**3.2 Influence of surface charge**

368 Non-Viral Gene Therapy

were investigated using liposomal formulations DC-Chol/DOPE, DC-Chol/DMPC, DOTAP/DOPC, DOTAP-DLPC, DOTAP/DOPE, prepared at different molar ratia. (Pozzi et al., 2009). It was shown that the most unstable lipoplexes (DOTAP/DOPC/DNA) rapidly release DNA, while the most stable ones (DC-Chol/DOPE/DNA) exhibit a lower degree of DNA release. Therefore, the results can be generalized as follows: the higher the structural stability, the lower the extent of DNA release. Using the SAXS technique it was demonstrated that the dilution of the DNA lattice takes place upon lipoplex interaction with anionic lipids, which is unequivocal proof of the charge neutralization of cationic lipids by anionic membranes (Banchelli et al., 2008; Lundqvist et al., 2008). Upon further interaction, disintegration of lipoplexes by anionic lipids as well as the formation of nonlamellar phases in lipoplex/anionic lipids mixtures are strongly affected by the shape coupling between lipoplexes and anionic lipids. Furthermore, coupling between the membrane charge densities of lipoplexes and anionic membranes contributes greatly to regulating the evolution of lipoplexes/anionic lipids mixtures and the release of plasmid

The data found in literature, describing the influence of the size of lipoplexes on the TE are contradictory. A number of researchers have argued that either size of the formed lipoplexes is not associated with TE, or that TE is not affected by initial lipoplex size (Han et al., 2008; Kearns et al., 2008; Malaekeh-Nikouei et al., 2009). Some researchers posit that large lipoplexes possess higher TE in comparison to the smaller ones. For instance, Ross et al. demonstrated that TE and cell uptake increased with the increase of the size of lipoplexes (Ross & Hui, 1999). The study of the CDAN(**18d**)/DOPE liposomes demonstrated the ability of the liposomes to form large complexes with plasmid DNA, which are characterized by the tendency for the sedimentation on the cell surface resulting in the increase of the TE (Keller et al., 2003). When siRNA was used as cargo for delivery into cells, the lipoplexes with a size between 60 and 400 nm were obtained and no influence of the lipoplex size on the efficacy of gene knockdown was observed (Spagnou et al., 2004). It was previously reported by Kawaura et al. that vesicles of a moderate size (0.4-1.4 micron) exhibit higher TE in terms of gene delivery (Kawaura et al., 1998). The data supporting the higher TE of the

When the formation of complexes has been performed in physiological ionic strength conditions, compared with 40 mM Tris buffer, the size of lipoplexes can be significantly increased (Kearns et al., 2008). In contrast, the presence of serum could slightly decrease the

 Conversely, some researchers have demonstrated that smaller lipoplexes were more efficient (Salvati et al., 2006). We studied the correlation between the size of the cationic lipids **6a-f**/nucleic acid complexes and their TE (Medvedeva, et al., 2009). The ability of the cationic lipids to deliver plasmid DNA is dependent upon the size of the lipids/DNA complexes formed in solution, which is consistent with that the maximum endocytosis by non-specialized cells requires that the particle size is below 100 nm (Chen et al., 2007). The lipid **6a** formed the smallest complexes with the plasmid DNA, characterized by a narrow size distribution; this lipid exhibited the highest TE. The lipids **6b** and **6d** formed with plasmid DNA the complexes with a wide size distribution and a large fraction of small particles inferior to 50 nm; these lipids display moderate TE. A reduction in the size of

large lipoplexes were also reported by other researchers (Ding et al., 2008).

DNA (Pozzi et al., 2009).

size of the lipoplexes (Han et al., 2008).

**3.1 Influence of size** 

The colloidal stability of the lipoplexes is determined by the surface charged of the particles, which can be expressed as zeta potential (ξ-potential). The value of ξ-potential can be changed from negative to positive depending on the N/P ratio (ratio describing the number of the negatively-charged phosphate groups of the nucleic acid to the positively charged groups of the amphiphile). N/P ration contributes significantly to the delivery of nucleic acids into cells. The complexes having a neutral charge are usually characterized by a large size and a low TE, as a result of a tendency to form aggregates and to precipitate (Salvati et al., 2006). A small redundant positive charge of the lipoplexes facilitates the efficient interaction with the negatively charged components of the cell membrane, as well as the transport *via* the cell membrane.

A direct correlation was observed between the value of ξ-potential and TE, when studying the transfection in vitro. It was demonstrated that the cationic liposomes formed by lipid **2а** had the highest ζ-potential in comparison with liposomes formed by lipids **1a,b, 2b,с** and **3а,**  and exhibited the highest TE with respect to HeLa, COS-7, and NIH 3T3 cells (Takeuchi et al., 1996). However the dependence of the structure of the lipid, ξ-potential and the TE was not always obvious (Kearns et al., 2008; Malaekeh-Nikouei et al., 2009).
