Probe chemistry and nanotechnology for molecular imaging and diagnostics
Hypoxia plays a key role in tumor resistance to radiotherapy (RT). It is important to study hypoxia dynamics during RT to improve treatment planning and prognosis. Here, we describe a luminescent nanoprobe, composed of a fluorescent semiconducting polymer and palladium (Pd) complex, for quantitative longitudinal imaging of tumor hypoxia dynamics during RT. The nanoprobe was designed to provide high sensitivity and reversible response for the subtle change in hypoxia over a narrow range (0-30 mmHg O2), which spans the oxygen range where tumors have limited radiosensitivity. Following intravenous administration, the nanoprobe efficiently accumulated in and distributed across the tumor, including the hypoxic region. The ratio between emissions at 700 and 800 nm provided quantitative mapping of hypoxia across the entire tumor. The nanoprobe has been applied to image the tumor hypoxia dynamics over 7 days during fractionated RT, revealing that high fractional dose (10 Gy) was more effective in improving tumor reoxygenation than low dose (2 Gy) and the effect tended to persist longer in smaller or more radiosensitive tumors. Our results also indicated the importance of the reoxygenation efficiency of the first fraction in the prediction of the radiation treatment outcome. In summary, this work has established a new nanoprobe for highly sensitive, quantitative and longitudinal imaging of tumor hypoxia dynamics following RT, and demonstrated its value for assessing the efficacy of RT and radiation treatment planning.
View details for DOI 10.1158/0008-5472.CAN-19-0530
View details for PubMedID 31311808
Tuberculosis (TB) remains a public health crisis and a leading cause of infection-related death globally. Although in high demand, imaging technologies that enable rapid, specific, and nongenetic labeling of live Mycobacterium tuberculosis (Mtb) remain underdeveloped. We report a dual-targeting strategy to develop a small molecular probe (CDG-DNB3) that can fluorescently label single bacilli within 1 hour. CDG-DNB3 fluoresces upon activation of the β-lactamase BlaC, a hydrolase naturally expressed in Mtb, and the fluorescent product is retained through covalent modification of the Mtb essential enzyme decaprenylphosphoryl-β-d-ribose 2'-epimerase (DprE1). This dual-targeting probe not only discriminates live from dead Bacillus Calmette-Guérin (BCG) but also shows specificity for Mtb over other bacterial species including 43 nontuberculosis mycobacteria (NTM). In addition, CDG-DNB3 can image BCG phagocytosis in real time, as well as Mtb in patients' sputum. Together with a low-cost, self-driven microfluidic chip, we have achieved rapid labeling and automated quantification of live BCG. This labeling approach should find many potential applications for research toward TB pathogenesis, treatment efficacy assessment, and diagnosis.
View details for PubMedID 30111644
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 PubMedID 24848238
View details for PubMedCentralID PMC4031611
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
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 PubMedID 24463363
View details for PubMedCentralID PMC3947658
Borylated poly(fluorene-benzothiadiazoles) (PF8-BT) are pi-conjugated polymers (CPs) with deep-red/near-infrared (NIR) absorption and emission profiles suitable for in vivo optical imaging. A fully borylated PF8-BT derivative (P4) was encapsulated in pegylated poly(lactic-co-glycolic acid) (PEG-PLGA) nanoparticles and compared with a reference NIR-emitting CP (PCPDTBT) or indocyanine green (ICG). All formulations satisfied quality requirements for parenterally administered diagnostics. P4 nanoparticles had higher quantum yield (2.3%) than PCPCDTBT (0.01%) or ICG nanoparticles (1.1%). The signal/background ratios (SBRs) of CP systems P4 and PCPDTBT in a phantom mouse (lambdaem = 820 nm) increased linearly with fluorophore mass (12.5-100 mug/mL), while the SBRs of ICG decreased above 25 mug/mL. P4 nanoparticles experienced <10% photobleaching over 10 irradiations (PCPDTBT: 25% and ICG: >44%). In a mouse tumor xenograft model, P4 nanoparticles showed a 5-fold higher SBR than PCPDTBT particles with fluorophore accumulation in the liver > spleen > tumor. Blood chemistry and tissue histology showed no abnormalities compared to untreated animals after a single administration.
View details for DOI 10.1021/acsami.9b17022
View details for PubMedID 31746180
PURPOSE: There is a clinical need for agents that target glioma cells for non-invasive and intraoperative imaging to guide therapeutic intervention and improve the prognosis of glioma. Matrix metalloproteinase (MMP)-14 is overexpressed in glioma with negligible expression in normal brain, presenting MMP-14 as an attractive biomarker for imaging glioma. In this study, we designed a peptide probe containing a near-infrared fluorescence (NIRF) dye/quencher pair, a positron emission tomography (PET) radionuclide, and a moiety with high affinity to MMP-14. This novel substrate-binding peptide allows dual modality imaging of glioma only after cleavage by MMP-14 to activate the quenched NIRF signal, enhancing probe specificity and imaging contrast.METHODS: MMP-14 expression and activity in human glioma tissues and cells were measured in vitro by immunofluorescence and gel zymography. Cleavage of the novel substrate and substrate-binding peptides by glioma cells in vitro and glioma xenograft tumors in vivo was determined by NIRF imaging. Biodistribution of the radiolabeled MMP-14-binding peptide or substrate-binding peptide was determined in mice bearing orthotopic patient-derived xenograft (PDX) glioma tumors by PET imaging.RESULTS: Glioma cells with MMP-14 activity showed activation and retention of NIRF signal from the cleaved peptides. Resected mouse brains with PDX glioma tumors showed tumor-to-background NIRF ratios of 7.6-11.1 at 4 h after i.v. injection of the peptides. PET/CT images showed localization of activity in orthotopic PDX tumors after i.v. injection of 68Ga-binding peptide or 64Cu-substrate-binding peptide; uptake of the radiolabeled peptides in tumors was significantly reduced (p < 0.05) by blocking with the non-labeled-binding peptide. PET and NIRF signals correlated linearly in the orthotopic PDX tumors. Immunohistochemistry showed co-localization of MMP-14 expression and NIRF signal in the resected tumors.CONCLUSIONS: The novel MMP-14 substrate-binding peptide enabled PET/NIRF imaging of glioma models in mice, warranting future image-guided resection studies with the probe in preclinical glioma models.
View details for DOI 10.1007/s00259-019-04607-x
View details for PubMedID 31773232
Multimodality imaging involves the use of more imaging modes to image the same living subjects and is now generally preferred in clinics for cancer imaging. Here we present multimodality-Magnetic Particle Imaging (MPI), Magnetic Resonance Imaging (MRI), Photoacoustic, Fluorescent-nanoparticles (termed MMPF NPs) for imaging tumor xenografts in living mice. MMPF NPs provide long-term (more than 2 months), dynamic, and accurate quantification, in vivo, of NPs and in real time by MPI. Moreover, MMPF NPs offer ultrasensitive MPI imaging of tumors (the tumor ROI increased by 30.6 times over that of preinjection). Moreover, the nanoparticle possessed a long-term blood circulation time (half-life at 49 h) and high tumor uptake (18% ID/g). MMPF NPs have been demonstrated for imaging breast and brain tumor xenografts in both subcutaneous and orthotopic models in mice via simultaneous MPI, MRI, fluorescence, and photoacoustic imaging with excellent tumor contrast to normal tissues.
