A stimuli-responsive and bioactive film based on blended polyvinyl alcohol and cashew gum polysaccharide

Prévia do material em texto

Materials Science and Engineering C 58 (2016) 927–934
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Materials Science and Engineering C
j ourna l homepage: www.e lsev ie r .com/ locate /msec
A stimuli-responsive and bioactive film based on blended polyvinyl
alcohol and cashew gum polysaccharide
Fábio E.F. Silva, Karla A. Batista, Maria C.B. Di-Medeiros, Cassio N.S. Silva, Bruna R. Moreira, Kátia F. Fernandes ⁎
Laboratório de Química de Polímeros, DBBM, ICB, Universidade Federal de Goiás, Brazil.
⁎ Corresponding author. Tel.: +55 62 3521 1492; fax: +
E-mail addresses: katia@icb.ufg.br, kfernandes.lqp@gm
http://dx.doi.org/10.1016/j.msec.2015.09.064
0928-4931/© 2015 Elsevier B.V. All rights reserved.
a b s t r a c t
a r t i c l e i n f o
Article history:
Received 24 June 2015
Received in revised form 4 September 2015
Accepted 14 September 2015
Available online 16 September 2015
Keywords:
Cashew gum polysaccharide
PEJU
Proteolytic enzyme
Biocompatible film
pH stimuli-responsive
In this study, a stimuli-responsive, biodegradable and bioactive filmwas produced by blending cashew gum
polysaccharide (CGP) and polyvinyl alcohol (PVA). The film presented malleability and mechanical proper-
ties enabling an easy handling. Wetting the film changed the optical property from opacity to levels of
transparency higher than 70% and resulted in up to 2-fold increase in its superficial area. Different swelling
indexes were obtained varying the pH of solvent, which allows classifying the CGP/PVA film as pH sensitive
stimuli-responsive material. The bioactivity was achieved through covalent immobilization of papain,
which remained active after storage of CGP/PVA-papain film for 24 h in the presence of buffer or in a dry
form. These results evidenced that CGP/PVA-papain film is a very promising material for biomedical
applications.
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
A biomaterial can be defined as amaterial intended to interfacewith
biological systems to evaluate, treat, augment or replace any tissue,
organ or function of the body [1]. The essential prerequisite to qualify
a material as a biomaterial is biocompatibility, which is the ability of a
material to perform with an appropriate host response in a specific ap-
plication. In the recent years, researches on biomaterials formedical and
related applications have moved from biostable biomaterials to biode-
gradable (hydrolytically or enzymatically degradable). This occurred
mainly because of the increasing demand for eco-friendly materials
leading to the search for alternative products obtained from renewable
sources [1]. One of the many approaches to produce eco-friendly mate-
rials is to blend biodegradable plastics with natural polymers [2–6].
Regarding the natural polymers, polysaccharides extracted from
Brazilian cashew gum have been chemically characterized as branched
heteropolysaccharides mainly composed by galactose (59.4–73%) glu-
cose (6.4–14%), arabinose (4.2–5.3%), rhamnose (2.4–4%), and glucu-
ronic acid (6.3–13.5%) [7–9].
Polyvinyl alcohol (PVA) is a non-toxic water-soluble synthetic poly-
mer with biodegradable properties [10]. Its water solubility is influ-
enced by its degree of hydrolysis, molecular weight, particle size
distribution and crystallinity [11]. PVA hydrogels present microporous
55 62 3521 1190.
ail.com (K.F. Fernandes).
structure with high hydrophilicity and high elasticity, and mechanical
properties of absorption and exudation of body fluid [10].
In previous studies, a biodegradable material was produced by
blending polyvinyl alcohol (PVA) and cashew gum polysaccharide
(CGP). This material was bioactivated through immobilization of a
pool of Trichoderma asperellum cell wall degrading enzymes (T-
CWDE) and successfully used to inhibit the growth of Penicillium
sp., Aspergillus niger and Sclerotinia sclerotiorum [3]. Nevertheless,
the mechanical properties and the opacity of this formulation re-
stricted a wide use of CGP/PVA films. Recently, deeper studies with
this formulation of CGP/PVA film evidenced that this biomaterial
presented the absence of cytotoxic against human PDL fibroblasts
[12], which allowed classifying it as a biocompatible material.
