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Development of stimuli-responsive polymeric hydrogels and their
applications
1
Dr. Pronob Gogoi and
2
Dr. Monalisha Boruah*
1
Department of Chemistry, Diphu Government College, Diphu, Karbi Anglong, Assam, 782462
2
Dimension Academy Senior Secondary School, Harigaon, Tezpur, Sonitpur, Assam, 784001
DOI:
https://doi.org/10.51583/IJLTEMAS.2026.150600232
Received: 10 July 2026; Accepted: 15 July 2026; Published: 28 July 2026
ABSTRACT
Electro-responsive nanocomposite hydrogels based on gelatin, partially neutralized acrylic acid (PAAc), and
acid-functionalized multiwalled carbon nanotubes (MWCNT-COOH) were developed and evaluated as potential
smart drug delivery systems. The successful functionalization of pristine MWCNTs was confirmed by the shift
in characteristic X-ray diffraction (XRD) peaks, while the presence of MWCNT-COOH peaks in the
nanocomposite hydrogel verified their effective incorporation into the polymer matrix. Scanning electron
microscopy (SEM) revealed distinct morphological changes following acid functionalization and demonstrated
the uniform dispersion of MWCNT-COOH within the hydrogel network, resulting in a smooth and homogeneous
surface. Swelling studies showed that increasing MWCNT-COOH content reduced the swelling percentage in
the absence of an electric field, whereas the application of a 10 V electric field significantly enhanced swelling,
confirming the electro-responsive nature of the hydrogels. In vitro hemolysis assays indicated excellent blood
compatibility, highlighting their suitability for biomedical applications. The electro-responsive release of
vitamin B
12
was strongly influenced by the ionic strength of the release medium, applied voltage, and MWCNT-
COOH concentration. Furthermore, drug release behavior was systematically optimized using response surface
methodology based on a central composite design. Overall, the developed Gelatin-g-PAAc/MWCNT-COOH
nanocomposite hydrogels exhibit favorable structural, swelling, biocompatible, and electro-responsive
characteristics, making them promising candidates for the design of advanced electrically controlled drug
delivery systems.
Keywords: Nanocomposite, hydrogel, electro-responsive, drug delivery, MWCNTs.
INTRODUCTION
In recent times, stimuli-responsive polymeric hydrogels constitute a new generation of biomaterials for a wide
range of applications such as drug delivery, tissue engineering, bioseparation, sensors, actuators etc.
1-3
Despite
many advantages of using conventional hydrogels, their applications are often limited due to their poor
mechanical and some restricted response properties.
4
So, attention has been given to improve the properties of a
hydrogel such as mechanical strength, thermal properties, response to a stimuli etc. In the current scenario of
drug delivery technologies, hydrogels have emerged an irreplaceable space because of their special three
dimensional network structure which can effectively provide a matrix for the entrapment of drugs.
5
It is obvious that the precise controls over the drug quantity and the release rate are required in order to optimize
the drug release. Now- a -days, pulsatile drug release devices are extensively developed which releases drug at
a predetermined time or in pulses of a predetermined sequence. These systems have been shown to alter their
rate of drug delivery in response to various stimuli.
6
Therefore, stimuli-responsive polymeric hydrogels have
been profoundly employed as intelligent drug delivery systems. Amongst various stimuli-responsive drug
delivery systems, electro-responsive hydrogels which carry free cations or anions are fascinating materials for
the design of electrically modulated drug-delivery systems.
7
The advantages of an electric field as an external
stimulus are accessibility of equipment, which imparts precise control with respect to the magnitude of the
current, duration of electric pulses, interval between pulses
8
etc. Electro-responsive behavior of the hydrogels
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can be significantly improved by the successful incorporation of different conducting polymers such as
polyaniline,
9
polypyrrol etc. or different carbon conducting fillers such as graphite,
10, 11
carbon fibers,
12
carbon
black
13
and carbon nanotubes
14
into the hydrogel matrix.
Both single-walled (SWCNT) and multi-wall (MWCNT) carbon nanotubes are promising nanomaterials with
great potential in the field of nanomedicine for both therapeutic and diagnostic applications.
15
They have also
been proved to be advanced multi-functional fillers in polymer-based nanocomposites.
16
But, incorporation of
CNTs into different polymeric systems becomes a technical challenge due to their poor dispersibility in water or
organic solvents.
17,18
So, this problem has been removed by chemical functionalization, in which some strong
oxidants are used to generate carboxyl group on the surface of CNTs.
19
Moreover, it has been already reported
that the biocompatibility of carbon nanotubes can be enhanced by the surface functionalization and introduction
of hydrophilic moieties to avoid unwanted cytotoxic responses and risks for carcinogenesis.
20
Gelatin is a low
cost material and has been extensively studied to be used as a biomaterial like artificial skin, bone grafts, and
scaffolds for tissue engineering
21-24
etc. Its physicochemical properties can be suitably modulated by developing
composites or nanocomposites through the introduction of some reinforcing fillers into its structure.
