silicon and arginyl-glycyl-aspartic-acid

This paper describes a simple strategy for covalent immobilization of the NHS-PEG-RGD peptide with the three different PEG lengths (8, 13, and 22) onto the amine-terminated monolayers with the subsequent investigation of fibroblast cellular response to the three derivatives of pegylated RGD peptides-modified substrates. First, acetamide-terminated monolayers were prepared on the hydride terminated silicon surface to protect NH2-terminated monolayers. This was followed by the removal of the protective groups, and the reaction of NHS-PEG8-RGD, NHS-PEG13-RGD and NHS-PEG22-RGD peptides with the NH2-terminated monolayers while reducing nonspecific protein adsorption. Analysis of X-ray photoelectron spectroscopy (XPS), Fourier Transform Infrared (ATR-FTIR) spectroscopy, and Ellipsometry measurements demonstrated that PEG13-RGD peptide forms relatively a more homogenous, thicker and stable structure compared with those of PEG8-RGD and PEG22-RGD peptide. The quantitative and qualitative assessment of cell adhesion, spreading, and proliferation indicated that relatively further elongated fibroblast cells attached on the PEG13-RGD peptide relative to those on the PEG8-RGD and PEG22-RGD peptide. The results presented here may offer a developed strategy based on the length of the spacer to regulate cellular behavior on the surface substrates.

Topics: Cell Adhesion; Fibroblasts; Humans; Oligopeptides; Polyethylene Glycols; Silicon; Spectroscopy, Fourier Transform Infrared; Surface Properties

The concept of high-throughput screening sheds new light on fabrication and analysis of materials. Herein, a combinatorial surface-modified platform with biochemical gradients was developed through thiol-ene "click" chemistry by adjusting the intensity of ultraviolet (UV) irradiation. Contact angle, X-ray photoelectron spectroscopy, and ellipsometry measurement results demonstrated that the sulfhydryl molecules including polyethylene glycol and RGD (arginine-glycine-aspartic acid) and REDV (arginine-glutamic acid-aspartic acid-valine) peptides can be directly attached onto alkene-modified substrates, in which the graft density can be well controlled by the intensity of UV irradiation. The multistep attachment of different molecules onto substrates is archived via the multistep UV-initiated thiol-ene "click" reaction. The high-throughput arrays with the gradient density of single ligand and the orthogonal gradient density of two ligands were rapidly fabricated via the one-step UV gradient irradiation and the two-step orthogonal UV gradient-initiated thiol-ene "click" reaction. Endothelial cells (ECs) and smooth muscle cells (SMCs) were cocultured on the array with the orthogonal gradient density of RGD and REDV to screen the peptide combination with high EC selectivity, which is essential for in situ endothelialization during stent implant. From 64, 8 × 8, combinations investigated, a special combinatorial surface representing the really high competitiveness of ECs over SMCs was screened. This platform puts forward a facile, high-throughput method to study the combinatorial variation of biochemical signals to cell behavior.

Topics: Alkenes; Cell Adhesion; Click Chemistry; Coculture Techniques; Glass; High-Throughput Screening Assays; Human Umbilical Vein Endothelial Cells; Humans; Myocytes, Smooth Muscle; Oligopeptides; Polyethylene Glycols; Proof of Concept Study; Silicon; Sulfhydryl Compounds; Ultraviolet Rays

The field of implantable electronics relies on using silicon materials due to the merits of a well-established fabrication process and favorable properties; of particular interest is the surface modification of such materials. In the present study, we introduce a surface modification technique based on coatings of functionalized Parylene on silicon substrates, where the modified layers provide a defined cell adhesion capability for the resultant silicon materials/devices. Functionalization of Parylene was achieved during a one-step chemical vapor deposition (CVD) polymerization process, forming NHS ester-functionalized Parylene, and subsequent RGD attachment was enabled via a conjugation reaction between the NHS ester and amine groups. The modification procedures additionally provided a clean and gentle approach to avoid thermal excursions, intense irradiation, chemicals, or solvents that might damage delicate structures or sensitive molecules on the devices. The modification layers exhibited excellent mechanical strength on the substrate, meeting the high standards of the American Society for Testing and Materials (ASTM), and the resultant cell adherence property was verified by a centrifugation assay and the analysis of attached cell morphologies; the results collectively demonstrated robust and sustainable modification layers of the NHS ester-functionalized Parylene and confirmed that the cell-adherence property imparted by using this facile modification technique was effective. The modification technology is expected to benefit the design of prospective interface properties for silicon-based devices and related industrial products.

