Preparation and biodistribution in mice of a new radiopharmaceutical-technetium-99m labeled methotrexate, as a tumor diagnostic agent

Our aim was to develop the procedure for radiolabeling of an anticancer drug e.g., methotrexate with (99m)Tc for tumors diagnosis. The study included the radiolabeling of methotrexate, in vitro stability of radiolabeled drug, in vitro binding of radiolabeled drug with plasma protein, partition coefficient and biodistribution of radiolabeled drug in mice. Results showed 98.2±0.5% radiolabeling of methotrexate with technetium-99m ((99m)Tc). In vitro stability was studied for 5h and 79.3±5% of the drug was bound with plasma proteins. Partition coefficient of the labeled drug showed that it was highly hydrophilic. Biodistribution study in tumor bearing mice exhibited high uptake in tumor cells which were further investigated by histopathological studies. In conclusion, our study indicates that technetium-99m labeled methotrexate is a potentially strong tumor diagnostic agent with low uptake in normal tissues.     

Difference in inhaled aerosol deposition patterns in the lungs due to three different sized aerosols.

Deposition patterns of inhaled aerosol in the lungs were studied in five normal subjects and 20 patients with lung disease by inhaling radioaerosols with three different particle size distributions. These aerosols were generated from BARC, UltraVent, and Mistogen-EN-142. Particle size distributions generated by these three nebulizers were 0.84, 1.04 and 1.93 microns in activity median aerodynamic diameter (AMAD) with its geometric standard deviation (sigma g) of 1.73, 1.71 and 1.52, respectively. Deposition patterns of inhaled aerosols were compared qualitatively and quantitatively by studying six different parameters: alveolar deposition ratio (ALDR), Xmax, Xmean, standard deviation (S.D.), skewness and kurtosis of the radioactive distribution in the lungs following inhalation. It has been found that aerosol deposition patterns varied with particle size. The unevenness of aerosol deposition, Xmax, Xmean and the number of 'hot spots' became more prominent with the increase in particle size, whereas values of ALDR and S.D. decreased as particle size increased. Knowing these deposition characteristics would facilitate a judicious application of aerosol inhalation to medical use.     

Beta-agonist aerosol distribution in respiratory syncytial virus bronchiolitis in infants.

Bronchodilator aerosols are frequently administered to infants with bronchiolitis but with little success. The efficacy of aerosol treatments depends mainly on adequate targeting of the aerosol particles to the inflamed airways. This study evaluated the lower respiratory tract distribution characteristics of nebulized bronchodilators in infants with acute bronchiolitis. METHODS: Twelve infants (mean age +/- SD, 8 mo +/- 4 mo) who were admitted for acute respiratory syncytial virus bronchiolitis were treated with (99m)Tc-albuterol aerosol. Gamma-scintigraphy was used to assess total body and lung deposition as well as pulmonary distribution of the medication. RESULTS: Of the total 6-min nebulized dose (i.e., drug aerosol dose leaving the nebulizer [not the nebulizer charge]), 1.5% +/- 0.7% reached the right lung, with only approximately one third of that (0.6%) penetrating to the peripheral lung zone. There was 7.8% +/- 4.9% deposition in the upper respiratory and gastrointestinal tracts and 10%-12% remained on the face. No correlation was found between any of the deposition indices and the clinical response data or any of the demographic parameters (e.g., height, weight, body surface area, or clinical score). CONCLUSION: Poor total aerosol deposition in infants may be related as much to their small conducting airways as to the disease state. There is considerable room for improvement in aerosol delivery in this age group, with greater emphasis on targeting narrowed peripheral airways with superfine aerosols.     

