Multilayer film assembled from charged derivatives of chitosan: physical characteristics and biological responses

Polyelectrolyte multilayer films were successfully assembled from each of the three charged derivatives of chitosan; N-[(2-hydroxyl-3-trimethylammonium)propyl]chitosan chloride (HTACC), N-succinyl chitosan (SCC) and N-sulfofurfuryl chitosan (SFC), paired with one of the two oppositely charged polyelectrolytes, poly(acrylic acid) (PAA) and poly(allylamine hydrochloride) (PAH) on surface-treated poly(ethylene terephthalate) (treated PET) substrates by alternate layer-by-layer adsorption. Surface coverage and wettability of the multilayer films were determined by AFM and water contact angle measurements, respectively. Analysis by quartz crystal balance with dissipation (QCM-D) has suggested that all multilayer films are relatively rigid and have a high water content associated within their structures, accounting for up to 85-90% (w/w) for films having 7-10 layers. In vitro cytocompatibility tests for the fibroblast-like L929 cell line revealed a slight dependency for cell adhesion and proliferation on the outermost layer. The multilayer film containing HTACC exhibited moderate antibacterial activity against E. coli and S. aureus. Bearing negative charges, the multilayer films terminating with SFC and having at least 10 layers were capable of suppressing the adsorption of plasma proteins and platelet adhesion at a comparable level to the multilayer film assembled from heparin, a well-known antithrombogenic polymer.     

Robust and Self-healable Antibiofilm Multilayer Coatings

The infection induced by implantation of biomedical materials may result from the biofilm formation after bacteria attachment.Hence, the antibiofilm surface coating represents a novel technique to improve the antibacterial activity of biomedical materials. The traditional antibiofilm surface coatings exhibited some disadvantages and provided a limited service life. In this work, we used polyethyleneimine grafted 3-maleimidopropionic acid(PEIM) and poly(acrylic acid) grafted 2-furfurylamine(PAAF) to achieve robust and self-healable crosslinked multilayer coatings, employing Layer-by-Layer(LbL) self-assembly technique and Diels-Alder reaction. Then, thiol-terminated poly((3-acrylamidopropyl)trimethylammonium chloride)(PAMPTMA-SH) was grafted onto the crosslinked multilayer coating by thiol-ene click reaction to form a novel multilayer coating(PEIM/PAAF)_(10)-PAMPTMA. We found that this coating showed robust and self-healable activity, and significantly inhibited the bacterial growth and biofilm formation after infection with Escherichia coli(E. coli) and Staphylococcus aureus(S. aureus) by in vitro and in vivo assays for 120 h. In addition, the multilayer coating did not induce significant hemolysis or affect the cell viability of red blood cells. In vivo studies also showed that(PEIM/PAAF)_(10)-PAMPTMA coating efficiently blocked the infiltration of inflammatory cells and gene expression in the mouse skin challenged with E. coli or S. aureus. Taken together, these results showed that the prepared multilayer coating exhibited strong antibiofilm activity and provided a new strategy for the application of highly efficient antibiofilm surface coating of biomedical materials.     

Linear Poly(methyl glycerol) and Linear Polyglycerol as Potent Protein and Cell Resistant Alternatives to Poly(ethylene glycol)

The nonspecific interaction of proteins with surfaces in contact with biofluids leads to adverse problems and is prevented by a biocompatible surface coating. The current benchmark material among such coatings is poly(ethylene glycol) (PEG). Herein, we report on the synthesis of linear polyglycerol derivatives as promising alternatives to PEG. Therefore, gold surfaces as a model system are functionalized with a self‐assembled monolayer (SAM) by a two‐step anhydride coupling and a direct thiol immobilization of linear poly(methyl glycerol) and polyglycerol. Surface plasmon resonance (SPR) spectroscopy reveals both types of functionalized surfaces to be as resistant as PEG towards the adsorption of the test proteins fibrinogen, pepsin, albumin, and lysozyme. Moreover, linear polyglycerols adsorb even less proteins from human plasma than a PEG‐modified surface. Additional cell adhesion experiments on linear poly(methyl glycerol) and polyglycerol‐modified surfaces show comparable cell resistance as for a PEG‐modified surface. Also, in the case of long‐term stability, high cell resistance is observed for all samples in medium. Additional in vitro cell‐toxicity tests add to the argument that linear poly(methyl glycerol) and polyglycerol are strong candidates for promising alternatives to PEG, which can easily be modified for biocompatible functionalization of other surfaces.     

