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pH-Sensitive Hydrogels Composed
of Chitosan and Polyacrylamide:
Enzymatic Degradation
P. BONINA, T. PETROVA, N. MANOLOVA* AND I. RASHKOV
Laboratory of Bioactive Polymers, Institute of Polymers,
Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
M. NAYDENOV
Department of Microbiology, Agricultural University, 4000 Plovdiv, Bulgaria
ABSTRACT: The enzymatic degradation of three types of pH-sensitive
hydrogels, composed of the natural polyaminosaccharide chitosan and polyacrylamide, was studied. The weight of the films that were made with netPAAm-ı-chitosan, net-chitosan-ı-PAAm and net-chitosan-net-PAAm decreased
in the presence of the Trichoderma viride enzyme complex; thus, the chitosan
in the composite retained its degradability after crosslinking. The rate of enzymatic degradation depended on the structure of the network, on the amount of
crosslinking agents, on the pH of the medium and on the temperature.
Crosslinked chitosan alone degraded slower than net-chitosan-ı-PAAm; this
was attributed to the facilitated penetration of enzyme by the water-soluble
PAAm in the semi-IPNs. T. viride embedded in chitosan/PAAm films or beads
developed and reproduced normally. However, T. viride embedded in net-chitosan-ı-PAAm developed considerably slower, and development was not
detected in the case of net-PAAm-ı-chitosan. All of the networks proved to be
appropriate carriers of Bacillus subtilis.
KEY WORDS: Chitosan, polyacrylamide, pH-sensitive hydrogels, semi-IPNs,
enzymatic degradation, T. viride, B. subtilis.
INTRODUCTION
n order to develop effective and ecologically safe devices for
modern agriculture, new polymer materials need to be developed
I
*Author to whom correspondence should be addressed. E-mail: [email protected]
Journal of BIOACTIVE AND COMPATIBLE POLYMERS,
Vol. 19 — May 2004
0883-9115/04/03 0197–12 $10.00/0
DOI: 10.1177/0883911504044455
© 2004 Sage Publications
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that are degradable under the action of enzymes produced by microorganisms. There are a considerable number of soil micro-organisms
that are good agents for the biocontrol of pathogenic fungi and insects
[1, 2]. Bacteria and fungi, for example, Penicillum, Trichoderma, Streptomyces, Bacillus, produce enzymes that are able to degrade natural
polymers such as chitin and its derivatives, which are construction elements of the cell walls of certain plant pathogenic fungi [3–6]. Some of
the enzymes produced by Trichoderma sp. possess endo-type chitinolytic
activity. They degrade chitosan to lower-molecular-weight products by
random scission of the macromolecular chains [6]. The biocontrol effect
of these useful micro-organisms results in crop increase and diminished
pollution of the environment with synthetic pesticides.
Recently, we have proposed new environmentally friendly systems
for plant protection, in which the biocontrol agent is embedded in films
or beads of chitosan or polymer complexes of chitosan and synthetic
water-soluble polymers, such as poly(2-acryloylamido-2-methylpropanesulfonic acid), poly(acrylic acid), polyoxyethylene, or poly(vinyl
alcohol) [7–10]. These polymer carriers play an active role in these
systems by maintaining the micro-organisms’ viability on storage, and
provide favorable conditions for normal development when in contact
with moist soil. Chitosan, the main component of these polymer carriers, is degraded by the enzyme action of the biocontrol agent to form
oligomers that can activate plant defense reactions.
Polyacrylamide (PAAm) and its crosslinked hydrogels are used as
soil conditioners without being harmful to warm-blooded animals and
plants [11]. It has been proposed that, as a first stage, polyacrylamide
is hydrolyzed under the action of some amidases to poly(acrylic acid)
and ammonia. The poly(acrylic acid) is degraded to carbon dioxide and
water and the ammonia takes part in the biosynthesis of amino acids
[12]. It has been shown that crosslinked hydrogels of PAAm also
undergo enzymatic degradation [13]. Two soil bacterium species,
Enterobacter agglomerans and Azomonas macrocytogenes, degrade
PAAm and use it as a source of nitrogen and hydrogen [14].