View details for DOI 10.1021/acsnano.9b01436
View details for PubMedID 31244043
View details for DOI 10.1038/s41565-019-0395-0
View details for Web of Science ID 000467053100016
View details for Web of Science ID 000473116801068
View details for DOI 10.1016/j.wneu.2019.01.180
View details for Web of Science ID 000466491700202
View details for Web of Science ID 000468965200420
Poly(ADP ribose) polymerase (PARP) enzymes generate poly(ADP ribose) post-translational modifications on target proteins for an array of functions centering on DNA and cell stress. PARP isoforms 1 and 2 are critically charged with the surveillance of DNA integrity and are the first line guardians of the genome against DNA breaks. Here we present a novel probe ([18F]-SuPAR) for noninvasive imaging of PARP-1/2 activity using positron emission tomography (PET). [18F]-SuPAR is a radiofluorinated nicotinamide adenine dinucleotide (NAD) analog that can be recognized by PARP-1/2 and incorporated into the long branched polymers of poly(ADP ribose) (PAR). The measurement of PARP-1/2 activity was supported by a reduction of radiotracer uptake in vivo following PARP-1/2 inhibition with talazoparib treatment, a potent PARP inhibitor recently approved by FDA for treatment of breast cancer, as well as ex vivo colocalization of radiotracer analog and poly(ADP ribose). With [18F]-SuPAR, we were able to map the dose- and time-dependent activation of PARP-1/2 following radiation therapy in breast and cervical cancer xenograft mouse models. Tumor response to therapy was determined by [18F]-SuPAR PET within 8 h of administration of a single dose of radiation equivalent to one round of stereotactic ablative radiotherapy.
View details for DOI 10.1021/acs.bioconjchem.9b00089
View details for Web of Science ID 000468368300008
View details for PubMedID 30973715
Magnetic particle imaging (MPI) is a novel radiation-free tomographic imaging method that provides a background-free, signal attenuation-free, direct quantification of the spatial distribution of superparamagnetic iron-oxide nanoparticles (SPIONs) with high temporal resolution (milliseconds), high spatial resolution (< 1 mm), and extreme sensitivity (mumol). The technique is based on non-linear magnetization of the SPIONs when exposed to an oscillating magnetic field. MPI was first described in 2001. Since then, the technique has been applied to experimental imaging of diseases affecting different organs in the human body. The aim of this paper is to review the potential applications of MPI in the field of neurosurgery. MPI has been used for the detection the loco-regional invasion of brain tumors, tracking and monitoring the viability of neural stem cells implanted for neuro-regenerative purposes, diagnosis of cerebral ischemia, and diagnosis and morpho-functional assessment of brain aneurysms. Currently, MPI is at a pre-clinical stage. In the future, human-sized MPI scanners, along with the optimal toxicity profile of SPIONs will allow diagnostic applications in neurosurgical diseases.
View details for PubMedID 30738942
View details for DOI 10.7150/ntno.35342
The condensation reaction between 6-hydroxy-2-cyanobenzothiazole (CBT) and cysteine has been shown for various applications such as site-specific protein labelling and in vivo cancer imaging. This report further expands the substrate scope of this reaction by varying the substituents on aromatic nitriles and amino thiols and testing their reactivity and ability to form nanoparticles for cell imaging. The structure-activity relationship study leads to the identification of the minimum structural requirement for the macrocyclization and assembly process in forming nanoparticles. One of the scaffolds made of 2-pyrimidinecarbonitrile and cysteine joined by a benzyl linker was applied to design fluorescent probes to image caspase-3/7 and β-galactosidase activity in live cells. These results demonstrate the generality of this system for imaging hydrolytic enzymes.
View details for DOI 10.1002/anie.201913314
View details for PubMedID 31828913
View details for Web of Science ID 000441803800181
View details for PubMedID 30075836
View details for DOI 10.1158/1538-7445.AM2018-LB-004
View details for Web of Science ID 000468818900177
Demarcation of malignant brain tumor boundaries is critical to achieve complete resection and to improve patient survival. Contrast-enhanced brain magnetic resonance imaging (MRI) is the gold standard for diagnosis and pre-surgical planning, despite limitations of gadolinium (Gd)-based contrast agents to depict tumor margins. Recently, solid metal-based nanoparticles (NPs) have shown potential as diagnostic probes for brain tumors. Gold nanoparticles (GNPs) emerged among those, because of their unique physical and chemical properties and biocompatibility. The aim of the present study is to review the application of GNPs for in vitro and in vivo brain tumor diagnosis.We performed a PubMed search of reports exploring the application of GNPs in the diagnosis of brain tumors in biological models including cells, animals, primates, and humans. The search words were "gold" AND "NP" AND "brain tumor." Two reviewers performed eligibility assessment independently in an unblinded standardized manner. The following data were extracted from each paper: first author, year of publication, animal/cellular model, GNP geometry, GNP size, GNP coating [i.e., polyethylene glycol (PEG) and Gd], blood-brain barrier (BBB) crossing aids, imaging modalities, and therapeutic agents conjugated to the GNPs.The PubMed search provided 100 items. A total of 16 studies, published between the 2011 and 2017, were included in our review. No studies on humans were found. Thirteen studies were conducted in vivo on rodent models. The most common shape was a nanosphere (12 studies). The size of GNPs ranged between 20 and 120 nm. In eight studies, the GNPs were covered in PEG. The BBB penetration was increased by surface molecules (nine studies) or by means of external energy sources (in two studies). The most commonly used imaging modalities were MRI (four studies), surface-enhanced Raman scattering (three studies), and fluorescent microscopy (three studies). In two studies, the GNPs were conjugated with therapeutic agents.Experimental studies demonstrated that GNPs might be versatile, persistent, and safe contrast agents for multimodality imaging, thus enhancing the tumor edges pre-, intra-, and post-operatively improving microscopic precision. The diagnostic GNPs might also be used for multiple therapeutic approaches, namely as "theranostic" NPs.
View details for PubMedID 29867737
As a new class of organic optical nanomaterials, semiconducting polymer nanoparticles (SPNs) have the advantages of excellent optical properties, high photostability, facile surface functionalization, and are considered to possess good biocompatibility for biomedical applications. This review surveys recent progress made on the design and synthesis of SPNs for molecular imaging and cancer phototherapy. A variety of novel polymer design, chemical modification and nanoengineering strategies have been developed to precisely tune up optoelectronic properties of SPNs to enable fluorescence, chemiluminescence and photoacoustic (PA) imaging in living animals. With these imaging modalities, SPNs have been demonstrated not only to image tissues such as lymph nodes, vascular structure and tumors, but also to detect disease biomarkers such as reactive oxygen species (ROS) and protein sulfenic acid as well as physiological indexes such as pH and blood glucose concentration. The potentials of SPNs in cancer phototherapy including photodynamic and photothermal therapy are also highlighted with recent examples. Future efforts should further expand the use of SPNs in biomedical research and may even move them beyond pre-clinical studies.
