**4.1 Hybrids of cationic liposomes and polymers**

Cationic polymers could combine with DNA to form a particulate complex, polyplex, capable of gene transfer into the targeted cells (El-Aneed, 2004), because most of them are

Cationic Liposomes in Different Structural Levels for Gene Delivery 305

Fig. 9. The role of chitosans on transfection efficiency of cationic liposomes. (A in Hela cells, A1: lipoplex, A2: polyplex, A3: lipopolyplex 1, A4: lipopolyplex 2; B in Hep2 cells, B1:

groups has been a major trend with other more biocompatible groups, such as peptides, in recent years, as the cationic lipids with quaternary ammonium head groups can become cytotoxic by interacting with critical enzymes such as PKC (Bottega & Epand, 1992). The commonly used headgroups are peptides consisting of amino acids, such as lysine, arginine, histine, ornigine and tryptophan. Therefore, the conjugates of peptides and lipids are much less toxic, whilst keeping the same transfection efficiency (Behr et al., 1989; Ahn et al., 2004). The first polypeptide cationic liposome prepared by a polycondensation reaction was described by Folda et al. (Folda et al., 1982). A subset of lipitoids with a repeated side chain trimer motif conjugated with dimyristoyl phosphatidyl-ethanolamine (DMPE) mediated DNA were also found to transfer cells with high efficiency (Huang et al., 1998). A compound which contained cholesterol and a dipeptide consisting of glycine and sterically protected arginine has been proved to be suitable for *in vitro* transfection in the presence of 10% sera more efficiently than other cholesterol derivatives (Sochanik et al., 2000). Peptide-based gemini surfactants GS could lead to an increase in levels of gene expression *in vitro*

Obata et al. (Obata et al., 2008) have proved that the lysine- or arginine-type lipids exhibited higher gene expression efficiencies than that of Lipofectamine2000, with COS-7 cells. A series of new lipophilic peptides possessing a cationic tripeptide headgroup were effective non-viral vectors for gene delivery. Then, they also synthesized a series of cationic amino acid-based lipids having a spacer between the cationic head group and hydrophobic moieties and examined the influence of the spacer on a liposome gene delivery system. (Obata et al., 2009). At present, they are investigating the ability of cationic liposomes composed of 1,5-dihexadecyl *N*-arginyl-L-glutamate (Arg-Glu2C16) to carry nucleic acids into neuronal cells. Arg-Glu2C16, as a model cationic amino acid-based lipid, had a high capability as a gene carrier, even for neuronal transfection (Obata et al., 2010). Coles et al. (Coles et al., 2010) has synthesized positively charged peptide-based carriers which could interact with DNA improved by performing isothermal titration calorimetry and particle size and zeta potential experiments. The particle sizes of the carrier/DNA complexes varied over the different charge ratios from 200-800nm. The utilization of lipophilic carriers is a

Some peptide head groups could endow additional functions to the lipids, such as membrane-disturbing ability. In a hybrid molecule, the covalent coupling of an amphipathic and membrane-disturbing peptide to a lipid moiety might create a stable and efficient

lipoplex, B2: lipopolyplex 1, B3: lipopolyplex 2, B4: lipopolyplex 3)

compared to well-established non-viral reagents (McGregor et al., 2001).

promising approach to improve the bioavailability of gene delivery.

completely soluble in water. Therefore, they have the obvious advantage of compressing DNA molecules to a relatively small size (Gershon et al., 1993; Ruponen et al., 1999). But they do not contain a hydrophobic moiety (Elouahabi & Ruysschaert, 2005), this may hinder the transfection efficiency and cause cytotoxicity to some degree.

