Introducing RGD peptides on PHBV films through PEG-containing cross-linkers to improve the biocompatibility

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biodegradable polyester, has been a good candidate of biomaterial employed in tissue engineering. However, the PHBV film is hydrophobic and has no recognition sites for cell attachment. In this study, PHBV films are activated by ammonia plasma treatment to produce amino groups on the surface, followed by sequential reactions with a heterobifunctional cross-linker containing a segment of poly(ethylene glycol) (PEG) and further with RGD-containing peptides. XPS analyses of modified surfaces after each reaction step reveal that the RGD-containing peptides have been covalently grafted onto PHBV films. The result of cell viability assay indicates that the RGD-modified PHBV films exhibit a distinctly improved cellular compatibility. Moreover, according to the results of serum adsorption tests by optical waveguide lightmode spectroscopy (OWLS) and fibrinogen adsorption tests by enzyme-linked immunosorbent assay (ELISA) on unmodified and modified PHBV surfaces, the introduced PEG chains can significantly decrease the nonspecific adsorption of proteins from serum and fibrinogen from plasma, thus decreasing the risk of thrombus formation and improving the blood compatibility of implanted materials.     

Engineering surfaces for bioconjugation: developing strategies and quantifying the extent of the reactions

This study presents two-step and multistep reactions for modifying the surface of plasma-functionalized poly(tetrafluoroethylene) (PTFE) surfaces for subsequent conjugation of biologically relevant molecules. First, PTFE films were treated by a radiofrequency glow discharge (RFGD) ammonia plasma to introduce amino groups on the fluoropolymer surface. This plasma treatment is well optimized and allows the incorporation of a relative surface concentration of approximately 2-3.5% of amino groups, as assessed by chemical derivatization followed by X-ray photoelectron spectroscopy (XPS). In a second step, these amino groups were further reacted with various chemical reagents to provide the surface with chemical functionalities such as maleimides, carboxylic acids, acetals, aldehydes, and thiols, that could be used later on to conjugate a wide variety of biologically relevant molecules such as proteins, DNA, drugs, etc. In the present study, glutaric and cis-aconitic anhydrides were evaluated for their capability to provide carboxylic functions to the PTFE plasma-treated surface. Bromoacetaldehyde diethylacetal was reacted with the aminated PTFE surface, providing a diethylacetal function, which is a latent form of aldehyde functionality. Reactions with cross-linkers such as sulfo-succinimidyl derivatives (sulfo-SMCC, sulfo-SMPB) were evaluated to provide a highly reactive maleimide function suitable for further chemical reactions with thiolated molecules. Traut reagent (2-iminothiolane) was also conjugated to introduce a thiol group onto the fluoropolymer surface. PTFE-modified surfaces were analyzed by XPS with a particular attention to quantify the extent of the reactions that occurred on the polymer. Finally, surface immobilization of fibronectin performed using either glutaric anhydride or sulfo-SMPB activators demonstrated the importance of selecting the appropriate conjugation strategy to retain the protein biological activity.     

Surface modification of chitosan films. Effects of hydrophobicity on protein adsorption

The surface of chitosan films was modified using acid chloride and acid anhydrides. Chemical composition at the film surface was analyzed by attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS). ATR-FTIR data verified that the substitution took place at the amino groups of chitosan, thus forming amide linkages, and the modification proceeded to the depth at least 1 microm. Choices of molecules substituted at the amino groups of the glucosamine units did affect the hydrophobicity of the film surface, as indicated by air-water contact angle analysis. The surface became more hydrophobic than that of non-modified film when a stearoyl group (C(17)H(35)CO-) was attached to the films. The reaction of chitosan films with succinic anhydride or phthalic anhydride, however, produced more hydrophilic films. Selected modified films were subjected to protein adsorption study. The amount of protein adsorbed, determined by bicinchoninic acid (BCA) assay, related to the types of attached molecules. The improved surface hydrophobicity affected by the stearoyl groups promoted protein adsorption. In contrast, selective adsorption behavior was observed in the case of the chitosan films modified with anhydride derivatives. Lysozyme adsorption was enhanced by H-bonding and charge attraction with the hydrophilic surface. While the amount of albumin adsorbed was decreased possibly due to negative charges that gave rise to repulsion between the modified surface and albumin. This study has demonstrated that it is conceivable to fine-tune surface properties which influence its response to bio-macromolecules by heterogeneous chemical modification.     

