(T1030-01-05) Flash Nanoprecipitation Permits Self-assembly of Ionizable Lipid Nanoparticles for Nucleic Acid Delivery

Purpose: Ionizable lipid nanoparticles (LNPs) are efficient in encapsulating hydrophilic, negatively charged nucleic acid therapeutics and releasing them precisely inside the cells. Ionizable lipids become positively charged at acidic pH such as in endolysosomes while remaining neutral at physiological pH, they remain neutral, and exhibit reduced toxicity as compared to fixed cationic lipids.^1-3 Given the recent clinical success of ionizable LNPs for the delivery of mRNA vaccines and siRNA therapeutics, preclinical research employing LNPs for prophylactic or therapeutic indications in several diseases, including genetic disorders, cancers, and infectious diseases is at an accelerated phase.^{4, 5} Microfluidic techniques like T-junction mixing, Microfluidic Hydrodynamic Flow Focusing, and Staggered Herringbone Mixing are most commonly utilized to self-assemble LNPs for nucleic acid delivery. However, these techniques involve either high costs associated with formulation setup or demand a large volume preparation. There is an urgent requirement to develop cost-effective alternative fabrication techniques to formulate LNPs that are suitable for the discovery phase and scalable production. Here, we introduce a flash nanoprecipitation (FNP) approach using the simple confined impingement (CIJ) mixer to rapidly self-assemble ionizable LNPs. Three clinically relevant ionizable lipids SM102, ALC0315, and DLin-MC3-DMA, were utilized to validate the FNP technique. SM102 and ALC0315 LNPs were tested for mRNA delivery both in vitro and in vivo, while DLin-MC3-DMA LNPs were tested for siRNA delivery in vitro. We envision that the FNP method using the CIJ mixer offers an efficient, cost-effective approach for producing nucleic acid-encapsulated ionizable LNPs, suitable for small-scale testing and scalable production.

Methods: For the FNP process, ionizable lipids, helper and structural lipid components, including DSPC, PEG lipid and cholesterol were dissolved in an organic phase containing ethanol. On the other hand, mRNA/siRNA payload was dissolved in an aqueous phase containing sodium citrate buffer (pH 4). The organic and aqueous phase was then impinged against each other in a CIJ mixer to form LNPs and ethanol was removed from the formulation through dialysis. The particle size and the surface charge of the LNPs were measured using dynamic light scattering and electrophoretic light scattering, respectively and the morphology of the LNPs was confirmed using Transmission Electron Microscopy (TEM). Intracellular delivery of the mCherry mRNA-loaded SM102 and ALC0315 LNPs was verified by confocal live-cell imaging using immortalized murine dendritic cells (DC 2.4). Subsequently, we performed a functional mRNA assay in DC 2.4 dendritic cells using OVA mRNA to showcase the antigen presentation abilities of LNPs. Finally, we verified the in vivo mRNA delivery in C57BL/6 mice with firefly luciferase mRNA loaded SM102 and ALC0315 LNPs and IVIS imaging. The cytoplasmic delivery of siRNA was confirmed in RAW 264.7 cells using Cy-5-conjugated siRNA-loaded DLin-MC3-DMA LNPs. Gene silencing efficacy of SilencerTM GFP siRNA LNPs was tested in GFP-expressing MDA-MB-231 cells and analyzed through flow cytometry.

Results: Ionizable LNPs were 133-137 nm in size with a surface neutral charge at physiological pH (7.4). TEM images confirm the spherical morphology of LNPs (Fig. 1B). The encapsulation efficiency of mRNA in SM102 and ALC0315 LNPs, and siRNA in DLin-MC3-DMA LNPs was found to be >96% (Fig. 1E). SM102 and ALC0315 LNPs successfully delivered mCherry mRNA into the cytoplasm, which was evident through translation to a red-colored mCherry protein signal inside the cells (Fig. 2A). The percentage of H-2Kb SIINFEKL positive cells with OVA mRNA-loaded SM102 or ALC0315 LNPs was comparable to OVA peptide-treated cells, indicating efficient antigen presentation (Fig. 2C). Both SM102 and ALC0315 LNP formulations effectively delivered mRNA to lymphoid organs, lymph nodes, and the spleen, which was verified using in vivo bioluminescence signal (Fig. 3). Furthermore, DLin-MC3-DMA LNPs delivered siRNA to the cytoplasm and was able to significantly reduce GFP expression in cells treated with GFP siRNA LNPs compared to control siRNA LNPs or untreated cells.

