The initial testing of the safety of a cellulose-heparinase hollow fiber device was assessed with respect to physical properties and in vitro biocompatibility. The material cleared urea and creatinine without passing albumin, even at high flow rates. The clearance of urea and creatinine by cellulose-heparinase was equal or slightly reduced in comparision to the cellulose device. The cellulose-neparinase device tolerance to now rates was also unchanged. In addition, scanning electron microscopy of the lumen established the uniformity of the material. The analysis of clearance rates and the scanning electron micrographs show there to be no damage to the cellulose membrane after tresyl chloride activation and heparinase immobilization. The investigation of biocompatibility in an in vitro test system with whole human blood indicated that there were no significant changes in the biocompatibility of cellulose with bound heparinase. There was no change in the level of red blood cells, white blood cells, or platelets over the course of in vitro whole blood perfusion through cellulose or cellulose-heparinase hollow fiber devices. Low levels of plasma hemoglobin and complement activation were observed with cellulose and cellulose-heparinase devices. Thus, the cellulose hollow fibers can be functionalized without any changes in in vitro performance.
The immobilization of heparinase to tresyl-chloride-activated cellulose hollow fibers for the removal of heparin from the bloodstream was examined. Whole blood can be circulated through cellulose hollow fibers without hemolysis and the tresyl chloride chemistry provides a strong linkage which limits the release of the enzyme from the support. The tresylation and immobilization methods were modified and optimized to improve the heparinase activity retained by cellulose. Pretreatment of the hollow fibers with 0.05/V sodium hydroxide increased the degree of tresylation and the immobilization yield by a factor of five. The use of triethylamine as the organic base in the tresyl chloride activation resulted in threefold greater activity retention by the support than when pyridine was used. Together, sodium hydroxide pretreatment and triethylamine enhanced the activity retained by cellulose to 26.2 +/- 7.0% of that bound to the support. The activity retention was also a function of the technique used for immobilization. The best results were achieved when the enzyme was applied to the activated fibers once every 12 to 24 h for a total of four times. The active enzyme loading on the fibers was 0.3 mg heparin degraded/h cm(2) when 4.5 microg protein/cm(2) was bound to the fibers.
Immobilized enzyme hollow fibers may be useful in the purification or treatment of whole blood under clinical conditions. In this study, catalytically pure heparinase was immobilized to cellulose to analyze the feasibility for the removal of heparin's anticoagulant activity from whole blood. The kinetics of catalytically pure heparinase immobilized to regenerated cellulose hollow fibers were quantified with respect to mass transfer coefficient and enzyme loading. The kinetic analysis showed that increases in the mass transfer coefficient of heparin in the fiber lumen decreased the apparent Michaelis constant while increases in enzyme activity immobilized to the fiber lumen increased the apparent Michaelis constant. The apparent Michaelis constant was an order of magnitude greater than the intrinsic K(m) value for the system. The intrinsic K(m) value for heparinase-cellulose is 0.4 +/- 0.3 mg/mL (N = 6) and it is the same order of magnitude as the K(m) value for soluble heparinase.
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