**5.7 Morphological analysis of the complexes with DNA 5.7.1 Formation of complex with DNA**

To elucidate the characteristics of N/P-dependence, formation of **DCP-spd**(PCL)/DNA and **DCP-spm**(PCL)/DNA lipoplexes was analyzed by agarose gel electrophoreses and DLS analysis. Electrophoretic analysis showed N/P ratio-dependent complexation; in the lower N/P range of 5–16, the open circular DNA band vanished and the supercoiled DNA band gradually faded for both of **DCP-spd**(PCL) and **DCP-spm**(PCL) and in the higher N/P range, 20–30, the latter DNA band totally disappeared. These observations indicate that the DNA molecules are completely entrapped within the lipoplex. Ethidium bromide (EtBr) replacement experiments also reveal condensation of DNA in the N/P range of 5–30 (Dewa et al., 2010).

The particle size of lipoplexes estimated by DLS analysis is summarized in Table 2. The diameters of the **DCP-spd**(PCL) and **DCP-spm**(PCL) alone are 158 ± 56, and 159 ± 30 nm, respectively (entries 1 and 7). Lipoplexes were larger and their size increased with increasing N/P up to 5 (entries 2 and 8 at N/P = 2 and entries 3 and 9 at N/P = 5). At N/P = 5, the PCL/DNA lipoplexes became larger with a broad distribution from 650 nm to over 1 μm (entries 3 and 9). In the higher N/P range (N/P = 16–24), sizes were reduced, converging at 261 ± 114 nm (**DCP-spd**(PCL), entry 5) and 256 ± 116 nm (**DCP-spm**(PCL), entry 11). ζ-potential measurements indicated the polarity of surface charge of lipoplexes inverts from negative in the lower N/P (5–11) to positive in the higher N/P (>16) regions.

AFM images revealed characteristic morphologies of lipoplexes in the both low and high N/P ranges (Figures 6A-D). When the **DCP-spd**(PCL)/DNA and **DCP-spm**(PCL)/DNA lipoplexes at N/P = 5 were put on PLL-treated mica (positively charged surface), large

Polyamine – Lipid Conjugates as Effective Gene Carriers:

Chemical Structure, Morphology, and Gene Transfer Activity 257

Fig. 6. AFM images of PCL/plasmid DNA (ColE1; 6646 bp) lipoplexes: (A), **DCPspd**(PCL)/DNA (N/P = 5); (B), **DCP-spm**(PCL)/DNA (N/P = 5); (C), **DCP-**

**DCP-PEI**(PCL)/DNA were taken from a different area of the bare mica surface.

**5.7.2 Effect of bilayer structure on the transfection activity** 

**spd**(PCL)/DNA (N/P = 24); (D), **DCP-spm**(PCL)/DNA (N/P = 24); (E and F), **DCP-**

**PEI**(PCL)/DNA complex (N/P = 24). Scale bars shown in the images are 500 nm (A, B, and D) and 1000 nm (C, E, and F). The PCL/DNA complexes were applied to PLL-mica (A and B) and bare mica (C, D, E, F). All images were taken under an ambient air conditions. Height profiles of the objects (i)-(iii) in C and (iv)-(v) in D are shown below these images. Arrows in (ii) and (iii) indicate step-like profiles discussed in the text. Images E and F for

