Biomaterials 26 (2005) 819826

Synthesis and in vitro evaluation of a novel thiolated chitosan

Krum Kafedjiiski
a
, Alexander H. Krauland
b
, Martin H. Hoffer
c
,
Andreas Bernkop-Schn. urch
a,
*
a
Department of Pharmaceutical Technology, Institute of Pharmacy, Leopold-Franzens-University Innsbruck, Innrain 52, Josef M. oller Haus,
A-6020 Innsbruck, Austria
b
Center of Pharmacy, Institute of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Althanstr. 14, A-1090 Vienna, Austria
c
MucoBiomer GmbH, Industriezeile 6, A-2100 Leobendorf, Austria
Received 4 November 2003; accepted 13 March 2004
Abstract
In order to achieve the same properties as chitosan4-thio-butyl-amidine and to overcome at the same time its insufcient
stability, the aim of this study was to evaluate the imidoester reaction of isopropyl-S-acetylthioacetimidate for the chemical
modication of chitosan and to study the properties of the resulting chitosanthioethylamidine (TEA) derivative. The
thioalkylamidine substitute was introduced without the formation of N-substituted non-thiol products. The resulting conjugates
exhibited 1.0570.17% or 139.68717.13 mmol immobilized free thiol groups per gram polymer and a total amount of reduced and
oxidized thiol groups of 1.8170.65% or 179.46767.95 mmol/g polymer. By the immobilization of thiol groups mucoadhesion was
strongly improved due to the formation of disulde bonds with mucus glycoproteins. ChitosanTEA was investigated regarding to
its mucoadhesive properties via tensile studies and the rotating cylinder method. In tensile studies the total work of adhesion of
chitosanTEA was increased 3.3-fold in comparison to unmodied chitosan. Results from the rotating cylinder method showed an
improvement ratio of 8.9 for chitosanTEA compared with unmodied chitosan. In spite of the immobilization of thiol groups onto
chitosan its swelling behavior in aqueous solutions was not signicantly altered. Cumulative release studies out of matrix tablets
comprising the chitosanTEA and the model compound uorescence labeled dextrane (FD
4
) demonstrated a controlled release over
3 h with a trend toward a pseudo-zero-order kinetic. Because of these features the new chitosan thioamidine conjugate might
represent a promising new polymeric excipient for various drug delivery systems.
r 2004 Elsevier Ltd. All rights reserved.
Keywords: Thiolated chitosan; Chitosanthioethylamidine conjugates; Isopropyl-S-acetylthioacetimidate.HCl; Mucoadhesion
1. Introduction
Chitosan is a natural, cationic aminopolysaccharide
copolymer of glucosamine and N-acetylglucosamine. It
is obtained by the alkaline, partial deacetylation of
chitin, which originates from shells of crustaceans such
as crabs and prawns [1]. Chitosan is a biodegradable,
biocompatible, less toxic and mucoadhesive biopolymer
[2]. Its use as a pharmaceutical excipient is meanwhile
well-established [3]. It has been reported to enhance
drug permeation across the intestinal, nasal and buccal
mucosa [46].
In order to further enhance the mucoadhesive proper-
ties of chitosan, different chitosan derivatives have been
developed based on various theories explaining the
mechanism of mucoadhesion [7]. By applying the known
concept of the immobilization of thiol groups to the
primary amino groups of chitosan, the above-mentioned
properties of the polymer were strongly improved [8
10]. In such a way chitosancysteine conjugates,
chitosanthioglycolic acid conjugates and chitosan
4thio-butyl-amidine conjugates (chitosanTBA) have
been obtained [1113]. Among them chitosanTBA
turned out to exhibit the most promising features. The
modifying reagent for chitosanTBA conjugates is 2-
iminothiolane (a cyclic thioimidate), which reacts with
amino groups and introduces a sulfhydryl residue via a
positively charged amidine substructure. Thus, when
2-iminothiolane is applied for the modication of
ARTICLE IN PRESS
*Corresponding author. Tel.: +43-512-507-53-83; fax: +43-512-
507-29-33.
Email-address: andreas.bernkop@uibk.ac.at
(A. Bernkop-Schn. urch).
0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2004.03.011
chitosan, the surface charge distribution of the polymer
