Preparation and characterization of reactive and stable glucose oxidase‐containing liposomes modulated with detergent

Abstract

Glucose oxidase-containing liposomes (GOL) as well as detergent-modulated glucose oxidase-containing liposomes were prepared and characterized, focusing not only on the reactivity of the liposomes upon external addition of glucose but also on the leakage of the entrapped glucose oxidase (GO) from the liposomes with the aim of developing a reactive and stable liposomal GO system. The membranes of the GOL prepared were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and modulated with either Triton X-100 or cholate. In the absence of added detergent, no GO leakage from the GOL was observed while its enzymatic activity was very low (low glucose permeability). As detergent-modulated liposomes, mixed POPC/Triton X-100 and mixed POPC/cholate liposomes (abbreviated as TL and CL, respectively) were prepared at different effective detergent/POPC molar ratios (R(e)) ranging from R(e) = 0 to R(e) = R(e) (sat) (R(e) (sat) is the critical value of R(e) at which the liposome membrane is saturated with detergent). The reactivity of GO-loaded TL (abbreviated as GOTL) or GO-loaded CL (GOCL) increased drastically with increase in the respective detergent content in the liposomes. In the case of GOTL, at R(e) (sat) = 0.40, a high reactivity was measured with a simultaneous high extent of GO leakage, suggesting that the observed enzymatic reaction was catalyzed mainly by leaked GO, caused by the interaction of Triton X-100 with the POPC membrane. On the other hand, GOCL prepared at R(e) (sat) = 0.43 showed relatively high reactivity with only a small extent of GO leakage, suggesting that most of the enzyme reaction was limited by the glucose permeation across the bilayers of GOCL. The GO leakage from GOCL was found to occur mostly during the rearrangement of the liposomal membrane during the preparation of the GOCL (mixing the GOL and cholate). Fluorescence polarization measurements of membrane-associated DPH (1,6-diphenyl-1,3,5-hexatriene) indicated that CL prepared by modifying POPC with cholate did not lead to a drastic change in membrane fluidity, indicating that the interacting cholate molecules did not penetrate deeply into the POPC bilayers. In summary, it was clearly shown that the membrane permeability of GOL can be quite simply modulated by mixing it with a certain amount of cholate to form highly reactive and stable GOCL with minimal enzyme leakage.

Novel immobilized liposomal glucose oxidase system using the channel protein OmpF and catalase

“…The time course of hydrogen peroxide produced by the GO-catalyzed oxidation of glucose was measured by the horseradish peroxidase-catalyzed formation of oxidized o-dianisidine, as a measure of GO activity. The detailed procedure for the assay is described in our previous works Yoshimoto et al, 2003).…”

“…There are at least two methods to increase the permeability of the liposome membrane towards substrates. One is to modify the membrane with low concentrations of a micelleforming detergent (Treyer et al, 2002;Yoshimoto et al, 2003). But this method is not applicable to the above bioreactor because of foam formation induced by the free (not membrane-bound) detergent present in the bulk solution.…”

Enhancement of apparent substrate selectivity of proteinase K encapsulated in liposomes through a cholate‐induced alteration of the bilayer permeability

“…To verify and quantify the formation of mixed POPC/cholate liposomes, R e sat and K (described below) were determined by turbidity measurements as described before (Paternostre et al, 1988;Lichtenberg, 1996;Treyer et al, 2002;Yoshimoto et al, 2003) The enzymatic activity of proteinase K was measured immediately after mixing a proteinase K solution with a cholate or POPC/cholate mixed micelle solution, or with a POPC suspension. The POPC/cholate mixed liposomes were first equilibrated for 15 -17 h at room temperature before mixing the proteinase K solution.…”

“…It has, however, recently been reported that the reactivity of an enzyme inside liposomes can be controlled by modulating the lipid membranes with detergents such as sodium cholate and Triton X-100 to form lipid/detergent mixed liposomes with minimal enzyme leakage (Annesini et al, 1997;Treyer et al, 2002;Yoshimoto et al, 2003). In this way, the apparent substrate selectivity of an enzyme inside liposomes could be controlled through an alteration of the membrane permeability caused by the detergent molecules.…”

“…Particular attention was required to minimize the leakage of proteinase K from the cholate-treated liposomes by carefully controlling the detergent content in the liposomal membranes, since proteinase K, with its relatively small molecular mass (M r = 28,900) was expected to leak from the liposomes more quickly than the larger enzymes studied before, such as glucose oxidase (M r i 180,000, Yoshimoto et al, 2003), polynucleotide phosphorylase (M r i 230,000, Treyer et al, 2002), and h-galactosidase (M r i 520,000, Annesini et al, 1997).…”