Biomolecule-based antibacterial coating on a stainless steel surface: multilayer film build-up optimization and stability study

The goal of this paper was to establish the durability profile of antibacterial multilayer thin films under storage and usage conditions. Thin films were built on stainless steel (SS) by means of a layer-by-layer process alternating a negatively charged polyelectrolyte, polyacrylic acid, with a cationic antibacterial peptide, nisin. SS coupons coated with the antibacterial film were challenged under environmental and usage conditions likely to be encountered in real-world applications. The change in antibacterial activity elicited by the challenge was used as an indicator of multilayer film resistance. Antibacterial SS samples could be stored for several weeks at 4°C in ambient air and antibacterial films were resistant to dipping and mild wiping in water and neutral detergent. The multilayer coating showed some weaknesses, however, that need to be addressed.     

Biomolecules in multilayer film for antimicrobial and easy-cleaning stainless steel surface applications

Microorganisms are able to attach to, grow on, and ultimately form biofilms on a large variety of surfaces, such as industrial equipment, food contact surfaces, medical implants, prostheses and operating rooms. Once organized into biofilms, bacteria are difficult to remove and kill, which increases the risk of cross-contamination and infection. One way to address the problem may thus be to develop antibacterial, anti-adhesion, ‘easy cleaning’ surfaces. In this study, stainless steel (SS) surfaces with antibacterial properties were created by embedding several antimicrobial peptides in a multilayer film architecture. The biocidal effect of these surfaces was demonstrated against both Gram-positive and Gram-negative bacteria according to two ISO tests. Also, coating SS surfaces with either mucin or heparin led to a reduction of S. epidermidis adhesion of almost 95% vs the bare substratum. Finally, by combining both antibacterial and anti-adhesion biomolecules in the same multilayer film, SS surfaces with better cleanability were produced. This surface coating property may help to delay the buildup of a dead bacterial layer which is known to progressively reduce exposure of the coating, leading to an undesirable decrease in the antibacterial effect of the surface.     

Inherent charge-shifting polyelectrolyte multilayer blends: a facile route for tunable protein release from surfaces

Recent research has highlighted degradable multilayer films that enable the programmed release of different therapeutics. Multilayers constructed by the layer-by-layer (LbL) deposition that can undergo disassembly have been demonstrated to be of considerable interest, particularly for biomedical surface coatings due to their versatility and mild aqueous processing conditions, enabling the inclusion of biologic drugs with high activity. In this study, we examine the controlled release of a protein using a different mechanism for film disassembly, the gradual dissociation of film interactions under release conditions. Poly(β-amino ester)s and poly(L-lysine) (PLL) were used as the positively charged multilayer components coassembled with a model negatively charged antigen protein, ovalbumin (Ova). The release of the protein from these multilayer films is dominated by the slow shift in the charge of components under physiological pH conditions rather than by hydrolytic degradative release. The time scale of release can be varied over almost 2 orders of magnitude by varying the ratio of the two polyamines in the deposition solution. The highly versatile and tunable properties of these films form a basis for designing controlled and sequential delivery of drug coatings using a variety of polyions.     

Manipulating the salt and thermal stability of DNA multilayer films via oligonucleotide length

DNA films are promising materials for diverse applications, including sensing, diagnostics, and drug/gene delivery. However, the ability to tune the stability of DNA films remains a crucial aspect for such applications. Herein, we examine the role of oligonucleotide length on the formation, and salt and thermal stability, of DNA multilayer films using oligonucleotides of homopolymeric diblocks (polyAG and polyTC), with each block (A, G, T, or C) ranging from 5 to 30 bases (10-, 20-, 30-, 40-, and 60-mer). Using a combination of quartz crystal microgravimetry, dual polarization interferometry, and flow cytometry, we demonstrate that at least 10 bases per hybridizing block in the DNA diblocks (that is, 20-mer) are required for successful hybridization and, hence, DNA multilayer film formation. Films assembled using longer oligonucleotide blocks were more stable in low salt conditions, with the DNA multilayer films assembled from the 60-mer oligonucleotides remaining intact in solutions of about 25 mM NaCl. A systematic increase in film melting temperature ( T m) was observed for the DNA multilayer films (assembled on colloids) with increasing oligonucleotide length, ranging from 38.5 degrees C for the 20-mer films to 53 degrees C for the 60-mer films. Further, an alternating trend in T m of the DNA multilayer films was observed with layer number (AG or TC); DNA multilayer films terminated with an AG layer exhibited a higher T m (44-49 degrees C) than films with an outermost TC layer (ca. 38 degrees C), suggesting a rearrangement of the film structure upon hybridization of the outermost layer. This work shows that the stability of DNA multilayer films can be tuned by varying the length of the oligonucleotide building blocks, thus providing a versatile means to tailor the salt and thermal stability of DNA films, which is necessary for the application of such films.     

