Design and optimization of RNAse free fusogenic ANANAS for the delivery of bioactive active RNA to target cell cytoplasm.

ANANAS (Avidin-Nucleic Acid NanoAssemblies) are polyavidins systems based on the interaction with nucleic acids and their high affinity for biotin. ANANAs can be functionalized with any element previously biotinylated. When decorated with biotin monoclonal antibodies targeting specific cell surface receptors, ANANAS nanoparticles enter the cells through the endosome lysosome pathway. Since the endosome/lysosome environment is toxic for nucleic acid based active agents, fusogenic formulations capable to lyse the endosomal membranes have been recently developed, paving the way toward nucleic acid delivery at the cytoplasm of target cells. However, when it comes to RNA delivery, a major formulation challenge stems from the intrinsic instability of the RNA cargo, which is due to both the intrinsic chemical nature of the molecule and the ubiquitous presence of RNAses in the environment. Consequently, dedicated operating procedures must be adopted in the lab to ensure the absence of RNAses along the entire NP production and manipulation process, and to avoid chemical destabilization of the RNA carrier as a result of inadequate manipulation and storage conditions. Optimizing RNAse free NP production/manipulation protocols requires sensitive tools for environmental RNASe detection. Commercial kits are available but very costly. Therefore, a laboratory-made procedure is desirable to be used along the complex RNASe-free NP preparation validation process. The general goal of this research thesis was to optimize RNAse-free fusogenic ANANAS formulations. To this end a sensitive protocol for RNAse detection was developed to be used along the nanoparticle preparation process and the RNAse levels of all ANANAS components was measured. A RNAse-free nanoparticle preparation process was then optimized and validated. Finally, the fusogenic potentials of EGFR+/Her2+ cell targeted ANANAS functionalized with two acid-sensitive membrane destabilizing moieities (Fus2 and Fus3) were investigated by fluorescent confocal microscopy. For this purpose, the formulations were made fluorescent by the addition of fluorophores (Atto 633 or Alexa 633), biotinylated by irreversible linker (Biotin-C 6-Atto 633) or reducible (Biotin-dPEG 4-SS-Atto 633) in the intracellular environment, while the vesicular compartment was highlighted thanks to a second fluorescent probe, calcein, present in the culture medium. While Fus3 did not show the expected activity, Fus2 proved effective, as nanoparticles functionalized with it were able to destabilize the membranes of 25% of cell vesicles. In summary, the combined use on the surface of ANANAS nanoparticles of a directional, the element Fus2 and the chemistry of disulfide for reversible loading of the active element appears a promising strategy for the delivery of nucleic acids to the cytoplasm of target cells