**3.3 Poly(L-lysine)**

Poly(L-lysine) (PLL) is one of mostly studied cationic polymers for non-viral gene delivery (G.Y. Wu & Wu, 1987). It is a linear polypeptide with L-lysine residues in repeat units. The commonly used PLL as a non-viral gene delivery vector is with the molecular weigh of 25.7 kDa. The transfection efficiency of PLL is much lower than that of PEI since it displays a low buffer capacity, which is not efficient for proton sponge effect. Another disadvantage of PLL is that transfection efficiency of PLL is significantly influenced by serum probably due to the

Bioreducible Cationic Polymers for Gene Transfection 91

degraded PAMAM, denoted as "fractured" dendrimer, could lead to enhanced transfection efficiency by 3 orders of magnitude compared to that of native PAMAM. This pronounced transfection efficiency is likely due to increased flexibility which results in apparent volume swelling of fractured dendrimer in the endosomes and thus efficient endosomal escape.

Chitosan is a naturally cationic polysaccharide and is degradable by lysozyme in the body. Chitosan is composed of β1→4 linked glucosamine, partly containing N-acetylglucosamine and has an apparent pKa value of 6.5. This indicates that chitosan could be used as non-viral vectors for gene delivery (W.G. Liu & Yao, 2002). Mumper *et al*. was the first to report chitosan as gene delivery vectors (Mumper et al., July 30-August 4, 1995). The molecular weight of chitosans influences their transfection efficiency. Generally, an optimal molecular weight is in the range of 10-50 kDa for efficient gene transfection. Also, both pH and serum largely influence the transfection efficiency of chitosan. It was shown that the transfection efficiency at pH 6.9 was higher than that at pH 7.6. Moreover, the transfection efficiency was 2 to 3 times higher in the presence of serum than that in the absence of serum. The enhancement with serum may be caused by the cell function raised by the addition of serum since major components in serum like albumin and globulin have little effect on the

Because chitosan is only soluble in acidic solution at pH 1~6, it readily self-aggregates under physiological conditions. A few chemical modifications on chitosan were thus performed to obtain improved solubility. One is to modify chitosan by introduction of a soluble moiety, for example, PEG, and the other is quaternization of the amine groups of chitosan (Thanou et al., 2002). A modification is also made to enhance the thermal stability of DNA by incorporating dodecylate chain into chitosan (F. Li et al., 2002). This modified chitosan displayed enhanced transfection capability with low cytotoxicity. However, the transfection efficiency of chitosan-

transfection efficiency of chitosan–based polyplexes (Sato et al., 2002).

based derivatives reported so far is normally not superior to that of PEI.

**4. Hydrolysable cationic polymers as non-viral gene delivery vectors** 

This section reviews hydrolysable cationic polymers as non-viral gene delivery vectors. In the past decade, hydrolysable cationic polymers (Figure 3) have been studied as non-viral gene delivery vectors since they display lower toxicity as compared to their non-degradable counterparts. These research works accelerate the availability of safe and efficient non-viral

Poly(trans-4-hydroxy-L-proline ester) (PHP) is the first hydrolysable cationic polymers for non-viral gene delivery (Lim et al., 1999). Since hydroxyproline is a component in collagen, gelatin, and other proteins, hydroxyproline-based materials are considered low cytotoxic. PHP ester were synthesized by the polymerization of cbz-protected 4-hydroxy-L-proline to generate poly(4-hydroxy-N-cbz-L-proline), followed by the treatment of formic acid and Pd/C. As expected, PHP is degradable under physiological conditions, but very fast with degradation half time of 2 hours. Moreover, PHP can efficiently bind DNA to form positive polyplexes with average diameter below 200 nm. The polyplexes could transfect CAPE cells

**3.5 Chitosan** 

gene delivery vectors.

