Maleimide-grafted cellulose nanocrystals as cross-linkers for bionanocomposite hydrogels

Article abstract of DOI:10.1016/j.carbpol.2016.04.091

This article deals with the preparation of bionanocomposite hydrogels from natural polymers and nanoentities, an emerging class of materials for biotechnological and biomedical applications. Herein, the applicability of the Diels-Alder "click" reaction to

Full text of DOI:10.1016/j.carbpol.2016.04.091

Carbohydrate Polymers 149 (2016) 94–101
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Maleimide-grafted cellulose nanocrystals as cross-linkers for
bionanocomposite hydrogels
∗
C. García-Astrain, K. González, T. Gurrea, O. Guaresti, I. Algar, A. Eceiza, N. Gabilondo
‘Materials + Technologies’ Group, Department of Chemical and Environmental Engineering, Polytechnic School, University of the Basque Country, Plaza
Europa 1, 20018 Donostia-San Sebastián, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
This article deals with the preparation of bionanocomposite hydrogels from natural polymers and
nanoentities, an emerging class of materials for biotechnological and biomedical applications. Herein,
the applicability of the Diels-Alder “click” reaction to the design of bionanocomposite hydrogels from
furan modified gelatin using maleimide-functionalized cellulose nanocrystals as multifunctional cocross-
linkers is demonstrated. The functionalization of cellulose nanocrystals with maleimide moieties was
confirmed by XPS. The swelling and rheological properties of the resulting bionanocomposite confirmed
the formation of hydrogel networks with covalently embedded nanoentities. The Diels-Alder reaction
resulted in the formation of stiffer networks with lower swelling ratios due to the formation of addi-
tional cross-linking points. The designed “click” strategy proved to be a promising candidate for the
formation of fully renewable bionanocomposite hydrogels.
Received 15 December 2015
Received in revised form 15 April 2016
Accepted 20 April 2016
Available online 23 April 2016
Keywords:
Bionanocomposite hydrogel
Diels-Alder
Cellulose nanocrystals
“Click” Chemistry
©
2016 Elsevier Ltd. All rights reserved.
1
. Introduction
electronic properties (García-Astrain, Ahmed et al., 2015; García-
Astrain, Chen et al., 2015; Gaharwar, Peppas, & Khademhosseini,
2014; Daniel-da-Silva, Salgueiro, & Trindade, 2013; Kamoun &
Menzel, 2012; Shin et al., 2012; Barbucci, Giani, Fedi, Bottari, &
Casolaro, 2012; Dvir et al., 2011; Narayana Reddy et al., 2011).
Moreover, in order to achieve superior environmental compat-
ibility as compared to inorganic nanofillers, renewable bionanoen-
tities are being incorporated into novel hydrogel formulations. In
particular, cellulose nanocrystals have proven to be effective rein-
forcements for various hydrogels (Mohammed, Grishkewich, Berry,
& Tam, 2015; Araki & Yamanaka, 2014; Dai & Kadla, 2009; McKee
et al., 2014; Wallenius et al., 2015). Cellulose nanocrystals (CNCs)
are nanoentities 100–500 nm in length and 5–30 nm in width which
can be obtained from the partial hydrolysis of a variety of cellulosic
materials (Lin & Dufresne, 2014; Yang et al., 2015). This natu-
ral nanoscaled material possesses numerous advantages including
rod-like morphology, high aspect ratio, crystallinity, non-toxicity,
biodegradability, high mechanical strength and surface reactivity
(Lin & Dufresne, 2014). Generally, bare CNCs are physically incor-
porated as reinforcements into the hydrogel matrix (Dai & Kadla,
Hydrogels represent an interesting class of materials with a
broad range of applications in the pharmaceutical and biomedical
field, as well as in agriculture and cosmetics. Among the dif-
ferent types of hydrogels, hydrogels from bio-derived polymers
are particularly interesting since they are easily made biodegrad-
able, promote tissue growth, mimic the extracellular matrix and
possess a renewable character (Yang, Han, Duan, Xu, & Sun,
2
013; Balakrishnan & Banerjee, 2011; Van Vlierberghe, Dubruel, &
Schacht, 2011). During the last decade, the design of bionanocom-
posite hydrogels including nanoentities such as carbon nanotubes,
metallic nanoparticles or clays have attracted great interest from
the scientific community (García-Astrain, Ahmed et al., 2015;
García-Astrain, Chen et al., 2015; Sabaa, Abdallah, Mohamed, &
Mohamed, 2015; Derkus, Emregul, & Emregul, 2015; Haraguchi,
2
007). The interactions between the nanoentities and the poly-
meric matrix at the nanoscale give rise to new materials with
enhanced mechanical, thermal, biological, magnetic, optical or
2
009; Mohammed et al., 2015; Wallenius et al., 2015). However,
recent work showed that surface-modified CNCs can act both as
reinforcement and cross-linking agent within a polymeric matrix.
