Abstract
Efficient delivery is a major obstacle in oligonucleotide therapeutics for the treatment of gliomas. Endogenous small vesicles known as extracellular vesicles (EVs) hold potential to act as oligonucleotide delivery vehicles given their unique properties, such as low immunogenicity, innate stability, and ability to cross the blood-brain barrier (BBB). However, their insufficient targeting ability upon intravenous administration limits the clinical advancement. In this study, neural stem cell line ReNcell was cultured to release EVs. The cyclo(Arg-Gly-Asp-D-Tyr-Lys) peptide [c(RGDyK)], which exhibits high affinity to integrin αvβ3 on tumor vascular endothelial cells, was conjugated on the isolated EVs via bio-orthogonal click chemistry. In the syngeneic graft glioma mice model, the c(RGDyK)-conjugated EVs (cRGD-EV) targeted glioma after intravenous administration. Then, priming with radiation enhanced cRGD-EV accumulation in the tumor and decreased their entrapment in the liver and spleen significantly. Furthermore, small interfering RNAs (siRNAs) for immune checkpoint molecules programmed death-ligand 1 (anti-PD-L1) and CD47 (anti-CD47) were conjugated with cholesterol, and loaded into EV membrane by hydrophobic interaction simultaneously. After intravenous injection, the knockdown of target proteins in the tumor was found. Also, the number of infiltrated CD4+ and CD8+ T cells and M1/M2 rate of macrophages were increased, showing the up-regulated immune response in tumor. The injection of siRNAs-loaded cRGD-EV induced the regression of tumor growth in immuno-competent mice and significantly prolonged survival. In addition, no obvious liver and lung toxicity or tissue damage was observed in the treated mice. These results suggest a targeting delivery strategy for glioma based on EVs, and have implications for cancer immunotherapy.