Background: Surgically induced nerve damage is a common but debilitating side effect. By developing tracers that specifically target the most abundant protein in peripheral myelin, namely myelin... Show moreBackground: Surgically induced nerve damage is a common but debilitating side effect. By developing tracers that specifically target the most abundant protein in peripheral myelin, namely myelin protein zero (P0), we intend to support fluorescence-guided nerve-sparing surgery. To that end, we aimed to develop a dimeric tracer that shows a superior affinity for P0. Methods: Following truncation of homotypic P0 protein-based peptide sequences and fluorescence labeling, the lead compound Cy5-P0(101-125) was selected. Using a bifunctional fluorescent dye, the dimeric Cy5-(P0(101-125))(2) was created. Assessment of the performance of the mono- and bi-labeled compounds was based on (photo)physical evaluation. This was followed by in vitro assessment in P0 expressing Schwannoma cell cultures by means of fluorescence confocal imaging (specificity, location of binding) and flow cytometry (binding affinity; K-D). Results: Dimerization resulted in a 1.5-fold increase in affinity compared to the mono-labeled counterpart (70.3 +/- 10.0 nM vs. 104.9 +/- 16.7 nM; p = 0.003) which resulted in a 4-fold increase in staining efficiency in P0 expressing Schwannoma cells. Presence of two targeting vectors also improves a pharmacokinetics of labeled compounds by lowering serum binding and optical stability by preventing dye stacking. Conclusions: Dimerization of the nerve-targeting peptide P0(101-125) proves a valid strategy to improve P0 targeting. Show less
Hoven, P. van den; Goncalves, L.N.; Quax, P.H.A.; Rijswijk, C.S.P. van; Schaik, J. van; Schepers, A.; ... ; Vorst, J.R. van der 2021
In assessing the severity of lower extremity arterial disease (LEAD), physicians rely on clinical judgements supported by conventional measurements of macrovascular blood flow. However, current... Show moreIn assessing the severity of lower extremity arterial disease (LEAD), physicians rely on clinical judgements supported by conventional measurements of macrovascular blood flow. However, current diagnostic techniques provide no information about regional tissue perfusion and are of limited value in patients with chronic limb-threatening ischemia (CLTI). Near-infrared (NIR) fluorescence imaging using indocyanine green (ICG) has been used extensively in perfusion studies and is a possible modality for tissue perfusion measurement in patients with CLTI. In this prospective cohort study, ICG NIR fluorescence imaging was performed in patients with CLTI and control patients using the Quest Spectrum Platform(R) (Middenmeer, The Netherlands). The time-intensity curves were analyzed using the Quest Research Framework. Fourteen parameters were extracted. Successful ICG NIR fluorescence imaging was performed in 19 patients with CLTI and in 16 control patients. The time to maximum intensity (seconds) was lower for CLTI patients (90.5 vs. 143.3, p = 0.002). For the inflow parameters, the maximum slope, the normalized maximum slope and the ingress rate were all significantly higher in the CLTI group. The inflow parameters observed in patients with CLTI were superior to the control group. Possible explanations for the increased inflow include damage to the regulatory mechanisms of the microcirculation, arterial stiffness, and transcapillary leakage. Show less
Linders, D.; Deken, M.; Valk, M. van der; Tummers, W.; Bhairosingh, S.; Schaap, D.; ... ; Hilling, D. 2021
Rectal cancer patients with a complete response after neoadjuvant therapy can be monitored with a watch-and-wait strategy. However, regrowth rates indicate that identification of patients with a... Show moreRectal cancer patients with a complete response after neoadjuvant therapy can be monitored with a watch-and-wait strategy. However, regrowth rates indicate that identification of patients with a pathological complete response (pCR) remains challenging. Targeted near-infrared fluorescence endoscopy is a potential tool to improve response evaluation. Promising tumor targets include carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), integrin alpha v beta 6, and urokinase-type plasminogen activator receptor (uPAR). To investigate the applicability of these targets, we analyzed protein expression by immunohistochemistry and quantified these by a total immunostaining score (TIS) in tissue of rectal cancer patients with a pCR. CEA, EpCAM, alpha v beta 6, and uPAR expression in the diagnostic biopsy was high (TIS > 6) in, respectively, 100%, 100%, 33%, and 46% of cases. CEA and EpCAM expressions were significantly higher in the diagnostic biopsy compared with the corresponding tumor bed (p < 0.01). CEA, EpCAM, alpha v beta 6, and uPAR expressions were low (TIS < 6) in the tumor bed in, respectively, 93%, 95%, 85%, and 62.5% of cases. Immunohistochemical evaluation shows that CEA and EpCAM could be suitable targets for response evaluation after neoadjuvant treatment, since expression of these targets in the primary tumor bed is low compared with the diagnostic biopsy and adjacent pre-existent rectal mucosa in more than 90% of patients with a pCR. Show less
