Polyelectrolyte Multilayer Films Research Articles

140 Development of nanoscale aptamer films for controlled release of encapsulated payloads

Aptamers are short, single-stranded nucleic acids that fold into well-defined 3D structures which bind to a single target molecule (from small molecules to cells) with affinities and specificities that can rival those of antibodies (Jeong et al., 2009). Unlike antibodies, aptamers can be chemically synthesized eliminating the need for animals or cell culture, which also allows for selection under non-physiological conditions and broadens potential targets to include toxic molecules (Banka & Stockley, 2006). The compatibility of aptamers with nanomaterials, in combination with their affinity, selectivity, and conformational changes upon target interaction, have allowed for the development of a large number of therapeutic and targeted delivery systems in recent years exploiting these properties. Despite this, many challenges still exist as unprotected DNA is readily degraded by nucleases prevalent in biological and environmental systems (Bouchard et al., 2010). Embedding aptamers within multilayer polyelectrolyte films could provide a biodegradable shelter, while allowing the detection of diffusible small molecules. An understanding of these materials will allow for the eventual encapsulation of relevant payloads into aptamer–polyelectrolyte microcapsules towards the development of a controlled release system. In this work, films composed of natural polyelectrolytes chitosan and hyaluronan are employed due to their biocompatibility, strong presence in current literature, and amiability to layer-by-layer film construction. Initial progress towards the development of an aptamer-embedded polyelectrolyte film system will be presented.

Electroactive properties of the multilayer films containing Prussian Blue nanoparticles

In our work we have focused on the incorporation of Prussian Blue nanoparticles (NP) into polyelectrolyte multilayer films (PEM). The main goal of presented studies was to obtain polymer/nanoparticles films with controlled electroactive properties. The amount and ordering of deposited nanoparticles depended on the adsorption conditions of the underlying polymer anchoring layer. In the case of fully charged polyelectrolyte layer, nanoparticles were distributed uniformly. When anchoring layer was not completely charged, strong aggregation of nanoparticles at the surface was observed, causing diminution of current response of studied nanocomposite films. Thus, the selection of the proper formation conditions of nanocomposite films may influence their properties and it may even enhance their response.

Donnan potential of polyelectrolyte multilayer films made from poly-l-glutamic/polyallylamine hydrochloride and the stability of hexacyanoferrate retained in the films

Polyelectrolyte multilayer films are a versatile surface functionalization method of solid-liquid interfaces and appear to be interesting reservoirs to load/release drugs and to act as permselective membranes. For the latter applications critical parameters are the porosity of the film and its Donnan potential. In this investigation the Donnan potential of PEI-(PGA-PAH)n (PEI, PGA and PAH stand for polyethyleneimine, poly-L-glutamic acid and polyallylamine) films will be determined as a function of the number of deposition steps and the concentration of the redox probe, hexacyanoferrate anions, for films made from 10 layer pairs. Complementarily, it will be shown that the retention of the redox probe in the films in the presence of 150 mM NaCl electrolyte depends on both the film thickness and the scan rates at which the electrochemical experiments are performed.

Gradients of physical and biochemical cues on polyelectrolyte multilayer films generated via microfluidics.

The cell microenvironment is a complex and anisotropic matrix composed of a number of physical and biochemical cues that control cellular processes. A current challenge in biomaterials is the engineering of biomimetic materials which present spatially controlled physical and biochemical cues. The layer-by-layer assembly of polyelectrolyte multilayers (PEM) has been demonstrated to be a promising candidate for a biomaterial mimicking the native extracellular matrix. In this work, gradients of biochemical and physical cues were generated on PEM films composed of hyaluronan (HA) and poly(l-lysine) (PLL) using a microfluidic device. As a proof of concept, four different types of surface concentration gradients adsorbed onto the films were generated. These included surface concentration gradients of fluorescent PLL, fluorescent microbeads, a cross-linker, and one consisting of a polyelectrolyte grafted with a cell adhesive peptide. In all cases, reproducible centimeter-long linear gradients were obtained. Fluorescence microscopy, Fourier transform infrared spectroscopy and atomic force microscopy were used to characterize these gradients. Cell responses to the stiffness gradient and to the peptide gradient were studied. Pre-osteoblastic cells were found to adhere and spread more along the stiffness gradient, which varied linearly from 200 kPa-600 kPa. Myoblast cell spreading also increased throughout the length of the increasing RGD-peptide gradient. This work demonstrates a simple method to modify PEM films with concentration gradients of non-covalently bound biomolecules and with gradients in stiffness. These results highlight the potential of this technique to efficiently and quickly determine the optimal biochemical and mechanical cues necessary for specific cellular processes.

