Mixing-sequence-dependent nucleic acid complexation and gene transfer efficiency by polyethylenimine

Abstract

Polyplexes, complexed nucleic acids by cationic polymers, are the most common forms of nonviral gene delivery vectors. In contrast to a great deal of efforts in synthesizing novel cationic polymers and exploring their extracellular and intracellular delivery pathways, polyplex preparation methods of mixing nucleic acids and cationic polymers are often overlooked. In this study, the mixing sequence, that is adding nucleic acids to polymers or vice versa, was found to greatly affect complexation of both plasmid DNA and siRNA, polyplexes' size, and polyplexes' surface charge, which all collaboratively affected the transfection efficiency and cytotoxicity. Adding polyethylenimine (PEI), the most conventionally used standard in nonviral gene delivery, to plasmid DNA and siRNA resulted in larger polyplexes, higher gene expression and silencing, but higher cytotoxicity than polyplexes prepared in the reverse order. Based on the experimental results, the authors developed a model that gradual addition of cationic polymers (e.g., PEI) to nucleic acids (e.g., plasmid DNA and siRNA) incorporates more copies of nucleic acids in larger polyplexes in a smaller number, results in higher gene expression and silencing levels in transfected cells, and generates higher cytotoxicity by leaving more free polymers upon complete mixing than the other mixing sequence. The proposed model can be explored using a broad range of cationic polymers and nucleic acids, and provide insightful information about how to prepare polyplexed nonviral vectors for efficient and safe gene delivery.

Structural variations in hyperbranched polymers prepared via thermal polycondensation of lysine and histidine and their effects on DNA delivery

“…These vectors offer advantages over viral vectors in immunogenicity and carcinogenicity, packing capacity, and possibility for scale‐up, even if many early examples exhibited lower transfection efficiency with transient gene expression and relatively high cytotoxicity (Godbey et al, ). More recent studies have utilised a very wide variety of functionalised cationic lipids (Viricel et al, , Altnoglu et al, , Semple et al, ) and cationic polymers, with increasing efficacy in transfecting target cells with reduced toxicity (Luo et al, , Blum et al, , Lee et al, , Cho et al, ). In this context, polymers based on polyamidoamine, polyethylenimine (PEI), and polylysine (PLL), synthesised into varying architectures from linear to branched and dendritic, have been designed and developed as efficient gene carriers (Khandare et al, , Ainalem et al, , Dufes et al, , Hardy et al, , Bansal et al, , Sun and Davis, ).…”

Synergistically enhanced selective intracellular uptake of anticancer drug carrier comprising folic acid-conjugated hydrogels containing magnetite nanoparticles

“…Hydrogels were relatively uniform in size and spherical in shape with diameter slightly less than 200 nm. Smaller particle size observed in dried samples of SEM and TEM than hydrated hydrogels in DLS measurement is a well-known phenomenon2425. As shown in Fig.…”

mRNA-Producing Pseudo-nucleus System

“…This mechanism, the so‐called the central dogma, derives its importance from the fact that it can be applied to metabolism‐mimicking synthetic cell designs in the same context. To date, however, the creation of synthetic cells has consisted of simply trapping exogenous genomes into polymer complexes and studying synthetic metabolism mimicry …”