针对细胞靶向递送和癌症治疗，靶向药物修饰系统的设计引起了广泛关注。然而，由于技术问题和繁琐的操作，目前对配体密度和结合效率的定量研究还很有限。本文制备了苯硼酸酯修饰的核壳磁性纳米颗粒（MSP-AOPB NPs）作为药物载体模型，并以位点定向方式将转铁蛋白（Tf）固定在纳米颗粒表面（Tf-MSP-AOPB NPs）。详细研究了制备条件以优化Tf的结合效率。适当的反应温度、时间或初始投料量可以显著增加Tf的结合量。MSP-AOPB NPs上的最大Tf结合量为184 mg g-1，通过调整反应条件可以很好地控制表面的靶向配体密度。体外研究表明，通过改变配体密度，优良的Tf介导的靶向能力和增强的细胞摄取效果得以实现。实现细胞摄取效率最高的最佳配体结合量为94 mg Tf/g，相应的配体结合密度约为0.05 Tf/nm2，结合效率超过90%。此外，通过位点定向偶联策略制备的Tf-MSP-AOPB NPs显示出最佳的细胞靶向能力，其在HepG2细胞中的细胞摄取量分别为物理吸附和EDC/NHS偶联反应的25倍和127倍。本研究为不同种类的抗体提供了一种简便的位点定向生物偶联技术，并能轻松获得适当的配体密度以增强细胞摄取效果，对于靶向递送和癌症治疗具有重要意义。

The design of targeting agent-conjugated systems is attracting much attention in cell targeted delivery and cancer therapy. However, quantitative study of the ligand density and binding efficiency is still limited due to the technical matters and tedious work involved. In this article, benzoboroxole-modified core-shell magnetic nanoparticles (MSP-AOPB NPs) as a drug carrier model were fabricated and transferrin (Tf) was immobilized on the nanoparticle surface in a site-oriented manner (Tf-MSP-AOPB NPs). The preparation conditions were investigated in detail to optimize the Tf binding efficiency. A suitable reaction temperature, time or initial feeding amount could significantly increase the Tf binding amount. The maximum Tf binding amount on the MSP-AOPB NPs was 184 mg g-1, and the targeting ligand density on the surface could be well controlled by simply adjusting the reaction conditions. In vitro studies demonstrated the excellent Tf-mediated targeting ability and enhanced cellular uptake efficacy by varying the ligand density. The optimal ligand binding amount for achieving the highest cellular uptake efficiency was 94 mg Tf/g, which corresponds to a ligand binding density of about 0.05 Tf/nm2, and the binding efficiency of conjugation was higher than 90%. Moreover, Tf-MSP-AOPB NPs prepared by a site-oriented conjugation strategy showed the best cell targeting ability, and their cellular uptake amount was 25 and 127 times higher than that of physical adsorption and EDC/NHS coupling reaction in HepG2 cells, respectively. This study provides a facile site-oriented bioconjugation technique for different kinds of antibodies, and a suitable ligand density can be easily attained to enhance the cellular uptake efficacy, which shows great significance for targeted delivery and cancer therapy.