Mitochondrial diseases, notably Leigh syndrome (LS), stand as significant concerns in inherited metabolic disorders, primarily affecting neurological functions linked to basal ganglia and brainstem dysfunction [1,2]. The diagnosis of LS requires the presence of clinical and radiological features, as well as biochemical evidence of abnormal energy metabolism. The underlying causes of these symptoms are commonly rooted in defects in the oxidative phosphorylation (OXPHOS) pathway or the pyruvate dehydrogenase complex (PDHc). Several genes linked to mitochondrial function, energy production, and LS have been identified, including NUDFA9, Complex II (CII)(flavoprotein subunit), Complex III (CIII)(cytochrome b subunit), and Complex IV (CIV) [3].

In the mitochondrial membrane, coenzyme Q10 (CoQ10) is a naturally occurring lipid-based benzoquinone that plays a critical role in electron transport as an electron acceptor, as well as protecting lipids from oxidation in the plasma membrane [4,5]. However, imbalanced CoQ10 levels can compromise mitochondrial function, resulting in reduced antioxidant activity and increased oxidative damage. This emphasizes the importance of maintaining optimal levels of CoQ10 [6]. Previous findings suggest that CoQ10 could be a promising therapy for patients with Leigh syndrome, and further research may be warranted to confirm these results [7,8].

DQAsomes, or dequalinium-based liposome-like vesicles, have emerged as a promising tool for mitochondrial targeting in the context of coenzyme Q10 (CoQ10) delivery. DQAsomes are known for their ability to target and fuse with the mitochondrial membrane, making them effective in delivering therapeutic agents to the mitochondria [9]. Furthermore, DQAsomes have been demonstrated to be effective in targeting mitochondria, as evidenced by their ability to enhance cancer cell death when used for the selective delivery of chemotherapeutic agents [10]. The positive charge of DQAsomes allows for easy condensation with the highly negatively charged backbone of DNA and DNA plasmids through electrostatic interactions, making them a potential candidate for mitochondrial gene delivery systems [11,12].

It has been established that arginine has potential advantages in patients with LS. Including acute arginine supplementation as a part of LS management can be a constructive approach to improve the overall therapeutic outcomes. The study that was conducted with Hristina et al. provided conclusive evidence that l-arginine can significantly enhance the activity of CoQ10 [13].

We focused on the respiratory chain complex I, specifically on the role played by the NDUFS4 gene, which encodes the NADH: ubiquinone oxidoreductase subunit S4 (NDUFS4) of complex I, including the subunit NDUFA9 [[14], [15], [16]]. To target this mutation-related disease, we developed CoQ10-loaded DQAsomes to solve the challenges of CoQ10's poor solubility and low bioavailability. We manufacture by using experimental design to evaluate formulation parameters. After characterization in terms of size, PDI, encapsulation efficiency %, morphologic formation of DQAsomes, and in-vitro release assays, we optimized formulations. To evaluate LS, NDUFS4 knockout mouse model was used. To investigate the cellular uptake of mitochondrial CoQ10, mitochondria were isolated from knockout mice's lungs. Subsequently, the combination of quantitative and qualitative assessments using Immunoblot and ELISA techniques to analyze the mitochondrial proteins NDUFA9, CII, CIII, and COX-II were employed. This multifaceted approach allowed us to comprehensively evaluate the efficacy and impact of CoQ10-loaded DQAsomes on both the micellar structure and mitochondrial function in the context of the NDUFS4 knockout mouse model.