25
Various approaches have been demonstrated in the literature describing the preparation of electro-responsive
drug delivery systems using hydrogels. Kulkarni et al. developed polyacrylamide-g-alginate based electrically
responsive hydrogel for drug delivery application. A pulsatile pattern of drug permeation was observed as
electric stimulus was switched on and off.
26
Banga et al. synthesized polyacrylamide hydrogels and demonstrated
the feasibility of the prepared hydrogels in iontophoretic release and transdermal delivery of three model
peptides, insulin, calcitonin and vasopressin under an applied electric field. It was observed that the release of
drug from a hydrogel matrix under action of electric field strength was more than the release in the absence of
electric field and the release can be precisely controlled.
27
Kim et al. investigated the electrically modulated
delivery of ionic drug cefazoline from electro-active Poly(vinyl alcohol)/Poly(acrylic Acid) IPN hydrogels. The
rapid release behaviors of cefazoline were observed when an electric stimulus was at “ON” state, whereas they
showed relatively slow release during the “OFF” state.
28
Tanaka et al. demonstrated that the partially hydrolyzed
polyacrylamide gel undergoes phase transition upon application of an electric field, and collapsed if the gel was
placed in a solvent such as 50% acetone-water binary mixture.
29
Modern optimization techniques using experimental designs are important assistance to the formulator, as they
help in developing the best possible formulation under a given set of conditions, consequently saving
considerable time, money and developmental effort.
30,31
Moreover, these systematic techniques are known to
provide a depth of understanding and ability to explore and defend the ranges for varied formulation and
processing factors. In this regard, Central composite design (CCD) has been frequently employed for the
optimization of controlled drug delivery systems.
32
The advantages of experimental design method over classical
experimental approach are, the reduction in the number of trials, ability to cover a large number of factors, the
detection of interactions between factors, detection of optimum reaction condition, a higher precision of the
response data and the empirical modeling of the data.
33
Statistical methods, such as response surface
methodology are used in developing and optimizing polymeric systems for a wide range of applications.
34
For
example, Li et al. performed the optimization of sustained release matrix tablet of metoprolol succinate using
central composite design. After investigating the effects of various parameters on drug release, a 2-factor, 5-
level central composite design was employed. Response surfaces were also established to obtain the matrix
ranges and the main factors affecting four responses. They predicted that this matrix combination can be used as
a good alternative to the commercially pellet technology, which was complicated, time-consuming and energy-
intensive.
35
Observing the significant applications of electro-responsive nanocomposite hydrogel, the present chapter
involves the development of a pH and electro-responsive nanocomposite hydrogel based on Gelatin-g-
poly(acrylic acid) and acid functionalized multiwall-carbon nanotubes. Physical characteristics such as
molecular structure and surface morphology of the prepared hydrogels were investigated thoroughly. We
examined the release behavior of vitamin B
12
at different applied voltages and ionic strength of the release media.
Also, the effect of time, MWCNT-COOH content and applied voltage on electro-responsive drug release
behavior was studied by response surface analysis. Vitamin B
12
is a water-soluble vitamin with a key role in the
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normal functioning of the nervous system and for the formation of blood.
36
Therefore, vitamin B
12
is chosen as
a model drug.
Experimental
Materials
Acrylic acid monomer (Aldrich) was distilled under reduced pressure prior to use. Gelatin A (300 Bloom),
ammonium persulfate (initiator), methylene bis-acrylamide (MBA) as a crosslinking agent and N, N, Nꞌ, Nꞌ-
tetramethylethylene diamine were supplied by Aldrich (A.R. grade). Multiwall carbon nanotubes (MWCNTs)
were supplied from Shenzhen Nanotech Port Co., Ltd., China. Nitric acid, sodium hydroxide and ethanol (A. R
grade) were purchased from Merck, Mumbai. Vitamin B
12
(C
63
H
88
CoN
14
O
14
P) was purchased from Merck,
Mumbai (M.W= 1355.4 g/mol).
Preparation of Gelatin-g-poly(acrylic acid)/MWCNTs nanocomposite hydrogel
Acid treatment of MWCNT
Functionalization of CNTs with different chemical groups is generally used to enhance their dispersibility in
water and the mostly used technique for this purpose is the oxidization with strong oxidants to generate
carboxyl group on the surface of CNTs. In this case, the carboxylic acid functionalized MWCNTs were
prepared according to the earlier described method.
37,38
60 mg of MWCNTs were added to a round-bottom
flask containing 50 ml of 60% HNO
3
aqueous solution. The mixture was refluxed for 6 hrs and then cooled to
room temperature. The mixture was diluted with 500 ml of deionized water and then collected by low speed
centrifugation. The obtained product was washed with deionized water until the pH of the filtrate reached 7
and was dried under vacuum for 24 hrs at 80
o
C.
