Effect of chitin nanowhiskers on mechanical and swelling properties of Gum Arabic hydrogels nanocomposites

Carbohydrate Polymers 266 (2021) 118116

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Effect of chitin nanowhiskers on mechanical and swelling properties of
Gum Arabic hydrogels nanocomposites
´tia S. Nunes a, Adley F. Rubira a, Edvani C. Muniz a, d, e, Andr´e
Antonio G.B. Pereira a, b, *, Ca
c, **
R. Fajardo
a

Grupo de Materiais Polim´ericos e Comp´
ositos (GMPC), Maring´
a State University, Av. Colombo 5790, 87020-900 Maring´
a, PR, Brazil
Laborat
orio de Biopolớmeros, Coordenaỗ
ao de Engenharia de Bioprocessos e Biotecnologia, Universidade Tecnol´
ogica Federal do Paran´
a (UTFPR- DV), Estrada para
Boa Esperanỗa, 85660-000 Dois Vizinhos, PR, Brazil
c
Laborat
orio de Tecnologia e Desenvolvimento de Comp´
ositos e Materiais Polim´ericos (LaCoPol), Federal University of Pelotas, Campus Cap˜
ao do Le˜
ao s/n, 96010-900

Pelotas, RS, Brazil
d
Departamento de Química, Universidade Federal do Piauớ, 64049-550 Teresina, PI, Brazil
e
Programa de P
os-graduaỗ
ao em Ciˆencia e Engenharia de Materiais, Universidade Tecnol´
ogica Federal do Paran´
a (UTFPR- LD), Avenida dos Pioneiros, 3131, 86036370 Londrina, PR, Brazil
b

A R T I C L E I N F O

A B S T R A C T

Keywords:
Chitin
Whiskers
Gum Arabic
Hydrogel
Composite
Swelling

Hydrogels based on biopolymers like Gum Arabic (GA) usually show low applicability due to weak mechanical
properties. To overcome this issue, (nano)fillers are utilized as reinforcing agents. Here, GA hydrogels were
reinforced by chitin nanowhiskers (CtNWs, aspect ratio of 14) isolated from the biopolymer chitin through acid
hydrolysis. Firstly, GA was chemically modified with glycidyl methacrylate (GMA), which allowed its cross­
linking by free radical reactions. Next, hydrogel samples containing different concentrations of CtNWs (0–10 wt
%) were prepared and fully characterized. Mechanical characterization revealed that 10 wt% of CtNWs promoted
an increase of 44% in the Young’s modulus and 96% the rupture force values compared to the pristine hydrogel.

Overall, all nanocomposites were stiffer and more resistant to elastic deformation. Due to this feature, the
swelling capacity of the nanocomposites decreased. GA hydrogel without CtNWs exhibited a swelling degree of
975%, whereas nanocomposites containing CtNWs exhibited swelling degrees under 725%.

1. Introduction
Polysaccharide nanoparticles have been extensively studied in the
last few years and present a huge potential of applications in different
areas including reinforcing phases in polymer composites (Eichhorn,
2011; Rodrigues et al., 2014; Tian et al., 2015). The second most
abundant polysaccharide, chitin or β(1 ⟶ 4)-linked N-acetyl-Dglucosamine polymer, is present in many organisms (e.g., arthropods,
nematodes, fungi, etc.) as a structural component in their exoskeletons
and cell walls and its highly crystalline structure is suitable for the
preparation of rod-like crystalline nanocrystals, or nanowhiskers
referred as CtNWs (Fan et al., 2012). In general, CtNWs present high
aspect ratio (length to width ratio), low density, high Young’s modulus
(~150 GPa and 15 GPa, for longitudinal and transversal, respectively)

(Zeng et al., 2012), and high dispersibility in acidified aqueous media.
Interesting biological properties attributed to CtNWs include biode­
gradability, biocompatibility, and antibacterial activity. The functional
groups at the surfaces of CtNWs and the high specific surface allow
dipolar interactions with other components as well as further chemical
modification (Ou et al., 2020). Therefore, because of such interesting
features, many potential applications of CtNWs are being unveiled.
The preparation of CtNWs was first reported by Marchessault et al. in
1959 and is well established nowadays (MARCHESSAULT et al., 1959).
Today, the methodology for preparing CtNWs is mainly based on the
acid hydrolysis, in which the differential hydrolysis of amorphous and
crystalline phases are kinetically controlled to produce whiskers with
diameters of 5–25 nm and lengths ranging from 150 nm up to 2 μm

(Pereira et al., 2014).

* Correspondence to: A. G. B. Pereira, Grupo de Materiais Polim´ericos e Comp´
ositos (GMPC), Maring´
a State University, Av. Colombo 5790, 87020-900 Maring´
a,
PR, Brazil.
** Corresponding author.
E-mail addresses: (A.G.B. Pereira), (A.R. Fajardo).
/>Received 14 October 2020; Received in revised form 5 April 2021; Accepted 18 April 2021
Available online 24 April 2021
0144-8617/© 2021 Elsevier Ltd. This article is made available under the Elsevier license ( />

A.G.B. Pereira et al.

Carbohydrate Polymers 266 (2021) 118116

The earliest report of CtNWs as reinforcement phase in the prepa­
ration of polymer nanocomposites was done by Paillet & Dufresne
(2001). The incorporation of CtNWs, at concentration as low as 1 wt%,
in polypropylene matrix allowed re-processed PP to recover its original
mechanical properties (de Sousa Mol & Or´
efice, 2016). The Young’s
modulus of poly(vinyl alcohol) films significantly increased from 28 to
50 GPa at 30 wt% CtNWs (Uddin et al., 2012). CtNWs also improved the
mechanical properties of maize films from 1.64 MPa to 3.69 MPa at 1 wt
% CtNWs, decreased the water vapor permeability to half of its original
value at 2 wt%, and induced antibacterial activity against Listeria mon­
ocytogenes (Qin et al., 2016). Poly(D,L-lactide) films were deep-coated
with CtNWs suspension to produce materials with improved tensile

