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Bioconjugated Quantum Dots for In Vivo Molecular and CellularImagingAndrew M. Smith, Hongwei Duan, Aaron M. Mohs, and Shuming Nie*1Departments of Biomedical Engineering and Chemistry, Emory University and Georgia Institute ofTechnology, 101 Woodruff Circle, Suite 2001, Atlanta, GA 30322, USA.AbstractSemiconductor quantum dots (QDs) are tiny light-emitting particles on the nanometer scale, and areemerging as a new class of fluorescent labels for biology and medicine. In comparison with organicdyes and fluorescent proteins, they have unique optical and electronic properties, with size-tunablelight emission, superior signal brightness, resistance to photobleaching, and broad absorption spectrafor simultaneous excitation of multiple fluorescence colors. QDs also provide a versatile nanoscalescaffold for designing multifunctional nanoparticles with both imaging and therapeutic functions.When linked with targeting ligands such as antibodies, peptides or small molecules, QDs can be usedto target tumor biomarkers as well as tumor vasculatures with high affinity and specificity. Here wediscuss the synthesis and development of state-of-the-art QD probes and their use for molecular andcellular imaging. We also examine key issues for in vivo imaging and therapy, such as nanoparticlebiodistribution, pharmacokinetics, and toxicology.KeywordsQuantum dots; nanocrystals; nanoparticles; nanotechnology; fluorescence; molecular imaging;cellular imaging; drug delivery; cancer; biomarkers; toxicology1. IntroductionThe development of biocompatible nanoparticles for molecular imaging and targeted therapyis an area of considerable current interest [1–9]. The basic rationale is that nanometer-sizedparticles have functional and structural properties that are not available from either discretemolecules or bulk materials [1–3]. When conjugated with biomolecular affinity ligands, suchas antibodies, peptides or small molecules, these nanoparticles can be used to target malignanttumors with high specificity [10–13]. Structurally, nanoparticles also have large surface areasfor the attachment of multiple diagnostic (e.g., optical, radioisotopic, or magnetic) andtherapeutic (e.g., anticancer) agents. Recent advances have led to the development ofbiodegradable nanostructures for drug delivery [14–18], iron oxide nanocrystals for magneticresonance imaging (MRI) [19,20], luminescent quantum dots (QDs) for multiplexed moleculardiagnosis and in vivo imaging [21–25], as well as nanoscale carriers for siRNA delivery [26,27].*Author to whom correspondence should be addressed; e-mail: firstname.lastname@example.com's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain.NIH Public AccessAuthor ManuscriptAdv Drug Deliv Rev. Author manuscript; available in PMC 2009 August 17.Published in final edited form as:Adv Drug Deliv Rev. 2008 August 17; 60(11): 1226–1240. doi:10.1016/j.addr.2008.03.015.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript
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Due to their novel optical and electronic properties, semiconductor QDs are being intenselystudied as a new class of nanoparticle probe for molecular, cellular, and in vivo imaging [10–24]. Over the past decade, researchers have generated highly monodispersed QDs encapsulatedin stable polymers with versatile surface chemistries. These nanocrystals are brightlyfluorescent, enabling their use as imaging probes both in vitro and in vivo. In this article, wediscuss recent developments in the synthesis and modification of QD nanocrystals, and theiruse as imaging probes for living cells and animals. We also discuss the use of QDs as ananoscale carrier to develop multifunctional nanoparticles for integrated imaging and therapy.In addition, we describe QD biodistribution, pharmacokinetics, toxicology, as well as thechallenges and opportunities in developing nanoparticle agents for in vivo imaging and therapy.2. QD Chemistry and Probe DevelopmentQDs are nearly spherical semiconductor particles with diameters on the order of 2–10nanometers, containing roughly 200–10,000 atoms. The semiconducting nature and the size-dependent fluorescence of these nanocrystals have made them very attractive for use inoptoelectronic devices, biological detection, and also as fundamental prototypes for the studyof colloids and the size-dependent properties of nanomaterials [28]. Bulk semiconductors arecharacterized by a composition-dependent bandgap energy, which is the minimum energyrequired to excite an electron to an energy level above its ground state, commonly through theabsorption of a photon of energy greater than the bandgap energy. Relaxation of the excitedelectron back to its ground state may be accompanied by the fluorescent emission of a photon.Small nanocrystals of semiconductors are characterized by a bandgap energy that is dependenton the particle size, allowing the optical characteristics of a QD to be tuned by adjusting itssize. Figure 1 shows the optical properties of CdSe QDs at four different sizes (2.2 nm, 2.9nm, 4.1 nm, and 7.3 nm). In comparison with organic dyes and fluorescent proteins, QDs areabout 10–100 times brighter, mainly due to their large absorption cross sections, 100–1000times more stable against photobleaching, and show narrower and more symmetric emissionspectra. In addition, a single light source can be used to excite QDs with different emissionwavelengths, which can be tuned from the ultraviolet [29], throughout the visible and near-infrared spectra [30–33], and even into the mid-infrared [34]. However QDs aremacromolecules that are an order of magnitude larger than organic dyes, which may limit theiruse in applications in which the size of the fluorescent label must be minimized. Yet, thismacromolecular structure allows the QD surface chemistry and biological functionality to bemodified independently from its optical properties.2.1. QD SynthesisQD synthesis was first described in 1982 by Efros and Ekimov [35,36], who grew nanocrystalsand microcrystals of semiconductors in glass matrices. Since this work, a wide variety ofsynthetic methods have been devised for the preparation of QDs in different media, includingaqueous solution, high-temperature organic solvents, and solid substrates [28,37,38]. Colloidalsuspensions of QDs are commonly synthesized through the introduction of semiconductorprecursors under conditions that thermodynamically favor crystal growth, in the presence ofsemiconductor-binding agents, which function to kinetically control crystal growth andmaintain their size within the quantum-confinement size regime.The size-dependent optical properties of QDs can only be harnessed if the nanoparticles areprepared with narrow size distributions. Major progress toward this goal was made in 1993 byBawendi and coworkers [39], with the introduction of a synthetic method for monodisperseQDs made from cadmium sulfide (CdS), cadmium selenide (CdSe), or cadmium telluride(CdTe). Following this report, the synthetic chemistry of CdSe QDs quickly advanced,generating brightly fluorescent QDs that can span the visible spectrum. As a result, CdSe hasbecome the most common chemical composition for QD synthesis, especially for biologicalSmith et al.Page 2Adv Drug Deliv Rev. Author manuscript; available in PMC 2009 August 17.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript
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applications. Many techniques have been implemented to post-synthetically modify QDs forvarious purposes, such as coating with a protective inorganic shell [40,41], surfacemodification to render colloidal stability [42,43], and direct linkage to biologically activemolecules [44,45]. QD production has now become an elaborate molecular engineeringprocess, best exemplified in the synthesis of polymer-encapsulated (CdSe)ZnS (core)shellQDs. In this method, CdSe cores are prepared in a nonpolar solvent, and a shell of zinc sulfide(ZnS) is grown on their surfaces. The QDs are then transferred to aqueous solution throughencapsulation with an amphiphilic polymer, which can then be cross-linked to biomoleculesto yield targeted molecular imaging agents.In the design of a QD imaging probe, the selection of a QD core composition is determined bythe desired wavelength of emission. For example, CdSe QDs may be size-tuned to emit in the450–650 nm range, whereas CdTe can emit in the 500–750 nm range. QDs of this compositionare then grown to the appropriate wavelength-dependent size. In a typical synthesis of CdSe,a room-temperature selenium precursor (commonly trioctylphosphine-selenide ortributylphosphine-selenide) is swiftly injected into a hot (~300°C) solution containing both acadmium precursor (dimethylcadmium or cadmium oleate) and a coordinating ligand(trioctylphosphine oxide or hexadecylamine) under inert conditions (nitrogen or argonatmosphere). The cadmium and selenium precursors react quickly at this high temperature,forming CdSe nanocrystal nuclei. The coordinating ligands bind to metal atoms on the surfacesof the growing nanocrystals, stabilizing them colloidally in solution, and controlling their rateof growth. This injection of a cool solution quickly reduces the temperature of the reactionmixture, causing nucleation to cease. The remaining cadmium and selenium precursors thencan grow on the existing nuclei at a slower rate at lower temperature (240–270°C). Once theQDs have reached the desired size and emission wavelength, the reaction mixture may becooled to room temperature to arrest growth. The resulting QDs are coated in aliphaticcoordinating ligands and are highly hydrophobic, allowing them to be purified through liquid-liquid extractions or via precipitation from a polar solvent.Because QDs have high surface area to volume ratios, a large fraction of the constituent atomsare exposed to the surface, and therefore have atomic or molecular orbitals that are notcompletely bonded. These "dangling" orbitals serve as defect sites that quench QDfluorescence. For this reason, it is advantageous to grow a shell of another semiconductor witha wider bandgap on the core surface after synthesis to provide electronic insulation. The growthof a shell of ZnS on the surface of CdSe cores has been found to dramatically enhancephotoluminescence efficiency [40,41]. ZnS is also less prone to oxidation than CdSe,increasing the chemical stability of the QDs, and greatly decreasing their rate of oxidativephotobleaching [46]. As well, the Zn2+ atoms on the surface of the QD bind more strongly thanCd2+ to most basic ligands, such as alkyl phosphines and alkylamines, increasing the colloidalstability of the nanoparticles [47]. In a typical shell growth of ZnS on CdSe, the purified coresare again mixed with coordinating ligands, and heated to an elevated temperature (140–240°C). Molecular precursors of the shell, usually diethylzinc and hexamethyldisilathiane dissolvedin TOP, are then slowly added [40]. The (CdSe)ZnS nanocrystals may then be purified justlike the cores.More recently, it has become possible to widely engineer the fluorescence of QDs by changingthe material composition while maintaining the same size. The technological advances thatmade this possible were the development of alloyed QDs [29,30] and type-II heterostructures[32]. For example, homogeneously alloying the semiconductors CdTe and CdSe in differentratios allows one to prepare QDs of 5 nm diameter with emission wavelengths of 620 nm forCdSe, 700 nm for CdTe, and 800 nm for the CdSe0.34Te0.67 alloy [30]. Alternatively, type-IIQDs allow one to physically separate the charge carriers (the electron and its cationiccounterpart, known as the hole) into different regions of a QD by growing an appropriatelySmith et al.Page 3Adv Drug Deliv Rev. Author manuscript; available in PMC 2009 August 17.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript
