Delivery of modified mRNA to damaged myocardium by systemic administration of lipid nanoparticles

Purpose: Modified mRNA (modRNA) is a promising cardiac regeneration therapeutic, but naked mRNA is large, negatively charged, and rapidly degraded. It is unknown whether systemically administered lipid nanoparticles (LNPs) can functionally deliver mRNA to ischemic/damaged myocardium after myocardial infarction.

Hypothesis: After ischemia-reperfusion injury, systemically administered LNPs will accumulate in the infarcted myocardium due to damage-induced vascular permeability and will functionally deliver modRNA to cells in the damaged heart, producing measurable protein expression.

Aims: Evaluate the biodistribution and circulation time of fluorescently labeled LNPs after myocardial ischemia-reperfusion injury. - Determine whether LNP-encapsulated firefly luciferase mRNA produces functional protein in the damaged myocardium after intravenous administration. - Identify which cardiac cell types take up and translate LNP-delivered Cre modRNA using Cre-reporter mice. - Assess dose-dependent delivery and preliminary liver toxicity of LNP-modRNA.

Delivery system: Platform: Lipid nanoparticles (LNPs) prepared by microfluidic mixing. - Composition: DLin-MC3-DMA, cholesterol, DSPC, PEG-DMG at molar ratio 50/38.5/10/1.5. - Payload: Modified Cre recombinase mRNA; unmodified firefly luciferase mRNA; fluorescently labeled LNPs using 0.2 mol% DSPE-Cy5.5 for biodistribution. - Encapsulation: mRNA/lipid weight ratio 10:1; encapsulation efficiency 95–99%. - Physicochemical properties: Size <100 nm; PDI <0.2; zeta potential approx. −4 to −8 mV. - Targeting: No active targeting ligand; systemic intravenous tail-vein administration.

Approach: Model: Murine myocardial ischemia-reperfusion injury via 60-min left anterior descending coronary artery ligation followed by reperfusion. - Animals: C57Bl/6 mice, Ai9 Cre-reporter mice; sham-operated and untreated controls. - Biodistribution: 50 µg LNP-mRNA IV; tissues collected at 4 h and 24 h; n = 4–8. - Functional delivery: 50 µg firefly luciferase mRNA IV 1 h after reperfusion; luciferase activity measured at 4 h; sham controls, n = 4. - Cell targeting: Cre modRNA doses 0, 5, 10, 25, 50, 100 µg IV; hearts analyzed 7 days later; n = 3 per dose. - Toxicity: H&E staining of liver at high doses.

Key methods: DLS for size and PDI; zeta potential; RiboGreen assay for encapsulation efficiency. - Whole-organ fluorescence imaging and tissue lysate fluorescence for LNP biodistribution. - Luciferase activity assay in tissue homogenates for functional mRNA delivery. - Immunofluorescence staining for tdTomato (Cre reporter), αSMA (fibroblasts), MF20 (cardiomyocytes), Mac3 (macrophages), CD45 (leukocytes). - H&E staining for liver toxicity.

Key results: LNPs were <100 nm with PDI <0.2 and 95–99% mRNA encapsulation. - After ischemia-reperfusion, fluorescent LNPs accumulated in the infarcted area of the heart at 4 h and 24 h, but not in sham or control hearts. - Myocardial luciferase activity was significantly higher in ischemia-reperfusion mice than sham-operated mice at 4 h, though still much lower than in liver and spleen. - Cre modRNA produced dose-dependent tdTomato+ cells in the infarct region and epicardial layer; most targeted cells in the infarct were cardiac fibroblasts (αSMA+), with some cardiomyocytes and macrophages. - No obvious liver toxicity was observed even at 100 µg modRNA.

Interpretation: The authors conclude that LNPs can functionally deliver modRNA to damaged myocardium after systemic administration, supporting further development of LNP-modRNA therapies for cardiac regeneration. However, cardiac expression remains low compared with liver and spleen, so optimization is needed to improve on-target delivery and reduce off-target expression.

Limitations: Cardiac mRNA expression was low relative to liver and spleen. - Fluorescent lipid labels may dissociate from LNPs and do not directly report mRNA biodistribution. - Cre-reporter readout is binary and does not quantify expression levels in targeted cells. - Delivery was tested only after acute ischemia-reperfusion injury, not in chronic heart failure without recent infarction. - No therapeutic efficacy endpoint; only reporter genes were used. - No large-animal validation. - Optimal timing of LNP administration relative to vascular leakage was not fully defined.