Screening sensibility and antifungal activity after topical application of a synthetic lactoferrin-derived antimicrobial peptide.

OBJECTIVE: Onychomycosis is the most common disease of the nails and constitutes about half of all nail abnormalities. Onychomycosis is usually caused by dermatophytes and incomparably less frequently by yeast-like fungi and non-dermatophyte molds. Current treatment options for onychomycosis are ineffective. METHODS: This study evaluated the performance of a commercial and CE-registered product containing antimicrobial peptide hLF1-11 in vitro for treating toenail onychomycosis. In a case-control setting, nail samples from 59 volunteers were obtained before and after treatment by a pedicurist and investigated for the presence of fungi by culturing, barcode sequencing, and MALDI-TOF-MS. RESULTS: Of 89 samples, T. rubrum (19%) and C. parapsilosis (17%) were cultured. In total, 47 samples (53%) were positive for culture. MALDI-TOF-MS could identify 28, but 19 remained unidentified; those species were not included in the commercial MALDI-TOF reference database library. A positive effect of treatment by the hLF1-11 product on 41 volunteers (1 placebo, 18 low doses, 22 high doses) was observed. No adverse effects of the peptide were observed or reported by the pedicurist or any of the participants. CONCLUSIONS: This study showed a positive therapeutic effect of a commercial product containing hLF1-11 in the case of 88.9% of the patients with onychomycosis. The present formulation of hLF1-11 into PBS is stable enough to permit storage at room temperature for at least two years.

Biodistribution of Radioactively Labeled Splice Modulating Antisense Oligonucleotides After Intracerebroventricular and Intrathecal Injection in Mice.

Antisense oligonucleotides (AONs) are promising therapeutic candidates, especially for neurological diseases. Intracerebroventricular (ICV) injection is the predominant route of administration in mouse studies, while in clinical trials, intrathecal (IT) administration is mostly used. There is little knowledge on the differences in distribution of these injection methods within the same species over time. In this study, we compared the distribution of splice-switching AONs targeting exon 15 of amyloid precursor protein pre-mRNA injected via the ICV and IT route in mice. The AON was labeled with radioactive indium-111 and mice were imaged using single-photon emission computed tomography (SPECT) 0, 4, 24, 48, 72, and 96 h after injection. In vivo SPECT imaging showed 111In-AON activity diffused throughout the central nervous system (CNS) in the first hours after injection. The 111In-AON activity in the CNS persisted over the course of 4 days, while signal in the kidneys rapidly decreased. Postmortem counting in different organs and tissues showed very similar distribution of 111In-AON activity throughout the body, while the signal in the different brain regions was higher with ICV injection. Overall, IT and ICV injection have very similar distribution patterns in the mouse, but ICV injection is much more effective in reaching the brain.

Insights into Peptidoglycan-Targeting Radiotracers for Imaging Bacterial Infections: Updates, Challenges, and Future Perspectives.

The unique structural architecture of the peptidoglycan allows for the stratification of bacteria as either Gram-negative or Gram-positive, which makes bacterial cells distinguishable from mammalian cells. This classification has received attention as a potential target for diagnostic and therapeutic purposes. Bacteria's ability to metabolically integrate peptidoglycan precursors during cell wall biosynthesis and recycling offers an opportunity to target and image pathogens in their biological state. This Review explores the peptidoglycan biosynthesis for bacteria-specific targeting for infection imaging. Current and potential radiolabeled peptidoglycan precursors for bacterial infection imaging, their development status, and their performance in vitro and/or in vivo are highlighted. We conclude by providing our thoughts on how to shape this area of research for future clinical translation.

The Development and Validation of Radiopharmaceuticals Targeting Bacterial Infection.

The International Atomic Energy Agency organized a technical meeting at its headquarters in Vienna, Austria, in 2022 that included 17 experts representing 12 countries, whose research spanned the development and use of radiolabeled agents for imaging infection. The meeting focused largely on bacterial pathogens. The group discussed and evaluated the advantages and disadvantages of several radiopharmaceuticals, as well as the science driving various imaging approaches. The main objective was to understand why few infection-targeted radiotracers are used in clinical practice despite the urgent need to better characterize bacterial infections. This article summarizes the resulting consensus, at least among the included scientists and countries, on the current status of radiopharmaceutical development for infection imaging. Also included are opinions and recommendations regarding current research standards in this area. This and future International Atomic Energy Agency-sponsored collaborations will advance the goal of providing the medical community with innovative, practical tools for the specific image-based diagnosis of infection.