Redox-controlled release of molecular payloads from multilayered organometallic polyelectrolyte films.

Organometallic poly(ferrocenylsilane)s (PFSs) featuring redox responsive ferrocene units in their main chain are prepared and characterized for redox triggered film disassembly and release of molecular payloads. Positively or negatively charged side groups render PFS water soluble and these polyelectrolytes also allow the use of an electrostatic self-assembly process for the fabrication of novel functional supermolecular nanostructures. The layer-by-layer (LbL) electrostatic fabrication approach is utilized to obtain films with different bilayer numbers. The electrochemical behaviour of the multilayers with different thicknesses is recorded by capturing cyclic voltammograms (CVs). Film disassembly (multilayer re-dispersed in water) is performed by exposing the multilayers to the different values of holding potentials, corresponding to partial or full oxidation of PFS. Disassembly kinetics is quantitatively monitored by determining the amount of polymer released from CV experiments, as well as from UV-VIS spectroscopy. The proposed PFS multilayer disassembly mechanism is presented. The morphology of the multilayer films as a function of holding times is monitored by AFM. The release of guest molecules such as fluorescent Alexa dyes incorporated at different depths in the multilayer films is studied by fluorescence spectroscopy.

Endothelialized and preconditioned natural umbilical arteries with long term patency open the route for future human uses

The major challenge of vascular tissue engineering is to develop a small calibre vascular graft with a high patency rate. In native vessels, the thrombosis is prevented by the endothelium located at the luminal site of the vessel. The aim of this study was to develop a resistant endothelial lining on the inner surface of vascular graft using a polyelectrolyte multilayers (PEM) film. Umbilical arteries were de-endothelialized, coated with 3.5 bilayers of poly(styrene sulfonate) (PSS)/poly(allylamine hydrochloride) (PAH) and then cellularized with endothelial cells. The grafts were cultured for a week in static condition and preconditioned by exposure to a shear stress of at 1 Pa for three hours before implantation on the rabbit carotid site. Histological and confocal microscopy in vitro investigations showed that PEMs films improve cell adhesion and retention on the luminal surface after shear stress preconditioning. In vivo Doppler data showed that graft preconditioning is a crucial factor for graft patency. Indeed, preconditioned grafts remained over the whole experimental period, whereas unpreconditioned grafts were obstructed after only one week of implantation. These results open the route toward the development of a new generation of vascular substitutes having a long term patency.

Anti-thrombogenicity by Layer-by-Layer

Anti-thrombogenic films with high durability were fabricated in a wet process. Anti-thrombogenicity was achieved with polyelectrolyte multilayer thin film prepared from poly(vinyl alcohol)-poly(acrylic acid)(PVA-PAA) blends, deposited in alternate layers with poly(allylamine hydrochloride) (PAH). Film durability, assessed by abrasion resistance and water resistance, was enhanced by forming crosslinks via amide bonds induced by heat treatment of the film. The film was found to be resistant to protein adsorption, as measured by the amount of fibrinogen adsorbed from an aqueous solution. Anti-thrombogenic efficacy was assessed in ex vivo experiments by the ability of stainless steel mesh, coated with the polyelectrolyte and inserted into a pig blood vessel, to inhibit thrombus formation. Mesh coated with the polyelectrolyte did not reduce blood flow over a period of 15 minutes, whereas with uncoated mesh blood flow stopped within 6 minutes because of blood vessel blockage by thrombus formation.

