(T1330-09-55) Early Development of Lipid Nanoparticles as Carriers for miRNA

Purpose: MicroRNAs (miRNAs) are endogenous, small noncoding RNAs that can serve as therapeutic agents by acting as mimics or inhibitors to modulate the activity of native miRNAs. miRNA-based therapies offer a promising alternative for the treatment of various diseases, including inflammatory disorders, viral infections, and cancer.^1–3 However, successful clinical translation of miRNA therapeutics requires the development of efficient and targeted delivery systems.⁴ In recent years, lipid nanoparticles (LNPs) have emerged as a powerful platform for the delivery of therapeutic RNAs.⁵ In this preliminary study, we evaluated two LNP formulations based on the Onpattro® lipid composition. The LNPs were prepared using either (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, MC3 (Cayman Chemicals, Ann Arbor, MI, USA) or a novel mono-4-hydroxy-piperidine-glycerol dialkyl glycerol tetraether (NAIL1), derived from the archaeal lipid glycerol dialkyl glycerol tetraether of Sulfolobus acidocaldarius (NovoArc GmbH, Vienna, Austria). The primary aim of this study was to compare the physicochemical properties and transfection efficiency of these two LNP formulations in delivering a model miRNA to human epithelial cells in vitro.

Methods: Model micro-RNA mimic, hsa-miR-147b-3p (Thermo Fisher Scientific, Whaltman, MA, USA),  was loaded into LNPs while manufacturing using a microfluidic mixer (NanoAssemblr™ Benchtop, Precision NanoSystems, Vancouver, BC, Canada.) with a staggered herringbone channel, at a total flow rate of 18 ml/min and flow rate ratio of aqueous and organic solution of 5:1. The LNPs were composed of ionizable lipid/DSPC/cholesterol/DMG-PEG2000 (DSPC – 1,2-distearoyl-sn-glycero-3-phosphocholine, DMG-PEG2000 – 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000) at a molar ratio of 50.0/10.0/38.5/1.5. MC3 or NAIL1 was used as an ionizable lipid in the formulations studied. Lipids were dissolved in ethanol (MC3) or ethanol/isopropanol mixture (NAIL1), depending on the ionizable lipid used, to achieve a final lipid concentration of 8 mM and mixed with miRNA solution in 25 mM sodium acetate buffer pH 4 with N/P ratio of 4. LNPs were dialyzed overnight using citric buffer (pH 6) and PBS (pH 7.4). The particle hydrodynamic diameter, PDI, and size distribution were determined by dynamic light scattering (intensity mode; Zetasizer Pro Red, Malvern Panalytical Ltd., Malvern, UK), using a disposable polystyrene cuvette filled with LNPs diluted in PBS. Encapsulation efficacy was calculated based on the measurements performed by the Quant-iT microRNA Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA).To assess transfection efficacy, human bronchial epithelial cells (16HBE) at a confluence of about 80% were transfected, involving four treatment groups: (1) miRNA147 mimic using Lipofectamine RNAiMAX reagent, (2) miRNA147 mimic in MC3-LNP, (3) miRNA147 mimic in NAIL1-LNP, and (4) non-transfected control cells. For both the RNAiMAX reagent and each LNP formulation tested, a negative miRNA control for miRNA 147 mimic was evaluated.Twenty-four hours after transfection, cells were harvested for RNA isolation using Qiazol reagent (Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocol. RNA samples were reverse-transcribed using the GoScript Reverse Transcription kit (Promega, Madison, WI, USA). Using the miRTarBase tool, the validated targets for hsa-mir-147b-3p, i.e., COL4A2 (Collagen Type IV Alpha 2 Chain) and SDHD (Succinate Dehydrogenase Complex Subunit D), were selected. Relative expression analysis was performed using quantitative reverse transcription PCR (qRT-PCR). GoTaq qPCR Master Mix (Promega, Madison, WI, USA) was used with specific primers and GAPDH as a reference gene. Calculations were carried out using the double delta method. Statistical analyses were performed with PQ Stat software (Student's t-tests for independent groups were used).

Results: The MC3-LNP and NAIL1-LNP were characterized by diameters of 47.6±1.0 nm and 67.4±1.1 nm, respectively, with PDI below 0.2, 0.136±0.004 (MC3-LNP) and 0.153±0.013 (NAIL1-LNP). Encapsulation efficacy of miRNA into tested formulations reached over 85% with more than 91% of miRNA incorporated into LNP. Expression of both target genes for hsa-miR-147b-3p was significantly lower in all groups transfected with a mimic for this miRNA, both from expression in non-transfected cells and from expression in cells transfected with miRNA negative control. This indicates that miRNA mimic transfection occurred in all examined groups. Comparing the expression of target genes between the group transfected with the RNAiMAX reagent and the groups transfected with MC3-LNP and NAIL1-LNP formulations (Figure 1), we did not observe significant differences in most cases, indicating the potential for using the tested formulations as miRNA carriers.

Conclusion: The results demonstrate the potential of MC3- and NAIL1-based LNPs as effective delivery carriers for miRNA. The particle size, size distribution, and miRNA encapsulation efficiency align with the characteristics of currently marketed RNA-based drug products. In vitro transfection efficacy of the LNPs, using either MC3 or NAIL1 as the ionizable lipid, is comparable to that of standard transfection reagents. Notably, NAIL1 achieves transfection performance comparable with MC3, the ionizable lipid used in the approved siRNA-based therapeutic Onpattro®. However, further in vivo studies using animal models are necessary to fully evaluate the therapeutic potential of these miRNA delivery systems.

References: 1. Hum, C. et al. (2021); MicroRNA Mimics or Inhibitors as Antiviral Therapeutic Approaches Against COVID-19. Drugs 81, 517–531. https://doi.org/10.1007/s40265-021-01474-5.
2. Langwiński, W. et al. (2022); Allergic inflammation in lungs and nasal epithelium of rat model is regulated by tissue-specific miRNA expression. Molecular Immunology 147, 115–125. https://doi.org/10.1016/j.molimm.2022.04.017.
3. Hassan, M. et al. (2023); MicroRNA-122 mimic/microRNA-221 inhibitor combination as a novel therapeutic tool against hepatocellular carcinoma. Non-coding RNA Research 8, 126–134. https://doi.org/10.1016/j.ncrna.2022.11.005.
4. Diener, C. et al. (2022); Emerging concepts of miRNA therapeutics: from cells to clinic. Trends in Genetics 38, 613–626. https://doi.org/10.1016/j.tig.2022.02.006.
5. Cullis, P.R., and Felgner, P.L. (2024); The 60-year evolution of lipid nanoparticles for nucleic acid delivery. Nat Rev Drug Discov 23, 709–722. https://doi.org/10.1038/s41573-024-00977-6.

Acknowledgements: The authors acknowledge the financial support from the Agencja Badań Medycznych (Medical Research Agency) project No 2024/ABM/03/KPO/KPOD.07.07-IW.07-0117/24-00.

Figure 1. Expression of COL4A2 (target gene for hsa-miR-147b-3p)
A – Treatment group 1 (miRNA147 mimic transfected with RNAiMAX reagent) vs treatment group 2 (miRNA147 mimic in MC3-LNP)
B - Treatment group 1 (miRNA147 mimic transfected with RNAiMAX reagent) vs treatment group 3 (miRNA147 mimic in NAIL1-LNP)