Clot-targeted magnetic hyperthermia permeabilizes blood clots to make them more susceptible to thrombolysis

Abstract

Background: Thrombolysis is a frontline treatment for stroke, which involves the application of tissue plasminogen activator (tPA) to trigger endogenous clot-degradation pathways. However, it is only effective within 4.5 h of symptom onset because of clot contraction preventing tPA permeation into the clot. Magnetic hyperthermia (MH) mediated by tumor-targeted magnetic nanoparticles is used to treat cancer by using local heat generation to trigger apoptosis of cancer cells.

Objectives: To develop clot-targeting magnetic nanoparticles to deliver MH to the surface of human blood clots, and to assess whether this can improve the efficacy of thrombolysis of contracted blood clots.

Methods: Clot-targeting magnetic nanoparticles were developed by functionalizing iron oxide nanoparticles with an antibody recognizing activated integrin αIIbβ3 (PAC-1). The magnetic properties of the PAC-1-tagged magnetic nanoparticles were characterized and optimized to deliver clot-targeted MH.

Results: Clot-targeted MH increases the efficacy of tPA-mediated thrombolysis in contracted human blood clots, leading to a reduction in clot weight. MH increases the permeability of the clots to tPA, facilitating their breakdown. Scanning electron microscopy reveals that this effect is elicited through enhanced fibrin breakdown and triggering the disruption of red blood cells on the surface of the clot. Importantly, endothelial cells viability in a three-dimensional blood vessel model is unaffected by exposure to MH.

Conclusions: This study demonstrates that clot-targeted MH can enhance the thrombolysis of contracted human blood clots and can be safely applied to enhance the timeframe in which thrombolysis is effective.

Keywords: blood clot permeation; clot-targeted magnetic hyperthermia; functionalized iron oxide nanoparticles; stroke; thrombolysis.

FIGURE 1

Iron oxide nanoparticles (IONPs) were successfully functionalized with PAC‐1 antibody (f‐IONPs) by covalent immobilization on nanoparticle surface. (A) Schematic representation of the functionalized iron oxide nanoparticle consisting of an iron oxide core, coating of citric acid, and conjugated with PAC‐1 antibody. (B) Transmission electron microscopy (TEM) of IONPs. (C) Confocal microscopies of IONPs and f‐IONPs after incubation with FITC‐Labeled anti‐IgM antibody. (D) Fluorescence measurements of IONPs and f‐IONPs after incubation with FITC‐labeled anti‐IgM antibody. (E) UV–vis spectroscopy of nonfunctionalized and functionalized nanoparticles. n = 3, **p < .01, cyan scale bar = 200 nm, black scale bar = 500 μm, error bars indicate SErM.

FIGURE 2

f‐IONPs labeled activated platelets and ex vivo blood clots and elicit hyperthermic responses upon stimulation with AMF. (A) Fluorescence measurements of resting (black and red bar) and activated (blue and green bar) incubated with f‐IONPs and, subsequently, with FITC‐labeled anti‐IgM antibody. (B) Representative AC susceptibility measurement of 200 μl PPP samples containing a human blood clot only, 0.33 g_Fe L⁻¹ μg of f‐IONPs, or blood clots and f‐IONPs together. (C) Representative confocal image of ex vivo‐derived human blood clot (black shadow) preincubated with fluo‐f‐IONPs (fluo‐f‐IONPs are shown in green). (D) AC magnetization loops of 2 g_Fe L⁻¹ of either IONPs or f‐IONPs measured at 100 kHz and 30 mT. (E) 3D representation of AMF generator system. (F) Specific absorption rate measurements of 2 g_Fe L⁻¹ IONPs dispersed in water at 105 kHz – 46 mT, 156 kHz – 42 mT, 205 kHz – 40 mT, and 306 kHz and 30 mT. (G) Heating curves of PPP alone or containing 0.021, 0.089, and 0.33 g_Fe L⁻¹ exposed to an AMF of 306 kHz and 30 mT for 30 min. n = 8 for platelet labelling measurements, n = 4 for confocal images with fluo‐IONPs, n = 3 for AC susceptometry and magnetometry measurements. *p < .05, Scale bar = 200 μm, error bars indicate SErM.

