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Synthesis of gelatin‐based biodegradable hydrogel nanocomposite and their

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Article  in  Advances in Polymer Technology · January 2018

DOI: 10.1002/adv.21938

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DOI: 10.1002/adv.21938

RESEARCH ARTICLE

Synthesis of gelatin-­based biodegradable hydrogel nanocomposite

and their application as drug delivery agent

Asghar Bakravi1  |  Yashar Ahamadian1  |  Hamed Hashemi1  |  Hassan Namazi1,2

1
Research Laboratory of Dendrimers and
Nanopolymers, Faculty of Chemistry,
Abstract
University of Tabriz, Tabriz, Iran In this work, gelatin was chemically cross-­linked using epichlorohydrin. Then, a se-
2
Research Center for Pharmaceutical ries of gelatin-­based hydrogel nanocomposites containing CuO nanoparticles were
Nanotechnology (RCPN), Tabriz University prepared by immersion of gelatin hydrogel in CuCl2 solution with different concen-
of Medical Science, Tabriz, Iran
trations. Gelatin was chosen, due to its cheapness, nontoxicity, and biocompatibility.
Correspondence The structure and morphology of hydrogel nanocomposites were characterized by a
Hassan Namazi, Research Laboratory of
number of techniques such as FT-IR, XRD, energy dispersive X-­ray spectroscopy,
Dendrimers and Nanopolymers, Faculty of
Chemistry, University of Tabriz, Tabriz, and scanning electron microscopy. Then, swelling behavior of synthesized hydrogel
Iran. nanocomposites in distilled water and different saline solutions was investigated.
Email: namazi@tabrizu.ac.ir
Their sensitivity to pH, biodegradability, and gel content was determined. Moreover,
Funding information
the water uptake and shrinking kinetics of hydrogel nanocomposites were evaluated.
University of Tabriz and Research Center
for Pharmaceutical Nanotechnology Finally, they were used as drug delivery agent, and the different concentrations effect
(RCPN); Tabriz University of Medical of CuO nanoparticles present within the hydrogel nanocomposites compared with
Science
neat hydrogel on mentioned tests was studied.

KEYWORDS
CuO nanoparticles, gelatin, hydrogel, nanocomposite

1  |   IN T RO D U C T ION limitations, numerous strategies have been developed such

By definition, hydrogels are a kind of cross-­linked hydro-
philic polymers which are able to absorb, swell, and retain a
large quantity of water, saline, and other physiological solu-
tions in their three-­dimensional structures, without being dis-
solved.[1,2] Hydrogels are made up of water-­soluble polymers
that are cross-­linked via chemical or physical bonds.[3,4] In
the recent two decades, considerable attention has been fo-
cused on these water-­swellable materials. As hydrogels have
a suitable sensitivity to environmental conditions variation,
for instance pH,[5,6] solvent composition,[7] and temperature,[8]
they have been widely used in many industrial and medical
fields.[9–11] Natural-­based hydrogels due to nontoxicity, bio-
degradability, and biocompatibility have been utilized in
medical, agriculture, horticulture, pharmaceutical, and baby
diapers.[12–18] But in practice, hydrogels are mechanically
weak because of their brittle and fragile networks after full
swelling which limited their applications. To overcome these
as using nanoparticles to form hydrogel nanocomposite.[19]
Agglomeration of nanoparticles to a polymer matrix usually
improves its mechanical properties[19,20] and it will also pro-
mote usage of hydrogel nanocomposites in various fields.[21,22]
Nanoparticles can either cross-­link the hydrogel networks or
they can absorb on the polymer chains when being entrapped
within the networks.[4] These networks are formed by both co-
valent and noncovalent bonds, by means of chemical or phys-
ical interactions. Oftentimes, the polymer and nanoparticles
work together synergistically in order to give unique prop-
erties to hydrogel such as mechanical, thermal, and optical.
These properties lead hydrogels to be used in the optics, elec-
tronics, catalyst, sensors, separation devices, and many other
applications.[23] Gelatin is a polypeptide with high molecular
weight. It is obtained from the controlled hydrolysis of col-
lagen. The most important feature of gelatin is that it can be
used to produce biocompatible materials.[24] Gelatin due to its
gelation capability is very attractive raw material for preparing