Liposome-mediated gene transfer could be improved by natural polycations such as protamine sulfate (PS), poly(L-lysine) (PLL), and spermine (Li & Huang, 1997; Cheng et al., 2009). The addition of poly(L-lysine) and protamine dramatically reduced the particle size of the complex formed between DNA and cationic liposomes and rendered DNA resistant to the nucleases (Gao & Huang, 1996). These polycations could form a complex with DNA and condense DNA from extended conformation to highly compact structure into 30-100 nm in size. A type of hybrid vectors were developed by Huang et al. (Gao & Huang, 1996; Lee & Huang, 1996) in which poly(lysine)-condensed DNA was entrapped into folate-targeted cationic liposomes (LPD). They found LPD vectors to be more efficient and less cytotoxic compared to conventional cationic liposomal vectors. Later, they modified LPDs through different cationic liposomes wherein LPDs were used to deliver antisense oligodeoxynucleotide and siRNA (Li & Huang, 2006; Chen et al., 2009; Gao & Huang, 2009). As a cationic polymer, PEI is commonly used for the delivery of genes. The hybrid usage

with cationic liposomes provides a promising way to the field of gene transfer. In a study, the combination of PEI and DOTAP-Chol caused more than 10-fold increase in the transfection efficiency and less toxicity in many cells compared with using polymer or liposome alone(Lee et al., 2003). Nearly at the same time, PEI2K-DNA-Dosper complexes showed much more cellular uptake of DNA than PEI2K-DNA complexes and two times higher transfection than Dosper-DNA complexes. It has been hypothesized that Dosper improved the cellular uptake of PEI2K-DNA complexes and PEI2K improved a transfer of the complexes from lysosomes to nucleus (Lampela et al., 2003).

In recent years, chitosan-based carriers have become one of the non-viral vectors that have gained increasing interest as a safer and cost-effective delivery system for gene materials, as they have beneficial qualities such as low toxicity, low immunogenicity, excellent biocompatibility as well as a high positive charge density (Zhang et al., 2007; Mao et al., 2010). Katas et al. (Katas & Alpar, 2006) may be the first group to investigate the use of chitosan to deliver siRNA *in vitro*. Two types of cell lines, CHO K1 and HEK 293 were used to reveal that preparation method of siRNA association to the chitosan played an important role on the silencing effect. Chitosan-TPP nanoparticles with entrapped siRNA were shown to be better vectors as siRNA delivery vehicles compared to chitosan-siRNA complexes possibly due to their high binding capacity and loading efficiency. We have combined chitosans and cationic liposomes to form a ternary lipopolyplex which could facilitate the delivery of genes into cells more efficiently than the utilization of a lipoplex or a polyplex alone (Fig. 9.). The confocal microscopy method proved that exogenous DNA molecules entered the nucleus through the nuclear membrane other than via the NPC. The results explored novel ways based on the hybrid vectors to enhance the pDNA delivery with chitosans and with further suitable intracellular mechanism, allowing the development of nonviral gene delivery and may provide the most exciting solution for hybrid biomaterials design used for beneficial candidates for gene therapy.

#### **4.2 Conjugates of peptides and lipids**

One of the most challenge things for gene delivery by cationic liposome method is the toxicity of cationic lipids originated in the cationic nature. The replacement of cationic head

completely soluble in water. Therefore, they have the obvious advantage of compressing DNA molecules to a relatively small size (Gershon et al., 1993; Ruponen et al., 1999). But they do not contain a hydrophobic moiety (Elouahabi & Ruysschaert, 2005), this may hinder

Liposome-mediated gene transfer could be improved by natural polycations such as protamine sulfate (PS), poly(L-lysine) (PLL), and spermine (Li & Huang, 1997; Cheng et al., 2009). The addition of poly(L-lysine) and protamine dramatically reduced the particle size of the complex formed between DNA and cationic liposomes and rendered DNA resistant to the nucleases (Gao & Huang, 1996). These polycations could form a complex with DNA and condense DNA from extended conformation to highly compact structure into 30-100 nm in size. A type of hybrid vectors were developed by Huang et al. (Gao & Huang, 1996; Lee & Huang, 1996) in which poly(lysine)-condensed DNA was entrapped into folate-targeted cationic liposomes (LPD). They found LPD vectors to be more efficient and less cytotoxic compared to conventional cationic liposomal vectors. Later, they modified LPDs through different cationic liposomes wherein LPDs were used to deliver antisense oligodeoxynucleotide and siRNA (Li & Huang, 2006; Chen et al., 2009; Gao & Huang, 2009). As a cationic polymer, PEI is commonly used for the delivery of genes. The hybrid usage with cationic liposomes provides a promising way to the field of gene transfer. In a study, the combination of PEI and DOTAP-Chol caused more than 10-fold increase in the transfection efficiency and less toxicity in many cells compared with using polymer or liposome alone(Lee et al., 2003). Nearly at the same time, PEI2K-DNA-Dosper complexes showed much more cellular uptake of DNA than PEI2K-DNA complexes and two times higher transfection than Dosper-DNA complexes. It has been hypothesized that Dosper improved the cellular uptake of PEI2K-DNA complexes and PEI2K improved a transfer of

