Development of a gelatin-g-poly(acrylic acid-co-acrylamide)–

montmorillonite superabsorbent hydrogels for

in vitro controlled release of vitamin B12
Jayashree Nath, Anupam Chowdhury, Ilias Ali, Swapan Kumar Dolui

ABSTRACT: Gelatin (Ge)-g-poly(acrylic acid-co-acrylamide) and montmorillonite (MMT)-clay-based nanocomposite hydrogels were fab-
ricated to study the controlled release of vitamin B12. Polymeric hydrogels were characterized with Fourier transform infrared (FTIR)
spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). FTIR spec-
troscopy confirmed the grafting of partially neutralized acrylic acid on a Ge backbone. The incorporation of MMT fillers inside the
nanocomposite hydrogels and their increased crystallinity were established by XRD analysis. The rough surface morphologies of the
composite hydrogels shown by SEM resulted from the assimilation of MMT inside the same. TEM confirmed the formation of nano-
sized composites. The average length and width of the MMT platelets were found to be 184.37 and 20.48 nm, respectively. The maxi-
mum swelling of the hydrogel was 375 g/g, and the results were established with Design-Expert software. The biodegradability of the
nanocomposite increased in comparison to that of the copolymer hydrogel. Biocompatibility and cytotoxicity studies were also per-
formed. During different time intervals, the controlled release of vitamin B12 in artificial gastric fluid (AGF) and artificial intestinal fluid
(AIF) was evaluated with a UV–visible spectrophotometer; this resulted in different controlled release curves. The release in AGF was
42%, and in AIF, the cumulative release was 80% over 6 h. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019, 136, 47596.

KEYWORDS: crosslinking; drug-delivery systems; gels

Received 17 August 2018; accepted 12 January 2019

DOI: 10.1002/app.47596

INTRODUCTION protein-based biodegradable polymer. The large number of func-

Three-dimensional (3D) crosslinked polymeric hydrogel network
structures possess high swelling and water-retention capacities.
Superabsorbent hydrogels with linear or branched chain polymers
with excellent hydrophilic properties have demonstrated wide
applications in drug delivery,1 hygiene products,2 wastewater
treatment,3 agriculture,4 cosmetics,5 and heavy-metal-ion removal.6
Hydrogel structures composed of synthetic polymers, namely,
acrylamide (AAm) and acrylic acid (AAc), are more commonly
used as superabsorbent materials.7–9 Superabsorbent hydrogels
based on natural polymers, such as chitosan,10,11 starch,12 colla-
gen,13 cellulose,14 sodium alginate,15,16 and guar gum,17 have drawn
much attention because of their nontoxic behavior,18 elevated
hydrophilic nature,19 and promising biodegradability.20 Hydrogels
with enhanced properties that can respond to changes in their envi-
ronment and thus act as smart or intelligent materials enable them
to be used in a more user-friendly way.21
The prerequisite of properties such as stimuli-responsive swellabil-
ity, biocompatibility, and drug encapsulation efficiency has limited
the number of polymers suitable for the controlled release of drugs.
Compared to natural polymers, synthetic polymers suffer from
numerous disadvantages. Therefore, such polymers are evaded in
biomedical research. Gelatin (Ge), derived from collagen, is a
tional groups present in Ge helps it in chemical crosslinking, and
its unique gelling capacity makes it greatly inspiring for the synthe-
sis of smart hydrogels.22 Cheng et al.23 synthesized biocompatible
chitosan–Ge based hydrogels, which were thermoresponsive, and
their application showed the sustained release of latanoprost
in vitro and in vivo.
Truong et al.24 performed another study on Ge-based injectable
hydrogels crosslinked with poly(ethylene glycol); this study revealed
its significant potency as a 3D culturing platform. This hydrogel
showed promising applications in the capture and release of cells and
cell delivery triggered by light. Another important aspect includes its
degradability under UV irradiation to allow the simple recovery of
encapsulated cells without any damage. Li et al.25 developed photo-
crosslinked Ge-based hydrogel systems to evaluate their efficiency for
focal corneal wound repair. The study demonstrated the hydrogel to
be a potential candidate for reducing the growing demand of cornea
implants. However, the lack of suitable mechanical and thermal
properties for drug-delivery applications minimize the utilization of
natural polymers in hydrogel synthesis. This leads to the vital step of
their structural modification and strength improvisation.
Recently, the incorporation of clay inside the hydrogel network
has appeared to be a broader area of study. Naturally abundant

© 2019 Wiley Periodicals, Inc.

