Preclinical evaluation of a 64Cu-labeled disintegrin for PET imaging of prostate cancer.

A novel recombinant disintegrin, vicrostatin (VCN), displays high binding affinity to a broad range of human integrins in substantial competitive biological advantage over other integrin-based antagonists. In this study, we synthesized a new 64Cu-labeled VCN probe and evaluated its imaging properties for prostate cancer in PC-3 tumor-bearing mice. Macrocyclic chelating agent 1,8-diamino-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosine (DiAmSar) was conjugated with PEG unit and followed by coupling with VCN. The precursor was then radiolabeled with positron emitter 64Cu (t1/2 = 12.7 h) in ammonium acetate buffer to provide 64Cu-Sar-PEG-VCN, which was subsequently subjected to in vitro studies, small animal PET, and biodistribution studies. The PC-3 tumor-targeting efficacy of 64Cu-Sar-PEG-VCN was compared to a cyclic RGD peptide-based PET probe (64Cu-Sar-RGD). 64Cu labeling was achieved in 75% decay-corrected yield with radiochemical purity of > 98%. The specific activity of 64Cu-Sar-PEG-VCN was estimated to be 37 MBq/nmol. MicroPET imaging results showed that 64Cu-Sar-PEG-VCN has preferential tumor uptake and good tumor retention in PC-3 tumor xenografts. As compared to 64Cu-Sar-RGD, 64Cu-Sar-PEG-VCN produces higher tumor-to-muscle (T/M) imaging contrast ratios at 2 h (4.66 ± 0.34 vs. 2.88 ± 0.46) and 24 h (4.98 ± 0.80 vs. 3.22 ± 0.30) post-injection (pi) and similar tumor-to-liver ratios at 2 h (0.43 ± 0.09 vs. 0.37 ± 0.04) and 24 h (0.57 ± 0.13 vs. 0.52 ± 0.07) pi. The biodistribution results were consistent with the quantitative analysis of microPET imaging, demonstrating good T/M ratio (2.73 ± 0.36) of 64Cu-Sar-PEG-VCN at 48 h pi in PC-3 tumor xenografts. For both microPET and biodistribution studies at 48 h pi, the PC-3 tumor uptake of 64Cu-Sar-PEG-VCN is lower than that of 64Cu-Sar-RGD. 64Cu-Sar-PEG-VCN has the potential for in vivo imaging of prostate cancer with PET, which may provide a unique non-invasive method to quantitatively localize and characterize prostate cancer.

Loss of ER retention motif of AGR2 can impact mTORC signaling and promote cancer metastasis.

Anterior gradient 2 (AGR2) is a member of the protein disulfide isomerase (PDI) family, which plays a role in the regulation of protein homeostasis and the unfolded protein response pathway (UPR). AGR2 has also been characterized as a proto-oncogene and a potential cancer biomarker. Cellular localization of AGR2 is emerging as a key component for understanding the role of AGR2 as a proto-oncogene. Here, we provide evidence that extracellular AGR2 (eAGR2) promotes tumor metastasis in various in vivo models. To further characterize the role of the intracellular-resident versus extracellular protein, we performed a comprehensive protein-protein interaction screen. Based on these results, we identify AGR2 as an interacting partner of the mTORC2 pathway. Importantly, our data indicates that eAGR2 promotes increased phosphorylation of RICTOR (T1135), while intracellular AGR2 (iAGR2) antagonizes its levels and phosphorylation. Localization of AGR2 also has opposing effects on the Hippo pathway, spheroid formation, and response to chemotherapy in vitro. Collectively, our results identify disparate phenotypes predicated on AGR2 localization. Our findings also provide credence for screening of eAGR2 to guide therapeutic decisions.

Effect of Androgen on Normal Biodistribution of [18F]-2'-Fluoro-5-methyl-1-beta-D-arabinofuranosyluracil (18F-FMAU) in Athymic Non-tumor-bearing Male Mice.

