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Cobalt-sarcophagine complexes exhibit high kinetic inertness under various stringent conditions, but there is limited literature on radiolabeling and in vivo positron emission tomography (PET) imaging using no carrier added 55 Co. To fill this gap, this study first investigates the radiolabeling of DiAmSar (DSar) with 55 Co, followed by stability evaluation in human serum and EDTA, pharmacokinetics in mice, and a direct comparison with [ 55 Co]CoCl 2 to assess differences in pharmacokinetics. Furthermore, the radiolabeling process was successfully used to generate the NTSR1-targeted PET agent [ 55 Co]Co-NT-Sarcage (a DSar-functionalized SR142948 derivative) and administered to HT29 tumor xenografted mice. The [ 55 Co]Co-DSar complex can be formed at 37 °C with purity and stability suitable for preclinical in vivo radiopharmaceutical applications, and [ 55 Co]Co-NT-Sarcage demonstrated prominent tumor uptake with a low background signal. In a direct comparison with [ 64 Cu]Cu-NT-Sarcage, [ 55 Co]Co-NT-Sarcage achieved a higher tumor-to-liver ratio but with overall similar biodistribution profile. These results demonstrate that Sar would be a promising chelator for constructing Co-based radiopharmaceuticals including 55 Co for PET and 58m Co for therapeutic applications.

The aim of this study is to evaluate a radioactive metal complex platform for brain tumor targeting. Herein, we introduce a new porphyrin derivative, 5,10,15,20-(tetra- N , N -dimethyl-4-aminophenyl)porphyrin (TDAP), in which four N , N -dimethyl-4- p -phenylenediamine (DMPD) moieties are conjugated to the porphyrin labeled with the radiometal 64 Cu. DMPD affected the pharmacokinetics of porphyrin in terms of retention time in vivo and tumor-targeting ability relative to those of unmodified porphyrin. [ 64 Cu]Cu-TDAP showed stronger enhancement than [ 64 Cu]Cu-porphyrin in U87MG glioblastoma cells, especially in the cytoplasm and nucleus, indicating its tumor-targeting properties and potential use as a therapeutic agent. In the subcutaneous and orthotopic models of brain-tumor-bearing mice, [ 64 Cu]Cu-TDAP was clearly visualized in the tumor site via positron emission tomography imaging and showed a tumor-to-brain ratio as high as 13. [ 64 Cu]Cu-TDAP deserves attention as a new diagnostic agent that is suitable for the early diagnosis and treatment of brain tumors.

The incorporation of non-covalent albumin binding moieties (ABMs) into radiotracers results in increased circulation time, leading to a higher uptake in the target tissues such as the tumor, and, in some cases, reduced kidney retention. We previously developed [ 18 F]AlF NOTA-K(ABM)-α v β 6 -BP, where α v β 6 -BP is a peptide with high affinity for the cell surface receptor integrin α v β 6 that is overexpressed in several cancers, and the ABM is an iodophenyl-based moiety. [ 18 F]AlF NOTA-K(ABM)-α v β 6 -BP demonstrated prolonged blood circulation compared to the non-ABM parent peptide, resulting in high, α v β 6 -targeted uptake with continuously improving detection of α v β 6 (+) tumors using PET/CT. To further extend the imaging window beyond that of fluorine-18 ( t 1/2 = 110 min) and to investigate the pharmacokinetics at later time points, we radiolabeled the α v β 6 -BP with copper-64 ( t 1/2 = 12.7 h). Two peptides were synthesized without ( 1 ) and with ( 2 ) the ABM and radiolabeled with copper-64 to yield [ 64 Cu] 1 and [ 64 Cu] 2 , respectively. The affinity of [ nat Cu] 1 and [ nat Cu] 2 for the integrin α v β 6 was assessed by enzyme-linked immunosorbent assay. [ 64 Cu] 1 and [ 64 Cu] 2 were evaluated in vitro (cell binding and internalization) using DX3puroβ6 (α v β 6 (+)), DX3puro (α v β 6 (-)), and pancreatic BxPC-3 (α v β 6 (+)) cells, in an albumin binding assay, and for stability in both mouse and human serum. In vivo (PET/CT imaging) and biodistribution studies were done in mouse models bearing either the paired DX3puroβ6/DX3puro or BxPC-3 xenograft tumors. [ 64 Cu] 1 and [ 64 Cu] 2 were synthesized in ≥97% radiochemical purity. In vitro , [ nat Cu] 1 and [ nat Cu] 2 maintained low nanomolar affinity for integrin α v β 6 (IC 50 = 28 ± 3 and 19 ± 5 nM, respectively); [ 64 Cu] 1 and [ 64 Cu] 2 showed comparable binding to α v β 6 (+) cells (DX3puroβ6: ≥70%, ≥42% internalized; BxPC-3: ≥19%, ≥12% internalized) and ≤3% to the α v β 6 (-) DX3puro cells. Both radiotracers were ≥98% stable in human serum at 24 h, and [ 64 Cu] 2 showed a 6-fold higher binding to human serum protein than [ 64 Cu] 1 . In vivo , selective uptake in the α v β 6 (+) tumors was observed with tumor visualization up to 72 h for [ 64 Cu] 2 . A 3-5-fold higher α v β 6 (+) tumor uptake of [ 64 Cu] 2 vs [ 64 Cu] 1 was observed throughout, at least 2.7-fold improved BxPC-3-to-kidney and BxPC-3-to-blood ratios, and 2-fold improved BxPC-3-to-stomach ratios were noted for [ 64 Cu] 2 at 48 h. Incorporation of an iodophenyl-based ABM into the α v β 6 -BP ([ 64 Cu] 2 ) prolonged circulation time and resulted in improved pharmacokinetics, including increased uptake in α v β 6 (+) tumors that enabled visualization of α v β 6 (+) tumors up to 72 h by PET/CT imaging.

Increasing numbers of lung tumors are identified at early disease stages by diagnostic imaging in screening programs, but difficulties in locating these during surgical intervention has prevented an improved treatment outcome. Surgical biomarkers that are visible on diagnostic images, and that provide the surgeon with real-time image guidance during the intervention are thus highly warranted to bridge diagnostic precision into enhanced therapeutic outcome. In this paper, a liquid soft tissue marker for near infrared fluorescence and radio-guidance is presented. The biocompatible marker is based on the carbohydrate ester, sucrose acetate isobutyrate, ethanol, and a multifunctional naphthalocyanine dye, which enable near infrared fluorescence image-guided resection at short, medium and long tissue depths. Naphthalocyanine dyes have high quantum yields and may further act as chelators of radionuclides. Upon injection of the liquid marker, a gel-like depot is formed in situ at the site of injection, wherein the fluorescent dye and radionuclide is retained. The radiolabeled markers were optimized for minimal fluorescence quenching and high retention of the positron emission tomography radionuclide 64 Cu. The performance of the radiolabeled marker was tested in vivo in mice, where it displayed high photostability over a period of 4 weeks, and high retention of 64 Cu for 48 hours. The retention and biodistribution of 64 Cu was quantified via PET/CT, and the fluorescence emission by an in vivo imaging system. The presented data demonstrate proof-of-concept for naphthalocyanine markers as multimodal imaging agents that can bridge the precision of diagnostic imaging into surgical interventions., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Purpose: The co-stimulatory molecules CD80 and CD86 are upregulated on activated antigen-presenting cells (APC). We investigated whether local APC activation, induced by subcutaneous (s.c.) inoculation of lipopolysaccharides (LPS), can be imaged by positron emission tomography (PET) with CD80/CD86-targeting 64 Cu-labelled abatacept., Procedures: Mice were inoculated s.c. with extracellular-matrix gel containing either LPS or vehicle (PBS). Immune cell populations were analysed by flow cytometry and marker expression by RT-qPCR. 64 Cu-NODAGA-abatacept distribution was analysed using PET/CT and ex vivo biodistribution., Results: The number of CD80 + and CD86 + immune cells at the LPS inoculation site significantly increased a few days after inoculation. CD68 and CD86 expression were higher at the LPS than the PBS inoculation site, and CD80 was only increased at the LPS inoculation site. CTLA-4 was highest 10 days after LPS inoculation, when CD80/CD86 decreased again. A few days after inoculation, 64 Cu-NODAGA-abatacept distribution to the inoculation site was significantly higher for LPS than PBS (4.2-fold). Co-administration of unlabelled abatacept or human immunoglobulin reduced tracer uptake. The latter reduced the number of CD86 + immune cells at the LPS inoculation site., Conclusions: CD80 and CD86 are upregulated in an LPS-induced local inflammation, indicating invasion of activated APCs. 64 Cu-NODAGA-abatacept PET allowed following APC activation over time.

The validation of metal-phenolic nanoparticles (MPNs) in preclinical imaging studies represents a growing field of interest due to their versatility in forming predesigned structures with unique properties. Before MPNs can be used in medicine, their pharmacokinetics must be optimized so that accumulation in nontargeted organs is prevented and toxicity is minimized. Here, we report the fabrication of MPNs made of a coordination polymer core that combines In(III), Cu(II), and a mixture of the imidazole 1,4-bis(imidazole-1-ylmethyl)-benzene and the catechol 3,4-dihydroxycinnamic acid ligands. Furthermore, a phenolic-based coating was used as an anchoring platform to attach poly(ethylene glycol) (PEG). The resulting MPNs, with effective hydrodynamic diameters of around 120 nm, could be further derivatized with surface-embedded molecules, such as folic acid, to facilitate in vivo targeting and multifunctionality. The prepared MPNs were evaluated for in vitro plasma stability, cytotoxicity, and cell internalization and found to be biocompatible under physiological conditions. First, biomedical evaluations were then performed by intrinsically incorporating trace amounts of the radioactive metals 111 In or 64 Cu during the MPN synthesis directly into their polymeric matrix. The resulting particles, which had identical physicochemical properties to their nonradioactive counterparts, were used to perform in vivo single-photon emission computed tomography (SPECT) and positron emission tomography (PET) in tumor-bearing mice. The ability to incorporate multiple metals and radiometals into MPNs illustrates the diverse range of functional nanoparticles that can be prepared with this approach and broadens the scope of these nanoconstructs as multimodal preclinical imaging agents.

