pH-Sensitive Genipin-Cross-Linked Chitosan Microspheres For

Heparin Removal
Kamil Kamin ski, Karolina Zazakowny, Krzysztof Szczubiaka,* and Maria Nowakowska
Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krako w, Poland
Received July 2, 2008; Revised Manuscript Received October 7, 2008
Chitosan hydrogel microspheres were obtained by cross-linking chitosan in its inverse emulsion using genipin as
cross-linker. The genipin-cross-linked chitosan microspheres (ChGp) swell signicantly in water at pH values
below 6.5 and shrink to a smaller extent at pH values above 6.5. ChGp microspheres bind heparin in water. The
kinetics of heparin binding was found to be pH dependent and was faster and more efcient at a lower pH. That
can be also controlled by the weight of ChGp microspheres used. Rate and efciency of heparin adsorption at pH
7.4, which is typical of blood, could be increased by quaternization of ChGp microspheres using glycidyltrim-
ethylammonium chloride (GTMAC). The polymeric material obtained thus can be potentially useful for heparin
removal in biomedical applications.
Introduction
Heparin (Figure 1), a substance discovered by McLean almost
a century ago,
1
has been in clinical use since 1937 and was the
rst polysaccharide-based drug to nd widespread application
in humans.
2
Heparin is a complex mixture of highly sulfated
glycosaminoglycan (GAG) polysaccharides that are produced
and stored in mast cells present in animal tissues (e.g., porcine
intestines or bovine lungs). (It has the highest negative charge
density of any known biological molecule
3
with an average of
2.7 negative charges per disaccharide repeating unit.
4
) It shows
powerful anticoagulant activity, although only one-third of the
heparin molecules have anticoagulant properties. It acts by
enhancing the ability of antithrombin (AT) to inactivate
thrombin and factor Xa, enzymes that promote coagulation.
5
Therefore, heparin is the drug of choice when a rapid antico-
agulant effect is required (e.g., during surgical procedures,
particularly to prevent thrombosis in devices used in extracor-
poreal therapy such as dialysers and oxygentators). It also has
many other therapeutic applications, for example, in unstable
angina and acute myocardial infarction.
However, the administration of heparin results in many side
effects,
6
among which the most frequent are bleeding, heparin-
induced thrombocytopenia (HIT),
7
and osteoporosis.
Therefore, it is often required to remove heparin from blood
after it has exerted its anticoagulant effect. Several methods of
its removal have been developed. The most common method
is based on the administration of protamine, a protein that was
introduced to clinical use as a heparin antagonist almost
simultaneously with heparin.
8
It is characterized by an excep-
tionally high content of basic amino acids (i.e., arginine, lysine,
and histidine), which may reach 80%.
9,10
In addition to
neutralizing the anticoagulant action of heparin, it is also used
for titrating heparin in vitro.
11
Poly-L-lysine is another polymer
used for heparin removal,
12-14
and it also amplies the action
of protamine.
15,16
Yet another approach to the problem in
heparin removal is its enzymatic degradation by immobilized
heparinase.
17,18
Unfortunately, the methods of heparin removal may produce
side effects. Protamine, if left in the bloodstream, is known to
provoke adverse reactions in 10% of patients.
19
The reactions
can be dramatic and often even lethal,
20
involving pulmonary
hypertension, systemic hypotension, anaphylactic shock, throm-
bocytopenia, granulocytopenia, complement activation, and
cytokine release.
21
Protamine-dependent heparin removal is
inaccurate and complicated by allergic reactions.
22
Many attempts to construct a heparin removal device, mostly
containingimmobilizedpoly-L-lysine, havebeenundertaken.
21,23-25
Despite the promising experimental data, there has been only
limited experience with such heparin removal devices, and none
of them have been clinically implemented until now. A method
frequently used to avoid complications brought about by free
heparin antagonists is their immobilization on polymeric sup-
ports. For example, protamine was immobilized as a bioligand
on the afnity matrix formed by grafting an acrylic polymer
on a cellulose backbone,
26
and a prototype heparin removal
system was developed by coupling protamine to the inner lumen
of cellulose hollow bers.
