Development and Validation of Nerve-Targeted Bacteriochlorin Sensors.

We report an innovative approach to producing bacteriochlorins (bacs) via formal cycloaddition by subjecting a porphyrin to a trimolecular reaction. Bacs are near-infrared probes with the intrinsic ability to serve in multimodal imaging. However, despite their ability to fluoresce and chelate metal ions, existing bacs have thus offered limited ability to label biomolecules for target specificity or have lacked chemical purity, limiting their use in bio-imaging. In this work, bacs allowed a precise and controlled appending of clickable linkers, lending the porphyrinoids substantially more chemical stability, clickability, and solubility, rendering them more suitable for preclinical investigation. Our bac probes enable the targeted use of biomolecules in fluorescence imaging and Cerenkov luminescence for guided intraoperative imaging. Bacs' capacity for chelation provides opportunities for use in non-invasive positron emission tomography/computed tomography. Herein, we report the labeling of bacs with Hs1a, a (NaV1.7)-sodium-channel-binding peptide derived from the Chinese tarantula Cyriopagopus schmidti to yield Bac-Hs1a and radiolabeled Hs1a, which shuttles our bac sensor(s) to mouse nerves. In vivo, the bac sensor allowed us to observe high signal-to-background ratios in the nerves of animals injected with fluorescent Bac-Hs1a and radiolabeled Hs1a in all imaging modes. This study demonstrates that Bac-Hs1a and [64Cu]Cu-Bac-Hs1a accumulate in peripheral nerves, providing contrast and utility in the preclinical space. For the chemistry and bio-imaging fields, this study represents an exciting starting point for the modular manipulation of bacs, their development and use as probes for diagnosis, and their deployment as formidable multiplex nerve-imaging agents for use in routine imaging experiments.

NaV1.7 targeted fluorescence imaging agents for nerve identification during intraoperative procedures.

Surgeries and trauma result in traumatic and iatrogenic nerve damage that can result in a debilitating condition that approximately affects 189 million individuals worldwide. The risk of nerve injury during oncologic surgery is increased due to tumors displacing normal nerve location, blood turbidity, and past surgical procedures, which complicate even an experienced surgeon's ability to precisely locate vital nerves. Unfortunately, there is a glaring absence of contrast agents to assist surgeons in safeguarding vital nerves. To address this unmet clinical need, we leveraged the abundant expression of the voltage-gated sodium channel 1.7 (NaV1.7) as an intraoperative marker to access peripheral nerves in vivo, and visualized nerves for surgical guidance using a fluorescently-tagged version of a potent NaV1.7-targeted peptide, Tsp1a, derived from a Peruvian tarantula. We characterized the expression of NaV1.7 in sensory and motor peripheral nerves across mouse, primate, and human specimens and demonstrated universal expression. We synthesized and characterized a total of 10 fluorescently labeled Tsp1a-peptide conjugates to delineate nerves. We tested the ability of these peptide-conjugates to specifically accumulate in mouse nerves with a high signal-to-noise ratio in vivo. Using the best-performing candidate, Tsp1a-IR800, we performed thyroidectomies in non-human primates and demonstrated successful demarcation of the recurrent laryngeal and vagus nerves, which are commonly subjected to irreversible damage. The ability of Tsp1a to enhance nerve contrast during surgery provides opportunities to minimize nerve damage and revolutionize standards of care across various surgical specialties.

PARP1: A Potential Molecular Marker to Identify Cancer During Colposcopy Procedures.

Despite efforts in prevention, cervical cancer still presents with a high worldwide incidence and remains a great problem in public health, especially in low-income countries. Screening programs, such as colposcopy with Papanicolaou testing, have greatly improved mortality rates. However, the agents currently used to delineate those lesions (topical application of acetic acid or Lugol iodine) lack specificity and sometimes can lead to unnecessary biopsies or even cervical excisions. A tool to enable in vivo histology to quickly and quantitatively distinguish between tumor, dysplastic tissue, and healthy tissue would be of great clinical interest. Methods: Here, we describe the use of PARPi-FL, a fluorescent inhibitor of poly[adenosine diphosphate-ribose]polymerase 1 (PARP1), which is a nuclear enzyme that is overexpressed in cancer when compared with the normal surrounding tissues. We exploit its use as an optical imaging agent to specifically target PARP1 expression, which was demonstrated to be higher in cervical cancer than the normal surrounding tissue. Results: After topical application of PARPi-FL on freshly excised cone biopsy samples, the nuclei of tumor cells emitted a specific fluorescent signal that could be visualized using a handheld fluorescence confocal microscope. Conclusion: This approach has the potential to improve in vivo identification of tumor cells during colposcopy examination, allowing a rapid, noninvasive, and accurate histopathologic assessment.