某铜矿山巷道内气溶胶浓度与粒径分布的测量和分析

目的 定性了解矿山巷道内气溶胶的浓度和粒径分布特性。方法 在巷道内的不同区间,分别用凝结核颗粒计数器和个人气溶胶测量仪巡测气溶胶的粒子数和质量浓度,并通过质量浓度的分级测量定性评价微米级气溶胶的粒径分布;在调度室内外,用金属丝网筛扩散法测量亚微米级气溶胶的粒径分布。结果 巷道内可吸入颗粒物(PM10)的平均质量浓度为0.42 mg/m³,其量值大小因工作断面而异,且受人工活动影响变化较大;巷道内粒径大于1.0 μm的颗粒物广泛存在,而粒子直径小于5 nm的气溶胶基本上未被检出。结论 矿山巷道内气溶胶特性因工作断面、人工活动和通风条件的不同而变化明显,在开展内照射剂量评价时应考虑粒径大于1.0 μm放射性气溶胶粒子的剂量贡献。     

Urine excretion of inhaled technetium-99m-DTPA: an alternative method to assess lung epithelial transport

Technetium-99m DTPA clearance (99mTc-DTPA) clearance measured by a gamma camera or a scintillation probe not only reflects epithelial transport, but is also influenced by an unknown amount of mucociliary clearance depending on particle size and aerosol deposition. This is confirmed by factor analysis of dynamic inhalation studies. Assessment of epithelial absorption by urinary excretion of inhaled 99mTc-DTPA is largely independent of aerosol lung deposition. Twenty-four-hour excretion reflects the amount of aerosol cleared by absorption, while two-hour excretion is a quantitative measure of the aerosol absorption rate from the epithelium into blood. Urinary 99mTc-DTPA excretion of two aerosols with different particle size correlated significantly (p less than 0.001) with analysis of lung clearance curves. A very similar regression in the form of a cumulative exponential function was found with both aerosols. Two-hour urine values of nonsmokers differed significantly from those of smokers or patients with active interstitial or infectious lung disease. This alternative procedure is suited as a bedside test and holds promise for patient monitoring and follow-up.     

气溶胶沉降模拟方法研究进展

对吸入颗粒物致病作用的评估,需要了解气溶胶颗粒在呼吸道中的沉积特征。由于气溶胶进入呼吸道引、发的呼吸道疾病致病机锏研究和临床药物气藩胶吸入治疗的需要,人们越发关注如气溶胶颗粒在呼吸道特定区域滞留率,研究气溶胶粒子在呼吸道中分布和沉降比例的关系。这些都需要借助沉降模型来模拟颗粒的区域沉积特征。沉降模型经过半个多世纪的发展,已经形成了成熟的分析模型。计算机流体动力学（CFD）模拟技术的出现,让沉降模拟进入了一个崭新的时代。该文综述了目前研究气溶胶沉降的主要方法和肺模型的发展趋势。     

Pyrazolyl conjugates of bombesin: a new tridentate ligand framework for the stabilization of fac-[M(CO)3]+ moiety

We have described the synthesis of tridentate pyrazolyl ligand frameworks for coordination to the fac-[*M(CO)(3)](+) metal fragment (*M=(186/188)Re or (99m)Tc). These ligands impart a degree of kinetic inertness on the metal center, warranting their study in biological systems. We herein report in vitro/in vivo radiolabeling investigations of a new series of pyrazolyl bombesin (BBN) conjugates radiolabeled via the Isolink kit. These new conjugates are based on the general structure [(99m)Tc-pyrazolyl-X-BBN[7-14]NH(2)], where X=beta-alanine, serylserylserine or glycylglycylglycine. The pyrazolyl ligand is a tridentate ligand framework that coordinates the metal center through nitrogen donor atoms. The results of these investigations demonstrate the ability of these new conjugates to specifically target the gastrin-releasing peptide receptor subtype 2, which is overexpressed on human prostate PC-3 cancerous tissues. Therefore, these studies suggest the tridentate pyrazolyl ligand framework to be an ideal candidate for the design and development of low-valent (99m)Tc-based diagnostic radiopharmaceuticals based on BBN or other targeting vectors.     