AIM: We assessed the association between the presence and absence of androgen on the normal biodistribution of the positron emission tomography (PET) cellular proliferation imaging biomarker, [18F]-2'-Fluoro-5-methyl-1-beta-D-arabinofuranosyluracil (18F-FMAU), in mice. MATERIALS AND METHODS: Non-castrated (n=4) and castrated (n=4) athymic non-tumor-bearing male mice served as models for presence and absence, respectively, of androgen. MicroPET-CT scans were performed 1 h following tail vein administration of 200 uCi of 18F-FMAU. Imaging was performed at baseline and then at 7-day intervals longitudinally for 35 days only in castrated mice following subcutaneous introduction of a 12.5 mg, 21-day release, dihydrotestosterone pellet. Mean standardized uptake values (SUVmean) were obtained for liver, heart, and muscle. Several two-group comparisons of average of SUVmean were performed. RESULTS: Pre-pellet baseline average SUVmean (±s.d.) values in castrated mice were significantly lower than baseline non-castrated values, increased on day 15 and reached peak values on day 28, at which time they were significantly higher than corresponding baseline levels in both non-castrated and pre-pellet castrated mice. The peak values decreased significantly following dihydrotestosterone withdrawal. CONCLUSION: There is a significant modulatory effect of androgen on normal 18F-FMAU uptake levels in mice liver, heart and muscle tissues.

A Nanoscale Tool for Photoacoustic-Based Measurements of Clotting Time and Therapeutic Drug Monitoring of Heparin.

Heparin anticoagulation therapy is an indispensable feature of clinical care yet has a narrow therapeutic window and is the second most common intensive care unit (ICU) medication error. The active partial thromboplastin time (aPTT) monitors heparin but suffers from long turnaround times, a variable reference range, limited utility with low molecular weight heparin, and poor correlation to dose. Here, we describe a photoacoustic imaging technique to monitor heparin concentration using methylene blue as a simple and Federal Drug Administration-approved contrast agent. We found a strong correlation between heparin concentration and photoacoustic signal measured in phosphate buffered saline (PBS) and blood. Clinically relevant heparin concentrations were detected in blood in 32 s with a detection limit of 0.28 U/mL. We validated this imaging approach by correlation to the aPTT (Pearson's r = 0.86; p < 0.05) as well as with protamine sulfate treatment. This technique also has good utility with low molecular weight heparin (enoxaparin) including a blood detection limit of 72 µg/mL. We then used these findings to create a nanoparticle-based hybrid material that can immobilize methylene blue for potential applications as a wearable/implantable heparin sensor to maintain drug levels in the therapeutic window. To the best of our knowledge, this is the first use of photoacoustics to image anticoagulation therapy with significant potential implications to the cardiovascular and surgical community.

A Periodic Diet that Mimics Fasting Promotes Multi-System Regeneration, Enhanced Cognitive Performance, and Healthspan.

Prolonged fasting (PF) promotes stress resistance, but its effects on longevity are poorly understood. We show that alternating PF and nutrient-rich medium extended yeast lifespan independently of established pro-longevity genes. In mice, 4 days of a diet that mimics fasting (FMD), developed to minimize the burden of PF, decreased the size of multiple organs/systems, an effect followed upon re-feeding by an elevated number of progenitor and stem cells and regeneration. Bi-monthly FMD cycles started at middle age extended longevity, lowered visceral fat, reduced cancer incidence and skin lesions, rejuvenated the immune system, and retarded bone mineral density loss. In old mice, FMD cycles promoted hippocampal neurogenesis, lowered IGF-1 levels and PKA activity, elevated NeuroD1, and improved cognitive performance. In a pilot clinical trial, three FMD cycles decreased risk factors/biomarkers for aging, diabetes, cardiovascular disease, and cancer without major adverse effects, providing support for the use of FMDs to promote healthspan.

Improved metabolic stability for 18F PET probes rapidly constructed via tetrazine trans-cyclooctene ligation.

