Renal clearance and degradation of glutathione-coated copper nanoparticles.

Degradation of inorganic nanoparticles (NPs) into small molecular complexes is often observed in the physiological environment; however, how this process influences renal clearance of inorganic NPs is largely unknown. By systematically comparing renal clearance of degradable luminescent glutathione coated copper NPs (GS-CuNPs) and their dissociated products, Cu(II)-glutathione disulfide (GSSG) complexes (Cu(II)-GSSG), we found that GS-CuNPs were eliminated through the urinary system surprisingly faster and accumulated in the liver much less than their smaller dissociation counterparts. With assistance of radiochemistry and positron emission tomography (PET) imaging, we found that the observed "nano size" effect in enhancing renal clearance is attributed to the fact that GS-CuNPs are more resistant to serum protein adsorption than Cu(II)-GSSG. In addition, since dissociation of GS-CuNPs follows zero-order chemical kinetics, their renal clearance and biodistribution also depend on initial injection doses and their dissociation processes. Quantitative understanding of size effect and other factors involved in renal clearance and biodistribution of degradable inorganic NPs will lay down a foundation for further development of renal-clearable inorganic NPs with minimized nanotoxicity.

Click-chemistry strategy for labeling antibodies with copper-64 via a cross-bridged tetraazamacrocyclic chelator scaffold.

We report a click-chemistry based modular strategy for antibody labeling with (64)Cu (t1/2 = 12.7 h; ß(+) 0.656 MeV, 17.4%; ß(-) 0.573 MeV, 39%; EC 43%) under ambient condition utilizing a cross-bridged tetraazamacrocyclic (CB-TE2A) analogue, which otherwise requires harsh conditions that make the CB-TE2A analogues under-utilized for protein labeling despite the fact that they form kinetically inert copper complexes with high in vivo stability. Our strategy involves prelabeling a CB-TE2A based scaffold (CB-TE2A-1C) with (64)Cu and its subsequent reaction with an antibody via the tetrazine-norbornene mediated click chemistry. The effectiveness of this strategy was demonstrated by labeling two monoclonal antibodies, an anti-PSMA antibody (YPSMA-1) and a chimeric anti-phosphatidylserine antibody (Bavituximab). The immunoreactivity of the antibodies remained unchanged after the tetrazine modification and click-chemistry (64)Cu labeling. To further demonstrate the practicality of the modular (64)Cu labeling strategy, we tested positron emission tomography (PET) imaging of tumor with the (64)Cu-labeled bavituximab in a mouse xenograft model. The tumor visualization and uptake of the labeled antibody exhibited the versatility of the click-chemistry strategy.

Molecular platform for design and synthesis of targeted dual-modality imaging probes.

We report a versatile dendritic structure based platform for construction of targeted dual-modality imaging probes. The platform contains multiple copies of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) branching out from a 1,4,7-triazacyclononane-N,N',N″-triacetic acid (NOTA) core. The specific coordination chemistries of the NOTA and DOTA moieties offer specific loading of (68/67)Ga(3+) and Gd(3+), respectively, into a common molecular scaffold. The platform also contains three amino groups which can potentiate targeted dual-modality imaging of PET/MRI or SPECT/MRI (PET: positron emission tomography; SPECT: single photon emission computed tomography; MRI: magnetic resonance imaging) when further functionalized by targeting vectors of interest. To validate this design concept, a bimetallic complex was synthesized with six peripheral Gd-DOTA units and one Ga-NOTA core at the center, whose ion T1 relaxivity per gadolinium atom was measured to be 15.99 mM(-1) s(-1) at 20 MHz. Further, the bimetallic agent demonstrated its anticipated in vivo stability, tissue distribution, and pharmacokinetic profile when labeled with (67)Ga. When conjugated with a model targeting peptide sequence, the trivalent construct was able to visualize tumors in a mouse xenograft model by both PET and MRI via a single dose injection.

