Lipid Nanoparticle-mediated Nucleic Acid Delivery for Microvascular Endothelial Cell Metabolic Modulation

RATIONALE: Pulmonary arterial hypertension (PAH) is a progressive disease marked by increased pulmonary vascular resistance, leading to right ventricular failure and death. In PAH, microvascular endothelial cells (MVECs) abnormally proliferate to form plexiform lesions, which occlude the microvasculature. MVECs in PAH display oncogenic-like behaviors such as increased intracellular calcium and endothelial-mesenchymal transition (EndMT). These maladaptive MVEC phenotypic changes are thought to be driven, in part, by pathogenic metabolic shifts. Increased fatty acid oxidation (FAO) and ketogenesis (i.e. production of the ketone body beta-hydroxybutyrate; BOHB) were recently observed in PAH MVECs 1, 2 . Furthermore, inhibiting BOHB production attenuated MVEC proliferation. These data suggest that BOHB production may represent a druggable target in PAH. We have demonstrated that increased activity of Bdh1, a key enzyme in ketone metabolism, accompanies BOHB production in MVECs derived from an experimental rodent PAH model. As nanoparticle-mediated strategies can enable the direct modulation of specific metabolites without globally disrupting metabolic pathways, we sought to develop a lipid nanoparticle (LNP) optimized for MVECs. Since MVECs implicated in PAH undergo dynamic acquisition and loss of cell surface markers through EndMT, we pursued a compositional-based screening approach, as opposed to an active-ligand based targeting strategy, to assess transfection efficiency. We have designed an LNP for cell preferential uptake in MVECs to modulate levels of metabolites differentially implicated in PAH. We hypothesize LNP-mediated Bdh1 downregulation can rescue endothelial cell function in PAH. Methods: We pursued a multi-step screening approach to identify optimized formulations for modulating cellular metabolism using reporter nucleic acid cargo. Employing an established library of 649 formulations with SM-102 as the ionizable lipid, helper lipids of cationic (DDAB, DOTAP), zwitterionic (DSPC, DOPE), and anionic charge (18BMP, 18PG), cholesterol and DMG-PEG-2000, we evaluated the transfection efficiency of Luciferase mRNA-LNPs in Human Umbilical Vein Endothelial Cells 3, 4 . We then stratified top formulations by cationic helper lipid charge to enhance lung tropism and evaluated reporter plasmid-DNA transfection (24 h) and siRNA transfection (48 h) in control rat lung MVECs isolated as described previously 1 .Transfection efficiency was assessed with a luminescence microplate reader, flow cytometry, and fluorescent microscopy. Bdh1 was depleted in MVECs with small interfering RNA (siBdh1), using non-targeting siRNA as control, and assessed with RT-qPCR. Results: Two plasmid-DNA DOTAP helper lipid formulations demonstrated nearly 60% transfection as evidenced by mCherry (+) cells using flow cytometry. We achieved efficient knock-down (KD) when delivering our LNP complexed with siRNA against Bdh1 in primary rat lung MVECs in vitro, in which over 80% KD of Bdh1 expression was achieved (RT-qPCR) when compared to non-targeting siRNA control and normalized to housekeeping gene, Beta-actin. Conclusion: We identified LNP formulations capable of delivering a range of payloads to MVECs. Future work will evaluate the preferential delivery and transfection efficiency of these formulations in vivo to determine the translational utility of metabolite modulation, notably BOHB, in PAH. REFERENCES: 1. Philip, N. et al. Am J Physiol Lung Cell Mol Physiol 326, L252–L265 (2024). 2. Lee, M. H. et al. Am J Physiol Lung Cell Mol Physiol 323, L355–L371 (2022). 3. Cheng, L. et al. ACS nano 18.42, 28735-28747(2024). 4. Zhu, Y. et al. Nat Commun 13.1, 4282 (2022). NSF GRFP DGE2139757 (A.G.), R01HL151530 (K.S.) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.