can be improved. However, storage stability studies
under N
2
showed an insufcient stability, which resulted
in a decrease of free thiol moieties. This might be due to
the formation of N-chitosanyl-substituted 2-iminothio-
lane structures. This undesired side-reaction occurs after
the derivatization of different amines with 2-iminothio-
lane. It involves the loss of ammonia and yields
recyclized N-substituted 2-iminothiolanes, as shown in
Fig. 1 [14].
As the degree of thioalkylamidine substitution with
B2% or B200 mmol SH groups/g polymer for chitosan
TBA is not sufcient for detection and proper identi-
cation via NMR analyses, the assumed structure was
conrmed by derivatization of (+)-d-glucosamine.HCl
with 2-iminothiolane under identical conditions as for
chitosan [15]. High-resolution mass spectroscopy (API
QSTAR Pulsar Hybrid-System
TM
/electrospray ioniza-
tion, positive mode) revealed indeed the presence of
N-glucosaminyl-2-iminothiolane [m/e (%) 264.08 (29),
M
+
+1] as well as glucosamine-TBA [m/e (%) 281.11
(1 0 0), M
+
+1].
According to these observations, a ligand, which is
truncated by two CH
2
groups and thus cannot recyclize,
will be stable. Reagents with similar structures, in
particular S-acetylmercaptoalkylthioimidates have been
investigated as heterobifunctional linkers for immuno-
toxin modulation (Fig. 2) [16].
In order to generate, on the one hand, a thiolated
chitosan exhibiting the same features as chitosanTBA
and to guarantee, on the other hand, also the stability of
this novel excipient, the objective of our study was to
synthesize the new chitosan amidine conjugates: chit-
osanthioethylamidine (TEA) (Fig. 3). It was achieved
by the modication of chitosan with the new reagent:
isopropyl-S-acetylthioacetimidate (i-PATAI.HCl). Apart
from the chemical modication, it was the purpose of
this study to evaluate the mucoadhesive properties and
swelling behavior of the new thiolated chitosan and to
prove whether a controlled release can be provided out
of such polymer conjugates when used as drug carrier
matrix.
2. Materials and methods
2.1. Materials
Chitosan (medium molecular mass: 400 kDa; degree
of deacetylation: 8385%) was purchased from Fluka
Chemie (Buchs, Switzerland). Isopropyl-S-acetylthioa-
cetimidate.HCl (i-PATAI) was kindly donated by
MucoBiomer (Leobendorf, Austria). Fluorescein iso-
thiocyanate-dextran (FD
4
av. mol wt 4000), l-cysteine
hydrochloride and 5,5
0
-dithiobis(2-nitrobenzoic acid)
were purchased from SIGMA (Austria). All chemicals
were of analytical grade.
2.2. Modication of chitosan with isopropyl-S-
acetylthioacetimidate
In order to obtain a 1% (w/v) polymer solution,
500 mg of chitosan was dissolved in 50 ml of 1% acetic
ARTICLE IN PRESS
S NH
2
+
Cl
S H
NH
2
+
N H
R
S N-R
R-NH
2
- NH
3
Fig. 1. Formation of an N-substituted 2-iminothiolane.
S
O
n
NH
2
+
S
Cl
n = 1 or 2
Fig. 2. S-acetylmercaptoalkylthioimidates.
H
H
H
NH
2
H
OH
CH
2
OH
H
O
O
O
S
O
NH.HCI
CH
2
OH
H
H
H
NH H
OH
H
O
O
HS
NH
2
+
Fig. 3. Synthetic pathway of chitosanTEA conjugates.
K. Kafedjiiski et al. / Biomaterials 26 (2005) 819826 820
acid and the mixture was stirred for 30 min. The pH was
adjusted to 67 with 1 m NaOH. After 30 min incubation
at room temperature, 500 mg isopropyl-S-acetylthioace-
timidate was added in ve aliquots over 1.5 h under
continuous stirring. The reaction mixture was stirred at
room temperature for one more hour. Deacetylation of
the mercapto-group occurred spontaneously in the
course of the reaction as already observed for S-
acetylmercaptoalkylthioimidates [16]. The addition of
0.05 m NH
2
OH solution at pH 67 as a common method
for deprotection of S-acetyl groups led to no further
increase of absorbance, thus deprotection was assumed
as being quantitative [16,17].
The resulting polymer conjugate was dialyzed rst
against 5 mm HCl, two times against 5 mm HCl contain-
ing 1% NaCl, once against 5 mm HCl and nally against
1 mm HCl. Controls were prepared in the same way but
omitting isopropyl-S-acetylthioacetimidate. Finally, the
aqueous polymer solutions (samples and controls) were
lyophilized at 30