In this way, the performance of the material is improved via the
covalent linkage which enhances the interfacial adhesion and rein-
forces the effect of the filler (Chen, Lin, Huang, & Dufresne, 2015).
∗ Corresponding author.
E-mail addresses: clara.garcia@ehu.eus (C. García-Astrain),
kizkitza.gonzalez@ehu.eus (K. González), taniagurrea@gmail.com (T. Gurrea),
olatz.guaresti@ehu.eus (O. Guaresti), itxaso.algar@ehu.eus (I. Algar),
arantxa.eceiza@ehu.eus (A. Eceiza), nagore.gabilondo@ehu.eus (N. Gabilondo).
http://dx.doi.org/10.1016/j.carbpol.2016.04.091
0
144-8617/© 2016 Elsevier Ltd. All rights reserved.

C. García-Astrain et al. / Carbohydrate Polymers 149 (2016) 94–101
95
Following this strategy, aldehyde-functionalized CNCs were used
as cross-linkers for carboxymethylcellulose/dextran hydrogels and
also for the preparation of injectable hyaluronic acid-based formu-
lations (Domingues et al., 2015; Yang, Bakaic, Hoare, & Cranston,
was observed. At the end of the whole process, an orange oil was
obtained. The solvent excess was removed under reduced pres-
sure and the product was washed with toluene (3 × 30 mL), which
was again removed under reduced pressure. The product was then
purified by flash chromatography using a silica column (DCM/ethyl
acetate 9:1). A white solid was obtained. Yield: 29.15%.
2
013). Xylan and gelatin-based hydrogels cross-linked with CNCs
or oxidized CNCs, respectively, have also been reported (Köhnke,
Elder, Theliander, & Ragauskas, 2014; Dash, Foston, & Ragauskas,
2
013). However, to the best of our knowledge, little work has been
2.3. CNCs functionalization (CNC-Mal)
done on the use of these renewable bionanofillers as hydrogel
cross-linkers via means of “click” chemistry.
CNCs were isolated from microcrystalline cellulose after acid
hydrolysis with sulfuric acid, removing the disordered or paracrys-
talline regions of cellulose and leaving crystalline regions intact
(González, Retegi, González, Eceiza, & Gabilondo, 2015). 5 g of CNC
We have recently reported the applicability of the Diels-Alder
(
DA) “click” reaction for the preparation of biopolymer-based
nanocomposite hydrogels (García-Astrain, Ahmed et al., 2015;
García-Astrain, Chen et al., 2015). Herein, we demonstrate the role
of maleimide-functionalized CNCs as cross-linkers for the forma-
tion of a completely renewable bionanocomposite hydrogel based
on gelatin and chondroitin sulfate (CS). Gelatin, composed of a large
variety of aminoacids, allows for a broad range of chemical mod-
ifications and, due to its sol-gel transition, is a suitable hydrogel
precursor (García-Astrain et al., 2014; Van Vlierberghe et al., 2011).
On the other hand, CS is a structural component of the extracellu-
lar matrix and its use is mainly focused on the synthesis of novel
biomaterials.