Label-free, disposable fiber-optic biosensors for DNA hybridization detection

A novel and highly sensitive fiber-optic DNA sensor based on a thin-core fiber modal interferometer (TCFMI) is demonstrated by using a layer-by-layer (LbL) self-assembly technology. Poly(ethylenimine) (PEI), poly(acrylic acid) (PAA) and single-stranded DNA (ssDNA) were used for the preparation of a polyelectrolyte multilayer film for DNA detection. The film thickness was measured through a surface profilometer. The surface morphologies of (PEI/PAA)4, (PEI/PAA)4.5 and (PEI/PAA)4(PEI/DNA)1 multilayer films were characterized by atomic force microscopy. The fabricated DNA sensors were tested with different types of target ssDNA solutions with a concentration of 1 μM. The results show that the sensitivity of the TCFMI-based ssDNA sensor is 0.27 nm/matched-base at the concentration of 1 μM and can even distinguish the number of matched bases of ssDNA chains.

Effect of RGD functionalization and stiffness modulation of polyelectrolyte multilayer films on muscle cell differentiation

Skeletal muscle tissue engineering holds promise for the replacement of muscle damaged by injury and for the treatment of muscle diseases. Although arginylglycylaspartic acid (RGD) substrates have been widely explored in tissue engineering, there have been no studies aimed at investigating the combined effects of RGD nanoscale presentation and matrix stiffness on myogenesis. In the present work we use polyelectrolyte multilayer films made of poly(l-lysine) (PLL) and poly(l-glutamic) acid (PGA) as substrates of tunable stiffness that can be functionalized by a RGD adhesive peptide to investigate important events in myogenesis, including adhesion, migration, proliferation and differentiation. C2C12 myoblasts were used as cellular models. RGD presentation on soft films and increasing film stiffness could both induce cell adhesion, but the integrins involved in adhesion were different in the case of soft and stiff films. Soft films with RGD peptide appeared to be the most appropriate substrate for myogenic differentiation, while the stiff PLL/PGA films induced significant cell migration and proliferation and inhibited myogenic differentiation. ROCK kinase was found to be involved in the myoblast response to the different films. Indeed, its inhibition was sufficient to rescue differentiation on stiff films, but no significant changes were observed on stiff films with the RGD peptide. These results suggest that different signaling pathways may be activated depending on the mechanical and biochemical properties of multilayer films. This study emphasizes the advantage of soft PLL/PGA films presenting the RGD peptide in terms of myogenic differentiation. This soft RGD-presenting film may be further used as a coating of various polymeric scaffolds for muscle tissue engineering.

Organic and Inorganic Dyes in Polyelectrolyte Multilayer Films

Polyelectrolyte multilayer films are a versatile functionalization method of surfaces and rely on the alternated adsorption of oppositely charged species. Among such species, charged dyes can also be alternated with oppositely charged polymers, which is challenging from a fundamental point of view, because polyelectrolytes require a minimal number of charges, whereas even monovalent dyes can be incorporated during the alternated adsorption process. We will not only focus on organic dyes but also on their inorganic counterparts and on metal complexes. Such films offer plenty of possible applications in dye sensitized solar cells. In addition, dyes are massively used in the textile industry and in histology to stain textile fibers or tissues. However, the excess of non bound dyes poses serious environmental problems. It is hence of the highest interest to design materials able to adsorb such dyes in an almost irreversible manner. Polyelectrolyte multilayer films, owing to their ion exchange behavior can be useful for such a task allowing for impressive overconcentration of dyes with respect to the dye in solution. The actual state of knowledge of the interactions between charged dyes and adsorbed polyelectrolytes is the focus of this review article.

In situ-ATR–FTIR analysis on the uptake and release of streptomycin from polyelectrolyte complex layers

