**2.1.5 Binding affinity and complexation thermodynamics**

Once the complex is released from the endosome, DNA must disassemble from its vector to be accessible to the cell machinery responsible for translating the enclosed information. For the case of polyplexes, the DNA decompaction (disassembly) is a process substantially dependent on i) the DNA-vector binding affinity, ii) the complexation thermodynamics, and iii) the solution conditions (Carlstedt et al., 2010; Prevette et al., 2007). Provided that acidic conditions demonstrated to be optimal for polyplex formation (see previous sections), we evaluated the DNA-chitosan binding affinity and complexation thermodynamics at a solution pH of 5.0. As depicted by isothermal titration calorimetry (ITC), lower valence chitosans demonstrated to have a higher binding affinity for DNA. Main results are described below.

Polycation-Mediated Gene Delivery: The Physicochemical Aspects Governing the Process 395

Table 2 summarizes the enthalpy, entropy, binding constant, and the stoichiometry of the

C(689) 29.9 + 0.75 0.27 + 0.03 -2.176 + 0.23 0.022 C(1652) 29.0 + 1.23 0.27 + 0.05 -2.712 + 0.12 0.020 C(2901) 4.82 + 0.08 0.73 + 0.01 -0.598 + 0.07 0.024

C(689) 0.095 + 0.01 0.86 + 0.02 -1.304 + 0.16 0.014 C(1652) 0.011 + 0.02 0.75 + 0.04 -1.357 + 0.12 0.014 C(2901) 0.009 + 0.01 0.62 + 0.07 -0.529 + 0.02 0.012

Reproduced from (Alatorre-Meda et al., 2011) with permission of Elsevier BV in the format Journal via

Table 2. Thermodynamic parameters of the DNA-chitosan binding process. Sub-indices next

As can be observed from this table, the DNA binding constants obtained for all chitosans are on the order of 105 to 106 and 103 to 104 M−1 for the first and second class of binding sites, respectively. These results are in good agreement with previously reported values for other systems including cationic polymers (Nisha et al., 2004; Prevette, et al., 2007) and proteins (Engler et al., 1997; Milev et al., 2005). On the other hand, the decreasing values of the binding constants with the chitosan valence reveal that lower valence chitosans have a higher binding affinity for DNA. This is indicative that chitosan chains may undergo steric restrictions as Mw (valence) increases, restrictions that in turn apparently hamper the interpolyelectrolyte interactions (Danielsen et al., 2004; Maurstad et al., 2007). Concerning the enthalpy, it is well known that it results from a combination of electrostatics, conformational changes (especially for second binding sites), and hydrogen bonding interactions; therefore, ΔH cannot be strictly related to any one contribution. However, and despite the experimental evidence demonstrating that the binding enthalpy ΔH was negative, the DNA–chitosan complexation was proved to be entropically driven. This result is in good agreement with other electrostatic, polyelectrolyte associations promoted by the release of counterions and solvent upon attraction (Matulis et al., 2000; Prevette, et al., 2007;

The potential of chitosans C(689) and C(2901) as DNA carriers towards HeLa cells was evaluated. Polyplexes and nanospheres with compositions in the range 1 ≤ N/P ≤ 18 were tested. In addition, a MEP:DNA lipoplex (4:1 μL:μg) and naked DNA were measured as positive and negative controls, respectively. Although considerably lower compared to that of the DNA–MEP lipoplex, the transfection efficiency of the polyplexes was found to increase with chitosan valence as depicted by the β-galactosidase and luciferase expression

Δ**H1 (kcal mol-1)** 

Δ**H2 (kcal mol-1)** 

Δ**S1 (kcal mol-1 K-1)** 

Δ**S2 (kcal mol-1 K-1)** 

DNA–chitosan interaction derived from the data fitting of figure 6.

**(M-1) n1**

**(M-1) n2**

to each parameter stand for the corresponding sites 1 and 2.

**chitosan K1** <sup>×</sup> **10-5**

**chitosan K2** <sup>×</sup> **10-5**

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Srinivasachari et al., 2007).

**2.1.6 Transfection efficiency** 

assays. Main findings are described below.

Reproduced from (Alatorre-Meda, et al., 2011) with permission of Elsevier BV in the format Journal via Copyright Clearance Center.

Fig. 6. Integrated heat of interaction of the titration of chitosan to DNA vs. N/P. Chitosans C(689) (squares), C(1652) (circles), and C(2901) (triangles) were titrated. Solid lines represent the two site model fitting to the experimental data.

