INNOVATIVE DESIGN OF RAFT-LIKE NANOSTRUCTURED LIPID CARRIERS FOR TARGETED NUCLEIC ACID DELIVERY

Gene therapy is based on the modification of gene expression or alteration of biological traits through the introduction of external genetic material, including deoxyribonucleic acid (DNA), messenger RNA (mRNA), or small interfering RNA (siRNA), into cells. It is currently being investigated as a potential treatment for a range of complex diseases, including cancer, cardiovascular, neurological, and immunological disorders. The success of mRNA-based vaccines for the treatment of SARS-CoV-2 (Pfizer and Moderna, 2020) has led to an intensified interest in RNA-based therapies due to the advantages they offer in terms of reversibility, specificity and improved delivery using lipid nanoparticles (LNPs) (1,2). They enhance the stability of RNA, improve target specificity and enhance transfection efficiency. Liposomes, an early version of LNPs, are a versatile nanomedicine delivery platform. A number of liposomal drugs have been approved and applied to medical practice. Subsequent generations of lipid nanocarriers, such as nanostructured lipid carriers (NLCs), exhibit more complex architectures and enhanced physical stabilities. Ongoing efforts to synthesize and screen innovative lipid nanomaterials by chemically optimizing their structures aim to achieve tuneable features. This approach supports the development of more versatile, efficient, and biocompatible drug delivery systems. In this context, the objective of this project is to develop an advance RNA delivery platform designing innovative nanostructured lipid carriers (NLCs). Innovative rafts-like NLCs composed of phosphatidylcholine (POPC), cholesterol, sphingomyelin, dimethyldioctadecylammonium bromide (DDAB) and 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)- N-(4-hydroxybutyl)hexan-1-aminium (ALC-0315) (50:25:10:10:5 molar ratio) will be synthesized through freeze and thaw cycles and by extrusion method (4). This composition allows the formation of cationic lipid rafts reconstituted in liposomes, useful to increase the loading efficiency of siRNA and mRNA (3), keeping the endosomal escape feature useful for nucleic acids release, and to increase the biocompatibility respect to liposomes currently used for gene delivery. These NLCs were characterized from a physicochemical point of view (size, charge, stability, loading capacity) and showed excellent biocompatibility with macrophages cell line RAW264.7. Efficient loading of RNA, both siRNA and mRNA, was confirmed by agarose gel retardation assays, demonstrated good binding affinity, suggesting a promising profile for combinatorial applications as well. Laurdan, a fluorescent probe that differentiates lipid phases (5), emission profile revealed the presence of ordered raft-like structure in NLCs. Transfection assay showed that NLCs were able to effectively deliver functional mRNA to in vitro cellular model and in vivo in healthy mice. The latter also confirmed the transient nature of the mRNA delivery, a phenomenon that was more visible in the spleen. The results obtained from this study offer a solid foundation for further in-depth research, thereby enhancing our comprehension of the stability, bioavailability, safety and functional efficacy of these innovative nanoparticles for RNA delivery in both in vitro and in vivo applications.