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Chemical modification of poly(vinyl chloride) resin using poly(ethylene glycol) to improve blood compatibility
ARTICLE IN PRESS
Biomaterials 26 (2005) 3495–3502 www.elsevier.com/locate/biomaterials
Chemical modification of poly(vinyl chloride) resin using poly(ethylene glycol) to improve blood compatibility Biji Balakrishnana, D.S. Kumarb, Yasuhiko Yoshidab, A. Jayakrishnana, a
Polymer Chemistry Division, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Satelmond Palace Campus, Poojapura, Trivandrum 695 012 Kerala, India b Department of Applied Chemistry, Bio-Nano Electronics Research Centre, Toyo University, Kawagoe, Saitama 350-8585, Japan Received 15 April 2004; accepted 20 September 2004
Abstract Poly(vinyl chloride) (PVC) was aminated by treating the resin with a concentrated aqueous solution of ethylenediamine. The aminated PVC was then reacted with hexamethylene diisocyanate to incorporate the isocyanate group onto the polymer backbone. The isocyanated PVC was further reacted with poly(ethylene glycol) (PEG) of molecular weight 600 Da. The modified polymer was characterized using infrared and X-ray photoelectron spectroscopy (XPS) and thermal analysis. Infrared and XPS spectra showed the incorporation of PEG onto PVC. The thermal stability of the modified polymer was found to be lowered by the incorporation of PEG. Contact angle measurements on the surface of polymer films cast from a tetrahydrofuran solution of the polymer demonstrated that the modified polymer gave rise to a significantly hydrophilic surface compared to unmodified PVC. The solid/ water interfacial free energy of the modified surface was 3.9 ergs/cm2 as opposed to 18.4 ergs/cm2 for bare PVC surface. Static platelet adhesion studies using platelet-rich plasma showed significantly reduced platelet adhesion on the surface of the modified polymer compared to control PVC. The surface hydrophilicity of the films was remarkably retained even in the presence of up to 30 wt% concentration of the plasticizer di-(2-ethylhexyl phthalate). The study showed that bulk modification of PVC with PEG using appropriate chemistry can give rise to a polymer that possesses the anti-fouling property of PEG and such bulk modifications are less cumbersome compared to surface modifications on the finished product to impart anti-fouling properties to the PVC surface. r 2004 Elsevier Ltd. All rights reserved. Keywords: Polyvinyl chloride; Polyethylene oxide; Surface modification; Surface energy; Platelet adhesion; Biocompatibility
1. Introduction Poly(vinyl chloride) (PVC) finds extensive application in the medical field [1]. Bags for the storage of blood and its components, tubings for extracorporeal circulation and endotracheal intubation and intravenous catheters are some of the medical devices wherein plasticized PVC is employed. PVC is not a blood-compatible polymer per se and additives such as plasticizers added to the polymer during processing to impart flexible character
Corresponding author. Fax: +91 471 2341814.
E-mail address: [email protected] (A. Jayakrishnan). 0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2004.09.032
to an otherwise rigid PVC also contribute to many adverse effects when used in contact with tissue or blood [2]. Many attempts to improve the biocompatibility of PVC have been reported in the literature. This includes polymer surface modification with endpoint attachment of heparin [3,4], immobilization of albumin [5], amination of PVC followed by complexation with heparin [6,7], grafting hydrophilic polymers onto PVC surface [8] and plasma modification [9]. In a recent study, it was shown that grafting poly(ethylene glycol) (PEG) onto PVC surface by the well-known Williamson reaction, taking advantage of the labile nature of chlorine atoms on PVC, can generate a protein and platelet repelling surface [10].
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Although surface modification is the key to altering the surface properties of the polymer without changing its physical and mechanical properties, the method has inherent disadvantages in that the modification has to be performed on the finished product. This is often undesirable, and modification of the polymer prior to processing would be more advantageous if the desirable surface properties are retained to a significant extent in the finished product. This is especially the case when the intended devices are disposable after single use. Most of the PVC-based devices used for medical applications fall into this category. Introduction of sophisticated chemical structures on the backbone of the polymer chain prior to processing is resorted to in many cases to produce the final product with the required physical, chemical and biological properties [11,12]. This work was undertaken in order to examine whether chemical modification of PVC resin using PEG can give rise to a more blood-compatible surface. This communication reports a procedure to chemically graft PEG onto PVC resin and examines the thermal behavior of the graft polymer and the surface properties of polymer films prepared by solution casting and its blood compatibility. The surface properties of solution cast films in the presence of different concentrations of the plasticizer di(2-ethylhexyl) phthalate (DEHP) were also examined in order to examine the suitability of modified PVC in reallife applications.
amine in distilled water and stirred magnetically in an oil bath maintained at 80 1C for 1 h. After the reaction, the resin was filtered and washed with copious amounts of tap water followed by distilled water to remove ethylenediamine and dried in an air oven. Reaction yield was quantitative and always over 95% (n47).
2. Materials and methods
2.2.3. Contact angle measurements Measurements were carried out on films using, captive air-in-water and octane-in-water methods using a goniometer (Rame’-Hart, Mountain Lakes, NJ, USA). Films were equilibrated overnight in distilled water before measurements. Values reported are the average of 8–10 measurements on different parts of the film. Surface energy parameters were calculated according to the method of Andrade et al. [13].
