A Receptor-targeted Fluorescent Radiopharmaceutical for Multireporter Sentinel Lymph Node Imaging

Purpose: To determine the imaging and receptor-binding properties of a multireporter probe designed for sentinel lymph node (SLN) mapping via nuclear and fluorescence detection. Materials and Methods: The animal experiments were approved by the institutional animal care and use committee. A multireporter probe was synthesized by covalently attaching cyanine 7 (Cy7), a near-infrared cyanine dye, to tilmanocept, a radiopharmaceutical that binds to a receptor specific to recticuloendothelial cells. In vitro binding assays of technetium 99m ((99m)Tc) -labeled Cy7 tilmanocept were conducted at 4°C by using receptor-bearing macrophages. Optical SLN imaging after foot pad administration was performed by using two molar doses of Cy7 tilmanocept. Six mice were injected with 0.11 nmol of (99m)Tc-labeled Cy7 tilmanocept (low-dose group); an additional six mice were injected with 31 nmol of (99m)Tc-labeled Cy7 tilmanocept (high-dose group) to saturate the receptor sites within the SLN. After 2.5 hours of imaging, the mice were euthanized, and the sentinel and distal lymph nodes were excised and assayed for radioactivity for calculation of SLN percentage of injected dose and extraction. Four mice were used as controls for autofluorescence. Standard optical imaging software was used to plot integrated fluorescence intensity against time for calculation of the SLN uptake rate constant and scaled peak intensity. Significance was calculated by using the Student t test. Results: In vitro binding assays showed subnanomolar affinity (mean dissociation constant, 0.25 nmol/L ± 0.10 [standard deviation]). Fluorescence imaging showed a detection sensitivity of 1.6 × 10(3) counts · sec(-1) · μW(-1) per picomole of Cy7. All four imaging metrics (percentage of injected dose, SLN extraction, SLN uptake rate constant, and expected peak fluorescence intensity) exhibited higher values (P = .005 to P = .042) in the low-dose group than in the high-dose group; this finding was consistent with receptor-mediated image formation. Conclusion: The multireporter probe (99m)Tc-labeled Cy7 tilmanocept exhibits in vitro and in vivo receptor-binding properties for successful receptor-targeted SLN mapping with nuclear and optical imaging. ? RSNA, 2012 Supplemental material: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.12120638/-/DC1.     

In vivo and in vitro evaluation of Cy5.5 conjugated epidermal growth factor receptor binding peptide

The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor and plays an important role in carcinogenesis. In this study, the epidermal growth factor receptor binding peptide (EGBP) was identified using a phage display method and evaluated in U87MG cells in order to investigate the possibility to target the EGFR using an optical imaging system. Cyanine dye 5.5 (Cy5.5) was conjugated with EGBP-GGG-SC, EGBP-AOC-SC, and EGBP-AM2BA-SC. Cellular binding study of EGBP-Linker-Cy5.5 conjugates or ¹²⁵I-EGBP-Linker compounds was performed in U87MG cells. Optical imaging studies were performed in U87MG bearing mice. Three of seven clones from the 12-mer peptide library showed a specific binding affinity to rhEGFR, and they encoded the same 12 amino acid peptide sequence, FPMFNHWEQWPP. Confocal images show that the fluorescent signal of EGBP-Linker-Cy5.5 conjugates was decreased in the order: EGBP-AOC-Cy5.5?EGBP-AM2BA-Cy5.5>EGBP-GGG-Cy5.5. EGBP-AOC-Cy5.5 appeared in cell cytoplasm and surface, and it was inhibited by free EGBP apparently. The cellular binding of EGBP-AOC-Cy5.5 exhibited a higher average radiance value than EGBP-GGG-Cy5.5 and EGBP-AM2BA-Cy5.5. Among various ¹²⁵I-EGBP-Linker compounds, EGBP-GGG showed a higher binding than other compounds. However, uptake of ¹²⁵I-EGBP-AOC was clearly inhibited by free EGBP in inhibition study. In an in vivo study, the fluorescent signal of EGBP-AOC-Cy5.5 treated mouse was mainly detected in the tumor and kidney. Among the three derivatives, EGBP-AOC-Cy5.5 was the optimized optical imaging agent for U87MG EGFR positive tumors in the animal model. This study demonstrated the EGBP-Linker-Cy5.5 conjugates may be useful as a potential EGFR target optical probe.     

