"Click-to-Clear": A Strategy to Minimize Radioactivity from the Blood Pool Utilizing Staudinger Ligation.

The availability of several bioorthogonal reactions that can proceed selectively and efficiently under physiologically relevant conditions has garnered the interest of biochemists and organic chemists alike. Bioorthogonal cleavage reactions represent the latest innovation in click chemistry. Here, we employed the Staudinger ligation reaction to release radioactivity from immunoconjugates, improving target-to-background ratios. In this proof-of-concept study, model systems, including the anti-HER2 antibody trastuzumab, radioisotope I-131, and a newly synthesized bifunctional phosphine, were used. Staudinger ligation occurred when biocompatible N-glycosyl azides reacted with this radiolabeled immunoconjugate, leading to cleavage of the radioactive label from the molecule. We demonstrated this click cleavage in vitro and in vivo. Biodistribution studies in tumor models showed that radioactivity was eliminated from the bloodstream, thereby improving tumor-to-blood ratios. SPECT imaging revealed that tumors could be visualized with enhanced clarity. Our simple approach represents a novel application of bioorthogonal click chemistry in the development of antibody-based theranostics.

Preclinical Evaluation of hnRNPA2B1 Antibody in Human Triple-Negative Breast Cancer MDA-MB-231 Cells via PET Imaging.

Triple-negative breast cancer (TNBC) does not express estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Because TNBC lacks the expression of commonly targeted receptors, it is challenging to develop a new imaging agent for this cancer subtype. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are RNA-protein complexes that have been linked to tumor development and progression. Considering the high expression of hnRNPA2B1, an hnRNP subtype, in TNBC MDA-MB-231 cells, this study aimed to develop a novel hnRNPA2B1 antibody-based nuclear imaging agent. The hnRNPA2B1-specific antibody was radiolabeled with 64Cu and evaluated in vitro and in vivo. The trans-cyclooctene (TCO) was functionalized on the antibody to obtain hnRNP-PEG4-TCO and reactive tetrazine (Tz) on the ultrastable bifunctional chelator PCB-TE2A-alkyne to yield PCB-TE2A-Tz for the inverse electron demand Diels-Alder reaction. The 64Cu-radiolabeled antibody was administered and imaged at 1-18 h time points for conventional imaging. Alternatively, the unlabeled antibody conjugate was administered, and 48 h later radiolabeled 64Cu-PCB-TE2A-Tz was administered to the same mice for the pretargeting strategy and imaged at the same time intervals for direct comparison. The tumor was successfully visualized in both strategies, and comparatively, pretargeting showed superior results. The 64Cu-PCB-TE2A-Tz was successfully clicked at the tumor site with hnRNP-PEG4-TCO and the non-clicked were concurrently eliminated. This led to increase the tumor uptake with extremely high tumor-to-background ratio manifested by positron emission tomography (PET) imaging and biodistribution studies.

In vivo detection of hydrogen sulfide in the brain of live mouse: application in neuroinflammation models.

PURPOSE: Hydrogen sulfide (H2S) plays important roles in brain pathophysiology. However, nuclear imaging probes for the in vivo detection of brain H2S in living animals have not been developed. Here, we report the first nuclear imaging probe that enables in vivo imaging of endogenous H2S in the brain of live mice. METHODS: Utilizing a bis(thiosemicarbazone) backbone, a fluorescent ATSM-FITC conjugate was synthesized. Its copper complex, Cu(ATSM-FITC) was thoroughly tested as a biosensor for H2S. The same ATSM-FITC ligand was quantitatively labeled with [64Cu]CuCl2 to obtain a radioactive [64Cu][Cu(ATSM-FITC)] imaging probe. Biodistribution and positron emission tomography (PET) imaging studies were performed in healthy mice and neuroinflammation models. RESULTS: The Cu(ATSM-FITC) complex reacts instantly with H2S to release CuS and becomes fluorescent. It showed excellent reactivity, sensitivity, and selectivity to H2S. Endogenous H2S levels in living cells were successfully detected by fluorescence microscopy. Exceptionally high brain uptake of [64Cu][Cu(ATSM-FITC)] (> 9% ID/g) was observed in biodistribution and PET imaging studies. Subtle changes in brain H2S concentrations in live mice were accurately detected by quantitative PET imaging. Due to its dual modality feature, increased H2S levels in neuroinflammation models were characterized at the subcellular level by fluorescence imaging and at the whole-body scale by PET imaging. CONCLUSION: Our biosensor can be readily utilized to study brain H2S function in live animal models and shows great potential as a novel imaging agent for diagnosing brain diseases.

