Materials Science and Engineering C 61 (2016) 457465

Contents lists available at ScienceDirect

Materials Science and Engineering C

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

Genipin-modied gelatin nanocarriers as swelling controlled drug

delivery system for in vitro release of cytarabine
Huda Khan a, R.N. Shukla a, A.K. Bajpai b,
a
b

Department of Chemistry, S.A.T.I., Vidhisha, Madhya Pradesh, India

Bose Memorial Research Laboratory, Department of Chemistry, Government Autonomous Science College, Jabalpur, Madhya Pradesh, India

a r t i c l e

i n f o

Article history:
Received 9 October 2015
Received in revised form 21 December 2015
Accepted 28 December 2015
Available online 30 December 2015
Keywords:
Gelatin
Nanocarriers
Cytarabine
Drug release

a b s t r a c t
The aim of the present investigation was to design biocompatible gelatin nanoparticles, capable of releasing the
cytarabine drug in a controllable way by regulating the extent of swelling of nanoparticles. In order to achieve the
proposed objectives, gelatin (Type A, derived from acid cured tissue) was modied by crosslinking with genipin
and nanoparticles of crosslinked gelatin were prepared using single water in oil (W/O) emulsion technique. The
nanoparticles were characterized by techniques like FTIR, SEM, TEM, particles size analysis, and surface potential
measurements. The nanoparticle chemical architecture was found to inuence drug-releasing capacity. The inuence of experimental conditions such as pH and simulated physiological uids as the release medium was also
investigated on the release proles of cytarabine. It is possible to fabricate high-performance materials, by designing of controlled size gelatin nanoparticles with good biocompatible properties along with desired drug release proles.
2015 Elsevier B.V. All rights reserved.

1. Introduction
In recent years, nanotechnology has shown great potential to be
used as a promising tool for several applications in vitro and in vivo. In
medicine, nanoparticles have been applied for diagnosis and therapy
and offer diversied applications like magnetic cell separation, and
magnetically targeted delivery of therapeutics. Moreover, they are also
used in magnetic resonance imaging (MRI) and magnetically induced
hyperthermia that are known modern clinical approaches of great relevance. The nanostructure materials have shown promise in treating
complex diseases like tumors and cancers. Recent investigations have
induced a variability of micro- and nanostructures with different materials, sizes, and specic surface chemistry [1]. In essence, nanostructure
materials are unique in their properties as well as performance in pharmaceutical and biomedical applications.
Modern synthetic organic chemistry has opened the new avenues to
design nanomaterials of various chemical architectures using different
materials sources. Although a large variety of synthetic polymers have
been employed for designing nanoparticles, however, biodegradable
and natural occurring polymers, like starch [2], chitosan [3], and
liposome's [4] have also been exhaustively used in fabrication of
nanomaterials for biomedical applications. Among various biopolymers
used for preparing nanoparticles, gelatin nanocarriers have been widely
evaluated as drug carriers in controlled drug-delivery technology. Gelatin nanoparticles represent a promising carrier system for controlled
Corresponding author.
E-mail address: akbmrl@yahoo.co.in (A.K. Bajpai).

http://dx.doi.org/10.1016/j.msec.2015.12.085
0928-4931/ 2015 Elsevier B.V. All rights reserved.

drug delivery applications as it has number of advantages over

other nanoparticle materials. Gelatin is known to be a natural biomacromolecule and completely nontoxic and non-carcinogenic in nature [5], and possesses low antigenicity. It has been extensively used
in parenteral formulations [6]. The biomedical and pharmaceutical applications of gelatin nanoparticles are richly documented in literature.
For example, Kaul and Amiji [7] prepared poly (ethylene glycol) modied gelatin nanoparticles for intracellular delivery and found them
quite benecial as long-circulating delivery systems in vivo. Employing
genipin as a crosslinking agent, gelatin nanospheres were prepared [8]
and their efciency as drug carriers was examined by the intramuscular
administration, both in vitro and in vivo conditions. Yan and Li [9] prepared gluteraldehyde crosslinked gelatin microspheres with an average
diameter of 70 m and loaded them with mitomycin C, an anticancer
drug, together with a radioisotope and studied their drug delivery
potential.
Apart from using gelatin as nanoparticles in drug delivery technology, its use in biomineralization constitutes another signicant area in
bio-inorganic chemistry. In an interesting study, Gashti and coworkers
[10] studied biomimicry of kidney stones by growing brushite crystals
within a host matrix of gelatin and glutamic acid and observed the effect
of presence of urea on the growth of crystals. Bhatnagar et al. [11]
showed that enzymatically crosslinked gelatin hydrogels were quite effective substrates for dental pulp stem cell (DPSC) differentiation toward odontoblasts. The authors found that the DPSC differentiation
and biomineralization were independent of hydrogel stiffness and presence of dexamethasone. In another study, the inuence of pH and
amino acids was investigated on biomineralization of hydroxyapatite

