Evaluation of [¹¹¹In]-labeled zinc-dipicolylamine tracers for SPECT imaging of bacterial infection.
ABSTRACT: PURPOSE:This study prepared three structurally related zinc-dipicolylamine (ZnDPA) tracers with [(111)In] labels and conducted biodistribution and single-photon emission computed tomography/computed tomography (SPECT/CT) imaging studies of a mouse leg infection model. PROCEDURES:Two monovalent tracers, ZnDPA-[(111)In]DTPA and ZnDPA-[(111)In]DOTA, each with a single zinc-dipicolylamine targeting unit, and a divalent tracer, Bis(ZnDPA)-[(111)In]DTPA, with two zinc-dipicolylamine units were prepared. Organ biodistribution and SPECT and CT imaging studies were performed on living mice with a leg infection created by injection of clinically relevant Gram positive Streptococcus pyogenes. Fluorescent and luminescent Eu(3+)-labeled versions of these tracers were also prepared and used to measure relative affinity for the exterior membrane surface of bacterial cells and mimics of healthy mammalian cells. RESULTS:All three (111)In-labeled radiotracers were prepared with a radiopurity of >90 %. The biodistribution studies showed that the two monovalent tracers were cleared from the body through the liver and kidney, with retained percentage injected dose for all organs of <8 % at 20 h and infected leg target to non-target ratio (T/NT) ratio of ?3.0. Clearance of the divalent tracer from the bloodstream was slower and primarily through the liver, with a retained percentage injected dose for all organs <37 % at 20 h and T/NT ratio rising to 6.2 after 20 h. The SPECT/CT imaging indicated the same large difference in tracer pharmacokinetics and higher accumulation of the divalent tracer at the site of infection. CONCLUSIONS:All three [(111)In]-ZnDPA tracers selectively targeted the site of a clinically relevant mouse infection model that could not be discerned by visual external inspection of the living animal. The highest target selectivity, observed with a divalent tracer equipped with two zinc-dipicolylamine targeting units, compares quite favorably with the imaging selectivities previously reported for other nuclear tracers that target bacterial cell surfaces. The tracer pharmacokinetics depended heavily on tracer molecular structure suggesting that it may be possible to rapidly fine tune the structural properties for optimized in vivo imaging performance and clinical translation.
Project description:This study reports the first set of synthetic molecules that act as broad spectrum agglutination agents and thus are complementary to the specific targeting of antibodies. The molecules have dendritic architecture and contain multiple copies of zinc(II)-dipicolylamine (ZnDPA) units that have selective affinity for the bacterial cell envelope. A series of molecular structures were evaluated, with the number of appended ZnDPA units ranging from four to thirty-two. Agglutination assays showed that the multivalent probes rapidly cross-linked ten different strains of bacteria, regardless of Gram-type and cell morphology. Fluorescence microscopy studies using probes with four ZnDPA units indicated a high selectivity for bacteria agglutination in the presence of mammalian cells and no measurable effect on the health of the cells. The high bacterial selectivity was confirmed by conducting in vivo optical imaging studies of a mouse leg infection model. The results suggest that multivalent ZnDPA molecular probes with dendritic structures have great promise as selective, broad spectrum bacterial agglutination agents for infection imaging and theranostic applications.
Project description:This study measured the antiplasmodial activity of nine zinc-dipicolylamine (ZnDPA) complexes against three strains of Plasmodium falciparum, the causative parasite of malaria. Growth inhibition assays showed significant activity against all tested strains, with 50% inhibitory concentrations between 5 and 600nM and almost no toxic effect against host cells including healthy red blood cells. Fluorescence microscopy studies with a green-fluorescent ZnDPA probe showed selective targeting of infected red blood cells. The results suggest that ZnDPA coordination complexes are promising antiplasmodial agents with potential for targeted malaria treatment.
Project description:The potential value of multiplexed positron emission tomography (PET) tracers in mice with turpentine-induced inflammation was evaluated and compared with 2-[¹?F]fluoro-2-deoxy-D-glucose ([¹?F]FDG) for glucose metabolism imaging. These PET tracers included [¹?F]fluoromethylcholine ([¹?F]FCH) for choline metabolism imaging, (S-[¹¹C]methyl)-D-cysteine ([¹¹C]DMCYS) for amino acid metabolism imaging, [¹¹C]bis(zinc(II)-dipicolylamine) ([¹¹C]DPA-Zn²?) for apoptosis imaging, 2-(4-N-[¹¹C]-methylaminophenyl)-6-hydroxybenzothiazole ([¹¹C]PIB) for ? amyloid binding imaging, and [¹?F]fluoride (¹?F?) for bone metabolism imaging. In mice with turpentine-induced inflammation mice, the biodistribution of all the tracers mentioned above at 5, 15, 30, 45, and 60 min postinjection was determined. Also, the time-course curves of the tracer uptake ratios for inflammatory thigh muscle (IM) to normal uninflammatory thigh muscle (NM), IM to blood (BL), IM to brain (BR), and IM to liver (LI) were acquired, respectively. Moreover, PET imaging with the tracers within 60 min postinjection on a clinical PET/CT scanner was also conducted. [¹?F]FDG and ¹?F? showed relatively higher uptake ratios for IM to NM, IM to BL, IM to BR, and IM to LI than [¹?F]FCH, [¹¹C]DPA-Zn²?, [¹¹C]DMCYS and [¹¹C]PIB, which were highly consistent with the results delineated in PET images. The results demonstrate that ¹?F? seems to be a potential PET tracer for inflammation imaging. [¹?F]FCH and [¹¹C]DMCYS, with lower accumulation in inflammatory tissue than [¹?F]FDG, are not good PET tracers for inflammation imaging. As a promising inflammatory tracer, the chemical structure of [¹¹C]DPA-Zn²? needs to be further optimized.