Imaging Strategy that Achieves Ultrahigh Contrast by Utilizing Differential Esterase Activity in Organs: Application in Early Detection of Pancreatic Cancer.

Most nanoparticles show much higher uptake in mononuclear phagocyte system (MPS) organs than in tumors, which has been a long-lasting dilemma in nanomedicine. Here, we report an imaging strategy that selectively decreases MPS organ uptakes by utilizing the differential esterase activity in tumors and other organs. When an esterase-labile radiotracer loaded liposome was injected into the body, radioactivity was rapidly excreted from the liver and spleen after breakage of the ester bond by esterase. However, the lipophilic radiotracer delivered to the tumor remained in the tumor with minimal bond cleavage. The underlying mechanism was fully characterized in vitro and in vivo in colon tumor models. As a proof of concept, the liposomal radiotracer was further optimized for the early detection of pancreatic cancer. The folate-coated liposomal radiotracer showed highly selective tumor uptake. At 4 h postinjection, a pancreatic tumor a few millimeters in size was unambiguously visualized in orthotopic tumor models by PET imaging. At 24 h, an exceptionally high tumor-to-background ratio was achieved, enabling the visualization of tumors alone with minimal background noise. More than 9% of the total radioactivity was found in the tumor. Utilizing our imaging strategy, various tumor imaging agents can be developed for sensitive detection with ultrahigh contrast.

Successful Application of CuAAC Click Reaction in Constructing 64Cu-Labeled Antibody Conjugates for Immuno-PET Imaging.

Immuno-positron emission tomography (immuno-PET) is a rapidly growing imaging technique in which antibodies are radiolabeled to monitor their in vivo behavior in real time. However, effecting the controlled conjugation of a chelate-bearing radioactive atom to a bulky antibody without affecting its immunoreactivity at a specific site is always challenging. The in vivo stability of the radiolabeled chelate is also a key issue for successful tumor imaging. To address these points, a facile ultra-stable radiolabeling platform is developed by using the propylene cross-bridged chelator (PCB-TE2A-alkyne), which can be instantly functionalized with various groups via the click reaction, thus enabling specific conjugation with antibodies as per choice. The PCB-TE2A-tetrazine derivative is selected to demonstrate the proposed strategy. The antibody trastuzumab is functionalized with the trans-cyclooctene (TCO) moiety in the presence or absence of the PEG linker. The complementary 64Cu-PCB-TE2A-tetrazine is synthesized via the click reaction and radiolabeled with 64Cu ions, which then reacts with the aforementioned TCO-modified antibody via a rapid biorthogonal ligation. The 64Cu-PCB-TE2A-trastuzumab conjugate is shown to exhibit excellent in vivo stability and to maintain a higher binding affinity toward HER2-positive cells. The tumor targeting feasibility of the radiolabeled antibody is evaluated in tumor models. Both 64Cu-PCB-TE2A-trastuzumab conjugates show high tumor uptakes in biodistribution studies and enable unambiguous tumor visualization with minimum background noise in PET imaging. Interestingly, the 64Cu-PCB-TE2A-PEG4-trastuzumab containing an additional PEG linker displays a much faster body clearance compared to its counterpart with less PEG linker, thus affording vivid tumor imaging with an unprecedentedly high tumor-to-background ratio.

Visualization and Quantification of Radiochemical Purity by Cerenkov Luminescence Imaging.

