**5. Morphological effect of polyamine-lipid/DNA complexes on their transfection activity**

As described above, when the polyamine-lipid conjugates were suspended in aqueous solution, they form micellar aggregates and exhibit moderate gene-transfer activity, the magnitude of which is relatively insensitive to the modification of the polyamine portion. Here we describe that preformed bilayer structure (as polycation liposomes, PCLs) significantly improves transfection efficacy compared with micellar aggregate form.

Polyamine – Lipid Conjugates as Effective Gene Carriers:

assay Kit (PIERCE) to give a relative light unit (RLU/mg protein).

activity, also had slightly higher toxicity than the former.

transfection activity of its complexes relative to those containing DPPC.

**5.4 Optimal structure of the conjugate molecules for gene transfer** 

**dicetyl phosphate derivatives of spermidine, sperimine, and polyamine** 

Chemical Structure, Morphology, and Gene Transfer Activity 253

of 5% CO2 in air. One day before a transfection experiment, 1×105 COS-1 cells were seeded onto each of several 35-mm dishes and incubated overnight in a CO2 incubator. Then, the cells were washed twice with DMEM, and a suspension of lipoplex (1 μg DNA) was added to them in the presence of 10% FBS-DMEM. After 3 h incubation (37C, 5% CO2), the cells were washed twice with DMEM and cultured for another 48 h in 10% FBS-DMEM. The cells in the 35-mm dishes were washed twice with phosphate-buffered saline at 37C, and 200 μL of cell lysis buffer (LC-β, TOYO B-Net Co. Ltd., Tokyo) were added. After 15 min incubation, the cells were collected with a cell scraper, frozen at -80 C, and then thawed at room temperature. The lysate was centrifuged at 15,000 rpm for 5 min at 4 C. The supernatant was subjected to the luciferase assay (Pica Gene, TOYO B-Net Co. Ltd., Tokyo) using a luminophotometer (Luminescencer-PSN AB-2200, ATTO). The observed intensity in instrument light units was normalized to the amount of protein determined by BCA protein

**5.3 Transfection efficacy of micellar aggregates and PCL vectors: Comparison of the** 

Figure 4A shows the transfection efficacy of the polyamine conjugate, **DCP-spd**, and its constituent molecules, DCP and spermidine (spd). The conjugate (in this case an aqueous micellar suspension) shows greatly increased efficacy relative to the constituent molecules, DCP and spermidine. For these polyamine/DNA complexes, the nitrogen/phosphorous ratio (N/P) was 16. The data of the figure indicate that coupling the lipophilic and cationic portions is essential to obtain gene transfer. Such an effect of conjugating these two moieties was also observed for the spermine conjugate, **DCP-spm** (Figure 4B). When the conjugates were further formulated with DOPE and cholesterol (conjugate/DOPE/cholesterol = 1/1/1 (mol/mol/mol)) to generate polycationic liposomes (PCL) (**DCP-spd**(PCL) and **DCPspm**(PCL)), efficacies were further enhanced by a factor of 2–3 relative to the micellar aggregate suspensions (**DCP-spd** and **DCP-spm**). The polycation PEI(1800) itself showed moderate activity and the conjugate, **DCP-PEI**, exhibited even greater activity. However, in contrast to the other conjugates, the activity of the liposomal form, **DCP-PEI**(PCL), was comparable to that of the micellar version, **DCP-PEI**. The cytotoxicity of the conjugates, both as micellar aggregates and PCLs, was low; the latter vectors, which exhibited higher

It was found that the DOPE-based PCL exhibits significantly greater activity than the DPPCbased compound for both of the **DCP-spd**(PCL) and the **DCP-spm**(PCL). This is indicative of a lipid-mediated gene transfer mechanism; DOPE is well-known as a "helper" lipid, which is believed to facilitate membrane fusion and endosomal escape of the DNA (Felgner et al., 1994). DOPE in the present PCL systems presumably also plays a role in the mechanism to expedite membrane fusion and destabilization of the endosomal membrane. DPPC, whose Tm is 41.5 C, renders the PCL more stable and more rigid than does DOPE. Thus, it is likely that the fusogenic property of DOPE is responsible for the enhanced

The facile synthetic route provides a variety of polycationic compounds that can be exploited to examine the effect of the polycationic and hydrophobic portions on transfection efficiency. We hence examined the effect on transfection activity of different polyamine

