**5. PEI-based non-viral vector systems**

with improved properties (Kim and Kim, 2009).

Polyethylene imine (PEI) is a simple repetition of the 43 Da CH2-CH2-NH ethylene imine motifs. It can be synthesized from ethylene imine (aziridine) via ring opening polymerization or by hydrolysis of poly(2-ethyl-2-oxazolium), leading to branched or linear polymeric backbones, respectively (Godbey *et al.*, 1999). PEI represents one of the most comprehensive investigated cationic polymer for gene delivery in vitro and in vivo (Godbey

Toxicity of Polymeric-Based Non-Viral Vector Systems for Pulmonary siRNA Application 487

25 kDa PEI was able to effective deliver siRNA. The authors explained the high activity of PEI25kDa/siRNA with good stability of polyplexes, small size, and positively surface

Succinylated PEI polymers for complexation of siRNA were introduced by Wagner and colleagues which showed 10-fold lower toxicity and higher knockdown efficacy compare to

Synthetic polymers and nanomaterials display selective phenotypic effects in cells and in the body that affect signal transduction mechanisms involved in inflammation, differentiation, proliferation, and apoptosis. When physically mixed or covalently conjugated with cytotoxic agents, bacterial DNA or antigens, polymers can drastically alter specific genetically controlled responses to these agents (Kabanov, 2006). These effects, in part, result from cooperative interactions of polymers and nanomaterials with plasma cell membranes and trafficking of polymers and nanomaterials to intracellular organelles. Cells and whole organism responses to these materials can be phenotype or genotype dependent. In selected cases, polymer agents can bypass limitations to biological responses imposed by the genotype, for example, phenotypic correction of immune response by polyelectrolytes. Overall, these effects are relatively benign as they do not result in cytotoxicity or major toxicities in the body. Collectively, however, these studies support the need for thoroughly assessing pharmacogenomic effects of polymer materials to maximize clinical outcomes and understand the pharmacological and toxicological effects of polymer formulations of biological agents, i.e. polymer genomics. In addition, it is well described in the literature that cationic nanoparticles disrupt lipid bilayers (Hong *et al.*, 2006; Leroueil *et al.*, 2008), induce oxidative stress inside the cell as a result of cell-type interplay and cause in some cases acute lung inflammation when administered intratracheally (Tan and Huang, 2002; Beyerle *et al.*, 2010b; Beyerle *et al*., 2011a and Beyerle *et al*., 2011c). Intensive efforts will have to focus on the issue of cytotoxicity to obtain more insight in the exact mechanisms behind, which are multidimensional and largely depend on the application route as well as the formulation that is delivered. Therefore, tissue specific toxicity profiles are still needed and represent a great implement in improving non-viral

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-

charge, but nevertheless the cytotoxicity was highest for PEI 25 kDa.

**7. Toxicity of PEI-based non-viral vector systems** 

pure PEI polyplexes (Zintchenko *et al.*, 2008).

delivery systems.

**8. General toxicity** 

*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 (Thomas *et al.*, 2005).
