The efficacy of immune checkpoint blockade (ICB), in the treatment of hepatocellular carcinoma (HCC), is limited due to low levels of tumor-infiltrating T lymphocytes and deficient checkpoint blockade in this immunologically "cool" tumor. Thus, combination approaches are needed to increase the response rates of ICB and induce synergistic antitumor immunity. Herein, we designed a pH-sensitive multifunctional nanoplatform based on layered double hydroxides (LDHs) loaded with siRNA to block the intracellular immune checkpoint NR2F6, together with the asynchronous blockade surface receptor PD-L1 to induce strong synergistic antitumor immunity. Moreover, photothermal therapy (PTT) generated by LDHs after laser irradiation modified an immunologically “cold” microenvironment to potentiate Nr2f6-siRNA and anti-PD-L1 immunotherapy. Flow cytometry was performed to assess the immune responses initiated by the multifunctional nanoplatform. Under the slightly acidic tumor extracellular environment, PEG detached and the re-exposed positively charged LDHs enhanced tumor accumulation and cell uptake. The accumulated siRNA suppressed the signal of dual protumor activity in both immune and H22 tumor cells by silencing the NR2F6 gene, which further reduced the tumor burden and enhanced systemic antitumor immunity. The responses include enhanced tumor infiltration by CD4+ helper T cells, CD8+ cytotoxic T cells, and mature dendritic cells; the significantly decreased level of immune suppressed regulator T cells. The therapeutic responses were also attributed to the production of IL-2, IFN-γ, and TNF-α. The prepared nanoparticles also exhibited potential magnetic resonance imaging (MRI) ability, which could serve to guide synergistic immunotherapy treatment. In summary, the three combinations of PTT, NR2F6 gene ablation and anti-PD-L1 can promote a synergistic immune response to inhibit the progression of primary HCC tumors and prevent metastasis. This study can be considered a proof-of-concept for the targeting of surface and intracellular immune checkpoints to supplement the existing HCC immunotherapy treatments.

中文翻译：

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超薄层状双氢氧化物介导的光热疗法联合异步阻断PD-L1和NR2F6抑制肝细胞癌

免疫检查点阻断 (ICB) 在治疗肝细胞癌 (HCC) 中的功效是有限的，因为这种免疫“冷”肿瘤中肿瘤浸润性 T 淋巴细胞水平低和检查点阻断不足。因此，需要联合方法来提高 ICB 的反应率并诱导协同抗肿瘤免疫。在此，我们设计了一种基于载有 siRNA 的层状双氢氧化物 (LDH) 的 pH 敏感多功能纳米平台，以阻断细胞内免疫检查点 NR2F6，并与异步阻断表面受体 PD-L1 一起诱导强协同抗肿瘤免疫。此外，激光照射后 LDHs 产生的光热疗法 (PTT) 改变了免疫学上的“冷”微环境，以增强 Nr2f6-siRNA 和抗 PD-L1 免疫疗法。进行流式细胞术以评估多功能纳米平台引发的免疫反应。在微酸性的肿瘤细胞外环境下，PEG脱离，重新暴露的带正电荷的LDHs增强了肿瘤的积累和细胞摄取。积累的 siRNA 通过沉默 NR2F6 基因来抑制免疫和 H22 肿瘤细胞中双重促癌活性的信号，从而进一步降低肿瘤负荷并增强全身抗肿瘤免疫。反应包括 CD4+ 辅助 T 细胞、CD8+ 细胞毒性 T 细胞和成熟树突状细胞增强的肿瘤浸润；免疫抑制调节 T 细胞水平显着降低。治疗反应也归因于 IL-2、IFN-γ 和 TNF-α 的产生。制备的纳米颗粒还表现出潜在的磁共振成像（MRI）能力，可用于指导协同免疫治疗。综上所述，PTT、NR2F6基因消融和抗PD-L1的三种组合可以促进协同免疫反应，抑制原发性HCC肿瘤的进展，防止转移。这项研究可以被认为是针对表面和细胞内免疫检查点的概念验证，以补充现有的 HCC 免疫治疗。