C and 0.01 mbar (Christ Beta 1-8k;

Osterode am Harz, Germany). Samples were stored at
4

C until further use.

2.3. Determination of the thiol group and disulde bond
content
The degree of modication, i.e. the amount of free
thiol groups immobilized on chitosan, was determined
photometrically by using Ellmans reagent. Initially,
0.5 mg of each conjugate was hydrated in 250 ml of
demineralized water. To aliquots (250 ml) of the con-
jugate solutions, 250 ml of 0.5 m phosphate buffer pH 8.0
and 500 ml of Ellmans reagent [3 mg of 5,5
0
-dithiobis(2-
nitrobenzoic acid) dissolved in 10 ml of 0.5 m phosphate
buffer pH 8.0] were added. Then the samples were
incubated for 2 h at room temperature. After removing
the precipitated polymer by centrifugation (24,000g;
5 min), 300 ml of the supernatant uid was transferred to
a microtitration plate and the absorbance was measured
at a wavelength of 450 nm with a microtitration-plate
reader (Anthos Reader; Salzburg, Austria). The amount
of free thiol groups was calculated from an according
standard curve obtained by solutions with increasing
concentrations of l-cysteine hydrochloride.
Disulde content was measured after reduction with
NaBH
4
and addition of 5,5
0
-dithiobis(2-nitrobenzoic
acid) as described by Habeeb [18]. To determine the
total amount of bound thiol functions 0.5 mg of the
conjugates was hydrated in 350 ml of demineralized
water and then 650 ml of 0.05 m phosphate buffer pH 6.8
was added. After a swelling process of 30 min, 1 ml of a
freshly prepared 4% solution of sodium-borohydride
was added to the polymer suspension. The mixture was
then incubated for 1 h in an oscillating water bath at
3770.5

C. Thereafter, 200 ml of 5 m HCl was added and

the reaction mixture was agitated for 10 min in order to
destroy the remaining sodium-borohydride. The solu-
tion was neutralized by the addition of 1 ml 1 m
phosphate buffer pH 8.5 and then 100 ml of 0.4% (m/v)
Ellmans reagent dissolved in 0.5 m phosphate buffer pH
8.0 was immediately added. After incubation for 1 h at
room temperature aliquots of 250 ml were transferred to
a microtitration plate and the absorbance was measured
at 450 nm with a microtitration-plate reader (Anthos
Reader 2001, Salzburg, Austria). The quantity of bound
TEA was calculated using a standard curve obtained
by the thiol group determination of a series of solu-
tions containing increasing concentrations of cysteine
hydrochloride. The amount of disulde bonds was
calculated by subtracting the quantity of free thiol
groups from the totality of thiol moieties present on the
polymer.
2.4. Evaluation of the swelling behavior
The water-absorbing capacity was determined by a
gravimetric method. Thirty milligrams of each of the
thiolated chitosans and the corresponding unmodied
chitosans were compressed (Hanseaten Type EI, Ham-
burg, Germany) to 5.0 mm diameter at-faced tablets.
The compaction pressure was kept constant during the
preparation of all tablets. Test tablets were xed to a
needle and placed in a beaker containing 100 mm
phosphate buffer pH 6.8 at 3770.5