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chosen material on the QD as a shell [32]. For example, both the valence and conduction bandenergy levels of CdSe are lower in energy than those of CdTe. This means that in aheterostructure composed of CdTe and CdSe domains, electrons will segregate to the CdSeregion to the lowest energy of the conduction band, whereas the hole will segregate to the CdTeregion, where the valence band is highest in energy. This will effectively decrease the bandgapdue to the smaller energy separating the two charge carriers, and emission will occur at a longerwavelength. By using different sizes of the core and different shell thicknesses, one canengineer QDs with the same size but different wavelengths of emission.2.2. Surface ModificationQDs produced in nonpolar solutions using aliphatic coordinating ligands are only soluble innonpolar organic solvents, making phase transfer an essential and nontrivial step for the QDsto be useful as biological reporters. Alternatively, QD syntheses have been performed directlyin aqueous solution, generating QDs ready to use in biological environments [48], but theseprotocols rarely achieve the level of monodispersity, crystallinity, stability, and fluorescentefficiency as the QDs produced in high-temperature coordinating solvents. Two generalstrategies have been developed to render hydrophobic QDs soluble in aqueous solution: ligandexchange, and encapsulation by an amphiphilic polymer. For ligand exchange, a suspensionof TOPO-coated QDs are mixed with a solution containing an excess of a heterobifunctionalligand, which has one functional group that binds to the QD surface, and another functionalgroup that is hydrophilic. Thereby, hydrophobic TOPO ligands are displaced from the QDthrough mass action, as the new bifunctional ligand adsorbs to render water solubility. Usingthis method, (CdSe)ZnS QDs have been coated with mercaptoacetic acid and (3-mercaptopropyl) trimethoxysilane, both of which contain basic thiol groups to bind to the QDsurface atoms, yielding QDs displaying carboxylic acids or silane monomers, respectively[44,45]. These methods generate QDs that are useful for biological assays, but ligand exchangeis commonly associated with decreased fluorescence efficiency and a propensity to aggregateand precipitate in biological buffers. More recently it has been shown that these problems canbe alleviated by retaining the native coordinating ligands on the surface, and covering thehydrophobic QDs with amphiphilic polymers [10,23,49]. This encapsulation method yieldsQDs that can be dispersed in aqueous solution and remain stable for long periods of time dueto a protective hydrophobic bilayer surrounding each QD through hydrophobic interactions.No matter what method is used to suspend the QDs in aqueous buffers, they should be purifiedfrom residual ligands and excess amphiphiles before use in biological assays, usingultracentrifugation, dialysis, or filtration. Also, when choosing a water solubilization method,it should be noted that many biological and physical properties of the QDs may be affected bythe surface coating, and the overall physical dimensions of the QDs are dependent on thecoating thickness. Typically the QDs are much larger when coated with amphiphiles, comparedto those coated with a monolayer of ligand.2.3. BioconjugationWater-soluble QDs may be cross-linked to biomolecules such antibodies, oligonucleotides, orsmall molecule ligands to render them specific to biological targets. This may be accomplishedusing standard bioconjugation protocols, such as the coupling of maleimide-activated QDs tothe thiols of reduced antibodies [22]. The reactivities of many types of biomolecules have beenfound to remain after conjugation to nanoparticles surfaces, although possibly at a decreasedbinding strength. The optimization of surface immobilization of biomolecules is currently anactive area of research [50,51]. The surfaces of QDs may also be modified with bio-inert,hydrophilic molecules such as polyethylene glycol, to eliminate possible nonspecific binding,or to decrease the rate of clearance from the bloodstream following intravenous injection. QDshave also emerged as a new class of sensor, mediated by energy transfer to organic dyes(fluorescence resonance energy transfer, FRET) [52–54]. It has also recently been reportedSmith et al.Page 4Adv Drug Deliv Rev. Author manuscript; available in PMC 2009 August 17.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript
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that QDs can emit fluorescence without an external source of excitation when conjugated toenzymes that catalyze bioluminescent reactions, due to bioluminescence resonance energytransfer (BRET) [55].Figure 2 depicts the most commonly used and technologically advanced QD probes.Biologically nonfunctional QDs may be prepared by using a variety of methods. As shownfrom left to right (top), QDs coated with a monolayer of hydrophilic thiols (e.g. mercaptoaceticacid) are generally stabilized ionically in solution [45]; QDs coated with a cross-linked silicashell can be readily modified with a variety of organic functionalities using well developedsilane chemistry [44]; QDs encapsulated in amphiphilic polymers form highly stable, micelle-like structures [23,49]; and any of these QDs may be modified to contain polyethylene glycol(PEG) to decrease surface charge and increase colloidal stability [56]. Also, water-soluble QDsmay be covalently or electrostatically bound to a wide range of biologically active moleculesto render specificity to a biological target. As shown in Figure 2 (middle), QDs conjugated tostreptavidin may be readily bound to many biotinylated molecules of interest with high affinity[23]; QDs conjugated to antibodies can yield specificity for a variety of antigens, and are oftenprepared through the reaction between reduced antibody fragments with maleimide-PEG-activated QDs [22,57]; QDs cross-linked to small molecule ligands, inhibitors, peptides, oraptamers can bind with high specificity to many different cellular receptors and targets [58,59]; and QDs conjugated to cationic peptides, such as the HIV Tat peptide, can quicklyassociate with cells and become internalized via endocytosis [60]. Further, QDs have been usedto detect the presence of biomolecules using intricate probe designs incorporating energydonors or acceptors. For example, QDs can be adapted to sense the presence of the sugarmaltose by conjugating the maltose binding protein to the nanocrystal surface (Figure 2,bottom) [53]. By initially incubating the QDs with an energy-accepting dye that is conjugatedto a sugar recognized by the receptor, excitation of the QD (blue) yields little fluorescence, asthe energy is nonradiatively transferred (grey) to the dye. Upon addition of maltose, thequencher-sugar conjugate is displaced, restoring fluorescence (green) in a concentration-dependent manner. QDs can also be sensors for specific DNA sequences [52]. By mixing thessDNA to be detected with (a) an acceptor fluorophores conjugated to a DNA fragmentcomplementary to one end of the target DNA and (b) a biotinylated DNA fragmentcomplementary to the opposite end of the target DNA, these nucleotides hybridize to yield abiotin-DNA-fluorophore conjugate. Upon mixing this conjugate with QDs, QD fluorescence(green) is quenched via nonradiative energy transfer (grey) to the fluorophore conjugate. Thisdye acceptor then becomes fluorescent (red), specifically and quantitatively indicating thepresence of the target DNA. Finally, QDs conjugated to the luciferase enzyme cannonradiatively accept energy from the enzymatic bioluminescent oxidation of luciferins on theQD surface, exciting the QDs without the need for external illumination [55].3. Live-Cell ImagingResearchers have achieved considerable success in using QDs for in vitro bioassays [61,62],labeling fixed cells [23] and tissue specimens [63,64], and for imaging membrane proteins onliving cells [58,65]. However, only limited progress has been made in developing QD probesfor imaging inside living cells. A major problem is the lack of efficient methods for deliveringmonodispersed (that is, single) QDs into the cytoplasms of living cells. A common observationis that QDs tend to aggregate inside cells, and are often trapped in endocytotic vesicles suchas endosomes and lysosomes.3.1. Imaging and Tracking of Membrane ReceptorsQD bioconjugates have been found to be powerful imaging agents for specific recognition andtracking of plasma membrane antigens on living cells. In 2002 Lidke et al. coupled red-lightemitting (CdSe)ZnS QDs to epidermal growth factor, a small protein with a specific affinitySmith et al.Page 5Adv Drug Deliv Rev. Author manuscript; available in PMC 2009 August 17.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript
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for the erbB/HER membrane receptor [58]. After addition of these conjugates to culturedhuman cancer cells, receptor-bound QDs could be identified at the single-molecule level (singleQDs may be distinguished from aggregates because the fluorescent intensity from discrete dotsis intermittent, or "blinking"). The bright, stable fluorescence emitted from these QDs allowedthe continuous observation of protein diffusion on the cellular membrane, and could even bevisualized after the proteins were internalized. Dahan et al. similarly reported that QDsconjugated to an antibody fragment specific for glycine receptors on the membranes of livingneurons allowed tracking of single receptors [65]. These conjugates showed superiorphotostability, lateral resolution, and sensitivity relative to organic dyes. These applicationshave inspired the use QDs for monitoring other plasma membrane proteins such as integrins[50,66], tyrosine kinases [67,68], G-protein coupled receptors [69], and membrane lipidsassociated with apoptosis [70,71]. As well, detailed procedures for receptor labeling andvisualization of receptor dynamics with QDs have recently been published [72,73], and newtechniques to label plasma membrane proteins using versatile molecular biology methods havebeen developed [74,75].3.2. Intracellular Delivery of QDsA variety of techniques have been explored to label cells internally with QDs, using passiveuptake, receptor-mediated internalization, chemical transfection, and mechanical delivery.QDs have been loaded passively into cells by exploiting the innate capacity of many cell typesto uptake their extracellular space through endocytosis [76–78]. It has been found that theefficiency of this process may be dramatically enhanced by coupling the QDs to membranereceptors. This is likely due to the avidity-induced increase in local concentration of QDs atthe surface of the cell, as well as an active enhancement caused by receptor-inducedinternalization [58,77,79]. However, these methods lead to sequestration of aggregated QDsin vesicles, showing strong colocalization with membrane dyes. Although these QDs cannotdiffuse to specific intracellular targets, this is a simple way to label cells with QDs, and an easymethod to fluorescently image the process of endocytosis. Nonspecific endocytosis was alsoutilized by Parak et al. to fluorescently monitor the motility of cells on a QD-coated substrate[78]. The path traversed by each cell became dark, and the cells increased in fluorescence asthey took up more QDs. Chemically-mediated delivery enhances