A Novel Radioimmune 99mTc-Labeled Tracer for Imaging Sphingosine 1-Phosphate Receptor 1 in Tumor Xenografts: An In Vitro and In Vivo Study.

Sphingosine-1-phosphate (S1P) is a phospholipid that regulates pleiotropic biological activities and exerts extracellular functions by binding to five specific G-protein-coupled receptors, S1P receptors (S1PR) 1-5. When activated by S1P, S1PR promote the proliferation and invasion of tumor cells by inducing the formation of new blood vessels. We developed and assessed a new monoclonal antibody imaging probe 99mTc-HYNIC-S1PR1mAb, to explore the feasibility of targeting the S1PR1 in vitro and in vivo. S1PR1mAb was prepared and followed by technetium-99m labeling with succinimidyl 6-hydraziniumnicotinate hydrochloride. Cell uptake and blocking studies were performed to investigate the binding specificity of 99mTc-HYNIC-S1PR1mAb in vitro. 99mTc-HYNIC-S1P1mAb was also tested in vivo in mice xenografted with SK-HEP-1 (high-expression of S1PR1) and MCF-7 (low-expression of S1PR1) using single-photon emission-computed tomography (SPECT). Ex vivo gamma counting of tissues from tumor-bearing mice was used to evaluate 99mTc-HYNIC-S1PR1mAb biodistribution. The biodistribution study results showed significantly higher uptake in SK-HEP-1 tumors than in MCF-7 tumors (P < 0.001). Reduced uptake of 99mTc-HYNIC-S1PR1mAb in SK-HEP-1 was observed in tumor-bearing nude mice pretreated with fingolimod, which binds competitively to the receptors, especially S1PR1. 99mTc-HYNIC-S1PR1mAb can be synthesized and specifically targeted to S1PR1 in vitro and in vivo, allowing S1PR1 expression assessment with SPECT imaging.

Microwave assisted synthesis of Fe3O4 stabilized ZrO2 nanoparticles - Free radical scavenging, radiolabeling and biodistribution in rabbits.

AIMS: In vivo biodistribution of radio labeled ZrO2 nanoparticles is addressed for better imaging, therapy and diagnosis. Nanoparticles are synthesized by microwave assisted sol-gel technique using Fe3O4 as a stabilizer. Antioxidant assay, hemolytic activity in human blood and biodistribution in rabbits was explored to study the therapeutical as well as in vivo targeted diagnostic applications of as synthesized nanoparticles. MAIN METHODS: Fe3O4 stabilized zirconia nanoparticles are synthesized using microwave assisted sol-gel method. Microwave (MW) powers are varied in the range of 100 to 1000 W. As synthesized nanoparticles are evaluated using different characterizations such as X-ray diffractometer, scanning electron microscope, Raman spectroscopy, impedance analyzer, Vickers micro hardness indenter, FTIR, and UV-Vis spectroscopy. In vitro activity of synthesized nanoparticles is checked in freshly extracted human blood serum. To study biodistribution of Fe3O4 stabilized zirconia nanoparticles in rabbit, technetium-99 m was used for labeling purpose. The labeling efficacy and stability of labeled nanoparticles are also measured with instant thin layer chromatography (ITLC) method. Intravenous injection of 99mTc-Fe3O4 stabilized zirconia nanoparticles (0.2 ml), containing 110 MBq of radioactivity, is performed to study the biodistribution; nanoparticles are injected into the ear vein of animal (rabbit). KEY FINDINGS: Zirconia (ZrO2) nanoparticles (NPs) are stabilized using Fe3O4 that were prepared by means of microwave assisted sol-gel method. Crystallite size (~20 nm) agrees well with the values required to stabilize tetragonal zirconia (t-ZrO2). Volume shrinkage results in high value of hardness (~1369). Dielectric constant values, compatible for biomedical application, are observed for tetragonally stabilized samples. Low value of hemolytic response is observed for Fe3O4 stabilized ZrO2 NPs. 99mTc radio labeled ZrO2 NPs proved to be potential candidate to study biodistribution. Biodistribution studies show stability of radiolabeled NPs in the original suspension as well as in blood serum. CT scan of rabbit is performed for several times to check the biodistribution of NPs with time and survival of rabbit. Results suggest that these NPs can also be used as targeted nanoparticles as well as variants of drug payload carrier. SIGNIFICANCE: Results signify that Fe3O4 stabilized ZrO2 nanoparticles synthesized by microwave assisted sol-gel method may be considered as "all-rounder" nanoplatform and are safe enough to be used in diagnostic as well as therapeutic purposes.