Adv Polym Technol. 2018;1–11. wileyonlinelibrary.com/journal/adv © 2018 Wiley Periodicals, Inc.     1 |

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2       BAKRAVI et al.

hydrogels. Moreover, owing to having a large number of from Merck (Darmstadt, Germany). Cephalexin was pur-
functional groups, it can be cross-­linked easily.[25] Gelatin chased from Dana Co of Iran. Deionized water was used
hydrogels are used as biodegradable materials in the medi- throughout this work.
cal and pharmaceutical.[26–29] As a result of exhibiting poor
mechanical strength, there are some limitations in the use of
2.2  |  Preparation of gelatin hydrogel
gelatin for producing hydrogels. The structure of gelatin needs
to be reinforced by some filler materials such as nanoparti-
2.2.1  |  Hydrogel of Gelatin
cles. Incorporating of nanoparticles, as multifunctional cross-­
linking agents, to gelatin-­based hydrogel matrices causes the About 100 ml of distilled water was introduced into a beaker
formation of strong networks with improved mechanical and and heated to 40°C and then 20 g of gelatin added and stirred
thermal properties.[30,31] Nano-­sized materials show signifi- vigorously until a homogeneous viscous mixture obtained.
cant properties compared to bulk ones, by reason of their high Afterward, 3 ml of epichlorohydrin as cross-­linker was added
surface-­to-­volume ratio. Copper oxide nanoparticles because dropwise under continuous stirring until mixture homog-
of easy preparation and unique physical and chemical proper- enized. Subsequently, this mixture was placed in warm water
ties are of great interest.[32] Copper oxide nanoparticles have bath at 80°C for 1 hr to remove unreacted chemicals; the
been used in various fields such as electric and magnetic stor- obtained hydrogel was washed with distilled water several
age devices, as a catalyst and solar energy transformation.[33] times, and finally dried in an oven at 50°C for 24 hr. Figure 1
As the copper ions are colorful, so the stage of their bind- represents the block diagram of gelatin hydrogel preparation
ing and converting to copper oxide nanoparticles can be vi- (Figure 1).
sually seen. Formerly, copper oxide nanoparticles have been
applied for preparing different nanocomposites, for example,
2.3  |  Preparation of hydrogel
carboxymethyl cellulose/CuO,[34] and polyurethane/CuO.[35]
nanocomposites
Over the past decades, natural polymers (biopolymers) have
frequently been used as raw materials for the design of effi-
2.3.1  |  Gelatin/CuO nanocomposites were
cient drug delivery systems (DDS).[36,37] The aim of DDS was
prepared by the following procedure
to provide a drug within the body to carry out some specific
therapeutic purposes. Among the different drug delivery sys- About 7 g of dried gelatin hydrogel was immersed in a
tems, hydrogels have particular properties which let them be CuCl2 solution with different concentrations (0/005, 0/01,
used as ideal drug delivery systems.[3] Because they are simi- 0/02, and 0/03 M) for 24 hr. Then, to remove unreacted
lar to body’s tissues, they have high water content and rubbery
consistency. Furthermore, hydrogels provide the possibility of
using in both dry and swollen networks for drug loading and
releasing.[38] This work deals with the preparation of new hy-
drogel nanocomposites through the in situ formation of CuO
nanoparticles within swollen gelatin hydrogel. By incorporat-
ing of copper oxide nanoparticles into the gelatin hydrogel, we
were to study the swelling, biodegradability properties, and
drug release profile of the nanocomposite hydrogel compared
to the neat hydrogel. After characterization, their swelling be-
haviors were investigated in detail. The o­ bjective of this arti-
cle was to prove whether the synthesized hydrogels have the
potential for applying for drug delivery purposes. Cephalexin
has been chosen as a model drug, being loaded in the hydrogel
nanocomposites. In vitro tests of drug delivery in conditions
simulating the gastrointestinal tract were carried out.

