DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY, 15(9), 1475-1494 (1989)

PREPARATION AND EVALUATION OF DRUG-CONTAINING

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CHITOSAN BEADS.

Roland Bodmeier, Kyoung-Hee Oh, and Yashoda Pramar

College of Pharmacy
The University of Texas at Austin, Austin, TX 78712
For personal use only.

ABSTRACT
Sulfadiazine beads were prepared by dropping drug-containing solutions of the
positively charged polysaccharide, chitosan, into tripolyphosphate (TPP) solutions.
The droplets instantaneously formed gelled spheres by ionotropic gelation,
entrapping the drug within a three-dimensional network of the ionically linked
polymer. To achieve maximum drug content, high payloads, short gelation times,
low TPP concentrations, and a low internal to external phase ratio were required.
The chitosan beads showed pH-dependent swelling and dissolution behavior. The
beads swelled and dissolved in 0.1N HC1, while they stayed intact in simulated
intestinal fluid. The release of sulfadiazine in 0.1N HCl decreased with increasing
concentration of TPP, but was independent of the TPP concentration in intestinal
fluids. The, morphology of the beads was investigated by scanning electron
microscopy. The porosity of the beads depended on the method of drying.

1475

Copyright @ 1989 by Marcel Dekker, Inc.

 1476 BODMEIER, OH, AND PRAMAR

I"
Chitosan ((1->4)-2-amino-2-deoxy-l3-D-glucan)is a hydrophilic, cationic
polyelectrolyte prepared by N-deacetylation of chitin (Figure 1). Chitin is the most
plentiful natural polymer next to cellulose and it is obtained from crab and shrimp
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shells. Chitosan and chitosan derivatives have been used in enzyme and cell
immobilization, as flocculants, in the preparation of reverse osmosis membranes, in
waste water treatment, and in personal care products (1,2). In addition, chitosan
has a variety of promising medical and pharmaceutical applications. Biomedical

r
For personal use only.

H NHCOCH 3 H NHCOCH
B
n

CHITOSAN

FIGURE 1
Structures of chitin and chitosan.
 DRUG-CONTAINING CHITOSAN BEADS 1477

applications of chitin and chitosan include wound and burn healing, soft and hard
contact lenses, and artificial kidney membranes (3). Chitosan has been used as a
direct-compressiondiluent (4), as a vehicle for sustained release of drugs (5-7), as
well as for the enhancement of the dissolution rate and bioavailability of water-
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insoluble drugs (4). Machida et al. studied the enzymatic degradation of chitosan
and hydroxypropyl-chitosanin-virro and in-vivo and concluded that chitosan might
be utilized as a biocompatible and biodegradable carrier for implantable drug
delivery systems (8). Heller et al. developed an enzyme-degradable, self-regulated
drug delivery system based on partially deacetylated chitin (9).
Sodium alginate beads were prepared by gelling the anionic polysaccharide
with CaC12 solutions to entrap cells (10). In the present investigation, cliitosan
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beads containing sulfadiazine or quinidine were prepared by dropping drug

containing chitosan solutions into tripolyphosphate solutions. The interaction of the
positively charged chitosan molecules with the anionic counterion,
tripolyphosphate, caused the formation of gelled spheres.
The objective of this study was to investigate variables affecting the
preparation of, the drug release from, and the morphology of the chitosan beads.
Potential pharmaceutical applications could include both sustained and enhanced
oral drug delivery systems, depending on the solubility characteristicsof the drug.

Materials
Three different grades of chitosan (Protan Laboratories, Redmond, WA) with
viscosities of 50,750, and 2790 cps (1% w/w in 1% v/v acetic acid) and degrees of
deacetylation of 81%, 84%, and 80.4% respectively, were used. The following
chemicals were obtained from commercial suppliers and used as received: caffeine
 1478 BODMEIER, OH, AND PRAMAR

(MCB Manufacturing Chemist, Inc., Gibbstown, N.J.), quinidine, salicylic acid,

sulfadiazine, and tripolyphosphate (Sigma Chemical Co., St.Louis, MO.).

