Colloids and Surfaces B: Biointerfaces 42 (2005) 147155

Chitosan based surfactant polymers designed to improve blood

compatibility on biomaterials
Sharon Sagnella, Katanchalee Mai-Ngam
National Metals and Materials Technology Center, 114 Paholyothin Rd, Klong Luang, Pathumthani, Thailand

Received 8 April 2004; accepted 11 July 2004

Abstract

We developed chitosan based surfactant polymers that could be used to modify the surface of existing biomaterials in order to improve their
blood compatibility. These polymers consist of a chitosan backbone, PEG side chains to repel non-specific protein adsorption, and hexanal
side chains to facilitate adsorption and proper orientation onto a hydrophobic substrate via hydrophobic interactions. Since chitosan is a
polycationic polymer, and it is thrombogenic, the surface charge was altered to determine the role of this charge in the hemocompatibility of
chitosan. Charge had a notable effect on platelet adhesion. The platelet adhesion was greatest on the positively charged surface, and decreased
by almost 50% with the neutralization of this charge. A chitosan surface containing the negatively charged SO3 exhibited the fewest number
of adherent platelets of all surfaces tested. Coagulation activation was not altered by the neutralization of the positive charge, but a marked
increase of 56 min in the plasma recalcification time (PRT) was displayed with the addition of the negatively charged species. Polyethylene
(PE) surfaces were modified with the chitosan surfactant resulting in a significant improvement in blood compatibility, which correlated to the
increasing PEG content within the polymer. Adsorption of the chitosan surfactants onto PE resulted in approximately an 8596% decrease
in the number of adherent platelets. The surfactant polymers also reduced surface induced coagulation activation, which was indicated by
the PEG density dependant increase in PRTs. These results indicate that surface modification with our chitosan based surfactant polymers
successfully improves blood compatibility. Moreover, the inclusion of either negatively charged SO3 groups or a high density of large
water-soluble PEG side chains produces a surface that may be suitable for cardiovascular applications.
2004 Elsevier B.V. All rights reserved.

Keywords: Chitosan; Platelet; Coagulation; Surfactant; Blood compatibility

1. Introduction protein adsorption in turn results in platelet adhesion and ac-

Chitosan is a polycationic biopolymer obtained by the al-
kaline deacetylation of chitin, a highly abundant natural poly-
mer [13]. The physical, chemical, and mechanical properties
of chitosan makes it ideal for use in a variety of biomed-
ical and pharmaceutical applications such as hemodialysis
membranes, artificial skin, wound dressings, and drug deliv-
ery systems, among others [1,46]. However, in the case of
blood contacting applications, chitosan has been shown to be
highly thrombogenic [1,2,79]. This is due to the fact that
the positively charged chitosan tends to attract circulating
plasma proteins, which adsorb to the material surface. This
Corresponding author. Tel.: +66 2564 6500x4444; fax: +66 2564 6446.
E-mail address: katancv@mtec.or.th (K. Mai-Ngam).
tivation on the surface of the material, thrombus formation,
and ultimately device failure.
Material surface properties such as charge density, chain
mobility, hydrophobicity/hydrophilicity, among others have
been shown to effect the composition and conformation of
the adsorbed protein layer [10,11]. Studies have demonstrated
that the surface modification of materials with large extended,
hydrophilic molecules such as dextran, maltose, polyethy-
lene glycol (PEG), and others provides a steric barrier that is
able to repel unwanted protein adsorption [1214]. However,
these polymers alone lack the ability to form a stable matrix
and therefore must be attached to a polymer with suitable
mechanical properties [14,15]. Due to the existence of reac-
tive amino and hydroxyl groups present in chitosan, it can
be easily altered chemically. Thus, attempts to improve the

