Gradient immobilization of a cell adhesion RGD peptide on thermal responsive surface for regulating cell adhesion and detachment

Using surface initiated atomic transfer radical polymerization (ATRP) and an injection method, a poly(N-isopropylacrylamide)-b-poly(acrylic acid)-g-RGD (PNIPAAm-b-PAA-g-RGD) gradient surface was prepared. First, a thermoresponsive surface with a constant thickness of PNIPAAm was fabricated, onto which the AA monomers were block copolymerized using the PNIPAAm macromolecules as initiators. During this process, a continuous injection method was employed to yield a molecular weight gradient of PAA on the underlying uniform PNIPAAm layer. RGD peptide was finally covalently immobilized onto the PAA gradient by carbodiimide chemistry. In vitro culture of HepG2 cells showed that immobilization of the RGD peptide could accelerate cell attachment, while the thermoresponsive layer beneath could effectively release the cells by simply lowering temperature. Thus, the PNIPAAm-b-PAA-g-RGD gradient surface, combining the thermal response with cell affinity properties, can well regulate the cell adhesion and detachment, which may thus be useful for investigation of cell-substrate interactions with a smaller number of samples.     

Scalable synthesis of an integrin-binding peptide mimetic for biomedical applications

A scalable, solution-phase synthesis of the selectively protected non-peptide RGD (arginine-glycine-aspartic acid) mimetic 6 is described. This synthesis serves as an alternative to the previously described solid-phase synthesis of this compound, thereby making this important integrin-binding mimetic readily accessible. The free carboxylic acid of 6 was conjugated to a protected diamine, followed by global deprotection to give a derivative 27, suitable for immobilization onto amine-reactive surfaces. The RGD mimetic 28 demonstrated superior biological activity in comparison to a native linear RGD peptide and the semi-synthetic cyclic cRGDfK peptide in a cell attachment inhibition assay.     

Electrically controlling cell adhesion,growth and migration

We have developed a neurochip to control the adhesion and outgrowth of individual neurons by electrochemical removal of protein repellent molecules from transparent electrodes. The neurochip architecture is based on three parallel indium-tin-oxide (ITO) electrodes on a SiO₂ substrate and a photoresist structure forming a landing spot for the neuron soma and two lateral outgrowth pathways for the neurites. The whole surface was turned protein and cell repellent with poly(ethylene glycol) grafted-poly(l-lysine) (PLL-g-PEG) before enabling neuron soma adhesion by selective PLL-g-PEG removal. After the neuron has settled down a potential was applied to the pathway electrodes to permit the neurite outgrowth along pathways formed by the SU8 structure. We also show the possibility to control cell migration by small pulsed currents. Myoblasts were therefore seeded on a chemical pattern of cell adhesive PLL and cell resistant PLL-g-PEG. The PLL-g-PEG was then removed electrochemically from the electrodes to permit migration onto the cell free electrodes. Electrodes without applied current were confluently overgrown within 24 h but a small pulsed current was able to inhibit cell growth on the bare ITO electrode for more than 72 h. With both techniques, cell adhesion, growth and migration can be controlled dynamically after the cells started to grow on the substrate. This opens new possibilities: we believe the key to control the development of topologically controlled neuron networks or more complex co-cultures is the combination of passive surface modifications and active control over the surface properties at any time of the experiment.     

Microelectrochemical approach to induce local cell adhesion and growth on substrates

The nature of an albumin-coated substrate that blocks protein adsorption and cell adhesion was rapidly switched to cell-adhesive by exposure to an oxidizing agent such as HBrO. This finding has enabled cellular pattern drawing even on a single-cell level by closely scanning a microelectrode above the substrate and electrochemically producing the agent at the tip of the electrode. The present microelectrochemical cell patterning is applicable even for a previously cell-patterned substrate and for a grooved substrate. These unique technical features will have impacts on a variety of cell-based studies that require the analysis of heterotypic cell-cell interactions and cellular arrangement on an uneven surface such as semiconductor devices.     

Modeling the interaction between integrin-binding peptide (RGD) and rutile surface: the effect of cation mediation on Asp adsorption