In-situ ATR–FTIR spectroscopy and line shape analysis of the diagnostic spectral region was used to quantify the bound amount and release of the antibiotic streptomycin (STRP) at polyelectrolyte (PEL) multilayers (PEM) of poly(ethyleneimine) (PEI) and poly(acrylic acid) (PAA) or PEI and sodium alginate (ALG). Unlike common concepts based on the drug enrichment of the release medium, this analytical concept allowed to measure quantitatively the drug depletion in the delivery matrix. The measured kinetic in situ ATR–FTIR data were analysed by a modified Korsmeyer–Peppas equation based on two characteristic release parameters k and n.As main experimental parameters the number of PEL layers (adsorption steps) z and the STRP/PEL ratio were varied. For z=8 the STRP/PEL ratio showed the most significant influence on release kinetics, whereby for STRP/PEL=1:25 slowest (n=0.77) and lowest (k=21.4%) and for STRP/PEL=1:5 most rapid (n=0.30) and highest (k=58.6%) drug releases were found. PEM-PEI/ALG-8 (STRP/PEL=1:5) revealed slower release rates (n=0.58) and lower released STRP amounts (k=17.1%) compared to PEI/PAA. UV–VIS data on time dependent STRP enrichment of the release medium showed a similar trend compared to respective ATR–FTIR data on STRP depletion in PEM. Released amounts of around 1–2mg from the herein introduced PEM films could be determined. The introduced analytical concept will be used as screening tool for other drugs, drug eluting films and bone substituting materials.

Carbaryl Detection by Gold Nanoparticles Electrodes for Fruit Application Model

A new method has been developed for the determination of carbaryl pesticides, , in water and fruit samples using gold nanoparticles electrode conductivity detection. The effects of pH on the binding between carbaryl and humic acid (HA) and number of layer coated electrode were investigated and optimized. HA modified the polyelectrolyte multilayer thin films (PEMs) coating electrode was used to detect carbaryl. The current was decreased 4.75% after dipping in HA for 10 minutes. The current was decreased around 7.28% from the initial value after HA binding with carbaryl. The results demonstrated that the newly developed method was an efficient procedure for the detection of carbamate pesticides in water.

Prolonging the Duration of Preventing Bacterial Adhesion of Nanosilver-Containing Polymer Films through Hydrophobicity

A superhydrophobic coating composed of silver nanoparticles was developed on copper from fluorinated multilayered polyelectrolyte films to examine its performance in preventing microbial adhesion. Antibacterial and antibiofouling experiments for this novel coating were conducted with SRB. From the disk diffusion tests (for 48 h), it was found that, compared to the traditional coating composed of nanosilver, this novel coating significantly improved antibacterial performance and long-term effectiveness. The oxidation states of the immobilized silver in polyelectrolyte multilayer films were investigated with X-ray photoelectron spectroscopy (XPS), and the stability of the immobilized silver was evaluated through a leaching test. It was found that if silver was exposed to aqueous environments some ionic silver species would be produced and released. The ion release kinetics showed that the duration of sustained release of antibacterial Ag ions from the novel coatings was prolonged, which was why they had more long-term antibacterial performance.

Deposition of Polyelectrolyte Multilayer Film on a Nanoporous Alumina Membrane for Stable Label-Free Optical Biosensing

The stabilization of inorganic nanostructures is a key challenge in developing label-free optical biosensors using inorganic nanoporous films. In the present study, a polyelectrolyte multilayer film (PMF) composed of poly(acrylic acid) and poly(allylamine hydrochloride) was deposited on porous anodic alumina (PAA) film (pore diameter = 40 nm) by the layer-by-layer technique. The characteristics of the PMF as a protective coating layer against dissolution of the PAA film were examined by means of in situ optical waveguide spectroscopy (OWS), ex situ scanning electron microscope/energy-dispersive spectroscopy (SEM/EDS), and infrared reflection–adsorption spectroscopy (IR-RAS). The results obtained by OWS and SEM/EDS indicated the formation of PMF at both the alumina pore surface and the PAA film surfaces. Processing the PMF-deposited PAA (PMF–PAA) film at 180 °C to promote thermal cross-linking allowed PMF to act as a strong protective coating layer in aqueous buffer solutions (pH 2.5–8.7) but also in common alumina matrix etchant. Residual amino groups in the cross-linked PMF could also be used to conjugate single-probe DNA at the PMF–PAA film for OWS-based DNA–DNA hybridization assay. From the results obtained in this study, it was concluded that the deposition of PMF followed by thermal cross-linking is potentially useful not only as a protective coating and but also for immobilizing biorecognition elements in the PAA film.

