Surface oxide net charge of a titanium alloy: modulation of fibronectin-activated attachment and spreading of osteogenic cells

In the current study, we have altered the surface oxide properties of a Ti6Al4V alloy using heat treatment or radiofrequency glow discharge (RFGD) in order to evaluate the relationship between the physico-chemical and biological properties of the alloy's surface oxide. The effects of surface pretreatments on the attachment of cells from two osteogenic cell lines (MG63 and MC3T3) and a mesenchymal stem cell line (C3H10T1/2) to fibronectin adsorbed to the alloy were measured. Both heat and RFGD pretreatments produced a several-fold increase in the number of cells that attached to fibronectin adsorbed to the alloy at a range of coating concentrations (0.001-10nM FN) for each cell line tested. An antibody (HFN7.1) directed against the central integrin binding domain of fibronectin produced a 65-70% inhibition of cell attachment to fibronectin-coated disks, indicating that cell attachment to the metal discs was dependent on fibronectin binding to cell integrin receptors. Both treatments also accelerated the cell spreading response manifested by extensive flattening and an increase in mean cellular area. The treatment-induced increases in the cell attachment activity of adsorbed fibronectin were correlated with previously demonstrated increases in Ti6Al4V oxide negative net surface charge at physiological pH produced by both heat and RFGD pretreatments. Since neither treatment increased the adsorption mass of fibronectin, these findings suggest that negatively charged surface oxide functional groups in Ti6Al4V can modulate fibronectin's integrin receptor activity by altering the adsorbed protein's conformation. Our results further suggest that negatively charged functional groups in the surface oxide can play a prominent role in the osseointegration of metallic implant materials.     

Electrochemical attachment of motile bacterial cells to gold

Selective attachment of Escherichia coli K-12 bacterial cells to charged gold surfaces was demonstrated. Electrostatic binding of E. coli K-12 bacterial cells to positively charged surfaces was observed starting at +750 mV. The binding of E. coli K-12 cells to positively charged gold surfaces is proposed to occur due to long-range electrostatic interactions between the negatively charged O-chain of lipopolysaccharide (LPS) molecules protruding the bacterial cell body and the electrode surface. Removing LPS alters the cellular surface charge and results in cellular attachment to negatively charged surfaces. Thus, applying an electrical potential allows for the direct, real time detection of live, dead or damaged bacterial cells. The attachment of E. coli K-12 bacterial cells to surfaces with an applied potential substantiates the hypothesis that an electrostatic interaction is responsible for the binding of bacterial cells to positively charged molecular assemblies on surfaces used for building bacterial microarrays.     

Entropic and electrostatic effects on the folding free energy of a surface-attached biomolecule: an experimental and theoretical study

Surface-tethered biomolecules play key roles in many biological processes and biotechnologies. However, while the physical consequences of such surface attachment have seen significant theoretical study, to date this issue has seen relatively little experimental investigation. In response we present here a quantitative experimental and theoretical study of the extent to which attachment to a charged-but otherwise apparently inert-surface alters the folding free energy of a simple biomolecule. Specifically, we have measured the folding free energy of a DNA stem loop both in solution and when site-specifically attached to a negatively charged, hydroxylalkane-coated gold surface. We find that whereas surface attachment is destabilizing at low ionic strength, it becomes stabilizing at ionic strengths above ~130 mM. This behavior presumably reflects two competing mechanisms: excluded volume effects, which stabilize the folded conformation by reducing the entropy of the unfolded state, and electrostatics, which, at lower ionic strengths, destabilizes the more compact folded state via repulsion from the negatively charged surface. To test this hypothesis, we have employed existing theories of the electrostatics of surface-bound polyelectrolytes and the entropy of surface-bound polymers to model both effects. Despite lacking any fitted parameters, these theoretical models quantitatively fit our experimental results, suggesting that, for this system, current knowledge of both surface electrostatics and excluded volume effects is reasonably complete and accurate.     

