Academic Appointments

Honors & Awards

  • Merck Fellow, Damon Runyon Cancer Research Fund (1999-2001)
  • Career Award at the Scientific Interface, Burroughs Wellcome (2002-2007)
  • Human Frontier Science Program Young Investigator, Human Frontier Science Program (2007-2010)

Professional Education

  • Ph.D., Harvard University, Chemistry (1999)

Current Research and Scholarly Interests

Probe chemistry and nanotechnology for molecular imaging and diagnostics

2014-15 Courses

Graduate and Fellowship Programs

Journal Articles

  • Bioorthogonal cyclization-mediated in situ self-assembly of small-molecule probes for imaging caspase activity in vivo NATURE CHEMISTRY Ye, D., Shuhendler, A. J., Cui, L., Tong, L., Tee, S. S., Tikhomirov, G., Felsher, D. W., Rao, J. 2014; 6 (6): 519-526


    Directed self-assembly of small molecules in living systems could enable a myriad of applications in biology and medicine, and already this has been used widely to synthesize supramolecules and nano/microstructures in solution and in living cells. However, controlling the self-assembly of synthetic small molecules in living animals is challenging because of the complex and dynamic in vivo physiological environment. Here we employ an optimized first-order bioorthogonal cyclization reaction to control the self-assembly of a fluorescent small molecule, and demonstrate its in vivo applicability by imaging caspase-3/7 activity in human tumour xenograft mouse models of chemotherapy. The fluorescent nanoparticles assembled in situ were imaged successfully in both apoptotic cells and tumour tissues using three-dimensional structured illumination microscopy. This strategy combines the advantages offered by small molecules with those of nanomaterials and should find widespread use for non-invasive imaging of enzyme activity in vivo.

    View details for DOI 10.1038/NCHEM.1920

    View details for Web of Science ID 000336897800014

  • Real-time imaging of oxidative and nitrosative stress in the liver of live animals for drug-toxicity testing. Nature biotechnology Shuhendler, A. J., Pu, K., Cui, L., Uetrecht, J. P., Rao, J. 2014; 32 (4): 373-380


    Current drug-safety assays for hepatotoxicity rely on biomarkers with low predictive power. The production of radical species, specifically reactive oxygen species (ROS) and reactive nitrogen species (RNS), has been proposed as an early unifying event linking the bioactivation of drugs to hepatotoxicity and as a more direct and mechanistic indicator of hepatotoxic potential. Here we present a nanosensor for rapid, real-time in vivo imaging of drug-induced ROS and RNS for direct evaluation of acute hepatotoxicity. By combining fluorescence resonance energy transfer (FRET) and chemiluminescence resonance energy transfer (CRET), our semiconducting polymer-based nanosensor simultaneously and differentially detects RNS and ROS using two optically independent channels. We imaged drug-induced hepatotoxicity and its remediation longitudinally in mice after systemic challenge with acetaminophen or isoniazid. We detected dose-dependent ROS and RNS activity in the liver within minutes of drug challenge, which preceded histological changes, protein nitration and DNA double-strand-break induction.

    View details for DOI 10.1038/nbt.2838

    View details for PubMedID 24658645

  • Semiconducting polymer nanoparticles as photoacoustic molecular imaging probes in living mice NATURE NANOTECHNOLOGY Pu, K., Shuhendler, A. J., Jokerst, J. V., Mei, J., Gambhir, S. S., Bao, Z., Rao, J. 2014; 9 (3): 233-239


    Photoacoustic imaging holds great promise for the visualization of physiology and pathology at the molecular level with deep tissue penetration and fine spatial resolution. To fully utilize this potential, photoacoustic molecular imaging probes have to be developed. Here, we introduce near-infrared light absorbing semiconducting polymer nanoparticles as a new class of contrast agents for photoacoustic molecular imaging. These nanoparticles can produce a stronger signal than the commonly used single-walled carbon nanotubes and gold nanorods on a per mass basis, permitting whole-body lymph-node photoacoustic mapping in living mice at a low systemic injection mass. Furthermore, the semiconducting polymer nanoparticles possess high structural flexibility, narrow photoacoustic spectral profiles and strong resistance to photodegradation and oxidation, enabling the development of the first near-infrared ratiometric photoacoustic probe for in vivo real-time imaging of reactive oxygen species-vital chemical mediators of many diseases. These results demonstrate semiconducting polymer nanoparticles to be an ideal nanoplatform for developing photoacoustic molecular probes.

    View details for DOI 10.1038/NNANO.2013.302

    View details for Web of Science ID 000332637200018

  • Self-luminescing BRET-FRET near-infrared dots for in vivo lymph-node mapping and tumour imaging NATURE COMMUNICATIONS Xiong, L., Shuhendler, A. J., Rao, J. 2012; 3


    Strong autofluorescence from living tissues, and the scattering and absorption of short-wavelength light in living tissues, significantly reduce sensitivity of in vivo fluorescence imaging. These issues can be tackled by using imaging probes that emit in the near-infrared wavelength range. Here we describe self-luminescing near-infrared-emitting nanoparticles employing an energy transfer relay that integrates bioluminescence resonance energy transfer and fluorescence resonance energy transfer, enabling in vivo near-infrared imaging without external light excitation. Nanoparticles were 30-40?nm in diameter, contained no toxic metals, exhibited long circulation time and high serum stability, and produced strong near-infrared emission. Using these nanoparticles, we successfully imaged lymphatic networks and vasculature of xenografted tumours in living mice. The self-luminescing feature provided excellent tumour-to-background ratio (>100) for imaging very small tumours (2-3?mm in diameter). Our results demonstrate that these new nanoparticles are well suited to in vivo imaging applications such as lymph-node mapping and cancer imaging.

    View details for DOI 10.1038/ncomms2197

    View details for Web of Science ID 000315992100028

    View details for PubMedID 23149738

  • Rapid point-of-care detection of the tuberculosis pathogen using a BlaC-specific fluorogenic probe NATURE CHEMISTRY Xie, H., Mire, J., Kong, Y., Chang, M., Hassounah, H. A., Thornton, C. N., Sacchettini, J. C., Cirillo, J. D., Rao, J. 2012; 4 (10): 802-809


    Early diagnosis of tuberculosis can dramatically reduce both its transmission and the associated death rate. The extremely slow growth rate of the causative pathogen, Mycobacterium tuberculosis (Mtb), however, makes this challenging at the point of care, particularly in resource-limited settings. Here we report the use of BlaC (an enzyme naturally expressed/secreted by tubercle bacilli) as a marker and the design of BlaC-specific fluorogenic substrates as probes for Mtb detection. These probes showed an enhancement by 100-200 times in fluorescence emission on BlaC activation and a greater than 1,000-fold selectivity for BlaC over TEM-1 ?-lactamase, an important factor in reducing false-positive diagnoses. Insight into the BlaC specificity was revealed by successful co-crystallization of the probe/enzyme mutant complex. A refined green fluorescent probe (CDG-OMe) enabled the successful detection of live pathogen in less than ten minutes, even in unprocessed human sputum. This system offers the opportunity for the rapid, accurate detection of very low numbers of Mtb for the clinical diagnosis of tuberculosis in sputum and other specimens.

    View details for DOI 10.1038/NCHEM.1435

    View details for Web of Science ID 000309154700012

    View details for PubMedID 23000993

  • Strategies for in vivo imaging of enzyme activity: an overview and recent advances CHEMICAL SOCIETY REVIEWS Razgulin, A., Ma, N., Rao, J. 2011; 40 (7): 4186-4216


    Imaging of enzyme activity in living subjects promises many applications in both basic and translational researches from helping elucidate the enzyme function and mechanism in biology to better disease detection and monitoring, but the complexity and dynamics of enzymatic reactions in living systems present unique challenges for probe design. This critical review examines the approaches in recent literature to in vivo imaging of the activity of a variety of enzyme targets with an emphasis on the chemical perspective of probe design, structure and function. Strategies for designing enzyme-activated probes based on a variety of molecular scaffolds including small molecules, organic and inorganic nanoparticles, and genetically encoded proteins for commonly used molecular imaging modalities--whole body optical (fluorescence, bioluminescence) imaging, magnetic resonance imaging, and radionuclide-based tomographic imaging, are critically evaluated. Recent advances in combining multiple modalities to imaging enzyme activity in living subjects are also highlighted (255 references).

