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Currently, positron emission tomography/computed tomography (PET/CT) is an important method for the discovery and diagnosis of digestive system tumors. However, the shortage of specific imaging tracer limits the effectiveness of PET. Triggering receptor expressed on myeloid cells 2 (TREM2) as an M2-type macrophage biomarker is receiving much attention considering its high abundance and specificity, which could be an ideal target for PET imaging. First, the expression of TREM2 in tumors and corresponding normal tissues was analyzed using a database and was verified by tissue microarrays and murine model slices, and we found that the expression of TREM2 in tumor tissues was significantly higher than that in normal tissues and enteritis tissues. Then, we established a macrophage co-culture system to obtain tumor-associated macrophages (TAMs). Compared with M1-type macrophages and tumor cells, TAMs had a higher expression level of TREM2. The novel radioligand

Gastric cancer (GC) is a common cancer worldwide, with high incidence and mortality rates. Therefore, early and precise diagnosis is critical to improving GC prognosis. Tumor-associated myeloid cells infiltrate the tumor microenvironment (TME) and can produce immunosuppressive effects in the early stage of the tumor. The surface integrin receptor CD11b is widely expressed in the specific subsets of myeloid cells, and it has the characteristics of high abundance, high specificity, and high potential for targeted immunotherapy. In this study, two strategies for labeling anti-CD11b, including

P2X7 receptors have been considered as a promising biomarker for vulnerable atherosclerotic plaques, which are highly expressed by that instability-associated factors such as macrophages. Thus, we aim to investigate the feasibility of using specific P2X7-targetedThe radioligandWe innovatively apply a new type P2X7-targeted PET probe (

This study is aimed at investigating the feasibility of cetuximab (Cet) F(ab')Cet-F(ab')HT29 cells had low EGFR expression while MGC803 cell exhibited the high EGFR expression. Cet-F(ab')SPECT/CT imaging using

Mutations in isocitrate dehydrogenase 1 (IDH1) are commonly found in various human malignancies. Inhibitors of several mutant IDH1 enzymes have entered clinical trials as target therapeutic drugs for the treatment of patients with IDH1 mutations. Herein, we report the synthesis and evaluation of two

The exceptional speed and biorthogonality of the inverse electron-demand Diels–Alder (IEDDA) click chemistry between 1,2,4,5-tetrazines and strained alkene dienophiles have made it promising in the realm of pretargeted imaging and therapy. During the past 10 years, the IEDDA-pretargeted strategies have been tested and have already proven capable of producing images with high tumor-to-background ratios and improving therapeutic effect. This review will focus on recent applications of click chemistry ligations in the pretargeted imaging studies of single photon emission computed tomography (SPECT), positron emission tomography (PET), and pretargeted radioimmunotherapy investigations. Additionally, the influence factors of stability, reactivity, and pharmacokinetic properties of TCO tag modified immunoconjugates and radiolabeled Tz derivatives were also summarized in this article, which should be carefully considered in the system design in order to develop a successful pretargeted methodology. We hope that this review will not only equip readers with a knowledge of pretargeted methodology based on IEDDA click chemistry but also inspire synthetic chemists and radiochemists to develop pretargeted radiopharmaceutical components in a more innovative way with various influence factors considered.

Previously, we successfully developed a pretargeted imaging strategy (atezolizumab-TCO/[99mTc]HYNIC-PEG11-Tz) for evaluating programmed cell death ligand-1 (PD-L1) expression in xenograft mice. However, the surplus unclicked [99mTc]HYNIC-PEG11-Tz is cleared somewhat sluggishly through the intestines, which is not ideal for colorectal cancer (CRC) imaging. To shift the excretion of the Tz-radioligand to the renal system, we developed a novel Tz-radioligand by adding a polypeptide linker between HYNIC and PEG11. Pretargeted molecular probes [99mTc]HYNIC-polypeptide-PEG11-Tz and cetuximab-TCO were synthesized. [99mTc]HYNIC-polypeptide-PEG11-Tz was evaluated for in vitro stability and in vivo blood pharmacokinetics. In vitro ligation reactivity of [99mTc]HYNIC-polypeptide-PEG11-Tz towards cetuximab-TCO was also tested. Biodistribution assay and imaging of [99mTc]HYNIC-polypeptide-PEG11-Tz were performed to observe its excretion pathway. Pretargeted biodistribution was measured at three different accumulation intervals to determine the optimal pretargeted interval time. Pretargeted (cetuximab-TCO 48 h/[99mTc]HYNIC-PEG11-Tz 6 h) and (cetuximab-TCO 48 h/[99mTc]HYNIC-Polypeptide-PEG11-Tz 6 h) imagings were compared to examine the effect of the excretion pathway on tumor imaging. [99mTc]HYNIC-polypeptide-PEG11-Tz showed favorable in vitro stability and rapid blood clearance in mice. SEC-HPLC revealed almost complete reaction between cetuximab-TCO and [99mTc]HYNIC-polypeptide-PEG11-Tz in vitro, with the 8:1 Tz-to-mAb reaction providing a conversion yield of 87.83 ± 3.27 %. Biodistribution and imaging analyses showed that the Tz-radioligand was cleared through the kidneys. After 24, 48, and 72 h of accumulation in HCT116 tumor, the tumor-to-blood ratio of cetuximab-TCO was 0.83 ± 0.13, 1.40 ± 0.31, and 1.15 ± 0.21, respectively. Both pretargeted (cetuximab-TCO 48 h/[99mTc]HYNIC-PEG11-Tz 6 h) and (cetuximab-TCO 48 h/[99mTc]HYNIC-polypeptide-PEG11-Tz 6 h) clearly delineated HCT116 tumor. Pretargeted imaging strategy using cetuximab-TCO/[99mTc]HYNIC-polypeptide-PEG11-Tz could be used for diagnosing CRC, as the surplus unclicked [99mTc]HYNIC-polypeptide-PEG11-Tz was cleared through the urinary system, leading to low abdominal uptake background. Our novel pretargeted imaging strategy (cetuximab-TCO/[99mTc]HYNIC-polypeptide-PEG11-Tz) was useful for imaging CRC, broadening the application scope of pretargeted imaging strategy. The pretargeted imaging strategy clearly delineated HCT116 tumor, showing that its use could be extended to selection of internalizing antibodies.

Site-specific imaging agents play a key role in tumor targeting, but only a few agents are currently available for inflammation targeting. Since the P2X7 receptor (P2X7R) is a promising molecular target for inflammation, we evaluated the potential value of the (18)F-labeled tracer (18)F-PTTP (5-{[2-Chloro-3-(trifluoromethyl)phenyl]carbonyl}-1-pyrimidin-2-yl-4,5,6,7-tetrahydro-1H-[1,2,3]triazolo[4,5-c]pyridin) for targeting P2X7Rs and thus differentiating inflammation from tumors. Methods: The radioligand (18)F-PTTP was achieved by a 1-step (18)F-trifluoromethylation reaction. The binding affinity of the ligand for P2X7R and its stability were evaluated in vitro. Blood pharmacokinetics tests and biodistribution studies were performed in vivo. Dynamic (18)F-PTTP small-animal PET/CT imaging was performed for 60 min on A549 tumor–bearing mice and inflammation-model mice for targeting differentiation. Results:(18)F-PTTP was afforded with decay-corrected radiochemical yields of 2.5%–7.0%, specific activity of 296–370 MBq/μmol, and radiochemical purity over 95%. (18)F-PTTP showed excellent stability in 0.9% NaCl and 0.1% bovine serum albumin, good affinity to RAW264.7 cells, and rapid blood clearance in mice. In inflammation-model mice, uptake of (18)F-PTTP peaked at 5 min after injection and kept at an imageable level till 30 min, whereas no significant radioactivity uptake was found in tumor grafts till 1 h after injection. The specificity of (18)F-PTTP was verified by blocking studies and histologic analysis. Conclusion: The current study provides compelling data that (18)F-PTTP is a novel radioligand targeting P2X7R and has potential to screen new drugs, quantify peripheral inflammation, and distinguish inflammation from certain solid tumors.