27,28
The results showed that at a blood
ow of 100 mL/min, the system removed >50% of administered
heparin in 10 min. Whereas a rapid protamine injection produced
profound hypotensive responses in dogs, the use of the heparin
removal system did not elicit any statistically signicant changes
in any of the hemodynamic parameters monitored. Another
report describes efcient heparin removal using alginate/poly-
L-lysine beads.
29
In this article, we propose an alternative polymeric system
that can be potentially useful for the construction of devices
for heparin removal from the bloodstream. It is based on the
application of polymeric microspheres obtained by the cross-
linking of chitosan using genipin. Although interactions between
chitosan and heparin have already been intensively studied
(Figure 2),
30-37
there are no reports on the application of
chitosan microspheres for heparin removal from blood or other
physiological liquids. A main advantage of the polymeric system
we propose is that all of the basic reagents used to prepare the
microspheres (chitosan, genipin, surfactants) are inexpensive and
nontoxic. The rate of heparin removal can be tuned by changing
the pH in a wide range. Moreover, to enable heparin removal
at higher pH values, we have cationically modied chitosan
* Corresponding author. E-mail: szczubia@chemia.uj.edu.pl. Tel: +48
12 6632020. Fax: +48 12 6340515.
Biomacromolecules 2008, 9, 31273132 3127
10.1021/bm800724q CCC: $40.75 2008 American Chemical Society
Published on Web 10/23/2008
microspheres, which resulted in a high rate of heparin removal,
even at the pH value that is typical of blood (ca. 7.4); this allows
even more medical applications of this material.
Experimental Section
Materials. Low-molecular-weight chitosan (Ch, Aldrich), genipin
(Gp, Challenge Bioproducts, 98%), glycidyltrimethylammonium chlo-
ride (GTMAC, Fluka, 90%), heparin sodium salt from bovine intestinal
mucosa (Sigma), Span 40 (Fluka), Span 80 (Fluka), Azure A (standard,
Fluka), potassium chloride (analytical grade, POCh), potassium dihy-
drogen phosphate (analytical grade, POCh), disodium hydrogen phos-
phate (analytical grade, POCh), sodium chloride (analytical grade,
POCh), cyclohexane (Lach-Ner, analytical grade), and acetic acid
(POCh, reagent grade) were used as received. Water was distilled twice
and puried using the Millipore SIMPLICITY system.
Apparatus. UV spectra were taken using an HP 8452A diode-array
spectrophotometer in 1 cm optical path quartz cuvettes. Microscopic
images were obtained using a Nikon TE-2000 uorescence microscope.
Synthesis of Genipin-Cross-Linked Chitosan Microspheres
(ChGp). Chitosan microspheres were prepared using a modied
procedure that was previously described.
38
In short, chitosan (0.5 g)
was dissolved in 35 mL of 0.7% v/v acetic acid and was then dialyzed
against water to remove acetic acid. After dialysis, the pH was 5.2.
The above chitosan solution was mixed with 250 mL of cyclohexane
in a 500 mL round-bottomed ask that contained 0.5 g Span 80 and
0.25 g Span 40. The mixture was emulsied while being stirred at 1200
rpm by a mechanical stirrer for 30 min. Then, 1 mL of 5% w/v aqueous
genipin solution in 70% v/v ethanol was added. The temperature of
the mixture was raised to 60 C, and the mixing of the emulsion was
continued. After 3 h, the aqueous phase became bluish, which indicated
Figure 1. Structure of heparin.
Figure 2. The complex between chitosan and heparin.
35
Figure 3. Structures of (a) chitosan and (b) genipin.
Figure 4. Optical microscope image of the ChGp microspheres.