Imaging Hydrogen Sulfide in Hypoxic Tissue with [99mTc]Tc-Gluconate.

Hydrogen sulfide (H2S) is the third gasotransmitter and is generated endogenously in hypoxic or inflammatory tissues and various cancers. We have recently demonstrated that endogenous H2S can be imaged with [99mTc]Tc-gluconate. In the present study, we detected H2S generated in hypoxic tissue, both in vitro and in vivo, using [99mTc]Tc-gluconate. In vitro uptake of [99mTc]Tc-gluconate was measured under hypoxic and normoxic conditions, using the colon carcinoma cell line CT26, and was higher in hypoxic cells than that in normoxic cells. An acute hindlimb ischemia-reperfusion model was established in BALB/c mice by exposing the animals to 3 h of ischemia and 3 h of reperfusion prior to in vivo imaging. [99mTc]Tc-gluconate (12.5 MBq) was intravenously injected through the tail vein, and uptake in the lower limb was analyzed by single-photon emission computed tomography/computed tomography (SPECT/CT). SPECT/CT images showed five times higher uptake in the ischemic limb than that in the normal limb. The standard uptake value (SUVmean) of the ischemic limb was 0.39 ± 0.03, while that of the normal limb was 0.07 ± 0.01. [99mTc]Tc-gluconate is a novel imaging agent that can be used both in vitro and in vivo for the detection of endogenous H2S generated in hypoxic tissue.

Renal Handling of 99mTc-Labeled Antibody Fab Fragments with a Linkage Cleavable by Enzymes on Brush Border Membrane.

The high and persistent renal radioactivity levels after injection of radiolabeled low-molecular-weight polypeptides constitute a significant problem for their diagnostic and therapeutic applications, especially when they are labeled with metallic radionuclides. To improve the renal radioactivity levels of technetium-99m (99mTc)-labeled Fab fragments, a mercaptoacetyltriglycine (MAG3)-based new bifunctional chelating agent with a cleavable glycyl-phenylalanyl-lysine (GFK) linkage, MAG3-GFK-suc-TFP, was designed, synthesized, and evaluated. 99mTc-labeled Fab was obtained by reacting MAG3-GFK-Fab conjugate with 99mTc-glucarate. The GFK linkage remained stable in plasma but was cleaved by enzymes on the renal brush border membrane. The comparative biodistribution studies with indium-111 (111In)-labeled Fab using SCN-CHX-A″-DTPA showed that while both radiolabeled Fabs exhibited similar elimination rates from the blood, [99mTc]Tc-MAG3-GFK-Fab registered much lower renal radioactivity levels from 30 min post-injection onward due to the release and subsequent urinary excretion of [99mTc]Tc-MAG3-Gly. However, [99mTc]Tc-MAG3-GFK-Fab showed an increase in the intestinal radioactivity levels with the time that was not observed with 111In-labeled Fab. The analysis of the intestinal contents suggested the redistribution of [99mTc]Tc-MAG3-Gly to the intestine. The retrospective comparison of [99mTc]Tc-MAG3-GFK-Fab with the radiolabeled Fabs so far prepared under the identical concept suggested that some portion of [99mTc]Tc-MAG3-Gly was generated after the coated vesicle formation and they were excreted into the blood, and subsequently redistributed in the intestine via hepatobiliary excretion. In conclusion, MAG3-GFK-suc-TFP provided 99mTc-labeled Fabs that exhibit low renal radioactivity shortly after injection by the post-labeling procedure. The present study indicated that, contrary to our earlier proposal, the generation of the radiometabolites would proceed not only during the internalization process of the parental antibody fragments but also after coated vesicle formation. This study also showed that the intracellular behaviors of radiometabolites played crucial roles in the elimination rates and the routes of the radioactivity from the kidney.

