Near-infrared Fluorescence Imaging Agent Research Articles

Azaphosphinate Dyes: A Low Molecular Weight Near-Infrared Scaffold for Development of Photoacoustic or Fluorescence Imaging Probes.

Near-infrared (NIR) dyes are desirable for biological imaging applications including photoacoustic (PA) and fluorescence imaging. Nonetheless, current NIR dyes are often plagued by relatively large molecular weights, poor water solubility, and limited photostability. Herein, we provide the first examples of azaphosphinate dyes which display desirable properties such as low molecular weight, absorption/emission above 750 nm, and remarkable water solubility. In PA imaging, an azaphosphinate dye exhibited a 4.1-fold enhancement in intensity compared to commonly used standards, the ability to multiplex with existing dyes in whole blood, imaging depths of 2.75 cm in a tissue model, and contrast in mice. An improved derivative for fluorescence imaging displayed a >10-fold reduction in photobleaching in water compared to the FDA-approved indocyanine green dye and could be visualized in mice. This new dye class provides a robust scaffold for the development of photoacoustic or NIR fluorescence imaging agents.

In Vivo Evaluation of a Gallium-68-Labeled Tumor-Tracking Cyanine Dye for Positron Emission Tomography/Near-Infrared Fluorescence Carcinoma Imaging, Image-Guided Surgery, and Photothermal Therapy.

Positron emission tomography (PET)/near-infrared fluorescence (NIRF) dual-modal imaging presents an enticing prospect for tumor diagnosis and surgical navigation. In this study, we developed a novel probe IR808-DOTA for tumor-targeted PET/NIRF imaging, image-guided surgery, and photothermal therapy. This construct had better water solubility and pharmacokinetics than IR808 and had similar photophysical properties, tumor targeting ability, and photothermal anticancer effect to IR808. By a simple labeling process, IR808-DOTA was labeled with gallium-68 and applied as a PET probe for tumor imaging in MCF-7 tumor xenografted mice. IR808-DOTA itself acted as an NIRF imaging agent in the following surgery for intraoperative navigation to aid surgeons in the delineation of tumor margins and visualizing sentinel lymph nodes to facilitate a more thorough tumor resection. Irradiation by laser, IR808-DOTA could prominently inhibit tumor growth in MCF-7 subcutaneous tumor model mice by directly ablating tumor cells, inhibiting tumor proliferation, and promoting tumor cell apoptosis. In summary, 68Ga-DOTA-IR808 could enable a convenient and user-friendly workflow for tumor imaging and guided surgery, and therefore, it may have great prospects for clinical translation as a PET/NIRF dual-modal probe.

Transbronchial real-time lung tumor localization with folate receptor–targeted near-infrared molecular imaging: A proof of concept study in animal models

ObjectiveThe diagnostic yield of bronchoscopy is not satisfactory, even with recent navigation technologies, especially for tumors located outside of the bronchial lumen. Our objective was to perform a preclinical assessment of folate receptor–targeted near-infrared imaging-guided bronchoscopy to detect peribronchial tumors. MethodsPafolacianine, a folate receptor-targeted molecular imaging agent, was used as a near-infrared fluorescent imaging agent. An ultra-thin composite optical fiberscope was used for laser irradiation and fluorescence imaging. Subcutaneous xenografts of KB cells in mice were used as folate receptor–positive tumors. Tumor-to-background ratio was calculated by the fluorescence intensity value of muscle tissues acquired by the ultra-thin composite optical fiberscope system and validated using a separate spectral imaging system. Ex vivo swine lungs into which pafolacianine-laden KB tumors were transplanted at various sites were used as a peribronchial tumor model. ResultsWith the in vivo murine model, tumor-to-background ratio observed by ultra-thin composite optical fiberscope peaked at 24 hours after pafolacianine injection (tumor-to-background ratio: 2.56 at 0.05 mg/kg, 2.03 at 0.025 mg/kg). The fluorescence intensity ratios between KB tumors and normal mouse lung parenchyma postmortem were 6.09 at 0.05 mg/kg and 5.08 at 0.025 mg/kg. In the peribronchial tumor model, the ultra-thin composite optical fiberscope system could successfully detect fluorescence from pafolacianine-laden folate receptor–positive tumors with 0.05 mg/kg at the carina and those with 0.025 mg/kg and 0.05 mg/kg in the peripheral airway. ConclusionsTransbronchial detection of pafolacianine-laden folate receptor–positive tumors by near-infrared imaging was feasible in ex vivo swine lungs. Further in vivo preclinical assessment is needed to confirm the feasibility of this technology.