According to Roy et al. [13], stimuli-responsive polymers can be clas-
sified as those that change their individual chaindimensions/size, second-
ary structure, solubility, or the degree of intermolecular association under
influence of the solvent. In most cases, the physical or chemical events
that cause these changes are limited to formation or destruction of sec-
ondary forces (hydrogenbonding, hydrophobic effects, electrostatic inter-
actions, etc.), simple reactions (i.e., acid–base reactions) of moieties
pendant to the polymer backbone; and/or osmotic pressure differentials
that result from such phenomena [14].
In this research, a stimuli-responsive CGP/PVA film with improved
mechanical and optical propertieswas produced and converted in a bio-
active material after immobilization of papain through covalent bond.
This material showed high physical and bioactivity stability with very
promising performance for biomedical applications.
Table 1
Experimental design and papain activity according to the CCRD.
Run pH
X1
Time (min)
X2
Temperature (°C)
X3
Papain activity
(U cm−2)
1 (−) 4.0 (−) 15 (−) 5 7.31 ± 0.01
2 (−) 4.0 (−) 15 (+) 25 1.43 ± 0.49
9 (−) 4.0 (0) 30 (0) 15 1.19 ± 0.54
3 (−) 4.0 (+) 60 (−) 5 11.11 ± 0.54
4 (−) 4.0 (+) 60 (+) 25 0.97 ± 0.28
11 (0) 6.0 (−) 15 (0) 15 2.45 ± 0.66
13 (0) 6.0 (0) 30 (−) 5 8.60 ± 0.47
15 (C) (0) 6.0 (0) 30 (0) 15 2.29 ± 0.91
16 (C) (0) 6.0 (0) 30 (0) 15 2.30 ± 0.72
14 (0) 6.0 (0) 30 (+) 25 1.98 ± 0.08
12 (0) 6.0 (+) 60 (0) 15 3.86 ± 0.63
5 (+) 8.0 (−) 15 (−) 5 3.40 ± 0.19
6 (+) 8.0 (−) 15 (+) 25 1.29 ± 0.50
10 (+) 8.0 (0) 30 (0) 15 1.46 ± 0.62
7 (+) 8.0 (+) 60 (−) 5 1.78 ± 0.42
8 (+) 8.0 (+) 60 (+) 25 1.43 ± 0.59
928 F.E.F. Silva et al. / Materials Science and Engineering C 58 (2016) 927–934
2. Material and methods
2.1. Origin and purification of cashew gum polysaccharides (CGP)
Samples of the cashew gumwere collected from trees of Anacardium
occidentale at CIALNE farm, Pacajus, Ceará, Brazil. Nodules were milled,
immersed in distilled water in a proportion of 20% (w/v), and kept at
room temperature (25 °C), for 24 h. The solution was filtered to remove
bark fragments and then precipitatedwith ethanol, in a ratio of 1:3 (v/v),
for 24 h. The precipitated cashew gum polysaccharide (CGP) was sepa-
rated by filtration, washed with ethanol, dried and stored at room tem-
perature (25 °C) in airtight vials.
2.2. Production of the biodegradable film
The biodegradable film was obtained by solvent casting procedure
after preparation of film-forming solutions. The film-forming solution
was prepared by mixing of 5 mL of 3 or 6% (w/v) acidic PVA solution
(pH 2.0; 5 mL of 3 or 6% (w/v) CGP aqueous solution; 0.6 mL of
1.0 mol L−1 sodium (meta)periodate solution; 1.5 mL of 1.0 mol L−1
phosphoric acid solution containing 10% (w/v) calcium chloride and
15mgofmannitol. This solutionwas casting on glassmolds and the sol-
vent evaporationwas left to occur at room temperature. The dried films
were peeled from the casting mold and exhaustively washed with
0.1 mol L−1 phosphate buffer (pH 8.0) for the pH neutralization and
complete removal of iodine excess. Then, filmswere dried at room tem-
perature and stored in plastic vials. Samples of CGP/PVA film were cho-
sen considering film thickness uniformity and the absence of defects
(i.e., air bubbles, holes, tears, flaws, etc.).
