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, 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]. The first step in the development of non-viral vectors is how to package long anionic DNA chains into a small particle, i.e., the DNA complexation and condensation. Previous study showed that the physical and colloidal characteristics of resultant polymer/DNA polyplexes are important for an effective delivery of gene [28–32], but results were controversial and not conclusive. Much of the past effort has been devoted to the synthesis of state-of-the-art polymers [33–37] and subsequent polyplex formation [38–41], rather than the fundamental understanding of how a non-viral vector promotes the intracellular trafficking of DNA besides its roles in DNA encapsulation and protection. The proposed proton-sponge effect has been well accepted and taken as granted by those who entered this field later [7, 42]. Using a combination of different methods to characterize the size, molar mass and surface charge of the DNA/PEI polyplexes formed under different conditions, we confirmed some of the previous literature results, but revealed that the average size and chain density of the resultant polyplexes are not that important in the gene