Background Surgically induced nerve damage is a common but debilitating side effect in oncological surgery. With the aim to use fluorescence guidance to enable nerve-sparing interventions in future... Show moreBackground Surgically induced nerve damage is a common but debilitating side effect in oncological surgery. With the aim to use fluorescence guidance to enable nerve-sparing interventions in future surgery, a fluorescent tracer was developed that specifically targets myelin protein zero (P0). Results Truncated homotypic P0 protein-based peptide sequences were C-terminally functionalized with the far-red cyanine dye Cy5. The lead compound Cy5-P0(101-125) was selected after initial solubility, (photo)physical and in vitro evaluation (including P0-blocking experiments). Cy5-P0(101-125) (K-D = 105 +/- 17 nM) allowed in vitro and ex vivo P0-related staining. Furthermore, Cy5-P0(101-125) enabled in vivo fluorescence imaging of the Sciatic nerve in mice after local intravenous (i.v.) administration and showed compatibility with a clinical fluorescence laparoscope during evaluation in a porcine model undergoing robot-assisted surgery. Biodistribution data revealed that i.v. administered [In-111]In-DTPA-P0(101-125) does not enter the central nervous system (CNS). Conclusion P0(101-125) has proven to be a potent nerve-specific agent that is able to target P0/myelin under in vitro, ex vivo, and in vivo conditions without posing a threat for CNS-related toxicity. Show less
Aim: Pre-targeting is a proven strategy for in vivo delivery of a diagnostic or therapeutic payload. The pre-targeting concept can be realized through various conjugation strategies, one of which... Show moreAim: Pre-targeting is a proven strategy for in vivo delivery of a diagnostic or therapeutic payload. The pre-targeting concept can be realized through various conjugation strategies, one of which is based on copper-free "click" chemistry. Copper-free click reactions have shown in vivo potential for imaging and radionuclide therapy, but this conjugation strategy has not yet been explored in combination with microspheres or unicellular organisms. This study aims to evaluate the in vivo efficacy of strain-promoted azide-alkyne cycloaddition (SPAAC) reactions to achieve imaging and targeting of azide-functionalized macro-aggregated albumin (MAA) microspheres and Staphylococcus aureus bacteria. Methods: MAA microspheres (diameter 10-90 mu m) were functionalized with a biorthogonal Cy5 fluorophore, bearing an azide functionality (N-3), to generate MAA-Cy5-N-3. S. aureus (diameter similar to 1 mu m) were functionalized with Tc-99m-UBI29-41-Cy5-N-3, generating S. aureus-Tc-99m-UBI29-41-Cy5-N-3. In situ and in vitro click conjugation on the -N-3 moieties was studied for 20 h using a radioactivity-based assay and fluorescence microscopy. For in vivo validation, both primary entities, radiolabeled with Tc-99m, were deposited into the microvasculature of the liver via intrasplenic injections. Secondary targeting was realized following the intravenous administration of indium-111-radiolabeled diethylenetriaminepentaacetic acid-dibenzocyclooctyne (In-111-DTPA-DBCO). To assess click reaction efficiency in vivo, Tc-99m and In-111-biodistributions were measured (SPECT and %ID g(-1)). Use of In-111-DTPA-DBCO in mice without MAA deposits or mice infected with non-functionalized S. aureus served as controls. Ex vivo confocal fluorescence imaging was carried out in excised tissues to confirm the presence of functionalized MAA and bacteria. Results: In vitro data confirmed effective click reactions on both the MAA particles and the bacterial membrane. SPECT imaging and biodistribution studies revealed significantly (p < 0.05) increased accumulation of In-111-DTPA-DBCO at the sites where MAA-Cy5-N-3 (7.5 +/- 1.5%ID g(-1)vs. 3.5 +/- 0.5%ID g(-1) in control mice) and S. aureus-Tc-99m-UBI29-41-Cy5-N-3 (9.3 +/- 1.3%ID g(-1)vs. 6.0 +/- 0.5%ID g(-1) in control mice) resided. Ex vivo fluorescence imaging confirmed the presence of either functionalized MAA or S. aureus in excised spleens and livers of mice. Conclusion: Copper-free click chemistry between a DBCO moiety and Cy5-N-3-functionalized microspheres or bacterial entities in the liver can be used to realize in vivo imaging and targeting. Show less
