Radiation-induced graft copolymerization of methacrylic acid on to poly(viny1 chloride) films and their thrombogenicity

Jagbir Sin&, Alok R. Ray, J.P. Singhal and Harpal Sin& Centre for Biomedical Engineering, Indian institute of Technology, Delhi Haur Khas New Delhi- 1 100 16, and All India Institute of Medical Sciences, New Delhi- 110029, India (Received 23 March 1989; revised 28 May 1989; accepted 10 September 1989)

Mathacrylic acid (MAA) was radiation grafted on to poly(vinyl chloride) (PVC) films to improve the blood compatibility of PVC. The thromoboganicity of MAA grafted PVC was evaluated by thrombus formation, platelet adhesion and haamolytic activity in vitro. The hydrophilicity of grafted PVC films was investigated by contact angle measurement. Mathacrylic acid grafted PVC film showed lower thromboganicity than that of PVC. It was found that the weight of thrombus formed on grafted PVC was less than that of PVC and glass and decreased with the increase in the graft level. The adhesion of platelets on grafted PVC was retarded after grafting with MAA hydrogals.

Keywords: Thrombogenesis, copolymers, polylvinyl chloride/

Polymers with hydrophilic and negatively charged surfaces have low adsorption of serum proteins and do not have strong interactions with blood cells’-4. Graft copolymerization is an effective method for improving the biocompatibility of polymers by introducing hydrophilicity and negative charges on the surface. Poly(vinyl chloride) (PVC), a typical hydro- phobic material, is presently in use in endotracheal tubes5, catheters6. ‘, oxygenators*, blood bagsg, lo, circulation tubing”.‘*, surgical dressing13, l4 and artificial skin15. Yamauchi” has grafted PVC tubing with Z-hydroxyethyl- methacrylate for blood circulation applications. Paul et a/.” have described the grafting of N-vinyl-N-methylacetamide hydrogels on to PVCfor improving blood compatibility. In the present studies poly(vinyl chloride) is grafted with meth- acrylic acid to make it more hydrophilic. The in vitro evaluation of blood compatibility of MAA grafted PVC is described.

MATERIALS

Food-grade PVC was obtained from Indian Petrochemicals Ltd, Baroda, India. MAA was obtained from E. Merck (Germany) and distilled at 80°C under vacuum of 10 mmHg before use. Dioctyl phthalate (DOP) was purchased from G.S.C. Chemicals, India. All other chemicals were obtained from Glaxo (India) and were used without further purification.

Correspondence to Dr H. Singh.

METHODS

Preparation of PVC films

PVC films were cast on glass plates from a solution of PVC and DOP (70:30, w/w) in cyclohexanone:toluene (60:40, v/v) solvent mixtures. These films were dried at room temperature for 24 h and then in a vacuum oven at 60°C for 8 h. They were stored in a dessicator over anhydrous calcium chloride.

Grafting procedure

Graft copolymerization was carried out in standard joint Corning tubes (12.0 x 3.0 cm) under nitrogen atmosphere. The weighed amount of plasticized PVC film (6.5 X 3.0 X 0.006 cm) was kept in a glass ampoule in an equivolume mixture of methanol-water for 24 h. MAA was added just before the exposure of samples. The ampoules were placed in the radiation chamber for the desired period. Most of the experiments were carried out under the following conditions unless otherwise specified: dose rate 56 rad/s, total dose 0.25 Mrad, monomer concentration 2.36 M, total volume of solvent 14 ml and DOP content of PVC film 30%. After irradiation, the films were removed from the external solution of homopolymer and thoroughly washed with hot water and then soaked in water at 50°C for several hours to remove homopolymer. Further extraction of homopolymer was done by Soxhlet extraction in methanol for 4 h and then

0 1990 Butterworth-Heinemann Ltd. 0142-961 Z/90/070473-04

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Thrombogenicity of MAA grafted PVC films: J. Singh et al.

films were washed with water and dried at 60°C at reduced pressure and weighed. Grafting was calculated as follows:

Gra~ing(mg/cm2) = W, - Wo/Ao

Where W, is the weight of grafted sample, and W, and A0 are the original weight and area, respectively, of the original sample. Effects of synthesis conditions on grafting of MAA on to PVC films and the characterization of grafted PVC have been reported in our previous papers’**“. ATRIR, SEM and ESCA have shown that grafting is mainly restricted to the surface of the WC.