**4.1 Poly(4-hydroxy-L-proline ester) (PHP)** 

rapid binding of PLL polyplexes with negatively-charged serum (C.H. Ahn et al., 2004). In PLL-mediated gene transfection against 293T cells, for example, the gene expression level is remarkably reduced by 10 times in the presence of 10% serum. The cytotoxicity profile of PLL is not satisfactory with about 60% cell viability at a tested concentration of 10 μg/mL. Thus, modification of PLL is needed to improve transfection ability and meanwhile decrease cytotoxicity.

Because the low transfection efficiency for PLL is attributed to its poor buffer capacity, Langer *et al*. introduced imidazole group (pKa ~6.5) into PLL to improve buffer capacity, thereby enhancing transfection efficiency (Putnam et al., 2001). The modified PLL was synthesized by the coupling of amino groups of PLL (34Ka) with 4-imidazoleacetic acid using an EDC/NHS activation. As expected, transfection efficiency of imidazole-modified PLL was increased with increasing amounts of imidazole groups and was much better than that of native PLL. The PLL with the highest imidazole content (86.5%) could mediate the best gene transfection, with gene expression level close to that of polyethylenimine. Low cytotoxicity is another merit for these imidazole-modified PLL (100% cell viability at 30 μg /mL). The PEGylation of PLL can also improve the transfection ability and cytotoxicity profile of PLL (C.H. Ahn et al., 2004). For example, a group of PLL-PEG multi-block copolymers were synthesized with molecular weight in the range from 32k to 65kDa. An optimal copolymer, PLL26-co-PEG32, was found highly efficient to transfect 293T cells. The low cytotoxicity of these copolymers was also observed with more than 95% cell viability. The PLL26-co-PEG32 copolymer could mediate almost the same transfection efficiency both in the absence and presence of 10% serum.

#### **3.4 Poly(amido amine) (PAMAM) dendrimer**

Poly(amido amine) dendrimers are a family of well-defined cationic polymers (Tomalia et al., 1990). Szoka *et al*. firstly investigated PAMAM cascade polymers as non-viral gene delivery vectors (Szoka, 1993). These polymers have an ammonia initiator core and different generation (G2-G10) of amido amine repeat units. In the transfection against CV-1 cells, an optimal level of gene expression (1x1010 RLU/mg protein) was observed for the sixth generation PAMAM (G6, MW 43451) at an N/P ratio of 6/1, but with 64% cell viability. However, 108 RLU/mg protein could be obtained for the PAMAM G5 at N/P=3, 6 or 10 with more than 90% cell viability. This high level of gene expression is due to a high buffer capacity of PAMAM since pKa value is 3.9 for internal tertiary amines and 6.9 for terminal amines. Another report showed that PAMAMs with an EDA initiator core can also mediate efficient gene transfection towards different mammalian cells (Kukowska-Latallo et al., 1996). The polyplexes of PAMAM G7 can transfect several types of cells with high gene transfection efficiency (≥1x1010 RLU/mg protein), which is even better than that that of Lipofectamine 2000. The cytotoxicity of these PAMAMs is however terrible. It appeared that the initiator core of PAMAM may influence their transfection efficiency.

In a further study, Szoka, *et al*. found that when intact PAMAM dendrimers were treated by heating in a solvent such as water or butanol, the resulting dendrimers can surprisingly induce higher transfection efficiency as compared to parent PAMAM (Tang et al., 1993). The efficiency is affected by the generation number of dendrimer and degree of degradation. For example, after the sixth-generation dendrimer initiated with tris(2-aminoehyl)amine (TAEA), termed as 6-TAEA, was degraded in the n-butanol for 43 hours, the resulting degraded PAMAM, denoted as "fractured" dendrimer, could lead to enhanced transfection efficiency by 3 orders of magnitude compared to that of native PAMAM. This pronounced transfection efficiency is likely due to increased flexibility which results in apparent volume swelling of fractured dendrimer in the endosomes and thus efficient endosomal escape.
