**3. Helper lipids and morphology of lipoplexes**

Neutrally charged helper lipids such as DOPE, DOPC (Fig. 8.), are often employed with cationic lipids in order to gain high transfection efficiency (Felgner & Ringold, 1989). When cationic liposomes are mixed with DNA, lipoplexes are formed with heterogeneous morphologies including beads on a string structure (Felgner & Ringold, 1989), spaghettis or meatballs structure (Sternberg et al., 1994), multilamellar structure LCα, inverted hexagonal phase structure HCII (Koltover et al., 1998), a map-pin structure (Sternberg et al., 1998) and a sliding columnar phase (O'Hern & Lubensky, 1998). Helper lipids play very important role during the formation of lipoplexes by combining cationic liposomes and genes, as they could determine the morphologies of lipoplexes. It has shown lipoplex size is very important for gene transfer to actively endocytosing cells (Ross & Hui, 1999), as such the influences on transfection efficiency: DNA ratio, types of liposomes, incubation time in polyanion containing media, and time of serum addition, are channeled mostly through their influences on lipoplex size.

Fig. 8. Chemical structure of DOPE and DOPC.

### **3.1 DOPE**

300 Non-Viral Gene Therapy

disconnecting the hydrophobic and hydrophilic portions of the lipoplex, and thereby to release DNA after entering endosomes in cell because of the pH decreasing (Liu et al., 2005a, 2008). We synthesized a series of carbamate-linked cationic lipids for liposome-mediated

It is familiar to chemists that compounds comprising redox-sensitive disulphide bonds is stable chemically as long as no reducing agents, and it is expected that these disulphidelinked lipids can keep stable in the circulation system while decomposing to release DNA after entering endosomes in cells (in a similar manner as the pH-sensitive systems) (Tang & Hughes, 1999b). Byk et al. (Byk et al., 1998, 2000) prepared a series of lipopolyamines that harbor a disulfide bridge within different positions in the backbone of the lipids as biosensitive function. They found that an early release of DNA during or after penetration into cells, probably promoted by reduction of a disulfide bridge placed between the

In addition, structural variations at the linker region such as length, the specific type of chemical bonds and the relative position of the hydrocarbon chains can affect the transfection efficiency, biodegradability and stability of cationic lipids (Fujiwara et al., 2000). The level of hydration and toxicity of the lipid can also be determined by the length of the linker (Floch et al., 2000). In a word, the use of linkers incorporating functional groups that are cleavable on shorter time scales and under specific stimuli is however of emerging interest, as DNA release may here be facilitated by a triggered decomplexation

From the chemistry point of view, the structure of cationic compounds is an important factor for their transfection activity and toxicity. Some common conclusions can be achieved by comparing the different structures and their transfection activity in the same family or different families of lipids. The transfection efficiency is not only determined by one domain of cationic lipids, but also depends on the combination of them (Tang & Hughes, 1999a). In general, it seems when researchers design cationic compounds for gene delivery, the balances between the opposite factors including fluidity and rigidity, symmetry and asymmetry, saturation and unsaturation, linearity and branching, short chain and long chain, hydrophilicity and lipophilicity of compounds should be taken into serious

Neutrally charged helper lipids such as DOPE, DOPC (Fig. 8.), are often employed with cationic lipids in order to gain high transfection efficiency (Felgner & Ringold, 1989). When cationic liposomes are mixed with DNA, lipoplexes are formed with heterogeneous morphologies including beads on a string structure (Felgner & Ringold, 1989), spaghettis or meatballs structure (Sternberg et al., 1994), multilamellar structure LCα, inverted hexagonal phase structure HCII (Koltover et al., 1998), a map-pin structure (Sternberg et al., 1998) and a sliding columnar phase (O'Hern & Lubensky, 1998). Helper lipids play very important role during the formation of lipoplexes by combining cationic liposomes and genes, as they could determine the morphologies of lipoplexes. It has shown lipoplex size is very important for gene transfer to actively endocytosing cells (Ross & Hui, 1999), as such the influences on transfection efficiency: DNA ratio, types of liposomes, incubation time in polyanion containing media, and time of serum addition, are channeled mostly through

gene delivery, which proved to have good gene transfection properties (Fig. 4.).

polyamine and the lipid, implied a total loss of transfection efficiency.

