Our existing biomaterials, although demonstrating generally satisfactory clinical performance, were developed based upon a trial-and-error optimization approach rather than being engineered to produce the desired interfacial reaction. Most biomaterials exhibit a nonspecific biological reaction, with sluggish kinetics and a broad spectrum of active processes simultaneously occurring. This article describes materials science nanotechnology, and molecular biology techniques that may permit the synthesis of precisely engineered surfaces. Such surfaces might demonstrate rapid, precise reactions with proteins and cells. This opens the question, "what type of specific surface bioreactions do we want?" New thoughts on biocompatibility are presented that may be helpful in the design of specific surfaces yielding precise, defined biological responses.

New ideas in biomaterials science--a path to engineered biomaterials.

Immobilization of Aspergillus oryzae β-galactosidase on tosylated cotton cloth

The invention relates to an apparatus useful for the separation of at least one preselected ligate present in a fluid. The invention also features an easily scaled-up membrane affinity separation process which is highly reliable, highly selective, provides a high yield of product, and is further characterized by a high volumetric throughput. The invention utilizes a substantially isotropic porous membrane, to which is associated a preselected ligand, which provides an optimum loading capacity and low dead volume while allowing high filtrate flow rates. Methods for isolating macromolecules having therapeutic value are described.

Membrane affinity apparatus and purification methods related thereto

The invention relates to methods for delivering polysaccharides by a pulmonary route to achieve local and systemic therapeutic effects. The polysaccharides may be formulated or unformulated and in some instances have an extremely fast absorption rate.

Methods and products related to pulmonary delivery of polysaccharides

Heparinase gene from flavobacterium heparinum

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 cellulose hollow fibers: I. Immobilization of heparinase

The ideal derivatized support for the clinical use of an immobilized enzyme system should irreversibly bind active enzyme. We have investigated the behavior of heparinase and bilirubin oxidase immobilized via cyanogen bromide, tresyl chloride, epoxide, or carbodiimidazole activated natural and synthetic matrices. The protein bound to each activated support was 90% for cyanogen bromide (CNBr) activated agarose, 50–80% for tresyl chloride activated agarose, and 50% for oxirane activated acrylic (Eupergit C). The activity retention of immobilized heparinase was greatest (50%) with CNBr activated agarose while for the immobilization of bilirubin oxidase, the activity retention was greatest (25–30%) with tresyl chloride activated agarose and oxirane activated acrylic.

The stability of the different covalent bonds was studied in vitro with radioiodinated enzymes. The leaching profiles showed the same trends for each support and chemistry. A plateau in portein leaching was reached after a few hours of incubatttion and the transient leaching period was well represented byu a logarithimic function of time. The amount of enzyme released from the least stable support (CNBr activated agarose) in 24 h was injected intravenously in New Zealand white rabbits. Using an indirect enzyme-linked immunnosorbant assay (ELISA), no immune responce was detected. The transient leaching profile was shortenend by washingthe enzyme-support conjugate with 1M hydroxylamine, pH8.5 intermolecular cross-linking with glutaraldehyde also improves the enzyme-support stability. Tresyl chloride and oxirane activated supports produce bonds with improved stability without adversely affecting enzymatic activity.

The influence of bond chemistry on immobilized enzyme systems for ex vivo use.

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.

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

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.

Immobilized enzyme cellulose hollow fibers: II. Kinetic analysis.

A method for the elimination of systemic heparin or low molecular weight derivatives of heparin wherein a blood filter containing an immobilized enzyme, including enzymes such as heparinase, glucuronate-reductase, aldose-reductase, beta-glucuronidase, O-sulfatase, and N-sulfatase, or other heparin degrading or neutralizing compound is constructed for use at the effluent of an extracorporeal device. The device meets specific stringent guidelines for clinical use. The first requirement is biocompatibility of the entire system. Secondly, the system is designed to remove clinical levels of heparin at variable flow rates, up to 4 liters blood/min for an adult to as low as 50 ml blood/min for an infant with perfusion times varying from hours up to between two and ten days for neonatal respiratory failure. Specific embodiments employing heparinase bound by tresyl chloride or CNBr techniques to reconstituted cellulose hollow fiber devices of the type used for kidney dialysis are disclosed. These include single or multistage devices suitable for heparin removal alone or in combination with dialysis or oxygenation. Also disclosed are analogous multiple membrane sheet devices.

Ann R. Comfort

Papers

Immobilized enzyme cellulose hollow fibers: I. Immobilization of heparinase

The influence of bond chemistry on immobilized enzyme systems for ex vivo use.

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

Immobilized enzyme cellulose hollow fibers: II. Kinetic analysis.

Neutralization of heparin.