Fibrin-Targeted and H2O2‑Responsive Nanoparticles as a Theranostics for Thrombosed Vessels Changsun Kang,† Sian Gwon,† ChulGyu Song,‡ Peter M. Kang,§ Seong-Cheol Park,∥ Jongho Jeon,⊥ Do Won Hwang,# and Dongwon Lee*,†,¶ †
Department of BIN Convergence Technology, ‡Department of Electronics Engineering, and ¶Department of Polymer·Nano Science and Technology, Chonbuk National University, Backje-daero 567, Jeonju, Chonbuk 561-756, Korea § Cardiovascular Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, United States ∥ Department of Polymer Engineering, Sunchon National University, Sunchon, Chonnam 540-950, Korea ⊥ Advanced Radiation Technology Institute, Atomic Energy Research Institute, Jeongeup, Chonbuk 580-185, Korea # Department of Nuclear Medicine, Seoul National University College of Medicine, Seoul 151-742, Korea S Supporting Information *
ABSTRACT: A thrombus (blood clot) is formed in injured vessels to maintain the integrity of vasculature. However, obstruction of blood vessels by thrombosis slows blood flow, leading to death of tissues fed by the artery and is the main culprit of various life-threatening cardiovascular diseases. Herein, we report a rationally designed nanomedicine that could specifically image obstructed vessels and inhibit thrombus formation. On the basis of the physicochemical and biological characteristics of thrombi such as an abundance of fibrin and an elevated level of hydrogen peroxide (H2O2), we developed a fibrin-targeted imaging and antithrombotic nanomedicine, termed FTIAN, as a theranostic system for obstructive thrombosis. FTIAN inhibited the generation of H2O2 and suppressed the expression of tumor necrosis factor-alpha (TNF-α) and soluble CD40 ligand (sCD40L) in activated platelets, demonstrating its intrinsic antioxidant, anti-inflammatory, and antiplatelet activity. In a mouse model of ferric chloride (FeCl3)-induced carotid thrombosis, FTIAN specifically targeted the obstructive thrombus and significantly enhanced the fluorescence/ photoacoustic signal. When loaded with the antiplatelet drug tirofiban, FTIAN remarkably suppressed thrombus formation. Given its thrombus-specific imaging along with excellent therapeutic activities, FTIAN offers tremendous translational potential as a nanotheranostic agent for obstructive thrombosis. KEYWORDS: thrombosis, nanomedicine, hydrogen peroxide, antiplatelet, theranostics
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thrombus forms in injured vessels to maintain the integrity of the circulatory system and seal the breach as a hemostatic process becomes activated.4 A thrombus is an aggregation of blood factors, composed of mainly activated platelets and water-insoluble fibrin. Thrombus formation is known to be initiated by thrombogenic materials such as collagen, which is exposed to the flowing blood and triggers the accumulation and activation of platelets, whose main function is attributed to hemostasis.5 However, obstruction of blood vessels due to
ne of the most common causes of vascular diseases is atherosclerosis, which is buildup of occlusive and lipid-rich plaques in the arterial wall that induces hardening of the artery and reduces blood flow through the vessel.1 Atherosclerotic plaques develop through the accumulation of lipid deposits and form cells (lipid-laden macrophages) in the arterial wall.2 In particular, vulnerable plaques rich in macrophages and form cells are prone to rupture due to the soft and weak fibrous cap.3 Vascular injury caused by rupture of vulnerable plaques exposes collagen and various plaque components to the circulation, leading to the formation of blood clots (thrombi) in the lumen. The primary trigger for arterial thrombosis is the rupture of atherosclerotic plaques.2 A © 2017 American Chemical Society
Received: April 3, 2017 Accepted: May 8, 2017 Published: May 8, 2017 6194
DOI: 10.1021/acsnano.7b02308 ACS Nano 2017, 11, 6194−6203
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Scheme 1. A schematic diagram of FTIAN as a thrombus-specific theranostic agent that is able to image thrombi and exert potent antithrombotic activity.