2.3. Film characterization
2.3.1. Transparency
Optical transmission measurements were performed according to
methodology described by Wang and Xiong [15]. Films samples were
analyzed by means of a UV–Vis Spectrophotometer (Biospectro SP-
220), by using a wavelength range between 350 and 750 nm, with a
step size of 5 nm.
2.3.2. Mechanical properties
Mechanical properties were determined in a Texture Analyzer TA1
(Lloyd), with a 50 N load cell equippedwith tensile grips (A/TG
model). Samples of film were cut into strips of 10 × 20 mm, according
to the ASTM D-638 M-93 standard [16]. The grip separation was set at
10 mm, with a crosshead speed of 5 mm min−1. Tensile strength (TS),
percentage of elongation at break (%E), and elastic modulus (EM)
were evaluated. Each sample used was previously inspected and those
containing any defect or showing average thickness variation higher
than 5% were rejected.
2.3.3. Swelling of films
For the determination of swelling index, pre-dried CGP/PVA films
were cut into strips of 1 × 1 cm, weighted, immersed in 5 mL of the dif-
ferent solutions and incubated at room temperature (25 °C). The sol-
vents tested were: (1) ultrapure water (Milli-Q); (2) 0.1 mol L−1
sodium acetate buffer pH 5.0; (3) 0.1 mol L−1 sodium citrate buffer
pH 5.; (4) 0.15mol L−1 saline solution; (5) 0.1 mol L−1 phosphate buff-
er pH 8.0; and (6) 0.1 mol L−1 Tris–HCl buffer pH 8.0.
The changes in the weight of the films were monitored until
reaching the stabilization of swelling. From time to time, the films
were withdrawn from the solvent solution and the excess of water
was removed with absorbent paper prior to weight measurements.
The equilibrium-swelling ratio of each sample at time t was obtained
using the following equation:
Swelling index ¼ Wt−W0ð Þ=W0
where,Wt is the weight of the swollen CGP/PVA film at time t andW0 is
the weight of the dried sample.
Samples soaked for 1 h, and subsequently dried, were also re-
swollen and the swelling index was again calculated using the equation
above.
Aiming to investigate the occurrence of dimensional changes during
swelling, strips of CGP/PVA films were cut and the area before and after
swelling was measured using a micrometer.
Furthermore, in order to evaluate the effect of solvents in the film
morphology, the swelling process was conducted using (1) ultrapure
water; (2) 0.15 mol L−1 NaCl solution; (3) 0.1 mol L−1 sodium ace-
tate buffer pH 5.0; and (4) 0.1 mol L−1 sodium phosphate buffer
pH 8.0. After the equilibrium swelling, samples were frozen, lyophi-
lized and then examined in a JEOL JSM 6610 scanning electron
microscope, using a secondary electron detector with 15 kV of
acceleration.
2.3.4. Fourier transform Infrared (FT-IR) spectroscopy
FT-IR spectra of CGP, PVA, CGP/PVA film and CGP/PVA-papain film
were acquired on a PerkinElmer FTIR spectrophotometer (PerkinElmer,
Inc., MA, USA) using potassium bromide (KBr) discs prepared from
powdered samples mixed with dry KBr. Spectrum was recorded (16
scans) in the transparent mode from 4000 to 400 cm−1, at 4 cm−1
resolution.
