(M1530-09-59) Solid-Phase Strategy for High-Yield Diversification and Functional Evaluation of Ionizable Triazine Lipids for mRNA Delivery

Purpose: Lipid nanoparticles (LNPs) have emerged as a cornerstone technology for the delivery of nucleic acid therapeutics, with ionizable cationic lipids playing a central role in enabling efficient encapsulation, cellular uptake, and endosomal escape. These lipids have dramatically improved the safety and transfection efficiency of LNP-based platforms, accelerating the clinical translation of mRNA vaccines and gene therapies (1). Despite their success, the rational design of next-generation lipids remains hindered by synthetic limitations, particularly the lack of efficient strategies to generate structurally diverse and tunable lipid libraries for systematic structure-activity relationship (SAR) studies (2). Previous work has demonstrated the potential of triazine-based lipids synthesized via solution-phase methods to enhance LNP performance, but the scope and throughput of this approach are constrained (3). This research addresses a critical gap by applying a solid-phase synthetic platform to rapidly assemble triazine-based ionizable lipids with modular headgroups and hydrophobic tails. This enables a more systematic exploration of chemical space to identify structure-function relationships that govern delivery efficacy and biocompatibility. By facilitating rapid lipid diversification combinatorially, this study aims to accelerate the discovery of optimized lipid formulations for both in vitro and in vivo applications.

Methods: To enable rapid diversification of ionizable lipid structures for SAR studies, we developed a solid-phase synthetic approach using a 4-nitrophenyl carbonate-functionalized resin as an acid-labile platform. This design allows stepwise, modular synthesis with simplified purification. The synthetic sequence begins with coupling a diamine headgroup to the resin, forming an amine-functionalized support. Cyanuric chloride is then introduced as a central triazine scaffold via nucleophilic aromatic substitution (SNAr), leveraging its chemoselective reactivity under thermally controlled conditions (4). At room temperature, we install various dialkylamines as hydrophobic lipid tails. A final substitution with structurally diverse amine nucleophiles at 80°C completes the triazine assembly, enabling wide chemical tunability across headgroup and tail domains (Figure 1). The cleaved products are released with trifluoroacetic acid, yielding highly pure lipids. Purity and identity are confirmed using ¹H NMR, ESI-MS, and HPLC. Only lipids exceeding 80% purity are selected for downstream biological evaluation. Formulated LNPs are generated via semi-automated microfluidic mixing, enabling consistent control over nanoparticle size, polydispersity index (PDI), and encapsulation efficiency. A Design of Experiment (DoE) framework is employed to systematically explore and optimize formulation parameters, such as lipid molar ratios, aqueous/ethanol phase ratio, N/P ratio, and total flow rate. This data-driven approach enables robust modeling of complex variable interactions while minimizing experimental runs. LNPs will be characterized for size, PDI, and zeta potential using a dynamic light scattering (DLS) instrument. Encapsulation efficiency of mRNA (e.g., FLuc) will be assessed via RiboGreen assay. Transfection efficiency will be quantified in vitro using luciferase reporter assays in HEK293 and immune-relevant cell lines (e.g., THP-1), while cytotoxicity will be evaluated using CellTiter-Glo.

Results: These data demonstrate that solid-phase synthesis using cyanuric chloride enables efficient generation of diverse ionizable lipids. In the first iteration, a C12 dialkylamine lipid tail was paired with eight distinct headgroups. Six of eight lipids achieved ~100% yield without additional purification, as confirmed by mass spectrometry and NMR. The remaining two lipids showed 80–90% conversion, likely due to steric hindrance from t-butyl-protected amines (Figure 2). This indicates that protecting groups can significantly reduce reaction efficiency. Based on these findings, a second iteration is underway to evaluate lipid tail diversity (C12, C14, C16, C18) while keeping headgroups constant. The goal is to define structure-activity relationships and optimize for delivery efficiency. Parallel formulation efforts using a multiplexed LNP-mRNA workflow have confirmed the scalability of this approach. A lead lipid from the initial screen was formulated into LNPs and assessed for particle size, encapsulation, transfection, and cytotoxicity (Figure 3). Several formulations demonstrated in vitro performance, specifically luciferase expression and cell viability, comparable to or exceeding that of a benchmark clinical LNP Pfizer-BioNTech formulation. These results support continued screening to identify candidates with strong potential for in vivo studies.

Conclusion: Solid-phase synthesis using cyanuric chloride enables rapid and modular generation of diverse ionizable lipids with high yield and purity. This platform allows precise control over headgroup and tail structures, accelerating the identification of structure-function relationships that influence LNP formulation, stability, and mRNA transfection efficiency. By integrating combinatorial chemistry with data-driven formulation and screening, this approach offers a scalable framework for optimizing next-generation delivery systems. The ability to streamline lipid design and formulation has broad implications for improving the development of mRNA therapeutics, and this work provides a foundation for advancing both in vitro and in vivo applications.

References: (1) Tang X, Zhang Y, Han X. Adv Nano Biomed Res. 2023;3. (2) Zhang Y, Sun C, Wang C, Jankovic KE, Dong Y. Chem Rev. 2021;121:12181–277. (3) Nardo , D; et al. (Venditto, VJ). RSC Adv, 2021, 24752-24761. (4) Simanek, E et al. 2009 Proc RSC A 466:1445-1468

Acknowledgements: This project is supported with grants from KSCHIRT (24-15) and the NIH (U01HL152392, R01HL152081). VJV is also supported by grants from the NIH (R01NS116068, R01NS126228) and an NIH Center for Biomedical Research Excellence (COBRE) in Pharmaceutical Research and Innovation (CPRI, P20GM130456). JAM is funded by the NIH Training Grant 5T32 NS077889, Neurobiology of CNS Injury and Repair, and by the Lyman T. Johnson Diversity Scholarship. We thank the James Brien Lab for training and access to the microfluidic formulation instrument.

Solid-phase synthesis of triazine-based lipids: Reaction scheme

Headgroup moiety addition- stacked HPLC traces of headgroup diversification of ionizable lipids via solid-phase synthetic strategy.

In vitro evaluation of lead triazine lipid (TZ3) LNP formulations for mRNA delivery. Low and high concentrations of TZ3 and Pfizer-BioNTech ionizable lipid (ALC-0315).