C. At scheduled
time intervals the swollen test tablets were taken out of
the incubation medium, excess water was removed and
the water uptake was determined gravimetrically [19].
2.5. Tensile studies
Thirty milligrams of lyophilized thiolated chitosan
and controls were compressed to tablets. Then the
tablets were attached to a freshly excised 4 mm thick
intestinal porcine mucosa in the following manner. The
tablet was attached to a stainless-steel at disc (8 mm in
diameter, 0.3 g of weight in the system), which was hung
by a nylon thread (15 cm) from a laboratory stand. The
porcine mucosa was xed on a glass support using a
cyanoacrylate adhesive. The support with the xed
tissue was completely immersed in 400 ml of 100 mm
phosphate-buffer saline pH 6.8. The beaker was placed
on a balance and was carefully raised by a mobile
platform until the mucosa came in contact with the
tablet. The contact was determined when the nylon
thread holding the tablet became bent. After 30 min
incubation at 25

C, the mucosa was pulled down from

the tablet at a rate of 0.1 mm/s. Data points were
collected every second by a personal computer (WIND-
WEDGE software; TAL technologies Inc., Philadel-
phia, PA) connected to the balance and then data were
transferred to EXCEL 97 (Microsoft, USA). The total
work of adhesion (TWA), representing the area under
ARTICLE IN PRESS
K. Kafedjiiski et al. / Biomaterials 26 (2005) 819826 821
the force/distance curve and the maximum detachment
force (MDF) representing the force needed for detach-
ment were calculated [19].
2.6. In vitro mucoadhesion studies with the rotating
cylinder method
In order to evaluate the adhesion time of thiolated
chitosan on the mucosa, a slightly modied method as
described previously has been used [8]. Thirty milligrams
of modied chitosan and control tablets were attached
to a freshly excised intestinal porcine mucosa, which was
xed on a stainless-steel cylinder (diameter: 4.4 cm;
height: 5.1 cm; apparatus 4-cylinder, USP). Thereafter,
the cylinder was placed in the dissolution apparatus
according to the USP containing 500 ml of 100 mm
phosphate buffer pH 6.8 at 3770.5

C and agitated
with 125 rpm. The detachment of the test tablets
was determined visually during an observation time of
36 h [13].
2.7. Preparation of a delivery system comprising
chitosanTEA conjugate
First, 75 mg of chitosanTEA conjugate was dissolved
in 15 ml of demineralized water and homogenized with
15 mg of FD
4
dissolved in 3 ml of demineralized water.
Samples were lyophilized at 30

C and 0.01 mbar

(Christ Beta 1-8k; Osterode am Harz, Germany).
Tablets were compressed out of the lyophilized poly-
mer/FD
4
mixture (30 mg, 5 mm diameter, Hanseaten
Type El, Hamburg, Germany).
2.8. Release studies
Release studies were performed with uorescein
isothiocyanate-dextran (FD
4
) used as a model com-
pound. The release rate of FD
4
from tablets as described
before was analyzed in vitro. Tablets were placed in a
beaker containing 10 ml of release medium (100 mm
phosphate buffer pH 6.8 at 3770.5

C). Beakers were

closed up and then placed on an oscillating water bath
(GFL 1092; 100 rev/min) and incubated at 3770.5

C.
Aliquots of 1 ml were withdrawn at 1 h intervals for 8 h
and replaced with an equal volume of release medium
equilibrated at 37

C. Sink conditions were maintained

throughout the study. Release rate of FD
4
was assayed
by measuring the uorescence intensity within each
sample (excitation wavelength (l
exc
): 485 nm; emission
wavelength (l
em
): 535 nm; SLT, Spectra Fluor, Tecan,
Austria). The amount of released FD
4
was calculated by
interpolation from a standard curve containing increas-
ing concentrations of FD
4
.
2.9. Statistical data analysis
Statistical data analyses were performed using the
t-test with po0:05 as the minimal level of signicance.
3. Results and discussion
3.1. Synthesis of chitosanthioethylamidine conjugates
Imidoesters (or imidates) are esters of hypothetical
imidic acids. They react with nucleophiles such as
amines or primary amino groups of amino acids thereby
forming amidines. Consequently, they were among the
rst used to modify protein structures and as cross-
linking agents [20].
In this work, the imidoester reaction scheme was used
for the rst time for the chemical modication of
chitosan (Fig. 3). The synthesized reagentisopropyl-S-
acetylthioacetimidate.HCl (i-PATAI) is also an S-group
carrier, resulting in a new thiolated chitosan with a
positive net electronic charge on it. It is important that
an imidoester reacts rapidly with an aminemaximum
for 1.5 h in comparison to the reaction with
2-iminothiolane which ends after 24 h under continuous
stirring at room temperature [13]. Besides the short
chain of i-PATAI excludes theoretically the possibility
of yielding cyclic non-thiol products. The nucleophilicity
of amino groups is dictated by the protonation state
making the reaction pH dependent. Usually, the
reaction is carried out under neutral to mild alkaline
conditions (pH 710) up to 30

