poly(L-lysine)/hyaluronic Acid Research Articles

Interaction of Lysozyme with Poly(L-lysine)/Hyaluronic Acid Multilayers: An ATR-FTIR Study

Polyelectrolyte multilayers (PEM) loaded with bioactive molecules such as proteins serve as excellent mimics of an extracellular matrix and may find applications in fields such as biomedicine and cell biology. A question which is crucial to the successful employment of PEMs is whether conformation and bioactivity of the loaded proteins is preserved. In this work, the polarized attenuated total reflection Fourier transform infrared (ATR-FTIR) technique is applied to investigate the conformation of the protein lysozyme (Lys) loaded into the poly(L-lysine)/hyaluronic acid (PLL/HA) multilayers. Spectra are taken from the protein in the PEMs coated onto an ATR crystal during protein adsorption and desorption. For comparison, a similar investigation is performed for the case of Lys in contact with the uncoated crystal. The study highlights the presence of both "tightly" and "poorly bound" Lys fractions in the PEM. These fractions differ in their conformation and release behavior from the PEM upon washing. Comparison of spectra recorded with different polarizations suggests preferential orientation of alpha helical structures, beta sheets and turns in the "tightly bound" Lys. In contrast, the "poorly bound" fraction shows isotropic orientation and its conformation is well preserved.

3D porous acellular cartilage matrix scaffold with surface mediated sustainable release of TGF-β3 for cartilage engineering

Acellular tissue matrix scaffolds are much closer to tissue’s complex natural structure and biological characteristics, thus assess great advantages in cartilage engineering. We used rabbit costal cartilage to prepare acellular microfilaments and further 3D porous acellular cartilage scaffold via crosslinking. Poly(l-lysine)/hyaluronic acid (PLL/HA) multilayer film was then built up onto the surface of the resulting porous scaffold. Furthermore, TGF-β3 was loaded into the PLL/HA multilayer film coated scaffold to obtain a 3D porous acellular cartilage scaffold with sustained releasing of TGF-β3 up to 60 days. The success of this project will provide a new way for the treatment of articular cartilage defects. Meanwhile, the anchoring and on-site sustained releasing of growth factors mediated by polyelectrolyte multilayered film can also provide a new method for improving the biocompatibility and the biofunctionality for other implanted biomaterials.

Mobility of lysozyme in poly(l-lysine)/hyaluronic acid multilayer films

The spatial and temporal control over presentation of protein-based biomolecules such as growth factors and hormones is crucial for in vitro applications to mimic the complex in vivo environment. We investigated the interaction of a model protein lysozyme (Lys) with poly(L-lysine)/hyaluronic acid (PLL/HA) multilayer films. We focused on Lys diffusion as well as adsorption and retention within the film as a function of the film deposition conditions and post-treatment. Additionally, an effect of Lys concentration on its mobility was probed. A combination of confocal fluorescence microscopy, fluorescence recovery after photobleaching, and microfluidics was employed for this investigation. Our main finding is that adsorption of PLL and HA after protein loading induces acceleration and reduction of Lys mobility, respectively. These results suggest that a charge balance in the film to a high extent governs the protein–film interaction. We believe that control over protein mobility is a key to reach the full potential of the PLL/HA films as reservoirs for biomolecules depending on the application demand.

Stretch-Induced Helical Conformations in Poly(l-lysine)/Hyaluronic Acid Multilayers.

We investigate the effect of stretching on the secondary structure of cross-linked poly(l-lysine)/hyaluronic acid (PLL/HA) multilayers. We show that stretching these films induces changes in the secondary structure of PLL chains. Our results suggest that not only α- but also 310-helices might form in the film under stretching. Such 310-helices have never been observed for PLL so far. These changes of the secondary structure of PLL are reversible, i.e., when returning to the nonstretched state one recovers the initial film structure. Using molecular dynamics simulations of chains composed of 20 l-lysine residues (PLL20), we find that these chains never adopt a helical conformation in water. In contrast, when the end-to-end distance of the chains is restrained to values smaller than the mean end-to-end distance of free chains, a distance domain rarely explored by the free chains, helical conformations become accessible. Moreover, the formation of not only α- but also 310-helices is predicted by the simulations. These results suggest that the change of the end-to-end distance of PLL chains in the stretched film is at the origin of the helix formation.

A biocompatible method of controlled retrieval of cell-encapsulating microgels from a culture plate.

We report a biocompatible method of selectively retrieving 3-D cell-encapsulating hydrogel microstructures from a culture substrate. First, poly(l-lysine)/hyaluronic acid (PLL/HA) polyelectrolyte multilayers (PEMs) with methacrylated chitosan (GMA-Chi) on top were formed on an ITO substrate. Then, a cell-encapsulating hydrogel micropattern was formed; a HepG2 cell-encapsulating heparin-based hydrogel micropattern was fabricated by thiol-ene photopolymerization. The application of an oxidative potential of 2 V resulted in the detachment of the cell-encapsulating hydrogels by the dissolution of PEMs. The time of complete retrieval of the hydrogels was controllable by modulating the number of PEM layers. The applied potential did not affect the viability or the function of the cells in the entire hydrogels. In contrast, when a reductive electrochemical potential (-1.8 V) was applied to a silane-modified ITO to release cell-encapsulating hydrogels by the desorption of silane [Chem. Commun., 2009, 5865], extensive cell death at the bottom of the hydrogel adjacent to the electrode was observed.