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in gelatin [12]. Thus, it is well established that gelatin is one of the promising materials in medicine and pharmacy; nevertheless it dissolves
rather rapidly in aqueous environments and, therefore, poses difculties
in production of long-term drug delivery systems. An effective remedy
to check this dissolution problem of gelatin is to carry out crosslinking
of this biopolymer using different chemical agents. For instance, Bigi
and coworkers [13] performed crosslinking of gelatin lms by glutaraldehyde to different extents and studied their thermal and mechanical
behaviors. In another study, Sung et al. [14] allowed crosslinking of a
gelatin-resorcinol mixture with formaldehyde and glutaraldehyde and
evaluated bioadhesive properties of the crosslinked materials. Patel
and coworkers [15] prepared microparticles of glutaraldehyde
crosslinked gelatin and studied controlled release of bone morphogenetic protein-2. Although the crosslinking agents described here are
quite efcient and effectively crosslink gelatin, however, the cytotoxicity aspects of these chemical crosslinkers cannot be ignored and must be
addressed. As an attempt to achieve nontoxic crosslinking of gelatin,
physical crosslinking such as dehydrothermal treatment and plasma
treatment have also been attempted [16], however the degree of
crosslinking was quite low. Recently, genipin, a naturally crosslinking
agent of gelatin has been proposed which is reported to be completely
nontoxic and effective and has been used to design crosslinked gelatin
microspheres as a biodegradable drug-delivery system for intramuscular administration [17].
It is known that the nature of drug is not only the factor that affects
the design of a drug delivery system, but how the drug is incorporated
into the drug carrier also matters a lot. The primary goal in drug incorporation is to integrate enough of the drug into the device to deliver effective doses of the drug. Another important consideration is that the
drug should not lose its biological activity upon incorporation. Out of
several approaches being adopted in drug incorporation, one method
involves addition of drug to the feed mixture during the preparation
or synthesis of the drug delivery system. Another method which is
more in practice is that the drug is present in the emulsion process during nanoparticles formation [18,19] or dissolved in the sol portion of a
thermally responsive hydrogel prior to gelation [20,21]. Alternatively,
the drug may be incorporated into the device after fabrication. For this
purpose nanoparticles may be soaked in a concentrate drug solution
to uptake drug [22,23]. The extent of drug loading depends on the nature of the drug, the design of the device, and the desired concentration
of the drug in the device.
Cytarabine (ara-C) is the most effective drug of acute leukemia that
interferes with DNA replication [24]. However, such drugs increase the
bone marrow insufciency and cause drastic secondary effects. When
ara-C doses are very high, complications like neurotoxicity and convulsions are observed. The administration of high dose of ara-C results partly from the short half life of the compound [25]. Accordingly, it appears
useful to develop drug delivery system that can reduce the toxicity of
ara-C by maintaining adequate drug levels in the body.
Thus, being motivated by the possible applications of degradable
nanoparticles as carriers for targeted release of anticancer drugs the
proposed investigation focus to study the dynamics of swelling controlled delivery of cytarabine (ara-C) from the genipin crosslinked
nanoparticles of gelatin (type A).
2. Experimental
2.1. Materials
Cytarabine (ara-C) was obtained from the Dabur Research Foundation (New Delhi, India) and used as received. Acid processed gelatin
(Type A, isoelectric point 7.6) in yellowish granular form, was supplied
by Loba Chemie, Mumbai, India and used without any pretreatment.
Type B gelatin (Bloom No. 240, isoelectric point 4.8) extracted from
human bone was purchased from E.Merck, India. Genipin was obtained
from Sigma Aldrich, USA. Polymethyl methacrylate (PMMA) (Sigma

Aldrich Co., USA, Average MW ~ 120,000 Da, inherent viscosity 0.20)

was used to prepare an oil phase. Other chemical and solvents were of
analytical reagent grade. Bi-distilled water was used throughout the
experiments.
2.2. Methods
2.2.1. Preparation of nanoparticles
The preparation methods of nanoparticles for pharmaceutical use
are divided broadly into two categories, those based on physicochemical properties such as phase separation [26] and solvent evaporation
[27], and these based on chemical reactions such as polymerization
and polycondensation. In the present study, emulsion crosslinking technique was followed which may briey be described as follows: Aqueous phase was prepared by dissolving a denite amount of gelatin
(A) in distilled water while for oil phase parafn oil was used. The
above two solutions were mixed with vigorous shaking (Shaking
speed 300 RPM, 0.5 HP Motor Capacity) (Toshniwal, India) for 30 min.
Thereafter, a suspension of genipin was prepared in 9 mL ethane diol
and 1 mL parafn oil and a xed volume of this suspension was added
to gelatin suspension with constant stirring. The crosslinking reaction
was allowed to take place for predetermined time period. Prepared
nanoparticles were separated using centrifugation. The separated gelatin nanoparticles were re-suspended three times in toluene and twice in
acetone for cleaning purpose. The nal product was dried at room temperature (25 C) to obtain a ne bluish powder which was stored in air
tight polyethylene bags at 4 C. The whole reaction scheme and mechanisms involved in crosslinking of gelatin with genipin may be depicted
as shown in Fig. 1.
2.2.2. Characterization
2.2.2.1. FTIR spectra. The IR spectra of cytarabine loaded gelatin
(A) nanoparticles was recorded on a FTIR Spectrophotometer (Shimadzu
8201 PC). Samples for the spectral analysis were prepared by mixing
nanoparticles and KBr in 1:10 proportion and the spectra were obtained
in the range of 4000 to 400 cm1 with a resolution of 2 cm1.
2.2.2.2. Scanning electron microcopy. Morphological studies of gelatin
nanoparticles were performed using SEM, Philips 515, ne coater
(Philips, Eindhoven, The Netherlands). Drops of the polymeric nanoparticles suspension were placed on a graphite surface and freeze-dried.
The freeze-drying process in presence of cryoprotectant, such as sucrose, prevents aggregation of nanoparticles and maintains their shape
and size. The sample was then coated with gold by ion sputter at
20 mA for 4 min, and observations were made at 10 kV.
2.2.2.3. Transmission electron microscopy study. The morphological features of genipin crosslinked gelatin nanoparticles were investigated by
recording transmission electron micrographs (TEM) (Hitachi Hu-11
B) with an accelerating voltage of 80 kV. The samples for the TEM measurements were prepared by dispersing a drop of the sample suspension on Formvar-coated C grids.
2.2.2.4. Surface potential measurements. In order to understand the nature of the cisplatingelatin nanoparticle interaction and stability of gelatin nanoparticles in suspension, surface potential studies were
performed with a digital pH meter (Systronics Model No. Digital pH
Meter MK VI, Ahmadabad, India). In a typical experiment 0.2 g nanoparticles were dispersed into 20 mL of respective pH solution and emf was
recorded using a compound electrode system. A similar experiment was
also repeated for drug loaded nanoparticles. The surface potential
measurements were also conducted at different temperature so that
the inuence of temperature on the stability of gelatin nanoparticles
suspension could also be assessed.