FIGURE 3

Clot‐targeted MH enhances the thrombolytic effect of highly contracted blood clots. (A) Pictures of ex vivo‐generated whole blood clots fabricated by 200 μl of human whole blood incubated with 0.33 g_Fe L⁻¹ f‐IONPs without further treatment (control), treated with 2 μg of tPA (tPa), or a combination of both tPA and f‐IONPs mediated MH (tPa + MH) for 30 min. (B) Absorbance measurements (415 nm) of the supernatant of the sample after the indicated treatment. Representative images of the supernatants are also shown. (C) Mean clot weight after treatment. AMF conditions: 306 kHz and 30 mT. The clots from the control group were kept 30 min at 37°C. n = 15. *p < .05 between groups; ***p < .001 between groups and control; error bars indicate SErM.

FIGURE 4

Magnetic hyperthermia increases the permeability of ex vivo‐derived thrombi. Thrombi were fabricated by ADP stimulation of platelet‐rich plasma (PRP). (A, C, E, G) Thrombi were treated with 0.33 g_Fe L⁻¹ f‐IONPs in absence of AMF or (B, D, F, H) exposed to an AMF of 306 kHz and 30 mT for 30 min. The thrombi were then exposed to an HBS solution containing 3 μM rhodamine‐B‐labeled 70 kDa dextran. (A, B) Bright field images of representative thrombi. (C, D) DiOC₆ fluorescent images of representative slides of the same thrombi. (E, F) Representative slides of combined bright field images and fluorescent dextran of clots. Red arrows indicate fluorescent dextran diffusing into the clots. (G, H) 3D reconstruction of the clot using DiOC₆ staining to indicate the outer edge of the clot (green) and permeation of the fluorescent dextran into the thrombi (red). n = 10, Scale bar = 500 μm.

FIGURE 5

Clot‐targeted MH enhances tPA‐mediated fibrin breakdown and triggers greater red blood cell release from the surface of highly contracted ex vivo‐generated blood clots. Representative SEM images of ex vivo whole blood clots surfaces of (A) nontreated or (B) treated with 0.33 g_Fe L⁻¹ f‐IONPs mediated MH, (C) 2 μg tPa, and (D1,2) a combined treatment with the same amount of tPa and 0.33 g_Fe L⁻¹ f‐IONPs mediated MH for 30 min. The control group was held at a temperature of 37°C for the same period as the MH treatment. Image D1 was obtained from the same donor as figures A–C and demonstrates the presence of spherical erythrocytes and a loss of fibrin on the surface of these clots. Image D2 is taken from another donor and shows the presence of erythrocytes with membrane blebbing (red arrows). AMF conditions: 306 kHz and 30 mT. n = 3.

FIGURE 6

MH using an optimized dose of f‐IONPs continues enhancing thrombolytic effect of highly contracted blood clots. (A) Pictures of ex vivo‐generated whole blood clots fabricated by 200 μl of human whole blood with no treatment (control), incubated with 0.021 g_Fe L⁻¹ f‐IONPs or 0.089 g_Fe L⁻¹ f‐IONPs, both in combination with 2 μg of tPA (tPa) only (‐MH) or with AMF exposure for 30 min (+MH). Representative images of the supernatants are also shown. (B) Absorbance measurements (415 nm) of the supernatant of the sample after the indicated treatment. (C) Mean clot weight after treatment. n = 11 *p < .05, **p < .05, ***p < .001; error bars indicate SErM.

FIGURE 7

MH using an optimized dose of f‐IONPs does not affect the viability of the surrounding endothelium. (A) Schematic representation of the experimental setup to assess HUVEC viability after exposure to magnetic hyperthermia. A 3D human tissue engineered intimal layer (TEIL) model was produced by growing a HUVEC monolayer on the surface of a 3D collagen hydrogel. The TEIL was then exposed to cell culture media in the presence or absence of either f‐IONPs and AMF.The hydrogels were then subsequently stained with a fluorescent live/dead cell stain kit. Viability of HUVECs was imaged and averaged over five different regions of interest on the surface of each TEIL sample as shown. (B, C) Representative images of the live/dead cell staining observed in TEIL treated with either nothing additional (control), an AMF alone (AMF), 0.089 g_Fe L⁻¹ f‐IONPs in the absence of an AMF (f‐IONPs), and 0.089 g_Fe L⁻¹ f‐IONPs in the presence on AMF to elicit MH either immediately after the experiments or (B) 24 h after (C). (D, E) Mean HUVEC viability of the TEIL immediately after the experiments (D) or 24 h after the treatment (E). AMF conditions: 306 kHz and 30 mT. n = 8, Scale bar = 200 μm, error bars indicate SErM.