0927-7765/$ see front matter 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2004.07.001
148 S. Sagnella, K. Mai-Ngam / Colloids and Surfaces B: Biointerfaces 42 (2005) 147155
hemocompatibility of chitosan have been made by coupling
large, water-soluble groups such as PEG to chitosan thereby
providing a steric barrier to undesirable protein adsorption
with some success [1,2,15].
In order to improve the blood compatibility of chitosan,
we have developed a series of novel surfactant polymers that
can easily modify the surface of existing hydrophobic bio-
materials. These surfactants consist of a chitosan backbone
with hexanal side chains to facilitate adsorption and proper
orientation on a hydrophobic substrate via hydrophobic in-
teractions, and PEG side chains to provide a steric barrier
preventing the adsorption of circulating plasma proteins onto
the material surface. In addition, we have altered the surface
charge of chitosan in order to establish the role of this charge
on platelet and coagulation activation. In vitro platelet adhe-
sion and the time required for clot formation on the material
surfaces were investigated by incubation of the materials with
platelet rich and -poor plasma.
2. Materials and methods
2.1. Reagents and chemicals
Chitosan with a degree of deacetylation of 95% and a
molecular weight of 45,000 was obtained from Seafresh
Lab (Bangkok, Thailand). Diethyl ether, methanol, and
acetone were provided by LabScan (Bangkok, Thailand).
Poly(ethylene glycol) monomethyl ether, 5-formyl-2-furan-
sulfonic acid (FFS), hexanal, sodium cyanoborohydride,
ammonium hydroxide, hydrochloric acid, and sodium
borohydride were purchased from Fluka BioChemical
(Singapore, Singapore). Sodium nitrite was obtained from
SigmaAldrich (Singapore, Singapore) and sodium hydrox-
ide from BDH chemical (Bangkok, Thailand). All aqueous
solutions were prepared with deionized water.
2.2. Chitosan lm preparation
Chitosan films were prepared by first dissolving chitosan
in 1% acetic acid creating a 2% w/v chitosan solution. An
amount of 65 ml of the solution was poured into a 60 mm
diameter polystyrene Petri dish and dried at 50 C until a
film was formed. The film was removed from the Petri dish
and punched to obtain 15 mm diameter circles. Prior to ex-
periments, chitosan was rinsed multiple times with PBS to
remove any residual acetic acid from the film. In order to
neutralize the positive charge on the surface of the chitosan,
films were soaked with a solution of methanol:ammonium hy-
droxide (70:30) for 10 min, rinsed three times with methanol,
and allowed to air dry.
2.3. Chitosan surfactant polymer synthesis
Prior to surfactant polymer synthesis, chitosan was de-
polymerized to obtain a lower molecular weight chitosan (Mn
5000). To do so, chitosan was first dissolved in 1% HCl.
Then, NaNO2 was added and the mixture was stirred at room
temperature for 180 min and the pH adjusted to 7 with NaOH.
Next, NaBH4 was added and the solution was stirred for 1 h at
room temperature. Chitosan was then precipitated in acetone
and filtered.
A series of chitosan based surfactant polymers with vary-
ing ratios of poly(ethylene glycol) (PEG):hexanal (Hex) were
synthesized to modify the surface of existing hydrophobic
biomaterials and improve blood compatibility. The molar
feed ratio of PEG to Hex was varied to produce surfaces
containing 0:1, 1:1, 1:4, and 1:8 PEG:Hex ratios. Depoly-
merized chitosan was first dissolved in 1% acetic acid. PEG
monomethyl ether was oxidized using acetic anhydride and
DMSO to create an aldehyde terminated PEG (PEG-CHO).
The desired feed ratio of PEG-CHO in methanol and hex-
anal was then added to the chitosan solution. A NaCNBH3
methanol solution was added, and the pH of the solution
was adjusted with NaOH to 56. The reaction was stirred at
room temperature for 24 h and then precipitated in 3:1 diethyl
ether:acetone. After washing with acetone, the surfactant was
filtered and weighed. In the case of the FFS containing surfac-
tant, chitosan surfactant containing no PEG (chitosan-Hex)
was dissolved in 2% acetic acid. FFS in methanol was added
followed by a NaCNBH3 methanol solution, and the mixture
was stirred for 24 h at room temperature. It was then precip-
itated in acetone, washed with methanol and filtered.
The structures of the chitosan based surfactants were char-
acterized by FT-IR (Perkin-Elmer) and 1 H NMR (Brunker
Avance). Surfactants for NMR analysis were placed in 0.5 ml
of 20% d-acetic acid in D2 O. In the case of the PEG contain-
ing chitosan surfactants, 120 l of 20% DCl was included
in the solvent. The final molar feed ratios were determined
by integration of proton peaks of the NMR spectra [16].
Polyethylene (PE) films used as control surfaces and for sur-
factant deposition were fabricated by extrusion of PE pellets.
Smoothness of the PE film was confirmed by scanning elec-
tron microscopy (JSM 5410; JEOL SEM, Japan). The sur-
factant (1 mg/ml, aqueous solution) was adsorbed onto PE at
room temperature for 24 h. Samples were removed from the
solution and allowed to air dry. Successful adsorption of the
surfactants onto PE was determined by water contact angles.
2.4. Platelet adhesion
Whole blood was drawn from aspirin free donors into
10 ml syringes containing 1 ml of 3.8% sodium citrate
(Sigma). The whole blood was centrifuged at 1000 rpm
for 15 min to obtain platelet rich plasma (PRP). Chitosan,
chitosan rinsed with NH4 OH, chitosan surfactants (no PEG,
1:1, 1:4, 1:8, and FFS) deposited on PE, and a PE control
surface were incubated with 500 l of PRP in 24 well plates
for 1 h at room temperature. At the end of 1 h, the PRP
was aspirated from the wells, and surfaces were rinsed with
phosphate buffered saline (PBS) to remove any non-adherent
platelets. They were then fixed for 1 h in 1% paraformalde-
 S. Sagnella, K. Mai-Ngam / Colloids and Surfaces B: Biointerfaces 42 (2005) 147155 149