Construction of a novel chimera consisting of a chelator-containing Tat peptide conjugated to a morpholino antisense oligomer for technetium-99m labeling and accelerating cellular kinetics

The attempt to target the limited copies of messenger RNA (mRNA) in vivo with radiolabeled nucleobase oligomers as antisense probes is challenging. Selecting an antisense molecule with superior properties, enhancing the cellular kinetics, and improving the radiolabeling chemistry would be the reasonable approach to accomplish this goal. The present study reports a method to construct a chimera of phosphorodiamidate morpholino nucleobase oligomer (MORF) covalently conjugated to a peptide containing a cell membrane transduction Tat peptide and an N(2)S(2) chelator for technetium-99m ((99m)Tc) radiolabeling (N(2)S(2)-Tat-MORF). The radiolabeling properties and cellular kinetics of (99m)Tc-N(2)S(2)-Tat-MORF were measured. As hypothesized, the preparation of (99m)Tc-N(2)S(2)-Tat-MORF could be achieved by an instant one-step method with labeling efficiency greater than 95%, and the (99m)Tc-N(2)S(2)-Tat-MORF showed distinct properties in cell culture from those of a control, the same MORF sequence without Tat but with mercaptoacetyltriglycine (MAG(3)) as chelator for (99m)Tc ((99m)Tc-MAG(3)-MORF). (99m)Tc-N(2)S(2)-Tat-MORF achieved maximum accumulation of about 35% within 2 h, while (99m)Tc-MAG(3)-MORF showed lower and steadily increasing accumulations but of less than 1% in 24 h. These preliminary results demonstrated that the proposed chimera has properties for easy labeling, and (99m)Tc-N(2)S(2)-Tat-MORF prepared by this method possesses enhanced cellular kinetics and merits further investigation for in vivo mRNA targeting.     

Imaging with radiolabeled antisence oligonucleotides for the detection of intracellular messenger RNA and cardiovascular disease

Conclusions  Most of the research efforts in nuclear medicine in the last two decades have focused on the development of radioactive ligands for the detection of extracellular antigens expressed on the membrane surface such as tumor markers, somatostatin receptors on tumor cells, or receptors that are more readily accessible than cytosolic or nuclear receptors. However, these general concepts of radiolabeling methods for these ligands have helped us significantly to embark on the next phase of development of gamma-emitting singlestranted ASON probes for studying gene activation. Previous studies have demonstrated the nuclease-sensitivity of ASON probes. Further modifications of phosphodiester backbone, ribose, and nucleotide subunits have identified derivatives with improved potency, desirable pharmacokinetics, and reduced toxicity. Encouraged by in vitro and in vivo inhibition studies, the oligonucleotides have been radiolabeled with nonmetallic and metallic radionuclides of I-123, In-111, and Tc-99m and demonstrated their use in imaging specific mRNA of oncogenes, c-myc, erb-b2, and telomerase present in breast tumors in the mouse model. Noninvasive imaging of mRNA of heat shock protein (HSP-70) has been shown in a pig model during an ischemic episode of cardiopulmonary bypass. The radiochemists have developed ASON probes for positron emission tomography imaging labeled whereby they were with carbon 11 and fluorine 18. The radiolabeled antisense probes may provide a novel method to image the amplified oncogenes in atherosclerotic plaques and heat shock genes in myocardial stress. The standard methods of radiolabeling and novel methods of charge neutralization may enhance the intracellular delivery for imaging a variety of activated genes in cardiovascular diseases. Development of radiolabeled probes and their applications indiagnostic imaging were funded by the University of Illinois Research Board (UI-1-2-69410     

Preparation and radiolabeling of human serum albumin (HSA)-coated magnetite nanoparticles for magnetically targeted therapy

In this paper, we describe the preparation of human serum albumin-coated magnetic particles of about 200 nm in diameter with narrow size distribution radiolabeled with ¹⁸⁸Re for the purpose of magnetically targeted therapy. The optimum radiolabeling conditions are: SnCl₂·2H₂O 8 mg/ml, citric acid 20 mg/ml, vitamin C 8 mg/ml, labeling volume 500 μl and a reaction time of 3 h. The stability of the radiolabeled particles is suitable for in vivo study.     