Design, synthesis and biological assessment of a triazine dendrimer with approximately 16 Paclitaxel groups and 8 PEG groups.

The synthesis and characterization of a generation three triazine dendrimer that displays a phenolic group at the core for labeling, up to eight 5 kDa PEG chains for solubility, and 16 paclitaxel groups is described. Three different diamine linkers--dipiperidine trismethylene, piperazine, and aminomethylpiperidine--were used within the dendrimer. To generate the desired stoichiometric ratio of 8 PEG chains to 16 paclitaxel groups, a monochlorotriazine was prepared with two paclitaxel groups attached through their 2'-hydroxyls using a linker containing a labile disulfide. This monochlorotriazine was linked to a dichlorotriazine with aminomethylpiperidine. The resulting dichlorotriazine bearing two paclitaxel groups could be reacted with the eight amines of the dendrimer. NMR and MALDI-TOF confirm successful reaction. The eight monochlorotriazines of the resulting material are used as the site for PEGylation affording the desired 2:1 stoichiometry. The target and intermediates were amenable to characterization by (1)H and (13)C NMR, and mass spectrometry. Analysis revealed that 16 paclitaxel groups were installed along with 5-8 PEG chains. The final construct is 63% PEG, 22% paclitaxel, and 15% triazine dendrimer. Consistent with previous efforts and computational models, 5 kDa PEG groups were essential for making the target water-soluble. Molecular dynamics simulations showed a high degree of hydration of the core, and a radius of gyration of 2.8 ± 0.2 nm. The hydrodynamic radius of the target was found to be 15.8 nm by dynamic light scattering, an observation indicative of aggregation. Drug release studies performed in plasma showed slow and identical release in mouse and rat plasma (8%, respectively). SPECT/CT imaging was used to follow biodistribution and tumor uptake. Using a two component model, the elimination and distribution half-lives were 2.65 h and 38.2 h, respectively. Compared with previous constructs, this dendrimer persists in the vasculature longer (17.33 ± 0.88% ID/g at 48 h postinjection), and showed higher tumor uptake. Low levels of dendrimer were observed in lung, liver, and spleen (~6% ID/g). Tumor saturation studies of small prostate cancer tumors (PC3) suggest that saturation occurs at a dose between 23.2 mg/kg and 70.9 mg/kg.

Beyond Colonoscopy: Exploring New Cell Surface Biomarkers for Detection of Early, Heterogenous Colorectal Lesions.

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths among both men and women in the United States. Early detection and surgical removal of high-risk lesions in the colon can prevent disease from developing and spreading. Despite implementation of programs aimed at early detection, screening colonoscopies fail to detect a fraction of potentially aggressive colorectal lesions because of their location or nonobvious morphology. Optical colonoscopies, while highly effective, rely on direct visualization to detect changes on the surface mucosa that are consistent with dysplasia. Recent advances in endoscopy techniques and molecular imaging permit microscale visualization of the colonic mucosa. These technologies can be combined with various molecular probes that recognize and target heterogenous lesion surfaces to achieve early, real-time, and potentially non-invasive, detection of pre-cancerous lesions. The primary goal of this review is to contextualize existing and emergent CRC surface biomarkers and assess each's potential as a candidate marker for early marker-based detection of CRC lesions. CRC markers that we include were stratified by the level of support gleaned from peer-reviewed publications, abstracts, and databases of both CRC and other cancers. The selected biomarkers, accessible on the cell surface and preferably on the luminal surface of the colon tissue, are organized into three categories: (1) established biomarkers (those with considerable data and high confidence), (2) emerging biomarkers (those with increasing research interest but with less supporting data), and (3) novel candidates (those with very recent data, and/or supportive evidence from other tissue systems). We also present an overview of recent advances in imaging techniques useful for visual detection of surface biomarkers, and discuss the ease with which these methods can be combined with microscopic visualization.

Neuropathological Effects of Chemotherapeutic Drugs.

