Controlled release of ooxacin from chitosanmontmorillonite hydrogel

Shuibo Hua
a,b
, Huixia Yang
a,b
, Wenbo Wang
a,b
, Aiqin Wang
a,

a
Center for Eco-material and Green Chemistry, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, PR China
b
Graduate University of the Chinese Academy of Sciences, Beijing 100049, PR China
a b s t r a c t a r t i c l e i n f o
Article history:
Received 11 August 2009
Received in revised form 7 July 2010
Accepted 12 July 2010
Available online 23 July 2010
Keywords:
Chitosan
Montmorillonite
Nanocomposite
Drug release
Ooxacin
A series of ooxacin/montmorillonite/chitosan (OFL/MMT/CTS) hydrogels were prepared by solution
intercalation and ionic crosslinking with sodium tripolyphosphate (TPP). The structure and drug loading
of the beads were characterized by Fourier transform infrared spectroscopy (FTIR). The formation of
intercalated nanocomposites was conrmed by X-ray diffraction (XRD). The swelling and degradation of the
beads were inuenced by the pH of test medium and the MMT content. Compared to pure CTS beads, the
incorporation of MMT enhanced the drug entrapment, improved the swelling behavior and reduced the
drug release. The CTS beads without MMT dissolved after a 3 h immersion in pH 1.2 solution. No notable
disintegration was visually observed for MMT-containing beads even after a 10 h immersion. The
observations suggested that the electrostatic interaction between CTS and MMT enhanced the stability of
the beads and showed good potential for the use as drug carriers for sustained release.
2010 Published by Elsevier B.V.
1. Introduction
Drug delivery systems recently received great interests because
they realized the effective and targeted delivery of drug and
minimized the side effects resulting from the traditional drug-dosage
form in the pharmaceutical eld. A large number of drug delivery
systems have been conceived and developed (Ding et al., 2002; Li
et al., 2004; Ginebra et al., 2006; Malmsten, 2006; Tomonori and
Hiroshi, 2008; Taa et al., 2009). Clay mineral composites as known
drug delivery systems showed unique hybrid properties superior to
the components and the capability to incorporate various drug
substances (Byrne and Deasy, 2005; Aguzzi et al., 2007; Lin et al.,
2007).
MMT exhibits enhanced gel strength, mucoadhesive capability to
cross the gastrointestinal (GI) barrier and adsorb bacterial and
metabolic toxins such as steroidal metabolites (Herrera et al., 2000).
MMT is a common ingredient as both the excipient and active
substance in pharmaceutical products (Takahashi et al., 2005).
Cationic drugs can be intercalated into MMT by ion exchange
(Zheng et al., 2007; Jung et al., 2008; Pongjanyakul et al., 2009). The
regulation of the amount and rate of drug release is difcult when the
drug is intercalated by ion exchange.
Chitosan (CTS) is an amine-bearing, linear polysaccharide derived
from the N-deacetylation and depolymerization of chitin (i.e., poly-N-
acetyl-glucosamine) (Agnihotri et al., 2004). As a unique cationic
polysaccharide, CTS shows gel and lm forming properties and found
potential in the pharmaceutical industry as a drug delivery system
(Thanou et al., 2000; Dang and Leong, 2006). CTS can form
nanocomposites with MMT, and there are several reports about the
drug release behavior from the CTS/MMT nanocomposite lms,
scaffolds and nanoparticles (Wang et al., 2008; Depan et al., 2009).
Fromthese points of view, the combination of MMT with CTS beads as
a drug carrier is attractive.
In our study, ooxacin (Fig. 1), the uoroquinolone antibiotic,
commonly used for treatment of prophylaxis of a variety of bacterial
diseases, was tested as the cationic model drug in in vitro experiments.
OFL/MMT nanocomposites were prepared by the solution intercala-
tion technique. Without ltering out the free drug, OFL/MMT
intercalated CTS beads were obtained through ionic crosslinking and
their morphology and structure were characterized by scanning
electron microscopy (SEM), FTIR and XRD. Finally, swelling and in
vitro drug release were studied in simulated gastric uids (SGF, pH
1.2) and simulated intestinal uids (SIF, pH 7.4).