2.4. Bioactivity studies
2.4.1. Immobilization of papain
The covalent immobilization of papain (Calbiochem, La Jolla, CA,
USA) onto CGP/PVA film was tested by adding 1 mL of papain solution
(5 mg mL−1) to a strip (1 cm2) of CGP/PVA previously activated with
0.1mol L−1 sodium (meta)periodate solution [3]. After immobilization,
the CGP/PVA-papain film was exhaustively washed with 0.1 mol L−1
Tris–HCl buffer (pH 8.0) in order to remove unbounded enzyme.
The optimum immobilization conditions were achieved using a
23 Central Composite Rotatable Design (CCRD). The parameters
and their levels selected for the study of papain immobilization
onto CGP/PVA film were pH (4.0, 6.0, and 8.0), immobilization
time (15, 30 and 60 min) and immobilization temperature (5 °C,
15 °C and 25 °C). In addition, a central point (pH 6.0, 30 min and
15 °C), with two replicates was also included for statistical evalua-
tion (Table 1).
Results from CCRD were analyzed by regression analysis coupled to
response surface methodology (RSM), using the software Statistica 6.0
(Statsoft Inc., Tulsa, USA, 1997). The adjustment of the experimental
Fig. 1. Fourier transform infrared spectra of CGP, PVA, CGP/PVA film and CGP/PVA-papain
film.
929F.E.F. Silva et al. / Materials Science and Engineering C 58 (2016) 927–934
data for the independent variables in the RSM was represented by the
second-order polynomial equation:
y ¼ β0 þ
X
j
β jx j þ
X
i≺ j
βi jxix j þ
X
j
β j jx
2
j þ e
where y is the dependent variable to be modeled; β0, βj, βij and βjj are
regression coefficients, xi and xj are independent variables and e is the
error. Themodel was simplified by dropping terms that were not statis-
tically significant (p N 0.05) by ANOVA.
2.4.2. Papain activity assay
The papain activity was determined according to the method of
Kunitz and modified by Arnon [17]. Briefly: to 0.1 mL of a papain so-
lution (5.0 mg mL−1) were added 0.9 mL of activator solution
(20 μmol L−1 EDTA and 50 μ mol L−1 cysteine in 0.1 mol L−1 Tris–
HCl buffer, pH 8.0). The mixture was incubated at 37 °C for 10 min
and then, 1 mL of a 1% (w/v) casein solution was added. The reaction
was stopped by the addition of 1.0 mL of 10% (w/v) trichloroacetic
acid (TCA) solution. After cooling to room temperature, the samples
were centrifuged (10,000 ×g, 5 min) and the hydrolysis extension
was measured by the readings of the absorbance of the supernatant
at 280 nm. One enzyme unit (U) was defined as the amount of papain
that produces an increase of 0.1 in the absorbance at 280 nm. The ac-
tivity of the containing-papain films (CGP/PVA-papain) was mea-
sured as described, except that papain solution was replaced by
1 cm2 of CGP/PVA-papain film suspended in 0.1mL of Tris–HCl buffer
pH 8.0. Blanks were done by adding TCA before casein in both free
and immobilized enzyme assay.
2.4.3. Effect of pH on the CGP/PVA-papain activity
To evaluate the effect of pH on the activity of immobilized papain,
tests were conducted replacing the standard assay buffer by
0.1 mol L−1 phosphate buffer for pH 6 and 7; 0.1 mol L−1 Tris–HCl
buffer for pH 8 and 9; and 0.1 mol L−1 glycine buffer for pH 10.
2.4.4. Storage stability
The stability of CGP/PVA-papain filmwas determined by assays con-
ducted on samples incubated for 24 h at 4 °C in different buffers. The
buffer solutions used were: (1) 0.1 mol L−1 glycine buffer
pH 3.6 + 0.6 mmol L−1 CaCl2; (2) 0.1 mol L−1 glycine buffer
pH 9.6 + 0.6 mmol L−1 CaCl2; (3) 0.1 mol L−1 Tris–HCl buffer pH 8.0;
(4) 0.1 mol L−1 Tris–HCl buffer pH 8.0 + 0.6 mmol L−1 CaCl2. In addi-
tion, the CGP/PVA-papain film was also stored at room temperature as
dried film.
