Effect of functionalization of multilayered polyelectrolyte films on motoneuron growth

We studied in vitro cell-substrate interaction of motoneurons with functionalized polylectrolyte films. Thin polylectrolyte films were built on glass by alternating polycations, poly(ethylene-imine) PEI, poly(L-lysine) PLL, or poly(allylamine hydrochloride) PAH, and polyanions, poly(sodium-4-styrenesulfonate) PSS or poly(L-glutamic acid) (PGA). These architectures were functionalized with Brain Derived Neurotrophic Factor (BDNF) or Semaphorin 3A (Sema3A). We used Optical Waveguide Lightmode Spectroscopy (OWLS) and Atomic Force Microscopy (AFM) to characterize the architectures. The viability of motoneurons was estimated by the acid phosphatase method, and morphometrical measures were performed to analyse the influence of different architectures on cell morphology. Motoneurons appeared to adhere and spread on all the architectures tested and preferentially on PSS ending films. The viability of motoneurons on polyelectrolyte multilayers was higher compared to polyelectrolyte monolayers. BDNF and Sema3A embedded in the films remained active and thereby create functionalized nanofilms.     

Gene delivery in vitro and in vivo from bioreducible multilayered polyelectrolyte films of plasmid DNA

Layer-by-layer (LbL) films were assembled on flexible stainless steel substrate using plasmid DNA and reducible hyperbranched poly(amido amine) (RHB) polycation. The films were characterized by XPS and their disassembly in reducing conditions confirmed by ellipsometry. Fibroblast and smooth muscle cell attachment and proliferation on DNA/RHB films were indistinguishable from those on control DNA/poly(ethylenimine) (PEI) films. In vitro transfection activity was evaluated using reporter plasmids encoding for secreted alkaline phosphatase (SEAP) and green fluorescent protein (GFP). DNA/RHB films showed higher and longer lasting transfection activity than control DNA/PEI films using SEAP plasmid. It was revealed through the use of GFP plasmid that DNA/RHB films transfected almost the entire cell population growing on the films. In vivo transfection activity was evaluated by subcutaneously implanting a stainless steel substrate coated with the DNA/RHB films containing SEAP plasmid DNA and measuring the levels of SEAP secreted into the blood circulation of rats. It was found that the plasma levels of SEAP peaked at ～160 ng SEAP/mL five days post-implantation.     

A facile method to construct hybrid multilayered films as a strong and multifunctional antibacterial coating

A multifunctional multilayered film containing TiO(2) nanoparticles as contact-active antibacterial agent and nanosilver as a release-active antibacterial agent was fabricated via layer-by-layer assembly. TiO(2) nanoparticles with the anatase crystalline dominant structure were synthesized via a sol-gel method. The QCM, AFM, and contact angle measurement results indicated that the TiO(2) nanoparticle-chitosan was successfully assembled with heparin via layer-by-layer assembly. The UV visible spectroscopy demonstrated that the silver ions could be loaded into the multilayers and in situ synthesize silver nanoparticles in the multilayers template. The short-term antibacterial assay showed the TiO(2)-chitosan/heparin multilayers loaded with nanosilver was bactericidal both in the low intensity UV light and in the dark. The long-term antibacterial assay indicated although the antibacterial in dark decreased with the PBS immersion time, the hybrid multilayered films sustained the long-term antibacterial in the low intensity UV light.     

Biologically active lipid A antagonist embedded in a multilayered polyelectrolyte architecture

Recently [Jessel N, Schwinte P, Donohue R, Lavalle P, Boulmedais F, Darcy R, et al. Pyridylamino-beta-cyclodextrin as a molecular chaperone for lipopolysaccharide embedded in a multilayered polyelectrolyte architecture. Adv Funct Mater 2004;14:963-9], we demonstrated the biological activity of a lipopolysaccharide from Escherichia coli incorporated into layer-by-layer films made of poly (l-lysine) and poly (l-glutamic acid) and containing a polycationic beta-cyclodextrin (CD) with chaperone properties. Here we develop innovative architectures containing a complex made of a charged beta-cyclodextrin and a lipid A antagonist (LAA) as potential systems for local endotoxin antagonistic activity. We examine the biological activity of these architectures. The CD-LAA complex adsorbed on top, or embedded into the polyelectrolyte films keeps its LPS antagonistic activity on both murine and human macrophages for at least 24h.     