E. coli adhesion to silica in the presence of humic acid

The influence of humic acid on the adhesion of Escherichia coli to silica particles or glass surfaces was investigated. After adsorbing various amounts of humic acid to the particles or surfaces, bacteria were added to the sample and allowed to adhere. For the silica particles the number of bacteria-particle couplets formed were counted from video microscopy images. For the glass surfaces, a differential electrophoresis force was applied, and the force required to detach the bacteria was quantified. These experiments showed a slight increase in the number of couplets formed in the presence of humic acid, and also showed a slight increase in the force required for detachment of the bacteria. Although an increase in adhesion number and strength was measured, the magnitude of the increase was small, indicating that humic acid plays a small role in bacterial adhesion to silica or glass surfaces.     

N-halamine biocidal coatings via a layer-by-layer assembly technique

Two N-halamine copolymer precursors, poly(2,2,6,6-tetramethyl-4-piperidyl methacrylate-co-acrylic acid potassium salt) and poly(2,2,6,6-tetramethyl-4-piperidyl methacrylate-co-trimethyl-2-methacryloxyethylammonium chloride) have been synthesized and successfully coated onto cotton fabric via a layer-by-layer (LbL) assembly technique. A multilayer thin film was deposited onto the fiber surfaces by alternative exposure to polyelectrolyte solutions. The coating was rendered biocidal by a dilute household bleach treatment. The biocidal efficacies of tested swatches composed of treated fibers were evaluated against Staphylococcus aureus and Escherichia coli. It was determined that chlorinated samples inactivated both S. aureus and E. coli O157:H7 within 15 min of contact time, whereas the unchlorinated control samples did not exhibit significant biocidal activities. Stabilities of the coatings toward washing and ultraviolet light exposure have also been studied. It was found that the stability toward washing was superior, whereas the UVA light stability was moderate compared to previously studied N-halamine moieties. The layer-by-layer assembly technique can be used to attach N-halamine precursor polymers onto cellulose surfaces without using covalently bonding tethering groups which limit the structure designs. In addition, ionic precursors are very soluble in water, thus promising for biocidal coatings without the use of organic solvents.     

Thickness and morphology of polyelectrolyte coatings on silica surfaces before and after protein exposure studied by atomic force microscopy

Analyte–wall interaction is a significant problem in capillary electrophoresis (CE) as it may compromise separation efficiencies and migration time repeatability. In CE, self-assembled polyelectrolyte multilayer films of Polybrene (PB) and dextran sulfate (DS) or poly(vinylsulfonic acid) (PVS) have been used to coat the capillary inner wall and thereby prevent analyte adsorption. In this study, atomic force microscopy (AFM) was employed to investigate the layer thickness and surface morphology of monolayer (PB), bilayer, (PB-DS and PB-PVS), and trilayer (PB-DS-PB and PB-PVS-PB) coatings on glass surfaces. AFM nanoshaving experiments providing height distributions demonstrated that the coating procedures led to average layer thicknesses between 1 nm (PB) and 5 nm (PB-DS-PB), suggesting the individual polyelectrolytes adhere flat on the silica surface. Investigation of the surface morphology of the different coatings by AFM revealed that the PB coating does not completely cover the silica surface, whereas full coverage was observed for the trilayer coatings. The DS-containing coatings appeared on average 1 nm thicker than the corresponding PVS-containing coatings, which could be attributed to the molecular structure of the anionic polymers applied. Upon exposure to the basic protein cytochrome c, AFM measurements showed an increase of the layer thickness for bare (3.1 nm) and PB-DS-coated (4.6 nm) silica, indicating substantial protein adsorption. In contrast, a very small or no increase of the layer thickness was observed for the PB and PB-DS-PB coatings, demonstrating their effectiveness against protein adsorption. The AFM results are consistent with earlier obtained CE data obtained for proteins using the same polyelectrolyte coatings.     