Methods
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The drug (1% w/w) was dissolved (caffeine, salicylic acid, or quinidine) or
dispersed (sulfadiazine) in a solution of chitosan (1% w/w) in dilute acetic acid (1%
v/v). The beads were formed by dropping the bubble-free solution or dispersion
(10 ml) through a disposable syringe onto a gently agitated tripolyphosphate
solution (100 ml). The chitosan beads were separated after one hour by filtration
and briefly rinsed with deionized water. The drying of the gel-like beads was
accomplished by either freeze-dryingor air-drying for 24 hours followed by oven-
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drying at 6OOC for 6 hours. All batches were prepared in triplicate.

The following preparative variables were investigated in this study: theoretical
sulfadiazine loading (10, 20, 30, 40, 50, 75, 90 %), stirring time (0.5, 1.0, 2.0,
3.0, 4.0 hours), TPP concentration (0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 10.0 %), and
amount of sulfadiazine added to 100 ml of 2% TPP solution (0, 100, 150, 200

mg).
The sulfadiazine or quinidine content of the beads was determined
spectrophotometricallyafter dissolving the beads in 0.1 N HC1, at h = 242nm or
251nm, respectively.
The solubilities of sulfadiazine and quinidine were determined by placing an
excess of drug in contact with the desired medium. Duplicate samples were shaken
for at least 48 hours at 22OC. The samples were filtered and solubilities were
obtained by measuring the concentration of the drugs spectrophotometricallyafter
appropriate dilution in 0.1 N HCI.
The release properties of the freeze-dried beads were studied in 0.1N HCl or
simulated intestinal fluid (I.F.) (USP XXI) using the USP XXI rotating paddle
 DRUG-CONTAINING CHITOSAN BEADS 1479

apparatus (500 ml, 37OC, 50 rpm). The beads (30 - 100 mg) were added to the
dissolution medium and samples of 3 ml were taken and replaced with fresh
medium at predetermined time intervals. The maximum concentration of
sulfadiazine in the release medium was less than 5% of its solubility. All samples
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were run in triplicate and assayed spectrophotometrically either directly or after

appropriate dilution with 0.1N HC1 or simulated intestinal fluid (sulfadiazine: h =
242nm in both 0.1N HC1 and I.F.).
Scanning electron microscopy (SEM)was used to characterize the surface and
cross sections of the chitosan beads before and after the dissolution study. Cross
sections were obtained by cutting the beads with a razor blade. The dried beads
were coated for 70 seconds under an argon atmosphere with gold-palladium (Pelco
Model 3 Sputter coater) and observed with a Jeol ISM 35C scanning electron
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microscope.

P
The polycationic polysaccharide, chitosan, forms gels with suitable
multivalent counterions. The ionic interactions between the positively charged
amino groups and the negatively charged counterion, tripolyphosphate, were used
in this study to prepare chitosan beads. The protonation of the amino groups
enables the dissolution of chitosan by a large number of strong and weak acids
(1 1). Solutions of chitosan in 1% acetic acid were dropped onto TPP solutions and
gelled spheres formed instantaneously by ionotropic gelation. The beads were
easily manufactured without any sophisticated equipment.
Chitosan is characterized by its degree of deacetylation and its viscosity in
1% (v/v) acetic acid solutions (1% w/w). The shape and preparation of the beads
were critically dependent on the viscosity of the chitosan solution. Three chitosan
samples with similar degrees of deacetylation but different viscosities were
 1480 BODMEIER, O H , AND PRAMAR

evaluated for bead formation. Only the high viscosity chitosan sample (2790 cps)
resulted in spherical and sufficiently strong beads at a concentration of 1% (w/w) in
dilute acetic acid. Beads could not be prepared from samples with viscosities of 50
and 750 cps. In comparison, perfect calcium alginate spheres were formed above a
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critical solution viscosity of only 30 cps (12).The anionic counterion, TPP, can
form either intermolecular or intramolecular linkages with the positively charged
amino groups. Kohn reported that stable interchain junction zones formed above a
minimum critical sequence length for calcium-alginate gels (13). The interactions
were individually weak and long arrays of non-covalent bonds acting together were
required for gel stability. The intermolecular linkages, which are responsible for the
successful formation of the beads, increase in number with increasing molecular
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weight. Normal polyelectrolyte or intramolecular binding was probably prevalent

