Scientific Bibliography

Nanobiology and Cancer Nanotechnology
Nanotechnology Devices and Nanomaterials
Nanotechnology-Enabled Therapeutics Development and Delivery
Cancer Diagnostics and Biosensors
Nanotechnologies in Advanced Imaging
Environment, Health and Safety

Nanobiology and Cancer Nanotechnology

2007  2006   2005   2004   2003   2002

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2007

Visual Recognition and Efficient Isolation of Apoptotic Cells with Fluorescent-Magnetic-Biotargeting Multifunctional Nanospheres.
Song EQ, Wang GP, Xie HY, Zhang ZL, Hu J, Peng J, Wu DC, Shi YB, Pang DW.
Clin Chem 2007 Oct 25; [Epub ahead of print].
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BACKGROUND: Fluorescent-magnetic-biotargeting multifunctional nanospheres are likely to find important applications in bioanalysis, biomedicine, and clinical diagnosis. We have been developing such multifunctional nanospheres for biomedical applications. METHODS: We covalently coupled avidin onto the surfaces of fluorescent-magnetic bifunctional nanospheres to construct fluorescent-magnetic-biotargeting trifunctional nanospheres and analyzed the functionality and specificity of these trifunctional nanospheres for their ability to recognize and isolate apoptotic cells labeled with biotinylated annexin V, which recognizes phosphatidylserine exposed on the surfaces of apoptotic cells. RESULTS: The multifunctional nanospheres can be used in combination with propidium iodide staining of nuclear DNA to identify cells at different phases of the apoptotic process. Furthermore, we demonstrate that apoptotic cells induced by exposure to ultraviolet light can be isolated simply with a magnet from living cells at an efficiency of at least 80%; these cells can then be easily visualized with a fluorescence microscope. CONCLUSIONS: Our results show that fluorescent-magnetic-biotargeting trifunctional nanospheres can be a powerful tool for rapidly recognizing, magnetically enriching and sorting, and simultaneously identifying different kinds of cells.

Lanthanide-containing polymer nanoparticles for biological tagging applications: nonspecific endocytosis and cell adhesion.
Vancaeyzeele C, Ornatsky O, Baranov V, Shen L, Abdelrahman A, Winnik MA.
J Am Chem Soc. 2007 Nov 7;129(44):13653-60.
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We describe the synthesis and characterization of element-encoded polystyrene nanoparticles with diameters on the order of 100 nm and a narrow size distribution. Individual particles contain ca. 10(3) chelated lanthanide ions, of either a single element or a mixture of elements. These particles were effectively internalized by nonspecific endocytosis into three cell lines associated with human leukemia. Using an assay based upon ICP-MS detection, we could monitor quantitatively cell adhesion induced by cell differentiation of THP-1 cells in response to phorbol ester stimulation (PMA) in single cell type or mixed cultures.

Nonfunctionalized nanocrystals can exploit a cell's active transport machinery delivering them to specific nuclear and cytoplasmic compartments.
Nabiev I, Mitchell S, Davies A, Williams Y, Kelleher D, Moore R, Gun'ko YK, Byrne S, Rakovich YP, Donegan JF, Sukhanova A, Conroy J, Cottell D, Gaponik N, Rogach A, Volkov Y.
Nano Lett. 2007 Nov;7(11):3452-61.
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We use high content cell analysis, live cell fluorescent imaging, and transmission electron microscopy approaches combined with inhibitors of cellular transport and nuclear import to conduct a systematic study of the mechanism of interaction of nonfunctionalized quantum dots (QDs) with live human blood monocyte-derived primary macrophages and cell lines of phagocytic, epithelial, and endothelial nature. Live human macrophages are shown to be able to rapidly uptake and accumulate QDs in distinct cellular compartment specifically to QDs size and charge. We show that the smallest QDs specifically target histones in cell nuclei and nucleoli by a multistep process involving endocytosis, active cytoplasmic transport, and entering the nucleus via nuclear pore complexes. Treatment of the cells with an anti-microtubule agent nocodazole precludes QDs cytoplasmic transport whereas a nuclear import inhibitor thapsigargin blocks QD import into the nucleus. These results demonstrate that the nonfunctionalized QDs exploit the cell's active transport machineries for delivery to specific intranuclear destinations.

Automatic Microtubule Tracking for QD-Based In Vivo Cell Imaging and Drug Efficacy Study.
Kong KY, Marcus AI, Hong JY, Giannakakou P, Wang MD.
Conf Proc IEEE Eng Med Biol Soc. 2006;1:3321-4.
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Microtubules (MT) are dynamic polymers that rapidly transition between states of growth, shortening, and pause. These dynamic events are critical for many microtubule functions such as intracellular trafficking and signaling. In addition, cancer chemotherapy drugs that target microtubules, such as the taxanes and the vinca alkaloids, are known to suppress microtubule dynamics at low doses, leading to mitotic arrest and cell death. Quantification of microtubule dynamics can be used as a read-out of anticancer-drug activity and can be a surrogate marker of drug sensitivity/resistance. The emerging nanotechnology such as quantum dots has provided properties such as less photo bleaching, higher probe imaging intensity, better specificity and sensitivity, which finally makes visualizing subcellular events over long enough time a possibility. But it also results in big increase in data acquisition. The traditional way of annotating MT manually is becoming a daunting task. Thus, the goal is to research and develop an efficient, reliable, and rapid MT tracking. In this paper, we describe active contour-based tracking methods to automatically track MT. We redefine the internal energy terms specifically for open snake, and examine different external energy terms for locating the end tips of a microtubule. This algorithm has been validated using simulated images, images of untreated MCF-7 breast cancer cells, and image of cells treated with the microtubule-targeting chemotherapeutic agent, Taxol.

Evaluation of poly (glycerol-adipate) nanoparticle uptake in an in vitro 3-D brain tumor co-culture model.
Meng W, Kallinteri P, Walker DA, Parker TL, Garnett MC.
Exp Biol Med (Maywood). 2007 Sep; 232(8):1100-8.
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Despite the inherent problems associated with in vivo animal models of tumor growth and metastases, many of the current in vitro brain tumor models also do not accurately mimic tumor-host brain interactions. Therefore, there is a need to develop such co-culture models to study tumor biology and, importantly, the efficacy of drug delivery systems targeting the brain. So far, few investigations of this nature have been published. In this paper we describe the development of a new model system and its application to drug delivery assessment. For our new model, a co-culture of DAOY cell brain tumor aggregates and organo-typic brain slices was developed. Initially, the DAOY aggregates attached to cerebellum slices and invaded as a unit. Single cells in the periphery of the aggregate detached from the DAOY aggregates and gradually replaced normal brain cells. This invasive behavior of DAOY cells toward organotypic cerebellum slices shows a similar pattern to that seen in vivo. After validation of the co-culture model using transmission electron microscopy, nanoparticle (NP) uptake was then evaluated. Confocal micrographs illustrated that DAOY cells in this co-culture model took up most of the NPs, but few NPs were distributed into brain cells. This finding corresponded with results of NP uptake in DAOY and brain aggregates reported elsewhere.

Carbon dots for multiphoton bioimaging.
Cao L, Wang X, Meziani MJ, Lu F, Wang H, Luo PG, Lin Y, Harruff BA, Veca LM, Murray D, Xie SY, Sun YP.
J Am Chem Soc. 2007 Sep 19;129(37):11318-9.
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Carbon nanoparticles upon simple surface passivation exhibit bright photoluminescence. Reported here is a new finding that these carbon dots are also strongly two-photon luminescent with pulsed laser excitation in the near-infrared. The experimentally measured two-photon absorption cross-sections are comparable to those of the high-performance semiconductor quantum dots already available in the literature. The two-photon luminescence microscopy imaging of human breast cancer cells with internalized carbon dots is demonstrated.

One at a time, live tracking of NGF axonal transport using quantum dots.
Cui B, Wu C, Chen L, Ramirez A, Bearer EL, Li WP, Mobley WC, Chu S.
Proc Natl Acad Sci U S A. 2007 Aug 21;104(34):13666-71.
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Retrograde axonal transport of nerve growth factor (NGF) signals is critical for the survival, differentiation, and maintenance of peripheral sympathetic and sensory neurons and basal forebrain cholinergic neurons. However, the mechanisms by which the NGF signal is propagated from the axon terminal to the cell body are yet to be fully elucidated. To gain insight into the mechanisms, we used quantum dot-labeled NGF (QD-NGF) to track the movement of NGF in real time in compartmentalized culture of rat dorsal root ganglion (DRG) neurons. Our studies showed that active transport of NGF within the axons was characterized by rapid, unidirectional movements interrupted by frequent pauses. Almost all movements were retrograde, but short-distance anterograde movements were occasionally observed. Surprisingly, quantitative analysis at the single molecule level demonstrated that the majority of NGF-containing endosomes contained only a single NGF dimer. Electron microscopic analysis of axonal vesicles carrying QD-NGF confirmed this finding. The majority of QD-NGF was found to localize in vesicles 50-150 nm in diameter with a single lumen and no visible intralumenal membranous components. Our findings point to the possibility that a single NGF dimer is sufficient to sustain signaling during retrograde axonal transport to the cell body.

