叶酸摄取主要由粒细胞、单核细胞等骨髓免疫细胞中的叶酸受体 (FR)β 介导，叶酸受体 (FR)β 由 FOLR2 基因编码，尤其是构成网状内皮系统 (RES) 并浸润肿瘤微环境的巨噬细胞中。由于骨髓免疫区室在肿瘤发生过程中动态变化，因此评估表达 FRβ 的骨髓细胞对肿瘤的浸润状态对于更好地确定叶酸功能化药物递送系统的靶向功效至关重要。另一方面，RES 的清除是叶酸修饰的纳米颗粒靶向功效的主要限制。因此，本研究的目的是（i）确定不同阶段浸润肿瘤的 FRβ 髓样细胞的数量和亚型，（ii）比较荷瘤和健康不同器官中 FRβ 髓样细胞的数量和亚型(iii) 测试原型叶酸功能化纳米颗粒的癌症靶向功效和生物分布是否与 FRβ 骨髓细胞的密度相关。在此，我们报告在小鼠乳腺癌模型中，在肿瘤发生的不同阶段，骨髓细胞浸润增强，并且 FRβ 上调。巨噬细胞的 CD206 子集高度表达 FRβ，在荷瘤小鼠和健康小鼠中均显着表达。在荷瘤小鼠中，肿瘤、肝、肺、脾、肾、淋巴结、腹膜腔、骨髓、心脏和脑中所有骨髓细胞（尤其是粒细胞）的数量显着增加。与巨噬细胞相比，粒细胞和单核细胞中的FRβ水平中等。肿瘤微环境中 FRβ 免疫细胞的密度与叶酸功能化环糊精纳米颗粒的肿瘤靶向功效没有直接关系。肺部被确定为叶酸功能化纳米颗粒的优先积累部位，其中 FRβ CD206 巨噬细胞显着吞噬环糊精纳米颗粒。总之，我们的结果表明，肿瘤的形成增加了 FR 水平，并改变了所有器官中骨髓免疫细胞的浸润和分布，这应被视为影响纳米粒子药物递送靶向功效的主要因素。

Folate uptake is largely mediated by folate receptor (FR)β, encoded by FOLR2 gene, in myeloid immune cells such as granulocytes, monocytes, and especially in macrophages that constitute the reticuloendothelial system (RES) and infiltrate the tumor microenvironment. Since the myeloid immune compartment dynamically changes during tumorigenesis, it is critical to assess the infiltration status of the tumors by FRβ-expressing myeloid cells to better define the targeting efficacy of folate-functionalized drug delivery systems. On the other hand, clearance by RES is a major limitation for the targeting efficacy of nanoparticles decorated with folate. Therefore, the aims of this study are (i) to determine the amount and subtypes of FRβ+ myeloid cells infiltrating the tumors at different stages, (ii) to compare the amount and subtype of FRβ+ myeloid cells in distinct organs of tumor-bearing and healthy animals, (iii) to test if the cancer-targeting efficacy and biodistribution of a prototypic folate-functionalized nanoparticle associates with the density of FRβ+ myeloid cells. Here, we report that myeloid cell infiltration was enhanced and FRβ was upregulated at distinct stages of tumorigenesis in a mouse breast cancer model. The CD206+ subset of macrophages highly expressed FRβ, prominently both in tumor-bearing and healthy mice. In tumor-bearing mice, the amount of all myeloid cells, but particularly granulocytes, was remarkably increased in the tumor, liver, lungs, spleen, kidneys, lymph nodes, peritoneal cavity, bone marrow, heart, and brain. Compared with macrophages, the level of FRβ was moderate in granulocytes and monocytes. The density of FRβ+ immune cells in the tumor microenvironment was not directly associated with the tumor-targeting efficacy of the folate-functionalized cyclodextrin nanoparticles. The lung was determined as a preferential site of accumulation for folate-functionalized nanoparticles, wherein FRβ+CD206+ macrophages significantly engulfed cyclodextrin nanoparticles. In conclusion, our results demonstrate that the tumor formation augments the FR levels and alters the infiltration and distribution of myeloid immune cells in all organs which should be considered as a major factor influencing the targeting efficacy of nanoparticles for drug delivery.