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In JoVE (1)

Other Publications (122)

Articles by Zhen Cheng in JoVE

 JoVE Clinical and Translational Medicine

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

1Department of Radiology and Bio-X Program Canary Cancer at Stanford for Cancer Early Detection, Stanford University


JoVE 4341

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

Other articles by Zhen Cheng on PubMed

Alpha-melanocyte-stimulating Hormone Peptide Analogs Labeled with Technetium-99m and Indium-111 for Malignant Melanoma Targeting

Previous studies have shown that the compact structure of a rhenium-cyclized alpha--melanocyte-stimulating hormone peptide analog, [Cys3410,D-Phe7]alpha-MSH(3--13), or Re-CCMSH, significantly enhanced its in vivo tumor uptake and retention. In this study, the metal chelate 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was coupled to the N-terminus of Re-CCMSH in order to develop a melanoma-targeting peptide that could be labeled with a wider variety of imaging and therapeutic radionuclides.

Modification of the Structure of a Metallopeptide: Synthesis and Biological Evaluation of (111)In-labeled DOTA-conjugated Rhenium-cyclized Alpha-MSH Analogues

Rhenium-cyclized CCMSH analogues are novel melanoma-targeting metallopeptides with high tumor uptake, long tumor retention, and low background in normal tissues, which make these metallopeptides an ideal structural motif for designing novel melanoma-targeting agents. ReCCMSH has been derivatized with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelate so that it can be labeled with a wide variety of radionuclides for imaging and therapeutic applications. This study involved optimization of the in vivo biological properties of DOTA-ReCCMSH (S), through modification of the structure of the metallopeptide. Several DOTA-ReCCMSH analogues, Ac-Lys(DOTA)-ReCCMSH (4) DOTA-ReCCMSH(Arg(11)) (6), DOTA-ReCCMSH-OH (8), and DOTA-ReCCMSH-Asp-OH (10), were synthesized using solid phase peptide synthesis followed by rhenium cyclization. The IC(50) values of the metallopeptides were determined through competitive binding assays against (125)I-(Tyr(2))-NDP. Radiolabeling of the DOTA-rhenium-cyclized peptides with (111)In was carried out in NH(4)OAc (0.1 M; pH 5.5)-buffered solution for 30 min at 70 degrees C. The stability of the radiolabeled complexes was evaluated in 0.01 M, pH 7.4, phosphate-buffered saline/0.1% bovine serum albumin solution. After separation of the radiolabeled peptide from the unlabeled peptide by reverse phase high-performance liquid chromatography, the biodistribution of the radiolabeled complex was performed in C57 mice bearing B16/F1 murine melanoma tumors. All radiolabeled complexes showed fast blood clearance (2 h postinjection (pi): (111)In-S, 0.07 +/- 0.03% ID/g; (111)In-4, 0.09 +/- 0.06% ID/g; (111)In-6, 0.21 +/- 0.08% ID/g; (111)In-8, 0.11 +/- 0.10% ID/g; and (111)In-10, 0.05 +/- 0.03% ID/g), and their clearance was predominantly through the urine (4 h pi: 93.5 +/- 1.7, 87.8 +/- 6.5, 89.8 +/- 4.2, 93.3 +/- 1.1, and 93.8 +/- 1.8 (% ID) for (111)In-labeled S, 4, 6, 8, and 10, respectively). Tumor uptake values of 9.45 +/- 0.90, 6.01 +/- 2.36, 17.41 +/- 5.61, 9.27 +/- 0.68, and 7.32 +/- 2.09 (% ID/g) for (111)In-labeled S, 4, 6, 8, and 10, respectively, were observed at 4 h pi. The kidney uptake was 9.27 +/- 2.65% ID/g for (111)In-S, 19.02 +/- 2.63% ID/g for (111)In-4, 7.37 +/- 1.13% ID/g for (111)In-6, 8.70 +/- 0.88% ID/g for (111)In-8, and 8.13 +/- 1.47% ID/g for (111)In-10 at 4 h pi. Complex 6 showed high melanoma uptake and lower kidney uptake than the corresponding Lys(11) analogues, supporting 6 for further investigations as a potential therapeutic radiopharmaceutical.

[Medical History from SARS to Pneumonia]

SARS is a new kind of pneumonia. From the end of 2002 to the beginning of 2003, SARS broke in Guangdong province, Hong Kong and Beijing, and then gradually spread to the world. SARS is extremely contagious. The symptoms of SARS progress very quickly. SARS smashes the people's tranquil life and many people live in horror, worry and anxiety. But if we review the medical history of pneumonia, we would have a better understanding of SARS. This article focuses the history of people's understanding of pneumonia on the historical documents, diagnosis, etiology and treatment. Through the epidemic of SARS, the author hopes to express that contagion will live with us for a long time, but it is not a deadly disease. It is preventable and good care is essential for contagious patients. As Chinese people, we should have the best use of TCM in our combat with contagion.

Radioiodination of Rhenium Cyclized Alpha-melanocyte-stimulating Hormone Resulting in Enhanced Radioactivity Localization and Retention in Melanoma

Radiohalogenated alpha-melanocyte-stimulating hormone (alpha-MSH) analogs were proposed for melanoma imaging and potential radiotherapy because alpha-MSH receptors are overexpressed on both mouse and human melanoma cell lines. However, biodistribution studies in tumor-bearing mice with radiohalogenated alpha-MSH peptides showed very rapid tumor radioactivity wash out due to lysosomal degradation of the radiohalogenated complex after internalization, which decreased the therapeutic efficacy significantly (R. Stein et al., Cancer Res., 55: 3132-3139, 1995; P. K. Garg et al., Bioconjugate Chem., 6: 493-501, 1995.). The melanoma-targeting metallopeptide ReO[Cys(3,4,10),D-Phe(7)]alpha-MSH(3-13) (ReCCMSH) was shown to possess high tumor uptake and retention properties (J. Chen et al., Cancer Res., 60: 5649-5658, 2000). Therefore, three peptides, Ac-Lys-ReCCMSH(Arg(11)), Ac-D-Lys-ReCCMSH(Arg(11)), and [Nle(4),D-Phe(7)]alpha-MSH (NDP) (for comparison), labeled with N-succinimidyl 4-[(125)I]iodobenzoate ((125)I-PIB), were prepared and evaluated in vitro and in vivo to develop radiohalogenated alpha-MSH peptide analogs with high tumor uptake, retention, and favorable biodistribution characteristics. In vitro cell binding and internalization data showed that approximately 90% of radioiodinated peptides were internalized at 2 h in cultured B16/F1 melanoma cells. Cellular retention studies showed that the receptor-bound radioiodinated linear alpha-MSH analog NDP was released from the cells into the medium very quickly, whereas significant amounts of cell-associated radioactivity remained in the cells for Ac-Lys((125)I-3- or 4-iodobenzoate (IBA))-ReCCMSH(Arg(11)) and Ac-D-Lys((125)I-IBA)-ReCCMSH(Arg(11)). The in vitro data clearly demonstrate that rhenium cyclization significantly enhanced peptide trapping in the cells, as did D-amino acid incorporation. The combination of these two effects resulted in a 2.9-fold increase in the retention of radioactivity for Ac-D-Lys((125)I-IBA)-ReCCMSH(Arg(11)) relative to (125)I-IBA-NDP at 4 h. In vivo studies also showed that Ac-D-Lys((125)I-IBA)-ReCCMSH(Arg(11)) exhibited extremely high radioactivity accumulation and prolonged retention in the tumor. Ac-D-Lys((125)I-IBA)-ReCCMSH(Arg(11)) and Ac-Lys((125)I-IBA)-ReCCMSH(Arg(11)) exhibited much higher tumor uptake at 24 h after injection compared with (125)I-IBA-NDP [7.18% injected dose/gram (ID/g), 4.92% ID/g, and 0.26% ID/g, respectively]. Ac-D-Lys((125)I-IBA)-ReCCMSH(Arg(11)) also showed very fast whole body clearance and low nonspecific radioactivity accumulation in normal tissues compared with (125)I-IBA-NDP and Ac-Lys((125)I-IBA)-ReCCMSH(Arg(11)). A tumor:blood ratio of 34.3 was observed for Ac-D-Lys((125)I-IBA)-ReCCMSH(Arg(11)) at 24 h postinjection, whereas values of 4.3 and 2.0 were observed for Ac-Lys((125)I-IBA)-ReCCMSH(Arg(11)) and (125)I-IBA-NDP, respectively. The biodistribution data clearly demonstrate that both rhenium cyclization and D-Lys incorporation enhanced the tumor localization and retention of the radiolabel. Therefore Ac-D-Lys-ReCCMSH(Arg(11)) is an excellent candidate for additional therapeutic studies.

Tests Involving Humans Subjects: Old and New in China

A New Strategy to Screen Molecular Imaging Probe Uptake in Cell Culture Without Radiolabeling Using Matrix-assisted Laser Desorption/ionization Time-of-flight Mass Spectrometry

Numerous new molecular targets for diseases are rapidly being identified and validated in the postgenomic era, urging scientists to explore novel techniques for accelerating molecular probe development. In this study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was investigated as a potential tool for high-throughput screening and characterization of molecular imaging probes. Specifically, MALDI-TOF-MS was used to screen a small library of phosphonium cations for their ability to accumulate in cells.

[99mTcOAADT]-(CH2)2-NEt2: a Potential Small-molecule Single-photon Emission Computed Tomography Probe for Imaging Metastatic Melanoma

Evaluation of [99mTc]oxotechnetium(V) complexes of the amine-amide-dithiol (AADT) chelates containing tertiary amine substituents as small-molecule probes for the diagnostic imaging of metastatic melanoma has shown that technetium-99m-labeled AADT-(CH2)2-NEt2 (99mTc-1) has the highest tumor uptake and other favorable biological properties. We have, therefore, assessed this agent in a more realistic metastatic melanoma model in which, after i.v. tail injection, a highly invasive melanoma cell line, B16F10, forms pulmonary tumor nodules in normal C57BL6 mice. Small melanotic lesions develop in the lungs and, on histologic examination, appear as small black melanoma colonies, increasing in size and number with time after tumor cell injection. Groups of mice received tumor cell inocula of 2 x 10(5), 4 x 10(5), or 8 x 10(5) B16F10 cells; 14 days later, 2 hours after 99mTc-1 administration, lung uptake of 2.83 +/- 0.21%, 3.63 +/- 1.07%, and 4.92 +/- 1.61% injected dose per gram of tissue (% ID/g), respectively, was observed, compared with normal lung uptake of 2.13 +/- 0.2% ID/g (P < 0.05). Additionally, a higher level of 99mTc-1 accumulation was seen 17 days after tumor cell inoculation as the lung lesions grew. These in vivo studies coupled with additional in vitro and ex vivo assessment show that 99mTc-1 has high and specific uptake in melanoma metastases in lungs and can potentially follow the temporal growth of these tumors.

MicroPET Imaging of Glioma Integrin {alpha}v{beta}3 Expression Using (64)Cu-labeled Tetrameric RGD Peptide

Integrin alpha(v)beta(3) plays a critical role in tumor-induced angiogenesis and metastasis and has become a promising diagnostic indicator and therapeutic target for various solid tumors. Radiolabeled RGD peptides that are integrin specific can be used for noninvasive imaging of integrin expression level as well as for integrin-targeted radionuclide therapy.

Reproducibility of 3'-deoxy-3'-(18)F-fluorothymidine MicroPET Studies in Tumor Xenografts in Mice

3'-Deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) has been used to image tumor proliferation in preclinical and clinical studies. Serial microPET studies may be useful for monitoring therapy response or for drug screening; however, the reproducibility of serial scans has not been determined. The purpose of this study was to determine the reproducibility of (18)F-FLT microPET studies.

Near-infrared Fluorescent RGD Peptides for Optical Imaging of Integrin Alphavbeta3 Expression in Living Mice

Near-infrared fluorescence optical imaging is a powerful technique for studying diseases at the molecular level in preclinical models. We recently reported that monomeric RGD peptide c(RGDyK) conjugated to the NIR fluorescent dye specifically targets integrin receptor both in cell culture and in living subjects. In this report, Cy5.5-conjugated mono-, di-, and tetrameric RGD peptides were evaluated in a subcutaneous U87MG glioblastoma xenograft model in order to investigate the effect of multimerization of RGD peptide on integrin avidity and tumor targeting efficacy. The binding affinities of Cy5.5-conjugated RGD monomer, dimer, and tetramer for alpha(v)beta(3) integrin expressed on U87MG cell surface were determined to be 42.9 +/- 1.2, 27.5 +/- 1.2, and 12.1 +/- 1.3 nmol/L, respectively. All three peptide-dye conjugates had integrin specific uptake both in vitro and in vivo. The subcutaneous U87MG tumor can be clearly visualized with each of these three fluorescent probes. Among them, tetramer displayed highest tumor uptake and tumor-to-normal tissue ratio from 0.5 to 4 h postinjection. Tumor-to-normal tissue ratio for Cy5.5-conjugated RGD monomer, dimer, and tetramer were found to be 3.18 +/- 0.16, 2.98 +/- 0.05, and 3.63 +/- 0.09, respectively, at 4 h postinjection. These results suggest that Cy5.5-conjugated monomeric, dimeric, and tetrameric RGD peptides are all suitable for integrin expression imaging. The multmerization of RGD peptide results in moderate improvement of imaging characteristics of the tetramer, compared to that of the monomer and dimeric counterparts.

[The Understanding of Western Medicine on Disease in the 18th Century.]

The concept of modern disease is formed by organic pathology, which was born in the 18(th) century and opened the studies in the history of taking disease as a core. This article expounds the level of western medicine on disease in the 18(th) century by discussing the formation of organic pathology, classification of disease, and method of diagnosis.

Medical Images Edge Detection Based on Mathematical Morphology

Medical images edge detection is an important work for object recognition of the human organs and it is an important pre-processing step in medical image segmentation and 3D reconstruction. Conventionally, edge is detected according to some early brought forward algorithms such as gradient-based algorithm and template-based algorithm, but they are not so good for noise medical image edge detection. In this paper, basic mathematical morphological theory and operations are introduced at first, and then a novel mathematical morphological edge detection algorithm is proposed to detect the edge of lungs CT image with salt-and-pepper noise. The experimental results show that the proposed algorithm is more efficient for medical image denoising and edge detection than the usually used template-based edge detection algorithms and general morphological edge detection algorithms.

Development of a Portable Blood Sugar Apparatus and GOD Enzyme Strip

A pocket blood sugar apparatus tested by enzyme electrode, which was designed using carbon and silver plasma as conducting materials. Both the work and reference electrodes are applied to the parts of enzyme electrode. The glucose oxidase is taken as the medium of blood sugar measuring. And the range of measuring glucose is about 50mg/dL - 500mgl/dL. It has better linear for the results and fit coefficient arrives at 0.985. Its sensitivity of measurement is higher than current glucose biosensor obviously. A single chip microcomputer, AD mu C812, is used for central control processor of the instrument parts. It measures the output of microampere level currency, which is conduced by blood sugar reacting with the glucose oxidase on the enzyme electrode. And at the same time, the microampere level currency is amplified, processed. Then the results are displayed on LCD. The apparatus are better for measuring blood sugar, and the results are consistent with what the large biochemical instruments get.

Imaging Chemically Modified Adenovirus for Targeting Tumors Expressing Integrin Alphavbeta3 in Living Mice with Mutant Herpes Simplex Virus Type 1 Thymidine Kinase PET Reporter Gene

The aim of this study was to change adenovirus tropism by chemical modification of the fiber knobs with PEGylated RGD peptide for targeting integrin alpha(v)beta(3) that is uniquely or highly expressed in tumor cells and neovasculature of tumors of various origins.