plasma membranetranslocation with the use of cationic lipids or peptides, and was originally developed for theintracellular delivery of a wide variety of drugs and biomolecules [60,80–83]. The efficacy ofthese carriers for the intracellular deliver of QDs is discussed below (Section 3.3 and Section3.4). Mechanical delivery methods include microinjection of QDs into individual cells, andelectroporation of cells in the presence of QDs. Microinjection has been reported to deliverQDs homogeneously into the cytoplasms of cells [49,83], however this method is of lowstatistical value, as careful manipulation of single cells prevents the use of large sample sizes.Electroporation makes use of the increased permeability of cellular membranes under pulsedelectric fields to deliver QDs, but this method was reported to result in aggregation of QDs inthe cytoplasm [83], and generally results in widespread cell death.Despite the current technical challenges, QDs are garnering interest as intracellular probes dueto their intense, stable fluorescence, and recent reports have demonstrated that intracellulartargeting is not far off. In 2004, Derfus et al. demonstrated that QDs conjugated to organelle-targeting peptides could specifically stain either cellular mitochondria or nuclei, followingmicroinjection into fibroblast cytoplasms [83]. Similarly, Chen et al. targeted peptide-QDconjugates to cellular nuclei, using electroporation to overcome the plasma membrane barrier[60]. These schemes have resulted in organelle-level resolution of intracellular targets for livingcells, yielding fluorescent contrast of vesicles, mitochondria, and nuclei, but not the ability tovisualize single molecules. Recently Courty et al. demonstrated the capacity to imageindividual kinesin motors in HeLa cells using QDs delivered into the cytoplasm via osmoticSmith et al. Page 6Adv Drug Deliv Rev. Author manuscript; available in PMC 2009 August 17.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript
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lysis of pinocytotic vesicles [84]. By incubating the cells in a hypertonic solution containingQDs, water efflux resulted in membrane invagination and pinocytosis, trapping extracellularQDs in endosomal vesicles. Then a brief incubation in hypotonic medium induced intracellularwater influx, rupturing the newly formed vesicles, and releasing single QDs into the cytosol.All of the QDs were observed to undergo random Brownian motion in the cytoplasm. Howeverif these QDs were first conjugated to kinesin motor proteins, a significant population of theQDs exhibited directional motion. The velocity of the directed motion and its processivity(average time before cessation of directed motion) were remarkably close to those observedfor the motion of these conjugates on purified microtubules in vitro. Although this workmanaged to overcome the plasma membrane diffusion barrier, it highlighted a differentproblem fundamental to intracellular imaging of living cells, which is the impossibility ofremoving probes that have not found their target. In this report, the behavior of the QDs wassufficient to distinguish bound QDs from those that were not bound, but this will not be thecase for the majority of other protein targets. Without the ability to wash away unbound probes,which is a crucial step for intracellular labeling of fixed, permeabilized cells, the need foractivateable probes that are 'off' until they reach their intended target is apparent. HoweverQDs have already found a niche for quantitative monitoring of motor protein transport and fortracking the fate of internalized receptors, allowing the study of downstream signalingpathways in real time with high signal-to-noise and high temporal and spatial resolution [58,67,68,85,86].3.3. Tat-QD ConjugatesCell-penetrating peptides are a class of chemical transfectants that have garnered widespreadinterest due to the high transfection efficiency of their conjugated cargo, versatility ofconjugation, and low toxicity. For this reason, cell-penetrating peptides such as polyarginineand Tat have been investigated for their capacity to deliver QDs into living cells [81,85,87],but the delivery mechanism and the behavior of intracellular QDs are still a matter of debate.Considerable effort has been devoted to understanding the delivery mechanism of thesecationic carrier, especially the HIV-1-derived Tat peptide, which has emerged as a widely usedcellular delivery vector [88–93]. The delivery process was initially thought to be independentof endocytosis because of its apparent temperature-independence [89–93]. However, laterresearch showed that the earlier work failed to exclude the Tat peptide conjugated cargos boundto plasma membranes, and was largely an artifact caused by cellular fixation. More recentstudies based on improved experimental methods indicate that Tat peptide-mediated deliveryoccurs via macropinocytosis [94], a fluid-phase endocytosis process that is initiated by thebinding of Tat-QD to the cell surface [90]. These new results, however, did not shed any lighton the downstream events or the intracellular behavior of the internalized cargo. This kind ofdetailed and mechanistic investigation would be possible with QDs, which are sufficientlybright and photostable for extended imaging and tracking of intracellular events. In addition,most previous studies on Tat peptide-mediated delivery are based on the use of small dyemolecules and proteins as cargo [89–93], so it is not clear whether larger nanoparticles wouldundergo the same processes of cellular uptake and transport. This understanding is needed forthe design and development of imaging and therapeutic nanoparticles for biology and medicine.Ruan et al. have recently used Tat peptide-conjugated QDs (Tat-QDs) as a model system toexamine the cellular uptake and intracellular transport of nanoparticles in live cells [95]. Theauthors used a spinning-disk confocal microscope for dynamic fluorescence imaging ofquantum dots in living cells at 10 frames per second. The results indicate that the peptide-conjugated QDs are internalized by macropinocytosis, in agreement with the recent work ofDowdy and coworkers [90]. It is interesting, however, that the internalized Tat-QDs aretethered to the inner surface of vesicles, and are trapped in intracellular organelles. Animportant finding is that the QD-loaded vesicles are actively transported by molecularSmith et al.Page 7Adv Drug Deliv Rev. Author manuscript; available in PMC 2009 August 17.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript
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machines (such as dyneins) along microtubule tracks to an asymmetric perinuclear regioncalled the microtubule organizing center (MTOC) [96]. Furthermore, it was found that Tat-QDs strongly bind to cellular membrane structures such as filopodia, and that large QD-containing vesicles are able to pinch off from the tips of filopodia. These results not onlyprovide new insight into the mechanisms of Tat peptide-mediated delivery, but also areimportant for the development of nanoparticle probes for intracellular targeting and imaging.3.4. QDs with Endosome-Disrupting CoatingsDuan and Nie [97] developed a new class of cell-penetrating quantum dots (QDs) based on theuse of multivalent and endosome-disrupting (endosomolytic) surface coatings (Figure 3).Hyperbranched copolymer ligands such as PEG-grafted polyethylenimine (PEI-g-PEG) werefound to encapsulate and stabilize luminescent quantum dots in aqueous solution through directligand binding to the QD surface. Due to the cationic charges and a "proton spongeeffect" [98–100] associated with multivalent amine groups, these QDs could penetrate cellmembranes and disrupt endosomal organelles in living cells. This mechanism arises from thepresence of a large number of weak bases (with buffering capabilities at pH 5–6), which leadto proton absorption in acidic organelles, and an osmotic pressure buildup across the organellemembrane [100]. This osmotic pressure causes swelling and/or rupture of the acidic endosomesand a release of the trapped materials into the cytoplasm. PEI and other polycations are knownto be cytotoxic, however the grafted PEG segment was found to significantly reduce the toxicityand improve the overall nanoparticle stability and biocompatibility. In comparison withprevious QDs encapsulated with amphiphilic polymers, the cell-penetrating QDs were smallerin size and exceedingly stable in acidic environments [56]. Cellular uptake and imaging studiesrevealed that these dots were rapidly internalized by endocytosis, and the pathways of the QDsinside the cells showed dependence on the number of PEG grafts of the polymer ligands. Whilehigher PEG content led to QD sequestration in vesicles, the QDs coated by PEI-g-PEG withfewer PEG grafts are able to escape from endosomes and release into the cytoplasm.Lovric et al. [101] recently reported that very small QDs (2.2 nm) coated with small moleculeligands (cysteamine) spontaneously translocated to the nuclei of murine microglial cellsfollowing cellular uptake through passive endocytosis. In contrast, larger QDs (5.5 nm) andsmall QDs bound to albumin remained in the cytosol only. This is fascinating because theseQDs could not only escape from endocytotic vesicles, but were also subjected to an unknowntype of active machinery that attracted the QDs to the nucleus. Nabiev et al. [102] studied asimilar trend of size-dependent QD segregation in human macrophages, and found that smallQDs may target nuclear histones and nucleoli after active transport across the nuclearmembrane. They found that the size cut-off for this effect was around 3.0 nm. Larger QDseventually ended up in vesicles in the MTOC region, although some QDs were found to befree in the cytoplasm. This group proposed that the proton sponge effect was also responsiblefor endosomal escape, as small carboxyl-coated QDs could buffer in the pH 5–7 range. Theseinsights are important for the design and development of nanoparticle agents for intracellularimaging and therapeutic applications.4. In Vivo Animal ImagingCompared to the study of living cells in culture, different challenges arise with the increase incomplexity to a multicellular organism, and with the accompanying increase in size. Unlikemonolayers of cultured cells and thin tissue sections, tissue thickness becomes a major concernbecause biological tissue attenuates most signals used for imaging. Optical imaging, especiallyfluorescence imaging, has been used in living animal models, but it is still limited by the poortransmission of visible light through biological tissue. It has been suggested that there is a near-infrared optical window in most biological tissue that is the key to deep-tissue optical imaging[103]. The rationale is that Rayleigh scattering decreases with increasing wavelength, and thatSmith et al. Page 8Adv Drug Deliv Rev. Author manuscript; available in PMC 2009 August 17.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript
