**3.1 Polyethylenimine**

Polyethylenimine (PEI) was investigated as a non-viral gene delivery vector in 1995 (Boussif et al., 1995). It is a high charge density polycation, in which every three atom is present with a protonable amino-nitrogen. Linear PEI only has secondary amino group that is almost protonated under physiological conditions. By contrast, branched PEI has not only the primary and secondary amine, but the tertiary amine. As such, only about two-thirds of amino groups in PEI are protonable under physiological conditions. It has been indicated that transfection ability of PEIs depends on their molecule weights, PEI nitrogen/DNA phosphate charge ratios (N/P) and cell types. For 800-Da PEI, it can mediate the delivery of pGL2-Luc gene into NIH 3T3 cells with an optimal gene expression level of 2X106 RLU/mg protein at an N/P of 8/1. However, for 25-kDa PEI, the level is increased to 109 RLU/mg protein at the same N/P ratio. The polyplexes of PEI may transfect different types of cell lines, with the levels of gene expression in the range from 105 (MCR-5 cells) to 108 RLU/mg protein (COS-7 cells).

Currently, high molecular weight PEI (e.g. 25kDa) is regarded as one of the most potent gene transfection agents. This superior gene transfection is explained by so-called "proton sponge" hypothesis (Boussif et al., 1995). In brief, the protonation of PEI in the endosomes induces a massive influx of chloride ions into the endosomes, which triggers the entry of water molecule into the endosome to balance the ion concentration. The entry of massive ions and water thus results in osmotic swelling of the endosome and subsequent membrane disruption. After that, genes are released into the cytoplasm. Buffer capacity (defined as the percentage of amino groups becoming protonated from pH 7.4 to 5.1) is regarded as an important parameter of cationic polymers to determine their ability to mediate endosomal escape, and is correlated with the pKa of protonable nitrogen in the polymers. Thus, cationic polymers containing protonable amino groups of a low pKa (5-7) commonly have good buffer capacity. This may explain why branched PEI can mediate better gene transfection than linear PEI because the former has one-third of protonable tertiary amino groups.

Also, a lot of investigations on biophysical properties of PEI-based polyplexes have been made to clarify PEI-mediated gene transfection (Sirirat et al., 2003). Dynamic light scatting and zeta-potential meters are typically applied to determine the particle size and zetapotential of polyplexes. In general, nano-scaled polyplexes below 150nm can be found with different molecular weight of PEI in the range of 2-25k at N/P ratios of 1-10. Notably, only

nuclear translocation process aided by the nuclear pore complex proteins in the nuclear membrane (Gorlich & Kutay, 1999; Ryan & Wente, 2000). When the genes are free from polyplexes in the nucleus, translation and transcription are conducted by gene expression

This section reviews typical non-degradable cationic polymers as non-viral vectors for gene delivery. Although these polymer systems normally have low transfection capability *in vitro* and/or high cytotoxicity, from the studies on these systems, a few fundamentals on gene delivery properties have been well understood, which are valuable in the design of safe and

Polyethylenimine (PEI) was investigated as a non-viral gene delivery vector in 1995 (Boussif et al., 1995). It is a high charge density polycation, in which every three atom is present with a protonable amino-nitrogen. Linear PEI only has secondary amino group that is almost protonated under physiological conditions. By contrast, branched PEI has not only the primary and secondary amine, but the tertiary amine. As such, only about two-thirds of amino groups in PEI are protonable under physiological conditions. It has been indicated that transfection ability of PEIs depends on their molecule weights, PEI nitrogen/DNA phosphate charge ratios (N/P) and cell types. For 800-Da PEI, it can mediate the delivery of pGL2-Luc gene into NIH 3T3 cells with an optimal gene expression level of 2X106 RLU/mg protein at an N/P of 8/1. However, for 25-kDa PEI, the level is increased to 109 RLU/mg protein at the same N/P ratio. The polyplexes of PEI may transfect different types of cell lines, with the levels of gene expression in the range from 105 (MCR-5 cells) to 108 RLU/mg

Currently, high molecular weight PEI (e.g. 25kDa) is regarded as one of the most potent gene transfection agents. This superior gene transfection is explained by so-called "proton sponge" hypothesis (Boussif et al., 1995). In brief, the protonation of PEI in the endosomes induces a massive influx of chloride ions into the endosomes, which triggers the entry of water molecule into the endosome to balance the ion concentration. The entry of massive ions and water thus results in osmotic swelling of the endosome and subsequent membrane disruption. After that, genes are released into the cytoplasm. Buffer capacity (defined as the percentage of amino groups becoming protonated from pH 7.4 to 5.1) is regarded as an important parameter of cationic polymers to determine their ability to mediate endosomal escape, and is correlated with the pKa of protonable nitrogen in the polymers. Thus, cationic polymers containing protonable amino groups of a low pKa (5-7) commonly have good buffer capacity. This may explain why branched PEI can mediate better gene transfection than linear PEI because the former has one-third of protonable tertiary amino groups.

Also, a lot of investigations on biophysical properties of PEI-based polyplexes have been made to clarify PEI-mediated gene transfection (Sirirat et al., 2003). Dynamic light scatting and zeta-potential meters are typically applied to determine the particle size and zetapotential of polyplexes. In general, nano-scaled polyplexes below 150nm can be found with different molecular weight of PEI in the range of 2-25k at N/P ratios of 1-10. Notably, only

**3. Non-degradable cationic polymers as non-viral gene delivery vectors** 

system to produce therapeutic proteins.

potent polymeric gene delivery vectors.

**3.1 Polyethylenimine** 

protein (COS-7 cells).

at the N/P ratios above 4/1, the polyplexes with a high surface charge (+10~35mV) can be obtained. Small particle sizes and positive surface charges are highly desirable for efficient cellular endocytosis, which may be the reason why PEI is potent for highly efficient gene transfection.

An inherent disadvantage of PEI is its high cytotoxicity *in vitro*. Depending on cell line type, the IC50 value of PEI is typically below 30 μg/mL. In PEI-mediated transfection process, a two-stage cytotoxicity mechanism is discovered (Godbey et al., 2001; Moghimi et al., 2005). In the first stage, free pEI may destabilize the cellular membrane, inducing necrosis-related cytotoxicity. The removal of free PEI from the polyplexes of PEI indeed can lead to lower cytotoxicity (Boeckle et al., 2004 ). In the second stage, free PEI that is dissociated from the polyplexes inside the cells can interact with negatively-charged mitochondrial membrane, inducing harmful cellular apoptosis. Thus, the cytotoxicity in this stage could be diminished after cationic polymers are intracellularly degraded into small pieces.
