Anti-degradation gelatin films crosslinked by active ester based on cellulose

Article abstract of DOI:10.1039/c5ra04808g

Functionalization of microcrystalline cellulose (MCC) with EDTA dianhydride (EDTAD) was first achieved using an esterification reaction. N-Hydroxysuccinimide-activated MCC-EDTAD ester (MEN), a novel macromolecule crosslinker based on MCC, was synthesized for the modification of gelatin films. The reaction between gelatin and MEN was verified by the residual free amino test, FTIR and XRD spectra. The introduction of MEN into gelatin decreased the film degradation ratio and increased its thermal stability, flexibility, hydrophobicity, light barrier performance and water uptake ability. Additionally, SEM images proved the successful surface grafting reaction and degradation phenomenon. This unique gelatin film material with advanced properties broke the limitation of the blending method for modification of gelatin with macromolecules and broadened its application as a novel sustained-release material.

Full text of DOI:10.1039/c5ra04808g

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Anti-degradation gelatin films crosslinked by active
ester based on cellulose†
Cite this: RSC Adv., 2015, 5, 52183
Chen Zhuang, Furong Tao and Yuezhi Cui*
Functionalization of microcrystalline cellulose (MCC) with EDTA dianhydride (EDTAD) was first achieved
using an esterification reaction. N-Hydroxysuccinimide-activated MCC-EDTAD ester (MEN), a novel
macromolecule crosslinker based on MCC, was synthesized for the modification of gelatin films. The
reaction between gelatin and MEN was verified by the residual free amino test, FTIR and XRD spectra.
The introduction of MEN into gelatin decreased the film degradation ratio and increased its thermal
stability, flexibility, hydrophobicity, light barrier performance and water uptake ability. Additionally, SEM
images proved the successful surface grafting reaction and degradation phenomenon. This unique
gelatin film material with advanced properties broke the limitation of the blending method for
modification of gelatin with macromolecules and broadened its application as a novel sustained-release
material.
Received 18th March 2015
Accepted 2nd June 2015
DOI: 10.1039/c5ra04808g
www.rsc.org/advances
17–22
23
montmorillonite, polyvinyl alcohol, zeolite, etc.
Jridi et al.
1
. Introduction
investigated the physical, structural, antioxidant and antimi-
Gelatin is a peptide-based polymeric material, obtained via the crobial properties of gelatin/chitosan composite lms and
hydrolytic treatment of collagen under acidic or alkaline chose the best proportion of the two components to be applied
24
conditions. The triple helix structure of collagen partially as a food packaging material; Li et al. prepared active gelatin-
separates and ruptures, and a non-uniform mixture of poly- based lms incorporating ve kinds of natural antioxidants and
1
peptides with different amino acids are formed. Because of its compared the effect of these extracts on the antioxidant, phys-
25
good biological properties and low toxicity, gelatin is widely ical and mechanical properties of the lms; Alves et al. studied
used for different kinds of materials, such as sponges, lms, the effect of three components (gelatin, cellulose, starch) on the
2
–6
microballoons, scaffolds, nanoparticles, bandages, etc.
However, the relatively weak thermal stability, poor mechanical properties of the starch/cellulose/gelatin nanocrystal lms
biodegradation, water vapor permeability and mechanical
26
properties and easily-degradable quality limit the potential using orthogonal experiments; Andrade et al. reported a new
application of gelatin as a practical material. Microcrystalline edible coating material containing gelatin and cellulose nano-
7
cellulose (MCC), a linear polysaccharide comprising b-glucoside bers, and evaluated the wettability of the coating lm on
units, is usually blended with gelatin to overcome the obstacles banana and eggplant epicarps. Unfortunately, the existing
8–10
of the biopolymer matrix.
Its excellent properties, such as modication of gelatin-based composite lms with natural
renewable origin, biodegradability of its components, and polymers, especially cellulose, is mostly achieved using a
environment-friendly and non-toxic character, further broaden blending method, in which the hydrogen bonding or electro-
10–13
its usages.
EDTAD) is commonly used as a chelating reagent. Its biode- of the polymer matrix. No exact chemical reaction occurs
Ethylenediamine tetraacetic dianhydride static interactions are used to explain the reaction mechanism
14
(
gradable behavior and special molecular structure, which between the gelatin and the original cellulose. Therefore,
consists of two anhydride groups that can react with hydroxyl or proper chemical modication of cellulose is needed to make the
2
7
amine groups, ensure its function in the modication of crosslinking reaction with gelatin possible. Cheng et al.
15,16
biomaterials.
In recent years, a great number of researchers worldwide ldehyde cellulose (DARC), which then reacted with collagen via
have been devoted to the modication of gelatin with various a Schiff base reaction between –NH in the collagen and –CHO
crude macromolecules, such as cellulose, chitosan, starch, in the DARC backbone to obtain DARC/Col composite lms; Li
oxidized cellulose by periodate oxidation to obtain 2,3-dia-
2
28
et al. employed the same oxidation process to oxidize car-
boxymethyl cellulose, and the product, with two aldehyde
Shandong Provincial Key Laboratory of Fine Chemicals, Qilu University of Technology,
Jinan 250353, P. R. China. E-mail: yuezhicui@163.com; Fax: +86 531 89631760; Tel: groups, reacted with gelatin to prepare an edible lm material.
