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Direct Imaging of Cerebral Thromboemboli Using Computed Tomography and Fibrin-targeted Gold Nanoparticles.

Kim JY, Ryu JH, Schellingerhout D, Sun IC, Lee SK, Jeon S, Kim J, Kwon IC, Nahrendorf M, Ahn CH, Kim K, Kim DE - Theranostics (2015)

Bottom Line: Glycol-chitosan-coated gold nanoparticles (GC-AuNPs) were synthesized and conjugated to fibrin-targeting peptides, forming fib-GC-AuNP.This targeted imaging agent and non-targeted control agent were characterized in vitro and in vivo in C57Bl/6 mice (n = 107) with FeCl3-induced carotid thrombosis and/or embolic ischemic stroke.Fibrin-binding capacity was superior with fib-GC-AuNPs compared to GC-AuNPs, with thrombi visualized as high density on microCT (mCT). mCT imaging using fib-GC-AuNP allowed the prompt detection and quantification of cerebral thrombi, and monitoring of tPA-mediated thrombolytic effect, which reflected histological stroke outcome.

View Article: PubMed Central - PubMed

Affiliation: 1. Molecular Imaging and Neurovascular Research Laboratory, Dongguk University College of Medicine, Goyang, South Korea;

ABSTRACT
Computed tomography (CT) is the current standard for time-critical decision-making in stroke patients, informing decisions on thrombolytic therapy with tissue plasminogen activator (tPA), which has a narrow therapeutic index. We aimed to develop a CT-based method to directly visualize cerebrovascular thrombi and guide thrombolytic therapy. Glycol-chitosan-coated gold nanoparticles (GC-AuNPs) were synthesized and conjugated to fibrin-targeting peptides, forming fib-GC-AuNP. This targeted imaging agent and non-targeted control agent were characterized in vitro and in vivo in C57Bl/6 mice (n = 107) with FeCl3-induced carotid thrombosis and/or embolic ischemic stroke. Fibrin-binding capacity was superior with fib-GC-AuNPs compared to GC-AuNPs, with thrombi visualized as high density on microCT (mCT). mCT imaging using fib-GC-AuNP allowed the prompt detection and quantification of cerebral thrombi, and monitoring of tPA-mediated thrombolytic effect, which reflected histological stroke outcome. Furthermore, recurrent thrombosis could be diagnosed by mCT without further nanoparticle administration for up to 3 weeks. fib-GC-AuNP-based direct cerebral thrombus imaging greatly enhance the value and information obtainable by regular CT, has multiple uses in basic / translational vascular research, and will likely allow personalized thrombolytic therapy in clinic by a) optimizing tPA-dosing to match thrombus burden, b) enabling the rational triage of patients to more radical therapies such as endovascular clot-retrieval, and c) potentially serving as a theranostic platform for targeted delivery of concurrent thrombolysis.

No MeSH data available.