properties and better cellular adhesion, proliferation, and osteogenic
differentiation of MC3T3-E1 strain (Liu et al., 2019). CtNWs and chi­
tosan whiskers (CsNWs) were used to modify the surfaces of cellulose
acetate electrospun nanofibers inducing antibacterial activity in the
prepared mats, which have the potential to be applied in the biomedical
field (Pereira et al., 2020).
Over the past few years, numerous studies focused on the develop­
ment of hydrogels have been reported in the literature (Curvello et al.,
2019; Du et al., 2020; Mohammadinejad et al., 2019). The success of
these soft materials can be credited to their several attractive properties,
which have stimulated their use in a wide range of applications (bio­
materials, delivery systems, soil conditioners, environmental remedia­
tion, among others). Hydrogels are characterized by three-dimensional
networks formed by crosslinked hydrophilic macromolecules. Such
characteristics endow hydrogels the ability to absorb and retain large
amounts of aqueous liquids without dissolving. Three-dimensional net­
works can be synthesized using different approaches, which depend on
the crosslinking process and the starting materials (Ahmed, 2015).
Therefore, hydrogels can be elaborated with specific features maxi­
mizing their action in a target application. Furthermore, the incorpo­
ration of other types of materials within the hydrogel network (resulting
in a composite) has been frequently reported as an efficient approach to
obtain hydrogels with superior properties (Feng et al., 2019; Thoniyot
et al., 2015). More recently, attention has been paid to filler materials
derived from polysaccharides, as CtNWs, mainly due to their reinforcing
action when associated with a hydrogel network. Hydrogel composites
synthesized with these fillers are an alternative to overcome the poor
mechanical properties reported to the hydrogels synthesized solely with
natural polymers. Many studies state that polysaccharide-based hydro­
gels have limited stretchability and are often brittle materials (Wang

et al., 2018). The presence of CtNWs in injectable chitosan-based
hydrogels improved the mechanical properties of the gel, promoted
fast gelation speed as it worked as a crosslinker, and favored biological
compatibility according to the MTT method (Wang et al., 2017). Simi­
larly, CtNWs have significant reinforcement effect on hydrogels of
gelatin (Ge et al., 2018), chitosan (Sun et al., 2018), chitosan/dextran
(Pang et al., 2020), and methylcellulose (Jung et al., 2019).
Gum Arabic (GA) is a dried exudate obtained from the stems and
branches of Acacia Senegal or Acacia seal consisting of complex and
branched polysaccharides structures. The hydrolysis of such carbohy­
drates yields mainly arabinose, galactose, rhamnose, and glucuronic
acid. GA is a water-soluble gum widely used in the food industry and,
currently, due to its attractive biological properties (antioxidant, he­
mostatic, nonhemolytic, antibacterial, among others) it has been used to
elaborate pharmaceutical and biomedical devices, as hydrogels (Gerola
et al., 2015; Li et al., 2017). GA-based hydrogels are often associated
with many benign and eco-friendly features (e.g., biocompatibility,
biodegradability, non-toxicity, among others). At the same time, this
kind of hydrogel is also disgraced due to their poor mechanical prop­
erties, which seems to be a huge shortcoming for their use in various
high-end applications. To overcome this obstacle, the introduction of
filler materials (from organic and/or inorganic sources) into the
hydrogel network seems to be a successful strategy. Most recently, the
use of nano-sized fillers has gained great attention, mainly because of

their astonishing ability to dissipate energy under mechanical stress (S.N. Li et al., 2020).
Up to date, few studies are devoted to synthesizing and characterize
hydrogel composites based on GA. To the best of our knowledge, this is
the first study devoted to evaluating the effect of different amounts of
CtNWs on the mechanical and swelling properties of hydrogels prepared

with GA. We hypothesize that CtNWs can act as an efficient reinforcing
agent for GA hydrogel.
2. Materials and methods
2.1. Materials
Chitin from shrimp shells (practical grade, high molecular weight >
300 kDa, CAS 1398-61-4), Gum Arabic (GA) from acacia trees (molec­
ular weight 250 kDa, high viscosity, CAS 9000-01-5), glycidyl methac­
rylate (GMA, molecular weight 142.15 g/mol, CAS 106–91-2), and
sodium persulfate (SPS, CAS 7775-27-1) were purchased from SigmaAldrich (USA). N,N′ -methylenebisacrylamide (MBA, CAS 110-26-9)
was purchased from Biorad (USA). Potassium hydroxide (KOH, 85%,
CAS 1310-58-3), sodium hydroxide (NaOH, 97%, CAS 1310-73-2), hy­
drochloric acid (HCl, 36.5%, CAS 7647-01-0), and pH 4 buffer acetate
(15% sodium acetate and 48% acetic acid) were purchased from Synth
(Brazil). Sodium chlorite (NaClO2, 80%, CAS 7758-19-2) was purchased
from Alfa Aesar (USA). Ethanol (99.5%, CAS 64-17-5) was purchased
from Nuclear (Brazil). Except for chitin, all chemicals were used without
further purification.
2.2. Isolation of chitin nanowhiskers
Chitin nanowhiskers (CtNWs) were isolated from chitin according to
previously reported protocols (Paillet & Dufresne, 2001; Pereira et al.,
2014) (Fig. 1a). Chitin (practical grade) was firstly purified by removing
residual proteins followed by bleaching. Proteins were removed by
heating 5 g of chitin in 150 mL of KOH solution (5 wt/v%) at boil under
vigorous stirring for 6 h. The suspension was kept under stirring at room
temperature for another 12 h, filtered, and washed with water. Next, the
collected solid was bleached in 150 mL of 1.7% NaClO2 in pH 4 buffer
acetate at 80 ◦ C for 2 h, then filtered and washed with water. The
bleaching reaction was performed twice. Finally, the bleached solid was
re-suspended in 150 mL of KOH solution (5 wt/v%) for 48 h, then
centrifuged to collect the purified chitin at 75% yield (~3.75 g).

CtNWs were obtained by hydrolyzing the purified chitin in 3 mol/L
HCl at boil for 90 min under stirring. The ratio chitin/volume of HCl
solution (g/mL) was fixed at 1:30 (Pereira et al., 2014). The reaction was
stopped by adding 50 mL of cold water and centrifuged (3400 rpm for
15 min). The precipitate was re-suspended in 200 mL of distilled water
followed by centrifugation. This procedure was repeated three times.
Next, the precipitate was re-suspended in distilled water and dialyzed
(molecular weight cut-off 12–14 kDa) against water up to neutral pH.
The suspension was sonicated (40% maximum amplitude) for a total of
20 min with 5 min of interval between every 5 min of sonication cycle,
followed by centrifugation (3000 rpm, 10 min) for removing any
remaining precipitate. Finally, the CtNWs suspension was stored at 8 ◦ C.
The yield of CtNWs (~65% or 2.44 g) was determined by gravimetric
analysis, in triplicate. For this, aliquots of the CtNWs suspension (1000
± 1 μL) were collected and the liquid phase was evaporated at 50 ◦ C.
Then, the residue was weighed and correlated to the total volume of the
nanowhiskers suspension. The CtNWs concentration in the suspension
was adjusted to be 5 wt% or 50 mg/mL. CtNWs were kept in the neverdried state prior to hydrogel preparation.
2.3. Characterization of CtNWs
CtNWs were characterized by zeta potential, Fourier Transform
Infrared Spectroscopy (FTIR), Thermogravimetric Analysis (TGA),
2


A.G.B. Pereira et al.