+
†
1
86 531 89631208
Recently, a novel crosslinker, N-hydroxysuccinimide (NHS)
active ester, which is synthesized via the reaction between
Electronic supplementary information (ESI) available. See DOI:
0.1039/c5ra04808g
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29
carboxylic acid and NHS in the presence of a carbodiimide,
(Shanghai). Glycerol (AR, 99%), DMF (AR, 99.5%), EDTA diso-
has attracted a substantial amount of attention, mainly due to dium salt (AR, 99%), acetic anhydride (AR, 98.5%) and other
3
0,31
its cytocompatibility, biocompatibility and availability.
agents were obtained from Tianjin Fu Yu Fine Chemical Co.,
32
Furthermore, Gil’s group has concentrated on modifying Ltd. All chemicals and reagents were used as received without
sugarcane bagasse, which is a raw material of cellulose, with further purication.
EDTAD to gain the ester group for its use as an absorbent
material. In light of this research, the hydroxyl and/or carboxyl
2
.2 Preparation of MEN
functional groups in these three biological polymers (gelatin,
cellulose and EDTAD) further guaranteed the chemical reaction
2.2.1 Synthesis of EDTA dianhydride (EDTAD). The EDTA
34
33
dianhydride was prepared using the method described by Gil
with EDTA disodium salt and acetic anhydride as ingredients.
5 g EDTA disodium salt was dissolved in 250 ml distilled water
to produce materials with new properties.
In this paper, microcrystalline cellulose was modied with
EDTAD to obtain a new type of cellulose ester, MCC-EDTAD (ME).
Then, a novel macromolecule crosslinker, N-hydroxysuccinimide
activated MCC-EDTAD ester (MCC-EDTAD-NHS, MEN), was
synthesized in the presence of 1-(3-dimethylaminopropyl)-3-
ethyl-carbodiimide hydrochloride (EDC) to react with gelatin
2
to obtain a clear solution, and then HCl was added dropwise
until precipitation of EDTA occurred. The precipitate was
vacuum ltered and rinsed with 99% EtOH and 99% diethyl
ether, subsequently dried in an oven at 70 C, and cooled in a
desiccator prior to use.
For the preparation of EDTA dianhydride, 18 g EDTA was
suspended in 50 ml pyridine and 25 ml acetic anhydride was
added. Then, the mixture was heated under reux and kept
stirring at 65 C for 24 h. Aer reaction, the solid obtained was
vacuum ltered, rinsed with diethyl ether and dried under
ꢀ
(Scheme 1), and a biological polymer lm with new qualities was
found. FTIR, XRD, TGA-DSC, mechanical properties, contact
angles and residual amino group testing were applied in our
present study. Additionally, in vitro degradation studies, light
barrier properties and water uptake measurements of the cross-
linked gelatin lms were investigated. On the basis of these
results, the comparison of the thermal stability and light barrier
properties between MEN-modied gelatin lms (Gel–MEN) and
cellulose blending lms (Gel/MCC) were explored.
ꢀ
ꢀ
vacuum at 50 C. The prepared EDTAD was characterized using
1
H-NMR spectroscopy (Bruker Advance 400 spectrometer) and
FTIR spectroscopy (Nicolet NEXUS 470 FT-IR spectrometer).
2.2.2 Synthesis of MCC-EDTAD (ME). The functionaliza-
tion of MCC with EDTAD was carried out according to the
35
method of Gil’s group, with slight modication. 9 g MCC and 3
2
. Experimental
g EDTAD were suspended in 100 ml DMF, and then the mixture
2.1 Materials
ꢀ
was shaken and heated under reux at 75 C for 24 h. The
Gelatin (type A, obtained from pigskin, with an approximate modied material was isolated by ltration under reduced
molecular weight of 50 000 and isoelectric point at pH ¼ 8, pressure, washed sequentially with DMF, distilled water, satu-
determined using uorescence measurements) was obtained rated NaHCO3 solution (in order to release carboxylate and
from Sinopharm Chemical Reagent Co., Ltd. MCC (extra pure, amine functions), distilled water, and then ethanol. Aer drying
ꢀ
average particle size 90 mm), NHS (AR, 98%) and EDC (AR, 99%) under vacuum at 50 C, the percent mass gain was calculated
were purchased from Energy Chemical Technology Co., Ltd using eqn (1).
m
modified ꢁ munmodified
Weight gain ð%Þ ¼
ꢂ 100
(1)
m
unmodified
2
.2.3 Synthesis of NHS MCC-EDTAD active ester (MEN).