The aim of this work was to explore the applicability of the Diels-
Alder reaction for the preparation of fully biobased nanocomposites
using modified CNCs as nanofillers. CNCs were surface-modified by
reaction with a maleimide-functionalized aminoacid and the Diels-
Alder cycloaddition was then employed as a mild covalent strategy
for their binding with furan-modified gelatin. In order to stabilize
the hydrogel, second cross-linking based on the amide coupling
between CS and gelatin was performed. The effect of functional-
ized CNCs on the swelling and viscoelastic properties was analyzed,
as well as the role of CNCs as stabilizer within this completely
renewable bionanocomposite formulation.
◦
were hydrolyzed in a water bath at 45 C using a 64% (w/w) sulfu-
ric acid solution for 30 min. After that time, the solution is poured
into a large excess of water and washed with successive centrifu-
gations. The solution was dialyzed against water until neutral pH
was reached and finally, nanocrystals were freeze-dried. For the
CNC functionalization, 0.5 g of CNC were suspended in 30 mL of
−
3
N,N-dimethylformamide (DMF) and 0.5 g of AMI (2.9 × 10 mol)
were added (Scheme 2) (Cateto & Ragauskas, 2011). The mixture
−
3
was cooled in ice and 0.399 g of DMAP (3.3 × 10 mol) were added
◦
at 0 C. Then, a 10% (w/w) DMF solution containing 0.567 g of EDC
−
3
(2.9 × 10 mol) was added dropwise. The mixture was stirred at
room temperature for 24 h. After that time, the surface function-
alized CNC (CNC-Mal) were precipitated in a dilute aqueous acid
solution and then dialyzed against water until neutral pH was
attained. The sample was washed several times with water, ethanol
and hydrochloric acid 0.5 M. Finally, the product was recovered
after freeze-drying.
2.4. Bionanocomposite preparation
For hydrogel formation, furan modified gelatin (GF, 70 mg) was
dissolved in a previously ultrasonicated 0.5 wt.% suspension of
maleimide-grafted CNCs. The final concentration of CNC-Mal in the
bionanocomposite was 6.3% wt. Finally, CS was incorporated to the
mixture (1:2 weight ratio with respect to gelatin) in the presence
2
. Materials and methods
2.1. Materials
−
3
−3
of EDC (2.3 × 10 mol) and NHS (2.0 × 10 mol). The mixture was
allowed to gel for 24 h (Scheme 3). Control hydrogels without CNCs
(G-CS hydrogel) or using bare CNCs (G-CS-CNC hydrogel) were also
prepared for comparison following the same procedure described
above.
Gelatin (from porcine skin Type A, 300 Bloom), furfuryl
glycidyl ether (FGE, 96.0%), chondroitin sulfate
salt from bovine trachea (CS, 60.0%), N-hydroxysuccinimide
NHS, 98.0%), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide
hydrochloride (EDC, 99.0%), ␤-alanine (99.0%), maleic anhydride
99.0%), 4-(dimethylamino)pyridine (DMAP, 99.0%) and microcrys-
A sodium
(
(
2.5. Methods
talline cellulose were purchased from Sigma-Aldrich. Phosphate
buffered saline (PBS) solution was prepared from PBS tablets
from Panreac (pH = 7.4). Acetic acid, sulphuric acid (96.0%) and
ethyl acetate were also purchased from Panreac and toluene and
dichloromethane from Lab Scan. Deionized water was employed
as solvent. All reagents and solvents were employed as received.
Gelatin was modified by reaction of its free ␧-amino groups with
furfuryl glycidyl ether (FGE) in aqueous solution as described in our
previous work (G-FGE) (García-Astrain et al., 2014).