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459

Fig. 1. Crosslinking reactions between gelatin and genipin. Scheme . Crosslinking reaction of gelatin by genipin with: (A) primary reaction through Michael addition to form stable
intermediate; and (B) secondary reaction with nucleophilic substitution of free lysine amine molecules into genipin activated ester.

2.2.2.5. Particle size analysis. The mean particle size (mean intensity
weighted diameter, z-average) and polydispersity index (width of the
size distribution, PI) by photon correlation spectroscopy (PCS), were determined to characterized the Genipin crosslinked gelatin nanoparticles
using a Zetasizer Nano ZS (Malvern Instruments, UK). For size determination, all samples were diluted vefold with MilliQ water.
2.2.3. Loading of cytarabine
Loading of cytarabine was performed by allowing 0.1 g of nanoparticles to swell in the freshly prepared drug solution till equilibrium, and
then drying to obtain the release device. The percent loading of drug
was calculated by the following eq.
% Loading

Wd Wo
x100
Wo

where Wd and Wo are the weights of loaded and unloaded nanoparticles, respectively.
2.2.4. In vitro drug release experiments
Release experiments were performed in phosphate buffer saline (PBS)
(pH 7.4, 1.2 mM KH2PO4, 1.15 mM Na2HPO4, 2.7 mM KCl, 1.38 mM NaCl)
In order to determine the released amount of the cytarabine, into 100 mg
of drug-loaded nanoparticles 8 mL of phosphate buffer saline (PBS) was
added as a release medium (pH 7.4) and the resulting suspension was
gently shaken for predetermined time period. After shaking was complete, the suspension was centrifuged; 3 mL of suspension was withdrawn, and assayed for cytarabine spetrophotometrically.

constant. In the withdrawn aliquots, the amount of cytarabine was determined spetrophotometrically.
For achieving mechanistic insights into the release process of
cytarabine, the following equation was used [28],
Wt
ktn
W

where Wt/W is the fractional release at time t and k is rate constant.

The exponent n, called as diffusion exponent, is an important indicator
of the mechanism of drug transport and, in general, has a value between
0.5 and 1. When n = 0.43, the release is taken to be Fickian. When n =
0.86, the release is zero order (Case II transport), and in between these
values, i.e. 0.43 b n b 0.86, the release is described as anomalous. When
Wt/W = 0.43, t becomes the half life, another extremely useful parameter in comparing systems.
2.2.6. Chemical integrity of drug
Chemical integrity of drug in acidic media (pH1.8) was judged by
UV spectrophotometric method (Double Beam UVVIS Spectrophotometer-2201, Ahmadabad, India) as explained elsewhere [29].
2.2.7. Statistical analysis
All measurements were done at least thrice and the gures and data
have been presented along with respective error bars and S.D.,
respectively.
3. Results and discussion
3.1. Characterization of nanoparticles

2.2.5. Kinetics of release process

For monitoring the progress of the release process, 3 mL of aliquots
were withdrawn from suspension at desired time intervals and instantly fresh 3 mL PBS was added to suspension to maintain its volume

3.1.1. FTIR spectral analysis

The FTIR spectral analysis is a simple tool to ascertain the structural
conrmation of a material. In the present study, the FTIR spectra of

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H. Khan et al. / Materials Science and Engineering C 61 (2016) 457465

native gelatin (A), genipin-cross linked gelatin nanoparticles and

drug (cytarabine) loaded genipin crosslinked gelatin nanoparticles
are shown in Fig. 2(a), (b) and (c), respectively. The respective spectra conrm the presence of gelatin, genipin, and cytarabine in the
drug loaded crosslinked gelatin nanoparticles. In the spectra (a) a
sharp peak at 3300 cm 1 may be attributed to NH stretching of
amide group of gelatin and is a characteristic Amide A band as reported elsewhere also [30], whereas Amide II band at 1531 cm-1
may be attributed to NH stretching and CN bending [31] vibrations. The observed peak at 2928 cm 1 may again be assigned as a
characteristic Amide B band of protein and it results from asymmetric stretching of CH2 groups of gelatin [32]. In the spectra (b) all the
bands mentioned previously are present and conrm the presence
of gelatin in the genipin crosslinked gelatin nanoparticles. However,
in the spectra (b), the Amide II band also appears at 1454 cm 1
which may be assigned to CH2 asymmetric bending. Furthermore,
the spectra also shows strong peak at 1080 cm 1 which may be
assigned to ring CH in plane bending and CO stretching mode of the
primary alcohol on the genipin molecule, respectively [33]. A strong
peak at 1370 cm 1 may be attributed to new bonds established between genipin and primary amine of lysine, hydroxylysine, or arginine
residues of gelatin [34]. Another signicant peak has been observed at
1740 cm1 which may be assigned to CO stretching of ketone group
in aromatic ring of genipin.
In the spectra (c) an Amide II band appears at 1399 cm1 which
may be attributed to COO-asymmetrical stretch. The spectra (c) not
only shows the above mentioned peaks of gelatin and genipin but
also exhibits strong peaks at 3477 cm1 and 798 cm1 which may
be due to NH and OH stretching, and CH bending of cytarabine
molecule.
3.1.2. SEM analysis
The morphological features of the prepared gelatin nanoparticles
have been investigated by the scanning electron microscopy (SEM)
analysis as shown in Fig. 3(a). It is clear from the observed image that
shape of particles is uniform and the size of the nanoparticles is estimated in the range of 25 to 75 nm.
3.1.3. TEM analysis
In order to look into more precise morphology of the gelatin
nanoparticles, TEM studies were also performed and the obtained
image is shown in Fig. 3(b). It is clear from the image that the particles are almost regular and spherical in shape and have sizes in the
range of 75 to 150 nm. A larger dimension of gelatin nanoparticles