2.1. Materials Medical grade PVC resin (60–140 mesh) having a k value of 70 was from Sriram Fibres Ltd., Kota, India. DEHP used in the manufacture of blood bags was procured from Indo-Nippon Chemical Co., Mumbai, India. PEG of average molecular weight 600 Da was from Central Drug House Ltd., Mumbai, India. Hexamethylene diisocyanate (HMDI) was from Merck–Shuchardt, Germany. Ethylenediamine, ethanol, benzene, tetrahydrofuran (THF) and hexane were from S.D. Fine Chemicals Ltd., Mumbai, India. THF was refluxed over sodium in the presence of benzophenone and distilled prior to use. Hexane was dried over sodium and distilled. Cephalin reagent and fibrinogen estimation kit (FIBRI–PRESTs) were from Diagnostica Stago, France. 2.2. Methods 2.2.1. Amination of PVC PVC was aminated by treating with a large excess of an 80% aqueous solution of ethylenediamine. Thus, 1 g resin was added to 10 ml of an 80% solution of the
2.2.2. Grafting of PEG onto aminated PVC Aminated PVC, 1 g, was dissolved in 25 ml dry THF in a 100 ml round-bottomed flask, 2 ml of HMDI was added, stoppered and the mixture was stirred magnetically for 1 h at room temperature. The resultant product was precipitated in dry hexane and washed with the same solvent over a fluted filter paper many times to remove the excess HMDI. It was then redissolved in THF and treated with 2.0 g of PEG for 15 min under magnetic stirring at room temperature. After the reaction, the product was precipitated in ethanol and extensively washed with the same solvent to remove the unreacted PEG and dried in vacuum. Yield: 95%. Films of modified PVC were cast from a 4% solution in THF in the absence and in the presence of 10, 20 and 30% DEHP (w/w) in glass Petri dishes. Solvent evaporation was allowed to take place slowly at room temperature by covering the dishes partially with a watch glass to obtain clear, transparent films slightly yellow in colour. Residual solvent, if any, in the films was removed in vacuum in a desiccator. Films were stored in a desiccator until further use.
2.2.4. Infrared and X-ray photoelectron spectroscopy (XPS) Infrared spectra of unmodified and modified PVC were recorded using thin films cast from a 4% THF solution of the polymers in a Fourier transform infrared spectrophotometer (Nicolet, Model Impact 410, Madison, WI, USA). XPS was done on films cast from a 4% solution in THF using an electron spectrometer (Shimadzu, Japan, Model ESCA 750) equipped with monochromatized X-ray source of Mg Kp radiation at 1253.6 eV. Operation conditions were set at 6 kV and 30 mA. 2.2.5. Thermal analysis Differential thermal analysis (DTA) and thermogravimetric analysis (TGA) of unmodified and modified
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PVC were carried out using a simultaneous DTA–TGA instrument (TA Instruments, Model SDT 2960, Delaware, USA) at a heating rate of 10 1C/min in an atmosphere of nitrogen. 2.2.6. Blood-compatibility evaluation All evaluations were made according to International Standard, ISO 10993-4 [14]. 2.2.6.1. Platelet adhesion studies. For platelet adhesion studies, films of 1.5 1.5 cm size were incubated for 1 h in phosphate-buffered saline (0.1 M, pH 7.4). Fresh human blood anticoagulated with acid citrate dextrose (ACD) was centrifuged at 2500 rpm for 5 min to obtain platelet-rich plasma (PRP). Platelet count was adjusted to 2–2.5 108/ml using platelet-poor plasma (PPP) obtained by centrifugation of anticoagulated blood at 4000 rpm for 15 min. The films were laid flat in small polystyrene Petri dishes, submerged with PRP and left at 37 1C for 1 h in an incubator. After washing gently with buffer many times to remove non-adhering platelets, fixing was done with 2.5% buffered glutaraldehyde solution overnight in the refrigerator at 4 1C. Specimens were then washed with distilled water, stained with Leishman stain and examined in an optical microscope (Leica, DMR, Germany). For electron microscopy, films after glutaraldehyde fixation and washing with distilled water were frozen in distilled water, lyophilized, coated with gold and examined in a scanning electron microscope (Jeol JSM 7400F, Japan). 2.2.6.2. Platelet count. Films (n=3, 1 1 cm) were incubated for 1 h in PBS and exposed to 3 ml of fresh human blood anticoagulated with ACD in siliconized test tubes under agitation at 100 rpm using Environshaker (Labline Instruments Inc., Illinois, USA) at 37 1C. After 1 min and 1 h exposure, platelet counts were made using an automated haematology analyzer (Cobas Minos, Roche Diagnostics, France). 2.2.6.3. Partial thromboplastin time (PTT). For evaluating the effect of material on intrinsic coagulation pathway, films (n ¼ 3; 1 1 cm) were incubated for 1 h in PBS, which were then exposed to fresh human blood anti-coagulated with ACD in siliconized test tubes under agitation at 100 rpm using the Environ-shaker at 37 1C. After 1 min and 1 h of exposure, the blood was centrifuged at 4000 rpm for 15 min to obtain PPP. Plasma was then mixed with cephalin reagent and incubated for 3 min before adding CaCl2 solution to initiate clotting. Clotting time was noted using an automated coagulation analyzer (Diagnostica Stago, France). 2.2.6.4. Fibrinogen concentration. The procedure adopted was the same as that used for determining
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PTT. Films were incubated for 1 h in PBS and exposed to fresh human blood anticoagulated with ACD. After 1 min and 1 h of exposure, blood was taken and centrifuged at 4000 rpm for 15 min to obtain PPP. When excess concentration of thrombin is added to diluted plasma, the clotting time is directly dependent on the fibrinogen concentration [15]. Using this principle, the fibrinogen concentration was determined by noting the clotting time of PPP after adding thrombin, using the automated coagulation analyzer. Calibration curve was generated using WHO-traceable control plasma by noting the clotting time and was stored in the memory of the instrument.
