Applied Surface Science 256 (2010) 3977–3981

Effects of L-arginine immobilization on the anticoagulant activity and hemolyticproperty of polyethylene terephthalate films

Yun Liu a,*, Yun Yang a, Feng Wu b

a Department of Chemistry, School of Science, Xi’an Jiaotong University, Xi’an 710049, Chinab Research Centre of Blood, College of Medicine, Xi’an Jiaotong University, Xi’an 710065, China

A R T I C L E I N F O

Article history:

Received 19 October 2009

Received in revised form 26 December 2009

Accepted 19 January 2010

Available online 25 January 2010

PACS:

87.85.jj

Keywords:

Surface modification

Polyethylene terephthalate

L-arginine

Anti-coagulant activity

Hemolysis

A B S T R A C T

Surface modification of polyethylene terephthalate (PET) films was performed with L-arginine (L-Arg) to

gain an improved anticoagulant surface. The surface chemistry changes of modified films were

characterized by X-ray photoelectron spectroscopy (XPS) and attenuated total reflection Fourier

transform infrared (ATR-FTIR) spectroscopy. The in vitro anticoagulant activities of the surface-modified

PET films were evaluated by blood clotting test, hemolytic test, and the measurement of clotting time

including plasma recalcification time (PRT), activated partial thromboplastin time (APTT), and

prothrombin time (PT). The data of blood coagulation index (BCI) for L-arginine modified PET films

(PET-Arg) was larger than that for PET at the same blood-sample contact time. The hemolysis ratio for

PET-Arg was less than that for PET and within the accepted standard for biomaterials. The PRT and APTT

for PET-Arg were significantly prolonged by 189 s and 25 s, respectively, compared to those for the

unmodified PET. All results suggested that the currently described modification method could be a

possible candidate to create antithrombogenic PET surfaces which would be useful for further medical

applications.

� 2010 Elsevier B.V. All rights reserved.

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1. Introduction

Polyethylene terephthalate (PET) has been widely used asimportant bio-medical devices in cardiovascular implants such asartificial heart valves sewing rings and artificial blood vesselsbecause of its excellent mechanical properties and moderatebiocompatibility. However, the occurrence of thrombus or bloodcoagulation on the surface of these devices is a significant clinicalproblem when coming into contact with blood. There is a growingrealization that the thrombus formed on the surface of polymers isgenerally associated with the surface chemistry of the material.Thus, many approaches of surface modification including graftingheparin and/or insulin, thiol-containing compounds and so onhave been developed for diminishing the risk of thrombusformation of PET [1–3]. There is a potential that the surfacemodification will lead to materials that can be used more widelyand efficiently in medical devices.

Based on the pivotal physiological role of nitric-oxide (NO) ininhibiting platelet adhesion, activation and aggregation [4], severaltypes of anticoagulant polymers that can release NO have beendeveloped in recent years [5–10]. Among them, one very mostpromising polymer can locally generate NO through endogenous

* Corresponding author. Tel.: +86 29 82655169.

E-mail addresses: liuy@tgrc.org, liuyun66@xjtu.edu.cn (Y. Liu).

0169-4332/$ – see front matter � 2010 Elsevier B.V. All rights reserved.

doi:10.1016/j.apsusc.2010.01.060

biochemical reaction [11]. For example, L-arginine (L-Arg), theprecursor of NO, can endogenously produce NO by the catalyticactivity of nitric-oxide synthase [12]. In addition, L-arginine itself iscapable of modifying plasmin generation and fibrinogenolysis andclot lysis [13]. It was consequently expected that L-argininemodified polymers would be of good antithrombogenicity. In thisresearch, L-arginine was covalently coupled with PET films usingglutaraldehyde as a cross-linker to gain an improved antithrom-bogenic surface. Our previous experiments [14] indicate that L-arginine modified PET films have the ability to reduce proteinadsorption, platelet adhesion, and thrombus formation to variousdegrees. To further understand potential polymer surfacesmodified with L-arginine, we investigated the anticoagulantactivities and hemolytic properties of the surface-modified PETfilms by blood clotting test, hemolytic test, and the assay of clottingtime including plasma recalcification time (PRT), activated partialthromboplastin time (APTT), and prothrombin time (PT).

