**4. Morphology of the micellar and complexes with DNA**

It was found that DNA (ColE1 plasmid DNA) complex with various polyamines and polyamine–lipid conjugates by monitoring decrease of fluorescence from EtBr initially intercalated into DNA. With an increase in the cation (N) /anion (P) ratio, defined as N/P, the relative fluorescence intensity decreased as a result of complexation of DNA and polyamine. The polymeric molecule, PEI(1800) (N ~ 42 per molecule) most efficiently forms a complex with DNA; the complexation was almost complete at N/P = 3. Spermidine (N = 3 per molecule) and spermine (N = 4 per molecule) are less effective than PEI(1800), however, with complexation being complete at around N/P~7. For polyamine-DCP conjugates, the tendency for the fluorescence intensity to decrease is due to the complexation with DNA in the same way as that of the free amines, that is, **DCP-spd** < **DCP-spm** < **DCP-PEI**. DPPA-polyamine conjugates exhibited similar DNA condensation manner to DCP-polyamines, however, the tendency of DNA condensation was opposite way. The EtBr replacement experiment suggested that the tendency of DNA condensation is **DPPA-spd** > **DPPA-spm** > **DPPA-PEI**; the order is opposite to that of DCP-conjugates with regard to the polyamine portions.

In Table 1, the size of the micellar and complex with DNA was summarized. The mean particle diameters of various polyamine-DCP conjugates in an aqueous suspension, as given by dynamic light scattering is summarized in Table 1: 155 ± 54 nm for **DCP-spd**, 173 ± 46 nm for **DCP-spm**, 128 ± 38 nm for **DCP-PEI**, 90 ± 8 nm for **DPPA-spd**, 106 ± 41 nm for **DPPAspm**, and 218 ± 35 nm for **DPPA-PEI**. AFM images for the suspension of these compounds exhibited spherical or ellipsoidal particles, whose sizes, defined according to diameter for the spheres and major × minor axes for the ellipsoid, were 132 nm for **DCP-spd**, 156 nm for **DCP-spm**, 209 × 145 nm for **DCP-PEI**, 96 nm for **DPPA-spd**, 111 nm for **DPPA-spm**, and 201 nm for **DPPA-PEI** (Figures 2A, B, C, D, E, and F, respectively). The particle sizes for these compounds as observed by AFM show good agreement with those obtained by DLS. The molecular shapes of these compounds are regarded to be the "cone" type, due to the attachment of the large polyamine moiety. We have endeavored to make liposome from these compounds, however, entrapment of a fluorescence probe, calcein, was impossible. Therefore, we assume that the particle consisting of polyamine-DCP conjugates is a micellelike aggregate.

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

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

It was found that DNA (ColE1 plasmid DNA) complex with various polyamines and polyamine–lipid conjugates by monitoring decrease of fluorescence from EtBr initially intercalated into DNA. With an increase in the cation (N) /anion (P) ratio, defined as N/P, the relative fluorescence intensity decreased as a result of complexation of DNA and polyamine. The polymeric molecule, PEI(1800) (N ~ 42 per molecule) most efficiently forms a complex with DNA; the complexation was almost complete at N/P = 3. Spermidine (N = 3 per molecule) and spermine (N = 4 per molecule) are less effective than PEI(1800), however, with complexation being complete at around N/P~7. For polyamine-DCP conjugates, the tendency for the fluorescence intensity to decrease is due to the complexation with DNA in the same way as that of the free amines, that is, **DCP-spd** < **DCP-spm** < **DCP-PEI**. DPPA-polyamine conjugates exhibited similar DNA condensation manner to DCP-polyamines, however, the tendency of DNA condensation was opposite way. The EtBr replacement experiment suggested that the tendency of DNA condensation is **DPPA-spd** > **DPPA-spm** > **DPPA-PEI**; the order is opposite to that of DCP-conjugates with

