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.
[ expand abstract ]

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.
[ expand abstract ]

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.
[ expand abstract ]

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.
[ expand abstract ]

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.
[ expand abstract ]

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.
[ expand abstract ]

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.
[ expand abstract ]

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 on the calcium phosphate gave a colloidal stabilisation. The efficiency of such multi-shell particles was significantly higher than that of simple DNA-coated calcium phosphate nanoparticles. The transfection efficiency of EGFP-encoding DNA was tested with different cell lines (T-HUVEC, HeLa, and LTK). The dispersions were stable and could be used for transfection after 2 weeks of storage at 4 degrees C without loss of efficiency.

Nano-titanium dioxide composites for the enrichment of phosphopeptides.
Liang SS, Makamba H, Huang SY, Chen SH.
J Chromatogr A. 2006 May 26;1116(1-2):38-45; Epub 2006 Mar 30.
[ expand abstract ]

Protein phosphorylation is one of the most important known posttranslational modifications and the strategy to enrich phosphopeptides becomes a critical issue for mapping protein phosphorylation sites. In this study, nano-titanium dioxide (TiO2) composites were synthesized, characterized, and demonstrated to have high loading capacity and high capture efficiency for enriching phosphopeptides. TiO2 nanoparticles were first silanized with methacryloxypropyltrimethoxysilane (MPTMS) and then were photopolymerized in the presence of a diacrylate crosslinker. The chemical bonds formed by the reaction were confirmed by both FT-IR and X-ray photoelectron spectroscopy (XPS). Scanning electron microscopy (SEM) further reveals that agglomeration of the particles was created by the crosslinking, which allowed the nanocomposites to be well retained within the cartridge and used as the chromatographic packing material. Titration with phenyl phosphate indicated that the TiO2 nanocomposites have two times as much phosphate binding capacity compared with 5 microm TiO2 particles. Moreover, based on repetitive analyses of the tryptic digest deduced from pure proteins as well as from protein mixtures containing phospho and non-phospho proteins, the capture efficiency of TiO2 nanocomposites was determined to be two to five times larger compared with 5 microm TiO2 particles. The cost for preparing nanocomposite TiO2 is low and it holds great promises to be used as chromatographic materials for phosphopeptide enrichment.

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 C, Pyo N, Higashitani K.
Biophys J. 2006 May 26; [Epub ahead of print] .
[ 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. In order to model various nano-particle 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 in the adhesive force upon 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 to malignant melanoma cells, and that the use of hydrophobic materials unexpectedly did not improve their affinity.

Single quantum dot-micelles coated with silica shell as potentially non-cytotoxic fluorescent cell tracers.
Zhelev Z, Ohba H, Bakalova R.
J Am Chem Soc. 2006 May 17;128(19):6324-5.
[ expand abstract ]

The present study describes a stabilization of single quantum dot (QD) micelles by hydrophobic silica precursors and an extension of the silica layer to form a silica shell around the micelle. The obtained product consists of up to 92% of single nanocrystals (CdSe, CdSe/ZnS, or CdSe/ZnSe/ZnS quantum dots) in the silica micelles, coated with silica shell. The thickness of silica shell could vary, starting from 3 to 4 nm. Increasing the shell thickness increases the photoluminescent characteristics of QDs in aqueous solution. The silica-shelled single CdSe/ZnS QD micelles possess a high quantum yield in aqueous solution, a controlled small size, sharp photoluminescence spectra (fwhm approximately 30 nm), an absence of aggregation, and a high transparency. The presence of a hydrophobic layer between the QD and silica shell ensures an incorporation of other hydrophobic molecules (with interesting properties) in the close proximity of nanocrystal. Thus, it is possible to combine the characteristics of hybrid material with the priority of small size. The nanoparticles are amino functionalized and ready for conjugation. A comparatively good biocompatibility is demonstrated. The nanoparticles show ability for intracellular delivery and are noncytotoxic during long-term incubation with viable cells in the absence of light exposure, which makes them appropriate for cell tracing and drug delivery.

Optically transparent au{111} substrates: flat gold nanoparticle platforms for high-resolution scanning tunneling microscopy.
Dahanayaka DH, Wang JX, Hossain S, Bumm LA.
J Am Chem Soc. 2006 May 10;128(18):6052-3.
[ expand abstract ]

We demonstrate a new type of Au{111} substrate that is both atomically flat and optically transparent, which consists of solution-grown flat gold nanoparticles (FGNPs) deposited on indium tin oxide (ITO)-coated glass. We show that FGNPs are atomically flat single-crystal plates with large {111} faces that expose only 2-4 atomic layers. These FGNPs are excellent platforms for alkanethiol self-assembled monolayers (SAMs) and for high-resolution scanning tunneling microscopy (STM). Our supported FGNPs are also low-cost Au{111} substrates, employing only basic wet chemical techniques in preparation. This approach should be broadly applicable to other types of substrates for scanning probe microscopies.

Nanoliter scale microbioreactor array for quantitative cell biology.
Lee PJ, Hung PJ, Rao VM, Lee LP.
Biotechnol Bioeng. 2006 May 5;94(1):5-14.
[ expand abstract ]

A nanoliter scale microbioreactor array was designed for multiplexed quantitative cell biology. An addressable 8 x 8 array of three nanoliter chambers was demonstrated for observing the serum response of HeLa human cancer cells in 64 parallel cultures. The individual culture unit was designed with a "C" shaped ring that effectively decoupled the central cell growth regions from the outer fluid transport channels. The chamber layout mimics physiological tissue conditions by implementing an outer channel for convective "blood" flow that feeds cells through diffusion into the low shear "interstitial" space. The 2 microm opening at the base of the "C" ring established a differential fluidic resistance up to 3 orders of magnitude greater than the fluid transport channel within a single mold microfluidic device. Three-dimensional (3D) finite element simulation were used to predict fluid transport properties based on chamber dimensions and verified experimentally. The microbioreactor array provided a continuous flow culture environment with a Peclet number (0.02) and shear stress (0.01 Pa) that approximated in vivo tissue conditions without limiting mass transport (10 s nutrient turnover). This microfluidic design overcomes the major problems encountered in multiplexing nanoliter culture environments by enabling uniform cell loading, eliminating shear, and pressure stresses on cultured cells, providing stable control of fluidic addressing, and permitting continuous on-chip optical monitoring. (c) 2005 Wiley Periodicals, Inc.

Microfluidic Single-Cell mRNA Isolation and Analysis.
Marcus JS, Anderson WF, Quake SR.
Anal Chem. 2006 May 1;78(9):3084-3089.
[ expand abstract ]

Single-cell gene expression analysis holds great promise for studying diverse biological systems, but methodology to process these precious samples in a reproducible, quantitative, and parallel fashion remains challenging. Here, we utilize microfluidics to isolate picogram and subpicogram mRNA templates, as well as to synthesize cDNA from these templates. We demonstrate single-cell mRNA isolation and cDNA synthesis, provide quantitative calibrations for each step in the process, and measure gene expression in individual cells. The techniques presented here form the foundation for highly parallel single-cell gene expression studies.

Designing a nano-interface in a microfluidic chip to probe living cells: Challenges and perspectives.
Helmke BP, Minerick AR.
Proc Natl Acad Sci U S A. 2006 Apr 25;103(17):6419-24; Epub 2006 Apr 17.
[ expand abstract ]

Nanotechnology-based materials are beginning to emerge as promising platforms for biomedical analysis, but measurement and control at the cell-chip interface remain challenging. This idea served as the basis for discussion in a focus group at the recent National Academies Keck Futures Initiative. In this Perspective, we first outline recent advances and limitations in measuring nanoscale mechanical, biochemical, and electrical interactions at the interface between biomaterials and living cells. Second, we present emerging experimental and conceptual platforms for probing living cells with nanotechnology-based tools in a microfluidic chip. Finally, we explore future directions and critical needs for engineering the cell-chip interface to create an integrated system capable of high-resolution analysis and control of cellular physiology.

Dendrimer FISH detection of single-copy intervals in acute promyelocytic leukemia.
Mora JR, Knoll JH, Rogan PK, Getts RC, Wilson GS.
Mol Cell Probes. 2006 Apr;20(2):114-20; [Epub 2006 Feb 7].
[ expand abstract ]

Acute promyelocytic leukemia (AML-M3) is characterized by a translocation between chromosomes 15 and 17 [t(15;17)]. The detection of t(15;17) at the single cell level, is commonly done by fluorescence in situ hybridization (FISH) using recombinant locus specific genomic probes greater than 14 kilobases kb in length. To allow a more thorough study of t(15;17), we designed small (0.9-3.6 kb), target-specific, single-copy probes from the human genome sequence. A novel detection approach was evaluated using moieties possessing more fluorophores, DNA dendrimers (up to 375 fluorophores per dendrimer). Two detection approaches were evaluated using the dendrimers: (1) dendrimers modified with anti-biotin antibodies for detection of biotinylated bound probes, and (2) dendrimers modified with 45-base long oligonucleotides designed from the single-copy probes, for direct detection of the target region. The selectivity of the probes was confirmed via indirect labeling with biotin/digoxigenin by nick translation, with detection efficiencies between 50 and 90%. Furthermore, the scFISH probes were successfully detected on metaphase cells with anti-biotin dendrimer conjugates and on interphase cells with 45-base modified dendrimers. Our results bring up the possibility to detect target regions of less than 1 kb, which will be a great contribution to high-resolution analysis of genomic sequences.

Microfluidic Techniques for Single-Cell Protein Expression Analysis.
Fitzpatrick E, McBride S, Yavelow J, Najmi S, Zanzucchi P, Wieder R.
Clin Chem. 2006 Mar 23; [Epub ahead of print].
[ expand abstract ]

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 integrin alpha5 expression induced by fibroblast growth factor-2 (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, indicating specificity. CONCLUSIONS: Immunofluorescence detection in a microfluidic channel has the potential for assaying surface protein expression in a few individual cells and could 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.

DNA-gold nanorod conjugates for remote control of localized gene expression by near infrared irradiation.
Chen CC, Lin YP, Wang CW, Tzeng HC, Wu CH, Chen YC, Chen CP, Chen LC, Wu YC.
J Am Chem Soc. 2006 Mar 22;128(11):3709-15.
[ expand abstract ]

Gold nanorods were attached to the gene of enhanced green fluorescence protein (EGFP) for the remote control of gene expression in living cells. The UV-vis spectroscopy, electrophoresis, and transmission electron microscopy (TEM) were used to study the optical and structural properties of the EGFP DNA and gold nanorod (EGFP-GNR) conjugates before and after femto-second near-infrared (NIR) laser irradiation. Upon NIR irradiation, the gold nanorods of EGFP-GNR conjugates underwent shape transformation that resulted in the release of EGFP DNA. When EGFP-GNR conjugates were delivered to cultured HeLa cells, induced GFP expression was specifically observed in cells that were locally exposed to NIR irradiation. Our results demonstrate the feasibility of using gold nanorods and NIR irradiation as means of remote control of gene expression in specific cells. This approach has potential applications in biological and medical studies.

Behavioral profiling of human transitional cell carcinoma ex vivo.
Estrada CR, Salanga M, Bielenberg DR, Harrell WB, Zurakowski D, Zhu X, Palmer MR, Freeman MR, Adam RM.
Cancer Res. 2006 Mar 15;66(6):3078-86.
[ expand abstract ]

Outcome studies of many types of cancer have revealed that tumors of indistinguishable histologic appearance may differ significantly in aggressiveness and in their response to therapy. A strategy that would enable early identification of patients at high risk for disease progression and allow screening of multiple therapeutic agents simultaneously for efficacy would improve clinical management. We have developed an orthotopic organ culture model of bladder cancer in which quantum dot-based fluorescent imaging approaches are used to obtain quantitative measurements of tumor cell behavior. Human transitional cell carcinoma (TCC) cells are labeled with quantum dot nanoparticles, and the cells instilled into the rat bladder in vivo, after which the bladder is excised and cultured ex vivo. Cell implantation, proliferation, and invasion into the organ wall are monitored using epifluorescence imaging and two-photon laser scanning confocal microscopy. Using this approach, we were able to assign distinct phenotypes to two metastatic bladder cancer cell lines based on different patterns of invasiveness into the bladder wall. We also showed that established tumor cell masses regressed following intravesical administration of the chemotherapeutic drug thiotepa. Collectively, these findings suggest that this assay system, which we have named EViTAS (for ex vivo tumor assay system), can recapitulate salient aspects of tumor growth in the host and is amenable to behavioral profiling of human cancer.

Nanotechnology for Cell-Substrate Interactions.
Sniadecki NJ, Desai RA, Ruiz SA, Chen CS.
Ann Biomed Eng. 2006 Mar 9; [Epub ahead of print].
[ expand abstract ]

In the pursuit to understand the interaction between cells and their underlying substrates, the life sciences are beginning to incorporate micro- and nanotechnology-based tools to probe and measure cells. The development of these tools portends endless possibilities for new insights into the fundamental relationships between cells and their surrounding microenvironment that underlie the physiology of human tissue. Here, we review techniques and tools that have been used to study how a cell responds to the physical factors in its environment. We also discuss unanswered questions that could be addressed by these approaches to better elucidate the molecular processes and mechanical forces that dominate the interactions between cells and their physical scaffolds.

Albumin-derived nanocarriers: Substrates for enhanced cell adhesive ligand display and cell motility.
Sharma RI, Pereira M, Schwarzbauer JE, Moghe PV.
Biomaterials. 2006 Mar 7; [Epub ahead of print].
[ expand abstract ]

Cell-adhesive ligands organized on nanoscale synthetic biomaterials can potentially recapitulate the nanoscale organization of extracellular matrix and the consequent effects of cell dynamics. In this study, 100nm albumin nanocarriers (ANC) were fabricated to serve as nanoscale organizational units for a well-defined ligand, the recombinant fragment from fibronectin comprised of the RGD-containing module 10 and the synergy-region-containing module 9. Conventional protein conjugation chemistry was employed to fabricate nanocarriers with increasing levels of displayed ligand. Presentation of ligand-functionalized ANCs adsorbed onto substrates was found to enhance keratinocyte attachment when compared to equivalent levels of adsorbed ligands, supported by ELISA data that the display of ligand on ANCs essentially increased the accessibility of the cell-binding domain and AFM data that the ligand was likely exposed due to ligand-ANC repulsion. The ligand presentation from ANCs converted the cellular morphology from a stationary phenotype to a motile phenotype, with the expression of filopodia-like microextensions, and a decrease in focal adhesions, indicating decreased cell adhesion strength. Consequently, cell motility was found to be significantly elevated on ligand-ANC substrates relative to substrates with equivalent levels of ligand. Overall, the ligand-functionalized albumin nanocarriers offer a unique model platform with two distinct properties: enhanced ligand exposure for enhancement of cell attachment to ligands at low concentrations; and enhanced cell detachment, motile phenotype, and migration kinetics.