An Aggressive Systematic Strategy for Acute Respiratory Distress Syndrome Caused by Severe Pneumonia After Renal Transplantation

Acute respiratory distress syndrome (ARDS) caused by pneumonia after renal transplantation was usually associated with overimmunosuppression and high mortality rate. We evaluated the efficacy of an aggressive systemic protocol including strategies improving body's immune function. Twenty-one recipients were enrolled in this study. Patients were subjected to a protocol including (i) withdrawal of most immunosuppressants, (ii) early use of immunoenhancers and continuous renal replacement therapy (CRRT), (iii) reasonable administration of antibiotic regimen, (iv) prompt mechanical ventilating strategy, and (v) adequate nutrition. Immunosuppressants were adjusted according to the value of CD4+, CD8+T lymphocytes in peripheral blood. CRRT was conducted at once when patients were admitted to the intensive care unit (ICU), regardless the graft function. Thirteen (62%) survived and eight died finally. This is a high survival rate for this kind of patients. Eighteen patients had received thymosin treatment. All patients who survived experienced renal allograft dysfunction during CRRT, but when CRRT stopped, the function of all grafts gradually recovered. No acute rejection episodes were documented during the treatment. The aggressive systemic protocol including strategies improving the body's immune function and CRRT can improve the outcome of patients with ARDS after renal transplantation. The count of CD4+, CD8+T lymphocytes of peripheral blood is useful in the adjustment of immunosuppressants and the prediction of patient outcome.

Near-infrared Fluorescent Deoxyglucose Analogue for Tumor Optical Imaging in Cell Culture and Living Mice

2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) has extensively been used for clinical diagnosis, staging, and therapy monitoring of cancer and other diseases. Nonradioactive glucose analogues enabling the screening of the glucose metabolic rate of tumors are of particular interest for anticancer drug development. A nonradioactive fluorescent deoxyglucose analogue may have many applications for both imaging of tumors and monitoring therapeutic efficacy of drugs in living animals and may eventually translate to clinical applications. We found that a fluorescent 2-deoxyglucose analogue, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), can be delivered in several tumor cells via the glucose transporters (GLUTs). We therefore conjugated D-glucosamine with a near-infrared (NIR) fluorphor Cy5.5 and tested the feasibility of the Cy5.5-D-glucosamine (Cy5.5-2DG) conjugate for NIR fluorescence imaging of tumors in a preclinical xenograft animal model. Cy5.5-2DG was prepared by conjugating Cy5.5 monofunctional N-hydroxysuccinimide ester (Cy5.5-NHS) and D-glucosamine followed by high-performance liquid chromatography purification. The accumulation of Cy5.5-2DG and Cy5.5-NHS in different tumor cell lines at 37 and 4 degrees C were imaged using a fluorescence microscope. Tumor targeting and retention of Cy5.5-2DG and Cy5.5-NHS in a subcutaneous U87MG glioma and A375M melanoma tumor model were evaluated and quantified by a Xenogen IVIS 200 optical cooled charged-coupled device system. Fluorescence microscopy imaging shows that Cy5.5-2DG and Cy5.5-NHS are taken up and trapped by a variety of tumor cell lines at 37 degrees C incubation, while they exhibit marginal uptake at 4 degrees C. The tumor cell uptake of Cy5.5-2DG cannot be blocked by the 50 mM D-glucose, suggesting that Cy5.5-2DG may not be delivered in tumor cells by GLUTs. U87MG and A375M tumor localization was clearly visualized in living mice with both NIR fluorescent probes. Tumor/muscle contrast was clearly visible as early as 30 min postinjection (pi), and the highest U87MG tumor/muscle ratios of 2.81 +/- 0.10 and 3.34 +/- 0.23 were achieved 24 h pi for Cy5.5-2DG and Cy5.5-NHS, respectively. While as a comparison, the micropositron emission tomography imaging study shows that [18F]FDG preferentially localizes to the U87MG tumor, with resulting tumor/muscle ratios ranging from 3.89 to 4.08 after 30 min to 2 h postadministration of the probe. In conclusion, the NIR fluorescent glucose analogues, Cy5.5-2DG and Cy5.5-NHS, both demonstrate tumor-targeting abilities in cell culture and living mice. More studies are warranted to further explore their application for optical tumor imaging. To develop NIR glucose analogues with the ability to target GLUTs/hexokinase, it is highly important to select NIR dyes with a reasonable molecular size.

Pulmonary Lophomonas Blattarum Infection in Patients with Kidney Allograft Transplantation

The aim of the study was to analyse the clinical manifestation and management of pulmonary Lophomonas blattarum infection in four allograft transplantation recipients retrospectively. Four patients with pulmonary L. blattarum infection were diagnosed by using Fiberoptic bronchoscopy (FOB) and bronchoalveolar lavage (BAL) examination. Their clinical manifestation and management are summarized. Four cases of pulmonary L. blattarum were found during the period from the second month to the third month after transplantation. Concurring infection by other pathogens was found in three of them. Common initial symptoms included fever (>38 degrees C) without cough and breathlessness. Lower lobe shadowing could be found on chest X-ray. Body temperature decreased to the normal range in three patients and to 37.5 degrees C in the other one, after intravenous injection of metronidazole and tapering immunosuppressant. Radiological examination confirmed improved health condition of the patients afterwards. Two patients received repeated FOB and only dead L. blattarum was found. Pulmonary L. blattarum infection in allograft transplant recipients carry relatively obscure initial symptoms. Possible L. blattarum infection needs to be screened in post-transplantation pulmonary infection patients with similar symptoms, especially in those who respond poorly to anti-infection treatment. Microscopic examination of BAL fluid can help to identify pulmonary L. blattarum infection and metronidazole is an ideal treatment choice.

In Vivo Bioluminescence Tumor Imaging of RGD Peptide-modified Adenoviral Vector Encoding Firefly Luciferase Reporter Gene

The goal of this study is to demonstrate the feasibility of chemically modified human adenovirus (Ad) vectors for tumor retargeting.

64Cu-labeled Alpha-melanocyte-stimulating Hormone Analog for MicroPET Imaging of Melanocortin 1 Receptor Expression

The alpha-melanocyte-stimulating hormone (alpha-MSH) receptor (melanocortin type 1 receptor, or MC1R) plays an important role in the development and growth of melanoma cells. It was found that MC1R was overexpressed on most murine and human melanoma, making it a promising molecular target for melanoma imaging and therapy. Radiolabeled alpha-MSH peptide and its analogs that can specifically bind with MC1R have been extensively explored for developing novel agents for melanoma detection and radionuclide therapy. The goal of this study was to evaluate a 64Cu-labeled alpha-MSH analog, Ac-Nle-Asp-His-D-Phe-Arg-Trp-Gly-Lys(DOTA)-NH2 (DOTA-NAPamide), as a potential molecular probe for microPET imaging of melanoma and MC1R expression in melanoma xenografted mouse models. 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated NAPamide was synthesized and radiolabeled with 64Cu (t1/2=12 h) in NH4OAc (0.1 M; pH 5.5) buffered solution for 60 min at 50 degrees C. Cell culture studies reveal rapid and high uptake and internalization of 64Cu-DOTA-NAPamide in B16F10 cells. Over 90% of receptor-bound tracer is internalized at 3 h incubation. A cellular retention study demonstrates that the receptor-bound 64Cu-DOTA-NAPamide is slowly released from the B16F10 cells into the medium; 66% of the radioactivity is still associated with the cells even after 3 h incubation. The biodistribution of 64Cu-DOTA-NAPamide was then investigated in C57BL/6 mice bearing subcutaneous murine B16F10 melanoma tumors with high capacity of MC1R and Fox Chase Scid mice bearing human A375M melanoma with a relatively low number of MC1R receptors. Tumor uptake values of 64Cu-DOTA-NAPamide are found to be 4.63 +/- 0.45% and 2.49 +/- 0.31% ID/g in B16F10 and A375M xenografted melanoma at 2 h postinjection (pi), respectively. The B16F10 tumor uptake at 2 h pi is further inhibited to 2.29 +/- 0.24% ID/g, while A375M tumor uptake at 2 h pi remains 2.20 +/- 0.41% ID/g with a coinjection of excess alpha-MSH peptide. MicroPET imaging of 64Cu-DOTA-NAPamide in B16F10 tumor mice clearly shows good tumor localization. However, low A375M tumor uptake and poor tumor to normal tissue contrast were observed. This study demonstrates that 64Cu-DOTA-NAPamide is a promising molecular probe for alpha-MSH receptor positive melanoma PET imaging as well as MC1R expression imaging in living mice.

Fluorescent Fructose Derivatives for Imaging Breast Cancer Cells

Breast cancer cells are known to overexpress Glut5, a sugar transporter responsible for the transfer of fructose across the cell membrane. Since Glut5 transporter is not significantly expressed in normal breast cells, fructose uptake can potentially be used to differentiate between normal and cancerous cells. Fructose was labeled with two fluorophores at the C-1 position: 7-nitro-1,2,3-benzadiazole (NBD) and Cy5.5. The labeling site was chosen on the basis of the presence and substrate specificity of the key proteins involved in the first steps of fructose metabolism. Using fluorescence microscopy, the uptake of the probes was studied in three breast cancer cell lines: MCF 7, MDA-MB-435, and MDA-MB-231. Both fluorescent fructose derivatives showed a very good uptake in all tested cell lines. The level of uptake was comparable to that of the corresponding glucose analogs, 2-NBDG and Cy5.5-DG. Significant uptake of 1-NBDF derivative was not observed in cells lacking Glut5 transporter, while the uptake of the 1-Cy5.5-DF derivative was independent of the presence of a fructose-specific transporter. While 1-NBDF showed Glut5-specific accumulation, the coupling of a large fluorophore such as Cy5.5 likely introduces big structural and electronic changes, leading to a fructose derivative that does not accurately describe the uptake of fructose in cells.

Small-animal PET of Melanocortin 1 Receptor Expression Using a 18F-labeled Alpha-melanocyte-stimulating Hormone Analog

(18)F-Labeled small synthetic peptides have emerged as attractive probes for imaging various molecular targets with PET. The alpha-melanocyte-stimulating hormone (alpha-MSH) receptor (melanocortin type 1 receptor [MC1R]) is overexpressed in most murine and human melanomas. It is a promising molecular target for diagnosis and therapy of melanomas. However, (18)F compounds have not been successfully developed for imaging the MC1R.

MicroPET-based Biodistribution of Quantum Dots in Living Mice

This study evaluates the quantitative biodistribution of commercially available CdSe quantum dots (QD) in mice.

Bisdeoxycoelenterazine Derivatives for Improvement of Bioluminescence Resonance Energy Transfer Assays

Breast Cancer Detection Based on Multi-frequency EIS Measurement

As a convenient, un-injurious and low cost method for women breast tumor diagnosis, Electrical Impedance Scanning (EIS) is being paid increasing attention. But EIS still has some problems. In theory the malignant tumor has higher electrical conductivity than normal tissue and benign tumor, so the cancer will be recognized in the EIS image as a bright spot. However, in practical the difference is not so obvious. In many cases it is difficult to judge whether a suspicious bright spot tells a malignant or benign tumor. This paper concerns a new method based on multi-frequency EIS measurement to solve this problem. A group of scanning results with various stimulating frequencies will be performed during an EIS measurement, which is automatically controlled by specially designed device and the location and pressure of the sensor will keep stable, so we will get a series of data at different stimulating frequencies through a measurement. Because the malignant and the benign tumor have different curves in electrical conductivity-frequency diagram, we can analyze the data at every frequency and find out the attribute of the tumor. Clinical trails have been carried out. Subjects were suspicious sufferers that were to receive histopathology examinations, and all analyses have been compared with histopathology results.

Real-time Intravital Imaging of RGD-quantum Dot Binding to Luminal Endothelium in Mouse Tumor Neovasculature

Nanoscale materials have increasingly become subject to intense investigation for use in cancer diagnosis and therapy. However, there is a fundamental dearth in cellular-level understanding of how nanoparticles interact within the tumor environment in living subjects. Adopting quantum dots (qdots) for their excellent brightness, photostability, monodispersity, and fluorescent yield, we link arginine-glycine-aspartic acid (RGD) peptides to target qdots specifically to newly formed/forming blood vessels expressing alpha vbeta 3 integrins. Using this model nanoparticle system, we exploit intravital microscopy with subcellular ( approximately 0.5 microm) resolution to directly observe and record, for the first time, the binding of nanoparticle conjugates to tumor blood vessels in living subjects. This generalizable method enabled us to show that in this model qdots do not extravasate and, unexpectedly, that they only bind as aggregates rather than individually. This level of understanding is critical on the path toward ensuring regulatory approval of nanoparticles in humans for disease diagnostics and therapeutics. Equally vital, the work provides a platform by which to design and optimize molecularly targeted nanoparticles including quantum dots for applications in living subjects.

Small-animal PET Imaging of Human Epidermal Growth Factor Receptor Type 2 Expression with Site-specific 18F-labeled Protein Scaffold Molecules

Human epidermal growth factor receptor type 2 (HER2) is a well-established tumor biomarker that is overexpressed in a wide variety of cancers and that serves as a molecular target for therapeutic intervention. HER2 also serves as a prognostic indicator of patient survival and as a predictive marker of the response to antineoplastic therapy. The development of (18)F-labeled biomolecules for PET imaging of HER2 (HER2 PET) is very important because it may provide a powerful tool for the early detection of HER2-positive tumor recurrence and for the monitoring of HER2-based tumor treatment.

Direct Site-specific Radiolabeling of an Affibody Protein with 4-[18F]fluorobenzaldehyde Via Oxime Chemistry

In this study, we introduce a methodology for preparing 18F-labeled Affibody protein, specifically 18F-Anti-HER2 dimeric Affibody (14 kDa), for in vivo imaging of HER2neu with positron emission tomography (PET).

Targeted Microbubbles for Imaging Tumor Angiogenesis: Assessment of Whole-body Biodistribution with Dynamic Micro-PET in Mice

To evaluate in vivo whole-body biodistribution of microbubbles (MBs) targeted to tumor angiogenesis-related vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) by using dynamic micro-positron emission tomography (PET) in living mice.

Carbon Nanotubes As Photoacoustic Molecular Imaging Agents in Living Mice

Photoacoustic imaging of living subjects offers higher spatial resolution and allows deeper tissues to be imaged compared with most optical imaging techniques. As many diseases do not exhibit a natural photoacoustic contrast, especially in their early stages, it is necessary to administer a photoacoustic contrast agent. A number of contrast agents for photoacoustic imaging have been suggested previously, but most were not shown to target a diseased site in living subjects. Here we show that single-walled carbon nanotubes conjugated with cyclic Arg-Gly-Asp (RGD) peptides can be used as a contrast agent for photoacoustic imaging of tumours. Intravenous administration of these targeted nanotubes to mice bearing tumours showed eight times greater photoacoustic signal in the tumour than mice injected with non-targeted nanotubes. These results were verified ex vivo using Raman microscopy. Photoacoustic imaging of targeted single-walled carbon nanotubes may contribute to non-invasive cancer imaging and monitoring of nanotherapeutics in living subjects.

[Real World Instantaneous Emission Simulation for Light-duty Diesel Vehicle]

Core architecture and input parameters of CMEM model were introduced to simulation the second by second vehicle emission rate on real world by taking a light-duty diesel car as a case. On-board test data by a portable emission measurement system were then used to validate the simulation results. Test emission factors of CO, THC, NO(x) and CO2 were respectively 0.81, 0.61, 2.09, and 193 g x km(-1), while calculated emission factors were 0.75, 0.47, 2.47, and 212 g x km(-1). The correlation coefficients reached 0.69, 0.69, 0.75, and 0.72. Simulated instantaneous emissions of the light duty diesel vehicle by CMEM model were strongly coherent with the transient driving cycle. By analysis, CO, THC, NO(x), and CO2 emissions would be reduced by 50%, 47%, 45%, and 44% after improving the traffic situation at the intersection. The result indicated that it is necessary and feasible to simulate the instantaneous emissions of mixed vehicle fleet in some typical traffic areas by the micro-scale vehicle emission model.