分子动力学模拟多巴在自组装膜上的黏附性

为了更好地理解贻贝在表面的黏附机理,实现水下胶黏,采用分子动力学方法研究了多巴在自组装膜上的黏附性:采用伞形取样和加权柱状图分析方法计算了多巴在不同自组装膜表面的黏附自由能,使用拉伸分子动力学模拟研究了多巴在不同自组装膜表面上黏附后的脱附力.结果表明,多巴在带负电的羧基自组装膜上的黏附能比在带正电的氨基自组装膜上的大,多巴更容易黏附到带负电表面;多巴在带电表面的黏附能比未带电表面的黏附能更强,表明在带电表面黏附更稳定.进一步分析了多巴在不同表面的取向分布,发现多巴与不同表面相互作用的方式不同:与疏水表面主要通过苯环相互作用;与亲水表面主要通过羟基相互作用;与负电表面主要通过氨基相互作用;与正电表面主要通过羧基相互作用.通过模拟比较了多巴在不同自组装膜上的脱附力,发现多巴在带电表面的脱附力比在未带电表面的大,与黏附能的趋势一致.对比4种非带电表面的脱附力,发现多巴在疏水性甲基自组装膜表面的脱附力最大,黏附更稳定,随着表面疏水性的增加,脱附力增大,黏附稳定性增强.本工作可为研发新型水下胶黏剂提供理论指导.     

Surface characterizations of mono-, di-, and tri-aminosilane treated glass substrates

The surface properties and structure of mono-, di-, and tri-aminosilane treated glass surfaces were investigated using surface analytical techniques including X-ray photoelectron spectroscopy (XPS), Raman spectroscopy, atomic force microscopy (AFM), and streaming potential. An optimized dip-coating process was demonstrated to produce roughly silane monolayer coverage on the glass surface. The surface charge measurements indicated that aminosilanization converts the glass surface from negative to positive potentials at neutral pH values. Higher positive streaming potential was observed for tri-compared with mono- and di-aminosilane treated glass surfaces. For all aminosilane treated glass samples, the high-resolution N 1s XPS spectra indicated a preferential orientation of the protonated amino-groups towards the glass surface whereas the free amino groups were protruding outward. This study aimed to obtain uniform, reproducibly thin, strongly adhering, internally cross-linked, and high positively charged aminosilane-coated glass surfaces for the attachment of DNA fragments used in microarraying experiments.     

Synthesis of positively charged silver nanoparticles via photoreduction of AgNO3 in branched polyethyleneimine/HEPES solutions

Branched polyethyleneimine (BPEI) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were used collaboratively to reduce silver nitrate under UV irradiation for the synthesis of positively charged silver nanoparticles. The effects of molar ratio of the ingredients and the molecular weight of BPEI on the particle size and distribution were investigated. The mechanism for the reduction of Ag+ ions in the BPEI/HEPES mixtures entails oxidative cleavage of BPEI chains that results in the formation of positively charged BPEI fragments enriched with amide groups as well as in the production of formaldehyde, which serves as a reducing agent for Ag+ ions. The resultant silver nanoparticles are positively charged due to protonation of surface amino groups. Importantly, these positively charged Ag nanoparticles demonstrate superior SERS activity over negatively charged citrate reduced Ag nanoparticles for the detection of thiocyanate and perchlorate ions; therefore, they are promising candidates for sensing and detection of a variety of negatively charged analytes in aqueous solutions using surface-enhanced Raman spectroscopy (SERS).     

Scanning Ion Conductance Microscopic Study for Cellular Uptake of Cationic Conjugated Polymer Nanoparticles

Positively charged conjugated polymer nanoparticles (CPNs) are emerging biomaterials exhibiting high levels of cellular entry. High rate of cellular entry efficiency is believed that the amphiphilic CPNs interact efficiently with the negatively charged hydrophobic cellular membranes. For the first time, the cell surface morphological changes of human cervical cancer cells treated with CPNs using a scanning probe microscopy technique, scanning ion conductance microscopy (SICM) are imaged. After 1 h of CPN incubation, distinct changes are observed in cell surface morphology such as interconnected protrusions and pits with sub‐micrometer sizes, which are not observed from cells treated with positively charged polyethyleneimine (PEI) under the same treatment conditions. The change on cell surface morphology is quantified by surface roughness ratio, which is increased as CPN concentration increases, while the ratio first increases and then decreases as the incubation time increases. These results suggest that cells respond actively toward CPN with both positive charges on the side chain and the hydrophobicity from rigid aromatic backbone, which leads to subsequent endocytosis. In conclusion, it is demonstrated that SICM is a suitable imaging technique to reveal the dynamic alternations on the cell surface morphology at the early stage of nanoparticles endocytosis with high resolution.