Figure 5. Swelling behavior of ChGp gel at different pH values.
3128 Biomacromolecules, Vol. 9, No. 11, 2008 Kamin ski et al.
that the cross-linking reaction with Gp began. The mixture was left
overnight without stirring at room temperature. The chitosan micro-
spheres obtained were washed with an excess of cyclohexane and
ltered on a Buchner funnel. The microspheres were blotted with lter
paper and stored in PBS buffer for no longer than 1 day. We veried
that storage did not change their adsorptive properties. A part of the
microspheres was dried at 40 C in a vacuum oven.
Synthesis of the Cationically Modied Chitosan Microspheres
(ChGpGl). About 0.25 g ChGp microspheres was transferred for 5 h
to 50 mL of the dilute solution of acetic acid at a pH of 5. Then, the
suspension of the microspheres in the above solution was placed in a
100 mL round-bottomed ask, and 10 mL of GTMAC was added. The
mixture was kept at 55 C while it was mixed with a magnetic stirrer.
The microspheres were ltered under reduced pressure and washed a
few times with an excess of methanol followed by pH 7.4 buffer, in
which further studies with the microspheres were carried out.
Determination of Heparin Concentration. Heparin concentration
in the solutions was determined by using the Azure A spectrophoto-
metric method. (See, e.g., ref 39.) In short, to 0.1 mL of the heparin
solution were added 0.9 mL of the respective buffer and 1 mL of
4.010
-5
M Azure A solution. The solution was mixed, and its UV-vis
absorption spectrum was measured. Heparin was assayed on the basis
of the intensity of the 630 nm band, which corresponds to monomeric
Azure A molecules.
Regeneration of ChGpGl Microspheres. ChGpGl microspheres (80
mg) were dispersed in 5 mL of pH 7.4 buffer containing 1 mg of
heparin. We spectrophotometrically veried that the concentration of
heparin dropped to almost 0 within 10 min. The suspension was then
centrifuged at 12 000 rpm for 3 min. The solution (2.5 mL) was
removed and replaced by the same volume of 2 M NaCl solution. The
suspension was mixed, sonicated for 5 min, and centrifuged for another
5 min. The solution from above the microspheres was taken, and the
concentration of heparin was determined by using the Azure A
spectrophotometric method.
Results and Discussion
We have chosen Ch and Gp (Figure 3) as starting materials
for obtaining polymeric microspheres by cross-linking the
chitosan aqueous solution dispersed in cyclohexane. Both of
these compounds display advantageous biomedical properties.
Chitosan is well known as a biodegradable and biocompatible
polymer.
40,41
It has many biomedical applications in the form
of a solution, lm, and microspheres. Genipin is a natural
nontoxic cross-linker that is extracted from Gardenia jasmi-
noides fruit. Cross-linking with Gp is easy to follow because
the cross-linked polymer turns intensively blue.
Also, the surfactants that are used to stabilize the emulsion
are known to be nontoxic. The microspheres obtained were quite
monodisperse with an average diameter of 30 m (Figure 4).
The swelling behavior of the ChGp gel has been studied as
a function of pH. The plot of the values of the swelling ratio,
S, dened as
S )
(W
s
-W
0
)
W
0
100%
where W
0
is the weight of dry gel and W
s
is the weight of
swollen gel, is shown in Figure 5.
It was found that the gel swells at pH values below 6.5,
whereas at higher pH values, it shrinks. This may be explained
by considering the protonation of chitosan chains at a low pH,
which results in their repealing from each other and favoring
penetration of the gel by water. The swelling of the gel is
reversible; for example, microspheres that are swollen at a pH
of 5.2 shrink when the pH value is increased.
The kinetics of gel swelling was investigated (Figure 6). We
found that the swelling was completed within 5 h (Figure 6a),
and S ts the linear dependence on ln(t) very well (Figure 6b).