The fast kinetics and bioorthogonal nature of the tetrazine trans-cyclooctene (TCO) ligation makes it a unique tool for PET probe construction. In this study, we report the development of an (18)F-labeling system based on a CF3-substituted diphenyl-s-tetrazine derivative with the aim of maintaining high reactivity while increasing in vivo stability. c(RGDyK) was tagged by a CF3-substituted diphenyl-s-tetrazine derivative via EDC-mediated coupling. The resulting tetrazine-RGD conjugate was combined with a (19)F-labeled TCO derivative to give HPLC standards. The analogous (18)F-labeled TCO derivative was combined with the diphenyl-s-tetrazine-RGD at µM concentration. The resulting tracer was subjected to in vivo metabolic stability assessment, and microPET studies in murine U87MG xenograft models. The diphenyl-s-tetrazine-RGD combines with an (18)F-labeled TCO in high yields (>97% decay-corrected on the basis of TCO) using only 4 equiv of tetrazine-RGD relative to the (18)F-labeled TCO (concentration calculated based on product's specific activity). The radiochemical purity of the (18)F-RGD peptides was >95% and the specific activity was 111 GBq/µmol. Noninvasive microPET experiments demonstrated that (18)F-RGD had integrin-specific tumor uptake in subcutaneous U87MG glioma. In vivo metabolic stability of (18)F-RGD in blood, urine, and major organs showed two major peaks: one corresponded to the Diels-Alder conjugate and the other was identified as the aromatized analog. A CF3-substituted diphenyl-s-tetrazine displays excellent speed and efficiency in (18)F-PET probe construction, providing nearly quantitative (18)F labeling within minutes at low micromolar concentrations. The resulting conjugates display improved in vivo metabolic stability relative to our previously described system.

Tumor targeting of a cell penetrating peptide by fusing with a pH-sensitive histidine-glutamate co-oligopeptide.

Cell penetrating peptides (CPPs) have been well established as potential carriers for intracellular delivery of protein/peptide therapeutics. However, their lack of selectivity impedes their application in vivo. In order to increase their specificity, a highly pH-sensitive histidine-glutamate (HE) co-oligopeptide was fused with a CPP, i.e. model amphipathic peptide (MAP), and was expressed as a fusion protein with glutathione S-transferase (GST) acting as a cargo protein. Compared with two other fusion proteins containing either HE or MAP, only the fused peptide (HE-MAP) could effectively deliver the cargo GST protein to cells at pH 6.5 or below, while maintaining low delivery to cells at pH 7.0 and above. Using a xenograft mouse model of human breast cancer, fluorescent imaging showed that only HE-MAP could effectively target GST to the tumor site, while reducing non-specific association of MAP in other organs. The data presented in this report demonstrate the diagnostic and/or therapeutic potential of the fused peptide, HE-MAP, for targeting the acidic tumor microenvironment. The concise design for this pH-sensitive peptide offers a simple way to overcome CPP's lack of selectivity, which could lead to increased application of CPPs and macromolecular therapeutics.

Efficient 18F labeling of cysteine-containing peptides and proteins using tetrazine-trans-cyclooctene ligation.

18F positron emission tomography (PET) has a number of attributes that make it clinically attractive, including nearly 100% positron efficiency, very high specific radioactivity, and a short half-life of ≈ 110 minutes. However, the short half-life of 18F and the poor nucleophilicity of fluoride introduce challenges for the incorporation of 18F into complex molecules. Recently, the tetrazine-trans-cyclooctene ligation was introduced as a novel 18F labeling method that proceeds with fast reaction rates without catalysis. Herein we report an efficient method for 18F labeling of free cysteines of peptides and proteins based on sequential ligation with a bifunctional tetrazinyl-maleimide and an 18F-labeled trans-cyclooctene. The newly developed method was tested for site-specific labeling of both c(RGDyC) peptide and vascular endothelial growth factor (VEGF)-SH protein. Starting with 4 mCi of 18F-trans-cyclooctene and only 10 µg of tetrazine-RGD (80-100 µM) or 15 µg of tetrazine-VEGF (6.0 µM), 18F-labeled RGD peptide and VEGF protein could be obtained within 5 minutes in 95% yield and 75% yield, respectively. The obtained tracers were then evaluated in mice. In conclusion, a highly efficient method has been developed for site-specific 18F labeling of cysteine-containing peptides and proteins. The special characteristics of the tetrazine-trans-cyclooctene ligation provide unprecedented opportunities to synthesize 18F-labeled probes with high specific activity for PET applications.