View details for PubMedID 29190479
View details for PubMedCentralID PMC5978728
Cellular apoptosis is an important criterion for evaluating the efficacy of cancer therapies. We have developed a new small molecule probe ([18F]C-SNAT) for positron emission tomography (PET) imaging of apoptosis. [18F]C-SNAT, when activated by caspase-3 and glutathione reduction, undergoes intramolecular cyclization followed by self-assembly to form nano-aggregates in apoptotic cells. This unique mechanism creates preferential retention of gamma radiation signals in targeted cells and thus enables the detection of apoptosis using PET, a sensitive and clinically practical technique. This protocol describes the chemical synthesis, radiolabeling and PET imaging of apoptosis using this probe.
View details for PubMedID 29858792
Iron oxides nanoparticles tailored for magnetic particle imaging (MPI) have been synthesized, and their MPI signal intensity is three-times that of commercial MPI contrast (Ferucarbotran, also called Vivotrax) and seven-times that of MRI contrast (Feraheme) at the same Fe concentration. MPI tailored iron oxide nanoparticles were encapsulated with semiconducting polymers to produce Janus nanoparticles that possessed optical and magnetic properties for MPI and fluorescence imaging. Janus particles were applied to cancer cell labeling and in vivo tracking, and as few as 250 cells were imaged by MPI after implantation, corresponding to an amount of 7.8 ng of Fe. Comparison with MRI and fluorescence imaging further demonstrated the advantages of our Janus particles for MPI-super sensitivity, unlimited tissue penetration, and linear quantitativity.
View details for PubMedID 29232142
Ongoing research into the application of nanotechnology for cancer treatment and diagnosis has demonstrated its advantages within contemporary oncology as well as its intrinsic limitations. The National Cancer Institute publishes the Cancer Nanotechnology Plan every 5 years since 2005. The most recent iteration helped codify the ongoing basic and translational efforts of the field and displayed its breadth with several evolving areas. From merely a technological perspective, this field has seen tremendous growth and success. However, an incomplete understanding of human cancer biology persists relative to the application of nanoscale materials within contemporary oncology. As such, this review presents several evolving areas in cancer nanotechnology in order to identify key clinical and biological challenges that need to be addressed to improve patient outcomes. From this clinical perspective, a sampling of the nano-enabled solutions attempting to overcome barriers faced by traditional therapeutics and diagnostics in the clinical setting are discussed. Finally, a strategic outlook of the future is discussed to highlight the need for next-generation cancer nanotechnology tools designed to address critical gaps in clinical cancer care.
View details for DOI 10.1021/acsnano.7b05108
View details for Web of Science ID 000423495200004
View details for PubMedID 29257865
A cascade amplification release nanoparticle (CARN) is constructed by the coencapsulation of β-lapachone and a reactive-oxygen-species (ROS)-responsive doxorubicin (DOX) prodrug, BDOX, in polymeric nanoparticles. Releasing β-lapachone first from the CARNs selectively increases the ROS level in cancer cells via NAD(P)H:quinone oxidoreductase-1 (NQO1) catalysis, which induces the cascade amplification release of DOX and overcomes multidrug resistance (MDR) in cancer cells, producing a remarkably improved therapeutic efficacy against MDR tumors with minimal side effects.
View details for PubMedID 28833669
Tuberculosis is a pulmonary disease with an especially high mortality rate in immuno-compromised populations, specifically children and HIV positive individuals. The causative agent, Mycobacterium tuberculosis (Mtb), is a very slow growing and difficult organism to work with, making both diagnosis and development of effective treatments cumbersome. We utilize a fiber-optic fluorescence microendoscope integrated with a whole-body imaging system for in vivo Mtb detection. The system exploits an endogenous enzyme of Mtb (β-lactamase, or BlaC) using a BlaC-specific NIR fluorogenic substrate. In the presence of BlaC, this substrate is cleaved and becomes fluorescent. Using intravital illumination of the lung to excite this probe, sensitivity of the optical system increases over trans- and epi-illumination methods of whole-body fluorescence imaging. We demonstrate that integration of these imaging technologies with BlaC-specific fluorescent reporter probe improves the level of detection to ∼100 colony forming units, a 100× increase in sensitivity in comparison to epi-illumination and a 10× increase in sensitivity in comparison to previous work in intravital excitation of tdTomato-expressing Mtb. This lower detection threshold enables the study of early stage bacterial infections with clinical strains of Mtb and longitudinal studies of disease pathogenesis and therapeutic efficacy with multiple time points in a single animal.
View details for PubMedID 27753271
View details for PubMedCentralID PMC5703064
As an emerging class of optical nanomaterials, semiconducting polymer nanoparticles (SPNs) are highly photostable, optically active and versatile in chemistry; these properties make them attractive as molecular imaging agents to enable imaging of biological events and functionalities at multiple scales. More recently, a variety of SPNs have been found to exhibit high photoacoustic properties, and further empowered photoacoustic imaging for contrast enhanced in vivo molecular imaging. Target-sensitive components can be incorporated in the SPNs to create activatable imaging probes to sense and monitor the target dynamics in living objects. Intrinsically biophotonic and biocompatible, SPNs can be further engineered for multimodal imaging and for real-time imaging of drug delivery. For further resources related to this article, please visit the WIREs website.
View details for DOI 10.1002/wnan.1418
View details for Web of Science ID 000397857100003
View details for PubMedCentralID PMC5192001
View details for DOI 10.1093/infdis/jiw298
View details for Web of Science ID 000397204700010
This work reports a novel caging strategy for designing fluorogenic probes to detect the activity of β-lactamases. The caging strategy uses a thiophenyl linker connected to a fluorophore caged by a good leaving group-dinitrophenyl. The uncaging proceeds in two steps through the sulfa-releasing and subsequent intramolecular substitution. The length of the linker has been examined and optimized to maximize the rate of intramolecular reaction and thus the rate of fluorescence activation. Finally based on this strategy, we prepared a green fluorogenic probe CAT-7 and validated its selectivity for detecting metallo-carbapenemases (VIM-27, IMP-1, NDM-1) in carbapenem-resistant Enterobacteriaceae (CRE) lysates.
View details for PubMedID 29568429
This work reports a novel caging strategy for designing fluorogenic probes to detect the activity of β-lactamases. The caging strategy uses a thiophenyl linker connected to a fluorophore caged by a good leaving group-dinitrophenyl. The uncaging proceeds in two steps through the sulfa-releasing and subsequent intramolecular substitution. The length of the linker has been examined and optimized to maximize the rate of intramolecular reaction and thus the rate of fluorescence activation. Finally based on this strategy, we prepared a green fluorogenic probe CAT-7 and validated its selectivity for detecting metallo-carbapenemases (VIM-27, IMP-1, NDM-1) in carbapenem-resistant Enterobacteriaceae (CRE) lysates.