裸鼠胃癌移植瘤的近红外荧光成像

目的：通过对非特异性探针Cy5.5在裸鼠体内分布及显像研究,探讨近红外荧光成像对胃癌的早期诊断及动态监测价值。方法：用MGC-803细胞株建立胃癌动物模型,进行早期、实时活体及离体显像实验。结果：近红外荧光成像可检测早期胃癌移植瘤的平均大小为2.807mm×3.045mm,与游标卡尺测得的肿瘤大小呈直线相关,r=0.924,P〈0.05。Cy5.5主要分布在肿瘤组织,主要代谢器官为肾脏;注入探针30min后,裸鼠肿瘤部位成像清晰,荧光寿命、荧光强度均高于对照部位（P〈0.05）。60min后,肿瘤区的荧光强度始高于血液（P〈0.05）。90min时达峰值。肿瘤部位的平均荧光寿命为（3.1376±0.9894）ns,明显高于对侧部位（P〈0.05）。结论：近红外荧光成像可用于胃癌的早期诊断及瘤体的动态监测。     

宽视场荧光内镜的构建及其初步应用

 目的 为探索胃癌分子成像技术的新方法,构建一套新型灵活的宽视场荧光内镜系统,并用于裸鼠胃癌移植瘤模型荧光成像研究。方法 通过合理的光路设计及光学组件机加工,装配宽视场荧光内镜系统,通过对不同浓度的2-DG-750荧光探针进行成像,分析系统性能;对尾静脉注射100 μl 2-DG-750荧光探针的裸鼠胃癌移植瘤模型进行在体荧光成像,对ROI的像素值和成像图进行分析。结果 体外成像ROI的像素值与荧光探针浓度存在较好的线性回归关系,在2-DG-750剂量为31.3 pmol时,该系统即可检测到荧光信号,提示该系统灵敏性好;在体成像显示用该系统联合2-DG-750荧光探针观察到了肿瘤组织的较高荧光信号强度。结论 宽视场荧光内镜系统具有很好的灵敏性,可以同时实现白光及荧光的双模式成像,具有实现光学分子成像诊断胃癌的潜在应用价值。     

Near-infrared fluorescence imaging of HER-2 protein over-expression in tumour cells

The aim of this study was to evaluate in vitro and in vivo imaging of HER-2-over-expressing tumours using near-infrared optical imaging. A fluorochrome probe was designed by coupling Cy5.5 to anti-HER-2 antibodies. Cells over-expressing (SK-BR-3 cells) or normally expressing (PE/CA-PJ34 cells) the HER-2 protein were incubated with the probe. After removing unbound probe molecules, fluorescence intensities were determined (a.u.: arbitrary units). Cells were additionally investigated using FACS and laser scanning microscopy. The probe was also injected intravenously into tumours bearing SK-BR-3 (n=3) or PE/CA-PJ34 (n=3). Whole-body fluorescence images were generated and analysed. The incubation of SK-BR-3 cells with the probe led to higher fluorescence intensities [2,133 (±143) a.u.] compared to controls [975 (±95) a.u.]. The results from FACS and immunocytochemical analysis were in agreement with these findings. A distinct dependency between the fluorescence intensity and the cell number used in the incubations was detected. In vivo, the relative fluorescence intensities in SK-BR-3 tumours were higher than in PE/CA-PJ34 tumours at 16–24 h after probe application. HER-2-over-expressing tumours were depictable in their original size. Labelling of HER-2 with Cy5.5 is suitable for in vitro and in vivo detection of HER-2-over-expressing tumour cells.     