Optical observation

MAA grafted PVC films were stained in gentian violet solution for 1 h. Sections of 5pm were cut from stained samples using a microtome after fixing the sample in wax. These sections were mounted on microscope slides and observed under the optical microscope at X 100 magnifi- cation.

Measurement of contact angle of water

The contact angles of water on the surfaces of the various samples were measured. Samples were immersed in water for 24 h and excess water was wiped off with a piece of tissue paper. Swollen samples were placed immediatelyon a contact angle chamber in NRL Goniometer (Rame-hart Inc., USA) and a drop of water was placed on the top of the film with a mi~rosyringe. The angle between the water drop and the surface, called the contact angle against water, was measured at 28°C.

Thrombus fonnulation in vitro

Thrombus formation of the PVC and MAA grafted PVC samples was evaluated by an in vitro method developed by lmai and Nose” using ACD human blood. ACD blood was prepared as described by lmai and Nose**. Before the clot test, the films (3 X 3 cm) were hydrated to equilibrium in saline water (0.9% NaCI) and kept at 37°C in a constant- temperature water bath in Petri dishes. ACD human blood (0.2 ml) was added to each sample. Reaction was started by addition of 0.02 ml of MA 0 calcium chloride to each film. Blood and samples were mixed with a Teflon stick. At appropriate time intervals, distilled water (5 ml) was added to stop the reaction and thrombus formed was separated and soaked in water for 5 min at room temperature and then fixed in 36% formaldehyde solution (5 ml) for 5 min. Fixed clot was placed in water for 5 min and then blotted between tissue paper for 3 min and weighed.

Platelet adhesion

AC0 human blood was used for platelet adhesion studies: 0,3 ml of human blood was taken in glass test-tube (5 X 50 mm). Polymer sample (25 mg) was added to each tube and platelet count was taken at 15 min intervals for 75 min using a saline water-type haemocytometer (Webers model BS 784, UK).

Hasmolysis assay

Citrated human blood (0.2 ml) was added to the test samples (3 X 3 cm) that had been equilibrated in normal saline for 30 min at 37°C. After IO min 4 ml of 0.9% NaCl saline was added to each sample to stop haemolysis and these samples were incubate for 60 min at 37°C. Positive

and negative controls were produced by adding 0.2 ml of human blood to 4 ml of distilled water and 0.9% NaCl saline respectively. After 60 min incubation all samples were centrifuge, supernatant was taken and the optical density (OD) of the supernatant was measured at 545 nm. The percentage haemolysis was calculated as follows:

96 haemolysis = OD test sample - OD (-) control

OD (+) control - OD (-) control x 100

RESULTS AND DISCUSSION

Effects of conditions of grafting have been reported in an earlier paper18. Tab/e 1 shows the grafting level of various samples used in the present study.

Optical obse~ation

Micrographs of the optical section of the stained samples are shown in Figure 7. It is clear from the micrographs that grafting of MAA on WC has occurred mainly on the surface of the films. Grafting is mainly restricted to the surface as PVC does not swells in methanol-water solvent medium and therefore monomer does not diffuse into the bulk. Samples with higher grafting percentage tend to break in the dry state during section cutting by microtome (Figure Ic). This may be due to the increase in the brittleness of highly grafted WC in the dry state. However, all grafted PVC samples are elastic in nature on swelling in water.

Contact angle measurements

Contact angle of water droplet in air on various samples was examined to establish the relationship between the degree of grafting and the hydrophilicity. The contact angle of water is measured on the films which were equilibrated in water for 8-10 h. It is clear from the Figure 2 that contact angle of the grafted PVC is appreciably lower than that of the ungrafted PVC and the angle decreases with the increasing extent of grafting, due to the hydrophilici~ of grafted WC. These observations are in accordance with the data on the contact angle of water on other system such as the ~ly(ethylene vinyl acelate)-g-ac~lamide system*‘.

