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 (≈ 1,000 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.

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 αVβ3. 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.

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” × 3” nano-layered 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-black (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.

Background There are currently no thymus-specific contrast agents for biomedical imaging. Thus, finding ectopic thymic tissue during certain surgeries 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 surgery. Methods After initial in vivo screening of a 315-compound NIR fluorophore library for thymic uptake, methylene blue and 5 different 700 nm emitting candidate molecules were injected into CD-1 mice for quantitation of the signal-to-background ratio (SBR) 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 Compound OX170 demonstrated the highest SBR (≥ 3) for thymic tissue relative to mediastinal fat, heart, lung, muscle, thyroid gland, and parathyroid gland, with peak SBR occurring 4 h after a single intravenous injection of a human equivalent dose of ≈ 7 mg. Simultaneous dual-channel NIR imaging permitted unambiguous identification of thymus from surrounding tissues, such as endocrine glands and lymph nodes. Conclusions In mouse and pig, NIR fluorescence imaging using OX170 permits high sensitivity, real-time identification of thymic tissue for surgical procedures requiring its resection or avoidance. The performance of OX170 for imaging human thymic tissue is currently not known.

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 organic solvent. The bead sorter employs a unique, reciprocating fluidic design capable of depositing 1 bead per 1.5 s with an average accuracy of 97%. We quantified the performance of this instrument by sorting over 8,500 beads followed by cleaving the conjugated compound and confirming the chemical identity of each by LC/MS. This instrument should enable more efficient screening of combinatorial small molecule libraries without the need to dry beads or otherwise change chemical environment.