Alginate/carboxymethyl scleroglucan hydrogels for controlled release of protein drugs
ABSTRACT Alginate/carboxymethyl scleroglucan (CMSG) hydrogels were suggested as a novel carrier for the controlled release of protein
drugs. The drug release characteristics of alginate hydrogels were improved by CMSG addition. Scleroglucan (Sclg) was carboxymethylated
using monochloroacetic acid in aqueous alkaline medium. Alginate/CMSG hydrogels were prepared by dropping the mixture solution
of alginate/CMSG into calcium chloride solution. The swelling behaviors and drug release characteristics of the hydrogels
were investigated in the buffers of pH 1.2 or 7.4. As the CMSG content increased in the hydrogels, the swelling ratio of the
alginate/CMSG hydrogel increased rapidly in the buffer of pH 7.4. At pH 1.2, however, the swelling ratio significantly decreased
compared to that at pH 7.4. According to in vitro release tests, only 15% of ovalbumin, investigated as a model protein drug,
was released from the alginate/CMSG hydrogels at pH 1.2 within 6 h. At pH 7.4, however, the drug release significantly increased
due to the rapid swelling of the hydrogels. The release and swelling behaviors of the hydrogels could be controlled by changing
the CMSG content in the hydrogels. These results supported the use of alginate/CMSG hydrogels as a suitable carrier for the
controlled release of protein drugs in a pH responsive manner.
Keywordsalginate–beads–carboxymethylation–drug delivery systems–ovalbumin–scleroglucan
- SourceAvailable from: Debasis Das[show abstract] [hide abstract]
ABSTRACT: This work was aimed to synthesis a newer guar based hydrogel and to characterize it for drug delivery application. Guar Gum Alkyl Amine (GGAA) the new polymeric material based on Guar Gum was synthesized in our laboratory. The hydrogel were characterized for its dynamic swelling behavior, responsiveness towards different ionic strength, pH responsive behavior, drug - polymer interaction (FTIR), thermal stability study (TGA), surface morphology (SEM), pay load and in vitro drug release. This newer hydrogel was found to be responsive towards salt of different ionic strength. The hydrogel also exhibited a pH dependent swelling. The cross-sectional morphology of the GGAA hydrogel reveals the porous structure. When the hydrogel was loaded with Ibuprofen the pay load was found to be 40%. There were no drug polymer compatibility issues and the hydrogel was found to be thermally more stable when compared with the native polymer. This drug loaded hydrogel exhibit no detectable release of drug in the gastric pH of 1.2 for first two hours. Then the release was conducted in the simulated intestinal pH of 7.4. In the alkaline environment 90% of the drug was released in 10 hrs. The release of drug followed first order kinetics and obeyed higuchi model.08/2013;
Macromolecular Research, Vol. 16, No. 5, pp 429-433 (2008)
*Corresponding Author. E-mail: email@example.com
Alginate/Carboxymethyl Scleroglucan Hydrogels for Controlled Release of Protein Drugs
Chang-Moon Lee and Hwan-Jeong Jeong
Department of Nuclear Medicine, Chonbuk National University Medical School, Jeonju, Jeollabuk-Do 561-712, Korea
Research Institute of Clinical Medicine, Chonbuk National University Medical School, Jeonju, Jeollabuk-Do 561-712, Korea
Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju, Jeollabuk-Do 561-712, Korea
Department of Clinical Pathology, Gwangyang Health College, Gwangyang, Jeonnam 545-703, Korea
Faculty of Applied Chemical Engineering and The Research Institute for Catalysis, Chonnam National University, Gwangju 500-757, Korea
Received November 23, 2007; Revised February 16, 2008; Accepted February 19, 2008
Abstract: Alginate/carboxymethyl scleroglucan (CMSG) hydrogels were suggested as a novel carrier for the con-
trolled release of protein drugs. The drug release characteristics of alginate hydrogels were improved by CMSG
addition. Scleroglucan (Sclg) was carboxymethylated using monochloroacetic acid in aqueous alkaline medium.
