Liposome-encapsulated enzyme

Kaszuba, M. and Jones, M. N. (1999). Hydrogen peroxide production from reactive liposomes encapsulating enzymes. Biochim. Biophys. Acta, 1419, 221-8. 

Liposomes have been used for years as components of drug delivery systems, and as transdermal carriers of active ingredients in the cosmetic industry (307, 308). More recently, liposomes have found use in the food and nutritional supplement industries. Keller (308) lists more than a dozen nutritional products on the market that have been formulated with novel liposome-based delivery systems. In the food area, hposomes have been studied for their ability to encapsulate and provide controlled release of enzymes (309, 310), and liposome-encapsulated enzymes have been used to accelerate the ripening of cheese (311). 

From the clinical point of view, the potential ability of liposome-encapsulated enzymes to enter the cytoplasm or lyso-somes of live cells is of primary importance for the treatment of inherited diseases caused by the abnormal functioning of some intracellular enzymes, especially in liver and CNS cells. The use of liposome-immobilized enzymes instead of their native precursors opens new opportunities for enz)une therapy especially in the treatment of diseases localized in liver cells that are natural targets for liposomes. Thus, the biodistribution of liposomes made of phosphatidylcholine, phospha-tidic acid, and cholesterol (in 7 1 2 molar ratio) and containing jS-fructofuranosidase has been studied. It was shown that within an hour, 50% of the administered enzyme can still be found in the circulation, and the enzyme preserves its activity for a long time — 25% of the administered activity can be found in the liver after 48 hours. Similar data have been obtained for intravenously administered liposome-encapsulated a-mannosidase and neuraminidase. ... 

Experimental in vitro investigations utilizing liposomal-encapsulated and polyethylene glycol-conjugated SOD and catalase have demonstrated the potential value of such means in countering oxidative asbestos-related diseases (Freeman etal., 1985 Mossman etal., 1986). In addition to using supplementary endogenous antioxidant enzymes, the use of iron chelators like desferrioxamine... 

Budai M, Chapela P et al (2009) Physicochemical characterization of stealth liposomes encapsulating an organophosphate hydrolyzing enzyme. J of Liposome Research 19(2) 163-168... 

To demonstrate polymerase activity in a model cell, Chakrabarti et al. [79] encapsulated polynucleotide phosphorylase in vesicles composed of dimyris-toylphosphatidylcholine (DMPC). This enzyme can produce RNA from nucleoside diphosphates such as adenosine diphosphate (ADP) and does not require a template, so it has proven useful for initial studies of encapsulated polymerase activity (Fig. 10a). Furthermore, DMPC liposomes are sufficiently permeable so that 5-10 ADP molecules per second enter each vesicle. Under these conditions, measurable amounts of RNA in the form of polyadenylic acid were synthesized and accumulated in the vesicles after several days incubation. The enzyme-catalyzed reaction could be carried out in the presence of a protease external to the membrane, demonstrating that the vesicle membrane protected the encapsulated enzyme from hydrolytic degradation. Similar behavior has been observed with monocarboxylic acid vesicles [80], and it follows that complex phospholipids are not required for an encapsulated polymerase system to function. 

From 1984, when they were first developed, DRV liposomes have been used for liposomal encapsulation of various active substances which may be divided into three main categories (1) Low MW drug molecules (mainly hydrophilic drugs) (3-20) (2) Proteins or peptides and enzymes (21-26), and (3) DNA or oligonucleotides (26-32). From these categories, the last two are primarily used as liposomal vaccines. Some examples of substances entrapped in DRV liposomes from the last 10 year literature are presented in Table 1. 

Van Rooijen N, Van Nieuwmegen R (1984) Elimination of phagocytic cells in the spleen after intravenous injection of liposome encapsulated dichloromethylene -diphosphonate. An enzyme-histochemical study. Cell Tissue Res 238 355... 

The term encapsulation has been used to distinguish entrapment preparations in which the biocatalyst environment is comparable to that of the bulk phase and where there is no covalent attachment of the protein to the containment medium (Fig. 6-1 D)[21J. Enzymes or whole cells may be encapsulated within the interior of a microscopic semi-permeable membranes (microencapsulation) or within the interior of macroscopic hollow-fiber membranes. Liposome encapsulation, a common microscopic encapsulation technique, involves the containment of an enzyme within the interior of a spherical surfactant bilayer, usually based on a phospholipid such as lecithin. The dimensions and shape of the liposome are variable and may consist of multiple amphiphile layers. Processes in which microscopic compart-mentalization (cf. living cells) such as multienzyme systems, charge transfer systems, or processes that require a gradient in concentration have employed liposome encapsulation. This method of immobilization is also commonly used for the delivery of therapeutic proteins. 

We prepared lipid-protein and lipid-polymer artificial cells to encapsulate biologically active materials. Later, Gregoriadis prepared concentric lipid manbrane liposomes containing enzymes. " Liposomes feature multiple lipid layers and onion-skin-like microspheres originally used by Bangham for basic membrane research. Workers in liposomes turned to preparing small submicron artificial... 

In an immunosensor, the antigen-antibody selective interaction is monitored. In order to improve the sensitivity, the indirect monitoring of the Ag-Ab interaction should be preferred, using competitive tracers or markers. Determination of alachlor, for example, can be achieved with increased sensitivity by using liposome encapsulated markers in a competitive binding reaction instead of enzymes. 

Enzymes may also be immobilized by microencapsulation. In this technique, which has medical applications, enzymes are enclosed by various types of semi-permeable membrane, e.g. polyamide, polyurethane, polyphenyl esters and phospholipids. Microcapsules of phospholipids are also called liposomes. The micro-encapsulated enzymes and proteins inside the micro-capsule cannot pass the membrane envelope, but low M, substrates can pass into it, and products can leave. Such encapsulated proteins do not elicit an antigenic response, and they are not attacked by proteases outside the microcapsule. They are therefore suitable for the delivery of enzymes for therapeutic purposes. This area of application is still at an early stage of development, but positive results have been reported from animal experiments and clinical studies, e.g. treatment of inherited catalase deficiency with encapsulated catalase. There are various methods of administration intramuscular, subcutaneous or intraperito-neal injection. However, their major area of application is outside the body. For example, microencapsulated urease can be employed as an artificial kidney in hemodiffusion (Rg.2). 

For instance, in the domain of antibacterial activity, glucose oxidase and HRP have been encapsulated into DPPC/PI liposomes by two different methods, namely extrusion and reverse-phase evaporation. The liposomes were characterized in terms of the content and activity of the encapsulated enzymes. Their enzymatic activity was probed... 

Liposome conjugates may be used in various immunoassay procedures. The lipid vesicle can provide a multivalent surface to accommodate numerous antigen-antibody interactions and thus increase the sensitivity of an assay. At the same time, it can function as a vessel to carry encapsulated detection components needed for the assay system. This type of enzyme-linked immunosorbent assay (ELISA) is called a liposome immunosorbent assay or LISA. One method of using liposomes in an immunoassay is to modify the surface so that it can interact to form biotin-avidin or biotin-streptavidin complexes. The avidin-biotin interaction can be used to increase detectability or sensitivity in immunoassay tests (Chapter 23) (Savage et al., 1992). 

Liposomes (see Chapter 9) have been used to deliver vaccines and have been observed to have immunostimulant activity. When administered orally liposomes with encapsulated antigens have been claimed to provide protection from the gastric proteolytic enzymes. Liposomes also have potential as mucosal delivery systems... 

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