Biodegradable Iodinated Polymeric Nanoparticle as a Computed Tomography Contrast Agent for Direct Imaging of Cerebral Thrombus - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search Log in Dashboard Publications Account settings Log out Search… Search NCBI Primary site navigation Search Logged in as: Dashboard Publications Account settings Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. 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Learn more: PMC Disclaimer | PMC Copyright Notice ACS Nano . 2026 Mar 31;20(14):11386–11396. doi: 10.1021/acsnano.6c01281 Search in PMC Search in PubMed View in NLM Catalog Add to search Biodegradable Iodinated Polymeric Nanoparticle as a Computed Tomography Contrast Agent for Direct Imaging of Cerebral Thrombus Hyun Jae Park Hyun Jae Park † Research Institute of Advanced Materials (RIAM), Department of Materials Science and Engineering, Seoul National University, Gwanak-ro 1, Gwanak-gu, Seoul 08826, Republic of Korea Find articles by Hyun Jae Park † , Subin Park Subin Park † Research Institute of Advanced Materials (RIAM), Department of Materials Science and Engineering, Seoul National University, Gwanak-ro 1, Gwanak-gu, Seoul 08826, Republic of Korea Find articles by Subin Park † , In Jae Chung In Jae Chung † Research Institute of Advanced Materials (RIAM), Department of Materials Science and Engineering, Seoul National University, Gwanak-ro 1, Gwanak-gu, Seoul 08826, Republic of Korea Find articles by In Jae Chung † , Hee Jeong Jang Hee Jeong Jang ‡ Department of Neurology, Dongguk University Ilsan Hospital, Goyang 10326, Republic of Korea Find articles by Hee Jeong Jang ‡ , Leesu Lee Leesu Lee ‡ Department of Neurology, Dongguk University Ilsan Hospital, Goyang 10326, Republic of Korea Find articles by Leesu Lee ‡ , Tae Hoon Kim Tae Hoon Kim ‡ Department of Neurology, Dongguk University Ilsan Hospital, Goyang 10326, Republic of Korea Find articles by Tae Hoon Kim ‡ , Dong-Eog Kim Dong-Eog Kim ‡ Department of Neurology, Dongguk University Ilsan Hospital, Goyang 10326, Republic of Korea Find articles by Dong-Eog Kim ‡, * , Cheol-Hee Ahn Cheol-Hee Ahn † Research Institute of Advanced Materials (RIAM), Department of Materials Science and Engineering, Seoul National University, Gwanak-ro 1, Gwanak-gu, Seoul 08826, Republic of Korea Find articles by Cheol-Hee Ahn †, * Author information Article notes Copyright and License information † Research Institute of Advanced Materials (RIAM), Department of Materials Science and Engineering, Seoul National University, Gwanak-ro 1, Gwanak-gu, Seoul 08826, Republic of Korea ‡ Department of Neurology, Dongguk University Ilsan Hospital, Goyang 10326, Republic of Korea * E-mail: [email protected] . * E-mail: [email protected] . Received 2026 Jan 21; Accepted 2026 Mar 24; Revised 2026 Mar 24; Collection date 2026 Apr 14. © 2026 The Authors. Published by American Chemical Society This article is licensed under CC-BY-NC-ND 4.0 PMC Copyright notice PMCID: PMC13085846 PMID: 41913724 Abstract Polymer-based organic nanoparticles enabling direct imaging of cerebral thrombus were developed, distinguishing them from conventional methods that depend on vascular imaging to identify thrombus localization. The HEATI (bis(2-hydroxyethyl) 5-amino-2,4,6-triiodoisophthalate) monomer, synthesized from ATIPA (5-amino-2,4,6-triiodoisophthalic acid), was engineered to contain 58.9 wt % iodine for enhanced X-ray attenuation, confirmed by 1 H NMR spectroscopy. The polymerization of HEATI with oxalyl chloride resulted in a ROS-degradable iodinated polymer (IP-HEATI), as evidenced by the disappearance of the hydroxyl hydrogen peak in the NMR spectrum and a GPC-measured average molecular weight of 2800 g mol –1 . Fib-GC@IP-HEATI nanoparticles were formed through nanoprecipitation, yielding a hydrodynamic diameter of 197.2 ± 64.0 nm and a zeta potential of +24.8 mV. The conjugation of fibrin-targeting peptides increased the size to 255.9 ± 64.2 nm. In vitro assays revealed that Fib-GC@IP-HEATI had superior fibrin-binding affinity compared to GC@IP-HEATI, demonstrated by lower IP-HEATI concentrations in the supernatant. Furthermore, GPC analysis showed that IP-HEATI degraded rapidly in the presence of H 2 O 2 , with complete degradation within 24 h. In vivo imaging confirmed effective thrombus visualization volume with both GC@IP-HEATI and Fib-GC@IP-HEATI at 30 min after intravenous injection. The imaging-derived thrombus visualization volume decreased substantially within 48 h, consistent with nanoparticle degradation. In conclusion, Fib-GC@IP-HEATI enables imaging of both carotid and cerebral thrombi while undergoing time-dependent degradation, outperforming conventional iodine-based small molecule agents that are unable to visualize thrombi and provide a quantitative assessment of their burden. Keywords: CT contrast agent, Iodinated nanoparticle, Direct thrombus imaging, Biodegradable polymer, Targeted delivery Thromboembolic events, particularly ischemic strokes, are major causes of morbidity and mortality worldwide. − Majority of ischemic strokes are caused by obstruction of cerebral blood flow by thrombus or emboli originating from atherosclerotic plaques or the heart. Accounting for about 87% of all stroke cases and affecting over 15 million individuals annually, ischemic stroke constitutes a critical medical emergency with substantial clinical and socioeconomic impact. The initial interruption of cerebral perfusion leads to potentially reversible neuronal dysfunction that, without timely reperfusion, progresses to infarction and irreversible injury. , Because neuronal death advances rapidly after ischemic onset, prompt diagnosis and intervention are essential. Current treatment guidelines recommend intravenous thrombolysis within 4.5 h and mechanical thrombectomy up to 24 h after onset