Quantification of primary amine groups available for subsequent biofunctionalization of polymer surfaces

Biocompatible polymers are commonly functionalized with specific moieties such as amino groups to modify their surface properties and/or to attach bioactive compounds. A reliable method is usually required to characterize amino group surface densities. In this study, aminated polyethylene terephthalate (PET) films were generated via an aminolysis reaction involving either ethylenediamine molecules (EtDA), in order to vary easily the amino group density on PET surfaces, or 25 kDa polyvinylamine (PVAm) as an alternative reagent preventing bulk damages resulting from the aminolysis reaction. Among commonly used dyes for amino group quantification, Orange II and Coomassie Brillant Blue (CBB) were selected to quantify the extent of amine grafting resulting from these derivatization procedures. Rapid and convenient colorimetric assays were compared to surface atomic compositions obtained from X-ray photoelectron spectroscopy (XPS) measurements. Orange II was found to be the most appropriate dye for quantifying primary amine groups in a reliable and specific way. Due to its unique negative charge and low steric hindrance compared to CBB, the Orange II dye was very sensitive and provided reliable quantification over a wide range of amino group surface densities (ca. 5 to at least 200 pmol/mm(2)). In order to further validate the use of the Orange II dye for amino group quantification, a heterobifunctional linker reacting with amino groups was then grafted on modified PET surfaces. Interestingly, the good correlation between the densities of adsorbed Orange II and covalently grafted linkers suggests that the Orange II method is a relevant, reliable, easy, and inexpensive method to predict the amount of amino groups available for subsequent functionalization of polymer surfaces.     

Human serum albumin adsorption onto a-SiC:H and a-C:H thin films deposited by plasma enhanced chemical vapor deposition

In the present paper, we report the study of the adsorption behavior of a model protein such as human serum albumin (HSA) onto surfaces of a-SiC:H and a-C:H thin films deposited by using the plasma-enhanced chemical vapor deposition (PECVD) technique. The surface composition and surface energy of the various substrates as well as the evaluation of the adsorbed amount of protein has been carried out by means of X-ray photoelectron spectroscopy (XPS) and contact angle measurements. It has been found that HSA tends to preferentially adsorb on Si-rich surfaces, as far as the relative amount of adsorbed HSA decreases with increasing S-C concentration. Preliminary elements of mechanistic models are proposed for the correlation between chemical factors and the observed protein adsorption behavior.     

Preparation and cell compatibility of acrylamide-grafted poly(3-hydroxyoctanoate)

Poly(3-hydroxyoctanoate) (PHO) films were treated with plasma of different discharge powers (10-50 W) and then treated with acryl amide solutions in order to prepare films with surfaces that contained different amounts of amide groups. The surfaces were characterized by contact angle measurement, attenuated total reflectance infrared spectroscopy, electron spectroscopy for chemical analysis, and scanning electron microscopy. Results from all these measurements indicated that amide groups were present on the surfaces. The amount of amide groups increased in proportion to the discharge power of the plasma. The interaction of Chinese hamster ovary cells with these grafted surfaces was investigated. The number of cells that adhered to and grew on the surfaces was highest for films grafted at 30 W of plasma discharge power, indicating that the moderate hydrophilicity was optimal for cells to adhere and grow. The present results support the suggestion that acryl amide-grafted PHO could be used as cell-compatible biomedical applications.     

