**2.3.1 MEP to DNA complexation ratio, (L/D)c**

404 Non-Viral Gene Therapy

(Alatorre-Meda et al., 2010b), the low transfection efficiency demonstrated by pDADMAC (even lower than that of chitosan) might be ascribed to i) the polycation barrier occurring in the core-shell structure proposed (see section 2.1.4), ii) the high binding affinity depicted by ITC (high binding constants), and iii) the high degree of DNA compaction exhibited by the

In this section we described our most important findings regarding the characterization of pDADMAC as DNA carrier. In general, the DNA-pDADMAC polyplexes exhibited good colloidal properties such as sizes in the range of 80 to 200 nm, ζ-potentials of about 12 mV, and stable toroidal structural conformations. However, the transfection efficiency was found to be even lower than that of the DNA-chitosan complexes. The influence of pDADMAC charge density and valence on the physicochemical properties of the polyplexes can be

pDADMAC charge density was found of play an important role on the DNA complexation ratio, (N/P)c. Our experiments demonstrated that the (N/P)c of pDADMACs polyplexes (pDADMAC charge density = 1) are lower than half the (N/P)c of the coDADMAC polyplex

pDADMAC valence was found to increase i) the size of the polyplexes, ii) the ratio from which the sizes remain practically constant, (N/P)\*, and iii) the DNA-pDADMAC binding affinity. In general, it is well accepted that higher valence polycations produce bulkier DNA polyplexes because of steric restrictions and solubility drops, giving support to our DLS results. However, our results demonstrate that the high water solubility and permanent cationic charge of pDADMAC apparently compensate such restrictions giving rise to higher binding affinities and lower (N/P)\* ratios as the valence increases. Such high DNApDADMAC interactions proved to reduce the transfection efficiency (at least compared to the chitosan-mediated complexes). Finally, in what time stability concerns, higher valence pDADMACs were found to provoke a polyplex size reduction with time. This structural rearrangement may be related to both the branching of the pDADMAC polymer chain,

Metafectene® Pro (MEP) is a liposomal formulation that encompasses a mixture of a polyamine-lipid as the cationic group (average molecular weight of the repeat unit of 272.26 g mol-1) and DOPE as the helper lipid. It belongs to a new class of transfection reagents based on the Repulsive Membrane Acidolysis technology (RMA) developed by Biontex laboratories GmbH (Bonetta 2005). Based on its high efficiency as transfection vector toward eukaryotic cells (Aluigi et al., 2007; Ibrahim & Kim, 2008; Kwon & Kim, 2008; Spinosa et al., 2008), MEP has been routinely used in our laboratories as a positive blank for DNA transfection assays. As observed in previous sections, compared to polyplexes formed with polycations chitosan and pDADMAC, the DNA-MEP lipoplexes yielded transfection rates markedly higher; therefore, it was of our interest to characterize the DNA-MEP complexation process from a physicochemical point of view attempting to elucidate the

The present section details the physical chemistry characterization of the interactions of MEP with DNA around the mass ratio recommended for transfection (L/D ~ 700). Aiming

expected to be present in a large extent, and the low stiffness of pDADMAC.

reason why of such a big difference in the transfection efficiencies.

polyplexes (see sections 2.2.1 and 2.2.4).

1. Role of pDADMAC charge density.

(coDADMAC charge density < 1). 2. Role of pDADMAC valence.

**2.3 The DNA-MEP system** 

**2.2.7 Particular conclusions** 

summarized as follows.

As done for chitosan polyplexes, the DNA-MEP complex formation was addressed via SLS. Depicted by a sharp increase in the scattering intensity, we determined the complexation ratio as (L/D)c ~ 600. Our main findings are described below.

Reproduced from (Alatorre-Meda et al., 2010a) by permission of the PCCP Owner Societies.