Swelling behavior of hyaluronic acid/polyallylamine hydrochloride multilayer films

The reversible swelling behavior of multilayer films containing hyaluronic acid and polyallylamine hydrochloride was investigated using in situ ellipsometry, since many of the natural functions and applied uses of hyaluronic acid are related to the extraordinary ability of this biopolymer to swell, and to respond conformationally to the local solution environment. This swelling was observed to be substantial, and depended strongly on the film thickness, the pH conditions used to prepare the films, and the swelling solution pH and ionic strength. The swelling results were also rationalized in terms of the dissociation behavior of the polyelectrolytes in the multilayer assemblies, measured by the zeta potential, on colloidal particles. The films were found to swell by as much as 8 times their dry thickness, and the extent of film hydration was observed to depend on the thickness of the films in a nonlinear fashion. This was related to the internal structure of the films, which is dictated by the assembly pH conditions. In addition, the swelling solution pH and ionic strength influence the electrostatic environment in the films and, in turn, have a substantial effect on the overall swelling behavior.     

不锈钢和氧化钛薄膜刺激内皮细胞释放NO和MCP-1的研究

目的:探讨不锈钢和氧化钛(Ti-O)薄膜对内皮细胞释放细胞因子的影响,评价生物材料表面的内皮细胞的功能.方法:用非平衡磁控溅射的方法制备Ti-O薄膜,提取人脐静脉内皮细胞(mrVEC)并种植到材料表面进行培养.第1、3、5天提取培养上清液,用NO检测试剂盒和酶联免疫吸附实验的方法进行一氧化氮(NO)和单核细胞趋化蛋白-1(Monocyte Chemotactic Protein 1,MCP-1)的检测,采用环境扫描电子显微镜(SEM)进行细胞形态观察.结果:内皮细胞在Ti-O薄膜表面上生长形态保持较好,其培养上清液NO和MCP-1因子释放量都要略小于不锈钢表面的.结论:Ti-O薄膜具有较好的促进内皮化和组织相容性,有望成为血管支架的新型生物材料.     

Preparation of spatially ordered multilayer thin films of antibody and their binding properties

Spatially ordered multilayer thin films containing anti-fluoresceinisothiocyanate (anti-FITC) were prepared on the surface of a quartz slide to study the binding properties of the multilayer films. A quartz slide was treated in solutions of avidin and biotin-labeled anti-FITC alternately and repeatedly to form multilayer thin films through a strong affinity between avidin and biotin. A spectrophotometric study revealed explicitly that the thin films thus prepared consisted of alternate monomolecular layers of avidin and biotin-labeled anti-FITC. The antibody retained its binding activity to antigen in the multilayer thin film, though the antigen could not access the antibody embedded deep in the multilayer film. Only the outermost four or five layers of antibody were involved in the binding of antigen.     

The location of cytochrome c on the surface of ultrathin lipid multilayer films using x-ray diffraction.