Preparation of Gelatin-g-PAAc/MWCNTs nanocomposite hydrogel
Gelatin-g-poly(acrylic acid) /MWCNT-COOH nanocomposite hydrogels were prepared by free radical
polymerization in distilled water. 2 g of gelatin was dissolved in 30 ml of distilled water at 60
o
C and partially
neutralized (60%) acrylic acid monomer was added to this solution. Also, MWCNT-COOH (0.1-0.6 wt%) was
added to gelatin-acrylic acid mixture and dispersed under the ultrasonic vibration for 30 min. Subsequently,
methylene bis-acrylamide (0.05-0.25 wt%), ammonium persulfate (1.0 wt%) and TEMED (0.05 ml) were
added to the mixture under continuous stirring. Nitrogen was used to remove dissolved oxygen from the
reactive solution. The reactive solution was first prepolymerizd at 60
o
C for 1 hr under stirring, and then poured
into a petri dish quickly. The post polymerization was carried out at 70
o
C for 3 hrs. When the reaction was
completed, the nanocomposite hydrogel was cut into (3×3 cm) blocks and immersed in repeatedly changed
deionized water for 72 hrs to remove the residual monomers. Tables 5.1-5.2 outline the feed compositions of
subsequently prepared hydrogels. Also, the preparation of nanocomposite hydrogel is shown schematically in
Fig 5.1.
Fig 5.1 Schematic representation for the formation of Gelatin-g-PAAc/MWCNT-COOH nanocomposite
hydrogel.
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Table 5.1 Recipe in the preparation of Gelatin-g-PAAc/MWCNT-COOH nanocomposite hydrogel with
different crosslinker (MBA) content:
Recipe
GM-1
GM-2
GM-3
GM-4
GM-5
Gelatin (g)
2
2
2
2
2
Acrylic acid (ml)
1.5
1.5
1.5
1.5
1.5
Methylene bis-acrylamide (wt%)
0.05
0.1
0.15
0.2
0.25
Gel fraction determined (%)
54
62
74
82
91
Ionic Conductivity (S/cm ×10
-2
)
2.50
1.94
1.72
1.58
1.37
*Tetramethylethylene diamine (ml) = 0.05 ml, MWCNT-COOH (wt%) = 0.4 and ammonium persulphate
(wt%) = 0.1.
Table 5.2 Recipe in the preparation of Gelatin-g-PAAc/MWCNT-COOH nanocomposite hydrogel with
different MWCNT-COOH content:
GA
GA-1
GA-2
GA-3
GA-4
GA-5
GA-6
2
2
2
2
2
2
2
1.5
1.5
1.5
1.5
1.5
1.5
1.5
0
0.1
0.2
0.3
0.4
0.5
0.6
0.92
1.65
2.2
2.85
3.41
3.92
4.52
*Tetramethylethylene diamine (ml) = 0.05ml, methylene bis-acrylamide (wt%) = 0.15 and ammonium
persulphate (wt%) = 0.1.
Characterization
Fourier transform infrared spectrometer (FTIR)
To gain insights into the structural information of prepared hydrogels, the IR spectra of the hydrogels were
recorded with a Nicolet Impact-410 IR spectrometer (USA) in KBr medium at room temperature in the region
4000–450 cm
-1
.
Poder X-ray diffraction (XRD)
Powder X-Ray diffraction (XRD) data were collected on a Rigaku Miniflex X-ray diffractometer (Tokyo, Japan)
with Cu Kα radiation (λ=0.15418 nm) at 30 kV and 15 mA with a scanning rate of 0.05
o
s
-1
in a 2θ ranges from
20
o
- 80
o
.
Morphological Analysis
The surface morphology of the composites was observed using a scanning electron microscope (SEM)
(Model- JSM-6390LV, JEOL, Japan). The surface of the sample was platinum coated before SEM analysis.
Further morphological analysis was done by Transmission electron microscope (JEM 2100) with an acceleration
voltage of 200 kv.
Swelling properties
To measure the degree of swelling the hydrogels, dried samples were placed in buffer media of different pH
values at room temperature until the hydrated gels reached a stable weight. The swelling behavior under an
applied electric field was determined by applying different electric potentials to the nanocomposite hydrogel
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in distilled water. The water absorbed on the surface of the hydrogels was removed using filter paper and the
weight noted.
Swelling % = W
s
-W
dry
/W
dry
× 100 (Eqn. 5.1)
where, W
s
is the weight of the hydrogel in swollen state and W
dry
is that of the hydrogel in dry state.
Gel fraction
The weight ratio of the dried hydrogels in rinsed and unrinsed conditions can be assumed as an index of degree
of crosslinking or gel fraction. The pieces of hydrogel samples (3×3 cm) were dried for 6 hrs at 50°C under
vacuum. Then the sample (3×3 cm) was immersed into excess of double distilled water for 4 days to rinse away
unreacted parts. The gels were dried again at 50°C under vacuum. The gel fraction percentage was calculated
by the following equation
39
-
Gel fraction % = W
f
/W
i
×100 (Eqn. 5.2)
where, W
i
= Initial weight before rinse and W
f
= Final weight after rinse.
Ionic conductivity
Impedance spectroscopy was used to assess the ionic conductivity by using a two electrode cell and an impedance
analyzer (HIOKI IM 3570). The impedance spectrum was measured in the frequency range 100 Hz to 1 MHz
with potential amplitude of 10 mV at room temperature. The ionic conductivity was calculated using the
following equation-
δ = l/R × A (Eqn.5.3)
where, δ is the ionic conductivity of the nanocomposite hydrogel (S.cm
-1
), l is the thickness of the film (cm), R
is the film resistance (Ω) and A is the surface area of the electrodes (cm
2
) prior to the measurement, the hydrogel
films (2.5 cm×2.5 cm) were immersed in distilled water at room temperature for 24 hrs and the surface water
was gently removed before the film were positioned between the electrodes in the measurement cell.