Conclusion: FNP technique rapidly self-assembled clinically relevant ionizable LNPs and stably encapsulated nucleic acid payloads. These LNPs were highly monodisperse and allowed precise delivery of mRNA/siRNA with superior functionality both in vitro and in vivo. Taken together, FNP could serve as an alternative approach to reproducibly producing LNPs to boost preclinical research efforts.

References: 1. Y. Xu, A. Golubovic, S. Xu, A. Pan and B. Li, Journal of Materials Chemistry B, 2023, DOI: 10.1039/D3TB00649B.
2. X. Han, H. Zhang, K. Butowska, K. L. Swingle, M.-G. Alameh, D. Weissman and M. J. Mitchell, Nature Communications, 2021, 12.
3. J. R. Melamed, S. S. Yerneni, M. L. Arral, S. T. LoPresti, N. Chaudhary, A. Sehrawat, H. Muramatsu, M.-G. Alameh, N. Pardi, D. Weissman, G. K. Gittes and K. A. Whitehead, Science Advances, 2023, 9, eade1444.
4. X. Hou, T. Zaks, R. Langer and Y. Dong, Nature Reviews Materials, 2021, 6, 1078-1094.
5. R. Tenchov, R. Bird, A. E. Curtze and Q. Zhou, ACS Nano, 2021, 15, 16982-17015.

Acknowledgements: This work was supported by the National Institutes of Health—National Institute of General Medical Sciences (NIH-NIGMS) Tumor Microenvironment Centers of Biomedical Research Excellence (TME CoBRE) grant [2P20GM121322-06].

Fig. 1. Fabrication and physicochemical characterization of lipid nanoparticles. A) Schematic showing the preparation of nucleic acid (RNA)-loaded Lipid Nanoparticle (LNP). LNPs were prepared by the Flash Nanoprecipitation (FNP) technique using a Confined Impingement Jet (CIJ) mixer. The LNP is composed of a helper phospholipid (Green), PEG lipid (Blue), and structural lipid Cholesterol (Yellow). Nucleic acids are entrapped with the help of the ionizable lipid (Violet). B) Negative stained transmission electron microscope (TEM) images for the SM102, ALC0315, and DLin-MC3-DMA ionizable LNPs. The TEM image scale is 100 nm. C) Particle size of LNPs was measured using dynamic light scattering, and the hydrodynamic diameter is reported as a Z average and reported in nm. D) Zeta potential was measured by electrophoretic light scattering and reported in millivolt (mV). E) Encapsulation efficiency of LNP-loaded mRNA or siRNA was measured using the QuantiFluor® RNA System. Data represented in C, D, and E are mean ± SD (n=3). The image was created with BioRender.com.

Fig. 2. Intracellular delivery of mRNA using LNPs in DC 2.4 dendritic cells. SM102 and ALC0315 LNPs were encapsulated with mCherry mRNA and incubated for 24 h with A) DC 2.4 cells. mCherry mRNA encoded into mCherry protein (red color) indicates successful cytoplasmic delivery of mRNA by LNPs. Live-cell images were taken using a confocal microscope with a 60X objective and red punctae inside the cells indicated by black arrows. The scale bar is 10 μm. B) Schematic showing functional SIINFEKL assay. DC 2.4 cells were treated with OVA-mRNA loaded SM102 and ALC0315 lipid nanoparticles or OVA peptide as control for 24 h, the dose of the OVA-mRNA was 1 µg/mL in each well. C) The expression level of the H-2kb antibody-bound OVA257-264 (SIINFEKL) was reported. Data represented as mean ± SD (n=3). Significant differences between each timepoint were determined by one-way ANOVA with Tukey’s multiple comparison test, ****p < 0.0001, **p = 0.002, *p = 0.0121 for ALC0315 LNPs and no treatment, and *p = 0.0214 for SM102 and ALC0315 LNP formulations.

Fig. 3. In vivo delivery of mRNA using LNPs in C57BL/6 mice. A) Schematic representation of firefly luciferase mRNA loaded LNPs, route of administration, and IVIS imaging of lymphoid organs. B) IVIS bioluminescent images of draining inguinal lymph nodes and the spleens of SM102 LNPs or ALC0315 LNPs or PBS only administered mice. C) Bioluminescence is represented as average radiance in draining lymph nodes and spleens of mice administered with SM102 and ALC0315 LNPs. Data represented as mean ± SD (n=3).