Reactive hydrogels grafted on gold surfaces

Three different strategies for the fixation of stable hydrogel coatings on gold surfaces, by the grafting‐to and grafting‐from techniques, are described. Monomeric or polymeric disulfide initiators are employed to start the photopolymerization of N‐[tris(hydroxy‐methyl)methyl]acrylamide 1 via decomposition of azo compounds or via a photoredox system. Different approaches, based on the post functionalization of the poly(1) films or on copolymerization of reactive monomers with 1 , are employed to bind potential receptor molecules bearing amino or thiol groups as anchor to the hydrogel. Thus, the functionalized coatings should allow the selective binding of particular analyzates. The overall goal is focused on hydrogel films for the preparation of biochips used in analytical devices such as Surface Plasmon Resonance (SPR), though our approach seems to be generally useful for related purposes.     

Cranberry derived proanthocyanidins reduce bacterial adhesion to selected biomaterials

Catheter associated urinary tract infections (CAUTI) linked with the uropathogens Escherichia coli (E. coli) and Enterococcus faecalis (E. faecalis) account for the majority of nosocomial infections acquired in the clinical environment. Because these infections develop following initial adhesion of the bacterial pathogens to the catheter surface, there is increased interest in developing effective methods to inhibit attachment of cells to biomaterials used in the manufacture of indwelling devices. High molecular weight proanthocyanidins (PAC) extracted from the North American cranberry (Vaccinium macrocarpon) were examined for their potential to reduce the initial adhesion of uropathogenic bacteria (E. coli CFT073 and E. faecalis 29212) to two model biomaterials, poly(vinyl chloride) (PVC) and polytetrafluoroethylene (PTFE). Well-controlled experiments conducted in a parallel-plate flow chamber (PPFC) demonstrated decreased attachment of both bacteria to PVC and PTFE when either the bacteria, biomaterial or both surfaces were treated with PAC. Most significant inhibition of bacterial adhesion was observed for the condition where both the bacteria and biomaterial surfaces were coated with PAC. Additional experiments conducted with nonbiological model particles demonstrate comparable extents of adhesion inhibition, supporting a nonbiospecific mechanism of PAC action. The results of this study are promising for the implementation of PAC in the clinical milieu for prevention of device associated infection as the proposed functional modification is independent of antibacterial mechanisms that may give rise to resistant strains.     

Bioinert solution-cross-linked hydrogen-bonded multilayers on colloidal particles

Bioinert polyelectrolyte multilayers comprised of poly(acrylic acid) and polyacrylamide were deposited on colloidal particles (1.7 microm in diameter) at low pH conditions by layer-by-layer assembly using hydrogen-bonding interactions. The multilayer films were coated uniformly on the colloidal particles without causing any flocculation of the colloids, and the deposited films were subsequently cross-linked by a single treatment of a carbodiimide aqueous solution. The lightly cross-linked multilayer films show excellent stability at physiological conditions (pH 7.4, phosphate-buffered saline), whereas untreated multilayer films dissolved. The multilayer-coated surfaces, both on flat substrates and on colloidal particles, exhibit excellent resistance toward mammalian cell adhesion. With this new solution-based cross-linking method, bioinert H-bonded multilayer coatings offer potential for biomedical applications.     

Coatings of polyethylene glycol for suppressing adhesion between solid microspheres and flat surfaces

This article describes the development and the examination of surface coatings that suppress the adhesion between glass surfaces and polymer microspheres. Superparamagnetic doping allowed for exerting magnetic forces on the microbeads. The carboxyl functionalization of the polymer provided the means for coating the beads with polyethylene glycol (PEG) with different molecular weight. Under gravitational force, the microbeads settled on glass surfaces with similar polymer coatings. We examined the efficacy of removing the beads from the glass surfaces by applying a pulling force of ~1.2 pN. The percent beads remaining on the surface after applying the pulling force for approximately 5 s served as an indication of the adhesion propensity. Coating of PEG with molecular weight ranging between 3 and 10 kDa was essential for suppressing the adhesion. For the particular substrates, surface chemistry and aqueous media we used, coatings of 5 kDa manifested optimal suppression of adhesion: that is, only 3% of the microbeads remained on the surface after applying the pulling magnetic force. When either the glass or the beads were not PEGylated, the adhesion between them was substantial. Addition of a noncharged surfactant, TWEEN, above its critical micelle concentrations (CMCs) suppressed the adhesion between noncoated substrates. The extent of this surfactant-induced improvement of the adhesion suppression, however, did not exceed the quality of preventing the adhesion that we attained by PEGylating both substrates. In addition, the use of surfactants did not significantly improve the suppression of bead-surface adhesion when both substrates were PEGylated. These findings suggest that such surfactant additives tend to be redundant and that covalently grafted coatings of PEGs with selected chain lengths provide sufficient suppression of nonspecific interfacial interactions.     