with the low viscosity or low molecular weight chitosan samples. This may have
prevented strong intermolecular crosslinking, and hence the formation of strong
beads.
Most microencapsulation techniques use organic solvents and/or heat (14). In
this method, the drugs were entrapped within the chitosan beads in a completely
aqueous environment under mild conditions. The pH values of chitosan solutions
(Iw/w% in I%v/v acetic acid) and of 4% TPP solutions were 4.1 and 8.9,
respectively. The acetic acid within the chitosan droplets was neutralized rapidly by
TPP, which diffused into the droplets, In addition, acetic acid diffused into the
external phase. The pH-indicators methyl red or bromothymol blue (0.1 ml of a
0.05% solution) were added to the chitosan solution (10 ml) prior to bead formation
in order to follow time-dependent pH-changes within the beads during the
preparation. The beads were cut at different time periods after bead formation and
the color changes of the pH-indicators within the beads were observed visually.
The color changes are shown in Table 1. The pH value inside the beads gradually
 DRUG-CONTAINING CHITOSAN BEADS 1481

TABLE 1
Time-dependentpH changes within the wet chitosan beads as visualized by color
changes of the pH indicators methyl red and bromothymol blue,
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time (min) color of wet beads (surface / interior)

methyl red (DHranee 4 3,- 6.2) bromothvmol bh e (DHr a w 6.0 - 7.6)

acid (red) - base (yellow) acid (yellow) - base (blue)

5 red / red yellow / yellow

10 yellow / red blue spots / yellow
15 yellow / red blue / yellow
20 yellow / yellow blue / yellow
25 blue / green
35 blue / blue
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/
changed from pH 4.1 (initial pH value of chitosan solution) to neutral pH values
within 20 to 35 minutes. One can conclude from these results that this time period
was necessary to obtain complete gelling across the beads.
Several drugs with different solubility characteristicswere chosen as model
/
compounds for the encapsulation within chitosan beads. This encapsulation
procedure works best for water-insoluble drugs. Water-soluble drugs such as
caffeine, salicylic acid or quinidine were lost to the TPP phase rapidly (Figure 2).
The drugs partitioned completely into the external TPP solution within 15 minutes.
To entrap water-soluble drugs, the chitosan spheres have to be separated from the
aqueous phase as early as possible.
Alternatively, in the case of ionizable drugs, the pH of the TPP phase could be
adjusted to minimize drug solubility. The solubility of quinidine in a 3% TPP
solution @H = 8.9) was only 0.09 a.However, the quinidine content in the beads
was insignificant (Table 2). Acetic acid diffused into the TPP phase during bead
 1482 BODMEIER, OH, AND PRAMAR

100
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-I 4
-A-
SALICYLIC ACID
QUlNlDlNE

0
0 10 20 30 40 50
time (min)

FIGURE 2
Time-dependent loss of water-soluble drugs to the external TPP (4%) phase during
For personal use only.

the preparation of chitosan beads.

preparation. The pH of the external phase dropped to pH 7.9 and the solubility of
quinidine increased to 1.15gA. Quinidine was lost completely, but it did not
precipitate in the external phase. The pH of the TPP phase was then raised to pH 11
by the addition of 1N NaOH. Although the final pH of the TPP phase was 8.97,
quinidine could not be entrapped. In contrast to the experiment with the 3% TPP

TABLE 2
Entrapment of quinidine within chitosan beads.
external phase pH of external phase quinidine content, wt%
initial - final (theoretical content = 50%)

3% TPP 8.94 - 7.89 1.3

3% TPP + 1N NaOH 11.06 - 8.97 1.6
3% TPP + pH 12 buffer 12.07 - 12.04 24.6
 DRUG-CONTAINING CHITOSAN BEADS 1483

solution, quinidine precipitated in the external phase. The pH of the solution close
to the bead surface initially dropped because of the loss of acetic acid. This caused
quinidine to diffuse out. The pH in the microenvironment increased with time and
caused the drug to precipitate. The drug was successfully encapsulated with a pH
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12 phosphate buffer, which kept the pH close to the bead surface at higher levels.