     

基于聚苯胺纳米纤维复合膜界面构建电化学细胞传感器及其对癌细胞的识别检测

合成了聚苯胺纳米纤维,直径在50～70 nm之间;基于静电作用构建聚苯胺纳米纤维-纳米金复合膜界面,并在此界面上层层组装修饰叶酸分子,构建叶酸功能化传感界面,基于叶酸分子与癌细胞表面过量表达的叶酸受体之间的特异性识别作用,将此传感界面应用于对癌细胞的识别和捕获。结果表明:叶酸功能化传感界面能够特异性识别和捕获叶酸受体过量表达的癌细胞。采用电化学阻抗技术,以HeLa细胞为模型,应用于对癌细胞的识别和检测,细胞在1.0×104～6.4×106cells/mL浓度范围内与阻抗变化值ΔRct呈良好的线性关系;检出限为2000 cells/mL。本方法简单、快速灵敏、重现性和稳定性良好;制备的传感器可以再生使用。     

The Effects of Radiofrequency Electromagnetic Radiation on the Adhesion Behavior of Aqueous Suspensions

The effect of radiofrequency treatment on the adhesion behavior of amidine and carboxylated polystyrene latexes was investigated. The isoelectric points pH(IEP) of copper and zinc were initially measured as 9.4 and 9.5, respectively, using a technique proposed by N. Kallay, Z. Torbic, M. Golic, and E. Matijevic [J. Phys. Chem. 95, 7028 (1991)] based on the attachment of charged colloids to metallic surfaces in an aqueous medium. Statistical analysis showed that the technique was repeatable with a coefficient of variation less than 6% and an accuracy greater than 95%. The effect of a radiofrequency signal (27 MHz) on adhesion behavior was evaluated and it was found that there was reduced attachment at pH values when the colloid and metallic surface were oppositely charged and enhanced attachment between similarly charged particles. It is proposed that this is due to a reduction in the surface potential of the charged particles due to thickening of the adsorbed layer by hydrogen and hydroxyl ions. Copyright 2000 Academic Press.     

Changes of conductance and compressibility of bilayer lipid membranes induced by oligonucleotide-cationic polyene antibiotic complexes

The positively charged polyene molecule amphotericin B 3-dimethylaminopropylamide (AMA) is an efficient agent for the delivery of antisense oligodeoxyribonucleotides (ODN) into target cells. In the present study, bilayer lipid membrane (BLM) conductance, elasticity modulus perpendicular to the membrane plane, surface potential and electrical capacitance were measured by conductance and electrostriction methods in the presence of AMA, pure or complexed to 20-mer single stranded ODN at different ratios. Pure AMA did not induce changes in conductance of cholesterol-containing BLM, but did induce an increase in elasticity modulus and surface potential. ODN/AMA complexes changed BLM properties depending on the charge ratio. The most pronounced effect on membrane conductance was observed for positively charged ODN/AMA complexes (charge ratio rho-/+=0.1), while for negatively charged complexes these changes were less marked/apparent, correlating to substantially lower binding constants. The effect of ODN/AMA complexes on elasticity modulus and charge potential was biphasic. After an increase in both values, a decrease was observed for higher incubation times and ODN/AMA concentrations. These results are interpreted as indicating that the membrane property changes result from the large AMA aggregates induced by the presence of the negatively charged ODN, which condensate on these aggregates. It is suggested that the decrease of elasticity modulus and surface potential in the presence of increasing incubation time and AMA concentration result from desorption of the complexes in the complex-free compartment of the BLM cell, or appearance of a non-linear conductance of the lipid bilayer. The first alternative would explain the AMA-induced transmembrane transfer of ODN.     

