Ionizable Lipid Nanoparticle-Mediated Delivery of Plasmid DNA in Cardiomyocytes

Purpose: Cardiomyocytes are hard-to-transfect cells, and gene therapy for cardiovascular disease is limited by insufficient delivery to cardiac tissue, nucleic acid degradation, and safety concerns with viral vectors. A safe, effective non-viral platform for pDNA delivery to cardiomyocytes is needed.

Hypothesis: Ionizable lipid nanoparticles (LNPs) can safely and effectively deliver plasmid DNA (pDNA) to cardiomyocytes, and optimization of LNP lipid composition and ionizable lipid:pDNA ratio will enhance transfection efficiency.

Aims: Develop a mini-library of pDNA-loaded LNPs with varying C12-200:pDNA ratios and lipid compositions. - Characterize LNPs and screen for pDNA delivery and transfection efficiency in cardiomyocytes in vitro. - Identify a lead LNP formulation and evaluate in vivo gene expression in heart tissue. - Assess toxicity, inflammation, and cardiac function after LNP treatment.

Delivery system: Platform: Ionizable lipid nanoparticles (LNPs). - Components: C12-200 ionizable lipid, DOPE, cholesterol, C14-PEG2000. - Payload: Plasmid DNA encoding ZsGreen (GFP). - Formulation: Eight LNP formulations with C12-200:pDNA ratios of 10:1 or 20:1 and varying DOPE/cholesterol molar ratios. - Lead formulation: LNP4 (10:1 C12-200:pDNA; 35% C12-200, 56.5% DOPE, 6% cholesterol, 2.5% PEG). - Targeting ligand: None. - Route: Intravenous tail-vein injection in mice. - Physicochemical properties: Size 91.8–122.6 nm; PDI 0.216–0.337; EE 71–94%; pKa 6.5 for LNP4.

Approach: In vitro: Neonatal rat ventricular cardiomyocytes; LNP screening at 0.4 and 0.8 µg pDNA; dose–response and time-course for lead LNP. - In vivo: C57BL/6 mice (n = 4); single IV injection of LNP4 (10 µg pDNA); heart harvested 7 days post-injection. - Controls: PBS-treated mice; non-transfected cells. - Toxicity assessment: Cell viability, cardiac contractility, Western blot for SERCA2/AKT/ERK, inflammatory cytokines, myeloid cell populations, hematology, liver enzymes, histopathology.

Key methods: DLS and Cryo-TEM for size, PDI, and morphology. - TNS assay for pKa determination. - Qubit dsDNA assay for encapsulation efficiency. - Fluorescence imaging for GFP expression and cellular uptake. - Resazurin assay for cell viability. - Confocal microscopy for LNP uptake and GFP expression. - Western blot for SERCA2, AKT, ERK1/2. - qRT-PCR for TNF-α and IL-6. - Flow cytometry for cardiac myeloid cell populations. - Hematology and liver enzyme measurements.

Key results: LNP4 was the top performer: ~1.3-fold higher GFP fluorescence than the second-best LNP8 and ~10-fold higher than the lowest performer LNP6. - LNP4 achieved >60% transfection efficiency at day 2 and >80% at day 4 in vitro. - LNP4 pKa was 6.5, closer to endosomal pH than LNP5 (7.2) and LNP6 (7.3). - In vivo, LNP4 induced a 2-fold increase in GFP expression in heart tissue compared to control. - LNP4 showed >90% cell viability in vitro; no significant changes in cardiac function, inflammatory cytokines, myeloid cell populations, hematology, or liver enzymes in vivo.

Interpretation: LNPs can deliver pDNA to cardiomyocytes with high transfection efficiency and negligible toxicity. Higher DOPE and lower cholesterol molar ratios enhance GFP expression. LNP4 holds promise as a platform for cardiovascular gene therapy, though further optimization for heart selectivity is needed.

Limitations: No active targeting ligand; selectivity for heart tissue not demonstrated. - Only a single IV injection and one time point (7 days) were tested in vivo. - Mechanism of uptake and intracellular trafficking not fully determined. - No disease model; only healthy mice were used. - Biodistribution not comprehensively assessed. - No large-animal validation. - Long-term expression, repeated dosing, and safety not evaluated. - pDNA delivery to non-cardiac tissues not fully characterized.