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The anionic cobaltabis (dicarbollide) [3,3′-Co(1,2-C2B9H11)2]−, [o-COSAN]−, is the most studied icosahedral metallacarborane. The sodium salts of [o-COSAN]− could be an ideal candidate for the anti-cancer treatment Boron Neutron Capture Therapy (BNCT) as it possesses the ability to readily cross biological membranes thereby producing cell cycle arrest in cancer cells. BNCT is a cancer therapy based on the potential of 10B atoms to produce α particles that cross tissues in which the 10B is accumulated without damaging the surrounding healthy tissues, after being irradiated with low energy thermal neutrons. Since Na[o-COSAN] displays a strong and characteristic ν(B-H) frequency in the infrared range 2.600–2.500 cm−1, we studied the uptake of Na[o-COSAN] followed by its interaction with biomolecules and its cellular biodistribution in two different glioma initiating cells (GICs), mesenchymal and proneural respectively, by using Synchrotron Radiation-Fourier Transform Infrared (FTIR) micro-spectroscopy (SR-FTIRM) facilities at the MIRAS Beamline of ALBA synchrotron light source. The spectroscopic data analysis from the bands in the regions of DNA, proteins, and lipids permitted to suggest that after its cellular uptake, Na[o-COSAN] strongly interacts with DNA strings, modifies proteins secondary structure and also leads to lipid saturation. The mapping suggests the nuclear localization of [o-COSAN]−, which according to reported Monte Carlo simulations may result in a more efficient cell-killing effect compared to that in a uniform distribution within the entire cell. In conclusion, we show pieces of evidence that at low doses, [o-COSAN]− translocates GIC cells' membranes and it alters the physiology of the cells, suggesting that Na[o-COSAN] is a promising agent to BNCT for glioblastoma cells. [ABSTRACT FROM AUTHOR]