    View details for DOI 10.1039/c1cs15035a

    View details for Web of Science ID 000291807600042

    View details for PubMedID 21552609

  • A biocompatible condensation reaction for controlled assembly of nanostructures in living cells Nature Chemistry Liang G, Ren H, Rao J 2010; 2 (1): 54-60
  • Self-illuminating quantum dot conjugates for in vivo imaging NATURE BIOTECHNOLOGY So, M. K., Xu, C. J., Loening, A. M., Gambhir, S. S., Rao, J. H. 2006; 24 (3): 339-343


    Fluorescent semiconductor quantum dots hold great potential for molecular imaging in vivo. However, the utility of existing quantum dots for in vivo imaging is limited because they require excitation from external illumination sources to fluoresce, which results in a strong autofluorescence background and a paucity of excitation light at nonsuperficial locations. Here we present quantum dot conjugates that luminesce by bioluminescence resonance energy transfer in the absence of external excitation. The conjugates are prepared by coupling carboxylate-presenting quantum dots to a mutant of the bioluminescent protein Renilla reniformis luciferase. We show that the conjugates emit long-wavelength (from red to near-infrared) bioluminescent light in cells and in animals, even in deep tissues, and are suitable for multiplexed in vivo imaging. Compared with existing quantum dots, self-illuminating quantum dot conjugates have greatly enhanced sensitivity in small animal imaging, with an in vivo signal-to-background ratio of > 10(3) for 5 pmol of conjugate.

    View details for DOI 10.1038/nbt1188

    View details for Web of Science ID 000235868600037

    View details for PubMedID 16501578

  • Magnetic resonance imaging of stem cell apoptosis in arthritic joints with a caspase activatable contrast agent. ACS nano Nejadnik, H., Ye, D., Lenkov, O. D., Donig, J. S., Martin, J. E., Castillo, R., Derugin, N., Sennino, B., Rao, J., Daldrup-Link, H. 2015; 9 (2): 1150-1160


    About 43 million individuals in the U.S. encounter cartilage injuries due to trauma or osteoarthritis, leading to joint pain and functional disability. Matrix-associated stem cell implants (MASI) represent a promising approach for repair of cartilage defects. However, limited survival of MASI creates a significant bottleneck for successful cartilage regeneration outcomes and functional reconstitution. We report an approach for noninvasive detection of stem cell apoptosis with magnetic resonance imaging (MRI), based on a caspase-3-sensitive nanoaggregation MRI probe (C-SNAM). C-SNAM self-assembles into nanoparticles after hydrolysis by caspase-3, leading to 90% amplification of (1)H MR signal and prolonged in vivo retention. Following intra-articular injection, C-SNAM causes significant MR signal enhancement in apoptotic MASI compared to viable MASI. Our results indicate that C-SNAM functions as an imaging probe for stem cell apoptosis in MASI. This concept could be applied to a broad range of cell transplants and target sites.

    View details for DOI 10.1021/nn504494c

    View details for PubMedID 25597243

  • Development of novel tumor-targeted theranostic nanoparticles activated by membrane-type matrix metalloproteinases for combined cancer magnetic resonance imaging and therapy. Small Ansari, C., Tikhomirov, G. A., Hong, S. H., Falconer, R. A., Loadman, P. M., Gill, J. H., Castaneda, R., Hazard, F. K., Tong, L., Lenkov, O. D., Felsher, D. W., Rao, J., Daldrup-Link, H. E. 2014; 10 (3): 566-?


    A major drawback with current cancer therapy is the prevalence of unrequired dose-limiting toxicity to non-cancerous tissues and organs, which is further compounded by a limited ability to rapidly and easily monitor drug delivery, pharmacodynamics and therapeutic response. In this report, the design and characterization of novel multifunctional "theranostic" nanoparticles (TNPs) is described for enzyme-specific drug activation at tumor sites and simultaneous in vivo magnetic resonance imaging (MRI) of drug delivery. TNPs are synthesized by conjugation of FDA-approved iron oxide nanoparticles ferumoxytol to an MMP-activatable peptide conjugate of azademethylcolchicine (ICT), creating CLIO-ICTs (TNPs). Significant cell death is observed in TNP-treated MMP-14 positive MMTV-PyMT breast cancer cells in vitro, but not MMP-14 negative fibroblasts or cells treated with ferumoxytol alone. Intravenous administration of TNPs to MMTV-PyMT tumor-bearing mice and subsequent MRI demonstrates significant tumor selective accumulation of the TNP, an observation confirmed by histopathology. Treatment with CLIO-ICTs induces a significant antitumor effect and tumor necrosis, a response not observed with ferumoxytol. Furthermore, no toxicity or cell death is observed in normal tissues following treatment with CLIO-ICTs, ICT, or ferumoxytol. These findings demonstrate proof of concept for a new nanotemplate that integrates tumor specificity, drug delivery and in vivo imaging into a single TNP entity through attachment of enzyme-activated prodrugs onto magnetic nanoparticles. This novel approach holds the potential to significantly improve targeted cancer therapies, and ultimately enable personalized therapy regimens.

    View details for DOI 10.1002/smll.201301456

    View details for PubMedID 24038954

  • Cancer therapy: development of novel tumor-targeted theranostic nanoparticles activated by membrane-type matrix metalloproteinases for combined cancer magnetic resonance imaging and therapy (small 3/2014). Small Ansari, C., Tikhomirov, G. A., Hong, S. H., Falconer, R. A., Loadman, P. M., Gill, J. H., Castaneda, R., Hazard, F. K., Tong, L., Lenkov, O. D., Felsher, D. W., Rao, J., Daldrup-Link, H. E. 2014; 10 (3): 417-?


    Cancer cells overexpress matrix-type metalloproteinases (MMPs, shown as pacmen). MMPs cleave the peptide linker connecting anticancer prodrug to the dextran coated magnetic nanoparticle. After the cleavage, the drug becomes toxic (active drug shown in purple). As J. Rao, H. E. Daldrup-Link, and co-workers describe on page 566, this tumor specific drug release reduces the side-effects of cancer therapy. The magnetic core of the nanoparticles allows for MRI monitoring of their distribution in the body.

    View details for DOI 10.1002/smll.201470016

    View details for PubMedID 24497471

  • Development of Novel Tumor-Targeted Theranostic Nanoparticles Activated by Membrane-Type Matrix Metalloproteinases for Combined Cancer Magnetic Resonance Imaging and Therapy SMALL Ansari, C., Tikhomirov, G. A., Hong, S. H., Falconer, R. A., Loadman, P. M., Gill, J. H., Castaneda, R., Hazard, F. K., Tong, L., Lenkov, O. D., Felsher, D. W., Rao, J., Daldrup-Link, H. E. 2014; 10 (3): 566-575
  • Caspase-responsive smart gadolinium-based contrast agent for magnetic resonance imaging of drug-induced apoptosis CHEMICAL SCIENCE Ye, D., Shuhendler, A. J., Pandit, P., Brewer, K. D., Tee, S. S., Cui, L., Tikhomirov, G., Rutt, B., Rao, J. 2014; 5 (10): 3845-3852

    View details for DOI 10.1039/c4sc01392a

    View details for Web of Science ID 000341195100020

  • Iron Administration before Stem Cell Harvest Enables MR Imaging Tracking after Transplantation RADIOLOGY Khurana, A., Chapelin, F., Beck, G., Lenkov, O. D., Donig, J., Nejadnik, H., Messing, S., Derugin, N., Chan, R. C., Gaur, A., Sennino, B., McDonald, D. M., Kempen, P. J., Tikhomirov, G. A., Rao, J., Daldrup-Link, H. E. 2013; 269 (1): 186-197


    Purpose:To determine whether intravenous ferumoxytol can be used to effectively label mesenchymal stem cells (MSCs) in vivo and can be used for tracking of stem cell transplants.Materials and Methods:This study was approved by the institutional animal care and use committee. Sprague-Dawley rats (6-8 weeks old) were injected with ferumoxytol 48 hours prior to extraction of MSCs from bone marrow. Ferumoxytol uptake by these MSCs was evaluated with fluorescence, confocal, and electron microscopy and compared with results of traditional ex vivo-labeling procedures. The in vivo-labeled cells were subsequently transplanted in osteochondral defects of 14 knees of seven athymic rats and were evaluated with magnetic resonance (MR) imaging up to 4 weeks after transplantation. T2 relaxation times of in vivo-labeled MSC transplants and unlabeled control transplants were compared by using t tests. MR data were correlated with histopathologic results.Results:In vivo-labeled MSCs demonstrated significantly higher ferumoxytol uptake compared with ex vivo-labeled cells. With electron microscopy, iron oxide nanoparticles were localized in secondary lysosomes. In vivo-labeled cells demonstrated significant T2 shortening effects in vitro and in vivo when they were compared with unlabeled control cells (T2 in vivo, 15.4 vs 24.4 msec; P < .05) and could be tracked in osteochondral defects for 4 weeks. Histologic examination confirmed the presence of iron in labeled transplants and defect remodeling.Conclusion:Intravenous ferumoxytol can be used to effectively label MSCs in vivo and can be used for tracking of stem cell transplants with MR imaging. This method eliminates risks of contamination and biologic alteration of MSCs associated with ex vivo-labeling procedures.© RSNA, 2013Supplemental material:

    View details for DOI 10.1148/radiol.13130858

    View details for Web of Science ID 000325000700021

  • Positron Emission Tomography Imaging of Drug-Induced Tumor Apoptosis with a Caspase-Triggered Nanoaggregation Probe ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Shen, B., Jeon, J., Palner, M., Ye, D., Shuhendler, A., Chin, F. T., Rao, J. 2013; 52 (40): 10511-10514


    Drug Design: An (18) F-labeled caspase-3-sensitive nanoaggregation positron emission tomography tracer was prepared and evaluated for imaging the caspase-3 activity in doxorubicin-treated tumor xenografts. Enhanced retention of the (18) F activity in apoptotic tumors is achieved through intramolecular macrocyclization and in situ aggregation upon caspase-3 activation.