2. Experimental

2.1. Materials

Polyethylene terephthalate films (PET, Malinex1, thick-ness = 0.2 mm) were generously provided by DuPont ChinaHolding Co., Ltd.-Shanghai Branch. L-arginine hydrochloride waspurchased from Shanghai Sangon Biological Engineering &

Y. Liu et al. / Applied Surface Science 256 (2010) 3977–39813978

Technology Co., Ltd. The test kit for determination of clotting timewas purchased from Dade Behring (Marburg, Germany). Ethyle-nediamine (EDA), glutaraldehyde (GA) and all other chemicalreagents were obtained from SinoPharm Chemical Reagent Co., Ltd.(Shanghai, China), which were of analytical grade and used asreceived unless otherwise stated.

2.2. Chemical modification of PET

PET films (1 cm � 2 cm) were modified by the scheme as shownin Fig. 1. First, the surface of PET film was aminolysed to introduceprimary amino groups in an ethylenediamine solution (50%, v/v)for 24 h at 40 8C. The aminated films (PET-NH2) were subsequentlyimmersed in a 1% (w/v) glutaraldehyde solution (pH 7.0) for40 min at room temperature under nitrogen atmosphere using themethod previously described [15]. The glutaraldehyde treatedfilms (PET-GA) were washed with distilled water and incubated ina 2% (w/v) L-arginine solution at pH 7.0 for 12 h at roomtemperature to form L-arginine modified PET films (PET-Arg).

2.3. Surface characterization

X-ray photoelectron spectra (XPS) measurements were per-formed using an Axis Ultra, Kratos (UK) spectrophotometerequipped with a monochromatic AlKa radiation source (150 W,15 kV, 1486.6 eV) to determine C, N, and O atoms present on thesamples. The pressure in the analysis chamber was maintained at10�9 Torr.

The FT-IR spectra of the surface-modified PET films wereobtained on a Nicolet Nexus-870 spectrophotometer in AttenuatedTotal Reflection (ATR) mode in the range of 400–4000 cm�1. Thespectra were recorded using Ge crystal at a 458 incident angle.

2.4. Density determination of immobilized L-arginine

Surface density of L-arginine immobilized on PET films wasdetermined as follows. Prior to L-arginine immobilization, the PET-GA films were washed three times with distilled water and thendried at a reduced pressure to a constant mass; the PET-Arg filmswere treated similarly. The density of L-arginine (DA) on PETsurface was defined as DA = (MGA �MArg)/S, where MGA and MArg

are the mass of the dry PET-GA and PET-Arg films, respectively; S isthe area of the film (cm2). The experiments were repeated ninetimes and the mean value was reported.

2.5. In vitro blood clotting test

The sample film (1 cm� 2 cm) was placed in a beaker and thenwarmed in a water bath at 37 8C for 5 min. Fresh human whole blood(0.25 mL) from aspirin-free adult donors (anti-coagulated with tri-sodium citrate in a 9:1 volumetric ratio) was dropped on the center

Fig. 1. Schematic representation for modifying the polyethylene terephthalate (PET)

films with L-Arg.

of the film with a micro syringe. Next 0.02 mL of CaCl2 (0.2 M) wasadded slowly to the blood sample and then the beaker was gentlyshaken to mix the CaCl2 solution with blood homogeneously. Thebeaker was further incubated at 37 8C for 5, 10, 20, 30, 40, 50, and60 min, it was then removed from the water bath and 50 mL ofdistilled water was carefully added by dripping water down theinside wall of the beaker. After 10 min gently rocking the beaker, thesupernatant was drawn to measure the relative absorbance at540 nm by spectrophotometer. The absorbance of 0.25 mL ofcitrated whole blood in 50 mL distilled at 540 nm was assumedto be 100 as a reference value. The blood clotting index (BCI) ofbiomaterials can be quantified by the following equation [16]:

BCI ¼Abs of blood which had been in

contact with sample at 540 nmAbs of citrated whole blood in water at 540 nm

� 100

(1)

The BCI values were plotted versus the sample-blood contactingtime. Each value is the average of three measurements. As the BCIvalue increases, blood clotting decreases.

