Synthesis and non-thrombogenicity of heparinoid polyurethaneureas

131

Synthesis and non-thrombogenicity of heparinoid polyurethaneureas Yoshihiro Ito, Yuichiro Iguchi and Yukio Imanishi Department of Polymer Chemistry, Faculty of Engineering, Kyoto University, Kyoto 606, Japan Unsaturated polyurethaneureas were synthesized from 4,4’-diphenylmethane diisocyanate, 1,2-diaminopropane and polybutadiene containing hydroxyl groups at both ends of a chain. The polyurethaneureas were cast as a film and subsequently treated with N-chlorosulphonyl isocyanate to produce sulphamate and carboxylate groups on the film surface. The level of in vitro non-thrombogenicity increased with increasing degree of modification. The high blood compatibility is attributed to the low level of platelet stimulation and to heparinoid activity. It was observed that platelet stimulation depends on the surface structure of the original polyurethaneurea, and that the heparinoid activity is related with the presence of sulphamate and carboxylate groups. Keywords:

Thrombogenicity,

polyurethanes,

platelets

Received 1 May 1990; revised 12 June 1990; accepted 28 October 1990

One of the most widely employed approaches in synthesizing blood compatible materials is heparinization, in which synthetic polymers are coated or immobilized with heparin, which is a natural polyanionic polysaccharidelm3. We have synthesized novel polyetherurethaneureas, which were ionically or covalently immobilized with heparin4-6. We have also investigated heparinized polyetherurethaneureas for in vitro antithrombogenicity, protein adsorption, platelet adhesion, fibroblast cell attachment and in viva antithrombogenicity4-‘3. These investigations have shown that heparin release is dispensable for the non-thrombogenicity of the materials, and that the heparinized polymers inhibit activation of the coagulation system and stimulation of platelets. Other researchers introduced sulphonate groups to synthetic polymers to enhance the blood compatibility of materials. For example, van der Does et al.14, Sederal et a1.15, Crassous et a1.16 and Gebelein and MurphyI modified unsaturated polymers with N-chlorosulphonyl isocyanate(CS1). Muzzarelli et al.*’ synthesized sulphated N-(carboxymethyl)chitosans as anticoagulants. Kammangne et al. lg and Mauzac and Jazefonvicz” investigated heparin-like activity of insoluble sulphonated polystyrene and dextran, respectively. Grasel and Cooper” and McCoy et aI.” evaluated the blood-contacting properties of polyurethanes grafted with varying amounts of propyl sulphonate groups and polyetherurethanes based on a sulphonic acid-containing diol. Ito et a1.23V24,however, reported the non-thrombogenicity of films which were glow discharged and graft copolymerized with sodium vinyl sulphonate. In the present study, the introduction of functional Correspondence

to Professor Y. Imanishi.

0 1992 Butterworth-Heinemann 0142-9612/92/030131-05

Ltd

groups found in heparin into polyurethaneureas was attempted to obtain non-thrombogenic polyurethaneureas. The reaction of polybutadiene with CSI, which was first investigated by van der Does et al.14 to produce blood-compatible polymers, was employed to produce sulphamate and carboxylate groups in unsaturated polyurethaneureas. It was expected that the surface of the modified polyurethaneureas would reduce platelet activation and suppress blood coagulation.

MATERIALS

AND METHODS

Materials Polybutadiene containing hydroxyl groups at both ends of a chain(Polybd, mol wt = 2800, trans-1,4:cis-1,4:vinyl1,2 = 80:20:20) was kindly presented by Idemitzu Petrochemical Industries. CSI (No. 14, 288-2) was purchased from Aldrich Chemical. Poly(tetramethylene glycol) (PTMG, mol wt = 1290), 4,4’-diphenylmethane diisocyanate(MD1) and 1,2_propanediamine(PDA) were purchased from Nacalai Tesque Co. Heparin isolated from porcine intestinal mucosa, which has an anticoagulant activity of 183.8 units mg-‘, was purchased from Nacalai Teague Co. Poly(oxyethylene) having a mol wt of 10 000 and poly(viny1 alcohol) having a mol wt of 20 000 were purchased from Wako Pure Chemical Industries. Methods Polymer synthesis Figure 1 shows the synthetic route to polyurethaneurea containing sulphamate and carboxylate groups on the film surface. Polybd(2.0 g) and MDI(0.42 g) were reacted Biomaterials

