Applied Clay Science 190 (2020) 105569

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Applied Clay Science

journal homepage: www.elsevier.com/locate/clay

Research Paper

Acrylic acid grafted gelatin/LDH based biocompatible hydrogel with pH- T

controllable release of vitamin B12
⁎
Jayashree Nath, Asfi Ahmed, Priyankamoni Saikia, Anupam Chowdhury, Swapan K. Dolui
Department of Chemical Sciences, Tezpur University, Napaam, Tezpur 784028, Assam, India

A R T I C LE I N FO A B S T R A C T

Keywords: In this work, gelatin-g-poly(acrylic acid) based pH-sensitive nanocomposite hydrogels incorporated with Layered
Hydrogel Double Hydroxide (LDH) were developed. Using various spectroscopic techniques such as FTIR, XRD, SEM, EDX
LDH and TEM, the polymeric hydrogels were characterized. FTIR confirmed grafting of partially neutralized Acrylic
Swelling Acid (AAc) on the backbone of Gelatin. Incorporation of LDH fillers inside the hydrogel matrix was established
pH-responsivity
from XRD data. Inclusion of LDH resulted wrinkled morphology of the surface of the hydrogel from the SEM
Drug release
images. Formation of nanocomposites were further evaluated by TEM analysis which displayed significant
change in the TEM morphology of the nanocomposite hydrogel. Using DLS, particle size for LDH were de-
termined further and was found to be around 100 nm. Swelling value of the hydrogel was around 34 g/g.
Biocompatibility results showed enhanced blood compatibility for the hydrogels. The controlled release studies
performed using the hydrogels demonstrated vitamin B12 was released in Artificial Gastric Fluid (AGF) and in
Artificial Intestinal Fluid (AIF) in a controlled manner. The release study was carried out for 7 h each and was
evaluated using UV–visible spectrophotometer. From the curves, the cumulative release in AGF and AIF was
found to be 24% and 31%, respectively.

1. Introduction drawbacks of synthetic polymers in the preparation of hydrogel, poly-

Polymers are able to crosslink to give rise to a three-dimensional
network structure, better known as hydrogel, which can retain a sig-
nificant amount of water within its network structure without getting
dissolved in it (Chen et al., 2018). While holding the water inside its
matrix, hydrogel does not lose its three-dimensional structure. The
properties of hydrogel make it suitable for employing as ‘intelligent’
material that can respond towards the environmental changes such as
pH (Nath et al., 2019), electric field (Boruah et al., 2014), temperature
(Li et al., 2018), magnetic field (Manjua et al., 2019), light (Zhu et al.,
2012), salt concentration (Zhao et al., 2005) etc. Thus according to the
stimuli applied on the hydrogels, they demonstrate changes in their
properties followed by promising application in various fields of re-
search (Tokarev and Minko, 2009). Hydrogels have found wide range of
applications in biomedical field mainly in site-specific drug delivery
applications (Ma et al., 2017).
Gelatin is a natural polymer extracted from collagen present inside
the body parts of animals. It is biodegradable and protein-based
polymer bearing a large number of functional groups in its side chain.
This endows gelatin with exceptional gelling capacity for synthesizing
‘intelligent’ hydrogels (Helminger et al., 2014). Due to various
mers based on natural sources have drawn quite a huge attention in
hydrogel synthesis followed by its application in different fields of re-
search (Fathi et al., 2013). Bakravi et al. developed gelatin based bio-
degradable hydrogel in presence of copper oxide (CuO) nanoparticles
for delivery of Cephalexin (Bakravi et al., 2018). Drug release from the
nanocomposite hydrogels was studied in pH 7.4 and in pH 1.2, which
results in a lower value of release in pH 1.2 than that of the former one.
This was due to decrease in the swelling value of the hydrogels in
pH 1.2. Lai et al. fabricated gelatin methacrylate/carboxybetaine me-
thacrylate biomimetic hydrogels to study the release behavior of
Fluorescein isothiocyanate-dextran (Lai et al., 2016). These hydrogels
showed enhanced cell viability with controlled release of Vascular
Endothelial Growth Factor (VEGF) and thereby displayed induced an-
giogenesis. Song et al. developed a thermosensitive hydrogel based on
gelatin and chitosan for ocular drug delivery of Timolol Maleate with
significant therapeutic effect (Song et al., 2018). These hydrogels pos-
sessed lower cytotoxicity and effective and long-lasting efficacy for
lowering intraocular pressure (IOP) up to 24 h than that of the con-
ventional eye drops. Hydrogel systems based on gelatin and polyvinyl
alcohol (PVA) was developed by Oun et al. to study the release of an-
ticancer drug Cisplatin placed within the Curcurbituril macrocycles