Synthesis and evaluation of 64Cu-labeled monomeric and dimeric NGR peptides for MicroPET imaging of CD13 receptor expression.

The NGR-containing peptides have been shown to bind specifically to CD13/aminopeptidase N (APN) receptor, one of the attractive tumor vasculature biomarkers. In this study, we evaluated (64)Cu-labeled monomeric and dimeric NGR peptides for microPET imaging of CD13 receptor expression in vivo. Western blot analysis and immunofluorescence staining were performed to identify CD13-positive and CD13-negative cell lines. NGR-containing peptides were conjugated with 1,4,7,10-tetraazadodecane-N,N',N″,N‴-tetraacetic acid (DOTA) and labeled with (64)Cu (t(1/2) = 12.7 h) in ammonium acetate buffer. The resulting monomeric ((64)Cu-DOTA-NGR1) and dimeric ((64)Cu-DOTA-NGR2) peptides were then subjected to in vitro stability, cell uptake and efflux, small animal micorPET, and biodistribution studies. In vitro studies demonstrated that CD13 receptors are overexpressed in human fibrosarcoma HT-1080 cells and negative in human colon adenocarcinoma HT-29 cells. The binding affinity of (64)Cu-DOTA-NGR2 to HT-1080 cells was measured to be within low nanomolar range and about 2-fold higher than that of (64)Cu-DOTA-NGR1. For small animal microPET studies, (64)Cu-DOTA-NGR2 displayed more favorable in vivo performance in terms of higher tumor uptake and slower tumor washout in CD13-positive HT-1080 tumor xenografts as compared to (64)Cu-DOTA-NGR1. As expected, significantly lower tumor uptake and poorer tumor/normal organ contrast were observed for both (64)Cu-DOTA-NGR1 and (64)Cu-DOTA-NGR2 in CD13-negative HT-29 tumor xenografts in comparison with those in the HT-1080 tumor xenografts. The CD13-specific tumor activity accumulation of both (64)Cu-DOTA-NGR1 and (64)Cu-DOTA-NGR2 was further demonstrated by significant reduction of tumor uptake in HT-1080 tumor xenografts with a coinjected blocking dose of cyclic NGR peptide [c(CNGRC)]. The biodistribution results were consistent with the quantitative analysis of microPET imaging. We concluded that both (64)Cu-DOTA-NGR1 and (64)Cu-DOTA-NGR2 have good and specific tumor uptake in CD13-positive HT-1080 tumor xenografts. (64)Cu-DOTA-NGR2 showed higher tumor uptake and better tumor retention than (64)Cu-DOTA-NGR1, presumably due to bivalency effect and increase in apparent molecular size. (64)Cu-DOTA-NGR2 is a promising PET probe for noninvasive detection of CD13 receptor expression in vivo.

[18F]-2'-Fluoro-5-methyl-1-beta-D-arabinofuranosyluracil (18F-FMAU) in prostate cancer: initial preclinical observations.

We hypothesized that imaging-based assessment of cellular proliferation in prostate cancer may improve tumor characterization. We therefore evaluated the biodistribution and effect of androgen on tumor uptake of the cellular proliferation imaging marker [(18)F]-2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil ((18)F-FMAU) in xenograft mouse models of human prostate cancer. Castrated and noncastrated athymic male mice were implanted with androgen-independent PC3 and androgen-sensitive CWR22 human prostate cancer cells. Dynamic micro-positron emission tomography (PET)/computed tomography was performed for 1 hour followed by 10-minute static scans at 2 and 3 hours. Animals were sacrificed after imaging for biodistribution studies and immunohistochemical staining of tumors for androgen receptor and Ki-67/MIB expression. (18)F-FMAU uptake was significantly higher in all major organs of the castrated animals in comparison with noncastrated mice, with the highest uptake in liver and the lowest uptake in muscle and bone. When compared to PC3 tumors, CWR22 xenografts showed significantly higher tumor to muscle (2.56 ± 0.30 vs 1.99 ± 0.30, p  =  .008) and tumor to liver (1.72 ± 0.12 vs 1.26 ± 0.17, p  =  .0003) uptake ratios in the noncastrated animal at the 3-hour time point. Androgen receptor and Ki-67/MIB expressions were higher in CWR22 than in PC3 xenografts. Our initial preclinical observations suggest that there may be an association between androgen signaling and thymidine metabolism and that (18)F-FMAU PET may be useful in prostate tumor characterization.

