Supplementary Material for: Mitochondria-Targeted SS-31 Conjugated Liposome Attenuate Oxidative Stress in Endothelial and Skeletal Muscle Cells

Background: Mitochondria play a pivotal role as therapeutic targets in a range of disorders, including metabolic and neurodegenerative diseases. SS31, a peptide engineered to target mitochondria, offers potent antioxidant activity, positioning it as a promising therapeutic option. Nevertheless, the hydrophilic profile of SS31 poses challenges such as reduced stability, suboptimal delivery, and poor mitochondrial localization in clinical applications.

Aim: This study was designed to develop a mitochondria-targeted liposomal carrier by conjugating SS31KRKC to the liposome surface (SS31-LP) and to investigate its biological effects in vitro.

Methods: A lysine-arginine-lysine-cysteine (KRKC) linker was incorporated with SS31 to facilitate surface conjugation to liposomes via thiol-based coupling. The hydrodynamic diameter and zeta potential of the resulting formulations were quantified to determine the optimal lipid-to-peptide ratio for mitochondrial targeting. The in vitro mitochondrial localization, cytotoxicity, antioxidant potential, and anti-apoptotic efficacy of SS31-LP were evaluated in HUVEC and C2C12 cells.

Results: SS31-LP demonstrated pronounced mitochondrial localization and showed variable cellular internalization based on zeta potential. Pretreatment with SS31-LP enhanced cellular viability, mitigated oxidative damage, and reduced apoptosis in response to oxidative stress caused by H₂O₂ or blue LEDs.

Conclusion: Overall, SS31-LP presents as a valuable therapeutic strategy for cellular protection against oxidative injury and may be an advantageous platform for targeted drug delivery applications.