36
MEN was synthesized using the method of Li, with a bit of
improvement. A mixed solution was prepared by dissolving 12.5
mmol ME, 50.0 mmol NHS, and 50.0 mmol EDC together in 200
ꢀ
ml distilled water and was gently stirred at 40 C for 1 h. Aer
the reaction, the solid was vacuum ltered, washed with
ꢀ
distilled water several times, then dried under vacuum at 50 C
to get puried MEN. The percent mass gain was once again
calculated using eqn (1). The obtained ME and MEN were
characterized using FTIR spectroscopy (Nicolet NEXUS 470 FT-
IR spectrometer), an Elemental Analyzer (Vario EL III, Ele-
mentar Analysensysteme, Germany) and TGA-DSC (Q600SDT,
TA, USA).
2
.3 Modication process and lm formation
Scheme 1 The formation process of crosslinked gelatin with MEN. (1)
The synthetic route of EDTA anhydride (EDTAD); (2) the preparation
path of gelatin modified with MEN.
A gelatin solution (3%, w/v) was prepared by dissolving gelatin
powder in distilled water and was then heated at 45 C for 2 h
ꢀ
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under continuous stirring. Glycerol was added as a plasticizer at 2.7 Scanning electron microscopy (SEM) of gelatin lms
a certain concentration (15% of dry gelatin weight). The dosage
of MEN was determined by the mass ratio with gelatin, i.e.,
The microstructures of the prepared lms were investigated
using a Quanta 200 environmental scanning electron micro-
scope (FEI Company, Holland). Before observation, the lm
surfaces were coated with Au using a SEM coating device. More
mMEN/mgelatin ¼ 0%, 5%, 10%, 15%, 20%, 25%, 30%. Therefore,
the corresponding modied gelatin samples were named as
Gel, Gel–5%MEN, Gel–10%MEN, Gel–15%MEN, Gel–20%MEN,
than ten micrographs were taken from different zones of each
Gel–25%MEN and Gel–30%MEN, respectively. The appropriate
surface lm.
weight of MEN powder was dissolved in distilled water under
stirring for 12 h at room temperature to produce a liquid
suspension. Then, the solution was added dropwise to the 2.8 Thermogravimetric analysis
gelatin liquid, and acetic acid (3% of water volume) was added
dropwise into the whole system to promote the start of the
The thermal stability of the gelatin lms was determined using
thermogravimetric analysis and differential thermal scanning
calorimetry synchronous apparatus (TGA-DSC, Q600SDT, TA,
USA). The gelatin lm samples (approximately 2.5 mg) were
interfacial reaction. These mixtures were gently stirred for 12 h
ꢀ
at 45 C.
To cast the lms, 30 g of each gelatin reaction solution was
weighed accurately into aluminium pans and sealed. The
transferred into a teon dish and placed at room temperature
ꢀ
endothermal curve of the crushed lm was recorded from 20 C
ꢀ
for 2 h, then put in an oven at 40 C until the lms dried. The
ꢀ
ꢀ
ꢁ1
to 500 C at a scanning rate of 10 C min under nitrogen
atmosphere. Additionally, the thermal stability of the Gel/MCC
blend lm was also studied and compared with the Gel–MEN
dried lms were peeled off and stored in a desiccator with
relative humidity #20%. In addition, one part of gelatin reac-
ꢀ
tion solution was freeze dried at ꢁ55 C, 70 Pa with a vacuum

lms.
freeze drier (FD-1A-50, Beijing, China) and the lyophilized
powder was characterized using FTIR spectroscopy (Nicolet
NEXUS 470 FT-IR spectrometer).
2.9 Mechanical testing
Prior to investigating the mechanical properties, the lms were
ꢀ
conditioned for 48 h at 20 C and 50 ꢃ 5% RH. Tensile strength
2.4 XRD analysis
(T ), elasticity modulus (E ) and elongation at break (Eab) were
s m
40
determined as described by Benjakul with a slight modica-
tion, using a Microcomputer Controlled Electronic Tensile
Testing Machine (WDL-005, Jinan, China) equipped with a
tensile load cell of 300 N. Samples with initial grip length of 25
mm were used for testing and the cross-head speed was set at 10
XRD analysis of the samples was performed on an X-ray
diffractometer (D8-ADVANCER, Bruker AXE, Germany) with a
thin lm attachment using Cu-Ka radiation (l ¼ 0.1541 nm) at a
current of 40 mA and an accelerating voltage of 40 kV. The
ꢀ
ꢀ
patterns were recorded from 10 to 60 .
ꢁ
1
mm min . The thickness of each lm was measured using a
Vernier Caliper (0.02 mm/150 mm, Shanghai, China).
2
.5 Determination of residual amino groups in gelatin
The residual –NH groups in the modied gelatin solution were
determined using the improved Van Slyke method at 45 C.
2.10 Contact angle measurement
2
ꢀ
37,38
The water contact angles (CAs) of all lms were measured using
Sample solutions were mixed with acetic acid and sodium the Sessile drop method with a DSA100 contact angle measuring
nitrite and stirred for 45 min. The residual primary amine (mol system from Kr u¨ ss. The gelatin reaction solution was coated on
ꢁ
1
g
) was calculated according to the volume of N
2
. All samples the surface of a glass sheet to obtain lms with thickness of
about 0.1 mm, and then stored in a desiccator with relative
humidity #20%.
were tested in triplicate.
