Background: Glioblastoma, the most common malignant primary brain tumor in adults, continues to present a major therapeutic challenge, with a median survival of only 12–15 months and a 5-year survival rate below 2%. Despite aggressive treatment—including maximal surgical excision, radiation, and temozolomide (TMZ) chemotherapy—recurrent disease is nearly universal due to the tumor’s infiltrative nature. Objectives: To address the critical need for improved diagnostic and therapeutic strategies for glioblastoma multiforme (GBM), we have developed an innovative theranostic molecule, [^99mTc]Tc-AGT-7. Methods: AGT-7 integrates diagnostic and therapeutic modalities comprising [^99mTc]Tc-TF (a nuclear medicine imaging agent) and TMZ. The diagnostic component has been tailored to selectively accumulate in glioma mitochondria. A chelating moiety enables radiolabeling with technetium-99m (^99mTc) for precise Single-Photon Emission Computed Tomography (SPECT) imaging. The therapeutic arm includes the tethering of a TMZ moiety for localized cytotoxicity. Conclusions: In vitro studies illustrated that AGT-7 has potent cytotoxic effects in GBM cell lines (T98 and U87), with greater efficacy than TMZ, and toxicity assays in zebrafish embryos indicated a favorable safety profile. Biodistribution studies in CFW mice demonstrated that [^99mTc]Tc-AGT-7 exhibited a ~10-fold lower heart uptake compared to [^99mTc]Tc-TF, implying reduced off-target cardiac localization. This significantly lowers the risk of cardiotoxicity and enhances AGT-7’s potential as a glioma-targeted theranostic agent.

Glioblastoma, the most frequent malignant primary brain tumor in adults, presents a significant challenge in cancer treatment, with a mean survival of merely 12-15 months and a 5-year survival rate of less than 2%. Despite extensive research and current treatments, involving maximal surgical excision, radiation, and chemotherapy mainly with temozolomide (TMZ), recurrence remains nearly universal. This is attributable to its infiltrative nature and tumor cells may be found far from the surgical resection cav-ity. To address the critical need for improved diagnostic and therapeutic strategies for glioblastoma multiform (GBM), we have developed an innovative theranostic molecule, AGT-7, based on tetrofosmin, a compound used in nuclear medicine bioimaging, and TMZ. AGT-7 was specifically designed to enable simultaneous tumor imaging and tar-geted chemotherapy. Its diagnostic component was inspired by the ability of tetrofosmin to selectively accumulate in glioma mitochondria, driven by its positive charge and the negative membrane potential of these organelles. A chelating moiety allows for radio-labeling with technetium-99m (⁹⁹ᵐTc), enabling precise SPECT imaging. The therapeutic arm comprises a tethered TMZ moiety, delivering localized cytotoxic activity. In vitro studies demonstrated that AGT-7 exerts potent cytotoxic effects in GBM cell lines (T98 and U87), with superior therapeutic efficacy compared to TMZ alone. Toxicity assess-ment in zebrafish embryos confirmed a favorable safety profile. Furthermore, biodis-tribution studies in CFW mice showed that [⁹⁹ᵐTc]Tc-AGT-7 exhibited approximately tenfold lower heart uptake than [⁹⁹ᵐTc]Tc-tetrofosmin, a standard cardiac imaging agent, indicating a significant reduction in off-target cardiac localization. This reduced heart uptake is particularly advantageous, as it minimizes potential cardiotoxicity and en-hances the suitability of AGT-7 as a glioma-targeted theranostic agent.