A Series of PSMA-Targeted Near-Infrared Fluorescent Imaging Agents.

We have synthesized a series of 10 new, PSMA-targeted, near-infrared imaging agents intended for use in vivo for fluorescence-guided surgery (FGS). Compounds were synthesized from the commercially available amine-reactive active NHS ester of DyLight800. We altered the linker between the PSMA-targeting urea moiety and the fluorophore with a view to improve the pharmacokinetics. Chemical yields for the conjugates ranged from 51% to 86%. The Ki values ranged from 0.10 to 2.19 nM. Inclusion of an N-bromobenzyl substituent at the ε-amino group of lysine enhanced PSMA+ PIP tumor uptake, as did hydrophilic substituents within the linker. The presence of a polyethylene glycol chain within the linker markedly decreased renal uptake. In particular, DyLight800-10 demonstrated high specific uptake relative to background signal within kidney, confirmed by immunohistochemistry. These compounds may be useful for FGS in prostate, renal or other PSMA-expressing cancers.

Bay Leaf Extract-Based Near-Infrared Fluorescent Probe for Tissue and Cellular Imaging.

The development of fluorescence dyes for near-infrared (NIR) fluorescence imaging has been a significant interest for deep tissue imaging. Among many imaging fluoroprobes, indocyanine green (ICG) and its analogues have been used in oncology and other medical applications. However, these imaging agents still experience poor imaging capabilities due to low tumor targetability, photostability, and sensitivity in the biological milieu. Thus, developing a biocompatible NIR imaging dye from natural resources holds the potential of facilitating cancer cell/tissue imaging. Chlorophyll (Chl) has been demonstrated to be a potential candidate for imaging purposes due to its natural NIR absorption qualities and its wide availability in plants and green vegetables. Therefore, it was our aim to assess the fluorescence characteristics of twelve dietary leaves as well as the fluorescence of their Chl extractions. Bay leaf extract, a high-fluorescence agent that showed the highest levels of fluorescence, was further evaluated for its tissue contrast and cellular imaging properties. Overall, this study confirms bay-leaf-associated dye as a NIR fluorescence imaging agent that may have important implications for cellular imaging and image-guided cancer surgery.

Tomographic Imaging and Correlation to Quantify Vascular and Inflammatory Changes in an Experimental Spinal Cord Injury.

Spinal cord injury (SCI) is a devastating condition causing the loss of sensory and motor functions. SCI pathology is multifaceted, encompassing inflammation, scarring, neuronal damage, and vascular and tissue remodeling. The dynamics of SCI rapidly transform from acute, sub-acute, and chronic phases. The rapidly changing environment necessitates the real-time monitoring of disease severity. Therefore, in this study, we used the IVIS spectrum, a noninvasive fluorescence imaging modality, to monitor the disease pathology in live animals. We used near-infrared fluorescence imaging agents including Angiosense 750 EX, a probe that detects vascular changes, and Cat B 680 FAST, a probe that detects inflammation at various day points post injury (DPI), that is, DPI-1, DPI-14, and DPI-28. We quantified the pathophysiological changes after SCI using IVIS in live animals. As a result, we observed distinct differences in the disease progression between injured and sham mice. Moreover, live imaging showed a good correlation with behavioral studies, protein expression, and immunohistological analysis. Hence, the goal of this study was to introduce a new optical imaging modality that offers a determination of disease severity and the advantage of accelerated imaging of the correlated biomarkers in a real-time and dynamic manner. This study concluded that Cat B 680 Fast and Angiosense 750 EX could be used to assess the disease severity after SCI. Furthermore, our study suggests that the noninvasive fluorescence optical imaging modality offers a unique approach in monitoring neuroinflammatory diseases in live animals.

A Molecularly Targeted Intraoperative Near-Infrared Fluorescence Imaging Agent for High-Grade Serous Ovarian Cancer.

