**1. Introduction**

84 Biomedicine

Zhang, B.; Zhang, Z.; Wang, C.(2010). Inhibitory effects on proliferation and invasion

Zhang, S.; Chen, W.; Lei Z.; Zou, X.; Zhao, P. (2008).A Report of Cancer Incidence from 37

Zhou, H.; Tang, Y.; Liang, X.; Yang, X.; Yang, J.; Zhu, G.; Zheng, M.; Zhang, C.(2009). RNAi

(November 2011), pp. 909-912, ISSN 1004-0242.

Vol.125, No.2, (July 2009), pp.453-462, ISSN 0020-7136

1001-9030

of lung cancer cells by RNAi-mediated knockdown of osteopontin, *Chinese Journal of experimental surgery,* Vol.27, No.2, (Febuary 2010), pp.230-231, ISSN

Cancer Registries in China, 2004, *Bulletin of Chinese Cancer*, Vol.17, No.11,

targeting urokinase-type plasminogen activator receptor inhibits metastasis and progression of oral squamous cell carcinoma in vivo, *International journal of cancer,* 

> Gene therapy holds substantial promise for the treatment of a broad class of overwhelming human diseases such as cancer and AIDS (Verma & Somia, 1997). An essential procedure in gene therapy program involves the delivery of encoded plasmid genes into the patient's somatic cells so as to express therapeutic proteins. An ideal strategy for successful gene delivery depends on safe and efficient gene delivery vectors (El-Aneed, 2004). Generally, gene delivery vectors are classified into two categories: viral vectors and non-viral vectors. Viral vectors are derived from natural viruses such as adenovirus and retrovirus with eliminated pathogenicity. Because of their unique capability in cell infection, viral vectors are most popular for gene delivery *in vitro* and *in vivo*. Unfortunately, clinical practice of viral vectors is seriously hampered by a few inherent issues including random insertion into the host genomes, immunogenicity, gene-carrying capacity limitation, and small-scale production (C.E. Thomas et al., 2003). In the past decades, these safety concerns on viral vectors have led to accelerated advancement in non-viral vectors (S. Li & Huang, 2000). Non-viral vectors such as lipids and polymers take more advantages over conventional viral vectors, including low immunogenicity after repeated administration, easy manufacture, large-scale production and low cost. However, current non-viral systems typically fail to give rise to as efficient gene transfection as powerful viral vectors (Pack et al., 2005). Thus, the availability of highly potent non-viral gene delivery vectors still remains a big challenge.

> Among different non-viral vectors, cationic polymers have received much attention because they can be prepared by different polymerization methods and easily modified to introduce different bio-functional groups (Luo & Saltzman, 2000). In the past two decades, a few traditional cationic polymers such as chitosan, polyethylenimine (pEI), poly(L-lysine) (pLL), polyamidoamine (PAMAM) dendrimer (Figure 1) have been studied widely as non-viral vectors for gene delivery (de Smedt et al., 2000). These cationic polymers can self-assemble with negatively-charged genes to form polymer/gene complexes (polyplexes) and induce detectable gene transfection efficiency *in vitro*. However, these first-generation polymeric gene vectors are not yet applied further for clinical practice, mainly due to low transfection efficiency and/or high cytotoxicity (Anwer et al., 2003; Merdan et al., 2002). In the past few years, extra- and intracellular gene delivery barriers have been identified that may seriously

Bioreducible Cationic Polymers for Gene Transfection 87

efficiently enter the cells. Moreover, naked gene is prone to degradation by enzymes in the cells. Cationic polymers are able to condense gene into nanoscale polyplexes, deliver the genes into the cells and protect DNA from the enzymatic degradation. Therefore, for the availability of safe and potent polymeric gene delivery vectors, it is essential to understand

+

**1. Formation of polyplexes by cationic polymer** 

**2. Uptake of polyplexes by absorptive or receptor-mediate endocytosis pathway 3. Endosomal escape via "proton sponge" effect**

*1*

**5. Degradation of DNA and polyplexes in endosomes or lysosomes**

**and DNA binding**

**4. Translocation in cytoplasm**

**6. Nuclear translocation**

cationic polymer-mediated gene delivery pathway.

