Hoon Hyun

Targeted zwitterionic near-infrared fluorophores for improved optical imaging

The signal-to-background ratio (SBR) is the key determinant of sensitivity, detectability and linearity in optical imaging. As signal strength is often constrained by fundamental limits, background reduction becomes an important approach for improving the SBR. We recently reported that a zwitterionic near-infrared (NIR) fluorophore, ZW800-1, exhibits low background. Here we show that this fluorophore provides a much-improved SBR when targeted to cancer cells or proteins by conjugation with a cyclic RGD peptide, fibrinogen or antibodies. ZW800-1 outperforms the commercially available NIR fluorophores IRDye800-CW and Cy5.5 in vitro for immunocytometry, histopathology and immunoblotting and in vivo for image-guided surgery. In tumor model systems, a tumor-to-background ratio of 17.2 is achieved at 4 h after injection of ZW800-1 conjugated to cRGD compared to ratios of 5.1 with IRDye800-CW and 2.7 with Cy5.5. Our results suggest that introducing zwitterionic properties into targeted fluorophores may be a general strategy for improving the SBR in diagnostic and therapeutic applications.

Synthesis and in vivo fate of zwitterionic near-infrared fluorophores.

A longstanding problem in the field of image-guided surgery is the development of ideal near-infrared (NIR) fluorophores. The heptamethine NIR fluorophore indocyanine green (ICG) has been used extensively for image-guided surgery because of clinical availability and safety.[1-3] However, ICG is far from ideal because it exhibits high uptake in the liver, contaminates the gastrointestinal (GI) tract, provides moderate optical properties,[4] is unstable in aqueous media,[3,5] and is unable to conjugate covalently to targeting ligands.[2] Although several classes of novel molecules have been described,[6-13] none to date exhibit simultaneous low background binding, bifunctionality, excellent optical properties, low protein binding, and high serum stability. Although it is intuitive that physicochemical properties, i.e., positive/negative charge density, hydrophilicity/lipophilicity, and charge distribution, will impact in vivo performance, chemical structures that exhibit ideal characteristics have not yet been defined.

Structure-inherent targeting of near-infrared fluorophores for parathyroid and thyroid gland imaging

The typical method for creating targeted contrast agents requires covalent conjugation of separate targeting and fluorophore domains. In this study, we demonstrate that it is possible to create near-infrared (NIR) fluorophores with different tissue specificities driven by their inherent chemical structures. Thus, a single compact molecule performs both targeting and imaging. We use this strategy to solve a major problem in head and neck surgery: the identification and preservation of parathyroid and thyroid glands. We synthesized 700-nm and 800-nm halogenated fluorophores that show high uptake into these glands after a single intravenous (IV) injection of 0.06 mg kg−1 in a pig. By using a dual-channel NIR imaging system, we observed—in real time and with high sensitivity—the unambiguous distinction of parathyroid and thyroid glands simultaneously in the context of blood and surrounding soft tissue. This novel technology lays a foundation for performing head and neck surgery with increased precision and efficiency along with potentially lower morbidity, and it provides a general strategy for developing targeted NIR fluorophores.

Phosphonated Near-Infrared Fluorophores for Biomedical Imaging of Bone†

The conventional method for creating targeted contrast agents is to conjugate separate targeting and fluorophore domains. A new strategy is based on the incorporation of targeting moieties into the non-delocalized structure of pentamethine and heptamethine indocyanines. Using the known affinity of phosphonates for bone minerals in a model system, two families of bifunctional molecules that target bone without requiring a traditional bisphosphonate are synthesized. With peak fluorescence emissions at approximately 700 or 800 nm, these molecules can be used for fluorescence-assisted resection and exploration (FLARE) dual-channel imaging. Longitudinal FLARE studies in mice demonstrate that phosphonated near-infrared fluorophores remain stable in bone for over five weeks, and histological analysis confirms their incorporation into the bone matrix. Taken together, a new strategy for creating ultra-compact, targeted near-infrared fluorophores for various bioimaging applications is described.

Near-Infrared Fluorescence Imaging for Noninvasive Trafficking of Scaffold Degradation

Biodegradable scaffolds could revolutionize tissue engineering and regenerative medicine; however, in vivo matrix degradation and tissue ingrowth processes are not fully understood. Currently a large number of samples and animals are required to track biodegradation of implanted scaffolds, and such nonconsecutive single-time-point information from various batches result in inaccurate conclusions. To overcome this limitation, we developed functional biodegradable scaffolds by employing invisible near-infrared fluorescence and followed their degradation behaviors in vitro and in vivo. Using optical fluorescence imaging, the degradation could be quantified in real-time, while tissue ingrowth was tracked by measuring vascularization using magnetic resonance imaging in the same animal over a month. Moreover, we optimized the in vitro process of enzyme-based biodegradation to predict implanted scaffold behaviors in vivo, which was closely related to the site of inoculation. This combined multimodal imaging will benefit tissue engineers by saving time, reducing animal numbers, and offering more accurate conclusions.