View details for DOI 10.1039/C7SC02416A
View details for PubMedCentralID PMC5849144
Glioblastoma (GBM) has a dismal prognosis. Evidence from preclinical tumor models and human trials indicates the role of GBM initiating cells (GIC) in GBM drug resistance. Here, we propose a new treatment option with tumor enzyme-activatable, combined therapeutic and diagnostic (theranostic) nanoparticles, which caused specific toxicity against GBM tumor cells and GICs. The theranostic cross-linked iron oxide nanoparticles (CLIO) were conjugated to a highly potent vascular disrupting agent (ICT) and secured with a matrix-metalloproteinase (MMP-14) cleavable peptide. Treatment with CLIO-ICT disrupted tumor vasculature of MMP-14 expressing GBM, induced GIC apoptosis and significantly impaired tumor growth. In addition, the iron core of CLIO-ICT enabled in vivo drug tracking with MR imaging. Treatment with CLIO-ICT plus temozolomide achieved tumor remission and significantly increased survival of human GBM bearing mice by more than 2 fold compared to treatment with temozolomide alone. Thus, we present a novel therapeutic strategy with significant impact on survival and great potential for clinical translation.
View details for PubMedID 28659432
Microglial activation is a key pathological feature of Alzheimer's disease (AD). PET imaging of translocator protein 18 kDa (TSPO) is a strategy to detect microglial activation in vivo. Here we assessed flutriciclamide ([(18)F]GE-180), a new second-generation TSPO-PET radiotracer, for its ability to monitor response to LM11A-31, a novel AD therapeutic in clinical trials. AD mice displaying pathology were treated orally with LM11A-31 for 3 months. Subsequent [(18)F]GE-180-PET imaging revealed significantly lower signal in cortex and hippocampus of LM11A-31-treated AD mice compared to those treated with vehicle, corresponding with decreased levels of TSPO immunostaining and microglial Iba1 immunostaining. In addition to detecting decreased microglial activation following LM11A-31 treatment, [(18)F]GE-180 identified activated microglia in AD mice with greater sensitivity than another second-generation TSPO radiotracer, [(18)F]PBR06. Together, these data demonstrate the promise of [(18)F]GE-180 as a potentially sensitive tool for tracking neuroinflammation in AD mice and for monitoring therapeutic modulation of microglial activation.
View details for DOI 10.7150/thno.17666
View details for PubMedID 28529627
Semiconducting polymer nanoparticles (SPNs) emerge as attractive molecular imaging nanoagents in living animals because of their excellent optical properties including large absorption coefficients, tunable optical properties and controllable dimensions, high photostability, and the use of organic and biologically inert components without toxic metals. This review summarizes the recent advances of these new organic nanoparticles in in vivo molecular imaging. The in vivo biocompatibility of SPNs is discussed first in details, followed by examples of their applications ranging from sentinel lymph node mapping and tumor imaging to long-term cell tracking, to drug toxicity and bacterial infection imaging for fluorescence, bioluminescence, chemiluminescence and photoacoustic imaging in living animals. The utility of SPNs for designing smart activatable probes for real-time in vivo imaging is also discussed.
View details for DOI 10.1016/j.jconrel.2016.01.004
View details for Web of Science ID 000386250700026
View details for PubMedID 26773769
View details for PubMedCentralID PMC4938792
As an emerging class of optical nanomaterials, semiconducting polymer nanoparticles (SPNs) are highly photostable, optically active and versatile in chemistry; these properties make them attractive as molecular imaging agents to enable imaging of biological events and functionalities at multiple scales. More recently, a variety of SPNs have been found to exhibit high photoacoustic properties, and further empowered photoacoustic imaging for contrast enhanced in vivo molecular imaging. Target-sensitive components can be incorporated in the SPNs to create activatable imaging probes to sense and monitor the target dynamics in living objects. Intrinsically biophotonic and biocompatible, SPNs can be further engineered for multimodal imaging and for real-time imaging of drug delivery. For further resources related to this article, please visit the WIREs website.
View details for DOI 10.1002/wnan.1418
View details for PubMedID 27346564
View details for PubMedCentralID PMC5192001
The illegal addition of β-lactamase (Bla) in milk to disguise β-lactam antibiotics has been a serious issue in the milk industry worldwide. Herein, we report a method for point-of-care detection of Bla based on a probe, Tokyo Green-tethered β-lactam (CDG-1), as a common substrate of various Blas (Bla A, B...) which can enzymatically convert CDG-1 (low fluorescence) to Tokyo Green (high fluorescence). This approach allows rapid screening of a broad spectrum of Blas in real milk samples within 15 min without any pretreatment. Combined with the immuno-magnetic separation, we achieved sensitive and quantitative detection of Bla (10(-5) U/mL), which provides a universal platform for screening and determining Blas in complex samples with high efficiency and accuracy.
View details for DOI 10.1021/acs.analchem.6b01122
View details for Web of Science ID 000377631000009
View details for PubMedID 27146449
View details for DOI 10.1126/scitranslmed.aac6117
View details for PubMedID 26491079
Personalized cancer medicine requires measurement of therapeutic efficacy as early as possible, which is optimally achieved by three-dimensional imaging given the heterogeneity of cancer. Magnetic resonance imaging (MRI) can obtain images of both anatomy and cellular responses, if acquired with a molecular imaging contrast agent. The poor sensitivity of MRI has limited the development of activatable molecular MR contrast agents. To overcome this limitation of molecular MRI, a novel implementation of our caspase-3-sensitive nanoaggregation MRI (C-SNAM) contrast agent is reported. C-SNAM is triggered to self-assemble into nanoparticles in apoptotic tumor cells, and effectively amplifies molecular level changes through nanoaggregation, enhancing tissue retention and spin-lattice relaxivity. At one-tenth the current clinical dose of contrast agent, and following a single imaging session, C-SNAM MRI accurately measured the response of tumors to either metronomic chemotherapy or radiation therapy, where the degree of signal enhancement is prognostic of long-term therapeutic efficacy. Importantly, C-SNAM is inert to immune activation, permitting radiation therapy monitoring.
View details for DOI 10.1038/srep14759
View details for Web of Science ID 000362259800001
View details for PubMedID 26440059
View details for PubMedCentralID PMC4594000
Materials with persistent luminescence are attractive for in vivo optical imaging since they have a long lifetime that allows the separation of excitation of fluorophores and image acquisition for time-delay imaging, thus eliminating tissue autofluorescence associated with fluorescence imaging. Persistently luminescent nanoparticles have previously been fabricated from toxic rare-earth metals. This work reports that nanoparticles made of the conjugated polymer MEH-PPV can generate luminescence persisting for an hour upon single excitation. A near-infrared dye was encapsulated in the conjugated polymer nanoparticle to successfully generate persistent near-infrared luminescence through resonance energy transfer. This new persistent luminescence nanoparticles have been demonstrated for optical imaging applications in living mice.