Microspheres as a Carrier System for Therapeutic Embolization Procedures: Achievements and Advances.

The targeted delivery of anti-cancer drugs and isotopes is one of the most pursued goals in anti-cancer therapy. One of the prime examples of such an application is the intra-arterial injection of microspheres containing cytostatic drugs or radioisotopes during hepatic embolization procedures. Therapy based on the application of microspheres revolves around vascular occlusion, complemented with local therapy in the form of trans-arterial chemoembolization (TACE) or radioembolization (TARE). The broadest implementation of these embolization strategies currently lies within the treatment of untreatable hepatocellular cancer (HCC) and metastatic colorectal cancer. This review aims to describe the state-of-the-art TACE and TARE technologies investigated in the clinical setting for HCC and addresses current trials and new developments. In addition, chemical properties and advancements in microsphere carrier systems are evaluated, and possible improvements in embolization therapy based on the modification of and functionalization with therapeutical loads are explored.

A radio- and fluorescently labelled tracer for imaging and quantification of bacterial infection on orthopaedic prostheses : a proof of principle study.

AIMS: Arthroplasty surgery of the knee and hip is performed in two to three million patients annually. Periprosthetic joint infections occur in 4% of these patients. Debridement, antibiotics, and implant retention (DAIR) surgery aimed at cleaning the infected prosthesis often fails, subsequently requiring invasive revision of the complete prosthetic reconstruction. Infection-specific imaging may help to guide DAIR. In this study, we evaluated a bacteria-specific hybrid tracer (99mTc-UBI29-41-Cy5) and its ability to visualize the bacterial load on femoral implants using clinical-grade image guidance methods. METHODS: 99mTc-UBI29-41-Cy5 specificity for Stapylococcus aureus was assessed in vitro using fluorescence confocal imaging. Topical administration was used to highlight the location of S. aureus cultured on femoral prostheses using fluorescence imaging and freehand single photon emission CT (fhSPECT) scans. Gamma counting and fhSPECT were used to quantify the bacterial load and monitor cleaning with chlorhexidine. Microbiological culturing helped to relate the imaging findings with the number of (remaining) bacteria. RESULTS: Bacteria could be effectively stained in vitro and on prostheses, irrespective of the presence of biofilm. Infected prostheses revealed bacterial presence on the transition zone between the head and neck, and in the screw hole. Qualitative 2D fluorescence images could be complemented with quantitative 3D fhSPECT scans. Despite thorough chlorhexidine treatments, 28% to 44% of the signal remained present in the locations of the infection that were identified using imaging, which included 500 to 2,000 viable bacteria. CONCLUSION: The hybrid tracer 99mTc-UBI29-41-Cy5 allowed effective bacterial staining. Qualitative real-time fluorescence guidance could be effectively combined with nuclear imaging that enables quantitative monitoring of the effectiveness of cleaning strategies.Cite this article: Bone Joint Res 2023;12(1):72-79.

Feasibility of fluorescence imaging at microdosing using a hybrid PSMA tracer during robot-assisted radical prostatectomy in a large animal model.

BACKGROUND: With the rise of prostate-specific membrane antigen (PSMA) radioguided surgery, which is performed using a microdosing regime, demand for visual target confirmation via fluorescence guidance is growing. While proven very effective for radiotracers, microdosing approaches the detection limit for fluorescence imaging. Thus, utility will be highly dependent on the tracer performance, the sensitivity of the fluorescence camera used, and the degree of background signal. Using a porcine model the ability to perform robot-assisted radical prostatectomy under fluorescence guidance using the bimodal or rather hybrid PSMA tracer (99mTc-EuK-(SO3)Cy5-mas3) was studied, while employing the tracer in a microdosing regime. This was followed by ex vivo evaluation in surgical specimens obtained from prostate cancer patients. RESULTS: T50% blood and T50% urine were reached at 85 min and 390 min, in, respectively, blood and urine. Surgical fluorescence imaging allowed visualization of the prostate gland based on the basal PSMA-expression in porcine prostate. Together, in vivo visualization of the prostate and urinary excretion suggests at least an interval of > 7 h between tracer administration and surgery. Confocal microscopy of excised tissues confirmed tracer uptake in kidney and prostate, which was confirmed with PSMA IHC. No fluorescence was detected in other excised tissues. Tumor identification based on ex vivo fluorescence imaging of human prostate cancer specimens correlated with PSMA IHC. CONCLUSION: Intraoperative PSMA-mediated fluorescence imaging with a microdosing approach was shown to be feasible. Furthermore, EuK-(SO3)Cy5-mas3 allowed tumor identification in human prostate samples, underlining the translational potential of this novel tracer. Trial registration Approval for use of biological material for research purposes was provided by the Translational Research Board of the Netherlands Cancer Institute-Antoni van Leeuwenhoek hospital (NKI-AvL) under reference IRBm19-273 (22/10/2019).

