Endothelial cell spreading on lipid bilayers with combined integrin and cadherin binding ligands.

Endothelial cells play a central role in the vascular system, where their function is tightly regulated by both cell-extracellular matrix (e.g., via integrins) and cell-cell interactions (e.g., via cadherins). In this study, we incorporated cholesterol-modified integrin and N-cadherin peptide binding ligands in fluid supported lipid bilayers. Human umbilical vein endothelial cell adhesion, spreading and vinculin localization in these cells were dependent on ligand density. One composition led to observe a higher extent of cell spreading, where cells exhibited extensive lamellipodia formation and a qualitatively more distinct N-cadherin localization at the cell periphery, which is indicative of N-cadherin clustering and a mimic of cell-cell contact formation. The results can be used to reconstitute the endothelial-pericyte interface on biomedical devices and materials.

Re- and Preconfigurable Multistable Visible Light Responsive Surface Topographies.

Light responsive materials that are able to change their shape are becoming increasingly important. However, preconfigurable bistable or even multi-stable visible light responsive coatings have not been reported yet. Such materials will require less energy to actuate and will have a longer lifetime. Here, it is shown that fluorinated azobenzenes can be used to create rewritable and pre-configurable responsive surfaces that show multi-stable topographies. These surface structures can be formed and removed by using low intensity green and blue light, respectively. Multistable preconfigured surface topographies can also be created in the absence of a mask. The method allows for full control over the surface structures as the topographical changes are directly linked to the molecular isomerization processes. Preliminary studies reveal that these light responsive materials are suitable as adaptive biological surfaces.

Photo-responsive Bioactive Surfaces Based on Cucurbit[8]uril-Mediated Host-Guest Interactions of Arylazopyrazoles.

A photoswitchable arylazopyrazole (AAP) derivative binds with cucurbit[8]uril (CB[8]) and methylviologen (MV2+ ) to form a 1:1:1 heteroternary host-guest complex with a binding constant of Ka =2×103 m-1 . The excellent photoswitching properties of AAP are preserved in the inclusion complex. Irradiation with light of a wavelength of 365 and 520 nm leads to quantitative E- to Z- isomerization and vice versa, respectively. Formation of the Z-isomer leads to dissociation of the complex as evidenced using 1 H NMR spectroscopy. AAP derivatives are then used to immobilize bioactive molecules and photorelease them on demand. When Arg-Gly-Asp-AAP (AAP-RGD) peptides are attached to surface bound CB[8]/MV2+ complexes, cells adhere and can be released upon irradiation. The heteroternary host-guest system offers highly reversible binding properties due to efficient photoswitching and these properties are attractive for designing smart surfaces.

Hydrolytically Labile Linkers Regulate Release and Activity of Human Bone Morphogenetic Protein-6.

Release of growth factors while simultaneously maintaining their full biological activity over a period of days to weeks is an important issue in controlled drug delivery and in tissue engineering. In addition, the selected strategy to immobilize growth factors largely determines their biological activity. Silica surfaces derivatized with glycidyloxy propyl trimethoxysilane and poly(glycidyl methacrylate) brushes yielded epoxide-functionalized surfaces onto which human bone morphogenetic protein-6 (hBMP-6) was immobilized giving stable secondary amine bonds. The biological activity of hBMP-6 was unleashed by hydrolysis of the surface siloxane and ester bonds. We demonstrate that this type of labile bonding strategy can be applied to biomaterial surfaces with relatively simple and biocompatible chemistry, such as siloxane, ester, and imine bonds. Our data indicates that the use of differential hydrolytically labile linkers is a versatile method for functionalization of biomaterials with a variety of growth factors providing control over their biological activity.

Electron Transfer Mediated by Surface-Tethered Redox Groups in Nanofluidic Devices.

