Anas El-Aneed is a doctoral student in biochemistry at the School of Pharmacy at Memorial University of Newfoundland.

Anas El-Aneed

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.	

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				

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) chain5,6 or directly onto the lipids.7 The former attachment methodology is used extensively and favoured because PEG serves as a spacer between the ligand and the liposomal surface, thereby providing easy access to the antibodies. PEG chains cause steric barriers when the mAb or Fab is attached to the lipids. It has been shown that PEG 2000 will mask the lipid-linked antibody to a lesser degree than will the longer PEG 5000 chain.8 In addition, a comparison study of PEG-linked and lipid-linked antibodies has shown that coupling is more efficient with the the use of PEG chains.9 Although the coupling reaction to PEG usually occurs after the preparation of the liposomes, anchor lipid molecules are attached to the antibody before assembly into the liposomal structure during preparation (in the case of direct linkage of the antibody to the lipids). However, a novel and simple prepa-ration method for immunoliposomes has been developed that involves transferring the lipid-conjugated mAb or Fab micelles to preformed, drug-loaded liposomes under specified conditions of temperature and pH.10-12 This method is referred to as the postinsertion technique.

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.	

Ligand-targeted liposomes have lower immunogenicity in comparison with immunoliposomes. Ligands vary according to the targeted tissues. One popular target is the liver, which is associated with many genetically based diseases such as haemophilia, lipoprotein receptor deficiency, 1-antitrypsin deficiency and liver cancer. Because many receptors, namely low-density lipoprotein (LDL) and asialoglycoprotein receptors, are expressed on the surface of the liver, the discussion in this section focusses on liver-targeted liposomes. 	

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	

Asialofetuin (AF) is a natural ligand for ASGP-R. It is a glycoprotein with several terminal galactose sugar chains35 and has been incorporated into the liposomal surface by covalently attaching a hydrophobic moiety (palmitic acid) as an anchor among the lipids of the liposomal vesicles (AF-lipo-somes).36,37 In one study, the AF-liposomal uptake by the liver in mice was increased 11 times in comparison with unmodified liposomes.38 Similarly, AF-liposomal-mediated transfection of the human alpha antitrypsin (hAAT) gene was significantly enhanced in comparison with regular liposomes. After one year of the treatment, hAAT mRNA in the liver was detected in all animals transfected with AF-liposomes compared with only 25% of those treated with regular liposomes, with more than a 4000-fold increase in the case of the AF-liposomes condition.39 In this study, AF was covalently linked to an anchor lipid on the surface of preformed liposomes. More recently, LPD was coated with the AF through charge–charge interactions, which significantly increased the HepG2 cells' uptake of the encapsulated DNA.40 AF, however, can induce immunogenic reaction. Therefore, simpler glycosylated liposomes were developed and evaluated for liver targeting.41 In another study, glycosylated cholesterol, for example, was synthesized and incorporated into the cationic liposomal vesicles, thereby resulting in a 10-fold increase in gene expression in the liver.42	

Liver cancer is another important target for gene delivery. HCC (hepatocellular carcinoma) is a leading cause of cancer-related deaths worldwide.43 Gene therapy can provide a new approach to treat this fatal disease. In addition to the various receptors on the hepatocyte cells, there are some receptors that are over-expressed in hepatoma cells. One example is transferrin (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 Tumour growth was inhibited as much as 70% in liver tumour xenografts after treatment with TF-liposomes containing the antiangiogenesis gene, endostatin.46 Linkages to the PEG terminal end of pegylated liposomes were also used for TF-liposome preparations.48,49	

Because cationic liposomes have a lower transfection efficiency than viruses, modifications that may increase transfection levels are always favoured 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 can also 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.				

1.	E. Benjamini, R. Coico and G. Sunshine, Immunology: A Short Course, 4th Edn (Wiley–Liss, New York, New York, USA, 2000).				

