Elisabetta Ada Cavalcanti-Adam

Induction of Cell Polarization and Migration by a Gradient of Nanoscale Variations in Adhesive Ligand Spacing

Cell interactions with adhesive surfaces play a vital role in the regulation of cell proliferation, viability, and differentiation, and affect multiple biological processes. Since cell adhesion depends mainly on the nature and density of the adhesive ligand molecules, spatial molecular patterning, which enables the modulation of adhesion receptor clustering, might affect both the structural and the signaling activities of the adhesive interaction. We herein show that cells plated on surfaces that present a molecularly defined spacing gradient of an integrin RGD ligand can sense small but consistent differences in adhesive ligand spacing of about 1 nm across the cell diameter, which is approximately 61 mum when the spacing includes 70 nm. Consequently, these positional cues induce cell polarization and initiate cell migration and signaling. We propose that differential positional clustering of the integrin transmembrane receptors is used by cells for exploring and interpreting their environment, at high spatial sensitivity.

Cell interactions with hierarchically structured nano-patterned adhesive surfaces

The activation of well-defined numbers of integrin molecules in predefined areas by adhesion of tissue cells to biofunctionalized micro-nanopatterned surfaces was used to determine the minimum number of activated integrins necessary to stimulate focal adhesion formation. This was realized by combining micellar and conventional e-beam lithography, which enabled deposition of 6 nm large gold nanoparticles on predefined geometries. Patterns with a lateral spacing of 58 nm and a number of gold nanoparticles, ranging from 6 to 3000 per adhesive patch, were used. For α(v) β(3)-integrin activation, gold nanoparticles were coated with c(-RGDfK-)-thiol peptides, and the remaining glass surface was passivated to prevent non-specific protein adsorption and cell adhesion. Results show that focal adhesion formation is dictated by the underlying hierarchical nanopattern. Adhesive patches with side lengths of 3000 nm and separated by 3000 nm, or with side lengths of 1000 nm and separated by 1000 nm, containing approximately 3007 ± 193 or 335 ± 65 adhesive gold nanoparticles, respectively, induced the formation of actin-associated, paxillin-rich focal adhesions, comparable in size and shape to classical focal adhesions. In contrast, adhesive patches with side lengths of 500, 250 or 100 nm, and separated from adjacent adhesive patches by their respective side lengths, containing 83 ± 11, 30 ± 4, or 6 ± 1 adhesive gold nanoparticles, respectively, showed a significant increase in paxillin domain length, caused by bridging the pattern gap through an actin bundle in order to mechanically, synergistically strengthen each single adhesion site. Neither paxillin accumulation nor adhesion formation was induced if less than 6 c(-RGDfK-)-thiol functionalised gold nanoparticles per adhesion site were presented to cells.

Cellular chemomechanics at interfaces: sensing, integration and response{

Living cells are complex entities whose remarkable, emergent capacity to sense, integrate, and respond to environmental cues relies on an intricate series of interactions among the cells macromolecular components. Defects in mechanosensing, transduction,or responses underlie many diseases such as cancers, immune disorders, cardiac hypertrophy, genetic malformations, and neuropathies. Here, we highlight micro- and nanotechnology-based tools that have been used to study how chemical and mechanical cues modulate the responses of single cells in contact with the extracellular environment. Understanding the physical aspects of these complex processes at the micro- and nanometer scale could produce profound and fundamental new insights into how the processes of cell migration, metastasis, immune function and other areas which are regulated by mechanical forces.

Cell adhesion and response to synthetic nanopatterned environments by steering receptor clustering and spatial location

During adhesion and spreading, cells form micrometer‐sized structures comprising transmembrane and intracellular protein clusters, giving rise to the formation of what is known as focal adhesions. Over the past two decades these structures have been extensively studied to elucidate their organization, assembly, and molecular composition, as well as to determine their functional role. Synthetic materials decorated with biological molecules, such as adhesive peptides, are widely used to induce specific cellular responses dependent on cell adhesion. Here, we focus on how surface patterning of such bioactive materials and organization at the nanoscale level has proven to be a useful strategy for mimicking both physical and chemical cues present in the extracellular space controlling cell adhesion and fate. This strategy for designing synthetic cellular environments makes use of the observation that most cell signaling events are initiated through recruitment and clustering of transmembrane receptors by extracellular‐presented signaling molecules. These systems allow for studying protein clustering in cells and characterizing the signaling response induced by, e.g., integrin activation. We review the findings about the regulation of cell adhesion and focal adhesion assembly by micro‐ and nanopatterns and discuss the possible use of substrate stiffness and patterning in mimicking both physical and chemical cues of the extracellular space.

