**2. Chemical structure of cationic compounds**

To understand the relationship between chemical structure and gene delivery, a large series of cationic lipids have been developed. In general, a cationic lipid used for gene therapy is constituted of three basic domains: a hydrophilic cationic headgroup, a hydrophobic domain, and a linker bond which joins the hydrophilic and hydrophobic regions (Fig. 1.) (Gao & Hui, 2001). The chemical structure of cationic lipids determines the physical parameters of the liposome and is an essential factor in both transfection activity and cytotoxicity levels. The effect of chemical structure modifications on gene delivery is discussed in detail through analyzing the large amount of literatures and exemplified thereinafter.

#### **2.1 Headgroup domain**

Since the first description of successful *in vitro* transfection with cationic lipid—DOTMA (Fig. 1.) by Felgner et al. in 1987 (Felgner et al., 1987), progress has been made in the design and analysis of each domain. The effect of transfection efficiency and cytotoxicity is associated with the cationic nature of the vectors, which is mainly determined by the structure of its hydrophilic headgroup. In general, the different types of headgroups fall into the following categories: primary, secondary, tertiary amines or quaternary ammonium salts (of which polyamines have often seen the most success) and guanidinium, amidine, as well as heterocyclic ring (Heyes et al., 2002). In addition, other charged groups that have been shown capable of binding plasmids have since been employed. For example, Floch et al. (Floch et al., 2000) showed that cationic lipids (Fig. 2.) characterized by a cationic charge carried by a phosphorus or arsenic atom instead of a nitrogen atom, led to an increased transfection efficiency (up to 7 times according to the cell lines tested) and reduced toxicity.

In hydrophilic headgroup, tertiary amine or quaternary ammonium groups are the most frequently used in many of the established cationic lipids (Felgner et al., 1987; Gao & Huang, 1991; Leventis & Silvius, 1990). A typical example that modified to the chemical

Cationic polymer, which condenses DNA by ionic interaction (at physiological pH), form a particulate complex, polyplex, capable of gene transfer into the targeted cells (El-Aneed, 2004). They can condense the negatively charged DNA to a relatively small size and reduce its susceptibility to nucleases so that they may be favorable for improving transfection efficacy. The introduction of polycations (such as poly-L-lysine and protamine) in cationic liposomes, as co-polymer may provide a synergistic effect on the transfection efficiency and a promising solution to the problem frustrating us (Gao & Huang, 1996; Li & Huang, 1997). In this chapter, we review the effect of cationic liposomes in different structural levels for gene delivery, and help furthering the understanding of the mechanism governing the formation and behaviour of cationic liposomes in gene delivery. The first level for studying on the structure–activity relationship of cationic lipids is the synthesis of new vectors, as well as attempts to improve transfection efficiencies and decrease cytotoxicity, through hydrophilic, hydrophobic and linker domain modifications. The second one is the study on morphologies of lipoplexes through the effects of helper lipids and particle sizes. The last one is the hybridized utilization of non-viral vectors including the complexes of cationic lipsomes and polymers, conjugates of lipids and peptides and of targeting moieties and

To understand the relationship between chemical structure and gene delivery, a large series of cationic lipids have been developed. In general, a cationic lipid used for gene therapy is constituted of three basic domains: a hydrophilic cationic headgroup, a hydrophobic domain, and a linker bond which joins the hydrophilic and hydrophobic regions (Fig. 1.) (Gao & Hui, 2001). The chemical structure of cationic lipids determines the physical parameters of the liposome and is an essential factor in both transfection activity and cytotoxicity levels. The effect of chemical structure modifications on gene delivery is discussed in detail through analyzing the large amount of literatures and exemplified

Since the first description of successful *in vitro* transfection with cationic lipid—DOTMA (Fig. 1.) by Felgner et al. in 1987 (Felgner et al., 1987), progress has been made in the design and analysis of each domain. The effect of transfection efficiency and cytotoxicity is associated with the cationic nature of the vectors, which is mainly determined by the structure of its hydrophilic headgroup. In general, the different types of headgroups fall into the following categories: primary, secondary, tertiary amines or quaternary ammonium salts (of which polyamines have often seen the most success) and guanidinium, amidine, as well as heterocyclic ring (Heyes et al., 2002). In addition, other charged groups that have been shown capable of binding plasmids have since been employed. For example, Floch et al. (Floch et al., 2000) showed that cationic lipids (Fig. 2.) characterized by a cationic charge carried by a phosphorus or arsenic atom instead of a nitrogen atom, led to an increased transfection efficiency (up to 7 times according to the cell lines tested) and