obtained from the TEM measurements could be attributed to the aggregation of nanoparticles.
3.1.4. Particle size analysis
The particle size distribution of genipin crosslinked gelatin nanoparticles has been carried out by dynamic light scattering measurements.
The results are depicted in Fig. 4 which clearly reveals that the size of
nanoparticles varies between 50 and 150 nm with majority of particles
having size of about 80 nm.
3.1.5. Surface potential measurements
The values of potential for unloaded, and drug loaded nanoparticles are summarized in Table 1. It is clear from the data that upon loading of the drug molecules on to nanoparticles, a net positive potential of
the particles surface increases at pH 1.8, 7.4 and 8.6. Since the isoelectric
point of gelatin is about 6.8, the gelatin bears a net positive charge at
pH 1.8 and slight negative charge at pH 7.4. On the other hand at
pH 8.6 the gelatin molecules possess a negative charge as pH 8.6 is
well above the isoelectric point. However, upon loading of cytarabine
the overall charge moves toward either more positive charge or less
negative values which suggest for a overall gain of positive charge on
the nanoparticles surfaces. The increase in positive surface charge at
pH 1.8 may be attributed to the reason that at pH 1.8, cytarabine molecules acquire net positive charge due to formation of NH+
3 groups on the
cytarabine molecule. However, at pH 7.4 and 8.6, when the gelatin molecules bear slight and more negative charge, respectively, loading of
cytarabine causes a reduction of negative charge due to positively
charged nature of cytarabine molecule.
4. Results on cytarabine release
The major objectives of the release experiments are to determine
how various experimental parameters affect the release of cytarabine
from genipin crosslinked gelatin nanoparticles. The effect of crosslinker,
gelatin, pH of the release medium, simulated physiological uids, and
type of gelatin was studied on the release proles of cytarabine.
4.1. Mechanism of drug release
In swelling controlled drug delivery systems, the amount of water
uptake determines the quantity of the released drug. If we visualize
swollen nanoparticles of genipin crosslinked gelatin, they may be considered like three dimensional structures of crosslinked gelatin macromolecules between the strands of which water permeation channels
are present [35]. When the drug loaded nanoparticles contact a thermodynamically favorable solvent, like water, the water invades the gelatin
chains and dissolves the drug molecules which diffuse out to the receptor medium. Yasuda and coworkers [36] developed a free volume theory according to which free volume of water molecules are always
available for diffusion of drug molecules in the nanoparticles network.
The theory reveals that free volumes in polymer network may be imagined as volume fraction of molecular sieve holes which are available for
drug molecules to undergo diffusion and subsequent release.
In the present study, the genipin crosslinked gelatin molecules build
up the nanoparticle network and drug molecules are present within the
nanoparticle structure. When the drug loaded gelatin nanoparticles
contact water, the glass transition temperature of gelatin is increased
and gelatin becomes soft and rubbery in nature. The rubbery nature of
gelatin allows relaxation of its chains and consequently the water molecules enter its network. The entered water dissolves the drug molecules and the drug comes out into the release medium.
4.2. Effect of percent loading on cytarabine release

Fig. 2. FTIR spectra of (a) native gelatin, (b) genipin crosslinked native gelatin, and
(c) cytarabine-loaded gelatin nanoparticles.

In the present study, physical loading was followed which involved

swelling of pre-weighed nanoparticles into the cytarabine solution of

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461

Fig. 3. (a) scanning electron micrograph (SEM) and (b) transmission electron micrograph (TEM) images of genipin crosslinked gelatin nanoparticles.

varying concentration thus yielding drug loaded nanoparticles from

48.5 to 88.4%. The loaded nanoparticles were allowed to release the
entrapped cytarabine drug into denite volume of the medium. The release proles of nanoparticles loaded to different extent are shown in
Fig. 5, which clearly indicate that the amount of released cytarabine
gradually increases with increasing percent loading. The observed increase in cytarabine release with increasing percent loading is due to
the reason that upon swelling of gelatin nanoparticles greater amount
of drug is released.

4.3. Effect of gelatin on cytarabine release

Drug release proles are often sensitive to chemical structure and
properties of the carriers as well as experimental conditions. The effect
of gelatin on release of cytarabine has been investigated by varying the
amount of gelatin from 1.0 to 7.0 g. Fig. 6 clearly shows that the cumulative release of cytarabine decreases with increasing amount of gelatin.
The decrease in cumulative release of cytarabine with increasing gelatin
is due to the fact that when gelatin is 1.0 g, the gelatin molecules are
highly crosslinked and, therefore, the gelatin nanoparticles are quite porous in nature. Due to high porosity, greater number of water molecules
enters the network and a large amount of drug is released due to
swelling.