1992, Vol. 13 No. 3

132

Heparinoid Polybd

PolybdUU

N=C=O A02Cl

N-C=0

I iO,Cl

J H

H

I

I

NaOH

-CH2-c-c-cCH2-

I

HN

I

PolybdUU-Sl

,S2,S3

C=O

1 6Na S03Na Figure1 Synthetic route to create polyurethaneureas N-sulphamate and carboxylate groups(PolybdUU-S).

having

in 100 ml of dioxane for 1 h at 50°C to form a prepolymer. PDA(4.16 X lo-'g)was then added gradually at 10°C to extend the polymer chain. The solution was poured into methanol to precipitate the polymer. The polymer obtained was dried in vacuum for 24 h. This polymer is represented as PolybdUU. The infrared spectrum of PolybdUU showed absorptions at 1710 and 1650 cm-’ which were ascribed to urethane and urea bond, respectively. PolybdUU was cast from dioxane solution(l0 wt%) on a glass plate under irradiation with infrared lamp. The reaction of CSI with the polymer film was carried out by a similar method to that reported by van der Does et al.14. The polymer film was immersed in toluene solution of CSI(5, 10 and 30 ~01%) for 1 min at 25’C under nitrogen atmosphere. The film was then immersed in 0.1 N NaOH solution for neutralization, Finally, the film was washed with distilled water until the washing liquid became neutral. These modified polymers are represented as PolybdUU-Sl, S2 and S3, corresponding to the CSI concentrations in the modification reaction being 5, 10 and 30 ~01% respectively. A conventional polyethrurethaneurea was synthesized as reported previously49 ‘, The polymer is composed of PTMG, MD1 and PDA in a molar ratio 1:2:1, and is represented as PEUU. Polybd was also treated with CSI as reported by van der Does et a1.14 and Sederal et a1.l’. Polybd(2.0 g) was dissolved in dioxane (500 ml). Various amounts of CSI(5.0 ~01% dioxane solution) were added dropwise to Biomaterials

1992, Vol. 13 No. 3

Y. MO ef al.

the Polybd solution. The mixture was continuously stirred at 50°C under nitrogen atmosphere for 3 h. After cooling to room temperature, 0.5 N NaOH solution was added to the solution for neutralization. The modified Polybd was purified by dialysis and is represented by Polybd-Sl, S2 and S3, corresponding to the molar ratio of CSI to the double bond of Polybd in the modification reaction being 1, 3 and 10, respectively. The extent of modification of the polymer was calculated from the sulphur content of elemental analysis to be 5.6, 10.6 and 13.6 mol%, respectively. ATR-FTIR spectra for the surface analysis of polymer films were obtained on a Digilab FTS-15E/D apparatus (resolution, 6 cm-‘; integration, 200 times: detector, triglycine sulphate). A germanium prism was used, and the incident beam angle was 45”.

1,2-propanediamine

Polyurethaneurea

polyurethaneureas:

