Preparation and Characterization of Theophylline-Chitosan Beads as an Aapproach
to Colon Delivery
Iranian Journal of Pharmaceutical Research (2004) 2:
Received: March 2004
Accepted: June 2004
Copyright ? 2004 by School of Pharmacy
Shaheed Beheshti University of Medical Sciences and Health Services
Preparation and Characterization of
Theophylline-Chitosan Beads as an Aapproach to Colon Delivery
Mohammad Reza Avadia,b, Amir Hossein
Ghassemic, Assal Mir Mohammad Sadeghib, Mohammad Erfana, Azim Akbarzadehd, Hamid Reza
Moghimia and Mortaza Rafiee-Tehranic*
Pharmacy, Shaheed Beheshti University of Medical Sciences and
Health Services, Tehran, Iran. bHakim Pharmaceutical Company, Tehran, Iran.
Pharmacy, Tehran University of Medical Sciences and Health
Services, Tehran, Iran. dPasteur Institute of Iran, Tehran, Iran
Chitosan with excellent biodegradable and
biocompatible characteristics has received attention as an oral
drug delivery vehicle for controlled-release formulations. In
this study an enteric-coated capsule containing
theophylline-chitosan beads based on 23 factorial designs was
prepared as a colon drug delivery system. The
theophylline-chitosan gel beads were formulated by adding the
drug-containing solution of chitosan into tripolyphosphate
solutions, dropwise. The obtained beads were washed with water
and freeze-dried before filling into the capsules. Eudragit® S100
was then used to enteric-coat the prepared capsules. Drug
entrapment efficiency and the effects of different variables
including: bead morphology, swelling behavior of the beads and
the release behavior of the system on these parameters were
investigated. Results showed that the highest and lowest
swelling ratio is obtained at pH 4.5 and 7.2, respectively.
These studies have shown that chitosan concentration and drug
polymer weight ratio significantly affect the drug entrapment.
Decreasing the drug solubility in external phase caused a
significant increase in drug loading. External phase saturation
with theophylline and tripolyphosphate, as well as decreasing
temperature, have increased drug loading. Furthermore, the
lowering of temperature had a significant effect on bead's
hardness. The release of theophylline from freeze-dried
beads filled in enteric-coated capsules was also investigated.
Release of theophylline was prolonged with saturation of both
drug and tripolyphosphate in the external phase. Results showed
that the release of theophylline from chitosan beads is
possibly due to more than one mechanism, possibly dissolution,
diffusion and relaxation of the polymer chains.
Bead; Drug delivery; Colon; Theophylline.
Colonic drug delivery (CDD) for either
local or systemic effects has been the subject of much research
over the last decade. This method of drug delivery has several
advantages including protection of drug from harsh environment
of stomach and small intestine, avoiding drug absorption from
upper GIT and increased bioavailability of some drugs. CDD is
performed using different polymers through various drug
delivery systems such as chitosan beads, which are the subject
of the present investigation.
Chitosan, a cationic polysaccharide
obtained from chitin, is a suitable polymer for drug delivery.
Chitin is one of the most important natural polysaccharides and
is found in crustaceous shells or in cell walls of fungi. This
polysaccharide has been used in food industry, medicine and
drug delivery systems. However, due to its low solubility in
many common solvents, chitin is not widely used for industrial
applications (1, 2). Chitosan, obtained by deacetylation of
chitin, is soluble in aqueous acidic media due to the presence
of amino groups. Bioadhesive properties, biocompatibility and
biodegradability of chitosan, have made this polymer a
potential and suitable carrier for biomedical and drug delivery
applications (3). This polymer has also been used in oral
sustained release formulations and implantable drug delivery
systems (3). In addition, it has been shown that chitosan can
interfere with dietary fat absorption (4).
