Recent advances in magnetic nanoparticle-based multi-modal imaging.

Magnetic nanoparticles have been extensively explored as a versatile platform for magnetic resonance imaging (MRI) contrast agents due to their strong contrast enhancement effects together with the platform capability for multiple imaging modalities. In this tutorial review, we focus on recent progress in the use of magnetic nanoparticles for MRI contrast agents and multi-mode imaging agents such as T1-T2 MRI, MRI-optical, and MRI-radioisotopes. This review also highlights emerging magnetic imaging techniques such as magnetic particle imaging (MPI), magneto-motive ultrasound imaging (MMUS), and magneto-photoacoustic imaging (MPA).

Synthesis and Evaluation of DNA Based Quantum Dot Fluorescence In Situ Hybridization (FISH) Probe for Telomere Detection.

Efficient oligonucleotide probe design and synthesis based on polymer-coated CdSe/ZnS quantum dot (QD) is demonstrated for detection of telomeres in human monocyte and Leishmania major, a protozoan pathogenic parasite. The highly photoluminescent polymer-coated QDs conjugated with various length of telomere probe sequences were prepared via carbodiimide chemistry and characterized. Specific detection of telomere was observed when DNA sequence was (CCCAAT)n (n = 5 or 3) probe sequence, rather than (GGGTTA)n (n = 3, 5, 8). The sensitivity and specificity were comparable with commercially available PNA probe for human telomere detection.

Synthesis, characterization and target protein binding of drug-conjugated quantum dots in vitro and in living cells.

Elucidation of unknown target proteins of a drug is of great importance in understanding cell biology and drug discovery. There have been extensive studies to discover and identify target proteins in the cell. Visualization of targets using drug-conjugated probes has been an important approach to gathering mechanistic information of drug action at the cellular level. As quantum dot (QD) nanocrystals have attracted much attention as a fluorescent probe in the bioimaging area, we prepared drug-conjugated QD to explore the potential of target discovery. As a model drug, we selected a well-known anticancer drug, methotrexate (MTX), which has been known to target dihydrofolate reductase (DHFR) with high affinity binding (K(d) = 0.54 nM). MTX molecules were covalently attached to amino-PEG-polymer-coated QDs. Specific interactions of MTX-conjugated QDs with DHFR were identified using agarose gel electrophoresis and fluorescence microscopy. Cellular uptake of the MTX-conjugated QDs in living CHO cells was investigated with regard to their localization and distribution pattern. MTX-QD was found to be internalized into the cells via caveolae-medicated endocytosis without significant sequestration in endosomes. A colocalization experiment of the MTX-QD conjugate with antiDHFR-TAT-QD also confirmed that MTX-QD binds to the target DHFR. This study showed the potential of the drug-QD conjugate to identify or visualize drug-target interactions in the cell, which is currently of great importance in the area of drug discovery and chemical biology.

Photochemical properties and shape evolution of CdSe QDs in a non-injection reaction.

Highly monodispersed CdSe quantum dots (QDs) were prepared without an injection procedure. A series of Cd salts of long chain fatty acids, including Cd-myristate (C14), Cd-palmitate (C16) and Cd-stearate (C18) was prepared, and all metallic precursors and surfactants were mixed together followed by increasing the temperature in a controlled manner. The reaction resulted in highly monodisperse and bright zinc blende QDs. In addition, the effects of specific ligands which have been known to lead anisotropic growth of the nanocrystals in the injection method were investigated. The use of alkyl phosphonic acid and alkyl amine was found to produce extremely monodisperse CdSe QDs with a high quantum yield. This procedure was proven to be able to yield a large quantity of zinc blende CdSe QDs (2 g) in a one-pot reaction. The use of a controlled amount of tetradecylphosphonic acid and octadecylamine resulted in tetrapod- and match-shaped QDs, the first reported by a non-injection method. These results clearly demonstrate that appropriate combination of precursors can provide high quality of CdSe nanocrystals in terms of quantum yield, monodispersity and shape control by a non-injection method.

Selective targeting of cellular nucleus using positively-charged quantum dots.

