Heparinized Styrene-Butadiene-Styrene Elastomers MATTHEUS F. A. GOOSEN AND MICHAEL V. SEFTON,* Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, M5S IA4 Summary A heparinized high-strength elastomer has been developed which is potentially useful as a nonthrombogenic vascular prosthesis. A surface hydroxylated styrene-butadiene-styrene (SBS) block copolymer with a t least 40% extent of reaction after glowdischarge cleaning was coated with a 2090 acetylated polyvinyl alcoholheparin mixture containing glutaraldehyde and magnesium chloride. After curing a t 80°C for 100 min, the polyvinyl alcohol, heparin, and hydroxylated SBS were covalently bound to each other by acetal bridges. The effects of the various substrate and coating parameters were optimized to achieve very strong adhesion between the coating layer and the surface hydroxylated SBS. Heparin was not leached from the surface of the new pg heparin/cm* material using 3M saline a t p H 7.4 despite a detection limit of min. Prolonged partial thromboplastin times of greater than 1200sec were observed (control: PTT = 120 sec). Preliminary ex uiua testing using a simple arteriovenous shunt in the leg of a rabbit showed good thromboresistance. The heparinized SBS shunt chamber remained patent for more than two hours without desorption of heparin. It was concluded that surface hydroxylated SBS heparinized by acetal coupling owed its thromboresistance to the heparin covalently bound to the surface and not to a microenvironment of heparin in solution a t the blood/material interface.
INTRODUCTION Over the past several years many implantable devices have been devised which aid the circulation of blood in diseased patients. One of the simplest of these is the vascular prosthesis, designed to act as a replacement or bypass for arteries or veins which have become occluded. While Dacron velour is most commonly used for these prostheses, the use of a velour limits the use of the prosthesis to those portions of the circulatory system ( principally the larger arteries) where the blood-flow rate is sufficiently fast to prevent buildup of thrombin to levels adequate to achieve fibrin formation. * To whom correspondence should be addressed.
Journal of Biomedical Materials Research, Vol. 13, 347-364 (1979) 01979John Wiley & Sons, Inc. 0021-9304/79/0013-0347$01.00
GOOSEN AND SEFTON
:148
As another example, chronic access to the bloodstream is one of the unsolved problems associated with the use of artificial organs, such as hemodialysers and the artificial endocrine pancreas. Clotting and thrombophlebitis around the implanted catheter are frequent complications. Much effort has been devoted to the development of nonthrombogenic materials, suitable for these prostheses, which would significantly extend the applicability of vascular replacement surgery. Expanded polytetrafluoroethylene,l ficin-digested bovine heterografts,2 segmented polyurethanes by themselves3 or as blends with silicone rubber? surface grafted hydrogels5or surfaces with microfiber scaffolds6 have all been developed largely for this purpose. Alternatively, heparin may be bound to suitably prepared substrates to produce thromboresistant materials. This approach has been used here to develop a high-strength elastomer with potential long-term thromboresistance. The material was prepared by covalent coupling of heparin by an acetal bond to the hydroxylated surface of a styrene-butadiene-styreneblock copolymer (SBS). Styrene-Butadiene-StyreneBlock Copolymers The unique properties of the styrene-butadiene-styreneblock copolymers make them particularly useful as biomaterial~.~ The block structure,
8
is produced by a “living polymer” anionic polymerization technique. This results in an ultrapure polymer containing no potentially toxic, elutable contaminants. The SBS copolymer containing 25 wt. % polystyrene satisfies the mechanical requirements of a vascular prosthesis, without further chemical modification. The polystyrene and polybutadiene blocks are thermodynamically incompatible and therefore there is a phase separation in the solid phase, with the formation, in a SBS triblock copolymer containing 25% polystyrene, of glassy domains of polystyrene dispersed in a continuum of the rubbery polybutadiene.