Nagai et al. (5) used chitosan with other
excipients for preparation of controlled release tablets and
found that the drug release rate was directly proportional to
the amount of chitosan within formulations. Nigalaye et al. (6)
prepared theophylline sustained release tablets using a
hydrocolloidal matrix system of chitosan, carbomer-934P and
citric acid and showed that at concentrations above 50% (w/w),
chitosan formed an insoluble non-erosive type matrix; whereas,
at lower concentrations (less than 33%) a fast-releasing matrix
system was obtained. Miyazaki et al. (7) found that the
addition of sodium alginate to chitosan-containing tablets
could improve their extended release property. Similar results
have also been published by Kawashima et al. (8), who suggested
that citric acid could form chitosan gel and, thereby, improve
the sustained release properties of the system.
Beads with spherical shape prepared by
complexation between positively charged macromolecules, such as
chitosan and negatively charged molecules, like
tripolyphosphate (TPP) has received attention as a controlled
release drug delivery system (9). Tripolyphosphate with
negative charge is able to interact with cationic chitosan
through electrostatic forces (10). Thus, not only reversible
physical crosslinking is substituted for chemical crosslinking,
the possible toxicity of reagents and other undesirable effects
could also be prevented.
Preparation of TPP/chitosan complex
through the addition of chitosan droplets into a
tripolyphosphate solution has been reported by Bodmeier et al.
(9). Aral and Akbuga (11) have produced strong and durable
TPP/chitosan beads by coating the bead's surface with sodium
alginate to form a polyelectrolyte complex film. Shu and Zhu
(12) reported a novel approach to prepare TPP/chitosan beads
for controlled release drug delivery. Their studies showed that
the prepared TPP/chitosan beads had a more homogeneous
structure and beads were strengthened greatly. Sezar and Akbuga
(13) studied the effect of different variables such as drug
concentration, type and concentration of chitosan, pH value of
TPP solution, volume of internal and external phase, gelation
time and drying condition on various properties of chitosan
beads. They showed that concentration of both chitosan and TPP
have an effects the drug loading. The structure and strength of
the beads might be dependent on the gelation time and drying
Certain polymers, with a charged moiety,
are pH-sensitive and can be used as coating agents to protect
contents of tablets, capsules or pellets from gastric fluid and
can, therefore, be used for colon targeting. A number of
commercially available methacrylic resins, popularly known as
Eudragit, are being used for colon-targeted formulations.
Methacrylic acid- methyl metacrylate copolymers, (Eudragit® S),
contains 30% methacrylic acid units and dissolves at pH values
higher than 7.0. This polymer is a suitable coating agent for
colon drug delivery system.
The main aim of the present investigation
was to prepare a suitable enteric-coated capsule containing
theophylline-chitosan beads for colon delivery. Furthermore,
the effect of some factors, such as the concentration of
chitosan and TPP, as well as the drug: polymer ratio on
drug loading of theophylline-chitosan beads was investigated by
utilizing the 23 factorial designs.
Chitosan (98% deacetylated, viscosity of
solution, 264 mPa.s) was a gift from Primex (Iceland).
Tripolyphosphate was purchased from Sigma (Vienna, Austria).
Eudragit® S100 was a gift from FMC. Other chemicals and
solvents were of pharmaceutical or analytical grades, and used
Spectrophotometer (Shimadzu 1201, Japan),
pH-meter (Corning 120, UK), freeze-drier (Rewart Edwards High
Vacuum 30P.2.T.S N114, UK).
Characterization of chitosan
Two different methods were used to
determine the degree of deacetylation (DD). According to a
modified acid-base titration method (14), chitosan (0.50 g) was
dissolved in 20.0 mL 0.10N HCl and titrated pH-metrically with
a standardized solution of a 0.10 N NaOH solution. The curve
constructed has two equivalent points related to the excess of
HCl and the protonated amino groups. DD was calculated based on
DD= 16.1 (Y-X) f/w (Eq.1)
Where Y and X are the consumed NaOH
volume at the equivalent points (mL), f is molarity of the NaOH
solution and w is the initial weight of chitosan (g).