Developing highly selective probes for subcellular regions such as nucleus and cytoplamic organelles is of great interest for cellular imaging and high content screening analysis for biology and medicine. Cytoplasmic delivery of QDs has been well-understood, while nuclear delivery of QDs has been a challenge due to the unique structural characteristics of cell nucleus. In this study, we systematically investigated nucleus penetrating properties of small-sized ligand-exchanged QDs with either positive or negative surface charges in the similar size range of hydrodynamic diameter (7-10 nm). We found that the positively-charged QDs efficiently stain the nucleus in fixed HeLa cells as well as label nucleolar compartments in live HeLa cells. In contrast, the negatively charged QDs with the similar size range stain only the cytoplam in either fixed or live cells. The charge-dependent labeling pattern allowed us to simultaneously perform multiplex imaging of nuclues and cytoplasm. This study offers an insight into efficient nuclear delivery of nanoparticles such as QDs of which surface charge and size are critical for intracelllar localization and delivery.

Fluorogenic quantum dot-gold nanoparticle assembly for beta secretase inhibitor screening in live cell.

We have developed a novel fluorogenic nanoprobe prepared from the assembly of CdSe/ZnS quantum dot (QD) and gold (Au) nanoparticles in which QD was conjugated with a specifically designed ß-secretase (BACE1) substrate peptide, which was allowed to bind to the Ni-nitrilotriacetate (Ni-NTA) modified Au nanoparticles. This coordination-mediated binding of the QD with Au nanoparticles via Ni-NTA-histidine (His) interaction resulted in highly efficient quenching of QD fluorescence through a distance-dependent fluorescence resonance energy transfer (FRET) phenomenon. The prequenched QD-Au assembly recovered the fluorescence in the presence of the BACE1 enzyme after incubation in vitro. The high quenching efficiency of AuNP and robust QD fluorescence signal recovery upon BACE1 enzymatic digestion enabled us to visualize BACE1 activity in living cells, which further allowed us to generate the half maximal inhibitory concentration (IC(50)) values for BACE1 inhibitors in the cell-based assay utilizing a high throughput system (HTS). These results suggest the potential application of QD-AuNP assembly toward the HTS drug screening system as a robust and efficient probe to identify active molecules in BACE1-related diseases such as Alzheimer's disease.

Quantum dot-based screening system for discovery of g protein-coupled receptor agonists.

Cellular imaging has emerged as an important tool to unravel biological complexity and to accelerate the drug-discovery process, including cell-based screening, target identification, and mechanism of action studies. Recently, semiconductor nanoparticles known as quantum dots (QDs) have attracted great interest in cellular imaging applications due to their unique photophysical properties such as size, tunable optical property, multiplexing capability, and photostability. Herein, we show that QDs can also be applied to assay development and eventually to high-throughput/content screening (HTS/HCS) for drug discovery. We have synthesized QDs modified with PEG and primary antibodies to be used as fluorescent probes for a cell-based HTS system. The G protein-coupled receptor (GPCR) family is known to be involved in most major diseases. We therefore constructed human osteosarcoma (U2OS) cells that specifically overexpress two types of differently tagged GPCRs: influenza hemagglutinin (HA) peptide-tagged κ-opioid receptors (κ-ORs) and GFP-tagged A3 adenosine receptors (A3AR). In this study, we have demonstrated that 1) anti-HA antibody-conjugated QDs could specifically label HA-tagged κ-ORs, 2) subsequent treatment of QD-tagged GPCR agonists allowed agonist-induced translocation to be monitored in real time, 3) excellent emission spectral properties of QD permitted the simultaneous detection of two GPCRs in one cell, and 4) the robust imaging capabilities of the QD-antibody conjugates could lead to reproducible quantitative data from high-content cellular images. These results suggest that the present QD-based GPCR inhibitor screening system can be a promising platform for further drug screening applications.

Detection of melanoma using antibody-conjugated quantum dots in a coculture model for high-throughput screening system.