2  |   EX P ER IME N TA L
2.1  | Materials
Gelatin with a molecular weight of 30,000 was obtained from
Aldrich. Epichlorohydrin (EPH) (99.5%), CuCl2, NaOH,
LiCl, KCl, MgCl2, NaCl, AlCl3, and HCl were purchased F I G U R E   1   Block diagram for gelatin hydrogel preparation
BAKRAVI et al.
copper ions from the surface of the hydrogels, copper ion-­
loaded hydrogels were taken out and washed with distilled
water. The washed hydrogels were poured in NaOH solu-
tion (100 ml of 0/2 M) for 15 min, to convert copper ions
to CuO nanoparticles. The obtained hydrogel nanocompos-
ites were washed with distilled water and dried in an oven
at 50°C for 48 hr. In this work, hydrogel nanocomposites
were marked with C0, C1, C2, C3, and C4, indicating
CuCl2 with 0/00, 0/005, 0/01, 0/02, and 0/03 M concentra-
tions, respectively.
2.4  | Characterization
The FT-IR spectra were obtained in KBr pellets using Bruker
Vector FT-IR spectrometer in the region of 400–4,000 cm−1.
X-­ray diffraction pattern was gotten using a Siemens diffrac-
tometer with Cu-­Ka radiation at 35 kV in the scan rate of 1°/
min and scan range of 2ϴ from 2° to 70°. All of the analyzed
samples were in powdery form. The morphology of dried sam-
ples and elemental analysis of the particles were taken using
scanning electron microscopy (SEM) (LEO 1430VP) operated
at 15 kV after coating the dried samples with gold and silver
films.
2.5  |  Equilibrium swelling measurement
To measure the equilibrium swelling (ES) of hydrogel na-
nocomposites in aqueous media, 0.2 g of dry samples was
immersed in 150 ml of distilled water at room temperature
for 24 hr. After reaching their maximum swelling capac-
ity, the hydrogel nanocomposites were taken out, dried,
and weighted. ES was calculated according to the equation
below:
w − wd
ES = s
wd (1)
where wd and ws are the weights of the initial and swollen
samples, respectively.
2.6  |  Swelling of different salt solutions
According to the swelling measurement in aqueous media,
absorption of the hydrogel nanocomposites was determined
in 0.1 M solution of NaCl, MgCl2, AlCl3, LiCl, and KCl. The
swelling capacity of samples was studied in NaCl solution
with different concentrations as well.
2.7  |  Absorbency at different pHs
  
   3
|
respectively. The pH values were precisely controlled by a pH
meter (consort/C533). Then, 0.1 g of each sample was used and
the swelling capacity evaluated according to Equation (1).
2.8  |  Gel Content measurement
To determine the gel content values of hydrogel nanocompos-
ites, 0.1 g of each sample in its dry state was poured in excess
distilled water at room temperature for 24 hr. Then, swol-
len samples were taken out, dried in an oven at 50°C, and re-
weighted. Gel content (Gel%) was calculated by Equation (2).
wf
Gel% = (2)
wi
where wf and wi are final and initial weights of sam-
ples, respectively. Table 1 gives the results of gel content
determination.
2.9  |  Determination of the
reswelling kinetics
Water uptake (WU) is used to determine the reswelling ki-
netics of different hydrogel nanocomposites after drying. To
evaluate WU, the dried gels were soaked again in distilled
water at room temperature. At consecutive time intervals, the
samples were taken out and after removing the excess solu-
tion on their surface, weighted. The WU values were deter-
mined according to Equation (3).
wt − wd
WU = × 100 (3)
ws − wd
where wt, ws, and wd are the weights of the wet hydrogel at
time t, wet hydrogel at equilibrium swelling at room tempera-
ture and dried hydrogel, respectively.
2.10  |  Determination of the
shrinking kinetics
Water retention is used to evaluate the deswelling kinetics of
hydrogels. Thus, the shrinking rate of a salt solution and water
retention of the hydrogel nanocomposites were studied as fol-
lows. Distilled water-­swollen hydrogel nanocomposites were
immersed in 0.1 M solution of NaCl. At consecutive time in-
tervals, the hydrogel nanocomposites were taken out and after
removing the excess solution on their surface, weighted. The
WR values were determined according to Equation (4).
wt − wd
WR = × 100 (4)
ws − wd

