Sorafenib is one of the few effective first-line drugs approved for the treatment of advanced hepatocellular carcinoma (HCC). However, the development of drug resistance is common among individuals with HCC. Recent evidence indicated that the anticancer activity of sorafenib mainly relies on the induction of ferroptosis. Furthermore, in this study, genes that suppress ferroptosis, especially GPX4 and DHODH, were enriched in sorafenib-resistant cells and primary tissues and were associated with poor prognosis of HCC patients who received sorafenib treatment. Therefore, a new ferroptosis inducer comprising a multiplex small interfering RNA (multi-siRNA) capable of simultaneously silencing GPX4 and DHODH was created. Then, exosomes with high multi-siRNA loading and HCC-specific targeting were established by fusing the SP94 peptide and the N-terminal RNA recognition motif (RRM) of U1-A with the exosomal membrane protein Lamp2b. The results from the in vitro and in vivo experiments indicate that this tumor-targeting nano-delivery system (ExoSP94-lamp2b-RRM-multi-siRNA) could enhance sorafenib-induced ferroptosis and overcome sorafenib resistance. Taken together, HCC-targeted exosomes (ExoSP94-Lamp2b-RRM) could specifically deliver multi-siRNA to HCC tissues, enhance sorafenib-induced ferroptosis by silencing GPX4 and DHODH expression and consequently increase HCC sensitivity to sorafenib, which opens a new avenue for clinically overcoming sorafenib resistance from the perspective of ferroptosis.
Efficient therapeutic cargo encapsulation into engineered exosomes expressing
SP94-Lamp2b-RRM fusion protein
(A) Schematic diagram of the process of multi-siRNA encapsulation into exosomes via the SP94-Lamp2b-RRM fusion protein in HEK-293T cells. The SP94-Lamp2b-RRM fusion protein recruits multi-siRNA containing the sequence “AUUGCAC” to exosomes via RNA-RRM recognition. (B) HEK-293T cells transiently transfected with FITC-tagged multi-siRNA#1 or FITC-tagged multi-siRNA#2. Then, exosomes were collected for the flow cytometry assay (multi-siRNA#1, contains si-GPX4#1, si-DHODH#1 and the RRM binding sequence; multi-siRNA#2, contains si-GPX4#1 and si-DHODH#1 without the RRM binding sequence). (C, D) HEK-293T cells transiently transfected with FITC-tagged multi-siRNAs were cocultured with HepG-2 cells for 24 h. (C) Schematic illustration of HepG-2 and HEK-293T cell coculturing. (D) Representative confocal images of FITC (green) and F-actin (red) staining in HepG-2 cells. The nuclei were counterstained with DAPI (blue) (scale bar, 10 µm). (E) HepG-2 cells were treated with functionalized exosomes derived from HEK-293 T cells and containing SP94-Lamp2b-RRM, and Western blot assays were conducted to detect GPX4 and DHODH expression in HepG-2 cells. Scramble: 1*106 HEK-293T cells were treated with the same amount of scramble siRNA; multi-siRNA#2: 1*106 HEK-293T cells were treated with the same amount of multi-siRNA#2; multi-siRNA#1: 1*106 HEK-293T cells were treated with the same amount of multi-siRNA#1. After 24 h, the exosomes were collected and added to HepG-2 cells.