    View details for DOI 10.1002/anie.201303422

    View details for Web of Science ID 000325091500023

  • Semiconducting Polymer Nanoprobe for In Vivo Imaging of Reactive Oxygen and Nitrogen Species ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Pu, K., Shuhendler, A. J., Rao, J. 2013; 52 (39): 10325-10329


    Semiconducting polymer nanoparticles are used as a free-radical inert and light-harvesting nanoplatform for in vivo molecular imaging of reactive oxygen and nitrogen species (RONS). This nanoprobe permits detection of RONS in the microenvironment of spontaneous bacterial infection (see picture; FRET=fluorescence resonance energy transfer).

    View details for DOI 10.1002/anie.201303420

    View details for Web of Science ID 000329141800033

    View details for PubMedID 23943508

  • Activatable Oligomerizable Imaging Agents for Photoacoustic Imaging of Furin-Like Activity in Living Subjects JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Dragulescu-Andrasi, A., Kothapalli, S., Tikhomirov, G. A., Rao, J., Gambhir, S. S. 2013; 135 (30): 11015-11022


    Photoacoustic (PA) imaging is continuing to be applied for physiological imaging and more recently for molecular imaging of living subjects. Owing to its high spatial resolution in deep tissues, PA imaging holds great potential for biomedical applications and molecular diagnostics. There is however a lack of probes for targeted PA imaging, especially in the area of enzyme-activatable probes. Here we introduce a molecular probe, which upon proteolytic processing is retained at the site of enzyme activity and provides PA contrast. The probe oligomerizes via a condensation reaction and accumulates in cells and tumors that express the protease. We demonstrate that this probe reports furin and furin-like activity in cells and tumor models by generating a significantly higher PA signal relative to furin-deficient and nontarget controls. This probe could report enzyme activity in living subjects at depths significantly greater than fluorescence imaging probes and has potential for molecular imaging in deep tumors.

    View details for DOI 10.1021/ja4010078

    View details for Web of Science ID 000322752900032

    View details for PubMedID 23859847

  • Synthesis of ligand-functionalized water-soluble [F-18]YF3 nanoparticles for PET imaging NANOSCALE Xiong, L., Shen, B., Behera, D., Gambhir, S. S., Chin, F. T., Rao, J. 2013; 5 (8): 3253-3256


    We report a simple, efficient synthesis of novel (18)F-labeled imaging agents based on YF3 nanoparticles. Targeting ligands and antitumor drug molecules can be introduced onto the YF3 nanoparticles in a one-pot synthesis. The (18)F-labeling reaction proceeds in aqueous solutions at room temperature with excellent radiolabeling yields (>80%) in a very short time (5-10 min). (18)F-labeled YF3 nanoparticles displayed high stability in mouse and human serum, and their application for mapping lymph nodes in live rats after local injection has also been demonstrated.

    View details for DOI 10.1039/c3nr00335c

    View details for Web of Science ID 000316959500019

    View details for PubMedID 23508229

  • Enzymatic activation of nitro-aryl fluorogens in live bacterial cells for enzymatic turnover-activated localization microscopy CHEMICAL SCIENCE Lee, M. K., Williams, J., Twieg, R. J., Rao, J., Moerner, W. E. 2013; 4 (1): 220-225

    View details for DOI 10.1039/c2sc21074f

    View details for Web of Science ID 000311971500023

  • A strategy to enhance the binding affinity of fluorophore-aptamer pairs for RNA tagging with neomycin conjugation CHEMICAL COMMUNICATIONS Jeon, J., Leez, K. H., Rao, J. 2012; 48 (80): 10034-10036


    Fluorogenic sulforhodamine-neomycin conjugates have been designed and synthesized for RNA tagging. Conjugates were fluorescently activated by binding to RNA aptamers and exhibited greater than 250-400 fold enhancement in binding affinity relative to corresponding unconjugated fluorophores.

    View details for DOI 10.1039/c2cc34498j

    View details for Web of Science ID 000308653800025

    View details for PubMedID 22951899

  • A Selenium Analogue of Firefly D-Luciferin with Red-Shifted Bioluminescence Emission ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Conley, N. R., Dragulescu-Andrasi, A., Rao, J., Moerner, W. E. 2012; 51 (14): 3350-3353


    A selenium analogue of amino-D-luciferin, aminoseleno-D-luciferin, is synthesized and shown to be a competent substrate for the firefly luciferase enzyme. It has a red-shifted bioluminescence emission maximum at 600 nm and is suitable for bioluminescence imaging studies in living subjects.

    View details for DOI 10.1002/anie.201105653

    View details for Web of Science ID 000302059400009

    View details for PubMedID 22344705

  • Immobilizing Reporters for Molecular Imaging of the Extracellular Microenvironment in Living Animals ACS CHEMICAL BIOLOGY Xia, Z., Xing, Y., Jeon, J., Kim, Y., Gall, J., Dragulescu-Andrasi, A., Gambhir, S. S., Rao, J. 2011; 6 (10): 1117-1126


    We report here an immobilization strategy using a collagen binding protein to deliver and confine synthetic reporters to the extracellular microenvironment in vivo for noninvasively imaging the activity of targets in the microenvironment. We show that the immobilization of reporters on collagens in the local microenvironment is highly efficient and physiologically stable for repetitive, long-term imaging. By using this strategy we successfully developed an immobilized bioluminescent activatable reporter and a dual-modality reporter to map and quantitatively image the activity of extracellular matrix metalloproteinases (MMP) in tumor-bearing mice. The inhibition of MMP activity by chemical inhibitor was also demonstrated in living subjects. We further demonstrated the general applicability of this immobilization strategy by imaging MMP activity at the inflammation site in a mouse model. Our results show that the in vivo immobilization of reporters can be used as a general strategy for probing the local extracellular microenvironment.

    View details for DOI 10.1021/cb200135e

    View details for Web of Science ID 000296208100018

    View details for PubMedID 21830814

  • MRI of Tumor-Associated Macrophages with Clinically Applicable Iron Oxide Nanoparticles CLINICAL CANCER RESEARCH Daldrup-Link, H. E., Golovko, D., Ruffell, B., DeNardo, D. G., Castaneda, R., Ansari, C., Rao, J., Tikhomirov, G. A., Wendland, M. F., Corot, C., Coussens, L. M. 2011; 17 (17): 5695-5704


    The presence of tumor-associated macrophages (TAM) in breast cancer correlates strongly with poor outcome. The purpose of this study was to develop a clinically applicable, noninvasive diagnostic assay for selective targeting and visualization of TAMs in breast cancer, based on magnetic resonanceI and clinically applicable iron oxide nanoparticles.F4/80-negative mammary carcinoma cells and F4/80-positive TAMs were incubated with iron oxide nanoparticles and were compared with respect to magnetic resonance signal changes and iron uptake. MMTV-PyMT transgenic mice harboring mammary carcinomas underwent nanoparticle-enhanced magnetic resonance imaging (MRI) up to 1 hour and 24 hours after injection. The tumor enhancement on MRIs was correlated with the presence and location of TAMs and nanoparticles by confocal microscopy.In vitro studies revealed that iron oxide nanoparticles are preferentially phagocytosed by TAMs but not by malignant tumor cells. In vivo, all tumors showed an initial contrast agent perfusion on immediate postcontrast MRIs with gradual transendothelial leakage into the tumor interstitium. Twenty-four hours after injection, all tumors showed a persistent signal decline on MRIs. TAM depletion via ?CSF1 monoclonal antibodies led to significant inhibition of tumor nanoparticle enhancement. Detection of iron using 3,3'-diaminobenzidine-enhanced Prussian Blue staining, combined with immunodetection of CD68, localized iron oxide nanoparticles to TAMs, showing that the signal effects on delayed MRIs were largely due to TAM-mediated uptake of contrast agent.These data indicate that tumor enhancement with clinically applicable iron oxide nanoparticles may serve as a new biomarker for long-term prognosis, related treatment decisions, and the evaluation of new immune-targeted therapies.