The effect of pH variations on swelling in the range of pH 2–12 T A B L E   1   Gel content values of gelatin hydrogel nanocomposites
was studied. Several solutions with acidic and basic pH values
Sample C0 C1 C2 C3 C4
were prepared. To achieve pH ≥ 6.0 and pH < 6.0, NaOH solu-
tion with pH 13.0 and HCl solution with pH 1.0 were diluted, Gel content (%) 94.11 88.5 92.3 97.5 99.2
 |
4       BAKRAVI et al.
where wt, ws, and wd are same as Equation (3).
2.11  |  Biodegradability measurement
To measure the biodegradability of hydrogel nanocompos-
ites, 0.5 g of each sample was poured in distilled water at
room temperature. At certain days, samples were taken out,
dried in an oven at 50°C, and their loss weights were calcu-
lated. Equation (5) is used to calculate the biodegradability of
hydrogel nanocomposites.
( )
wi − wf
Percent remaining = 1 − × 100
(5)
wi
where wi and wf are weights of initial and dried hydrogels,
respectively.
2.12  |  Drug loading
In this work, cephalexin was used as a model drug. The drug
loading was carried out by immersing the hydrogels in ce-
phalexin solution. Experimentally, 0.2 g of each sample was
suspended in 50 ml of the drug solution (50 mg/50 ml of
distilled water) at 37°C for 48 hr. Then, drug-­bearing hydro-
gels were taken out, washed, and dried. The amount of drug
loaded by the hydrogels was determined using UV-­Vis spec-
troscopy at 262 nm. The results of drug loading were evalu-
ated by Equation (6).
Amount of drug in beads
Drug loading (g/g) = (6)
Amount of beads
2.13  |  In vitro drug release studies
The drug release behaviors of hydrogels were studied
in pH 1.2 (gastric fluid) and pH 7.4 (intestinal fluid).
Experimentally, 0.2  g of the drug-­ bearing hydrogels
was immersed in 50 ml of the chosen release medium at
37°C and under magnetic stirring (50 rpm). At appropri-
ate time intervals, 5 ml of the solution was taken out, and
the amount of cephalexin released from the hydrogels was
determined using UV spectrophotometer at 262 nm. To
release medium being constant, the measured sample was
added back to the solution. The results of drug release are
presented in Figures 13 and 14.
3  |  RESULTS AND DISCUSSION
3.1  |  Formation of gelatin hydrogel
At first, it was tried to cross-­link gelatin by physical cross-­
linking agents, but the obtained hydrogel decomposed in
the stage of converting copper ions to copper oxide nano-
particles (NPs) by NaOH, so the chemical cross-­linking
agent (epichlorohydrin) was employed. Gelatin has many
functional groups in its structure, so it can easily react with
epichlorohydrin.[26] Epichlorohydrin has been widely used
in the preparation of different hydrogels for drug delivery
purposes.[1] Epichlorohydrin is toxic for prolonged high-­
level oral consumption.[4] But as it is connected to gelatin
from both sides by strong covalent bonds,[5] and the excess
of epichlorohydrin was removed by washing using distilled
water, no specific toxicity was expected. The formation
mechanism of gelatin hydrogel is presented in Scheme 1.
In this process, amine groups on the chain of gelatin can
react with epichlorohydrin from both sides to yield hydro-
gel (Scheme 1).
3.2  |  In situ formation of CuO Nanoparticles
Scheme 2 presents the formation of copper oxide nanopar-
ticles in the swollen polymer network. A two-­step process
was applied to prepare copper oxide nanoparticles. In the
first step, gelatin hydrogel was soaked in CuCl2 solution with
different concentrations. Then, carbonyl, amine, and ani-
onic groups of the gelatin hydrogel bound the copper (Cu+2)
ions via electrostatic interactions.[39] Consequently, its color
changed from bright yellow (neat hydrogel) to blue. When
these hydrogels were suspended in the suitable basic agent
(NaOH solution), Cu+2 ions converted to copper oxide nano-
particles. This followed visually by a change in color from
blue to green, denoting the formation of copper oxide nano-
particles inside the gelatin matrix. This process was simple
and economical, for did not need to heat or any other tools
(Scheme 2).
S C H E M E   1   Formation mechanism of
gelatin hydrogel by epichlorohydrin
BAKRAVI et al.
S C H E M E   2   In situ formation of
copper oxide nanoparticles within gelatin
hydrogel
3.3  |  FT-­IR analysis
Figure 2 displays the FT-­IR spectra of neat hydrogel (a)
and hydrogel nanocomposite (b). In Figure 2a,b, character-
istic peaks at 3,420 and 3,422 cm−1 are assigned to N–H
stretching on the backbone of gelatin. The band appearing
at 1,637 cm−1 indicates amide I band; furthermore, peak at
1,449 cm−1 is attributed to aliphatic C–H bending vibrations.
The peak at 1,718 cm−1 represents absorption of carbonyl
groups. The asymmetric stretching vibration of carboxy-
late groups is observed at 1,560 cm−1. Peaks at 2,924 and
2,926 cm−1 are related to aliphatic C–H stretching, while
peak at 1,458 indicates aliphatic C–H bending vibration.
The peak at 1,339 cm−1 shows stretching vibration of C–N
bond.[18] Moreover, there is no clear peak at 750 cm−1 which
indicates all of Cl groups in epichlorohydrin have completely
been replaced and hydrogel contains no residual epichlorohy-
drin. In Figure 1b, peaks in the region of 400–600 cm−1 are
specific to Cu–O bond that proves the formation of copper
oxide nanoparticles[40] (Figure 2).
3.4  |  XRD analysis
The XRD profile of gelatin hydrogel/CuO nanocomposite is
given in Figure 3. XRD was used to characterize the existence
F I G U R E   2   FT-­IR spectra of (a) neat hydrogel, (b) gelatin/copper
oxide hydrogel nanocomposite
  