2.6. In vitro hemolysis ratio measurement

The samples were cut into small bars of 0.5 cm� 2 cm and rinsedthree times with distilled water and physiologic saline (0.9%, w/v),respectively. The sample bars were put into a glass tube andequilibrated with 10 mL of saline at 37 8C for 30 min. Then 0.2 mL ofdiluted citrated whole blood (8 mL of citrated whole blood wasdiluted by adding 10 mL of physiologic saline) was dropped into thetube for the sample bars to soak in the mixtures of citrated blood andsaline at 37 8C for another 60 min. Next, the mixtures were aspiratedand centrifuged at 1000 rpm for 5 min, and the absorbance of theclear supernatant was measured at 540 nm using a spectrophotom-eter. The hemolysis ratio (HR) was defined as HR = 100 � (AS � AN)/(AP� AN), where AS is the absorbance of sample supernatant; APand AN are the absorbance of the positive control (mixtures of 10 mLdistilled water and 0.2 mL diluted citrated blood) and the negativecontrol (mixtures of 10 mL saline and 0.2 mL diluted citrated blood),respectively [17]. All samples were run in triplicate and a mean valuewas calculated.

2.7. Determination of blood coagulation time

The citrated human whole blood was centrifuged at 3000 rpmfor 10 min to obtain platelet-poor plasma (PPP). Assays ofcoagulation time were performed to evaluate surface-inducedabnormalities in the intrinsic and extrinsic coagulation pathway.The end-point for these assays was the duration for the onset offibrin (clot) formation when PPP contacted with the samples [18].

2.7.1. Plasma recalcification time (PRT)

The plasma recalcification time was measured to comparesample-induced delay in clotting of PPP following activation ofprothrombin (Factor II) in the presence of Ca2+. 0.1 mL of PPP wasplaced on the sample sheet (1 cm� 1 cm) attached to a cultural dishand incubated in water bath at 37 8C for 30 min. 0.1 mL of CaCl2solution (0.025 M) was then added to the PPP and the mixture wasmonitored for clotting by manually dipping a stainless-steel hookinto the mixture to detect fibrin threads. Clotting time was recordedat the first signs of any fibrin formation on the hook. The experimentwas conducted in quadruplicate and a mean value was calculated.

2.7.2. Determination of activated partial thromboplastin time (APTT)

and prothrombin time (PT)

Activated partial thromboplastin time (APTT) is a simple andhighly reliable measurement of the capacity of blood to coagulate

Fig. 2. XPS survey-scan spectra of (a) PET, (b) PET-NH2, and (c) PET-Arg.

Table 1Surface chemical composition of untreated and treated PET films.

Sample Elemental atomic concentration Ratio

%C1s %O1s %N1s N/C

PET 72.58 27.42

PET-NH2 71.40 26.05 2.55 0.036

PET-Arg 70.41 24.27 5.32 0.076

Y. Liu et al. / Applied Surface Science 256 (2010) 3977–3981 3979

through the intrinsic coagulation mechanism and the effect of thebio-material on possible delay of the process, and Prothrombintime (PT) is measured to assess material-induced deferment orinterdiction of the extrinsic coagulation pathway [18]. A samplesheet (1 cm � 1 cm) was firstly incubated in 0.5 mL of PPP at 37 8Cfor 1 h, the APTT and PT of PPP were then determined using anautomated blood coagulation analyzer (LG-Paber-II, Steellex Corp.,Beijing, China). All measurements were repeated in quadruplicateand a mean value calculated.