Tc-99m-galactosyl-neoglycoalbumin: in vivo characterization of receptor-mediated binding to hepatocytes

The biodistribution and kinetics of a receptor-binding hepatic radiopharmaceutical, Tc-99m-galactosyl-neoglycoalbumin (Tc-NGA), were investigated using mammalian and avian models. The radiopharmaceutical exhibited four significant features associated with receptor-mediated binding at the hepatocyte membrane in mammals: (a) high tissue specificity, (b) high molecular specificity, (c) affinity-dependent uptake, and (d) dose-dependent uptake. Diminished hepatic uptake by the avian model illustrated low nonspecific binding. The kinetic sensitivity to ligand-receptor affinity and stoichiometry illustrated the principal feature of receptor-binding radio-pharmaceuticals, namely, quantitative assessment of tissue function based upon the biochemical interaction of a ligand and its specific receptor.     

Diagnosis of arthritis using near-infrared fluorochrome Cy5.5

PURPOSE: Near-infrared range fluorescence (NIRF) imaging is a potential tool to diagnose biologic processes in vivo. This applicability study sought to define whether imaging with fluorochrome Cy5.5 can identify arthritis in murine antigen-induced arthritis (AIA). MATERIALS AND METHODS: On day 7 of AIA (n = 9 mice), fluorescence intensities in inflamed and contralateral knee joints (the latter as internal control) were measured before and after intravenous injection of Cy5.5 (until 72 hours). Cy5.5 joint deposition was verified by confocal laser-scanning microscopy. Dye phagocytosis was evaluated in cultured macrophages (cell line PMJ2-R) by FACS analysis. Cy5.5 binding to serum protein was tested by NIRF scanning and gel electrophoresis. RESULTS: Between 2 and 72 hours, the arthritic knee joints showed significantly higher fluorescence intensities compared with contralateral joints. Microscopy confirmed Cy5.5 deposition in the synovial membrane. Cultured macrophages actively phagocytosed Cy5.5. Cy5.5 bound mainly to albumin as the main serum protein. CONCLUSION: NIRF imaging with Cy5.5 can identify arthritic joints in vivo, likely due to nonspecific deposition.     

Catheter-based in vivo imaging of enzyme activity and gene expression: feasibility study in mice

PURPOSE: To construct and evaluate an interventional catheter-based imaging system for intravital monitoring of molecularly sensitive near-infrared fluorescent probes and optical marker genes. MATERIALS AND METHODS: An imaging device that was based on a miniaturized fiberoptic sensor (MIFS) was built in which images created with a 2.7-F fiberoptic catheter were relayed through a dichroic mirror, through a bandpass filter, and on two independent cameras. This system permitted simultaneous recording of white-light and fluorescent images. Spatial resolution, spectral transmissions, and sensitivity were determined in vitro. In vivo testing was performed in nude mice bearing intraperitoneal tumors that express green fluorescent protein and in a mouse model of ovarian carcinoma with enzyme-activatable near-infrared probes sensitive to tumoral protease activity. Signal intensity on images of tumors and that on images of normal tissue were measured and compared with t test. RESULTS: The catheter, which was advanced through an 18-gauge sheath, showed resolution of 7 line pairs per millimeter and detection limit for fluorochrome Cy5.5 of 1-10 pmol. Detection of endogenous green fluorescent protein gene expression was feasible in tumor nodules smaller than 1 mm in diameter (mean tumor signal intensity, 153.26 +/- 26.45 [SD], compared with that of adjacent nontumoral tissue of 36.73 +/- 11.69; P <.008). Similarly, activation of the near-infrared probe by tumoral proteases could be detected in peritoneal tumor seeds of ovarian cancer model with mean tumor signal intensity of 246.33 +/- 7.77 compared with that of adjacent nontumoral tissue of 41.56 +/- 18.64 (P <.001). Mean contrast-to-noise ratio in the near-infrared channel exceeded white-light contrast-to-noise ratio by a factor of 6.7 (P <.02). CONCLUSION: With this system, in vivo MIFS imaging of gene expression, enzyme activity, and potentially other molecular events is feasible, through direct interventional access to several organs and body cavities and potentially through transvascular approaches.     