Determination of radiochemical purity is essential for characterization of all radioactive compounds, including clinical radiopharmaceuticals. Radio-thin layer chromatography (radio-TLC) has been used as the gold standard for measurement of radiochemical purity; however, this method has several limitations in terms of sensitivity, spatial resolution, two-dimensional scanning, and quantification accuracy. Here, we report a new analytical technique for determination of radiochemical purity based on Cerenkov luminescence imaging (CLI), whereby entire TLC plates are visualized by detection of Cerenkov radiation. Sixteen routinely used TLC plates were tested in combination with three different radioisotopes (131I, 124I, and 32P). All TLC plates doped with a fluorescent indicator showed excellent detection sensitivity with scanning times of less than 1 min. The new CLI method was superior to the traditional radio-TLC scanning method in terms of sensitivity, scanning time, spatial resolution, and two-dimensional scanning. The CLI method also showed better quantification features across a wider range of radioactivity values compared with radio-TLC and classical zonal analysis, especially for ß--emitters such as 131I and 32P.

High in Vivo Stability of 64Cu-Labeled Cross-Bridged Chelators Is a Crucial Factor in Improved Tumor Imaging of RGD Peptide Conjugates.

Although the importance of bifunctional chelators (BFCs) is well recognized, the chemophysical parameters of chelators that govern the biological behavior of the corresponding bioconjugates have not been clearly elucidated. Here, five BFCs closely related in structure were conjugated with a cyclic RGD peptide and radiolabeled with Cu-64 ions. Various biophysical and chemical properties of the Cu(II) complexes were analyzed with the aim of identifying correlations between individual factors and the biological behavior of the conjugates. Tumor uptake and body clearance of the 64Cu-labeled bioconjugates were directly compared by animal PET imaging in animal models, which was further supported by biodistribution studies. Conjugates containing propylene cross-bridged chelators showed higher tumor uptake, while a closely related ethylene cross-bridged analogue exhibited rapid body clearance. High in vivo stability of the copper-chelator complex was strongly correlated with high tumor uptake, while the overall lipophilicity of the bioconjugate affected both tumor uptake and body clearance.

Anti-inflammatory effects of ursolic acid-3-acetate on human synovial fibroblasts and a murine model of rheumatoid arthritis.

Ursolic acid (UA), a pentacyclic triterpenoid, is a common natural substance known to be effective in the treatment of inflammation, oxidative stress, and ulcers in arthritis. This study examined the effects of ursolic acid-3-acetate (UAA), a derivative of UA, on rheumatoid arthritis (RA) and verified the underlying mechanism of action by using a type-II collagen-induced arthritis (CIA) mice model and tumor necrosis factor (TNF)-α-stimulated RA synovial fibroblasts. The oral administration of UAA showed a decrease in clinical arthritis symptoms, paw thickness, histologic and radiologic changes, and serum IgG1 and IgG2a levels. UAA administration reduced Th1/Th17 phenotype CD4+ T lymphocyte expansion and inflammatory cytokine production in draining lymph nodes. In addition, UAA effectively reduced the expression and production of inflammatory mediators, including cytokines and matrix metalloproteinase-1/3 in the knee joint tissue and RA synovial fibroblasts, through the downregulation of IKKα/ß, ΙκBα, and nuclear factor-κB. Our findings showed that UAA modulated helper T cell immune responses and matrix-degrading enzymes. The effects of UAA were comparable with those of the positive control drug, dexamethasone. In summary, all the evidence presented in this paper suggest that UAA could be a therapeutic candidate for the treatment of RA.

Immobilization of the Gas Signaling Molecule H2 S by Radioisotopes: Detection, Quantification, and In Vivo Imaging.

Hydrogen sulfide (H2 S) has multifunctional roles as a gas signaling molecule in living systems. However, the efficient detection and imaging of H2 S in live animals is very challenging. Herein, we report the first radioisotope-based immobilization technique for the detection, quantification, and in vivo imaging of endogenous H2 S. Macrocyclic (64) Cu complexes that instantly reacted with gaseous H2 S to form insoluble (64) CuS in a highly sensitive and selective manner were prepared. The H2 S concentration in biological samples was measured by a thin-layer radiochromatography method. When (64) Cu-cyclen was injected into mice, an elevated H2 S concentration in the inflamed paw was clearly visualized and quantified by Cerenkov luminescence and positron emission tomography (PET) imaging. PET imaging was also able to pinpoint increased H2 S levels in a millimeter-sized infarcted lesion of the rat heart.