Nanometer-sized diamond particle as a probe for biolabeling.
Chao JI, Perevedentseva E, Chung PH, Liu KK, Cheng CY, Chang CC, Cheng CL.
Biophys J. 2007 Sep 15;93(6):2199-208.
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A novel method is proposed using nanometer-sized diamond particles as detection probes for biolabeling. The advantages of nanodiamond's unique properties were demonstrated in its biocompatibility, nontoxicity, easily detected Raman signal, and intrinsic fluorescence from its natural defects without complicated pretreatments. Carboxylated nanodiamond's (cND's) penetration ability, noncytotoxicity, and visualization of cND-cell interactions are demonstrated on A549 human lung epithelial cells. Protein-targeted cell interaction visualization was demonstrated with cND-lysozyme complex interaction with bacteria Escherichia coli. It is shown that the developed biomolecule-cND complex preserves the original functions of the test protein. The easily detected natural fluorescent and Raman intrinsic signals, penetration ability, and low cytotoxicity of cNDs render them promising agents in multiple medical applications.

Antitumour drugs impede DNA uncoiling by topoisomerase I.
Koster DA, Palle K, Bot ES, Bjornsti MA, Dekker NH.
Nature. 2007 Jul 12;448(7150):213-7.
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Increasing the ability of chemotherapeutic drugs to kill cancer cells is often hampered by a limited understanding of their mechanism of action. Camptothecins, such as topotecan, induce cell death by poisoning DNA topoisomerase I, an enzyme capable of removing DNA supercoils. Topotecan is thought to stabilize a covalent topoisomerase-DNA complex, rendering it an obstacle to DNA replication forks. Here we use single-molecule nanomanipulation to monitor the dynamics of human topoisomerase I in the presence of topotecan. This allowed us to detect the binding and unbinding of an individual topotecan molecule in real time and to quantify the drug-induced trapping of topoisomerase on DNA. Unexpectedly, our findings also show that topotecan significantly hinders topoisomerase-mediated DNA uncoiling, with a more pronounced effect on the removal of positive (overwound) versus negative supercoils. In vivo experiments in the budding yeast verified the resulting prediction that positive supercoils would accumulate during transcription and replication as a consequence of camptothecin poisoning of topoisomerase I. Positive supercoils, however, were not induced by drug treatment of cells expressing a catalytically active, camptothecin-resistant topoisomerase I mutant. This combination of single-molecule and in vivo data suggests a cytotoxic mechanism for camptothecins, in which the accumulation of positive supercoils ahead of the replication machinery induces potentially lethal DNA lesions.

Intercalating gold nanoparticles as universal labels for DNA detection.
Mehrabi M, Wilson R.
Small. 2007 Sep;3(9):1491-5.
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No abstract available. (Citation link)

Fueling protein DNA interactions inside porous nanocontainers.
Cisse I, Okumus B, Joo C, Ha T.
Proc Natl Acad Sci U S A. 2007 Sep 11;104(37):14878.
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Vesicle encapsulation offers a biologically relevant environment for many soluble proteins and nucleic acids and an optimal immobilization medium for single-molecule fluorescence assays. Furthermore, the confinement of biomolecules within small volumes opens up new avenues to unique experimental configurations. Nevertheless, the vesicles' impermeability, even toward ions and other small molecules such as ATP, hinders more general applications. We therefore developed methods to induce pores into vesicles. Porous vesicles were then used to modulate the interaction between Escherichia coli RecA proteins and ssDNA by changing the extravesicular nucleotides. Repetitive binding and dissociation of the same RecA filament on the DNA was observed with a rebinding rate two orders of magnitude greater than in the absence of confinement, suggesting a previously unreported nucleation pathway for RecA filament. This method provides a biofriendly and simple alternative to surface tethering that is ideal for the study of transient and weakly interacting biological complexes.

Peptide-conjugated gold nanorods for nuclear targeting.
Oyelere AK, Chen PC, Huang X, El-Sayed IH, El-Sayed MA.
Bioconjug Chem. 2007 Sep-Oct;18(5):1490-7.
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Resonant electron oscillations on the surface of noble metal nanoparticles (Au, Ag, Cu) create the surface plasmon resonance (SPR) that greatly enhances the absorption and Rayleigh (Mie) scattering of light by these particles. By adjusting the size and shape of the particles from spheres to rods, the SPR absorption and scattering can be tuned from the visible to the near-infrared region (NIR) where biologic tissues are relatively transparent. Further, gold nanorods greatly enhance surface Raman scattering of adsorbed molecules. These unique properties make gold nanorods especially attractive as optical sensors for biological and medical applications. In the present work, gold nanorods are covalently conjugated with a nuclear localization signal peptide through a thioalkyl-triazole linker and incubated with an immortalized benign epithelial cell line and an oral cancer cell line. Dark field light SPR scattering images demonstrate that nanorods are located in both the cytoplasm and nucleus of both cell lines. Single cell micro-Raman spectra reveal enhanced Raman bands of the peptide as well as molecules in the cytoplasm and the nucleus. Further, the Raman spectra reveal a difference between benign and cancer cell lines. This work represents an important step toward both imaging and Raman-based intracellular biosensing with covalently linked ligand-nanorod probes.

Interfacing silicon nanowires with mammalian cells.
Kim W, Ng JK, Kunitake ME, Conklin BR, Yang P.
J Am Chem Soc. 2007 Jun 13;129(23):7228-9.
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We present the first demonstration of a direct interface of silicon nanowires with mammalian cells such as mouse embryonic stem (mES) cells and human embryonic kidney (HEK 293T) cells without any external force. The cells were cultured on a silicon (Si) substrate with a vertically aligned SiNW array on it. The penetration of the SiNW array into individual cells naturally occurred during the incubation. The cells survived up to several days on the nanowire substrates. The longevity of the cells was highly dependent on the diameter of SiNWs. Furthermore, successful maintenance of cardiac myocytes derived from mES cells on the wire array substrates was observed, and gene delivery using the SiNW array was demonstrated. Our results suggest that the nanowires can be potentially utilized as a powerful tool for studying intra- and intercellular biological processes.

Horseradish peroxidase embedded in polyacrylamide nanoparticles enables optical detection of reactive oxygen species.
Poulsen AK, Scharff-Poulsen AM, Olsen LF.
Anal Biochem. 2007 Jul 1;366(1):29-36.
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We have synthesized and characterized new nanometer-sized polyacrylamide particles containing horseradish peroxidase and fluorescent dyes. Proteins and dyes are encapsulated by radical polymerization in inverse microemulsion. The activity of the encapsulated enzyme has been examined and it maintains its ability to catalyze the oxidation of guaiacol with hydrogen peroxide as the electron acceptor, although at a slightly lower rate compared to that of the free enzyme in solution. The embedded enzyme is also capable of catalyzing the peroxidase-oxidase reaction. However, the rate is decreased by a factor of 2-3 compared to that of the free enzyme. The reduced rate is probably due to limitation of diffusion of substrates and products into and out of the particles. The catalytic activity of horseradish peroxidase in the polyacrylamide matrix demonstrates that the particles have pores which are large enough for substrates to enter and products to leave the polymer matrix containing the enzyme. The polymer matrix protects the embedded enzyme from proteolytic digestion, which is demonstrated by treating the particles with a mixture of the two proteases trypsin and proteinase K. The particles allow for quantification of hydrogen peroxide and other reactive oxygen species in microenvironments, and we propose that the particles may find use as nanosensors for use in, e.g., living cells.

Use of lanthanide-grafted inorganic nanoparticles as effective contrast agents for cellular uptake imaging.
Voisin P, Ribot EJ, Miraux S, Bouzier-Sore AK, Lahitte JF, Bouchaud V, Mornet S, Thiaudière E, Franconi JM, Raison L, Labrugère C, Delville MH.
Bioconjug Chem. 2007 Jul-Aug;18(4):1053-63.
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The improvement of commonly used Gd3+ -based MRI agents requires the design of new systems with optimized in vivo efficacy, pharmacokinetic properties, and specificity. To design these contrast agents, two parameters are usually considered: increasing the number of coordinated water molecules or increasing the rotational correlation time by increasing molecular weight and size. This has been achieved by noncovalent or covalent binding of low-molecular weight Gd3+ chelates to macromolecules or polymers. The grafting of these high-spin paramagnetic gadolinium chelates on metal oxide nanoparticles (SiO2, Al2O3) is proposed. This new synthetic strategy presents at least two main advantages: (1) a high T1-relaxivity for MRI with a 275% increase of the MRI signal and (2) the ability of nanoparticles to be internalized in cells. Results indicate that these new contrast agents lead to a huge reconcentration of Gd3+ paramagnetic species inside microglial cells. This reconcentration phenomenon gives rise to high signal-to-noise ratios on MR images of cells after particle internalization, from 1.4 to 3.75, using Al2O3 or SiO2 particles, respectively. The properties of these new particles will be further used to get new insight into gene therapy against glioma, using microglial cells as vehicles to simultaneously transport a suicide gene and contrast agents. Since microglia are chemoattracted to brain tumors, the presence of these new contrast agents inside the cells will lead to a better MRI determination of the in vivo location, shape, and borders of the tumors. These Gd3+-loaded microglia can therefore provide effective localization of tumors by MRI before applying any therapeutic treatment. The rate of carcinoma remission following a suicide gene strategy is also possible.