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montmorillonite (MMT) is a smectite group of clay without any
toxicity.26 Kevadiya et al.27 developed a Ge–ciprofloxacin–MMT
composite hydrogel demonstrating the controlled release of drugs
up to 150 h and application in wound dressing. Following the sol–
gel route, Jesus et al.28 synthesized ureasil–poly(ethylene oxide)
hybrid hydrogels incorporating sodium MMT inside the gel matrix
to report the sustained release of sodium diclofenac. This hybrid
hydrogel is considered to be applicable for prolonged arthritis,
migraines, sprains, gout, and pain after surgical procedures. Another
hydrogel synthesized on the basis of poly(vinyl alcohol) and chito-
san containing MMT clay and honey was demonstrated to be effi-
cient in wound healing. The release of honey was responsive toward
changes in pH and temperature. The proposed system revealed bio-
compatibility and more than 99% antibacterial activity.29 Boruah
et al.30 developed a hydrogel based on carboxymethylcellulose and
AAc with incorporated organically modified MMT clay to demon-
strate the in vitro blood compatibility of the nanocomposite hydro-
gels. These hydrogels showed increased mechanical strength with
elevated crosslinker content followed by pH-responsive drug deliv-
ery up to 10 h with promising applications in the biomedical field.
Wang et al.31 fabricated a hydrogel based on 2-hydroxypropyltri-
methyl ammonium chloride chitosan to study the release behavior
of ofloxacin. The incorporated halloysite nanotubes inside the
hydrogel matrix affected the surface roughness remarkably,
accelerating the release of ofloxacin and then improving the bio-
availability of the drug. Ozay32 developed a hydrogel based on poly
(2-hydroxylethyl methacrylate-co-N-allyl succinamic acid) to study
the in vitro release of vitamin B12 and rhodamine 6G as model
drugs in three different media; this resulted in a 93.7
4.4% drug-
release ratio. Maheswari et al.33 synthesized a thermoresponsive
hydrogel based on poly(N-isopropylacrylamideco-N-vinyl-2-pyrroli-
dinone) to demonstrate 91% and 85% releases of a methylene blue
dye solution and vitamin B12, respectively. The proposed non-
Fickian diffusion and relaxation-controlled diffusion of the release
mechanism established the hydrogel as an appropriate candidate for
delivery systems in therapeutics. Fan et al.34 prepared nanocompo-
site hydrogel beads to study the release behavior of diclofenac
sodium with a release rate of 9.19 mg g−1 h−1. The incorporation of
appropriate amounts of hydroxyapatite and halloysite nanotubes
inside the beads increased the entrapment efficiency from 62.85