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the major chromophores in mammals, hemoglobin and water, have local minima in absorptionin this window. Few organic dyes are available that emit brightly in this spectral region, andthey suffer from the same photobleaching problems as their visible counterparts, although thishas not prevented their successful use as contrast agents for living organisms [104]. One of thegreatest advantages of QDs for imaging in living tissue is that their emission wavelengths canbe tuned throughout the near-infrared spectrum by adjusting their composition and size,resulting in photostable fluorophores that are stable in biological buffers [24].4.1 Biodistribution of QDsFor most in vivo imaging applications using QDs and other nanoparticle contrast agents,systemic intravenous delivery into the bloodstream will be the main mode of administration.For this reason, the interaction of the nanoparticles with the components of plasma, the specificand nonspecific adsorption to blood cells and the vascular endothelium, and the eventualbiodistribution in various organs are of great interest. Immediately upon exposure to blood,QDs may be quickly adsorbed by opsonins, in turn flagging them for phagocytosis. In addition,platelet coagulation may occur, the complement system may be activated, or the immunesystem can be stimulated or repressed (Figure 4). Although it is important for each of thesepotential biological effects to be addressed in detail, so far there are no studies that directlyexamine blood or immune system biocompatibility of QDs in vivo or ex vivo. However, a recentreview article by Dobrovolskaia and McNeil addresses the immunological properties ofpolymeric, liposomal, carbon-based, and magnetic nanoparticles [105]. Considering the manyfactors that may affect systemically administered QDs, such as size, shape, charge, targetingligands, etc., the two most important parameters that affect biodistribution are likely size andthe propensity for serum protein adsorption.The number of papers published on quantum dot pharmacokinetics and biodistribution islimited, but several common trends can be identified. It has been consistently reported thatQDs are taken up nonspecifically by the reticuloendothelial system (RES), including the liverand spleen, and the lymphatic system [106–108]. These findings are not necessarily intrinsicto QDs, but are strictly predicated upon the size of the QDs and their surface coatings. Ballouand coworkers reported that (CdSe)ZnS QDs were rapidly removed from the bloodstream intoorgans of the RES, and remained there for at least 4 months with detectable fluorescence[107]. TEM of these tissues revealed that these QDs retained their morphology, suggesting thatgiven the proper coating, QDs are stable in vivo for very long periods of time withoutdegradation into their potentially toxic elemental components. A complimentary work byFischer, et al. showed that nearly 100% of albumin-coated QDs were removed from circulationand sequestered in the liver within hours after a tail vein injection, much faster than QDs thatwere not bound to albumin [108]. Within the liver, QDs conjugated to albumin were primarilyassociated with Kupffer cells (resident macrophages). From a clinical perspective, it may bepossible to completely inhibit the accumulation of QDs and avoid potential toxic effects if theyare within the size range of renal excretion. Recent publications have focused on this insight.Frangioni and coworkers demonstrated that the renal clearance of quantum dots is closelyrelated to the hydrodynamic diameter of the nanoparticle and the renal filtration threshold (~5–6 nm) [109]. Of equal importance to the QD size, is that the surface does not promote proteinadsorption, which could significantly increase QD size above that of the renal threshold, andpromote phagocytosis. However, it is unlikely that even small QDs could be entirely eliminatedfrom the kidneys, as it has also been found that small QDs (~9 nm) may directly extravasateout of blood vessels, into interstitial fluid [110].For targeted imaging, specific modulation of the biodistribution of QD contrast agents is themain goal. One way to increase the probability of bioaffinity ligand-specific distribution is toincrease the circulation time of the contrast agent in the bloodstream. QD structure and surfaceSmith et al.Page 9Adv Drug Deliv Rev. Author manuscript; available in PMC 2009 August 17.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript
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properties have been found to strongly impact the plasma half-life. It was demonstrated byBallou et al. [107] that the lifetime of anionic, carboxylated QDs in the bloodstream of mice(4.6 minutes half-life) is significantly increased if the QDs are coated with PEG polymer chains(71 minutes half-life). This effect has also been documented for other types of nanoparticlesand small molecules, in part due to decreased nonspecific adsorption of the nanoparticles, anincrease in size, and decreased antigenicity [111]. In a more recent study using perfused porcineskin in vitro, Lee, et al. demonstrated that carboxylated QDs were extracted more rapidly fromcirculation, and had greater tissue deposition than PEG coated QDs [112]. It is important tonote that a bioaffinity molecule may also be prone to RES uptake, despite a strong affinity forits intended target. For example, Jayagopal et al. reported that QD-antibody conjugates havea significantly longer circulation time if the Fc antibody regions (non-antigen binding domains)are immunologically shielded to reduce nonspecific interactions [113].4.2. In Vivo Vascular ImagingOne of the most immediately successful applications of QDs in vivo has been their use ascontrast agents for the two major circulatory systems of mammals, the cardiovascular systemand the lymphatic system. In 2003, Larson et. al demonstrated that green-light emitting QDsremained fluorescent and detectable in capillaries of adipose tissue and skin of a living mousefollowing intravenous injection [114]. This work was aided by the use of near-infrared two-photon excitation for deeper penetration of excitation light, and by the extremely large two-photon cross-sections of QDs, 100–20,000 times that of organic dyes [115]. In other work,Lim et al. used near-infrared QDs to image the coronary vasculature of a rat heart [116], andSmith et al. imaged the blood vessels of chicken embryos with a variety of near-infrared andvisible QDs [117]. The later report showed that QDs could be detected with higher sensitivitythan traditionally used fluorescein-dextran conjugates, and resulted in a higher uniformity inimage contrast across vessel lumena. Jayagopal et al. [113] recently demonstrated the potentialfor QDs to serve as molecular imaging agents for vascular imaging. Spectrally distinct QDswere conjugated to three different cell adhesion molecules (CAMs), and intravenously injectedin a diabetic rat model. Fluorescence angiography of the retinal vasculature revealed CAM-specific increases in fluorescence, and allowed imaging of the inflammation-specific behaviorof individual leukocytes, as they freely floated in the vessels, rolled along the endothelium,and underwent leukostasis. The unique spectral properties of QDs allowed the authors tosimultaneously image up to four spectrally distinct QD tags.For imaging of the lymphatic system, the overall size of the probe is an important parameterfor determining biodistribution and clearance. For example, Kim et al. [24] intradermallyinjected ~16–19 nm near-infrared QDs in mice and pigs. QDs translocated to sentinel lymphnodes, likely due to a combination of passive flow in lymphatic vessels, and active migrationof dendritic cells that engulfed the nanoparticles. Fluorescence contrast of these nodes couldbe observed up to 1 cm beneath the skin surface. It was found that if these QDs were formulatedto have a smaller overall hydrodynamic size (~9 nm), they could migrate further into thelymphatic system, with up to 5 nodes showing fluorescence [110]. This technique could havegreat clinical impact due to the quick speed of lymphatic drainage and the ease of identificationof lymph nodes, enabling surgeons to fluorescently identify and excise nodes draining fromprimary metastatic tumors for the staging of cancer. This technique has been used to identifylymph nodes downstream from the lungs [106,118], esophagus [119], and from subcutaneoustumors [120]. Recently the multiplexing capabilities of QDs have been exploited for mappinglymphatic drainage networks. By injection of QDs of different color at different intradermallocations, these QDs could be fluorescently observed to drain to common nodes [121], or upto 5 different nodes in real time [122]. A current problem is that a major fraction of the QDsremain at the site of injection for an unknown length of time [123].Smith et al.Page 10Adv Drug Deliv Rev. Author manuscript; available in PMC 2009 August 17.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript
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4.3. In Vivo Tracking of QD-Loaded CellsCells can also be loaded with QDs in vitro, and then administered to an organism, providinga means to identify the original cells and their progeny within the organism. This was firstdemonstrated on a small organism scale by microinjecting QDs into the cytoplasms of singlefrog embryos [49]. As the embryos grew, the cells divided, and each cell that descended fromthe original labeled cell retained a portion of the fluorescent cytoplasm, which could befluorescently imaged in real time under continuous illumination. In reports by Hoshino et al.[124] and Voura et al. [82], cells loaded with QDs were injected intravenously into mice, andtheir distributions in the animals were later determined through tissue dissection, followed byfluorescence imaging. Also Gao et al. loaded human cancer cells with QDs, and injected thesecells subcutaneously in an immune-compromised mouse [10]. The cancer cells divided to forma solid tumor, which could be visualized fluorescently through the skin of the mouse. Rosenet al. recently reported that human mesenchymal stem cells loaded with QDs could beimplanted into an extracellular matrix patch for use as a regenerative implant for canine heartswith a surgically-induced defect [125]. Eight weeks following implantation, it was found thatthe QDs remained fluorescent within the cells, and could be used to track the locations andfates of these cells. This group also directly injected QD-labeled stem cells into the caninemyocardium, and used the fluorescence signals in cardiac tissue sections to elaboratelyreconstruct the locations of these cells in the heart. With reports that cells may be labeled withQDs at a high degree of specificity [80,81], it is foreseeable that multiple types of cells maybe simultaneously monitored in living organisms, and also identified using their distinct opticalcodes.4.4. In Vivo Tumor ImagingImaging of tumors presents a unique challenge not only because of the urgent need for sensitiveand specific imaging agents of cancer, but also because of the unique biological attributesinherent to cancerous tissue. Blood vessels are abnormally formed during tumorinducedangiogenesis, having erratic architectures and wide endothelial pores. These pores are largeenough to allow the extravasation of large macromolecules up to ~400 nm in size, whichaccumulate in the tumor microenvironment due to a lack of effective lymphatic drainage[126–129]. This "enhanced permeability and retention" effect (EPR effect) has inspired thedevelopment of a variety of nanotherapeutics and nanoparticulates for the treatment andimaging of cancer (Figure 5). Because cancerous cells are effectively exposed to theconstituents of the bloodstream, their surface receptors may also be used as active targets ofbioaffinity molecules. In the case of imaging probes, active targeting of cancer antigens(molecular imaging) has become an area of tremendous interest to the field of medicine becauseof the potential to detect early stage cancers and their metastases. QDs hold great promise forthese applications mainly due to their intense fluorescent signals and multiplexing capabilities,which could allow a high degree of sensitivity and selectivity in cancer imaging with multipleantigens.The first steps toward this goal were undertaken in 2002 by Akerman et al., who conjugatedQDs to peptides with affinity for various tumor cells and their vasculatures [130]. Afterintravenous injection of these probes into tumor-bearing mice, microscopic fluorescenceimaging of tissue sections demonstrated that the QDs specifically homed to the tumorvasculature. In 2004 Gao et al. demonstrated that tumor targeting with QDs could generatetumor contrast on the scale of whole-animal imaging [10]. QDs were conjugated to an antibodyagainst the prostate-specific membrane antigen (PSMA), and intravenously injected into micebearing subcutaneous human prostate cancers. Tumor fluorescence was significantly greaterfor the actively targeted conjugates compared to nonconjugated QDs, which also accumulatedpassively though the EPR effect. Using similar methods, Yu et al. were able to actively targetand image mouse models of human liver cancer with QDs conjugated to an antibody againstSmith et al.Page 11Adv Drug Deliv Rev. Author manuscript; available in PMC 2009 August 17.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript
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alpha-fetoprotein [131], and Cai et al. showed that labeling QDs with RGD peptidesignificantly increased their uptake in human glioblastoma tumors [132].The development of clinically relevant QD contrast agents for in vivo imaging is certain toencounter many roadblocks in the near future (see Section 5), however QDs can currently beused as powerful imaging agents for the study of the complex anatomy and pathophysiologyof cancer in animal models. Stroh et al. [133] demonstrated that QDs greatly enhance currentintravital microscopy techniques for the imaging of tumor microenvironment. The authors usedQDs as fluorescent contrast agents for blood vessels using two-photon excitation, andsimultaneously captured images of extracellular matrix from autofluorescent collagen, andperivascular cell contrast from fluorescent protein expression. The use of QDs allowed starkcontrast between the tumor constituents due to their intense brightness, tunable wavelengths,and reduced propensity to extravasate into the tumor, compared to organic dye conjugates. Inthis work, the authors also used QD-tagged beads with variable sizes to model the size-dependent distribution of various nanotherapeutics in tumors. As well, the authorsdemonstrated that bone marrow lineage-negative cells, which are thought to be progenitors forneovascular endothelium, were labeled ex vivo with QDs and imaged in vivo as they flowedand adhered to tumor blood vessels following intravenous administration. More recently, Tadaet al. used QDs to study the biological processes involved in active targeting of nanoparticles.The authors used QDs labeled with an antibody against human epidermal growth factorreceptor 2 (HER2) to target human breast cancer in a mouse model [134]. Through intravitalfluorescence microscopy of the tumor following systemic QD administration, the authors coulddistinctly observe individual QDs as they circulated in the bloodstream, extravasated into thetumor, diffused in extracellular matrix, bound to their receptors on tumor cells, and thentranslocated into the perinuclear region of the cells. The combination of sensitive QD probeswith powerful techniques like intravital microscopy and in vivo animal imaging could soonlead to major breakthroughs in the current understanding of tumor biology, improve earlydetection schemes, and guide new therapeutic designs.5. Nanoparticle ToxicityGreat concern has been raised over the use of quantum dots in living cells and animals due totheir chemical composition of toxic heavy metal atoms (e.g. Cd, Hg, Pb, As, Pb). Presently themost commonly used QDs contain divalent cadmium, a nephrotoxin in its ionic form. Althoughthis element is incorporated into a nanocrystalline core, surrounded by biologically inert zincsulfide, and encapsulated within a stable polymer, it is still unclear if these toxic ions willimpact the use of QDs as clinical contrast agents. It may be of greater concern that QDs, andmany other types of nanoparticles, have been found to aggregate, bind nonspecifically tocellular membranes and intracellular proteins, and induce the formation of reactive oxygenspecies. As previously stated, QDs larger than the renal filtration threshold quickly accumulatein the reticuloendothelial system following intravenous administration. The eventual fate ofthese nanoparticles is of vital importance, but so far has yet to be elucidated.5.1. Cadmium ToxicityIn the only long-term, quantitative study on QD biodistribution to date, Yang, et al. showedthat after intravenous administration of cadmium-based QDs, the concentration of cadmiumin the liver and kidneys gradually increased over the course of 28 days, as determined via ICP-MS [135]. The cadmium levels in the kidneys eventually reached nearly 10% of the injecteddose, compared to 40% in the liver. Although it was not apparent if the cadmium was in theform of a free ion, or remained in the nanocrystalline form, fluorescence microscopy revealedthe presence of intact QDs in both the liver and kidneys. However the redistribution of thecadmium over time may signify the degradation of QDs in vivo, since the natural accumulationsites of Cd2+ ions are the liver and kidneys [79,136,137]. In acute exposures, free cadmiumSmith et al.Page 12Adv Drug Deliv Rev. Author manuscript; available in PMC 2009 August 17.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript
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also may be redistributed to the kidneys via hepatic production of metallothionein [138].Whether or not this is the specific mechanism observed in this report should be the focus ofdetailed in vivo validation studies. Nevertheless, these findings stress that (a) QD size andnonspecific protein interaction should be minimized to allow renal filtration, or else QDs willinevitably accumulate in organs and tissues of the RES, lung, and kidney, and (b) the potentialrelease of the elements of the QD and their distribution in specific organs, tissues, cell types,and subcellular locations must be well understood.In general, most in vitro studies on the exposure of cells to QDs have attempted to relatecytotoxic events to the release of potentially toxic elements and/or to the size, shape, surface,and cellular uptake of QDs. Because the toxicity of Cd2+ ions is well documented, a significantbody of work has focused on the intracellular release of free cadmium from the QDs. Cd2+ions can be released through oxidative degradation of the QD, and may then bind to sulfhydrylgroups on a variety of intracellular proteins, causing decreased functionality in manysubcellular organelles [139]. Several groups have investigated methods to quantify the amountof free Cd2+ ions released from QDs, either intracellularly or into culture media, by ICP-MSor fluorometric assays, leading to the conclusion that Cd2+ release correlates with cytotoxicmanifestations [79,140,141]. Derfus, et al. facilitated oxidative release of cadmium ions fromthe surface of CdSe QDs by exposure to air or ultraviolet irradiation [79]. Under theseconditions, CdSe QD cores coated with small thiolate ligands were toxic. Capping these QDswith ZnS shells or coating with BSA rendered the QD cores less susceptible to oxidativedegradation and less toxic to primary rat hepatocytes, implicating the potential role of cadmiumin QDs cytotoxicity. The decrease in QD cytotoxicity of CdSe QDs with the overgrowth of aZnS shell has since been verified in several reports [139,142]. If it is revealed in the future thatCd2+ release is a major hindrance for the use of QDs in cells and in animals, several new typesof QDs that have no heavy metals atoms may be useful for advancing this field [143,144].5.2. Toxicity Induced by Colloidal InstabilityPresently it is nearly impossible to drawing firm conclusions about the toxicity of QDs incultured cells due to (a) the immense variety of QDs and variations of surface coatings usedby different labs and (b) a technical disparity in experimental conditions, such as the durationof the nanoparticle exposure, use of relevant cell lines, media choice (e.g. with or withoutserum), and even the units of concentration (e.g. mg/ml versus nM). Nonetheless, thecytotoxicity of QDs reported in the literature has strongly correlated with the stability andsurface coatings of these nanoparticles, which can be separated into three categories. (1) CoreCdTe QDs that are synthesized in aqueous solution and stabilized by small thiolate ligands(e.g. mercaptopropionic acid or mercaptoacetic acid). These QDs have been widely used dueto their ease of synthesis, low cost, and immediate utility in biological buffers. However,because these QDs are protected only by a weakly bound ligand, they are highly prone todegradation and aggregation, and their cytotoxicity toward cells in culture has been widelyreported [140,145]. (2) Core/shell CdSe/ZnS QDs synthesized in nonpolar solvents andtransferred to water using thiolate ligands. CdSe is less prone to oxidation than CdTe, and ZnSis even more inert, and therefore these QDs are much more chemically stable. With directcomparison to CdTe QDs, these nanocrystals are significantly less cytotoxic, although highconcentrations have been found to illicit toxic responses from cells [140]. Because these QDsare coated with a ZnS shell, the origin of this cytotoxicity is still unclear, whether it is fromdegradation of the shell, leading to cadmium release, or if it is caused by other effects. Whencoated with small ligands, these QDs have similar surface chemistries compared to aqueousCdTe QDs, burdened by significant dissociation of ligands from the QDs, rendering thenanoparticles colloidally unstable [146]. This propensity for aggregation may contribute totheir cytotoxicity, even if free cadmium is not released. Importantly for the comparison betweenCdSe/ZnS QDs and their cadmium-only counterparts (CdSe or CdTe core QDs), thiolateSmith et al.Page 13Adv Drug Deliv Rev. Author manuscript; available in PMC 2009 August 17.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript
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ligands bind more strongly to zinc than to cadmium, which may contribute colloidal stability.(3) Core/shell CdSe/ZnS QDs synthesized in nonpolar solvents and transferred to water viaencapsulation in amphiphilic polymers or cross-linked silica. These QDs have been found tobe significantly more stable colloidally, chemically, and optically when compared to theircounterparts coated in small ligands [56]. For this reason, they have been found to be nearlybiologically inert in both living cells and living animals [10,24,49,60,79,107,114,147]. Onlywhen exposed to extreme conditions or when directly injected into cells at immensely highconcentrations have these QDs been found to elicit toxic or inflammatory responses [49,142].It is feasible that a significant amount of toxicological data obtained for QDs thus far has beenconsiderably influenced by the colloidal nature of these nanoparticles. The tendency fornanoparticles to aggregate, precipitate on cells in culture, nonspecifically adsorb tobiomolecules, and catalyze the formation of reactive oxygen species (ROS) may be just asimportant as heavy metal toxicity contributions to toxicity. For example, Kircher et al. foundthat CdSe/ZnS QDs coated with an amphiphilic polymer shell induced the detachment ofhuman breast cancer cells from their cell culture substrate [139]. This effect was found to alsooccur for biologically inert gold nanoparticles coated with the same polymer, thus ruling outthe possibility of heavy metal atom poisoning. Microscopic examination of the cells revealedthat the nanoparticles precipitated on the cells, causing physical harm. Indeed, carbonnanotubes, which are entirely composed of harmless carbon, have been found to be capable ofimpaling cells and causing major problems in the lungs of mammals [148]. Nonspecificadsorption to intracellular proteins may also impair cellular function, especially for very smallQDs (3 nm and below), which can invade the cellular nucleus [101], binding to histones andnucleosomes [102], and damage DNA in vitro [149,150]. QDs are also known to catalyze theformation of ROS [145,151], especially when exposed to ultraviolet radiation. In fact, Cho etal. exposed cells to CdTe QDs in cell culture and determined that their cytotoxicity could onlybe accounted for with the effects of ROS generation, as there was no dose-dependentrelationship with intracellular Cd2+ release, as determined with a cadmium-reactive dye[140]. However, protection of the surface of QDs with a thick ZnS shell may greatly reduceROS production [152,153]. Despite a significant surge of interest in the cytotoxicity ofnanoparticles, there is still much to learn about the cytological and physiological mediators ofnanoparticle toxicology. If it is determined that heavy metal composition plays a negligiblerole in QD toxicity, QDs will have as good of a chance as any other nanoparticle at being usedas clinical contrast agents.6. Dual-Modality QDs for Imaging and TherapyIn comparison with small organic fluorophores, QDs have large surfaces that can be modifiedthrough versatile chemistry. This makes QDs convenient scaffolds to accommodate multipleimaging (e.g., radionuclide-based or paramagnetic probes) and therapeutic agents (e.g.anticancer drugs), through chemical linkage or by simple physical immobilization. This mayenable the development of a nearly limitless library of multifunctional nanostructures formultimodality imaging, as well as for integrated imaging and therapy.6.1. Dual-Modality ImagingThe applications of QDs described above for in vivo imaging are limited by tissue penetrationdepth, quantification problems, and a lack of anatomic resolution and spatial information. Toaddress these limitations, several research groups have led efforts to couple QD-based opticalimaging with other imaging modalities that are not limited by penetration depth, such as MRI,positron emission tomography (PET) and single photon emission computed tomography(SPECT) [154–158]. For example, Mulder et al. [154] developed a dual-modality imagingprobe for both optical imaging and MRI by chemically incorporating paramagnetic gadoliniumcomplexes in the lipid coating layer of QDs [154,155]. In vitro experiments showed thatSmith et al.Page 14Adv Drug Deliv Rev. Author manuscript; available in PMC 2009 August 17.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript
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labeling of cultured cells with these QDs led to significant T1 contrast enhancement with abrightening effect in MRI, as well as an easily detectable fluorescence signal from QDs.However, the in vivo imaging potential of this specific dual-modality contrast agent is uncertaindue to the unstable nature of the lipid coating that was used. More recently, Chen and coworkersused a similar approach to attach the PET-detectable radionuclide 64Cu to the polymeric coatingof QDs through a covalently bound chelation compound [158]. The use of this probe fortargeted in vivo imaging of a subcutaneous mouse tumor model was achieved by also attachingαvβ3 integrin-binding RGD peptides on the QD surface. The quantification ability and ultrahighsensitivity of PET imaging enabled the quantitative analysis of the biodistribution and targetingefficacy of this dual-modality imaging probe. However, the full potential of in vivo dual-modality imaging was not realized in this study, as fluorescence was only used as an ex vivoimaging tool to validate the in vivo results of PET imaging, primarily due to the lower sensitivityof optical imaging in comparison with PET. This imbalance in sensitivity is fundamental tothe differences in the physics of these imaging modalities, and points to an inherent difficultyin designing useful multimodal imaging probes. The majority of these probes are still at anearly stage of development. The clinical relevance of these nanoplatforms still needs furtherimprovement in sensitivity and better integration of different imaging modalities, as well asvalidation of their biocompatibility and safety.It is also noteworthy that recent advances in the synthesis of QDs containing paramagneticdopants, such as manganese, have led to a new class of QDs that are intrinsically fluorescentand magnetic [159,160]. However the utility of these new probes for bioimaging applicationis unclear because they are currently limited to the ultraviolet and visible emission windows,and their stability (e.g., photochemical and colloidal) and biocompatibility have yet to besystematically investigated [144]. As well, inorganic heterodimers of QDs and magneticnanoparticles have generated dual-functional nanoparticles [161,162]. Although these newmaterials are of great interest, they are still in development and have only recently shownapplicability in cell culture, but not yet in living animals [160,163].6.2. Integration of Imaging and TherapyDrug-containing nanoparticles have shown great promise for treating tumors in animal modelsand even in clinical trials [157]. Both passive and active targeting of nanotherapeutics havebeen used to increase the local concentration of chemotherapeutics in the tumor. Due to thesize and structural similarities between imaging and therapeutic nanoparticles, it is possiblethat their functions can be integrated to directly monitor therapeutic biodistribution, to improvetreatment specificity, and to reduce side effects. This synergy has become the principlefoundation for the development of multi-functional nanoparticles for integrated imaging andcancer treatment. Most studies are still at a proof-of-concept stage using cultured cancer cells,and are not immediately relevant to in vivo imaging and treatment of solid tumors. However,these studies will guide the future design and optimization of multifunctional nanoparticleagents for in vivo imaging and therapy [164–167].In one example, Farokhzad et al. reported a ternary system composed of a QD, an aptamer,and the small molecular anticancer drug doxorubicin (Dox) for in vitro targeted imaging,therapy and sensing of drug release [165]. As illustrated in Figure 6, aptamers were conjugatedto QDs to serve as targeting units, and Dox was attached to the stem region of the aptamers,taking advantage of the nucleic acid binding ability of doxorubicin. Two donor-quencher pairsof fluorescence resonance energy transfer occurred in this construct, as the QD fluorescencewere quenched by Dox, and Dox was quenched by the double-stranded RNA aptamers. As aresult, gradual release of Dox from the conjugate was found to "turn on" the fluorescence ofboth QDs and Dox, providing a means to sense the release of the drug. However it is clear thatSmith et al.Page 15Adv Drug Deliv Rev. Author manuscript; available in PMC 2009 August 17.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript
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Agatha Christie Collection - Peter Ustinov
These adaptation of Agatha Christie's Collection was very disappointing to us. Had we not seen these movies previously w/David Sachet as the star, I suppose we would have liked these more. However, we felt the acting by all concerned was not up to standard. I would not recommend this series.
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Originally posted on Fox Sports North | Last updated 2/21/13
It didn't take long for the national media to try to wrangle an answer from Minnesota Vikings general manager Rick Spielman on Thursday regarding receiver Percy Harvin. It took less time for Spielman to reiterate Minnesota's position on its talented but temperamental receiver: "We have no intent to trade Percy Harvin." They were the same words uttered by Spielman last week when he met with local reporters for his annual pre-Combine question-and-answer session. Spielman took to the podium again Thursday in Indianapolis and the first question was about Harvin, his unhappiness with the team that drafted him No. 22 overall in the 2009 and whether the Vikings might look to trade a player who is entering the final season of his rookie contract. Harvin is set to make 2.9 million, well below the average of the league's top receivers. "As I stated earlier, we have no intent to trade Percy Harvin," Spielman said at the Combine. "Anything related to his contract or any discussions will all ...
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A top-down approach to implementing shared services can exacerbate difficulties for employees trying to adapt, says ATF CIO Richard Holgate. (FCW file photo)
The push for shared services is often framed as a technical challenge, but to Customs and Border Patrol CTO Wolf Tombe, the first step is always the people. And the resistance within his agency often resembles the four stages of grief.
"You can never discount the cultural resistance factor," said Tombe, who along with several others discussed shared services on May 20 at the ACT-IAC Management of Change Conference. "You have to account for it, and plan for how you're going to manage it."
Tombe's bureau is the largest user of the Department of Homeland Security's shared email service, and he said his office is pushing a wide range of shared services for other IT needs. But every time a service is proposed, he said, there are those in his agency who swear that they "will never use that new service."
"That's denial," Tombe said. "Then they come back and say, 'Okay, I'll do this, but can we wait until next year?' That's negotiation."
Deborah Johnson, a consultant who works with intelligence agencies on shared services, recognized the pattern, and noted: "Once you get to the negotiating phase, they're starting to see the light."
And acceptance rarely comes without at least a brief stop in the anger phase, Tombe said. "A lot of people get explosively angry. You can't take that personally -- it's a stage they're going through."
Successfully implementing shared services requires more than grief counseling, of course, but Dale Luddeke, senior vice president and chief growth officer for TASC, stressed that "shared services is more a people problem than a technology problem."
Richard Holgate, CIO of the Bureau of Alcohol, Tobacco, Firearms and Explosives, agreed, and argued that a top-down approach to shared services can exacerbate those people problems. When an agency identifies an available service that it does not currently have, as was the case with ATF and human resources management systems nearly a decade ago, IT leaders often become customers of another agency's service -- in that instance, the Treasury Department's H.R. system.
But when a department-wide mandate comes from on high, Holgate said, agencies or offices are disinclined to give up their own solutions for a shared service that may be seen as a lowest common denominator.
"Up until a year ago, we had 23 different email systems in DOJ," Holgate said. "Many of which had 1,000 or fewer users -- prime candidate for consolidation. But we had to make sure all the stakeholders had their questions and concerns addressed. We did a good job, but didn't cover all the bases."