3. Results and discussion

3.1. Surface analysis

To prove the attachment of L-arginine onto PET films, XPSsurvey scan spectra of PET, PET-NH2, and PET-Arg were performed(shown in Fig. 2). The XPS spectrum of PET showed two peakscorresponding to C1s (binding energy, 284.8 eV) and O1s (binding

Fig. 3. N1s scan spectra of (a)

energy, 531.6 eV). In addition to peaks C1s and O1s, PET-NH2 andPET-Arg showed one another peak corresponding to N1s (bindingenergy, 397.9 eV). Furthermore, in the spectrum of PET-Arg N1speak increased sharply. In agreement with the XPS survey scanspectra, the surface chemical composition of treated and untreatedPET films was calculated and is listed in Table 1. The results showthat carbon and oxygen contents decreased in the order of PET,PET-NH2, and PET-Arg, and the nitrogen atomic concentrations onPET-NH2 and PET-Arg increased by 2.55% and 5.32%, respectively,in comparison with PET. As well, a higher nitrogen-to-carbon ratiowas determined on the surface of PET-Arg than on that of PET-NH2.

Fig. 3 shows the N1s high-resolution spectra for PET-NH2 andPET-Arg. The shape of N1s high-resolution spectrum for PET-Argwas different from that for PET-NH2, which suggests theappearance of a new peak of N55C chemical binding on the surfaceof PET-Arg. However, it was still difficult to distinguish the peak ofN55C from the peaks of N–C55O and NH2 in the N1s spectrum ofPET-Arg, because these peaks were almost present at the sameposition and probably overlapped with each other.

Fig. 4 shows the FT-IR spectra of PET and PET-Arg. In thespectrum of PET-Arg, a broad and strong peak was detected at�3432 cm�1 due to the N–H stretching vibration. The band at1652 cm�1 was attributed to imine bond (C55N) or the N–Hbending vibration. The peak at 1541 cm�1 was attributed to theasymmetrical stretching vibration of carboxyl anion (–COO�1). Itshould be clarified that the carboxyl anion may be present here dueto the transfer of a proton from carboxyl group to the imine bond ofguanidino during washing the films with distilled water. Hence,there existed both a carbonium ion and carboxyl anion on thesurface of the modified PET films (see Fig. 1). Because PET-NH2 canreact with ninhydrin-alcohol solution (1%, w/v) under heating toform a purple color, but PET and PET-Arg cannot. PET-NH2 was thusqualitatively distinguished from PET and PET-Arg by a simpleninhydrin test, so the FT-IR spectrum of PET-NH2 was notperformed in our experiments. These results together with theanalysis of XPS indicated that L-arginine was successfully

PET-NH2 and (b) PET-Arg.

Fig. 4. ATR-FTIR spectra of (a) PET and (b) PET-Arg.

Table 2Results of hemolytic test of untreated and treated PET

films (mean� SD, n = 3).

Sample Hemolysis ratio (%)

PET 2.3�0.2

PET-NH2 2.7�0.2

PET-Arg 1.7�0.1

Y. Liu et al. / Applied Surface Science 256 (2010) 3977–39813980

immobilized on the surface of PET according to the modificationscheme described above.

The surface density of L-arginine immobilized on PET films was4.35 mg/cm2, approximately, 2.5 � 10�8 mol/cm2 in average.

3.2. Blood clotting properties of surface-modified PET

The blood clotting test reflects the antithrombogenic activity ofa biomaterial. The antithrombogenic activity can be quantitativelyexpressed by a relative parameter known as the blood clottingindex. A larger BCI value represents an enhancement of antith-rombogenic activity. Fig. 5 shows the effects of sample-bloodcontact time on BCI values. The results indicated that all the BCIvalues for PET, PET-NH2, and PET-Arg declined with increasedsample-blood contact time. The BCI values for PET-Arg not onlydecreased slowly with the length of contact time compared withPET and PET-NH2 but also were clearly greater than those for PETand PET-NH2 at the same blood-sample contact time. This resultwas ascribed to L-arginine immobilized onto the surface of PET. Italso demonstrated that L-arginine modified PET exhibited animproved antithrombogenic activity.

3.3. Hemolysis ratio (HR)

Hemolysis essentially results from the increase in osmoticpressure of red blood cells (RBCs) and occurs with the rupture ofRBCs and the release of hemoglobin from RBCs. Hemolysis ratio

Fig. 5. Effects of blood-sample contact time on blood clotting index (BCI).

represents the extent of RBCs broken by the sample in contact withwhole blood. The more the number of the broken red cells, thelarger the value of HR is. The smaller the HR value, the better theblood compatibility of the biomaterial is. It is known that the HRvalue for a desired biomaterial must be below 5 [16]. The HR values(listed in Table 2) for PET, PET-NH2, and PET-Arg were 2.3, 2.7, and1.7, respectively, and were all within acceptable standard forbiomaterials. Furthermore, the HR value for PET-Arg was slightlyless than those for PET and PET-NH2. The results demonstrated thatL-arginine modified films led to little hemolysis when exposed toblood.

