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Author's personal copy
Carbohydrate Polymers 83 (2011) 1207–1212
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Genipin-cross-linked kappa-carrageenan/carboxymethyl cellulose beads
and effects on beta-carotene release
Ida Idayu Muhamad ∗ , Lim Shu Fen, Ng Hui Hui, Noor Anis Mustapha
Dept. of Bioprocess Engineering, Faculty of Chemical Engineering, Universiti Teknologi Malaysia, Johor Bahru 81310, Johor, Malaysia
a r t i c l e
i n f o
Article history:
Received 25 July 2010
Received in revised form 27 August 2010
Accepted 14 September 2010
Available online 18 September 2010
Keywords:
Hydrogel
Cross-linking
Genipin
Kappa-carrageenan/carboxymethyl
cellulose beads
Swelling ratio
a b s t r a c t
Beads of kappa-carrageenan/sodium carboxymethylcellulose were prepared based on different blend
formulations using genipin, a natural and non-toxic cross-linking reagent. Different genipin concentrations (0.5, 1.0, 1.5 mM) were used to study the effects on swelling ratio of the beads in different pH
values under simulated gastrointestinal tract condition (pH 1.2 and 7.4). Results have shown that the
cross-linked beads possess lower swelling ability in all pH conditions and swelling ratio decreases with
increasing genipin concentration (95.24% in pH 1.2; 100% in pH 7.4 at 0.5 mM genipin; 76.2% in pH 1.2;
85.71% in pH 7.4 at 1.5 mM genipin). It was also found the beads released beta-carotene slower and lesser
after being cross-linked. Microstructure study shows that cross-linked beads exhibited smoother surface
and more spherical shape compared to the native beads. This indicates that cross-linking of genipin has
enhanced the beads network stability and their structure to be applied as suitable hydrogel.
© 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Hydrogels are polymeric materials that have a threedimensional network structure and can swell considerably in
aqueous medium without dissolution. They can be prepared in a
wide-variety of physical forms: foams, films, sheets, beads, powder, tubes and blocks (Graham, 1986). In response to stimuli such as
pH, temperature, and presence of chemical species, solvent, light or
pressure, hydrogels have the properties and ability to change their
shapes or volumes (Gupta & Raghava, 2008). Qui and Park (2001)
has reported that this environmental sensitive hydrogel has been
studied for biomedical and pharmaceutical application in protecting drug and deliver it in response to the pH and temperature in
human gastro-intestinal tract.
Recent advance in hydrogel technology have focused on finding
more biocompatible, non-toxic material intended for pharmaceutical, biomedical or even in food application. Hydrogels formed from
polysaccharides, such as carrageenan, are good candidates for drug
release systems, owing to their nontoxicity and acceptance by regulating authorities and most importantly, to their easy gelling ability,
thermo reversibility of the gel network and appropriate viscoelastic
properties (Liu, Li, & Cai, 2006) that able to undergo harsh condition.
In order to increase the time frame and stability of hydrogel carrier for drug delivery, the polymeric material need to be
cross-linked. Several cross-linking reagents have been used for
∗ Corresponding author. Tel.: +60 7 5535541; fax: +60 7 5581463.
E-mail address: idayu@fkkksa.utm.my (I.I. Muhamad).
0144-8617/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2010.09.021
cross-linking such as glutaraldehyde, tripolyphosphate, ethylene
glycol, diglycidyl ether and diisocyanate. Genipin, a natural crosslinker is found to be 10000 times less toxicity than the common
used cross linking agent, glutaraldehyde is utilized to cross-link
the hydrogels with minimum cytotoxic effect. Studies have shown
that cross-linked hydrogels exhibit different properties as compared to uncross-linked hydrogels (Yuan et al., 2007). Cross-linked
hydrogels may have changes in their swelling properties, mechanical strength, degradation rate depending on the degree of cross
linking and more other factors (Moffat & Marra, 2005). Muzzarelli
(2009) reported the most important applications of genipin in conjunction with chitosan are the preparation of elastic and resistant
gels such as the cartilage substitutes, the manufacture of drug carriers for controlled release, the encapsulation of biological products
and living cells, and the medication of wounds in animals and
humans. Genipin might replace glutaraldehyde with the advantages of stability and biocompatibility of the crosslinked products
whose quality assessment and manipulation would be easier.