3. Results
3.1. CGP/PVA films
CGP/PVA films were produced through oxidation of PVA and CGP
with sodium (meta)periodate, resulting in reactive sites in both poly-
meric chains, and formation of a stable covalently linked network. The
presence of considerable amounts of uronic acids in the CGP structure
confer to this material a polyanionic character, depending on the pH
of the solvent [18]. This character might be enhanced by some
unreacted carboxyl groups generated during sodium (meta)periodate
treatment.
The FTIR spectra of the film components showed the characteristics
bands of CGP and PVA (Fig. 1). As can be observed in the Fig. 1a, PVA
spectrum presented a typical band at 1734 cm−1 related to the
stretching of –C_O, a common group in the PVA molecule. The spec-
trum of the CGP evidenced a band commonly assigned to stretching of
hydroxyl groups of polysaccharides in the region of 1639 cm−1 [19,
20]. After blending CGP and PVA, it was detected a reduction in the
band intensity in the region of 3500–3200 cm−1 related to the
stretching vibration of hydroxyl groups from PVA chain and hydroxyls
from the hexoses of CGP. During the film forming, the hydroxyls groups
were involved in the bounding between the polymers, which
occasioned the lower band intensity. In addition, it can be observed a
lower intensity in the band at 1734 cm−1 (related to the PVAmolecule)
and the absence of the band at 1639 cm−1 (related to the CGP
molecule).
3.2. CGP/PVA films characterization
3.2.1. Transparency
At macroscopic scale, CGP/PVA films were homogenous, with an
opaque appearance when dried, whereas became transparent when
wet (Fig. 2).
The transparency is a relevant property related to the functional-
ity of films due to theirlarge impact on the appearance of the final
product [21]. It is known that the film components can interfere
with transparency, since the presence of roughness on the surface
of the material would be detrimental to transparency because of
the scattering of light [22]. The high transparency of CGP/PVA film
was confirmed in tests of visible light transmittance, as shown in
Fig. 3. As can be seen, the transmittance average was 60.7%, with
Fig. 2.Macroscopic appearance of CGP/PVA film: (a) dried and (b) wet forms.
930 F.E.F. Silva et al. / Materials Science and Engineering C 58 (2016) 927–934
the minimum value obtained at 380 nm (50.1%) andmaximum value
at 550 nm (70%).
3.2.2. Mechanical properties
Mechanical properties of films are largely associated with distribu-
tion and density of inter and intramolecular interactions created in the
network of the polymeric material [23]. The tensile strength (TS) can
be defined as the capacity of resistance to rupture presented by the ma-
terialwhen submitted to pressure force. On the other hand, the percent-
age of elongation (E%) is related to the elasticity of a material since it is
measured through the extension under traction [24]. Elastic modulus
(EM) is a measure related to properties of molecular flexibility of the
polymer chain, evidencing the ability of a polymer to absorb and dis-
perse energy without deformation of its tridimensional network.
Our results evidenced that changing in the content of CGP and PVA
changed the mechanical properties of the films. Tensile strength was
enhanced from 3.71 to 7.56 kgf cm−2 when PVA and CGP content
were increased from 3 to 6%. This effect can be due to the formation of
a more compact network between CGP and PVA in the film containing
Fig. 3. Optical transmittance of CGP/PVA fil
6% of each polymer. On the other hand, it was observed a 29% reduction
in the percentage of elongation (E%) in the CGP/PVA film producedwith
the highest polymers concentration (263.2% for 3% CGP/PVA; 186.6% for
6% CGP/PVA). The increase in the polymers concentration in the film
probably resulted in higher hydrogen and covalent bonds between
CGP and PVA, leading to the formation of a more cohesive network
with less elasticity. Despite the reduction in the percentage of elonga-
tion, the value of elasticmodulus (EM) of the 6% CGP/PVAfilm increased
to 49,904 kgf whereas the 3% CGP/PVA film presented value of elastic
modulus of 12,135 kgf. This result indicates that 6% CGP/PVA material
is more resistant to deformation of its tridimensional network.
