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

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

Polyamine – Lipid Conjugates as Effective Gene Carriers:

new nonviral transfection strategies.

(A) (B)

nm (D).

**conditions** 

Chemical Structure, Morphology, and Gene Transfer Activity 259

observed by AFM imaging (Figure 7) and by electrophoretic analyses. Upon acidification (down to pH4), the extent of protonation of the polyamine portion is increased. Assuming that the lipoplex forms smectic lamella, electrostatic repulsion between layers must be increased with such protonation, resulting in the disruption of the lipoplex. For lipoplexes composed of **DCP-spd**(PCL) and **DCP-spm**(PCL), disruption accompanying DNA release has been confirmed. In sharp contrast, the size and shape of micellar aggregate/DNA complexes composed of **DCP-spd** and **DCP-spm** are insensitive to acidification (Table 2, entries 6 and 12). This would be due to their amorphous structure, which does not respond to pH change. Thus, the disassembly of the lipoplex composed of the bilayer-structured PCL is essential in effective gene transfer, especially in the process of endosomal escape, where lipid exchange and flip-flop are involved in disrupting the membrane and leading to DNA release (Xu & Szoka, 1996). Although the acidity of this experimental condition (pH4) seems higher than that in endosome (pH5.5), such a protonation process on the polyamines should be involved in the endosomal environment, because the protonation on the polyamines whose pKa values are > 8 may proceed rather gradually in the acidic region, especially in the self-assembled lamellar structure, where polyamines are densely packed. This finding suggests a strategy for molecular design—especially in the polyamine portion—in which a morphological transformation of lipoplexes is taken advantage of for

2000 nm 1000 nm 500 nm 1000 nm

Fig. 7. AFM images of disassembled lipoplexes, **DCP-spd**(PCL)/DNA (A and B) and **DCPspm**(PCL)/DNA (C and D) by acidification at pH 4. The acid-treated suspensions of complexes were put on bare mica (A, C) and PLL-mica (B and D). After removal of the solution the images were acquired. Scale bars: 2000 nm (A), 1000 nm (B), 500 nm (C), 1000

Detachable conjugates between cationic and hydrophobic lipid portions may facilitate the DNA release from complexes in intracellular reductive environment. This is a promising strategy for improvement of efficacy of non-viral gene delivery. Such approaches have been reported using disulfide-linked polymer (Oba et al., 2008) and gemini lipids (Behr). Recently, we have successfully synthesized oligo-arginine bearing phospholipids via disulfide linkage, DPPE-SS-R*n* (Scheme 2). Oligo-arginines, e.g., TAT peptide, are well known for effective carriers across cell membranes (Futaki et al., 2001). The reaction scheme is shown in Scheme 2. In brief, 1,2-dipalmitoyl-*sn*-glycero-3-phospho-ethanolamine (DPPE) was reacted with a heterobifunctional coupling agent, *N*-succinimidyl-3-(2-pyridyldithio)-propionate

**6. Cleavable peptide-phospholipid conjugates under physiological** 

(C) (D)

and DNA molecules likely aggregate randomly (Table 2 entry 5). This may be one of the reasons for the higher activity of PCL-based lipoplex, whose size is more favorable to transfection.

The linear dependence of activity on N/P (Figure 5B) is related to the morphology of the lipoplex. The electrophoresis experiment and AFM images (Figures 6A-D) suggest a reasonable explanation of the dependence, namely, the following: In the low N/P range (~5), the PCLs inadequately condense DNA molecules, giving the bead-like structures (Figures 6A and B). The DNA molecules loosely packed in the complex are readily released during electrophoresis. Such a complex, whose ζ-potential is negative, is too large to be introduced into the cell membrane via endocytosis; therefore the transfection level is low. With increasing N/P ratio, the morphology of the lipoplex transforms from large bead-like structure into smaller particles, wherein DNA molecules are condensed more tightly (Figures 6C and D). The size of the lipoplexes, whose ζ-potential is positive, is 150–400 nm, is more favorable for cellular uptake via endocytosis (Koynova, Wang & MacDonald, 2006). Given that the lamellar assembly in the lipoplex is responsible for its effectiveness as a gene carrier (Koltover et al., 1998; Koynova, Wang & MacDonald, 2006), the population of active species for gene transfer would increase with increasing in the N/P. Although highly positive-charged carriers are generally toxic, the PCL described here exhibit low cytotoxicity, an advantage for in vitro and in vivo applications.

