Statistical Copolymers that Mimic Aspects of Mussel Adhesive Proteins: Access to Robust Adhesive-Domains for Non-Covalent Surface PEGylation.

Reconstructing functional sequence motifs of proteins, using statistical copolymers greatly reduces the information content, but simplifies synthesis significantly. Key amino acid residues involved in the adhesion of mussel foot proteins are identified. The side-chain functionalities of Dopa, lysine, and arginine are abstracted and incorporated into acrylate monomers to allow controlled radical polymerization. The resulting Dopa-acrylate (Y*-acr), arginine-acrylate (R-acr), and lysine-acrylate (K-acr) monomers are polymerized in different monomer ratios and compositions by reversible addition fragmentation transfer polymerization with a poly(ethylene glycol) (PEG) macrochain transfer agent. This results in two sets of PEG-block-copolymers with statistical mixtures and different monomer ratios of catechol/primary amine and catechol/guanidine side-chain functionalities, both important pairs for mimicking π-cation interactions. The coating behavior of these PEG-block-copolymers is evaluated using quartz crystal microbalance with dissipation energy monitoring (QCM-D), leading to non-covalent PEGylation of the substrates with clear compositional optima in the coating stability and antifouling properties. The coatings prevent non-reversible albumin or serum adsorption, as well as reduce cellular adhesion and fungal spore attachment.

Photothermal Control of Heat-Shock Protein Expression at the Single Cell Level.

Laser heating of individual cells in culture recently led to seminal studies in cell poration, fusion, migration, or nanosurgery, although measuring the local temperature increase in such experiments remains a challenge. Here, the laser-induced dynamical control of the heat-shock response is demonstrated at the single cell level, enabled by the use of light-absorbing gold nanoparticles as nanosources of heat and a temperature mapping technique based on quadriwave lateral shearing interferometry (QLSI) measurements. As it is label-free, this approach does not suffer from artifacts inherent to previously reported fluorescence-based temperature-mapping techniques and enables the use of any standard fluorescent labels to monitor in parallel the cell's response.

Adhesion forces and cortical tension couple cell proliferation and differentiation to drive epidermal stratification.

To establish and maintain organ structure and function, tissues need to balance stem cell proliferation and differentiation rates and coordinate cell fate with position. By quantifying and modelling tissue stress and deformation in the mammalian epidermis, we find that this balance is coordinated through local mechanical forces generated by cell division and delamination. Proliferation within the basal stem/progenitor layer, which displays features of a jammed, solid-like state, leads to crowding, thereby locally distorting cell shape and stress distribution. The resulting decrease in cortical tension and increased cell-cell adhesion trigger differentiation and subsequent delamination, reinstating basal cell layer density. After delamination, cells establish a high-tension state as they increase myosin II activity and convert to E-cadherin-dominated adhesion, thereby reinforcing the boundary between basal and suprabasal layers. Our results uncover how biomechanical signalling integrates single-cell behaviours to couple proliferation, cell fate and positioning to generate a multilayered tissue.

Light-Assisted Solvothermal Chemistry Using Plasmonic Nanoparticles.

Solvothermal synthesis, denoting chemical reactions occurring in metastable liquids above their boiling point, normally requires the use of a sealed autoclave under pressure to prevent the solvent from boiling. This work introduces an experimental approach that enables solvothermal synthesis at ambient pressure in an open reaction medium. The approach is based on the use of gold nanoparticles deposited on a glass substrate and acting as photothermal sources. To illustrate the approach, the selected hydrothermal reaction involves the formation of indium hydroxide microcrystals favored at 200 °C in liquid water. In addition to demonstrating the principle, the benefits and the specific characteristics of such an approach are investigated, in particular, the much faster reaction rate, the achievable spatial and time scales, the effect of microscale temperature gradients, the effect of the size of the heated area, and the effect of thermal-induced microscale fluid convection. This technique is general and could be used to spatially control the deposition of virtually any material for which a solvothermal synthesis exists.

