Reteplase Encapsulated in Minocycline‐Containing Nanoparticles Retains Thrombolytic Activity

Abstract

Thrombolytic therapy is a critical potion of the treatment regimen for a number of life‐threatening conditions, including cardiovascular disease and stroke. Although thrombolytic drugs have shown profound effect on dissolving clots, short half‐life (t1/2 of minutes) and hemorrhagic complications are significant challenges associated with their use. Nanotechnology has shown to prolong the blood circulation time of drugs and to improve their efficacy and safety. However, leakage of thrombolytic drug‐loaded nanocarriers (NC) from intravascular compartment into brain parenchyma due to compromised blood‐brain barrier integrity following ischemia can still lead to adverse effects. Therefore, it is necessary to combine neuroprotection with thrombolysis therapy in ischemic stroke treatment. Here we have developed NC that contains reteplase, an engineered tissue‐type plasminogen activator, and minocycline, a neuroprotective agent. The formation of the NC is based on metal ion‐assisted self‐assembly of complexes using negatively charged heparin and minocycline. The reteplase is incorporated into the NC through a protein binding site on minocycline. The entrapment efficiency of reteplase and minocycline were 91.8% and 78.3%, respectively. By tracing radiolabeled reteplase, our results showed that the NC significantly increased the blood circulation time of reteplase compared to free reteplase (Panel A). In the in vitro clot lysis assay, the free reteplase or reteplase‐loaded NC (NC(ret)) were incubated with pre‐formed plasma clots and the results indicated that the NC formation did not compromise the thrombolytic function of reteplase (Panel B). In the pulmonary embolism model, free reteplase or NC(ret) was injected 10 min after 125I‐labeled microemboli. Blood and organs were collected 1 hr‐post injection of microemboli to study the retention of clot in the lung after the treatment. Our results showed that the reteplase‐loaded NC significantly reduced the clot retention in the lung (Panel C). All together, we have developed NC containing neuroprotective agent that maintain the function of thrombolytic drug. Future studies will be focused on evaluating the neuroprotective properties of the NC, relative to free drug.Support or Funding InformationNIH (RO1 HL128398‐02 and RO1 HL 143806‐01), Cardiovascular Institute of Philadelphia (400‐4119‐2)Characteristics of Reteplase‐Containing Nanocarriers. A: Blood pharmacokinetics of free reteplase (ret) and NC‐encapsulated reteplase (NC(ret)). Comparisons by students t‐test. B: In vitro fibrinolytic activity of ret and NC(ret). C: Lysis of pulmonary emboli following injection of ret and NC(ret). Comparisons by 1‐way ANOVA with Dunnett's test. * denotes p<0.05, ** p<0.01, *** p<0.001.Figure 1