2. Materials and methods
2.1. Materials
Chitosan (CTS, deacetylation degree, 81%; mass average molar
mass, 910
5
) from shrimp shell was purchased from Yuhuan Ocean
Biochemical Co. (Taizhou, China). Ooxacin (OFL) was obtained from
Kunshan Double-crane Pharmaceutical Co., Ltd, China. High purity
montmorillonite (MMT, the reference substance for scientic re-
search; the content of montmorillonite is 99% determined by XRD
technology) was purchased from Zhejiang Sanding Technology Co.,
Ltd (Shaoxing, China), and the contents of SiO
2
and Al
2
O
3
are
Applied Clay Science 50 (2010) 112117
Corresponding author. Tel.: +86 931 4968118; fax: +86 931 8277088.
E-mail address: aqwang@licp.cas.cn (A. Wang).
0169-1317/$ see front matter 2010 Published by Elsevier B.V.
doi:10.1016/j.clay.2010.07.012
Contents lists available at ScienceDirect
Applied Clay Science
j our nal homepage: www. el sevi er. com/ l ocat e/ cl ay
59.48 mass% and 21.93 mass%, respectively. Tripolyphosphate (TPP)
was purchased from Sinopharm Chemical Reagent Co., Ltd, China.
Simulated gastric uid (SGF, pH 1.2) containing 21.25 mL HCl, 11.18 g
KCl in 3000 mL distilled water and simulated intestinal uid (SIF,
phosphate buffer solutions, PBS, pH 7.4), containing 20.4 g K
2
HPO
4
and 4.8 g NaOHin 3000 mL distilled water were prepared as described
in US Pharmacopoeia 30. All other chemicals were of analytical grade
and used as received.
2.2. Preparation of OFL/MMT/CTS hydrogel beads
The hydrogel beads were prepared according to an improved
technique using TPP as the gelling counterion (Sezer and Akbuga,
1995). A series of the samples with different amounts of MMT were
prepared according to the following procedure. 0.2 g MMT was
dispersed in 20 mL of 2 mass% acetic acid solution, then 0.20 g OFL
were added into the dispersion, followed by a continuous stirring at
roomtemperature for 1 h. CTS (0.60 g) was dissolved in the OFL/MMT
mixture to reach the nal concentration of 3% (mass/v) and stirred for
0.5 h. The beads were formed by dropping the bubble-free solution or
dispersion through a disposable syringe onto a gently agitated TPP
solution (50 mL, 5 mass%; pH 5.0, adjusted with standard 1 mol/L HCl
solution). After 0.5 h, the obtained beads were ltrated with a 100-
mesh stainless screen and rinsed with distilled water for three times.
Finally, the gel-like beads were air-dried for 24 h, followed by drying
at 70 C for 6 h. Similar procedures were used to prepare OFL-free
placebo beads. The beads corresponded to 0, 0.10, 0.20, 0.30, 0.40 and
0.50 g MMT were denoted as M0, M1, M2, M3, M4 and M5. The M0
and M4 beads without adding OFL were termed as M0C and M4C
(Table 1).
2.3. Entrapment efciency
The amount of OFL entrapped in the beads was calculated by
measuring the content of OFL in the gelling medium. The oating
small MMT particles were removed through a 0.45 m membrane
lter. The clear supercial solution was analyzed by HPLC (Waters
TM
600 Pump, 2998 Photodiode Array Detector) using the C18 column.
The mixture of acetonitrile and buffer solutions (25:75, v/v) was used
as the mobile phase. OFL was detected at the wavelength of 294 nm.
The buffer solution was prepared according to USP 30: 1300 mL of an
aqueous solution containing 4.0 g ammonium acetate and 7.0 g
sodium perchlorate were adjusted to pH 2.2 with phosphoric acid.
The drug content was determined by comparing with the standard
curve of OFL which was achieved from OFL solutions in PBS 7.4 with
concentration from 0.002 to 0.01 g/L. The drug entrapment efciency
(EE) is expressed as follows:
EE = Practical drug loading = Theoretical drug loading 100%
1
2.4. Swelling studies
Swelling studies and in vitro degradation of the beads were carried
out in two aqueous media: 0.1 mol/L HCl solution (pH 1.2) as
simulated gastric uid and phosphate buffer solutions (pH 7.4) as
simulated intestinal uid. Beads (100 mg) were placed at 37 C in the
dissolution test apparatus (ZRS-8G, Tianjing University Wireless
factory, China) containing 500 mL of the medium at 50 rpm. At
regular intervals, the beads were reweighed after carefully wiping off
excess liquid with a tissue paper. The mass change of the wet beads
Q
w
and of the dry beads Q
d
with respect to time was determined as
follows:
Q
w
= W
w
W
0
= W
0
2
Q
d
= W
d
W
0
= W
0
3
where W
w
and W
d
are the wet and dry mass of the beads at time t,
respectively; W
o
is the initial mass of beads.
