Elucidating the Berberine-Induced Epigenetic Regulation of Nrf2 Signaling Axis and Its Targeted Delivery via Folate- Functionalized Bovine Serum Albumin Nanocarriers Against Glioblastoma

Abstract

Glioblastoma multiforme (GBM) is recognized as one of the most aggressive and fatal forms of brain cancer, characterized by rapid proliferation, invasive nature, and a poor prognosis. The conventional treatment typically encompasses a combination of surgical resection, radiotherapy, and subsequent chemotherapy using primarily temozolomide. Therapeutic resistance and limited treatment efficacy highlight the need for alternatives, with natural phytochemicals showing promise for their anti-cancer properties. Among phytochemicals, berberine (BER), a naturally occurring isoquinoline alkaloid, has exhibited considerable anti-cancer activity. Our study demonstrates that berberine’s mode of action involves multiple pathways, including triggering apoptosis, inhibiting cel migration, inducing cell cycle arrest, and causing DNA damage, which is primarily mediated through mitochondrial dysfunction facilitated by reactive oxygen species generation. Further, it was found that berberine downregulates the Nrf2 antioxidant signaling pathway in GBM cells. The mechanistic analysis reveals that berberine decreases the expression of KDM6B, resulting in an increased occupancy of H3K27me3 at the Nrf2 promoter, suppressing downstream signaling of the Nrf2 pathway. These findings highlight berberine's potential to promote ROS-mediated cell death and inhibit metastasis- associated pathways, establishing it as a promising therapeutic candidate for GBM treatment. Although berberine exhibits notable anti-cancer potential, its clinical application is significantly constrained by limited solubility, rapid metabolic degradation, and poor bioavailability. A novel drug delivery system employing bovine serum albumin nanoparticles (BSA NPs) was developed to encapsulate berberine, improving its stability and bioavailability. The FESEM and TEM analysis of these BER-BSA NPs revealed a spherical morphology, and further characterizations indicated the successful encapsulation of berberine within the BSA matrix. Hemocompatibility assays confirmed the formulation's biocompatibility, exhibiting no adverse responses with blood components, while in vitro release studies revealed a sustained drug release profile, ensuring gradual release of BER over time. Further, in vitro cell culture evaluations in LN229 cells indicated that BER-BSA NPs produced more significant cytotoxic effects than free berberine, suggesting that the nanoparticle formulation enhances the therapeutic potency of berberine. Additionally, enhanced inhibition of cell migration, increased apoptosis, nuclear condensation, reduction of mitochondrial membrane potential, and elevated ROS production were observed. To enhance the glioblastoma-specific targeting capability of the Sayantan Ghosh (519LS1009), Ph.D. Thesis, NIT Rourkela delivery system, folic acid (FA) was conjugated to BSA nanoparticles, taking advantage of the overexpression of folate receptors in glioblastoma cells. The resulting folate-conjugated nanoparticles (FA-BER-BSA NPs) maintained a spherical shape, and the successful conjugation of folic acid to BER-BSA NPs was validated through FTIR and UV-Vis spectroscopy, with XRD and DSC confirming their amorphous nature. The in vitro evaluations on LN229 cells showed that these FA-BER-BSA NPs possessed superiorcytotoxic effects compared to their non-targeted ones. The enhanced cytotoxicity was further corroborated with enhanced inhibition of cell migration, nuclear condensation, ROS generation, mitochondrial membrane damage, apoptosis induction, and cell cycle arrest. Subsequently, enhanced cellular uptake of FA-BER-BSA NPs was evidenced by fluorescence microscopy and flow cytometry analysis conducted with LN229 monolayers and three-dimensional tumor spheroids. The study demonstrates the efficacy and safety of FA-BER-BSA nanoparticles as a targeted drug delivery system, combining folic acid’s precision targeting with berberine’s anti-cancer activity, offering a promising approach for glioblastoma treatment in precision oncology.