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Colorimetric Screening of DNA-Binding Molecules with Gold Nanoparticle Probes.
Han MS, Lytton-Jean AK, Oh BK, Heo J, Mirkin CA.
Angew Chem Int Ed Engl. 2006 Mar 6;45(11):1807-1810.
[ expand abstract ]

No abstract available

Detection of MicroRNAs Using Electrocatalytic Nanoparticle Tags.
Gao Z, Yang Z.
Anal Chem. 2006 Mar 1;78(5):1470-7.
[ expand abstract ]

An ultrasensitive microRNA (miRNA) assay employing electrocatalytic nanoparticle tags to meet the need of miRNA expression analysis is described in this report. The assay utilizes an indium tin oxide electrode on which oligonucleotide capture probes are immobilized. After hybridization with periodate-treated miRNA, the nanoparticle tags, isoniazid-capped OsO(2) nanoparticles, are brought to the electrode through a condensation reaction to chemically amplify the signal. The resulting electrode exhibits electrocatalytic activity toward the oxidation of hydrazine at -0.10 V, reducing the oxidation overpotential by as much as 900 mV. The effect of experimental variables on the amperometric response is investigated and optimized. A detection limit of 80 fmol/L in 2.5-muL droplets and a linear current-concentration relationship up to 200 pmol/L are obtained following a 60-min hybridization. Successful attempts are made in miRNA expression analysis of HeLa cells.

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Qdot nanocrystal conjugates conjugated to bombesin or ANG II label the cognate G protein-coupled receptor in living cells.
Young SH, Rozengurt E.
Am J Physiol Cell Physiol. 2006 Mar;290(3):C728-32.
[ expand abstract ]

Quantum dots (Qdot Nanocrystal Conjugates; Quantum Dot, Hayward, CA) exhibit high fluorescence and low photobleaching compared with organic dyes, properties that should enhance their detection at low densities. In view of the properties of Qdots and the biological and pharmaceutical importance of G protein-coupled receptors (GPCRs), we attempted to use Qdots to label GPCRs in a variety of live cell types. An agonist consisting of biotinylated bombesin or ANG II was conjugated to Qdot Nanocrystal Conjugates coated with streptavidin through a biotin-streptavidin linkage (Qdot agonist). Herein we demonstrate that Qdot-bombesin conjugate can label the bombesin-preferring GPCR in living mouse Swiss 3T3 cells and in Rat-1 cells. Similarly, we used the Qdot-ANG II conjugate to label GPCR in intact rat intestinal epithelial cells (IEC)-18 and in a human pancreatic adenocarcinoma cell line of ductal origin, HPAF-II cells. We demonstrate that Qdot-ANG II is brighter and more photostable than agonist labeled with the organic dye Cy3. Our results demonstrate that Qdot technology can be adapted to monitor ligand binding to GPCRs. Combined with the narrow and symmetric emission profile of Qdot Nanocrystal Conjugates, this information suggests the potential for a new multiplex strategy to determine the effect of agonists and/or antagonists on agonist binding to several GPCRs simultaneously in living cells.

A two-photon excitation fluorescence cross-correlation assay for a model ligand-receptor binding system using quantum dots.
Swift JL, Heuff R, Cramb DT.
Biophys J. 2006 Feb 15;90(4):1396-410.
[ expand abstract ]

Two-photon excitation fluorescence cross-correlation spectroscopy (TPE-XCS) is a very suitable method for studying interactions of two distinctly labeled fluorescent molecules. As such, it lends itself nicely to the study of ligand-receptor interactions. By labeling the ligand with one color of fluorescent dye and the receptor with another, it is possible to directly monitor ligand binding rather than inferring binding by monitoring downstream effects. One challenge of the TPE-XCS approach is that of separating the signal due to the receptor from that of the ligand. Using standard organic fluorescent labels there is almost inevitably spectral cross talk between the detection channels, which must be accounted for in TPE-XCS data analysis. However, using quantum dots as labels for both ligand and receptor this limitation can be alleviated, because of the dot's narrower emission spectra. Using solely quantum dots as fluorescent labels is a novel approach to TPE-XCS, which may be generalizable to many pairs of interacting biomolecules after the proof of principle and the assessment of limitations presented here. Moreover, it is essential that relevant pharmacological parameters such as the equilibrium dissociation constant, K(d), can be easily extracted from the XCS data with minimal processing. Herein, we present a modified expression for fractional occupancy based on the auto- and cross-correlation decays obtained from a well-defined ligand-receptor system. Nanocrystalline semiconductor quantum dots functionalized with biotin (lambda(em) = 605 nm) and streptavidin (lambda(em) = 525 nm) were used for which an average K(d) value of 0.30 +/- 0.04 x 10(-9) M was obtained (cf. native system approximately 10(-15)). Additionally, the off-rate coefficient (k(off)) for dissociation of the two quantum dots was determined as 5 x 10(-5) s(-1). This off-rate is slightly larger than for native biotin-streptavidin (5 x 10(-6) s(-1)); the bulky nature of the quantum dots and restricted motion/orientation of functionalized dots in solution can account for differences in the streptavidin-biotin mediated dot-dot binding compared with those for native streptavidin-biotin

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Dendrimer FISH detection of single-copy intervals in acute promyelocytic leukemia.
Mora JR, Knoll JH, Rogan PK, Getts RC, Wilson GS.
Mol Cell Probes. 2006 Feb 3; [Epub ahead of print] .
[ expand abstract ]

Acute promyelocytic leukemia (AML-M3) is characterized by a translocation between chromosomes 15 and 17 [t(15;17)]. The detection of t(15;17) at the single cell level, is commonly done by fluorescence in situ hybridization (FISH) using recombinant locus specific genomic probes greater than 14kilobases kb in length. To allow a more thorough study of t(15;17), we designed small (0.9-3.6kb), target-specific, single-copy probes from the human genome sequence. A novel detection approach was evaluated using moieties possessing more fluorophores, DNA dendrimers (up to 375 fluorophores per dendrimer). Two detection approaches were evaluated using the dendrimers: (1) dendrimers modified with anti-biotin antibodies for detection of biotinylated bound probes, and (2) dendrimers modified with 45-base long oligonucleotides designed from the single-copy probes, for direct detection of the target region. The selectivity of the probes was confirmed via indirect labeling with biotin/digoxigenin by nick translation, with detection efficiencies between 50 and 90%. Furthermore, the scFISH probes were successfully detected on metaphase cells with anti-biotin dendrimer conjugates and on interphase cells with 45-base modified dendrimers. Our results bring up the possibility to detect target regions of less than 1kb, which will be a great contribution to high-resolution analysis of genomic sequences.

Microfluidic system for measuring neutrophil migratory responses to fast switches of chemical gradients.
Irimia D, Liu SY, Tharp WG, Samadani A, Toner M, Poznansky MC.
Lab Chip. 2006 Feb;6(2):191-8.
[ expand abstract ]

Experimental systems that provide temporal and spatial control of chemical gradients are required for probing into the complex mechanisms of eukaryotic cell chemotaxis. However, no current technique can simultaneously generate stable chemical gradients and allow fast gradient changes. We developed a microfluidic system with microstructured membranes for exposing neutrophils to fast and precise changes between stable, linear gradients of the known chemoattractant Interleukin-8 (IL-8). We observed that rapidly lowering the average concentration of IL-8 within a gradient, while preserving the direction of the gradient, resulted in temporary neutrophil depolarization. Fast reversal of the gradient direction while increasing or decreasing the average concentration also resulted in temporary depolarization. Neutrophils adapted and maintained their directional motility, only when the average gradient concentration was increased and the direction of the gradient preserved. Based on these observations we propose a two-component temporal sensing mechanism that uses variations of chemokine concentration averaged over the entire cell surface and localized at the leading edge, respectively, and directs neutrophil responses to changes in their chemical microenvironment.

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Characterization of a microfluidic dispensing system for localised stimulation of cellular networks.
Kraus T, Verpoorte E, Linder V, Franks W, Hierlemann A, Heer F, Hafizovic S, Fujii T, de Rooij NF, Koster S.
Lab Chip. 2006 Feb;6(2):218-29.
[ expand abstract ]

We present a 3-D microfluidic device designed for localized drug delivery to cellular networks. The device features a flow cell comprising a main channel for nutrient delivery as well as multiple channels for drug delivery. This device is one key component of a larger, fully integrated system now under development, based upon a microelectrode array (MEA) with on-chip CMOS circuitry for recording and stimulation of electrogenic cells (e.g. neurons, cardiomyocytes). As a critical system unit, the microfluidics must be carefully designed and characterized to ensure that candidate drugs are delivered to specific regions of the culture at known concentrations. Furthermore, microfluidic design and functionality is dictated by the size, geometry, and material/electrical characteristics of the CMOS MEA. Therefore, this paper reports on the design considerations and fabrication of the flow cell, including theoretical and experimental analysis of the mass transfer properties of the nutrient and drug flows, which are in good agreement with one another. To demonstrate proof of concept, the flow cell was mounted on a dummy CMOS chip, which had been plated with HL-1 cardiomyocytes. A test chemical compound was delivered to the cell culture in a spatially resolved manner. Envisioned applications of this stand-alone system include simultaneous toxicological testing of multiple compounds and chemical stimulation of natural neural networks for neuroscience investigations.

Study of osteoblastic cells in a microfluidic environment.
Leclerc E, David B, Griscom L, Lepioufle B, Fujii T, Layrolle P, Legallaisa C.
Biomaterials. 2006 Feb;27(4):586-95.
[ expand abstract ]

Bone tissue engineering consists of culturing osteoblastic cells onto synthetic three-dimensional (3D) porous scaffolds. The organization of bone cells into 3D scaffolds is crucial for ex vivo tissue formation. Diffusional rates of nutrients could be greatly improved by perfusing media through the 3D microporous scaffolds. However, bone cells cultured in vitro are responsive to a variety of different mechanical signals including fluid flow and shear stresses. In this work, we attempt to study osteoblastic cells behaviour cultured within microdevices allowing continuous and homogenous feeding of cells. We have fabricated polydimethylsiloxane PDMS microdevices with a 3D microstructured channel network. Mouse calvarial osteoblastic cells MC3T3-E1 were seeded at 2x10(6)cells/ml and cultured into the microdevices under flow rates of 0, 5, 35 microl/min. Cells attached and proliferated well in the designed microdevices. Cell viability was found around 85% up to 1 to 2 weeks for shear stress value under 5 mPa. The alkaline phosphatase (ALP) activity was enhanced 3- and 7.5-fold inside the microdevices under static and dynamic flow of 5 microl/min as compared to flat static cultures in PDMS coated Petri dishes. Therefore, osteoblastic cells could be successfully cultured inside the microdevices under dynamic conditions and their ALP activity was enhanced. These results are promising for bone cell growth and differentiation as well as future tissue regeneration using larger 3D microfluidic microdevices.

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A two-photon excitation fluorescence cross-correlation assay for a model ligand-receptor binding system using quantum dots.
Swift JL, Heuff R, Cramb DT.
Biophys J. 2006 Feb;90(4):1396-410.
[ expand abstract ]

Two-photon excitation fluorescence cross-correlation spectroscopy (TPE-XCS) is a very suitable method for studying interactions of two distinctly labeled fluorescent molecules. As such, it lends itself nicely to the study of ligand-receptor interactions. By labeling the ligand with one color of fluorescent dye and the receptor with another, it is possible to directly monitor ligand binding rather than inferring binding by monitoring downstream effects. One challenge of the TPE-XCS approach is that of separating the signal due to the receptor from that of the ligand. Using standard organic fluorescent labels there is almost inevitably spectral cross talk between the detection channels, which must be accounted for in TPE-XCS data analysis. However, using quantum dots as labels for both ligand and receptor this limitation can be alleviated, because of the dot's narrower emission spectra. Using solely quantum dots as fluorescent labels is a novel approach to TPE-XCS, which may be generalizable to many pairs of interacting biomolecules after the proof of principle and the assessment of limitations presented here. Moreover, it is essential that relevant pharmacological parameters such as the equilibrium dissociation constant, K(d), can be easily extracted from the XCS data with minimal processing. Herein, we present a modified expression for fractional occupancy based on the auto- and cross-correlation decays obtained from a well-defined ligand-receptor system. Nanocrystalline semiconductor quantum dots functionalized with biotin (lambda(em) = 605 nm) and streptavidin (lambda(em) = 525 nm) were used for which an average K(d) value of 0.30 +/- 0.04 x 10(-9) M was obtained (cf. native system approximately 10(-15)). Additionally, the off-rate coefficient (k(off)) for dissociation of the two quantum dots was determined as 5 x 10(-5) s(-1). This off-rate is slightly larger than for native biotin-streptavidin (5 x 10(-6) s(-1)); the bulky nature of the quantum dots and restricted motion/orientation of functionalized dots in solution can account for differences in the streptavidin-biotin mediated dot-dot binding compared with those for native streptavidin-biotin.

Gold glyconanoparticles for mimics and measurement of metal ion-mediated carbohydrate-carbohydrate interactions.
Reynolds AJ, Haines AH, Russell DA.
Langmuir. 2006 Jan 31;22(3):1156-63.
[ expand abstract ]

To mimic and measure calcium ion-mediated carbohydrate-carbohydrate interactions, four lactose derivatives have been synthesized for assembly on gold nanoparticles. The series of lactose derivatives varied by the length of the thiolated ethylene glycol anchor chain [O(CH2CH2O)(m)CH2CH2SH; where m = 0, 1, 2, and 3] used to self-assemble the carbohydrates to the preformed gold nanoparticles of ca. 16 nm diameter. Upon addition of calcium ions to the lactose-stabilized nanoparticles, rapid carbohydrate-carbohydrate interactions were visualized and subsequently measured using UV-visible spectrometry and transmission electron microscopy (TEM). The nanoparticle aggregates formed via metal-mediated carbohydrate-carbohydrate interactions could be readily redispersed through the addition of EDTA. Multiple reaggregation and redispersion cycles were achieved, confirming that the aggregation process was due to metal ion-mediated carbohydrate interactions rather than calcium chelation by residual citrate ions on the particle surface. The essential involvement of the lactose moiety in Ca2+ complexation was shown by control measurements on related D-glucose-derivatized nanoparticles, where a significantly reduced aggregation response was obtained only at high ion concentrations. Other group 2 metal ions with radii larger than that of calcium, viz., barium and strontium, were also shown to mediate the aggregation of the lactose-stabilized nanoparticles. The induced aggregation of the lactose nanoparticles was determined to be quantitatively dependent upon the calcium ion concentration. Furthermore, the analytical sensitivity of the calcium-induced aggregation and the linear dynamic range were dependent on the length of the ethylene glycol anchor chain. The shortest ethylene glycol chain (m = 0) gave the most sensitive response with the optimum limit of detection (0.8 mM Ca2+), whereas the longest ethylene glycol chain (m = 3) provides a measurement of calcium ion concentration over the largest linear dynamic range (10-35 mM Ca2+). This work has shown that the self-assembled deposition of lactose derivatives on gold nanoparticles provides multivalent carbohydrate surfaces that can be used as mimics for the measurement of biologically relevant carbohydrate-carbohydrate interactions. Additionally, this study has highlighted the importance of the structure and length of the ligand that anchors the carbohydrate sugar to the gold particle surface to facilitate such carbohydrate interactions and for "tuning" the analytical characteristics of bioassays developed using metal nanoparticle technology.

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Optical detection of DNA conformational polymorphism on single-walled carbon nanotubes.
Heller DA, Jeng ES, Yeung TK, Martinez BM, Moll AE, Gastala JB, Strano MS.
Science. 2006 Jan 27;311(5760):508-11.
[ expand abstract ]

The transition of DNA secondary structure from an analogous B to Z conformation modulates the dielectric environment of the single-walled carbon nanotube (SWNT) around which it is adsorbed. The SWNT band-gap fluorescence undergoes a red shift when an encapsulating 30-nucleotide oligomer is exposed to counter ions that screen the charged backbone. The transition is thermodynamically identical for DNA on and off the nanotube, except that the propagation length of the former is shorter by five-sixths. The magnitude of the energy shift is described by using an effective medium model and the DNA geometry on the nanotube sidewall. We demonstrate the detection of the B-Z change in whole blood, tissue, and from within living mammalian cells.