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

In this work, we have developed multiple-armed DNA tetrahedral nanostructures (TDNs) for dual-modality in vivo imaging using near-infrared (NIR) fluorescence and single-photon emission computed tomography (SPECT). We found that the presence of arm strands in TDNs remarkably enhanced their in vitro stability, allowing them to stay intact for at least 12 h in serum. By using NIR fluorescence imaging, we evaluated in mice the pharmacokinetics of TDNs, which exhibited distinctly different in vivo biodistribution patterns compared with those of double-stranded (ds)DNA. We also noticed that TDNs had twofold longer circulation time in the blood system than that of dsDNA. With the use of multiple-armed TDNs, we could precisely anchor an exact number of functional groups including tumor-targeting folic acid (FA), NIR emitter Dylight 755, and radioactive isotope (99m)Tc on prescribed positions of TDNs, which showed the capability of targeted imaging ability in cancer cells. Furthermore, we realized noninvasive tumor-targeting imaging in tumor-bearing mice by using both NIR and SPECT modalities.

Previously, we demonstrated specific accumulation into bacteria of a 12-mer phosphorodiamidate morpholino (MORF) oligomer complementary to a ribosomal RNA (rRNA) segment found in all bacteria using the universal probe called Eub338 (Eub). Here, two MORF oligomers Eco and Kpn with sequences specific to the rRNA of Escherichia coli (Eco) and Klebsiella pneumoniae (Kpn) were investigated along with Eub and control (nonEub).To determine bacterial rRNA binding, oligomers were tagged with Alexa Fluor 633 (AF633) for fluorescence in situ hybridization (FISH) and fluorescence microscopy, and radiolabeled with technetium-99m (Tc-99m) for biodistribution and SPECT imaging in infected mice.By both FISH and fluorescence microscopy, Eub showed a positive signal in both E. coli and K. pneumoniae as expected, and Kpn showed significantly higher accumulation in K. pneumoniae with near background in E. coli (p 0.01). Conversely, Eco was positive in both E. coli and K. pneumoniae, hence nonspecific. As determined by biodistribution, the accumulation of [(99m)Tc]Kpn was higher in the thigh infected with live K. pneumoniae than with live E. coli (p = 0.05), and significantly higher than with heat-killed K. pneumoniae (p = 0.02) in the target thigh. By SPECT imaging, the accumulation of [(99m)Tc]Kpn was obviously higher in its specific target of K. pneumoniae compared to an E. coli infected thigh.Kpn complementary to the rRNA of K. pneumoniae, labeled with Tc-99m or AF633, demonstrated specific binding to fixed and live K. pneumoniae in culture and in infected mice such that Tc-99m-labeled Kpn as the MORF oligomer may be useful for K. pneumoniae infection detection through imaging.

Inflammation and calcification concomitantly drive atherosclerotic plaque progression and rupture and are the compelling targets for identifying plaque vulnerability. However, current imaging modalities for vulnerable atherosclerotic plaques are often limited by inadequate specificity and sensitivity. Here, we show that natural H-ferritin nanocages radiolabeled with technetium-99m (99mTc-HFn) can identify and accurately localize macrophage-rich, atherosclerotic plaques in living mice using combined SPECT and CT. Focal 99mTc-HFn uptake was observed in the atherosclerotic plaques with multiple high-risk features of macrophage infiltration, active calcification, positive remodeling, and necrosis on histology and in early active ongoing lesions with intense macrophage infiltration. The uptake of 99mTc-HFn in plaques enabled quantitative measuring of the dynamic changes of inflammation during plaque progression and anti-inflammation treatment. This strategy lays the foundation of using bioengineered endogenous human ferritin nanocages for the identification of vulnerable and early active plaques as well as potential assessment of anti-inflammation therapy.

A novelWith Hynic as the chelating agent,micro-SPECT/CT imaging of nude mice transplanted with H1993 and H292 tumors usingmicro-SPECT/CT imaging through

This study attempted to evaluate the feasibility of 99m Tc-labeled glucarate ( 99m Tc-GLA) imaging in non-small cell lung cancer (NSCLC) and the potential tumor uptake mechanism. Cell lysates from two NSCLC cell lines, H292 and H1975, were immunoblotted with anti-glucose transporter 5 (GLUT5) antibody for Western blotting. Thereafter, the two cell lines were used to examine cellular uptake of 99m Tc-GLA with or without fructose. SPECT/CT imaging studies were performed on small animals bearing H292 and H1975 tumors. Biodistribution studies were also conducted to achieve accurate tissue uptake of this tracer in two tumor models. Hematoxylin & eosin (H&E) staining and GLUT5, Ki67 and cytokeratin-7 (CK-7) immunohistochemistry (IHC) analysis were further investigated on tumor tissues. In Western blotting, H292 cells showed higher levels of GLUT5 compared to the H1975 cells. Meanwhile, the in vitro cell assays indicated GLUT5-dependent uptake of 99m Tc-GLA in H292 and H1975 cells. The fructose competition assays showed a significant decrease in 99m Tc-GLA uptake by H292 and H1975 cells when fructose was added. The 99m Tc-GLA accumulation was as much as two-fold higher in H292 implanted tumors than in H1975 implanted tumors. 99m Tc-GLA exhibited rapid clearance pharmacokinetics and reasonable uptake in human NSCLC H292 (1.69±0.37 ID%/g) and H1975 (0.89±0.06 ID%/g) implanted tumors at 30min post injection. Finally, the expression of GLUT5, Ki67 and CK-7 on tumor tissues also exhibited positive correlation with the in vitro cell test results and in vivo SPECT/CT imaging results in xenograft tumors. Both in vitro and ex vivo studies demonstrated that the uptake of 99m Tc-GLA in NSCLC is highly related to GLUT5 expression. Imaging and further IHC results support that 99m Tc-GLA could be a promising SPECT imaging agent for NSCLC diagnosis and prognosis evaluation.

Apoptosis is a key factor in unstable plaques. The aim of this study is to evaluate the utility of visualizing atherosclerotic plaques with radiolabeled duramycin and Annexin V.ApoE[This stable, fast clearing, and highly specific [

It has been reported that the positive detection rate of Fluorine-18-fluorodeoxyglucose (The TGF-β1 antibody was obtained from Bioss Inc. The Huh-7 cell line (Liver Cancer Institute, Zhongshan Hospital, Fudan University) is a HCC cell line with high metastatic potential. Each mouse was subcutaneously injected with 5×10The average percentage of injected dose per gram of tissue (ID%) was 1,3% and 2,4%. The tumor was strongly positive for TGF-β1.This pilot study provides an experimental basis for further exploration of the feasibility of TGF-β1 receptor as a target in HCC imaging and in other cancers.