(1) Background: doxorubicin is a potent chemotherapeutic agent, but it has limitations regarding its side effects and therapy resistance. Hydrogels potentially deal with these problems, but several... Show more(1) Background: doxorubicin is a potent chemotherapeutic agent, but it has limitations regarding its side effects and therapy resistance. Hydrogels potentially deal with these problems, but several characterizations need to be optimized to better understand how hydrogel assisted chemotherapy works. Poloxamer 407 (P407) hydrogels were mixed with doxorubicin and physico-chemical, biological, and pharmacological characterizations were considered. (2) Methods: hydrogels were prepared by mixing P407 in PBS at 4 degrees C. Doxorubicin was added upon solutions became clear. Time-to-gelation, hydrogel morphology, and micelles were studied first. The effects of P407-doxorubicin were evaluated on MC-38 colon cancer cells. Furthermore, doxorubicin release was assessed and contrasted with non-invasive in vivo whole body fluorescence imaging. (3) Results: 25% P407 had favorable gelation properties with pore sizes of 30-180 mu m. P407 micelles were approximately 5 nm in size. Doxorubicin was fully released in vitro from 25% P407 hydrogel within 120 h. Furthermore, P407 micelles strongly enhanced the anti-neoplastic effects of doxorubicin on MC-38 cells. In vivo fluorescence imaging revealed that hydrogels retained fluorescence signals at the injection site for 168 h. (4) Conclusions: non-invasive imaging showed how P407 gels retained drug at the injection site. Doxorubicin P407 micelles strongly enhanced the anti-tumor effects. Show less
Leeuwen, F.W.B. van; Schottelius, M.; Brouwer, O.R.; Vidal-Sicart, S.; Achilefu, S.; Klode, J.; ... ; Buckle, T. 2020
Introduction: Adequate signal to background ratios are critical for the implementation of fluorescence-guided surgery technologies. While local tracer administrations help to reduce the chance of... Show moreIntroduction: Adequate signal to background ratios are critical for the implementation of fluorescence-guided surgery technologies. While local tracer administrations help to reduce the chance of systemic side effects, reduced spatial migration and non-specific tracer diffusion can impair the discrimination between the tissue of interest and the background. To combat background signals associated with local tracer administration, we explored a pretargeting concept aimed at quenching non-specific fluorescence signals. The efficacy of this concept was evaluated in an in vivo neuronal tracing set-up.Methods: Neuronal tracing was achieved using a wheat germ agglutinin (WGA) lectin. functionalized with an azide-containing Cy5 dye (N-3-Cy5-WGA). A Cy7 quencher dye (Cy7-DBCO) was subsequently used to yield Cy7-Cy5-WGA, a compound wherein the Cy5 emission is quenched by Forster resonance energy transfer to Cy7. The photophysical properties of N-3-Cy5-WGA and Cy7-Cy5-WGA were evaluated together with deactivation kinetics in situ, in vitro (Schwannoma cell culture), ex vivo (muscle tissue from mice; used for dose optimization), and in vivo (nervus ischiadicus in THY-1 YFP mice).Results: In situ, conjugation of Cy7-DBCO to N-3-Cy5-WGA resulted in >90% reduction of the Cy5 fluorescence signal intensity at 30 minutes after addition of the quencher. In cells, pretargeting with the N-3-Cy5-WGA lectin yielded membranous staining, which could efficiently be deactivated by Cy7-DBCO over the course of 30 minutes (91% Cy5 signal decrease). In ex vivo muscle tissue, administration of Cy7-DBCO at the site where N-3-Cy5-WGA was injected induced 80-90% quenching of the Cy5-related signal after 10-20 minutes, while the Cy7-related signal remained stable over time. In vivo, Cy7-DBCO effectively quenched the non-specific background signal up to 73% within 5 minutes, resulting in a 50% increase in the signal-to-background ratio between the nerve and injection site.Conclusion: The presented pretargeted fluorescence-quenching technology allowed fast and effective reduction of the background signal at the injection site, while preserving in vivo nerve visualization. While this proof-of-principle study was focused on imaging of nerves using a fluorescent WGA-lectin, the same concept could in the future also apply to applications such as sentinel node imaging. Show less
Mezzanotte, L.; Iljas, J.D.; Que, I.; Chan, A.; Kaijzel, E.; Hoeben, R.; Lowik, C. 2017
