The potential of receptor-mediated fluorescence-based image-guided surgery tracers is generally linked to the near-infrared emission profile and good-manufacturing-production availability of fluorescent dyes. Surprisingly, little is known about the critical interaction between the structural composition of the dyes and the pharmacokinetics of the tracers. In this study, a dual-modality tracer design was used to systematically and quantitatively evaluate the influence of elongation of the polymethine chain in a fluorescent cyanine dye on the imaging potential of a targeted tracer. As a model system, the integrin marker αβ was targeted using arginylglycylaspartisc acid [RGD]-based vectors functionalized with a In-diethylenetriaminepentaacetic acid (DTPA) chelate and a fluorescent dye: (Cy3-(SO)methyl-COOH [emission wavelength (λ), 580 nm], Cy5-(SO)methyl-COOH [λ, 680 nm], or Cy7-(SO)methyl-COOH [λ, 780 nm]). Tracers were analyzed for differences in photophysical properties, serum protein binding, chemical or optical stability, and signal penetration through tissue. Receptor affinities were evaluated using saturation and competition experiments. In vivo biodistribution (SPECT imaging and percentage injected dose per gram of tissue) was assessed in tumor-bearing mice and complemented with in vivo and ex vivo fluorescence images obtained using a clinical-grade multispectral fluorescence laparoscope. Two carbon-atom-step variations in the polymethine chain of the fluorescent cyanine dyes were shown to significantly influence the chemical and photophysical characteristics (e.g., stability, brightness, and tissue penetration) of the hybrid RGD tracers. DTPA-Cy5-(SO)methyl-COOH-c[RGDyK] structurally outperformed its Cy3 and Cy7 derivatives. Radioactivity-based evaluation of in vivo tracer pharmacokinetics yielded the lowest nonspecific uptake and highest tumor-to-background ratio for DTPA-Cy5-(SO)methyl-COOH-c[RGDyK] (13.2 ± 1.7), with the Cy3 and Cy7 analogs trailing at respective tumor-to-background ratios of 5.7 ± 0.7 and 4.7 ± 0.7. Fluorescence-based assessment of tumor visibility revealed a similar trend. These findings underline that variations in the polymethine chain lengths of cyanine dyes have a profound influence on the photophysical properties, stability, and in vivo targeting capabilities of fluorescent imaging tracers. In a direct comparison, the intermediate-length dye (Cy5) yielded a superior c[RGDyK] tracer, compared with the shorter (Cy3) and longer (Cy7) analogs.

Background Bacterial infections are still a major global healthcare problem. To combat the increasing antimicrobial resistance, early diagnosis of bacterial infections-including the identification of bacterial species-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., 99m Tc-and 68 Galabelled UBI 29-41 , 99m Tc-vancomycin, m-[ 18 F]-fluoro-PABA, [methyl-11 C]-D-methionine, [ 18 F]-FDS, [ 18 F]-maltohexaose and [ 18 F]-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.

Prostate cancer surgery is currently being revolutionized by the use of prostate-specific membrane antigen (PSMA)-targeted radiotracers, for example, 99m Tc-labeled PSMA tracer analogs for radioguided surgery. The purpose of this study was to develop a second-generation 99m Tc-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-(SO 3)Cy5-mas 3 , EuK-Cy5(SO 3)-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 99m Tc, 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 • 10 4 M −1 • 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 [IC 50 ] 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, 99m Tc-EuK-(SO 3)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 (99m Tc-EuK-(SO 3)Cy5-mas 3) yielded the most promising tracer candidate for imaging of PSMA.

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. 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).

