**3. Transfection efficacy of various polyamine-lipid conjugates as micellar carriers**

First, we describes trasnsfection efficacy of these polyamine-lipid conjugates. Figure 1A shows the efficacy of these carriers using β-galactosidase activity (milli-unit/well), in the absence of FBS. Compared with the commercially available transfection reagent, *O*-ethyl DOPC (E-DOPC) (MacDonald et al., 1999), which produced 0.1 milli-unit/well transgene expression, the compounds, **DCP-spd**, **DCP-spm**, **and DCP-PEI**, exhibit moderate efficiency, ~30 to 50 % of the efficiency of E-DOPC. The transfection efficiencies of the components themselves, *i.e*., DCP, spermidine, spermine, and PEI(1800) were almost negligible. Thus, the transfection activity results from the conjugation of two moieties, a hydrophilic polyamine and a hydrophobic DCP. Compound **DCP-PEI** shows the highest efficiency at 3/1 (w/w) of **DCP-PEI** /DNA, whereas the efficiency of the other compounds are comparable and insensitive to the compound/DNA ratio within the range of error. In

Polyamine – Lipid Conjugates as Effective Gene Carriers:

Complex with DNA

**DCP-PEI** 128 ± 38 115 ± 40 ellipsoid (209 ×

Micellar aggregate

**DPPAspm** 

DNA was carried out in water.

DNA suspended in aqueous solution a)

Chemical Structure, Morphology, and Gene Transfer Activity 249

**DCP-spd** 155 ± 54 409 ± 115 sphere (132) 21 aggregate (~1000 × 437) 16 **DCP-spm** 173 ± 46 237 ± 127 sphere (156) 24 aggregate (569 × 317) 20

**DPPA-spd** 90 ± 8 140 ± 40 sphere (96) 24 sphere (129) 21

**DPPA-PEI** 218 ± 35 205 ± 39 aggregate (201) 12 aggregate (201) 17

a) The ratio of polyamine conjugate / DNA was 3/1 (w/w). Complexation of polyamine conjugate with

b) Sample solution was spread on a mica surface and dried. AFM images were obtained under dry condition. Width and height of the complexes were estimated from the AFM images in Figs 2 and 3. Table 1. Estimated size and shape of polyamine-lipid micelles and complexes with ColE1

D E F

200 nm. The object indicated by the arrow is discussed in the text.

Fig. 2. AFM images of arrays of compounds, **DCP-spd** (A), **DCP-spm** (B), **DCP-PEI** (C), **DPPA-spd** (D), **DPPA-spm** (E), and **DPPA-PEI** (F). The compound was suspended in distilled water then dropped onto a mica surface by spin coating. All scale bars represent

Particle sizes of the conjugate/DNA (3/1: w/w) complex evaluated by DLS is 409 ± 115 nm for **DCP-spd**, 237 ± 127 nm for **DCP-spm**, 115 ± 40 nm for **DCP-PEI**, 140 ± 40 nm for **DPPAspd**, 109 ± 40 nm for **DPPA-spm**, and 205 ± 39 nm for **DPPA-PEI**, respectively (Table 1). With increase in the size of polyamine portion in DCP-conjugates, the particle size significantly decreases, whereas opposite tendency was observed in DPPA-conjugates. AFM images support the tendency. Figure 3 shows an AFM image of DNA (A) and of the complexes it forms with various polyamines (B – I). Figure 3A reveals a clear image of partially-coiled ColE1 plasmid DNA (6646 bp), whose size is estimated to be 300~ 400 nm.

A B C

145)

106 ± 41 109 ± 40 sphere (112) 16 sphere (172) 37

Micelle aggregate Complex with DNA

12 sphere (120) 20

Compound DLS (nm) AFM / shape (size (nm)) and height (nm) b)

Figure 1B is shown the transfection efficiency in the presence of 20% FBS. The efficiency of these compounds was not influenced by the presence of 20% FBS, retaining 80–100 % of the activity (except compound **DCP-PEI** at 3/1 (w/w)). Such serum-resistant activity was also observed for the PCL gene transfection system previously reported (Matsuura et al., 2003). It is well known that serum often inhibits transfection; such inhibition is due to binding of negatively charged serum proteins to the cationic transfection reagents resulting in forming aggregates ineffective to the transfection. Although it is not clear why the polyamine-DCP conjugates are not influenced by the presence of the serum, the polyamine part may be assumed to efficiently interact with DNA via electrostatic interactions.

Polyamine-DPPA derivatives also exhibited transfection activity, whose extent is almost comparable to the polyamine-DCP derivatives (Figure 1C). The tendency of the activity is **DPPA-spd** ≥ **DPPA-spm** > **DPPA-PEI**. In the following section, we will discuss on the relationship between gene transfer activity and morphology of polyamine-lipid complexes.
