**3. Physico-chemical properties of lipoplexes and their influence on the transfection efficiency**

It is commonly known, that the efficiency of liposome-mediated gene delivery is determined not only by the structure of cationic and helper lipids or properties of the transfected plasmids, but also by the size of the lipoplex and its ζ-potential. The structure of the supramolecular DNA–lipid complexes is dependent upon both the external (pH, degree of hydration, temperature, and the presence of doubly charged cations, i.e., Ca2+, Mg2+) and internal factors. The physico-chemical properties can alter the lifetime, distribution, and the TE efficacy of lipoplexes. Thus, in order to shed more light on the mechanism of transfection and to elucidate the structure-activity relationships, it is necessary to investigate a number of physico-chemical parameters of the lipoplexes.

Using a set of physico-chemical methods, it was demonstrated that condensation and compactization of DNA by DC-Chol (**1a**)/DOPE cationic liposomes is a result of a strong entropically-driven surface electrostatic interactions. Fluorescence anisotropy results have revealed that low cationic lipid contents in the liposomes tend to favor more fluid bilayers; which in turn are potential advantages for transfecting cells. DC-Chol/DOPE-DNA lipoplexes are represented by supramolecular complexes with a different morphology: DNA-coated unilamellar lipoplexes, lipoplex nanostructures with thickened, flattened, and deformed walls, and also multilamellar lipoplexes with or without open bilayers (Rodriguez-Pulido et al., 2008). The effects of hydration and temperature on the structure of DC-Chol/DOPE–DNA lamellar lipoplexes were also investigated (Pozzi et al., 2006). The DNA complexation and condensation properties of two established cationic liposome formulations, CDAN (**18d**)/DOPE (50:50, *m*/*m*; TrojeneTM) and DC-Chol (**1a**)/DOPE (60:40, *m*/*m*), were studied by means of biophysical methods (Keller et al., 2003). The results provide a suitable framework for the understanding of why CDAN/DOPE cationic liposomes are exceptionally efficient, in comparison with other cationic lipid-based systems, at mediating cell transfection. The liposomes CDAN (**18d**)/DOPE formed the metastable lipoplexes, exhibiting greater transfection efficiency *in vitro* in the presence of 10% serum, in comparison to DC-Chol/DOPE liposomes. This metastability may be related to the unusually low p*K*a value of 5.7 of amino groups. In addition, it was supposed that CDAN (**18d**)/DOPE-pDNA particles may have a greater tendency to interact with negatively charged serum components and facilitate the DNA release from endosomes (Keller et al., 2003).

A critical factor in the lipid-mediated gene delivery is the structural and phase evolution of lipoplexes upon interaction and mixing with anionic cellular lipids (Tarahovsky et al., 2004; Koynova et al., 2005, 2006; Koynova & MacDonald, 2007). Such a structural rearrangement is supposed to play a central role in the DNA escape process; i.e. how DNA dissociates from lipoplexes and is released into the cytoplasm. The structural and phase evolution of lipoplexes upon interaction with lipid mixtures similar to real membranes and DNA release

Non-Viral Gene Delivery Systems Based on

(Gullotti & Yeo, 2009).

**3.2 Influence of surface charge** 

transport *via* the cell membrane.

Cholesterol Cationic Lipids: Structure-Activity Relationships 369

liposomes, might facilitate their penetration through the physiological barriers, after the administration *in vivo*, in addition to the possibility of passively targeting the tumor sites, which is facilitated by the enhanced permeability of blood vessels and retention (EPR) effect

It was demonstrated that the size of latex particles has a significant effect upon the efficiency of cell uptake and the mode of the endocytic pathway (Rejman et al., 2004). Particles that have a size of 500 nm penetrated into the cell via the caveolae-mediated endocytosis. On the contrary, the microspheres having a diameter of 200 nm or less and preferred the clathrinmediated endocytosis, were found to accumulate in the lysosomal compartment. The precise control of the size of lipoplexes is important for further intracellular fate of lipoplexes that

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

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

The low transfection efficiency *in vivo* is one of factors that hinders the design of an efficient liposomal gene delivery systems. Negatively charged components of serum could interact with cationic liposome and compete with DNA for cationic liposome binding, leading to a decrease in TE. Other destructive effects of serum components attributed to its interaction with lipoplexes and early release of DNA from lipid shielding bilayer that reduces the TE. Moreover, the released nucleic acids could be recognized by Toll-like receptor expressed in B cells and dendritic cells; resulting in toxicity *via* induction of the cytokine production (Tousignant et al., 2000). Lipoplexes were reported to be generally more than 100 nm in diameter, as well as tend to self-aggregate in the blood stream, which resulting in limited

The difference between the optimal transfection parameters of *in vitro* and *in vivo* due to the profound difference of the biochemical characteristics between the cells and the organism, is the most severe problem associated with the use and practical implementation of cationic lipid or liposomes for the treatment of genetic and acquired diseases. In the case of monocationic lipids there is a single reference that the *in vivo* result corresponded to the *in* 

determine by-turn their transfection efficiencies (Rejman et al., 2006)*.* 

not always obvious (Kearns et al., 2008; Malaekeh-Nikouei et al., 2009).

**3.3 Influence of physico-chemical parameters on the TE** *in vivo*

passage through the vessel walls (Pouton & Seymour, 2001).

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 DNA (Pozzi et al., 2009).

#### **3.1 Influence of size**

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

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

 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 liposomes, might facilitate their penetration through the physiological barriers, after the administration *in vivo*, in addition to the possibility of passively targeting the tumor sites, which is facilitated by the enhanced permeability of blood vessels and retention (EPR) effect (Gullotti & Yeo, 2009).

It was demonstrated that the size of latex particles has a significant effect upon the efficiency of cell uptake and the mode of the endocytic pathway (Rejman et al., 2004). Particles that have a size of 500 nm penetrated into the cell via the caveolae-mediated endocytosis. On the contrary, the microspheres having a diameter of 200 nm or less and preferred the clathrinmediated endocytosis, were found to accumulate in the lysosomal compartment. The precise control of the size of lipoplexes is important for further intracellular fate of lipoplexes that determine by-turn their transfection efficiencies (Rejman et al., 2006)*.* 