碳酸酐酶在聚甲基戊烯中空纤维膜表面的固定及其性能分析

采用两步法将碳酸酐酶共价键合在聚甲基戊烯(Polymethyl-pentene,PMP)膜式氧合器表面以提高其清除血液中CO2的能力。首先采用等离子体处理法将羟基引入PMP表面,然后用偶联剂溴化氰(CNBr)将碳酸酐酶(Carbonic anhydrase,CA)固定在PMP膜表面。采用XPS、表面接触角测定仪对等离子体处理后材料表面的物理化学性质进行了表征。以对硝基苯酚乙酸酯(p-nitrophenyl acetate,p-NPA)为底物,采用紫外分光光度计测定了接枝CA的活性、浓度、重复利用性、储存稳定性。结果表明,等离子体处理方法能将羟基成功引入PMP表面;CA能被成功地偶联在无活性官能团聚合物表面,在保持酶活性的同时获得较高的接枝效率;共价接枝CA(Covalently immobilized CA,CACI)的浓度随CNBr浓度的增加而增加,最大可达理论单分子层接枝量的73%;CACI比物理吸附的CA(Physically adsorbed CA,CAPA)具更好的重复利用性;37oC下,CACI比CA溶液表现出更好的储存稳定性。本方法有望应用在膜式氧合器上以提高其对血液中CO2的排除效率。     

Bioactive polymer fibers to direct endothelial cell growth in a three-dimensional environment

This study reports the fabrication of bioactive polymer fibers onto which signaling molecules can control and direct cell responses. To encourage and control directional biological responses, GRGDS peptides were immobilized onto the surface of 100 microm diameter poly(ethylene terephtalate) (PET) fibers (monofilaments). PET fiber surfaces were first coated with a thin polymeric interfacial bonding layer bearing amine groups by plasma polymerization. Carboxy-methyl-dextran (CMD) was covalently grafted onto the surface amine groups using water-soluble carbodiimide chemistry. GRGDS were covalently immobilized onto CMD-coated fiber surfaces. X-ray photoelectron spectroscopy (XPS) analyses enabled characterization of the multilayer fabrication steps. Human umbilical vein endothelial cells were seeded and grown on fibers to investigate cell patterning behavior (i.e., adhesion, spreading, cytoskeleton organization, and cell orientation). Cell adhesion was reduced on CMD-coated fibers, whereas amine- and GRGDS-coated fibers promoted cell adhesion and spreading. Cell adhesion was enhanced as the GRGDS concentration increased. Epifluorescence microscopic visualization of cells on RGD-coated substrates showed well-defined stress fibers and sharp spots of vinculin, typical of focal adhesions. In comparison to plasticware commonly used in cell cultures, fiber curvature promoted cell orientation along the fiber axis.     

Chemical analysis of a cornstarch film surface modified by SF6 plasma treatment

SF₆ plasmas were employed to improve the water repellency of cornstarch films by producing physical as well as chemical modifications of the film surface. Samples were placed in the cathode of a capacitively coupled plasma enhanced chemical vapor deposition (PECVD) reactor. Local surface modifications resulting from SF₆ plasma treatment were evaluated using atomic force microscopy (AFM) and scanning electron microscopy (SEM). Photoelectron spectroscopy (XPS) and Raman spectroscopy were used to characterize sulfur and fluoride incorporation on the surface as well as changes in the chemical state of carbon. The results indicate that fluoride and sulfur incorporation is dependent on the self bias, and fluoride is preferentially incorporated at self-biases higher than 100 V. The carbon chemical state changed, and an amorphous-like layer was formed upon treatment. Surface reticulation was observed, indicated by the formation of a structure that resembled starch recrystallization. Optimized treatment conditions led to water contact angles over 120°.     

Physicochemical and blood compatibility characterization of polypyrrole surface functionalized with heparin

A surface modification technique was developed for the covalent immobilization of heparin onto electrically conductive polypyrrole (PPY) film. The PPY film was first graft copolymerized with poly(ethylene glycol) methacrylate (PEGMA) and then activated with cyanuric chloride (CC). Heparin was then immobilized onto the film through the reaction between the chloride groups of CC and the amine and/or hydroxyl groups of heparin. X-ray photoelectron spectroscopy (XPS) was used to characterize the surface-modified film after each stage. The biocompatibility of the surface-modified PPY was evaluated using plasma recalcification time (PRT) and platelet adhesion. After surface modification, the film had improved wettability while retaining significant electrical conductivity. With immobilized heparin, platelet adhesion and platelet activation on PPY film was significantly suppressed, and the PRT was significantly prolonged. Electrical stimulation also plays a positive role in decreasing platelet adhesion and increasing PRT on pristine and surface-modified PPY films.     