Processive movement of single kinesins on crowded microtubules visualized using quantum dots.
Seitz A, Surrey T.
EMBO J. 2006 Jan 25;25(2):267-77.
[ expand abstract ]

Kinesin-1 is a processive molecular motor transporting cargo along microtubules. Inside cells, several motors and microtubule-associated proteins compete for binding to microtubules. Therefore, the question arises how processive movement of kinesin-1 is affected by crowding on the microtubule. Here we use total internal reflection fluorescence microscopy to image in vitro the runs of single quantum dot-labelled kinesins on crowded microtubules under steady-state conditions and to measure the degree of crowding on a microtubule at steady-state. We find that the runs of kinesins are little affected by high kinesin densities on a microtubule. However, the presence of high densities of a mutant kinesin that is not able to step efficiently reduces the average speed of wild-type kinesin, while hardly changing its processivity. This indicates that kinesin waits in a strongly bound state on the microtubule when encountering an obstacle until the obstacle unbinds and frees the binding site for kinesin's next step. A simple kinetic model can explain quantitatively the behaviour of kinesin under both crowding conditions.

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Cellular transfer and AFM imaging of cancer cells using Bioimprint.
Muys JJ, Alkaisi MM, Evans JJ, Melville DO, Nagase J, Parguez GM, Sykes P.
J Nanobiotechnology. 2006 Jan 22;4(1):1 .
[ expand abstract ]

A technique for permanently capturing a replica impression of biological cells has been developed to facilitate analysis using nanometer resolution imaging tools, namely the atomic force microscope (AFM). The method, termed Bioimprint, creates a permanent cell ;footprint' in a non-biohazardous Poly(dimethylsiloxane) (PDMS) polymer composite. The transfer of nanometer scale biological information is offered as an alternative imaging technique at a resolution beyond those available by current optical based techniques. By transferring cell topology into a rigid medium more suited for AFM imaging many of the limitations associated with scanning of biological specimens can be overcome. Potential for this technique is demonstrated by analyzing Bioimprint replicas created from human endometrial cancer cells. The high resolution transfer of this process is further detailed by imaging membrane morphological structures consistent with exocytosis. The integration of soft lithography to replicate biological materials presents an enhanced method for the study of biological systems at the nanoscale.

Processive movement of single kinesins on crowded microtubules visualized using quantum dots.
Seitz A, Surrey T.
EMBO J. 2006 Jan 12; [Epub ahead of print].
[ expand abstract ]

Kinesin-1 is a processive molecular motor transporting cargo along microtubules. Inside cells, several motors and microtubule-associated proteins compete for binding to microtubules. Therefore, the question arises how processive movement of kinesin-1 is affected by crowding on the microtubule. Here we use total internal reflection fluorescence microscopy to image in vitro the runs of single quantum dot-labelled kinesins on crowded microtubules under steady-state conditions and to measure the degree of crowding on a microtubule at steady-state. We find that the runs of kinesins are little affected by high kinesin densities on a microtubule. However, the presence of high densities of a mutant kinesin that is not able to step efficiently reduces the average speed of wild-type kinesin, while hardly changing its processivity. This indicates that kinesin waits in a strongly bound state on the microtubule when encountering an obstacle until the obstacle unbinds and frees the binding site for kinesin's next step. A simple kinetic model can explain quantitatively the behaviour of kinesin under both crowding conditions.

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Multicolor FRET Silica Nanoparticles by Single Wavelength Excitation.
Wang L, Tan W.
Nano Lett. 2006 Jan;6(1):84-8.
[ expand abstract ]

Fluorescent nanoparticles with multiple emission signatures by a single wavelength excitation are needed in multiplex bioanalysis and molecular imaging. We have prepared silica nanoparticles encapsulated with three organic dyes using a modified Stober synthesis method. By varying the doping ratio of the three tandem dyes, fluorescence resonance energy transfer (FRET)-mediated emission signatures can be tuned to have the nanoparticles exhibit multiple colors under one single wavelength excitation. These nanoparticles are intensely fluorescent, highly photostable, uniform in size, and biocompatible. The acceptor emission of the FRET nanoparticles has generated a large Stokes shift, which implicates broad applications in biological labeling and imaging. Molecular recognition moieties, such as biotin, can be covalently attached to the nanoparticle surface to allow for specific binding to target molecules. These multicolor FRET silica nanoparticles can be used as barcoding tags for multiplexed signaling. By using these NPs, one can envision a dynamic, multicolor, colocalization methodology to follow proteins, nucleic acids, molecular machines, and assemblies within living systems.

Intracellular uptake of CdSe-ZnS/polystyrene nanobeads.
Zhang Y, Huang N.
J Biomed Mater Res B Appl Biomater. 2006 Jan;76(1):161-8.
[ expand abstract ]

The ideal optical properties of quantum dots (QDs) offer the possibility of using them as fluorescent probes in biological staining and diagnostics. Some techniques have been developed to incorporate QDs into polymer beads, to solve the problems relating to QDs' surface chemistry, such as water solubility, biocompatibility, chemical stability in physiological media, and so on, or to pack different combinations of QDs and produce QD encoded polymer beads. However, the QD encoded polymer beads that have been reported so far are above 100 nm; therefore they are very useful for multiplexed bioassays, but not suitable for staining or labeling of subcellular components or intracellular measurements because of the relatively big size of the beads. There is great need for QD encoded polymer beads smaller than 100 nm. In this work, luminescent CdSe-ZnS QDs were incorporated into polystyrene (PS) beads grafted with carboxyl groups with the use of an emulsion polymerization method, and separation of nanoscale QD encoded PS beads (30 nm) was performed through centrifugation at high speed in viscous solution. The nanobeads were further surface modified with folic acid and their intracellular delivery into NIH-3T3 and HT-29 cell lines was investigated with the use of confocal microscope. The longevity of QDs allows the intracellular delivery of the nanobeads to be tracked over a certain time period.

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2005

Carbon Nanotubes as Intracellular Transporters for Proteins and DNA: An Investigation of the Uptake Mechanism and Pathway.
Kam NW, Liu Z, Dai H.
Angew Chem Int Ed Engl. 2005 Dec 13; [Epub ahead of print].
[ expand abstract ]

No abstract available

Nanoparticle decorated surfaces with potential use in glycosylation analysis.
Fromell K, Andersson M, Elihn K, Caldwell KD.
Colloids Surf B Biointerfaces. 2005 Dec 10;46(2):84-91.
[ expand abstract ]

A majority of all biologically active proteins are glycosylated and various diseases have proven to correlate with alterations in protein glycosylation. Sensitive identification of different glycoprotein glycoforms is therefore of great diagnostic value. Here we describe a method with potential for glycoprotein profiling, based on lectins as capture probes immobilized on particulate substrates in the nm-range. The nanoparticles present high concentrations of attachment sites for specific ligands and cause minimal steric hindrance to binding. In the present model study the mannose-binding lectin ConA has been coupled to polystyrene nanoparticles via a poly(ethyleneoxide) linker which protects the protein conformation and activity and prevents unspecific protein adsorption. The ConA-coated particles are accommodated at different spots on the analytical surface via oligonucleotide linkage. This attachment, which relies on the hybridization of complementary oligonucleotides, allows firm fixation of the particles at specific positions. The ConA attached to the particles has retained conformation and activity and binds selectively to a series of different glycoproteins. The results indicate the potential for using a multi-lectin nanoparticle array in glycoprotein mapping.

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Growth of gold nanoparticles in human cells.
Anshup A, Venkataraman JS, Subramaniam C, Kumar RR, Priya S, Kumar TR, Omkumar RV, John A, Pradeep T.
Langmuir. 2005 Dec 6;21(25):11562-7.
[ expand abstract ]

Gold nanoparticles of 20-100 nm diameter were synthesized within HEK-293 (human embryonic kidney), HeLa (human cervical cancer), SiHa (human cervical cancer), and SKNSH (human neuroblastoma) cells. Incubation of 1 mM tetrachloroaurate solution, prepared in phosphate buffered saline (PBS), pH 7.4, with human cells grown to approximately 80% confluency yielded systematic growth of nanoparticles over a period of 96 h. The cells, stained due to nanoparticle growth, were adherent to the bottom of the wells of the tissue culture plates, with their morphology preserved, indicating that the cell membrane was intact. Transmission electron microscopy of ultrathin sections showed the presence of nanoparticles within the cytoplasm and in the nucleus, the latter being much smaller in dimension. Scanning near field microscopic images confirmed the growth of large particles within the cytoplasm. Normal cells gave UV-visible signatures of higher intensity than the cancer cells. Differences in the cellular metabolism of cancer and noncancer cells were manifested, presumably in their ability to carry out the reduction process.

Proteolytic cleavage reveals interaction patterns between silica nanoparticles and two variants of human carbonic anhydrase.
Lundqvist M, Andresen C, Christensson S, Johansson S, Karlsson M, Broo K, Jonsson BH.
Langmuir. 2005 Dec 6;21(25):11903-6.
[ expand abstract ]

To characterize the sites on the protein surface that are involved in the adsorption to silica nanoparticles and the subsequent rearrangements of the protein/nanoparticle interaction, a novel approach has been used. After incubation of protein with silica nanoparticles for 2 or 16 h, the protein was cleaved with trypsin and the peptide fragments were analyzed with mass spectrometry. The nanoparticle surface area was in 16-fold excess over available protein surface to minimize the probability that the initial binding would be affected by other protein molecules. When the fragment patterns obtained in the presence and absence of silica nanoparticles were compared, we were able to characterize the protein fragments that interact with the surface. This approach has allowed us to identify the initial binding sites on the protein structure and the rearrangement of the binding sites that occur upon prolonged incubation with the surface.

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Cantilever sensor for nanomechanical detection of specific protein conformations.
Mukhopadhyay R, Sumbayev VV, Lorentzen M, Kjems J, Andreasen PA, Besenbacher F.
Nano Lett. 2005 Dec;5(12):2385-8.
[ expand abstract ]

A fast, label-free, and multiplexed method based on piezoresistive cantilevers is reported for the detection of specific protein conformations at the nanoscale level. The ligand-binding domain of the human oestrogen receptor (ERalpha-LBD) is used as the experimental model system, and ERalpha-LBD with or without oestradiol (E2) is detected using the conformation-specific peptides alpha/betaI (Ser-Ser-Asn-His-Gln-Ser-Ser-Arg-Leu-Ile-Glu-Leu-Leu-Ser-Arg, which recognizes E2-bound ER) and alpha/betaII (Ser-Ala-Pro-Arg-Ala-Thr-Ile-Ser-His-Tyr-Leu-Met-Gly-Gly, which recognizes E2-free ER). Target-specific signals are obtained in situ at protein concentrations of 2.5-20 nM. The in-build electrical readout of the piezoresistive cantilevers provides a convenient alternative to the conventional optical detection, and the presented method offers the possibility of detecting protein conformational changes using miniaturized microarrays.

Tuning compliance of nanoscale polyelectrolyte multilayers to modulate cell adhesion.
Thompson MT, Berg MC, Tobias IS, Rubner MF, Van Vliet KJ.
Biomaterials. 2005 Dec;26(34):6836-45.
[ expand abstract ]

It is well known that mechanical stimuli induce cellular responses ranging from morphological reorganization to mineral secretion, and that mechanical stimulation through modulation of the mechanical properties of cell substrata affects cell function in vitro and in vivo. However, there are few approaches by which the mechanical compliance of the substrata to which cells adhere and grow can be determined quantitatively and varied independent of substrata chemical composition. General methods by which mechanical state can be quantified and modulated at the cell population level are critical to understanding and engineering materials that promote and maintain cell phenotype for applications such as vascular tissue constructs. Here, we apply contact mechanics of nanoindentation to measure the mechanical compliance of weak polyelectrolyte multilayers (PEMs) of nanoscale thickness, and explore the effects of this tunable compliance for cell substrata applications. We show that the nominal elastic moduli E(s) of these substrata depend directly on the pH at which the PEMs are assembled, and can be varied over several orders of magnitude for given polycation/polyanion pairs. Further, we demonstrate that the attachment and proliferation of human microvascular endothelial cells (MVECs) can be regulated through independent changes in the compliance and terminal polyion layer of these PEM substrata. These data indicate that substrate mechanical compliance is a strong determinant of cell fate, and that PEMs of nanoscale thickness provide a valuable tool to vary the external mechanical environment of cells independently of chemical stimuli

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Simultaneous determination of glutathione and reactive oxygen species in individual cells by microchip electrophoresis.
Ling YY, Yin XF, Fang ZL.
Electrophoresis. 2005 Nov 9; [Epub ahead of print].
[ expand abstract ]

A microchip electrophoresis method was developed for simultaneous determination of reactive oxygen species (ROS) and reduced glutathione (GSH) in the individual erythrocyte cell. In this method, cell sampling, single-cell loading, docking, lysing, and capillary electrophoretic separation with LIF detection were integrated on a microfluidic chip with crossed channels. ROS was labeled with dihydrorhodamine 123 in the intact cell, while GSH was on-chip labeled with 2,3-naphthalene-dicarboxaldehyde, which was included in the separation medium. On-chip electrical lysis, characterized by extremely fast disruption of the cellular membrane (<40 ms), was exploited to minimize enzymatic effects on analyte concentrations during the determination. The microfluidic network was optimized to prevent cell leaking from the sample reservoir (S) into separation during the separation phase. The structure of the S was modified to avoid blockage of its outlet by deposited cells. Detection limits of 0.5 and 6.9 amol for ROS and GSH, respectively, were achieved. The average cell throughput was 25 cells/h. The effectiveness of the method was demonstrated in the simultaneous determination of GSH and ROS in individual cells and the variations of cellular GSH and ROS contents in response to external stimuli.

Fluorescent Detection of Apoptotic Cells by Using Zinc Coordination Complexes with a Selective Affinity for Membrane Surfaces Enriched with Phosphatidylserine.
Hanshaw RG, Lakshmi C, Lambert TN, Johnson JR, Smith BD.
Chembiochem. 2005 Nov 7; [Epub ahead of print].
[ expand abstract ]

The appearance of phosphatidylserine on the membrane surface of apoptotic cells (Jurkat, CHO, HeLa) is monitored by using a family of bis(Zn(2+)-2,2'-dipicolylamine) coordination compounds with appended fluorescein or biotin groups as reporter elements. The phosphatidylserine affinity group is also conjugated directly to a CdSe/CdS quantum dot to produce a probe suitable for prolonged observation without photobleaching. Apoptosis can be detected under a wide variety of conditions, including variations in temperature, incubation time, and binding media. Binding of each probe appears to be restricted to the cell membrane exterior, because no staining of organelles or internal membranes is observed.