In recent years, chitosan-based carriers have become one of the non-viral vectors that have gained increasing interest as a safer and cost-effective delivery system for gene materials, as they have beneficial qualities such as low toxicity, low immunogenicity, excellent biocompatibility as well as a high positive charge density (Zhang et al., 2007; Mao et al., 2010). Katas et al. (Katas & Alpar, 2006) may be the first group to investigate the use of chitosan to deliver siRNA *in vitro*. Two types of cell lines, CHO K1 and HEK 293 were used to reveal that preparation method of siRNA association to the chitosan played an important role on the silencing effect. Chitosan-TPP nanoparticles with entrapped siRNA were shown to be better vectors as siRNA delivery vehicles compared to chitosan-siRNA complexes possibly due to their high binding capacity and loading efficiency. We have combined chitosans and cationic liposomes to form a ternary lipopolyplex which could facilitate the delivery of genes into cells more efficiently than the utilization of a lipoplex or a polyplex alone (Fig. 9.). The confocal microscopy method proved that exogenous DNA molecules entered the nucleus through the nuclear membrane other than via the NPC. The results explored novel ways based on the hybrid vectors to enhance the pDNA delivery with chitosans and with further suitable intracellular mechanism, allowing the development of nonviral gene delivery and may provide the most exciting solution for hybrid biomaterials

One of the most challenge things for gene delivery by cationic liposome method is the toxicity of cationic lipids originated in the cationic nature. The replacement of cationic head

the transfection efficiency and cause cytotoxicity to some degree.

the complexes from lysosomes to nucleus (Lampela et al., 2003).

design used for beneficial candidates for gene therapy.

**4.2 Conjugates of peptides and lipids** 

Fig. 9. The role of chitosans on transfection efficiency of cationic liposomes. (A in Hela cells, A1: lipoplex, A2: polyplex, A3: lipopolyplex 1, A4: lipopolyplex 2; B in Hep2 cells, B1: lipoplex, B2: lipopolyplex 1, B3: lipopolyplex 2, B4: lipopolyplex 3)

groups has been a major trend with other more biocompatible groups, such as peptides, in recent years, as the cationic lipids with quaternary ammonium head groups can become cytotoxic by interacting with critical enzymes such as PKC (Bottega & Epand, 1992). The commonly used headgroups are peptides consisting of amino acids, such as lysine, arginine, histine, ornigine and tryptophan. Therefore, the conjugates of peptides and lipids are much less toxic, whilst keeping the same transfection efficiency (Behr et al., 1989; Ahn et al., 2004).

The first polypeptide cationic liposome prepared by a polycondensation reaction was described by Folda et al. (Folda et al., 1982). A subset of lipitoids with a repeated side chain trimer motif conjugated with dimyristoyl phosphatidyl-ethanolamine (DMPE) mediated DNA were also found to transfer cells with high efficiency (Huang et al., 1998). A compound which contained cholesterol and a dipeptide consisting of glycine and sterically protected arginine has been proved to be suitable for *in vitro* transfection in the presence of 10% sera more efficiently than other cholesterol derivatives (Sochanik et al., 2000). Peptide-based gemini surfactants GS could lead to an increase in levels of gene expression *in vitro* compared to well-established non-viral reagents (McGregor et al., 2001).

