Molecularly Engineered Theranostic Nanoparticles for Thrombosed

Dec 19, 2017 - A thrombus (blood clot), composed mainly of activated platelets and fibrin, obstructs arteries or veins, leading to various life-threat...
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Molecularly Engineered Theranostic Nanoparticles for Thrombosed Vessels: H2O2‑Activatable Contrast-Enhanced Photoacoustic Imaging and Antithrombotic Therapy Eunkyeong Jung,† Changsun Kang,† Jeonghun Lee,† Donghyuck Yoo,† Do Won Hwang,‡ Dohyun Kim,‡ Seong-Cheol Park,§ Sang Kyoo Lim,∥ Chulgyu Song,⊥ and Dongwon Lee*,†,# †

Department of BIN Convergence Technology, Chonbuk National University, Baekjedaero 567, Jeonju, Chonbuk 54896, Republic of Korea ‡ Department of Nuclear Medicine, Seoul National University, Seoul 03083, Republic of Korea § Department of Polymer Engineering, Sunchon National University, Sunchon, Chonnam 57922, Republic of Korea ∥ Division of Nano & Energy Convergence Research, Daegu Gyeongbuk Institute of Science and Technology, Daegu 42988, Republic of Korea ⊥ Department of Electronic Engineering, Chonbuk National University, Baekjedaero 567, Jeonju, Chonbuk 54896, Republic of Korea # Department of Polymer·Nano Science and Technology, Chonbuk National University, Baekjedaero 567, Jeonju, Chonbuk 54896, Republic of Korea S Supporting Information *

ABSTRACT: A thrombus (blood clot), composed mainly of activated platelets and fibrin, obstructs arteries or veins, leading to various life-threatening diseases. Inspired by the distinctive physicochemical characteristics of thrombi such as abundant fibrin and an elevated level of hydrogen peroxide (H2O2), we developed thrombus-specific theranostic (T-FBM) nanoparticles that could provide H2O2-triggered photoacoustic signal amplification and serve as an antithrombotic nanomedicine. T-FBM nanoparticles were designed to target fibrin-rich thrombi and be activated by H2O2 to generate CO2 bubbles to amplify the photoacoustic signal. In the phantom studies, T-FBM nanoparticles showed significant amplification of ultrasound/photoacoustic signals in a H2O2-triggered manner. T-FBM nanoparticles also exerted H2O2-activatable antioxidant, anti-inflammatory, and antiplatelet activities on endothelial cells. In mouse models of carotid arterial injury, T-FBM nanoparticles significantly enhanced the photoacoustic contrast specifically in thrombosed vessels and significantly suppressed thrombus formation. We anticipate that T-FBM nanoparticles hold great translational potential as nanotheranostics for H2O2-associated cardiovascular diseases. KEYWORDS: thrombus, photoacoustic imaging, nanoparticles, theranostic, antithrombotic penetration from selective optical absorption.4−6 The only biologically safe mechanism to generate sufficient photoacoustic signal is thermoelastic expansion.5 In thermal expansion-based

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recently emerging biomedical imaging modality is photoacoustic imaging, which relies on the broadband acoustic waves generated from the interaction between nanosecond pulsed light and photoabsorbers in tissues.1−3 Photoacoustic imaging shares a common signal detection regimen with ultrasound imaging and therefore could combine high spatial resolution and traditional ultrasound depth © XXXX American Chemical Society

Received: September 15, 2017 Accepted: December 19, 2017 Published: December 19, 2017 A

DOI: 10.1021/acsnano.7b06560 ACS Nano XXXX, XXX, XXX−XXX

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Scheme 1. Schematic illustration of T-FBM nanoparticles as a thrombus-specific nanotheranostic agent. T-FBM nanoparticles target a fibrin-rich thrombus and serve as a H2O2-triggered photoacoustic signal amplifier but also an antithrombotic nanomedicine.

imaging.2 However, vaporization-based photoacoustic imaging with photoabsorber-containing nanodroplets has limitations such as poor stability of bubbles, short lifetime of signals, and precise timing control with laser irradiation.12,18−20 In this study, we developed H2O2-triggered bubble-generating fluorescent nanoparticles as self-contrast enhanced ultrasound/ photoacoustic imaging agents, which do not require gas precursors (perfluorocarbon) and external pulsed laser. Nearinfrared fluorescent-dye-conjugated boronated maltodextrin (FBM) was employed as a platform for H2O2-triggered bubblegenerating nanoparticles. FBM was prepared by conjugating borylbenzyl carbonate and fluorescent IR780 to maltodextrin. Borylbenzyl carbonate and IR780 were exploited as a H2O2triggered bubble-generating moiety and a photoabsorber, respectively. In chemical terms, aryl boronate is rapidly oxidized by H2O2 to generate phenol, which exists in equilibrium with phenolate.21 The phenolate of borylbenzyl carbonate undergoes rapid 1,6-elimination to produce quinone methide, leading to CO2 generation through subsequent decarboxylation of carbonate. Borylbenzyl carbonate and IR780 provide FBM with hydrophobic nature to confer solubility in organic solvents, allowing for nanoparticle formulation by conventional emulsion techniques. We therefore hypothesized that borylbenzyl carbonate of FBM nanoparticles reacts with H2O2 to form CO2 bubbles, which could amplify both ultrasound and photoacoustic signals, with a mechanism similar to laser-activatable microbubble-generating nanodroplets. In addition, quinone methide generated from H2O2-triggered oxidation of aryl boronate rapidly reacts with nucleophilic H2O to generate hydroxybenzyl alcohol (HBA).21,22 HBA is known to exert antioxidant and anti-inflammatory activity and also play protective roles in oxidative stress-related conditions such as ischemic heart injury.23−25 Therefore, FBM is expected to exert therapeutic activities by releasing antioxidant and anti-inflammatory HBA in a H2O2-triggered manner. Moreover, FBM could scavenge H2O2 through oxidation of boronate ester. On the basis of the outstanding features of rationally designed FBM such as