Beta Cell Imaging-From Pre-Clinical Validation to First in Man Testing.

There are presently no reliable ways to quantify human pancreatic beta cell mass (BCM) in vivo, which prevents an accurate understanding of the progressive beta cell loss in diabetes or following islet transplantation. Furthermore, the lack of beta cell imaging hampers the evaluation of the impact of new drugs aiming to prevent beta cell loss or to restore BCM in diabetes. We presently discuss the potential value of BCM determination as a cornerstone for individualized therapies in diabetes, describe the presently available probes for human BCM evaluation, and discuss our approach for the discovery of novel beta cell biomarkers, based on the determination of specific splice variants present in human beta cells. This has already led to the identification of DPP6 and FXYD2ga as two promising targets for human BCM imaging, and is followed by a discussion of potential safety issues, the role for radiochemistry in the improvement of BCM imaging, and concludes with an overview of the different steps from pre-clinical validation to a first-in-man trial for novel tracers.

Chromosome aberrations during in-vitro labeling of red blood cells with 99mTc: a study with cytokinesis block micronucleus assay in human peripheral blood lymphocytes.

Radiopharmaceuticals are special medicines composed by a radionuclide and a non-radioactive compound characterized by non-pharmacodynamic effects, low prevalence of side effects, and a possible risk of oncogenesis, since its administration to patients supposes a radiation dose to organism. Over these years, radiation damage induced by diagnosis radiopharmaceuticals has been evaluated, including the radiolabeled autologous cells, a group of radiopharmaceuticals where blood cells extracted from patients are labeled in-vitro and readministered for diagnosis. There is not a consensus about the possibility of increasement of risk for malignancies associated with the radiolabeled blood cells, so for a more accurate evaluation of the potential oncogenic risk related to the administration of [Tc]Tc labeled red blood cells, radiation dose received by the cells during the labeling process is studied by means of the cytokinesis-blocked micronucleus (CBMN) assay and a dose-response curve constructed by in-vitro external irradiation of blood samples. Our work enables to establish the range of activity to be added during the in-vitro labeling of red blood cells with [Tc]Tc pertechnetate to avoid radiation damage to cells. Activities recommended for blood volume determination and angiography do not increase the risk of malignancies, whilst activities of 370 MBq show chromosome aberrations in lymphocytes. Evaluation of the radiation damage related to the in-vitro labeling is recommended to estimate the potential oncogenic risk and minimize it.

Cardiac Scintigraphy With Technetium-99m-Labeled Bone-Seeking Tracers for Suspected Amyloidosis: JACC Review Topic of the Week.

Technetium-labeled cardiac scintigraphy (i.e., Tc-PYP scan) has been repurposed for the diagnosis of transthyretin amyloid cardiomyopathy (ATTR-CM). Validated in cohorts of patients with heart failure and echocardiographic and/or cardiac magnetic resonance imaging findings suggestive of cardiac amyloidosis, cardiac scintigraphy can confirm the diagnosis of ATTR-CM only when combined with blood and urine testing to exclude a monoclonal protein. Multisocietal guidelines support the nonbiopsy diagnosis of ATTR-CM using cardiac scintigraphy, yet emphasize its use in the appropriate clinical context and the crucial need to rule out light chain amyloid cardiomyopathy. Although increased awareness of ATTR-CM and the advent of effective therapy have led to rapid adoption of diagnostic scintigraphy, there is heterogeneity in adherence to consensus guidelines. This perspective outlines clinical scenarios wherein findings on technetium-labeled cardiac scintigraphy have been misinterpreted, reviews causes of false-negative and false-positive results, and provides strategies to avoid costly and potentially fatal misdiagnoses.

Superior Diagnostic Performance of the GLP-1 Receptor Agonist [Lys40(AhxHYNIC-[99mTc]/EDDA)NH2]-Exendin-4 over Conventional Imaging Modalities for Localization of Insulinoma.