3.2.3. Swelling index measurements
Swelling is one of the most important properties of polysaccharide
films, which characterize its use for biomedical applications [25–27].
Results from swelling studies are shown in Fig. 4.
As can be seen in Fig. 4a, the CGP/PVA films presented a similar
swelling behavior, characterized by a high increase in the swelling
index in the first 10 min of soaking in all solvents, except for acetate
m in the visible range of the spectrum.
Fig. 4. Swelling properties of CGP/PVA film during soaking in different solvents: (a) swelling index and (b) superficial area.
931F.E.F. Silva et al. / Materials Science and Engineering C 58 (2016) 927–934
buffer, which presented increases in the swelling index up to 30 min. It
was possible to observe a difference in the swelling index as function of
the pH of the soaking solvent. In general, the films soaked in neutral or
alkaline solutions presented higher swelling index. On the contrary, the
films soaked in acidic solutions presented lower swelling indexes.
Generally, the changes observed in pH/dimensional sensitive mate-
rials are related to the shift from hydrophobic to hydrophilic interac-
tions in the polymeric network. In the case of CGP/PVA film, the
glucuronic acid residues on CGP and the carboxyl groups resultant
from oxidative action of sodium (meta)periodate on PVA and sugar res-
idues of CGPwere protonatedwhen filmswere swollen in acid solution.
Under this condition, the hydrophobic interaction among the polymer
chains was predominant and consequently, the CGP/PVA film shrunk
and has absorbed small amounts of solvent. The swelling process of
CGP/PVA film in neutral or basic solutions promoted deprotonation of
those carboxyl groups. These negatively charged groups in the polymer
chains caused repulsion among them, increasing the area of CGP/PVA
film and consequent solvent entrance into the film structure.
Indeed, SEM photomicrographs of swollen films has shown that
these materials present quite different morphology as function of the
solvent used in the soaking procedure (Fig. 5).
The soaking process in phosphate buffer resulted in a material with
large and well-defined pores (Fig. 5a) while soaking in acetate buffer
produced a rough material with few and very small pores (Fig. 5d).
Table 2 shows the average pore size of the films soaked in different
solvents. As can be seen, films soaked in neutral or alkaline solutions
presented higher values of pore size, which can explain the higher
swelling index obtained in these materials.
According to Chen and Chang [14] solvent-active composites (SAC)
are a class of stimuli-responsive materials (SRM) that can be obtained
Fig. 5. Scanning electron micrographs of CGP/PVA soaked in (a) 0.1 mol L−1 sodium phosphate buffer pH 8.0; (b) 0.15 mol L−1 NaCl solution; (c) ultrapure water (Milli-Q); and
(d) 0.1 mol L−1 acetate buffer pH 5.0.
932 F.E.F. Silva et al. / Materials Science and Engineering C 58 (2016) 927–934
on deformed polymers because solvent molecules cause swelling on
polymeric materials and increase the flexibility of the macromolecule
chains of the polymers. Considering the CGP/PVA films, the switching
capacity in the conformation of their polymer chains resulted in switch-
able properties of transparency and swelling/area. The possibility of
controlling the film network expansion depending on the nature of
the solvent or pH dictates different applications for this material,
which comprises pharmaceutical and biomedical possibilities.
Compared to other films, CGP/PVA presented a rapid diffusion of
water and saline solution into the film structure, which can be due to
the hydrophilic nature of CGP/PVA film. The presence of amino in CGP
as well as the hydroxyl groups in the both CGP and PVA interacted
with hydrophilic groups in the water/saline. Despite of similar profile
of swelling, the CGP/PVA films soaked in saline solution has shown
swelling indexes 17% higher than those soaked in water (1.79). In addi-
tion, the swollen CGP/PVA films presented areas 2-fold higher than
those of dried samples, independently of soaking solution (Fig. 4b).