Fabrication of micropatterned arrays of gold nanoparticles for photothermal manipulation of living cells.

The fabrication of micro/nanostructured surfaces functionalized with stimulus-responsive chemical groups proved to be an interesting approach to simultaneously confine cell adhesion and manipulate cell-substrate interactions down to the single cell level. However, reversibility of stimulus-triggered systems is often not possible or exhibits slow switching kinetics. In contrast to such setups, gold nanoparticles have the properties to efficiently and reversibly generate heat under illumination at their plasmon resonance band. Thus, photo-induced heating could be used to directly and locally interface living cells and dynamically tailor the interactions to their adhesive environment. In the present chapter, we will first detail the preparation of micropatterned and functionalized gold nanoparticles immobilized on glass coverslips, and then report how to reliably characterize the photothermal properties of such substrates that enable the dynamic manipulation of cells.

Photoinduced heating of nanoparticle arrays.

The temperature distribution throughout arrays of illuminated metal nanoparticles is investigated numerically and experimentally. The two cases of continuous and femtosecond-pulsed illumination are addressed. In the case of continuous illumination, two distinct regimes are evidenced: a temperature confinement regime, where the temperature increase remains confined at the vicinity of each nanosource of heat, and a temperature delocalization regime, where the temperature is uniform throughout the whole nanoparticle assembly despite the heat sources' nanometric size. We show that the occurrence of one regime or another simply depends on the geometry of the nanoparticle distribution. In particular, we derived (i) simple expressions of dimensionless parameters aimed at predicting the degree of temperature confinement and (ii) analytical expressions aimed at estimating the actual temperature increase at the center of an assembly of nanoparticles under illumination, preventing heavy numerical simulations. All these theoretical results are supported by experimental measurements of the temperature distribution on regular arrays of gold nanoparticles under illumination. In the case of femtosecond-pulsed illumination, we explain the two conditions that must be fulfilled to observe a further enhanced temperature spatial confinement.

A molecular toolkit for the functionalization of titanium-based biomaterials that selectively control integrin-mediated cell adhesion.

We present a click chemistry-based molecular toolkit for the biofunctionalization of materials to selectively control integrin-mediated cell adhesion. To this end, α5ß1-selective RGD peptidomimetics were covalently immobilized on Ti-based materials, and the capacity to promote the selective binding of α5ß1 was evaluated using a solid-phase integrin binding assay. This functionalization strategy yielded surfaces with a nine-fold increased affinity for α5ß1, in comparison to control samples, and total selectivity against the binding of the closely related integrin αvß3. Moreover, our methodology allowed the screening of several phosphonic acid containing anchoring units to find the best spacer-anchor moiety required for establishing an efficient binding to titanium and to promote selective integrin binding. The integrin subtype specificity of these biofunctionalized surfaces was further examined in vitro by inducing selective adhesion of genetically modified fibroblasts, which express exclusively the α5ß1 integrin. The versatility of our molecular toolkit was proven by shifting the cellular specificity of the materials from α5ß1- to αvß3-expressing fibroblasts by using an αvß3-selective peptidomimetic as coating molecule. The results shown here represent the first functionalization of Ti-based materials with α5ß1- or αvß3-selective peptidomimetics that allow an unprecedented control to discriminate between α5ß1- and αvß3-mediated adhesions. The role of these two integrins in different biological events is still a matter of debate and is frequently discussed in literature. Thus, such bioactive titanium surfaces will be of great relevance for the study of integrin-mediated cell adhesion and the development of new biomaterials targeting specific cell types.

ß1- and αv-class integrins cooperate to regulate myosin II during rigidity sensing of fibronectin-based microenvironments.