in patients with salvageable tissue. However, existing imaging approaches depend on angiographic visualization of vessel obstruction or indirect filling defects, rather than direct identification of the thrombus itself. This limitation underscores the need for direct thrombus imaging technologies capable of accurately localizing and characterizing thrombi in vivo , thereby improving diagnostic precision and therapeutic decision-making. Imaging modalities, particularly magnetic resonance imaging (MRI) and computed tomography (CT), are fundamental tools for the diagnosis of ischemic stroke. MRI is limited by restricted availability in emergency settings, longer acquisition times, and higher costs. Conversely, CT is widely used as the first-line modality because of its rapid acquisition and broad accessibility. − Iodinated contrast agents, widely used in CT imaging due to their high atomic number, are typically based on 1,3,5-triiodobenzene derivatives. Hydrophilic functional groups, such as hydroxyl groups, are incorporated to improve water solubility and reduce nonspecific receptor interactions. However, these small-molecular iodinated contrast agents are rapidly cleared by the kidneys, resulting in short imaging windows. Their fast excretion, coupled with the need for large injection volumes, leads to high osmolality and increases the risk of contrast-induced nephropathy. Moreover, because these agents distribute nonspecifically across both intravascular and extravascular spaces, thrombi are visualized only indirectly as vascular filling defects. The indirect approach limits accurate thrombus tracking and makes it difficult to distinguish thrombi from underlying atherosclerotic plaques, both of which can cause vascular occlusion or stenosis. To overcome these shortcomings, recent studies have explored targeted contrast agents for direct thrombus imaging. Kim et al. developed a first gold nanoparticle–based CT contrast agent capable of selective thrombus targeting. , However, because rigid metallic nanoparticles must be smaller than 6 nm to allow renal clearance, − the gold nanoparticles used in that study measured 127 ± 2.7 nm, precluding renal excretion and limiting clinical applicability. Conversely, nanoparticles below 6 nm provide insufficient CT contrast due to their low X-ray attenuation. , Therefore, there is a critical need for a CT contrast agent that offers strong in vivo contrast owing to its submicron size yet can degrade into fragments smaller than 6 nm to permit renal clearance after imaging. Such dual properties may be realized using degradable organic nanomaterials. It is well established that reactive oxygen species (ROS) are abundantly generated within thrombotic environments. − Damaged endothelial cells release ROS, promoting the recruitment of inflammatory cells and amplifying oxidative stress. In addition, thrombi activate leukocytesparticularly neutrophilswhich generate ROS as part of the innate immune response, contributing both to pathogen clearance and to local inflammation. − Given the elevated ROS concentrations at thrombotic sites, biomaterials that respond to ROS through oxidative degradation have attracted significant interest. Such materials incorporate ROS-cleavable chemical bonds, enabling controlled biodegradation and subsequent elimination. Several ROS-degradable linkages have been reported, including thioether, selenium/tellurium, thioketal, amino acrylate, boronic ester, peroxalate ester, and polyproline moieties. Among these, peroxalate esters exhibit particularly high reactivity toward ROS, ,, decomposing into biocompatible byproducts such as carbon dioxide. Notably, peroxalate bonds have been shown to undergo efficient degradation under thrombotic conditions. In this study, we developed a novel biodegradable CT contrast agent for direct imaging of arterial thrombus. A high-iodine-content monomer, bis(2-hydroxyethyl) 5-amino-2,4,6-triiodoisophthalate (HEATI), was synthesized from 5-amino-2,4,6-triiodoisophthalic acid (ATIPA) to enhance X-ray attenuation and enable condensation polymerization with oxalyl chloride through its hydroxyl groups. The resulting polymer, termed IP-HEATI, was designed to degrade under ROS-rich conditions. To achieve systemic circulation and thrombus targeting, IP-HEATI was encapsulated within glycol chitosan nanoparticles (GC@IP-HEATI). For specific thrombus binding, a fibrin-targeting peptide (Fib) was conjugated to the nanoparticle surface (Fib-GC@IP-HEATI), leveraging fibrin’s abundance in clots and absence in normal vessels to minimize off-target accumulation. ,, The peptide EP-2104R, previously used in gadolinium-based MRI agents, was employed for fibrin targeting. , The nanoparticles were characterized for ROS-responsive degradation, fibrin affinity, and X-ray attenuation in vitro , followed by in vivo micro CT imaging in mouse models of carotid artery thrombosis and embolic stroke. Thrombus localization and visualization volume were monitored up to 24 h postinjection, including assessment of newly induced thrombi to evaluate nanoparticle persistence. The results demonstrated that Fib-GC@IP-HEATI enabled precise, real-time thrombus visualization and quantitative volumetric assessment using CT alone. This approach offers a promising platform to accelerate diagnosis and treatment of ischemic stroke, potentially reducing associated morbidity and mortality. Results Characterization of Thrombus Targeting Biodegradable CT Contrast Nanoparticles (Fib-GC@IP-HEATI) The HEATI monomer was designed with a high iodine content to enhance X-ray attenuation and included two hydroxyl groups to facilitate polymerization ( Figure ). HEATI was synthesized from ATIPA, a precursor commonly used in iodine-based contrast agents. The structure of the HEATI monomer was confirmed by 1 H NMR spectroscopy ( Figure S1A ), with characteristic peaks at 4.25 ppm, corresponding to protons on the ester group. Based on the molecular weight of the iodinated monomer and the number of iodine atoms, the iodine content of HEATI was calculated to be 58.9 wt %, which is sufficient to provide effective X-ray attenuation compared to commercial iodine-based contrast agents such as Iopamidol (49.0 wt %) and Iodixanol (49.1 wt %). To synthesize a ROS-degradable iodinated polymer (IP-HEATI), HEATI was polymerized with oxalyl chloride via condensation polymerization. This process involved the reaction of the hydroxyl groups of HEATI with the acyl chloride groups of oxalyl chloride. The 1 H NMR spectra of IP-HEATI showed a shift of the protons adjacent to the hydroxyl group of HEATI from 3.7 ppm (as seen in Figure S1A ) to 4.6 ppm, which corresponds to the formation of an ester bond after reaction with oxalyl chloride. This shift confirms the successful polymerization ( Figure S1B ). GPC provided additional evidence of polymer formation, showing that the number-average molecular weight ( M n ) of IP-HEATI was 2800 g mol –1 with a polydispersity of 1.41 ( Figure S2 ). 1. Open in a new tab Synthetic routes of IP-HEATI and schematic illustration for preparation of Fib-GC@IP-HEATI The preparation of Fib-GC@IP-HEATI is illustrated in Figure . Nanoprecipitation was used to encapsulate IP-HEATI within glycol chitosan, which acted as a stabilizer due to its hydrophilicity and stability under physiological conditions. Glycol chitosan also served as a source of amine groups, facilitating conjugation with fibrin-targeting peptides. To verify complete coating of glycol chitosan on GC@IP-HEATI, hydrodynamic diameter and zeta potential measurements were taken using dynamic light scattering (DLS). DLS results showed that the size of GC@IP-HEATI was 197.2 ± 64.0 nm ( Figure S3A ), with a positive zeta potential of +24.8 mV ( Figure S3C ), attributable to the primary amine groups on the glycol chitosan surface. Fibrin-targeting peptides were conjugated to the GC@IP-HEATI via EDC/NHS coupling, reacting the primary amine groups on the glycol chitosan surface with the carboxylic acid groups on the C-terminus of the peptide. After peptide conjugation, Fib-GC@IP-HEATI exhibited a slight increase in size (255.9 ± 64.2 nm, Figure S3B ) and a slight decrease in zeta potential (+23.6 mV, Figure S3C ). In Vitro Evaluation of Thrombus Targeting CT Contrast Performance of Fib-GC@IP-HEATI Nanoparticles The in vitro fibrin clot binding capability of Fib-GC@IP-HEATI was assessed using a fibrin gel conjugation assay. Fibrin hydrogels were formed by reacting fibrinogen with thrombin in saline containing CaCl 2 . Solutions of GC@IP-HEATI and Fib-GC@IP-HEATI were introduced into the fibrin gel, and the supernatant was collected after incubation for 1 h. The binding affinity to fibrin clots was evaluated by measuring the residual amount of IP-HEATI in the supernatant, as quantified by UV–vis spectroscopy at 325 nm, corresponding to IP-HEATI. The concentration of free GC@IP-HEATI in the supernatant was found to be 7.15 times higher than that of Fib-GC@IP-HEATI, which demonstrated that Fib-GC@IP-HEATI had a higher fibrin-binding affinity than GC@IP-HEATI ( Figure A). 2. Open in a new tab (A) In vitro experiments to show a fibrin-binding capacity of GC@IP-HEATI and Fib-GC@IP-HEATI. (B) IP-HEATI degradation mechanism (C) Degradation of GC@IP-HEATI in 37 °C PBS buffer using GPC (D) Degradation of GC@IP-HEATI in 37 °C PBS buffer containing 10 mM H 2 O 2 using GPC. IP-HEATI was designed to degrade in ROS-rich environments, releasing its degradation product of HEATI monomers at the thrombus site. The degradation of peroxalate ester groups into carbon dioxide by H 2 O 2 involves the cyclic 1,2-dioxetanedione as a reactive intermediate ( Figure B). , To evaluate the effect of H 2 O 2 on the degradation rate, GC@IP-HEATI nanoparticles were incubated in PBS buffer at 37 °C with or without 10 mM H 2 O 2 . GPC analysis showed that, regardless of the presence of H 2 O 2 , oligomer peaks began to appear at an elution time of 25–30 min up to 9 h, indicating similar degradation behavior. However, after 24 h for incubation, the sample with 10 mM H 2 O 2 clearly exhibited a monomer peak at an elution time of around 32 min ( Figure D)., whereas the sample without H 2 O 2 showed predominating oligomer peaks ( Figure C). Based on the fact that HEATI monomer peaks appeared after 48 h in PBS without H 2 O 2 , IP-HEATI hydrolyzes under physiological conditions and the degradation is significantly accelerated by H 2 O 2 . This ROS-triggered degradation supports the rapid disintegration of the nanoparticles after accumulation at the thrombus site. The high iodine content in GC@IP-HEATI conferred strong X-ray attenuation properties, making it well-suited for CT imaging. In solutions with GC@IP-HEATI concentrations of 5 mg/mL or higher, clear contrast relative to water was observed, and concentrations exceeding 20 mg/mL produced markedly enhanced CT signals ( Figure S4A ). A linear correlation between CT signal intensity and GC@IP-HEATI concentration was confirmed (Figure S4B), supporting its potential for quantitative imaging analysis. Micro CT Visualization of Carotid Artery Thrombus with Fib-GC@IP-HEATI In vivo thrombus imaging was conducted in male C57BL/6 mice with carotid artery thrombi by intravenous administration of GC@IP-HEATI (200 μL, 30 mg/mL; n = 3), Fib-GC@IP-HEATI (200 μL, 30 mg/mL; n = 3), or Fib-GC@Gold (200 μL, 2.5 mg/mL; n = 3). Fib-GC@Gold, a fibrin-targeted gold nanoparticle imaging agent previously shown to effectively visualize carotid and cerebral thrombi, served as a positive control. For comparison with a clinically used small-molecule iodinated contrast agent, Ultravist 370 was administered via continuous