The binding of a negatively charged residue, aspartic acid (Asp) in tripeptide arginine-glycine-aspartic acid, onto a negatively charged hydroxylated rutile (110) surface in aqueous solution, containing divalent (Mg(2+), Ca(2+), or Sr(2+)) or monovalent (Na(+), K(+), or Rb(+)) cations, was studied by molecular dynamics (MD) simulations. The results indicate that ionic radii and charges will significantly affect the hydration, adsorption geometry, and distance of cations from the rutile surface, thereby regulating the Asp/rutile binding mode. The adsorption strength of monovalent cations on the rutile surface in the order Na(+) > K(+) > Rb(+) shows a "reverse" lyotropic trend, while the divalent cations on the same surface exhibit a "regular" lyotropic behavior with decreasing crystallographic radii (the adsorption strength of divalent cations: Sr(2+) > Ca(2+) > Mg(2+)). The Asp side chain in NaCl, KCl, and RbCl solutions remains stably H-bonded to the surface hydroxyls and the inner-sphere adsorbed compensating monovalent cations act as a bridge between the COO(-) group and the rutile, helping to "trap" the negatively charged Asp side chain on the negatively charged surface. In contrast, the mediating divalent cations actively participate in linking the COO(-) group to the rutile surface; thus the Asp side chain can remain stably on the rutile (110) surface, even if it is not involved in any hydrogen bonds with the surface hydroxyls. Inner- and outer-sphere geometries are all possible mediation modes for divalent cations in bridging the peptide to the rutile surface.     

Thermoresponsive copolymer nanofilms for controlling cell adhesion, growth, and detachment

This study reports the development and use of a novel thermoresponsive polymeric nanofilm for controlling cell adhesion and growth at 37 °C, and then cell detachment for cell recovery by subsequent temperature drop to the ambient temperature, without enzymatic cleavage or mechanical scraping. A copolymer, poly(N-isopropylacrylamide-co-hydroxypropyl methacrylate-co-3-(trimethoxysilyl)propyl methacrylate) (abbreviated PNIPAAm copolymer), was synthesized by free radical polymerization. The thermoresponses of the copolymer in aqueous solution were demonstrated by dynamic light scattering (DLS) through detecting the sensitive changes of copolymer aggregation against temperature. The DLS measurements revealed the lower critical solution temperature (LCST) at approximately 30 °C. The PNIPAAm film stability and robustness was provided through silyl cross-linking within the film and with the hydroxyl groups on the substrate surface. Film thickness, stability, and reversibility with respect to temperature switches were examined by spectroscopic ellipsometry (SE), atomic force microscopy (AFM), and contact angle measurements. The results confirmed the high extent of thermosensitivity and structural restoration based on the alterations of film thickness and surface wettability. The effective control of adhesion, growth, and detachment of HeLa and HEK293 cells demonstrated the physical controllability and cellular compatibility of the copolymer nanofilms. These PNIPAAm copolymer nanofilms could open up a convenient interfacial mediation for cell film production and cell expansion by nonenzymatic and nonmechanical cell recovery.     

Effect of linker and spacer on the design of a fibronectin-mimetic peptide evaluated via cell studies and AFM adhesion forces

The design of a fibronectin-mimetic peptide that specifically binds to the alpha 5beta 1 integrin has been widely studied because of this integrin's participation in many physiological and pathological processes. A promising design for such a peptide includes both the primary binding site RGD and the synergy site PHSRN connected by a linker and extended off of a surface by a spacer. Our original hypothesis was that the degree of hydrophobicity/hydrophilicity between the two sequences (RGD and PHSRN) in fibronectin is an important parameter in designing a fibronectin-mimetic peptide (Mardilovich, A.; Kokkoli, E. Biomacromolecules 2004, 5, 950-957). A peptide-amphiphile, PR_b, that was previously designed in our laboratory employed a hydrophobic tail connected to the N terminus of a peptide headgroup that was composed of a spacer, the synergy site sequence, a linker mimicking both the distance and hydrophobicity/hydrophilicity present in the native protein fibronectin (thus presenting an overall "neutral" linker), and finally the primary binding sequence. Even though our previous work (Mardilovich, A.; Craig, J. A.; McCammon, M. Q.; Garg, A.; Kokkoli, E. Langmuir 2006, 22, 3259-3264) demonstrated that PR_b is a promising sequence compared to fibronectin, this is the first study that tests our hypothesis by comparing PR_b to other peptides with hydrophobic or hydrophilic linkers. Furthermore, different peptide-amphiphiles were designed that could be used to study the effect of building blocks systematically, such as the peptide headgroup linker length and hydrophobicity/hydrophilicity as well as the headgroup spacer length on integrin adhesion. Circular dichroism spectroscopy was first employed, and the collected spectra demonstrated that only one peptide-amphiphile exhibited a secondary structure. Their surface topography was evaluated by taking atomic force microscopy (AFM) images of Langmuir-Blodgett peptide-amphiphile membranes supported on mica. Their adhesion was first evaluated with AFM force measurements between the different sequences and an AFM tip functionalized with purified integrins. The amphiphiles were further characterized via 1-12 h cell studies that examined human umbilical vein endothelial cell adhesion and extracellular matrix fibronectin production. The AFM studies were in good agreement with the cell studies. Overall, the adhesion studies validated our hypothesis and demonstrated for the first time that a "neutral" linker, which more closely mimics the cell adhesion domain of fibronectin, supports higher levels of adhesion compared to other peptide designs with a hydrophobic or hydrophilic linker or even fibronectin. Neutral linker lengths that were within the distance found between PHSRN and RGD in fibronectin performed equally well. However, the 10 amino acid neutral linker gave slightly better cell adhesion than did the control fibronectin at all times. Also, a short spacer was shown to give higher adhesion than other sequences with no spacer or a longer spacer, suggesting that a short spacer is necessary to extend the sequence further away from the interface. In conclusion, this work outlines a logical approach that can be applied for the rational design of any protein-mimetic peptide with two binding sites.     