We proposed an effective strategy for evaluating the targeting specificity of an antibody-conjugated quantum dot (QD) nanoprobe in a coculture system mimicking an in vivo-like tumor microenvironment in which cancer cells grow with normal cells. Analysis of the images was performed with automated confocal microscopy. We have employed a melanoma-melanocyte coculture model to assess the specific binding of QDs conjugated with melanoma antibodies. Conjugation of antibodies to the QD significantly improved the melanoma specificity, while unconjugated antibody alone suffered from non-specific binding to melanocytes. Concentration-dependent binding and competitive inhibition studies with QD-antibody conjugates reproducibly proved the specificity to melanoma cells against melanocytes. The specificity and targeting efficiency of nanoprobes evaluated in a simple coculture model may provide a reasonable assessment for the in vitro diagnosis of early stage melanoma development before in vivo studies. Further, a rapid and sensitive cancer cell detection system demonstrated herein may allow for the development of high-throughput screening platforms for early cancer diagnosis and anti-cancer therapeutics.

Intracellular protein target detection by quantum dots optimized for live cell imaging.

Imaging of specific intracellular target proteins in living cells has been of great challenge and importance for understanding intracellular events and elucidating various biological phenomena. Highly photoluminescent and water-soluble semiconductor nanocrystal quantum dots (QDs) have been extensively applied to various cellular imaging applications due to the long-term photostability and the tunable narrow emission spectra with broad excitation. Despite the great success of various bioimaging and diagnostic applications, visualization of intracellular targets in live cells still has been of great challenge. Nonspecific binding, difficulty of intracellular delivery, or endosomal trapping of nanosized QDs are the main reasons to hamper specific target binding in live cells. In this context, we prepared the polymer-coated QDs (pcQD) of which the surface was optimized for specific intracellular targeting in live cells. Efficient intracellular delivery was achieved through PEGylation and subsequent cell penetrating peptide (i.e., TAT) conjugation to the pcQD in order to avoid significant endosomal sequestration and to facilitate internalization of the QDs, respectively. In this study, we employed HEK293 cell line overexpressing endothelin A receptor (ET(A)R), a family of G-protein coupled receptor (GPCR), of which the cytosolic c-terminal site is genetically engineered to possess green fluorescent protein (GFP) as our intracellular protein target. The fluorescence signal of the target protein and the well-defined intracellular behavior of the GPCR help to evaluate the targeting specificity of QDs in living cells. To test the hypothesis that the TAT-QDs conjugated with antibody against intracellular target of interest can find the target, we conjugated anti-GFP antibody to TAT-PEG-pcQD using heterobifunctional linkers. Compared to the TAT-PEG-pcQD, which was distributed throughout the cytoplasm, the antiGFP-functionalized TAT-PEG-pcQD could penetrate the cell membrane and colocalize with the GFP. An agonist (endothelin-1, ET-1) treatment induced GFP-ET(A)R translocation into pericentriolar region, where the GFP also significantly colocalized with antiGFP-TAT-PEG-pcQD. These results demonstrate that stepwise optimization of PEG-pcQD conjugation with both a cell penetrating peptide and an antibody against a target of interest allows specific binding to the intracellular target protein with minimized nonspecific binding.

Quantum dots: a new tool for anti-malarial drug assays.

BACKGROUND: Malaria infects over 300 million people every year and one of the major obstacles for the eradication of the disease is parasite's resistance to current chemotherapy, thus new drugs are urgently needed. Quantum dot (QD) is a fluorescent nanocrystal that has been in the spotlight as a robust tool for visualization of live cell processes in real time. Here, a simple and efficient method using QD to directly label Plasmodium falciparum-infected erythrocytes (iRBCs) was searched in order to use the QD as a probe in an anti-malarial drug-screening assay. METHODS: A range of QDs with different chemical coatings were tested for their ability to specifically bind iRBCs by immunofluorescence assay (IFA). One QD was selected and used to detect parasite growth and drug sensitivity by flow cytometry. RESULTS: PEGylated-cationic QD (PCQD) was found to specifically label infected erythrocytes preferentially with late stage parasites. The detection of QD-labelled infected erythrocytes by flow cytometry was sensitive enough to monitor chloroquine anti-malarial toxicity with a drug incubation period as short as 24 h (EC50 = 113nM). A comparison of our assay with another widely used anti-malarial drug screening assay, the pLDH assay, showed that PCQD-based assay had 50% improved sensitivity in detecting drug efficacy within a parasite life cycle. An excellent Z-factor of 0.8 shows that the QD assay is suitable for high-throughput screening. CONCLUSIONS: This new assay can offer a rapid and robust platform to screen novel classes of anti-malarial drugs.