Surface functionalization of hyaluronic acid hydrogels by polyelectrolyte multilayer films

Hyaluronic acid (HA), an anionic polysaccharide, is one of the major components of the natural extracellular matrix (ECM). Although HA has been widely used for tissue engineering applications, it does not support cell attachment and spreading and needs chemical modification to support cellular adhesion. Here, we present a simple approach to functionalize photocrosslinked HA hydrogels by deposition of poly(l-lysine) (PLL) and HA multilayer films made by the layer-by-layer (LbL) technique. PLL/HA multilayer film formation was assessed by using fluorescence microscopy, contact angle measurements, cationic dye loading and confocal microscopy. The effect of polyelectrolyte multilayer film (PEM) formation on the physicochemical and mechanical properties of hydrogels revealed polyelectrolyte diffusion inside the hydrogel pores, increased hydrophobicity of the surface, reduced equilibrium swelling, and reduced compressive moduli of the modified hydrogels. Furthermore, NIH-3T3 fibroblasts seeded on the surface showed improved cell attachment and spreading on the multilayer functionalized hydrogels. Thus, modification of HA hydrogel surfaces with multilayer films affected their physicochemical properties and improved cell adhesion and spreading on these surfaces. This new hydrogel/PEM composite system may offer possibilities for various biomedical and tissue engineering applications, including growth factor delivery and co-culture systems.     

Endothelial cell--interactions with polyelectrolyte multilayer films

The seeding of endothelial cells (ECs) on biomaterial surfaces became a major challenge, allowing to improve the non-thrombogenic properties of these surfaces. Recently, the use of polyelectrolyte films has been suggested as a new versatile technique of surface modification aimed at tissue engineering. In this study, we evaluate the adhesion properties of ECs on two types of polyelectrolyte films ending either by poly(D-lysine) (PDL), or poly(allylamine hydrochloride) (PAH), and compared them to data obtained on PDL or PAH monolayers, glass and fibronectin (Fn)-coated glass. ECs seeded on polyelectrolyte films showed a good morphology, allowing ECs to resist physiological shear stress better compared to ECs seeded on glass or Fn. The expression of beta1 integrins was slightly lower on polyelectrolyte films than on control surfaces. However, the phosphorylation of focal adhesion kinase, involved in the transduction of adhesion signal, was not modified on PAH ending films compared to control surfaces; whereas it became lower on PDL ending films. Finally, PAH ending films improve strongly ECs adhesion without disturbing the adhesion mechanism, necessary for the development of a new endothelium. These types of films or similar build-ups could thus be used in the future as a way to modify surfaces for vascular tissue engineering.     

Engineering muscle tissues on microstructured polyelectrolyte multilayer films

The use of surface coating on biomaterials can render the original substratum with new functionalities that can improve the chemical, physical, and mechanical properties as well as enhance cellular cues such as attachment, proliferation, and differentiation. In this work, we combined biocompatible polydimethylsiloxane (PDMS) with a biomimetic polyelectrolyte multilayer (PEM) film made of poly(L-lysine) and hyaluronic acid (PLL/HA) for skeletal muscle tissue engineering. By microstructuring PDMS in grooves of a different width (5, 10, 30, and 100?μm) and by modulating the stiffness of the (PLL/HA) films, we guided skeletal muscle cell differentiation into myotubes. We found optimal conditions for both the formation of parallel-oriented myotubes and their maturation. Significantly, the myoblasts were collectively prealigned to the grooves before their differentiation. Before fusion, the highest aspect ratio and orientation of nuclei were observed for the 5 and 10?μm wide micropatterns. The formation of myotubes was observed regardless of the size of the micropatterns, and we found that their typical width was 10-12?μm. Their maturation was characterized by the immunolabeling of type II isomyosin. The amount of myosin striation was not affected by the topography, except for the 5?μm wide micropatterns. We highlighted the spatial constraints that led to an important nuclei deformation and further impairment of maturation within the 5?μm grooves. Altogether, our results show that the PEM film combined with PDMS is a powerful tool that is used for skeletal muscle engineering. This work opens perspectives for the development of skeletal muscle tissue in contact with films containing bioactive peptides or growth factors as well as for the study of pathogenic myotubes.     

