Targeted
Cationic Liposomes,
Technologies, and
Developments
Anas El-Aneed
PHOTODISC, INC.
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, anas@pharm.mun.ca.
58 Pharmaceutical Technology DECEMBER 2003
C
ationic liposomes are widely used to transfer genetic ma-
terials into specific cells. A good liposomal formulation
for gene therapy should encapsulate and protect the nu-
cleic acid materials, escape endosomal degradation, and
reach the tumor site. The last goal can be achieved by incorpo-
rating a tumor-specific ligand that can deliver DNA to the tar-
geted 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 struc-
ture, ligand attachment strategies also are similar, and any dis-
cussion 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., im-
munoliposomes) or the attachment of a tissue-specific ligand
to the surface of the liposomes. This article briefly reviews liver-
targeted and tumor-specific liposomes as examples of targeted
liposomes. This article also describes liposome–ligand attach-
ment techniques.
Immunoliposomes
Antibodies are soluble proteins that are produced by B cells of
the immune system to bind to the antigens mediating their de-
struction. 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 at-
taching them directly to lipids (2,3). Because of their short half
lives, immunoliposomes are used mainly in long circulating pe-
gylated liposomes (4). Antibodies can be attached to the sur-
face of the pegylated vesicles either at the terminal end of a poly-
ethylene glycol (PEG) chain (5,6) or directly onto the lipids (7).
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The former attachment methodology is used ex- In the case of hepatocyte cells, the main
tensively and favored because PEG serves as a spacer
Abbreviations challenge is to divert the liposomes from
between the ligand and the liposomal surface, DNA:deoxyribonucleic acid the lung “trap.” A conventional lip-
thereby providing easy access to the antibodies. PEG mAb:monoclonal antibody 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
attached to the lipids. It has been shown that PEG EGFR:epidermal growth factor receptor (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
degree than will the longer PEG 5000 chain (8). In LPD:liposomes/ Protamine/ DNA in the lungs where transfection may occur.
addition, a comparison study of PEG-linked and LDL:low density lipoprotein Liver accumulation of lipoplexes can be
lipid-linked antibodies has shown that coupling is ASGP-R:asialoglycoprotein receptors 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
curs after the preparation of the liposomes, anchor HCC:hepatocellular carcinoma 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 en-
the 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 circum-
liposomes 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 gly-
intravenous injection of immunoliposomes (15). coprotein with several terminal galactose sugar chains (35) and
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 4000-
with 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 signif-
lently (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 cancer-
liver 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 transfer-
60 Pharmaceutical Technology DECEMBER 2003 www.phar mtech.com
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 sur-
rounding normal tissues. This process can be exaggerated by
hepatic arterial injection of TF-liposomes mediating DNA de-
livery (45). TF-liposome complexes are typically prepared
through charge–charge interactions by simple mixing and in-
cubation (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).
Closing remarks
Because cationic liposomes have a lower transfection efficiency
than viruses, modifications that may increase transfection lev-
els are always favored for the development of liposomal prepa-
rations for gene transfer. One possible alteration is the intro-
duction 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, pro-
teins, and sugar moieties have been explored as potential tar-
geting ligands. Targeting specific tissues by altering the physi-
cal 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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