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

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 can enumerate the following physicochemical aspects.

#### **1.4.1 Size and surface charge**

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

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 lethal damage to the cell.