View details for DOI 10.1002/anie.201502736
View details for PubMedID 26223794
View details for PubMedCentralID PMC4575640
An early readout of tumor response to therapy through measurement of drug or radiation-induced cell death may provide important prognostic indications and improved patient management. It has been shown that the uptake of (18)F-C-SNAT can be used to detect early response to therapy in tumors by positron emission tomography (PET) via a mechanism of caspase-3-triggered nanoaggregation.Here, we compared the preclinical utility of (18)F-C-SNAT for the detection of drug-induced cell death to clinically evaluated radiotracers, (18)F-FDG, (99m)Tc-Annexin V, and (18)F-ML-10 in tumor cells in culture, and in tumor-bearing mice in vivo.In drug-treated lymphoma cells, (18)F-FDG, (99m)Tc-Annexin V, and (18)F-C-SNAT cell-associated radioactivity correlated well to levels of cell death (R(2) > 0.8; P < 0.001), with no correlation measured for (18)F-ML-10 (R(2) = 0.05; P > 0.05). A similar pattern of response was observed in two human NSCLC cell lines following carboplatin treatment. EL-4 tumor uptake of (99m)Tc-Annexin V and (18)F-C-SNAT were increased 1.4- and 2.1-fold, respectively, in drug-treated versus naïve control animals (P < 0.05), although (99m)Tc-Annexin V binding did not correlate to ex vivo TUNEL staining of tissue sections. A differential response was not observed with either (18)F-FDG or (18)F-ML-10.We have demonstrated here that (18)F-C-SNAT can sensitively detect drug-induced cell death in murine lymphoma and human NSCLC. Despite favorable image contrast obtained with (18)F-C-SNAT, the development of next-generation derivatives, using the same novel and promising uptake mechanism, but displaying improved biodistribution profiles, are warranted for maximum clinical utility. Clin Cancer Res; 21(17); 3896-905. ©2015 AACR.
View details for DOI 10.1158/1078-0432.CCR-14-3176
View details for PubMedID 25972517
View details for PubMedCentralID PMC4558304
Diketopyrrolopyrrole-based semiconducting polymer nanoparticles with high photostability and strong photoacoustic brightness are designed and synthesized, which results in 5.3-fold photoacoustic signal enhancement in tumor xenografts after systemic administration.
View details for DOI 10.1002/adma.201502285
View details for PubMedID 26247171
View details for PubMedCentralID PMC4567488
Early detection of tumor response to therapy is crucial to the timely identification of the most efficacious treatments. We recently developed a novel apoptosis imaging tracer, (18)F-C-SNAT (C-SNAT is caspase-sensitive nanoaggregation tracer), that undergoes an intramolecular cyclization reaction after cleavage by caspase-3/7, a biomarker of apoptosis. This caspase-3/7-dependent reaction leads to an enhanced accumulation and retention of (18)F activity in apoptotic tumors. This study aimed to fully examine in vivo pharmacokinetics of the tracer through PET imaging and kinetic modeling in a preclinical mouse model of tumor response to systemic anticancer chemotherapy.Tumor-bearing nude mice were treated 3 times with intravenous injections of doxorubicin before undergoing a 120-min dynamic (18)F-C-SNAT PET/CT scan. Time-activity curves were extracted from the tumor and selected organs. A 2-tissue-compartment model was fitted to the time-activity curves from tumor and muscle, using the left ventricle of the heart as input function, and the pharmacokinetic rate constants were calculated.Both tumor uptake (percentage injected dose per gram) and the tumor-to-muscle activity ratio were significantly higher in the treated mice than untreated mice. Pharmacokinetic rate constants calculated by the 2-tissue-compartment model showed a significant increase in delivery and accumulation of the tracer after the systemic chemotherapeutic treatment. Delivery of (18)F-C-SNAT to the tumor tissue, quantified as K1, increased from 0.31 g⋅(mL⋅min)(-1) in untreated mice to 1.03 g⋅(mL⋅min)(-1) in treated mice, a measurement closely related to changes in blood flow. Accumulation of (18)F-C-SNAT, quantified as k3, increased from 0.03 to 0.12 min(-1), proving a higher retention of (18)F-C-SNAT in treated tumors independent from changes in blood flow. An increase in delivery was also found in the muscular tissue of treated mice without increasing accumulation.(18)F-C-SNAT has significantly increased tumor uptake and significantly increased tumor-to-muscle ratio in a preclinical mouse model of tumor therapy. Furthermore, our kinetic modeling of (18)F-C-SNAT shows that chemotherapeutic treatment increased accumulation (k3) in the treated tumors, independent of increased delivery (K1).
View details for DOI 10.2967/jnumed.115.155259
View details for PubMedID 26045308
This paper describes a method for the quantitative detection of cells expressing BlaC, a β-lactamase naturally expressed by Mycobacterium tuberculosis, intended for the diagnosis of tuberculosis. The method is based on the compartmentalization of bacteria in picoliter droplets at limiting dilutions such that each drop contains one or no cells. The co-encapsulation of a fluorogenic substrate probe for BlaC allows the quantification of bacteria by enumerating the number of fluorescent drops. Quantification of 10 colony forming units per milliliter is demonstrated. Furthermore, the encapsulation of single cell in drops maintains the specificity of the detection scheme even when the concentration of bacteria that do not express BlaC exceeds that expressing BlaC by one million-fold.
View details for DOI 10.1063/1.4928879
View details for PubMedID 26339319
View details for PubMedCentralID PMC4545073
Ultrasound induced microbubble cavitation can cause enhanced permeability across natural barriers of tumors such as vessel walls or cellular membranes, allowing for enhanced therapeutic delivery into the target tissues. While enhanced delivery of small (<1nm) molecules has been shown at acoustic pressures below 1MPa both in vitro and in vivo, the delivery efficiency of larger (>100nm) therapeutic carriers into cancer remains unclear and may require a higher pressure for sufficient delivery. Enhanced delivery of larger therapeutic carriers such as FDA approved pegylated poly(lactic-co-glycolic acid) nanoparticles (PLGA-PEG-NP) has significant clinical value because these nanoparticles have been shown to protect encapsulated drugs from degradation in the blood circulation and allow for slow and prolonged release of encapsulated drugs at the target location. In this study, various acoustic parameters were investigated to facilitate the successful delivery of two nanocarriers, a fluorescent semiconducting polymer model drug nanoparticle as well as PLGA-PEG-NP into human colon cancer xenografts in mice. We first measured the cavitation dose produced by various acoustic parameters (pressure, pulse length, and pulse repetition frequency) and microbubble concentration in a tissue mimicking phantom. Next, in vivo studies were performed to evaluate the penetration depth of nanocarriers using various acoustic pressures, ranging between 1.7 and 6.9MPa. Finally, a therapeutic microRNA, miR-122, was loaded into PLGA-PEG-NP and the amount of delivered miR-122 was assessed using quantitative RT-PCR. Our results show that acoustic pressures had the strongest effect on cavitation. An increase of the pressure from 0.8 to 6.9MPa resulted in a nearly 50-fold increase in cavitation in phantom experiments. In vivo, as the pressures increased from 1.7 to 6.9MPa, the amount of nanoparticles deposited in cancer xenografts was increased from 4- to 14-fold, and the median penetration depth of extravasated nanoparticles was increased from 1.3-fold to 3-fold, compared to control conditions without ultrasound, as examined on 3D confocal microscopy. When delivering miR-122 loaded PLGA-PEG-NP using optimal acoustic settings with minimum tissue damage, miR-122 delivery into tumors with ultrasound and microbubbles was 7.9-fold higher compared to treatment without ultrasound. This study demonstrates that ultrasound induced microbubble cavitation can be a useful tool for delivery of therapeutic miR loaded nanocarriers into cancer in vivo.
