Enzymes inside lipid vesicles: Preparation, reactivity and applications

There are a number of methods that can be used for the preparation of enzym e-containing lipid vesicles (liposomes) which are lipid dispersions that co ntain water-soluble enzymes in the trapped aqueous space. This has been sho wn by many investigations carried out with a variety of enzymes. A review o f these studies is given and some of the main results are summarized. With respect to the vesicle-forming amphiphiles used, most preparations are base d on phosphatidylcholine, either the natural mixtures obtained from soybean or egg yolk, or chemically defined compounds, such as DPPC (1,2-dipalmitoy l-sn-glycero-3-phosphocholine) or POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-p hosphocholine). Charged enzyme-containing lipid vesicles are often prepared by adding a certain amount of a negatively charged amphiphile (typically d icetylphosphate) or a positively charged lipid (usually stearylamine). The presence of charges in the vesicle membrane may lead to an adsorption of th e enzyme onto the interior or exterior site of the vesicle bilayers. If (i) the high enzyme encapsulation efficiencies; (ii) avoidance of the use of o rganic solvents during the entrapment procedure; (iii) relatively monodispe rse spherical vesicles of about 100 nm diameter; and (iv) a high degree of unilamellarity are required, then the use of the so-called 'dehydration-reh ydration method', followed by the 'extrusion technique has shown to be supe rior over other procedures. In addition to many investigations in the field of cheese production-there are several studies on the (potential) medical and biomedical applications of enzyme-containing lipid vesicles (e.g. in th e enzyme-replacement therapy or for immunoassays)-including a few in vivo s tudies. In many cases, the enzyme molecules are expected to be released fro m the vesicles at the target site, and the vesicles in these cases serve as the carrier system. For (potential) medical applications as enzyme carrier s in the blood circulation, the preparation of sterically stabilized Lipid, vesicles has proven to be advantageous. Regarding the use of enzyme-contain ing vesicles as submicrometer-sized nanoreactors, substrates are added to t he bulk phase. Upon permeation across the vesicle bilayer(s), the trapped e nzymes inside the vesicles catalyze the conversion of the substrate molecul es into products. Using physical (e.g. microwave irradiation) or chemical m ethods (e.g. addition of micelle-forming amphiphiles at sublytic concentrat ion), the bilayer permeability can be controlled to a certain extent. A det ailed molecular understanding of these (usually) submicrometer-sized biorea ctor systems is still not there. There are only a few approaches towards a, deeper understanding and modeling of the catalytic activity of the entrapp ed enzyme molecules upon externally added substrates. Using micrometer-size d vesicles (so-called 'giant vesicles') as simple models for the lipidic ma trix of biological cells, enzyme molecules can be microinjected inside indi vidual target vesicles, and the corresponding enzymatic reaction, can be mo nitored by fluorescence microscopy using appropriate fluorogenic substrate molecules. (C) 2001 Elsevier Science B.V. All rights reserved.