photoacoustic imaging, light absorbed by the tissue chromophores is converted into heat to cause localized volume heating, leading to transient thermoelastic expansion and consequently broadband acoustic waves.7−9 However, unfortunately, thermoelastic expansion is the least efficient mechanism in terms of the efficiency of light−sound energy conversion and amplitude of acoustic waves.7 Various light-absorbing nanoconstructs such as gold nanorods and carbon nanotubes have been extensively used as a photoacoustic imaging agent because of their outstanding photothermal and optical scattering behaviors.3,10−14 However, the generation of photoacoustic signal from these nanoconstructs is still directed by thermoelastic expansion.2 Perfluorocarbon-filled microbubbles have long been widely used as an ultrasound contrast agent due to their high acoustic scattering properties.15−17 Interestingly, microbubbles could also produce enhanced photoacoustic responses beyond the traditionally used mechanism, thermoelastic expansion. Several lines of evidence have demonstrated that the contrast-enhanced photoacoustic imaging can be achieved through vaporization of nanodroplets consisting of perfluorocarbon and gold nanorods.2,6 Under laser irradiation, perfluorocarbon with boiling temperature below body temperature could become easily superheated and undergo liquid−gas transition to form microbubbles, which induce one-time, high-amplitude photoacoustic signal.6,18 Therefore, upon laser irradiation, photoabsorbercontaining nanodroplets could provide combined photoacoustic signals via two mechanisms: vaporization of perfluorocarbon by liquid−gas phase transition and traditional thermoelastic expansion caused by photoabsorbers. Photoacoustic imaging through vaporization of nanodroplets, therefore, could display significantly higher signal amplitude than those through thermoelastic expansion. Furthermore, once vaporization is initiated, the resulting microbubbles of nanodroplets also provide significant acoustic impedance mismatch between gas bubbles and the surrounding media, thereby enhancing ultrasound contrast. Therefore, photoabsorber-containing nanodroplets would generate contrast-enhanced dual ultrasound/photoacoustic B

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Figure 1. Characterization of H2O2-activatable FBM nanoparticles. (a) UV−vis absorption of FBM. (b) Fluorescence emission spectra of FBM. (c) Size distribution of FBM nanoparticles suspended in PBS. The inlet is the scanning electron microscope (SEM) image of FBM nanoparticles. (d) Release kinetics of HBA from FBM nanoparticles in the presence or absence of H2O2. Values are mean ± SD (n = 4). (e) Ability of FBM nanoparticles to scavenge H2O2. Values are mean ± SD (n = 4). (f) H2O2-triggered CO2 generation of FBM nanoparticles over time.

It was also found that 1 mg of FBM nanoparticles could release ∼200 μg of HBA. During incubation in H2O2 solutions, FBM nanoparticles readily scavenged H2O2 (Figure 1e), indicating that boronate linkages of FBM undergo H2O2-triggered oxidation to deplete H2O2. CO2 gas chromatography was employed to verify the generation of CO2 from FBM in the presence of H2O2. FBM nanoparticles generated CO2 in a H2O2-triggered manner (Figure 1f). A noticeable increase in the level of CO2 was observed from 30 min after the addition of H2O2 (500 μM), not immediately probably because CO2, which was entrapped in the core of nanoparticles and/or formed bubbles bound to their surface, was not collected for analysis. The amount of CO2 generated from FBM nanoparticles gradually increased for 6 h in the presence of H2O2. However, in the absence of H2O2, no noticeable change in the level of CO2 was observed for 24 h, demonstrating that H2O2-triggered boronate oxidation of FBM expedites decarboxylation of carbonate to generate CO2 (Figure S3). Effects of H2O2-triggered CO2 bubble generation on the size and morphology of FBM nanoparticles were studied by dynamic light scattering and transmission electron microscopy (TEM). In the absence of H2O2, FBM nanoparticles in aqueous solutions containing serum proteins (10%) displayed slight increase in their hydrodynamic diameter, but remained intact for 7 days (Figure S4a). However, when exposed to H2O2 (1 mM), FBM nanoparticles showed a rapid and gradual increase in their size and eventually dissociated completely because FBM became water soluble after the cleavage of hydrophobic borylbenzyl carbonates (Figure S4b). A H2O2-triggered change in the structure of FBM nanoparticles was also evidenced by TEM images. It seems that CO2 generated in the core of FBM nanoparticles diffuses to the surface and forms bubbles on their surfaces, evidenced by the “flower-like” appearance (Figure S4c,d). H2O2-triggered dissociation of FBM nanoparticles was further confirmed by the dramatic change in the turbidity of boronated maltodextrin nanoparticles (Figure S5). FBM nanoparticles