infusion using a syringe pump (total volume: 400 μL) during real-time micro-CT acquisition. Micro CT imaging 30 min after injection demonstrated complete thrombus visualization volume in mice receiving GC@IP-HEATI and Fib-GC@IP-HEATI, with imaging performance comparable to that observed in the Fib-GC@Gold group ( Figure A, C). In contrast, Ultravist provided intravascular contrast during real-time infusion but did not yield thrombus-specific enhancement under the same imaging conditions ( Figure A). The optimal concentration of Fib-GC@IP-HEATI was determined by varying injection doses and measuring carotid thrombus visualization volume on imaging ( Figure S5 ). In line with previous findings that 2.5 mg/mL of Fib-GC@Gold enabled complete carotid artery thrombus imaging, Fib-GC@IP-HEATI at 30 mg/mL achieved a comparable level of carotid thrombus visualization volume, as reflected by similar thrombus volumes quantified from imaging. These results demonstrate that the proposed organic nanoparticle platform enables direct in vivo thrombus imaging. Notably, even in the absence of fibrin-targeting peptides, GC@IP-HEATI was able to visualize large carotid thrombi. This observation is likely attributable to electrostatic interactions between the positively charged glycol chitosan backbone and negatively charged components within thrombi, including red blood cells and platelets. A similar charge-mediated accumulation phenomenon was previously observed in our gold nanoparticle–based system (GC@Gold), where nontargeted glycol chitosan–coated nanoparticles also exhibited passive accumulation in carotid artery thrombi. However, while such electrostatic and hemodynamic effects may be sufficient to enable imaging of large, highly occlusive carotid thrombi, fibrin targeting is expected to play a more critical role in ensuring stable retention and sensitive detection of smaller or intracranial thrombi, where passive accumulation mechanisms and nanoparticle–thrombus contact probability are inherently limited. 3. Open in a new tab In vivo coronal micro-CT images of thrombus visualization in a carotid artery thrombosis model. (A) Representative coronal CT images acquired at 30 min postinjection comparing Fib-GC@IP-HEATI (30 mg mL-1, 200 μL), nontargeted GC@IP-HEATI (30 mg mL-1, 200 μL), nondegradable Fib-GC@Gold (2.5 mg mL-1, 200 μL),, and a clinically used iodinated contrast agent (Ultravist 370). Ultravist 370 was continuously administered via a syringe pump (400 μL total volume) with real-time CT acquisition, whereas nanoparticle-based agents were imaged after bolus injection. Arrows indicate the thrombus location. (B) Coronal CT images acquired at 24 h postinjection for Fib-GC@IP-HEATI, GC@IP-HEATI, and Fib-GC@Gold.(C) Quantitative analysis of thrombus visualization volume at 30 min and 24 h postinjection ( n = 3 per group).(D) Time-course coronal CT images of thrombus visualization following Fib-GC@IP-HEATI injection over 48 h, demonstrating time-dependent signal attenuation. The red box indicates the visualized thrombus region. Scale bar = 1 mm. In Vivo Degradation of Fib-GC@IP-HEATI in the Thrombotic Environment At 24 h after injection, thrombus visualization volume signal intensity decreased by approximately 70% in both the fibrin targeted and nontargeted GC@IP-HEATI groups, whereas the Fib-GC@Gold group showed no significant signal loss, indicating signal attenuation associated with nanoparticle degradation ( Figure C). The degradation of oxalate-containing IP-HEATI moieties by thrombus-associated ROS likely caused the intended nanoparticle disintegration and the expected loss of imaging capability. The degradation behavior of Fib-GC@IP-HEATI was monitored over a 48 h period by tracking changes in the imaging-derived thrombus visualization volume within the carotid artery ( Figure D). A 60% reduction in the thrombus visualization volume was observed within 12 h, and by 48 h the thrombus visualization volume signal fell below the detectable threshold on CT imaging, consistent with extensive nanoparticle degradation and the consequent loss of imaging capability. Notably, this time-dependent degradation profile closely mirrors the degradation behavior observed in vitro in PBS buffer containing H 2 O 2 , supporting the role of ROS in mediating IP-HEATI degradation. To determine whether the observed reduction in thrombus visualization volume resulted from degradation of the polymer within the nanoparticles leading to nanoparticle disintegration, rather than from simple detachment of thrombus-bound nanoparticles due to blood flow, an additional in vivo degradation study was conducted ( Figure A). As in prior experiments, thrombi were first induced in the left carotid artery of mice, followed by intravenous administration of Fib-GC@IP-HEATI (200 μL, 30 mg/mL; n = 3) or Fib-GC@Gold (200 μL, 2.5 mg/mL; n = 3) via the tail vein. Micro CT imaging was performed 30 min postinjection. After 24 h, a new thrombus was induced in the right carotid artery, and a second CT scan was acquired. If the administered nanoparticles had remained in circulation, the newly formed right carotid thrombus would have been visualized in the second scan. Conversely, the absence of enhancement in the new thrombus visualization volume indicates nanoparticle degradation and loss of imaging function. 