Smart4Vir - Smart surfactant-based nanovectors for RNA anti-viral therapeutics

The development of efficient therapeutics for viral diseases stands currently among the biggest scientific challenges in global health. The serious societal problems caused by the recent pandemic originated by the SARSCov-2 virus are a stark reminder of this pressing and continuous need. Among key strategies to fight viruses is the development of smart nanocarriers that efficiently transport therapeutic RNA into cells to activate the immune system. RNA-based treatments hold the promise of achieving de novo/increased expression of specific therapeutic proteins (exogenous mRNA), like viral antigens, or silencing of pathological genes (with small interfering RNA, siRNA). The bottom-up design, at molecular level, of efficient nanocarriers using the concepts and experimental tools of surface and colloid chemistry is key to a successful strategy, but it is also a challenging endeavor due to some critical obstacles to overcome. A successful RNA delivery system should encapsulate RNA efficiently, protect it from degradation, deliver it to cells and facilitate its endosomal escape for gene expression or gene silencing. The nanovector efficiency depends on the formulation constituents, nanocarriers' physicochemical features and interfacial behavior with the tissues
and cells. Amphiphile-based nanocarriers (i.e. surfactants and/or lipids) have been extensively validated for RNA delivery, for their loading efficiency but importantly for their generally favorable safety profile, and several are already available in the clinic, including as antiviral vaccines. Nonetheless, there is still need for improvement specially regarding stability, safety, efficacy and, inevitably, cost. The aim of this project is to develop versatile and stimuli-responsive (hence, smart) surfactant-based nanocarriers for RNA anti-viral therapeutics. Our approach is based on a coherent and multidisciplinary combination of organic synthesis, colloid chemistry and biological studies. The surfactants are novel and bioinspired, derivatized from amino acids, and possess a gemini structure, which allows for structural versatility. Gemini surfactants contain two polar headgroups linked by a covalent spacer, and their aggregation properties depend inter alia on the spacer length. Cationic surfactants are typically used in nucleic acid delivery (electrostatic-driven complexation), together with helper lipids, whose basic role is to facilitate cell entry and/or decrease possible surfactant cytotoxicity. In our project, (i) the charge state of the gemini surfactants is pH-dependent (can be neutral or cationic, with +2 or +4 valence), due to presence of ionizable amine groups in their polar headgroup, and (ii) they also have cleavable chemical bonds, ester and/or disulfide bonds, both labile and dependent on local pH, redox environment and enzymatic activity. The rationale is that these molecular features will allow the surfactant-based RNA nanocarriers to change sequentially their properties (namely charge and chemical stability) along the delivery path to be effective in the different critical steps: initial RNA complexation/encapsulation; nanovector stability in the systemic circulation; cell internalization; and endosomal escape leading to RNA expression.