Ovarian cancer is the fifth leading cause of cancer deaths among women, accounting for more deaths than any other cancer of the female reproductive system. The foundation of its management consists of cytoreductive surgery (CRS) followed by systemic chemotherapy, with the completeness of surgical resection consistently identified as one of the most important prognostic factors for the disease. The goal of our investigation is the development of a near-infrared fluorescence (NIRF) imaging agent for the intraoperative imaging of high-grade serous ovarian cancer (HGSOC). As surgeons are currently limited to the visual and manual assessment of tumor tissue during CRS, this technology could facilitate more complete resections as well as serve important functions at other points in the surgical management of the disease. Elevated levels of cancer antigen 125 (CA125) have proven a useful biomarker of HGSOC, and the CA125-targeting antibody B43.13 has shown potential as a platform for immunoPET imaging in murine models of ovarian cancer. Herein, we report the development of a NIRF imaging agent based on B43.13: ssB43.13-IR800. We site-specifically modified the heavy chain glycans of B43.13 with the near-infrared dye IRDye 800CW using a chemoenzymatic approach developed in our laboratories. SDS-PAGE analysis confirmed the specificity of the conjugation reaction, and flow cytometry, immunostaining, and fluorescence microscopy verified the specific binding of ssB43.13-IR800 to CA125-expressing OVCAR3 human ovarian cancer cells. NIRF imaging studies demonstrated that ssB43.13-IR800 can be used to image CA125-expressing HGSOC tumors in subcutaneous, orthotopic, and patient-derived xenograft mouse models. Finally, ex vivo analyses confirmed that ssB43.13-IR800 can bind and identify CA125-expressing cells in primary tumor and metastatic lymph node samples from human patients with HGSOC.

A Conserved Molecular Signaling Target for Fluorescence Imaging of Solid Tumors

HomeRadiology: Imaging CancerVol. 2, No. 3 PreviousNext Research HighlightsFree AccessA Conserved Molecular Signaling Target for Fluorescence Imaging of Solid TumorsGary D. LukerGary D. LukerGary D. LukerPublished Online:May 29 2020https://doi.org/10.1148/rycan.2020204014MoreSectionsPDF ToolsImage ViewerAdd to favoritesCiteTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinked InEmail Take-Away Points■ Major Focus: To develop a peptide-based, near-infrared fluorescence imaging agent to detect abnormal signaling in tumors.■ Key Result: Discovered an octapeptide imaging agent that binds to a phosphorylated cell surface protein, Annexin A2.■ Impact: Sensitive detection of a wide range of solid tumors for image-guided surgery and targeted drug delivery.Molecular imaging agents and targeted therapies commonly rely on cell surface proteins selectively or preferentially overexpressed by malignant cells. The limited number of cancer-selective cell surface targets typically apply only to subtypes of one or a few cancers, necessitating repeated development and testing of new imaging agents. To overcome this challenge, Shen et al focused on imaging a protein modified by activation of a signaling process, phosphorylation, commonly dysregulated in most solid tumors. The investigators designed an octapeptide, near-infrared fluorescence imaging agent that bound with high affinity to the phosphorylated form of Annexin A2, a protein that localizes to the cell surface and drives invasion of cancer cells only when phosphorylated. The imaging agent bound to phosphorylated Annexin A2 in cancer cell lines, mouse models of cancer, and human cancer tissues from malignancies including breast cancer, pancreatic cancer, and soft-tissue sarcoma. Cancer cells also caused phosphorylation of Annexin A2 in adjacent fibroblasts and macrophages, allowing imaging of invasive margins of tumors. Normal fibroblasts and macrophages did not bind the imaging agent. In mouse models of breast cancer, the imaging probe detected lymph node metastases and tumor margins for highly effective image-guided surgery. When conjugated to the chemotherapy drug doxorubicin, the same octapeptide targeting agent also improved drug delivery to tumors while sparing dose-limiting toxicity to the heart. By targeting aberrant signaling in cancer, the investigators developed a new imaging agent with potentially broad applications for imaging and image-guided therapy of many solid tumors. The research team next plans to advance this imaging agent to clinical testing.Highlighted ArticleShen D, Xu B, Liang K, et al. Selective imaging of solid tumours via the calcium-dependent high-affinity binding of a cyclic octapeptide to phosphorylated Annexin A2. Nat Biomed Eng 2020;4(3):298–313. doi: https://doi.org/10.1038/s41551-020-0528-7Highlighted ArticleShen D, Xu B, Liang K, et al. Selective imaging of solid tumours via the calcium-dependent high-affinity binding of a cyclic octapeptide to phosphorylated Annexin A2. Nat Biomed Eng 2020;4(3):298–313. doi: https://doi.org/10.1038/s41551-020-0528-7 Crossref, Medline, Google ScholarArticle HistoryPublished online: May 29 2020 FiguresReferencesRelatedDetailsRecommended Articles Cancer Genomics and Important Oncologic Mutations: A Contemporary Guide for Body ImagersRadiology2017Volume: 283Issue: 2pp. 314-340Cryoablation and Immunotherapy for Breast Cancer: Overview and Rationale for Combined TherapyRadiology: Imaging Cancer2021Volume: 3Issue: 2High-Resolution Photoacoustic Tomography for Early-Stage Cancer Detection and Its Clinical TranslationRadiology: Imaging Cancer2020Volume: 2Issue: 3Breast Cancer Tissue Markers, Genomic Profiling, and Other Prognostic Factors: A Primer for RadiologistsRadioGraphics2018Volume: 38Issue: 7pp. 1902-1920Nanotechnology for Cancer Imaging: Advances, Challenges, and Clinical OpportunitiesRadiology: Imaging Cancer2021Volume: 3Issue: 3See More RSNA Education Exhibits Imaging Biomarkers in Targeted Therapies: From Quantitative Imaging to RadiomicsDigital Posters2019Breast Health in the Elderly: Implications for ImagingDigital Posters2020American Joint Committee on Cancer’s Staging System for Breast Cancer, Eighth Edition: Pictorial Guide for RadiologistsDigital Posters2020 RSNA Case Collection Locally Advanced Breast CancerRSNA Case Collection2021Male Breast CancerRSNA Case Collection2021Secondary Angiosarcoma of the BreastRSNA Case Collection2021 Vol. 2, No. 3 Metrics Downloaded 142 times Altmetric Score PDF download