⁎
Corresponding author.
E-mail address: skdolui1970@gmail.com (S.K. Dolui).

https://doi.org/10.1016/j.clay.2020.105569
Received 26 September 2019; Received in revised form 12 March 2020; Accepted 17 March 2020
0169-1317/ © 2020 Elsevier B.V. All rights reserved.
J. Nath, et al.
(Oun et al., 2014). The variation in disintegration, swelling and release
properties were calculated based on the varying concentration of PVA
contents. For inhibition of tumour growth, in vivo study of the hydrogels
under skin of nude mice demonstrated that lower dosage of Cisplatin
(30 μg equivalent of drug) containing hydrogel was effective as com-
pared to free Cisplatin (150 μg). Raafat et al. prepared gelatin and ac-
rylic acid based pH-responsive biodegradable hydrogel that was cross-
linked in presence of gamma radiation (Raafat, 2010). The hydrogel
samples were utilized for evaluating in vitro release of Ketoprofen
(model drug). Both the composition of the hydrogel and pH of the
media effect the release profile of the drug.
Recently, usage of fillers inside the hydrogels has come up to be an
emerging area of study (Utech and Boccaccini, 2016). Among them,
clay (Peng et al., 2016), layered double hydroxide (LDH) (Nath and
Dolui, 2018), graphene oxide (GO) (Nath et al., 2018), multiwalled
carbon nanotube (MWCNT) (Liu et al., 2010), metal nanoparticles
(Merino et al., 2015) etc. play a vital role in enhancing the properties of
the hydrogels. Brucite-like [M(OH)2] sheets possessing LDH are versa-
tile and flexible in composition. These are termed as anionic clays and
the anion-exchange property with biocompatibility of LDH encourages
the researchers for developing nanocomposite hydrogels with improved
properties (Xiang et al., 2009). Incorporation of LDH inside numerous
natural polymers in fabricating hydrogels has been studied in a vast
dimension very recently. Green hydrogels synthesized based on ise-
thionate intercalated LDH nanosheets by Hu et al. showed enhanced
stability towards flowing shear forces, heating, acids/alkalis (Hu and
Chen, 2014). Alcantara et al. prepared LDH-biopolymer nanocompo-
sites based on LDH and two biopolymers (a protein and a poly-
saccharide). The system proved to be an effective drug delivery system
(DDS) for releasing Ibuprofen (Alcantara et al., 2010). Li et al. de-
scribed the synthesis of a hydrogel-based on biocompatible poly-
urethane and PVA incorporating Mg-Al-LDH inside the hydrogel matrix
(Li et al., 2019). These hydrogels were utilized for double carrier wound
dressing and drug delivery system by intercalating quinolone anti-
biotics Enoxacin (ENO) inside LDH by ion-exchange method. They
showed enhanced mechanical property as well as wound healing
ability. Zhang et al. synthesized thermoresponsive hydrogels based on
LDH and N-isopropylacrylamide having excellent mechanical proper-
ties and swelling/deswelling behavior (Zhang et al., 2018). These hy-
drogels were expected to have potential application in the field of drug
delivery vehicle, tissue engineering etc. Barkhordari et al. developed
pH-responsive hydrogel beads with LDH and carboxymethyl cellulose
(CMC) to study the release behavior of Ibuprofen by crosslinking with
Fe3+ cations (Barkhordari et al., 2014). Their release behavior was
studied in the gastrointestinal tract to establish the effectiveness of such
hydrogels as drug delivery system in the form of beads.
Several studies have already been carried out to study the release
behavior of vitamin B12 using stimuli-responsive hydrogels (Gong et al.,
2009; Yun and Kim, 2012; Nizam El-Din and El-Naggar, 2014;
Maheswari et al., 2014; Ozay, 2014). Following the above considera-
tions and performing the literature survey have inspired us to design
hydrogel system containing gelatin and acrylic acid (AAc) as monomers
incorporating LDH as filler inside the matrix for studying the release
