**2. Synthetic strategy of various polyamine-lipid conjugates via facile synthetic routes**

In many cases, polycationic compounds have been synthesized through multi step reactions including protection/deprotection reactions on polyamine moieties. Our polyamine-lipid

Polyamine – Lipid Conjugates as Effective Gene Carriers:

Chemical Structure, Morphology, and Gene Transfer Activity 247

Fig. 1. Transfection efficacy of polyamine-DCP (A and B) and polyamine-DPPA conjugates (C) on VSMC, in the absence (A and C) and the presence of 20% of FBS (B). The ratios of compound/β-galactosidase plasmid DNA (w/w) were 1.5/1, 3/1, and 6/1, respectively.

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

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

**carriers**

compounds can be prepared via two-step reactions without such protection/deprotection reactions. The synthetic schemes are shown in Scheme 1, where two types of polyaminelipid conjugate; dialkyl phosphate- and diacylphosphatidic acid-based compounds, are described. In this section, we showed a synthetic strategy for polyamine-lipid conjugates via facile routes (Dewa et al., 2004a,b, 2010).