Electrochemistry provides a powerful sensor transduction and amplification mechanism that is highly suited for use in integrated, massively parallelized assays. Here, the cyclic voltammetric detection of flexible, linear poly(ethylene glycol) polymers is demonstrated, which have been functionalized with redox-active ferrocene (Fc) moieties and surface-tethered inside a nanofluidic device consisting of two microscale electrodes separated by a gap of <100 nm. Diffusion of the surface-bound polymer chains in the aqueous electrolyte allows the redox groups to repeatedly shuttle electrons from one electrode to the other, resulting in a greatly amplified steady-state electrical current. Variation of the polymer length provides control over the current, as the activity per Fc moiety appears to depend on the extent to which the polymer layers of the opposing electrodes can interpenetrate each other and thus exchange electrons. These results outline the design rules for sensing devices that are based on changing the polymer length, flexibility, and/or diffusivity by binding an analyte to the polymer chain. Such a nanofluidic enabled configuration provides an amplified and highly sensitive alternative to other electrochemical detection mechanisms.

Assessment of Cooperativity in Ternary Peptide-Cucurbit[8]uril Complexes.

Evaluating cooperativity for cucurbit[8]uril (CB[8])-mediated ternary complexation is required for understanding and advancing designs of such ternary self-assembled systems. A key issue is to dissect the contributions of the binding steps of the first and second guest molecules to the overall ternary complex formation energy. This is addressed by performing concentration-dependent titrations between CB[8] and guests by means of concentration-dependent calorimetric and 1 H-NMR titrations. The sensitivity of the fitting of the cumulative heat of complexation of the calorimetric titrations is evaluated in terms of fitting error and enthalpy-entropy compensation and, together with the NMR spectroscopic analysis of the separate species, non-cooperative binding is conceived to be the most probable binding scenario. The binding behavior of CB[8] homoternary complexes is similar to CB[8] heteroternary complexes, with an enthalpy-driven tight fit of the guests in the CB[8] cavity overcoming the entropic penalty. Also for these types of complexes, a non-cooperative binding is the most probable.

Electron Transfer Processes in Ferrocene-Modified Poly(ethylene glycol) Monolayers on Electrodes.

Electrochemistry is a powerful tool to study self-assembled monolayers. Here, we modified cystamine-functionalized electrodes with different lengths of linear poly(ethylene glycol) (PEG) polymers end-functionalized with a redox-active ferrocene (Fc) group. The electron transport properties of the Fc probes were studied using cyclic voltammetry. The Fc moiety attached to the shortest PEG (Mn = 250 Da) behaved as a surface-confined species, and the homogeneous electron transfer rate constants were determined. The electron transfer of the ferrocene group on the longer PEGs (Mn = 3.4, 5, and 10 kDa) was shown to be driven by diffusion. For low surface densities, where the polymer exists in the mushroom conformation, the diffusion coefficients (D) and rate constants were increasing with polymer length. In the loose brush conformation, where the polymers are close enough to interact with each other, the thickness of the layers (e) was unknown and a parameter D1/2/e was determined. This parameter showed no dependence on surface density and an increase with polymer length.

Cell Adhesion on RGD-Displaying Knottins with Varying Numbers of Tryptophan Amino Acids to Tune the Affinity for Assembly on Cucurbit[8]uril Surfaces.

Cell adhesion is studied on multivalent knottins, displaying RGD ligands with a high affinity for integrin receptors, that are assembled on CB[8]-methylviologen-modified surfaces. The multivalency in the knottins stems from the number of tryptophan amino acid moieties, between 0 and 4, that can form a heteroternary complex with cucurbit[8]uril (CB[8]) and surface-tethered methylviologen (MV2+). The binding affinity of the knottins with CB[8] and MV2+ surfaces was evaluated using surface plasmon resonance spectroscopy. Specific binding occurred, and the affinity increased with the valency of tryptophans on the knottin. Additionally, increased multilayer formation was observed, attributed to homoternary complex formation between tryptophan residues of different knottins and CB[8]. Thus, we were able to control the surface coverage of the knottins by valency and concentration. Cell experiments with mouse myoblast (C2C12) cells on the self-assembled knottin surfaces showed specific integrin recognition by the RGD-displaying knottins. Moreover, cells were observed to elongate more on the supramolecular knottin surfaces with a higher valency, and in addition, more pronounced focal adhesion formation was observed on the higher-valency knottin surfaces. We attribute this effect to the enhanced coverage and the enhanced affinity of the knottins in their interaction with the CB[8] surface. Collectively, these results are promising for the development of biomaterials including knottins via CB[8] ternary complexes for tunable interactions with cells.