**3. Helper lipids and morphology of lipoplexes** 

mechanism.

consideration.

their influences on lipoplex size.

DOPE often presents a super synergistic effect when used in cationic liposomes, because DOPE destabilized lipid bilayers, and it was believed to be involved in endosomal disruption (Litzinger & Huang, 1992), allowing the release of DNA into the cytosol (Farhood et al., 1995) and leading to mixed bilayers (Scarzello et al., 2005). Most studies have shown that lipoplexes containing the non-bilayer-phase-preferring lipid DOPE or cholesterol would promote HCII organization (Zuhorn et al., 2005). A transition from the LCα phase to the HCII phase could be expected by increasing weight fraction of DOPE, via controlling the spontaneous radius of curvature "Ro" of the lipid layers, favored by the elastic free energy (Safinya, 2001). Another helper lipid, cholesterol, could also promote HIIC organization as DOPE. It has been proved that *in vivo* applications cholesterol was a more effective helper lipid than DOPE (Lasic, 1997).

Koltover et al. (Koltover et al., 1998) disclosed the reason in the level of phase transition through synchrotron small-angle X-ray scattering (SAXS) and optical microscopy to show the phase transition from LαC to HIIC induced by DOPE via controlling the spontaneous curvature *C*o = 1/*R*o of the lipid monolayer. It has been concluded that DOPE facilitates endosomal escape by forming an unstable inverted hexagonal phase at the endosomal pH that destabilizes both the complex and the endosomal membrane. But in a recent study (Leal et al., 2010), they developed CL-siRNA complexes with a novel cubic phase nanostructure exhibiting efficient silencing at low toxicity by using glycerol monooleate other than DOPE as the helper lipid. The inverse bicontinuous gyroid cubic nanostructure was unequivocally established from synchrotron X-ray scattering data, while fluorescence microscopy revealed colocalization of lipid and siRNA in complexes.

Tubes of lipoplexes containing DOTAP/MOG, DOTAP or DOTAP/PC, and DOTAP/DOPE were observed in freeze-fracture electron micrographs. The tubes were extremely short and appeared bead-like in lipoplexes containing DOTAP/MOG, slightly longer in those containing DOTAP or DOTAP/PC, and extensively elongated in DOTAP/DOPE lipoplexes (Xu et al., 1999). The spaghetti-like structures, occurring at DNA: lipid concentrations which were used during transfection and their diameter came closest to the diameter of the nuclear pores, may be the active cationic lipoplexes (Zhdanov et al., 2002). In the study of the structure and morphology of DC-Chol–DOPE/DNA complexes it was found the existence of cluster-like aggregates made of multilamellar DNA/lipid domains coexisting with other multilamellar lipoplexes or, alternatively, with DNA-coated vesicles (Amenitsch et al., 2010). The further study showed that DC-Chol-DOPE/DNA lipoplexes preferentially used a raft mediated endocytosis, while DOTAP-DOPC/DNA systems were mainly internalized by not specific fluid phase macropinocitosys. Most efficient multicomponent lipoplexes, incorporating different lipid species in their lipid bilayer, can use multiple endocytic pathways to enter cells. Their data demonstrated that efficiency of endocytosis was

Cationic Liposomes in Different Structural Levels for Gene Delivery 303

lipoplexs on the transfection activity so far, most transfection complexes fall within an average size range of 100–300nm. The lipoplex particles can be categorized as small (≤100nm), medium (100–200nm), large (200–1000nm) or giant (≥1000nm). (Donkuru