In the present work, the degradation of chitosan as a component of
semi-interpenetrating networks (semi-IPNs) (i.e. net-PAAm-ı-chitosan
and net-chitosan-ı-PAAm) and of an interpenetrating network (IPN)
(i.e. net-chitosan-net-PAAm) in the presence of Trichoderma viride
enzyme complex was evaluated. The macroscopic changes of the hydrogels and the degradation of chitosan component were followed as a
function of the network structure, the degree of crosslinking and the
conditions of the medium (pH and temperature). The possibility of
using these hydrogels as carriers of biocontrol agents was examined.
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MATERIALS AND METHODS
Chitosan (MW 4.105 with 80% deacetylation, estimated according to
Baxter [15]) (Fluka, Biochemika) and acrylamide (AAm) (Fluka) were
used. Glutaraldehyde (50% aqueous solution) and N,N'-methylenebisacrylamide (MBAAm) (Fluka) were used as crosslinking agents.
The salts used for the redox-initiator system, (NH4)2S2O8 and
N,N,N',N'-tetramethylethylenediamine, were obtained from Fluka. All
buffer solutions and chemicals used were analytical grade.
Polymer Synthesis
Polyacrylamide (PAAm) was prepared by radical polymerization of
AAm (8% aqueous solution) in the presence of K2S2O8 (2 g/L) as initiator at 50°C under inert atmosphere for 2 h. Poly(2-acryloylamido-2methylpropanesulfonic acid) (PAMPS) was prepared by radical
polymerization in 20% aqueous solution of AMPS at 25°C for 15 h with
the redox-initiator system Fe(NH4)2(SO4)2 0.05 g/L, Na2SO3 2.2 g/L,
and
(NH4)2S2O8 2.2 g/L. The viscosity
average molar masses of PAAm
—
—
(M v = 1.3.106) and of PAMPS (M v = 5.105) were calculated from the
intrinsic viscosity, measured at 25°C with an Ubbelohde viscometer in
water and in 5M NaCl, respectively. The Mark–Houwink constants
values were α = 0.66, K = 6.8 × 104 [16], and α = 0.80, K = 2.11 × 105
[17], respectively. The methods and the conditions for the preparation
of the net-PAAm-ı-chitosan, net-chitosan-ı-PAAm and net-chitosan-netPAAm, as well as the determination of the equilibrium degree of
swelling, have been described elsewhere [18].
Enzyme Preparations
A culture suspension of T. viride was cultivated at 28°C for 3 days in
200 mL liquid medium containing 2.5% glucose and 2.5% corn extract.
A culture suspension from B. subtilis was cultivated at 28°C for 2 days
in 200 mL liquid medium, containing Triptic soy broth. The culture
supernatant obtained by centrifugation (30 min, 4200 rpm) of the suspension was used as a crude enzyme complex for the enzymatic degradation of the networks.
The enzyme activity was determined by a modified method
described by Miller [19] using the concentration of reducing sugar liberated during the hydrolysis of 1% colloidal chitin as a substrate. Colloidal chitin was prepared according to a published procedure [20]. One
unit enzyme activity (U) was defined as the amount of enzyme that
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could produce 1 µmol of reducing sugar/min, using N-acetylglucosamine as a standard.
Film Preparation
Films for the enzymatic degradation studies of net-PAAm-ı-chitosan, net-chitosan-ı-PAAm and net-chitosan-net-PAAm were prepared
as described elsewhere [18]. For all of the hydrogels, the molar ratio
was [chitosan] : [AAm] = 2. Samples (20 mm × 10 mm × 0.05 mm) were
cut from the films obtained, immersed in 5 mL buffer solutions at pH 4
(or pH 6), ionic strength 0.1, and thermostated at 25°C (or 35°C). After
reaching equilibrium swelling, the samples were immersed into a
buffered solution of crude T. viride enzyme complex (a mixture of 5 mL
buffer solution and 5 mL of the crude enzyme complex with an enzyme
activity of 0.012 U/mL). Samples were removed from the solution at
fixed time intervals and dried to constant weight. The enzymatic
degradation was estimated from the weight loss.
The dynamic viscosity of 2% aqueous solutions of PAAm or chitosan
in the presence of a crude enzyme complex of T. viride was measured
using a Brookfield LVT viscometer equipped with a small sample thermostating adapter, spindle and chamber SC4–18/13R.
