**1.4.2 Structural organization**

384 Non-Viral Gene Therapy

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

Gene transfer to eukaryotic cells is a long process encompassing several successive steps. Plasmid DNA must be packaged into complexes/particles first. Next, the DNA-containing complexes/particles must associate with cells and become internalized into them by cellular uptake processes. Following uptake, DNA-containing complexes/particles must escape the endosomal compartment into the cytoplasm and release their DNA-cargo. Finally, DNA must translocate into the cell nucleus to be transcribed into mRNA and subsequently translated into protein antigen (Nguyen et al., 2009). For all these steps to successfully occur, the final characteristics of the lipo- and polyplexes to be employed must, undoubtedly, be studied and tailored. Of especial importance for complex formation and gene delivery, we

The ability of cationic vectors to condense DNA into nano-sized complexes is believed to be crucial for gene therapy (Sahay et al., 2010; Tros de Ilarduya et al., 2010). DNA compaction, also known as condensation, is a reversible coil to globule transition favored by the binding of cationic vectors to the negatively charged DNA phosphate groups (Bloomfield, 1997). When the number of neutralized charges reaches a critical value, DNA undergoes localized bending or distortion, which facilitates the formation of complexes with sizes much smaller (in the range of nanometers) than that of the DNA coil conformation (in the range of microns) (De Smedt et al., 2000; Wilson & Bloomfield, 1979). Analyzing the impact of experimental conditions on the resulting complex dimensions, several studies have demonstrated that parameters like type, size and modification of the cationic vector, the carrier/DNA charge ratio, and also the protocol of complex formation, can all exert a strong influence (Ogris et al., 1998). On the other hand, with respect to the role of complex size on the cellular internalization mechanism, it has been demonstrated that lipo- and polyplexes with sizes up to 200 nm are taken up by the clathrin-dependent pathway, whereas aggregates larger than 500 nm are internalized via clathrin-independent mechanisms

Apart from a reduction in size, binding of cationic vectors to DNA also imparts a positive charge. Although this positive charge is important for both cellular-binding and internalization, it might also be a cause for concern for in vivo applications since cationic complexes readily bind with serum proteins such as serum albumin, promoting aggregation and blood clearance (Tros de Ilarduya et al., 2010). Additionally, an excess of positive charge, commonly reflected by complexes formed at high vector to DNA ratios, might lead to cytotoxicity provided that negatively charged cell membranes are prone to be damaged in the presence of cationic, extracellular compounds (Thomas & Klibanov, 2003). Thus, a successful gene delivery procedure, pursuing a high transfection efficiency at the lowest possible cytotoxicity, should find a delicate balance in the complex surface charge, that is, the complex must possess a high enough positive charge so as to ensure a proper cellcomplex association, but at the same time this necessary positive charge must not cause a

polyplexes from endosomes, and the transfection efficiency (Behr 1997).

**1.4 Physicochemical aspects of relevance for gene therapy** 

can enumerate the following physicochemical aspects.

**1.4.1 Size and surface charge**

(Rejman et al., 2004; Sahay et al., 2010).

lethal damage to the cell.

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; Danielsen et al., 2004).

### **1.4.3 Binding affinity**

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