Controlling mammalian cell interactions on patterned polyelectrolyte multilayer surfaces

A newly discovered class of cell resistant surfaces, specifically engineered polyelectrolyte multilayers, was patterned with varying densities of adhesion ligands to control attachment of mammalian cells and to study the effects of ligand density on cell activity. Cell adhesive patterns were created on cell resistant multilayer films composed of poly(acrylic acid) and polyacrylamide through polymer-on-polymer stamping of poly(allylamine hydrochloride) PAH and subsequent reaction of the amine functional groups with an adhesion ligand containing RGD (Arg-Gly-Asp). These cell patterns demonstrated great promise for long-term applications since they remained stable for over 1 month, unlike ethylene glycol functional surfaces. By changing the stamping conditions of PAH, it was possible to alter the number of available functional groups in the patterned regions, and as a result, control the ligand density. Cell spreading, morphology, and cytoskeletal organization were compared at four different RGD densities. The highest RGD density, approximately 152 000 molecules/microm2, was created by stamping PAH at a pH of 11.0. Lowering the stamping ink pH led to patterns with lower ligand surface densities (83 000 molecules/microm2 for pH 9.0, 53,000 molecules/ microm2 for pH 7.0, and 25 000 molecules/microm2 for pH 3.5). An increasing number of cells attached and spread as the RGD density of the patterns increased. In addition, more cells showed well-defined actin stress fibers and focal adhesions at higher levels of RGD density. Finally, we found that pattern geometry affected cytoskeletal protein organization. Well-formed focal adhesions and cell-spanning stress fibers were only found in cells on wider line patterns (at least 25 microm in width).     

F-Actin reassembly during focal adhesion impacts single cell mechanics and nanoscale membrane structure

Focal adhesions play an important role in cell spreading,migration,and overall mechanical integrity.The relationship of cell structural and mechanical properties was investigated in the context of focal adhesion processes.Combined atomic force microscopy(AFM) and laser scanning confocal microscopy(LSCM) was utilized to measure single cell mechanics,in correlation with cellular morphology and membrane structures at a nanometer scale.Characteristic stages of focal adhesion were verified via confocal fluorescent studies,which confirmed three representative F-actin assemblies,actin dot,filaments network,and long and aligned fibrous bundles at cytoskeleton.Force-deformation profiles of living cells were measured at the single cell level,and displayed as a function of height deformation,relative height deformation and relative volume deformation.As focal adhesion progresses,single cell compression profiles indicate that both membrane and cytoskeleton stiffen,while spreading increases especially from focal complex to focal adhesion.Correspondingly,AFM imaging reveals morphological geometries of spherical cap,spreading with polygon boundaries,and elongated or polarized spreading.Membrane features are dominated by protrusions of 41-207 nm tall,short rods with 1-6 μm in length and 10.2-80.0 nm in height,and long fibrous features of 31-246 nm tall,respectively.The protrusion is attributed to local membrane folding,and the rod and fibrous features are consistent with bilayer decorating over the F-actin assemblies.Taken collectively,the reassembly of F-actin during focal adhesion formation is most likely responsible for the changes in cellular mechanics,spreading morphology,and membrane structural features.     

Effects of the expression level of epidermal growth factor receptor on the ligand-induced restructuring of focal adhesions: a QCM-D study

Epidermal growth factor receptor (EGFR) plays a major role in cell migration and invasion and is considered to be the primary source of activation of various malignant tumors. To gain insight into how elevated levels of EGFR influence cellular function, particularly cell motility, we used a quartz crystal microbalance with dissipation monitoring (QCM-D) to examine restructuring of focal adhesions in MCF-10A cells induced by epidermal growth factor. Engineered cells that overexpress epidermal growth factor receptor (EGFR) exhibited a very different kinetic profile from wildtype MCF-10A cells that have a lower level of EGFR with a higher rate for the initial disassembly of focal adhesion and a much lower rate for the later reassembly of focal adhesions. It is conceivable that these effects exhibited by EGFR-overexpressing cells may promote the initiation and maintenance of a more favorable adhesion state for cell migration. This study has demonstrated the capability of the dissipation monitoring function of the QCM-D to quantitatively assess kinetic aspects of cellular processes with a high temporal resolution and sensitivity.

