Simultaneous silencing of the A2aR and PD-1 immune checkpoints by siRNA-loaded nanoparticles enhances the immunotherapeutic potential of dendritic cell vaccine in tumor experimental models

Following various immunotherapies, lack of proper anti-tumor immune responses is considered a significant problem in novel cancer therapeutic approaches. The expression of inhibitory checkpoint molecules on tumor infiltrating T cells is one of the main reasons for the ineffectiveness of various immunotherapies. Therefore, we decided to inhibit two of the most important immune checkpoints expressed on tumor-associated T cells, PD-1 and A2aR. Ligation of PD-1 with PD-L1 and A2aR with adenosine significantly suppress T cell responses against tumor cells. Whitin tumors, specific inhibition of these molecules on T cells is of particular importance for successful immunotherapy as well as the elimination of treatment-associated side-effects. Thus, in this study, superparamagnetic iron oxide (SPION) nanoparticles (NPs) were covered by chitosan lactate (CL), functionalized with TAT peptide, and loaded with siRNA molecules against PD-1 and A2aR. Appropriate physicochemical properties of the prepared NPs resulted in efficient delivery of siRNA to tumor-derived T cells and suppressed the expression of A2aR and PD-1, ex vivo. T cell functions such as cytokine secretion and proliferation were considerably enhanced by the downregulation of these molecules which led to an increase in their survival time. Interestingly, treatment of CT26 and 4T1 mouse tumors with siRNA-loaded NPs not only inhibited tumor growth but also markedly increased anti-tumor immune responses and survival time. The results strongly support the efficacy of SPION-CL-TAT NPs loaded with anti-PD-1/A2aR siRNAs in cancer therapy and their further devel-opment for cancer patients in the near future.