Optimization of lipoplexes functionalized with a sialic acid mimetic (F9-PEG) to target the C1858T PTPN22 variant for preclinical assessment of a novel immunotherapy in endocrine autoimmunity

Background: The C1858T PTPN22 variant is strongly associated with type 1 diabetes and autoimmune thyroid disease. Current treatment is substitutive hormonal administration, which does not target the disease pathogenetic mechanism. We previously implemented a novel immunotherapy, employing siRNA directed against the C1858T variant of PTPN22 delivered via functionalized lipoplexes, in order to halt autoimmune disease progression. Objectives: The objective of this study was to optimize lipoplex formulations functionalized with F9-PEG (a Siglec-10's ligand) to facilitate targeted delivery by investigating their physical and chemical properties to warrant the best performance in in vivo studies. Methods: The effectiveness of siRNA liposome binding was evaluated by varying the relative lipid/siRNA charge ratio and analyzing the stability of the different formulations with respect to the methods of F9-PEG addition and ATTO740 fluorescent labeling by electrophoresis, dynamic and dielectrophoretic light scattering (DLS and DELS), and high-performance liquid chromatography (HPLC). Results: The optimal charge ratio of +2/-1 (lipid/siRNA) ensured a greater stability of lipoplexes and complete complexation of siRNA. Stability was improved by selecting a protocol of preparation that envisages functionalization with F9-PEG and the addition of ATTO740 lipid in the lipid film preparation step. HPLC confirmed the integrity of siRNA after preparation. Conclusions: The results of this study lead to formulations of F9-PEG lipoplexes with optimal properties that could be used for biodistribution and safety/efficacy studies in mice. Lipoplexes functionalized with F9-PEG could therefore represent a promising personalized nanotherapeutic platform for targeted siRNA delivery in endocrine C1858T patients.