Development and In Vitro Characterization of Milk-Derived Extracellular Vesicle-Mithramycin Formulations for Potential Glioma Therapy

• Published in: Molecular pharmaceutics Vol. 22; no. 6; pp. 2881 - 2894
• Main Authors: Patnam, Sreekanth, Singh, Anula Divyash, Ali, Mohammad Sadik, Thakur, Basant Kumar, Rengan, Aravind Kumar, Manda, Sasidhar Venkata
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 02.06.2025
• Subjects: Animals; Brain Neoplasms - drug therapy; Cell Line, Tumor; Drug Compounding - methods; Drug Delivery Systems - methods; Extracellular Vesicles - chemistry; Glioblastoma - drug therapy; Glioma - drug therapy; Humans; Milk - chemistry; Plicamycin - administration & dosage; Plicamycin - analogs & derivatives; Plicamycin - chemistry; Plicamycin - pharmacology; polyethylene glycol; glioma; mithramycin; milk-derived extracellular vesicles
• ISSN: 1543-8384; 1543-8392; 1543-8392
• DOI: 10.1021/acs.molpharmaceut.4c01189

Glioblastoma (GBM) is a highly aggressive brain tumor with resistance to conventional therapies. Mithramycin (Mit-A), a potent antitumor agent, has shown promise in several tumor types including, GBM. However, its clinical application is limited by toxicity. To address this, we explored the use of milk-derived extracellular vesicles (mEVs) as a delivery system to enhance the therapeutic efficacy of Mit-A. In this study, mEVs were isolated using a 3000 PEG precipitation method and confirmed their size, morphology, and stability through dynamic light scattering (DLS), transmission electron microscopy (TEM), and atomic force microscopy (AFM). The isolated vesicles with a size of 125.6 ± 2.78 nm, a polydispersity index (PDI) of 0.083 ± 0.02, and a ζ-potential of 15 ± 0.57 mV. The presence of typical EV markers such as TSG101, HSP70, and CD63 confirmed their purity. Encapsulation of Mit-A within mEVs led to a slight increase in size to 131.8 ± 6.9 nm, a PDI of 0.081 ± 0.006, and a decrease in ζ-potential to −17 ± 2.0 mV, with an encapsulation efficiency of 58% by the freeze–thaw method. The in vitro transepithelial transport assay revealed that mEV­(Mit-A) transported Mit-A more effectively than free Mit-A. The mEV­(Mit-A) formulation demonstrated excellent stability in simulated salivary and gastrointestinal fluids, with a sustained release of Mit-A observed over 24 h in vitro in PBS (pH 6.8). Furthermore, mEV­(Mit-A) formulations significantly inhibited glioma cell growth, and migration, and induced apoptosis, showing a 2-fold lower IC50 than free Mit-A, indicating superior efficacy. These findings suggest that mEVs represent a promising delivery vehicle for Mit-A, enhancing its potential as an effective treatment for glioblastoma.