2.	L.D. Leserman et al., "Targeting to Cells of Fluorescent Liposomes Covalently Coupled with Monoclonal Antibody or Protein A," Nature 288(5791), 602–604 (1980).	

3.	T.D. Heath, R.T. Fraley and D. Papahdjopoulos, "Antibody Targeting of Liposomes: Cell Specificity Obtained by Conjugation of F(ab')2 to Vesicle Surface," Science 210(4469), 539–541 (1980).	

4.	G. Bendas, "Immunoliposomes: A Promising Approach to Targeting Cancer Therapy," Bio. Drugs 15(4), 215–224 (2001).	

5.	N. Shi, R.J. Boado and W.M. Pardridge, "Receptor-Mediated Gene Targeting to Tissues In Vivo Following Intravenous Administration of Pegylated Immunoliposomes," Pharm. Res. 18(8), 1091–1095 (2001).	

6.	T.M. Allen et al., "A New Strategy for Attachment of Antibodies to Sterically Stabilized Liposomes Resulting in Efficient Targeting to Cancer Cells," Biochim. Biophys. Acta. 1237(2), 99–108 (1995).	

7. 	A.L. Klibanov et al., "Activity of Amphipathic Poly(ethylene glycol) 5000 to Prolong the Circulation Time of Liposomes Depends on the Liposome Size and is Unfavorable for Immunoliposome Binding to Target," Biochim. Biophys. Acta. 1062(2), 142–148 (1991).	

8.	A. Mori et al., "Influence of the Steric Barrier Activity of Amphipathic Poly(ethyleneglycol) and Ganglioside GM1 on the Circulation Time of Liposomes and on the Target Binding of Immunoliposomes In Vivo," FEBS Lett. 284(2), 263–266 (1991).	

9.	C.B. Hansen et al., "Attachment of Antibodies to Sterically Stabilized Liposomes: Evaluation, Comparison and Optimization of Coupling Procedures," Biochim. Biophys. Acta. 1239(2), 133–144 (1995).	

10.	T.M. Allen, P. Sapra and E. Moase, "Use of the Postinsertion Method for the Formation of Ligand-Coupled Liposomes," Cell Mol. Bio. Lett. 7(3), 889–894 (2002).	

11.	D.L. Iden and T.M. Allen, "In Vitro and In Vivo Comparison of Immunoliposomes Made by Conventional Coupling Techniques with Those Made by a New Postinsertion Approach," Biochim. Biophys. Acta. 1513(2), 207–216 (2001).	

12.	T. Ishida, D.L. Iden and T.M. Allen, "A Combinatorial Approach to Producing Sterically Stabilized (Stealth) Immunoliposomal Drugs," FEBS Lett. 460(1), 129–133 (1999).	

13.	C.H. Lee et al., "Enhanced Gene Delivery to Her-2-Overexpressing Breast Cancer Cells by Modified Immunolipoplexes Conjugated with the Anti-Her-2 Antibody," J. Biomed. Sci. 10(3), 337–344 (2003).	

14.	G.Y. Kao, L.J. Change and T.M. Allen, "Use of Targeted Cationic Liposomes in Enhanced DNA Delivery to Cancer Cells," Cancer Gene Ther. 3(4), 250–256 (1996).	

15.	Y. Zhang, C. Zhu and W.M. Pardridge, "Antisense Gene Therapy of Brain Cancer with an Artificial Virus Gene Delivery System," Mol. Ther. 6(1), 67–72 (2002).	

16.	J. Yoshida and M. Mizuno, "Simple Preparation and Characterization of Cationic Liposomes Associated with Monoclonal Antibody Against Glioma-Associated Antigen (Immunoliposomes)," J. Liposome Res. 5(4), 981–995 (1995).	

17.	M. Mizuno and J. Yoshida, "Repeated Exposure to Cationic Immunoliposomes Activates Effective Gene Transfer to Human Glioma Cells," Neurol. Med. Chir. 36(3), 141–144 (1996).	