Therefore in this research, natural cross-linking agent—genipin
is selected to study the effect of cross linking on the formulated
kappa-carrageenan/carboxymethylcellulose hydrogel beads.
2. Experimental
2.1. Material
Sodium carboxymethyl cellulose (NaCMC) with average molecular weight of 250,000 was purchased from Acros Organic and
-carrageenan (C) was purchased from Sigma–Aldrich. Genipin
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was purchased from Challenge Bioproducts Co., Ltd. (Taiwan). All
chemicals are used as received without further purification.
2.2. Preparation of beads
2.2.1. Control blend
Control blend of -carrageenan/NaCMC beads are formulated
based on the C:NaCMC ratio of 60:40, 70:30, 80:20 and 90:10. The
hot carrageenan solution was blend with NaCMC solution according
to the ratio as mentioned. The homogeneous beads were made by
conventional dripping method into cold potassium chloride (KCl)
hardening solution layered with cold rape seed oil. The solution
was maintained at 10 ◦ C for 5 h to let the beads harden. Beads then
washed with diluted KCl solution to remove remaining oil layer.
Finally, beads were dried in 37 ◦ C oven overnight.
2.2.2. Cross-linking
The most suitable C:NaCMC blend ratio was chosen to be
cross-inked with three concentrations of genipin—0.5 mM, 1.0 mM
and 1.5 mM. Genipin stock solutions were prepared by dissolving
genipin powder in 10 percent of ethanol with continuous stirring.
Genipin solution was then added to the blend of C/NaCMC hot
solution and dripped into KCl solution to form cross-linked beads.
The formulated beads were then dried in oven at 37.5 ◦ C for 24 h.
2.2.3. Swelling ratio measurement
The swelling ratio of native and cross-linked beads were determined by immersion in solution of pH 1.2 (0.1 N HCl) and pH 7.4
buffer solution. Subsequently, the diameter of the swollen bead
(Dt ) was examined under microscope and the swelling ratio was
calculated according to the equation as follows:
Swelling ratio (%) =
D − D
t
0
D0
× 100
(1)
where Dt is the diameter of swollen beads at time t and D0 is the
initial diameter of dried beads. The experiment was performed in
triplicate and represented as a mean value.
2.2.4. Immobilization of ˇ-carotene in beads
0.5 mg/ml of -carotene that dissolved in ethanol was added
to the blend solution of -carrageenan/NaCMC. Different concentrations of genipin were then added to the hot solution to form
cross-linked beta carotene loaded beads by using dripping method.
All -carotene loaded beads were kept in hardening solution for
30 min and release test was performed immediately after hardening.
2.2.5. Release study of ˇ-carotene
The -carotene release study of the beads from each
cross-linked concentration was performed in the simulated gastrointestinal condition by the pH change method at 37 ◦ C. One gram
of beads was enclosed in the teabag and placed into beaker that
contained 50 ml of the dissolution medium. The beaker was placed
and incubated at 37 ± 2 ◦ C water bath. The pH of the dissolution
medium was kept at 0.1 N HCl for first 10 min, then changed to
buffer solution pH 6.6 and finally pH 7.4 up to minutes 30. 5 ml
of dissolution medium was withdrawn every 2 min and was then
assayed by UV–vis spectrophotometer at 446 nm. All experiments
were performed in triplicate. The amount of released -carotene
was calculated by interpolation from the -carotene standard curve
at 446 nm. A cumulative correction was made for the previously
removed sample to determine the total -carotene release.
2.2.6. Microstructure of hydrogel beads
The surface morphology of the native (uncross-linked), crosslinked bead was examined by using a Philips XL 40 scanning
Fig. 1. Swelling ratio versus time for different blend ratios of beads in buffer solution
pH 7.4.
electron microscope operates at 25 kV accelerating voltage. In
preparation of SEM examination, the samples were mounted on
metal grids and coated by gold using gold sputter coater under
vacuum before observation. The photomicrographs were taken at
different magnifications.
3. Results and discussion
3.1. Preparation of beads
-Carrageenan/NaCMC beads were formulated based on the
C:NaCMC ratio of 60:40, 70:30, 80:20 and 90:10. Significantly
spherical beads with homogeneous surface were produced and the
diameter of the beads formed is approximately 0.016–0.018 mm.