The enzyme histidine decarboxylase (Hdc), which generates histamine, is highly expressed in CD11bHdc-EGFP bacterial artificial chromosome (BAC) transgenic reporter mice (Hdc-EGFP) were used to track Hdc expression during the development of atherosclerosis. The expression of EGFP fluorescence was examined by immunofluorescence staining in a variety of adult tissues. Wild-type (WT), Apoe knockout (ApoeUsing Hdc-EGFP transgenic mice, we demonstrated that HdcModulation of histamine and Stat6-signaling may represent an attractive therapeutic strategy for the prevention or treatment of atherosclerosis.

Aim To evaluate the targeting property in vitro and in vivo of two tumor-associated glycoprotein 72 (TAG-72) binding peptides, previously identified in this laboratory by phage selection using different elution conditions. Materials and methods The peptides GGVSCMQTSPVCENNL (A2-6) and NPGTCKDKWEICLLNGG (A3-10) were radiolabeled with technetium-99m ((99m)Tc) using N-hydroxysuccinimidyl-S-acetyl-mercaptoacetyltriglycine (NHS-MAG(3)) as a chelator or were biotinylated. The specificity of the two peptides for the TAG-72 positive LS-174T cancer cells was demonstrated in vitro both by flow cytometry analysis using the biotinylated peptides and by competitive binding using the (99m)Tc-labeled peptides. The in-vivo biodistributions of the peptides were evaluated in TAG-72 positive LS-174T tumor-bearing mice by small-animal single photon emission computed tomography/computed tomography imaging. Results As evidence of specific binding, both peptides showed a significant increase in percentage binding with increasing peptide concentration by flow cytometry analysis to LS-174T cells, but not to TAG-72 negative HT-29 cells. The (99m)Tc-labeled A2-6 peptide bound LS-174T cells with an inhibition constant at 50% of 46.5 nmol/l compared with 420 nmol/l for the A3-10 peptide. In mice, accumulation of both peptides was highest in kidneys and gallbladder. Tumors were clearly visible by single photon emission computed tomography imaging for both (99m)Tc-labeled peptides through 60 min, although the tumor accumulation was higher for the A3-10 peptide. Conclusion The A3-10 peptide, with lower, yet reasonable binding affinity compared with the A2-6 peptide, showed sufficiently favorable specific binding and tumor accumulation to be considered further as a potential imaging agent for TAG-72 positive cancers.

Our objective was to compare the performance of a micro-single photon emission computed tomography (micro-SPECT) with that of a micro-positron emission tomography (microPET) in a Her2+ tumored mice using an anti-Her2 nanoparticle radiolabeled with (99m)Tc and (18)F. Camera performance was first compared using phantoms; then a tumored mouse administered the (99m)Tc-nanoparticle was imaged on a Bioscan NanoSPECT/CT, while another tumored mouse received the identical nanoparticle, labeled now with (18)F, and was imaged on a Philips Mosaic HP PET camera. The nanoparticle was radiolabeled with (99m)Tc via MAG(3) chelation and with (18)F via SFB as an intermediate. Phantom imaging showed that the resolution of the SPECT camera was clearly superior, but even with 4 heads and multipinhole collimators, detection sensitivity was 15-fold lower. Radiolabeling of the nanoparticle by chelation with (99m)Tc was considerably easier and safer than manual covalent attachment of (18)F. Both cameras provided accurate quantitation of radioactivity over a broad range. In conclusion, when deciding between (99m)Tc vs (18)F, an advantage rests with the chelation of (99m)Tc over covalent attachment of (18)F, achieved manually or otherwise, but with these small animal cameras, this choice also results in trading lower sensitivity for higher resolution.

Trastuzumab (Herceptin™) is often internalized following binding to Her2+ tumor cells. The objective of this study was to investigate whether trastuzumab can be used as a specific carrier to deliver antisense oligomers into Her2+ tumor cells both in vitro and in vivo.A biotinylated MORF oligomer antisense to RhoC mRNA and its biotinylated sense control were labeled with either lissamine for fluorescence detection or 99mTc for radioactivity detection and were linked to biotinylated trastuzumab via streptavidin. The nanoparticles were studied in SUM190 (RhoC+, Her2+) study and SUM149 (RhoC+, Her2−) control cells in culture and as xenografts in mice.As evidence of unimpaired Her2+ binding of trastuzumab within the nanoparticle, accumulations were clearly higher in SUM190 compared to SUM149 cells and, by whole-body imaging, targeting of SUM190 tumor was similar to that expected for a radiolabeled trastuzumab. As evidence of internalization, fluorescence microscopy images of cells grown in culture and obtained from xenografts showed uniform cytoplasm distribution of the lissamine-MORF. An invasion assay showed decreased RhoC expression in SUM190 cells when incubated with the antisense MORF nanoparticles at only 100 nM.Both in cell culture and in animals, the nanoparticle with trastuzumab as specific carrier greatly improved tumor delivery of the antisense oligomer against RhoC mRNA into tumor cells overexpressing Her2 and may be of general utility.

A simple and rapid method for measuring the hybridization stability of duplexes of DNAs and other oligomers in different environments is described. When added to an oligomer duplex, the thiazole orange (TO) dye intercalates and in this state is fluorescent. Therefore, when duplex dissociation occurs, the release of TO results in a detectable change in fluorescence intensity. This assay was developed primarily to screen antisense oligomer duplexes that are stable in serum and in the cytoplasm but unstable in the presence of their target messenger RNA (mRNA).The two antisense oligomers of this investigation were both 25 mer phosphorothioate (PS) DNAs, one directed against the RIalpha mRNA and the other directed against the mdr1 mRNA. The former duplex was first used in the solution studies, in most cases duplexed with a 16 mer phosphodiester (PO) complementary DNA (i.e., PS-DNA25/PO-cDNA16). Both duplexes were then tested in a series of cell studies using SK-BR-3 (RIalpha+), KB-G2 (mdr1++), and KB-31 (mdr1+/-) cells.Preliminary measurements in solution showed that maximum fluorescence was achieved when more than ten TO molecules were bound to each duplex. When a 25 mer PO-DNA or PO-RNA with the base sequence of the RIalpha mRNA was added, the dramatic change in fluorescence intensity that followed signified dissociation of the antisense DNA from the study duplex and reassociation with the target DNA. Kinetic measurements showed that this process was completed in about 3 min. Fluorescent measurements of SK-BR-3 (RIalpha+) cells incubated at 37 degrees C with the anti-RIalpha study duplex over time showed a maximum at the point where the loss of fluorescence due to dissociation of the study duplex, probably by an antisense mechanism, began to dominate over the increasing fluorescence due to continuing cellular accumulation. A similar result was observed in the KB-G2 (mdr1+) cells incubated with the anti-mdr1 study duplex.When study duplexes shown to be stable in serum were incubated with their target cells, the assay successfully detected evidence of dissociation, most likely by an antisense mechanism. Thus, a TO fluorescence assay has been developed that is capable of detecting the dissociation of DNA duplexes.