0.29% to 74.63
1.65%, and the burst release of diclofenac sodium
was overcome. Shi and Wang35 synthesized a superporous hydro-
xyethyl cellulose based hydrogel with superabsorbent properties
along with smart pH- and salt-responsive behaviors. In distilled
water and 0.9 wt % NaCl solution, the hydrogel showed the highest
equilibrium swelling ratio with a rapid swelling rate.
Sharma et al.36 synthesized a biodegradable superabsorbent gum
ghatti-co-poly(AAc–aniline) hydrogel composite for colon-
specific controlled drug delivery. The hydrogel showed efficient
controlled release behavior in alkaline medium for the controlled
release of the drug. However, the swelling percentage value for
the hydrogel was 1294%. Hosseinzadeh37 studied controlled
release of diclofenac sodium from pH-sensitive carrageenan-g-
poly(AAc) superabsorbent hydrogels. These hydrogels also
showed a swelling percentage around 10,050%.37 No work has
been reported so far on the fabrication of a nanocomposite
hydrogel formed by the grafting of AAc and AAm on Ge in the
47596 (2 of 11)
presence of MMT having a higher swelling percentage to study
release of vitamin B12. Natural polymers such as Ge are antici-
pated to endow lower toxicities to synthesized hydrogels and to
facilitate higher swelling in the presence of grafted monomers
along with the clay and crosslinker.
In this study, we proposed the development of superabsorbent
hydrogels based on Ge, AAc, AAm, and MMT clay as a filler for
studying the controlled release of vitamin B12 and the hydrogels’
biodegradability, biocompatibility, and cytotoxicity. The swelling
behavior of the hydrogels was studied with Design-Expert soft-
ware to evaluate the highest value of swelling with the changes in
the concentrations of the filler, crosslinker, and initiator. In this
study, we investigated the plausible application of the synthesized
hydrogels as carriers for vitamin B12 in artificial gastric fluid
(AGF) and artificial intestinal fluid (AIF).
EXPERIMENTAL
Materials
AAc monomer was received from Sigma-Aldrich and was dis-
tilled under reduced pressure before it was used for reaction pur-
poses. AAm was obtained from Fluka and was used as
purchased. Ge (from porcine skin), obtained from Himedia, was
used as purchased. Ammonium peroxydisulfate (APS; analytical
grade) and MMT (aluminum pillared clay) were obtained from
Aldrich and were used as received. Citric acid monohydrate
(CA) was obtained from Rankem (analytical-reagent grade). Vita-
min B12 (C63H88CoN14O14P) was supplied by Merck (Mumbai).
A volume of 200 mL of AGF with a pH value 1.2 was prepared
with 1.5 mL of hydrochloric acid and 0.74 g of potassium chlo-
ride. To prepare the AIF, desired amounts of potassium dihydro-
gen phosphate and sodium dihydrogen phosphate were dissolved
in deionized water with volume adjustment up to 250 mL to
obtain a solution with a pH of 7.4.
Synthesis of the Superabsorbent Hydrogel Nanocomposites
An amount of 2 g of Ge was placed in 10 mL of hot distilled
water and stirred for 45 min at 800 rpm to obtain a clear solu-
tion. A certain amount of MMT clay was placed in distilled water
and ultrasonicated for 60 min for uniform dispersion. Ge solu-
tion was placed in two-necked, round-bottom flask and stirred
under an N2 atmosphere at 60  C for 15 min. The initiator APS
at different percentages of total monomer weight was added and
stirred for 15 min. Partially neutralized AAc and AAm, CA cross-
linker, and MMT clay (at different percentages of total monomer
weight) were added all together in the solution and stirred for
another 15 min. The solution became a little viscous and was
transferred to a petri dish for postpolymerization around 60  C
for 10 h. The acquired hydrogel was divided into sizes of equal
dimensions (2–3 cm) by cutting with a knife. We extracted resid-
ual monomers from the pieces by keeping them dipped in DI
water, and the water was repeatedly refilled every 6 h for 72 h.
Thus, the final product was dried at room temperature and stud-
ied for further characterization.
Characterization of the Hydrogels
The sample for Fourier transform infrared (FTIR) spectroscopy
was prepared with KBr pellets, and the spectra were recorded
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with an Impact 410 instrument (Nicolet). In a 2θ range from
10 to 80 and with a scanning rate of 0.05 s−1, powder X-ray
diffraction (XRD) data were acquired on a Rigaku Miniflex X-ray
diffractometer (Tokyo, Japan) with Cu Kα radiation (λ = 0.15418
nm) at 30 kV and 15 mA. With scanning electron microscopy
(SEM; model JSM-6390LV, JEOL, Japan), the surface morphology
of the composite was illustrated. Platinum coating was done on
the sample surface before SEM analysis. With a JEOL JEM 1400
instrument (Japan) with an acceleration voltage of 120 kV, trans-
mission electron microscopy (TEM) images were recorded. Swell-
ing studies with various precursor concentrations were studied
with Design-Expert software (version 6.0.11, Stat-Ease). A biode-
gradability study was carried out with a spectrophotometer
(Thermo-Fisher Scientific). A Pseudomonas aeruginosa MTCC
2297 culture was grown with nutrient broth. The test hydrogels were
cut in the dimensions of 6 × 5 mm2. The hydrogels were inoculated
with 100 μL containing 0.1 mL of a 0.4 optical density (OD) bacte-
rial culture in 15 mL of mineral salt medium. The samples were
incubated at 37  C for 18 h and centrifuged at 140 rpm.
