European Journal of Pharmaceutical Sciences 24 (2005) 77–84

Preparation of chitosan beads by simultaneous

cross-linking/insolubilisation in basic pH
Rheological optimisation and drug loading/release behaviour
R. Barreiro-Iglesias, R. Coronilla, A. Concheiro, C. Alvarez-Lorenzo∗
Departamento de Farmacia y Tecnologı́a Farmacéutica, Facultad de Farmacia, Universidad de Santiago de Compostela,
15872-Santiago de Compostela, Spain

Received 12 May 2004; received in revised form 27 September 2004; accepted 29 September 2004
Available online 19 November 2004

Abstract

A one-step procedure to prepare chitosan beads by simultaneous cross-linking with glutaraldehyde and insolubilisation in 1.5 M NaOH
solution has been developed. The optimisation of the procedure was carried out by monitoring the evolution of the loss and storage moduli
of chitosan solutions (1.5% (w/v), in acetic acid 0.2 M) in the presence of different proportions of glutaraldehyde. Increasing the chitosan
molecular weight, glutaraldehyde concentration and/or process temperature from 20 to 37 ◦ C, a reduction of time to reach the gel point was
observed. The diameter of freshly prepared swollen beads was 3.2 ± 0.4 mm and, after drying 0.48 ± 0.18 mm. Swollen or previously dried
beads were loaded with metronidazole by immersion in 0.1% (w/v), drug solution in a phosphate buffer pH 7.5, purified water, 0.2 M acetic acid
or 0.1 M HCl. Beads synthesised at 37 ◦ C experimented faster swelling than the ones prepared at 20 ◦ C and even disintegrated in acetic acid.
The amounts of metronidazole loaded (ranging from 1 to 286 mg/g dried beads) increased with swelling capacity of beads. The release studies
carried out in 0.1 M HCl indicated that, regardless of the medium used to load the beads, all of them released the dose in less than 30 min. In
summary, applying this one-step procedure and choosing the adequate glutaraldehyde proportion, it is possible to obtain particles of chitosan
cross-linked with itself, which exhibit pH-sensitive swelling and which are able to release all the drug quickly into an acidic environment
such as the stomach. The results obtained also highlight the importance of the pH of the medium for modulating the amount of drug loaded
(it is remarkably greater at lower pHs) and the influence of temperature at which the beads are prepared on their tendency to disintegrate.
© 2004 Elsevier B.V. All rights reserved.

Keywords: Chitosan deacetylation; Chitosan cross-linking; Oscillatory rheometry; Metronidazole; Glutaraldehyde; Microparticles

1. Introduction capability have shown an enormous potential as the basis

Cationic polysaccharides, such as cationic celluloses,
cationic guar gums, amino-modified starches and chitosans,
are large-scale commercial products that have many useful
characteristics such as hydrophilicity, biocompatibility, and
bioadhesivity (Luyen and Huong, 1996). The development
of procedures to cross-link cationic polysaccharides has in-
creased their possible uses. Polymer hydrogels that combine
adequate mechanical properties with a high drug loading
∗ Corresponding author. Tel.: +34 981 563 100; fax: +34 981 547 148.
E-mail address: ffrusdog@usc.es (C. Alvarez-Lorenzo).
for controlled drug delivery systems (Peppas et al., 2000;
Alvarez-Lorenzo and Concheiro, 2003, 2004). In the case
of chitosan, the amino groups of the polymer may allow
the establishment of different types of interactions with both
non-ionic and ionic drugs (Kumbar et al., 2002) and also
provide pH-sensitive systems, which swell at gastric con-
ditions allowing a site-specific release (Hejazi and Amiji,
2003). Microparticulate systems based on chitosan have been
proposed for local treatment of gastric infections since they
can cover a wide mucose area and release the drug instan-
taneously (Remuñán-López et al., 2000; Hejazi and Amiji,
2004).