Topics: 3T3 Cells; Animals; Cell Adhesion; Cell Line; Cell Proliferation; Cell Survival; Coated Materials, Biocompatible; Electronics, Medical; Epithelial Cells; Esters; Mice; Oligopeptides; Polymers; Prostheses and Implants; Silicon; Structure-Activity Relationship; Surface Properties; Volatilization; Xylenes

Graphene derivatives have immense potential in stem cell research. Here, we report a three-dimensional graphene/arginine-glycine-aspartic acid (RGD) peptide nanoisland composite effective in guiding the osteogenesis of human adipose-derived mesenchymal stem cells (ADSCs). Amine-modified silica nanoparticles (SiNPs) were uniformly coated onto an indium tin oxide electrode (ITO), followed by graphene oxide (GO) encapsulation and electrochemical deposition of gold nanoparticles. A RGD-MAP-C peptide, with a triple-branched repeating RGD sequence and a terminal cysteine, was self-assembled onto the gold nanoparticles, generating the final three-dimensional graphene-RGD peptide nanoisland composite. We generated substrates with various gold nanoparticle-RGD peptide cluster densities, and found that the platform with the maximal number of clusters was most suitable for ADSC adhesion and spreading. Remarkably, the same platform was also highly efficient at guiding ADSC osteogenesis compared with other substrates, based on gene expression (alkaline phosphatase (ALP), runt-related transcription factor 2), enzyme activity (ALP), and calcium deposition. ADSCs induced to differentiate into osteoblasts showed higher calcium accumulations after 14-21 days than when grown on typical GO-SiNP complexes, suggesting that the platform can accelerate ADSC osteoblastic differentiation. The results demonstrate that a three-dimensional graphene-RGD peptide nanoisland composite can efficiently derive osteoblasts from mesenchymal stem cells.

Topics: Adipose Tissue; Cell Differentiation; Cells, Cultured; Gold; Graphite; Humans; Mesenchymal Stem Cells; Metal Nanoparticles; Nanocomposites; Oligopeptides; Osteogenesis; Silicon

In this work, we report on a novel approach to develop hierarchically-structured cell culture platforms incorporating functionalized gold nanoparticles (AuNPs). In particular, the hierarchical substrates comprise primary pseudo-periodic arrays of silicon microcones combined with a secondary nanoscale pattern of homogeneously deposited AuNPs terminated with bio-functional moieties. AuNPs with various functionalities (i.e. oligopeptides, small molecules and oligomers) were successfully attached onto the microstructures. Experiments with PC12 cells on hierarchical substrates incorporating AuNPs carrying the RGD peptide showed an impressive growth and NGF-induced differentiation of the PC12 cells, compared to that on the NP-free, bare, micropatterned substrates. The exploitation of the developed methodology for the binding of AuNPs as carriers of specific bio-functional moieties onto micropatterned culture substrates for cell biology studies is envisaged.

Topics: Animals; Biocompatible Materials; Cell Differentiation; Cell Proliferation; Gold; Metal Nanoparticles; Nanostructures; Oligopeptides; PC12 Cells; Rats; Silicon; Surface Properties

Ocular neovascularization can result in devastating diseases that lead to marked vision impairment and eventual visual loss. In clinical implementation, neovascular eye diseases are first diagnosed by fluorescein angiography and then treated by multiple intravitreal injections, which nevertheless involves vision-threatening complications, as well as lack of real-time monitoring disease progression and timely assessment of therapeutic outcomes. To address this critical issue, we herein present a kind of theranostic agents made of peptide-functionalized silicon nanoparticles (SiNPs), suitable for simultaneous ocular neovascularization imaging and therapy. Typically, in addition to negligible toxicity and high specific binding ability to human retinal microvascular endothelial cells tube formation, the cyclo-(Arg-Gly-Asp-d-Tyr-Cys) ( c-(RGDyC))-conjugated SiNPs (SiNPs-RGD) features efficacious antiangiogenic ability in wound healing migration, transwell migration, transwell invasion, and tube formation assays. Taking advantage of these unique merits, we further employ the SiNPs-RGD for labeling angiogenic blood vessels and neovascularization suppression, demonstrating obvious inhibition of new blood vessels formation in mouse corneas. These results suggest the SiNPs-RGD as a novel class of high-quality theranostic probes is suitable for simultaneous diagnosis and treatment in ocular neovascular diseases.