Oleanolic acid acetate inhibits rheumatoid arthritis by modulating T cell immune responses and matrix-degrading enzymes.

Rheumatoid arthritis (RA) is a chronic autoimmune disease associated with a combination of synovium joint inflammation, synovium hyperplasia, and destruction of cartilage and bone. Oleanolic acid acetate (OAA), a compound isolated from Vigna angularis, has been known to possess pharmacological activities, including anti-inflammation and anti-bone destruction. In this study, we investigated the effects of OAA on RA and the underlying mechanisms of action by using a type-II collagen-induced arthritis (CIA) mouse model and tumor necrosis factor (TNF)-α-stimulated RA synovial fibroblasts. Oral administration of OAA decreased the clinical arthritis symptoms, paw thickness, histologic and radiologic changes, and serum total and anti-type II collagen IgG, IgG1, and IgG2a levels. OAA administration reduced Th1/Th17 phenotype CD4(+) T lymphocyte expansions and inflammatory cytokine productions in T cell activated draining lymph nodes and spleen. OAA reduced the expression and production of inflammatory mediators, such as cytokines and matrix metalloproteinase (MMP)-1/3, in the ankle joint tissue and RA synovial fibroblasts by down-regulating Akt, mitogen-activated protein kinases, and nuclear factor-κB. Our results clearly support that OAA plays a therapeutic role in RA pathogenesis by modulating helper T cell immune responses and matrix-degrading enzymes. The immunosuppressive effects of OAA were comparable to dexamethasone and ketoprofen. We provide evidences that OAA could be a potential therapeutic candidate for RA.

Phosphonate Pendant Armed Propylene Cross-Bridged Cyclam: Synthesis and Evaluation as a Chelator for Cu-64.

A propylene cross-bridged macrocyclic chelator with two phosphonate pendant arms (PCB-TE2P) was synthesized from cyclam. Various properties of the synthesized chelator, including Cu-complexation, Cu-complex stability, (64)Cu-radiolabeling, and in vivo behavior, were studied and compared with those of a previously reported propylene cross-bridged chelator (PCB-TE2A).

Synthesis and Evaluation of New Generation Cross-Bridged Bifunctional Chelator for (64)Cu Radiotracers.

Bifunctional chelators have been successfully used to construct (64)Cu-labeled radiopharmaceuticals. Previously reported chelators with cross-bridged cyclam backbones have various essential features such as high stability of the copper(II) complex, high efficiency of radiolabeling at room temperature, and good biological inertness of the radiolabeled complex, along with rapid body clearance. Here, we report a new generation propylene-cross-bridged chelator with hybrid acetate/phosphonate pendant groups (PCB-TE1A1P) developed with the aim of combining these key properties in a single chelator. The PCB-TE1A1P was synthesized from cyclam with good overall yield. The Cu(II) complex of our chelator showed good robustness in kinetic stability evaluation experiments, such as acidic decomplexation and cyclic voltammetry studies. The Cu(II) complex of PCB-TE1A1P remained intact under highly acidic conditions (12 M HCl, 90 °C) for 8 d and showed quasi-reversible reduction/oxidation peaks at -0.77 V in electrochemical studies. PCB-TE1A1P was successfully radiolabeled with (64)Cu ions in an acetate buffer at 60 °C within 60 min. The electrophoresis study revealed that the (64)Cu-PCB-TE1A1P complex has net negative charge in aqueous solution. The biodistribution and in vivo stability study profiles of (64)Cu-PCB-TE1A1P indicated that the radioactive complex was stable under physiological conditions and cleared rapidly from the body. A whole body positron emission tomography (PET) imaging study further confirmed high in vivo stability and fast clearance of the complex in mouse models. In conclusion, PCB-TE1A1P has good potential as a bifunctional chelator for (64)Cu-based radiopharmaceuticals, especially those involving peptides.

