**3.2.2 Chemical gene delivery methods**

Because DNA cannot pass through cellular membranes alone, various chemicals have been designed to aid the transfer of therapeutic genes into cells. The chemicals used in chemical gene delivery function to enhance the stability of the DNA molecule, to increase the efficiency of cellular uptake and intracellular trafficking, or to alter the distribution of the transferred DNA in the cells. These methods are very successful in terms of transferring genes into cells and are currently the most widely used methods. Also, chemical gene delivery methods are the easiest and most effective among various non-viral gene delivery methods developed thus far.

The most well-studied and effective approach for non-viral gene delivery is the use of cationic lipids. Positively charged cationic lipids naturally bind to negatively charged DNA in solution to condense DNA so that the DNA molecules and cationic lipids form complexes called lipoplexes. After lipoplexes are formed, the positively charged cationic lipids of the lipoplexes interact with cell membranes to allow cells to take up the lipoplexes by endocytosis. In typical cell physiology, endosomes that are formed as the result of endocytosis will fuse with lysosomes to degrade the lipoplexes containing the DNA. An

physical gene delivery methods. The easiest method to deliver genes into cells is to draw naked DNA into a microneedle and then inject the microneedle into cells to transfer the naked DNA directly to the cells. Though gene transfer efficiency by this method is very efficient, the method is very slow and laborious. The main drawback of this method is that microinjection can be only performed on one cell at a time, which means that this approach cannot be used for typical gene therapeutic approaches. The approach is limited to use for

Currently, the most popular physical methods for gene delivery into cells are electroporation and sonoporation. The cellular membrane is punctured by an electric pulse (electroporation) (Neumann et al., 1982) or ultrasonic wave (sonoporation) (Yizhi et al., 2007). The pores in the cellular membrane are only temporarily formed, and DNA molecules pass through during the short period of time when the pores open. These methods are generally efficient and work well across a broad range of cell types. However, a high rate of cell death limits their use, especially in gene therapy. These methods are widely used for gene delivery of immortal cells in which cell viability is not a critical issue during gene

Another popular method for physical gene delivery is the use of particle bombardment. In this method, gold particles (gene gun) (Gan et al., 2000) or magnetic particles (magnetofection) (Scherer et al., 2002) are coated with naked DNA. In the gene gun method, the DNA-coated gold particles are shot into the cell using high pressure gas, and the particles pass through the cellular membrane to introduce the particles inside the cells. In the magnetofection method, a magnet is placed underneath the tissue culture dish to attract DNA-coated magnetic particles. Then, the DNA-coated magnetic particles come into contact with a cell monolayer to introduce the particles inside the cells. These methods yield reasonably high efficiency gene transfers, but do not yield better efficiencies compared to other non-viral gene transfer methods, despite the requirement for expensive equipment. Also, it is quite difficult to control the DNA entry pathway, and the metal particles in the cells following gene transfer could negatively affect cells. Therefore, these methods are not

Because DNA cannot pass through cellular membranes alone, various chemicals have been designed to aid the transfer of therapeutic genes into cells. The chemicals used in chemical gene delivery function to enhance the stability of the DNA molecule, to increase the efficiency of cellular uptake and intracellular trafficking, or to alter the distribution of the transferred DNA in the cells. These methods are very successful in terms of transferring genes into cells and are currently the most widely used methods. Also, chemical gene delivery methods are the easiest and most effective among various non-viral gene delivery

The most well-studied and effective approach for non-viral gene delivery is the use of cationic lipids. Positively charged cationic lipids naturally bind to negatively charged DNA in solution to condense DNA so that the DNA molecules and cationic lipids form complexes called lipoplexes. After lipoplexes are formed, the positively charged cationic lipids of the lipoplexes interact with cell membranes to allow cells to take up the lipoplexes by endocytosis. In typical cell physiology, endosomes that are formed as the result of endocytosis will fuse with lysosomes to degrade the lipoplexes containing the DNA. An

gene delivery into germ-line cells to produce transgenic organisms.

transfer.

widely used.

**3.2.2 Chemical gene delivery methods** 

methods developed thus far.

exogenous gene in the lipoplexes would not have a chance to be released into the cytoplasm for gene expression if the endosomes are stable. Therefore, helper lipids are added to form lipoplexes to facilitate the endosomal escape of the exogenous gene (Herringson et al., 2009a, 2009b; Savva et al., 2005). This approach is very successful because it increases the transfection efficiency dramatically. There are various combinations of cationic lipids and helper lipids available. More than 40 products are commercially available for cationic lipidbased gene delivery, including LipoTAXI (Agilent Technologies), LipofectaminTM (Invitrogen), NanoJuice® (Merck), Transfectam® (Promega), and LipoJet TM (SignaGen Laboratories). The cationic lipid-based gene delivery shows a very high transfection efficiency of up to 90 in *in vitro* cell culture. Because the cationic lipid-based lipoplexes are not stably maintained in the blood, these methods are best for *ex vivo* gene therapy. However, cationic lipid-based lipoplexes show a very poor transfection efficiency with primary cells, such as stem cells, indicating that new methodological developments are required for the practical application of *ex vivo* gene therapy.

Other than cationic lipids, several different positively charged materials are used as a base material for non-viral DNA delivery, such as cationic polymers (Segura & Shea, 2001), cationic peptides consisting of poly-L-Lysine (D'Haeze et al., 2007; Mullen et al., 2000; Niidome et al., 1997), or other types of cationic proteins (De Lima et al., 1999; Jean et al., 2009; Lam et al., 2004; Lee et al., 2003; Oliveira et al., 2009; Vighi et al., 2007). These approaches produce DNA carrying complexes that are more stable. However, the transfection efficiency of this method is not better than cationic lipid-based lipoplexes. Therefore, most of these methods are designed for *in vivo* gene therapy.
