尽管基于纳米颗粒的淋巴药物递送系统有望更好地治疗癌症、传染病和免疫疾病，但其临床转化受到递送效率低和转运机制不明确的限制。在这里，我们采用了三维（3D）淋巴管芯片，其特征是工程淋巴管（LV）能够排出包括纳米粒子在内的间质液。我们使用淋巴芯片装置测试了不同尺寸（30、50 和 70 nm）的 PLGA-b-PEG 纳米颗粒 (NP) 的淋巴引流。在这项研究中，我们发现较小的纳米颗粒（30 和 50 nm）比较大的纳米颗粒（70 nm）通过间隙空间的运输速度更快，正如预期的那样，但较小的纳米颗粒被淋巴内皮细胞（LEC）捕获并在其胞质内积累，延迟 NP 转运至淋巴管腔，这在较大的 NP 中未观察到。为了检查大小依赖性 NP 转运的机制，我们使用了四种抑制剂：dynasore、制霉菌素、阿米洛利和肾上腺髓质素，选择性阻断动力蛋白、小窝蛋白、巨胞饮介导的内吞作用和细胞连接介导的旁细胞转运。使用 dynasore 抑制动力增强了较小 NP（30 和 50 nm）进入淋巴管腔的转运，最大限度地减少了胞质积累，但对较大 NP 转运没有影响。有趣的是，制霉菌素对小窝蛋白的抑制减少了较大纳米颗粒的淋巴转运，而不影响较小纳米颗粒的转运，表明不同大小的纳米颗粒使用不同的内吞机制。阿米洛利对巨胞饮作用的抑制不会改变所有尺寸纳米颗粒的引流；然而，肾上腺髓质素的细胞旁运输抑制作用阻碍了各种大小的纳米颗粒的淋巴运输。我们进一步发现，较小的纳米颗粒被捕获在 Rab7 阳性的晚期淋巴内体中，以延迟其淋巴引流，而这可以通过动力抑制来逆转，这表明 Rab7 是增强较小纳米颗粒的淋巴递送的潜在靶标。我们的 3D 芯片淋巴管模型共同揭示了淋巴药物输送中依赖于尺寸的 NP 转运机制。

Although nanoparticle-based lymphatic drug delivery systems promise better treatment of cancer, infectious disease, and immune disease, their clinical translations are limited by low delivery efficiencies and unclear transport mechanisms. Here, we employed a three-dimensional (3D) lymphatics-on-a-chip featuring an engineered lymphatic vessel (LV) capable of draining interstitial fluids including nanoparticles. We tested lymphatic drainage of different sizes (30, 50, and 70 nm) of PLGA-b-PEG nanoparticles (NPs) using the lymphatics-on-a-chip device. In this study, we discovered that smaller NPs (30 and 50 nm) transported faster than larger NPs (70 nm) through the interstitial space, as expected, but the smaller NPs were captured by lymphatic endothelial cells (LECs) and accumulated within their cytosol, delaying NP transport into the lymphatic lumen, which was not observed in larger NPs. To examine the mechanisms of size-dependent NP transports, we employed four inhibitors, dynasore, nystatin, amiloride, and adrenomedullin, to selectively block dynamin-, caveolin-, macropinocytosis-mediated endocytosis-, and cell junction-mediated paracellular transport. Inhibiting dynamin using dynasore enhanced the transport of smaller NPs (30 and 50 nm) into the lymphatic lumen, minimizing cytosolic accumulation, but showed no effect on larger NP transport. Interestingly, the inhibition of caveolin by nystatin decreased the lymphatic transport of larger NPs without affecting the smaller NP transport, indicating distinct endocytosis mechanisms used by different sizes of NPs. Macropinocytosis inhibition by amiloride did not change the drainage of all sizes of NPs; however, paracellular transport inhibition by adrenomedullin blocked the lymphatic transport of NPs of all sizes. We further revealed that smaller NPs were captured in the Rab7-positive late-stage lymphatic endosomes to delay their lymphatic drainage, which was reversed by dynamin inhibition, suggesting that Rab7 is a potential target to enhance the lymphatic delivery of smaller NPs. Together, our 3D lymphatics-on-a-chip model unveils size-dependent NP transport mechanisms in lymphatic drug delivery.