   5
|
of copper oxide nanoparticles within the hydrogel network. As
shown in Figure 3, a broad peak at 20° is related to polymer
network. Four other peaks at 36°, 39°, 49°, and 62° are assigned
to (110), (111), (202), and (113) planes of copper oxide nano-
particles, respectively.[41] The broad reflections show the nano-
structure of copper oxide particles (Figure 3).
3.5  |  Scanning electron microscopy
Scanning electron microscopy was used to show surface
morphology of the hydrogels. SEM images of the neat gela-
tin hydrogel and gelatin/CuO hydrogel nanocomposite have
been depicted in Figure 4. In this figure, (a) gives a uniform
and homogeneous surface morphology for neat hydrogel.
Figure 4b and 4c are related to 0.01 and 0.03 M concentra-
tions of copper oxide nanoparticles, respectively. As it can
be seen, distribution of the nanoparticles within hydrogels is
acceptable and has small size about 25–30 nm. In the case
of 0.03 M concentration of copper oxide nanoparticles, some
aggregation is observed (Figure 4).
3.6  |  Energy dispersive X-­ray spectroscopy
The chemical composition of gelatin hydrogel nanocompos-
ite was analyzed by energy dispersive X-­ray spectroscopy
F I G U R E   3   XRD pattern of gelatin/copper oxide hydrogel
nanocomposite
 |
6       BAKRAVI et al.
(a)
(b)
(c)
F I G U R E   4   Scanning electron microscopy images of (a) neat
hydrogel, (b), and (c) gelatin/copper oxide hydrogel nanocomposite
(EDX). Figure 5 shows EDX pattern of gelatin/copper oxide
hydrogel nanocomposite. The presence of carbon was at-
tributed to gelatin. The signals for copper in Figure 5 con-
firmed the formation of copper oxide nanoparticles. The
copper oxide nanoparticles appeared in acceptable content in
the prepared hydrogel nanocomposites matrix as seen in the
EDX pattern. The peaks at 1.75–2.25 keV are identified to
gold, which was used for sample coating (Figure 5).
3.7  |  Swelling behavior in distilled water
In aqueous medium, swelling strongly depends on cross-­linking
degree but in saline solutions, the order of swelling is a little dif-
ferent[6] because in saline solutions, ionic strength of surrounding
F I G U R E   5   Energy dispersive X-­ray spectroscopy analysis of
gelatin/copper oxide hydrogel nanocomposite
F I G U R E   6   Equilibrium swelling of gelatin hydrogel
nanocomposites in distilled water at room temperature
solution and screening effect play a vital role in swelling.[7] For
example, C0 has the lowest swelling as a result of screening
effect. But in hydrogel nanocomposites, the presence of copper
oxide nanoparticles diminishes the screening effects, the higher
nanoparticles the lesser screening effects. In C1 and C2, nano-
particles with repulsive forces expanded hydrogel network and
swelling increased, but in C3 and C4, attractive forces shrank
hydrogel network and swelling decreased. In Figure 6, swelling
of the hydrogel nanocomposites in distilled water is given. As it
is observed, C1 and C2 have more swelling than neat hydrogel
(C0), due to electrostatic repulsion between copper oxide nano-
particle and the interfacial chain,[42] expanding hydrogel pores
and absorbing more water molecules. In the cases of C3 and C4,
swelling decreased compared with C0. It means copper oxide
nanoparticles acted as cross-­linker that reduced expansion and
relaxation of molecular chains (Figure 6).
3.8  |  Swelling in various salt solutions
Figures 7–9 show the swelling behavior of hydrogels in
different salt solutions. Hydrogels are polyelectrolyte;
BAKRAVI et al.   
   7
|