Novel treatments, screening, and detection methods have prolonged the lives of numerous cancer patients worldwide. Unfortunately, existing and many promising new chemotherapeutics can cause deleterious, off-target side effects in normal tissue and organ systems. The central and peripheral nervous systems are widely recognized as frequent off-target effectors of anticancer drugs which can produce persistent neurological and neuropsychiatric symptoms collectively termed "chemobrain". Following chemotherapy, patients report several forms of cognitive impairment occurring acutely and sometimes persisting years after treatment. There are no effective treatments for cognitive decline induced by chemotherapeutics, and the underlying molecular mechanisms are poorly characterized and understood. In this study, we find that chronic treatment with two common chemotherapeutic agents, cisplatin and gemcitabine, impairs brain region-specific metabolism, hippocampus-dependent memory formation, and stress response behavior. This corresponds to reduced hippocampal synaptic excitability, altered neuronal signal transduction, and neuroinflammation. These findings underline that a better understanding of the basic pathological consequences of chemotherapy-induced cognitive impairment is the first step toward improving cancer treatment survivorship.

Hyperpolarized MRI with silicon micro and nanoparticles: Principles and applications.

Silicon-based micro and nanoparticles are ideally suited for use as biomedical imaging agents because of their biocompatibility, biodegradability, and simple surface chemistry that facilitates drug loading and targeting. A method to hyperpolarize silicon particles using dynamic nuclear polarization (DNP), which increases magnetic resonance (MR) imaging signals by several orders-of-magnitude through enhanced nuclear spin alignment, was developed to allow silicon particles to function as contrast agents for in vivo magnetic resonance imaging. In this review, we describe the application of the DNP technique to silicon particles and nanoparticles for background-free real-time molecular MR imaging. This review provides a summary of the state-of-the-science in silicon particle hyperpolarization with a detailed protocol for hyperpolarizing silicon particles. This information will foster awareness and spur interest in this emerging area of nanoimaging and provide a path to new developments and discoveries to further advance the field. This article is categorized under: Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Therapeutic Approaches and Drug Discovery > Emerging Technologies.

Theranostic Nanoseeds for Efficacious Internal Radiation Therapy of Unresectable Solid Tumors.

Malignant tumors are considered "unresectable" if they are adhere to vital structures or the surgery would cause irreversible damages to the patients. Though a variety of cytotoxic drugs and radiation therapies are currently available in clinical practice to treat such tumor masses, these therapeutic modalities are always associated with substantial side effects. Here, we report an injectable nanoparticle-based internal radiation source that potentially offers more efficacious treatment of unresectable solid tumors without significant adverse side effects. Using a highly efficient incorporation procedure, palladium-103, a brachytherapy radioisotope in clinical practice, was coated to monodispersed hollow gold nanoparticles with a diameter about 120 nm, to form (103)Pd@Au nanoseeds. The therapeutic efficacy of (103)Pd@Au nanoseeds were assessed when intratumorally injected into a prostate cancer xenograft model. Five weeks after a single-dose treatment, a significant tumor burden reduction (>80%) was observed without noticeable side effects on the liver, spleen and other organs. Impressively, >95% nanoseeds were retained inside the tumors as monitored by Single Photon Emission Computed Tomography (SPECT) with the gamma emissions of (103)Pd. These findings show that this nanoseed-based brachytherapy has the potential to provide a theranostic solution to unresectable solid tumors.

A Transistor-like pH Nanoprobe for Tumour Detection and Image-guided Surgery.

Because of profound genetic and histological differences in cancerous tissue, it is challenging to detect a broad range of malignant tumours at high resolution. Here, we report the design and performance of a fluorescent nanoprobe with transistor-like responses (transition pH = 6.9) for the detection of the deregulated pH that drives many of the invasive properties of cancer. The nanoprobe amplifies fluorescence signal in the tumour over that in the surrounding normal tissues, resulting in a discretized, binary output signal with spatial resolution smaller than 1 mm. The nanoprobe allowed us to image a broad range of tumours in mouse models using a variety of clinical cameras, and to perform real-time tumour-acidosis-guided detection and surgery of occult nodules (< 1 mm3) in mice bearing head-and-neck or breast tumours, significantly lengthening mice survivability. We also show that the pH nanoprobe can be used as a reporter in a fast, quantitative assay to screen for tumour-acidosis inhibitors. The binary delineation of pH achieved by the nanoprobe promises to improve the accuracy of cancer detection, surveillance and therapy.