    View details for DOI 10.1158/1078-0432.CCR-10-3420

    View details for Web of Science ID 000294477600020

    View details for PubMedID 21791632

  • Real-Time Imaging of Rab5 Activity Using a Prequenched Biosensor ACS CHEMICAL BIOLOGY Zhan, K., Xie, H., Gall, J., Ma, M., Griesbeck, O., Salehi, A., Rao, J. 2011; 6 (7): 692-699


    A key regulator of receptor-mediated endocytosis, Rab5, plays a pivotal role in cargo receptor internalization, endosomal maturation, and transduction and degradation of internalized signaling molecules and recycling cargo receptor. Stressful conditions within cells lead to increased Rab5 activation, and increasing evidence correlates Rab5 activity abnormalities with certain diseases. Current antibody-based imaging methods cannot distinguish active Rab5 from total Rab5 population and provide dynamic information on magnitude and duration of Rab5 activation in cellular events and pathogenesis. We report here novel molecular imaging probes that specifically target GTP-bound Rab5 associated with the early endosome membrane in live cells and fixed mouse brain tissues. Our Rab5 activity fluorescent biosensor (RAFB) contains the Rab5 binding domain of the Rab5 effector Rabaptin 5, a fluorophore (a quantum dot or fluorescent dye) and a cell-penetrating peptide for live-cell delivery. The quantum dot conjugated RAFB was able to image the elevated Rab5 activity in both the cortex and hippocampi tissues of a Ts65Dn mouse. A prequenched RAFB based on fluorescence resonance energy transfer (FRET) can image cytosolic active Rab5 in single live cells. This novel method should enable imaging of the biological process in which Rab5 activity is regulated in various cellular systems.

    View details for DOI 10.1021/cb100377m

    View details for Web of Science ID 000292850900004

    View details for PubMedID 21506516

  • Controlling Intracellular Macrocyclization for the Imaging of Protease Activity ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Ye, D., Liang, G., Ma, M. L., Rao, J. 2011; 50 (10): 2275-2279

    View details for DOI 10.1002/anie.201006140

    View details for Web of Science ID 000288036300011

    View details for PubMedID 21351335

  • Controlled Self-Assembling of Gadolinium Nanoparticles as Smart Molecular Magnetic Resonance Imaging Contrast Agents ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Liang, G., Ronald, J., Chen, Y., Ye, D., Pandit, P., Ma, M. L., Rutt, B., Rao, J. 2011; 50 (28): 6283-6286

    View details for DOI 10.1002/anie.201007018

    View details for Web of Science ID 000292642600012

    View details for PubMedID 21618367

  • Superresolution Imaging of Targeted Proteins in Fixed and Living Cells Using Photoactivatable Organic Fluorophores JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Lee, H. D., Lord, S. J., Iwanaga, S., Zhan, K., Xie, H., Williams, J. C., Wang, H., Bowman, G. R., Goley, E. D., Shapiro, L., Twieg, R. J., Rao, J., Moerner, W. E. 2010; 132 (43): 15099-15101


    Superresolution imaging techniques based on sequential imaging of sparse subsets of single molecules require fluorophores whose emission can be photoactivated or photoswitched. Because typical organic fluorophores can emit significantly more photons than average fluorescent proteins, organic fluorophores have a potential advantage in super-resolution imaging schemes, but targeting to specific cellular proteins must be provided. We report the design and application of HaloTag-based target-specific azido DCDHFs, a class of photoactivatable push-pull fluorogens which produce bright fluorescent labels suitable for single-molecule superresolution imaging in live bacterial and fixed mammalian cells.

    View details for DOI 10.1021/ja1044192

    View details for Web of Science ID 000283621700003

    View details for PubMedID 20936809

  • Combining SELEX Screening and Rational Design to Develop Light-Up Fluorophore-RNA Aptamer Pairs for RNA Tagging ACS CHEMICAL BIOLOGY Lee, J., Lee, K. H., Jeon, J., Dragulescu-Andrasi, A., Xiao, F., Rao, J. 2010; 5 (11): 1065-1074


    We report here a new small molecule fluorogen and RNA aptamer pair for RNA labeling. The small-molecule fluorogen is designed on the basis of fluorescently quenched sulforhodamine dye. The SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure and fluorescence screening in E. coli have been applied to discover the aptamer that can specifically activate the fluorogen with micromolar binding affinity. The systematic mutation and truncation study on the aptamer structure determined the minimum binding domain of the aptamer. A series of rationally modified fluorogen analogues have been made to probe the interacting groups of fluorogen with the aptamer. These results led to the design of a much improved fluorogen ASR 7 that displayed a 33-fold increase in the binding affinity for the selected aptamer in comparison to the original ASR 1 and an 88-fold increase in the fluorescence emission after the aptamer binding. This study demonstrates the value of combining in vitro SELEX and E. coli fluorescence screening with rational modifications in discovering and optimizing new fluorogen-RNA aptamer labeling pairs.

    View details for DOI 10.1021/cb1001894

    View details for Web of Science ID 000284437800009

    View details for PubMedID 20809562

  • Facile Synthesis, Silanization, and Biodistribution of Biocompatible Quantum Dots SMALL Ma, N., Marshall, A. F., Gambhir, S. S., Rao, J. 2010; 6 (14): 1520-1528


    A facile strategy for the synthesis of silica-coated quantum dots (QDs) for in vivo imaging is reported. All the QD synthesis and silanization steps are conducted in water and methanol under mild conditions without involving any organometallic precursors or high-temperature, oxygen-free environments. The as-prepared silica-coated QDs possess high quantum yields and are extremely stable in mouse serum. In addition, the silanization method developed here produces nanoparticles with small sizes that are difficult to achieve via conventional silanization methods. The silica coating helps to prevent the exposure of the QD surface to the biological milieu and therefore increases the biocompatibility of QDs for in vivo applications. Interestingly, the silica-coated QDs exhibit a different biodistribution pattern from that of commercially available Invitrogen QD605 (carboxylate) with a similar size and emission wavelength. The Invitrogen QD605 exhibits predominant liver (57.2% injected dose (ID) g(-1)) and spleen (46.1% ID g(-1)) uptakes 30 min after intravenous injection, whereas the silica-coated QDs exhibit much lower liver (16.2% ID g(-1)) and spleen (3.67% ID g(-1)) uptakes but higher kidney uptake (8.82% ID g(-1)), blood retention (15.0% ID g(-1)), and partial renal clearance. Overall, this straightforward synthetic strategy paves the way for routine and customized synthesis of silica-coated QDs for biological use.

    View details for DOI 10.1002/smll.200902409

    View details for Web of Science ID 000280633900011

    View details for PubMedID 20564726

  • Imaging tuberculosis with endogenous beta-lactamase reporter enzyme fluorescence in live mice PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kong, Y., Yao, H., Ren, H., Subbian, S., Cirillo, S. L., Sacchettini, J. C., Rao, J., Cirillo, J. D. 2010; 107 (27): 12239-12244


    The slow growth rate and genetic intractability of tubercle bacilli has hindered progress toward understanding tuberculosis, one of the most frequent causes of death worldwide. We overcame this roadblock through development of near-infrared (NIR) fluorogenic substrates for beta-lactamase, an enzyme expressed by tubercle bacilli, but not by their eukaryotic hosts, to allow real-time imaging of pulmonary infections and rapid quantification of bacteria in living animals by a strategy called reporter enzyme fluorescence (REF). This strategy has a detection limit of 6 +/- 2 x 10(2) colony-forming units (CFU) of bacteria with the NIR substrate CNIR5 in only 24 h of incubation in vitro, and as few as 10(4) CFU in the lungs of live mice. REF can also be used to differentiate infected from uninfected macrophages by using confocal microscopy and fluorescence activated cell sorting. Mycobacterium tuberculosis and the bacillus Calmette-Guérin can be tracked directly in the lungs of living mice without sacrificing the animals. Therapeutic efficacy can also be evaluated through loss of REF signal within 24 h posttreatment by using in vitro whole-bacteria assays directly in living mice. We expect that rapid quantification of bacteria within tissues of a living host and in the laboratory is potentially transformative for tuberculosis virulence studies, evaluation of therapeutics, and efficacy of vaccine candidates. This is a unique use of an endogenous bacterial enzyme probe to detect and image tubercle bacilli that demonstrates REF is likely to be useful for the study of many bacterial infections.

    View details for DOI 10.1073/pnas.1000643107

    View details for Web of Science ID 000279572100037

    View details for PubMedID 20566877

  • Near-Infrared Light Emitting Luciferase via Biomineralization JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Ma, N., Marshall, A. F., Rao, J. 2010; 132 (20): 6884-?


