Nanodiamonds Interact with Primary Human Macrophages and Dendritic Cells Evoking a Vigorous Interferon Response

Nanodiamonds (NDs) display several attractive features rendering them useful for medical applications such as drug delivery. However, the interactions between NDs and the immune system remain poorly understood. Here, we investigated amino-, carboxyl-, and poly(ethylene glycol) (PEG)-terminated NDs with respect to primary human immune cells. We applied cytometry by time-of-flight (CyToF) to assess the impact on peripheral blood mononuclear cells at the single-cell level, and observed an expansion of plasmacytoid dendritic cells (pDCs) which are critically involved in antiviral responses. Subsequent experiments demonstrated that the NDs were actively internalized, leading to a vigorous type I interferon response involving endosomal Toll-like receptors. ND-NH2 and ND-COOH were more potent than ND-PEG, as evidenced by using TLR reporter cell lines. Computational studies demonstrated that NDs interacted with the ligand-binding domains of TLR7 and TLR9 with high affinity though this was less pronounced for ND-PEG. NDs with varying surface functionalities were also readily taken up by macrophages. To gain further insight, we performed RNA sequencing of a monocyte-like cell line exposed to NDs, and found that the phagosome maturation pathway was significantly affected. Indeed, evidence for lysosomal hyperacidification was obtained in dendritic cells and macrophages exposed to NDs. Moreover, using a reporter cell line, NDs were found to impinge on autophagic flux. However, NDs did not affect viability of any of the cell types studied. This study has shown that NDs subvert dendritic cells leading to an antiviral-like immune response. This has implications not only for drug delivery but also for anticancer vaccines using NDs.纳米金刚石（Nanodiamonds, NDs）具备多项优异特性，使其可用于药物递送等医疗领域。然而，目前学界对纳米金刚石与免疫系统的相互作用机制仍知之甚少。本研究针对表面修饰氨基、羧基及聚乙二醇（poly(ethylene glycol), PEG）的纳米金刚石，探究其与原代人免疫细胞的相互作用。研究采用飞行时间流式细胞术（Cytometry by Time-of-Flight, CyToF）在单细胞层面评估其对外周血单个核细胞的影响，观测到浆细胞样树突状细胞（plasmacytoid dendritic cells, pDCs）发生扩增——这类细胞在抗病毒免疫应答中发挥关键作用。后续实验证实，纳米金刚石可被细胞主动摄取，进而引发强烈的I型干扰素应答，该过程依赖于内体Toll样受体。通过Toll样受体报告细胞系实验可知，氨基修饰纳米金刚石（ND-NH₂）与羧基修饰纳米金刚石（ND-COOH）的活性显著强于聚乙二醇修饰纳米金刚石（ND-PEG）。计算模拟研究表明，纳米金刚石可与Toll样受体7（TLR7）和Toll样受体9（TLR9）的配体结合结构域高亲和力结合，但聚乙二醇修饰纳米金刚石的该结合作用相对较弱。不同表面功能化的纳米金刚石同样可被巨噬细胞高效摄取。为进一步阐明作用机制，本研究对暴露于纳米金刚石的单核细胞样细胞系开展RNA测序，发现吞噬体成熟通路受到显著影响。此外，在暴露于纳米金刚石的树突状细胞与巨噬细胞中，研究人员观测到溶酶体过度酸化的现象。进一步利用报告细胞系实验发现，纳米金刚石会干扰自噬流。值得注意的是，本研究涉及的所有细胞类型的细胞活力均未受纳米金刚石影响。本研究证实，纳米金刚石可调控树突状细胞功能，诱导类抗病毒免疫应答，这一发现不仅为药物递送应用提供参考，也为基于纳米金刚石的抗癌疫苗研发带来启示。