0928-0987/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejps.2004.09.013
78 R. Barreiro-Iglesias et al. / European Journal of Pharmaceutical Sciences 24 (2005) 77–84
Several procedures to prepare hydrogels (Berger et al.,
2004a,b) and microparticles (Peniche et al., 2003; Sinha
et al., 2004) from chitosan have recently been revised. Most
of the methods involve the use of organic solvents and the
formation of emulsions or coacervates; the cross-linking of
the polymer taking place in a second step (Koseva et al.,
1999; Kumbar et al., 2002). However, despite the numerous
cross-linking methods described, few papers have focused
on the characterisation of the kinetics of the forming net-
works (Mi et al., 2000) and on how the different variables of
production influence the drug loading/release performance
of the cross-linked systems (Gupta and Ravi Kumar, 2000;
Dini et al., 2003; Ko et al., 2003; Alsarra et al., 2004). The
cross-linking processes that involve covalent bonding to the
amino groups, can alter the pH sensitivity of the network and
its capability to interact with oppositely charged molecules
and surfaces (Berger et al., 2004a). Thus, the degree of cross-
linking is a key parameter for chitosan systems. Additionally,
since most covalent cross-linkers can have toxic effects above
a certain level (Ghanem and Ghaly, 2004), the improvement
of the yield of the cross-linking process—i.e. how to reach
a certain degree of cross-linking with the minimum amount
of cross-linking molecules—would be useful to enhance the
safety of these systems.
The aim of this work was to develop a one-step proce-
dure to obtain chitosan beads by simultaneous covalent cross-
linking and precipitation in an aqueous medium by a simple
change in pH. To characterise the reaction with the cross-
linker, the gelation of chitosan solutions with glutaraldehyde
was monitored by the evolution of their viscoelastic prop-
erties through oscillatory rheometry analysis at low angular
frequencies. Then, chitosan beads were synthesised under
various conditions selected from the rheological experiments.
Finally, the swelling and drug loading in different pH media
were analysed to elucidate the most adequate procedure to
obtain beads with a high drug dose and able to release all of
it in acidic environment.
2. Materials and methods
2.1. Materials
Two commercially available chitosans were used: Chit-F
from Georges S. Daras S.A. (France, chitosan 222, batch
991012) and Chit-I from Marine Chemicals (India, batch
MI/OA/270/FC/99/4294). Glutaraldehyde (25% (v/v), in pu-
rified water) and metronidazole were from Merck Co. (Ger-
many) and Sigma–Aldrich (USA), respectively. Water puri-
fied by reverse osmosis (MilliQ® , Millipore Spain) with a
resistivity above 1.82 M
cm was used.
2.2. Structural characterisation of the chitosans
Degree of deacetylation: The elemental composition of
each chitosan was determined using a Carlo-Erba 1108 El-
emental Analyzer (Fisons Instruments, UK). The degree
of acetylation was calculated from the carbon/nitrogen ra-
tio (C/N). The latter varies from 5.145 in completely N-
deacetylated chitosan to 6.861 in the fully N-acetylated chitin
(Kasaai et al., 2000). The degree of deacetylation was there-
fore calculated according to:


C/N − 5.145
DD = 100 − × 100 . (1)
6.861 − 5.145
Intrinsic viscosity and molecular weight: The viscosity of
chitosan dispersions in 0.5 M acetic acid/0.25 M NaCl (0.02,
0.04, 0.06, 0.08 or 0.10 g dL−1 ) at 25 ◦ C was measured in
a Cannon-Fenske capillary viscometer (six determinations
per concentration). Intrinsic viscosity ([η], dL g−1 ) was esti-
mated by fitting Martin’s equation to the results thus obtained:
ηsp
log = log[η] + k[η]c (2)
C
In this equation, c represents the polymer concentration in
g dL−1 and k a constant.
Mean molecular weights, M, were then estimated from the
[η] values using the Mark–Houwink equation:
[η] = KM a (3)
with constants K and a set to 2.14 × 10−3 and 0.657, respec-
tively (Rege and Block, 1999).
Infrared spectroscopy: IR spectra were recorded over the
range 400–4000 cm−1 , in a Bruker IFS 66V FT-IR spectrom-
eter (Germany), using the potassium bromide pellet tech-
nique.
X-ray spectroscopy: X-ray diffraction spectra of chitosan
were recorded in a Philips PW 1710 (Germany) at a rate of
1.5◦ 2θ/min, in the range between 5 and 40 ◦ 2θ, using Cu
K␣ radiation.
2.3. Rheological characterisation of the cross-linking
process
Chitosan solutions (1.5% (w/v)) were prepared by dis-
persing the required amount of dry polymer in 100 mL of
0.2 M acetic acid under stirring. The system was left to stand
for 24 h at room temperature for complete hydration of the
polymer and removal of the bubbles. Glutaraldehyde (0.02 to
0.12 mL of commercial solution) was added to 10 mL of the
polymer solution at 20 ◦ C, stirred for two minutes, sonicated
30 s and, immediately, a portion was used for the rheologi-
cal analysis and the other portion transferred to a test tube,
hermetically closed, and kept at 20 ◦ C. The rheological be-
haviour of the dispersion spread on the Peltier plate of the
Rheolyst AR-1000N rheometer (TA Instruments, New Cas-
tle DE, USA) was evaluated in triplicate at 20 ◦ C and at 37 ◦ C
using a cone-plate (2◦ , 6 cm diameter) measuring geometry.
The evolution in time of the storage and loss moduli (G and
G , respectively) and of tan δ (=G /G ) was recorded for an
angular frequency of 0.1 rad/s and a shear stress of 0.1 Pa,
by measuring these parameters for the initial 60–120 min of
 R. Barreiro-Iglesias et al. / European Journal of Pharmaceutical Sciences 24 (2005) 77–84 79