Pentamethine cyanine (Cy5) fluomphores have proven to be versatile imaging agents (i.e., tracers) for a range of micro- and macroscopic imaging applications, including image-guided surgery. In this... Show morePentamethine cyanine (Cy5) fluomphores have proven to be versatile imaging agents (i.e., tracers) for a range of micro- and macroscopic imaging applications, including image-guided surgery. In this study the relationship between the structure of asymmetric Cy5 fluorophores and their photophysical properties was studied. To this end, seven Cy5 analogues, bearing orthogonal N-indole substituents (H, SC3-, or benzene), were synthesised and evaluated. In-depth analysis revealed that introduction of sulfonates enhanced the fluorescence brightness and photostability, while reducing the lipophilicity, serum binding and stacking tendency. The addition of benzene moieties induced a bathochromic shift of 10-20 nm, increased the lipophilicity (LogP = -1.56-1.23) and serum binding (67.3-93.8% bound), as well as negatively impacted the brightness (0.74-42.9 . 10(3) M-1 cm(-1)), photostability (24.4-90.6% remaining), and stacking tendency. Chemical stability was uninfluenced by the substitution pattern. Additionally, the generation of a c[RGDyK]-based hybrid tracer based on one of these fluomphores in combination with a diethylenetriaminepentaacetic acid (DTPA) chelate and an In-111-isotope was reported. This compound was evaluated in vitro using alpha(v)beta(3)-overexpressing Ge beta 3 cells and in vivo using a 4T1 mouse tumour model. Overall, the presented results imply that alterations of the asymmetrical orthogonal Cy5 fluomphore structure have impact on the (photo)physical properties. Furthermore, the orthogonal Cy5 fluorophore framework can readily be applied in tracer development. Show less
Hensbergen, A.W.; Willigen, D.M. van; Beurden, F. van; Leeuwen, P.J. van; Buckle, T.; Schottelius, M.; ... ; Leeuwen, F.W.B. van 2020
Expressed on virtually all prostate cancers and their metastases, the transmembrane protein prostate-specific membrane antigen (PSMA) provides a valuable target for the imaging of prostate cancer.... Show moreExpressed on virtually all prostate cancers and their metastases, the transmembrane protein prostate-specific membrane antigen (PSMA) provides a valuable target for the imaging of prostate cancer. Not only does PSMA provide a target for noninvasive diagnostic imaging, e.g., PSMA-positron emission tomography (PSMA PET), it can also be used to guide surgical resections of PSMA-positive lesions. The latter characteristic has led to the development of a plethora of PSMA-targeted tracers, i.e., radiolabeled, fluorescent, or hybrid. With image-guided surgery applications in mind, this review discusses these compounds based on clinical need. Here, the focus is on the chemical aspects (e.g., imaging label, spacer moiety, and targeting vector) and their impact on in vitro and in vivo tracer characteristics (e.g., affinity, tumor uptake, and clearance pattern). Show less
Hensbergen, A.W.; Buckle, T.; Willigen, D.M. van; Schottelius, M.; Welling, M.M.; Wijk, F.A. van der; ... ; Leeuwen, F.W.B. van 2020