    A new strategy based on biomineralization is presented to rationally tune the emission wavelength of luciferase. In this study luciferase (Luc8) was used as a template to direct the synthesis of near-infrared (NIR) light emitting PbS quantum dots (QDs) at ambient conditions to form a Luc8-PbS nanocomplex. The as-synthesized PbS QDs exhibited photoluminescence in the range of 800-1050 nm, and the Luc8 enzyme remained active within the Luc8-PbS complex. Upon the addition of the substrate coelenterazine, the energy produced by Luc8 was nonradiatively transferred to PbS QDs via bioluminescence resonance energy transfer (BRET) and enabled the complex to emit NIR light. This is the first study to form dual functional bioinorganic hybrid nanostructures via active enzyme-templated synthesis of inorganic nanomaterials.

    View details for DOI 10.1021/ja101378g

    View details for Web of Science ID 000277999700009

    View details for PubMedID 20441172

  • Bioluminescent nanosensors for protease detection based upon gold nanoparticle-luciferase conjugates CHEMICAL COMMUNICATIONS Kim, Y., Daniel, W. L., Xia, Z., Xie, H., Mirkin, C. A., Rao, J. 2010; 46 (1): 76-78


    This communication reports the use of click chemistry to site-specifically conjugate bioluminescent Renilla luciferase proteins to gold nanoparticles (Au NPs) for sensing protease activity. The bioluminescent emission from luciferase was efficiently quenched by Au NPs, but significantly recovered after the proteolytic cleavage.

    View details for DOI 10.1039/b915612g

    View details for Web of Science ID 000272679200009

    View details for PubMedID 20024298

  • In Vivo Bioluminescence Imaging of Furin Activity in Breast Cancer Cells Using Bioluminogenic Substrates BIOCONJUGATE CHEMISTRY Dragulescu-Andrasi, A., Liang, G., Rao, J. 2009; 20 (8): 1660-1666


    Furin, a proprotein convertases family endoprotease, processes numerous physiological substrates and is overexpressed in cancer and inflammatory conditions. Noninvasive imaging of furin activity will offer a valuable tool to probe furin function over the course of tumor growth and migration in the same animals in real time and directly assess the inhibition efficacy of drugs in vivo. Here, we report successful bioluminescence imaging of furin activity in xenografted MBA-MB-468 breast cancer tumors in mice with bioluminogenic probes. The probes are conjugates of furin substrate, a consensus amino acid motif R-X-K/R-R (X, any amino acid), with the firefly luciferase substrate D-aminoluciferin. In the presence of the luciferase reporter, the probes are unable to produce bioluminescent emission without furin activation. Blocking experiments with a furin inhibitor and control experiments with a scrambled probe showed that the bioluminescence emission in the presence of firefly luciferase is furin-dependent and specific. After furin activation, a 30-fold increase in the bioluminescent emission was observed in vitro, and on average, a 7-8-fold contrast between the probe and control was seen in the same tumor xenografts in mice. Direct imaging of furin activity may facilitate the study of furin function in tumorigenicity and the discovery of new drugs for furin-targeted cancer therapy.

    View details for DOI 10.1021/bc9002508

    View details for Web of Science ID 000269042100029

    View details for PubMedID 19642690

  • Semiconductor Quantum Dots for Biosensing and In Vivo Imaging IEEE TRANSACTIONS ON NANOBIOSCIENCE Xing, Y., Xia, Z., Rao, J. 2009; 8 (1): 4-12


    Semiconductor quantum dots (QDs) have captivated researchers in the biomedical field over the last decade. Compared to organic dyes and fluorescent proteins, QDs have unique optical properties such as tunable emission spectra, improved brightness, superior photostability, and simultaneous excitation of multiple fluorescence colors. Since the first successful reports on the biological use of QDs a decade ago, QDs and their bioconjugates have been successfully applied to various imaging applications including fixed cell labeling, live-cell imaging, in situ tissue profiling, fluorescence detection and sensing, and in vivo animal imaging. In this review, we will briefly survey the optical properties of QDs, the biofunctionalization strategies, and focus on their biosensing and in vivo imaging applications. We conclude with a discussion on the issues and perspectives on QDs as biosensing probes and in vivo imaging agents.

    View details for DOI 10.1109/TNB.2009.2017321

    View details for Web of Science ID 000268040200002

    View details for PubMedID 19304495

  • Biosensing and imaging based on bioluminescence resonance energy transfer CURRENT OPINION IN BIOTECHNOLOGY Xia, Z., Rao, J. 2009; 20 (1): 37-44


    Bioluminescence resonance energy transfer (BRET) operates with biochemical energy generated by bioluminescent proteins to excite fluorophores and offers additional advantages over fluorescence energy transfer (FRET) for in vivo imaging and biosensing. While fluorescent proteins are frequently used as BRET acceptors, both small molecule dyes and nanoparticles can also serve as acceptor fluorophores. Semiconductor fluorescent nanocrystals or quantum dots (QDs) are particularly well suited for use as BRET acceptors due to their high quantum yields, large Stokes shifts and long wavelength emission. This review examines the potential of QDs for BRET-based bioassays and imaging, and highlights examples of QD-BRET for biosensing and imaging applications. Future development of new BRET acceptors should further expand the multiplexing capability of BRET and improve its applicability and sensitivity for in vivo imaging applications.

    View details for DOI 10.1016/j.copbio.2009.01.001

    View details for Web of Science ID 000266535200006

    View details for PubMedID 19216068

  • CNOB/ChrR6, a new prodrug enzyme cancer chemotherapy MOLECULAR CANCER THERAPEUTICS Thorne, S. H., Barak, Y., Liang, W., Bachmann, M. H., Rao, J., Contag, C. H., Matin, A. 2009; 8 (2): 333-341


    We report the discovery of a new prodrug, 6-chloro-9-nitro-5-oxo-5H-benzo(a)phenoxazine (CNOB). This prodrug is efficiently activated by ChrR6, the highly active prodrug activating bacterial enzyme we have previously developed. The CNOB/ChrR6 therapy was effective in killing several cancer cell lines in vitro. It also efficiently treated tumors in mice with up to 40% complete remission. 9-Amino-6-chloro-5H-benzo(a)phenoxazine-5-one (MCHB) was the only product of CNOB reduction by ChrR6. MCHB binds DNA; at nonlethal concentration, it causes cell accumulation in the S phase, and at lethal dose, it induces cell surface Annexin V and caspase-3 and caspase-9 activities. Further, MCHB colocalizes with mitochondria and disrupts their electrochemical potential. Thus, killing by CNOB involves MCHB, which likely induces apoptosis through the mitochondrial pathway. An attractive feature of the CNOB/ChrR6 regimen is that its toxic product, MCHB, is fluorescent. This feature proved helpful in in vitro studies because simple fluorescence measurements provided information on the kinetics of CNOB activation within the cells, MCHB killing mechanism, its generally efficient bystander effect in cells and cell spheroids, and its biodistribution. The emission wavelength of MCHB also permitted its visualization in live animals, allowing noninvasive qualitative imaging of MCHB in mice and the tumor microenvironment. This feature may simplify exploration of barriers to the penetration of MCHB in tumors and their amelioration.

    View details for DOI 10.1158/1535-7163.MCT-08-0707

    View details for Web of Science ID 000263397300008

    View details for PubMedID 19190118

  • Particle Size, Surface Coating, and PEGylation Influence the Biodistribution of Quantum Dots in Living Mice SMALL Schipper, M. L., Iyer, G., Koh, A. L., Cheng, Z., Ebenstein, Y., Aharoni, A., Keren, S., Bentolila, L. A., Li, J., Rao, J., Chen, X., Banin, U., Wu, A. M., Sinclair, R., Weiss, S., Gambhir, S. S. 2009; 5 (1): 126-134


    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.

    View details for DOI 10.1002/smll.200800003

    View details for Web of Science ID 000262895300019

    View details for PubMedID 19051182

  • A Biocompatible Condensation Reaction for the Labeling of Terminal Cysteine Residues on Proteins ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Ren, H., Xiao, F., Zhan, K., Kim, Y., Xie, H., Xia, Z., Rao, J. 2009; 48 (51): 9658-9662

    View details for DOI 10.1002/anie.200903627

    View details for Web of Science ID 000273093700011

    View details for PubMedID 19924746

  • Imaging Target mRNA and siRNA-Mediated Gene Silencing In Vivo with Ribozyme-Based Reporters CHEMBIOCHEM So, M., Gowrishankar, G., Hasegawa, S., Chung, J., Rao, J. 2008; 9 (16): 2682-2691


    Noninvasive imaging of specific mRNAs in living subjects promises numerous biological and medical applications. Common strategies use fluorescently or radioactively labelled antisense probes to detect target mRNAs through a hybridization mechanism, but have met with limited success in living animals. Here we present a novel molecular imaging approach based on the group I intron of Tetrahymena thermophila for imaging mRNA molecules in vivo. Engineered trans-splicing ribozyme reporters contain three domains, each of which is designed for targeting, splicing, and reporting. They can transduce the target mRNA into a reporter mRNA, leading to the production of reporter enzymes that can be noninvasively imaged in vivo. We have demonstrated this ribozyme-mediated RNA imaging method for imaging a mutant p53 mRNA both in single cells and noninvasively in living mice. After optimization, the ribozyme reporter increases contrast for the transiently expressed target by 180-fold, and by ten-fold for the stably expressed target. siRNA-mediated specific gene silencing of p53 expression has been successfully imaged in real time in vivo. This new ribozyme-based RNA reporter system should open up new avenues for in vivo RNA imaging and direct imaging of siRNA inhibition.

