**2.3.4 Structural organization**

406 Non-Viral Gene Therapy

chitosan complexation process (see section 2.1.1). In that case, the increment in the scattering was attributed to the change in the particle structure, where the intensity of light scattered by the collapsed polymeric chains was confirmed to be higher than that scattered by the linear chains of DNA and chitosan before mixing. Nevertheless, in the present case the hydrodynamic radius of MEP scarcely changed after its mixing with DNA (see the following section), suggesting no change either in its vesicular conformation or in the coil conformation of DNA. Therefore, in our opinion the increase in intensity in the zone of L/D ≥ 600 can only be explained in terms of a constructive interference that presumably arises when liposomes are connected one to each other by DNA coils (see section 2.3.4), where contrary to moving freely they start to move in ensemble. This assumption becomes clearer when the normalized intensity of the lipoplexes, (I\* = Ilipoplex/(IDNA + IMEP)), is

The inset shows two well differentiated regions, one for L/D < 600 where DNA and MEP are expected not to interact, and other for (L/D) ≥ 600 where, in good agreement with the zone suggested by Biontex Laboratories GmbH and demonstrated by transfection assays (Aluigi, et al., 2007; Ibrahim & Kim, 2008; Kwon & Kim, 2008; Spinosa et al., 2008), complexation occurs. On the other hand, the lowest intensity exhibited by pure DNA, as aforementioned, is a behavior characteristic of linear molecules in solution which are hardly

Particle sizes of both MEP and lipoplexes were measured via DLS in order to be compared. We found that the size of the MEP vesicles was equivalent to that of the lipoplexes, with the latter ones being slightly smaller (ca. 135 nm). It appears then, that as DNA comes in contact with MEP, the polyanion acts as a stabilizer of the liposomes, a result that has been observed for other polymer-vesicle interactions (Antunes et al., 2009; Rodriguez-Pulido et al., 2008). Very importantly, compared to the other DNA–cationic vector formulations here studied, in particular to the DNA–chitosan system (RH up to 450 nm), the sizes depicted by the DNA– MEP complexes are considerably lower. This is believed to facilitate the cellular uptake

To check the stability of the lipoplexes, we measured the time evolution of RH of samples with L/D ≥ (L/D)c. The magnitude of RH during the testing time (7 days) changed less than a 10% in all cases, with the mean value and standard deviation lowering as the value of L/D

In order to elucidate the lipoplex charge at the transfection conditions, we studied the ζpotential of the lipoplexes around the mass ratio recommended for transfection. To our surprise, the ζ-potential of the lipoplexes at the transfection conditions resulted to be negative (data not shown). This striking result finds support on the lipoplex structural conformation we detected by TEM and AFM (see next section) showing non-complexed DNA segments. Alternatively, as reported by others (Dias et al., 2002; Radler et al., 1997; Salditt et al., 1997), there must be a coexistence of DNA and lipoplexes in which, provided

Compared to cationic lipoplexes, negatively charged ones should offer advantages of decreased cytotoxicity and increased serum compatibility (Thakor et al., 2009); however, as

increased (data not shown). Thus, the lipoplexes were validated as stable.

the negative ζ-potential, DNA is expected to be in excess.

plotted as a function of the mass ratios (inset in Fig. 10).

detected by SLS (Drifford & Dalbiez, 1984).

**2.3.2 Size and time stability**

(Tros de Ilarduya, et al., 2010).

**2.3.3 Surface charge**

Figure 11 presents typical TEM (A and B) and AFM (C and D) images obtained for lipoplexes at L/D = 1000. This figure depicts non aggregated liposomes with DNA coils coming out from their surfaces seemingly connecting them; a feature that is more easily observed in the zooms shown in panels B and D. Such a morphology, referred to as the ''beads on a string'' conformation, has been observed not only for DNA–vesicle systems but also for DNA–micellar aggregates (Ruozi et al., 2007; Wang et al., 2007). In general, this structural conformation, occurring at low lipid to DNA ratios, is believed to appear because of packing and bending constraints on the long DNA molecules (Dan, 1998). Of importance for gene therapy, the exposed DNA sections are covered by a metastable, cylindrical lipid bilayer that protects DNA from inactivation or degradation (Sternberg et al., 1994).