Self-assembly and bioactivity of a polymer/peptide conjugate containing the RGD cell adhesion motif and PEG

The self-assembly and bioactivity of the peptide–polymer conjugate DGRFFF–PEG3000 containing the RGD cell adhesion motif has been examined, in aqueous solution. The conjugate is designed to be amphiphilic by incorporation of three hydrophobic phenylalanine residues as well as the RGD unit and a short poly(ethylene glycol) (PEG) chain of molar mass 3000 kg mol⁻¹. Above a critical aggregation concentration, determined by fluorescence measurements, signals of β-sheet structure are revealed by spectroscopic measurements, as well as X-ray diffraction. At high concentration, a self-assembled fibril nanostructure is revealed by electron microscopy. The fibrils are observed despite PEG crystallization which occurs on drying. This suggests that DGRFFF has an aggregation tendency that is sufficiently strong not to be prevented by PEG crystallization. The adhesion, viability and proliferation of human corneal fibroblasts was examined for films of the conjugate on tissue culture plates (TCPs) as well as low attachment plates. On TCP, DGRFFF–PEG3000 films prepared at sufficiently low concentration are viable, and cell proliferation is observed. However, on low attachment surfaces, neither cell adhesion nor proliferation was observed, indicating that the RGD motif was not available to enhance cell adhesion. This was ascribed to the core–shell architecture of the self-assembled fibrils with a peptide core surrounded by a PEG shell which hinders access to the RGD unit.     

Stimulation of human vascular endothelial cell growth by a platelet-derived growth factor and thrombin

Repair of a vascular wound is mediated by migration and subsequent replication of the endothelial cells that form the inner lining of blood vessels. We have measured the growth response of human umbilical vein endothelial cells (HuE) to two polypeptides that are transiently produced in high concentrations at the site of a wound; the platelet-derived growth factor (PDGF) and the protease thrombin. When 10(4) HuE cells are seeded as a dense island (2-mm diameter) in the center of a 16-mm tissue culture well in medium containing 20% human serum derived from platelet-poor plasma (PDS), no increase in cell number or colony size is observed. With the addition of 0.5 ng/ml partially purified PDGF, colony size increases and the number of cells after 8 days is 4.8 X 10(4). When human thrombin (1 microgram/ml) is added along with the PDGF, the cell number rises to 9.2 X 10(4). Thrombin alone stimulates no increase in cell number. Although partially purified PDGF stimulates endothelial cells maintained in PDS as well as those maintained in whole blood serum (WBS), pure PDGF is active only when assayed in medium that contains WBS and is supplemented with thrombin. These results suggest the existence of a second class of platelet-derived factors that enable HuE cells to respond to the mitogenic activity of the purified platelet mitogen and thrombin.     

The expression patterns of vascular endothelial growth factor and thrombospondin 2 after corneal alkali burn

The aim of the investigation was to explore the expression patterns of VEGF and TSP2 after corneal alkali burn in vivo. After the model of corneal alkali burn was established in mice, the expression levels of VEGF and TSP2 were determined by immunohistochemistry (IHC), RT-PCR, image analysis and statistical evaluation. Compared with control group, the expression level of VEGF increased significantly at 6h after alkali burn and reached its maximum at 12h. Then, it increased again till the second peak appeared at 96h and 192h. The VEGF-positive reaction mainly gathered in the stroma of cornea. On the other hand, the expression of TSP2 enhanced at 3h and attained two peaks at 6h and 96h, respectively, with the process of wound healing. TSP2 was expressed mainly in the base of epithelial layer. The expression patterns of VEGF and TSP2 reflect the complicated interaction with many factors including promoted and inhibited vascularization in vivo. Moreover, it might provide a novel method for controlling vascular hyperplasia in future clinical work according to the data of VEGF and TSP2.     