Cartilage-Specific Near-Infrared Fluorophores for Biomedical Imaging†

A novel class of near-infrared fluorescent contrast agents was developed. These agents target cartilage with high specificity and this property is inherent to the chemical structure of the fluorophore. After a single low-dose intravenous injection and a clearance time of approximately 4 h, these agents bind to all three major types of cartilage (hyaline, elastic, and fibrocartilage) and perform equally well across species. Analysis of the chemical structure similarities revealed a potential pharmacophore for cartilage targeting. Our results lay the foundation for future improvements in tissue engineering, joint surgery, and cartilage-specific drug development.

Insulin-loaded microcapsules for in vivo delivery.

Microencapsulation of insulin has been difficult, due to the high sensitivity of insulin to the harsh conditions that can occur during the microencapsulation process. We have developed a method of preparing insulin-loaded microcapsules by using a monoaxial ultrasonic atomizer to form microdroplets of insulin in aqueous solution surrounded by poly(lactic-co-glycolic acid) (PLGA) solution. Administration of these insulin-loaded microcapsules to type 1 diabetic rats maintained plasma insulin concentrations for 30 days, due to the sustained insulin release properties of the microcapsules. In contrast, plasma insulin concentrations after subcutaneous injection of insulin solution reached near zero levels within 2 days. Insulin solution showed only an immediate pharmacological effect, with no reduction of glycemia after 3 days, whereas insulin-loaded microcapsules maintained blood glucose levels at 100-200 mg/dL for 55 days. Molecular imaging using fluorescein isothiocyanate (FITC)-insulin-loaded microcapsules showed in vivo sustained release of the FITC-insulin in microcapsules. Using insulin-loaded microcapsules, we observed inflammation only immediately after injection, indicating that the rats adapted to long-term insulin release. In conclusion, insulin-loaded microcapsules may reduce nonrepetitive insulin administration and show sustained pharmacological performance.

Prototype Nerve-Specific Near-Infrared Fluorophores

Nerve preservation is an important issue during most surgery because accidental transection or injury results in significant morbidity, including numbness, pain, weakness, or paralysis. Currently, nerves are still identified only by gross appearance and anatomical location during surgery, without intraoperative image guidance. Near-infrared (NIR) fluorescent light, in the wavelength range of 650-900 nm, has the potential to provide high-resolution, high-sensitivity, and real-time avoidance of nerve damage, but only if nerve-specific NIR fluorophores can be developed. In this study, we evaluated a series of Oxazine derivatives to highlight various peripheral nerve structures in small and large animals. Among the targeted fluorophores, Oxazine 4 has peak emission near into the NIR, which provided nerve-targeted signal in the brachial plexus and sciatic nerve for up to 12 h after a single intravenous injection. In addition, recurrent laryngeal nerves were successfully identified and highlighted in real time in swine, which could be preserved during the course of thyroid resection. Although optical properties of these agents are not yet optimal, chemical structure analysis provides a basis for improving these prototype nerve-specific NIR fluorophores even further.

Tailored Near-Infrared Contrast Agents for Image Guided Surgery

The success of near-infrared (NIR) fluorescence to be employed for intraoperative imaging relies on the ability to develop a highly stable, NIR fluorescent, nontoxic, biocompatible, and highly excreted compound that retains a reactive functionality for conjugation to a cancer-recognizing peptide. Herein, systematic modifications to previously detailed fluorophore ZW800-1 are explored. Specific modifications, including the isosteric replacement of the O atom of ZW800-1, include nucleophilic amine and sulfur species attached to the heptamethine core. These novel compounds have shown similar satisfactory results in biodistribution and clearance while also expressing increased stability in serum. Most importantly, all of the synthesized and evaluated compounds display a reactive functionality (either a free amino group or carboxylic acid moiety) for further bioconjugation. The results obtained from the newly prepared derivatives demonstrate that the central substitution with the studied linking agents retains the ultralow background in vivo performance of the fluorophores regardless of the total net charge.

Pancreas-Targeted NIR Fluorophores for Dual-Channel Image-Guided Abdominal Surgery

Objective: Pancreas-related complications are some of the most serious ones in abdominal surgery. The goal of this study was to develop and validate novel near-infrared (NIR) fluorophores that would enable real-time pancreas imaging to avoid the intraoperative pancreatic injury. Design: After initial screening of a large NIR fluorophore library, the performance of 3 selected pancreas-targeted 700 nm NIR fluorophores, T700-H, T700-F, and MB, were quantified in mice, rats, and pigs. Dose ranging using 25 and 100 nmol, and 2.5 µmol of T700-F, and its imaging kinetics over a 4 h period were tested in each species. Three different 800 nm NIR fluorophores were employed for dual-channel FLARE™ imaging in pigs: 2 μmol of ZW800-1 for vessels and kidney, 1 μmol of ZW800-3C for lymph nodes, and 2 μmol of ESNF31 for adrenal glands. Results: T700-F demonstrated the highest signal to background ratio (SBR), with peak SBR at 4 h postinjection in mice. In pigs, T700-F produced an SBR ≥ 2 against muscle, spleen, and lymph nodes for up to 8 h after a single intravenous injection. The combination of T700-F with each 800 nm NIR fluorophore provided simultaneous dual-channel intraoperative imaging of pancreas with surrounding organs in real time. Conclusion: Pancreas-targeted NIR fluorophores combined with the FLARE dual-channel imaging system enable the real-time intraoperative pancreas imaging which helps surgeons perform safer and more curative abdominal surgeries.