Related in: MedlinePlus

Electron microscopy study demonstrates that targeted fib-GC-AuNPs are more densely clustered in cerebral thrombus than the non-targeted equivalent, correlating well with the more sensitive mCT visualization of thrombus by the targeted compound. A-J, Axial mCT image (A; 2 mm thickness), ex vivo Cy5.5 near-infrared fluorescent (NIRF) thrombus image (B) and visible light image (C) of a representative C57Bl/6 mouse brain with the Y-shape embolic clot at the left distal internal carotid artery bifurcation area after injection with fib-GC-AuNP. (D-F) Coronal reformations of the mCT (D) in the same plane as sectioned (at red-colored brain regions in A and B) brain (2mm thickness) imaged optically for Cy5.5 (E), and gross brain digitally photographed (F). Red reticles in D-F contain the anteromedial portion of the Y-shape embolic clot, overlaid by the red-color bars in A and B. (G) The gross brain photographed after removing the red reticle area in D-F (left), Cy5.5 NIRF thrombus image of the brain section (middle), and 2,3,5-triphenyl-tetrazolium chloride (TTC)-stained brain section (right). (H) Autofluorescent green channel image of the removed (red reticle area) brain tissue (left) and Cy5.5 NIRF thrombus image (right). (I) Coronal mCT image (0.053 mm thickness) that corresponds to the green line crossing the removed thrombotic area in H. The inset shows a magnified view of the green-squared thrombotic area. (J) Electron microscopy images captured in the 60 nm thick toluidine blue-stained section of the removed tissue (J1-4), which corresponds to the green-squared area of I and has cerebral thrombus occluding the left middle cerebral artery. Please note that fib-GC-AuNPs are clustered within the thrombus; and most of the nanoparticles are not aggregated, suggesting that they individually diffused into the clot and were captured. A'-J', The same data for an animal injected with non-targeted GC-AuNPs. Please note that non-targeted GC-AuNPs are much less dense in the cerebral thrombus (J'1-4). mCT images show correspondingly weak (A') or absent (D' and I') thrombus-related hyperdense lesions. Scale bars = 2 mm (A-I); 100 μm (insets of I, J); 1 μm (J1-4, left panel); 0.2 μm (J1-4, right panel).
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Figure 4: Electron microscopy study demonstrates that targeted fib-GC-AuNPs are more densely clustered in cerebral thrombus than the non-targeted equivalent, correlating well with the more sensitive mCT visualization of thrombus by the targeted compound. A-J, Axial mCT image (A; 2 mm thickness), ex vivo Cy5.5 near-infrared fluorescent (NIRF) thrombus image (B) and visible light image (C) of a representative C57Bl/6 mouse brain with the Y-shape embolic clot at the left distal internal carotid artery bifurcation area after injection with fib-GC-AuNP. (D-F) Coronal reformations of the mCT (D) in the same plane as sectioned (at red-colored brain regions in A and B) brain (2mm thickness) imaged optically for Cy5.5 (E), and gross brain digitally photographed (F). Red reticles in D-F contain the anteromedial portion of the Y-shape embolic clot, overlaid by the red-color bars in A and B. (G) The gross brain photographed after removing the red reticle area in D-F (left), Cy5.5 NIRF thrombus image of the brain section (middle), and 2,3,5-triphenyl-tetrazolium chloride (TTC)-stained brain section (right). (H) Autofluorescent green channel image of the removed (red reticle area) brain tissue (left) and Cy5.5 NIRF thrombus image (right). (I) Coronal mCT image (0.053 mm thickness) that corresponds to the green line crossing the removed thrombotic area in H. The inset shows a magnified view of the green-squared thrombotic area. (J) Electron microscopy images captured in the 60 nm thick toluidine blue-stained section of the removed tissue (J1-4), which corresponds to the green-squared area of I and has cerebral thrombus occluding the left middle cerebral artery. Please note that fib-GC-AuNPs are clustered within the thrombus; and most of the nanoparticles are not aggregated, suggesting that they individually diffused into the clot and were captured. A'-J', The same data for an animal injected with non-targeted GC-AuNPs. Please note that non-targeted GC-AuNPs are much less dense in the cerebral thrombus (J'1-4). mCT images show correspondingly weak (A') or absent (D' and I') thrombus-related hyperdense lesions. Scale bars = 2 mm (A-I); 100 μm (insets of I, J); 1 μm (J1-4, left panel); 0.2 μm (J1-4, right panel).

Mentions: There was a clear difference between targeted and non-targeted nanoparticles on cerebral CT imaging (Figure 3), with excellent thrombus demonstration on CT with targeted nanoparticles, and excellent matching to corresponding histological findings, while CT demonstration failed for non-targeted nanoparticles, even though thrombi were clearly present on histology. After intravenous administration of fib-GC-AuNPs in mice with embolic stroke, in vivo mCT imaging detected hyperdense lesions in the brain, and these lesions co-localized with Cy5.5 fluorescent thromboemboli on ex vivo NIRF images of the whole brain tissue and 2 mm thick sections (Figure 3 and Supplementary Material: Figure S4). NIRF microscopic imaging of 10 μm thick cryosections of the tissue confirmed thrombi filling in cerebral arteries (Figure 3). However, non-targeted GC-AuNPs could not always visualize cerebral thrombus in the COW, even when these thrombi could be clearly imaged by ex vivo NIRF imaging of the brain tissues and NIRF microscopic imaging of the brain sections. Histological findings clearly showed that mCT signal corresponded to cerebral arterial thrombi on H&E-stained or fibrin immune-stained brain sections. Electron microscopy studies confirmed that numerous fib-GC-AuNPs were scattered within the thrombi, whereas GC-AuNPs were only sparsely observed (Figure 4). Target-specific and localized accumulation of fib-GC-AuNPs in millimeter-sized carotid or cerebral thrombi may have allowed successful mCT demonstration of the thrombi even at the low doses administered (2 or 12 mg/kg vs. 20 or 120 mg/kg, respectively).