Obata et al. (Obata et al., 2008) have proved that the lysine- or arginine-type lipids exhibited higher gene expression efficiencies than that of Lipofectamine2000, with COS-7 cells. A series of new lipophilic peptides possessing a cationic tripeptide headgroup were effective non-viral vectors for gene delivery. Then, they also synthesized a series of cationic amino acid-based lipids having a spacer between the cationic head group and hydrophobic moieties and examined the influence of the spacer on a liposome gene delivery system. (Obata et al., 2009). At present, they are investigating the ability of cationic liposomes composed of 1,5-dihexadecyl *N*-arginyl-L-glutamate (Arg-Glu2C16) to carry nucleic acids into neuronal cells. Arg-Glu2C16, as a model cationic amino acid-based lipid, had a high capability as a gene carrier, even for neuronal transfection (Obata et al., 2010). Coles et al. (Coles et al., 2010) has synthesized positively charged peptide-based carriers which could interact with DNA improved by performing isothermal titration calorimetry and particle size and zeta potential experiments. The particle sizes of the carrier/DNA complexes varied over the different charge ratios from 200-800nm. The utilization of lipophilic carriers is a promising approach to improve the bioavailability of gene delivery.

Some peptide head groups could endow additional functions to the lipids, such as membrane-disturbing ability. In a hybrid molecule, the covalent coupling of an amphipathic and membrane-disturbing peptide to a lipid moiety might create a stable and efficient

Cationic Liposomes in Different Structural Levels for Gene Delivery 307

Later, they used galactosylated cationic liposomes to target liver cell asialoglycoprotein receptors *in vivo* (Kawakami et al., 2000a). Many groups also reported a few other glycosylated cationic bolaamphiphiles similar to the compound (Letrou-Bonneval et al., 2008). Brunelle et al. (Brunelle et al., 2009) synthesized a new series of dissymmetric hemifluorocarbon bolaamphiphiles (Fig. 10.), and the dissymmetric functionalization of diiodoperfluorooctane led to bolaamphiphile molecules composed of a partially fluorocarbon core end-capped with a glycoside and an ammonium salt. They found that the incorporation of two fluorinated segments in the molecular structure of the bolaamphiphiles

is detrimental for an efficient DNA condensation.

Fig. 10. A set of dissymmetric hemifluorocarbon bolaamphiphiles.

The mannose receptor (MR) is found on the surface of macrophages and dendritic cells can recognize complex carbohydrates that are located on glycoproteins that are a part of many different biological processes, and bind terminal mannoses found on vector. Kawakami et al. (Kawakami et al., 2000b) have developed a novel mannosylated cholesterol derivative, Man-C4-Chol, consisting of modified cationic liposomes with mannose moieties for NPCselective gene delivery via mannose receptors on NPC. The mannosylated cationic

peptide-based gene transfer system. The luciferase activity induced by the dioleoylmelittin/DNA complex was 5-500-fold higher than that induced by a cationic lipid/DNA complex, depending on the cationic lipid and the cell-line (Legendre et al., 1997). Later, a membrane-disrupting peptide derived from the influenza virus was covalently linked to different polymethacrylates using N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) as a coupling agent to increase the transfection efficiency of polyplexes based on these polymers. *In vitro* transfection and toxicity were tested in COS-7 cells, and these experiments showed that the polyplexes with grafted peptides had a substantially higher transfection activity than the control polyplexes, while the toxicity remained unchanged (Funhoff et al., 2005).

#### **4.3 Conjugates of targeting moieties and lipids**

For a number of gene therapy applications, targeted gene delivery systems have attracted great attention due to their potential in directing the therapeutic genes to the target cells, and it may help minimize adverse effects such as cytotoxicity or immune reactions, as well as maximizing the efficacy of the therapeutic response. The targeted delivery of the lipoplexes may be achieved through the addition of targeting moieties (e.g., ligands) into liposomes by direct formulation, with no covalent bond to any lipid (Seol et al., 2000); conjugated to helper lipid (Dauty et al., 2002), or connected directly to the cationic lipids (Kawakami et al., 2000a; Gaucheron et al., 2001c). For targeted lipoplexes, modified with a targeting moiety such as folate (Dauty et al., 2002), galactose(Kawakami et al., 2000a; Gaucheron et al., 2001c), mannose (Kawakami et al., 2000b), antibodies (Duan et al., 2008) and transferrin (Seol et al., 2000; Sakaguchi et al., 2008), the uptake can be receptor mediated and enhanced (Zhang et al., 2010).