In vivo imaging of integrin ανβ<Subscript>3</Subscript> expression using fluorescence-mediated tomography

Purpose Optical imaging would be desirable for cancer diagnostics since it can potentially resolve relevant oncological target structures in vivo. We therefore synthesised an αvβ₃ targeted fluorochrome and imaged tumour xenografts with different αvβ₃ expression levels using both planar and tomographic optical imaging methods. Methods An αvβ₃-targeted RGD peptide was labelled with a cyanine dye (Cy 5.5). Binding of the optical tracer was tested on M21 melanoma (n = 5), HT-1080 fibrosarcoma (n = 6) and MCF-7 adenocarcinoma (n = 5) cells and their tumour xenografts. All optical imaging studies were performed using two-dimensional planar fluorescence reflectance imaging (FRI) technology and three-dimensional fluorescence-mediated tomography (FMT). Results In vitro, the peptide-dye conjugate showed a clear binding affinity to αvβ₃-positive M21 and HT-1080 cells while αvβ₃-negative MCF-7 cells and pre-dosing with the free RGD peptide revealed little to no fluorescence. In vivo, tumour xenografts were clearly visualised by FRI and FMT up to 24 h post injection. FMT allowed quantification of the fluorochrome distribution in deeper tissue sections showing an average fluorochrome concentration of 417.61 ± 105.82 nM Cy 5.5 (M21), 353.68 ± 54.02 nM Cy 5.5 (HT-1080) and 262.83 ± 155.36 nM Cy 5.5 (MCF-7) in the target tissue 60 min after tracer administration. Competition with the free RGD peptide resulted in a reduction in the fluorochrome concentration in M21 tumour tissue (294.35 ± 84.27 nM). Conclusion RGD-Cy 5.5 combined with novel tomographic optical imaging methods allows non-invasive imaging of tumour-associated αvβ₃ expression and may thus be a promising strategy for sensitive evaluation of tumour target expression.     

Fluorescence reflectance imaging of macrophage-rich atherosclerotic plaques using an alphavbeta3 integrin-targeted fluorochrome

Macrophages play an important role during the development and progression of atherosclerotic plaques. alphavbeta3 integrins are highly expressed by macrophages; thus, targeting alphavbeta3 may allow targeting of culprit macrophage-loaded atherosclerotic lesions in vivo. METHODS: An alphavbeta3-targeted Arg-Gly-Asp (RGD) peptide was labeled with the cyanine 5.5 (Cy 5.5) dye and applied to image atherosclerotic plaques in apolipoprotein E-deficient mice. RESULTS: The peptide-dye conjugate binds to alphavbeta3 integrin-positive RAW264.7 macrophages with high affinity. Competition experiments confirmed binding specificity of the probe. A significant fluorochrome accumulation in atherosclerotic plaques was demonstrated 24 h after injection by fluorescence reflectance imaging, which was blocked with high efficiency by competition with the unlabeled peptide. Conversely, the nonconjugated dye revealed only a minor fluorescence signal in the plaques. Fluorescence microscopy revealed colocalization of the probe with macrophages in the plaque of a mouse model for accelerated atherosclerosis, which was corroborated in human carotid artery specimens. In addition to macrophage-associated signals, binding of the probe to the neointima or elastica of the arteries was observed. CONCLUSION: RGD-Cy 5.5, combined with near-infrared optical imaging methods, allows the specific imaging of alphavbeta3-integrin expression on macrophages recruited to vascular lesions and may serve to estimate macrophage-bound inflammatory activity of atherosclerotic lesions.     