Blood compatibility by hemolytic activity assay
The hemolytic test was performed following the protocol of Das Purkayastha, M. et al, with slight modification.
40
Briefly, fresh goat blood from a slaughterhouse was collected in a centrifuge tube containing anticoagulant,
trisodium citrate (3.2%), and was centrifuged at 2500 rpm for 10 min. The supernatant was discarded, and only
the erythrocytes were collected. The erythrocytes were further washed three times with PBS (pH 7.4). A 5%
(v/v) suspension of erythrocytes in PBS was prepared; 0.95 ml of this erythrocyte solution was placed in a 1.5
ml centrifuge tube and 0.05 ml of sample (0.5 mg dissolved in 1 ml of 0.5% DMSO) was added to it. The tubes
were then incubated for 1 hr at 37°C. Triton X-100 (0.2 %) and PBS were taken as the positive and negative
controls, respectively, for comparison. After incubation, the tubes were subjected to centrifugation at 2500 rpm
for 10 min. Then, 0.2 ml of the supernatant was added to 96-well plate, and finally absorbance was taken at 570
nm in a UV−visible spectrophotometer (Shimadzu UV-2550 UV–visible spectrophotometer).
Hemolysis % = [(Sample O.D – Negative control O.D)/ (Positive control O.D
– Negative control O.D] X 100 (Eqn. 5.4)
Loading of vitamin B
12
The method of soaking or equilibration was employed for vitamin B
12
loading. In this method, the amount
of buffer necessary for complete swelling of the nanocomposite hydrogel was determined.
41
Dry hydrogel was
placed in the drug solution at a concentration of 0.125 wt%, prepared in the buffer solution of pH 7.0 and left
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until all the drug solution was sucked up. Then the completely swollen hydrogel loaded with the vitamin B
12
was placed in an oven at 30
o
C for drying overnight.
Drug release experiment under electric field
To investigate the release behavior of vitamin B
12
under electric stimulus, drug loaded hydrogels were placed in
the middle of two carbon electrodes in a desired release medium as shown in the Fig 5.1. Then, the amount of
drug released into the respective medium was monitored by varying factors such as ionic strength of the release
medium and the MWCNT-COOH content in the nanocomposite hydrogel. The pulsatile release of vitamin B
12
under the applied electric field was also determined by applying pulses of electric current at 5 V, 30 min ‘ON’
and 30 min ‘OFF’.
Fig 5.2 Apparatus for electro-responsive drug delivery.
At scheduled time intervals, 5 ml solution was withdrawn and assayed spectrophotometrically by using a UV-
visible spectrophotometer (UV-2001Hitachi, Japan) for the determination of the cumulative amount of drug
release from the absorbance at 362 nm. To maintain a constant volume, 5 ml of the same solution was returned
to the container. The amount of vitamin B
12
released from the nanocomposite hydrogel was calculated from a
calculated from a previously calibrated standard curve.
42
Experimental Design
Central composite design (face-centered) was used in this study and 2 factors were evaluated, each at 3 levels;
experimental trials were performed at all 13 possible combinations to investigate influence of the parameters
such as time, MWCNT-COOH content and applied voltage on release behaviour of the drug. Two central
composite designs (with electric field and without electric field) were constructed to study the effect of two
different factors in each study on drug release behavior. In each case, the two factors, X
1
(time) and X
2
(MWCNT-COOH content); X
1
(time) and X
2
(applied voltage) were varied as required by the experimental
design and the factor levels were suitably coded (Table 5.3-5.4).
Suppose, we code the levels in standardized units so that the values taken by each of the two variables X
1
and
X
2
are -1, 0 and +1.Coded values were obtained by the following formula
43
:
Z = (X-X
o
)/ΔX (Eqn. 5.5)
Where, Z is the coded values, X is the corresponding natural value, X
o
is the natural value in the center of the
domain and ΔX is the increment of X corresponding to one unit of Z.
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Table 5.3 Central Composition Design Conditions (without electric field):
Run
Coded factor levels
X
1
X
2
1
1
1
2
0
1
3
0
0
4
0
0
5
0
0
6
0
0
7
-1
1
8
1
-1
9
0
-1
10
-1
0
11
-1
-1
12
0
0
13
0
0
Translation of coded levels in actual units
Coded level
-1
0
+1
X
1
: Time (hr)
0
5
10
X
2
: MWCNT-COOH (wt%)
0.2
0.4
0.6
Table 5.4 Central Composition Design Conditions (with electric field):
Run
Coded factor levels
X
1
X
2
1
1
0
2
1
-1
3
-1
-1
4
1
1
5
0
1
6
-1
1
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7
-1
0
8
0
-1
9
0
0
10
0
0
11
0
0
12
0
0
13
0
0
Translation of coded levels in actual units
Coded level
-1
0
+1
X
1
: Time (hr)
0
5
10
X
2
: Applied voltage (V)
0
5
10
Again, data were analyzed by multiple regressions to fit the following second order equation:
kk
o i i ij i j
i=1 i=1
Y = B + B X + B X X

where, Y = predicted response.