Background: The accumulation of liposome encapsulated chemotherapy in solid cancers is dependent on the presence of the enhanced permeability and retention (EPR) effect. Positron emission tomography (PET) imaging with a liposome encapsulated radioisotope, such as liposome encapsulated Cu-64 ( 64 Cu-liposome) may help to identify tumors with high liposome accumulation, and thereby stratify patients based on expected benefit from liposomal chemotherapy. However, intravenous administration of liposomes without a cytotoxic content is complicated by the accelerated blood clearance (ABC) phenomenon for succeeding therapeutic liposome dosing. Alternative markers for assessing the tumor's EPR level are therefore warranted., Materials and Methods: To increase our understanding of EPR variations and to ultimately identify an alternative marker for the EPR effect, we investigated the correlation between 64 Cu-liposome PET/CT (EPR effect) and 68 Ga-RGD PET/CT (neoangiogenesis), 18 F-FDG PET/CT (glycolysis), diffusion-weighted MRI (diffusivity) and interstitial fluid pressure in two experimental cancer models (CT26 and COLO 205)., Results: 64 Cu-liposome and 68 Ga-RGD SUV max displayed a significant moderate correlation, however, none of the other parameters evaluated displayed significant correlations. These results indicate that differences in neoangiogenesis may explain some EPR variability, however, as correlations were only moderate and not observed for SUV mean , 68 Ga-RGD is probably insufficient to serve as a stand-alone surrogate marker for quantifying the EPR effect and stratifying patients., Competing Interests: Financial support was provided by the Danish Strategic Research Council (NABIIT) ref. 2106-07-0033, the Lundbeck Foundation and the European Council (ERC grant). Annemarie Thuri Kristensen reports grants from Danish Technical University, during the conduct of the study; grants from Novo Nordisk A/S, grants from Copenhagen ZOO, grants from Independent Research Fund Denmark, outside the submitted work. The authors report no other potential conflicts of interest in this work., (© 2020 Børresen et al.)

Background: Macrophages, innate immune cells that reside in all organs, defend the host against infection and injury. In the heart and vasculature, inflammatory macrophages also enhance tissue damage and propel cardiovascular diseases., Methods: We here use in vivo positron emission tomography (PET) imaging, flow cytometry, and confocal microscopy to evaluate quantitative noninvasive assessment of cardiac, arterial, and pulmonary macrophages using the nanotracer 64 Cu-Macrin-a 20-nm spherical dextran nanoparticle assembled from nontoxic polyglucose., Results: PET imaging using 64 Cu-Macrin faithfully reported accumulation of macrophages in the heart and lung of mice with myocardial infarction, sepsis, or pneumonia. Flow cytometry and confocal microscopy detected the near-infrared fluorescent version of the nanoparticle ( VT680 Macrin) primarily in tissue macrophages. In 5-day-old mice, 64 Cu-Macrin PET imaging quantified physiologically more numerous cardiac macrophages. Upon intravenous administration of 64 Cu-Macrin in rabbits and pigs, we detected heightened macrophage numbers in the infarcted myocardium, inflamed lung regions, and atherosclerotic plaques using a clinical PET/magnetic resonance imaging scanner. Toxicity studies in rats and human dosimetry estimates suggest that 64 Cu-Macrin is safe for use in humans., Conclusions: Taken together, these results indicate 64 Cu-Macrin could serve as a facile PET nanotracer to survey spatiotemporal macrophage dynamics during various physiological and pathological conditions. 64 Cu-Macrin PET imaging could stage inflammatory cardiovascular disease activity, assist disease management, and serve as an imaging biomarker for emerging macrophage-targeted therapeutics.

Background: Molecular imaging is of great benefit to early disease diagnosis and timely treatment. One of the most striking innovations is the development of multimodal molecular imaging technology, which integrates two or more imaging modalities, largely in view of making the best of the advantages of each modality while overcoming their respective shortcomings. Hence, engineering a versatile and easily prepared nanomaterial with integrating multimodal molecular imaging function holds great promise, but is still a great challenge., Materials and Methods: We firstly designed and synthesized a BDT-DPP conjugated polymer and then noncovalent self-assembly with phospholipid-polyethylene glycol endowed BDT-DPP with water solubility and biocompatibility. Followed by [Cu] labeling, the acquired multifunctional nanoparticles (NPs) were studied in detail for the photophysical property. The cytotoxicity and biocompatibility of DPP-BDT NPs were examined through MTT assay and H&E stained analysis. In addition, we investigated the accumulation of the NPs in HepG2 tumor models by positron emission tomography (PET) and photoacoustic (PA) dual-mode imaging., Results and Discussion: The DPP-BDT NPs exhibited excellent optical stability, strong near-infrared (NIR) light absorption as well as fine biocompatibility. After tail vein injection into the living mice, the PA signals in the neoplastic tissues were gradually increased and reached to the maximum at the 4-h post-injection, which was consistent with the PET analysis. Such strong PA and PET signals were attributed to the efficient NPs accumulation resulting from the enhanced permeability and retention (EPR) effect., Conclusion: The biocompatible DPP-BDT NPs demonstrated to be strong NIR absorption property and PAI sensitivity. Besides, these novel DPP-BDT NPs can act not only as a PA imaging contrast agent but also as an imaging agent for PET., Competing Interests: The authors report no conflicts of interest in this work., (© 2020 Li et al.)

Polymeric micellar nanoparticles represent versatile and biocompatible platforms for targeted drug delivery. However, tracking their biodistribution, stability, and clearance profile in vivo is challenging. The goal of this study was to prepare surface-modified micelles with peptide GE11 for targeting the epidermal growth factor receptor (EGFR). In vitro fluorescence studies demonstrated significantly higher internalization of GE11 micelles into EGFR-expressing HCT116 colon cancer cells versus EGFR-negative SW620 cells. Azo coupling chemistry of tyrosine residues in the peptide backbone with aryl diazonium salts was used to label the micelles with radionuclide 64 Cu for positron emission tomography (PET) imaging. In vivo analysis of 64 Cu-labeled micelles showed prolonged blood circulation and predominant hepatobiliary clearance. The biodistribution profile of EGFR-targeting GE11 micelles was compared with nontargeting HW12 micelles in HCT116 tumor-bearing mice. PET revealed increasing tumor-to-muscle ratios for both micelles over 48 h. Accumulation of GE11-containing micelles in HCT116 tumors was higher compared to HW12-decorated micelles. Our data suggest that the efficacy of image-guided therapies with micellar nanoparticles could be enhanced by active targeting, as demonstrated with cancer biomarker EGFR.

Adeno-associated viruses (AAVs) are typically single-stranded deoxyribonucleic acid (ssDNA) encapsulated within 25-nm protein capsids. Recently, tissue-specific AAV capsids (e.g. PHP.eB) have been shown to enhance brain delivery in rodents via the LY6A receptor on brain endothelial cells. Here, we create a non-invasive positron emission tomography (PET) methodology to track viruses. To provide the sensitivity required to track AAVs injected at picomolar levels, a unique multichelator construct labeled with a positron emitter (Cu-64, t 1/2  = 12.7 h) is coupled to the viral capsid. We find that brain accumulation of the PHP.eB capsid 1) exceeds that reported in any previous PET study of brain uptake of targeted therapies and 2) is correlated with optical reporter gene transduction of the brain. The PHP.eB capsid brain endothelial receptor affinity is nearly 20-fold greater than that of AAV9. The results suggest that novel PET imaging techniques can be applied to inform and optimize capsid design.

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 64 Cu-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 64 Cu (t 1/2  = 12.7 h) in ammonium acetate buffer to provide 64 Cu-Sar-PEG-VCN, which was subsequently subjected to in vitro studies, small animal PET, and biodistribution studies. The PC-3 tumor-targeting efficacy of 64 Cu-Sar-PEG-VCN was compared to a cyclic RGD peptide-based PET probe ( 64 Cu-Sar-RGD). 64 Cu labeling was achieved in 75% decay-corrected yield with radiochemical purity of  > 98%. The specific activity of 64 Cu-Sar-PEG-VCN was estimated to be 37 MBq/nmol. MicroPET imaging results showed that 64 Cu-Sar-PEG-VCN has preferential tumor uptake and good tumor retention in PC-3 tumor xenografts. As compared to 64 Cu-Sar-RGD, 64 Cu-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 64 Cu-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 64 Cu-Sar-PEG-VCN is lower than that of 64 Cu-Sar-RGD. 64 Cu-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.