STYRENE-BUTADIENE-STYRENE ELASTOMERS
349
Besides keeping the polybutadiene entanglements in place, the polystyrene domains act as particles of reinforcing filler to give the copolymer, at 37”C,the properties of a high-strength elastomer (initial modulus, 650 psi; tensile strength, 3500 psi).8 Nevertheless, SBS is still a thermoplastic. By raising the temperature above the glass transition temperature of polystyrene (-lOOOC), the elastomer can be easily extruded or molded into any desired shape or form. Polybutadiene is apparently stable to biological degradation and is only subject to oxidative degradation in the presence of ultraviolet light or ozone, neither of which are present inside the body.
Heparinization Numerous methods have been used to couple heparin, ionically or covalently, to polymer surfaces. Ionic bonding, whether by direct quaternization of suitable functional groups on the polymer surfacegJO by the addition of quaternary ammonium-heparin c ~ m p l e x e s l ’ -or ~~ by the copolymerization of cationic or tertiary amine m o n o m e r ~ ~ ~ J ~ results in materials with short-term compatibility. Ion exchange of the heparin with blood components renders this approach unsuitable for long-term implants. Covalent coupling,16J7on the other hand, has typically resulted in the “denaturation” of the heparin, resulting in only very limited thromboresistance. However, the covalent coupling of heparin to a polyvinyl alcohol through an acetal bridge has resulted in materials which retain long-term blood compatibility in arteriovenous shunts in Similar thromboresistance has been suggested for cyanogen bromide bonded heparin, although this observation may be complicated by the continuous loss of heparin from these surfaces.20,21 To make the surface of SBS thromboresistant, heparin was coupled to the surface using a modified form of the acetal bridge technique of Merrill and Wong.lg To provide reactive sites for the bonding of heparin, the surface was hydroxylated by reaction with peracetic acid (Fig. 1). By limiting the reaction to the material surface, the desirable bulk properties of the SBS elastomer were retained. The technique is similar to that used by Sefton and M e ~ ~ i l l . ~ A technique was developed whereby films of surface hydroxylated styrene-butadiene-styrenewere covered with a thin heparin-polyvinyl alcohol (PVA) coating. The hydroxyl groups on the SBS surface were bound to those of heparin and the PVA by reaction with a mixture
GOOSEN AND SEFTON
:350 - CH
= CH -
peracetic acid +
-HC-CH\
ALKENE
/
0
EPOXIDE
- CH - CH I I
HO
OH
GLYCOL
- CH I
HO
-
CH I
OOCCH3
HYDROXYACETATE
Fig. 1. Hydroxylation reaction scheme. Peracetic acid reacts wit,h the residual unsaturation in the polybutadiene block to produce epoxide rings. The epoxide rings are cleaved by acid catalysis to produce a mixture of 1,2 glycol and hydroxylacetate addition products. The secondary hydroxyls are active sites for the acetal coupling of heparin.
of aldehydes (Fig. 2). The PVA ensures an adequate number of hydroxyl groups available for binding to heparin and acts as an intermediate between the heparin and the SBS surface.
MATERIALS AND METHODS Experimental work was directed towards obtaining strong adhesion between the thin PVA-heparin coating and the surface hydroxylated elastomer. Furthermore, the stability of the bound heparin and the amount of heparin remaining in the film after washing was determined. Biological tests were then performed on the films to give an indication of their blood compatibility.
and others
H
R = H or ICH 214-
C
=0
Fig. 2. Acetal coupling reaction. The mono- and dialdehydes react with the hydroxyls of heparin, polyvinyl alcohol and surface hydroxylated SBS to produce acetal bonds among these three species.
STYRENE-BUTADIENE-STYRENE ELASTOMERS
351
Surface Hydroxylation Films of SBS TR-41-2443 (research-grade styrene-butadienestyrene block copolymer, Kranton, Shell Chemical Co., Houston, Texas; see Table I) were suspended in a flask in a reaction medium composed of peracetic acid, sulfuric acid, acetic acid and water, all maintained at a fixed temperature (45°C). The technique was similar to that used by Sefton and Merrill.7 After sufficient time the films were removed from the bath and washed free of acid overnight in distilled water. The films were not further hydrolyzed in 2N KOH. Delamination in chloroform was used to determine the extent of reaction. Unreacted SBS dissolves in chloroform while SBS-OH does not; the percent reaction refers to the fraction of the film which does not dissolve in chloroform. Extent of reaction was varied by varying the length of time from 50 to 200 min that the 200-pm-thick films were in the reaction bath.