2.5. In vitro drug release
The in vitro drug release tests were carried out using the USP 30
NO.2 dissolution test apparatus xed with six rotating paddles. The
release medium is a simulated intestinal uid (pH 7.4) or simulated
gastric uid (pH1.2) (500 mL, 37.50.5 C), and the speed of rotation
was 501 rpm. 50 mg samples were placed into each of the six cups,
and 5 mL solution was collected from the release medium at regular
intervals. After each sample collection, the equal amount of fresh
release medium at the same temperature was added. The amount of
drug released was monitored by a UVvis spectrophotometer
(SPECORD 200, Analytik Jera AG) at 294 nm based on the established
UV standard absorbance curve for OFL. In the concentration range
(2.5~12.510
5
mol/L) investigated, the UV absorbance obeyed the
Beer's law.
2.6. Characterization
FTIR spectra were recorded on a FTIR spectrophotometer (Thermo
Nicolet, NEXUS, TM) in the range of 4000400 cm
1
using KBr pellets.
The surface morphology was observed by scanning electron micros-
copy (JSM-5600LV, JEOL) after coating the samples with gold lm
using an acceleration voltage of 20 kV. Powder XRD analyses were
performed using a diffractometer with Cu anode (PAN analytical
X'pert PRO), running at 40 kV and 30 mA, scanning from 3 to 40 at
3/min.
2.7. Statistical analysis
Each experiment was carried out in triplicate and the results were
averaged. The inuence of MMT content on the entrapment and the
release of the drug from beads were statistically analyzed by one-way
ANOVA. The data were considered to be signicantly different at
pb0.05.
Fig. 1. Structure of OFL.
Table 1
Feed composition and drug content of the samples.
Code CTS (g) OFL (g) MMT (g) OFL content (mass%)
M0 0.6 0.2 0 7.81
M1 0.6 0.2 0.1 10.00
M2 0.6 0.2 0.2 10.92
M3 0.6 0.2 0.3 12.02
M4 0.6 0.2 0.4 12.74
M5 0.6 0.2 0.5 12.83
M0C 0.6 0 0
M4C 0.6 0 0.4
113 S. Hua et al. / Applied Clay Science 50 (2010) 112117
3. Results and discussion
3.1. Morphology of the beads
When the dispersion of CTS in acetic acid was dropped into TPP
solution, gelled spheres formed instantaneously due to the electro-
static interaction between positively charged CTS and negatively
charged TPP. Generally, the wet beads were spherical with a diameter
of about 5 mm and possessed a smooth surface. After air drying, the
diameter of test beads decreased to about 2 mm but still kept the
spherical shape. For any batches of nanocomposite beads with various
amounts of MMT, there was no obvious variation of the bead size. The
large size of wet beads suggested high swelling and water retention
ability. The surface morphology showed severe wrinkles, which was
caused by partial collapsing of the polymer network during drying
(Fig. 2). Compared to placebo M0C beads, little particles appeared on
the surface of the OFL-loaded beads (M0) due to the leakage of OFL
crystals from the network space of beads. However, the OFL crystals
disappeared with increasing MMT content, suggesting that the
loading of OFL was remarkably improved due to the incorporation
of MMT.
3.2. FTIR analysis
The intense characteristic bands of CTS at 1651 and 1602 cm
1
(Fig. 3) were ascribed to the absorptions of amide I and amide
II (Papadimitriou et al., 2008), which shifted to 1640 cm
1
and
1557 cm
1
after the formation of CTS beads with TPP addition. This
information indicated that the amide formed ionic bonds. This
interaction reduced the solubility of CTS and contributed to the
separation of CTS from the solution in the form of beads. The
absorption band of MMT at 3623 cm
1
(stretching vibration of
the interlayer OH groups) almost disappeared and the band at
1714 cm
1
(characteristic absorption of the C=O group of OFL) was
weakened in the spectra of the beads (M4). This result indicated
electrostatic interaction between OFL and MMT. The FTIR results
conrmed that CTS was crosslinked with TPP in the beads and OFL
was encapsulated in the beads or stabilized in the interlayer space of
MMT.
