Antimicrobial Nanoplexes meet Model Bacterial Membranes: the key role of Cardiolipin

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

Supported fluid lipid bilayer as a scaffold to direct assembly of RNA nanostructures

RNA architectonics offers the possibility to design and assemble RNA into specific shapes, such as nanoscale 3D solids or nanogrids. Combining the minute size of these programmable shapes with precise positioning on a surface further enhances their potential as building blocks in nanotechnology and nanomedicine. Here we describe a bottom-up approach to direct the arrangement of nucleic acid nanostructures by using a supported fluid lipid bilayer as a surface scaffold. The strong attractive electrostatic interactions between RNA polyanions and cationic lipids promote RNA adsorption and self-assembly. Protocol steps for the characterization of assembled RNA complexes via several complementary methods (QCM-D, ellipsometry, confocal fluorescence microscopy, AFM) are also provided. Due to their tunable nature, lipid bilayers can be used to organize RNA laterally on the micrometer scale and thus facilitate the building of more complex 3D structures. The bilayer-based approach can be extended to other programmable RNA or DNA objects to construct intricate structures, such as 2D grids or 3D cages, with high precision