C. In view of the fact that

chitosan is insoluble at this pH, the reaction was carried
out at pH 6.57 at which pH value the oxidation process
of thiol groups is decreased [16]. The efcacy of the
purication method described here could be veried by
corresponding controls which were prepared in exactly
the same way as the polymer conjugates but omitting
i-PATAI during the reaction, exhibiting no thiol groups.
The lyophilized thiolated chitosan or so-called chitosan
TEA conjugates appeared as white, odorless powder of
brous structure.
The conjugates produced by this method exhibited
1.0570.17% or 139.68717.13 mmol immobilized free
thiol groups per gram polymer and a total amount of
reduced and oxidized thiol groups of 1.8170.65% or
179.46767.95 mmol/g polymer. The amount of disulde
bonds could be calculated by subtracting the quantity of
free thiol groups from the totality of thiol moieties
present on the polymer.
The results indicate that the amount of covalently
bound thiol groups can be increased approximately
twice as much as by allowing the reaction to go in a way
that prevents the oxidation of thiol groups. This can be
achieved by carrying out the reaction under inert
conditions or by introducing an additional step in the
ARTICLE IN PRESS
K. Kafedjiiski et al. / Biomaterials 26 (2005) 819826 822
synthesis of the product, i.e. the treatment of the
obtained polymer with suitable reducing agents.
The described synthetic pathway for chitosanTEA
conjugates is one effective, industrially applicable,
reproducible method. In contrast, when 2-iminothiolane
is used to modify amino groups, the mechanism includes
apart from the immobilization of a thiol group with
amidine functionality the shift of equilibrium toward the
formation of an N-substituted non-thiol product [14], as
illustrated in Fig. 1. This leads to complications
encountered with the standardization and stability of
chitosanTBA.
3.2. Swelling behavior
The swelling behavior of mucoadhesive polymers has
a considerable inuence on their adhesive properties and
cohesiveness. The hydration theory postulates that
mucoadhesive polymers take water from the underlying
mucosal tissue by absorbing, swelling and capillary
effects, leading to a considerably strong adhesion [21].
Mucoadhesive drug delivery system can be attached in
dry form to buccal, ocular, nasal or vaginal mucosa. A
prolonged residence time of the system on mucosa leads
to an extended period of absorption and consequently of
improved bioavailability. When it is directed to the
small intestine, it reaches its target already in at least
partially hydrated form. Therefore, slow swelling is a
requisite to avoid the formation of an over hydrated
form that loses its mucoadhesive properties before
reaching the target [22].
In order to evaluate the correlation between the
swelling behavior and mucoadhesion, the study was
carried out in 100 mm phosphate buffer pH 6.8 at
3770.5