Near-infrared optical imaging in glioblastoma xenograft with ligand-targeting α3 integrin

Purpose  Patients with glioblastoma usually have a very poor prognosis. Even with a combination of radiotherapy plus temozolomide, the median survival of these patients is only 14.6 months. New treatment approaches to this cancer are needed. Our purpose is to develop new cell surface-binding ligands for glioblastoma cells and use them as targeted imaging and therapeutic agents for this deadly disease. Methods  One-bead one-compound combinatorial cyclic peptide libraries were screened with live human glioblastoma U-87MG cells. The binding affinity and targeting specificity of peptides identified were tested with in vitro experiments on cells and in vivo and ex vivo experiments on U-87MG xenograft mouse model. Results  A cyclic peptide, LXY1, was identified and shown to be binding to the α3 integrin of U-87MG cells with moderately high affinity (K _d = 0.5 ± 0.1 μM) and high specificity. Biotinylated LXY1, when complexed with streptavidin–Cy5.5 (SA–Cy5.5) conjugate, targeted both subcutaneous and orthotopic U-87MG xenograft implants in nude mice. The in vivo targeting specificity was further verified by strong inhibition of tumor uptake of LXY1–biotin–SA–Cy5.5 complex when intravenously injecting the animals with anti-α3 integrin antibody or excess unlabeled LXY1 prior to administrating the imaging probe. The smaller univalent LXY1–Cy5.5 conjugate (2,279 Da) was found to have a faster accumulation in the U-87MG tumor and shorter retention time compared with the larger tetravalent LXY1–biotin–SA–Cy5.5 complex (approximately 64 kDa). Conclusions  Collectively, the data reveals that LXY1 has the potential to be developed into an effective imaging and therapeutic targeting agent for human glioblastoma. Standard single letter amino acids are used for all natural l-amino acids.     

Optical imaging of spontaneous breast tumors using protease sensing 'smart' optical probes

OBJECTIVE: The objective of this study was to determine if spontaneous breast cancer lesions can be detected by fluorescence reflectance imaging (FRI) and fluorescence mediated tomography (FMT) using protease-sensing optical probes. MATERIALS AND METHODS: Transgenic (FVB/N-TgN (WapHRAS)69Lin Y)) mice, which spontaneously develop breast cancer, were injected intravenously with a cathepsin-sensing fluorescent imaging probe. FRI and FMT were performed 24 hours after probe injection and region of interest (ROI) analysis was performed. Magnetic resonance images were acquired for anatomic coregistration with the FMT data. Moreover, correlative immunohistochemistry and fluorescence microscopy were performed. RESULTS: All tumor nodules were clearly delineated by FRI showing an average signal intensity of 380 +/- 106 AU. Similarly, tumors were clearly detected by FMT imaging. Immunohistochemistry confirmed cathepsin-B expression of primary tumors and fluorescence microscopy revealed a strong Cy 5.5 deposition in the tissue. CONCLUSIONS: FRI and FMT using "smart" protease sensing probes permits detection of experimental spontaneous breast cancers. Because the expression levels of various proteases correlate with patient outcome, this technique may not only help to detect, but also to differentiate breast cancers noninvasively.     