The response of bone cells to titanium surfaces modified by simvastatin-loaded multilayered films

Abstract

The aim of this study was to enhance cytocompatibility of titanium substrates by loading a multilayer film of chitosan (Chi), gelatin (Gel) and simvastatin (SV). This was fabricated using a spin-assisted layer-by-layer (LBL) technique. The surface properties of the different substrates were characterized by field emission scanning electron microscopy (FE-SEM), atomic force microscope (AFM), X-ray photoelectron spectroscopy (XPS) and contact angle measurement, respectively. Simvastatin release in vitro was measured by ultraviolet-visible spectrophotometer. A well morphology with filopodia extensions was observed in mesenchymal stem cells (MSCs) grown on simvastatin loaded multilayered films-modified titanium substrates. After 7, 14 and 21 days of culture, the simvastatin loaded multilayered films increased cell proliferation, improved osteoblastic differentiation of alkaline phosphatase (ALP) and mineralization. Additionally, osteoclast diffentiation marker tartrate-resistant acid phosphatase (TRAP) was decreased in simvastatin loaded multilayered films. This study provides a new insight for the fabrication of titanium-based implants to enhance osseointegration especially for osteoporosis patients in orthopedic application.     

Tunable dual growth factor delivery from polyelectrolyte multilayer films

A promising strategy to accelerate joint implant integration and reduce recovery time and failure rates is to deliver a combination of certain growth factors to the integration site. There is a need to control the quantity of growth factors delivered at different times during the healing process to maximize efficacy. Polyelectrolyte multilayer (PEM) films, built using the layer-by-layer (LbL) technique, are attractive for releasing controlled amounts of potent growth factors over a sustained period. Here, we present PEM films that sequester physiological amounts of osteogenic rhBMP-2 (recombinant human bone morphogenetic protein-2) and angiogenic rhVEGF??? (recombinant human vascular endothelial growth factor) in different ratios in a degradable [poly(β-amino ester)/polyanion/growth factor/polyanion] LbL tetralayer repeat architecture where the biologic load scaled linearly with the number of tetralayers. No burst release of either growth factor was observed as the films degraded. The release of rhBMP-2 was sustained over a period of 2 weeks, while rhVEGF??? eluted from the film over the first 8 days. Both growth factors retained their efficacy, as quantified with relevant in vitro assays. rhBMP-2 initiated a dose dependent differentiation cascade in MC3T3-E1S4 pre-osteoblasts while rhVEGF??? upregulated HUVEC proliferation, and accelerated closure of a scratch in HUVEC cell cultures in a dose dependent manner. In vivo, the mineral density of ectopic bone formed de novo by rhBMP-2/rhVEGF??? PEM films was approximately 33% higher than when only rhBMP-2 was introduced, with a higher trabecular thickness, which would indicate a decrease in the risk of osteoporotic fracture. Bone formed throughout the scaffold when both growth factors were released, which suggests more complete remodeling due to an increased local vascular network. This study demonstrates a promising approach to delivering precise doses of multiple growth factors for a variety of implant applications where control over spatial and temporal release profile of the biologic is desired.     