PURPOSE: Insulinomas are the most common functioning neuroendocrine neoplasms of the pancreas, typically diagnosed due to characteristic symptoms. In the vast majority, the treatment is surgical and curative, requiring accurate localization of the tumour; conventional imaging, including somatostatin receptor molecular imaging, is negative in up to 10 % of cases. Recently, labelled glucagon-like peptide receptor (GLP-1R) analogues were introduced as a sensitive diagnostic method for localization of insulinomas. The aim of this study was to assess the diagnostic accuracy of a Tc-99m-labelled GLP-1R agonist [Lys40(AhxHYNIC-[99mTc]EDDA)NH2]-exendin-4 for localization of occult insulinoma. PROCEDURES: Eight patients (all females; age range 35-75 years) with biochemically proven insulinoma and with negative or inconclusive conventional imaging (consisting of somatostatin receptor scintigraphy, computed tomography, endoscopic ultrasound and magnetic resonance imaging) were enrolled. Whole-body single-photon emission tomography/computed tomography (SPECT/CT) imaging was performed 4 h post-injection of 740 MBq of [Lys40(AhxHYNIC-[99mTc]EDDA)NH2]-exendin-4. Surgical treatment was performed based on imaging findings. Histology of the removed lesions and biochemical and clinical symptom resolution was considered as the gold standard for analysis of the imaging results. RESULTS: Focal uptake of [Lys40(AhxHYNIC-[99mTc]EDDA)NH2]-exendin-4 was found in all patients, leading to successful removal of the offending lesion and complete biochemical and symptomatic resolution. Histological analysis confirmed insulinoma in all included patients. CONCLUSIONS: [Lys40(AhxHYNIC-[99mTc]EDDA)NH2]-exendin-4 SPECT/CT appears to be an excellent molecular imaging method for preoperative localization of an occult insulinoma, surpassing conventional imaging methods. If routinely available, it could be considered as a method of choice due to its favorable combination of imaging characteristics.

99mTc-Labeled Native RBC Scintigraphy in Distinguishing Polysplenia From Abdominal Masses in a Patient With Situs Inversus Totalis.

A 39-year-old previously healthy woman presented possible hematuria. An ultrasound examination showed right adrenal mass and suggested pheochromocytoma. A Tc-HYNIC-TOC SPECT/CT was performed, which incidentally detected situs inversus totalis and suspicion of polysplenia without definite normal spleen in the right upper abdomen. In order to differentiate the polysplenia from other etiologies, a heat-damaged Tc RBC scintigraphy was performed. The images showed significant activity in the multiple soft tissue nodules in the posterior right abdomen, consistent with ectopic polysplenia.

SPECT/CT imaging of apoptosis in aortic aneurysm with radiolabeled duramycin.

The objective of this research was to estimate whether a [99mTc]duramycin probe can be used for apoptosis imaging in patients with aortic aneurysm (AA). Vascular smooth muscle cell (SMC) apoptosis has an important influence on AA development. Thus, non-invasive imaging of SMC apoptosis may be able to evaluate AA progress and risk stratification. SMCs were treated with hydrogen peroxide (H2O2; 200 µΜ) or culture medium as a control. Apoptosis was measured using flow cytometry and [99mTc]duramycin to detect the binding efficiency to apoptotic SMCs. C57/BL6 mice were administered angiotensin-II and beta-aminopropionitrile (BAPN) subcutaneously to establish an AA model, or saline for controls. Aortic specimens underwent pathological evaluation and their aortic diameters were measured after 6 weeks. Micro-SPECT/CT scanning of [99mTc]duramycin and 18F-FDG PET detection were performed. SMCs treated with H2O2 showed more apoptosis compared with the control group (67.2 ± 3.8% vs. 16.1 ± 0.6%, P < 0.01). The experimental group showed a high rate of AA formation (70%) compared with no AA formation in the control group. The average aorta diameter was higher and [99mTc]duramycin uptake at the AA site was higher in the experimental group compared with the control group. Compared with the normal aorta in the control group, AA in experiment group had more severe medial degeneration, elastic fiber reduction and fracture, and collagen degeneration. TUNEL staining verified the higher apoptosis rate at the AA site in experiment group compared with the control group (63.9 ± 3.7% in ascending AA, 66.4 ± 4.0% in thoracic AA, vs. 3.5 ± 0.3% in normal aorta, P < 0.01). [99mTc]Duramycin may be an effective probe to evaluate apoptosis in AA.