Particle Size, Surface Coating, and PEGylation Influence the Biodistribution of Quantum Dots in Living Mice

This study evaluates the influence of particle size, PEGylation, and surface coating on the quantitative biodistribution of near-infrared-emitting quantum dots (QDs) in mice. Polymer- or peptide-coated 64Cu-labeled QDs 2 or 12 nm in diameter, with or without polyethylene glycol (PEG) of molecular weight 2000, are studied by serial micropositron emission tomography imaging and region-of-interest analysis, as well as transmission electron microscopy and inductively coupled plasma mass spectrometry. PEGylation and peptide coating slow QD uptake into the organs of the reticuloendothelial system (RES), liver and spleen, by a factor of 6-9 and 2-3, respectively. Small particles are in part renally excreted. Peptide-coated particles are cleared from liver faster than physical decay alone would suggest. Renal excretion of small QDs and slowing of RES clearance by PEGylation or peptide surface coating are encouraging steps toward the use of modified QDs for imaging living subjects.

A Novel Method for Direct Site-specific Radiolabeling of Peptides Using [18F]FDG

We have used the well-accepted and easily available 2-[(18)F]fluoro-2-deoxyglucose ([(18)F]FDG) positron emission tomography (PET) tracer as a prosthetic group for synthesis of (18)F-labeled peptides. We herein report the synthesis of [(18)F]FDG-RGD ((18)F labeled linear RGD) and [(18)F]FDG-cyclo(RGD(D)YK) ((18)F labeled cyclic RGD) as examples of the use of [(18)F]FDG. We have successfully prepared [(18)F]FDG-RGD and [(18)F]FDG-cyclo(RGD(D)YK) in 27.5% and 41% radiochemical yields (decay corrected) respectively. The receptor binding affinity study of FDG-cyclo(RGD(D)YK) for integrin alpha(v)beta(3), using alpha(v)beta(3) positive U87MG cells confirmed a competitive displacement with (125)I-echistatin as a radioligand. The IC(50) value for FDG-cyclo(RGD(D)YK) was determined to be 0.67 +/- 0.19 muM. High-contrast small animal PET images with relatively moderate tumor uptake were observed for [(18)F]FDG-RGD and [(18)F]FDG-cyclo(RGD(D)YK) as PET probes in xenograft models expressing alpha(v)beta(3) integrin. In conclusion, we have successfully used [(18)F]FDG as a prosthetic group to prepare (18)F]FDG-RGD and [(18)F]FDG-cyclic[RGD(D)YK] based on a simple one-step radiosynthesis. The one-step radiosynthesis methodology consists of chemoselective oxime formation between an aminooxy-functionalized peptide and [(18)F]FDG. The results have implications for radiolabeling of other macromolecules and would lead to a very simple strategy for routine preclinical and clinical use.

Engineered Knottin Peptides: a New Class of Agents for Imaging Integrin Expression in Living Subjects

There is a critical need for molecular imaging agents to detect cell surface integrin receptors that are present in human cancers. Previously, we used directed evolution to engineer knottin peptides that bind with high affinity ( approximately 10 to 30 nmol/L) to integrin receptors that are overexpressed on the surface of tumor cells and the tumor neovasculature. To evaluate these peptides as molecular imaging agents, we site-specifically conjugated Cy5.5 or (64)Cu-1,4,7,10-tetra-azacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) to their N termini, and used optical and positron emission tomography (PET) imaging to measure their uptake and biodistribution in U87MG glioblastoma murine xenograft models. NIR fluorescence and microPET imaging both showed that integrin binding affinity plays a strong role in the tumor uptake of knottin peptides. Tumor uptake at 1 hour postinjection for two high-affinity (IC(50), approximately 20 nmol/L) (64)Cu-DOTA-conjugated knottin peptides was 4.47% +/- 1.21% and 4.56% +/- 0.64% injected dose/gram (%ID/g), compared with a low-affinity knottin peptide (IC(50), approximately 0.4 mumol/L; 1.48 +/- 0.53%ID/g) and c(RGDyK) (IC(50), approximately 1 mumol/L; 2.32 +/- 0.55%ID/g), a low-affinity cyclic pentapeptide under clinical development. Furthermore, (64)Cu-DOTA-conjugated knottin peptides generated lower levels of nonspecific liver uptake ( approximately 2%ID/g) compared with c(RGDyK) ( approximately 4%ID/g) 1 hour postinjection. MicroPET imaging results were confirmed by in vivo biodistribution studies. (64)Cu-DOTA-conjugated knottin peptides were stable in mouse serum, and in vivo metabolite analysis showed minimal degradation in the blood or tumor upon injection. Thus, engineered integrin-binding knottin peptides show great potential as clinical diagnostics for a variety of cancers.

Engineered Two-helix Small Proteins for Molecular Recognition

Less is more: By starting with a high-affinity HER2-binding 3-helix affibody molecule, we successfully developed 2-helix small protein binders with 5 nM affinities by using a combination of several different strategies. Our efforts clearly suggest that 2-helix small proteins against important tumor targets can be obtained by rational protein design and engineering.

Photoactivable Bioluminescent Probes for Imaging Luciferase Activity

A set of stable and efficient photoactivable bioluminescent probes for imaging luciferase activity has been developed, which displayed robust bioluminescent signals upon brief UV illumination in buffer, cells and living animals.

Visualizing Implanted Tumors in Mice with Magnetic Resonance Imaging Using Magnetotactic Bacteria

To determine if magnetotactic bacteria can target tumors in mice and provide positive contrast for visualization using magnetic resonance imaging.

Application of the Indirect Fluorescent Antibody Assay in the Study of Malaria Infection in the Yangtze River Three Gorges Reservoir, China

China Yangtze Three Gorges Project (TGP) is one of the biggest construction projects in the world. The areas around the Three Gorge Dam has a history of tertian malaria and subtertian malaria epidemic, but there are no overall data about malaria epidemics before the completion of the project. The objective of this study was to get a reliable baseline on malaria infection in the Yangtze River Three Gorges reservoir area and to provide reference data for future studies about the impact of the project on malaria epidemics.

A 2-helix Small Protein Labeled with 68Ga for PET Imaging of HER2 Expression

Affibody molecules are a class of scaffold proteins being developed into a generalizable approach to targeting tumors. Many 3-helix-based Affibody proteins have shown excellent in vivo properties for tumor imaging and therapy. By truncating one alpha-helix that is not responsible for receptor recognition in the Affibody and maturating the protein affinity through synthetic strategies, we have successfully identified in our previous research several small 2-helix proteins with excellent binding affinities to human epidermal growth factor receptor type 2 (HER2). With preferential properties such as faster blood clearance and tumor accumulation, lower immunogenic potential, and facile and economically viable synthetic schemes, we hypothesized that these 2-helix protein binders could become excellent molecular imaging probes for monitoring HER2 expression and modulation.

Melanin-targeted Preclinical PET Imaging of Melanoma Metastasis

Dialkylamino-alkyl-benzamides possess an affinity for melanin, suggesting that labeling of such benzamides with (18)F could potentially produce melanin-targeted PET probes able to identify melanotic melanoma metastases in vivo with high sensitivity and specificity.

PET of Malignant Melanoma Using 18F-labeled Metallopeptides

Melanocortin type 1 receptor (MC1R), also known as alpha-melanocyte-stimulating hormone (alpha-MSH) receptor, is an attractive molecular target for melanoma imaging and therapy. An (18)F-labeled linear alpha-MSH peptide ((18)F-FB-Ac-Nle-Asp-His-d-Phe-Arg-Trp-Gly-Lys-NH(2) [NAPamide]) shows promising melanoma imaging properties but with only moderate tumor uptake and retention. A transition metal rhenium-cyclized alpha-MSH peptide, ReO[Cys(3,4,10),d-Phe(7),Arg(11)]alpha-MSH(3-13) (ReCCMSH(Arg(11))), has shown high in vitro binding affinity to MC1R and excellent in vivo melanoma-targeting profiles when labeled with radiometals. Therefore, we hypothesized that ReCCMSH(Arg(11)) could be a good platform for the further development of an (18)F-labeled probe for PET of MC1R-positive malignant melanoma.

An Engineered Knottin Peptide Labeled with 18F for PET Imaging of Integrin Expression

Knottins are small constrained polypeptides that share a common disulfide-bonded framework and a triple-stranded beta-sheet fold. Previously, directed evolution of the Ecballium elaterium trypsin inhibitor (EETI-II) knottin led to the identification of a mutant that bound to tumor-specific alpha(v)beta(3) and alpha(v)beta(5) integrin receptors with low nanomolar affinity. The objective of this study was to prepare and evaluate a radiofluorinated version of this knottin (termed 2.5D) for microPET imaging of integrin positive tumors in living subjects. Knottin peptide 2.5D was prepared by solid-phase synthesis and folded in vitro, and its free N-terminal amine was reacted with N-succinimidyl-4-18/19F-fluorobenzoate (18/19F-SFB) to produce the fluorinated peptide 18/19F-FB-2.5D. The binding affinities of unlabeled knottin peptide 2.5D and 19F-FB-2.5D to U87MG glioblastoma cells were measured by competition binding assay using 125I-labeled echistatin. It was found that unlabeled 2.5D and 19F-FB-2.5D competed with 125I-echistatin for binding to cell surface integrins with IC(50) values of 20.3 +/- 7.3 and 13.2 +/- 5.4 nM, respectively. Radiosynthesis of 18F-FB-2.5D resulted in a product with high specific activity (ca. 100 GBq/micromol). Next, biodistribution and positron emission tomography (PET) imaging studies were performed to evaluate the in vivo behavior of 18F-FB-2.5D. Approximately 3.7 MBq 18F-FB-2.5D was injected into U87MG tumor-bearing mice via the tail vein. Biodistribution studies demonstrated that 18F-FB-2.5D had moderate tumor uptake at 0.5 h post injection, and coinjection of a large excess of the unlabeled peptidomimetic c(RGDyK) as a blocking agent significantly reduced tumor uptake (1.90 +/- 1.15 vs 0.57 +/- 0.14%ID/g, 70% inhibition, P < 0.05). In vivo microPET imaging showed that 18F-FB-2.5D rapidly accumulated in the tumor and quickly cleared from the blood through the kidneys, allowing excellent tumor-to-normal tissue contrast to be obtained. Collectively, 18F-FB-2.5D allows integrin-specific PET imaging of U87MG tumors with good contrast and further demonstrates that knottins are excellent peptide scaffolds for development of PET probes with potential for clinical translation.

Comparative Study on Separation and Purification of Isoflavones from the Seeds and Sprouts of Chickpea by HSCCC

Chickpea is known as a plant that is rich in protein, carbohydrates, and nutrition, and its seeds and sprouts have been processed into various health foods. In the present study, four isoflavones were purified from the seeds and sprouts of chickpea by high speed countercurrent chromatography (HSCCC) using two biphasic solvent systems composed of n-hexane-ethyl acetate-methanol-water (5:5:5:5, v/v) and ethyl acetate-water (1:1 v/v). The results indicated that 14.2 mg of formononetin, 15.7 mg of biochanin A, 9.1 mg of ononin, 11.3 mg of biochanin A-7-O-β-D-glucoside were obtained from 150 mg of sprout extracts with the purity of 92.26%, 95.86%, 95.32%, and 96.56%, respectively. Compared with the sprouts, separation of seed extracts yielded less amounts of biochanin A-7-O-β-D-glucoside and biochanin A with lower purity. The results indicate that four main isoflavones in chickpea, i.e., isoflavones, formononetin, biochanin A, ononin, and biochanin A-7-O-β-D-glucoside, are substantially increased by biosynthesis during the seed germination.

64Cu-labeled Affibody Molecules for Imaging of HER2 Expressing Tumors

INTRODUCTION: The development of molecular probes based on novel engineered protein constructs is under active investigation due to the great potential of this generalizable strategy for imaging a variety of tumor targets. DISCUSSION: In this report, human epidermal growth factor receptor type 2 (HER2)-binding Affibody molecules were radiolabeled with (64)Cu and their imaging ability was further evaluated in tumor mice models to understand the promise and limitations of such probes. The anti-HER2 Affibody molecules in monomeric (Z(HER2:477)) and dimeric [(Z(HER2:477))(2)] forms were site specifically modified with the maleimide-functionalized chelator, 1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid)-10-acetate mono (N-ethylmaleimide amide) (Mal-DOTA). The resulting DOTA-Affibody conjugates were radiolabeled with (64)Cu and evaluated in nude mice bearing subcutaneous SKOV3 tumors. Biodistribution experiments showed that tumor uptake values of (64)Cu-DOTA-Z(HER2:477) and (64)Cu-DOTA-(Z(HER2:477))(2) were 6.12 +/- 1.44% and 1.46 +/- 0.50% ID/g, respectively, in nude mice (n = 3 each) at 4 h postinjection. Moreover, (64)Cu-labeled monomer exhibited significantly higher tumor/blood ratio than that of radiolabeled dimeric counterpart at all time points examined in this study. MicroPET imaging of (64)Cu-DOTA-Z(HER2:477) in SKOV3 tumor mice clearly showed good and specific tumor localization. This study demonstrates that (64)Cu-labeled Z(HER2:477) is a promising targeted molecular probe for imaging HER2 receptor expression in living mice. Further work is needed to improve the excretion properties, hence dosimetry and imaging efficacy, of the radiometal-based probe.

Ultrasmall Near-infrared Non-cadmium Quantum Dots for in Vivo Tumor Imaging

The high tumor uptake of ultrasmall near-infrared quantum dots (QDs) attributed to the enhanced permeability and retention effect is reported. InAs/InP/ZnSe QDs coated by mercaptopropionic acid (MPA) exhibit an emission wavelength of about 800 nm (QD800-MPA) with very small hydrodynamic diameter (<10 nm). Using 22B and LS174T tumor xenograft models, in vivo and ex vivo imaging studies show that QD800-MPA is highly accumulated in the tumor area, which is very promising for tumor detection in living mice. The ex vivo elemental analysis (Indium) using inductively coupled plasma (ICP) spectrometry confirm the tumor uptake of QDs. The ICP data are consistent with the in vivo and ex vivo fluorescence imaging. Human serum albumin (HSA)-coated QD800-MPA nanoparticles (QD800-MPA-HSA) show reduced localization in mononuclear phagocytic system-related organs over QD800-MPA plausibly due to the low uptake of QD800-MPA-HSA in macrophage cells. QD800-MPA-HSA may have great potential for in vivo fluorescence imaging.

Evaluation of a (64)Cu-labeled Cystine-knot Peptide Based on Agouti-related Protein for PET of Tumors Expressing Alphavbeta3 Integrin

Recently, a truncated form of the agouti-related protein (AgRP), a 4-kDa cystine-knot peptide of human origin, was used as a scaffold to engineer mutants that bound to alpha(v)beta(3) integrin with high affinity and specificity. In this study, we evaluated the potential of engineered integrin-binding AgRP peptides for use as cancer imaging agents in living subjects.