X-ray diffraction and spectroscopic techniques were used to characterize ultrathin fatty acid multilayers having a bound surface layer of cytochrome c. Three to six monolayers of arachidic acid were deposited onto an alkylated glass surface, using the Langmuir-Blodgett method. These fatty acid multilayer films were stored either in a 1 mM NaHCO3 pH 7.5 solution or a buffered 10 microM cytochrome c solution, pH 7.5. After washing extensively with buffer, these multilayer films were assayed for bound cytochrome c by optical spectroscopy. It was found that the cytochrome c bound only to the odd-numbered monolayer films (which have hydrophilic surfaces). The theoretical number of cytochrome c molecules bound to the ultrathin multilayer films having three or five monolayers was calculated as N = 1.2 x 10(13)/cm2 (assuming a hexagonally close-packed monolayer of protein), which would produce an optical density of 0.002 at a wavelength of 550 nm; for a three or five monolayer ultrathin film that was incubated with cytochrome c, OD550 approximately equal to 0.002. The protein was released from the film when treated with greater than 100 mM KCl solution, as would be expected for an electrostatic interaction. Meridional x-ray diffraction data were collected from the arachidic acid films with and without a bound cytochrome c layer. A box refinement technique, previously shown to be effective in deriving the profile structures of nonperiodic ultrathin films, was used to determine the multilayer electron density profiles. The electron density profiles and their autocorrelation functions showed that bound cytochrome c resulted in an additional electron dense feature on the multilayer surface, consistent with a bound cytochrome c monolayer. The position of the bound protein relative to the multilayer surface was independent of the number of fatty acid monolayers in the multilayer. Future studies will use these methods to investigate the structures of membrane protein complexes bound directly to the surface of multilayer films.     

The Effect of Heparin-VEGF Multilayer on the Biocompatibility of Decellularized Aortic Valve with Platelet and Endothelial Progenitor Cells

The application of polyelectrolyte multilayer films is a new, versatile approach to surface modification of decellularized tissue, which has the potential to greatly enhance the functionality of engineered tissue constructs derived from decellularized organs. In the present study, we test the hypothesis that Heparin- vascular endothelial growth factor (VEGF) multilayer film can not only act as an antithrombotic coating reagent, but also induce proliferation of endothelial progenitor cells (EPCs) on the decellularized aortic heart valve. SEM demonstrated the adhesion and geometric deformation of platelets. The quantitative assay of platelet activation was determined by measuring the production of soluble P-selectin. Binding and subsequent release of heparin and VEGF from valve leaflets were assessed qualitatively by laser confocal scanning microscopy and quantitatively by ELISA methods. Human blood derived EPCs were cultured and the adhesion and growth of EPCs on the surface modified valvular scaffolds were assessed. The results showed that Heparin-VEGF multilayer film improved decellularized valve haemocompatibility with respect to a substantial reduction of platelet adhesion. Release of VEGF from the decellularized heart valve leaflets at physiological conditions was sustained over 5 days. In vitro biological tests demonstrated that EPCs achieved better adhesion, proliferation and migration on the coatings with Heparin-VEGF multilayer film. Combined, these results indicate that Heparin-VEGF multilayer film could be used to cover the decellularized porcine aortic valve to decrease platelet adhesion while exhibiting excellent EPCs biocompatibility.     

Orientation and lateral mobility of cytochrome c on the surface of ultrathin lipid multilayer films.

We have previously shown that cytochrome c can be electrostatically bound to an ultrathin multilayer film having a negatively charged hydrophilic surface; furthermore, x-ray diffraction and absorption spectroscopy techniques indicated that the cytochrome c was bound to the surface of these ultrathin multilayer films as a molecular monolayer. The ultrathin fatty acid multilayers were formed on alkylated glass, using the Langmuir-Blodgett method. In this study, optical linear dichroism was used to determine the average orientation of the heme group within cytochrome c relative to the multilayer surface plane. The cytochrome c was either electrostatically or covalently bound to the surface of an ultrathin multilayer film. Horse heart cytochrome c was electrostatically bound to the hydrophilic surface of fatty acid multilayer films having an odd number of monolayers. Ultrathin multilayer films having an even number of monolayers would not bind cytochrome c, as expected for such hydrophobic surfaces. Yeast cytochrome c was covalently bound to the surface of a multilayer film having an even number of fatty acid monolayers plus a surface monolayer of thioethyl stearate. After washing extensively with buffer, the multilayer films with either electrostatically or covalently bound cytochrome c were analyzed for bound protein by optical absorption spectroscopy; the orientation of the cytochrome c heme was then investigated via optical linear dichroism. Polarized optical absorption spectra were measured from 450 to 600 nm at angles of 0 degrees, 30 degrees, and 45 degrees between the incident light beam and the normal to the surface plane of the multilayer. The dichroic ratio for the heme alpha-band at 550 nm as a function of incidence angle indicated that the heme of the electrostatically-bound monolayer of cytochrome c lies, on average, nearly parallel to the surface plane of the ultrathin multilayer. Similar results were obtained for the covalently-bound yeast cytochrome c. Furthermore, fluorescence recovery after photobleaching (FRAP) was used to characterize the lateral mobility of the electrostatically bound cytochrome c over the monolayer plane. The optical linear dichroism and these initial FRAP studies have indicated that cytochrome c electrostatically bound to a lipid surface maintains a well-defined orientation relative to the membrane surface while exhibiting measurable, but highly restricted, lateral motion in the plane of the surface.     

