Small Molecules for Multi-Wavelength Near-Infrared Fluorescent Mapping of Regional and Sentinel Lymph Nodes in Colorectal Cancer Staging.

Assessing lymph node (LN) status during tumor resection is fundamental for the staging of colorectal cancer. Current guidelines require a minimum of 12 LNs to be harvested during resection and ultra-staging regional lymph nodes by sentinel lymph node (SLN) assessment is being extensively investigated. The current study presents novel near-infrared (NIR) fluorescent dyes for simultaneous pan lymph node (PanLN; regional) and SLN mapping. PanLN-Forte was intravenously injected in mice and assessed for accumulation in regional LNs. SLN800 was injected intradermally in mice, after which the collection and retention of fluorescence in SLNs were measured using indocyanine green (ICG) and its precursor, SLN700, as references. LNs in the cervical, inguinal, jejunal, iliac, and thoracic basins could clearly be distinguished after a low dose intravenous injection of PanLN-Forte. Background fluorescence was significantly lower compared to the parent compound ZW800-3A (p < 0.001). SLN700 and SLN800 specifically targeted SLNs with fluorescence being retained over 40-fold longer than the current clinically used agent ICG. Using SLN700 and SLN800, absolute fluorescence in SLN was at least 10 times higher than ICG in second-tier nodes, even at 1 hour post-injection. Histologically, the fluorescent signal localized in the LN medulla (PanLN-Forte) or sinus entry (SLN700/SLN800). PanLN-Forte and SLN800 appear to be optimal for real-time NIR fluorescence imaging of regional and SLNs, respectively.

A dual-labeled cRGD-based PET/optical tracer for pre-operative staging and intraoperative treatment of colorectal cancer.

cRGD peptides target integrins associated with angiogenesis (e.g., αvß3) and cancer, and have been used as binding ligands for both positron emission tomography (PET) and near-infrared fluorescence (NIRF) optical imaging. This study introduces the hybrid tracer cRGD-ZW800-1-Forte-[89Zr]Zr-DFO, which is based on a novel zwitterionic fluorophore structure that reduces non-specific background uptake during molecular imaging of tumors. An in vitro binding assay was used to validate tracer performance. 10 nmol ZW800F-cRGD-Zr-DFO was injected in mice (n=7) bearing orthotopic human colorectal tumors (HT29-luc2) for tumor detection with NIRF imaging. Subsequently, ZW800F-cRGD-Zr-DFO was loaded with 89Zr and 10 nmol cRGD-ZW800-1-Forte-[89Zr]Zr-DFO (3 MBq) was injected in mice (n=8) for PET/CT imaging. Imaging and biodistribution was performed at 4 and 24 h. NIRF imaging was performed up to 168 h after administration. Sufficient fluorescent signals were measured in the tumors of mice injected with ZW800F-cRGD-Zr-DFO (emission peak ~800 nm) compared to the background. The signal remained stable for up to 7 days. The fluorescence signal of cRGD-ZW800-1-Forte-[89Zr]Zr-DFO remained intact after labeling with 89Zr. PET/CT permitted clear visualization of the colorectal tumors at 4 and 24 h. Biodistribution at 4 h showed the highest uptake of the tracer in kidneys and sufficient uptake in the tumor, remaining stable for up to 24 h. A single molecular imaging agent, ZW800F-cRGD-[89Zr]Zr-DFO, permits serial PET and NIRF imaging of colorectal tumors, with the latter permitting image-guided treatment intraoperatively. Due to its unique zwitterionic structure, the tracer is rapidly renally cleared and fluorescent background signals are low.

Real-time near-infrared fluorescence imaging using cRGD-ZW800-1 for intraoperative visualization of multiple cancer types.

Incomplete resections and damage to critical structures increase morbidity and mortality of patients with cancer. Targeted intraoperative fluorescence imaging aids surgeons by providing real-time visualization of tumors and vital structures. This study evaluated the tumor-targeted zwitterionic near-infrared fluorescent peptide cRGD-ZW800-1 as tracer for intraoperative imaging of multiple cancer types. cRGD-ZW800-1 was validated in vitro on glioblastoma (U-87 MG) and colorectal (HT-29) cell lines. Subsequently, the tracer was tested in orthotopic mouse models with HT-29, breast (MCF-7), pancreatic (BxPC-3), and oral (OSC-19) tumors. Dose-ranging studies, including doses of 0.25, 1.0, 10, and 30 nmol, in xenograft tumor models suggest an optimal dose of 10 nmol, corresponding to a human equivalent dose of 63 µg/kg, and an optimal imaging window between 2 and 24 h post-injection. The mean half-life of cRGD-ZW800-1 in blood was 25 min. Biodistribution at 4 h showed the highest fluorescence signals in tumors and kidneys. In vitro and in vivo competition experiments showed significantly lower fluorescence signals when U-87 MG cells (minus 36%, p = 0.02) or HT-29 tumor bearing mice (TBR at 4 h 3.2 ± 0.5 vs 1.8 ± 0.4, p = 0.03) were simultaneously treated with unlabeled cRGD. cRGD-ZW800-1 visualized in vivo all colorectal, breast, pancreatic, and oral tumor xenografts in mice. Screening for off-target interactions, cRGD-ZW800-1 showed only inhibition of COX-2, likely due to binding of cRGD-ZW800-1 to integrin αVß3. Due to its recognition of various integrins, which are expressed on malignant and neoangiogenic cells, it is expected that cRGD-ZW800-1 will provide a sensitive and generic tool to visualize cancer during surgery.