Developing a multi-systemic fall prevention model, incorporating the physical environment, the care process and technology: a systematic review.

AIMS: This paper reports a review that assessed the effectiveness and characteristics of fall prevention interventions implemented in hospitals. A multi-systemic fall prevention model that establishes a practical framework was developed from the evidence. BACKGROUND: Falls occur through complex interactions between patient-related and environmental risk factors, suggesting a need for multifaceted fall prevention approaches that address both factors. DATA SOURCES: We searched Medline, CINAHL, PsycInfo and the Web of Science databases for references published between January 1990 and June 2009 and scrutinized secondary references from acquired papers. REVIEW METHODS: Due to the heterogeneity of interventions and populations, we conducted a quantitative systematic review without a meta-analysis and used a narrative summary to report findings. RESULTS: From the review, three distinct characteristics of fall prevention interventions emerged: (1) the physical environment, (2) the care process and culture and (3) technology. While clinically significant evidence shows the efficacy of environment-related interventions in reducing falls and fall-related injuries, the literature identified few hospitals that had introduced environment-related interventions in their multifaceted fall intervention strategies. CONCLUSION: Using the multi-systemic fall prevention model, hospitals should promote a practical strategy that benefits from the collective effects of the physical environment, the care process and culture and technology to prevent falls and fall-related injuries. By doing so, they can more effectively address the various risk factors for falling and therefore, prevent falls. Studies that test the proposed model need to be conducted to establish the efficacy of the model in practice.

Characterization and cancer cell specific binding properties of anti-EGFR antibody conjugated quantum dots.

Synthesis of biologically active antibody conjugated quantum dots (QDs) has been of great importance in cellular imaging and diagnostics. Cetuximab (or Erbitux) is the first monoclonal antibody drug which targets the epidermal growth factor receptor (EGFR) overexpressed in most cancer cells. In the present work, we investigated three different conjugation strategies to obtain the biologically functional QD-cetuximab conjugates for the tumor-specific imaging. Successful conjugation of cetuximab to QDs was achieved using PEG conjugated polymer-coated QDs and two long-chain heterobifunctional linkers, sulfo-LC-SPDP and sulfo-SMCC. The dissociation constant of the QD-cetuximab conjugates to EGFR was determined to be 0.61 +/- 0.28 nM. The cancer cell-specific binding ability of the QD-cetuximab conjugates was evaluated in vitro, and the cellular internalization of the QD-cetuximab conjugates was clearly demonstrated in live cells by confocal microscopy. The cellular imaging experiments using the QD-cetuximab conjugates showed a clear endocytosis pathway, which was evidenced by the colocalization of the QD-cetuximab conjugates with dye-labeled transferrin. These results suggest that the QD-cetuximab conjugates as an imaging modality for tumor EGFR overexpression can be expected to provide important information on the expression levels of EGFR on the cancer cells.

Compact and versatile nickel-nitrilotriacetate-modified quantum dots for protein imaging and Förster resonance energy transfer based assay.

The generation of compact quantum dots (QDs) probes is of critical importance for visualizing molecular interaction occurring in biological context, particularly when using the Förster resonance energy transfer (FRET) approach. This Article reports novel water-soluble compact CdSe/ZnS QDs prepared by ligand exchange reaction using thiolated nitrilotriacetate (NTA). The resulting NTA-QDs revealed higher stability and remarkable conjugation efficiency compared to the other QDs prepared with different ligands by using the ligand exchange method. The Ni-NTA group is a well-known binding moiety for the detection and purification of oligohistidine-tagged recombinant proteins. We demonstrated that NiNTA-QDs prepared by Ni(2+) complexation exhibited highly specific binding ability toward 6-histidine (His)-tagged peptides present in various experimental conditions (buffer solution, agarose beads, and HEK cells). Importantly, the compact NiNTA-QDs serve as an efficient FRET donor. These results suggest that the stable and highly selective multifunctional NTA-QDs can be useful for labeling and tracking molecular interactions within biological context.