Intravascular Molecular Imaging to Detect High-Risk Vulnerable Plaques: Current Knowledge and Future Perspectives

To describe vulnerable plaque pathobiology and summarize potential targets for molecular imaging with a focus on intravascular near-infrared fluorescence (NIRF) and its translatable applications. Structural imaging alone is unable to precisely identify high-risk plaques in patients with coronary artery disease (CAD). Intravascular near-infrared fluorescence (NIRF) imaging is an emerging translational approach that can image specific in vivo molecular processes and cells that characterize vulnerable plaques. High-priority NIRF targets imaged by intravascular NIRF imaging thus far include macrophages, cathepsin protease activity, oxidized low-density lipoprotein (oxLDL), and abnormal endothelial permeability. The newest generation of NIRF catheters is multimodal in nature and combines NIRF with either IVUS or OCT, providing simultaneous co-registered morphological and pathobiological assessment of atherosclerotic plaques. While most intravascular NIRF studies have been performed in a preclinical environment, a first-in-human NIR autofluorescence-OCT trial has recently been performed. These developments suggest that clinical intravascular NIRF molecular imaging will be available within the next 3 years. Molecular imaging is a powerful approach to enhance our understanding of atherosclerosis pathophysiology. Intravascular NIRF/OCT and NIRF/IVUS molecular imaging is nearing its use in atherosclerosis patients and will initially leverage indocyanine green (ICG) as an FDA-approved NIRF agent that reports on abnormal plaque permeability. Clinical trials are needed to assess the value of intravascular NIRF imaging using ICG as well as other novel NIRF imaging agents to better understand vulnerable plaque pathobiology, event prediction, and to enable personalized pharmacotherapy of high-risk plaques and patients.

Applications of near infrared and surface enhanced Raman scattering techniques in tumor imaging: A short review.

Imaging technologies play a vital role in clinical oncology and have undergone massive growth over the past few decades. Research in the field of tumor imaging and biomedical diagnostics requires early detection of physiological alterations so as to provide curative treatment in real time. The objective of this review is to provide an insight about near infrared fluorescence (NIRF) and surface enhanced Raman scattering (SERS) imaging techniques that can be used to expand their capabilities for the early detection and diagnosis of cancer cells. Basic setup, principle and working of the instruments has been provided and common NIRF imaging agents as well as SERS tags are also discussed besides the analytical advantages/disadvantages of these techniques. This review can help researchers working in the field of molecular imaging to design cost effective fluorophores and SERS tags to overcome the limitations of both NIRF as well as SERS imaging technologies.

Matrix metallopeptidase 2 targeted delivery of gold nanostars decorated with IR-780 iodide for dual-modal imaging and enhanced photothermal/photodynamic therapy