64Cu Labeled AmBaSar-RGD2 for micro-PET Imaging of Integrin αvβ3 Expression

Integrin αvβ3 plays a critical role in tumor-induced angiogenesis and metastasis. Previously, a 64Cu-AmBaSar- RGD monomer with high in vivo stability compared with 64Cu-DOTA-RGD was developed for integrin αvβ3 PET imaging. It has been established that dimeric RGD peptides have higher receptor-binding affinity and superior in vivo kinetics compared with monomeric RGD peptides due to the polyvalency effect. In this context, we synthesized and evaluated 64Cu-labeled AmBaSar dimeric RGD conjugates (64Cu-AmBaSar-RGD2) for PET imaging of integrin αvβ3 expression. The dimeric RGD peptide was conjugated with a cage-like chelator AmBaSar and labeled with 64Cu. Cell binding, microPET imaging, receptor blocking, and biodistribution studies of 64Cu-AmBaSar-RGD2 were conducted in the U87MG human glioblastoma xenograft model. AmBaSar-RGD2 conjugate was obtained in reasonable yield (45.0 ± 2.5%, n= 4) and the identity was confirmed by HPLC and MS (found 1779.8, calculated m/z for [M+H]+ M: C81H125N27O19 1779.9). 64Cu-AmBaSar-RGD2 was obtained with high radiochemical yield (92.0 ± 1.3%) and purity (≥ 98.0%) under mild conditions (pH 5.0~5.5, 23~37 °C) in 30 min. The specific activity of 64Cu-AmBaSar-RGD2 was estimated to be 15-22 GBq/μmol at the end of synthesis. Based on microPET imaging and biodistribution studies, 64Cu-AmBaSar-RGD2 has demonstrated higher tumor uptake at selected time points than 64Cu-AmBaSar-RGD. At 20 h p.i., the tumor uptake reached 0.65 ± 0.05 %ID/g for 64Cu-AmBaSar-RGD and 1.76 ± 0.38 %ID/g for 64Cu-AmBaSar-RGD2, respectively. The integrin αvβ3 targeting specificity was confirmed by blocking experiments. Therefore, the new tracer 64Cu-AmBaSar- RGD2 exhibited better tumor-targeting efficacy and more favorable in vivo pharmacokinetics than the 64Cu labeled RGD monomer due to the polyvalency effect.     

Preparation and biological evaluation of 2-amino-6-[18F]fluoro-9-(4-hydroxy-3-hydroxy-methylbutyl) purine (6-[18F]FPCV) as a novel PET probe for imaging HSV1-tk reporter gene expression

INTRODUCTION: 2-Amino-6-[(18)F]fluoro-9-(4-hydroxy-3-hydroxy-methylbutyl) purine (6-[(18)F]FPCV) was prepared via a one-step nucleophilic substitution and evaluated as a novel probe for imaging the expression of herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene. METHODS: Log P of 6-[(18)F]FPCV was calculated in octanol/phosphate-buffered saline (PBS). Stability studies were performed in PBS and bovine serum albumin (BSA). Cell uptake was performed at various time points in wild-type cells and transduced cells. For in vivo studies, tumors were grown in nude mice by inoculation with C6 cells, wild type and tk positive. The radiotracer was intravenously injected to animals, and micro-PET imaging was performed. Biodistribution of 6-[(18)F]FPCV was performed on another group of animals at different time points. RESULTS: Log P of 6-[(18)F]FPCV was -0.517. 6-[(18)F]FPCV was fairly stable in PBS and BSA at 6 h. The tracer uptake in C6-tk cells was 5.5-18.8 times higher than that in wild-type cells. The plasma half-life of 6-[(18)F]FPCV was as follows: alpha t(1/2)=1.2 min and beta t(1/2)=73.7 min. The average ratio of tumor uptake between the transduced tumor and the wild-type tumor was 1.69 at 15 min. CONCLUSION: Biological evaluation showed that 6-[(18)F]FPCV is a potential probe for imaging HSV1-tk gene expression. However, its in vivo defluorination may limit its application in PET imaging of gene expression.     