thrombosis rapidly slows or stops blood flow, leading to the death of tissues fed by the artery. Obstruction of blood vessels to critical organs such as the heart, brain, and lung is a leading cause of death (nearly a third of the deaths each year) and longterm disability in the Western world.1,6,7 Tissue plasminogen activator is known to degrade fibrin and lyse the offending thrombus at the site of cerebral artery occlusion and is considered as the only approved therapy for acute ischemic stroke.8 Tirofiban, a glycoprotein IIb/IIIa receptor inhibitor, is also an effective antiplatelet agent in the management of acute coronary syndromes.9 These antithrombotic drugs are effective at reducing arterial thrombosis in patients with cardiovascular diseases. However, their clinical applications are limited by the narrow therapeutic window and adverse side effect such as bleeding at the local site of clinical intervention.2 Therefore, there has been great interest in the development of strategies that are able to detect specifically obstructed vessels and inhibit thrombosis effectively and rapidly.6,7,10,11 In the interrupted endothelium, platelet activation stimulates additional platelet recruitment, promoting thrombus formation.10,12 Platelet activation is associated with a burst of H2O2, which is more stable than other reactive oxygen species (ROS) and is therefore the most abundant and most involved in signaling in the vasculature.12−15 Several lines of evidence indicate that thrombus formation elevates oxidative stress, which plays a causal role in platelet pathophysiology.16,17 H2O2 derived from activated platelets and other vascular sources serves as a key mediator of platelet activation and aggregation. H2O2 mediates endothelial expression of inflammatory proteins and activates platelet−endothelium interactions, facilitating
inflammation and development of obstructive vascular diseases.18−20 Therefore, H2O2-mediated platelet activation is one of the mechanisms leading to prothrombotic phenotypes.5,12,21−23 In this regard, depletion of H2O2 and prevention of platelet activation would be a promising antithrombotic therapeutic strategy for the treatment of various vascular thrombotic diseases.16,24 Fibrin, a clotted plasma protein, is produced upon cleavage of the fibrinogen by thrombin and is one of the main components of a thrombus.1 Fibrin exists in the form of a cross-linked mesh deposited both inside and on the surface of the thrombus and plays important roles in the stabilization of the thrombus.25 Cross-linking of fibrin by factor XIII, a transglutaminase, stabilizes the clot, protecting it from mechanical stress and proteolytic attack. Therefore, fibrin has been widely explored as a target for site-specific delivery of anticlotting/anticoagulating agents and imaging agents to thrombi.1,26−29 Photoacoustic imaging is a functional optical imaging technique that relies on the light absorption, thermal expansion, and broadband acoustic waves generated from the interaction between nanosecond pulsed light and photoabsorbers in tissues.23,30,31 Photoacoustic imaging provides the advantages of both high spatial resolution and deep penetration of ultrasound imaging and chemical specificity of optical imaging and therefore has recently emerged as a complementary imaging modality.32,33 A number of photoabsorbers have been developed as a contrast agent for photoacoustic imaging to detect various pathological conditions.23,34 The capability of photoacoustic imaging to visualize vasculature has been shown extensively and has recently been translated to in vivo animal models.33 In particular, intravascular photoacoustic imaging is 6195
DOI: 10.1021/acsnano.7b02308 ACS Nano 2017, 11, 6194−6203
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Figure 1. Design and characterization of FTIAN. (a) UV−vis absorbance of fBAP. (b) Fluorescence spectra of fBAP at various concentrations. (c) Determination of critical micelle concentration of fBAP. The intensity ratio (I384/I373) for pyrene-loaded fBAP micelles is plotted against the concentration of fBAP. (d) Representative dynamic light scattering of FTIAN in PBS. (e) Representative transmission electron micrograph of FTIAN. (f) Changes in the hydrodynamic diameter of FTIAN under physiological conditions. H2O2 (1 mM) was added at a given time point. (g) H2O2-scavenging activity of FTIAN. Values are mean ± SD (n = 4). (h) Representative fluorescence image of IR820 and FTIAN at various concentrations. (i) Representative photoacoustic image of FTIAN embedded in agarose gel (1.0%). The photoacoustic signals were obtained during pulsed laser (808 nm) irradiation and overlaid onto the ultrasound images.