Electron-Transfer Rates in Host-Guest Assemblies at ß-Cyclodextrin Monolayers.

The effect of the distance between a ß-cyclodextrin (ßCD) host core and a conductive substrate on the electron-transfer rate of complexed guests as well as of free-diffusing electrochemically active probes has been studied. First we have evaluated a set of short-tethered ßCD adsorbates bearing different anchoring groups in order to get a reliable platform for the study of short-distance electron transfer. An electrochemically active trivalent guest was immobilized on these host monolayers in a selective and reversible manner, providing information about the packing density. Iodine- and nitrile-functionalized ßCD monolayers gave coverages close to maximum packing. Electron transfer in the presence of Fe(CN)63-/4- studied by impedance spectroscopy revealed that the electron transfer of the diffusing probe was 3 orders of magnitude faster than when the ßCD cores were separated from the surface by undecyl chains. When an electrochemically active guest was immobilized on the surface, electron-transfer rate measurements by cyclic voltammetry and capacitance spectroscopy showed differences of up to a factor of 8 for different ßCD monolayers. These results suggest that increasing the distance between the ßCD core and the underlying conductive substrate leads to a diminishing of the electron-transfer rate.

Modulating the Nucleated Self-Assembly of Tri-ß(3) -Peptides Using Cucurbit[n]urils.

The modulation of the hierarchical nucleated self-assembly of tri-ß(3) -peptides has been studied. ß(3) -Tyrosine provided a handle to control the assembly process through host-guest interactions with CB[7] and CB[8]. By varying the cavity size from CB[7] to CB[8] distinct phases of assembling tri-ß(3) -peptides were arrested. Given the limited size of the CB[7] cavity, only one aromatic ß(3) -tyrosine can be simultaneously hosted and, hence, CB[7] was primarily acting as an inhibitor of self-assembly. In strong contrast, the larger CB[8] can form a ternary complex with two aromatic amino acids and hence CB[8] was acting primarily as cross-linker of multiple fibers and promoting the formation of larger aggregates. General insights on modulating supramolecular assembly can lead to new ways to introduce functionality in supramolecular polymers.

Self-Assembly of Proteins: Towards Supramolecular Materials.

The study of protein self-assembly has attracted great interest over the decades, due to the important role that proteins play in life. In contrast to the major achievements that have been made in the fields of DNA origami, RNA, and synthetic peptides, methods for the design of self-assembling proteins have progressed more slowly. This Concept article provides a brief overview of studies on native protein and artificial scaffold assemblies and highlights advances in designing self-assembling proteins. The discussions are focused on design strategies for self-assembling proteins, including protein fusion, chemical conjugation, supramolecular, and computational-aided de novo design.

Effects of Variations in Ligand Density on Cell Signaling.

Multiple simultaneous interactions between receptors and ligands dictate the extracellular and intracellular activities of cells. The concept of programmable ligand display is generally used to study the interaction between ligands, displayed on surfaces at various densities, with receptors present on cell surfaces. Various strategies are discussed here to display ligands on surfaces to study their effect on cell behavior. Only very few strategies have been reported where this display combines precise control over density with lateral spacing of ligands on surfaces. In this review, selected examples of strategies to control ligand density and spacing and their implications for biological functions of cells are discussed.

Photoresponsive Cucurbit[8]uril-Mediated Adhesion of Bacteria on Supported Lipid Bilayers.

In this work, the development of a photoresponsive platform for the presentation of bioactive ligands to study receptor-ligand interactions has been described. For this purpose, supramolecular host-guest chemistry and supported lipid bilayers (SLBs) have been combined in a microfluidic device. Quartz crystal microbalance with dissipation monitoring (QCM-D) studies on methyl viologen (MV)-functionalized oligo ethylene glycol-based self-assembled monolayers, gel and liquid-state SLBs have been compared for their nonfouling properties in the case of ConA and bacteria. In combination with bacterial adhesion test, negligible nonspecific bacterial adhesion is observed only in the case of methyl-viologen-modified liquid-state SLBs. Therefore, liquid-state SLBs have been identified as most suitable for studying specific cell interactions when MV is incorporated as a guest on the surface. The photoswitchable supramolecular ternary complex is formed by assembling cucurbit[8]uril (CB[8]) and an azobenzene-mannose conjugate (Azo-Man) onto MV-functionalized liquid-state SLBs and the assembly process has been characterized using QCM-D and fluorescence techniques. Mannose has been found to enable binding of E. coli via cell-surface receptors on the nonfouling supramolecular SLBs. Optical switching of the azobenzene moiety allows us to "erase" the bioactive surface after bacterial binding, providing the potential to develop reusable sensors. Localized photorelease of bacterial cells has also been shown indicating the possibility of optically guiding cellular growth, migration, and intercellular interactions.