Figure 6 shows the heat of interaction resulting from the titration of chitosan to DNA as a function of N/P. Supported on calorimetric measurements, it is well accepted that polyelectrolyte complex formation and coacervation are mainly entropically driven through the release of condensed counterions via the ion-exchange process in which an endothermic signal is recorded during the complex formation (de Kruif et al., 2004; Matulis et al., 2000). By contrast, in a result most commonly observed in the formation of protein–ligand complexes, it can be observed from figure 6 that for all experiments the injection of chitosan appears as a markedly negative signal at the beginning of the binding process followed by a gradual decrease in the released heat up until thermal equilibrium, that is, the complexation is exothermic. A similar decrease in the quantity of heat released on successive injections of titrant has been interpreted as an indicative of the progressive neutralization of charges in the reservoir molecule; meanwhile, the zone of the thermogram in which a plateau in the heat released is reached might be attributed to the complete DNA compaction (Bharadwaj et al., 2006). In this context, comparing the onset of the plateau yielded for the three chitosans during the DNA compaction it is clear that for C(2901) N/P∼1, whereas for C(689) and C(1652) N/P∼0.5. This result is consistent with those we observed by SLS (section 2.1.1) and ζ-potential (previous section) indicating that the use of higher valence chitosans, even at low pHs, apparently demands larger amounts of cationic polymer for DNA compaction. This is also supported by the 4–5-fold smaller enthalpic contribution rendered by C(2901) compared to its lower valence homologues (discussed below).


Table 2 summarizes the enthalpy, entropy, binding constant, and the stoichiometry of the DNA–chitosan interaction derived from the data fitting of figure 6.

Reproduced from (Alatorre-Meda et al., 2011) with permission of Elsevier BV in the format Journal via Copyright Clearance Center.

Table 2. Thermodynamic parameters of the DNA-chitosan binding process. Sub-indices next to each parameter stand for the corresponding sites 1 and 2.

As can be observed from this table, the DNA binding constants obtained for all chitosans are on the order of 105 to 106 and 103 to 104 M−1 for the first and second class of binding sites, respectively. These results are in good agreement with previously reported values for other systems including cationic polymers (Nisha et al., 2004; Prevette, et al., 2007) and proteins (Engler et al., 1997; Milev et al., 2005). On the other hand, the decreasing values of the binding constants with the chitosan valence reveal that lower valence chitosans have a higher binding affinity for DNA. This is indicative that chitosan chains may undergo steric restrictions as Mw (valence) increases, restrictions that in turn apparently hamper the interpolyelectrolyte interactions (Danielsen et al., 2004; Maurstad et al., 2007). Concerning the enthalpy, it is well known that it results from a combination of electrostatics, conformational changes (especially for second binding sites), and hydrogen bonding interactions; therefore, ΔH cannot be strictly related to any one contribution. However, and despite the experimental evidence demonstrating that the binding enthalpy ΔH was negative, the DNA–chitosan complexation was proved to be entropically driven. This result is in good agreement with other electrostatic, polyelectrolyte associations promoted by the release of counterions and solvent upon attraction (Matulis et al., 2000; Prevette, et al., 2007; Srinivasachari et al., 2007).

#### **2.1.6 Transfection efficiency**

394 Non-Viral Gene Therapy

**0.0 0.5 1.0 1.5 2.0 2.5 3.0**

*N/P*

Reproduced from (Alatorre-Meda, et al., 2011) with permission of Elsevier BV in the format Journal via

Fig. 6. Integrated heat of interaction of the titration of chitosan to DNA vs. N/P. Chitosans C(689) (squares), C(1652) (circles), and C(2901) (triangles) were titrated. Solid lines represent

Figure 6 shows the heat of interaction resulting from the titration of chitosan to DNA as a function of N/P. Supported on calorimetric measurements, it is well accepted that polyelectrolyte complex formation and coacervation are mainly entropically driven through the release of condensed counterions via the ion-exchange process in which an endothermic signal is recorded during the complex formation (de Kruif et al., 2004; Matulis et al., 2000). By contrast, in a result most commonly observed in the formation of protein–ligand complexes, it can be observed from figure 6 that for all experiments the injection of chitosan appears as a markedly negative signal at the beginning of the binding process followed by a gradual decrease in the released heat up until thermal equilibrium, that is, the complexation is exothermic. A similar decrease in the quantity of heat released on successive injections of titrant has been interpreted as an indicative of the progressive neutralization of charges in the reservoir molecule; meanwhile, the zone of the thermogram in which a plateau in the heat released is reached might be attributed to the complete DNA compaction (Bharadwaj et al., 2006). In this context, comparing the onset of the plateau yielded for the three chitosans during the DNA compaction it is clear that for C(2901) N/P∼1, whereas for C(689) and C(1652) N/P∼0.5. This result is consistent with those we observed by SLS (section 2.1.1) and ζ-potential (previous section) indicating that the use of higher valence chitosans, even at low pHs, apparently demands larger amounts of cationic polymer for DNA compaction. This is also supported by the 4–5-fold smaller enthalpic contribution rendered by C(2901) compared to its lower valence

**-3000**

homologues (discussed below).

the two site model fitting to the experimental data.

