**Cationic Liposomes in Different Structural Levels for Gene Delivery**

Yinan Zhao, Defu Zhi and Shubiao Zhang *SEAC-ME Key Laboratory of Biochemical Engineering College of Life Science, Dalian Nationalities University China* 

### **1. Introduction**

292 Non-Viral Gene Therapy

Zhang, S. H., Shen, S. C., Chen, Z., Yun, J. X., Yao, K. J., Chen, B. B., and Chen, J. Z. (2008)

*Engineering Journal*, Vol. 144, pp 324-328, ISSN 1385-8947.

Preparation of solid lipid nanoparticles in co-flowing microchannels, *Chemical* 

Over the last few decades, as a promising strategy for the treatment of many refractory diseases, such as inherited diseases (Martin-Rendon & Blake, 2003) and acquired immunodeficiency syndrome (AIDS) (Fanning et al., 2003), gene therapy, the objective to allow a gene to express the protein coded in the target cells and consequently to treat disease by the protein secreted from cells transfected, has become an invaluable experimental tool to study gene function and its regulation (El-Aneed, 2004). The success of gene therapy critically depends on suitable transfection vectors, which have the high efficiency transfer of genes to target cells as well as a favorable safety profile. Broadly, these vectors are mainly classified into two categories: viral and non-viral (Liu & Huang, 2002). Currently, viral vectors based on many different viruses such as adenovirus and retrovirus have achieved some success particularly in cancer gene therapy (Williams et al., 2010), and their performance and pathogenicity have been evaluated in animal models. However, several issues including difficulty in production, limited opportunity for repeated administrations due to acute inflammatory response, and delayed humeral or cellular immune responses need to be addressed, so that their clinical potential can be fully realized (Love et al., 2010). Thus it is necessary to develop more efficient and flexible security system in the category of vectors for gene delivery. Synthetic non-viral vectors are potential alternatives to viral vectors, and may help to overcome some of these problems (Zhang et al., 2010). Among these, cationic compounds (mainly including cationic lipids and cationic polymers) are believed to cause less safety problems due to their relative simplicity, and have been the most extensively studied.

Since the first description of successful *in vitro* transfection with cationic lipid by Felgner et al. in 1987 (Felgner et al., 1987), numerous cationic lipids have been synthesised and used for delivery of nucleic acids into cells during the last 20 years. Cationic liposomes are composed of lipid constituents, and have improved the gene delivery efficacy owing to their typical bilayer structure. Some helper lipids such as dioleylphosphatidyl choline (DOPC) or dioleylphosphatidyl ethanolamine (DOPE), typically neutrally lipids (Zuhorn et al., 2005), are often employed with cationic lipids, and play very important role during the formation of lipoplexes by combining cationic liposomes and genes, as they could determine the morphologies of lipoplexes.

Cationic Liposomes in Different Structural Levels for Gene Delivery 295

structure of headgroup was described by Felgner et al. (Felgner et al., 1994). They synthesized a series of 2,3-dialkyloxy quaternary ammonium compounds containing a hydroxyl moiety (Fig. 3.), which were more efficient in transfection compared with DOTMA that lacks a hydroxyl group on the quaternary amine. In our previous work, we also synthesized a series of cationic lipids containing a hydroxyl moiety on the quaternary amine for liposome-mediated gene delivery (Fig. 4.). Several cationic liposomes with relatively higher transfection efficiency were selected after *in vitro* transfection studies of the prepared cationic liposomes, whose biological performance was superior or parallel to that of the commercial transfection agents, Lipofectamine2000 and DOTAP. It is suggested that the headgroup hydration can be decreased by the incorporation of a hydroxyalkyl chain capable of hydrogen bonding to neighbouring headgroups while it improves the compaction of DNA by several mechanisms, for example, DNA can form hydrogen bonds with the lipid, and the hydroxyl group can enhance the membrane hydration. Accordingly, several groups have synthesised cationic lipids that varied the chain length of the hydroxyalkyl moiety, while keeping the remaining structure unchanged, and observed that the activity of lipid increased with the decrease in the hydroxyalkyl chain length. It shows that a decrease in the number of carbon atoms in the hydroxyalkyl chain, providing more rigidity to the terminal hydroxyl group, are very efficient in compacting DNA, which is responsible for the more

observed transfection activity ( Felgner et al., 1994; Bennett et al.,1997).

Fig. 1. Representative structure of the cationic lipid DOTMA. Examples of cationic lipid structural components: hydrophilic headgroup, linker bond, and hydrophobic domain.

Cationic polymer, which condenses DNA by ionic interaction (at physiological pH), form a particulate complex, polyplex, capable of gene transfer into the targeted cells (El-Aneed, 2004). They can condense the negatively charged DNA to a relatively small size and reduce its susceptibility to nucleases so that they may be favorable for improving transfection efficacy. The introduction of polycations (such as poly-L-lysine and protamine) in cationic liposomes, as co-polymer may provide a synergistic effect on the transfection efficiency and a promising solution to the problem frustrating us (Gao & Huang, 1996; Li & Huang, 1997). In this chapter, we review the effect of cationic liposomes in different structural levels for gene delivery, and help furthering the understanding of the mechanism governing the formation and behaviour of cationic liposomes in gene delivery. The first level for studying on the structure–activity relationship of cationic lipids is the synthesis of new vectors, as well as attempts to improve transfection efficiencies and decrease cytotoxicity, through hydrophilic, hydrophobic and linker domain modifications. The second one is the study on morphologies of lipoplexes through the effects of helper lipids and particle sizes. The last one is the hybridized utilization of non-viral vectors including the complexes of cationic lipsomes and polymers, conjugates of lipids and peptides and of targeting moieties and lipids.
