**2.2.4 Structural organization**

400 Non-Viral Gene Therapy

compaction provided that the amount of positive charges in the polycation chain is lower, as also demonstrated for other systems encompassing non-ionic copolymers grafted to polycationic segments (Toncheva et al., 1998). Alternatively, the high hydrophilic capacity of acrylamide (AM) (Nuno-Donlucas et al., 2004) may allow larger amounts of water to be

On the other hand, the time stability of the DNA-pDADMAC polyplexes was measured by following the time evolution of RH for polyplexes at an N/P ratio of 10, as mentioned before. We observed that the sizes of the polyplexes formed with lower valence polymers remained practically constant during 7 days (ca. 85 nm). However, contrary to what we found with the chitosan systems, the DNA-p(1, 1703), -p(1, 2786), and -p(0.26, 668) polyplexes, whose initial sizes were above 100 nm, apparently underwent a structural change with time, resulting in a size reduction (data not shown). This structural rearrangement appeared to be valence-dependent since for the DNA-p(1,1703) system the size stabilization occurred from day 2 on, while for the DNA-p(1,2786) one it occurred from day 3 on. In general, it is theorized that both the branching of the pDADMAC polymer chain, expected to be present in a large extent (Wandrey et al., 1999), and the low stiffness of pDADMAC (Jaeger et al., 1989) are the main causes of such a behavior. Anyway, the final sizes of the homopolymer-

In the present study, the ζ-potential characterization was done in the range 0.2 ≤ N/P ≤ 10 for all polyplexes. Results are summarized in table 5. All polyplexes presented a positive, stable ζ-potential from N/P ratios as low as (N/P)c. As concluded for chitosan complexes,

this result suggests a complete DNA compaction. Main findings are discussed below.

**N/P p(1, < 619) p(1, 929) p(1, 1703) p(1, 2786) p(0.26, 668) 0.2** -45.0 + 3.2 -44.3 + 1.9 -43.1 + 0.7 -39.9 + 3.0 -41.4 + 2.3 **0.4** -44.6 + 1.4 -33.3 + 1.4 -35.4 + 2.2 -41.2 + 3.5 -35.5 + 1.3 **0.6** -35.3 + 1.4 -34.3 + 3.5 -33.6 + 2.0 -40.6 + 1.3 -34.0 + 6.0 **0.8** -29.0 + 5.5 -24.0 + 5.4 -16.4 + 12.0 -22.3 + 2.6 -24.0 + 1.6 **1** 7.7 + 0.8 10.5 + 0.8 10.1 + 0.5 11.8 + 1.1 -19.9 + 1.9 **2** 11.8 + 0.7 12.2 + 0.1 11.2 + 0.4 11.8 + 0.6 12.7 + 0.3 **4** 11.7 + 1.3 10.5 + 0.8 13.1 + 0.6 12.0 + 0.7 11.5 + 0.2 **6** 12.2 + 0.5 12.0 + 0.5 12.0 + 0.3 11.9 + 0.3 10.6 + 0.5 **10** 11.8 + 0.3 12.0 + 0.3 11.9 + 0.4 12.8 + 0.1 12.3 + 0.1

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

Table 5. ζ-potential of the DNA-pDADMAC polyplexes (in mV) measured at different N/P

in the format Journal via Copyright Clearance Center.

ratios.

housed in the complex interior, resulting in bulkier polyplexes.

and copolymer-based complexes were of ca. 85 and 120 nm, respectively.

**2.2.3 Surface charge**

In order to illustrate the morphology of the polyplexes, tapping mode AFM in air was conducted. Figure 8 shows images of DNA polyplexes made with p(0.26, 668) (A), and with p(1, 2786) (B) at a constant ratio N/P = 10.

Reproduced from (Alatorre-Meda et al., 2010b) with permission of AMERICAN CHEMICAL SOCIETY in the format Journal via Copyright Clearance Center.

Fig. 8. Height AFM images of DNA polyplexes made with p(0.26, 668) (A), and with p(1, 2786) (B) at N/P = 10. Bars next to images represent the Z scale in nm.

Both figures depict high populations of well-defined toroids with sizes ranging from 125 to 250 nm and 80 to 200 nm for the DNA-p(0.26, 668) and the –p(1, 2786) polyplexes, respectively. As mentioned before, the toroidal conformation suggests a maximum DNA compaction. For the case of the pDADMAC polyplexes, this maximum DNA compaction seems logical given the permanent cationic charge of the polymer; however, for the case of the coDADMAC polyplex, such a high DNA compaction appears to be somehow counterintuitive in view of the fact that three molecules of non-ionic AM are present per each molecule of cationic charged DADMAC (Alatorre-Meda et al., 2010b). Concerning the smaller sizes depicted by AFM as compared to those displayed by DLS, it should be recalled that for the former technique the samples were dried before the measurement, that is, polyplexes apparently became dehydrated.