However, quinidine was still lost, and precipitated. A buffer with a higher capacity
may further increase the drug content. The presence of TPP was required for bead
formation. A white precipitate, but no droplets were formed when the chitosan-
quinidine solution was dropped in the pH 12 buffer. The increase in pH within the
beads probably caused quinidine to precipitate in the interior of the beads during the
preparation. Although quinidine could be successfully entrapped with this modified
technique, the resulting beads were weak. The positively charged amino groups of
For personal use only.

chitosan were probably converted to the unionized state at the higher pH values.
This resulted in reduced ionic interactions or crosslinking with the anionic
counterion, TPP.
Sulfadiazine, the water-insoluble model drug, was suspended in the chitosan
solution and entrapped successfully.The effect of sulfadiazinepayload on the actual
sulfadiazine content in the chitosan spheres is shown in Figure 3. The sulfadiazine
content increased with increasing payload. High drug loadings up to 80% were
achieved. Sulfadiazine was continuously lost to the external tripolyphosphate phase
during the preparation of the beads (Figure 4). The pH and the solubility of
sulfadiazine within the beads increased because of TPP diffusing into the beads.
Sulfadiazine has a solubility of 0.07mg/d in 1% acetic acid and of 3.5 mg/ml in
3% TPP solution. The sulfadiazine crystals initially dissolved from the surface of
the beads, and then with increasing time periods, more drug dissolved from the
central regions. The TPP solution to chitosan solution ratio should be kept to a
minimum for maximum drug entrapment. More drug was lost with increasing
 1484 BODMEIER, OH, AND PRAMAR
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0 20 40 60 80 100

theoretical sulfadiazine content (%)

HGURE 3
Effect of sulfadiazinepayload on the actual sulfadiazinecontent in the chitosan
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beads (3% TPP).

1 I I I

0 1 2 3 4

stirring time (hours)

FIGURE 4
Effect of stirring time on sulfadiazine content in the chitosan beads (2%TPP).
 DRUG-CONTAINING CHITOSAN BEADS 1485

35 -

30 -

25 -
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20 -

15
10
1
;
0
I

2 4
1 I

6
-m freeze drying
4 air drying

8
I I
10

TPP solution (%)

FIGURE 5
Effect of the concentrationof tripolyphosphate (TPP) and of the method of drying
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on the sulfadiazine content in the chitosan beads.

volume of the external phase. The drug content was independent of the total volume
used, as long as the phase ratio was kept constant.
The effect of TPP concentration and of the method of drying on drug content
is shown in Figure 5. The sulfadiazine content within the beads decreased with
increasing TPP concentration. The solubility of sulfadiazine in the external phase
and hence the amount of drug lost to the external TPP phase increased with
increasing mpolyphosphate concentration (solubility of sulfadiazine in 1% TPP =
2.8 mg/ml, in 3% TPP = 3.5 mg/ml, and in 5% TPP = 3.8 mg/ml). Freeze-drying
resulted in higher drug contents within the beads when compared to &-dried beads.
The beads shrank significantly during air-drying. Water was exuded and
accumulated on the surface of the beads before evaporation. Dissolved sulfadiazine
migrated with the water to the outside of the beads. After complete drying,
sulfadiazine crystals were visible on the exterior of the beads. During the freeze-
 1486 BODMEIER, O H , AND PRAMAR

drying process, the beads rapidly solidified. Water transport did not occur and
sulfadiazine could not diffuse to the outside of the beads under these conditions.
The sulfadiazinecontent within the beads could be further increased by adding drug
to the external TPP phase as shown in Figure 6.
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The effect of sulfadiazine loading on drug release in 0.1N HC1 and simulated
intestinal fluid is shown in Figures 7 and 8. The release rate decreased with
increasing drug content in both media. Ground mixtures of chitin or chitosan with
drugs have been prepared to improve the dissolution properties and bioavailability

of poorly soluble drugs such as griseofulvin, phenytoin, and prednisolone(15-17).