Blood Compatibility Study
The hemolytic activity assay was performed with a protocol
described by Das Purkayastha et al.38 with a slight modification.
In brief, in a centrifuge tube containing trisodium citrate (3.2%),
fresh goat blood was collected. At 4  C, it was centrifuged at
2500 rpm for 15 min. The red blood corpuscles (or erythrocytes)
obtained by discarding the supernatant were washed three times
with phosphate-buffered saline (PBS; pH 7.4). From a 10% v/v
erythrocyte suspension prepared in PBS, 1.9 mL of the suspen-
sion was placed in a 2 mL centrifuge tube; this was followed by
the addition of 1 mg of samples in the corresponding tubes. At
37  C, the tubes were incubated for 1 h. As positive and negative
controls, 1% Triton X-100 and PBS were taken, respectively. The
tubes containing the samples and suspension were again centri-
fuged after incubation at 2500 rpm for 15 min at 40  C. With a
UV–visible spectrophotometer, the absorbance of the supernatant
was taken at 540 nm.
Cytotoxicity Study
To evaluate the cytotoxicity of the sample, an MMT (MTT is
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay
that is utilized for studying cell toxicity) reduction colorimetric
assay was used on a CaCO2 cell line to evaluate the cytotoxicity of
the prepared samples. Dulbecco’s modified Eagle’s medium supple-
mented with 20% fetal bovine serum was used to culture all of the
cells. Primarily, 1 × 104 cells were developed in each well of a 96 well
plate at 37  C and 5% CO2 for 24 h. Then, for another 24 h, the
cells were treated with 1 mg of different samples. The MTT solution
(5 mg/mL in PBS) was added to it after treatment. The cells were
incubated at 37  C for 3 h in a cell culture incubator. Dimethyl
sulfoxide was used to dissolve the formazan crystals, and the
absorbance was recorded at 570 nm in a 96 well plate reader
(Thermo Scientific Multiskan GO). With the following formula,
the cell viability was calculated:
Viable cell ð%Þ = OD of drug− treated sample=
OD of the untreated sampleÞ × 100
47596 (3 of 11)
Loading of Vitamin B12
A soaking or equilibration method was implemented for vitamin
B12 loading. The first step of the method followed the determina-
tion of the amount of buffer necessary for the equilibrium swell-
ing of the nanocomposite hydrogel. A vitamin B12 solution with
a concentration of 1.25 wt % was prepared at pH 7.0, and dried
hydrogel samples were immersed in it to load the vitamin. The
loaded hydrogel samples were dried at 40  C in an oven for 15 h
after they soaked up the vitamin solution completely.
In Vitro Drug-Release Studies
Buffer solutions (30 mL, pH ≈ 1.2 and 7.4) were used to immerse
the vitamin B12 loaded hydrogels. To calculate the cumulative
release of vitamin B12, a 5 mL aliquot was taken out after every
1 h and spectrophotometrically assessed at 362 nm with a UV–
visible spectrophotometer (UV-2001, Hitachi, Japan). A volume
of 5 mL of similar buffer solution was added to maintain a con-
stant volume. A previously calibrated standard curve was used to
establish the amount of vitamin B12 released from the nanocom-
posite hydrogel.39
RESULTS AND DISCUSSION
Mechanism of Hydrogel Formation
AAc and AAm were grafted onto the Ge backbone, and grafting
was carried out in the presence of the CA crosslinker and MMT
filler. The APS free-radical initiator decomposed on heating and
generated sulfate anionic radicals, which extracted hydrogen from
the functional groups present in the side chains of the Ge backbone.
This gave rise to a persulfate–saccharide redox system and resulted
in the formation of active centers. These centers then started initiat-
ing the polymerization process radically. The end tricarboxylic
groups of the CA crosslinking agent may have reacted with polymer
chains during the chain propagation. Thus, after completion of the
three steps, the copolymer was obtained with a crosslinked structure.
With the incorporation of the filler, the composite structure of the
hydrogel was fabricated (Scheme 1).
FTIR Analysis
The fabrication of the hydrogel samples was investigated with the
help of the FTIR spectra of the precursors, as illustrated in
Figure 1. The number of scans was four, and the spectra wave
number was in the range 4000–400 cm−1. The strong absorption
peaks at 3446 and 2924 cm−1 for Ge were attributed to the pres-
ence of ─NH and ─CH2 groups, respectively. The presence of
─NH stretching was established by the peak at 1648 cm−1, and
the peak observed at 1458 cm−1 was due to sp3 C─H bending.
The strong absorption peaks at 3370 and 1675 cm−1 for AAm
were characteristic of the presence of NH2 and C O groups,
respectively. The peaks at 988 and 962 cm−1 confirmed the pres-
ence of alkene sp2 C─H bending. For CA, the strong absorption
peaks at 3421 and 1729 cm−1 arose because of the presence of
the OH and C O groups of carboxylic acid, respectively, whereas
the peak at 1418 cm−1 was attributed to sp3 C─H bending. The
FTIR spectrum of the copolymer hydrogel demonstrated a peak
at 3436 cm−1, which was due to ─NH stretching vibrations, and
the one at 2925 cm−1 was attributed to the presence of ─CH2
groups. The peak at 1633 cm−1 was attributed to C O bending
(amide I). There were differences in the peak intensities of the
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Scheme 1. Formation of the polymeric nanocomposite hydrogel. [Color figure can be viewed at wileyonlinelibrary.com]