    View details for DOI 10.1002/cbic.200800370

    View details for Web of Science ID 000261001900019

    View details for PubMedID 18972511

  • Shedding Light on Tumors Using Nanoparticles ACS NANO Rao, J. 2008; 2 (10): 1984-1986


    The scaffold of nanoparticles (broadly defined as having a size range of 1-100 nm) presents a convenient platform to incorporate multiple functionalities into one single particle for cancer imaging and therapeutics. Whether hollow inside or not, a single nanoparticle can encapsulate a large payload of imaging probes, anticancer drug molecules, or both. On the surface, tumor-specific targeting molecules (e.g., receptor-binding ligands or antibodies) may be immobilized to facilitate active tumor targeting and drug delivery. This versatile nanoplatform promises more efficient delivery of payloads to tumors for improving cancer detection and treatment.

    View details for DOI 10.1021/nn800669n

    View details for Web of Science ID 000260503100003

    View details for PubMedID 19206441

  • HaloTag protein-mediated specific labeling of living cells with quantum dots BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS So, M., Yao, H., Rao, J. 2008; 374 (3): 419-423


    Quantum dots emerge as an attractive alternative to small molecule fluorophores as fluorescent tags for in vivo cell labeling and imaging. This communication presents a method for specific labeling of live cells using quantum dots. The labeling is mediated by HaloTag protein expressed at the cell surface which forms a stable covalent adduct with its ligand (HaloTag ligand). The labeling can be performed in one single step with quantum dot conjugates that are functionalized with HaloTag ligand, or in two steps with biotinylated HaloTag ligand first and followed by streptavidin coated quantum dots. Live cell fluorescence imaging indicates that the labeling is specific and takes place at the cell surface. This HaloTag protein-mediated cell labeling method should facilitate the application of quantum dots for live cell imaging.

    View details for DOI 10.1016/j.bbrc.2008.07.004

    View details for Web of Science ID 000259108800004

    View details for PubMedID 18621022

  • Improved QD-BRET conjugates for detection and imaging BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Xing, Y., So, M., Koh, A. L., Sinclair, R., Rao, J. 2008; 372 (3): 388-394


    Self-illuminating quantum dots, also known as QD-BRET conjugates, are a new class of quantum dot bioconjugates which do not need external light for excitation. Instead, light emission relies on the bioluminescence resonance energy transfer from the attached Renilla luciferase enzyme, which emits light upon the oxidation of its substrate. QD-BRET combines the advantages of the QDs (such as superior brightness and photostability, tunable emission, multiplexing) as well as the high sensitivity of bioluminescence imaging, thus holding the promise for improved deep tissue in vivo imaging. Although studies have demonstrated the superior sensitivity and deep tissue imaging potential, the stability of the QD-BRET conjugates in biological environment needs to be improved for long-term imaging studies such as in vivo cell tracking. In this study, we seek to improve the stability of QD-BRET probes through polymeric encapsulation with a polyacrylamide gel. Results show that encapsulation caused some activity loss, but significantly improved both the in vitro serum stability and in vivo stability when subcutaneously injected into the animal. Stable QD-BRET probes should further facilitate their applications for both in vitro testing as well as in vivo cell tracking studies.

    View details for DOI 10.1016/j.bbrc.2008.04.159

    View details for Web of Science ID 000256941300002

    View details for PubMedID 18468518

  • Quantum dot bioconjugates for in vitro diagnostics & in vivo imaging CANCER BIOMARKERS Xing, Y., Rao, J. 2008; 4 (6): 307-319


    Semiconductor quantum dots are tiny light-emitting nanocrystals (2-10 nm) that have captivated researchers in the biomedical field in the last decade. Compared to organic dyes and fluorescent proteins, quantum dots (QDs) have unique optical properties such as tunable emission spectra, improved brightness, superior photostability, and simultaneous excitation of multiple fluorescence colors. Since the first successful reports on biological use of QDs a decade ago, QDs and their bioconjugates have been successfully applied in various imaging applications including fixed cell labeling, imaging of live cell dynamics, in situ tissue profiling, fluorescence detection, sensing and in vivo animal imaging. In this review, we will cover the optical properties of QDs, the biofunctionization strategies, their in vitro diagnostic applications and in vivo imaging applications. In addition, we will discuss the making of a new class of QDs--the self-illuminating QDs and their in vivo imaging and sensing applications. We will conclude with the issues and perspectives on QDs as in vivo imaging probes.

    View details for Web of Science ID 000262451900003

    View details for PubMedID 19126959

  • Quantum dot imaging for embryonic stem cells BMC BIOTECHNOLOGY Lin, S., Xie, X., Patel, M. R., Yang, Y., Li, Z., Cao, F., Gheysens, O., Zhang, Y., Gambhir, S. S., Rao, J. H., Wu, J. C. 2007; 7


    Semiconductor quantum dots (QDs) hold increasing potential for cellular imaging both in vitro and in vivo. In this report, we aimed to evaluate in vivo multiplex imaging of mouse embryonic stem (ES) cells labeled with Qtracker delivered quantum dots (QDs).Murine embryonic stem (ES) cells were labeled with six different QDs using Qtracker. ES cell viability, proliferation, and differentiation were not adversely affected by QDs compared with non-labeled control cells (P = NS). Afterward, labeled ES cells were injected subcutaneously onto the backs of athymic nude mice. These labeled ES cells could be imaged with good contrast with one single excitation wavelength. With the same excitation wavelength, the signal intensity, defined as (total signal-background)/exposure time in millisecond was 11 +/- 2 for cells labeled with QD 525, 12 +/- 9 for QD 565, 176 +/- 81 for QD 605, 176 +/- 136 for QD 655, 167 +/- 104 for QD 705, and 1,713 +/- 482 for QD 800. Finally, we have shown that QD 800 offers greater fluorescent intensity than the other QDs tested.In summary, this is the first demonstration of in vivo multiplex imaging of mouse ES cells labeled QDs. Upon further improvements, QDs will have a greater potential for tracking stem cells within deep tissues. These results provide a promising tool for imaging stem cell therapy non-invasively in vivo.

    View details for DOI 10.1186/1472-6750-7-67

    View details for Web of Science ID 000252448600001

    View details for PubMedID 17925032

  • MicroPET-based biodistribution of quantum dots in living mice JOURNAL OF NUCLEAR MEDICINE Schipper, M. L., Cheng, Z., Lee, S., Bentolila, L. A., Iyer, G., Rao, J., Chen, X., Wu, A. M., Weiss, S., Gambhir, S. S. 2007; 48 (9): 1511-1518


    This study evaluates the quantitative biodistribution of commercially available CdSe quantum dots (QD) in mice.(64)Cu-Labeled 800- or 525-nm emission wavelength QD (21- or 12-nm diameter), with or without 2,000 MW (molecular weight) polyethylene glycol (PEG), were injected intravenously into mice (5.55 MBq/25 pmol QD) and studied using well counting or by serial microPET and region-of-interest analysis.Both methods show rapid uptake by the liver (27.4-38.9 %ID/g) (%ID/g is percentage injected dose per gram tissue) and spleen (8.0-12.4 %ID/g). Size has no influence on biodistribution within the range tested here. Pegylated QD have slightly slower uptake into liver and spleen (6 vs. 2 min) and show additional low-level bone uptake (6.5-6.9 %ID/g). No evidence of clearance from these organs was observed.Rapid reticuloendothelial system clearance of QD will require modification of QD for optimal utility in imaging living subjects. Formal quantitative biodistribution/imaging studies will be helpful in studying many types of nanoparticles, including quantum dots.

    View details for DOI 10.2967/jnumed.107.040071

    View details for Web of Science ID 000252894700039

    View details for PubMedID 17704240

  • Chemical labeling of protein in living cells CHEMBIOCHEM Dragulescu-Andrasi, A., Rao, J. 2007; 8 (10): 1099-1101

    View details for DOI 10.1002/cbic.200700158

    View details for Web of Science ID 000248067100002

    View details for PubMedID 17492742

  • Fluorescence imaging in vivo: recent advances CURRENT OPINION IN BIOTECHNOLOGY Rao, J., Dragulescu-Andrasi, A., Yao, H., Yao, H. 2007; 18 (1): 17-25


    In vivo fluorescence imaging uses a sensitive camera to detect fluorescence emission from fluorophores in whole-body living small animals. To overcome the photon attenuation in living tissue, fluorophores with long emission at the near-infrared (NIR) region are generally preferred, including widely used small indocarbocyanine dyes. The list of NIR probes continues to grow with the recent addition of fluorescent organic, inorganic and biological nanoparticles. Recent advances in imaging strategies and reporter techniques for in vivo fluorescence imaging include novel approaches to improve the specificity and affinity of the probes and to modulate and amplify the signal at target sites for enhanced sensitivity. Further emerging developments are aiming to achieve high-resolution, multimodality and lifetime-based in vivo fluorescence imaging.