Non-cardiac uptake of technetium-99m pyrophosphate in transthyretin cardiac amyloidosis.

BACKGROUND: Technetium-based bone scintigraphy is rapidly becoming the most common non-invasive imaging tool in the diagnosis of Transthyretin cardiac amyloidosis (ATTR). Skeletal muscle uptake has been described with technetium-99m-3,3-diphosphono-1,2-propanodicarboxylic acid (TcDPD), and may account for masking of bony uptake. We sought to investigate skeletal muscle uptake of technetium-99m-pyrophosphate (TcPYP) in patients with ATTR. METHODS AND RESULTS: This was a retrospective analysis of 57 patients diagnosed with ATTR who underwent TcPYP scintigraphy. Cardiac uptake was assessed on whole-body planar imaging using a semiquantitative scale (grades 0 to 3) and on single-photon emission computed tomography (SPECT) with CT attenuation correction using total myocardial counts per voxel after a 3-hour incubation. Skeletal muscle (psoas and biceps), vertebral body, LV myocardium, and blood pool mean counts were calculated. In the cohort (age 78 ± 9 years, 77% male, and 30% hereditary ATTR), there was no visualized tracer uptake in skeletal muscle or soft tissue on qualitative SPECT assessment. Total and blood pool-corrected uptake in the muscle groups were significantly less than myocardium and bone (P < 0.001). Blood pool-corrected muscle uptake was not associated with semiquantitative grade 3 vs 2 uptake (psoas P = 0.66, biceps P = 0.13) or presence of hereditary ATTR (psoas P = 0.43, biceps P = 0.69). As bony uptake decreased, there was no corresponding increase in skeletal muscle uptake. CONCLUSIONS: In patients with ATTR cardiac amyloidosis, skeletal muscle uptake of TcPYP is minimal when assessed by qualitative and quantitative metrics, and is not significantly different in patients with grade 2 vs 3 semiquantitative uptake. The properties of this tracer may be different than TcDPD with respect to non-cardiac uptake.

Preferential Cleavage of a Tripeptide Linkage by Enzymes on Renal Brush Border Membrane To Reduce Renal Radioactivity Levels of Radiolabeled Antibody Fragments.

The obstructive renal radioactivity after injection of antibody fragments/constructs labeled with metallic radionuclides would be improved by liberating a radiometal chelate of urinary excretion from the antibody molecules by enzymes on the renal brush border membrane (BBM). A tripeptide GFK sequence was newly evaluated as an enzyme-cleavable linkage and conjugated to a 99mTc chelate of an isonicotinic acid derivative of 2-picolylglycine (99mTc-IPG). 99mTc-IPG-glycine was liberated from 99mTc-IPG-GFK by the enzymes, while 99mTc-IPG-GK (where the tripeptide GFK was substituted with a dipeptide GK) did not. When injected into mice, 99mTc-IPG-GFK-conjugated Fab exhibited lower renal radioactivity levels than directly radioiodinated Fab shortly after injection without reducing the tumor radioactivity levels, due to a release and excretion of 99mTc-IPG-glycine by enzymes present on the renal BBM. These findings would provide insights to develop antibody fragments/constructs labeled with metallic radionuclides of the clinical relevance for improved renal radioactivity levels.

Lung uptake during 99mTc-hydroxymethylene diphosphonate scintigraphy in patient with TTR cardiac amyloidosis: An underestimated phenomenon.