Controllable surface modification of poly(lactic-co-glycolic acid) (PLGA) by hydrolysis or aminolysis I: physical, chemical, and theoretical aspects

While biodegradable, biocompatible polyesters such as poly (lactic-co-glycolic acid) (PLGA) are popular materials for the manufacture of tissue engineering scaffolds, their surface properties are not particularly suitable for directed tissue growth. Although a number of approaches to chemically modify the PLGA surface have been reported, their applicability to soft tissue scaffolds, which combine large volumes, complex shapes, and extremely fine structures, is questionable. In this paper, we describe two wet-chemical methods, base hydrolysis and aminolysis, to introduce useful levels of carboxylic acid or primary and secondary amine groups, respectively, onto the surface of PLGA with minimal degradation. The effects of temperature, concentration, pH, and solvent type on the kinetics of these reactions are studied by following changes in the wettability of the PLGA using contact angle measurements. In addition, the treated surfaces are studied using X-ray photoelectron spectroscopy (XPS) to determine the effect on the surface chemical structure. Furthermore, we show using XPS analysis that these carboxyl and amine groups are readily activated to allow the covalent attachment of biological macromolecules.     

The investigation of protein adsorption behaviors on different functionalized polymers films

The adsorption of BSA and fibrinogen onto plasma-polymerized di-(ethylene glycol) vinyl ether, allylamine, and maleic anhydride films were investigated in detail by surface plasmon resonance spectroscopy (SPR). The chemical properties of the plasma polymers were initially determined by the plasma deposition conditions during the generation procedure. The analysis of the chemical structure of the films and the refractive index of plasma polymers in aqueous solution was carried out using Fourier transform infrared spectroscopy and waveguide mode spectroscopy, respectively. Using water contact angle measurement, the surface wettability of plasma polymers was also characterized. These properties have a critical influence on the behavior of protein adsorption on the surface of the plasma polymers. Protein adsorption was found to depend not only on the types of functionalized groups, but also on the plasma polymer thickness since the protein molecules penetrate into the plasma polymer network bulk. According to the size of protein molecules in aqueous solution and the amount of adsorbed proteins observed by SPR, the conformational changes of proteins could be deduced.     

Laccase-induced grafting on plasma-pretreated polypropylene

A new environmentally friendly strategy for the sustainable functionalization of inert man-made polymer surfaces is mapped out for the first time using a combination of plasma pretreatment and enzymatic postgrafting. The efficiency of enzymatic covalent binding is investigated by grafting methacrylate monomers possessing different amino groups, primary, tertiary, and quaternary, onto a polypropylene surface using plasma pretreatment. Subsequent enzymatic grafting, using laccase and guaiacol sulfonic acid (GSA), is determined by surface analytical techniques, such as attenuated total reflectance Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. The grafting of GSA in the presence of a laccase is proven by a 10-fold increase in sulfur compared to the control. The covalent coupling between GSA and primary amine groups is determined by HPLC-MS using hexylamine as a model substrate. The advantage of technology is in the strong covalent binding of functional groups onto the synthetic polymer's surface, which could then be suitably tailored by enzymes possessing substrate specificity and regional selectivity.     

Micropattern printing of adhesion, spreading, and migration peptides on poly(tetrafluoroethylene) films to promote endothelialization

We report here the development of an original multistep micropatterning technique for printing peptides on surfaces, based on the ink-jet printer technology. Contrary to most micropatterning methods used nowadays, this technique is advantageous because it allows displaying 2D-arrays of multiple biomolecules. Moreover, this low cost procedure allies the advantages of computer-aided design with high flexibility and reproducibility. A Hewlett-Packard printer was modified to print peptide solutions, and Adobe Illustrator was used as the graphic-editing software to design high-resolution checkerboard-like micropatterns. In a first step, PTFE films were treated with ammonia plasma to introduce amino groups on the surface. These chemical functionalities were reacted with heterobifunctional cross-linker sulfo-succinimidyl 4-(N-maleimidomethyl)cycloexane-1-carboxylate (S-SMCC) to allow the subsequent surface covalent conjugation of various cysteine-modified peptides to the polymer substrate. These peptidic molecules containing RGD and WQPPRARI sequences were selected for their adhesive, spreading, and migrational properties toward endothelial cells. On one hand, our data demonstrated that the initial cell adhesion does not depend on the chemical structure and combination of the peptides covalently bonded either through conventional conjugation or micropatterning. On the other hand, spreading and migration of endothelial cells is clearly enhanced while coconjugating the GRGDS peptide in conjunction with WQPPRARI. This behavior is further improved by micropatterning these peptides on specific areas of the polymer surface.     