In vitro blood clotting test was carried out as reported previously4. Canine blood was collected by gravity through a 19-gauge scalp vein needle into a polypropylene bag containing one part of ACD solution for nine parts of blood. The ACD solution contains anhydrous Dglucose, sodium citrate dihydrate and citric acid monohydrate in distilled water. The ACD blood was placed on the polymer film, which was kept at 37°C in a constant temperature bath. The clotting reaction was started by adding 0.1 M aqueous CaCl, solution to the blood on the film. After 20 min, distilled water was added to stop the coagulation reaction. The thrombus formed was fixed with formaldehyde and dried. The weight of thrombus formed was determined. Four measurements were carried out for each sample and averaged. Platelet adhesion experiments were also carried out as reported previouslyg. Canine platelet-rich plasma (PRP) was prepared from the supernatant solution of the ACD blood after centrifugation at 700 rev min-’ for 10 min. Platelets in PRP were labelled by incubation with Nai’CrO, for 30 min at 37’C. Radioisotope-labelled PRP (200 ~1) was placed on the polymer film (diameter 1.6 cm) and incubated for 10 min at 37”C, after which the polymer film was washed with phosphate-buffered saline,pH7.4. The radioactivity of “Cr of the washed polymerwas counted using an Alloka JDC-751 automatic scintillation counter. Activated partial thromboplastin time (APTT) was measured as follows. Platelet-poor plasma (PPP) was prepared from the supernatant solution of the canine ACD blood after centrifugation at 3000 rev min-’ for 15 min. Keeping the temperature at 37’C, PPP was placed in a test tube. Soluble polymer or insoluble polymer film and cephalin were then added into the PPP. After adding 0.01 M CaCl, solution to the mixture, a needle was put in it and moved once for 1 s. The time needed for the formation of fibrous product at the top of the needle from the CaCI, addition was measured.

RESULTS Polymer modification The infrared spectra of Polybd-S3 and PolybdUU-S3 show absorptions at 1300 and 1050 cm-l which were assigned to the asymmetric and symmetric vibration of

133

Y. Ifo et al.

Heparinoid polyurethaneureas:

0= S= 0, respectively. These absorptions are not seen in the spectra of Polyhd and PolybdUU, as shown in Figure 2. To investigate the effect of CSI on urethane and urea linkages, PEUU was treated with dioxane solution of CSI(30 vol%), However, the ATR-FTIR spectrum of the

polymer film had not changed. These observations confirm that only the reaction product of C=C and CSI are produced on the surface of PolybdUU-S films. The elemental analysis showed an absence of sulphur in the treated PEUU. The chemical modification of PolybdUU and Polybd could be controlled, as shown in X&es 2 and 2, respectively. The degree of modification increased with increasing CSI concentrations.

/

Non-thromhogenicity

Results of a blood-clotting test on PolybdUU-S, which contains different amounts of sulphamate and carboxylate groups, are shown in Z&Me1. Th~mbus formation on the polymers was less than that on the glass. The nonthrombogenicity of PolybdUU was slightly lower than that of PEUU. However, the non-thrombogenicity of the PolybdUU-S was enhanced, with increasing content of functional groups found in heparin. Platelet adhesion on the polymer films was more suppressed than that on glass, as shown in Z’We I. However, the level of platelet adhesion on PolybdUU-S was nearly the same as that on PEUU, and almost independent of the quantity of functional groups introduced. This observation indicates that either the sulphamate or carboxylate group does not influence the platelet adhesion. APTT of PolybdUU-S was definitely prolonged, with increasing amounts of functional groups introduced. This elongation of APTT was also observed in the case of water-soluble Polybd-S, as shown in Figure 3. The APTT of the Polybd-S increased linearly with concentration. The slopes of the straight lines illustrate the anticoagulant activities and are summa~zed in Table 2. Although poly(oxyethylene) and poly(viny1 alcohol) are ineffective in anticoagulant activity, PolybdS have some activity. The non-thrombogenic activity of Polybd-S3 reached one twenty-third that of heparin.

\

Potybd

\;;~

of materials

Polybd-S3

DISCUSSION

1

I

S

10 3#0

1

I!