Advancing intraoperative magnetic tracing using 3D freehand magnetic particle imaging.

PURPOSE: Sentinel lymph node biopsy is a routine procedure for nodal staging in penile cancer. Most commonly, this procedure is guided by radioactive tracers, providing various forms of preoperative and intraoperative guidance. This is further extended with fluorescence imaging using hybrid radioactive-fluorescence tracers. Alternatively, a magnetic-based approach has become available using superparamagnetic iron-oxide nanoparticles (SPIONs). This study investigates a novel freehand magnetic particle imaging and navigation modality (fhMPI) for intraoperative localization, along with a hybrid approach, combining magnetic and fluorescence guidance. MATERIALS AND METHODS: The fhMPI set-up was built with a surgical navigation device, optical tracking system and magnetometer probe. A dedicated reconstruction software based on a look-up-table method was used to reconstruct a superficial 3D volume of the SPION distribution in tissue. For fluorescence guidance, indocyanine green (ICG) was added to the SPIONs. The fhMPI modality was characterized in phantoms, ex vivo human skin and in vivo porcine surgery. RESULTS: Phantom and human skin explants illustrated that the current fhMPI modality had a sensitivity of 2.2 × 10-2 mg/mL SPIONs, a resolving power of at least 7 mm and a depth penetration up to 1.5 cm. Evaluation during porcine surgery showed that fhMPI allowed for an augmented reality image overlay of the tracer distribution in tissue, as well as 3D virtual navigation. Besides, using the hybrid approach, fluorescence imaging provided a visual confirmation of localized nodes. CONCLUSION: fhMPI is feasible in vivo, providing 3D imaging and navigation for magnetic nanoparticles in the operating room, expanding the guidance possibilities during magnetic sentinel lymph node procedures. Furthermore, the integration of ICG provides the ability to visually refine and confirm correct localization. Further clinical evaluation should verify these findings in human patients as well.

Cyclodextrin/Adamantane-Mediated Targeting of Inoculated Bacteria in Mice.

Cyclodextrin (CD)-based host-guest interactions with adamantane (Ad) have demonstrated use for functionalizing living cells in vitro. The next step in this supramolecular functionalization approach is to explore the concept to deliver chemical cargo to living cells in vivo, e.g., inoculated bacteria, in order to study their dissemination. We validated this concept in two rodent Staphylococcus aureus models. Bacteria (1 × 108 viable S. aureus) were inoculated by (1) intramuscular injection or (2) intrasplenic injection followed by dissemination throughout the liver. The bacteria were prefunctionalized with 99mTc-UBI29-41-Ad2 (primary vector), which allowed us to both determine the bacterial load and create an in vivo target for the secondary host-vector (24 h post-inoculation). The secondary vector, i.e., chemical cargo delivery system, made use of a 111In-Cy50.5CD9PIBMA39 polymer that was administered intravenously. Bacteria-specific cargo delivery as a result of vector complexation was evaluated by dual-isotope SPECT imaging and biodistribution studies (111In), and by fluorescence (Cy5); these evaluations were performed 4 h post-injection of the secondary vector. Mice inoculated with nonfunctionalized S. aureus and mice without an infection served as controls. Dual-isotope SPECT imaging demonstrated that 111In-Cy50.5CD9PIBMA39 colocalized with 99mTc-UBI29-41-Ad2-labeled bacteria in both muscle and liver. In inoculated muscle, a 2-fold higher uptake level (3.2 ± 1.0%ID/g) was noted compared to inoculation with nonfunctionalized bacteria (1.9 ± 0.4%ID/g), and a 16-fold higher uptake level compared to noninfected muscle (0.2 ± 0.1%ID/g). The hepatic accumulation of the host-vector was nearly 10-fold higher (27.1 ± 11.1%ID/g) compared to the noninfected control (2.7 ± 0.3%ID/g; p < 0.05). Fluorescence imaging of the secondary vector corroborated SPECT-imaging and biodistribution findings. We have demonstrated that supramolecular host-guest complexation can be harnessed to achieve an in vivo cargo delivery strategy, using two different bacterial models in soft tissue and liver. This proof-of-principle study paves a path toward developing innovative drug delivery concepts via cell functionalization techniques.