2.6 In vitro degradation studies
2.11 Light barrier properties and transparency
The degradation study of gelatin lms was carried out in vitro by
incubating in phosphate buffer (pH 7.40) at 37 C for different
The ultraviolet and visible light barrier properties of the lms (1
cm ꢂ 2 cm) were measured using an ultraviolet-visible spec-
trophotometer (UV-7504C, Shanghai, China) at selected wave-
lengths from 200 to 800 nm following Fang’s method. The
transparency values of the lms were calculated using eqn (3),
where T is the transmission (%) at each wavelength and x is the
ꢀ
intervals (1, 3, 5, 7, 9, 12 and 24 h), which was developed from
39
ꢀ
the method of Haroun. The gelatin lms were dried at 60 C to
a constant weight prior to use and marked as m . Aer different
41
0
degradation times, the samples were washed with distilled
ꢀ
water aer ltering under vacuum and dried at 60 C to a

lm thickness (mm). According to the equation, high trans-
constant weight (m ). The degradability performance was
t
parency values indicate good light barrier performance.
examined from the weight remaining using eqn (2).
m
t
Transparency value ¼ ꢁlog T/x
(3)
Weight remaining ð%Þ ¼
ꢂ 100
(2)
m
0
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ꢀ
2.12 Water uptake measurement
transition temperature (T
g
) and the char residue at 500 C of
MCC, ME and MEN are recorded in Table 1 (Fig. S4†). The T
i
The water uptake of the lms was determined similarly to
Kavoosi and Tang, with a little development. Rectangular
ꢀ
ꢀ
42
43
values of MCC and ME were 309.10 C and 271.45 C, respec-
ꢀ
tively, while that of MEN was 222.54 C, which suggested a
specimens sized 15 mm ꢂ 10 mm with a thickness of 0.1 mm
ꢀ
reduction in thermal stability. This can be related to the reac-
tivity of the three materials with –NH in gelatin, which was in
2
accord with the results of the residual amino groups, below. To
summarize, the difference of each item further veried the
introduction of EDTA and NHS into MCC, which agreed with
the FTIR and elemental analysis.
were prepared. The samples were conditioned at 20 C in a
desiccator containing silica gel (RH 20% ꢃ 5%) for three days,
i
to constant weight (W ). Then, the lm samples were transferred
into desiccators at 100% relative humidity (super-saturated salt
ꢀ
solution of CuSO $5H O) at 20 C for eleven days to absorb
4
2
water until the weight reached equilibrium. The weights of the
samples at the adsorption time t were noted as W . The amount
t
of water adsorbed at different intervals and at equilibrium were
calculated using eqn (4). All tests are the means of at least three
measurements.
3
.2 Conrmation of MEN crosslinking with gelatin
.2.1 FTIR spectra analysis of MEN, gelatin and Gel–MEN
lm. FTIR spectra of the pristine gelatin (curve a), pristine MEN
3

W
t
(curve b), and Gel–25%MEN lm (curve c) are compared in
Water absorption ð%Þ ¼
ꢂ 100
(4)
W
i
Fig. 1. In the case of pristine gelatin, the C]O stretching
vibration appearing at 1664 cm demonstrated the amide I
band, while the amide band II indicating the N–H bending
vibration was observed at 1535 cm . In addition, aliphatic C–H
ꢁ
1
3
. Results and discussion
3.1 Characterization of MEN
ꢁ
1
1
3
.1.1 Spectra of EDTAD. H NMR (400 MHz, DMSO,
Fig. S1†): d 3.691 (s, 8H), 2.657 (s, 4H), 3.080 (s, DMSO), 2.496–
.488 (m, DMSO), which is in accord with the characteristic
ꢁ1
bending vibrations were observed at 1450 cm and bands at
ꢁ1
1331 and 1230 cm showed the C–N bond stretching vibra-
2
tions. Gel–MEN showed all the characteristic peaks of gelatin
and MEN, such as those at 1643 and 1546 cm , which indi-
cated the successful reaction between gelatin and crosslinker
MEN, along with a representative peak at 1741 cm , which
clearly indicated the amidation reaction between –NH
gelatin and the active ester base in MEN.
peaks of H in the ideal product. The FTIR spectrum (Fig. S2†)
further proved the dianhydride structure, with two groups of
splitting peaks. The peaks in the high frequency region split at
ꢁ1
ꢁ1
ꢁ
1
ꢁ1
1
813, 1759 and 1689 cm , with a gap of 60 cm between
2
in
adjacent peaks. The low frequency groups split at 1139, 1074,
ꢁ
1
and 991 cm , with the same interval. Additionally, the bands at
3.2.2 X-ray diffraction studies of MEN, gelatin and Gel–
ꢁ
1
1
245 and 1400 cm , related to C–O and C–N stretching,
respectively, were also evidence of the EDTAD structure.