F I G U R E   7   Effect of cation radius on swelling of gelatin F I G U R E   8   Effect of cation charge on swelling of gelatin
hydrogel nanocomposites hydrogel nanocomposites

hence, they are sensitive to the ionic strength of external

solution.[43] As well-­known when ionic strength of salt so-
lution increases, swelling of the hydrogels will decrease,
owing to reducing osmotic pressure difference between the
hydrogel and its surrounding salt solution. In Figure 7, the
effect of cation radius on the swelling capacity of hydrogels
is exhibited. As shown in Figure 7, when cation radius in-
creased, swelling accordingly increased as interaction of the
anionic carboxylate with large cations is weaker than with
small cations.[39] In Figure 8, the effect of cation charge
on swelling is observed. As it is obvious in Figure 8, with
an increase in cation charge, swelling decreased because
the cross-­linking degree between carboxylate groups and
cation was increased. In Figure 9, the effect of salt solution
F I G U R E   9   Effect of salt solution concentration on swelling of
concentration on the swelling is presented. When concen-
gelatin hydrogel nanocomposites
tration of salt solution increased, swelling decreased. for
example, in the salt solution, the ionic strength increased,
consequently osmotic pressure difference between hydro- efficient repulsion, ended in low swelling. The swelling be-
gel and external solution reduced. This caused low water tween pH 4 and 10 was high and approximately constant[44,45]
uptake (Figures 7–9). (Figure 10) .

3.9  |  Hydrogel sensitivity to pH variations 3.10  |  Gel content of hydrogels (%Gel)

To determine the sensitivity of hydrogels to pH variations,
their swelling was evaluated in different pH solutions in the
range of 2–12. Figure 10 exhibits the effect of different pH
values on the swelling. As shown in this Figure, in acidic
media (pH 2–5), amine groups (–NH2) on the backbone of
gelatin hydrogel could be protonated. This led to high repul-
sion between positively charged amine groups (−NH+3 ) and
swelling increased. In very acidic media (pH < 2), positive
charges of the ammonium cations were shielded by Cl− ions
and prevented significant repulsion; therefore, swelling was
low. At higher pH values (pH 5–10), swelling is attributed
to efficient repulsion between carboxylate groups (–COO−).
At high basic media, charge screening effect induced by Na+
ions shielded the anion charge of carboxylate and prevented
Gel content of the hydrogels is presented in Table 1.
According to the results, with an increase in copper oxide na-
noparticles concentration, gel content increased. The reason
is that, in more concentrations of nanoparticles, many parts of
the gelatin hydrogel network involved with nanoparticles and
gel content increased (Table 1).
3.11  |  Reswelling kinetics of hydrogels
Figure 11 shows the reswelling capability of dried hydrogel
nanocomposites in distilled water at room temperature. The
reswelling of hydrogels decreased with increasing concen-
tration of copper oxide nanoparticles. The water uptake of
dried C1 reached 90%, whereas that of dried C0 exhibited
 |
8       BAKRAVI et al.

oxide nanoparticles, water retention increased, as interactions

between carboxylate groups (–COO−) and nanoparticles in-
creased and shielded anion charge of the carboxylate groups,
as a consequence, screening effects reduced and water re-
tention increased. Thus, screening effect for hydrogel nano-
composites was in the order of C0 > C1 > C2 > C3 > C4
(Figure 12).

