白细胞介素 12 过表达纳米颗粒通过诱导 ICD 和激活树突状细胞、CD8 T 细胞和 CD4 T 细胞来抑制黑色素瘤的增殖。

IL-12-Overexpressed Nanoparticles Suppress the Proliferation of Melanoma Through Inducing ICD and Activating DC, CD8 T, and CD4 T Cells.

机构信息

Department of Biochemistry and Molecular Biology, Binzhou Medical University, YanTai, Shandong, 264003, People's Republic of China.

Shandong Laboratory of Advanced Materials and Green Manufacturing, Yantai, Shandong, 264000, People's Republic of China.

出版信息

Int J Nanomedicine. 2024 Mar 18;19:2755-2772. doi: 10.2147/IJN.S442446. eCollection 2024.

DOI:10.2147/IJN.S442446

PMID:38525008

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10959451/

Abstract

PURPOSE

The drug resistance and low response rates of immunotherapy limit its application. This study aimed to construct a new nanoparticle (CaCO-polydopamine-polyethylenimine, CPP) to effectively deliver interleukin-12 (IL-12) and suppress cancer progress through immunotherapy.

METHODS

The size distribution of CPP and its zeta potential were measured using a Malvern Zetasizer Nano-ZS90. The morphology and electrophoresis tentative delay of CPP were analyzed using a JEM-1400 transmission electron microscope and an ultraviolet spectrophotometer, respectively. Cell proliferation was analyzed by MTT assay. Proteins were analyzed by Western blot. IL-12 and HMGB1 levels were estimated by ELISA kits. Live/dead staining assay was performed using a Calcein-AM/PI kit. ATP production was detected using an ATP assay kit. The xenografts in vivo were estimated in C57BL/6 mice. The levels of CD80/CD86, CD3/CD4 and CD3/CD8 were analyzed by flow cytometry.

RESULTS

CPP could effectively express EGFP or IL-12 and increase ROS levels. Laser treatment promoted CPP-IL-12 induced the number of dead or apoptotic cell. CPP-IL-12 and laser could further enhance CALR levels and extracellular HMGB1 levels and decrease intracellular HMGB1 and ATP levels, indicating that it may induce immunogenic cell death (ICD). The tumors and weights of xenografts in CPP-IL-12 or laser-treated mice were significantly reduced than in controls. The IL-12 expression, the CD80/CD86 expression of DC from lymph glands, and the number of CD3/CD8T or CD3/CD4T cells from the spleen increased in CPP-IL-12-treated or laser-treated xenografts compared with controls. The levels of granzyme B, IFN-γ, and TNF-α in the serum of CPP-IL-12-treated mice increased. Interestingly, CPP-IL-12 treatment in local xenografts in the back of mice could effectively inhibit the growth of the distant untreated tumor.

CONCLUSION

The novel CPP-IL-12 could overexpress IL-12 in melanoma cells and achieve immunotherapy to melanoma through inducing ICD, activating CD4 T cell, and enhancing the function of tumor-reactive CD8 T cells.

摘要

目的

免疫疗法的耐药性和低反应率限制了其应用。本研究旨在构建一种新的纳米颗粒（CaCO-聚多巴胺-聚乙烯亚胺，CPP），通过免疫疗法有效递送白细胞介素 12（IL-12）并抑制癌症进展。

方法

使用 Malvern Zetasizer Nano-ZS90 测量 CPP 的粒径分布及其 zeta 电位。使用 JEM-1400 透射电子显微镜和紫外分光光度计分别分析 CPP 的形态和电泳迁移率。通过 MTT 分析检测细胞增殖。通过 Western blot 分析蛋白质。通过 ELISA 试剂盒估计 IL-12 和 HMGB1 水平。使用 Calcein-AM/PI 试剂盒进行死活染色试验。使用 ATP 测定试剂盒检测 ATP 产生。在 C57BL/6 小鼠中评估异种移植物。通过流式细胞术分析 CD80/CD86、CD3/CD4 和 CD3/CD8 的水平。

结果

CPP 可以有效地表达 EGFP 或 IL-12 并增加 ROS 水平。激光处理促进 CPP-IL-12 诱导死亡或凋亡细胞的数量增加。CPP-IL-12 和激光还可以进一步提高 CALR 水平和细胞外 HMGB1 水平，并降低细胞内 HMGB1 和 ATP 水平，表明其可能诱导免疫原性细胞死亡（ICD）。与对照组相比，CPP-IL-12 或激光处理的小鼠异种移植物的肿瘤和重量明显减少。与对照组相比，CPP-IL-12 处理或激光处理的异种移植物中淋巴结来源的 DC 的 IL-12 表达、CD80/CD86 表达以及脾中 CD3/CD8T 或 CD3/CD4T 细胞的数量增加。CPP-IL-12 处理小鼠血清中的颗粒酶 B、IFN-γ 和 TNF-α 水平增加。有趣的是，CPP-IL-12 处理小鼠背部局部异种移植物可有效抑制未处理的远处肿瘤的生长。