.1.2 Spectra of MCC, ME and MEN. The FTIR spectra
Fig. S3†) fully depicted the functional groups of MCC, ME and
MEN. Compared with MCC, the appearance of a strong band at
MEN lm. In order to examine the effect of MEN on the crystal
structure and crystallinity of gelatin, the XRD patterns of the
freeze-dried gelatin lms are investigated. Data on the 25%
MEN formulation are presented as a representative example. As
shown in Fig. 2, curve (a) was the XRD pattern of MEN, which
displayed the typical XRD pattern of the native cellulose with
3
(
ꢁ1
1
741 cm in ME can be attributed to axial deformation of the
ꢁ
1
ester bond, and bands at 1633 and 1406 cm are attributed to
asymmetric and symmetric axial deformations of the carbox-
ylate. These bands conrmed the successful functionalization
of MCC with EDTAD via the formation of ester linkages. For
MEN, absorption peaks at 1706, 1210 and 811 cm represented
g-dicarbonyl stretching vibration, C–N stretching and C–C
vibration respectively. In particular, the reinforcement of the
ester carbonyl band at 1742 cm and the weakening of the
carboxy carbonyl band at 1600 cm further proved the struc-
ture of the active ester.
ꢀ
ꢀ
ꢀ
the main diffraction signals at around 14.9 , 16.2 , 22.5 and
ꢀ
44
34.3 . Curve (b) only showed an extensively broadened peak in
ꢀ
the 2q range of 15–25 , which was a typical XRD pattern of pure
gelatin, originating from the a-helix and triple-helical struc-
ture.
Fig. 4(c), in which the characteristic peaks of MEN (22.6 ) and
the characteristic broad diffraction peak of gelatin were each
observed. This suggests that the gelatin was modied with MEN
aer the crosslinking reaction, which is consistent with the
FTIR results.
ꢁ1
45,46
The XRD pattern of the Gel–25%MEN lm is given in
ꢀ
ꢁ1
ꢁ
1
3.1.3 Elemental analysis and thermal properties of MCC,
3
.2.3 Free –NH in Gel–MEN lm formation solution. Fig. 3
2
ME and MEN. As can be seen in Table 1, there was a consid-
erable increase in nitrogen content of 1.92% aer functionali-
zation of MCC with EDTAD. Accompanied by the signicant
weight gain of 72.50%, the modied material (ME) with EDTAD
incorporated was obtained. Similarly, the content of N
increased to 2.48% in MEN, 0.50% higher than that of ME,
which shows that the esterication reaction occurred between
ME and NHS, with linkage of a ve-membered nitrogenous ring.
Also, the weight gain of 30.80% further proved this.
shows the changing curve (a) of residual primary amino groups
in the Gel–MEN lm formation solution and gelatin liquid
blend with ME (Gel/ME, curve (b)) against the ratio d (d ¼ m(MEN/ME)
(dry gelatin)). For Gel–MEN, the dosage of crosslinker played an
important role in the content of free –NH , while the free –NH
/
m
2
2
changed little no matter how much ME was added. This sug-
gested the stability of the ester group in ME, which was not
active enough to react with –NH in gelatin. This means that the
2
amine groups in gelatin did not act as nucleophiles to break the
ester bonds in ME but could break the ester bonds in MEN. All
of these results conrmed that the reaction process (Scheme 1)
The initial decomposition temperature at 5% weight loss
(T ), the maximum weight loss temperature (T ), the glass
i m
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Table 1 Elemental analysis and thermal property values of MCC, ME and MEN
Weight gain
(%)
Residue
(%)
o
o
o
Materials
C (%)
H (%)
N (%)
T
i
( C)
T
m
( C)
g
T ( C)
MCC
ME
MEN
42.21
42.29
42.97
6.40
6.45
6.65
0.11
1.92
2.48
—
72.50
30.80
309.10
271.45
222.54
364.08
331.50
377.31
340.68
342.70
364.09
15.28
33.01
10.45
glycidol and the maximum –NH conversion rate was 42%, the
2
–
NH conversion rate in this work was 28% higher than that
2
reported.
3.3 Performance of Gel–MEN lms
3.3.1 Degradation properties in vitro. As sustained-release
materials, the composite lms are expected to degrade at a
proper rate to match particular needs and maintain activity
within their service life. The degradation behavior of the lms
in a physiological environment plays an important role in their
application as sustained-release materials. The in vitro degra-
dation performance of the Gel and Gel–MEN lms in phosphate
buffered saline (PBS, pH 7.40) at different intervals was inves-
tigated. As shown in Fig. 4, the blank gelatin degraded rapidly
because of the large number of hydrophilic amino and carboxyl
groups in the gelatin backbone. In addition, the physical
structure of gelatin, which possesses higher porosity and a
thinner pore-wall, contributed to the minimum remaining
weight of 15% at 24 h. The composite polymer Gel–MEN
degraded proportionally slowly because of the incorporation of
the cellulose-based crosslinker, MEN. The remaining weights of
Gel–25%MEN and Gel–30%MEN at 24 h were 57% and 58%,
respectively, 40% greater than that of the original gelatin lms.
The amido bond formed between MEN and gelatin was stable
enough to resist adverse external factors. It was reasonable to
consider that the strong hydrogen bonding and electrostatic
interaction between the gelatin polypeptide and the hydrophilic
hydroxyl or carboxylic groups in MEN also depressed the
Fig. 1 The FTIR spectra of MEN, gelatin and Gel–MEN.