hyde (PFA). Surfaces were washed three times (5 min each) Table 1
with PBS prior to staining. Platelets were stained with a Characterization of chitosan based surfactant polymers including molar feed
ratios and measured ratios
fluorescently labelled antibody to the glycoprotein IIb/IIIa
receptor, CD41a FITC (Dako). Surfaces with adherent PEG:Hex PEG:Hex FFS:Hex
platelets were submerged into a solution containing a 1:100 Feed ratio 1:1 1:4 1:8 0:1
dilution of antibody:staining medium (PBS with 4% fetal Measured ratio 1:2.5 1:6.3 1:11.2 0:1 1:0.67
bovine serum) and incubated in the dark for 1 h. They were Composition (mol %)
then again rinsed three times with PBS, and mounted using GlcNH-PEG 9.9 6.3 4.0 0
GlcNH-Hex 25.1 39.7 44.9 42 39.98
gelmount (Biomeda Corp., Foster City, CA).
Glc-FFS 59.76
Samples were examined by confocal microscopy using a Glc-NH2 65.0 50.0 51.1 58.0 0.26
Zeiss LSM microscope, with a mercury arc lamp light source NMR used to measure ratios.
and a 488 nm filter. Surfaces were scanned from left to right
with at least five images acquired per sample. Platelet ad-
hesion was quantified by direct enumeration. In the case of
activation. Neutralization of the positive charge on chitosan
aggregates, the pixel area of the aggregate was determined
was confirmed by FT-IR analysis. The FT-IR transmission
and then divided by the pixel area of a standard singular
spectra showed the disappearance of a peak at 1410 cm1
platelet in the same field to obtain equivalent platelet num-
present in the positively charged chitosan films. This resulted
bers for the aggregates. All samples were tested in at least
in the unmasking of a CH2 bend present at 1376 cm1 indi-
triplicate.
cating a neutralization of the charge. FT-Raman spectroscopy
(Perkin-Elmer) confirmed the successful incorporation of
2.5. Plasma recalcication time
the negatively charged FFS group into a chitosan surfactant,
thereby creating a negatively charged chitosan surface.
Plasma recalcification time (PRT) is an indicator of in- 1 H NMR was used to obtain a quantitative analysis of
trinsic coagulation cascade activation, and therefore a use-
the final composition of all chitosan based surfactants. For
ful marker of biomaterial induced coagulation activation.
the FFS and chitosan-Hex, there was no overlapping of
For PRT measurements, human whole blood was collected
chitosan (H1H6; 2.54.3), hexanal (H1H13; 0.31.4), or
in 10 ml syringes containing 1 ml of 3.8% sodium citrate.
FFS protons peaks (H1H2; 6.26.5), so simply integrating
Platelet poor plasma (PPP) was obtained by centrifuging
the peaks provided a quantitative molar composition of each
whole blood at 3000 rpm for 15 min. PRT was measured for
component. The addition of PEG, however, created an over-
PPP in contact with chitosan, chitosan rinsed with NH4 OH,
lap between the protons of PEG and the protons of chitosan.
chitosan surfactants, and PE. To measure PRT, 500 l of PPP
Therefore, a solvent containing DCl was used to separate out
was mixed with 250 l of 0.05M CaCl2 and incubated on the
one of the chitosan proton peaks and allow for quantitative
surface of the different materials in a water bath at 37 C.
analysis. The addition of DCl caused a shift in one of the
Samples were occasionally removed from the water bath and
chitosan proton peaks to 4.85.2, thereby allowing integra-
gently stirred. The time until the PPP clouded and fibrin clot
tion of the single peak and determination of the amount of
formation initially began was recorded as the PRT. Experi-
chitosan in the sample. The amount of chitosan was then
ments were run in triplicate.
subtracted out of the PEG/chitosan peak to obtain the amount
of PEG present in the surfactant. The final measured molar
2.6. Statistical analysis
composition of all surfactants is shown in Table 1. Actual
compositions were slightly less than actual feed ratios.
Statistical analysis was performed in Microsoft Excel. A
Typical water contact angles for PE were around 95 .
students t-test was performed to compare the differences in
These decrease considerably after adsorption of the surfac-
platelet adhesion and PRT on the different surfaces. Signifi-
tants onto the PE surface as shown in Table 2.
cance was considered a P-value less than 0.05.