Infrared spectroscopy was also used for
determining DD according to a previously reported method (15).
Molecular weight determination
For determination of the chitosan average
molecular weight (MW), five various concentrations of chitosan
solution in acetic acid-sodium acetate buffers were prepared.
The relative viscosity was obtained with a capillary viscometer
at 30 ± 0.05ºC. Next, the intrinsic viscosity was
determined and the molecular weight of chitosan was calculated
based on the Mark-Houwink equation (16)
Where k= 1.64 10-30 DD14
and a= -1.02 10-2 DD +1.82.
Factorial design experiments
Chitosan beads were obtained based on the
23 factorial design. Chitosan concentration (X1),
tripolyphosphate concentration (X2) and drug:polymer weight
ratio (X3) were selected as independent variables (Table 1).
The drug entrapment efficiency of the beads (Y) is the response
parameter or the dependent variable (Table 2). Furthermore,
release studies as well as the saturation effect of external
phase with tripolyphosphate or theophylline on drug loading
Preparation of chitosan beads
Initially, 200 mg chitosan was dissolved
in 10 ml of a 1% acetic acid solution under stirring for 20 min
at room temperature. Then, theophylline was dispersed in this
solution and finally theophylline-chitosan mixture was added
dropwise the into tripolyphosphate aqueous solution at room
temperature, using a syringe. The formed beads were allowed to
stand in the tripolyphosphate solution for 15 min to be cured.
The beads were separated with paper filter, then washed twice
with water and dried by freeze- drying.
Dried beads (500 mg) were placed in hard
capsules (size 1) and coated using the pan coating procedure.
First, Eudragit® S100 was dissolved in acetone, then
1% triethyl citrate (as plastisizer) was added and
stirred to obtain a homogeneous solution. Spray coating was
carried out using a coater made of stainless steel with an
atomizing nozzle of 0.5 mm in diameter. Compressed air with a
pressure of 2 bars was used to atomize the coating solution
with a spray rate of 0.8 g/min. The inlet temperature and
coating time were 50ºC and 30 min, respectively. Coating
weight and thickness were determined to be 45±3 mg and
0.32±0.01 mm, respectively (data are Mean±SD, n=
Particle size determination
For each formulation, diameter of 100
beads were measured with a micrometer and the mean particle
size was determined.
Beads were initially broken in pH 1.2 HCl
and, after filtration; their theophylline-contents assayed
spectrophotometrically at 272nm.
Drug release studies
The release of theophylline from
freeze-dried beads filled in enteric-coated capsules was
studied using the USP basket method (Apparatus I) at 50 rpm and
in 900 mL of dissolution fluid at 37 ± 0.5ºC. Six
coated capsules were tested in pH 1.2 simulated gastric fluid
(SGF) for the first 1.5 h, pH 6.0 phosphate buffer for the
second 1.5 h and pH 7.2 phosphate buffer for the remaining
period of time (4.5 h). At set intervals, 5 ml samples were
removed and replaced with equal volumes of the buffer solution.
The amount of drug released was measured spectrophotometrically
at 272 nm. The amount of theophylline released was plotted
against time in different media.
Results and discussion
The average molecular weight and
intrinsic viscosity of the starting chitosan calculated from a
DD-dependent Mark-Houwink relationship (16), was determined to
be 10.26 105 g/mol and 1050 cm3/g, respectively.