Photoelectrochemical, photophysical and morphological studies of electrostatic layer-by-layer thin films based on poly(p-phenylenevinylene) and single-walled carbon nanotubes

The preparation of multilayer films based on poly(p-phenylenevinylene) (PPV) and carboxylic-functionalized single-walled carbon nanotubes (SWNT-COOH) by electrostatic interaction using the layer-by-layer (LbL) deposition method is reported herein. The multilayer build-up, monitored by UV-Vis and photoluminescence (PL) spectroscopies, displayed a linear behavior with the number of PPV and SWNT-COOH layers deposited that undergo deviation and spectral changes for thicker films. Film morphology was evaluated by AFM and epifluorescence microscopies showing remarkable changes after incorporation of SWNT-COOH layers. Films without SWNT show roughness and present dispersed grains; films with SWNT-COOH layers are flatter and some carbon nanotube bundles can be visualized. The photoinduced charge transfer from the conducting polymer to SWNT-COOH was analyzed by PL quenching either by the decrease of the emission intensity or by the presence of dark domains in the epifluorescence micrographs. Photoelectrochemical characterization was performed under white light and the films containing SWNT-COOH displayed photocurrent values between 2.0 μA cm(-2) and 7.5 μA cm(-2), as the amount of these materials increases in the film. No photocurrent was observed for the film without carbon nanotubes. Photocurrent generation was enhanced and became more stable when an intermediate layer of PEDOT:PSS was interposed between the active layer and the ITO electrode, indicating an improvement in hole transfer to the contacts. Our results indicate that these multilayer films are promising candidates as active layers for organic photovoltaic cells.     

Elasticity of native and cross-linked polyelectrolyte multilayer films

Mechanical properties of polyelectrolyte multilayer films were studied by nanoindentation using the atomic force microscope (AFM). Force-distance measurements using colloidal probe tips were systematically obtained for supported films of poly(L-lysine) and hyaluronan that are suited to bio-application. Both native and covalently cross-linked films were studied as a function of increasing layer number, which increases film thickness. The effective Young's modulus perpendicular to the film, Eperpendicular, was determined to be a function of film thickness, cross-linking, and sample age. Thick PEM films exhibited a lower Eperpendicular than thinner PEM, whereas the Young's modulus of cross-linked films was more than 10-fold larger than native films. Moduli range from approximately 20 kPa for native films up to approximately 800 kPa for cross-linked ones. Young's moduli increased slightly with sample age, plateauing after approximately 4 weeks. Spreading of smooth muscle cells on these substrates with pre-attached collagen proved to be highly dependent on film rigidity with stiffer films giving greater cell spreading.     

Multilayer biomimetics: reversible covalent stabilization of a nanostructured biofilm

Designed polypeptides and electrostatic layer-by-layer self-assembly form the basis of promising research in bionanotechnology and medicine on development of polyelectrolyte multilayer films (PEMs). We show that PEMs can be formed from oppositely charged 32mers containing several cysteine residues. The polypeptides in PEMs become cross-linked under mild oxidizing conditions. This mimicking of disulfide (S-S) bond stabilization of folded protein structure confers on the PEMs a marked increase in resistance to film disassembly at acidic pH. The reversibility of S-S bond stabilization of PEMs presents further advantages for controlling physical properties of films, coatings, and other applications involving PEMs.     