Moreover, compared to otherfilmswith biomedical applications, CGP/
PVA film showed lower time of soaking as well as higher swelling index
when compared with chitosan films [25,28]. Furthermore, the CGP/PVA
film presented higher swelling index than that presented by Meher
et al. [29] for cellulose-poly(methyl methacrylate) mucoadhesive film.
Table 2
Pore size in films swollen in different solvents.
Pore size
(μm)
Phosphate buffer 19.6
Saline solution 9.64
Ultrapure water 9.49
Tris buffer 7.69
Acetate buffer 3.05
In order to evaluate if the huge swelling was an effect arising from
the structure of the film and if its intrinsic structure was not affected,
the films were soaked for 1 h, dried and re-soaked. The results evi-
denced that swelling powerwasmaintained after re-swollen of samples
evidencing the stability of the chemical structure, which remained unal-
tered upon swelling and subsequent drying.
The short time required in maximizing swelling and the mainte-
nance of swelling capacity of CGP/PVA film is an important finding
since in vitro tests have indicated that rapid swelling behavior in mate-
rials assists in cell adhesion and growth [27].
3.3. Papain immobilization onto CGP/PVA film
It is known that the enzymatic removal of the necrotic tissue
from injured areas by papain promotes wound healing [30]. Thereare numerous commercial pharmaceutical formulations containing
papain, however they lack stability and quickly lose their activity
due to the autolytic enzyme action. Therefore, the inclusion of pa-
pain onto CGP/PVA film is an interesting approach aiming to pro-
duce a bioactive material with extended action compared to free
enzyme formulations.
Table 1 shows the factors investigated in the CCRD, as well as the
coded and decoded levels, and the means of papain activity. The multi-
variate analysis for papain immobilization (Fig. 6a) showed that the
main factor influencing the immobilization process is the temperature
(X3), followed by interaction between pH and time (X1/X3). The linear
term for temperature (X3) negatively affected immobilization whereas
the interaction factor pH/time (X1/X3) positively affected the papain ac-
tivity (p b 0.05).
The regression analysis showed an adequate fit of experimental
values to the second-order polynomial model as a function of the signif-
icant factors. Consequently, the mathematical model describing the
Fig. 6. Pareto chart and surface response plots for papain immobilization.
933F.E.F. Silva et al. / Materials Science and Engineering C 58 (2016) 927–934
correlation between the response and the variables is presented as
follows:
Papain activity Uð Þ ¼ 9:16−0:39X2
1−1:48X3 þ 0:02X2
3
þ 0:08X1X3 r2 ¼ 0:89
� �
:
The relationships between immobilization factors and the papain ac-
tivity can be better understood by examining the series of response sur-
face plots. Fig. 6b shows de effect of immobilization temperature, pH
and their mutual interaction on the papain immobilization. Apparently,
increasing the temperature and pH of immobilization decreases the pa-
pain activity. In addition, the correlation analysis evidenced that tem-
perature had the most pronounced effect, negatively affecting the
enzyme activity (-0.67).
In order to achieve the best conditions for papain immobilization,
the desirability function method was used. Desirability is an optimiza-
tion method that combines all variables into a single objective function,
which represents the relationship of all responses obtained during opti-
mization. As can be seen in Fig. 6c, the calculated desirability decreased
with the increase of temperature and pH. That is, optimal desirability
could be obtained at lower temperature (5 °C) and pH (4.0) and inter-
mediate immobilization time (60 °C). The higher the desirability, the
better are the optimal conditions. After numerical optimization based
on highest desirability, the immobilization conditions were established
as pH 4.0, 5 °C and 60 min (d = 0.80). Triple verifying experiments at
optimal conditionswere done to confirm this prediction. The papain ac-
tivity was about 11.1 U, which was approximately equal to calculated
11.49 U according to the regression model.
After immobilization, the CGP/PVA-papain film was analyzed using
infrared spectroscopy, and the FTIR spectrum is shown in Fig. 1b. As can
be observed, the CGP/PVA film containing papain presented a broad
band at 3300 cm−1 stronger than that present in the CGP/PVA film.
