**5.1 Preparation of bioreducible cationic polymers as non-viral gene vectors**

Bioreducible cationic polymers can be designed and synthesized that contain disulfide bond either in polymer main chain or side chain. In earlier studies, the disulfide was introduced in the polymer side chain to conceptually confirm the role of the disulfide in gene delivery. A typical synthesis route is the preparation of cationic polymers with pyridyldithio residue, which is then modified with suitable thiol compounds via an exchange reaction (Figure 5a). By this method, the pLL containing disulfide linkages in the polymer side chains (termed as poly[Lys- (AEDTP)]) was prepared through chemical modification of the primary amines in pLL with Nsuccinimidyl-3-(2-pyridyldithio)propionate, followed by an exchange reaction with mercapthoethylamine (Pichon et al., 2002). The polyplexes of poly[Lys-(AEDTP)] can transfect HeLa cells with a level of gene expression 10-fold higher than that of parent pLL. This thus implies that disulfide linker plays a pivotal role in improved gene transfection. In another work, PAEs with pyridyldithio groups in the polymer side chains were synthesized via Michael-type addition reaction between diacrylates and 2-(pyridyldithio)-ethylamine. These polymers were further modified with mercaptoethylamine or thiol peptide such as RGD, yielding the PAEs with disulfide linkers in the side chains (SS-PAEs) (Zugates et al., 2006). The polyplexes of SS-PAEs could transfect HCC cells with the efficiency comparable to that of 25-kDa PEI.

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.

> **self assembly**

> > **disulfide cleavage**

**+**

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

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,

Bioreducible cationic polymers can be designed and synthesized that contain disulfide bond either in polymer main chain or side chain. In earlier studies, the disulfide was introduced in the polymer side chain to conceptually confirm the role of the disulfide in gene delivery. A typical synthesis route is the preparation of cationic polymers with pyridyldithio residue, which is then modified with suitable thiol compounds via an exchange reaction (Figure 5a). By this method, the pLL containing disulfide linkages in the polymer side chains (termed as poly[Lys- (AEDTP)]) was prepared through chemical modification of the primary amines in pLL with Nsuccinimidyl-3-(2-pyridyldithio)propionate, followed by an exchange reaction with mercapthoethylamine (Pichon et al., 2002). The polyplexes of poly[Lys-(AEDTP)] can transfect HeLa cells with a level of gene expression 10-fold higher than that of parent pLL. This thus implies that disulfide linker plays a pivotal role in improved gene transfection. In another work, PAEs with pyridyldithio groups in the polymer side chains were synthesized via Michael-type addition reaction between diacrylates and 2-(pyridyldithio)-ethylamine. These polymers were further modified with mercaptoethylamine or thiol peptide such as RGD, yielding the PAEs with disulfide linkers in the side chains (SS-PAEs) (Zugates et al., 2006). The polyplexes of SS-

of the polyplex, and (c) intracellular DNA release from the degraded polymer.

**5.1 Preparation of bioreducible cationic polymers as non-viral gene vectors** 

PAEs could transfect HCC cells with the efficiency comparable to that of 25-kDa PEI.

unique biophysical properties of the polyplexes based on the polymers.

**HS**

**HS**

**SH**

**SH**

**SH**

**SH**

**SH**

**DNA release**

**SH**

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

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 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

Bioreducible Cationic Polymers for Gene Transfection 97

P(CBA-ABOL), a homogeneous dispersed fluorescence was observed both in the cytoplasm and the nucleus. By contrast, for the p(BAP-ABOL) lacking the disulfide linkages, many micro-sized aggregated clumps were found in the perinuclear space and only a few weak fluorescence was observed in the nucleus. These results may serve as an indication that P(CBA-ABOL) is intracellularly degradable faster by reducing cleavage of the disulfide bonds, resulting in a diffuse distribution of fluorescently labeled polymer fragments inside the cells. Slow degradation of p(BAP-ABOL) may, however, contributes

**N**

**HO**

Fig. 7. Intracellular distribution of the polyplexes from bioreducible P(CBA-AOBL) (left) and non-degradable P(BAP-ABOL) lacking disulfide bonds (right), observed under confocal

line/animal

pCMV-Luc CHO Comparable to

NIH-3T3/ Balb/c mouse

pCMV-Luc Mice enhanced plasmid

laser scanning microscopy. The polymers are shown in green and the nucleus in red.

Plasmid Cell

pCMV-Luc, 32P-labeled plasmid

**HO** *P(CBA-ABOL)*

*P(CBA-ABOL)*

**n n N N O O**

Transfection Ref.

(Gosselin et al., 2001)

(Neu et al., 2007a)

(Neu et al., 2007b)

25-kDa PEI

60 min.

blood levels up to

3-fold higher than 25-kDa PEI, considerable gene expression in the liver and lung

**N**

to the formation of the aggregation.

**S**

**S**

**N H** **O**

**N H**

**O**

Disulfide-based polymers

DTSP or DTBPcrosslinked PEI

PEGylated PEI crosslinked with

Crosslinked PEI with

DTSP

DSP

et al., 2001; Gosselin et al., 2002). Recently, disulfide-containing poly(amido amine) (SS-PAA) (co)polymers were synthesized through Michael-type addition reaction of CBA to primary amines, secondary diamines or PEI oligoamines. The structural effects of these SS-PAAs on gene delivery properties were systematically investigated. It was shown that the SS-PAA with the hydroxybutyl or hydroxypentyl side groups led to higher transfection efficiencies and lower cytotoxicity in COS-7 cells than 25-kDa branched PEI. Herein, we summarize those typical bioreducible cationic polymers in Table 1 and their performance in gene transfection efficiency against different cell lines.