In hydrophilic headgroup, tertiary amine or quaternary ammonium groups are the most frequently used in many of the established cationic lipids (Felgner et al., 1987; Gao & Huang, 1991; Leventis & Silvius, 1990). A typical example that modified to the chemical

lipids.

thereinafter.

reduced toxicity.

**2.1 Headgroup domain** 

**2. Chemical structure of cationic compounds** 

structure of headgroup was described by Felgner et al. (Felgner et al., 1994). They synthesized a series of 2,3-dialkyloxy quaternary ammonium compounds containing a hydroxyl moiety (Fig. 3.), which were more efficient in transfection compared with DOTMA that lacks a hydroxyl group on the quaternary amine. In our previous work, we also synthesized a series of cationic lipids containing a hydroxyl moiety on the quaternary amine for liposome-mediated gene delivery (Fig. 4.). Several cationic liposomes with relatively higher transfection efficiency were selected after *in vitro* transfection studies of the prepared cationic liposomes, whose biological performance was superior or parallel to that of the commercial transfection agents, Lipofectamine2000 and DOTAP. It is suggested that the headgroup hydration can be decreased by the incorporation of a hydroxyalkyl chain capable of hydrogen bonding to neighbouring headgroups while it improves the compaction of DNA by several mechanisms, for example, DNA can form hydrogen bonds with the lipid, and the hydroxyl group can enhance the membrane hydration. Accordingly, several groups have synthesised cationic lipids that varied the chain length of the hydroxyalkyl moiety, while keeping the remaining structure unchanged, and observed that the activity of lipid increased with the decrease in the hydroxyalkyl chain length. It shows that a decrease in the number of carbon atoms in the hydroxyalkyl chain, providing more rigidity to the terminal hydroxyl group, are very efficient in compacting DNA, which is responsible for the more observed transfection activity ( Felgner et al., 1994; Bennett et al.,1997).

Fig. 1. Representative structure of the cationic lipid DOTMA. Examples of cationic lipid structural components: hydrophilic headgroup, linker bond, and hydrophobic domain.

Cationic Liposomes in Different Structural Levels for Gene Delivery 297

Guanidine and its salts are important intermediates for organic synthesis and medicine; they have also been proposed for making cationic lipids for gene delivery. Yingyongnarongkul et al. (Yingyongnarongkul et al., 2004) studied a library of aminoglycerol–diamine conjugatebased cationic lipids with urea linkage between varying length of diamines and hydrophobic chains, and found two compounds with bis-guanidinium and one tail had transfection activity superior to that of the commercial lipid transfection reagent effective and merit further investigation. In addition, several cationic lipids containing guanidine (Floch et al., 2000), amidine (Lensink et al., 2009), or cyclic guanidine (Frederic at al., 2000) in

As a way to not only bind DNA but to compact DNA, polyvalent cationic lipids known as lipopolyamines and lipospermine were synthesized, e.g., DOSPA, DOGS (Behr et al., 1989), are claimed to be more efficient than single-charged lipids such as DOTMA, DOTAP, DC-Chol, DMRIE (Ferrari et al., 1998). The choice of headgroup may depend on the desired and specific function of the cationic lipid, but for gene transfection it is essential that the headgroups of cationic lipid interact strongly with the minor groove of DNA via its multivalent headgroup, and has the ability to efficiently condense DNA. In addition, a few parameters, such as the shape (linear, T-shaped, globular, branched) and the length of the multivalent headgroup, and the distance between two consecutive nitrogen atoms in the polyamine, are also important in transfection efficiency and

In summary, the choice of cationic headgroups has expanded into the use of natural architectures and functional groups with recognized DNA binding modes. Multivalent cationic lipids are more likely to be the most efficient in compacting DNA; however, they are prone to formation of micelles typically contributing to increased toxicity (Pedroso de Lima et al., 2003). The incorporation of a heteroatomic group as the substitution of the liner amine headgroup, such as pyridinium and guanidine, can improve the compaction of DNA by the positive charge of the cationic head, and then transfection efficiency is increased and

