Representative histograms of the size distribution and spherical morphologies, as observed using SEM and a particle size analyzer, are given in figure 1C, D. siRNA, as a delivery platform to silence HIF-C2 immune checkpoints. This study used the TC-1 and EG7 tumor models to determine the potential therapeutic efficacy of the PLGA (PD-L1 siRNA+PD-1 siRNA)-NPs, on administration twice per week for 4 weeks. Moreover, we observed combination effect of PLGA (PD-L1 siRNA+PD-1 siRNA)-NPs and PLGA (antigen+adjuvant)-NPs using TC-1 and EG7 tumor-bearing mouse models. Results PLGA (PD-L1 siRNA+PD-1 siRNA)-NPs boosted the host immune reaction by restoring CD8+ T?cell function and promoting cytotoxic CD8+ T?cell responses. We demonstrated that this combination of NP-based therapeutic vaccine and PLGA (siRNA)-NPs resulted in significant inhibition of tumor growth compared with the control and antibody-based treatments (p 0.001). The proposed system significantly inhibited tumor growth compared with the antibody-based approaches. Conclusion Our findings suggest a potential combination approach for cancer immunotherapy using PLGA (PD-L1 siRNA+PD-1 siRNA)-NPs and PLGA (antigen+adjuvant)-NPs KLRD1 as novel immune checkpoint silencing brokers. strong class=”kwd-title” Keywords: vaccination, tumor microenvironment, immunotherapy, adoptive Background Cancer immunotherapy is an exciting therapeutic approach that has seen tremendous advances in recent years for various types of cancer.1 These approaches have focused on improving the HIF-C2 immunological function of cytotoxic T cells.2 Among novel immunotherapeutic strategies, immune checkpoint inhibitors such as antibody-based programmed death ligand 1 (anti-PD-L1) and programmed cell death 1 (anti-PD-1) have shown effectiveness against a large number of cancer types.3 PD-L1 is expressed on the surface of various cells, including macrophages and dendritic cells (DCs).4 In particular, PD-L1 is abundantly expressed in various tumor cells such as lung,5 colon,6 melanoma,7 and leukemic cells,8 and contributes to immune escape through its conversation with PD-1 on cytotoxic T cells.2 Moreover, recent studies have revealed the intrinsic expression of PD-1 in tumor cells. PD-1 can activate the expression of PD-L1 in tumor cells by means of cross-reactive stimulation, leading to the promotion of cell growth regardless of adaptive immunity. 9 10 Although anti-PD-L1 or anti-PD-1 blockade is currently approved to treat cancers, the overall response rates are limited to 20% of patients.11 More importantly, PD-L1 and PD-1 can be secreted from tumor cells into the tumor microenvironment in a soluble form, which may lead to reduced therapeutic efficacy for antibody blockades.12 The immunosuppressive function of secreted PD-L1 in blood circulation has been highly correlated with poor prognosis in multiple cancers.13 These secreted PD-L1 increase the complexity and diversity of the PD-1/PD-L1 signaling pathway composition.12 Eventually, the secretion of PD-1 or PD-L1 from tumor cells or T cells competitively interrupts the neutralizing activity of antibody-based blockade and induces HIF-C2 resistance.14 To overcome these hurdles, we hypothesized that immune checkpoint silencing might be a better strategy for enhancing therapeutic efficacy than immune checkpoint blocking. Therefore, in this study, we propose a small interfering RNA (siRNA)-based immune checkpoint silencing system. The advantages of the siRNA approach include target-specific gene silencing compared with other small molecules or antibody-based approaches.15 Despite the therapeutic potential of siRNA, siRNA delivery has led to issues in clinical applications due to its rapid degradation after intravenous injection. Therefore, an effective delivery platform is essential for the use of siRNA.16 We selected the poly(lactic-co-glycolic acid) (PLGA) nanoparticle (NP) system as the siRNA delivery platform, which is a particularly attractive option for clinical and biological applications, because of its low toxicity, low immunogenicity, biocompatibility, and biodegradability.17 18 To extend our concept, we combined the PLGA-NP-based therapeutic vaccine system with tumor antigens and adjuvants.19 20 This approach was selected because of the increased efficiency of intracellular delivery of tumor antigens and adjuvants to DCs, induction of DC maturation, and activation of cytotoxic CD8+ T cells HIF-C2 via antigen-specific cross-presentation, leading to increased tumor-specific cytotoxic CD8+ T?cell responses. In.