Molecular imaging with targeted perfluorocarbon nanoparticles: quantification of the concentration dependence of contrast enhancement for binding to sparse cellular epitopes.
Marsh JN, Partlow KC, Abendschein DR, Scott MJ, Lanza GM, Wickline SA.
Ultrasound Med Biol. 2007 Jun;33(6):950-8.
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Targeted, liquid perfluorocarbon nanoparticles are effective agents for acoustic contrast enhancement of abundant cellular epitopes (e.g., fibrin in thrombi) and for lower prevalence binding sites, such as integrins associated with tumor neovasculature. In this study, we sought to delineate the quantitative relationship between the extent of contrast enhancement of targeted surfaces and the density (and concentration) of bound perfluorocarbon (PFC) nanoparticles. Two dramatically different substrates were utilized for targeting. In one set of experiments, the surfaces of smooth, flat, avidin-coated agar disks were exposed to biotinylated nanoparticles to yield a thin layer of targeted contrast. For the second set of measurements, we targeted PFC nanoparticles applied in thicker layers to cultured smooth muscle cells expressing the transmembrane glycoprotein "tissue factor" at the cell surface. An acoustic microscope was used to characterize reflectivity for all samples as a function of bound PFC (determined via gas chromatography). We utilized a formulation of low-scattering nanoparticles having oil-based cores to compete against high-scattering PFC nanoparticles for binding, to elucidate the dependence of contrast enhancement on PFC concentration. The relationship between reflectivity enhancement and bound PFC content varied in a curvilinear fashion and exhibited an apparent asymptote (approximately 16 dB and 9 dB enhancement for agar and cell samples, respectively) at the maximum concentrations (approximately 150 microg and approximately 1000 microg PFOB for agar and cell samples, respectively). Samples targeted with only oil-based nanoparticles exhibited mean backscatter values that were nearly identical to untreated samples (<1 dB difference), confirming the oil particles' low-scattering behavior. The results of this study indicate that substantial contrast enhancement with liquid perfluorocarbon nanoparticles can be realized even in cases of partial surface coverage (as might be encountered when targeting sparsely populated epitopes) or when targeting surfaces with locally irregular topography. Furthermore, it may be possible to assess the quantity of bound cellular epitopes through acoustic means.

Weighing of biomolecules, single cells and single nanoparticles in fluid.
Burg TP, Godin M, Knudsen SM, Shen W, Carlson G, Foster JS, Babcock K, Manalis SR.
Nature. 2007 Apr 26;446(7139):1066-9.
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Nanomechanical resonators enable the measurement of mass with extraordinary sensitivity. Previously, samples as light as 7 zeptograms (1 zg = 10(-21) g) have been weighed in vacuum, and proton-level resolution seems to be within reach. Resolving small mass changes requires the resonator to be light and to ring at a very pure tone-that is, with a high quality factor. In solution, viscosity severely degrades both of these characteristics, thus preventing many applications in nanotechnology and the life sciences where fluid is required. Although the resonant structure can be designed to minimize viscous loss, resolution is still substantially degraded when compared to measurements made in air or vacuum. An entirely different approach eliminates viscous damping by placing the solution inside a hollow resonator that is surrounded by vacuum. Here we demonstrate that suspended microchannel resonators can weigh single nanoparticles, single bacterial cells and sub-monolayers of adsorbed proteins in water with sub-femtogram resolution (1 Hz bandwidth). Central to these results is our observation that viscous loss due to the fluid is negligible compared to the intrinsic damping of our silicon crystal resonator. The combination of the low resonator mass (100 ng) and high quality factor (15,000) enables an improvement in mass resolution of six orders of magnitude over a high-end commercial quartz crystal microbalance. This gives access to intriguing applications, such as mass-based flow cytometry, the direct detection of pathogens, or the non-optical sizing and mass density measurement of colloidal particles.

A p53-derived apoptotic peptide derepresses p73 to cause tumor regression in vivo.
Bell H, Dufes C, O’Prey J, Crighton D, Bergamaschi D, Lu X, Schätzlein A, Vousden K, Ryan K.
J. Clin. Invest. 2007 Apr;117 (4):1008-1018.
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The tumor suppressor p53 is a potent inducer of tumor cell death, and strategies exist to exploit p53 for therapeutic gain. However, because about half of human cancers contain mutant p53, application of these strategies is restricted. p53 family members, in particular p73, are in many ways functional paralogs of p53, but are rarely mutated in cancer. Methods for specific activation of p73, however, remain to be elucidated. We describe here a minimal p53-derived apoptotic peptide that induced death in multiple cell types regardless of p53 status. While unable to activate gene expression directly, this peptide retained the capacity to bind iASPP — a common negative regulator of p53 family members. Concordantly, in p53-null cells, this peptide derepressed p73, causing p73-mediated gene activation and death. Moreover, systemic nanoparticle delivery of a transgene expressing this peptide caused tumor regression in vivo via p73. This study therefore heralds what we believe to be the first strategy to directly and selectively activate p73 therapeutically and may lead to the development of broadly applicable agents for the treatment of malignant disease.

Quantum dot labeling and tracking of human leukemic, bone marrow and cord blood cells.
Garon EB, Marcu L, Luong Q, Tcherniantchouk O, Crooks GM, Koeffler HP.
Leuk Res. 2007 May;31(5):643-51.
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Quantum dots (QDs) are nanometer scale fluorescent semiconductors that are increasingly used as labeling tools in biological research. These nanoparticles have physical properties, such as high quantum yield and resistance to photobleaching, that make them attractive molecular probes for tracking hematologic cells. Here, we show that QDs attached to a transporter protein effectively label all hematologic cells tested, including cell lines and malignant and non-malignant patient samples. We demonstrate that dividing cells can be tracked through at least four cell divisions. In leukemic cell lines, some cells remain labeled for 2 weeks. We show that QDs can be used to follow cells as they differentiate. QDs are seen in monocyte-like and neutrophil-like progeny of labeled HL-60 myeloblasts exposed to Vitamin D analogues and DMSO, respectively. QDs are also observed in monocytes generated from labeled CD34+ cells. In addition, QDs attached to streptavidin can target cells with differing cell surface markers, including CD33. In summary, QDs have the ability to bind to specific cells of interest, be taken up by a diverse range of hematologic cells, and followed through many divisions and through differentiation. These results establish QDs as extremely useful molecular imaging tools for the study of hematologic cells.

Exploiting nanotechnology to target cancer.
Sengupta S, Sasisekharan R.
Br J Cancer. 2007 May 7;96(9):1315-9.
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Nanotechnology is increasingly finding use in the management of cancer. Nanoscale devices have impacted cancer biology at three levels: early detection using, for example, nanocantilevers or nanoparticles; tumour imaging using radiocontrast nanoparticles or quantum dots; and drug delivery using nanovectors and hybrid nanoparticles. This review addresses some of the major milestones in the integration of nanotechnology and cancer biology, and the future of nanoscale approaches for cancer management.

Nanotechnology applications in cancer.
Nie S, Xing Y, Kim GJ, Simons JW.
Annu Rev Biomed Eng. 2007;9:257-88.
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Cancer nanotechnology is an interdisciplinary area of research in science, engineering, and medicine with broad applications for molecular imaging, molecular diagnosis, and targeted therapy. The basic rationale is that nanometer-sized particles, such as semiconductor quantum dots and iron oxide nanocrystals, have optical, magnetic, or structural properties that are not available from molecules or bulk solids. When linked with tumor targeting ligands such as monoclonal antibodies, peptides, or small molecules, these nanoparticles can be used to target tumor antigens (biomarkers) as well as tumor vasculatures with high affinity and specificity. In the mesoscopic size range of 5-100 nm diameter, nanoparticles also have large surface areas and functional groups for conjugating to multiple diagnostic (e.g., optical, radioisotopic, or magnetic) and therapeutic (e.g., anticancer) agents. Recent advances have led to bioaffinity nanoparticle probes for molecular and cellular imaging, targeted nanoparticle drugs for cancer therapy, and integrated nanodevices for early cancer detection and screening. These developments raise exciting opportunities for personalized oncology in which genetic and protein biomarkers are used to diagnose and treat cancer based on the molecular profiles of individual patients.

Nanotechnology platforms and physiological challenges for cancer therapeutics.
Kim KY.
Nanomedicine. 2007 Jun;3(2):103-10.
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Nanotechnology is considered to be an emerging, disruptive technology that will have significant impact in all industrial sectors and across-the-board applications in cancer research. There has been tremendous investment in this area and an explosion of research and development efforts in recent years, particularly in the area of cancer research. At the National Institutes of Health, nanomedicine is one of the priority areas under its Roadmap Initiatives. Moreover, in 2005 the National Cancer Institute alone committed $144.3 million over 5 years for its Alliance for Nanotechnology in Cancer program. Much research and development is progressing in the areas of cancer diagnostics, devices, biosensors, and microfluidics, but this review will focus on therapeutics. Current nanotechnology platforms for cancer therapeutics encompass a vast array of nanomaterials and nanodevices. This review will focus on six of the most prominent and most widely studied: nanoshells, carbon nanotubes, dendrimers, quantum dots, superparamagnetic nanoparticles, and liposomes. All of these nanotechnology platforms can be multifunctional, so they are frequently touted as "smart" or "intelligent." This review will discuss the shared approaches in the design and development of these nanotechnology platforms that bestow such characteristics to the nanoparticles. Finally, the review will raise awareness of the physiological challenges for the application of these therapeutic nanotechnologies, in light of some recent advances in our understanding of tumor biology.