The hydrophobic domains represent the non-polar hydrocarbon moieties of cationic lipids and are usually made of two types of hydrophobic moieties—aliphatic chains, steroid domain. Transfection efficiency and toxicity of cationic lipids can be affected by structural variations in the hydrophobic domain such as length, the specific type of chemical bonds,

Cationic lipids with aliphatic chains have been very thoroughly researched. The chains are either linear and saturated or linear and mono-unsaturated and used in liposomal vectors

and the relative position of the hydrocarbon chains (Zhi at al., 2010).

Fig. 5. Heterocyclic cationic lipids with morpholine or piperazine polar heads.

the head group have been studied.

toxicity is decreased significantly.

**2.2 Hydrophobic domain** 

cytotoxicity (Byk et al., 1998; Fujiwara et al., 2000).

Fig. 2. Alternative cations in the lipid headgroup.

Fig. 3. Double-chain cationic lipids containing a hydroxyl moiety on the quaternary amine.

Fig. 4. Transfection comparisons of lipids synthesized and commercial reagents.

A number of cationic lipids that spread the positive charge of the cationic head by delocalizing it into a heterocyclic ring have been designed in an attempt to improve transfection efficacy and to lower toxicity. Heterocyclic cationic lipids with morpholine or piperazine polar heads conjugated to cholesterol directly or via a spacer (Fig. 5.) have been reported to compare favourably with linear polyamine head groups with similar or greater number of charges (Gao & Hui, 2001). And then, the use of imidazole or pyridine rings have been also reported to display higher transfection efficiency and reduced cytotoxicity when compared with classical transfection systems (Ilies et al., 2003; Medvedeva et al., 2009).

Fig. 3. Double-chain cationic lipids containing a hydroxyl moiety on the quaternary amine.

2/1 3/1 4/1 5/1 6/1 8/1 Lipo DOTAP Liposomes

Fig. 4. Transfection comparisons of lipids synthesized and commercial reagents.

A number of cationic lipids that spread the positive charge of the cationic head by delocalizing it into a heterocyclic ring have been designed in an attempt to improve transfection efficacy and to lower toxicity. Heterocyclic cationic lipids with morpholine or piperazine polar heads conjugated to cholesterol directly or via a spacer (Fig. 5.) have been reported to compare favourably with linear polyamine head groups with similar or greater number of charges (Gao & Hui, 2001). And then, the use of imidazole or pyridine rings have been also reported to display higher transfection efficiency and reduced cytotoxicity when compared with classical transfection systems (Ilies et al., 2003;

Lipid 1/DOPE=1:1 Lipid 2/DOPE=1:1 Lipid 3/DOPE=1:1 Lipofectamine 2000

DOTAP

Fig. 2. Alternative cations in the lipid headgroup.

0

Medvedeva et al., 2009).

100000

200000

RLU/mg Z

300000

400000

500000

600000

Fig. 5. Heterocyclic cationic lipids with morpholine or piperazine polar heads.

Guanidine and its salts are important intermediates for organic synthesis and medicine; they have also been proposed for making cationic lipids for gene delivery. Yingyongnarongkul et al. (Yingyongnarongkul et al., 2004) studied a library of aminoglycerol–diamine conjugatebased cationic lipids with urea linkage between varying length of diamines and hydrophobic chains, and found two compounds with bis-guanidinium and one tail had transfection activity superior to that of the commercial lipid transfection reagent effective and merit further investigation. In addition, several cationic lipids containing guanidine (Floch et al., 2000), amidine (Lensink et al., 2009), or cyclic guanidine (Frederic at al., 2000) in the head group have been studied.

