Abstract
Graphene oxide (GO), a two-dimensional material with a high aspect ratio and polar functional groups, can physically adsorb single-strand DNA through different types of interactions, such as hydrogen bonding and π-π stacking, making it an attractive nanocarrier for nucleic acids. In this work, we demonstrate a strategy to target exosites I and II of thrombin simultaneously by using programmed hybrid-aptamers for enhanced anticoagulation efficiency and stability. The targeting ligand is denoted as Supra-TBA15/29 (supramolecular TBA15/29), containing TBA15 (a 15-base nucleotide, targeting exosite I of thrombin) and TBA29 (a 29-base nucleotide, targeting exosite II of thrombin), and it is designed to allow consecutive hybridization of TBA15 and TBA29 to form a network of TBAs (i.e., supra-TBA15/29). The programmed hybrid-aptamers (Supra-TBA15/29) were self-assembled on GO to further boost anticoagulation activity by inhibiting thrombin activity, and thus suppress the thrombin-induced fibrin formation from fibrinogen. The Supra-TBA15/29-GO composite was formed mainly through multivalent interaction between poly(adenine) from Supra-TBA15/29 and GO. We controlled the assembly of Supra-TBA15/29 on GO by regulating the preparation temperature and the concentration ratio of Supra-TBA15/29 to GO to optimize the distance between TBA15 and TBA29 units, aptamer density, and aptamer orientation on the GO surfaces. The dose-dependent thrombin clotting time (TCT) delay caused by Supra-TBA15/29-GO was >10 times longer than that of common anticoagulant drugs including heparin, argatroban, hirudin, and warfarin. Supra-TBA15/29-GO exhibits high biocompatibility, which has been proved by in vitro cytotoxicity and hemolysis assays. In addition, the thromboelastography of whole-blood coagulation and rat-tail bleeding assays indicate the anticoagulation ability of Supra-TBA15/29-GO is superior to the most widely used anticoagulant (heparin). Our highly biocompatible Supra-TBA15/29-GO with strong multivalent interaction with thrombin [dissociation constant (Kd) = 1.9 × 10−11 M] shows great potential as an effective direct thrombin inhibitor for the treatment of hemostatic disorders.
Highlights
Stable and uninterrupted blood flow is important for maintaining a healthy life style
The sequences of A20h15T5TBA15T5h15 (sTBA15) and sTBA29 comprised of three blocks, a 20-polyadenine (A20) for anchoring on Graphene oxide (GO), two 15-base sequences (h15) for consecutive hybridization, a 5-repeat thymidine (T5) as a linker, and a TBA sequence providing functionality
Atomic force microscopy (AFM) images showed that the Supra-TBA15/29-GO was dispersed as 2D nanosheets
Summary
Stable and uninterrupted blood flow is important for maintaining a healthy life style. Many diseases are caused by the formation of thrombus (Previtali et al, 2011; Otsuka et al, 2016). In a normal coagulation system, complex interactions of coagulation factors, platelets, cofactors, and regulators maintain homeostasis for the systematic regulation of bleeding and thrombus formation (Palta et al, 2014). Targeting thrombin activity could be an effective strategy to control thrombosis. Heparin may cause an immune-mediated coagulation side effect of heparin-induced thrombocytopenia (HIT) (Pollak et al, 2011). HIT causes abnormal coagulation of platelets and triggers thrombosis symptoms, and the incidence is higher for high-molecular-weight heparin than that of low-molecularweight heparin. Developing an anticoagulant with high stability and low side effects is an important and challenging issue
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