Two different methods, pH-metric
titration and infrared spectroscopy were used to determine the
percentage of chitosan deacetylation. The value resulted from
the pH-metric titration (DD 0.94) was inagreement with the FTIR
(DD 0.91) spectroscopic method, both matching the value
reported by the manufacturer (0.98). In the IR spectrum (Figure
1), the amide bond at 1655 cm-1, representing the N-acetyl
group content, and the hydroxyl bond at 3450 cm-1, as an
internal standard, were used to determine the percentage of
acetylated amine groups. The percentage of acetylated amine
groups were calculated by equations 3 and 4, as follows: (15)
After freeze-drying, all of the beads
were found to be spherical. The mean particle size of eight
different formulations were between 0.642±0.019 to
0.825±0.011 mm. The particle size of different
formulations is depicted in Figure 2. Scanning electron
microscopy (SEM) was used to investigate the morphology of
chitosan beads (formulation F8). The surface of dried beads
seemed to be smooth and did not shrink during the freeze-drying
process (Figure 3).
Chitosan, with a polycationic
characteristic, forms gel beads with the negatively charged
tripolyphosphate counterion. These studies have shown that the
shape and preparation of the beads were critically dependent on
the viscosity of the chitosan, as well as the concentration of
tripolyphosphate solution. When a 1% chitosan solution was
used, no beads were formed. However, smooth beads were obtained
upon dropping 1.5 and 2% chitosan solution into different
concentrations of tripolyphosphate solutions.
Bodmeier et al. (9) examined the effect
of pH on drug loading of some model drugs. They showed that pH
changes the drug entrapment efficiency due to the increase in
drug solubility within the external phase. However,
theophylline with a pKa of 8.77 did not show significant
changes by changing the pH of the external phase between 4 and
To study the swelling ratio of the
formulations prepared, beads were dispersed in various
solutions with different pH and their swelling property was
investigated visually. The highest swelling ratio was obtained
at pH 4.5, whereas at pH 7.2, beads swelled slowly but they
were broken after 2-3 h (Figure 4).
Various formulations (F1-F8) were
prepared using the 23 factorial design procedure. An optimum
drug loading of 40.15% ± 0.64 (n=6) was obtained with
formulation 8 (Table 2). Three more formulations (F9-F11) were
also prepared to increase the drug entrapment efficiency by
different methods. In formulation 9, the external phase was
saturated with tripolyphosphate and as result the loading
increased up to 51.37%±0.45. Surprisingly, saturation of
theophylline in the external phase caused a tremendous shift in
drug loading, up to 91.71%±1.2 in formulation 10.
Furthermore, decreasing the external phase temperature
increased the drug entrapment to 78.61%±0.78
(formulation 11). Table 3 shows the results of ANOVA for the 23 factorial
design experiments. All factors and interactions have
significant effect on drug loading (P<0.005). Increasing the
chitosan concentration caused an increase in drug loading,
possibly due to the higher ability of gel formation.
Tripolyphosphate concentration also showed a significant effect
on drug content at drug/ polymer weight ratio of 0.5:1. This
might explain why more tripolyphosphate was needed to obtain
the required gel strength when the drug:polymer ratio was
increased. Furthermore, saturation of the external phase with
tripolyphosphate or theophylline caused a significant
increasing in drug loading. This could be due to reduced
concentration gradient and, therefore, reduced diffusion of
drug toward beads surface and thereby dissolution in the
external phase. Reduction of external phase temperature also
caused an increase in drug entrapment in formation F11,
possibly due to the reduction in of theophylline solubility
within the external phase. Nevertheless, lowering the
temperature could also increase the stiffness of the beads.
Freeze-dried beads showed a higher drug loading in comparison
to the air-dried beads. This may be due to the migration of
theophylline in the air-dried method. Theophylline could
migrate with water to the surface of the beads in the air-dried
method and the beads could shrink after water evaporation. In
contrary, in the freeze-drying process, since just the frozen
water molecules sublimate, the drug could not migrate to the
surface and beads are intactly solidified.