The efficient synthesis and biological evaluation of novel bi-functionalized sarcophagine for (64)cu radiopharmaceuticals.

Purpose We and others have reported that Sarcophagine-based bifunctional chelators could be effectively used in the syntheses of (64)Cu radiopharmaceuticals. The resulted (64)Cu-Sarcophagine complexes demonstrated great in vivo stability. The goal of this study was to further derivatize Sarcophagine cage with amino and maleimide functional groups for conjugation with bioligands.Methods Starting from DiAmSar, three novel chelators (AnAnSar, BaMalSar, and Mal(2)Sar) with two functional groups have been synthesized. Among those, BaMalSar and Mal(2)Sar have been conjugated with cyclic peptide c(RGDyC) (denoted as RGD) and the resulted conjugates, BaMalSar-RGD and Mal(2)Sar-RGD(2) have been labeled with (64)Cu. The tumor targeting efficacy of (64)Cu-labeled RGD peptides were evaluated in a subcutaneous U87MG glioblastoma xenograft model.Results The conjugates, BaMalSar-RGD and Mal(2)Sar-RGD(2) could be labeled with (64)CuCl(2) in 10 min with high purity (>98%) and high radiochemical yield (>90%). Both (64)Cu-BaMalSar-RGD and (64)Cu-Mal(2)Sar-RGD(2) exhibited high tumor uptake and tumor-to-normal tissue ratios.Conclusion Three novel chelators with two functional groups have been developed based on Sarcophagine cage. The platform developed in this study could have broad applications in the design and synthesis of( 64)Cu-radiopharmaceuticals.

Efficient construction of PET/fluorescence probe based on sarcophagine cage: an opportunity to integrate diagnosis with treatment.

PURPOSE: Due to the shortage of established platforms/methods for multimodality probe construction, in this study, we developed a heterofunctional chelator, BaAn(Boc)Sar, from sarcophagine cage as a general platform for dual-modality probe construction. PROCEDURES: A dual-modality probe for positron-emission tomography (PET) and fluorescence imaging was synthesized using the developed BaAn(Boc)Sar chelator. The c(RGDyK)(2) peptide (denoted as RGD(2)) and fluorescence dye Cy5.5 were conjugated with BaAn(Boc)Sar to form BaAnSar-RGD(2)-Cy5.5. Then, BaAnSar-RGD(2)-Cy5.5 was labeled with (64)Cu in ammonium acetate buffer. PET and fluorescent imaging were carried out to evaluate (64)Cu-BaAnSar-RGD(2)-Cy5.5 in nude mice bearing U87MG glioblastoma xenograft. RESULTS: The BaAnSar-RGD(2)-Cy5.5 was labeled with (64)Cu very efficiently in 0.1 M NH(4)OAc buffer within 10 min at 37 °C in the yield of 86.7 ± 4.4 % (n = 3). The specific activity of (64)Cu-BaBaSar-RGD(2) was controlled at 50-200 mCi/µmol for the consideration of both PET and optical imaging. MicroPET quantification analysis shows that the U87MG tumor uptake is 6.41 ± 0.28, 6.51 ± 1.45, and 5.92 ± 1.57 %ID/g at 1, 4, and 20 h postinjection, respectively. Good correlation was obtained between the tumor to muscle ratios measured by the radioactivity and fluorescence intensity. As a proof of concept, an animal surgery study demonstrated that this dual-modality probe would greatly benefit the patients because the PET moiety could be used for tumor detection, and the fluorescent moiety would allow image-guided surgery. CONCLUSIONS: Our findings demonstrated the effectiveness and feasibility of preparing dual-modality imaging probes based on the sarcophagine scaffold. The resulting PET and fluorescent imaging probe also holds a great potential for clinical translation.

