(L-446) Optimizing Ionizable Lipid Tails for Liver and Non-Liver Delivery of mRNA Lipid Nanoparticles

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, a gold standard IL, as a model, we designed a library of ILs with varying tail lengths and numbers of tails and evaluated their potency in vivo. We determined that small changes in lipophilicity can drastically increase or decrease 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.

An IL library of varying tail lengths and number of tails was synthesized (Fig. 1A). The polyamine core, N1-(2-(4-(2-aminoethyl)piperazin-1-yl)ethyl)ethane-1,2-diamine (lipid core 200) was reacted with a varying number of the corresponding epoxide-terminated alkanes of length C6 to C16. 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 um 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 pK_a determination (TNS assay), and ζ potential. LNPs were screened in vivo by injecting them intravenously BALB/cJ mice (n=3) at a dose of 0.1 mg/kg. After 12 h, 200 uL of luciferin was injected intraperitoneally at a dose of 15 mg/kg, and full body and organ luminescence was evaluated using IVIS imaging.

IL synthesis was confirmed to be successful based on structural characterization and purity determination using NMR and LC-MS. C10-200 outperformed C12-200 in mRNA LNP delivery to the liver (Fig. 1B). A 10-fold increase in mRNA delivery to the liver was achieved by reducing the tail lengths of C12-200 by two carbons each. Given that C12-200 is a gold-standard IL for liver delivery, it is significant that a simple change to its tail structure led to significantly higher mRNA transfection. C12-200 analogues with differing numbers of tails resulted in different organ biodistribution patterns (Fig. 1C). 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% of transfected mRNA being trafficked 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.

Characterization of LNP physical properties was performed to determine if a physical explanation for these differences. No significant differences were found for pKa, ζ potential, hydrodynamic diameter, or encapsulation efficiency compared to C12-200, thus the differences in mRNA translation are likely not related to physiochemical characteristics. We are currently investigating potential protein corona and endosomal escape mechanisms to explain these results.

When designing structures for ionizable lipids, considering ionizable lipid tail length and number of tails provides an opportunity to control delivery efficacy and organ tropism. Small changes in tail length drastically impact mRNA LNP delivery to the liver. Further, within a single tail length, varying the number of tails impacts organ biodistribution. We are currently examining the generalizability of these results by screening similar structures in other polyamine cores. Additionally, we are formulating the LNPs with mRNAs of different lengths to ascertain the relationship between lipophilicity and mRNA size.