A Dual-Labeled Knottin Peptide for PET and Near-Infrared Fluorescence Imaging of Integrin Expression in Living Subjects

Previously, we used directed evolution to engineer mutants of the Ecballium elaterium trypsin inhibitor (EETI-II) knottin that bind to alpha(v)beta(3) and alpha(v)beta(5) integrin receptors with low nanomolar affinity, and showed that Cy5.5- or (64)Cu-DOTA-labeled knottin peptides could be used to image integrin expression in mouse tumor models using near-infrared fluorescence (NIRF) imaging or positron emission tomography (PET). Here, we report the development of a dual-labeled knottin peptide conjugated to both NIRF and PET imaging agents for multimodality imaging in living subjects. We created an orthogonally protected peptide-based linker for stoichiometric coupling of (64)Cu-DOTA and Cy5.5 onto the knottin N-terminus and confirmed that conjugation did not affect binding to alpha(v)beta(3) and alpha(v)beta(5) integrins. NIRF and PET imaging studies in tumor xenograft models showed that Cy5.5 conjugation significantly increased kidney uptake and retention compared to the knottin peptide labeled with (64)Cu-DOTA alone. In the tumor, the dual-labeled (64)Cu-DOTA/Cy5.5 knottin peptide showed decreased wash-out leading to significantly better retention (p < 0.05) compared to the (64)Cu-DOTA-labeled knottin peptide. Tumor uptake was significantly reduced (p < 0.05) when the dual-labeled knottin peptide was coinjected with an excess of unlabeled competitor and when tested in a tumor model with lower levels of integrin expression. Finally, plots of tumor-to-background tissue ratios for Cy5.5 versus (64)Cu uptake were well-correlated over several time points post injection, demonstrating pharmacokinetic cross validation of imaging labels. This dual-modality NIRF/PET imaging agent is promising for further development in clinical applications where high sensitivity and high resolution are desired, such as detection of tumors located deep within the body and image-guided surgical resection.

Molecular Optical Imaging with Radioactive Probes

Optical imaging (OI) techniques such as bioluminescence and fluorescence imaging have been widely used to track diseases in a non-invasive manner within living subjects. These techniques generally require bioluminescent and fluorescent probes. Here we demonstrate the feasibility of using radioactive probes for in vivo molecular OI.

In Vivo Tumor-targeted Fluorescence Imaging Using Near-infrared Non-cadmium Quantum Dots

This article reported the high tumor targeting efficacy of RGD peptide labeled near-infrared (NIR) non-cadmium quantum dots (QDs). After using poly(ethylene glycol) to encapsulate InAs/InP/ZnSe QDs (emission maximum at about 800 nm), QD800-PEG dispersed well in PBS buffer with the hydrodynamic diameter (HD) of 15.9 nm and the circulation half-life of approximately 29 min. After coupling QD800-PEG with arginine-glycine-aspartic acid (RGD) or arginine-alanine-aspartic acid (RAD) peptides, we used nude mice bearing subcutaneous U87MG tumor as models to test tumor-targeted fluorescence imaging. The results indicated that the tumor uptake of QD800-RGD is much higher than those of QD800-PEG and QD800-RAD. The semiquantitative analysis of the region of interest (ROI) showed a high tumor uptake of 10.7 +/- 1.5%ID/g in mice injected with QD800-RGD, while the tumor uptakes of QD800-PEG and QD800-RAD were 2.9 +/- 0.3%ID/g and 4.0 +/- 0.5%ID/g, respectively, indicating the specific tumor targeting of QD800-RGD. The high reproducibility of bioconjunction between QDs and the RGD peptide and the feasibility of QD-RGD bioconjugates as tumor-targeted fluorescence probes warrant the successful application of QDs for in vivo molecular imaging.

Near-infrared Quantum Dots As Optical Probes for Tumor Imaging

Molecular imaging plays a key role in personalized medicine, which is the goal and future of patient management. Among the various molecular imaging modalities, optical imaging may be the fastest growing area for bioanalysis, and the major reason is the research on fluorescence semiconductor quantum dots (QDs) and dyes have evolved over the past two decades. The great efforts on the synthesis of QDs with fluorescence emission from UV to near-infrared (NIR) regions speed up the studies of QDs as optical probes for in vitro and in vivo molecular imaging. For in vivo applications, the fluorescent emission wavelength ideally should be in a region of the spectrum where blood and tissue absorb minimally and tissue penetration reach maximally, which is NIR region (typically 700-1000 nm). The goal of this review is to provide readers the basics of NIR-emitting QDs, the bioconjugate chemistry of QDs, and their applications for diagnostic tumor imaging. We will also discuss the benefits, challenges, limitations, perspective, and the future scope of NIR-emitting QDs for tumor imaging applications.

Assessing the Effects of Acetochlor on Soil Fungal Communities by DGGE and Clone Library Analysis

We assessed the potential eco-toxicological risks of the herbicide acetochlor on fungal communities in the microcosm of black soil using 28S rRNA gene-PCR-DGGE and clone library analysis. The acetochlor was applied to black soil at four concentrations (0-control, 50, 150, and 250 mg/kg). The DGGE fingerprint patterns indicated that acetochlor stimulated fungal communities at day 7 after application, after which there was a suppression effect. The fungal communities in acetochlor-treated soil gradually became more like that of the control during the 60-day experimental period. Diversity indices in the 50 and 150 mg/kg acetochlor treatments changed more rapidly than in the 250 mg/kg acetochlor treatment. The cluster analysis indicated a significant change in fungal community structure after application of acetochlor. The impacts were markedly greater in the 150 and 250 mg/kg acetochlor treatments compared with the 50 mg/kg acetochlor treatment. Sequencing of clones showed that acetochlor application resulted in an increase in pathogenic and non-cultivatable fungal populations, which could increase the risk of plant disease outbreaks.

Small-animal PET Imaging of Human Epidermal Growth Factor Receptor Positive Tumor with a 64Cu Labeled Affibody Protein

Epidermal growth factor receptor (EGFR) has become an attractive target for cancer molecular imaging and therapy. Affibody proteins against EGFR have been reported, and thus, we were interested in evaluating their potential for positron emission tomography (PET) imaging of EGFR positive cancer. An Affibody analogue (Ac-Cys-Z(EGFR:1907)) binding to EGFR was made through conventional solid phase peptide synthesis. The purified protein was site-specifically coupled with the 1,4,7,10-tetraazacyclododecane-1,4,7-tris-aceticacid-10-maleimidethylacetamide (maleimido-mono-amide-DOTA) to produce the bioconjugate, DOTA-Z(EGFR:1907). (64)Cu labeled probe (64)Cu-DOTA-Z(EGFR:1907) displayed a moderate specific activity (5-8 MBq/nmol, 22-35 microCi/microg). Cell uptake assays by pre-incubating without or with 300 times excess unlabeled Ac-Cys-Z(EGFR:1907) showed high EGFR-specific uptake (20% applied activity at 0.5 h) in A431 epidermoid carcinoma cancer cells. The affinity (K(D)) of (64)Cu-DOTA-Z(EGFR:1907) as tested by cell saturation analysis was 20 nM. The serum stability test showed excellent stability of the probe with >95% intact after 4 h of incubation in mouse serum. In vivo small-animal PET imaging showed fast tumor targeting, high tumor accumulation (approximately 10% ID/g at 1 h p.i.), and good tumor-to-normal tissue contrast of (64)Cu-DOTA-Z(EGFR:1907) spiked with a wide dose range of Ac-Cys-Z(EGFR:1907). Bio-distribution studies further demonstrated that the probe had high tumor, blood, liver, and kidney uptakes, while blood radioactivity concentration dropped dramatically at increased spiking doses. Co-injection of the probe with 500 microg of Ac-Cys-Z(EGFR:1907) for blocking significantly reduced the tumor uptake. Thus, (64)Cu-DOTA-Z(EGFR:1907) showed potential as a high tumor contrast EGFR PET imaging reagent. The probe spiked with 50 microg of Ac-Cys-Z(EGFR:1907) improved tumor imaging contrast which may have important clinical applications.

Radiation-luminescence-excited Quantum Dots for in Vivo Multiplexed Optical Imaging

Quantum Dots: Small 10/2010

Molecular Probes for Malignant Melanoma Imaging

Malignant melanoma represents a serious public health problem and is a deadly disease when it is diagnosed at late stage. Though (18)F-fluorodeoxyglucose ((18)F-FDG) positron emission tomography (PET) has been widely used clinically for melanoma imaging, other approaches to specifically identify, characterize, monitor and guide therapeutics for malignant melanoma are still needed. Consequently, many probes targeting general molecular events including metabolism, angiogenesis, hypoxia and apoptosis in melanoma have been successfully developed. Furthermore, probes targeting melanoma associated targets such as melanocortin receptor 1 (MC1R), melanin, etc. have undergone active investigation and have demonstrated high melanoma specificity. In this review, these molecular probes targeting diverse melanoma biomarkers have been summarized. Some of them may eventually contribute to the improvement of personalized management of malignant melanoma.

In Vivo Near-infrared Fluorescence Imaging of Cancer with Nanoparticle-based Probes

The use of in vivo near-infrared fluorescence (NIRF) imaging techniques for sensitive cancer early detection is highly desirable, because biological tissues show very low absorption and autofluorescence in the NIR spectrum window. Cancer NIRF molecular imaging relies greatly on stable, highly specific and sensitive molecular probes. Nanoparticle-based NIRF probes have overcome some of the limitations of the conventional NIRF organic dyes, such as poor hydrophilicity and photostability, low quantum yield, insufficient stability in biological system, low detection sensitivity, etc. Therefore, a lot of efforts have been made to actively develop novel NIRF nanoparticles for in vivo cancer molecular imaging. The main focus of this article is to provide a brief overview of the synthesis, surface modification, and in vivo cancer imaging applications of nanoparticle-based NIRF probes, including dye-containing nanoparticles, NIRF quantum dots, and upconversion nanoparticles.

Cy5.5-labeled Affibody Molecule for Near-infrared Fluorescent Optical Imaging of Epidermal Growth Factor Receptor Positive Tumors

Affibody protein is an engineered protein scaffold with a three-helical bundle structure. Affibody molecules of small size (7 kD) have great potential for targeting overexpressed cancer biomarkers in vivo. To develop an Affibody-based molecular probe for in vivo optical imaging of epidermal growth factor receptor (EGFR) positive tumors, an anti-EGFR Affibody molecule, Ac-Cys-Z(EGFR:1907) (7 kD), is site-specifically conjugated with a near-IR fluorescence dye, Cy5.5-mono-maleimide. Using fluorescent microscopy, the binding specificity of the probe Cy5.5-Z(EGFR:1907) is checked by a high-EGFR-expressing A431 cell and low-EGFR-expressing MCF7 cells. The binding affinity of Cy5.5-Z(EGFR:1907) (K(D)) to EGFR is 43.6+/-8.4 nM, as determined by flow cytometry. For an in vivo imaging study, the probe shows fast tumor targeting and good tumor contrast as early as 0.5 h postinjection (p.i.) for A431 tumors, while MCF7 tumors are barely visible. An ex vivo imaging study also demonstrates that Cy5.5-Z(EGFR:1907) has high tumor, liver, and kidney uptakes at 24 h p.i.. In conclusion, Cy5.5-Z(EGFR:1907) shows good affinity and high specificity to the EGFR. There is rapid achievement of good tumor-to-normal-tissue contrasts of Cy5.5-Z(EGFR:1907), thus demonstrating its potential for EGFR-targeted molecular imaging of cancers.

HSA Coated MnO Nanoparticles with Prominent MRI Contrast for Tumor Imaging

We report in this Communication a facile, two-step surface modification strategy to achieve manganese oxide nanoparticles with prominent MRI T1 contrast. In a U87MG glioblastoma xenograft model, we confirmed that the particles can accumulate efficiently in tumor area to induce effective T1 signal alteration.

Dynamic Visualization of RGD-quantum Dot Binding to Tumor Neovasculature and Extravasation in Multiple Living Mouse Models Using Intravital Microscopy

Near-infrared Fluorescent Nanoprobes for Cancer Molecular Imaging: Status and Challenges

Near-infrared fluorescence (NIRF) imaging promises to improve cancer imaging and management; advances in nanomaterials allow scientists to combine new nanoparticles with NIRF imaging techniques, thereby fulfilling this promise. Here, we present a synopsis of current developments in NIRF nanoprobes, their use in imaging small living subjects, their pharmacokinetics and toxicity, and finally their integration into multimodal imaging strategies. We also discuss challenges impeding the clinical translation of NIRF nanoprobes for molecular imaging of cancer. Whereas utilization of most NIRF nanoprobes remains at a proof-of-principle stage, optimizing the impact of nanomedicine in cancer patient diagnosis and management will probably be realized through persistent interdisciplinary amalgamation of diverse research fields.

Effects of Nanoparticle Size on Cellular Uptake and Liver MRI with Polyvinylpyrrolidone-coated Iron Oxide Nanoparticles

The effect of nanoparticle size (30-120 nm) on magnetic resonance imaging (MRI) of hepatic lesions in vivo has been systematically examined using polyvinylpyrrolidone (PVP)-coated iron oxide nanoparticles (PVP-IOs). Such biocompatible PVP-IOs with different sizes were synthesized by a simple one-pot pyrolysis method. These PVP-IOs exhibited good crystallinity and high T(2) relaxivities, and the relaxivity increased with the size of the magnetic nanoparticles. It was found that cellular uptake changed with both size and surface physiochemical properties, and that PVP-IO-37 with a core size of 37 nm and hydrodynamic particle size of 100 nm exhibited higher cellular uptake rate and greater distribution than other PVP-IOs and Feridex. We systematically investigated the effect of nanoparticle size on MRI of normal liver and hepatic lesions in vivo. The physical and chemical properties of the nanoparticles influenced their pharmacokinetic behavior, which ultimately determined their ability to accumulate in the liver. The contrast enhancement of PVP-IOs within the liver was highly dependent on the overall size of the nanoparticles, and the 100 nm PVP-IO-37 nanoparticles exhibited the greatest enhancement. These results will have implications in designing engineered nanoparticles that are optimized as MR contrast agents or for use in therapeutics.

PET Imaging of Tumor Neovascularization in a Transgenic Mouse Model with a Novel 64Cu-DOTA-knottin Peptide

Due to the high mortality of lung cancer, there is a critical need to develop diagnostic procedures enabling early detection of the disease while at a curable stage. Targeted molecular imaging builds on the positive attributes of positron emission tomography/computed tomography (PET/CT) to allow for a noninvasive detection and characterization of smaller lung nodules, thus increasing the chances of positive treatment outcome. In this study, we investigate the ability to characterize lung tumors that spontaneously arise in a transgenic mouse model. The tumors are first identified with small animal CT followed by characterization with the use of small animal PET with a novel 64Cu-1,4,7,10-tetra-azacylododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-knottin peptide that targets integrins upregulated during angiogenesis on the tumor associated neovasculature. The imaging results obtained with the knottin peptide are compared with standard 18F-fluorodeoxyglucose (FDG) PET small animal imaging. Lung nodules as small as 3 mm in diameter were successfully identified in the transgenic mice by small animal CT, and both 64Cu-DOTA-knottin 2.5F and FDG were able to differentiate lung nodules from the surrounding tissues. Uptake and retention of the 64Cu-DOTA-knottin 2.5F tracer in the lung tumors combined with a low background in the thorax resulted in a statistically higher tumor to background (normal lung) ratio compared with FDG (6.01±0.61 versus 4.36±0.68; P<0.05). Ex vivo biodistribution showed 64Cu-DOTA-knottin 2.5F to have a fast renal clearance combined with low nonspecific accumulation in the thorax. Collectively, these results show 64Cu-DOTA-knottin 2.5F to be a promising candidate for clinical translation for earlier detection and improved characterization of lung cancer.