3. Results and discussion PEG has attracted considerable attention as a biomaterial in recent years because of its protein and cell-repelling characteristics, and there are numerous reports in the literature on the anti-fouling properties of PEG-rich surfaces [16,17]. The amination of chlorine containing polymers using alkyl amines has been reported in the literature. Dragen et al. [18,19] reported the formation of soluble and cross-linked polymers by reacting chloromethylated polystyrene with tris-(2-hydroxyethyl) amine. Ferruti et al. [6,7] aminated PVC using a concentrated aqueous solution of bis-(2-aminoethyl) amine. The aminated PVC was then coupled onto acrylamido end-capped poly(amido–amine), which was used to complex heparin to generate an antithrombogenic surface on PVC. Although reaction of alkyl halides with ammonia or primary amines can result in the formation of quaternary salts in the presence of a large excess of amine, the reaction can yield primary and secondary amines. The polymeric species will be less reactive toward the amine as is the case of PVC. The use of a bifunctional amine such as ethylenediamine can yield a cross-linked product on prolonged reaction with the polymer. Indeed, such cross-linking was observed when PVC was reacted with ethylenediamine for prolonged periods. The products thus obtained were found to be partially or completely insoluble in good solvents for PVC. Therefore, one has to judiciously monitor the time of reaction in order to obtain soluble reaction products. Reaction for a period of 1 h yielded the aminated resin, which was found to be completely soluble in THF. The aminated PVC was reacted with HMDI to introduce the highly reactive isocyanate groups onto the polymer. HMDI is also a bifunctional reagent and the formation of a soluble product is contingent on the extent of the reaction. Prolonged reaction with HMDI was also found to yield a product insoluble in THF indicating cross-linking. The optimum period for the reaction was 1 h giving rise to the modified resin, which was soluble in THF. Reaction
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of the isocyanate-modified PVC with PEG resulted in grafting. The reaction was carried out for 15 min duration to obtain a product soluble in THF. Prolonged reaction times at this stage also resulted in a cross-linked product insoluble in THF and other good solvents for PVC, such as cyclohexanone. The reactions are outlined in Scheme 1. Fig. 1 shows the infrared spectra of unmodified and PEG-grafted PVC films cast from a 4% solution in THF. The peak at 3300 cm1 is due to the hydroxyl groups of PEG and the peak at 1600 cm1 is due to the carbonyl group from the isocyante moiety, both of which are absent in PVC. Thus, the infrared spectra unambiguously confirm the incorporation of PEG into the PVC backbone. Since step 1, which is amination of PVC, is the crucial step in determining the extent of modification, it was of interest to examine the extent of reaction in this step. Fig. 2(a) shows the wide scan XPS spectrum of PVC resin cast into films from THF solution where the C1s peak appears at 285 eV followed by the Cl 2p peak at 200 eV and Cl 2s peak at 269 eV. A small signal of O1s at 533.3 seen in the spectrum is attributed to the presence of a trace amount of moisture trapped in the film. Resin aminated for 1 h shows the presence of N 1s at 400 eV confirming the amination of PVC (Fig. 2b). Fig. 2(c) shows the spectrum of PEGylated PVC wherein the intense O1s signal at 533.3 eV can clearly be identified. Based on quantitative estimation of the percentage of N in the aminated specimen, under the justifiable presumption that the surface concentration will be more or less equal to the bulk concentration (since measurements were done on the films cast by dissolution of the modified resin in THF), the degree of substitution of chlorine was calculated to be 3.5, 11.8 and 16%, respectively, for 15, 30 and 60 min amination. The air-in-water and octane-in-water contact angles on bare and modified PVC surface are given in Table 1 along with the surface energy parameters calculated. The solid/water surface free energy of the modified PVC
Scheme 1. Grafting of PEG onto PVC.
Fig. 1. FTIR spectrum of unmodified PVC (a) and PEGylated PVC (b) films cast from THF.
surface is nearly one-fourth of the unmodified surface indicating considerable hydrophilicity on the modified surface. Determination of equilibrium uptake of water by immersion of specimens for 24 h in distilled water at room temperature showed that the PEGylated sample incorporated about 12% water while unmodified PVC had only about 1% (Fig. 3). When PEG was grafted onto the surface of plasticized PVC by the Williamson reaction, we found that the solid/water free energy of the modified surface was virtually zero [10]. In surface modification, there was a uniform coverage of the PEG on the polymer surface. It should be recalled that the measurements reported in the present study are on films of the modified polymer prepared by solvent casting. All the PEG incorporated into the polymer by grafting will not be exposed onto the surface on contact with water. Even then, the decrease in contact angle and solid/water free energy of the surface is striking. When PEGylated PVC cast into films in the presence of different concentrations of DEHP was examined for surface hydrophilicity by contact angle measurements, it was seen that the surface hydrophilicity was retained to a significant extent even in the presence of DEHP (see Table 1). Therefore, the bulk modification of PVC with PEG still exerts its effect on the surface properties even in the presence of significant concentrations of the oily plasticizer. Figs. 4a and b show the DTA–TGA trace of unmodified and PEGylated PVC, respectively. The Tg of PVC is around 83 1C and the decomposition of the polymer starts around 300 1C. In the case of PEGylated PVC, the Tg is lowered to 25–30 1C and the decomposition temperature is lowered to about 250 1C indicating reduced thermal stability of the modified polymer due to the incorporation of PEG on the polymer backbone as expected. Nevertheless, the reduced thermal stability is not expected to affect significantly the processing of the modified polymer. Fig. 5 shows the optical and electron micrographs of platelet adhesion on PVC and PEGylated PVC films. As can be seen from the photomicrographs, the adhesion is
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Fig. 2. (a) Wide scan XPS spectrum of PVC resin cast as a film from a THF solution; (b) XPS spectrum of PVC aminated for 60 min; (c) Wide scan XPS spectrum of PEGylated PVC. Table 1 Contact angles and surface energy parameters of unmodified and PEG-modified PVC films. (all angles and standard deviations are rounded to the nearest whole number) Surface
yair (deg)
Foctane (deg)
gds=v (erg/cm2)
gps=v (erg/cm2)
gs=w (erg/cm2)
Bare PVC PEG–PVC PEG–PVC–10%DEHP PEG–PVC–20%DEHP PEG–PVC–30%DEHP
7771 4771 5773 5473 5473
11571 15371 13574 13472 13272
8.53 7.9 6.89 11.07 11.6
25.7 45.6 37.66 37.21 36.34
18.4 3.9 7.4 5.4 5.31
significantly less on the film prepared from the modified polymer compared to control. In fact, on electron microscopy examination, the surface looked almost clean. This observation was further corroborated by automated platelet count using whole human blood with
no change in count after 1 h of exposure on PEGmodified PVC films, as opposed to 1974% decrease for unmodified PVC. To examine whether PEGylated PVC containing DEHP still resisted platelet adhesion, films cast in the
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Fig. 3. Percentage equilibrium water content of unmodified and PEGylated PVC.
