Reinforcing the induction of immunogenic cell death by silibinin-loaded nanocarriers

Abstract

Introduction: Induction of Immunogenic cell death (ICD) is a potential approach for modulating immunosuppression in the tumor microenvironment (TME) and induction of potent antitumor immunity. Silibinin, a natural compound, causes cancer cells to undergo apoptosis and ICD. The development of drug-loaded nano-delivery systems has been used as a vital strategy for improving the tumor-targeted delivery and bioavailability of poorly water-soluble agents such as silibinin. Furthermore, encapsulation of ICD-inducing chemotherapeutics agents in nano-carriers has been shown to potentiate ICD induction by these agents. Based on these findings, encapsulation of silibinin in nano-micellar formulation is expected to enhance its effects on the induction of ICD and subsequent antitumor immune responses. Aim: The present study intended to examine whether silibinin-loaded nano-carriers can enhance ICD induction by silibinin in the B16F10 cancer cell line. Methods: Poly (ethylene glycol)-poly (benzyl-ε-caprolactone) (PEG-PBCL)-based micelles were incorporated with silibinin using the co-solvent evaporation method. Free and nanoparticulate silibinin were evaluated for their growth-inhibitory effects using the MTT assay. For the analysis of apoptosis, Annexin-PI was used. Calreticulin (CRT) exposure was measured using the flow cytometry technique. Western blotting was conducted to examine the levels of elf2α, which plays a role in the ICD pathway. HSP90 and ATP levels were detected by their specific detection kit.Results: Compared to the free drug, silibinin-loaded nanocarriers significantly increased the induction of ICD and apoptosis in B16F10 cells. Because of these changes, we observed an increase in damage-associated molecular patterns (DAMPs) levels, including CRT, HSP90, and ATP. We also detected an increased expression of p-elf-2α/ elf-2α in B16F10 cells treated with silibinin-loaded nanocarriers compared to the group receiving free silibinin. Conclusion: Based on our research, encapsulation of silibinin in polymeric micelles can potentiate the effects of this drug in induction of ICD and apoptosis in B16F10 melanoma cells.