Collagenase Immobilized on Cellulose Acetate Membranes

Abstract

Collagenase covalently attached to cellulose acetate membranes, following periodate activation of the membrane, maintained its enzymatic activity and permanence of attachment for more than 6 weeks. The length of spacer between the enzyme and the membrane was studied with enzymes attached directly, through ethylenediamine and through ethylenediamine plus succinnic anhydride. Enzyme activity was measured by determining the degradation rate of a pigskin gelatin solution, and stability of the bond was measured by observing continued degradation after the enzyme-membrane was removed from the solution. The longest spacer resulted in the highest enzyme activity. I nterest is growing rapidly in supplying blood glucose control in diabetics through the use of chambers in which Islets of Langerhans are interfaced with the blood supply through a permeable membrane. The object is to provide adequate insulin release in response to blood glucose, while isolating the cells from immune response. Consideration is being given to both intravascular and extravascular islet-transplantation chambers. A problem that arises in the consideration of an extravascular device is that synthetic materials placed in the body tissues rapidly become covered with cells (polymorphonuclear leukocytes and macrophages) and surrounded with relatively impervious fibrous tissue. This could decrease the diffusion rates of glucose and insulin, eventually rendering a chamber useless. One possible solution to this problem is the attachment of proteolytic enzymes to the surface of the chamber to prevent formation and attachment of fibrous tissue at the interface. It was reported by Jolley (1 ) that a cellulosic chamber (Millipore Corporation) coated with covalently bound collagenase and implanted in the rat

Islet immuno‐isolation: The use of hybrid artificial organs to prevent islet tissue rejection

“…In an attempt to reduce the host fibroblastic response, Jolly et al [24] described transient success with dog islets in rats using membranes coated with proteases. Our group modified Jolly's technique and bound collagenase to the outside of the membranes of our diffusion chambers [25]. While we could demonstrate the successful binding of collagenase to the surface of these membranes, we still observed a host fibroblastic response after implantation into rats.…”

Covalent Enzyme Immobilization

“…In line with this idea, collagenase was immobilized on oxidized cellulose acetate membranes, via a spacer, with high retention of activity, after derivatization with a diaminoalkane [466]. In contrast, the oxidized enzyme carrying aldehyde groups can also be immobilized on amino carriers.…”

“…Aldehyde can be converted to carboxyl groups by reaction with ethylenediamine and, subsequently, with succinic acid anhydride to introduce a spacer [466].…”

“…Although some active carrier functionality, for example the epoxy group in Eupergit C [57,148,468], the aldehyde [466] group in oxidized dextran, or isourea functionality of cyanogen bromide activated polysaccharide [377] can be used for direct binding of enzyme molecules, direct binding of enzymes to these active carriers often leads to undesired results, for example poor retention of activity, lower stability, or poor selectivity, for reasons which include unmatched surface chemistry, steric hindrance, unstable linkage, inappropriate microenvironment, and, particularly, modification of the essential amino acid residues involved in the active centre -for example modification of the active centre of pectin esterase on Enzacryl AA (diazotization) led to complete loss of activity [467].…”