4. Open in a new tab (A) Schematic illustration of in vivo thrombus visualization and nanoparticles degradation experiment procedure. In vivo cross section CT image of carotid artery thrombosis model acquired at 30 min and 24 h after inducing thrombosis in left carotid artery, followed by inducing thrombosis in right carotid artery. (B) Fib-GC@IP-HEATI (30 mg mL –1 , 200 μL), (C) Fib-GC@Gold (2.5 mg mL –1 , 200 μL) The red squares indicate the thrombus in the left carotid artery formed immediately after particle injection, while the yellow squares indicate the newly formed thrombus in the right carotid artery observed 24 h later. In vivo coronal view CT images of the carotid artery thrombosis model at 30 min and 24 h after inducing thrombosis in left carotid artery and after inducing thrombosis in right carotid artery. (D) Fib-GC@IP-HEATI (30 mg mL –1 , 200 μL), (E) Fib-GC@Gold (2.5 mg mL –1 , 200 μL), Scale bar = 1 mm, (F) Quantitative analysis of the thrombus visualization volume. ( n = 3/group) In the Fib-GC@Gold group, only a slight reduction in contrast was noted in the original thrombus, and the newly formed thrombus in the right carotid artery remained clearly visualized ( Figure C). Cross-sectional micro CT images further illustrated the distinct visualization patterns between the two nanoparticle formulations in the newly formed thrombus ( Figure E). Quantitative analysis showed that Fib-GC@Gold achieved near-complete visualization of the right carotid thrombi (85.3% of clot volume) 24 h postinjection ( Figure F). In the Fib-GC@IP-HEATI group, however, a marked decrease in the contrast-enhanced volume of the pre-existing left carotid thrombus was observed 24 h after injection, and the newly induced right carotid thrombus was not visualized ( Figure B,D). These results suggest that the signal loss observed with Fib-GC@IP-HEATI primarily arises from degradation of the oxalate linkages within IP-HEATI, triggered by ROS and hydrolytic conditions at the thrombus site, leading to nanoparticle disintegration. In contrast, Fib-GC@Gold nanoparticles remained structurally intact and continued image thrombi over the same period, reflecting their nondegradable naturea property advantageous for repeated animal imaging but less desirable for clinical translation. Micro CT Visualization of Cerebral Thrombus with Fib-GC@IP-HEATI To determine whether Fib-GC@IP-HEATI can visualize cerebral thrombi, a thromboembolic stroke model was prepared and followed by intravenous injection of Fib-GC@IP-HEATI. Embolic stroke was induced by delivering a fibrin clot prelabeled with a Cy5.5 fluorescent probe to the middle cerebral artery (MCA)–anterior cerebral artery (ACA) bifurcation via catheter. Successful thrombus formation was confirmed by a measurable decrease in cerebral blood flow on laser Doppler flowmetry. At 30 min postinjection, in vivo micro-CT imaging clearly visualized a Y-shaped thrombus at the MCA–ACA bifurcation ( Figure A). The contrast-enhanced thrombus volume observed on CT closely matched the thrombus region identified by ex vivo fluorescence imaging of the excised brains, confirming accurate localization and direct visualization of the cerebral thrombus. Following initial CT imaging, the animals were euthanized for ex vivo near-infrared fluorescence (NIRF) imaging, which further confirmed thromboembolic occlusion at the MCA–ACA junction with a well-defined Y-shaped morphology ( Figure B,C). Of the two mice, one succumbed shortly after CT imaging due to infarction, while the other survived for 4 h. During this period, the imaging-derived thrombus visualization volume decreased by approximately 60%, consistent with progressive degradation of Fib-GC@IP-HEATI in vivo ( Figure D,E). Collectively, these findings demonstrate that Fib-GC@IP-HEATI can visualize both large carotid artery thrombi and smaller cerebral thrombi and that it undergoes time-dependent degradation after thrombus localization. 5. Open in a new tab Representative coronal CT image (A), ex vivo Cy5.5 near-infrared fluorescent (NIRF) thrombus image (B) and visible light image (C) of thrombo-embolic stroke model with the Y-shaped embolic clot at the left distal internal carotid artery bifurcation 30 min after intravenous injection of Fib-GC@IP-HEATI. (D) Representative coronal CT images of thrombo-embolic stroke model acquired at 30 min and 4 h after injection of Fib-GC@IP-HEATI (50 mg mL –1 , 300 μL., n = 2) (E) Quantitative analysis of contrasted-enhanced Y-shaped thrombus volume at different time points. Discussion In this study, we demonstrate that Fib-GC@IP-HEATI enables effective in vivo CT visualization of thrombi through a combination of fibrin targeting, intravascular accessibility, and environmentally responsive degradation. Unlike conventional small-molecule iodinated contrast agents that rely solely on transient intravascular distribution, this nanoparticle-based platform is designed to interact directly with pathological components of thrombi, thereby achieving thrombus-specific contrast enhancement. Fibrin was selected as the targeting motif because it is not a constituent of normal physiological tissues and is predominantly confined to pathological coagulation environments. Although transient fibrin deposition may occur in certain pathological conditions such as acute inflammation or wound healing, these deposits are typically sparse, heterogeneous, and rapidly remodeled, lacking the dense and highly organized fibrin networks characteristic of mature intravascular thrombi. , Moreover, such extra-thrombotic fibrin deposition often occurs in extravascular or poorly accessible regions, limiting sustained accumulation of circulating agents. These features collectively support the thrombus selectivity of fibrin-targeted nanoparticles and reduce the likelihood of meaningful off-target imaging signals. The observed thrombus contrast was not solely dependent on fibrin targeting. Even nontargeted GC@IP-HEATI achieved substantial contrast in large carotid artery thrombi, which can be attributed to passive accumulation mechanisms arising from disturbed hemodynamics and electrostatic interactions. Specifically, the positively charged glycol chitosan backbone can interact with negatively charged components within thrombi, including red blood cells and platelets. A similar charge-mediated