How different integrins that bind to the same type of extracellular matrix protein mediate specific functions is unclear. We report the functional analysis of ß1- and αv-class integrins expressed in pan-integrin-null fibroblasts seeded on fibronectin. Reconstitution with ß1-class integrins promotes myosin-II-independent formation of small peripheral adhesions and cell protrusions, whereas expression of αv-class integrins induces the formation of large focal adhesions. Co-expression of both integrin classes leads to full myosin activation and traction-force development on stiff fibronectin-coated substrates, with αv-class integrins accumulating in adhesion areas exposed to high traction forces. Quantitative proteomics linked αv-class integrins to a GEF-H1-RhoA pathway coupled to the formin mDia1 but not myosin II, and α5ß1 integrins to a RhoA-Rock-myosin II pathway. Our study assigns specific functions to distinct fibronectin-binding integrins, demonstrating that α5ß1integrins accomplish force generation, whereas αv-class integrins mediate the structural adaptations to forces, which cooperatively enable cells to sense the rigidity of fibronectin-based microenvironments.

Micropatterning thermoplasmonic gold nanoarrays to manipulate cell adhesion.

The ability to reversibly control the interactions between the extracellular matrix (ECM) and cell surface receptors such as integrins would allow one to investigate reciprocal signaling circuits between cells and their surrounding environment. Engineering microstructured culture substrates functionalized with switchable molecules remains the most adopted strategy to manipulate surface adhesive properties, although these systems exhibit limited reversibility and require sophisticated preparation procedures. Here, we report a straightforward protocol to fabricate biofunctionalized micropatterned gold nanoarrays that favor one-dimensional cell migration and function as plasmonic nanostoves to physically block and orient the formation of new adhesion sites. Being reversible and not restricted spatiotemporally, thermoplasmonic approaches will open new opportunities to further study the complex connections between ECM and cells.

Thermal imaging of nanostructures by quantitative optical phase analysis.

We introduce an optical microscopy technique aimed at characterizing the heat generation arising from nanostructures, in a comprehensive and quantitative manner. Namely, the technique permits (i) mapping the temperature distribution around the source of heat, (ii) mapping the heat power density delivered by the source, and (iii) retrieving the absolute absorption cross section of light-absorbing structures. The technique is based on the measure of the thermal-induced refractive index variation of the medium surrounding the source of heat. The measurement is achieved using an association of a regular CCD camera along with a modified Hartmann diffraction grating. Such a simple association makes this technique straightforward to implement on any conventional microscope with its native broadband illumination conditions. We illustrate this technique on gold nanoparticles illuminated at their plasmonic resonance. The spatial resolution of this technique is diffraction limited, and temperature variations weaker than 1 K can be detected.

Benzyl alcohol and block copolymer micellar lithography: a versatile route to assembling gold and in situ generated titania nanoparticles into uniform binary nanoarrays.

Simultaneous synthesis and assembly of nanoparticles that exhibit unique physicochemical properties are critically important for designing new functional devices at the macroscopic scale. In the present study, we report a simple version of block copolymer micellar lithography (BCML) to synthesize gold and titanium dioxide (TiO(2)) nanoarrays by using benzyl alcohol (BnOH) as a solvent. In contrast to toluene, BnOH can lead to the formation of various gold nanopatterns via salt-induced micellization of polystyrene-block-poly(vinylpyridine) (PS-b-P2VP). In the case of titania, the use of BCML with a nonaqueous sol-gel method, the "benzyl alcohol route", enables the fabrication of nanopatterns made of quasi-hexagonally organized particles or parallel wires upon aging a (BnOH-TiCl(4)-PS(846)-b-P2VP(171))-containing solution for four weeks to grow TiO(2) building blocks in situ. This approach was found to depend mainly on the relative lengths of the polymer blocks, which allows nanoparticle-induced micellization and self-assembly during solvent evaporation. Moreover, this versatile route enables the design of uniform and quasi-ordered gold-TiO(2) binary nanoarrays with a precise particle density due to the absence of graphoepitaxy during the deposition of TiO(2) onto gold nanopatterns.

Integrin-linked kinase controls microtubule dynamics required for plasma membrane targeting of caveolae.