Radiofluorinated Rhenium Cyclized α-MSH Analogues for PET Imaging of Melanocortin Receptor 1

In order to accomplish in vivo molecular imaging of melanoma biomarker melanocortin 1 receptor (MC1R), several α-melanocyte-stimulating hormone (α-MSH) analogues have been labeled with N-succinimidyl-4-¹⁸F-fluorobenzoate (¹⁸)F-SFB) and studied as positron emission tomography (PET) probes in our recent studies. To further pursue a radiofluorinated α-MSH peptide with high clinical translation potential, we utilized 4-nitrophenyl 2-¹⁸F-fluoropropionate (¹⁸F-NFP) to radiofluorinate the transition metal rhenium cyclized α-MSH metallopeptides for PET imaging of MC1R positive malignant melanoma. Metallopeptides Ac-d,Lys-ReCCMSH(Arg¹¹) (two isomers, namely RMSH-1 and RMSH-2) were synthesized using conventional solid phase peptide synthesis chemistry and rhenium cyclization reaction. The two isomers were then conjugated with ¹⁹F-NFP or ¹⁸F-NFP. The resulting cold or radiofluorinated metallopeptides, (¹⁸/¹⁹)F-FP-RMSH-1 and (¹⁸/¹⁹)F-FP-RMSH-2, were further evaluated for their in vitro receptor binding affinities, in vivo biodistribution, and small-animal PET imaging properties. The binding affinities of ¹⁹F-FP-RMSH-1 and ¹⁹F-FP-RMSH-2 were determined to be within low nanomolar range. In vivo studies revealed that both F-labeled metallopeptides possessed good tumor uptake in the B16F10 murine model with high MC1R expression, while possessing much lower uptake in A375M human melanoma xenografts. Moreover, ¹⁸F-FP-RMSH-1 displayed more favorable in vivo performance in terms of higher tumor uptake and much lower accumulation in the kidney and liver, when compared to that of ¹⁸F-FP-RMSH-2 at 2 h postinjection (p.i.). ¹⁸F-FP-RMSH-1 also displayed lower liver and lung uptake when compared with that of the same peptide labeled with ¹⁸F-SFB (named as ¹⁸F-FB-RMSH-1). Small animal PET imaging of ¹⁸F-FP-RMSH-1 in mice bearing B16F10 tumors at 1 and 2 h showed good tumor imaging quality. As expected, much lower tumor uptake and poorer tumor/normal organ contrast were observed for A375M model compared to those of the B16F10 model. ¹⁸F-FP-RMSH-1 also exhibited higher tumor uptake and better tumor retention when compared with ¹⁸F-FB-RMSH-1. ¹⁸F-FP-RMSH-1 demonstrates significant advantages over ¹⁸F-FB-RMSH-1 and ¹⁸F-FP-RMSH-2. It is a promising PET probe for imaging MC1R positive melanoma and MC1R expression in vivo.

Optical Imaging of Reporter Gene Expression Using a Positron-emission-tomography Probe

Reporter gene∕reporter probe technology is one of the most important techniques in molecular imaging. Lately, many reporter gene∕reporter probe systems have been coupled to different imaging modalities such as positron emission tomography (PET) and optical imaging (OI). It has been recently found that OI techniques could be used to monitor radioactive tracers in vitro and in living subjects. In this study, we further demonstrate that a reporter gene∕nuclear reporter probe system [herpes simplex virus type-1 thymidine kinase (HSV1-tk) and 9-(4-(18)F-fluoro-3-[hydroxymethyl] butyl) guanine ([(18)F]FHBG)] could be successfully imaged by OI in vitro and in vivo. OI with radioactive reporter probes will facilitate and broaden the applications of reporter gene∕reporter probe techniques in medical research.

Protein Scaffold-based Molecular Probes for Cancer Molecular Imaging

Protein scaffold molecules are powerful reagents for targeting various cell signal receptors, enzymes, cytokines and other cancer-related molecules. They belong to the peptide and small protein platform with distinct properties. For the purpose of development of new generation molecular probes, various protein scaffold molecules have been labeled with imaging moieties and evaluated both in vitro and in vivo. Among the evaluated probes Affibody molecules and analogs, cystine knot peptides, and nanobodies have shown especially good characteristics as protein scaffold platforms for development of in vivo molecular probes. Quantitative data obtained from positron emission tomography, single photon emission computed tomography/CT, and optical imaging together with biodistribution studies have shown high tumor uptakes and high tumor-to-blood ratios for these probes. High tumor contrast imaging has been obtained within 1 h after injection. The success of those molecular probes demonstrates the adequacy of protein scaffold strategy as a general approach in molecular probe development.

Ferritin Enhances SPIO Tracking of C6 Rat Glioma Cells by MRI

To investigate the effect of ferritin protein overexpression on superparamagnetic iron oxide (SPIO) particle labeling of C6 rat glioma cells, and track the labeled cells in vivo using magnetic resonance imaging (MRI).

De Novo Development of Circulating Anti-endothelial Cell Antibodies Rather Than Pre-existing Antibodies is Associated with Post-transplant Allograft Rejection

Anti-endothelial cell antibodies (AECAs) are thought to be involved in the development of renal allograft rejection. To explore this further, we determine whether AECAs play a role both in predicting the incidence of allograft rejection and long-term outcomes by analysis of serum samples from 226 renal allograft recipients for AECAs pre- and post-transplant. Surprisingly, the presence of pre-existing AECAs was not associated with either an increased risk of rejection or a detrimental impact on recipient/graft survival. Subsequent de novo AECAs, however, were associated with a significantly increased risk of early acute rejection. Moreover, these rejections tended to be more severe with a significantly increased incidence of both steroid-resistant and multiple episodes of acute rejection. The acute rejections associated with de novo AECAs did not correlate with C4d deposition at the time of renal biopsy, but did demonstrate an association with the presence of glomerulitis and peritubular capillary inflammation. Significantly more patients with de novo AECAs developed graft dysfunction. Thus, our prospective study suggests the emergence of de novo AECAs is associated with transplant rejection that may lead to allograft dysfunction.

In Vivo Imaging of Embryonic Stem Cell Therapy

Embryonic stem cells (ESCs) have the most pluripotent potential of any stem cell. These cells, isolated from the inner cell mass of the blastocyst, are "pluripotent," meaning that they can give rise to all cell types within the developing embryo. As a result, ESCs have been regarded as a leading candidate source for novel regenerative medicine therapies and have been used to derive diverse cell populations, including myocardial and endothelial cells. However, before they can be safely applied clinically, it is important to understand the in vivo behavior of ESCs and their derivatives. In vivo analysis of ESC-derived cells remains critically important to define how these cells may function in novel regenerative medicine therapies. In this review, we describe several available imaging modalities for assessing cell engraftment and discuss their strengths and limitations. We also analyze the applications of these modalities in assessing the utility of ESCs in regenerative medicine therapies.

Reproducibility Study of [(18)F]FPP(RGD)2 Uptake in Murine Models of Human Tumor Xenografts

An (18)F-labeled PEGylated arginine-glycine-aspartic acid (RGD) dimer {[(18)F]FPP(RGD)(2)} has been used to image tumor α(v)β(3) integrin levels in preclinical and clinical studies. Serial positron emission tomography (PET) studies may be useful for monitoring antiangiogenic therapy response or for drug screening; however, the reproducibility of serial scans has not been determined for this PET probe. The purpose of this study was to determine the reproducibility of the integrin α(v)β(3)-targeted PET probe, [(18)F]FPP(RGD)(2,) using small animal PET.

Affibody-based Nanoprobes for HER2-expressing Cell and Tumor Imaging

This article reports the affibody-based nanoprobes specifically target and image human epidermal growth factor receptor type 2 (HER2)-expressing cells and tumors. The affibody molecules are a promising class of targeting ligands with simple, robust, and precise structure and high affinity. Using near-infrared (NIR) quantum dots (QDs) and iron oxide (IO) nanoparticles as two representative nanomaterials, we designed anti-HER2 affibody molecules with a N-terminus cysteine residue (Cysteine-Z(HER2:342)) and precisely conjugated with maleimide-functionalized nanoparticles to make nanoparticle-affibody conjugates. The in vitro and in vivo study showed the conjugates are highly specific to target and image HER2-expressing cells and tumors. This work indicated the nanoparticle-affibody conjugates may be excellent candidates as targeting probes for molecular imaging and diagnosis.

Preliminary Evaluation of (177)Lu-labeled Knottin Peptides for Integrin Receptor-targeted Radionuclide Therapy

Cystine knot peptides (knottins) 2.5D and 2.5F were recently engineered to bind integrin receptors with high affinity and specificity. These receptors are overexpressed on the surface of a variety of malignant human tumor cells and tumor neovasculature. In this study, 2.5D and 2.5F were labeled with a therapeutic radionuclide, (177)Lu, and the resulting radiopeptides were then evaluated as potential radiotherapeutic agents in a murine model of human glioma xenografts.

Radiolabeled Affibody-albumin Bioconjugates for HER2-positive Cancer Targeting

Affibody molecules have received significant attention in the fields of molecular imaging and drug development. However, Affibody scaffolds display an extremely high renal uptake, especially when modified with chelators and then labeled with radiometals. This unfavorable property may impact their use as radiotherapeutic agents in general and as imaging probes for the detection of tumors adjacent to kidneys in particular. Herein, we present a simple and generalizable strategy for reducing the renal uptake of Affibody molecules while maintaining their tumor uptake. Human serum albumin (HSA) was consecutively modified by 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DOTA-NHS ester) and the bifunctional cross-linker sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (Sulfo-SMCC). The HER2 Affibody analogue, Ac-Cys-Z(HER2:342), was covalently conjugated with HSA, and the resulting bioconjugate DOTA-HSA-Z(HER2:342) was further radiolabeled with ⁶⁴Cu and ¹¹¹In and evaluated in vitro and in vivo. Radiolabeled DOTA-HSA-Z(HER2:342) conjugates displayed a significant and specific cell uptake into SKOV3 cell cultures. Positron emission tomography (PET) investigations using ⁶⁴Cu-DOTA-HSA-Z(HER2:342) were performed in SKOV3 tumor-bearing nude mice. High tumor uptake values (>14% ID/g at 24 and 48 h) and high liver accumulations but low kidney accumulations were observed. Biodistribution studies and single-photon emission computed tomography (SPECT) investigations using ¹¹¹In-DOTA-HSA-Z(HER2:342) validated these results. At 24 h post injection, the biodistribution data revealed high tumor (16.26% ID/g) and liver (14.11% ID/g) uptake but relatively low kidney uptake (6.06% ID/g). Blocking studies with coinjected, nonlabeled Ac-Cys-Z(HER2:342) confirmed the in vivo specificity of HER2. Radiolabeled DOTA-HSA-Z(HER2:342) Affibody conjugates are promising SPECT and PET-type probes for the imaging of HER2 positive cancer. More importantly, DOTA-HSA-Z(HER2:342) is suitable for labeling with therapeutic radionuclides (e.g., ⁹⁰Y or ¹⁷⁷Lu) for treatment studies. The approach of using HSA to optimize the pharmacokinetics and biodistribution profile of Affibodies may be extended to the design of many other targeting molecules.

Affibody-functionalized Gold-silica Nanoparticles for Raman Molecular Imaging of the Epidermal Growth Factor Receptor

The affibody functionalization of fluorescent surface-enhanced Raman scattering gold-silica nanoparticles as multimodal contrast agents for molecular imaging specific to epidermal growth factor receptor (EGFR) is reported. This nanoparticle bioconjugate reports EGFR-positive A431 tumors with a signal nearly 35-fold higher than EGFR-negative MDA-435S tumors. The low-level EGFR expression in adjacent healthy tissue is 7-fold lower than in the positive tumors. Validation via competitive inhibition reduces the signal by a factor of six, and independent measurement of EGFR via flow cytometry correlates at R(2) = 0.92.

Functional Mutation of Multiple Solvent-exposed Loops in the Ecballium Elaterium Trypsin Inhibitor-II Cystine Knot Miniprotein

The Ecballium elaterium trypsin inhibitor (EETI-II), a 28-amino acid member of the knottin family of peptides, contains three interwoven disulfide bonds that form multiple solvent-exposed loops. Previously, the trypsin binding loop of EETI-II has been engineered to confer binding to several alternative molecular targets. Here, EETI-II was further explored as a molecular scaffold for polypeptide engineering by evaluating the ability to mutate two of its structurally adjacent loops.

Structure-based Repurposing of FDA-approved Drugs As TNF-α Inhibitors

Structure-based Optimization of FDA-approved Drug Methylene Blue As a C-myc G-quadruplex DNA Stabilizer

G-quadruplexes are non-canonical DNA secondary structures putatively present in the promoter regions of oncogenes in the human genome. The targeting of promoter G-quadruplex structures to repress oncogene transcription represents a potential anticancer strategy. Here, we have used high-throughput virtual screening to identify FDA-approved drug methylene blue (MB) as a promising scaffold for binding the c-myc oncogene G-quadruplex DNA. Based on molecular docking analysis of MB to the c-myc G-quadruplex, we designed and screened 50 MB derivatives containing side chains that could interact with the G-quadruplex grooves. As a proof-of-concept, the highest-scoring compounds were synthesized and the interactions with the c-myc G-quadruplex were investigated using the FID assay. The results showed that the methylene blue derivatives 6a-c were able to bind to the c-myc G-quadruplex with greater binding affinity compared to the known G-quadruplex binding ligand, crystal violet. The activity of the most potent compound identified from the FID assay, 6b, as an inhibitor for polymerase-drive DNA extension was examined using a PCR-stop assay and compared against that of the parent compound methylene blue. The results of the PCR-stop assay showed that the addition of the side chain improved the activity of the derivatives as an inhibitor compared to the parent compound. The MB derivative 6b was shown to be highly selective towards c-myc G-quadruplex over double-stranded DNA and other biologically relevant G-quadruplexes using UV-visible spectroscopy and mass spectrometry, respectively. The MB derivative 6b could induce or stabilize c-myc G-quadruplex formation in both cell-free and cellular biological models, and displayed higher cytoxicity against human hepatocarcinoma cells compared to the parent compound, MB.

Macrocyclic Chelator Assembled RGD Multimers for Tumor Targeting

Macrocyclic chelators have been extensively used for complexation of metal ions. A widely used chelator, DOTA, has been explored as a molecular platform to assemble multiple bioactive peptides in this paper. The multivalent DOTA-peptide bioconjugates demonstrate promising tumor targeting ability.

Enzyme-responsive Multifunctional Magnetic Nanoparticles for Tumor Intracellular Drug Delivery and Imaging

Enzyme-responsive, hybrid, magnetic silica nanoparticles have been employed for multifunctional applications in selective drug delivery and intracellular tumor imaging. In this study, doxorubicin (Dox)-conjugated, enzyme-cleavable peptide precursors were covalently tethered onto the surface of uniform silica-coated magnetic nanoparticles through click chemistry. This enzyme-responsive nanoparticle conjugate demonstrated highly efficient Dox release upon specific enzyme interactions in vitro. It also exhibits multiple functions in selective tumor intracellular drug delivery and imaging in the tumor cells with high cathepsin B expression, whereas it exhibited lower cytotoxicity towards other cells without enzyme expression.

Synthesis and Radioluminescence of PEGylated Eu(3+) -doped Nanophosphors As Bioimaging Probes

Elevated Expression of CUEDC2 Protein Confers Endocrine Resistance in Breast Cancer

Endocrine resistance is a major obstacle to hormonal therapy for breast cancers. Although reduced expression of estrogen receptor-α (ER-α) is a known contributing factor to endocrine resistance, the mechanism of ER-α downregulation in endocrine resistance is still not fully understood. Here we report that CUE domain-containing protein-2 (CUEDC2), a ubiquitin-binding motif-containing protein, is a key factor in endocrine resistance in breast cancer. We show that CUEDC2 modulates ER-α protein stability through the ubiquitin-proteasome pathway. Through the study of specimens from a large cohort of subjects with breast cancer, we found a strong inverse correlation between CUEDC2 and ER-α protein expression. Notably, subjects with tumors that highly expressed CUEDC2 had poor responsiveness to tamoxifen treatment and high potential for relapse. We further show that ectopic CUEDC2 expression impaired the responsiveness of breast cancer cells to tamoxifen. Therefore, our findings suggest that CUEDC2 is a crucial determinant of resistance to endocrine therapies in breast cancer.