Development of a nanotechnology based low-LET multi-microbeam array single cell irradiation system.
Chang S, Zhang J, Bordelon D, Schreiber E, Cox A, Zhou O.
Radiat Prot Dosimetry. 2006;122(1-4):323-6.
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A novel single cell irradiation system using carbon nanotube (CNT) based field emission technology is proposed. The system can produce electron microbeam at a large range of pulsation frequencies and dose rates with energy between 20 and 60 keV. Different from any existing single beam microbeam device, the CNT-based system can have 10,000 microbeam pixels, each is approximately 10 microm in size and individually controlled. Microscope imaging will be used for targeting cell(s) and the coordinate(s) identification. A single cell or large number of individually selected cells can be simultaneously irradiated under real time microscope observation. This poster reports our preliminary results in the initial stage of the CNT multipixel microbeam array development-prototype single pixel CNT microbeam device development.

Honokiol, a natural plant product, inhibits the bone metastatic growth of human prostate cancer cells.
Shigemura K, Arbiser JL, Sun SY, Zayzafoon M, Johnstone PA, Fujisawa M, Gotoh A, Weksler B, Zhau HE, Chung LW.
Cancer. 2007 Apr 1;109(7):1279-89.
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BACKGROUND: Honokiol, a soluble nontoxic natural product derived from Magnolia spp., has been shown to induce apoptosis in malignant cells. The effect of honokiol and the combined therapy with docetaxel on prostate cancer (PCa) growth and bone metastasis was investigated in experimental models. METHODS: The in vitro proapoptotic effects of honokiol on human androgen-dependent and -independent PCa, bone marrow, bone marrow-derived endothelial, and prostate stroma cells were investigated. Honokiol-induced activation of caspases was evaluated by Western blot and FACS analysis. To confirm the cytotoxicity of honokiol, mice bone was inoculated in vivo with androgen-independent PCa, C4-2 cells and the effects of honokiol and/or docetaxel on PCa growth in bone were evaluated. Daily honokiol (100 mg/kg) and/or weekly docetaxel (5 mg/kg) were injected intraperitoneally for 6 weeks. PCa growth in mouse bone was evaluated by radiography, serum prostate-specific antigen (PSA) and tissue immunohistochemistry. RESULTS: Honokiol induced apoptosis in all cell lines tested. In PCa cells honokiol induced apoptosis via the activation of caspases 3, 8, and 9 and the cleavage of poly-adenosine diphosphate ribose polymerase in a dose- and time-dependent manner. Honokiol was shown to inhibit the growth and depress serum PSA in mice harboring C4-2 xenografts in the skeleton and the combination with docetaxel showed additive effects that inhibited further growth without evidence of systemic toxicity. Immunohistochemical staining confirmed honokiol exhibited growth-inhibitory, apoptotic, and antiangiogenic effects on PCa xenografts. CONCLUSIONS: The combination of honokiol and low-dose docetaxel may be used to improve patient outcome in androgen-independent prostate cancer with bone metastasis.

Single-Molecule Fluorescence Analysis of Cellular Nanomachinery Components.
Peters R.
Annu Rev Biophys Biomol Struct. 2007 Feb 8; [Epub ahead of print].
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Recent progress in proteomics suggests that the cell can be conceived as a large network of highly refined, nanomachine-like protein complexes. This working hypothesis calls for new methods capable of analyzing individual protein complexes in living cells and tissues at high speed. Here, we examine whether single-molecule fluorescence (SMF) analysis can satisfy that demand. First, recent technical progress in the visualization, localization, tracking, conformational analysis, and true resolution of individual protein complexes is highlighted. Second, results obtained by the SMF analysis of protein complexes are reviewed, focusing on the nuclear pore complex as an instructive example. We conclude that SMF methods provide powerful, indispensable tools for the structural and functional characterization of protein complexes. However, the transition from in vitro systems to living cells is in the initial stages. We discuss how current limitations in the nanoscopic analysis of living cells and tissues can be overcome to create a new paradigm, nanoscopic biomedicine.

Characterization and application of single fluorescent nanodiamonds as cellular biomarkers.
Fu CC, Lee HY, Chen K, Lim TS, Wu HY, Lin PK, Wei PK, Tsao PH, Chang HC, Fann W.
Proc Natl Acad Sci USA. 2007 Jan 16;104(3):727-32.
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Type Ib diamonds emit bright fluorescence at 550-800 nm from nitrogen-vacancy point defects, (N-V)(0) and (N-V)(-), produced by high-energy ion beam irradiation and subsequent thermal annealing. The emission, together with noncytotoxicity and easiness of surface functionalization, makes nano-sized diamonds a promising fluorescent probe for single-particle tracking in heterogeneous environments. We present the result of our characterization and application of single fluorescent nanodiamonds as cellular biomarkers. We found that, under the same excitation conditions, the fluorescence of a single 35-nm diamond is significantly brighter than that of a single dye molecule such as Alexa Fluor 546. The latter photobleached in the range of 10 s at a laser power density of 10(4) W/cm(2), whereas the nanodiamond particle showed no sign of photobleaching even after 5 min of continuous excitation. Furthermore, no fluorescence blinking was detected within a time resolution of 1 ms. The photophysical properties of the particles do not deteriorate even after surface functionalization with carboxyl groups, which form covalent bonding with polyL-lysines that interact with DNA molecules through electrostatic forces. The feasibility of using surface-functionalized fluorescent nanodiamonds as single-particle biomarkers is demonstrated with both fixed and live HeLa cells.

Microarray-based kinase inhibition assay by gold nanoparticle probes.
Sun L, Liu D, Wang Z.
Anal Chem. 2007 Jan 15;79(2):773-7.
[ expand abstract ]

We report on the development of a new class of kinase microarray for the detection of kinase inhibition based on marking peptide phosphorylation/biotinylation events by attachment of gold nanoparticles followed by silver deposition for signal enhancement. The alpha-catalytic subunit of cyclic adenosine 5'-monophosphate-dependent protein kinase (PKA), and its well-known substrate, kemptide, were used for the purpose of monitoring phosphorylation and inhibition. As expected, highly selective inhibition of PKA is demonstrated with the four inhibitors: H89, HA1077, mallotoxin, and KN62. Furthermore, an inhibition assay demonstrates the ability to detect kinase inhibition as well as derive IC50 (half-maximal inhibitory concentration) plots.

Luminescent Quantum Dots Fluorescence Resonance Energy Transfer-Based Probes for Enzymatic Activity and Enzyme Inhibitors.
Shi L, Rosenzweig N, Rosenzweig Z.
Anal Chem. 2007 Jan 1;79(1):208-214.
[ expand abstract ]

The paper describes the development and characterization of analytical properties of quantum dot-based probes for enzymatic activity and for screening enzyme inhibitors. The luminescent probes are based on fluorescence resonance energy transfer (FRET) between luminescent quantum dots that serve as donors and rhodamine acceptors that are immobilized to the surface of the quantum dots through peptide linkers. Peptide-coated CdSe/ZnS quantum dots were prepared using a one-step ligand exchange process in which RGDC peptide molecules replace trioctylphosphine oxide (TOPO) molecules as the capping ligands of the quantum dots. The peptide molecules were bound to the surface of the CdSe/ZnS quantum dots through the thiol group of the peptide cysteine residue. The peptide-coated quantum dots were labeled with rhodamine to form the FRET probes. The emission quantum yield of the quantum dot FRET probes was 4-fold lower than the emission quantum yield of TOPO-capped quantum dots. However, the quantum dot FRET probes were sufficiently bright to enable quantitative enzyme and enzyme inhibition assays. The probes were used first to test the enzymatic activity of trypsin in solution based on FRET signal changes of the quantum dot-based enzymatic probes in the presence of proteolytic enzymes. For example, exposure of the quantum dot FRET probes to 500 &mgr;g/mL trypsin for 15 min resulted in 60% increase in the photoluminescence of the quantum dots and a corresponding decrease in the emission of the rhodamine molecules. These changes resulted from the release of rhodamine molecules from the surface of the quantum dots due to enzymatic cleavage of the peptide molecules. The quantum dot FRET-based probes were used to monitor the enzymatic activity of trypsin and to screen trypsin inhibitors for their inhibition efficiency.

A microfluidic system in combination with optical tweezers for analyzing rapid and reversible cytological alterations in single cells upon environmental changes.
Eriksson E, Enger J, Nordlander B, Erjavec N, Ramser K, Goksor M, Hohmann S, Nystrom T, Hanstorp D.
Lab Chip. 2007 Jan;7(1):71-6.
[ expand abstract ]

We report on the development of an experimental platform where epi-fluorescence microscopy and optical tweezers are combined with a microfluidic system to enable the analysis of rapid cytological responses in single cells. The microfluidic system allows two different media to be merged in a Y-shaped channel. Microscale channel dimensions ensure purely laminar flow and, as a result, an environmental gradient can be created between the two media. Optical tweezers are used to move a single trapped cell repeatedly between the different environments. The cell is monitored continuously by fluorescence microscopy during the experiment. In a first experiment on yeast (Saccharomyces cerevisiae) we observed changes in cell volume as the cell was moved between environments with different osmolarity. This demonstrated that the platform allowed analysis of cytological alterations on a time scale shorter than 0.2 s. In a second experiment we observed the spatial migration of the Yap1p transcription factor fused to GFP as a cell was moved from an environment of low to high oxidative capacity. The system is universal allowing the response to numerous environmental changes to be studied on the sub second time scale in a variety of model cells. We intend to use the platform to study how the age of cells, their progression through the cell cycle, or their genetic landscape, alter their capacity (kinetics and amplitude) to respond to environmental changes.