Fourier Transform Infrared Spectroscopy (FTIR) was performed
in a Nicolet Nexus spectrophotometer at room temperature. KBr
−
1
−1
pellets were used in the range from 4000 to 400 cm , with a 4 cm
1
resolution. Proton and carbon Nuclear Magnetic Resonance ( H and
13
C NMR) spectra were recorded with an Avance Bruker equipped
with BBO z-gradient probe. Experimental conditions were as fol-
lows: (a) for 13C NMR: 125.75 MHz, number of scans 14 000,
1
spectral window 25 000 Hz, and recovery delay 2s; (b) for H NMR:
5
00 MHz, number of scans 64, spectral window 5000 Hz, and recov-
ery delay 1s. The solvent employed in all cases was D O. X-ray
Photoelectron Spectroscopy (XPS) was carried out using a SPECS
2
.2. Synthesis of 3-(2,5-dioxo-2H-pyrrol-1(5H)-yl)propanoic acid
2
(
AMI)
(Berlin, Germany) system equipped with a Phoibos analyzer 150
AMI was prepared following a reported procedure with some
1D-DLD and a monochromatic source Al-K␣ (1486.7 eV). An initial
analysis of the present elements was performed (wide scan: step
energy 1 eV, dwell time 0.1 s, pass energy 80 eV) and detailed analy-
sis of the present elements was carried out (detail scan: step energy
0.1 eV, dwell time 0.1 s, pass energy 50 eV) with a take-off angle of
90 for the photoelectron analyser. Curve fitting was performed
using a Gaussian–Lorentzian peak shape function with a straight
base line throughout the analysis using a CasaXPS software 2.3.16.
modifications (Scheme 1) (Mantovani et al., 2005). An acetic acid
solution of maleic anhydride (5.00 g in 50 mL) was added dropwise
to an acetic acid solution of ␤-alanine (4.54 g in 50 mL). The mixture
was stirred for 3 h at room temperature and a white suspension
was obtained. After that period, 70 mL of AcOH were added, the
◦
◦
temperature was raised until 115 C and the mixture was stirred
overnight. After one hour of reaction a limpid colourless solution

9
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C. García-Astrain et al. / Carbohydrate Polymers 149 (2016) 94–101
Scheme 1. AMI synthesis from ␤-alanine and maleic anhydride.
Scheme 2. CNC functionalization using AMI.
Scheme 3. Diels-Alder reaction between furan-modified gelatin (GF) and CNC-Mal and picture of the hydrogel G-CS-CNC-Mal.
Atomic Force Microscopy (AFM) measurements were performed
in tapping mode using a Veeco Multimode scanning probe micro-
scope equipped with a Nanoscope IIIa Controller. All measurements
were recorded using TESP type silicon tips having a resonance fre-
quency of approximately 340 kHz and a cantilever spring constant
voltage. Freeze-dried samples were sputter-coated with approx-
imately 8 nm of Pt/Au layer to reduce electron charging effects.
Dynamic rheological behavior of the hydrogels was analyzed with
a Rheometric Scientific Advanced Rheometric Expansion System
(ARES), using parallel plate geometry (25 mm diameter). Fre-
−1
◦
about 40 N m . Scanning Electron Microscopy (SEM) experiments
were performed by a Hitachi S-2700 SEM at 15 kV accelerating
quency sweep measurements were performed at 37 C from 0.1
−
1
to 500 rad s
at a fixed strain in the linear viscoelastic region,

C. García-Astrain et al. / Carbohydrate Polymers 149 (2016) 94–101
97
Fig. 1. (a) 1H NMR spectra of AMI in D2O, (b) 13C NMR spectra of AMI in D2O and (c) FTIR spectra of AMI.
previously assessed by strain sweep experiments. Hydrogel sam-
ples were prepared in the form of disks of 25 mm diameter.
Swelling capacity of freeze-dried hydrogels was studied by a gen-
◦
eral gravimetric method. Samples (n = 3) were incubated at 37 C
in simulated intestinal fluid (phosphate buffer (PBS), pH = 7.4)
and water, at selected time intervals the swollen hydrogels were
removed, the excess of water absorbed with a filter paper, and
weighed. The samples were then dried until constant weight. The
swelling ratio (SR) was calculated using Eq. (1):
SR(%) = (Ws − W )/W × 100
(1)
d
d
where Ws is the weight of the swollen sample and W is the weight
d
of the dried hydrogel samples. The equilibrium swelling was con-
sidered to be achieved when the weight of the hydrogels no longer
increased. For degradation studies weighted hydrogel freeze-dried
◦
samples were immersed in PBS and incubated at 37 C for 24 and
4
8 h. After those times the samples were removed from PBS, dried
and weighted. The hydrogel degradation was calculated using Eq.