In Table 1, the size of the micellar and complex with DNA was summarized. The mean particle diameters of various polyamine-DCP conjugates in an aqueous suspension, as given by dynamic light scattering is summarized in Table 1: 155 ± 54 nm for **DCP-spd**, 173 ± 46 nm for **DCP-spm**, 128 ± 38 nm for **DCP-PEI**, 90 ± 8 nm for **DPPA-spd**, 106 ± 41 nm for **DPPAspm**, and 218 ± 35 nm for **DPPA-PEI**. AFM images for the suspension of these compounds exhibited spherical or ellipsoidal particles, whose sizes, defined according to diameter for the spheres and major × minor axes for the ellipsoid, were 132 nm for **DCP-spd**, 156 nm for **DCP-spm**, 209 × 145 nm for **DCP-PEI**, 96 nm for **DPPA-spd**, 111 nm for **DPPA-spm**, and 201 nm for **DPPA-PEI** (Figures 2A, B, C, D, E, and F, respectively). The particle sizes for these compounds as observed by AFM show good agreement with those obtained by DLS. The molecular shapes of these compounds are regarded to be the "cone" type, due to the attachment of the large polyamine moiety. We have endeavored to make liposome from these compounds, however, entrapment of a fluorescence probe, calcein, was impossible. Therefore, we assume that the particle consisting of polyamine-DCP conjugates is a micelle-

assumed to efficiently interact with DNA via electrostatic interactions.

**4. Morphology of the micellar and complexes with DNA** 

complexes.

regard to the polyamine portions.

like aggregate.


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

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 DNA suspended in aqueous solution a)

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

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.

Polyamine – Lipid Conjugates as Effective Gene Carriers:

accompanied with cetyl-PEI enters into nucleus.

text.

**transfection activity** 

Chemical Structure, Morphology, and Gene Transfer Activity 251

DNA/polyamine complex to be transported into nucleous. As previous report, DNA

A B C

G H I

Fig. 3. AFM images of polyamine-ColE1 plasmid DNA complexes: (A), ColE1 plasmid DNA alone on polylysine-treated mica; (B), spermine/DNA; (C), PEI(1800)/DNA; (D), **DCPspd**/DNA; (E), **DCP-spm**/DNA; (F), **DCP-PEI**/DNA; (G), **DPPA-spd**/DNA; (H), **DPPAspm**/DNA; (I), **DPPA-PEI**/DNA. The polyamine/DNA ratio was 3/1 (w/w). Scale bars inserted in these images represent (A) 200, (B) 1000, (C) 200, (D) 500, (E) 500, (F) 200, (G) 500, (H) 500, and (I) 200 nm, respectively. The objects indicated by arrow(s) are discussed in the

In this section, we described novel types of polyamine-dialkyl phosphate conjugates that have moderate gene transfection activity for β-galactosidase assay. These conjugates are easy to prepare via a novel synthetic intermediate, dimerized DCP anhydride, **1**. The synthetic approaches described herein are flexible and possess potential for the rationale design of highly efficient gene carriers with single or narrow ranges of molecular weight.

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)

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

significantly improves transfection efficacy compared with micellar aggregate form.