Preclinical Evaluation of Raman Nanoparticle Biodistribution for Their Potential Use in Clinical Endoscopy Imaging

Raman imaging offers unsurpassed sensitivity and multiplexing capabilities. However, its limited depth of light penetration makes direct clinical translation challenging. Therefore, a more suitable way to harness its attributes in a clinical setting would be to couple Raman spectroscopy with endoscopy. The use of an accessory Raman endoscope in conjunction with topically administered tumor-targeting Raman nanoparticles during a routine colonoscopy could offer a new way to sensitively detect dysplastic lesions while circumventing Raman's limited depth of penetration and avoiding systemic toxicity. In this study, the natural biodistribution of gold surface-enhanced Raman scattering (SERS) nanoparticles is evaluated by radiolabeling them with (64) Cu and imaging their localization over time using micropositron emission tomography (PET). Mice are injected either intravenously (IV) or intrarectally (IR) with approximately 100 microcuries (μCi) (3.7 megabecquerel (MBq)) of (64) Cu-SERS nanoparticles and imaged with microPET at various time points post injection. Quantitative biodistribution data are obtained as % injected dose per gram (%ID g(-1)) from each organ, and the results correlate well with the corresponding microPET images, revealing that IV-injected mice have significantly higher uptake (p < 0.05) in the liver (5 h = 8.96% ID g(-1); 24 h = 8.27% ID g(-1)) than IR-injected mice (5 h = 0.09% ID g(-1); 24 h = 0.08% ID g(-1)). IR-injected mice show localized uptake in the large intestine (5 h = 10.37% ID g(-1); 24 h = 0.42% ID g(-1)) with minimal uptake in other organs. Raman imaging of excised tissues correlate well with biodistribution data. These results suggest that the topical application of SERS nanoparticles in the mouse colon appears to minimize their systemic distribution, thus avoiding potential toxicity and supporting the clinical translation of Raman spectroscopy as an endoscopic imaging tool.

One-step Radiosynthesis of ¹⁸F-AlF-NOTA-RGD₂ for Tumor Angiogenesis PET Imaging

One of the major obstacles of the clinical translation of (18)F-labeled arginine-glycine-aspartic acid (RGD) peptides has been the laborious multistep radiosynthesis. In order to facilitate the application of RGD-based positron emission tomography (PET) probes in the clinical setting we investigated in this study the feasibility of using the chelation reaction between Al(18)F and a macrocyclic chelator-conjugated dimeric RGD peptide as a simple one-step (18)F labeling strategy for development of a PET probe for tumor angiogenesis imaging.

Adipose Tissue-derived Stem Cells Display a Proangiogenic Phenotype on 3D Scaffolds

Ischemic heart disease is the leading cause of death worldwide. Recent studies suggest that adipose tissue-derived stem cells (ASCs) can be used as a potential source for cardiovascular tissue engineering due to their ability to differentiate along the cardiovascular lineage and to adopt a proangiogenic phenotype. To understand better ASCs' biology, we used a novel 3D culture device. ASCs' and b.END-3 endothelial cell proliferation, migration, and vessel morphogenesis were significantly enhanced compared to 2D culturing techniques. ASCs were isolated from inguinal fat pads of 6-week-old GFP+/BLI+ mice. Early passage ASCs cells (P3-P4), PKH26-labeled murine b.END-3 cells or a co-culture of ASCs and b.END-3 cells were seeded at a density of 1 × 10(5) on three different surface configurations: (a) a 2D surface of tissue culture plastic, (b) Matrigel, and (c) a highly porous 3D scaffold fabricated from inert polystyrene. VEGF expression, cell proliferation, and tubulization, were assessed using optical microscopy, fluorescence microscopy, 3D confocal microscopy, and SEM imaging (n = 6). Increased VEGF levels were seen in conditioned media harvested from co-cultures of ASCs and b.END-3 on either Matrigel or a 3D matrix. Fluorescence, confocal, SEM, bioluminescence revealed improved cell, proliferation, and tubule formation for cells seeded on the 3D polystyrene matrix. Collectively, these data demonstrate that co-culturing ASCs with endothelial cells in a 3D matrix environment enable us to generate prevascularized tissue-engineered constructs. This can potentially help us to surpass the tissue thickness limitations faced by the tissue engineering community today.

Cdk1-phosphorylated CUEDC2 Promotes Spindle Checkpoint Inactivation and Chromosomal Instability

Aneuploidy and chromosomal instability are major characteristics of human cancer. These abnormalities can result from defects in the spindle assembly checkpoint (SAC), which is a surveillance mechanism for accurate chromosome segregation through restraint of the activity of the anaphase-promoting complex/cyclosome (APC/C). Here, we show that a CUE-domain-containing protein, CUEDC2, is a cell-cycle regulator that promotes spindle checkpoint inactivation and releases APC/C from checkpoint inhibition. CUEDC2 is phosphorylated by Cdk1 during mitosis. Depletion of CUEDC2 causes a checkpoint-dependent delay of the metaphase-anaphase transition. Phosphorylated CUEDC2 binds to Cdc20, an activator of APC/C, and promotes the release of Mad2 from APC/C-Cdc20 and subsequent APC/C activation. CUEDC2 overexpression causes earlier activation of APC/C, leading to chromosome missegregation and aneuploidy. Interestingly, CUEDC2 is highly expressed in many types of tumours. These results suggest that CUEDC2 is a key regulator of mitosis progression, and that CUEDC2 dysregulation might contribute to tumour development by causing chromosomal instability.

A Novel 18F-labeled Two-helix Scaffold Protein for PET Imaging of HER2-positive Tumor

Two-helix scaffold proteins (~ 5 kDa) against human epidermal growth factor receptor type 2 (HER2) have been discovered in our previous work. In this research we aimed to develop an (18)F-labeled two-helix scaffold protein for positron emission tomography (PET) imaging of HER2-positive tumors.

Molecular Image-guided Theranostic and Personalized Medicine

Direct Analysis of Oligosaccharides and Alpha Hydroxy Acids in Fruits Using Electrosonic Spray Ionization Mass Spectrometry

Electrosonic spray ionization (ESSI) is a derivative technique of electrospray ionization (ESI) for mass spectrometry (MS) in which droplets are charged in the course of sonic spray. In this study, we applied ESSI MS to direct analysis of oligosaccharides and alpha hydroxy acids (AHAs) in fruits. The components were extracted from fruit fleshes by a feasible method prior to ESSI MS analysis, but the fruit juices were analyzed without further pretreatment. The results demonstrate that mainly alkali metal adducts of oligosaccharides are favorably produced in positive ion mode, while deprotonated AHAs and oligosaccharides are produced in negative ion mode. Compared with mass spectra obtained using electrospray droplet impact/secondary ion mass spectrometry (EDI/SIMS), mass spectra using ESSI make the identification of oligosaccharides more straightforward in positive ion mode than in negative ion mode.

[American Surgeon J. P. Webster and the Beginning of Plastic Surgery in China]

To investigate the role of American plastic surgeon Jerome P. Webster in the history of plastic surgery in China.

Cobalt-doped Cadmium Selenide Colloidal Nanowires

Co(2+)-doped CdSe colloidal nanowires with tunable size and dopant concentration have been prepared by a solution-liquid-solid (SLS) approach for the first time. These doped nanowires exhibit anomalous photoluminescence temperature dependence in comparison with undoped nanowires.

In Vivo Targeting of HER2-positive Tumor Using 2-helix Affibody Molecules

Molecular imaging of human epidermal growth factor receptor type 2 (HER2) expression has drawn significant attention because of the unique role of the HER2 gene in diagnosis, therapy and prognosis of human breast cancer. In our previous research, a novel cyclic 2-helix small protein, MUT-DS, was discovered as an anti-HER2 Affibody analog with high affinity through rational protein design and engineering. MUT-DS was then evaluated for positron emission tomography (PET) of HER2-positive tumor by labeling with two radionuclides, (68)Ga and (18)F, with relatively short half-life (t (1/2) < 2 h). In order to fully study the in vivo behavior of 2-helix small protein and demonstrate that it could be a robust platform for labeling with a variety of radionuclides for different applications, in this study, MUT-DS was further radiolabeled with (64)Cu or (111)In and evaluated for in vivo targeting of HER2-positive tumor in mice. Design 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated MUT-DS (DOTA-MUT-DS) was chemically synthesized using solid phase peptide synthesizer and I(2) oxidation. DOTA-MUT-DS was then radiolabeled with (64)Cu or (111)In to prepare the HER2 imaging probe ((64)Cu/(111)In-DOTA-MUT-DS). Both biodistribution and microPET imaging of the probe were evaluated in nude mice bearing subcutaneous HER2-positive SKOV3 tumors. DOTA-MUT-DS could be successfully synthesized and radiolabeled with (64)Cu or (111)In. Biodistribution study showed that tumor uptake value of (64)Cu or (111)In-labeled DOTA-MUT-DS was 4.66 ± 0.38 or 2.17 ± 0.15%ID/g, respectively, in nude mice bearing SKOV3 xenografts (n = 3) at 1 h post-injection (p.i.). Tumor-to-blood and tumor-to-muscle ratios for (64)Cu-DOTA-MUT-DS were attained to be 3.05 and 3.48 at 1 h p.i., respectively, while for (111)In-DOTA-MUT-DS, they were 2.04 and 3.19, respectively. Co-injection of the cold Affibody molecule Z(HER2:342) with (64)Cu-DOTA-MUT-DS specifically reduced the SKOV3 tumor uptake of the probe by 48%. (111)In-DOTA-MUT-DS displayed lower liver uptake at all the time points investigated and higher tumor to blood ratios at 4 and 20 h p.i., when compared with (64)Cu-DOTA-MUT-DS. This study demonstrates that the 2-helix protein based probes, (64)Cu/(111)In DOTA-MUT-DS, are promising molecular probes for imaging HER2-positive tumor. Two-helix small protein scaffold holds great promise as a novel and robust platform for imaging and therapy applications.

Harnessing the Power of Radionuclides for Optical Imaging: Cerenkov Luminescence Imaging

Over the past several years, nuclear imaging modalities such as PET and SPECT have received much attention because they have been instrumental not only in preclinical cancer research but also in nuclear medicine. Yet nuclear imaging is limited by high instrumentation cost and subsequently low availability to basic researchers. Cerenkov radiation, a relativistic physical phenomenon that was discovered 70 years ago, has recently become an intriguing subject of study in molecular imaging because of its potential in augmenting nuclear imaging, particularly in preclinical small-animal studies. The intrinsic capability of radionuclides emitting luminescent light from decay is promising because of the possibility of bridging nuclear imaging with optical imaging-a modality that is much less expensive, is easier to use, and has higher throughput than its nuclear counterpart. Thus, with the maturation of this novel imaging technology using Cerenkov radiation, which is termed Cerenkov luminescence imaging, it is foreseeable that advances in both nuclear imaging and preclinical research involving radioisotopes will be significantly accelerated in the near future.

Non-invasive Imaging of Cysteine Cathepsin Activity in Solid Tumors Using a 64Cu-labeled Activity-based Probe

The papain family of cysteine cathepsins are actively involved in multiple stages of tumorigenesis. Because elevated cathepsin activity can be found in many types of human cancers, they are promising biomarkers that can be used to target radiological contrast agents for tumor detection. However, currently there are no radiological imaging agents available for these important molecular targets. We report here the development of positron emission tomography (PET) radionuclide-labeled probes that target the cysteine cathepsins by formation of an enzyme activity-dependent bond with the active site cysteine. These probes contain an acyloxymethyl ketone (AOMK) functional group that irreversibly labels the active site cysteine of papain family proteases attached to a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) tag for labeling with (64)Cu for PET imaging studies. We performed biodistribution and microPET imaging studies in nude mice bearing subcutaneous tumors expressing various levels of cysteine cathepsin activity and found that the extent of probe uptake by tumors correlated with overall protease activity as measured by biochemical methods. Furthermore, probe signals could be reduced by pre-treatment with a general cathepsin inhibitor. We also found that inclusion of a Cy5 tag on the probe increased tumor uptake relative to probes lacking this fluorogenic dye. Overall, these results demonstrate that small molecule activity-based probes carrying radio-tracers can be used to image protease activity in living subjects.

PET Imaging of Integrin Positive Tumors Using F Labeled Knottin Peptides

Purpose: Cystine knot (knottin) peptides, engineered to bind with high affinity to integrin receptors, have shown promise as molecular imaging agents in living subjects. The aim of the current study was to evaluate tumor uptake and in vivo biodistribution of (18)F-labeled knottins in a U87MG glioblastoma model.Procedures: Engineered knottin mutants 2.5D and 2.5F were synthesized using solid phase peptide synthesis and were folded in vitro, followed by radiolabeling with 4-nitrophenyl 2-(18)F-fluoropropionate ((18)F-NFP). The resulting probes, (18)F-FP-2.5D and (18)F-FP-2.5F, were evaluated in nude mice bearing U87MG tumor xenografts using microPET and biodistribution studies.Results: MicroPET imaging studies with (18)F-FP-2.5D and (18)F-FP-2.5F demonstrated high tumor uptake in U87MG xenograft mouse models. The probes exhibited rapid clearance from the blood and kidneys, thus leading to excellent tumor-to-normal tissue contrast. Specificity studies confirmed that (18)F-FP-2.5D and (18)F-FP-2.5F had reduced tumor uptake when co-injected with a large excess of the peptidomimetic c(RGDyK) as a blocking agent.Conclusions: (18)F-FP-2.5D and (18)F-FP-2.5F showed reduced gallbladder uptake compared with previously published (18)F-FB-2.5D. (18)F-FP-2.5D and (18)F-FP-2.5F enabled integrin-specific PET imaging of U87MG tumors with good imaging contrasts. (18)F-FP-2.5D demonstrated more desirable pharmacokinetics compared to (18)F-FP-2.5F, and thus has greater potential for clinical translation.

[Professor CHENG Zhifan and PUMHS Department of Medical History]

Professor Zhifan Cheng is a notable expert on medical history in modern China. Since 1950 when he graduated from Peking University Medical School, Prof. Cheng was working in the Department of Medical History until he retired in 2002. During the Cultural Revolution, he was sent to the TCM Department of Bei Da Hospital (Now Peking University First Hospital). Professor Cheng devoted himself to teaching medical history, exploring the aim and methods of teaching in China, writing and editing textbooks, developing postgraduate education, training teachers and promoting the research of medical history in academic communications. Prof. Cheng, working for over a half century in the department, had made every effort for the development of this office.

Luminescent Detection of DNA-binding Proteins

Transcription factors play a central role in cell development, differentiation and growth in biological systems due to their ability to regulate gene expression by binding to specific DNA sequences within the nucleus. The dysregulation of transcription factor signaling has been implicated in the pathogenesis of a number of cancers, developmental disorders, inflammation and autoimmunity. There is thus a high demand for convenient high-throughput methodologies able to detect sequence-specific DNA-binding proteins and monitor their DNA-binding activities. Traditional approaches for protein detection include gel mobility shift assays, DNA footprinting and enzyme-linked immunosorbent assays (ELISAs) which tend to be tedious, time-consuming, and may necessitate the use of radiographic labeling. By contrast, luminescence technologies offer the potential for rapid, sensitive and low-cost detection that are amenable to high-throughput and real-time analysis. The discoveries of molecular beacons and aptamers have spear-headed the development of new luminescent methodologies for the detection of proteins over the last decade. We survey here recent advances in the development of luminescent detection methods for DNA-binding proteins, including those based on molecular beacons, aptamer beacons, label-free techniques and exonuclease protection.