Spatiotemporal control of cell adhesion on a self-assembled monolayer having a photocleavable protecting group

Control of cell adhesion is a key technology for cell-based drug screening and for analyses of cellular processes. We developed a method to spatiotemporally control cell adhesion using a photochemical reaction. We prepared a cell-culturing substrate by modifying the surface of a glass coverslip with a self-assembled monolayer of an alkylsiloxane having a photocleavable 2-nitrobenzyl group. Bovine serum albumin (BSA) was adsorbed onto the substrate to make the surface inert to cell adhesion. When exposed to UV light, the alkylsiloxane underwent a photocleavage reaction, leading to the release of BSA from the surface. Fibronectin, a protein promoting cell adhesion, was added to cover the irradiated regions and made them cell-adhesive. Seeding of cells on this substrate resulted in their selective adhesion to the illuminated regions. By controlling the sizes of the illuminated regions, we formed cell-adhesive spots smaller than single cells and located focal adhesions of the cells. Moreover, by subsequently illuminating the region alongside the cells patterned on the substrate in advance, we released their geometrical confinements and induced migration and proliferation. These manipulations were conducted under a conventional fluorescence microscope without any additional instruments. The present method of cell manipulation will be useful for cell biological studies as well as for the formation of cell arrays.     

Assessing the spatial resolution of cellular rigidity sensing using a micropatterned hydrogel-photoresist composite

The biophysical machinery that permits a cell to sense substrate rigidity is poorly understood. Rigidity sensing of adherent cells likely involves traction forces applied through focal adhesions and measurement of resulting deformation. However, it is unclear if this measurement takes place underneath single focal adhesions, over local clusters of focal adhesions, or across the length of the entire cell. To address this question, we developed a composite, chip-based material containing many arrays of 6.5 μm × 6.5 μm rigid adhesive islands, with an edge-edge distance of 8 μm, grafted onto the surface of a non-adhesive polyacrylamide hydrogel. This material is thus rigid within single islands while long-range rigidity is determined by the hydrogel. On soft gels, most NIH 3T3 cells spread only across two islands in a given dimension forming small stress fibers and focal adhesions. On stiff gels, cell spreading, stress fibers, and focal adhesions were indistinguishable from those on regular culture surfaces. We conclude that rigidity sensing is dictated by material compliance across the cell length and that responses to rigidity may be inhibited at any point when large substrate strain is encountered during spreading. Our finding may serve as a guideline for the design of biomaterials for tissue engineering.     

Turning Cell Adhesions ON or OFF with High Spatiotemporal Precision Using the Green Light Responsive Protein CarH

Spatiotemporal control of integrin-mediated cell adhesions to extracellular matrix regulates cell behavior with has numerous implications for biotechnological applications. In this work, two approaches for regulating cell adhesions in space and time with high precision are reported, both of which utilize green light. In the first design, CarH, which is a tetramer in the dark, is used to mask cRGD adhesion-peptides on a surface. Upon green light illumination, the CarH tetramer dissociates into its monomers, revealing the adhesion peptide so that cells can adhere. In the second design, the RGD motif is incorporated into the CarH protein tetramer such that cells can adhere to surfaces functionalized with this protein. The cell adhesions can be disrupted with green light, due to the disassembly of the CarH-RGD protein. Both designs allow for photoregulation with noninvasive visible light and open new possibilities to investigate the dynamical regulation of cell adhesions in cell biology.     

Multifunctional Fluorinated Lubricant-Infused Poly(4-Hydroxybutyrate) (P4HB) Membranes for Full-Thickness Abdominal Wall Defect Repair