Combining optical tweezers and scanning probe microscopy to study DNA-protein interactions.
Huisstede JH, Subramaniam V, Bennink ML.
Microsc Res Tech. 2007 Jan;70(1):26-33.
[ expand abstract ]

We present the first results obtained with a new instrument designed and built to study DNA-protein interactions at the single molecule level. This microscope combines optical tweezers with scanning probe microscopy and allows us to locate DNA-binding proteins on a single suspended DNA molecule. A single DNA molecule is stretched taut using the optical tweezers, while a probe is scanned along the molecule. Interaction forces between the probe and the sample are measured with the optical tweezers. The instrument thus enables us to correlate mechanical and functional properties of bound proteins with the tension within the DNA molecule. The typical friction force between a micropipette used as probe and a naked DNA molecule was found to be <1 pN. A 16 mum DNA molecule with approximately 10-15 digoxygenin (DIG) molecules located over a 90 nm range in the middle of the DNA was used as a model system. By scanning with an antidigoxygenin (alpha-DIG) antibody-coated pipette we were able to localize these sites by exploiting the high binding affinity between this antibody-antigen pair. The estimated experimental resolution assuming an infinitesimally thin and rigid probe and a single alpha-DIG/DIG bond was 15 nm.

Single-cell manipulation and analysis using microfluidic devices.
Roman GT, Chen Y, Viberg P, Culbertson AH, Culbertson CT.
Anal Bioanal Chem. 2007 Jan;387(1):9-12.
[ expand abstract ]

No abstract available

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2006

Nanoliter microfluidic hybrid method for simultaneous screening and optimization validated with crystallization of membrane proteins.
Li L, Mustafi D, Fu Q, Tereshko V, Chen DL, Tice JD, Ismagilov RF.
Proc Natl Acad Sci USA. 2006 Dec 19;103(51):19243-8.
[ expand abstract ]

High-throughput screening and optimization experiments are critical to a number of fields, including chemistry and structural and molecular biology. The separation of these two steps may introduce false negatives and a time delay between initial screening and subsequent optimization. Although a hybrid method combining both steps may address these problems, miniaturization is required to minimize sample consumption. This article reports a "hybrid" droplet-based microfluidic approach that combines the steps of screening and optimization into one simple experiment and uses nanoliter-sized plugs to minimize sample consumption. Many distinct reagents were sequentially introduced as approximately 140-nl plugs into a microfluidic device and combined with a substrate and a diluting buffer. Tests were conducted in approximately 10-nl plugs containing different concentrations of a reagent. Methods were developed to form plugs of controlled concentrations, index concentrations, and incubate thousands of plugs inexpensively and without evaporation. To validate the hybrid method and demonstrate its applicability to challenging problems, crystallization of model membrane proteins and handling of solutions of detergents and viscous precipitants were demonstrated. By using 10 microl of protein solution, approximately 1,300 crystallization trials were set up within 20 min by one researcher. This method was compatible with growth, manipulation, and extraction of high-quality crystals of membrane proteins, demonstrated by obtaining high-resolution diffraction images and solving a crystal structure. This robust method requires inexpensive equipment and supplies, should be especially suitable for use in individual laboratories, and could find applications in a number of areas that require chemical, biochemical, and biological screening and optimization.

Single nanoparticle photothermal tracking (SNaPT) of 5-nm gold beads in live cells.
Lasne D, Blab GA, Berciaud S, Heine M, Groc L, Choquet D, Cognet L, Lounis B.
Biophys J. 2006 Dec 15;91(12):4598-604.
[ expand abstract ]

Tracking individual nano-objects in live cells during arbitrary long times is a ubiquitous need in modern biology. We present here a method for tracking individual 5-nm gold nanoparticles on live cells. It relies on the photothermal effect and the detection of the Laser Induced Scattering around a NanoAbsorber (LISNA). The key point for recording trajectories at video rate is the use of a triangulation procedure. The effectiveness of the method is tested against single fluorescent molecule tracking in live COS7 cells on subsecond timescales. We further demonstrate recordings for several minutes of AMPA receptors trajectories on the plasma membrane of live neurons. Single Nanoparticle Photothermal Tracking has the unique potential to record arbitrary long trajectory of membrane proteins using nonfluorescent nanometer-sized labels.

Nanoliter microfluidic hybrid method for simultaneous screening and optimization validated with crystallization of membrane proteins.
Li L, Mustafi D, Fu Q, Tereshko V, Chen DL, Tice JD, Ismagilov RF.
Proc Natl Acad Sci USA. 2006 Dec 11; [Epub ahead of print].
[ expand abstract ]

High-throughput screening and optimization experiments are critical to a number of fields, including chemistry and structural and molecular biology. The separation of these two steps may introduce false negatives and a time delay between initial screening and subsequent optimization. Although a hybrid method combining both steps may address these problems, miniaturization is required to minimize sample consumption. This article reports a "hybrid" droplet-based microfluidic approach that combines the steps of screening and optimization into one simple experiment and uses nanoliter-sized plugs to minimize sample consumption. Many distinct reagents were sequentially introduced as approximately 140-nl plugs into a microfluidic device and combined with a substrate and a diluting buffer. Tests were conducted in approximately 10-nl plugs containing different concentrations of a reagent. Methods were developed to form plugs of controlled concentrations, index concentrations, and incubate thousands of plugs inexpensively and without evaporation. To validate the hybrid method and demonstrate its applicability to challenging problems, crystallization of model membrane proteins and handling of solutions of detergents and viscous precipitants were demonstrated. By using 10 microl of protein solution, approximately 1,300 crystallization trials were set up within 20 min by one researcher. This method was compatible with growth, manipulation, and extraction of high-quality crystals of membrane proteins, demonstrated by obtaining high-resolution diffraction images and solving a crystal structure. This robust method requires inexpensive equipment and supplies, should be especially suitable for use in individual laboratories, and could find applications in a number of areas that require chemical, biochemical, and biological screening and optimization.

Nanoparticle-Based Energy Transfer for Rapid and Simple Detection of Protein Glycosylation.
Oh E, Lee D, Kim YP, Cha SY, Oh DB, Kang HA, Kim J, Kim HS.
Angew Chem Int Ed Engl. 2006 Dec 4;45(47):7959-7963.
[ expand abstract ]

No abstract available

Cellular uptake of magnetic nanoparticle is mediated through energydependent endocytosis in A549 cells.
Kim JS, Yoon TJ, Yu KN, Noh MS, Woo M, Kim BG, Lee KH, Sohn BH, Park SB, Lee JK, Cho MH.
J Vet Sci. 2006 Dec;7(4):321-6.
[ expand abstract ]

Biocompatible silica-overcoated magnetic nanoparticles containing an organic fluorescence dye, rhodamine B isothiocyanate (RITC), within a silica shell [50 nm size, MNP@SiO(2)(RITC)s] were synthesized. For future application of the MNP@SiO(2)(RITC)s into diverse areas of research such as drug or gene delivery, bioimaging, and biosensors, detailed information of the cellular uptake process of the nanoparticles is essential. Thus, this study was performed to elucidate the precise mechanism by which the lung cancer cells uptake the magnetic nanoparticles. Lung cells were chosen for this study because inhalation is the most likely route of exposure and lung cancer cells were also found to uptake magnetic nanoparticles rapidly in preliminary experiments. The lung cells were pretreated with different metabolic inhibitors. Our results revealed that low temperature disturbed the uptake of magnetic nanoparticles into the cells. Metabolic inhibitors also prevented the delivery of the materials into cells. Use of TEM clearly demonstrated that uptake of the nanoparticles was mediated through endosomes. Taken together, our results demonstrate that magnetic nanoparticles can be internalized into the cells through an energy-dependent endosomal-lysosomal mechanism.

Optical measurement of cell membrane tension.
Popescu G, Ikeda T, Goda K, Best-Popescu CA, Laposata M, Manley S, Dasari RR, Badizadegan K, Feld MS.
Phys Rev Lett. 2006 Nov 24;97(21):218101.
[ expand abstract ]

Using a novel noncontact technique based on optical interferometry, we quantify the nanoscale thermal fluctuations of red blood cells (RBCs) and giant unilamellar vesicles (GUVs). The measurements reveal a nonvanishing tension coefficient for RBCs, which increases as cells transition from a discocytic shape to a spherical shape. The tension coefficient measured for GUVs is, however, a factor of 4-24 smaller. By contrast, the bending moduli for cells and vesicles have similar values. This is consistent with the cytoskeleton confinement model, in which the cytoskeleton inhibits membrane fluctuations.

A quantitative observation and imaging of single tumor cell migration and deformation using a multi-gap microfluidic device representing the blood vessel.
Chaw KC, Manimaran M, Tay FE, Swaminathan S.
Microvasc Res. 2006 Nov;72(3):153-60.
[ expand abstract ]

A microfluidic device was developed for quantifying the migratory and deformability capabilities of a single tumor cell using direct imaging. It was fabricated using photolithography and is made of polydimethysiloxane. Chemotaxis approach was used for directing cell movement, using 10 mum microgaps to restrict the migration to a single cell. Each cell's migration rate is quantified as a measure of its distance traveled over time taken. Real-time recording of cell deformation under physiological flow was performed, and the elongation index and surface area change of the cells were compared. Three human tumor cell lines viz. HepG2, HeLa and MDA-MB-435S were used to verify the operation and methodology of the device. Their migration rates ranged from 5 to 15 mum/h, consistent with other scientific reports. By reducing the microgap width to 3 mum, it was found that the cells moved along the row of microgaps but were unable to migrate across the microgaps. Subsequent deformation of the cells through the gaps further showed that their migratory capability might be governed by their deformation ability and the deformation stress on their membranes. The strategy of targeting cancer cell membrane for rupture may provide a therapy for metastasis. Being a valuable tool for rapid quantification of a single cell's migratory capability, this device should be helpful for pharmacologic and drug screening, investigation of factors that regulate cell migration and deformation.