Direct Imaging of Cerebral Thromboemboli Using Computed Tomography and Fibrin-targeted Gold Nanoparticles.

Kim JY, Ryu JH, Schellingerhout D, Sun IC, Lee SK, Jeon S, Kim J, Kwon IC, Nahrendorf M, Ahn CH, Kim K, Kim DE - Theranostics (2015)

Electron microscopy study demonstrates that targeted fib-GC-AuNPs are more densely clustered in cerebral thrombus than the non-targeted equivalent, correlating well with the more sensitive mCT visualization of thrombus by the targeted compound. A-J, Axial mCT image (A; 2 mm thickness), ex vivo Cy5.5 near-infrared fluorescent (NIRF) thrombus image (B) and visible light image (C) of a representative C57Bl/6 mouse brain with the Y-shape embolic clot at the left distal internal carotid artery bifurcation area after injection with fib-GC-AuNP. (D-F) Coronal reformations of the mCT (D) in the same plane as sectioned (at red-colored brain regions in A and B) brain (2mm thickness) imaged optically for Cy5.5 (E), and gross brain digitally photographed (F). Red reticles in D-F contain the anteromedial portion of the Y-shape embolic clot, overlaid by the red-color bars in A and B. (G) The gross brain photographed after removing the red reticle area in D-F (left), Cy5.5 NIRF thrombus image of the brain section (middle), and 2,3,5-triphenyl-tetrazolium chloride (TTC)-stained brain section (right). (H) Autofluorescent green channel image of the removed (red reticle area) brain tissue (left) and Cy5.5 NIRF thrombus image (right). (I) Coronal mCT image (0.053 mm thickness) that corresponds to the green line crossing the removed thrombotic area in H. The inset shows a magnified view of the green-squared thrombotic area. (J) Electron microscopy images captured in the 60 nm thick toluidine blue-stained section of the removed tissue (J1-4), which corresponds to the green-squared area of I and has cerebral thrombus occluding the left middle cerebral artery. Please note that fib-GC-AuNPs are clustered within the thrombus; and most of the nanoparticles are not aggregated, suggesting that they individually diffused into the clot and were captured. A'-J', The same data for an animal injected with non-targeted GC-AuNPs. Please note that non-targeted GC-AuNPs are much less dense in the cerebral thrombus (J'1-4). mCT images show correspondingly weak (A') or absent (D' and I') thrombus-related hyperdense lesions. Scale bars = 2 mm (A-I); 100 μm (insets of I, J); 1 μm (J1-4, left panel); 0.2 μm (J1-4, right panel).
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Figure 4: Electron microscopy study demonstrates that targeted fib-GC-AuNPs are more densely clustered in cerebral thrombus than the non-targeted equivalent, correlating well with the more sensitive mCT visualization of thrombus by the targeted compound. A-J, Axial mCT image (A; 2 mm thickness), ex vivo Cy5.5 near-infrared fluorescent (NIRF) thrombus image (B) and visible light image (C) of a representative C57Bl/6 mouse brain with the Y-shape embolic clot at the left distal internal carotid artery bifurcation area after injection with fib-GC-AuNP. (D-F) Coronal reformations of the mCT (D) in the same plane as sectioned (at red-colored brain regions in A and B) brain (2mm thickness) imaged optically for Cy5.5 (E), and gross brain digitally photographed (F). Red reticles in D-F contain the anteromedial portion of the Y-shape embolic clot, overlaid by the red-color bars in A and B. (G) The gross brain photographed after removing the red reticle area in D-F (left), Cy5.5 NIRF thrombus image of the brain section (middle), and 2,3,5-triphenyl-tetrazolium chloride (TTC)-stained brain section (right). (H) Autofluorescent green channel image of the removed (red reticle area) brain tissue (left) and Cy5.5 NIRF thrombus image (right). (I) Coronal mCT image (0.053 mm thickness) that corresponds to the green line crossing the removed thrombotic area in H. The inset shows a magnified view of the green-squared thrombotic area. (J) Electron microscopy images captured in the 60 nm thick toluidine blue-stained section of the removed tissue (J1-4), which corresponds to the green-squared area of I and has cerebral thrombus occluding the left middle cerebral artery. Please note that fib-GC-AuNPs are clustered within the thrombus; and most of the nanoparticles are not aggregated, suggesting that they individually diffused into the clot and were captured. A'-J', The same data for an animal injected with non-targeted GC-AuNPs. Please note that non-targeted GC-AuNPs are much less dense in the cerebral thrombus (J'1-4). mCT images show correspondingly weak (A') or absent (D' and I') thrombus-related hyperdense lesions. Scale bars = 2 mm (A-I); 100 μm (insets of I, J); 1 μm (J1-4, left panel); 0.2 μm (J1-4, right panel).
Mentions: There was a clear difference between targeted and non-targeted nanoparticles on cerebral CT imaging (Figure 3), with excellent thrombus demonstration on CT with targeted nanoparticles, and excellent matching to corresponding histological findings, while CT demonstration failed for non-targeted nanoparticles, even though thrombi were clearly present on histology. After intravenous administration of fib-GC-AuNPs in mice with embolic stroke, in vivo mCT imaging detected hyperdense lesions in the brain, and these lesions co-localized with Cy5.5 fluorescent thromboemboli on ex vivo NIRF images of the whole brain tissue and 2 mm thick sections (Figure 3 and Supplementary Material: Figure S4). NIRF microscopic imaging of 10 μm thick cryosections of the tissue confirmed thrombi filling in cerebral arteries (Figure 3). However, non-targeted GC-AuNPs could not always visualize cerebral thrombus in the COW, even when these thrombi could be clearly imaged by ex vivo NIRF imaging of the brain tissues and NIRF microscopic imaging of the brain sections. Histological findings clearly showed that mCT signal corresponded to cerebral arterial thrombi on H&E-stained or fibrin immune-stained brain sections. Electron microscopy studies confirmed that numerous fib-GC-AuNPs were scattered within the thrombi, whereas GC-AuNPs were only sparsely observed (Figure 4). Target-specific and localized accumulation of fib-GC-AuNPs in millimeter-sized carotid or cerebral thrombi may have allowed successful mCT demonstration of the thrombi even at the low doses administered (2 or 12 mg/kg vs. 20 or 120 mg/kg, respectively).