presence of 10, 20 and 30 wt% DEHP were exposed to PRP and examined microscopically for platelet adhesion. Even in the presence of 30% DEHP, very few platelets were found to adhere onto the surface (Fig. 6) as compared to control PVC demonstrating that the bulk modification still exerts its influence on the surface properties in the presence of DEHP. For further evaluating the blood compatibility of the material, we determined PTT and amount of fibrinogen in the plasma after exposure to PEGylated PVC for 1 h. PTT is standard test for analyzing the material’s effect on intrinsic coagulation pathway, which can be triggered by variety of stimuli including the contact of blood with glass or other materials. To avoid the effect of glass, we conducted all the experiments in siliconized glass test tubes. PEG-modified PVC does not impart any change in clotting time (Fig. 7), whereas, there is a reduction of 1472 s in the case of unmodified PVC films. This showed that the material did not have any adverse effect on the intrinsic coagulation pathway pointing to better blood compatibility. Fibrinogen content of the PPP was same for PEGylated PVC before and after exposure, whereas, it decreased by 1476% in case of unmodified PVC films after 1 h of exposure to blood. The decrease in the fibrinogen content for unmodified PVC can be attributed to its adsorption on the surface. The presence of platelet-adhesive proteins such as fibrinogen results in the adhesion of platelets as seen in the case of unmodified PVC films (Fig. 5), which then induces thrombus formation [20]. It is well known that PEG repels proteins and the absence of proteins having affinity for the platelets is responsible for the reduced cell adhesion that is seen in PEG-rich surfaces. Although the modified polymer is not expected to be covered uniformly by PEG unlike surface grafting, the reduced platelet adhesion seen on the modified polymer surface is a pointer to the enhanced blood compatibility of the surface. PVC is extensively used as endotracheal tubes and a major problem in its use is the extensive colonization of bacteria [21]. PEG-rich surfaces are known to adhere less bacteria [22,23], and it is reasonable to expect less bacterial colonization on the modified polymer.
4. Conclusions
Fig. 4. (a) DTA–TGA of PVC and (b) PEGylated PVC.
The results obtained in this study show that although surface modification of PVC using PEG results in high grafting density on the surface, even bulk modification by appropriate chemistry will be able to change the surface properties of the polymer to a significant extent toward better blood compatibility. Chemical modification of PVC using appropriate chemistry is a feasible proposition due to the labile nature of the chlorine atoms present on the polymer
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Fig. 5. Optical and scanning electron micrographs of platelet adhesion onto unmodified PVC and PEGylated PVC films. (a,c) Unmodified PVC; (b,d) PEGylated PVC.
Fig. 6. Optical photomicrograph of platelet adhesion onto PVC film containing 30% DEHP.
[24]. It would also be interesting to look at the possibility of radiation grafting of PEG-like molecules onto resin and examine the surface properties of films prepared from such modified resin. Bulk modification of polymers such as PVC to prepare more blood and tissue-compatible devices will be more cost effective than surface modification of finished products especially when such devices are disposable after single use.
Fig. 7. Histogram showing the change in clotting time obtained from PTT measurements for PVC and PEGylated PVC after 1 h of exposure to blood.
Acknowledgement A.J. thanks the Director, SCTIMST, for permission to publish this manuscript and the Japan Society for
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Promotion of Science for an Invitation Fellowship to Toyo University, Saitama, Japan. References [1] Blass CR, Jones C, Courtney JM. Biomaterials for blood tubing: the application of plasticized poly(vinyl chloride). Int J Artif Organs 1992;15:200–3. [2] Latini G. Potential hazards of exposure to di-(2-ethylhexyl)phthalate (DEHP) in babies: a review. Biol Neonate 2000;78: 269–76. [3] Larm O, Larson R, Olsson P. A new non-thrombogenic surface prepared by selective covalent binding of heparin via a modified reducing terminal residue. Biomater Med Dev Artif Organs 1983;11:161–73. [4] Yu JL, Johansson S, Ljungh A. Fibronectin exposes different domains after adsorption to a heparinized and unheparinized poly(vinyl chloride) surface. Biomaterials 1997;18:421–7. [5] De Queiroz AAA, Barrak ER, Gil HA, Higa OZ. Surface studies of albumin immobilized onto PE and PVC films. J Biomat Sci Polym Ed 1997;8:667–81. [6] Ferruti P, Barbucci R, Danzo N, Torrisi A, Puglisi O, Pigataro S, Spartano P. Preparation and ESCA characterization of poly(vinyl chloride) surface-grafted with heparin complexing poly(amidoamine) chains. Biomaterials 1982;3:33–7. [7] Barbucci R, Casini G, Ferruti P, Tempesti F. Surface grafted heparinizable materials. Polymer 1985;26:1349–52. [8] Krishnan VK, Jayakrishnan A, Francis JD. Radiation grafting of hydrophilic monomers onto plasticized poly(vinyl chloride) II. Migration behavior of the plasticizer from N-vinyl pyrrolidone grafted sheets. Biomaterials 1991;12:482–9. [9] Ishikawa Y, Honda K, Sasakawa S, Hatada K, Kobayashi H. Prevention of leakage of di-(2-ethylhexyl)phthalate from blood bags by glow discharge treatment and its effect on aggregability of stored platelets. Vox Sang 1983;45:68–76. [10] Lakshmi S, Jayakrishnan A. Migration resistant, blood-compatible plasticized poly(vinyl chloride) for medical and related applications, Artif Organs 1998;22:222–9. [11] Carraher Jr CE, Tsuda M, editors. Modification of polymers, ACS Symposium Series, vol. 121. Washington, DC: Am Chem Soc; 1980. [12] Moore JA, editor. Reactions on polymers. Dordrecht: Reidel; 1973.