Apoptosis imaging studies in various animal models using radio-iodinated peptide.

Apoptosis has a role in many medical disorders and treatments; hence, its non-invasive evaluation is one of the most riveting research topics. Currently annexin V is used as gold standard for imaging apoptosis. However, several drawbacks, including high background, slow body clearance, make it a suboptimum marker for apoptosis imaging. In this study, we radiolabeled the recently identified histone H1 targeting peptide (ApoPep-1) and evaluated its potential as a new apoptosis imaging agent in various animal models. ApoPep-1 (CQRPPR) was synthesized, and an extra tyrosine residue was added to its N-terminal end for radiolabeling. This peptide was radiolabeled with (124)I and (131)I and was tested for its serum stability. Surgery- and drug-induced apoptotic rat models were prepared for apoptosis evaluation, and PET imaging was performed. Doxorubicin was used for xenograft tumor treatment in mice, and the induced apoptosis was studied. Tumor metabolism and proliferation were assessed by [(18)F]FDG and [(18)F]FLT PET imaging and compared with ApoPep-1 after doxorubicin treatment. The peptide was radiolabeled at high purity, and it showed reasonably good stability in serum. Cell death was easily imaged by radiolabeled ApoPep-1 in an ischemia surgery model. And, liver apoptosis was more clearly identified by ApoPep-1 rather than [(124)I]annexin V in cycloheximide-treated models. Three doxorubicin doses inhibited tumor growth, which was evaluated by 30-40% decreases of [(18)F]FDG and [(18)F]FLT PET uptake in the tumor area. However, ApoPep-1 demonstrated more than 200% increase in tumor uptake after chemotherapy, while annexin V did not show any meaningful uptake in the tumor compared with the background. Biodistribution data were also in good agreement with the microPET imaging results. All of the experimental data clearly demonstrated high potential of the radiolabeled ApoPep-1 for in vivo apoptosis imaging.

Propylene cross-bridged macrocyclic bifunctional chelator: a new design for facile bioconjugation and robust (64)Cu complex stability.

The first macrocyclic bifunctional chelator incorporating propylene cross-bridge was efficiently synthesized from cyclam in seven steps. After the introduction of an extra functional group for facile conjugation onto the propylene cross-bridge, the two carboxylic acid pendants could contribute to strong coordination of Cu(II) ions, leading to a robust Cu complex. The cyclic RGD peptide conjugate of PCB-TE2A-NCS was prepared and successfully radiolabeled with (64)Cu ion. The radiolabeled peptide conjugate was evaluated in vivo through a biodistribution study and animal PET imaging to demonstrate high tumor uptake with low background.

Vivid tumor imaging utilizing liposome-carried bimodal radiotracer.

By developing a new bimodal radioactive tracer that emits both luminescence and nuclear signals, a trimodal liposome for optical, nuclear, and magnetic resonance imaging is efficiently prepared. Fast clearance of the radiotracer from reticuloendothelial systems enables vivid tumor imaging with minimum background.

pH-responsive gold nanoparticles-in-liposome hybrid nanostructures for enhanced systemic tumor delivery.

We report a pH-responsive gold nanoparticles-in-liposome hybrid nanostructure, which effectively combines the pH-responsive assembly and surface plasmon property changes of 'smart' gold nanoparticles and enhanced systemic circulation and tumor accumulation of the PEG-grafted liposomes.

New bifunctional chelator for 64Cu-immuno-positron emission tomography.

A new tetraazamacrocyclic bifunctional chelator, TE2A-Bn-NCS, was synthesized in high overall yield from cyclam. An extra functional group (NCS) was introduced to the N-atom of TE2A for specific conjugation with antibody. The Cu complex of TE2A-Bn-NCS showed high kinetic stability in acidic decomplexation and cyclic voltammetry studies. X-ray structure determination of the Cu-TE2A-Bn-NH2 complex confirmed octahedral geometry, in which copper atom is strongly coordinated by four macrocyclic nitrogens in equatorial positions and two carboxylate oxygen atoms occupy the elongated axial positions. Trastuzumab was conjugated with TE2A-Bn-NCS and then radiolabeled with 64Cu quantitatively at room temperature within 10 min. Biodistribution studies showed that the 64Cu-labeled TE2A-Bn-NCS-trastuzumab conjugates maintain high stability in physiological conditions, and NIH3T6.7 tumors were clearly visualized up to 3 days by 64Cu-immuno-positron emission tomography imaging in animal models.

pH-responsive assembly of gold nanoparticles and "spatiotemporally concerted" drug release for synergistic cancer therapy.