Nanotheranostics has gained increasing interest, as it offers a great potential to realize personalized diagnostics and therapy. In this work, we report a facile approach of the fabrication of gold nanostars (GNS) attached with matrix metalloproteinases (MMP2) polypeptides (Ac-GPLGIAGQ) and IR-780 iodide through bovine serum albumin (BSA) for targeted dual-modal photoacoustic (PA)/near-infrared (NIR) fluorescence imaging and enhanced photothermal therapy (PTT)/photodynamic therapy (PDT) for lung cancer. MMP2 polypeptides served as the targeting ligand, IR-780 iodide functioned as the NIR fluorescence imaging agent as well as PTT/PDT agent, and GNS acted as the carrier of IR-780 molecules and performed PA imaging and PTT. DLS and CCK-8 assay demonstrated that the nanoprobes (GNS@BSA/I-MMP2) exhibited excellent stability and biocompatibility under physiological conditions. Subsequent in vitro studies verified that GNS@BSA/I-MMP2 nanoparticles (NPs) were effectively internalized by A549 cancer cells and exhibited remarkable antitumor efficacy. Furthermore, GNS@BSA/I-MMP2 NPs could specifically target the tumor and significantly suppress the tumor growth, and their antitumor effects were mainly through the synergistic effects of PDT and PTT based on IR-780 and GNS. These findings imply the potential of GNS@BSA/I-MMP2 NPs as a targeting PA/NIR probe in tumor diagnosis and combined therapy with a single light source. Statement of SignificanceWe reported a convenient and facile approach to load IR-780 iodides in gold nanostars (GNS). This material could simultaneously perform near-infrared imaging/photoacoustic imaging and thermotherapy/photodynamic therapy.MMP2 coating on the surface of GNS@BSA/IR-780 promoted the prepared nanoparticles (GNS@BSA/I-MMP2) to target the tumor region.The heat generated by the synergistic effect of the GNS and IR-780 molecules resulted in the high temperature of the GNS@BSA/I-MMP2 NPs, which efficiently suppressed the growth of tumor, and the tumor volume decreased by 93% compared with that in the PBS groups with laser irradiation.

NIRF Optical/PET Dual-Modal Imaging of Hepatocellular Carcinoma Using Heptamethine Carbocyanine Dye.

Combining near-infrared fluorescence (NIRF) and nuclear imaging techniques provides a novel approach for hepatocellular carcinoma (HCC) diagnosis. Here, we report the synthesis and characteristics of a dual-modality NIRF optical/positron emission tomography (PET) imaging probe using heptamethine carbocyanine dye and verify its feasibility in both nude mice and rabbits with orthotopic xenograft liver cancer. This dye, MHI-148, is an effective cancer-specific NIRF imaging agent and shows preferential uptake and retention in liver cancer. The corresponding NIRF imaging intensity reaches 109/cm2 tumor area at 24 h after injection in mice with HCC subcutaneous tumors. The dye can be further conjugated with radionuclide 68Ga (68Ga-MHI-148) for PET tracing. We applied the dual-modality methodology toward the detection of HCC in both patient-derived orthotopic xenograft (PDX) models and rabbit orthotopic transplantation models. NIRF/PET images showed clear tumor delineation after probe injection (MHI-148 and 68Ga-MHI-148). The tumor-to-muscle (T/M) standardized uptake value (SUV) ratios were obtained from PET at 1 h after injection of 68Ga-MHI-148, which was helpful for effectively capturing small tumors in mice (0.5 cm × 0.3 cm) and rabbits (1.2 cm × 1.8 cm). This cancer-targeting NIRF/PET dual-modality imaging probe provides a proof of principle for noninvasive detection of deep-tissue tumors in mouse and rabbit and is a promising technique for more accurate and early detection of HCC.

Metal-Organic Frameworks@Polymer Composites Containing Cyanines for Near-Infrared Fluorescence Imaging and Photothermal Tumor Therapy.

As a noninvasive treatment method, photothermal therapy (PTT) has been widely investigated for cancer therapy. In this work, metal-organic frameworks@polymer composites (UiO-66@CyP) with bioimaging and PTT activity were prepared by introducing cyanine-containing polymer (CyP) via multicomponent Passerini reaction in the presence of Zr-based nanoscale metal-organic frameworks (UiO-66). As-prepared UiO-66@CyP not only possesses uniformed size, controllable morphology, and excellent dispersibility in aqueous media, but also indicates strong near-infrared absorption and high photothermal conversion efficiency. Due to these combined merits, UiO-66@CyP appears to be an excellent phototherapy agent for ablation of tumor cells under a low-power laser irradiation and near-infrared fluorescence imaging agent. This work might open up a new avenue to develop multifunctional composites by integrating metal-organic frameworks with carboxyl, aldehyde, and isocyano-containing materials.

Prolactin Receptor-Mediated Internalization of Imaging Agents Detects Epithelial Ovarian Cancer with Enhanced Sensitivity and Specificity.