Caveolae are specialized compartments of the plasma membrane that are involved in signaling, endocytosis, and cholesterol transport. Their formation requires the transport of caveolin-1 to the plasma membrane, but the molecular mechanisms regulating the transport are largely unknown. Here, we identify a critical role for adhesion-mediated signaling through ß1 integrins and integrin-linked kinase (ILK) in caveolae formation. Mice lacking ß1 integrins or ILK in keratinocytes have dramatically reduced numbers of plasma membrane caveolae in vivo, which is due to impaired transport of caveolin-1-containing vesicles along microtubules (MT) to the plasma membrane. Mechanistically, ILK promotes the recruitment of the F-actin binding protein IQGAP1 to the cell cortex, which, in turn, cooperates with its effector mDia1 to locally stabilize MTs and to allow stable insertion of caveolae into the plasma membrane. Our results assign an important role to the integrin/ILK complex for caveolar trafficking to the cell surface.

Immobilized chemokine fields and soluble chemokine gradients cooperatively shape migration patterns of dendritic cells.

Chemokines orchestrate immune cell trafficking by eliciting either directed or random migration and by activating integrins in order to induce cell adhesion. Analyzing dendritic cell (DC) migration, we showed that these distinct cellular responses depended on the mode of chemokine presentation within tissues. The surface-immobilized form of the chemokine CCL21, the heparan sulfate-anchoring ligand of the CC-chemokine receptor 7 (CCR7), caused random movement of DCs that was confined to the chemokine-presenting surface because it triggered integrin-mediated adhesion. Upon direct contact with CCL21, DCs truncated the anchoring residues of CCL21, thereby releasing it from the solid phase. Soluble CCL21 functionally resembles the second CCR7 ligand, CCL19, which lacks anchoring residues and forms soluble gradients. Both soluble CCR7 ligands triggered chemotactic movement, but not surface adhesion. Adhesive random migration and directional steering cooperate to produce dynamic but spatially restricted locomotion patterns closely resembling the cellular dynamics observed in secondary lymphoid organs.

Adaptive force transmission in amoeboid cell migration.

The leading front of a cell can either protrude as an actin-free membrane bleb that is inflated by actomyosin-driven contractile forces, or as an actin-rich pseudopodium, a site where polymerizing actin filaments push out the membrane. Pushing filaments can only cause the membrane to protrude if the expanding actin network experiences a retrograde counter-force, which is usually provided by transmembrane receptors of the integrin family. Here we show that chemotactic dendritic cells mechanically adapt to the adhesive properties of their substrate by switching between integrin-mediated and integrin-independent locomotion. We found that on engaging the integrin-actin clutch, actin polymerization was entirely turned into protrusion, whereas on disengagement actin underwent slippage and retrograde flow. Remarkably, accelerated retrograde flow was balanced by an increased actin polymerization rate; therefore, cell shape and protrusion velocity remained constant on alternating substrates. Due to this adaptive response in polymerization dynamics, tracks of adhesive substrate did not dictate the path of the cells. Instead, directional guidance was exclusively provided by a soluble gradient of chemoattractant, which endowed these 'amoeboid' cells with extraordinary flexibility, enabling them to traverse almost every type of tissue.

Templated nanocrystal-based porous TiO(2) films for next-generation electrochemical capacitors.

The advantages in using nanoscale materials for electrochemical energy storage are generally attributed to short diffusion path lengths for both electronic and lithium ion transport. Here, we consider another contribution, namely the charge storage from faradaic processes occurring at the surface, referred to as pseudocapacitive effect. This paper describes the synthesis and pseudocapacitive characteristics of block copolymer templated anatase TiO(2) thin films synthesized using either sol-gel reagents or preformed nanocrystals as building blocks. Both materials are highly crystalline and have large surface areas; however, the structure of the porosity is not identical. The different titania systems are characterized by a combination of small- and wide-angle X-ray diffraction/scattering, combined with SEM imaging and physisorption measurements. Following our previously reported approach, we are able to use the voltammetric sweep rate dependence to determine quantitatively the capacitive contribution to the current response. Considerable enhancement of the electrochemical properties results when the films are both made from nanocrystals and mesoporous. Such materials show high levels of capacitive charge storage and high insertion capacities. By contrast, when mesoscale porosity is created in a material with dense walls (rather than porous walls derived from the aggregation of nanocrystals), insertion capacities comparable to templated nanocrystal films can be achieved, but the capacitance is much lower. The results presented here illustrate the importance of pseudocapacitive behavior that develops in high surface area mesoporous oxide films. Such systems provide a new class of pseudocapacitive materials, which offer increased charge storage without compromising charge storage kinetics.