Some times large lipoplexes sizes could be more efficient to transfer genes because large particles lead to maximum contact with cells (Kennedy et al., 2000), the formation of large intracellular vesicles which are more easily disrupted, thus releasing DNA into the cytoplasm (Escriou et al., 1998), phagocytic activity accompanied by endosomal escape (Xu et al., 1999) and faster sedimentation and better cellular trafficking (Lee et al., 2003). At the same time, some reports supported that particles with smaller size would gain high transfection efficiency (Pitard et al., 1997; Kneuer et al., 2006). The requirement for efficient transfection may be different *in vivo* and *in vitro*. Compared with *in vitro* delivery, small particles tend to have high transfection efficiency *in vivo* because of the ability of small particles to traverse narrow capillary networks. Large particles typically have low *in vivo* transfection efficiencies, while 200-400nm is the optimal size for lipoplexes *in vitro* (Zhdanov et al., 2002; Kennedy et al., 2000). Measurement of the endosomal uptake of fluorescent dextran beads of various sizes clarified that particles smaller than 200 nm were predominantly taken up by means of clathrin mediated endocytosis; with increasing the size, a shift to another mechanism occurred, so that particles larger than 500 nm were

taken up predominantly by caveolae mediated pathways (Rejman et al., 2004).

controllable assembly of lipoplexes may provide a solution to this problem.

**4. Hybrid vectors based on cationic lipids** 

**4.1 Hybrids of cationic liposomes and polymers** 

Carriere et al. (Carriere et al., 2002) have proved that lipofection inhibition by serum was largely due to the serum inhibition of lipoplex size growth, and may be overcome by using large, stable lipoplexes. Lipoplexes of over 700 nm mean diameter induced efficient transfection in the presence or absence of serum (Turek et al., 2000), but lipoplexes of less than 250 nm in size showed efficient transfection only in the absence of serum. It was reported that the particle sizes may be one of the factors that were contributed to serum resistance of EDL (ethanol-dried lipid-DNA) lipoplexes, and the large cationic lipoplexes may delay the dissociation of DNA with lipid, thereby enhancing DNA transfection

Although a general rule is not obtained until now, there is no doubt that high transfection would be gained from large lipoplexes when endocytosis is dominant, because large particles facilitate membrane contact and fusion. When cells are not actively endocytosing, either small particles may have high transfection efficiency, or lipoplex sizes don't correlate with lipofection efficiency. The possibility of a final agreement on the lipoplexes size effect may be very small, as the other conditions of every transfection case could be different. The

The hybridized utilization of non-viral vectors also provides an alternative solution to the delivery of genes. We could hybridize cationic lipsomes and polymers; introduce peptides and targeting moieties into lipids for approaching the requirements of gene therapy (Zhang

Cationic polymers could combine with DNA to form a particulate complex, polyplex, capable of gene transfer into the targeted cells (El-Aneed, 2004), because most of them are

et al., 2010).

efficiency (Lian & Ho, 2003).

et al., 2010).

regulated by shape coupling between lipoplex and membrane lipids to suggest that such a shape-dependent coupling regulated efficient formation of endocytic vesicles thus determining the success of internalization (Marchini et al., 2010).

Kato et al. (Kato et al., 2010) observed the effect of phase separation of the membrane by changing PE from DOPE to dipalmitoylethanolamine (DPPE), which corresponded to a change from a homogeneous single phase to two segregated phases of liquid-ordered and liquid-disordered states on the membrane. This study further proved that helper lipids could change the morphologies of lipoplexes through the mutual interaction with DNA based on their chemical structures. Several helper lipids such as dilauroylphosphatidylethanolamine (C12:0), dimiristoylphosphatidylethanolamine (C14:0), dipalmitoylphosphatidylethanolamine (C16:0), diphytanoylphosphatidylethanolamine (C16:0, branched), distearoylphosphatidylethanolamine (DSPE, C18:0) were compared with DOPE (C18:1) to show that the branched and unsaturated species combined with cationic lipids acted in physical synergism to increase transfection efficiency (Heinze et al., 2010).