#### **5.7.3 Disassembly of the lipoplexes and DNA release**

When the **DCP-spd**(PCL)/DNA and **DCP-spm**(PCL)/DNA lipoplexes (N/P = 24) were incubated in acidic solution (down to pH 4), the particle sizes measured by DLS became significantly larger and exhibited broad distributions (Table 2, entries 6 and 12). AFM imaging revealed morphological transformation of the PCL/DNA complexes upon acidification. When the dispersion of **DCP-spd**(PCL)/DNA lipoplexes (N/P = 24) was acidified at pH 4 for 1 h by addition of acetic acid, deformed structures were observed on bare mica (Figure 7A). Relative to the original structure (Figure 6C), the complex is decisively deformed by the acid treatment. Although some of flat-topped sphere complexes remain, the predominant morphology is particles connected with strings. The height of the clusters is 25–53 nm and they are connected with string portions that are 6–10 nm high. When the acid-treated complex solution was put on PLL-mica, additional deformed objects appeared on the surface (Figure 7B). A "beads on a string" deformed structure is composed of very small particles (50~100 nm in diameter) and string parts (~70 nm in width and 2–5 nm in height). The beads on a string structure observed on the positively charged surface must consist of DNA-rich fragments associated with some lipid components. The **DCPspm**(PCL)/DNA lipoplex maintains its spherical structure on bare mica (Figure 7C). On the PLL-mica, on the other hand, deformed structures were observed as in the case of **DCPspd**(PCL)/DNA lipoplex (Figure 7D). Such morphological changes upon acidification result from disassembly of the lipoplexes and the accompanying DNA release. Gel electrophoretic analysis provided evidence for the DNA release; the released plasmid band increased with the acidification from pH 8 to pH 4. In sharp contrast, such a morphological change was not observed for the micellar aggregate **DCP-spd**. DLS analysis indicates an insensitivity of the micellar aggregates to acidification (Table 2, entries 6 and 12).

Facile escape from the acidic endosomal compartment is necessary for efficient gene transfer. Disassembly of the lipoplex associated with DNA release has been clearly

and DNA molecules likely aggregate randomly (Table 2 entry 5). This may be one of the reasons for the higher activity of PCL-based lipoplex, whose size is more favorable to

The linear dependence of activity on N/P (Figure 5B) is related to the morphology of the lipoplex. The electrophoresis experiment and AFM images (Figures 6A-D) suggest a reasonable explanation of the dependence, namely, the following: In the low N/P range (~5), the PCLs inadequately condense DNA molecules, giving the bead-like structures (Figures 6A and B). The DNA molecules loosely packed in the complex are readily released during electrophoresis. Such a complex, whose ζ-potential is negative, is too large to be introduced into the cell membrane via endocytosis; therefore the transfection level is low. With increasing N/P ratio, the morphology of the lipoplex transforms from large bead-like structure into smaller particles, wherein DNA molecules are condensed more tightly (Figures 6C and D). The size of the lipoplexes, whose ζ-potential is positive, is 150–400 nm, is more favorable for cellular uptake via endocytosis (Koynova, Wang & MacDonald, 2006). Given that the lamellar assembly in the lipoplex is responsible for its effectiveness as a gene carrier (Koltover et al., 1998; Koynova, Wang & MacDonald, 2006), the population of active species for gene transfer would increase with increasing in the N/P. Although highly positive-charged carriers are generally toxic, the PCL described here exhibit low