[13] Andrade JD, King RN, Gregonis DE, Coleman DL. Surface characterization of poly(hydroxyethyl methacrylate) and related polymers. I. Contact angle methods in water. J Polym Sci Polym Symp 1979;66:313–36. [14] ISO 10993-4. Biological evaluation of medical devices— Part 4: selection of tests for interactions with blood, 2nd ed. 2002. [15] Koepke JA, Gilmer PR, Filip Jr DJ, Eckstein JD, Eckstein JD, Sibley CA. Studies of fibrinogen measurement in the CAP survey program. Am J Clin Pathol 1975;63:984–9. [16] Harris JM, editor. Poly(ethylene glycol) chemistry: biotechnical and biomedical applications. New York: Plenum; 1992. [17] Amiji M, Park K. Surface modification of polymeric biomaterials with poly(ethylene oxide). In: Shalaby SW, editor. Polymers of biological and biomedical significance. Washington, DC: American Chemical Society; 1994. p. 135–46. [18] Dragan S, Luca C, Petrariu I, Dima M. Cationic polyelectrolytes I. Soluble polymers prepared from reaction of chloromethylated polystyrene with tris(2-hydroxyethyl) amine. J Polym Sci Polym Chem Ed 1980;18:544–65. [19] Luca C, Dragan S, Barboiu V, Dima M. Chloromethylated polystyrene reaction with tris(2-hydroxyethyl) amine I. Crosslinked polymers prepared by chloromethylated polystyrene with tris(2-hydroxyethyl) amine. J Polym Sci Polym Chem Ed 1980;18:449–54. [20] Shultz JS, Lindenauer SM, Penner JA. Thrombus formation on surfaces in contact with blood. In: Cooper SL, Peppas NA, editors. Biomaterials: interfacial phenomena and applications, vol. 199. Washington, DC: American Chemical Society; 1982. p. 43–58. [21] Jones DS, McGovern JG, Woolfson DA, Gorman SP. Role of physiological conditions in the oropharynx on the adherence of respiratory bacterial isolates to endotracheal tube poly(vinyl chloride). Biomaterials 1997;18:503–10. [22] Desai NP, Hossainy SFA, Hubbel JA. Surface-immobilized polyethylene oxide for bacterial resistance. Biomaterials 1992;13: 417–20. [23] Park JH, Cho YW, Kwon IC, Jeong SY, Bae YH. Assessment of PEO/PTMO multiblock copolymer/segmented polyurethane blends as coating materials for urinary catheters: in vitro bacterial adhesion and encrustation behavior. Biomaterials 2002;23: 3991–4000. [24] Jayakrishnan A, Lakshmi S. Immobile plasticizer in flexible PVC. Nature 1998;396:638.
Biomaterials 26 (2005) 3495–3502 www.elsevier.com/locate/biomaterials
Chemical modification of poly(vinyl chloride) resin using poly(ethylene glycol) to improve blood compatibility Biji Balakrishnana, D.S. Kumarb, Yasuhiko Yoshidab, A. Jayakrishnana, a
Polymer Chemistry Division, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Satelmond Palace Campus, Poojapura, Trivandrum 695 012 Kerala, India b Department of Applied Chemistry, Bio-Nano Electronics Research Centre, Toyo University, Kawagoe, Saitama 350-8585, Japan Received 15 April 2004; accepted 20 September 2004
Abstract Poly(vinyl chloride) (PVC) was aminated by treating the resin with a concentrated aqueous solution of ethylenediamine. The aminated PVC was then reacted with hexamethylene diisocyanate to incorporate the isocyanate group onto the polymer backbone. The isocyanated PVC was further reacted with poly(ethylene glycol) (PEG) of molecular weight 600 Da. The modified polymer was characterized using infrared and X-ray photoelectron spectroscopy (XPS) and thermal analysis. Infrared and XPS spectra showed the incorporation of PEG onto PVC. The thermal stability of the modified polymer was found to be lowered by the incorporation of PEG. Contact angle measurements on the surface of polymer films cast from a tetrahydrofuran solution of the polymer demonstrated that the modified polymer gave rise to a significantly hydrophilic surface compared to unmodified PVC. The solid/ water interfacial free energy of the modified surface was 3.9 ergs/cm2 as opposed to 18.4 ergs/cm2 for bare PVC surface. Static platelet adhesion studies using platelet-rich plasma showed significantly reduced platelet adhesion on the surface of the modified polymer compared to control PVC. The surface hydrophilicity of the films was remarkably retained even in the presence of up to 30 wt% concentration of the plasticizer di-(2-ethylhexyl phthalate). The study showed that bulk modification of PVC with PEG using appropriate chemistry can give rise to a polymer that possesses the anti-fouling property of PEG and such bulk modifications are less cumbersome compared to surface modifications on the finished product to impart anti-fouling properties to the PVC surface. r 2004 Elsevier Ltd. All rights reserved. Keywords: Polyvinyl chloride; Polyethylene oxide; Surface modification; Surface energy; Platelet adhesion; Biocompatibility
1. Introduction Poly(vinyl chloride) (PVC) finds extensive application in the medical field [1]. Bags for the storage of blood and its components, tubings for extracorporeal circulation and endotracheal intubation and intravenous catheters are some of the medical devices wherein plasticized PVC is employed. PVC is not a blood-compatible polymer per se and additives such as plasticizers added to the polymer during processing to impart flexible character
Corresponding author. Fax: +91 471 2341814.
E-mail address: [email protected] (A. Jayakrishnan). 0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2004.09.032
to an otherwise rigid PVC also contribute to many adverse effects when used in contact with tissue or blood [2]. Many attempts to improve the biocompatibility of PVC have been reported in the literature. This includes polymer surface modification with endpoint attachment of heparin [3,4], immobilization of albumin [5], amination of PVC followed by complexation with heparin [6,7], grafting hydrophilic polymers onto PVC surface [8] and plasma modification [9]. In a recent study, it was shown that grafting poly(ethylene glycol) (PEG) onto PVC surface by the well-known Williamson reaction, taking advantage of the labile nature of chlorine atoms on PVC, can generate a protein and platelet repelling surface [10].