1

2800 2200

i

I

VC?O

t

I

1300

t@tllJ

900

400

Wave number (cm-‘) Figure2 Transmission infrared (IR) spectra of Polybd and Polybd-S3, and ATR IR spectra of PolybdUU and PolybdUUs3. Table 1

The reaction of C = C with CSI was applied to synthesize heparinoid polymers14-‘7. However, unsaturated polymers have been confined to polybutadiene, polyisoprene and their block copolymers with polystyrene. In the present investigation, the same method was applied to unsaturated polyurethaneureas with successful results in synthesizing heparinoid polymers. The heparinoid polyurethaneureas synthesized, PolybdUU-S, had a high non-thrombogenicity as the

Synthesis and antithrombogenici~ of polyurethanes containing sulphamate and carboxylate groups

Materials

PolybdUU PolybdUU-St PolybdUU-SP PolybdUU-S3 PEUU Glass

CSI concentration (vol%)

Sulphur content (wt%)

Thrombus formeda (%)

Platelet adhesionb (%)

APTT (s)

0 0.24 0.82 1.64

84 83 64 48 75 100

87 76 80 74 77 100

104 IL 4 1i9t3 130f4 164t3 110+3 60 f 5

+ 3 I!I 5 + 4 f 3 + 5 + 3

ztr2 + 5 +- 5 f 5 Ifi 5 rt 4

‘The weight of thrombus formed on the glass is taken as 100%. bThe number of platelets adhered on the glass is taken as 100% --Biomaterials

1992,

Vol.

13 No. 3

134 Table 2

Heparinoid Properties

of modified Molar ratio CSI/C=C

Polymers

Polybd-Sl : Polybd-S2 Polybd-S3 10 Heparin Poly(oxyethylene) Poly(vinyl alcohol)-

polybutadiene Sulphur content (wt%)

AnticoaQuiant activity (units/mg)

0.95 1.74 2.13

1.5 2.2 8.1 183.8 0.1 0.1

REFERENCES 1 2

4

5

6

7

8

9

I

0.5

1.0

Concentration

1.5

10

2.0

{g/l) 11

Figure3 Activated partial thromboplastin time (APTT) of Polybd-S. 0, Polybd-Sl (sulphur content, O.Q5wt%); 0, Polybd-S2 (sulphur content, 1.7 wt%); A, Polybd-S3 (sulphur content, 2.13 wt%).

result of reduced adhesion of platelets and anticoagulant activity. The reduction of platelet adhesion could be attributed to the surface morphology of the polyurethaneurea film”, because the extent of chemical modification did not strongly affect the platelet adhesion. Grafting with poly(sodium vinyl sulphonate) did not reduce platelet adhesion with increasing quantityz3* 24. The inhibition of thrombin activity assessed by APTT could have arisen from the heparinoid structure of the film surface. That soluble Polybd-S behaved similarly supports strongly that consideration. Heparin release has been considered indispensable for non-thrombogenicity of heparin-adsorbed polymers. However, this and our previous work’ demonstrated that it is not always necessary. That non-thrombogenicity is extended by covalently immobilized heparin, and even by sulphonate and carboxylate groups on the surface of Biomate~als

1992, Vol. 13 No. 3

Y. Ito et al.

an insoluble film, will be a useful clue for the development of novel non-thrombogenic materials.

3

0

polyurethaneureas:

12

13

14

15

16

17

18

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in respect

chronically AV-shunted canines, J. Biomed. Mater. Res. 1990, 24,107-129 Ito, Y., Iguchi, Y., Kashiwagi, T. and Imanishi, Y., Synthesis and nonthrombogenicity of polyetherurethaneurea film grafted with poly(sodium vinyl sulphonate), J. Biomed. Mater. Res. 1991, 25, 1349-1361 Ito, Y., Liu, L.S. and Imanishi, Y., Interactions of poly(sodium vinyl sulphonate) and its surface graft with anti-thrombin III, J. Biomed. Mater. Res. 1991, 25, 99-115 Lyman, D.J., Knutson, K., McNeil, B. and Shibatani, K., The effects of chemical structure and surface properties of synthetic polymers on the coagulation of blood. IV. The relationship between polymer morphology and protein adsorption, 7kans. ASAZO 1975, 21, 49-54

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1992, Vol. 13 No. 3