    View details for DOI 10.1016/j.copbio.2007.01.003

    View details for Web of Science ID 000244593000004

    View details for PubMedID 17234399

  • Visualizing RNA splicing in vivo MOLECULAR BIOSYSTEMS Gowrishankar, G., Rao, J. 2007; 3 (5): 301-307


    Ribozymes are RNA molecules capable of associating with other RNA molecules through base-pairing and catalyzing various reactions involving phosphate group transfer. Of particular interest to us is the well known ribozyme from Tetrahymena thermophila capable of catalyzing RNA splicing in eukaryotic systems, chiefly because of its potential use as a gene therapy agent. In this article we review the progress made towards visualizing the RNA splicing mediated by the Tetrahymena ribozyme in single living mammalian cells with the beta-lactamase reporter system and highlight the development made in imaging RNA splicing with the luciferase reporter system in living animals.

    View details for DOI 10.1039/b617574k

    View details for Web of Science ID 000246156700001

    View details for PubMedID 17460789

  • Quantum dot/bioluminescence resonance energy transfer based highly sensitive detection of proteases ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Yao, H., Zhang, Y., Xiao, F., Xia, Z., Rao, J. 2007; 46 (23): 4346-4349

    View details for DOI 10.1002/anie.200700280

    View details for Web of Science ID 000247130400024

    View details for PubMedID 17465433

  • A bioluminogenic substrate for in vivo imaging of beta-lactamase activity ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Yao, H., So, M., Rao, J. 2007; 46 (37): 7031-7034

    View details for DOI 10.1002/anie.200701931

    View details for Web of Science ID 000249752200012

    View details for PubMedID 17676567

  • How molecular imaging is speeding up antiangiogenic drug development MOLECULAR CANCER THERAPEUTICS Cai, W., Rao, J., Gambhir, S. S., Chen, X. 2006; 5 (11): 2624-2633


    Drug development is a long process that generally spans about 10 to 15 years. The shift in recent drug discovery to novel agents against specific molecular targets highlights the need for more robust molecular imaging platforms. Using molecular probes, molecular imaging can aid in many steps of the drug development process, such as providing whole body readout in an intact system, decreasing the workload and speeding up drug development/validation, and facilitating individualized anticancer treatment monitoring and dose optimization. The main focus of this review is the recent advances in tumor angiogenesis imaging, and the targets include vascular endothelial growth factor and vascular endothelial growth factor receptor, integrin alpha(v)beta(3), matrix metalloproteinase, endoglin (CD105), and E-selectin. Through tumor angiogenesis imaging, it is expected that a robust platform for understanding the mechanisms of tumor angiogenesis and evaluating the efficacy of novel antiangiogenic therapies will be developed, which can help antiangiogenic drug development in both the preclinical stage and the clinical settings. Molecular imaging has enormous potential in improving the efficiency of the drug development process, including the specific area of antiangiogenic drugs.

    View details for DOI 10.1158/1535-7163.MCT-06-0395

    View details for Web of Science ID 000242138000004

    View details for PubMedID 17121909

  • Protease-modulated cellular uptake of quantum dots NANO LETTERS Zhang, Y., So, M. K., Rao, J. 2006; 6 (9): 1988-1992


    Quantum dots (QDs) are often cell-impermeable and require transporters to facilitate crossing over cell membranes. Here we present a simple and versatile method that utilizes enzymes, matrix metalloprotease 2 (MMP-2) and MMP-7, to modulate the cellular uptake of QDs. QD-peptide conjugates could be efficiently taken up into cells after the MMP treatment. This enzyme-modulated cellular uptake of QDs may be applied to other nanoparticles for biological imaging and selective drug delivery into tumor cells.

    View details for DOI 10.1021/nl0611586

    View details for Web of Science ID 000240465100027

    View details for PubMedID 16968013

  • A self-assembled quantum dot probe for detecting beta-lactamase activity BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Xu, C., Xing, B., Rao, H. 2006; 344 (3): 931-935


    This communication describes a quantum dot probe that can be activated by a reporter enzyme, beta-lactamase. Our design is based on the principle of fluorescence resonance energy transfer (FRET). A biotinylated beta-lactamase substrate was labeled with a carbocyanine dye, Cy5, and immobilized on the surface of quantum dots through the binding of biotin to streptavidin pre-coated on the quantum dots. In assembling this nanoprobe, we have found that both the distance between substrates and the quantum dot surface, and the density of substrates are important for its function. The fluorescence emission from quantum dots can be efficiently quenched (up to 95%) by Cy5 due to FRET. Our final quantum dot probe, assembled with QD605 and 1:1 mixture of biotin and a Cy5-labeled lactam, can be activated by 32microg/mL of beta-lactamase with 4-fold increase in the fluorescence emission.

    View details for DOI 10.1016/j.bbrc.2006.030225

    View details for Web of Science ID 000237585000033

    View details for PubMedID 16631595

  • Detection of mRNA in mammalian cells with a split ribozyme reporter CHEMBIOCHEM Hasegawa, S., Gowrishankar, G., Rao, J. 2006; 7 (6): 925-928

    View details for DOI 10.1002/cbic.200600061

    View details for Web of Science ID 000238171400011

    View details for PubMedID 16671127

  • Modulating the splicing activity of Tetrahymena ribozyme via RNA self-assembly FEBS LETTERS Hasegawa, S., Rao, J. H. 2006; 580 (6): 1592-1596


    The internal guiding sequence (IGS) is normally located at the 5' end of trans-splicing ribozymes that are derived from the Tetrahymena group I intron, and is required for the recognition of substrate RNAs and for trans-splicing reactions. Here, we separated the Tetrahymena group I intron at the L2 loop to produce two fragments: the IGS-containing substrate, and the IGS-lacking ribozyme. We show here that two fragments can complex not through the IGS interaction but under the guidance of appended interacting nucleotides, and perform trans-splicing. The splicing reactions took place both in vitro and in mammalian cells, and the spliced mRNA product from the self-assembled ribozyme complex can be translated into functional proteins in vivo. The splicing efficiency was dependent on the length of appending nucleotides.

    View details for DOI 10.1016/j.febslet.2006.01.090

    View details for Web of Science ID 000236058200011

    View details for PubMedID 16472807

  • HaloTag protein-mediated site-specific conjugation of bioluminescent proteins to quantum dots ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Zhang, Y., So, M., Loening, A. M., Yao, H., Gambhir, S. S., Rao, J. 2006; 45 (30): 4936-4940

    View details for DOI 10.1002/anie.200601197

    View details for Web of Science ID 000239543300010

    View details for PubMedID 16807952

  • Creating self-illuminating quantum dot conjugates NATURE PROTOCOLS So, M., Loening, A. M., Gambhir, S. S., Rao, J. 2006; 1 (3): 1160-1164


    Semiconductor quantum dots are inorganic fluorescent nanocrystals that, because of their unique optical properties compared with those of organic fluorophores, have become popular as fluorescent imaging probes. Although external light excitation is typically required for imaging with quantum dots, a new type of quantum dot conjugate has been reported that can luminesce with no need for external excitation. These self-illuminating quantum dot conjugates can be prepared by coupling of commercially available carboxylate-presenting quantum dots to the light-emitting protein Renilla luciferase. When the conjugates are exposed to the luciferase's substrate coelenterazine, the energy released by substrate catabolism is transferred to the quantum dots through bioluminescence resonance energy transfer, leading to quantum dot light emission. This protocol describes step-by-step procedures for the preparation and characterization of these self-illuminating quantum dot conjugates. The preparation process is relatively simple and can be done in less than 2 hours. The availability of self-illuminating quantum dot conjugates will provide many new possibilities for in vivo imaging and detection, such as monitoring of in vivo cell trafficking, multiplex bioluminescence imaging and new quantum dot-based biosensors.

    View details for DOI 10.1038/nprot.2006.162

    View details for Web of Science ID 000251155400012

    View details for PubMedID 17406398

  • Cell-permeable near-infrared fluorogenic substrates for imaging beta-lactamase activity JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Xing, B., Khanamiryan, A., Rao, J. H. 2005; 127 (12): 4158-4159


    This communication describes a design of cell-permeable near-infrared fluorogenic substrates for imaging beta-lactamase expression in living mammalian cells. This design is based on fluorescence energy transfer resonance and utilizes a peracetylated d-glucosamine to facilitate the transport of the near-infrared probe across cell membranes. This new type of fluorogenic probe may also be applied to image gene expression in living animals.

