Synthetic polymer PEI represents the most extensively studied cationic polymer for nonviral gene delivery, especially with respect to pulmonary application.19 The transfection efficiency and cytotoxicity of PEI are affected by many factors, such as molecular weight, degree of branching, cationic charge density, molecular structure, sequence, and the ratio between PEI and siRNA. The highly cationic property and the powerful ability to compact nucleic acid and trigger endosomal release through proton sponge effect make PEI the “gold standard” for in vitro gene delivery. PEI is often used in proof-of-principle studies or setups where a method is optimized. Gene expression in lung epithelium downregulated by the polyplexes of siRNA and PEI has also been demonstrated in vivo.20 Despite the excellent performance of PEI, its clinical application is severely hampered by its poor biocompatibility and cytotoxicity. Linking PEI with other polymers, mostly polyethylene glycol (PEG), with the possibility of further conjugation to other polymers is the common strategy to reduce the cytotoxicity of PEI. With respect to siRNA delivery to the lungs, it would appear that the higher the degree of PEGylation and the shorter the PEG chain length, the fewer the cytotoxic effects that were observed.21 Subsequently, Beyerle et al.22

compared lung toxicity, inflammation, and efficiency of siRNA nanocomplex with two types of PEG-modified PEI derivatives on ubiquitously enhanced green fluorescent protein (EGFP) expressing transgenic in BALB/c mice models after intratracheal administration. It was found that the PEG modification reduced cytotoxicity but elevated the immune response and proinflammatory effects. Therefore, further chemical modifications on PEI backbone, aimed at increasing the biocompatibility, specificity, and efficacy of PEI-based nanocarriers, are needed in future studies.

To address the issue of poor physical stability of polyplex, Kedinger et al. modified the structure of siRNA and designed the so-called sticky siRNAs that polymerize via their complementary 3' overhangs. The polyplexes formed between “sticking” siRNAs and linear PEI exhibited better stability than usually obtained siRNA/PEI complexes and showed enhanced gene silencing efficiency both in vitro and in vivo.23 For longterm storage, Terry et al. proposed a lyophilization method in which the two components (polymer and siRNA) were flash frozen and lyophilized as separate droplets in the same reaction tube and rapidly formed polyplexes upon addition of water. This formulation step did not have any adverse effects on the particle size of polyplexes or transfection efficiency and the knockdown efficiency of up to 90% was maintained.15

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