**1.3 Cationic-liposomes and -polymers for gene therapy**

382 Non-Viral Gene Therapy

binding energetics, vi) the morphology, and vii) the transfection efficiency. The physicochemical characterization was carried out by means of different experimental techniques including dynamic and static light scattering (DLS and SLS), electrophoretic mobility, isothermal titration calorimetry (ITC), transmission electron- and atomic force microscopy (TEM and AFM), and conductometry at 25 ºC. Transfection experiments were conducted at standard culture conditions and evaluated by means of the β-galactosidase (βgal) and luciferase assays at 25 ºC. Outstanding results concerning the electrochemical and energetic features of the complexes with higher transfection efficiencies are fully discussed.

The possibility to treat diseases by the insertion of genes into human cells and tissues has proposed gene therapy as the therapy of the 21st century (Verma & Somia, 1997). With the first clinical trial reported in the early 1990s (Anderson, 1990), protocols for several, diverse disorders have been conducted and promising results have been obtained (O'Connor & Crystal, 2006); however, a single protocol suitable to be applied as a routinely means to treat

In practice, the entrance of naked, exogenous DNA to the cell nucleus results problematic due to different extra and intracellular barriers. On the one hand, systemic circulation of DNA is hindered by nuclease degradation (Nguyen et al., 2009). On the other hand, the electrolytic nature of DNA gives rise to electrostatic repulsions as DNA approaches to cells provided that both DNA and cell membranes are negatively charged (due to the phosphate groups distributed by the outside of the polymer helices and the several proteoglycans constituting the cell membranes) (Tros de Ilarduya et al., 2010). Also, once inside cells, steric restrictions are expected to hamper the DNA transportation to the cell nucleus given that mobility of free DNA based on diffusion in the cytoplasm is negligible (Dowty et al., 1995), possibly due to cytoskeletal elements within the cytoplasm that function as molecular sieves and prevent the diffusion of large molecules (Lubyphelps et al., 1987). Thus, for exogenous DNA to be properly transferred to living cells (and tissues), all these extra and intracellular

Current gene transfer protocols rely on the use of natural and synthetic DNA complexing agents, referred to as vectors or gene carriers, to compact, protect, and provoke a charge inversion of DNA, surpassing, by this way, the previously cited biological barriers. Vectors are either viral or non-viral. Viral delivery, also known as transduction, involves the packaging of DNA (or in some cases RNA) into a virus particle (Mancheño-Corvo & Martín-Duque, 2006). This procedure is, by far, the most effective one considering the high transfection efficiencies it renders; however, fundamental problems associated with viral vectors, including toxicity and immunogenicity, among others, have encouraged the investigation of safer gene

Compared to viruses, non-viral transfection vectors possess many important advantages such as safety, versatility, ease of preparation, and, in some cases, the possibility to transfect DNA fragments of unlimited sizes (Orth et al., 2008). The first approaches using non-viral vectors as gene carriers come from the late 1980s when Felgner and coworkers started to try with cationic liposomes (Felgner et al., 1987). Cationic liposome-DNA complexes, also referred to as lipoplexes, form spontaneously after electrostatic interactions between the positively charged liposomes and the negatively charged DNA, producing physically stable formulations suitable to transfect relatively high amounts of plasmid DNA to cells in culture

delivery alternatives such as non-viral vectors (Verma & Somia, 1997).

**1.2 Gene therapy**

any particular disease is far to be achieved.

barriers must be circumvented.

Polycations and cationic liposomes are the non-viral vectors most commonly studied for gene therapy due to the outstanding characteristics they present. In addition to the potential safety benefits, they offer, for instance, a great structural and chemical versatility for manipulating their physicochemical properties, vector stability upon storage and reconstitution, and a larger gene capacity to transfer DNA as compared to their viral and non-viral counterparts (Dias et al., 2002; Midoux et al., 2009; Tros de Ilarduya et al., 2010). As mentioned before, cationic liposomes were the first class of non-viral vectors showing satisfactory transfection efficiencies. In the first work reporting on lipofection (the lipidmediated DNA transfection process), Felgner and coworkers employed N-[1-(2,3 dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), a synthetic cationic lipid, to transfect plasmid DNA to different cells lines in culture (Felgner et al., 1987). Major advantages of utilizing DOTMA containing liposomes were that DNA entrapment inside the lipoplexes was found to be of a 100%. Also, as suggested by fluorescence microscopy data, DOTMA demonstrated to facilitate fusion of the complexes with the plasma membrane of the studied culture cells, resulting in DNA transfer rates from 5- to >100-fold more effective (depending on the transfected cell line) than previous procedures such as the calcium phosphate or the DEAE-dextran transfection techniques (Kucherlapati & Skoultchi, 1984). However, a major drawback was that these liposomes were found to be cytotoxic (Felgner et al., 1987). Following the trail of this pioneering work, other cationic lipids and surfactants have also been tested over the years (Simberg et al., 2004). Unfortunately, the general perspective is that excessive positive charges, facilitating the electrostatic interactions with negatively charged DNA, also promote cytotoxicity. As a result, zwitterionic lipids such as dioleoylphosphatidylethanolamine (DOPE) and cholesterol are nowadays commonly implemented (Tros de Ilarduya et al., 2010).