BACKGROUND: Full body scintigraphy using bone tracers plays an important role in defining the type of amyloidosis and in diagnosing the heart involvement (cardiac amyloidosis, CA). No study has been conducted to explore lung retention (LR) in CA and its correlation to heart retention (HR).We evaluated LR in patients undergoing 99mTc-HMDP scintigraphy during evaluation for suspected CA. METHODS AND RESULTS: We enrolled 93 suspected CA patients. Patients underwent a complete diagnostic work up. After diagnostic process 82 patients resulted affected by certain CA (20 AL and 62 TTR), while 11 subjects showed left ventricular hypertrophy (LVH) not caused by CA. 99mTc-HMDP cardiac uptake was evaluated using the Perugini visual score while the modified Janssen score was used for LR estimation (grade 0 no uptake, grade 1 less than ribs, grade 2 more than ribs). RESULTS: 99mTc-HMDP LR was observed in 1/20 AL patient (5%), while 36/62 (58%) TTR patients showed LR with 29 grade 1 (47%) and 7 grade 2 (11%). No LR was observed in patients with LVH and no CA. LR was not evident in patients without HR, present in 1/3 (33%) of the patients with Perugini 1 HR and 11/24 (46%) and 26/36 (72%) of the patients showing respectively a Perugini 2 and a Perugini 3. CONCLUSION: 99mTc-HMDP scintigraphy shows LR in about 60% of TTR subjects, related to the grade of HR. In AL amyloidosis LR is less frequent than in TTR amyloidosis suggesting an aetiological tropism that seems comparable to the already known TTR related cardiac tropism.

Coordination-Mediated Synthesis of Purification-Free Bivalent 99mTc-Labeled Probes for in Vivo Imaging of Saturable System.

In the synthesis of technetium-99m (99mTc) labeled target-specific ligands, the presence of a large excess of unlabeled ligands over 99mTc in the injectate hinders target accumulation of 99mTc-labeled ligands by competing for target molecules. To circumvent the problem, we recently developed a concept of the metal coordination-mediated multivalency, and proved the concept with a 99mTc-labeled trivalent compound [99mTc(CO)3(CN-RGD)3]+. In this study, D-penicillamine (Pen) was selected as a chelating molecule and a cyclic RGDfK peptide was conjugated to Pen via a hexanoic linkage (Pen-Ahx-c(RGDfK)). 99mTc complexation reaction, and the stability, integrin αvß3 binding affinity, and biodistribution of the 99mTc-labeled probe were investigated to evaluate the applicability of the concept to bivalent probes. 99mTc-[Pen-Ahx-c(RGDfK)]2 was obtained over 95% radiochemical yields under low Pen-Ahx-c(RGDfK) concentration (50 µM). 99mTc-[Pen-Ahx-c(RGDfK)]2 showed approximately 10-times higher integrin αvß3 binding affinity than the monovalent compounds, Pen-Ahx-c(RGDfK) and c(RGDyV). In biodistribution studies, the tumor accumulation of 99mTc-[Pen-Ahx-c(RGDfK)]2 was decreased to 77% and 43% of HPLC-purified (Pen-Ahx-c(RGDfK)-free) 99mTc-[Pen-Ahx-c(RGDfK)]2 by the presence of 5 nmol of unlabeled Pen-Ahx-c(RGDfK) and Re-[Pen-Ahx-c(RGDfK)]2, respectively. 99mTc-[Pen-Ahx-c(RGDfK)]2 provided tumor image without removing unlabeled ligand, while a 99mTc-labeled monovalent probe prepared from a monovalent ligand could not. These findings indicate the availability of the design concept to prepare 99mTc-labeled bivalent probes with a variety of 99mTc core and other metallic radionuclides of clinical relevance.

Technetium-99m complexes of l-arginine derivatives for targeting amino acid transporters.

Although relevant from the clinical point of view, radiotracers targeting cationic amino acid transporters are relatively unexplored and, in particular, no metal-based radiotracers are known. The rare examples of complexes recognized by amino acid transporters, namely by the Na+-independent neutral l-type amino acid transporter 1 (LAT1), are 99mTc(i)/Re(i) compounds. Herein, we describe conjugates comprising a pyrazolyl-diamine chelating unit and the cationic amino acid l-arginine (l-Arg) linked by a propyl (L1) or hexyl linker (L2), which allowed the preparation of stable complexes of the type fac-[99mTc(CO)3(k3-L)]+ (Tc1, L = L1; Tc2, L = L2) and of the respective surrogates Re1 and Re2. Interestingly, complex Tc2 exhibited moderate levels of time-dependent internalization in three human tumoural cell lines, with approximately 3% of total applied activity internalized, corresponding to 21% of the cell-associated activity. A putative mechanism of retention in the cytoplasm of cells could be the interaction of the complex with inducible nitric oxide synthase (iNOS), which is the enzyme responsible for the catalytic oxidation of l-Arg to citrulline and nitric oxide. However, the surrogate complex Re2 does not recognize iNOS, as demonstrated by the in vitro assays with purified iNOS and in studies with lipopolysaccharide(LPS)-activated macrophages. Preliminary mechanistic studies suggest that the internalization of Tc2 is linked to the cationic amino acid transporters, namely system y+. This finding might open the way towards the development of novel families of metal-based radiotracers for probing metabolically active cancer cells.

