Mechanochemical activation and patterning of an adhesive surface toward nanoparticle deposition.

Mechanical pulling of adhesive tape creates radicals on the tape's surface. These radicals are capable of reducing metal salts to the corresponding metal nanoparticles. In this way, the mechanically activated tape can be decorated with various types of nanoparticles, including Au, Ag, Pd, or Cu. While retaining their mechanical properties and remaining "sticky," the tapes can exhibit new properties derived from the presence of metal nanoparticles (e.g., bacteriostaticity, increased electrical conductivity). They can also be patterned with nanoparticles only at selective locations of mechanical activation.

Microtubule guidance tested through controlled cell geometry.

In moving cells dynamic microtubules (MTs) target and disassemble substrate adhesion sites (focal adhesions; FAs) in a process that enables the cell to detach from the substrate and propel itself forward. The short-range interactions between FAs and MT plus ends have been observed in several experimental systems, but the spatial overlap of these structures within the cell has precluded analysis of the putative long-range mechanisms by which MTs growing through the cell body reach FAs in the periphery of the cell. In the work described here cell geometry was controlled to remove the spatial overlap of cellular structures thus allowing for unambiguous observation of MT guidance. Specifically, micropatterning of living cells was combined with high-resolution in-cell imaging and gene product depletion by means of RNA interference to study the long-range MT guidance in quantitative detail. Cells were confined on adhesive triangular microislands that determined cell shape and ensured that FAs localized exclusively at the vertices of the triangular cells. It is shown that initial MT nucleation at the centrosome is random in direction, while the alignment of MT trajectories with the targets (i.e. FAs at vertices) increases with an increasing distance from the centrosome, indicating that MT growth is a non-random, guided process. The guided MT growth is dependent on the presence of FAs at the vertices. The depletion of either myosin IIA or myosin IIB results in depletion of F-actin bundles and spatially unguided MT growth. Taken together our findings provide quantitative evidence of a role for long-range MT guidance in MT targeting of FAs.

Controlled pH stability and adjustable cellular uptake of mixed-charge nanoparticles.

Nanoparticles functionalized with mixed self-assembled monolayers (m-SAMs) comprising positively and negatively charged thiols are stable at both low and high pH but precipitate sharply at the pH where the charges on the particle are balanced (pH(prec)). By adjusting the proportion of the positively and negatively charged ligands in the m-SAM or changing particle size, pH(prec) can be varied flexibly between ~4 and ~7. In addition, changes in the SAMs' composition and particles' net charge translate into different degrees of cellular uptake. Remarkably, the presence of the positively charged thiols allows for the uptake of particles having net negative charge.

Carboxybetaine methacrylate polymers offer robust, long-term protection against cell adhesion.

Films of poly(carboxybetaine methacrylate), poly(CBMA), grafted onto microetched gold slides are effective in preventing nonspecific adhesion of cells of different types. The degree of adhesion resistance is comparable to that achieved with the self-assembled monolayers, SAMs, of oligo(ethylene glycol) alkanethiolates. In sharp contrast to the SAMs, however, substrates protected with poly(CBMA) can be stored in dry state without losing their protective properties for periods up to 2 weeks.

Lévy-like movement patterns of metastatic cancer cells revealed in microfabricated systems and implicated in vivo.

Metastatic cancer cells differ from their non-metastatic counterparts not only in terms of molecular composition and genetics, but also by the very strategy they employ for locomotion. Here, we analyzed large-scale statistics for cells migrating on linear microtracks to show that metastatic cancer cells follow a qualitatively different movement strategy than their non-invasive counterparts. The trajectories of metastatic cells display clusters of small steps that are interspersed with long "flights". Such movements are characterized by heavy-tailed, truncated power law distributions of persistence times and are consistent with the Lévy walks that are also often employed by animal predators searching for scarce prey or food sources. In contrast, non-metastatic cancerous cells perform simple diffusive movements. These findings are supported by preliminary experiments with cancer cells migrating away from primary tumors in vivo. The use of chemical inhibitors targeting actin-binding proteins allows for "reprogramming" the Lévy walks into either diffusive or ballistic movements.

Microfabricated Systems and Assays for Studying the Cytoskeletal Organization, Micromechanics, and Motility Patterns of Cancerous Cells.

Cell motions are driven by coordinated actions of the intracellular cytoskeleton - actin, microtubules (MTs) and substrate/focal adhesions (FAs). This coordination is altered in metastatic cancer cells resulting in deregulated and increased cellular motility. Microfabrication tools, including photolithography, micromolding, microcontact printing, wet stamping and microfluidic devices have emerged as a powerful set of experimental tools with which to probe and define the differences in cytoskeleton organization/dynamics and cell motility patterns in non-metastatic and metastatic cancer cells. In this review, we discuss four categories of microfabricated systems: (i) micropatterned substrates for studying of cell motility sub-processes (for example, MT targeting of FAs or cell polarization); (ii) systems for studying cell mechanical properties, (iii) systems for probing overall cell motility patterns within challenging geometric confines relevant to metastasis (for example, linear and ratchet geometries), and (iv) microfluidic devices that incorporate co-cultures of multiple cells types and chemical gradients to mimic in vivo intravasation/extravasation steps of metastasis. Together, these systems allow for creating controlled microenvironments that not only mimic complex soft tissues, but are also compatible with live cell high-resolution imaging and quantitative analysis of single cell behavior.