99mTc(CO)3-VIP analogues: preparation and evaluation as tumor imaging agent.

Vasoactive intestinal peptide (VIP) receptors are expressed abundantly on many types of tumors and, hence, radiolabeled VIP analogues are being explored for tumor imaging and therapy. Here, we report synthesis of three VIP analogues and their radiolabeling with (99m)Tc via a novel tricarbonyl synthon. The radiolabeled product could be prepared in high yields (>95%) and stability. In vitro studies showed significant uptake of (99m)Tc(CO)((3))-VP05 in human colon carcinoma cells. Biodistribution studies in animal tumor model showed 0.4-1%ID/g tumor uptake.     

Effects of temperature on radiochemical purity and immunoreactivity of radiolabeled monoclonal antibody 1H10

OBJECTIVE: This study was undertaken to investigate the effects of temperature on preserving the radiochemical purity and immunoreactivity of 125I- and 131I-labeled monoclonal antibody (MAb) 1H10--an antibody against human cervical carcinoma cell-surface antigen. METHODS: An antibody-irrelevant human melanoma cell line, H2269, served as the control group. Iodine-125 and 131I radiolabeling of MAbs 1H10 and H2669 was performed by the chloramine-T method. All the prepared MAbs were divided into aliquots and stored at 4, -20, and -70 degrees C for 2-14 d. The radiochemical purity and immunoreactivity of the labeled antibodies in set conditions were measured by thin-layer chromatography and a modified index, respectively, after a single freeze-and-thaw cycle. RESULTS: Reduced release of free radioiodide and better preservation of immunoreactivity were observed in the radiolabeled MAbs stored at -70 degrees C than in those stored at -20 degrees C or 4 degrees C. The extent of free iodide dissociation and immunologic binding degradation of 125I-labeled MAb 1H10 appeared milder than that of 131I-labeled MAb under the same conditions. However, both 125I- and 131I-labeled MAb stored at -70 degrees C or -20 degrees C retained more than 90% radiochemical purity for at least 3d. CONCLUSION: Freezing provides an appropriate alternative for reducing radiolysis and preserving immunoreactivity of radioiodinated MAbs. MAb 1H10, labeled with either 125I or 131I and stored at temperatures of -20 degrees C or below for 3 d after labeling, appeared stable in both radiolabeling and binding studies in vitro and was still acceptable for in vivo use.     

Lung ventilation studies with technetium-99m Pseudogas

Technetium-99m Pseudogas is an ultrafine near monodisperse aerosol of 0.12-microgram diam particle size. This report describes initial clinical experiences with 27 patients referred for investigation of suspected pulmonary embolism, and in whom Pseudogas ventilation images were compared with a high quality commercial aerosol. An additional group of ten patients with severe COPD was examined in a comparative trial of Pseudogas with 81mKr. Pseudogas was better than a conventional aerosol in reaching a diagnosis of pulmonary embolism using a simple blinded comparison with coded images. In addition, bronchial deposition was minimal unless COPD was severe. Moderately well patients had no difficulty inhaling the necessary activity in one or two breaths, and even severely ill and frail aged persons could accomplish the passive breathing maneuver in less than a minute. Clearance of Pseudogas was directly to the systemic circulation with a half-time of 10 min in normal subjects extending up to 100 min in patients with airways disease.     