Polycations (i.e., positively charged polyelectrolytes) are macromolecules attractive to gene therapy for various reasons. Firstly, provided the high charge density they bear, they are considered as the most efficient nucleic acid-condensing agents. Different to other kinds of non-viral vectors like trivalent ions and cationic surfactants, interacting with a few consecutive DNA monomers (bases) (Sarraguca & Pais, 2006), polycations interact with DNA bases that are significantly far apart, promoting bridging between different sites in the DNA chain or between different DNA chains (Dias et al., 2003).

Secondly, because of the strong DNA-polycation interactions, DNA-polycation complexes (polyplexes) are specially effective in what DNA charge masking and extracellular protection concerns (De Smedt et al., 2000). And thirdly, given that they can be functionalized, copolymerized or structurally modified, polycation constructs can be tailored to improve cell-specific therapeutic efficacy with reduced side effects (Ke & Young, 2010). Polycations most frequently studied as gene carriers include poly(L-lysine) (PLL) (G. Y. Wu & C. H. Wu, 1987), polyethylenimine (PEI) (Boussif et al., 1995), chitosan (Mumper et al., 1995), poly(β-amino ester)s (Lynn & Langer, 2000), and poly(amido amine) (PAMAM) dendrimers (Haensler & Szoka, 1993). Generally speaking, the basicity

Polycation-Mediated Gene Delivery: The Physicochemical Aspects Governing the Process 385

The DNA ordering inside lipoplexes has been reported in the form of four main conformations: one with a short-range lamellar structure composed of flat lipid bilayers and DNA packed between them (Battersby et al., 1998; Dias et al., 2002; Lasic et al., 1997; Radler et al., 1997; Salditt et al., 1997), another where the DNA molecules are encapsulated inside a lipid bilayer forming cylindrical complexes that are closely packed on a hexagonal network (Koltover et al., 1998), another where positively charged vesicles attach to the extended DNA molecule, the so-called ''beads on a string'' model (Felgner et al., 1987; Gershon et al., 1993; Ruozi et al., 2007; Sternberg et al., 1994), and a final one where DNA is expected to collapse and attach in the form of a globule into the outer surface of positively charged vesicles (Miguel et al., 2003). What can be drawn from all four cases, whatever the nature of the interactions, is that at dilute concentrations no structural change in the systems is present, namely vesicle or bilayer disruption, whereas at high concentrations the situation becomes different with vesicles tending to disrupt and flocculate (Dias et al., 2002; Radler et al., 1997; Salditt et al., 1997;). Concerning polyplexes, rod-like, globular, and toroidal DNA condensates are the morphologies most commonly observed (Carnerup et al., 2009;

The capability of lipo- and polyplexes to avoid premature dissociation and promote the release of genetic material to the cytoplasm once inside the cell is strongly related to the binding affinity between the DNA and the vector in question (Prevette et al., 2007). Indeed, a strong binding affinity between DNA and its carrier entails a high DNA compaction and protection against degradation in the extracellular environment. However, following their escape from endosomes, the complexes need to approach the nucleus, as well as dissociate; as such, a high DNA-vector binding affinity might constitute a limiting step for transfection considering the difficulty in the separation of the DNA from its gene carrier (Tros de Ilarduya et al., 2010). For the case of polyplexes, presenting by far the highest degree of DNA binding affinity (and condensation), it is well accepted that the molecular weight (Mw) of the polycation (directly related to the cationic valence) is a key factor controlling the DNA-vector binding affinity and subsequent transfection. In general, lower Mw polycations yield higher DNA transfection efficiencies (Ziady et al., 1999) as the DNA dissociation from

As already mentioned, the present chapter concerns with the physicochemical characterization of the DNA compaction and transfection mediated by polycations and cationic liposomes. Two families of polycations, Chitosan and Poly (diallyldimethylammonium chloride) (pDADMAC), and one cationic lipid formulation extensively used worldwide in transfection assays, Metafectene® Pro (MEP), are characterized as DNA vectors. Important for the DNA complexation, the structural, electrochemical, and energetic aspects are assessed. Particular attention is paid to the effect of polycations charge density and valence on complex parameters such as i) the hydrodynamic radius, RH, ii) the stability with time, iii) the vector to DNA ratio at which complexation takes place, iv) the ζ-potential, v) the energetics of binding, vi) the

**1.4.2 Structural organization**

Danielsen et al., 2004).

**1.4.3 Binding affinity** 

them is faster (Schaffer et al., 2000).

morphology, and vii) transfection efficiency.

**2. Polycation-mediated gene delivery: Our main results** 

and degree of protonation of polycationic vectors depend on their amount of primary, secondary, and tertiary amines, which greatly influences the cell toxicity, the escape of polyplexes from endosomes, and the transfection efficiency (Behr 1997).