accumulation phenomenon was previously observed in our glycol chitosan–coated gold nanoparticle (GC@Gold) system, supporting the generality of this effect. However, such passive mechanisms are inherently limited in smaller vessels and intracranial thrombi, where nanoparticle–thrombus contact probability is reduced. In these settings, fibrin targeting is expected to play a critical role in ensuring stable retention and sensitive detection, as demonstrated by the superior performance of fibrin-targeted systems in embolic stroke models. From a safety perspective, no mortality, significant body weight loss, or abnormal behavior was observed in mice following nanoparticle administration under the dosing conditions used in this study, suggesting the absence of severe acute toxicity. Furthermore, the degradation products of IP-HEATI include ATIPA-based structures, which are well-established synthetic precursors for all clinically approved nonionic iodinated CT contrast agents. This structural and historical context supports the expectation of low intrinsic toxicity of the degradation products, although comprehensive toxicological, immunogenicity, and clearance studies will be required as the platform advances toward formal preclinical development. Beyond diagnostic imaging, this work aligns with a broader emerging paradigm in which nanomaterials are designed to function as active interfaces with pathological microenvironments through multiple physical and biological interactions. Recent studies have demonstrated that multifunctional nanomaterial platforms can integrate imaging, signal modulation, and tissue repair within complex in vivo environments. Although developed in different pathological contexts, such advances highlight the potential for nanomaterial-based systems to move beyond passive contrast enhancement toward integrated diagnostic and therapeutic applications. − While the present study focuses on thrombus imaging, the modular design of Fib-GC@IP-HEATI suggests opportunities for future expansion into theranostic strategies. The translational advancement of polymeric and nanoparticle-based contrast agents is inevitably accompanied by regulatory and manufacturing challenges. Compared with small-molecule agents, regulatory evaluation requires stringent batch-to-batch control over critical quality attributes such as particle size distribution, iodine content, surface chemistry, stability, and degradation behavior, as well as comprehensive assessment of biodistribution, clearance pathways, and immunological responses. Scalable manufacturing and sterilization strategies that preserve product consistency will also be essential. While these considerations remain important challenges, they will require systematic investigation during the chemistry, manufacturing, and controls (CMC) stage, including rigorous definition of critical quality attributes, scalable manufacturing protocols, and standardized quality control strategies as part of formal nonclinical development. Finally, although ischemic stroke was selected as the primary disease model in this study due to its urgent clinical need and diagnostic challenges, fibrin is a universal structural component of thrombi regardless of anatomical location. The fibrin-targeting peptide employed here (EP-2104R) has previously demonstrated feasibility for thrombus imaging in other thrombotic conditions, suggesting that this platform may be extendable to additional thrombotic diseases such as deep vein thrombosis and pulmonary embolism. Disease-specific validation will be required to confirm performance under distinct hemodynamic and pathological conditions, and such applications are therefore proposed as future research directions. In addition, it should be acknowledged that the embolic stroke model used in this study relies on the introduction of externally prepared clots, which provides a controlled and reproducible framework for evaluating thrombus imaging performance but does not fully recapitulate the complex pathophysiology of spontaneous ischemic stroke. In clinically relevant settings, thrombi form in situ under dynamic hemodynamic, inflammatory, and biochemical conditions that may influence fibrin organization and nanoparticle–thrombus interactions. Accordingly, further validation in endogenous ischemic stroke modelswhere thrombosis arises naturally without exogenous clot implantationwill be essential to more rigorously assess targeting specificity, imaging sensitivity, and translational relevance. Such studies are planned as an important next step toward advancing this platform into preclinical development. Conclusions In this study, we report the first organic material-based contrast agent capable of directly imaging carotid and cerebral thrombi. An iodine-rich monomer designed for X-ray attenuation (HEATI) was synthesized and polymerized with oxalyl chloride to form a ROS-triggered degradable oxalate linker (IP-HEATI). Degradation of IP-HEATI under H 2 O 2 conditions was confirmed by a decrease in molecular weight. The IP-HEATI was encapsulated into glycol chitosan-coated nanoparticles (GC@IP-HEATI) via a nanoprecipitation process, producing particles with a uniform diameter of approximately 300 nm. To confer thrombus targeting capability, fibrin-targeting peptidesspecifically expressed in blood clotswere conjugated to the glycol chitosan surface, yielding Fib-GC@IP-HEATI. These nanoparticles successfully visualized thrombi in both extracranial carotid artery and intracranial arteries, accurately delineating thrombus volume and morphology in mice. Over time, the imaging-derived thrombus visualization volume progressively decreased, consistent with degradation of the oxalate linkages within IP-HEATI and subsequent nanoparticle disintegration. The development of this organic nanoparticle