[(68) Ga]-HP-DO3A-nitroimidazole: a promising agent for PET detection of tumor hypoxia.

The goal of this study is to evaluate a new (68) Ga-based imaging agent for detecting tumor hypoxia using positron emission tomography (PET). The new hypoxia targeting agent reported here, [(68) Ga]-HP-DO3A-nitroimidazole ([(68) Ga]-HP-DO3A-NI), was constructed by linking a nitroimidazole moiety with the macrocyclic ligand component of ProHance®, HP-DO3A. The hypoxia targeting capability of this agent was evaluated in A549 lung cancer cells in vitro and in SCID mice bearing subcutaneous A549 tumor xenografts. The cellular uptake assays showed that significantly more [(68) Ga]-HP-DO3A-NI accumulates in hypoxic tumor cells at 30, 60 and 120 min than in the same cells exposed to 21% O2 . The agent also accumulated in hypoxic tumors in vivo to give a tumor/muscle ratio (T/M) of 5.0 ± 1.2 (n = 3) as measured by PET at 2 h post-injection (p.i.). This was further confirmed by ex vivo biodistribution data. In addition, [(68) Ga]-HP-DO3A-NI displayed very favorable pharmacokinetic properties, as it was cleared largely through the kidneys with little to no accumulation in liver, heart or lung (%ID/g < 0.5%) at 2 h p.i. The specificity of the agent for hypoxic tissues was further validated in a comparative study with a control compound, [(68) Ga]-HP-DO3A, which lacks the nitroimidazole moiety, and by PET imaging of tumor-bearing mice breathing air versus 100% O2 . Given the commercial availability of cGMP (68) Ge/(68) Ga generators and the ease of (68) Ga labeling, the new agent could potentially be widely applied for imaging tumor hypoxia prior to radiation therapy.

Differential expression of cell cycle regulators in CDK5-dependent medullary thyroid carcinoma tumorigenesis.

Medullary thyroid carcinoma (MTC) is a neuroendocrine cancer of thyroid C-cells, for which few treatment options are available. We have recently reported a role for cyclin-dependent kinase 5 (CDK5) in MTC pathogenesis. We have generated a mouse model, in which MTC proliferation is induced upon conditional overexpression of the CDK5 activator, p25, in C-cells, and arrested by interrupting p25 overexpression. Here, we identify genes and proteins that are differentially expressed in proliferating versus arrested benign mouse MTC. We find that downstream target genes of the tumor suppressor, retinoblastoma protein, including genes encoding cell cycle regulators such as CDKs, cyclins and CDK inhibitors, are significantly upregulated in malignant mouse tumors in a CDK5-dependent manner. Reducing CDK5 activity in human MTC cells down-regulated these cell cycle regulators suggesting that CDK5 activity is critical for cell cycle progression and MTC proliferation. Finally, the same set of cell cycle proteins was consistently overexpressed in human sporadic MTC but not in hereditary MTC. Together these findings suggest that aberrant CDK5 activity precedes cell cycle initiation and thus may function as a tumor-promoting factor facilitating cell cycle protein expression in MTC. Targeting aberrant CDK5 or its downstream effectors may be a strategy to halt MTC tumorigenesis.

Enantiopure bifunctional chelators for copper radiopharmaceuticals--does chirality matter in radiotracer design?