the gelation process. The time at which G and G crossover Table 1
Structural characteristics of the chitosans
(tan δ = 1) was arbitrarily chosen as the gelation time (tgel )
(Vogelsberger et al., 2000; Mours and Winter, 2000). Chitosan N (%) C/N DD (%) Intrinsic Molecular
viscosity weight
(dL g−1 )* (Da)*
2.4. Preparation of chitosan beads
Chit-F 7.07 5.55 76.2 9.55 (1.00) 359150
(11569)
To prepare the beads, 10 g of chitosan dispersion (1.5% Chit-I 7.84 5.41 84.6 5.90 (0.91) 172242
(w/v), in acetic acid 0.2 M) were mixed with 0.090 mL glu- (9971)
taraldehyde solution (25% (v/v), in purified water) for 30 s * Mean values (standard deviation); n = 6.
and then, were added drop wise (21-gauge needle) to a 1.5 M
NaOH solution (100 mL) equilibrated at 20 ◦ C (cold beads) DD and a greater molecular weight than Chit-I. The dif-
or 37 ◦ C (hot beads). After 15 min in this solution, the beads ferences in DD were also clearly seen in the FT-IR spectra
were washed for 3 days, without stirring, with purified wa- (Fig. 1); Chit-F featuring sharper amide bands at 1659 cm−1
ter at 20 ◦ C (cold beads) or 37 ◦ C (hot beads), changing the and at 1559 cm−1 , and a less intense primary amine band at
medium each 12 h in order to remove the unreacted glu- 1597 cm−1 . Chitosans may show three different morpholo-
taraldehyde and the excess of NaOH. gies: non-crystalline, hydrated crystalline, and anhydrous
crystalline, which can be identified by X-ray diffraction. The
2.5. Characterisation of chitosan beads peak at around 10◦ 2θ that appears in the Chit-F diffrac-
togram, in addition to the peak at 20◦ 2θ (Fig. 2), is charac-
2.5.1. Degree of swelling teristic of the hydrated crystalline structure and also of chitin
The swelling rates of desiccated beads (oven at 40 ◦ C) (Luyen and Huong, 1996), which may be related to its lower
in different aqueous media were followed using a zoom DD. The lack of this peak in the Chit-I is compatible with
stereo microscope Olympus (SZ-6045-TR-F) equipped with the anhydrous crystalline structure. Additionally, Chit-F par-
a colour videocamera DP12 (Olympus, Japan). The degree of ticles were clearer (completely white compared to the yellow
swelling was estimated as the ratio of the area of the projected
surface of the particles, analysed with DP-SOFT software,
compared to that of freshly prepared swollen beads.

2.5.2. Drug loading

Chitosan beads were loaded with metronidazole by im-
mersion of freshly prepared swollen beads or oven dried
beads (40 ◦ C for 2 days) in 0.1% (w/v) metronidazole solu-
tions in phosphate buffer pH 7.4, purified water, 0.2 M acetic
acid or 0.1 M HCl for 90 min (or for 15 min in the case of
Chit-F swollen beads, prepared at 37 ◦ C, that disintegrated in
acid medium after this time). Additionally, in a third expe-
rience, dried beads were immersed in each medium without
drug for 15 min and then the medium was exchanged by the
drug solution. Finally, in all cases, the beads were desiccated
at 40 ◦ C until equilibrium (constant weight). The amount of Fig. 1. FT-IR spectra of the chitosans.
drug loaded was estimated from the total amount released
after 4 h in 0.1 M HCl.

