1.3 Vectors for hepatocyte-directed gene transfer
1.3.2 Non-viral methods
1.3.2.2 Chemical methods
Chemical methods of gene delivery, by broad definition, entail the construction of complexes with therapeutic nucleic acids (Dani, 1999). While such strategies encompass a variety of carriers, polymer and lipid-based systems, modified with hepatocyte-specific homing devices (refer to section 1.4), have been widely investigated for application to liver-directed gene therapy (Pathak et al., 2008).
Several natural and synthetic polymers have been explored as potential gene carriers (Dang and Leong, 2006; Kundu and Sharma, 2008). The most promising thereof, as illustrated in Figure 1.2, bear functional groups that are protonated at physiological pH. Therefore such polymers possess the cationic property required to electrostatically bind and condense nucleic acids into microspheric particles, known as polyplexes (Dang and Leong, 2006).
10 1 Liposomes
Figure 1.2: Cationic polymeric nucleic acid carriers, a) linear polyethylenimine (PEI); b) branched PEI (Lungwitz et al., 2005); c) chitosan (Martinez-Huitle et al., 2009); d) poly-L- lysine (PLL) (Tang and Szoka, 1997); e) polyamidoamine (PAMAM) dendrimer; and f) polypropylenimine (PPI) dendrimer (Pathak et al., 2009).
a)
b)
c)
d)
e)
f)
11 While the primary amino functions of polymers such as PEI, PLL and dendrimers facilitate effective binding of nucleic acids, these groups are believed to contribute, by and large, to the cytotoxicity associated with these carriers (Pathak et al., 2009). In addition, recent studies have presented evidence that short-term gene expression afforded by PEI and PLL, arguably the most widely documented cationic, polymeric carriers, may be attributed to their induction of apoptosis in several human cell lines (Hunter, 2006). Of particular interest to the present discussion are reports which demonstrate that PEI and PLL have induced toxic effects in hepatocytes (Bandyopadhyay et al., 1998). It is therefore evident that the performance of such carriers is yet to be optimised (Lutz, 2006). Consequently, chemical manipulation has been explored as a means of attenuating undesired effects (Pathak et al., 2009).
In spite of difficulties associated with many chemical methods, lipid-based vehicles, the liposomes, have maintained interest in this area (Lasic and Templeton, 1996). In fact,
liposomes have been given greater recognition than any other non-viral gene transfer system in clinical trials (Tu et al., 2010). Therefore the application of this vector to the field of hepatocyte-specific gene transfer merits further discussion.
1.3.2.2.1 Liposomes
Liposomes are defined by Schuber and colleagues (1998) as “spherical structures consisting of single or multiple concentric bilayers resulting from the self-assembly of amphiphilic molecules, such as phospholipids, in an aqueous medium,” (Figure 1.3). The scientific community was first introduced to liposomes by Bangham and coworkers (1965), who observed that lipids extracted from egg yolk naturally organised into micro-spheres upon introduction to water. The membranes encompassing these spheres closely resembled biological membranes; therefore, liposomes were initially studied as model membrane systems (Schuber et al., 1998).
12 Figure 1.3: Formation of conventional liposomes from the spontaneous arrangement of phospholipids (http://www.nanolifenutra.com/liposome_technology.html).
Studies initially conducted by Hoffman and colleagues (1978), highlighted the nucleic acid carrying potential of liposomes. This group encapsulated DNA of high molecular weight within liposomes consisting of egg lecithin, which rendered the DNA resistant to the degradative action of DNase. Such liposomes, prepared from neutral and anionic lipids that are either naturally occurring or synthetic, are termed conventional liposomes (Lasic, 1997).
However early attempts at hepatocyte-directed gene transfer using conventional liposomes demonstrated poor transfection efficiency, mainly due to massive accumulation of the administered liposomes in the lung. This phenomenon and the technical difficulties associated with DNA encapsulation limited the use of conventional liposomes in gene transfer applications (Ledley, 1996; Li and Huang, 1999). However, it was the advent of the cationic liposome, in conjunction with targeting strategies, which potentiated more feasible liposome-mediated gene transfer systems.
1.3.2.2.1.1 Cationic liposomes
Cationic liposomes, as the name suggests, possess a net positive charge on the outer surface of the bilayer, due to the incorporation of cationic lipids, termed cytofectins. Nucleic acids may therefore be electrostatically bound to the surface of such a liposome, following
13 incubation of the nucleic acids with liposomes. As such condensed nanostructures, known as lipoplexes, are formed (Lasic, 1997). Often, lipoplexes are constructed to bear a net positive charge as this encourages cellular uptake by way of their affinity for anionic biological surfaces (Felgner et al., 1994). While several mechanisms for the cellular entry of lipoplexes have been proposed (Figure 1.4), according to Zhdanov and colleagues (2002), the lipoplexes predominantly enter via endocytosis or direct membrane fusion, following adherence to the plasma membrane.
