Highly efficient control of thrombin activity by multivalent nanoparticles

We have demonstrated that the incorporation of sulfated galactose acid (sulf-Gal) into thrombin-binding-aptamer (TBA)-conjugated gold nanoparticles (TBA-AuNPs) enables highly effective inhibition of thrombin activity toward fibrinogen. AuNP bioconjugates (TBA ₁₅/TBA ₂₉/sulf-Gal- AuNPs) were prepared from 13 nm AuNPs, 15-mer thrombin-binding aptamer (TBA ₁₅), 29-mer thrombin-binding aptamer (TBA ₂₉), and sulf-Gal. The numbers of TBA and sulf-Gal molecules per AuNP proved to have a strong impact on inhibitory potency. The best results were observed for 15-TBA ₁₅/TBA ₂₉/sulf-Gal-AuNPs (with 15 TBA ₁₅ and 15 TBA ₂₉ molecules per AuNP), which, because of their particularly flexible conformation and multivalency, exhibited ultrahigh binding affinity toward thrombin (K _d=3.4Ã-10 ^-12 M) and thus extremely high anticoagulant (inhibitory) potency. Compared to the case without inhibitors (the "normal" value), their measured thrombin clotting time (TCT) was 91 times longer, whereas for TBA ₁₅ alone it was only 7.2 times longer. Their anticoagulant activity was suppressed by TBA-complementary-sequence (cTBA)-modified AuNPs (cTBA ₁₅/cTBA ₂₉-AuNPs) at a rate that was 20 times faster than that of free cTBA ₁₅/cTBA ₂₉. Thus, easily prepared, low-cost, multivalent AuNPs show great potential for biomedical control of blood clotting.