Rapid sequestration and prolonged retention of intravenously injected nanoparticles by the liver and spleen (reticuloendothelial system (RES)) presents a major barrier to effective delivery to the target site and hampers clinical translation of nanomedicine. Inspired by biological macromolecular drugs, synthesis of ultrasmall (diameter ≈12-15 nm) porous silica nanoparticles (UPSNs), capable of prolonged plasma half-life, attenuated RES sequestration, and accelerated hepatobiliary clearance, is reported. The study further investigates the effect of tumor vascularization on uptake and retention of UPSNs in two mouse models of triple negative breast cancer with distinctly different microenvironments. A semimechanistic mathematical model is developed to gain mechanistic insights into the interactions between the UPSNs and the biological entities of interest, specifically the RES. Despite similar systemic pharmacokinetic profiles, UPSNs demonstrate strikingly different tumor responses in the two models. Histopathology confirms the differences in vasculature and stromal status of the two models, and corresponding differences in the microscopic distribution of UPSNs within the tumors. The studies demonstrate the successful application of multidisciplinary and complementary approaches, based on laboratory experimentation and mathematical modeling, to concurrently design optimized nanomaterials, and investigate their complex biological interactions, in order to drive innovation and translation., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Molecular probes targeting bacteria provide opportunities to target bacterial infections in vivo for both imaging and therapy. In the current study, we report the development of positron emission tomography (PET) probes for imaging of live bacterial infection based on the small molecules HLys-DOTA, a polycationic peptide synthesized as the D-isomer (RYWVAWRNRG) conjugated to 1, 4, 7, 10-tetraazacyclododecane-N',N″,N‴,N-tetraacetic acid (DOTA) and AB1-HLys-DOTA, which includes an unnatural amino acid AB1 that preferentially binds to bacteria membrane lipids with amine groups via formation of iminoboronates. HLys-DOTA and AB1-HLys-DOTA peptides were radiolabeled with 64 Cu and investigated as PET imaging agents to track bacterial infection in vitro and in intramuscularly infected (IM) mice models. Cell uptake studies at 37°C in Staphylococcus aureus (SA) show higher uptake of 64 Cu-AB1-HLys-DOTA; 98.47 ± 3.54% vs 64 Cu-HLys-DOTA; 39.12 ± 3.27% at 24 h. Standard uptake values (SUV) analysis of the PET images resulted in mean SUV of 0.70 ± 0.08, 0.49 ± 0.04, and 0.31 ± 0.01 for 64 Cu-AB1-HLys-DOTA and 0.17 ± 0.06, 0.16 ± 0.02, and 0.13 ± 0.01 for 64 Cu-HLys-DOTA at 1, 4, and 24 h post injection, respectively, in the infected muscles. Similarly, in the biodistribution studies, dose uptake in the infected muscles was 4 times higher in the targeted 64 Cu-AB1-HLys-DOTA group than in the 64 Cu-HLys-DOTA group and 2-3 times higher than in the PBS control group at 1, 4, and 24 h post injection. 64 Cu-AB1-HLys-DOTA was able to distinguish between SA-infected muscle and Pseudomonas aeruginosa (PA) infected muscle with lower mean SUV of 0.28 ± 0.10 at 1 h post injection. This illustrates the utility of the AB1 covalently targeting group in synergy with the HLys peptide, which noncovalently binds to bacterial membranes. These results suggest that 64 Cu-labeled AB1-HLys-DOTA peptide could be used as an imaging probe for detection of bacterial infection in vivo with specificity for Gram-positive bacteria., Competing Interests: The authors do not have any conflicts of interest to declare., (Copyright © 2019 Tolulope A. Aweda et al.)

Although positron emission tomography (PET) imaging with 18-Fluorodeoxyglucose ( 18 F-FDG) is a promising technique in multiple myeloma (MM), the development of other radiopharmaceuticals seems relevant. CD138 is currently used as a standard marker for the identification of myeloma cells and could be used in phenotype tumor imaging. In this study, we used an anti-CD138 murine antibody (9E7.4) radiolabeled with copper-64 ( 64 Cu) or zirconium-89 ( 89 Zr) and compared them in a syngeneic mouse model to select the optimal tracers for MM PET imaging. Then, 9E7.4 was conjugated to TE2A-benzyl isothiocyanate (TE2A) and desferrioxamine (DFO) chelators for 64 Cu and 89 Zr labeling, respectively. 64 Cu-TE2A-9E7.4 and 89 Zr-DFO-9E7.4 antibodies were evaluated by PET imaging and biodistribution studies in C57BL/KaLwRij mice bearing either 5T33-MM subcutaneous tumors or bone lesions and were compared to 18 F-FDG-PET imaging. In biodistribution and PET studies, 64 Cu-TE2A-9E7.4 and 89 Zr-DFO-9E7.4 displayed comparable good tumor uptake of subcutaneous tumors. On the bone lesions, PET imaging with 64 Cu-TE2A-9E7.4 and 89 Zr-DFO-9E7.4 showed higher uptake than with 18 F-FDG-PET. Comparison of both 9E7.4 conjugates revealed higher nonspecific bone uptakes of 89 Zr-DFO-9E7.4 than 64 Cu-TE2A-9E7.4. Because of free 89 Zr's tropism for bone when using 89 Zr-anti-CD138, 64 Cu-anti-CD138 antibody had the most optimal tumor-to-nontarget tissue ratios for translation into humans as a specific new imaging radiopharmaceutical agent in MM.

Albumin is one of the most attractive nanoplatforms for targeted imaging and drug delivery due to its biocompatibility and long circulation half-life. However, previously reported albumin-based nanoplatforms have shown inconsistent blood circulation half-life according to the modified methods, and the affecting factors were not well evaluated, which could hamper the clinical translation of albumin-based nanoplatforms. Herein, we developed a finely tuned click-chemistry based albumin nanoplatform (CAN) with a longer circulation half-life and an efficient tumor targeting ability. Methods: CAN was synthesized in two steps. First, albumin was conjugated with ADIBO-NHS (albumin-ADIBO) by reacting albumin with various molar ratios of ADIBO. The number of attached ADIBO moieties was determined using matrix-assisted laser desorption ionization time of flight (MALDI-TOF). Second, the desired modalities including azide-functionalized chelator, a fluorescence dye, and folate were incorporated into albumin-ADIBO using strain-promoted alkyne-azide cycloaddition reaction (SPAAC reaction). The biodistribution and targeting efficiency of functionalized CANs were demonstrated in mice. Results: The degree of functionalization (DOF) and resulting in vivo biodistribution was controlled precisely using the click chemistry approach. Specifically, the numbers of attached azadibenzocyclooctyne (ADIBO) moieties on albumin, the DOF, were optimized by reacting albumin with varying molar ratios of ADIBO with a high reproducibility. Furthermore, we developed a simple and efficient method to estimate the DOF using UV - visible spectrophotometry (UV-vis), which was further validated by matrix-assisted laser desorption ionization time of flight (MALDI-TOF). The biodistribution of CAN could be controlled by DOF, and CAN with an optimized DOF showed a long circulation half-life (> 18 h). CAN was further functionalized using a simple click chemistry reaction with an azide functionalized chelator, a fluorescence dye, and folate. 64 Cu- and folate-labeled CAN ( 64 Cu-CAN-FA) showed effective and specific folate receptor targeting in vivo , with an over two-fold higher uptake than the liver at 24 h post-injection. Conclusions: Our development from the precisely controlled DOF demonstrates that an optimized CAN can be used as a multifunctional nanoplatform to obtain a longer half-life with radioisotopes and ligands, and provides an effective method for the development of albumin-based tumor theranostic agents., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Carbonic anhydrase IX (CAIX) plays an important role in glioma cell proliferation, invasion, metastasis, and resistance to radiotherapy and chemotherapy. An effective and noninvasive PET molecular imaging agent targeting CAIX would help its diagnosis and treatment but is not currently available. Recently, a low-molecular-weight (LMW) CAIX targeting agent, [ 64 Cu]XYIMSR-06, was reported to have significantly improved properties for targeting clear cell renal cell carcinoma (ccRCC). We are encouraged to investigate the feasibility of adapting this agent for the diagnosis and treatment of CAIX-overexpressing malignant glioma. In vitro cell uptake and binding affinity assays were used to verify the binding capacity of [ 64 Cu]XYIMSR-06 to U87 MG tumor cells in which CAIX overexpression was confirmed. The U87 MG tumor-bearing mouse (in situ and subcutaneous) model was built, and mice were injected with the radiotracer and/or coinjected with acetazolamide (0.2 g/kg) as a blocking agent for noninvasive micro-PET imaging. Micro-PET imaging was performed at 2, 4, and 8 h postinjection. ROI (region of interest)-based semiquantification was performed in an orthotopic glioma tumor model. Biodistribution throughout each organ was performed at 2, 4, 4 h block, 8, and 24 h postinjection. Hematoxylin and eosin (HE) staining and immunofluorescence or immunohistochemistry (IF/IHC) staining were implemented postimaging to assess the expression of CAIX in tumor organs. In vitro, [ 64 Cu]XYIMSR-06 exhibits greater uptake in glioma cells (high CAIX expression) than in HCT116 cells (low CAIX expression). The binding affinity of [ 64 Cu]XYIMSR-06 to U87 MG cell lines reaches up to 4.22 nM. Both orthotopic and subcutaneous tumors were clearly visualized at 2-8 h postinjection. Biodistribution studies demonstrated a maximum tumor uptake of 3.13% ID/g at 4 h postinjection, and the tumor to brain ratio (T/brain) was 6.51 at 8 h postinjection. The ROI-based T/brain values were 7.03 and 5.46 at 2 and 8 h postinjection, respectively. Histopathological analysis confirmed the overexpression of CAIX in gliomas, and the area of CAIX-positive IF staining is extremely consistent with the morphology on micro-PET imaging. In this study, [ 64 Cu]XYIMSR-06 demonstrated specific accumulation in CAIX-expressing U87 MG glioma tumors, indicating that the radiotracer has the potential for noninvasively monitoring and guiding personalized treatment of malignant glioma and other tumors overexpressing CAIX.