Table 2
3. Results
Advancing water contact angles for polyethylene and chitosan surfactant
polymers
3.1. Characterization of chitosan and chitosan based
Material Contact angle
surfactant polymers
PE 96.1 1.8
Chitosan-Hex 63.0 1.4
Chitosan based surfactants that could modify existing FFS 30.2 1.8
hydrophobic biomaterials such as PE were designed to l:8 (PEG:Hex) 12.0 2.6
improve blood compatibility. In addition, since chitosan l:4 (PEG:Hex) 11.9 1.8
itself is naturally thrombogenic, the charge on chitosan was l:l (PEG:Hex) 13.0 2.4
altered to help determine its role in platelet and coagulation Contact angles were measure by the sessile drop method.
150 S. Sagnella, K. Mai-Ngam / Colloids and Surfaces B: Biointerfaces 42 (2005) 147155

3.2. Platelet adhesion as a function of charge density 3.3. Platelet adhesion and PEG density

To investigate the effect of charge on platelet adhesion
to chitosan based polymers, four surfaces were examined:
a positively charged chitosan film (chitosan), a positively
charged chitosan surfactant polymer with no PEG (chitosan-
Hex), a chitosan film with the charge neutralized via rinsing
with NH4 OH (chitosan + NH4 OH), and a chitosan surfactant
polymer with a negative charge (FFS). Of the chitosan
surfaces, the chitosan film with the positive charge showed a
significantly greater number of adherent platelets and platelet
aggregates than the other three surfaces as shown in Fig. 1.
Interestingly, the neutrally charged chitosan + NH4 OH film
and the positively charged chitosan-Hex surfactant showed
similar numbers of adherent platelets, and the chitosan +
NH4 OH had significantly more aggregates present than the
chitosan-Hex (Fig. 2). The FFS surface showed very little
platelet adhesion and almost no aggregates. Fig. 3(BD and
F) shows the images of platelets on the positive, negative, and
neutrally charged chitosan surfaces. Platelets on the different
chitosan surfaces show only small aggregates or singular
platelets. Platelets adherent to chitosan, chitosan-Hex, and
chitosan + NH4 OH are mostly spread and active, but only
small aggregates are present. The FFS surface shows very
few platelets, and those attached display little to no spreading
and activation.
Platelet adhesion was examined on chitosan based sur-
factant polymers adsorbed on PE with differing densities
of PEG side chains ranging from 1:1 to 1:8 PEG:Hex.
The number of adherent platelets decreased significantly
on the PE following surfactant deposition, and decreased
even further with an increasing number of PEG side
chains as shown in Fig. 4. The chitosan surfactant poly-
mers (with or without PEG) had significantly fewer ad-
herent platelets than the chitosan films. The addition of
PEG to the surfactant resulted in a significant decrease in
platelet adhesion. Of the PEG containing surfactants, the
1:1 PEG:Hex showed significantly lower platelet adhesion
than the other two surfaces with only about 715 adher-
ent platelets per mm2 . The 1:8 surface had a significantly
greater number of adherent platelets (2150 platelets/mm2 )
than either the 1:4 (1400 platelets/mm2 ) or 1:1 sur-
faces, and slightly but not significantly fewer platelets than
the surfactant containing no PEG (2690 platelets/mm2 ).
Large aggregates were commonly seen on the PE sur-
face, which decreased significantly following surfactant
modification (Figs. 3 and 5Figs. 3(A and E) and 5). All
three PEG chitosan surfactants had very few aggregates,
and significantly fewer aggregates than the chitosan-Hex
surface.