3.3. XRD analysis
The reection of CTS at around 20 (Fig. 4) was characteristic of the
hydrated crystalline structure (Liu et al., 2008), but its intensity was
largely weakened after forming the beads. Crystalline CTS was not
observed. OFL showed characteristic intense reections at 6 and
10.9 due to the presence of OFL crystals. These reections
disappeared in the pattern of M4, which indicated that OFL was
dispersed at the molecular level in the polymer matrix or intercalated
for organic-nanoclay hybrid systems (Zheng et al., 2007; Depan et al.,
2009) and no crystals were found in the drug-loaded matrices. The
XRD pattern of MMT showed the basal reection at about 6.68 with a
basal spacing of 1.32 nm. The basal spacing increased from1.32 nm to
2.14 nm after OFL addition indicating the intercalation of OFL.
However, due to its coiled or helicoidal structures, CTS was only
adsorbed on the surface of MMT or led to the very less intercalation to
MMT as the basal spacing only increased from 1.32 nm to 1.36 nm
(M4C). This result was in agreement with a previous study (Gnister
et al., 2007).
3.4. Entrapment efciency
The EE of the beads increased with the amount of MMT added
(Fig. 5). The entrapment of OFL in beads without MMT (M0) was 31.2,
whereas the corresponding entrapment in the MMT-containing beads
varied from 45.3 to 83.5. A comparison of EE with the different MMT
contents was evaluated by ANOVA and the F value was 906.41
(df =17, p=0). Thus, the incorporation of MMT in the beads very
effectively improved the entrapment of OFL.
Fig. 2. SEM images of (a) M0C, (b) M0, (c) M2, and (d) M4.
114 S. Hua et al. / Applied Clay Science 50 (2010) 112117
During the reaction with TPP, it is likely that a part of the drug
wouldleak into the solution. The additionof MMT obviously decreased
the leakage of OFL (Fig. 2). This signicant improvement can be best
ascribed to the following facts. MMT with the large specic area
adsorbs OFL not only at the external surfaces but also in the interlayer
space. On the other hand, the addition of MMT increased the viscosity
of the dispersions and retarded the diffusion of OFL (Fig. 2).
Sriamornsak et al. previously studied the rheological properties, size
of the aggregates, and the zeta potential of CTSMAS (magnesium
aluminum silicate) dispersions (Sriamornsak et al., 2007). They
considered that the electrostatic interactions between the surface
charges of MAS and the positively charged amino groups (NH
3
+
) of
CTS may affect the zeta potential and the ow behavior of the
dispersion.
3.5. Swelling studies
In order to simulate the possible effect of pHon drug release rate, a
swelling study was conducted in simulated gastric uid (pH 1.2) and
simulated intestinal uid (pH 7.4) at physiological temperature of
37 C (Fig. 6). The swelling ratio at pH 7.4 was very small (about 30%).
At pH 1.2, with increasing the MMT content, the initial swelling ratio
decreased (b1.2 h), but the swelling ratio within 10 h increased
signicantly (pb0.05). The mass of M0 beads increased by 4.68 fold
for the initial 1 h, and then was sharply reduced until the beads were
disintegrated for the subsequent 1 h immersion. However, the mass of
M2 beads increased by 6 fold for the rst 4 h, and then reduced to 4.3
times of their initial values for the subsequent 6 h immersion. For
swelling of M4 beads, the mass of M4 beads increased by 12 fold
within 10 h.
A greater extent of crosslinking of the beads restricted the mobility
of the polymer chains and thus limited the swelling. The swelling was
mainly inuenced by ionic interactions between CTS chains and MMT
interlayers, which depended on the crosslinking density. The
incorporation of MMT strengthened the network of prepared beads.
Liu et al. (2008) studied the drug release fromCTSMMT hydrogels by
electrostimulation, and considered that exfoliated MMT layers can act
as crosslinkers between CTS and MMT (Liu et al., 2008). In our study,
some MMT layers could also act as crosslinker between the CTS
chains. However, in contrast to decreased swelling by increasing MMT
content as reported by Liu et al. (2008), a greater swelling at pH1.2 for
10 h incubation was obtained, probably because the crosslinking
between CTS and TPP was broken. Meanwhile, electrostatic repulsion
occurred between protonated amine groups on CTS causing higher
water uptake as in the simulated gastric uid. However, no such
repulsion was effective in the simulated intestinal uid, which caused
only a small swelling at pH 7.4. If the crosslinking density became too
small, interactions were no longer strong enough to avoid disinte-
gration and the beads decomposed completely within 3 h (M0).