Fig. 2 The XRD patterns of MEN, gelatin and Gel–MEN.
we proposed was correct. Interestingly, aer activation with
NHS, the active ester MEN could consume –NH in gelatin and
was dose dependent. The amount of free –NH decreased
2
2
sharply when the ratio d increased from 0 to 25%, and then
decreased slightly when the ratio further increased. In partic-
ular, the amount of free –NH decreased down to a minimum
2
ꢁ4
ꢁ1
value of about 1.74 ꢂ 10 mol g when d ¼ 30%. All of these
results proved that the whole system overcame the inhibition of
the interfacial reaction. Compared with the former interface
47
Fig. 3 Residual amino group content of gelatin solution modified with
MEN (a) and ME (b) at different dosages.
reaction study by Xu, in which gelatin was modied with
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diffusion of the PBS medium and protected the gelatin poly-
peptide chains from degradation. Meanwhile, the presence of
MEN, a macromolecule crosslinker based on cellulose, also
served as physical crosslinking sites, which enhanced the
stability of the network. To conclude, MEN improved the anti-
degradation performance of the gelatin lms and this guaran-
tees its potential usage as a sustained-release material in many

elds, such as food packaging, medical engineering, controlled-
release fertilizer in agriculture, and so on.
.3.2 Morphology evaluation. SEM photographs of the
3
blank lms revealed a dense, smooth and compact structure
without any embossing or holes, as shown in Fig. 5(a). The
magnication of the rst row (a
1 1 1
, b , c ) is lower than that of
1 2 1 2
Fig. 5 Film surface morphology of Gel (a , a ), Gel–25%MEN (b , b )
second row (a , b , c ). The introduction of the cellulose-based and Gel–25%MEN after 1 h degradation (c , c ).
2
2
2
1
2
crosslinker MEN destroyed the homogeneous lm surface,
with slice-like or rod-like macromolecules graed on the
covering of gelatin (Fig. 5(b)). The inset of Fig. 5(b
displays the features of the MEN.
2
) clearly
as a plasticizer in the blank gelatin lm. The DTG patterns of
Gel–MEN presented three steps for weight loss at temperatures
In addition, the SEM images provided very good evidence in
favor of the in vitro degradation of the test sample (Gel–25%
MEN). It can be seen from Fig. 5(b) and (c) that the lm surface
was almost planar and even, though combined with some sags
and crests, before the degradation started. Aer one hour of
degradation, a porous structure with irregularities and aper-
tures can be observed on the surface of the composite lm,
which conrmed that the internal structure of the Gel–MEN
polymeric lm had started to degrade in the liquid medium. It
can be assumed that the degradation of the lms was gradually
ꢀ
ꢀ
ꢀ
of around 100 C, 250.07 C and 320–350 C, involving one
strong and two weak endothermic peaks. The rst weight loss at
ꢀ
the temperature around 100 C and the second weight loss at
ꢀ
about 250.07 C were similar to those of curve b. The third
ꢀ
weight loss, with a strong endothermic peak at 320–350 C, was
due to the incorporation of the active ester MEN into gelatin,
and exhibited a positive correlation with the dosage of cross-
linker. This demonstrated that the crosslinking effect of the
cellulose-based crosslinker improved the thermal stability of
the material to some degree, as found in the literature. On the
one hand, the crosslinking reaction between gelatin and MEN
with formation of amido bonds made the macromolecule
structure more stable and impregnable. On the other hand, the
hydrogen bonding and electrostatic interaction of the func-
tional groups in gelatin and MEN further strengthened the
structure. All of these factors provided an effective reinforce-
48
penetrating deeper from the surface.
.3.3 Thermal stability. The thermogravimetric analysis
TGA) and differential thermogravimetric curves (DTG) of the
3
(
composite lms, as shown in Fig. 6, are used to investigate the
thermal stability. Curve a (gel) and curve b (gel/glycerol) were
almost similar, with two representative peaks at 190.00–
ꢀ
ꢀ
2
20.00 C and 321.44 C, which corresponded to the initial
ment layer to endure thermal degradation. As Fig. 6-1 and 6-2
decomposition temperature at 5% weight loss (T ) and
show, Tm reached a maximum of 349 0.26 ꢀC when d (d ¼
i
maximum weight loss temperature (T
peak at 250.07 C in curve b was due to the blending of glycerol
m
) of gelatin. The special
m(MEN)/m(dry gelatin)) was 25%.
ꢀ
The thermal properties of Gel–25%MEN and Gel/25%MCC
in the presence of glycerol were compared in Fig. 6-3 and 6-4, in
which curve b consisted of four decomposition stages. The four
ꢀ
peaks at 104.42, 192.23, 250.72 and 359.12 C were resolved into
four different components of water, gelatin, glycerol and MCC,
ꢀ
respectively. The obvious peak at 192.23 C that almost dis-
appeared in curve a indicated the severe phase separation in the
Gel/MCC system. However, compared with the typical T
m
ꢀ
49
(
309.10 C) of MCC, the increased decomposition temperature
ꢀ
of 359.12 C in curve b may caused by the hydrogen bonds
formed between gelatin and MCC, which increased the thermal
stability of the Gel/MCC lms.