The light spectrum above 650 nm allows for good tissue penetration depths, far-red and nearinfrared fluorescent dyes are therefore popular fluorophores applied in (bio)medical diagnostics, including image-guided surgery. However, near-infrared fluorescent dyes often suffer from instability and limited brightness, two important features that, together with the labelling efficiency (e.g., non-one-or di-conjugated products) and serum-dye interactions are key elements that drive in vivo characteristics. Due to the fact that stability and brightness of far-red fluorophores are often superior over near-infrared dyes, interest in the use of dyes such as Cy5 is increasing. As there are clear indications that the influence of the chemical structure on the (photo)physical properties of a dye is dye-structure-dependent, the (photo)physical properties of ten structural variants of asymmetrical Cy5-(R1)R2-(R3)COOH (R representing the varied substituents) were extensively studied, While stacking in solution was not induced in most of the Cy5 far-red fluorophores, multimers and 2 stacking characteristics were observed in protein conjugates. And although all dye variants were shown to be stable towards photobleaching, clear differences in brightness and serum interactions were found. Combined, these findings indicate that the chemical substituents prominently influence the photophysical properties of Cy5 dyes, a feature that should be considered when using fluorescent dyes in future tracer development.

There is a need to develop diagnostic and analytical tools that allow noninvasive monitoring of bacterial growth and dissemination in vivo. For such cell-tracking studies to hold translational value to controlled human infections, in which volunteers are experimentally colonized, they should not require genetic modification, and they should allow tracking over a number of replication cycles. To gauge if an antimicrobial peptide tracer, 99mTc-UBI29–41-Cy5, which contains both a fluorescent and a radioactive moiety, could be used for such in vivo bacterial tracking, we performed longitudinal imaging of a thigh-muscle infection with 99mTc-UBI29–41-Cy5-labeled Staphylococcus aureus. Mice were imaged using SPECT and fluorescence-imaging modalities at various intervals during a 28 h period. Biodistribution analyses were performed to quantitate radioactivity in the abscess and other tissues. SPECT and fluorescence imaging in mice showed clear retention of the 99mTc-UBI29–41-Cy5-labeled bacteria following inoculation in the thigh muscle. Despite bacterial replication, the signal intensity in the abscess only modestly decreased within a 28 h period: 52% of the total injected radioactivity per gram of tissue (%ID/g) at 4 h postinfection (pi) versus 44%ID/g at 28 h pi (15% decrease). After inoculation, a portion of the bacteria disseminated from the abscess, and S. aureus cultures were obtained from radioactive urine samples. Bacterial staining with 99mTc-UBI29–41-Cy5 allowed noninvasive bacterial-cell tracking during a 28 h period. Given the versatility of the presented bacterial-tracking method, we believe that this concept could pave the way for precise imaging capabilities during controlled-human-infection studies.

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 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-P0101–125 was selected after initial solubility, (photo)physical and in vitro evaluation (including P0-blocking experiments). Cy5-P0101–125 (KD = 105 ± 17 nM) allowed in vitro and ex vivo P0-related staining. Furthermore, Cy5-P0101–125  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 [111In]In-DTPA-P0101–125 does not enter the central nervous system (CNS). Conclusion P0101–125 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.

Background Today, both radioactive SPECT and PET imaging radiopharmaceuticals are used for clinical diagnosis of bacterial infections. Due to the possible applications in image-guided surgery, fluorescent imaging of infections has gained interest. We here present the highlights and recent developments in the use of fluorescence imaging for bacterial infections. In this overview, we also include the latest developments in multimodal bacterial imaging strategies that combine radioactive and fluorescent imaging. Based on this literature, we present our future perspectives for the field including the translational potential. Methods In the current review, we complement earlier reports with the most recent fluorescent and multimodal radiopharmaceuticals for bacterial infection imaging. Where possible, in this review, the chemical structure of the compounds and clinical images were shown. Results A total of 35 out of 77 original articles on pre-clinical and clinical imaging of bacterial infections with fluorescent tracers and multimodality radiopharmaceuticals were included for reviewing. Conclusion In our view, the highest translational potential lies with compounds that are based on targeting vectors that are specific for bacteria: e.g., fluorescently labelled UBI 29-41 , polymyxin B, vancomycin, ZnDPA and a M. tuberculosis-specific β-lactamase-cleavable linker CNIP800. Multimodal concepts using dually labelled UBI 29-41 , vancomycin, and ZnDPA help connect optical imaging to the more traditional use of radiopharmaceuticals in infectious diseases. Multimodal bacterial imaging is a promising strategy not only to diagnose bacterial infections but also to evaluate the effectivity of surgical treatment for infections.