3.13  |  Biodegradability of hydrogels

F I G U R E   1 0   Effect of pH variations on swelling of gelatin
hydrogel nanocomposites
Figure 13 presents the biodegradability of hydrogel nano-
composites in distilled water at room temperature. According
to Figure 13, C1 has maximum and C4 minimum biodegrada-
bility, indicating that with an increase inf copper oxide nano-
particles concentration, biodegradability decreased. It means
that at higher concentrations of copper oxide nanoparticles,
gel strength of gelatin hydrogel improved. Biodegradability
in gelatin hydrogel nanocomposites is considered to be by
reason of hydrolyzing amide bonds by water molecules.[48]
There are different amide bonds on the backbone of gelatin

F I G U R E   1 1   Reswelling kinetics of gelatin hydrogel

nanocomposites

a low value of 45%. Because strong intermolecular hydro- F I G U R E   1 2   Deswelling kinetics of gelatin hydrogel
gen bonding interactions occurred in C0 during the desicca- nanocomposites
tion process, the relaxation and expansion of the molecular
chains greatly decreased.[46] In the cases of C1 and C2, as a
result of having electrostatic repulsion between copper oxide
nanoparticle and the interfacial chain,[3] a network of the
hydrogels expanded and water uptake increased. But in C3
and C4, copper oxide nanoparticles acted as cross-­linker, and
with reducing expansion of molecular chains, water uptake
decreased (Figure 11).

3.12  |  Deswelling kinetics of hydrogels

Figure 12 displays the shrinking kinetics of gelatin hydrogel/
CuO nanocomposites in NaCl aqueous solution at room tem-
perature. All of the swollen hydrogels tended to shrink and
lose water when transferred in NaCl solution.[47] The water
retention of hydrogels reduced from 85% for C4 to 60% for F I G U R E   1 3   Biodegradability of gelatin hydrogel
C0, indicating that with increasing concentration of copper nanocomposites
BAKRAVI et al.   
|
   9

that may be attacked by water molecules. The more water

molecules enter into hydrogel network, the more hydrolysis
of amide bonds occurs. Thus, biodegradability will increase.
As it can be seen in Figure 13, biodegradability in the gelatin
hydrogel nanocomposites has direct relation with swelling
and reverse relation with cross-­ linking degree. As men-
tioned above, with increasing concentration of copper oxide
nanoparticles, cross-­linking degree in the hydrogel network
increased; therefore, fewer water molecules entered into hy-
drogel network, but when swelling increased, more water
molecules entered into hydrogel network (Figure 13).

