Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29, Yoshidashimoadachi-cho, Sakyo-ku, Kyoto, 606-8501, Japan.
Mol Pharm. 2010 Apr 5;7(2):533-42. doi: 10.1021/mp900247d.
To improve the transfection efficiency of plasmid DNA (pDNA) into cells, various types of cationic liposome have been used to prepare pDNA/cationic liposome complexes (lipoplexes). It is well-known that lipoplexes induce a large amount of proinflammatory cytokines because unmethylated CpG dinucleotides (CpG motifs) abundantly present in pDNA are recognized by Toll-like receptor-9 (TLR9) expressed in immune cells such as macrophages and dendritic cells. This nonspecific cytokine production is problematic in nonviral gene therapy. Moreover, recent studies have demonstrated that lipoplexes induce not only proinflammatory cytokines but also another type of cytokine, type I interferons (IFNs), irrespective of the frequency of CpG motifs in DNA and the expression of TLR9. To gain more insight into the CpG motif- and TLR9-independent induction of type I IFNs and proinflammatory cytokines by lipoplex, macrophage activation was evaluated in vitro using various cationic liposomes complexed with pDNA containing no CpG motifs. The production of IFN-beta, TNF-alpha and IL-6 by lipoplex was confirmed to be induced independently of the interaction between CpG DNA and TLR9 in macrophages from TLR9-knockout mice. Then, the release of the cytokines, the mRNA expression of Z-DNA binding protein-1 (Zbp1), a cytosolic double-stranded DNA sensor, and the cellular uptake of pDNA were examined in a macrophage-like cell line, RAW264.7. The level of cytokine production and the increase in the Zbp1 mRNA varied depending on the type of cationic liposome used. A good correlation was observed between the cytokine level and the Zbp1 mRNA. A confocal microscopic study using fluorescently labeled pDNA complexes showed that the complexes that released a lot of cytokines showed an enhanced distribution of pDNA-derived fluorescence into the cytosol. These results suggest that different intracellular trafficking derived from the type of liposomes determines the recognition of pDNA by ZBP1 after uptake of lipoplexes by the macrophages, followed by the release of type I IFNs and inflammatory cytokines. The present study demonstrates that cationic liposomes should be selected based on these findings for optimization of DNA-based therapies using lipoplexes.
为了提高质粒 DNA（pDNA）转染细胞的效率，已经使用了各种类型的阳离子脂质体来制备 pDNA/阳离子脂质体复合物（脂质体）。众所周知，脂质体诱导大量促炎细胞因子的产生，因为 pDNA 中大量存在未甲基化的 CpG 二核苷酸（CpG 基序）被巨噬细胞和树突状细胞等免疫细胞表达的 Toll 样受体 9（TLR9）识别。这种非特异性细胞因子的产生在非病毒基因治疗中是有问题的。此外，最近的研究表明，脂质体不仅诱导促炎细胞因子，还诱导另一种细胞因子，即 I 型干扰素（IFN），而与 DNA 中的 CpG 基序的频率和 TLR9 的表达无关。为了更深入地了解脂质体诱导 I 型 IFN 和促炎细胞因子的 CpG 基序和 TLR9 非依赖性，使用不含 CpG 基序的 pDNA 与各种阳离子脂质体复合，在体外评估巨噬细胞的激活。证实脂质体诱导 IFN-β、TNF-α 和 IL-6 的产生与 TLR9 敲除小鼠巨噬细胞中 CpG DNA 与 TLR9 的相互作用无关。然后，在巨噬细胞样细胞系 RAW264.7 中检查细胞因子的释放、Z-DNA 结合蛋白-1（Zbp1）的 mRNA 表达，一种胞质双链 DNA 传感器，以及 pDNA 的细胞摄取。细胞因子的产生水平和 Zbp1 mRNA 的增加取决于所使用的阳离子脂质体的类型。细胞因子水平和 Zbp1 mRNA 之间观察到良好的相关性。使用荧光标记的 pDNA 复合物的共聚焦显微镜研究表明，释放大量细胞因子的复合物显示出 pDNA 衍生荧光向细胞质的分布增强。这些结果表明，不同的脂质体类型衍生的细胞内运输决定了脂质体摄取后巨噬细胞对 pDNA 的 ZBP1 识别，随后释放 I 型 IFN 和炎症细胞因子。本研究表明，阳离子脂质体应该根据这些发现进行选择，以优化使用脂质体的 DNA 治疗。