Abdominal wall defect caused by surgical trauma, congenital rupture, or tumor resection may result in hernia formation or even death. Tension-free abdominal wall defect repair by using patches is the gold standard to solve such problems. However, adhesions following patch implantation remain one of the most challenging issues in surgical practice. The development of new kinds of barriers is key to addressing peritoneal adhesions and repairing abdominal wall defects. It is already well recognized that ideal barrier materials need to have good resistance to nonspecific protein adsorption, cell adhesion, and bacterial colonization for preventing the initial development of adhesion. Herein, electrospun poly(4-hydroxybutyrate) (P4HB) membranes infused with perfluorocarbon oil are used as physical barriers. The oil-infused P4HB membranes can greatly prevent protein attachment and reduce blood cell adhesion in vitro. It is further shown that the perfluorocarbon oil-infused P4HB membranes can reduce bacterial colonization. The in vivo study reveals that perfluoro(decahydronaphthalene)-infused P4HB membranes can significantly prevent peritoneal adhesions in the classic abdominal wall defects’ model and accelerate defect repair, as evidenced by gross examination and histological evaluation. This work provides a safe fluorinated lubricant-impregnated P4HB physical barrier to inhibit the formation of postoperative peritoneal adhesions and efficiently repair soft-tissue defects.     

Evaluation of the stability of nonfouling ultrathin poly(ethylene glycol) films for silicon-based microdevices

The creation of nonfouling surfaces is one of the major prerequisites for microdevices for biomedical and analytical applications. Poly(ethylene glycol) (PEG), a water soluble, nontoxic, and nonimmunogenic polymer has the unique ability of reducing nonspecific protein adsorption and cell adhesion and, therefore, is generally coupled with a wide variety of surfaces to improve their biocompatibility. The performance of these modified surfaces for long-term biomedical applications largely depends on the stability of these PEG films. To this end, we have investigated the stability of covalently coupled ultrathin PEG films on silicon in aqueous in vivo like conditions for a period of 4 weeks. The PEG-modified silicon substrates were incubated in PBS (37 degrees C, pH 7.4, 5% CO2) for different periods of time and then characterized using the techniques of ellipsometry, contact angle measurement, X-ray photoelectron spectroscopy, and atomic force microscopy. The ability of the PEG-modified surfaces to control protein fouling was examined by protein adsorption studies using fluorescein isothiocyanate labeled bovine serum albumin and ellipsometry. Furthermore, the ability of these films to control fibroblast adhesion was examined. Studies suggest that the PEG-modified surfaces retain their protein and cell repulsive nature even though the PEG film thickness decreases for the period of investigation.     

Combination of integrin-binding peptide and growth factor promotes cell adhesion on electron-beam-fabricated patterns

Understanding and controlling cell adhesion on engineered scaffolds is important in biomaterials and tissue engineering. In this report we used an electron-beam (e-beam) lithography technique to fabricate patterns of a cell adhesive integrin ligand combined with a growth factor. Specifically, micron-sized poly(ethylene glycol) (PEG) hydrogels with aminooxy- and styrene sulfonate-functional groups were fabricated. Cell adhesion moieties were introduced using a ketone-functionalized arginine-glycine-aspartic acid (RGD) peptide to modify the O-hydroxylamines by oxime bond formation. Basic fibroblast growth factor (bFGF) was immobilized by electrostatic interaction with the sulfonate groups. Human umbilical vein endothelial cells (HUVECs) formed focal adhesion complexes on RGD- and RGD and bFGF-immobilized patterns as shown by immunostaining of vinculin and actin. In the presence of both bFGF and RGD, cell areas were larger. The data demonstrate confinement of cellular focal adhesions to chemically and physically well-controlled microenvironments created by a combination of e-beam lithography and "click" chemistry techniques. The results also suggest positive implications for addition of growth factors into adhesive patterns for cell-material interactions.     

Factors affecting the stability of O/W emulsion in BSA solution: stabilization by electrically neutral protein at high ionic strength

Bovine serum albumin (BSA) was used as an emulsifier to disperse corn oil in aqueous media with various protein concentration, pH, and ionic strength. Quantitative estimation was made on the homogenizing activity of BSA and dispersion stability of oil particles by measuring particle size, turbidity, and creaming rate. Dispersion stability strongly depended on pH and became a minimum around pH 5.0 which was the isoelectric point of BSA. The interfacial tension between BSA solution and corn oil was minimized at pH 5.0. Interesting results were obtained concerning the ionic-strength dependence of stability. When the ionic strength was set below 30 mM, the emulsions became more stable with the increase of BSA concentration at pH 6.7 but the opposite behavior (enhanced destabilization) was confirmed at pH 5.0 with the BSA content. In high ionic strength conditions (ca. > or = 80 mM NaCl), however, BSA-stabilized emulsions became fairly stable even at pH 5.0. These results suggested that BSA molecules having no net charge induced some attractive interactions (e.g., bridging or depletion) in low ionic strength but steric stabilization in high ionic strength, respectively.     