Beads were prepared by capillary extrusion of chitosan/PAAm ([Chitosan] : [PAAm] = 2) containing 50 mg of biomass per 1 g polymer. A
portion of the beads was coated with the polyelectrolyte complex chitosan/PAMPS. Films of chitosan-PAAm, net-PAAm-ı-chitosan, net-chitosan-ı-PAAm and net-chitosan-net-PAAm containing T. viride or B.
subtilis were prepared. The culture suspensions were mixed with
appropriate amounts of the macromolecular carrier to obtain 79 mg of
biomass of T. viride/g of carrier and 57 mg of biomass of B. subtilis/g of
carrier. The ability of the embedded micro-organisms to develop was
tested by inoculating the beads or film discs (14 mm diameter) on the
surface of Rose-Bengal Chloramphenicol Agar (Oxoid) at 25°C and
28°C for T. viride and for B. subtilis, respectively.
RESULTS AND DISCUSSION
Previously we described the preparation and characterization of
three types of hydrogels, net-PAAm-ı-chitosan, net-chitosan-ı-PAAm
and net-chitosan-net-PAAm, using glutaraldehyde to crosslink chitosan
and N,N'-methylenebisacrylamide to crosslink PAAm. It was shown
that the hydrogels were pH-sensitive at specific ratios of [chitosan] :
[PAAm] ≥ 1 [18]. The equilibrium degree of swelling of net-PAAm-ı-
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chitosan, net-chitosan-ı-PAAm and net-chitosan-net-PAAm hydrogels
depended on the pH of the medium, on the ionic strength (I) and on
the degree of crosslinking. The highest degree of swelling for the three
types of networks was observed at pH 4, and the lowest was at pH 9
(I = 0.1). On increasing the crosslinking, as well as on increasing the
ionic strength, the equilibrium swelling decreased. Chitosan, due to its
polyelectrolytic nature, was the determinant in the swelling of the
three types of networks.
Before studying the effect of the crude T. viride enzyme complex on
the semi-interpenetrating and interpenetrating chitosan-PAAm networks, we tested the behavior of PAAm and chitosan. The viscosity of
the PAAm solution alone did not change under the action of the
enzyme complex; in contrast, however, the enzyme degradation of chitosan caused a significant decrease in the chitosan solution viscosity
within 60–70 min. Films of crosslinked PAAm retained their weight
while in contact with the enzyme complex for one week (pH 6, 25°C).
These results imply that the enzyme complex of T. viride does not alter
PAAm nor the crosslinked PAAm in the time scale of the present
study.
Effect of the Enzyme Complex Produced by Trichoderma
viride on net-PAAm-ı-chitosan
Networks of net-PAAm-ı-chitosan were prepared with three different molar ratios of the crosslinking agent MBAAm to AAm monomer
{[MBAAm] : [AAm] = 1 : 5; 1 : 25; 1 : 50}. All three of the networks
obtained were pH-sensitive. The equilibrium swelling (αeq) for
[MBAAm] : [AAm] = 1 : 50 at pH 4 (I = 0.1) was 1130% (25°C), while at
pH 6 it was only 420% (I = 0.1). On increasing the degree of crosslinking or on increasing the ionic strength, αeq decreased, for example, the
αeq value for [MBAAm] : [AAm] = 1:5 was 120% at pH 4 and I = 1.
Degradation of chitosan began after the networks came into contact
with the enzyme complex of T. viride. As the degradation progressed,
oligomeric chitosan fractions appeared. As the chitosan oligomers left
the PAAm network, the rest of chitosan macromolecules were more
accessible for the enzymes. The penetration of the enzymes into the
hydrogel was facilitated by the higher αeq values. The crosslinked
PAAm did not degrade under the action of the enzyme complex, thus
the hydrogels retained their macroscopic appearance. Although the
polymer network films lost up to 80% of their initial weight, the
swollen films retained their integrity. As seen in Figure 1, the weight
losses strongly depended on the crosslinking ratio.
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Figure 1. Weight loss during enzymatic degradation of net-PAAm-ı-chitosan; molar
ratios [MBAAm] : [AAm] = 1 : 50 (), 1 : 25 () and 1 : 5 (); pH 4, 25°C.