H2O2-triggered CO2 bubble generation and H2O2-activable therapeutic activities, we reasoned that FBM nanoparticles serve as theranostic agents for H2O2-associated cardiovascular diseases such as thrombosis (Scheme 1). Thrombosis occurs when arteries or veins form blood clots that are excessively large and obstruct blood flow.26 A thrombus is a complex structure that is composed mainly of activated platelets and a mesh of water-insoluble fibrin.22,27 Platelet activation during thrombotic events is closely associated with a high level of H2O2, which mediates expression of proinflammatory proteins in endothelial cells and stimulates platelet−endothelium interactions.28−30 Excessively generated H2O2 exceeding 200 μM is known to induce endothelial damage.31 Therefore, on the basis of the close relationship between H2O2 and thrombus formation and H2O2-responsiveness of FBM, we sought to develop FBM nanoparticles as H2O2triggered contrast-enhanced imaging agents and therapeutic agents for thrombosed vessels. In this work, we report the rational design and translational potential of FBM nanoparticles as theranostic agents for thrombosis.

RESULTS AND DISCUSSION Preparation and Characterization of FBM Nanoparticles. FBM was designed to have borylbenzyl carbonate and IR780 as a side chain (Figure S1). The chemical structure of FBM was confirmed by NMR, and its molecular weight was determined to be ∼30 000 Da. The degree of substitution of borylbenzyl carbonate and IR780 was determined to be ∼60% and ∼5%, respectively. FBM showed a green color with strong absorption at ∼780 nm and fluorescence emission at ∼820 nm (Figure 1a,b). H2O2-triggered release of HBA from FBM was also confirmed by NMR (Figure S2). Using a conventional single-emulsion method, hydrophobic FBM was easily formulated into spherical nanoparticles with a smooth surface and a mean diameter of ∼320 nm (Figure 1c). FBM nanoparticles exhibited H2O2-dependent release kinetics of HBA (Figure 1d). C

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Figure 2. H2O2-triggered ultrasound/photoacoustic signal amplification of FBM nanoparticles. (a) H2O2-dependent echogenicity of FBM nanoparticles over time. (b) Photoacoustic imaging of FBM nanoparticles at various concentrations. (c) Linear photoacoustic signal dependence on the concentration of FBM nanoparticles. (d) H2O2-triggered photoacoustic signal amplification of FBM nanoparticles over time.

nanoparticles is not able to generate ultrasound contrast signal under the acoustic field. The effects of H2O2-triggered bubble generation on the photoacoustic signal of FBM nanoparticles were investigated using a Vevo LAZR photoacoustic imaging system. In the absence of H2O2, FBM nanoparticles exhibited photoacoustic signals, but their signal intensity was dependent only on their concentration (Figure 2b,c). The results indicate that the photoacoustic response of FBM nanoparticles is attributed only to thermoelastic expansion caused by IR780. However, in the presence of a biologically relevant level of H2O2,31,32 FBM nanoparticles showed time-dependent and H2O2 concentrationdependent photoacoustic signals (Figure 2d). At 500 μM H2O2, FBM nanoparticles showed a significantly increased photoacoustic signal, with a maximum intensity at 15 min, which is ∼5 fold higher than those obtained without H2O2 (Figure S6b). The signal intensity then gradually decreased to its steady-state level, which is attributed to the IR780-induced thermoelastic expansion. The duration of the enhanced photoacoustic signal was in agreement with those of ultrasound signal. In order to further confirm the H2O2-triggered photoacoustic signal enhancement of FBM nanoparticles, PLGA, poly(lactic-coglycolic acid), was used as a control, which does not generate bubbles. We prepared IR780-loaded PLGA particles, in which the content of IR780 was the same as those in FBM

remained as an opaque suspension in the absence of H2O2, but became transparent in the presence of H2O2 after 12 h, demonstrating that FBM nanoparticles undergo H2O2-triggered dissociation. Ultrasound and Photoacoustic Signal Amplification. The echogenicity of H2O2-triggered CO2 bubble-generating FBM nanoparticles was evaluated using an agarose gel phantom (Figure 2a). Under the ultrasound field, FBM nanoparticles suspended in phosphate buffer (pH 7.4) showed negligible contrast without H2O2. However, in the presence of H2O2, the echo intensity of FBM nanoparticles gradually increased in a H2O2 concentration-dependent manner and was significantly higher than those obtained without H2O2. With 1 mM H2O2, FBM nanoparticles displayed a ∼5-fold higher signal at 10 min, and the enhanced echo signal could last for ∼2 h (Figure S6a). These results demonstrate that CO2-bubble-generating FBM nanoparticles provide significant acoustic impedance mismatch with the surrounding environment and act as ultrasound contrast agents. However, despite continuous CO2 generation for ∼6 h in the presence of H2O2 (Figure 1f), FBM nanoparticles could display ultrasound contrast enhancement for ∼2 h. The discrepancy can be explained by the rationales that small degrading nanoparticles could not accommodate CO2 bubbles and CO2 that diffuses away through the defects of the degrading D