The wet beads were then dried in oven (37.5 ◦ C) overnight before
the swelling test. Swelling ratio measurement was done to determine the suitable blend ratio for further study.
3.2. Swelling ratio measurement
3.2.1. Control blend
Figs. 1 and 2 show the swelling trend for 60:40, 70:30, 80:20
and 90:10 blends when immersed in pH 7.4 buffer solution and pH
1.2 acidic medium for 60 min respectively. Dried bead with initial
diameter of 0.007 mm start to swell once immersed in the medium
and have reached the equilibrium of swelling after 40 min immersion in both medium. From both Figs. 1 and 2, the swelling ratio of
C/NaCMC beads displayed a systematic trend in accordance with
weight fraction, in which the degree of swelling increases with the
ratio of carrageenan. As in 60:40 blends, the beads contain highest
weight fraction of NaCMC, therefore the swelling in both medium
is the lowest. From the plot of swelling ratio versus time for both
Fig. 2. Swelling ratio over time for different blend ratios of bead in 0.1 N HCl (pH
1.2).
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I.I. Muhamad et al. / Carbohydrate Polymers 83 (2011) 1207–1212
Fig. 3. Comparison between swelling ratio of native beads and beads cross-linked
with different concentrations of genipin in pH 1.2 medium (0.1 N HCl) after equilibrium state.
pH, the 90:10 blend beads swell the fastest among all, followed by
80:20, 70:30 and the slowest 60:40.
NaCMC is considered as in weakly negative while carrageenan
is in negative states. NaCMC is a weak polyelectrolyte behaves
almost neutral polymer as in buffer solution pH 7.4. Therefore,
cross-linking point among polymer was coming from the sulfonate
group (SO3− ) in carrageenan. As the weight fraction of carrageenan
increases in the blend, i.e. in 90:10 ratios, the counterions in the
solution (SO3− ) also increase. The increases of SO3− ion contribute
to stronger electrostatic repulsion between the SO3− groups and
therefore the swelling of the polymer also increases (Mistsumata,
Suemitsu, Fujii, Fujii, & Taniguchi, 2003). Besides, more SO3− ion
causes the osmotic pressure to increase and finally the degree of
swelling increases as well.
In order to know the respond of hydrogel swelling on pH, the
swelling of beads was tested in pH 1.2 and pH 7.4 medium. By
comparing the swelling ratio of beads in both medium, most blend
ratio of beads exhibit better swelling in pH 7.4 medium than acidic
medium. In blend ratio of 70:30 beads, the swelling degree of bead
after equilibrium state is 109% and 100% respectively in pH 7.4
medium and acidic medium. At low pH, the carboxylate COONa
changes to acid form COOH, therefore most carboxymethyl groups
are in the form of COOH that is less ionized. Table 1 shows the comparison of beads characteristics for the different blend ratio beads.
As the pH increases, the carboxylic groups become ionized, and the
resulting repulsion in the network will cause the beads to swell.
Collapsing is observed in the 60:40 blend ratio beads (in acidic
medium) due to hydrogen bond formation, where the beads dissolved in the medium. This happened when the 60:40 blend beads
were immersed in pH 1.2 medium longer than 60 min.
Consequently, beads with blend ratio of 70:30 were chosen for
further investigation in cross-linking with genipin. Beads in this
ratio have not much different in degree of swelling as compared to
the blend ratio 80:20 beads. Although beads with blend ratio 80:20
and 90:10 have better swelling degree than beads with 70:30 ratio,
they were not suitable for the formation of beads as it did not produce spherical beads. According to Sankalia, Mashru, Sankalia, and
Sutrariya (2006), higher carrageenan concentrations in the ratio did
not produce spherical beads, probably due to the high viscosity of
the dripping solution. In addition, increase in dripping solution viscosity will lead to formation of larger beads that is less suitable for
nutraceutical study. Beads with blend ratio 60:40 were not chosen
as its gel structure is not strong and might dissolve in the medium
during study.