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**-2500**

**-2000**

**-1500**

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

**-1000**

**-500**

**0**

The potential of chitosans C(689) and C(2901) as DNA carriers towards HeLa cells was evaluated. Polyplexes and nanospheres with compositions in the range 1 ≤ N/P ≤ 18 were tested. In addition, a MEP:DNA lipoplex (4:1 μL:μg) and naked DNA were measured as positive and negative controls, respectively. Although considerably lower compared to that of the DNA–MEP lipoplex, the transfection efficiency of the polyplexes was found to increase with chitosan valence as depicted by the β-galactosidase and luciferase expression assays. Main findings are described below.

Polycation-Mediated Gene Delivery: The Physicochemical Aspects Governing the Process 397

The luciferase assay was conducted in order to confirm the results obtained by the βgalactosidase assay. The transfection efficiency for all cases was similar to that observed in the β-galactosidase method. In general, naked DNA and complexes regardless of the N/P ratio and structure yielded a luminescence three orders of magnitude lower than that of the DNA–MEP lipoplex. Concerning the protein content determined by the BCA assay, the polyplexes and nanospheres rendered protein contents slightly higher than that of the DNA–MEP lipoplex (data not shown). In consequence, the transfection efficiency of

In general, the low capacity of DNA to escape from complexes is regarded as one of the major limitations for the transfection efficiency of polyplexes (Midoux et al., 2009; Tros de Ilarduya et al., 2010). This feature has been ascribed to a number of factors. While some authors support that an excess of polycation, even in the presence of chloroquine, limits the protein expression due to in vitro cytotoxicity (Fischer et al., 2004), other authors affirm that the low tolerance of DNA to dissociate from polyplexes is presumably due to the bulky form they adopt in solution (Izumrudov et al., 1999). In our case the low transfection efficiency of the DNA–chitosan polyplexes compared to that of the lipoplex is most likely related to both the colloidal properties they exhibit and the DNA-chitosan binding affinity. On the one hand, the morphological structure depicted by TEM and AFM in conjunction with the markedly positive ζ-potentials obtained suggest a core–shell like polyplex structure with chitosan occupying the outer part of the complex (A. V. Kabanov & V. A. Kabanov, 1998). On the other hand, as mentioned before, chitosans presenting higher DNA binding affinities were found to yield lower transfection

In general, the DNA-chitosan polyplexes exhibited good colloidal properties such as sizes in the range of 180 to 250 nm, ζ-potentials of about 16 mV, and a stable core-shell like structural conformation. The influence of chitosan charge density and valence on all these

Chitosan charge density was found to play an important role on the complexation of DNA. Namely, as the solution pH got close to the chitosan pKa, the neutralization of the amino groups, entailing a decrease of chitosan charge density, resulted in a higher amount of chitosan needed for complexation. That is, the acidic conditions were found to be favorable

Chitosan valence was found to be related to both the polyplex size and, more importantly, to the transfection efficiency. On the one hand, the hydrodynamic radii of complexes increased linearly with the chitosan Mw demonstrating that larger chitosan chains produce bulkier and less soluble polyplexes. On the other hand, in what we conceive as the main contribution of our investigations, we found that higher valence chitosans, exhibiting lower

As stated before, cationic polymers most commonly studied as gene carriers include chitosan, PEI, PLL, poly(β-amino ester)s, and poly-(amidoamine) dendrimers. In addition,

polyplexes was confirmed to be low compared to that of the lipoplex.

efficiencies.

**2.1.7 Particular conclusions**

for complex formation. 2. Role of chitosan valence.

1. Role of chitosan charge density.

**2.2 The DNA-pDADMAC system**

physicochemical properties can be summarized as follows.

DNA binding affinities, yielded higher DNA transfection efficiencies.

Figure 7 shows the transfection efficiency of the polyplexes and controls as well as the protein content of the wells after lysation. Two features are observed from this figure. On the one hand, it is clear that polyplexes within the whole range of ratios rendered levels of βgalactosidase expression slightly higher than that of the negative control (DNA without polymer) with transfection efficiency being increased with chitosan valence. This result is somehow logical taking into account the lower binding affinity depicted by ITC assays for the DNA–C(2901) complex (see Table 2); that is, the DNA release from this complex in the interior of the cell is expected to be favored. On the other hand, compared to that of the DNA–MEP lipoplex, the transfection efficiency of the polyplexes is considerably lower.