3.4. Assay of blood clotting time

The plasma recalcification time (PRT) reflects the delay in theintrinsic coagulation process. The PRT is generally delayed in theabsence of coagulation factors, such as fibrinogen, thrombinogenand so on, or in the presence of anti-coagulant substance.Therefore, the longer the PRT for a biomaterial, the better is theanticoagulant activity of the biomaterial. The PRTs for PET, PET-NH2, and PET-Arg are shown in Fig. 6. The PRT for L-Arg modifiedfilms was 451 � 8 s, it was significantly prolonged by about 189 scompared to that of PET. The result showed that L-arginine modifiedfilms could delay the process of intrinsic coagulation and thusenhance the anticoagulant activities of PET films.

The blood coagulation cascade pathway includes intrinsic,extrinsic, and common pathway. The intrinsic and extrinsicpathways lead to the formation of fibrin clots [19]. The APTT fora biomaterial reflects the duration in which the intrinsic pathway,especially the Factor XII, is activated over the contact with blood.The longer the time, the more difficult the onset of the intrinsicpathway is. So, a prolonged APTT suggests an improved anticoag-ulant activity of a biomaterial; whereas the PT represents theduration in which the extrinsic pathway is started, and a prolongedPT also signifies an enhanced anticoagulant activity of abiomaterial. The values of APTT and PT for PET were 37.5 � 0.5 sand 15.8 � 0.3 s, respectively, and those for PET-Arg were 62.2 � 1.2 sand 19.5 � 0.4 s, respectively (shown in Fig. 6). The L-arginine

Fig. 6. PRT, APTT, and PT of treated and untreated films. Data are presented as

mean � standard deviation of four experiments.

Y. Liu et al. / Applied Surface Science 256 (2010) 3977–3981 3981

modified films prolonged the APTT significantly (by 25 s) but the PTslightly (by 3.7 s) compared to those of PET. The results indicated thatPET-Arg films greatly affected the intrinsic pathway but had littleeffects on the extrinsic pathway. The results were attributed to theinhibition of L-arginine to hemostasis activation [20]. The values ofPRT and APTT further suggested that the L-arginine modified PET filmsimproved the anticoagulant activities mainly through the intrinsiccoagulation cascade pathway.

With the improved antithrombogenicity of the L-Arg modifiedfilms determined, the mechanisms of antithrombogenicity of thecorresponding films were dealt with. It was showed that NO couldstimulate the soluble guanylate cyclase (sGC) by interacting withferrohemo center of the enzyme and thus increased the levels of30,50-cyclic monophosphate (cGMP), subsequently inhibitingplatelet adhesion and thrombus formation [21,22]. In the currentmodification method, the free guanidino group present on theimmobilized L-Arg might locally generate NO under the catalysis ofnitric-oxide synthase, thus imparting antithromboresistant prop-erties to the L-Arg modified PET surfaces.

4. Conclusion

PET films were modified by covalently immobilizing L-Arg usingglutaraldehyde as a cross-linker. The results of blood clotting test,hemolytic test, and blood clotting time demonstrated that L-Argmodified PET films exhibited good antithrombogenicity. Thesignificantly prolonged PRT and APTT further indicated that L-Arg modified PET films enhanced the anticoagulant activitiesprincipally through the intrinsic coagulation cascade pathway. Thepresented modification method could be a possible candidate to

obtain antithrombogenic PET surfaces for medical uses. Otherrelevant research works, such as measuring the amount of NOreleased from L-Arg modified PET and platelet cGMP formationwhen contacting with blood components, as well as furtheroptimizing the amount of L-Arg immobilized on PET filmsaccording to NO releasing, etc., are under study in our laboratory.

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