Recombinant Tissue Plasminogen Activator Conjugated Nanoparticles For Targeting Thrombolysis

Thrombosis is the primary cause of death and complications worldwide. Thrombolytic treatment to rapidly achieve reperfusion of the ischemic tissues is the most effective therapy, restoring blood to save the tissues on the verge of death. Although some progresses have been made in thrombolytic treatment, the hemorrhagic complications and secondary reocclusions remain the major obstacles. Hitherto, recombinant tissue plasminogen activator (rtPA) is the only FDA-approved drug for the treatment of acute ischemic stroke. But it is urgent to improve the efficacy and safety of rtPA due to its low bioavailability and requirement of large dose administration.One intriguing approach is to focus on targeted drug delivery system. Recently, nanomedicine is a new field that uses nanoparticles to transfer diagnostic or therapeutic agents to the targeted lesions. Nanoparticles can also be engineered to have multiple functions, which provide a potential advantage over conventional drugs.In a strategy for thrombosis combination therapy, a fibrin targeting nanoparticle (NP) drug delivery system was developed by conjugating recombinant tissue plasminogen activator (rtPA) to poly(ethylene glycol)-poly(ε-caprolactone) (PEG-PCL) nanoparticles (rtPA-NP). In the first part, the construction and characterization of rtPA-NP was desciribed. PEG-PCl nanoparticles were prepared by single-emulsion and solvent evaporation method and covalently conjugated with rtPA through COOH-PEG by EDC/NHS method. The Physicochemical characteristics such as the particle size, rtPA loading amount and storage stability were investigated. The particle size of rtPA-NP was 129±3.8nm and rtPA-NP have an acceptable polydispersity index (PDI<0.25). Based on the TEM photograph, both NP and rtPA-NP exhibited the morphology of spherical particles. The rtPA loading amount was 49.2±3.1ug/mg NP. RtPA-NP can be stored at 4℃for one week without loss of enzymatic activity.In order to evaluate the pharmacokinetics of rtPA-NP, in the second part,6-coumarin labeled rtPA-NP was prepared. Rat were injected with injected with 1 mg/kg free rtPA, 20mg/kg rtPA-NP separately. Blood was collected at each time point to determine the blood concentration of rtPA or nanoparticle. Results showed that intravenously injected rtPA-NP retained rtPA activity for 3 hours; In contrast, there is a marked reduction of rtPA concentration after an hour in the group treated with free rtPA; Based on the rtPA-NP blood concentration-time curve in blood, the half-life of rtPA-NP (92.3±14.0min) was approximately 18 times longer than that of free rtPA (n=5).The third part described rtPA-NP has fibrin-targeting thrombolysis ability both in vitro and in vivo. Fibrin clots and rat middle cerebral occlusion model were prepared. When compared with non-targeting nanoparticles, a significantly higher amount of 6-coumarin-labeled rtPA-conjugated nanoparticles bound non-passively to fibrin clots in vitro (p<0.001) and to fibrin thrombi in a rat model of transient middle cerebral arterial occlusion. Its thrombolytic activity was also evaluated in fibrin clots and in a rat middle cerebral artery occlusion model through infracts volume and neurological deficit test. RtPA-NP, equivalently to 10% of a typical dose of rtPA, can dissolve fibrin clots and mediate neuroprotection after focal cerebral ischemia in terms of both infarct volume and neurological deficit evaluation (p<0.001). No significant difference was found between the normal saline and rtPA-NP treated groups for the PT, APTT, Fbg level (n=4).These results showed that rtPA-NP retained the activity of rtPA and prolonged the half-life of rtPA, endowing rtPA-NP with thrombus-targeting ability and thrombolysis activity. RtPA-NP did not influence hemostasis or coagulation system. These experiments suggested that rtPA conjugated PEG-PCL nanoparticles were promising in fibrin targeting delivery system for combination therapy of thrombosis disease due to its novel bioavailability and safety. In addition, This strategy is promising to combination with other antithrombotic drug for maximizing thrombolysis effectiveness and minimizing adverse effects, such as bleeding, reocclusion, etc.