Evidence of surface charge at the air/water interface from thin-film studies on polyelectrolyte-coated substrates

The stability of thin water films on silicon substrates coated with cationic and anionic polyelectrolytes was investigated by the thin film pressure balance technique. Depending on the surface charge of the substrate, the water films are either stable (on negatively charged wafers) or rupture rapidly (on positively charged wafers). It is supposed that this behavior is due to a negative surface charge of the free water surface. The underlying assumption that the films' stability is due to electrostatic interactions is supported by measurements of the disjoining pressure on silicon wafers with a native oxide layer, which indicates a decrease of the film thickness, and thus decreasing repulsive interaction between the two film interfaces, with increasing ionic strength.     

Effect of glutamate on the electrical properties of cationic solid lipid nanoparticles containing stearylamine and dioctadecyldimethyl ammonium bromide

Electrical properties, including electrophoretic mobility, zeta potential, total surface charge density, and surface charge density resulting from primary amino groups, of cationic solid lipid nanoparticles (CSLNs) were investigated in the present study. Cationic lipids including stearylamine (SA) and dioctadecyldimethyl ammonium bromide (DODAB) were covered on the external cores of CSLNs. The influences of glutamate concentration in the medium, composition of cationic lipids, and surfactant species were especially analyzed. The results indicated that an increase in the mole ratio of SA in the cationic lipid caused an increase in the average diameter of CSLNs. Also, the average diameter of Span 20-stabilized CSLNs was larger than that of Tween 80-stabilized CSLNs. The electrostatic traits of CSLNs were reduced as the mole ratio of SA increased, and the electricity of Span 20-stabilized CSLNs was weaker than that of Tween 80-stabilized CSLNs. An increase in the glutamate concentration in the medium led to a decrease in electrophoretic mobility, zeta potential, and total surface charge density of CSLNs. As the glutamate concentration increased, surface charge density resulting from primary amino groups increased, and that from quaternary amino groups decreased as a result of the adsorption of negatively charged glutamate on CSLN surfaces. Ohshima's soft particle theory was adopted to describe the electrical behavior of CSLNs, and the deviations of zeta potential predicted by the Smoluchowski, Happel, and Kuwabara models were normally greater than 10%.     

Preparation of Amino-functionalized Multiwall Carbon Nanotube/Gold Nanoparticle Composites

Multiwall carbon nanotubes (MWNT) were modified orderly with carboxyl groups and amino groups. The MWNT/gold nanoparticle composites were formed when the amino‐functionalized MWNT was interacted with gold colloids. The functionalized MWNT was characterized using Fourier transform infrared spectroscopy and X‐ray photoelectron spectroscopy. The amino‐functionalized MWNT allows further attaching gold nanoparticles through electrostatic interaction between the negatively charged gold nanoparticles and amino groups on the surface of the MWNT. The composite of gold nanoprticles and amino‐functionalized MWNT was characterized by transmission electron microscopy. This method decorating carbon nanotubes can be used to identify the location of functional groups, i.e. defect sites on carbon nanotubes.     

Prediction of the orientations of adsorbed protein using an empirical energy function with implicit solvation

When simulating protein adsorption behavior, decisions must first be made regarding how the protein should be oriented on the surface. To address this problem, we have developed a molecular simulation program that combines an empirical adsorption free energy function with an efficient configurational search method to calculate orientation-dependent adsorption free energies between proteins and functionalized surfaces. The configuration space is searched systematically using a quaternion rotation technique, and the adsorption free energy is evaluated using an empirical energy function with an efficient grid-based calculational method. In this paper, the developed method is applied to analyze the preferred orientations of a model protein, lysozyme, on various functionalized alkanethiol self-assembled monolayer (SAM) surfaces by the generation of contour graphs that relate adsorption free energy to adsorbed orientation, and the results are compared with experimental observations. As anticipated, the adsorbed orientation of lysozyme is predicted to be dependent on the discrete organization of the functional groups presented by the surface. Lysozyme, which is a positively charged protein, is predicted to adsorb on its 'side' on both hydrophobic and negatively charged surfaces. On surfaces with discrete positively charged sites, attractive interaction energies can also be obtained due to the presence of discrete local negative charges present on the lysozyme surface. In this case, 'end-on' orientations are preferred. Additionally, SAM surface models with mixed functionality suggest that the interactions between lysozyme and surfaces could be greatly enhanced if individual surface functional groups are able to access the catalytic cleft region of lysozyme, similar to ligand-receptor interactions. The contour graphs generated by this method can be used to identify low-energy orientations that can then be used as starting points for further simulations to investigate conformational changes induced in protein structure following initial adsorption.     

