Balakirev M, Schoehn G, Chroboczek J
Institute de Biologie Structurale, Grenoble, France.
Chem Biol. 2000 Oct;7(10):813-9. doi: 10.1016/s1074-5521(00)00030-2.
Intracellular release of free DNA from the vector complex is one of the critical steps limiting the efficiency of non-viral gene delivery. The complex should be stable enough to prevent DNA degradation but it should be destabilized inside the cell to allow DNA release and transcription. Destabilization and degradation of synthetic vectors is also required to reduce their cytotoxicity and augment the life-time of transfected cells.
Here we describe new cationic amphiphiles made from the natural pro-vitamin, lipoic acid, that reversibly binds and releases DNA, depending on the redox state of the lipoate moieties. In the oxidized state these amphiphiles condense DNA into homogeneous spherical particles, which, upon reduction, swell into DNA toroids with subsequent release of free DNA. Complex reduction and DNA release can be induced by various thiols as well as enzymatically, by thioredoxin reductase. Transfection with amphiphile-DNA complexes in vitro shows a several fold increase of transgene expression compared with DOTAP, and can be further augmented by attachment of the nucleus-targeting peptide to the amphiphile. The increase of transfection efficiency results from GSH- and NAD(P)H-dependent complex reduction and release of free DNA inside the cells.
The present work demonstrates the principle of a redox-controlled gene delivery system that uses the reversibility of thiol-disulfide exchange reaction. Our data suggest that the efficiency of synthetic vectors can be augmented by their controlled destabilization inside the cells. Being formed from the natural non-toxic compound lipoic acid, these cationic amphiphiles provide a new promising class of synthetic vectors for gene delivery.
载体复合物中游离DNA的细胞内释放是限制非病毒基因递送效率的关键步骤之一。该复合物应足够稳定以防止DNA降解，但在细胞内又应不稳定以允许DNA释放和转录。合成载体的不稳定和降解对于降低其细胞毒性和延长转染细胞的寿命也是必需的。
在此我们描述了由天然维生素前体硫辛酸制成的新型阳离子两亲物，其根据硫辛酸部分的氧化还原状态可逆地结合和释放DNA。在氧化状态下，这些两亲物将DNA浓缩成均匀的球形颗粒，在还原时，这些颗粒膨胀成DNA环，随后释放出游离DNA。复合物的还原和DNA释放可由各种硫醇诱导，也可由硫氧还蛋白还原酶酶促诱导。与DOTAP相比，在体外转染两亲物-DNA复合物显示转基因表达增加了几倍，并且通过将核靶向肽连接到两亲物上可进一步增强。转染效率的提高源于细胞内谷胱甘肽和NAD(P)H依赖性复合物的还原和游离DNA的释放。
本研究证明了一种利用硫醇-二硫键交换反应可逆性的氧化还原控制基因递送系统的原理。我们的数据表明，合成载体的效率可通过其在细胞内的可控不稳定来提高。这些阳离子两亲物由天然无毒化合物硫辛酸形成，为基因递送提供了一类新的有前景的合成载体。
