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Adsorption of Human Serum Albumin (HSA) On Modified PET Films Monitored by QCM-D, XPS and AFM
Adsorption of Human Serum Albumin (HSA) On Modified PET Films Monitored by QCM-D, XPS and AFM
Adsorption of Human Serum Albumin (HSA) On Modified PET Films Monitored by QCM-D, XPS and AFM
a r t i c l e i n f o a b s t r a c t
Article history: The adsorption behavior of human serum albumin (HSA) on differently modified poly(ethylene tereph-
Received 4 December 2009 thalate) (PET) model film surfaces was studied using the quartz crystal microbalance (QCM), X-ray
Received in revised form 23 February 2010 photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) techniques. The aim was to eval-
Accepted 1 March 2010
uate the influence of different modifications of PET surfaces with selected polysaccharides (chitosan,
Available online 7 March 2010
fucoidan, chitosan sulfate) on HSA adsorption.
As a first step, PET hydrophilicity was increased by alkaline hydrolysis. From these foils model PET
Keywords:
films were prepared by the spin coating technique on a quartz crystal. Selected polysaccharides (chitosan,
QCM-D
PET films
fucoidan and chitosan sulfate) were adsorbed from aqueous solutions on the PET surfaces. Adsorption
Human serum albumin (HSA) of HSA on the films was monitored with QCM-D. The surface composition and morphology of the HSA
Chitosan sulfate covered PET films was analyzed using XPS and AFM.
Fucoidan It was found that due to steric repulsion the chitosan, fucoidan and chitosan sulfate adlayers reduced
HSA adsorption, especially in case of chitosan/fucoidan and chitosan/chitosan sulfate covered films.
© 2010 Elsevier B.V. All rights reserved.
0927-7757/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfa.2010.03.003
T. Indest et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 360 (2010) 210–219 211
expected. Hence protein HSA adsorption was monitored with a was adjusted to 7.4 with NaOH. The solution was stored at 8 ◦ C and
quartz crystal microbalance with dissipation unit (QCM-D) on mod- used within a week.
ified PET systems; PET-H/chitosan, PET-H/chitosan/fucoidan and
PET-H/chitosan/chitosan sulfate.
QCM is an efficient, high-resolution mass sensing technique 2.1.7. Adsorption
which can additionally provide information about energy dissi- The adsorption of chitosan and fucoidan onto PET-H films was
pating properties of the bound surface mass. It is one of a few measured at pH 5, and of chitosan sulfate at pH 7.4, in all cases
techniques that can be used to give a direct observation of the from 140 mM NaCl solutions. The resulting adlayers of polysaccha-
adsorption process in situ. Thus, it is a suitable method for protein rides were then rinsed with phosphate buffer saline (PBS; 6.44 mM
adsorption studies at the solid/liquid interface [16,17]. KH2 PO4 , 8 mM Na2 HPO4 , 140 mM NaCl; pH 7.4).
Before HSA adsorption, the initial baseline was established with
the adlayers on the quartz crystal immersed in PBS. Then a solution
2. Materials and methods
of 0.05 g/L HSA in PBS (pH 7.4) was introduced into the QCM cell
and adsorption was allowed to proceed until equilibrium uptake
2.1. Materials
was reached. After this, the surface was rinsed with PBS solution in
order to test the stability of the adsorbed protein layer. The same
2.1.1. PET foil
procedure was used also when measuring HSA adsorption on PET
The foil was Mylar® polyethylene terephthalate (PET) foil, of
surfaces without adlayers.
thickness 175 m.
Table 2
The results of high-resolution spectra fittings of carbon (C1, C2, C3, C4) and oxygen (O1, O2) for non-hydrolyzed (PET) and hydrolyzed (PET-H) films.