Figure 1.4: Possible liposome-cell interactions (Lasic, 1997).
Positively charged lipids, except for sphingosine and a few lipids in primitive organisms, do not exist in nature. Therefore, the positive charge was initially conferred upon liposomes by incorporating cationic detergents into the bilayer. However, the toxicity of such formulations severely limited their use (Lasic, 1997). In 1987, as a result of studies by Felgner and
coworkers, the concept of the cationic lipid as an agent of transfection was made practical.
This group successfully demonstrated the transfection of cultured cells using a liposome formulation prepared from equimolar quantities of the synthetic cationic lipid DOTMA (N- [1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) and neutral lipid DOPE (L- α-dioleoylphosphatidylethanolamine) (Felgner et al., 1987). The lack of tedious nucleic acid- encapsulation procedures, as well as the chemical flexibility, targeting potential and low toxicity of such a system; have contributed to the popularity of the cationic liposome as a non-viral vector (Lasic, 1997).
14 In the years to follow, much progress in the field of cationic liposome-mediated gene transfer has occurred in parallel with advances in cytofectin design (de Lima et al., 2001). A typical cationic amphiphile for use in transfection studies, as represented in Figure 1.5, consists of a hydrocarbon anchor for stable insertion into the liposomal bilayer; a hydrophilic headgroup that is protonated at physiological pH in order to bind and condense nucleic acids; a linker bond and spacer between the aforementioned components.
CATIONIC HEADGROUP
O NH
O CH2
CH2 NH
H3C H3C
SPACER LINKER
BOND
LIPID ANCHOR
Figure 1.5: The four functional domains of a cytofectin, illustrated using 3β[N-(N′,N′- dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) as an example (Lasic and Templeton, 1996).
Studies have demonstrated that the nature of the respective functional domains contribute to critical features of the vector such as nucleic acid-binding capacity, stability, biodegradability and toxicity; all of which ultimately influence its transfection capabilities (Karmali and Chaudhury, 2007; Rao, 2010). Therefore, in an attempt to optimise cationic liposome- mediated transfection, libraries of novel cationic lipids, of which a few examples are
provided in Figure 1.6, have been synthesised from varying combinations of the four domains (Cao et al., 2006). In fact, Sherman and colleagues (1998) have reported that several cationic lipids are undergoing safety and efficacy evaluation for use in clinical trials.
15
O Cl
O
N
O O
N OH
Br
Cl N CH3
NH
NH
H3N
O H2N
NH3
Figure 1.6: Cationic lipids for use in gene transfer, a) N-[1-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA) (de Lima et al., 2001); b) (±)-N-(2-hydroxyethyl)- N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (DMRIE) (de Lima et al., 2001); c) N-methyl-4-(dioleyl)methylpyridinium (SAINT-2) (Hoekstra et al., 2007); and d) Spermine cholesterol (Sper-Chol) (Lasic and Templeton, 1996).
a)
b)
c)
d)
16 Despite continuing reports of promising transfection studies achieved with cationic liposome technology; according to Wu and coworkers (2002), liposomal gene transfer to the liver remains significantly more challenging than to other organs, such as the lungs. Several factors, presented in Figure 1.7, which hamper the successful transfer of genes to hepatocytes using cationic liposomes, both in vitro and in vivo, have been identified. While the genetic modification of rat hepatocytes has been achieved by cationic liposome-mediated transfection ex vivo, current research seeks to avoid surgical procedures, as these are associated with significant mortality or adverse effects on the patient in the long term (Rangarajan et al., 1997). Therefore, in order to achieve the eventual application of systemically administered cationic liposomes to routine treatment of liver disease, researchers have attempted to address these concerns by exploring numerous strategies, and combinations thereof. The discussion to follow focuses on strategies aimed at adapting cationic liposomal systems towards
hepatocyte-directed gene transfer, with emphasis on overcoming the problems of poor cell- specificity, lipoplex aggregation, recognition by the immune system and damage due to endosomal processing.
Figure 1.7: Biological barriers to hepatocyte-directed, cationic liposome-mediated gene transfer (adapted from Pathak et al., 2009; Wiethoff and Middaugh, 2003; Wu et al., 2002).
17