A dendrimer-based nanosized contrast agent dual-labeled for magnetic resonance and optical fluorescence imaging to localize the sentinel lymph node in mice

PURPOSE: To preoperatively and intraoperatively localize the sentinel lymph node (SLN), a single hybrid probe for MR and near infrared (NIR) optical imaging was synthesized and tested. MATERIALS AND METHODS: A macromolecular MR/NIR optical contrast agent was synthesized based on a approximately 191 gadolinium-labeled contrast agent using generation-6 polyamidoamine dendrimer (G6), which is also labeled with 2 Cy5.5, an NIR fluorophore. After establishing the optimal dose, the agent was injected into mammary glands of 10 normal mice to examine the lymphatic drainage from the breast using a 3T clinical scanner. Immediately after the MRI scan, NIR optical imaging and image-guided surgery were performed to compare the two imaging modalities. RESULTS: To consistently identify the SLNs, we needed to inject 25 microL of 30 mM [Gd] G6-Cy5.5. All SLNs could be easily identified and resected under NIR optical imaging-guided surgery. Although external NIR optical imaging failed to identify SLNs close to the injection site due to shinethrough, MR lymphography (MRL) consistently identified all SLNs regardless of their location. CONCLUSION: We have successfully synthesized and tested a dual labeled MR/NIR optical hybrid contrast agent, G6-Cy5.5 for reoperative and intraoperative localization of SLNs.     

Radiolabelled peptides for oncological diagnosis

Radiolabelled receptor-binding peptides targeting receptors (over)expressed on tumour cells are widely under investigation for tumour diagnosis and therapy. The concept of using radiolabelled receptor-binding peptides to target receptor-expressing tissues in vivo has stimulated a large body of research in nuclear medicine. The (111)In-labelled somatostatin analogue octreotide (OctreoScan) is the most successful radiopeptide for tumour imaging, and was the first to be approved for diagnostic use. Based on the success of these studies, other receptor-targeting peptides such as cholecystokinin/gastrin analogues, glucagon-like peptide-1, bombesin (BN), chemokine receptor CXCR4 targeting peptides, and RGD peptides are currently under development or undergoing clinical trials. In this review, we discuss some of these peptides and their analogues, with regard to their potential for radionuclide imaging of tumours.     

Validation of in vivo receptor measurements via in vitro radioassay: technetium-99m-galactosyl-neoglycoalbumin as prototype model

Hepatic binding protein (HBP) is a hepatocyte-specific receptor for serum asialoglycoproteins. The receptor also recognizes a synthetic glycoprotein that has been developed as a radiopharmaceutical, technetium-99m-galactosyl-neoglycoalbumin (99mTc-NGA). This report describes the correlation between receptor parameters measured in vivo via kinetic modeling of 99mTc-NGA and those measured by in vitro radioassay of biopsied liver tissue. Eleven patients with diffuse hepatic disease underwent percutaneous liver biopsy followed by a 99mTc-NGA functional imaging study. In vivo measurements of HBP quantity Ro and forward binding rate constant kb obtained from the kinetic analysis of 99mTc-NGA liver and blood time-activity data were compared to total receptor quantity and the HBP-99mTc-NGA association constant KA as measured by Scatchard binding assay of the biopsied tissue. The correlation coefficients between in vivo and in vitro measurements were 0.73 (df = 8, p = 0.015) and 0.98 (df = 8, p less than 0.01) for Ro and kb, respectively. The in vivo measurements of HBP biochemistry via kinetic analysis of the radiopharmaceutical time-activity data reflect the average concentration and affinity of the receptor. This study further substantiates the validity of 99mTc-NGA as a quantitative probe for the HBP receptor.     

Optical imaging of matrix metalloproteinase-2 activity in tumors: feasibility study in a mouse model.