C. Ch-TEA is very poorly soluble at pH 6.8 and

for the time of the test it has been partially dissolved as
well as erosion of the tablet was observed.
The swelling behavior of chitosanTEA conjugates
and unmodied chitosan is shown in Fig. 4. Results
from the test showed an improvement swelling ratio
(swelling weight relative to the initial weight) of 1.2 for
chitosanTEA compared with unmodied chitosan,
respectively. The values were obtained in the same
range for chitosanTBA and chitosanthioglycolic acid
conjugates dependent on the degree of modication
[19,22].
In spite of the immobilization of thiol groups on
chitosan its swelling behavior in aqueous solutions was
not signicantly altered. The determined difference was
of no statistical signicance demonstrating that the
covalent attachment of thioacetimidate had no inuence
on the swelling behavior of chitosan. Therefore, the
swelling behavior is not responsible for the improved
mucoadhesive properties of the conjugates.
3.3. Mucoadhesion studies
Tensile studies represent the most widely employed
in vitro test method for the assessment of the adhesive
strength of mucoadhesives. In the present study, the
MDF and the TWA were low for chitosan controls
compared to chitosanTEA conjugates, demonstrating
the high afnity of the thiolated chitosan for mucosal
tissue (Figs. 5 and 6). TWA of chitosanTEA was
increased 3.3-fold in comparison to the control-unmo-
died chitosan.
The data were in the same range compared to other
thiolated chitosans depending on the degree of mod-
ication with thiol bearing moieties and the molecular
mass of chitosan. For example, chitosanthioglycolic
acid conjugate exhibiting almost twice as many thiol
groups as the chitosanTEA conjugate demonstrated a
6.3-fold increase in TWA compared to unmodied
chitosan [19].
These results are explained by the evidential theory of
the enhanced mucoadhesive properties of thiolated
chitosans in view of the formation of covalent bonds
between thiol groups of the polymer and cysteine-rich
subdomains of glycoproteins in the mucus layer [9,22].
The resulting disulde bond is a bridging structure most
commonly encountered in biological systems. In con-
trast, the traditional mucoadhesive polymers are only
able to form non-covalent bonds via ionic interactions
and hydrogen bonds within the mucus layer.
In order to conrm the results of tensile studies,
mucoadhesion studies were also carried out with the
rotating cylinder method (Fig. 7). The contact time or
the duration of adhesion is a critical factor in the design
and formulation of an effective mucoadhesive dosage
form. Results from the rotating cylinder method
showed an improvement ratio of 8.9 for chitosanTEA
ARTICLE IN PRESS
0
50
100
150
200
250
0 20 40 60 80
Time [min]
U
p
t
a
k
e
n

w
a
t
e
r

[
m
g
]
Fig. 4. Swelling behavior of tablets based on chitosanTEA () and
unmodied chitosan (B) in 100 mm phosphate buffer pH 6.8 at 37

C;
indicated values are means (7S.D.) of at least three experiments.
K. Kafedjiiski et al. / Biomaterials 26 (2005) 819826 823
compared with unmodied chitosan. The improvement
ratio was calculated by the adhesion time of conjugates
versus adhesion time of the control [19].
The novel conjugates combine two different mechan-
ism of mucoadhesion: ionic interactions between the
cationic groups of chitosan and anionic moieties
provided by sialic acid and sulfonic acid substructures
within the mucus layer, and the formation of disulde
bonds due to the introduction of thiol groups.
3.4. Drug release studies
Another advantage of chitosanTEA in drug delivery
systems is a controlled drug release, which can be
reached by using the polymer as a carrier matrix. Recent
studies have shown that nally total drug release in a
standard phosphate buffer of pH 6.8 for unmodied
chitosan occurs very quickly: plateau is reached within
30 min [23]. Fig. 8 shows a cumulative controlled release
over 3 h of the model compound FD
4
out of matrix
tablets based on chitosanTEA with a trend toward a
pseudo-zero-order kinetic. In addition, tablets did not
disintegrate and showed no erosion during the study.
One reason for the relatively slow release may be the
possibility of the formation of disulde bonds within the
matrix tablet [24]. This cross-linking process leads to
lowering the velocity of diffusion of drug molecules. The
polymer matrix probably combines two major types of
ARTICLE IN PRESS
0
20
40
60
80
100
120
140
160
Chitosan Chitosan-TEA
T
W
A

(

J
)
Fig. 6. Comparison of the adhesive properties of chitosanTEA
conjugates and unmodied chitosan. Represented values are means
(7S.D.; n 325) of the TWA determined in tensile studies at pH 6.8.
0
5
10
15
20
25
Chitosan Chitosan-TEA
T
i
m
e