behavior of a vitamin. In presence of grafted AAc, gelatin with non-
toxic characteristic is expected to increase the feasibility of developing
a pH-sensitive hydrogel in presence of non-toxic Mg-Al-LDH with en-
hanced value of swelling. Therefore in this study, we have demon-
strated the viability of incorporating LDH inside gelatin and AAc based
hydrogel to study the pH-controllable release of vitamin B12 in the
environment mimicking that of the stomach and the intestine. The
blood compatibility of the hydrogels were also evaluated.
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Applied Clay Science 190 (2020) 105569
2. Materials and methods
2.1. Materials
Gelatin, from procine skin, was acquired from Himedia and was
used as received. Acrylic Acid (AAc) monomer was obtained from
Sigma-Aldrich and was distilled under reduced pressure before use. Mg
(NO3)2·6H2O, Al(NO3)3·9H2O and NaOH were obtained from Merck.
Ammonium persulfate (APS, A.R. grade) and N, N/-methylenebis(ac-
rylamide) (MBA) were acquired from Sigma Aldrich (A.R. grade).
Vitamin B12 (C63H88CoN14O14P) was purchased from Merck, Mumbai.
To prepare 200 ml of Artificial Gastric Fluid (AGF) of pH = 1.2,
desired amounts of Hydrochloric Acid (HCl) and Potassium Chloride
(KCl) were dissolved in deionised water by adjusting the volume to the
required quantity. Certain amounts of Potassium Dihydrogen Phosphate
(KH2PO4) and Sodium Dihydrogen Phosphate (NaH2PO4) were dis-
solved in deionised water to prepare 250 ml of Artificial Intestinal Fluid
(AIF) adjusting the pH of the same to 7.4.
2.2. Preparation of Mg-Al-LDH
Following a co-precipitation method, Mg-Al-LDH was synthesized.
33.5 mmol of Mg(NO3)2·6H2O and 16.5 mmol of Al(NO3)3·9H2O were
dissolved in 50 ml of deionised water followed by addition of 50 ml of
2.0 M NaOH solution dropwise. During addition, constant stirring was
maintained under N2 atmosphere. The acquired slurry was kept for
aging at 95°C for 24 h and the pH was maintained between 9 and 10.
The precipitate was centrifuged and washed with double-distilled water
followed by drying the sample in vacuum oven at 50°C for 24 h.
2.3. Preparation of LDH cross-linked gelatin nanocomposite hydrogels
Free radical polymerization was utilized for fabricating gelatin-g-
PAAc/LDH hydrogel in distilled water. A certain amount of gelatin was
dissolved in 15 ml of double-distilled water. Then AAc was added to the
above solution and the overall solution was stirred for 15 min. 5 ml of
LDH dispersion was prepared by keeping it under ultrasonic vibration
for 2 h. The solutions were mixed well and afterward MBA was added
followed by APS to it under continuous stirring condition. For 15 min,
the overall solution was kept under Nitrogen purging to remove dis-
solved oxygen while maintaining the stirring for pre-polymerization at
70°C for 30 min. At 65°C, post-polymerization was carried out for 15 h.
The dried nanocomposite hydrogel thus acquired was cut into equal-
sized pieces (3–4 cm). Hydrogel pieces were then put in deionised water
to remove excess residual monomers and refilled with water after every
72 h followed by final drying of the same in an oven at a temperature
65°C. (See Tables 1-3.)
2.4. Characterization of the nanocomposite hydrogels
Using Impact 410 instrument (Nicolet, USA), Fourier Transform
Infrared (FTIR) spectra were collected. The spectra wave number was in
the range 4000–400 cm−1 and the number of scans was four. Powder X-
ray Diffraction (XRD) data were recorded using Rigaku Miniflex X-ray
diffractometer (Tokyo, Japan) with Cu Kα radiation having wavelength
(λ) value 0.15418 nm at 30 kV and 15 mA. 2θ value ranges between 7o-
Table 1
Recipe for the preparation of gelatin-g-PAAc/LDH nanocomposite hydrogel
with varying initiator (APS) amounts.
Gelatin (g) 2 2 2
AAc (ml) 1.5 1.5 1.5
APS (wt%) 1 3 5
MBA (wt%) 1 1 1
LDH (wt%) 5 5 5
J. Nath, et al. Applied Clay Science 190 (2020) 105569

