PLGA-Encapsulated Mitochondrial Hydrogen Sulphide Donor, AP39, Resolve Endothelial Inflammation via Mitochondria-Targeted Bioenergetic and Redox Modulation

Vascular inflammation and endothelial dysfunction are key drivers in the development of cardiovascular and neurovascular diseases. Mitochondrial dysfunction and oxidative stress further amplify inflammatory cascades, emphasising the need for targeted strategies that restore endothelial homeostasis at the subcellular level. Hydrogen sulphide (H2S) donors, such as AP39, offer cytoprotective benefits but are limited by short half-life and rapid release of the active compound, H2S. We developed poly(lactic-co-glycolic acid) (PLGA) nanoparticles encapsulating AP39 (PLGA-AP39) to achieve sustained, mitochondria-targeted H2S delivery. Nanoparticles were characterised by size, polydispersity, zeta potential, encapsulation efficiency, and in vitro release kinetics. Human umbilical vein endothelial cells (HUVEC) were exposed to TNF-α to induce inflammation, followed by treatment with free AP39 or PLGA-AP39. Anti-inflammatory effects were assessed by measuring IL-6, IL-8, and TGF-β levels. Mitochondrial function was evaluated using a Seahorse XFe24 Analyser, membrane potential assays, and mitochondrial ROS detection. Moreover, we investigated vascular function by analysing capillary-like tube formation and wound closure in response to treatments. PLGA-AP39 nanoparticles displayed a uniform size (~227 nm), low PDI, and high encapsulation efficiency (>78%). Sustained AP39 release was observed over seven days. Treatment with PLGA-AP39 significantly restored TNF-α-induced endothelial dysfunction and reduced TNF-α-induced release of IL-6, IL-8, and TGF-β compared to untreated controls. Seahorse analysis revealed restoration of maximal respiration and increased spare respiratory capacity. Encapsulated AP39 also preserved mitochondrial membrane potential and reduced mitochondrial ROS production, demonstrating enhanced protection against inflammation-induced metabolic dysfunction. This work establishes a novel nanoparticle-based strategy for prolonged, mitochondria-specific H2S delivery to counteract vascular inflammation and enhance endothelial bioenergetics. The results from this work are pioneering in the generation of a novel delivery method for H2S donors employing PLGA and represent a promising therapeutic avenue for treating chronic vascular inflammatory disorders.