Folate receptor-α targeted delivery of berberine <i>via</i> folate-functionalized bovine serum albumin nanocarriers to enhance intracellular internalization and suppress 3D spheroid formation as a pioneering therapeutic modality for glioblastoma

Glioblastoma multiforme, the most aggressive brain malignancy, exhibits poor prognosis and intrinsic resistance to standard therapies, often limited by systemic toxicity and suboptimal efficacy. Berberine, a bioactive isoquinoline alkaloid, exhibits potent anti-glioma activity; however, its clinical utility is hindered by rapid metabolism, low bioavailability, and poor penetration across the blood-brain barrier. To overcome these challenges, we designed folic acid-functionalized BSA nanoparticles for targeted and sustained delivery of berberine to glioma cells. Folic acid modification facilitated receptor-mediated endocytosis <i>via</i> overexpressed folate receptors in glioblastoma cells, enhancing specificity and therapeutic potency. FA-BER-BSA NPs were synthesized <i>via</i> desolvation method using EDC instead of glutaraldehyde to mitigate potential human and environmental toxicity. The resulting nanoparticles exhibited a spherical morphology with an average diameter of 120-140 nm, as confirmed by FESEM and TEM analyses. Comprehensive physicochemical characterization, conducted through XRD, DSC, FTIR, and UV-vis spectroscopy, confirmed the successful conjugation of folic acid to the nanoparticles. <i>In vitro</i> studies in LN229 cells demonstrated that FA-BER-BSA NPs exhibited higher cytotoxicity compared to BER-BSA NPs, accompanied by enhanced inhibition of cell migration, ROS generation, nuclear condensation, mitochondrial membrane potential disruption, apoptosis induction, and cell cycle arrest. Fluorescence microscopy and flow cytometry confirmed efficient internalization of FA-BER-BSA NPs in (FR)+ LN229 cells and 3D tumor spheroids, with negligible effects on (FR)- HaCaT cells. Notably, FA-BER-BSA NPs significantly suppressed the growth of 3D spheroids compared to BER. These findings highlight the potential of the nanoparticles as a promising targeted therapeutic agent for glioblastoma, offering enhanced tumor-specific delivery and improved anticancer efficacy.