2):
Fig. 2. FTIR spectra of (a) CNCs and (b) CNC-Mal.
(
Degradation(%) = (mt/m ) × 100
(2)
CH CH bond of the maleimide ring observed in the 1H NMR
spectra (Fig. 1a) (Wei et al., 2010). In the C NMR the peaks cor-
responding to the carbonyl groups and the double bond of the
maleimide ring appeared at 173.15 and 134.86 ppm, respectively
0
13
Gel content was determined after swollen hydrogel samples
were dried to constant weight, and the percentage conversion was
evaluated using Eq. (3):
(
Fig. 1b).
Furthermore, at 176.11 ppm the peak corresponding to the car-
Gel Content(%) = (Wt/W ) × 100
(3)
d
bonyl group of the carboxylic acid can also be observed (Mantovani
et al., 2005). The success of the ␤-alanine modification was also con-
firmed by FTIR spectroscopy (Fig. 1c). Carbonyl stretching vibration
where Wd is the initial weight of the dry hydrogel sample and Wt
is the dry weight of the hydrogel at time t (after incubation in PBS
for time t).
−1
was observed at 1770 and 1709 cm . Moreover, the presence of
the maleimide ring was revealed by the appearance of bands at
−
1
−1
3
. Results and discussion
1452 cm and at 696 cm corresponding to the C C stretching
vibration and alkene bending, respectively (Wei et al., 2010).
The modified aminoacid (AMI) was then used for the surface
functionalizaton of the extracted cellulose nanocrystals. The suc-
cess of the functionalization reaction of CNCs was confirmed by
FTIR spectroscopy. Fig. 2 shows the FTIR spectra obtained for CNC
and CNC-Mal. It can be observed that the bands corresponding to
the stretching vibration of the carbonyl groups present in CNC-Mal
3.1. Functionalization of CNC-Mal
CNCs were functionalized with maleimide moieties for the
cross-linking of furan-modified gelatin. Maleimide-functionalized
CNC-Mal nanocrystals were obtained after the reaction of the
hydroxyl groups of CNCs with the carboxylic groups of AMI (Cateto
−
1
&
Ragauskas, 2011; Kalaskar et al., 2008). Maleimide-modified
appeared at 1709 and 1777 cm (Wei et al., 2010). Furthermore,
−
1
aminoacid derivatives have been employed as bifunctional cross-
linkers for the preparation of hydrogels via Diels-Alder reaction
two bands appeared at 1570 and 1400 cm corresponding to the
C and H stretching maleimide vibrations, respectively.
Further confirmation of CNC modification was acquired by XPS
analysis. Fig. 3 shows the XPS spectra for non-modified CNC and
C
C
(Wei et al., 2010). The incorporation of the maleimide into the
AMI structure was confirmed by the peak at 6.81 ppm due to the

9
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C. García-Astrain et al. / Carbohydrate Polymers 149 (2016) 94–101
Finally, CNCs size and morphology before and after functional-
ization reaction was studied by AFM. Fig. 4 shows the AFM images
of spin-coated aqueous solutions of CNCs and CNC-Mal. As it can
be observed, both nanocrystals displayed a fibrilar geometry with
a diameter within the nanometric scale (8.5 ± 1.9 nm) and vari-
able length, which indicates that the nanocrystals maintained their
integrity. It is worth noting that, as it can be observed in the
AFM images, after functionalization, the interactions between the
nancocrystals seemed to decrease, since CNC-Mal appeared to be
better dispersed with less aggregated entities. This result confirmed
the surface maleimide-functionalization which probably hinders
hydrogen bonding strong interactions between nanocrystals and,
thus, reduces the number of aggregates.