The resulting data were fitted into Design Expert Software (Version 6.0.10 Stat-Ease, USA) and the developed
models were adopted for the multiple correlation coefficient (R
2
), the adjusted multiple correlation coefficient
(adjusted R
2
) and corresponding ‘P’ value provided by analysis of variance (ANOVA). The greater values of R
2
and adjusted R
2
are preferable and it was considered significant when the corresponding ‘P’ value was less than
0.05. The data were also subjected to 3D response surface methodology to determine the influence of each factor
on electro-responsive release behaviour of vitamin B
12
. Effectiveness of the model is evaluated based on the
calculated coefficient of determination (R
2
) by the program.
RESULTS AND DISCUSSION
FTIR analysis
FTIR spectra of pristine MWCNT, acid functionalized MWCNT, Gelatin-g-PAAc copolymer and Gelatin-g-
PAAc/MWCNT-COOH nanocomposite hydrogels are shown in the Fig 5.3. The characteristic bands due to
generated polar functional groups on the MWCNT are observed in the FT-IR spectrum of the MWCNTs after
chemical oxidation in HNO
3
acid. The FT-IR spectrum of MWCNT–COOH shows absorption peak at 2910
cm
-1
corresponding to the C-H symmetric stretching vibration. Also, the appearance of peak at 1531 cm
-1
confirms the existence of carbon double bonding (C=C), which again reveals the integrity of hexagonal
structure on the pristine MWCNT.
44
Appearance of peak at 1731 cm
-1
assigns carbonyl (C=O) stretching
vibration of carboxyl groups which is not present in pristine MWCNTs. It confirms the carboxylation on the
surfaces of functionalized MWCNTs (Fig 5.3a).
(Eqn. 5.6)
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Fig 5.3 FTIR spectra of (a) MWCNT-pristine, (b) MWCNT-COOH, (c) Gelatin-g-PAAc/MWCNT-
COOH nanocomposite hydrogels and (d) Gelatin-g-PAAc hydrogel.
FT-IR spectrum of Gelatin-g-PAAc shows absorption peaks at 3391 cm
-1
and 2950 cm
-1
which are attributed to
the N-H stretching vibration and C-H stretching vibration in –CH
3
group. The peaks at 1630 cm
-1
and 1520 cm
-
1
are due to the amide I (C=O stretching vibration) and amide II (-NH bending vibration) respectively. The
absorption peak at 1126 cm
-1
is due to the C-N stretching vibration of amide III. Also, the characteristic peaks
at 3450 cm
-1
, 1735 cm
-1
and 613 cm
-1
are due to the -OH stretching vibration of PAAc, C=O stretching vibration
and the -OH out of plane vibration of the carboxylic groups of PAAc, which confirms the grafting reaction. Fig
5.3c represents the FT-IR spectrum of Gelatin-g-PAAc/MWCNT-COOH nanocomposite hydrogel which shows
characteristics peaks of MWCNT–COOH at 1565 cm
-1
which is ascribed to the C=C bonding that forms the
framework of the carbon nanotube sidewall. The peak at 1050 cm
-1
indicates the presence of –COOH groups of
carboxylated MWCNT and the peaks present in 500-1000 cm
-1
range indicates the presence of hexagonal carbon
atom. Presence of all these characteristics peaks indicates the incorporation of MWCNT–COOH into the Gelatin-
g-PAAc hydrogel matrix.
XRD analysis
The diffraction peaks with 2θ values of 25.54
o
and 45.70
o
are found for pristine MWCNTs and the diffraction
peaks at 26.08
o
and 43.08
o
are attributed to the graphite structure (002) and (100) planes of the MWCNT-
COOH as shown in the Fig 5.4.
Fig 5.4 XRD patterns of (a) MWCNT-pristine and (b) MWCNT-COOH.
The shifting of characteristic peak position in the XRD pattern of MWCNT-COOH shows the perfect
modification of pristine MWCNTs. There is no drastic change in the position of characteristic peaks of pristine
MWCNTs and MWCNT-COOH was observed, which suggest that MWCNTs are well retained their original
structure even after functionalization.
45
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Fig 5.5 depicts the XRD patterns of Gelatin-g-PAAc copolymer hydrogel and Gelatin-g-PAAc/MWCNT-
COOH nanocomposite hydrogels respectively. The XRD pattern of Gelatin-g-PAAc doesn’t possess any
characteristics peaks, but some minor peaks were observed around 21 and 22°, which could be attributed to
minor crystallites of grafted polyacrylic acid chains. On the other hand, the XRD pattern of Gelatin-g-
PAAc/MWCNT-COOH shows the characteristics peaks of MWCNT-COOH with low intensity which
confirms the incorporation of acid functionalized multiwall carbon nanotubes into the Gelatin-g-PAAc
hydrogels.
Fig 5.5 XRD patterns of (a) Gelatin-g-PAAc hydrogel and (b) Gelatin-g-PAAc/MWCNT-COOH
nanocomposite hydrogels.