Niemann-Pick C disease (NPC) is an autosomal recessive lysosomal storage disorder resulting from mutations in the NPC1 (95% of cases) or NPC2 genes. Disturbance of copper homeostasis has been reported in NPC1 disease. In this study we have used whole-body positron emission tomography (PET) and brain electronic autoradiography with copper-64 ( 64 Cu), in the form of the copper(II) bis(thiosemicarbazonato) complex 64 Cu-GTSM, to image short-term changes in copper trafficking after intravenous injection in a transgenic mouse model of NPC1 disease. 64 Cu-GTSM is taken up in all tissues and dissociates rapidly inside cells, allowing monitoring of the subsequent efflux and redistribution of 64 Cu from all tissues. Significantly enhanced retention of 64 Cu radioactivity was observed in brain, lungs and blood at 15 h post-injection in symptomatic Npc1 -/- transgenic mice compared to wildtype controls. The enhanced retention of 64 Cu in brain was confirmed by electronic autoradiography, particularly in the midbrain, thalamus, medulla and pons regions. Positron emission tomography imaging with 64 Cu in selected chemical forms could be a useful diagnostic and research tool for the management and understanding of NPC1 disease.

Copper-64-Diacetyl-bis(N 4 -methylthiosemicarbazone) [ 64 Cu][Cu(ATSM)] is a hypoxia-targeting PET tracer with applications in oncology and cardiology. Upon entering a hypoxic cell, [ 64 Cu][Cu(II)(ATSM)] is reduced to a putative [ 64 Cu][Cu(I)(ATSM)] - species which dissociates to deposit radiocopper, thereby providing hypoxic contrast. This process may be dependent upon protonation arising from intracellular acidosis. Since acidosis is a hallmark of ischemic tissue and tumors, the hypoxia specificity of [ 64 Cu][Cu(ATSM)] may be confounded by changes in intracellular pH. We have therefore determined the influence of intracellular pH on [ 64 Cu][Cu(ATSM)] pharmacokinetics. Using isolated perfused rat hearts, acidosis was induced using an ammonium pre-pulse method, with and without hypoxic buffer perfusion. Cardiac [ 64 Cu][Cu(ATSM)] pharmacokinetics were determined using NaI detectors, with intracellular pH and cardiac energetics monitored in parallel by 31 P NMR. To distinguish direct acidotic effects on tracer pharmacokinetics from acidosis-induced hypocontractility, parallel studies used lidocaine perfusion to abolish cardiac contraction. Hypoxic myocardium trapped [ 64 Cu][Cu(ATSM)] despite no evidence of it being acidotic when characterised by 31 P NMR. Independent induction of tissue acidosis had no direct effect on [ 64 Cu][Cu(ATSM)] pharmacokinetics in either normoxic or hypoxic hearts, beyond decreasing cardiac oxygen consumption to alleviate hypoxia and decrease tracer retention, leading us to conclude that tissue acidosis does not mediate the hypoxia selectivity of [ 64 Cu][Cu(ATSM)].

Background: 68Ga-PSMA is a widely useful PET/CT tracer for prostate cancer imaging. Being a transmembrane protein acting as a glutamate carboxypeptidase enzyme, PSMA is highly expressed in prostate cancer cells. PSMA can also be labeled with 64Cu, offering a longer half-life and different resolution imaging. Several studies documented bio-distribution and pitfalls of 68Ga-PSMA as well as of 64Cu- PSMA. No data are reported on differences between these two variants of PSMA. Our aim was to evaluate physiological distribution of these two tracers and to analyze false positive cases., Methods: We examined tracer bio-distribution in prostate cancer patients with negative 68Ga-PSMA PET/CT (n=20) and negative 64Ga-PSMA PET/CT (n=10). A diagnostic pitfall for each tracer was documented., Result: Bio-distribution of both tracers was similar, with some differences due to renal excretion of 68Ga- PSMA and biliary excretion of 64Cu-PSMA. 68Ga-PSMA uptake was observed in sarcoidosis while 64Cu- PSMA uptake was recorded in pneumonitis., Discussion: Both tracers may present similar bio-distribution in the human body, with similar uptake in exocrine glands and high intestinal uptake. Similarly to other tracers, false positive cases cannot be excluded in clinical practice., Conclusion: The knowledge of difference in bio-distribution between two tracers may help in interpretation of PET data. Diagnostic pitfalls can be documented, due to the possibility of PSMA uptake in inflammation. Our results are preliminary to future studies comparing diagnostic accuracies of 68Ga-PSMA and 64Cu-PSMA., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Human carbonic anhydrase IX is a membrane enzyme that is significantly expressed in some types of cancer cells, while copper radioisotopes offer wide range of diagnostic, therapeutic and theranostic properties. The work was focused on a new approach to the labelling of antibody IgG M75 for epitope human carbonic anhydrase IX with copper radioisotopes 61 Cu and 64 Cu and its in vivo testing in mice with inoculated colorectal cancer. Monoclonal antibody IgG M75 for epitope human carbonic anhydrase IX was successfully conjugated with copper-specific chelator "phosphinate" and labelled with 61 Cu and 64 Cu The obtained molecule has considerable potential as a radioimmuno pharmaceutical suitable for imaging of tumours expressing carbonic anhydrase IX by positron emission tomography (PET)., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Here, we describe immuno-Cerenkov luminescence imaging (immuno-CLI) with a specific monoclonal antibody-based tracer for the detection of prostate tumors, which is used in preclinical positron emission tomography (PET) imaging. As PET isotopes generate a continuous spectrum of light in the ultraviolet/visible (UV/vis) wavelength range (Cerenkov luminescence, CL) in dielectric materials and consequently inside living tissues, these isotopes can also be detected by luminescence imaging performed with optical imaging (OI) systems. Imaging tumors with tracers that are specifically binding to a tumor-associated antigen can increase diagnostic accuracy, enables monitoring of treatment efficacy, and can be advantageous compared to radiolabeled small molecules used in PET-oncology such as 2-deoxy-2-[ 18 F]-fluoro-D-glucose ([ 18 F]FDG; glucose metabolism) or [ 11 C]choline (membrane synthesis) which was used to image prostate cancer. In this study, we compared on three consecutive days immuno-CLI and -PET of the applied 64 Cu-labeled and well described monoclonal antibody 3/F11 in prostate-specific membrane antigen (PSMA)-positive (C4-2, PSMA + ) and -negative (DU 145, PSMA - ) prostate tumor xenografts, inoculated in SCID mice. In vivo immuno-CLI and -PET measurements demonstrated linear correlation of both modalities, in line with ex vivo analysis performed with CLI and γ-counting. As CLI is also able to trace radioisotopes used for theranostic approaches, immuno-CLI could be an interesting, low-cost imaging alternative to immuno-PET., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

We describe a simple and effective bioconjugation strategy to extend the plasma circulation of a low molecular weight targeted phototheranostic agent, which achieves high tumor accumulation (9.74 ± 2.26%ID/g) and high tumor-to-background ratio (10:1). Long-circulating pyropheophorbide (LC-Pyro) was synthesized with three functional building blocks: (1) a porphyrin photosensitizer for positron-emission tomography (PET)/fluorescence imaging and photodynamic therapy (PDT), (2) a urea-based prostate-specific membrane antigen (PSMA) targeting ligand, and (3) a peptide linker to prolong the plasma circulation time. With porphyrin's copper-64 chelating and optical properties, LC-Pyro demonstrated its dual-modality (fluorescence/PET) imaging potential for selective and quantitative tumor detection in subcutaneous, orthotopic, and metastatic murine models. The peptide linker in LC-Pyro prolonged its plasma circulation time about 8.5 times compared to its truncated analog. High tumor accumulation of LC-Pyro enabled potent PDT, which resulted in significantly delayed tumor growth in a subcutaneous xenograft model. This approach can be applied to improve the pharmacokinetics of existing and future targeted PDT agents for enhanced tumor accumulation and treatment efficacy.

Introduction: Siliceous nanoparticles (NPs) have been extensively studied in nanomedicine due to their high biocompatibility and immense biomedical potential. Although numerous technologies have been developed, the synthesis of siliceous NPs for biomedical applications mainly relies on a few core technologies predominantly intended to produce spherical-shaped NPs., Methods: In this context, the impact of different morphologies of siliceous NPs on biodistribution in vivo is limited. In the present study, we developed a novel technique based on an aerosol silane reactor to produce sintered silicon NPs of similar size but different surface areas due to distinct spherical subunits. Silica-converted particles were functionalized for radiolabeling with copper-64 ( 64 Cu) to systematically analyze their behavior in the passive targeting of A431 tumor xenografts in mice after intravenous injection., Results: While low nonspecific uptake was observed in most organs, the majority of particles were accumulated in the liver, spleen, and lung. Depending on the morphologies and function-alization, significant differences in the uptake profiles of the particles were observed. In terms of tumor uptake, spherical shapes with lower surface areas showed the highest accumulation and tumor-to-blood ratios of all investigated particles., Conclusion: This study highlights the importance of shape and fuctionalization of siliceous NPs on organ and tumor accumulation as significant factors for biomedical applications., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Bifunctional derivatives of bis(phosphinate)-bearing cyclam (BPC) chelators bearing a carboxylate, amine, isothiocyanate, azide, or cyclooctyne in the BP side chain were synthesized. Conjugations required no protection of phosphinate or ring secondary amine groups. The ring amines were not reactive (proton protected) at pH < ∼8. For isothiocyanate coupling, oligopeptide N-terminal α-amines were more suitable than alkyl amines, e.g., Lys ω-amine (p K a ∼7.5-8.5 and ∼10-11, respectively) due to lower basicity. The Cu-64 labeling was efficient at room temperature (specific activity ∼100 GBq/μmol; 25 °C, pH 6.2, ∼100 ligand equiv, 10 min). A representative Cu-64-BPC was tested in vivo showing fast clearance and no nonspecific radioactivity deposition. The monoclonal anti-PSCA antibody 7F5 conjugates with thiocyanate BPC derivative or NODAGA were radiolabeled and studied in PC3-PSCA tumor bearing mice by PET. The radiolabeled BPC conjugate was accumulated in the prostate tumor with a low off-target uptake, unlike Cu-64-labeled NODAGA-antibody conjugate. The BPC chelators have a great potential for theranostic applications of the Cu-64/Cu-67 matched pair.

