Development of lipoplexes with enhanced crossing of cell barriers and biocompatibility

Oligonucleotides have recently been studied as therapeutic agents for various pathologies such as genetic disorders, viral infections and cancer. Among the different types of oligonucleotides used for therapeutic purposes siRNA is raising greater interest, because its action applies the natural mechanism of RNAi, which can silence the expression of pathologically overexpressed or mutated proteins. To exploit the therapeutic action of the oligonucleotides is necessary to develop a dedicated delivery system that allows them to reach intact and at high concentration the action site minimizing the exposure of other tissues. A possible delivery system for oligonucleotides can be the formulation of lipoplexes, which consists of complexes between cationic liposomes and oligonucleotides. Liposomes composed of cationic lipids allow a great loading of nucleic acid via electrostatic interactions and generate complexes in which the genetic materials are protected and targeted to the desired site. Within this thesis project, lipoplexes were formulated for the delivery of oligonucleotides and were prepared using an innovative oligocationic enhancer synthesized by our group. This lipid consisted of a dendrimeric portion functionalized by four arginines, which provide cationic charges, and a lipid anchor, which favors the insertion into the liposome bilayer. Using a 4% (mol/mol) OCE ratio, the effect of the lipid saturation on the lipoplexes was investigated using saturated phospholipid HSPC or unsaturated phospholipid EPC. Independently from the lipid saturation lipoplexes were characterized by sizes around 200 nm, low polydispersity index (< 0.2) and high positive surface charge (~ +40 mV). Other than the formulations generated with the excess of DOPE, namely HSPC/DOPE (1:3 mol/mol), HSPC/DOPE (1:2 mol/mol) and their EPC counterparts, lipoplexes were characterized with high loading efficiency (> 80%) independently from the lipid composition. Even though EPC-based lipoplexes have shown a similar particle size, polydispersity index, zeta potential and loading efficiency, they were excluded from the study for their very fast release profile. These screening studies allowed the identification of suitable formulations namely 1:1 HSPC/DOPE, 2:1 HSPC/DOPE, 2:1:1 HSPC/Cholesterol/DOPE and 2:1 HSPC/Cholesterol lipoplexes with 4% OCE and generated at N/P ratio of 10:1. PEGylation is a common process used for formulations that are thought to be administered intravenously, since it can avoid the formation of a protein corona around the lipoplexes. In this project the PEGylation of the four suitable formulations was provided by post instertion with different PEG ratio, namely 1, 3, 5 and 7% (mol/mol). The PEGylated lipoplexes were then characterized in terms of size, PDI, zeta potential, loading efficiency and capacity. Their colloidal stability and release profiles were also investigated and in the end the 5% (mol/mol) PEG ratio was selected to conduct in vitro studies. The formulation parameters were then transferred for the assembly of lipoplexes loaded with biologically active siRNA. In-vitro cytotoxicity studies conducted by MTT and LDH assays showed that siRNA-loaded lipoplexes induced negligible toxicity within the siRNA concentration range used in the studies.