Fig. 1. Platelet adhesion as a function of surface charge on chitosan and chitosan-Hex (+), chitosan + NH4 OH (neutral), and FFS (). The positively charged
chitosan surface shows the greatest number of adherent platelets, while minimal platelet adhesion is seen on the negatively charged surface. The neutrally
charged chitosan film has significantly fewer platelets on the surface than the positively charge chitosan film, but similar numbers to the positively charged
chitosan surfactant.
 S. Sagnella, K. Mai-Ngam / Colloids and Surfaces B: Biointerfaces 42 (2005) 147155 151

Fig. 2. Platelet aggregates as a function of surface charge, chitosan and chitosan-Hex (+), chitosan + NH4 OH (neutral), and FFS (). The positively charged
chitosan film shows the greatest number of aggregates, while almost no aggregates are present on the FFS. The neutrally charged chitosan + NH4 OH has
significantly fewer aggregates than the chitosan, but more than the positively charge chitosan-Hex surfactant.

3.4. Plasma recalcication times Table 3

Plasma recalcification times on the different biomaterial surfaces
Plasma recalcification time, a measurement of intrinsic Material Time (min)
coagulation cascade activation, indicates the time required PE 13.6 0.8
for fibrin clot formation once calcium has been introduced l:8 (PEG:Hex) 15.8 0.8
into sodium citrate anticoagulated plasma. Contact activa- Chitosan-Hex 16.6 1.5
Chitosan 17.3 1.5
tion of the intrinsic cascade in plasma will vary with the type Chitosan + NH4 OH 17.4 2.7
of surface, and thus plasma recalcification can be used as an l:4 (PEG:Hex) 17.6 2.0
indicator of bloodbiomaterial interactions. Plasma recalcifi- 1:1 (PEG:Hex) 18.1 1.8
cation time on PE (approximately 13.6 min) was significantly FFS 21.2 1.1
shorter than on all other surfaces as shown in Table 3. Neu- CaCl2 was added to platelet poor plasma containing a sodium citrate antico-
tralization of the positive charge on chitosan did not have a agulant, and the time required for the initiation of a fibrin clot was recorded.
significant effect on PRT, since the chitosan, chitosan-Hex,
and chitosan + NH4 OH all had similar PRTs. However, the 4. Discussion
negatively charge FFS had a PRT of about 21.2 min, which
was significantly longer than on any other surface. Similar to The current study aimed to design a surface modification
platelet adhesion, the PRT increased with increasing density method that could both exploit the desirable biomedical prop-
of PEG in the chitosan surfactants. The 1:8 and the chitosan- erties of chitosan while improving the hemocompatibility of
Hex surfactant polymers had similar PRTs of about 15.8 and existing hydrophobic biomaterials. To this end, we created
16.6 min, respectively. The PRTs for the 1:4 (17.6 min) and surfactant polymers consisting of a chitosan backbone and
1:1 (18.1 min) surfactants were also similar, but both were hexanal side chains to facilitate adsorption and proper ori-
significantly greater than the 1:8 or chitosan-Hex. entation onto hydrophobic substrates. In addition, PEG side
152 S. Sagnella, K. Mai-Ngam / Colloids and Surfaces B: Biointerfaces 42 (2005) 147155

Fig. 3. Images of adherent platelets on (A) PE, (B) chitosan, (C) chitosan-Hex, (D) chitosan + NH4 OH, (E) 1:1 PEG:Hex, and (F) FFS. Many large aggregates
are present on the PE surface, and the surface is covered extensively with platelets. Platelets on the chitosan, chitosan-Hex, and chitosan + NH4 OH surfaces
show some small aggregates, but noticeably less adherent platelets than the PE. Many platelets are spread and active, but some are more rounded. Very few
platelets are seen on the 1:1 PEG:Hex surface, but those that adhere to the surface are mostly spread and active. The FFS surface shows the smallest number of
adherent platelets, and those that are on the surface maintain a fairly inactive morphology. (Original magnification 100.)