Studies on Heparin Adsorption by ChGp Micro-
spheres. We studied the adsorption of heparin by the micro-
spheres by adding the microspheres to the buffered aqueous
heparin solutions and following the changes in heparin con-
centration using a colorimetric method with Azure A as the
titrant. We found that in 5 mL of buffer solution of pH 6.0 the
concentration of heparin decreased from an initial value of 200
g/mL to almost 0 within 50 min of the addition of 40 mg
ChGp microspheres (Figure 7). The kinetics of heparin adsorp-
tion were different from that found for heparin removal with
protamine; it follows a rst-order exponential curve.
42
Because
the degree of protonation of chitosan is greater at a lower pH
(and so is the positive charge density), it could be anticipated
that the degree of adsorption of negatively charged heparin on
microspheres is lower for higher pH values. That assumption
was conrmed by measurements of heparin adsorption in the
pH range of 6.0 to 8.0. The amount of heparin adsorbed by the
Figure 6. Swelling kinetics of ChGp gel in water.
Figure 7. Dependence of relative heparin concentration (c
0
) 200
g/mL, V ) 5 mL) on time after the addition of 40 mg ChGp
microspheres in pH (b) 6.0, (9) 6.8, ([) 7.4, and (2) 8.0 buffer.
Microspheres For Heparin Removal Biomacromolecules, Vol. 9, No. 11, 2008 3129
microspheres drastically changes with pH changes in that range.
Whereas at pH 6.8, heparin can be practically completely
removed from 5 mL of the solution containing heparin (c )
200 g/mL) by 40 mg of ChGp microspheres, at pH 7.4 to 8.0,
only 20% of heparin could be adsorbed under the same
conditions.
We have next studied how the amount of the ChGp
microspheres used inuences the adsorption prole of heparin
and whether the amount of heparin adsorbed at higher pH values
could be increased by increasing the content of ChGp micro-
spheres. The results are shown in Figure 8.
We found that at a pH of 6.8, the amount of ChGp
microspheres that was added strongly inuences the rate of
heparin adsorption. The increase in the weight of ChGp
microspheres from 40 to 150 mg decreases the time that is
required for a 50% reduction of heparin concentration from 25
to 10 min. At a pH of 7.4, heparin adsorption is much slower,
less efcient, and less sensitive to the amount of ChGp
microspheres added. The addition of as much as 80 mg ChGp
microspheres could reduce the heparin concentration to 60%
of its initial value within 50 min.
Studies on Heparin Adsorption by ChGpGl Micro-
spheres. Because the degree and rate of heparin adsorption at
pH 7.4 (the value characteristic of blood) were found to be quite
low, we have attempted to improve the efciency and rate of
heparin adsorption by functionalization of chitosan with GT-
MAC. GTMAC does not undergo pH-dependent protonation;
therefore, it imparts a positive charge on the chitosan macro-
molecule, even at higher pH values. Chitosan quaternized with
GTMAC was found to be nontoxic,
43
and it shows enhanced
antibacterial
44
and antifungal
45
activity compared with the native
chitosan. The microscope images have shown that the reaction
of ChGp microspheres with GTMAC only slightly affected their
shape. However, the diameter of the microspheres signicantly
decreased 3 to 4 m (Figure 9).
We expect that the glycidyl group of GTMAC reacted with
the primary amino groups of Ch (Figure 10).
This was conrmed by the FT-IR spectra of ChGp and
ChGpGl microspheres (Figure 11). In the IR spectrum of
ChGpGl microspheres, a band at 1483 cm
-1
appears, which
can be attributed to an asymmetric angular bending of the methyl
groups of GTMAC. This band is not present in the spectrum of
ChGp microspheres. Moreover, the band that corresponds to
the primary amine group deformation vibrations at 1560 cm
-1
that are present in the ChGp microspheres spectrum is much
weaker in the ChGpGl spectrum because of the substitution in
the amine group. Similar features of the IR spectra were
observed in other studies on quaternized chitosan.