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Figure 3. Therapeutic activity of FBM nanoparticles in vitro. (a) Protective effect of FBM nanoparticles on H2O2-stimulated endothelial cells. Values are mean ± SD (n = 4). ***p < 0.001 relative to H2O2 only; †††p < 0.001 relative to H2O2+ HBA. (b) Confocal fluorescent images of H2O2-stimulated endothelial cells treated with FBM nanoparticles. (c) Effects of FBM nanoparticles on the expression of TNF-α (c) and IL-1β (d) in H2O2-stimulated endothelial cells. Values are mean ± SD (n = 4). ***p < 0.001 relative to H2O2 only; †††p < 0.001 relative to H2O2 + HBA (200 μM). Effects of FBM nanoparticles on the level of sCD40L (e) and H2O2 (f) in activated platelets. Values are mean ± SD (n = 4). ***p < 0.001 relative to thrombin + CaCl2; †††p < 0.001 relative to H2O2 + HBA (200 μM).

Figure S9, more HBA was detected in H2O2-activated cells than nonactivated cells, demonstrating that FBM undergoes H2O2triggered oxidation of boronate ester to release HBA. Antiplatelet activity of FBM nanoparticles was evaluated using platelets activated by the cotreatment of thrombin and CaCl2. Activated platelets showed a significantly elevated level of soluble CD40 ligand (sCD40L), which is known to circulate and interact with CD40 on immune cells or endothelial cells to mediate thrombotic and inflammatory processes.33,34 The level of sCD40L in activated platelets was significantly reduced by FBM nanoparticles, suggesting that FBM nanoparticles exert highly potent antiplatelet activities (Figure 3e). Activated platelets are also known to generate a large amount of H2O2 to facilitate their adhesion to endothelium and promote thrombus formation.35,36 We therefore evaluated the effects of FBM nanoparticles on the level of H2O2 in activated platelets (Figure 3f). Activated platelets displayed a significantly elevated level of H2O2. While equivalent HBA showed moderate inhibitory effects on H2O2 generation, FBM nanoparticles significantly suppressed H2O2 generation in activated cells. The strong antioxidant, anti-inflammatory, and antiplatelet activities of FBM nanoparticles could be attributed to the combined effects of H2O2-scavenging boronate and intrinsic activity of HBA released after H2O2-triggered oxidation. Preparation and Characterization of T-FBM Nanoparticles. Encouraged by the promising results, FBM was formulated into nanoparticles with a lipopeptide, GPRPPC− PEG (polyethylene glycol)−DSPE (distearoylphosphatidylethanolamine), to establish theranostic agents for thrombosis. A pentapeptide, GPRPP, was employed as a thrombus-targeting ligand because of its high affinity to fibrin, one of major components of a thrombus.37−39 GPRPP-decorated FBM nanoparticles

nanoparticles (Figure S7). As expected, IR780-loaded PLGA nanoparticles (FPLGA) showed measurable photoacoustic signals generated by IR780-induced thermoelastic expansion, but the photoacoustic signal intensity was H2O2-independent and time-independent. These results confirm that H2O2-triggered bubble generation of FBM nanoparticles significantly amplifies the photoacoustic signal with a mechanism similar to lasertriggered microbubble-generating nanodroplets, and CO2 bubble formation is the predominant mechanism to generate high photoacoustic signals. However, unlike laser-triggered microbubblegenerating nanodroplets, FBM nanoparticles could display great photoacoustic signal amplification without gas precursors and laser irradiation. In Vitro Therapeutic Activity of FBM Nanoparticles. Cell culture studies were performed to evaluate the cytotoxicity and therapeutic activities of FBM nanoparticles. FBM nanoparticles induced negligible toxicity to arterial endothelial cells and RAW264.7 cells (Figure 3a and Figure S8a). Stimulation of cells with H2O2 caused a large generation of ROS (reactive oxygen species) and elevated the level of pro-inflammatory TNF-α (tumor necrosis factor-alpha) and IL (interleukin)-1β. However, FBM nanoparticles significantly suppressed ROS generation and inhibited the expression of TNF-α and IL-1β in stimulated cells, protecting cells against H2O2-induced toxicity (Figure 3b−d and Figure S8b−e). HBA exerted noticeable anti-inflammatory activities at 100 μM, which is almost equivalent to 50 μg/mL of FBM nanoparticles. However, FBM nanoparticles exhibited significantly higher therapeutic activities than equivalent HBA. We also investigated whether HBA is released from FBM nanoparticles in cells in a H2O2-triggered manner using LC-MS/ MS (liquid chromatography−mass spectroscopy). As shown in E

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Figure 4. Imaging of thrombosed vessels with T-FBM nanoparticles. (a) Schematic illustration of a mouse model of FeCl3-treated carotid arterial injury. (b) Fluorescence imaging of carotid artery after injection of FBM and T-FBM nanoparticles at 15 min after injection. (c) Time course of fluorescence imaging of carotid artery using T-FBM nanoparticles. (d) Quantification of fluorescence intensity in thrombosed vessels. Values are mean ± SD (n = 4). ***p < 0.001 relative to FBM; †††p < 0.001 relative to GPRPP with T-FBM. (e) Ratio of fluorescence intensity between FeCl3treated carotid artery and noninjured artery as a function of time. Values are mean ± SD (n = 4). (f) Ratio of photoacoustic signal intensity between FeCl3-treated carotid artery and noninjured artery as a function of time. Values are mean ± SD (n = 4). (g) Representative photoacoustic imaging of carotid artery of mice treated with FBM nanoparticles or T-FBM nanoparticles at 20 min after injection. (h) Time course of photoacoustic imaging of carotid artery. Arrows indicate a carotid artery.