contrast agent enables direct and quantitative imaging of cerebral thrombi, outperforming conventional iodine-based small molecule agents that are unable to visualize thrombi and provide quantitative assessment of their burden. Materials and Methods Materials Potassium fluoride (KF, 99%), chloroethanol (99%), oxalyl chloride (98%), triethyl amine (TEA, 99%), N , N -dimethylformamide (DMF, anhydrous, 99.8%), glycol chitosan (GC) ( M w = 350 kDa), N -(3-(dimethylamino)propyl)- N ′-ethylcarbodiimide (EDC, 97%), N -hydroxysuccinimide (NHS, 98%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 5-Amino-2,4,6-triiodoisophthalic acid (ATIPA, 96%) were commercially available from Tokyo Chemical Industry Co., Ltd. (Japan). Dimethyl sulfoxide (DMSO, 99.5%), potassium carbonate (K 2 CO 3 , 99.5%), sodium chloride (NaCl, 99%), magnesium sulfate (MgSO 4 , 99%), ethyl acetate (EA, 99.5%), hexane (95%), hydrogen peroxide (H 2 O 2 , 30%) and tetrahydrofuran (THF, 99.5%) were obtained from Daejung Chemicals and Metals Co. (Korea). THF was distilled from sodium benzophenone and TEA was dried over calcium hydride. All other chemicals were used as received without further purification. Water was double distilled before use. Instruments 1 H NMR analysis was performed using Bruker Advance 300 MHz spectrometer in CDCl 3 or DMSO- d 6 at room temperature. UV–vis spectra were recorded from UV-2450 by Shimadzu (Tokyo, Japan) scanned over the range of 300–600 nm. Gel permeation chromatography (GPC) was taken with CTO-10A (Shimadzu) at 40 °C using 10 mM LiBr in N , N -dimethylformamide as an eluent at the rate of 1.0 mL/min and the molecular weight was calculated with polystyrene standards. Measurements of particle size and zeta potential were carried out using Anton-paar Litesizer 500 instrument equipped with semiconductor laser at a wavelength of 658 nm. EDS analysis were performed with Field Emission Scanning Electron Microscope of JEOL Ltd. JSM-7800F Prime instrument. In vitro and in vivo radiopacity of samples were investigated with a micro CT instrument, NFR Polaris-G90 (NanoFocusRay, Jeonju, Korea). Synthesis of Bis(2-hydroxyethyl) 5-Amino-2,4,6-triiodoisophthalate (HEATI) 80 mL DMF dissolving ATIPA (10.0 g, 17.9 mmol) were charged to a 2-neck round-bottom flask under nitrogen atmosphere. KF (2.1 g, 35.8 mmol) and chloroethanol (7.2 g, 89.5 mmol) were introduced into the mixture and the reaction was continued at 40 °C for 12 h. After the reaction, the reaction mixture was cooled to room temperature. 300 mL water were poured into the reaction mixture and solution was extracted by EA (3 × 200 mL) and the organic layer was collected. The organic layer was extracted by 10% K 2 CO 3 aqueous solution (2 × 300 mL), saturated NaCl solution (2 × 300 mL) and dried over MgSO 4 . After removing the volatiles in vacuo, the residues was purified by column chromatography (EA: Hexane = 2:1) to give ATIPA-Ester-diol as a viscous oil (Yield: 56.1%). 1 H NMR (300 MHz, DMSO- d 6 , δ): 5.69 (s, 2 H), 4.97–4.93 (t, J = 1.8 Hz, 1 H), 4.28–4.25 (t, J = 1.9 Hz, 2 H), 3.73–3.68 (q, J = 1.8 Hz, 2 H) Polymerization of ATIPA-Ester-diol (IP-HEATI) HEATI (5.0 g, 7.7 mmol) and THF (50 mL) were charged into a 100 mL 2-neck round-bottom flask which placed in an ice bath under nitrogen atmosphere. Oxalyl chloride (1.0 g, 7.7 mmol) were added to reaction mixture, followed by dropwise addition of TEA (0.8 g, 8.1 mmol). After the addition was complete, the temperature was raised up to room temperature and stirred for 5 h. After the reaction, the reaction mixture was precipitated in 300 mL EA to give IP-HEATI as a white powder (Yield: 86.8%). 1 H NMR (300 MHz, DMSO- d 6 , δ): 5.81–5.67 (m, 11 H), 4.71–4.53 (m, 11 H), 4.35–4.21 (m, 11 H), 3.81–3.65 (m, 2 H) Preparation of Glycol Chitosan Coated Polyoxalate Nanoparticles (GC@IP-HEATI NPs) and Fibrin Targeted Glycol Chitosan Coated Polyoxalate Nanoparticles (Fib-GC@IP-HEATI NPs) GC@IP-HEATI NPs were prepared by nanoprecipitation method. IP-HEATI in DMSO (5 mL, 10 mg/mL) was added dropwise (0.2 mL/min) into GC aqueous solution (50 mL, 1 mg/mL) under sonication using probe-type sonicator for 30 min under an ice bath and centrifuged 2 times (14,000 rpm, 60 min) to remove unreacted GC. Fib-GC@IP-HEATI NPs (3.8 mL, 5 mg/mL) and EP-2104R (1.2 mg, 8.4 μmol) were introduced into 10 mL Schlenk tube and stirred at room temperature for 1 h. EDC (3.9 mg, 20.4 μmol) and NHS (2.4 mg, 20.4 μmol) were transferred into colloid. After 4 h, Fib-GC@PO NPs were centrifuged (14,000 rpm, 60 min) and washed 2 times with water to remove remnants of the peptide. In Vitro CT Imaging of GC@IP-HEATI GC@IP-HEATI NPs were dispersed in distilled water at different concentrations (1.3, 2.6, 5.2, 10.4, 20.8 mg I/mL) and loaded into eppendorf tube. The CT images were captured using a clinical positron emission tomography/CT scanner (Gemini; Philips Medical Systems, Cleveland, OH. USA; values in Hounsfield Unit) as well as micro CT scanner (NFR Polaris-G90; NanoFocusRay, Jeonju, Korea; values in arbitrary unit). CT parameters were as follows; 500 ms/frame, 65 kVp, 60 μA., In Vitro GC@IP-HEATI NPs Degradation Test GC@IP-HEATI NPs which dispersed in PBS buffer and PBS buffer containing 10 mM H 2 O 2 were collected at the predetermined time points (3, 6, 9, 24, 48 h) and lyophilized. Molecular weights of each sample were evaluated by gel permeation chromatography. Fibrin Clot Binding Test with Fibrin Gel and Fib-GC@IP-HEATI NPs Fibrin clot binding test were followed the previously reported method. Fibrin gel was formed with fibrinogen (4 mg) and thrombin (0.3 U) in 1 mL of pH 7.4 normal saline containing 2.5 mM CaCl 2 . After incubation of reaction mixture at 37 °C for 1 h, fibrin gel was immersed in 1 mL of GC@IP-HEATI NPs and Fib-GC@IP-HEATI NPs (2.5 mg/mL). The mixture was shaken at 37 °C for 1 