Sample C–C (C1) [at%] C–O (C2) [at%] C O (C3) [at%] O–C O (C4) [at%] (O C–O) O1 [at%] (O C–O) O2 [at%]
Table 3
The results of surface elemental concentrations obtained from XPS wide spectra of modified PET-H films with chitosan (PET-HC (0.2)), chitosan/fucoidan (PET-HCF(0.2)) and
chitosan/chitosan sulfate (PET-HCSH(0.03)).
explanation for this might be found in the conformation of HSA of how the new added mass affects the adsorbed layer struc-
molecules in the adlayer or in binding/trapping of more water ture. The spacing of data points becomes more distant when the
[14,17,24] in the HSA film adsorbed on hydrolyzed PET (PET-H). adsorption kinetics is faster [17]. For both samples there was ini-
However, it is more likely that both events contributed to the dis- tial slow adsorption of HSA, as indicated by the high density of
sipation changes. data points, then the distance between data points increased, indi-
The larger frequency shift and the smaller shift in dissipation cating faster adsorption up to about f = −6 Hz for adsorption of
for PET/HSA sample indicate that more adsorbed mass was more HSA on PET-H sample and slightly above f = −12 Hz for adsorp-
rigidly distributed and denser packed on the surface than on the tion on PET/HSA. Then there was again a slower process of diffusion
PET-H/HSA sample. during which f and D slowly increased. The slope of the curve
The D/f curves in Fig. 2 compare the behavior of HSA layer for PET-H/HSA is slightly steeper and hence the protein layer was
on PET and PET-H surfaces. The D/f plot gives an indication more loosely bound to the surface than on the PET/HSA surface
Fig. 1. Change in frequency (third overtone) (a) and dissipation changes (third overtone) (b) as a function of time for HSA (0.05 g/L HSA, pH 7.4) adsorption on non-hydrolyzed
(PET) and hydrolyzed PET (PET-H) films. The points where the rinsing with phosphate buffer (154 mM salts, pH 7.4) started are marked with “PBS”.
214 T. Indest et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 360 (2010) 210–219
Fig. 2. Change in the dissipation factor as a function of change in frequency (third overtone) occurring at multiple adsorption steps of HSA (0.05 g/L, pH 7.4) on PET(grey line)
and PET-H (black line) film.
where the protein layer thus was more rigid and more densely ference can be explained by referring to the previously published
packed. [19] AFM analysis of chitosan-covered hydrolyzed PET (PET-HC),
which shows that chitosan does not cover the whole PET-H sur-
3.3.2. Adsorption of HSA on modified spin-coated PET films face, but rather forms some kind of blobs on its surface (see Fig. 4c).
The adsorption mechanism and the stability of adsorbed lay- Thus, HSA was adsorbed on chitosan only partly covering PET-H
ers of chitosan, fucoidan and chitosan sulfate on PET films was surface as well as on uncovered parts of PET-H film. This arguably
investigated in detail earlier [18,19]. The general conclusions from explains why such a small difference in frequency change after HSA
these studies of importance for the adsorption of HSA are that even adsorption between the PET-H and PET-HC surfaces was observed.
at high ionic strength (up to 140 mM NaCl) electrostatic interac- In case of fucoidan (PET-HCF) the frequency shifts after HSA
tions are not totally screened, but high ionic strength and high adsorption amounted to f = −7.1 Hz at adsorption equilibrium
concentrations of polymer result in denser and thicker adlayers. (∼65 min). Thus, only a small amount of HSA was adsorbed on this
Chitosan/fucoidan films are thinner and more compressed than chi- surface. Previously published AFM results [19] show that fucoidan
tosan/chitosan sulfate layers, in which large amounts of adsorbed covers the PET-HC surface homogenously. Hence the protein was
chitosan sulfate form a loose and thick adsorbed layer. adsorbed on a fucoidan layer covering the PET surface.
Fig. 3 shows the changes in frequency (Fig. 3a) and changes in In the case of chitosan sulfate (PET-HCSH) there was almost neg-
dissipation (Fig. 3b) as a function of time for protein HSA adsorp- ligible change in the frequency by HSA adsorption (∼30 min). This
tion on non-hydrolyzed (PET), hydrolyzed PET film (PET-H) and indicates that protein did not adsorb to the sample’s surface. It was
films modified by adsorption of chitosan (PET-HC), fucoidan (PET- found previously by Indest et al. [18], in an investigation of chi-
HCF) and chitosan sulfate (PET-HCSH). The values for the changes tosan sulfate adsorption on PET-H, that a large amount of chitosan
in frequency and dissipation at adsorption equilibrium for HSA sulfate is adsorbed, forming a thick and loose adlayer fully cover-
adsorption on PET, PET-H and on modified PET-H films (PET-HC, ing the sample’s surface, thus leaving no sites available for protein
PET-HCF, PET-HCSH) are summarized in Table 4. HSA to adsorb. The results show that both fucoidan and chitosan
Clear differences can be observed between HSA adsorption sulfate when adsorbed on chitosan reduce the amount of protein
behavior on hydrolyzed and non-hydrolyzed PET substrates, as adsorbed, but there are differences in their efficiency.