FTIAN and its translational potential as a nanotheranostic agent for obstructed thrombotic diseases.
able to image atherosclerotic plaque structure and composition.23,35 However, to our best knowledge, there have been no reports on the noninvasive photoacoustic imaging of thrombi using contrast agents that could specifically target thrombi and also exert intrinsic antithrombotic activity. By taking advantage of the physicochemical and biological characteristics of a thrombus, such as the abundance of fibrin and the elevated level of H2O2, we developed a fibrin-targeted imaging and antithrombotic nanomedicine, termed FTIAN (Scheme 1). FTIAN was designed to target fibrin specifically to image thrombi, scavenge H2O2, and prevent platelet activation, leading to the inhibition of thrombus formation in injured vasculature. FTIAN was prepared from near-infrared (NIR) fluorescent dye-conjugated boronate antioxidant polymers (fBAP) and fibrin-targeting lipopeptides. As shown in Scheme 1, fBAP and lipopeptides could self-assemble under aqueous conditions to form nanoparticles with fibrin-targeting peptides on their surface. When desired, FTIAN could also serve as a drug carrier to occlusive thrombi. Anti-inflammatory and antiplatelet activities of FTIAN were evaluated using cell culture models and a mouse model of FeCl3-induced carotid arterial injury. In vivo imaging studies indicated that FTIAN accumulates in a thrombus specifically and provides significantly high-contrast-enhanced photoacoustic imaging of the thrombus. In the present work, we report the rational design of
RESULTS AND DISCUSSION Synthesis and Characterization of FTIAN. To establish FTIAN, we first synthesized a fluorescent dye-conjugated H2O2-scavenging polymer, fBAP, as a framework of FTIAN. In the design, fBAP covalently incorporates H2O2-scavenging boronate and fluorescent IR820 and could release hydroxybenzyl alcohol (HBA) after H2O2-mediated oxidation of boronate. HBA is a major component of Gastrodia elata, which exerts anti-inflammatory and antiplatelet activities and has been used for the treatment of oxidative stress-associated diseases.36 However, HBA is unable to scavenge H2O2 and has poor bioavailability due to short half-life in circulation. In order to enhance its therapeutic efficacy and expand its clinical applications, we synthesized H2O2-responsive boronate antioxidant polymer (BAP) as a polymeric prodrug of HBA, which is able to rapidly scavenge H2O2 and release HBA in a H2O2triggered manner. As shown in Figure S1, H2O2-activatable antioxidant prodrug 1 was first synthesized, which would be activated by H2O2 to not only release HBA but also scavenge H2O2.37 1 was then reacted with acryloyl chloride to give a diacrylate compound, 2. BAP was obtained from Michael addition polymerization of poly(ethylene glycol) (PEG)acrylate, 2-(3,4-dimethoxyphenyl)ethanamine, tyramine, and 6196
DOI: 10.1021/acsnano.7b02308 ACS Nano 2017, 11, 6194−6203
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Figure 2. Antioxidant, anti-inflammatory, and antiplatelet activities of FTIAN. Given amounts of HBA or FTIAN were added to cells cultured in 1 mL of medium. (a) Cytotoxicity of FTIAN against arterial endothelial cells. (b) Protective effects of FTIAN on the H2O2-induced toxicity against arterial endothelial cells. (c) Representative optical micrographs of arterial endothelial cells activated by H2O2 in the presence of FTIAN. Scale bar: 50 μm. (d) Level of TNF-α in the arterial endothelial cells activated by H2O2. (e) Level of H2O2 in platelets activated by thrombin and CaCl2. (f) Level of sCD40L in the activated platelets. Values are mean ± SD (n = 4). *