A challenge in using plasmonic nanostructure-drug conjugates for thermo-chemo combination cancer therapy lies in the huge size discrepancy; the size difference can critically differentiate their biodistributions and hamper the synergistic effect. Properly tuning the plasmonic wavelength for photothermal therapy typically results in the nanostructure size reaching ∼100 nm. We report a new combination cancer therapy platform that consists of relatively small 10 nm pH-responsive spherical gold nanoparticles and conjugated doxorubicins. They are designed to form aggregates in mild acidic environment such as in a tumor. The aggregates serve as a photothermal agent that can selectively exploit external light by their collective plasmon modes. Simultaneously, the conjugated doxorubicins are released. The spatiotemporal concertion is confirmed at the subcellular, cellular, and organ levels. Both agents colocalize in the cell nuclei. The conjugates accumulate in cancer cells by the rapid phagocytic actions and effective blockage of exocytosis by the increased aggregate size. They also effectively accumulate in tumors up to 17 times over the control because of the enhanced permeation and retention. The conjugates exhibit a synergistic effect enhanced by nearly an order of magnitude in cellular level. The synergistic effect is demonstrated by the remarkable reductions in both the therapeutically effective drug dosage and the photothermal laser threshold. Using an animal model, effective tumor growth suppression is demonstrated. The conjugates induce apoptosis to tumors without any noticeable damage to other organs. The synergistic effect in vivo is confirmed by qRT-PCR analysis over the thermal stress and drug-induced growth arrest.

Non-cross-bridged tetraazamacrocyclic chelator for stable (64)cu-based radiopharmaceuticals.

N-mono/dimethylated TE2A tetraazamacrocycles (MM-TE2A and DM-TE2A) were synthesized in high yields. Both Cu-MM/DM-TE2A complexes showed increased kinetic stability compared to that of Cu-TE2A, whereas Cu-DM-TE2A showed even higher in vitro stability than that of Cu-ECB-TE2A. MM-TE2A and DM-TE2A were quantitatively radiolabeled with (64)Cu ions and showed rapid clearance from the body to emerge as a potential efficient bifunctional chelator.

Synthesis and evaluation of a radioiodinated bladder cancer specific peptide.

Bladder cancer is the second most common cancer of the urinary tract, however the invasive cystoscopy is still the standard technique for diagnosis and surveillance of bladder cancer. Herein, we radiolabel bladder cancer specific peptide with radioactive iodine ((131/124)I) and evaluate its potential as a new radiopharmaceutical for the non-invasive diagnosis of bladder cancer. A 9-mer bladder cancer specific peptide (BP) was conjugated with tyrosine and cyclized by disulfide bond formation to give Y-BP, which was further radioiodinated to give [(131/124)I]Y-BP in good radiochemical yield. The biodistribution data showed the high selectivity of [(124)I]Y-BP in HT1376 human bladder cancer xenograft models with a tumor-to-muscle ratio of 6.2. This tumor targeting was not observed in control B16F10 melanoma tumor models. In microPET studies, while the control scrambled peptide, [(124)I]Y-sBP, did not accumulate in either the bladder cancer or melanoma, [(124)I]Y-BP showed high tumor uptake only in animals with HT1376 bladder cancer cells. Furthermore, [(124)I]Y-BP showed superior bladder cancer uptake even compared to most commonly used cancer imaging tracer, [(18)F]FDG. The experimental results suggest the potential of [(124)I]Y-BP as a new radiopharmaceutical for the non-invasive diagnosis of bladder cancer with high binding affinity and selectivity.