To reduce accumulation in the abdomen by MORF/cMORF pretargeting, 111In was compared to 99mTc as the radiolabel.After receiving either 99mTc (MAG3)-cMORF or 111In (DTPA)-cMORF, normal mice were imaged and killed for pharmacokinetics. Thereafter, tumored mice were pretargeted withMORF-antibody, 48 h later were given an injection of 99mTc- or 111In-cMORF, and finally were imaged repeatedly.The cMORF biodistribution in both normal and pretargeted tumored mice was influenced by its radiolabel. While excretion of both 99mTc-cMORF and 111In-cMORF was rapid and mainly through the kidneys, about 2% of 99mTc accumulated in the intestines compared toessentially no intestinal accumulation for 111In at any time. Tumor accumulation was unchanged.In applications of MORF/cMORF pretargeting intended to image organs deep within the abdomen such as the pancreas, radiolabeling with 111In may be superior to 99mTc.

It remains challenging to predict the risk of rupture for a specific atherosclerotic plaque timely, a thrombotic trigger tightly linked to inflammation. CD11b, is a biomarker abundant on inflammatory cells, not restricted to monocytes/macrophages. In this study, we fabricated a probe named as 99mTc-MAG3-anti-CD11b for detecting inflamed atherosclerotic plaques with single photon emission computed tomography/computed tomography (SPECT/CT). The ApoE-knockout (ApoE−/−) mice were selected to establish animal models, with C57BL/6J mice used for control. A higher CD11b+-cell recruitment with higher CD11b expression and more serious whole-body inflammatory status were identified in ApoE−/− mice. The probe showed high in vitro affinity and specificity to the Raw-264.7 macrophages, as well as inflammatory cells infiltrated in atherosclerotic plaques, either in ex vivo fluorescent imaging or in in vivo micro-SPECT/CT imaging, which were confirmed by ex vivo planar gamma imaging, Oil-Red-O staining and CD11b-immunohistochemistry staining. A significant positive relationship was identified between the radioactivity intensity on SPECT/CT images and the CD11b expression in plaques. In summary, this study demonstrates the feasibility of anti-CD11b antibody mediated noninvasive SPECT/CT imaging of inflammatory leukocytes in murine atherosclerotic plaques. This imaging strategy can identify inflammation-rich plaques at risk for rupture and evaluate the effectiveness of inflammation-targeted therapies in atheroma.

Multi functional probes possessing magnetic resonance imaging and single-photon emission computed tomography properties are favorable for the molecular imaging of cancers. In this study, ultra small super paramagnetic iron oxide nanoparticles, about 3.5 nm in size, were synthesized by the polyol method. The particles were functionalized using c(RGDyC) peptides and labeled with 99mTc to prepare molecular imaging probes for detecting tumor angiogenesis. The probes demonstrated good T1 (r1 = 8.2 s(-1) mM(-1)) and reasonable T2 contrast effects (r2 = 20.1 s(-1) mM(-1)) and could specifically target avβ3-positive cells, inducing more cell ingestion, unlike that in case of the control probes [functionalized with scrambled c(RADyC) peptides]. After the probes were injected into the mice bearing H1299 lung tumors, T1/T2-weighted magnetic resonance imaging and single-photon emission computed tomography revealed that they addressed tumor angiogenic vessels, which were distributed mainly in the peripheral region of tumors. Biodistribution studies indicated that tumor accumulation of the probes was significant [13.8 ± 9.6%ID/g (p0.01), which is more than that of the control probes, 4.5 ± 1.9%ID/g], and could be inhibited by free RGD peptides (6.0 ± 2.8%ID/g, p0.01). Our study demonstrated that the dual-contrast (T1/T2) magnetic resonance and dual-modal imaging probe based on ultra small superparamagnetic iron oxide nanoparticles is very promising for the molecular imaging of tumor angiogenesis.

In an effort to develop a peptide-based radiopharmaceutical for the detection of tumors overexpressed vasoactive intestinal peptide receptors with positron emission tomography, we have prepared a novel [R(8,15,21), L17]-VIP peptide for 18F-labeling. This peptide inhibited 125I-VIP binding to rats lung membranes with high affinity [half-maximal inhibitory concentrations (IC50) of 0.12 nm]. Additionally, [R(8,15,21), L17]-VIP showed higher stability than native vasoactive intestinal peptide in vivo of mice. With N-succinimidyl 4-[18F] fluorobenzoate as labeling prosthetic group, [18F]FB-[R(8,15,21), L17]-VIP was obtained in99% radiochemical purity within 100 min in decay-for-corrected radiochemical yield of 33.6 +/- 3% (n = 5) and a specific radioactivity 255 GBq/micromol at the end of synthesis. Stability of [18F]FB-[R(8,15,21), L17]-VIP in vitro and in vivo were investigated. Biodistribution of this trace was carried out in mice with induced C26 colorectal tumor. Fast clearance of [18F]FB-[R(8,15,21), L17]-VIP from non-target tissues and specific uptakes by tumors realized higher tumor-to-muscle ratio (3.55) and tumor-to-blood ratio (2.37) 60 min postinjection. Clear difference was observed between the blocking and unblocking experiments in biodistribution and whole body radioautography. [18F]FB-[R(8,15,21), L17]-VIP has demonstrated its potential for diagnosing tumors overexpressed vasoactive intestinal peptide receptors both in vitro and in vivo.

Atherosclerosis (AS), especially the vulnerable AS plaque rupture-induced acute obstructive vascular disease, is a leading cause of death. Accordingly, there is a need for an effective method to draw accurate predictions about AS progression and plaque vulnerability. Herein we report on an approach to constructing a hybrid nanoparticle system using a single-photon-emission computed tomography (SPECT)/magnetic resonance imaging (MRI) multimodal probe, aiming for a comprehensive evaluation of AS progression by achieving high sensitivity along with high resolution. Ultrasmall superparamagnetic iron oxide (USPIO) was covered by aminated poly(ethylene glycol) (PEG) and carboxylated PEG simultaneously and then functionalized with diethylenetriaminepentacetate acid for (99m)Tc coordination and subsequently Annexin V for targeting apoptotic macrophages abundant in vulnerable plaques. The in vivo accumulations of imaging probe reflected by SPECT and MRI were consistent and accurate in highlighting lesions. Intense radioactive signals detected by SPECT facilitated focus recognization and quantification, while USPIO-based T2-weighted MRI improved the focal localization and volumetry of AS plaques. For subsequent ex vivo planar images, targeting effects were further confirmed by immunohistochemistry, including CD-68 and TUNEL staining; meanwhile, the degree of concentration was proven to be statistically correlated with the Oil Red O staining results. In conclusion, these results indicated that the Annexin V-modified hybrid nanoparticle system specifically targeted the vulnerable AS plaques containing apoptotic macrophages and could be of great value in the invasively accurate detection of vulnerable plaques.