#### **3.2 DOPC**

Ewert et al. (Ewert et al., 2004) demonstrated that *σ*Μ, the average membrane charge density of the CL-vector, was a key universal parameter that governed the transfection behavior of LαC complexes in cells. DOPC favors the formation of LαC type of lipoplexes, in which, a system of DOPC/DOTAP-DNA lipoplex showed a strong dependence on the molar fraction of neutral lipid DOPC (*Φ*DOPC) and therefore membrane charge density σΜ. The transfection efficiency started low for 0.5 < *Φ*DOPC < 0.7 and increased dramatically to a similar value, at *Φ*DOPC = 0.2, with HIIC lipoplex achieved by the DOPE/DOTAP-DNA. In contrast to LαC complexes, HIIC complexes containing DOPE exhibited no dependence on σM. The transfection efficiency increased exponentially with a linear increase of *σ*<sup>Μ</sup> for the MVL5/DOPC/DNA lipoplex bearing Lα<sup>C</sup> (Ewert et al., 2002). And then, they found that the curve of transfection efficiency versus *σ*Μ assumed a bell-shape with increasing *σ*Μ using MVL type of cationic lipids (Ahmad et al., 2005). Ewert et al. (Ewert et al., 2006) also found that hexagonally arranged tubular lipid micelles (HI C) surrounded by DNA rods were formed though DOPC was used in the dendritic lipid-based cationic liposome.

Later it has been proved that the enhanced transfection efficiency was supported by a mesoscale computer modeling of cationic lipid lipoplexes in LαC phase at high concentrations of cationic lipid (Farago et al., 2006). Recently, a study (Kedika & Srilakshmi, 2011) showed that DOPC was a more efficacious colipid than DOPE. The difference in the transfection efficiencies of lipoplexes in the presence of colipids DOPE and DOPC was explained as the uptake of the lipoplexes in the presence of DOPE took place mainly from the fusion of the lipoplexes with the plasma membrane, whereas "endocytosis" facilitated uptake in the presence of DOPC. Many researchers have agreed membrane charge density *σ*Μ is a universal parameter governing the transfection efficiency of LCα lipoplexs (Ewert et al., 2005a, 2005b; Lin, 2003). But for the question, which morphology among LαC governed main by DOPC and HIIC governed mainly by DOPE is favored in terms of transfection efficiency, we still need to carry out more research.

#### **3.3 Lipoplex sizes**

Another parameter of morphologies affecting transfection efficiency is lipoplex sizes, for the important role of lipoplex sizes in determining the nature of the entry pathway by endocytosis (Wasungu & Hoekstra, 2006). Though it is difficult to unify the size effect of

regulated by shape coupling between lipoplex and membrane lipids to suggest that such a shape-dependent coupling regulated efficient formation of endocytic vesicles thus

Kato et al. (Kato et al., 2010) observed the effect of phase separation of the membrane by changing PE from DOPE to dipalmitoylethanolamine (DPPE), which corresponded to a change from a homogeneous single phase to two segregated phases of liquid-ordered and liquid-disordered states on the membrane. This study further proved that helper lipids could change the morphologies of lipoplexes through the mutual interaction with DNA based on their chemical structures. Several helper lipids such as dilauroylphosphatidylethanolamine (C12:0), dimiristoylphosphatidylethanolamine (C14:0), dipalmitoylphosphatidylethanolamine (C16:0), diphytanoylphosphatidylethanolamine (C16:0, branched), distearoylphosphatidylethanolamine (DSPE, C18:0) were compared with DOPE (C18:1) to show that the branched and unsaturated species combined with cationic lipids acted in physical synergism to increase transfection efficiency (Heinze et al., 2010).

Ewert et al. (Ewert et al., 2004) demonstrated that *σ*Μ, the average membrane charge density of the CL-vector, was a key universal parameter that governed the transfection behavior of LαC complexes in cells. DOPC favors the formation of LαC type of lipoplexes, in which, a system of DOPC/DOTAP-DNA lipoplex showed a strong dependence on the molar fraction of neutral lipid DOPC (*Φ*DOPC) and therefore membrane charge density σΜ. The transfection efficiency started low for 0.5 < *Φ*DOPC < 0.7 and increased dramatically to a similar value, at *Φ*DOPC = 0.2, with HIIC lipoplex achieved by the DOPE/DOTAP-DNA. In contrast to LαC complexes, HIIC complexes containing DOPE exhibited no dependence on σM. The transfection efficiency increased exponentially with a linear increase of *σ*<sup>Μ</sup> for the MVL5/DOPC/DNA lipoplex bearing Lα<sup>C</sup> (Ewert et al., 2002). And then, they found that the curve of transfection efficiency versus *σ*Μ assumed a bell-shape with increasing *σ*Μ using MVL type of cationic lipids (Ahmad et al., 2005). Ewert et al. (Ewert et al., 2006) also found

C) surrounded by DNA rods were

determining the success of internalization (Marchini et al., 2010).

that hexagonally arranged tubular lipid micelles (HI

we still need to carry out more research.