Three-dimensional reconstruction of cell nuclei, internalized quantum dots and sites of lipid peroxidation.
Funnell WR, Maysinger D.
J Nanobiotechnology. 2006 Oct 20;4:10.
[ expand abstract ]

ABSTRACT: BACKGROUND: The purpose of the study was to develop and illustrate three-dimensional (3-D) reconstruction of nuclei and intracellular lipid peroxidation in cells exposed to oxidative stress induced by quantum dots. Programmed cell death is characterized by multiple biochemical and morphological changes in different organelles, including nuclei, mitochondria and lysosomes. It is the dynamics of the spatio-temporal changes in the signalling and morphological adaptations which will ultimately determine the 'shape' and fate of the cell. RESULTS: We present new approaches to the 3-D reconstruction of organelle morphology and biochemical changes in confocal live-cell images. We demonstrate the 3-D shapes of nuclei, the 3-D intracellular distributions of QDs and the accompanying lipid-membrane peroxidation, and provide methods for quantification. CONCLUSION: This study provides an approach to 3-D organelle and nanoparticle visualization in the context of cell death; however, this approach is also applicable more generally to investigating changes in organelle morphology in response to therapeutic interventions, stressful stimuli and internalized nanoparticles. Moreover, the approach provides quantitative data for such changes, which will help us to better integrate compartmentalization of subcellular events and to link morphological and biochemical changes with physiological outcomes.

Applications of microfluidics in chemical biology.
Weibel DB, Whitesides GM.
Curr Opin Chem Biol. 2006 Oct 20; [Epub ahead of print].
[ expand abstract ]

This review discusses the application of microfluidics in chemical biology. It aims to introduce the reader to microfluidics, describe characteristics of microfluidic systems that are useful in studying chemical biology, and summarize recent progress at the interface of these two fields. The review concludes with an assessment of future directions and opportunities of microfluidics in chemical biology.

Quantum dot labeling and tracking of human leukemic, bone marrow and cord blood cells.
Garon EB, Marcu L, Luong Q, Tcherniantchouk O, Crooks GM, Koeffler HP.
Leuk Res. 2006 Oct 5; [Epub ahead of print] .
[ expand abstract ]

Quantum dots (QDs) are nanometer scale fluorescent semiconductors that are increasingly used as labeling tools in biological research. These nanoparticles have physical properties, such as high quantum yield and resistance to photobleaching, that make them attractive molecular probes for tracking hematologic cells. Here, we show that QDs attached to a transporter protein effectively label all hematologic cells tested, including cell lines and malignant and non-malignant patient samples. We demonstrate that dividing cells can be tracked through at least four cell divisions. In leukemic cell lines, some cells remain labeled for 2 weeks. We show that QDs can be used to follow cells as they differentiate. QDs are seen in monocyte-like and neutrophil-like progeny of labeled HL-60 myeloblasts exposed to Vitamin D analogues and DMSO, respectively. QDs are also observed in monocytes generated from labeled CD34+ cells. In addition, QDs attached to streptavidin can target cells with differing cell surface markers, including CD33. In summary, QDs have the ability to bind to specific cells of interest, be taken up by a diverse range of hematologic cells, and followed through many divisions and through differentiation. These results establish QDs as extremely useful molecular imaging tools for the study of hematologic cells.

Distinct Effects of Annexin A7 and p53 on Arachidonate Lipoxygenation in Prostate Cancer Cells Involve 5-Lipoxygenase Transcription.
Torosyan Y, Dobi A, Naga S, Mezhevaya K, Glasman M, Norris C, Jiang G, Mueller G, Pollard H, Srivastava M.
Cancer Res. 2006 Oct 1;66(19):9609-16.
[ expand abstract ]

Tumor suppressor function for Annexin A7 (ANXA7; 10q21) is based on cancer-prone phenotype in Anxa7(+/-) mouse and ANXA7 prognostic role in human cancers. Because ANXA7-caused liposome aggregation can be promoted by arachidonic acid (AA), we hypothesized that the phospholipid-binding tumor suppressor ANXA7 is associated with AA cascade. In a comparative study of ANXA7 versus canonical tumor suppressor p53 effects on AA lipoxygenation pathway in the p53-mutant and androgen-insensitive DU145 prostate cancer cells, both tumor suppressors altered gene expression of major 5-lipoxygenase (LOX) and 15-LOXs, including response to T helper 2 (Th2)-cytokine [interleukin-4 (IL-4)] and endogenous steroids (mimicked by dexamethasone). Wild-type and mutant ANXA7 distinctly affected expression of the dexamethasone-induced 15-LOX-2 (a prostate-specific endogenous tumor suppressor) as well as the IL-4-induced 15-LOX-1. On the other hand, wild-type p53 restored 5-LOX expression in DU145 to levels comparable to benign prostate epithelial cells. Using mass spectrometry of DNA affinity-enriched nuclear proteins, we detected different proteins that were bound to adjacent p53 and estrogen response elements in the 5-LOX promoter in DU145 cells introduced with ANXA7 versus p53. Sex hormone regulator 17-beta hydroxysteroid dehydrogenase 4 was identified under p53 introduction, which induced the 5-LOX expression. Meantime, nuclear proteins bound to the same 5-LOX promoter site under introduction of ANXA7 (that was associated with the repressed 5-LOX) were identified as zinc finger proteins ZNF433 and Aiolos, pyrin domain-containing NALP10, and the p53-regulating DNA repair enzyme APEX1. Thus, ANXA7 and p53 can distinctly regulate LOX transcription that is potentially relevant to the AA-mediated cell growth control in tumor suppression. (Cancer Res 2006; 66(19): 9609-16).

Single-molecule mountains yield nanoscale cell images.
Moerner WE.
Nat Methods. 2006 Oct;3(10):781-2.
[ expand abstract ]

No abstract available

Intracellular labeling method for chip-based capillary electrophoresis fluorimetric single cell analysis using liposomes.
Sun Y, Lu M, Yin XF, Gong XG.
J Chromatogr A. 2006 Sep 25; [Epub ahead of print] .
[ expand abstract ]

An intracellular derivatization method mediated by liposome was developed for single cell analysis with chip-based capillary electrophoresis (CE) and laser-induced fluorescence (LIF) detection. Liposomes with an average diameter of 100nm were produced from phosphatidylcholine to encapsulate fluorescent dyes by an ultrasonic method. The encapsulation yield and the vesicle density were determined to be 46+/-5% and 8.8x10(14)/mL, respectively. The amount of fluorescent dye that entered the cells was dependent on the duration of incubating cells with liposomes, liposome density, and concentration of the dye solution encapsulated in liposomes. The described method introduced cell membrane nonpermeable fluorescent dyes into living cells without reducing cell viability. Single cell analysis using microfluidic chip-based CE revealed that liposome-membrane fusion occurred after entrance of liposomes into the cells, with release of encapsulated fluorescence dyes and labeling of intracellular species.

Single Nanoparticle Photothermal Tracking (SNaPT) of 5 nm gold beads in live cells.
Lasne D, Blab GA, Berciaud S, Heine M, Groc L, Choquet D, Cognet L, Lounis B.
Biophys J. 2006 Sep 22; [Epub ahead of print] .
[ expand abstract ]

Tracking individual nano-objets in live cells during arbitrary long times is an ubiquitous need in modern biology. We present here a method for tracking individual 5 nm gold nanoparticles on live cells. It relies on the photothermal effect and the detection of the Laser Induced Scattering around a NanoAbsorber (LISNA). The key point for recording trajectories at video rate is the use of a triangulation procedure. The effectiveness of the method is tested against Single fluorescent Molecule Tracking in live COS7 cells on subsecond time scales. We further demonstrate recordings for several minutes of AMPA receptors trajectories on the plasma membrane of live neurons. SNaPT has the unique potential to record arbitrary long trajectory of membrane proteins using non-fluorescent nanometer sized labels.

Atomic force microscopy study of the specific adhesion between a colloid particle and a living melanoma cell: Effect of the charge and the hydrophobicity of the particle surface.
McNamee CE, Pyo N, Higashitani K.
Biophys J. 2006 Sep 1;91(5):1960-9.
[ expand abstract ]

We investigated the effect of the charge and the hydrophobicity of drug delivery system (DDS) carriers on their specificity to living malignant melanoma B16F10 cells with the atomic force microscope. To model various nanoparticle DDS carriers, we used silica particles that were modified with silane coupling agents. We then measured the compression and decompression forces between the modified colloid probes and the living B16F10 cell in a physiological buffer as a function of their separation distances. The maximum adhesive force on decompression was related to the strength of the specificity of the DDS to the malignant cell. A comparison of the average maximum adhesive force of each functionality group surprisingly showed that negatively charged surfaces and hydrophobic modified surfaces all had similar low values. Additionally, we saw the unexpected result that there was no observable dependence on the degree of hydrophobicity of the probe surface to a B16F10 cell. Only the positively charged particle gave a strong adhesive force with the B16F10 cell. This indicated that DDS carriers with positive charges appeared to have the highest affinity for malignant melanoma cells and that the use of hydrophobic materials unexpectedly did not improve their affinity.