copolymer hydrogel. The FTIR peaks of the nanocomposite XRD Analysis

hydrogel revealed the transformation of the peak intensities and
locations, which signified the successful grafting of AAc and
AAm in the Ge crosslinked by CA. The peak at 3641 cm−1 disap-
peared in the nanocomposite hydrogel, and prominent peaks
were observed at 878 and 461 cm−1; these signified the presence
of clay inside the gel matrix.
The XRD patterns of the MMT, nanocomposite hydrogel, and
copolymer hydrogel are shown in Figure 2. MMT showed a sharp
peak at 2θ around 21.87 ; this was the diffraction peak for the
(001) plane. The sharp peaks in MMT indicated the crystalline
diffraction peaks. In the case of the superabsorbent hydrogel
nanocomposite, the peak was shifted to 2θ = 23.08 . The

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Figure 1. FTIR spectra of the (a) reactants and (b,a) nanocomposite and (b,b) copolymer hydrogels. [Color figure can be viewed at wileyonlinelibrary.com]

intensities of the peaks of the nanocomposite hydrogels were
reduced in comparison to those of the pristine nanoclay; this sig-
nified the intercalation of MMT between the hydrogel network.
The presence of low-intensity peaks indicated an increase in the
crystallinity in the case of the superabsorbent hydrogel in the
presence of clay, whereas no such peak was observed in case of
the copolymer hydrogel.
SEM
SEM was performed to investigate the morphology of the hydro-
gel samples. The SEM micrographs of the MMT, copolymer, and
nanocomposite hydrogels are shown in Figure 3. It is evident that
the micrographs show a smooth surface for the copolymer hydro-
gel. The presence of clay inside the nanocomposite hydrogel was
confirmed in the micrograph from the rough whitish surfaces
accompanied by some pores and gaps. Thus, the change in the
exterior morphology established the incorporation of MMT in
the hydrogel matrix, which thereby improved its overall surface
structure. Figure 3(c) verifies that the synthesized hydrogels had
a porous structure. The pores facilitated the adherence of the
drug to the interpenetrating network of hydrogel. The porous
structures were interconnected and could also act as water chan-
nels. Hence, large amounts of water were taken up by the hydro-
gel; this may have led to an increased swelling ratio.40 It was
supposed that the pores were areas for the permeation of water
and could also act as interaction sites between the external stim-
uli and hydrophilic groups of superabsorbent hydrogels. The
existence of holes between the fine particles permitted the easy
absorbance of water by the hydrogel because it could possess a
high specific surface area. In particular, for large particle sizes of
superabsorbent, the characteristic surface morphology was signif-
icant enough to absorb water.
TEM
TEM analysis was performed to demonstrate the presence of
MMT clay inside the hydrogel matrix. The sizes of the clay plate-
lets were also calculated from the TEM image shown in Figure 4
(a). The average length and width of the clay platelets were found
to be 184.37 and 20.48 nm, respectively. The unveiled gray portion
in the TEM image of the hydrogel in Figure 4(b) indicates the
polymer matrix. Clay layers incorporated into small monomer
molecules underwent polymerization during the polymerization
reaction to give rise to high-molecular-weight polymers. A clay
gallery can be exfoliated to a better distribution of clay by the
formed polymer molecules if the polymers exert an elastic force
higher than the external force provided by the clay layer.41 The
darker parts in Figure 4(b) were attributed to the layers of MMT
clay platelets, which were dispersed within the polymer matrix.

