Decoding Nano-Bio Interactions: Gene Regulatory Networks in Advanced Drug Loading

Conventional treatments often face challenges such as the limited ability to penetrate the blood-brain barrier (BBB). The Doxorubicin-loaded graphene oxide/magnetite (DOX/GO/Fe₃O₄) nanocomplex offers a promising platform due to GO's high surface area and pH-sensitive release, and Fe₃O₄'s magnetic properties. This protocol describes the methodology for evaluating the cytotoxicity of free DOX versus the DOX/GO/Fe₃O₄ nanocomplex in the A-172 glioblastoma cell line, followed by advanced bioinformatics analysis to identify gene networks and indirect pathways that enhance the nanomaterial's biocompatibility. The methodology integrates the MTT assay, real-time PCR for apoptosis genes (Casp3, Bax, and Bcl-2), and advanced analysis, including protein-protein interaction (PPI) networking, clustering, and promoter motif analysis. The analysis indicated that miR-92a-2-5p is a potential therapeutic target for preventing myocardial damage and enhancing biocompatibility. The findings highlight key regulatory pathways that indirectly boost nanodrug biocompatibility through the modulation of secondary components like miRNAs and cellular stress mechanisms.