Alginate/CMSG hydrogels were prepared by dropping the mixture solution of alginate/CMSG into calcium chloride
solution. The swelling behaviors and drug release characteristics of the hydrogels were investigated in the buffers of
pH 1.2 or 7.4. As the CMSG content increased in the hydrogels, the swelling ratio of the alginate/CMSG hydrogel
increased rapidly in the buffer of pH 7.4. At pH 1.2, however, the swelling ratio significantly decreased compared
to that at pH 7.4. According to in vitro release tests, only 15% of ovalbumin, investigated as a model protein drug,
was released from the alginate/CMSG hydrogels at pH 1.2 within 6 h. At pH 7.4, however, the drug release signifi-
cantly increased due to the rapid swelling of the hydrogels. The release and swelling behaviors of the hydrogels
could be controlled by changing the CMSG content in the hydrogels. These results supported the use of alginate/
CMSG hydrogels as a suitable carrier for the controlled release of protein drugs in a pH responsive manner.
Keywords: alginate, beads, carboxymethylation, drug delivery systems, ovalbumin, scleroglucan.
Administration of pharmaceutically active drugs through
an oral route is the most preferred route for therapy of vari-
ous diseases. However, oral drugs are readily degraded by
the low pH of gastric medium in the stomach and absorbed
poorly into action sites.1,2 Therefore the drugs should be pro-
tected from harsh environment such as inactivation, degra-
dation, and instability in the stomach.3 The problems can be
eliminated by incorporating the drugs into natural polymers.
Natural polymers, such as sodium alginate, chitosan, cel-
lulose, gelatin, and pectin have received much attention for
an oral delivery system due to their protecting effect of the
drugs from harsh environment in the stomach. They have
also low cytotoxicity, biocompatibility, and biodegradabil-
ity. Among these polymers, sodium alginate is very promis-
ing and has been widely exploited in the design for oral
delivery of protein or peptide drugs. Sodium alginate is a
natural polysaccharide derived from algae and a heteropoly-
mer made of β-D-mannuronic acid (M block) and α-L-gulu-
ronic acid (G block) residues in a linear form. Interestingly,
it has a gel-forming property in the presence of multivalent
cations such as calcium ions. The gel-formation of sodium
alginate takes place mainly at junctions in the G-G sequence
rich chain region known as the ‘egg box junctions’.4 The
hydrogel has been used as a delivery system for live cell,
protein, or peptide drugs because the gel-forming process is
carried out under mild conditions as compared with the
delivery system using poly(lactide-co-glycolide), poly(lac-
tide-co-glycolide), and poly(ε-caprolactone). In particulate,
the biological activity and the therapeutic effect of these
drugs loaded in the hydrogel can be still retained.5 However,
the drug release from the alginate hydrogel may be limited
at the absorption site due to the relatively strong ionic inter-
action between the carboxylic groups of alginate and cal-
cium ions.6 In order to eliminate the limited drug release,
C.-M. Lee et al.
430Macromol. Res., Vol. 16, No. 5, 2008
many attempts have been tried.7-9 For example, Lin et al.
prepared the chitosan/alginate hydrogel for oral drug delivery.10
They reported that release characteristics of the protein drug
from alginate hydrogel including chitosan could be improved
with pH sensitive pattern.
In this study, a carboxymethyl scleroglucan (CMSG) was
selected to improve the limited drug release from the algi-
nate hydrogel. Scleroglucan (Sclg) produced by micro-
organisms consists of a main chain of (1→3)-β-D-glucopyr-
anosyl units, bearing a branch chain of (1→6)-β-D-glucopy-
ranosyl monomer.11 Recently, Sclg has been proposed for
the sustained-drug release and the pH-controlled drug deliv-
ery through an oral route.12 CMSG, a derivative of Sclg hav-
ing a carboxymethyl substituent, relatively forms a weaker
hydrogel by ionic interaction with calcium ions than the alg-
inate hydrogel. Therefore, the combination of alginate and
CMSG will show the desirable swelling ratio and release
profile. The aim of this study is to prepare the alginate/
CMSG hydrogel for oral delivery of protein drugs. The
influence of CMSG on drug release and swelling properties
of the hydrogel was also investigated. Ovalbumin was used
as the protein drug. It is used to induct the mucosal immu-
nity in mucosal vaccine delivery. Thus, we selected ovalbu-
min as a model drug to evaluate the alginate/CMSG hydrogel
as an oral drug delivery carrier.
Materials. Sodium alginate of low viscosity (250 cps for
a 2% (w/v) solution at 25oC), ovalbumin (OVA), calcium
chloride, and Bradford reagent were purchased from Sigma
Chemical Co. (St. Louis, MO, U.S.A.). Scleroglucan (Sclg)
was presented from Deggusa Co. (Trostberg, Germany). All
chemicals were used without further purification.