Embedded silver ions-containing liposomes in polyelectrolyte multilayers: cargos films for antibacterial agents

A new antibacterial coating made of poly(L-lysine)/hyaluronic acid (PLL/HA) multilayer films and liposome aggregates loaded with silver ions was designed. Liposomes filled with an AgNO 3 solution were first aggregated by the addition of PLL in solution. The obtained micrometer-sized aggregates were then deposited on a PLL/HA multilayer film, playing the role of a spacer with the support. Finally, HA/PLL/HA capping layers were deposited on top of the architecture to form a composite AgNO 3 coating. Release of encapsulated AgNO 3 from this composite coating was followed and triggered upon temperature increase over the transition temperature of vesicles, found to be equal to 34 degrees C. After determination of the minimal inhibitory concentration (MIC) of AgNO 3 in solution, the antibacterial activity of the AgNO 3 coating was investigated against Escherichia coli. A 4-log reduction in the number of viable E. coli cells was observed after contact for 120 min with a 120 ng/cm (2) AgNO 3 coating. In comparison, no bactericidal activity was found for PLL/HA films previously dipped in an AgNO 3 solution and for PLL/HA films with liposome aggregates containing no AgNO 3 solution. The strong bactericidal effect could be linked to the diffusion of silver ions out of the AgNO 3 coating, leading to an important bactericidal concentration close to the membrane of the bacteria. A simple method to prepare antibacterial coatings loaded with a high and controlled amount of AgNO 3 is therefore proposed. This procedure is far superior to that soaking AgNO 3 or Ag nanoparticles into a coating. In principle, other small bactericidal chemicals like antibiotics could be encapsulated by this method. This study opens a new route to modify surfaces with small solutes that are not permeating phospholipid membranes below the phase transition temperature.     

Biomimetic hemocompatible coatings through immobilization of hyaluronan derivatives on metal surfaces

Biomimetic coatings offer exciting options to modulate the biocompatibility of biomaterials. The challenge is to create surfaces that undergo specific interactions with cells without promoting nonspecific fouling. This work reports an innovative approach toward biomimetic surfaces based on the covalent immobilization of a carboxylate terminated PEGylated hyaluronan (HA-PEG) onto plasma functionalized NiTi alloy surfaces. The metal substrates were aminated via two different plasma functionalization processes. Hyaluronan, a natural glycosaminoglycan and the major constituent of the extracellular matrix, was grafted to the substrates by reaction of the surface amines with the carboxylic acid terminated PEG spacer using carbodiimide chemistry. The surface modification was monitored at each step by X-ray photoelectron spectroscopy (XPS). HA-immobilized surfaces displayed increased hydrophilicity and reduced fouling, compared to bare surfaces, when exposed to human platelets (PLT) in an in vitro assay with radiolabeled platelets (204.1 +/- 123.8 x 10 (3) PLT/cm (2) vs 538.5 +/- 100.5 x 10 (3) PLT/cm (2) for bare metal, p < 0.05). Using a robust plasma patterning technique, microstructured hyaluronan surfaces were successfully created as demonstrated by XPS chemical imaging. The bioactive surfaces described present unique features, which result from the synergy between the intrinsic biological properties of hyaluronan and the chemical composition and morphology of the polymer layer immobilized on a metal surface.     

