2. Revisiting Complexation Between DNA and Polyethylenimine: The Effect of Uncomplexed Chains Free in the Solution Mixture on Gene Transfection

In comparison with viral vectors, more efforts have recently been spent on the development of non-viral vectors because of few fatal accidents in clinical trials of viral carriers [1–3]. It has been well recognized that non-viral vectors have their own advantages, such as low immune toxicity, construction flexibility and facile fabrication, in the gene transfection, especially for clinical applications [4–6]. However, they are still much less efficient than their viral counterparts. Among thousands of experimentally tested non-viral vectors, cationic polyethylenimine (PEI) is still considered as one of the most efficient candidates to deliver genes and often served as a “golden standard” [7–9]. Previously, PEI has been chemically modified in different ways so that additional functions were introduced for a better gene delivery, including the incorporation of intracellular biodegradable linkers, [10, 11] the PEGylation to improve the serum stability during circulation [12, 13] and the attachment of some functional molecules to target specific cells or tissues [12–15]. Less attention, however, has been paid to why PEI remains one of the best non-viral carriers and how it facilitates the intracellular trafficking [16–27].