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Microfluidic device for electric field-driven single-cell capture and activation.
Toriello NM, Douglas ES, Mathies RA.
Anal Chem. 2005 Nov 1;77(21):6935-41.
[ expand abstract ]

A microchip that performs directed capture and chemical activation of surface-modified single cells has been developed. The cell capture system is comprised of interdigitated gold electrodes microfabricated on a glass substrate within PDMS channels. The cell surface is labeled with thiol functional groups using endogenous RGD receptors, and adhesion to exposed gold pads on the electrodes is directed by applying a driving electric potential. Multiple cell types can thus be sequentially and selectively captured on desired electrodes. Single-cell capture efficiency is optimized by varying the duration of field application. Maximum single-cell capture is attained for the 10-min trial, with 63 +/- 9% (n = 30) of the electrode pad rows having a single cell. In activation studies, single M1WT3 CHO cells loaded with the calcium-sensitive dye fluo-4 AM were captured; exposure to the muscarinic agonist carbachol increased the fluorescence to 220 +/- 74% (n = 79) of the original intensity. These results demonstrate the ability to direct the adhesion of selected living single cells on electrodes in a microfluidic device and to analyze their response to chemical stimuli.

Colloidal gold nanoparticle modified carbon paste interface for studies of tumor cell adhesion and viability.
Du D, Liu S, Chen J, Ju H, Lian H, Li J.
Biomaterials. 2005 Nov;26(33):6487-95.
[ expand abstract ]

A non-toxic biomimetic interface for immobilization of living cells and electrochemical exogenous effect study of cell viability was constructed by mixing colloidal gold nanoparticles in carbon paste. A new approach to study the effects of anti-tumor drug and other exogenous factors on cell viability was proposed. The nanoparticles were efficient for preserving the activity of immobilized living cells and preventing their leakage from the electrode surface. The immobilized living AsPC-1 cells (pancreatic adenocarcinoma cells derived from ascites) exhibited an irreversible voltammetric response related to the oxidation of guanine. The presence of guanine was verified by liquid chromatography-mass spectrometry. The contents of guanine in cytoplasm of each AsPC-1 and normal pancreatic cell were detected to be 370 and 22amol, respectively. The cytotoxic effect of adriamycin resulted in a decrease in peak current of guanine. The optimal exogenous factors that affected cell viability, including pH, temperature and salt concentration of electrolyte, were just consistent with cell growth conditions in culture. This simple and rapid method could be applied for the electrochemical investigation of exogenous effect and characterization of the viability of living cells.

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Microarray gene expression analysis free of reverse transcription and dye labeling.
Sun Y, Fan WH, McCann MP, Golovlev V.
Anal Biochem. 2005 Oct 15;345(2):312-9.
[ expand abstract ]

A new microarray system has been developed for gene expression analysis using cationic gold nanoparticles with diameters of 250nm as a target detection reagent. The approach utilizes nonlabeled target molecules hybridizing with complementary probes on the array, followed by incubation in a colloidal gold solution. The hybridization signal results from the precipitation of nanogold particles on the hybridized spots due to the electrostatic attraction of the cationic gold particles and the anionic phosphate groups in the target DNA backbone. In contrast to conventional fluorescent detection, this nanoparticle-based detection system eliminates the target labeling procedure. The visualization of hybridization signals can be accomplished with a flatbed scanner instead of a confocal laser scanner, which greatly simplifies the process and reduces the cost. The sensitivity is estimated to be less than 2pg of DNA molecules captured on the array surface. The signal from hybridized spots quantitatively represents the amount of captured target DNA and therefore permits quantitative gene expression analysis. Cross-array reproducibility is adequate for detecting twofold or less signal changes across two microarray experiments.

Nanoscale Imaging of Buried Structures via Scanning Near-Field Ultrasound Holography.
Shekhawat GS, Dravid VP.
Science. 2005 Oct 7;310(5745):89-92.
[ expand abstract ]

A nondestructive imaging method, scanning near-field ultrasound holography (SNFUH), has been developed that provides depth information as well as spatial resolution at the 10- to 100-nanometer scale. In SNFUH, the phase and amplitude of the scattered specimen ultrasound wave, reflected in perturbation to the surface acoustic standing wave, are mapped with a scanning probe microscopy platform to provide nanoscale-resolution images of the internal substructure of diverse materials. We have used SNFUH to image buried nanostructures, to perform subsurface metrology in microelectronic structures, and to image malaria parasites in red blood cells.

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Intracellular uptake of CdSe-ZnS/polystyrene nanobeads.
Zhang Y, Huang N.
J Biomed Mater Res B Appl Biomater. 2005 Oct 5; [Epub ahead of print].
[ expand abstract ]

The ideal optical properties of quantum dots (QDs) offer the possibility of using them as fluorescent probes in biological staining and diagnostics. Some techniques have been developed to incorporate QDs into polymer beads, to solve the problems relating to QDs' surface chemistry, such as water solubility, biocompatibility, chemical stability in physiological media, and so on, or to pack different combinations of QDs and produce QD encoded polymer beads. However, the QD encoded polymer beads that have been reported so far are above 100 nm; therefore they are very useful for multiplexed bioassays, but not suitable for staining or labeling of subcellular components or intracellular measurements because of the relatively big size of the beads. There is great need for QD encoded polymer beads smaller than 100 nm. In this work, luminescent CdSe-ZnS QDs were incorporated into polystyrene (PS) beads grafted with carboxyl groups with the use of an emulsion polymerization method, and separation of nanoscale QD encoded PS beads (30 nm) was performed through centrifugation at high speed in viscous solution. The nanobeads were further surface modified with folic acid and their intracellular delivery into NIH-3T3 and HT-29 cell lines was investigated with the use of confocal microscope. The longevity of QDs allows the intracellular delivery of the nanobeads to be tracked over a certain time period.

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Microfluidic devices for the analysis of apoptosis.
Qin J, Ye N, Liu X, Lin B.
Electrophoresis. 2005 Oct;26(19):3780-8.
[ expand abstract ]

Apoptosis is the outcome of a metabolic cascade that results in cell death in a controlled manner. Due to its important role in maintaining balance in organisms, in mechanisms of diseases, and tissue homeostasis, apoptosis is of great interest in the emerging fields of systems biology. Research into cell death regulation and efforts to model apoptosis processes have become powerful drivers for new technologies to acquire ever more comprehensive information from cells and cell populations. The microfluidic technology promises to integrate and miniaturize many bioanalytical processes, which offers an alternative platform for the analysis of apoptosis. This review aims to highlight the recent developments of microfluidic devices in measuring the hallmarks as well as the dynamic process of cellular apoptosis. The potential capability and an outlook of microfluidic devices for the study of apoptosis are addressed.

Rare cancer cell analyzer for whole blood applications: Microcytometer cell counting and sorting subcircuits.
Lancaster C, Kokoris M, Nabavi M, Clemmens J, Maloney P, Capadanno J, Gerdes J, Battrell CF.
Methods. 2005 Sep 28; [Epub ahead of print] .
[ expand abstract ]

We demonstrate sorting of rare cancer cells from blood using a thin ribbon monolayer of cells within a credit-card sized, microfluidic laboratory-on-a-card ("lab card") structure. This enables higher cell throughput per minute thereby speeding up cell interrogation. In this approach, multiple cells are viewed and sorted, not individually, but as a whole cell row or section of the ribbon at a time. Gated selection of only the cell rows containing a tagged rare cell provides enrichment of the rare cell relative to background blood cells. We also designed the cell injector for laminar flow antibody labeling within 20s. The approach combines rapid laminar flow cell labeling with monolayer cell sorting thereby enabling rare cell target detection at sensitivity levels 1000 to 10,000 times that of existing flow cytometers. Using this method, total cell labeling and data acquisition time on card may be reduced to a few minutes compared to 30-60min for standard flow methods.

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Magnetic force probe for nanoscale biomolecules.
Koenig A, Hebraud P, Gosse C, Dreyfus R, Baudry J, Bertrand E, Bibette J.
Phys Rev Lett. 2005 Sep 16;95(12):128301.
[ expand abstract ]

We present a new technique to measure the mechanical properties of small biomolecules. This technique uses long range repulsive colloidal forces together with magnetic attraction as a force probing tool. The biomolecules are grafted between superparamagnetic particles, which are regularly spaced within long chains maintained by an external magnetic field. Varying the magnetic field results in compression or extension of the molecules between the particles. In order to demonstrate this technique we use, as a size controlled model molecule, a short double stranded DNA (151 base pairs) for which the force-extension law is determined and found in agreement with existing predictions

Single cell manipulation, analytics, and label-free protein detection in microfluidic devices for systems nanobiology.
Hellmich W, Pelargus C, Leffhalm K, Ros A, Anselmetti D.
Electrophoresis. 2005 Sep 9; [Epub ahead of print] .
[ expand abstract ]

Single cell analytics for proteomic analysis is considered a key method in the framework of systems nanobiology which allows a novel proteomics without being subjected to ensemble-averaging, cell-cycle, or cell-population effects. We are currently developing a single cell analytical method for protein fingerprinting combining a structured microfluidic device with latest optical laser technology for single cell manipulation (trapping and steering), free-solution electrophoretical protein separation, and (label-free) protein detection. In this paper we report on first results of this novel analytical device focusing on three main issues. First, single biological cells were trapped, injected, steered, and deposited by means of optical tweezers in a poly(dimethylsiloxane) microfluidic device and consecutively lysed with SDS at a predefined position. Second, separation and detection of fluorescent dyes, amino acids, and proteins were achieved with LIF detection in the visible (VIS) (488 nm) as well as in the deep UV (266 nm) spectral range for label-free, native protein detection. Minute concentrations of 100 fM injected fluorescein could be detected in the VIS and a first protein separation and label-free detection could be achieved in the UV spectral range. Third, first analytical experiments with single Sf9 insect cells (Spodoptera frugiperda) in a tailored microfluidic device exhibiting distinct electropherograms of a green fluorescent protein-construct proved the validity of the concept. Thus, the presented microfluidic concept allows novel and fascinating single cell experiments for systems nanobiology in the future.

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Microfluidic devices for the analysis of apoptosis.
Qin J, Ye N, Liu X, Lin B.
Electrophoresis. 2005 Sep 9; [Epub ahead of print] .
[ expand abstract ]

Apoptosis is the outcome of a metabolic cascade that results in cell death in a controlled manner. Due to its important role in maintaining balance in organisms, in mechanisms of diseases, and tissue homeostasis, apoptosis is of great interest in the emerging fields of systems biology. Research into cell death regulation and efforts to model apoptosis processes have become powerful drivers for new technologies to acquire ever more comprehensive information from cells and cell populations. The microfluidic technology promises to integrate and miniaturize many bioanalytical processes, which offers an alternative platform for the analysis of apoptosis. This review aims to highlight the recent developments of microfluidic devices in measuring the hallmarks as well as the dynamic process of cellular apoptosis. The potential capability and an outlook of microfluidic devices for the study of apoptosis are addressed.

New reagents for phosphatidylserine recognition and detection of apoptosis.
Hanshaw RG, Smith BD.
Bioorg Med Chem. 2005 Sep 1;13(17):5035-42.
[ expand abstract ]

The phospholipid bilayer surrounding animal cells is made up of four principle phospholipid components, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM). These four phospholipids are distributed between the two monolayers of the membrane in an asymmetrical fashion, with PC and SM largely populating the extracellular leaflet and PE and PS restricted primarily to the inner leaflet. Breakdown in this transmembrane phospholipid asymmetry is a hallmark of the early to middle stages of apoptosis. The consequent appearance of PS on the extracellular membrane leaflet is commonly monitored using dye-labeled Annexin V, a 36 kDa, Ca2+-dependent PS binding protein. Substitutes for Annexin V are described, including small molecules, nanoparticles, cationic liposomes, and other proteins that can recognize PS in a membrane surface. Particular attention is given to the use of these reagents for detecting apoptosis.

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A reagentless biosensor of nitric oxide based on direct electron transfer process of cytochrome c on multi-walled carbon nanotube.
Zhao GC, Yin ZZ, Wei XW.
Front Biosci. 2005 Sep 1;10:2005-10.
[ expand abstract ]

Direct electron transfer between Cytochrome c (Cyt.c) and electrode can be achieved through immobilizing Cyt.c on the surface of multi-walled carbon nanotubes (MWNTs). Under the condition of cyclic potential scans, Cyt.c can be adsorbed on the surface of MWNTs that were modified on a glassy carbon (GC) electrode to form an approximate monolayer. The redox characteristic and bioactivity of Cyt.c could be remained after it was adsorbed on MWNTs' surface. This provides a way to construct a new biosenser based on the activity of Cyt.c. Further investigation displayed that Cyt.c adsorbed on MWNTs showed an enzyme-like activity to catalyze the reduction of nitric oxide (NO). Due to catalyzing by Cyt.c, the reduction of NO in aqueous solution was achieved, which reductive potential appeared at -0.747V (vs. SCE). The peak currents were linearly proportional to concentration of NO in the range from 2 to 48 micromol/l with a limit of detection of 1.3 microM. The biosensor showed a good stability and excellent repeatability.

Nanoscale imaging and quantification of local proteolytic activity.
Kusick S, Bertram H, Oberleithner H, Ludwig T.
J Cell Physiol. 2005 Sep;204(3):767-74.
[ expand abstract ]

Proteolytic cleavage of extracellular matrix (ECM) is a critical feature of tumor cell invasion, and affects cancer cell growth, differentiation, apoptosis, and migration. Malignant cells secrete most proteases as inactive proenzymes that undergo proteolytic cleavage for activation, and proteolytic activity is elevated in close proximity to these cells. Therefore, local activity rather than protease concentration determines ECM proteolysis. Precise quantification of local proteolytic activity, functional investigation, and high resolution imaging of morphological ECM alterations have proven difficult. In this study, we present a novel approach for measuring proteolytic activity in the microenvironment of cells by using atomic force microscopy (AFM). Amelanotic melanoma cells (A7-clone) were seeded on fluorescent gelatin or collagen-IV coatings. Proteolysis reduced fluorescence of these coatings. Fluorescence microscopy (FM) in combination with AFM was used to maneuver the AFM-tip to tumor cell induced proteolytic spots. AFM enabled nanoscale volume measurement, three-dimensional reconstruction of single proteins and demonstrated that ECM cleavage is restricted to the proteolytic microenvironment of cancer cells. This method detected significant decreases in molecular weight of protein clusters (-76.6%), matrix volume (-46.6%), and height (-38.1%) between intact and proteolyzed gelatin. Similar parameter changes were demonstrated without FM, by AFM-scanning gelatin in close proximity to invasive cells. Furthermore, AFM depicted significantly stronger local degradation of gelatin than collagen-IV by A7-cells. Taken together, AFM allows specific quantification and imaging of local proteolytic processes at a nanometer level, thus providing a unique method for the functional evaluation of invasiveness and metastatic potential of tumor cells in small scale samples. (c) 2005 Wiley-Liss, Inc.

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Nanotechnology for the biologist.
McNeil SE.
J Leukoc Biol. 2005 Sep;78(3):585-94.
[ expand abstract ]

Nanotechnology refers to research and technology development at the atomic, molecular, and macromolecular scale, leading to the controlled manipulation and study of structures and devices with length scales in the 1- to 100-nanometers range. Objects at this scale, such as "nanoparticles," take on novel properties and functions that differ markedly from those seen in the bulk scale. The small size, surface tailorability, improved solubility, and multifunctionality of nanoparticles open many new research avenues for biologists. The novel properties of nanomaterials offer the ability to interact with complex biological functions in new ways-operating at the very scale of biomolecules. This rapidly growing field allows cross-disciplinary researchers the opportunity to design and develop multifunctional nanoparticles that can target, diagnose, and treat diseases such as cancer. This article presents an overview of nanotechnology for the biologist and discusses "nanotech" strategies and constructs that have already demonstrated in vitro and in vivo efficacy.