Fig. 3. FTIR spectra of CTS, OFL, MMT, M0C and M4 beads.
Fig. 4. XRD patterns of OFL, MMT, CTS, M0C, M4 and M4C beads. Fig. 5. Entrapment efciency of OFL at different MMT contents of the beads.
115 S. Hua et al. / Applied Clay Science 50 (2010) 112117
The dry masses of M0, M2andM4 beads at pH7.4decreasedrapidly
within the rst hour due to the leakage of the loaded beads. No notable
disintegration was visually observed, and the spherical shape of the
beads was retained even after 10 h. However, the dry mass of M0, M2
and M4 beads at pH 1.2 decreased strongly and disintegration was
clearly observed. Formation of more stable gels and slower disinte-
gration due to the addition of MMT was also reported with diclofenac/
magnesiumaluminumsilicate/Ca-alginate beads (Puttipipatkhachorn
et al., 2005) and lms of chitosan-g-lactic acid and MMT (Depan et al.,
2009). From these results, a possible model for the nanocomposite
beads is shown in Fig. 7. The physical crosslinking between CTS and
MMT layers formed the network structure of the beads accompanied
by the ionic crosslinking between CTS and TPP and thus enhanced the
stability of the beads.
3.6. In vitro release studies
In vitro release of OFL from the beads with various MMT contents
at pH 7.4 and 1.2 are shown in Figs. 8 and 9. The drug released at pH
1.2 was relatively faster than at pH 7.4. The noticeably higher release
rate of OFL at pH1.2 can be attributed to the higher solubility of OFL in
acidic medium (Cui et al., 2008) and the degradation behavior at pH
1.2 (Figs. 6 and 10). The drug release was correlated to the swelling
and degradation of the beads. The entrapped OFL in M0 bead was
released almost completely within 2 h because of the almost complete
degradation of the beads in SGF. The swelling of M4 beads in SGF,
especially for the initial 4 h, caused about 66% of entrapped OFL to
migrate out. For the subsequent 6 h, the bead swelling was minimal
and only about 13% of entrapped OFL was further released.
At pH1.2, the cumulative amount of OFL released for the test beads
without MMT addition (M0) was about 90%. In contrast, at any time of
incubation, the level of release was higher in the order of M0, M1, M2,
M3, M4 and M5, which clearly indicated that increased amount of
MMT reduced the extent of drug release (pb0.05). After 10 h, none of
the MMT-containing samples (M1 to M5) completely released the
encapsulated drug. Also, prolonged time did not markedly increase
the release (Nunes et al., 2007). This fact was more signicant in the
release prole of M5, where the release was below 35% and 70% in SIF
and SGF within 10 h. Based on the above analyses, the addition of a
certain amount of MMT not only improved the drug EE, but also
provided a slower and continuous drug release. Thus, the incorpora-
tion of MMT particles into biopolymers may be useful for the
sustained delivery formations of drugs and other bioactive molecules.
4. Conclusions
CTS/OFL/MMT hydrogel beads were prepared and characterized by
SEM, FTIR and XRD techniques. The beads showed improved drug
loading and controlled release. These properties were highly
dependent on the MMT content. The disintegration of the pure CTS
beads at pH 1.2 was overcome by introducing MMT, which made this
systemmore useful in targeting at lowpH. The drug release rate of the
Fig. 6. Mass change of wet beads (M0, M2 and M4) at pH 7.4 and 1.2.
Fig. 7. Schematic illustration of interactions between CTS, OFL and MMT.
Fig. 8. Effect of MMT content on the release prole at pH 7.4.
Fig. 9. Effect of MMT content on the release prole at pH 1.2.
116 S. Hua et al. / Applied Clay Science 50 (2010) 112117
beads was also inuenced by pH of the medium. The cumulative drug
release of OFL from the nanocomposite beads at pH 7.4 was slower
than that at pH 1.2, which decreased with increasing MMT content. A
sustained release system can be prepared by incorporating MMT into
the nanocomposite beads.
Acknowledgement
The authors would like to thank the Western Action Project of CAS
(No. KGCX2-YW-501) and the Special Research Fund of Scholarship of
the Dean of CAS for nancial support of this research.
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Fig. 10. Mass change of dry beads (M0, M2 and M4) at pH 7.4 and 1.2.
117 S. Hua et al. / Applied Clay Science 50 (2010) 112117