Table 2 2.7. Loading of vitamin B12

Recipe for the preparation of gelatin-g-PAAc/LDH nanocomposite hydrogel
with varying crosslinker (MBA) amounts. Vitamin B12 was loaded in the hydrogel samples employing soaking
Gelatin (g) 2 2 2 or equilibrium method. The hydrogel samples were completely swelled
AAc (ml) 1.5 1.5 1.5 inside the buffer solution having pH = 7.0 and thus the exact amount of
MBA (wt%) 1 3 5 the buffer necessary for the same was determined. A solution at a
APS (wt%) 5 5 5
concentration of 1.25 wt% was prepared and dry hydrogel sample was
LDH (wt%) 5 5 5
placed inside the same till complete absorption of the solution by the
gel. Fully swollen drug-loaded hydrogel was then taken out afterward
Table 3 and was dried in a vacuum oven at 40°C.
Recipe for the preparation of gelatin-g-PAAc/LDH nanocomposite hydrogel
with varying LDH. 2.8. In vitro drug release studies

Gelatin (g) 2 2 2
AAc (ml) 1.5 1.5 1.5
LDH (wt%) 1 3 5 taken out from each solution and vitamin B12 loaded hydrogel samples
MBA (wt%) 1 1 1 were kept submerged in the solutions. From the solutions, 5 ml of the
APS (wt%) 5 5 5 aliquot was taken after every 1 h. To maintain a constant volume of the
80o and the scanning rate of the same was 0.05o s−1. Using Scanning
Electron Microscopy (SEM, Model-JSM-6390LV, JEOL, Japan), the
surface morphology of the hydrogel samples were studied. The sample
was sputter-coated with platinum before carrying out the SEM analysis.
Using SEM (JEOL-JSM-6390LV) coupled with energy dispersive X-ray
detector, Energy-Dispersive X-ray Spectroscopy (EDX) of the hydrogel
samples were carried out followed by the surface structure determina-
tion. With an acceleration voltage of 120 kV, Transmission Electron
Microscopy (JEOL JEM 1400, Japan) images were recorded. With
Nanotrac Wave-Particle Size analyzer, hydrodynamic volume was cal-
culated. Using Shimadzu TA-60 thermogravimetric analysis, thermal
analysis was performed. The platinum pan was loaded with a pre-
weighed amount of latex and heated till 600°C at a rate of 10°C/min
under N2 atmosphere.
2.5. Swelling of the hydrogels
20–25 mg dehydrated gel samples were immersed in distilled water
and buffer solutions to measure the swelling ratios. Gravimetric
methods were utilized for determining the swelling percentage of hy-
drogel. It was calculated using the following formula:
Ws − Wd
Swelling % = × 100
Wd (1)
where, Ws and Wd are the weights of the swollen and dry samples, re-
spectively. Eq. (1) gives the swelling percentage values of the hydrogels
both in distilled water, AGF and AIF which mimics the environment
similar to that of the gastrointestinal tract.
2.6. Blood compatibility study
Following the protocol described by Das Purkayastha et al., hemo-
lytic activity assay was performed with slight modification (Das et al.,
2013). Briefly, fresh goat blood was collected in a centrifuge tube
containing trisodium citrate (3.2%). It was then centrifuged for 15 min
at 2500 rpm at 4°C. Red blood corpuscles (or erythrocytes) were ob-
tained by discarding the supernatant and the same was washed with
phosphate-buffered saline (PBS; pH 7.4) for three times. In PBS, 10% v/
v erythrocyte suspension was prepared and 1.9 ml of the same was kept