3.14  |  Drug loading and release behavior F I G U R E   1 4   Drug release behavior of gelatin hydrogel
nanocomposites in pH 7.4
Generally, drugs are incorporated into hydrogels by physical
entrapment.[9] It was reported that there is a physical interac-
tion between cephalexin and gelatin.[9] The carboxylic acid
and amine groups in cephalexin are converted to carboxy-
late and ammonium groups, respectively, and consequently,
it will easily attract by oppositely charged gelatin backbone.
According to similar study about the drug loading efficiency
in functionalized gelatin, the drug encapsulation efficiency
was reported to be 64%.[49]
The drug release behavior was studied to prove the capabil-
ity of gelatin/copper oxide hydrogel nanocomposites as drug
carriers. According to the previous studies about the drug
release behavior of gelatin-­g-­poly(N-­isopropylacrylamide),
it was proposed that drug release occurred in four steps: in
the first stage or initial burst release stage, high drug con- F I G U R E   1 5   Drug release behavior of gelatin hydrogel
centration released in the first 30 min, that is most probably nanocomposites in pH
due to the discharge of localized drug molecules on the car-
rier surface; in the second-­stage effect of diffusion, the drug
concentration remains almost constant from 1 to 4 hr, and it of pH 1.2, because of having more swelling. So, there is a
is believed that the hydrophilic properties of gelatin allow hy- direct correlation between drug release and swelling. When
drogel to swell more that consequently facilitate more drug the hydrogels swell, a transition from dry (glassy) to the
diffusion due to a loose polymeric network;[50,51] in stage swollen (rubbery) state takes place, substantially increasing
three or the combined diffusion and degradation stage, the the mobility of drug molecules in their softened matrix and
biodegradation of hydrogel network increased the drug con- causing drug to release. So, mechanism for drug release is
centration. In the last stage, the effect of polymer degrada- swelling-­controlled. In this mechanism, the rate of drug re-
tion, the level of drug concentration decreased slowly.[51] The lease depends on the rate of hydrogel swelling.[52–56] With
in vitro drug release behaviors of hydrogel nanocomposites increasing swelling, the penetration of water molecules into
were analyzed in pH 1.2 (gastric fluid) and pH 7.4 (intesti- the hydrogel network increased, resulted in breaking more
nal fluid). The degree of drug release from hydrogel nano- hydrogen bonding interactions between drug molecules and
composites as a function of time is plotted in Figure 14 (pH the hydrogel and releasing hydrophilic drug molecules into
7.4) and Figure 15 (pH 1.2). According to these figures, in the buffer medium (Figures 14 and 15).
the C1 and C2 samples, CuO nanoparticles expand the hy-
drogel network due to the existence of repulsion forces. as
the amount of water molecules increased the drug release 4  |  CONCLUSIONS
increased. however, in the case of C3 and C4 samples, the
presence of attractive forces, shrank the hydrogel network. Novel gelatin-­based hydrogel nanocomposites in the presence
indeed in the presence of few amounts of water molecules, of copper oxide nanoparticles were successfully prepared.
the drug release decreased. Also the amount of drug release Their swelling properties were studied in detail. Cephalexin
from hydrogel nanocomposites in pH 7.4 is more than that was used as a model drug, after loading its release behaviors
 |
10       BAKRAVI et al.
in pH 1.2 (gastric fluid) and pH 7.4 (intestinal fluid) were
investigated. X-­ ray diffraction and EDX patterns proved
the formation of copper oxide nanoparticles in the hydrogel
network. SEM micrograph of the hydrogel nanocomposites
showed the existence of copper oxide nanoparticles with a
size range of 25–30 nm within the hydrogel matrix. Measured
swelling in various salt solutions was less than in distilled
water induced by screening effects. With an increase in salt
concentration and cation charge and reduction in cation ra-
dius, swelling decreased. It was observed that at high acidic
and basic media, the swelling was low but between pH 4 and
10 and swelling was high and approximately constant. As the
concentration of CuO nanoparticles increased, more parts of
hydrogel was engaged with nanoparticles and consequently
the gel content increased. The reswelling of hydrogels de-
creased at high concentrations of copper oxide nanoparticles;
consequently, the water uptake of dried C1 reached 90%,
whereas that of dried C0 exhibited a low value of 45%. The
water retention of hydrogels reduced from 95% for C4 to 60%
for C0; hence, with decreasing concentration of copper oxide
nanoparticles, water retention decreased. The rate of biodeg-
radability at higher concentrations of CuO nanoparticles was
low, thus gel strength of gelatin hydrogel improved. Finally,
the amount of drug release from hydrogel nanocomposites
in pH 7.4 was more than in pH 1.2, because of having more
swelling.
ACKNOWLEDGMENTS
This work was supported by the University of Tabriz and
Research Center for Pharmaceutical Nanotechnology
(RCPN), Tabriz University of Medical Science. We thank our
colleagues from the chemistry department of the University
of Tabriz who provided insight and expertise that greatly as-
sisted the research.
ORCID
Hassan Namazi  http://orcid.org/0000-0002-1977-4677
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How to cite this article: Bakravi A, Ahamadian Y,
Hashemi H, Namazi H. Synthesis of gelatin-­based
biodegradable hydrogel nanocomposite and their
application as drug delivery agent. Adv Polym
Technol. 2018;00:1–11.
https://doi.org/10.1002/adv.21938