Abstract

Background: The worldwide prevalence of Parkinson’s disease (PD) is increasing continuously, not only in elders but in youngers also. The PD prevalence is growing in developed as well as underdeveloped countries, and the present therapy lacks a stable cure. However, the golden drug of ‘levodopa’ is also sparked with several side effects including ‘on-off fluctuations.’ To overcome this barrier now researchers were focusing on formulation development process. Various sustained release formulation of levodopa has been developed to reduce its side effect and enhance duration of action. Therefore, we have the gold-containing medication auranofin (AUF) in its neuroprotective form. Its neuroprotective function in Alzheimer's disease and the rotenone model has been noted Previously. But lacuna associated is its low bioavailability. Therefore, research has developed AUF-loaded hybrid nanoparticles (AUFHNPs).

Objective: To explore the neuroprotective potential of AUFHNPs against the in-silico and in-vivo model of PD.

Methods: The CDOCKER software was utilized to perform the in-silico study and binding affinity of AUF with P13K (5DXT), AKT (1UNQ), GSK-3β (1Q41), Nrf2 (2DYH), and HO-1 (1N45). Then, AUFHNPs were prepared using ethanol ejection method and characterized for various parameters such as particle size, zeta potential, SEM/TEM and In-Vitro drug release assay. Following that in-vivo study was conducted on rats, intra-striatal unilateral injection of 6-OHDA (10μg/2μl) was given with the help of stereotaxic surgery. To confirm the occurrence of PD, an amphetamine challenge test was performed on 21st day. Then animals were treated with AUF (10mg/kg) and AUFHNPs (5 & 10mg/kg) for 21 days. All the behaviour (open field test, grip strength test, narrow beam walk) parameters were performed on the last day, and animals were sacrificed by cervical dislocation; brains were isolated and homogenized in PBS to perform various biochemical (GSH, SOD, and MDA), neuroinflammatory (IL-6, NFκB), apoptotic (caspase 3, Caspase 9), histological (H&E staining), immunohistochemistry (GFAP) and western blot (PI3K/AKT/ GSK-3β)  analysis.

Results: The In-Silico study of AUF results in the highest binding score of -61.1231 with GSK-3β, while also offered a good binding score with PI3K/AKT. The π-π interaction, hydrogen bonding, π-carbonyl bonding was observed between AUF and amino acid residues of various targets. The nanoparticles were successfully prepared and characterized; all the parameters were within specified limit. The In-vivo administration of AUF and AUFHNPs significantly restored all the motor and behaviour alterations observed in open field tests, narrow beam walks, and grip strength meter. It also restored nuclear morphology and GFAP expression in response to 6-OHDA. Also, increased expression of pPI3K/pAKT, followed by phosphorylation of GSK-3β.

Conclusion: The research found that the AUFHNPs were more significant than AUF alone and exerted neuroprotection via modulation of PI3K/AKT/GSK-3β signaling pathways.

Keywords: Auranofin, 6-OHDA, Parkinson’s disease, Nanoparticles, PI3K/AKT signaling