Synthesis of Carboxymethyl Scleroglucan (CMSG). CMSG
was synthesized according to the procedure previously
described by de Nooy et al. with some modifications.11 In
briefly, 1 g of Sclg was suspended in 40 mL of isopropyl
alcohol and then the solution was stirred for 30 min at room
temperature. After adding 4 mL of NaOH solution (10 M),
the mixture was strongly stirred for 1 h. 1.8 g of monochlo-
roacetic acid was added into the reaction mixture. The final
solution was stirred for 3 h at 50oC and then neutralized
with acetic acid. After washing with aqueous methanol
solution and dialysis against water, the solid product was
freeze-dried. The synthesized CMSG was confirmed by the
Preparation of Alginate/CMSG Hydrogels. The alginate/
CMSG hydrogels were prepared by dropping aqueous algi-
nate-CMSG into calcium chloride solution. Alginate/CMSG
solutions at distinct compositions (alginate:CMSG (weight-%
ratio) = 2:0 (AS 0), 1.5:0.5 (AS 1), 1:1 (AS 2), 0.5:1.5 (AS 3),
0:2 (AS 4), 2:2 (AS 5), and 1.5:1.5 (AS 6)) were prepared.
The prepared alginate-CMSG solutions were dropped into
a stirred 0.1 M of calcium chloride solution through a
syringe needle. The hydrogels were rinsed with distilled
water to remove unreacted calcium chloride and then dried
at 37oC for 24 h. The hydrogels were examined using an
optical digital camera (Optical zoom DSC-T1, Sony Co.,
Swelling Properties of Alginate/CMSG Hydrogels. The
swelling properties of the alginate-CMSG hydrogels were
evaluated by immersing dried the hydrogels to swell in 1 mL
of HCl buffer (0.1M, pH1.2) or PBS buffer (0.1 M, pH 7.4)
at 37oC. At specific time intervals, the immersed hydrogels
were removed from the swelling buffer and excess water on
the surface of the samples was absorbed with Whatman paper.
The swelling (Qs, %) were expressed according to the fol-
Where Ws is the weight of the swollen hydrogels and Wd is
the weight of the dried hydrogels. The data was represented
by the mean ± S.D. of three independent experiments.
Release Studies of OVA from the Alginate/CMSG
Hydrogels. In order to prepare drug-loaded beads, OVA
with a final concentration of 1% (w/v) was added to algi-
nate/CMSG solution and the mixture was then dropped into
a calcium chloride solution (0.1 M). The rest of the proce-
dure was similar to those used in the preparation of the algi-
To investigate the release profiles of OVA from the hydro-
gels, dried samples were immersed in a buffer with pH
value of 1.2 or 7.4 at 37oC. At predetermined time point,
100 μL of the solution from release medium was taken out
and the released drug was analyzed by the Bradford method
at 595nm using a UV spectrophotometer (UV-1201, Shimadzu,
Kyoto, Japan). The percentage of cumulative amount of released
OVA was calculated from the standard calibration curve pre-
Results and Discussion
Characteristics of CMSG. Figure 1 shows the FT-IR
spectra of native scleroglucan and CMSG. The intensity
increase of the peak at 1067 cm-1 in CMSG indicates C-O
stretch. The peak of 1605 cm-1 in CMSG is a characteristic
band for the asymmetrical carboxylic acid stretching, sug-
gesting that carboxymethyl groups exist on CMSG. The
band at 1432 is assigned to the stretching vibration modes
of the symmetrical carboxylic acid. This result shows that
carboxymethyl groups were substituted to Sclg.
Characteristic of Alginate/CMSG Hydrogels. Alginate
has been used to encapsulate cells and drugs since it forms
an ionic hydrogel in the presence of calcium ions under mild
conditions.13 When CMSG solution was added into calcium
chloride solution, hydrogel formation was observed. This
Alginate/CMSG Hydrogels for Controlled Release of Protein Drugs
Macromol. Res., Vol. 16, No. 5, 2008 431
phenomenon indicates that ionic interaction between the car-
boxylate ions of CMSG and calcium ions are presence.14 The
physical strength of the CMSG hydrogel by calcium ions
was relatively weak compare with that of the alginate
hydrogel (Figure 2).