in a centrifuge tube (2 ml). To the tubes containing suspension, 1 mg of
the samples were added in each. Incubation of the tubes was then
carried out for 1 h at 37°C. 1% Triton was taken as positive control and
PBS was taken as a negative control. After the incubation was done,
sample and suspension bearing tubes were centrifuged again at 40°C for
15 min at 2500 rpm. The absorbance of the supernatant was recorded
with UV–visible spectrophotometer at 540 nm.
In the prepared pH solutions (1.2 and 7.4), 30 ml of the same was
same, a solution with the same pH was added to the vessel. Previously
collected aliquot was spectrophotometrically assayed at 362 nm with
an UV–Visible spectrophotometer (UV-2001, Hitachi, Japan).
Cumulative release of vitamin B12 from the hydrogel samples were
measured from a standard curve calibrated previously (Hu and Chen,
2014).
3. Results and discussion
3.1. Mechanism of hydrogel formation
Graft polymerization was followed to prepare the desired gelatin
and AAc based hydrogel in presence of LDH and APS as free radical
initiator. Hydrogen was extracted from the functional groups found in
gelatin by sulphate anionic radical upon heating. Thus, persulfate-sac-
charide redox system was generated and the same gave rise to active
centres for initiating the polymerization process by the radicals. In
presence of MBA crosslinker, the end vinyl groups of the crosslinking
agent were expected to react with the polymer chains in the propaga-
tion of the chain. After completion of the final step of the reaction, the
end product obtained was a crosslinked structure of a copolymer hy-
drogel followed by composite structure of the hydrogel acquired by the
addition of LDH inside the copolymer matrix. Scheme 1 demonstrates
the plausible mechanism of formation of hydrogel.
3.2. FTIR spectra analysis
FTIR spectra of LDH and hydrogels were shown in Fig. 1 and
characteristic bands were found to corroborate with the reported data.
The broad band at 3444 cm−1 for the FTIR spectrum of LDH was due to
–OH stretching of hydroxyl groups and water molecules present inside
the space of LDH. Bending mode of the absorbed water for –OH group
could be confirmed from the absorption band at 1625 cm−1. In addition
to this, the spectrum for LDH showed band at 1378 cm−1 for the an-
ionic NO3 groups present inside the layers of LDH. The formation of the
same was further confirmed by the bands occurring in the region
400–800 cm−1 for M–O (M = Al and Mg) stretching within the LDH
layers.
For the copolymer hydrogel as shown in Fig. 1(b), the bands at
3436 cm−1 and 2930 cm−1 could be attributed to –NH stretching vi-
brations and presence of –CH2 groups, respectively. The band at around
1740 cm−1 was due to the C]O stretching vibration. The out-of-plane
vibration of –OH of the carboxylic groups of PAAc could be ascribed as
the reason for the same. The characteristic bands occurring at 1633 and
1452 cm−1 were due to the asymmetrical and symmetrical stretching of
COO– groups.
In Fig. 1(c), for the nanocomposite hydrogel, there was an increase
in the intensity of the band at 1647 cm−1 which was due to the
asymmetric stretching vibration band of the carboxylate groups in the