43,44,46
The adsorptive capability of ChGpGl microspheres toward
heparin, both in terms of the rate of adsorption and the amount
of heparin adsorbed per unit weight of microspheres, was
dramatically improved by quaternization of ChGp microspheres.
Figure 12 shows a comparison of heparin adsorption by the same
amounts of ChGp and ChGpGl microspheres. Whereas at pH
7.4, 40 mg ChGp microspheres can adsorb only 20% of
heparin in 5 mL of a solution containing 200 g/mL heparin,
ChGpGl microspheres can bind 90% of heparin under the same
experimental conditions. More importantly, the process is very
fast; the 50% decrease in heparin concentration occurs within
5 min. A comparable rate of heparin adsorption on ChGp
microspheres could be achieved at only a considerably lower
pH and by using a few times greater amount of microspheres.
The experiment on heparin binding using various amounts
of ChGpGl microspheres was also carried out to nd whether
the rate and degree of heparin adsorption can be adjusted by
changing the amount of microspheres used (Figure 13). The
results indicate that the rate of heparin binding can be improved
by increasing the amount of ChGpGl microspheres.
Desorption of heparin bound to ChGpGl microspheres was
also attempted. We noted that the addition of concentrated NaCl
solution to the suspension of ChGpGl microspheres containing
adsorbed heparin resulted in heparin desorption, which was
evidenced by the Azure A spectrophotometric method. We
found that in 1 M NaCl solution 55% of bound heparin is
desorbed from ChGpGl microspheres. Therefore, the ChGpGl
microspheres may be potentially reused for heparin adsorption.
Their properties and ability to rebind heparin are the subject of
our current studies.
Therefore, it can be expected that ChGpGl microspheres can
be effectively used to remove excess heparin from blood or other
physiological uids. When the consequences of heparin removal
Figure 8. Dependence of relative heparin concentration (c
0
) 200 g/mL, v ) 5 mL) on time for (a) (b) 40 and (9) 150 mg of ChGp microspheres
added at pH 6.8 and for (b) (b) 20, (9) 40, and ([) 80 mg of ChGp microspheres added at pH 7.4.
Figure 9. Microscopic image of ChGpGl microspheres.
3130 Biomacromolecules, Vol. 9, No. 11, 2008 Kamin ski et al.
from blood with this method are considered, the well-known
hemostatic effect of chitosan
47
should also be taken into account.
It has been recently reported that chitosan microspheres can
shorten the clotting time and may induce the activation and
adhesion of platelets.
48
It was speculated that amino groups of
chitosan may induce adhesion of erythrocytes.
49
Moreover, it
was found that chitosan has a synergistic effect on warfarin,
another anticoagulant drug,
50
which was ascribed to the adsorp-
tion by chitosan of vitamin K, which plays an important role in
coagulation of blood. Thus, one may expect that chitosan would
potentiate the effect of heparin removal. Therefore, the chitosan
microspheres obtained will be further studied in vitro with blood
samples.
Conclusions
Genipin-cross-linked chitosan microspheres were found to
bind heparin in the aqueous solution. The rate and efciency
of binding are dependent on pH and decrease with rising pH of
the solution. The adsorption of heparin could be enhanced by
increasing the weight of the microspheres. However, at pH 7.4,
which is characteristic of blood, heparin binding is slow and
inefcient. At pH 7.4, microspheres obtained by quaternization
of ChGp microspheres with GTMAC show a dramatically
enhanced rate and efciency of heparin binding. The rate of
that process can be adjusted accordingly by using the required
amount of microspheres. Thus, both types of chitosan micro-
spheres may be used for the effective removal of heparin from
solution at various pH values.
Acknowledgment. We gratefully acknowledge nancial
support from the Ministry of Science and Higher Education in
the form of grant no. N205 027 32/1510.
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