PREPARATION AND CHARACTERIZATION OF LIPID NANOPARTICLE FORMULATIONS FOR siRNA DELIVERY BY A MICROFLUIDIC APPROACH

The marketing authorizations of vaccines against SARS-Cov-2 have speed up the application of Lipid Nanoparticles (LNPs) to nucleic acid delivery for the treatment of rare diseases, diabetes, and cancer. In comparison to non-viral vehicles, LNPs provide a safer and more effective tool with no immunogenic responses. Meanwhile, LNPs are able to encapsulate different genetic materials as small interfering RNA (siRNA), messenger RNA (mRNA), or plasmids and are extremely tuneable to changes in the formulation composition and chemical modification. Herein, LNPs encapsulating siRNA were manufactured through a microfluidic system, starting from a mixture of ionizable amino lipid DLin-MC3-DMA, DSPC, cholesterol, PEG-DMG in a molar ratio of 50:10:38.5:1.5, respectively, with a final total lipid concentration of 12.5 mM [1, 2]. The organic phase of lipids dissolved in ethanol and the aqueous phase containing siRNA in acetate buffer (pH 4) were combined at a 1:3 volume with a final flow rate of 12 mL/min. After testing different N/P (Nitrogen/nucleic acid Phosphate) charge ratios, the 3:1 proportion was selected as the best balance for formulating siRNA-loaded LNPs. The resultant siRNA-LNPs were then dialyzed against phosphate-buffered saline (PBS, pH 7.4) using GeBaFlex dialysis membranes (14 kDa MWCO) for 3 h at 4 °C to remove ethanol, and subsequently filtered through a 0.2 µm filters. Specifically, two different non-specific negative-control si-RNAs were employed leading to siRNA-NC1-LNPs and siRNA-NC2-LNPs. The two formulations, prepared under nuclease free conditions, showed comparable features with a mean particle size of about 50 nm, a polydispersity index of 0.127 and a near neutral surface charge at physiological pH of 7.4. Both the colloidal suspensions were stable up to 30 days during storage at 4 °C. Lipid concentration was determined by the measurement of cholesterol content using an enzymatic colorimetric method. To minimize the waste of expensive materials (i.e. RNA) and thus optimize the process in view of a manufacturing development, the encapsulation efficiency (EE%) was calculated taken into consideration the amount of RNA added in the preparative mixture (input RNA) rather than the total RNA amount measured after the processing steps, as traditionally reported [3]. RNA encapsulation efficiency (EE%) was calculated upon measurement by Quant-iT Ribogreen RNA assay of the whole LNPs (unencapsulated RNA) and the LNPs lysed with 0.1% Tryton (encapsulated RNA). EE% was calculated by taking the ratio of encapsulated RNA to the input RNA. The siRNA-NC1-LNP and siRNA-NC2-LNP formulations provided an EE% of about 90%. At selected time points (0-7-15-21-30 days) physical and chemical features of LNPs as well as a possible leakage of RNA from LNPs (unencapsulated RNA) were measured confirming a great stability of LNPs over time and siRNA retention. In conclusion, we demonstrated that for better guiding the design of future RNA therapeutics, an in-depth analysis of the synthesis parameters can provide a useful insight for process optimization to reduce RNA loss and the associated cost.