Characterizing the Structure and Photocycle of PR 2D Crystals with CD and FTIR Spectroscopy†

We present here a study on proteorhodopsin (PR) 2D crystals with analytical ultracentrifugation, circular dichroism and Fourier transform infrared (FTIR) spectroscopy. The aim of our experiments was to test the activity of 2D crystal sample preparations and to gain further insight in PR structure, stability and function with these techniques. Our results demonstrate higher stability compared to detergent‐solubilized or reconstituted samples. For different pH values, low pH 2D crystals tend to form bigger aggregates and are less stable than at basic pH. The pH 9 sample shows a sharp phase transition during heat denaturation and there is also evidence for protein–protein interaction due to the close proximity of the proteins in the 2D crystals. In the FTIR measurements at cryogenic temperatures (77 K), we characterized the first step in the PR photocycle. At pH 9, the K intermediate could be observed and the samples showed no orientation effects. At pH 5, we could trap the K/L intermediate, characterized by its negative IR signal at 1741 cm^?1. In rapid‐scan FTIR experiments, we could also identify the M intermediate of the photocycle at basic pH. We conclude that the PR 2D crystals exhibit a fully functional photocycle and are therefore well suited for further studies on the proton transport mechanism of PR.     

Imaging Cell‐Matrix Adhesions and Collective Migration of Living Cells by Electrochemiluminescence Microscopy

Cell‐matrix adhesions play essential roles in a variety of biological processes. Herein, we report a label‐free method to map cell‐matrix adhesions of single living cells on an electrode surface by electrochemiluminescence (ECL). An indium tin oxide electrode modified with a silica nanochannel membrane was used as the substrate electrode, at which the ECL generation from freely diffusing luminophores provided a distinct visual contrast between adhesion sites and noncontacted domains, thus selectively revealing the former in a label‐free manner. With this methodology, we studied the spatial distribution, as well as dynamic variation, of cell‐matrix adhesions and the adhesion strength at the subcellular level. Cell‐matrix adhesions of an advancing cell sheet were finally imaged to study the movement of cells in collective migration. A statistical analysis suggests that cells on the far side of leading edge also have the propensity to migrate and do not act as just passive followers.     

Structural adaptation of the subunit interface of oligomeric thermophilic and hyperthermophilic enzymes

Enzymes from thermophilic and, particularly, from hyperthermophilic organisms are surprisingly stable. Understanding of the molecular origin of protein thermostability and thermoactivity attracted the interest of many scientist both for the perspective comprehension of the principles of protein structure and for the possible biotechnological applications through application of protein engineering. Comparative studies at sequence and structure levels were aimed at detecting significant differences of structural parameters related to protein stability between thermophilic and hyperhermophilic structures and their mesophilic homologs. Comparative studies were useful in the identification of a few recurrent themes which the evolution utilized in different combinations in different protein families. These studies were mostly carried out at the monomer level. However, maintenance of a proper quaternary structure is an essential prerequisite for a functional macromolecule. At the environmental temperatures experienced typically by hyper- and thermophiles, the subunit interactions mediated by the interface must be sufficiently stable. Our analysis was therefore aimed at the identification of the molecular strategies adopted by evolution to enhance interface thermostability of oligomeric enzymes. The variation of several structural properties related to protein stability were tested at the subunit interfaces of thermophilic and hyperthermophilic oligomers. The differences of the interface structural features observed between the hyperthermophilic and thermophilic enzymes were compared with the differences of the same properties calculated from pairwise comparisons of oligomeric mesophilic proteins contained in a reference dataset. The significance of the observed differences of structural properties was measured by a t-test. Ion pairs and hydrogen bonds do not vary significantly while hydrophobic contact area increases specially in hyperthermophilic interfaces. Interface compactness also appears to increase in the hyperthermophilic proteins. Variations of amino acid composition at the interfaces reflects the variation of the interface properties.     