Polypeptide multilayer film co-delivers oppositely-charged drug molecules in sustained manners

The current state-of-the-art for drug-carrying biomedical devices is mostly limited to those that release a single drug. Yet there are many situations in which more than one therapeutic agent is needed. Also, most polyelectrolyte multilayer films intended for drug delivery are loaded with active molecules only during multilayer film preparation. In this paper, we present the integration of capsules as vehicles within polypeptide multilayer films for sustained release of multiple oppositely charged drug molecules using layer-by-layer nanoassembly technology. Calcium carbonate (CaCO(3)) particles were impregnated with polyelectrolytes, shelled with polyelectrolyte multilayers, and then assembled onto polypeptide multilayer films using glutaraldehyde. Capsule-integrated polypeptide multilayer films were obtained after decomposition of CaCO(3) templates. Two oppositely charged drugs were loaded into capsules within polypeptide multilayer films postpreparation based on electrostatic interactions between the drugs and the polyelectrolytes impregnated within capsules. We determined that the developed innovative capsule-integrated polypeptide multilayer films could be used to load multiple drugs of very different properties (e.g., opposite charges) any time postpreparation (e.g., minutes before surgical implantation inside an operating room), and such capsule-integrated films allowed simultaneous delivery of two oppositely charged drug molecules and a sustained (up to two weeks or longer) and sequential release was achieved.     

Enhanced resistance of polyelectrolyte multilayer microcapsules to pepsin erosion and release properties of encapsulated indomethacin

Polyelectrolyte multilayer films were prepared through layer-by-layer (LbL) self-assembly using polysaccharide sodium alginate (ALG) and chitosan (CHI). After incubation in an enzyme pepsin solution, the multilayer film was partially destroyed as detected by the decrease in fluorescent intensity because of the enzymatic degradation of CHI. The enzymatic desorption was also observed from the microcapsule wall made of the ALG/CHI multilayer film directly deposited on indomethacin (IDM) microcrystals through LbL self-assembly. After pepsin erosion, the IDM release from the microcapsules monitored by UV absorbance was obviously accelerated because of desorption. To enhance the stability of the ALG/CHI multilayer film to the enzymatic erosion, some physical and chemical methods were established to increase film thickness or to cross-link the polysaccharides within the film. Increasing the layer number and raising the deposition temperature effectively slowed down the enzymatic desorption and release rate. Especially, increasing deposition temperature was more effective because of producing a more perfect structure in the ALG/CHI multilayer film. Cross-linking the neighboring layers of ALG and CHI with 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide in the ALG/CHI multilayer film significantly reduced the enzymatic desorption and release rate. Therefore, increasing deposition temperature and cross-linking neighboring layers are effective methods to protect the multilayer film fabricated using LbL assembly from the enzymatic erosion and to prolong the release of the encapsulated drug.     

Morphological and optical characterization of polyelectrolyte multilayers incorporating nanocrystalline cellulose

Aqueous layer-by-layer (LbL) processing was used to create polyelectrolyte multilayer (PEM) nanocomposites containing cellulose nanocrystals and poly(allylamine hydrochloride). Solution-dipping and spin-coating assembly methods gave smooth, stable, thin films. Morphology was studied by atomic force microscopy (AFM) and scanning electron microscopy (SEM), and film growth was characterized by X-ray photoelectron spectroscopy (XPS), ellipsometry, and optical reflectometry. Relatively few deposition cycles were needed to give full surface coverage, with film thicknesses ranging from 10 to 500 nm. Films prepared by spin-coating were substantially thicker than solution-dipped films and displayed radial orientation of the rod-shaped cellulose nanocrystals. The relationship between film color and thickness is discussed according to the principles of thin film interference and indicates that the iridescent properties of the films can be easily tailored in this system.     

Enzymatic desorption of layer-by-layer assembled multilayer films and effects on the release of encapsulated indomethacin microcrystals

Polyelectrolyte multilayer films were prepared through layer-by-layer (LbL) self-assembly of chitosan (CHI) and pyrene labeled poly(2-acrylamido-2-methylpropanesulfonic acid) (APy). After incubation in an enzyme pepsin solution, multilayer films were partially destroyed as detected by a decrease in fluorescence intensity due to enzymatic degradation of CHI and desorption of APy. The multilayer desorption rate was the highest at pH 4.0. Increasing temperature from 20 degrees C to 60 degrees C accelerated desorption. The enzymatic desorption was also observed from microcapsule walls made of CHI/alginate (ALG) multilayer films directly deposited on indomethacin (IDM) microcrystals by LbL self-assembly. After pepsin erosion, the IDM release from the microcapsule monitored by UV absorbance was obviously accelerated due to desorption. The influence of incubation time, pH, and temperature of the pepsin solution on the IDM release was investigated. The release rate was the fastest after incubation in the pepsin solution at pH 4.0 due to the highest activity of pepsin. Increasing incubation temperature from 20 degrees C to 60 degrees C, however, slowed down the release rate, which was considered to be due to the formation of more perfect and compact multilayer films through the chain rearrangement at higher temperatures. The CHI/ALG multilayer film was found to maintain its barrier function to the IDM diffusion even after 6-h incubation in the pepsin solution.     