Topics: Animals; Biocompatible Materials; Cell Line; Drug Stability; Fluorescent Dyes; Humans; MCF-7 Cells; Mice; Nanoparticles; Neovascularization, Pathologic; Oligopeptides; Optical Imaging; Retina; Silicon; Theranostic Nanomedicine; Time Factors; Tissue Distribution

Gastrin-releasing-peptide (GRP)-receptors and αvβ3-integrins are widely discussed as potential target structures for oncological imaging with positron emission tomography (PET). Favored by the overexpression of receptors on the surface of tumor cells good imaging characteristics can be achieved with highly specific radiolabeled receptor ligands. PEGylated bombesin (PESIN) derivatives as specific GRP receptor ligands and RGD (one-letter codes for arginine-glycine-aspartic acid) peptides as specific αvβ3 binders were synthesized and tagged with a silicon-fluorine-acceptor (SiFA) moiety. The SiFA synthon allows for a fast and highly efficient isotopic exchange reaction at room temperature giving the [(18)F]fluoride labeled peptides in up to 62% radiochemical yields (d.c.) and ≥99% radiochemical purity in a total synthesis time of less than 20 min. Using nanomolar quantities of precursor high specific activities of up to 60 GBq μmol(-1) were obtained. To compensate the high lipophilicity of the SiFA moiety various hydrophilic structure modifications were introduced leading to significantly reduced logD values. Competitive displacement experiments with the PESIN derivatives showed a 32 to 6 nM affinity to the GRP receptor on PC3 cells, and with the RGD peptides a 7 to 3 μM affinity to the αvβ3 integrins on U87MG cells. All derivatives proved to be stable in human plasma over at least 120 min. Small animal PET measurements and biodistribution studies revealed an enhanced and specific accumulation of the RGD peptide (18)F-SiFA-LysMe3-γ-carboxy-d-Glu-RGD (17) in the tumor tissue of U87MG tumor-bearing mice of 5.3% ID/g whereas the PESIN derivatives showed a high liver uptake and only a low accumulation in the tumor tissue of PC3 xenografts. Stability studies with compound 17 provided further information on its metabolism in vivo. These results altogether demonstrate that the reduction of the overall lipophilicity of SiFA tagged RGD peptides is a promising approach for the generation of novel potent (18)F-labeled imaging agents.

Topics: Animals; Bombesin; Female; Fluorine Radioisotopes; Humans; Male; Mice; Mice, Nude; Mice, SCID; Molecular Imaging; Molecular Probes; Molecular Structure; Neoplasms, Experimental; Oligopeptides; Positron-Emission Tomography; Silicon; Tumor Cells, Cultured

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.

Topics: Biocompatible Materials; Cell Adhesion; Electrons; Extracellular Matrix; Fibroblast Growth Factors; Focal Adhesions; Human Umbilical Vein Endothelial Cells; Humans; Integrins; Ligands; Methacrylates; Oligopeptides; Polyethylene Glycols; Silicon

Organised nanotopography mimicking the natural extracellular matrix can be used to control morphology, cell motility, and differentiation. However, it is still unknown how specific cell types react with specific patterns. Both initial adhesion and preferential cell migration may be important to initiate and increase cell locomotion and coverage with cells, and thus achieve an enhanced wound healing response around an implantable material. Therefore, the aim of this study was to evaluate how MC3T3-E1 osteoblast initial adhesion and directional migration are influenced by nanogrooves with pitches ranging from 150 nm up to 1000 nm. In this study, we used a multi-patterned substrate with five different groove patterns and a smooth area with either a concentric or radial orientation. Initial cell adhesion measurements after 10 s were performed using atomic force spectroscopy-assisted single-cell force spectroscopy, and demonstrated that nascent cell adhesion was highly induced by a 600 nm pitch and reduced by a 150 nm pitch. Addition of RGD peptide significantly reduced adhesion, indicating that integrins and cell adhesive proteins (e.g. fibronectin or vitronectin) are key factors in specific cell adhesion on nanogrooved substrates. Also, cell migration was highly dependent on the groove pitch; the highest directional migration parallel to the grooves was observed on a 600 nm pitch, whereas a 150 nm pitch restrained directional cell migration. From this study, we conclude that grooves with a pitch of 600 nm may be favourable to enhance fast wound closure, thereby promoting tissue regeneration.