View details for DOI 10.1016/j.jconrel.2015.02.018
View details for Web of Science ID 000351696600011
View details for PubMedID 25687306
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
Site specificity is pivotal in obtaining homogeneously labeled proteins without batch-to-batch variations. More importantly, precisely controlled modification at specific sites avoids potential pitfalls that could otherwise interfere with protein folding, structure, and function. Inspired by the chemical synthesis of D-luciferin, we have developed an efficient strategy (second-order rate constant k 2 = 9.2 M(-1) s(-1)) for labeling of proteins containing 1,2-aminothiol via reaction with 2-cyanobenzothiazole (CBT). In addition, the CBT condensation enjoys the convenience of protein engineering, as production of N-terminal cysteine-containing proteins has been well developed for native chemical ligation. This protocol describes the preparation of Renilla luciferase (rLuc) with 1,2-aminothiol at either its N- or C-terminus, and site-specific labeling of rLuc with fluorescein or (18)F via CBT condensation.
View details for DOI 10.1007/978-1-4939-2272-7_5
View details for PubMedID 25560068
The epidermal growth factor receptor (EGFR) serves as an attractive target for cancer molecular imaging and therapy. Our previous positron emission tomography (PET) studies showed that the EGFR-targeting affibody molecules (64)Cu-DOTA-ZEGFR:1907 and (18)F-FBEM-ZEGFR:1907 can discriminate between high and low EGFR-expression tumors and have the potential for patient selection for EGFR-targeted therapy. Compared with (64)Cu, (18)F may improve imaging of EGFR-expression and is more suitable for clinical application, but the labeling reaction of (18)F-FBEM-ZEGFR:1907 requires a long synthesis time. The aim of the present study is to develop a new generation of (18)F labeled affibody probes (Al(18)F-NOTA-ZEGFR:1907 and (18)F-CBT-ZEGFR:1907) and to determine whether they are suitable agents for imaging of EGFR expression. The first approach consisted of conjugating ZEGFR:1907 with NOTA and radiolabeling with Al(18)F to produce Al(18)F-NOTA-ZEGFR:1907. In a second approach the prosthetic group (18)F-labeled-2-cyanobenzothiazole ((18)F-CBT) was conjugated to Cys-ZEGFR:1907 to produce (18)F-CBT-ZEGFR:1907. Binding affinity and specificity of Al(18)F-NOTA-ZEGFR:1907 and (18)F-CBT-ZEGFR:1907 to EGFR were evaluated using A431 cells. Biodistribution and PET studies were conducted on mice bearing A431 xenografts after injection of Al(18)F-NOTA-ZEGFR:1907 or (18)F-CBT-ZEGFR:1907 with or without coinjection of unlabeled affibody proteins. The radiosyntheses of Al(18)F-NOTA-ZEGFR:1907 and (18)F-CBT-ZEGFR:1907 were completed successfully within 40 and 120 min with a decay-corrected yield of 15% and 41% using a 2-step, 1-pot reaction and 2-step, 2-pot reaction, respectively. Both probes bound to EGFR with low nanomolar affinity in A431 cells. Although (18)F-CBT-ZEGFR:1907 showed instability in vivo, biodistribution studies revealed rapid and high tumor accumulation and quick clearance from normal tissues except the bones. In contrast, Al(18)F-NOTA-ZEGFR:1907 demonstrated high in vitro and in vivo stability, high tumor uptake, and relative low uptake in most of the normal organs except the liver and kidneys at 3 h after injection. The specificity of both probes for A431 tumors was confirmed by their lower uptake on coinjection of unlabeled affibody. PET studies showed that Al(18)F-NOTA-ZEGFR:1907 and (18)F-CBT-ZEGFR:1907 could clearly identify EGFR positive tumors with good contrast. Two strategies for (18)F-labeling of affibody molecules were successfully developed as two model platforms using NOTA or CBT coupling to affibody molecules that contain an N-terminal cysteine. Al(18)F-NOTA-ZEGFR:1907 and (18)F-CBT-ZEGFR:1907 can be reliably obtained in a relatively short time. Biodistribution and PET studies demonstrated that Al(18)F-NOTA-ZEGFR:1907 is a promising PET probe for imaging EGFR expression in living mice.
View details for DOI 10.1021/mp5003043
View details for PubMedID 24972326
Non-invasive detection of caspase-3/7 activity in vivo has provided invaluable predictive information regarding tumor therapeutic efficacy and anti-tumor drug selection. Although a number of caspase-3/7 targeted fluorescence and positron emission tomography (PET) imaging probes have been developed, there is still a lack of gadolinium (Gd)-based magnetic resonance imaging (MRI) probes that enable high spatial resolution detection of caspase-3/7 activity in vivo. Here we employ a self-assembly approach and develop a caspase-3/7 activatable Gd-based MRI probe for monitoring tumor apoptosis in mice. Upon reduction and caspase-3/7 activation, the caspase-sensitive nano-aggregation MR probe (C-SNAM: 1) undergoes biocompatible intramolecular cyclization and subsequent self-assembly into Gd-nanoparticles (GdNPs). This results in enhanced r1 relaxivity-19.0 (post-activation) vs. 10.2 mM(-1) s(-1) (pre-activation) at 1 T in solution-and prolonged accumulation in chemotherapy-induced apoptotic cells and tumors that express active caspase-3/7. We demonstrate that C-SNAM reports caspase-3/7 activity by generating a significantly brighter T1-weighted MR signal compared to non-treated tumors following intravenous administration of C-SNAM, providing great potential for high-resolution imaging of tumor apoptosis in vivo.
View details for PubMedID 25429349
Current methods for the detection of Mycobacterium tuberculosis (Mtb) are either time consuming or require expensive instruments and are thus are not suitable for point-of-care diagnosis. The design, synthesis, and evaluation of fluorogenic probes with high specificity for BlaC, a biomarker expressed by Mtb, are described. The fluorogenic probe CDG-3 is based on cephalosporin with substitutions at the 2 and 7 positions and it demonstrates over 120,000-fold selectivity for BlaC over TEM-1 Bla, the most common β-lactamase. CDG-3 can detect 10 colony-forming units of the attenuated Mycobacterium bovis strain BCG in human sputum in the presence of high levels of contaminating β-lactamases expressed by other clinically prevalent bacterial strains. In a trial with 50 clinical samples, CDG-3 detected tuberculosis with 90% sensitivity and 73% specificity relative to Mtb culture within one hour, thus demonstrating its potential as a low-cost point-of-care test for use in resource-limited areas.
