The preparation of a t-PA/nanoparticle conjugate via bio-affinity ligation

Abstract

Event Abstract Back to Event The preparation of a t-PA/nanoparticle conjugate via bio-affinity ligation Xiaoli Liu1, Zengchao Tang1, Dan Li1* and Hong Chen1* 1 Soochow University, China Introduction: Tissue plasminogen activator (t-PA) is commonly used as a thrombolytic agent in the treatment of thrombotic diseases such as acute myocardial infarction and ischemic stroke. A major issue in the therapeutic use of t-PA is its very short half-life in the circulation (2–6 min) due to the effects of inhibitors, enzymes and antibodies in blood. Efforts have been made to overcome this deficiency by conjugating t-PA to synthetic materials to limit access of inhibitors or to materials with thrombus-targeted ability.[1] However, the linkages between t-PA and synthetic materials are commonly based on covalent bonding or electrostatic interactions, which are “invasive” and generally accompanied by loss of protein activity and immunogenic responses. Bio-affinity ligation based on the specific interactions between ligands and receptors has many advantages over covalent bonding and other nonspecific interactions for the conjugation of proteins to synthetic materials since it is benign to protein, highly specific and efficient, proceeds under physiological conditions and has no unwanted side reactions. Most proteins possess specific domains that can be recognized by some affinity ligand, so the interaction between such a domain and its affinity ligand can be utilized to link protein to materials. For t-PA, there is a high affinity lysine binding site in one of its two kringle domains that mediates its binding to carboxy-terminal lysine residues (ε-lysines) exposed on the surface of fibrin. Therefore ε-lysine is a natural affinity ligand that can be utilized to link t-PA to a synthetic material. Moreover, t-PA conjugated through ε-lysine mimics the one bound on the surface of fibrin in physiological fibrinolytic process, which is advantageous for limiting access of inhibitors. In this study, a t-PA/gold nanoparticle (t-PA/AuNP) conjugate was prepared via bio-affinity ligation. Materials and Methods: Here AuNPs were used as the model nanoparticles and modified with ε-lysine-terminated polyvinyl pyrrolidone (PVP), referred to as AuNPs-PVP-Lys. PVP functions as a bio-inert moiety to prevent the conjugate from interacting nonspecifically with other components in blood. t-PA was conjugated to AuNPs-PVP-Lys under physiological conditions through affinity interactions between ε-lysine exposed on AuNP surface and the specific domain in t-PA. Results and Discussion: Activity of the conjugated t-PA was compared with free t-PA both in vitro and in vivo. It is found that the t-PA/AuNP conjugate can not only maintain almost all enzyme activity of t-PA but also reduce the inhibition effect, leading to a prolonged circulation time (20.5 min). Conclusions: In conclusion, a t-PA/AuNPs-PVP-Lys conjugate was prepared under physiological conditions via bio-affinity ligation based on the bio-affinity interactions under physiological conditions. Compared with free t-PA, the conjugate retained almost full enzyme activity and clot-dissolving efficiency, and the t-PA was protected from inhibition by PAI-1 to some extent. Moreover, the conjugate showed prolonged circulation time in vivo. It will be of interest to apply this bio-affinity ligation more generally to conjugate synthetic materials with other proteins, especially those with important biomedical functions. National Natural Science Foundation of China (21204055, 21304062, 21334004); National Natural Science Foundation Fund for Distinguished Young Scholars (21125418); The Natural Science Foundation of the Jiangsu Higher Education Institutions of China (13KJB430020); China Postdoctoral Science Foundation (2013M541714)