"It's a challenge," Holgate admitted. Whereas shared-service email entails a per-user fee, "a lot of our smaller components supported their email internally, so the cost was staff. And it felt to them that the cost was zero."
Tombe cited similar challenges at DHS. "We didn't know how much it cost to provide email," he said, "and it was really hard to find out."
Ultimately, Tombe said, the department turned to an outside consultant to determine DHS' baseline costs -- and confirmed that the hardware and software expenses were "a fraction" of the staffing costs were. Yet "the fine-grained analysis took about a year and a half to do," he said.
Despite those difficulties, Tombe said that fiscal realities are helping to overcome even the staunchest resistance. Four years ago, he said, CBP's IT budget was $1.2 billion, but it's now $650 million and dropping."
"We cannot possibly fund the current operating infrastructure we have now," Tombe declared.
Holgate agreed. "The current fiscal environment is a big motivator," he said.
About the Author
Troy K. Schneider is editor-in-chief of FCW and GCN. Connect with him on Twitter: @troyschneider.
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The outgoing speaker helped push forward data and transparency measures that have opened up the House of Representatives to the American people.
The Census Bureau announced plans to fold in new technology and third party data into its 2020 population count, but questions remain about the viability of the Bureau's enterprise IT.
A technology roadmap can help agencies reap the benefits FITARA has to offer. Here are the four stages of a successful effort.
In the newest Federal Employee Viewpoint Survey, feds show they're feeling better than they have over the past few years – but there's still room for improvement, especially in mission-critical IT.
The General Services Administration's Enterprise Infrastructure Solutions contract will anchor the agency's NS 2020 telecommunications strategy.
House and Senate both approve a measure to maintain funding at fiscal 2015 levels through Dec. 11.
The aviation regulator missed a Congressional deadline to develop rules of the sky-ways for drones.
Agency heads, acquisition officers and even CIOs don't need to be hard-core techies. FCW's four-part look at the expertise that's really required to make federal IT run.
FCW recognizes 14 individuals having an outsized impact early in their careers.
Call it for what is. A way to reduce staff by picking the low hanging fruit. And never complain how hard it is to attract talent down the road.
I think that the case for shared services, which is obvious, would be better served if CIOs hadn't spent all their political capital ignoring the actual mission of the agency and confusing that with what is commodity IT infrastructure. Make the plumbing go away and don't destroy the ability of program offices to actually execute program. Its one of the biggest moral downers for Feds right now - program resources gobbled up by CIOs under the guise of shared services. Then blank stares from CIO when asked where the program services are...
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David Stefan Mach
1 portrait
© Iain Clark / National Portrait Gallery, London
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David Stefan Mach
by Iain Clark
Lambda C-type colour print, 8 September 2011
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Purchased, 2013
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Photographed in David Mach's temporary studio at the City Arts Centre Edinburgh during his exhibition, David Mach - Precious Light: a celebration of the King James Bible 1611-2011 (July-October 2011). The image was commissioned as part of a series of portraits for Clark's exhibition This is the way we make our work held at the Glasgow Art Club in May 2012.
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South Florida beats Connecticut 13-10 - 0 Comments
EAST HARTFORD, Conn. — Marvin Kloss hit two field goals, and Aaron Lynch returned a fumble 44 yards for a touchdown as South Florida beat Connecticut 13-10 on Saturday.
It was the Bulls second consecutive win without an offensive touchdown. USF (2-4) is 2-0 in the American Athletic Conference after beating Cincinnati last week.
Kloss' 44-yard field goal with just over 4 minutes left capped a more than 7 minute fourth-quarter drive, and was the game winner. He has converted on nine consecutive attempts, including a 47-yarder in the first half.


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Tomorrow will be a down day, with no activities planned. I am certain Chris will take some time to show us around Charlevoix, and I'll make certain I have my camera with me. Wednesday is a busy day, with a trip up to famous Mackinac Island AND the premier of Sharknado 3! I'm not sure how we'll fit all of this excitement into one day, but we're sure going to try!
'Night, all!
Finally an RV Resort that is truly a Resort!
... for us it was National Fudge day and the island's oldest fudge maker Murdick's Fudge was giving away free fudge so of course we got some free and bought more - just like they wanted! Fudge had its beginnings on the island in the 1887's. There were quite a lot of fudge shops to choose from.
The Chuck Wagon was a great place for lunch with their claim to have the best hamburgers on the island - which must have been true ...
Bill's Final Obituary
... will take place on Saturday, September 27, 2014 from 2:00 PM to 6:00 PM at the Reynold-Jonkhoff Funeral Home at 305 6th St., Traverse City, MI. (231) 947-6347.
In lieu of flowers, donations would be appreciated for the new Cowell Family Cancer Center being built at Munson Hospital. Contact Des Worthington at (231) 935-6509 or firstname.lastname@example.com.
The family is being served by the Reynolds-Jonkhoff Funeral Home and Cremation Services.
Day 50 Macinaw City to Boyne City, MI Mlie: 2,671
... about two million years ago, glacial action scraped the earth and spread the fossils across the northern Lower Peninsula, depositing major concentrations in the Petoskey area. The prehistoric fossil, unique to the Traverse Group rock strata, is called the Petoskey Stone and is Michigan's official state stone.
Beautiful People Do Not Just Happen
... by Michigan law. He was only in the hospital for one night, two days. We were able to attend Bill's niece's wedding downstate last Saturday, which Bill really enjoyed. A team of doctors met to give us options going forward. Initially, they thought both tumors could be treated with radiation. However, early this morning, our Radiation Oncologist (who was on vacation last week) called to say they now recommend surgery ...


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Hoops Season to Get Underway at Gopher All-Star Friday Night
Sept. 26, 2012
The University of Minnesota men's and women's basketball programs will officially begin the season by hosting "Gopher All-Star Friday Night" on Oct. 12 at Williams Arena. The event, formally known as "Midnight Madness," "Tubby's Tip-off" or "Hoopla" will begin at 7:00 p.m. The entire night's schedule is below. The Williams Arena doors will open at 7:00 p.m. in preparation for the evening's first event.
The Gopher women will open 2012-13 regular season play on Nov. 10 against Washington State in the Best Buy Classic. The Gophers also host a pair of exhibition match-ups against Concordia-St. Paul (Oct. 31) and Minnesota State, Mankato (Nov. 4).
GOPHER ALL-STAR FRIDAY NIGHT TIMELINE (Times are Approximate)
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8/10/2015Gophers in Spain: Aug. 10-20
7/22/2015Top Ten Moments of 2014-15: #1
7/20/2015Meet the Men's Basketball Newcomers
06/03/2015Men's Basketball Feature: The Future
05/26/2015Gophers Complete Class with Fitzgerald
05/20/2015Gophers Sign Jordan Murphy


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You are a fan of from lifted ? Come to discover the best forums from lifted on the internet and share your experience with a community fan of from lifted . You can also build a forum from lifted and create your online discussion.
A forum for all who have and love their rides! Every truck, from small to the over sized, even farm equipment!
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Necromancing the Stone
Metal Blade Records is proud to announce the signing of Necromancing the Stone! Upon unleashing their upcoming debut, the band plans to take their unique brand of no-nonsense, traditional heavy metal out on the road.Read More
REDEMPTION's sixth album, 'The Art of Loss', has been completed and will be released early in 2016 by Metal Blade Records.Read More
On October 30th, Whitechapel will unleash their new feature length video release, 'The Brotherhood of the Blade', which follows the sextet on tour and at home, and dives deep into the hearts and minds of one of metal's brightest acts.Read More
Six Feet Under
American Death Metal legends SIX FEET UNDER return to mainland Europe early December for the 'X-Mas in Hell Tour 2015'!Read More
Twitching Tongues
Southern California's rising underground metal/hardcore band Twitching Tongues has unveiled a new song called 'Sacrifice Me' today at NPR.org.Read More
Mother Feather
Mother Feather has released the title track from their full length debut, 'Mother Feather'! The song is available now on iTunes, and the video is streaming at metalblade.com/motherfeather.Read More
New Releases
Upcoming Releases
Latest News
BELOW to head out for European tour in February of 2015!
- 25/11/14 – Swedish Doomster BELOW are heading out for the...Read More
SIX FEET UNDER confirmed as headliners for HATEFEST tour 2015 featuring Marduk and Vader among others!
- 25/11/14 – April 2015 will see the return of the HATEFEST Tour. The...Read More
PRIMORDIAL release official videoclip for 'Babel's Tower'!
- 24/11/14 – New album "Where Greater Men Have Fallen" out...Read More
ENSIFERUM announce special guests for 'One Man Army' European Tour!
- 20/11/14 – ENSIFERUM has completed work on their upcoming new album,...Read More
CRIPPER launches third single for new album 'Hyena'!
- 19/11/14 – "Hyëna" to be released this week...Read More
PORTRAIT release RAM cover 'Blessed To Be Cursed'!
- 17/11/14 – "Under Command" split album out in two...Read More
ENSIFERUM announce album title and release date for 2015!
- 17/11/14 – ENSIFERUM has completed work on their upcoming new album,...Read More
THOMAS GILES debuts 'Siphon the Bad Blood' video on AllMusic.com!
- 14/11/14 – THOMAS GILES, the solo project from Between the Buried and...Read More


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Don't Miss
- Spachts honored for years of service
- Lititz women's chorus seeking new members
- MCFEE Family Breakfast set for Oct. 24
- Cavalcade of Bands set for Halloween
- The Rooster Crows in Lititz
- Art about town
- More Chocolate Walk stops revealed
- Lowe's, Aaron's Acres team to upgrade Manheim park
- Flying high for fun — for now
- Countdown to Chocolate Walk
Movin' on! Expansion project
By Lititz Record on November 6, 2013
Marleise Emrhein's hat trick and two goals from Liz Wenger added up to a 5-0 win over Owen J. Roberts in the first round of States. Full coverage as only Bruce Morgan can provide is on page B1.
What's going on at Warwick Ambulance? Find out on page A16.


