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

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

Bioreducible Cationic Polymers for Gene Transfection 93

versatile, a large number of PAEs with different functional groups can be designed (Lynn & Langer, 2000). Due to the presence of multiple ester bonds in polymer main chain, PAEs are degradable and normally possess relatively low cytotoxicity. A particular example is that Langer et al. examined a library containing more than 2000 PAEs for non-viral gene delivery (Anderson et al., 2003). Also, the structure-activity relationships were investigated. The conclusions from the study are: 1) Bisacrylate monomers with strongly hydrophobic residues are almost always present in the 50 best-performing PAEs; 2) Linear, bis(secondary amines) are over represented in the hit structures; 3) Mono- or dialcohol side group in PAE is an important functional entity for efficient gene transfection. One PAE from this library showed transfection ability, with the level of gene expression 5-fold higher than that of 25- kDa PEI against 3T3 cell lines under optimal conditions. The studies on PAEs strongly support the idea that degradable cationic polymers are very promising for safe and efficient gene delivery.

Poly(2-amioethyl propylene phosphate) (PPE-EA) is a degradable cationic polymer which can yield ultimate low-toxic degradation products including α-propylene glycol, phosphate, and ethanolamine. This polymer is synthesized through ring-opening polymerization of 4 methyl-2-oxo-2-hydro-1,3,2-dioxaphospholane, followed by two-step chemical modification (Wang et al., 2001). The transfection of PPE-EA (30 kDa) gives 100-fold higher levels of gene expression at an N/P ratio of 6 as compared to that of 27- kDa PLL at an N/P ratio of 5. Importantly, PPE-EA has low cytotoxicity towards COS-7 cells with more than 80% cell viability up to a tested concentration of 1000 μg/mL. However, this polymer has poor ability of endosomal escape since the presence of chloroquine (100 μM), a reagent known to disrupt endosomal membrane, may lead to remarkably enhanced transfection efficiency.

Polyphosphazenes (PPAs) are cationic polymers derived from poly(dichloro)phosphazene (Luten et al., 2003). They are degradable slowly under physiological conditions with a halflife of more than 10 days, but relatively faster at pH 5 with a half-life of 4-5 days. The PPAs (>100 kDa) can condense genes into nanoscale polyplexes at low polymer/DNA mass ratios of 3~5, with a high positive surface charge (+ 40mV). The transfection of PPA was comparable with that of PEI towards COS-7 cells. The optimal transfection efficiency of PPA was observed at a polymer/DNA mass ratio of 6, however, with a high cytotoxicity profile (~50% cell viability). The pronounced cytotoxicity could be due to high molecular weight

**5. Bioreducible cationic polymers as non-viral gene delivery vectors** 

In the design of hydrolysable cationic polymers for non-viral gene delivery, a contradiction has to be found that the polymers are expected to be rapidly degradable intracellularly as one hand, but chemically stable extracellularly as another hand. In order to avoid this issue, disulfide bond as a bioreducible linker has received much attention in recent years. The disulfide bond is chemically stable in the blood plasma, but intracellularly bio-cleavable by reducing enzymes like glutathione reductase and sulfhydryl components like glutathione since the concentration of these reducing species is much higher in the cytoplasm than the

**4.4 Polyphosphoester (PPE-EA)** 

**4.5 Polyphosphazene (PPA)** 

and slow degradation profile inside the cells.

at a high polymer/DNA ratio of 50/1(w/w) with transfection efficiency of 1.5 times higher than that of PLL. Importantly, this polymer has very low cytotoxicity compared to 25-kDa PEI. It is worthy pointing out that the transfection of PHP is not influenced by serum, indicating this polymer is biocompatible for non-viral gene delivery.

Fig. 3. Typical examples of hydrolysable cationic polymers as non-viral vectors

#### **4.2 Poly[α-(4-aminobutyl)-L-glycolic acid] (PAGA)**

Poly[α-(4-aminobutyl)-L-glycolic acid] (PAGA, 3.3kDa) is an analogue of PLL (Lim et al., 2000). Since the amide bonds in PLL are replaced with ester bonds, PAGA is degradable under physiological conditions. PAGA is rapidly degraded at pH 7.4 and 37°C with the degradation half time of 30 min. The presence of primary amine in the side chain of PAGA renders this polymer highly efficient for gene binding. As such, at a low N/P ratio of 5/1, nanoscale polyplexes from PAGA/DNA (~326nm) can be formed [34]. The transfection efficiency of PAGA is comparable with that of PLL. At an optimal N/P of 40/1, the transfection efficiency of the polyplexes of PAGA is 3-fold higher compared to those of 4 kDa PLL. The cytotoxicity of PAGA is much low in comparison with that of PLL (100% vs. 20% cell viability). Although PAGA is not as efficient as PEI for gene transfection, the study on the PAGA indicates that biodegradable cationic polymers are relatively safer due to low cytotoxicity as compared to non-degradable cationic polymers.