Electrostatic attachment of gold and poly(lactic acid) nanoparticles onto omega-aminoalkanoic acid self-assembled monolayers on 316L stainless steel

The assembly of poly(lactic acid) (PLA) nanoparticles on a 12-aminodecanoic acid (ADA) self-assembled monolayer (SAM) is described. Assembly is accomplished through electrostatic interactions between the positively charged SAM and the negatively charged PLA nanoparticles. The strategy used involves two steps in which a preliminary electrochemical coating of the ADA SAM is followed by a second step that involves immersing the SAM in a solution containing gold or PLA nanoparticles. The SAM was characterized by using cyclic voltammetry (CV), X-ray photoelectron spectroscopy (XPS), FTIR spectroscopy, and contact angle measurements, whereas scanning electron microscopy (SEM) was used to image the nanoparticles after electrostatic attachment was achieved. We found that the surface coverage of the nanoparticles could be controlled by modulating the electrostatic interactions between the negatively charged particles and the positively charged SAM surface by varying the pH of the nanoparticle solution, the immersion time, and the number of cyclic voltammetry scans under which the SAM was formed.     

Synthesis and capping of water-dispersed gold nanoparticles by an amino acid: bioconjugation and binding studies

We report a novel strategy for the synthesis of aqueous stable, carboxylated gold nanoparticles (GNPs) by using glutamic acid as the reducing agent. The ratio of chloroaurate ions, AuCl(-)(4) to glutamic acid was optimized in the reaction medium to obtain monodispersed GNPs. Glutamic acid reduced gold nanoparticles were characterized by UV-visible, FTIR, dynamic light scattering and transmission electron microscopy, which demonstrated high stability in aqueous solution over a period of time indicating stabilization via surface-bound amino acid. Functionalized nanoparticles were conjugated with protein molecules through electrostatic attraction between the surface-terminated negatively charged carboxylate groups (COO(-)) of glutamic acid and the positively charged amino groups (NH(+)(3)) of the protein. The conjugation efficiency of the GNP:protein conjugates was confirmed qualitatively and quantitatively through gel electrophoresis and critical flocculation concentration analysis. The interaction between functionalized GNPs with protein molecules was investigated using fluorescence spectroscopy showing the fluorescence quenching of the tryptophan residues of protein molecules after conjugation. Circular dichroism (CD) studies of the conjugates confirmed that the protein undergoes a more flexible conformational state on the boundary surface of GNPs after conjugation. There was substantial conformational transition from alpha-helix to beta-sheet structure after conjugation of protein to GNPs.     

Preparation of a Novel Amino-Phosphate Zwitterionic Stationary Phase for Hydrophilic Interaction Chromatography

A new stationary phase which contains both negatively charged phosphate groups and positively charged amino groups was successfully synthesized by modification of amino-functionalized silica particles with trichlorophosphine oxide (POCl₃) for hydrophilic interaction chromatography (HILIC). The composition of the surface grafts was determined by Fourier transform infrared spectroscopy and elemental analysis. Various parameters, such as column temperature, water content, pH values and ionic strength of the mobile phase were investigated to study the retention mechanism. The results demonstrated that the stationary phase involved a complex retention process including surface adsorption, partitioning and electrostatic interactions. Under optimized conditions, the separation of nucleobases and nucleosides, water-soluble vitamins, organic acids on the novel stationary phase could be achieved in the HILIC mode.     