PURPOSE: To develop an optical imaging method to determine the expression level of tumoral matrix metalloproteinase-2 (MMP-2) in vivo. MATERIALS AND METHODS: An optical contrast agent was developed that was highly activatable by means of MMP-2-induced conversion. Signal characteristics of the probe were quantified ex vivo with a recombinant enzyme. Animal tumor models were established with MMP-2-positive (human fibrosarcoma cell line, n = 4) and MMP-2-negative (well-differentiated mammary adenocarcinoma, n = 4) tumor cell lines. Both tumors were implanted into nude mice and were optically imaged after intravenous administration of the MMP-2-sensitive probe. RESULTS: The MMP-2-sensitive probe was activated by MMP-2 in vitro, producing up to an 850% increase in near-infrared fluorescent signal intensity. This activation could be blocked by MMP-2 inhibitors. MMP-2-positive tumors were easily identified as high-signal-intensity regions as early as 1 hour after intravenous injection of the MMP-2 probe, while contralateral MMP-2-negative tumors showed little to no signal intensity. A nonspecific control probe showed little to no activation in MMP-2-positive tumors. CONCLUSION: It is feasible to image MMP-2 enzyme activity in vivo by using near-infrared optical imaging technology and "smart" matrix metalloproteinase-sensitive probes.     

microPET of tumor integrin alphavbeta3 expression using 18F-labeled PEGylated tetrameric RGD peptide (18F-FPRGD4).

In vivo imaging of alpha(v)beta(3) expression has important diagnostic and therapeutic applications. Multimeric cyclic RGD peptides are capable of improving the integrin alpha(v)beta(3)-binding affinity due to the polyvalency effect. Here we report an example of (18)F-labeled tetrameric RGD peptide for PET of alpha(v)beta(3) expression in both xenograft and spontaneous tumor models. METHODS: The tetrameric RGD peptide E{E[c(RGDyK)](2)}(2) was derived with amino-3,6,9-trioxaundecanoic acid (mini-PEG; PEG is poly(ethylene glycol)) linker through the glutamate alpha-amino group. NH(2)-mini-PEG-E{E[c(RGDyK)](2)}(2) (PRGD4) was labeled with (18)F via the N-succinimidyl-4-(18)F-fluorobenzoate ((18)F-SFB) prosthetic group. The receptor-binding characteristics of the tetrameric RGD peptide tracer (18)F-FPRGD4 were evaluated in vitro by a cell-binding assay and in vivo by quantitative microPET imaging studies. RESULTS: The decay-corrected radiochemical yield for (18)F-FPRGD4 was about 15%, with a total reaction time of 180 min starting from (18)F-F(-). The PEGylation had minimal effect on integrin-binding affinity of the RGD peptide. (18)F-FPRGD4 has significantly higher tumor uptake compared with monomeric and dimeric RGD peptide tracer analogs. The receptor specificity of (18)F-FPRGD4 in vivo was confirmed by effective blocking of the uptake in both tumors and normal organs or tissues with excess c(RGDyK). CONCLUSION: The tetrameric RGD peptide tracer (18)F-FPRGD4 possessing high integrin-binding affinity and favorable biokinetics is a promising tracer for PET of integrin alpha(v)beta(3) expression in cancer and other angiogenesis related diseases.     

Novel membrane‐permeable contrast agent for brain tumor detection by MRI

One of the key challenges hindering the clinical intervention against brain cancer is defined by the inability to detect brain tumors at an early enough stage to permit effective therapy. Furthermore, the rapid growth and severe lethality of this form of cancer predicate the vital importance of monitoring the development of the pathology and its outcome after therapeutic intervention. With this in mind, we designed a novel membrane‐permeant contrast agent, MN‐MPAP‐Cy5.5, which consists of a superparamagnetic iron oxide core, for MRI conjugated to myristoylated polyarginine peptides, as a membrane translocation module and labeled with the near‐infrared dye Cy5.5 for correlative microscopy. This probe showed a remarkable uptake by U‐87 human glioma cells in vitro and localized and delineated stereotactically injected tumor in vivo by MRI. Our findings suggest that the agent mediates its effects by translocation of the magnetic nanoparticles label across the leaky tumor vasculature, followed by enhanced accumulation in tumor cells. The noninvasive detection of brain tumors when they are still small represents a formidable challenge from an imaging standpoint. Our study describes an improved strategy to detect brain lesions by utilizing a contrast agent with membrane translocation properties. Magn Reson Med, 2010. © 2010 Wiley‐Liss, Inc.