**1. Introduction**

242 Non-Viral Gene Therapy

Zheng XZ, Li HL, Du LF, Wang HP, Gu Q. (2009) Comparative analysis of gene transfer to

Zhou S, Li S, Liu Z, Tang Y, Wang Z, Gong J, Liu C. (2010) Ultrasound-targeted

Zhou XY, Liao Q, Pu YM, Tang YQ, Gong X, Li J, Xu Y, Wang ZG. (2009) Ultrasound-

Zhou Y, Kumon RE, Cui J, Deng CX. (2009) The size of sonoporation pores on the cell

Zhou Y, Zhou XY, Wang ZG, Zhu YF, Li P. (2010) Elevation of plasma membrane

treats hepatoma in mice. *J Exp Clin Cancer Res* 29: 170.

cavitation activities. *Biophysical J* 94: L51-53.

membrane. *Ultrasound Med Biol* 35: 1756 - 1760.

*Basic Med Sci* 9: 174-181.

25: 587-594.

human and rat retinal pigment epithelium cell line by a combinatorial use of recombinant adeno- associated virus and ultrasound or/and microbubbles. *Bosn J* 

microbubble destruction mediated herpes simplex virus-thymidine kinase gene

mediated microbubble delivery of pigment epithelium-derived factor gene into retina inhibits choroidal neovascularization. *Chinese Medical J* 122: 2711-2717. Zhou Y, Cui J, Deng CX. (2008) Dynamics of sonoporation correlated with acoustic

permeability upon laser irradiation of extracellular microbubbles. *Lasers Med Sci* 

Development of more efficient and safer gene carriers using nonviral compounds is one of the most challenging aspects of gene therapy (Kay, 1997; Lasic, 1997). Compared to viral carrier systems, nonviral gene carrier systems have advantages in simplicity of use, lack of specific immune response, and ease of mass production due to the low cost of preparation; however, they have the disadvantage of low transfection efficiency, which needs to be overcome (Miller, 1998; Li & Huang, 2000). To improve the efficiency of nonviral carriers, many synthetic organic compounds, including cationic lipids (Felgner & Ringold, 1989; MacDonald et al., 1999; Felgner et al., 1987; Behr et al., 1989; Meyer et al., 1998), polycations (Boussif et al., 1995; Petersen et al., 2002; Koide et al., 2006; Russ et al., 2008; Haensler & Szoka, 1993; Shim & Kwon, 2009), and combinations thereof (Guillot-Nieckowski et al., 2007; Wu et al., 2001; Ewert et al., 2006; Takahashi et al., 2007; Matsui et al., 2006; Mustapa et al., 2009; Kogure et al., 2008), have been developed as nonviral gene carriers (Mintzer, M. A. & Simanek, E. E., 2009; also references cited therein). Substantial research has been reported on structure–activity relationships for cationic amphiphiles concerning the cationic and hydrophobic portions (Remy et al., 1994; Geall et al., 1999; Ewert et al, 2002; Byk et al., 1998; McGregor et al., 2001). Such amphiphiles form self-assembling micelles and liposomes in an aqueous phase, the structures of which have been investigated using small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), and atomic force microscopy (AFM) to gain knowledge about structure–activity relationship, particularly those involving ordered structures (lamellar, inverted hexagonal, and cubic phases) and their morphological changes (Koltover et al., 1998; Koynova, Wang & MacDonald, 2006) as well as about the size of complexes (Aoyama et al., 2003).

The mechanism of gene delivery by such cationic carriers probably involves an endosomal pathway (Wrobel & Collins (1995)): (i) cellular uptake via endocytosis, (ii) DNA release from endosome, and (iii) entry into the nucleus. Many researchers have devised cationic compounds that facilitate the process, for example, ligand-conjugated molecules targeting a receptor such as integrin (Mustapa et al., 2007; Varga, Wickham & Lauffenburger, 2000), pH-responsive or cleavable molecules that enable escape of DNA from endosome (Russ et al, 2008; Oupicky, Parker & Seymour, 2002; Dauty et al., 2001; Miyake et al., 2004; Anderson,

Polyamine – Lipid Conjugates as Effective Gene Carriers:

Scheme 1. Synthetic Strategy for Polyamine-lipid Conjugates.