Primary Focal Segmental Glomerulosclerosis in Nephrotic Patients: Common Complications and Risk Factors

Background: Focal segmental glomerulosclerosis (FSGS) presents a range of potentially serious complications, including acute kidney injury (AKI), infection and thromboembolism. This study aimed to find out the incidence rates and risk factors for these complications in FSGS patients. Methods: Patients with biopsy-proven primary FSGS and nephrotic-range proteinuria were included in this study. A short-term (16-week) follow-up was performed to observe the aforementioned complications. Clinical characteristics of patients were recorded upon enrollment. AKI was diagnosed as an absolute increase in serum creatinine of >=0.3 mg/dL or a percentage increase of >=50% within 48 hours; infection, by a combination of clinical manifestations, laboratory tests and imaging examinations; and thromboembolism, by imaging methods. Risk factors for complications were evaluated by logistic regression model. Results: The study population included 90 FSGS patients (63 males, mean age 28.9 ± 12.9 years). The incidences of AKI, infection and thromboembolism were 44.4%, 25.6% and 12.2%, respectively. Patients with AKI were more likely to be male, with lower serum albumin, greater proteinuria and more severe acute tubulointerstitial damage. Patients with infection had higher proteinuria and lower serum albumin, globulin and IgG. Circulating endothelial cells (CECs) and von Willebrand factor were higher in patients with thromboembolism. Logistic regression showed that increased urine retinol-binding protein, decreased serum albumin and IgG, and increased CECs and hemoglobin were independent risk factors for AKI, infection and thromboembolism, respectively. Conclusions: AKI, infection and thromboembolism are common among FSGS patients. Awareness of risk factors and prevention of these complications are important for the prognosis of these patients.

A Novel Clinically Translatable Fluorescent Nanoparticle for Targeted Molecular Imaging of Tumors in Living Subjects

The use of quantum dots (QDs) in biomedical research has grown tremendously, yet successful examples of clinical applications are absent due to many clinical concerns. Here, we report on a new type of stable and biocompatible dendron-coated InP/ZnS core/shell QD as a clinically translatable nanoprobe for molecular imaging applications. The QDs (QD710-Dendron) were demonstrated to hold several significant features: near-infrared (NIR) emission, high stability in biological media, suitable size with possible renal clearance, and ability of extravasation. More importantly, a pilot mouse toxicity study confirmed that QD710-Dendron lacks significant toxicity at the doses tested. The acute tumor uptake of QD710-Dendron resulted in good contrast from the surrounding nontumorous tissues, indicating the possibility of passive targeting of the QDs. The highly specific targeting of QD710-Dendron-RGD(2) to integrin α(v)β(3)-positive tumor cells resulted in high tumor uptake and long retention of the nanoprobe at tumor sites. In summary, QD710-Dendron and RGD-modified nanoparticles demonstrate small size, high stability, biocompatibility, favorable in vivo pharmacokinetics, and successful tumor imaging properties. These features satisfy the requirements for clinical translation and should promote efforts to further investigate the possibility of using QD710-Dendron-based nanoprobes in the clinical setting in the near future.

Optical Imaging with Her2-targeted Affibody Molecules Can Monitor Hsp90 Treatment Response in a Breast Cancer Xenograft Mouse Model

To determine whether optical imaging can be used for in vivo therapy response monitoring as an alternative to radionuclide techniques. For this, we evaluated the known Her2 response to 17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride (17-DMAG) treatment, an Hsp90 inhibitor.

Comparative Evaluation of Light-trap Catches, Electric Motor Mosquito Catches and Human Biting Catches of Anopheles in the Three Gorges Reservoir

The mosquito sampling efficiency of light-trap catches and electric motor mosquito catches were compared with that of human biting catches in the Three Gorges Reservoir. There was consistency in the sampling efficiency between light-trap catches and human biting catches for Anopheles sinensis (r = 0.82, P<0.01) and light-trap catches were 1.52 (1.35-1.71) times that of human biting catches regardless of mosquito density (r = 0.33, P>0.01), while the correlation between electric motor mosquito catches and human biting catches was found to be not statistically significant (r = 0.43, P>0.01) and its sampling efficiency was below that of human biting catches. It is concluded that light-traps can be used as an alternative to human biting catches of Anopheles sinensis in the study area and is a promising tool for sampling malaria vector populations.

In vitro and in Vivo Uncaging and Bioluminescence Imaging by Using Photocaged Upconversion Nanoparticles

Proof-of-concept Study of Monitoring Cancer Drug Therapy with Cerenkov Luminescence Imaging

Cerenkov luminescence imaging (CLI) has emerged as a less expensive, easier-to-use, and higher-throughput alternative to other nuclear imaging modalities such as PET. It is expected that CLI will find many applications in biomedical research such as cancer detection, probe development, drug screening, and therapy monitoring. In this study, we explored the possibility of using CLI to monitor drug efficacy by comparisons against PET. To assess the performance of both modalities in therapy monitoring, 2 murine tumor models (large cell lung cancer cell line H460 and prostate cancer cell line PC3) were given bevacizumab versus vehicle treatments. Two common radiotracers, 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) and (18)F-FDG, were used to monitor bevacizumab treatment efficacy.

Human Serum Albumin Conjugated Biomolecules for Cancer Molecular Imaging

Molecular imaging is a fast growing field in biomedical research. The discovery, development and continual improvement of molecular probes are important for ongoing research efforts in molecular imaging. Human serum albumin (HSA) offers favorable characteristics and opportunities as a platform protein for molecular imaging probe discovery and optimization. It has many advantages, including alternation of biodistribution and pharmacokinetic properties of molecular imaging probes, enhancing the blood half-life of bio-molecules, and making these molecules multivalent, all of which make HSA a promising carrier for cancer-targeted imaging and therapy. Numerous studies have focused on the development and application of HSA-based cancer imaging and treatment. This review gives a brief account of albumin-based molecular probes, focusing on their applications in cancer molecular imaging, such as PET/SPECT, MRI and optical imaging.

Biomarkers of Endothelial Dysfunction in Patients with Primary Focal Segmental Glomerulosclerosis

Endothelial dysfunction occurs in nephrotic syndrome (NS) and may constitute a link between NS and vascular complications. Focal segmental glomerulosclerosis (FSGS) is a common cause of NS. This study aimed to assess endothelial markers at different stages of FSGS and define whether they were associated with thromboembolic complications and disease activity.

Analysis of in Situ and Ex Vivo αVβ3 Integrin Expression During Experimental Carotid Atherogenesis

Mural inflammation has been shown to contribute to the development of plaque, with the α(V)β(3) integrin highly expressed in atherosclerotic plaques. We herein examined α(V)β(3) integrin expression as a function of carotid atherosclerosis formation in the apolipoprotein E-deficient (apoE(-/-)) mouse.

A Multimodality Reporter Gene for Monitoring Transplanted Stem Cells

The aim of this study is to explore the feasibility of a triple-fused reporter gene, termed TGF [herpes simplex virus type 1 thymidine kinase (HSV1-tk), enhanced green fluorescent protein (eGFP) and firefly luciferase (Fluc)], to monitor stem cells using multimodality molecular imaging.

A Label-free G-quadruplex-based Switch-on Fluorescence Assay for the Selective Detection of ATP

A G-quadruplex-based, label-free, switch-on fluorescence detection method has been developed for the selective detection of ATP in aqueous solution using crystal violet as a G-quadruplex-selective probe. The assay is highly simple and rapid, and does not require the use of fluorescent labeling.

177Lu-DO3A-HSA-Z EGFR:1907: Characterization As a Potential Radiopharmaceutical for Radionuclide Therapy of EGFR-expressing Head and Neck Carcinomas

Epidermal growth factor receptor 1 (EGFR) is an attractive target for radionuclide therapy of head and neck carcinomas. Affibody molecules against EGFR (Z(EGFR)) show excellent tumor localizations in imaging studies. However, one major drawback is that radiometal-labeled Affibody molecules display extremely high uptakes in the radiosensitive kidneys which may impact their use as radiotherapeutic agents. The purpose of this study is to further explore whether radiometal-labeled human serum albumin (HSA)-Z(EFGR) bioconjugates display desirable profiles for the use in radionuclide therapy of EGFR-positive head and neck carcinomas. The Z(EFGR) analog, Ac-Cys-Z(EGFR:1907), was site-specifically conjugated with HSA. The resulting bioconjugate 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A)-HSA-Z(EGFR:1907) was then radiolabeled with either (64)Cu or (177)Lu and subjected to in vitro cell uptake and internalization studies using the human oral squamous carcinoma cell line SAS. Positron emission tomography (PET), single photon emission computed tomography (SPECT), and biodistribution studies were conducted using SAS-tumor-bearing mice. Cell studies revealed a high (8.43 ± 0.55 % at 4 h) and specific (0.95 ± 0.09 % at 4 h) uptake of (177)Lu-DO3A-HSA-Z(EGFR:1907) as determined by blocking with nonradioactive Z(EGFR:1907). The internalization of (177)Lu-DO3A-HSA-Z(EGFR:1907) was verified in vitro and found to be significantly higher than that of (177)Lu-labeled Z(EFGR) at 2-24 h of incubation. PET and SPECT studies showed good tumor imaging contrasts. The biodistribution of (177)Lu-DO3A-HSA-Z(EGFR:1907) in SAS-tumor-bearing mice displayed high tumor uptake (5.1 ± 0.44 % ID/g) and liver uptake (31.5 ± 7.66 % ID/g) and moderate kidney uptake (8.5 ± 1.08 % ID/g) at 72 h after injection. (177)Lu-DO3A-HSA-Z(EGFR:1907) shows promising in vivo profiles and may be a potential radiopharmaceutical for radionuclide therapy of EGFR-expressing head and neck carcinomas.

In Vivo Biodistribution and Small Animal PET of (64)Cu-Labeled Antimicrobial Peptoids

Peptoids are a rapidly developing class of biomimetic polymers based on oligo-N-substituted glycine backbones, designed to mimic peptides and proteins. Inspired by natural antimicrobial peptides, a group of cationic amphipathic peptoids has been successfully discovered with potent, broad-spectrum activity against pathogenic bacteria; however, there are limited studies to address the in vivo pharmacokinetics of the peptoids. Herein, (64)Cu-labeled DOTA conjugates of three different peptoids and two control peptides were synthesized and assayed in vivo by both biodistribution studies and small animal positron emission tomography (PET). The study was designed in a way to assess how structural differences of the peptidomimetics affect in vivo pharmacokinetics. As amphipathic molecules, major uptake of the peptoids occurred in the liver. Increased kidney uptake was observed by deleting one hydrophobic residue in the peptoid, and (64)Cu-3 achieved the highest kidney uptake of all the conjugates tested in this study. In comparison to peptides, our data indicated that peptoids had general in vivo properties of higher tissue accumulation, slower elimination, and higher in vivo stability. Different administration routes (intravenous, intraperitoneal, and oral) were investigated with peptoids. When administered orally, the peptoids showed poor bioavailability, reminiscent of that of peptide. However, remarkably longer passage through the gastrointestinal (GI) tract without rapid digestion was observed for peptoids. These unique in vivo properties of peptoids were rationalized by efficient cellular membrane permeability and protease resistance of peptoids. The results observed in the biodistribution studies could be confirmed by PET imaging, which provides a reliable way to evaluate in vivo pharmacokinetic properties of peptoids noninvasively and in real time. The pharmacokinetic data presented here can provide insight for further development of the antimicrobial peptoids as pharmaceuticals.

111In-labeled Cystine-knot Peptides Based on the Agouti-related Protein for Targeting Tumor Angiogenesis

Agouti-related protein (AgRP) is a 4-kDa cystine-knot peptide of human origin with four disulfide bonds and four solvent-exposed loops. The cell adhesion receptor integrin α(v)β(3) is an important tumor angiogenesis factor that determines the invasiveness and metastatic ability of many malignant tumors. AgRP mutants have been engineered to bind to integrin α(v)β(3) with high affinity and specificity using directed evolution. Here, AgRP mutants 7C and 6E were radiolabeled with (111)In and evaluated for in vivo targeting of tumor integrin α(v)β(3) receptors. AgRP peptides were conjugated to the metal chelator 1, 4, 7, 10-tetra-azacyclododecane- N, N', N″, N'''-tetraacetic acid (DOTA) and radiolabeled with (111)In. The stability of the radiopeptides (111)In-DOTA-AgRP-7C and (111)In-DOTA-AgRP-6E was tested in phosphate-buffered saline (PBS) and mouse serum, respectively. Cell uptake assays of the radiolabeled peptides were performed in U87MG cell lines. Biodistribution studies were performed to evaluate the in vivo performance of the two resulting probes using mice bearing integrin-expressing U87MG xenograft tumors. Both AgRP peptides were easily labeled with (111)In in high yield and radiochemical purity (>99%). The two probes exhibited high stability in phosphate-buffered saline and mouse serum. Compared with (111)In-DOTA-AgRP-6E, (111)In-DOTA-AgRP-7C showed increased U87MG tumor uptake and longer tumor retention (5.74 ± 1.60 and 1.29 ± 0.02%ID/g at 0.5 and 24 h, resp.), which was consistent with measurements of cell uptake. Moreover, the tumor uptake of (111)In-DOTA-AgRP-7C was specifically inhibited by coinjection with an excess of the integrin-binding peptidomimetic c(RGDyK). Thus, (111)In-DOTA-AgRP-7C is a promising probe for targeting integrin α(v)β(3) positive tumors in living subjects.

The Manipulation of Natural Killer Cells to Target Tumor Sites Using Magnetic Nanoparticles

The present work demonstrates that Cy5.5 conjugated Fe(3)O(4)/SiO(2) core/shell nanoparticles could allow us to control movement of human natural killer cells (NK-92MI) by an external magnetic field. Required concentration of the nanoparticles for the cell manipulation is as low as ~20 μg Fe/mL. However, the relative ratio of the nanoparticles loaded NK-92MI cells infiltrated into the target tumor site is enhanced by 17-fold by applying magnetic field and their killing activity is still maintained as same as the NK-92MI cells without the nanoparticles. This approach allows us to open alternative clinical treatment with reduced toxicity of the nanoparticles and enhanced infiltration of immunology to the target site.

Evaluation of Four Affibody-Based Near-Infrared Fluorescent Probes for Optical Imaging of Epidermal Growth Factor Receptor Positive Tumors

The epidermal growth factor receptor 1 (EGFR) has become an attractive target for cancer molecular imaging and therapy. An Affibody protein with strong binding affinity for EGFR, Z(EGFR:1907), has been reported. We are interested in translating Affibody molecules to potential clinical optical imaging of EGFR positive cancers. In this study, four anti-EGFR Affibody based near-infrared (NIR) fluorescent probes were thus prepared, and their in vivo performance was evaluated in the mice bearing EGFR positive subcutaneous A431 tumors. Methods: The Affibody analogue, Ac-Cys-Z(EGFR:1907), was synthesized using solid-phase peptide synthesis method. The purified small protein was then site-specifically conjugated with four NIR fluorescent dyes, Cy5.5-monomaleimide, Alex-Fluor-680-maleimide, SRfluor680-maleimide, or IRDye-800CW-maleimide, to produce four optical probes-Cy5.5-Z(EGFR:1907), Alexa680-Z(EGFR:1907), SR680-Z(EGFR:1907), and 800CW-Z(EGFR:1907). The EGFR binding property and specificity of the four NIR fluorescent Affibody probes were studied by fluorescence microscopy using high EGFR expressing A431 cells and low expressing MCF7 cells. The binding affinities of the probes (K(D)) to EGFR were further determined by flow cytometry. In vivo optical imaging of the four probes was performed in the mice bearing subcutaneous A431 tumors. Results: The four NIR optical probes were prepared in high purity. In vitro cell imaging studies demonstrated that all of them could specifically bind to EGFR positive A431 cells while showing minimum uptake in low EGFR expressing MCF7 cells. Flow cytometry showed that Cy5.5-Z(EGFR:1907) and Alexa680-Z(EGFR:1907) possessed high binding affinity in low nanomolar range (43.6 ± 8.4 and 28.3 ± 4.9, respectively). In vivo optical imaging of the four probes revealed that they all showed fast tumor targeting ability and good tumor-to-normal tissue contrast as early as 0.5 h postinjection (p.i.). The tumor-to-normal tissue ratio reached a peak at 2 to 4 h p.i. by regional of interest (ROI) analysis of images. Ex vivo studies further demonstrated that the four probes had high tumor uptakes. Particularly, Cy5.5-Z(EGFR:1907) and Alex680-Z(EGFR:1907) displayed higher tumor-to-normal tissue ratios than those of the other two probes. Conclusion: This work demonstrates that Affibody proteins can be modified with different NIR fluorescent dyes and used for imaging of EGFR expressing tumors. Different NIR fluorescent dyes have variable impact on the in vitro binding and in vivo performance of the resulting Affibody based probes. Therefore, selection of an appropriate NIRF label is important for optical probe development. The probes developed are promising for further tumor imaging applications and clinical translation. Particularly, Alex680-Z(EGFR:1907) and Cy5.5-Z(EGFR:1907) are excellent candidates as EGFR-targeted probes for optical imaging.