*2 3*

*4*

*6*

*5*

Fig. 2. Schematic illustration on cationic polymer-mediated gene delivery

A schematic gene delivery mediated by a cationic polymer is illustrated in Figure 2. First, cationic polymers bind DNA via electrostatic self-assembly to form compact polymer/DNA complexes (polyplexes). An excess amount of cationic polymer is normally needed to neutralize negative DNA and cause resulting polyplexes with net positive surface charge. Then, the positive polyplexes can interact with cellular membrane and are internalized into the cells through adsorptive endocytosis or receptor-mediated endocytosis. After they enter cells, the polyplexes normally undergo an undesirable degradation pathway from the early to later endosomes and finally locate in the lysosomes. DNA is easily degraded by enzymes in the acidic endosomes or lysosomes (pH 5~6). In this situation, cationic polymeric vectors can protect DNA from degradation and induce efficient endosomal escape by a mechanism like "proton sponge" effect (Boussif et al., 1995). After endosomal escape, polyplexes stay in the cytoplasm and move towards the nucleus by passive diffusion. At this stage, it is still unclear whether the genes should be unloaded in the cytoplasm. The polyplexes with the particle size below 25 nm may freely diffuse through the nuclear pore in the nuclear membrane (Suh et al., 2003), but the polyplexes with bigger particle size have to undergo a

hamper efficient gene transfection (Nishikawa & Huang, 2001; Wiethoff & Middaugh, 2003). To overcome these barriers, on-going research works are devoted to molecular design of cationic polymers with multiple properties for circumventing the gene delivery barriers. It has been aware that the structures of polymers play an important role in gene transfection efficiency (Jeong et al., 2007).

Fig. 1. Typical examples of current cationic polymers as non-viral gene delivery vectors

A lot of evidences have indicated that biodegradable cationic polymers are new-generation polymeric gene vectors because of their favourable low cytotoxicity profiles (Luten et al., 2008). Particularly, bioreducible cationic polymers, containing the disulfide bond as a bioreducible linker in polymeric main chain or side chain, are of interest. It has been well known that disulfide bond is relatively chemically stable in the extracellular environment, but can be rapidly biodegradable inside the cells due to the presence of a high amount of reducing enzymes and sulfhydryl components such as glutathione (Ganta et al., 2008; G. Wu et al., 2004). By intracellular biodegradation, smart disulfide-based cationic polymers are able to efficiently unload genes in the nucleus (Soundara & Oupicky, 2006), thereby giving rise to high levels of gene expression. Meanwhile, the biodegradation also induces relatively low cytotoxicity by avoiding the accumulation of high molecular weight cationic polymer inside cells. These efforts are now actively striving to reach safe and potent polymeric gene vectors.

In this chapter, we aim to contribute the understanding of current status on biodegradable cationic polymers for non-viral gene therapy. Fundamental knowledge on the mechanism of polymer-mediated gene delivery is described briefly. Then, first-generation polymeric gene vectors and their pros and cons are outlined. Bioreducible polymers are finally reviewed to highlight current advancement and the challenge in near feature.

### **2. Cationic polymer-mediated gene delivery pathway**

DNA is a flexible, negatively-charged biomacromolecule under physiological conditions. It can be electrostatically repelled by negatively-charged cellular membranes and thus fails to

hamper efficient gene transfection (Nishikawa & Huang, 2001; Wiethoff & Middaugh, 2003). To overcome these barriers, on-going research works are devoted to molecular design of cationic polymers with multiple properties for circumventing the gene delivery barriers. It has been aware that the structures of polymers play an important role in gene transfection

**N**

**N**

**HN**

**O**

**O**

**NH N**

**O NH**

**NH2**

**HN O**

**N**

**O NH**

**NH O**

**NH2**

**NH2**

**NH2**

**O**

**NH**

**O**

**HN N**

**N**

**NH O**

**NH2**

**NH <sup>O</sup>**

**NH2**

**NH O**

> **NH O**

efficiency (Jeong et al., 2007).

**\***

*n*

**HN**

**\* <sup>N</sup> H**

**<sup>N</sup> <sup>N</sup>**

*linear PEI*

**NH2**

**O**

**OH**

**OH**

*chitosan*

**NH2**

highlight current advancement and the challenge in near feature.

**2. Cationic polymer-mediated gene delivery pathway** 

**O \***

*n*

**H2N**

**H2N**

*branched PEI PAMAM dendrimer (G2)* Fig. 1. Typical examples of current cationic polymers as non-viral gene delivery vectors