When the **DCP-spd**(PCL)/DNA and **DCP-spm**(PCL)/DNA lipoplexes (N/P = 24) were incubated in acidic solution (down to pH 4), the particle sizes measured by DLS became significantly larger and exhibited broad distributions (Table 2, entries 6 and 12). AFM imaging revealed morphological transformation of the PCL/DNA complexes upon acidification. When the dispersion of **DCP-spd**(PCL)/DNA lipoplexes (N/P = 24) was acidified at pH 4 for 1 h by addition of acetic acid, deformed structures were observed on bare mica (Figure 7A). Relative to the original structure (Figure 6C), the complex is decisively deformed by the acid treatment. Although some of flat-topped sphere complexes remain, the predominant morphology is particles connected with strings. The height of the clusters is 25–53 nm and they are connected with string portions that are 6–10 nm high. When the acid-treated complex solution was put on PLL-mica, additional deformed objects appeared on the surface (Figure 7B). A "beads on a string" deformed structure is composed of very small particles (50~100 nm in diameter) and string parts (~70 nm in width and 2–5 nm in height). The beads on a string structure observed on the positively charged surface must consist of DNA-rich fragments associated with some lipid components. The **DCPspm**(PCL)/DNA lipoplex maintains its spherical structure on bare mica (Figure 7C). On the PLL-mica, on the other hand, deformed structures were observed as in the case of **DCPspd**(PCL)/DNA lipoplex (Figure 7D). Such morphological changes upon acidification result from disassembly of the lipoplexes and the accompanying DNA release. Gel electrophoretic analysis provided evidence for the DNA release; the released plasmid band increased with the acidification from pH 8 to pH 4. In sharp contrast, such a morphological change was not observed for the micellar aggregate **DCP-spd**. DLS analysis indicates an insensitivity of the

Facile escape from the acidic endosomal compartment is necessary for efficient gene transfer. Disassembly of the lipoplex associated with DNA release has been clearly

cytotoxicity, an advantage for in vitro and in vivo applications.

micellar aggregates to acidification (Table 2, entries 6 and 12).

**5.7.3 Disassembly of the lipoplexes and DNA release** 

transfection.

observed by AFM imaging (Figure 7) and by electrophoretic analyses. Upon acidification (down to pH4), the extent of protonation of the polyamine portion is increased. Assuming that the lipoplex forms smectic lamella, electrostatic repulsion between layers must be increased with such protonation, resulting in the disruption of the lipoplex. For lipoplexes composed of **DCP-spd**(PCL) and **DCP-spm**(PCL), disruption accompanying DNA release has been confirmed. In sharp contrast, the size and shape of micellar aggregate/DNA complexes composed of **DCP-spd** and **DCP-spm** are insensitive to acidification (Table 2, entries 6 and 12). This would be due to their amorphous structure, which does not respond to pH change. Thus, the disassembly of the lipoplex composed of the bilayer-structured PCL is essential in effective gene transfer, especially in the process of endosomal escape, where lipid exchange and flip-flop are involved in disrupting the membrane and leading to DNA release (Xu & Szoka, 1996). Although the acidity of this experimental condition (pH4) seems higher than that in endosome (pH5.5), such a protonation process on the polyamines should be involved in the endosomal environment, because the protonation on the polyamines whose pKa values are > 8 may proceed rather gradually in the acidic region, especially in the self-assembled lamellar structure, where polyamines are densely packed. This finding suggests a strategy for molecular design—especially in the polyamine portion—in which a morphological transformation of lipoplexes is taken advantage of for new nonviral transfection strategies.

Fig. 7. AFM images of disassembled lipoplexes, **DCP-spd**(PCL)/DNA (A and B) and **DCPspm**(PCL)/DNA (C and D) by acidification at pH 4. The acid-treated suspensions of complexes were put on bare mica (A, C) and PLL-mica (B and D). After removal of the solution the images were acquired. Scale bars: 2000 nm (A), 1000 nm (B), 500 nm (C), 1000 nm (D).