A similar enhancing effect was seen in this study. Sulfadiazine is a poorly soluble
drug and its solubilities in gastric and intestinal fluids were 0.5 mg/ml and 0.37
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mg/ml, respectively. The entrapment of the sulfadiazine crystals within the chitosan
matrix rendered the crystal surfaces more hydrophilic. This improved the wettability

tan
25 -
20 1 I I I 1

0 50 100 150 200

sulfadlazine added to 2% TPP solution (mg)

FIGURE 6
Effect of the addition of sulfadiazine to the external tripolyphosphate phase on the
sulfadiazine content in the chitosan beads.
 DRUG-CONTAINING CHITOSAN BEADS 1487

100

80

60
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* 2.7%
40 * 8.4%
4 12.0%
* 25.2%
20 -m 49.8%

n I I I I

0 3 6 9 12
time (hours)

FIGURE 7
Effect of sulfadiazineloading on drug release from chitosan beads in 0.1NHCl.
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100

80

60

40 4 2.7%
+ 8.4%
4 12.0%
+ 25.2%
20 * 49.8%

0
0 3 6 9 12
time (hours)

FIGURE 8
Effect of sulfadiazine loading on drug release from chitosan beads in simulated
intestinal fluid.
 1488 BODMEIER, OH, AND PRAMAR

of the crystals by the dissolution media and resulted in the enhancing effect of
chitosan on the sulfadiazine release.
The drug release from the chitosan beads depended on the penetration of the
dissolution medium into the beads, the eventual swelling and dissolution of the
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chitosan matrix, and the dissolution and subsequent diffusion of the drug through
the swollen or unswollen chitosan matrix. The swelling of the chitosan beads was
dependent on the pH of the dissolution medium. The beads when wetted by the
acidic dissolution medium swelled extensively, and formed a hydrogel matrix
before they dissolved completely. They did not swell or dissolve in simulated
intestinal fluid.
Figures 9 and 10 show the effect of the concentration of the gelling agent
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(TPP) on the sulfadiazine release in 0.1N HC1 and intestinal fluid. The anionic
counterion, TPP,acts as an ionic crosslinking agent. Drug release from hydrogels

0 3 6 9 12
time (hours)

FIGURE 9
Effect of the concentration of tripolyphosphate on the sulfadiazine release from
chitosan beads in 0.1N HC1.
 DRUG-CONTAINING CHITOSAN BEADS 1489

100 1
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0 3 6 9 12

time (hours)

FIGURE 10
Effect of the concentration of tripolyphosphate on the sulfadiazine release from
For personal use only.

chitosan beads in simulated intestinal fluid.

has been controlled by the degree of crosslinking (18). Increased crosslinking

reduced the degree of swelling and the rate of drug release. The same tendency was
seen in this study. The sulfadiazinerelease decreased with increasing concentration
of TPP in 0.1N HCI. The dissolution of the drug crystals and subsequent diffusion
through the mamx increased as a result of the swelling which increased the free
volume of the mamx. Ionization of the free amino groups in 0.1N HCl caused
hydration and swelling of the beads prior to the dissolution of chitosan. On the
contrary, the beads did not swell or dissolve in simulated intestinal fluid and the
effect of the crosslinking agent on the release of sulfadiazine was insignificant.
Except for the 1% TPP concentration, the release varied insignificantly with
increasing TPP concentrations.
Another interesting property of the beads was the ability to float on the
dissolution medium. Sustained release oral delivery has been achieved by using
 1490 BODMEIER, OH, AND PRAMAR

A
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B
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x120 X780

FIGURE 11
Scanning electron micrographs of cross sections of air-dried (1 1A-D) and freeze-dried
(1 1E) chitosan beads containing different amounts of sulfadiazine: A, 12.2%; B,
25.2%; C, 57.2%; D, 25.2% after dissolution study in intestinal fluid; E, 50.4%
(freeze-dried).
 Drug Development and Industrial Pharmacy Downloaded from informahealthcare.com by University of Guelph on 08/17/12
For personal use only.