The weight loss after 96 h was 80% and 70% for the hydrogels with
lower degrees of crosslinking, [MBAAm] : [AAm] = 1 : 50 and 1 : 25,
respectively. The net-PAAm-ı-chitosan hydrogels contained 83% chitosan. The loss of 70–80% from the initial weight shows that most of
the chitosan had degraded to oligomers and was able to leave the
network by diffusion into the aqueous medium. Generally 3–13% of the
chitosan remained in the network, at least up to 168 h. The weight loss
of the net-PAAm-ı-chitosan hydrogels decreased with decreases in αeq
(1130, 905 and 350%) and reached 3–4% in the case of the hydrogel
with [MBAAm] : [AAm] = 1 : 5. In this case, the degree of crosslinking
may have attained a “critical” value, at which the main part of chitosan included in the network was practically inaccessible to the
enzymes.
At a higher temperature (35°C), the enzymatic degradation of chitosan proceeded faster. The difference in the rate of the enzymatic
degradation may be estimated by comparing the time τ1/2, at which the
hydrogel loses 50% of its initial weight. For example, for hydrogels
obtained at ratios of [MBAAm] : [AAm] = 1 : 25 and 1 : 50, the τ1/2 was
reached at 25°C in 48 and 28 h, respectively, while at 35°C it was
reached in 8 and 24 h. respectively. Since at 25°C and 35°C the αeq
values were rather close (905 and 1130% for 25°C; 944 and 1150% for
35°C), the higher rate of degradation at 35°C could be attributed to the
higher enzyme activity at this temperature. It is noteworthy that, in
this series of experiments, the weight loss of strongly crosslinked networks remained rather low, and did not exceed 7–8%.
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At higher pH values, the rate of enzymatic hydrolysis was significantly lower. For the hydrogel with the lowest degree of crosslinking,
the τ1/2 was reached in ~110 h at pH 6. This could be due to a combination of pH effects on both the enzyme activity and the equilibrium
swelling of the hydrogel. The optimum pH for the degradation of chitosan under the action of the enzyme complex of T. viride is about 4
[7] and the hydrolysis rate decreased with increasing pH. At the
highest pH, in this study pH 6, the αeq values were low (αeq = 220, 340
and 420%) for hydrogels obtained at ratios [MBAAm] : [AAm] = 1 : 5,
1 : 25, 1 : 50, respectively.
Degradation of net-Chitosan-ı-PAAm Hydrogels
The net-chitosan-ı-PAAm hydrogels consisted of a crosslinked chitosan network in which PAAm was included. These were prepared
using glutaraldehyde at molar ratios of [NH2] : [CHO] = 30 : 1 and 50 : 1.
The role of the hydrophilic non-ionic component included in the chitosan network was evaluated and compared to pure chitosan networks.
After putting this semi-IPN in contact with the enzyme complex of T.
viride, the degradation of the chitosan network began. In contrast to
the former type of semi-IPNs, in the case of net-chitosan-ı-PAAm,
PAAm is not crosslinked and, at a given degree of degradation, diffusion of the water-soluble PAAm chains into the liquid medium was
possible. Due to the degradation of the chitosan component, the films
changed in shape during enzymatic degradation; at a certain amount
of time they were completely dissolved in the liquid medium.
The weight loss during the enzymatic degradation of crosslinked
chitosan and of the network polymers net-chitosan-ı-PAAm is presented in Figure 2. The weight loss of the net-chitosan-ı-PAAm
occurred much faster with time than crosslinked chitosan alone. The
presence of the water-soluble and high segment mobility of PAAm in
the net-chitosan-ı-PAAm probably contributed to easier enzyme penetration and resultant interaction. Two clearly differentiated stages, an
initial slow stage and a second faster stage, were observed during the
enzymatic degradation of all the networks. At a specific time, degradation of the chitosan network was at a much higher rate than in the
initial stages. This could be due to the combination of two effects, the
degradation of the chitosan network and the loss of chitosan oligomers
from the film, which would provide enzyme access to the crosslinked
chitosan. After a time, the chitosan network became free enough so
that it no longer hampered the diffusion of the chitosan oligomers. In the
case of net-chitosan-ı-PAAm hydrogels, the degradation also produced
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Figure 2. Weight loss during enzymatic degradation of net-chitosan-ı-PAAm; molar
ratios [NH2] : [CHO] 50 : 1 () and 30 : 1 (); and of crosslinked chitosan, molar ratios
[NH2] : [CHO] 50 : 1 () and 30 : 1 (); pH 4, 25°C.
water-soluble PAAm for diffusion. At the end of the enzymatic degradation, the hydrogels were completely dissolved due to the degradation
of the chitosan network.