Figure 3 shows the photographs of the alginate/CMSG
hydrogels prepared at distinct compositions. In all the com-
positions, the hydrogel could be formed with a regular
shape and a useful strength. The diameters of the hydrogels
were approximately 0.3-0.4 mm. The photographs of the
alginate/CMSG hydrogels in dried state and swelled state
were shown in Figure 4. In the formation of the hydrogels
using only CMSG without alginate, their shape was spherical.
However, the shape with an irregular form was observed
after drying. On the other hand, when alginate content in the
hydrogels increased, the spontaneous formation of spherical
gel with strong strength was observed. This phenomenon
demonstrates that ionic interaction between CMSG and cal-
cium ions occurs, but the cross-linking degree in the hydro-
gels decreases significantly as the CMSG content increased
in the alginate/CMSG hydrogel.10 The cross-linking density
was improved and controlled by addition of alginate. These
results may be due to the difference of cross-linking proper-
ties by calium ions in the formation of the alginate/CMSG
hydrogels. Alginate had a strong cross-linking by calium
ions, while CMSG formed relatively a weakly cross-linked
Swelling Ability of Alginate/CMSG Hydrogels. Drug
release profiles from a hydrogel depend upon their swelling
behaviors. Swelling behaviors of alginate/CMSG hydrogel
were investigated in the PBS buffer (pH 7.4) and HCl buffer
(pH 1.2) at 37oC. Figure 5 shows the swelling behaviors of
the alginate/CMSG hydrogels. The swelling ability was
dependent on the CMSG content in the hydrogels. As the
CMSG content increased in the composition of the hydro-
gels, the rapid swelling showed in the buffer of pH 7.4. The
hydrogels of AS 1 and 2 attained to maximum swelling per-
Figure 1. FT-IR spectra of slceroglucan and carboxymethylsclero-
Figure 3. Photographs of the alginate/CMSG hydrogels cross-
linked with calcium ions. (a) AS 0, (b) AS 1, (c) AS 2, and (d) AS 3.
Figure 4. Photographs of the aliginate/CMSG hydrogels in dried
state and swelled state.
Figure 2. Schematically illustration of the physical structures of
the alginate/CMSG hydrogels cross-linked with calcium ions.
C.-M. Lee et al.
432Macromol. Res., Vol. 16, No. 5, 2008
cent of 982.3±58.5% and 1167.3±60.8% within only 1h.
After attaining maximum swelling percent, the hydrogels
began to lose weight and finally dissolve. The hydrogels of
AS 4 exhibited maximum swelling percent of 1630 ± 42.0%
for 150 min in the buffer of pH 7.4. These swelling behav-
iors may be explained as following. It is because CMSG is
attributed to the poor mechanical strength of the hydrogels
by their weak cross-linking network.15 On the other hand, in
the buffer of pH 1.2, the swelling percent of the hydrogels
showed the significant difference as compared to those in
the buffer of pH 7.4. At pH 1.2, the hydrogels swelled rap-
idly within first 15 min and then did not swell further. The
present of CMSG in the hydrogels may be attributed to high
swelling percent and it affects the mechanical strength of
the hydrogel. In the design of oral delivery system for peptide
or protein drugs, pH responsive hydrogels have attracted
increasing attention because drugs administered orally are
absorbed through the small intestine. In this study, the swelling
of the alginate/CMSG hydrogel in the stomach can be mini-
mal and thus the drug release can also be minimal. As the
hydrogels passed down the intestine tract, the release ratio
of the drugs from the hydrogels will increase with pH
responsive pattern. Therefore, the alginate/CMSG hydro-
gels are a suitable carrier for the intestinal delivery of drugs
through an oral route.4 Figure 6 shows swelling behaviors of
the hydrogels prepared with the alginate-to-CMSG weight
ratio of 1:1. In the buffer of pH 7.4, as the total concentra-
tion of alginate-CMSG increased, the swelling ratio of the
hydrogels decreased. The hydrogels of AS 2 with the algi-
nate-CMSG composition of 1%:1% (w/v) showed rapid
swelling ratio and stared to disintegrate within 1 h in the
buffer of pH 7.4. In contrast, the hydrogels of AS 5 and 6
with the alginate-CMSG composition of 1.5%:1.5% (w/v)
and 2%:2% (w/v) swelled slowly and attained to the maxi-
mum swelling percent at 150 min. These results may be due
to the tight ionic cross-link between alginate and calcium
ions.10 Swelling behaviors of the alginate/CMSG hydrogels
could be controlled by changing the content of alginate and
CMSG, and it may be influenced on drug release from the
Release Profiles of OVA from Alginate/CMSG Hydrogels.