Prostate cancer surgery is currently being revolutionized by the use of prostate-specific membrane antigen (PSMA)-targeted radiotracers, for example, (99)mTc-labeled PSMA tracer analogs for... Show moreProstate cancer surgery is currently being revolutionized by the use of prostate-specific membrane antigen (PSMA)-targeted radiotracers, for example, (99)mTc-labeled PSMA tracer analogs for radioguided surgery. The purpose of this study was to develop a second-generation (99)mTc-labeled PSMA-targeted tracer incorporating a fluorescent dye. Methods: Several PSMA-targeted hybrid tracers were synthesized: glutamic acid- urea-lysine (EuK)-Cy5-mas(3), EuK-(SO3)Cy5-mas(3), EuK-Cy5(SO3)-mas(3), EuK-(Ar)Cy5-mas(3), and EuK-Cy5(Ar)-mas(3); the Cy5 dye acts as a functional backbone between the EuK targeting vector and the 2-mercaptoacetyl-seryl-seryl-seryl (mas(3)) chelate to study the dye's interaction with PSMA's amphipathic entrance funnel. The compounds were evaluated for their photophysical and chemical properties and PSMA affinity. After radiolabeling with (99)mTc, we performed in vivo SPECT imaging, biodistribution, and fluorescence imaging on BALB/c nude mice with orthotopically transplanted PC346C tumors. Results: The dye composition influenced the photophysical properties (brightness range 0.3-1.5 x 10(4) M-1 x cm(-1)), plasma protein interactions (range 85.0% +/- 2.3%-90.7%+/- 1.3% bound to serum, range 76%+/- 0%-89%+/- 6% stability in serum), PSMA affinity (half-maximal inhibitory concentration [IC50] range 19.2 +/- 5.8-175.3 +/- 61.1 nM) and in vivo characteristics (tumorto-prostate and tumor-to-muscle ratios range 0.02 +/- 0.00-154.73 +/- 28.48 and 0.46 +/- 0.28-5,157.50 +/- 949.17, respectively; renal, splenic, and salivary retention). Even though all tracer analogs allowed tumor identification with SPECT and fluorescence imaging, (99)mTc-EuK(SO3)Cy5-mas(3) had the most promising properties (e.g., half-maximal inhibitory concentration, 19.2 +/- 5.8, tumor-to-muscle ratio, 5,157.50 +/- 949.17). Conclusion: Our findings demonstrate the intrinsic integration of a fluorophore in the pharmacophore in PSMA-targeted smallmolecule tracers. In this design, having 1 sulfonate on the indole moiety adjacent to EuK ((99)mTc-EuK-(SO3)Cy5-mas(3)) yielded the most promising tracer candidate for imaging of PSMA. Show less
Background Bacterial infections are still a major global healthcare problem. To combat the increasing antimicrobial resistance, early diagnosis of bacterial infection-including the identification... Show moreBackground Bacterial infections are still a major global healthcare problem. To combat the increasing antimicrobial resistance, early diagnosis of bacterial infection-including the identification of bacterial specie-is needed to improve antibiotic stewardship and to help reduce the use of broad-spectrum antibiotics. To aid successful targeted antibiotic treatment, specific detection and localisation of infectious organisms is warranted. Nuclear medicine imaging approaches have been successfully used to diagnose bacterial infections and to differentiate between pathogen induced infections and sterile inflammatory processes.Aim In this comprehensive review we present an overview of recent developments in radiolabelled bacterial imaging tracers.Methods The PubMed/MEDLINE and Embase (OvidSP) literature databases were systematically searched for publications on SPECT and PET on specific imaging of bacterial using specific guidelines with MeSH-terms, truncations, and completion using cross-references. Tracers in literature that was extensively reviewed before 2016 were not included in this update. Where possible, the chemical structure of the radiolabelled compounds and clinical images were shown.Results In 219 original articles pre-clinical and clinical imaging of bacterial infection with new tracers were included. In our view, the highest translational potential lies with tracers that are specific to target the pathogens: e.g., Tc-99m- and Ga-68-labelled UBI29-41, Tc-99m-vancomycin, m-[F-18]-fluoro-PABA, [methyl-C-11]-D-methionine, [F-18]-FDS, [F-18]-maltohexaose and [F-18]-maltotriose. An encouraging note is that some of these tracers have already been successfully evaluated in clinical settings.Conclusion This review summarises updates in tracer development for specific (pre-clinical and clinical) imaging of bacterial infections. We propsed some promising tracers that are likely to become innovative standards in the clinical setting in the near feature. Show less
Spa, S.J.; Hensbergen, A.W.; Wal, S. van der; Kuil, J.; Leeuwen, F.W.B. van 2019