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Although surface modification is the key to altering the surface properties of the polymer without changing its physical and mechanical properties, the method has inherent disadvantages in that the modification has to be performed on the finished product. This is often undesirable, and modification of the polymer prior to processing would be more advantageous if the desirable surface properties are retained to a significant extent in the finished product. This is especially the case when the intended devices are disposable after single use. Most of the PVC-based devices used for medical applications fall into this category. Introduction of sophisticated chemical structures on the backbone of the polymer chain prior to processing is resorted to in many cases to produce the final product with the required physical, chemical and biological properties [11,12]. This work was undertaken in order to examine whether chemical modification of PVC resin using PEG can give rise to a more blood-compatible surface. This communication reports a procedure to chemically graft PEG onto PVC resin and examines the thermal behavior of the graft polymer and the surface properties of polymer films prepared by solution casting and its blood compatibility. The surface properties of solution cast films in the presence of different concentrations of the plasticizer di(2-ethylhexyl) phthalate (DEHP) were also examined in order to examine the suitability of modified PVC in reallife applications.
amine in distilled water and stirred magnetically in an oil bath maintained at 80 1C for 1 h. After the reaction, the resin was filtered and washed with copious amounts of tap water followed by distilled water to remove ethylenediamine and dried in an air oven. Reaction yield was quantitative and always over 95% (n47).
2. Materials and methods
2.2.3. Contact angle measurements Measurements were carried out on films using, captive air-in-water and octane-in-water methods using a goniometer (Rame’-Hart, Mountain Lakes, NJ, USA). Films were equilibrated overnight in distilled water before measurements. Values reported are the average of 8–10 measurements on different parts of the film. Surface energy parameters were calculated according to the method of Andrade et al. [13].
2.1. Materials Medical grade PVC resin (60–140 mesh) having a k value of 70 was from Sriram Fibres Ltd., Kota, India. DEHP used in the manufacture of blood bags was procured from Indo-Nippon Chemical Co., Mumbai, India. PEG of average molecular weight 600 Da was from Central Drug House Ltd., Mumbai, India. Hexamethylene diisocyanate (HMDI) was from Merck–Shuchardt, Germany. Ethylenediamine, ethanol, benzene, tetrahydrofuran (THF) and hexane were from S.D. Fine Chemicals Ltd., Mumbai, India. THF was refluxed over sodium in the presence of benzophenone and distilled prior to use. Hexane was dried over sodium and distilled. Cephalin reagent and fibrinogen estimation kit (FIBRI–PRESTs) were from Diagnostica Stago, France. 2.2. Methods 2.2.1. Amination of PVC PVC was aminated by treating with a large excess of an 80% aqueous solution of ethylenediamine. Thus, 1 g resin was added to 10 ml of an 80% solution of the
2.2.2. Grafting of PEG onto aminated PVC Aminated PVC, 1 g, was dissolved in 25 ml dry THF in a 100 ml round-bottomed flask, 2 ml of HMDI was added, stoppered and the mixture was stirred magnetically for 1 h at room temperature. The resultant product was precipitated in dry hexane and washed with the same solvent over a fluted filter paper many times to remove the excess HMDI. It was then redissolved in THF and treated with 2.0 g of PEG for 15 min under magnetic stirring at room temperature. After the reaction, the product was precipitated in ethanol and extensively washed with the same solvent to remove the unreacted PEG and dried in vacuum. Yield: 95%. Films of modified PVC were cast from a 4% solution in THF in the absence and in the presence of 10, 20 and 30% DEHP (w/w) in glass Petri dishes. Solvent evaporation was allowed to take place slowly at room temperature by covering the dishes partially with a watch glass to obtain clear, transparent films slightly yellow in colour. Residual solvent, if any, in the films was removed in vacuum in a desiccator. Films were stored in a desiccator until further use.
2.2.4. Infrared and X-ray photoelectron spectroscopy (XPS) Infrared spectra of unmodified and modified PVC were recorded using thin films cast from a 4% THF solution of the polymers in a Fourier transform infrared spectrophotometer (Nicolet, Model Impact 410, Madison, WI, USA). XPS was done on films cast from a 4% solution in THF using an electron spectrometer (Shimadzu, Japan, Model ESCA 750) equipped with monochromatized X-ray source of Mg Kp radiation at 1253.6 eV. Operation conditions were set at 6 kV and 30 mA. 2.2.5. Thermal analysis Differential thermal analysis (DTA) and thermogravimetric analysis (TGA) of unmodified and modified
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PVC were carried out using a simultaneous DTA–TGA instrument (TA Instruments, Model SDT 2960, Delaware, USA) at a heating rate of 10 1C/min in an atmosphere of nitrogen. 2.2.6. Blood-compatibility evaluation All evaluations were made according to International Standard, ISO 10993-4 [14]. 2.2.6.1. Platelet adhesion studies. For platelet adhesion studies, films of 1.5 1.5 cm size were incubated for 1 h in phosphate-buffered saline (0.1 M, pH 7.4). Fresh human blood anticoagulated with acid citrate dextrose (ACD) was centrifuged at 2500 rpm for 5 min to obtain platelet-rich plasma (PRP). Platelet count was adjusted to 2–2.5 108/ml using platelet-poor plasma (PPP) obtained by centrifugation of anticoagulated blood at 4000 rpm for 15 min. The films were laid flat in small polystyrene Petri dishes, submerged with PRP and left at 37 1C for 1 h in an incubator. After washing gently with buffer many times to remove non-adhering platelets, fixing was done with 2.5% buffered glutaraldehyde solution overnight in the refrigerator at 4 1C. Specimens were then washed with distilled water, stained with Leishman stain and examined in an optical microscope (Leica, DMR, Germany). For electron microscopy, films after glutaraldehyde fixation and washing with distilled water were frozen in distilled water, lyophilized, coated with gold and examined in a scanning electron microscope (Jeol JSM 7400F, Japan). 2.2.6.2. Platelet count. Films (n=3, 1 1 cm) were incubated for 1 h in PBS and exposed to 3 ml of fresh human blood anticoagulated with ACD in siliconized test tubes under agitation at 100 rpm using Environshaker (Labline Instruments Inc., Illinois, USA) at 37 1C. After 1 min and 1 h exposure, platelet counts were made using an automated haematology analyzer (Cobas Minos, Roche Diagnostics, France). 2.2.6.3. Partial thromboplastin time (PTT). For evaluating the effect of material on intrinsic coagulation pathway, films (n ¼ 3; 1 1 cm) were incubated for 1 h in PBS, which were then exposed to fresh human blood anti-coagulated with ACD in siliconized test tubes under agitation at 100 rpm using the Environ-shaker at 37 1C. After 1 min and 1 h of exposure, the blood was centrifuged at 4000 rpm for 15 min to obtain PPP. Plasma was then mixed with cephalin reagent and incubated for 3 min before adding CaCl2 solution to initiate clotting. Clotting time was noted using an automated coagulation analyzer (Diagnostica Stago, France). 2.2.6.4. Fibrinogen concentration. The procedure adopted was the same as that used for determining
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PTT. Films were incubated for 1 h in PBS and exposed to fresh human blood anticoagulated with ACD. After 1 min and 1 h of exposure, blood was taken and centrifuged at 4000 rpm for 15 min to obtain PPP. When excess concentration of thrombin is added to diluted plasma, the clotting time is directly dependent on the fibrinogen concentration [15]. Using this principle, the fibrinogen concentration was determined by noting the clotting time of PPP after adding thrombin, using the automated coagulation analyzer. Calibration curve was generated using WHO-traceable control plasma by noting the clotting time and was stored in the memory of the instrument.
