Ionizable Lipids with Reduced Numbers of Tails Influence Lipid Nanoparticle Organ Tropism

Introduction: RNA-based therapeutics have gained traction for the prevention and treatment of a variety of diseases. However, a significant barrier for the delivery of these therapeutics is their large size and negative charge, which prevents them from entering cells and leads to rapid degradation and immune recognition. This has necessitated the use of nanomaterial carriers. Lipid nanoparticles (LNPs) have emerged as the preeminent delivery vehicle for RNA therapies, most recently as the carriers for the mRNA COVID-19 vaccines. LNPs consist of an ionizable lipid (IL), a phospholipid, cholesterol, and a PEGylated lipid, with the IL playing an essential role in RNA delivery. ILs are protonated in the endosome due to acidic conditions, which prompts cargo release into the cytosol. Currently, there is growing evidence that the structure of IL lipid tails significantly impacts the efficacy of LNP-mediated mRNA translation, but this idea has been underexplored. As such, this project examines the structure-function relationship between the lipid tails of ILs and the efficacy of mRNA delivery of the corresponding LNPs. Using C12-200 and C14-494, two gold standard ILs, as models, we designed a library of ILs with varying numbers of tails and evaluated their potency in vivo. We determined that small changes in lipophilicity can drastically influence mRNA translation. Excitingly, we also discovered that the biodistribution of LNPs can be altered by changing the number of tails. We envision that the results of this project can be utilized as future design criteria for the next generation of LNPs.

Materials and

Methods: An IL library of varying tail lengths and number of tails was synthesized (Fig. 1A). The polyamine lipid cores, 200 and 494, were reacted with varying equivalents of the corresponding epoxide-terminated alkanes of length C10, C12, and C14. The reaction mixture was stirred for 48 h at 80 °C in ethanol. The resulting product was purified using flash chromatography and analyzed using 1H NMR and LC-MS.

Following the synthesis of the library, LNPs were formulated with each IL and luciferase mRNA. The mRNA was dissolved in an aqueous phase, and the ionizable lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (C14PEG-2000) were dissolved in ethanol in a 35:16:46.5:2.5 molar ratio, respectively. The two phases were mixed together using a microfluidic device, and the mixture then dialyzed in a microdialysis cassette for 2 h in 1X PBS prior to filtering the particles through a 0.22 µm filter. The mRNA concentration of each LNP was measured using a Quant-iT RiboGreen assay.

LNP physical property analysis consisted of encapsulation efficiency (RiboGreen), hydrodynamic diameter (dynamic light scattering), relative pKa determination (TNS assay), and ζ potential. BALB/cJ mice (n=3) were injected with LNPs at a dose of 0.1 mg/kg via the lateral tail vein. After 12 h, 200 µL of luciferin was injected intraperitoneally at a dose of 15 mg/kg, and full body and organ luminescence was evaluated using IVIS imaging.

Results, Conclusions, and Discussions: Here, we developed a simple synthetic scheme for varying the number of IL tails using a stoichiometric approach. IL synthesis was confirmed to be successful based on structural characterization and purity determination using NMR and LC-MS. C12-200 analogues with differing numbers of tails resulted in different organ biodistribution patterns (Fig. 1B). While C12-200 primarily targets the liver, its 3- and 4-tailed analogues resulted in increased delivery to the spleen and lungs. C12-200 resulted in 97% trafficking of transfected mRNA to the liver, compared to 25% and 8% for C12,4-200 and C12,3-200 respectively. In terms of spleen delivery, 42% and 67% of transfected mRNA was trafficked to the spleen in the C12,4-200 and C12,3-200 treatment groups respectively. Both C12,4-200 and C12,3-200 achieved significant relative lung delivery, at 27% and 23% of transfected mRNA respectively. These results show that organ tropism is dramatically altered by reducing the number of tails on one IL structure. Additionally, organ tropism varied between the analogues of the C10-200 and C12-200 ILs, demonstrating that IL structures with the same core and different tail lengths further mediate this effect. Last, analogues of C12-494 and C14-494, two 5-tail ILs that primarily target the liver, with reduced numbers of tails were tested. The 3- and 4-tail 494-core analogues demonstrated increased relative liver delivery compared to the corresponding 200-core analogues, demonstrating altered tropism with different polyamine cores.

When designing new IL structures, considering the number of tails provides an opportunity to control organ tropism of the corresponding LNPs. We are currently applying our synthetic scheme to additional cores and tail lengths to investigate a greater variety of reduced tail number analogues. Then, we hope to identify patterns and mechanisms behind our results, which will enable translation of our findings into clear and generalizable design rules. Additionally, while our current analogues contain a particular number of tails positioned randomly on the core, we are now investigating regiospecific analogues in which IL tails are selectively attached to specific core positions. Lastly, we plan to utilize our reduced tail number structures for clinically relevant applications of LNP delivery to the lung, liver, and spleen.

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