PVA
cr
ip t
Figure 4
d
Page 23 of 25
Ac
ce
pt
ed
M
an
us
cr
i
Figure 5
Page 24 of 25
Ac ce p
te
d
M
an
us
cr
ip t
Figure 6
Page 25 of 25