**synthetic routes** 

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

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

cytosolic condition.

Chemical Structure, Morphology, and Gene Transfer Activity 245

dynamic light scattering (DLS) and AFM observation. Furthermore we introduce (5) our recent effort for synthesis of cleavable polyamine-lipid conjugates under a reductive

Lynn & Lange, 2003), and conjugation of nuclear localization signal peptides (NLS) (Zanta et al., 1999; Manickam & Oupicky, 2006) for steps (i)-(iii), respectively. For cationic lipids/DNA complexes (lipoplexes), it has been proposed that a morphological change from lamellar to inverted hexagonal phase in the acidic endosomal environment facilitates the endosomal release and escape of DNA (Bell et al., 2003; Xu & Szoka, 1996). In addition to investigation of intracellular trafficking of polycation–DNA complexes (polyplexes and lipoplexes), observation of morphology and metamorphosis of the complexes is very important to shed light on the mechanism of gene transfer and provide information for development of novel synthetic carriers (Koynova, Wang & MacDonald, 2006; Wan et al., 2008; Tarahovsky, Koynova & MacDonald, 2004).

We have reported that polycationic liposomes (PCL) containing cetylated polyethylenimine (cetyl-PEI) possess high gene transfer activity (Yamazaki et al., 2000; Oku et al., 2001; Matsuura et al., 2003). The cetyl-PEI molecule is anchored by the hydrophobic cetyl portion and is distributed over the liposomal surface. In our previous report, we proposed a possible mechanism of PCL-mediated gene transfer wherein PCL/DNA complexes are uptaken by endosomal pathway; this was based on tracking of fluorescence-labeled components, PCL lipid, cetyl-PEI, and DNA, which the release and transfer of cetyl PEI / DNA complex into the nucleus via the cytosol (Sugiyama et al., 2004). Compaction of DNA is therefore crucial, and both electrostatic and hydrophobic interactions in the cetyl PEI / DNA complex are responsible for its effective compaction.

PEI is used as a gene transfer vector by itself, however, it has inherent disadvantages, *i.e.,* cytotoxicity and polydispersity. We have previously reported successful syntheses of a series of polyamine–dicetyl phosphate (DCP) conjugates via reaction of a novel synthetic intermediate, dimerized DCP anhydride (compound **1** in Scheme 1), with various polyamines, spermidine, spermine, and PEI (Scheme 1) (Dewa et al., 2004a). Since spermidine and spermine are naturally occurring polyamines, we expected low cytotoxicity. When 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. We also observed the morphology of the conjugate / DNA complex by using atomic force microscopy (AFM). We discuss briefly the relation between the assembling structure of the conjugate/DNA and their transfection efficiency.

In this report, we describe (1) facile synthetic strategy of polyamine-lipid conjugates in brief and (2) evidence demonstrating that the transgene activity is dramatically enhanced when the conjugates are assembled into liposomes containing cholesterol and phospholipid and that the activity is susceptible to the chemical modification of the conjugate both in the polyamine and in the hydrophobic chain portions (Scheme 1). We show further that (3) gene transfer activity of the corresponding PCLs strongly depends on the type of polyamine in the conjugate, with notable differences between the lower molecular weight polyamines (spermidine and spermine) on one hand and the polymer type (PEI(1800)) on the other.