Figure 2. XRD patterns of the (a) MMT and the (b,a) copolymer and (b) nanocomposite hydrogels. [Color figure can be viewed at wileyonlinelibrary.com]

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Figure 3. SEM morphologies of the (a) MMT and (b) copolymer and (c) nanocomposite hydrogels.

Experimental Design
Central composite (quadratic) design was used in this study, and
three factors were evaluated, each at three levels; experimental tri-
als were performed on all 20 possible combinations to investigate
the influence of the parameters, including the initiator, clay con-
tent, and crosslinker on the swelling of the hydrogels. The
amount of initiator, clay, and crosslinker were varied from 2 to
10, 0.1 to 1.5, and 1 to 4%, respectively. The ratio of AAc to
AAm was 3:1 (Table I).
The resulting data were fitted into Design-Expert software (version
6.0.11 Stat-Ease), and the developed models were adopted for
the multiple correlation coefficients (R2s), the adjusted multiple
correlation coefficients (adjusted R2), and corresponding p values
provided by analysis of variance. Greater values of R2 and adjusted
R2 are preferable, and it is considered significant when the corre-
sponding p value is less than 0.05. The data were also subjected to
3D response surface methodology to determine the influence of
each on the swelling of the hydrogels. The effectiveness of the

Figure 4. TEM micrographs of the (a) MMT clay and (b) nanocomposite hydrogel.

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Table I. Optimized Values of the Factors A, B and C

Factor Name Unit Type Low actual High actual Low coded High coded
A Initiator % Numeric 3.62 8.38 −1 1
B Clay % Numeric 0.38 1.22 −1 1
C Crosslinker % Numeric 1.61 3.39 −1 1

model was evaluated on the basis of the calculated coefficient of The final equation in terms of the coded factors is as follows:
determination (R2) by the program.
Swelling = 17,813:81− 6766:62A −2154:10B + 3396:75C + 531:43A2
Optimization experiments were conducted with the set of values
shown in Table II. + 611:64B2 + 1002:32C 2 + 1138:20AB
−1106:95AC − 1161:45BC
For Response: Swelling.. An analysis of variance was used for
the response surface quadratic model analysis of variance table The final equation in terms of the actual factors was
(partial sum of squares), as shown in Table III.
Swelling = 34,453:81502− 3587:60141 × Initiator − 9901:18483
The model F value of 55.34 implied that the model was significant. × Clay + 3142:3326 × Crosslinker + 93:94496
Values of p > F of less than 0.0500 indicated that the model terms
× ðInitiatorÞ2 + 3530:56998 × ðClayÞ2
were significant. In the optimization experiment, the A (initiator
percentage), B (clay percentage), C (crosslinker percentage), C2, + 1259:99080 × ðCrosslinkerÞ2 + 1149:75941
AB, AC, and BC factors (AB denotes the factor for the combined × Initiator × Clay − 521:82300 × Initiator
influence of initiator and clay on the swelling of hydrogel, BC × Crosslinker −3128:65456 × Clay × Crosslinker
denotes the factor for the combined influence of clay and crosslin-
ker on the swelling of hydrogel, and AC denotes the factor for the The final results are plotted in Figures 5–7.
combined influence of initiator and crosslinker on the swelling of From the 3D surface plots, it was evident that with increasing
hydrogel) were significant model terms and had a direct influence crosslinker, the swelling properties of the hydrogels increased.
on swelling. However, with increasing amount of clay and initiator, there was
a decrease in the swelling value. Being a tricarboxylic acid, the
Table II. Optimization Experiments with Various Precursor Contents CA crosslinker was capable of introducing high amounts of
hydrophilic carboxylic acid groups during the formation of the
Equilibrium hydrogel samples. These groups were responsible for the forma-
Sample A B C swelling (%) tion of the polyelectrolyte network and the increase in the water
1 Block 1 6 1.5 2.5 15,796
sorption by trapping the water molecules within the crosslinking
network; this thereby resulted in enhanced swelling.
2 Block 1 3.62 1.22 3.39 26,486
3 Block 1 6 0.8 2.5 18,452
4 Block 1 8.38 0.38 1.61 12,113 Biodegradation Study
Biodegradation studies were carried out for the Ge-g-(AAc–
5 Block 1 6 0.8 2.5 18,415
AAm)–MMT hydrogels to investigate their role in biodegradation.
6 Block 1 6 0.1 2.5 21,784
Nanocomposite hydrogels and copolymer hydrogels were exposed
7 Block 1 3.62 0.38 3.39 37,563.6 to P. aeruginosa bacteria in a nutrient broth culture medium for
8 Block 1 6 0.8 2.5 16,945.6 the biodegradation study, as shown in Table IV. The incubation
9 Block 1 10 0.8 2.5 7714.28 was performed for the samples at 37  C for 18 h; this was followed
10 Block 1 8.38 1.22 1.61 10,234 by centrifugation at 140 rpm for the proper circulation of O2. The
11 Block 1 6 0.8 1 14,275 hydrogel samples functioned as catabolite for the bacteria. After
12 Block 1 3.62 0.38 1.61 24,514
10 days of incubation of the samples in broth culture medium, the
growth of bacteria, and the biodegradation rate were clearly detect-
13 Block 1 3.62 1.22 1.61 21,365
able. The bacterial growth of the samples with respect to time is
14 Block 1 8.38 1.22 3.39 14,210
shown in Figure 8. The higher rate of bacterial growth in hydrogels
15 Block 1 6 0.8 4 25,515 as compared to the control samples was due to the presence of car-
16 Block 1 6 0.8 2.5 18,023 bon in all of the monomers along with the amide and OH groups
17 Block 1 8.38 0.38 3.39 17,452 present in the Ge backbone. In the enhancement of bacterial
18 Block 1 2 0.8 2.5 29,412 growth, the cellulolytic and pectinolytic activity of the bacteria
19 Block 1 6 0.8 2.5 16,429.4 may have played an important role. In the presence of individually
biodegradable biobased components such as CA, Ge enhanced the
20 Block 1 6 0.8 2.5 18,876.5
biodegradability of the gel samples. With the increase in the con-
See Table I for the factor definitions. tent of such components, there was an increase in the hydrolysable