Motility efficiency and spatiotemporal synchronization in non-metastatic vs. metastatic breast cancer cells.

Metastatic breast cancer cells move not only more rapidly and persistently than their non-metastatic variants but in doing so use the mechanical work of the cytoskeleton more efficiently. The efficiency of the cell motions is defined for entire cells (rather than parts of the cell membrane) and is related to the work expended in forming membrane protrusions and retractions. This work, in turn, is estimated by integrating the protruded and retracted areas along the entire cell perimeter and is standardized with respect to the net translocation of the cell. A combination of cross-correlation, Granger causality, and morphodynamic profiling analyses is then used to relate the efficiency to the cell membrane dynamics. In metastatic cells, the protrusions and retractions are highly "synchronized" both in space and in time and these cells move efficiently. In contrast, protrusions and retractions formed by non-metastatic cells are not "synchronized" corresponding to low motility efficiencies. Our work provides a link between the kinematics of cell motions and their energetics. It also suggests that spatiotemporal synchronization might be one of the hallmarks of invasiveness of cancerous cells.

Tomography and static-mechanical properties of adherent cells.

A tomography approach is used to reconstruct 3D cell shapes and, simultaneously, the shapes/positions of the nuclei within these cells. Subjecting the cells to well-defined microconfinements of various diameters allow for relating the steady-state shapes of cells to their static-mechanical properties. The observed shapes show striking regularities between different cell types and all fit to a model that takes into account the cell membrane, cortical actin, and the nucleus.

Antibacterial nanoparticle monolayers prepared on chemically inert surfaces by cooperative electrostatic adsorption (CELA).

Cooperative electrostatic adsorption (CELA) is used to deposit monolayer coatings of silver nanoparticles on relatively chemically inert polymers, polypropylene, and Tygon. Medically relevant components (tubing, vials, syringes) coated by this method exhibit antibacterial properties over weeks to months with the coatings being stable under constant-flow conditions. Antibacterial properties of the coatings are due to a slow release of Ag(+) from the particles. The rate of this release is quantified by the dithiol-precipitation method coupled with inductively coupled plasma optical emission spectrometer (ICP-OES) analysis.

Nanoparticle-based solution deposition of gold films supporting bioresistant SAMs.

Thin films of gold on glass are prepared by solution deposition of functionalized gold nanoparticles followed by thermal treatment. The processed films adhere strongly to glass without any adhesion layers and can be micropatterned/microetched without delamination from the substrate. The formation of self-assembled monolayers (SAMs) of oligo(ethylene glycol) alkane thiols (EG SAMs) renders the films resistant to cell adhesion and allows for cell patterning.

The chemopreventive bioflavonoid apigenin inhibits prostate cancer cell motility through the focal adhesion kinase/Src signaling mechanism.

Prostate cancer mortality is primarily attributed to metastatic rather than primary, organ-confined disease. Acquiring a motile and invasive phenotype is an important step in development of tumors and ultimately metastasis. This step involves remodeling of the extracellular matrix and of cell-matrix interactions, cell movement mediated by the actin cytoskeleton, and activation of focal adhesion kinase (FAK)/Src signaling. Epidemiologic studies suggest that the metastatic behavior of prostate cancer may be an ideal target for chemoprevention. The natural flavone apigenin is known to have chemopreventive properties against many cancers, including prostate cancer. Here, we study the effect of apigenin on motility, invasion, and its mechanism of action in metastatic prostate carcinoma cells (PC3-M). We found that apigenin inhibits PC3-M cell motility in a scratch-wound assay. Live cell imaging studies show that apigenin diminishes the speed and affects directionality of cell motion. Alterations in the cytoskeleton are consistent with impaired cell movement in apigenin-treated cells. Apigenin treatment leads to formation of "exaggerated filopodia," which show accumulation of focal adhesion proteins at their tips. Furthermore, apigenin-treated cells adhere more strongly to the extracellular matrix. Additionally, apigenin decreases activation of FAK and Src, and phosphorylation of Src substrates FAK Y576/577 and Y925. Expression of constitutively active Src blunts the effect of apigenin on cell motility and cytoskeleton remodeling. These results show that apigenin inhibits motility and invasion of prostate carcinoma cells, disrupts actin cytoskeleton organization, and inhibits FAK/Src signaling. These studies provide mechanistic insight into developing novel strategies for inhibiting prostate cancer cell motility and invasiveness.

Characterization and optimization of gold nanoparticle-based silver-enhanced immunoassays.

Silver-enhanced nanoparticle-labeled immunoassays provide a simple, low-cost, and effective way of detecting antigens in dilute solutions. The physical mechanisms behind their operation, however, have not been fully investigated. We present a semiquantitative approach for optimizing sandwich nanoparticle immunoassays using an adsorption-controlled kinetic model. Primary antibodies were immobilized on a solid substrate to bind the target antigens in solution. An optical signal was measured by secondary labeling of antigens with gold nanoparticles and their enhancement by silver nucleation. The opacity of the silver-enhanced spots was quantified by densitometry. The selectivity of the sandwich immunoassays was adequately high, and antigen concentrations as low as 0.1 microg cm(-3) (4 ng total) were detected reproducibly. The role of mass transfer was investigated, and a model was developed to optimize the performance of immunoassays by correlating the opacities of silver spots to the concentration and incubation times of antigens and gold nanoparticles. The results could allow the development of more rapid and reliable nanoparticle immunoassays.