MICROFLUIDIC FORMULATION OF NANOSYSTEMS FOR NUCLEIC ACID DELIVERY

Oligonucleotides have recently emerged as promising therapeutic agents for a wide range of medical conditions, including genetic disorders, viral infections, and cancer. Among these oligonucleotides, siRNA has garnered particular attention due to its ability to harness the natural RNA interference (RNAi) mechanism, which can effectively reduce the expression of pathologically overexpressed or mutated proteins. To harness the therapeutic potential of genetic material, it is essential to develop targeted delivery systems capable of protecting the nucleic acid cargo and ensuring its arrival at the desired site. One common strategy for precise gene material delivery involves the formation of lipoplexes, which are complexes formed between cationic lipids and nucleic acids. Liposomes containing cationic lipids facilitate electrostatic interactions with negatively charged nucleic acids, resulting in high-loading systems. When encapsulated within lipid carriers, nucleic acids are shielded from enzymatic degradation and can effectively reach their intended destination. Cationic liposomes offer several advantages for gene delivery, accommodating various nucleic acids such as DNA plasmids, antisense oligonucleotides, siRNAs, and mRNAs. This research project focuses on the development of a microfluidic method for the creation of liposomes with a specific lipid composition (HSPC:DOPE:OCE in a ratio of 48:48:4) to enhance the delivery of genetic material. Within these liposomal formulations, a novel oligocationic enhancer (OCE) synthesized by the Padua research group is introduced. This cationic lipid features a dendrimeric structure functionalized with four arginines and an anchor lipid designed for insertion into the liposome bilayer. OCE is incorporated into the liposomal formulations for its dual role in condensing oligonucleotides and serving as a cell penetration enhancer, thereby facilitating the accessibility of lipoplexes to target cells. Additionally, the fusogenic lipid DOPE is integrated into the formulation to ensure the successful traversal of the final intracellular barrier and subsequent entry into the cytosol. Subsequently, these liposomes were compared with other lipid nanosystems to evaluate their chemical and physical characteristics and therapeutic efficiency. The lipoplexes exhibited consistent characteristics, including a size of approximately 170 nm, a low polydispersity index, a high positive surface charge and high loading efficiency. To evaluate the therapeutic activity of siRNA-loaded lipoplexes, silencing efficiency was measured as an indicator. Lipoplexes were loaded with biologically active GFP-siRNA and demonstrated to have a high silencing efficiency in RAW 264.7 cell line. In summary, the findings underscored that OCE-enhanced lipoplexes exhibited promising characteristics for efficient oligonucleotide delivery with minimal cytotoxicity and potent silencing capabilities.