Poor prognosis of ovarian cancer, the deadliest of the gynecologic malignancies, reflects major limitations associated with detection and diagnosis. Current methods lack high sensitivity to detect small tumors and high specificity to distinguish malignant from benign tissue, both impeding diagnosis of early and metastatic cancer stages and leading to costly and invasive surgeries. Tissue microarray analysis revealed that >98% of ovarian cancers express the prolactin receptor (PRLR), forming the basis of a new molecular imaging strategy. We fused human placental lactogen (hPL), a specific and tight binding PRLR ligand, to magnetic resonance imaging (gadolinium) and near-infrared fluorescence imaging agents. Both in tissue culture and in mouse models, these imaging bioconjugates underwent selective internalization into ovarian cancer cells via PRLR-mediated endocytosis. Compared with current clinical MRI techniques, this targeted approach yielded both enhanced signal-to-noise ratio from accumulation of signal via selective internalization and improved specificity conferred by PRLR upregulation in malignant ovarian cancer. These features endow PRLR-targeted imaging with the potential to transform ovarian cancer detection. Cancer Res; 77(7); 1684-96. ©2017 AACR.

ZW800-1 for Assessment of Blood-Brain Barrier Disruption in a Photothrombotic Stroke Model.

Background: Since it is known that serum albumin-bound dyes can cross the blood-brain barrier (BBB) after ischemia, Evans Blue dye is commonly used to assess BBB disruption because of its rapid binding to serum albumin. In addition, indocyanine green (ICG), a clinically available dye, binds to serum proteins that could also be used for assessment of BBB impairment. Unlike these near-infrared (NIR) dyes, zwitterionic NIR fluorophore (ZW800-1) shows no serum binding, ultralow non-specific tissue uptake, and rapid elimination from the body via renal filtration. In this study, we report the use of ZW800-1 as a NIR fluorescence imaging agent for detecting BBB disruption in rat stroke models.Methods: Three types of NIR fluorophores, Evans Blue, ICG, and ZW800-1, were administered intraperitoneally into rat photothrombotic stroke models by using 4% concentration of each NIR dye. The NIR fluorescence signals in the infarcted brain tissue and biodistribution were observed in real-time using the Mini-FLARE® imaging system up to 24 h post-injection.Results: ZW800-1 provided successful visualization of the ischemic injury site in the brain tissue, while the remaining injected dye was clearly excreted from the body within a certain period of time. Although Evans Blue and ICG provided mapping of the infarcted brain lesions, they exhibited high non-specific uptake in most of the tissues and organs and persisted in the body over 24 h post-injection.Conclusion: Our results suggest the promising application of ZW800-1 as a new strategy in BBB experiments and future therapeutic development.

Everolimus-eluting stents stabilize plaque inflammation in vivo: assessment by intravascular fluorescence molecular imaging.

Inflammation drives atherosclerosis complications and is a promising therapeutic target for plaque stabilization. At present, it is unknown whether local stenting approaches can stabilize plaque inflammation in vivo. Here, we investigate whether everolimus-eluting stents (EES) can locally suppress plaque inflammatory protease activity in vivo using intravascular near-infrared fluorescence (NIRF) molecular imaging. Balloon-injured, hyperlipidaemic rabbits with atherosclerosis received non-overlapping EES and bare metal stents (BMS) placement into the infrarenal aorta (n = 7 EES, n = 7 BMS, 3.5 mm diameter x 12 mm length). Four weeks later, rabbits received an injection of the cysteine protease-activatable NIRF imaging agent Prosense VM110. Twenty-four hours later, co-registered intravascular 2D NIRF, X-ray angiography and intravascular ultrasound imaging were performed. In vivo EES-stented plaques contained substantially reduced NIRF inflammatory protease activity compared with untreated plaques and BMS-stented plaques (P = 0.006). Ex vivo macroscopic NIRF imaging of plaque protease activity corroborated the in vivo results (P = 0.003). Histopathology analyses revealed that EES-treated plaques showed reduced neointimal and medial arterial macrophage and cathepsin B expression compared with unstented and BMS-treated plaques. EES-stenting stabilizes plaque inflammation as assessed by translational intravascular NIRF molecular imaging in vivo. These data further support that EES may provide a local approach for stabilizing inflamed plaques.

