It was recently reported that polyanion/DNA/polycation ternary polyplex markedly improved gene transfection activity in comparison to the original DNA/polycation binary polyplex. In this study, to explore the influence of polyanion on physicochemical properties and biological activities of polyanion/pDNA/polycation ternary polyplex, four types of biocompatible polyanions were selected mainly based on their acid strengths of anionic function groups and molecular rigidity to form ternary polyplexes with PEI 25 kDa and DNA. That polyanions loosened DNA polyplex, weakened adsorption of serum proteins and improved cellular uptake, which are thought of as the important factors leading to the high transfection efficiency of DNA ternary polyplex, were specifically investigated. Electrophoresis retardation analysis indicated that the loosening capacities of polyanions depended on the pKa values of the functional anion groups as well as the flexibility of polyanions. The low pKa and flexible structure of polyanions tended to easily loosen the compact DNA polyplex. The thermodynamic analysis with isothermal titration calorimetry provided the direct evidences about the serum proteins and DNA ternary polyplex interactions. The polyanion/pDNA/polycation ternary polyplex exhibited obviously lower binding affinities and less adsorption to serum protein, compared with the original DNA/polycation binary polyplex. These relatively stable DNA ternary polyplexes in serum containing medium maintained the high levels of cellular uptake and intracellular accumulation that was correlated with their high transfection efficiency. In contrast, their original pDNA/polycation binary polyplex formed clustered polyplex by strong adsorption of large amounts of serum proteins, leading to the sharp reduction of cellular uptake and intracellular accumulation, and then low gene transfer efficiency. These results provided a basis of the development of polyanion/DNA/polycation ternary polyplex for polyfection.
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