Microfluidics as A Tool to Understand the Build‐Up Mechanism of Exponential‐Like Growing Films

Microfluidics is used here for the first time to efficiently tune the growth conditions for understanding the build-up mechanism of exponentially growing polyelectrolyte (PE) films. The velocity of PE supply and time of interaction can be successfully altered during the layer-by-layer assembly. Another advantage of this method is that the deposition of poly-L-lysine/hyaluronic acid (PLL/HA) films in microchannels can be monitored online by fluorescence microscopy. The study demonstrates that PE mass transport to the film surface and diffusion in the film are key parameters affecting PLL/HA film build-up. Increase of PE supply rate results in a change in the "transition" (exponential-to-linear growth) towards higher number of deposition steps, thus indicating a mass transport-mediated growth mechanism.

Anisotropic multicompartment micro- and nano-capsules produced via embedding into biocompatible PLL/HA films

We present a novel strategy to fabricate anisotropic multicompartment Janus capsules by embedding larger containers into a soft poly-L-lysine/hyaluronic acid (PLL/HA) polymeric film, followed by adsorption of smaller containers on top of their unmasked surface. This research is also attractive for developing substrates for cell cultures.

Hole formation induced by ionic strength increase in exponentially growing multilayer films

Polyelectrolyte multilayer (PEM) films consist of polyanion/polycation super-structures that are sensitive to various stresses like ionic strength changes. We investigate the swelling process of the exponentially growing poly(L-lysine)/hyaluronic acid (PLL/HA) films induced by changes of the ionic strength of the contact solution. We show that above a first critical ionic strength the swelling is accompanied by a release of both polyelectrolytes constituting the film, leading to its subsequent dissolution. At a second critical ionic strength, the swelling of the multilayer is so important that, in addition to this polyelectrolyte release, formation of spherical holes is observed inside the film. The presence of dissolved PLL and HA chains in these holes leads to an increase of the concentration of their counterions inside of them, and thus induces an extra osmotic pressure. This in turn favors the size increase of the holes before they coalesce. The release of both polyelectrolytes from the film into the supernatant ultimately allows a decrease of the osmotic pressure inside the PLL/HA film, which finally leads to the disappearance of the holes and concomitantly to a complete dissolution of the film. When the release of polyelectrolytes into the solution is prevented by a poly(diallyldimethyl ammonium chloride)/poly(styrene sulfonate) (PDADMAC/PSS) capping film, the holes appear at a smaller critical ionic strength compared to uncapped films. Here too the formation of the holes is attributed to an increase of the osmotic pressure inside the film. As soon as the capping barrier ruptures because of the swelling of the film, both PLL and HA chains can diffuse out of the film and the holes decrease in size and disappear, as does the film.

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.

Transfection Ability and Intracellular DNA Pathway of Nanostructured Gene-Delivery Systems

Considerable efforts have been devoted to the design of structured materials with functional properties. Polyelectrolyte multilayer films are now a well-established nanostructured concept with numerous potential applications, in particular as biomaterial coatings. This technique allows the preparation of nanostructured architectures exhibiting specific properties for cell-activation control and local drug delivery. In this study, we used a multilayered system made of poly-(l-lysine)/hyaluronic acid (PLL/HA) as a reservoir for active DNA complexes with nonviral gene-delivery vectors, PLL, beta-cyclodextrin (CD), and PLL-CD. When embedded into the multilayered films, the transfection efficiencies of the DNA complexes and the cell viability were improved. The highest transfection efficiency was obtained with the PLL-CD/plasmid DNA (pDNA) complexes. We found that this high transfection efficiency was related to an efficient internalization of the complexes in the cell cytoplasm and selected nuclei domains through a nonendocytotic pathway. For the first time, we report the intracellular pathway of the pDNA in complexes incorporated into the multilayered system.

Micro-stratified architectures based on successive stacking of alginate gel layers and poly(l-lysine)-hyaluronic acid multilayer films aimed at tissue engineering.

A micro-stratified 3D scaffold was designed by successive stacking of alginate gel layers (AGLs) and poly(l-lysine)-hyaluronic acid (PLL-HA) multilayer films. AGLs are obtained by complexation of alginate by Ca2+ ions. Alginate solutions are first sprayed onto a solid substrate inclined such that the excess of solution be removed by natural drainage. A CaCl2 solution is then either sprayed onto the substrate or the alginate covered substrate is dipped into a CaCl2 solution. The spraying of the CaCl2 solution leads to micro-porous AGLs, whereas the dipping in a CaCl2 aqueous solution leads to a more homogeneous gel layer without porosity. The second process also allows the formation of AGLs with a controlled thickness. With the goal of stacking different AGLs and PLL-HA films, the influence of a PLL-HA precursor film on the formation of AGLs is firstly investigated. It is found that when an alginate solution is sprayed on a PLL-HA multilayer built in the presence of CaCl2, the multilayer plays the role of reservoir of Ca2+ ions and of PLL chains, which both diffuse out of the multilayer film and complex alginate chains. This leads to the formation of a "pre-alginate gel". When this film is further dipped in the CaCl2 solution, an additional AGL forms, which is, however, free of PLL chains. Finally after the build-up of a PLL-HA film on the top of AGL, we succeeded in designing micro-stratified 3D scaffolds constituted by alternating strata of AGLs and PLL-HA films. This micro-stratified gel provides a new scaffold design with a perfectly controlled build-up: AGL aims to be a 3D scaffold for cell culture, and the PLL-HA multilayers should act as reservoirs for biologically active molecules.