3
J. Nath, et al.
Scheme 1. Plausible mechanism of hydrogel formation.
nanocomposite hydrogel. This could be attributed to the formation of
an anionic bond between the negative carboxylate groups and the po-
sitively charged LDH sheets. A characteristic band at 1543 cm−1 was
reconfirmed by the band at 1457 cm−1, which was due to the C]O
asymmetric stretching in the carboxylate anion. The occurrence of the
grafting reaction was confirmed from the band observed at 612 cm−1
which is due to the out-of-plane vibration of –OH of carboxylic groups
of PAAc. AAc monomer and the –OH of LDH could react with each other
and the chemical bond formation might have taken place between the
4
Applied Clay Science 190 (2020) 105569
LDH particles and the polymer chains to form the gelatin/PAAc/LDH
network. For the nanocomposite hydrogel, the shifting of location of the
bands and arising of new bands were the evidence of the formation of
AAc grafted LDH incorporated gelatin hydrogels.
3.3. XRD analysis
XRD patterns for prepared Mg-Al-LDH and the hydrogels were de-
monstrated in Fig. 2. Mg-Al-LDH displayed characteristic hydrotalcite-
J. Nath, et al.
Fig. 1. FTIR spectra of (a) LDH, (b) copolymer hydrogel and (c) nanocomposite hydrogel.
like reflections. The reflection observed for LDH at around 10° was due
to d003 spacing and it could be attributed to an enlarged basal spacing of
0.84 nm (Gao et al., 2009). This peak demonstrated the formation of
desired Mg-Al-LDH. For the LDH, the peak occurring at 20° was due to
the presence of (006) plane. This peak demonstrated the formation of
desired Mg-Al-LDH. Reflection arising at around 43° and 62° were due
to (015) and (110) planes, respectively. In Fig. 2(b), the diffraction peak
at around 20° arising due to the plane (009) could be attributed to the
amorphous nature of gelatin combined with the peak of LDH at that
position. This peak was comparatively sharper than the peak at the
same position for the copolymer hydrogel.
3.4. Scanning electron microscopy and energy-dispersive X-ray spectroscopy
With Scanning Electron microscope, the morphology of the pre-
pared LDH and hydrogels were studied. LDH demonstrated platelet like
structure with stacking up of the layers on one another. For the copo-
lymer hydrogel, the smooth surface morphology demonstrated became
rough on incorporation of LDH inside the hydrogel matrix. This change
in the morphology could be attributed to the distribution of LDH within
the hydrogel structure. The cross-sectional SEM image for the nano-
composite hydrogel could be demonstrated in Fig. 3(d). Introduction of
LDH into the hydrogel was further confirmed by the elemental mapping
Fig. 2. XRD patterns of LDH, (a) copolymer hydrogel and (b) nanocomposite hydrogel.
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Applied Clay Science 190 (2020) 105569
images of Aluminium, Magnesium, Nitrogen, and Oxygen. (See Fig. 4.)
3.5. Transmission electron microscopy
To demonstrate the presence of LDH inside the hydrogel matrix,
TEM analysis was carried out as shown in Fig. 5. Sheet-like structures of
LDH occurred in the nanocomposite hydrogel which was not present in
case of the copolymer hydrogel. TEM analysis disclosed the crystalline
behavior of LDH nanosheets.
3.6. Dynamic light scattering and Tyndall effect
Intensity weighted average particle size was measured with DLS.
The particle size distribution of LDH in here was determined to be
100 nm. The size indicated to be in the nano range of the synthesized
Mg-Al-LDH particles. The nanocomposite hydrogel was thus obtained
by incorporating the synthesized LDH.(See Fig. 6.)
Inset showed the optical photograph of water suspension of LDH
particles. Light beam was incident from the side of the vial to show
Tyndall effect. Possible exfoliation of the LDH sheets was indicated by
the Tyndall effect. This effect demonstrated a clearly visible path of
scattered light. The individual particles suspended in the solution
scattered and reflected the light and thus the beam became visible.
J. Nath, et al.
Fig. 3. SEM morphology of (a) LDH (b) copolymer hydrogel (c) nanocomposite hydrogel; (d) Cross-section of nanocomposite hydrogel
Fig. 4. EDX mapping of the nanocomposite hydrogel
When a solution containing LDH dispersion was irradiated with a laser
beam, it displayed the colloidal nature of the solution. Tyndall effect
supported the existence of colloidal LDH nanosheets in the dispersion
sample. Bulk LDH got exfoliated into the single layers and hence,
Tyndall effect provided good indication of the same (Song and Hu,
2014).
3.7. Swelling studies
Swelling behavior of the nanocomposite hydrogels were studied in