D E F

The height is ~ 2 nm, corresponding to the diameter of B-form DNA, ca. 2.4 nm. When DNA was complexed with compound **DCP-spd**, a "spider nest"-like structure was observed (Figure 3D). The size of the "quasi-ellipsoidal core region" is ~1000 × 437 nm and the height is ~16 nm. The height of the radiating peripheral "nest" region is ~ 2 nm, suggesting the nest region consists of free DNA. The height of the core region (~16 nm) is clearly larger than that of the DNA part, therefore, the core region of the structure must consist of the **DCP-spd**/DNA complex. Thus, complexation of compound **DCP-spd** and DNA gives rise to a segregated array, having complex and free DNA portions. The compound **DCP-spm** forms a similar but distinguishably different complex structure with DNA (Figure 3E), which resembles a "pearl necklace-like" aggregate (Yoshikawa et al., 1996), 569 × 317 nm in the plane of the substrate and 4~20 nm in height. The size of the pearl parts is 120 ~ 170 nm in diameter and 11 ~ 20 nm in height. These parts are connected by a region that is 4 ~ 5 nm in height. It appears from the image that the assembly consists of a tightly packed **DCPspm**/DNA complex and regions of partially compacted DNA parts. Pronounced compaction of the complex was observed for the **DCP-PEI**/DNA, which formed a spherical cluster (120 nm in width and ~20 nm in height) (Figure 3F). The order of increasing compaction of DNA, **DCP-spd** < **DCP-spm** < **DCP-PEI**, is consistent with the extent of intercalation of EtBr as mentioned above, that is, the more tightly packed was the DNA complex, the lower was the extent of intercalation of EtBr. The tendency of size of the complex and the DNA condensation for DPPA-polyamine conjugates, that is, **DPPA-spd** (G) > **DPPA-spm** (H) > **DPPA-PEI** (I). One may wonder why the morphologies of the complexes, DCP- and DPPA-based conjugates, as well as **DCP-spd** and **DCP-spm** with DNA are so different despite the structural difference in cationic portion seems very little. It might come from the difference in delicate balance of hydrophilic/hydrophobic factor and/or molecular shapes which reflect a packing parameter.

Upon comparison, of the observed DNA complex with free polyamines, spermine (Figure 3B) and PEI (Figure 3C), the complex size and shape are obviously distinguishable from the corresponding conjugate forms; much larger complexes form with these free polyamines. The dimension of the spermine/DNA complex (Figure 3B) is ~3 μm; the compaction of DNA is obviously incomplete, judging from the presence of the "nest" portion of DNA that are similar to peripheral part of the **DCP-spd**/DNA complex (Figure 3D). The PEI/DNA complex is smaller (416 × 218 nm by 22 nm high) than the former complex, suggesting that the greater cationic charge makes the complex smaller. When one considers the effect of the hydrophobic portion in the polyamine compound on the size of the complex, it is clear that hydrophobic alkyl parts in the conjugates play an important role in the compaction of DNA, which is most prominently observed in conjugate **DCP-PEI** (Figure 3C vs 3F). From these results, the prominent transfection efficiency of **DCP-PEI** likely results from the more efficient compaction of DNA in the complex **DCP-PEI**/DNA, whereas the lower activity of **DCP-spd** and **DCP-spm** is likely due to their much weaker compaction.

Although the precise mechanism remains to be clarified, the compaction by these polyamine compounds probably plays an important role because entry into the nucleus is thought to be a key step and a smaller complex is likely to be associated with more efficient transfection. The conjugates in this study possibly decompose into separate polyamine and DCP portions in endosome. We assume that two critically important factors are involved in the present transfection system; compaction of DNA by polyamine part and with hydrophobic dialkyl portion and decomposition of the complex in endosome so as to liberate the

The height is ~ 2 nm, corresponding to the diameter of B-form DNA, ca. 2.4 nm. When DNA was complexed with compound **DCP-spd**, a "spider nest"-like structure was observed (Figure 3D). The size of the "quasi-ellipsoidal core region" is ~1000 × 437 nm and the height is ~16 nm. The height of the radiating peripheral "nest" region is ~ 2 nm, suggesting the nest region consists of free DNA. The height of the core region (~16 nm) is clearly larger than that of the DNA part, therefore, the core region of the structure must consist of the **DCP-spd**/DNA complex. Thus, complexation of compound **DCP-spd** and DNA gives rise to a segregated array, having complex and free DNA portions. The compound **DCP-spm** forms a similar but distinguishably different complex structure with DNA (Figure 3E), which resembles a "pearl necklace-like" aggregate (Yoshikawa et al., 1996), 569 × 317 nm in the plane of the substrate and 4~20 nm in height. The size of the pearl parts is 120 ~ 170 nm in diameter and 11 ~ 20 nm in height. These parts are connected by a region that is 4 ~ 5 nm in height. It appears from the image that the assembly consists of a tightly packed **DCPspm**/DNA complex and regions of partially compacted DNA parts. Pronounced compaction of the complex was observed for the **DCP-PEI**/DNA, which formed a spherical cluster (120 nm in width and ~20 nm in height) (Figure 3F). The order of increasing compaction of DNA, **DCP-spd** < **DCP-spm** < **DCP-PEI**, is consistent with the extent of intercalation of EtBr as mentioned above, that is, the more tightly packed was the DNA complex, the lower was the extent of intercalation of EtBr. The tendency of size of the complex and the DNA condensation for DPPA-polyamine conjugates, that is, **DPPA-spd** (G) > **DPPA-spm** (H) > **DPPA-PEI** (I). One may wonder why the morphologies of the complexes, DCP- and DPPA-based conjugates, as well as **DCP-spd** and **DCP-spm** with DNA are so different despite the structural difference in cationic portion seems very little. It might come from the difference in delicate balance of hydrophilic/hydrophobic factor