View details for DOI 10.1002/anie.201405243
View details for Web of Science ID 000342676100046
View details for PubMedID 24989449
Controlled self-assembly of small molecule gadolinium (Gd) complexes into nanoparticles (GdNPs) is emerging as an effective approach to design activatable magnetic resonance imaging (MRI) probes and amplify the r₁ relaxivity. Herein, we employ a reduction-controlled macrocyclization reaction and self-assembly to develop a redox activated Gd-based MRI probe for sensing a reducing environment. Upon disulfide reduction at physiological conditions, an acyclic contrast agent 1 containing dual Gd-chelates undergoes intramolecular macrocyclization to form rigid and hydrophobic macrocycles, which subsequently self-assemble into GdNPs, resulting in a ∼60% increase in r₁ relaxivity at 0.5 T. Probe 1 has high r₁ relaxivity (up to 34.2 mM(-1) s(-1) per molecule at 0.5 T) upon activation, and also shows a high sensitivity and specificity for MR detection of thiol-containing biomolecules.
View details for DOI 10.1021/bc500254g
View details for PubMedID 24992373
Despite the pressing need to noninvasively monitor transplanted cells in vivo with fluorescence imaging, desirable fluorescent agents with rapid labeling capability, durable brightness, and ideal biocompatibility remain lacking. Here, phosphorylcholine-coated near-infrared (NIR) fluorescent semiconducting polymer nanoparticles (SPNs) are reported as a new class of rapid, efficient, and cytocompatible labeling nanoagents for in vivo cell tracking. The phosphorylcholine coating results in efficient and rapid endocytosis and allows the SPN to enter cells within 0.5 h in complete culture medium apparently independent of the cell type, while its NIR fluorescence leads to a tissue penetration depth of 0.5 cm. In comparison to quantum dots and Cy5.5, the SPN is tolerant to physiologically ubiquitous reactive oxygen species (ROS), resulting in durable fluorescence both in vitro and in vivo. These desirable physical and physiological properties of the SPN permit cell tracking of human renal cell carcinoma (RCC) cells in living mice at a lower limit of detection of 10 000 cells with no obvious alteration of cell phenotype after 12 d. SPNs thus can provide unique opportunities for optimizing cellular therapy and deciphering pathological processes as a cell tracking label.
View details for DOI 10.1002/adhm.201300534
View details for PubMedID 24668903
View details for PubMedCentralID PMC4134769
Controlled self-assembly of small molecule gadolinium (Gd) complexes into nanoparticles (GdNPs) is emerging as an effective approach to design activatable magnetic resonance imaging (MRI) probes and amplify the r₁ relaxivity. Herein, we employ a reduction-controlled macrocyclization reaction and self-assembly to develop a redox activated Gd-based MRI probe for sensing a reducing environment. Upon disulfide reduction at physiological conditions, an acyclic contrast agent 1 containing dual Gd-chelates undergoes intramolecular macrocyclization to form rigid and hydrophobic macrocycles, which subsequently self-assemble into GdNPs, resulting in a ∼60% increase in r₁ relaxivity at 0.5 T. Probe 1 has high r₁ relaxivity (up to 34.2 mM(-1) s(-1) per molecule at 0.5 T) upon activation, and also shows a high sensitivity and specificity for MR detection of thiol-containing biomolecules.
View details for DOI 10.1021/bc500254g
View details for Web of Science ID 000340735900020
View details for PubMedCentralID PMC4140571
Reported herein is the design of fluorogenic probes specific for carbapenem-resistant Enterobacteriaceae (CRE) and they were designed based on stereochemically modified cephalosporin having a 6,7-trans configuration. Through experiments using recombinant β-lactamase enzymes and live bacterial species, these probes demonstrate the potential for use in the specific detection of carbapenemases, including metallo-β-lactamases in active bacterial pathogens.
View details for DOI 10.1002/anie.201402012
View details for PubMedID 24764125
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 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
View details for DOI 10.1002/smll.201301456
View details for PubMedID 24038954
Non-invasive detection of caspase-3/7 activity in vivo has provided invaluable predictive information regarding tumor therapeutic efficacy and anti-tumor drug selection. Although a number of caspase-3/7 targeted fluorescence and positron emission tomography (PET) imaging probes have been developed, there is still a lack of gadolinium (Gd)-based magnetic resonance imaging (MRI) probes that enable high spatial resolution detection of caspase-3/7 activity in vivo. Here we employ a self-assembly approach and develop a caspase-3/7 activatable Gd-based MRI probe for monitoring tumor apoptosis in mice. Upon reduction and caspase-3/7 activation, the caspase-sensitive nano-aggregation MR probe (C-SNAM: 1) undergoes biocompatible intramolecular cyclization and subsequent self-assembly into Gd-nanoparticles (GdNPs). This results in enhanced r1 relaxivity-19.0 (post-activation) vs. 10.2 mM(-1) s(-1) (pre-activation) at 1 T in solution-and prolonged accumulation in chemotherapy-induced apoptotic cells and tumors that express active caspase-3/7. We demonstrate that C-SNAM reports caspase-3/7 activity by generating a significantly brighter T1-weighted MR signal compared to non-treated tumors following intravenous administration of C-SNAM, providing great potential for high-resolution imaging of tumor apoptosis in vivo.
View details for DOI 10.1039/c4sc01392a
View details for Web of Science ID 000341195100020
View details for PubMedCentralID PMC4241271
Recent advances in molecular imaging and nanotechnology are providing new opportunities for biomedical imaging with great promise for the development of novel imaging agents. The unique optical, magnetic, and chemical properties of materials at the scale of nanometers allow the creation of imaging probes with better contrast enhancement, increased sensitivity, controlled biodistribution, better spatial and temporal information, multi-functionality and multi-modal imaging across MRI, PET, SPECT, and ultrasound. These features could ultimately translate to clinical advantages such as earlier detection, real time assessment of disease progression and personalized medicine. However, several years of investigation into the application of these materials to cancer research has revealed challenges that have delayed the successful application of these agents to the field of biomedical imaging. Understanding these challenges is critical to take full advantage of the benefits offered by nano-sized imaging agents. Therefore, this article presents the lessons learned and challenges encountered by a group of leading researchers in this field, and suggests ways forward to develop nanoparticle probes for cancer imaging. Published by Elsevier Ltd.
View details for DOI 10.1016/j.nantod.2013.06.001
View details for Web of Science ID 000326995500005
View details for PubMedCentralID PMC4240321
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: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.13130858/-/DC1.