Fluorescence detection of enzymatic activity within a liposome based nano-biosensor.
Vamvakaki V, Fournier D, Chaniotakis NA.
Biosens Bioelectron. 2005 Aug 15;21(2):384-8.
[ expand abstract ]

The encapsulation of enzymes in microenvironments and especially in liposomes, has proven to greatly improve enzyme stabilization against unfolding, denaturation and dilution effects. Combining this stabilization effect, with the fact that liposomes are optically translucent, we have designed nano-sized spherical biosensors. In this work liposome-based biosensors are prepared by encapsulating the enzyme acetylcholinesterase (AChE) in L-a phosphatidylcholine liposomes resulting in spherical optical biosensors with an average diameter of 300+/-4nm. Porins are embedded into the lipid membrane, allowing for the free substrate transport, but not that of the enzyme due to size limitations. The enzyme activity within the liposome is monitored using pyranine, a fluorescent pH indicator. The response of the liposome biosensor to the substrate acetylthiocholine chloride is relatively fast and reproducible, while the system is stable as has been shown by immobilization within sol-gel

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Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry.
Tao WA, Wollscheid B, O'brien R, Eng JK, Li XJ, Bodenmiller B, Watts JD, Hood L,
Aebersold R.
Nat Methods. 2005 Aug;2(8):591-8.
[ expand abstract ]

We present a robust and general method for the identification and relative quantification of phosphorylation sites in complex protein mixtures. It is based on a new chemical derivatization strategy using a dendrimer as a soluble polymer support and tandem mass spectrometry (MS/MS). In a single step, phosphorylated peptides are covalently conjugated to a dendrimer in a reaction catalyzed by carbodiimide and imidazole. Modified phosphopeptides are released from the dendrimer via acid hydrolysis and analyzed by MS/MS. When coupled with an initial antiphosphotyrosine protein immunoprecipitation step and stable-isotope labeling, in a single experiment, we identified all known tyrosine phosphorylation sites within the immunoreceptor tyrosine-based activation motifs (ITAM) of the T-cell receptor (TCR) CD3 chains, and previously unknown phosphorylation sites on total 97 tyrosine phosphoproteins and their interacting partners in human T cells. The dynamic changes in phosphorylation were quantified in these proteins

Ion Transport Through Membrane-Spanning Nanopores Studied by Molecular Dynamics Simulations and Continuum Electrostatics Calculations.
Peter C, Hummer G.
Biophys J. 2005 Jul 8; [Epub ahead of print].
[ expand abstract ]

Narrow hydrophobic regions are a common feature of biological channels, with possible roles in ion-channel gating. We study the principles that govern ion transport through narrow hydrophobic membrane pores by molecular dynamics (MD) simulation of model membranes formed of hexagonally packed carbon nanotubes (CNTs). We focus on the factors that determine the energetics of ion translocation through such nonpolar nanopores and compare the resulting free-energy barriers for pores with different diameters corresponding to the gating regions in closed and open forms of potassium channels. Our model system also allows us to compare the results from molecular dynamics simulations directly to continuum electrostatics calculations. Both simulations and continuum calculations show that sub-nanometer wide pores pose a huge free energy barrier for ions, but a small increase in the pore diameter to ~1 nm nearly eliminates that barrier. We also find that in those wider channels the ion mobility is comparable to that in the bulk phase. By calculating local electrostatic potentials, we show that the long range Coulomb interactions of ions are strongly screened in the wide water-filled channels. Whereas continuum calculations capture the overall energetics reasonably well, the local water structure, which is not accounted for in this model, leads to interesting effects such as the preference of hydrated ions to move along the pore wall rather than the center of the pore.

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Transport of nanoparticles across an in vitro model of the human intestinal follicle associated epithelium.
Rieux AD, Ragnarsson EG, Gullberg E, Preat V, Schneider YJ, Artursson P.
Eur J Pharm Sci. 2005 July - August;25(4-5):455-465.
[ expand abstract ]

An in vitro model of the human follicle associated epithelium (FAE) was characterized and the influence of nanoparticle properties on the transcellular transport across the in vitro model was investigated. The model was established by co-culturing Caco-2 and Raji cells, with Caco-2 cells alone as control. The conversion of Caco-2 cells to follicle associated epithelium (FAE) like cells was monitored by following the surface expression of beta1-integrins (immunofluorescence) and nanoparticle transport (flow cytometry). The influence of the nanoparticle concentration at the apical side, temperature, size and surface properties of nanoparticles on transport was evaluated, as well as the influence of transport conditions. The conversion of Caco-2 cells into FAE-like cells occurred. The transport was concentration, temperature and size-dependent. Aminated nanoparticles were more efficiently transported than carboxylated nanoparticles, suggesting a role of nanoparticle surface functional groups and hydrophobicity, possibly leading to a different pattern of protein adsorption at their surface. In conclusion, this in vitro model is a promising tool to study the role of M cells in transintestinal nanoparticle transport, as well as to evaluate new drug delivery systems.

Three-dimensional Imaging of the Intracellular Localization of Growth Hormone and Prolactin and Their mRNA Using Nanocrystal (Quantum Dot) and Confocal Laser Scanning Microscopy Techniques.
Matsuno A, Itoh J, Takekoshi S, Nagashima T, Osamura RY.
J Histochem Cytochem. 2005 Jul;53(7):833-8.
[ expand abstract ]

Semiconductor nanocrystals (Quantum dots, Qdots) have recently been used in biological research, because they do not fade on exposure to light, and they enable us to obtain multicolor imaging because of a narrow emission peak that can be excited via a single wavelength of light. There have been no reports of simultaneous localization of mRNA and protein using Qdots. We successfully applied these advantages of Qdot and confocal laser scanning microscopy (CLSM) to three-dimensional images of the intracellular localization of growth hormone and prolactin and to their mRNA. In situ hybridization and immunohistochemistry using Qdots combined with CLSM can optimally illustrate the relationship between protein and mRNA simultaneously in three dimensions. Such an approach enables us to visualize functional images of proteins in relation with mRNA synthesis and localization.

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Quantum dot-based cell motility assay.
Gu W, Pellegrino T, Parak WJ, Boudreau R, Le Gros MA, Gerion D, Alivisatos AP, Larabell CA.
Sci STKE. 2005 Jun 28;2005(290):pl5.
[ expand abstract ]

Because of their favorable physical and photochemical properties, colloidal CdSe/ZnS-semiconductor nanocrystals (commonly known as quantum dots) have enormous potential for use in biological imaging. In this report, we present an assay that uses quantum dots as markers to quantify cell motility. Cells that are seeded onto a homogeneous layer of quantum dots engulf and absorb the nanocrystals and, as a consequence, leave behind a fluorescence-free trail. By subsequently determining the ratio of cell area to fluorescence-free track area, we show that it is possible to differentiate between invasive and noninvasive cancer cells. Because this assay uses simple fluorescence detection, requires no significant data processing, and can be used in live-cell studies, it has the potential to be a powerful new tool for discriminating between invasive and noninvasive cancer cell lines or for studying cell signaling events involved in migration.

Detection of nanometer-sized particles in living cells using modern fluorescence fluctuation methods.
Edetsberger M, Gaubitzer E, Valic E, Waigmann E, Kohler G.
Biochem Biophys Res Commun. 2005 Jun 24;332(1):109-16.
[ expand abstract ]

Nanosized materials are increasingly used in medicine and biotechnology but originate also from various aerosol sources. A detailed understanding of their interaction with cells is a prerequisite for specific applications and appraisal of hazardous effects. Fluorescence fluctuation methods are applied to follow the time-course of the translocation and distribution of fluorescent 20 nm polystyrene nanoparticles with negative surface charges in HeLa cells under almost physiological conditions. The experimental results demonstrate that singular particles enter the cell without significant contribution by endocytotic mechanisms and are distributed within the cytoplasm. Subsequently aggregation is observed, which can be blocked by cytotoxins, like Genistein and Cytochalasin B, interfering with cellular uptake processes. The observed non-active uptake is due to non-specific interactions with the cell surface and could be responsible for distribution of nanometer-sized materials in tissue.

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Nanogel-quantum dot hybrid nanoparticles for live cell imaging.
Hasegawa U, Nomura SM, Kaul SC, Hirano T, Akiyoshi K.
Biochem Biophys Res Commun. 2005 Jun 17;331(4):917-21.
[ expand abstract ]

We report here a novel carrier of quantum dots (QDs) for intracellular labeling. Monodisperse hybrid nanoparticles (38 nm in diameter) of QDs were prepared by simple mixing with nanogels of cholesterol-bearing pullulan (CHP) modified with amino groups (CHPNH2). The CHPNH2-QD nanoparticles were effectively internalized into the various human cells examined. The efficiency of cellular uptake was much higher than that of a conventional carrier, cationic liposome. These hybrid nanoparticles could be a promising fluorescent probe for bioimaging.

Nanoparticle labels in immunosensing using optical detection methods.
Seydack M.
Biosens Bioelectron. 2005 Jun 15;20(12):2454-69.
[ expand abstract ]

Efforts to improve the performance of immunoassays and immunosensors by incorporating different kinds of nanostructures have gained considerable momentum over the last decade. Apart from liposomes, which will not be discussed here, most groups focus on artificial, particulate marker systems, both organic and inorganic. The underlying detection procedures may be based either on electro-magnetical or optical techniques. This review will be confined to the latter only, comprising nanoparticle applications generating signals as diverse as static and time-resolved luminescence, one- and two-photon absorption, Raman and Rayleigh scattering as well as surface plasmon resonance and others. In general, all endeavors cited are geared to achieve one or more of the following goals: lowering of detection limits (if possible, down to single-molecule level), parallel integration of multiple signals (multiplexing), signal amplification by several orders of magnitude and prevention of photobleaching effects with concomitant maintenance of antigen binding specificity and sensitivity. Inorganic nanoparticle labels based on noble metals, semiconductor quantum dots and nanoshells appear to be the most versatile systems for these bioanalytical applications of nanophotonics.

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Unusual inflammatory and fibrogenic pulmonary responses to single walled carbon nanotubes in mice.
Shvedova AA, Kisin ER, Mercer R, Murray AR, Johnson VJ, Potapovich AI, Tyurina YY, Gorelik O, Arepalli S, Schwegler-Berry D, Hubbs AF, Antonini J, Evans DE, Ku BK, Ramsey D, Maynard A, Kagan VE, Castranova V, Baron P.
Am J Physiol Lung Cell Mol Physiol. 2005 Jun 10; [Epub ahead of print].
[ expand abstract ]

Single walled carbon nanotubes (SWCNT) are new materials of emerging technological importance. As SWCNT are introduced into the life cycle of commercial products, their effects on human health and environment should be addressed. We demonstrated that pharyngeal aspiration of SWCNT elicited unusual pulmonary effects in C57BL/6 mice that combined a robust but acute inflammation with early onset yet progressive fibrosis and granulomas. A dose-dependent increase in the protein, lactate dehydrogenase (LDH), and g-glutamyl transferase (GGT) activities in BAL were found along with accumulation of 4-hydroxynonenal (oxidative biomarker), and depletion of glutathione in lungs. An early neutrophils accumulation (day 1), followed by lymphocyte (day 3) and macrophage (day 7) influx were accompanied by early elevation of pro-inflammatory cytokines (TNF-alpha, IL-1beta, day 1) followed by fibrogenic TGF-beta1 (peaked on day 7). A rapid progressive fibrosis found in mice exhibited two distinct morphologies: (1) SWCNT-induced granulomas mainly associated with hypertrophied epithelial cells surrounding SWCNT aggregates and (2) diffuse interstitial fibrosis and alveolar wall thickening likely associated with dispersed SWCNT. In vitro exposure of murine RAW264.7 macrophages to SWCNT triggered TGF-beta1 production similarly to zymosan but generated less TNF-alpha and IL-1beta. SWCNT did not cause superoxide or NO(.) production, active SWCNT engulfment, or apoptosis in RAW264.7 macrophages. Functional respiratory deficiencies and decreased bacterial clearance (Listeria monocytogenes) were found in mice treated with SWCNT. Equal doses of ultrafine carbon black particles (UfCB) or fine crystalline silica (SiO2) did not induce granulomas, alveolar walls thickening, and caused a significantly weaker pulmonary inflammation and damage.

Colloidal gold nanoparticle modified carbon paste interface for studies of tumor cell adhesion and viability.
Du D, Liu S, Chen J, Ju H, Lian H, Li J.
Biomaterials. 2005 Jun 9; [Epub ahead of print].
[ expand abstract ]

A non-toxic biomimetic interface for immobilization of living cells and electrochemical exogenous effect study of cell viability was constructed by mixing colloidal gold nanoparticles in carbon paste. A new approach to study the effects of anti-tumor drug and other exogenous factors on cell viability was proposed. The nanoparticles were efficient for preserving the activity of immobilized living cells and preventing their leakage from the electrode surface. The immobilized living AsPC-1 cells (pancreatic adenocarcinoma cells derived from ascites) exhibited an irreversible voltammetric response related to the oxidation of guanine. The presence of guanine was verified by liquid chromatography-mass spectrometry. The contents of guanine in cytoplasm of each AsPC-1 and normal pancreatic cell were detected to be 370 and 22amol, respectively. The cytotoxic effect of adriamycin resulted in a decrease in peak current of guanine. The optimal exogenous factors that affected cell viability, including pH, temperature and salt concentration of electrolyte, were just consistent with cell growth conditions in culture. This simple and rapid method could be applied for the electrochemical investigation of exogenous effect and characterization of the viability of living cells.

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Transport of nanoparticles across an in vitro model of the human intestinal follicle associated epithelium.
Rieux AD, Ragnarsson EG, Gullberg E, Preat V, Schneider YJ, Artursson P.
Eur J Pharm Sci. 2005 Jun 7; [Epub ahead of print].
[ expand abstract ]

An in vitro model of the human follicle associated epithelium (FAE) was characterized and the influence of nanoparticle properties on the transcellular transport across the in vitro model was investigated. The model was established by co-culturing Caco-2 and Raji cells, with Caco-2 cells alone as control. The conversion of Caco-2 cells to follicle associated epithelium (FAE) like cells was monitored by following the surface expression of beta1-integrins (immunofluorescence) and nanoparticle transport (flow cytometry). The influence of the nanoparticle concentration at the apical side, temperature, size and surface properties of nanoparticles on transport was evaluated, as well as the influence of transport conditions. The conversion of Caco-2 cells into FAE-like cells occurred. The transport was concentration, temperature and size-dependent. Aminated nanoparticles were more efficiently transported than carboxylated nanoparticles, suggesting a role of nanoparticle surface functional groups and hydrophobicity, possibly leading to a different pattern of protein adsorption at their surface. In conclusion, this in vitro model is a promising tool to study the role of M cells in transintestinal nanoparticle transport, as well as to evaluate new drug delivery systems.

Within the cell: analytical techniques for subcellular analysis.
Olson KJ, Ahmadzadeh H, Arriaga EA.
Anal Bioanal Chem. 2005 Jun 1; [Epub ahead of print] .
[ expand abstract ]

This review covers recent developments in the preparation, manipulation, and analyses of subcellular environments. In particular, it highlights approaches for (1) separation and detection of individual organelles, (2) preparation of ultra-pure organelle fractions, and (3) utilization of novel labeling strategies. These approaches, based on innovative technologies such as microfluidics, immunoisolation, mass spectrometry and electrophoresis, suggest that subcellular analyses will soon become as commonplace as single cell and bulk cellular assays.