Bottom Line: Glycol-chitosan-coated gold nanoparticles (GC-AuNPs) were synthesized and conjugated to fibrin-targeting peptides, forming fib-GC-AuNP.This targeted imaging agent and non-targeted control agent were characterized in vitro and in vivo in C57Bl/6 mice (n = 107) with FeCl3-induced carotid thrombosis and/or embolic ischemic stroke.Fibrin-binding capacity was superior with fib-GC-AuNPs compared to GC-AuNPs, with thrombi visualized as high density on microCT (mCT). mCT imaging using fib-GC-AuNP allowed the prompt detection and quantification of cerebral thrombi, and monitoring of tPA-mediated thrombolytic effect, which reflected histological stroke outcome.

View Article: PubMed Central - PubMed

Affiliation: 1. Molecular Imaging and Neurovascular Research Laboratory, Dongguk University College of Medicine, Goyang, South Korea;

ABSTRACT
Computed tomography (CT) is the current standard for time-critical decision-making in stroke patients, informing decisions on thrombolytic therapy with tissue plasminogen activator (tPA), which has a narrow therapeutic index. We aimed to develop a CT-based method to directly visualize cerebrovascular thrombi and guide thrombolytic therapy. Glycol-chitosan-coated gold nanoparticles (GC-AuNPs) were synthesized and conjugated to fibrin-targeting peptides, forming fib-GC-AuNP. This targeted imaging agent and non-targeted control agent were characterized in vitro and in vivo in C57Bl/6 mice (n = 107) with FeCl3-induced carotid thrombosis and/or embolic ischemic stroke. Fibrin-binding capacity was superior with fib-GC-AuNPs compared to GC-AuNPs, with thrombi visualized as high density on microCT (mCT). mCT imaging using fib-GC-AuNP allowed the prompt detection and quantification of cerebral thrombi, and monitoring of tPA-mediated thrombolytic effect, which reflected histological stroke outcome. Furthermore, recurrent thrombosis could be diagnosed by mCT without further nanoparticle administration for up to 3 weeks. fib-GC-AuNP-based direct cerebral thrombus imaging greatly enhance the value and information obtainable by regular CT, has multiple uses in basic / translational vascular research, and will likely allow personalized thrombolytic therapy in clinic by a) optimizing tPA-dosing to match thrombus burden, b) enabling the rational triage of patients to more radical therapies such as endovascular clot-retrieval, and c) potentially serving as a theranostic platform for targeted delivery of concurrent thrombolysis.

No MeSH data available.


Related in: MedlinePlus