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.
Trang 1Carbohydrate Polymers 266 (2021) 118116
Available online 24 April 2021
0144-8617/© 2021 Elsevier Ltd This article is made available under the Elsevier license (http://www.elsevier.com/open-access/userlicense/1.0/)
Effect of chitin nanowhiskers on mechanical and swelling properties of
Gum Arabic hydrogels nanocomposites
Antonio G.B Pereiraa,b,*, C´atia S Nunesa, Adley F Rubiraa, Edvani C Muniza,d,e, Andr´e
R Fajardoc,**
aGrupo de Materiais Polim´ericos e Comp´ositos (GMPC), Maring´a State University, Av Colombo 5790, 87020-900 Maring´a, PR, Brazil
bLaborat´orio de Biopolímeros, Coordenaç˜ao de Engenharia de Bioprocessos e Biotecnologia, Universidade Tecnol´ogica Federal do Paran´a (UTFPR- DV), Estrada para
Boa Esperança, 85660-000 Dois Vizinhos, PR, Brazil
cLaborat´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
dDepartamento de Química, Universidade Federal do Piauí, 64049-550 Teresina, PI, Brazil
ePrograma de P´os-graduaç˜ao em Ciˆencia e Engenharia de Materiais, Universidade Tecnol´ogica Federal do Paran´a (UTFPR- LD), Avenida dos Pioneiros, 3131, 86036-
370 Londrina, PR, Brazil
A R T I C L E I N F O
Keywords:
Chitin
Whiskers
Gum Arabic
Hydrogel
Composite
Swelling
A B S T R A C T 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-D-
glucosamine 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
(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: antoniog@utfpr.edu.br (A.G.B Pereira), andre.fajardo@pq.cnpq.br (A.R Fajardo)
Contents lists available at ScienceDirect Carbohydrate Polymers
https://doi.org/10.1016/j.carbpol.2021.118116
Received 14 October 2020; Received in revised form 5 April 2021; Accepted 18 April 2021
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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 Sigma-
Aldrich (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 never-
dried state prior to hydrogel preparation
2.3 Characterization of CtNWs
CtNWs were characterized by zeta potential, Fourier Transform Infrared Spectroscopy (FTIR), Thermogravimetric Analysis (TGA),
A.G.B Pereira et al
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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)
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
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 GA- GMA 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
Fig 1 (a) Isolation of CtNWs from raw chitin and (b) synthesis of GA-GMA followed by the synthesis of the hydrogel nanocomposite
Table 1
Hydrogels composition
Sample GA-
GMA (g)
CtNWs (wt/wt
%)
CtNWs suspension (mL)
MBA (g) SPS (g) Final volume
(mL) GA-GMA/
GA-GMA/
GA-GMA/
GA-GMA/
CtNWs10 5 10 10 0.1 0.1 25
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fixed at 10 wt/wt% because higher concentrations showed a poor
dispersion into the polymer solution before hydrogel formation
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 1 H NMR
(model 300, UK) For this, 10 wt/wt% solutions in D2O (raw GA and GA-
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
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
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 (https://imagej.nih.gov/ij)
2.6 Swelling experiments
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) pre-
moistened 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):
Swelling (%) =(w t− w o)
w o
where wt is the swollen hydrogel weight at a specific time (t) and wo is its
dry weight
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 cross- sectional area of 126 mm2 The Young’s modulus was calculated per
Eq (2):
Young′s modulus = F.L1
where F is the necessary force (N) to compress the sample, A is the
sample transversal area (m2), L 1, and L 2 are the initial and compressed sample thicknesses (mm), respectively For each sample, 5 measure-ments were done
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+⇋ R–NH3+), which explains this high positive charge density Indeed, 1H 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
3.2 Chemical modification of GA with GMA
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 GA- GMA 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
degree of substitution (DS) on GA after reacting with GMA The DS was
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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)
DS (%) =
[
(I δ6.20+I δ6.16) ×0.5
I δ5.41
]
After a careful baseline treatment, the areas of the resonance signals
were measured and the values found were Iδ 6.20 ≈1.0, Iδ 6.16 ≈1.21, and
I δ5.41 ≈9.05, respectively Using these values, the DS calculated for GA-
GMA was 12.21% Although the DS was slightly lower than other reports
(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
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
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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-metric and sym(asym-metric C––O stretching of carboxylic groups), and 1288
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
bands at 1718 cm− 1 and 1634 cm− 1 (C––O and C––C stretching of ester
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
small band at 1718 cm− 1 and a shoulder-type band at 1634 cm− 1 (C––O
and C––C groups of GMA) confirm the modification of GA, as previously
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
cm− 1 (C––O stretching, amide I band), at 1560 cm− 1 (a combination of
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;
Pereira et al., 2014, 2020) The absorbance ratio at 1663 cm− 1 (C––O
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
1718 cm− 1 (C––O stretching of GMA moiety), and bands in the range 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 asymmetric C––O stretching of carboxylic groups of GA-GMA from 1607
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
bonds (around 1230 cm− 1) can be attributed to MBA, used as the crosslinker agent (Huang et al., 2013) These particularities in the GA- GMA 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 shoulder- type 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 GA- GMA/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 H- bond 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
Fig 4 FTIR spectra obtained for GMA, raw GA, and GA-GMA
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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 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
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
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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 GA-
GMA/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
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 dipole- dipole 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 GA- GMA/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
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
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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 (Cˆandido 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:
W t
/
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 /W eq)
as a function of time (t) (Fig 7b), while the values of n are calculated
from the plot log (Wt /W eq) 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 /W eq) = − 0.175 +
0.376 log t (R2 =0.970), log (Wt /W eq) = − 0.153 + 0.434 log t (R2 =
0.983), log (W t /W eq ) = − 0.218 + 0.348 log t (R2 =0.965) and log (W t /
W eq) = − 0.245 + 0.415 log t (R2 =0.988) for GA-GMA/CtNWs0, GA-
respectively
According to the empirical power-law model used to fit the data
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
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
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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 reducconcentra-tion 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
(wt/wt%) Maximum swelling (%) Mechanical properties Young’s modulus (KPa) Rupture Ref
force (N)
Strain at rupture (%)
aAbbreviations: PAAc - poly(acrylic acid); PVA - poly(vinyl alcohol); MOF - metal organic framework; PAAm - poly(acrylamide)
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