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Highly efficient molecular delivery into mammalian cells using carbon nanotube spearing.
Cai D, Mataraza JM, Qin ZH, Huang Z, Huang J, Chiles TC, Carnahan D, Kempa K, Ren Z.
Nat Methods. 2005 Jun;2(6):449-54.
[ expand abstract ]

Introduction of exogenous DNA into mammalian cells represents a powerful approach for manipulating signal transduction. The available techniques, however, are limited by low transduction efficiency and low cell viability after transduction. Here we report a highly efficient molecular delivery technique, named nanotube spearing, based on the penetration of nickel-embedded nanotubes into cell membranes by magnetic field driving. DNA plasmids containing the enhanced green fluorescent protein (EGFP) sequence were immobilized onto the nanotubes, and subsequently speared into targeted cells. We have achieved an unprecedented high transduction efficiency in Bal17 B-lymphoma, ex vivo B cells and primary neurons with high viability after transduction. This technique may provide a powerful tool for highly efficient gene transfer into a variety of cells, especially the hard-to-transfect cells.

Quantum dots spectrally distinguish multiple species within the tumor milieu in vivo.
Stroh M, Zimmer JP, Duda DG, Levchenko TS, Cohen KS, Brown EB, Scadden DT, Torchilin VP, Bawendi MG, Fukumura D, Jain RK.
Nat Med. 2005 Jun;11(6):678-82.
[ expand abstract ]

A solid tumor is an organ composed of cancer and host cells embedded in an extracellular matrix and nourished by blood vessels. A prerequisite to understanding tumor pathophysiology is the ability to distinguish and monitor each component in dynamic studies. Standard fluorophores hamper simultaneous intravital imaging of these components. Here, we used multiphoton microscopy techniques and transgenic mice that expressed green fluorescent protein, and combined them with the use of quantum dot preparations. We show that these fluorescent semiconductor nanocrystals can be customized to concurrently image and differentiate tumor vessels from both the perivascular cells and the matrix. Moreover, we used them to measure the ability of particles of different sizes to access the tumor. Finally, we successfully monitored the recruitment of quantum dot-labeled bone marrow-derived precursor cells to the tumor vasculature. These examples show the versatility of quantum dots for studying tumor pathophysiology and creating avenues for treatment.

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Microreactor Microfluidic Systems with Human Microsomes and Hepatocytes for use in Metabolite Studies.
Biomed Microdevices. 2005 Jun;7(2):117-25.
Zguris JC, Itle LJ, Hayes D, Pishko MV.
[ expand abstract ]

In the area of drug discovery, high-speed synthesis has increased the number of drug candidates produced. These potential drugs need to be evaluated for their adsorption, distribution, metabolism, elimination, and toxicology (ADMET) properties as early in the drug development stage as possible. Previously, a potential drug's ADMET properties have been found out by using monolayer cell cultures and live animals. These methods can be costly, time-intensive, and impractical for screening the large amount of potential drugs created by combinatorial chemistry. A quick, small, inexpensive, and highly parallel device would be desirable to determine a drug candidate's properties (i.e., metabolism of the drug). Here we fabricate a microfluidic device entrapping human microsomes within poly(ethylene) glycol hydrogels thereby generating an in situ microreactor to assess a drug candidate's metabolic properties that can be coupled to analysis equipment. We show that microsomes can be entrapped without the loss of enzymatic activity during photopolymerization. Additionally, a microreactor utilizing hepatocytes was also created for comparison with the microsome microreactor.

A methodology to study intracellular distribution of nanoparticles in brain endothelial cells.
Garcia-Garcia E, Andrieux K, Gil S, Kim HR, Doan TL, Desmaele D, d'Angelo J, Taran F, Georgin D, Couvreur P.
Int J Pharm. 2005 May 26; [Epub ahead of print].
[ expand abstract ]

Cell internalisation and intracellular distribution of PEG-coated polyhexadecylcyanoacrylate (PEG-PHDCA) nanoparticles in rat brain endothelial cells (RBEC) have been investigated. A cell fractionation method has been developed based on the selective permeabilisation of RBEC plasma membrane by digitonin. By interacting with membrane cholesterol, digitonin creates pores allowing the release of soluble and diffusible species outside the cell. The selectivity of plasma membrane permeabilisation was controlled by using compartment markers such as lactate dehydrogenase (LDH) for cytoplasm and cathepsin B for lysosomes. An optimal digitonin concentration of 0.003% (w/v) has been identified to induce a pattern of membrane permeabilisation corresponding to the extraction of 72% LDH and less than 15% of Cathepsin B. Membrane permeabilisation at this digitonin concentration allows one to distinguish between the cell cytoplasm and its endo/lysosomal fraction. This methodology was applied to investigate the intracellular distribution of the nanoparticles after their incubation with the RBEC. The results showed that PEG-PHDCA nanoparticles were able to be internalised to a higher extent than PHDCA nanoparticles (after 20min incubation). Additionally, these nanoparticles displayed different patterns of intracellular capture, depending on their specific surface composition: PEG-PHDCA nanoparticles were 48% in the plasma membrane, 24% in the cytoplasm, 20% in vesicular compartments and 8% associated with the fraction of the nucleus, the cytoskeleton and caveolae suggesting that PEG-PHDCA nanoparticle uptake by RBEC is specific and presumably due to endocytosis. Confocal microscopy studies confirmed the cellular uptake of PEG-PHDCA nanoparticles

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Quantum dots spectrally distinguish multiple species within the tumor milieu in vivo.
Stroh M, Zimmer JP, Duda DG, Levchenko TS, Cohen KS, Brown EB, Scadden DT, Torchilin VP, Bawendi MG, Fukumura D, Jain RK.
Nat Med. 2005 May 8; [Epub ahead of print].
[ expand abstract ]

A solid tumor is an organ composed of cancer and host cells embedded in an extracellular matrix and nourished by blood vessels. A prerequisite to understanding tumor pathophysiology is the ability to distinguish and monitor each component in dynamic studies. Standard fluorophores hamper simultaneous intravital imaging of these components. Here, we used multiphoton microscopy techniques and transgenic mice that expressed green fluorescent protein, and combined them with the use of quantum dot preparations. We show that these fluorescent semiconductor nanocrystals can be customized to concurrently image and differentiate tumor vessels from both the perivascular cells and the matrix. Moreover, we used them to measure the ability of particles of different sizes to access the tumor. Finally, we successfully monitored the recruitment of quantum dot-labeled bone marrow-derived precursor cells to the tumor vasculature. These examples show the versatility of quantum dots for studying tumor pathophysiology and creating avenues for treatment.

Fabrication of quantum dot-lectin conjugates as novel fluorescent probes for microscopic and flow cytometric identification of leukemia cells from normal lymphocytes.
Zhelev Z, Ohba H, Bakalova R, Jose R, Fukuoka S, Nagase T, Ishikawa M, Baba Y.
Chem Commun (Camb). 2005 Apr 21;(15):1980-2.
[ expand abstract ]

The present study describes a synthesis of QD-lectin conjugates and their application for identification of leukaemia cells from normal lymphocytes using fluorescent confocal microscopy and flow cytometry. The results are compared with commercially available FITC-lectin.

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Protein biosensors based on biofunctionalized conical gold nanotubes.
Siwy Z, Trofin L, Kohli P, Baker LA, Trautmann C, Martin CR.
J Am Chem Soc. 2005 Apr 13;127(14):5000-1.
[ expand abstract ]

There is increasing interest in the concept of using nanopores as the sensing elements in biosensors. The nanopore most often used is the alpha-hemolysin protein channel, and the sensor consists of a single channel embedded within a lipid bilayer membrane. An ionic current is passed through the channel, and analyte species are detected as transient blocks in this current associated with translocation of the analyte through the channel-stochastic sensing. While this is an extremely promising sensing paradigm, it would be advantageous to eliminate the very fragile lipid bilayer membrane and perhaps to replace the biological nanopore with an abiotic equivalent. We describe here a new family of protein biosensors that are based on conically shaped gold nanotubes embedded within a mechanical and chemically robust polymeric membrane. While these sensors also function by passing an ion current through the nanotube, the sensing paradigm is different from the previous devices in that a transient change in the current is not observed. Instead, the protein analyte binds to a biochemical molecular-recognition agent at the mouth of the conical nanotube, resulting in complete blockage of the ion current. Three different molecular-recognition agents, and correspondingly three different protein analytes, were investigated: (i) biotin/streptavidin, (ii) protein-G/immunoglobulin, and (iii) an antibody to the protein ricin with ricin as the analyte.

Establishment and implications of a characterization method for magnetic nanoparticle using cell tracking velocimetry and magnetic susceptibility modified solutions.
Zhang H, Moore LR, Zborowski M, Williams PS, Margel S, Chalmers JJ.
Analyst. 2005 Apr;130(4):514-27.
[ expand abstract ]

Magnetic micro and nanoparticles conjugated to affinity labels have become a significant, commercial reagent. It has been demonstrated that the performance of cell separation systems using magnetic labels is a function of the magnitude of the magnetic force that can be generated through labeling. This magnetic force is proportional to the number of magnetic particles bound to the cell, the magnetic energy gradient, and the particle-field interaction parameter. This particle-field interaction parameter, which is the product of the relative volumetric, magnetic susceptibility and the volume of the micro or nanoparticle, is a fundamental parameter which can be used to characterize the magnetic particles. An experimental technique is presented which measures the volumetric magnetic susceptibility of particles through the use of susceptibility modified solutions and an experimental instrument, Cell Tracking Velocimetry, CTV. Experimental studies were conducted on polystyrene microspheres alone and those bound to four different magnetic nanoparticles. The experimentally determined values of the magnetic susceptibility of the polystyrene microspheres are consistent with values found from literature. Consequently, magnetic susceptibility measurements of these polystyrene microspheres bound with the magnetic nanoparticles combined with particle size measurements using commercial dynamic light scattering instrument allowed estimates of the particle-field interaction parameter to be made for four commercial, magnetic nanoparticles. The value found for MACS([trade mark sign]) beads is close to what is reported from an independent study. The values for MACS([trade mark sign]) beads and Imag([trade mark sign]) beads are found to agree with what is observed from experiments. Finally, an experimental demonstration of the impact that differences in this field interaction parameter has on the labeling of human lymphocytes is presented.

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FRET measurements of cell-traction forces and nano-scale clustering of adhesion ligands varied by substrate stiffness.
Kong HJ, Polte TR, Alsberg E, Mooney DJ.
Proc Natl Acad Sci U S A. 2005 Mar 22;102(12):4300-5.
[ expand abstract ]

The mechanical properties of cell adhesion substrates regulate cell phenotype, but the mechanism of this relation is currently unclear. It may involve the magnitude of traction force applied by the cell, and/or the ability of the cells to rearrange the cell adhesion molecules presented from the material. In this study, we describe a FRET technique that can be used to evaluate the mechanics of cell-material interactions at the molecular level and simultaneously quantify the cell-based nanoscale rearrangement of the material itself. We found that these events depended on the mechanical rigidity of the adhesion substrate. Furthermore, both the proliferation and differentiation of preosteoblasts (MC3T3-E1) correlated to the magnitude of force that cells generate to cluster the cell adhesion ligands, but not the extent of ligand clustering. Together, these data demonstrate the utility of FRET in analyzing cell-material interactions, and suggest that regulation of phenotype with substrate stiffness is related to alterations in cellular traction forces.

Serum exposed to nanoparticle carbon black displays increased potential to induce macrophage migration.
Barlow PG, Donaldson K, MacCallum J, Clouter A, Stone V.
Toxicol Lett. 2005 Mar 15;155(3):397-401.
[ expand abstract ]

OBJECTIVE: To assess whether fine and ultrafine particles (nanoparticles) have the capacity to activate factors in serum that would induce macrophage migration. This is a model previously reported to investigate complement activation by other respirable particles and fibres. METHOD: Foetal bovine serum was exposed to varying doses of fine and nanoparticle carbon black as well as the oxidant tert-butyl hydroperoxide (tBHP). The subsequent potential of the serum to induce macrophage migration was measured using a macrophage chemotaxis assay. RESULTS: Treatment of serum with 10 mg/ml of nanoparticle carbon black generated substances that induced a 1.8-fold increase in macrophage migration (P<0.001) compared with untreated serum. This effect was partially inhibited by antioxidant intervention. Serum treated with an equivalent mass of fine carbon black did not display any chemotactic potential. tBHP treatment of the serum did not result in the generation of macrophage chemotactic factors. CONCLUSIONS: High doses of nanoparticle carbon black have the capacity to cause chemotactic factor generation in serum, by a mechanism involving ROS generation, although ROS alone, in the form of tBHP are not adequate to generate chemotactic factors in serum.

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Multi-walled carbon nanotube interactions with human epidermal keratinocytes.
Monteiro-Riviere NA, Nemanich RJ, Inman AO, Wang YY, Riviere JE.
Toxicol Lett. 2005 Mar 15;155(3):377-84.
[ expand abstract ]

Carbon nanotubes have widespread applications in multiple engineering disciplines. However, little is known about the toxicity or interaction of these particles with cells. Carbon nanotube films were grown using a microwave plasma enhanced chemical vapor deposition system. Human epidermal keratinocytes (HEK) were exposed to 0.1, 0.2, and 0.4 mg/ml of multi-walled carbon nanotubes (MWCNT) for 1, 2, 4, 8, 12, 24 and 48 h. HEK were examined by transmission electron microscopy for the presence of MWCNT. Here we report that chemically unmodified MWCNT were present within cytoplasmic vacuoles of the HEK at all time points. The MWCNT also induced the release of the proinflammatory cytokine interleukin 8 from HEKs in a time dependent manner. These data clearly show that MWCNT, not derivatized nor optimized for biological applications, are capable of both localizing within and initiating an irritation response in a target epithelial cell that composes a primary route of occupational exposure for manufactured nanotubes.

Labeling of cells with quantum dots.
Parak WJ, Pellegrino T, Plank C.
Nanotechnology 2005; 16 R9-R25..
[ expand abstract ]

Colloidal quantum dots are semiconductor nanocrystals well dispersed in a solvent. The optical properties of quantum dots, in particular the wavelength of their fluorescence, depend strongly on their size. Because of their reduced tendency to photobleach, colloidal quantum dots are interesting fluorescence probes for all types of labeling studies. In this review we will give an overview on how quantum dots have been used so far in cell biology. In particular we will discuss the biologically relevant properties of quantum dots and focus on four topics: labeling of cellular structures and receptors with quantum dots, incorporation of quantum dots by living cells, tracking the path and the fate of individual cells using quantum dot labels, and quantum dots as contrast agents.

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Labeling Ribonuclease S with a 3 nm Au Nanoparticle by Two-Step Assembly.
Aubin ME, Morales DG, Hamad-Schifferli K.
Nano Lett. 2005 Mar 9;5(3):519-522.
[ expand abstract ]

We label ribonuclease S with a 3 nm Au nanoparticle (NP) by utilizing its two-piece structure. One portion, S-peptide, is mutated with a unique NP attachment site. NP-peptide self-assembles with the other portion, S-protein, to form an active enzyme. NP mobility decreases with peptide labeling and S-protein association. Surface plasmon shifts support conjugation. Higher S-peptide coverages on the NP surface reduce nonspecific adsorption, while sterically hindering assembly of RNaseS. Thiols displace nonspecific adsorption, maximizing site-specific labeling.

