Novel piperazine‐based ionizable lipid nanoparticles allow the repeated dose of mRNA to fibrotic lungs with improved potency and safety

Figures

• 

  FIGURE 1. The illustration of newly developed ionizable lipid nanoparticles (LNPs) for targeted delivery of mRNA to the lungs of idiopathic pulmonary fibrosis (IPF) mice. The new biodegradable LNP can deliver mRNA safely and efficiently to the pulmonary fibrosis‐induced lungs of mice by systemic administration.

• 

  FIGURE 2. The 244cis lipid nanoparticles (LNPs) show improved potency of mRNA delivery and translation without immunogenicity. (a) The graph represents the potency versus immunogenicity of three different LNPs (244cis, SM‐102, and MC3). On the y‐axis, serum expression level of human erythropoietin (hEPO) protein is plotted against the serum level of monocyte chemoattractant protein 1 (MCP‐1) levels on the x‐axis. Three LNPs were systemically injected at the dose of 0.1 mg/kg of hEPO mRNA. After 6 h injection, the serum level of hEPO and MCP‐1 was analyzed by ELISA (n = 5, mean ± standard deviation [SD]). (b) The expression profile of hEPO by three LNPs at different time points. Mice were injected with LNPs containing 0.1 mg/kg of hEPO mRNA via systemic administration. SM‐102 and MC3 were used as controls (n = 4, mean ± SD).

• 

  FIGURE 3. Optimization of lung targeting LNPs with the replacement of helper lipid (a) Chemical structures of four lipid components and the molar ratio of the lung targeting LNPs. LNPs were formulated with 20 or 40 mol% of cationic lipids instead of DOPE. (b) Evaluation of delivery efficacy to the lungs by firefly luciferase expression. The LNPs were loaded with fLuc mRNA and injected systemically into C57BL/6 mice at the mRNA dose of 0.2 mg/kg. After 6 h of injection, bioluminescence was analyzed by ex vivo organ imaging (n = 3, mean ± SD). (c) The lung targeting LNPs containing 20% of DOTAP accumulated in the lungs selectively. (d) Evaluation of serum MCP‐1 expression upon the administration of lung targeting LNPs as compared to their native LNPs. The LNPs were loaded with hEPO mRNA and injected systemically into BALB/c mice at an mRNA dose of 0.1 mg/kg. After 6 h of injection, the blood was collected, and MCP‐1 cytokines were analyzed by ELISA (n = 3–4, mean ± SD).

• 

  FIGURE 4. Evaluation of cell type‐specific delivery of Cre mRNA in the lungs. (a) The schematic illustration shows that the deletion of the stop cassette by the delivery of Cre mRNA activating the expression of tdTomato. (b) The lung targeting LNPs carrying 0.3 mg/kg Cre mRNA were systemically administrated, resulting in the expression of tdTomato fluorescence in the lungs. Mice were sacrificed, and five major organs were imaged for tdTomato fluorescence 72 h after IV injection. (c) Flow cytometry analysis of cell type‐specific delivery of Cre mRNA using 244cis or SM‐102 LNPs in nonpulmonary fibrosis (PF) and (d) PF models to quantify the percentage of tdTom+ cells (n = 3, mean ± SD, ****p < 0.0001, one‐way analysis of variance [ANOVA]). (e) Flow cytometry analysis of tdTom+ cells in the subtypes of fibroblasts (n = 3, mean ± SD).