#### **4.3 Poly(β-aminoester)s (PAE)**

Poly(β-amino ester)s (PAEs) are a family of degradable cationic polymers that are prepared via Michael-type addition between bisacrylates and amines. Since these reactive monomers are

at a high polymer/DNA ratio of 50/1(w/w) with transfection efficiency of 1.5 times higher than that of PLL. Importantly, this polymer has very low cytotoxicity compared to 25-kDa PEI. It is worthy pointing out that the transfection of PHP is not influenced by serum,

indicating this polymer is biocompatible for non-viral gene delivery.

Fig. 3. Typical examples of hydrolysable cationic polymers as non-viral vectors

Poly[α-(4-aminobutyl)-L-glycolic acid] (PAGA, 3.3kDa) is an analogue of PLL (Lim et al., 2000). Since the amide bonds in PLL are replaced with ester bonds, PAGA is degradable under physiological conditions. PAGA is rapidly degraded at pH 7.4 and 37°C with the degradation half time of 30 min. The presence of primary amine in the side chain of PAGA renders this polymer highly efficient for gene binding. As such, at a low N/P ratio of 5/1, nanoscale polyplexes from PAGA/DNA (~326nm) can be formed [34]. The transfection efficiency of PAGA is comparable with that of PLL. At an optimal N/P of 40/1, the transfection efficiency of the polyplexes of PAGA is 3-fold higher compared to those of 4 kDa PLL. The cytotoxicity of PAGA is much low in comparison with that of PLL (100% vs. 20% cell viability). Although PAGA is not as efficient as PEI for gene transfection, the study on the PAGA indicates that biodegradable cationic polymers are relatively safer due to low

Poly(β-amino ester)s (PAEs) are a family of degradable cationic polymers that are prepared via Michael-type addition between bisacrylates and amines. Since these reactive monomers are

**4.2 Poly[α-(4-aminobutyl)-L-glycolic acid] (PAGA)** 

cytotoxicity as compared to non-degradable cationic polymers.

**4.3 Poly(β-aminoester)s (PAE)** 

versatile, a large number of PAEs with different functional groups can be designed (Lynn & Langer, 2000). Due to the presence of multiple ester bonds in polymer main chain, PAEs are degradable and normally possess relatively low cytotoxicity. A particular example is that Langer et al. examined a library containing more than 2000 PAEs for non-viral gene delivery (Anderson et al., 2003). Also, the structure-activity relationships were investigated. The conclusions from the study are: 1) Bisacrylate monomers with strongly hydrophobic residues are almost always present in the 50 best-performing PAEs; 2) Linear, bis(secondary amines) are over represented in the hit structures; 3) Mono- or dialcohol side group in PAE is an important functional entity for efficient gene transfection. One PAE from this library showed transfection ability, with the level of gene expression 5-fold higher than that of 25- kDa PEI against 3T3 cell lines under optimal conditions. The studies on PAEs strongly support the idea that degradable cationic polymers are very promising for safe and efficient gene delivery.

#### **4.4 Polyphosphoester (PPE-EA)**

Poly(2-amioethyl propylene phosphate) (PPE-EA) is a degradable cationic polymer which can yield ultimate low-toxic degradation products including α-propylene glycol, phosphate, and ethanolamine. This polymer is synthesized through ring-opening polymerization of 4 methyl-2-oxo-2-hydro-1,3,2-dioxaphospholane, followed by two-step chemical modification (Wang et al., 2001). The transfection of PPE-EA (30 kDa) gives 100-fold higher levels of gene expression at an N/P ratio of 6 as compared to that of 27- kDa PLL at an N/P ratio of 5. Importantly, PPE-EA has low cytotoxicity towards COS-7 cells with more than 80% cell viability up to a tested concentration of 1000 μg/mL. However, this polymer has poor ability of endosomal escape since the presence of chloroquine (100 μM), a reagent known to disrupt endosomal membrane, may lead to remarkably enhanced transfection efficiency.

#### **4.5 Polyphosphazene (PPA)**

Polyphosphazenes (PPAs) are cationic polymers derived from poly(dichloro)phosphazene (Luten et al., 2003). They are degradable slowly under physiological conditions with a halflife of more than 10 days, but relatively faster at pH 5 with a half-life of 4-5 days. The PPAs (>100 kDa) can condense genes into nanoscale polyplexes at low polymer/DNA mass ratios of 3~5, with a high positive surface charge (+ 40mV). The transfection of PPA was comparable with that of PEI towards COS-7 cells. The optimal transfection efficiency of PPA was observed at a polymer/DNA mass ratio of 6, however, with a high cytotoxicity profile (~50% cell viability). The pronounced cytotoxicity could be due to high molecular weight and slow degradation profile inside the cells.