Immunoglobulin G (IgG)-Based Imaging Probe Accumulates in M1 Macrophage-Infiltrated Atherosclerotic Plaques Independent of IgG Target Molecule Expression.

PURPOSE: Vulnerable plaques are key factors for ischemic diseases. Thus, their precise detection is necessary for the diagnosis of such diseases. Immunoglobulin G (IgG)-based imaging probes have been developed for imaging biomolecules related to plaque formation for the diagnosis of atherosclerosis. However, IgG accumulates nonspecifically in atherosclerotic regions, and its accumulation mechanisms have not yet been clarified in detail. Therefore, we explored IgG accumulation mechanisms in atherosclerotic lesions and examined images of radiolabeled IgG for the diagnosis of atherosclerosis. PROCEDURES: Mouse IgG without specificity to biomolecules was labeled with technetium-99m via 6-hydrazinonicotinate to yield [99mTc]IgG. ApoE-/- or C57BL/6J mice were injected intravenously with [99mTc]IgG, and their aortas were excised 24 h after injection. After radioactivity measurement, serial aortic sections were autoradiographically and histopathologically examined. RAW264.7 macrophages were polarized into M1 or M2 and then treated with [99mTc]IgG. The radioactivities in the cells were measured after 1 h of incubation. [99mTc]IgG uptake in M1 macrophages was also evaluated after the pretreatment with an anti-Fcγ receptor (FcγR) antibody. The expression levels of FcγRs in the cells were measured by western blot analysis. RESULTS: [99mTc]IgG accumulation levels in the aortas were significantly higher in apoE-/- mice than in C57BL/6J mice (5.1 ± 1.4 vs 2.8 ± 0.5 %ID/g, p < 0.05). Autoradiographic images showed that the accumulation areas highly correlated with the macrophage-infiltrated areas. M1 macrophages showed significantly higher levels of [99mTc]IgG than M2 or M0 (nonpolarized) macrophages [2.2 ± 0.3 (M1) vs 0.5 ± 0.1 (M2), 0.4 ± 0.1 (M0) %dose/mg protein, p < 0.01] and higher expression levels of FcγRI and FcγRII. [99mTc]IgG accumulation in M1 macrophages was suppressed by pretreatment with the anti-FcγR antibody [2.2 ± 0.3 (nonpretreatment) vs 1.2 ± 0.2 (pretreatment) %ID/mg protein, p < 0.01]. CONCLUSIONS: IgG accumulated in pro-inflammatory M1 macrophages via FcγRs in atherosclerotic lesions. Thus, the target biomolecule-independent imaging of active inflammation should be taken into account in the diagnosis of atherosclerosis using IgG-based probes.

Novel 99mTc-Radiolabeled Anionic Linear Globular PEG-Based Dendrimer-Chlorambucil: Non-Invasive Method for In-Vivo Biodistribution.

Chlorambucil (CBL) is an alkylating agent, which widely use in the treatment of various types of tumors. The main purpose of this study is to evaluate the in-vivo biodistribution of CBL conjugated to the anionic dendrimer, which has a great ability to labeled with 99mTc through binding to carboxylate terminate groups. Whole body scans were used to analyze the percentage of the injected dose in different times. Radiochemical purity (RCP) and in-vivo biodistribution were also calculated with the SPECT/CT instrument. Our study proposes a new method for RCP determination and shows that this carrier is a promising agent to complex with the 99mTc and biological assessment.

SPECT-CT Comparison of Lung Deposition using a System combining a Vibrating-mesh Nebulizer with a Valved Holding Chamber and a Conventional Jet Nebulizer: a Randomized Cross-over Study.

