Lipos

Targeted Cationic Liposomes, Technologies, and Developments Anas El-Aneed

PHOTODISC, INC.

C Cationic liposomes are widely used in gene therapy as a safe alternative to highly immunogenic viral vectors. Attachment of a tissue-specific ligand to the surface of the liposomes can increase specificity and reduce undesired transfection. Targeted liposomes can be categorized as either immunoliposomes or ligand-targeted liposomes. The author provides a brief review of tumor-specific and liver-targeted cationic liposomes and the strategies for the development of liposome–ligand complexes. Anas El-Aneed is a doctoral student in biochemistry at the School of Pharmacy at Memorial University of Newfoundland, 300 Prince Philip Dr., St. John’s, NL, A1B 3V6 Canada, tel. 709.737.4331, fax 709.777.7044, [email protected].

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ationic 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 tumor site. The last goal can be achieved by incorporating a tumor-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 also can be applied when using targeted cationic liposomes. Because cationic and noncationic liposomes have similar composition and structure, ligand attachment strategies also are 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) (i.e., immunoliposomes) or the attachment of a tissue-specific ligand to the surface of the liposomes. This article briefly reviews livertargeted and tumor-specific liposomes as examples of targeted liposomes. This article also 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), which are fragments responsible for antigen recognition, and Fc (fragment crystallizable), which are fragments that play a role in biological activity (1). Immunoliposomes are studied because of their relative ease of preparation and high specificity. In the early 1980s, researchers first linked antibodies or Fab fragments to liposomes by attaching them directly to lipids (2,3). Because of their short half lives, immunoliposomes are used mainly in long circulating pegylated liposomes (4). Antibodies can be attached to the surface of the pegylated vesicles either at the terminal end of a polyethylene glycol (PEG) chain (5,6) or directly onto the lipids (7). www.phar mtech.com