Multiple Strata of Exponentially Growing Polyelectrolyte Multilayer Films

Polyelectrolyte multilayers are potential candidates for bioactive coatings with time scheduled biological activity. One way to achieve this goal is to use coatings constituted of several successive multilayers. We demonstrate in the present study that the buildup of successively two exponentially growing multilayers, namely a poly(l-lysine)/poly(l-glutamic acid) (PLL/PGA)n film on top of a poly(l-lysine)/hyaluronic acid (PLL/HA)m precursor film is possible. The growth process of (PLL/PGA)n remains exponential and the presence of the underneath (PLL/HA)m multilayers enhances the film growth when compared with a (PLL/PGA)n film constructed on a solid substrate. PGA also diffuses into the entire (PLL/HA)m multilayer and forms complexes with free PLL chains present in the precursor architecture. As the buildup of (PLL/PGA)n progresses, the PLL chains deposited on top of the film loose gradually their ability to diffuse into the (PLL/HA)m film. These results should have far reaching consequences on the buildu...

Polyelectrolyte Multilayer Films as Substrates for Photoreceptor Cells

Reconstruction of extracellular matrix substrates for delivery of functional photoreceptors is crucial in pathologies such as retinal degeneration and age-related macular degeneration. In this study, we assembled polyelectrolyte films using the layer-by-layer deposition method. The buildup of three different films composed of poly(L-lysine)/chondroitin sulfate (PLL/CSA), poly(L-lysine)/poly(styrenesulfonate) (PLL/PSS), or poly(L-lysine)/hyaluronic acid (PLL/HA) was followed by means of quartz crystal microbalance measurements, optical waveguide light mode spectroscopy, confocal microscopy, and atomic force microscopy. The exponential growth regime and the diffusion of PLL chains from the bulk through the PLL/CSA, PLL/PSS, and PLL/HA films was examined. Evaluation of photoreceptor cell viability was optimal on one layer of PLL (PLL(1)), followed by 10 bilayers of PLL/HA [(PLL/HA)(10)] and 10 bilayers of PLL/CSA [(PLL/CSA)(10)]. The number of bilayers and the type of terminating layer also had a significant influence on the number of photoreceptor cells attached. Functionalized polyelectrolyte multilayer films were obtained by adsorbing basic fibroblastic factor (bFGF) or the insoluble fraction of interphotoreceptor matrix (IPM) on or within polyelectrolyte multilayers. bFGF and IPM adsorption on top of the (PLL/CSA)(10)/PLL polyelectrolyte films increased the number of photoreceptor cells attached and maintained the differentiation of rod and cone cells.

Polyelectrolyte Multilayers and Degradable Polymer Layers as Multicompartment Films

Polyelectrolyte multilayers are now a well established concept with numerous potential applications in particular as biomaterial coatings. To timely control the biological activity of cells in contact with a substrate, multicompartment films made of different polyelectrolyte multilayers deposited sequentially on the solid substrate constitute a promising new approach. In a first paper (Langmuir 2004, 20, 7298) we showed that such multicompartment films can be designed by alternating exponentially growing polyelectrolyte multilayers acting as reservoirs and linearly growing ones acting as barriers. In the present study, we first demonstrate however that these barriers composed of synthetic polyelectrolytes are not degraded despite the presence of phagocytic cells. We propose an alternative approach where exponentially growing poly(L-lysine)/hyaluronic acid (PLL/HA) multilayers, used as reservoirs, are alternated with biodegradable polymer layers consisting in poly(lactic-co-glycolic acid) (PLGA) and acting as barriers for PLL chains that diffuse within the PLL/HA reservoirs. We first show that these PLGA layers can be deposited alternatively with PLL/HA multilayers leading to polyelectrolyte multilayer/hydrolyzable polymeric layer films and acting as a reservoirs/barriers system. Bone marrow cells seeded on these films ending by a PLL/HA reservoir rapidly degrade it and internalize the PLL chains confined in this reservoir. Then the cells degraded locally the PLGA barrier and internalize the PLL localized in a lower (PLL/HA) compartment after 5 days of seeding. By changing the thickness of the PLGA layer, we hope to be able to tune the time delay of degradation. Such mixed architectures made of polyelectrolyte multilayers and hydrolyzable polymeric layers could act as coatings allowing us to induce a time scheduled cascade of biological activities. We are currently working on the use of comparable films with compartments filled by proteins or peptides and in which the degradation of the barriers results from a hydrolysis over tunable time scales.