A Cy5.5-labeled phage-displayed peptide probe for near-infrared fluorescence imaging of tumor vasculature in living mice.

Near-infrared (NIR) fluorescence optical imaging is an emerging imaging technique for studying diseases at the molecular level. Optical imaging with a NIR emitting fluorophore for targeting tumor vasculature offers a noninvasive method for early detection of tumor angiogenesis and efficient monitoring of response to anti-tumor vasculature therapy. The previous in vitro results demonstrated that the GX1 peptide, identified by phage-display technology, is a tumor vasculature endothelium-specific ligand. In this report, Cy5.5-conjugated GX1 peptide was evaluated in a subcutaneous U87MG glioblastoma xenograft model to investigate tumor-targeting efficacy. The in vitro flow cytometry results revealed dose-dependent binding of Cy5.5-GX1 peptide to U87MG glioma cells. In vivo optical imaging with the Cy5.5-GX1 probe exhibited rapid U87MG tumor targeting at 0.5 h p.i., and high tumor-to-background contrast at 4 h p.i. Tumor specificity of Cy5.5-GX1 was confirmed by effective blocking of tumor uptake in the presence of unlabeled GX1 peptide (20 mg/kg). Ex vivo imaging further confirmed in vivo imaging findings, and demonstrated that Cy5.5-GX1 has a tumor-to-muscle ratio (15.21 ± 0.84) at 24 h p.i. for the non-blocked group and significantly decreased ratio (6.95 ± 0.75) for the blocked group. In conclusion, our studies suggest that Cy5.5-GX1 is a promising molecular probe for optical imaging of tumor vasculature.

Evaluation of 64Cu labeled GX1: a phage display peptide probe for PET imaging of tumor vasculature.

PURPOSE: Molecular imaging using positron emission tomography (PET) radiotracers targeted to tumor vasculature offers a noninvasive method for early detection of tumor angiogenesis and efficient monitoring of response to anti-tumor vasculature therapy. The previous in vitro results demonstrated that the GX1 peptide, identified by phage display technology, is a tumor vasculature endothelium-specific ligand. In this study, we evaluated a 64Cu-labeled GX1 peptide as a potential radiotracer for microPET imaging of tumor vasculature in a U87MG tumor xenografted mouse model. METHODS: Macrocyclic chelating agent 1,4,7,10-tetraazacyclododecane-N, N', N'', N'''-tetraacetic acid (DOTA)-conjugated GX1 peptide was synthesized and radiolabeled with 64Cu (t(1/2) = 12.7 h) in ammonium acetate buffer. The 64Cu-labeled GX1 peptide was then subjected to in vitro tumor cell uptake study, small animal PET and direct tissue sampling biodistribution studies in a U87MG tumor xenografted mouse model. RESULTS: The in vitro experiment demonstrated that 64Cu-DOTA-GX1 is stable in PBS with more than 91% of 64Cu-DOTA-GX1 peptide remaining intact after 24 h of incubation. Cellular uptake and retention studies revealed (64)Cu-DOTA-GX1 binds to U87MG glioma cells and has good tumor cell retention. For small animal PET imaging studies, the U87MG tumors were all clearly visible with high contrast to contralateral background at all measured time points after injection of 64Cu-DOTA-GX1 while high accumulation in liver and kidneys were also observed at early time points. The U87MG tumor uptake was determined to be the highest (7.97 ± 0.75%ID/g) at 24 h pi. The blocking experiment was achieved by co-injection of 64Cu-DOTA-GX1 with non-radiolabeled GX1 peptide (20 mg/kg) at 24 h pi, suggesting 64Cu-DOTA-GX1 is a target-specific tracer. Furthermore, the biodistribution results were consistent with the quantification of microPET imaging, demonstrating the highest ratio (16.09 ± 1.21) of tumor/muscle uptake of 64Cu-DOTA-GX1 at 24 h pi for non-blocking group and significant decreased ratio (6.57 ± 0.58) for blocking group. Finally, metabolic studies suggested that 64Cu-DOTA-GX1 is stable in mouse blood and urine in vivo at early time point while the metal transchelation may also occur in mouse liver and kidneys. CONCLUSION: Our studies demonstrate that 64Cu-DOTA-GX1 is a promising radiotracer for imaging tumor vasculature.