Carbohydrate Polymers 266 (2021) 118116

Fig. 1. (a) Isolation of CtNWs from raw chitin and (b) synthesis of GA-GMA followed by the synthesis of the hydrogel nanocomposite.

Differential Scanning Calorimetry (DSC), X-ray Diffraction (XRD),
Scanning Electron Microscopy (SEM), Transmission Electron Micro­
scopy (TEM), 1H, 13C, and 2D HSQC (Heteronuclear Single Quantum
Coherence) Nuclear Magnetic Resonance (NMR). A complete descrip­
tion of these analyses has already been reported in previous papers
(Pereira et al., 2014, 2015).

added to the reaction system under stirring. The resulting solution was
heated to 70 ◦ C and this temperature was kept up to the hydrogel for­
mation (Fig. 1b). The as-synthesized hydrogel was recovered, soaked in
distilled water, and oven-dried (40 ◦ C, 24 h). A set of hydrogel com­
posites containing CtNWs were synthesized using a similar procedure;
however, specific volumes of CtNWs suspensions were added to the GAGMA solution prior to the hydrogel formation. The compositions of each
hydrogel sample as well as their respective labels are detailed in Table 1.
It is important to inform that the limiting concentration of CtNWs was

2.4. Synthesis of the hydrogel composite containing CtNWs
Before hydrogel synthesis, raw GA was chemically modified with
glycidyl methacrylate (GMA) adapting the methodology reported pre­
viously by Paulino et al. (2012) (Fig. 1b). Briefly, 30 g of GA were sol­
ubilized in 1 L of distilled water at 65 ◦ C under magnetic stirring.
Thereafter, the pH of the solution was adjusted to 3.5 by adding a few
drops of concentrated HCl (36.5 wt/v%). Next, 3 mL of GMA were
added, and the reaction system was kept under stirring at 65 ◦ C for 24 h.
The chemically modified GA was recovered by precipitation using
ethanol. The precipitate (denoted as GA-GMA) was rinsed with abun­
dant ethanol to eliminate the unreacted chemicals, centrifuged and
oven-dried (40 ◦ C) for 24 h.
The synthesis of the GA-GMA hydrogels was performed as follows; 5
g of GA-GMA were solubilized in 25 mL of HCl acidified water (pH ~3).

Next, 0.1 g of MBA (crosslinker) and 0.1 g of SPS (radical initiator) were

Table 1
Hydrogels composition.

3

Sample

GAGMA
(g)

CtNWs
(wt/wt
%)

GA-GMA/
CtNWs0
GA-GMA/
CtNWs1
GA-GMA/
CtNWs5
GA-GMA/
CtNWs10

5

0

5

CtNWs
suspension
(mL)

MBA
(g)

SPS
(g)

Final
volume
(mL)

0

0.1

0.1

25

1

1

0.1

0.1

25

5

5

5

0.1

0.1

25

5

10

10

0.1

0.1

25


A.G.B. Pereira et al.

Carbohydrate Polymers 266 (2021) 118116

fixed at 10 wt/wt% because higher concentrations showed a poor
dispersion into the polymer solution before hydrogel formation.

2.7. Mechanical properties
The mechanical properties of as-synthesized hydrogels were exam­
ined by compressive tests performed in a Texturometer analyzer (TA.
XTplusC - Stable Micro Systems, UK) equipped with a 50 N load cell.
Swollen samples were cut in cubic shapes (10 × 10 × 10 mm) prior to
the tests. Experiments were performed at 25 ◦ C under controlled hu­
midity conditions (55%). Other texturometer parameters: 5-mm inden­
tation depth, 1 mm/s downward probe velocity, and initial crosssectional area of 126 mm2. The Young’s modulus was calculated per
Eq. (2):

2.5. Characterization techniques
All reactants used for the following characterizations were ACS grade
(purity ≥ 99%), suitable for NMR and FTIR analysis.
2.5.1. 1H NMR
1
H NMR spectra were recorded on Varian 300 MHz spectrometer
(model 300, UK). For this, 10 wt/wt% solutions in D2O (raw GA and GAGMA) and CDCl3 (for GMA) were prepared in 5 mm tubes. Tetrame­
thylsilane (TMS) was used as an internal standard and it was suppressed
from the spectra. Data were collected under the following conditions: 12
K data points; 30 s relaxation delay; angle pulse of 90◦ ; acquisition time
of 5 s; temperature of 298 K; 32 scans.

′

Young s modulus =

F.L1
A.(L2 − L1 )

(2)

where F is the necessary force (N) to compress the sample, A is the
sample transversal area (m2), L1, and L2 are the initial and compressed
sample thicknesses (mm), respectively. For each sample, 5 measure­
ments were done.

2.5.2. FTIR
FTIR spectra were recorded on a spectrophotometer (model MB-100
spectrometer, Bomen, Quebec, Canada) operating in the region from
4000 to 500 cm− 1 with a resolution of 4 cm− 1 and 64 scan acquisitions.
Before the spectra acquisition, the dried samples were mixed with KBr
powder and pressed into pellets.

3. Results and discussion
3.1. Isolation of CtNWs
TEM images of CtNWs obtained from casting of dilute aqueous sus­
pension are presented in Fig. 2. The acid hydrolysis of purified chitin
yielded chitin nanorods or nanowhiskers (CtNWs) with averages length
and width of 219 ± 42 nm and 16 ± 5 nm, giving an aspect ratio of 14.
The CtNWs yield was approximately 65% of the mass of the purified
chitin. This 35% initial mass loss was attributed to hydrolysis of chitin
and dissolution of smaller nano-chitins into soluble molecules (oligomer
and monomer). Full characterization of CtNWs has been published
elsewhere by our group (Pereira et al., 2014, 2015, 2020), and only the
main features are discussed in the present paper.