Surface structure of chitosan and hybrid chitosan-amylose films-restoration of the antibacterial properties of chitosan in the amylose film

The surface structure of films prepared by casting aqueous solutions of mixtures of water soluble chitosan (WSC) and amylose as well as a fully deacetylated chitosan was studied. Zeta potential measurements indicated that the surface of WSC and fully deacetylated chitosan films is positively charged but very weakly, whereas, a film of amylose blended with WSC exhibited an obvious positive charge. X-ray photoelectron spectra of these films suggest that less amino groups are exposed on the surface of WSC and fully deacetylated chitosan films, whereas, more amino groups are exposed on the surface of a WSC film blended with amylose. A sheet structure in which free amino groups are less exposed on the surface of the film of WSC or fully deacetylated chitosan is proposed. This accounts for the loss of antibacterial activity of chitosan on the WSC film surface. When blended with amylose, the morphology of the film may be disrupted, resulting in strong antibacterial properties.     

pH-responsive properties of multilayered poly(L-lysine)/hyaluronic acid surfaces

Multilayer films have been prepared by the sequential electrostatic adsorption of poly(L-lysine) and hyaluronic acid onto charged silicon surfaces from dilute aqueous solutions under various pH conditions. Microelectrophoresis was used to examine the local acid-base equilibria of the polyelectrolytes within the films as a function of the total number of layers in the film and the assembly solution pH. The acid-base dissociation constants were observed to deviate significantly from dilute solution values upon adsorption, to be layer dependent only within the first 3-4 layers, and to show sensitivity to the assembly solution pH. As a result, some of the physicochemical properties of the films have also been found to exhibit pH-responsive behavior. For example, the thickest films result when at least one of the polyelectrolytes is only partially dissociated in solution. As well, the pH-dependent degree of dissociation of the surface functional groups can be used to vary the contact angle of a water droplet by as much as 25 degrees and the coefficient of friction by up to an order of magnitude. In addition, the extent to which PLL/HA films can be made to swell in solution can be varied by a factor of 7 depending on the assembly solution and swelling solution pH. The anomalies found in the dissociation constants account for the trends in these pH-dependent properties. Here, we demonstrate that knowledge of the acid-base dissociation behavior in multilayer films is key to understanding and controlling the physical properties of the films, particularly surface friction and wettability, which are fundamentally important factors for many biomaterials applications. For example, we outline a mechanism whereby biopolymer thin films can be electrostatically adsorbed under highly charged "sticky" conditions and then quickly transformed into stable low-friction films simply by altering the pH environment.     

Imaging cell interactions with native and crosslinked polyelectrolyte multilayers

The adhesion of primary chondrocytes to polyelectrolyte multilayer films, made of poly(l-lysine) (PLL) and hyaluronan (HA), was investigated for native and crosslinked films, either ending by PLL or HA. Crosslinking the film was achieved by means of a water-soluble carbodiimide in combination with N-hydroxysulfosuccinimide. The adhesion of macrophages and primary chondrocytes was investigated by microscopical techniques (optical, confocal, and atomic), providing useful information on the cell/film interface. Native films were found to be nonadhesive for the, primary chondrocytes, but could be degraded by macrophages, as could be visualized by confocal laser scanning microscopy after film labeling. Confocal microscopy images show that these films can be deformed by the condrocytes and that PLL diffuses at the chondrocyte membrane. In contrast, the cells adhered and proliferated well on the crosslinked films, which were not degraded by the macrophages. These results were confirmed by a MTT test over a 6-d period and by atomic force microscopy observations. We thus prove that chemical crosslinking can dramatically change cell adhesion properties, the cells being more stably anchored on the crosslinked films. Both authors kcontributed equally.