3.3.4 Mechanical properties. Mechanical properties, espe-
cially elasticity modulus (E ) and elongation at break (E ), are
m
ab
particularly crucial for sustained-release materials in many
elds. Table 2 shows the thickness, tensile strength (T ), E and
ab of the Gel–MEN composite lms. The decreased T indicated
that the modied lms yielded at lower stress than the pure

s
m
E
s
Fig. 4 Effect of macromolecule crosslinker on in vitro degradation of gelatin lm. The tensile strength increased with added
the Gel–MEN composite films.
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Fig. 6 TGA and DTG curves of modified gelatin films with different dosages of crosslinker (6-1 and 6-2) and comparison curves between Gel–
5%MEN and Gel/25%MCC blend films (6-3 and 6-4).
2
crosslinker from Gel–5%MEN to Gel–20%MEN, but decreased groups in the gelatin polypeptide, and the hydrophilic groups
in Gel–25%MEN and Gel–30%MEN. The results were in accord could form hydrogen bonds. All the newly formed bonds
50
with the work reported by Azeredo. This may caused by the fact weakened the protein–protein interactions, which was effective
that when the amount of the macromolecule crosslinker was for stabilizing the gelatin network. In addition, MCC, the base
high, adding it to the lm may induce the development of a of the crosslinker MEN, has been demonstrated to be an
heterogeneous structure with the presence of discontinuous effective nano-reinforcement for biopolymer lms that can
areas, which produced lower tensile strength. Similarly, Mar- drastically inuence the mechanical properties of biomate-
51
50
tucci reported that the addition of dialdehyde starch in gelatin rials. All of these factors contributed to better exibility of the
resulted in lower T values than the control lm and explained new biological polymer matrix.
this apparently anomalous behavior. The polymeric nature of 3.3.5 Hydrophobicity analysis using contact angles.
s
the dialdehyde starch did not introduce severe restrictions Gelatin is a kind of hydrophilic material because of its func-
within the gelatin matrix, as usually occurrs with short chain tional groups, amino, carboxyl, hydroxyl, and so on. Its water-
dialdehydes such as formaldehyde or glutaraldehyde, and some sensitive properties have limited its application in many elds
21
degree of phase separation in the gelatin–dialdehyde starch and hydrophobization was needed. Ninan reported a new
lms could also reduce the T . Fortunately, E and Eab tended to material based on a gelatin/zeolite porous scaffold and the

s
m
ꢀ
ꢀ
predict better elasticity and exibility, indicating that the new contact angle was found to increase from 88.6 C to 108.0 C
material was not fragile anymore. The Eab of Gel–25%MEN was with increasing concentration of zeolites in the gelatin. The
31.96%, thirty times that of the blank gelatin lm, while the E
m
hydrophobic effect of the crosslinker MEN on gelatin was
of Gel–25%MEN was 448.72 MPa, a quarter that of the blank conrmed by the results of the water contact angle measure-

lm, which was 1736.11 MPa. The reason was that the active ments (Fig. S5†). The pure gelatin lm (Fig. S5a†) had a typical
ꢀ
ester group in MEN could form covalent bonds with the amino contact angle of 77.8 because of the hydrophilic groups
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Table 2 Mechanical performance of different Gel–MEN films
Thickness
(mm)
Tensile strength
(MPa)
Elongation at
break (%)
Elasticity modulus
(MPa)
Films
Gel
0.10
0.18
0.20
0.20
0.28
0.26
0.20
24.17
15.28
16.17
18.25
18.10
13.97
14.08
1.84
7.64
1736.11
435.73
468.75
595.24
525.21
448.72
476.19
Gel–5%MEN
Gel–10%MEN
Gel–15%MEN
Gel–20%MEN
Gel–25%MEN
Gel–30%MEN
11.52
12.44
28.64
31.96
83.08
exposed on the gelatin chains. Aer being crosslinked by MEN, (%) reached the maximum among all the MEN crosslinked
the Gel–MEN lms (Fig. S5b and c†) presented a sharp increase gelatin lms. This was attributed to the increase in pore size of
ꢀ
ꢀ
to 125.1 and 135.5 , respectively. This was due to the replace- the gelatin lm with the presence of MEN. Additionally, the
ment of some surface amino groups in the gelatin polypeptide Gel–MEN composites showed an increase in the swelling
with active ester groups in MEN. In addition, the hydrogen capacity up to the 11th day, which indicated a better water
bonds formed between the hydrophilic groups of the gelatin absorption ability. By comparison, the original gelatin lm
backbone and the MCC-based crosslinker also contributed to acquired its maximum swelling capacity on the 10th day and
good hydrophobicity of the modied gelatin lms. The modi- thereaer the percentage of water uptake was found to be

ed lms, with perfect hydrophobicity, overcame the perma- invariant, or even declining. This event suggested that intro-
nent weakness of water-sensitivity in the application of gelatin. duction of MEN into gelatin provided an effective channel for
The advanced properties broaden its usage as different kinds of water molecules to diffuse into the polymer matrix, and thereby
material.