We also examined (4) the morphology of the lipoplexes by AFM and discuss the relationship between the structure of lipoplexes and their transfection efficiency. AFM analysis has a considerable advantage for observation of lipoplex morphology, especially for less ordered structures (Oberle et al., 2000), however, until now little clear evidence has been reported on the relationship between morphological change and DNA release. In this research, DNA release as a result of disassociation of the complex was revealed by AFM (Dewa et al., 2010). We discuss morphology–activity relationships on the basis of electrophoresis analysis,

Lynn & Lange, 2003), and conjugation of nuclear localization signal peptides (NLS) (Zanta et al., 1999; Manickam & Oupicky, 2006) for steps (i)-(iii), respectively. For cationic lipids/DNA complexes (lipoplexes), it has been proposed that a morphological change from lamellar to inverted hexagonal phase in the acidic endosomal environment facilitates the endosomal release and escape of DNA (Bell et al., 2003; Xu & Szoka, 1996). In addition to investigation of intracellular trafficking of polycation–DNA complexes (polyplexes and lipoplexes), observation of morphology and metamorphosis of the complexes is very important to shed light on the mechanism of gene transfer and provide information for development of novel synthetic carriers (Koynova, Wang & MacDonald, 2006; Wan et al.,

We have reported that polycationic liposomes (PCL) containing cetylated polyethylenimine (cetyl-PEI) possess high gene transfer activity (Yamazaki et al., 2000; Oku et al., 2001; Matsuura et al., 2003). The cetyl-PEI molecule is anchored by the hydrophobic cetyl portion and is distributed over the liposomal surface. In our previous report, we proposed a possible mechanism of PCL-mediated gene transfer wherein PCL/DNA complexes are uptaken by endosomal pathway; this was based on tracking of fluorescence-labeled components, PCL lipid, cetyl-PEI, and DNA, which the release and transfer of cetyl PEI / DNA complex into the nucleus via the cytosol (Sugiyama et al., 2004). Compaction of DNA is therefore crucial, and both electrostatic and hydrophobic interactions in the cetyl PEI /

PEI is used as a gene transfer vector by itself, however, it has inherent disadvantages, *i.e.,* cytotoxicity and polydispersity. We have previously reported successful syntheses of a series of polyamine–dicetyl phosphate (DCP) conjugates via reaction of a novel synthetic intermediate, dimerized DCP anhydride (compound **1** in Scheme 1), with various polyamines, spermidine, spermine, and PEI (Scheme 1) (Dewa et al., 2004a). Since spermidine and spermine are naturally occurring polyamines, we expected low cytotoxicity. When 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. We also observed the morphology of the conjugate / DNA complex by using atomic force microscopy (AFM). We discuss briefly the relation between the

In this report, we describe (1) facile synthetic strategy of polyamine-lipid conjugates in brief and (2) evidence demonstrating that the transgene activity is dramatically enhanced when the conjugates are assembled into liposomes containing cholesterol and phospholipid and that the activity is susceptible to the chemical modification of the conjugate both in the polyamine and in the hydrophobic chain portions (Scheme 1). We show further that (3) gene transfer activity of the corresponding PCLs strongly depends on the type of polyamine in the conjugate, with notable differences between the lower molecular weight polyamines (spermidine and spermine) on one hand and the polymer

We also examined (4) the morphology of the lipoplexes by AFM and discuss the relationship between the structure of lipoplexes and their transfection efficiency. AFM analysis has a considerable advantage for observation of lipoplex morphology, especially for less ordered structures (Oberle et al., 2000), however, until now little clear evidence has been reported on the relationship between morphological change and DNA release. In this research, DNA release as a result of disassociation of the complex was revealed by AFM (Dewa et al., 2010). We discuss morphology–activity relationships on the basis of electrophoresis analysis,

assembling structure of the conjugate/DNA and their transfection efficiency.

2008; Tarahovsky, Koynova & MacDonald, 2004).

DNA complex are responsible for its effective compaction.

type (PEI(1800)) on the other.

dynamic light scattering (DLS) and AFM observation. Furthermore we introduce (5) our recent effort for synthesis of cleavable polyamine-lipid conjugates under a reductive cytosolic condition.

Scheme 1. Synthetic Strategy for Polyamine-lipid Conjugates.