[
h
]
Fig. 7. Comparison of the adhesion time of chitosanTEA conjugates
and unmodied chitosan on the rotating cylinder. The indicated time
of adhesion represents the mean (7S.D.) of at least three experiments.
0
50
100
150
200
250
300
350
400
450
0 1 2 3 4 5 6
time [h]
F
D
4

r
e
l
e
a
s
e
d

[

g
/
m
l
]
Fig. 8. Release prole of FD
4
from tablets comprising the chitosan
TEA conjugate. Dissolution studies were performed in 100 mm
phosphate buffer pH 6.8 at 37

C; indicated values are means

(7S.D.) of at least three experiments.
0
2
4
6
8
10
12
Chitosan Chitosan-TEA
M
D
F

(
m
N
)
Fig. 5. Comparison of the adhesive properties of chitosanTEA
conjugates and unmodied chitosan. Represented values are means
(7S.D.; n 325) of the MDF determined in tensile studies at pH 6.8.
K. Kafedjiiski et al. / Biomaterials 26 (2005) 819826 824
mechanisms for drug release: controlled diffusion and
swelling.
3.5. Views on chitosanTEA conjugates
In view of drug delivery, these improved mucoadhe-
sive properties along with a controlled release out of the
polymer matrix should render chitosanTEA as a
promising tool, which might increase drug bioavail-
ability by adhering to mucosal tissues. Because of these
properties, the evaluation of chitosanTEA should be
further continued as well as its application in various
drug delivery systems should be investigated. It has been
shown that the presystemic metabolism of orally given
peptide drugs can be reduced, if the delivery system
provides an intimate contact with the intestinal mucosa
[19]. For instance, in vivo studies showed that the use of
a chitosanTBA carrier matrix provides a signicantly
enhanced oral bioavailability of salmon calcitonin used
as a model drug [25,26]. According to this, similar
features might be observed for chitosanTEA conju-
gates representing an essential step forward in the
development of this new generation of polymeric
excipients.
4. Conclusions
The chemical modication of chitosan with isopropyl-
S-acetylthioacetimidate.HCl leads to conjugates exhibit-
ing improved mucoadhesive properties and good swel-
ling behavior. In addition, a controlled drug release can
be achieved. In contrast to chitosanTBA conjugates
unintended cyclization side reactions can be excluded.
Because of these characteristics, the chitosanthioethy-
lamidine conjugate appears to be a promising novel
excipient for the development of various drug delivery
systems.
Acknowledgements
This work was partly supported by Grant Impul-
sprojekt E 71 from the Fonds zur F. orderung der
wissenschaftlichen Forschung (FWF) to M. Hoffer and
MucoBiomer GmbH. The authors thank Mr. Str. obel
and co-workers from the slaughterhouse Totzenbach for
the supply of porcine intestinal mucosa.
References
[1] Muzzarelli RAA. Chitin. In: Muzzarelli RAA, editor. Natural
chelating polymers: alginic acid, chitin, and chitosan. New York:
Pergamon Press; 1973. p. 83252.
[2] Lehr CM, Bouwstra JA, Schacht EH, Junginger HE. In vitro
evaluation of mucoadhesive properties of chitosan and some
other natural polymers. Int J Pharm 1992;78:438.
[3] Illum L. Chitosan and its use as a pharmaceutical excipient.
Pharm Res 1998;15:132631.
[4] Aspden TJ, Illum L, Skaugrad O. Chitosan as a nasal delivery
system: evaluation of insulin absorption enhancement and effect
of nasal membrane integrity using rat models. Eur J Pharm
Biopharm 1996;4:2331.
[5] Senel S, Kremer MJ, Kas S, Wertz PW, Hincal AA, Squier CA.
Enhancing effect of chitosan on peptide drug delivery across
buccal mucosa. Biomaterials 2000;21:206771.
[6] Dodane Y, Khan MA, Merwin JR. Effect of chitosan on