Control of drug accessibility on functional polyelectrolyte multilayer films

A surface coating based on polylysine/hyaluronic acid multilayers was designed and acted as a reservoir for an antiproliferative agent, paclitaxel (Taxol). Absolutely no chemical modification of polyelectrolytes or of the drug was needed and the final architecture was obtained in an extremely simple way using the layer-by-layer method. The paclitaxel dose available for human colonic adenocarcinoma cells HT29 seeded on the films could be finely tuned. Moreover, the accessibility of the drugs was controlled by adding on the top of the drug reservoir a capping made of synthetic polyelectrolyte multilayers. This capping was also required to allow adhesion of HT29 cells. Paclitaxel activity was maintained after embedding in the polyelectrolyte multilayers and cellular viability could be reduced by about 80% 96 h after seeding. The strategy described in this paper could be valuable for various other drug/cell systems.     

Construction and enzymatic degradation of multilayered poly-l-lysine/DNA films

The layer-by-layer (LbL) self-assembly of poly-l-lysine (PLL) and deoxyribonucleic acid (DNA) was used to construct the enzymatic biodegradable multilayered films. The LbL build up of DNA multilayers was monitored by UV-vis spectrometry, and atomic force microscopy (AFM). AFM, UV-vis spectrometry and fluorescence spectrometry measurements indicated that 90% of DNA within the films was released almost linearly under 5 U mL(-1)alpha-chymotrypsin in PBS at 37 degrees C in 35 h. TEM and zeta potential experiments revealed that the released DNA molecules were condensed into the slight positive complexes with size from 20 to several hundred nanometers. The well-structured, easy processed enzymatic biodegradable multilayered film may have great potential for gene applications in tissue engineering, medical implants, etc.     

Layer-by-layer assembly of polyelectrolyte films improving cytocompatibility to neural cells

The using of layer-by-layer assembly polyelectrolyte (PE) films has been suggested as a new versatile technique for surface modification aimed at tissue engineering and cell-based chips. In this study, we investigated the surface morphology of the hyaluronic acid (HA)-based PE films deposited on the amino-functionalized glass slides using atomic force microscopy. These thin films (bilayer number <9) were measured to have nanoscale roughness ranging from 10 to 100 nm. Then the primary hippocampal and cortical neural cells were cultured on the PE films, respectively. After 5 days of culturing, the cytocompatibility to neural cells was evaluated by cellular morphology, neurite outgrowth, and microtubule-associated protein 2 expressions. From the present results, the HA-based PE films were found to be able to support neural cell adhesion and neurite development, especially for the polycation-ending films. It is suggested these HA-based multilayer PE films or similar build-ups could thus be used in the future as a way to modify surfaces for nerve scaffolds and neuron-based chips.     

Fibrillar films obtained from sodium soap fibers and polyelectrolyte multilayers

An objective of tissue engineering is to create synthetic polymer scaffolds with a fibrillar microstructure similar to the extracellular matrix. Here, we present a novel method for creating polymer fibers using the layer-by-layer method and sacrificial templates composed of sodium soap fibers. Soap fibers were prepared from neutralized fatty acids using a sodium chloride crystal dissolution method. Polyelectrolyte multilayers (PEMs) of polystyrene sulfonate and polyallylamine hydrochloride were deposited onto the soap fibers, crosslinked with glutaraldehyde, and then the soap fibers were leached with warm water and ethanol. The morphology of the resulting PEM structures was a dense network of fibers surrounded by a nonfibrillar matrix. Microscopy revealed that the PEM fibers were solid structures, presumably composed of polyelectrolytes complexed with residual fatty acids. These fibrillar PEM films were found to support the attachment of human dermal fibroblasts.     

Defect formation in thin polyelectrolyte films on polycrystalline NiTi substrates