Project description: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 ?v?3-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.
Project description:Cutaneous leishmaniasis is a neglected tropical disease that causes painful lesions and severe disfigurement. Modern treatment relies on a few chemotherapeutics with serious limitations, and there is a need for more effective alternatives. This study describes the selective targeting of zinc(II)-dipicolylamine (ZnDPA) coordination complexes toward Leishmania major, one of the species responsible for cutaneous leishmaniasis. Fluorescence microscopy of L. major promastigotes treated with a fluorescently labeled ZnDPA probe indicated rapid accumulation of the probe within the axenic promastigote cytosol. The antileishmanial activities of eight ZnDPA complexes were measured using an in vitro assay. All tested complexes exhibited selective toxicity against L. major axenic promastigotes, with 50% effective concentration values in the range of 12.7 to 0.3 ?M. Similar toxicity was observed against intracellular amastigotes, but there was almost no effect on the viability of mammalian cells, including mouse peritoneal macrophages. In vivo treatment efficacy studies used fluorescence imaging to noninvasively monitor changes in the red fluorescence produced by an infection of mCherry-L. major in a mouse model. A ZnDPA treatment regimen reduced the parasite burden nearly as well as the reference care agent, potassium antimony(III) tartrate, and with less necrosis in the local host tissue. The results demonstrate that ZnDPA coordination complexes are a promising new class of antileishmanial agents with potential for clinical translation.
Project description:Here, the expression of F4/80 on the cell surface of murine macrophages was exploited to develop a novel imaging tracer that could visualize macrophages in vivo.The immunoreactive fraction and IC50 of anti-F4/80-A3-1, conjugated with diethylenetriaminepentaacetic acid (DTPA) and radiolabelled with (111)In, were determined in vitro using murine bone marrow-derived macrophages. In vivo biodistribution studies were performed with (111)In-anti-F4/80-A3-1 and isotype-matched control antibody (111)In-rat IgG2b at 24 and 72 h post-injection (p.i.) in SCID/Beige mice bearing orthotopic MDA-MB-231 xenografts. In some studies mice were also treated with liposomal clodronate. Macrophage content in tissues was determined immunohistochemically. Micro-single photon emission computed tomography (SPECT)/CT images were also acquired.In vitro binding assays showed that (111)In-anti-F4/80-A3-1 specifically binds F4/80 receptor-positive macrophages. The immunoreactivity of anti-F4/80-A3-1 was 75 % and IC50 was 0.58 nM. In vivo, injection of 10 or 100 ?g (111)In-anti-F4/80-A3-1 resulted in splenic uptake of 78 %ID/g and 31 %ID/g, respectively, and tumour uptake of 1.38 %ID/g and 4.08 %ID/g, respectively (72 h p.i.). Liposomal clodronate treatment reduced splenic uptake of 10 ?g (111)In-anti-F4/80-A3-1 from 248 %ID/g to 114 %ID/g and reduced (111)In-anti-F4/80-A3-1 uptake in the liver and femur (24 h p.i.). Tracer retention in the blood and tumour uptake increased (24 h p.i.). Tumour uptake of (111)In-anti-F4/80-A3-1 was visualized by microSPECT/CT. Macrophage density in the spleen and liver decreased in mice treated with liposomal clodronate. Uptake of (111)In-rat IgG2b was lower in the spleen, liver and femur when compared to (111)In-anti-F4/80-A3-1.Radiolabelled anti-F4/80-A3-1 antibodies specifically localize in tissues infiltrated by macrophages in mice and can be used to visualize tumours. The liver and spleen act as antigen sink organs for macrophage-specific tracers.