Strain-Promoted Catalyst-Free Click Chemistry for Rapid Construction of (64)Cu-Labeled PET Imaging Probes.

A rapid, efficient, and catalyst-free click chemistry method for the construction of (64)Cu-labeled PET imaging probes was reported based on the strain-promoted aza-dibenzocyclooctyne ligation. This new method was exemplified in the synthesis of (64)Cu-labeled RGD peptide for PET imaging of tumor integrin αvß3 expression in vivo. The catalyst-free click chemistry reaction proceeded with a fast rate and eliminated the contamination problem of the catalyst Cu(I) ions interfering with the (64)Cu radiolabeling procedure under the conventional Cu-catalyzed 1,3-dipolar cycloaddition condition. The new strategy is simple and robust, and the resultant (64)Cu-labeled RGD probe was obtained in an excellent yield and high specific activity. PET imaging and biodistribution studies revealed significant, specific uptake of the "click" (64)Cu-labeled RGD probe in integrin αvß3-positive U87MG xenografts with little uptake in nontarget tissues. This new approach is versatile, which warrants a wide range of applications for highly diverse radiometalated bioconjugates for radioimaging and radiotherapy.

Tetrazine-trans-cyclooctene ligation for the rapid construction of integrin αvß3 targeted PET tracer based on a cyclic RGD peptide.

Labeling biomolecules with (18)F is usually done through coupling with prosthetic groups, which generally requires several time-consuming radiosynthetic steps resulting in low labeling yield. Recently, the tetrazine-trans-cyclooctene ligation has been introduced as a method of bioconjugation that proceeds with fast reaction rates without need for catalysis. Herein, we report the development of an extremely fast and efficient method for generating (18)F labeled probes based on the tetrazine-trans-cyclooctene ligation. Starting with only 30 µg (78 µM) of a tetrazine-RGD conjugate and 2 mCi (5 µM) of (18)F-trans-cyclooctene, the (18)F labeled RGD peptide could be obtained in more than 90% yield within five minutes. The (18)F labeled RGD peptide demonstrated prominent tumor uptake in vivo. The receptor specificity was confirmed by blocking experiments. These results successfully demonstrate that the tetrazine-trans-cyclooctene ligation serves as an efficient labeling method for PET probe construction.

Trackable and Targeted Phage as Positron Emission Tomography (PET) Agent for Cancer Imaging.