Objective This study explores a new, non-invasive imaging method for the specific diagnosis of insulinoma by providing an initial investigation of the use of 125I-labelled molecules of the glucagon-like peptide-1 (GLP-1) analogue liraglutide for in vivo and in vitro small-animal SPECT/CT (single-photon emission computed tomography/computed tomography) imaging of insulinomas. Methods Liraglutide was labelled with 125I by the Iodogen method. The labelled 125I-liraglutide compound and insulinoma cells from the INS-1 cell line were then used for in vitro saturation and competitive binding experiments. In addition, in a nude mouse model, the use of 125I-liraglutide for the in vivo small-animal SPECT/CT imaging of insulinomas and the resulting distribution of radioactivity across various organs were examined. Results The labelling of liraglutide with 125I was successful, yielding a labelling rate of approximately 95% and a radiochemical purity of greater than 95%. For the binding between 125I-liraglutide and the GLP-1 receptor on the surface of INS-1 cells, the equilibrium dissociation constant (Kd) was 128.8±30.4 nmol/L(N = 3), and the half-inhibition concentration (IC50) was 542.4±187.5 nmol/L(N = 3). Small-animal SPECT/CT imaging with 125I-liraglutide indicated that the tumour imaging was clearest at 90 min after the 125I-liraglutide treatment. An examination of the in vivo distribution of radioactivity revealed that at 90 min after the 125I-liraglutide treatment, the target/non-target (T/NT) ratio for tumour and muscle tissue was 4.83±1.30(N = 3). Our study suggested that 125I-liraglutide was predominantly metabolised and cleared by the liver and kidneys. Conclusion The radionuclide 125I-liraglutide can be utilised for the specific imaging of insulinomas, representing a new non-invasive approach for the in vivo diagnosis of insulinomas.

The farnesoid-X-receptor (FXR) is a member of the nuclear hormone receptor superfamily. The FXR has critical functions in maintaining bile acid synthesis and homeostasis, liver regeneration and tumorigenesis, intestinal diseases, intestinal tumorigenesis, cholesterol gallstone disease, cholestasis, and atherosclerosis. FXR expression is strongly downregulated in liver fibrosis, hepatocellular adenoma and hepatocellular carcinoma compared to expression levels in adjacent normal tissues. Chenodeoxycholic acid (CDCA) is the most potent physiological ligand for FXR. CDCA was radiolabeled with (18)F based on the efficiency click reaction of 1,3-dipolar cycloaddition of terminal alkynes and organic azides for noninvasively evaluating the relationship between FXR and FXR-related disease. The PET tracer [(18)F]8 was produced by 'click' labeling and showed a high non-decay corrected radiochemical yield (end of synthesis (EOS) yield=42±3% (n=5) from aqueous [(18)F]fluoride), high radiochemical purity ( >99%), and high specific activity (>320GBq/μmol). [(18)F]8 had a high metabolic stability in vitro and in vivo. PET imaging studies in nude mice indicated a rapid uptake of the tracer into liver tissue with uniform distribution of radioactivity in the liver. Significant accumulation of radioactivity was found in the liver, gallbladder, and intestine, while no obvious uptake was observed in other organs, such as the bladder, heart, and brain. Thus, this PET tracer represents a novel tool for early detection of abnormalities in the liver and staging of neoplasms.

Insulinoma is a neuroendocrine tumor derived from the β cells of pancreatic islets. They are usually relatively inaccessible for surgical intervention. High expression levels of glucagon-like peptide-1 (GLP-1) receptor have been detected in insulinoma.The aim of the study was to evaluate the potential of F-radiolabeled GLP-1 analog exendin-4 for the diagnosis of insulinoma using PET/computed tomography imaging.The GLP-1 receptor-specific molecular probe [F]FB-exendin-4 was prepared by the conjugation of exendin-4 and N-succinimidyl-4-[F] fluorobenzoate ([F]SFB). High expression of GLP-1 by the RIN-m5f insulinoma line and GLP-1 receptor specificity were evaluated by determining the saturation curve for in-vitro binding of I-radiolabeled exendin-4 and by investigation of the competitive binding between I-radiolabeled and unlabeled exendin-4. Further, the in-vivo biodistribution and micro-PET/computed tomography images of insulinoma-bearing mice were studied.An overall radiochemical yield of 35.6±2.3% (decay corrected, n=5) and specific radioactivity of around 30 GBq/µmol were achieved for [F]FB-exendin-4, and the radiochemical purity was over 98%. Both in-vitro and in-vivo studies confirmed the specificity of [F]FB-exendin-4 to insulinoma cells.[F]FB-exendin-4 has been found to be an effective molecular imaging probe for detecting insulinomas.

Radiolabeled oligomers complementary to the 16S rRNA in bacteria were investigated as bacterial infection imaging agents.Identical sequences with backbones phosphorodiamidate morpholino (MORF), peptide nucleic acid (PNA), and phosphorothioate DNA (PS-DNA) were (99m)Tc-labeled and evaluated for binding to bacterial RNA. MORF binding to RNA from Escherichia coli strains SM101 and K12 was 4- and 150-fold higher compared to PNA and PS-DNA, respectively. Subsequently MORF oligomer in fluorescence in situ hybridization showed a stronger signal with study MORF compared to control in fixed preparations of two E. coli strains and Klebsiella pneumoniae. Flow cytometry analysis showed study MORF accumulation to be 8- and 80-fold higher compared to the control in live K. pneumoniae and Staphylococcus aureus, respectively. Further, fluorescence microscopy showed increased accumulation of study MORF over control in live E. coli and K. pneumonia. Binding of (99m)Tc-study MORF to RNA from E. coli SM101 and K12 was 30.4 and 117.8pmol, respectively, per 10(10) cells. Mice with K. pneumoniae live or heat-killed (sterile inflammation) in one thigh at 90min for both (99m)Tc-study MORF and control showed higher accumulation in target thighs than in blood and all other organs expect for kidneys and small intestine. Accumulation of (99m)Tc-study MORF was significantly higher (p=0.009) than that of the control in the thigh with sterile inflammation.A (99m)Tc-MORF oligomer complimentary to the bacterial 16S rRNA demonstrated binding to bacterial RNA in vitro with specific accumulation into live bacteria. Radiolabeled MORF oligomers antisense to the bacterial rRNA may be useful to image bacterial infection.

As large amount of vasoactive intestinal peptide (VIP) receptors are expressed in various tumors and VIP-related diseases, radiolabeled VIP provides a potential PET imaging agent for VIP receptor. However, structural modification of VIP is required before being radiolabeled and used for VIP receptor imaging due to its poor in vivo stability. As a VIP analogue, [R8, 15, 21, L17]-VIP exhibited improved stability and receptor specificity in preliminary studies. In this study, F-18 labeled [R8,15,21, L17]-VIP was produced with the radiochemical yield being as high as33.6%±3%(decay-for-corrected,n=5) achieved within 100 min, a specific activity of 255 GBq/μmol, and a radiochemical purity as high as 99% as characterized by radioactive HPLC, TLC, and SDS-Page radioautography. A biodistribution study in normal mice also demonstrated fast elimination of F-18 labeled [R8,15,21, L17]-VIP in the blood, liver, and gastrointestinal tracts. A further micro-PET imaging study in C26 colon carcinoma bearing mice confirmed the high tumor specificity, with the tumor/muscle radioactivity uptake ratio being as high as 3.03 at 60 min following injection, and no apparent radioactivity concentration in the intestinal tracts. In addition, blocking experiment and Western Blot test further confirmed its potential in PET imaging of VIP receptor-positive tumor.