H. Loughrey, M.B. Bally and P.R. Cullis, "A Noncovalent Method of Attaching Antibodies to Liposomes," Biochim. Biophys. Acta. 901(1), 157–160 (1987).	

19.	M. Verhoeyen, C. Milstein and G. Winter, "Reshaping Human Antibodies: Grafting an Antilysozyme Activity," Science 239(4847), 1534–1536 (1988).	

20.	P.T. Jones et al., "Replacing the Complementarity-Determining Regions in a Human Antibody with Those from a Mouse," Nature 321(6069), 522–525 (1986).	

21.	M. Iyer et al., "Noninvasive Imaging of Cationic Lipid-Mediated Delivery of Optical and PET Reporter Genes in Living Mice," Mol. Ther. 6(4), 555–562 (2002).	

22.	J.W. McLean et al., "Organ-Specific Endothelial Cell Uptake of Cationic Liposome-DNA Complexes in Mice," Am. J. Physiol. 273(1 Pt 2), H387–404 (1997).	

23.	G. Osaka et al., "Pharmacokinetics, Tissue Distribution and Expression Efficiency of Plasmid [33P] DNA Following Intravenous Administration of DNA–Cationic Lipid Complexes in Mice: Use of a Novel Radionuclide Approach," J. Pharm. Sci. 85(6), 612–618 (1996).	

24.	Z. Ma et al., "Lipid-Mediated Delivery of Oligonucleotide to Pulmonary Endothelium," Am. J. Respir. Cell Mol. Biol. 27(2), 151–159 (2002).	

25.	B. Li et al., "Lyophilization of Cationic Lipid–Protamine–DNA (LPD) Complexes," J. Pharm. Sci. 89(3), 355–364 (2000).	

26.	S. Li and L. Huang, "In Vivo Gene Transfer via Intravenous Administration of Cationic Lipid–Protamine–DNA (LPD) Complexes," Gene Ther. 4(9), 891–900 (1997).	

27.	S.H. Hwang et al., "Liver-Targeted Gene Transfer into a Human Hepatoblastoma Cell Line and In Vivo by Sterylglucoside-Containing Cationic Liposomes," Gene Ther. 8(16), 1276–1280 (2001).	

28.	S.F. Alino et al., "Human Alpha 1-Antitrypsin Gene Transfer to In Vivo Mouse Hepatocytes," Hum. Gene Ther. 7(16), 531–536 (1996).	

29.	J. Wu and M.A. Zern, "Modification of Liposomes for Liver Targeting," J. Hepatol. 24(6), 757–763 (1996).	

30.	R.I. Mahato et al., "Biodistribution and Gene Expression of Lipid–Plasmid Complexes after Systemic Administration," Hum. Gene Ther. 9(14), 2083–2099 (1998).	

31.	L. Liu et al., "Poly(cationic lipid)-Mediated In Vivo Gene Delivery to Mouse Liver," Gene Ther. 10(2), 180–187 (2003).	

32.	D.C. Litzinger et al., "Fate of Cationic Liposomes and Their Complex with Oligonucleotide In Vivo," Biochim. Biophys. Acta. 1281(2), 139–149 (1996).	

33.	F. Roerdink et al., "The Involvement of Parenchymal, Kupffer and Endothelial Liver Cells in the Hepatic Uptake of Intravenously Injected Liposomes. Effects of Lanthanum and Gadolinium Salts," Biochim. Biophys. Acta. 677(1), 79–89 (1981).	

34.	J. Wu, M.H. Nantz and M.A. Zern, "Targeting Hepatocytes for Drug and Gene Delivery: Emerging Novel Approaches and Applications," Front. Biosci. 7, 717–725 (1 March 2002).	

Articles

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 tumour-specific and liver-targeted cationic liposomes and strategies for the development of liposome?ligand complexes.

Targeted Cationic Liposomes: Technologies and Developments

The author provides a brief review of tumor-specific and liver-targeted cationic liposomes and the strategies for the development of liposome-ligand complexes.