Abstract
Recombinant tissue plasminogen activator (rtPA) is the only thrombolytic agent that has been approved by the FDA for treatment of ischemic stroke. However, a high dose intravenous infusion is required to maintain effective drug concentration, owing to the short half-life of the thrombolytic drug, whereas a momentous limitation is the risk of bleeding. We envision a dual targeted strategy for rtPA delivery will be feasible to minimize the required dose of rtPA for treatment. For this purpose, rtPA and fibrin-avid peptide were co-immobilized to poly(lactic-co-glycolic acid) (PLGA) magnetic nanoparticles (PMNP) to prepare peptide/rtPA conjugated PMNPs (pPMNP-rtPA). During preparation, PMNP was first surface modified with avidin, which could interact with biotin. This is followed by binding PMNP-avidin with biotin-PEG-rtPA (or biotin-PEG-peptide), which was prepared beforehand by binding rtPA (or peptide) to biotin-PEG-maleimide while using click chemistry between maleimide and the single –SH group in rtPA (or peptide). The physicochemical property characterization indicated the successful preparation of the magnetic nanoparticles with full retention of rtPA fibrinolysis activity, while biological response studies underlined the high biocompatibility of all magnetic nanoparticles from cytotoxicity and hemolysis assays in vitro. The magnetic guidance and fibrin binding effects were also confirmed, which led to a higher thrombolysis rate in vitro using PMNP-rtPA or pPMNP-rtPA when compared to free rtPA after static or dynamic incubation with blood clots. Using pressure-dependent clot lysis model in a flow system, dual targeted pPMNP-rtPA could reduce the clot lysis time for reperfusion by 40% when compared to free rtPA at the same drug dosage. From in vivo targeted thrombolysis in a rat embolic model, pPMNP-rtPA was used at 20% of free rtPA dosage to restore the iliac blood flow in vascular thrombus that was created by injecting a blood clot to the hind limb area.
Highlights
Within the two ischemic and hemorrhagic categories of stroke, the ischemic stroke accounts for ~87% of all cases, according to the American Stroke Association [1]
In this study, poly(lactic-co-glycolic acid) (PLGA) was used as a polymer matrix to encapsulate hydrophobic oleic acid-coated iron oxide MNP (OMNP) and form PLGA magnetic nanoparticles (PMNP) in a single oil-in-water (O/W) emulsion step in order to prevent aggregation of iron oxide MNP
The bond formation between biotin and avidin is very rapid and the formed bond is unaffected by pH, organic solvents, or temperature, which facilitates the conjugation of recombinant tissue plasminogen activator (rtPA) to PLGA MNP (PMNP)-avidin
Summary
Within the two ischemic and hemorrhagic categories of stroke, the ischemic stroke accounts for ~87% of all cases, according to the American Stroke Association [1]. We have extensively studied different MNP-based polymeric nano-carrier for magnetically targeted delivery of rtPA, being prepared either by immobilization of rtPA on the particle surface or by encapsulation within the polymeric matrix, and demonstrated the improved thrombolytic efficacy both in vitro and in vivo [13,14,15]. The ligands used for rtPA delivery targeting thrombus include peptide targeting FXIII [18], fibrin antibody targeting fibrin [19], Arg-Gly-Asp (RGD) [20], cyclicRGD [21], or other peptides [22] targeting GP IIb/IIIa, and fucoidan targeting p-selectin [23] Those targeted thrombolysis strategies have demonstrated promising results in improving thrombolytic efficiency with shorter clot lysis time, which makes possible the use of reduced drug dosage to circumvent associated side effects, such as the bleeding risk [24]. The clot thrombolysis efficiency of pPMNP-rtPA was tested in vivo in a rat embolic model
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