The helminth glycoprotein omega-1 improves metabolic homeostasis in obese mice through type 2 immunity-independent inhibition of food intake.

Type 2 immunity plays an essential role in the maintenance of metabolic homeostasis and its disruption during obesity promotes meta-inflammation and insulin resistance. Infection with the helminth parasite Schistosoma mansoni and treatment with its soluble egg antigens (SEA) induce a type 2 immune response in metabolic organs and improve insulin sensitivity and glucose tolerance in obese mice, yet, a causal relationship remains unproven. Here, we investigated the effects and underlying mechanisms of the T2 ribonuclease omega-1 (ω1), one of the major S mansoni immunomodulatory glycoproteins, on metabolic homeostasis. We show that treatment of obese mice with plant-produced recombinant ω1, harboring similar glycan motifs as present on the native molecule, decreased body fat mass, and improved systemic insulin sensitivity and glucose tolerance in a time- and dose-dependent manner. This effect was associated with an increase in white adipose tissue (WAT) type 2 T helper cells, eosinophils, and alternatively activated macrophages, without affecting type 2 innate lymphoid cells. In contrast to SEA, the metabolic effects of ω1 were still observed in obese STAT6-deficient mice with impaired type 2 immunity, indicating that its metabolic effects are independent of the type 2 immune response. Instead, we found that ω1 inhibited food intake, without affecting locomotor activity, WAT thermogenic capacity or whole-body energy expenditure, an effect also occurring in leptin receptor-deficient obese and hyperphagic db/db mice. Altogether, we demonstrate that while the helminth glycoprotein ω1 can induce type 2 immunity, it improves whole-body metabolic homeostasis in obese mice by inhibiting food intake via a STAT6-independent mechanism.

Imaging Bacteria with Radiolabelled Probes: Is It Feasible?

Bacterial infections are the main cause of patient morbidity and mortality worldwide. Diagnosis can be difficult and delayed as well as the identification of the etiological pathogen, necessary for a tailored antibiotic therapy. Several non-invasive diagnostic procedures are available, all with pros and cons. Molecular nuclear medicine has highly contributed in this field by proposing several different radiopharmaceuticals (antimicrobial peptides, leukocytes, cytokines, antibiotics, sugars, etc.) but none proved to be highly specific for bacteria, although many agents in development look promising. Indeed, factors including the number and strain of bacteria, the infection site, and the host condition, may affect the specificity of the tested radiopharmaceuticals. At the Third European Congress on Infection/Inflammation Imaging, a round table discussion was dedicated to debate the pros and cons of different radiopharmaceuticals for imaging bacteria with the final goal to find a consensus on the most relevant research steps that should be fulfilled when testing a new probe, based on experience and cumulative published evidence.

A Supramolecular Platform Technology for Bacterial Cell Surface Modification.

In an era of antimicrobial resistance, a better understanding of the interaction between bacteria and the sentinel immune system is needed to discover new therapeutic targets for combating bacterial infectious disease. Sentinel immune cells such as macrophages phagocytose intact bacteria and thereby initiate ensuing immune responses. The bacterial surface composition is a key element that determines the macrophage signaling. To study the role of the bacterial cell surface composition in immune recognition, we developed a platform technology for altering bacterial surfaces in a controlled manner with versatile chemical scaffolds. We show that these scaffolds are efficiently loaded onto both Gram-positive and -negative bacteria and that their presence does not impair the capacity of monocyte-derived macrophages to phagocytose bacteria and subsequently signal to other components of the immune system. We believe this technology thus presents a useful tool to study the role of bacterial cell surface composition in disease etiology and potentially in novel interventions utilizing intact bacteria for vaccination.

Extending the Hybrid Surgical Guidance Concept With Freehand Fluorescence Tomography.

