**6. Modifications of PEI**

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

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 al.*, 2011a).

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

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