Niosomal co-delivery of metformin and telaglenastat: A targeted nanotherapeutic strategy for breast cancer treatment and metastasis suppression in 2D and 3D models.

Overexpression of glucose transporter-1 (GLUT-1) and glutaminase-1 (GLS-1) underlies the metabolic dependence of breast cancer cells on glucose and glutamine. Exploiting this vulnerability, a dual-drug niosomal delivery system was developed for the encapsulation of metformin (MET) and telaglenastat (TEL) to concurrently modulate these metabolic pathways. The optimized formulation, prepared at a cholesterol:Span 60 ratio of 1:1, exhibited nanosized vesicles (79.7-494.6 nm) with low polydispersity (PDI < 0.3), confirming a uniform and suitable size distribution for efficient cellular uptake. The encapsulation efficiency (EE%) was high for both agents, notably 84.34 % for TEL and 67.93 % for MET. Flow-cytometric profiling revealed elevated GLUT-1 and GLS-1 expression relative to unstained controls, validating the rationale for dual-pathway targeting. Functionally, the niosomal formulations outperformed free and single-drug treatments across both two- and three-dimensional models. In 3D spheroids, free TEL (200 µM) and free MET (200 mM) resulted in cell viabilities of 44.97 % and 54.00 %, respectively, whereas TEL-NP and MET-NP reduced viability to 9.49 % and 22.14 %, representing 4.7-and 2.4-fold enhancements in cytotoxic efficacy. The dual formulation promoted extensive spheroid disintegration and maintained intratumoral penetration for up to 72 h. In wound-healing assays, significant inhibition of migration was observed, with residual wound areas of 97.07 % (MET-NP), 87.43 % (TEL-NP), and 93.49 % (combined MET-NP + TEL-NP). Metabolic analyses further substantiated the mechanistic effect of dual inhibition. Glutamine-glutamate quantification demonstrated a marked shift from the untreated profile (glutamate 87.77 % / glutamine 12.23-59.89 % / 40.11 %) following combined nanoparticle treatment, consistent with suppression of GLS-1-mediated glutaminolysis. Correspondingly, ROS-Glo™ H₂O₂ assays revealed decreased intracellular hydrogen peroxide in all treated groups, with the most pronounced reduction observed in the dual-NP combination, indicating attenuation of oxidative stress and restoration of redox balance. In conclusion, coordinated inhibition of GLUT-1- and GLS-1-driven metabolism via MET/TEL-loaded niosomes achieves enhanced cytotoxicity, durable spheroid penetration, and strong anti-migratory and metabolic modulatory effects. This stable co-delivery platform represents a promising nanotherapeutic strategy to overcome metabolic adaptability and treatment resistance in breast cancer.