Study On The Preparation And Properties Of Fibrinolytic Materials

Thrombosis induced by foreign materials remains the most serious issue for the successful application of blood-contacting devices. Traditional strategies for preparing anti-thrombotic materials are frequently focused on the ‘inhibition of fibrin formation’ through surface modification. No materials, however, has proved to be thrombosis-free in contact with blood. The concept of “fibrinolytic surface” provides an opportunity to endow the surface with clot-lysing property. The best way to realize the concept is to modify material with ε-lysine so as to bind preferentially from blood the essential components of the fibrinolytic system, namely plasminogen(Plg) as well as tissue plasminogen activator(t-PA). Such a material would therefore be able to lyse the fibrin clot formed on the surface before any harms caused. Over the past ten years, the feasibility of fibrinolytic surface has been extensively demonstrated and different strategies have been developed to build such surfaces. However, the methods for surface immobilization of ε-lysine usually involve complicated and multi-step processes and require rigorous conditions. This restricts the development of fibrinolytic surface towards commercial exploitation.In this thesis, methods for preparing lysinization-based fibrinolytic surface were optimized, aiming to simplify the procedures of surface modification and improve the controllability and repeatability over the whole process. In addition, based on the specific interactions between t-PA and its affinity ligands, t-PA was conjugated to nanoparticle, polymer and material surface), in order to improve the fibrinolytic efficacy of t-PA and t-PA-immobilized surfaces in blood-contacting applications.With the aim to simplify the process for fabricating the lysinized surfaces, a vinyl-containing lysine monomer was synthesized and subsequently copolymerized with 2-hydroxylethyl methacrylate(HEMA) to prepare the lysine-containing copolymer(P(HEMA-co-Lys)). The composition of the copolymer could be adjusted by changing the monomer feed ratio. Copolymers prepared could be easily casted into polymer films. Lysine density on the film surface could be tuned by adjusting the copolymer composition and reached up to 63.9 nmol/cm2. The copolymer surfaces were shown to reduce fibrinogen(Fg) adsorption while binding Plg selectively from plasma. The amount of Plg adsorbed increased as the surface lysine density increased with the highest value up to 1.8 μg/cm2. Upon activation by t-PA, the Plg bound on the copolymer surfaces was able to lyse the fibrin clot in vitro; the more Plg was adsorbed, the rapider clot was lysed. In further study, lysinized PU surfaces were prepared via surface graft copolymerization of lysine monomer and HEMA. Surface lysine density and Plg uptake could be tailored by changing the monomer feed ratio. The approach for surface lysinization developed in this study is advantageous in good controllability and repeatability without involving severe reactions.Based on the affinity interactions between t-PA and ε-lysine, a complex of t-PA with lysinized gold nanoparticle was prepared aiming to improve activity preservation and to prolong the circulation time of t-PA. A lysine-containing RAFT agent was synthesized and subsequently utilized for the preparation of thiol- and ε-lysine-terminated polyvinyl pyrrolidone(SH-PVP-Lys). Surface of gold nanoparticles(Au NPs) was grafted with SH-PVP-Lys and was shown to resist non-specific protein adsorption and specifically to bind t-PA due to the affinity interactions with ε-lysine. The t-PA/Au NPs-PVP-Lys complex retained almost the full enzymatic activity and fibrinolytic efficacy as compared with free t-PA. Moreover, t-PA was shown to be protected from being inhibited by PAI-1 to some extent by binding with Au NPs-PVP-Lys. The half-life of the complex in mouse was found to be 3 folds as long as that of free t-PA. Compared with typical methods for protein conjugation, binding based on affinity interactions was more promising in retaining the enzymatic activity of t-PA in blood environment.The very short half-life of t-PA in circulation is primarily caused by the rapid inhibition by its physiological inhibitor PAI-1. Inspired by the mechanism of t-PA/PAI-1 binding, a PAI-1-derived affinity ligand for t-PA was exploited. A peptide sequence(ARMAPE) on PAI-1 involved in the binding with t-PA was used as an affinity ligand for the immobilization of t-PA with polymers and material surfaces. First, the peptide-terminated poly(oligo(ethylene glycol) methacrylate)(Pep-POEGMA) was prepared and subsequently conjugated to t-PA. The conjugate remained the full activity of t-PA and was shown to be able to resist the access of PAI-1 since the PAI-1 binding site on t-PA was occupied by the surface-tethered peptide. The peptide was then introduced to material surfaces via POEGMA as the spacer. The resulting surface showed a high t-PA loading capacity(560 ng/cm2) which was much higher than that achieved on the lysine-modified surface(280 ng/cm2). Compared to the conjugation approaches based on nonspecific covalent bonding or physical adsorption, t-PA immobilization via affinity interactions with the peptide was more superior in retaining the activity of t-PA. Even in the presence of PAI-1, 80% of the original activity of peptide-bound t-PA was still retained. With all favourable factors in retaining activity, the peptide-immobilized t-PA endows the surface with fibrinolytic activity by showing clot-lysis in a plasma recalcification assay. The affinity conjugation of t-PA based on PAI-1-derived ligand is advantageous over other methods due to the effective occupation of PAI-1 binding site in t-PA by the peptide limits the access of PAI-1.