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A proposed fencing ordinance in Prospect is being called too intrusive and unenforceable.
Members of the Prospect city council are have been formulating new guidelines for fences since winter.
Residents' latest concerns stem from two provisions in the ordinance.
It states temporary fences are allowable for seasonal vegetable gardens, but shouldn't be visible from the road.
Resident Peggy Mealey-Miles said with some effort, most backyards are visible from the street.
A La Grange police officer is injured and another man is hospitalized after a crash on I-71.
According to police, a pickup truck pulling a small trailer jackknifed and flipped on its side about 1:20 a.m. Sunday on I-71 southbound between milemarkers 20 and 21.
Two officers from the Oldham County Police Department and La Grange Police Officer Jeremy Ferguson responded to the scene and prepared to close a section of the interstate.
OCPD Lt. Col. Billy Way said Ferguson exited his vehicle and popped the trunk to retrieve emergency equipment and a reflective vest.
The Oldham Era is gearing up for this weekend's Kickoff Classic with our special preview section filled with stats, schedules, analyses and photos of North Oldham, Oldham and South Oldham high school football players.
Warrant arrests
Timothy Allen Carpenter Jr., 23, 2701 W. Ky. 22, Crestwood, arrested by OCSO Aug. 10, conditional release violation and failure to comply.
Robert K. Johnson, 47, 510 E. Riverside Drive, Evansville, Ind., arrested by OCSO Aug. 8, third-degree criminal trespassing.
Police are investigating the cause of a Sunday morning house fire after firefighters rescued a woman from the house.
Neighbors noticed smoke coming from a bi-level home in the 7300 block of Autumn Bent Way in Orchard Grass Hills subdivision about 8:30 a.m. Sunday.
When firefighters arrived at the scene, witnesses told police they believed a 41-year-old woman and her daughter could still be inside.
Firefighters encountered heavy smoke inside the house, and it took about 20 minutes to locate the woman.
The joy of seeing their two small dogs rescued from a house fire became short-lived for a Ballardsville family.
When Ballardsville Fire Department Lt. Ryan Baker emerged from the Kinser family's home carrying two Shih Tzus, Kathy Kinser started hugging and thanking everyone in sight.
Kinser had waited more than an hour Aug. 12 hoping to see her dogs alive after an electrical fire ripped through her home.
"Everything else can be replaced," she said that afternoon.
Oldham Collision Center of Crestwood has become an ecological ambassador using AkzoNobel's environmentally-friendly waterborne products.
The company teamed up with AkzoNobel to become an environmental ambassador for the collision repair industry.
California law makers are requiring all collision repair shops to implement waterborne paint technology by the end of 2009 and other states are also considering similar legislation.
The Oldham County Chamber of Commerce is currently accepting applications for the 2009-10 class of Leadership Oldham County.
Interested participants must live and/or work in Oldham County, or must be an Oldham County Chamber member.
About 15 Oldham County business leaders and residents participate in this program annually. Leadership Oldham County is designed to educate members about the services, amenities and challenges in Oldham County.
The new classroom is nice – really nice – but for middle school science teacher Gary Webb, the best part of the new Saint Mary Academy building is that there's only one.
For the past two school years, since Mother of Good Council and Immaculate Conception schools merged to form Saint Mary Academy, Webb made the 18-mile drive between the two every day like various other teachers and administrators.
Police are investigating reports of an armed robbery at a cell tower Monday in the 2400 block of Liberty Lane in Goshen.
According to police, two men approached a T-Mobile technician working at the tower about 11:40 a.m. and asked for directions to Oldham County Middle School.
Seconds later, one man brandished a small handgun and demanded money and tools.
Witnesses said the men fled the scene in a 1970s gold- or copper-colored Plymouth Fury with missing hubcaps and drove north headed toward U.S. 42.
Copyright © 2015
The Oldham Era is your source for local news, sports, events, and information in Oldham County and LaGrange, Kentucky, and the surrounding area.


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O'Keefe offers hope for the Church, but not the hope found in the cliches of the past, rather in the hope of the future. He challenges the dated misconceptions of the institutionalized versions of Church and calls into the future, to inform our now. He invites us to question the evolution of leadership throughout the history of the Church and to seek more influential alternatives. This book is a caffeinated clarion call to something better, an informed and insightful engagement that is a must have page-turner.
George Elerick,
Cultural Theorist, Human Rights Worker
Author of Jesus Bootlegged
If O'Keefe ever changes careers, he should consider bartending. He's a storyteller, but also pulls a story out of you. He has advice, but it's the kind that makes you think for yourself. He invites you to sit, stay for a bit, release your inhibitions, be honest, enjoy the company, and talk about what really matters. I want more. I hope he sticks with writing. But, if he ever opens that bar, I hope it's in my town. People need real bartenders who know the real Jesus here.
This book does not suck. Thanks, John.
John Voelz
The Curator; Westwinds Church, Jackson, MI
Author of Broken Record: Enjoying the Music of Relationship Through the Hisses, Pops, and Scratches
I've been in church leadership for over 16 years and for the first time in a long time I've found a leadership book that speaks my language. boneYard challenges church leadership here in USAmerica (no matter how big or how small your faith community) to explore healthier ways to engage the culture of the here and now by inviting a left brain institution into a right brain world.
Phil Shepherd aka Whiskey Preacher
Co-pastor/communal architect of the Eucastrophe in Fort Worth TX.
"John O'Keefe has crafted an insightful argument for living into a both/and world of what is becoming a conceptual age. He invites his readers to see the church not as a static organization but as a living organism that can bring people to life. He suggests six personas for living into God's creativity and grace that will transform and bring life to the church. This is a must read book for someone looking to step out into a radical way of doing church and life."
Jonathan Brink,
Author of Discovering The God Imagination: Reconstructing A Whole New Christianity
John O'Keefe's "Boneyard" will delightfully and convincingly educate readers on the recent shifts in the cultural mindset and how Christians must comprehend and adjust to those shifts in order to communicate the gospel effectively and intelligibly.
Frank Viola
Author of From Eternity to Here, Reimagining Church, and Finding Organic Church, ptmin.org
Albert Einstein observed, "We can't solve problems by using the same kind of thinking we used when we created them." Insert John O'Keefe and his book, Boneyard: Creatives Will Change The Way We Lead In The Church. John represents a different way of thinking that no longer sees church as a business to run but an experience to live, a tribe to belong to, and a creative reality that spontaneously unfolds along the everyday paths of life. John O'Keefe's mind runs on a MAC and he is more likely to find Burning Man as a relevant model for being the church than a Fortune 500 company business plan. What do you get when you combine a poet, choreographer, tattoo artist, photographer, and adventure guide? The Kind of thinking John says will revolutionize what church is. John O'Keefe may be the Pierre Omidyar (EBAY) as it relates to the way church exists in the future and the good news is that you will find this genius his book Boneyard.
Jim Palmer
Author of Divine Nobodies, and Wide Open Spaces
What Others Say About John
"Not only does John have a bright vision of the church; he has initiated strategies and networks to help the church itself become visionary. One of the consultants out there I trust the most and learn the most from."
Leonard Sweet, Senior Professor, Drew University
"I met John through his superb web site, and have interacted with him on e-mail. John is highly thought of, and quoted and referred to regularly. As a person and for his web resources I recommend him regularly, and highly."
Jason Clark, Founding Pastor, Vineyard Church
"As an entrepreneur, leader, and communicator, John is a catalyst for the kingdom of God in our emerging culture. His unique and trusted voice has been highly influential in my own life and countless others."
Jim Palmer, Author
"[Lindner] offers examples of prominent EC [Emerging Church] bloggers: John O'Keefe, founder of ginkworld.net, "an emerging/postmodern site exploring what it means to be a follower of Jesus in today's world..."
NCC's 2006 Yearbook {cites two 21st century trends: blogging and the Emergent Church}
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Who discovered the new world with Christopher Columbus?
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This book is for all those that wondered what was wrong with the church or have been asked to leave the church. Read morePublished on November 28, 2010 by Thomas A. Wallek
This book opened my eyes, head, and heart to the world around me in a new and fresh light. On the surface I knew things were changing in the world but I never stopped to really... Read morePublished on October 16, 2010 by Shellene Evans


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Yoko Ono, Thurston Moore & Kim Gordon are releasing a Limited edition "Early in the Morning" 10" vinyl record will be available in stores in US June 19th and Europe July 3rd. Beautifully pressed on one-sided 10" vinyl with an etching by Yoko on the flip side.
Justin Jones' new album Fading Light is an Americana opus, full of deep emotion, musical mood swings, and heartfelt playing.
Band of Skulls return to the states on August 3rd for Lollapalooza and for select shows with Jane's Addiction.
Dark Star Orchestra has been gearing up for their inaugural Dark Star Jubilee Festival, taking place in Thornville, Ohio, August 31st - September 2nd, 2012. In prepartion for the Jubilee, DSO has compiled a Dark Star Jubilee FAQ:
What are the camping options?
Camping Option
Each year since 1976, the New Jersey Jazz Society has presented Jazzfest, one of the best and oldest mainstream jazz festivals in the entire northeast.
Stern Grove Festival will be celebrating their 75th Season of Concerts this year as well. In celebration of their anniversary, Preservation Hall Jazz Band have been commissioned to compose and perform a piece of music.
Danielle Anderson, who makes a living writing and performing folk songs as Danielle Ate the Sandwich, today releases her new album, Like a King (June 5, 2012
There are those things they teach you in school about poetry, things like rhyme schemes and stresses and metrical feet. Things like sonnets and pastoral poetry and the epic. Regardless of all those rules and terms though, poetry can be one of the most free-form arts, allowing the writer to take on a poetic license, which is really to say that the author can do whatever he pleases.
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Bosco is caught on a security camera having sex in his squad car with the daughter of his Precinct Captain. Meanwhile Doc drags Carlos around the city on a personal mission to find the owner of a watch, found in the ambulance.
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