E
D
C

X60
DRUG-CONTAINING CHITOSAN BEADS

FIGURE 11 C-E
x200
1491
 1492 BODMEIER, OH, AND PRAMAR

formulations which float on the gastric juice (19). Most other ionic gel-forming
macromolecules such as sodium alginate and polyacrylic acid are of anionic
character and dissolve or swell at higher pH values.
The morphology of the beads was investigated by scanning electron
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microscopy. The matrix structure depended on the method of drying. SEM-

photographs of air-dried chitosan spheres containing different amounts of drugs are
shown in Figure 11A-C. The beads shrank during air-drying. The sulfadiazine
crystals were embedded in a dense chitosan matrix. The increasing amount of
sulfadiazine crystals within the beads was clearly visible. The large openings
within the cross sections resulted from entrapped air bubbles incorporated during
the preparation of the chitosan-sulfadiazine suspension. Figure 11D shows the
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crystal-free cross section of a bead following a dissolution study in simulated

intestinal fluid. The freeze-dried beads were significantly larger and had a much
more porous internal structure when compared to the air-dried beads as shown in
Figure 11E.The beads were frozen instantaneously and did not shrink during the
freeze-dryingprocess.
In summary, drug containing chitosan beads were successfully prepared by
gelling the cationic polysaccharide with the anionic counterion, tripolyphosphate.
As with most encapsulation techniques, the drug solubility in the external phase has
to be minimized to maximize drug entrapment. To achieve maximum drug content,
high payloads, short stirring times, low TPP concentrations, and a low internal to
external phase ratio were required. The beads showed pH-dependent swelling and
dissolution behavior. They swelled and dissolved in 0.1NHC1, but stayed intact in
simulated intestinal fluid. The method of drying determined the porosity of the
beads.
 CYTOSINE ARABINOSIDE RELEASE 1493

REFERENCES
1. R.A.A. Muzzarelli, "Chitin," Pergamon Press, Oxford, 1977
2. F. Lim, Appl. Biochem. Biotech., 10,81 (1984)
3. G.G. Allen, L.C. Altman, R.E. Bensinger, D.K.Ghosh, Y.Hirabayashi,
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A.N. Neogi, and S. Neogi, in "Chitin, Chitosan, and Related Enzymes," J.P.
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4. T, Nagai, Y. Sawayanagi, and N. Nambu, in "Chitin, Chitosan, and Related

Enzymes," J.P. Zikakis, ed., Academic Press, Inc., 1984, p. 21.
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Pharm. Bull., 33, 2107 (1985)
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For personal use only.

J. Pharm. Sci., 74,264 (1985)

8. Y. Machida, T. Nagai, M. Abe, and T. Sannan, Proceedings of the 4th
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1 1. Protan Laboratories, publication PLI-002,"Chitin and Chitosan--General
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12. M.F.A. Goosen, G.M. OShea, H.M. Gharapetian, S. Chou, and A.M. Sun,
Biotechnol. Bioeng., 27, 146 (1985)
13. R. Kohn, Pure Appl. Chem., 42,371 (1975)
 1494 BODMEIER, OH, AND PRAMAR

14. P.B. Deasy, "Microencapsulation and Related Active Ingredient Processes,"

Marcel Dekker, Inc, New York, 1984
15. Y. Sawayanagi, N. Nambu, and T. Nagai, Chem. Pharm. Bull., 30,4464
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16. Y. Sawayanagi, N. Nambu, and T. Nagai, Chem. Pharm. Bull., 31,2064

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For personal use only.