Heparinization The films were subject to glow-discharge or ultrasonic cleaning prior to adhesive coating. For glow-discharge cleaning the surface hydroxylated films (SBS-OH) were dried under vacuum overnight and then cleaned for 10 min a t 500 X Torr in a radio frequency glow-discharge apparatus (Plasma Cleaner, Harrick Scientific, Ossining, N.Y.). TABLE I Characterization of SBS TR-41-244322
Block Molecular Weight 16,000-85,000-17,000 Composition Polystyrene 27.7 wt. % (25.1 vol. %) Triblock 100% Polybutadiene Microstructure cis 1,4 trans 1.4 1,2
40% 49% 11%
352
GOOSEN AND SEFTON
The composition of a typical coating solution is shown in Table 11. Sodium heparin without further purification (153 U/mg, Canada Packers Ltd., Toronto) and "S-heparin (94 mCilg, Amersham Corp., Oakville, Ontario) were added as required. Completely deacetylated polyvinyl alcohol (Elvanol71-30, DuPont of Canada, Toronto) or 20% acetylated PVA (Gelvatol 20-60, Monsanto Canada Ltd., Toronto) was used to evaluate the effect of coating surface tension on adhesive strength. After cleaning, the SBS-OH films were dipped in this coating solution for 5 min. A 15-min drying time was allowed between coats. For subsequent coats the films were dipped for only a few seconds. The films were allowed to dry for 3 hr in the fume hood before being reacted a t 80°C for 100 min in an oven.
Adhesive Strength The qualitative adhesive strength was determined by scratching the surface of the coated film with a pair of fine tweezers and observing how difficult it was to peel back the coating. Substrate effects, surface cleaning, and composition of the coating solution were evaluated to provide for maximum adhesive strength between the heparin-polyvinyl alcohol coating and the surface hydroxylated SBS-OH.
Heparin Stability After coating and curing the heparinized films were washed with saline in a flow circuit a t a nominal Reynolds number of 3300. The films were washed for 160 hr. Isotonic saline was used for the first 24 hr; 3M saline was used for the duration. The amounts of 35STABLE I1 Typical Adhesive Coating Solution wt. Yo ~~
Polyvinyl Alcohol Formaldehyde Glutaraldehyde Glycerol MgCly6Hz0 Sodium Heparin Water
~~
10 2.2 0.25 4.0 5.0 0.05-2.0 (to make 100%)
STYRENE-BUTADIENE-STYRENE ELASTOMERS
353
heparin leached from the films was determined using the Beckman LS 8000 liquid scintillation spectrometer and Aquasol cocktail (New England Nuclear). The detection limit of the analytical procedure Fg/ml of 35S-heparin. From the amount of 35Swas
INTRODUCTION Over the past several years many implantable devices have been devised which aid the circulation of blood in diseased patients. One of the simplest of these is the vascular prosthesis, designed to act as a replacement or bypass for arteries or veins which have become occluded. While Dacron velour is most commonly used for these prostheses, the use of a velour limits the use of the prosthesis to those portions of the circulatory system ( principally the larger arteries) where the blood-flow rate is sufficiently fast to prevent buildup of thrombin to levels adequate to achieve fibrin formation. * To whom correspondence should be addressed.
