Injectable thermo-responsive hydrogel scaffold for enhanced delivery of second-generation induced neural stem cells for treatment of glioblastoma

Glioblastoma (GBM) remains a highly aggressive brain tumor with poor prognosis despite surgical resection and standard chemoradiation. Induced neural stem cell (iNSC)-based therapies offer a promising strategy owing to their inherent tumor-homing ability and capacity to deliver therapeutics selectively to the tumor site; however, their poor retention within the tumor resection cavity limits clinical potential. Herein, we evaluated second-generation TRAIL-secreting iNSCs (hiNeuroS) in combination with a biodegradable, thermo-responsive chitosan hydrogel for localized and sustained delivery of high densities of hiNeuroS. Direct comparisons with first-generation iNSCs (hiNSCs) demonstrated that hiNeuroS achieved faster and more extensive tumor cell kill across multiple tumor-to-therapeutic cell ratios in both U87 and patient-derived GBM8 models. Injectable chitosan scaffolds supported stem cell densities up to 2 \(\:\times\:\)10⁷ cells/mL, maintained > 90% viability, preserved scaffold microstructure, and maintained rapid gelation at physiological conditions. Gravimetric analysis revealed stable 30-day mass change profiles, with minimal net mass loss indicating preserved scaffold integrity. Moreover, encapsulated hiNeuroS retained their migratory capacity and sustained TRAIL secretion, inducing significant tumor cell death in both GBM models. Collectively, these findings demonstrate that hiNeuroS maintain functional potency in GBM and are compatible with scaffold-based delivery. This work provides a foundation for future in vivo studies to assess scaffold-mediated retention, persistence, and therapeutic efficacy in an established GBM mouse model, supporting the development of an injectable hydrogel platform for next-generation cell-based therapies for treatment of GBM and other malignant tumors.