Assessment and Comparison of Magnetic Nanoparticles As MRI Contrast Agents in a Rodent Model of Human Hepatocellular Carcinoma

The purpose of this study was to synthesize, characterize and tailor the surface properties of magnetic nanoparticles with biocompatible copolymer coatings and to evaluate the efficiency of the resulting nanoconjugates as magnetic resonance imaging (MRI) contrast agents for liver imaging. Magnetic nanoparticles with core diameters of 10 and 30 nm were synthesized by pyrolysis and were subsequently coated with a copolymer containing either carboxyl (SHP) or methoxy groups as termini. All four formulas, and ferumoxides (Feridex I.V.(®)), were individually injected intravenously into separate, normal Balb/C mice (at 2.5, 1.0 and 0.56 mg Fe kg(-1)), and the animals underwent T(2)-weighted MRI at multiple time points post injection (p.i.) to evaluate the hepatic uptake and clearance. Furthermore, we compared the abilities of the new formulas and Feridex to detect tumors in an orthotropic Huh7 tumor model. Transmission electron microscopy (TEM) revealed a narrow size distribution of both the 10 and 30 nm nanoparticles, in contrast to a wide size distribution of Feridex. MTT, apoptosis and cyclin/DNA flow cytometry assays showed that the polymer coated nanoparticles had no adverse effect on cell growth. Among all the tested formulas, including Feridex, SHP-30 showed the highest macrophage uptake at the in vitro level. In vivo MRI studies on normal mice confirmed the superiority of SHP-30 in inducing hypointensities in the liver tissue, especially at clinical dose (0.56 mg Fe kg(-1)) and 3 T field. SHP-30 showed better contrast-to-noise ratio than Feridex on the orthotropic Huh7 tumor model. SHP-30 was found to be an efficient contrast agent for liver MR imaging. The success of this study suggests that, by improving the synthetic approach and by tuning the surface properties of IONPs, one can arrive at better formulas than Feridex for clinical practice.

PET of EGFR Expression with an 18F-labeled Affibody Molecule

Epidermal growth factor receptor (EGFR) is often overexpressed in a variety of human cancers, and its expression is associated with poor prognosis for many cancer types. However, an accurate technique to noninvasively image EGFR expression in vivo is not available in the clinical setting. In this research, an Affibody analog, anti-EGFR Ac-Cys-Z(EGFR:1907), was successfully site-specifically (18)F-labeled for PET of EGFR expression.

Discovery of a Natural Product Inhibitor Targeting Protein Neddylation by Structure-based Virtual Screening

NEDD8-activating enzyme (NAE) controls the specific degradation of proteins regulated by cullin-RING ubiquitin E3 ligase, and has been considered as an attractive molecular target for the development of anti-cancer drugs. We report herein the identification of the dipeptide-conjugated deoxyvasicinone derivative (1) as an inhibitor of NAE by virtual screening of over 90,000 compounds from the ZINC database of natural products. Molecular modelling results suggested that 1 may be a non-covalent competitive inhibitor of NAE by blocking the ATP-binding domain. Compound 1 was able to inhibit NAE activity in both cell-free and cell-based assay with potencies in the micromolar range and selectivity over analogous E1 enzymes UAE and SAE. We envisage that the identification and molecular docking analysis of this bioactive scaffold as an NAE inhibitor would provide the scientific community with useful information in order to generate more potent analogues.

Radioluminescent Nanophosphors Enable Multiplexed Small-animal Imaging

We demonstrate the ability to image multiple nanoparticle-based contrast agents simultaneously using a nanophosphor platform excited by either radiopharmaceutical or X-ray irradiation. These radioluminescent nanoparticles emit optical light at unique wavelengths depending on their lanthanide dopant, enabling multiplexed imaging. This study demonstrates the separation of two distinct nanophosphor contrast agents in gelatin phantoms with a recovered phosphor separation correlation of -0.98. The ability to distinguish the two nanophosphors and a Cerenkov component is then demonstrated in a small animal phantom. Combined with the high-resolution potential of low-scattering X-ray excitation, this imaging technique may be a promising method to probe molecular processes in living organisms.

Endoscopic Imaging of Cerenkov Luminescence

We demonstrate feasibility of endoscopic imaging of Cerenkov light originated when charged nuclear particles, emitted from radionuclides, travel through a biological tissue of living subjects at superluminal velocity. The endoscopy imaging system consists of conventional optical fiber bundle/ clinical endoscopes, an optical imaging lens system, and a sensitive low-noise charge coupled device (CCD) camera. Our systematic studies using phantom samples show that Cerenkov light from as low as 1 µCi of radioactivity emitted from (18)F-Fluorodeoxyglucose (FDG) can be coupled and transmitted through conventional optical fibers and endoscopes. In vivo imaging experiments with tumor bearing mice, intravenously administered with (18)F-FDG, further demonstrated that Cerenkov luminescence endoscopy is a promising new tool in the field of endoscopic molecular imaging.

A Metal-based Inhibitor of Tumor Necrosis Factor-α

Staying in the pocket: A cyclometalated iridium(III) biquinoline complex targets the protein-protein interface (see picture; C yellow, N blue, Ir dark green) of the tumor necrosis factor-α (TNF-α) trimer. Molecular-modeling studies confirm the nature of this interaction. Both enantiomers of the iridium complex display comparable in vitro potency to the strongest small-molecule inhibitor of TNF-α.

Activatable Near-infrared Fluorescent Probe for in Vivo Imaging of Fibroblast Activation Protein-alpha

Fibroblast activation protein-alpha (FAPα) is a cell surface glycoprotein which is selectively expressed by tumor-associated fibroblasts in malignant tumors but rarely on normal tissues. FAPα has also been reported to promote tumor growth and invasion and therefore has been of increasing interest as a promising target for designing tumor-targeted drugs and imaging agents. Although medicinal study on FAPα inhibitors has led to the discovery of many FAPα-targeting inhibitors including a drug candidate in a phase II clinical trial, the development of imaging probes to monitor the expression and activity of FAPα in vivo has largely lagged behind. Herein, we report an activatable near-infrared (NIR) fluorescent probe (ANP(FAP)) for in vivo optical imaging of FAPα. The ANP(FAP) consists of a NIR dye (Cy5.5) and a quencher dye (QSY21) which are linked together by a short peptide sequence (KGPGPNQC) specific for FAPα cleavage. Because of the efficient fluorescence resonance energy transfer (FRET) between Cy5.5 and QSY21 in ANP(FAP), high contrast on the NIR fluorescence signal can be achieved after the cleavage of the peptide sequence by FAPα both in vitro and in vivo. In vitro assay on ANP(FAP) indicated the specificity of the probe to FAPα. The in vivo optical imaging using ANP(FAP) showed fast tumor uptake as well as high tumor to background contrast on U87MG tumor models with FAPα expression, while much lower signal and tumor contrast were observed in the C6 tumor without FAPα expression, demonstrating the in vivo targeting specificity of the ANP(FAP). Ex vivo imaging also demonstrated ANP(FAP) had high tumor uptake at 4 h post injection. Collectively, these results indicated that ANP(FAP) could serve as a useful NIR optical probe for early detection of FAPα expressing tumors.

Intraoperative Imaging of Tumors Using Cerenkov Luminescence Endoscopy: a Feasibility Experimental Study

Cerenkov luminescence imaging (CLI) is an emerging new molecular imaging modality that is relatively inexpensive, easy to use, and has high throughput. CLI can image clinically available PET and SPECT probes using optical instrumentation. Cerenkov luminescence endoscopy (CLE) is one of the most intriguing applications that promise potential clinical translation. We developed a prototype customized fiberscopic Cerenkov imaging system to investigate the potential in guiding minimally invasive surgical resection.

A Novel Radiofluorinated Agouti-related Protein for Tumor Angiogenesis Imaging

A novel protein scaffold based on the cystine knot domain of the agouti-related protein (AgRP) has been used to engineer mutants that can bind to the α(v)β(3) integrin receptor with high affinity and specificity. In the current study, an (18)F-labeled AgRP mutant (7C) was prepared and evaluated as a positron emission tomography (PET) probe for imaging tumor angiogenesis. AgRP-7C was synthesized by solid phase peptide synthesis and site-specifically conjugated with 4-nitrophenyl 2-(18/19)F-fluoropropionate ((18/19)F-NFP) to produce the fluorinated peptide, (18/19)F-FP-AgRP-7C. Competition binding assays were used to measure the relative affinities of AgRP-7C and (19)F-FP-AgRP-7C to human glioblastoma U87MG cells that overexpress α(v)β(3) integrin. In addition, biodistribution, metabolic stability, and small animal PET imaging studies were conducted with (18)F-FP-AgRP-7C using U87MG tumor-bearing mice. Both AgRP-7C and (19)F-FP-AgRP-7C specifically competed with (125)I-echistatin for binding to U87MG cells with half maximal inhibitory concentration (IC(50)) values of 9.40 and 8.37 nM, respectively. Non-invasive small animal PET imaging revealed that (18)F-FP-AgRP-7C exhibited rapid and good tumor uptake (3.24 percentage injected dose per gram [% ID/g] at 0.5 h post injection [p.i.]). The probe was rapidly cleared from the blood and from most organs, resulting in excellent tumor-to-normal tissue contrasts. Tumor uptake and rapid clearance were further confirmed with biodistribution studies. Furthermore, co-injection of (18)F-FP-AgRP-7C with a large molar excess of blocking peptide c(RGDyK) significantly inhibited tumor uptake in U87MG xenograft models, demonstrating the integrin-targeting specificity of the probe. Metabolite assays showed that the probe had high stability, making it suitable for in vivo applications. (18)F-FP-AgRP-7C exhibits promising in vivo properties such as rapid tumor targeting, good tumor uptake, and excellent tumor-to-normal tissue ratios, and warrants further investigation as a novel PET probe for imaging tumor angiogenesis.

Structural Characterization of Synthetic Polymers Using Thermal-assisted Atmospheric Pressure Glow Discharge Mass Spectrometry

With the development of material science and the practical needs of the polymer industry, rapid characterization of synthetic polymers using mass spectrometry is of sustainable interest. Herein a new method for characterizing synthetic polymers using thermal-assisted atmospheric pressure glow discharge mass spectrometry (TA-APGD-MS) is established. After illustration of the mechanism of ion formation, typical polymer samples such as polystyrene (PS), polyoxymethylene (POM) and poly (butanediol succinate) (PBS) were directly characterized at the molecular level using TA-APGD-MS. The thermal degradation products of synthetic polymers including monomer units and/or other fragments were rapidly detected by tandem mass spectrometry, providing rich information about the chemical composition for the structural characterization of homo- and co-polymers. The result suggests that TA-APGD-MS allows direct and rapid analysis of both synthetic homo-polymers and co-polymers under ambient conditions without any sample pretreatment. This method features high throughput, high sensitivity and rich information, showing promising applications in polymer science.

Novel, Cysteine-Modified Chelation Strategy for the Incorporation of [M(I)(CO)(3)](+) (M = Re, (99m)Tc) in an α-MSH Peptide

Engineering peptide-based targeting agents with residues for site-specific and stable complexation of radionuclides is a highly desirable strategy for producing diagnostic and therapeutic agents for cancer and other diseases. In this report, a model N-S-N(Py) ligand (3) and a cysteine-derived α-melanocyte stimulating hormone (α-MSH) peptide (6) were used as novel demonstrations of a widely applicable chelation strategy for incorporation of the [M(I)(CO)(3)](+) (M = Re, (99m)Tc) core into peptide-based molecules for radiopharmaceutical applications. The structural details of the core ligand-metal complexes as model systems were demonstrated by full chemical characterization of fac-[Re(I)(CO)(3)(N,S,N(Py)-3)](+) (4) and comparative high-performance liquid chromatography (HPLC) analysis between 4 and [(99m)Tc(I)(CO)(3)(N,S,N(Py)-3)](+) (4a). The α-MSH analogue bearing the N-S-N(Py) chelate on a modified cysteine residue (6) was generated and complexed with [M(I)(CO)(3)](+) to confirm the chelation strategy's utility when applied in a peptide-based targeting agent. Characterization of the Re(I)(CO)(3)-6 peptide conjugate (7) confirmed the efficient incorporation of the metal center, and the (99m)Tc(I)(CO)(3)-6 analogue (7a) was explored as a potential single photon emission computed tomography (SPECT) compound for imaging the melanocortin 1 receptor (MC1R) in melanoma. Peptide 7a showed excellent radiolabeling yields and in vitro stability during amino acid challenge and serum stability assays. In vitro B16F10 melanoma cell uptake of 7a reached a modest value of 2.3 ± 0.08% of applied activity at 2 h at 37 °C, while this uptake was significantly reduced by coincubation with a nonlabeled α-MSH analogue, NAPamide (3.2 μM) (P < 0.05). In vivo SPECT/X-ray computed tomography (SPECT/CT) imaging and biodistribution of 7a were evaluated in a B16F10 melanoma xenografted mouse model. SPECT/CT imaging clearly visualized the tumor at 1 h post injection (p.i.) with high tumor-to-background contrast. Blocking studies with coinjected NAPamide (10 mg per kg of mouse body weight) confirmed the in vivo specificity of 7a for MC1R-positive tumors. Biodistribution results with 7a yielded a moderate tumor uptake of 1.20 ± 0.09 percentage of the injected radioactive dose per gram of tissue (% ID/g) at 1 h p.i. Relatively high uptake of 7a was also seen in the kidneys and liver at 1 h p.i. (6.55 ± 0.36% ID/g and 4.44 ± 0.17% ID/g, respectively), although reduced kidney uptake was seen at 4 h p.i. (3.20 ± 0.48% ID/g). These results demonstrate the utility of the novel [M(I)(CO)(3)](+) chelation strategy when applied in a targeting peptide.

Continuous Sensing of Tumor-targeted Molecular Probes with a Vertical Cavity Surface Emitting Laser-based Biosensor

ABSTRACT. Molecular optical imaging is a widespread technique for interrogating molecular events in living subjects. However, current approaches preclude long-term, continuous measurements in awake, mobile subjects, a strategy crucial in several medical conditions. Consequently, we designed a novel, lightweight miniature biosensor for in vivo continuous optical sensing. The biosensor contains an enclosed vertical-cavity surface-emitting semiconductor laser and an adjacent pair of near-infrared optically filtered detectors. We employed two sensors (dual sensing) to simultaneously interrogate normal and diseased tumor sites. Having established the sensors are precise with phantom and in vivo studies, we performed dual, continuous sensing in tumor (human glioblastoma cells) bearing mice using the targeted molecular probe cRGD-Cy5.5, which targets αVβ3 cell surface integrins in both tumor neovasculature and tumor. The sensors capture the dynamic time-activity curve of the targeted molecular probe. The average tumor to background ratio after signal calibration for cRGD-Cy5.5 injection is approximately 2.43±0.95 at 1 h and 3.64±1.38 at 2 h (N=5 mice), consistent with data obtained with a cooled charge coupled device camera. We conclude that our novel, portable, precise biosensor can be used to evaluate both kinetics and steady state levels of molecular probes in various disease applications.

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