chains were incorporated to provide a steric barrier to unde-
sirable protein adsorption, thereby improving blood compat-
ibility.
PE was successfully modified by the surfactant polymers
as indicated by the drop in water contact angles. The
surfactants altered the surface wettability of PE from
hydrophobic to hydrophilic. Chitosan itself is hydrophilic,
and the incorporation of the large PEG molecules into the
chitosan surfactants resulted in a surface that was even more
hydrophilic. The actual molar composition of the chitosan
surfactants was a bit different than the values predicted from
the feed ratios. This difference is most likely due to the size of
the PEG molecule, preventing close packing of the molecules
onto the chitosan backbone. The PEG surfactants had a
significant number of unreacted amine groups ranging from
about 5065%, with the highest percentage present in the
1:1 PEG:Hex. The size of the PEG limits the number of PEG
molecules that can be incorporated into the backbone, and the
amount of PEG in the 1:1 PEG:Hex is approaching this limit.
In order to maintain consistency, the chitosan-Hex surfactant
was synthesized so that it would contain a similar number of
hexanal side chains as the PEG containing surfactants and
thus possessed about 58% unreacted amine groups in the
backbone.
Platelet activation and adhesion to polymers is a complex
process that is dependant on a variety of properties includ-
ing surface chain mobility, surface chemical composition,
hydrogen bonding properties, charge density, hydrophoboc-
ity/hydrophilicity, among others [10,11]. These properties
will effect kinetics, composition, and conformation of the
adsorbed protein layer thereby effecting cellular activation
and adhesion on biomaterials. In the case of charge, stud-
ies have shown that addition of negatively charged species
such as SO3 tend to be much less platelet activating result-
ing in very few adherent platelets [1,1719]. Many biolog-
ical molecules such as glycosaminoglycans, phospholipids,
plasma proteins, and extracellular matrix proteins can carry
a negative charge. Therefore, electrostatic repulsion may be
one mechanism by which the negatively charged biomate-
rial surfaces maintain their hemocompatibility. In this study,
the SO3 containing FFS chitosan surfactant displayed the
fewest number of adherent platelets, the least amount of
platelet aggregation, and the longest PRT of any of the sur-
faces examined. Alternatively to repulsion of plasma pro-
teins, it has been suggested that fibrinogen binds with high
affinity to sulfonated polymers in a conformation such the
platelet binding sight is not accessible [1,1820]. In addi-
tion, it has been observed that fibrinogen adsorbed on sul-
fonated polymers cannot be displaced by other plasma pro-
teins [1,18,19]. The tight binding of fibrinogen to the surface
would both prevent platelets from adhering to the surface and
prevent other plasma proteins from interacting with the sur-
faces thereby inhibiting activation of the intrinsic coagulation
cascade.
 S. Sagnella, K. Mai-Ngam / Colloids and Surfaces B: Biointerfaces 42 (2005) 147155 153

Fig. 4. Platelet adhesion as a function of amount of PEG present in surfactant polymer as compared to an uncoated PE film. The surfactant polymers show
significantly less platelet adhesion then the uncoated PE. In addition, the surfactant with the greatest amount of PEG present (1:1) had significantly fewer
platelets on the surface than any of the other surfactants. Decreasing the amount of PEG in the polymer resulted in an increase in platelet adhesion, and while 1:4
PEG:Hex showed significantly less platelets than 1:8 PEG:Hex or the chitosan surfactant with no PEG, there was no significant difference in platelet adhesion
on the 1:8 PEG:Hex and the chitosan-Hex.

Conversely, chitosan exhibits a cationic nature, which al-
lows for electrostatic interactions with the aforementioned
negatively charged biological molecule. A number of stud-
ies have indicated that chitosan promotes surface induced
thrombosis, which can be attributed in part to these electro-
static interactions [1,2,8]. While neutralization of the positive
charge on chitosan resulted in a decrease in the number of
adherent platelets and number of aggregates present, it did
not have a significant effect on coagulation activation. Dur-
ing platelet activation, the plasma membrane of the platelet
undergoes an inversion such that an abundance of negatively
charged phosphatidyl serine is present on the outer mem-
brane. Thus an electrostatic attraction between chitosan and
activated platelets is possible that would not occur on the
neutrally charged surface [10,11]. The lack of difference be-
tween the positively charged surface and the neutrally charge
surface in terms of PRT time indicates that the positive charge
most likely does not exert as strong of an influence on coagu-
lation activation as it does on platelet adhesion. In addition to
charge, both the hydrophilic nature of chitosan and its hydro-
gen bonding ability due to the presence of OH and NH2 can
effect the adsorbed protein layer. In the case of the positively
charged chitosan surfactant, there is approximately a 42% re-
duction in the number of positively charge amine groups due
to the attachment of the hexanal side chains to the polymer
via these groups. This reduction in positive charge resulted
in platelet adhesion and aggregation results closer to those
seen for the neutral chitosan film. This suggests that a high
positive charge density in chitosan is necessary to cause an
increase in platelet adhesion and aggregation. Therefore, due
to the lower density of positive charge in our chitosan surfac-
tant polymers, they are more ideal for use in blood contacting
applications.
Modification of PE with the chitosan surfactant polymers
had a dramatic effect of both platelet adhesion and PRT
times. PE had the shortest PRT time and the greatest number
of adherent platelets and platelet aggregates. Adsorption of
the chitosan onto the PE surface resulted in a drastic decrease
in platelet numbers, a decrease in the number of aggregates,
and an increase in PRT time. Addition of PEG side chains to
the surfactant resulted in a PEG density dependant drop in
platelet numbers and increase in PRT time. There was a de-
154 S. Sagnella, K. Mai-Ngam / Colloids and Surfaces B: Biointerfaces 42 (2005) 147155