In situ visualization of gene expression using polymer-coated quantum-dot-DNA conjugates.

Imaging of specific mRNA targets in cells is of great importance in understanding gene expression and cell signaling processes. Subcellular localization of mRNA is known as a universal mechanism for cells to sequester specific mRNA for high production of required proteins. Various gene expressions in Drosophila cells are studied using quantum dots (QDs) and the fluorescence in situ hybridization (FISH) method. The excellent photostability and highly luminescent properties of QDs compared to conventional fluorophores allows reproducible obtainment of quantifiable mRNA gene expression imaging. Amine-modified oligonucleotide probes are designed and covalently attached to the carboxyl-terminated polymer-coated QDs via EDC chemistry. The resulting QD-DNA conjugates show sequence-specific hybridization with target mRNAs. Quantitative analysis of FISH on the Diptericin gene after lipopolysaccharide (LPS) treatment shows that the intensity and number of FISH signals per cell depends on the concentration of LPS and correlates well with quantitative real-time PCR results. In addition, our QD-DNA probes exhibit excellent sensitivity to detect the low-expressing Dorsal-related immunity factor gene. Importantly, multiplex FISH of Ribosomal protein 49 and Actin 5C using green and red QD-DNA conjugates allows the observation of cellular distribution of the two independent genes simultaneously. These results demonstrate that highly fluorescent and stable QD-DNA probes can be a powerful tool for direct localization and quantification of gene expression in situ.

Positively charged compact quantum Dot-DNA complexes for detection of nucleic acids.

Novel QD-DNA complexes are prepared by simple electrostatic interaction between pegylated amine-functionalized CdSe/ZnS quantum dots (QDs) and DNA. The cationic nature of the amine functionality on the QD surface allows for formation of an electrostatic complex with negatively charged DNA. The presence of polyethylene glycol (PEG5000) molecules on the QD leads to enhanced stability and decreased nonspecific adsorption of DNA on the QD surface. Unlike assembly of QD-DNA based on hydrogen bonding, the present QD probes tend to be more strongly stabilized during the hybridization process by increasing the overall negative charges. In addition, the DNA loading efficiency can be modulated by changing the pH of the reaction medium. The fluorescence of the QD is quenched up to 90% by complexation with 5'-TAMRA-modified oligonucleotide (TAMRA=carboxytetramethylrhodamine) through fluorescence resonance energy transfer (FRET). With the FRET pair we selected, the R(0) value was calculated to be 5.5 nm and r is about 5 nm. This quenching of QD fluorescence is then reversed on binding of unlabeled target DNA. The maximum recovery of QD fluorescence is 60%. The QD-DNA probe (5DNA/QD) exhibits selective photoluminescence (PL) recovery in the presence of target oligonucleotide with a PL ratio of 3 for complementary versus noncomplementary. The present QD-DNA probes also show the capability to detect the synthetic 100-mer oligonucleotide derived from H5N1 influenza virus when present at concentrations as low as 200 nM in the solution.

The Effect of Light on Sleep and Sleep-Related Physiological Factors Among Patients in Healthcare Facilities: A Systematic Review.

OBJECTIVES: Lighting is one of the environmental factors which can improve patient sleep in healthcare environments. Due to the high degree of variation in study designs and results on this topic, the implications have been difficult to interpret. This review consolidates studies on the impact of bright light exposure on sleep to identify lighting conditions that can be applied and researched in future healthcare environments. METHODS: We searched for peer-reviewed articles on the impact of light on sleep or sleep-related outcomes in healthcare settings. We provided detailed analysis of the direct links between light and sleep, and a more cursory analysis of links between light and sleep-related factors, from 34 articles which met our inclusion criteria. RESULTS: The current state of the literature includes evidence on how various durations and intensities of morning, midday, and evening bright light exposure, as well as whole-day light exposure interventions can improve specific aspects of sleep. Lighting interventions differed in all attributes (illuminance levels, exposure time, exposure duration, and spectral qualities) but showed promising results in improving patients' sleep. CONCLUSIONS: Short-term bright light exposure in the morning, up to 2 hr of moderate (3,000-10,000 lux) morning exposures, up to 4 hr of moderate evening exposure, and whole-day exposures to lower illuminance levels (<3,000 lux) can improve patient sleep outcomes. Based on new findings on the mechanism through which light impacts sleep, future studies should be more specific about the spectral qualities of light sources.

