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Shear-Activated Nanotherapeutics for Drug Targeting to Obstructed Blood Vessels

Science  10 Aug 2012:
Vol. 337, Issue 6095, pp. 738-742
DOI: 10.1126/science.1217815

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  1. Fig. 1

    Microscale SA-NTs disperse into NPs only when exposed to pathological shear stresses. (A) Scanning electron micrographs of the microscale (~2 to 5 μm) SA-NTs (left) and the PLGA NPs (~180 nm) used to produce them (right). Scale bar, 2 μm. (B) Fluorescence micrographs demonstrating intact SA-NTs (top) and NPs dispersed after their exposure to 1000 dyne/cm2 for 10 min by using a rheometer (bottom). Scale bar, 10 μm. (C) Quantification of release of fluorescent NPs from the SA-NTs as a function of shear revealed that exposure to pathological levels of shear (≥100 dyne/cm2 for 1 min) caused large increase in the breakup of the microscale aggregates into NPs compared with physiological levels of shear (1 or 10 dyne/cm2) (*P < 0.005). (D) CFD simulations comparing fluidic shear stress in a normal coronary artery (left) and a stenotic vessel with a 60% lumen obstruction (right). Left inset shows the corresponding angiogram of the stenotic left coronary artery in a 63-year-old male patient.

  2. Fig. 2

    Shear-induced dissociation of SA-NTs and NP targeting under hemodynamic conditions in microfluidic devices. (A) A microfluidic vascular stenosis model showing how SA-NTs (large spheres) should remain intact in the prestenotic region but then break up into NPs (small spheres) when they flow through a constriction (90% lumen occlusion) and can accumulate in endothelial cells lining the bottom of the channel. (B) A photograph of the microdevice that mimics vascular stenosis fabricated in PDMS. (C) CFD simulations of the microfluidic device shown in (B) demonstrating that a physiological inlet shear rate of 1000 s−1 (10 dyne/cm2) upstream from the constriction increases to a pathological level of ~100,000 s−1 (1000 dyne/cm2) in the region displaying 90% lumen occlusion. (D) Graph shows a >10-fold increase in release of fluorescent NPs from SA-NTs when they are perfused through the channel shown in (B) compared with flow through an unconstricted channel (*P < 0.005). Fluorescent micrographs compare the NPs collected in the outflow from the control channel (top) versus the constricted channel (bottom). Scale bar, 2 μm. (E) Graph demonstrates that many more fluorescent NPs accumulate in endothelial cells lining the downstream area (poststenosis) of the constriction relative to an upstream area (P < 0.005). Fluorescence microscopic images show cells from regions before (left) and after (right) the constriction. Scale bar, 20 μm.

  3. Fig. 3

    Shear-targeting of a thrombolytic drug in a mouse arterial thrombosis model using SA-NTs. (A) Schematic of the experimental strategy. Ferric chloride injury initiates formation of a thrombus (top) that grows to partially obstruct blood flow (top middle). Intraveneously injected SA-NTs dissociate into NPs at the thrombus site because of the rise in local shear stress (bottom middle). Accumulation of tPA-coated NPs and binding to the clot at the occlusion site progressively dissolve the obstruction (bottom). (B) Sequential intravital fluorescence microscopic images of a thrombus in a partially occluded mesenteric artery recorded over a 5-min period beginning after bolus injection of fluorescent tPA-coated SA-NTs (1 mg NPs; 50 ng tPA) 8 min after injury initiation. Scale bar, 100 μm. The NPs accumulate at the clot, first visualizing its location and then demonstrating clearance of the clot within 5 min after injection at the bottom (movie S1). (C) Sequence of intravital fluorescence microscopic images recorded over a 5-min period showing fluorescently labeled platelets accumulated within a forming thrombus that partially occludes a mesenteric artery 8 min after injury. Thrombosis is then treated with injection of either tPA-carrying SA-NTs (50 ng tPA) (left) or PBS (right). Scale bar, 100 μm. The clot on the left is greatly reduced in size within 5 min after SA-NTs injection (movie S2), whereas the control vessel on the right fully occludes over the same time period. (D) Graph showing that a bolus injection of SA-NTs carrying 50 ng tPA (tPA-SA-NT) significantly delayed the time to full vascular occlusion in FeCl-injured vessels (***P < 0.0005), whereas administration of the same concentration of soluble tPA (free tPA), uncoated SA-NTs (bare SA-NT), tPA-coated NPs that were artificially dissociated from SA-NTs before injection (dispersed tPA-NPs), and heat-fused NP microaggregates with tPA coating that do not dissociate (fused SA-NT) did not produce any significant delay in thrombosis.

  4. Fig. 4

    Shear-targeting of a thrombolytic drug to vascular emboli in vitro and therapeutic delivery in a mouse pulmonary embolism model. (A) Time-lapse fluorescence (top) and bright field (bottom) views of artificial microemboli (~250 μm) in a microfluidic channel before (0 min) and 1 or 60 min after injection of SA-NTs coated with tPA (50 ng/ml), showing progressive lysis of the clots over time (movie S3). Scale bar, 100 μm. (B) Graph showing enhanced emboli lysis kinetics induced by tPA-coated SA-NTs (50 ng/ml, blue line) compared with soluble tPA (red line). (C) Fluorescence (top) and phase contrast (bottom) views of histological sections of normal (left) versus obstructed (right) pulmonary arteries showing local accumulation of fluorescent NPs within the obstructing emboli in a mouse ex vivo lung ventilation-perfusion model. Scale bar, 100 μm. (D) Graph showing almost a 20-fold increase (P < 0.005) in accumulation of fluorescent NPs in regions of obstructed versus nonobstructed vessels, as detected with microfluorimetry. (E) Real-time measurements of pulmonary artery pressure in the ex vivo pulmonary embolism model showing that the tPA-coated SA-NTs (blue line) reversed pulmonary artery hypertension within approximately 1 hour, whereas the same concentration (50 ng/ml) of free tPA was ineffective (red line). (F) Graph showing that tPA carrying SA-NTs normalize pulmonary artery pressure within 1 hour, whereas the same concentration of free tPA (50 ng/ml) or a 10-times-higher dose (500 ng/ml) did not reduce pulmonary artery pressure (*P < 0.005); only a 100-fold-higher dose (5000 ng/ml) produced similar effects. (G) Survival curve showing that almost all (86%) of the mice injected with the tPA-coated SA-NTs survived, whereas all control mice died within 45 min after injection of fibrin clots that caused acute emboli formation.