Although CtNWs have its core composed mostly of α-chitin, the
surface is not completely acetylated, as indicated by the positive charges
(+30 mV) noticed from the zeta potential measurements performed at
pH 3. Under the acidic condition, the deacetylated amino groups
available on the CtNWs surfaces become protonated (R–NH2 + H3O+ ⇋
1
R–NH+
3 ), which explains this high positive charge density. Indeed, H
NMR analysis indicated that only 56% of CtNWs surface remains acet­
ylated, while 44% of the amino groups are deacetylated corroborating
the zeta potential data (Pereira et al., 2014). It is worthy to mention that
this high value of zeta potential measured under acidic conditions is
appreciated since it ensures the stability of CtNWs suspension.

2.5.3. XRD
XRD patterns were recorded from powder samples using a Shimadzu
diffractometer (model XRD 6000, Japan) using Ni-filtered Cu-Kα radi­
ation (λ = 1.5406 Å) at a 30 kV anode voltage and a 20 mA current. The
scanning angle (2θ) was ranged from 5◦ to 40◦ at a scan rate of 2◦ /min.
2.5.4. Thermogravimetric analysis (TGA)
TGA analyses were performed from 30 to 600 ◦ C at a heating rate of
10 ◦ C/min and dynamic N2 atmosphere (50 mL/min), in a thermogra­
vimetric analyzer (Netzch, model STA 409 PG/4/G Luxx, USA).
2.5.5. TEM images
TEM images were recorded using a JEOL microscope (JEM-1200EX,
Japan) using an acceleration voltage of 80 kV. Before the TEM imaging,
the selected hydrogel sample was embedded in epoxy resin, fully dried
and then, cut into 80–100 nm thick transverse sections using an ultra­
microtome. The sections were placed onto carbon-coated copper grids
and doubly stained with (3 wt/v%) uranyl acetate before imaging.

The widths and lengths of CtNWs were collected from TEM images
and the averages were calculated from measuring over 100 individual
samples using ImageJ software ( />2.6. Swelling experiments

3.2. Chemical modification of GA with GMA

The swelling behavior of the synthesized hydrogels was investigated
in distilled water at room temperature using a gravimetric method. For
this, known amounts of dried samples (particle size 16–18 mesh or
0.18–1.00 mm) were placed in 30 mL filter crucibles (porosity no. 0) premoistened with a dry outer wall. This set (crucible + hydrogel sample)
was immersed in distilled water allowing the hydrogel to be completely
submerged. At specific time intervals, the system was removed from the
water, the external wall dried and weighed. The swelling rate at a spe­
cific time interval was calculated per Eq. (1):

The 1H NMR spectrum of modified GA (GA-GMA) exhibited the
typical resonance signals of GA accompanied by the appearance of three
new signals (Fig. 3). Two of them, at δ 6.20 and δ 6.17 ppm (denoted as a
and a′), are ascribed to the hydrogen atoms bonded to the vinyl group of
GMA, while the signal at δ 2.21 ppm (denoted as b) is due to hydrogen
atoms of the methyl group. All the resonance signals observed in the GAGMA spectrum agree with previous reports (Reis et al., 2006). Overall,
the chemical modification of polysaccharides with GMA may occur by a
distinct reaction mechanism according to the solvent used (Reis et al.,
2009). In other words, low-rate and irreversible epoxide ring-opening
occur in protic solvents, such as water, while transesterification hap­
pens in an aprotic solvent (e.g., dimethyl sulfoxide, DMSO) yielding the
methacrylated polysaccharide and glycidol as a by-product. This last
mechanism is faster, however, reversible (Reis et al., 2009). Therefore,
as the water was used as the solvent, epoxide ring-opening is the most
likely modification mechanism of GA.

The 1H NMR spectrum of GA-GMA was also used to quantify the
degree of substitution (DS) on GA after reacting with GMA. The DS was

Swelling (%) =

(wt − wo )
× 100
wo

(1)

where wt is the swollen hydrogel weight at a specific time (t) and wo is its
dry weight.

4


A.G.B. Pereira et al.

Carbohydrate Polymers 266 (2021) 118116

Fig. 2. TEM images (left - bright field mode; right - dark field mode) of CtNWs isolated from chitin by acid hydrolysis (conditions: 3 mol/L HCl, 1 g/30 mL chitin/HCl
solution, at boil, 90 min).

Fig. 3. 1H NMR spectra of GMA, raw GA, and GA-GMA.

calculated per Eq. (3) that relates the total areas of the two resonance
signals ascribed to the vinyl hydrogens (δ 6.20 and δ 6.16 ppm) observed
in the GA-GMA spectrum and the resonance signal at δ 5.41 ppm in the
GA spectrum. This signal can be associated with the hydrogen atom

bond to the anomeric carbon in the glucose unit of GA (Fan et al., 2013).
[
]
(Iδ6.20 + Iδ6.16 ) × 0.5
DS (%) =
× 100
(3)
Iδ5.41

(Gerola et al., 2016), as a result of the experimental conditions chosen, it
is important to highlight that polymers highly functionalized with GMA
moieties (i.e., high DS values) usually result in hydrogels with high
crosslinking density. In general, the vinylic groups (–C=CH) from GMA
react with MBA (the crosslinker) through radical reactions resulting in
covalent bonds that hold the polymer chains together. So, high DS values
imply that the concentration of vinylic groups on the polymer backbone
is also high, which in certain aspects can boost the crosslinking process
by reacting either with MB or with other vinylic groups from neigh­
boring molecules. Hydrogels with high crosslinking densities may show
accentuated stiffness and low swelling ability, which impairs their use in
some application fields (e.g., biomaterials, adsorption, among others).
The chemical nature of the GMA, raw GA, and GA-GMA was also

After a careful baseline treatment, the areas of the resonance signals
were measured and the values found were Iδ6.20 ≈ 1.0, Iδ6.16 ≈ 1.21, and
Iδ5.41 ≈ 9.05, respectively. Using these values, the DS calculated for GAGMA was 12.21%. Although the DS was slightly lower than other reports

5

A.G.B. Pereira et al.