RGD Peptides Immobilized on a Mechanically Deformable Surface Promote Osteoblast Differentiation

The major objective of this work was to attach bone cells to a deformable surface for the effective transmission of force. We functionalized a silastic membrane and treated it with 3‐aminopropyltriethoxysilane (APTS). A minimal RGD peptide was then covalently linked to the aminated surface. MC3T3‐E1 osteoblast‐like cells were cultured on the arginine‐glycine‐aspartic acid (RGD)‐treated membrane for 3–15 days and cell attachment and proliferation was evaluated. We observed that cells were immediately bound to the membrane and proliferated. After 8 days on the material surface, osteoblasts exhibited high levels of ALP staining, indicating that the cells were undergoing maturation. Alizarin red staining and Fourier transform infrared (FTIR) analysis showed that the mineral formed by the cells was a biological apatite. The second objective was to apply a mechanical force to cells cultured on the modified silicone membrane. Dynamic equibiaxial strain, 2% magnitude, and a 0.25‐Hz frequency were applied to bone cells for 2 h. Osteoblasts elicited increased phalloidin fluorescence, suggesting that there was reorganization of the cytoskeleton. Furthermore, the applied strain elicited increased expression of the αvβ3 integrin receptor. We concluded that the covalent binding of RGD peptides to a silicone membrane provides a compatible surface for the attachment and subsequent differentiation of osteoblasts. Moreover, the engineered surface transduces applied mechanical forces directly to the adherent cells via integrin receptors.

Polymeric substrates with tunable elasticity and nanoscopically controlled biomolecule presentation

Despite tremendous progress in recent years, nanopatterning of hydrated polymeric systems such as hydrogels still represents a major challenge. Here, we employ block copolymer nanolithography to arrange gold nanoparticles on a solid template, followed by the transfer of the pattern to a polymeric hydrogel. In the next step, these nanoparticles serve as specific anchor points for active biomolecules. We demonstrate the engineering of poly(ethylene glycol) hydrogel surfaces with respect to elasticity, nanopatterning, and functionalization with biomolecules. For the first time, biomolecule arrangement on the nanometer scale and substrate stiffness can be varied independently from each other. Youngs moduli, a measure of the compliance of the substrates, can be tuned over 4 orders of magnitude, including the values for all of the different tissues found in the human body. Structured hydrogels can be used to pattern any histidine-tagged protein as exemplified for his-protein A as an acceptor for immunoglobulin. When cell-adhesion-promoting peptide cRGDfK is selectively coupled to gold nanoparticles, the surfaces provide cues for cell-surface interaction and allow for the study of the modulation of cellular adhesion by the mechanical properties of the environment. Therefore, these substrates represent a unique multipurpose platform for studying receptor/ligand interactions with adhering cells, mechanotransduction, and cell-adhesion-dependent signaling.

Functionalizing αvβ3- or α5β1-selective integrin antagonists for surface coating: a method to discriminate integrin subtypes in vitro

