Targeted Association and Intracellular Delivery of Nanocargoes into Primary T Lymphocytes via Interleukin-2 Receptor-Mediated Endocytosis,Bioconjugate Chemistry

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Targeted Association and Intracellular Delivery of Nanocargoes into Primary T Lymphocytes via Interleukin-2 Receptor-Mediated Endocytosis
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2021-07-09 , DOI: 10.1021/acs.bioconjchem.1c00212
V S S Abhinav Ayyadevara ₁ , Armin Ahmadi ₂ , Kyung-Ho Roh _{1,

2}

Affiliation

Despite the tremendous progress in immunotherapy regimens using T cells, efforts to modulate the functions of T cells are still significantly hampered by the lack of reliable methods to deliver various cargoes into the T cells. This ongoing challenge originates from the intrinsic resistance of T cells in taking up exogenous materials. Here, we strategically aimed to hijack the natural endocytosis of Interleukin-2 (IL2) by the activated T cells for the targeted association and intracellular delivery of cargoes in varying sizes. First, we carefully characterized the fluctuations in the expression levels of IL2 receptor (IL2R) subunits (CD25, CD122, and CD132) during the murine primary T cell cultures over 12 days. We identified the highest fraction of T cells that would express the high-affinity trimeric IL2R on Day 3. By examining the association and uptake efficiencies of IL2 molecules that are biotinylated via either random lysine-targeting chemical reaction (using NHS-PEG4-Biotin) or site-specific enzymatic modification (using Avitag sequence), we demonstrated that the most efficient delivery of cargo can be achieved by C-terminal conjugation. Upon confirmation of successful delivery of a small model cargo, streptavidin, we employed superparamagnetic iron oxide nanoparticles (SPIONs) as bigger model cargoes having core diameters of 50, 100, and 200 nm. We examined the association and intracellular delivery of the IL2-conjugated nanocargoes using flow cytometry, confocal laser scanning microscopy, and transmission electron microscopy. While cargoes of all tested sizes were successfully associated with the IL2R-expressing T cells in comparable efficiencies, the uptake efficiencies were inversely proportional to the sizes of the cargoes. Nevertheless, our current definitive report confirms that nanocargoes with a practical maximum size limit around 100–200 nm can be intracellularly delivered into activated primary T cells using IL2R-mediated endocytosis, which opens a new horizon for engineering and manufacturing of various T cell immunotherapeutics.

中文翻译：

纳米货物通过白细胞介素 2 受体介导的内吞作用靶向结合和细胞内递送至原代 T 淋巴细胞

尽管使用 T 细胞的免疫治疗方案取得了巨大进展，但由于缺乏将各种货物输送到 T 细胞中的可靠方法，调节 T 细胞功能的努力仍然受到严重阻碍。这一持续的挑战源于 T 细胞在吸收外源材料方面的固有阻力。在这里，我们战略性地旨在通过激活的 T 细胞劫持白细胞介素 2 (IL2) 的自然内吞作用，用于不同大小的货物的靶向结合和细胞内递送。首先，我们仔细表征了 12 天小鼠原代 T 细胞培养过程中 IL2 受体 (IL2R) 亚基（CD25、CD122 和 CD132）表达水平的波动。我们确定了在第 3 天表达高亲和力三聚体 IL2R 的 T 细胞比例最高。通过检查通过随机赖氨酸靶向化学反应（使用 NHS-PEG4-生物素）或位点特异性酶促修饰（使用 Avitag 序列）生物素化的 IL2 分子的结合和吸收效率，我们证明了最有效的货物输送可以通过 C 端结合来实现。在确认成功交付小模型货物链霉亲和素后，我们采用超顺磁性氧化铁纳米粒子 (SPION) 作为更大的模型货物，其核心直径为 50、100 和 200 nm。我们使用流式细胞术、共聚焦激光扫描显微镜和透射电子显微镜检查了 IL2 偶联纳米货物的关联和细胞内递送。虽然所有测试大小的货物都以可比的效率成功地与表达 IL2R 的 T 细胞相关联，吸收效率与货物的大小成反比。尽管如此，我们目前的最终报告证实，实际最大尺寸限制在 100-200 nm 左右的纳米货物可以使用 IL2R 介导的内吞作用在细胞内递送到活化的原代 T 细胞中，这为各种 T 细胞免疫治疗剂的工程和制造开辟了新的视野。

更新日期：2021-08-19

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