Immobilized enzyme cellulose hollow fibers: III. Physical properties and in vitro biocompatibility

Abstract

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.

Heparinase I from Flavobacterium heparinum

“…Earlier studies suggested the importance of basic residues in heparinase I activity (Comfort et al, 1989;Leckband and Langer, 1991). Recently, we found that cysteine 135 of heparinase I is catalytically active .…”

Heparinase I from Flavobacterium heparinum: The Role of the Cysteine Residue in Catalysis as Probed by Chemical Modification and Site-Directed Mutagenesis

“…The effects of sulfhydryl modifications demonstrated that cysteine-reactive probes resulted in loss of enzymatic activity and the presence of a free cysteine (Leckband & Langer, 1991). In addition, earlier biochemical studies reported that heparinase I contained three to four cysteines (Yang et al, 1985) and a disulfide bond (Comfort et al, 1989). Taking all the above results together we set to address the number, the states, and the role of cysteines in heparinase I.…”

“…Heparinase I is believed to bind heparin through the lysine residues on the enzyme surface (Yang et al, 1985;Linhardt et al, 1982Linhardt et al, , 1990). The observation that modification by amine-reactive reagents and immobilization of heparinase I on amine-reactive supports result in extensive activity losses suggested the importance of lysines (Bernstein et al, 1988;Comfort et al, 1989;Leckband & Langer, 1991). In addition, evidence for an electrostatic nature of the interaction lies in the pH and ionic strength dependence of heparinase I activity (Yang et al, 1985).…”

“…In addition, evidence for an electrostatic nature of the interaction lies in the pH and ionic strength dependence of heparinase I activity (Yang et al, 1985). These observations, however, enzymatic activity, thus suggesting lysine involvement in enzyme activity (Comfort et al, 1989;Leckband & Langer, 1991). The effects of sulfhydryl modifications demonstrated that cysteine-reactive probes resulted in loss of enzymatic activity and the presence of a free cysteine (Leckband & Langer, 1991).…”

A Novel In Situ Self-Dissolving Needle Web Based on Medicated Cellulose Hollow Fibres with Drug Delivery Features

“…By the end of the 1980s, heparinase was immobilized within cellulosic hollow fibres to remove heparin's anticoagulant activity in blood filters [33][34][35]. Our fibres were endowed by simple capillary forces with two biotherapeutic model drugs that were not immobilized, but subsequently freeze-dried: The debriding enzyme Krillase ® , a protease of the antarctic krill (Euphasia superba) and antibiotic bacteriophages.…”