Morphological analysis
The morphological images of the copolymer hydrogel and MWCNT-COOH incorporated hydrogels were
studied by SEM and are shown in Fig 5.6. SEM micrographs indicate the change in the surface morphology of
the prepared hydrogels after the incorporation of MWCNT-COOH.
Fig 5.6 SEM images of (a) Gelatin-g-PAAc hydrogel, (b) Gelatin-g-PAAc/MWCNT-COOH
nanocomposite hydrogels, (c) MWCNT-pristine and (d) MWCNT-COOH.
A rough surface morphology is observed and some pores can be observed in the micrograph of Gelatin-g-PAAc
hydrogel. But, comparatively a smooth surface was observed after the impregnation of MWCNT-COOH into
the hydrogel. This observation implies that MWCNT-COOH is uniformly dispersed within the hydrogel. Also,
scanning electron microscopy was used to investigate possible MWCNTs fragmentation occurred during
treatment. SEM images of MWCNT-COOH and pristine MWCNT are also shown in Fig 5.6. From both the
micrographs, a significant difference was observed. A sharp surface was observed for the functionalized
MWCNT, which was apparently smooth in case of pristine MWCNT. Also, the transparency of nanotubes has
been decreased after the functionalization as compared to the pristine MWCNTs which possibly related the
introducing of functional groups.
46,47
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Further functionalized MWCNTs morphology characterization was conducted using TEM analysis and Fig 5.7
represents the TEM micrograph of MWCNT-COOH. No structural damage occurred after acid
functionalization of MWCNTs. The MWCNTs retained their original morphology even after the acid
treatment.
Fig 5.7 TEM image of acid functionalized multiwall carbon nanotubes (MWCNTs).
Ionic Conductivity
Impedance spectroscopy provides a relatively straightforward and rapid technique to assess ionic
conductivity. Ionic conductivities of the prepared hydrogels were determined with the variation of crosslinker
(MBA) and MWCNT-COOH content in Gelatin-g-PAAc/MWCNT-COOH nanocomposite hydrogel. It was
observed that ionic conductivities decrease with an increase in crosslinker (MBA) concentration. It may be due
to the decreased proton mobility resulting from increased methylene bis-acrylamide concentration. As the
crosslinking density is increased, more crosslinking points will be developed forming a dense network, which
will restrict the ionic mobilities of the ionic groups present in the nanocomposite hydrogel. On the other hand,
the ionic conductivity was found to be increased with MWCNT-COOH content in the nanocomposite hydrogel.
This may be due to the improved ionic transport in presence of MWCNT-COOH in the hydrogel.
48
Ionic
conductivities values with the variation of crosslinker and MWCNT-COOH content are given in the Table 5.1-
5.2.
Swelling study
Effects of pH
The hydrogels have shown tremendous promise in different biomaterial applications because of their unique
water holding capacity. Hence, swelling behavior of a particular hydrogel material should be investigated in
order to confirm its utility in different biomedical areas. Fig 5.8 represents the swelling behavior of the prepared
nanocomposite hydrogels with different crosslinker amount in acidic (pH=4) and basic (pH=7.4). The swelling
behavior was found to be increased with an increase in pH from 4 to 7.4. The gelatin structure is ionizable
because the basic dissociation constant (pK
b
) of the NH
3
+
is about 6.5 and the acid dissociation constant (pK
a
)
of the COOH is about 4.7 in gelatin.
49
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Fig 5.8 Influence of pH of the medium on the swelling behaviours of the nanocomposite hydrogels: (a)
pH = 4 and (b) pH = 7.4.
So, this improved swelling behavior is due to the presence of the hydrophilic functional groups (mainly –COO
-
) in the gelatin structure. Moreover, at higher pH, ionization of the carboxylic acid groups occurs, resulting in
electrostatic repulsion between the carboxylate (–COO
-
) groups as well as expansion of the space network
thereby increasing the swelling percentage.
50
Effect of MWCNT-COOH content
Fig 5.9a depicts the effect of MWCNT-COOH content on the swelling behavior of Gelatin-g-PAAc hydrogels.
It was observed that the extent of swelling was decreased with an increase in the concentration of MWCNT-
COOH. It may be mainly due to the hydrophobic nature of MWCNT-COOH. The result is consistent with other
reports in the literature.
51
Moreover, functionalized MWCNT in Gelatin-g-PAAc may acts as like some
crosslinking sites which make the diffusion of water into the hydrogel more difficult. So, interaction between
MWCNT-COOH and hydrogels contributed to lower the swelling percentage of the nanocomposite hydrogels
than for the native gel.
Effect of applied electric field
The response of an electro-responsive hydrogel in presence of an external electric field depends on the shape
and position of the gel between the electrodes.
52
When the gel is placed at a fixed position away from the
electrodes, as in our experiment, the swelling behaviour is observed. So, the swelling behavior of the
nanocomposite hydrogels with various MWCNT-COOH content were measured as a function of time in
distilled water under the applied electric potential of 10V as shown in Fig 5.9b.