Journal of Biomedical Materials Research, Vol. 13, 347-364 (1979) 01979John Wiley & Sons, Inc. 0021-9304/79/0013-0347$01.00
GOOSEN AND SEFTON
:148
As another example, chronic access to the bloodstream is one of the unsolved problems associated with the use of artificial organs, such as hemodialysers and the artificial endocrine pancreas. Clotting and thrombophlebitis around the implanted catheter are frequent complications. Much effort has been devoted to the development of nonthrombogenic materials, suitable for these prostheses, which would significantly extend the applicability of vascular replacement surgery. Expanded polytetrafluoroethylene,l ficin-digested bovine heterografts,2 segmented polyurethanes by themselves3 or as blends with silicone rubber? surface grafted hydrogels5or surfaces with microfiber scaffolds6 have all been developed largely for this purpose. Alternatively, heparin may be bound to suitably prepared substrates to produce thromboresistant materials. This approach has been used here to develop a high-strength elastomer with potential long-term thromboresistance. The material was prepared by covalent coupling of heparin by an acetal bond to the hydroxylated surface of a styrene-butadiene-styreneblock copolymer (SBS). Styrene-Butadiene-StyreneBlock Copolymers The unique properties of the styrene-butadiene-styreneblock copolymers make them particularly useful as biomaterial~.~ The block structure,
8
is produced by a “living polymer” anionic polymerization technique. This results in an ultrapure polymer containing no potentially toxic, elutable contaminants. The SBS copolymer containing 25 wt. % polystyrene satisfies the mechanical requirements of a vascular prosthesis, without further chemical modification. The polystyrene and polybutadiene blocks are thermodynamically incompatible and therefore there is a phase separation in the solid phase, with the formation, in a SBS triblock copolymer containing 25% polystyrene, of glassy domains of polystyrene dispersed in a continuum of the rubbery polybutadiene.
STYRENE-BUTADIENE-STYRENE ELASTOMERS
349
Besides keeping the polybutadiene entanglements in place, the polystyrene domains act as particles of reinforcing filler to give the copolymer, at 37”C,the properties of a high-strength elastomer (initial modulus, 650 psi; tensile strength, 3500 psi).8 Nevertheless, SBS is still a thermoplastic. By raising the temperature above the glass transition temperature of polystyrene (-lOOOC), the elastomer can be easily extruded or molded into any desired shape or form. Polybutadiene is apparently stable to biological degradation and is only subject to oxidative degradation in the presence of ultraviolet light or ozone, neither of which are present inside the body.
Heparinization Numerous methods have been used to couple heparin, ionically or covalently, to polymer surfaces. Ionic bonding, whether by direct quaternization of suitable functional groups on the polymer surfacegJO by the addition of quaternary ammonium-heparin c ~ m p l e x e s l ’ -or ~~ by the copolymerization of cationic or tertiary amine m o n o m e r ~ ~ ~ J ~ results in materials with short-term compatibility. Ion exchange of the heparin with blood components renders this approach unsuitable for long-term implants. Covalent coupling,16J7on the other hand, has typically resulted in the “denaturation” of the heparin, resulting in only very limited thromboresistance. However, the covalent coupling of heparin to a polyvinyl alcohol through an acetal bridge has resulted in materials which retain long-term blood compatibility in arteriovenous shunts in Similar thromboresistance has been suggested for cyanogen bromide bonded heparin, although this observation may be complicated by the continuous loss of heparin from these surfaces.20,21 To make the surface of SBS thromboresistant, heparin was coupled to the surface using a modified form of the acetal bridge technique of Merrill and Wong.lg To provide reactive sites for the bonding of heparin, the surface was hydroxylated by reaction with peracetic acid (Fig. 1). By limiting the reaction to the material surface, the desirable bulk properties of the SBS elastomer were retained. The technique is similar to that used by Sefton and M e ~ ~ i l l . ~ A technique was developed whereby films of surface hydroxylated styrene-butadiene-styrenewere covered with a thin heparin-polyvinyl alcohol (PVA) coating. The hydroxyl groups on the SBS surface were bound to those of heparin and the PVA by reaction with a mixture
GOOSEN AND SEFTON
:350 - CH
= CH -
peracetic acid +
-HC-CH\
ALKENE
/
0
EPOXIDE
- CH - CH I I
HO
OH
GLYCOL
- CH I
HO
-
CH I
OOCCH3
HYDROXYACETATE
Fig. 1. Hydroxylation reaction scheme. Peracetic acid reacts wit,h the residual unsaturation in the polybutadiene block to produce epoxide rings. The epoxide rings are cleaved by acid catalysis to produce a mixture of 1,2 glycol and hydroxylacetate addition products. The secondary hydroxyls are active sites for the acetal coupling of heparin.
of aldehydes (Fig. 2). The PVA ensures an adequate number of hydroxyl groups available for binding to heparin and acts as an intermediate between the heparin and the SBS surface.
