Design and investigation of targeting agent orientation and density on nanoparticles for enhancing cellular uptake efficiency.

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/nm 2 , 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.