Effects of Various Cell Surface Engineering Reactions on the Biological Behavior of Mammalian Cells

Cell surface engineering technologies can regulate cell function and behavior by modifying the cell surface. Previous studies have mainly focused on investigating the effects of cell surface engineering reactions and materials on cell activity. However, they do not comprehensively analyze other cellular processes. This study exploits covalent bonding, hydrophobic interactions, and electrostatic interactions to modify the macromolecules succinimide ester-methoxy polyethylene glycol (NHS-mPEG), distearoyl phosphoethanolamine-methoxy polyethylene glycol (DSPE-mPEG), and poly-L -lysine (PLL), respectively, on the cell surface. This work systematically investigates the effects of the three surface engineering reactions on the behavior of human umbilical vein endothelial cells (HUVECs) and human skin fibroblasts, including viability, growth, proliferation, cell cycle, adhesion, and migration. The results reveals that the PLL modification method notably affects cell viability and G2/M arrest and has a short modification duration. However, the DSPE-mPEG and NHS-mPEG modification methods have little effect on cell viability and proliferation but have a prolonged modification duration. Moreover, the DSPE-mPEG modification method highly affects cell adherence. Further, the NHS-mPEG modification method can significantly improve the migration ability of HUVECs by reducing the area of focal adhesions. The findings of this study will contribute to the application of cell surface engineering technology in the biomedical field.     

Imaging Cell-Matrix Adhesions and Collective Migration of Living Cells by Electrochemiluminescence Microscopy

Cell-matrix adhesions play essential roles in a variety of biological processes. Herein, we report a label-free method to map cell-matrix adhesions of single living cells on an electrode surface by electrochemiluminescence (ECL). An indium tin oxide electrode modified with a silica nanochannel membrane was used as the substrate electrode, at which the ECL generation from freely diffusing luminophores provided a distinct visual contrast between adhesion sites and noncontacted domains, thus selectively revealing the former in a label-free manner. With this methodology, we studied the spatial distribution, as well as dynamic variation, of cell-matrix adhesions and the adhesion strength at the subcellular level. Cell-matrix adhesions of an advancing cell sheet were finally imaged to study the movement of cells in collective migration. A statistical analysis suggests that cells on the far side of leading edge also have the propensity to migrate and do not act as just passive followers.     

Affinity adhesion of carbohydrate particles and yeast cells to boronate-containing polymer brushes grafted onto siliceous supports

Cross-linked agarose particles (Sepharose CL-6B) and baker's yeast cells were found to adhere to siliceous supports end-grafted with boronate-containing copolymers (BCCs) of N,N-dimethylacrylamide at pH> or =7.5, due to boronate interactions with surface carbohydrates of the particles and the cells. These interactions were registered both on macroscopic and on molecular levels: the BCCs spontaneously adsorbed on the agarose gel at pH> or =7.5, with adsorption increasing with pH. Agarose particles and yeast cells stained with Procion Red HE-3B formed stable, monolayer-like structures at pH 8.0, whereas at pH 7.0-7.8 the structures on the copolymer-grafted supports were less stable and more random. At pH 9.0, 50 % saturation of the surface with adhering cells was attained in 2 min. Stained cells formed denser and more stable layers on the copolymer-grafted supports than they did on supports modified with self-assembled organosilane layers derivatized with low-molecular-weight boronate, presumably due to a higher reactivity of the grafted BCCs. Quantitative detachment of adhered particles and cells could be achieved by addition of 20 mM fructose--a strong competitor for binding to boronates--at pH 7.0-9.0. Regeneration of the grafted supports allowed several sequential adhesion and detachment cycles with stained yeast cells. Affinity adhesion of micron-sized carbohydrate particles to boronate-containing polymer brushes fixed on solid supports is discussed as a possible model system suggesting a new approach to isolation and separation of living cells.     