UNLABELLED: The recent advancement of nanotechnology has provided unprecedented opportunities for the development of nanoparticle enabled technologies for detecting and treating cancer. Here, we reported the construction of a PET trackable organic nanoplatform based on phage particle for targeted tumor imaging. METHOD: The integrin α(v)ß(3) targeted phage nanoparticle was constructed by expressing RGD peptides on its surface. The target binding affinity of this engineered phage particle was evaluated in vitro. A bifunctional chelator (BFC) 1,4,7,10-tetraazadodecane-N,N',N",N"'-tetraacetic acid (DOTA) or 4-((8-amino-3,6,10,13,16,19-hexaazabicyclo [6.6.6] icosane-1-ylamino) methyl) benzoic acid (AmBaSar) was then conjugated to the phage surface for (64)Cu(2+) chelation. After (64)Cu radiolabeling, microPET imaging was performed in U87MG tumor model and the receptor specificity was confirmed by blocking experiments. RESULTS: The phage-RGD demonstrated target specificity based on ELISA experiment. According to the TEM images, the morphology of the phage was unchanged after the modification with BFCs. The labeling yield was 25 ± 4% for (64)Cu-DOTA-phage-RGD and 46 ± 5% for (64)Cu-AmBaSar-phage-RGD, respectively. At 1 h time point, (64)Cu-DOTA-phage-RGD and (64)Cu-AmBaSar-phage-RGD have comparable tumor uptake (~ 8%ID/g). However, (64)Cu-AmBaSar-phage-RGD showed significantly higher tumor uptake (13.2 ± 1.5 %ID/g, P<0.05) at late time points compared with (64)Cu-DOTA-phage-RGD (10 ± 1.2 %ID/g). (64)Cu-AmBaSar-phage-RGD also demonstrated significantly lower liver uptake, which could be attributed to the stability difference between these chelators. There is no significant difference between two tracers regarding the uptake in kidney and muscle at all time points tested. In order to confirm the receptor specificity, blocking experiment was performed. In the RGD blocking experiment, the cold RGD peptide was injected 2 min before the administration of (64)Cu-AmBaSar-phage-RGD. Tumor uptake was partially blocked at 1 h time point. Phage-RGD particle was also used as the competitive ligand. In this case, the tumor uptake was significantly reduced and the value was kept at low level consistently. CONCLUSION: In this report, we constructed a PET trackable nanoplatform based on phage particle and demonstrated the imaging capability of these targeted agents. We also demonstrated that the choice of chelator could have significant impact on imaging results of nano-agents. The method established in this research may be applicable to other receptor/ligand systems for theranostic agent construction, which could have an immediate and profound impact on the field of imaging/therapy and lay the foundation for the construction of next generation cancer specific theranostic agents.

Regulation of mitochondrial glutathione redox status and protein glutathionylation by respiratory substrates.

Brain and liver mitochondria isolated by a discontinuous Percoll gradient show an oxidized redox environment, which is reflected by low GSH levels and high GSSG levels and significant glutathionylation of mitochondrial proteins as well as by low NAD(P)H/NAD(P) values. The redox potential of brain mitochondria isolated by a discontinuous Percoll gradient method was calculated to be -171 mV based on GSH and GSSG concentrations. Immunoblotting and LC/MS/MS analysis revealed that succinyl-CoA transferase and ATP synthase (F(1) complex, α-subunit) were extensively glutathionylated; S-glutathionylation of these proteins resulted in a substantial decrease of activity. Supplementation of mitochondria with complex I or complex II respiratory substrates (malate/glutamate or succinate, respectively) increased NADH and NADPH levels, resulting in the restoration of GSH levels through reduction of GSSG and deglutathionylation of mitochondrial proteins. Under these conditions, the redox potential of brain mitochondria was calculated to be -291 mV. Supplementation of mitochondria with respiratory substrates prevented GSSG formation and, consequently, ATP synthase glutathionylation in response to H(2)O(2) challenges. ATP synthase appears to be the major mitochondrial protein that becomes glutathionylated under oxidative stress conditions. Glutathionylation of mitochondrial proteins is a major consequence of oxidative stress, and respiratory substrates are key regulators of mitochondrial redox status (as reflected by thiol/disulfide exchange) by maintaining mitochondrial NADPH levels.

Determination of GSH, GSSG, and GSNO using HPLC with electrochemical detection.

GSNO is an important intermediate in nitric oxide metabolism and mediates many ()NO-mediated signaling pathways through the post-translational modification of redox-sensitive proteins. The detection of GSNO in biological samples has been hampered by a lack of sensitive and simple assays. In this work, we describe the utilization of HPLC with electrochemical detection for the identification and quantification of GSNO in biological samples. GSNO requires a high potential (>700 mV) for its electrochemical detection, similar to that of GSSG. A simple isocratic HPLC system can be used to separate and simultaneously detect GSH, GSSG, and GSNO electrochemically. This HPLC system can be utilized to measure the redox profile of biological samples and applied for the measurement of GSNO reductase activity in cells. Proper sample preparation is essential in GSNO measurements, because artifactual formation of GSNO occurs in acidic conditions due to the reaction between GSH and nitrite. Treatment of samples with ammonium sulfamate or N-ethylmaleimide (NEM) can prevent the artifactual formation of GSNO and accurately detect GSNO in biological samples. Overall, the HPLC with electrochemical detection is a powerful tool to measure redox status in cells and tissues.