**5. Conclusion**

Cationic lipisomes-mediated gene transfer has shown to be a safe and effective way to transfer genes for gene therapy, and will gain clinical application in the near future. Some traditional cationic lipids have been used in gene transfer for a long time; many of them have trademarks such as lipofectin, lipofectamine and transfectam. These cationic lipids, however, may not be enough for the application in clinical trials; on the other hand, they have some shortcomings for a special application field. Therefore, many novel cationic lipids in chemical structure nature have been researching to meet the requirements with respect to gene therapy. Besides the lipid structure the next level should be the formulations of cationic liposomes, as in order to increase transfection efficiency and to decrease cytotoxicity other ingredients such as heper lipids could be chosen. Different formulations may cause various morphologies of lipoplexes formed by the combination between cationic liposomes and genes. It shows that the purposeful design of morphologies could increase transfection efficiency. The hybrid utilization of non-viral vectors based on cationic liposomes provides another solution to gene delivery. Much research has shown the promising combination between cationic lipsomes and polymers, the favorable conjugation between lipids and peptides and between targeting moieties and lipids.

#### **6. Acknowledgement**

The study was supported by the National Natural Science Foundation of China (20876027 and 21046008) and Program for New Century Excellent Talents in University (NCET-08- 0654) and the Fundamental Research Funds for the Central Universities (DC10020103).

#### **7. References**

Ahmad, A.; Evans, H.M.; Ewert, K.; George, C.X.; Samuel, C.E. & Safinya1, C.R. (2005). New Multivalent Cationic Lipids Reveal Bell Curve for Transfection Efficiency Versus

liposomes can deliver pDNA to liver nonparenchymal cells (Kawakami et al., 2000b) and

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

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

Cationic lipisomes-mediated gene transfer has shown to be a safe and effective way to transfer genes for gene therapy, and will gain clinical application in the near future. Some traditional cationic lipids have been used in gene transfer for a long time; many of them have trademarks such as lipofectin, lipofectamine and transfectam. These cationic lipids, however, may not be enough for the application in clinical trials; on the other hand, they have some shortcomings for a special application field. Therefore, many novel cationic lipids in chemical structure nature have been researching to meet the requirements with respect to gene therapy. Besides the lipid structure the next level should be the formulations of cationic liposomes, as in order to increase transfection efficiency and to decrease cytotoxicity other ingredients such as heper lipids could be chosen. Different formulations may cause various morphologies of lipoplexes formed by the combination between cationic liposomes and genes. It shows that the purposeful design of morphologies could increase transfection efficiency. The hybrid utilization of non-viral vectors based on cationic liposomes provides another solution to gene delivery. Much research has shown the promising combination between cationic lipsomes and polymers, the favorable conjugation

The study was supported by the National Natural Science Foundation of China (20876027 and 21046008) and Program for New Century Excellent Talents in University (NCET-08- 0654) and the Fundamental Research Funds for the Central Universities (DC10020103).

Ahmad, A.; Evans, H.M.; Ewert, K.; George, C.X.; Samuel, C.E. & Safinya1, C.R. (2005). New

Multivalent Cationic Lipids Reveal Bell Curve for Transfection Efficiency Versus

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splenic DCs and improve immune activation in DNA vaccines (Hattori et al., 2004).

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**14** 

*Italy* 

**Neutral Liposomes and DNA Transfection** 

Non viral gene transfer vectors for human gene therapy (HGT) applications represent today one of the widest fields of chemical, biological and medical research. Some authors (Kostaleros & Miller, 2005) have expressed the opinion that the future of a safe and efficient gene therapy will depend on suitable synthetic vectors of genetic material, rather than on viruses. The reason for this preference is based on the consideration that viruses, although characterized by high transfection efficiency of genetic material, may suffer from some serious disadvantages such as immune response (Marshall, 2000) and potential oncogenic activity (Hacein-Bey-Abina et al., 2003), as well as a high cost of preparation of the transferring system. On the contrary, synthetic vectors have many potential advantages, such as lack of immunogenicity and oncogenicity, no limits to the size of nucleic acids to be carried inside the cells (Harrington et al., 1997; Roush, 1997; Willard, 2000) and finally preparation procedures cheap and easy to perform. Nevertheless an awkward problem that accompanies their use is the low efficiency of the transfections *in vivo*. Among the synthetic vectors, the most explored by the researchers are the cationic liposomes (CLs), after Felgner and collaborators (Felgner et al., 1997) described the synthesis of the first cation lipid, *N*-[1- (2,3-dioleoyloxy)propyl]-*N*,*N*,*N*-trimethylammonium chloride (DOTMA) and demonstrated that it was able to bind DNA and transfect it both *in vitro* and *in vivo* experiments. Other cationic lipids followed and became popular, such as the commercially widely used N-[1- (2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), dimethyldioctadecylammonium bromide (DDAB) and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol); many others were synthesized during the past years and are still being synthesized. A few years after the Felgner's discovery scientists' interest was focused also on cationic polymers (Wu, G.Y. & Wu; C.H., 1987), that became popular after the discovery of polyethylenimines (Boussif, 1995) and are still the object of great interest. Both classes of cationic compounds owe their interest to the formation of stable complexes with DNA, called lipoplexes and polyplexes respectively, formed through an electrostatic interaction between the cationic head of lipids and the negative phosphates of DNA. The great amount of data and experiments reported in the literature with cationic vectors and some encouraging results in *in vivo* transfection experiments have led to the significant milestone of 20% of the ongoing clinical trials run with synthetic vectors (Edelstein et al., 2004): however the goal of a higher efficiency is still a problem to be solved. As recently stated (Safinya et al., 2006), the long-term target of research in this area is to develop a general

**1. Introduction**

Michela Pisani, Giovanna Mobbili and Paolo Bruni

*Marche Polytechnic University* 