PURPOSE: To compare in vivo the total and regional pulmonary deposition of aerosol particles generated by a new system combining a vibrating-mesh nebulizer with a specific valved holding chamber and constant-output jet nebulizer connected to a corrugated tube. METHODS: Cross-over study comparing aerosol delivery to the lungs using two nebulizers in 6 healthy male subjects: a vibrating-mesh nebulizer combined with a valved holding chamber (Aerogen Ultra®, Aerogen Ltd., Galway, Ireland) and a jet nebulizer connected to a corrugated tube (Opti-Mist Plus Nebulizer®, ConvaTec, Bridgewater, NJ). Nebulizers were filled with diethylenetriaminepentaacetic acid labelled with technetium-99 m (99mTc-DTPA, 2 mCi/4 mL). Pulmonary deposition of 99mTc-DTPA was measured by single-photon emission computed tomography combined with a low dose CT-scan (SPECT-CT). RESULTS: Pulmonary aerosol deposition from SPECT-CT analysis was six times increased with the vibrating-mesh nebulizer as compared to the jet nebulizer (34.1 ± 6.0% versus 5.2 ± 1.1%, p < 0.001). However, aerosol penetration expressed as the three-dimensional normalized ratio of the outer and the inner regions of the lungs was similar between both nebulizers. CONCLUSIONS: This study demonstrated the high superiority of the new system combining a vibrating-mesh nebulizer with a valved holding chamber to deliver nebulized particles into the lungs as comparted to a constant-output jet nebulizer with a corrugated tube.

The Use of Technetium-99m for Intravital Tracing of Transplanted Multipotent Stromal Cells.

We studied the possibility of in vivo tracing of multipotent mesenchymal stromal cells labeled with a radiophermaceutic preparation based on metastable isotope Technetium-99m and injected to rats with modeled traumatic brain injury. Accumulation of labeled cells occurred primarily in the liver and lungs. The cells distribution in internal organs greatly varied depending on the administration route. Cell injection into the carotid artery led to their significant accumulation in the damaged brain hemisphere, while intravenous injection was followed by diffuse cell distribution in all brain structures. Scintigraphy data were confirmed by magnetic resonance imaging and histological staining of cells. Visualization of stem cells labeled with Technetium-99m-based preparation by scintigraphy is an objective and highly informative method allowing real-time in vivo cell tracing in the body.

Feasibility of imaging of epidermal growth factor receptor expression with ZEGFR:2377 affibody molecule labeled with 99mTc using a peptide-based cysteine-containing chelator.

The epidermal growth factor receptor (EGFR) is overexpressed in a number of malignant tumors and is a molecular target for several specific anticancer antibodies and tyrosine kinase inhibitors. The overexpression of EGFR is a predictive biomarker for response to several therapy regimens. Radionuclide molecular imaging might enable detection of EGFR overexpression by a non-invasive procedure and could be used repeatedly. Affibody molecules are engineered scaffold proteins, which could be selected to have a high affinity and selectivity to predetermined targets. The anti-EGFR ZEGFR:2377 affibody molecule is a potential imaging probe for EGFR detection. The use of the generator-produced radionuclide 99mTc should facilitate clinical translation of an imaging probe due to its low price, availability and favorable dosimetry of the radionuclide. In the present study, we evaluated feasibility of ZEGFR:2377 labeling with 99mTc using a peptide-based cysteine-containing chelator expressed at the C-terminus of ZEGFR:2377. The label was stable in vitro under cysteine challenge. In addition, 99mTc-ZEGFR:2377 was capable of specific binding to EGFR-expressing cells with high affinity (274 pM). Studies in BALB/C nu/nu mice bearing A431 xenografts demonstrated that 99mTc-ZEGFR:2377 accumulates in tumors in an EGFR-specific manner. The tumor uptake values were 3.6±1 and 2.5±0.4% ID/g at 3 and 24 h after injection, respectively. The corresponding tumor-to-blood ratios were 1.8±0.4 and 8±3. The xenografts were clearly visualized at both time-points. This study demonstrated the potential of 99mTc-labeled ZEGFR:2377 for imaging of EGFR in vivo.