Morphology and chemical characterization of Ti surfaces modified for biomedical applications

The aim of the present work is to characterize in detail the chemical composition and morphology of titanium surfaces subjected to various environments. Modifications consisted of exposure of Ti to acidic, alkaline or polymer solutions. Such modifications result in chemical and/or morphological changes in the Ti surface. Special attention has been given to identifying the factors influencing cell adhesion and growth. SEM examinations provided morphological characterization of the Ti samples. Surface analytical techniques such as AES or XPS combined with Ar(+) ion sputtering allowed examination of the chemical properties of the Ti surface after chemical pretreatments and investigating the chemical composition of the Ti oxide layer. Raman spectroscopy investigations allowed determination of the crystalline phases of the Ti-oxide layers and characterization of the dextran-modified surface. The results show large differences in the morphology of Ti pretreated with different procedures whereas only minor differences in the chemistry of the surfaces were found. High-resolution Auger investigations have revealed that all the chemical modifications of Ti surfaces resulted in the formation of a titanium oxide layer. XPS confirmed that TiO(2) is the main component of the chemically modified Ti surface. The Raman spectroscopy investigations showed that the titanium surface with a dextran coating is rich in hydroxyl groups. All the surfaces investigated exhibit a hydrophilic character. The possible influence of various surface features on surface biocompatibility is discussed.     

Enzyme-linked immunosorbent assay of Escherichia coli O157:H7 in surface enhanced poly(methyl methacrylate) microchannels

A novel surface treatment method was developed to enhance polymer-based microchannel enzyme-linked immunosorbent assay (ELISA) for Escherichia coli O157:H7 detection. By applying an amine-bearing polymer, poly(ethyleneimine) (PEI), onto poly(methyl methacrylate) (PMMA) surface at pH higher than 11, PEI molecules were covalently attached and their amine groups were introduced to PMMA surface. Zeta potential analysis and X-ray photoelectron spectroscopy (XPS) demonstrated that the alkali condition is preferable for PEI attachment onto the PMMA surface. The amine groups on the PMMA surface were then functionalized with glutaraldehyde, whose aldehyde groups served as the active sites for binding the antibody by forming covalent bonds with the amine groups of the protein molecules. This surface modification greatly improved antibody binding efficiency and the microchannel ELISA for E. coli O157:H7 detection. Compared with untreated PMMA microchannels, approximately 45 times higher signal and 3 times higher signal/noise ratio were achieved with the PEI surface treatment, which also shortened the time required for cells to bind to the microchannel surface to approximately 2 min, much less than that usually required for the same ELISA carried out in 96-well plates. The detection in the microchannel ELISA only required 5-8 cells per sample, which is also better than 15-30 cells required in multi-well plates. With the high sensitivity, short assay time, and small reagent consumption, the microchannel ELISA can be economically used for fast detection of E. coli O157:H7.     

Minimization of protein adsorption on poly(vinylidene fluoride)

Surfaces covered with polyethylene glycol (PEG) have been shown to be biocompatible because PEG yields nonimmunogenicity, nonantigenicity and protein rejection. To produce a biocompatible surface coating, we have developed a method for grafting PEG onto modified poly(vinylidene fluoride) (PVDF) films. The first step was to create carboxy groups on the PVDF surface following covalente coupling of polyethylenimine (PEI) to achieve high density of amino groups. These surface amines were reacted with formyl-terminated PEG's with various molecular weight. The modified PVDF surface was characterized by means of static contact angle measurements, infrared (IR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The influence of the chain length on lysozyme repellence was investigated by means of surface-MALDI-Tof mass spectrometry (Surface-MALDI-Tof-MS). Lysozyme adsorption was significantly suppressed on the PEG 5000 modified PVDF surface.     