Supramolecular Surface Immobilization of Knottin Derivatives for Dynamic Display of High Affinity Binders.

Knottins are known as a robust and versatile class of miniprotein scaffolds for the presentation of high-affinity binding peptides; however, to date their application in biomaterials, biological coatings, and surface applications have not been explored. We have developed a strategy to recombinantly synthesize a ß-trypsin inhibitory knottin with supramolecular guest tags that enable it to adhere to self-assembled monolayers of the supramolecular host cucurbit[8]uril (CB[8]). We have described a strategy to easily express knottins in E. coli by conjugating them to a fluorescent protein after which they are cleaved and purified. Knottin constructs that varied in the number and position of the supramolecular tag at either the N- or C-termini or at both ends have been verified for their trypsin inhibitory function and CB[8]-binding properties in solution and on surfaces. All of the knottin constructs showed strong inhibition of trypsin with inhibition constants between 10 and 30 nM. Using microscale thermophoresis, we determined that the supramolecular guest tags on the knottins bind CB[8] with a Kd of ∼6 µM in solution. At the surface, strong divalent binding has been determined with a Kd of 0.75 µM in the case of the knottin with two supramolecular guest tags, whereas only weak monovalent binding occurred when only one guest tag was present. We also show successful supramolecular surface immobilization of the knottin using CB[8] and prove that they can be used to immobilize ß-trypsin at the surface.

Supramolecular Protein Immobilization on Lipid Bilayers.

Protein immobilization on surfaces, and on lipid bilayers specifically, has great potential in biomolecular and biotechnological research. Of current special interest is the immobilization of proteins using supramolecular noncovalent interactions. This allows for a reversible immobilization and obviates the use of harsh ligation conditions that could denature fragile proteins. In the work presented here, reversible supramolecular immobilization of proteins on lipid bilayer surfaces was achieved by using the host-guest interaction of the macrocyclic molecule cucurbit[8]uril. A fluorescent protein was successfully immobilized on the lipid bilayer by making use of the property of cucurbit[8]uril to host together a methylviologen and the indole of a tryptophan positioned on the N-terminal of the protein. The supramolecular complex was anchored to the bilayer through a cholesterol moiety that was attached to the methylviologen tethered with a small polyethylene glycol spacer. Protein immobilization studies using a quartz crystal microbalance (QCM) showed the assembly of the supramolecular complexes on the bilayer. Specific immobilization through the protein N-terminus is more efficient than through protein side-chain events. Reversible surface release of the proteins could be achieved by washing with cucurbit[8]uril or buffer alone. The described system shows the potential of supramolecular assembly of proteins and provides a method for site-specific protein immobilization under mild conditions in a reversible manner.

About supramolecular systems for dynamically probing cells.

This article reviews the state of the art in the development of strategies for generating supramolecular systems for dynamic cell studies. Dynamic systems are crucial to further our understanding of cell biology and are consequently at the heart of many medical applications. Increasing interest has therefore been focused recently on rendering systems bioactive and dynamic that can subsequently be employed to engage with cells. Different approaches using supramolecular chemistry are reviewed with particular emphasis on their application in cell studies. We conclude with an outlook on future challenges for dynamic cell research and applications.

On-chip electrophoresis in supported lipid bilayer membranes achieved using low potentials.

