系统评价枯否细胞在清除PEG化纳米载体中的作用

Kupffer cells are the largest number of tissue resident macrophages, accounting for 80%-90% of tissue macrophages, and have the characteristic of self-renewal. Kupffer cells play a very important role in the removal of the foreign materials and homeostasis maintainability. Based on the fact that mannose and fucose receptors are highly expressed on the surface of Kupffer cells, different molecular weight PEG lipid derivatives with mannose and fucose were synthesized and modified on the surface of liposomes to specific target Kupffer cells. The effect of PEG molecular weight, PEG modified density, the ratio of mannose and fucose derivatives, and particle size on the targeting ability of liposomes to Kupffer cells were investigated. Then, bisphosphonate drugs were loaded in the liposomes that exhibited the most targeting ability and this preparation was used to establish Kupffer cells depletion animal model. PEGylated nanocarriers (liposomes, emulsions, solid lipid nanoparticles, and micelles) with different particle sizes were intravenously injected in this model to compare their pharmacokinetics and biodistributions in normal and tumor-bearing animals, and analyse the effect of Kupffer cells on the clearance and antitumor activity of PEGylated nanocarriers. The memory characteristic of Kupffer cells was also exploratorily studied to provide a guidance for the clinical application of PEGylated nanocarriers and a novel way for cancer treatments, vaccine development, and liver diseases (such as liver fibrosis) treatments.

枯否细胞是机体内数量最为庞大的组织驻留性巨噬细胞，占全身组织巨噬细胞总数的80%-90%，具有自我更新的特征，在清除异物及维护内环境稳定方面有着非常重要的作用。利用枯否细胞表面高度表达的甘露糖和岩藻糖受体，设计合成不同PEG分子量的甘露糖与岩藻糖脂质衍生物，构建特异性靶向枯否细胞载体，考察PEG分子量、PEG修饰密度、甘露糖和岩藻糖脂质衍生物配比及粒径等因素对其靶向性的影响。选择靶向性最佳的脂质体，装载膦酸类药物，建立枯否细胞耗竭的动物模型。静脉注射不同粒径的PEG化纳米载体（脂质体、乳剂、固体脂质纳米粒、胶束），比较研究各载体在枯否细胞耗竭的正常动物及荷瘤动物体内的药动学行为和组织分布，进一步分析枯否细胞对不同性质的PEG化纳米载体清除与抗肿瘤效果的影响。探索性研究枯否细胞的免疫记忆特征，为PEG化纳米载体的合理应用提供指导，为肿瘤治疗、疫苗研制与肝脏疾病（如肝纤维化）防治等提供新思路。

免疫系统与药物递送系统（drug delivery system，DDS）之间的相互作用，不仅影响DDS在体内的靶向效率与治疗效果，也会影响免疫系统的行为，本研究从这两方面评价了肝脏枯否细胞（Kupffer cells，KCs）在清除PEG化纳米载体中的作用。本课题组首次合成了PEG化岩藻糖脂质衍生物（DSPE-PEG2000-APF，DPF）和PEG化甘露糖脂质衍生物（DSPE-PEG2000-APM，DPM），修饰于阿仑膦酸钠（AD）脂质体表面，以Wistar大鼠为动物模型、小鼠永生化KCs细胞株为细胞模型，筛选出最优的KCs靶向制剂（AD-MFPL5-5），成功构建KCs耗竭大鼠模型。药动学结果显示，KCs的耗竭导致加速血液清除（accelerated blood clearance，ABC）现象增强，证明KCs是PEG化纳米载体最终清除的主要执行者，而且在ABC现象的诱导过程中发挥重要作用。利用ABC现象，通过KCs靶向制剂（MFPL5-5）药动学模型、以及首次肝门静脉与尾静脉注射PEG化乳剂（PE）药动学对比实验证明，PEG化纳米载体可诱导KCs对二次注射PEG化纳米载体摄取能力增强，且增强程度与刺激程度呈正相关，推测KCs对PEG化纳米载体产生了免疫记忆。首次在动物水平和细胞水平，以KCs二次摄取量为评价指标，证明了PE首次刺激KCs后，会诱导7 ~ 14 d后KCs对二次刺激的PEG化纳米载体摄取能力非应激性增强，即产生了短时固有免疫记忆。以Wistar大鼠和裸鼠为模型，证明了该记忆可以经由过继转移传递至未刺激的个体中。通过非PEG化普通乳剂（CE）和PEG化胶束（PM）的体内药动学和体外细胞摄取实验发现，相同磷脂浓度下的各制剂均可诱导KCs产生免疫记忆，但强度各不相同，说明KCs对纳米载体的记忆具有一定的非特异性。本课题组通过细胞摄取、细胞回输和透射电镜等手段，确证了中性粒细胞协助KCs清除PEG化纳米载体，说明了即刻固有免疫阶段在PEG化纳米载体清除过程中的重要作用。首次将DSPE-PG610的多羟基结构改造为多羧基磷脂衍生物（PGC），并通过大鼠模型证明PGC修饰乳剂不会诱导产生ABC现象，找到一种消除ABC现象的材料。

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