(T1030-01-02) Development of Novel Ionizable Lipid Nanoparticles for Enhanced siRNA Delivery

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. However, the commercial application of siRNA-LNPs is mostly restricted to delivering nucleic acids to the liver. Achieving targeted delivery to tumor tissues and facilitating the release of cargo from endosomes after cellular internalization remains a significant challenge. We developed a novel ionizable lipid, NLD, with the goal of enhancing siRNA LNP internalization and releasing siRNA from endosome to improve cancer therapy.

Methods: We used anti-eGFP siRNA as a model siRNA. LNP formulations were developed comprising of NLD:DSPC:Cholesterol:DMG-c-PEG 2000 at a molar ratio of 50:10:38.5:1.5. These LNPs were manufactured by NanoGenerator Flex-M microfluidic mixing of pH 4 acetate buffer and ethanol phases containing the nucleic acid and total lipids, respectively. They were further dialyzed against pH 7.4 phosphate buffer for ethanol removal and buffer exchange. LNPs were tested for their particle size, polydispersity index, zeta potential, and morphology using Dynamic Light Scattering and Transmission Electron Microscopy techniques. Encapsulation efficiency was calculated using ribogreen assay. Fluorescent dye 6-(p-Toluidino)-2-naphthalenesulfonic acid (TNS) mixed with buffers was used to determine pKa of the novel ionizable LNP. MDA-MB-231 GFP cell viability study established the 50% inhibitory concentration (IC₅₀) of these novel LNPs. Further, fluorescent labelled siRNA loaded in LNPs were developed to study cell uptake and endosomal escape in a lung cancer cell line, A549. Confocal microscopy was used to track the cell uptake, endo-lysosomal escape, and in vitro transfection of these novel LNPs. Cell uptake and silencing efficiency were also evaluated using flow cytometry.

Results: The recovery of manufactured LNPs by microfluidic mixing was consistently 92±3%. The particle size of the preliminary LNPs was between 150-180 nm, PdI 0.15-0.27, zeta potential of 2-10 mV after dialysis, with statistically significant difference between blank and loaded LNPs. Difference in the blank and loaded LNPs shows successful electrostatic interaction between the cargo and novel ionizable cationic lipid at pH 4. Encapsulation efficiency was calculated to be between 86-91% based on ribogreen assay demonstrating the successful ionizable property of the lipid. pKa stands as a pivotal determinant influencing the capability of ionizable lipids to release cargo within the acidic environment of endo-lysosomes. pKa of the novel blank and siRNA loaded LNP was calculated to be 5.7 and 5.97, respectively. IC₅₀ was calculated to be 133.33 µg/mL, showcasing its safety at employed concentrations for the experiments. Time dependent cell uptake showed that LNPs were significantly internalized in A549 cancer cell as early as 2 hours after treatment, and up to 47% after 4 hours of treatment. Lysotracker Red helped to visualize the LNP outside lysosome with some dispersed green signal, representing our siRNA LNP, in the cytoplasm showcasing endo-lysosomal escape. Reduction in fluorescence intensity was observed under confocal microscope. A significant reduction in green fluorescent protein (GFP) expression was seen with our novel LNPs in comparison to the untreated control. Transfection efficiency of our LNPs was superior to that of the positive control, Lipofectamine-siRNA sample at the same concentration. These silencing studies further demonstrated the advantage of the system compared with conventional ionizable lipid nanoparticle and transfecting agent lipofectamine.

Conclusion: In conclusion, our anti-eGFP siRNA loaded novel LNP formulations show excellent physicochemical properties and promising results in cell-based studies. Our novel LNP platform holds great potential for efficient nucleic acid delivery, with implications for future targeted cancer therapies.


Figure 1. Overlay of cell uptake after 2, 3, 4 hours of treatment of anti-eGFP siRNA LNP using flow cytometry


Figure 2. Endo-lysosomal escape of anti-eGFP siRNA LNP after 4 hours of treatment using confocal microscopy.