T-FBM Nanoparticles as a Fibrin-Targeting Imaging Agent. The potential of T-FBM nanoparticles as contrastenhanced photoacoustic imaging agents and therapeutic agents for thrombosis was investigated using a mouse model of FeCl3induced arterial thrombosis (Figure 4a). Fluorescence imaging of thrombotic carotid was first performed to determine whether T-FBM nanoparticles could specifically target thrombi and how fast they accumulate in thrombosed vessels (Figure 4b,c). While FBM nanoparticles produced a background level of fluorescence in the FeCl3-treated artery, T-FBM nanoparticles exhibited significantly strong fluorescence in the injured artery from 5 min after injection. The fluorescence signal gradually increased for 30 min and thereafter did not increase further. When free GPRPP was preinjected, T-FBM nanoparticles produced significantly reduced fluorescence signal in the injured artery, demonstrating that T-FBM nanoparticles specifically target fibrin-rich thrombi within 30 min (Figure 4d).

were termed T-FBM nanoparticles and expected to specifically target fibrin-rich thrombi. PEG (3.4k) would serve as a surface corona to prevent nonspecific interaction of T-FBM nanoparticles with plasma proteins. T-FBM nanoparticles were spherical in shape with a mean diameter of ∼260 nm (Figure S10). The ability of T-FBM nanoparticles to target specifically thrombi was evaluated using blood clots that were induced by incubating platelet-rich plasma with thrombin/ CaCl2. FBM nanoparticles or T-FBM nanoparticles were added to plasma 5 min after the onset of clot formation. Blood clots treated with T-FBM nanoparticles showed a significantly (∼5 fold) higher fluorescence intensity than those treated with FBM nanoparticles, which have no fibrin-targeting ligand (Figure S11). Pretreatment of blood clots with free GPRPP significantly suppressed targeting of T-FBM particles, evidenced by the significantly reduced fluorescence intensity. These results suggest that T-FBM nanoparticles specifically target fibrin-rich thrombi. F

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Figure 5. Therapeutic activity of T-FBM nanoparticles on thrombosed vessels. Effects of T-FBM nanoparticles on the expression of TNF-α (a) and sCD40L (b) in the FeCl3-treated carotid artery. Values are mean ± SD (n = 4). ***p < 0.001 relative to FeCl3 only; †††p < 0.001 relative to GPRPP with T-FBM. (c) Histological examination of FeCl3-treated carotid artery stained with H&E, Masson’s trichrome, and DHE.

reactions with endogenous H2O2, without external laser irradiation. To our best knowledge, T-FBM nanoparticles are the first photoacoustic contrast agent that specifically targets fibrin-rich thrombi and exhibits stimulus-triggered photoacoustic contrast amplification. Antithrombotic Activity of T-FBM Nanoparticles. The therapeutic activity of T-FBM nanoparticles on thrombosis was evaluated by measuring the level of TNF-α and sCD40L. FeCl3 treatment significantly elevated the level of TNF-α in the artery and circulation (Figure 5a,b and Figure S12a,b). While equivalent HBA exhibited moderate effects on the expression of inflammatory TNF-α, T-FBM nanoparticles significantly suppressed the expression of TNF-α. The level of sCD40L was also significantly elevated by the FeCl3 treatment. T-FBM nanoparticles exhibited significantly stronger inhibitory effects on the expression of sCD40L than HBA, demonstrating that T-FBM nanoparticles exert potent anti-inflammatory and antiplatelet activities. The therapeutic effects of T-FBM nanoparticles were further assessed by histological examination of carotid artery (Figure 5c). FeCl3 treatment caused the formation of a large thrombus. While FBM nanoparticles slightly suppressed the FeCl3-induced thrombus formation, T-FBM nanoparticles significantly inhibited thrombus formation. Strong DHE (dihydroethidium) fluorescence was observed in the thrombus and endothelium of the FeCl3-treated artery, indicating that massive ROS was produced by activated platelets that were recruited to the thrombosed vessel. T-FBM nanoparticles significantly suppressed ROS generation, concentration dependently, which is in good agreement with Figure 3b. These findings suggest that T-FBM nanoparticles specifically target the