4.4. Effect of genipin

Certain reagents have been used for crosslinking gelatin such as glutaraldehyde, tripolyphosphate, formaldehyde and carbodiimide hydrochloride, however, studies have shown that the synthetic crosslinking
reagents are more or less cytotoxic and may affect biocompatibility of
the drug delivery system. Hence, it is desirable to use a crosslinking reagent for biomedical applications that has low cytotoxicity and forms
stable and biocompatible crosslinked products. Thus, genipin could be
a relevant option as a crosslinker of gelatin. It is worth to mention that
besides the empirical data of the traditional medicine; today some information on the benecial effects of genipin is available in biochemical
terms [37].
The effect of crosslinker on the release proles of cytarabine has
been investigated by varying the concentration of genipin in the range
0.1 to 0.6 wt.% of gelatin. The results are shown in Fig. 7, which clearly
reveal that the fractional release of cytarabine increases with increasing
genipin up to 0.3 wt.% while beyond it a fall in the released amount of
cytarabine is noticed. The results of crosslinker on the amount of
released cytarabine can be explained by the fact that since genipin is a
hydrophilic crosslinker, its increasing number of linkages in the nanoparticles enhances their hydrophilicity which, in turn, will allow increased number of water molecules to enter into the nanoparticles
and obviously the swelling ratio will increase. Thus, increased swelling
will permit greater number of cytarabine molecules to diffuse out and
the release of cytarabine increase. However, when the concentration
of genipin is beyond 0.3 wt.%, the size of nanoparticles will decrease
due to enhanced crosslinking density of the nanoparticles and as a result; therefore, both swelling and release will fall. Another explanation
for the observed decrease in the cytarabine release may be that increasing the crosslinker concentration lowers the molecular weight between
the crosslinks and this, consequently, reduces the free volumes accessible to the penetrant water molecules. Similar types of results have also
been reported by other workers [38]. Some authors [39], however, have
reported that due to the crosslinking of polymer matrix, it's glass

Table 1
Surface potentials of unloaded and drug loaded gelatin nanoparticles.
Particles

Medium

Potential (mV)a

Unloaded

1.8 pH
7.4 pH
8.6 pH
1.8 pH
7.4 pH
8.6 pH

283 12
38.4 9
184.5 0.28
304.5 10
31.7 7
168.1 3

Nanoparticles
Drug
Loaded nanoparticles
Fig. 4. Particle size analysis of genipin crosslinked gelatin nanoparticle.

The results have been expressed as mean S.D. of three measurements.

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H. Khan et al. / Materials Science and Engineering C 61 (2016) 457465

Fig. 5. Effect of % loading of cytarabine on its release prole for a denite composition of
nanoparticles [gelatin] = 1.0 g, [genipin] = 0.1326 mM, pH = 7.4, Temp. = 25 0.2 C.

transition temperature (Tg) is reduced. Due to the reduced Tg the polymer behaves like a glass at experimental temperature and the mobility
of polymer chains (gelatin) is restrained. This clearly results in a lower
degree of swelling of nanoparticles and reduced cytarabine release.

4.5. Effect of pH on cytarabine release

In the present investigation, the release dynamics of the cytarabine
has been observed under varying pH conditions as found in the gastrointestinal tract (GIT) e.g., stomach (gastric juice) 1.0, and small intestine
(7.58.6). The wide range of pH allows a specic drug to be delivered to
a targeted site only. For example, the pH in the stomach is quite different from the neutral pH of the intestine and this pH difference could
be used to prevent release of foul-tasting drugs into the neutral pH environment of the mouth while using polycationic hydrogels as drug carrier [40]. Similarly a polyanion hydrogel, which shows a minimal
swelling at acidic pH (such as in stomach), could be of potential use as
increase in pH will lead to ionization of the carboxylic groups [41].

Fig. 6. Effect of varying amounts of gelatin in nanoparticles on release proles of

cytarabine for a denite composition of nanoparticles [genipin] = 0.3 wt.% of gelatin,
pH = 7.4, Temp. = 25 0.2 C, %Loading = 88.4.

Fig. 7. Effect of varying amounts of genipin (crosslinker) on release proles of cytarabine

for a denite nanoparticle composition [gelatin] = 1.0 g, pH = 7.4, Temp. = 25 0.2 C,
%Loading = 88.4.

The results obtained in the present study are depicted in Fig. 8,

which clearly indicate that the cumulative release is 12.4 mg/mL at
pH 1.8, 8.3 mg/mL at pH 7.4 and 9.7 at pH 8.5. Thus, the cumulative release is minimum at pH 7.4 which is very close to isoelectric point of the
gelatin (B). It is clear from the results that the cumulative release of
cytarabine is signicantly different at pH values of the experiments.
When the cytarabine loaded nanoparticles are placed at pH 1.8, the
cytarabine molecules entrapped within the nanoparticles remain in
protonated state as shown below:

Fig. 8. Variation in released amount of cytarabine with varying pH of the release medium
for a denite nanoparticle composition [gelatin] = 1.0 g, [genipin] = 0.3 wt.% of gelatin,
Temp. = 25 0.2 C, %Loading = 88.4.