Purpose Invasive aspergillosis is a major cause of infectious morbidity and mortality in immunocompromised patients. The fungus Aspergillus fumigatus (A. fumigatus) is the primary causative agent of invasive aspergillosis. However, A. fumigatus infections remain difficult to diagnose particularly in the early stages due to the lack of a rapid, sensitive and specific diagnostic approach. In this study, we investigated 99m Tc labeled MORF oligomers targeting fungal ribosomal RNA (rRNA) for the imaging detection of fungal infections. Procedures Three phosphorodiamidate morpholino (MORF) oligomer (a DNA analogue) probes were designed: AGEN, complementary to a sequence of the fungal 28S ribosomal RNA (rRNA) of Aspergillus, as a genus-specific probe; AFUM, complementary to the 28S rRNA sequence of A. fumigatus, as a fungus species-specific probe; and cMORF, irrelevant to all fungal species, as a control probe. The probes were conjugated with Alexa Fluor 633 carboxylic acid succinimidyl ester (AF633) for fluorescence imaging or with NHS-mercaptoacetyl triglycine (NHS-MAG3) for nuclear imaging with 99m Tc and then evaluated in vitro and in vivo. Results The specific binding of AGEN and AFUM to fungal total RNA was confirmed by dot blot hybridization while specific binding of AGEN and AFUM in fixed and live A. fumigatus was demonstrated by both fluorescent in situ hybridization (FISH) analysis and accumulation in live cells. SPECT imaging of BALB/c mice with pulmonary A. fumigatus infections and administered 99m Tc labeled AGEN and AFUM showed immediate and obvious accumulation in the infected lungs, while no significant accumulation of the control 99m Tc-cMORF in the infected lung was observed. Compared to non-infected mice, with sacrifice at 1h, the accumulation of 99m Tc-AGEN and 99m Tc-AFUM in the lungs of mice infected with A. fumigatus was 2 and 2.7 fold higher respectively. Conclusions In vivo targeting fungal ribosomal RNA with 99m Tc labeled MORF probes AGEN and AFUM may be useful for A. fumigatus infection imaging and may provide a new strategy for the noninvasive diagnosis of invasive aspergillosis and other fungal infections.

Introduction A zinc-dipicolylamine analog (Zn-DPA) conjugated with a fluorophore (PSVue®794) has been shown to image bacterial infections in mice. However, radiolabeled Zn-DPA has not previously been considered for nuclear imaging of infection. Methods Both 111 In-labeled DOTA-biotin and Zn-DPA-biotin were combined using streptavidin (SA) as a noncovalent linker. Mice injected intramuscularly with Streptococcus pyogenes (infection model) or with lipopolysaccharide (LPS) (inflammation model) were coinjected intravenously with 6 μg of DPA as PSVue794 and as 111 In-DOTA-biotin/SA/biotin-Zn-DPA. Periodic fluorescent and SPECT (single photon emission computed tomography)/CT (computed tomography) images were acquired, and biodistributions were obtained at 22 h. Results Histological examination confirmed the validity of both the infection and inflammation animal models. Both the whole-body optical and nuclear images showed obvious accumulations in the target thigh in both models at all time points. At 22 h, the average target thigh accumulation of 111 In was 1.66%ID/g (S.D. 0.15) in the infection mice compared to 0.58%ID/g (S.D. 0.07) in the inflammation mice ( P 111 In target/normal thigh ratio was 2.8 fold higher in the infection animals compared to the inflammation animals. Conclusions These preliminary results show that Zn-DPA within streptavidin targets S. pyogenes -infected mice similarly to its free fluorescent analogue. The significantly higher accumulation in the live bacterial infection thigh compared to that of the LPS-induced inflammation thigh suggests that Zn-DPA may be a promising imaging agent to distinguish between bacterial infections and sterile inflammations.

Noninvasive measurement of human islet cell mass in pancreas or following islet transplantation by nuclear imaging has yet to be achieved. It has been shown using mouse tumor models that pretargeting imaging strategies are sensitive and can greatly increase target to nontarget signal ratios. The objective now is to demonstrate the specific pretargeting of human islet cells in mice. Our pretargeting strategy uses an anti-human islet cell antibody HPi1, conjugated to a phosphorodiamidate morpholino oligomer (MORF) that binds specifically to a (99m)Tc labeled complementary MORF (cMORF). Sensitivity and specificity of the pretargeting were first validated in culture using a human beta cell line (betalox5) and a negative control human cell line (HEK293). Pretargeting was then used to target and visualize these two cell lines and human islets transplanted subcutaneously in NOD-scid IL2rγ(null) mice. In culture, (99m)Tc accumulation on the betalox5 cells pretargeted by MORF-HPi1 was 100-fold higher than on untreated betalox5 cells or following treatment with native HPi1 and much higher than on the MORF-HPi1 pretargeted control HEK293 cells. Small animal imaging readily localized the transplanted betalox5 cells and human islets, but not the HEK293 cells. Ex vivo counting demonstrated 3-fold higher (99m)Tc accumulation in the transplanted betalox5 cells and human islets than in the control HEK293 cells. The target accumulation was also shown to increase linearly with increased numbers of the implanted betalox5 cells. These results demonstrate specific binding of radioactivity and successful imaging of human betalox5 cells and human islets transplanted in mice. Thus MORF/cMORF pretargeting may be useful to measure noninvasively human islet cell mass within the pancreas or following islet transplantation.

Recent advances in small-animal molecular imaging instrumentation combined with well characterized antibody-labeling chemistry have enabled detailed in vivo measurements of antibody distribution in mouse models. This article reviews the strengths and limitations of in vivo antibody imaging methods with a focus on positron emission tomography and single-photon emission computed tomography and a brief discussion of the role of optical imaging in this application. A description of the basic principles behind the imaging techniques is provided along with a discussion of radiolabeling methods relevant to antibodies. Practical considerations of study design and execution are presented through a discussion of sensitivity and resolution tradeoffs for these techniques as defined by modality, signaling probe (isotope or fluorophore) selection, labeling method, and radiation dosimetry. Images and analysis results from a case study are presented with a discussion of output data content and relevant informatics gained with this approach to studying antibody pharmacokinetics.

Phage display was used to select novel peptides that specifically bind the TAG-72 antigen and with properties suitable for imaging TAG-72 positive cancers.After three rounds of selection against TAG-72 and using two different elution conditions including a long elution, the consensus sequences FRERCDKHPQKCTKFL and DPRHCQKRVLPCPAWL were expressed on phages G3-15 and T3-15 respectively. ELISA, fluorescence-activated cell sorting analysis and fluorescence microscopy provided evidence that both phages specifically bound TAG-72 in vitro. Both peptides are stable in 37oC serum. By a cell binding competition assay, the IC50 for T3-15 was measured as 0.29 nM and therefore 36-fold higher affinity than G3-15 at 10.32 nM. The biodistribution in mice carrying LS-174T tumors in one thigh were similar for both 99mTc-peptides at 30 min, but at 90 min the 99mTc-T3-15 peptide accumulated almost three times higher in the tumor. The SPECT/CT images were consistent with the biodistribution results.The f88-4/Cys6 phage library and two different elution conditions were used to identify two new higher affinity binding peptides for the TAG-72 antigen. One, was a single brief elution with pH 2.2 glycine buffer, and the second began with the glycine elution but was followed with a longer elution with Tris buffered saline (TBS) at pH 7.4. The phages that bound TAG-72 were evaluated by fluorescence-activated cell sorting analysis using TAG-72 positive LS-174T cells and confirmed by immunofluorescence imaging. The consensus peptides displayed on the selected phages were synthesized and conjugated with NHS-MAG3 for radiolabeling with 99mTc. The IC50 for TAG-72 binding was evaluated by cell binding competition in vitro while binding affinity was evaluated in vivo by necropsy and SPECT/CT imaging in a tumor mouse model.We have identified a peptide with a sub nanomolar inhibition constant for the TAG-72 antigen that may have application in cancer imaging.