well as between adlayers of chitosan, chitosan/fucoidan and chi- To produce protein-resistant biomaterial surfaces, it is clear
tosan/chitosan sulfate. that one must design systems where repulsive interactions are
The frequency shift for HSA at adsorption equilibrium on the maximized and attractive ones are minimized [25]. The purpose
PET-HC layer amounts to f = −17.4 Hz, which is only slightly lower of using chitosan as a sublayer was to improve the adsorption
than the value f = −18.5 Hz for PET-H/HSA. This rather minor dif- of the two anionic polysaccharides to the PET-H surface. Both
fucoidan and chitosan sulfate carry negative (sulfate) charges that
can be expected to contribute to repulsion of the approaching neg-
Table 4 atively charged HSA molecules, at least at low or moderate ionic
The average values of frequency shift (third overtone, f3 [Hz]) and energy dis-
sipation changes (third overtone, D3 ) at adsorption equilibrium are presented
strength. Another well-known repulsive mechanism is steric repul-
for protein adsorption on non-hydrolzed (PET), hydrolyzed film (PET-H) and on sion, which requires that a solvent-swollen adsorbed polymer layer
modified PET films by chitosan (PET-HC), chitosan/fucoidan (PET-HCF) and chi- interacts more strongly with the solvent than with the adsorbate
tosan/chitosan sulfate (PET-HCSH):. molecules, and also that the adsorbed layer is thick enough so that
Sample f3 (Hz) max D3 × 10−6 van der Waals interactions between the underlying surface and
the adsorbate are small [25,26]. QCM-D studies of chitosan sulfate
PET/HSA −27.1 0.92
PET-H/HSA −18.5 1.3 adsorbed on chitosan [18] show that under the conditions used in
PET-HC(0.2)/HSA −17.4 0.7 this investigation, chitosan sulfate forms a water-swollen layer that
PET-HCF (0.2)/HSA −7.1 0.3 is expected to well fulfill these conditions, while the fucoidan layer
PET-HCSH (0.03)/HSA −1.3 −0.7 is thinner. Nevertheless, although as noted above, electrostatic
T. Indest et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 360 (2010) 210–219 215
Fig. 3. Change in frequency (f3 , third overtone) (a) and change in dissipation (D3 ) (b) as a function of time for HSA adsorption (0.05 g/L HSA, PBS solution) on non-
hydrolyzed (PET)—grey line, hydrolyzed PET film (PET-H)—black line, and modified films with chitosan (PET-HC, 0.2 g/L)—violet line, fucoidan (PET-HCF, 0.03 g/L)—blue line;
and chitosan sulfate (PET-HCSH, 0.03 g/L)—green line (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.).
interactions may play some part even at the high ionic strength tion value might indicate that, although small amount of HSA is
used, it is obvious from comparison of the three surfaces that steric adsorbed on chitosan sulfate sample, HSA forms a compact layer
repulsion is a predominant factor preventing the adsorption of with sulfated chitosan from which water is expelled.
