**2.2.2 Time stability and size**

398 Non-Viral Gene Therapy

because of its permanent cationic charge, poly(diallyldimethylammonium chloride) (pDADMAC) has recently been explored as well (Fischer et al., 2004; Krajcik et al., 2008). pDADMAC is a water soluble cationic polymer. It is composed of mainly configurational isomers of pyrrolidinium rings and a small amount of pendant double bonds (Dautzenberg et al., 1998; Jaeger et al., 1996). With the pendent allylic double bonds being less reactive than those of the monomer, strictly linear macromolecules are formed at low conversions, but branching can proceed at high conversions as was demonstrated for commercial samples (Wandrey et al., 1999). Because of its physical structure pDADMAC is a highly flexible polymer compared to other polycations such as chitosan (Marcelo et al., 2005; Trzcinski et al., 2002). pDADMAC has been widely used in technical applications as a flocculant agent and as a composite for biosensors, which is because of its pH-independent

Homopolymers < 100 p(1, < 619)

Copolymer 250 p(0.26, 668)

In the present section we summarize outstanding results obtained in our laboratory describing relevant physicochemical characteristics of the DNA-pDADMAC complexes (Alatorre-Meda et al., 2010b). As done for chitosan in the previous section, we highlight the role of pDADMAC charge density and valence. Four homo-polymers (charge density = 1, with different valences) and one co-polymer, p(acrylamide-co-diallyldimethylammonium chloride) (coDADMAC) (charge density < 1, equivalent in valence to one of the homopolymers), were employed. Table 3 lists the cationic polymers characterized as gene

Different to the chitosan system, the DNA-pDADMAC polyplexes exhibited two distinct characteristic ratios: the previously observed (N/P)c (i.e., the ratio from which DNA is compacted) and (N/P)\*, an additional ratio from which the polyplexes adopt the most compact structure. Similarly to the DNA-chitosan polyplexes, (N/P)c was found to be dependent on the polycationic charge density, whereas (N/P)\* proved to be a function of pDADMAC valence. Table 4 summarizes the characteristic ratios for each DNA-pDADMAC system together with the average size of the polyplexes formed (discussed in the next

(N/P)c was determined by means of conductometry. pDADMAC aliquots were injected to DNA and buffer solutions and the change in conductivity was recorded. Successive pDADMAC injections produced exactly the same outcome observed for the chitosan system; namely, a linear increment in conductivity for the buffer solution and an inflection

Table 3. pDADMACs employed. In p(x,y), x and y stand for charge density and valence,

**Mw (kDa) Label** 

150 p(1, 929) 275 p(1, 1703) 450 p(1, 2786)

cationic charge (Dautzenberg et al., 1998; Jaeger et al., 1996).

carriers along with the nomenclature cited throughout this section.

**2.2.1 DNA-pDADMAC characteristic ratios, (N/P)c and (N/P)\***

section). Our main findings can be described as follows.

respectively.

The characterization of the DNA-pDADMAC polyplexes in terms of size and time stability was carried out by means of DLS. The study was done in two steps. First, the hydrodynamic radii of the polyplexes, RH, were determined at 0.2 ≤ N/P ≤ 10. And second, the evolution of RH with time was followed for the polyplexes at N/P = 10. RH results are depicted in table 4. It is observed from table 4, that similarly to the chitosan systems, the size of the polyplexes was found to increase with pDADMAC valence, that is, the general assumption that the electrostatic interactions are outweighed to a certain extent by a decrease in the polycation solubility is confirmed (MacLaughlin et al., 1998; Mumper et al., 1995). Concerning the coDADMAC polyplex, it is clear that its size is ca. twice as big as those of the pDADMAC polyplexes. This outcome can be a consequence of an expected lower degree of DNA

Polycation-Mediated Gene Delivery: The Physicochemical Aspects Governing the Process 401

The data in table 5 reveal that all polyplexes, regardless of pDADMAC valence, present a constant ζ-potential value of around 12 mV at N/P > (N/P)c, which is in good agreement with the chitosan systems. Of special interest for gene therapy is the fact that the presence of AM, expected to improve the polyplex biocompatibility, does not cause a decrease in the positive charge recommended for transfection. Very importantly, these constant ζ-potential values, irrespective of further addition of pDADMAC, are also suggestive of a polyplex core-shell conformation, as observed for the chitosan-mediated complexes (see section 2.1.3).

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

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

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,

As done for the DNA-chitosan polyplexes, ITC was performed to evaluate the DNApDADMAC binding affinity and complexation thermodynamics. Striking results were obtained as compared to the chitosan systems. Firstly, the DNA binding affinity of pDADMACs was found to be favored with valence; secondly, the complexation process was

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.

**2.2.4 Structural organization**

p(1, 2786) (B) at a constant ratio N/P = 10.

in the format Journal via Copyright Clearance Center.

polyplexes apparently became dehydrated.

**2.2.5 Binding affinity and complexation thermodynamics** 

completed in three successive stages. Main results are discussed below.

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 housed in the complex interior, resulting in bulkier polyplexes.

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

#### **2.2.3 Surface charge**

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.


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

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

The data in table 5 reveal that all polyplexes, regardless of pDADMAC valence, present a constant ζ-potential value of around 12 mV at N/P > (N/P)c, which is in good agreement with the chitosan systems. Of special interest for gene therapy is the fact that the presence of AM, expected to improve the polyplex biocompatibility, does not cause a decrease in the positive charge recommended for transfection. Very importantly, these constant ζ-potential values, irrespective of further addition of pDADMAC, are also suggestive of a polyplex core-shell conformation, as observed for the chitosan-mediated complexes (see section 2.1.3).