View details for DOI 10.1148/radiol.13130858
View details for PubMedID 23850832
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 PubMedID 23881906
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 PubMedID 23943508
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 PubMedID 23859847
View details for Web of Science ID 000318694100376
View details for Web of Science ID 000314691400062
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
View details for PubMedCentralID PMC3645980
Recent advances in molecular imaging and nanotechnology are providing new opportunities for biomedical imaging with great promise for the development of novel imaging agents. The unique optical, magnetic, and chemical properties of materials at the scale of nanometers allow the creation of imaging probes with better contrast enhancement, increased sensitivity, controlled biodistribution, better spatial and temporal information, multi-functionality and multi-modal imaging across MRI, PET, SPECT, and ultrasound. These features could ultimately translate to clinical advantages such as earlier detection, real time assessment of disease progression and personalized medicine. However, several years of investigation into the application of these materials to cancer research has revealed challenges that have delayed the successful application of these agents to the field of biomedical imaging. Understanding these challenges is critical to take full advantage of the benefits offered by nano-sized imaging agents. Therefore, this article presents the lessons learned and challenges encountered by a group of leading researchers in this field, and suggests ways forward to develop nanoparticle probes for cancer imaging. Published by Elsevier Ltd.
View details for PubMedID 25419228
Many modern super-resolution imaging methods based on single-molecule fluorescence require the conversion of a dark fluorogen into a bright emitter to control emitter concentration. We have synthesized and characterized a nitro-aryl fluorogen which can be converted by a nitroreductase enzyme into a bright push-pull red-emitting fluorophore. Synthesis of model compounds and optical spectroscopy identify a hydroxyl-amino derivative as the product fluorophore, which is bright and detectable on the single-molecule level for fluorogens attached to a surface. Solution kinetic analysis shows Michaelis-Menten rate dependence upon both NADH and the fluorogen concentrations as expected. The generation of low concentrations of single-molecule emitters by enzymatic turnovers is used to extract subdiffraction information about localizations of both fluorophores and nitroreductase enzymes in cells. Enzymatic Turnover Activated Localization Microscopy (ETALM) is a complementary mechanism to photoactivation and blinking for controlling the emission of single molecules to image beyond the diffraction limit.
View details for DOI 10.1039/c2sc21074f
View details for Web of Science ID 000311971500023
View details for PubMedCentralID PMC3722058
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
View details for PubMedCentralID PMC3527090
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 β-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
An efficient method based on a rapid condensation reaction between 2-cyanobenzothiazole (CBT) and cysteine has been developed for (18)F-labeling of N-terminal cysteine-bearing peptides and proteins. An (18)F-labeled dimeric cRGD ([(18)F]CBTRGD(2)) has been synthesized with an excellent radiochemical yield (92% based on radio-HPLC conversion, 80% decay-corrected, and isolated yield) and radiochemical purity (>99%) under mild conditions using (18)F-CBT, and shown good in vivo tumor targeting efficiency for PET imaging. The labeling strategy was also applied to the site-specific (18)F-labeling of a protein, Renilla lucifierase (RLuc8) with a cysteine residue at its N-terminus. The protein labeling was achieved with 12% of decay-corrected radiochemical yield and more than 99% radiochemical purity. This strategy should provide a general approach for efficient and site-specific (18)F-labeling of various peptides and proteins for in vivo molecular imaging applications.
View details for DOI 10.1021/bc300273m
View details for Web of Science ID 000308833600021
View details for PubMedID 22845703
View details for PubMedCentralID PMC3447118
View details for Web of Science ID 000307019800188
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 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
View details for PubMedCentralID PMC3494413
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
View details for PubMedCentralID PMC3199358
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 αCSF1 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
View details for PubMedCentralID PMC3166957
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
Optical imaging is emerging as a powerful tool to study physiological, neurological, oncological, cell biological, molecular, developmental, immunological, and infectious processes. This unit describes the use of fluorescent reporters for biological organisms, components, or events. We describe the application of fluorescence imaging to examination of infectious processes, in particular subcutaneous and pulmonary bacterial infections, but the same approaches are applicable to nearly any infectious route. The strategies described use mycobacterial infections as an example, but nearly identical systems can be used for Pseudomonas, Legionella, Salmonella, Escherichia, Borrelia, and Staphylococus, suggesting that the approaches are generally applicable to nearly any infectious agent. Two strategies for fluorescence imaging are described: the first method uses reporter enzyme fluorescence (REF), and the second uses fluorescent proteins for fluorescence imaging. Methods are described in detail to facilitate successful application of these emerging technologies to nearly any experimental system.
View details for DOI 10.1002/9780471729259.mc02c03s21
View details for PubMedID 21538304
Bioluminescence imaging is a powerful technique to visualize and monitor biological processes in numerous systems. This unit describes two strategies for bioluminescence imaging that can be used to study bacterial infection in mice. One method is to express a luciferase gene in the bacteria; the second method is to use bacteria that express both a luciferase and β-lactamase along with a substrate containing caged luciferin, which is released by β-lactamase hydrolysis and reacts with luciferase to generate light. For both strategies, bioluminescent signals are imaged using an IVIS live animal imaging system (Caliper Life Sciences). The bioluminescence images are analyzed to localize bioluminescent bacteria, quantify signal, and determine the wavelengths of the signals produced. The correlation of bacterial numbers with signal intensity in vivo can be determined, allowing a quantitative measure of bacterial numbers in mice in real time. Methods are described in detail to facilitate successful application of these emerging technologies in nearly any experimental system.
View details for DOI 10.1002/9780471729259.mc02c04s21
View details for PubMedID 21538305
View details for DOI 10.1002/anie.201007018
View details for Web of Science ID 000292642600012
View details for PubMedID 21618367
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
View details for Web of Science ID 000295901600503
View details for DOI 10.1002/anie.201006140
View details for Web of Science ID 000288036300011
View details for PubMedID 21351335
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 PubMedID 20936809
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
View details for PubMedCentralID PMC3044212
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
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
View details for PubMedCentralID PMC2901431
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
View details for PubMedCentralID PMC2892383
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
View details for PubMedCentralID PMC3930333
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
View details for PubMedCentralID PMC2888877
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
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
View details for PubMedCentralID PMC2680468
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
View details for PubMedCentralID PMC2670992
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
View details for PubMedCentralID PMC3084659
View details for DOI 10.1002/anie.200903627
View details for Web of Science ID 000273093700011
View details for PubMedID 19924746
View details for PubMedCentralID PMC4878437
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
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
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
View details for PubMedCentralID PMC2553894
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
View details for PubMedCentralID PMC2529157
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
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
View details for PubMedCentralID PMC2174930
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 PubMedID 17704240
View details for DOI 10.1002/cbic.200700158
View details for Web of Science ID 000248067100002
View details for PubMedID 17492742
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
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
View details for DOI 10.1002/anie.200700280
View details for Web of Science ID 000247130400024
View details for PubMedID 17465433
View details for DOI 10.1002/anie.200701931
View details for Web of Science ID 000249752200012
View details for PubMedID 17676567
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
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
View details for PubMedCentralID PMC2553895
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
View details for DOI 10.1002/cbic.200600061
View details for Web of Science ID 000238171400011
View details for PubMedID 16671127
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
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 PubMedID 16501578
View details for DOI 10.1002/anie.200601197
View details for PubMedID 16807952
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 PubMedID 17406398
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
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
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
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
View details for Web of Science ID 000086204700003
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
View details for Web of Science ID 000079363300049
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 (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
View details for Web of Science ID A1997YD81500005
View details for Web of Science ID A1997YA23200002