As a way to not only bind DNA but to compact DNA, polyvalent cationic lipids known as lipopolyamines and lipospermine were synthesized, e.g., DOSPA, DOGS (Behr et al., 1989), are claimed to be more efficient than single-charged lipids such as DOTMA, DOTAP, DC-Chol, DMRIE (Ferrari et al., 1998). The choice of headgroup may depend on the desired and specific function of the cationic lipid, but for gene transfection it is essential that the headgroups of cationic lipid interact strongly with the minor groove of DNA via its multivalent headgroup, and has the ability to efficiently condense DNA. In addition, a few parameters, such as the shape (linear, T-shaped, globular, branched) and the length of the multivalent headgroup, and the distance between two consecutive nitrogen atoms in the polyamine, are also important in transfection efficiency and cytotoxicity (Byk et al., 1998; Fujiwara et al., 2000).

In summary, the choice of cationic headgroups has expanded into the use of natural architectures and functional groups with recognized DNA binding modes. Multivalent cationic lipids are more likely to be the most efficient in compacting DNA; however, they are prone to formation of micelles typically contributing to increased toxicity (Pedroso de Lima et al., 2003). The incorporation of a heteroatomic group as the substitution of the liner amine headgroup, such as pyridinium and guanidine, can improve the compaction of DNA by the positive charge of the cationic head, and then transfection efficiency is increased and toxicity is decreased significantly.

#### **2.2 Hydrophobic domain**

The hydrophobic domains represent the non-polar hydrocarbon moieties of cationic lipids and are usually made of two types of hydrophobic moieties—aliphatic chains, steroid domain. Transfection efficiency and toxicity of cationic lipids can be affected by structural variations in the hydrophobic domain such as length, the specific type of chemical bonds, and the relative position of the hydrocarbon chains (Zhi at al., 2010).

Cationic lipids with aliphatic chains have been very thoroughly researched. The chains are either linear and saturated or linear and mono-unsaturated and used in liposomal vectors

Cationic Liposomes in Different Structural Levels for Gene Delivery 299

(Lee et al., 1996). Other steroid compounds used as hydrophobic moieties for cationic lipids include vitamin D (Ren et al., 2000), bile acids (Randazzo et al., 2009), antibiotic (Kichler et

To summarise, the hydrophobic domain of cationic lipids mainly includes aliphatic chains and steroid domain, which determines the phase transition temperature and the fluidity of the bilayer, and influences the stability and toxicity of liposomes, the DNA protection from

For lipids without a backbone, the linker bond that acts as a connector between the hydrophobic and cationic headgroup domains can affect the transfection efficiency, biodegradability and stability of cationic lipids. Linker bonds are commonly ether, ester (Leventis & Silvius, 1990), amide (Behr et al., 1989) or urethane (or carbamate) (Lee et al., 1996) groups (Koynova & Tenchov, 2010), but other groups such as redox-sensitive disulphide (Byk et al., 1998, 2000) have also been employed (Fig. 1.). Cationic lipids with ether bonds such as DOTMA in the linker domain generally render better transfection efficiency, but they are too stable to be biodegraded thus may cause higher toxicity. Compared with ether bonds, although cationic lipids with ester bonds such as DOTAP are more biodegradable and associated with less cytotoxicity in cultured cells (Leventis & Silvius, 1990; Choi et al., 2001), those with ester may also decrease the stability of liposomes

The chemistry of the linker has most often been of the carbamate or amide variety, both of which are chemically stable and biodegradable, and cationic lipids with these linkers could be used as efficient gene delivery carriers (Ren et al., 2001; Liu et al., 2005a, 2005b, 2008). A typical example of cationic lipid with carbamate linker is DC-Chol, which was the first lipid used in clinical trials because of its combined properties of transfection efficiency, stability, and low toxicity (Gao & Huang, 1995). As well known, when incorporating a carbamate group into the linker, it may therefore be hoped that the pH drop will act as a trigger,

al., 2005), cholestane and litocholic acid (Fujiwara et al., 2000).

Fig. 6. Glycosylated analogue of lipopolyamines.

Fig. 7. Chemical structure of GL-67.

**2.3 Linker bond** 

in systemic circulation.

nucleases, and the DNA release from complex.

ranging from C5:0 to C18:1, but oleyl, lauryl, myristyl, palmityl and stearyl, have been the most researched ones (Niculescu-Duvaz et al., 2003). A common variation is the use of branched (Ferrari et al., 2002), acetylenic (Fletcher et al., 2006) chains and *cis*monounsaturated alkyl chains (Bennett at al., 1997).