Imaging Early-Stage Metastases Using an 18F-Labeled VEGFR-1-Specific Single Chain VEGF Mutant.

PURPOSE: Metastatic breast cancer is the second leading cause of cancer-related death in women. The 5-year survival rate for metastatic breast cancer has remained near 26.9 % for over a decade. The recruitment of hematopoietic stem cells with high expression of the vascular endothelial growth factor receptor 1 (VEGFR-1) has been implicated in early stages of metastasis formation. We propose the use of an 18F-labeled single-chain version of VEGF121, re-engineered to be selective for VEGFR-1 (scVR1), as a positron emission tomography (PET) imaging agent to non-invasively image early-stage metastases. PROCEDURES: scVR1 was 18F-labeled via a biorthogonal click reaction between site-specifically trans-cyclooctene functionalized scVR1 and an Al18F labeled tetrazine-NODA (1,4,7-triazacyclononane-1,4-diiacetic acid). The [18F]AlF-NODA-scVR1 was purified using a PD10 column and subsequently analyzed on HPLC to determine radiochemical purity. Animal experiments were performed in 6-8-week-old female BALB/c mice bearing orthotopic primary 4T1 breast tumors or 4T1 metastatic lesions. The [18F]AlF-NODA-scVR1 tracer was administered via tail vein injection; PET imaging and ex vivo analysis was performed 2 h post-injection. RESULTS: The [18F]AlF-NODA-scVR1 was prepared with a 98.2 ± 1.5 % radiochemical purity and an apparent molar activity of 7.5 ± 1.2 GBq/µmol. The specific binding of scVR1 to VEGFR-1 was confirmed via bead-based assay. The ex vivo biodistribution showed tumor uptake of 3.5 ± 0.5 % ID/g and was readily observable in PET images. Metastasis formation was detected with [18F]AlF-NODA-scVR1 tracer showing colocalization with bioluminescent imaging as well as ex vivo autoradiography and immunofluorescent staining of VEGFR-1. CONCLUSIONS: The diagnostic capabilities of the [18F]AlF-NODA-scVR1 PET tracer was confirmed in both orthotopic and metastatic murine cancer models. These results support the potential use of [18F]AlF-NODA-scVR1 as a PET tracer that could image metastases, providing clinicians with an additional tool to assess a patient's need for adjuvant therapies.

Fluorescence labeling of a NaV1.7-targeted peptide for near-infrared nerve visualization.

BACKGROUND: Accidental peripheral nerve injury during surgical intervention results in a broad spectrum of potentially debilitating side effects. Tissue distortion and poor visibility can significantly increase the risk of nerve injury with long-lasting consequences for the patient. We developed and characterized Hs1a-FL, a fluorescent near-infrared molecule for nerve visualization in the operating theater with the aim of helping physicians to visualize nerves during surgery. Hs1a was derived from the venom of the Chinese bird spider, Haplopelma schmidti, and conjugated to Cy7.5 dye. Hs1a-FL was injected intravenously in mice, and harvested nerves were imaged microscopically and with epifluorescence. RESULTS: Hs1a-FL showed specific and stable binding to the sodium channel NaV1.7, present on the surface of human and mouse nerves. Hs1a-FL allowed epifluorescence visualization of sciatic mouse nerves with favorable nerve-to-muscle contrast. CONCLUSIONS: Fluorescent NaV1.7-targeted tracers have the potential to be adopted clinically for the intraoperative visualization of peripheral nerves during surgery, providing guidance for the surgeon and potentially improving the standard of care.

A systematic review of validated tools assessing functional and aesthetic outcomes following fibula free flap reconstruction of the mandible.

BACKGROUND: Segmental mandibulectomy impairs health-related quality of life (QoL), by altering speech, mastication, swallowing, and facial aesthetics. Fibula free flap (FFF) used for mandible reconstruction is known to improve outcomes; however, minimal information exists in the literature regarding patient-reported outcomes. We aim to assess how current studies evaluate patient perception following segmental mandibulectomy and FFF mandible reconstruction. METHODS: Following Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, a search was conducted for publications involving FFF mandible reconstruction from 2005 to 2017 using PubMed, Cochrane, EMBASE, Web of Science, and PsychInfo. RESULTS: Of 2212 articles identified initially, only 7 studies were deemed suitable. Six studies used the University of Washington Quality of Life questionnaire, 3 Oral Health Impact Profile, and 1 used European Organization for Research and Treatment of Cancer Head and Neck (EORTC-H&N35). CONCLUSIONS: There is a paucity of information in published reports on QoL outcomes following mandible reconstruction with FFF. In the era of patient-centered health care, observations warrant attention from researchers for physician-assessed patient-reported measures to factor in QoL expectation during surgical decision-making about the choice of reconstruction.