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Cost-effective and High-Performance Ionizable Lipids for mRNA-LNP Vaccines: One-Pot Ugi-4CR Strategy and Preclinical Evaluation

16 July 2025

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The COVID-19 pandemic underscored the enormous potential of mRNA vaccine technology for infectious disease prevention. mRNA vaccines require efficient delivery vehicles—typically lipid nanoparticles (LNPs)—to enable translation of antigenic proteins and induction of humoral and cellular immunity. Ionizable lipids (ILs) are the pivotal component in LNPs, critically influencing mRNA encapsulation, endosomal escape, biodistribution, and overall vaccine efficacy. However, most commercial ILs are produced via multi-step organic syntheses, resulting in elevated costs and quality control challenges. The development of a simplified, cost-effective, and robust synthetic approach to ILs is crucial for the large-scale, global deployment of mRNA vaccine technologies.

A recent study titled "Cost-effective yet high-performance ionizable lipids for mRNA-lipid nanoparticle vaccines" developed a one-pot Ugi four-component reaction (Ugi-4CR) approach to rapidly synthesize a structurally diverse library of 161 novel ILs under mild conditions, with high yields. Six representative ILs, featuring three distinct ionizable head groups (R1, R2, R3) and two hydrophobic tails with different unsaturation degrees (U1, U2), were selected for detailed investigation. These were combined into six ILs (R1U1, R1U2, R2U1, R2U2, R3U1, R3U2) and incorporated into LNP formulations for the delivery of mRNA encoding antigens such as firefly luciferase (mFluc), SARS-CoV-2 spike protein (mSpike), and ovalbumin (mOVA).

Major Findings
1. Efficient Synthesis and Biocompatibility:

Figure 1. Construction and Characterization of Six Ionizable Lipid-Based mFluc-LNPs

All six ILs were synthesized in a one-pot Ugi four-component reaction (Ugi-4CR) with high yield, and were then incorporated into lipid nanoparticles (LNPs) for mRNA firefly luciferase (mFluc) delivery. The resulting mRNA-LNPs had uniform particle size (~100 nm) and suitable zeta potential (0 to −10 mV) for lymph node targeting. Cytotoxicity assays confirmed excellent cell compatibility across multiple cell types and doses.

2. R2U2 Outperforms in Delivery and Immune Response:

Figure 2. R2U2-Based mRNA Lipid Nanoparticles: Enhanced Expression and Improved Retention in Lymph Nodes and Spleen

Among the ILs tested, R2U2-based LNPs exhibited the highest mRNA delivery efficiency and the most robust and durable antigen expression in both spleen and draining lymph nodes (dLNs) following intramuscular injection in mice.

Figure 3. Humoral and Cellular Immune Responses Induced by R2U2-Based mRNA Lipid Nanoparticles

This led to:
·Potent induction of spike-specific IgG after two and three immunizations.
·Significantly enhanced cellular immune responses (elevated IFN-γ–producing splenocytes; higher TNF-α–secreting CD4+/CD8+ T cells).
·Comparable or superior immune responses to the commercial ALC-0315-based LNP benchmark, as confirmed in both mice and cynomolgus macaques.

3. Safety Evaluation:

Figure 4. Safety Assessment of R2U2-Based mRNA Lipid Nanoparticles

The safety profile of R2U2-based mRNA lipid nanoparticles (LNPs) was rigorously assessed in both murine and cynomolgus macaque models. Transient increases in body temperature and minor reductions in body weight were observed in mice following vaccination, but these changes were fully reversible during the recovery phase. Hematological analyses—including white blood cell counts, lymphocyte and neutrophil proportions, and liver function markers—revealed only mild and temporary deviations from baseline, with all parameters returning to normal shortly after vaccination. Histopathological examination of major organs (heart, liver, spleen, lung, and kidney) demonstrated no evidence of tissue damage or pathological alterations. Parallel studies in cynomolgus macaques confirmed the absence of acute or chronic adverse effects. Furthermore, in vitro experiments utilizing human induced pluripotent stem cell (hiPSC)-derived liver organoids demonstrated that R2U2-LNPs maintained high cellular viability at therapeutically relevant concentrations, with only mild membrane perturbation observed at the highest tested doses. Collectively, these comprehensive safety data provide strong preclinical support for the further clinical development of R2U2-based LNP formulations.

4. Robust Mucosal Immunity:

Figure 5. Mucosal Immunity Induction Capability of R2U2-Based mRNA Lipid Nanoparticles

Notably, R2U2-based mRNA-LNPs achieved efficient mRNA expression and antigen-specific secretory IgA (sIgA) induction in mucosal tissues (nasal cavity, vagina, and bronchoalveolar lavage fluid) following intranasal and intratracheal administration, highlighting their potential for respiratory pathogen vaccines.

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In this study, Y-27632 was utilized during the process of human induced pluripotent stem cell (hiPSC) dissociation and maintenance, specifically for the generation of liver organoids:
·During the dissociation of human induced pluripotent stem cells (hiPSCs) into single cells, 10 μM Y-27632 was added to the EDTA solution to improve cell recovery. ·After dissociation, cells were maintained in a passage medium supplemented with 10 μM Y-27632 to preserve cell viability and state during the induction of 3D human liver organoids for safety assessment of R2U2-based LNPs.

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References

[1]Zhan Gao, Haihong Yang, Zepeng He, et al. Cost-effective yet high-performance ionizable lipids for mRNA-lipid nanoparticle vaccines. Biomaterials, 2025, 323, 123421.

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