**8. General toxicity**

486 Non-Viral Gene Therapy

*et al.*, 1999; Fischer *et al.*, 2002; Brus *et al.*, 2004; Neu *et al.*, 2005; Gary *et al.*, 2007). PEI 25 kDa serves as gold standard for in vitro transfection experiments (Godbey *et al.*, 2000). The mechanism of cell entry and action for gene delivery is intensively analyzed. To enhance the endosomal release of endocytosed polyplexes PEI uses the so-called "proton-sponge" effect (Boussif *et al.*, 1995; Behr, 1997) Due to the high buffer capacity of PEI amino groups in PEI molecules will be protonated at lower pHs like in the endosomal-lysosomal environment, additional chloride influx into the vesicles increases the osmolarity and the vesicles begin to swell and under the increased osmotic pressure the vesicle will be disrupted and the nucleic acid protected from PEI will be released into the cytoplasm (Godbey *et al.*, 1999; Akinc *et al.*, 2005; Nel *et al.*, 2009). PEI has been used for many years to facilitate nucleic acid delivery (Demeneix and Behr, 2005). However, due to toxicity and variable performance a lot of research is undertaken to reduce the toxicity of PEI and maintain or improve the efficacy and specificity by modification PEI backbone and/or conjugation of hydrophilic molecules like polyethylene glycol (PEG) (Petersen *et al.*, 2002a; Petersen *et al.*, 2002b), disulfide linkages (Breunig *et al.*, 2008), or for specific targeting molecules like transferrin, galactose, TAT-peptide, RGD-motifs (Ogris *et al.*, 1999; Kunath *et al.*, 2003a; Kunath *et al.*, 2003b; Kleemann *et al.*, 2005). Other approaches are reduction of the molecular weight of PEI 25 kDa or purification of PEI 25 kDa via gel filtration (Boeckle *et al.*, 2004; Urban-Klein *et al.*, 2005; Werth, 2006; Fahrmeir *et al.*, 2007) or using instead of the branched PEI 25 kDa the linear form PEI22kDa (Breunig *et al.*, 2005). Thomas and colleagues showed that full deacylation of linear PEI dramatically improves the efficacy but on cost of increased cytotoxicity due to increased numbers of protonatable nitrogens in the PEI molecule

Modifications of PEI with the hydrophilic poly(ethylene glycol) (PEG) reduces dramatically the cytotoxicity of PEI 25 kDa but in part on cost of efficacy and increased immunomodulatory and proinflammatory effects (Kichler *et al.*, 2002; Petersen *et al.*, 2002b; Mao *et al.*, 2005; Glodde *et al.*, 2006; Beyerle *et al.*, 2010a; Beyerle *et al.*, 2010b). PEG provides polyplexes with improved solubility, lower surface charge, diminished aggregation, lower

Glodde et al. synthesized a series of PEG-PEI copolymers and found that the molecular weight of PEG was found to be the major determinant of polyplex size, via its influence on particle aggregation and polyplex stability (Glodde *et al.*, 2006). Transfection efficiency was correlated to polyplex stability and low molecular weight PEI 2 kDa grafted with PEG showed higher activity than their counterparts with high molecular weight PEI 25 kDa (Williams *et al.*, 2006). In contrast, Petersen and Mao showed good transfection efficiencies for PEI 25 kDa - PEG copolymers with high molecular weight PEG and low numbers of grafting on PEI backbone compare to low molecular weight PEG with high grafting numbers on PEI 25 kDa (Mao *et al.*, 2005; Merkel *et al.*, 2009; Beyerle *et* 

Grayson and colleagues investigated the siRNA transfection efficacy of different PEI polymers (branched 800 Da, branched 25 kDa and linear 22 kDa) in HeLa derivative cell line (Grayson *et al.*, 2006). They showed that the siRNA delivery and activity was mainly dependent on the biophysical and structural characteristics of the polyplexes and only

cytotoxicity, and possibly improved "stealth effect" in the bloodstream.

(Thomas *et al.*, 2005).

*al.*, 2011a).