Upon comparison, of the observed DNA complex with free polyamines, spermine (Figure 3B) and PEI (Figure 3C), the complex size and shape are obviously distinguishable from the corresponding conjugate forms; much larger complexes form with these free polyamines. The dimension of the spermine/DNA complex (Figure 3B) is ~3 μm; the compaction of DNA is obviously incomplete, judging from the presence of the "nest" portion of DNA that are similar to peripheral part of the **DCP-spd**/DNA complex (Figure 3D). The PEI/DNA complex is smaller (416 × 218 nm by 22 nm high) than the former complex, suggesting that the greater cationic charge makes the complex smaller. When one considers the effect of the hydrophobic portion in the polyamine compound on the size of the complex, it is clear that hydrophobic alkyl parts in the conjugates play an important role in the compaction of DNA, which is most prominently observed in conjugate **DCP-PEI** (Figure 3C vs 3F). From these results, the prominent transfection efficiency of **DCP-PEI** likely results from the more efficient compaction of DNA in the complex **DCP-PEI**/DNA, whereas the lower activity of

Although the precise mechanism remains to be clarified, the compaction by these polyamine compounds probably plays an important role because entry into the nucleus is thought to be a key step and a smaller complex is likely to be associated with more efficient transfection. The conjugates in this study possibly decompose into separate polyamine and DCP portions in endosome. We assume that two critically important factors are involved in the present transfection system; compaction of DNA by polyamine part and with hydrophobic dialkyl portion and decomposition of the complex in endosome so as to liberate the

and/or molecular shapes which reflect a packing parameter.

**DCP-spd** and **DCP-spm** is likely due to their much weaker compaction.

DNA/polyamine complex to be transported into nucleous. As previous report, DNA accompanied with cetyl-PEI enters into nucleus.

Fig. 3. AFM images of polyamine-ColE1 plasmid DNA complexes: (A), ColE1 plasmid DNA alone on polylysine-treated mica; (B), spermine/DNA; (C), PEI(1800)/DNA; (D), **DCPspd**/DNA; (E), **DCP-spm**/DNA; (F), **DCP-PEI**/DNA; (G), **DPPA-spd**/DNA; (H), **DPPAspm**/DNA; (I), **DPPA-PEI**/DNA. The polyamine/DNA ratio was 3/1 (w/w). Scale bars inserted in these images represent (A) 200, (B) 1000, (C) 200, (D) 500, (E) 500, (F) 200, (G) 500, (H) 500, and (I) 200 nm, respectively. The objects indicated by arrow(s) are discussed in the text.

In this section, we described novel types of polyamine-dialkyl phosphate conjugates that have moderate gene transfection activity for β-galactosidase assay. These conjugates are easy to prepare via a novel synthetic intermediate, dimerized DCP anhydride, **1**. The synthetic approaches described herein are flexible and possess potential for the rationale design of highly efficient gene carriers with single or narrow ranges of molecular weight.