Topics: Animals; Biocompatible Materials; Cell Adhesion; Cell Adhesion Molecules; Cell Movement; Cells, Cultured; Integrins; Mice; Microscopy, Atomic Force; Nanostructures; Oligopeptides; Osteoblasts; Silicon; Surface Properties; Tissue Engineering; Wound Healing

Multifunctionality is gaining more and more importance in the field of improved biomaterials. Especially peptides feature a broad chemical variability and are versatile mediators between inorganic surfaces and living cells. Here, we synthesized a unique peptide that binds to SiO(2) with nM affinity. We equipped the peptide with the bioactive integrin binding c[RGDfK]-ligand and a fluorescent probe by stepwise Diels-Alder reaction with inverse electron demand and copper(I) catalyzed azide-alkyne cycloaddition. For the first time, we report the generation of a multifunctional peptide by combining these innovative coupling reactions. The resulting peptide displayed an outstanding binding to silicon oxide and induced a significant increase in cell spreading and cell viability of osteoblasts on the oxidized silicon surface.

Topics: Alkynes; Azides; Biocompatible Materials; Biotin; Catalysis; Cell Line, Tumor; Cell Survival; Click Chemistry; Copper; Cycloaddition Reaction; Drug Design; Enzyme-Linked Immunosorbent Assay; Fluorescent Dyes; Humans; Integrin alphaVbeta3; Oligopeptides; Osteoblasts; Peptides, Cyclic; Silicon; Silicon Dioxide; Surface Properties

We report the use of copper(I)-catalyzed alkyne-azide cycloaddition reaction (CuAAC) to selectively functionalize the internal and external surfaces of mesoporous materials. Porous silicon rugate filters with narrow line width reflectivity peaks were employed to demonstrate this selective surface functionalization approach. Hydrosilylation of a dialkyne species, 1,8-nonadiyne, was performed to stabilize the freshly fabricated porous silicon rugate filters against oxidation and to allow for further chemical derivatization via "click" CuAAC reactions. The external surface was modified through CuAAC reactions performed in the absence of nitrogen-based Cu(I)-stabilizing species (i.e., ligand-free reactions). To subsequently modify the interior pore surface, stabilization of the Cu(I) catalyst was required. Optical reflectivity measurements, water contact angle measurements, Fourier transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS) were used to demonstrate the ability of the derivatization approach to selectively modify mesoporous materials with different surface chemistry on the exterior and interior surfaces. Furthermore, porous silicon rugate filters modified externally with the cell-adhesive peptide Gly-Arg-Gly-Asp-Ser (GRGDS) allowed for cell adhesion via formation of focal adhesion points. Results presented here demonstrate a general approach to selectively modify mesoporous silicon samples with potential applications for cell-based biosensing.

Topics: Acetylene; Animals; Azides; Biosensing Techniques; Catalysis; Cattle; Cell Adhesion; Cells, Immobilized; Click Chemistry; Copper; Endothelial Cells; Hydrogen; Oligopeptides; Organometallic Compounds; Porosity; Silicon; Surface Properties

Mesoporous porous silicon (PSi) microcavity sensors are used to conduct conventional solid-phase peptide synthesis. The sensor optical response provides a convenient means to monitor the synthesis reaction in a nondestructive manner. Measurements indicate that peptide synthesis occurs only when the PSi sensor/scaffold is amine-terminated using, for example, the amino silane or deprotected acid-labile Rink linker. Equivalent coupling efficiencies of the first amino acid to both amine terminations are observed. Kinetic studies indicate that coupling reactions are 90% complete in 1 h. Quantitative analysis of the optical response following the synthesis of homo-oligopeptides (4-mers) suggests that coupling efficiencies and/or optical thickness changes depend on the peptide length. The synthesis of the cell adhesive oligopeptide (RGD) was monitored by the optical sensor response and validated by the cell culture of primary dermal fibroblasts. Secondary ion mass spectrometry (SIMS) analysis successfully detected peptide on the silicon wafer adjacent to the PSi. Our findings suggest the potential to exploit the high surface area, efficient coupling, and intrinsic optical detection properties of PSi for label-free high-throughput screening.

