(M0930-06-38) Engineering Next-Generation Ionizable Lipid Nanoparticles for siRNA Therapeutics: Insights from a DoE Optimization Strategy

Purpose: FDA approved siRNA drug and COVID-19 mRNA vaccines have put the spotlight on the lipid nanoparticles (LNPs) utilization to enhance the delivery of nucleic acids by protecting them from degradation and aiding in their delivery to target tissues. LNPs can successfully encapsulate siRNA in its core due to electrostatic interaction between the ionizable cationic lipid and anionic siRNA at acidic pH.  A novel ionizable lipid, NLD, was used to improve siRNA encapsulation, enhance endosomal escape, and achieve better delivery efficiency compared to existing commercial lipid, Dlin-DMA-MC3. We optimize the formulation components of siRNA-loaded lipid nanoparticles (LNPs) incorporating a novel ionizable lipid in order to maximize delivery efficiency, enhance siRNA stability, minimize cytotoxicity, and ensure consistent nanoparticle characteristics for improved therapeutic performance. A Design of Experiments (DOE) approach was used to efficiently optimize multiple formulation variables and identify key factors influencing the performance of novel ionizable lipid-based siRNA nanoparticles.

Methods: A full factorial design was employed with 12 formulations to screen two structural lipids, cholesterol and Beta-sitosterol, three helper lipids, DSPC, DOPC, DOPE, and two stealth lipids, DMG-c-PEG 2000, DSPE PEG 2000 (Table 1). Formulations were labelled as F1-F12. Formulations were prepared using a 5:1 aqueous-to-organic phase flow rate ratio and a total flow rate of 3 mL/min, maintaining a constant molar ratio of 50:38.5:10:1.5 for the novel ionizable lipid (NLD), structural lipid, helper lipid, and stealth lipid, respectively. These LNPs were manufactured by NanoGenerator Flex-M microfluidic mixing of pH 4 25mM acetate buffer and ethanol phases containing the nucleic acid and total lipids, respectively. They were tested for size, polydispersity index (PdI), surface charge, and morphology using Dynamic/Electric Light Scattering and Transmission Electron Microscopy. Encapsulation efficiency (EE) was determined using ribogreen assay. Apparent pKa profiles of these LNPS were evaluated using 6-(p-Toluidino)-2-naphthalenesulfonic acid (TNS) assay. Flow cytometry and qPCR were utilized to study the in vitro transfection efficiency on MDA-MB-231 GFP cells. All formulations were tested for their inherent cytotoxicity using CellTitre-Glo® assay.

Results: A high recovery of 90±3% was achieved for the prepared LNPs consistently. F6 showed lipid incompatibility, hence eliminated from the statistical analysis. All formulations’ particle size ranged from 90 nm to 140 nm, PdI 0.05 to 0.29, surface charge of 3 mV to -9.6 mV. No statistically significant impact of different lipids was observed across F1- F12 however, all the results were within the desired range. Encapsulation efficiency was calculated to be more than 90% for all formulations proving our novel ionizable lipid, NLD, could efficiently interact and encapsulate siRNA within the LNP (Figure 1). Uniform spherical morphology was observed for all F1-F12 formulations. The apparent pKa values of the LNP govern its ionization state in the acidic vesicles like late endosome and lysosome. This property helps in the fusion of the LNP with the endosomal membrane leading to the release of siRNA in the cytosol to participate in the RNAi pathway. The apparent pKa of all formulations was between 5.3 to 6.4 with helper lipids significantly impacting the apparent pKa profile, due to the cylindrical to conical geometries assumed by the LNPs. Scrambled formulations showed no impact on the mRNA and protein expression compared to the untreated control demonstrating the gene specific silencing effect of all formulations. All formulations, except F5, F11, F12, performed significantly better than the untreated control (Figure 2). F8 performed best with 92% and 80% knockdown in target mRNA and protein expression, respectively similar to the positive control, Lipofectamine RNAiMAX. Stealth lipids significantly reduced mRNA and protein expression: DMG-c-PEG 2000 > DSPE PEG 2000. PEG shedding and protein corona formation were identified as the critical factors impacting the efficiency of the LNPs. No inherent toxicity was associated with any formulations at the tested dose.

Conclusion: DoE study helped to optimize the LNP composition and understand the impact of different components on product efficacy. Our optimized NLD LNP formulation shows superior physicochemical properties and promising in vitro performance. These novel LNPs hold great potential for efficient nucleic acid delivery, with implications for targeted cancer therapies.


Table 1. Description of F1-F12 formulations


Figure 1. Over 90% encapsulation efficiency of F1-F12 showcasing the ionization capacity of the novel lipid, NLD.


Figure 2. mRNA expression of the tested formulations with 8 out of 12 formulations significantly different than the untreated control MDA-MB-231 cells (UT) and F8, F9 similar to positive control Lipofectamine RNAiMAX (LRM).