Synthesis and functional evaluation of DNA-assembled polyamidoamine dendrimer clusters for cancer cell-specific targeting.

We sought to produce dendrimers conjugated to different biofunctional moieties (fluorescein [FITC] and folic acid [FA]), and then link them together using complementary DNA oligonucleotides to produce clustered molecules that target cancer cells that overexpress the high-affinity folate receptor. Amine-terminated, generation 5 polyamidoamine (G5 PAMAM) dendrimers are first partially acetylated and then conjugated with FITC or FA, followed by the covalent attachment of complementary, 5'-phosphate-modified 34-base-long oligonucleotides. Hybridization of these oligonucleotide conjugates led to the self-assembly of the FITC- and FA-conjugated dendrimers. In vitro studies of the DNA-linked dendrimer clusters indicated specific binding to KB cells expressing the folate receptor. Confocal microscopy also showed the internalization of the dendrimer cluster. These results demonstrate the ability to design and produce supramolecular arrays of dendrimers using oligonucleotide bridges. This will also allow for further development of DNA-linked dendrimer clusters as imaging agents and therapeutics.

Environmental affordances: designing for family presence and involvement in patient care.

OBJECTIVE: This comparative study in two ICUs examines the impact of the patient-centered unit design on family involvement, operationalized as percentages of family presence and family-patient/family-staff interaction in patient rooms. BACKGROUND: As hospitals have become more patient-centered, there has been a trend toward including a family area inside the patient area to promote family presence, support, and involvement in patient care. There is growing evidence that family members play an important role in supporting patient care, and that the physical environment affects family involvement. However, few empirical studies have attempted to show the effectiveness of the patient-centered design on family members' presence and their behavior. METHODS: This study compared the degree of family presence and family-patient and family-staff interactions in two intensive care units (ICUs) with different physical environmental conditions, but housing patients of similar acuity and disease type. RESULTS: The analysis identified a significant difference in family presence in patient rooms (t = -2.176; df = 79.0; p = 0.03) between the traditional and the patient-centered units. Patients in the family-centered care unit (M = 37.77; SD = 34.02) spent significantly more time with their family members in patient rooms than did patients in the traditional unit (M = 23.89; SD = 21.90). Patient-related variables other than unit design had no significant impact on family presence and interactions. CONCLUSIONS: Findings demonstrated that the patient-centered unit (5K) was associated with increased family presence in the patient rooms and increased family interaction with patients, when compared with the traditionally designed unit. KEYWORDS: Critical care/intensive care, evidence-based design, patient-centered care, quality care, social support.

Fluorogenic assay and live cell imaging of HIV-1 protease activity using acid-stable quantum dot-peptide complex.

A novel QD-peptide complex for detecting HIV-1 protease activity was prepared from simple one step electrostatic interaction. Fluorescence recovery of the pre-quenched QD through fluorescence resonance energy transfer allowed for in vitro assay and live cell imaging of the protease activity in HIV-1 transfected cells, proving the potential for cell-based protease inhibitor screening.

Controlled stoichiometric synthesis of DNA-quantum dot conjugates using Ni-mediated coordination chemistry.

An oligonucleotide modified with Ni-nitrilotriacetate (NTA) was successfully synthesized and used for the stoichiometric functionalization of QDs. This synthetic approach allowed for the facile preparation of DNA-QD conjugates with a defined DNA/QD ratio using well-known Ni-histidine coordination chemistry. A FRET based DNA-QD nanoprobe was prepared using this method highlighting the great potential of this synthetic strategy.