Reproduced from (Alatorre-Meda et al., 2011) with permission of Elsevier BV in the format Journal via Copyright Clearance Center.

Fig. 7. Transfection efficiency of the DNA-chitosan complexes (columns) and protein content in wells after lysation (squares) vs. N/P.

Speculating that the low transfer rate of the polyplexes (compared to that of the lipoplex) might be related to cytotoxicity effects, the protein concentration was determined via the BCA assay. From figure 7 (right hand side axis) a comparable level of protein content for all formulations including the blanks was observed. Even if not totally conclusive regarding a measure of the cytotoxicity, this result shows that the cells proliferated approximately in the same way; consequently, the poor transfection efficiency shown by the DNA–chitosan polyplexes cannot be ascribed to cytotoxicity.

In the light of the transfection efficiency depicted by the polyplexes, a second kind of experiment was implemented. DNA–chitosan nanospheres were prepared following the protocol described by Mao and coworkers (Mao et al., 2001). The transfection efficiency results for C(689) are also plotted in figure 7. Compared to the polyplexes, no transfection improvement was observed, on the contrary, nanospheres in general showed a decreasing in gene expression yielding results only comparable to the naked DNA administration.

The luciferase assay was conducted in order to confirm the results obtained by the βgalactosidase assay. The transfection efficiency for all cases was similar to that observed in the β-galactosidase method. In general, naked DNA and complexes regardless of the N/P ratio and structure yielded a luminescence three orders of magnitude lower than that of the DNA–MEP lipoplex. Concerning the protein content determined by the BCA assay, the polyplexes and nanospheres rendered protein contents slightly higher than that of the DNA–MEP lipoplex (data not shown). In consequence, the transfection efficiency of polyplexes was confirmed to be low compared to that of the lipoplex.

In general, the low capacity of DNA to escape from complexes is regarded as one of the major limitations for the transfection efficiency of polyplexes (Midoux et al., 2009; Tros de Ilarduya et al., 2010). This feature has been ascribed to a number of factors. While some authors support that an excess of polycation, even in the presence of chloroquine, limits the protein expression due to in vitro cytotoxicity (Fischer et al., 2004), other authors affirm that the low tolerance of DNA to dissociate from polyplexes is presumably due to the bulky form they adopt in solution (Izumrudov et al., 1999). In our case the low transfection efficiency of the DNA–chitosan polyplexes compared to that of the lipoplex is most likely related to both the colloidal properties they exhibit and the DNA-chitosan binding affinity. On the one hand, the morphological structure depicted by TEM and AFM in conjunction with the markedly positive ζ-potentials obtained suggest a core–shell like polyplex structure with chitosan occupying the outer part of the complex (A. V. Kabanov & V. A. Kabanov, 1998). On the other hand, as mentioned before, chitosans presenting higher DNA binding affinities were found to yield lower transfection efficiencies.

### **2.1.7 Particular conclusions**

396 Non-Viral Gene Therapy

Figure 7 shows the transfection efficiency of the polyplexes and controls as well as the protein content of the wells after lysation. Two features are observed from this figure. On the one hand, it is clear that polyplexes within the whole range of ratios rendered levels of βgalactosidase expression slightly higher than that of the negative control (DNA without polymer) with transfection efficiency being increased with chitosan valence. This result is somehow logical taking into account the lower binding affinity depicted by ITC assays for the DNA–C(2901) complex (see Table 2); that is, the DNA release from this complex in the interior of the cell is expected to be favored. On the other hand, compared to that of the DNA–MEP lipoplex, the transfection efficiency of the polyplexes is considerably lower.

**MEP 4:1**

in wells after lysation (squares) vs. N/P.

polyplexes cannot be ascribed to cytotoxicity.