**6. Modifications of PEI** 

Hornung et al. described that any rupture or leakage of the endosomal or lysosomal membrane will release cathepsin B, which leads to an inflammasome activation associated with IL-1 production and apoptosis (Hornung *et al.*, 2008). Beyerle et al. found that application PEI/siRNA complexes caused release of proinflammatory cytokines like IL-6, G-CSF, TNF-a, IP-10 in murine lung cell lines (Beyerle *et al.*, 2010a; Beyerle *et al.*, 2010b; Beyerle *et al.*, 2011a and Beyerle *et al.*, 2011c). Cytokine release upon PEI/nucleic acid polyplex treatment has been also described by Gautam and Kawakami et al. (Gautam *et al.*, 2001; Kawakami *et al.*, 2006). Cubillos-Ruis and co-workers investigated linear PEI/siRNA complexes for antitumor immunity and identified linear PEI as TLR 5 agonist of mouse and human. They found that linear PEI/siRNA complexes induced a pattern of inflammatory cytokines which are triggered in vivo by flagellin in a TLR5 dependent manner (Cubillos-

Toxicity of Polymeric-Based Non-Viral Vector Systems for Pulmonary siRNA Application 489

Fig. 1. Structure-function-relationships of PEG-PEI copolymers

investigated molecules.

Overview of the structure-function relationships of PEG modified PEI copolymers (B-C) in comparisonto PEI 25kDa (A) with regard to cytotoxic (v,w), oxidative stress (x,y) and proinflammatory responses (z). Arrows represent the up- or downregulation of the

able to pass through biological membranes/barriers and transfer the desired information to target sites with minimal impact on the integrity of the target cell or tissue (Forrest and Pack, 2002; Omidi *et al.*, 2008). Viral vectors possess high efficacy accompanied by stimulation of the immune systems which is a limitation of these systems to deliver nucleic acids and human use. Therefore, non-viral vector systems should overcome these adverse side effects and represent safer and more efficient alternatives with improved bioavailability and reduced cellular toxicity in the clinics (Akhtar *et al.*, 2000; Somia and Verma, 2000; Panyam and Labhasetwar, 2003). It has been shown that cationic polymers and lipid-based transfection reagents could elicit cellular gene expression changes and complexation with siRNA increased these changes (Omidi *et al.*, 2003; Omidi *et al.*, 2005; Fedorov *et al.*, 2006; Hollins *et al.*, 2007; Tagami *et al.*, 2007; Tagami *et al.*, 2008). Beyerle et al. analyzed the expression changes of genes related to cytotoxicity, inflammation and oxidative stress in a pathway focused qRT-PCR array system upon treatment with different PEI-PEG copolymers in murine lung epithelial cells (LA-4 cell line) and could show that PEGylated PEI copolymers altered the gene expression profile on cost of upregulation of genes involved in inflammatory and oxidative stress processes while PEI 25 kDa mainly induced genes related to cytotoxicity and apoptosis (Beyerle *et al.*, 2010a). In addition, the potential of PEI and PEI-PEG copolymers to induce DNA damage and therefore their genotoxic potential was investigated in a lung epithelial cell line derived from the MutaMouse, but no indication for

Ruiz *et al.*, 2009). Thus, for in vivo use a lot of effort should be made to avoid the high proinflammatory effects caused by the rupture or leakage of the endosome caused by PEI. Godbey classified PEI-mediated toxicity in an immediate toxicity, associated with free PEI and a delayed form, connected with cellular processing of PEI/DNA polyplexes (Godbey *et al.*, 2001). To form stable and protective PEI nucleic acid polyplexes an excess of PEI polymer is needed, 60-80% PEI remains in a free form after nucleic acid escape and is mainly attributed to PEI toxicity. The high positively charged PEI molecule is able to disrupt cell membranes, disruption of the endosome is on one hand favourable with respect to the intended cytoplasmatic delivery, but on the other hand disruption of other cell membranes (e.g., lysosomal membranes, mitochondrial membrane, plasma membrane) is not favourable as it will cause stress responses or even apoptotic or necrotic cell death. In this context it has been shown that PEI causes apoptosis in an unspecific manner in all kinds of cells (Beyerle *et al.*, 2010a; Merkel *et al.*, 2011) which should be avoided with regard to human use. Therefore, a purification approach of the PEI polymer before and after complexation with nucleic acid is one possibility to reduce PEI-related toxicity (Boeckle *et al.*, 2004; Werth, 2006; Fahrmeir *et al.*, 2007).