With the advent of positron emission tomography/magnetic resonance imaging (PET/MRI) scanner, PET/MRI dual-modal imaging will play more and more important role in the diagnosis of cancers and other diseases. Until now, there is no an approved PET/MRI dual-modal imaging probe. The goal of this work is to design and synthesize potential PET/MRI dual-modal imaging probe based on superparamagnetic manganese ferrite nanoparticles. We have developed superparamagnetic nanoparticles that have uniform size with 5 nm and can be further functionalized through surface coating with dopamine and polyethylene glycol derivatives, which provide functional groups for conjugating tumor-targeting biomolecules and bifunctional chelators. The nanoparticles conjugated with integrin α v β 3 over-expressed targeting cyclic arginine-glycine-aspartic acid (RGD)-peptide and labeled with positron radionuclide copper-64 were intravenously injected into glioblastoma xenograft nude mice. In vivo MRI and PET imaging of mice implied that the PET/MRI dual-modal imaging probe can precisely locate the tumor site with α v β 3 over expression., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The goal of this study was to establish the feasibility of integrating focused ultrasound (FUS)-mediated delivery of 64 Cu-integrated gold nanoclusters ( 64 Cu-AuNCs) to the pons for in vivo quantification of the nanocluster brain uptake using positron emission tomography (PET) imaging. FUS was targeted at the pons for the blood-brain barrier (BBB) disruption in the presence of systemically injected microbubbles, followed by the intravenous injection of 64 Cu-AuNCs. The spatiotemporal distribution of the 64 Cu-AuNCs in the brain was quantified using in vivo microPET/CT imaging at different time points post injection. Following PET imaging, the accumulation of radioactivity in the pons was further confirmed using autoradiography and gamma counting, and the gold concentration was quantified using inductively coupled plasma-mass spectrometry (ICP-MS). We found that the noninvasive and localized BBB opening by the FUS successfully delivered the 64 Cu-AuNCs to the pons. We also demonstrated that in vivo real-time microPET/CT imaging was a reliable method for monitoring and quantifying the brain uptake of 64 Cu-AuNCs delivered by the FUS. This drug delivery platform that integrates FUS, radiolabeled nanoclusters, and PET imaging provides a new strategy for noninvasive and localized nanoparticle delivery to the pons with concurrent in vivo quantitative imaging to evaluate delivery efficiency. The long-term goal is to apply this drug delivery platform to the treatment of pontine gliomas., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Hybrid positron emission tomography (PET)-magnetic resonance imaging (MRI) systems have been taken in use as new clinical diagnostic tools including detection and therapy planning of cancer. To reduce the amount of contrast agents injected in patients while fully benefitting both modalities, dual-modality probes are required., Material and Methods: This study was first aimed at developing a hybrid PET-MRI probe by labeling superparamagnetic iron oxide nanoparticles (SPIONs) with 64 Cu using a fast and chelator-free conjugation method, and second, to demonstrate the ability of the agent to target sentinel lymph nodes (SLNs) in vivo using simultaneous PET-MRI imaging., Results: High labeling efficiency of 97% produced within 10-15 min was demonstrated at room temperature. 64 Cu-SPIONs were chemically stable in mouse serum for 24 h and after intradermal injection in the hind paw of C57BL/6J mice, demonstrated specific accumulation in the SLN. Simultaneous PET-MRI clearly demonstrated visualization of 64 Cu-SPIONs, in dynamic and static imaging sequences up to 24 h after administration., Conclusion: The use of a single hybrid probe and simultaneous hybrid imaging provides an efficient, complementary integration of quantitation and is expected to improve preoperative planning and intraoperative guidance of cancer treatments.

Bombesin receptor 2 (BB 2 ) and integrin α v β 3 receptor are privileged targets for molecular imaging of cancer because of their overexpression in a number of tumor tissues. The most recent developments in heterodimer-based radiopharmaceuticals concern BB 2 - and integrin α v β 3 -targeting compounds, consisting of bombesin (BBN) and cyclic arginine-glycine-aspartic acid peptides (RGD), connected through short length linkers. Molecular imaging probes based on RGD-BBN heterodimer design exhibit improved tumor targeting efficacy compared to the single-receptor targeting peptide monomers. However, their application in clinical study is restricted because of inefficient synthesis or unfavorable in vivo properties, which could depend on the short linker nature. Thus, the aim of the present study was to develop a RGD 2 -BBN heterotrimer, composed of (7-14)BBN-NH 2 peptide (BBN) linked to the E[ c(RGDyK)] 2 dimer peptide (RGD 2 ), bearing the new linker type [Pro-Gly] 12 . The heterodimer E[c(RGDyK)] 2 -PEG 3 -Glu-(Pro-Gly) 12 -BBN(7-14)-NH 2 (RGD 2 -PG 12 -BBN) was prepared through conventional solid phase synthesis, then conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODA-GA). In 64 Cu labeling, the NODA-GA chelator showed superior radiochemical characteristics compared to DOTA (70% vs 40% yield, respectively). Both conjugates displayed dual targeting ability, showing good α v β 3 affinities and high BB 2 receptor affinities which, in the case of the NODA-GA conjugate, were in the same range as the best RGD-BBN heterodimer ligands reported to date ( K i = 24 nM). 64 Cu-DOTA and 64 Cu-NODA-GA probes were also found to be stable after 1 h incubation in mouse serum (>90%). In a microPET study in prostate cancer PC-3 xenograft mice, both probes showed low tumor uptake, probably due to poor pharmacokinetic properties in vivo. Overall, our study demonstrates that novel RGD-BBN heterodimer with long linker can be prepared and they preserve high binding affinities to BB 2 and integrin α v β 3 receptor binding ability. The present study represents a step forward in the design of effective heterodimer or heterotrimer probes for dual targeting.

The Gp2 domain is a protein scaffold for synthetic ligand engineering. However, the native protein function results in a heterogeneous distribution of charge on the conserved surface, which may hinder further development and utility. We aim to modulate charge, without diminishing function, which is challenging in small proteins where each mutation is a significant fraction of protein structure. We constructed rationally guided combinatorial libraries with charge-neutralizing or charge-flipping mutations and sorted them, via yeast display and flow cytometry, for stability and target binding. Deep sequencing of functional variants revealed effective mutations both in clone-dependent contexts and broadly across binders to epidermal growth factor receptor (EGFR), insulin receptor, and immunoglobulin G. Functional mutants averaged 4.3 charge neutralizing mutations per domain while maintaining net negative charge. We evolved an EGFR-targeted Gp2 mutant that reduced charge density by 33%, maintained net charge, and improved charge distribution homogeneity while elevating thermal stability ( T m = 87 ± 1 °C), improving binding specificity, and maintaining affinity ( K d = 8.8 ± 0.6 nM). This molecule was conjugated with 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid for 64 Cu chelation and evaluated for physiological distribution in mice with xenografted A431 (EGFR high ) and MDA-MB-435 (EGFR low ) tumors. Excised tissue gamma counting and positron emission tomography/computed tomography imaging revealed good EGFR high tumor signal (4.7 ± 0.5%ID/g) at 2 h post-injection and molecular specificity evidenced by low uptake in EGFR low tumors (0.6 ± 0.1%ID/g, significantly lower than for non-charge-modified Gp2, p = 0.01). These results provide charge mutations for an improved Gp2 framework, validate an effective approach to charge engineering, and advance performance of physiological EGFR targeting for molecular imaging.

Receptor-mediated internalization followed by trafficking and degradation of antibody-conjugates (ACs) via the endosomal-lysosomal pathway is the major mechanism for delivering molecular payloads inside target tumor cells. Although a mainstay for delivering payloads with clinically approved ACs in cancer treatment and imaging, tumor cells are often able to decrease intracellular payload concentrations and thereby reduce the effectiveness of the desired application. Thus, increasing payload intracellular accumulation has become a focus of attention for designing next-generation ACs. We developed a composite compound (ChAcNLS) that enables ACs to escape endosome entrapment and route to the nucleus resulting in the increased intracellular accumulation as an interleukin-5 receptor α-subunit (IL-5Rα)-targeted agent for muscle invasive bladder cancer (MIBC). We constructed 64 Cu-A14-ChAcNLS, 64 Cu-A14-NLS, and 64 Cu-A14 and evaluated their performance by employing mechanistic studies for endosome escape coupled to nuclear routing and determining whether this delivery system results in improved 64 Cu cellular accumulation. ACs consisting of ∼20 ChAcNLS or NLS moieties per 64 Cu-A14 were prepared in good yield, high monomer content, and maintaining high affinity for IL-5Rα. Confocal microscopy analysis demonstrated ChAcNLS mediated efficient endosome escape and nuclear localization. 64 Cu-A14-ChAcNLS increased 64 Cu cellular accumulation in HT-1376 and HT-B9 cells relative to 64 Cu-A14 and 64 Cu-A14-NLS. In addition, we tested 64 Cu-A14-ChAcNLS in vivo to evaluate its tissue distribution properties and, ultimately, tumor uptake and targeting. A model of human IL-5Rα MIBC was developed by implanting NOD/SCID mice with subcutaneous HT-1376 or HT-B9MIBC tumors, which grow containing high and low IL-5Rα-positive tumor cell densities, respectively. ACs were intravenously injected, and daily blood sampling, biodistribution at 48 and 96 h, and positron emission tomography (PET) at 24 and 48 h were performed. Region of interest (ROI) analysis was also performed on reconstructed PET images. Pharmacokinetic analysis and biodistribution studies showed that 64 Cu-A14-ChAcNLS had faster clearance rates from the blood and healthy organs relative to 64 Cu-A14. However, 64 Cu-A14-ChAcNLS maintained comparable tumor accumulation relative to 64 Cu-A14. This resulted in 64 Cu-A14-ChAcNLS having superior tumor/normal tissue ratios at both 48 and 96 h biodistribution time points. Visualization of AC distribution by PET and ROI analysis confirmed that 64 Cu-A14-ChAcNLS had improved targeting of MIBC tumor relative to 64 Cu-A14. In addition, 64 Cu-A14 modified with only NLS had poor tumor targeting. This was a result of poor tumor uptake due to extremely rapid clearance. Thus, the overall findings in this model of human IL-5Rα-positive MIBC describe an endosome escape-nuclear localization cholic-acid-linked peptide that substantially enhances AC cellular accumulation and tumor targeting.