**0.00**

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**0.01**

**0.02**

**0.03**

**Transfection efficiency, mg-1 s-1**

**0.04**

**0.05**

**0.06**

**--**

**N/P 1**

**N/P 2**

**N/P 3**

**N/P 6**

**N/P 12**

**N/P 18**

**--**

**N/P 1**

Reproduced from (Alatorre-Meda et al., 2011) with permission of Elsevier BV in the format Journal via

Fig. 7. Transfection efficiency of the DNA-chitosan complexes (columns) and protein content

Speculating that the low transfer rate of the polyplexes (compared to that of the lipoplex) might be related to cytotoxicity effects, the protein concentration was determined via the BCA assay. From figure 7 (right hand side axis) a comparable level of protein content for all formulations including the blanks was observed. Even if not totally conclusive regarding a measure of the cytotoxicity, this result shows that the cells proliferated approximately in the same way; consequently, the poor transfection efficiency shown by the DNA–chitosan

In the light of the transfection efficiency depicted by the polyplexes, a second kind of experiment was implemented. DNA–chitosan nanospheres were prepared following the protocol described by Mao and coworkers (Mao et al., 2001). The transfection efficiency results for C(689) are also plotted in figure 7. Compared to the polyplexes, no transfection improvement was observed, on the contrary, nanospheres in general showed a decreasing in gene expression yielding results only comparable to the naked DNA administration.

**N/P 2**

**N/P 3**

**nanospheres DNA-C(689) DNA-C(2901)**

**N/P 6**

**N/P 12**

**N/P 18**

**--**

**N/P 1**

**N/P 6**

DNA-**C(689)**

**N/P 18**

**--**

**DNA**

**0**

**25**

**50**

 **Protein content per well,** 

μ**g** 

**75**

**100**

**125**

In general, the DNA-chitosan polyplexes exhibited good colloidal properties such as sizes in the range of 180 to 250 nm, ζ-potentials of about 16 mV, and a stable core-shell like structural conformation. The influence of chitosan charge density and valence on all these physicochemical properties can be summarized as follows.

1. Role of chitosan charge density.

Chitosan charge density was found to play an important role on the complexation of DNA. Namely, as the solution pH got close to the chitosan pKa, the neutralization of the amino groups, entailing a decrease of chitosan charge density, resulted in a higher amount of chitosan needed for complexation. That is, the acidic conditions were found to be favorable for complex formation.

2. Role of chitosan valence.

Chitosan valence was found to be related to both the polyplex size and, more importantly, to the transfection efficiency. On the one hand, the hydrodynamic radii of complexes increased linearly with the chitosan Mw demonstrating that larger chitosan chains produce bulkier and less soluble polyplexes. On the other hand, in what we conceive as the main contribution of our investigations, we found that higher valence chitosans, exhibiting lower DNA binding affinities, yielded higher DNA transfection efficiencies.

#### **2.2 The DNA-pDADMAC system**

As stated before, cationic polymers most commonly studied as gene carriers include chitosan, PEI, PLL, poly(β-amino ester)s, and poly-(amidoamine) dendrimers. In addition,

Polycation-Mediated Gene Delivery: The Physicochemical Aspects Governing the Process 399

in the conductivity plot for the DNA one (plots not shown). The ratio at which the conductivity inflection occurred is reported as (N/P)c (see section 2.1.1 for a complete

It is clear from table 4 that (N/P)c is governed by the charge density provided that polyplexes formed with pDADMAC homopolymers have complexation ratios lower than that of the polyplex formed with coDADMAC. This result confirms what we observed for chitosan not only with respect to the role of charge density but also regarding the (N/P)c values obtained which are very similar for both chitosan- and pDADMAC-based polyplexes

**polymer (N/P)c (N/P)\* RH (nm)** 

**p(1, < 619)** 0.7 4 79.4 + 2.1

**p(1, 929)** 0.6 2 87.0 + 6.9

**p(1, 1703)** 0.7 2 108.9 + 12.5

**p(1, 2786)** 0.6 1 112.6 + 9.7

**p(0.26, 668)** 1.5 2 199.8 + 23.5

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

Table 4. Characteristic N/P ratios and RH of the DNA-pDADMAC polyplexes. (N/P)c and (N/P)\* stand for the DNA compaction ratio (determined by conductometry) and for the ratio from which the size of the polyplexes remain constant (determined by DLS), respectively. RH is the average of the recorded values in the range (N/P)\* ≤ N/P ≤ 10.

(N/P)\* was found by DLS as a characteristic ratio from which the polyplexes adopt the most compact structure. As observed from table 4, the value of (N/P)\* follows a decreasing trend with pDADMAC valence of the polyplexes, with p(1,619) and p(1,2786) showing the highest and the lowest (N/P)\* values of 4 and 1, respectively. The interplay between (N/P)c and (N/P)\* can be reasoned in terms of the different complexation states of DNA mediated by

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

in the format Journal via Copyright Clearance Center.

pDADMAC (Fischer et al., 2004).

**2.2.2 Time stability and size** 

explanation).

(see section 2.1.1).

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


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

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 carriers along with the nomenclature cited throughout this section.