Corona ions from powerlines and increased exposure to pollutant aerosols

PURPOSE: To investigate corona ions emitted from high-voltage powerlines and assess their effects on exposure to environmental pollutant aerosols. MATERIALS AND METHODS: The charge density of ions in air required to produce a given change in the observed DC field at ground level was calculated. A DC field mill meter was used to map the DC fields near 132, 275 and 400 kV powerlines. RESULTS: The Earth's natural DC field of around approximately 100 Vm(-1) was significantly modified near powerlines in 8 out of 14 cases. Typically, downwind of the powerlines the field direction was reversed giving values to -340 V m(-1). In one case, the effect extended more than 500 m from a 275 kV line. DISCUSSION: These results can be analysed in terms of the charge density present. This analysis suggests that typically 2000 excess negative charges per cm3 are required to match the measured DC fields. Such space charge will result in unipolar aerosol charging in excess of the normal bipolar steady state charge distribution of pollutant aerosols. This may lead to increased lung deposition on inhalation.     

Increased exposure to pollutant aerosols under high voltage power lines

PURPOSE: To assess increased exposure to airborne pollutants near power lines by investigating theoretically and experimentally the behaviour of 222Rn decay product marker aerosols in the 50 Hz electric field under power lines. MATERIALS AND METHODS: The behaviour of aerosols in outdoor air including those carrying 222Rn decay products was modelled theoretically in the presence of an AC field. TASTRAK alpha-particle spectroscopy was used to characterize 218Po and 214Po aerosols outdoors. Sampling points were chosen along a line at right angles up to 200 m from a number of high voltage power (transmission) lines. Each sampling point comprised an arrangement of mutually orthogonal TASTRAK detectors. Exposures were carried out at different power line locations in various weather conditions. RESULTS: The model predicts a two- to three-fold increase in deposition of aerosols on spherical surfaces mimicking the human head under high voltage power lines. Experimental measurements using detectors mounted on grounded metal spheres showed an enhanced deposition of both 218Po and 214Po aerosols. Enhanced 218Po deposition on 400 kV lines ranged from 1.96+/-0.15 to 2.86+/-0.32. Enhanced 214Po deposition on 275 kV and 132 kV lines were 1.43+/-0.07 and 1.11+/-0.21, respectively, where the latter value was not significant. CONCLUSIONS: The observations demonstrate a mode of increased exposure to pollutant aerosols under high voltage power lines by increased deposition on the body. The total (indoor + outdoor) 218Po and 214Po dose to the basal layer of facial skin is estimated to be increased by between 1.2 and 2.0 for 10% of time spent outdoors under high voltage power lines.     

Reduction of background activity through radiolabeling of antifibrin Fab' with 99mTc-dextran.

Scintigraphic detection of occult disease is limited by background activity in the blood and in the extravascular space that reduces target-specific contrast. To lower nonspecific background activity, we have studied the in vivo biodistribution kinetics of a clot-targeting molecule (MH1 Fab') attached to (99m)Tc-dextran. We tested the hypothesis that the complex will have better background clearance than the directly radiolabeled clot-targeting molecule. METHODS: Fab' fragments of MH1 Fab' antifibrin antibody were coupled to (99m)Tc-sulfhydryl dextran through disulfide exchange, and clot binding bioreactivity was tested in vitro and in vivo in a rabbit jugular vein thrombus model. To assess the background clearance kinetics and extravascular leakage, we studied (99m)Tc-dextran, (99m)Tc-MH1 Fab', and the (99m)Tc-dextran-labeled MH1 Fab' complexes in rats. RESULTS: (99m)Tc-radiolabeled dextran derivatives were radiochemically stable and retained clot-binding bioreactivity in vivo. In the rat model, blood and tissue clearance of the (99m)Tc-dextran MH1 Fab' constructs was substantially improved relative to directly radiolabeled MH1 Fab'. At 1 h, total and extravascular tracer localizations in lung and muscle were significantly lower for 99mTc-dextranradiolabeled MH1 Fab' than for (99m)Tc-MH1 Fab' (P < 0.05). CONCLUSION: The study observations suggest that radiolabeling through a (99m)Tc-dextran moiety may improve the detection of pulmonary emboli and other clinically important fixed intravascular targets by lowering nonspecific background activity.     

