Procarta Biosystems Ltd, Norwich Innovation Centre, Norwich, UK.
Department of Chemistry "Ugo Schiff" and CSGI, University of Florence, Firenze, Italy.
Sci Rep. 2017 Jan 25;7:41242. doi: 10.1038/srep41242.
Antimicrobial resistance to traditional antibiotics is a crucial challenge of medical research. Oligonucleotide therapeutics, such as antisense or Transcription Factor Decoys (TFDs), have the potential to circumvent current resistance mechanisms by acting on novel targets. However, their full translation into clinical application requires efficient delivery strategies and fundamental comprehension of their interaction with target bacterial cells. To address these points, we employed a novel cationic bolaamphiphile that binds TFDs with high affinity to form self-assembled complexes (nanoplexes). Confocal microscopy revealed that nanoplexes efficiently transfect bacterial cells, consistently with biological efficacy on animal models. To understand the factors affecting the delivery process, liposomes with varying compositions, taken as model synthetic bilayers, were challenged with nanoplexes and investigated with Scattering and Fluorescence techniques. Thanks to the combination of results on bacteria and synthetic membrane models we demonstrate for the first time that the prokaryotic-enriched anionic lipid Cardiolipin (CL) plays a key-role in the TFDs delivery to bacteria. Moreover, we can hypothesize an overall TFD delivery mechanism, where bacterial membrane reorganization with permeability increase and release of the TFD from the nanoplexes are the main factors. These results will be of great benefit to boost the development of oligonucleotides-based antimicrobials of superior efficacy.
抗生素耐药性是医学研究的一个关键挑战。寡核苷酸疗法，如反义寡核苷酸或转录因子诱饵（TFD），有可能通过作用于新的靶点来规避当前的耐药机制。然而，要将它们完全转化为临床应用，需要高效的传递策略和对其与靶细菌细胞相互作用的基本理解。为了解决这些问题，我们使用了一种新型的阳离子双偶联剂，它能与 TFD 高亲和力结合形成自组装复合物（纳米复合物）。共焦显微镜显示纳米复合物能有效地转染细菌细胞，这与动物模型上的生物学功效一致。为了了解影响传递过程的因素，我们用不同组成的脂质体（作为模型合成双层膜）挑战纳米复合物，并利用散射和荧光技术进行了研究。由于对细菌和合成膜模型的结果进行了综合分析，我们首次证明了富含原核生物的阴离子脂质心磷脂（CL）在 TFD 向细菌的传递中起着关键作用。此外，我们可以假设一个总的 TFD 传递机制，其中细菌膜的重组导致通透性增加，以及纳米复合物中 TFD 的释放是主要因素。这些结果将极大地促进高效基于寡核苷酸的抗生素的开发。