Quantum dots based probes conjugated to annexin v for photostable apoptosis detection and imaging.
Le Gac S, Vermes I, van den Berg A.
Nano Lett. 2006 Sep;6(9):1863-9.
[ expand abstract ]

Quantum dots (Qdots) are nanoparticles exhibiting fluorescent properties that can be used for cell staining. We present here the development of quantum dots conjugated to Annexin V for specific targeting of apoptotic cells, for both apoptosis detection and staining of apoptotic "living" cells. For that purpose, Qdots Streptavidin Conjugates are coupled to biotinylated Annexin V, a 35-kDa protein which specifically recognizes and binds to phosphatidylserine (PS) moieties present on the outer membrane of apoptotic cells and not on healthy or necrotic cells. By using Annexin V, our Qdots probes are made specific for apoptotic cells. Staining of apoptotic cells was checked using fluorescence and confocal microscopy techniques and nonfixed cells. It is shown here that Qdots are insensitive to bleaching after prolonged exposure as opposed to organic dyes. This makes Qdots excellent candidates to continuously follow fast changes occurring at the membrane of apoptotic cells and facilitates time-lapse imaging as they alleviate any bleaching issue.

Atomic force microscopy-based cell nanostructure for ligand-conjugated quantum dot endocytosis.
Pan YL, Cai JY, Qin L, Wang H.
Acta Biochim Biophys Sin (Shanghai). 2006 Sep;38(9):646-52.
[ expand abstract ]

While it has been well demonstrated that quantum dots (QDs) play an important role in biological labeling both in vitro and in vivo, there is no report describing the cellular nanostructure basis of receptor-mediated endocytosis. Here, nanostructure evolution responses to the endocytosis of transferrin (Tf)-conjugated QDs were characterized by atomic force microscopy (AFM). AFM-based nanostructure analysis demonstrated that the Tf-conjugated QDs were specifically and tightly bound to the cell receptors and the nanostructure evolution is highly correlated with the cell membrane receptor-mediated transduction. Consistently, confocal microscopic and flow cytometry results have demonstrated the specificity and dynamic property of Tf-QD binding and internalization. We found that the internalization of Tf-QD is linearly related to time. Moreover, while the nanoparticles on the cell membrane increased, the endocytosis was still very active, suggesting that QD nanoparticles did not interfere sterically with the binding and function of receptors. Therefore, ligand-conjugated QDs are potentially useful in biological labeling of cells at a nanometer scale.

Nanoparticle-based sensing of glycan-lectin interactions.
Dai Z, Kawde AN, Xiang Y, La Belle JT, Gerlach J, Bhavanandan VP, Joshi L, Wang J.
J Am Chem Soc. 2006 Aug 9;128(31):10018-9.
[ expand abstract ]

Here we present the first report on nanoparticle-based biosensing of glycan markers of diseases. The protocol relies on the competition between a nanocrystal (CdS)-tagged sugar and the target sugar for the binding sites of surface-confined lectin and monitoring the extent of competition through highly sensitive electrochemical detection of the captured nanocrystal. This development is expected to allow decentralized detection of carbohydrate moieties and lectin-carbohydrate interactions to be performed more rapidly, sensitively, inexpensively, and reliably.

Co-culture of human embryonic stem cells with murine embryonic fibroblasts on microwell-patterned substrates.
Khademhosseini A, Ferreira L, Blumling J 3rd, Yeh J, Karp JM, Fukuda J, Langer R.
Biomaterials. 2006 Aug 8; [Epub ahead of print] .
[ expand abstract ]

Human embryonic stem (hES) cells are generally cultured as cell clusters on top of a feeder layer formed by mitotically inactivated murine embryonic fibroblasts (MEFs) to maintain their undifferentiated state. This co-culture system, which is typically used to expand the population of undifferentiated hES cells, presents several challenges since it is difficult to control cell cluster size. Large cell clusters tend to differentiate at the borders, and clusters with different sizes may lead to heterogeneous differentiation patterns within embryoid bodies. In this work, we develop a new approach to culture hES cells with controlled cluster size and number through merging microfabrication, and biomaterials technologies. Polymeric microwells were fabricated and used to control the size and uniformity of hES cell clusters in co-culture with MEFs. The results show that it is possible to culture hES cells homogeneously while keeping their undifferentiated state as confirmed by the expression of stem cell markers octamer binding protein 4 (Oct-4) and alkaline phosphatase (ALP). In addition, these clusters can be recovered from the microwells to generate nearly homogeneous cell aggregates for differentiation experiments.

The smart Petri dish: a nanostructured photonic crystal for real-time monitoring of living cells.
Schwartz MP, Derfus AM, Alvarez SD, Bhatia SN, Sailor MJ.
Langmuir. 2006 Aug 1;22(16):7084-90.
[ expand abstract ]

The intensity of light scattered from a porous Si photonic crystal is used to monitor physiological changes in primary rat hepatocytes. The cells are seeded on the surface of a porous Si photonic crystal that has been filled with polystyrene and treated with an O2 plasma. Light resonant with the photonic crystal is scattered by the cell layer and detected as an optical peak with a charge-coupled-device spectrometer. It is demonstrated that exposure of hepatocytes to the toxins cadmium chloride or acetaminophen leads to morphology changes that cause a measurable increase in scattered intensity. The increase in signal occurs before traditional assays are able to detect a decrease in viability, demonstrating the potential of the technique as a complementary tool for cell viability studies. The scattering method presented here is noninvasive and can be performed in real time, representing a significant advantage compared to other techniques for in vitro monitoring of cell morphology.

Tracking Individual Kinesin Motors in Living Cells Using Single Quantum-Dot Imaging.
Courty S, Luccardini C, Bellaiche Y, Cappello G, Dahan M.
Nano Lett. 2006 Jul 12;6(7):1491-1495.
[ expand abstract ]

We report a simple method using semiconductor quantum dots (QDs) to track the motion of intracellular proteins with a high sensitivity. We characterized the in vivo motion of individual QD-tagged kinesin motors in living HeLa cells. Single-molecule measurements provided important parameters of the motor, such as its velocity and processivity, as well as an estimate of the force necessary to carry a QD. Our measurements demonstrate the importance of single-molecule experiments in the investigation of intracellular transport as well as the potential of single quantum-dot imaging for the study of important processes such as cellular trafficking, cell polarization, and division.

Optimization of a Microfluidic Mixer for Studying Protein Folding Kinetics.
Hertzog DE, Ivorra B, Mohammadi B, Bakajin O, Santiago JG.
Anal Chem. 2006 Jul 1;78(13):4299-4306.
[ expand abstract ]

We have applied an optimization method in conjunction with numerical simulations to minimize the mixing time of a microfluidic mixer developed for protein folding studies. The optimization method uses a semideterministic algorithm to find the global minimum of the mixing time by varying the mixer geometry and flow conditions. We describe the minimization problem and constraints and give a brief overview of the optimization algorithm. We present results of the optimization, including the optimized geometry and parameter sensitivities, and we demonstrate the improvement in mixing performance with experiments using microfabricated mixers. The dye-quenching experiments of the original and optimized mixer designs show respective mixing times of 7 and 4 mus, a 40% reduction. The new design also provides more uniform mixing across streamlines that enter the mixer. The optimized mixer is the fastest reported continuous flow mixer for protein folding.

Proteolytic activity monitored by fluorescence resonance energy transfer through quantum-dot-peptide conjugates.
Medintz IL, Clapp AR, Brunel FM, Tiefenbrunn T, Tetsuo Uyeda H, Chang EL, Deschamps JR, Dawson PE, Mattoussi H.
Nat Mater. 2006 Jul;5(7):581-9; Epub 2006 Jun 25.
[ expand abstract ]

Proteases are enzymes that catalyse the breaking of specific peptide bonds in proteins and polypeptides. They are heavily involved in many normal biological processes as well as in diseases, including cancer, stroke and infection. In fact, proteolytic activity is sometimes used as a marker for some cancer types. Here we present luminescent quantum dot (QD) bioconjugates designed to detect proteolytic activity by fluorescence resonance energy transfer. To achieve this, we developed a modular peptide structure which allowed us to attach dye-labelled substrates for the proteases caspase-1, thrombin, collagenase and chymotrypsin to the QD surface. The fluorescence resonance energy transfer efficiency within these nanoassemblies is easily controlled, and proteolytic assays were carried out under both excess enzyme and excess substrate conditions. These assays provide quantitative data including enzymatic velocity, Michaelis-Menten kinetic parameters, and mechanisms of enzymatic inhibition. We also screened a number of inhibitory compounds against the QD-thrombin conjugate. This technology is not limited to sensing proteases, but may be amenable to monitoring other enzymatic modifications.

HPLC analysis of functionalized poly(amidoamine) dendrimers and the interaction between a folate-dendrimer conjugate and folate binding protein.
Shi X, Bi X, Ganser TR, Hong S, Myc LA, Desai A, Holl MM, Baker JR.
Analyst. 2006 Jul;131(7):842-8; Epub 2006 Jun 8.
[ expand abstract ]

Poly(amidoamine) (PAMAM) dendrimers of different generations with carboxyl, acetyl, and hydroxyl terminal groups and a folic acid (FA)-dendrimer conjugate were separated and analyzed using reverse-phase high performance liquid chromatography (HPLC). Analysis of both the individual PAMAM derivatives and the separation of mixed generations can be achieved using a linear gradient 0-50% acetonitrile (ACN) (balance water) within 40 min. We also show that PAMAMs with defined acetylation and carboxylation degrees can be analyzed using HPLC. Furthermore, a generation 5 dendrimer-FA conjugate (G5.75Ac-FA(4); Ac denotes acetyl) was analyzed and its specific binding with a bovine folic acid binding protein (FBP) was monitored. The HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results indicate the formation of three complexes after the binding of G5.75Ac-FA(4) with FBP. Dendrimers with FA moieties show much higher specific binding capability with FBP than those without FA moieties. Findings from this study indicate that HPLC is an effective technique not only for characterization and separation of functionalized PAMAM dendrimers and conjugates but also for investigation of the interaction between dendrimers and biomolecules.