Thrombus formation

The weights of thrombus formed atdifferent time intervals in in vitro studies are shown in Table 2 (each value is an average

Table 1 Graft level of various PVC samples

Sample code Grafted WC sample Graft level (mg/cm’)

A WC-g-MU 0.599 B PVC-g-MA/% 1.080 c WC-g-Mu 1.630 D WC-g-MAA 2.630 E WC-g-MAA 3.100

Table 2 Weight of thrombus formed on various samples

Samples Weight of thrombus formed (mg)

5 min 1Omin 15min

WC 40.0 51.0 59.0 8 13.0 20.0 40.0

c 10.0 16.5 31.0

D 8.0 14.0 25.0 Glass 47.0 56.0 71.0

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Thrombogenicity of MAA grafted PVC films: J. Singh et al.

Figure 1 Micrographs of the optical observation X 100: (a) PVC-g-Ma graft level 1.08 mg/cm’; (b) PVC-g-MAR graft level 1.63 mg/cm’: (cl PVC-g-MAA

graft /eve/ 3.10 mg/cm2.

0 1 .o 2.0 3.0 4.0

Grafting (nqicm21

Figure 2 Dependence of contact angle against water on extent of grafiing.

of three experiments). The weight of the clot formed for a particular time is highest for glass followed by poly(vinyl chloride) and MAA grafted PVC. The clot formed increased with time for all samples. The thrombus formation on PVC surface decreases significantly after grafting with MAA due to the repulsion of negative charges present on MAAgrafted PVC samples as well as on blood components. It was also observed that clot formation decreases with increasing graft level on PVC, which might be due to the hydrophilici~ of the grafted PVC22-25.

Platelet adhesion

Figure 3 shows the results of platelet adhesion on various PVC films. Platelet adhesion to unmodified PVC surface is greater than that of the MAA grafted PVC. It was also observed that the percentage adhesion is lower in the grafted PVC. There is no charge on unmodified PVC while MAA grafted PVC film possesses negative charges on the surface. The repulsion between negatively charged platelets22 and MMA grafted PVC might explain the lower amount of platelet adhesion to MAA grafted PVC. The percentage adhesion of platelet increases as the time of exposure

increases in all the samples. The effect of degree of grafting on platelet adhesion is given in Figure 4. An attempt is also made to correlate the platelet adhesion and we~abili~ (contact angle) of various polymers and results are shown in Figure 5. It can be concluded from Figure 5 that platelet adhesion decreases with the decrease in contact angle.

Haemalysis assay Haemolytic activity of the PVC and MAA grafted PVC is performed with titrated human blood by measuring the optical density of free haemoglobin spectraphotometrically

1 1 I I I I I 1 I 0 10 20 30 40 50 60 70 80

Time (min)

Figure 3 Platelet adhesion versus rime for various samples: A, PVC; l , MAA grafted PVC wifh graft level of 1.63 mglcm’; 3, MAA grafted PVCwifh graft level of 2.63 mg/cm2.

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Throm&ogenic& of MAA graftad PVC films: J. Singh et al.

80

20

10

8 I i I

0 1.0 2.0 3.0 4.0

Crafting (mg/cm2)

figure 4 Effect of grafting on percentage platelet adhesion fur different time intervals: a, 15 min; X, 30 m/n; 0, 45 min; A, 60 min: 0. 75 min.

60 80 100

Contact angle (degrees)

Figure 5 Relationship between platelet adhesion and contact angle.

Table 3 Optical density of supematant of haemolysed blood for various samples

Sample Optical density at 545 nm Percentage haemolysis

Water 4.90 +Control Saline 0.01 -Control Glass 0.08 1.43 PVC 0.05 0.8 B 0.03 0.4 D 0.015 0.1

at 545 nm of supernatant of blood in contact with various samples (Table 3). A noticeable difference was observed in the haemolytic activity of the PVC and the grafted PVC while glass had more pronounced haemol~i~ activity.

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