Control of the surface properties of micro-fluidic devices for external power-free biochip application

Micro-fluidic or nano-fluidic devices have been received a great attraction in chemical and biological applications. Due to the increase of the surface/volume ratio, control of the surface properties of these devices is highly important. Polymer-based materials have been newly developed in micro-fluidic or nano-fluidic devices due to the great needs of easy processing, cost-effectiveness and clarity for the material. However, it is still challenging to control of the surface properties of these devices on demand. Most of the current materials in use for micro-fluidic devices are relatively hydrophobic; therefore, they usually need an extra assistance to transport aqueous samples within the microfluidic chip, such as micro-pump or any relevant mechanical or electrical apparatus. This makes the micro-fluidic device bulky and difficult to be portable. Polyelectrolyte multilayer (PEM) films assembled by layer-by-layer are very practical coatings to modify surfaces of various substrates. Layer-byLayer deposition (LbL) is a versatile technique whereby ultrathin films are assembled from the repetitive, sequential adsorption of oppositely charged polyelectrolytes from dilute aqueous solution. Moreover, the functional groups which remained reactive after the film deposition allow further chemical reactions such as polymer micro-contact printing, selective photo-crosslinking and controlled nanoparticle synthesis. In this paper, we utilized this method to modify the surface of the micro-channel of the device in order to control cellular interactions inside of the channel. We have been studied cellular interactions on various polyelectrolyte multilayer films on open surfaces. Multilayer films comprised of weak polyelectrolytes exhibit different surface properties as they were assembled at different pH conditions. Therefore, the wettability of the PEM-coated surface also can be tuned by changing the assembly condition. We applied various PEM films on the channel surface of microfluidic device and tested the wetting property and capillary force build-up in the micro-channel. Considering biological applications, weak polyelectrolytes including biocompatible polymer such as poly(hyaluronic acid) (HA) were investigated for this purpose. And the preliminary results were obtained in the study of surface-cell interaction using these coatings.

Polyelectrolyte Multilayers Studied by Electron Paramagnetic Resonance (EPR) Spin-Label Technique

A nitroxide spin label has been covalently attached to the polyelectrolyte poly(ethylene-alt-maleic acid) (P(E-alt-MA)) to study the interaction between this weak polyanion, the oppositely charged strong polycation poly(diallyldimethylammonium chloride) (PDADMAC) and water in swollen polyelectrolyte multilayers (PEM) by electron paramagnetic resonance (EPR) spectroscopy. If the spin-labeled polyanion has been used for the preparation of every double layer, the growth of the PEM film can be monitored by quantitative EPR. On the other hand, if the spin-labeled polyanion has been selectively placed in different layers in the PEM film the influence of the environment such as pH of the swelling medium of the mobility of the polyelectrolyte molecules positioned in the selected layer can be investigated.

Effect of Linear Elongation on Carbon Nanotube and Polyelectrolyte Structures in PDMS-Supported Nanocomposite LbL Films

Polyelectrolyte (PE) multilayer (PEM) thin films prepared by layer-by-layer self-assembly on flexible substrates are exposed to elongation in many fields of technology. Upon elongation, these types of films are showing interesting, but not understood, phenomena, such as controlled wetting, stimuli-responsive nanovalves, and lithography-free surface structuring. To investigate the mechanisms causing these interesting phenomena, we employed spectroscopic investigations of supported PEM films that were prepared from polystyrene sulfonate (PSS)-wrapped single-walled carbon nanotubes (SWNTs) or pyrene-labeled PSS (PSS-PY) and polydiallyldiammonium chloride. Our results show that the SWNTs agglomerated upon deposition into the PEM and showed a strong change in orientation upon uniaxial elongation of the PEM. Upon release of elongation, the resulting wrinkling pattern was changing its wavelength upon time, in the case of the SWNT-containing PEM. Fluorescence measurements of the PSS-PY in the PEM showed that the PEs changed their orientation due to constant mechanical force from elongation up to a time scale of 2 days after beginning the elongation. The results prove that elongated and released PEM films, until now considered static structures, possess strong kinetics, which has to be taken into account for their application.