Electrokinetic and thermodynamic analysis of the adsorption process of N-cetylpyridinium chloride on polyester fabric

An electrokinetic and thermodynamic analysis of the adsorption process of N-cetylpyridinium chloride on polyester fabric is described in the present work. The electrokinetic study was performed by means of electrophoretic mobility measurements of the polyester-surfactant system. The most significant result is the increase in electrokinetic potential, zeta, toward more positive values as the surfactant concentration in the dispersion medium is raised. Given the molecular structure of N-cetylpyridinium chloride (N-CP-Cl), which contains a pyridinium group, positively charged, it is feasible that such increase in |zeta| is due to the electrostatic attraction between the carboxyl groups of polyester, ionized at pH 8.5, and the pyridinium group of the surfactant. The uptake of N-CP-Cl by the fiber is experimentally determined at four temperatures: the strong increase in the amount of the surfactant incorporated onto the fiber as the initial concentration of N-CP-Cl is larger shows that the electrostatic attraction between the fiber and the surfactant is the main mechanism of the adsorption of the surfactant onto the fiber. The obtained data on the kinetics and thermodynamics of adsorption of N-cetylpyridinium chloride onto the polyester, standard free energy, enthalpy, and entropy related to the process of adsorption are in accordance with our hypothesis on the mechanisms of adsorption. From a different point of view, the efficient coverage of polyester by N-CP-Cl is also demonstrated by the changes experienced by the surface free energy of polyester upon treatment with N-CP-Cl.     

Water-soluble nonstoichiometric polyelectrolyte complexes of modified chitosan

A modified polysaccharide that, in each deacetylated unit, carries a functional secondary amino group and a quaternized amino group that provides a positive charge and solubility to the polymer throughout the pH range is prepared by the alkylation of primary amino groups of chitosan with glycidyltrimethylammonium chloride. The mixing of modified chitosan solutions with solutions of polystyrenesulfonate or polymethacrylate anions in neutral solutions gives rise to negatively charged nonstoichiometric polyelectrolyte complexes soluble and stable under physiological conditions. The effects of pH, ionic strength, the degree of polymerization, the nature of the lyophilizing polyanion, and the charge-to-charge ratio of components on the boundaries of existence of soluble complexes are ascertained. The collected experimental data may serve as a basis for designing biocompatible and biodegradable means useful for the delivery of genetic material and drugs to living cells.     

Tissue engineering of heart valves by recellularization of glutaraldehyde-fixed porcine valves using bone marrow-derived cells

To increase the biocompatibility and durability of glutaraldehyde (GA)-fixed valves, a biological coating with viable endothelial cells (ECs) has been proposed. However, stable EC layers have not been formed successfully on GA-fixed valves due to their inability to repopulate. In this study, to improve cellular adhesion and proliferation, the GA-fixed prostheses were detoxified by treatment with citric acid to remove free aldehyde groups. Canine bone marrow mononuclear cells (MNCs) were differentiated into EC-like cells and myofibroblast-like cells in vitro. Detoxified prostheses were seeded and recellularized with differentiated bone marrow- derived cells (BMCs) for seven days. Untreated GA-fixed prostheses were used as controls. Cell attachment, proliferation, metabolic activity, and viability were investigated and cell-seeded leaflets were histologically analyzed. On detoxified GA-fixed prostheses, BMC seeding resulted in uninhibited cell proliferation after seven days. In contrast, on untreated GA-fixed prostheses, cell attachment was poor and no viable cells were observed. Positive staining for smooth muscle a-actin, CD31, and proliferating cell nuclear antigen was observed on the luminal side of the detoxified valve leaflets, indicating differentiation and proliferation of the seeded BMCs. These results demonstrate that the treatment of GA-fixed valves with citric acid established a surface more suitable for cellular attachment and proliferation. Engineering heart valves by seeding detoxified GA-fixed biological valve prostheses with BMCs may increase biocompatibility and durability of the prostheses. This method could be utilized as a new approach for the restoration of heart valve structure and function in the treatment of end-stage heart valve disease.