Department of Bioengineering, University of Maryland College Park, College Park, 8278 Paint Branch Drive, MD 20742, USA.
Department of Bioengineering, University of Maryland College Park, College Park, 8278 Paint Branch Drive, MD 20742, USA.
Acta Biomater. 2022 Jun;145:146-158. doi: 10.1016/j.actbio.2022.03.054. Epub 2022 Apr 2.
Lymphatic vessels have recently been shown to effectively deliver immune modulatory therapies to the lymph nodes, which enhances their therapeutic efficacy. Prior work has shown that lymphatics transport 10-250 nm nanoparticles from peripheral tissues to the lymph node. However, the surface chemistry required to maximize this transport is poorly understood. Here, we determined the effect of surface poly(ethylene glycol) (PEG) density and size on nanoparticle transport across lymphatic endothelial cells (LECs) by differentially PEGylated model polystyrene nanoparticles. Using an established in-vitro lymphatic transport model, we found PEGylation improved the transport of 100 and 40 nm nanoparticles across LECs 50-fold compared to the unmodified nanoparticles and that transport is maximized when the PEG is in a dense brush conformation or high grafting density (Rf/D = 4.9). We also determined that these trends are not size-dependent. PEGylating 40 nm nanoparticles improved transport efficiency across LECs 68-fold compared to unmodified nanoparticles. We also found that PEGylated 100 nm and 40 nm nanoparticles accumulate in lymph nodes within 4 h after intradermal injection, while unmodified nanoparticles accumulated minimally. Densely PEGylated nanoparticles traveled the furthest distance from the injection site and densely PEGylated 40 nm nanoparticles had maximum accumulation in the lymph nodes compared to low density PEGylated and unmodified nanoparticles. Finally, we determined that nanoparticles are transported via both paracellular and transcellular mechanisms, and that PEG conformation modulates the cellular transport mechanisms. Our results suggest that PEG conformation is crucial to maximize nanoparticle transport across LECs and into lymphatic vessels, making PEG density a crucial design. Optimizing PEG density on nanoparticle formulations has the potential to enhance immunotherapeutic and vaccine outcomes. STATEMENT OF SIGNIFICANCE: Lymphatic vessels are an emerging target for drug delivery both in the context of modulating immune responses and enhancing bioavailability by avoiding first pass hepatic metabolism after oral delivery. Lymphatic vessels are the natural conduits from peripheral tissues to the lymph nodes, where the adaptive immune response is shaped, and eventually to systemic circulation via the thoracic duct. Lymphatics can be targeted via nanoparticles, but the surface chemistry required to maximize nanoparticle transport by lymphatics vessels remains poorly understood. Here, we demonstrate that coating nanoparticles with hydrophilic polyethylene glycol (PEG) effectively enhances their transport across lymphatic endothelial cells in vitro and in vivo and that both paracellular and micropinocytosis mechanisms underly this transport. We found that dense PEG coatings maximize lymphatic transport of nanoparticles, thus providing new material design criteria for lymphatic targeted drug delivery.
淋巴管最近被证明可以有效地将免疫调节疗法递送到淋巴结，从而增强其治疗效果。先前的工作表明，淋巴管将 10-250nm 纳米颗粒从外周组织输送到淋巴结。然而，最大限度地提高这种输送的表面化学性质还了解甚少。在这里，我们通过对不同聚乙二醇（PEG）化模型聚苯乙烯纳米颗粒的研究，确定了表面 PEG 密度和尺寸对纳米颗粒穿过淋巴管内皮细胞（LEC）的运输的影响。使用已建立的体外淋巴管运输模型，我们发现与未修饰的纳米颗粒相比，PEG 化将 100nm 和 40nm 纳米颗粒穿过 LEC 的运输提高了 50 倍，并且当 PEG 处于密集刷状构象或高接枝密度（Rf/D=4.9）时，运输达到最大值。我们还确定这些趋势与尺寸无关。与未修饰的纳米颗粒相比，PEG 化的 40nm 纳米颗粒穿过 LEC 的运输效率提高了 68 倍。我们还发现，与未修饰的纳米颗粒相比，经 PEG 化的 100nm 和 40nm 纳米颗粒在皮内注射后 4 小时内即可积聚在淋巴结中，而未修饰的纳米颗粒则很少积聚。高度 PEG 化的纳米颗粒从注射部位移动的距离最远，与低浓度 PEG 化和未修饰的纳米颗粒相比，高度 PEG 化的 40nm 纳米颗粒在淋巴结中的积聚最多。最后，我们确定纳米颗粒是通过细胞旁和跨细胞机制进行运输的，PEG 构象调节细胞运输机制。我们的结果表明，PEG 构象对于最大化纳米颗粒穿过 LEC 并进入淋巴管的运输至关重要，这使得 PEG 密度成为一个关键的设计因素。优化纳米颗粒制剂中的 PEG 密度有可能增强免疫治疗和疫苗的效果。
淋巴管是药物输送的新兴靶点，无论是在调节免疫反应方面，还是在避免口服给药后首过肝代谢以提高生物利用度方面都是如此。淋巴管是外周组织到淋巴结的天然通道，在淋巴结中，适应性免疫反应得到塑造，并最终通过胸导管进入全身循环。可以通过纳米颗粒靶向淋巴管，但仍不清楚最大限度地提高淋巴管输送纳米颗粒所需的表面化学性质。在这里，我们证明了将亲水性聚乙二醇（PEG）涂覆在纳米颗粒上可有效增强其在体外和体内穿过淋巴管内皮细胞的运输，并且细胞旁和微胞饮作用均为这种运输的基础。我们发现，密集的 PEG 涂层可最大程度地促进纳米颗粒的淋巴管运输，从而为靶向淋巴药物输送提供了新的材料设计标准。