Within image-guided surgery, 'hybrid' guidance technologies have been used to integrate the complementary features of radioactive guidance and fluorescence guidance. Here, we explore how the generation of a novel freehand fluorescence (fhFluo) imaging approach complements freehand SPECT (fhSPECT) in a hybrid setup. Near-infrared optical tracking was used to register the position and the orientation of a hybrid opto-nuclear detection probe while recording its readings. Dedicated look-up table models were used for 3D reconstruction. In phantom and excised tissue settings (i.e., flat-surface human skin explants), fhSPECT and fhFluo were investigated for image resolution and in-tissue signal penetration. Finally, the combined potential of these freehand technologies was evaluated on prostate and lymph node specimens of prostate cancer patients receiving prostatectomy and sentinel lymph node dissection (tracers: indocyanine green (ICG) +99m Tc-nanocolloid or ICG-99mTc-nanocolloid). After hardware and software integration, the hybrid setup created 3D nuclear and fluorescence tomography scans. The imaging resolution of fhFluo (1 mm) was superior to that of fhSPECT (6 mm). Fluorescence modalities were confined to a maximum depth of 0.5 cm, while nuclear modalities were usable at all evaluated depths (<2 cm). Both fhSPECT and fhFluo enabled augmented- and virtual-reality navigation toward segmented image hotspots, including relative hotspot quantification with an accuracy of 3.9% and 4.1%. Imaging in surgical specimens confirmed these trends (fhSPECT: in-depth detectability, low resolution, and fhFluo: superior resolution, superficial detectability). Overall, when radioactive and fluorescent tracer signatures are used, fhFluo has complementary value to fhSPECT. Combined the freehand technologies render a unique hybrid imaging and navigation modality.

Hybrid Tracers Based on Cyanine Backbones Targeting Prostate-Specific Membrane Antigen: Tuning Pharmacokinetic Properties and Exploring Dye-Protein Interaction.

Prostate cancer surgery is currently being revolutionized by the use of prostate-specific membrane antigen (PSMA)-targeted radiotracers, for example, 99mTc-labeled PSMA tracer analogs for radioguided surgery. The purpose of this study was to develop a second-generation 99mTc-labeled PSMA-targeted tracer incorporating a fluorescent dye. Methods: Several PSMA-targeted hybrid tracers were synthesized: glutamic acid-urea-lysine (EuK)-Cy5-mas3, EuK-(SO3)Cy5-mas3, EuK-Cy5(SO3)-mas3, EuK-(Ar)Cy5-mas3, and EuK-Cy5(Ar)-mas3; the Cy5 dye acts as a functional backbone between the EuK targeting vector and the 2-mercaptoacetyl-seryl-seryl-seryl (mas3) 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 99mTc, 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 × 104 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 [IC50] range 19.2 ± 5.8-175.3 ± 61.1 nM) and in vivo characteristics (tumor-to-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, 99mTc-EuK-(SO3)Cy5-mas3 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 small-molecule tracers. In this design, having 1 sulfonate on the indole moiety adjacent to EuK (99mTc-EuK-(SO3)Cy5-mas3) yielded the most promising tracer candidate for imaging of PSMA.

Click Chemistry in the Design and Production of Hybrid Tracers.