h to assess the interaction between fibrin and nanoparticles. After the reaction, supernatant was collected and added DMSO (water: DMSO, 1:2 by volume) to dissolve Fib-GC@PO NPs. The UV absorbance of the resulting solution was measured at the wavelength of 322 nm to determine the conjugation ratio of Fib-GC@IP-HEATI NPs to fibrin gel. Mouse Carotid Artery Model All following animal procedures demonstrated have been reviewed and approved by the Dongguk University Ilsan Hospital Animal Care and Use Committee (IACUC 2020–01195) and conducted in accordance with the principles and procedures outlined in the NIH Guide for the Care and Use of Animals. Thrombus formation in the carotid artery followed the previously reported method. , 10-week-old C57BL/6 mice were anesthetized using an Anesthesia machine (Royal medical; Seoul, Korea). Strips of filter paper (grade 42; Whatman; Oxon, UK; 1 × 1 mm 2 ) soaked in 10% FeCl3 were applied to the surgically exposed common carotid artery for 10 min. The incision site was closed and 200 μL of nanoparticles were injected intravenously via the tail vein. In vivo micro CT imaging was performed under the following protocol: 65 kVp, 60 μA, 26.7 × 26.7 mm 2 field of view, 0.053 × 0.053 × 0.054 mm 3 voxel size, 500 ms per frame, 360 views, 512 × 512 reconstruction matrix, and 600 slices. Micro CT data were converted to the Digital Imaging and Communications in Medicine (DICOM) format to make 3-dimensionally rendered imaging using a software package (Lucion, MeviSYS, Seoul, Korea). Concentration of nanoparticles was from 1 to 50 mg/mL for Fib-GC@IP NPs, 30 mg/mL for GC@IP-HEATI and 2.5 mg/mL for Fib-GC@AuNP. ( N = 3 for each concentration) For comparison with a clinically used iodinated contrast agent, Ultravist 370 was continuously administered via a syringe pump (total volume: 400 μL) during real-time micro-CT acquisition. CT image was taken and the volume of visualized thrombus was measured by using image analysis software, ImageJ (NIH, Bethesda, MD, USA). Carotid micro CT images were analyzed, and a representative longitudinal section image (0.053 mm thickness) was reconstructed to encompass the full length of the carotid thrombus. For animals that underwent serial imaging, follow-up images were prepared in the same manner as the corresponding baseline images by selecting DICOM files in the same orientation and position, referencing nearby bony structures. In Vivo Fib-GC@IP-HEATI NPs Degradation Test Thrombus was formed in the carotid artery as mentioned above and nanoparticles were injected intravenously via the tail vein at 30 mg/mL for Fib-GC@IP-HEATI and GC@IP-HEATI and 2.5 mg/mL for Fib-GC@AuNP. ( N = 3 for each nanoparticle) CT images were taken at 30 min and 24 h after injection. The volume of visualized thrombus was measured and compared between nanoparticles. For Fib-GC@IP-HEATI, degradation was observed with further experiments. The illustration of the experiment scheme is in Figure (a). Thrombus was formed in the left carotid artery and Fib-GC@IP-HEATI was injected intravenously via tail vein at 30 mg/mL. ( N = 3) After 30 min, a CT image was taken. Then, 24 h after injection, the mouse was anesthetized and a thrombus was formed in the right carotid artery. CT image was taken right immediately without additional injection of nanoparticles. The same procedure was applied for Fib-GC@AuNP instead of Fib-GC@IP-HEATI. In vivo degradation of Fib-GC@IP-HEATI was observed over 48 h. Thrombus was formed in the carotid artery and Fib-GC@IP-HEATI was injected intravenously via tail vein at 30 mg/mL ( N = 3). CT image was taken at predetermined time points and the volume of visualized thrombus is measured. Mouse Embolic Stroke Model Thromboembolic stroke for in vivo imaging was induced by following the previously reported method. An exogenously formed thrombus which is labeled with fluorescence marker C15 probe is prepared by the method mentioned in the reference above. Then, 10-week-old C57BL/6 mice were anesthetized. Laser Doppler Flowmetry (Omefawave, Tokyo, Japan) was used to monitor cerebral blood flow. Using a sterile silk suture, the proximal common carotid artery and distal/proximal external carotid artery were ligated. Then a small hole was made between the ligated sites of the external carotid artery using a microscissor. A thrombus-containing catheter was inserted into the external carotid artery. Using an electrical cautery (Bovie, NY, USA), the superficial temporal artery, pterygopalatine artery, and ligated external carotid artery was cauterized. The inserted catheter was aligned to the direction of the internal carotid artery and advanced 9 mm to place the thrombus into the middle cerebral artery-anterior cerebral artery bifurcation area. After the thrombus was placed cerebral blood flow was monitored for 40 min and only mice that showed a decrease in cerebral blood flow below 40% of baseline were used ( N = 2). Fib-GC@IP-HEATI was injected intravenously into thromboembolic stroke induced mouse via tail vein at 50 mg/mL. Then, a CT image was taken right after the injection. One of the 2 mice died right after the CT image was taken and the other died after 4 h. Then the brain was resected and NIRF imaging was performed. Supplementary Material nn6c01281_si_001.pdf (521.8KB, pdf) Acknowledgments This research was supported by the Korea Drug Development Fund, funded by the Ministry of Science and ICT, the Ministry of Trade, Industry, and Energy, and the Ministry of Health and Welfare (2710086751/RS-2025-02223267, Republic of Korea), and Korea Basic Science Institute (National Research Facilities and Equipment Center) grant, funded by the Ministry of Education (grant No. RS-2025-02314069). The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.6c01281 . 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