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463

At this pH, the gelatin molecules also possess a net positive charge
due to predominance of the protonated amine groups (NH+
3 ) over carboxylate ions (COO) of their amino acids and cause repulsion among
the entrapped drug molecules within the nanoparticles. This results in
a widening of the mesh sizes of the nanoparticle network, which consequently facilitate the release of the cytarabine molecules into the release
medium. Similarly, at pH 8.6, which is quite above the isoelectric point
of the gelatin, the gelatin chains will acquire negative charge due to ionization of carboxylic acid groups present along the gelatin chains and
will repeal due to electrostatic repulsion. This will clearly bring about
an increase in the release of cytarabine drug.
However, at pH 7.4, which is near to the isoelectric point of the gelatin, the gelatin macromolecules will be electrically neutral and will not
cause any repulsion among gelatin chains. Thus, cytarabine molecules
will not be released much and cumulative release will be minimum.
4.6. Chemical integrity of drug
In order to ascertain the chemical integrity of cytarabine in highly
acidic medium such as gastric juice, the drug was left in simulated
gastric juice medium and its UV spectra was scanned and compared
to that of the cytarabine in the aqueous medium. The spectra are
shown in Fig. 9 which clearly indicate that they are nearly identical
to each other.
The results of chemical integrity of drug suggest that even after remaining in highly acidic media, the chemical nature of cytarabine does
not change. Moreover, it was also found that even gelatin nanoparticles
do not undergo any cleavage or dissolution in gastric juice medium. This
clearly explains the chemical integrity of drug carrier system in highly
acidic media.
4.7. Effect of type of gelatin on cytarabine release
Gelatin is a natural biopolymer that is extracted from collagen by alkaline or acidic pretreatment and thermal denaturation [42]. Depending
on the pretreatment two types of gelatin can be distinguished, A and B.
Gelatin A is extracted from porcine skin, and processed by acidic pretreatment, while gelatin B is extracted from bovine skin, and processed
by alkaline pretreatment. The alkaline pretreatment converts glutamine
and aspargine residues into glutamic acid and aspartic acid, which
results in a higher carboxylic acid content for gelatin B (118/1000
amino acids) than for gelatin A (77/1000 amino acids) [43]. The effect
of type of gelatin on the release prole of cytarabine has been investigated by using gelatin nanoparticles of type A and B and determining

Fig. 10. Effect of type of gelatin on the released amount of cytarabine for denite
nanoparticle compositions [gelatin] = 1.0 g, [genipin] = 0.3 wt.% of gelatin, pH = 7.4,
Temp. = 25 0.2 C, %Loading = 88.4.

the amounts of released cytarabine. The results clearly indicate that

drug cumulative release is quite higher in the case of gelatin type B
(9.6 mg/mL) compared to A (8.4 mg/mL) (Fig. 10).
The type of gelatin exerts a pronounced inuence on the release proles of the cytarabine. The obtained results may be explained by the fact
that at the experimental pH (7.4) (which is above the isoelectric point
4.8) the gelatin B molecules will possess a net negative charge due to
COO groups in the molecule. Thus, the cytarabine molecules, which
are almost fully ionized at pH 7.4, will attach to these negatively charged
centers present along the gelatin molecules and result in a greater percent loading. When the cytarabine loaded type B nanoparticles are
placed in the release medium, the COO groups present along the gelatin chains repel each other, thus producing a greater relaxation of network chains in the nanoparticles. This obviously results in a larger
swelling of the loaded nanoparticles which, in turn, produces greater release of cytarabine from the type B gelatin nanoparticles. Similar types
of results have also been published elsewhere [44].

4.8. Effect of simulated physiological uids on cytarabine release

Fig. 9. UV spectra showing the chemical stability of cytarabine in its (a) pure solution,
(b) released medium.

During the administration of drug loaded nanocarriers, the nanoparticles come across several biological uids in different parts of the body.
It is, therefore, necessary to study the inuence of biouids on the released amount of cytarabine when the drug loaded gelatin nanoparticles contact these biouids. The effect of nature of the medium on the
released amount of cytarabine has been investigated by performing release experiments in various simulated physiological uids such as saline, glucose solution, urea and articial urine. The results are depicted
in Fig. 11 which reveals that the released amount of cytarabine is significantly suppressed in physiological uids in comparison to that in the
PBS.
In the case of biouids the possible reason for the lower release
and swelling of cytarabine may be that the presence of salt ions in
the release medium lowers the rate of penetration of water molecules into the loaded nanoparticles, thus resulting in a fall in the released amount of cytarabine. In the case of urea, its capacity to break
hydrogen bonds between water molecules and gelatin chains may be
responsible for the lower water uptake and consequently, lower release of cytarabine.

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H. Khan et al. / Materials Science and Engineering C 61 (2016) 457465

Fig. 11. Inuence of nature of release media on the released amount of cytarabine.

