Receptor-ligand interactions on cell membrane play key roles in many physiological and pathological processes, especially in the nanodrug targeted delivery system. Hyaluronic acid (HA) is the main ligand for binding CD44 to modulate the targeted delivery of nanodrugs; however, the corresponding receptor clustering mechanism remains unclear. The differential effects of HA on CD44 clustering are closely associated with the number of disaccharide units. Herein, the binding dynamic parameters between CD44 and HA containing various disaccharide units were analyzed at the single-molecule level. Then, the clustering effect induced by HA containing various disaccharide units was evaluated and the optimal clustering effect was verified. Furthermore, the clustering effect on cell entry dynamic parameters of HA targeting nanodrugs was assessed based on different cell lines at the single particle level. These results demonstrate that the clustering effect will enhance the entry cell efficiency of HA targeting nanodrugs, and the effect is more obvious on the cell line with low expression level of CD44. This study offers a new way to evaluate the cell membrane receptor clustering and the corresponding effect on cellular uptake, which will provide potential strategy for designing appropriate targeting nanodrugs with high delivery efficiency tailored to different cancers.

细胞膜上的受体-配体相互作用在许多生理和病理过程中起关键作用，特别是在纳米药物靶向递送系统中。透明质酸（HA）是结合CD44的主要配体，用于调节纳米药物的靶向递送；然而，相应的受体聚集机制尚不清楚。HA对CD44聚集的不同影响与其二糖单元的数量密切相关。在此，我们分析了含不同二糖单元的HA与CD44之间的单分子水平上的动态结合参数。然后评估了由含不同二糖单元的HA诱导的聚集效应，并验证了最佳聚集效果。此外，基于不同的细胞系，在单颗粒水平上评估了HA靶向纳米药物进入细胞的动力学参数变化下的聚集效应。这些结果表明，聚集效应对提高HA靶向纳米药物的入胞效率有增强作用，且在CD44表达水平较低的细胞系中效果更明显。该研究为评价细胞膜受体聚集及其对细胞摄取的影响提供了一种新方法，并为此类设计适应不同癌症类型的高递送效率靶向纳米药物提供了潜在策略。