3. Results and discussion PEG has attracted considerable attention as a biomaterial in recent years because of its protein and cell-repelling characteristics, and there are numerous reports in the literature on the anti-fouling properties of PEG-rich surfaces [16,17]. The amination of chlorine containing polymers using alkyl amines has been reported in the literature. Dragen et al. [18,19] reported the formation of soluble and cross-linked polymers by reacting chloromethylated polystyrene with tris-(2-hydroxyethyl) amine. Ferruti et al. [6,7] aminated PVC using a concentrated aqueous solution of bis-(2-aminoethyl) amine. The aminated PVC was then coupled onto acrylamido end-capped poly(amido–amine), which was used to complex heparin to generate an antithrombogenic surface on PVC. Although reaction of alkyl halides with ammonia or primary amines can result in the formation of quaternary salts in the presence of a large excess of amine, the reaction can yield primary and secondary amines. The polymeric species will be less reactive toward the amine as is the case of PVC. The use of a bifunctional amine such as ethylenediamine can yield a cross-linked product on prolonged reaction with the polymer. Indeed, such cross-linking was observed when PVC was reacted with ethylenediamine for prolonged periods. The products thus obtained were found to be partially or completely insoluble in good solvents for PVC. Therefore, one has to judiciously monitor the time of reaction in order to obtain soluble reaction products. Reaction for a period of 1 h yielded the aminated resin, which was found to be completely soluble in THF. The aminated PVC was reacted with HMDI to introduce the highly reactive isocyanate groups onto the polymer. HMDI is also a bifunctional reagent and the formation of a soluble product is contingent on the extent of the reaction. Prolonged reaction with HMDI was also found to yield a product insoluble in THF indicating cross-linking. The optimum period for the reaction was 1 h giving rise to the modified resin, which was soluble in THF. Reaction
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of the isocyanate-modified PVC with PEG resulted in grafting. The reaction was carried out for 15 min duration to obtain a product soluble in THF. Prolonged reaction times at this stage also resulted in a cross-linked product insoluble in THF and other good solvents for PVC, such as cyclohexanone. The reactions are outlined in Scheme 1. Fig. 1 shows the infrared spectra of unmodified and PEG-grafted PVC films cast from a 4% solution in THF. The peak at 3300 cm1 is due to the hydroxyl groups of PEG and the peak at 1600 cm1 is due to the carbonyl group from the isocyante moiety, both of which are absent in PVC. Thus, the infrared spectra unambiguously confirm the incorporation of PEG into the PVC backbone. Since step 1, which is amination of PVC, is the crucial step in determining the extent of modification, it was of interest to examine the extent of reaction in this step. Fig. 2(a) shows the wide scan XPS spectrum of PVC resin cast into films from THF solution where the C1s peak appears at 285 eV followed by the Cl 2p peak at 200 eV and Cl 2s peak at 269 eV. A small signal of O1s at 533.3 seen in the spectrum is attributed to the presence of a trace amount of moisture trapped in the film. Resin aminated for 1 h shows the presence of N 1s at 400 eV confirming the amination of PVC (Fig. 2b). Fig. 2(c) shows the spectrum of PEGylated PVC wherein the intense O1s signal at 533.3 eV can clearly be identified. Based on quantitative estimation of the percentage of N in the aminated specimen, under the justifiable presumption that the surface concentration will be more or less equal to the bulk concentration (since measurements were done on the films cast by dissolution of the modified resin in THF), the degree of substitution of chlorine was calculated to be 3.5, 11.8 and 16%, respectively, for 15, 30 and 60 min amination. The air-in-water and octane-in-water contact angles on bare and modified PVC surface are given in Table 1 along with the surface energy parameters calculated. The solid/water surface free energy of the modified PVC
Scheme 1. Grafting of PEG onto PVC.