distilled water and in solutions of pH 1.2 and 7.4 as shown in Fig. 7. In
acidic pH, the swelling of the hydrogel was lower than that of the
swelling values of the same in basic pH. At pH 7.4, swelling value in-
creased. Carboxylic acid groups were ionised to a higher extent at al-
kaline pH (pH=7.4) compared to of acidic pH (pH=1.2). These ionised
carboxylic acid groups enhanced the swelling of the hydrogels. The
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Applied Clay Science 190 (2020) 105569
presence of the same also initiated hydrophobic-to-hydrophilic transi-
tion of the hydrogel backbones followed by the electronic repulsion
among the negatively charged chains. As the amount of PAAc in-
creased, swelling value increased for a given value of pH, whereas
lower amount of gelatin contributed towards lower crosslinking causing
higher values of swelling. LDH containing hydrogels possessed higher
swelling in comparison to the hydrogels having montmorillonite clay
inside their matrix (Boruah et al., 2015). The swelling values were
found to be more or around 2000% which was higher than the hy-
drogels prepared using montmorillonite clay. The swelling values of the
copolymer hydrogels were found to be lesser than the nanocomposite
hydrogels. Incorporation of LDH inside the hydrogel matrix helped in-
creasing the swelling of hydrogels. The synthesized gel possessed better
swelling compared to many other reported articles.
3.8. Thermogravimetric analysis
Thermogravimetric analysis (TGA) was performed as shown in
Fig. 8 to study the thermal stability of the hydrogels. Both the TGA
thermograms showed weight loss at 100°C due to the loss of water
molecules inside the matrix. The decomposition for copolymer arising
at 300°C which had a lesser value for the nanocomposite hydrogel. At
temperature around 450°C, thermogram for the copolymer hydrogel
was showing higher weight loss compared to that of the composite
hydrogel at the same temperature. The weight loss for the nano-
composite hydrogel could be attributed to the decomposition of the AAc
anionic species and the polymeric chains, and lamellar hydroxyl group
loss (dehydroxylation) of the LDH layers (la Rosa-Guzmán et al., 2019).
Hence, it could be conferred that the incorporation of LDH could en-
hance the thermal stability providing superior insulation and mass
transport barrier for the volatile compounds that were generated during
the decomposition of polymer.
J. Nath, et al.
Fig. 5. TEM images of (a) copolymer hydrogel (b) nanocomposite hydrogel
Fig. 6. Particle size measurement of LDH and Tyndall effect (inset)
3.9. Biocompatibility studies
To study the in vitro biocompatibility of the prepared hydrogel,
hemolysis tests were performed with the synthesized hydrogel spe-
cimen. For designing biomaterials with novel applications, various ef-
forts were made by the research groups continuously. The Involvement
of human or other animals in several biomedical applications en-
couraged the researchers to study the blood compatibility study of the
hydrogels. With the help of hemolysis, in vitro blood compatibility
studies were carried out for the hydrogel samples. Low hemolysis ac-
tivity was demonstrated for the gel samples as shown in Fig. 9 thereby
contributing towards its utilisation in drug delivery system. Due to the
biological response to the different metal cations present in LDH
structure, biocompatibility occurred in the overall nanocomposite
structure. The codes COPH (U) and COPH (L) denoted drug unloaded
copolymer hydrogel and loaded copolymer hydrogel, respectively. Si-
milarly the codes NCH (U) and NCH (L) denoted drug unloaded nano-
composite hydrogel and drug loaded nanocomposite hydrogel, respec-
tively.
Willems et al. prepared thermoreversible celecoxib loaded poly-N-
isopropylacrylamide MgFe-layered double hydroxide hydrogel and
studied the same for biocompatible studies. After performing the in vivo
dose-response study, the hydrogels were found to be safe and bio-
compatible (Willems et al., 2015). The works carried out by Cunha et al.
also demonstrated that the LDH triggered a good tissue repair with
significant biocompatibility and also promoted the deposition of
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Applied Clay Science 190 (2020) 105569
collagen. This also established that the hydrogels containing LDH were
biocompatible in nature (Cunha et al., 2016).
3.10. In vitro drug release
Vitamin B12 release from the nanocomposite hydrogels were in-
vestigated and the reaction media resembled that of the stomach and
the intestine. The loaded drug inside the gel started to diffuse into the