Surface immobilization of galactose onto aliphatic biodegradable polymers for hepatocyte culture

A novel surface modification method of biodegradable polymers was investigated for inducing the attachment of specific cells onto the polymer surface via ligand-receptor interactions. Galactose, a targeting ligand specific to asialoglycoprotein receptors present on cell membrane of hepatocytes, was introduced on the surface of poly(D,L-lactic-co-glycolic acid) (PLGA) films. A terminal end group of carboxylic acid in PLGA was activated by dicyclohexylcarbodiimide and N-hydroxysuccinimide for the direct conjugation of lactose by reductive amination reaction. Di-block copolymers of PLGA-b-poly(ethylene glycol) (PEG) having a free terminal amine group were also synthesized and used for the conjugation of galactose for the introduction of a PEG spacer between PLGA and galactose. The presence of galactose moieties on the blend film surface was characterized by measuring water contact angle and X-ray photon spectroscopy, and the amount of galactose was indirectly determined by a specific lectin-binding assay. With increasing the galactose concentration on the blend film surface, the initial attachment as well as the cell viability of hepatocyates concomitantly increased. The introduction of PEG spacer reduced the cell attachment and viability. Albumin secretion rate from hepatocytes was enhanced for galactose modified surfaces, whereas it was reduced for the surfaces not having galactose moieties.     

Ammonia plasma treatment of polystyrene surfaces enhances proliferation of primary human mesenchymal stem cells and human endothelial cells

The control of surface properties is a substantial step in the development and improvement of biomaterials for clinical applications as well as for their use in tissue engineering. Interaction of the substrate surface with the biochemical or biological environment is crucial for the outcome of the applied biomaterial and therefore should meet specific requirements regarding the chemical composition, wettability, elasticity, and charge. In this study, we examined the effect of chemical groups introduced by low pressure plasma treatments of polystyrene surfaces on the cell behavior of primary human mesenchymal stem cells (hMSCs) and dermal microvascular endothelial cells (hDMECs). X-ray photoelectron spectroscopy analysis and contact angle measurements were employed to evaluate ammonia-, carbon dioxide-, and acrylic acid-plasma modifications to substrate surfaces. HMSCs and hDMECs were analyzed simultaneously to identify the most suitable surface functionalization for each cell type. Significantly higher cell proliferation was detected on ammonia plasma-treated surfaces. Cell-material interaction could be shown on all created interfaces as well as the expression of typical cell markers. Hence, the applied plasma treatment presents a suitable tool to improve culture condition on polystyrene for two important cell types (hMSCs and hDMECs) in the field of tissue engineering.     

Synthesis and characterization of novel organic/inorganic hybrid material with short peptide brushes generated on the surface

A novel route to synthesize an organic/inorganic hybrid material containing short peptide chains attached on the surface (e.g., oligo(S-benzyl-L-cysteine)) was developed. Poly[N-(beta-aminoethylene)acrylamide] (PAEA) adsorbed onto silica particles surface (main diameter between 15 and 40 microm) was irreversibly fixed by the reaction between the accessible primary amino groups of the PAEA and 3,3',4,4'-benzophenone tetracarboxylic dianhydride (BTCDA). After the deposition of PAEA from a salt-free aqueous solution onto microporous silica particles and stabilization by a cross-linking reaction with BTCDA, five repeated coupling reactions of boc-S-benzyl-L-cysteine were performed. Changes in surface charges during the polyelectrolyte adsorption were studied by electrokinetic measurements. The cross-linking degree was a tool to control the surface charge of the PAEA/silica hybrid particles. X-ray photoelectron spectroscopy (XPS) was employed to obtain information about the amount of the adsorbed polyelectrolyte as well as the amount of the amino acid S-benzyl-L-cysteine that was covalently bound to the hybrid particle surface and polycondensed there. In the XPS spectra, the sulfur peaks (S 2p3/2, S 2p1/2, and S 2s) qualitatively and quantitatively indicated the presence of the amino acid on the hybrid material surface. After each step of coupling, the intensity of the S 2s peak was increased by a constant value. This indicates the oligopeptide growth. The novel hybrid material offers possibilities for subsequent derivatization reactions such as coupling other amino acids, peptides, obtaining hybrid ion exchange resins, and so forth.