#### **5.1 Preparation of Micellar Aggregates, PCL Vectors, and their complexes with DNA**

Polycation liposome suspensions were typically prepared as follows: polyamine conjugate, phospholipid, 1,2-dioleoyl-*sn*-glycero-3-phosphoethanolamine (DOPE), and cholesterol (1/1/1 as a molar ratio) were dissolved in t-butyl alcohol. After removal of the solvent under reduced pressure, the residual solvent was removed by freeze-drying overnight. The lyophilized powder was hydrated with Tris-HCl buffer (20 mM, pH 8.0) followed by three freeze–thaw cycles and the resultant suspension was then subsequently extruded through polycarbonate membranes of 0.4, 0.2, and 0.1-μm pore diameter at room temperature. A suspension of the polyamine conjugate alone was prepared in the Tris-HCl buffer (20 mM, pH 8.0) by ultrasonication for 3 min. For convenience, particles so prepared are termed "micellar aggregates". Hereafter, polycationic liposomes (PCL) composed of the conjugates are described as "**conjugate**(PCL)", such as **DCP-spd**(PCL). Otherwise, micellar aggregates are described as just the name of conjugate, such as **DCP-spd**.

A plasmid encoding luciferase gene, pCAG-luc3 (6480 bp, a gift of DNAVEC Institute, Tsukuba, Japan), was amplified in E. coli JM109 (Nippon Gene, Toyama, Japan) and purified as described before (Matsuura et al., 2003). One microgram of the plasmid DNA in a TE buffer was added to a suspension of PCL containing 1 mM of polyamine conjugate so as to give the desired nitrogen/phosphate ratio, N/P. The mixture was incubated for 20 min at room temperature when used for transfection.

Fig. 4. Transfection efficacy of polyamine–lipid conjugates, **DCP-spd** (A), **DCP-spm** (B), and **DCP-PEI** (C), and their constituents, DCP (A) and polyamines, spermidine (spd, A), spermine (spm, B) and PEI1800 (PEI, C), on COS-1 cells. Efficacy was evaluated with the luciferase activity. The observed values for spermidine and spermine indicated by the asterisks (\*) were apparently negligible on the activity scale shown. The conjugates **DCPspd**, **DCP-spm**, and **DCP-PEI** represent their micellar aggregate forms, and **DCP-spd**(PCL), **DCP-spm**(PCL), and **DCP-PEI**(PCL) represent the conjugate-based PCLs (conjugate/DOPE/cholesterol = 1/1/1 (mol/mol/mol)). The nitrogen/phosphate (N/P) ratio was 16/1 for the polyamine conjugate/DNA complexes. For the monoanionic DCP, the molar ratio, DCP/nucleotide = 16/1, was applied as a negative control experiment. Transfection was conducted in the presence of 10% FBS.

#### **5.2 Transfection procedure**

COS-1 cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Japan Bioserum Co. Ltd.) under a humidified atmosphere

**5.1 Preparation of Micellar Aggregates, PCL Vectors, and their complexes with DNA**  Polycation liposome suspensions were typically prepared as follows: polyamine conjugate, phospholipid, 1,2-dioleoyl-*sn*-glycero-3-phosphoethanolamine (DOPE), and cholesterol (1/1/1 as a molar ratio) were dissolved in t-butyl alcohol. After removal of the solvent under reduced pressure, the residual solvent was removed by freeze-drying overnight. The lyophilized powder was hydrated with Tris-HCl buffer (20 mM, pH 8.0) followed by three freeze–thaw cycles and the resultant suspension was then subsequently extruded through polycarbonate membranes of 0.4, 0.2, and 0.1-μm pore diameter at room temperature. A suspension of the polyamine conjugate alone was prepared in the Tris-HCl buffer (20 mM, pH 8.0) by ultrasonication for 3 min. For convenience, particles so prepared are termed "micellar aggregates". Hereafter, polycationic liposomes (PCL) composed of the conjugates are described as "**conjugate**(PCL)", such as **DCP-spd**(PCL). Otherwise, micellar aggregates

A plasmid encoding luciferase gene, pCAG-luc3 (6480 bp, a gift of DNAVEC Institute, Tsukuba, Japan), was amplified in E. coli JM109 (Nippon Gene, Toyama, Japan) and purified as described before (Matsuura et al., 2003). One microgram of the plasmid DNA in a TE buffer was added to a suspension of PCL containing 1 mM of polyamine conjugate so as to give the desired nitrogen/phosphate ratio, N/P. The mixture was incubated for 20 min at

Fig. 4. Transfection efficacy of polyamine–lipid conjugates, **DCP-spd** (A), **DCP-spm** (B), and

(conjugate/DOPE/cholesterol = 1/1/1 (mol/mol/mol)). The nitrogen/phosphate (N/P) ratio was 16/1 for the polyamine conjugate/DNA complexes. For the monoanionic DCP, the molar ratio, DCP/nucleotide = 16/1, was applied as a negative control experiment.