epithelial permeability and structure. Int J Pharm 1999;182:
2132.
[7] Thanou M, Verhoef JC, Junginger HE. Trimethylated chitosan
derivatives are effective and safe penetration enhancers for oral
peptide drug delivery and absorption. STP Pharm Sci
2000;10:3159.
[8] Bernkop-Schn. urch A, Schwarz V, Steininger S. Polymers with
thiol groups: a new generation of mucoadhesive polymers? Pharm
Res 1999;16:87681.
[9] Leitner VM, Walker GF, Bernkop-Schn. urch A. Thiolated
polymers: evidence for the formation of disulphide bonds with
mucus glycoproteins. Eur J Pharm Biopharm 2003;56:20714.
[10] Bernkop-Schn. urch A. Mucoadhesive polymers. In: Dumitriu S,
editor. Polymeric biomaterials. 2nd ed. New York: Marcel
Dekker; 2000. p. 14765.
[11] Bernkop-Schn. urch A, Brandt UM, Clausen AE. Synthesis and
in vitro evaluation of chitosancysteine conjugates. Sci Pharm
1999;67:196208.
[12] Bernkop-Schn. urch A, Hopf TE. Synthesis and in vitro evaluation
of chitosanthioglycolic acid conjugates. Sci Pharm 2001;69:
10918.
[13] Bernkop-Schn. urch A, Hornof M, Zoidl T. Thiolated polymers
thiomers: synthesis and in vitro evaluation of chitosan2-
iminothiolane conjugates. Int J Pharm 2003;260:22937.
[14] Singh R, Kats L, Blattler WA, Lambert JM. Formation of N-
substituted 2-iminothiolanes when amino groups in proteins and
peptides are modied by 2-iminothiolane. Anal Biochem
1996;236:11425.
[15] Guggi D. Development of non-invasive peptide drug delivery
systems. Thesis, University of Vienna, 2003.
[16] Delprino L, Giacomotti M, Dosio F, Brusa P, Ceruti M, Grosa
G, Cattel L. Toxin-targeted design for anticancer therapy. I:
synthesis and biological evaluation of new thioimidate hetero-
bifunctional reagents. J Pharm Sci 1993;82:50612.
[17] Duncan RJS, Weston PD, Wrigglesworth R. A new reagent,
which may be used to introduce sulfhydryl groups into proteins,
and its use in the preparation of conjugates for immunoassay.
Anal Biochem 1983;132:6873.
[18] Habeeb AF. A sensitive method for localization of disulde
containing peptides in column efuents. Anal Biochem
1973;56:605.
[19] Kast CE, Bernkop-Schn. urch A. Thiolated polymersthiomers:
development and in vitro evaluation of chitosanthioglycolic
acids conjugates. Biomaterials 2001;22:234552.
[20] Whitely NM, Berg HC. Amidination of the outer and inner
surfaces of the human erthrocyte membrane. J Mol Biol
1974;87:54161.
[21] Mortazavi SA, Smart JD. An investigation into the role of water
movement and mucus gel dehydration in mucoadhesion.
J Control Rel 1993;25:197203.
[22] Roldo M, Hornof M, Caliceti P, Bernkop-Schn. urch A. Improve-
ment in the mucoadhesive properties of chitosan by derivatisation
with 2-iminothiolane. Eur J Pharm Biopharm 2004;57:11521.
ARTICLE IN PRESS
K. Kafedjiiski et al. / Biomaterials 26 (2005) 819826 825
[23] Khoo CGL, Frantzich S, Rosinski S, Sjostrom M, Hoogstraate J.
Oral gingival delivery systems from chitosan blend with hydro-
philic polymers. Eur J Pharm Biopharm 2003;55:4756.
[24] Kast CE, Valenta C, Leopold M, Bernkop-Schn. urch A. Design
and in vitro evaluation of a novel bioadhesive vaginal drug
delivery system for clotrimazole. J Control Rel 2002;81:34754.
[25] Bernkop-Schn. urch A, Kast CE, Guggi D. Permeation enhancing
polymers in oral delivery of hydrophilic macromolecules: thiomer/
GSH systems. J Control Rel 2004;93:95103.
[26] Guggi D, Krauland AH, Bernkop-Schn. urch A. Systemic peptide
delivery via the stomach: in vivo evaluation of an oral dosage
form for salmon calcitonin. J Control Rel 2003;92:12535.
ARTICLE IN PRESS
K. Kafedjiiski et al. / Biomaterials 26 (2005) 819826 826