Wilson's disease is a genetic disorder that causes excessive accumulation of copper in the body, leading to toxic damage, especially in the liver and nervous system. The current treatment cause burdensome side effects. We describe the use of chemically modified biopolymer carriers based on microcrystalline cellulose and chitosan containing the highly specific copper chelator 8-hydroxyquinoline as a new type of therapy for Wilson's disease. The chelators can scavenges copper ions released from food during digestion and copper ions present in secretions in the gastrointestinal tract. Because the chelator is covalently bound to indigestible biopolymer carriers (crosslinked chitosan or modified cellulose), it is not taken up by the gastrointestinal tract and it can be eliminated through the feces, avoiding unwanted side effects. This concept was tested on Wistar rats, which received a radioactive 64 CuCl 2 solution together with the polymers with covalently bound 8-hydroxyquinoline through a gastric probe. 64 Copper complex uptake from the gastrointestinal tract was significantly inhibited by both chelating polymers. With the modified polymers, the presence of 64 Cu was detected mostly in the gastrointestinal tract, not in the internal organs. These findings indicate modified cellulose and crosslinked chitosan, with covalently bound 8-hydroxyquinoline exhibited the potential to be excellent therapeutics for treating Wilson's disease., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Specific oncology diagnostics requires new types of the selective radiopharmaceuticals, particularly those suitable for the molecular PET imaging. The aim of this work is to present a new, specific PET-immunodiagnostic radiopharmaceutical based on the monoclonal antibody IgG M75 targeting human carbonic anhydrase IX labelled with 64 Cu (T ½ = 12.70h) and its in vitro and in vivo evaluation. The antibody IgG M75 was conjugated with a non-commercial copper-specific chelator "phosphinate" and then labelled with the positron emitter 64 Cu. Stability of the labelled conjugated was tested in human serum. The immunoreactivity of the labelled conjugate was evaluated in vitro on a suitable cell cultures of the colorectal carcinoma (HT-29) and its imaging properties were estimated in vivo on a mouse model with inoculated colorectal carcinoma HT-29 imaged on a µPET/CT. The tested radioimmunoconjugate was obtained in a specific activity of 0.25-0.5 MBq/µg. In vitro uptake experiments revealed specific binding to the HT-29 cells (45 ± 2.8% of the total added activity) and the measured K D value was found to be 9.2nM. Imaging clearly demonstrated significant uptake of the labelled monoclonal antibody in the tumour at 18h post administration. The radioimmunoconjugate 64 Cu-PS-IgG M75 seems to be a suitable candidate for PET diagnostics of hypoxic tumours expressing human carbonic anhydrase IX., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

As a growing number of patients with multiple myeloma (MM) respond to upfront therapies while eventually relapsing in a time frame that is often unpredictable, attention has increasingly focused on developing novel diagnostic criteria to also account for disease dissemination. Positron emission tomography/computed tomography (PET/CT) is often used as a noninvasive monitoring strategy to assess cancer cell dissemination, but because the uptake of the currently used radiotracer 18fluorodeoxyglucose ( 18 F-FDG) is a function of the metabolic activity of both malignant and nonmalignant cells, the results frequently lack sufficient specificity. Radiolabeled antibodies targeting MM tissue may detect disease irrespective of cell metabolism. Hence, we conjugated the clinically significant CD38-directed human antibody daratumumab (Darzalex [Dara]) to the DOTA chelator and labeled it with the positron-emitting radionuclide copper 64 ( 64 Cu; 64 Cu-DOTA-Dara). Here, we show that 64 Cu-DOTA-Dara can efficiently bind CD38 on the surface of MM cells and was mainly detected in the bones associated with tumor in a MM murine model. We also show that PET/CT based on 64 Cu-DOTA-Dara displays a higher resolution and specificity to detect MM cell dissemination than does 18 F-FDG PET/CT and was even more sensitive than were bioluminescence signals. We therefore have supporting evidence for using 64 Cu-DOTA-Dara as a novel imaging agent for MM., (© 2018 by The American Society of Hematology.)

Liposomes are nanoparticles used in drug delivery that distribute over several days in humans and larger animals. Radiolabeling with long-lived positron emission tomography (PET) radionuclides, such as manganese-52 ( 52 Mn, T½=5.6days), allow the imaging of this biodistribution. We report optimized protocols for radiolabeling liposomes with 52 Mn, through both remote-loading and surface labeling. For comparison, liposomes were also remote-loaded and surface labeled with copper-64 ( 64 Cu, T½=12.7h) through conventional means. The chelator DOTA was used in all cases. The in vivo stability of radiometal chelates is widely debated but studies that mimic a realistic in vivo setting are lacking. Therefore, we employed these four radiolabeled liposome types as platforms to demonstrate a new concept for such in vivo evaluation, here of the chelates 52 Mn-DOTA and 64 Cu-DOTA. This was done by comparing "shielded" remote-loaded with "exposed" surface labeled variants in a CT26 tumor-bearing mouse model. Remote loading (90min at 55°C) and surface labeling (55°C for 2h) of 52 Mn gave excellent radiolabeling efficiencies of 97-100% and 98-100% respectively, and the liposome biodistribution was imaged by PET for up to 8days. Liposomes with surface-conjugated 52 Mn-DOTA exhibited a significantly shorter plasma half-life (T ½ =14.4h) when compared to the remote-loaded counterpart (T ½ =21.3h), whereas surface-conjugated 64 Cu-DOTA cleared only slightly faster and non-significantly, when compared to remote-loaded (17.2±2.9h versus 20.3±1.2h). From our data, we conclude the successful remote-loading of liposomes with 52 Mn, and furthermore that 52 Mn-DOTA may be unstable in vivo whereas 64 Cu-DOTA appears suitable for quantitative imaging., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Radiation therapy may affect several important parameters in the tumor microenvironment and thereby influence the accumulation of liposomes by the enhanced permeability and retention (EPR)-effect. Here we investigate the effect of single dose radiation therapy on liposome tumor accumulation by PET/CT imaging using radiolabeled liposomes. Head and neck cancer xenografts (FaDu) and syngenic colorectal (CT26) cancer models were investigated. Radiotherapy displayed opposite effects in the two models. FaDu tumors displayed increased mean accumulation of liposomes for radiation doses up to 10 Gy, whereas CT26 tumors displayed a tendency for decreased accumulation. Tumor hypoxia was found negatively correlated to microregional distribution of liposomes. However, liposome distribution in relation to hypoxia was improved at lower radiation doses. The study reveals that the heterogeneity in liposome tumor accumulation between tumors and different radiation protocols are important factors that need to be taken into consideration to achieve optimal effect of liposome based radio-sensitizer therapy., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Alzheimer's disease can involve brain copper dyshomeostasis. We aimed to determine the effect of AD-like pathology on 64 Cu trafficking in mice, using positron emission tomography (PET imaging), during 24 hours after intravenous administration of ionic 64 Cu (Cu(ii) acetate) and 64 Cu-GTSM (GTSMH 2 = glyoxalbis(thiosemicarbazone)). Copper trafficking was evaluated in 6-8-month-old and 13-15 month-old TASTPM transgenic and wild-type mice, by imaging 0-30 min and 24-25 h after intravenous administration of 64 Cu tracer. Regional 64 Cu distribution in brains was compared by ex vivo autoradiography to that of amyloid-β plaque. 64 Cu-acetate showed uptake in, and excretion through, liver and kidneys. There was minimal uptake in other tissues by 30 minutes, and little further change after 24 h. Radioactivity within brain was focussed in and around the ventricles and was significantly greater in younger mice. 64 CuGTSM was taken up in all tissues by 30 min, remaining high in brain but clearing substantially from other tissues by 24 h. Distribution in brain was not localised to specific regions. TASTPM mice showed no major changes in global or regional 64 Cu brain uptake compared to wildtype after administration of 64 Cu acetate (unlike 64 Cu-GTSM) but efflux of 64 Cu from brain by 24 h was slightly greater in 6-8 month-old TASTPM mice than in wildtype controls. Changes in copper trafficking associated with Alzheimer's-like pathology after administration of ionic 64 Cu are minor compared to those observed after administration of 64 Cu-GTSM. PET imaging with 64 Cu could help understand changes in brain copper dynamics in AD and underpin new clinical diagnostic imaging methods.