2.5.3. Drug release

Dissolution studies were carried out in 0.1 M HCl (5 beads
in 5 mL dissolution medium). The amount of metronidazole
released was quantified spectrophotometrically at 277 nm
(HP Agilent 8453, Germany).

3. Results and discussion

3.1. Polymer structure

Some structural parameters of the two chitosans selected

for the study are shown in Table 1. Chit-F presents a lower Fig. 2. X-ray spectra of the chitosans.
80 R. Barreiro-Iglesias et al. / European Journal of Pharmaceutical Sciences 24 (2005) 77–84
Table 2
Influence of the volume of cross-linker solution (25% (w/v), glutaraldehyde
in purified water) added to 10 mL of chitosan solution (1.5% (w/v), in 0.2 M
acetic acid) on the rate of cross-linking at 20 ◦ C
Glutaraldehyde added Gel time (min)
(mL/10 mL)
Chit-F*
0.06 35 (1.5)
0.08 27 (1.1)
0.10 25 (0.8)
0.12 18 (0.5)
* Mean values (standard deviation); n = 3.
colour of the Chit-I particles) and readily dispersible in acetic
acid, which suggest a more regular distribution of the acety-
lated groups (Luyen and Huong, 1996). All these structural
features, especially the proportion of free amino groups and
the molecular weight of the chains, may dictate the reactivity
with cross-linking agents and the interaction properties with
drugs as well as the swelling behaviour of the beads.
3.2. Cross-linking process
The cross-linking process of chitosan with glutaraldehyde
was followed using oscillatory rheology. This procedure does
not alter the structure of the growing network and can provide
a direct measurement, through the elastic modulus values, of
the number of junction points at different times (Vogelsberger
et al., 2000; Rodrı́guez et al., 2003). Polymer concentration
(1.5% (w/v) in acetic acid 0.2 M) was chosen to be above the
entanglement concentration (estimated as 10/[η]), which is
required for cross-linking among different polymer chains.
Otherwise, the cross-linking agent only favours intramolec-
ular junctions (Capitan et al., 2000; Merkovich et al., 2001).
In the absence of glutaraldehyde, Chit-F dispersions had loss
modulus (G ) greater than the storage modulus (G ), and the
values of both moduli were above those of Chit-I disper-
sions, probably owing to the greater molecular weight of the
former. In the temperature-sweep experiments at the angular
frequency of 0.1 rad/s, G and G values remained practically
constant in time. This angular frequency was chosen consid-
ering that: (a) in this region, both chitosan dispersions showed
mainly viscous behaviour (values of G lower than G ) and,
in consequence, the effect of the cross-linking could be seen
more clearly; and (b) this angular frequency may make it pos-
sible to record enough data points about the evolution of G
and G without distorting the forming network (Rodrı́guez
et al., 2003).
When 0.02 or 0.04 ml of the commercial glutaraldehyde
solution (25% (w/v) in purified water) were added to the
10 mL of chitosan solutions at 20 ◦ C, a slight increase in
G and G values was observed, but no gel was obtained in
the 90 min of the experiment. In contrast, in the presence of
0.06 mL or greater volumes of cross-linking solution, G in-
creased faster than G , being greater than G after a time, and
finally reaching a plateau region (Fig. 3). Table 2 shows the
time at which the crossover of G and G occurred (tan δ = 1).
Chit-I*
58 (1.6)
45 (0.5)
36 (0.9)
20 (0.5)
Fig. 3. Effects of glutaraldehyde concentration on the time dependence of
the storage (G , solid symbols) and loss (G , open symbols) moduli for the
chitosan dispersions (1.5% (w/v)) prepared in 0.2 M acetic acid. Different
volumes of glutaraldehyde solution (25% (v/v) in purified water) were added
to 10 mL chitosan solutions at 20 ◦ C. The data plotted are the mean values
of three measurements.
This time can be considered the gel time in the cross-linking
process of chitosan with glutaraldehyde (Rodrı́guez et al.,
2003).
For each chitosan, the gel time decreased strongly when
glutaraldehyde concentration increased and was also shorter
for the chitosan with greater molecular weight, Chit-F. These
results indicate that both glutaraldehyde concentration and
chain length influence the cross-linking process, and agree
with the pattern of the viscosity data obtained by Mi et al.
(2000) in flow experiments.
It is interesting to note that, considering the DD of Chit-F
and assuming that one glutaraldehyde molecule reacts with
two amine groups to form two Schiff bases, the addition of
0.06 mL of glutaraldehyde solution provides the possibility
of reacting with approximately the half of the amino groups
free in this chitosan. This same volume contains glutaralde-
hyde enough to react with only 43% of the Chit-I deacetylated
amino groups. The gels prepared in test tubes with any chi-
tosan and this glutaraldehyde proportion, in parallel experi-
ments, were difficult to handle owing to their relatively low
consistency. Following the same reasoning, the addition of
0.12 mL of glutaraldehyde solution provides an almost exact