**3.3 Lipoplex sizes** 

formed though DOPC was used in the dendritic lipid-based cationic liposome.

Later it has been proved that the enhanced transfection efficiency was supported by a mesoscale computer modeling of cationic lipid lipoplexes in LαC phase at high concentrations of cationic lipid (Farago et al., 2006). Recently, a study (Kedika & Srilakshmi, 2011) showed that DOPC was a more efficacious colipid than DOPE. The difference in the transfection efficiencies of lipoplexes in the presence of colipids DOPE and DOPC was explained as the uptake of the lipoplexes in the presence of DOPE took place mainly from the fusion of the lipoplexes with the plasma membrane, whereas "endocytosis" facilitated uptake in the presence of DOPC. Many researchers have agreed membrane charge density *σ*Μ is a universal parameter governing the transfection efficiency of LCα lipoplexs (Ewert et al., 2005a, 2005b; Lin, 2003). But for the question, which morphology among LαC governed main by DOPC and HIIC governed mainly by DOPE is favored in terms of transfection efficiency,

Another parameter of morphologies affecting transfection efficiency is lipoplex sizes, for the important role of lipoplex sizes in determining the nature of the entry pathway by endocytosis (Wasungu & Hoekstra, 2006). Though it is difficult to unify the size effect of

**3.2 DOPC** 

lipoplexs on the transfection activity so far, most transfection complexes fall within an average size range of 100–300nm. The lipoplex particles can be categorized as small (≤100nm), medium (100–200nm), large (200–1000nm) or giant (≥1000nm). (Donkuru et al., 2010).

Some times large lipoplexes sizes could be more efficient to transfer genes because large particles lead to maximum contact with cells (Kennedy et al., 2000), the formation of large intracellular vesicles which are more easily disrupted, thus releasing DNA into the cytoplasm (Escriou et al., 1998), phagocytic activity accompanied by endosomal escape (Xu et al., 1999) and faster sedimentation and better cellular trafficking (Lee et al., 2003). At the same time, some reports supported that particles with smaller size would gain high transfection efficiency (Pitard et al., 1997; Kneuer et al., 2006). The requirement for efficient transfection may be different *in vivo* and *in vitro*. Compared with *in vitro* delivery, small particles tend to have high transfection efficiency *in vivo* because of the ability of small particles to traverse narrow capillary networks. Large particles typically have low *in vivo* transfection efficiencies, while 200-400nm is the optimal size for lipoplexes *in vitro* (Zhdanov et al., 2002; Kennedy et al., 2000). Measurement of the endosomal uptake of fluorescent dextran beads of various sizes clarified that particles smaller than 200 nm were predominantly taken up by means of clathrin mediated endocytosis; with increasing the size, a shift to another mechanism occurred, so that particles larger than 500 nm were taken up predominantly by caveolae mediated pathways (Rejman et al., 2004).

Carriere et al. (Carriere et al., 2002) have proved that lipofection inhibition by serum was largely due to the serum inhibition of lipoplex size growth, and may be overcome by using large, stable lipoplexes. Lipoplexes of over 700 nm mean diameter induced efficient transfection in the presence or absence of serum (Turek et al., 2000), but lipoplexes of less than 250 nm in size showed efficient transfection only in the absence of serum. It was reported that the particle sizes may be one of the factors that were contributed to serum resistance of EDL (ethanol-dried lipid-DNA) lipoplexes, and the large cationic lipoplexes may delay the dissociation of DNA with lipid, thereby enhancing DNA transfection efficiency (Lian & Ho, 2003).

Although a general rule is not obtained until now, there is no doubt that high transfection would be gained from large lipoplexes when endocytosis is dominant, because large particles facilitate membrane contact and fusion. When cells are not actively endocytosing, either small particles may have high transfection efficiency, or lipoplex sizes don't correlate with lipofection efficiency. The possibility of a final agreement on the lipoplexes size effect may be very small, as the other conditions of every transfection case could be different. The controllable assembly of lipoplexes may provide a solution to this problem.