Carbohydrate Polymers 266 (2021) 118116

– O stretching of GMA moiety), and bands in the range
1718 cm− 1 (C–
1200–1000 cm− 1 are due to the motions of the glycosidic skeleton
(Espinosa-Andrews et al., 2010; Stefanovic et al., 2013). Additionally,
these spectra also exhibited a shifting of the band ascribed to the
– O stretching of carboxylic groups of GA-GMA from 1607
asymmetric C–
cm− 1 to 1659 cm− 1, which could be caused by the crosslinking reaction.
Similarly, the increase of intensity noticed to the bands associated with
the N–H bending (around 1550 cm− 1) and C–N stretching of amine
bonds (around 1230 cm− 1) can be attributed to MBA, used as the
crosslinker agent (Huang et al., 2013). These particularities in the GAGMA and GA-GMA/CtNWs10 spectra confirm the success of the cross­
linking process. Furthermore, the spectrum of GA-GMA/CtNWs10 also
exhibited two new bands at 1663 cm− 1 and 1556 cm− 1, which are
assigned to the amide I and II vibrational modes of CtNWs. The shouldertype band at 3264 cm− 1 (N–H stretching) also can be attributed to the
presence of CtNWs into the hydrogel matrix. Although these bands are
discreet due to low CtNWs concentration, their presence confirms the
nanocomposite formation. Herein, the filler remained embedded into
the hydrogel matrix without chemical interactions (i.e., there is no ev­
idence of a covalent bond between the CtNWs and GA-GMA chains). On
the other hand, comparing the spectra of GA-GMA/CtNWs0, and GAGMA/CtNWs10, a slight shift of the band attributed to the O–H
stretching (hydroxyl groups) from 3437 cm− 1 from 3442 cm− 1 could be
observed. The interactions among the CtNWs and GA-GMA chains by Hbond explain such behavior. Also, this finding indicates compatibility
between the hydrogel matrix and CtNWs (the filler material).
The XRD pattern recorded for CtNWs exhibited the typical diffrac­
tions peaks of α-chitin in the 2θ range of 5◦ to 40◦ (Goodrich & Winter,

2007). The most important diffraction peaks are at 2θ ≈ 9.3◦ ; 19.2◦ ;
20.8◦ ; 23.3◦ , and 26.3◦ and they are consistent with the crystallographic
planes (020), (110), (101), (130) and (013) attributed to the α allo­
morph of chitin (Fig. 5b). The data corroborates with FTIR analysis
demonstrating that the controlled acid hydrolysis, based on the differ­
ential kinetics of amorphous and crystalline phases, did not affect the
crystal structure of chitin, but generated highly crystalline CtNWs (86%)
(Liu et al., 2015; Pereira et al., 2014). In contrast, the GA-GMA/CtNWs0
XRD exhibited a halo-shaped pattern (with a maximum of around 2θ ≈
19.7◦ ) evidencing the amorphous nature of this hydrogel sample (Pau­
lino et al., 2010). On the other hand, the GA-GMA/CtNWs10 XRD
exhibited the characteristic diffraction signals proceeding from CtNWs
at 2θ ≈ 9.3◦ , 19.2◦ , and 26.3◦ , indicating that the GA-GMA hydrogel was
successfully embedded with CtNWs. Worth of mention, the peaks
attributed to CtNWs in the composite did not shift from CtNWs, indi­
cating the crystalline domains were maintained.
Thermogravimetry (TGA/DTG) was performed to investigate the
thermal stability of the synthesized hydrogels and the effect of CtNWs on
the matrix, as shown in Fig. 5c. The thermal profile of the isolated
CtNWs exhibited two main weight loss stages at the temperature range
of 30 ◦ C to 600 ◦ C. The first weight loss stage (25–120 ◦ C) related to the
evaporation of water was minimal showing the hydrophobic nature of
CtNWs. The second stage (220–440 ◦ C), with a maximum temperature at
379 ◦ C, was due to the thermal degradation of the chitin backbone
(Salaberria et al., 2017). Similarly, the TGA/DTG curves of GA-GMA/
CtNWs0 also exhibited two weight loss stages. The first (25–180 ◦ C)
was due to the evaporation of water adsorbed on the hydrogel (~10% of
weight loss) and the second (190–600 ◦ C) was associated with the
thermal decomposition of the hydrogel matrix (~70% of weight loss). At
600 ◦ C, the residue of GA-GMA/CtNWs0 was 21% of its initial weight.

Overall, the GA-GMA/CtNWs10 nanocomposite exhibited a thermal
behavior comparable to that presented by the pristine hydrogel; how­
ever, some differences can be pointed out. For example, the first stage
due to the water evaporation resulted in a lower weight loss percentage
(~8%), which may suggest that the introduction of CtNWs affected the
hydrophilicity of the hydrogels nanocomposites. Moreover, a third
weight-loss stage can be noticed in the TGA/DTG curves of GA-GMA/
CtNWs0. This additional weight loss stage with a maximum

investigated by FTIR, and the obtained spectra were shown in Fig. 4. The
spectrum of raw GA exhibited a broad band centered at 3424 cm− 1
(O–H stretching of hydroxyl groups) and other typical bands at 2928
cm− 1 (C–H stretching of CHx groups), at 1607 and 1424 cm− 1 (asym­
– O stretching of carboxylic groups), and 1288
metric and symmetric C–
cm− 1 (C–OH stretching). The bands observed in the range 1180–1000
cm− 1 are due to the C–O–C and C–O stretching of glycosidic bonds
(Espinosa-Andrews et al., 2010). GMA exhibited typical bands in the
range 3065–2930 cm− 1 (C–H stretching of –
– CH and –CHx groups) and
– O and C–
– C stretching of ester
bands at 1718 cm− 1 and 1634 cm− 1 (C–
conjugated system), at 908 cm− 1 (C–O–C stretching of epoxide ring)
(Pereira et al., 2013). After modification with GMA, the GA-GMA
spectrum exhibited the typical bands of GA accompanied by some
wavenumber shifting and changes in intensity. Also, the appearance of
new bands was noticed in the GA-GMA spectrum. The band associated
with the OH stretching of hydroxyl groups was broadened and shifted to
3385 cm− 1, while bands in region 1180–1000 cm− 1 exhibited an in­