Fig. 1. FTIR spectrum of unmodified PVC (a) and PEGylated PVC (b) films cast from THF.
surface is nearly one-fourth of the unmodified surface indicating considerable hydrophilicity on the modified surface. Determination of equilibrium uptake of water by immersion of specimens for 24 h in distilled water at room temperature showed that the PEGylated sample incorporated about 12% water while unmodified PVC had only about 1% (Fig. 3). When PEG was grafted onto the surface of plasticized PVC by the Williamson reaction, we found that the solid/water free energy of the modified surface was virtually zero [10]. In surface modification, there was a uniform coverage of the PEG on the polymer surface. It should be recalled that the measurements reported in the present study are on films of the modified polymer prepared by solvent casting. All the PEG incorporated into the polymer by grafting will not be exposed onto the surface on contact with water. Even then, the decrease in contact angle and solid/water free energy of the surface is striking. When PEGylated PVC cast into films in the presence of different concentrations of DEHP was examined for surface hydrophilicity by contact angle measurements, it was seen that the surface hydrophilicity was retained to a significant extent even in the presence of DEHP (see Table 1). Therefore, the bulk modification of PVC with PEG still exerts its effect on the surface properties even in the presence of significant concentrations of the oily plasticizer. Figs. 4a and b show the DTA–TGA trace of unmodified and PEGylated PVC, respectively. The Tg of PVC is around 83 1C and the decomposition of the polymer starts around 300 1C. In the case of PEGylated PVC, the Tg is lowered to 25–30 1C and the decomposition temperature is lowered to about 250 1C indicating reduced thermal stability of the modified polymer due to the incorporation of PEG on the polymer backbone as expected. Nevertheless, the reduced thermal stability is not expected to affect significantly the processing of the modified polymer. Fig. 5 shows the optical and electron micrographs of platelet adhesion on PVC and PEGylated PVC films. As can be seen from the photomicrographs, the adhesion is
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Fig. 2. (a) Wide scan XPS spectrum of PVC resin cast as a film from a THF solution; (b) XPS spectrum of PVC aminated for 60 min; (c) Wide scan XPS spectrum of PEGylated PVC. Table 1 Contact angles and surface energy parameters of unmodified and PEG-modified PVC films. (all angles and standard deviations are rounded to the nearest whole number) Surface
yair (deg)
Foctane (deg)
gds=v (erg/cm2)
gps=v (erg/cm2)
gs=w (erg/cm2)
Bare PVC PEG–PVC PEG–PVC–10%DEHP PEG–PVC–20%DEHP PEG–PVC–30%DEHP
7771 4771 5773 5473 5473
11571 15371 13574 13472 13272
8.53 7.9 6.89 11.07 11.6
25.7 45.6 37.66 37.21 36.34
18.4 3.9 7.4 5.4 5.31
significantly less on the film prepared from the modified polymer compared to control. In fact, on electron microscopy examination, the surface looked almost clean. This observation was further corroborated by automated platelet count using whole human blood with
no change in count after 1 h of exposure on PEGmodified PVC films, as opposed to 1974% decrease for unmodified PVC. To examine whether PEGylated PVC containing DEHP still resisted platelet adhesion, films cast in the
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Fig. 3. Percentage equilibrium water content of unmodified and PEGylated PVC.
presence of 10, 20 and 30 wt% DEHP were exposed to PRP and examined microscopically for platelet adhesion. Even in the presence of 30% DEHP, very few platelets were found to adhere onto the surface (Fig. 6) as compared to control PVC demonstrating that the bulk modification still exerts its influence on the surface properties in the presence of DEHP. For further evaluating the blood compatibility of the material, we determined PTT and amount of fibrinogen in the plasma after exposure to PEGylated PVC for 1 h. PTT is standard test for analyzing the material’s effect on intrinsic coagulation pathway, which can be triggered by variety of stimuli including the contact of blood with glass or other materials. To avoid the effect of glass, we conducted all the experiments in siliconized glass test tubes. PEG-modified PVC does not impart any change in clotting time (Fig. 7), whereas, there is a reduction of 1472 s in the case of unmodified PVC films. This showed that the material did not have any adverse effect on the intrinsic coagulation pathway pointing to better blood compatibility. Fibrinogen content of the PPP was same for PEGylated PVC before and after exposure, whereas, it decreased by 1476% in case of unmodified PVC films after 1 h of exposure to blood. The decrease in the fibrinogen content for unmodified PVC can be attributed to its adsorption on the surface. The presence of platelet-adhesive proteins such as fibrinogen results in the adhesion of platelets as seen in the case of unmodified PVC films (Fig. 5), which then induces thrombus formation [20]. It is well known that PEG repels proteins and the absence of proteins having affinity for the platelets is responsible for the reduced cell adhesion that is seen in PEG-rich surfaces. Although the modified polymer is not expected to be covered uniformly by PEG unlike surface grafting, the reduced platelet adhesion seen on the modified polymer surface is a pointer to the enhanced blood compatibility of the surface. PVC is extensively used as endotracheal tubes and a major problem in its use is the extensive colonization of bacteria [21]. PEG-rich surfaces are known to adhere less bacteria [22,23], and it is reasonable to expect less bacterial colonization on the modified polymer.
4. Conclusions
Fig. 4. (a) DTA–TGA of PVC and (b) PEGylated PVC.
The results obtained in this study show that although surface modification of PVC using PEG results in high grafting density on the surface, even bulk modification by appropriate chemistry will be able to change the surface properties of the polymer to a significant extent toward better blood compatibility. Chemical modification of PVC using appropriate chemistry is a feasible proposition due to the labile nature of the chlorine atoms present on the polymer
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Fig. 5. Optical and scanning electron micrographs of platelet adhesion onto unmodified PVC and PEGylated PVC films. (a,c) Unmodified PVC; (b,d) PEGylated PVC.
Fig. 6. Optical photomicrograph of platelet adhesion onto PVC film containing 30% DEHP.
[24]. It would also be interesting to look at the possibility of radiation grafting of PEG-like molecules onto resin and examine the surface properties of films prepared from such modified resin. Bulk modification of polymers such as PVC to prepare more blood and tissue-compatible devices will be more cost effective than surface modification of finished products especially when such devices are disposable after single use.
Fig. 7. Histogram showing the change in clotting time obtained from PTT measurements for PVC and PEGylated PVC after 1 h of exposure to blood.
Acknowledgement A.J. thanks the Director, SCTIMST, for permission to publish this manuscript and the Japan Society for
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