3.2. Hydrogel characterization
As explained before, hydrogels were prepared by reactive
Fig. 3. XPS spectra of CNC and CNC-Mal.
mixture of Gel-FGE and chondroitin sulfate, incorporating bare
nanocrystals for controls or maleimide-functionalized nanocrystals
in case of Diels-Alder nanocomposite hydrogels. Simultaneously,
amide coupling between the free amine groups of gelatin, which
have not reacted with FGE, and the carboxylic groups of chon-
droiting sulfate in the presence of EDC and NHS took place. After
the reaction, stable hydrogels were obtained. The microstructure
of the nanocomposite hydrogels and the control was studied by
means of SEM (Fig. 5a–c). For both G-CS-CNC-Mal and G-CS-CNC
similar homogeneous compact structures with pits and grooves
were observed. In G-CS hydrogel bumps and striated regions in
combination with smooth areas were observed. Hydrogels were
then characterized in terms of rheological behavior and swelling
capacity.
modified CNC-Mal. The XPS spectrum of non-modified CNC pre-
sented two peaks in the region of 532 and 286 eV corresponding
to oxygen (O) and carbon (C), respectively. After the reaction with
AMI, the CNC-Mal samples a third peak near 401 eV attributable to
the presence of nitrogen at the surface of the modified nanocrystals
(Cateto & Ragauskas, 2011). As expected, after grafting, an increase
in the concentration of carbon and nitrogen was observed, which
is a clear indication of the grafting of AMI to the CNC surface. XPS
data give rise to the determination of the degree of substitution
of the surface (DSS) (Missoum, Bras, & Belgacem, 2012). With this
technique the degree of substitution of the first nanometers layers
can be deduced and the DSS was calculated on basis of nitrogen
content (Rueda et al., 2011). From the XPS data, the number of
surface-grafted maleimide moieties per glucose unit was estimated
and resulted to be 2.2.
The viscoelastic behavior of the bionanocomposite hydrogel was
evaluated by means of frequency sweep rheological experiments.
G-CS-CNC-Mal, G-CS-CNC and G-CS hydrogels were submitted to
◦
frequency sweep tests at 37 C at a fixed strain in order to assess
Fig. 4. AFM images of CNC (a) phase image and (c) height image, AFM images of CNC-Mal (b) phase image and (d) height image and (e) height profile of CNC-Mal nanocrystals.

C. García-Astrain et al. / Carbohydrate Polymers 149 (2016) 94–101
99
Fig. 5. SEM pictures of a) G-CS-CNC-Mal, b) G-CS-CSN, c) G-CS and b) Frequency sweep of hydrogels (G’ (filled symbols) and G” (empty symbols). ᭿ G-CS-CNC-Mal, ꢀG-CS-CNC
and ꢁ G-CS hydrogels.).
the rheological behavior of the samples. The storage modulus (G’)
resulted to be higher than the loss modulus (G”) and remained
almost constant in the frequency range studied, a characteristic of
chemically cross-linked networks (Fig. 5). As expected, comparing
the different hydrogels, the G’ and G” moduli of G-CS were lower
than those of CNCs containing hydrogels. In addition, the modu-
lus of G-CS-CNC-Mal bionanocomposite was higher than that of
G-CS-CNC, indicating that the covalent attachment of CNC-Mal to
furan-modified gelatin resulted in the formation of more cross-
linking points and, thus, stiffer networks.
However, as it can be observed in Fig. 5, the mere incorporation
of CNCs to the hydrogel formulation resulted in an increase in mod-
uli values as a result of the strong hydrogen bonding interactions
between nanocrystals and the two hydrogel forming biopolymers.
However, those differences were only appreciated at low frequen-
cies, whereas at higher values both hydrogels (G-CS and G-CS-CNC)
presented almost identical storage moduli values. On the contrary,
G-CS-CNC-Mal hydrogel presented higher G’ values in the entire
range of frequency, indicating the formation of stiffer hydrogels
that maintained their improved properties due to the participation
of the covalently clicked nanocrystals. The cross-linking density
Kabiri, 2013; Jiang, Su, Mather, & Bunning, 1999; Shet Hui Wong,
Ashton, & Dodou, 2015):
ꢀ
G = ꢀcRT
(3)
where R is the gas constant and T the temperature. The resulting
−
3
values were 1.55, 1.04 and 1.09 mol m for G-CS-CNC-Mal, G-CS-
CNC and G-CS, respectively. As it can be observed, the used of
functionalized CNCs in G-CS-CNC-Mal resulted in the formation
of more cross-linked networks due to the covalent attachment of
CNC-Mal to furan-modified gelatin.