Hybrid tracers containing both fluorescent and radioactive imaging labels have demonstrated clinical potential during sentinel lymph node procedures. To combine these two labels on a single targeting vector that allows tumor-targeted imaging, end-labeling strategies are often applied. For αvß3-integrin-targeting hybrid tracers, providing an excellent model for evaluating tracer development strategies, end-labeling-based synthesis provides a rather cumbersome synthesis strategy. Hence, the aim of this study was to investigate the use of heterobifunctional cyanine dyes in a click-chemistry-based synthesis strategy for RGD-based hybrid tracers. The triazole-based hybrid tracers DTPA.DBCO.N 3 (SO 3 )-Cy5-c[RGDyK] and DTPA.BCN.N 3 (SO 3 )-Cy5-c[RGDyK] were obtained in fewer steps than DTPA-Lys(Cy5(SO 3 )methyl)-Cys-c[RGDyK] and had partition coefficients of log P (o/w) = -2.55 ± 0.10, -1.45 ± 0.03, and -2.67 ± 0.12, respectively. Both tracers were chemically stable, and the brightnesses of DTPA.DBCO.N 3 (SO 3 )-Cy5-c[RGDyK] and DTPA.BCN.N 3 (SO 3 )-Cy5-c[RGDyK] were, respectively, 23 × 103 and 40 × 103 M-1 cm-1; lower than that of the reference tracer DTPA-Lys(Cy5(SO 3 )methyl)-Cys-c[RGDyK] (50 × 103 M-1 cm-1). Assessment of serum protein binding revealed no statistically significant difference (44 ± 2 and 40 ± 2% bound for DTPA.DBCO.N 3 (SO 3 )-Cy5-c[RGDyK] and DTPA.BCN.N 3 (SO 3 )-Cy5-c[RGDyK], respectively; 36 ± 5% bound for DTPA-Lys(Cy5(SO 3 )methyl)-Cys-c[RGDyK]; p > 0.05). DTPA.DBCO.N 3 (SO 3 )-Cy5-c[RGDyK] (K D = 17.5 ± 6.0) had a statistically significantly higher affinity than the reference compound DTPA-Lys(Cy5(SO 3 )methyl)-Cys-c[RGDyK] (K D = 30.3 ± 5.7; p < 0.0001), but DTPA.BCN.N 3 (SO 3 )-Cy5-c[RGDyK] had a statistically significantly lower affinity (K D = 76.5 ± 18.3 nM; p < 0.0001). Both [ 111 In]DTPA.DBCO.N 3 (SO 3 )-Cy5-c[RGDyK] and [ 111 In]DTPA.BCN.N 3 (SO 3 )-Cy5-c[RGDyK] enabled in vivo visualization of the 4T1 tumor via fluorescence and single-photon emission computed tomography (SPECT) imaging. Biodistribution data (% ID/g) revealed a significant increase in nonspecific uptake in the kidney, liver, and muscle for both [ 111 In]DTPA.DBCO.N 3 (SO 3 )-Cy5-c[RGDyK] and [ 111 In]DTPA.BCN.N 3 (SO 3 )-Cy5-c[RGDyK]. As a result of the higher background activity, the tumor-to-background ratio of the click-labeled RGD analogues was twofold lower compared to the end-labeled reference compound. The use of click chemistry labeling did not yield a pronounced negative effect on serum protein binding, in vitro stability, and receptor affinity; and tumors could still be visualized using SPECT and fluorescence imaging. However, quantitative in vivo biodistribution data suggest that the triazole and strained cyclooctyne moieties associated with this type of click chemistry negatively influence the pharmacokinetics of RGD peptides. Nevertheless, the design might still hold promise for other targets/targeting moieties.

Recent advances in nuclear and hybrid detection modalities for image-guided surgery.

Introduction: Radioguided surgery is an ever-evolving part of nuclear medicine. In fact, this nuclear medicine sub-discipline actively bridges non-invasive molecular imaging with surgical care. Next to relying on the availability of radio- and bimodal-tracers, the success of radioguided surgery is for a large part dependent on the imaging modalities and imaging concepts available for the surgical setting. With this review, we have aimed to provide a comprehensive update of the most recent advances in the field. Areas covered: We have made an attempt to cover all aspects of radioguided surgery: 1) the use of radioisotopes that emit γ, ß+, and/or ß- radiation, 2) hardware developments ranging from probes to 2D cameras and even the use of advanced 3D interventional imaging solutions, and 3) multiplexing solutions such as dual-isotope detection or combined radionuclear and optical detection. Expert opinion: Technical refinements in the field of radioguided surgery should continue to focus on supporting its implementation in the increasingly complex minimally invasive surgical setting, e.g. by accommodating robot-assisted laparoscopic surgery. In addition, hybrid concepts that integrate the use of radioisotopes with other image-guided surgery modalities such as fluorescence or ultrasound are likely to expand in the future.

Multimodal Tracking of Controlled Staphylococcus aureus Infections in Mice.

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.

In vivo stability of supramolecular host-guest complexes monitored by dual-isotope multiplexing in a pre-targeting model of experimental liver radioembolization.