Fig 5.9 Effect of MWCNT-COOH content on the swelling behavior of Gelatin-g-PAAc hydrogels: (a) at
0V and (b) at 10V.
The swelling percentage of Gelatin-g-PAAc/MWCNT-COOH gradually increased with increasing MWCNT-
COOH content. MWCNT-COOH provides the efficient pathway of electric field. Therefore, MWCNTs could
contribute to the increase in swelling percentage of the nanocomposites by increasing the extent of ionization
of the functional groups in the nanocomposite hydrogel under electric voltage applied. This behavior is
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completely opposite to the swelling character under normal condition where swelling percentage decreases
with increase in MWCNT-COOH content.
Gel fraction
A typical dependency of gel fraction to the quantity of crosslinker incorporated into hydrogels is given in the
Table 5.1. As seen, the gel fraction of samples is increased by increasing the amounts of crosslinker from
0.05% up to 0.25% by weight. The gel fraction data reveal that the increase in crosslinker amount within the
three dimensional network of hydrogel causes an increase in crosslinking density, thus creates more entangled
structure.
Blood compatibility studies
Numerous efforts have been given to design novel biomaterials with superior blood compatibility by various
research groups. Hemocompatibility is a prime requirement for biomedical applications such as drug delivery,
tissue engineering etc. intended for direct or indirect blood exposure. Fig 5.10 represents the data obtained from
hemolysis test as described in Section 5.3.7. The hemolysis test was performed for the nanocomposite hydrogels
with 0.1 and 0.6 wt% of MWCNT-COOH content with different concentrations. For all samples, in contact with
blood showed a mean hemolysis value less than 0.5 %.The test showed very low hemolysis activity and the data
obtained are at the permissible limit as shown in the Fig 5.10a. Photographs showing precipitated RBCs at the
end of the hemolysis experiment are also given.
Fig 5.10 Hemolysis results: (a) Hemolysis percentage of the nanocomposite hydrogels with 0.6 wt% (GA-
6) and 0.1 wt% (GA-6) MWCNT-COOH content, (b) Photographs of RBCs treated with different
samples (GA-6 and GA-1).
Electro-responsive release behavior of vitamin B
12
from Gelatin-g-PAAc/MWCNT-COOH
nanocomposite hydrogels:
Effect of applied voltage
The effect of applied electric potential on release behavior of vitamin B
12
from Gelatin-g-PAAc/MWCNT-
COOH nanocomposite hydrogel is shown in Fig 5.11. It can be observed that the drug release rate was much
higher under the influence of an electrical stimulus than without electric stimulus. A systematic increase in the
drug release rate was observed when the applied voltage was increased from 5 V to 10 V. According to
Sawahata et al. drug transport occurs only if the applied electrical current is sufficiently high to induce
dimensional changes in the hydrogel. Under higher applied voltage, the ionizable groups are more ionized
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which results in improved swelling behavior of the nanocomposite hydrogel. The improved swelling will
enhance the release of drug from the nanocomposite hydrogel through diffusion.
53
Fig 5.11 Drug release behavior of Gelatin-g-PAAc/MWCNT-COOH nanocomposite hydrogels- (a) At
different electric voltage applied: 0 V, 5V and 10 V, (b) Drug release behavior as a function of applied
voltage of 0V and 5V, which was altered at 30-min time intervals.
Moreover, the rapid release behaviors of vitamin B
12
were observed when an electric stimulus was at ‘ON’
state, whereas they showed a relatively slow release rate during the ‘OFF’ state while applied electric potential
was maintained at 0 and 5V alternately. The plausible reason for this switching pattern of the release of drug
molecules is due to the electrically induced changes in osmotic pressure within the gel and local pH gradient
attributed to water electrolysis. This could also affect the swelling behaviour of the nanocomposite hydrogel
under electric field as well as release of drug from the Gelatin-g-PAAc/MWCNT-COOH nanocomposite
hydrogel. In ‘OFF’ stage (when no electric field), normal diffusion controlled release of drug is taking place
which is quite slow.
Effect of ionic strength of the release medium
The ionic strength of the release medium exerts an influence on the release rates of drugs. In our study, the
release of vitamin B
12
was studied in media containing two different ionic strengths, 0.1 M and 0.2 M NaCl
solutions. Fig 5.12 exhibits the release behavior of the drug as a function of ionic strength and at a fixed electric
potential of 10 V. It was observed that at higher ionic strengths, the drug release rate was slower with time than
in the lower ionic strength media.
Fig 5.12 Drug release behavior of Gelatin-g-PAAc/MWCNT-COOH nanocomposite hydrogels
depending on the ionic strength of the release medium (0.1M and 0.2M NaCl solution).
It may be attributed to the charge screening effect of the additional anions causing a non perfect electrostatic
repulsion, which leads to a decreased osmotic pressure difference between the hydrogel network and the
external solution. Thereby the shrinkage of the gel was observed instead of swelling. Moreover, the drug
solubility may also decrease with increasing ionic strength of the medium, which may also affect in lowering
of the drug release rate. Similar behavior was observed by Agnihotri et al.
while investigated the electrically
modulated release behavior of drug from sodium alginate and carbopol hydrogels. They found that the drug
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transport followed the switch-on and switch-off pattern in a pulsatile manner and release rate was significantly
decreased with increase in ionic strength of the release medium.