Various carbonaceous nanomaterials, including fullerene, carbon nanotube, graphene, and carbon dots, have attracted increasing attention during past decades for their potential applications in biological imaging and therapy. In this study, we have developed a fullerene-based tumor-targeted positron emission tomography (PET) imaging probe. Water-soluble functionalized C 60 conjugates were radio-labeled with 64 Cu and modified with cyclo (Arg-Gly-Asp) peptides (cRGD) for targeting of integrin α v β 3 in glioblastoma. The specificity of fluorescein-labeled C 60 conjugates against cellular integrin α v β 3 was evaluated in U87MG (integrin α v β 3 positive) and MCF-7 cells (integrin α v β 3 negative) by confocal fluorescence microscopy and flow cytometry. Our results indicated that cRGD-conjugated C 60 derivatives showed better cellular internalization compared with C 60 derivatives without the cRGD attachment. Moreover, an interesting finding on intra-nuclei transportation of cRGD-conjugated C 60 derivatives was observed in U87MG cells. In vivo serial PET studies showed preferential accumulation of cRGD-conjugated C 60 derivatives at in U87MG tumors. In addition, the pharmacokinetic profiles of these fullerene-based nanoparticles conjugated with cRGD and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) fit well with the three compartment model. The renal clearance of C 60 -based nanoparticles is remarkably fast, which makes this material very promising for safer cancer theranostic applications., Statement of Significance: Safety is one of the major concerns for nanomedicine and nanomaterials with fast clearance profile are highly desirable. Fullerene is a distinct type of zero-dimensional carbon nanomaterial with ultrasmall size, uniform dispersity, and versatile reactivity. Here we have developed a fullerene-based tumor-targeted positron emission tomography imaging probe using water-soluble functionalized C 60 conjugates radio-labeled with 64 Cu and modified with cyclo (Arg-Gly-Asp) peptides (cRGD) for glioblastoma targeting. The improved tumor targeting property along with fast renal clearance behavior of C 60 -based nanoparticles makes this material very promising for future safer cancer theranostic applications., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Developing tumor-homing nanoparticles with integrated diagnostic and therapeutic functions, and meanwhile could be rapidly excreted from the body, would be of great interest to realize imaging-guided precision treatment of cancer. In this study, an ultrasmall coordination polymer nanodot (CPN) based on the coordination between tungsten ions (W VI ) and gallic acid (W-GA) was developed via a simple method. After polyethylene glycol (PEG) modification, PEGylated W-GA (W-GA-PEG) CPNs with an ultrasmall hydrodynamic diameter of 5 nm were rather stable in various physiological solutions. Without the need of chelator molecules, W-GA-PEG CPNs could be efficiently labeled with radioisotope 64 Cu 2+ , enabling positron emission tomography (PET) imaging, which reveals efficient tumor accumulation and rapid renal clearance of W-GA-PEG CPNs upon intravenous injection. Utilizing the radio-sensitizing function of tungsten with strong X-ray absorption, such W-GA-PEG CPNs were able to greatly enhance the efficacy of cancer radiotherapy in inhibiting the tumor growth. With fast clearance and little long-term body retention, those W-GA-PEG CPNs exhibited no appreciable in vivo toxicity. This study presents a type of CPNs with excellent imaging and therapeutic abilities as well as rapid renal clearance behavior, promising for further clinic translation.

Positron emission tomography (PET) imaging agents that detect amyloid plaques containing amyloid beta (Aβ) peptide aggregates in the brain of Alzheimer's disease (AD) patients have been successfully developed and recently approved by the FDA for clinical use. However, the short half-lives of the currently used radionuclides 11 C (20.4 min) and 18 F (109.8 min) may limit the widespread use of these imaging agents. Therefore, we have begun to evaluate novel AD diagnostic agents that can be radiolabeled with 64 Cu, a radionuclide with a half-life of 12.7 h, ideal for PET imaging. Described herein are a series of bifunctional chelators (BFCs), L 1 -L 5 , that were designed to tightly bind 64 Cu and shown to interact with Aβ aggregates both in vitro and in transgenic AD mouse brain sections. Importantly, biodistribution studies show that these compounds exhibit promising brain uptake and rapid clearance in wild-type mice, and initial microPET imaging studies of transgenic AD mice suggest that these compounds could serve as lead compounds for the development of improved diagnostic agents for AD.

Poly(ADP-ribose) polymerase (PARP) has emerged as an important molecular target for the treatment of several oncological diseases. A couple of molecular probes based on Olaparib scaffold have been developed by incorporation of F-18 or fluorophore for positron emission tomography (PET) or optical imaging in several types of tumor. PARP has been reported overexpressed in mesothelioma. We hereby synthesized an analogue of Olaparib containing DOTA moiety and radiolabeled it with Cu-64 to evaluate its utility of PET tracer for mesothelioma. The Cu-64 labeling was conveniently achieved at 90% yield with final compound at >99% radiochemistry purity. The biodistribution and PET imaging were performed at 0.5, 1, 2 and 18h to confirm the in vivo tumor targeting. The tumor uptake in study group was significant higher than that in control group (3.45±0.47% ID/g vs 2.26±0.30% ID/g) and tumor were clearly detected by PET imaging. These results suggest the feasibility to develop an Olaparib-based theranostic agent for mesothelioma., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Atherosclerosis is an inflammatory disorder characterized by the progressive thickening of blood vessel walls eventually resulting in acute vascular syndromes. Here, intravenously injectable hybrid nanoconstructs are synthesized for tempering immune cell inflammation locally and systemically. Lipid and polymer chains are nanoprecipitated to form 100 nm spherical polymeric nanoconstructs (SPNs), loaded with methotrexate (MTX) and subsequently labeled with Cu 64 and fluorescent probes for combined nuclear/optical imaging. Upon engulfment into macrophages, MTX SPNs intracellularly release their anti-inflammatory cargo significantly lowering the production of proinflammatory cytokine (interleukin 6 and tumor necrosis factor α) already at 0.06 mg mL -1 of MTX. In ApoE -/- mice, fed with high-fat diet up to 17 weeks, nuclear and optical imaging demonstrates specific accumulation of SPNs within lipid-rich plaques along the arterial tree. Histological analyses confirm SPN uptake into macrophages residing within atherosclerotic plaques. A 4-week treatment with biweekly administration of MTX SPNs is sufficient to reduce the plaque burden in ApoE -/- mice by 50%, kept on high-fat diet for 10 weeks. Systemic delivery of MTX to macrophages via multifunctional, hybrid nanoconstructs constitutes an effective strategy to inhibit atherosclerosis progression and induce, potentially, the resorption of vascular lesions., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This work aimed at estimating the kinetic parameters, and hence cumulated activity (A C ), of a diagnostic/therapeutic convergence radiopharmaceutical, namely 64 Cu-/ 177 Lu-labeled antibody ( 64 Cu-/ 177 Lu-cetuximab), that acts as anti-epidermal growth factor receptor. Methods: In mice bearing esophageal squamous cell carcinoma tumors, to estimate uptake (K), release rate constant (k R ), and hence A C , a kinetic model analysis was applied to recently published biodistribution data of immuno-PET imaging with 64 Cu-cetuximab and of small-animal SPECT/CT imaging with 177 Lu-cetuximab, including blood and TE-8 tumor. Results: K, k R , and A C were estimated to be 0.0566/0.0593 g⋅h -1 ⋅g -1 , 0.0150/0.0030 h -1 , and 2.3 × 10 10 /4.1 × 10 12 disintegrations (per gram of TE-8 tumor), with an injected activity of 3.70/12.95 MBq, for 64 Cu-/ 177 Lu-cetuximab, respectively. Conclusion: A model is available for comparing kinetic parameters and A C of the companion diagnostic/therapeutic 64 Cu-/ 177 Lu-cetuximab that may be considered as a step for determining whether one can really use the former to predict dosimetry of the latter., (© 2017 by the Society of Nuclear Medicine and Molecular Imaging.)

Heterodimerization of EGFR with HER2 coexpressed in breast cancer (BC) promotes tumor growth, and increased EGFR expression is associated with trastuzumab resistance. Our aim was to construct 64 Cu-labeled bispecific radioimmunoconjugates (bsRIC) composed of trastuzumab Fab, which binds HER2 linked through a polyethylene glycol (PEG 24 ) spacer to EGF, and to compare their pharmacokinetic, biodistribution, and tumor imaging characteristics by positron-emission tomography (PET). bsRICs were generated by linking maleimide modified trastuzumab Fab with thiolated EGF through a thioether bond. HER2 and EGFR binding were assessed in vitro in MDA-MB-231 (EGFR mod /HER2 low ), MDA-MB-468 (EGFR high /HER2 neg ), MDA-MB-231-H2N (EGFR mod /HER2 mod ), and SKOV3 (EGFR low /HER2 high ) cells by competition and saturation cell binding assays to estimate the dissociation constant (K d ). The elimination of the 64 Cu-NOTA-trastuzumab Fab-PEG 24 -EGF bsRICs from the blood of Balb/c mice was compared to monospecific 64 Cu-NOTA-trastuzumab Fab and 64 Cu-NOTA-EGF. MicroPET/CT imaging was performed in NOD/SCID mice bearing subcutaneous MDA-MB-468, MDA-MB-231/H2N, or SKOV3 human BC xenografts at 24 and 48 h postinjection (p.i.) of bsRICs. Tumor and normal tissue uptake were quantified by biodistribution studies and compared to monospecific agents. The binding of bsRICs to MDA-MB-231 cells was decreased to 24.5 ± 5.2% by excess EGF, while the binding of bsRICs to SKOV3 cells was decreased to 38.6 ± 5.4% by excess trastuzumab Fab, demonstrating specific binding to both EGFR and HER2. 64 Cu-labeled bsRICs incorporating the PEG 24 spacer were eliminated more slowly from the blood than 64 Cu-bsRICs without the PEG spacer and were cleared much more slowly than 64 Cu-NOTA-Fab or 64 Cu-NOTA-EGF. All three tumor xenografts were visualized by microPET/CT at 24 and 48 h p.i. of bsRICs. Biodistribution studies at 48 h p.i. in NOD/SCID mice with MDA-MB-231/H2N tumors demonstrated significantly greater tumor uptake of 64 Cu-NOTA-Fab-PEG 24 -EGF (4.9 ± 0.4%ID/g) than 64 Cu-NOTA-Fab (1.9 ± 0.3%ID/g; P < 0.0001) and 64 Cu-NOTA-EGF (0.7 ± 0.2%ID/g; P < 0.0001). Furthermore, preadministration of an excess of trastuzumab Fab or trastuzumab Fab-PEG 24 -EGF significantly decreased the tumor uptake of 64 Cu-NOTA-Fab-PEG 24 -EGF in SK-OV-3 and MDA-MB-468 xenografts by 4.4-fold (P = 0.0012) and 1.8-fold (P = 0.0031), respectively. 64 Cu-labeled bsRICs bound HER2 or EGFR and were taken up specifically in vivo in tumor xenografts expressing one or both receptors. The PEG 24 linker prolonged the blood residence time contributing to the higher tumor uptake of the bsRICs than monospecific agents.