The former attachment methodology is used exIn the case of hepatocyte cells, the main Abbreviations tensively and favored because PEG serves as a spacer challenge is to divert the liposomes from between the ligand and the liposomal surface, DNA:deoxyribonucleic acid the lung “trap.” A conventional lipmAb:monoclonal antibody thereby providing easy access to the antibodies. PEG some–DNA complex (lipoplex) (21–23) chains cause steric barriers when the mAb or Fab is PEG:polyethylene glycol and the novel liposomal preparation LPD EGFR:epidermal growth factor receptor attached to the lipids. It has been shown that PEG (liposomes–protamine–DNA) (24–26) 2000 will mask the lipid-linked antibody to a lesser mRNA:messenger ribonucleic acid tend to be trapped by capillary embolism LPD:liposomes/ Protamine/ DNA degree than will the longer PEG 5000 chain (8). In in the lungs where transfection may occur. LDL:low density lipoprotein addition, a comparison study of PEG-linked and Liver accumulation of lipoplexes can be ASGP-R:asialoglycoprotein receptors lipid-linked antibodies has shown that coupling is enhanced by manipulating the size of the more efficient with the the use of PEG chains (9). AF:asialofetuin particles (27–29) or lowering the complex Although the coupling reaction to PEG usually oc- hAAT:human alpha antitrypsin surface charge (30). Recent studies have HCC:hepatocellular carcinoma curs after the preparation of the liposomes, anchor shown that transfection occurs mainly in lipid molecules are attached to the antibody before the liver with the development of the assembly into the liposomal structure during preparation (in serum resistant poly(cationic lipid) (31). However, one must enthe case of direct linkage of the antibody to the lipids). How- sure that liposomes have actually reached the parenchymal liver ever, a novel and simple preparation method for immunolipo- cells rather than the phagocytic kupffer cells. Some studies have somes has been developed that involves transferring the lipid- shown that kupffer cells were actually the main destination for conjugated mAb or Fab micelles to preformed, drug-loaded liposomes in the liver (32,33). This problem may be circumliposomes under specified conditions of temperature and pH vented by attaching a receptor-specific ligand to the surface of (10–12). This method is referred to as the postinsertion tech- the liposomes. In such a case, the ligand binds to its receptors nique. on the parenchymal cells before internalization occurs. In two cancer gene therapy studies, researchers significantly Asialoglycoprotein receptors (ASGP-R) are abundant on the enhanced gene expression in tumors using immunoliposome mammalian parenchymal liver cells. Their major role is to clear technology instead of conventional liposomes (13,14). In an- glycoproteins and lipoproteins from circulation. The receptor other study, the life span of mice bearing aggressive brain tu- contains a carbohydrate-recognition domain that can bind to mors was increased by 100% after treatment with epidermal galactose derivatives (34). growth factor receptor (EGFR) antisense mRNA delivered by Asialofetuin (AF) is a natural ligand for ASGP-R. It is a glycoprotein with several terminal galactose sugar chains (35) and intravenous injection of immunoliposomes (15). In those studies, antibodies were covalently linked to the li- has been incorporated into the liposomal surface by covalently posomal surface. However, noncovalent linkages also have been attaching a hydrophobic moiety (palmitic acid) as an anchor used by simple mixing of the antibody with the liposomal vesi- among the lipids of the liposomal vesicles (AF-liposomes) cles resulting in two- to four-fold increases in the transfection (36,37). In one study, the AF-liposomal uptake by the liver in efficiency of the reporter gene in a glioma cell line (16,17). More- mice was increased 11 times in comparison with unmodified efficient noncovalent linkages were obtained through avidin– liposomes (38). Similarly, AF-liposomal-mediated transfection biotin binding. Biotinated lipids were bound to strepavidin, of the human alpha antitrypsin (hAAT) gene was significantly which contains four biotin binding sites, and the system then enhanced in comparison with regular liposomes. After one year was attached to biotinated mAb by simple incubation (9,18). of the treatment, hAAT mRNA in the liver was detected in all Immunogenicity is the main concern associated with im- animals transfected with AF-liposomes versus only 25% with munoliposome applications. This drawback was minimized those treated with regular liposomes, with more than a 4000with the use of Fab subunits instead of the whole antibodies or fold increase in the case of the AF-liposomes condition (39). In the fully humanized mAb first produced in the 1980s (19,20). this study, AF was covalently linked to an anchor lipid on the The linkage techniques of Fab fragments are identical to those surface of preformed liposomes. More recently, LPD was coated applied on the complete mAb, covalently (3,13) or noncova- with the AF through charge–charge interactions, which signiflently (17,18). These liposome–ligand attachment methods also icantly increased the HepG2 cells’ uptake of the encapsulated are applicable on all other peptide and protein ligands. DNA (40). AF, however, can induce immunogenic reaction. Therefore, simpler glycosylated liposomes were developed and Ligand-targeted liposomes evaluated for liver targeting (41). In another study, glycosylated Ligand-targeted liposomes have lower immunogenicity in com- cholesterol, for example, was synthesized and incorporated into parison with immunoliposomes. Ligands vary according to the the cationic liposomal vesicles, thereby resulting in a 10-fold targeted tissues. One popular target is the liver, which is asso- increase in gene expression in the liver (42). ciated with many genetically based diseases such as hemophilia, Liver cancer is another important target for gene delivery. lipoprotein receptor deficiency, 1-antitrypsin deficiency, and HCC (hepatocellular carcinoma) is a leading cause of cancerliver cancer. Because many receptors, namely low-density related deaths worldwide (43). Gene therapy can provide a new lipoprotein (LDL) and asialoglycoprotein receptors, are ex- approach to treat this fatal disease. In addition to the various pressed on the surface of the liver, the discussion in this section receptors on the hepatocyte cells, there are some receptors that focuses on liver-targeted liposomes. are over-expressed in hepatoma cells. One example is transfer60

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rin (TF) receptors, which are also elevated in other malignant cells (44). TF-liposomes will not only target the cancerous cells, but will also reduce undesired transfection levels in the surrounding normal tissues. This process can be exaggerated by hepatic arterial injection of TF-liposomes mediating DNA delivery (45). TF-liposome complexes are typically prepared through charge–charge interactions by simple mixing and incubation (45–47). Tumor growth was inhibited as much as 70% in liver tumor xenografts after treatment with TF-liposomes containing the antiangiogenesis gene, endostatin (46). Linkages to the PEG terminal end of pegylated liposomes also were used for TF-liposome preparations (48,49).

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Closing remarks Because cationic liposomes have a lower transfection efficiency than viruses, modifications that may increase transfection levels are always favored for the development of liposomal preparations for gene transfer. One possible alteration is the introduction of a tissue-specific ligand on the surface of the liposomes. This modification can enhance liposomal specificity and reduce the undesired delivery associated with toxic effects. Ligands vary according to the targeted tissues. Peptides, proteins, and sugar moieties have been explored as potential targeting ligands. Targeting specific tissues by altering the physical properties of liposomes also can be combined with ligand attachment for optimum targeting outcomes. Research in this area is expected to expand and increasingly enter the stage of clinical evaluation in humans.

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