Project description:Cell death is a central process in developmental biology and also an important indicator of disease status and treatment efficacy. Two related fluorescent probes are described that are molecular conjugates of one or two zinc dipicolylamine (ZnDPA) coordination complexes with an appended solvatochromic benzothiazolium squaraine dye. The probes were designed to target the anionic phospholipid, phosphatidylserine (PS), that is exposed on the surface of dead and dying cells. A series of spectrometric and microscopy studies using liposomes and red blood cell ghosts as models showed that the probe with two ZnDPA targeting units produced higher affinity, stronger fluorescence "turn-on" effect, and better image contrast than the probe with one ZnDPA. Both fluorescent probes enabled "no-wash" time-lapse microscopic imaging of mammalian cell death within a culture. The probe with two ZnDPA units was used for non-invasive time-lapse imaging of cell death during the development of Xenopus laevis (frog) embryos. In vivo fluorescence micrographs revealed probe accumulation within the embryo tail, head and spine regions that were undergoing regression and apoptosis during growth and maturation. These new fluorescent probes are likely to be useful for time-resolved, non-invasive in vivo imaging of cell death process in range of living organisms. From a broader perspective, it should be possible to utilize the negative solvatochromism exhibited by benzothiazolium squaraine dyes for development of various "turn-on" deep-red fluorescent probes and materials that target cell surface biomarkers for in vitro and in vivo imaging.
Project description:(111)In (typically as [(111)In]oxinate3) is a gold standard radiolabel for cell tracking in humans by scintigraphy. A long half-life positron-emitting radiolabel to serve the same purpose using positron emission tomography (PET) has long been sought. We aimed to develop an (89)Zr PET tracer for cell labelling and compare it with [(111)In]oxinate3 single photon emission computed tomography (SPECT).[(89)Zr]Oxinate4 was synthesised and its uptake and efflux were measured in vitro in three cell lines and in human leukocytes. The in vivo biodistribution of eGFP-5T33 murine myeloma cells labelled using [(89)Zr]oxinate4 or [(111)In]oxinate3 was monitored for up to 14 days. (89)Zr retention by living radiolabelled eGFP-positive cells in vivo was monitored by FACS sorting of liver, spleen and bone marrow cells followed by gamma counting.Zr labelling was effective in all cell types with yields comparable with (111)In labelling. Retention of (89)Zr in cells in vitro after 24 h was significantly better (range 71 to >90%) than (111)In (43-52%). eGFP-5T33 cells in vivo showed the same early biodistribution whether labelled with (111)In or (89)Zr (initial pulmonary accumulation followed by migration to liver, spleen and bone marrow), but later translocation of radioactivity to kidneys was much greater for (111)In. In liver, spleen and bone marrow at least 92% of (89)Zr remained associated with eGFP-positive cells after 7 days in vivo.[(89)Zr]Oxinate4 offers a potential solution to the emerging need for a long half-life PET tracer for cell tracking in vivo and deserves further evaluation of its effects on survival and behaviour of different cell types.
Project description:?(v)?(3) integrin is involved in (tumor-induced) angiogenesis and is a promising candidate for the specific visualization of both primary tumors and of their distant metastases. Combination of radioactive and fluorescent imaging labels in a single multimodal, or rather hybrid, RGD-based imaging agent enables integration of pre-, intra-, and postoperative angiogenesis imaging. A hybrid imaging agent targeting the ?(v)?(3) integrin--(111)In-MSAP-RGD (MSAP = multifunctional single-attachment-point reagent), which contains a targeting moiety, a pentetic acid (DTPA) chelate, and a cyanine dye--was evaluated for its potential value in combined lesion detection and interventional molecular imaging in a 4T1 mouse breast cancer model. SPECT/CT and fluorescence imaging were used to visualize the tumor in vivo. Tracer distribution was evaluated ex vivo down to the microscopic level. The properties of (111)In-MSAP-RGD were compared with those of (111)In-DTPA-RGD. Biodistribution studies revealed a prolonged retention and increased tumor accumulation of (111)In-MSAP-RGD relative to (111)In-DTPA-RGD. With (111)In-MSAP-RGD, identical features could be visualized preoperatively (SPECT/CT) and intraoperatively (fluorescence imaging). As well as the primary tumor, (111)In-MSAP-RGD also enabled detection and accurate excision of distant metastases in the head and neck region of the mice. Therefore, the hybrid RGD derivative (111)In-MSAP-RGD shows potential in preoperative planning and fluorescence-based surgical intervention.
Project description:Magnetic particle imaging (MPI) is an emerging tracer-based medical imaging modality that images non-radioactive, kidney-safe superparamagnetic iron oxide (SPIO) tracers. MPI offers quantitative, high-contrast and high-SNR images, so MPI has exceptional promise for applications such as cell tracking, angiography, brain perfusion, cancer detection, traumatic brain injury and pulmonary imaging. In assessing MPI's utility for applications mentioned above, it is important to be able to assess tracer short-term biodistribution as well as long-term clearance from the body. Here, we describe the biodistribution and clearance for two commonly used tracers in MPI: Ferucarbotran (Meito Sangyo Co., Japan) and LS-oo8 (LodeSpin Labs, Seattle, WA). We successfully demonstrate that 3D MPI is able to quantitatively assess short-term biodistribution, as well as long-term tracking and clearance of these tracers in vivo.