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Table III. Response Surface Quadratic Model Analysis

Source Sum of squares DF Mean square F p>F

Model 8.98 × 108 9 99,742,881 55.34 <0.0001 Significant
8 8
A 6.25 × 10 1 6.25 × 10 346.9 <0.0001
B 63,369,704 1 63,369,704 35.16 0.0001
C 1.58 × 108 1 1.58 × 108 87.42 <0.0001
2
A 4,070,050 1 4,070,050 2.258 0.1638
B2 5,391,315 1 5,391,315 2.991 0.1144
C2 14,478,137 1 14,478,137 8.032 0.0177
AB 10,364,062 1 10,364,062 5.75 0.0375
AC 9,802,773 1 9,802,773 5.438 0.0419
BC 10,791,799 1 10,791,799 5.987 0.0344
Residual 18,024,913 10 1,802,491
Lack of fit 13,423,896 5 2,684,779 2.918 0.1324 Not significant
Pure error 4,601,017 5 920,203.4
Cor total 9.16 × 108 19

See Table I for the factor definitions. Cor is the quantified variability around the mean of observations.

linkages, and this led to an increased rate of biodegradation.

Therefore, the increase in the biodegradable segments increased
the weight loss because of biodegradation. The higher biodegrada-
tion rate in the nanocomposites was mainly due to the catalytic
role of the clay in hydrolysis of various groups present in the
monomer chains.42 After absorbing water in the presence of
microbes, the unreacted terminal hydroxyl groups of the clay
caused heterogeneous hydrolysis, which took a little bit time for
initiation. All of the samples were tested in duplicate, and the con-
trol samples were placed in the absence of the hydrogel. The code
GOP7wc denotes the copolymer hydrogel, and the code GOP7
denotes the nanocomposite hydrogel.

Blood Compatibility and Cell Toxicity Study

To design novel biomaterials such as hydrogels with superior
blood compatibility, constant efforts have been carried out by Figure 6. Response surface plot of the swelling of the superabsorbent nano-
various research groups. Biomedical applications, such as drug composite hydrogel (effects of the clay and crosslinker on the swelling per-
delivery and tissue engineering involve the use of humans or centage). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5. Response surface plot of the swelling of the superabsorbent nano- Figure 7. Response surface plot of the swelling of the superabsorbent nano-
composite hydrogel (effects of the clay and initiator on the swelling percent- composite hydrogel (effects of the crosslinker and initiator on the swelling
age). [Color figure can be viewed at wileyonlinelibrary.com] percentage). [Color figure can be viewed at wileyonlinelibrary.com]

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ARTICLE WILEYONLINELIBRARY.COM/APP

Table IV. Hydrogel Samples Kept in Bacteria P. aeruginosa in a Nutrient medium. The cell viability percentage value indicated a very low
Broth Culture Medium cytotoxicity of the nanocomposite hydrogel, which could
enhance its utilization in biomedical applications.43 Further
Sample Day 1 Day 10 detailed in vivo testing and studies with human cell lines are
Copolymer hydrogel (T1) 0.07 0.116
needed to establish this potential.
Copolymer hydrogel (T2) 0.062 0.103
Hydrogel (T1) 0.077 0.144
In Vitro Drug Release
Figure 10 demonstrates the pH-dependent in vitro release behav-
Hydrogel (T2) 0.086 0.108
ior of vitamin B12 from the nanocomposite hydrogels in an envi-
Control (T1) 0.092 0.045
ronment mimicking that of the inside of the stomach (AGF) and
Control (T2) 0.047 0.042 intestine (AIF). After the diffusion mechanism, the drug particles
T1 represents sample and T2 represents duplicate sample.
started to diffuse into the AGF and AIF from the loaded gel sam-
ples as soon as the samples were immersed in the solutions. The
release study was performed for 6 h in both AGF and AIF. The
other animals. Therefore, it is very important to study their bio-
hydrogel samples swelled readily in AIF; this resulted in the fast
compatibility with blood and cell toxicity. The in vitro blood
diffusion of vitamin B12 in the solution. For the hydrogel sam-
compatibility studies were carried out for the hydrogel samples
ples, the pH-dependent cumulative release (percentage) of vita-
with hemolysis and cytotoxicity tests, as described earlier. A low
min B12 was observed.
hemolysis activity was observed for the gel samples from the
hemolysis test shown in Figure 9(a). This indicated its efficiency In the prepared AGF having a pH value 1.2, there was a lower
for use as a drug-delivery system. To evaluate the cytotoxicity number of negative charges present; this led to the ionization of
activity of the nanocomposite hydrogels, as shown in Figure 9 carboxylic acid groups of the acrylate structure to a poorer extent.
(b), we observed that the absorbance of the medium cultured This hindered the rapid swelling of the hydrogel samples and
with nanocomposite hydrogel was nearer to that of the control confirmed inadequate swelling behavior. An increase in the pH