3.2.2. Cross-linking
The most suitable blend ratio—70:30 blends were cross-linked
with different genipin concentrations: 0.5 mM, 1.0 mM and 1.5 mM
to study the effect of cross-linker concentration on swelling. One of
the cross-linking effects can be obviously seen from Figs. 3 and 4,
1209
where all the beads have shown a decrease in swelling ratio after
crosslinked with genipin. From observation in both mediums, beads
sample cross-linked with lowest concentration of genipin linked
to the highest percentage increase in its swelling diameter. It can
be seen from both Figs. 3 and 4, beads crosslinked with 0.5 mM
genipin have the swelling of approximately 95.24% and 100% in pH
1.2 and pH 7.4 medium respectively. In contrast, beads crosslinked
with highest concentration of genipin (1.5 mM) show least percent
increase in diameter, approximately 76.2% in pH 1.2 medium and
85.71% in pH 7.4 medium. Native beads without cross-linking have
the highest swelling in both medium. This may due to the fact that
high concentration of genipin could result in great extent of chemical crosslinking of the C/NaCMC chains. This restricts the mobility
and hydration of the macromolecular chain in the beads and lead to
less swelling in diameter. For each crosslinked beads sample investigated, the swelling ratio in pH 1.2 medium was lower than the
swelling ratio in a pH 7.4 medium. This maybe attribute to the formation of hydrogen bond between the carboxymethyl cellulose due
to the existence of carboxylic group (–COOH) at low pH. At pH 7.4,
the carboxylic acid groups on the crosslinked beads became progressively ionized (–COO–). So the resultant beads swelled more
significantly due to a large swelling force induced by the electrostatic repulsion between the ionized groups (Song, Li, Yang, & Li,
2009).
Mi, Sung, Shyu, Su, and Peng (2003) who synthesized novel
chitosan gel beads by concurrent ionic and covalent crosslinking mechanisms involving tripolyphosphate (TPP) and genipin
reported that the co-crosslinking depends on pH where the covalent crosslinking dominates at pH values 7.0 and 9.0, whilst ionic
crosslinking dominates at pH 1.0, 3.0 and 5.0. There are evident
effects on the swelling and the enzymatic degradation of genipin
crosslinked chitosan derivatives. Chen et al. (2004) studied on N,Ocarboxymethyl chitosan/alginate hydrogel crosslinked by genipin
reported that the swelling ratio was higher at high pH than at low
pH.
3.3. Immobilization of ˇ-carotene in beads
Immobilization of -carotene in beads crosslinked with different genipin concentrations was then carried out to investigate
their characteristics. The beads with successful immobilization of
-carotene are easy to be recognize as it turned into transparent
orange in color beads compared to the original transparent beads.
Higher concentration of genipin cross-linked beads has darker
orange color and the beads are more elastic compared to the lower
concentration of genipin cross-linked beads. Moura, Figueiredo,
and Gil (2007) also concluded that under physiological conditions
the viscoelastic features of a chitosan solution and its gelling ability
could be tuned by changing the genipin concentration (very low in
all cases); relatively strong elastic gels were obtained.
Fig. 4. Comparison between swelling ratio of native beads and beads crosslinked
with different concentrations of genipin in buffer solution pH 7.4 after equilibrium
state.
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Table 1
Comparison of bead characteristics for the different blend ratio beads.
Blend ratio (C:NaCMC)
60:40
70:30
80:20
90:10
Swelling ratio (%)
pH 7.4
pH 1.2
85.71
109.52
114.29
150.00
85.71
100.00
114.29
133.33
Fig. 5. Standard curve of -carotene.
3.4. Release of ˇ-carotene
In this section, the ability of the beads to hold -carotene
was investigated by determining the release of -carotene from
each type of beads. To determine the concentration of -carotene
released, standard curve representing the absorbance at wavelength of 446 nm of different concentrations of -carotene was
constructed as shown in Fig. 5. The relation correlating this curve
is calculated using Eq. (2) (from -carotene standard curve):
Absorbance = concentration (mg/mL) × 21.355
(2)
From this relation, a concentration of unknown sample can be
determined. The accumulated release of -carotene was carried
out as a function of time at a pH change system in order to simulate
the gastrointestinal tract (GIT) conditions. The release rate of 1 g
native and crosslinked C/NaCMC beads was measured in 30 min
time.
From the accumulated release profile of -carotene as shown in
Fig. 6, the release of -carotene from the native beads was faster
compared to the release from crosslinked beads. At the first 2 min
immersion in pH 1.2 medium, the release of -carotene from native
beads was 7% while for 0.5 mM crosslinked beads was only 4%.
