Cationic liposomes are widely used to transfer genetic materials into specific cells. A good liposomal formulation for gene therapy should encapsulate and protect the nucleic acid materials, escape endosomal degradation and reach the tumour site. The last goal can be achieved by incorporating a tumour-specific ligand that can deliver DNA to the targeted tissue.

The same strategies that are applied when using anionic liposomes to develop tissue-specific formulations can be applied when using targeted cationic liposomes. Because cationic and non-cationic liposomes have a similar composition and structure, ligand attachment strategies are also similar, and any discussion in this area cannot separate the two liposomal groups. The two main strategies in developing targeted liposomes are the attachment of a monoclonal antibody (mAb), that is, immunoliposomes, or the attachment of a tissue-specific ligand to the surface of the liposomes. This article briefly reviews liver-targeted and tumour-specific liposomes as examples of targeted liposomes, and describes liposome–ligand attachment techniques.

Immunoliposomes Antibodies are soluble proteins that are produced by B cells of the immune system to bind to the antigens mediating their destruction. This process is accomplished either directly or with the help of other immune-system components-namely, Fab (fragment antigen binding) fragments which are responsible for antigen recognition, and Fc (fragment crystallizable) fragments that play a role in biological activity.1

In two cancer gene therapy studies, researchers significantly enhanced gene expression in tumours using immunoliposome technology instead of conventional liposomes.13,14 In another study, the life span of mice bearing aggressive brain tumours was increased by 100% after treatment with epidermal growth factor receptor (EGFR) antisense mRNA delivered by intravenous injection of immunoliposomes.15

In those studies, antibodies were covalently linked to the liposomal surface. However, non-covalent linkages have also been used by simple mixing of the antibody with the liposomal vesicles resulting in two- to four-fold increases in the transfection efficiency of the reporter gene in a glioma cell line.16,17 More efficient non-covalent linkages were obtained through avidin–biotin binding. Biotinated lipids were bound to streptavidin, which contains four biotin binding sites, and the system was then attached to biotinated mAb by simple incubation.9,18

Immunogenicity is the main concern associated with immunoliposome applications. This drawback was minimized with the use of Fab subunits instead of the whole antibodies or the fully humanized mAb first produced in the 1980s.19,20 The linkage techniques of Fab fragments are identical to those applied on the complete mAb, covalently3,13 or non-covalently.17,18 These liposome–ligand attachment methods are also applicable on all other peptide and protein ligands.

Abbreviations

In the case of hepatocyte cells, the main challenge is to divert the liposomes from the lung "trap." A conventional liposome–DNA complex (lipoplex)21–23 and the novel liposomal preparation LPD (liposomes/protamine/DNA)24–26 tend to be trapped by capillary embolism in the lungs where transfection may occur. Liver accumulation of lipoplexes can be enhanced by manipulating the size of the particles27–29 or lowering the complex surface charge.30 Recent studies have shown that transfection occurs mainly in the liver with the development of the serum-resistant poly(cationic lipid).31 However, one must ensure that liposomes have actually reached the parenchymal liver cells rather than the phagocytic Kupffer cells. Some studies have shown that Kupffer cells were actually the main destination for liposomes in the liver.32,33 This problem may be circumvented by attaching a receptor-specific ligand to the surface of the liposomes. In such a case, the ligand binds to its receptors on the parenchymal cells before internalization occurs.

Asialoglycoprotein receptors (ASGP-R) are abundant on the mammalian parenchymal liver cells. Their major role is to clear glycoproteins and lipoproteins from circulation. The receptor contains a carbohydrate-recognition domain that can bind to galactose derivatives.34