5. Conclusions
Crosslinking of gelatin B emulsion by genipin forms small size nanoparticles which act as a swelling controlled drug release system. The anticancer drug cytarabine is adequately loaded on to the nanoparticles
and released in a desirably controlled manner. The crosslinking of gelatin chains by genipin is well conrmed by the FTIR spectral analysis. The
SEM and TEM analysis of nanoparticles suggest for regular shaped nanoparticles with an average size of 75 nm. The particle size analysis also
conrms that most of the nanoparticles are below 100 nm. The surface
charge measurements reveal that upon loading of cytarabine the negative charge of the nanoparticles is reduced. It is found that release proles of cytarabine are greatly inuenced by % loading of cytarabine
and when the percent loading increases from 48.5 to 88.4, the cumulative release of cytarabine also increases. In the case of gelatin, the released amount of cytarabine decreases when the amount of gelatin
increases from 1.0 g to 7.0 g. The released amount of cytarabine increases with increasing genipin content up to 0. 3 wt.% of gelatin whereas the released amount of cytarabine decreases beyond 0.3 wt.%. It is
noticed that the release behavior is directly regulated by the extent of
swelling of gelatin nanoparticles. Type of gelatin also has a profound effect on the release potential of nanoparticles and it is found that type B
gelatin nanoparticles show a greater drug delivery than that by type A
nanoparticles. An optimum drug release is obtained near physiological
pH (7.4) while lower release is observed in basic pH range. The physiological uids suppress the extent of release of cytarabine. The UV spectral study suggests that the drug loaded gelatin nanoparticles are quite
stable in even highly acidic medium and chemical integrity of the drug
is fairly maintained.
References
[1] C. Alexiou, R. Jurgons, C. Seliger, H. Iro, Medical applications of magnetic nanoparticles, J. Nanosci. Nanotechnol. 6 (2006) 27622768.
[2] G. Hamidi, G. Ponchel, D. Duchene, An original method for studying in vitro the enzymatic degradation of cross-linked starch microspheres, J. Control. Release 55 (2
3) (1998) 193201.
[3] A.K. Andrianov, L.G. Payne, Polymeric carriers for oral uptake of microparticulates,
Adv. Drug Deliv. Rev. 34 (23) (1998) 155170.
[4] R. Singh, S.P. Vyas, Topical liposomal system for localized and controlled drug delivery, J. Dermatol. Sci. 13 (2) (1996) 107111.
[5] R.K. Stevens, J.N. Einerson, A.J. Burmania, J.W. Kao, In vivo biocompatibility of
gelatin-based hydrogels and interpenetrating networks, J. Biomater. Sci. Polym.
Ed. 13 (12) (2002) 13531366.
[6] Y. Marios, N. Chakfe, X. Weng, M. Marios, T. Haw, W. King, R. Guidoin, Carbodiimide
cross-linked gelatin: a new coating for porous polyester arterial prostheses, Biomaterials 16 (15) (1995) 11311139.
[7] G. Kaul, M. Amiji, Long-circulating poly (ethylene glycol)-modied gelatin nanoparticles for intracellular delivery, Pharm. Res. 19 (7) (2002) 10611067.