In principle, a DNA duplex consisting of an antisense fluorophore-conjugated major strand hybridized to a shorter complementary inhibitor-conjugated minor strand should provide fluorescence only in the tumor after intravenous administration if designed to remain intact except in the presence in tumor of its mRNA target. While we have obtained impressive tumor images in mice using this approach, there remains some background fluorescence. In this study, tissue homogenates of selected mouse organs were incubated with a test duplex and the kinetics of duplex dissociation in normal tissues were measured. In this manner we were able to identify the liver as the likely major source responsible for the duplex dissociation providing this fluorescence background. Thereafter liver homogenates were used to screen a series of duplex candidates with variable-length minor strands, and dissociation was measured by gel electrophoresis. The selected fluorophore/inhibitor duplex with improved stability displayed an insignificant (P > 0.05) background fluorescence after administration to SKH-1 normal mice and apparently without affecting target mRNA binding in vitro in cell culture or in vivo in tumor bearing mice.

The utility of MORF/cMORF pretargeting for the radiotherapy of cancer requires further validation in tumored mice before clinical trials. We now report on a therapeutic study in mice pretargeted with MORF-CC49 (the anti-TAG-72 antibody CC49 conjugated with MORF, a phosphorodiamidate morpholino oligomer) and then targeted by 188Re-cMORF (a 188Re labeled complementary MORF). Before the dose-escalating therapeutic study, a pretargeting study in LS174T tumored mice was performed at tracer levels. By both necropsy and imaging, the tracer study showed that the whole body radioactivity was largely restricted to tumor in the mice pretargeted 48 h earlier with MORF-CC49 and the tumor radioactivity was retained over 90 h. After decay correction, a best-fit to the biodistribution provided the areas under the radioactivity curves (AUCs) used for the radiation dose estimates. The tumor to normal organ AUC ratios in all cases were greater than unity and ranged from 3 (kidneys) to 48 (muscle). Tumor growth was inhibited in the therapy study. At the highest 188Re dose of 1.40 mCi, a complete but temporary tumor remission was evident in three out of the five animals. Histological examination of tissues from these animals showed no evidence of cytotoxicity to normal tissues but obvious radiation damage to tumor. In conclusion, effective radiotherapy was achieved in a mouse model by MORF/cMORF pretargeting using 188Re as the therapeutic radionuclide and CC49 as the pretargeting antibody.

Objective: Recently 99mTc-glucarate, a radiolabeled glucose analogue, has been considered as a SPECT alternative to 18F-FDG and PET for non-invasive detection of certain tumors. Thus far there have been few studies on 99mTcglucarate for tumor imaging and fewer, if any, studies comparing 99mTc-glucarate with 18F-FDG. As a preliminary indication of the properties of 99mTc-glucarate as a possible substitute for 18F-FDG in animal studies, we have imaged mice bearing xenografts of four tumor types with 99mTc-glucarate and have compared in two mice with one of these tumor types the 99mTc and 18F biodistributions. Methods: Two mice bearing SUM190 breast cancer xenografts received 1 mCi of 99mTc-glucarate and were imaged on a NanoSPECT/CT small animal camera. One day later, the same animals received 1 mCi of 18F-FDG and were imaged on a MosaicHP PET small animal camera. In addition, 0.5-1 mCi of 99mTc-glucarate only was administered to mice bearing xenografts induced by BxPC3 pancreatic cancer cells, HEK-293 renal cell carcinomas cells or HCT-116 colorectal tumor cells. NanoSPECT/CT acquisitions were performed in these mice to evaluate tumor accumulations. Results: In the SUM190 xenografted mice, the average tumor accumulation was 1.4 % (ID%/cm3) for 99mTc-glucarate and 2.1 % (ID%/cm3) for 18F-FDG. While slightly higher than 99mTc-glucarate, the tumor accumulation of 18F-FDG was accompanied by higher bone marrow and muscle accumulations at levels that could interfere with the tumor image depending upon location. The whole body clearance of 99mTc-glucarate was faster than that of 18F-FDG. Tumor accumulation of 99mTc-glucarate varied among tumor types but the tumors were readily visible in all images. Conclusion: In a direct comparison in the same two SUM190 tumored animals, SPECT images obtained with 99mTcglucarate compared favorably with PET images obtained with 18F-FDG. Tumor images with 99mTc-glucarate were also positive in three additional tumor mouse models. While further comparison studies are necessary, we conclude that 99mTcglucarate may be a more convenient and less expensive alternative to 18F-FDG for tumored mouse studies.

Purpose: The incidence of bone metastasis in advanced breast cancer (BrCa) exceeds 70%. Bortezomib, a proteasome inhibitor used for the treatment of multiple myeloma, also promotes bone formation. We tested the hypothesis that proteasome inhibitors can ameliorate BrCa osteolytic disease. Experimental Design: To address the potentially beneficial effect of bortezomib in reducing tumor growth in the skeleton and counteracting bone osteolysis, human MDA-MB-231 BrCa cells were injected into the tibia of mice to model bone tumor growth for in vivo assessment of treatment regimens before and after tumor growth. Results: Controls exhibited tumor growth, destroying trabecular and cortical bone and invading muscle. Bortezomib treatment initiated following inoculation of tumor cells strikingly reduced tumor growth, restricted tumor cells mainly to the marrow cavity, and almost completely inhibited osteolysis in the bone microenvironment over a 3- to 4-week period as shown by [18F]fluorodeoxyglucose positron emission tomography, micro–computed tomography scanning, radiography, and histology. Thus, proteasome inhibition is effective in killing tumor cells within the bone. Pretreatment with bortezomib for 3 weeks before inoculation of tumor cells was also effective in reducing osteolysis. Our in vitro and in vivo studies indicate that mechanisms by which bortezomib inhibits tumor growth and reduces osteolysis result from inhibited cell proliferation, necrosis, and decreased expression of factors that promote BrCa tumor progression in bone. Conclusion: These findings provide a basis for a novel strategy to treat patients with BrCa osteolytic lesions, and represent an approach for protecting the entire skeleton from metastatic bone disease. Clin Cancer Res; 16(20); 4978–89. ©2010 AACR.