Hydrophobic surfaces for enhanced differentiation of embryonic stem cell-derived embryoid bodies.

With their unique ability to differentiate into all cell types, embryonic stem (ES) cells hold great therapeutic promise. To improve the efficiency of embryoid body (EB)-mediated ES cell differentiation, we studied murine EBs on the basis of their size and found that EBs with an intermediate size (diameter 100-300 microm) are the most proliferative, hold the greatest differentiation potential, and have the lowest rate of cell death. In an attempt to promote the formation of this subpopulation, we surveyed several biocompatible substrates with different surface chemical parameters and identified a strong correlation between hydrophobicity and EB development. Using self-assembled monolayers of various lengths of alkanethiolates on gold substrates, we directly tested this correlation and found that surfaces that exhibit increasing hydrophobicity enrich for the intermediate-size EBs. When this approach was applied to the human ES cell system, similar phenomena were observed. Our data demonstrate that hydrophobic surfaces serve as a platform to deliver uniform EB populations and may significantly improve the efficiency of ES cell differentiation.

Growth and assembly of crystalline tungsten oxide nanostructures assisted by bioligation.

Here, we present a systematic study on the influence of the bioligand deferoxamine mesylate on the crystallization and assembly behavior of tungsten oxide in a soft-chemistry process. Without deferoxamine mesylate, this approach yields pseudo-single crystalline tungstite nanoplatelets consisting of a large number of crystallographically almost perfectly aligned primary crystallites. In the presence of a constant amount of deferoxamine, the particle morphology drastically changes with temperature, ranging from wormlike organic-inorganic hybrid nanostructures to single-crystalline tungsten oxide nanowires, highlighting the role of the bioligand in controlling the crystal growth and assembly behavior. The nanowires have a uniform diameter of about 1.3 nm, an aspect ratio of more than 500, and the structural flexibility of tungsten oxide. The presented process is based on the combination of biomimetic construction principles with nonaqueous sol-gel chemistry, thus combining the advantages of both tools, excellent control over particle morphology and high crystallinity at low temperature.

Ligand functionality as a versatile tool to control the assembly behavior of preformed titania nanocrystals.

Nanoparticle powders composed of surface-functionalized anatase crystals with diameters of about 3 nm self-organize into different structures upon redispersion in water. The assembly is directed by a small amount of a low-molecular-weight functional ligand (the "assembler") adsorbed on the surface of the nanoparticles. The ligand functionality determines the anisotropy of the resulting structures. Multidentate ligands, such as trizma ((HOCH(2))(3)CNH(2)) and serinol ((HOCH(2))(2)CNH(2)), with a chargeable terminal group preferentially induce the formation of anisotropic nanostructures several hundreds of nanometers in total length, whereas all the other investigated ligands (ethanolamine H(2)N(CH(2))(2)OH, glycine hydroxamate H(2)NCH(2)CONHOH, dopamine (OH)(2)C(6)H(3)(CH(2))(2)NH(3)Cl, tris (HOCH(2))(3)CCH(3)) mainly lead to uncontrolled agglomeration. Experimental data suggests that the anisotropic assembly is a consequence of the water-promoted desorption of the organic ligands from the {001} faces of the crystalline building blocks together with the dissociative adsorption of water on these crystal faces. Both processes induce the preferred attachment of the titania nanoparticles along the [001] direction. The use of polydentate and charged ligands to functionalize the surface of nanoparticles thus provides a versatile tool to control their arrangement on the nanoscale.