Liposomes biotinylated

Biotinylated liposomes usually are created by modification of PE components with an amine-reactive biotin derivative, for example NHS-LC-Biotin (Chapter 11, Section 1). The NHS ester reacts with the primary amine of PE residues, forming an amide bond linkage (Figure 22.19). A better choice of biotinylation agent may be to use the NHS-PEG -biotin compounds (Chapter 18), because the hydrophilic PEG spacer provides better accessibility in the aqueous environment than a hydrophobic biotin spacer. Since the modification occurs at the hydrophilic end of the phospholipid molecule, after vesicle formation the biotin component protrudes out from the liposomal surface. In this configuration, the surface-immobilized biotins are able to bind (strept)avidin molecules present in the outer aqueous medium. 

However, since many of the traditional biotinylation reagents, such as NHS-LC-biotin contain hydrophobic spacers, their use with amphipathic liposomal constructions may not be entirely appropriate. A better choice may be to use a hydrophilic PEG-based biotin compound that creates a water-soluble biotin modification on the outer aqueous surface of the liposome bilayer. Biotinylation reagents of this type are discussed in Chapter 18, Section 3. 

Biotinylation may be done before or after liposome formation, but having a stock supply of biotin-modified PE is an advantage, since it can then be used to test a number of liposomal recipes. In addition, only a very small percent of the total lipid should be biotinylated to prevent avidin-induced aggregation in the absence of antigen. It is difficult to control precisely... 

Figure 22.18 Biotinylated liposomes may be used in immunoassay systems to enhance the signal for detection or measurement of specific analytes. The liposome components may be constructed to include fluorescent molecules to facilitate detection of antigens within tissue sections.

The following method for the formation of a biotinylated liposome is adapted from Plant et al., 1989). It assumes prior production of B-PE. 

Prepare a biotinylated liposome construct by first dissolving in chloroform, the lipids DMPC cholesterol dicetylphosphate (Sigma) at mole ratios of 5 4 1, and adding to this solution 0.1 mol percent B-PE. Larger mole ratios of B-PE to total lipid may result in nonspecific aggregation of liposomes in the presence of avidin. Maintain all lipids under an inert atmosphere to prevent oxidation. 

Figure 22.19 Biotinylated liposomes may be formed using biotinylated PE. Reaction of NHS-LC-biotin with PE results in amide bond linkages and a long spacer arm terminating in a biotin group.

The biotinylated liposomes prepared by this procedure may be stored under an inert-gas atmosphere at 4°C for long periods without degradation. 

Figure 22.21 Antibodies may be conjugated to liposomes using an indirect approach incorporating a (strept)avidin-biotin system. Biotinylated liposomes may be complexed with biotinylated antibodies using (strept)avidin as a bridging molecule or may be complexed with an antibody-(strept)avidin conjugate.

Hashimoto, K., Loader, J.E., and Kinsky, S.C. (1986) Iodoacetylated and biotinylated liposomes Effect of spacer length on sulfbydryl ligand binding and avidin precipitability. Biochim. Biophys. Acta 856, 556-565. 

Fig. 20. Targeting of the endothelial integrin a P3 as a specific angiogenesis marker. The receptor is recognized by a biotinylated antibody that is then bound by an avidin moiety bearing a Gd(III) loaded liposome.

Rosenberg, M.B., X.O. Breakefield, and E. Hawrot, Targeting of liposomes to cells bearing nerve growth factor receptors mediated by biotinylated nerve growth factor. J Neurochem, 1987. 48(3) 865-75. 

Biotinylation may be done before or after liposome formation, but having a stock supply of biotin-modified PE is an advantage, since it can then be used to test a number... 

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