crease of intensity caused by the introduction of C–O bonds in the GA
backbone. Similarly, the increment in the intensity of the bands that
occur in the range 1450–1350 cm− 1 can be ascribed to additional CH2
and CH3 groups proceeding form GMA. Finally, the appearance of a
–O
small band at 1718 cm− 1 and a shoulder-type band at 1634 cm− 1 (C–
– C groups of GMA) confirm the modification of GA, as previously
and C–
reported by studies on the modification of polysaccharides with GMA
(Gerola et al., 2016; Pereira et al., 2013).
3.3. Characterization of the synthesized hydrogels
Vibrational spectroscopy was also used to characterize the chemical
nature of the isolated CtNWs and synthesized hydrogels and composites
(with CtNWs), as shown in Fig. 5a. The main absorption bands of CtNWs
were observed at 3446 cm− 1 (O–H stretching of hydroxyl groups), at
3264 cm− 1 and 3105 cm− 1 (N–H stretching), at 1663 cm− 1 and 1627
– O stretching, amide I band), at 1560 cm− 1 (a combination of
cm− 1 (C–
C–N–H stretching and N–H bending, amide II band), and at 1028
cm− 1 (C–O stretching of chitin skeletons) (Goodrich & Winter, 2007;
–O
Pereira et al., 2014, 2020). The absorbance ratio at 1663 cm− 1 (C–
stretching) and 3446 cm− 1 (O–H stretching) (A1663/A3446 × 115) was
proposed by Baxter et al. to indicate the degree of N-acetylation of
CtNWs (Baxter et al., 1992). Herein, the overall N-acetylation degree (in
the bulk phase) was calculated to be around 81%.
The FTIR spectra recorded from the pristine hydrogel (GA-GMA/
CtNWs0) and hydrogel nanocomposite containing 10 wt% of CtNWs
(GA-GMA/CtNWs10) showed to be very similar and exhibited the
characteristic bands of GA-GMA at 2932 cm− 1 (C–H stretching), at

Fig. 4. FTIR spectra obtained for GMA, raw GA, and GA-GMA.
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Carbohydrate Polymers 266 (2021) 118116

Fig. 5. (a) FTIR spectra (b) XRD diffraction patterns and (c) TGA (solid lines)/DTG (dash lines) curves of CtNWs, GA-GMA/CtNWs0, and GA-GMA/CtNWs10.

temperature at 374 ◦ C is due to the thermal decomposition of the CtNWs
embedded into the hydrogel matrix. Finally, at 600 ◦ C there are more
residues for GA-GMA/CtNWs10 (~28%) than GA-GMA/CtNWs0, likely
due to the presence of CtNWs into the nanocomposite. It should be noted
that the weight loss stage associated with the thermal decomposition of
the hydrogel matrix (at 294 ◦ C) did not varied, suggesting that the
introduction of CtNWs did not affect the stability of the GA-GMA
hydrogel. Although the FTIR analysis indicated the physical interac­
tion between CtNWs and GA-GMA matrix (H-bonds), this physical

interaction was unable to increase the temperature of GA-GMA/
CtNWs10 thermal decomposition. The lack of chemical bonds between
CtNWs and the hydrogel, the low concentration of CtNWs (≤10 wt%)
and the similar polysaccharide backbone (with similar thermal stability)
could explain the observed behavior.
For TGA analysis, the aqueous CtNWs suspension was dried prior to
analysis. Upon drying, CtNWs self-assemble into tens of micron thick
sheet-like layers with much smaller sub-micron fibrillated network
structures in between, as we have showed (Pereira et al., 2014). In the

Fig. 6. Schematic illustration of the GA-GMA hydrogel nanocomposite containing CtNWs and TEM image of the GA-GMA/CtNWs10. The white arrows in the TEM
image indicate the CtNWs dispersed through the hydrogel matrix.
7


A.G.B. Pereira et al.

Carbohydrate Polymers 266 (2021) 118116

900% before 5 h, indicating a superabsorbent ability that can be
explained by the great hydrophilicity of GA chains (Khan et al., 2020).
After this, the liquid uptake slows down, and the swelling equilibrium
(976%) was eventually reached after 10 h.
CtNWs introduced into the hydrogel matrices exerted a significant
effect on their swelling profile for long-term liquid uptake. Overall, after
the first hour of the experiment, the swelling rate for the nano­
composites slow down and the absorption depended on the concentra­
tion of CtNWs, Fig. 7a, however, there was no linear relationship
between the concentration of CtNWs in the nanocomposite and water
uptake behavior. At equilibrium, the swelling rates of GA-GMA/
CtNWs1, GA-GMA/CtNWs5, and GA-GMA/CtNWs10 were calculated
to be around 601%, 802%, and 725%, respectively. These results reveal
that the presence of CtNWs reduces the liquid uptake capacity of the
nanocomposites as compared to the pristine hydrogel. This trend can be
explained by two main reasons: (i) the abundant hydroxyl and amino
groups at the CtNWs surfaces can interact (via H-bond and/or dipoledipole interactions) with the functional groups of GA causing an addi­
tional physical crosslinking of the hydrogel matrix; and (ii) these in­
teractions limit the interaction between the hydrogel matrix and water
molecules. Higher density of crosslinking means higher rigidity of the

polymeric matrix, preventing the expansion of the hydrogel volume as
well as the water adsorption. Additionally, the reduction of hydrophilic
groups due to the filler-matrix interaction impairs the anchorage of
water molecules. This discussion corroborates the TGA analysis, which
demonstrated that GA-GMA/CtNWs10 is less hydrophilic than GAGMA/CtNWs0. Furthermore, the interaction between CtNWs and the
GA-GMA matrix also aids to explain the non-linear behavior observed
for the nanocomposite samples regarding their swelling capacity. Earlier
studies demonstrated that hydrogel nanocomposites experienced an
increment of their swelling capacity with the increase of filler concen­
tration (Spagnol et al., 2012). However, this increment is limited to a

composites, the whiskers were well dispersed and immobilized in the GA
hydrogel, and did not aggregate or self-assemble upon drying to form a
compact structure as in the case of pure CtNWs. Therefore, although
CtNWs presented higher thermal stability than GA-GMA/CtNWs0, its
presence in GA-GMA/CtNWs10 as individual well dispersed whiskers
and at low concentration explain the similar thermal behavior of
hydrogels with and without CtNWs. Hence, the thermal stability of GAGMA/CtNWs10 is little influenced by CtNWs at 10 wt%.
TEM images recorded of the GA-GMA/CtNWs10 nanocomposite
revealed that the nanowhiskers are randomly and uniformly dispersed
through the hydrogel matrix, Fig. 6. This well dispersed system cor­
roborates the FTIR data indicating compatibility between CtNWs and
the GA chains (due to the H-bonds), which could improve the hydrogel
mechanical properties as well as the water uptake kinetics, for instance.
3.4. Swelling behavior
The ability of absorb and retain large amounts of aqueous fluids is
the most notorious feature of hydrogels. Overall, this ability has assured
a wide spectrum of potential applications for hydrogels, such as the
adsorption of pollutants, soil conditioning, drug release, and scaffolding
in tissue engineering, among others (Curvello et al., 2019; Du et al.,