Targeted Near-Infrared Fluorescence Imaging of Atherosclerosis: Clinical and Intracoronary Evaluation of Indocyanine Green

ObjectivesThis study sought to determine whether indocyanine green (ICG)–enhanced near-infrared fluorescence (NIRF) imaging can illuminate high-risk histologic plaque features of human carotid atherosclerosis, and in coronary atheroma of living swine, using intravascular NIRF-optical coherence tomography (OCT) imaging. BackgroundNew translatable imaging approaches are needed to identify high-risk biological signatures of atheroma. ICG is a U.S. Food and Drug Administration–approved NIRF imaging agent that experimentally targets plaque macrophages and lipid in areas of enhanced endothelial permeability. However, it is unknown whether ICG can target atheroma in patients. MethodsEight patients were enrolled in the BRIGHT-CEA (Indocyanine Green Fluorescence Uptake in Human Carotid Artery Plaque) trial. Five patients were injected intravenously with ICG 99 ± 25 min before clinically indicated carotid endarterectomy. Three saline-injected endarterectomy patients served as control subjects. Excised plaques underwent analysis by intravascular NIRF-OCT, reflectance imaging, microscopy, and histopathology. Next, following ICG intravenous injection, in vivo intracoronary NIRF-OCT and intravascular ultrasound imaged 3 atheroma-bearing coronary arteries of a diabetic, cholesterol-fed swine. ResultsICG was well tolerated; no adverse clinical events occurred up to 30 days post-injection. Multimodal NIRF imaging including intravascular NIRF-OCT revealed that ICG accumulated in all endarterectomy specimens. Plaques from saline-injected control patients exhibited minimal NIRF signal. In the swine experiment, intracoronary NIRF-OCT identified ICG uptake in all intravascular ultrasound–identified plaques in vivo. On detailed microscopic evaluation, ICG localized to plaque areas exhibiting impaired endothelial integrity, including disrupted fibrous caps, and within areas of neovascularization. Within human plaque areas of endothelial abnormality, ICG was spatially related to localized zones of plaque macrophages and lipid, and, notably, intraplaque hemorrhage. ConclusionsThis study demonstrates that ICG targets human plaques exhibiting endothelial abnormalities and provides new insights into its targeting mechanisms in clinical and experimental atheroma. Intracoronary NIRF-OCT of ICG may offer a novel, clinically translatable approach to image pathobiological aspects of coronary atherosclerosis. (Indocyanine Green Fluorescence Uptake in Human Carotid Artery Plaque [BRIGHT-CEA]; NCT01873716)

Synthesis and Characterization of Water-Soluble Conjugated Oligoelectrolytes for Near-Infrared Fluorescence Biological Imaging.

Near-infrared (NIR) fluorophores attract increasing attention as a molecular marker (or probe) for in vivo and in vitro biological fluorescence imaging. Three types of new NIR fluorescent conjugated oligoelectrolytes (COEs: Q-FlTBTTFl, Q-FlBBTFl, and Q-FlTBBTTFl) are synthesized with quaternized ammonium ionic groups in their side-chains for water solubility. The emission wavelength is modulated in the range 600-1300 nm, by adjusting the intramolecular charge transfer in the molecular backbone based on the electron-rich fluorene (and/or thiophene) and electron-deficient benzo[2,1,3]thiadiazole (or benzo[1,2-c:4,5-c']bis[1,2,5]thiadiazole) moieties. The COEs show a remarkably larger Stokes shift (147-276 nm) compared to commercial rhodamine and cyanine dyes in water, avoiding self-quenching and interference from the excitation backscattered light. The photoluminescence (PL) quantum efficiency is improved substantially by up to 27.8% in water by fabricating a vesicular complex, COE/v, with a block ionomer, poly[(ethylene oxide)-block-(sodium 2-acrylamido-2-methyl-1-propanesulfonate)]. In vitro cellular uptake images with the COEs are obtained with good biocompatibility by confocal single-photon and two-photon microscopy. The ex vivo and in vivo images of a mouse xenograft model treated with the Q-FlBBTFl/v exhibit a substantially stronger fluorescence signal at the tumor site than at the other organs, highlighting the potential of the COE/v as an NIR fluorescent imaging agent for the diagnosis of cancer.

Predicting Response to Therapy for Autoimmune and Inflammatory Diseases Using a Folate Receptor-Targeted Near-Infrared Fluorescent Imaging Agent.

Although current therapies for many inflammatory/autoimmune diseases are effective, a significant number of patients still exhibit only partial or negligible responses to therapeutic intervention. Since prolonged use of an inadequate therapy can result in both progressive tissue damage and unnecessary expense, methods to identify nonresponding patients are necessary. Four murine models of inflammatory disease (rheumatoid arthritis, ulcerative colitis, pulmonary fibrosis, and atherosclerosis) were induced, treated with anti-inflammatory agents, and evaluated for inflammatory response. The mice were also injected intraperitoneally with OTL0038, a folate receptor-targeted near-infrared dye that accumulates in activated macrophages at sites of inflammation. Uptake of OTL0038 in inflamed lesions was then correlated with clinical measurements of disease severity. OTL0038 accumulated at sites of inflammation in all four animal models. More importantly, changes in lesion-associated OTL0038 preceded changes in clinical symptoms in mice treated with all anti-inflammatory drugs examined. OTL0038 has the ability to predict responses to multiple therapies in four murine models of inflammation.