As FBM nanoparticles were designed to provide H2O2triggered ultrasound/photoacoustic signal enhancement, we first determined the level of H2O2 in the blood clots using the Amplex red assay. Blood clots induced by thrombin showed a significantly higher level of H2O2 compared to the untreated plasma (Figure S11c), indicating that H2O2 is able to serve as a thrombus-specific trigger to generate CO2 bubbles and amplify the ultrasound/photoacoustic signal of T-FBM nanoparticles. We next evaluated the translational potential of T-FBM nanoparticles as contrast-enhanced photoacoustic imaging agents for thrombosed vessels (Figure 4e−h). One minute after the treatment of carotid artery with FeCl3, mice were injected intravenously with FBM nanoparticles or T-FBM nanoparticles at a dose of 5 mg/kg. While FBM nanoparticles generated no noticeable photoacoustic signal in the FeCl3-treated carotid artery, T-FBM nanoparticles exhibited a strong photoacoustic signal in the injured artery from 15 min after injection. Interestingly, the photoacoustic signal intensity in the injured artery gradually increased for 30 min and then decreased to their steady-state level. The photoacoustic response of T-FBM nanoparticles in vivo is in good agreement with the agarose phantom test. IR780-loaded PLGA nanoparticles decorated with GPRPP (T-FPLGA) also showed a noticeable photoacoustic signal, but the signal intensity was significantly lower than that of T-FBM particles and was constant for 1 h of observation. These observations demonstrate that T-FBM nanoparticles amplify the photoacoustic signal in the injured artery through H2O2-triggered CO2 bubble generation. Unlike nanodroplets encapsulating gas precursors, T-FBM nanoparticles provide significantly elevated photoacoustic signals in thrombi by chemical G

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resin (Merck Chemicals Ltd., Nottingham, UK). For each coupling step, the Fmoc-protected amino acids and coupling reagents were added in a 5-fold molar excess to a resin concentration. After cleavage and precipitation of peptides, the cloud peptides were purified via reversed phase high-performance liquid chromatography (RP-HPLC) with a Waters C18 column (19 × 300 mm). The purified GPRPPC peptide was dissolved in dimethylformamide (DMF) and mixed with DSPEPEG(3.4k)-maleimide at 2:1 molar ratio. The conjugation was allowed for 24 h at 25 °C, and the reaction mixture was dialyzed against distilled water. GPRPPC-PEG-DSPE was obtained after lyophilizataion and stored at −20 °C. Preparation and Characterization T-FBM Nanoparticles. One hundred milligrams of FBM dissolved in 1 mL of DCM was added in 10 mL of 5 (w/v) % PVA solution. The mixture was sonicated with a sonicator (Fisher Scientific, Sonic Dismembrator 500) for 1 min and then homogenized with a homogenizer (PRO Scientific, PRO 200) for 2 min. The primary emulsion was emulsified in 15 mL of PVA solution (0.5 w/v %) using a homogenizer for 1.5 min. The organic solvent was removed by a rotary evaporator, and the FBM nanoparticle suspension was ultracentrifuged at 12000g at 4 °C for 6 min and washed with deionized water. For the preparation of T-FBM nanoparticles, GPRPPC-PEG-DSPE (10 mg) was added in 1 mL of DCM solution containing FBM (90 mg). The procedure for T-FBM nanoparticle formulation was the same for the empty FBM nanoparticle formulation. The morphology was observed by a scanning electron microscope (JSM-6400, JEOL, Japan) with an accelerating voltage of 1 kV and a transmission electron microscope (JEOL-2010, Japan). The size distribution of nanoparticles was measured using a particle size analyzer (90Plus, Brookhaven Instrument Corp., Holtsville, NY, USA). H2O2 Scavenging by FBM Nanoparticles. The ability of FBM nanoparticles to scavenge H2O2 was evaluated based on peroxalate chemiluminescence. FBM nanoparticles were added to 1 mL of H2O2 solutions (500 μM). The level of remaining H2O2 in the mixture was determined by measuring the chemiluminescence intensity after the reaction with diphenyl oxalate solution containing rubrene. The chemiluminescence intensity was measured using a luminometer (FB 12, Berthold Detection Systems, Germany). CO2 Gas Chromatography. FBM nanoparticles (100 mg) were added into 20 mL of H2O2 solution (500 μM) in a vial capped with a septum. A syringe needle was inserted into the vial to take the air containing CO2 at appropriate time intervals, and the level of CO2 was measured using gas chromatography (6890N GC, Agilent Technologies, Wilmington, DE, USA). Ultrasound and Photoacoustic Imaging of FBM Nanoparticles. Agarose gel was used to as a phantom to simulate body conditions. A 250 μL Eppendorf tube or a 2 mm glass capillary was embedded in an agarose solution (3 wt %) and then carefully removed after the gel cooled to make a well. FBM nanoparticles were suspended in phosphate-buffered saline (PBS) at a concentration of 5 mg/mL with or without H2O2 (0.5 or 1 mM), and 200 μL of an FBM nanoparticles suspension was placed into wells of a phantom gel. Ultrasound images (B-mode) of FBM nanoparticles were made using an ultrasound instrument (Sonix TOUCH, Ultrasonix, Canada). Photoacoustic imaging of FBM nanoparticles (1 mg/mL) was made with a Vevo LAZR photoacoustic imaging system (Visualsonics, Toronto, Canada) with an LZ550 transducer (32−55 MHz) at an excitation wavelength (808 nm). Cell Culture Studies. Endothelial cells were isolated from the abdominal aorta of 8-week-old mice. After anaesthetization, the middle of the abdominal aorta was cut and perfused with 1 mL of 1000 U/mL of heparin from the left ventricle. The aorta was excised and immersed in DMEM (Dulbecco’s modified Eagle’s medium) containing 20% FBS (fetal bovine serum) and 1000 U/mL of heparin. The aorta was filled with collagenase II solution and incubated at 37 °C for 45 min. Endothelial cells were isolated by flushing of DMEM containing 20% FBS and centrifugation. Endothelial cells were cultured in DMEM with 20% FBS. RAW 264.7 cells were obtained from the Korean Cell Line Bank (Korea) and cultured in DMEM containing 10% serum protein. A standard MTT (3-(4,5-dimethylthiazil-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to evaluate the viability of RAW 264.7 and arterial endothelial cells. Cells were treated with 200 μM

