Huang Yingbin, Li Yihu, He Rui, Dong Shuyi, Zhao Zheng, Jiao Xingyuan
Organ Transplantation Center, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China.
Department of Hepatobiliary Surgery, The Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, China.
Front Bioeng Biotechnol. 2024 Nov 4;12:1433126. doi: 10.3389/fbioe.2024.1433126. eCollection 2024.
Cytotoxic agents have shown limited benefits in hepatocellular carcinoma (HCC), mediated in part by the lack of targeting. As cell-penetrating peptides (CPPs) are capable of delivering various biologically active molecules into cells, including protein, peptides, small chemo-drugs, and nucleic acid with or without targeting, we developed T22-PE24, a CXCR4-targeted self-assembling cytotoxic nanotoxin, to effectively induce HCC pyroptosis.
T22 incorporating EGFP) or PE24 was purified from DE3 bacterial cells and characterized using transmission electron microscopy, the Zetasizer Nano, and SEC-HPLC. The internalization effect of T22-EGFP was detected by flow cytometry system (FCS) in CXCR4/LM3() HCC cells. The CCK8, lactate dehydrogenase (LDH) release, Western blot, and nude mice HCC models were used to estimate the cell viability of T22-PE24. The complete-immunity HCC tumor-bearing mice model was used to assess the immune response of T22-PE24.
The round shape under transmission electron microscopy, 49.4 nm hydrodynamic diameter, and -33.33 mV zeta potential indicated that T22-PE24 self-assembled into nanoparticles. T22 incorporating EGFP selectively internalized in CXCR4 HCC cells and showed no accumulation in CXCR4-knockout HCC cells. The T22-PE24 nanotoxin induced HCC pyroptosis via the caspase-3/GSDME signaling pathway and suppressed tumor growth in the absence of histological alterations in normal organs. Using the complete-immunity HCC tumor-bearing mice model, we found that T22-PE24 nanotoxin effectively induces the global reprogramming of cell components of the immune tumor microenvironment, leading to enhanced antitumor effects compared to those observed in immunodeficient mice.
Our findings demonstrate the activation of the innate immune response in HCC by inducing pyroptosis with T22-PE24 nanotoxin treatment and support an implementation of this strategy for HCC treatment.
细胞毒性药物在肝细胞癌（HCC）中的益处有限，部分原因是缺乏靶向性。由于细胞穿透肽（CPPs）能够将各种生物活性分子递送至细胞内，包括蛋白质、肽、小分子化学药物以及有或无靶向性的核酸，我们开发了T22-PE24，一种靶向CXCR4的自组装细胞毒性纳米毒素，以有效诱导肝癌细胞焦亡。
从DE3细菌细胞中纯化出包含EGFP的T22或PE24，并使用透射电子显微镜、Zetasizer Nano和SEC-HPLC对其进行表征。通过流式细胞仪系统（FCS）在CXCR4/LM3（）肝癌细胞中检测T22-EGFP的内化效果。使用CCK8、乳酸脱氢酶（LDH）释放、蛋白质印迹法以及裸鼠肝癌模型来评估T22-PE24的细胞活力。使用完全免疫的荷肝癌小鼠模型来评估T22-PE24的免疫反应。
透射电子显微镜下呈圆形、流体动力学直径为49.4nm以及ζ电位为-33.33mV表明T22-PE24自组装成纳米颗粒。包含EGFP的T22在CXCR4肝癌细胞中选择性内化，在CXCR4基因敲除的肝癌细胞中无积累。T22-PE24纳米毒素通过半胱天冬酶-3/GSDME信号通路诱导肝癌细胞焦亡，并在正常器官无组织学改变的情况下抑制肿瘤生长。使用完全免疫的荷肝癌小鼠模型，我们发现T22-PE24纳米毒素有效地诱导免疫肿瘤微环境中细胞成分的整体重编程，与免疫缺陷小鼠相比，导致抗肿瘤作用增强。
我们的研究结果表明，通过用T22-PE24纳米毒素治疗诱导细胞焦亡可激活肝癌中的先天免疫反应，并支持将该策略用于肝癌治疗。