Metalloproteinase and cytokine production by THP-1 macrophages following exposure to chitosan-DNA nanoparticles.
Chellat F, Grandjean-Laquerriere A, Le Naour R, Fernandes J, Yahia L, Guenounou M, Laurent-Maquin D.
Biomaterials. 2005 Mar;26(9):961-70.
[ expand abstract ]

The use of nanoparticles for gene therapy is gaining more and more interest for medical applications. Chitosan is among the candidate polymers that have a potential application as a gene delivery system. Before using chitosan-DNA nanoparticles in vivo, one must study their interaction and cell's behavior. Since macrophages play an important role in inflammatory processes, this study was performed to investigate the effects of chitosan-DNA nanoparticles on human THP-1 cell line. Cytokine (TNF-alpha, IL-1beta, IL-6 and IL-10) and metalloproteinase (MMP-2 and MMP-9) release as well as their inhibitors (TIMP-1 and TIMP-2) were assessed after time course incubation with different amount of nanoparticles. Their secretion was quantified by enzyme-linked immunosorbent assay. Gelatinolytic activity of MMP-2 and MMP-9 was determined by zymography in cell supernatants and lysates. Cytokine secretion was not detected even in the presence of high amount of nanoparticles. On the contrary, the secretion of MMP-9 in cell supernatants increased significantly after 24 and 48 h in comparison with non-treated cells. MMP-2 secretion was augmented only after 48 h for the highest concentrations of nanoparticles (10 and 20 microg/ml DNA content). However, zymography studies showed that the secreted MMPs were in the proactive forms, while the active form of MMP-9, but not MMP-2, was detected in cell lysates when 10 and 20 microg/ml DNA containing nanoparticles were used. In conclusion, exposure of THP-1 macrophages to Ch-DNA nanoparticles did not induce release of proinflammatory cytokines. The presence of active MMP-9 within the macrophages could possibly be related to nanoparticle phagocytosis and degradation rather than to inflammatory reactions.

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Cancer nanotechnology: opportunities and challenges.
Ferrari M.
Nat Rev Cancer. 2005 Mar;5(3):161-71.
[ expand abstract ]

Nanotechnology is a multidisciplinary field, which covers a vast and diverse array of devices derived from engineering, biology, physics and chemistry. These devices include nanovectors for the targeted delivery of anticancer drugs and imaging contrast agents. Nanowires and nanocantilever arrays are among the leading approaches under development for the early detection of precancerous and malignant lesions from biological fluids. These and other nanodevices can provide essential breakthroughs in the fight against cancer.

Effects of gold nanoparticle and electrode surface properties on electrocatalytic silver deposition for electrochemical DNA hybridization detection.
Lee TM, Cai H, Hsing IM.
Analyst. 2005 Mar;130(3):364-9. Epub 2005 Jan 13.
[ expand abstract ]

In this paper we report the catalytic effects of various gold nanoparticles for silver electrodeposition on indium tin oxide (ITO)-based electrodes, and successfully apply this methodology for signal amplification of the hybridization assay. The most widely used gold nanoparticle-based hybridization indicators all promote silver electrodeposition on the bare ITO electrodes, with decreasing catalytic capability in order of 10 nm gold, DNA probe-10 nm gold conjugate, streptavidin-5 nm gold, and streptavidin-10 nm gold. Of greater importance, these electrocatalytic characteristics are affected by any surface modifications of the electrode surfaces. This is illustrated by coating the ITO with an electroconducting polymer, poly(2-aminobenzoic acid)(PABA), as well as avidin molecules, which are promising immobilization platforms for DNA biosensors. The catalytic silver electrodeposition of the gold nanoparticles on the PABA-coated ITO surfaces resembles that on the bare surfaces. With avidin covalently bound to the PABA, it is interesting to note that the changes in electrocatalytic performance vary for different types of gold nanoparticles. For the streptavidin-5 nm gold, the silver electrodeposition profile is unaffected by the presence of the avidin layer, whereas for both the 10 nm Au and DNA probe-10 nm gold conjugate, the deposition profiles are suppressed. The streptavidin-5 nm gold is employed as the hybridization indicator, with avidin-modified (via PABA) ITO electrode as the immobilization platform, to enable signal amplification by the silver electrodeposition process. Under the conditions, this detection strategy offers a signal-to-noise ratio of 20. We believe that this protocol has great potential for simple, reproducible, highly selective and sensitive DNA detection on fully integrated microdevices in clinical diagnostics and environmental monitoring applications.

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Nanomedicine: current status and future prospects.
Moghimi SM, Hunter AC, Murray JC.
FASEB J. 2005 Mar;19(3):311-30.
[ expand abstract ]

Applications of nanotechnology for treatment, diagnosis, monitoring, and control of biological systems has recently been referred to as "nanomedicine" by the National Institutes of Health. Research into the rational delivery and targeting of pharmaceutical, therapeutic, and diagnostic agents is at the forefront of projects in nanomedicine. These involve the identification of precise targets (cells and receptors) related to specific clinical conditions and choice of the appropriate nanocarriers to achieve the required responses while minimizing the side effects. Mononuclear phagocytes, dendritic cells, endothelial cells, and cancers (tumor cells, as well as tumor neovasculature) are key targets. Today, nanotechnology and nanoscience approaches to particle design and formulation are beginning to expand the market for many drugs and are forming the basis for a highly profitable niche within the industry, but some predicted benefits are hyped. This article will highlight rational approaches in design and surface engineering of nanoscale vehicles and entities for site-specific drug delivery and medical imaging after parenteral administration. Potential pitfalls or side effects associated with nanoparticles are also discussed.

Immobilized carbon nanotubes as matrix for MALDI-TOF-MS analysis: Applications to neutral small carbohydrates.
Ren SF, Zhang L, Cheng ZH, Guo YL.
J Am Soc Mass Spectrom. 2005 Mar;16(3):333-339.
[ expand abstract ]

In this work, we reported on the advantages of immobilized carbon nanotubes as a novel MALDI-matrix. Recently, carbon nanotubes have been reported to be an effective MALDI matrix for small molecules (Anal. Chem.2003, 75, 6191), as it can eliminate the interfering matrix peaks as well as form a web morphology to fully disperse the analyte and allow strong ultraviolet absorption for enhanced pulsed laser desorption and ionization. In our study, to overcome the problem that the carbon nanotube matrix may fly off from the target, a type of polyurethane adhesive, NIPPOLAN-DC-205, is introduced to immobilize carbon nanotubes on the target, which enables widespread application of carbon nanotubes as matrix for MALDI-MS analysis. At the same time, the properties of the carbon nanotubes as an efficient matrix remained after immobilization. The presence of NIPPOLAN-DC-205 increases the time for analysis at a particular desorption spot by minimizing the time-consuming search for "hot spots" and facilitating experiments such as post source decay (PSD) which need longer-lasting signals. Moreover, NIPPOLAN-DC-205 produces no interference peaks and can easily be cleaned with acetone. Fast evaporation technology may be used to enhance signal reproducibility in MALDI analysis using carbon nanotubes as matrix. Consequently, the applicability of the carbon nanotube as matrix for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of low molecular mass analytes is highly improved. The feasibility of the method employing polyurethane is demonstrated by comparison of the results produced from the carbon nanotube matrix with and without immobilization. In addition, neutral small carbohydrates, which are difficult to be ionized normally, can be cationized with high efficiency by MALDI-TOF-MS using the immobilized carbon nanotube matrix. The method was further applied to analyze peptides and detect urine glucose successfully.

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2004

A systematic characterization of mitochondrial proteome from a human T leukemia cells.
Rezaul K, Wu L, Mayya V, Hwang SI, Han DK.
Mol Cell Proteomics. 2004 Dec 14; [Epub ahead of print].
[ expand abstract ]

Global understanding of tissue specific differences in mitochondrial signal transduction requires comprehensive mitochondrial protein identification from multiple cell and tissue types. Here, we explore the feasibility and efficiency of protein identification using the one dimensional-gel electrophoresis in combination with the nano liquid-chromatography tandem mass spectrometry (GeLC-MS/MS). The use of only 40 microg of purified mitochondrial proteins and data analysis using stringent scoring criteria and the molecular weight validation of the gel slices enables the identification of 227 known mitochondrial proteins (membrane and soluble) and 453 additional proteins likely to be associated with mitochondria. Replicate analyses of 60microg of mitochondrial proteins on the faster scanning LTQ mass spectrometer validate all the previously identified proteins and most of the single hit proteins except the 81 single hit proteins. Among the identified proteins, 466 proteins are known to functionally participate in various processes such as respiration, tricarboxylic acid cycle (TCA cycle), amino acid and nucleotide metabolism, glycolysis, protection against oxidative stress, mitochondrial assembly, molecular transport, protein biosynthesis, cell cycle control, and many known cellular processes. The distribution of identified proteins in terms of size, pI, and hydrophobicity reveal that the present analytical strategy is largely unbiased and very efficient. Thus, we conclude that this approach is suitable for characterizing sub-cellular proteomes form multiple cells and tissues.

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Tracking metastatic tumor cell extravasation with quantum dot nanocrystals and fluorescence emission-scanning microscopy.
Voura EB, Jaiswal JK, Mattoussi H, Simon SM.
Nat Med. 2004 Sep;10(9):993-8. Epub 2004 Aug 29.
[ expand abstract ]

Metastasis is an impediment to the development of effective cancer therapies. Our understanding of metastasis is limited by our inability to follow this process in vivo. Fluorescence microscopy offers the potential to follow cells at high resolution in living animals. Semiconductor nanocrystals, quantum dots (QDs), offer considerable advantages over organic fluorophores for this purpose. We used QDs and emission spectrum scanning multiphoton microscopy to develop a means to study extravasation in vivo. Although QD labeling shows no deleterious effects on cultured cells, concern over their potential toxicity in vivo has caused resistance toward their application to such studies. To test if effects of QD labeling emerge in vivo, tumor cells labeled with QDs were intravenously injected into mice and followed as they extravasated into lung tissue. The behavior of QD-labeled tumor cells in vivo was indistinguishable from that of unlabeled cells. QDs and spectral imaging allowed the simultaneous identification of five different populations of cells using multiphoton laser excitation. Besides establishing the safety of QDs for in vivo studies, our approach permits the study of multicellular interactions in vivo.

Confocal fluorescence detection expanded to UV excitation: the first continuous fluorimetric assay of human steroid sulfatase in nanoliter volume.
Billich A, Bilban M, Meisner NC, Nussbaumer P, Neubauer A, Jager S, Auer M
Assay Drug Dev Technology 2004 Feb;2(1):21-30.
[ expand abstract ]

Steroid sulfatase is an enzyme that currently enjoys considerable interest as a potential drug target in the treatment of estrogen- and androgen-dependent diseases, in particular breast cancer. We have purified human steroid sulfatase to apparent homogeneity from recombinant Chinese hamster ovary cells, and we established an assay with a new fluorogenic substrate, 3,4-benzocoumarin-7-O-sulfate (1). Substrate 1 features a K(m) value of 22.5 microM, which is close to the value for the natural substrate dehydroepiandrosterone sulfate (26 microM) and much lower than the K(m) values of other synthetic substrates (276-736 microM). Importantly, the cleavage of substrate 1 can be monitored continuously during the enzymatic cleavage, since a change in fluorescence intensity is detectable at the pH where the enzyme is active; in contrast, all other synthetic substrates described so far require alkalization to reveal a measurable absorbance or fluorescence signal. The adaptation of the assay to the 96-well format allows continuous monitoring of multiple wells in a microplate fluorescence reader. Applications of the assay for the determination of IC(50) and K(i) values of novel steroid sulfatase inhibitors are presented. Most importantly the assay was transferred to the nanoscale format (1-microl assay volume) in 2080-well plates with confocal fluorescence detection. This miniaturization will permit screening with a minimum throughput of 20000 compounds per day. The system presented demonstrates that the confocal detection platform used for nanoscreening can be successfully adapted to assays for which conventional ultraviolet dyes like coumarins are necessary. This strongly broadens the application range of confocal readers in drug screening.

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Applications of T-lymphoma labeled with fluorescent quantum dots to cell tracing markers in mouse body.
Hoshino A, Hanaki K, Suzuki K, Yamamoto K.
Biochem Biophys Res Commun. 2004 Jan 30;314(1):46-53.
[ expand abstract ]

Photoluminescent semiconductor quantum dots (QDs) are novel nanometer-size probes that have found bioimaging. Here we imaged a cell line of mouse lymphocytes. QDs were actively taken into the target cells by endocytotic pathways. The fluorescence of QDs held in the endosomes could be studied for more than a week and remained stable luminescence against cell activation induced by concanavalin A, phytohemagglutinin, phorbol myristate acetate, and calcium ionophore A23187. These results suggested that QD-labeling was stable and did not affect either cell activation or cell function. When QD-labeled cells were intravenously injected into mouse, they remained in the peripheral blood in a concentration of approximately 10% up to 5 days after injection using both fluorescence microscopy and flow cytometry. In addition, approximately 20% of QDs were detected in the kidneys, liver, lung, and spleen and could still be observed 7 days after injection. These results suggested that fluorescent probes of QDs might be useful as bioimaging tools for tracing target cells over the period of a week in vivo.

Biocompatible fluorescent nanocrystals for immunolabeling of membrane proteins and cells
Sukhanova A, Devy J, Venteo L, Kaplan H, Artemyev M, Oleinikov V, Klinov D, Pluot M, Cohen JH, Nabiev I
Anal Biochem 2004 Jan 1;324(1):60-7
[ expand abstract ]

A methodology for simple convenient preparation of bright, negatively or positively charged, water-soluble CdSe/ZnS core/shell nanocrystals (NCs) and their stabilization in aqueous solution is described. Single NCs can be detected using a standard epifluorescent microscope, ensuring a detection limit of one molecule coupled with an NC. NCs solubilized in water by DL-Cys were stabilized, to avoid aggregation, by poly(allylamine) and conjugated with polyclonal anti-mouse antibodies (Abs). NC-Abs conjugates were tested in dot-blots and exhibited retention of binding capacity within several nanograms of antigen detected. We further demonstrated the advantages of NC-Abs conjugates in the immunofluorescent detection and three-dimensional (3D) confocal analysis of p-glycoprotein (p-gp), one of the main mediators of the MDR phenotype, overexpressed in the membrane of MCF7r breast adenocarcinoma cells. Immunolabeling of p-gp with NC-Abs conjugates was 4200-, 2600-, and 420-fold more resistant to photobleaching than its labeling with fluorescein isothiocyanate-Abs, R-phycoerythrin-Abs, and AlexaFluor488-Abs, respectively. The labeling of p-gp with NC-Abs conjugates was highly specific, and the data were used for confocal reconstruction of 3D images of the p-gp distribution in the MCF7r cell membrane. Finally, we demonstrated the applicability of NC-Abs conjugates obtained by the method described to specific detection of antigens in paraffin-embedded formaldehyde-fixed cancer tissue specimens, using immunostaining of cytokeratin in skin basal carcinoma as an example. We conclude that the NC-Abs conjugates may serve as easy-to-do, highly sensitive, photostable labels for immunofluorescent analysis, immunohistochemical detection, and 3D confocal studies of membrane proteins and cells.