Fig. 6. Typical examples of bioreducible cationic polymers as non-viral gene delivery vectors

#### **5.2 Intracellular fate of disulfide-based polymeric gene vectors**

It is assumed that higher efficient transfection induced by disulfide-based cationic polymers is at least partly due to intracellular degradation via the cleavage of the disulfides in the reducing intracellular environment. In order to obtain experimental evidences, polyplexes of fluorescently labelled P(CBA-ABOL) containing disulfide bonds and P(BAPABOL) lacking disulfide bonds (Figure 7) were used for gene transfection against COS-7 cells at the same polymer/DNA mass ratio of 12/1. Dynamic light scattering and zeta-potential measurement showed that polyplexes of P(CBA-ABOL) and p(BAP-ABOL) had comparable average particle size and surface charge (128 nm vs. 82 nm; +20.2 mV vs. +19.2 mV), allowing good comparison of the transfection activity of both types of polyplexes. The intracellular distributions of the two polymers, labelled by a Rhodamine dye, are clearly different under fluorescence microscopy (Figure 7). For

et al., 2001; Gosselin et al., 2002). Recently, disulfide-containing poly(amido amine) (SS-PAA) (co)polymers were synthesized through Michael-type addition reaction of CBA to primary amines, secondary diamines or PEI oligoamines. The structural effects of these SS-PAAs on gene delivery properties were systematically investigated. It was shown that the SS-PAA with the hydroxybutyl or hydroxypentyl side groups led to higher transfection efficiencies and lower cytotoxicity in COS-7 cells than 25-kDa branched PEI. Herein, we summarize those typical bioreducible cationic polymers in Table 1 and their performance in

Fig. 6. Typical examples of bioreducible cationic polymers as non-viral gene delivery vectors

It is assumed that higher efficient transfection induced by disulfide-based cationic polymers is at least partly due to intracellular degradation via the cleavage of the disulfides in the reducing intracellular environment. In order to obtain experimental evidences, polyplexes of fluorescently labelled P(CBA-ABOL) containing disulfide bonds and P(BAPABOL) lacking disulfide bonds (Figure 7) were used for gene transfection against COS-7 cells at the same polymer/DNA mass ratio of 12/1. Dynamic light scattering and zeta-potential measurement showed that polyplexes of P(CBA-ABOL) and p(BAP-ABOL) had comparable average particle size and surface charge (128 nm vs. 82 nm; +20.2 mV vs. +19.2 mV), allowing good comparison of the transfection activity of both types of polyplexes. The intracellular distributions of the two polymers, labelled by a Rhodamine dye, are clearly different under fluorescence microscopy (Figure 7). For

**5.2 Intracellular fate of disulfide-based polymeric gene vectors** 

gene transfection efficiency against different cell lines.

P(CBA-ABOL), a homogeneous dispersed fluorescence was observed both in the cytoplasm and the nucleus. By contrast, for the p(BAP-ABOL) lacking the disulfide linkages, many micro-sized aggregated clumps were found in the perinuclear space and only a few weak fluorescence was observed in the nucleus. These results may serve as an indication that P(CBA-ABOL) is intracellularly degradable faster by reducing cleavage of the disulfide bonds, resulting in a diffuse distribution of fluorescently labeled polymer fragments inside the cells. Slow degradation of p(BAP-ABOL) may, however, contributes to the formation of the aggregation.

Fig. 7. Intracellular distribution of the polyplexes from bioreducible P(CBA-AOBL) (left) and non-degradable P(BAP-ABOL) lacking disulfide bonds (right), observed under confocal laser scanning microscopy. The polymers are shown in green and the nucleus in red.


Bioreducible Cationic Polymers for Gene Transfection 99

It appears that disulfide degradation mainly proceeds in the cytoplasm and in the nucleus. However, a few recent studies showed that, depending on the cell line type and the polymer constructs, the disulfide could also be degradable in those microenvironments such as the cellular surface, the endosomes and the lysosomes (Blacklock et al., 2009; Morre & Morre, 2003). Thus, further studies are certainly needed to understand the factors influencing the

Cationic polymers with multiple functionalities are promising as non-viral vectors for gene transfection. Since more and more extracellular and intracellular gene delivery barriers are identified that seriously hamper efficient gene transfection, a number of cationic polymers have been designed that are capable of overcoming one or more gene delivery barriers, thus leading to detectable gene transfection efficiency. From those conventional non-degradable cationic polymers to current bioreducible cationic polymers, peoples have more and more reached virus-like, safe and potent polymeric gene delivery vectors. Further understanding on structure-activity relationships of cationic polymers and their intracellular fate should be indispensable, in order to achieve polymer systems that can exhibit multiple gene delivery

This work was financially supported by the Innovation Program of Shanghai Municipal Education Commission (No. 10ZZ26), the Program for Young Excellent Talents in Tongji University (No. 2009KJ077), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry, and the National Natural Science Foundation of

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**6. Conclusion** 

**7. Acknowledgment** 

China (No. 20904041).

0168-3659

**8. References** 


Table 1. A summary of bioreducible cationic polymers and their transfection efficiencies in different types of cell lines.

It appears that disulfide degradation mainly proceeds in the cytoplasm and in the nucleus. However, a few recent studies showed that, depending on the cell line type and the polymer constructs, the disulfide could also be degradable in those microenvironments such as the cellular surface, the endosomes and the lysosomes (Blacklock et al., 2009; Morre & Morre, 2003). Thus, further studies are certainly needed to understand the factors influencing the degradation at specific locations.