99mTc(CO)3-DTMA bombesin conjugates having high affinity for the GRP receptor

INTRODUCTION: Targeted diagnosis of specific human cancer types continues to be of significant interest in nuclear medicine. 99mTc is ideally suited as a diagnostic radiometal for in vivo tumor targeting due to its ideal physical characteristics and diverse labeling chemistries in numerous oxidation states. METHODS: In this study, we report a synthetic approach toward design of a new tridentate amine ligand for the organometallic aqua-ion [99mTc(H2O)3(CO)3]+. The new chelating ligand framework, 2-(N,N'-Bis(tert-butoxycarbonyl)diethylenetriamine) acetic acid (DTMA), was synthesized from a diethylenetriamine precursor and fully characterized by mass spectrometry and nuclear magnetic resonance spectroscopy (1H and 13C). DTMA was conjugated to H2N-(X)-BBN(7-14)NH2, where X=an amino acid or aliphatic pharmacokinetic modifier and BBN=bombesin peptide, by means of solid phase peptide synthesis. DTMA-(X)-BBN(7-14)NH2 conjugates were purified by reversed-phase high-performance chromatography and characterized by electrospray-ionization mass spectrometry. RESULTS: The new conjugates were radiolabeled with [99mTc(H2O)3(CO)3]+ produced via Isolink radiolabeling kits to produce [99mTc(CO)3-DTMA-(X)-BBN(7-14)NH2]. Radiolabeled conjugates were purified by reversed-phase high-performance chromatography. Effective receptor binding behavior was evaluated in vitro and in vivo. CONCLUSIONS: [99mTc(CO)3-DTMA-(X)-BBN(7-14)NH2] conjugates displayed very high affinity for the gastrin releasing peptide receptor in vitro and in vivo. Therefore, these conjugates hold some propensity to be investigated as molecular imaging agents that specifically target human cancers uniquely expressing the gastrin releasing peptide receptor subtypes.     

Corona ions from powerlines and increased exposure to pollutant aerosols

Purpose: To investigate corona ions emitted from high-voltage powerlines and assess their effects on exposure to environmental pollutant aerosols. Materials and methods: The charge density of ions in air required to produce a given change in the observed DC field at ground level was calculated. A DC field mill meter was used to map the DC fields near 132, 275 and 400kV powerlines. Results: The Earth's natural DC field of around ~100Vm -1 was significantly modified near powerlines in 8 out of 14 cases. Typically, downwind of the powerlines the field direction was reversed giving values to -340Vm -1. In one case, the effect extended more than 500m from a 275kV line. Discussion: These results can be analysed in terms of the charge density present. This analysis suggests that typically 2000 excess negative charges per cm 3 are required to match the measured DC fields. Such space charge will result in unipolar aerosol charging in excess of the normal bipolar steady state charge distribution of pollutant aerosols. This may lead to increased lung deposition on inhalation.     

