Bio produced nanoparticles load into extracellular vesicles and support horizontal transfer of therapeutic antimiR

The extracellular vesicles (EVs) serve for cell-to-cell communication and delivery of signaling molecules and they represent interesting approach in targeted therapy. This study focuses on an EV-mediated transfer of functionalized nanoparticles into recipient tumor cells. We used biologically produced gold nanoparticles (AuNPs) with known physical-chemical properties and decorated them with an inhibitory RNA targeting oncogenic microRNA-135b (AuNPs-antimiR 135 b) and transferrin (AuNPs-antimiR 135 b-Tf). The AuNPs were added to the breast cancer 4T1 cell line at a non-toxic dose 0.03 µg/µL. The 4T1-autologous EVs (with or without the AuNPs) were extracted and analyzed. The amount of endocytosed and excreted AuNPs was determined by graphite furnace atomic absorption spectrometry. The protein content was analyzed using sodium dodecyl-sulfate polyacrylamide gel electrophoresis and liquid chromatography with mass spectrometry (LC–MS). The fate of EVs and/or AuNPs was characterized using a Transmission Electron Microscopy and Zetasizer. The target-specific silencing effect of the antimir-135b was analyzed with a quantitative real-time PCR (qPCR). The AuNPs produced by Fusarium oxysporum possessed round shape, size of 5 nm and a zeta potential of -33 mV. We observed two types of EV populations: 60 nm and 250 nm. Out of the initial 58.7 µg of AuNPs added to the cells, 52.68 ± 0.65 µg were internalized and 38.64 ± 0.29 µg were found inside the EVs. The protein content in EV-AuNPs was higher compared to control EVs. LC-MS analysis showed that the AuNPs could alter the protein expression profile of the cancer cell. qPCR results revealed both EV-AuNPs-antimiR 135 b and EV-AuNPs-antimiR 135 b-Tf silenced target microRNA-135b, but higher efficacy was observed in the EV-AuNPs-antimiR 135 b-Tf group. Obtained data proved EV-mediated transportation of biological AuNPs bearing effector RNA among tumor cells. We also found the efficacy of loaded EVs is higher using AuNPs decorated with a tumor-specific ligand such as transferrin.