Figure 8. Growth of bacilli (OD = 600 nm) with time (days). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 9. (a) Hemolysis percentage and (b) cytotoxicity study of the hydrogel samples. [Color figure can be viewed at wileyonlinelibrary.com]

47596 (9 of 11) J. APPL. POLYM. SCI. 2019, DOI: 10.1002/APP.47596

ARTICLE WILEYONLINELIBRARY.COM/APP
Figure 10. Cumulative percentage release of vitamin B12 at pH (a) 1.2 (AGF) and (b) 7.4 (AIF).
value aided the carboxylic acid groups to get more ionized, and
this led to significant electrostatic repulsion between the COO−
groups. This repulsion enhanced the expansion within the net-
works of the hydrogel and improved the swelling.44 Swelling reg-
ulated the controlled release of vitamin B12 in the release study
and emphasized increasing swelling behavior with elevated pH
value. Therefore, this led to enhanced vitamin B12 release from
the gel samples. This phenomenon signified the cause behind the
increased drug release of the clay-incorporated hydrogel in
increased pH media. The release behavior in both AIF and AGF
increased with time for the nanocomposite hydrogels. At pH 7.4
(AIF), enhanced protonation took place for the carboxylic acid
groups; this led to around an 80% release of the drug in 6 h. On
the other hand, poorer ionization indicated the lesser drug-
release behavior, around 42% in 6 h, of the clay-incorporated
hydrogel in acidic media as compared to AIF.
CONCLUSIONS
In this study, we synthesized a series of nanocomposite hydrogels
based on acrylate-grafted Ge and AAc in the presence of MMT
clay via free-radical graft polymerization. The prepared nanocom-
posite hydrogels showed better and highly enhanced swellability in
distilled water. The value of the swelling percentage was found to
be around 37,563%; this was very much higher than that of the
fabricated nanocomposite hydrogel. In vitro vitamin B12 delivery
was carried out with the prepared hydrogels. In the medium mim-
icking the small intestine environment, vitamin B12 release from
the hydrogels was selectively enhanced. The vitamin B12 release
behavior exhibited a significant dependence on the pH of the
medium. The release behavior was found to be greater in the basic
medium (pH 7.4) than in the acidic medium (pH 1.2). Thus, the
clay-incorporated nanocomposite hydrogel could be considered a
potential candidate for the development of pH-responsive drug-
delivery systems with higher values of swelling.
ACKNOWLEDGMENTS
The authors sincerely acknowledge the University Grants Com-
mission for providing a National Fellowship for Other Backward
Classes (contract grant number F./2017-18/NFO-2017-18-OBC-
ASS-50336). The authors thank the Sophisticated Analytical
47596 (10 of 11)
Instrumentation Centre of Tezpur University and Manabendra
Mandal, Pritam Bardhan, and Kuldeep Gupta of the Department
of Molecular Biology and Biotechnology of Tezpur University for
their analytical support. The authors acknowledge Jitendra
Kumar and Vinod Khanna of the Transmission Electron Micros-
copy Facility of the Central Instruments Facility of the Indian
Institute of Technology Delhi, Naminita Gogoi Lahon of Bio-
Scope Catalyst Atomic Force Microscopy (BioAFM) of the Cen-
tral Instruments Facility of the Indian Institute of Technology
Delhi, and S. Wazed Ali of the Department of Textile Technology
of the Indian Institute of Technology Delhi.
REFERENCES
1. Nath, J.; Dolui, S. K. Appl. Clay Sci. 2018, 155, 65.
2. Ahmed, E. M. J. Adv. Res. 2015, 6, 105.
3. Liu, J.; Su, D.; Yao, J.; Huang, Y.; Shao, Z.; Chen, X.
J. Mater. Chem. A. 2017, 5, 4163.
4. Sabadini, R. C.; Silva, M. M.; Pawlicka, A.; Kanicki, J. Appl.
Polym. Sci. 2018, 135, 45636.
5. White, C. J.; McBride, M. K.; Pate, K. M.; Tieppo, A.;
Byrne, M. E. Biomaterials. 2011, 32, 5698.
6. Zhang, M.; Song, L.; Jiang, H.; Li, S.; Shao, Y.; Yanga, J.;
Li, J. J. Mater. Chem. A. 2017, 5, 3434.
7. Pourjavadi, A.; Mahdavinia, G. R.; Zohuriaan Mehr, M. J.
J. Appl. Polym. Sci. 2003, 90, 3115.
8. Dai, H.; Huang, H. J. Agric. Food Chem. 2017, 65, 565.
9. Pourjavadi, A.; Bardajee, G. R.; Soleyman, R. J. Appl.
Polym. Sci. 2009, 112, 2625.
10. Karimi, A. R.; Rostaminezhad, B.; Khodadadi, A. J. Appl.
Polym. Sci. 2018, 135, 46167.
11. Wang, Q.; Zhang, J.; Wang, A. Carbohydr. Polym. 2009,
78, 731.
12. Qiao, D.; Liu, H.; Yu, L.; Bao, X.; Simon, G. P.;
Petinakis, E.; Chen, L. Carbohydr. Polym. 2016, 147, 146.
13. Xing, R.; Liu, K.; Jiao, T.; Zhang, N.; Ma, K.; Zhang, R.;
Zou, Q.; Ma, G.; Yan, X. Adv. Mater. 2016, 28, 3669.
14. Peng, N.; Hu, D.; Zeng, J.; Li, Y.; Liang, L.; Chang, C. ACS
Sustain. Chem. Eng. 2016, 4, 7217.
J. APPL. POLYM. SCI. 2019, DOI: 10.1002/APP.47596
ARTICLE WILEYONLINELIBRARY.COM/APP
15. Ma, G.; Ran, F.; Yang, Q.; Feng, E.; Lei, Z. RSC Adv. 2015,
66, 53819.
16. Wang, Q.; Xie, X.; Zhang, X.; Zhang, J.; Wang, A. Int.
J. Biol. Macromol. 2010, 46, 356.
17. Wang, W.; Wang, A. J. Compos. Mater. 2009, 43, 2805.
18. Sudarsan, S.; Franklin, D. S.; Sakthivel, M.; Guhanathan, S.
Carbohydr. Polym. 2016, 148, 206.
19. Li, T.; Shen, J.; Zhang, Z.; Wang, S.; Wei, D. RSC Adv.
2016, 6, 40656.
20. Sun, T.; Zhu, C.; Xu, J. Soft Matter. 2018, 14, 921.
21. Shi, Y.; Ma, C.; Peng, L.; Yu, G. Adv. Funct. Mater. 2015, 25,
1219.
22. Navaei, A.; Saini, H.; Christenson, W.; Sullivan, R. T.;
Ros, R.; Nikkhah, M. Acta Biomater. 2016, 41, 133.
23. Cheng, Y.-H.; Hung, K.-H.; Tsai, T.-H.; Lee, C.-J.; Ku, R.-
Y.; Chiu, A. W.; Chiou, S.-H.; Liu, C. J. Acta Biomater.
2014, 10, 4360.
24. Truong, V. X.; Tsang, K. M.; Simon, G. P.; Boyd, R. L.;
Evans, R. A.; Thissen, H.; Forsythe, J. S. Biomacromolecules.
2015, 16, 2246.
25. Li, L.; Lu, C.; Wang, L.; Chen, M.; White, J.; Hao, X.;
McLean, K. M.; Chen, H.; Hughes, T. C. ACS Appl. Mater.
Interfaces. 2018, 10, 13283.
26. Garcia-Hernandez, A.; Lobato-Calleros, C.; Vernon-
Carter, E. J.; Sosa-Hernandez, E.; Alvarez-Ramirez, J.
J. Appl. Polym. Sci. 2017, 134, 44517.
27. Kevadiya, B. D.; Rajkumar, S.; Bajaj, H. C.;
Chettiar, S. S.; Gosai, K.; Brahmbhatt, H.; Bhatt, A. S.;
Barvaliya, Y. K.; Dave, G. S.; Kothari, R. K. Colloids Surf.
B. 2014, 122, 175.
47596 (11 of 11)
28. Jesus, C. R. N.; Molina, E. F.; Pulcinelli, S. H.; Santilli, C. V.
ACS Appl. Mater. Interfaces. 2018, 10, 19059.
29. Noori, S.; Kokabi, M.; Hassan, Z. M. J. Appl. Polym. Sci.
2018, 135, 46311.
30. Boruah, M.; Gogoi, P.; Manhar, A. K.; Khannam, M.;
Mandal, M.; Dolui, S. K. RSC Adv. 2014, 4, 43865.
31. Wang, Q.; Zhang, J.; Mu, B.; Fan, L.; Wang, A. Carbohydr.
Polym. 2014, 102, 877.
32. Ozay, O. J. Appl. Polym. Sci. 2013, 131, 39660.
33. Maheswari, B.; Babu, P. E. J.; Agarwal, J. Biomater. Sci.
Polym. Ed. 2014, 25, 269.
34. Fan, L.; Zhang, J.; Wang, A. J. Mater. Chem. B. 2013, 1, 6261.
35. Shi, X.; Wang, A. J. Appl. Polym. Sci. 2015, 132, 42027.
36. Sharma, K.; Kumar, V.; Chaudhary, B.; Kaith, B. S.;
Kalia, S.; Swart, H. C. Polym. Degrad. Stab. 2016, 124, 101.
37. Hosseinzadeh, H. J. Chem. Sci. 2010, 122, 651.
38. Das Purkayastha, M.; Das, S.; Manhar, A. K.; Deka, D.;
Mandal, M.; Mahanta, C. L. J. Agric. Food Chem. 2013, 61,
10746.
39. Hu, Z.; Chen, G. Adv. Mater. 2014, 26, 5950.
40. Marandi, G. B.; Hosseinzadeh, H. Polym. Polym. Compos.
2007, 15, 395.
41. Banerjee, S. L.; Khamrai, M.; Kundu, P. P.; Singha, N. K.
RSC Adv. 2016, 6, 81654.
42. Pandey, J. K.; Reddy, K. R.; Kumar, A. P.; Singh, R. P.
Polym. Degrad. Stab. 2005, 88, 234.
43. Sirousazar, M.; Kokabi, M.; Hassan, Z. M. J. Biomater. Sci.
Polym. Ed. 2011, 22, 1023.
44. Wang, L.; Zhang, J.; Wang, A. Colloids Surf. A. 2008, 322, 47.
J. APPL. POLYM. SCI. 2019, DOI: 10.1002/APP.47596