The affinity of a synthetic polymer nanoparticle (NP) to a target biomacromolecule is determined by the association and dissociation rate constants (k(on), k(off)) of the interaction. The individual rates and their sensitivity to local environmental influences are important factors for the on-demand capture and release a target biomacromolecule. Positively charged NPs for small interfering RNA (siRNA) delivery is a case in point. The knockdown efficacy of siRNA can be strongly influenced by the binding kinetics to the NP. Here, we show that k(on) and k(off) of siRNA to NPs can be individually engineered by tuning the chemical structure and composition of the NP. N-Isopropylacrylamide-based NPs functionalized with hydrophobic and amine monomers were used. k(off) decreased by increasing the amount of amine groups in the NP, whereas k(on) did not change. Importantly, NPs showing a low k(off) at pH 5.5 together with a high k(off) at pH 7.4 showed high knockdown efficiency when NP/siRNA complexes were packaged in lipid nanoparticles. These results provide direct evidence for the premise that the efficacy of an siRNA delivery vector is linked with the strong affinity to the siRNA in the endosome and low affinity in the cytoplasm.