2020; Mohammadinejad et al., 2019). The amount of liquid absorbed by
a dry hydrogel mass is generally computed by a parameter known as
swelling capacity or swelling ratio. As reported, the swelling capacity
depends on several factors, including the nanocomposite formation and
kind of filler used (Ebrahimi, 2019). Swelling experiments were per­
formed to investigate the effect of different CtNWs concentrations on
GA-GMA/CtNWs nanocomposites. The hydrogels presented fast initial
swelling rate, but the pristine hydrogel (GA-GMA/CtNWs0) displayed
greater swelling rate and higher water uptake at equilibrium than the
composites. The swelling rate computed to GA-GMA/CtNWs0 achieved

Fig. 7. (a) Swelling kinetics of nanocomposites synthesized with different CtNWs concentrations, (b) Diffused water rate, (c) Power-law plot, and (d) values of
exponent n (diffusion coefficient) calculated from the power-law model as function of CtNWs concentration.
8


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Carbohydrate Polymers 266 (2021) 118116

maximum filler concentration, that when exceeded causes the impair­
ment of the swelling. The excess of filler lead to higher filler-filler and
filler-matrix interactions, negatively affecting the liquid uptake and the
swelling of the nanocomposite (Cˆ
andido et al., 2012; Carsi et al., 2019).
Indeed, the increase of the filler-filler interactions at CtNWs concen­
trations higher than 5 wt/wt% can trigger a phase separation process
reducing the surface area and the hydrophilicity of the nanocomposite.
Previous work has shown that polysaccharide rod-like nanoparticles (e.
g., cellulose nanowhiskers) undergo a concentration dependent phase

transition under aqueous media (Khandelwal & Windle, 2013).
To better understand the mechanism that drives the liquid uptake,
the power-law model was applied to the swelling data (Brannon-Peppas
& Peppas, 1990). This widely known mathematical model is valid for
liquid uptakes below 60% of the equilibrium, and it is given by the
following equation:
/
Wt
= ktn
(4)
Weq

depicted in Fig. 7a, the values of n can be correlated to different physical
mechanisms, such as Fickian diffusion, non-Fickian diffusion (known as
anomalous), or Case II (relaxation-controlled) transport, that controls
liquid uptake by a swellable polymer matrix (Carbinatto et al., 2014;
Ganji et al., 2010). Considering the geometry of the hydrogel nano­
composite synthesized in this work, n = 0.45 indicates Fickian diffusion,
0.45 < n < 0.89 indicates non-Fickian diffusion, and n > 0.89 implies
Case II transport. The values of n calculated using the power-law model
are displayed in Fig. 7d and are around 0.45 indicating that the liquid
uptake mechanism for these hydrogels is consistent with a Fickian
diffusional process. In this case, the liquid diffusion rate is much lower
than the polymer relaxation rate, which means that the liquid molecules
diffuse easily through the hydrogel matrices because of the high flexi­
bility of the polymer chains. Interestingly, the introduction of CtNWs
even at different concentrations did not impede the access of water in­
ward the hydrogel matrices; however, the filler affected their liquid
uptake capacities. These observations are consistent with earlier studies
that investigated the swelling properties of hydrogel (nano)composites

(Toledo et al., 2018). It is important to mention that the power-law
model was able to fit adequately the experimental swelling data since
the coefficients of determination (R2) were higher than 0.965.

where Wt and Weq are the absorbed water weights in the nanocomposites
at a specific time (t) and at equilibrium, respectively. k is a swelling
constant associated with the hydrogel network. The exponent n is known
as the diffusion coefficient and its value is used to describe the liquid
uptake mechanism. The swelling profile of hydrogels and hydrogels
nanocomposite has been assessed by plotting the water uptake (Wt/Weq)
as a function of time (t) (Fig. 7b), while the values of n are calculated
from the plot log (Wt/Weq) versus log t using simple linear regression
(Fig. 7c). The parameters computed from the experimental swelling data
and equations from the power-low model are: log (Wt/Weq) = − 0.175 +
0.376 log t (R2 = 0.970), log (Wt/Weq) = − 0.153 + 0.434 log t (R2 =
0.983), log (Wt/Weq) = − 0.218 + 0.348 log t (R2 = 0.965) and log (Wt/
Weq) = − 0.245 + 0.415 log t (R2 = 0.988) for GA-GMA/CtNWs0, GAGMA/CtNWs1,
GA-GMA/CtNWs5,
and
GA-GMA/CtNWs10,
respectively.
According to the empirical power-law model used to fit the data

3.5. Mechanical properties
Hydrogel nanocomposites were synthesized using different concen­
trations of CtNWs (0–10 wt%) and their mechanical properties were
examined through compressive tests. The computed mechanical prop­
erties are shown in Fig. 8.
According to the data displayed in Fig. 8a, a remarkable increase of
Young’s modulus (from 6.23 to 8.95 KPa) was observed as the CtNWs

concentration increased from 0 to 10 wt%, which represents an
improvement of 45% compared to the pristine hydrogel. In general, this
mechanical parameter denotes the stiffness or resistance of the hydrogel

Fig. 8. Mechanical properties of nanocomposite hydrogels embedded with CtNWs. (a) Young’s modulus, (b) rupture force, and (c) maximum compressive strain
at rupture.
9


A.G.B. Pereira et al.

Carbohydrate Polymers 266 (2021) 118116

towards elastic deformation under load. Therefore, the introduction of
10 wt% CtNWs allows increasing the ability of the GA-GMA/CtNWs10 to
resist compressive deformation better than GA-GMA/CtNWs0. Simi­
larly, the force requested to crack the nanocomposites samples (or
rupture force) increased from 1.07 to 2.10 N (96% of increment) when
the GA-GMA/CtNWs0 and GA-GMA/CtNWs10 samples are compared.
Notoriously, these mechanical enhancements were only noticed to the
samples embedded with at least 5 wt% CtNWs. As assessed, the nano­
composites with the lowest concentration of CtNWs did not show a
significant increase in Young’s modulus or rupture force value. Araki
et al. stated that for reinforcing the hydrogel network, the CtNWs must
connect (or interact) to at least more than two crosslinking points per
single nanocrystal, as otherwise, CtNWs do not bear mechanical stress
nor strength the hydrogel (Araki et al., 2012). According to the swelling
data, the introduction of 1 wt% CtNWs in the hydrogel matrix affected
considerably the liquid uptake capacity of GA-GMA/CtNWs1 as
compared to GA-GMA/CtNWs0, however, this effect did not reflect an