thrombosed vessel and exert highly potent antithrombotic effects. The toxicity of T-FBM nanoparticles was evaluated by histological examinations of organs after 1 week of daily administration (5 mg/kg). There was no noticeable accumulated toxicity in the organs (Figure S13a). In addition, T-FBM nanoparticle-treated groups showed no significant difference in the level of alanine transaminase (ALT), compared with the untreated group (Figure S13b), indicating that T-FBM nanoparticles have excellent safety profiles. However, further studies including dose, pharmacodynamics, and toxicology are warranted to maximize the translational potential of T-FBM nanoparticles for clinical settings.

CONCLUSIONS The rationally designed T-FBM nanoparticles were developed as theranostic agents for thrombosis that amplify photoacoustic contrast through H2O2-triggered CO2-bubble formation and exert potent therapeutic effects. Despite several challenges in the animal studies such as highly scattering tissues and high levels of background photoacoustic signal, T-FBM nanoparticles displayed significantly enhanced photoacoustic contrast in the FeCl3-induced thrombosed carotid artery. Beyond H2O2triggered photoacoustic signal amplification, a single injection of T-FBM nanoparticles exerted highly potent antioxidant, antiinflammatory, and antiplatelet activities to suppress thrombus formation in FeCl3-treated carotid artery. Given their thrombustargeting ability, H2O2-triggered photoacoustic signal amplification, and H2O2-activatable therapeutic action, T-FBM nanoparticles hold tremendous translational potential as nanotheranostic agents for various life-threatening H2O2-associated cardiovascular diseases. METHODS AND MATERIALS Materials. Maltodextrin, 1,1′-carbonyldiimidazole, 4-(hydroxymethyl)phenylboronic acid pinacol ester, poly(vinyl alcohol) (PVA, MW = 13 000−23 000 Da), and IR780 were purchased from SigmaAldrich (St. Louis, MO, USA). DSPE-PEG(3.4k)-maleimide was purchased from NOF Corporation (Japan). All materials were used without further purification. Preparation and Characterization of FBM. Borylbenzylsubstituted imidazole, 1, was synthesized from the reaction of 1,1′-carbonyldiimidazole (2.07 g) and 4-(hydroxymethyl)phenylboronic acid pinacol ester (2.0 g) in 20 mL of dichloromethane at room temperature for 2 h. 1 was obtained from silica gel chromatography (ethyl acetate/hexane, 1:1). The chemical structure was confirmed by 1H NMR (JEOL JNM, 400 MHz, CDCl3). Maltodextrin and 1 were dissolved in dimethyl sulfoxide, and the conjugation reaction was allowed for 24 at room temperature. The degree of substitution of borylbenzyl carbonate to maltodextrin was determined by measuring the molecular weight with gel permeation chromatography. Boronated maltodextrin was obtained through precipitation in cold water. For conjugation of IR780, boronated maltodextrin and IR780 were dissolved in dimethyl sulfoxide (5 mL)-containing triethylamine. FBM was obtained through extraction using water/dichloromethane, followed by vacuum drying. UV−vis absorbance of FBM dissolved in dimethyl sulfoxide was measured using a spectrometer (S-3100 Scinco, Korea). FBM and IR780 were dissolved in dimethyl sulfoxide, and their fluorescence was measured using a fluorospectrometer (FP-6500, Jasco, Japan) at peak emission after 785 nm excitation. The weight content of IR780 in FBM was determined from a calibration curve of free IR780 using a spectrometer. Synthesis of GPRPPC-PEG-DSPE. GPRPPC peptide was prepared using a Liberty Microwave Peptide synthesizer (CEM Co., Matthews, NC, USA) with 9-fluorenylmethyloxy carbonyl (Fmoc)-protected amino acids (CEM Co.) and a Rink amide MBHA (4-methylbenzhydrylamine) H