It is well recognized that carbon chirality plays a critical role in the design of drug molecules. However, very little information is available regarding the effect of stereoisomerism of macrocyclic bifunctional chelators (BFC) on biological behaviors of the corresponding radiopharmaceuticals. To evaluate such effects, three enantiopure stereoisomers of a copper radiopharmaceutical BFC bearing two chiral carbon atoms were synthesized in forms of R,R-, S,S-, and R,S-. Their corresponding peptide conjugates were prepared by coupling with a model peptide sequence, c(RGDyK), which targets the αvß3 integrin for in vitro and in vivo evaluation of their biological behaviors as compared to the racemic conjugate. Despite the chirality differences, all the conjugates showed a similar in vitro binding affinity profile to the αvß3 integrin (106, 108, 85 and 100 nM for rac-H2-1, RR-H2-1, SS-H2-1, and RS-H2-1 respectively with all p values > 0.05) and a similar level of in vivo tumor uptake (2.72 ± 0.45, 2.60 ± 0.52, 2.45 ± 0.48 and 2.88 ± 0.59 for rac-(64)Cu-1, RR-(64)Cu-1, SS-(64)Cu-1, and RS-(64)Cu-1 at 1 h p.i. respectively). Furthermore, they demonstrated a nearly identical biodistribution pattern in major organs (e.g. 2.07 ± 0.21, 2.13 ± 0.58, 1.70 ± 0.20 and 1.90 ± 0.46 %ID/g at 24 h p.i. in liver for rac-(64)Cu-1, RR-(64)Cu-1, SS-(64)Cu-1, and RS-(64)Cu-1 respectively; 1.80 ± 0.46, 2.30 ± 1.49, 1.73 ± 0.31 and 2.23 ± 0.71 at 24 h p.i. in kidneys for rac-(64)Cu-1, RR-(64)Cu-1, SS-(64)Cu-1, and RS-(64)Cu-1 respectively). Therefore we conclude that the chirality of BFC plays a negligible role in αvß3-targeted copper radiopharmaceuticals. However, we believe it is still worthwhile to consider the chirality effects of BFCs on other targeted imaging or therapeutic agents.

Use of Fc-Engineered Antibodies as Clearing Agents to Increase Contrast During PET.

UNLABELLED: Despite promise for the use of antibodies as molecular imaging agents in PET, their long in vivo half-lives result in poor contrast and radiation damage to normal tissue. This study describes an approach to overcome these limitations. METHODS: Mice bearing human epidermal growth factor receptor type 2 (HER2)-overexpressing tumors were injected with radiolabeled ((124)I, (125)I) HER2-specific antibody (pertuzumab). Pertuzumab injection was followed 8 h later by the delivery of an engineered, antibody-based inhibitor of the receptor, FcRn. Biodistribution analyses and PET were performed at 24 and 48 h after pertuzumab injection. RESULTS: The delivery of the engineered, antibody-based FcRn inhibitor (or Abdeg, for antibody that enhances IgG degradation) results in improved tumor-to-blood ratios, reduced systemic exposure to radiolabel, and increased contrast during PET. CONCLUSION: Abdegs have considerable potential as agents to stringently regulate antibody dynamics in vivo, resulting in increased contrast during molecular imaging with PET.

Lin28b is sufficient to drive liver cancer and necessary for its maintenance in murine models.

Lin28a/b are RNA-binding proteins that influence stem cell maintenance, metabolism, and oncogenesis. Poorly differentiated, aggressive cancers often overexpress Lin28, but its role in tumor initiation or maintenance has not been definitively addressed. We report that LIN28B overexpression is sufficient to initiate hepatoblastoma and hepatocellular carcinoma in murine models. We also detected Lin28b overexpression in MYC-driven hepatoblastomas, and liver-specific deletion of Lin28a/b reduced tumor burden, extended latency, and prolonged survival. Both intravenous siRNA against Lin28b and conditional Lin28b deletion reduced tumor burden and prolonged survival. Igf2bp proteins are upregulated, and Igf2bp3 is required in the context of LIN28B overexpression to promote growth. Therefore, multiple murine models demonstrate that Lin28b is both sufficient to initiate liver cancer and necessary for its maintenance.