HSA. Dissipation indicates that rigidly attached adsorbed layers of
Washing with PBS solution was performed after each HSA HSA were formed on PET and PET-HC samples but less so on PET-
adsorption experiment and did not result in frequency increase, H sample. The rather small dissipation changes on PET-HCF and
indicating that no desorption of HSA occurred from any of the PET-HCSH samples correspond to minor frequency shifts, and con-
modified PET samples. sequently almost no adsorbed mass of HSA on these samples. As
Fig. 3b shows the dissipation changes (D) as a function of time a rule, dissipation changes less than 1 × 10−6 indicate that the
for HSA adsorption on PET, PET-H and films modified with chitosan adsorbed layer is sufficiently rigid so that the adsorbed mass can
(PET-HC), fucoidan (PET-HCF) and chitosan sulfate (PET-HCSH). The be calculated from Sauerbrey equation (Eq. (1)) [27,28].
dissipation change at equilibrium was slightly larger for the PET- Since the results in Table 4 indicate that these conditions are
H/HSA layer than for PET/HSA, and was lower than both for the fulfilled for the HSA layers except for PET-H/HSA, the adsorbed
chitosan (PET-HC/HSA) layer (Table 4). In case of fucoidan (PET- mass of HSA on the other layers was calculated using Sauerbrey
HCF/HSA) sample the dissipation change after protein adsorption equation, yielding the frequency shift for PET sample corresponds
was very low and for the chitosan sulfate sample (PET-HCSH/HSA) to 1.6 mg/m2 HSA on PET, 1.03 mg/m2 on PET-HC, 0.42 mg/m2 on
the dissipation decreased to a −0.7 × 10−6 . The negative dissipa- PET-HCF and 0.04 mg/m2 on PET-HCSH.
216 T. Indest et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 360 (2010) 210–219
Fig. 4. Height (left) and Phase (right) contrast AFM images of (a) spin-coated PET-H sample [19], (b) protein HSA adsorbed on PET-H, (c) PET-HC (0.2 g/L) sample [19] (d)
protein HSA adsorbed on PET-HC, (e) PET-HCF (0.2) sample [18], (f) protein HSA adsorbed on PET-HCF (g) PET-HCSH (0.03) sample [18], and (h) protein HSA adsorbed on
PET-HCSH sample.
T. Indest et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 360 (2010) 210–219 217
Fig. 4. (Continued ).
218 T. Indest et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 360 (2010) 210–219
Table 5 with fucoidan and chitosan sulfate. The coverage of the surface
The average results of elemental surface concentration from XPS survey spectra
has proven to be an important factor for protein adsorption and
of hydrolyzed PET film (PET-H) and protein HSA adsorbed onto PET-H film (PET-
H/HSA). when the surface is fully covered especially by chitosan sulfate,
protein adsorption does not occur. Thus it can be concluded that
Sample O 1s [%] C 1s [%] N 1s [%] Na 1s [%] Si 2p [%] Cl 2p [%]
surfaces, modified with negatively charged sulfated polysaccha-
PET-H 27.0 72.6 – – 0.2 0.2
PET-H/HSA 23.3 71.4 2.5 0.2 2.2 0.4
rides (fucoidan, chitosan sulfate), are effective in preventing HSA
adsorption due to steric repulsion between polysaccharides and
HSA. The obtained reduced HSA adsorption is in agreement with
3.4. Characterization of the adsorbed HSA on modified PET films biocompatibility definition by which a protein adsorption should
be reduced in order to improve biocompatibility. Therefore the
3.4.1. Surface chemical composition decrease in non-specific protein adsorption can be the measure for
The elemental composition of PET-H and PET-H film with improved biocompatibility.
adsorbed HSA (PET-H/HSA) was determined by XPS analysis. The
results from XPS survey spectra of both samples are shown in Acknowledgements
Table 5. There are clear differences in atomic composition between
pure PET-H film and film with adsorbed HSA. In XPS analysis nitro- We gratefully acknowledge the support of Dr. Monika Österberg
gen is the most important peak for determination of proteins, such regarding interpretation of AFM images, Ritva Kivelä and Marja
as HSA. The absence of nitrogen in the PET-H film and a signifi- Kärkkäinen are acknowledged for performing AFM measurements.
cant amount of nitrogen (2.5%) in the PET-H/HSA film confirm that Special thank goes to Dr. Leena-Sisko Johansson and to Dr. Joseph
protein was adsorbed on the PET-H film surface. As expected, the Campbell for performing the XPS measurements and to Petri Myl-
XPS results (survey spectra) of PET films modified with chitosan, lytie for the support regarding spin coating.
fucoidan and chitosan sulfate; summarized in Table 3, indicate high
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