Radiolabeled bombesin analogs for prostate cancer diagnosis: preclinical studies

INTRODUCTION: Radionuclide imaging can be a useful tool for the diagnosis of prostate cancer. Bombesin (BBN) is a molecule with high affinity for gastrin releasing peptide (GRP) receptors which are over-expressed in that tumor. This report compares (99m)Tc-HYNIC-betaAla-BBN(7-14)NH2 [(99m)Tc-HYNIC-BBN] and (99m)Tc identical withN(PNP6)-Cys-betaAla-BBN(7-14)NH2 [(99m)TcN(PNP6)-Cys-BBN] with regard to labeling procedures as well as in vitro and in vivo evaluation (biodistribution and scintigraphic imaging). METHODS: Peptide synthesis was performed in an automated peptide synthesizer. HYNIC-BBN was radiolabeled with pertechnetate using tricine and ethylenediamine diacetic acid (EDDA) as coligands. Cys- BBN was radiolabeled in a two-step procedure with the preparation of the precursor (99m)Tc-Nitrido first and then introducing diphosphine (PNP6). Radiochemical evaluation of conjugates, as well as studies of stability, transchelation toward cysteine, and partition coefficient were done. Biological studies included internalization, biodistribution in healthy animals and in animals bearing PC3 cancer cells with acquisition of images from the tumor-bearing animals. RESULTS: Both complexes showed a high radiochemical yield along with good stability. Biodistribution studies pointed out strong renal excretion for the former complex due to its hydrophilic profile and marked hepatobiliary excretion for the latter, corresponding to observed lipophilicity. Tumor uptake was higher for (99m)Tc-HYNIC-BBN and the same occurred with internalization findings, which exceeded those of (99m)TcN(PNP6)-BBN. Blocking studies in mice bearing PC-3 tumor cells revealed significantly reduced pancreas and tumor uptake, demonstrating receptor specificity of the conjugates. CONCLUSION: The best radiotracer was (99m)Tc-HYNIC-BBN on the basis of high radiochemical yield, fast radiolabeling procedure without need for a purification step, and more consistent tumor uptake.     

Tumor imaging using a standardized radiolabeled adapter protein docked to vascular endothelial growth factor.

Direct radiolabeling of proteins can result in the loss of targeting activity, requires highly customized procedures, and yields heterogeneous products. Here we describe a novel imaging complex comprised of a standardized (99m)Tc-radiolabeled adapter protein noncovalently bound to a "Docking tag" fused to a "Targeting protein". The assembly of this complex is based on interactions between human 109-amino acid (HuS) and 15-amino acid (Hu-tag) fragments of ribonuclease I, which serve as an "Adapter protein" and a Docking tag, respectively. METHODS: HuS modified with hydrazinonicotinamide (HYNIC) was radiolabeled using (99m)Tc-tricine to a specific activity of 3.4-7.4 MBq/microg. Protein complexes were then formed by mixing (99m)Tc-HuS with equimolar amounts of either Hu-tagged VEGF(121) (Hu-VEGF [vascular endothelial growth factor]) or Hu-tagged anti-VEGFR-2 single-chain antibody (Hu-P4G7) and incubating on ice for 15 min. 4T1 luc/gfp luciferase-expressing murine mammary adenocarcinoma cells (1 x 10(4)) were implanted subcutaneously or injected intravenously into BALB/c mice. Bioluminescent imaging (BLI) was performed 10 d later. Immediately after BLI visualization of tumor, 18.5-37 MBq of tracer (5-10 microg of protein) were injected via tail vein. One hour later planar or SPECT images were obtained, followed by killing the mice. RESULTS: There was significantly (P = 0.0128) increased uptake of (99m)Tc-HuS/Hu-VEGF (n = 10) within subcutaneous tumor as compared with (99m)Tc-HuS/Hu-P4G7 (n = 5) at biodistribution assay (2.68 +/- 0.75 vs. 1.8 +/- 0.21; tumor-to-subcutaneous tissue [ratio of specific activities], respectively), despite similar molecular weights. The focal (99m)Tc-HuS/Hu-VEGF uptake seen on planar images (3.44 +/- 1.16 [tumor to soft-tissue background]) corresponded directly to the locations of tumor observed by BLI. Region of interest analyses of SPECT images revealed a significant increase of (99m)Tc-HuS/Hu-VEGF (n = 5) within the lungs with BLI-detectable pulmonary tumor nodules as compared with controls (n = 4) (right: 4.47 +/- 2.07 vs. 1.79 +/- 0.56; left: 3.66 +/- 1.65 vs. 1.62 +/- 0.45, tumor lung [counts/pixel]/normal lung [counts/pixel], respectively). CONCLUSION: (99m)Tc-HuS/Hu-VEGF complex is stable for at least 1 h in vivo and can be effectively used to image mouse tumor neovasculature in lesions as small as several millimeters in soft tissue. We expect that a similar approach can be adapted for in vivo delivery of other targeting proteins of interest without affecting their bioactivity.