Topics: Amino Acids; Cells, Cultured; Electrodes; Fibroblasts; Humans; Kinetics; Oligopeptides; Optics and Photonics; Particle Size; Porosity; Silicon; Spectrometry, Mass, Secondary Ion; Surface Properties

We demonstrate for the first time the formation of a fluid lipid bilayer membrane on mesoporous silicon substrates for bioapplications. Using fluorescence recovery after photobleaching, the diffusion coefficients for the bilayers supported on oxidized, amino-, and biotin-functionalized mesoporous silicon were determined. The biodetection of a single human umbilical vein endothelial cell was accomplished using confocal microscopy and exploiting Foerster resonance energy transfer effects after the incorporation of RGD covalently linked lipid soluble dyes, with fluorescence donor and acceptor components, within the fluid membrane. A signal response of greater than 100% was achieved via the clustering of RGD peptides binding with areas of high integrin density on the surface of a single cell. These results are a testament to the usefulness of such functional molecular assemblies, based on mobile receptors, mimicking the cell membrane in the development of a new generation of biosensors.

Topics: Biosensing Techniques; Endothelial Cells; Humans; Lipid Bilayers; Oligopeptides; Photobleaching; Porosity; Silicon; Umbilical Veins

Recent efforts in our laboratory have focused on developing methods for immobilizing bioactive peptides to low cell-adhesive dextran monolayer coatings and promoting biospecific cell adhesion for biomaterial implant applications. In the current study, this dextran-based bioactive coating technology was developed for silicon, polyimide, and gold, the base materials utilized to fabricate our prototype neural implants. Chemical composition of all modified surfaces was verified by X-ray photoelectron spectroscopy (XPS). We observed that surface-immobilized dextran supported very little cell adhesion in vitro (24-h incubation with serum-supplemented medium) on all base materials. Inactive nonadhesion-promoting Gly-Arg-Ala-Asp-Ser-Pro peptides immobilized on dextran-coated materials promoted adhesion and spreading at low levels not significantly different from dextran-coated substrates. Arg-Gly-Asp (RGD) peptide-grafted surfaces were observed to promote substantial fibroblast and glial cell adhesion with minimal PC12 (neuronal cell) adhesion. In contrast, dextran-coated materials with surface-grafted laminin-based, neurite-promoting Ile-Lys-Val-Ala-Val (IKVAV) peptide promoted substantial neuron cell adhesion and minimal fibroblast and glial cell adhesion. It was concluded that neuron-selective substrates are feasible using dextran-based surface chemistry strategies. The chemical surface modifications could be utilized to establish a stable neural tissue-implant interface for long-term performance of neural prosthetic devices.

Topics: Amino Acid Sequence; Biocompatible Materials; Dextrans; Neurons; Oligopeptides; Prostheses and Implants; Silicon; Surface Properties

The ability of biomaterial surfaces to regulate cell behavior requires control over surface chemistry and microstructure. One of the greatest challenges with silicon-based biomedical microdevices such as those recently developed for neural stimulation, implantable encapsulation, biosensors, and drug delivery, is to improve biocompatibility and tissue integration. This may be achieved by modifying the exposed silicon surface with bioactive peptides. In this study, Arg-Gly-Asp (RGD) peptide conjugated surfaces were prepared and characterized. The effect of these surfaces on fibroblast adhesion and proliferation was examined over 4 days. Silicon surfaces coupled with a synthetic RGD peptide, as characterized with X-ray photoelectron spectroscopy and atomic force microscopy, display enhanced cell proliferation and bioactivity. Results demonstrate an almost three-fold greater cell attachment! proliferation on RGD immobilized surfaces compared to unmodified (control) silicon surfaces. Modulating the biological response of inorganic materials such as silicon will allow us to design more appropriate interfaces for implantable diagnostic and therapeutic silicon-based microdevices.

Topics: Animals; Cell Adhesion; Cell Division; Cells, Cultured; Electron Probe Microanalysis; Fibroblasts; Microscopy, Atomic Force; Models, Chemical; Oligopeptides; Peptides; Protein Binding; Rats; Rats, Sprague-Dawley; Silanes; Silicon; Time Factors