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

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 morphology, and vii) transfection efficiency.

Polycation-Mediated Gene Delivery: The Physicochemical Aspects Governing the Process 387

It is well accepted that linear, highly charged polyelectrolytes, at the dilute and semi-dilute regimes, can interact via a Coulombic repulsive potential which is strong enough to keep the polymer chains elongated and widely separated, although these interactions be partly screened by non-condensed counterions in solution (Manning, 1978). Polyelectrolytes in such concentration regimes produce in consequence very small scattering signals when irradiated with any source of light (Drifford & Dalbiez, 1984). The situation is rather distinct when polyelectrolytes interact one with each other or are complexed with external agents; in such a case they scatter higher amounts of light when irradiated (Drifford & Dalbiez, 1984). Based on these foundations we analyzed our polyplexes via SLS in order to follow the complexation process. SLS has proved to be a suitable tool to detect structural changes in linear biopolymers expected to be compacted provided that upon structural changes (such as the coil-globule transition observed during DNA compaction) they must scatter light to

To determine (N/P)c, we tested numerous chitosan/DNA formulations with varying N/P ratios (at a constant DNA concentration) in terms of the chitosan charge density and valence. What we found by SLS was that there exist, indeed, a molar ratio from which the structural conformation of the polyplexes remains apparently constant independently of further addition of chitosan (i.e., presenting non-accentuated changes in light scattering intensity). That molar ratio, defined as (N/P)c, proved to be strongly influenced by the chitosan charge density adopting values of around 1.5, 2.0, and 6.0 for the pHs of 5.0, 6.0, and 6.5, respectively. Our estimations, inferred from figure 1, can be discussed as follows. Figure 1 presents the light scattering intensity of the DNA-C(1652) polyplexes as a function of the N/P ratio for the three studied pHs. This figure reflects various features worth analyzing. Interestingly, the system revealed plots similar in shape, but different in I values. The plots collected at pH 5.0 and 6.0 have I values one close to the other, whereas the plot at pH 6.5 has higher I values over the whole range of N/P studied. In all three plots three distinct regions can be identified, namely at N/P = 0, at 0 < N/P ≲ 2, and at N/P >2. At N/P = 0, the system containing pure DNA shows the intensity at least five times lower than the samples at N/P > 2, indicating no aggregation. However, upon addition of chitosan to DNA, the intensity increases sharply with the maximum at N/P around 1-2 to finally level off at N/P > 2. This peculiar and interesting behavior observed when chitosan concentration in the system relative to DNA is around 1-2, is suggestive of the formation of some kind of complex structures between DNA and chitosan at this region, large in size, possibly aggregates that are responsible for the dispersion of higher amounts of light. Finally at N/P > 2, the intensity I reaches a constant value revealing the presence of well-formed, stable DNA-chitosan polyplexes with regular sizes. The N/P ratio marking the onset of the constant value in I is denoted as the (N/P)c. Very importantly, these results suggest that as the pH of the medium increases larger amounts of chitosans are required to completely compact the given amount of DNA. This phenomenon can be explained by the fact that at pHs close to its pKa (6.3–6.5), chitosan undergoes a decrease in its charge density due to the neutralization of its amino groups (Kumar et al., 2004), a feature that becomes even more pronounced for chitosans with higher molecular weights (higher valences) (MacLaughlin et al., 1998). Therefore, and having in mind that one of the driving forces of polyelectrolyte complexation is the release of counterions from the polyanion–polycation pair (Manning, 1978; Matulis et al., 2000), it is not strange that the binding affinity between DNA and chitosan lowered as the pH increased and got close to 6.5. Likewise, the highest intensities

a. Determination of the (N/P)c via static light scattering (SLS)

different extents.

It has to be noted that our characterizations were conducted as a function of two distinct cation-to-anion ratios reported in either molar (i.e., the N/P ratio, sections 2.1 and 2.2) or mass units (i.e., the L/D ratio, section 2.3). To get a complete description of the sample preparation, experimental procedures, and data analysis of the results here exposed, the reader is encouraged to consult our published papers (Alatorre-Meda et al., 2009; Alatorre-Meda et al., 2010a, 2010b; Alatorre-Meda et al., 2011). Outstanding results are presented below.