A micro supported lipid bilayer (SLB) electrophoresis method was developed, which functions at low potentials and appreciable operating times. To this end, (hydroxymethyl)-ferrocene (FcCH2OH) was employed to provide an electrochemical reaction at the anode and cathode at low applied potential to avoid electrolysis of water. The addition of FcCH2OH did not alter the SLB characteristics or affect biomolecule function, and pH and temperature variations and bubble formation were eliminated. Applying potentials of 0.25-1.2 V during flow gave homogeneous electrical fields and a fast, reversible, and strong build-up of a charged dye-modified lipid in the direction of the oppositely charged electrode. Moreover, streptavidin mobility could be modulated. This method paves the way for further development of analytical devices.

A supramolecular host-guest carrier system for growth factors employing V(H)H fragments.

A supramolecular strategy is presented for the assembly of growth factors employing His6-tagged single-domain antibodies (VHH). A combination of orthogonal supramolecular interactions of ß-cyclodextrin (ßCD)-adamantyl (Ad) host-guest and N-nitrilotriacetic acid (NTA)-histidine (His) interactions was employed to generate reversible and homogeneous layers of growth factors. A single-domain antibody V(H)H fragment was identified to bind to the human bone morphogenetic protein-6 (hBMP6) growth factor and could be recombinantly expressed in E. coli. The V(H)H fragment was equipped with a C-terminal hexahistidine (His6) tether to facilitate the assembly on ßCD surfaces using a linker that contains an Ad group to bind to the ßCD receptors and an NTA moiety to interact with the His6-tag upon cocomplexation of Ni(2+) ions. After exploring the thermodynamic and kinetic stability of the V(H)H assemblies on ßCD surfaces using a variety of experimental techniques including microcontact printing (µCP), surface plasmon resonance (SPR), microscale thermophoresis (MST), and theoretical models for determining the thermodynamic behavior of the system, hBMP6 was assembled onto the V(H)H-functionalized surfaces. After analyzing the immobilized hBMP6 using immunostaining, the biological activity of hBMP6 was demonstrated in cell differentiation experiments. Early osteogenic differentiation was analyzed in terms of alkaline phosphatase (ALP) activity of KS483-4C3 mouse progenitor cells, and the results indicated that the reversibly immobilized growth factors were functionally delivered to the cells. In conclusion, the supramolecular strategy used here offers the necessary affinity, reversibility, and temporal control to promote biological function of the growth factors that were delivered by this strategy.

Locked-in biomimetic surface gradients that are tunable in size, density and functionalization.

Tuneable and stable surface-chemical gradients in supported lipid bilayers (SLBs) hold great promise for a range of applications in biological sensing and screening. Yet, until now, no method has been reported that provides temporal control of SLB gradients. Herein we report on the development of locked-in SLB gradients that can be tuned in space, time and density by applying a process to control lipid phase behaviour, electric field and temperature. Stable gradients of charged Texas-Red-, serine- or biotin-terminated lipids have been prepared. For example, the Texas-Red surface density was varied from 0 to 2 mol %, while the length was varied between several tens to several hundreds of microns. At room temperature the gradients are shown to be stable up to 24 h, while at 60 °C the gradients could be erased in 30 min. Covalent and non-covalent chemical modification of the gradients is demonstrated, for example, by FITC, hexahistidine-tagged proteins, and SAv/biotin. The amenability to various (bio)chemistries paves the way for novel SLB-based gradients, useful in sensing, high-throughput screening and for understanding dynamic biological processes.

Dual stimuli-responsive self-assembled supramolecular nanoparticles.

Supramolecular nanoparticles (SNPs) encompass multiple copies of different building blocks brought together by specific noncovalent interactions. The inherently multivalent nature of these systems allows control of their size as well as their assembly and disassembly, thus promising potential as biomedical delivery vehicles. Here, dual responsive SNPs have been based on the ternary host-guest complexation between cucurbit[8]uril (CB[8]), a methyl viologen (MV) polymer, and mono- and multivalent azobenzene (Azo) functionalized molecules. UV switching of the Azo groups led to fast disruption of the ternary complexes, but to a relatively slow disintegration of the SNPs. Alternating UV and Vis photoisomerization of the Azo groups led to fully reversible SNP disassembly and reassembly. SNPs were only formed with the Azo moieties in the trans and the MV units in the oxidized states, respectively, thus constituting a supramolecular AND logic gate.