Targeting of the folate receptor (FR) had received much attention in recent years, since the folate receptor is a tumor marker over expressed in large numbers of cancer cells, including cancers of the ovary, kidney, uterus, testis, brain, colon, and in addition, folic acid is a relatively small molecule (MW 441 Da), therefore, it has the advantages of being stable and nonimmunogenic compared to monoclonal antibodies (Kane et al., 1986), and still having a relatively high receptor affinity. In a study, the folate moiety was attached to a lipid membrane anchor via a cysteinyl-PEG3400 spacer, which greatly increased specific cellular uptake to FR overexpressing cancer cells in comparison with unmodified cationic liposome and can significantly improve the transfection efficiency of a cationic liposomal formulation (Reddy et al., 2002). Recently, Yoshizawa et al. (Yoshizawa et al., 2008) developed a folate-linked nanoparticle (NP-F), which was composed of cholesteryl-3βcarboxyamidoethylene-N-hydroxyethylamine, Tween 80 and folate-poly(ethylene glycol) distearoyl-phosphatidylethanolamine conjugate (f-PEG2000-DSPE), and was delivered synthetic siRNA with high transfection efficiency and selectivity into nasopharyngeal tumor KB cells.

The asialoglycoprotein receptor (ASPGR), present at the surface of hepatocytes, could recognize and bind to β-D-galactoside terminated glycoproteins for the targeting of cationic vector-based gene delivery systems (Ashwell & Harford, 1982). Kawakami et al. (Kawakami et al., 1998) have studied liposomal with asialoglycoprotein receptor gene carrier systems for gene delivery to hepatocytes, which was a novel galactosylated cholesterol derivatives, cholesten-5-yloxy-N-(4-((1-imino-2-β-D-thiogalactosylethyl)amino)alkyl) formamide. In human hepatoma cells (HepG2), the liposomes containing this galactolipid showed higher transfection activities than DC-Chol liposomes based on a receptor-mediated mechanism.

peptide-based gene transfer system. The luciferase activity induced by the dioleoylmelittin/DNA complex was 5-500-fold higher than that induced by a cationic lipid/DNA complex, depending on the cationic lipid and the cell-line (Legendre et al., 1997). Later, a membrane-disrupting peptide derived from the influenza virus was covalently linked to different polymethacrylates using N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) as a coupling agent to increase the transfection efficiency of polyplexes based on these polymers. *In vitro* transfection and toxicity were tested in COS-7 cells, and these experiments showed that the polyplexes with grafted peptides had a substantially higher transfection activity than the control polyplexes, while the toxicity remained unchanged

For a number of gene therapy applications, targeted gene delivery systems have attracted great attention due to their potential in directing the therapeutic genes to the target cells, and it may help minimize adverse effects such as cytotoxicity or immune reactions, as well as maximizing the efficacy of the therapeutic response. The targeted delivery of the lipoplexes may be achieved through the addition of targeting moieties (e.g., ligands) into liposomes by direct formulation, with no covalent bond to any lipid (Seol et al., 2000); conjugated to helper lipid (Dauty et al., 2002), or connected directly to the cationic lipids (Kawakami et al., 2000a; Gaucheron et al., 2001c). For targeted lipoplexes, modified with a targeting moiety such as folate (Dauty et al., 2002), galactose(Kawakami et al., 2000a; Gaucheron et al., 2001c), mannose (Kawakami et al., 2000b), antibodies (Duan et al., 2008) and transferrin (Seol et al., 2000; Sakaguchi et al., 2008), the uptake can be receptor mediated