This paper reports on the mechanical properties of ultrathin PAA/PAH (polyacrylic acid/polyallylamine hydrochloride) polyelectrolyte films deposited by a layer-by-layer technique on a polycrystalline Nickel–Titanium (NiTi) substrate. Since thin polyelectrolyte films are potentially suitable coatings to reduce the Ni release in biomedical applications, the mechanical properties of the thin films were determined by applying monotonic and cyclic tensile strains of 5% and 3%, respectively. While single tensile strains up to 5% revealed the amazing strain to failure of the applied coating, cyclic strains resulted in defect formation within the polyelectrolytes.To provide a better understanding of the mechanisms that are determining the defect formation, macroscopic and microscopic defect localizations were determined by digital image correlation–and EBSD (electron back-scattered diffraction)–techniques. Defects emerged particularly within areas of elevated local strain differences and were predominantly observed in the vicinity of grain boundaries. To relate these findings to the transformation behavior of polycrystalline NiTi considering strain localizations and intergranular constraints, crystallographic data obtained from the EBSD measurements were correlated with the defect distribution. EBSD data revealed a distinct dependence of defect formation on misorientation of neighboring grains.     

Spatial control of cellular adhesion using photo-crosslinked micropatterned polyelectrolyte multilayer films

Cellular patterning on biomaterial surfaces is important in fundamental studies of cell–cell and cell–substrate interactions, and in biomedical applications such as tissue engineering, cell-based biosensors, and diagnostic devices. In this study, we combined the layer-by-layer polyelectrolyte multilayer deposition and photolithographic technique to create an easy and versatile technique for cell patterning. Poly(acrylic acid) (PAA) conjugated with 4-azidoaniline was interwoven in PAA/polyacrylamide (PAM) multilayer films. After UV irradiation through a photo mask, the UV-exposed areas were crosslinked and the unexposed areas were rinsed away by alkaline water, resulting in micropatterns. Cell patterns were formed when the cell adhesion was limited to the base substrate, but not on the multilayer films. The stability of cell patterns could be modulated by simply modification of the surface chemistry of base substrate and PEM films with conjugation of bioactive macromolecules. This technique can be also applied to other PEM systems with proper rinsing protocol, and many types of substrates. Cell co-culture systems can be also achieved by this technique.     

Apatite coating on anionic and native collagen films by an alternate soaking process

The present study focuses on apatite coating on collagen films, with various different densities of carboxyl groups, using an alternate soaking process. Anionic collagen (AC), which has different densities of carboxylic groups compared to native collagen (NC), was obtained by hydrolysis of carboxyamides of asparagine and glutamine residues. From X-ray diffraction analysis, apatite was found to be coated on AC and NC films. Peaks ascribed to apatite were observed at 26 degrees and 32 degrees in the diffraction patterns of hydroxyapatite crystals. The amount of apatite coated on both AC and NC collagen films continued to increase up to 100 reaction cycles. However, there is a significant difference in apatite coating between the two films. The amount of apatite formed on the surface of AC film increased 1.24 times faster than on NC film. The scanning electron photomicrograph images of the mineralized NC and the AC film coatings formed after 100cycles show that regular porous apatite coating had formed within the collagen fibrils. These results suggest that the higher content of carboxyl groups in AC plays an effective role in the heterogeneous nucleation of apatite in the body environment.     

High-throughput cellular screening of engineered ECM based on combinatorial polyelectrolyte multilayer films

The capacity to engineer the extracellular matrix is critical to better understand cell function and to design optimal cellular environments to support tissue engineering, transplantation and repair. Stacks of adsorbed polymers can be engineered as soft wet three dimensional matrices, with properties tailored to support cell survival and growth. Here, we have developed a combinatorial method to generate coatings that self assemble from solutions of polyelectrolytes in water, layer by layer, to produce a polyelectrolyte multilayer (PEM) coating that has enabled high-throughput screening for cellular biocompatibility. Two dimensional combinatorial PEMs were used to rapidly identify assembly conditions that promote optimal cell survival and viability. Conditions were first piloted using a cell line, human embryonic kidney 293 cells (HEK 293), and subsequently tested using primary cultures of embryonic rat spinal commissural neurons. Cell viability was correlated with surface energy (wettability), modulus (matrix stiffness), and surface charge of the coatings.Our findings indicate that the modulus is a crucial determinant of the capacity of a surface to inhibit or support cell survival.     