INTRODUCTION: Cyclodextrin (CD)-based supramolecular interactions have been proposed as nanocarriers for drug delivery. We previously explored the use of these supramolecular interactions to perform targeted hepatic radioembolization. In a two-step procedure the appropriate location of the diagnostic pre-targeting vector can first be confirmed, after which the therapeutic vector will be targeted through multivalent host-guest interactions. Such a procedure would prevent therapeutic errors that come from a mismatch between diagnostic and therapeutic procedures. In the current study we explored the use of dual-isotope imaging to assess the in vivo stability of the formed complex and individual components. METHODS: Dual-isotope imaging of the host and guest vectors was performed after labeling of the pre-targeted guest vector, being adamantane (Ad) functionalized macro-aggregated albumin (MAA) particles, with technetium-99 m (99mTc-MAA-Ad). The host vector, Cy50.5CD9PIBMA39, was labeled with indium-111 (111In-Cy50.5CD9PIBMA39). The in situ stability of both the individual vectors and the resulting [MAA-Ad-111In-Cy50.5CD9PIBMA39] complexes was studied over 44 h at 37 °C in a serum protein-containing buffer. In vivo, the host vector 111In-Cy50.5CD9PIBMA39 was administered two hours after local deposition of 99mTc-MAA-Ad in mice. Dual-isotope SPECT imaging and quantitative biodistribution studies were performed between 2 and 44 h post intravenous host vector administration. RESULTS: The individual vectors portrayed <5% dissociation of the radioisotope over the course of 20 h. Dissociation of [MAA-Ad-111In-Cy50.5CD9PIBMA39] complexes remained within a 10-20% range after incubation in serum. In vivo dual-isotope SPECT imaging of host-guest interactions revealed co-localization of the tracer components. Quantitative assessment of the biodistribution revealed that the hepatic accumulation of the host vector nearly doubled between 2 h and 44 h post-injection (from 14.9 ±â€¯6.1%ID/g to 26.2 ±â€¯2.1%ID/g). CONCLUSIONS: Assessment of intra-hepatic host-guest complexation was successfully achieved using dual isotope multiplexing, underlining the complex stability that was found in situ (up to 44 h in serum). Overall, the results obtained in this study highlight the potential of supramolecular chemistry as a versatile platform that could advance the field of nanomedicine.

A Supramolecular Approach for Liver Radioembolization.

Hepatic radioembolization therapies can suffer from discrepancies between diagnostic planning (scout-scan) and the therapeutic delivery itself, resulting in unwanted side-effects such as pulmonary shunting. We reasoned that a nanotechnology-based pre-targeting strategy could help overcome this shortcoming by directly linking pre-interventional diagnostics to the local delivery of therapy. Methods: The host-guest interaction between adamantane and cyclodextrin was employed in an in vivo pre-targeting set-up. Adamantane (guest)-functionalized macro albumin aggregates (MAA-Ad; d = 18 µm) and (radiolabeled) Cy5 and ß-cyclodextrin (host)-containing PIBMA polymers (99mTc-Cy50.5CD10PIBMA39; MW ~ 18.8 kDa) functioned as the reactive pair. Following liver or lung embolization with (99mTc)-MAA-Ad or (99mTc)-MAA (control), the utility of the pre-targeting concept was evaluated after intravenous administration of 99mTc-Cy50.5CD10PIBMA39. Results: Interactions between MAA-Ad and Cy50.5CD10PIBMA39 could be monitored in solution using confocal microscopy and were quantified by radioisotope-based binding experiments. In vivo the accumulation of the MAA-Ad particles in the liver or lungs yielded an approximate ten-fold increase in accumulation of 99mTc-Cy50.5CD10PIBMA39 in these organs (16.2 %ID/g and 10.5 %ID/g, respectively) compared to the control. Pre-targeting with MAA alone was shown to be only half as efficient. Uniquely, for the first time, this data demonstrates that the formation of supramolecular interactions between cyclodextrin and adamantane can be used to drive complex formation in the chemically challenging in vivo environment. Conclusion: The in vivo distribution pattern of the cyclodextrin host could be guided by the pre-administration of the adamantane guest, thereby creating a direct link between the scout-scan (MAA-Ad) and delivery of therapy.

Tracers for Fluorescence-Guided Surgery: How Elongation of the Polymethine Chain in Cyanine Dyes Alters the Pharmacokinetics of a Dual-Modality c[RGDyK] Tracer.

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. Methods: As a model system, the integrin marker αvß3 was targeted using arginylglycylaspartisc acid [RGD]-based vectors functionalized with a 111In-diethylenetriaminepentaacetic acid (DTPA) chelate and a fluorescent dye: (Cy3-(SO3)methyl-COOH [emission wavelength (λem), 580 nm], Cy5-(SO3)methyl-COOH [λem, 680 nm], or Cy7-(SO3)methyl-COOH [λem, 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. Results: 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-(SO3)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-(SO3)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. Conclusion: 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.