Targeting of the folate receptor (FR) had received much attention in recent years, since the folate receptor is a tumor marker over expressed in large numbers of cancer cells, including cancers of the ovary, kidney, uterus, testis, brain, colon, and in addition, folic acid is a relatively small molecule (MW 441 Da), therefore, it has the advantages of being stable and nonimmunogenic compared to monoclonal antibodies (Kane et al., 1986), and still having a relatively high receptor affinity. In a study, the folate moiety was attached to a lipid membrane anchor via a cysteinyl-PEG3400 spacer, which greatly increased specific cellular uptake to FR overexpressing cancer cells in comparison with unmodified cationic liposome and can significantly improve the transfection efficiency of a cationic liposomal formulation (Reddy et al., 2002). Recently, Yoshizawa et al. (Yoshizawa et al., 2008) developed a folate-linked nanoparticle (NP-F), which was composed of cholesteryl-3βcarboxyamidoethylene-N-hydroxyethylamine, Tween 80 and folate-poly(ethylene glycol) distearoyl-phosphatidylethanolamine conjugate (f-PEG2000-DSPE), and was delivered synthetic siRNA with high transfection efficiency and selectivity into nasopharyngeal

The asialoglycoprotein receptor (ASPGR), present at the surface of hepatocytes, could recognize and bind to β-D-galactoside terminated glycoproteins for the targeting of cationic vector-based gene delivery systems (Ashwell & Harford, 1982). Kawakami et al. (Kawakami et al., 1998) have studied liposomal with asialoglycoprotein receptor gene carrier systems for gene delivery to hepatocytes, which was a novel galactosylated cholesterol derivatives, cholesten-5-yloxy-N-(4-((1-imino-2-β-D-thiogalactosylethyl)amino)alkyl) formamide. In human hepatoma cells (HepG2), the liposomes containing this galactolipid showed higher transfection activities than DC-Chol liposomes based on a receptor-mediated mechanism.

(Funhoff et al., 2005).

and enhanced (Zhang et al., 2010).

tumor KB cells.

**4.3 Conjugates of targeting moieties and lipids** 

Later, they used galactosylated cationic liposomes to target liver cell asialoglycoprotein receptors *in vivo* (Kawakami et al., 2000a). Many groups also reported a few other glycosylated cationic bolaamphiphiles similar to the compound (Letrou-Bonneval et al., 2008). Brunelle et al. (Brunelle et al., 2009) synthesized a new series of dissymmetric hemifluorocarbon bolaamphiphiles (Fig. 10.), and the dissymmetric functionalization of diiodoperfluorooctane led to bolaamphiphile molecules composed of a partially fluorocarbon core end-capped with a glycoside and an ammonium salt. They found that the incorporation of two fluorinated segments in the molecular structure of the bolaamphiphiles is detrimental for an efficient DNA condensation.

Fig. 10. A set of dissymmetric hemifluorocarbon bolaamphiphiles.

The mannose receptor (MR) is found on the surface of macrophages and dendritic cells can recognize complex carbohydrates that are located on glycoproteins that are a part of many different biological processes, and bind terminal mannoses found on vector. Kawakami et al. (Kawakami et al., 2000b) have developed a novel mannosylated cholesterol derivative, Man-C4-Chol, consisting of modified cationic liposomes with mannose moieties for NPCselective gene delivery via mannose receptors on NPC. The mannosylated cationic

Cationic Liposomes in Different Structural Levels for Gene Delivery 309

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liposomes can deliver pDNA to liver nonparenchymal cells (Kawakami et al., 2000b) and splenic DCs and improve immune activation in DNA vaccines (Hattori et al., 2004).

Since theirs early use for the targeting to erythroblasts (Zhang et al., 2010), targeting proteins such as antibodies or transferrin have become one of the most widely used ligands for targeting of synthetic vectors, and been used in conjunction with cationic lipoplexes (Rao, 2010) to mediate uptake of plasmid DNA and antisense oligonucleotides. Another important class of cell proteins with promise for targeted gene delivery is growth factor receptor, which is a most commonly single pass transmembrane protein with an extracellular ligand binding domain and an intracellular region with enzymatic activity, usually a tyrosine kinase domain, which transmits a growth factor signal from the cell's environment to its interior. It has been used to target polylysine complexes (Cristiano & Roth, 1996), liposomes (Kikuchi, 1996), PEI polyplexes (Cristiano & Roth, 1996), and adenovirus-derived peptides (Medina-Kauwe et al., 2001) to receptor-positive cells.

Targeting provides a generic strategy to improve the specificity of a pharmaceutical formulation independently of the specificity of the drug or gene itself, primarily through a modulation of the carriers' biodistribution, so that a dose differential is created between healthy and diseased tissue.
