Bio-inspired Solid Lipid Nanoparticles for therapeutic siRNA delivery targeting the LRRK2 G2019S mutation in a Parkinson’s disease cell model.

Abstract

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons and the presence of Lewy bodies. A significant genetic contributor to PD is the LRRK2 G2019S mutation, which leads to increased kinase activity and cellular dysfunction. Current treatments for PD are primarily palliative, necessitating an innovative gene delivery system adapted to a curative function. This study aimed to develop a therapeutic strategy using siRNA-mediated gene silencing to target the LRRK2 G2019S mutation and mitigate its pathogenic effects. A preliminary study demonstrated the proof of principle using Ginkgo biloba leaf extract (EGB)-functionalized sphingomyelin-cholesterol solid lipid nanoparticles (EGB-PLL-SLNPs). These SLNPs were successfully bio-synthesized using EGB, facilitating a dual therapy with favourable properties from the EGB and lipid components. Safety, biocompatibility, cytotoxicity, and cellular uptake efficiency were assessed through caspase and 3-[4,5-dimetylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assays. The cellular uptake was assessed using the BLOCK-iT™Fluorescent Oligo to provide a means of visualization through its ability to fluoresce on the wild-type SH-SY5Y (neuroblastoma) and embryonic kidney (HEK293) cells, confirming biocompatibility and efficient internalization, with cell viability exceeding 90%. We further evaluated the therapeutic efficacy of EGB-PLL-SLNPs and compared them with the SLNPs without EGB (H2O-PLL-SLNPs) in LRRK2 G2019S mutated SH-SY5Y cells. The EGB-PLL-SLNPs reduced ROS (+) levels by 51.39%, compared to a 31.27% reduction by their H2O-based counterparts. The colour-based DNA damage assay showed that EGB-PLLSLNPs decreased total DNA damage by 49.90%, whereas the H2O-PLL-SLNPs achieved a 33.17% reduction. Additionally, EGB-PLL-SLNPs reduced kinase activity by 52.65%, surpassing the H2O-PLL-SLNPs. The LRRK2 G2019S gene silencing efficiency of the siRNAcomplexed EGB-PLL-SLNPs was particularly notable, significantly decreasing the kinase activity and associated cellular dysfunction. The consistent efficacy of the EGB-PLL-SLNPs can be attributed to the bioactive components in EGB, which provided additional antioxidant and DNA repair benefits. These components enhance the LNPs' properties, facilitating better cellular uptake and siRNA delivery, highlighting the dual therapeutic capabilities of EGB and the therapeutic siRNA. The pathogenic effects of the LRRK2 G2019S mutation were effectively mitigated, with the findings suggesting that the EGB-PLL-SLNPs are highly effective nano vehicles for managing PD, particularly in cases involving the LRRK2 G2019S mutation. This study proposes a novel strategy for using EGB-PLL-SLNPs as a promising treatment for PD and emphasizes the importance of natural products in advancing personalized medicine. The environmental safety, low toxicity, and cost-effectiveness of using EGB can make advanced therapeutics more accessible and sustainable for a broader range of neurodegenerative and genetic disorders.