Fig. 10. Average intensity of light scattered, I, by lipoplexes (filled squares) and by MEP (empty squares) as a function of the mass ratio L/D, and of the concentration L, respectively. The average intensity of light scattered by DNA is also included. The inset shows the average intensity scattered by lipoplexes normalized to the sum of the intensities scattered by MEP and by DNA in separate. The dotted line is a guide for the eye.

Figure 10 depicts the average intensity of the light scattered by lipoplexes as a function of the mass ratio L/D, and by MEP solutions in absence of DNA as a function of the concentration L. This figure shows that at L/D ~ 600 the intensity of light scattered by lipoplexes is roughly 50% lower than that scattered by their corresponding MEP solutions, whereas for (L/D) ≥ 600 this trend shifts, becoming higher the intensities scattered by the lipoplexes. An increment like this in the light scattered was observed along the DNA-

Polycation-Mediated Gene Delivery: The Physicochemical Aspects Governing the Process 407

mentioned before, their cell internalization is problematic. Cell internalization of negatively charged particles often requires the presence of cell-specific ligands (attached to particle surface) for endocytosis to occur. Such ligands build the "bridge" between cellular membranes and particles otherwise absent in view of the electrostatic repulsions (Kono et al., 2001; Sahay et al., 2010; Simoes et al., 1998). By contrast, anionic particles not bearing

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

cell-specific ligands are expected to enter cells during mitosis3 (Khalil et al., 2006).

bilayer that protects DNA from inactivation or degradation (Sternberg et al., 1994).

As observed in previous sections, compared to polyplexes formed with polycations chitosan and pDADMAC, the DNA-MEP lipoplex yielded transfection rates markedly higher. We speculate that the higher transfection efficiency of the MEP lipoplex must be related to a successful endosomal escape that is promoted simultaneously by a repulsive membrane acidolysis process and different conformational transitions adopted by DOPE upon pH changes (Bonetta 2005; Khalil et al., 2006; Tros de Ilarduya et al., 2010). Concerning the cell entrance mechanism our lipoplexes should display, we hypothesize mitosis as the most probable option given both the negative ζ-potential of the system and that, to our best knowledge, MEP does not contain any kind of cell-receptors. In this context, HeLa cells (the cells we worked with) are recognized as highly proliferating ones (Ota, et al.,2010). Importantly, as part of our protocols we seeded the cells at an 80-90% optical confluence so that transfection was practiced with the maximum possible number of healthy cells,

Emphasizing the importance of studying the lipoplex formation under the same conditions at which transfection is practiced, our results point to a ''beads on a string'' complex conformation as depicted by i) the TEM and AFM micrographs revealing coils of DNA coming out from vesicle surface, ii) the ζ-potential results showing that the transfection mass ratios are well below isoneutrality, and iii) the practically constant vesicle sizes after complexation depicted by DLS. On the other hand, a sharp increase in the intensity of light

3 Mitosis is also accepted as an important factor in the nuclear translocation of transgenes given that the integrity of the nuclear membrane is transiently lost, allowing their entrance (Khalil et al, 2006). 4 It has to be noted that this very reasoning should be applied to the chitosan- and pDADMAC-

**2.3.4 Structural organization**

**2.3.5 Transfection efficiency** 

assuring mitosis.4

mediated polyplexes.

**2.3.6 Particular conclusions** 

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

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 detected by SLS (Drifford & Dalbiez, 1984).

#### **2.3.2 Size and time stability**

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

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

#### **2.3.3 Surface charge**

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

Compared to cationic lipoplexes, negatively charged ones should offer advantages of decreased cytotoxicity and increased serum compatibility (Thakor et al., 2009); however, as mentioned before, their cell internalization is problematic. Cell internalization of negatively charged particles often requires the presence of cell-specific ligands (attached to particle surface) for endocytosis to occur. Such ligands build the "bridge" between cellular membranes and particles otherwise absent in view of the electrostatic repulsions (Kono et al., 2001; Sahay et al., 2010; Simoes et al., 1998). By contrast, anionic particles not bearing cell-specific ligands are expected to enter cells during mitosis3 (Khalil et al., 2006).