Influence of surface hydroxylation on 3-aminopropyltriethoxysilane growth mode during chemical functionalization of GaN Surfaces: an angle-resolved X-ray photoelectron spectroscopy Study

A comparative study of the chemical functionalization of undoped, n- and p-type GaN layers grown on sapphire substrates by metal-organic chemical vapor deposition was carried out. Both types of samples were chemically functionalized with 3-aminopropyltriethoxysilane (APTES) using a well-established silane-based approach for functionalizing hydroxylated surfaces. The untreated surfaces as well as those modified by hydroxylation and APTES deposition were analyzed using angle-resolved X-ray photoelectron spectroscopy. Strong differences were found between the APTES growth modes on n- and p-GaN surfaces that can be associated with the number of available hydroxyl groups on the GaN surface of each sample. Depending on the density of surface hydroxyl groups, different mechanisms of APTES attachment to the GaN surface take place in such a way that the APTES growth mode changes from a monolayer to a multilayer growth mode when the number of surface hydroxyl groups is decreased. Specifically, a monolayer growth mode with a surface coverage of approximately 78% was found on p-GaN, whereas the formation of a dense film, approximately 3 monolayers thick, was observed on n-GaN.     

Protein adhesion on silicon-supported hyperbranched poly(ethylene glycol) and poly(allylamine) thin films

Hyperbranching poly(allylamine) (PAAm) and poly(ethylene glycol) (PEG) on silicon and its effect on protein adhesion was investigated. Hyperbranching involves sequential grafting of polymers on a surface with one of the components having multiple reactive sites. In this research, PAAm provided multiple amines for grafting PEG diacrylate. Current methodologies for generating PEG surfaces include PEG-silane monolayers or polymerized PEG networks. Hyperbranching combines the nanoscale thickness of monolayers with the surface coverage afforded by polymerization. A multistep approach was used to generate the silicon-supported hyperbranched polymers. The silicon wafer surface was initially modified with a vinyl silane followed by oxidation of the terminal vinyl group to present an acid function. Carbodiimide activation of the surface carboxyl group allowed for coupling to PAAm amines to form the first polymer layer. The polymers were hyperbranched by grafting alternating PEG and PAAm layers to the surface using Michael addition chemistry. The alternating polymers were grafted up to six total layers. The substrates remained hydrophilic after each modification. Static contact angles for PAAm (32-44 degrees) and PEG (33-37 degrees) were characteristic of the corresponding individual polymer (30-50 degrees for allylamine, 34-42 degrees for PEG). Roughness values varied from approximately 1 to 8 nm, but had no apparent affect on protein adhesion. Modifications terminating with a PEG layer reduced bovine serum albumin adhesion to the surface by approximately 80% as determined by ELISA and radiolabel binding studies. The hyperbranched PAAm and PEG surfaces described in this paper are nanometer-scale, multilayer films capable of reducing protein adhesion.     

Interaction and adhesion properties of polyelectrolyte multilayers

The growth, morphology, and interaction/adhesion properties of supported poly(sodium 4-styrenesulfonate)/poly(allylamine hydrochloride) (PSS/PAH) and DNA/PAH multilayers were investigated by means of surface plasmon resonance spectroscopy, atomic force microscope (AFM) imaging, and AFM-related force measurements. Multilayers were assembled on a prelayer of poly(ethylenimine) (PEI) both with and without drying. SPR results showed a linear growth of the assembly in the case of PSS/PAH multilayers and nonlinear growth for DNA/PAH multilayers. Measurements of forces acting between a bare glass sphere and a multilayer-coated surface indicated repulsive or attractive forces, depending on surface charge, which suggests that, on approach, electrostatic forces dominate. On separation, we observed large pull-off forces in the case of positively charged multilayers and weak pull-off forces in the case negatively charged multilayers. Multiple adhesions and plateau regions observed on separation were interpreted in terms of a bridging of multiple polymer chains between the glass particle and the multilayer and a stretching of the polyelectrolyte loops. The dependence of the pull-off force on the number of deposited layers shows regular oscillations.     