54
Effect of MWCNT-COOH content
The release of vitamin B
12
was found to be profoundly dependent on the MWCNT-COOH content of the
nanocomposite hydrogels. It can be seen from Fig 5.13a, that the drug release rate was decreased as the content
of MWCNT-COOH increased when no electric field was applied. On the other hand, a reverse release behavior
was observed when an electric potential of 10V was applied to the nanocomposite hydrogels. The release of
vitamin B
12
was found to be increased with increase in MWCNT-COOH content, as predicted from the swelling
behavior of nanocomposites in presence of an electric field displayed in Fig 5.13b.
Fig 5.13 Drug release behavior of Gelatin-g-PAAc/MWCNT-COOH nanocomposite hydrogels with
various MWCNT-COOH content and at different electric voltage applied: (a) 0 V and (b) 10 V
Response Surface Analysis
The cumulative release % of vitamin B
12
with respect to two sets of factors was regressed using the Design
Expert Software. The multiple regression equation for cumulative drug release % (Y
1
) with respect to time (hr)
and MWCNT-COOH content:
Final Equation in Terms of Coded Factors
Cumulative drug release % (Y
1
) = 52.25+29.20*X
1
+4.58*X
2
-22.13*X
1
2
-1.38* X
2
2
+
3.43*X
1
*X
2
(Eqn. 5.7)
Final Equation in Terms of Actual Factors
Cumulative drug release % (Y
1
) = -6.92011+13.32241*X
1
+33.41236*X
2
-0.88524*X
1
2
-
34.52586*X
2
2
+3.42500*X
1
*X
2
(Eqn. 5.8)
The multiple regression equation for cumulative drug release % (Y
2
) with respect to time (hr) and applied
voltage:
Final Equation in Terms of Coded Factors
Cumulative drug release % (Y
2
) = 49.32+8.35*X
1
+20.54*X
2
-7.48*X
1
2
-14.13*X
2
2
+6.53*X
1
*X
2
(Eqn. 5.9)
Final Equation in Terms of Actual Factors
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Cumulative drug release % (Y
2
) = -5.34788+3.35723*X
1
+8.45369*X
2
-0.29915*X
1
2
-
0.56515*X
2
2
+0.26100*X
1
*X
2
(Eqn. 5.10)
The results are plotted in Fig 5.14. These results show that the electro-responsive release behaviour of the
nanocomposite hydrogels was more significantly influenced by the release time and applied voltage than
MWCNT-COOH content in the prepared hydrogel. It can be observed from Table 5.5 that R
2
is high for all
responses, which indicates a high degree of correlation between the experimental and predicted responses. In
addition, the predicted R
2
value is in good agreement with the adjusted R
2
value, resulting in reliable models.
Fig 5.14 3D response surface plot of drug release behaviour of Gelatin-g-PAAc/MWCNT-COOH
nanocomposite hydrogel; (a) showing the effect of amount of MWCNT-COOH (wt%) and time (hr) on
release behaviour of vitamin B
12
and (b) showing the effect of applied voltage (V) and time (hr) on
release behaviour of vitamin B
12
.
Table 5.5 Regression statistics table:
Regression Statistics
Y
1
Y
2
Predicted-R
0.9742
0.6721
R -square
0.9973
0.9538
Adjusted-R
0.9954
0.9209
Standard deviation
1.64
6.29
Observations
13
13
CONCLUSION
Nanocomposite hydrogels based on gelatin, partially neutralized acrylic acid and acid functionalized multiwall
carbon nanotubes (MWCNT-COOH) have been developed. The shifting of characteristic peak position in the
XRD pattern of MWCNT-COOH shows the perfect modification of pristine MWCNTs. Also, the characteristics
peaks of MWCNT-COOH in the XRD pattern of Gelatin-g-PAAc/MWCNT-COOH confirm the incorporation
of acid functionalized multiwall carbon nanotubes into the Gelatin-g-PAAc hydrogels. SEM studies
demonstrated the difference in surface morphology of pristine MWCNTs and after the acid functionalization of
MWCNTs along with the smooth surface of the nanocomposite hydrogel, which implies the uniform dispersion
of MWCNT-COOH within the nanocomposite hydrogel. Swelling percentage was found to be decreased with
an increase in MWCNT-COOH content, when no electric field was applied; but reversed swelling behavior was
observed when an external electric field (10 V) was applied. In vitro experiments of percentage hemolysis reveal
that the prepared nanocomposite hydrogels possess proficient blood compatibility suitable for biomedical
applications. The electro-responsive release behavior of vitamin B
12
exhibited a considerable dependence on
ionic strength of the medium, applied voltage and MWCNT-COOH content of the nanocomposite hydrogels.
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Drug release behavior was further investigated by response surface methodology and by applying a central
composite design. So, the results obtained in this work lead us to the conclusion that Gelatin-g-PAAc/MWCNT-
COOH nanocomposite hydrogels can be a promising platform for the development of electro-responsive drug
delivery systems.
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