Secondary Structure of rhBMP-2 in a Protective Biopolymeric Carrier Material

Efficient delivery of growth factors is one of the great challenges of tissue engineering. Polyelectrolyte multilayer films (PEM) made of biopolymers have recently emerged as an interesting carrier for delivering recombinant human bone morphogenetic protein 2 (rhBMP-2 noted here BMP-2) to cells in a matrix-bound manner. We recently showed that PEM made of poly(l-lysine) and hyaluronan (PLL/HA) can retain high and tunable quantities of BMP-2 and can deliver it to cells to induce their differentiation in osteoblasts. Here, we investigate quantitatively by Fourier transform infrared spectroscopy (FTIR) the secondary structure of BMP-2 in solution as well as trapped in a biopolymeric thin film. We reveal that the major structural elements of BMP-2 in solution are intramolecular β-sheets and unordered structures as well as α-helices. Furthermore, we studied the secondary structure of rhBMP-2 trapped in hydrated films and in dry films since drying is an important step for future applications of these bioactive films onto orthopedic biomaterials. We demonstrate that the structural elements were preserved when BMP-2 was trapped in the biopolymeric film in hydrated conditions and, to a lesser extent, in dry state. Importantly, its bioactivity was maintained after drying of the film. Our results appear highly promising for future applications of these films as coatings of biomedical materials, to deliver bioactive proteins while preserving their bioactivity upon storage in dry state.

Polyelectrolyte multilayer films made from polyallylamine and short polyphosphates: Influence of the surface treatment, ionic strength and nature of the electrolyte solution

The present article focuses on the influence of the salt concentration as well as on the influence of the nature of the salt on the deposition of (PAH-PSP)n films in the case where PSP is made from only 24 monomer units on average. The two main messages of this article are that: (i) for salt concentrations lower than about 0.2M, the film thickness increases linearly with the number of adsorption cycles after the deposition of a coating with a critical thickness necessary to prime the deposition of the film. In addition, for salt concentrations lower than 0.2M, there is no effect of the influence of the supporting electrolyte on the film thickness but a marked influence on the film composition as revealed from XPS measurements. (ii) For salt concentrations higher than about 0.2M, the films are extremely rough, and we demonstrate that the use of a homogeneous and isotropic model is not suited anymore to calculate the film thickness and its refractive index from the measured ellipsometric angles.Finally we try to rationalize all the data obtained so far on the (PAH-PSP)n films on the basis of counterion condensation of Na+ cations on the PSP chains.

Surface induced porous morphological transition of the organic self-assembled monolayer hybridized polyelectrolyte thin films

Surface coating is used for materials to achieve a desired surface property without changing the whole material. Polyelectrolyte multilayer (PEM) deposition may be one of the most versatile techniques to create conformal surface coating in large area with a great control over the film thickness in nano-meter scale. These multilayer films are assembled through, so called, the layer-by-layer (LbL) deposition process which is the repetitive, sequential dipping of the substrate into the oppositely charged polyelectrolytes solutions. PEM films can be used for various substrates (glass slides, silicon wafer, plastics, etc.) without any difficult treatment. Moreover, the functional groups which remained reactive after the film deposition allow further chemical reactions such as the polymer micro-contact printing and the selective crosslinking. Due to these advantages, many studies using the LbL technique have conducted intensively to create functional coatings for electro-optical and biomedical applications. Other frequently used method of surface coating is self-assembled monolayer (SAM) formation. SAM can form organic interfaces with the properties largely controlled by the end groups of the molecules comprising of the film. Certainly, these two methods are the most competitive fabrication techniques of surface coating. While the surface properties of most SAMs are presented by the nature of the end groups of SAM molecules, PEM films exhibit their surface properties that are orchestrated by the chemical compositions of the polyelectrolyte film and physical factors such as surface morphology, thickness, etc.. If these two very different materials meet, what phenomena will occur at the interface? How would they compensate their discrepancy? In this paper, we present a study of the coating system that was fabricated as a mixture of PEM and SAM. The hybrid films were prepared by polyelectrolyte multilayers comprised of hydrophilic polyelectrolytes assembled atop of hydrophobic SAM films. We have observed a unique phase separation of the SAM/PEM hybrid film, which lead the formation of nano-pores of the film surface. Surface properties including wetting and biological interaction behaviors of the hybrid films are also discussed in this paper.