This increase in the band intensity can be due to the additional stretching
vibrations of –NH from amide groups present in papain, which in associ-
ation with the stretching vibrations of –OH from CGP/PVA produced a
broad stronger band. In addition, a band at 1657 cm−1, characteristic of
Amide I, commonly present in the protein structure, evidenced the
immobilization of papain onto CGP/PVA film [31].
3.3.1. Effect of pH on the papain activity
Changes in pH profile are frequently reported for immobilized en-
zymes, most of them caused by the chemical modifications in the cata-
lytic microenvironment surrounding the support. Considering that
devices for biomedical applications must be active in a narrow range
of pH, tests were conducted to evaluate if the immobilization of papain
onto CGP/PVA film changed its pH profile.
As can be seen in Fig. 7, while free enzyme presented a plateau of ac-
tivity around pH 7–8, CGP/PVA-papain presented maximum activity at
pH 8. This modification in the pH profile of CGP/PVA-papain compared
to the free papain might be attributed to pH changes in the domain of
the immobilized enzymes particles, arising from the chemical proper-
ties of the polymer.
The narrowing of the optimal pH for the immobilized papain was
probably due to the gradual ionization of the surface of CGP/PVA film
until pH 7, maintaining the pH in the neighborhood of the enzyme
more acidic than the bulk. After complete ionization of the support,
the pH in the microenvironment of papain rapidly increases favoring
the ionization of amino acids in the papain active site, which leads to
an increase of the proteolytic activity.
Although the pH profile of CGP/PVA-papain has changed, the
immobilized enzyme presented satisfactory retention of activity near
to physiologic values of pH (6–7), allowing the use of CGP/PVA-papain
as a bioactive material in the biomedical area.
3.3.2. Storage stability
Storage stability is one of themost important criteria for the applica-
tion of an enzyme on a commercial scale. In this study, the CGP/PVA-
papain stored in different buffers presented activity retention between
78% and 100% after 24 h of storage. The best stability was obtained by
storing the CGP/PVA films in Tris–HCl buffer pH 8 + CaCl2 (96.8%),
followed by glycine buffer pH 3.6 + CaCl2 (80.9%), Tris–HCl buffer
(80%) and glycine buffer pH 9.6 + CaCl2 (78.6%).
Nevertheless, the best stability was obtained when CGP/PVA-papain
filmwas dried and stored at room temperature. As reported in the liter-
ature, in general immobilized enzymes lost their activity after drying,
probably due to the loss of the intrinsic mobility of the enzyme after
Fig. 7. pH profile of free and CGP/PVA-papain film.
934 F.E.F. Silva et al. / Materials Science and Engineering C 58 (2016) 927–934
immobilization, which is necessary to reordering of the polypeptide
chain during water removal [32]. In our study was demonstrated that
after drying and storage at room temperature the enzyme activity
remained unchanged (100% activity), evidencing that immobilization
process did not restrained the structural mobility of the papain.
4. Conclusions
Compared to other CGP/PVA formulations [3,12,24], the CGP/PVA
film produced in this study presented superior mechanical properties
(tensile strength and percentage of elongation), resulting in a stablema-
terial of ease handling. This material presented switchable optical and
structural properties as function of the solvent used to swell the film, as-
suming high transparency and considerable increase in the area after
wetting. The area increase may be controlled by the pH of the solvent
used to wet the film, since the film is a pH stimuli-responsive material.
The film became bioactive after papain immobilization, which is an en-
zyme frequently used for treatment of wounds. The biocompatibility,
bioactivity and the short time for maximum swelling and the high
swelling index presented by CGP/PVA film allows proposing its use for
biomedical applications.
Acknowledgments
This study was supported by a grant provided by CNPq (Process
number 402468/2013-9). Fábio E.F. Silva, Bruna R. Moreira and Cassio
N.S. Silva thank CNPq for the fellowship support. Karla A. Batista and
Maria C. B. Di-Medeiros thank CAPES for the fellowship support.
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