The amount of -carotene released from the crosslinked beads was
found to be less than that from the native beads. The lower the concentration of the genipin used, the larger amount of -carotene was
Shape (spherical)
Beads strength
Yes
Yes
No
No
Poor (dissolved in swelling medium)
Good
Too viscous
Too viscous
released. For lower concentration of cross-linker used, the crosslinking degree will decrease. According to Bachtsi and Kiparissides
(1995), as the degree of polymer cross-linking decreases, the density of the polymer network also decreases. Consequently, as the
available free space for drug diffusion increases, the rate of drug
release also increases. Meanwhile, an increase of the degree of
polymer cross-linking increases the polymer density and therefore
decreases the available free space for drug diffusion which results
in a decrease in drug release rates.
It is also noticed that the release of -carotene from beads in
acidic medium was slower compared to buffer solution pH 7.4
and buffer solution pH 6.0. This can be observed from the accumulated release profile where -carotene concentration from t = 0
to t = 10 min (when immersed in acidic medium) was low and
increase fast after t = 10 min (immersed in pH 6.0 medium) and
fastest after t = 20 min (when immersed in pH 7.4 medium). This
can be explained by the swelling behavior of the beads in which the
beads swell better in buffer solution pH 7.4 as compared to acidic
condition. Therefore the medium could easily diffuse into the beads
and hence a faster and higher amount of -carotene was released.
In short, native beads have the highest release rate if compared to
cross-linked beads. This means that cross-linking has changed the
mechanical characteristics and properties of the beads as it is now
able to hold the content in it for longer period. This agrees with Chen
et al. (2004) who found that the amount of albumin released from
their genipin-crosslinked NOCC/alginate hydrogel at pH 1.2 was
relatively low (20%), while at pH 7.4 it increased significantly (80%).
Another substituted chitosan, the carboxymethyl-hexanoyl chitosan amphiphatic hydrogel with excellent water-absorption and
water-retention capacity under neutral conditions was crosslinked
with genipin and then employed as a carrier for delivering ibuprofen and other amphiphatic agents (Liu et al., 2006).
3.5. Microstructure of hydrogel beads
The observation of shape and surface topography of the native
and crosslinked beads was done by scanning electron microscope.
Fig. 7(A)–(D) shows the micrograph of native, cross linked beads
with genipin concentration of 0.5, 1.0, and 1.5 mM respectively
Fig. 6. Accumulate release profile of -carotene under in vitro release condition (pH 1.2, followed by pH 6.6 and pH 7.4 medium).
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I.I. Muhamad et al. / Carbohydrate Polymers 83 (2011) 1207–1212
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Fig. 7. Scanning electron micrograph (100×) and (300×), (A) native bead, (B) 0.5 mM genipin crosslinked, (C) 1.0 mM genipin crosslinked, and (D) 1.5 mM genipin crosslinked.
at magnification of 100× and 300×. From the micrograph, it can
be observed that the shapes of the beads were not completely
spherical and the surface was rough and folded. These phenomena
are due to the beads shrunk during the drying process. Fig. 7(A)
shows that the surface of the native beads was irregular and rough
moon-like surface. Meanwhile, as for Fig. 7(B)–(D), the surface of
cross linked beads exhibited more spherical shape and smoother as
the cross-linker concentration increases. The change in the surface
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topography might be due to the change in molecular arrangement
after crosslinked with genipin. In view of this more stable behavior
the formulations were deemed suitable for encapsulation of cells
and bioactive compounds. This phenomenon genipin crosslinked
polymers results in highly swelling microgels with a variation in
volume of more than 100% between shrunken state and swollen
state in dilute solutions which can be of tremendous interest for
targeted release of the microgels contents in the gastrointestinal
tract.
ditions. The results suggest that the genipin-crosslinked C/NaCMC
beads may be used as a suitable carrier for nutraceuticals.
Acknowledgements
The authors gratefully acknowledge the financial support provided by FRGS from Ministry of Higher Education Malaysia, and
Research Management Centre Universiti Teknologi Malaysia.
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4. Conclusion
In this study, the most suitable blend ratio of 70:30 was chosen
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