amount of cross-linking agent to react with all the free amino
groups of Chit-F, and around 90% of the Chit-I ones. With this
last glutaraldehyde proportion, the cross-linking process oc-
 R. Barreiro-Iglesias et al. / European Journal of Pharmaceutical Sciences 24 (2005) 77–84
curs faster and the values of loss and storage moduli obtained
after 90 min are considerably higher (Fig. 3). As a result of
the increase in the cross-linking junctions, the gels formed
in the test tubes were stiff, but exhibited a brittle charac-
ter. Therefore, an intermediate proportion of glutaraldehyde
(0.090 mL/10 mL) was used to prepare the beads. This pro-
portion can mean reaching the gel point in a relatively short
time at 20 ◦ C (30–40 min) and an even shorter time at 37 ◦ C
(less than 5 min).
It is interesting to note that despite the fact that it has been
reported that the reaction with glutaraldehyde is favoured in a
neutral medium (Goycoolea et al., 2003), the results obtained
prove that in an acidic medium the reaction also progresses
properly. Since acetic acid is a better solvent for chitosan, the
gels are expected to have a more homogeneous structure than
if they were prepared at a higher pH.
3.3. Beads
The procedure developed provided spherical beads of ho-
mogeneous surface with no tendency to aggregate (Fig. 4).
Immersion in NaOH gives the forming beads a hydrophobic
surface that avoids the dispersion of chitosan in the medium.
Compared to commonly used methods, this one-step proce-
dure avoids the use of organic solvents and emulsions, and
is notably simpler and faster since the cross-linking occurs
as beads are being formed, not in a second step (Koseva et
Fig. 4. Chitosan beads as freshly prepared (A) and detail of the surface (B).
81
al., 1999; Kumbar et al., 2002). Taking into account the rhe-
ological data obtained during chitosan cross-linking process,
it is foreseeable that, in the 15 min the beads remained in
NaOH solution, those prepared at 37 ◦ C would have reached
a greater cross-linking density than those formed at 20 ◦ C.
During the subsequent washing in purified water, the reac-
tion with glutaraldehyde may also progress while some unre-
acted glutaraldehyde molecules can diffuse out of the beads.
The diameter of fully swollen beads in purified water, after
cross-linking and washing, was 3.2 ± 0.4 mm. No differences
in appearance were observed between the beads obtained at
20 ◦ C and at 37 ◦ C, which indicates that the size is mainly
determined by the diameter of the needle used for dripping
the chitosan dispersion.
To analyse the influence of the solvent on the amount of
metronidazole loaded, the beads (freshly prepared or previ-
ously dried) were immersed in drug solutions prepared in
phosphate buffer pH 7.4, purified water, 0.2 M acetic acid or
0.1 M HCl. Significant changes in the degree of swelling were
then observed for each type of chitosan beads depending on
the temperature at which they were prepared.
Immersion in acidic medium prompted a further swelling
of the freshly prepared beads (2–3 times the diameter they
showed in purified water), especially in 0.2 M acetic acid,
due to ionisation of the neutralised hydrophobic surface of
chitosan beads. The degree of swelling was always greater
for the beads prepared at 37 ◦ C than for those obtained at
20 ◦ C.
In contrast, when the beads were removed from puri-
fied water, dried (final diameter 0.48 ± 0.12 mm and weight
0.37 ± 0.01 mg, each), and then re-immersed in purified wa-
ter, they just recovered approximately 10% (Chit-I beads) to
20% (Chit-F beads) of the initial volume (Fig. 5). This de-
crease in swelling may be attributed to the loss of water and
shrinking of the beads during the desiccation process at 40 ◦ C
that cause a strong reduction in the average distance among
cross-linking sites. This may promote a substantial incre-
ment in the actual cross-linking density of the beads, proba-
bly through additional interpolymeric hydrogen bonding, and
a change in the morphology of the network due to a decrease
in porosity (Capitan et al., 2000; Ghanem and Ghaly, 2004).
As a result, after the drying stage, the beads cannot swell in
purified water as much as freshly prepared ones. However,
when immersed in an acidic medium, chitosan amino and
imine groups begin to protonise as the medium penetrates
from the surface of the beads towards their core. Both 0.1 M
HCl and 0.2 M acetic acid solutions, despite their different
pH (around 1 and 5, respectively), provide an acidic envi-
ronment below the pKa of chitosan, 6.2–6.7 (Kasaai et al.,
2000). The ionisation causes the breakage of the hydrogen
bonds among the amino groups and an increase in osmotic
pressure inside the beads that induces a faster uptake of the
aqueous medium (Gupta and Ravi Kumar, 2000). According
to the Flory–Huggins theory, equilibrium degree of swelling
in a good solvent is controlled by the competition between the
factors that promote the swelling (such as volume interaction
82 R. Barreiro-Iglesias et al. / European Journal of Pharmaceutical Sciences 24 (2005) 77–84

