**2.2.6 Transfection efficiency**

402 Non-Viral Gene Therapy

Figure 9 shows the heat of interaction resulting from the titration of pDADMACs to DNA as a function of N/P. Three consecutive processes along the DNA-pDADMAC binding are observed. The first phase of binding occurred at N/P molar ratios lower than ~1, drawing a biphasic nature of the binding profiles for all polymers (except for p(1,<619)). As the polymer chains began to saturate the DNA, the slightly endothermic binding enthalpy decreased and reached an exothermic minimum at an N/P ratio of ~0.5. Similar slight endothermic heats have been ascribed to entropy-driven binding processes (Matulis et al., 2000; Srinivasachari et al., 2007) and ligand interactions with the DNA minor groove (Privalov et al., 2007) Concerning the reduction in the heat of interaction Q, it might result from the combination of both a decreased accessibility of binding sites to polymer molecules due to partial saturation of DNA molecules and from the dipole-dipole interactions between water molecules oriented favorably on adjacent DNA and polymer molecules (Strey et al., 1998). At this stage, bending of single DNA strand, bridging of neighboring DNA molecules by polymer chains and hydration can contribute to DNA

The second phase of binding was characterized by an additional post-transition endothermic heat which finished in a maximum for pDADMACs at an N/P molar ratio of ~0.75, with a subsequent decrease in enthalpy due to phosphate saturation. This endothermic heat increment with a subsequent peak or discontinuity has been suggested to represent the DNA collapse (Matulis et al., 2000). The increase in Q, from the zone in which DNA chains are partially saturated (the exothermic minimum) up to the observed maximum before phosphate saturation, is attributed to a binding of further polymer molecules to the partially saturated DNA. Finally, the third phase of binding was characterized by exothermic post-transition heats after complete phosphate saturation.

**0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0**

*N/P* Reproduced from (Alatorre-Meda et al., 2010b) with permission of AMERICAN CHEMICAL SOCIETY

Fig. 9. Integrated heats of interaction of the titration of pDADMAC to DNA vs. N/P. Polymers

employed. The solid line in the inset represents the data fitting of the DNA-p(1, 1703) interaction.

p(1, <619) squares, p(1, 919) circles, p(1,1703) triangles, and p(1, 2786) diamonds were

**-150 -100 -50 0 50 100 150**

*Q***, cal mol-1**

**0.0 0.2 0.4 0.6 0.8 1.0 -200**

*N/P*

collapse (Rau & Parsegian, 1992).

**-400 -300 -200 -100**

in the format Journal via Copyright Clearance Center.

**0**

*Q***, cal mol-1**

To determine the transfection efficiency of the DNA-pDADMAC polyplexes, we performed exactly the same protocols as those described for the chitosan systems (see section 2.1.6). Similarly to the chitosan complexes, the transfer rate of the pDADMAC polyplexes was very low compared to that of the DNA-MEP lipoplex. Even worse, polyplexes within the whole range of ratios rendered levels of β-galactosidase and luciferase expressions comparable to that of naked DNA (data not shown). Given that no cytotoxic effects can be argued

Polycation-Mediated Gene Delivery: The Physicochemical Aspects Governing the Process 405

to establish a more general conclusion about the DNA-polyelectrolyte interactions, the experimental conditions implemented (salt concentration in buffer, pH, and temperature) were chosen to be similar to the other systems previously studied (Alatorre-Meda et al., 2010a). For simplicity and in order to use units consistent with the protocols established for transfection, all characterizations presented in this section are expressed in terms of the

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

**0 200 400 600 800 1000**

*L/D*

**0 4 8 12 16 20**

*L***, mg g-1**

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

(empty squares) as a function of the mass ratio L/D, and of the concentration L,

scattered by MEP and by DNA in separate. The dotted line is a guide for the eye.

Fig. 10. Average intensity of light scattered, I, by lipoplexes (filled squares) and by MEP

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

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-

**0.5 0.6 0.7 0.8 0.9 1.0 1.1**

*I\**

**200 400 600 800 1000**

*L/D*

liposome to DNA mass ratio, L/D. Main results are exposed below.

ratio as (L/D)c ~ 600. Our main findings are described below.

**MEP**

**Lipoplexes**

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

**DNA**

*I***, kC s-1**

(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 polyplexes (see sections 2.2.1 and 2.2.4).

#### **2.2.7 Particular conclusions**

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 summarized as follows.

1. Role of pDADMAC charge density.

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 (coDADMAC charge density < 1).

2. Role of pDADMAC valence.

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

#### **2.3 The DNA-MEP system**

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

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 to establish a more general conclusion about the DNA-polyelectrolyte interactions, the experimental conditions implemented (salt concentration in buffer, pH, and temperature) were chosen to be similar to the other systems previously studied (Alatorre-Meda et al., 2010a). For simplicity and in order to use units consistent with the protocols established for transfection, all characterizations presented in this section are expressed in terms of the liposome to DNA mass ratio, L/D. Main results are exposed below.