Abstract B25: Development of 5B1, an anti-CA19.9 monoclonal antibody, as a near-infrared fluorescent probe for intraoperative imaging of pancreatic cancer

Abstract Background: For patients diagnosed with pancreatic ductal adenocarcinoma (PDAC), resection offers the best chance of survival. However, for those 20% of patients who qualify for resection, the 5-year survival rate is only 25% due to the high incidence of undiscovered metastases. Along with identifying metastases, properly defining the margin between tumor and healthy tissue is also a crucial determinant of the success of resection. The intraoperative use of near-infrared fluorescence (NIRF) imaging agents to facilitate the identification of previously undetected metastases and to define proper resection margins has the potential to improve the outcomes in patients. Monoclonal antibodies (mABs) are one promising platform for the development of NIRF imaging agents due to their high-specificity and low background in healthy tissues. CA19.9 is a well-established biomarker for PDAC, and 5B1, a fully human, anti-CA19.9 antibody has already shown promise both as a potential therapeutic and as a platform for development of a positron emission tomography agent. Based on previous studies we investigated 5B1’s potential as a NIRF imaging agent for use in an intraoperative setting. Methods: IRDye800CW-NHS and IRDye650-NHS were conjugated to 5B1 and a mouse IgG via lysine residues to generate 5B1-FL800, 5B1-FL650, and IgG-FL800. Each construct and the free acid of the IR800Dye were characterized using in vitro binding assays with two CA19.9-positive cell pancreatic cancer derived lines (BxPC3 and L3.6pl) and a negative cell line (PanC-1). The in vivo dose needed achieve an optimal signal to background ratio was determined in female, athymic nude mice with varying doses of 5B1-FL800. The potential of the constructs for use as NIRF agents for image-guided tumor identification and surgery was examined in a subcutaneous xenograft model using the previously mentioned cell lines as well as an L3.6pl orthotopic pancreas xenograft model. Images were obtained at 24, 48, 96, and 144 hours (750nm excitation/800nm emission). Post mortem, NIRF image-guided surgery was performed to identify and remove the tumor tissue and the relative uptake was compared to healthy tissue. Immunohistochemistry (IHC) as well as hematoxylin and eosin staining (H&E) were performed on excised tumors to confirm specific uptake in tumor tissue. Results: The NIRF dyes were conjugated to 5B1 and a non-specific mouse IgG with a dye:mAB ratio of 1.1−3.2 (>98% purity). In vitro binding assays showed negligible binding to any cell line by the NIRF-labeled IgG and unconjugated dye. The two NIRF 5B1 constructs bound to both CA19.9 positive cell lines with low nanomolar affinity while no specific binding to PanC1 was observed. Specific binding could be inhibited by addition of excess 5B1. The binding was visualized in BxPC3 and L3.6pl cells by fluorescence microscopy. Pilot imaging studies in a small cohort of mice showed that 5B1-FL800 was superior to 5B1-FL650 in achieving a high signal-to-background ratio in vivo and that 0.5 nmol per mouse dose was an optimal dose for in vivo imaging. Imaging studies showed excellent tumor uptake in CA19.9-positive tumor bearing mice with an optimal tumor:background ratio being achieved at 96 hours post injection. The ratio of NIRF signal in the excised tumors was 8-30x higher than any healthy organ. The 5B1-800FL signal was used to successfully identify and guide the removal of tumor tissue from mice, including small portions of the tumor that were invisible to the naked eye. IHC and HE staining confirmed specific binding of the NIRF-conjugated 5B1 constructs to CA19.9 positive tumor tissue. Conclusions: IRDye800CW and IRDye650 were conjugated to the anti-CA19.9, fully-human mAB, 5B1, which was successfully used for in vivo NIRF imaging in both subcutaneous and orthotopic xenograft models of PDAC. 5B1 demonstrated exceptional potential for use in delineation of pancreatic tumor margins, NIRF guided-resection, and identification of metastases overlooked by other imaging techniques. Citation Format: Jacob L. Houghton, Dalya A. Abdel-Atti, Ritsuko Sawada, Wolfgang W. Scholz, Jason S. Lewis. Development of 5B1, an anti-CA19.9 monoclonal antibody, as a near-infrared fluorescent probe for intraoperative imaging of pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B25.