(formed at 37 ◦ C). Therefore, the temperature reached during

Fig. 5. Swelling profiles of dried chitosan beads prepared at 20 ◦ C (open
symbols) and at 37 ◦ C (solid symbols). The experiments were carried out by
triplicate.
of polymer segments, interaction with the solvent, and/or a
gain in entropy of counter ions) and the elastic component
of the system, which takes in the entropic restrictions to the
swelling, since at the swollen state less conformations can be
adopted (Alvarez-Lorenzo and Concheiro, 2002). The cross-
linking density contributes to enhance this last factor. From
the swelling results, acetic acid seems to be a better solvent
for chitosan beads than HCl. As a result, the strong stress
caused by the uncoiling of chitosan chains produces the con-
siderably higher swelling of the least cross-linked and more
flexible beads (those obtained at 20 ◦ C) but induces the dis-
integration of the greater cross-linked and more brittle beads
bead synthesis and drying plays an important role in the final
performance of the beads. The significantly greater degree
of swelling of Chit-F beads in any medium may be related
to its greater molecular weight and to its more hydrophilic
character. The results obtained show that it is possible to
obtain particles of chitosan cross-linked with itself that ex-
hibit pH-sensitive swelling. The lack of pH responsiveness
found for other chitosan cross-linked systems (Berger et al.,
2004a) may be attributed to an inadequate cross-linking den-
sity, since usually the cross-linking agent is added in excess.
The disintegrating behaviour in acidic medium of the dried
beads prepared at 37 ◦ C made it impossible to continue their
metronidazole loading process in HCl or acetic acid solu-
tions. For all other conditions, it was observed that the greater
the swelling, the greater the amount of drug loaded (Fig. 5,
Table 3). For the loading, we have also considered a third
situation in which the dried beads were immersed in each
medium without drug for 15 min and then, when rehydrated,
the drug was added to the medium.
The amounts of drug loaded were estimated as the amounts
released in 0.1 M HCl at 4 h, time enough to release all dose
since (a) the beads completely swelled or even, disintegrated
in a shorter time (Fig. 5), (b) no chemical interactions (elec-
trostatic, hydrogen-bonding) are expected in the aqueous
medium between the non-ionic metronidazole and the fully
ionised amino groups of chitosan beads, and (c) the volume
of the medium was enough to be under sink conditions.
The amount of metronidazole loaded strongly depended
on the composition of the beads and on the conditions used
for loading. Freshly prepared swollen beads showed a signif-
icantly greater loading of metronidazole (more than 30 times
greater) than those previously dried (Fig. 6, Table 3).
Regarding the dried beads, those previously rehydrated in
each medium presented an even lower capacity to take up the
drug, especially in the acidic medium. For example, Chit-F
beads synthesised at 20 ◦ C, dried, rehydrated in 0.1 M HCl or
in 0.2 M acetic acid, and finally immersed in the correspond-
ing drug solution, loaded about 0.04 or 0.06 g/g-dried bead,
respectively. Those same dried beads, directly immersed in

Table 3
Amounts of metronidazole loaded by the beads (mg/g) in different media for 90 min
Chitosan State of the beads when loading Metronidazole solution medium