A lot of evidences have indicated that biodegradable cationic polymers are new-generation polymeric gene vectors because of their favourable low cytotoxicity profiles (Luten et al., 2008). Particularly, bioreducible cationic polymers, containing the disulfide bond as a bioreducible linker in polymeric main chain or side chain, are of interest. It has been well known that disulfide bond is relatively chemically stable in the extracellular environment, but can be rapidly biodegradable inside the cells due to the presence of a high amount of reducing enzymes and sulfhydryl components such as glutathione (Ganta et al., 2008; G. Wu et al., 2004). By intracellular biodegradation, smart disulfide-based cationic polymers are able to efficiently unload genes in the nucleus (Soundara & Oupicky, 2006), thereby giving rise to high levels of gene expression. Meanwhile, the biodegradation also induces relatively low cytotoxicity by avoiding the accumulation of high molecular weight cationic polymer inside cells. These efforts are now actively striving to reach safe and potent polymeric gene vectors. In this chapter, we aim to contribute the understanding of current status on biodegradable cationic polymers for non-viral gene therapy. Fundamental knowledge on the mechanism of polymer-mediated gene delivery is described briefly. Then, first-generation polymeric gene vectors and their pros and cons are outlined. Bioreducible polymers are finally reviewed to

DNA is a flexible, negatively-charged biomacromolecule under physiological conditions. It can be electrostatically repelled by negatively-charged cellular membranes and thus fails to

**O**

*n*

**\***

**\***

**H <sup>N</sup> \***

**H2N**

*PLL*

efficiently enter the cells. Moreover, naked gene is prone to degradation by enzymes in the cells. Cationic polymers are able to condense gene into nanoscale polyplexes, deliver the genes into the cells and protect DNA from the enzymatic degradation. Therefore, for the availability of safe and potent polymeric gene delivery vectors, it is essential to understand cationic polymer-mediated gene delivery pathway.

Fig. 2. Schematic illustration on cationic polymer-mediated gene delivery

A schematic gene delivery mediated by a cationic polymer is illustrated in Figure 2. First, cationic polymers bind DNA via electrostatic self-assembly to form compact polymer/DNA complexes (polyplexes). An excess amount of cationic polymer is normally needed to neutralize negative DNA and cause resulting polyplexes with net positive surface charge. Then, the positive polyplexes can interact with cellular membrane and are internalized into the cells through adsorptive endocytosis or receptor-mediated endocytosis. After they enter cells, the polyplexes normally undergo an undesirable degradation pathway from the early to later endosomes and finally locate in the lysosomes. DNA is easily degraded by enzymes in the acidic endosomes or lysosomes (pH 5~6). In this situation, cationic polymeric vectors can protect DNA from degradation and induce efficient endosomal escape by a mechanism like "proton sponge" effect (Boussif et al., 1995). After endosomal escape, polyplexes stay in the cytoplasm and move towards the nucleus by passive diffusion. At this stage, it is still unclear whether the genes should be unloaded in the cytoplasm. The polyplexes with the particle size below 25 nm may freely diffuse through the nuclear pore in the nuclear membrane (Suh et al., 2003), but the polyplexes with bigger particle size have to undergo a

Bioreducible Cationic Polymers for Gene Transfection 89

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

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

Low molecular weight PEI (below 2 kDa) normally displays lower cytotoxicity, but inferior transfection capability as compared to high molecular weight counterparts. Klibanov *et al*. modified the primary amines of 2k-Da PEI with dodecyl or hexadecyl iodides (M. Thomas & Klibanov, 2002). The transfection efficiencies of these alkylated 2k-Da PEI are surprising. In the transfection towards COS-7 cells, dodecylated or hexadecylated 2k-Da PEI can induce a high level of gene expression in the presence of serum, that is, 5-fold higher than that of 25k-Da PEI. The cytotoxicity of these alkylated PEI is much lower as compared to 25k-Da PEI

The incorporation of poly(ethylene glycol) (PEG) into PEI may yield PEGylated PEI with reduced cytotoxicity (C.-H. Ahn et al., 2002). PEGylated PEI copolymers can be synthesized by coupling activated PEG (2000 Da) with low molecule weight PEI (600, 1200 or 1800 Da). An optimal PEI-PEG copolymer is found that has 87 units of PEI1800 and 100 units of PEG2000. Again, the copolymer can efficiently bind plasmid DNA to form nanoscale polyplexes with positively surface charge (+20~+40mV) (average diameter 120~150nm) at N/P ratios from 1/1 to 4/1. The transfection efficiency of these polyplexes towards 293T cell is 3-fold higher than that of parent PEI1800. The cytotoxicity is very low with 80% cell viability. It should be noted that PEGylation often leads to reduced transfection efficiency *in vitro*. This is because PEGylated PEI-based polyplexes have low surface charges which impair efficient cellular internalization and also efficient endosomal escape of polyplexes (Mishra et al., 2004). Thus, the molecular weight of PEG and the composition ratio between

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

after cationic polymers are intracellularly degraded into small pieces.

transfection.

**3.2 Polyethylenimine derivatives** 

(100% *vs*. 80% cell viability).

PEI and PEG must be optimized.

**3.3 Poly(L-lysine)** 

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 system to produce therapeutic proteins.