In the series of polyamines tested in this study, the low molecular weight polyamines were found to be more effective gene carriers when these conjugates were assembled into PCLs. The transfection activity of the PCL was ~3 times larger than as the corresponding micellar aggregate (Figure 4). The effect of the helper lipid, DOPE, was clearly substantial when compared with DPPC used instead of DOPE. DOPE, a predominantly non-lamellar lipid, is thought to facilitate fusion and destabilization of the endosomal membrane after uptake of cationic lipid/DNA complexes into a cell. In our previous report, we described intercellular trafficking of PCL (composed of cetyl-PEI and DOPE)–DNA complexes, which were taken up into cells by endosomal pathway *(*Sugiyama et al., 2004), followed by endosomal escape. In fact, transfection activity of the present PCLs was inhibited by nigericin, which is able to dissipate the pH gradient across the endosomal membranes, by 30-50%, suggesting that endosomal pathway is likely involved in the mechanism in the present lipoplex system. It appears therefore, that the mechanism of the lipofection by the compounds in this study may be similar to that of this and other agents known to be enhanced by DOPE. The lipoplexes made from the bilayer-structured PCLs evidently involve lamellar assemblies, given that AFM images reveal the presence of step-like profiles ((ii) and (iii) in Figure 6). The step-like profiles imply a lamellar complex, in which DNA rods (2 nm in diameter) are laminated between bilayers (4 nm thickness). Such an intrinsic bilayer structure may predispose lipoplexes to interact with cell and endosomal membranes. This is not the case for micellar aggregates, whose morphology is large spheres, in which polyamine conjugate

aggregates in the sub-~micrometer size range (600–1200 nm, Figures 6A and B) were observed. These structures resembled by bead-like aggregates (Yoshikawa et al., 1996) composed of small particles (80–120 nm in diameter, 8–20 nm in height) connected to one another. Such aggregates were not observed on a negatively-charged bare mica. This is understandable since the lipoplex at N/P = 5 is negatively charged, and the lipoplex must be adsorbed on the PLL-mica surface through electrostatic interaction to be imaged. The outer periphery of the large aggregates is rich in DNA molecules, presumably those were so loosely attached that they were liberated from the aggregates during electrophoresis.


Table 2. DLS analysis of various polyamine–dicetyl phosphate conjugate/DNA complexes

At the high N/P = 24, spherical complexes were observed for **DCP-spd**(PCL) and **DCPspm**(PCL) complexes; their diameters were 200–400 nm for **DCP-spd**(PCL)/DNA (C) and 150–250 nm for **DCP-spm**(PCL)/DNA lipoplexes (D), and their heights were 12–30 and 27– 60 nm, respectively. The size of the complexes is in good agreement with the values observed by DLS. The detailed topography of these **DCP-spd**(PCL)/DNA and **DCPspm**(PCL)/DNA lipoplexes showed flat-topped spheres and spherical structures (line profiles (i)–(iii) for (C) and (iv)–(vi) for (D)). The profiles (ii) and (iii) are characteristically "step-like" (arrows). The heights indicated in (ii) are 6, 10, and 14 nm. Considering the thickness of lipid bilayer (4 nm) and the diameter of DNA (2 nm), these three values correspond to one bilayer (4 nm) + DNA (2 nm) = 6 nm, a double bilayer (8 nm) + DNA = 10 nm, and a triple bilayer (12 nm) + DNA = 14 nm, respectively. Thus, the step-like structure is reasonably indicative of a smectic lamellar assembly, where DNA molecules are laminated between bilayers.

Compared with **DCP-spd**(PCL) and **DCP-spm**(PCL) lipoplexes, the size of the **DCP-PEI**(PCL)/DNA lipoplex (N/P = 24) is large and has a broad distribution (1062 ± 783 nm, Table 2, entry 14). AFM images of the **DCP-PEI**(PCL)/DNA lipoplex show aggregates with heterogeneous and featureless shapes (Figure 6E and F). EtBr replacement experiments revealed DNA condensation in a similar manner to that of **DCP-spd**(PCL) and **DCPspm**(PCL).

aggregates in the sub-~micrometer size range (600–1200 nm, Figures 6A and B) were observed. These structures resembled by bead-like aggregates (Yoshikawa et al., 1996) composed of small particles (80–120 nm in diameter, 8–20 nm in height) connected to one another. Such aggregates were not observed on a negatively-charged bare mica. This is understandable since the lipoplex at N/P = 5 is negatively charged, and the lipoplex must be adsorbed on the PLL-mica surface through electrostatic interaction to be imaged. The outer periphery of the large aggregates is rich in DNA molecules, presumably those were so

loosely attached that they were liberated from the aggregates during electrophoresis.

