**3.1.4 Lipopolyplexes**

528 Non-Viral Gene Therapy

a hydrophobic moiety and are completely soluble in water. A wide variety of cationic polymers that transfect cells *in vitro* have been characterized (Midoux et al., 2008). A key determinant of polyplex gene transfer efficiency is the positive (on amine nitrogen atoms in the polymer) to negative charge ratio or the related negative to positive (N/P) ratio. Given their polymeric nature, cationic polymers can be synthesized in different lengths, with different geometry (linear versus branched), and with substitutions or additions of functional groups with relative ease and flexibility, which opens the way to extensive

> \* <sup>N</sup> H

> > N

O

P O O O

Fig. 2. Examples of commonly used lipids (DOPE and DOTAP) and polymers (PEI and poly-

Polymer-based nanoparticles are now widely used for gene and drug delivery and targeted therapy. One of the most widely applied cationic polymers used for DNA transfections is polyethyleneimine (Choosakoonkriang et al. 2003). DNA complexation with PEI, has not been found to result in an alteration of DNA conformation, remaining essentially in the B form, and the utility of PEI as a gene delivery vector has been demonstrated in numerous studies (Jere et al. 2009;Moore et al. 2009). High molecular weight PEI has been shown to be one of the most successful polymeric vectors due to the large number of protonatable amine groups that result in an enhanced ability to escape from the endosome following uptake by the cell via the so-called "proton sponge" effect. This benefit is contrasted by the high level of cellular toxicity also imparted by the number of amine groups within the polymer. Attempts to overcome this increased level of toxicity have involved using low molecular weight PEI; however, transfection efficiencies are directly correlated with decreases in molecular weight while the tendency to aggregate can increase with decreasing polymer molecular weight. Another successful strategy has involved the shielding of the polyethylenimine/DNA core with a shell of polyethylene glycol (PEG). This approach results in the formation of a dense hydrophilic

H3N

NH2

H <sup>N</sup> <sup>N</sup> H

O

NH2

H O O

> H O O

O

O

O

O

O

\*

O

NH2

structure/function relationship studies.

H

DOTAP (N-[1-(2,3-dioleyl)propyl]-N,N,N-trimethylammonium chloride)

DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine)

L-lysine) in gene therapy.

N NH2

NH2

<sup>N</sup> NH2

Branched polyethyleneimine Poly-L-lysine

H2N <sup>N</sup> <sup>N</sup> <sup>N</sup>

<sup>N</sup> H2N NH2

H2N

Lipopolyplexes (lipid-polymer-DNA complexes or LPDs) combine plasmid DNA with both a cationic polymer and liposomes via electrostatic interactions. In general, these vectors are compact particles that exhibit superior colloidal stability, reduced cytotoxicity, and provide elevated transfection efficiency compared to either polyplexes or lipoplexes alone. The cationic polymer may be covalently linked to the liposomes (e.g. lipopolylysine) or be noncovalently incorporated into a ternary lipid–polymer–DNA complex by a charge-mediated self-assembly process. The polycation component facilitates the optimal condensation of plasmid DNA, whereas lipidic components, to which targeting ligands can be attached, further stabilize the vector formulation and mediate the efficient endosomal escape of the vector following cellular internalization. LPD particles prepared using protamine as the cationic polymer and DOTAP/Chol cationic liposomes have been reported to inhibit tumor growth following i.v. administration in mice (Whitmore et al. 1999). Both *in vitro* and *in vivo* studies have demonstrated improved outcomes of (liposomes/protamine/DNA) LPDmediated gene transfer over conventional liposomes (El-Aneed 2004) . It is believed that the small size of LPD (100 to 250 nm, which is almost three to five times less than conventional lipoplexes) will facilitate endocytosis and increase the *in vivo* circulating half life.