## **5. Bioreducible cationic polymers as non-viral gene delivery vectors**

In the design of hydrolysable cationic polymers for non-viral gene delivery, a contradiction has to be found that the polymers are expected to be rapidly degradable intracellularly as one hand, but chemically stable extracellularly as another hand. In order to avoid this issue, disulfide bond as a bioreducible linker has received much attention in recent years. The disulfide bond is chemically stable in the blood plasma, but intracellularly bio-cleavable by reducing enzymes like glutathione reductase and sulfhydryl components like glutathione since the concentration of these reducing species is much higher in the cytoplasm than the

Bioreducible Cationic Polymers for Gene Transfection 95

**S S**

**R**

**N H** **+**

**H**

**H**

**NH2 <sup>+</sup> Cl-**

**O**

+ **PEI <sup>N</sup>**

+ **PEI <sup>N</sup>**

Fig. 5. Typical methods for the preparation of bioreducible cationic polymers as non-viral

to disulfide-containing copolymers with multiple functionalities (Read et al., 2005).

A simple approach for the availability of disulfide-based cationic polymers is the chemical coupling of amine compounds with disulfide-containing reagents, such as cystamine bisacrylamide (CBA) in a Michael addition reaction (Lin et al., 2006, 2007a; Lin et al., 2007b; Lin et al., 2008; Lin & Engbersen, 2008) (Figure 5c), and dithiobis(succinimidyl propionate) (DTSP) or dithiobispropionimidate (DTBP) in a polycondensation reaction (Figures 5d&e). These reactions can generate linear or branched disulfide-containing cationic polymers with different molecular structures (Figure 6). Lee *et al*. firstly prepared disulfide-containing branched pEI by the crosslinking of low molecular weight PEI with DTSP or DTBP (Gosselin

Alternatively, one route to generate bioreducible cationic polymers is the polyoxidation of dithiol-based monomers having amino groups (Figure 5b). Typical examples are disulfidecontaining cationic polymers based on pEI , pLL and pDMAEMA (SS-PEI, SS-PLL and SS-PDMAEMA, respectively, in Figure 5). In general, the preparation of these dithiol-based oligoamines is time-consuming and these compounds can not be stored for long term due to oxidation of thiol groups by air. As typical examples, Park *et al*. reported on the synthesis of dithiol-containing oligoamines via organic synthesis involving protection and deprotection of amino groups (Lee et al., 2007). Oupický *et al*. described the preparation of well-defined dithiol-based PDMAEMA oligomers via reversible addition-fragmentation chain transfer polymerization (You et al., 2007). Seymour *et al*. produced dithiol-based oligopeptides (Cys-Lys10-Cys) via solid-phase organic synthesis (Oupicky et al., 2002). These dithiol-based oligoamines can be oxidized by DMSO as an oxidant agent to yield disulfide-containing cationic polymers. Also, different dithiol-bearing groups, e.g. nuclear localization sequences comprising two cysteine residues, can be incorporated in the oxidation reaction, giving rise

**S**

**<sup>S</sup> <sup>N</sup> H**

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

**S HN**

**<sup>S</sup> <sup>S</sup> O H**

**<sup>S</sup> <sup>S</sup>**

**PEI PEI**

**PEI PEI**

**O**

**N O**

**H N NH2 <sup>+</sup> Cl-**

**<sup>R</sup>** *<sup>n</sup>*

**N H**

**<sup>O</sup> <sup>S</sup> <sup>S</sup> O**

**<sup>O</sup> <sup>S</sup> <sup>S</sup> NH2 <sup>+</sup> Cl-**

**O**

**S S N**

**O**

**O**

**b)**

**a)**

**c)**

**d)**

**e)**

gene delivery vectors

**S**

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

**DTSP**

**DTBP**

**CBA**

+

**<sup>S</sup> <sup>N</sup> H** **O**

**HS SH \* <sup>S</sup> <sup>S</sup> \***

**HS**

**R**

polymer main chain polymer main chain

**O O**

**O**

**O NH2 <sup>+</sup> Cl-**

**O**

<sup>+</sup> **NH2 R**

blood plasma (intracellular vs. extracellular glutathione concentration, 0.5-10 mM vs. 2-20 μM) (G. Wu et al., 2004). Thus, this feature makes the disulfide very valuable in the design of biodegradable cationic polymers for triggered gene delivery. Figure 4 shows a schematic illustration on intracellular gene delivery mediated by disulfide-based cationic polymers.

Fig. 4. A conceptual illustration of DNA binding and subsequent intracellular release: (a) formation of the polyplexes of bioreducible cationic polymers, which are relatively stable in the extracellular environment, (b) intracellular cleavage of disulfide linkages in the polymer of the polyplex, and (c) intracellular DNA release from the degraded polymer.

This section reviews current progress in disulfide-based cationic polymers as non-viral gene delivery vectors. The topics are focused on the synthesis of bioreducible cationic polymers, unique biophysical properties of the polyplexes based on the polymers.