Studying protein-drug interaction by microfluidic chip affinity capillary electrophoresis with indirect laser-induced fluorescence detection.
Liu X, Liu X, Liang A, Shen Z, Zhang Y, Dai Z, Xiong B, Lin B.
Electrophoresis. 2006 Jun 29; [Epub ahead of print].
[ expand abstract ]

We developed a microfluidic chip-affinity CE method based on indirect LIF detection to study protein-drug interactions. The interaction between heparin and BSA was quantitatively studied, as a model system. In our method, sodium fluorescein was chosen as background, and redistilled water as marker to monitor EOF. The electrophoretic mobility changes of BSA were measured, with various concentrations of heparin added to the running buffer. Each run was completed within 80 s. The binding constant was determined to be (1.24 +/- 0.05)x10(3) M(-1), which was in good agreement with that reported in the literature.

Structures of DNA-linked nanoparticle aggregates.
Park SY, Lee JS, Georganopoulou D, Mirkin CA, Schatz GC.
J Phys Chem B Condens Matter Mater Surf Interfaces Biophys. 2006 Jun 29;110(25):12673-81.
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The room-temperature structure of DNA-linked gold nanoparticle aggregates is investigated using a combination of experiment and theory. The experiments involve extinction spectroscopy measurements and dynamic light scattering measurements of aggregates made using 60 and 80 nm gold particles and 30 base-pair DNA. The theoretical studies use calculated spectra for models of the aggregate structures to determine which structure matches the observations. These models include diffusion-limited cluster-cluster aggregation (DLCA), reaction-limited cluster-cluster aggregation (RLCA), and compact (nonfractal) cluster aggregation. The diameter of the nanoparticles used in the experiments is larger than has been considered previously, and this provides greater sensitivity of spectra to aggregate structure. We show that the best match between experiment and theory occurs for the RLCA fractal structures. This indicates that DNA hybridization takes place under irreversible conditions in the room-temperature aggregation. Some possible structural variations which might influence the result are considered, including the edge-to-edge distance between nanoparticles, variation in the diameter of the nanoparticles, underlying lattice structures of on-lattice compact clusters, and positional disorders in the lattice structures. We find that these variations do not change the conclusion that the room-temperature structure of the aggregates is fractal. We also examine the variation in extinction at 260 nm as temperature is increased, showing that the decrease in extinction at temperatures below the melting temperature is related to a morphological change from fractal toward compact structures.

Enzyme-mediated individual nanoparticle release assay.
Glass JR, Dickerson JC, Schultz DA.
Anal Biochem. 2006 Jun 15;353(2):209-16; Epub 2006 Mar 31.
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Numerous methods have been developed to measure the presence of macromolecular species in a sample; however, the number of methods that detect functional activity or modulators of that activity is more limited. To address this limitation, an approach was developed that uses the optical detection of nanoparticles as a measure of enzyme activity. Nanoparticles are increasingly being used as biological labels in static binding assays; here, we describe their use in a release assay format, where the enzyme-mediated liberation of individual nanoparticles from a surface is measured. A double-stranded fragment of DNA is used as the initial tether to bind the nanoparticles to a solid surface. The nanoparticle spatial distribution and number are determined using dark-field optical microscopy and digital image capture. Site-specific cleavage of the DNA tether results in nanoparticle release. The methodology and validation of this approach for measuring enzyme-mediated, individual DNA cleavage events, rapidly, with high specificity, and in real-time are described. This approach was used to detect and discriminate between nonmethylated and methylated DNA, and demonstrates a novel platform for high-throughput screening of modulators of enzyme activity.

Novel multi-depth microfluidic chip for single cell analysis.
Yue S, Xue-Feng Y.
J Chromatogr A. 2006 Jun 9;1117(2):228-33; Epub 2006 Apr 18.
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A novel multi-depth microfluidic chip was fabricated on glass substrate by use of conventional lithography and three-step etching technology. The sampling channel on the microchip was 37mum deep, while the separation channel was 12mum deep. A 1mm long weir was constructed in the separation channel, 300mum down the channel crossing. The channel at the weir section was 6mum deep. By using the multi-depth microfluidic chip, human carcinoma cells, which easily aggregate, settle and adhere to the surface of the channel, can be driven from the sample reservoir to the sample waste reservoir by hydrostatic pressure generated by the difference of liquid level between sample and sample waste reservoirs. Single cell loading into the separation channel was achieved by applying a set of pinching potentials at the four reservoirs. The loaded cell was stopped by the weir and precisely positioned within the separation channel. The trapped cell was lysed by sodium dodecyl sulfate (SDS) containing buffer solution in 20s. This approach reduced the lysing time and improved the reproducibility of chip-based electrophoresis separations. Reduced glutathione (GSH) and reactive oxygen species (ROS) were used as model intracellular components in single human carcinoma cells, and the constituents were separated by chip-based electrophoresis and detected by laser-induced fluorescence (LIF). A throughput of 15samples/h, a migration time precision of 3.1% RSD for ROS and 4.9% RSD for GSH were obtained for 10 consecutively injected cells.

Microfluidic techniques for single-cell protein expression analysis.
Fitzpatrick E, McBride S, Yavelow J, Najmi S, Zanzucchi P, Wieder R.
Clin Chem. 2006 Jun;52(6):1080-8; Epub 2006 Mar 23.
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BACKGROUND: The analysis of single cells obtained from needle aspirates of tumors is constrained by the need for processing. To this end, we investigated two microfluidic approaches to measure the expression of surface proteins in single cancer cells or in small populations (<50 cells). METHODS: One approach involved indirect fluorescence labeling of cell-surface proteins and channeling of cells in a microfluidic device past a fluorescence detector for signal quantification and analysis. A second approach channeled cells in a microfluidic device over detection zones coated with ligands to surface proteins and measured rates of passage and of retardation based on transient interactions between surface proteins and ligands. RESULTS: The fluorescence device detected expression of integrin alpha5 induced by basic fibroblast growth factor (FGF-2) treatment in MCF-7 cells and that of Her-2/neu in SK-BR-3 cells compared with controls. Experiments measuring passage retardation showed significant differences in passage rates between FGF-2-treated and untreated MCF-7 cells over reaction regions coated with fibronectin and antibody to integrin alpha5beta1 compared with control regions. Blocking peptides reversed the retardation, demonstrating specificity. CONCLUSIONS: Immunofluorescence detection in a microfluidic channel demonstrates the potential for assaying surface protein expression in a few individual cells and will permit the development of future iterations not requiring cell handling. The flow retardation device represents the first application of this technology for assessing cell-surface protein expression in cancer cells and may provide a way for analyzing expression profiles of single cells without preanalytical manipulation.

Cell trapping in microfluidic chips.
Johann RM.
Anal Bioanal Chem. 2006 Jun;385(3):408-12.
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No abstract available

A parallel-gradient microfluidic chamber for quantitative analysis of breast cancer cell chemotaxis.
Saadi W, Wang SJ, Lin F, Jeon NL.
Biomed Microdevices. 2006 Jun;8(2):109-18.
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Growth factor-induced chemotaxis of cancer cells is believed to play a critical role in metastasis, directing the spread of cancer from the primary tumor to secondary sites in the body. Understanding the mechanistic and quantitative behavior of cancer cell migration in growth factor gradients would greatly help in future treatment of metastatic cancers. Using a novel microfluidic chemotaxis chamber capable of simultaneously generating multiple growth factor gradients, we examined the migration of the human metastatic breast cancer cell line MDA-MB-231 in various conditions. First, we quantified and compared the migration in two gradients of epidermal growth factor (EGF) spanning different concentrations: 0-50 ng/ml and 0.1-6 ng/ml. Cells showed a stronger response in the 0-50 ng/ml gradient. However, the fact that even a shallow gradient of EGF can induce chemotaxis, and that EGF can direct migration over a large dynamic range of gradients, confirms the potency of EGF as a chemoattractant. Second, we investigated the effect of antibody against the EGF receptor (EGFR) on MDA-MB-231 chemotaxis. Quantitative analysis indicated that anti-EGFR antibody impaired both motility and directional orientation (CI = 0.03, speed = 0.71 microm/min), indicating that cell motility was induced by the activation of EGFR. The ability to compare, in terms of quantitative parameters, the effects of different pharmaceutical inhibitors, as well as subtle differences in experimental conditions, will aid in our understanding of mechanisms that drive metastasis. The microfluidic chamber described in this work will provide a platform for cell-based assays that can be used to compare the effectiveness of different pharmaceutical compounds targeting cell migration and metastasis.

Effective transfection of cells with multi-shell calcium phosphate-DNA nanoparticles.
Sokolova VV, Radtke I, Heumann R, Epple M.
Biomaterials. 2006 Jun;27(16):3147-53.
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Coated calcium phosphate nanoparticles were prepared for cell transfection. A calcium phosphate nanoparticle served as core which was then coated with DNA for colloidal stabilisation. The efficiency of transfection could be considerably increased by adding another layer of calcium phosphate on the surface, thereby incorporating DNA into the particle and preventing its degradation within the cell by lysosomes. A subsequent outermost layer of DNA o