Since formulations F8-F11 had the highest
drug entrapment efficiencies, they were selected for release
studies. Release profiles of theophylline from the prepared
chitosan beads (formulations 8, 9, 10 and 11) are depicted in
Figure 5. Since Eudragit® S100 is a pH-dependent
polymer and is not sensitive to low pH, the release of
theophylline from the enteric-coated capsules in simulated
gastric medium and pH 6.0 phosphate buffer solution was not
significant. However, coated capsules were degraded and the
beads began to release their theophylline after a short lag
time and with a fast rate, when exposed to pH 7.2. phosphate
buffer solution. The release profile (Figure 5) shows a
lag-phase and two different release phases. The lag-phase
reveals a delay in release, as was expected from the
pH-dependent system used. The other two phases are related to
the release of drug from the matrix. During the first release
phase, nearly 70% of the entrapped drug was released over a
period of less than 40 min, which might suggest a fast
phenomena like diffusion in porous system, dissolution of drug
or dissociation of drug-polymer molecular complexes. As
analysis of data shows that 0.5<n<1.0, combination of the
above-mentioned mechanisms are expected, which all are highly
possible in our system before swelling. After this first step,
release rate decreased several times. This might be due to
swelling of the polymers, which is expected to reduce the
release rate. The swelling observed with the system (data not
shown) supports this suggestion. As the system shows a constant
release rate, the decreased rate is surely not due to the
decreased thermodynamic activity or concentration gradient.
After swelling, either diffusion through throws a swollen
matrix or the process of swelling itself could be the rate
limiting step. Our analysed data for this part shows that n is nearly 1.0
(data not shown), revealing that most of the release is being
controlled by swelling, which is expected for the polymer. Gupta
et al. Investigated the drug release behavior of chitosan beads
(17). Their studies showed that the release of diclofenac sodium
depends greatly on the swelling of the beads. Furthermore, at pH
7.2-7.4, there is a very limited swelling; thus the drug
entrapped within the beads can not be released easily.
As shown in Figure 5, saturation of the
external phase with tripolyphosphate in formulation No. 9
decreases drug release in comparison to formulation F8; which
could be due to an increased degree of crosslinking.
Furthermore, an increment of bead hardness in formulation 11
caused a decrease in dissolution rate, possibly due to the
slower penetration of the medium in to this formulation.
Fickian and non-Fickian (anamolous)
behaviors have been used for determining the mechanism of drug
release from polymeric systems. It is difficult to determine the
exact release kinetic in such complex systems. However, we tried
to analyze the drug release data, using a general equation as
Mt/M?= ktn (5)
where Mt is the amount of drug released in a given time,
M? is the total amount of theophylline within the
beads, k and n are equation constants and t is the time. Also a
logarithmic form of this equation could be used in these systems
(18). The initial section of the release curves (Figure 5) (Mt/M?<0.6) was analyzed by this equation and the
equation constants were determined (data are presented in Table
4). For all formulations, the values of the exponent n were
between 0.5 and 1, indicating a non-Fickian transport. This
suggests that transport is possibly controlled by diffusion
and/or relaxation of the polymer chains.
Similar results have also been reported by
takka et al. (19), who investigated the release mechanism of
nicardipine-alginate gel beads. Their investigation showed that
values of the exponent n lies between 0.5 and 1.0, which
indicate a non-Fickian transport controlled by diffusion and
relaxation of the polymer.
Chitosan, a natural biocompatible and
biodegradable polymer, was used as a vehicle for the preparation
of theophylline beads and the effect of different formulation
variables on properties of system prepared was investigated.
Results have indicated that the saturation of the external phase
with theophylline, tripolyphosphate and a decrease in
temperature of external phase could affect the properties of the
system. It was also shown that the release of theophylline from
chitosan beads was governed with more than one mechanism
(possibly dissolution, diffusion and relaxation of the polymer
chains). Data obtained shows that all variables should be taken
into account during the formulation of such a system.
We are grateful to Mr. S. Assadi and Dr.
M. Parnianpour (executive members of Hakim Pharmaceutical
Company) for their supports. Also the technical assistance of
Ms. R. Nosrati is appreciated.