Biomedical significance of endothelial cell specific growth factor, angiopoietin

Until recently, vascular endothelial growth factor (VEGF) was the only growth factor proven to be specific and critical for blood vessel formation. Other long-known factors, such as the fibroblast growth factors (FGFs), platelet-derived growth factor, or transforming growth factor-beta, had profound effects in endothelial cells. But such factors were nonspecific, in that they could act on many other cells, and it seemed unlikely that these growth factors would be effective targets for treatment of endothelial cell diseases. A recently discovered endothelial cell specific growth factor, angiopoietin, has greatly contributed to our understanding of the development, physiology, and pathology of endothelial cells (Davis et al., 1996; Yancopoulos et al., 2000). The recent studies that identified and characterized the physiological and pathological roles of angiopoietin have allowed us to widen and deepen our knowledge about blood vessel formation and vascular endothelial function. Therefore, in this review, we describe the biomedical significance of these endothelial cell growth factors, the angiopoietins, in the vascular system under normal and pathological states.     

Recombinant tetra-cell adhesion motifs supports adhesion, migration and proliferation of keratinocytes/fibroblasts, and promotes wound healing

An extracellular matrix protein plays an important role in skin wound healing. In the present study, we engineered a recombinant protein encompassing the 9(th) and 10(th) type III domains of fibronectin, and 4(th) FAS1 domain of betaig-h3. This recombinant protein, in total, harbors four known-cell adhesion motifs for integrins: Pro-His-Ser-Arg-Asn (PHSRN) and Arg-Gly-Asp (RGD) in 9(th) and 10(th) type III domains of fibronectin, respectively, and Glu-Pro-Asp-Ile-Met (EPDIM) and Try-His (YH) in 4(th) FAS1 domain of betaig-h3, were designated to tetra-cell adhesion motifs (T-CAM). In vitro studies showed T-CAM supporting adhesion, migration and proliferation of different cell types including keratinocytes and fibroblasts. In an animal model of full-thickness skin wound, T-CAM exhibited excellent wound healing effects, superior to both 4(th) FAS1 domain of betaig-h3 or 9(th) and 10(th) type III domains of fibronectin. Based on these results, T-CAM can be applied where enhancement of cell adhesion, migration and proliferation are desired, and it could be developed into novel wound healing drug.     

Inflammatory mimetic microfluidic chip by immobilization of cell adhesion molecules for T cell adhesion

Leukocyte adhesion to adhesion molecules on endothelial cells is important in immune function, cancer metastasis and inflammation. This cell-cell binding is mediated via cell adhesion molecules such as E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) found on endothelial cells. Because these adhesion molecules on endothelial cells vary significantly across several disease conditions such as autoimmune diseases, inflammation or cancer metastasis, investigations of therapeutic agents that down-regulate leukocyte-endothelial interactions have been based on in vitro models using endothelial cell lines. Here we report a new model, an inflammatory mimetic microfluidic chip, which emulates leukocyte binding to cell adhesion molecules (CAM) by controlling the types and ratio of adhesion molecules. In our model, E-selectin was essential for the synergic binding of Jurkat T cells. Immunosuppressive drugs, such as tacrolimus (FK506) and cyclosporine A (CsA), were used to inhibit T cell interactions under the physiologic model of T cell migration at a ratio of 5?:?4.3?:?3.9 (E-selectin?:?ICAM-1?:?VCAM-1). Our results support the potential usefulness of the inflammatory mimetic microfluidic chip as a T cell adhesion assay tool with modified adhesion molecules for applications such as immunosuppressive drug screening. The inflammatory mimetic microfluidic chip can also be used as a biosensor in clinical diagnostics, drug efficacy tests and high throughput drug screening due to the dynamic monitoring capability of the microfluidic chip.     

Regulation of charged groups and laminin patterns for selective neuronal adhesion

Primary neuronal cultures on substrates patterned with extracellular matrix proteins such as laminin have yielded much information regarding the physiological characteristics of neuronal cells in vitro. Surface charge also influences neuronal adherence, and a positive charge can have stimulatory effects. The attraction between laminin patterns and polycation films are of interest in the study of neuronal adhesion. We cultured primary hippocampal neurons on poly(ethylenimine) (PEI) films with laminin grids and evaluated their viability and morphology by means of fluorescent microscopy after 5–7 days. The results showed that the neurons did not form networks on the laminin grids. It is inferred that the PEI films were more favourable for neuronal adhesion than the laminin grid.     

Protein adsorption and cell adhesion on RGD-functionalized silicon substrate surfaces

A method was developed to modify silicon surfaces with good protein resistance and specific cell attachment. A silicon surface was initially deposited using a block copolymer of N-vinylpyrrolidone (NVP) and 2-hydroxyethyl methacrylate (HEMA) (PVP-b-PHEMA) film through surface-initiated atom transfer radical polymerization and then further immobilized using a short arginine-glycine-aspartate (RGD) peptide. Our results demonstrate that the RGD-modified surfaces (Si-RGD) can suppress non-specific adsorption of proteins and induce the adhesion of L929 cells. The Si-RGD surface exhibited higher cell proliferation rates than the unmodified silicon surface. This research established a simple method for the fabrication of dual-functional silicon surface that combines antifouling and cell attachment promotion.