应用PAMAM dendrimers作为DNA运送载体的体外研究

Application of Starburst^(TM) PAMAM Dendrimers as DNA Carriers In vitro

StarburstTMPAMAMdendrimers分子是一类新型的高分枝、辐射状对称的树状高分子,在生理条件下其表面具有高密度的正电荷,可以通过静电相互作用与核酸形成复合物后,介导遗传物质进入细胞.研究了G3,G3 5,G5,G7,G7 5,G9各代dendrimers分子与DNA结合后介导其转染细胞的能力,并初步评价这种复合物转染对细胞活力的影响.实验证实,全代的PAMAMdendrmers皆可与DNA结合,并可在体外培养的细胞中介导高效的DNA转染.PAMAMdendrimer/DNA复合物很稳定,在较大的pH值变化范围内(pH 2～ 10 )不解离.PAMAMdendrimers可保护与之复合的DNA分子免受限制性内切酶的降解.在一定的电荷比范围内,高代数的dendrimers分子与DNA形成的复合物对培养细胞的转染效率高于低代数dendrimer分子,复合物所介导的转染效率在不同的细胞系之间也有差异.在有效作用浓度范围内(≤ 1 3× 10 -1g/L),PAMAMdendrimers/DNA复合物对被转染细胞无毒性.但是,未与DNA复合的dendrimers分子在较低浓度时则表现出毒性,表明StarburstTM PAMAMdendrimers分子可作为新型的低毒非病毒DNA载体,用于介导DNA对体外培养细胞的转染.这些前期观察。

Starburst(TM) PAMAM dendrimers are novel polymers with a molecular architecture characterized by regular, dentritic branching with radial symmetry. Having high density of positive charges on their surfaces in physiological condition because of the protonization of amino groups on the surfaces, and complexing with genetic materials on the basis of electrostatic interactions, those Starburst(TM) PAMAM dendrimers deliver genes into alive cells. In order to characterize the potential effects of Starburst(TM) PAMAM dendrimers as a carrier for DNA transfection, six different types generations of Starburst(TM) PAMAM dendrimers were investigated for their capabilities in binding DNA, and the effects on both DNA transfection and maintenance of cell viability was evaluated in vitro. The experiments demonstrated that it was the full generations but not the half generations of Starburst(TM) PAMAM dendrimer could transfect eukaryotic cells efficiently. The dendrimer/DNA complexes were very steady, no dissociation of the complexes was detectable in a large scope of pH (2 similar to 10). The complexation of Starburst(TM) PAMAM dendrimer and DNA prevent the reaction that endonuclease dissociates the DNA. In a certain range of dendrimers to DNA charge ratios, the Starburst(TM) PAMAM dendrimer with higher generations showed much better transfection efficiency than those with lower generations. The transfection efficiency was also variable in different cell lines. Starburst(TM) PAMAM dendrimers complexing with DNA have no or very low cytotoxicity at the concentrations effective for DNA transfection (less than or equal to 1. 3 x 10(-1) g/L). However, the cytotoxicity of Starburst(TM) PAMAM dendrimers without binding DNA could be detected at a lower concentration. The results demonstrated that Starburst(TM) PAMAM dendrimers, as a novel type of low toxicity, non-viral DNA delivery vehicle, had promising potential to mediate DNA transfection in vitro. It provide primary experimental basis for the application of the nanometer material-Starburst(TM) PAMAM dendrimers in vivo as DNA delivery carrier.