Intraoperative Near-Infrared Fluorescence Imaging of Thymus in Preclinical Models.

BACKGROUND: There are currently no thymus-specific contrast agents for biomedical imaging. Thus, finding ectopic thymic tissue during certain operations is extremely difficult. The purpose of the present study was to determine if near-infrared (NIR) fluorescence imaging could provide high sensitivity, real-time identification of thymic tissue during the operation. METHODS: After initial in vivo screening of a 315-compound NIR fluorophore library for thymic uptake, methylene blue and five different 700-nm emitting candidate molecules were injected into CD-1 mice for quantitation of the signal-to-background ratio as a function of kinetics and dosing. Results were confirmed in 35-kg Yorkshire pigs. Dual-channel NIR imaging was also performed using a variety of 800-nm emitting NIR fluorophores targeted to various tissues in the mediastinum and neck. RESULTS: The compound Oxazine 170 demonstrated the highest signal-to-background ratio (≥3) for thymic tissue relative to mediastinal fat, heart, lung, muscle, thyroid gland, and parathyroid gland, with peak signal-to-background ratio occurring 4 h after 1 intravenous injection of a human equivalent dose of approximately 7 mg. Simultaneous dual-channel NIR imaging permitted unambiguous identification of the thymus from surrounding tissues, such as endocrine glands and lymph nodes. CONCLUSIONS: In mouse and pig, NIR fluorescence imaging using Oxazine 170 permits high sensitivity, real-time identification of thymic tissue for surgical procedures requiring its resection or avoidance. The performance of Oxazine 170 for imaging human thymic tissue is currently not known.

High-Throughput Sorting and Placement of One-Bead-One-Compound (OBOC) Libraries from Bulk to Single Wells in Organic Solvent.

One-bead-one-compound (OBOC) solid-phase combinatorial chemistry has been used extensively in drug discovery. However, a major bottleneck has been the sorting of individual beads, while still swollen in organic solvent, into individual wells of a microwell plate. To solve this problem, we have constructed an automated bead sorting system with integrated quality control that is capable of sorting and placing large numbers of beads in bulk to single wells of a 384-well plate, all in an organic solvent. The bead sorter employs a unique, reciprocating fluidic design capable of depositing 1 bead every 1.5 s, with an average accuracy of 97%. We quantified the performance of this instrument by sorting over 8500 beads, followed by cleaving the conjugated compound and confirming the chemical identity of each by liquid chromatography/mass spectrometry (LC/MS). This instrument should enable more efficient screening of combinatorial small molecule libraries without the need to dry beads or otherwise change the chemical environment.

High-throughput screening of small molecule ligands targeted to live bacteria surface.

The discovery of small molecule ligands targeted to the surface of live pathogenic bacteria would enable an entirely new class of antibiotics. We report the development and validation of a microarray-based high-throughput screening platform for bacteria that exploits 300 µm diameter chemical spots in a 1 in. × 3 in. nanolayered glass slide format. Using 24 model compounds and 4 different bacterial strains, we optimized the screening technology, including fluorophore-based optical deconvolution for automated scoring of affinity and cyan-magenta-yellow-key (CMYK) color-coding for scoring of both affinity and specificity. The latter provides a lossless, one-dimensional view of multidimensional data. By linking in silico analysis with cell binding affinity and specificity, we could also begin to identify the physicochemical factors that affect ligand performance. The technology we describe could form the foundation for developing new classes of antibiotics.

cGMP-Compatible preparative scale synthesis of near-infrared fluorophores.

Image-guided surgery using optical imaging requires the availability of large quantities of clinical-grade fluorophores. We describe the cGMP-compatible synthesis of the zwitterionic heptamethine indocyanine near-infrared fluorophore ZW800-1 at the 10 g scale (~1000 patient doses) using facile and efficient solvent purification, and without the need for column chromatography. ZW800-1 has >90% yield at the final step and >99% purity as measured by fluorescence and evaporative light scatter detection. We describe an analytical framework for qualifying impurities, as well as a detailed analysis of counterion identities. Finally, we report the unique in vivo properties of ZW800-1 in large animals approaching the size of humans, thus laying the foundation for rapid clinical translation of these methods.