It is commonly believed that cationic lipids have one to four hydrocarbon chains. Several studies have also shown that incorporating aliphatic chains with different numbers can improve transfection efficiency potentially by promoting endosomal escape (Felgner et al., 1987; Tang & Hughes, 1999a; Gaucheron at al., 2002; Zhi et al., 2010). Cationic lipids with double-chain hydrocarbons in the hydrophobic domain represent the majority of cationic lipids synthesized so far. Cationic lipids containing two aliphatic chains such as DOTMA and DOTAP, are among the most active lipids for systemic gene delivery. However, Tang et al. (Tang & Hughes, 1999a) demonstrated that 6-lauroxyhexyl ornithinate (LHON) with one tail was more efficient and of lower cytotoxicity compared to DOTAP. Generally speaking, for aliphatic chains, single-tailed and three-tailed cationic lipids are better known as surfactants because of their ability to form micelles in solution, but they are more toxic and less efficient than their double-tailed counterparts. Usually, cationic lipids with doubletailed hydrocarbons are capable of forming liposomes by themselves or with a helper phospholipid. Therefore, most of the aliphatic chains in the cationic lipids are double-tailed.

It is generally agreed that the length and saturation of the aliphatic chains incorporated into cationic lipids significantly affect their transfection efficiency. In order to gain chain length– activity correlation, Felgner et al. (Felgner et al., 1994) studied a series of hydroxyethyl quaternary ammonium lipids with myristoyl (diC14:0, DMRIE), palmitoyl (diC16:0, DPRIE), stearoyl (diC18:0, DSRIE), and oleoyl (diC18:1, DORIE) chains. They observed that a comparison of vectors based solely on the lengths of the two aliphatic chains led to identify the order C14:0 > C18:1 > C16:0 > C18:0. Our study on double-chain cationic lipids also showed increasing transfection efficiency with decrease of the chain length (Liu et al., 2008). It was therefore proposed that cationic lipids with shorter chain length (for saturated chains) were generally important for acquiring high transfection efficiency, since they are responsible for membrane fluidity and good lipid mixing within the bilayer. Beyond that, the best chains in terms of benefit to transfection are frequently the unsaturated ones. The overwhelming majority of results showed that the unsaturated C18:1 oleyl was the optimal aliphatic chain, which was frequently the best choice for good transfection (Fletcher et al., 2006).

Some new vectors were designed to covalently connect some special moieties in the hydrophobic chains, in order to get the relationship between hydrophobic chains and transfection efficiency. Jacopin et al. (Jacopin et al., 2001) synthesized a glycosylated analogue (Fig. 6.) of the dialkylamidoglycylcarboxyspermines, which formed stable particles at low charge ratio and was efficient for gene delivery. Many groups also reported a few other glycosylated cationic bolaamphiphiles similar to the compound (Fabio et al., 2003; Brunelle et al., 2009). In addition, the fluorinated part of the hydrophobic chain can also influence the transfection efficiency of cationic lipids *in vivo* and *in vitro.* Many varieties of fluorinated cationic lipids have been developed as transfecting agents, which are very efficient in compacting DNA and delivering genes into cells *in vivo* and *in vitro* (Gaucheron et al., 2001a, 2001b, 2001c, 2001d).

In the steroid groups, cholesterol is by far the most frequently encountered and used as an alternative to aliphatic chains because of its rigidity, as well as its endogenous biodegradability and fusion activity. An example is cationic lipid 'GL-67' (Fig. 7.), which has been found to be particularly efficient for gene transfer to cultured cells and in murine lungs (Lee et al., 1996). Other steroid compounds used as hydrophobic moieties for cationic lipids include vitamin D (Ren et al., 2000), bile acids (Randazzo et al., 2009), antibiotic (Kichler et al., 2005), cholestane and litocholic acid (Fujiwara et al., 2000).

Fig. 6. Glycosylated analogue of lipopolyamines.

Fig. 7. Chemical structure of GL-67.

To summarise, the hydrophobic domain of cationic lipids mainly includes aliphatic chains and steroid domain, which determines the phase transition temperature and the fluidity of the bilayer, and influences the stability and toxicity of liposomes, the DNA protection from nucleases, and the DNA release from complex.