In connection with pretargeting, an amine-derivatized morpholino phosphorodiamidate oligomer (NH(2)-cMORF) was conjugated conventionally with p-isothiocyanate benzyl-DTPA (p-SCN-Bn-DTPA). However, after (111)In radiolabeling, unexpected label instability was observed. To understand this instability, the NH(2)-cMORF and, as control, the native cMORF without the amine were conjugated in the conventional manner. Surprisingly, the (111)In labeling of the native cMORF conjugate was equally effective as that of the NH(2)-cMORF conjugate (>95%) despite the absence of the amine group. Furthermore, heating the radiolabeled NH(2)-cMORF and native cMORF conjugates resulted in a 35% loss and a complete loss of the label, respectively. Since the (111)In labeled DTPA is known to be stable, the instability in both cases must be due to some unstable association of DTPA to the cMORF, presumably unstable association to some endogenous sites in cMORF. Based on this assumption, a postconjugation-prepurification heating step was introduced, and labeling efficiency and stability were again investigated. By introducing the heating step, the side products were dissociated, and after purification and labeling, the NH(2)-cMORF conjugate provided a stable label and high labeling efficiency with no need for postlabeling purification. The biodistribution of this radiolabeled conjugate in normal mice showed significantly lower backgrounds compared with the labeled unstable native cMORF conjugate. In conclusion, the conventional conjugation procedure to attach the p-SCN-Bn-DTPA to NH(2)-cMORF resulted in side product(s) that were responsible for the (111)In label instability. Adding a postconjugation-prepurification heating step dissociated the side products, improved the label stability and lowered tissue backgrounds in mice.

Combining two or more different imaging modalities in the same agent can be of considerable value in molecular imaging. We describe the use of streptavidin nanoparticle-based complexes as multimodality imaging agents to achieve tumor detection in a mouse model by both fluorescence and nuclear imaging. Up to four biotinylated functionalities can be readily attached to these streptavidin nanoparticles without apparent influence on their properties and with reasonable pharmacokinetics and therefore may be ideally suited for multimodality imaging. By binding a biotinylated anti-Her2 Herceptin antibody to provide tumor targeting, a biotinylated DOTA chelator labeled with (111)ln and a biotinylated Cy5.5 fluorophore to a streptavidin nanoparticle, we demonstrated multimodality imaging in SUM190 (Her2+) tumor bearing mice on both an IVIS fluorescence camera and a NanoSPECT/CT small animal nuclear camera. The imaging results show high tumor accumulation and strong tumor-to-normal tissue contrast by both fluorescence and nuclear imaging. The subsequent biodistribution study confirmed the specific tumor accumulation in that tumor accumulation of radioactivity at 40 h was 21 ID%/g and therefore much higher than all other tissues including liver, heart, kidney, spleen, and muscle that accumulated 8.7, 2.5, 6.9, 7.2, and 1.9 ID%/g, respectively. In conclusion, the streptavidin nanoparticle under development in this laboratory was used effectively for multimodality imaging of tumor in mice by fluorescence and nuclear detection. Presumably, other imaging modalities could also be considered.

Fluorescent conjugated DNA oligonucleotides for antisense targeting of mRNA has the potential of improving tumor/normal tissue ratios over that achievable by nuclear antisense imaging. By conjugating the Cy5.5 emitter to the 3' equivalent end of a 25 mer phosphorothioate (PS) antisense major DNA and hybridizing with a shorter 18 mer phosphodiester (PO) complementary minor DNA (cDNA) with the Black Hole inhibitor BHQ3 on its 5' end (i.e., PS DNA25-Cy5.5/PO cDNA18-BHQ3), we previously achieved antisense optical imaging in mice as a proof of this concept. In a process of optimization, we have now evaluated the stability of a small series of duplexes with variable-length minor strands. From these results, a new study anti-mdr1 antisense duplex was selected with a 10 mer minor strand (i.e., PS DNA25-Cy5.5/PO cDNA10-BHQ3). The new study duplex shows stability in serum environments at 37 degrees C and provides a dramatically enhanced fluorescence in KB-G2 (pgp++) cells when compared with KB-31 (pgp+/-) as evidence of antisense dissociation at its mdr1 mRNA target. The duplex was also administered to KB-G2 tumor bearing mice, and when compared to the duplex used previously, the fluorescence from the tumor thigh was more obvious and the tumor-to-background fluorescence ratio was improved. In conclusion, by a process designed to optimize the duplex for optical antisense tumor targeting, the fluorescence signal was improved both in cells and in tumored mice.

When antisense oligomers are intracellular, they migrate to and are retained in the nucleus of tumor cells and therefore may be used to carry Auger electron–emitting radionuclides such as 111In for effective tumor radiotherapy. Methods: Our nanoparticle consists of streptavidin that links 3 biotinylated components: the antiHer2 antibody trastuzumab (to improve pharmacokinetics), the tat peptide (to improve cell membrane transport), and the 111In-labeled antiRIα messenger RNA antisense morpholino (MORF) oligomer. Results: As evidence of unimpaired function, tumor cell and nuclear accumulations were orders of magnitude higher after incubation with 99mTc-MORF/tat/trastuzumab than after incubation with free 99mTc-MORF and significantly higher with the antisense than with the sense MORF. In mice, tumor and normal-tissue accumulations of the 99mTc-MORF/tat/trastuzumab nanoparticle were comparable to those of free 99mTc-trastuzumab, confirming the improved pharmacokinetics due to the trastuzumab component. Although kidneys, liver, and other normal tissues also accumulated the nanoparticle, immunohistochemical evaluation of tissue sections in mice receiving the Cy3-MORF/tat/trastuzumab nanoparticle showed evidence of nuclear accumulation only in tumor tissue. In a dose escalation study, as measured by the surviving fraction, the nanoparticle significantly increased the kill of SK-BR-3 breast cancer Her2+/RIα+ cells, compared with all controls. Conclusion: Significant radiation-induced antisense-mediated cytotoxicity of tumor cells in vitro was achieved using an Auger electron–emitting antisense MORF oligomer administered as a member of a 3-component streptavidin-delivery nanoparticle.

BACKGROUND Radiolabelled vasoactive intestinal peptide (VIP) and its analogues have shown their potential as imaging agents for diagnosing tumours expressing VIP receptor. However, the fast proteolytic degradation in vivo has limited their clinical use. AIM To prepare the 18F-labelled (R8,15,21, L17)-VIP analogue in a convenient way and to evaluate its potential as an imaging agent for VIP receptor-positive tumours. METHODS Radiolabelled (R8,15,21, L17)-VIP was obtained by conjugation with N-succinimidyl 4-([18F]fluoromethyl) benzoate and purified by HPLC. Radiochemical purity and specific radioactivity were measured by analytical HPLC. In-vitro stability of the product was carried out in HSA solution and analysed by HPLC. Biodistribution study was carried out in mice bearing C26 colorectal tumours. RESULTS 18F-(R8,15,21, L17)-VIP was obtained in greater than 99% radiochemical purity within 60 min in decay-for-corrected radiochemical yields of 21.8+/-4.7% (n=5) and a specific activity of 17.76 GBq x mumol(-1) at the end of synthesis (EOS). Results of in-vitro studies demonstrated a high stability in human serum albumin (HSA) solution. Biodistribution data showed a rapid blood clearance and specific binding towards receptor-positive tumours. CONCLUSION 18F-(R8,15,21, L17)-VIP was prepared by a convenient method. Preliminary biodistribution results showed its potential for imaging tumours over-expressing VIP receptors and encouraged further investigation.

Introduction 2-Amino-6-[18F]fluoro-9-(4-hydroxy-3-hydroxy-methylbutyl) purine (6-[18F]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-[18F]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-[18F]FPCV was performed on another group of animals at different time points. Results Log P of 6-[18F]FPCV was −0.517. 6-[18F]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-[18F]FPCV was as follows: α t1/2=1.2 min and β t1/2=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-[18F]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.