Figure 7 shows release profiles of OVA from the alginate/
CMSG hydrogels. As shown in the figure, in the buffer of
pH 1.2, the cumulative amount of OVA released from the
hydrogels was less than 15% within 6 h. In contrast, as the
CMSG content in the hydrogels increased, the OVA amount
released from the hydrogels significantly increased in the
buffer of pH 7.4. After 1 h, the hydrogels of AS 4 released
47.3 ± 7.5% of total OVA loaded in the hydrogels, but the
amount of OVA released from the hydrogels of AS 1 was
approximately 100% for the same time. This result shows
that the drug release behaviors from the hydrogels are
Figure 6. Swelling behavior of the alginate/CMSG hydrogels for
AS 2 (triangle), AS 5 (hexagon), and AS 6 (diamond) at 37oC in
the buffer of pH 1.2 (closed symbol) and 7.4 (open symbol).
Figure 7. Release properties of OVA from the alginate/CMSG
hydrogels for AS 0 (square), AS 1 (triangle), AS 2 (circle), and
AS 3 (diamond) at 37oC in the buffer of pH 1.2 (closed symbol)
and 7.4 (open symbol).
Figure 5. Swelling behavior of the alginate/CMSG hydrogels for
AS 0 (square), AS 1 (triangle), AS 2 (circle), and AS 3 (diamond)
at 37oC in the buffer of pH1.2 (closed symbol) and 7.4 (open symbol).
Alginate/CMSG Hydrogels for Controlled Release of Protein Drugs
Macromol. Res., Vol. 16, No. 5, 2008433
matched to the swelling behaviors of the hydrogels.
OVA release profiles from the hydrogels prepared with
the alginate-to-CMSG weight ratio of 1:1 were shown in
Figure 8. In the buffer of pH 7.4, as the total concentration
of alginate-CMSG increased, the release of OVA compara-
tively decreased. The hydrogels of AS 5 and 6 with the algi-
nate-CMSG composition of 1.5%:1.5% and 2%:2% released
83.4 ± 3.7% and 71.9 ± 1.8% of the total of OVA loaded in
the hydrogels within 150 min. The hydrogel with the algi-
nate-CMSG composition of 1%:1% exhibited more the
rapid release characteristics within the same time. OVA
release from the hydrogels could be controlled by changing
the content of alginate and CMSG in the hydrogels. As our
expectation, the limited drug release properties from the algi-
nate hydrogels could be improved by addition of CMSG.
Recently, George and Abraham reported the pH sensitive
alginate-guar gum hydrogel for the controlled delivery of
protein drugs.16 The alginate-guar gum hydrogel was required
the use of cross-linking procedures by glutaraldehyde treat-
ment. However, the use of cross-linking agents may lead to
toxic side effects. In contrast, the hydrogel presented in this
study was prepared under mild condition and it is biocom-
patible without toxic side effects. In the design of oral deliv-
ery system for peptide or protein drugs, pH responsive hydrogels
have attracted increasing attention because drugs adminis-
tered orally are absorbed through the small intestine. In this
study, the swelling of the alginate/CMSG hydrogel in the
stomach can be minimal and thus the drug release can also be
minimal. As the hydrogels passed down the intestine tract,
the release ratio of the drugs from the hydrogels will increase
with pH responsive pattern. Therefore, the alginate/CMSG
hydrogels are a suitable carrier for the intestinal delivery of
drugs through an oral route.
It is concluded that the alginate/CMSG hydrogels cross-
linked calcium can be a suitable delivery carrier for a protein
drug. The main advantage of this system is to improve the
limited swelling and release properties of the alginate hydro-
gels. In this study, the swelling behaviors were dependent
on the content of CMSG in the alginate/CMSG hydrogels and
the release of OVA from the alginate/CMSG hydrogels was
controlled by the changing the content of CMSG. Finally,
these results clearly suggest that the hydrogel prepared with
alginate and CMSG is a good candidate as a protein delivery
Acknowledgements. This research was supported by the
Program for the Training of Graduate Students in Regional
Innovation which was conducted by the Ministry of Commerce,
Industry and Energy of the Korea Government.
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Figure 8. Release properties of OVA from the alginate/CMSG
hydrogels for AS 2 (circle), AS 5 (triangle), and AS 6 (hexagon)
at 37oC in the buffer of pH 1.2 (closed symbol) and 7.4 (open