Synergistic metabolic inhibition with 2-DG and metformin loaded electrospun scaffolds disrupts glioblastoma redox balance and glycolytic output

Glioblastoma (GBM) displays exceptional metabolic plasticity. Beyond the canonical Warburg Effect, which demonstrates preferential reliance on aerobic glycolysis despite sufficient oxygen, GBM can upregulate oxidative phosphorylation to maintain proliferation and redox balance under glycolytic stress. Targeting both pathways simultaneously represents a promising strategy to overcome this resilience. This study evaluates the synergistic effects of 2-deoxy-D-glucose (2-DG), a glycolytic inhibitor, and metformin, a mitochondrial complex I modulator, delivered through electrospun polycaprolactone scaffolds engineered for localized release at the tumor resection margin.Concentration range-finding was conducted by quantifying total metabolic activity via PrestoBlue and normalizing to total DNA content via CyQUANT. Mechanistic assays included mitochondrial membrane potential (ΔΨm) via JC-1 and reactive oxygen species via CellROX. Viability was assessed using Trypan Blue. Scaffolds were electrospun de novo, drug-loaded, and evaluated for structural integrity to determine feasibility for sustained local delivery. Parallel studies are extending this work to primary human astrocytes to assess differential sensitivity in noncancerous glial tissue.Dual treatment produced a synergistic reduction in metabolic index and viability that surpassed monotherapy and exceeded the response to temozolomide. Mechanistically, combined inhibition induced a hyperpolarized ΔΨm consistent with stress-driven proton-gradient accumulation under impaired ATP synthesis rather than improved mitochondrial function. Reactive oxygen species measurements indicated redox imbalance characteristic of mitochondrial stress. Trypan Blue analysis confirmed near-complete loss of viable GBM cells by day five. Electrospun scaffolds incorporated both agents effectively and retained fiber morphology compatible with controlled release.These findings indicate that simultaneous inhibition of glycolysis and mitochondrial respiration precipitates bioenergetic failure in glioblastoma cells through coordinated disruption of ATP production, ΔΨm homeostasis, and redox stability. Integrating this strategy into drug-releasing electrospun scaffolds offers a platform for localized metabolic therapy at resection margins while minimizing systemic exposure. Ongoing studies include astrocyte toxicity profiling, scaffold release kinetics, and application to three-dimensional tumorsphere models.