[8] H.C. Liang, W.H. Chang, K.J. Lin, H.W. Sung, Genipin-crosslinked gelatin microspheres as a drug carrier for intramuscular administration: in vitro and in vivo studies, J. Biomed. Mater. Res. A65 (2) (2003) 125310.
[9] Y. Changhong, L. Xiongwei, C. Xiaoli, D. Wang, Z. Dachang, T. Tan, H. Kitano, Anticancer gelatin microspheres with multiple functions, Biomaterials 12 (7) (2001)
640644.
[10] M.P. Gashti, M. Bourquin, M. Stir, J. Hulliger, Glutamic acid inducing kidney stone
biomimicry by a brushite/gelatin composite, J. Mater. Chem. B 1 (2013) 15011508.
[11] D. Bhatnagar, A.K. Bherwani, M. Simon, M.H. Rafailovich, Biomineralization on enzymatically cross-linked gelatin hydrogels in the absence of dexamethasone, J. Mater.
Chem. B 3 (2015) 52105219.
[12] S. Eiden-Amann, M. Viertelhaus, A. Hei, K.A. Hoetzer, J. Felsche, The inuence of
amino acids on the biomineralization of hydroxyapatite in gelatin, J. Inorg. Biochem.
91 (3) (2002) 481486.
[13] A. Bigi, G. Cojazzi, S. Panzavolta, K. Rubine, N. Roven, Mechanical and thermal properties of gelatin lms at different degrees of glutaraldehyde crosslinking, Biomaterials 22 (8) (2001) 763768.
[14] H.-W. Sung, D.-M. Huang, W.-H. Chang, R.-N. Huang, J.-C. Hus, Evaluation of gelatin
hydrogel crosslinked with various crosslinking agents as bioadhesives: in vitro
study, J. Biomed. Mater. Res. 46 (4) (1999) 520530.
[15] Z.S. Patel, M. Yamamoto, H. Ueda, Y. Tabata, A.G. Mikos, Biodegradable gelatin microparticles as delivery systems for the controlled release of bone morphogenetic
protein-2, Acta Biomater. 4 (5) (2008) 11261128.
[16] J. Ratanvaraporn, R. Rangkupan, H. Jeeratawatchai, S. Kanokpanont, S. Damrangsokkul,
Inuences of physical and chemical crosslinking techniques on electrospun type A and
B gelatin ber mats, Int. J. Biol. Macromol. 47 (4) (2010) 431438.
[17] H.-C. Liang, C. Wen-Hsiang, K.-J. Lin, S. Hsing-Wen, Genipin-crosslinked gelatin microspheres as a drug carrier for intramuscular administration: in vitro and in vivo
studies, J. Biomed. Mater. Res. A 65A (2) (2003) 271282 (J. Biomedical Materials
Res. Part B: Applied Biomaterials Published Online: 20 Mar 2003).
[18] S.K. Jain, et al., Calcium silicate based microspheres of repaglinide for gastroretentive
oating drug delivery: preparation and in vitro characterization, J. Control. Release
107 (2) (2005) 300309.
[19] C. Gomez, et al., Cytarabine release from comatrices of albumin microspheres in a
poly (lactide-co-glycolide) lm: in vitro and in vivo studies, Eur. J. Pharm. Biopharm.
57 (2) (2004) 225233.
[20] Z. Liu, et al., Study of an alginate/HPMC-based in situ gelling ophthalmic delivery
system for gatioxacin, Int. J. Pharm. 315 (12) (2006) 1217.
[21] N. Bhattarai, et al., PEG-grafted chitosan as an injectable thermosensitive hydrogel
for sustained protein release, J. Control. Release 103 (3) (2005) 609624.
[22] F. Iemma, et al., pH-sensitive hydrogel based on bovine serum albumin for oral drug
delivery, Int. J. Pharm. 312 (12) (2006) 151157.
[23] M. Changez, et al., Studies on biodegradation and release of gentamicin sulphate
from interpenetrating network hydrogel based on poly(acrylic acid) and gelatin:
in vitro and in vivo, Biomaterials 25 (1) (2004) 139146.
[24] M.D. Blaneo, R.M. Trigo, C. Teijon, C. Gomez, J.M. Teijon, In vitro evaluation of a ph
sensitive hydrogel for control of GI drug delivery from silicon-base matrices, Int. J.
Pharm. 130 (1) (1996) 8392.
[25] M.D. Blanco, C. Gomez, O. Gareia, J.M. Teijon, Polym. Gels Netw. 6 (1998) 57.
[26] H. Murakami, Y. Kawashima, T. Niw, T. Hin, H. Takeuchi, M. Kobayashi, Inuence of
the degrees of hydrolyzation and polymerization of poly (vinyl alcohol) on the
preparation and properties of poly (DL-lactide-co-glycolide) nanoparticles, Int. J.
Pharm. 149 (1997) 4349.
[27] A.M. Le Ray, M. Vert, J.C. Gautier, J. Benoit, End-chain radio labeling and in vitro stability studies of radio labeled poly (hydroxy acid) nanoparticles, J. Pharm. Sci. 83
(1994) 845851.
[28] G.W.R. Davidson, N.A. Peppas, Solute and penetrant diffusion in swellable polymers
V relaxation controlled transport in P(HEMA-Co MMA) copolymers, J. Control. Release 3 (14) (1986) 243258.
[29] A. Wang, Li-Fang, W.B. Chen, Tzu-Yichen, Shui Chunlu, J. Biomater. Sci. Polym. Ed. 14
(1) (2003) 27.
[30] G.K. Pal, T. Nidheesh, P.V. Suresh, Comparative study on characteristics and in vitro
bril formation ability of acid and pepsin soluble collagen, from the skin of catla
(Catla catla) and rohu (Labeo rohita), Food Res. Int. 76 (3) (2015) 804812.
[31] F. Pati, B. Adhikari, S. Dhara, Isolation and characterization of sh scale collagen of
higher thermal stability, Bioresour. Technol. 101 (10) (2010) 37373742.
[32] Y.J. Yin, K.D. Yao, G.X. Cheng, J.B. Ma, Properties of polyelectrolyte complex lms of
chitosan and gelatin, Polym. Int. 48 (1999) 428.
[33] N.Y. Wiley, in: G. Socrates (Ed.), Infrared Characteristics Group Frequencies,
Tables and Charts, 1996.
[34] Y.M. Liu, C. Jiaa, H. Do, A. Eltoukhy, Evaluation of amorphous carbon nitride thin
lms for magnetic rigid thin lms, IEEE Trans. Magn. 33 (5) (1997) 31063108.
[35] A.J. Kuijpers, G.H.M. Engbers, P.B. Van Wachem, J. Krijgsveld, S.A.J. Zaat, J. Dankert,
et al., Controlled delivery of antibacterial proteins from biodegradable matrices, J.
Control. Release 53 (1998) 235247.
[36] A.Y. Nosaka, Tanzawa, H.H-NMR studies on water in methacrylate hydrogels, J. Appl.
Polym. Sci. 43 (6) (1991) 11651170.
[37] I. Mie, M. Satoshi, M. Atsushi, F. Kenji, Japanese herbal medicine inchin-koto as a
therapeutic drug for liver brosis, J. Hepatol. 41 (2004) 584591.
[38] D. Cohn, M. Aronhime, B. Abdo, Poly (urethane)-crosslinked poly (HEMA)
hydrogels, J. Macromol. Sci. Pure Appl. Chem. 29A (1992) 841.
[39] B. Ramaraj, G. Radhakrishnan, Modication of the dynamic swelling behavior of
poly (2-hydroxyethyl methacrylate) hydrogels in water through interpenetrating
polymer network, Polymer 35 (1994) 21672173.
[40] P.J. Flory, Statistical thermodynamics of random networks, Proc. R. Soc. Lond. Ser. A
351 (1976) 351380.

H. Khan et al. / Materials Science and Engineering C 61 (2016) 457465

[41] A. Bilia, V. Corelli, G.D. Colo, Nannipieri, In vitro evaluation of a ph sensitive hydrogel
for control of GI drug delivery from silicon-base matrices, Int. J. Pharm. 130 (1)
(1996) 8392.
[42] P.I. Rose, H.F. Mark, N.M. Bikales, C.G. Overberger, G. Menges, J.I. Eds Kroschwitz, Encyclopedia of Polymer Science and Engineering, John Wiley, New York, 1987 488513.

465

[43] P. Johns, A. Courts, A.G. Ward, A. Courts, In Relationship Between Collagen and
GelatinEds. The Science and Technology of Gelatin, Academic Press, London 1977,
pp. 138177.
[44] Y. Qiu, K. Park, Environment sensitive hydrogel for drug delivery, Adv. Drug Deliv.
Rev. 53 (3) (2001) 321339.