solution when the samples were dipped in AGF and AIF. This followed a
diffusion mechanism. The release study for the drug was performed in
both the media for 7 h each. Cumulative release (%) of vitamin B12 from
the nanocomposite hydrogels was found to be pH dependent. This was
because, fast diffusion of drug was displayed from the hydrogel samples
in the environment mimicking that of the intestine via swelling.
At pH 1.2, presence of lower negative charges lowered the ioniza-
tion of the carboxylic acid groups of the acrylate moiety. This led to
poorer swelling of the hydrogel samples indicating lower diffusion.
Increase in the pH value of the solution increased the ionization of the
carboxylic acid (–COO‐) groups. This indicated increase in the electro-
static repulsion followed by the expansion of the space network be-
tween the groups to improve the swelling behavior further. Swelling of
the gel samples mainly governed the controlled release of the drug.
With increasing pH, swelling increased contributing towards the in-
crease in the drug release behavior of the hydrogel.
In LDH incorporated nanocomposite hydrogel, carboxylic acid
groups got protonated at pH = 7.4 which led to the release of vitamin
B12 around 31% in 7 h, whereas the same showed release around 24%
in 7 h at pH = 1.2. This signified that the incorporation of LDH in-
creased drug release in controlled manner in the nanocomposite hy-
drogel as shown in Fig. 10. Several researchers carried out works on
drug release profile incorporating different types of LDH inside the
hydrogels (Barkhordari et al., 2014; Nath et al., 2018; San Román et al.,
2013; Li et al., 2019; Lee and Chen, 2004).
For the hydrogel without LDH, it could be demonstrated that there
was increase in the drug release behaviour with increase in the pH of
the medium. Due to the change in the pH of the solution, this behaviour
took place. In such hydrogel, the increase in the protonation of more
carboxylic acid groups at pH = 7.4 led towards 24% release of the drug
in 7 h. At pH = 1.2, the drug release behaviour of the hydrogel could be
found to be around 12% in 7 h. In both the cases, amount of vitamin B12
was less as compared to the hydrogels with LDH in Fig. 10. This could
be attributed to the fact that absence of LDH inside the hydrogel matrix
caused less swelling leading towards lesser drug release behaviour as
shown in Fig. 11 (Lee and Lee, 2006).
J. Nath, et al.
Fig. 7. Swelling behavior of nanocomposite hydrogel in acidic buffer, distilled water and basic buffer
Fig. 8. Thermogravimetric analysis (a) copolymer hydrogel and (b) nano-
composite hydrogel
4. Conclusion
LDH incorporated gelatin and PAAc based pH-responsive hydrogels
were prepared via free radical polymerization. With increasing pH,
swelling percentage of the hydrogels also increases. In comparison to
CMC-g-PAAc/OMMT, the synthesized hydrogels demonstrated
8
Applied Clay Science 190 (2020) 105569
Fig. 9. Hemolysis percentage study of copolymer hydrogel (with and without
drug loading) and nanocomposite hydrogel (with and without drug loading)
enhanced values for swelling. The swelling of the hydrogel samples
were found to be pH responsive which was established in acidic and
basic media. Oral delivery of vitamin B12 was tested using the prepared
hydrogel samples. In the environment resembling that of the small in-
testine, vitamin B12 release from the hydrogels was stimulated which
signifies their noticeable dependency on the pH of the medium. Release
of vitamin B12 was found to have higher value in basic medium
J. Nath, et al.
Fig. 10. In vitro drug release studies for nanocomposite hydrogels in (a) pH=1.2 and (b) pH=7.4
Fig. 11. In vitro drug release studies for hydrogels without LDH in (a) pH=1.2 and (b) pH=7.4
(pH = 7.4) than in acidic medium (pH = 1.2). Therefore, gelatin-g-
PAAc/LDH nanocomposite hydrogels can be used as probable candidate
for developing pH dependent drug delivery system.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgement
Author would like to acknowledge University Grants Commission
for providing National Fellowship for Other Backward Classes (contract
grant number F./2017-18/NFO-2017-18-OBC-ASS-50336). The authors
also sincerely thank Sophisticated Analytical Instrumentation Centre
(SAIC), Tezpur University, India and Manbendra Mandal and Kuldeep
Gupta of Department of Molecular Biology & Biotechnology, Tezpur
University for analytical support. The authors also acknowledge S.
Wazed Ali, Department of Textile Technology and Central Instruments
Facility (CIF) of Indian Institute of Technology Delhi, India.
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