enhancement of GA-GMA/CtNWs1 mechanical properties. Analyzing
the swelling and mechanical data, which are convergent properties of
hydrogels, it can be assumed that the introduction of a low concentra­
tion of CtNWs only causes a reduction of hydrophilic groups within the
hydrogel matrix or an increase of hydrophobicity, whereas the cross­
linking was not affected. Therefore, the results depicted in Fig. 8a and b
suggest that an additional crosslinking effect is only noticed to the
nanocomposite samples synthesized with at least 5 wt% CtNWs. Indeed,
these finds corroborate with other studies that demonstrated that
hydrogel matrices are reinforced by CtNWs only when concentrations
higher than 1 wt% are used (Araki et al., 2012). Finally, although the
introduction of CtNWs in the hydrogel matrix enhanced their mechan­
ical properties, it does not have a significant effect on the maximum
strain computed at the rupture point for the nanocomposite samples
(Fig. 8c). This is an interesting result since it stands out that the ability to
strain of the nanocomposites is preserved even after the reinforcement
with CtNWs, which can be an advantage from practical and handling
viewpoints.
A comparative table presenting swelling and mechanical data of this
study and others reported for GA-based hydrogels (composites or not) is
presented in Table 2. In general lines, the composite hydrogels show an
antagonistic behavior between swelling capacity and mechanical prop­
erties. Although the introduction of filler materials within the hydrogel
network improves mechanical properties, the swelling capacity ends up
being affected. Depending on the target application, the impairment of
the swelling capacity is a limiting factor. As noticed, the hydrogels
synthesized in this study exhibited maximum swelling capacity

comparable to or even superior to other hydrogels based on GA. In
contrast, the simple numeric comparison of the mechanical properties

data revealed that GA-GMA/CtNWs0 and GA-GMA/CtNWs10 are
slightly inferior to the other examples. Herein, one must be aware of the
comparison must be done with care since these hydrogels have different
formulations and/or were prepared using different protocols. These two
variables (i.e., composition and preparation) exert a straight effect on
the morphology and mechanical properties of the resulting hydrogels.
Moreover, due to inconsistencies in the data reported in the literature, it
is difficult to compare the mechanical properties of this kind of hydrogel
as they are estimated at different analytical conditions. Beyond these
constraints, few studies focused on the synthesis of GA-based hydrogels
have devoted time to investigate the mechanical properties of such
materials. Due to this, a trustable data comparison is not a simple task.
In summary, it was demonstrated here for the first time that GA-GMA
hydrogels can be mechanically reinforced by CtNWs. Due to the large
aspect ratio, high mechanical strength, and unique rod-like shape, these
nanowhiskers were easily dispersed through the hydrogel matrix
resulting in a noticeable enhancement on the mechanical properties of
the conventional GA-GMA hydrogel. Surely, these novel nano­
composites can broaden the range of applications of this kind of
hydrogel, which include biomedical (e.g., tissue engineering, wound
healing, and delivery systems) and environmental (e.g., wastewater
remediation and soil conditioning) applications.
4. Conclusion
Nanocomposites were efficiently synthesized by introducing
different amounts of CtNWs in a GA-GMA hydrogel matrix. CtNWs were
efficiently isolated from raw chitin and GA was modified with cross­
linkable functional groups using GMA. Although chemical bond be­
tween GA-GMA and CtNWs were not evidenced by FTIR analysis, the
physical interaction through H-bond was enough to provide well
dispersed and distributed CtNWs throughout the hydrogel matrix.

Overall, the nanocomposites showed mechanic and swelling properties
dependent on the amount of CtNWs; however, no linear correlation
between these properties and the amount of CtNWs was observed.
CtNWs increased the rigidity and reduced the swelling capacity of
hydrogel that could be modulated by adjusting the amount of CtNWs.
This is an attractive advantage since it widens the range of functionality
and applicability of these nanocomposites based on abundant and nat­
ural polymers. It is expected that further studies investigate the appli­
cability of these soft materials in the biomedical field (biomaterials or
biomedical devices), for example.

Table 2
Comparison of maximum swelling and mechanical properties of various GA-based hydrogels and composited hydrogels.
Hydrogela

GA-GMA/CtNWs0
GA-GMA/CtNWs10
GA-g-PAAc/pinus residue
GA-g-PAAc/eucaliptus residue
Oxidized GA-g-PVA
GA-GMA-g-PAAc
GA-GMA-g-PAAc/graphene
GA-GMA-g-PAAc/MOF-UIO66
GA-gelatin-AAM
GA/O-carboxymethyl chitosan
GA‑sodium alginate-chitosan
GA-g-PAAm
GA-GMA/magnetite
GA/starch nanocrystals
GA-gelatin/cellulose whiskers

a

Filler
(wt/wt%)

Maximum swelling (%)

–
10.0
10.0
10.0
–
–
–
0.1
–
–
–
–
5.5
33.3
5.0

976
725
98
102
210
1000
280

800
865
750
208
1428
148
200
500

Mechanical properties

Ref.

Young’s modulus (KPa)

Rupture
force
(N)

Strain at rupture
(%)

6.23
8.94
–
–
–
26.60
66.90
183.40

40.0
–
–
–
–
–
–

1.07
2.10
–
–
–
–
–
–
–
–
–
–
–
–
–

58
60
–
–
75
–

–
–
1050
–
–
–
–
–
–

Abbreviations: PAAc - poly(acrylic acid); PVA - poly(vinyl alcohol); MOF - metal organic framework; PAAm - poly(acrylamide).
10

This study
This study
(de Souza et al., 2019)
(de Souza et al., 2019)
(Pandit et al., 2019)
(Fan et al., 2013)
(Fan et al., 2013)
(Ribeiro et al., 2019)
(Wang et al., 2019)
(Huang et al., 2016)
(Younis et al., 2018)
(Kaith & Ranjta, 2010)
(Paulino et al., 2010)
(Alwaan et al., 2019)
(Favatela et al., 2021)

A.G.B. Pereira et al.

Carbohydrate Polymers 266 (2021) 118116

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Antonio G.B. Pereira: Conceptualization, Formal analysis, Meth­
´tia S. Nunes: Formal analysis.
odology, Writing – original draft. Ca
´ R.
Adley F. Rubira: Supervision. Edvani C. Muniz: Supervision. Andre
Fajardo: Methodology, Writing – original draft.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
The authors thank CNPq for its financial support and for the PQ
fellowship to A.R.F. (Grant number 303873/2019-5). This study was
financed in part by the Coordenaỗ
ao de Aperfeiỗoamento de Pessoal de
Nớvel Superior, Brazil (CAPES), Finance Code 001. The authors are
thankful to INOMAT (Chemistry Institute, UNICAMP, SP, Brazil) for its
technical support regarding TEM microscopy.
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