Buffer pH 7.5* Purified water* HCl 0.1 M* Acetic acid 0.2 M*

Chit-F Swollen (20 ◦ C) 54 (3) 45 (2) 154 (3) 225 (8)
Swollen (37 ◦ C) 34 (1) 31 (1) 43 (1)** 51 (2)**
Dried (20 ◦ C) 10.5 (1) 5.4 (0.5) 62 (2) 101 (4)
Dried and re-hydrated (20 ◦ C) 5.2 (0.5) 8.8 (0.5) 43 (1) 81 (2)
Chit-I Swollen (20 ◦ C) 53 (2) 55 (2) 120 (4) 245 (6)
Swollen (37 ◦ C) 44 (2) 44 (2) 163 (3) 286 (4)
Dried (20 ◦ C) n.d. 1.7 (0.5) 3.9 (0.5) 8.5 (0.5)
Dried and re-hydrated (20 ◦ C) n.d. 1.4 (0.5) 3.2 (0.5) 7.3 (0.5)
The temperature at which the beads were prepared is shown in parenthesis in the second column. (n.d.: not detected).
∗ Mean value (standard deviation); n = 3.
∗∗ Immersed in the loading solution for only 15 min to avoid disintegration).
 R. Barreiro-Iglesias et al. / European Journal of Pharmaceutical Sciences 24 (2005) 77–84
Fig. 6. Metronidazole release profiles in 0.1 M HCl at 37 ◦ C from dried Chit-
I beads that were loaded under different conditions: (A) freshly prepared, in
the swollen state, in purified water (triangles), 0.1 M HCl (circles) or 0.2 M
acetic acid (squares); the solid symbols represent data from beads prepared
at 37 ◦ C, while open symbols correspond to the beads obtained at 20 ◦ C;
(B) previously dried beads, obtained at 20 ◦ C, that were directly immersed
in the drug solution (solid symbols) or firstly rehydrated (open symbols) in
purified water (triangles), 0.1 M HCl (circles) or 0.2 M acetic acid (squares).
The experiments were carried out by triplicate.
each drug solution, incorporated a 20–30% greater amount
of metronidazole. Similar results were observed for Chit-I
beads, although these beads showed a lower loading capacity
owing to their smaller degree of swelling after being dried.
The dependence of the amount loaded on the procedure
of immersion of the dried beads can be understood as an en-
hancing of the drug penetration inside the bead during the
absorption of the medium and swelling. The drug molecules
are swept along by the flux of fluid towards the core of the
bead. In contrast, once swollen, the bead is loaded by a par-
titioning equilibrium of the drug between the outer solution
and the liquid that hydrates the bead. This loading process is,
in general, less efficient (Table 3).
In any case, the release studies carried out in 0.1 M HCl
indicated that, independently of the conditions used to load
the beads, all of them released the entire dose in less than
30 min (Fig. 6).
4. Conclusions
Oscillatory rheometry tests carried out on chitosan/
glutaraldehyde dispersions provide information useful to se-
83
lect the optimum conditions for the cross-linking process
during bead preparation. An amount of cross-linker enough
to react with 70–75% of the chitosan amino groups is ade-
quate to prepare consistent but yet flexible beads with pH-
sensitive swelling. The temperatures at which the beads
are obtained and dried are also key factors in the perfor-
mance of the beads owing to their influence on the ki-
netics and final degree of cross-linking. Dried beads syn-
thesised at 37 ◦ C exhibited faster swelling rates than the
ones prepared at 20 ◦ C and, even, disintegrated after 30 min
in 0.2 M acetic acid. The amount of metronidazole loaded
strongly depends on the composition of the medium and
the thermal-hydration history of the beads. Better results
were obtained with 0.2 M acetic acid > 0.1 M HCl > purified
water > pH 7.5 buffer phosphate, and with freshly prepared
swollen beads > dried beads > rehydrated dried beads. These
results highlight the importance of considering both the in-
herent and the external factors that influence the degree of
swelling in order to optimise the loading capacity of chitosan
beads, which can be obtained by a simple procedure and are
able to release the whole dose in less than 30 min in an acidic
environment.
Acknowledgments
This work was financed by Xunta de Galicia, Spain
(PGIDIT02BTF20302PR and an equipment grant DOG
04/06/97) and by Ministerio de Ciencia y Tecnologı́a, Spain
(SAF2002-03440; RYC 2001/8). R. B.-I. acknowledges a re-
search contract supported by Xunta de Galicia. The authors
express their gratitude to George S. Daras (France) for pro-
viding free samples of chitosan.
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