Fig. 5. Platelet aggregates as a function of amount of PEG present in surfactant polymer as compared to an uncoated PE film. The surfactant polymers showed
significantly fewer platelet aggregates than the PE. Addition of PEG to the surfactant polymers resulted in a significant decrease in the number of aggregates
than the surfactant polymer with no PEG. However, no difference was seen in aggregate number among the three PEG surfactants.

crease in the number of aggregates with the addition of PEG, 5. Conclusions

but this was not PEG density dependant. PEG has been used
extensively to improve the hemocompatibility of biomaterials
due to its ability to effectively repel unwanted protein adsorp-
tion [1214]. PEG is a large, water-soluble polymer that can
extend into solution in a brush like conformation and steri-
cally repel protein adsorption similar to the polysaccharides
present in cell membranes. Therefore, it is not surprising that
an increase in the amount of PEG present in the surfactant re-
sulted in improved blood compatibility. For PEG to be effec-
tive it must shield the entire surface [13,21]. The effect of PEG
density was not as pronounced with respect to PRT times,
however there was a significant decrease in PRT on the 1:8
PEG:Hex surface. In the case of the 1:4 PEG:Hex and particu-
larly the 1:8 PEG:Hex, the amount of PEG may not be enough
to accomplish complete surface coverage, which would
explain the increase in platelet numbers and decrease in PRT
time with the decrease in the amount of PEG present. In addi-
tion, poor surfactant coating could expose the underlying PE
substrate, thereby resulting in a slightly more thrombogenic
surface.
Despite the desirable properties of chitosan in relation
to certain biomedical applications such as its antibacterial
properties, low toxicity, and suitable physical and mechani-
cal properties [1,4,5,22,23], its thombogenicity limits its use
in blood contacting applications. Here, we have demonstrated
that this is at least in part due to the high density of positive
charge in chitosan. Therefore, we have created a surfactant
polymer system containing chitosan that can be used to mod-
ify the surface of otherwise thrombogenic biomaterials. The
chitosan-Hex surfactant was considerably less thrombogenic
than the chitosan film, showing similar in vitro blood com-
patibility to a chitosan film with a neutral charge due to the
decrease in positive charge density in the surfactant polymers.
We have demonstrated here that the addition of the chitosan
surfactant to a PE surface improves the blood compatibility of
the PE. Moreover, through the inclusion of PEG side chains,
we effectively improve the surface hemocompatibility even
further. Since the presence of the negatively charged SO3
resulted in the greatest improvement in blood compatibility
 S. Sagnella, K. Mai-Ngam / Colloids and Surfaces B: Biointerfaces 42 (2005) 147155
of the chitosan containing polymers, combining this chemi-
cal group with the PEG side chains could create an even more
passivating surface. The major advantage of our chitosan sur-
factant is the variety of molecules can be easily incorporated
into our surfactant polymer in order to alter its interaction
with a biological environment. Studies have used chitosan
in tissue engineering application with some favorable results
in relation to cellular adhesion and growth [2426]. With
our polymer, we could further improve these cell/chitosan
interactions through the inclusion of cell binding peptide or
growth factors side chains into our polymer. Thus, we can
exploit the desirable properties of chitosan, while catering
the polymer system to the desired application.
Acknowledgements
Financial support for this study was provided by the Na-
tional Metal and Materials Technology Center, Thailand.
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