Investigating the different functions of distinct surface receptors is essential to understand the complex interactions between cells and their extracellular environment. Cells use specific transmembrane receptors of the integrin family to anchor and respond to extracellular matrix (ECM) proteins. In doing so, integrins are capable of regulating cell migration, survival, cell cycle progression, and differentiation, which are essential tasks for the development of all multicellular organisms. Integrins are often classified according to their binding specificity for extracellular ligands. For instance, arginine–glycine–aspartate (RGD)-containing proteins, such as fibronectin (Fn), exhibit high binding affinity to 8 of the 24 integrins expressed in mammals. Among the fibronectin-binding integrins, avb3 and a5b1 play crucial roles during embryogenesis, angiogenesis, and in pathology. Despite the wealth of information on integrin biology, it is unclear how cell functions and responses are regulated by a single integrin subtype. To address this relevant issue, the design of new active molecules that are able to selectively recognize distinct integrin subtypes is essential for in vitro studies and the development of selective drugs for disease therapy (personalized medicine). Moreover, the conjugation of functional groups to such ligands without affecting their affinity and selectivity for cell surface receptors remains a difficult task to achieve. Herein, we report a strategy to synthesize avb3or a5b1specific ligands for the functionalization of nanostructured gold surfaces and demonstrate that cell adhesion can be selectively mediated by a single integrin subtype. The adhesion of either avb3or a5b1-expressing cells was clearly mediated or blocked through coating of gold nanoarrays with these molecules, thus giving striking evidence for their selectivity. These compounds are a powerful tool to elucidate the difference between avb3 and a5b1 integrin-mediated cell adhesion. The interaction of integrins with ECM proteins leads to the clustering of integrins and the recruitment of intracellular proteins to the integrin cytoplasmic domains. The recruited proteins, collectively called the adhesome, play crucial roles in transducing integrin-mediated processes and are thus also involved in biological processes, such as angiogenesis and tumor development. They are therefore interesting targets for pharmacological research. 9] Although the functions of integrins in cell adhesion and formation of focal contacts have been studied with unselective cyclic RGD peptides and other ECM mimetics, the exact role of avb3 and a5b1 integrin subtypes played in these processes remains to be elucidated. Natural integrin ligands are not well suited for those studies owing to the lack of integrin subtype selectivity. Coating of surfaces with peptidic integrin ligands for biophysical cell adhesion studies, to develop implant materials, or to identify binding motifs was realized in various cases. 12] However, it turned out that peptides, in contrast to peptidomimetics, were not able to achieve high activity and sufficient selectivity. Nowadays, it is still challenging to functionalize selective peptidomimetics for this purpose without losing activity.

Block copolymer micelle nanolithography on non-conductive substrates

A new lithographic technique has been developed and applied to cell adhesion studies and electro-optical material development. Attachment of 6 nm Au particles, in periodic and non-periodic pattern, onto non-conductive substrates has been achieved. This was performed via a combination of diblock copolymer self-assembly and electron beam lithographic techniques. To optimize e-beam resolution on non-conductive materials, an additional carbon layer was thread-coated onto the substrates. This carbon coating and the diblock copolymer used in the self-assembly step were simultaneously removed by a final hydrogen plasma treatment to reveal Au nanodot patterns of unprecedented pattern quality. These optically transparent substrates (glass cover slips) were bio-functionalized via the Au-dot patterns to yield a platform for unique cell adhesion studies. The same Au-dot patterning technique was applied to sapphire substrates, which were subsequently employed to nucleate electro-optically active ZnO nanopost growth.

A comprehensive evaluation of the activity and selectivity profile of ligands for RGD-binding integrins

Integrins, a diverse class of heterodimeric cell surface receptors, are key regulators of cell structure and behaviour, affecting cell morphology, proliferation, survival and differentiation. Consequently, mutations in specific integrins, or their deregulated expression, are associated with a variety of diseases. In the last decades, many integrin-specific ligands have been developed and used for modulation of integrin function in medical as well as biophysical studies. The IC50-values reported for these ligands strongly vary and are measured using different cell-based and cell-free systems. A systematic comparison of these values is of high importance for selecting the optimal ligands for given applications. In this study, we evaluate a wide range of ligands for their binding affinity towards the RGD-binding integrins αvβ3, αvβ5, αvβ6, αvβ8, α5β1, αIIbβ3, using homogenous ELISA-like solid phase binding assay.

Surface immobilization of bone morphogenetic protein 2 via a self-assembled monolayer formation induces cell differentiation.

Bone extracellular matrix consists of a network of proteins in which growth factors, like bone morphogenetic protein 2 (BMP-2), are embedded and released upon matrix turnover and degradation. Recombinant human (rh)BMP-2 shows promise in enhancing bone fracture repair, although issues regarding finding a suitable delivery system still limit its extensive clinical use. The aim of this study is to determine which cell activities are triggered by the presentation of immobilized rhBMP-2. For this purpose gold surfaces were first decorated with a self-assembled monolayer consisting of a hetero-bifunctional linker. rhBMP-2 was covalently bound to the surfaces via this linker and used to investigate the cellular responses of C2C12 myoblasts. We show that covalently immobilized rhBMP-2 (iBMP-2) initiates short-term signaling events. Using a BMP-responsive reporter gene assay and western blotting to monitor phosphorylation of Smad1/5/8 we prove that iBMP-2 activates BMP-dependent signal transduction. Furthermore, we demonstrate that iBMP-2 suppresses myotube formation and promotes the osteoblast phenotype in C2C12 cells. The bioactivity of surface-bound rhBMP-2 presented in this study is not due to its release into the medium. As such, our simple approach paves the way for the controlled local presentation of immobilized growth factors, limiting degradation while still maintaining biological activity.