1 **DCP-spd** — 8 158 ± 56 249 ± 106

3 5 8 651 ± 501 1181 ± 1057

5 24 8 261 ± 114 1791 ± 1630

6 24 4 985 ± 1437 1624 ± 1566 7 **DCP-spm** — 8 159 ± 30 225 ± 102

9 5 8 764 ± 378 321 ± 181

11 24 8 256 ± 116 1767 ± 1284

12 24 4 1033 ± 1060 1209 ± 949 13 **DCP-PEI** — 8 214 ± 90 275 ± 184 14 24 8 1062 ± 783 317 ± 243 Table 2. DLS analysis of various polyamine–dicetyl phosphate conjugate/DNA complexes At the high N/P = 24, spherical complexes were observed for **DCP-spd**(PCL) and **DCPspm**(PCL) complexes; their diameters were 200–400 nm for **DCP-spd**(PCL)/DNA (C) and 150–250 nm for **DCP-spm**(PCL)/DNA lipoplexes (D), and their heights were 12–30 and 27– 60 nm, respectively. The size of the complexes is in good agreement with the values observed by DLS. The detailed topography of these **DCP-spd**(PCL)/DNA and **DCPspm**(PCL)/DNA lipoplexes showed flat-topped spheres and spherical structures (line profiles (i)–(iii) for (C) and (iv)–(vi) for (D)). The profiles (ii) and (iii) are characteristically "step-like" (arrows). The heights indicated in (ii) are 6, 10, and 14 nm. Considering the thickness of lipid bilayer (4 nm) and the diameter of DNA (2 nm), these three values correspond to one bilayer (4 nm) + DNA (2 nm) = 6 nm, a double bilayer (8 nm) + DNA = 10 nm, and a triple bilayer (12 nm) + DNA = 14 nm, respectively. Thus, the step-like structure is reasonably indicative of a smectic lamellar assembly, where DNA molecules are

Compared with **DCP-spd**(PCL) and **DCP-spm**(PCL) lipoplexes, the size of the **DCP-PEI**(PCL)/DNA lipoplex (N/P = 24) is large and has a broad distribution (1062 ± 783 nm, Table 2, entry 14). AFM images of the **DCP-PEI**(PCL)/DNA lipoplex show aggregates with heterogeneous and featureless shapes (Figure 6E and F). EtBr replacement experiments revealed DNA condensation in a similar manner to that of **DCP-spd**(PCL) and **DCP-**

PCL Micellar aggregate

entry compound N/P ratio pH size (nm)

2 2 8 231 ± 89

4 16 8 190 ± 83

8 2 8 293 ± 112

10 16 8 296 ± 168

laminated between bilayers.

**spm**(PCL).

Fig. 6. AFM images of PCL/plasmid DNA (ColE1; 6646 bp) lipoplexes: (A), **DCPspd**(PCL)/DNA (N/P = 5); (B), **DCP-spm**(PCL)/DNA (N/P = 5); (C), **DCPspd**(PCL)/DNA (N/P = 24); (D), **DCP-spm**(PCL)/DNA (N/P = 24); (E and F), **DCP-PEI**(PCL)/DNA complex (N/P = 24). Scale bars shown in the images are 500 nm (A, B, and D) and 1000 nm (C, E, and F). The PCL/DNA complexes were applied to PLL-mica (A and B) and bare mica (C, D, E, F). All images were taken under an ambient air conditions. Height profiles of the objects (i)-(iii) in C and (iv)-(v) in D are shown below these images. Arrows in (ii) and (iii) indicate step-like profiles discussed in the text. Images E and F for **DCP-PEI**(PCL)/DNA were taken from a different area of the bare mica surface.