Molecular imaging can report on the status of the tumor immune microenvironment and guide immunotherapeutic strategies to enhance the efficacy of immune modulation therapies. Imaging agents that can rapidly report on targets of immunomodulatory therapies are few. The programmed death ligand 1 (PD-L1) is an immune checkpoint protein over-expressed in several cancers and contributes to tumor immune suppression. Tumor PD-L1 expression is indicative of tumor response to PD-1 and PD-L1 targeted therapies. Herein, we report a highly specific peptide-based positron emission tomography (PET) imaging agent for PD-L1. We assessed the binding modes of the peptide WL12 to PD-L1 by docking studies, developed a copper-64 labeled WL12 ([ 64 Cu]WL12), and performed its evaluation in vitro, and in vivo by PET imaging, biodistribution and blocking studies. Our results show that [ 64 Cu]WL12 can be used to detect tumor PD-L1 expression specifically and soon after injection of the radiotracer, to fit within the standard clinical workflow of imaging within 60 min of administration., Competing Interests: COI: The authors declare no conflicts of interest., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We previously reported that microSPECT/CT imaging with 111 In-labeled pertuzumab detected decreased HER2 expression in human breast cancer (BC) xenografts in athymic mice associated with response to treatment with trastuzumab (Herceptin). Our aim was to extend these results to PET/CT by constructing F(ab') 2 of pertuzumab modified with NOTA chelators for complexing 64 Cu. The effect of the administered mass (5-200 µg) of 64 Cu-NOTA-pertuzumab F(ab') 2 was studied in NOD/SCID mice engrafted with HER2-positive SK-OV-3 human ovarian cancer xenografts. Biodistribution studies were performed in non-tumor bearing Balb/c mice to predict radiation doses to normal organs in humans. Serial PET/CT imaging was conducted on mice engrafted with HER2-positive and trastuzumab-sensitive BT-474 or trastuzumab-insensitive SK-OV-3 xenografted mice treated with weekly doses of trastuzumab. There were no significant effects of the administered mass of 64 Cu-NOTA-pertuzumab F(ab') 2 on tumor or normal tissue uptake. The predicted total body dose in humans was 0.015 mSv/MBq, a 3.3-fold reduction compared to 111 In-labeled pertuzumab. MicroPET/CT images revealed specific tumor uptake of 64 Cu-NOTA-pertuzumab F(ab') 2 at 24 or 48 h post-injection in mice with SK-OV-3 tumors. Image analysis of mice treated with trastuzumab showed 2-fold reduced uptake of 64 Cu-NOTA-pertuzumab F(ab') 2 in BT-474 tumors after 1 week of trastuzumab normalized to baseline, and 1.9-fold increased uptake in SK-OV-3 tumors after 3 weeks of trastuzumab, consistent with tumor response and resistance, respectively. We conclude that PET/CT imaging with 64 Cu-NOTA-pertuzumab F(ab') 2 detected changes in HER2 expression in response to trastuzumab while delivering a lower total body radiation dose compared to 111 In-labeled pertuzumab.

Objectives: The aim of the study is to evaluate the pharmacokinetics and microbiodistribution of Cu-labeled collagen-binding peptides., Methods: The affinity constant (KD), association (ka), and dissociation rate constant (kd) for the peptide collagelin or its analog (named CRPA) binding to collagen were measured by biolayer interferometric analysis. Rats (n=4-5) with myocardial infarction or normal were injected intravenously with the Cu-labeled peptides or Cu-DOTA as a control. Dynamic PET imaging was performed for 60 min at 7-8 weeks after infarct. Fluorine-18 fluorodeoxyglucose PET imaging was performed to identify the viable myocardium. To validate the PET images, slices of heart samples from the base to the apex were analyzed using autoradiography and histology., Result: The peptides bound to collagen with a KD of ∼0.9 µmol/l. The Cu-peptides and Cu-DOTA accumulated in the infarct area (confirmed by autoradiography and histology images) within 1 min of injection and were excreted rapidly through the renal system. The blood clearance curves were biphasic with elimination half-lives of 21.9±2.4, 26.2±4.6, and 21.2±2.1 min for Cu-CRPA, Cu-collagelin, and the control Cu-DOTA, respectively. The clearance half-lives from the focal fibrotic tissue (24.1±1.5, 25.6±8.0, and 21.4±1.3 min, respectively) and remote myocardium (20.8±0.7, 21.0±5.5, and 19.1±2.4 min, respectively) were not significantly different. The uptake ratios of infarct-to-remote myocardium (1.93±0.18, 2.15±0.38, and 1.88±0.08, respectively) for Cu-CRPA, Cu-collagelin, and Cu-DOTA remained stable for the time period between 10 and 60 min., Conclusion: The distribution of the Cu-collagelin probes corresponds to the heterogeneous distribution of expanded extracellular space in the setting of myocardial infarction. The overall washout rate from the fibrous tissue was determined by the slow washout rate (t1/2≥20 min) of the peptides from the extracellular space to the vasculature, not by the dissociation rate (t1/2<2 min) of the Cu-peptides from collagen.

Antibody-based therapies gain momentum in clinical therapy, thus the need for accurate imaging modalities with respect to target identification and therapy monitoring are of increasing relevance. Cerenkov luminescence imaging (CLI) are a novel method detecting charged particles emitted during radioactive decay with optical imaging. Here, we compare Position Emission Tomography (PET) with CLI in a multimodal imaging study aiming at the fast and efficient screening of monoclonal antibodies (mAb) designated for targeting of the neuroblastoma-characteristic epitope disialoganglioside GD2. Neuroblastoma-bearing SHO mice were injected with a 64Cu-labeled GD2-specific mAb. The tumor uptake was imaged 3 h, 24 h and 48 h after tracer injection with both, PET and CLI, and was compared to the accumulation in GD2-negative control tumors (human embryonic kidney, HEK-293). In addition to an in vivo PET/CLI-correlation over time, we also demonstrate linear correlations of CLI- and γ-counter-based biodistribution analysis. CLI with its comparably short acquisition time can thus be used as an attractive one-stop-shop modality for the longitudinal monitoring of antibody-based tumor targeting and ex vivo biodistribution.These findings suggest CLI as a reliable alternative for PET and biodistribution studies with respect to fast and high-throughput screenings in subcutaneous tumors traced with radiolabeled antibodies. However, in contrast to PET, CLI is not limited to positron-emitting isotopes and can therefore also be used for the visualization of mAb labeled with therapeutic isotopes like electron emitters.

As peripheral arterial disease (PAD) results in muscle ischemia and neovascularization, it has been claimed that nanoparticles can passively accumulate in ischemic tissues through the enhanced permeability and retention (EPR) effect. At this time, a quantitative evaluation of the passive targeting capabilities of nanoparticles has not been reported in PAD. Using a murine model of hindlimb ischemia, we quantitatively assessed the passive targeting capabilities of (64)Cu-labeled PEGylated reduced graphene oxide - iron oxide nanoparticles ((64)Cu-RGO-IONP-PEG) through the EPR effect using positron emission tomography (PET) imaging. Serial laser Doppler imaging was performed to monitor changes in blood perfusion upon surgical induction of ischemia. Nanoparticle accumulation was assessed at 3, 10, and 17 days post-surgery and found to be highest at 3 days post-surgery, with the ischemic hindlimb displaying an accumulation of 14.7 ± 0.5% injected dose per gram (%ID/g). Accumulation of (64)Cu-RGO-IONP-PEG was lowest at 17 days post-surgery, with the ischemic hindlimb displaying only 5.1 ± 0.5%ID/g. Furthermore, nanoparticle accumulation was confirmed by photoacoustic imaging (PA). The combination of PET and serial Doppler imaging showed that nanoparticle accumulation in the ischemic hindlimb negatively correlated with blood perfusion. Thus, we quantitatively confirmed that (64)Cu-RGO-IONP-PEG passively accumulated in ischemic tissue via the EPR effect, which is reduced as the perfusion normalizes. As (64)Cu-RGO-IONP-PEG displayed substantial accumulation in the ischemic tissue, this nanoparticle platform may function as a future theranostic agent, providing both imaging and therapeutic applications., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In vivo imaging of β-amyloid (Aβ) aggregates consisting of Aβ(1-40) and Aβ(1-42) peptides by positron emission tomography (PET) contributes to the diagnosis and therapy for Alzheimer's disease (AD). Because (64)Cu (t1/2=12.7h) is a radionuclide for PET with a longer physical half-life than (11)C (t1/2=20min) and (18)F (t1/2=110min), it is an attractive radionuclide for the development of Aβ imaging probes that are suitable for routine use. In the present study, we designed and synthesized two novel (64)Cu labeled benzofuran derivatives and evaluated their utility as PET imaging probes for Aβ aggregates. In an in vitro binding assay, 6 and 8 showed binding affinity for Aβ(1-42) aggregates with a Ki value of 33 and 243nM, respectively. In addition, these probes bound to Aβ plaques deposited in the brain of an AD model mouse in vitro. In a biodistribution experiment using normal mice, these probes showed low brain uptake (0.33% and 0.36% ID/g) at 2min post-injection. Although refinement to enhance brain uptake is needed, [(64)Cu]6 and [(64)Cu]8 demonstrated the feasibility of developing novel PET probes for imaging Aβ aggregates., (Copyright © 2016 Elsevier Ltd. All rights reserved.)