Engineered Flagellin Biomaterial as a Pattern Recognition Receptor Signal Activating Immunostimulants and Controlled Release of Boronated Anti-Cancer Drugs

Targeting glioblastoma, a highly aggressive brain tumor with limited treatment options, demands innovative strategies that address tumor growth and its immunosuppressive microenvironment. Glioblastoma exhibits high levels of toll-like receptor 5 (TLR5) expression, making it a promising target for therapeutic intervention. This study investigates flagellin, a TLR5 agonist and the primary component of bacterial flagella, as a platform for immune activation and targeted drug delivery. TLR5 activation by flagellin triggers the MyD88-dependent signaling pathway, promoting antigen-presenting cell maturation and proinflammatory cytokine secretion. A pH-sensitive catechol functional group was genetically engineered into the system via misaminoacylation to enhance therapeutic efficacy. This modification facilitated the self-assembly of the flagellin derivative and enabled efficient covalent conjugation with the boron-based anticancer drug Ixazomib, allowing for targeted drug release under physiological conditions. Receptor specificity revealed that congener monomeric Flagellin derivative demonstrated a strong binding affinity to TLR5 (<i>K</i>_d = 0.22 μM), while its polymeric form remained inactive. This selective activation reduces off-target immune responses, highlighting the potential of this bioengineered system for targeted therapeutic applications. TLR5 activation by a monomeric ligand was validated through upregulation of TLR5 pathway genes and significant inhibition of glioblastoma cell viability in vitro. Further, 3D tumor spheroid validation of ligand-Ixazomib conjugate exhibited efficient drug release in the acidic tumor microenvironment. It resulted in a 70% reduction in 3D tumor spheroid viability and an 80% inhibition of 20S proteasome activity. This dual-action approach of selective immune activation and precise drug delivery highlights the potential of flagellin derivative-based therapeutics for glioblastoma and other TLR5-expressing tumors.