the swelling ability increased. Uncontrolled swelling properties
3
.3.6 Water uptake studies. The hydrophilic property of can badly affect the mechanical properties, so it is advantageous
52
gelatin can be controlled in two ways. One is the hydro- to tune the swelling capacity.
phobization referred to above, and the other is the control of the
3.3.7 Light resistance performance. Much research has
expected absorption of water molecules. This occurs because of indicated that ultraviolet radiation is one of the main causes of
two benecial structural features of gelatin. One advantage of skin damage, light-stimulated aging and skin cancer. Hence,
gelatin-based materials is its highly hygroscopic nature, due to the low light transmission also gives active gelatin lms some
24
which it swells and transforms into any shape easily in a humid health function. Light transmission at selected wavelengths
environment. The other is the porosity in the network that from 200 to 800 nm in the UV and visible ranges and the
allows more water to enter, because of which the porous gelatin transparency values of the gelatin lms are shown in Table 3 (&

lms show higher swelling capacity. The water uptake (%) Fig. S6†). Comparison of the results with the control lms
values of the pure gelatin lms and Gel–MEN composites tested revealed that lower light transmittance (T) was found in the Gel–
over 11 days are shown in Fig. 7. The water uptake (%) can be MEN composite lms and the lms with 30%MEN displayed
controlled by incorporating different dosages of MEN in the the lowest values among them. This revealed that the addition
polymer matrix. In the case of Gel–15%MEN, the water uptake of MEN improved the UV barrier properties of the gelatin lms,
Fig. 7 Water uptake properties of gelatin films incorporated with different dosage of MEN.
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Table 3 Light transmission and transparency values of different Gel–MEN films
Transparency
value
Wavelength (nm)
Films
200
280
350
400
450
500
550
600
700
800
280
600
Gel
2.2
1.5
0.6
0.2
0.1
0
0
1.9
1.4
1.5
5.8
2.6
0.2
0.2
0
0
9.3
5.3
43.2
29.8
12.4
4.4
2.2
0.6
0.4
34.6
26.6
55.0
34.4
14.4
5.8
3.0
1.0
0.6
39.6
31.7
59.7
35.4
15.1
6.3
3.5
1.3
0.9
40.9
33.0
62.6
36.4
15.5
6.6
3.6
1.4
1.0
42.3
34.4
64.4
37.0
15.8
6.7
3.7
1.5
1.1
43.3
35.5
66.5
37.8
16.2
7.1
3.7
1.6
1.1
45.1
36.9
67.6
38.4
16.4
7.1
3.7
1.6
1.1
46.5
38.0
68.5
39.0
16.4
7.2
3.7
1.6
1.1
47.7
39.3
10.13
7.73
11.32
13.49
13.49
—
—
10.32
12.78
0.98
2.64
5.65
5.74
7.16
8.98
9.79
3.46
4.32
Gel–MEN5%
Gel–MEN10%
Gel–MEN15%
Gel–MEN20%
Gel–MEN25%
Gel–MEN30%
Gel/MCC15%
Gel/MCC25%
resulting from the amido bonds from the Schiff base formation composites exhibited serious phase separation. The mechanical
between the active ester groups in MEN and the amino groups properties changed to some degree, with higher Eab and lower
of the lysine or hydroxylysine side chains in gelatin. Based on
E
m
, which suggested better exibility and shatter-proong. The
ꢀ
the transparency values (T , Table 3), more crosslinker led to contact angles, with a highest value of 135.5 , indicated good
v
greater T , which represented the opacity of the resultant lms. hydrophobic properties. The swelling ability aer absorbing
v
The opacity was highly inuenced by the crystalline content of a water could be regulated by adding different weights of cross-
sample: more compact polymer chains made it more difficult linker. The light barrier performance was improved with the
for light to pass through, and the opacity of the lms was introduction of MEN compared with both pure gelatin lm and
53
increased. All of these results indicated that the protein-based Gel/MCC composites. Given the potential applications of
lms exhibit high UV barrier properties, owing to their high gelatin, our study is an extension of the existing NHS cross-
content of aromatic amino acids, which absorb UV light. linking technique and will broaden the application of gelatin

Table 3 also displays the light barrier properties of Gel/15% lms as a sustained-released material in the food industry,
MCC and Gel/25%MCC blend lms, as compared with the medicine, agriculture, and so on.
corresponding mass ratio of Gel–MEN lms. The Gel/MCC
blend lms exhibited better transparency but worse light
resistance performance. This fact may be an indication that
MCC nanoparticles were homogeneously distributed in the
Acknowledgements
We gratefully acknowledge the promotive research fund for
young and middle-aged scientists of Shandong Province
(BS2014NJ012), the National Natural Science Foundation of
China (no. 21276149) and the Program for Scientic Research
Innovation Team in Colleges and Universities of Shandong
Province.
matrix because they are white, and light incident on the lm
surface was reected in a larger quantity due to the white
particles. The light barrier properties of the lms are relatively
important when used as sustained-release materials for food
packaging or food coating. The polymer matrix in this work
matches these needs.
25
4
. Conclusion
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