Finally, the swelling capacity (SR) of the hydrogels was assessed
◦
as a function of the incubation time in PBS and water at 37 C
(Fig. 6a). It is well known that the swelling ratio of hydrogels
depends on the cross-linking density of the polymeric network,
the hydrophilic character of the polymers and their concentration
(García-Astrain et al., 2014). During hydrogel swelling, the poly-
meric chains imbibe the solution which starts to permeate inside
the hydrogel, this process being restricted by the network cross-
linking. As it can be seen, the lowest SR value was recorded for
the G-CS-CNC-Mal hydrogel both in water and PBS (2200 ± 210%
and 862 ± 28%, respectively). Similar SR values were recorded for
G-CS and G-CS-CNC hydrogels in PBS (1100 ± 18% and 1156 ± 19%,
respectively). In water, G-CS-CNC SR was higher than that of G-CS,
probably due to the hydrophilic nature of the CNCs (3220 ± 224%
and 2463 ± 31%, respectively). These results confirmed again the
(
␳c) of the different hydrogels was calculated from the rheologi-
cal data, since the storage modulus is related to the cross-linking
density according to the already reported Eq. (3) (Hajighasem &

1
00
C. García-Astrain et al. / Carbohydrate Polymers 149 (2016) 94–101
via Diels-Alder “click” cycloaddition to furan-modified gelatin. The
role of CNCs as cross-linkers for gelatin was confirmed by swelling
and rheological measurements. Typically, higher storage moduli
values were recorded for the cross-linked bionanocomposite when
comparing with the controls. Moreover, changes in the swelling
properties of the hybrid hydrogels were attributed to the formation
of additional cross-linking points due to the presence of functional-
ized CNCs. This synthetic strategy proved to be a suitable alternative
for the preparation of biobased hydrogels with tailored proper-
ties and obtained completely from renewable resources which may
lead to materials with potential applications in the biomedical field.
Acknowledgements
Financial support from the GV/EJ Grupos Consolidados (IT-
776-13) and Domingo Martínez Foundation is acknowledged. C.
García-Astrain wishes to acknowledge the Universidad del País
Vasco/Euskal Herriko Unibertsitatea (Ayudas para la Formación de
Personal Investigador) for PhD grant PIFUPV10/034. Technical and
human support provided by SGIker (UPV/EHU, MICINN, GV/EJ, ERDF
and ESF) is also gratefully acknowledged.
◦
Fig. 6. Equilibrium SR of hydrogels in H2O and PBS at 37 C.
Table 1
◦
Degradation of the different hydrogel samples in PBS at 37 C.
Sample
Degradation (%)
4 h
2
48 h
G-CS-CNC-Mal
G-CS-CNC
G-CS
13
28
28
77
70
41
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important role of CNC-Mal as cross-linker for gelatin chains, since
more cross-linked networks usually display lower swelling ratios
García-Astrain, Ahmed et al., 2015).
The gel contents of the different hydrogels were found in the
range of 87–72%; this indicated the applicability of an effective
cross-linking method for hydrogel preparation. The swelling ratio
(
◦
in PBS solution at 37 C was also recorded for longer time periods
24 and 48 h) in order to evaluate the stability against hydrolytic
(
degradation of the bionanocomposites. The degradation results,
calculated using Eq. (2), are shown in Table 1. The results suggested
that the incorporation of reactive functionalized CNCs resulted in
more stable hydrogels after shorter incubation periods, when com-
paring with hydrogels containing only bare CNCs. However, after
longer incubation periods, the incorporation of both bare CNCs
and maleimide-modified CNCs favoured the degradation. The high
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