Antifungal activity of DNA-lipid complexes and DNA-lipid films against Candida species

In this study amphiphilic lipids, DNA-lipid complexes, and DNA-lipid films were prepared, and their antifungal activity against Candida species was examined. The amphiphilic lipids were synthesized from a reaction of glycine or L-alanine with n-alkyl alcohol in the presence of p-toluene sulfonic acid. DNA-lipid complexes, which were prepared by the simple mixing of DNA and amphiphilic lipids, were insoluble in water. Self-standing, water-insoluble DNA-lipid films were prepared by casting the DNA-lipid complexes from a chloroform/ethanol solution. The antifungal activities of the lipids and DNA-lipid complexes against the Candida species were evaluated by minimum inhibitory concentrations (MICs); those of DNA-lipid films were evaluated by the disk diffusion method. The seven kinds of lipids, DNA-lipid complexes, and DNA-lipid films showed antifungal activity, and no differences were seen in the antifungal activities between glycine and L-alanine derivatives. The lipids, DNA-lipid complexes, and DNA-lipid films, which have shorter alkyl chain length in lipids, showed antifungal activity against all Candida species. However, the effect of antifungal activity against Candida species decreased with increased alkyl chain length in lipids. In this study, it was found that lipids, DNA-lipid complexes, and films with a decyl or dodecyl group exhibit more favorable antifungal activity.     

Controlled degradation of multilayered poly(lactide-co-glycolide) films using electron beam irradiation

The ability to undergo predictable and controlled degradation allows biopolymers to release prescribed dosages of drugs locally over a sustained period. However, the bulk or homogeneous degradation of some of these polymers like poly(L-lactide) (PLLA) and poly(lactide-co-glycolide) (PLGA) work against a better controlled release of the drugs. Inducing the polymers to undergo surface erosion or layer-by-layer degradation could provide a better process of controlled drug release from the polymers. This study has demonstrated that surface erosion degradation of PLGA is possible with the use of a multilayer film system, with PPdlLGA [plasticized poly(D,L-lactide-co-glycolide) (PdlLGA)] as the surface layers and poly(L-lactide-co-glycolide) as the center layer. The use of the more hydrophilic PPdlLGA as the surface layer resulted in a faster degradation of the surface layers compared to the center layer, thus giving a surface erosion degradation effect. The rate of surface degradation could also be controlled with electron beam (e-beam) radiation, where e-beam irradiation was shown to alter the degradation time and onset of polymer mass loss. It was also shown that the more highly irradiated PPdlLGA surface layers had an earlier onset of mass loss, which resulted in a faster reduction in overall film thickness. The ability to control the rate of film thickness reduction with different radiation dose promises a better controlled release of drugs from this multilayer PLGA film system.     

Cytotoxicity of polyethyleneimine (PEI), precursor base layer of polyelectrolyte multilayer films

Polyethyleneimine (PEI) is a synthetic polymer commonly used as precursor base layer in polyelectrolyte multilayer films. However, the biological properties of this cationic macromolecule are poorly understood. The aim of this experimental investigation was to evaluate in vitro the biocompatibility of PEI towards two different human cell lines. The experimental investigation was undertaken on pure titanium (Ti) and nickel-titanium (NiTi) alloy samples with an average surface roughness of Ra=0.3microm. A biological study was undertaken at day 0 (2h after seeding), day 2, day 4 and day 7 to observe the cellular response of fibroblasts and osteoblasts cell lines in terms of morphology, adhesion (as observed by scanning electron microscopy), and viability (Mosmann's test). The results showed that PEI can be successfully deposited onto Ti or NiTi alloy, but generates a detrimental cellular response on both substrates as illustrated by a decrease of both fibroblast and osteoblast adhesion and proliferation over a 7-day culture period. These results suggest that PEI is potentially cytotoxic and may not be biocompatible enough in clinical applications using high molecular weight. As a consequence, polyelectrolyte multilayer films, which are promising in prosthesis and implantology fields, could not be coated with PEI at a high molecular weight. A lower molecular weight should be considered or a more biocompatible molecular base as precursor layer of polyelectrolyte multilayer films would be better to use for a good human bio-integration.