COS-1 cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Japan Bioserum Co. Ltd.) under a humidified atmosphere

**DCP-PEI** (C), and their constituents, DCP (A) and polyamines, spermidine (spd, A), spermine (spm, B) and PEI1800 (PEI, C), on COS-1 cells. Efficacy was evaluated with the luciferase activity. The observed values for spermidine and spermine indicated by the asterisks (\*) were apparently negligible on the activity scale shown. The conjugates **DCPspd**, **DCP-spm**, and **DCP-PEI** represent their micellar aggregate forms, and **DCP-spd**(PCL),

**DCP-spm**(PCL), and **DCP-PEI**(PCL) represent the conjugate-based PCLs

Transfection was conducted in the presence of 10% FBS.

**5.2 Transfection procedure** 

are described as just the name of conjugate, such as **DCP-spd**.

room temperature when used for transfection.

of 5% CO2 in air. One day before a transfection experiment, 1×105 COS-1 cells were seeded onto each of several 35-mm dishes and incubated overnight in a CO2 incubator. Then, the cells were washed twice with DMEM, and a suspension of lipoplex (1 μg DNA) was added to them in the presence of 10% FBS-DMEM. After 3 h incubation (37C, 5% CO2), the cells were washed twice with DMEM and cultured for another 48 h in 10% FBS-DMEM. The cells in the 35-mm dishes were washed twice with phosphate-buffered saline at 37C, and 200 μL of cell lysis buffer (LC-β, TOYO B-Net Co. Ltd., Tokyo) were added. After 15 min incubation, the cells were collected with a cell scraper, frozen at -80 C, and then thawed at room temperature. The lysate was centrifuged at 15,000 rpm for 5 min at 4 C. The supernatant was subjected to the luciferase assay (Pica Gene, TOYO B-Net Co. Ltd., Tokyo) using a luminophotometer (Luminescencer-PSN AB-2200, ATTO). The observed intensity in instrument light units was normalized to the amount of protein determined by BCA protein assay Kit (PIERCE) to give a relative light unit (RLU/mg protein).

#### **5.3 Transfection efficacy of micellar aggregates and PCL vectors: Comparison of the dicetyl phosphate derivatives of spermidine, sperimine, and polyamine**

Figure 4A shows the transfection efficacy of the polyamine conjugate, **DCP-spd**, and its constituent molecules, DCP and spermidine (spd). The conjugate (in this case an aqueous micellar suspension) shows greatly increased efficacy relative to the constituent molecules, DCP and spermidine. For these polyamine/DNA complexes, the nitrogen/phosphorous ratio (N/P) was 16. The data of the figure indicate that coupling the lipophilic and cationic portions is essential to obtain gene transfer. Such an effect of conjugating these two moieties was also observed for the spermine conjugate, **DCP-spm** (Figure 4B). When the conjugates were further formulated with DOPE and cholesterol (conjugate/DOPE/cholesterol = 1/1/1 (mol/mol/mol)) to generate polycationic liposomes (PCL) (**DCP-spd**(PCL) and **DCPspm**(PCL)), efficacies were further enhanced by a factor of 2–3 relative to the micellar aggregate suspensions (**DCP-spd** and **DCP-spm**). The polycation PEI(1800) itself showed moderate activity and the conjugate, **DCP-PEI**, exhibited even greater activity. However, in contrast to the other conjugates, the activity of the liposomal form, **DCP-PEI**(PCL), was comparable to that of the micellar version, **DCP-PEI**. The cytotoxicity of the conjugates, both as micellar aggregates and PCLs, was low; the latter vectors, which exhibited higher activity, also had slightly higher toxicity than the former.

It was found that the DOPE-based PCL exhibits significantly greater activity than the DPPCbased compound for both of the **DCP-spd**(PCL) and the **DCP-spm**(PCL). This is indicative of a lipid-mediated gene transfer mechanism; DOPE is well-known as a "helper" lipid, which is believed to facilitate membrane fusion and endosomal escape of the DNA (Felgner et al., 1994). DOPE in the present PCL systems presumably also plays a role in the mechanism to expedite membrane fusion and destabilization of the endosomal membrane. DPPC, whose Tm is 41.5 C, renders the PCL more stable and more rigid than does DOPE. Thus, it is likely that the fusogenic property of DOPE is responsible for the enhanced transfection activity of its complexes relative to those containing DPPC.