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Real-time nucleic acid sequence-based amplification in nanoliter volumes.
Gulliksen A, Solli L, Karlsen F, Rogne H, Hovig E, Nordstrom T, Sirevag R
Anal Chem 2004 Jan 1;76(1):9-14.
[ expand abstract ]

Real-time nucleic acid sequence-based amplification (NASBA) is an isothermal method specifically designed for amplification of RNA. Fluorescent molecular beacon probes enable real-time monitoring of the amplification process. Successful identification, utilizing the real-time NASBA technology, was performed on a microchip with oligonucleotides at a concentration of 1.0 and 0.1 microM, in 10- and 50-nL reaction chambers, respectively. The microchip was developed in a silicon-glass structure. An instrument providing thermal control and an optical detection system was built for amplification readout. Experimental results demonstrate distinct amplification processes. Miniaturized real-time NASBA in microchips makes high-throughput diagnostics of bacteria, viruses, and cancer markers possible, at reduced cost and without contamination.

Novel technologies and recent advances in metastasis research.
Crnic I, Christofori G.
Int J Dev Biol. 2004;48(5-6):573-81.
[ expand abstract ]

In this review we have attempted to summarize some of the recent developments in using novel technologies to unravel the molecular mechanisms of tumor progression, in particular the formation of tumor metastasis. In order to push forward the frontiers in cancer research, it is obvious that several fields have to be further developed and interconnected: (1) clinical, epidemiological and pathological studies which mainly use innovative technologies, including microarray technology and nanotechnology to determine as many parameters as possible, (2) the development of improved and suitable bioassays and better animal models and (3) the use of novel computation and bioinformatics methods to sample and integrate the exponentially growing sets of data coming from such investigations. Fashionable as scientists are, this new endeavor may be called systems biology.

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Mortalin imaging in normal and cancer cells with quantum dot immuno-conjugates.
Kaul Z, Yaguchi T, Kaul SC, Hirano T, Wadhwa R, Taira K.
Cell Res. 2003 Dec;13(6):503-7.
[ expand abstract ]

Quantum dots are the nanoparticles that are recently emerging as an alternative to organic fluorescence probes in cell biology and biomedicine, and have several predictive advantages. These include their i) broad absorption spectra allowing visualization with single light source, ii) exceptional photo-stability allowing long term studies and iii) narrow and symmetrical emission spectrum that is controlled by their size and material composition. These unique properties allow simultaneous excitation of different size of quantum dots with a single excitation light source, their simultaneous resolution and visualization as different colors. At present there are only a few studies that have tested quantum dots in cellular imaging. We describe here the use of quantum dots in mortalin imaging of normal and cancer cells. Mortalin staining pattern with quantum dots in both normal and cancer cells mimicked those obtained with organic florescence probes and were considerably stable.

Quantum dot-based cell motility assay.
Pellegrino T, Parak WJ, Boudreau R, Le Gros MA, Gerion D, Alivisatos AP, Larabell CA.
Differentiation. 2003 Dec;71(9-10):542-8.
[ expand abstract ]

Motility and migration are measurable characteristics of cells that are classically associated with the invasive potential of cancer cells, but in vitro assays of invasiveness have been less than perfect. We previously developed an assay to monitor cell motility and migration using water-soluble CdSe/ZnS nanocrystals; cells engulf the fluorescent nanocrystals as they crawl across them and leave behind a fluorescent-free trail. We show here that semiconductor nanocrystals can also be used as a sensitive two-dimensional in vitro invasion assay. We used this assay to compare the behavior of seven different adherent human cell lines, including breast epithelial MCF 10A, breast tumor MDA-MB-231, MDA-MB-435S, MCF 7, colon tumor SW480, lung tumor NCI H1299, and bone tumor Saos-2, and observed two distinct behaviors of cancer cells that can be used to further categorize these cells. Some cancer cell lines demonstrate fibroblastic behaviors and leave long fluorescent-free trails as they migrate across the dish, whereas other cancer cells leave clear zones of varying sizes around their periphery. This assay uses fluorescence detection, requires no processing, and can be used in live cell studies. These features contribute to the increased sensitivity of this assay and make it a powerful new tool for discriminating between non-invasive and invasive cancer cell lines.

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Oligonucleotides targeted against a junction oncogene are made efficient by nanotechnologies.
Maksimenko A, Malvy C, Lambert G, Bertrand JR, Fattal E, Maccario J, Couvreur P.
Pharm Res. 2003 Oct;20(10):1565-7.
[ expand abstract ]

PURPOSE: Antisense oligonucleotides (AON) against junction EWS-Fli-1 oncogene (which is responsible for the Ewing Sarcoma) are particularly interesting for targeting chromosomal translocations that are only found in tumor cells. However, these AON have proved in the past to be ineffective in vivo because of their susceptibility to degradation and their poor intracellular penetration. The aim of this study was to improve the delivery of these molecules through the use of nanotechnologies. METHOD: Two different AONs, and their controls, both targeted against the junction area of the fusion gene EWS-Fli-1 were used. Nanocapsules were employed to deliver a phosphorothioate AON and its control. The nanospheres were used to deliver a chimeric phosphorothioate, phosphodiester AON, with 5 additional bases in 5' which allow this AON to be structured with a loop. These formulations were injected intratumorally to nude mice bearing the experimental EWS-Fli-1 tumor. The tumour volume was estimated during the experiments by two perpendicular measurements length (a) and width (b) of the tumour and was calculated as ab(2)/2. Northern blot analysis was also performed after removing the tumors 24 h after the treatment with a single dose of AON either free or associated with nanotechnologies. RESULTS: This study shows for the first time that AON against EWS-Fli-1 oncogene may inhibit with high specificity the growth of an EWS-Fli-1 dependent tumor grafted to nude mice provided they are delivered by nanocapsules or nanospheres. In this experience, the antisense effect was confirmed by the specific down regulation of EWS-Fli-1 mRNA. CONCLUSION: Thus, both nanocapsules and nanospheres may be considered as promising systems for AON delivery in vivo.

Effects of hydroxyapatite nanoparticles on proliferation and apoptosis of human hepatoma BEL-7402 cells.
Liu ZS, Tang SL, Ai ZL.
World J Gastroenterol. 2003 Sep;9(9):1968-71.
[ expand abstract ]

AIM: To study the effect of hydroxyapatite (HAP) nanoparticles on human hepatoma cell line BEL-7402 in vitro. METHODS: The human hepatoma cell line BEL-7402 was cultured and treated with HAP nanoparticles at various concentrations. Growth suppression was detected with MTT colorimetric assay, cell apoptotic alterations were evaluated by cytochemical staining (Hoechst 33258), transmission electron microscopy (TEM), and flow cytometry (FCM). RESULTS: HAP nanoparticles inhibited the growth of hepatoma cells in a dose-dependent manner, with IC(50) values of 29.30 mg/L. Treated with 50-200 mg/L HAP nanoparticles for 48 h, BEL-7402 cells apoptosis with nuclear chromatin condensation and fragmentation as well as cell shrinkage and the formation of apoptotic bodies were observed under cytochemical staining and transmission electron microscopy. FCM analysis showed hypodiploid peaks on histogram, the apoptotic rates at the concentrations of 50, 75, 100, 150 and 200 mg/L of HAP nanoparticles were 20.35+/-2.23 %, 25.35+/-1.92 %, 29.34+/-4.61 %, 44.92+/-3.78 % and 53.64+/-3.49 %, respectively, which were all significantly higher than that of control group 2.23+/-0.14 %. There was a significant correlation between HAP nanoparticle concentration and apoptotic rate (r=0.994, P<0.01). CONCLUSION: HAP nanoparticles not only inhibit proliferation but also induce apoptosis of human hepatoma cell line BEL-7402 in vitro.

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Ratiometric optical PEBBLE nanosensors for real-time magnesium ion concentrations inside viable cells.
Park EJ, Brasuel M, Behrend C, Philbert MA, Kopelman R.
Anal Chem. 2003 Aug 1;75(15):3784-91.
[ expand abstract ]

This paper presents the development and characterization of a highly selective magnesium fluorescent optical nanosensor, made possible by PEBBLE (probe encapsulated by biologically localized embedding) technology. A ratiometric sensor has been developed by co-immobilizing a dye that is sensitive to and highly selective for magnesium, with a reference dye in a matrix. The sensors are prepared via a microemulsion polymerization process, which entraps the sensing components inside a polymer matrix. The resultant spherical sensors are approximately 40 nm in diameter. The Coumarin 343 (C343) dye, which by itself does not enter the cell, when immobilized in a PEBBLE is used as the magnesium-selective agent that provides the high and necessary selectivity over other intracellular ions, such as Ca2+, Na+, and K+. The dynamic range of these sensors was 1-30 mM, with a linear range from 1 to 10 mM, with a response time of <4 s. In contrast to free dye, these nano-optodes are not perturbed by proteins. They are fully reversible and exhibit minimal leaching and photobleaching over extended periods of time. In vitro intracellular changes in Mg2+ concentration were monitored in C6 glioma cells, which remained viable after PEBBLE delivery via gene gun injection. The selectivity for Mg2+ along with the biocompatibility of the matrix provides a new and reliable tool for intracellular magnesium measurements.

Collection of trace amounts of DNA/mRNA molecules using genomagnetic nanocapturers.
Zhao X, Tapec-Dytioco R, Wang K, Tan W.
Anal Chem. 2003 Jul 15;75(14):3476-83.
[ expand abstract ]

The collection and then the separation of rare DNA/mRNA targets with single-base mismatches in a complex matrix is critically important in human disease diagnostics, gene expression studies, and gene profiling. The major result of this work is the development and application of a novel genomagnetic nanocapturer (GMNC) for the collection, separation, and detection of trace amounts of DNA/RNA molecules with one single-base difference. The GMNC is constructed by bioconjugating molecular beacon DNA probes onto magnetic nanoparticle surfaces. We have successfully applied the GMNC in artificial buffer solution samples and in cancer cell samples, both containing different proteins and random DNA sequences. Our method has three distinctly useful features: highly efficient collection of trace amount of DNA/mRNA samples down to femtomolar (10(-15) M) concentrations; excellent ability to differentiate single-base-mismatched DNA/mRNA samples by combining the exceptional specificity of molecular beacons and the separation power of magnetic nanoparticles; and real-time monitoring and confirmation of the collected gene products. The newly developed genomagnetic nanocapturers will be highly useful for the collection of trace amounts of DNA/mRNA targets in a variety of sample sources in forensic, medical, and biotechnological fields.

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Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots.
Wu X, Liu H, Liu J, Haley KN, Treadway JA, Larson JP, Ge N, Peale F, Bruchez MP.
Nat Biotechnol. 2003 Jan;21(1):41-6.
[ expand abstract ]

Semiconductor quantum dots (QDs) are among the most promising emerging fluorescent labels for cellular imaging. However, it is unclear whether QDs, which are nanoparticles rather than small molecules, can specifically and effectively label molecular targets at a subcellular level. Here we have used QDs linked to immunoglobulin G (IgG) and streptavidin to label the breast cancer marker Her2 on the surface of fixed and live cancer cells, to stain actin and microtubule fibers in the cytoplasm, and to detect nuclear antigens inside the nucleus. All labeling signals are specific for the intended targets and are brighter and considerably more photostable than comparable organic dyes. Using QDs with different emission spectra conjugated to IgG and streptavidin, we simultaneously detected two cellular targets with one excitation wavelength. The results indicate that QD-based probes can be very effective in cellular imaging and offer substantial advantages over organic dyes in multiplex target detection.

Microphysiological testing for chemosensitivity of living tumor cells with multiparametric microsensor chips.
Otto AM, Brischwein M, Niendorf A, Henning T, Motrescu E, Wolf B.
Cancer Detect Prev. 2003;27(4):291-6.
[ expand abstract ]

A constraint in the reliability of predictive chemosensitivity assays is linked to the fact that they analyze only a single cellular or biochemical parameter. A multiparametric test system using microsensor chips has been developed which can detect online microphysiological changes in living cells. Tumor cells were grown directly on glass- or silicon-based electronic sensor chips. Changes in extracellular pH and pO(2), reflecting metabolic activities, and changes in impedance, reflecting morphological properties, were monitored. In this study, colon and breast cancer cells as well as doxorubicin-sensitive and doxorubicin-resistant sarcoma cell lines were exposed to cytochalasin B, chloroacetaldehyde, or doxorubicin. Results show (1) reduction in medium acidification, (2) marked and rapid changes in O(2) consumption, and (3) modulations in impedance correlating with morphological changes observed in the microscope. Drug-resistant cells do not show these changes. Therefore, this microphysiological monitoring is a versatile tool for chemosensitivity testing of tumor cells.

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Nanosensor for in vivo measurement of the carcinogen benzo[a]pyrene in a single cell.
Kasili PM, Cullum BM, Griffin GD, Vo-Dinh T.
J Nanosci Nanotechnol.2002 Dec;2(6):653-8.
[ expand abstract ]

This work describes the fabrication and the application of an antibody-based fiber-optic nanosensor for in situ measurements of the carcinogen benzo[a]pyrene (BaP) in a single cell. This antibody-based spectroscopic nanosensor is miniaturized enabling the detection of fluorescent analytes in single cells. In addition to measuring fluorescent analytes in single cells, the nanosensor has the potential to be applied for both diagnostic and proteomics purposes. In this work, the human breast carcinoma cell line, MCF-7, was used as the model system to perform BaP measurements in single cells. A standard concentration curve for BaP was established and used to perform quantitative analyses of BaP in individual cells. From these analyses, it was estimated that the concentration of BaP in the individual cells investigated was approximately 3.61 x 10(-10) M. The results obtained demonstrate the application of antibody-based nanosensors for performing in situ measurements inside a single cell.

Structures of the cancer-related Aurora-A, FAK, and EphA2 protein kinases from nanovolume crystallography.
Nowakowski J, Cronin CN, McRee DE, Knuth MW, Nelson CG, Pavletich NP, Rogers J, Sang BC, Scheibe DN, Swanson RV, Thompson DA.
Structure (Camb). 2002 Dec;10(12):1659-67.
[ expand abstract ]

Protein kinases are important drug targets in human cancers, inflammation, and metabolic diseases. This report presents the structures of kinase domains for three cancer-associated protein kinases: ephrin receptor A2 (EphA2), focal adhesion kinase (FAK), and Aurora-A. The expression profiles of EphA2, FAK, and Aurora-A in carcinomas suggest that inhibitors of these kinases may have inherent potential as therapeutic agents. The structures were determined from crystals grown in nanovolume droplets, which produced high-resolution diffraction data at 1.7, 1.9, and 2.3 A for FAK, Aurora-A, and EphA2, respectively. The FAK and Aurora-A structures are the first determined within two unique subfamilies of human kinases, and all three structures provide new insights into kinase regulation and the design of selective inhibitors.

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Nanometer-scale pores: potential applications for analyte detection and DNA characterization.
Kasianowicz JJ.
Dis Markers. 2002;18(4):185-91.
[ expand abstract ]

Several classes of transmembrane protein ion channels function in vivo as sensitive and selective detection elements for analytes. Recent studies on single channels reconstituted into planar lipid bilayer membranes suggest that nanometer-scale pores can be used to detect, quantitate and characterize a wide range of analytes that includes small ions and single stranded DNA. We briefly review here these studies and identify leaps in technology that, if realized, might lead to innovations for the early detection of cancer.