Structural and antitumor properties of the YSNSG cyclopeptide derived from tumstatin

We previously demonstrated that the NC1[alpha3(IV)185-191] CNYYSNS peptide inhibited in vivo tumor progression. The YSNS motif formed a beta turn crucial for biological activity. The aim of the present study was to design a YSNSG cyclopeptide with a constrained beta turn on the YSNS residues more stable than CNYYSNS. By nuclear magnetic resonance and molecular modeling, we demonstrated that the YSNSG cyclopeptide actually adopted the expected beta-turn conformation. It promoted melanoma cell adhesion and prevented their adhesion to the native peptide. It inhibited in vitro cell proliferation and migration through Matrigel by downregulating proteolytic cascades. Moreover, intraperitoneal administration of the YSNSG cyclopeptide inhibited melanoma progression far more efficiently than the native peptide. The increased solubility and stability at low pH of the YSNSG cyclopeptide suggest this peptide as a potent antitumor therapeutic agent.     

Effect of pH on thermal- and chemical-induced denaturation of GFP

Green fluorescent protein (GFP) is an unusually stable autofluorescent protein that is increasingly being exploited for many applications. In this report, we have used fluorescence spectroscopy to study the effect of pH on the denaturation of GFP with sodium dodecyl sulfate (SDS), urea, and heat. Surprisingly, SDS (up to 0.5%) did not have any significant effect on the fluorescence of GFP at pH 7.5 or 8.5 buffers; however, at pH 6.5, the protein lost all fluorescence within 1 min of incubation. Similarly, incubation of GFP with 8 M urea at 50°C resulted in time dependent denaturation of GFP, but only in pH 6.5 buffer. At higher pH values (pH 7.5 and pH 8.5), the GFP was quite stable in 8 M urea at 50°C, showing only a slight decrease in fluorescence. Heat denaturation of GFP was found to be pH dependent as well, with the denaturation being fastest at pH 6.5 as compared to pH 7.5 or pH 8.5. Like the denaturation studies, renaturation of heat-denatured GFP was most efficient at pH 8.5, followed by pH 7.5, and then pH 6.5. These results suggests that GFP undergoes a structural/stability shift between pH 6.5 and pH 7.5, with the GFP structure at pH 6.5 being very sensitive to denaturation by SDS, urea, and heat.     

A highly stable nonbiofouling surface with well-packed grafted zwitterionic polysulfobetaine for plasma protein repulsion

An ideal nonbiofouling surface for biomedical applications requires both high-efficient antifouling characteristics in relation to biological components and long-term material stability from biological systems. In this study we demonstrate the performance and stability of an antifouling surface with grafted zwitterionic sulfobetaine methacrylate (SBMA). The SBMA was grafted from a bromide-covered gold surface via surface-initiated atom transfer radical polymerization to form well-packed polymer brushes. Plasma protein adsorption on poly(sulfobetaine methacrylate) (polySBMA) grafted surfaces was measured with a surface plasmon resonance sensor. It is revealed that an excellent stable nonbiofouling surface with grafted polySBMA can be performed with a cycling test of the adsorption of three model proteins in a wide range of various salt types, buffer compositions, solution pH levels, and temperatures. This work also demonstrates the adsorption of plasma proteins and the adhesion of platelets from human blood plasma on the polySBMA grafted surface. It was found that the polySBMA grafted surface effectively reduces the plasma protein adsorption from platelet-poor plasma solution to a level superior to that of adsorption on a surface terminated with tetra(ethylene glycol). The adhesion and activation of platelets from platelet-rich plasma solution were not observed on the polySBMA grafted surface. This work further concludes that a surface with good hemocompatibility can be achieved by the well-packed surface-grafted polySBMA brushes.     

Protein stability prediction: a Poisson-Boltzmann approach

Most proteins are only marginally stable at physiological temperatures. Thus a common defect due to mutation is the loss of protein stability, resulting in loss of their well-defined structures and functions at their functioning temperatures. Quantification of protein stability change upon mutation has attracted a large number of experimental and theoretical studies. In this work, we have extended the Poisson-Boltzmann theory that is originally used for predicting stability changes of charged mutations to predicting stability changes of all mutations. To achieve this, we have proposed a free energy model covering both electrostatic and hydrophobic interactions. A G?-like model for the denatured state that incorporates both nativeness and randomness of the denatured state has been used to calculate the hydrophobic contribution to protein stability. The new model is computationally simple and fast, and performs well for charged and hydrophobic mutations for all four tested proteins. Future directions for extending the method into pH-dependent effect and more accurate prediction for polar mutations are discussed.