DOI: 10.1021/acsnano.7b06560 ACS Nano XXXX, XXX, XXX−XXX

Article

ACS Nano H2O2 for 2 h and then given FBM nanoparticles. Each well was given MTT solution (100 μL) and incubated for 4 h. Dimethyl sulfoxide was added to each well to dissolve the resulting formazan crystals. The absorbance at 570 nm was measured using a microplate reader (Biotek Instruments, Winooski, VT, USA), and the cell viability was determined by comparing the absorbance of treated cells with that of control cells. For the detection of ROS in H2O2-stimulated cells, DCFH-DA (Sigma-Aldrich) was used as an ROS probe. Cells were treated with 5 μM DCFH-DA for 15 min at 37 °C in the dark and added with 400 μL of 1× binding buffer. The stained cells were analyzed by flow cytometry (FACS Caliber, Becton Dickinson, San Jose, CA, USA). Cells were also observed under confocal laser scanning microscope (LSM 510 Meta, Carl Zeiss, Inc., Germany). LC-MS/MS Analysis. RAW264.7 cells cultured in three T-75 flasks were treated with FBM nanoparticles. After 24 h of incubation, cells were washed with new medium and collected after centrifugation. Cell pellets were added with 1 mL of the mixture of methanol/water and thoroughly vortexed to dissolve HBA. The nondissolved materials were removed by centrifugation (10000g) for 10 min, and the supernatant was carefully collected for analysis. For LC-MS/MS analysis, 5 μL of analyte was injected into an Ultra Performance liquid chromatograph (Xevo TQ-S, Waters). The LC-MS-MS was controlled by MassLynx software (version 4.1). The UPLC column was a Phenomenex Hydro RP 80 Å (150 × 2 mm) Waters PhACQUITY UPLCBEH C8 (2.1 × 100 mm, 1.7 μm). The mobile phase was 50 mM ammonium formate in deionized water (buffer A, pH 8.0) and 50 mM ammonium formate in acetronitrile (buffer B, pH 8.0). The flow rate was 0.5 mL/min, and the gradient was made from 5% to 100% B buffer for 10 min. The MS was equipped with an ESI (electrospray ionization) interface operating at an ionization voltage of +3000 V and a source temperature of 380 °C. Ions were generated in negative ionization mode using an ESI interface. The capillary voltage, con voltage, and source offset were set at 3 kV, 30 kV, and 30 V, respectively. Tandem MS analysis of HBA was performed using the multireaction-monitoring mode by monitoring the transition pair of m/z 122.93 → 104.77. Blood Clot Assay. Platelet-rich plasma was obtained from mice. Clot formation was induced by incubating platelet-rich plasma (180 μL) in the presence of thrombin (0.1 U/μL) and CaCl2 (0.4 M) in a 96-well assay plate. After 90 min after the onset of clot formation, FBM or T-FBM nanoparticles (100 μg) were added on the top of clots and allowed to incubate for 1 min. The clots were washed three times with fresh saline solution, and then the plates were analyzed using a fluorescence imaging system (FOBI, Neoscience, Korea). Determination of the Level of H2O2 in Platelets. Platelet-rich plasma obtained from the blood of mice was treated with thrombin (0.1 U/μL) and CaCl2 (0.4 M) in a 96-well plate. After 3 min of treatment, FBM nanoparticles were added to the platelets. The level of H2O2 in the plasma was determined using an Amplex red assay. Mouse Model of FeCl3-Induced Carotid Thrombosis. Anaesthetized mice (8 weeks old) were placed in supine position using tape to secure the hindlimbs and lower body. A midline incision was made between the sternum and chin, and the carotid arteries were exposed. Thrombus formation was induced by placing a filter paper saturated with FeCl3 (10%) on the top of the left carotid artery. Immediately after the initiation of thrombus formation, T-FBM nanoparticles were injected intravenously. Fluorescence imaging of thrombosed vessels was done with a fluorescence imaging system (FOBI, Neoscience, Korea). Photoacoustic imaging of thrombus was done with a Vevo LAZR photoacoustic imaging system (Visualsonics, Toronto, Canada) with an LZ550 transducer (32−55 MHz) at an excitation wavelength (808 nm). For histological examination of thrombosed vessels, carotid tissues were fixed with paraformaldehyde (4%) and embedded in optimal cutting temperature (OCT) compound blocks. The tissue blocks were sectioned and stained with H&E and Masson’s trichrome using standard histological techniques. For DHE staining, tissues were cryosectioned with a thickness of 7 μm and incubated with DHE (5 μM) at 37 °C for 30 min in a humidified chamber in the dark. Then, the sections were incubated with 4,6-diamidino-2-phenylindole. The image of carotid tissues was acquired with a confocal fluorescence microscope. The antiinflammatory and antiplatelet activities of T-FBM nanoparticles were

evaluated by measuring the level of TNF-α and sCD40L in the carotid artery and blood with a mouse sCD40L (ab119517, Abcam, Cambridge, UK) and a mouse TNF-α ELISA kit (eBioscience, San Diego, CA, USA) as advised by the manufacturer. In Vivo Toxicity of T-FBM Nanoparticles. T-FBM nanoparticles were administrated to mice at a dose of 5 mg/kg every day for 7 days. Livers were removed from the mice and homogenized in PBS. The activity of serum ALT was determined with an ALT enzymatic assay kit (Asan Pharma, Seoul, Korea) using a microplate reader (Synergy MX, BioTek Instruments, Inc.). For histological examination, liver, lung, kidney, spleen, and heart tissues were fixed with paraformaldehyde (4%) and embedded in OCT compound blocks. The tissue blocks were sectioned and stained with H&E. Statistical Analyses. Values were expressed as mean ± SD (standard deviation). Comparisons between and within groups were conducted with unpaired Student’s t tests and repeated-measures ANOVA using GraphPad Prism 5.0 (San Diego, CA, USA), respectively. Probability (p) values of