At a higher temperature (35°C), the degradation rate of net-chitosan-ıPAAm increased, while an increase in the medium pH to 6 led to a
decrease of the rate of degradation. Again the semi-IPNs degraded faster
than chitosan networks alone. This is due to the lower enzyme activity at
pH 6 and to the hindered diffusion of the degradation products to the
solution due to the lower equilibrium swelling of the hydrogel at this pH.
Degradation of net-PAAm-net-Chitosan Hydrogels
The enzymatic degradation of net-PAAm-net-chitosan films is shown
in Figure 3. For the higher crosslinked chitosan and PAAm network
which resulted in lower equilibrium swelling (αeq = 400%), insignificant
degradation occurred (3–4%). In the case of network with the lower
degree of crosslinking and with a higher αeq (αeq = 1200%), the enzymatic degradation proceeded much faster and the weight loss was 93%
after 72 h. The fact that this weight loss was higher than the initial
amount of chitosan (83%) was attributed to micro-fragmentation of the
hydrogel as well as to the presence of a small fraction of noncrosslinked chitosan or PAAm.
The degradation results of the three types of hydrogels showed that
degradation occurred in the presence of the enzyme complexes pro-
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Figure 3. Weight loss during enzymatic degradation of interpenetrating networks netPAAm-net-chitosan; molar ratios, [MBAAm] : [AAm]; and [NH2] : [CHO] = 1 : 5 and 30 : 1
(), 1 : 50 and 50 : 1 (), respectively; pH 4, 25°C.
duced by the soil fungus T. viride when chitosan was included in the
networks. The PAAm in the network retained the macroscopic shape
of the hydrogels since it was not degraded by the enzyme complex of T.
viride during the reaction time of this study.
Testing the Hydrogels as Carriers of Biocontrol Agents
Based on microbiological tests, when T. viride was embedded in chitosan-PAAm films or beads, the fungus developed and reproduced normally (Figure 4.1). Good proliferation was also observed after coating
the films or beads with PAMPS (Figure 4.2). The T. viride, embedded
in net-chitosan-ı-PAAm developed significantly slower and, in the case
of net-PAAm-ı-chitosan, fungal development was not observed. To
explain this peculiarity it would be necessary to evaluate the fungistatic and fungicide activity of AAm and the crosslinking agents in the
concentration ranges used in this study. The microbiological tests
showed that Bacillus subtilis, embedded in net-PAAm-ı-chitosan, netchitosan-ı-PAAm and net-chitosan-net-PAAm, all developed and reproduced normally (Figures 4.3 and 4.4).
CONCLUSION
The semi-IPNs and IPNs hydrogels composed of chitosan and
PAAm can be partially or completely degraded by the action of the
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(1)
(3)
(2)
(4)
Figure 4. Chitosan/PAAm/T. viride beads (1) and PAMPS-coated chitosan/PAAm/T.
viride beads (2), 5 days, 28°C; net-chitosan-ı-PAAm/B. subtilis films (3) and net-chitosannet-PAAm/B. subtilis films (4), 3 days, 25°C.
enzyme complex produced by the soil fungus T. viride, by degrading
chitosan as well as crosslinked chitosan. The time for the disintegration of hydrogel films and beads depends on the structure of the
network, on the amount of the crosslinking, on pH of the reaction
medium and on the temperature. All of the hydrogels in this study
would be appropriate carriers for Bacillus subtilis, but not all of them
would be suitable carriers for T. viride.
ACKNOWLEDGMENT
Financial support from the Bulgarian National Fund for Scientific
Research (Grant CH-1105) is gratefully acknowledged.
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