    View details for Web of Science ID 000227895500021

    View details for PubMedID 15783183

  • Single-cell detection of trans-splicing ribozyme in vivo activity JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Hasegawa, S., Choi, J. W., Rao, J. H. 2004; 126 (23): 7158-7159


    The Tetrahymena trans-splicing ribozyme can edit RNA in a sequence-specific manner, but its efficiency needs to be improved for any functional rescues. This communication describes a simple method that uses a bacterial enzyme beta-lactamase to report trans-splicing activity of Tetrahymena ribozyme in single living mammalian cells by fluorescence microscopy and flow cytometry. This enzyme-based single-cell detection method is highly sensitive and compatible with living cell flow cytometry, and should allow a cell-based systematic screening of a vast library of ribozymes for better trans-spliced ribozyme variants.

    View details for DOI 10.1021/ja049144u

    View details for Web of Science ID 000221963600002

    View details for PubMedID 15186136

  • Imaging Tetrahymena ribozyme splicing activity in single live mammalian cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hasegawa, S., Jackson, W. C., Tsien, R. Y., Rao, J. 2003; 100 (25): 14892-14896


    Tetrahymena ribozymes hold promise for repairing genetic disorders but are largely limited by their modest splicing efficiency and low production of final therapeutic proteins. Ribozyme evolution in intact living mammalian cells would greatly facilitate the discovery of new ribozyme variants with high in vivo activity, but no such strategies have been reported. Here we present a study using a new reporter enzyme, beta-lactamase, to report splicing activity in single living cells and perform high-throughput screening with flow cytometry. The reporter ribozyme constructs consist of the self-splicing Tetrahymena thermophila group I intron ribozyme that is inserted into the ORF of the mRNA of beta-lactamase. The splicing activity in single living cells can be readily detected quantitatively and visualized. Individual cells have shown considerable heterogeneity in ribozyme activity. Screening of Tetrahymena ribozymes with insertions in the middle of the L1 loop led to identification of better variants with at least 4-fold more final in vivo activity than the native sequence. Our work has provided a new reporter system that allows high-throughput screening with flow cytometry of single living mammalian cells for a direct and facile in vivo selection of desired ribozyme variants.

    View details for DOI 10.1073/pnas.2036553100

    View details for Web of Science ID 000187227200053

    View details for PubMedID 14645710

  • Novel fluorogenic substrates for imaging 6-lactamase gene expression JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Gao, W. Z., Xing, B. G., Tsien, R. Y., Rao, J. H. 2003; 125 (37): 11146-11147


    A new class of small nonfluorescent fluorogenic substrates becomes brightly fluorescent after beta-lactamase hydrolysis with up to 153-fold enhancement in the fluorescence intensity. Less than 500 fM of beta-lactamase in cell lysates can be readily detected, and beta-lactamase expression in living cells can be imaged with a red fluorescence derivative. These new fluorogenic substrates should find uses in clinical diagnostics and facilitate the applications of beta-lactamase as a biosensor.

    View details for DOI 10.1021/ja036126o

    View details for Web of Science ID 000185341800005

    View details for PubMedID 16220906

  • Design, synthesis, and characterization of a high-affinity trivalent system derived from vancomycin and L-Lys-D-Ala-D-Ala JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Rao, J. H., Lahiri, J., Weis, R. M., Whitesides, G. M. 2000; 122 (12): 2698-2710
  • Binding of a dimeric derivative of vancomycin to L-Lys-D-Ala-D-lactate in solution and at a surface CHEMISTRY & BIOLOGY Rao, J. H., Yan, L., Lahiri, J., Whitesides, G. M., Weis, R. M., Warren, H. S. 1999; 6 (6): 353-359


    The emergence of bacteria that are resistant to vancomycin (V), a glycopeptide antibiotic, results from the replacement of the carboxy-terminal D-Ala-D-Ala of bacterial cell wall precursors by D-Ala-D-lactate. Recently, it has been demonstrated that covalent dimeric variants of V are active against vancomycin-resistant enterococci (VRE). To study the contribution of divalency to the activities of these variants, we modeled the interactions of V and a dimeric V with L-Lys-D-Ala-D-lactate, an analog of the cell-wall precursors of the vancomycin-resistant bacteria.A dimeric derivative of V (V-Rd-V) was found to be much more effective than V in inhibiting the growth of VRE. The interactions of V and V-Rd-V with a monomeric lactate ligand - diacetyl-L-Lys-D-Ala-D-lactate (Ac2KDADLac) - and a dimeric derivative of L-Lys-D-Ala-D-lactate (Lac-R'd-Lac) in solution have been examined using isothermal titration calorimetry and UV spectroscopy titrations; the results reveal that V-Rd-V binds Lac-R'd-Lac approximately 40 times more tightly than V binds Ac2KDADLac. Binding of V and of V-Rd-V to Nalpha-Ac-L-Lys-D-Ala-D-lactate presented on the surface of mixed self-assembled monolayers (SAMs) of alkanethiolates on gold indicates that the apparent off-rate for dissociation of V-Rd-V from the surface is much slower than that of V from the same surface.The results are compatible with the hypothesis that divalency is responsible for tight binding, which correlates with small values of minimum inhibitory concentrations of V and V-Rd-V.

    View details for Web of Science ID 000084001000005

    View details for PubMedID 10375541

  • Using surface plasmon resonance to study the binding of vancomycin and its dimer to self-assembled monolayers presenting D-Ala-D-Ala JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Rao, J. H., Yan, L., Xu, B., Whitesides, G. M. 1999; 121 (11): 2629-2630
  • A trivalent system from vancomycin center dot D-Ala-D-Ala with higher affinity than avidin center dot biotin SCIENCE Rao, J. H., Lahiri, J., Isaacs, L., Weis, R. M., Whitesides, G. M. 1998; 280 (5364): 708-711


    Tris(vancomycin carboxamide) binds a trivalent ligand derived from D-Ala-D-Ala with very high affinity: dissociation constant (Kd) approximately 4 x 10(-17) +/- 1 x 10(-17) M. High-affinity trivalent binding and monovalent binding are fundamentally different. In trivalent (and more generally, polyvalent) binding, dissociation occurs in stages, and its rate can be accelerated by monovalent ligand at sufficiently high concentrations. In monovalent binding, dissociation is determined solely by the rate constant for dissociation and cannot be accelerated by added monomer. Calorimetric measurements for the trivalent system indicate an approximately additive gain in enthalpy relative to the corresponding monomers. This system is one of the most stable organic receptor-ligand pairs involving small molecules that is known. It illustrates the practicality of designing very high-affinity systems based on polyvalency.

    View details for Web of Science ID 000073415600038

    View details for PubMedID 9563940

  • Affinity capillary electrophoresis: A physical-organic tool for studying interactions in biomolecular recognition ELECTROPHORESIS Colton, I. J., Carbeck, J. D., Rao, J., Whitesides, G. M. 1998; 19 (3): 367-382


    Affinity capillary electrophoresis (ACE) is a technique that is used to measure the binding affinity of receptors to neutral and charged ligands. ACE experiments are based on differences in the values of electrophoretic mobility of free and bound receptor. Scatchard analysis of the fraction of bound receptor, at equilibrium, as a function of the concentration of free ligand yields the dissociation constant of the receptor-ligand complex. ACE experiments are most conveniently performed on fused silica capillaries using a negatively charged receptor (molecular mass < 50 kDa) and increasing concentrations of a low molecular weight, charged ligand in the running buffer. ACE experiments that involve high molecular weight receptors may require the use of running buffers containing zwitterionic additives to prevent the receptors from adsorbing appreciably to the wall of the capillary. This review emphasizes ACE experiments performed with two model systems: bovine carbonic anhydrase II (BCA II) with arylsulfonamide ligands and vancomycin (Van), a glycopeptide antibiotic, with D-Ala-D-Ala (DADA)-based peptidyl ligands. Dissociation constants determined from ACE experiments performed with charged receptors and ligands can often be rationalized using electrostatic arguments. The combination of differently charged derivatives of proteins - protein charge ladders - and ACE is a physical-organic tool that is used to investigate electrostatic effects. Variations of ACE experiments have been used to estimate the charge of Van and of proteins in solution, and to determine the effect of the association of Van to Ac2KDADA on the value of pKa of its N-terminal amino group.

    View details for Web of Science ID 000072716000002

    View details for PubMedID 9551788

  • Tight binding of a dimeric derivative of vancomycin with dimeric L-Lys-D-Ala-D-Ala JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Rao, J. H., Whitesides, G. M. 1997; 119 (43): 10286-10290
  • Using capillary electrophoresis to study the electrostatic interactions involved in the association of D-Ala-D-Ala with vancomycin JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Rao, J. H., Colton, I. J., Whitesides, G. M. 1997; 119 (40): 9336-9340