Surface functionalization of silicone rubber for permanent adhesion improvement

The surface properties of poly(dimethyl siloxane) (PDMS) layers screen printed onto silicon wafers were studied after oxygen and ammonia plasma treatments and subsequent grafting of poly(ethylene -alt-maleic anhydride) (PEMA) using X-ray photoelectron spectroscopy (XPS), roughness analysis, and contact angle and electrokinetic measurements. In the case of oxygen-plasma-treated PDMS, a hydrophilic, brittle, silica-like surface layer containing reactive silanol groups was obtained. These surfaces indicate a strong tendency for "hydrophobic recovery" due to the surface segregation of low-molecular-weight PDMS species. The ammonia plasma treatment of PDMS resulted in the generation of amino-functional surface groups and the formation of a weak boundary layer that could be washed off by polar liquids. To avoid the loss of the plasma modification effect and to achieve stabilization of the mechanically instable, functionalized PDMS top layer, PEMA was subsequently grafted directly or after using gamma-APS as a coupling agent on the plasma-activated PDMS surfaces. In this way, long-time stable surface functionalization of PDMS was obtained. The reactivity of the PEMA-coated PDMS surface caused by the availability of anhydride groups could be controlled by the number of amino functional surface groups of the PDMS surface necessary for the covalent binding of PEMA. The higher the number of amino functional surface groups available for the grafting-to procedure, the lower the hydrophilicity and hence the lower the reactivity of the PEMA-coated PDMS surface. Additionally, pull-off tests were applied to estimate the effect of surface modification on the adhesion between the silicone rubber and an epoxy resin.     

Analysis of Dynamic and Thermodynamic Adsorption of Nanoparticles on Solid Surfaces by Dark‐Field Light Scattering Measurements

We have constructed a dark‐field light scattering microscope using a very low‐cost digital camera to investigate the adsorption of gold nanoparticles (AuNPs) on four different substrates at various pH values. The substrates used are glass, polycarbonate (PC), poly(dimethylsiloxane) (PDMS), and poly(methyl methacrylate) (PMMA). The coverage of AuNPs on hydrophobic substrates such as PDMS is greater than that on hydrophilic substrates like glass. The adsorption and aggregation of AuNPs on a particular substrate increased upon decreasing the pH (from 9.0 to 4.0). A greater coverage percentage of AuNPs, but less aggregation, occurs on glass treated with poly(diallyldimethylammonium) (PDDA) than on bare glass. The scattering intensity increases upon increasing the number of layers of adsorbed AuNPs on glass that was treated sequentially with AuNPs and PDDA. When compared to UV‐Vis absorption, dark‐field microscope provides greater sensitivity and qualitative surface information.     

Do cationic polymer coatings retain their biocidal activity after washing with water?

Aqueous solutions of poly(diallyldimethylammonium chloride) and its electrostatic complexes with sodium polyacrylate were deposited onto glass surfaces. Upon successive washing cycles, they formed thin stable coatings that exhibited antimicrobial activity towards gram-positive and gram-negative bacteria. The obtained results are valuable for the development of antibacterial coatings.     

The zeta potential of surface-functionalized metallic nanorod particles in aqueous solution

Metallic nanoparticles suspended in aqueous solutions and functionalized with chemical and biological surface coatings are important elements in basic and applied nanoscience research. Many applications require an understanding of the electrokinetic or colloidal properties of such particles. We describe the results of experiments to measure the zeta potential of metallic nanorod particles in aqueous saline solutions, including the effects of pH, ionic strength, metallic composition, and surface functionalization state. Particle substrates tested include gold, silver, and palladium monometallic particles as well as gold/silver bimetallic particles. Surface functionalization conditions included 11-mercaptoundecanoic acid (MUA), mercaptoethanol (ME), and mercaptoethanesulfonic acid (MESA) self-assembled monolayers (SAMs), as well as MUA layers subsequently derivatized with proteins. For comparison, we present zeta potential data for typical charge-stabilized polystyrene particles. We compare experimental zeta potential data with theoretically predicted values for SAM-coated and bimetallic particles. The results of these studies are useful in predicting and controlling the aggregation, adhesion, and transport of functionalized metallic nanoparticles within microfluidic devices and other systems.     

塑料表面载银微凝胶层层组装膜的制备及抗菌活性

以载银聚烯丙基胺盐酸盐-葡聚糖微凝胶与聚苯乙烯磺酸钠为构筑基元,利用层层组装技术制备了一种可直接沉积在疏水的塑料基底表面的载银抗菌微凝胶膜. 研究结果表明,该载银抗菌微凝胶膜具有很好的抗菌能力,并且其抗菌活性可以通过控制载银微凝胶膜的组装层数方便地进行调控. 这种制备在塑料表面的载银抗菌微凝胶膜具有良好的稳定性和基底粘附力,能够保障其长效抗菌的实现.