MATERIALS AND METHODS Experimental work was directed towards obtaining strong adhesion between the thin PVA-heparin coating and the surface hydroxylated elastomer. Furthermore, the stability of the bound heparin and the amount of heparin remaining in the film after washing was determined. Biological tests were then performed on the films to give an indication of their blood compatibility.
and others
H
R = H or ICH 214-
C
=0
Fig. 2. Acetal coupling reaction. The mono- and dialdehydes react with the hydroxyls of heparin, polyvinyl alcohol and surface hydroxylated SBS to produce acetal bonds among these three species.
STYRENE-BUTADIENE-STYRENE ELASTOMERS
351
Surface Hydroxylation Films of SBS TR-41-2443 (research-grade styrene-butadienestyrene block copolymer, Kranton, Shell Chemical Co., Houston, Texas; see Table I) were suspended in a flask in a reaction medium composed of peracetic acid, sulfuric acid, acetic acid and water, all maintained at a fixed temperature (45°C). The technique was similar to that used by Sefton and Merrill.7 After sufficient time the films were removed from the bath and washed free of acid overnight in distilled water. The films were not further hydrolyzed in 2N KOH. Delamination in chloroform was used to determine the extent of reaction. Unreacted SBS dissolves in chloroform while SBS-OH does not; the percent reaction refers to the fraction of the film which does not dissolve in chloroform. Extent of reaction was varied by varying the length of time from 50 to 200 min that the 200-pm-thick films were in the reaction bath.
Heparinization The films were subject to glow-discharge or ultrasonic cleaning prior to adhesive coating. For glow-discharge cleaning the surface hydroxylated films (SBS-OH) were dried under vacuum overnight and then cleaned for 10 min a t 500 X Torr in a radio frequency glow-discharge apparatus (Plasma Cleaner, Harrick Scientific, Ossining, N.Y.). TABLE I Characterization of SBS TR-41-244322
Block Molecular Weight 16,000-85,000-17,000 Composition Polystyrene 27.7 wt. % (25.1 vol. %) Triblock 100% Polybutadiene Microstructure cis 1,4 trans 1.4 1,2
40% 49% 11%
352
GOOSEN AND SEFTON
The composition of a typical coating solution is shown in Table 11. Sodium heparin without further purification (153 U/mg, Canada Packers Ltd., Toronto) and "S-heparin (94 mCilg, Amersham Corp., Oakville, Ontario) were added as required. Completely deacetylated polyvinyl alcohol (Elvanol71-30, DuPont of Canada, Toronto) or 20% acetylated PVA (Gelvatol 20-60, Monsanto Canada Ltd., Toronto) was used to evaluate the effect of coating surface tension on adhesive strength. After cleaning, the SBS-OH films were dipped in this coating solution for 5 min. A 15-min drying time was allowed between coats. For subsequent coats the films were dipped for only a few seconds. The films were allowed to dry for 3 hr in the fume hood before being reacted a t 80°C for 100 min in an oven.
Adhesive Strength The qualitative adhesive strength was determined by scratching the surface of the coated film with a pair of fine tweezers and observing how difficult it was to peel back the coating. Substrate effects, surface cleaning, and composition of the coating solution were evaluated to provide for maximum adhesive strength between the heparin-polyvinyl alcohol coating and the surface hydroxylated SBS-OH.
Heparin Stability After coating and curing the heparinized films were washed with saline in a flow circuit a t a nominal Reynolds number of 3300. The films were washed for 160 hr. Isotonic saline was used for the first 24 hr; 3M saline was used for the duration. The amounts of 35STABLE I1 Typical Adhesive Coating Solution wt. Yo ~~
Polyvinyl Alcohol Formaldehyde Glutaraldehyde Glycerol MgCly6Hz0 Sodium Heparin Water
~~
10 2.2 0.25 4.0 5.0 0.05-2.0 (to make 100%)
STYRENE-BUTADIENE-STYRENE ELASTOMERS
353
heparin leached from the films was determined using the Beckman LS 8000 liquid scintillation spectrometer and Aquasol cocktail (New England Nuclear). The detection limit of the analytical procedure Fg/ml of 35S-heparin. From the amount of 35Swas
