Novel AP39-Loaded Liposomes Sustain the Release of Hydrogen Sulphide, Enhance Blood-Brain Barrier Permeation, and Abrogate Oxidative Stress-Induced Mitochondrial Dysfunction in Brain Cells

Mohamad Anas Al Tahan,1,* Mandeep Kaur Marwah,1,* Mandheer Dhaliwal,2 Lorena Diaz Sanchez,1 Hala Shokr,3 Manjit Kaur,4 Shakil Ahmad,1 Raj KS Badhan,5 Irundika HK Dias,1 Lissette Sanchez-Aranguren1 1Aston Medical School, Aston University, Birmingham, UK; 2School of Biosciences, Aston University, Birmingham, UK; 3Pharmacy Division, University of Manchester, Manchester, UK; 4School of Health and Care, Coventry University, Coventry, UK; 5School of Pharmacy, Aston University, Birmingham, UK*These authors contributed equally to this workCorrespondence: Lissette Sanchez-Aranguren, Email [email protected]: Neurodegenerative diseases are often linked to oxidative stress (OS), which worsen neuroinflammation and cause neuronal damage. Managing OS with gasotransmitters such as hydrogen sulphide (H2S) is a promising therapeutic approach to protecting brain cells from oxidative damage. AP39, a mitochondria-targeted H2S donor, has shown neuroprotective potential by reducing OS and improving mitochondrial function. However, its clinical application is limited due to poor stability and rapid release, necessitating a drug delivery system to enhance therapeutic efficacy.Purpose: This study aimed to develop a novel AP39-loaded liposomal formulation to provide controlled H2S release, facilitate AP39 permeation across the blood-brain barrier (BBB), and assess functional effects in mitigating oxidative stress and preserving mitochondrial function.Methods: AP39-loaded unilamellar liposomes were prepared via ethanol injection and characterised for size, polydispersity, and zeta potential. Entrapment efficiency was determined using HPLC, while cytotoxicity was assessed in human vein endothelial (HUVEC) and neuroblastoma (SHSY5Y) cells. Liposomal permeability, AP39 release kinetics, and cellular uptake were evaluated using a microvasculature BBB model. Mitochondrial function under oxidative stress was assessed using a Seahorse XFe24 Analyzer.Results: AP39-loaded liposomes had an average size of 135.92 ± 10.05 nm, a zeta potential of 17.35 ± 3.40 mV, and an entrapment efficiency of 84.48% ± 4.7. Cytotoxicity studies showed no adverse effects after 4 h. Cellular uptake of encapsulated AP39 was significantly higher (7.13 ± 0.28 μg) than the free form (5.8 ± 0.31 μg). The BBB model demonstrated sustained AP39 release (7.28 μg/mL vs 6.44 μg/mL for free AP39). Mitochondrial assays confirmed liposomal AP39 preserved H2S antioxidant properties and enhanced oxygen consumption.Conclusion: Our novel liposomal formulation encapsulating AP39 improves stability, promotes sustained release, and enhances BBB permeability while preserving antioxidant effects. These findings indicate that liposomal AP39 is a suitable therapeutic approach to further investigate in the treatment of neurodegenerative diseases. Keywords: liposomes, hydrogen sulphide, AP39, reactive oxygen species, mitochondrial-targeted compounds, mitochondrial dysfunction